impaginato foreword throughout the nineteenth century and for much of the twentieth century, the definition of secondary metabolites simply meant that these substances, while present in plants, did not participate in the metabolic processes essential to the life of the organism. they were defined as by­products of primary metabolism or as excretory products or final products of metabolism, always pointing out that their irregular presence on plants meant that they were not indispensable. over the last 50 years, knowledge in this area has been greatly expanded, especially following the development of new isolation, separation and structural identification technologies (spectroscopy, nuclear magnetic resonance and mass spectrometry). the ease of structural identification has expanded the number of secondary metabolites to reach the unthinkable number of 200000 for many of which, however, remain uncertainty about their biochemical and physiological role. in recent years, however, many researches have progressively clarified the role of these substances and some of them are known to have a fundamental function. thus it has been for shikimic acid, for many years considered a simple metabolite of illicium anisatum, then revealed as a key molecule for synthesis of aromatic amino acids and, in turn, precursors of phenylpropanoids (flavonoids, coumarin, tannins and lignin). certainly these are not secondary functions! primary metabolism is essential to plants for growth and development, and secondary metabolism helps plants to interact with the environment. many plant metabolites are also industrially important (e.g.: the physiologically active alkaloids used in modern medicine). these metabolites are produced by plants through complex metabolic pathways. among the researchers in this area it is inevitable to remember j.b. harborne, who has been devoting some 50 years to the study of flavonoids and, more generally, to chemical ecology and plant chemosystematics. harborne’s work has called for experimentation on all other classes of secondary metabolites, alkaloids, non­protein amino acids, glucosinolates, lectins, terpenes, steroids, tannins, flavonoids, phenylpropanoids, lignins, coumarin, waxes, etc. plant secondary metabolites are critical to various biological processes. the plant’s specific organization has led it to produce a large amount of secondary metabolites and probably selective pressure has left those molecules capable of conferring specific benefits to the plant: defense role against animal, fungal and bacterial parasites, but also those molecules that allow to establish relationships between plants of the same species or between different species. numerous contributions to this special number of ahs are devoted to ecological biochemistry and that explains the “non­ secondary” role of so many metabolites; it is also hoped that the molecular biological tools will spread to the labs in order to have accurate information on the genes involved in the various secondary metabolism. plant genomes are variously estimated to contain 20000­60000 genes, and perhaps 15­25% of these genes encode enzymes for secondary metabolism. still a lot of research work has to be done both physiologically and biochemically on the secondary metabolism of many species, but i believe it is essential that laboratories use data availability about the genes of arabidopsis, an extraordinary model species, involved in secondary metabolism. thus, our knowledge of secondary metabolites, which have already made great advances over the last decades, will make further progress in both physiological and biochemical and technological terms, confirming that their traditional placement as “secondary metabolism products” was only due to lack of knowledge. amedeo alpi ahs vol. 31 no. 2 (2017): special issue on "plant and food metabolomic" 111 book reviews peppers. botany, production and uses. russo vincent m. (ed.). cab international, wallingford, oxfordshire, uk, 2012, pp. 280. isbn 978-1-84593-767-6. € 110.00. the purpose of the book (which contains 19 chapters) is to provide a general description of the world of peppers. even though difficulties exist with pepper taxonomy, the book presents a good introduction where all disputes are clearly elucidated and discussed. botany and cultivation techniques are widely described and throughout the book several unanswered questions relating to the lack of information about peppers emerge, keeping up the reader’s interest. some information can result a bit redundant in the chapters, which are written by different authors, but this offers the reader the opportunity to easily jump from one part to another. moreover, all the chapters are clearly written and present a thorough bibliography, making the book suitable for both scientists and common devotees of peppers. the book can be used as a manual for cultivation, starting from a single plant to a massive production of peppers, with both biotic and abiotic stresses properly described. particular attention is given to cultivation problems such as pests and weed control and for descriptions and reports, causes and effects of various types of diseases are also listed. other common problems relating to peppers and analyzed in the book include irrigation, fertilization and soil composition, and economic and logistic problems regarding field and greenhouse production of plants. from the molecular side, the book is a good anthology of the majority of molecules involved in peppers and underlying their functions and physiology. while a large part of the work is fully dedicated to field production, a brief chapter is dedicated to tissue culture of peppers despite their recalcitrant nature. finally, germination and transplant techniques are illustrated, making this book complete and useful for a wide audience. diego comparini stolo. bibliographische findmitell zur gartenkultur. band i. italien. strumento bibliografico sulla cultura dei giardini. volume 1. italia. (bibliographic tools for the culture of gardens. volume 1. italy) schneider u., and g. gröning. wernersche verlgasgesellschaft mbh, worms, germany, 2009. pp. 576. isbn 978-3-88462-248-3 this volume, dedicated to italy, is the first of nine in the series stolo on the history and cultural theory of gardens and related topics in the center-west of europe. each volume treats a single country and can be consulted individually, as the material presented pertains to the subject country. at the same time, the ample literature reported opens up international connections, presenting to the reader the geographic impact and extension of the treated topic. the work required to compile the bibliographic material was carried out between 2004 and 2007, and required numerous trips to public and private libraries. in its complex, this work by stolo is a notable tool to access the history of gardens in various european countries, and it is presented in a way that is easy-to-use and in-depth. this particular work on italy is divided into eight chapters: sull’importanza sistematica delle conclusioni letterarie nella cultura dei giardini. (the systematic importance of literary conclusions in terms of garden culture) 2. strumenti bibliografici generali e documentazione letteraria. (general bibliographic tools and literary documentation) 3. bibliografie specifiche e strumenti bibliografici sulla cultura dei giardini e materie affini. (specific bibliographies and bibliographic tools regarding the culture of gardens and related topics) 4. manuali e opere generali sulla cultura dei giardini come contributi e temi speciali. (manuals and general works on the culture of gardens as contributions and special themes) 5. materiale primario. (primary material) 6. periodici e collane (periodicals and series) 7. ricerca in internet. (internet research) 8. organizzazioni, federazioni, associazioni, biblioteche nella cultura dei giardini. (organizations, federations, associations and libraries for the culture of gardens). enrico rinaldelli 152 1. introduction in recent years, consumer demand for ready-to-eat fruits and vegetable products, has increased considerably due to product added value and easy consumption. a major challenge facing this industry is maintaining and preserving the quality of minimally processed produce because the physical damage caused by preparation accelerates the metabolism with associated increases in certain biochemical reactions responsible for quality loss (sabir et al., 2011). table grape is a non-climacteric fruit with a low rate of physiological activity that is very sensitive to water loss and fungal infection (botrytis cinerea) during postharvest handling and cold storage. gray mold is the most aggressive post harvest disease because of its ability to develop at low temperatures. for this reason, it is standard practice to fumigate table grapes with sulfur dioxide (so 2 ) immediately after packing (crisosto and mitchell, 2002). however, sulphite residue is an important consumer problem. to avoid it, different technologies have been developed such as controlled atmosphere (ca), storage with high co 2 (15-25%), and hot water and ethanol (crisosto et al., 2002; karabulut et al., 2004), although injuries occur, like rachis browning and off-flavors. one of the most effective technologies in terms of quality commodity preservation is the combination of modified atmosphere and packaging (map) that protects from water loss and browning, and reduces chilling injury symptoms (kader et al., 1989). martinez-romero et al. (2003) showed that the use of map maintained table grape berry quality. the aim of the present work was to study the effects of map on quality parameters of minimally processed white ‘vittoria’ and red ‘red globe’ table grape cultivars during 28 days of cold storage. 2. materials and methods plant material white and red table grapes (cv. vittoria and cv. red globe) were harvested from a commercial vineyard, located in mazzarrone italy (37° 05’ n, 14° 34’ e, 128 m asl, ct). the vineyard has a row spacing of 2.8 m × 2.8 m (with 1275 plants/ha) with “tendone” training system, covered with net. white ‘vittoria’ (july) and red ‘red globe’ (september) table grapes were hand-picked at the commercially ripe stage (≥12.5°brix), suitable for the fresh fruit market. immediately after harvest, table grape quality parameters were analyzed on three clusters per cultivar. sample preparation and packaging after harvest, ‘vittoria’ and ‘red globe’ clusters were immediately transported to the laboratory and selected on effects of modified atmosphere packaging on quality parameters of minimally processed table grapes during cold storage g. liguori, g. sortino(*), c. de pasquale, p. inglese dipartimento di scienze agrarie e forestali, università degli studi di palermo, viale delle scienze, 90128 palermo, italy. key words: fresh-cut, map, postharvest performance, v. vinifera. abstract: ‘vittoria’ and ‘red globe’ table grapes were packed in microperforated polypropylene films (passive map) and non-perforated polyethylene (active map: 1) 20% co2 + air; 2) 5% o2 + 15% co2 + n) and stored at 5 °c for 28 days. microperforated polypropylene packages had the highest postharvest performance in both cultivars until 14 days of cold storage, with reduced weight losses, rachis and berry decay. total soluble solids content slightly increased in non-perforated polyethylene boxes as a consequence of the higher weight losses, particularly in active map with 5% o2 + 15% co2 + n, while no significant differences were found for titratable acidity. non-perforated polyethylene packages showed excessively high co2 concentrations (30-60%) in headspace gas composition, causing berry and rachis decay. the highest post harvest performances in terms of weight losses, total soluble solids, titratable acidity, crunchiness, juiciness and berry decay were found with micro-perforated polypropylene packages after 14 d of 5 °c cold storage, and 6 days of shelf-life (15-18°c). adv. hort. sci., 2015 29(2-3): 152-154 (*) corresponding author: giuseppe.sortino@unipa.it received for publication 26 september 2014 accepted for publication 26 may 2015 153 liguori et al., effects of modified atmosphere packaging on quality parameters of minimally processed table grapes during cold storage the basis of uniform size, color, firmness, lack of diseases, health and greenish rachises. table grape clusters were then cut with sanitized scissors to obtain a 200 g cluster. the samples were sterilized with sodium hypochlorite solution (150 ppm active chlorine) and were irradiated with ultraviolet-c (uv-c) to control microbial spoilage. after washing, each 200 g cluster sample was packaged in a 15×8 cm rigid polypropylene retail box and sealed with two different films: microperforated polypropylene and non-microperforated polyethylene. the boxes sealed with microperforated polypropylene film were packaged in air (passive map), while, the boxes sealed with nonmicroperforated polyethylene film were packaged under two different initial headspace gas compositions (active map: 1) 20% co 2 + air; 2) 5% o 2 + 15% co 2 + n). the sealed boxes were then stored at 5°c with a relative humidity of 90% for 28 days, with quality evaluation performed on days 0, 7, 14, 21, and 28, after the shelf-life (5 days at 15-18°c). quality parameters: firmness, soluble solids, titratable acidity and decay for each treatment, 12 randomly chosen boxes, were taken for shelf life at each sampling date (7, 14, 21, 28 days) and analyzed for weight loss, soluble solid content (ssc), ph, titratable acidity (ta) and decay. berry weight loss was calculated as a percentage of its fresh weight (at the beginning of each shelf life). total soluble solids (tss) were determined by digital refractometer (palette pr-32, atago co., ltd) and titratable acidity (ta) by titration of 10 ml homogenized berry flesh juice with 0.1 n naoh to an endpoint of ph 8.1 and expressed as the percentage of tartaric acid. (mod. s compact titrator, crison instruments. headspace gas composition o 2 and co 2 contents of the packages were measured using an o 2 and co 2 portable analyzer (checkpoint, dansensor italia, segrate, milano, italy) during each shelf-life, after 7, 14, 21, 28 days at 5°c. statistical analysis data were submitted to one-way analysis of variance (anova) and means were separated with tukey’s test at p≤0.05. the statistical analysis was carried out using systat 10 (systat, usa). 3. results and discussion weight loss (fig. 1a) of ‘red globe’ packaged with non-microperforated polyethylene sealed boxes (active map: 20% co 2 +air (pet1); 5% o 2 +15% co 2 +n (pet2) increased sharply during storage, reaching values lower (8% and 14 % respectively) than those measured at the beginning of the shelf-life. weight losses of ‘red globe’ grape packaged with microperforated polypropylene boxes (passive map (pp) were significantly lower than pet1 and pet2, with weight losses of 4% during shelf-life. tss content did not significantly change (p<0.05) in ‘red globe’ table grapes wrapped with pp (passive map) at the end of the shelf-life (fig. 1b), while a significant increase (p<0.05) in tss was found in table grapes packaged in both active pet1 and pet2 (active map), with an increase of 4% and 6% at the end of the shelf-life (fig. 1b). non-perforated polyethylene packages (pet1 and pet2) showed excessively high co 2 concentrations (from 30 to 60%) in headspace gas composition of ‘red globe’ and ‘vittoria’ table grapes, causing berry and rachis decay (fig. 1c, 2c). non-microperforated polyethylene sealed boxes of both cultivars (pet and pet2), showed a similar trend in fig. 1 changes in weight loss (a), soluble solid content (b) and headspace co 2 concentration (c) of minimally processed ‘red globe’ grape berries during shelf life after 14 days of cold storage (5°c): microperforated polypropylene packaging in air (pp); non-microperforated polyethylene packaging in 20% co 2 +air (pet1); non-microperforated polyethylene packaging in 5% o 2 +15% co 2 +n (pet2). data are mean±se. * indicate significant differences for values (p≤0.05). 154 adv. hort. sci., 2015 29(2-3): 152-154 terms of weight loss. indeed, berries had 9% and 14% decrease of weight loss during the shelf-life while pp table grape boxes showed a significantly lower decrease (4%) compared to pet1 and pet2. (fig. 2a). tss of pet1 and pet2 ‘vittoria’ berries were significantly higher (p<0.05) than those of pp treatments, with an increase of 5% and 8% respectively at the end of the shelf-life (fig. 2b). a good visual aspect and no off-flavor were detected until 21 days of cold storage in ‘vittoria’ pp table grapes (data not shown). packaging did not affect ta in either of the cultivars. microperforated polypropylene packages (pp) had the highest postharvest performance in both cultivars until 14 days of cold storage, also in terms of rachis and berry decay. 4. conclusions ‘vittoria’ and red globe’ grapes showed the best postharvest performances in terms of weight losses, tss, ta, crunchiness, juiciness and berry decay when packed with micro-perforated polypropylene packages after 14 days of 5°c cold storage and 6 days of shelf-life (1518°c). this study has demonstrated that active map (pet1 and pet2) did not affect the postharvest performances of minimally processed table grapes. as costa et al. (2011) reported, the conditions created in the package to modify the initial headspace composition probably compromised the mass loss and the sensory acceptability of the fresh produce. ‘vittoria’ berries and rachises from pp boxes remained healthy and green, even after 21 days of 5°c storage, which could be useful for consumers interested in readyto-eat products. acknowledgements the work reported in this paper has been presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 2223 may 2014. references costa c., lucera a., conte a., mastromatteo m., speranza b., antonacci a., del nobile m.a., 2011 effects of passive and active modified atmosphere packaging conditions on ready-to-eat table grape. j. of food eng. 102: 115-121. crisosto c.h., garner d., crisosto g., 2002 carbon dioxide-enriched atmospheres during cold storage limit losses from botrytis but accelerate rachis browning of ‘red globe’ table grapes. postharvest biol. technol., 26: 181-189. crisosto c.h., mitchell f.g., 2002 postharvest handling systems: table grapes, pp. 357-363. in: kader a.a. (ed.) postharvest technology of horticultural crops. university of california, usa, pp. 535. kader a.a., zagory d., kerbel e.l., 1989 modified atmosphere packaging of fruits and vegetables. crit. rev. food sci. nutr., 28: 1-30. karabulut o.a., gabler f.m., mansour m., smilanick j.l., 2004 postharvest ethanol and hot water treatments of table grapes to control gray mold. postharvest biol. technol., 34: 169-177. martinez-romero d., guillen f., castillo s., valero d., serrano m., 2003 modified atmosphere packaging maintains quality of table grapes. j. food sci. 68: 1838-1843. sabir a., sabir f.k., kara z., 2011 effects of modified atmosphere packing and honey dip treatments on quality maintenance of minimally processed grape cv. razaki (v. vinifera l.) during cold storage. j. food sci. technol., 48(3): 312-318. fig. 2 changes in weight loss (a), soluble solid content (b) and headspace co 2 concentration (c) of minimally processed ‘vittoria’ grape berries during shelf life after 14 days of cold storage (5°c): microperforated polypropylene packaging in air (pp); non-microperforated polyethylene packaging in 20% co 2 + air (pet1); non-microperforated polyethylene packaging in 5% o 2 +15% co 2 +n (pet2). data are mean±se. * indicate significant differences for values (p≤0.05). 25 1. introduction tomato is the leading vegetable crop grown throughout the world and it is also the number one vegetable crop in jordan. the statistics of the ministry of agriculture (moa, 2012) reveal that the total tomato area cultivated is 12344.5 ha producing an average of 73.4 tons per hectare. in addition, the statistics indicate that the total number of plastic houses is extensively increasing. in jordan, the use of various types of mulches has become a well-established practice over recent years. it is well known that mulching helps to maintain healthy vegetable crops. the benefits of using several types of mulches have been extensively studied and recognized. however, with the global call for organic agricultural production and reduction of pesticide and fertilizer use, mulches will continue to be used. peet (1992) summarized the environmental, cultural, and anatomical factors that can increase the incidence of cracking as: irregular watering, high temperatures and light, fruit anatomy, excessive rapid fruit growth, genetical differences among cultivars, high differences between day and night temperatures, and high humidity. peet and willits (1995) concluded that growers should reduce watering tomato cultivars that are crack-prone, particularly when yielding from the upper clusters. the cuticular membrane in the outer epidermal periclinal walls of both resistant and intermediate cultivars was thicker when compared to cultivars that exhibit cracking (matas et al., 2004). cracking is mainly caused by extreme changes in fruit growth rate caused by moisture fluctuations (swiader et al., 1992). thus, the growth rate is affected by mulching material by means of manipulating the microclimate (bender et al., 2008; abubaker, 2013). differences in the microclimate, depending on the mulch material, will influence the growth rate and hence could affect fruit cracking incidence. therefore, the objective of this study was to investigate the effect of different types of mulches on ‘newton’ tomato yield and fruit cracking incidence under plastic house conditions. 2. materials and methods the research was conducted at al-balqa’ applied university research station during the 2011/2012 growing season under a green plastic house (25 m long, 9 m wide, and with a height of 2.7 m) covered with a single glaze of 200 micron clear polyethylene film. during the summer season 2011, soil solarization was applied against soil borne pests. soil was then disked and prepared for laying the types of mulches used in the study. seven types of mulches were used: black plastic, tuff gravel (thickness of covering 6-7 cm), clear plastic, compost (thickness of covering 6-7 cm), crushed stone (thickness of covering 6-7 cm), shredded wood (thickness of covering 6-7 cm), and the control (no effect of different types of mulches on ‘newton’ tomato yields and fruit cracking under plastic greenhouse conditions s. abubaker (1), i. qrunfleh, m. hasan department of plant production and protection, faculty of agricultural technology, al-balqa’ applied university, al-salt 19117, jordan. key words: lycopersicon esculentum m., mulching, tomato cracking. abstract: this study was conducted at al-balqa` applied university research station to investigate the effect of different types of mulches on ‘newton’ tomato yields and fruit cracking incidence under greenhouse conditions. the experiment consisted of seven treatments (black plastic, tuff gravel, clear plastic, compost, crushed stone, shredded wood, and the control); a randomized complete block design with three replicates was used. different mulch types showed significant effects on early, medium, late, and total yields/ha of the tomato fruits. higher early and medium yields were obtained using black and clear plastic. compost resulted with the highest total yield. results of this study clearly showed that mulching improves total tomato yields under greenhouse conditions. in addition, larger fruits were obtained by applying mulching. tomato cracking was also slightly affected by the mulch types used in this study. adv. hort. sci., 2014 28(1): 25-28 (1) corresponding author: samih_abubaker@yahoo.com received for publication 26 march 2014 accepted for publication 12 may 2014 26 mulch). these were arranged in a randomized complete block design with three replicates for each mulch type. five to six-week-old seedlings of the commercial indeterminate tomato (lycopersicon esculentum m. var. ‘newton’), with an average of 12 cm height, were transplanted in december 2011. each experimental unit consisted of a 3 m row length, with a 1 m space between every two rows. seedling distances were 30 cm between plants with ten plants per plot. the soil surface was covered with the designated type of mulch just before transplanting and it remained covered until the end of the growing season. tomato plants were then trained to one stem by continuous removal of auxiliary shoots. soil moisture content was monitored using a mpkit-160 soil moisture meter. a drip irrigation system was employed to irrigate and fertilize the plants according to local commercial tomato, plastic-house grower practices. weeds were removed by hand and pest management control practices were applied throughout the study. growth parameters (plant height, stem diameter, total number of leaves, and dry matter) were recorded (abubaker, 2013). total yield of ripe fruits was determined by recording the consecutive weights of 24 hand-harvested fruits from 6 march to 10 june 2012. total yield was subdivided into three categories: early, medium, and late yields consisting of eight harvests each. the total number of cracked fruits and the percentages were recorded for the three harvest dates. all statistical analyses were performed using sas/stat version 9.2 and analysis of variance was conducted by the proc glimmix procedure. means were separated following the fisher’s protected least significant difference (lsd) test. 3. results and discussion the different mulch types showed significant effects on early, medium, late, and total yields (t/ha) of the tomato fruits (table 1). tomato plants grown under black, clear plastic, and shredded wood mulches resulted in the highest early yields, which were significantly higher than that of the control (table 1). however, tuff gravel, crushed stone, and the plants grown using compost resulted in higher early yields compared to the control but they were not significantly different. swiader et al. (1992) mentioned that black, gray, and transparent mulches raise soil temperatures. thus, the rate of growth for plants grown under those mulches will increase. the mean yields showed fewer differences between the various types of mulches (table 1). black and clear plastic resulted in higher mean yields, however they were not significantly different from that of the control. compost also resulted in higher mean yields that were not significantly different from the control and the slow release of nutrients from the organic material early in the season may be a possible explanation. tomato plants that were grown using shredded wood and tuff gravel as a mulches recorded the lowest mean yields: 56.1 and 57.4 t/ha, respectively (table 1). however, these yields were not significantly different compared to the control. late yields showed less difference among the various types of mulches (table 1), although the highest were recorded with tuff gravel (60. 2 t/ha) and compost (59.7 t/ha). these yields were significantly different compared to the yields of plants growing without mulch. shredded wood also resulted in higher late yields that were significantly compared to the control. in addition, the control yields (48.1 t/ha) showed higher late yields but they were not significantly different compared to the clear plastic (46.4 t/ha), black plastic (43.5 t/ha), and crushed stone (43.3 t/ha). the highest overall yields were obtained using compost (table 1). the descending order from highest to lowest total yields was found to be: compost, black plastic, clear plastic, tuff gravel, shredded wood, control, and finally crushed stones. the total yields of plants grown under compost, black plastic, clear plastic, and tuff gravel were significantly different compared to the control but were not significantly different compared to the shredded wood mulch. crushed stone mulch gave the lowest total yields (144.2 t/ha), which were not significantly different compared to the control and shredded wood much. the soil temperature-increasing effect of black mulch and the late season release of organic material from the compost explain the ability of such mulches to give superior production compared to the other types. our results coincide with kayum et al. (2008) and bay (2011). both researchers showed that table 1 yield (early, medium, late, and total) and average fruit weight of greenhouse tomato ‘newton’ under different types of mulches mulch type early yield (t/ha) medium yield (t/ha) late yield (t/ha) total yield (t/ha) fruit weight (g) black plastic 46.5 a 70.1 a 43.5 b 160.1 a 173.4 ab tuff gravel 38.7 bc 57.4 b 60.2 a 156.3 a 169.5 abc clear plastic 45.2 a 66.3 a 46.4 b 157.9 a 174.7 a compost 36.7 c 66.0 a 59.7 a 162.4 a 175.1 a crushed stone 37.7 bc 62.2 ab 43.3 b 144.2 b 150.5 d shredded wood 43.3 ab 56.1 b 56.2 a 155.6 ab 160.8 bcd control 33.2 c 63.3 ab 48.1 b 144.6 b 160.2 cd lsd 0.05 6.29 8.11 7.45 11.48 13.15 different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 27 mulching significantly affected yield components, increased yield, and improved fruit quality. regarding fruit weight, the largest fruits were obtained using compost, clear and black plastic with average weights of 175.1, 174.7, and 173.4 g/fruit (table 1). these weights were not significantly different compared to tuff gravel. the lowest fruit weights were achieved when crushed stone was used, followed by the control and shredded wood. there were no significant differences with regard to the lowest fruit weights. dry matter contents of leaves and stems were significantly affected by mulch types (table 2). tomato dry matter was highest when grown using the compost as a mulching material (19.4%); differences with crushed stone (18.8%) and black plastic (18.3%) were not significant. abubaker (2013) attributed similar findings to the higher amounts of available minerals released from the compost which also manifested favorable effects on available water content during the growing season, directly affecting vegetative growth. regarding fruit cracking, average values are presented in figure 1 and the average percentage of cracked fruits are presented in figure 2. the results clearly indicate no significant differences between the seven mulch types used in our study. however, the average number of cracked fruits was highest when compost was used, and it is worth mentioning again that the highest overall yields were obtained using this type of mulch. however, slight differences were observed in the average percentage of cracked fruits with regard to the total number of fruits, particularly between the clear plastic and control treatments but these differences were not significantly different. our results are in agreement with suwwan et al. (1988) who indicated that seasonal cracking was not affected by the five mulch types they studied (i.e. silver plastic, black plastic, paper, white/black plastic and black/white plastic). 4. conclusions mulching improved growth parameters and yields of ‘newton’ tomato grown under plastic house conditions. the highest overall yields were obtained using compost followed by black plastic. no significant differences among types of mulches were observed regarding the number of cracked fruits. however slight, though not significant, differences were found when considering the average number of cracked fruits compared to the total number of fruits. references: abubaker s., 2013 effect of different types of mulch on performance of tomato (lycopersicon esculentum m.) under plastic house conditions. journal of food, agriculture & environment, 11(2): 132-134. bay b., 2011 effect of mulching and amount of water on the yield of tomato under drip irrigation. journal of horticulture and forestry, 3(7): 200-206. bender i., raudseping m., vabrit s., 2008 effect of organic mulches on the growth of tomato plants and quality of fruits in organic cultivation. acta horticulturae, 779: 341-346. kayum m.a., asaduzzaman m., haque m., 2008 eftable 2 dry matter percentages of ’newton’ tomato leaves and stems grown under different types of mulches mulch type dry matter of leaves and stems (%) black plastic 18.3 ab tuff gravel 17.8 b clear plastic 17.6 b compost 19.4 a crushed stone 18.8 ab shredded wood 17.7 b control 17.6 b lsd 0.05 1.30 different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 0 5 10 15 20 25 bp g cp c no s w n u m b er o f c ra ck ed f ru it s mulch type fig. 1 the average number of cracked `newton` tomato fruits for the three harvest dates for each mulch type. bp= black plastic, g= gravel, cp= clear plastic, c= compost, no= no mulch (control), s= stone, and w= wood. 0 5 10 15 20 25 bp g cp c no s w p er ce n ta g e o f c ra ck ed f ru it s mulch type fig. 2 the percentage of cracked `newton` tomato fruits for the three harvest dates for each mulch type. bp= black plastic, g= gravel, cp= clear plastic, c= compost, no= no mulch (control), s= stone, and w= wood. 28 fects of indigenous mulches on growth and yield of tomato. journal of agriculture & rural development, 6(1&2): 1-6. matas a.j., cobb e., paolillo d., niklas k., 2004 crack resistance in cherry tomato fruit correlates with cuticular membrane thickness. hortscience, 39(6): 1354-1358. moa, 2012 annual report. ministry of agriculture, amman, jordan. peet m.m., 1992 fruit cracking in tomato. horttechnology, 2(2): 216-223. peet m.m., willits d., 1995 role of excess water in tomato fruit cracking. hortscience, 30(1): 65-68. suwwan m., akkawi m., al-musa a.m., mansour a., 1988 tomato performance and incidence of tomato yellow leaf curl (tylc) virus as affected by type of mulch. scientia horticulturae, 37(1-2): 39-45. swiader j., ware g., mccollum j., 1992 producing vegetable crops. fourth edition, interstate publishers, inc., danville, illinois, usa 116 1. introduction camellia japonica l. is an important ornamental shrub commonly used as potted plant and for landscaping (accati et al., 2006; scariot et al., 2007). generally, camellia flowers on the plants last for seven to ten days, and flower senescence is mainly characterised by browning regions on the petals and by floral organ abscission. on the contrary, when flowers are removed from the plant, they usually wilt quickly unless they are held at low temperatures and high humidity (bonner and honda, 1950). for this reason the use of camellia as cut flower has been restricted to situations where longevity is not especially important, even if the deep and bright green foliage with a high number of flower buds make camellias potentially appreciated also as cut flowering branches. only a few studies have been performed on this topic. in the 1950’s considerable efforts were made to find treatments to extend camellia cut flower vase life by cothran (1958). this author showed that poor water relations lead to early wilting of cut camellia blooms. therefore, most of the efforts were directed towards the reduction of vapour pressure deficit or to prevent water loss from the petals. for better conservation, threlkeld (1962) suggested harvesting flowers with no stem, holding them at high humidity. more recently, doi and reid (1996) reported that in the first day of vase life, the camellia flower is unable to acquire sufficient water from the vase solution to supply the needs of petal expansion and transpiration. the role of ethylene in camellia senescence have been also envisaged. woolf et al. (1992) reported that abscission of camellia flower buds was induced by a foliar spray with 2-4 ml l-1 ethephon [(2-chloroethyl)phosphonic acid] and that the vegetative buds and leaves were less sensitive to ethephon than flower buds. doi and reid (1996) used silver thiosulphate (sts) to prevent flower abscission. the negative effects of ethylene can be significantly delayed by treatment with inhibitors of ethylene action, such as 1-methylcyclopropene (1-mcp) (serek et al., 2006). the possible value of such treatment in improving the life of cut camellia flowering branches has not been explored. recently, a non-volatile formulation of 1-mcp in β-cyclodextrinbased nanosponges (β-cd-ns 1:8) was developed (seglie et al., 2011 a, b). this structure, supplied in the conservation solution, prolonged the vase life of several cut flowers better than gaseous 1-mcp (seglie et al., 2013). since there is a lack of knowledge on postharvest characteristics of c. japonica cut flowers and foliage, the objectives of this study were to evaluate the role of ethylene in postharvest flower and leaf abscission of seven cultivars ethylene and the postharvest performance of cut camellia flowering branches m. caser 1, l. seglie 1, r. bizioli 2, v. scariot 1(*) 1 dipartimento di scienze agrarie, forestali e alimentari, università di torino, largo paolo braccini, 2, 10095 grugliasco (to), italy. 2 fiori tipici del lago maggiore e del biellese s.c.r.l., s.s. del sempione, 4, 28831 baveno (vb), italy. key words: ethylene antagonist, ethylene production, flower longevity, senescence, vase life. abstract: camellias are potentially appreciated as cut branches due to their deep and bright green foliage with a high number of flower buds. the present research investigated the role of ethylene in postharvest flower and leaf abscission of seven cultivars of camellia japonica and evaluated the effect of inhibitors of ethylene action on their vase life. cut flowering branches were shipped from private companies, mimicking a long and short supply chain. plant materials were treated with commercial chrysal, gaseous 1-mcp, and 1-mcp included in β-cyclodextrin-based nanosponges and compared to cut branches exposed to exogenous ethylene (1 ± 0.2 µl l-1) for 24 h and control in tap water. visual checks for symptoms of variation in senescence, flower opening stage, flower abscission, leaf chlorophyll content (spad) and gloss, in addition to ethylene production levels were monitored daily. cut branches placed in tap water lost ornamental value after ca 5.5 days of vase life and reached complete senescence after about 10 days in all the studied cultivars, regardless of supply chain, with the exception of ‘il tramonto’ with 10 and 20 days, respectively. exogenous ethylene application reduced camellia cut branch longevity in ‘debbie’ and ‘r.h. wheeler’, while the other cultivars lasted as long as the control. anti-ethylene treatments prolonged the vase life only in ‘korun koku’ and ‘il tramonto’. adv. hort. sci., 2015 29(2-3): 116-120 (*) corresponding author: valentina.scariot@unito.it received for publication 26 september 2014 accepted for publication 8 june 2015 117 caser et al., ethylene and the postharvest performance of cut camellia flowering branches of c. japonica and investigate the effect of inhibitors of ethylene action (commercial chrysal, commercial 1-mcp and 1-mcp included in β-cyclodextrin-based nanosponges) on camellia flowering branch vase life. 2. materials and methods flower budded branches (50 cm in length) of seven c. japonica cultivars were supplied to the postharvest laboratory of the department of agricultural, forest and food sciences (disafa) at the university of torino (italy) as listed in table 1. the long supply chain (three days long) was simulated as follows: (1) cut branches were harvested at verbania (italy) at two companies (f.li savioli and società cooperativa val intrasca) and kept in cold storage overnight; (2) transportation to the flower market of sanremo (imperia, italy) by refrigerated truck; and (3) shipment to disafa the next day. in the short supply chain (one day long), cut branches were collected at verbania and then directly transferred to disafa. at disafa, branches were recut to 20 cm length and ten branches per treatment were used. three treatments were compared with a tap water control: chrysal professional 2 (10 ml l-1) for 24 h followed by the application of chrysal professional 3 (10 mg l-1) for all the experiment; commercial gaseous 1-mcp (3.3% ai, smartfreshtm, agrofresh inc., usa) (0.25 µl l-1) for 6 h in a gas-tight cabinet (112 l); and β-cyclodextrin-based nanosponge 1-mcp complex (β-cd-ns 1:8) (0.25 µl l-1). ten branches were also sealed in a box (112 l) and exposed to exogenous ethylene (1±0.2 µl l-1) for 24 h. subsequently, six branches per cultivar were individually enclosed in sealed tubes (1 l) containing 300 ml tap water, to quantify the daily production of endogenous ethylene. the study was undertaken during spring 2012 in a controlled room at 20±2°c day/night temperature, 60% relative humidity, and 46 µmol m-2 s-1 cool white light (meter model ht307; ht, faenza, italy). every day, the postharvest performance of the branches was evaluated considering both flowers and foliage characteristics, according to the following factors: visual check for symptoms of variation in senescence, flower opening stage, flower abscission, leaf chlorophyll content (spad), gloss, and endogenous ethylene production. variation in senescence was rated on a scale from 0 to 2, in which 0 = no visible senescence, 1 = initial senescence, and 2 = complete senescence. the loss of ornamental value of branches was considered when they reached level 1 on the scale (seglie et al., 2010). variation in flower opening stage was evaluated on the basis of the following scale: 1 = initial opening, 2 = half opening, and 3 = full opening (guo et al., 2004, modified). chlorophyll content was indirectly measured in leaves through a chlorophyll meter spad-502 instrument (konica minolta sensing inc., osaka, japan). gloss variation was measured in leaves using a spectrophotometer cm-2600 (konica minolta sensing inc., osaka, japan). ethylene concentration was monitored by a digital agilent technologies gas chromatograph, 6890n network gc system (santa clara, california). the gas carrier was n 2 at 40 ml min-1, and column temperature was 60°c. photo-documentation at different stages of senescence was carried out. data were registered until all the cut branches appeared completely damaged. petal, flower, and bud abscission percentages were mathematically transformed using arcsin√p/100. differences were initially subjected to the homogeneity of the variance test and then the analysis of variance (anova) was established using ryan-einot-gabriel-welsch-f (regw-f) post-hoc test. the critical value for statistical significance was p < 0.05. all the data were computed by means of the spss statistical package (version 21.0; spss inc., chicago, illinois). 3. results camellia cut flowering branches placed in tap water lost ornamental value after approximately 5.5 days of vase life and reached complete senescence after about 10 days in all studied cultivars, regardless of supply chain, with the exception of ‘il tramonto’ (fig. 1) in which senescence symptoms started to appear after 10 days and complete senescence was reached at day 20. exogenous ethylene application reduced camellia cut branch longevity in ‘debbie’ and ‘r.h. wheeler’, while the other treated cultivars lasted as long as the control. only in ‘charles cobb’ did cut branches treated with exogenous ethylene last longer, reaching complete senescence at day 14. the two antiethylene treatments (1-mcp and β-cd-ns) similarly prolonged vase life up to 10 days, slightly better than chrysal in ‘korun koku’ and ‘colonel firey’. on the contrary, chrysal outperformed the anti-ethylene treatments in ‘il tramonto’, prolonging its vase life up to 27 days. flowers fully opened on branches of ‘korun koku’ treated with β-cd-ns, ‘debbie’ treated with both chrysal and 1-mcp, ‘bonomiana’ in tap water, ‘colonel firey’ treated with both exogenous ethylene and 1-mcp, and ‘il tramonto’ in all treatments with the exception of β-cdtable 1 the studied camellia japonica cultivars and their supply chain length cultivar supply chain ´korun koku´ long ´debbie´ long ´bonomiana´ long ´colonel firey´ long ´charles cobb´ short ´r.h. wheeler´ short ´il tramonto´ short long= 3 days; short= 1 day. 118 adv. hort. sci., 2015 29(2-3): 116-120 ns (fig. 2). branches of ‘charles cobb’ and ‘r.h. wheeler’ reached, at maximum, stage 1 (initial opening). exogenous ethylene increased petal, flower, and bud abscission more than control in ‘korun koku’ (16.67% of flowers), ‘bonomiana’ (100% of buds), ‘colonel firey’ (100% of petals and 100% of flowers), and ‘il tramonto’ (50% of buds) (table 2). among the other treatments, an increase in abscission was noted in branches of ‘bonomiana’ and ‘colonel firey’ treated with β-cd-ns (25 % of flowers and 75 % of petals, respectively), in ‘charles cobb’ and in ‘il tramonto’ treated with chrysal (25 and 75% of flowers, respectively), and in ‘il tramonto’ also treated with 1-mcp (50% of buds). however, no correlations were found between abscission levels and applied treatments. generally, leaf spad and gloss values were not affected by treatments. only in ‘korun koku’, ‘r.h. wheeler’, and ‘il tramonto’ were slight differences found. in ‘korun koku’ chrysal outperformed the anti-ethylene treatments, and conversely in ‘r.h. wheeler’ and ‘il tramonto’. figure 3 shows differences in ethylene production among the studied camellia cultivars. a general trend was observed during the experiment with an ethylene increase until day 11. ‘korun koku’, ‘colonel firey’, and ‘bonomiana’ produced up to 1.42, 1.10, and 1.04 µl l-1, respectively. on the contrary, in ‘charles cobb’, ‘debbie’, and ‘r.h. wheeler’, 0.75, 0.67 and 0.66 µl l-1 were recorded, respectively. ‘il tramonto’ followed a similar but postponed trend. this latter cultivar started to produce ethylene at day 10 until day 24. also the ethylene production was much higher (2.10 µl l-1 at day 20). fig. 1 cut flowering branch longevity of the studied camellia japonica cultivar according to five treatments and two supply chains (a. long, b. short). the number of days to reach the initial stage of senescence (stage 1, black bar) and complete senescence (stage 2, grey bar) was measured. mean separation within bars of stage 1 (lower case) and stage 2 (upper case) were computed by the ryan-einot-gabriel-welsch’s multiple stepdown f (regw-f) test, p ≤ 0.05. fig. 2 variation in flower opening stages (stage 1 =  initial opening, stage 2 = half opening, and stage 3 = full opening) of the studied camellia japonica cultivar according to five treatments and two supply chains (a. long, b. short). fig. 3 mean values of the endogenous ethylene production in the studied cut flowering branches of camellia japonica cultivars placed in tap water. 119 caser et al., ethylene and the postharvest performance of cut camellia flowering branches 4. discussion and conclusions camellias are important ornamental species worldwide. their morphological (corneo and remotti, 2003) and genetic (ueno et al., 1999; caser et al., 2010) diversity as well as multiplication techniques and cultivation practices (larcher et al., 2011; berruti and scariot, 2013) were previously evaluated for their breeding and production. in this study, we investigated camellia attitude to be used as cut flowering branches. in 2001, van doorn stated that senescence performance and ethylene sensitivity are highly species related. here, we observed a great variability among cultivars, similarly to what was found in buttercup by kenza et al. (2000), scariot et al. (2009), and seglie et al. (2013), in rose by chanami et al. (2005), in peony by hoffman et al. (2010), and in bluebell by scariot et al. (2008). exogenous ethylene application in this study anticipated the senescence only in ‘debbie’ and ‘r.h. wheeler’. anti-ethylene treatments prolonged the vase life only in ‘korun koku’ and ‘il tramonto’. therefore, precautions against exposure to ethylene cannot be generalised for camellia cultivars. the provided data could be of benefit to growers and those involved in shipping. in conclusion, ‘il tramonto’ was the most interesting genotype as cut branches. this cultivar kept its ornamental value up to 27 days (when treated with chrysal), its flowers fully opened, and its foliage maintained colour and brightness during the vase life. this information could be useful to bring new produce to the flower market and to stimulate further research on postharvest techniques for camellia flowering branches. acknowledgements the authors gratefully acknowledge the flower market of sanremo and f.li savioli and società cooperativa val intrasca companies for their supply of plant material. this research was funded by mipaaf, project “netflor mettere in rete la produzione nazionale mediante un network di operatori italiani”. references accati e., corneo a., hillebrand p., lombardo d.m., merlo f., pisoni c.a., remotti d., scariot v., 2006 le antiche camelie dei rovelli. grossi (eds.) domodossola, italy, pp. 174. berruti a., scariot v., 2013 efficacy of flurprimidol and peat alternatives on growth control of potted camellia. nz j. crop hortic. sci., 41: 230-239. bonner j., honda s., 1950 treatments for increasing the life of cut camellia flowers. camellia res., 45. caser m., torello marinoni d., scariot v., 2010 microsatellite based genetic relationships in the genus cameltable 2 petal, flower, and bud abscission of the studied camellia japonica cultivars subjected to five treatments and measured at the end of the experiment cultivar treatments petal abscission (%) flower abscission (%) bud abscission (%) ´korun koku´ chrysal 35.71 b (z) 0.00 b 35.71 b ethylene 100.00 a 16.67 a 83.33 a 1-mcp 60.00 ab 0.00 b 60.00 ab β-cd-ns 66.67 ab 0.00 b 66.67 ab control 44.44 ab 0.00 b 44.44 ab p * ** * ´debbie´ chrysal 66.67 a 16.67 ab 50.00 ab ethylene 100.00 a 66.67 a 33.33 ab 1-mcp 20.00 b 0.00 b 20.00 b β-cd-ns 87.50 a 4.17 b 83.33 a control 100.00 a 37.50 ab 62.50 ab p ** * * ´bonomiana´ chrysal 27.27 27.27 b 0.00 b ethylene 100.00 0.00 b 100.00 a 1-mcp 18.75 0.00 b 18.75 b β-cd-ns 25.00 25.00 a 0.00 b control 14.29 0.00 b 14.29 b p ns ** ** ´colonel firey´ chrysal 11.11 b 0.00 b 11.11 ethylene 100.00 a 100.00 a 0.00 1-mcp 25.00 b 0.00 b 25.00 β-cd-ns 75.00 a 0.00 b 75.00 control 12.50 b 0.00 b 12.50 p ** * ns ´charles cobb´ chrysal 75.00 25.00 a 50.00 ethylene 80.00 0.00 b 80.00 1-mcp 100.00 0.00 b 100.00 β-cd-ns 100.00 0.00 b 100.00 control 100.00 0.00 b 100.00 p ns * ns ´r.h. wheeler´ chrysal 100.00 a 10.00 90.00 a ethylene 37.50 b 0.00 37.50 b 1-mcp 41.67 b 0.00 41.67 b β-cd-ns 54.55 b 0.00 54.44 b control 100.00 a 0.00 100.00 a p *** ns ** ´il tramonto´ chrysal 75.00 75.00 a 0.00 b ethylene 50.00 0.00 c 50.00 a 1-mcp 75.00 25.00 b 50.00 a β-cd-ns 33.33 33.33 b 0.00 b control 20.00 20.00 b 0.00 b p ns * ** (z) different letter indicates significant differences at the 0.05 level, ryan-einot-gabriel-welsch (f) post hoc test. 120 adv. hort. sci., 2015 29(2-3): 116-120 lia potential for improving cultivars. genome, 53: 384-399. chanami e., khalighi a., joyce d.c., irving d.e., zamani z.a., mostofi y., kafi m., 2005 ethylene and anti-ethylene treatment effects on cut ‘first red’ rose. j. appl. hortic., 7: 3-7. corneo a., remotti d., 2003 camelie dell’ottocento nel verbano. regione piemonte, torino, italy. cothran c.d., 1958 a discussion of methods for increasing the life of cut flowers, pp. 199-207. in: tourje e.c. (ed.) camellia culture. macmillan company, new york, ny, usa. doi m., reid m.s., 1996 postharvest characteristics of cut camellia japonica l. ‘kumasaka’. postharvest biol. technol., 7: 331-340. guo w., chen r., dong r., 2004 the postharvest characteristics and water balance of some cultivars of tree-peony cut flowers. sci. silvae sin., 40: 89-93. hoffman g.d., mattinson d.s., fellman j.k., 2010 1-mcp shortens peony vase life. acta horticulturae, 857: 169-178. kenza m., umiel n., borochov a., 2000 the involvement of ethylene in senescence of ranunculus cut flowers. postharvest biol. technol., 19: 287-290. larcher f., berruti a., gullino p., scariot v., 2011 reducing peat and growth regulator input in camellia pot cultivation. hortic. sci., 38: 35-42. scariot v., larcher f., caser m., costa e., beruto m., devecchi m., 2009 flower longevity in ten cultivars of cut ranunculus asiaticus l., as affected by ethylene and ethylene inhibitors. eur. j. hortic. sci., 74: 137-142. scariot v., lombardo d.m., merlo f., 2007 camelie dell’ottocento. vol. ii. uno studio tra verbano e canton ticino. “quaderni della regione piemonte agricoltura, supplemento al n. 54, turin, italy, pp. 184. scariot v., seglie l., caser m., devecchi m., 2008 evaluation of ethylene sensitivity and postharvest treatments to improve the vase life of four campanula species. eur. j. hortic. sci., 73: 166-170. seglie l., devecchi m., trotta f., scariot v., 2013 β-cyclodextrin-based nanosponges improve 1-mcp efficacy in extending the postharvest quality of cut flowers. sci. hortic., 159: 162-165. seglie l., martina k., devecchi m., roggero c., trotta f., scariot v., 2011 a the effect of 1-mcp in cyclodextrin-based nanosponges to improve the vase life of dianthus caryophyllus cut flowers. postharvest biol. technol., 59(2): 200-205. seglie l., martina k., devecchi m., roggero c., trotta f., scariot v., 2011 b β-cyclodextrin-based nanosponges as carriers for 1-mcp in extending the postharvest longevity of carnation cut flowers. an evaluation of different degrees of cross-linking plant growth regul., 65: 505-511. seglie l., sisler e.c., mibus h., serek m., 2010 use of a non-volatile 1-mcp formulation, n,n-dipropil(1-cyclopropenylmethyl)amine, for improvement of postharvest quality of ornamental crops. postharvest biol. technol., 56: 117-122. serek m., sisler e.c., frello s., sriskandarajah s., 2006 postharvest technologies for extending the shelf life of ornamental crops. int. j. postharvest technol. inn., 1: 69-75. threlkeld j., 1962 camellias for corsages, wreaths and arrangements, pp. 94-103. in: threlkeld j. the camellia book. d. van nostrand company, inc., new york, ny, pp. 204. ueno s., yoshimaru h., tomaru n., yamamoto s., 1999 development and characterization of microsatellite markers in camellia japonica l. mol. ecol., 8: 335-338. van doorn w.g., 2001 categories of petal senescence and abscission: a re-evaluation. ann. bot., 87: 447-456. woolf a.b., clemens j., plummer j.a., 1992 selective removal of floral buds from camellia with ethephon. hortscience, 27: 32-34. 176 1. introduction petty-clark’s law of economics (formally known as petty’s law) (tsuchiya, 1993) suggests that the proportion of primary industries declines as the economy of a country develops, and in turn, those of secondary and tertiary industries must increase (kawata, 2011). today in japan, agricultural, fishery, and forestry workers make up about 3.51% of the total working population in the nation. in the past six decades, the agricultural force in japan has been markedly weakened and thinned, partly due to a nation-wide decline in birth rates and a decrease in new employment in the field (http://www.stat.go.jp/data/roudou/ longtime/03roudou.htm). these factors eventually caused highly advanced aging of the agricultural population in japan. from a macroscopic point of view, many may expect that the decreasing trend in the agricultural working population may continue based on predictions by the petty-clark’s law (nakamura, 2009). in contrast, there would be microscopic growth in the working population in specific forms of agriculture. one of the authors of this paper (tk) is currently proposing two models in which agriculture can be converted to higher industries, i.e. as leisure and as a high-tech food production industry (kawano, 2014). in the last two decades, prototypic high-tech agricultural farms (so-called “plant factories”) aimed at automating the production of fresh produce, chiefly vegetables, under precisely controlled environments have been developed (ikeda, 2011; nishida et al., 2012; nishimura et al., 2012). today, such commercially launched “plant factories” cover sprouts, leafy vegetables, and tomatoes (morimoto, 2005; shimizu et al., 2008). in order to expand the scope of “plant factories” and enable the automated cultivation of a wide range of agriculturally important plant species, the agricultural and horticultural processes which are a successive series of necessary maintenance steps must be translated or re-documented into automatable protocols based on the records of both plant physiological and mechanical data (kawano et al., 2012). despite their agricultural importance, twining plants have not been included in the trends of “plant factory” development, possibly due to the belief that handling of growing vines must be achieved manually by experienced specialists (kawano et al., 2012). ipomoea cultivars are examples of such twining crops, whose aerial parts are herbaceous perennial vines and bear alternate heart-shaped green leaves and sympetalous flowers (sher et al., 2001). in addition, root vegetables or crops bearing root products belowground have not been produced in “plant factories” yet, despite the fact that the growth of roots are central non-invasive acoustic sensing of tuberous roots of sweet potato (ipomoea batatas) growing belowground j. iwase1, y. sato(*)1, d. comparini2, 3, e. masi 3, s. mancuso3,, t. kawano2, 4 1 collaboration center, kyushu institute of technology, kitakyushu 808-0196, japan. 2 faculty and graduate school of environmental engineering, the university of kitakyushu, kitakyushu 808-0135, japan. 3 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 4 universitee paris diderot, sorbonne paris cité, paris interdisciplinary energy research institute (pieri), paris, france. key words: sweet potatoes, undersoil detection, water-proof speaker. abstract: the study applies acoustic means for the detection of belowground tuberous roots of sweet potato by developing a novel non-invasive sensing technology based on propagation of frequency-modulated sound through the soil and its detection with acoustic band-pass filtering devices. the presence of tuberous roots hidden in the bed of sandy soil was successfully detected with the proposed acoustic approach, which is still primitive. however, this investigation may induce further studies and developments for belowground sensing and imaging techniques as novel ecological research tools and to support the introduction of vegetables into so-called “plant factories”. adv. hort. sci., 2015 29(4): 176-180 (*) corresponding author: sato@lsse.kyutech.ac.jp; kawanotomi@kitakyu-u.ac.jp received for publication 08 january 2015 accepted for publication 21 july 2015 short note 177 iwase et al., non-invasive acoustic sensing of tuberous roots of sweet potato growing belowground to the physiology and ecology of higher terrestrial plants including forest trees and agricultural crops. in soilor sand-based conventional agricultural fields, a variety of root vegetables are produced. such vegetables having highly developed roots include beet (beta vulgaris ssp. vulgaris), turnip known as kabu in japan (brassica rapa l.), carrot (daucus carota l.), radish and daikonjapanese white radish (raphanus sativus l.), chinese yam known as nagaimo in japan (dioscorea oppositifolia l.), and burdock known as gobō in japan (arctium lappa l.). among the root vegetables, sweet potato (ipomoea batatas l.), cassava (manihot esculenta crantz), and yacón or so-called andes potato (smallanthus sonchifolius) are typical examples of vegetables bearing starchy tuberous roots. in addition to the above true root vegetables, there are groups of undersoil vegetables that develop root-like organs as storage of starch, sugars, and nutrition. economically important crops with undersoil organs such as corms of taro (colocasia esculenta), tubers of potato (solanum tuberosum), and rhizomes of lotus (nelumbo nucifera), ginger (zingiber officinale), turmeric (curcuma longa), and ginseng (panax ginseng), are examples of vegetables with specialized stems. furthermore, vegetables producing bulbs can be included in the undersoil vegetables, mostly belonging to allium such as garlic (allium sativum l.), onion, and shallot (allium cepa l.). today, soils or sands are hardly manipulated in sensor-equipped “plant factories” while hydroponic cultures are mostly favored. in order to introduce such root vegetables in “plant factories”, it is necessary to monitor the belowground properties and processes where plant roots take place, mainly spatial occupation by the growing root system and changes in water, salts, and other elements that can influence productivity and functioning of the soil ecosystem. however, dynamic growth and development of a root system are poorly understood since roots growing belowground are invisible to conventional optical means. before facing the challenges to growing under-soil vegetables in the controlled environment inside “plant factories”, appropriate non-invasive sensing technologies allowing real-time monitoring of belowground plant growth must be developed and applied. due to the light absorbing nature of the soils, optical measurements are difficult to apply. three-dimensional computed tomography with xray scanning technology might be a candidate technology if plants are grown in laboratory-scaled small pots (we have developed non-invasive scanning protocols for garlic bulbs, data not shown). however, x-ray scanning cannot be employed on-site in plant producing facilities. in this study, we attempted to detect the presence of tuberous roots which were hidden in the sandy bed. for this purpose, a novel instrumental set-up was designed for modulation and demodulation of sound signals passed through soil or sand with and without growing plant tissues. sweet potato, known as satsuma-imo in japanese, is a dicotyledonous crop that belongs to the family of convolvulaceae. due to its starchy and sweet-tasting tuberous roots, this plant is often considered both a starch producing crop and a fiber-rich root vegetable (purseglove, 1968; woolfe, 1992). 2. materials and methods acoustic sensing system frequency-modulated sound signals propagated through sandy soil containing plant tissue samples or not were monitored using a pair of acoustic probes, a flat sound radiator, and sensing microphones (fig. 1a, b). in order to eliminate the background sound noise, a signal from the reference (ground) microphone was used. to generate the modulated sound signal at the frequency of interest, a function generator (dfg-6020 20mhz dds, ez digital co. ltd., gwangju, korea) was used (fig. 1c). signals received by the sensing microphone were amplified by a at-ma2 microphone amplifier (audio-technica, tokyo, japan) and band-filtered to demodulate using an acoustic band-pass filter (multi-function filter 3611 nf corporation, yokohama, japan), and the amplitude of the sound at specific frequency captured through the system was analyzed using an oscilloscope (tbs 1064, tektronix inc., or, usa). fig. 1 equipment for the acoustic sensing of belowground plant tissues. (a) experimental configuration. modulation and demodulation of acoustic signal propagated in a sandy soil bed were preliminarily tuned with and without wooden stick. (b) a pair of acoustic probes designed for emitting and receiving the modulated and band-pass filtered sound signals in the soil or sand. left, sensing and reference microphones. right, flat-shaped sound radiator with directional sound propagation. (c) sweet potato tuberous roots used for demonstration. (d) experimental design. 178 adv. hort. sci., 2015 29(4): 176-180 experimental procedure an experimental set-up (fig. 1) inside the sand-filled container of polystylene foam with or without model tissues embeded in the sand, was used for acoustic measurements. band-modulated sound signals were emitted and detected at a constant distance of 100 mm, by a pair of acoustic probes, namely, a directional sound radiator and receivers (microphones), both positioned in the bed of dry (ca. 1% of water content) or wet (ca. 10% of water content) sands. the given sound frequency was in the range between 100 and 20,000 hz. changes in the height of voltage signal between the peaks and valleys of the recorded sound waves were recorded (fig. 2a) and expressed as the extent of signal intensity, reflecting the perception of band-pass filtered sound after propagation of band-modulated sound. mean values of the height of voltage signal obtained at each frequency (n=5) were used to obtain a series of regression lines with the least-square method. data were recorded in the absence (blank control) and presence of tubers. plant materials since ipomoea plants naturally grow in sandy fields, and sand-culture is often employed for cultivation of sweet potatoes (leonard et al., 1948; spence and humphries, 1971), commercially available river-sand mixture was chosen as tuber root supporting medium. assuming there is the necessity to monitor tuberous roots inside a plant growth container, a planter made of foamed polystyrene filled with dry or wet river-sand mixture was employed as a model platform for acoustic sensing demonstrations. inside the container, two types of sandy soil (volume: 4l; area: 220 mm x 262 mm, depth: 100 mm) with known water content (dry at ca. 1 or wet at ca. 10% w/w) were tested. portions or intact sweet potato tuberous roots of different sizes were detected and compared with the absence of any vegetable tissue (blank control). in particular, two intact tuberous roots with a central swelled portion (45 and 82 mm in diameter) (fig. 1c) were obtained from a local market and used as model vegetable tissues. as a model for sampling tissues with smaller mass, a cut plug (cylindrical section of 20 mm in diameter) of tuberous root was prepared and used for acoustic measurements. 3. results and discussion sound signal detection the intensity of sound signals propagated through sand differed noticeably depending on the sound frequency (fig. 2). fractuations of signal intensities were shown to be higher in the relatively lower range of frequency examined (1001,600 hz) in both sand samples. compared to the dry sand, the wet sand likely enhanced the yield of sound signals in the presence of sweet potato tissues, especially at the higher range of frequency examined (3,200-20,000 hz). figure 2a reports typical raw records of sound signals showing a sizedependent enhancement at 6.4 khz in the wet condition. thus, data from the wet sand bed was used for further analysis to elucidate the effect of tissue size on signal acquisition. sound signal relationships relationships between sound signal frequency and yield in the beds of dry and wet sands were compared by obtaining a series of regression lines with the least-square method (fig. 3a). correlation coefficients (r2) for four regressions were between 0.8825 and 0.9991. to evaluate tissue sizedependent changes in the yield of sound signals in the wet sand bed, two indices (i.e. index of subtraction and index of ratio) were proposed and the processed data were used to obtain the linear regressions and were compared with other linear regressions obtained for raw signal data (fig. 3b, c). with the index of subtraction (sample-blank) (fig. 3b), correlation coefficients (r2) for three regressions were between 0.7148 and 0.9623. with the index of ratio (sample / blank) (fig. 3c), correlation coefficients (r2) for three regressions were between 0.7829 and 0.8885. the regressions obtained for the index of subtraction highlighted the relationship between the signal yield and tissue size increases since the coefficients for x in the linear regressions increased with size: 2·105 x for the ø20 mm sample, 3·105 x for the ø45 mm sample, and 5·105 x for the ø82 mm sample (fig. 3b). further studies during frequency-modulated sensing of plant tissues, the impact of acoustic signals on plant growth must be fig. 2 detection of sound signals propagated in the sand layer with and without sweet potato tuberous toots. (a) typical raw records of signal modulated at ca. 6.4 khz (6.386-6.413 khz). changes in signal intensity (voltage changes between the peaks and valleys of the waves) were monitored. relationship between the frequency and yield of sound signal in the beds of dry sand (b; water content, ca. 1%) and wet sand (c; water content, ca. 10%) were compared. arrows in (b) and (c) indicate signal yields at 6.4 khz. error bars, s.d. (n= 5). 179 iwase et al., non-invasive acoustic sensing of tuberous roots of sweet potato growing belowground minimized, as recently demonstrated by gagliano et al. (2012 a, b) who suggested that the growing tips of plant roots could be attracted by acoustic signals with specific spectra raging between 200 and 400 hz, if plant roots were continuously exposed to the sound stimulus. one possible way to avoid such influence during acoustic sensing is to use signals in a frequency range higher or lower than the plant activating range of sound (<200 hz, >400 hz), or to use sound only for short periods when required. since the range of frequency recommended for monitoring belowground plant tissues was shown to be between 3,200 and 20,000 hz (fig. 3), the impact of applied sound on plant growth could be avoided. in the present work we have developed a novel, noninvasive sensing technology for the detection of belowground plant tissues based on sound propagation in the soil. we employed tuberous root tissue of sweet potato as model material to test the instrumental set-up which was specifically designed for modulation and demodulation of sound signals through soil or sand with and without plant tissues. it is well known that growth of tuberous roots of sweet potato is highly sensitive to changes in temperature (spence and humphries, 1971), moisture (spence and humphries, 1971; eguchi et al., 2012), nutrient (leonard et al., 1948), phytohormone levels (spence and humphries, 1971), and oxygen supplies (eguchi and yoshida, 2011) in the supporting sand. however, to date, underground growth of sweet potatoes’ tuberous root was rarely studied with noninvasive real-time monitoring approaches. further studies are still needed, although there are several technical problems to be overcome, chiefly, sensitivity and resolution of the sensing units. ecologists, ecohydrologists, and biogeochemists need detailed insights into plant properties and processes where plant roots reside, including changes in water, salts, and other elements that can influence ecosystem productivity and functioning (jayawickreme et al., 2014). however, limitations in data and some confusion over terminology, together with a strong dependence on a small set of conceptual frameworks, have limited the exploration of root function in terrestrial models (smithwick et al., 2014). relying on traditional mechanical sampling and observation techniques can be costly, time consuming, and poorly feasible, especially if the spatial scales involved are large (jayawickreme et al., 2014). therefore, non-invasive realtime monitoring may largely benefit both the ecological research community and the field of “plant factory” research and development. although the present study succeeded in only the primary test case, we hope it will stimulate further studies and development by scientists and engineers. references eguchi t., ito y., yoshida s., 2012 periodical wetting increases α-tocopherol content in the tuberous roots of sweetpotato (ipomoea batatas (l.) lam.). environ. control biol., 50: 297-303. fig. 3 signals for belowground tuberous roots differed in size based on the acoustic recording at the higher range of frequency (between 3,200 and 20,000 hz). data recorded in the absence (blank control) and presence of tuberous roots of sweet potato (20, 45 and 85 mm in diameter) were plotted. mean values obtained at each frequency were used (n = 5). (a) plots and linear regression of raw signal. (b) evaluation of the signal with the subtraction index (sample-blank). 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(1975) examined the proline amount of zoysia japonica steud. ‘meyer’ during the months from october to march and found that there is an increase in proline from october to december. it has been shown that for the period of adaptation to cold, sod and cat activity in agrostis stolonifera l., poa pratensis l. and lolium perenne l. significantly increased (sarkar and bhowmik, 2009). the main objective of the present study was to investigate the effects of low temperature stress on biochemical and physiological responses of tall fescue and common bermudagrass. to the best of our knowledge, this is the first report on how these turfgrasses counter cold stress. 2. materials and methods plant materials and experimental conditions the experiment was conducted in a growth chamber (gallenkamp, germany) at the department of horticultural sciences at the college of agriculture, shiraz university (29°36’ n and 52°32’ e, elevation 1810 m). seeds of common bermudagrass (cynodon dactylon [l.] pers. ‘california origin’) and tall fescue (festuca arundinacea schreb. ‘starlett’) were cultivated in 5 l plastic pots containing a biochemical and physiological adjustments in common bermudagrass (cynodon dactylon [l.] pers.) and tall fescue (festuca arundinacea schreb.) under low temperature stress r. manuchehri, h. salehi(1), a. jowkar department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: antioxidant enzymes activity, common bermudagrass, low temperature stress, tall fescue, turfgrasses. abstract: low temperature is a restrictive factor for turfgrass growth and development in temperate regions. a study was conducted with the purpose of examining the physiological and antioxidant response of two turf species, festuca arundinacea schreb. ‘starlett’ and cynodon dactylon [l.] pers. ‘california origin’ to cold stress in a growth chamber at the college of agriculture, shiraz university. five temperatures (25, 15, 7.5, 0 and -7.5°c) in four replicates were examined in a completely randomized design experiment. it was revealed that under low temperature stress, soluble sugar contents, proline, malondialdehyde (mda) and hydrogen peroxide (h2o2) were increased in both turfgrasses. antioxidant enzyme activity, particularly catalase (cat, ec 1.11.1.6) and superoxide dismutase (sod, ec 1.15.1.1), was increased as a result of temperature reduction from 25°c to 0°c. tall fescue is thought to be better adapted to cold stress than common bermudagrass due to higher soluble sugar contents, proline, malondialdehyde and antioxidant enzyme activity. the results show that scavenging enzymes have a direct effect in cold season tolerance of turfgrass and improve the defense mechanism of plants, but their exact role merits further investigation. adv. hort. sci., 2014 28(1): 9-13 (1) corresponding author: hsalehi@shirazu.ac.ir received for publication 6 december 2013 accepted for publication 18 march 2014 short note 10 mixture of 1:2 (v/v) of loamy soil/decomposed farmyard manure. irrigation was carried out on a daily schedule. established turfs were clipped from 3 cm above ground by a hand mower and were transferred to the growth chambers prior to the application of treatments. all treatments received a constant light intensity of 3000 lux, relative humidity of 65±5% and a 12 h photoperiod. low temperatures were maintained at 25, 15, 7.5, 0 and -7.5°c for 48 h. experimental design and data analysis experiment factors were arranged in a completely randomized design with four replications. data were analyzed using sas software (ver. 9.1.3) and means were compared using the least significant difference (lsd) test at p<0.05. reducing sugars and proline content phenol-sulfuric acid reactions were used to determine the reducing sugar content. shoot samples were oven dried at 60°c for 48 h and then ground to a fine powder using an electric mill. samples (0.2 g) were diluted with 80% ethanol and centrifuged at 13500 rpm. supernatant was further diluted to 25 ml by 80% ethanol. then, 1 ml of extract was mixed with 1 ml of 5% phenol. five ml of concentrated sulfuric acid were added to tubes and immediately stirred. light absorption was measured by a spectrophotometer (biochrome, uk) at 490 nm wavelength (dubois et al., 1956). proline was determined according to the method used by bates et al. (1973) using a spectrophotometer at 520 nm wavelength. measurement of antioxidant enzyme activity to extract antioxidant enzymes, fresh leaf samples (0.5 g) were collected and ground to a fine powder in a mortar by adding liquid nitrogen and then homogenized with an ice cold enzyme extraction buffer containing 0.5% polyvinylpyrrolidone (pvp), 3 mm edta, and 0.1 m potassium phosphate buffer (ph=7.5). the extracted samples were centrifuged for 10 min at 13500 rpm and 2-4°c and stored on ice until used. the resulting supernatants were used for enzyme analysis. cat activity was determined according to the procedure used by dhindsa et al. (1981) and sod activity was determined as described by beauchamp and fridovich (1971). malondialdehyde (mda) as for h 2 o 2 , 0.25 g of leaf samples were ground in a mortar containing 5 ml tca (0.1%). leaf extracts were centrifuged at 10000 rpm for 5 min. supernatants (250 µl) were mixed with 1 ml mda solution containing 20% tca and 0.5% thiobarbituric acid. the mixtures were warmed at 95°c for 30 min and then immediately cooled on ice. sample tubes were centrifuged at 10000 rpm for 10 min. absorption of light was measured by a spectrophotometer at 532 nm wavelength according to heath and packer (1969). hydrogen peroxide (h 2 o 2 ) leaf samples (0.25 g) were ground in a mortar containing 5 ml trichloroacetic acid (tca) (0.1%). extracts were centrifuged at 10000 rpm for 5 min. supernatants (250 µl) were mixed with 250 µl phosphate buffer (100 mm) and 500 µl potassium iodide (1 m). absorption of light was measured by a spectrophotometer at 390 nm wavelength according to alexieva et al. (2001). 3. results with a decrease of temperature from 25°c to -7.5°c, reducing sugars increased considerably, with tall fescue showing a greater increase than common bermudagrass. the highest soluble sugar content in tall fescue was formed at -7.5°c and the highest reducing sugar content produced in common bermudagrass was detected at 0°c (table 1). there was no significant difference between the turfgrasses for proline content. the highest proline content was observed at 0°c and the lowest proline content was seen at 25°c. the interaction of temperature and turf species showed that tall fescue at 25°c had the lowest proline content, while tall fescue at -7.5°c had the highest (table 1). it was found that as temperature decreased from 25°c to 7.5°c, cat activity increased. the greatest cat activity was observed in tall fescue at 7.5°c and the least was seen in bermudagrass at -7.5°c (table 1). comparison of the means showed that sod activity in tall fescue is greater, but not significantly different from common bermudagrass. maximum sod activity in bermudagrass was detected at 0°c, while the minimum was found at 25°c in tall fescue. as the temperature diminished from 25°c to -7.5°c, mda amassed continuously in the plants. mda accumulated significantly more in common bermudagrass with the highest amount built up at -7.5°c (table 2). h2 o 2 increased in plants as the temperature lowered to 0°c. the most h 2 o 2 was produced in common bermudagrass at 0°c, whilst the lowest was observed in tall fescue at 25°c (table 2). 4. discussion and conclusions as the temperature decreased from 25°c to -7.5°c, soluble sugars and proline content increased, which tall fescue had higher amounts at -7.5°c (table 1). a similar behavior was found in saltgrass (distichlis spicata l.), centipedegrass (eremochloa ophiuroides [munro]), annual bluegrass (poa annua l.) and buffalograss (bouteloua dactyloides [nutt.]) (fry, 1993; shahba et al., 2003). generally, one of the first reactions by these plants to counter the chilling stress of winter is a buildup of sugar (fry, 1993; ball et al., 2002), whilst amino acids help adapt the plants to low temperature (guy, 1990). proline and reducing sugars serve as cryoprotectants through increasing the concentration of cell content and reducing the water potential (ball et al., 2002). comparable results were observed in zoysiagrass (zoysia japonica steud.) and annual bluegrass (dionne et al., 2001). 11 the increase in cat and sod activity found in this study is assumed to protect the cells from oxidative damage caused by cold stress as seen in other plants (matsumura et al., 2002; larkindale and huang, 2004; jiang et al., 2011). sod converts superoxide (o 2 .-) to h 2 o 2 and cat detoxifies the latter to water and oxygen (fuchs et al., 1997; polidoros and scandalios, 1999). antioxidant enzymes help maintain cell homeostasis under severe low temperatures by scavenging as well as signaling, although their definite function should be further elucidated (polle, 1997). higher antioxidant enzyme activity in tall fescue could be attributed to better cold tolerance compared to common bermudagrass. mda and h 2 o 2 increase in the turfgrasses in this research is dependent on the cold stress received (table 2). mda and h 2 o 2 are produced by lipid peroxidation of plants under chilling stress (leshem, 1987; wise and naylor, 1987). these two sensitive indicators are considered to point toward the extent of low temperature stress and damage inflicted to the plant (xu et al., 2006). greater amounts of these two substances in common bermudagrass compared to tall fescue could be interpreted as a greater sensitivity to and injury from low temperatures (table 2), which is consistent with the reports in manila grass (zoysia matrella l.) (wang et al., 2009). overall, cold stress produces large amounts of ros which causes oxidative damage to plants through vast destruction of proteins, carbohydrates, lipids, cellular membranes, dna and major decline of atp reserve, and finally cell death (dionne et al., 2001; gill and tuteja, 2010). since ros has multifunctional roles, it is essential for the cells to control the level of ros tightly to avoid any oxidative injury and not to eliminate them table 1 effects of cold stress on biochemical changes [reducing sugar, proline content, catalase (cat) and superoxide dismutase (sod) activity] in the two turfgrasses used in this study turfgrass temperature (°c) mean -7.5 0 +7.5 +15 +25 reducing sugar (mg·g-¹ d.w.) tall fescue 176.4±60.1 a 160.8±16.3 ab 130.2±27.3 bcd 114.8±13.2 cd 104.3±22.4 dz 137.4 a bermudagrass 121.9±11.5 bcd 158.9±21.0 abc 140.9±18.7 a-d 117.3±19.8 bcd 96.5±14.7 d 127.1 a mean 149.2 a 159.8 a 135.5 ab 116.1 bc 100.4 c proline content (µg·g-¹ d.w.) tall fescue 35.8±3.7 a 35.1±3.4 a 27.2±2.7 b 13.7±3.1 cd 9.2±0.9 d 24.2 a bermudagrass 26.4±1.7 b 31.8±1.5 ab 27.3±5.1 b 17.3±4.5 c 12.7±1.3 cd 23.1 a mean 31.1 a 33.5 a 27.2 b 15.5 c 11.0 d cat (u g·g-¹ d.w.) tall fescue 36.3±5.4 c 46.7±6.1 ab 52.8±9.8 a 37.5±3.6 c 32.8±5.7 c 41.2 a bermudagrass 31.5±4.5 c 39.5±4.4 bc 47.5±2.9 ab 41.5±3.9 bc 36.9±5.8 c 39.3 a mean 35.4 c 43.1 b 50.1 a 39.5 bc 34.8 c sod (u g·g-¹ d.w.) tall fescue 179.6±17.6 abc 161.6±33.1 a-d 186.6±30.5 ab 126.0±19.0 de 106.6±17.0 e 152.1 a bermudagrass 127.3±23.1 cde 193.3±25.1 a 145.0±42.7 a-e 137.6±58.6 b-e 120.6±11.7 de 144.8 a mean 153.5 ab 177.5 a 165.8 ab 131.8 bc 113.6 c z in each variable, data followed by the same letters±sd (small letters for interactions and capital letters for means) are not significantly different at 5% level of probability using lsd test. table 2 effects of cold stress on malondialdehyde (mda) and hydrogen peroxide (h 2 o 2 ) in the two turfgrasses used in this study turfgrass temperature (°c) mean -7.5 0 +7.5 +15 +25 mda (µmol g-1 f.w.) tall fescue 7.5±1.6 cd 7.8±1.3 bc 6.8±0.8 cde 4.4±0.7 fg 3.9±0.7 gz 6.1 b bermudagrass 11.6±0.7 a 9.5±0.7 b 7.7±0.8 c 5.9±1.0 def 5.1±0.7 efg 8.0 a mean 9.6 a 8.6 a 7.3 b 5.1 c 4.5 c h 2 o 2 (µmol g-1 f.w.) tall fescue 5.1±0.9 a 4.7±0.7 a 3.5±0.6 b 2.9±0.4 b 2.7±0.4 b 3.8 b bermudagrass 5.0±0.3 a 5.3±0.4 a 4.9±0.7 a 3.5±0.5 b 3.1±0.7 b 4.3 a mean 5.0 a 5.0 a 4.2 b 3.2 c 2.9 c (z) in each variable, data followed by the same letter ± sd (small letters for interactions and capital letters for means) are not significantly different at 5% level of probability using lsd test. 12 entirely (sharma et al., 2012). it is concluded that both turfgrasses increase reducing sugars, proline, cat, sod, mda and h 2 o 2 in response to lower temperatures, but tall fescue has a better defense mechanism than common bermudagrass and is more tolerant to cold stress. the results show that scavenging enzymes have a direct effect in cold season tolerance of turfgrass and improve the defense mechanism of plants, but their exact role merits further investigation. acknowledgements the authors wish to thank the administration of the students’ scientific association of shiraz university for their financial support. references alexieva v., sergiev i., mapelli s., karanov e., 2001 the effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. plant cell and environment, 24: 1337-1344. ball s., qian y.l., stushnoff c., 2002 soluble carbohydrates in two buffalograss cultivars with contrasting freezing tolerance. j. amer. soc. hortic. sci., 127(1): 45-49. bates l.s., waldern r.p., teave i.d., 1973 rapid determination of free proline for water stress standies. plant and soil, 39: 107-205. beauchamp c., fridovich i., 1971 superoxide dismutases: improved assays and an assay predictable to acrylamide gels. analytical bioch., 44: 276-287. carrow r., 1994 zoysiagrass cultivar performance as affected by traffic and n-level. agronomy abstracts, asa, madison, wi, usa, p. 183. dhindsa r.s., plumb-dhindsa p., thorpe t.a., 1981 leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. j. exp. bot., 32(1): 93-101. dionne j., castonguay y., nadeau p., desjardins y., 2001 freezing tolerance and carbohydrate changes during cold acclimation of green-type annual bluegrass (poa annua. l) ecotypes.crop science, 41: 443-451. dubois m., gilles k.a., hamilton j.k., rebers p.a., smith f., 1956 colorimetric method for determination of suger and related substances. analytical bioch., 28: 350-356. fry j.d., 1993 freezing tolerance and carbohydrate content of low-temperature-acclimated and nonacclimated centipedegrass. crop science, 33: 1051-1055. fuchs d., bitterlich g.b., wede i., wachter h., 1997 reactive oxygen and apoptosis, pp. 139-167. in: scandalios j.g. (eds.) oxidative stress and the molecular biology of antioxidant defenses. cold spring harbor laboratory press, cold spring harbor, ny, usa, pp. 890. gill s.s., tuteja n., 2010 reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. plant physiology and biochemistry, 48: 909-930. gulzar s.s., shabir h.w., wasim h., singh n.b., 2011 engineering cold stress tolerance in crop plants. current genomics, 12: 30-43. guy c.l., 1990 molecular mechanism of cold acclimation, pp. 35-61. in: katterman f. (eds.) environmental injury to plants. academic press, san diego, ca, usa, pp. 302. heath r.l., packer l., 1969 photoperoxidation in isolated chloroplast; kinetics and stoichiometry of fatty acid peroxidation. arch. bioch. biophy., 125: 189-198. jiang x., song y., xi x., guo b., ma k., wang z., li b., zhang d., an x., zhang z., 2011 physiological and biochemical responses to low temperature stress in hybrid clones of populususs uriensis kom.× p. deltoides bartr. african j. biotech., 10: 19011-19024. jiang y.w., carrow r.n., duncan r.r., 2005 physiological acclimation of seashore paspalum and bermudagrass to low light. scientia hort., 105: 101-115. larkindale j., huang b., 2004 thermotolerance and antioxidant systems in agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene. j. plant physiol., 161(4): 405-413. leshem y.y., 1987 membrane phospholipids catabolism and ca2+ activity in control of senescence. plant physiology, 69: 551-559. levitt j., 1980 responses of plants to environmental stress. volume 1. chilling, freezing, and high temperature stress. academic press, new york, pp. 497. matsumura t., tabayashi n., kamagata y., souma c., saruyama h., 2002 wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress. plant physiology, 116: 317-327. polidoros n.a., scandalios, j.g., 1999 role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione s-transferase gene expression in maize (zea mays l.). plant physiology, 106: 112-120. polle a., 1997 defense against photooxidative damage in plants, pp. 623-666. in: scandalios j.g. (eds.) oxidative stress and the molecular biology of antioxidant defenses. cold spring harbor laboratory press. plainview, ny, usa, pp. 890. rogers r.a., dunn j.h., nelson c.j., 1975 cold hardening and carbohydrate composition of meyer zoysia. agronomy journal, 67: 836-838. sakai a., larcher w., 1987 frost survival of plants: response and adaptation to freezing stress. springer-verlag, berlin. sarkar d., bhowmik p. c., 2009 cold acclimation responses of three cool-season turfgrasses and the role of proline-associated pentose phosphate pathway.journal of the american society for horticultural science, 134: 210-220. shahba m.a., qian y.l., hughes h.g., koski a.j., christensen d., 2003 relationship of carbohydrates and cold hardinesss in six saltgrass accessions. crop science, 43: 2148-2153. sharma p., jha a.b., dubey r.s., pessarakli m., 2012 reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. journal of botany, pp. 26. doi: 10.1155/2012/217037. wang y., yang z.m., zhang q.f., li j.l., 2009 enhanced 13 chilling tolerance in zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine. biologia plantarum, 53: 179-182. wise r.r., naylor a.w., 1987 chilling-enhanced photooxidation, evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. plant physiology, 83: 278-282. xu s., li j.l., zhang x.q., wei h., cui l.j., 2006 effects of heat acclimation pretreatment on changes of membrane lipid peroxidation, antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turf grass species under heat stress. environ. exp. bot., 56: 274-285. impaginato 11 1. introduction the polyamines as natural compounds are present ubiquitously in almost all living organisms. the main polyamines in significant amounts are putrescine, spermidine, and spermine which are crucial for the growth and development of plant and fruit as well as stress responses (valero and serrano, 2010). they are known as anti-senescent agents that decrease the rate of fruit softening and senescence by suppression of ethylene production (kramer et al., 1991). reduced values of polyamines have been attributed with enhanced ethylene production and vice versa (walden et al., 1997). this mechanism is correlated to a competition between polyamine and ethylene for the common precursor s-adenosyl methionine (sam) (pandey et al., 2000). the use of polyamines has been claimed to decrease ethylene synthesis in a wide range of plants by decreasing acc synthase (acs) and acc oxidase (aco) enzymes activities (ke and romani, 1988; kakkar and rai, 1993; lee et al., 1997; martinez-romero et al., 2001; bregoli et al., 2002; perez-vicente et al., 2002; serrano et al., 2003; petkou et al., 2004; malik and singh, 2005; de dios et al., 2006; khan et al., 2007). in several investigations putrescine applied exogenously have been reported to increase storage life and quality attributes of mango (razzaq et al., 2014), pear (franco-mora et al., 2005), apricot (martinezromero et al. , 2002), strawberry (zokaee khosroshahi et al., 2007), plum (abu-kpawoh et al., 2002; pérez-vicente et al., 2002), grapes (harindra champa et al., 2015; mirdehghan and rahimi, 2016), pomegranate (mirdehghan et al., 2007; barman et al., 2011) and litchi (jiang and chen, 1995). therefore, the aim of this study was to investigate the role of preharvest putrescine treatment on maintaining postharvest quality of pear fruit cv. spadona. 2. materials and methods the experiments were conducted on pear trees (p. communis cv. spadona) in the center of horticultural research of the university of tehran, karaj, iran. eighteen 16-year-old trees (250 cm height) were selected in terms of uniformity in size and fruit load then sprayed with putrescine at 0.5, 1 and 2 mm (3.5 adv. hort. sci., 2017 31(1): 11-17 doi: 10.13128/ahs-20720 effect of pre-harvest putrescine treatment on quality and postharvest life of pear cv. spadona m.s. hosseini (*), z. fakhar, m. babalar, m.a. askari department of horticulture science, college of agriculture and natural resources, university of tehran, 31587 karaj, iran. key words: cold storage, color, fruits, quality assessment. abstract: the study was conducted to determine the effect of pre-harvest foliar spraying with putrescine (at 0.5, 1 and 2 mm) on quality and postharvest life of pyrus communis cv. spadona during cold storage. fruit quality assessment such as weight loss, firmness, total soluble solids (tss), titratable acidity (ta), flavor index, skin color (l*, hue angle), vitamin c total phenol (tp), and total antioxidant activity (taa) were made at harvest and after 3, 6, 9, 12, 15, 18 and 21 weeks of storage at 0±1°c, 80-85% relative humidity. weight loss, fruit softening, tss and ph increased during storage but the rate of changes was significantly lower in fruit treated with putrescine at 1 and 2 mm. putrescine application maintained higher levels of ta, vitamin c, tp, taa, l*, hue angle and reduced decay incidence compared to control. furthermore, higher doses of putrescine were effective in terms of prolonging the storage and marketability of fruits more than 127142 days. in conclusion, pre-harvest application of putrescine could be an effective means for extending the postharvest life of pear cv. spadona. (*) corresponding author: m.hosseini79@yahoo.com received for publication 28 august 2016 accepted for publication 12 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 11-17 12 l per tree) at different stages of fruit development in may, june, and july. six trees sprayed with water (3.5 l per tree) were used as control. fruits were harvested manually and transported to the postharvest laboratory, and selected for absence of visual symptoms of disease or blemishes, then stored (5 fruits per basket) at 0±1°c, 80-85% rh for 21 weeks. quality attributes were measured in five fruits of each replicate at harvest and after 3, 6, 9, 12, 15, 18 and 21 weeks of cold storage. fruit quality assessments fruit color changes were calculated at two opposite sides of fruit with a minolta chroma meter cr400 (osaka, japan). the values of l*(0 black; 100 white), a* (green to red), b* (blue to yellow) and hue angle (h°=180 + tan-1 b*/a*, if a* < 0) were recorded (fernando et al., 2007; pek et al., 2010). the percentage of weight loss was recorded by using following equation: % weight loss = (a-b)/b × 100 in which a was the initial fruit weight and b was the final fruit weight. fruit firmness was determined using a penetrometer ft327 (gffeci, italy) fitted with an 8 mm tip on the equatorial position of fruit. the results were expressed in newton (n). total soluble solids (tss) in the extracted juice of each treatment were measured by a refractometer (atago n1, japan) at 20°c and the result was recorded as percentage. five ml of diluted juice titrated against 0.1 n naoh to ph 8.2 to assess ta. phenolphthalein was used as an indicator. the ta was expressed as malic acid percentage (saini et al., 2001). the ph of fruit juice was calculated using a mtt65 (japan) ph meter calibrated by ph 4 and 7 buffer solutions. flavor index was estimated by dividing tss with the corresponding ta value. vitamin c was measured using the procedures of tian et al. (2002). total phenol (tp) content and total antioxidant activity (taa) tp and taa were assessed according to koushesh saba et al. (2012). decay incidence determination fruit decay was determined based on the procedure of khademi and ershadi (2013). scales from 1 to 5 were given to individual treatment group whereas: 1= normal (without decay), 2= (up to 5 % decay), 3= (5-20 % decay), 4= (20-50% decay) and 5= (more than 50% of fruits skin was decayed). statistical analysis this experiment was conducted in a randomized experimental design with three levels of putrescine (0.5, 1 and 2 mm), using plants sprayed with water as control in three replications and two trees in each experimental unit. to estimate storability of pear fruit cv. spadona, a factorial design in completely randomized were carried out and the experimental data analyzed using sas statistical software package 9.4 for windows and mean comparisons were conducted using duncan’s multiple range tests. 3. results and discussion color a high rate of color changes was observed in control fruits and 0.5 mm putrescine treated fruits, whereas, they exhibited lower l* and hue angel than others during storage (fig. 1 a and b). therefore, the conversion rate of green to yellow and degradation fig. 1 the effect of putrescine at different concentrations on l* (a) and hue angle (b) of pear cv. spadona along the storage. values are the mean ± se. hosseini et al. putrescine application for improving storage life of pear 13 of chlorophyll were shown slower in putrescine treated fruits by 1 and 2 mm. the effect of putrescine in retarding skin color changes throughout the storage by decreasing senescence rate has also been reported in table grape (harindra champa et al., 2015), and pomegranate (barman et al., 2011). weight loss and firmness the weight loss increased in all fruit samples during the 21 weeks cold storage. as shown in figure 2 a, putrescine at 1 and 2 mm reduced the weight loss value than control at the end of storage. however, fruit treated with 2 mm putrescine showed inferior weight loss which started at the third sampling date (6th week), while it was not seen in those treated with 1 mm before the fifth sampling date (12th week). reduction of weight loss in putrescine treated fruits can be ascribed to conjugation of polyamines to the cell membrane phospholipids and consequently stabilization as well as consolidation of both cell integrity and permeability (barman et al., 2011; mirdehghan and rahimi, 2016). irrespective of treatments, fruit firmness decreased with the advancement of storage but putrescine treatment at 1 and 2 mm maintained highest fruit firmness compared to control (fig. 2b). it is suggested that polyamines maintain fruit firmness by their cross-linkage to the pectin substances carboxyl groups in the cell wall and lead to strengthening of cell wall and consequently decreasing cell wall degrading enzymes activities of pectin methyl esterase (pme), pectin esterase (pe) and polygalactouronase (pg) (valero et al., 2002). the role of putrescine in reducing weight loss and maintaining fruit firmness has been reported for peach (zokaee khosroshahi and esna-ashari, 2008) and pear (franco-mora et al., 2005). tss, ta, ph and flavor index the contents of tss (in the first 12 weeks of storage), ph and flavor index increased in all treated and untreated fruits while ta showed reverse trend along the storage. however, the lowest tss, ph and flavor index were observed in treated fruits by 1 and 2 mm (fig. 3 a, b, c and d). the role of putrescine on maintaining tss, ta and ph in treated fruits would be attributed to the reduction of respiration rate (valero et al., 2002), ethylene synthesis (barman et al., 2011) and subsequently retarding the ripening process. similar results have been reported in peach (zokaee khosroshahi and esna-ashari, 2008) and apricot (enas et al., 2010). vitamin c vitamin c significantly declined as the storage advanced. however, this trend was slower in 1 and 2 putrescine treated fruits (fig. 4). this effect can be associated with the property of putrescine on reducing or delaying the activity of ascorbate oxidase and consequently maintaining vitamin c (ascorbic acid) content (ishaq et al., 2009). similar results have been reported in mango (razzaq et al., 2014) and apricot (davarynejad et al., 2013). total phenol (tp) and total antioxidant activity (taa) measurement irrespective of treatments, total phenolic content and total antioxidant activity decreased at the end of storage; while these decreases were significantly higher at 1 and 2 mm putrescine treated fruits (fig. 5 a and b). in spite of taa, the tp changes were not constant during storage, it reached the highest value at the 9th week in fruits treated with 1 and 2 mm with the maximal values of 28 and 31 mg of gae/100 g of fw at the 9th week respectively, then followed by reducing tp during the rest of storage period. fig. 2 the effect of putrescine at different concentrations on weight loss (a) and firmness (b) of pear cv. spadona along the storage. values are the mean ± se. adv. hort. sci., 2017 31(1): 11-17 14 fig. 3 the effect of putrescine at different concentrations on tss (a), ta (b), ph (c) and flavor index (d) of pear cv. spadona along the storage. values are the mean ± se. fig. 4 the effect of putrescine at different concentrations on vitamin c of pear cv. spadona along the storage. values are the mean ± se. fig. 5 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on total phenol content (a) and total antioxidant activity (b) of pear cv. spadona along the storage. values are the mean ± se. hosseini et al. putrescine application for improving storage life of pear 15 the changes in the level of tp content may be associated to the breakdown of cell structure and subsequently senescence (ghasemnezhad et al., 2010). the role of putrescine treatment to maintain tp could be ascribed to the delay of senescence process (arora et al., 2002; razzaq et al., 2014). as shown in figure 5, the value of taa decreased along with a decrease of tp during storage. it may be ascribed to a direct correlation among tp content and taa (razzaq et al., 2014). however, putrescine treatment at 1 and 2 mm maintained taa compared to control during storage. similar results demonstrated a positive correlation among tp and taa in mango (palafox-carlos et al., 2012) and apricot (ghasemnezhad et al., 2010). decay incidence the lowest rate of fruit decay percentage was observed in fruits treated with 1 and 2 mm putrescine contrary to control at the end of storage (fig. 6). it is suggested that polyamines have all requirements of an alternative approach for management of postharvest decay (romanazzi et al., 2012). storage life the application of putrescine at higher doses (1 and 2 mm) extended storage life of pear fruits, and consequently they were suitable to be exposed in the market more than 127-142 days after the beginning of storage in comparison to control (109 days) (fig. 7). 4. conclusions the pre-harvest application of 1 and 2 mm putrescine treatment 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27-32. impaginato 225 1. introduction the market of pomegranate fruits is noticeably expanding worldwide. the increasing interest towards this photophilous and moderately xerophytic species relies essentially on it nutritional and nutraceutical properties, being considered a functional product of relevant benefit for the prevention of human diseases (martinez et al., 2012; kotsiou and tesseromatis, 2015). punica granatum l. is native to iran (teixeira da silva et al., 2013), nevertheless a western area (which includes part of anatolia, the caucasus and iran) and an eastern area (southeastern turkmenistan, southern tajikistan, afghanistan, northern pakistan, kashmir, northern india and nepal) are generally recognized (alam, 2011). due to its hardiness pomegranate naturalizes quite easily and many local landraces and varieties can be found in the area of the mediterranean basin, in middle east and in the arabic peninsula (al-sadi et al., 2015). iran embrace a rich germplasm of pomegranate, with 760 specimens, 24 genotypes and various commercially important cultivars listed in different studies, nevertheless also india, israel, egypt, tunisia, morocco, spain and italy hold valuable genetic resources of punica granatum (holland et al., 2009). more than 20 varieties of pomegranate (locally called ‘anar’), among which ‘bedana’ (soft seeded), ‘khog dandar’, ‘tarwah’, ‘boocha’, ‘pastakai’, and ‘soor’, were grown in afghanistan during the second half of the xx century (alam, 2011). more recently, 48 cultivars of pomegranate from various provinces of afghanistan were described and characterized in relation to fruit colour, flavour and seed hardiness adv. hort. sci., 2016 30(4): 225-230 doi: 10.13128/ahs-20348 short note selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions e. giordani 1, m. berti 2, m. rauf yaqubi 3, s. stanikzai 3, a. amad 3, b. zadran3, a. saeedi 3, m. ghous3 1 dipartimento di scienze delle produzioni agroforestali e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 indipendent consultant. 3 afghanistan national horticulture development organisation, kabul, afghanistan. key words: genetic resources, phenotype, punica granatum. abstract: during the period 2008-09 and in the frame of phdp 58 afghan and 18 foreign pomegranate accessions have been collected and grown under homogeneous environmental and cultivation practice conditions in ex situ collections. standardized procedures were adopted to describe mature trees, leaves, flowers and fruits for a total of 30 phenotypic traits. within the national collection of pomegranate of afghanistan, coefficients of variability ranged from 8.8 to 31.7% for fruit diameter and weight of non edible part, respectively. principal component analysis revealed the absence of correlated variables among different organs. the whole set of accessions resulted discriminated on the basis of the studied morphological parameters and all the accessions were grouped into 3 sub-sets by hierarchical cluster analysis. local accessions resulted distributed in the 3 clusters, nevertheless the largest one held all the foreign varieties while the second one included all the accessions collected under the putative name of ‘bedana’. the adopted morphological studies allowed to identify one true-to-type ‘bedana’ accession, considered the best afghan variety for fresh consume due to its very soft seed, and to solve the cases of homonymy. analogously, various accessions originally collected from different regions of afghanistan under the name of ‘kandahari’ were identified and renamed. (*) corresponding author: edgardo.giordani@unifi.it received for publication 11 january 2016 accepted for publication 27 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 225-230 226 (samadi, 2008); among them, ‘kandahari’ and ‘bedana’ were considered excellent cultivars (glozer and fergusson, 2008). in the frame of the perennial horticulture development project (phdp phase i), several pomegranate producing areas of afghanistan were surveyed during the period 2007-2008 and a set of the most valuable varieties was collected (giordani et al., 2014). the in situ pomegranate collection resulted comprehensive of 59 local accessions which were propagated by cuttings. the obtained saplings were planted in ex situ collections, together with 19 imported cultivars in kandahar and jalalabad, thus representing the national collection of fruits and nut of afghanistan (saeedi et al., 2012). morphological characterisation of native germplasm accessions is the first step for their rational introduction in modern and efficient production chains and a standardized description of phenotypic traits based on the adoption of specific descriptor lists is a pre-requisite for this purpose (frankel, 1970). the present study aims at describing and analysing the pomegranate accessions belonging to the national collection of varieties of fruits and nuts of afghanistan. 2. materials and methods plant material data were collected during 2013 and 2014 from self-rooted 4 years old trees grown in the repository of the national collection of fruits and nuts of afghanistan located in kandahar (latitude 31°36’ n and longitude 65°42′ e; altitude 1009 m a.s.l.). for each accession flowers (10 samples), fully expanded leaves (10 samples) and fruits (20 samples) were randomly collected for two consecutive years (20132014) from trees (6 samples) grown following standardized cultural practices. data collection traits were described by rating and coding procedures based on the ec 29 genres project pomegranate descriptor list and guidelines (genres, 1998; bellini et al., 2008). quantitative variables were measured and weighed adopting a manual calliper and a precision (0.01 g) electronic balance, respectively. colour parameters were visually determined using a specifically designed colour chart; other qualitative characteristics were attributed by using illustrated charts. data of 13 qualitative characteristics (tree habit, vigour and suckering tendency; shoot thorniness; leaf apex shape; number of flowering periods; flower calyx and corolla colour; colour change of calyx after petal fall; fruit juiciness and fruit skin colour and thickness; seed colour) and 17 biometrical variables (table 1) were collected for all the studied accessions. data processing and statistical analysis average and mode were calculated for all qualitative and quantitative parameters respectively; such values were used for the attribution of each accession to one class, regardless the year of data collection. classes and relative notes were defined for each quantitative measured trait. after average value standardization, multivariate principal component analysis and hierarchical cluster analysis (euclidean distance and ward’s agglomeration method) were performed on xls stat software. 3. results and discussion studied accessions the survey allowed to collect 58 afghan accessions (fig. 1) in stands represented by orchards and home gardens distributed in the eastern (kapisa, nangarhar; 29 accessions), southern (kandahar and farah; 16 accessions) and northern (takahr, balkh and sar-e-pul; 13 accessions) areas, at altitudes ranging from 422 m a.s.l. to 1513 m a.s.l. such accessions variable mean range cv (%) standard deviation leaf total length (mm) 68.4 52.6-86.2 11.5 7.9 leaf blade length (mm) 63.2 48.0-78.5 11.5 7.3 leaf blade width (mm) 20.7 16.2-26.7 11.7 2.4 flower petal length (mm) 26.1 19.2-30.8 9.1 2.4 flower petal width (mm) 19.9 14.3-30.2 13.4 2.7 flower length of calyx (mm) 38.5 29.5-46.7 10.2 3.9 flowerwidth of calyx (mm) 13.8 9.1-24.3 14.4 2.0 fruit equatorial diameter (mm) 84.5 64.9-99.2 8.1 6.8 fruit diameter of calyx (mm) 21.1 11.6-35.3 19.6 4.1 fruit height without calyx (mm) 76.4 56.7-94.0 8.8 6.7 fruit calyx height (mm) 23.2 14.8-34.6 18.6 4.3 fruit total weight (g) 309.3 134.8-490.1 24.3 75.1 fruit weight of non edible part (g) 144.6 76.6-323.1 31.7 45.8 fruit edible/total weight (%) 53.8 18.7-70.8 13.5 7.3 seed weight (g) 0.3 0.2-0.4 18.8 0.1 seed length (g) 10.3 8.0-13.6 8.9 0.9 seed width (mm) 6.8 4.9-8.4 12.2 0.8 table 1 mean values, ranges and coefficients of variation for 17 quantitative traits of pomegranate accessions of the national fruit and nuts collection of afghanistan giordani et al. afghanistan selected pomegranate germplasm. morphological variability and relationship 227 were grown together with 19 imported varieties including the international reference cultivar ‘wonderful’. morphologic and phenological trait variation descriptive statistical values for continuous biometric data of the whole set of 77 accessions are reported in table 1. leaf size showed a relatively low variation (cv% ≈ 11.5 for total leaf length, length and width blade), as well as flower biometric traits, while higher values of variations were observed in fruit and seed traits. namely, minimum and maximum mean of fresh fruit weight (≈ 134-490 g) fall within the range of analogous values observed by okatan et al. (2015), caliskan and bayazit (2013) in pomegranate collections from turkey and by mars and marrakchi (1999) in a set of 16 accessions collected in tunisia. on the other hand, orhan et al. (2014), mondragòn jacobo (2012), bartual et al. (2012), ferrara et al. (2012), russo et al. (2012), and legua et al. (2012) observed higher mean values of fruit weight in accessions collected in coruh valley (turkey), in mexico, in elche (spain), in apulia (italy) and in morocco, respectively. a narrower range of variability for this parameter (204-288 g) was observed in a study on iranian accessions (theranifar et al., 2010). seed (aril) fresh weight (mean 0.33 g; range 0.17-0.33 g), resulted higher than the values reported by okatan et al. (2015) (0.28 g; range 0.14-0.41 g) for turkish accessions and by mondragòn jacobo (2012)(0.20 g; range 0.12-0.26 g) for mexican accessions, but lower than those found out by caliskan and bayazit (2013) in turkish pomegranate accessions (0.42 g; range 17-67 g) and by ferrara et al. (2012) (0.38 g; range 0.270.51) in apulian pomegranates in italy. the ratio between the edible part of the fruit (arils) and the total fresh fruit weight ranged from 18.7% to 70.8%, with an average of 53.8%, lower than the value (55.6%) found by legua et al. (2012) in accessions from morocco. the beginning of flowering time ranged from 104 to 115 days from the beginning of the year while day 253 resulted the earliest date of the beginning of ripening time (fig.1 a, b). the range of flowering and ripening time of the investigated accessions resulted of 11 and 44 days, with a discontinuous frequency for ripening time between days 269 and 284 (fig. 1 b). the ripening time period (end of septemberoctober) resulted similar to the spanish and turkish one; no significant correlation has been found between fruit size and ripening time, a pattern observed by different authors in the germplasm of other areas (holland et al., 2008; martínez et al., 2012; caliskan and bayazit, 2013). fig. 1 distribution (%) of the collected pomegranate accessions in relation to: a) beginning of flowering time (day of the year); b) beginning of ripening time (day of the year); c) fruit skin color and d) fleshy part of seed (aril) color. adv. hort. sci., 2016 30(4): 225-230 228 attaining to colour fruit traits, which are important quality attributes in pomegranate marketing (mena et al., 2011), most of accessions presented fruits with yellowish skin (53.1%), followed by reddish colouration (45.7%), and only 1.2% of accessions showed totally black skin colour. fleshy part of seeds was in most cases white (39.2% of accessions), while pinkish and red-white colour shared the same percentage (≈30%) of accessions and only 1.4 % of varieties showed dark red arils (fig. 1 c, d). relationship between the described accessions principal component analysis based on the averages of the discrete scores of the morphological qualitative and quantitative variables showed a low correlation between the utilized variables, with 12 principal components with eigenvalue higher than 1, all together accounting only for 76.8% of the variability. taking into account the sub-set of 19 biometric continuous variables regarding leaves, fruits, and seeds, the 6 principal components with eigenvalue>1 accounted for 78% of the whole variability, confirming a very low correlation among variables for the same set of accessions. higher levels of correlation among the biometric variables have been reported for different groups of turkish (caliskan and bayazit, 2013; okatan et al., 2015) and tunisian (mars and marrakchi, 1999) local varieties. the average euclidean distance of the generated proximity matrix based on the 17 biometric variables resulted equal to 5.96±1.21, ranging from 1.99 observed in the pair ‘bedana samashkeli jan mohammadi’ up to 11.67 for the couple ‘sorkhaksakerdze’. the dendrogram generated by the cluster analysis is reported in figure 2, where three main significant clusters (c1, c2 and c3) can be observed. cluster c1 resulted the largest with 35 accessions, 19 of which represented by foreign non afghan accessions, including the international cultivar ‘wonderful’. cluster c2 was constituted by 19 accessions, 6 of which were originally collected as ‘bedana’, a typical afghan variety appreciated by the presence of very soft seeds. albeit this easily appreciable and discriminating trait, the standardized characterisation of the presumed ‘bedana’ accessions showed that only one accession could be identified as belonging to the true ‘bedana’ type. the accession collected in kandahar and entered in the collection as ‘bedana-afg0383’ showed heavy and hard seeds and other relevant differences from the ‘bedana’ type, such as big calyx, pale skin colour and low juiciness, and it has been fig. 2 dendrogram (euclidean distance; ward's agglomerating method) obtained by analyzing 30 multi-state discrete morphological variables of 58 pomegranate accessions of afghan origin and 19 imported varieties belonging to the national collection of fruits and nuts of afghanistan (bold: imported cultivars; bold cursive: putative ‘bedana’ type; cursive: putative ‘kandahari’ type). giordani et al. afghanistan selected pomegranate germplasm. morphological variability and relationship 229 identified as ‘mirwais khani’ (fig. 2). similarly the accession originally denominated ‘bedana-afg0762’, brought medium hard seeds, white-pink arils, and was renamed as ‘abasi’. also ‘bedana-afg5018’, collected in tabag district of kapisa province, and ‘bedana afg6061’, originated from sherzad district of jalalabad province, did not show the typical characteristics of the true-to-type ‘bedana’, and were renamed as ‘ghani kheli’ and ‘jan mohammadi’ respectively. finally, the presumed ‘bedana’ type ‘afg6053’, originated in the district of jalalabad, notwithstanding its high morphological similarity with the true-to-type ‘bedana’ (soft seeds, juicy and good taste), showed low productivity and was denominated ‘bedana samaskheli’. cluster c3, holding 23 local accessions, is not evidencing any special relationship among previously known cultivars. during the survey, a part from ‘bedana’ name, another recurrent denomination attributed by farmers to local accessions was ‘kandahari’ (from kandahar). indeed ‘kandahari’ accessions were collected from different provinces of afghanistan and they showed different and unique phenotypic traits during the characterisation hence they have been considered as different varieties. for instance, ‘kandahari afg6066’ originated from sher zad district of nangarhar province, had thin fruit skin and hard seeds, and it was renamed as ‘rahmani’; ‘saifi’ was the accepted name for the former ‘kandahari afg5019’, collected in tagab district (kapisa province) producing fruits with hard seeds, reddish arils and low juiciness. ‘mukhtari’ was originally ‘kandahari afg0862’ from khulam district (balkh province) with medium hard seeds and white arils. actually ‘kandahari’ name was kept only for the accession collected as ‘kandahari afg6057’ originated from tagab district (kapisa province) and producing large fruits with medium hard skin, medium hard seeds and red arils. 4. conclusions this study was the first of its kind carried on for pomegranate in afghanistan in terms of standardized methodology; comparative analysis with previous data collected from local afghan varieties of pomegranate trees grown in different environmental conditions and as unique individuals resulted not completely reliable. the results revealed a wide variability of morphological traits within the afghan pomegranate collected accessions; this finding is consistent with similar researches carried on local pomegranate accessions in countries where p. granatum is present since ancient time. such variation can be considered as a result of the combined effect of sexual reproduction and asexual propagation. on the other hand, many cases of homonyms have been observed, hence confirming a common trend in the denomination of varieties by farmers of countries where perennial species are grown traditionally. more specific morphological studies associated to the evaluation of tree productivity and fruit biochemical and commercial traits would be the next step in order to better classify the afghan pomegranate accessions in relation to their main suitability for different uses (fresh fruit consume, juice production, food supplements, etc.). acknowledgements activity founded by the european commission europeaid program. a special appreciation to the staff of the ministry of agriculture, irrigation and livestock of afghanistan for the availability and support. an unique everlasting memory for mohammad ghous and monica berti. references alam m., 2011 trees and shrubs of afghanistan. a dendrological guide. rossolis, musée botanique cantonal lausanne, switzerland, pp. 512. al-sadi a.m., al-fahdi a.r., al-yahyai r.a, al-ghaithi a.g., al-said f.a, soleiman m.j., 2015 genetic analysis suggests a shared origin of punica granatum cultivars in oman with cultivars from the center of origin, iran. genet resour. crop evol., 62: 815-821. bartual j., valdes g., andreu j., lozoya a., garcia j., badenes m.l., 2012 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2008 pomegranate varieties of afghanistan. business directory. pomegranate agfair-world fair, ahmad printing press, kabul, afghanistan. teixeira da silva j.a., rana t.s., narzary d., verma n., meshram d.t., ranade s.a., 2013 pomegranate biology and biotechnology: a review. sci hortic., 160: 85-107. theranifar a., zarei m., nemati z., esfandiyari b., vazifeshenas m.r., 2010 investigation of physicochemical properties and antioxidant activity of twenty iranian pomegranate (punica granatum l.) cultivars. sci. hort., 26: 180-185. impaginato 135 1. introduction in animal cells, the role of plasma membrane behavior is deeply investigated, and it may reflect health status. cellular defense against stress is a key function in which the plasma membrane redox system is involved. moreover, some of the plasma membrane redox system enzymes produce reactive oxygen species as well as play a protective role against them. moreover, evidences underline as redox state of the cell and, accordingly, plasma membrane electron transport contribute to control cell growth, development and apoptosis (ly and lawen, 2003). in animal cells, virus infection can lead to variation in membrane potential (akeson et al., 1992). these effects may occur even during early pathogen recognition events in plant-microbe interaction. as reviewed by elmore and coaker (2011), plasma membrane h+-atpases are dynamically regulated during plant immune responses and quantitative proteomics studies suggest complex spatial and temporal modulation of plasma membrane h+-atpase activity during early pathogen recognition events, even if no data relative to virus were available. anyway, an important role is played by h+-atpases in bacterial infection, where the enzyme cooperate with plant immune signaling protein (namely rin4) to regulate stomatal apertures during pest invasion of leaf tissue. these enzymes are ubiquitous and are involved in plant immune responses. moreover, they may are targeted by pathogens to increase plant susceptibility, even if these evidences are related to fungi or bacteria. thus shabala et al. (2010) affirm that little is known about the ion fluxes generated during plantvirus interactions, despite significant losses caused by viruses to agricultural crops. viruses represent a major threat to agricultural production, mainly due to the lack of effectiveness treatments, and preventative measures represent the main tools available to protect plants. while it is widely believed that in many seed-propagated crops virus the threat is of limited importance, the health status of plants subjected to vegetative propagation is critical. although the viruses that infect woody plants raised more adv. hort. sci., 2016 30(3): 135-139 doi: 10.13128/ahs-20249 insight on trans-plasma membrane behavior of virus-infected plant cells a. luvisi 1 (*), e. rinaldelli 2 1 dipartimento di scienze e tecnologie biologiche ed ambientali, università del salento, via provinciale lecce monteroni, 73100 lecce, italy. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: grapevine, potassium channel, tobacco, tmv. abstract: little is known about the ion fluxes generated during plant-virus interactions, despite significant losses caused by viruses to agricultural crops. changes in average ion currents were identifying an early event in the signal transduction pathway related to virus/host interaction. while significant decrease in the average inward currents, mainly due to ca2+ moving into the cell was observed, the role of potassium may be significant. host specific k+ efflux with a concomitant decrease in the intracellular k+ was observed in tobacco plants during the early minutes after infection, suggesting many hypothesis about the role of potassium in host-virus interaction. in the last years, trans-plasma membrane potential was evaluated for some viruses, observing as effect on membrane was different in relation to virus infection and host. conversely, settle virus infection generally lead to an increase of activity in trans-plasma membrane electron transport. (*) corresponding author: andrea.luvisi@unisalento.it received for publication 21 june 2016 accepted for publication 8 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 135-139 136 health concerns, trials are longer and more difficult to manage compared to test with herbaceous plants. 2. viruses and hosts: a poorly investigated binomial in trans-plasma membrane events few study investigated the trans-plasma membrane behavior of virus-infected plants, thus few viruses were evaluated in trials, mainly focused on herbaceous plant-related virus. tobacco mosaic virus (tmv) is a widespread virus difficult to eradicate or control from plant and soils (luvisi et al., 2012 a; panattoni et al., 2013 a; luvisi et al., 2015 a). tmv was frequently chosen as “model virus” in host-virus interaction tests. it was also chosen in first studies in trans-plasma membrane behavior of plants (schvarzstein, 1997). cucumber mosaic virus (cmv) (rinaldelli et al., 2012), papaya mosaic virus (pmv) (schvarzstein, 1997), potato virus x (pvx) (shabala et al., 2010) and tobacco ringspot virus (trsv) (stack and tattar, 1978) were also investigated. among virus affecting woody plants, grapevine viruses (rizzo et al., 2012, 2015) were the most investigated. grapevine leafroll associated virus 1 or 3 (glrav-1, glrav-3), grapevine fanleaf nepovirus (gflv), grapevine fleck maculavirus (gfkv), grapevine vitivirus a (gva) and arabis mosaic virus (armv) were evaluated during antiviral tests or in settle infections (panattoni et al., 2013 b; rinaldelli et al., 2014). 3. trans-plasma membrane behavior in early virus infection events few data are available for early events in plantvirus interaction. stack and tattar (1978) observed as trsv-infected cells of cowpea showed altered transmembrane electropotentials and responded differently from healthy cells when subjected to the metabolic inhibitor sodium azide in their ability to recover. changes in average ion currents were also observed in protoplast of gomphrena globosa artificially infected by pmv and tmv, identifying an early event in the signal transduction pathway related to virus/host interaction (schvarzstein, 1997). in g. globosa, necrotic lesions surrounded by a chlorotic ring developed in inoculated leaves are thought to be the results of a non-host hypersensitive response (hr) that ultimately defend the plants from virus infection. schvarzstein (1997), using patch-clamp techniques, elucidated the early events occurring during pmv infection. whole cell recording indicate a significant decrease in the average inward currents, mainly due to ca2+ moving into the cell or gluconate moving out of the cell. in the same time, an increase in the outward currents was also observed, due to clmoving into the cell and k+ moving out of the cell. the outward currents were correlated to pmv concentration, and a similar behavior was observed during tmv infection. the studies of schvarzstein were the first one in which ion fluxes were characterized during virus-plant interaction and unfortunately, very few studies following. the phenomenon of rapid alteration of ion fluxes in host cells during the early phase of viral infection was reported in chlorella (neupartl et al., 2008). chlorella viruses tests suggested a key-role of potassium, due to an observed host specific k+ efflux with a concomitant decrease in the intracellular k+. the use of blockers of the viral-encoded k(+) channel (kcv) reduced this k+ efflux, suggesting as depolarization and k+ efflux are at least partially mediated by kcv. the k+ efflux seems virus-triggered and it occurs in the same time frame as host cell wall degradation and ejection of viral dna. therefore, neupartl et al. (2008) supposed that loss of k+ and associated water fluxes from the host lower the pressure barrier to aid ejection of dna from the virus particles into the host. even if chlorella shared some metabolic pathway with plants, this interesting findings cannot directly transferred to plants without further studies. moreover, dna-based viruses are uncommon pathogens in plants. anyway, the key-role of potassium in plant-virus interaction was confirmed by shabala et al. (2010), moving the focus from calcium. the role of ion fluxes in plant defense signaling is well documented and elicitor-induced transient ca2+ influx from the external environment into the cytosol has always been named as a key element of the signaling cascade and appears to be crucial in the induction of plant defense against pathogens (scheel, 1998; zimmermann et al., 1999; blume et al., 2000; grant et al., 2000), but this studies are not related to viruses. shabala et al. (2010) observed as an addition of the purified pvx to the mesophyll tissue of tobacco plants caused no changes in the rate of ca2+ transport across the plasma membrane. these evidences were supported by no significant changes in concentration of ca2+ in cytosol for at least 50 min after pvx treatment. this behavior indicated that ca2+ release luvisi and rinaldelli trans-plasma membrane behavior of virus-infected plant cells 137 from internal stores was also not a part of the signal transduction mechanism in plant-viral interaction. authors suggest that, contrary to bacterial pathogens, rapid ca2+ signaling may not be essential for the viral perception and initiation of downstream transduction pathway. conversely, massive k+ efflux was measured as early as 10 min after virus inoculation and this behavior in host-related. prolonged exposure virus caused lower concentration of ca2+ in cytosol compared with control plants, suggesting of the role of ca2+ efflux systems in downstream cascades of the plant responses to viral infection. k+ efflux was partially reduced by blockers of kcv channels such as cs+ (shabala et al., 2010), similarly to chlorella tests. the antiviral drug mycophenolic acid (mpa) (panattoni et al., 2014; guazzelli et al., 2015) was also indicated as interferent with katp channel activity in grapevine, as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by mpa (luvisi et al., 2015 b). nowadays, the physiological role of the observed viral-induced k+ efflux was not known. neupartl et al. (2008) suggest that the efflux of k+ and the associated water efflux from the chlorella cell may be needed to reduce turgor and lower the pressure barrier, to aid ejection of dna from the virus particles into the host, but adding several known blockers of k+ efflux channels to the buffer media during pvx inoculation in tobacco plants did not ameliorate infection symptoms, probably because the blocking effect of pharmacological agents was only partial (shabala et al., 2010). further hypothesis may be related to role of potassium homeostasis in plant adaptive responses to environment (shabala et al., 2007), including programmed cell death (shabala, 2009). interestingly, the observed viral-induced activation of k+ efflux systems appears to be a highly hostspecific process, indicating that the observed k+ fluxes may be linked to the plant’s ability to recognize compatible viral infection and activate defense pathways (shabala et al., 2010). 4. trans-plasma membrane behavior in settle virus infection membrane potential membrane potential of grapevine cells were monitored during antiviral drugs treatments (luvisi et al., 2012 b). the complex of results obtained in this research highlights, first of all, that membrane electrical response of the tested antiviral drugs is supported by the metabolism of plant cell, and no differences in δ em were found glrav-1-infected grapevine leaves compared to virus-free leaves. however the behavior of membrane of plants treated with antiviral drugs was different in other hosts, as reported in tobacco plants infected by cmv, where trans-plasma membrane potential activity seemed to be influenced by virus presence, acting differently in infected or healthy samples during drug uptake by cells (rinaldelli et al., 2012). anyway, drugs may hindered or enhance virus effects of membrane behavior and specific trials in settle virus infection were carried out in grapevine (rinaldelli et al., 2014). samples infected by leafroll viruses showed no difference in membrane potential values compared to healthy samples, confirming the similar behavior of glrav-1 or -3 infected samples and healthy ones observed during antiviral drug treatments. gflv, or armv infected tissues led to plasma membrane hyperpolarization with higher values compared to healthy samples, while gfkv and gva infected tissues showed plasma membrane depolarization significantly lower than the control (rinaldelli et al., 2014). according to the role of h+atpase activity in generating trans-plasma membrane potential gradient (sondergaard et al., 2004), gflv and armv infected cells could be considered more energized compared to others. conversely, gfkv and gva infection was also associated to increasing difficulty of cell membrane measurements, as occurs under stress conditions (rawyler et al., 2002). electron transport rubinstein and luster (1993) indicated that that trans-plasma membrane electron transport (t-pmet) occurs in all types of organisms, including plants. tpmet allows reduction of extracellular oxidants at the expense of intracellular reducing equivalents that may derive from nadh or nadph (del principe et al., 2011). the t-pmet enzymes have been investigated based on their ability to reduce external artificial impermeant electron acceptors. ferricyanide (fe3+) has been commonly used as electron acceptor in assays performed with intact cells; ferricyanide is converted to ferrocyanide (fe2+), and the rate of this reduction can be monitored. using a carbon fibre microelectrode (cfme) it is possible to map oxidoreductase activity using impermeant electron acceptors or donors (taylor and chow, 2001). this techniques adv. hort. sci., 2016 30(3): 135-139 138 were also used to evaluate in vivo estimation of the inosine monophosphate dehydrogenase inhibition caused by antiviral drugs (panattoni et al., 2015). the t-pmet was observed during antiviral treatments in cmv-infected tobacco plants (rinaldelli et al., 2012). infected samples were less sensitive to antiviral treatments considering t-pmet. this effect may be due to the concurrent entry of drug within the symplast that, as indicated by membrane potential, was lower in infected samples and that can lead to lower inhibition of nad+/nadh conversion by drug and to the following increase of fe3+ conversion. this virus-related behavior was confirmed in glrav-1 and -3 tests (panattoni et al., 2013 b). virusinfected samples exhibited elevated t-pmet activity compared to healthy samples. the [fe2+] in healthy samples was set at 26 µm, while glrav-1and glrav-3-infected samples showed 34-49 µm [fe2+]. these data can be linked to the nadh content in glrav-1 and glrav-3 samples, that was set at 1.2 times higher than healthy samples. this trans-plasma membrane behavior was partially confirmed for other grapevine viruses (rinaldelli et al., 2014). in virus-infected samples, while the [fe2+] produced by gfkv and gva infected tissues were similar to healthy tissues, the samples infected by gflv, armv and leafroll viruses showed higher t-pmet activity. the higher nadh content due to virus infection was used differently by infected samples during their tpmet activity. samples whose infectious status did not interfere negatively with membrane potential, such as armv, gflv, glrav-1, and -3 showed higher t-pmet activity compared to healthy samples, in agreement with the higher nadh availability. 5. discussion the high level of specialization attained by many viruses due to their replication and pathogenetic mechanisms towards the host make them an extremely variable and complex target. this complexity is the background upon which a defensive strategy can be optimized. therefore, the outcome of therapeutic action is strongly influenced by the ontological properties of the virus to be eliminated as well as the characteristics expressed by the plant as well as the trans-membrane transport of drugs. in human pathology, trans-membrane alteration may represent 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cell. mol. life sci., 55: 183-203. 73 1. introduction floral thermogenesis occurs in several plant taxa including gymnosperms (cycadaceae), as well as eudicots (nymphaeaceae) and monocots (araceae). thermogenesis begins when these plants bloom, and heat production terminates when pollen is released from the anthers. one thermoregulatory plant, the eastern asian skunk cabbage (symplocarpus renifolius), can keep the spadix temperature between 22-26°c for several days even when the ambient temperature falls below freezing (fig. 1a) (knutson, 1974; uemura et al., 1993; seymour, 2004). other thermoregulatory plants studied to date include phillodendron sellom (nagy et al., 1972; seymour et al., 1983) and nelumbo nucifera (seymour and schultze-motel, 1998; seymour et al., 1998). many species, which are thermogenic but not thermoregulatory, are generally able to produce heat for only 24 h at best. the robust thermoregulation observed in s. renifolius and other species makes these plants great models for unraveling the mechanism underlying floral thermogenesis. in several species of araceae, floral thermogenesis has been proposed to serve the physiological role of spreading odor to attract pollinators (meeuse and raskin, 1988), whereas thermoregulation in s. renifolius is not closely associated with cross-pollination (seymour and blaylock, 1999). s. renifolius produces only a faint aroma in early spring when few insects are active. thus, heating may promote early flowering or protect the s. renifolius inflorescence from damage by freezing. in s. renifolius, thermogenesis is closely associated with three stages of inflorescence development: female, bisexual, and male (fig. 1b). at the female stage, which lasts until the stamens emerge from the surface of the spadix, the spadix can produce massive amounts of heat. at the bisexual stage, the stamens begin to release pollen and thermogenesis fluctuates. finally, at the male stage, pollen is released from nearly all stamens and thermogenesis is undetectable. microscopic analysis revealed that structural changes in the stamen are significant, and extensive anther development occurs during inflorescence development (ito-inaba et al., 2009 a). in addition to the structural changes in stamens, the ultrastructure of petals and pistils also significantly change. these tissues accumulate a larger number of mitochondria during the female stage than during the male stage. also, large cytoplasmic vacuoles develop during the male stage. in our recent gene expression analysis, expression of genes involved in cellular respiration and mitochondrial function was significantly enhanced during the thermogenic female stage, whereas genes involved in stress responses and protein degradation were significantly up-regulated during the non-thermogenic male stage (ito-inaba et al., 2012 a). therefore, changes in the intracellular structure observed in thermogenesis in skunk cabbage (symplocarpus renifolius): new insights from the ultrastructure and gene expression profiles y. ito-inaba organization for promotion of tenure track, university of miyazaki, 1-1 gakuenkibanadai-nishi, miyazaki 889-2192, japan. key words: floral thermogenesis, inflorescence, low temperature, mitochondria, respiration, transcriptome. abstract: floral thermogenesis has been found in several plant species. the spadix of one thermoregulatory plant, the eastern asian skunk cabbage (symplocarpus renifolius), can maintain its temperature at approximately 22-26°c for several days, even when the ambient temperature falls below freezing. there are two major stages in skunk cabbage inflorescence development: the thermogenic female stage and the non-thermogenic male stage; in the former the spadix can produce massive amounts of heat, whereas in the latter, thermogenesis is undetectable. based on previous studies, there is a positive correlation between heat production and the abundance of mitochondria in plant tissues and cells, and genes involved in cellular respiration and mitochondrial function are significantly enhanced at the female stage. taken together, these findings suggest that the increased respiration or mitochondrial abundance observed in thermogenic tissues may be attributable to the high expression of specific genes. this review summarizes new insights into the changes in intracellular structures and gene expression profiles of skunk cabbage spadices during the female-male transition and proposes possible processes that are essential for each stage during floral development. adv. hort. sci., 2014 28(2): 73-78 received for publication 31 march 2014 accepted for publication 18 june 2014 74 petals or pistils during the female-male transition are well supported by changes in the transcriptome during inflorescence development. two processes may be important for thermoregulation in skunk cabbage (ito-inaba et al., 2012 a). first, shortterm mechanisms that depend on increased cellular respiration with the help of energy dissipating proteins, such as alternative oxidase (aox) or uncoupling protein (ucp), may play an essential role in which aox may have a more major function than ucp. secondly, long-term effects of mitochondrial biogenesis on the number and structure of mitochondria probably are also involved. following much effort to characterize the activity or expression of aox during floral development, the pivotal role of this enzyme in floral thermogenesis was revealed (watling et al., 2006; grant et al., 2008; wagner et al., 2008; ito-inaba et al., 2009 b; miller et al., 2011). however, the presence of additional genes that are co-expressed with aox and that may function directly or indirectly in thermogenesis remains to be clarified. in addition, although it has been hypothesized that heat-producing floral tissues contain many mitochondria, quantitative and comparative studies on mitochondrial content are lacking. in this review, we summarize our recent progress in describing changes in the ultrastructure and gene expression profiles during skunk cabbage floral development. 2. thermogenesis and mitochondrial abundance in mammalian cells, the positive correlation between metabolic activity and the number and size of mitochondria within a tissue is well established (ghadially, 1988). mammalian brown adipose tissue (bat), which is the main site for non-shivering thermogenesis, contains considerable numbers of large mitochondria with abundant cristae. in contrast, these relationships are not well characterized in plants, and there are very few published papers that have examined the intracellular structure of thermogenic tissues by electron microscopy. in a well-known thermogenic plant, sauromatum guttatum (voodoo lily), ultrastructural changes in the inflorescence during the transition from the preto post-thermogenic stages were extensively studied, and clear details of mitochondrial morphology were obtained (skubatz et al., 1993). in addition, during the thermogenic stage of s. guttatum floral development, mitochondria accumulated osmophilic material between the inner and outer membranes (skubatz and kunkel, 2000). in another thermogenic plant, philodendron selloum, large lipid bodies present in sterile florets before heating were progressively depleted during heat generation, and the mitochondria often contained enlarged cristae during maximum heating (walker et al., 1983). however, there are no conclusive data indicating a relationship between heating in plant tissues and mitochondrial features, such as content or morphology. we first analyzed the detailed changes in mitochondrial content and morphology during floral development of thermogenic skunk cabbage, s. renifolius (ito-inaba et al., 2009 a). as shown in figure 2a, petal cells at the female stage contained a large number of mitochondria. by contrast, petal cells at the male stage contained only a small number of mitochondria but had large central vacuoles. in the pistil cells, likewise, a large number of mitochondria were present at the female stage but few mitochondria persisted to the male stage. furthermore, stamens at the female stage, especially in the microspore and plasmodium, had high densities of mitochondria. the sizes and morphologies of mitochondria observed in all tissues varied. to evaluate the mitochondrial content quantitatively between the female and male stages in each floral tissue, the average mitochondrial density (mitochondrial numbers µm-2 cytosol) in thin sections of cells were analyzed in five to 10 cells. these data also revealed that both petals and pistils at the female stage contained larger numbers of mitochondria compared with the male stage. details of the ultrastructure and the quantitative fig. 1 thermoregulation in skunk cabbage (s. renifolius). (a) skunk cabbages were photographed using a camera in the visible (left panel) and infrared spectra (right panel). the thermal image was taken with thermotracer sc620 (flir). heat production was observed in the spadix during the female stage of floral development. (b) the sequential changes in spadix (red) and air (blue) temperatures during floral development from the female to the male stage. spadices at the female stage can maintain internal temperature at approximately 22-26°c, whereas spadices at the male stage cannot produce heat. spadices at the bisexual stage between the female and male stages show unstable thermogenesis. photographs of a femaleand a male-stage spadix, are shown in the upper right and lower right panels, respectively. (b) was partially extracted from figure 1 in our previous paper (ito-inaba et al., 2009 a). 75 data on mitochondrial content are described in our previous paper (ito-inaba et al., 2009 a). we next compared the mitochondrial protein content recovered from thermogenic and non-thermogenic stages or tissues (ito-inaba et al.. 2009 a, b). as shown in figure 2b, the mitochondrial protein content of female-stage spadices (0.54 mg g-1) was two-fold higher than that of males (0.29 mg g-1), a value consistent with our electron microscopic data. in addition, mitochondrial protein content of non-thermogenic skunk cabbage, lysichiton camtschatcensis (0.011 mg g-1), was much lower than that of s. renifolius. since l. camtschatcensis has no ability to produce heat but has a close relationship with s. renifolius in morphology and phylogeny, this result suggests that a lower mitochondrial content may correlate with the lack of thermogenesis in l. camtschatcensis. taken together, these results reveal that there is a positive correlation between heat production and the abundance of mitochondria in plant tissues and cells. these are the first quantitative data indicating differences in mitochondrial content between thermogenic and non-thermogenic stages or tissues. therefore, plants might produce the massive heat from their tissues by increasing their mitochondrial density in a manner similar to mammalian bat. 3. the quantitative gene expression profile in femaleand male-stage spadices of s. renifolius to understand the molecular basis of floral thermogenesis, we examined the gene expression profiles of female and male-stage spadices of s. renifolius. since the complete genome sequence of s. renifolius is not available, we took advantage of the super serial analysis of gene expression (supersage) methodology as this method can provide quantitative and comprehensive gene expression profiles (ito-inaba et al., 2012 a). in our study, 26 bp tags (supersage tags) expressed from femaleand male-stage spadices were prepared and sequenced using a 454 life sciences genome sequencer 20 system. since the length of 26 bp tags is sufficient to identify the origin of a tag using cdna databases (matsumura et al., 2003, 2011), each 26 bp tag was annotated based on our cdna database of the female-stage spadices using the bioedit program. the gene expression profiles obtained were subjected to cluster analysis to identify candidate sets of co-regulated genes directly or indirectly associated with the process of femaleand male-stage spadices, and were qualified as a group of femaleor male-stage specific genes. to further assess the function of each gene, agi codes of arabidopsis orthologs corresponding to the identified genes were obtained from the database of the arabidopsis information resource (http://www.arabidopsis.org/index.jsp), and the identified genes were classified based on gene ontology (go) terms using the agi codes. this analysis allowed us to predict the localization and function of the orthologs in s. renifolius. each gene was weighted according to the number of corresponding supersage tags that reflected the expression level of each gene. based on these methods, transcripts were assigned to specific cellular components or biological processes (itoinaba et al., 2012 a) and the major transcriptional changes are shown in figure 3. it was of particular interest that genes encoding mitochondrial proteins were actively transcribed in female spadices but not in male spadices (fig. 3a). in addition, the activity of genes related to electron transport or energy pathways decreased significantly during the transition from the female to the male stage (fig. 3b). these results suggest that mitochondrial function fig. 2 abundant mitochondria are present in the spadix of thermogenic skunk cabbage. (a) female spadix cells (petal tissues) contain many mitochondria. this photograph was adapted from fig. 5a in our previous paper (ito-inaba et al., 2009a). in this study, large numbers of mitochondria were also observed in pistils and in several tissues in stamens. (b) quantitative comparison of mitochondrial protein amount from thermogenic and non-thermogenic stages or tissues. female-stage spadices (♀) in s. renifolius (sr) contain 2-fold and 50-fold higher content of mitochondria than male spadices (♂) and or spadices from l. camtschatcensis (lc), respectively. data was extracted from table 2 and table 1 in our previous papers (ito-inaba et al., 2009 a, b, respectively). fig. 3 examples of transcriptional changes of cellular components and biological processes of femaleand male-stage spadices of s. renifolius. genes encoding proteins localized in mitochondria (a) or that play roles in electron transport or the energy pathway (b) are highly expressed at the female stage, but not at the male stage of floral development. genes encoding stress-responsive proteins (c) were highly expressed at the male stage, but not at the female stage. (a) was partially extracted from figure 3a (the cellular component data) in our previous paper (ito-inaba et al., 2012 a). (b) and (c) were also partially extracted from figure 3b (the biological process data) in the same paper. 76 and/or cellular respiration play a key role in floral thermogenesis. this finding is consistent with our electron microscopic observation that the thermogenic female-stage spadix accumulates a large number of mitochondria and has an increased oxygen consumption rate. furthermore, genes classified as stress responsive were highly expressed in male spadices (fig. 3c). of these genes, a gene encoding a cysteine protease in s. renifolius, designated as srcpa, was the most abundant transcript in the spadices, and levels increased significantly during the female-male transition (ito-inaba et al., 2012 b). this class of cysteine protease is involved in programmed cell death (beyene et al., 2006) and stress responses (stevens et al., 1996) in other organisms. since our previous studies suggested that a parallel relationship exists between the increase in cp transcripts and vacuolar development in each of the various spadix tissues during the female-male transition, the high level of srcpa expression may be correlated with vacuolar development in male-stage spadices. in addition, several stress-responsive genes and genes encoding degradative enzymes or ubiquitin-proteasome system components had increased expression levels at the post-thermogenic stage. therefore, we hypothesize that cysteine protease and other degradative enzymes that leak from the vacuole may degrade mitochondria, thereby terminating thermogenesis at the male stage. 4. conclusions and perspectives our previous electron microscopic study revealed that intracellular structures within the individual tissues change significantly during the transition from the female to the male-stage spadix in s. renifolius. the mitochondrial content is reduced, especially in the petals and pistils, whereas the vacuolar volume increases during the femalemale transition. consistent with this cellular change, gene expression profiles analyzed using supersage methods indicated that the genes involved in cellular respiration and mitochondrial function are up-regulated in female-stage spadices, whereas the genes involved in stress responses and protein degradation are up-regulated in male-stage spadices. these observations suggest that the maintenance and termination of floral thermogenesis in the femaleand the male-stage spadices, respectively, may be explained as shown in figure 4. at the female stage, the high expression levels of genes related to cellular respiration and mitochondrial function induce significant oxygen consumption and mitochondrial biogenesis, and activate cellular metabolism leading to substantial heat production. in contrast, at the male stage, the high expression levels of genes related to protein degradation and vacuolar metabolism induce senescence, programmed cell death, and vacuolar development, leading to the termination of heat production. after thermogenesis, the expression of several stress response genes, such as cold-inducible genes, increase because the spadix cannot produce any heat. with exposure to the cold air, the spadix cells proceed to senescence. more than 200 years ago, pioneering studies on floral thermogenesis were undertaken in the european arum (araceae) by lamarck (1778). since then, heat production by the reproductive organs of several plants has been investigated. we anticipate that the numbers of plants known to produce heat will increase in the future as the subtle temperature differences between the air and plant bodies can be measured by technical advances in temperature probes or thermography. to study the molecular mechanisms underlying floral thermogenesis, two energy dissipating systems, an alternative oxidase (aox) and uncoupling protein (ucp), have been the principal subjects of investigation (vanlerberghe and mcintosh, 1997; vercesi et al., 2006; zhu et al., 2011). because of the correlation between heat production and aox concentration, as well as activity in several thermogenic plants (grant et al., 2008; ito-inaba et al., 2009 b; miller et al., 2011), aox rather than ucp has been assumed to control plant thermogenesis. recently, the crystal structure of a trypanosomal aox was reported (shiba et al., 2013). since the post-translational regulation of aox has been hypothesized to regulate the thermogenic capacity of this protein (grant et al., 2009), revealing the structural features of aox may open the door to elucidating the mechanisms underlying the post-translational regulation of aox. furthermore, we anticipate that recent advances in next generation sequence (ngs) technology will uncover additional genes, besides aox and ucp, that are involved in floral thermogenesis. in s. renifolius, the gene expression profile has already been studied using ngs technology combined with the supersage method and could provide valuable information to define the identity of femaleand male-stage spadices at the molecular level (ito-inaba et al., 2012 a). as far as we know, this was the first study in which the molecular mechanism underlying floral thermogenesis was analyzed using ngs technology. quite recently, the genome of the sacred lotus (nelumbo nucifera), a well-known thermogenic plant, was sequenced (ming et al., 2013). we also expect that this advance will accelerate study of the molecular mechanism underlying heat production in the reproductive organ development of fig. 4 a proposed model of the possible processes in femaleand male-stage spadices to maintain and terminate thermogenesis, respectively. 77 sacred lotus. s. renifolius is a monocot thermogenic plant, whereas n. nucifera is a eudicot thermogenic plant. thus, comparative studies of these plants will reveal general and diverse aspects of floral thermogenesis. acknowledgements this work was supported by the naito foundation, grants-in-aid for research activity start-up (no. 24880027), the program to 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53: 7-13. 145 1. introduction consumption patterns have considerably changed over recent years. consumers are progressively more and more aware of the issues related to food and impacts on the economy and the environment. they understand the close relationship between food quality , the environment and the wellbeing of society in general, so nowadays they are turning gradually toward those food products which are an expression of this interaction. consumers perceive, evaluate, and choose each product on the basis of the range of food/environmental/social characteristics that it demonstrates (lancaster, 1971), sometimes not all explicitly recognizable or conveyed by the producer. however, the process of consumption is not homogeneous: not all consumers have the same values and want the same features, however, despite agreeing with certain issues (for e.g. sustainability, social justice goals, etc.), they do not necessarily change their consumption behavior (weinstein, 1988). a lack of information (i.e. information asymmetry) between producers and consumers, might prohibit consumers from making informed purchase decisions and not allow them to have insights into the implications of their purchase decisions on the food supply chain (aprile et al., 2012). indeed, food safety issues often arise from problems of asymmetric information between consumers and food producers with regard to product-specific attributes or characteristics (ortega et al., 2011). food labels become the only tool for consumers to acquire additional information about products for their purchase decisions; in fact, studies have shown that there is a relationship between the objective characteristics of the label and the reactions of consumers (cavicchi, 2008; bialkova and van trijp, 2010; di pasquale, 2011; grunert, 2011; veneziani et al., 2012; vianelli and marzano, 2012; siriex et al., 2013). each label conveys a set of characteristics (such as text, color, shape, etc.) that provides information about the product; however, the space available is always limited by the size of the package as well as by the regulations set out by law. in today’s modern, globalized market, these limitations can be partially overcome by using mobile marketing, such as the qr code (quick response code), that combines the possibility to provide information with that of promoting and enhancing the value of the product and/ importance of food labeling as a means of information and traceability according to consumers s. bacarella, l. altamore (*), v. valdesi, s. chironi, m. ingrassia dipartimento di scienze agrarie e forestali, università degli studi di palermo, viale delle scienze, 90128 palermo, italy. key words: consumer behavior, consumer profiles, food quality, multidimensional scaling, qr code. abstract: consumption patterns have considerably changed over recent years. we are witnessing more and more frequently to a lack of information (i.e. information asymmetry) between food producers and consumers, this generate in the consumer the latter need to access information related to processes of production of food and food distribution. decisionmaking, in absence of further information, leads consumers to pay attention to food labeling. food labels become the only tool for consumers to acquire additional information about products in order to make the purchase decision. in today’s modern and globalized market, labels limitations can be partially overcome by using mobile marketing tools, such as the qr code (quick response code). therefore the objective of this study is: (1) categorize profiles of consumers according to the importance given to various information patterns shown on food labeling; (2) discover consumer behaviors when making a purchasing decision based on food information they require in the label; (3) discover consumer profiles with regards to food quality and the use qr code to acquire further information about food products. two italian regional capitals were chosen, as representative of northern and southern italy, basing on the geographical division of the country made by the istat (istituto nazionale di statistica). the interviews were carried out by telephone, using a questionnaire. data collected were processed by multidimensional scaling we discovered eight profiles of consumers with different purchasing behaviors. some consumer profiles, with lifestyles typical of contemporary life, use the qr code to obtain additional information about food products. the innovative purchasing behavior identifies a consumer who is particularly interested in food quality and safety. agri-food businesses that focus on quality productions could target the innovative profile and communicate further information trough the use of modern communication technologies. adv. hort. sci., 2015 29(2-3): 145-151 (*) corresponding author: luca.altamore@unipa.it received for publication 26 september 2014 accepted for publication 30 july 2015 146 adv. hort. sci., 2015 29(2-3): 145-151 or of the brand, thanks to new communication systems. qr mobile marketing, used for traceability of food products, transforms the physical identifiers (adhesives and labels on products, packaging, price tags, etc.) into something new and interactive, which can provide much information about the product’s production process and general information about the company (shiang-yen et al., 2010; kwak, 2013). this system, applied to labels of food products, whether fresh or processed, can become a tool for the consumer to acquire more information about traceability, provenance, producer’s background, production, breeding and cultivation techniques used, etc. the purpose of this study was to determine what information, of that possibly provided on the labels of food products, consumers are particularly interested in knowing and what information consumers are looking for as added value as a guarantee of food quality and safety, e.g. traceability, absence of gmo, organic production, etc. more particularly, our aim was to (1) categorize profiles of consumers according to the importance given to various information patterns shown on food labeling; (2) discover consumer behaviors when making a purchasing decision based on food information they require in the label; (3) discover consumer profiles with regards to food quality and the use of qr code to acquire further information about food products. each consumer profile corresponds to a different, homogeneous market segment for similar purchasing behavior and sociodemographic characteristics. 2. materials and methods sampling design for this research, two italian regional capitals were chosen, as representative of northern and southern italy, based on the geographical division of the country made by the istat (istituto nazionale di statistica): milan for the north (n 1 ) and palermo for the south (n 2 ). a sample of consumers equal to n = 267 (with p = 95% and ε = 7%) was extracted using the stratified-proportional sampling schema (with random extraction from each stratum) from the population of residents in the two cities. population size was equal to n 1 + n 2 = n = 1,917,088 (istat, 2012), where n 1 = 1,262,101 and n 2 = 654,987 (proportional subsample sizes are n 1 = 176 and n 2 = 91). the sample units (i.e. respondents) were extracted randomly from the telephone directories of the two cities. respondents had to be aged between 20 and 80 years, and those who did not fit with this requirement were asked not to continue the interview (table 1). education and income levels of respondents had to be proportionally equal among consumers in the sample. measurements the interviews were carried out by telephone, using a proper questionnaire divided into two sections. the first section contained questions on consumer socio-demographic characteristics; the second listed 20 food attributes usually displayed in food labels (i.e. information about foods) and respondents were asked to score, using a scale of 1 (not at all) to 10 (a lot), the attributes in relation to their personal preferences (i.e. subjective importance) based on what they wanted to find in food labels. to evaluate preferences, consumers had to subsequently order the 20 scored attributes with the aim of ranking of them. the attributes were selected based on caswell’s classification of food attributes (caswell, 2000) (table 2). these attributes were categorized basing on consumer purchasing behavior variables (i.e. purchasing style, perceived quality); categorization is reported in table 3. data analysis multidimensional scaling (mds) is a set of data analysis techniques that display the structure of distance-like data as a geometrical picture (young, f., 1985). mds has its origins in psychometrics but now has become a general data analysis technique used in a wide variety of fields (schiffman et al., 1981) including marketing studies. mds portrays the structure of a set of objects from data that approximate the distance between pairs of the objects. the data, which are called similarities, dissimilarities, distances, or proximities reflect the amount of similarity (or dissimilarity) between pairs of objects or events (i.e. in this study the agreement in judgments between pairs of consumers). the graphical output is a representation which consists of a geometric configuration of points on a map (cox and cox, 2005). each point in the configuration corresponds to one of the objects. two points near each other indicate that there are similarities in the attributes for which the similarity (or dissimilarity) has been calculated (rebollar et al., 2012). this configuration reflects the “hidden structure” in the data, and often makes data much easier to comprehend (kruskal and wish, 1984). in addition, the map of stimuli can be interpreted based on just a few dimensions corresponding to the attribute measured (cox and cox, 2005; rebollar et al., 2012). the space is usually a two (or three) dimensional euclidean space (but may be not euclidean). classic mds may be table 1 sample stratification and sub-samples by range of age and cities (based on population strata size) range of age cities %milan (p 1 = 1.262.101; n 1 = 176) palermo (p 2 = 654.987; n 2 = 91) 20-30 15 8 8.79 30-40 33 17 18.68 40-50 45 24 26.37 50-60 41 21 23.08 60-70 30 15 16.48 70-80 12 6 6.59 total 176 91 100 147 bacarella et al., importance of food labeling as a means of information and traceability table 2 attributes selected caswell’s classification our classification intrinsic quality attributes   1. food safety attributes   • foodborne • pathogens   • heavy metals and toxins   • pesticide or drug residues   • soil and water contaminants   • food additives, preservatives   • physical hazards   • spoilage and botulism   • irradiation and fumigation other   2. nutrition attributes   • calories • calories/kilojoules content • fat and cholesterol content • nutritional information • sodium and minerals • ingredients • carbohydrates and fiber content • probiotic product • protein • nutritional properties • vitamins • other 3. sensory/organoleptic attributes • taste and tenderness   • color   • appearance/blemishes   • freshness   • softness   • smell/aroma   • other   4. value/function attributes   • compositional integrity • shelf life and best before dates • size   • style   • preparation/convenience   • package materials   • keepability   • other   5. process attributes   • animal welfare • organic products • authenticity of process/place of origin • production zone • traceability • traceability • biotechnology/biochemistry • genetically modified organism (gmo) • organic/environmental impact • worker safety • other extrinsic quality indicators and cues 1. test/measurement indicators • quality management • organic certification • systems • quality certification (pod, pgi, doc, etc.) • certification • records • labeling • minimum quality standards • occupational licensing • other 2. cues   • price • price • brand name • brand • manufacturer name • territory of origin of the product • store name • green economy product • packaging • traditional product • advertising • easy to cook product • country of origin • information on promotions and discounts • distribution outlet • food preservation methods • warranty • reputation • past purchase experience • other information provided 148 adv. hort. sci., 2015 29(2-3): 145-151 metric or nonmetric. in non metric mds data can be at the ordinal level of measurement (ordinal data), moreover data may be complete or incomplete, symmetric or asymmetric, and it is possible to measure similarities or dissimilarities. for these reasons in this study nonmetric mds was the most appropriate. the nonmetric mds uses quantitative data models to describe qualitative data. nonmetric mds tries to find a configuration of points that minimizes the squared differences between the optimally scaled proximities and the distances between the points. in other words, coordinates have to be found that minimize the so-called stress, which. mds programs automatically do to obtain the mds solution. among the different versions of stress in literature, kruskal (1964) provided some guidelines for the interpretation of the stress value with respect to the goodness of fit of the solution. according to kruskal’s findings, stress decreases as the number of dimensions increases (thus a two-dimensional solution always has more stress than a three-dimensional one). there are two additional techniques commonly used for judging the adequacy of an mds solution: the screen plot and the shepard diagram. among the different mds techniques, the alternating least squares scaling (alscal) method (takane et al., 1977) was used in this study to analyze the data obtained. this model appeared the most suitable in this case because it can analyze data that are nominal, ordinal, complete, or with missing observations, conditional or unconditional, symmetric or asymmetric, replicated or un-replicated, continuous or discrete (takane et al., 1977). the alscal method uses the euclidean model as a basis to compute optimal distances between objects in a n-dimensional stimulus space (kruskal and wish, 1978). to determine the badness-of-fit between the hypothesized structure and the original data, alscal method minimizes the loss function called s-stress (ss), which is minimized using an alternating least squares algorithm (cox and cox, 2005). a value of zero indicates a perfect fit (kruskal and wish, 1978), but kruskal and wish (1984) consider the solution to be acceptable when the s-stress values are less than 0.1 (rebollar et al.,, 2012). r-square is a squared correlation index that indicates the proportion of variance of the optimally scaled data that can be accounted for by the mds procedure; according to literature, values of 0.60 or better are considered acceptable. while r-square is a measure of goodness-of-fit, stress measures badness-of-fit, or the proportion of variance of the optimally scaled data that is not accounted for by the mds model (cil, 2012). stress values of less than 10% are considered acceptable. the vector model (davison, 1983) was used to interpret the dimensions of preference in function of observable attributes. this model helps to interpret the dimension of the space of similarities using the attributes which make up the similarities between the stimuli. following the explanation of the vector model made by rebollar et al. (2012), the attribute-vector is displayed as a line in the space representing consumer preferences based on personal purchasing behavior which the projection of each stimulus corresponds to with the degree of importance assigned by respondents. if two stimuli (i.e. points or objects) are strongly related to each other, then the stimuli projections will coincide very closely and the correlation between them will be quite high. when two objects lie in the same direction, this also indicates a high correlation between the two. when the points that represent the vector are close to a dimension and far from the centre, these are important for explaining that dimension. if a point is midway between two dimensions, this indicates that this attribute explains both dimensions. if an object is close to the center of the stimulus space, this means that it is not important in the explanation of the dimension in this space (i.e. it does not depend on either of the two dimensions). this configuration reflects the “hidden structure” in the data, and often makes data much easier to comprehend (kruskal and wish, 1984). this model allowed consumer’s preferences (i.e. information contents required in food labels) to be ordered based on the respondents’ attribute evaluations. it also made it possible to determine which attributes present a high correlation in the evaluation of the stimuli. furthermore, it was possible to group the objects on the basis of the distances on the map, highlighting characteristics that were statistically correlated, directly or inversely (kruskal, 1964; young et al., 1978). in this study these groups represent different consumer’s profiles correlated by the information required in food labels (preferences). table 3 variable description and variable code n. variable description variable code 1 shelf-life and best before dates shelf_life_bestbefore_dates 2 ingredients ingredients 3 nutritional information nutritional_information 4 production zone production_zone 5 genetically modified organism (gmo) genetically_modified_organism_gmo 6 organic products organic_product 7 green economy product green_economy_product 8 probiotic product probiotic_products 9 food preservation methods food_preservation_methods 10 kalories/kilojoules content kalories_kilojoules_content 11 price price 12 brand brand 13 nutritional properties nutritional_properties 14 territory of origin of the product product_territory_of_origin 15 traceability traceability 16 organic certification organic_certification 17 quality certification (pod, pgi, doc, ecc.) quality_certification 18 traditional product traditional_product 19 easy to cook product easy_to_cook 20 information on promotions and discounts promotions_discounts 149 bacarella et al., importance of food labeling as a means of information and traceability the data was processed using the statistical software spss (version 21). 3. results the preference classification of food attributes based on respondents’ ranking is shown in figure 1. the s-stress of the configuration in the space of the first two dimensions was 0.09102, indicating a good fit in those dimensions. the r-square value was 96%, confirming the good fit in the two dimensions. the figure shows how food attributes required by consumers in food labels were classified according to the dimensions. dimension 1 differentiates the attributes regarding quality information required by respondents; dimension 2 is particularly important in this space because it differentiates attributes according to the respondents’ purchasing styles. if we look at correlation values in the figure it is possible to graphically analyze similarities (and dissimilarities) of different points and groups of them representing preferences. different groups of preferences represent similarities in respondents’ rankings, and therefore these segments of preferences, similar (homogeneous) inside, are the discovered profiles of consumers. these profiles are characterized by quality information required (namely “quality”) and purchasing styles (namely “behavior”). dimension 1 shows two major groups of variables, one in the left part of figure 1, with respect to the vertical axis (located at zero point), and another in the right part of the axis. also dimension 2 has two major groups, one in the lower region and the other in the upper region with respect to the horizontal axis passing through the zero point. the results highlight four main aggregations of points, derived from the combination of variations of the two dimensions. these aggregations have been named as: 1. extra; 2. regular; 3. classic; 4. innovative (see fig. 1 and table 4). furthermore, it’s possible to note four other sub-groups of variables identified in figure 1 and table 4: 5. traditional; 6. basic; 7. niche; 8. prime. two of these sub-groups, namely niche and prime, seem particularly interesting because variables are independent from dimension 2, and correlation is almost equal to zero. 4. discussion and conclusions analysis of the results revealed eight profiles of consumers homogeneous for similar preferences of food product attributes required in food labels. profiles with characteristics and purchasing behaviors are listed in table 4. according to this classification, the first profile, “1. extra,” includes consumers interested in nutritional/health aspects (e.g. traceability, organic cultivation, gmo-free, etc.) and other attributes like area of production and origin of the product. profile “2. regular” represents consumers that base their purchases on regular and general information about food; information about nutrition and product origin are also important. the “3. classic” profile refers to a consumer who purchases according to brand, price, ease of food preparation, food preservation methods, and health features such as calorie content. the “4. innovative” profile is interested in personal health and issues like environmental protection and animal welfare; this consumer looks for food quality, food certifications, food safety, and traceability. profile “5. traditional” requires information on energy content of food, food preservation methods, and promotional information. profile “6. basic” is a consumer who chooses exclusively on the basis of brand and product price. profile “7. niche” chooses mainly traditional products that are well-known and familiar; probiotics and healthy/functional foods are of interest. the profile “8. prime” is a consumer who is interested in primary elements of food products, like best-before date and ingredients, but these primary elements also include other attributes (variables) of food which are closely related to the “made-in” concept, i.e. “territory of origin” and “traditional product” which may provide benefits to health. the classic purchasing behavior (profile 3) identifies a consumer who is less interested in innovation. in contrast, the innovative purchasing behavior (profile 4) identifies a consumer who is particularly interested in food quality and safety. it is interesting to see that innovative behavior represents consumers who have developed a new concept of product quality: not only intrinsic attributes but also ethics and health attributes, and information regarding quality certification and traceability. agri-food businesses that focus on quality productions could therefore target the innovative profile and communicate further information through the use of modern communication technologies that facilitate the transfer of information via qr code. this system can be used by producers, distributors, and retailers and it represents a new way to interact with the consumer. thanks to a website link or to fig. 1 preference classification of food attributes based on respondents’ ranking. 150 adv. hort. sci., 2015 29(2-3): 145-151 videos with information about the food production process, food production area, traceability or alternative ways to use and store the product, this system allows producers to survey consumers’ interest toward their products. references aprile m.c., caputo v., nayga jr r.m., 2012 consumers’ valuation of food quality labels: the case of the european geographic indication and organic farming labels. international journal of consumer studies, 36: 158-165. bialkova s., van trijp h., 2010 what determines consumer attention to nutrition labels? food quality and preference, 21: 1042-1051. caswell j.a., 2000 analysing quality and quality assurance (including labeling) for gmos. agbio forum, 3(4): 225-230. cavicchi a., 2008 qualità alimentare e percezione del consumatore. agriregionieuropa, 15. cil i., 2012, consumption universes based supermarket layout through association rule mining and multidimensional table 4 characteristics of discovered profiles profile variables quality information required (quality) purchase styles (behavior) behavior 1. extra probiotic product traditional product organic product organic certification gmo quality certification traceability calories/kilojoules content food preservation methods information on promotion and discount extra from classic to innovative purchase mainly certified food products 2. regular price brand ingredients nutritional information nutritional properties territory of origin of the product production zone regular from classic to innovative purchase basing on regular and general information 3. classic price brand calories/kilojoules content food preservation methods information on promotion and discount easy to cook probiotic product from regular to extra classic little time available for preparing meals but health-conscious 4. innovative nutritional information nutritional properties organic certification organic product gmo quality certification traceability green economy product production zone from regular to extra innovative living well in harmony with the environment 5. traditional calories/kilojoules content food preservation 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subtropical areas and as cut flowers they are among the most important for flower bouquets, baskets and wreaths (kendirli and cakmak, 2007). the florets have a very sweet fragrance and are widely cultivated in india and france as a source of essential oils for the perfume industry. polianthes is also a common garden plant in the spring and it flowers during the summer and early autumn (de hertogh and le nard, 1993). two major cultivars, white-colored ‘single’ and ‘double’, are for commercial production (shen et al., 2003). in tuberose, fewer than 50% of the buds normally open after harvest and florets and buds usually drop off after a few days in vase. postharvest performance is worse in tuberose which has been shipped to distant markets (waithaka et al., 2001). keeping quality of spikes is only three days for florets and vase life of flowers is only a few days. since it has delicate flowers and sellers and customers are keen to extend its vase life, it is necessary to improve its postharvest life (anjum et al., 2001). senescence in cut flowers is affected by three main parameters: the water balance, the supply of carbohydrates, and susceptibil ity to ethylene (cortes et al . , 2011). treatment with gibberellic acid has also been shown to enhance postharvest life and quality of gerbera cut flowers. using ga3 at different concentrations improved membrane stability index, leading to better flower vase life of gerbera cut flowers (emongor, 2004). similar effects on membrane stability index have been reported in gladiolus with ba and ga3 (singh et al., 2008). ga3 treatment of sandersonia flowers delays the senescence-associated increase in protease activity, which by implication delays the breakdown of senescence-associated proteins (eason, 2002). calcium (ca) is an important element which is found in 3% of the earth’s crust. it is essential to living organisms and to plant growth and development. some of these benefits include stronger cell walls, increased postharvest life of flowering plants, and increased disease resistance (robichaux, 2005). calcium spray increased the life of rose petals by increasing the relative water content (rwc), maintaining turgidity of leaf cells, avoiding cell wall deformation, and decreasing electrolyte leakage from cells of cut flowers by increasing cell adv. hort. sci., 2016 30(2): 69-74 doi: 10.13128/ahs-19131 some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid s.n. mortazavi (*), f. bagheri, m. bahadoran collage of agriculture, department of horticulture, university of zanjan, zanjan, iran. key words: bulbous plant, cut flower, ethylene, longevity, postharvest. abstract: in the present study the effect of gibberellic acid (ga3) and calcium chloride (cacl2) sprays (0, 150, 300 and 450 ml l-1), applied 25 and 15 days before harvesting, on physiological and morphological characteristics of tuberose ‘pearl double’ was studied. cut flowers were harvested and transported to the laboratory where they were placed in distilled water. the experiment considered some parameters for evaluation, such as relative water content of leaves and petals, water intake, percentage of open florets, electrolyte leakage, ethylene production, chlorophyll and carotenoid content. results indicate that the best treatment was the combination of 150 ml l-1 cacl2 and 450 ml l-1 ga3 for most of parameters. (*) corresponding author: mortazavi46@gmail.com received for publication 20 january 2016 accepted for publication 1 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 69-74 70 wall integrity and stability (mortazavi et al., 2007). floret abscission and short vase life of tuberose are common problems, thus the aim of this study was to determine the effect of foliar application of gibberellic acid and cacl2 treatments on some morpho-physical parameters that affect the vase life of cut tuberose. 2. materials and methods this experiment was conducted in summer 2012 at the commercial tuberose (double cultivar) farm located in tarom county of zanjan province, iran; latitude 36° 57΄ north and longitude 48° 54΄ east. about 500 m2 of this farm is used for experimental treatments. the plants received as foliar spray cacl2 (from merck), with molar mass 147.01 g m-1, and gibberellic acid (from merck), with molar mass 346.37 g m-1, 15 and 25 days before harvest of flowers. cacl2 (0, 150, 300 and 450 ml l-1) (0, 1, 2 and 3 mm) and gibberellic acid (0, 150, 300 and 450 ml l-1) (0, 0.4, 0.8 and 1.2 mm) were used. before application, leaves of plants sprayed with distill water, one hour after distil water spired, cacl2 treatment (four liters) and two hours after that gibberellic acid treatments (four liters) were done until runoff. when most of the two or three lowest inflorescence florets opened, flowers were harvested and immediately transferred to the postharvest laboratory at the department of horticulture, faculty of agriculture, zanjan university. flowering stems then were cut to 65 cm, weighted (fresh weight) and placed in 400 ml distilled water. all experiments were performed in a postharvest room equipped with a controlled environment maintained at 22±1°c, 45±5% relative humidity and light intensity for 12 h/day by cool-white fluorescent lamps. the following parameters were measured: fresh weight of flowers and numbers of open florets of inflorescences before being placed in distilled water, rate of opening and abscission of florets every three days, water uptake every four days, relative water content (rwc) of leaves and petals, cell membrane injury (cmi) of petals, chlorophyll and carotenoid content of plant tissue, and ethylene production of flowers. vase life longevity was recorded when at least four open florets on the inflorescence were present. to measure the rwc, 1 g of petal tissue (fresh weight, f.w.) was immersed in distilled water for 24 h and weighed again (turgid weight, t.w.), then dried at 80°c inside an oven for 48 h (dry weight, d.w.). rwc was calculated using the following equation (turner, 1981): rwc= (f.w.-d.w.)/(t.w.-d.w.)×100 cell membrane injury was measured following the isacc and urban (1995) method; after seeing the first flowers of inflorescence, measurements were done. one g of petal tissue of top open bud flowers weighted of each plant and washed with distilled water, then were immerged in glass container containing 10 ml of distilled water and were placed inside a benmary (gemmy ind. corp., taiwan) at 30°c for 60 min and ec was measured (ec1), then were placed inside a autoclave at 120°c for 20 min, and ec was measured again (ec2), and cmi was calculated using the following equation: cmi =1-(ec1/ec2) ×100 ethylene production of flowers 3, 6, 12 and 24 h after placement in 500 ml distilled water at 23oc was measured by an ethylene biosynthesis bioconservation device model icna56. treated flowering inflorescence was in distilled water in the laboratory with the same conditions inside the container and packaging (volume 2.5 liters) were (almost all the flowers were the same size). flowers in the tank without meeting with any special hole for injection or sampling were used and thus ethylene concentrations were prepared by flowers. chlorophyll and carotenoid content were determined by spectrophotometeric method (arnon, 1949). the present study was carried out in a complete randomized design with factorial arrangements including 16 treatments and three replications. data were analyzed by mstatc software and means were compared using lsd test at 5% level. 3. results and discussion effect of cacl2 and gibberellic acid on relative water content of tuberose leaves (rwcl) and florets (rwcf) results indicated that interaction of 450 ml l-1 gibberellic acid and 150 ml l-1 cacl2 had a significant effect at 5% level on the relative water content of florets compared with the control (table 1). our results agree with those of cortes et al. (2011) on rosa x hybrid cv. grand gala, mortazavi et al. (2007) on rosa x hybrida cv. iliona and abdolmaleki et al. (2015) on cut rose cv. dolce vita. gibberellic acid reducing water loss via transpiration, increase water mortazavi et al. some characteristics of tuberose as affected by pre harvest application 71 uptake in plant tissues (emongor, 2004). calcium spraying increased the life of petals by increasing the rwc, maintaining leaf cell turgidity and avoiding cell wall deformation. gibberellic acid application together with cacl2 might increase the efficiency of ca use in plants. effect of cacl2 and gibberellic acid on water uptake of tuberose (wu) results indicated that 450 ml l-1 cac2 + 450 ml l-1 gibberellic acid gave maximum water uptake (table 1). these results agree with results of vijaya et al. (1999) on cut tuberose, cortes and et al. (2011) regarding rosa hybrid cv. grand gala, dansheng (2003) on cut rose, and sosa nan (2007) on sunflower. calcium interacts with polygalacturonic acid (pga) groups, forming a structure known as an “egg box”, which causes the contraction of pectins in the pit borders, increasing the diameter and, consequently, water flow (cortes et al. , 2011). the increased reducing sugars in flower heads and stems of gerbera cut flowers may increase the osmotic potential of flower head and stem, thus improving their ability to absorb water and maintain their turgidity (emongor, 2004). water uptake improved in tuberose by foliar application of gibberellic acid and cacl2. effect of cacl2 and gibberellic acid on opening florets of tuberose (op) interaction of 150 ml l-1 cacl2 and 450 ml l-1 gibberellic acid yielded the maximum amount of opening florets (table 1). this result concurred with halevy et al. (2001) who reported that cacl2 treatment promoted bud opening and delayed senescence in rose cut flowers. the treated flowers stayed turgid and continued their initial postharvest growth for longer periods. treatment with ga3 is useful for improving the vase life of cut n. tazetta var. chinensis flowers (ichimura and goto, 2000). spraying with 200 ml l-1 ga3 increased plant height, early flowering, spike length and number, rachis length, flower weight and length, and total flower yield in a study carried out by bharathi and kumar (2009), which was the same as our results. gibberellins increase hydrolysis of starch, fructans and sucrose into glucose and fructose which are utilized by the flowers for disc floret opening (emongor, 2004). effect of cacl2 and gibberellic acid on floret abscission of tuberose (af) the lowest percentage of floret abscission (9.83%) was found at 300 ml l-1 gibberellic acid while the highest (23.45%) was found at 150 ml l-1 cacl2 (table 1). our results were similar to those of uthairatanakij (2005) who reported cacl2 significantly reduced the postharvest dropping of orchid buds flower compared to control. calcium treatment probably increases the strength of cell walls. the abscission of leaves, flowers, and fruits is presumed to be brought about through the weakening of the cell walls in the abscission zone. this weakening may have two components: a solubilizing of the cell wall cementing subtable 1 means of interaction of cac2 and gibberellic acid on relative water content (rwc) of leaves and florets (%), water uptake (ml), opening and abscission of florets, number of florets, fresh weight of flower (g), vase life (day), chlorophyll content a, b and total chlorophyll content (mg), and carotenoid content (µg) of cut tuberose (polianthes tuberosa l.) after first flower opening of inflorescence cacl 2 (ml l-1) gibberellic acid (ml l-1) rwc of least (%) rwc of floret (%) water uptake (ml) opening florets (%) abscission of florets (%) number of florets fresh weight of flower (g) vase life longevity (day) chlorophyll content b (mg) total chlorophyll content (mg) carotenoid content (µg) 0 0 82.48 g* 76.12 de 111.7 bc 20.77 g 12.24 fgh 35.33 ab 78.46 ef 8.50 h 0.04600 bcdef 0.03267 efg 0.01300 g 150 92.67 b 70.54 fg 105.6 bcdef 29.43 def 11.14 fgh 32.67 bcd 78.28 ef 9.30 g 0.03767 cdef 0.04867 cde 0.02333 cd 300 87.51 def 76.66 de 98.89 f 30.15 def 09.83 h 36.33 a 88.27 ab 9.77 def 0.05400 bcdef 0.03567 defg 0.01300 g 450 94.91 a 91.58 b 112.2 b 37.66 ab 10.70 gh 32.67 bcd 82.46 cde 11.37 b 0.07333 bc 0.01037 a 0.01900 de 150 0 79.39 h 85.82 c 110.6 bcd 31.02 def 23.45 a 33.00 bcd 78.64 def 10.80 c 0.03133 def 0.01733 g 0.01000 h 150 96.66 a 70.73 fg 103.9 cdef 36.27 abc 18.75 bc 34.00 abc 79.74 def 9.43 fg 0.02100 f 0.02367 fg 0.01267 g 300 82.27 g 73.75 ef 110.6 bcd 34.50 bcd 16.70 cde 32.00 cd 88.75 a 9.57 efg 0.04600 bcdef 0.03500 defg 0.01833 e 450 73.99 i 96.10 a 113.3 b 40.74 a 13.72 efg 35.33 ab 82.01 cde 11.83 a 0.16260 a 0.05933 c 0.02233 ab 300 0 86.55 ef 79.45 d 103.9 cdef 30.74 def 20.92 ab 35.00 ab 80.92 def 10.13 d 0.06767 bcd 0.03333 efg 0.01067 h 150 85.58 f 77.06 de 100.0 ef 27.94 ef 20.22 b 34.67 ab 83.57 bcd 9.90 de 0.03300 def 0.04700 cdef 0.01967 de 300 89.02 cd 73.29 ef 110.0 bcd 29.82 def 14.23 def 36.00 a 77.50 ef 9.53 efg 0.04233 bcdef 0.05700 cd 0.02133 bc 450 90.27 c 86.30 c 113.3 b 32.40 cde 13.75 efg 31.00 d 77.77 ef 11.00 bc 0.08000 b 0.08100 b 0.02367 a 450 0 86.48 ef 74.44 ef 103.3 def 29.83 def 17.16 cd 31.00 d 79.24 def 9.47 fg 0.06700 bcde 0.01700 g 0.01333 g 150 88.33 cde 68.17 g 106.1 bcdef 26.56 f 16.61 cde 35.00 ab 75.88 f 8.70 h 0.02933 ef 0.02533 efg 0.01300 g 300 87.68 def 71.28 fg 107.8 bcde 30.32 def 12.33 fgh 36.33 a 86.15 abc 9.30 g 0.04333 bcdef 0.03167 efg 0.01667 f 450 89.49 cd 90.72 b 123.3 a 40.70 a 11.54 fgh 33.67 abc 86.35 abc 11.30 b 0.04833 bcdef 0.03433 defg 0.01633 f means in the same column followed by the same letter are not significantly different using lsd test level 5%. adv. hort. sci., 2016 30(2): 69-74 72 stances, and a hydrolysis of the structural components of the wall. a major part of the cementing properties of walls is presumed to be through the binding of pectic substances by double salt formation with ca ions (poovaiah and leopold, 1973). gibberellic acid delays flower abscission by decreasing the amount of dry matter (khan and chaudhry, 2006). effect of cacl2 and gibberellic acid on number of tuberose florets (nf) our results indicate that the interaction of 300 ml l-1 gibberellic acid and 450 ml l-1 cacl2 yielded the maximum number of florets, while the interaction of 300 ml l-1 cacl2 and 450 ml l-1 gibberllic acid gave the fewest (table 1). our results were similar to those found by parmar et al . (2009) on spider l i ly, mukhopadhyay and bankar (1983) on tuberose, and singh et al. (1991) on african marigold (tagetes erecta l.), who reported an increase in number of florets because of role of gibberellic acid on cell elongation and division. effect of cacl2 and gibberellic acid on fresh weight of tuberose florets (fw) our results show that the interaction of 150 ml l-1 cacl2 and 300 ml l-1 gibberellic acid led to the maximum fresh weight of florets (table 1), findings which are in agreement with those of cortes et al. (2011) and dansheng (2003) regarding rose, sosa nan (2007) working on sunflower, and vijaya et al. (1999) tuberose. the effect of gibberellic acid on the fresh weight of florets may be a result of its role on increasing cell division (arun et al., 2000). effect of cacl2 and gibberellic acid on vase life of tuberose (vl) application of 150 ml l-1 cacl2 and 450 ml l-1 gibberellic acid had significant effect on vase life parameter of tuberose (table 1). cortes et al. (2011) found that using cacl2 in the vase water of rose cv. grand gala gave maximum fresh weight. loss of cell membrane integrity is characteristic of senescence in plants. calcium protects the membranes from lipid degradation probably through several mechanisms. calcium can stabilize the plasma lemma by binding to the negatively charged head groups of pl, which become less prone to degradation by lipolytic enzymes (cheour et al., 1992). analogous results were found by uthairatanakij et al. (2005) regarding spraying cacl2 on dendrobium orchid, and by robichaux (2005) regarding the effect of calcium chloride, sulfate or nitrate spray on the vase life of rose and poinsettia. gibberellic acid increases water absorption and relative water content, resulting in vase life longevity. our results also agree with the findings of su et al. (2001) on tuberose and emongor (2004) on gerbera flower. effect of cacl2 and gibberellic acid on cell membrane injury of tuberose (cmi) results reveal that the minimum cell membrane injury was at 300 and 450 ml l-1 gibberellic acid and with interaction of 450 ml l-1 cacl2 and 450 ml l-1 gibberellic acid (fig. 1). the enhancing effect of the application of ca can be explained on the basis of its role in cell membrane structure. it may be noted that ca stabilizes cell membranes by connecting various proteins and lipids at membrane surfaces, influences the ph of cells and prevents solute leakage from cytoplasm and increase shoot elongation (al-whaibi et al., 2010). if low ca makes the membrane more permeable, it should follow that elevated concentrations make the membrane less permeable (hepler, 2005). effect of cacl2 and gibberellic acid on ethylene production of tuberose the lowest values of ethylene production after 3 h were found in 450 ml l-1 gibberellic acid and in of 300 ml l-1 cacl2 + 450 ml l-1 gibberellic acid; these values were significant compared to control and most of the other treatments. ethylene production after 6 h of treatment with 300 ml l-1 gibberellic acid and after 12 h with 300 ml l-1 cacl2 combined with 150, 300 and 450 ml l-1 gibberellic acid, respecitvely, showed the lowest values. after 24 h of treatment, the lowest levels were found with 450 ml l-1 cacl2 + 150, 300 fig. 1 means of interaction of cacl 2 and gibberellic acid on cell membrane injury (cmi) of leaves of cut tuberose. mortazavi et al. some characteristics of tuberose as affected by pre harvest application 73 and 450 ml l-1 gibberellic acid. highest ethylene production was found at 3 and 12 hours with treatments of 150 ml l-1 cacl2 + 150 ml l-1 gibberellic acid, at 6 hours with treatment 150 ml l-1 cacl2, and 150 ml l-1 cacl2 + 150 ml l-1 gibberellic acid at 24 hours (fig. 2). pre-harvest treatment of cacl2 decreased ethylene production, which agrees with the results of uthairatanakij et al. (2005) on orchid and cortes et al. (2011) on rose. calcium decreased activity and effect of ethylene on cell walls and affected senescence with inhibition of cell membrane injury. the application of calcium spraying gave the result to improve the strength of plant cell wall and delayed the senescence processes by inhibition of ethylene synthesis. in addition, calcium ions also seem to affect ethylene action on cell membranes by inhibiting ion leakage and reducing the effect of ethylene on senescence (asfanani et al., 2008). gibberellic acid treatments decreased ethylene production compared with the control, findings that agree with the results of ichimura and goto (2000) on narcissus and lers et al. (1998) on parsley. inhibition of ethylene production by gibberellic acid is related to the ethylene production enzyme. gibberellic acid inhibited acc enzyme activity and resulted in inhibition of ethylene production (ben-arie and ferguson, 1991). effect of cacl2 and gibberellic acid on chlorophyll and carotenoid of tuberose results of this study indicate that the interaction effect of 300 ml l-1 cacl2 and 450 ml l-1 gibberellic acid on chlorophyll a content was significant. also 150 ml l-1 cacl2 and 450 ml l-1 gibberellic acid had a significant effect on chlorophyll b content. the lowest chlorophyll b content was found in treatment with 150 ml l-1 of gibberellic acid and cacl2. total chlorophyll content was highest with 450 ml l-1 gibberellic acid. the maximum carotenoid content was recorded at 300 ml l-1 cacl2 and 450 ml l-1 gibberellic acid, with the lowest level was found at 150 and 300 ml l-1 cacl2 (table 1). calcium treatment caused the leaves to grow greener in color and the stems to grow more (asfanani et al., 2008). according to the results of aharoni (1989), lers et al. (1998), ichimura and goto (2000), ferrante et al. (2002), and khan and chaudhry (2006), yellowing of leaves destruction and decrease of chlorophyll can be delayed by gibberellic acid treatments. our results reveal that using cacl2 and gibberellic acid together was better at increasing chlorophyll a, b, and total content than using each one alone. the same results were observed for the content of chlorophyll a, b, and total of faba bean (vicia faba l.) cv. taraby (al-whaibi et al., 2010). 4. conclusions pre-harvest treatments with cacl2 and gibberellic acid improved some morphological and physiological parameters as well as vase life of cut tuberose. combining cacl2 and gibberellic acid had significant effects on some parameters. floret abscission and low vase life of tuberose are common problems that can be improved by using cacl2 and gibberellic acid before harvest. acknowledgements the authors of this research with to thank the department of horticulture of zanjan university for its support of this experiment. references abdolmaleki m., khosh-khui m., eshghi s., ramezanian a., 2015 improvement in vase life of cut rose cv. “dolce vita” by preharvest foliar application of calcium chloride and salicylic acid. int. j. hort. sci. techn., 2: 55-66. aharoni n., 1989 interrelationship between ethylene and plant growth regulator in the senescence of lettuce leaf discs. plant growth regulation, 8: 307-317. al-whaibi m.h., siddiqui m.h., al-amri a., basalah m.o., 2010 performance of faba bean under calcium and gibberellic acid application. int. j. p. dev. bio., 4: 60-63. anjum m.a., naveed f., shakeel f., amin s., 2001 effect of some chemicals on keeping quality and vaselife of tuberose (polianthes tuberosa l.) cut flowfig. 2 means of interaction of cacl 2 and gibberellic acid on ethylene production after 1, 2, 3 and 4 hours (nl g-1 h-1) of cut tuberose. adv. hort. sci., 2016 30(2): 69-74 74 ers. j. of res. 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2001 calcium in regulation of postharvest life of flowers. acta horticulturae, 543: 345-351. hepler p.k., 2005 calcium: a central regulator of plant growth and developent. plant cell, 17(8): 2142-2155. ichimura k., goto r., 2000 effect of gibberellin a 3 on leaf yellowing and vase life of cut narcissus tazetta var. chinensis flowers. j. jap. soc. hort. sci., 69: 423-427. isacc s., urban l., 1995 effect of electrical conductivity and supply rate of the nutrient on stomatal conductance of rose plant leaves. acta horticulturae, 424: 131-134. kendirli b., cakmak b., 2007 economics of cut flower in greenhouse: case study from turkey. agri. j., 2: 499502. khan a.s., chaudhry n.y., 2006 ga 3 improves flower yield in some cucurbits treated with lead and mercury. afr. j. bio., 5: 149-153. lers a., jiang w., lomaniec e., aharoni n., 1998 gibberellic acid and co 2 additive effect in retarding postharvest senescence of parsley. j. f. sci., 63: 66-68. mortazavi n., naderi r., khlighi a., babalar m., allizadeh h., 2007 the effect of cytokin and calcium on cut flowers quality in rose (rosa hybrida l.) cv. iliona. j. f. agr. and environ., 5: 311-313. mukhopadhyay a., bankar g.j., 1983 regulation of growth and flowering in polianthes tuberosa l. with gibberellic acid and ethrel spray. sci. hort., 19: 149-152. parmar a.b., patel h.c., chavda j.c., parmar j.r., 2009 effect of plant growth regulators on growth and flowering of spider lily (hymenocallis speciosa l.). a. j. hort., 4: 170-172. poovaiah b.w., leopold a.c., 1973 inhibition of abscission by calcium. plant. physiol., 51: 848-851. robichaux m.b., 2005 the effect of calcium or silicon on potted miniature roses or poinsettias. m. sc. thesis. b.s., university of louisiana at lafayette, usa. shen t.m., huang k.l., shen r.s., du b.s., 2003 breeding of dwarf tuberose (polianthes tuberosa l.). acta horticulturae, 624: 73-76. singh a., kumar j., kumar p., 2008 effect of plant growth regulators and sucrose on post harvest physiology, membrane stability and vase life of cut spikes of gladiolus. j. plant growth regul., 55: 221-229. singh m.p., singh r.p., singh g.n., 1991 effect of ga 3 and ethrel on growth and flowering of african marigold (tagetes erecta l.). progr. hort., 24: 92-95. sosa nan s.j., 2007 effects of preand postharvest calcium supplementation on longevity of sunflower (helianthus annuus cv. superior sunset). m.sc. thesis, lousiana state university, usa. su w.r., huang k.l., chang p.s., chen w.s., 2001 improvement of postharvest vase life and flower bud opening in polianthes tuberose using gibberellic acid and sucrose. a. j. exp. agr., 41: 1227-1230. turner n.c., 1981 techniques and experiment approaches for the measurement of plant water status. plant and soil, 58: 339-366. uthairatanakij a., jansri s., jitareerat p., kanlayanarat s., 2005 effect of preharvest calciums spraying on gamma irradiate inflorescences of ‘walter oumae 4n’ dendrobium. international symposium “new frontier of irradiated food and non-food products” 2223 september, kmutt, bangkok, thailand. vijaya b., venkata v., rao p., reddy y.n., 1999 effect of minerals on post-harvest vase-life of cut tuberose (polianthes tuberosa l.) cv. double. indian journal of horticulture, 56(4): 257-261. waithaka k., reid m.s., dodge l.l., 2001 cold storage and flower keeping quality of cut tuberose (polianthes tuberosa l.). j. hort. sci. biot., 76: 271-275. 133 1. introduction asparagus (asparagus officinalis l.) is a highly perishable product with a very short shelf-life due to its high respiration rate, which leads to a rapid degradation of chemical compounds and toughening of the spears (kader, 1992). during storage, physiological and compositional changes include loss of sugar, vitamins and water, toughening, degradation of pigments and bract opening that reduce spears quality (villanueva et al., 2005). among these, changes in texture, colour and brightness are the main factors which affect asparagus acceptance. environmental humidity is also an important factor in determining asparagus freshness; weight loss of 3-6% makes the product unacceptable (albanese et al., 2007). rapid cooling after harvest and low storage temperature notably reduce postharvest changes in quality characteristics. modified atmosphere packaging (map), combined with low temperatures, has been used to extend the shelf-life of minimally processed vegetables by reducing respiration rate, retarding the maturation and senescence, reducing microbial proliferation and quality deterioration (king et al., 1986; zagory and kader, 1988; gontard et al., 1996; fonseca et al., 2002). these effects are related to the surrounding atmosphere of products: the depletion of oxygen and accumulation of carbon dioxide inside the packaging. all these factors are affected by metabolic activity, storage temperature and film gas diffusion characteristics. the purpose of the present study was to evaluate the effect of two film-packaging materials on maintaining the quality of fresh cut green asparagus stored at 2°c (recommended storage and transport temperature) or 10°c (to simulate final display) (king et al., 1993). 2. materials and methods plant material asparagus (asparagus officinalis l. cv. grande) spears were harvested from a commercial greenhouse located in alghero (sardinia). commercially mature spears (25 cm long) were randomly harvested with some standing stalk in april and were immediately transported to the laboratory and refrigerated at 2°c and 10°c. effect of film packaging and storage temperature on physical and chemical changes in fresh-cut green asparagus a. palma (*), m. schirra, s. d’aquino istituto di scienze delle produzioni, cnr, via de mille, 48, 07100 sassari, italy. key words: antioxidants, modified atmosphere, quality attributes, toughness. abstract: the effect of two packaging materials, film 1 (polyvinylchloride film, manually extensible, 12 µm thickness, o2 permeability of 22,000 cm3/m2/24 h/atm) and film 2 (polyethylene film, 13 µm thickness, heat shrinkable, o2 permeability of 8,500 cm3/m2/24 h/atm) on changes in oxygen, carbon dioxide and ethylene concentrations within film packaging, weight losses, chemical parameters and textural properties of fresh-cut green asparagus (asparagus officinalis l.) were evaluated during three weeks of storage at 2 or 10°c. during the first two days of storage, in-package carbon dioxide and ethylene concentration increased progressively, while oxygen level decreased. an overall decrease in ph, sucrose and fructose content was observed while an increase in titratable acidity was observed in non-packaged asparagus. a significant increase in total phenols and total soluble solids was recorded, while in film 2 at 10°c significant decreases were detected in total soluble solids. antioxidant activity did not change in asparagus packaged at 2°c while in unpackaged and in film 1 and 2 at 10°c there were significant decreases. ascorbic acid contents declined rapidly after storage in all samples. weight loss increased markedly in non-packaged asparagus; in asparagus packaged with film 1 at 10°c significant differences were detected with respect to the other packaged treatments. both packaging materials preserved rheological properties of spears whereas un-packaged asparagus lost crispness rapidly. the overall results showed that the best storage conditions to extend the shelf-life of fresh-cut green asparagus were achieved by combining packaging and storage at 2°c. adv. hort. sci., 2015 29(2-3): 133-140 (*) corresponding author: amedeo.palma@ispa.cnr.it received for publication 26 september 2014 accepted for publication 16 june 2015 134 adv. hort. sci., 2015 29(2-3): 133-140 preparation of samples and storage conditions the asparagus samples were selected according to diameter and cut at a length of about 20 cm. samples of about 300 g each were then un-packaged or packaged with omni film (a polyvinylchloride film manually extensible, 12 µm thickness, o 2 permeability of 22,000 cm3/m2/24 h/ atm and water vapour transmission rate of 515 g/m2/24 h/1 atm.) (film 1) or with bolphane bx polyethylene film (bolloré plastic films division, dayville, france, 13 µm thickness, heat shrinkable, o 2 permeability of 8,500 cm3/ m2/24 h/atm and water vapour transmission rate of 16 g/ m2/24 h/1 atm) (film 2). both films were applied tightly to each asparagus bunch and sealed by a hand-wrapping machine to stretch the film (sw-500 e, sambo tech corporation lovero, gyeonggi-do, korea) in the case of film 1 and heat-shrunk by a bellpack packaging machine (tecnopack packaging equipment, livorno, italy) in the case of film 2. in both packaged treatments, in-package gas atmosphere was achieved passively. all samples were stored for 21 days at 2°c (recommended storage and transport temperature) or 10°c to simulate retail outlet display. quality characteristics were determined at harvest and after 7, 15 or 21 days. these included weight loss, toughness, ph, titratable acidity, total soluble solid (tss), sucrose, glucose, fructose, total phenols, antioxidant activity and ascorbic acid. chemical analyses were determined on asparagus juice obtained using a domestic juicer and results were referred to 100 g of fresh weight (fw). crude juice was centrifuged in an eppendorf tube using a k3 system centrifuge (centurion scientific ltd, west sussex, england) at 12,000 rpm for 20 min; the clear supernatant was filtered through a 0.45 mm acetate cellulose filter (sun et al., 2005). analyses were performed in triplicate. all reagents were of analytical or better grade. modified atmosphere packaging the atmosphere composition (o 2 , co 2 and c 2 h 4 ) inside the packages was determined after 1, 3, 7, 14 and 21 days of storage. gas samples (1 ml) were withdrawn from each package with a gas-tight syringe. ethylene was determined using a varian 3300 gc (varian analytical instruments, walnut creek, ca, usa) equipped with a flame ionisation detector (fid), carbowax 20 m 80/120 mesh carbograph 1 aw 30 column (alltech, milan, italy), column temperature of 60°c, injector 110°c, and detector 180°c. oxygen and co 2 concentrations were determined with a agilent 6890 gc system equipped with a thermal conductivity detector (tcd), ctr i column 6’x1/4’ outer & 6’x1/8’ inner (alltech, milan, italy); column temperature was 60°c, injector 120°c, and detector 160°c. helium was used as carrier gas. assessment of physical parameters the weight loss of each lot was determined by a precision scale (sartorius cp 22025-oce, gottingen, germany). textural properties were measured using two different methods based on a cutting test at 7.5 and 15 cm from the tip using a 1.2-mm thickness blade (speed 3 mm s-1) and a puncture test at 15 cm from the tip using a 2-mm needle (speed 1 mm s-1, depth 3 mm) (rodrıguez et al., 2002 a). both test were accomplished using a texturometer interfaced with a computerized system with specific software (do-fb0.5ts, zwick roell, ulm, germany). chemical analysis titratable acidity was determined by titrating 5 ml of juice using a potentiometric titrator (titrino 720 sm, metrohm, herisau, switzerland) with 0.1 n naoh till ph 8.1. the ph was measured by a ph meter (orion 720a). total soluble solids (tss) were determined using a digital refractometer (pr-101, atago, tokyo, japan) and expressed as °brix; ascorbic acid by extraction with metaphosphoric acid and determined volumetrically by titration with 2,6-dichlorophenolindophenol (aoac method 967.21). total phenolic content was analyzed according to the folin-ciocalteau colorimetric method (singleton and rossi, 1965). total phenols were expressed as gallic acid equivalent. antioxidant activity was assessed using the free radical 2,2’-diphenyl-1-picrylhydrazyl (dpph) (bonded et al., 1997). the mixture, containing 3 ml of a methanol solution of 0.16 mm dpph and 0.025 ml of asparagus extract, was allowed to react in a cuvette and the absorbance of the dpph solution was determined at 515 nm after 15 min of reaction. trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) was used as a reference to compare the antioxidant activity. the activity was expressed as mm trolox equivalent antioxidant activity (teac) related to 100 g of fresh weight. analysis of carbohydrate was performed by coupling a liquid chromatograph system consisting of a d-7000 manager, l-7100 pump, l7200 auto-sampler (lachrom, merck-hitachi ltd., tokyo, japan) with an evaporative light scattering detector (elsd sedex 60lt, alfortville, france). a bio-rad aminex fast carbohydrate column (100 mm x 7.8 mm, 9 µm, lead form, bio-rad, milan, italy) with a guard column (bio-rad micro-guard carbo-p aminex cation exchange resin, lead form) thermostated at 80°c, was employed. the isocratic mobile phase was h 2 o ultra-pure and a flow rate of 0.8 ml/min was employed. the elsd detector was set as follows: drift tube temperature 45°c; nebulizer gas (air) pressure, 2.5 bar; and photomultiplier 8. stock standard solutions of each carbohydrate were prepared in ultra-pure water and their quantification in asparagus juice was calculated according to the linear calibration curves of standard compounds. the analyses were conducted in triplicate for all parameters. visual assessment the external appearance of spears (turgidity, formation of longitudinal striation, colour changes and presence of off-flavour) was assessed by a panel of six untrained technicians. spear quality was evaluated using a 1 to 5 subjective scale (1= unacceptable; 2= acceptable; 3= good; 4= very good; 5= excellent, freshly harvested appearance). 135 palma et al., effect of film packaging and storage temperature in fresh-cut green asparagus statistical analysis statistical analysis was carried out using statgraphics software (timberlake, version 5 professional, 2000). separation of means was performed by the lsd test, p≤0.05. 3. results modified atmosphere packaging the levels of in-package co 2 and o 2 were significantly affected by storage temperature (fig. 1). at 2°c, co 2 partial pressure was always lower than at 10°c (fig. 1a), while o 2 levels were higher (fig. 1b). in-package co 2 increased steadily in all treatments during the first 3-7 days, thereafter it increased at a lower rate in packages stored at 10°c, while it was quite stable or increased slightly in those stored at 2°c. in contrast, in-package o 2 partial pressure decreased gradually in all packages with final values in the range of 13.8-16.5 kpa. irrespective of the type of film, when asparagus spears were stored at 10°c, ethylene rates were slightly higher than samples stored at 2°c during the first 7 days, thereafter a dramatic increase occurred in both films, especially in samples sealed with film 2 which always displayed levels significantly higher than those sealed with film 1 (fig. 1c). weight losses weight losses increased markedly in non-packaged asparagus, slightly in samples sealed with film 1 and stored at 10°c but with values always significantly higher than in the other packages, and were negligible in samples sealed with film 2 at both storage temperature and in those sealed with film 1 stored at 2°c. in particular, at the end of storage weight losses averaged 1% and 4% in asparagus spears packaged with film 1, 0.3% and 0.8% in those packaged with film 2 and 12 and 30 % in un-packaged spears stored at 2°c or 10°c, respectively (fig. 2). fig. 1 influence of film packaging (film 1, omnifilm pvc film; film 2, bolphane bx polyolefinic film) and storage conditions on inpackage co 2 (a), o 2 (b) and ethylene levels (c). vertical bars represent the standard deviation. lsd are given at the 5% level. fig. 2 influence of film packaging and storage conditions on weight loss in green asparagus. vertical bars represent lsd, p ≤ 0.05. 136 adv. hort. sci., 2015 29(2-3): 133-140 texture figure 3 illustrates results with regard to changes in maximum shear stress to cut and displacement of f max of asparagus spears. over the storage period, the profile of the curves and f max following cutting at 7.5 (fig. 3a) and 15 cm (fig. 3b) from the tip were very similar. the maximum shear force required for cutting (figs. 3a, 3b) and force displacement (deformation l (mm) at f max) (figs. 3c, 3d) was affected by packaging, storage time and temperature. changes at harvest time (time 0) were notably higher in un-packaged asparagus, especially in samples cut at 15 cm of both storage temperatures and in those cut at 7.5 cm and stored at 10°c. the smallest changes occurred in packages stored at 2°c, where the breaking force and displacement at f max were significantly lower than all other treatments, perhaps due to the lower weight loss. for both parameters, differences between the two films were negligible. overall results of puncture test revealed minor changes both in f max (fig. 4a) and deformation (fig. 4b) in packaged samples, especially in those stored at 2°c; in contrast, both parameters increased in unpackaged asparagus, especially in those stored at 10°c. chemical analysis in table 1 the influence of film packaging and storage condition on ph, tss, ta, sucrose and fructose is reported. with respect to harvest time, ph decreased at both storage temperatures. titratable acidity increased significantly in unpackaged asparagus stored at 10°c; changes were obvious after 14 days of storage when the asparagus spears were in advanced decay, while in the other treatments values were fairly stable. a slight increase in tss fig. 3 effect of film packaging (control, un-packaged; film 1, omnifilm pvc film; film 2, bolphane bx polyolefinic film) and storage conditions (2°c and 10°c) on cutting force (f max) and force displacement (l at f max) in green asparagus during 21 days of storage at 7.5 cm (a and c) and 15 cm (b and d) of the tip. vertical bars represent the standard deviation. lsd are given at the 5% level. 137 palma et al., effect of film packaging and storage temperature in fresh-cut green asparagus table 1 influence of film packaging (film 1, omnifilm pvc film; film 2, bolphane bx polyolefinic film) and storage conditions on ph, total soluble solids (tss), titratable acidity (ta), sucrose and fructose in asparagus spears storage period ph sst (°brix) ta (g/100g as citric acid) glucose (g/100g) fructose (g/100g) 7 days storage harvest 6.15 a 5.20 bc 0.11 abc 1.29 a 1.51 a un-packaged 2°c 5.93 c 5.23 bc 0.12 ab 1.19 b 1.40 abc film 1 2°c 6.04 b 5.86 a 0.09 c 1.19 b 1.42 ab film 2 2°c 6.18 a 5.16 c 0.11 abc 1.03 c 1.30 cd un-packaged 10°c 5.84 d 5.33 bc 0.13 a 1.04 c 1.31 bcd film 1 10°c 6.04 b 5.40 b 0.10 bc 0.93 d 1.35 bcd film 2 10°c 6.07 b 5.30 bc 0.11 abc 0.96 cd 1.27 d 14 days storage harvest 6.15 a 5.20 bc 0.11 b 1.29 a 1.51 a un-packaged 2°c 5.88 d 5.53 ab 0.12 b 1.19 b 1.31 b film 1 2°c 6.07 b 5.80 a 0.11 b 1.21 b 1.24 b film 2 2°c 5.95 c 5.30 bc 0.09 c 1.04 c 1.28 b un-packaged 10°c 5.90 cd 5.10 c 0.15 a 1.02 c 0.98 c film 1 10°c 6.06 b 5.23 bc 0.11 b 0.84 d 1.04 c film 2 10°c 6.07 b 4.80 d 0.11 b 0.75 e 1.02 c 21 days storage harvest 6.15 a 5.20 b 0.11 b 1.29 a 1.51 a un-packaged 2°c 5.70 e 5.73 a 0.12 b 1.01 c 1.28 b film 1 2°c 5.93 c 5.73 a 0.11 b 1.18 b 1.07 c film 2 2°c 5.86 d 5.13 b 0.10 b 1.00 c 1.05 c un-packaged 10°c 5.88 cd 5.67 a 0.23 a 0.88 d 0.85 d film 1 10°c 6.03 b 5.03 bc 0.12 b 0.84 d 0.98 d film 2 10°c 6.07 b 4.70 c 0.12 b 0.81 d 0.75 e values in columns followed by unlike letters of each storage period and harvesting time are significantly different according to the duncan’s test of the least significant difference at p≤ 0.05. fig. 4 influence of film packaging (control, un-packaged; film 1, omnifilm pvc film; film 2, bolphane bx polyolefinic film) and storage conditions (2°c and 10°c) on force puncture test (f max) (a) and displacement of force (l at f max) (b) in green asparagus during storage. vertical bars represent the standard deviation. lsd are given at the 5% level. 138 adv. hort. sci., 2015 29(2-3): 133-140 after storage occurred in unpackaged asparagus or in samples packaged with film 1 at 2°c, while in film 1 at 10°c a significant decrease was detected; few changes occurred in the other samples. glucose and fructose were the predominant sugars, with slightly higher levels of fructose with respect to glucose (table 1), while sucrose was detected in traces with concentrations lower than 0.3 g/100 g fw (data not shown). at harvest the concentrations of glucose and fructose were 1.29 and 1.51 g/100 g, respectively. during storage their content gradually declined, especially in samples stored at 10°c, which always showed higher losses. the effect of film wrapping was not so evident. significant differences detected at some sampling dates were not confirmed in others, denoting an inconsistent and overall not significant effect of the films. with regard to ascorbic acid, differences were observed among all samples (table 2). the obtained data show that ascorbic acid content was higher at harvest and then decreased quickly during storage. the kinetics of degradation of ascorbic acid content during storage fit an exponential function of the following kind: y = a e-bx, where the coefficient “a” represents the initial concentration and coefficient “b” refers to the rate of ascorbic acid degradation of stored asparagus (fig. 5). the percentages of ascorbic acid retention after 14 days of storage at 2°c were 63.5, 57.7 and 45.8% while at 10°c they were 34.9, 29.7 and 20.5% in normal atmosphere, packaged with film 1 and film 2 respectively. total phenolic compounds at harvest were determined to be 69.16 mg/100 g fw (table 2). over the storage period, significant increases were detected in packaged and unpackage asparagus spears stored at 2°c and a similar trend was observed in asparagus packaged and stored at 10°c, although the increase was less than at 2°c, while in unpackaged spears stored at 10°c total phenols remained similar to those at harvest. antioxidant activity was fairly stable in asparagus stored at the lower temperature, while in asparagus stored at 10 °c it decreased more than samples stored at 2°c; most of the decline occurred during the first week of storage and with the highest losses detected in unwrapped spears stored at 10°c. (table 2). visual assessment after 7 days of storage at the test temperatures, packaged spears were judged as excellent, unpackaged ones scored 3 at 2°c and 2 at 10°c (data not shown). the main alterations detected in unpackaged samples were longitudinal striations due to excessive water loss, especially toward the base. these phenomena increased during storage and were more evident in spears stored at 10°c. as a result, after 14 days unpackaged asparagus stored at 10°c were unmarketable (score 1) (villanueva et al., 2005). after 21 days, spears stored at 2°c and packaged with film 2 and film 1 were scored 3 and 2, respectively, with the main defect being a loss of green color, which also occurred in unpackaged samples. after 21 days of storage even unpackaged samples stored at 2°c were unmarketable, while packaged spears, with only slight discoloration, were still marketable. table 2 influence of film packaging (film 1, omnifilm pvc film; film 2, bolphane bx polyolefinic film) and storage conditions on ascorbic acid, total phenols and antioxidant activity in asparagus spears storage period ascorbic acid (mg/100g) total phenols (mg/100g) antioxidant activity (mm teac) 7 days storage harvest 22.70 a 69.16 cd 1.71 ab un-packaged 2°c 18.23 b 69.98 bcd 1.61 b film 1 2°c 17.20 b 74.19 a 1.77 a film 2 2°c 13.57 c 72.93 abc 1.79 a un-packaged 10°c 11.20 d 68.44 d 1.41 c film 1 10°c 11.00 d 74.48 a 1.47 c film 2 10°c 8.30 e 73.72 ab 1.46 c 14 days storage harvest 22.70 a 69.16 c 1.71 ab un-packaged 2°c 14.43 b 80.35 a 1.63 bc film 1 2°c 13.10 b 78.79 a 1.73 ab film 2 2°c 10.47 c 82.21 a 1.79 a un-packaged 10°c 7.93 d 70.60 bc 1.35 d film 1 10°c 6.77 d 74.05 b 1.44 c film 2 10°c 4.67 e 73.84 b 1.45 c 21 days storage harvest 22.70 a 69.16 c 1.71 a un-packaged 2°c 11.30 b 84.14 a 1.63 b film 1 2°c 9.68 c 83.27 a 1.73 a film 2 2°c 9.43 c 85.97 a 1.76 a un-packaged 10°c 7.63 d 69.52 c 1.35 d film 1 10°c 5.17 e 76.91 b 1.49 c film 2 10°c 4.63 e 75.10 b 1.48 c values in columns followed by unlike letters of each storage period and harvesting time are significantly different according to the duncan’s test of the least significant difference at p≤0.05. fig. 5 kinetic model of ascorbic acid degradation during storage. 139 palma et al., effect of film packaging and storage temperature in fresh-cut green asparagus 4. discussion and conclusions this study confirms a delay of the processes of physical and chemical deterioration in asparagus spears in response to storage temperature and modified atmosphere resulting from two different packaging solutions. the response was evident in visual appearance, weight loss, and some chemical parameters. modified atmospheres are known to reduce the metabolic activity of harvested products. in the present experiment the modified atmosphere was passively created by the accumulation of co 2 released by respiration and its composition was affected by storage temperature and film permeability to gases. the final partial pressures of co 2 and o 2 were within the tolerance limits for this commodity, which are above 10k pa for o 2 and below 15 kpa for co 2 (berrang et al., 1990). ethylene concentration was generally higher at 10°c than at 2°c, especially, after 7 days. moreover, when asparagus were stored at 10°c, differences in ethylene concentrations were also affected by the type of film. nevertheless, the in-package gas composition was remarkably effective in all conditions at delaying chemical and physical compositional changes, regardless of storage temperature. it is likely that the potential negative effect of ethylene, especially in packages stored at 10°c, was counteracted by co 2 , which notoriously can mitigate the negative effect of ethylene by interacting with its receptors (burg, 2004). the rate of weight loss was dependent on storage temperature, packaging, and type of film used. prevention of weight loss due to the maintenance of high relative humidity is a major advantage of packaging, and the beneficial effect of this practice was particularly evident in this study. however, the different permeability to water vapor of the two films led to significant differences in weight loss between film 1 and film 2, especially at 10°c. as a result, overall appearance of asparagus stored at 2°c and sealed with film 2 (the less permeable) declined at a slower rate and allowed better maintenance of market quality. asparagus toughening has generally been associated with the lignification process (siomos et al., 2000). postharvest texture changes are influenced by numerous biochemical modifications of plant tissues, such as compositional modifications of cell walls, lignification of the pericyclic fibers, increases in phenolic compounds such as ferulic acid and p-coumaric acid, deposition of polysaccharides, mainly xylan, and storage conditions (rodriguez et al., 2002 b; 2004; 2005). the results of the present study are in agreement with previous findings (waldron and selvendran, 1990; rodriguez et al., 1999 a, b). moreover, textural properties were influenced by water loss; toughness increases as a consequence of turgor reduction. the increased titrable acidity detected in unpackaged asparagus stored at 10°c might be induced by the advanced decaying process, which did not occur in packaged samples. a slight increase in tss after storage occurred in unpackaged or packaged asparagus with film 1 at 2°c, while in film 1 at 10°c a significant decrease was recorded; few changes occurred in the other samples. packaged and unpackaged spears showed significant reductions in glucose and fructose content during storage, particularly in those stored at 10°c. changes in tss, glucose and fructose generally denoted marked differences between unpackaged and packaged samples and, above all, between the two storage temperatures. the few differences in chemical composition between packaged and non-packaged asparagus, in contrast with the marked differences detected in weight loss and textural properties, may indicate that the overall beneficial effect of packaging is a consequence of the reduced transpiration occurring in packaged asparagus rather than the physiological effect due to the increased levels of co 2 and reduced availability of o 2 . as for ascorbic acid content, the results reflect a significant decrease with storage which is more accentuated in unpackaged samples and in those stored at 10°c (esteve et al., 1995). the kinetics of degradation confirmed this trend, in fact the highest rate of degradation was approximately 0.4 mg ascorbic acid/100 g/day and 0.8 mg ascorbic acid/100 g/ day for packaged asparagus and 0.3 mg ascorbic acid/100 g/day and 0.6 mg ascorbic acid/100 g/day for unpackaged samples, refrigerated at 2°c and 10°c, respectively. total phenolic content increased during storage: changes were clear after 14 days in packaged spears and stored at 2°c. this trend can be ascribed to an increase of phenols in asparagus cell walls (rodriguez et al., 2002 b). however, in unpackaged spears stored at 10°c no change occurred in phenols content when they were in advanced decay, most likely because decaying cells are no longer able to synthesize phenolic compounds. antioxidant capacity was fairly stable during storage in asparagus spears packaged with film 1 and film 2 stored at 2°c. in contrast, antioxidant capacity of packaged and unpackaged spears stored at 10°c declined during both periods with values significantly lower than at harvest. the antioxidant activity in foods depends on the content of components such as phenolic compounds, carotenoids and ascorbic acid and their fate during processing or storage. in this study the antioxidant capacity might have been balanced by the decreasing trend of ascorbic acid and the increasing tendency of total phenols. as a result, higher levels of antioxidant activity was detected in packaged asparagus stored at 2°c, which contained more total polyphenols and lost vitamin c at a slower rate than all other treatments. however, considerations about changes in chemical parameters should take account the effect of transpiration, as a loss of water inevitably leads to an increase of solute concentration, and this effect might be particularly marked in unpackaged samples where weight loss over the storage period ranged between about 6 and 32 %. the overall results of this study show the positive effect on quality retention of cold-stored asparagus, both from a market point of view and from the chemical and nutraceutical perspective. in particular, the best results were achieved with the less permeable film (film 2) and storage at 2°c. 140 adv. hort. sci., 2015 29(2-3): 133-140 acknowledgements we thank domenico mura (ctre cnr ispa) for his contribution with chemical and physical analyses. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references albanese d., russo l., cinquanta l., brasiello a., di matteo m., 2007 physical and chemical changes in minimally processed green asparagus during cold-storage. food chem., 101: 274-280. aoac, 2007 official methods of analysis, 18th ed. association of official analytical chemists international, gaithersburg, md, usa. berrang m.e., brackett r.e., beuchat l.r., 1990 microbial, colour and textural qualities of fresh asparagus, broccoli and coliflower stored under controlled atmosphere. food prot., 53(5): 391-395. bonded v., brand-williams w., berset c., 1997 kinetics and mechanism of antioxidant activity using the dpph free radical method. leb.-wiss. tech., 30: 609-615. burg s.p., 2004 ethylene, pp. 132–239. in: burg s.p. (ed.) postharvest physiology and hypobaric storage of fresh produce. cabi publishing, wallingford, uk. esteve m.j., farrè r., frigola a., 1995 changes in ascorbic acid content of green asparagus during the harvesting period and storage. j. agr. food chem., 43: 2058-2061. fonseca s.c., oliveira f.a.r., brecht j.k., 2002 modelling respiration rate of fresh fruits and vegetables for modified atmosphere packages: a review. j. food eng., 52: 99-119. gontard n., thibault r., cuq b., guilbert s., 1996 influence of relative humidity and film composition on oxygen and carbon dioxide permeabilities of edible films. j. agr. food chem., 44: 1064-1069. kader a.a., 1992 postharvest biology and technology: an overview. postharvest technology of horticultural crops, university of california, vol. 3311. king g.a., henderson k.g., lill r.e., 1986 asparagus: effect of controlled atmosphere storage on shelf-life of four cultivars. new zealand j. experimental agr., 14: 421424. king g.a., hurst p.l., irving 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chem., 91: 609-619. waldron k.w., selvendran r., 1990 composition of the cell walls of different asparagus (asparagus officinalis) tissues. physiol. plantarum, 80: 568-575. zagory d., kader a.a., 1988 modified atmosphere packaging of fresh produce. food technology, 9: 70-77. impaginato 503 adv. hort. sci., 2018 32(4): 503-510 doi: 10.13128/ahs-22083 allelopathic activities of celery extract and its fractions against corchorus olitorius, echinochloa crusgalli and portulaca oleracea weeds r. el-mergawi (*), e.r. el-desoki botany department, national research centre, 12622 cairo, egypt. key words: apium graveolens, germination, natural herbicide, phenolic acids. abstract: aqueous extract of celery (2.5-20 g l-1, w/v) was evaluated for its phytotoxicity against three weed species, under laboratory and greenhouse conditions. celery extract had a strong inhibitory effect on germination and seedling growth of corchorus olitorius, echinochloa crusgalli and portulaca oleracea seeds. from dose response curves of tested seeds, lc50 were calculated to be in the range from 6.3 to 8.3 g l-1 for germination percent, from 7.2 to 8.0 g l-1 for shoot length and from 1.7 to 3.6 g l-1 for root length. completely inhibition of root growth was exhibited c. olitorius and p. oleracea at 7.5 g l-1 corresponded with 15 g l-1 for e. crusgalli seed. total phenolics in celery extract at 20 g l-1 constituted 201 mg l-1. ten phenolic acids were identified in extract by hplc, among of them p-coumaric acid and p-hydroxybenzoic acid were presented in high amounts. aqueous extract was partitioning between three solvents, hexane, methylene chloride, ethyl acetate. generally, water residue after partitioning aqueous extract with the three solvents had the most phytotoxic effect on seedling growth of target seeds. in greenhouse trial, foliar spray of aqueous extract of celery (30, 60 and 90 g l-1) and its fractions did not produce any significant effect on growth of two-weeks-old c. olitorius, or e. crusgalli or p. oleracea weeds. 1. introduction worldwide, weeds caused about 34% yield losses among the major crops. herbicide application is the most reliable weed control methods. negative impacts of herbicides on environmental human health and herbicide resistant weeds were considered the two problems faced in weed management with herbicides (jabran et al., 2015). thus, manipulating the allelopathy can help to improve weed control in agriculture and increase the acceptance of agricultural products in today´s demanding consumer markets (trezzi et al., 2016). allelopathy has been used as the basis for identifying plant species which may contain phytotoxic chemicals. these natural compounds can (*) corresponding author: ragabelmergawi05@hotmail.com citation: el-mergawi r., el-desoki e.r., 2018 allelopathic activities of celery extract and its fractions against corchorus olitorius, echinochloa crusgalli and portulaca oleracea weeds. adv. hort. sci., 32(4): 503-510 copyright: © 2018 el-mergawi r., el-desoki e.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 november 2017 accepted for publication 28 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 503-510 504 offer excellent potential for new herbicidal solutions, or lead compounds for new natural herbicides (duke et al., 2000; vyvyan, 2002). the main purposes of research on allelopathy include the application of the allelopathic effects to agricultural production, reduction of the input of chemical pesticides and consequent environmental pollution, and provision of effective methods for the sustainable development of agricultural production and ecological systems (han et al., 2013; jabran et al., 2015). knowledge concerning allelopathy can also be a key component in supporting organic farming, for which weed control is a major problem (trezzi et al., 2016) organic farming can involve reduced weed infestation by using plant extracts or intercropping plant species with an allelopathic potential (bajwa et al., 2015). plant extracts are the way of using allelochemicals for weed control in agroecosystems, as they have been already used as post-emergence natural herbicides in some countries. in pakistan, for example, an aqueous extract deriving from sorghum shoots with a 10% concentration was left to ferment for several weeks and was subsequently sprayed post emergence for weed control. this fermented water extract, known as “sorgaab”, reduced weed density and weed dry weight up to 50% in field trials (cheema and khaliq, 2000; cheema et al., 2002). celery (apium graveolens l.; apiaceae) has been cultivated for the last 3000 years, notably in egypt, and was known in china in the fifth century bc. it has been used as a popular aromatic herb and spice (chevallier, 1998). earlier studies of a. graveolens led to isolation of some phenolic compounds as phthalides (tang et al., 1990; momin and nair, 2001) and furocoumarins (garg et al., 1979). these compounds are reported for their insecticidal, nematicidal, antifungal and phytotoxic activities (kato et al., 1977; momin and nair, 2001; pavela and vrchotová, 2013). recently, sbai et al. (2017) reported that the aqueous extract (10-50 g/l) of a. graveolens had great inhibitory effect on root growth of germinated seeds of lettuce (in the range between 80% and 90%). they isolated six compounds which included three phthalides [senkyunolide a, (3s)-butylphthalide and sedanolide], two furanocoumarins (bergapten and scopoletin) and one phenyl propanoid (p-hydroxyphenethyltrans ferulate). senkyunolide a compound was the most toxic on lettuce germination and shoot growth, however, p-hydroxyphenethyl trans-ferulate was the most toxic on root growth. however, in spite of the wide range of biological activity of celery extract, but information concerning the herbicidal activity of this extract is rarely available. therefore, the main objective of this study was to evaluate the herbicidal activity of the aqueous celery extract and its fractions against corchorus olitorius, echinochloa crusgalli and portulaca oleracea weeds, with the goal of developing an effective plant derived herbicide. moreover, the phenolic acids that considered the main source of all bioactive phenolic substances were identified in celery extract via hplc. 2. materials and methods plant material plants of celery (a. graveolens l. var. dulce) were purchased from a local market in cairo, egypt. identification of celery based on morphological traits that extensive observation of mature plants. the leaves located at the top of the leaf stalks were collected and dried in hot-air oven at 50°c for 72 h, powdered and used for extraction. preparation of aqueous extract different concentrations (w/v) of extracts were prepared by soaking known weight of dried leaves in known volume from distilled water at room temperature, and shaken for 24 h. the extracts were filtered through a whatman no. 1 filter paper and kept at 4°c in the dark until use. germination bioassay mature seeds of corchorus olitorius, echinochloa crusgalli and portulaca oleracea were collected from plants growing in fields of the experimental station of the national research centre (research and production station, nubaria region, behaira governorate, egypt. uniform healthy seeds were selected. seeds were surface-sterilized with sodium hypochlorite (0.1%, w/v) for 2 min, washed under running tap water for 5 min followed by distilled water for 2 min, and stored for further use. twenty seeds were placed in a 9-cm plastic petri dish lined with a single whatman no. 1 filter paper, and then 4 ml of each extract concentration (2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 g l-1) was added onto filter paper. distilled water (4 ml) was applied to a petri dish to serve as a control. four replicates of petri dishes of each treatment were placed, in a completely randomized manner, in a growth chamber at 25°c. after 5 days, germination percent, shoot length and root length of germinated seeds were determined. el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 505 extract concentrations required to cause 50% inhibition of different germination criteria (germination percentage, root length and shoot length) were calculated by probit analysis. determination of total phenolics in aqueous extract extract of celery at 20 g l-1 was subjected to determine total contents of phenolics using folinciocalteu reagent (singleton and rossi, 1965). analysis of phenolic acids in aqueous extract by highperformance liquid chromatography (hplc) phenolic acids in aqueous celery extract at a concentration 20 g l-1 were hydrolyzed with sodium hydroxide (mckeehen et al., 1999) and subjected to hplc analysis. approximately 15 ml of aqueous extract was added to 15 ml of 8 n naoh in a 50 ml pyrex centrifuge tube, purged with nitrogen, shaken for 2 h in the dark with a shaker and acidified with ice-cold 6 n hcl to reduce ph to 2. sample was centrifuged at 3000g, and the supernatant was decanted into separatory funnel. the supernatant was extracted with ethyl acetate (3 x 50 ml) with shaking for 10 s, and the mixture was allowed to settle for 5 min between extractions. the phenolic acids rich ethyl acetate fraction was dried by addition of anhydrous sodium sulfate and concentrated using rotary evaporator at 40°c to dryness. the residue was re-solubilized in 2.5 ml of methanol and filtered through a 0.2 μm ptfe filter prior to analysis. hplc analysis was performed using equipment from shimadzu (japan): a shimadazu lc-2010a liquid chromatograph, a shimadazu spdm10a diode array detector and a shimadzu class-vp v6.12 sp4 offline processing system. phenolics were analyzed using a luna rp-c18 (2) column (250×4.6 mm i.d, 5 μm, phenomenx). the mobile phase consisted of a mixture of acetate buffer: acetonitrile (9:1, v/v). acetate buffer was prepared by dissolving 6.35 g sodium acetate in one-liter h2o and 20 ml acetic acid. the detecting wavelength was 260 nm. standard phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, syringic, ferulic and pcoumaric) were purchased from sigma aldrich and vanillic, caffeic, salicylic and cinnamic were purchased from fluka. greenhouse bioassay another experiment was performed to study the effect of the aqueous. celery extract on the growth of two-weeks-old c. olitorius, e. crusgalli and p. oleracea weeds grown under controlled conditions in an experimental greenhouse (25±3°c, 12 h photoperiod). aqueous extracts at four concentrations (0, 30, 60 and 90 g l-1) were prepared as previously mentioned and used in a greenhouse bioassay. plants were raised from the collected seeds in 12-cm diameter earthenware pots. the pots were filled with 750 g soil (sand:peat moss = 3:1, w/w) and 20 seeds of each plant species were sown per pot. two weeks after emergence, plants were sprayed with plant extracts at 0, 30, 60 and 90 g l-1 concentrations. extract solutions were applied to shoots of tested weeds using a epoca sprayer (italy). the solution was sprayed evenly over the entire surface of the plant, including the adaxial and abaxial surface of leaves. a total of 48 pots were maintained (i.e. 4 extract concentrations × 3 weeds × 4 replicates) in a completely randomized design. seven days after extract spraying, the plants were examined for visible injury levels and the percent of chlorotic and necrotic areas were recorded. fractionation of extract one hundred milliliter of high extract concentration that used either in germination trial (20 g l-1, w/v) or in greenhouse trial (90 g l-1, w/v) was subsequently partitioned with organic solvents with increasing polarity. the extract was partitioned three times with 200 ml aliquots of each solvent: hexane, methylene chloride (mecl2) and ethyl acetate (etoac) using separatory funnel. solvents were dried by addition of anhydrous, sodium sulfate, filtered and evaporated using buchi rotary evaporator at 40°c to dryness. the residues of hexane, mecl2 and etoac fractions were dissolved in 100 ml of dmso-water solution (0.1%, v/v). these three fractions and water residue (h2o fraction) were subjected to bioassay as previously mentioned. statistical analysis all data was subjected to anova analysis using costat software to evaluate the effect of extract rates upon germination and growth parameters of tested weeds. after anova, the parameters that were statistically significant (a. graveolens extracts with p≤0.05), were subjected to pro-bit analysis using ldp line. pro-bit curves were derived by plotting the extract concentration (on the x-axis) and inhibition % (on the y-axis). with the curves obtained, lc50 value was calculated for each parameter. this value represents the extract concentration at which 50% inhibiadv. hort. sci., 2018 32(4): 503-510 506 tion in different germination parameter occurs. 3. results effect of celery extract on germination percentage of seeds as shown in table 1, aqueous extract of celery had a strong inhibitory effect on germination percentage of the three tested seeds and the inhibition increased with concentration. increasing extract concentration above 5 g l-1 produced gradual decrease in germination percentage of c. olitorius. the highest reduction effect was produced by extract at highest concentrations, 17.5 and 20 g l-1 (89% and 93%, respectively). also, germination percentage of e. crusgalli and p. oleracea was reduced by the extract at all tested concentrations. the reduction percentage varied from 37% and 9%, respectively at the lowest concentration to 96% and 73%, respectively at the highest ones. effect of celery extract on seedling growth of seeds aqueous extract of celery exhibited a great inhibitory effect on shoot length of the three germinated seeds (table 2). the extract improved the shoot growth of e. crusgalli and p. oleracea at the lowest concentrations. the progressive increase in extract concentration followed by progressive reduction in shoot length of all tested seeds. more than 90% reduction in shoot length of three germinated seeds was obtained by soaking seeds in celery extract at 17.5 and 20 g l-1. an inhibition of root growth was observed in presence of all extract concentrations and roots more sensitive than shoots (table 2). root length of three target seeds varied in their response to different extract concentrations and e. crusgalli was less effective one. soaking seeds in celery extract at the lowest concentration produced great reduction in root elongation of all treated seeds ranged between 36% and 71%, relative to control. using extract at 7.5 g l-1 and above completely inhibited root growth of both p. oleracea and c. olitorius, whereas complete inhibition of e. crusgalli roots was obtained at 15 g l-1. extract concentrations required to cause 50% inhibition (lc50) inhibition percent and levels of lc50 of all germination parameters of tested weeds were calculated and dose-response curves were illustrated in figure 1. the three tested weeds varied in their inhibition percentages as affected with celery extract. among tested seeds, germination percent of p. oleracea was conconcentration (g l-1) germination % c. olitorius e. crusgalli p. oleracea control 100±0 a 100±0 a 100±0 a 2.5 95±5 a 63±7 b 91±9 b 5 93±2 a 67±4 b 84±4 b 7.5 49±5 b 52±2 c 73±3 c 10 41±4 c 43±5 c 74±7 c 12.5 23±3 d 39±7 c 60±2 d 15 11±5 e 25±4 d 40±2 e 17.5 11±5 e 14±5 d 37±2 e 20 7±2 e 4±1 e 27±3 f table 1 effect of aqueous celery extract on germination percentage of three weed species values are given as means of three replicates ± standard error. means with the same letters in a column are not significantly different at p<0.05. table 2 effect of aqueous celery extract on shoot and root lengths of three weed species values are given as means of three replicates ± standard error. means with the same letters in a column are not significantly different at p<0.05. rate (g l-1) shoot length (cm) root length (cm) c. olitorius e. crusgalli p. oleracea c. olitorius e. crusgalli p. oleracea control 2.56±0.14 a 3.35±0.35 a 2.17±0.13 b 2.41±0.08 a 1.83 ±0.26 a 1.95±0.23 a 2.5 2.74±0.25 a 2.91±0.34 b 2.50±0.23 a 0.81±0.17 b 1.17 ±0.12 b 0.57±0.07 b 5 2.21±0.19 b 2.43±0.19 c 2.19±0.60 b 0.51±0.11 c 0.65 ±0.20 c 0.29±0.06 c 7.5 1.38±0.40 c 1.73±0.33 d 0.58±0.40 c 0.00 d 0.51 ±0.17 c 0.00 d 10 0.76±0.28 d 0.79±0.20 e 0.60±0.06 c 0.00 d 0.25 ±0.06 d 0.00 d 12.5 0.36±0.06 e 0.77±0.20 e 0.56±0.31 c 0.00 d 0.22 ±0.01 d 0.00 d 15 0.10±0.02 e 0.53±0.31 ef 0.15±0.08 d 0.00 d 0.00 e 0.00 d 17.5 0.05±0.03 e 0.27±0.14 ef 0.14±0.14 d 0.00 d 0.00 e 0.00 d 20 0.00 f 0.06±0.03 f 0.11±0.06 d 0.00 d 0.00 e 0.00 d el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 507 sidered a less sensitive to celery extract. since, value of lc50 of p. oleracea constituted 13.4 g l-1 corresponded with 8.3 and 6.3 g l-1 for c. olitorius and e. crusgalli, respectively. whereas, inhibition percent of shoot length did not produce a great variation among tested seeds and lc50 calculated to be 7.8, 7.2 and 8 g l-1 for c. olitorius, e. crusgalli and p. oleracea seeds, respectively. as shown in figure 1, root length exhibited the maximum inhibition effect as affected by celery extract when compared with germination percent and shoot length. moreover, root length of tested seeds varied in their inhibition percent as affected with celery extract. depending on lc50 levels, root length of p. oleracea (1.65 g l-1) was considered the most sensitive, followed by c. olitorius (1.8 g l-1) and e. crusgalli (3.6 g l-1). total phenolics and phenolic acids content in aqueous extract in this study, we determine total phenolics for celery extract at a concentration 20 g l-1 and found that the extract contained high amounts of phenolics config. 1 dose-response curves showing the effect of celery extract (inhibition % and lc50) on percent germination and seedling growth of three tested seeds. 508 adv. hort. sci., 2018 32(4): 503-510 stituted 201 mg l-1, as gallic acid. using hplc, ten phenolic acids were detected in this aqueous. extract, of which six compounds consisted of benzoic acid derivatives and four of cinnamic acid derivatives (table 3). among the identified phenolic acids, phydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in high amounts (18.72, 30.32 and 11.12 mg l-1, respectively). except the minor quantity of cinnamic acid, moderate concentrations from other phenolic acids were determined in celery sample. effect of fractions on germination of weeds as shown in table 4, fractions obtained from the aqueous extract of celery varied in their effects on germination criteria of target seeds. as for germination percentage, the significant reduction effect was recorded only for mecl2 fraction on p. oleracea (15%) as well as for h2o residue on both c. olitorius (30%) and p. oleracea (36%). but, all examined fractions did not produce any significant effect on germination percentage of e. crusgalli seeds. whereas, h2o fraction induced a great reduction on shoot elongation of c. olitorius, e. crusgalli and p. oleracea seeds, reached 64%, 47% and 74%, respectively relative to control. with less extent, hexane and mecl2 fractions reduced shoot length of e. crusgalli. with few exceptions, all fractions reduced root elongation of target seeds. water fraction was the most toxic, inducing an inhibition of 99%, 94% and 50% in root length of p. oleracea, c. olitorius and e. crusgalli, respectively. whereas, the other fractions, hexane, mecl2 and etoac fractions reduced root length of e. crusgalli and p. oleracea between 17% and 43%, relative to control. effect of celery extract and its fraction on growth of weeds data presented table 5 revealed that spraying aqueous celery extract and its fractions did not produce any significant effect on growth of two-weeks old of either c. olitorius, or e. crusgalli or p. oleracea weeds. table 3 phenolic acids in aqueous extract of celery at concentration 20 g l-1 means with the same letters in a raw are not significantly different at p<0.05. table 4 effect of fractions obtained from aqueous extract of celery on germination of three weeds (% of control) acid derivatives concentration (g l-1) benzoic acid derivatives p-hydroxybenzoic acid 18.72 gallic acid 8.56 vanillic acid 8.8 syringic acid 6.4 protocatechuic acid 9.28 salicylic acid 7.84 cinnamic acid derivatives cinnamic acid 0.64 caffeic acid 9.52 p-coumaric acid 3.032 ferulic acid 11.12 table 5 effect of aqueous celery extract and its fractions on the growth of two-weeks-old of three weeds criteria weed species fractions control hexane mecl2 etoac h2o germination (%) c. olitorius 100 a 104 a 96 a 102 a 70 b e. crusgalli 100 a 98 a 104 a 102 a 95 a p. oleracea 100 a 107 a 85 b 109 a 64 c shoot length c. olitorius 100 a 97 a 102 a 102 a 36 b e. crusgalli 100 a 86 b 83 b 95 a 53 c p. oleracea 100 a 95 a 100 a 87 a 26 b root length c. olitorius 100 a 101 a 74 b 103 a 6 c e. crusgalli 100 a 57 c 83 b 79 b 50 c p. oleracea 100 a 61 b 76 b 63 b 1 c treatments injuries ( % of control) c. olitorius e. crusgalli p. oleracea 30 g l-1 2±0 a 0±0 a 1±0 a 60 g l-1 3±1 a 2±5 a 3±2 a 90 g l-1 3±2 a 3±1 a 3±1 a hexane fraction 2±1 a 3±2 a 4±2 a mecl 2 fraction 4±2 a 1±0 a 1±0 a etoac fraction 3±1 a 2±1 a 3±1 a h 2 o residue 4±1 a 1±0 a 1±0 a values are given as means of three replicates± standard error. means with the same letters in a column are not significantly different at p<0.05. 509 el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 4. discussions and conclusions aqueous extract of celery was evaluated for its phytotoxicity against three plant species typically present as weeds in summer crops, under laboratory and greenhouse conditions. the extract displayed a great inhibition on germination percentage and seedling growth of target seeds. in line of these results, sbai et al. (2017) reported that lettuce germination was completely inhibited by celery extract at concentration above 20 g l-1. they extracted and identified the allelochemicals compounds that responsible of toxicity namely phthalides, among of them senkyunolide a was the most toxic in lettuce germination. current study revealed that the inhibitory effect of celery extract on germination percent varied between tested seeds, and p. oleracea possessed the least sensitivity. variation between different plant species in their sensitivity to plant extracts was previously observed by many investigators (al-humid and el-mergawi, 2014; han et al., 2008). this study showed that root growth was the most sensitive to extract than shoot growth. since, lc50 of shoot length of three target seeds ranged between 7.2 g l-1 and 8 g l-1 whereas, values of lc50 for root growth are calculated to be in the range of 1.7 g l-1 and 3.6 g l-1. these results are in agreement with sbai et al. (2017), who reported that celery extract had more pronounced effects on roots of lettuce, rather than shoots. generally, in germination bioassay, water extract of allelopathic plants have more pronounced effect on root rather than shoot growth (inderjit and dakshini, 1995; muhammad et al., 2011). this may be attributed to the fact that roots are the first to absorb the phytotoxic compounds (turk and tawaha, 2002). in order to identify and distribute the chemical groups of toxic allelochemical constituents, celery aqueous. extract was partitioning between three organic solvents varied in their polarities. generally, the most phytotoxic compounds were represented water residue (h2o fraction). it can be suggested that the high toxicity compounds in celery extract may be related to presence of more polar compounds. these results are in general agreement with the results obtained by sbai et al. (2017). who observed that extraction of celery with non-polar solvents, petroleum ether or chloroform had no significant effect on lettuce germination, contrarily to the high toxicity effect of methanol extract (polar solvent). the high toxicity of celery extracts may be attributed to the present water soluble compounds as saponins, glycosides, hormones or enzyme which could affect growth directly or by altering the mobilization of storage compounds during germination (chaves and escudero, 1997; el-khatib, 1997). for the greatest inhibition effect of aqueous celery extract, we analyzed phenolics and phenolic acids in the extract at a concentration 20 g l-1. the results indicated the presence of high amount of phenolic compounds (201 mg l-1 as gallic acid) may explain the greatest effect of celery extract. in line of these results, jung et al. (2011) found high levels of phenolic in celery (51.09 mg g-1 dw, as gallic acid). phenolic acids are the precursor of all phenolic constituents in plants as well as the bioactive constituents in celery extract. identification and determination the concentration of the phenolic acids in the aqueous extract was conducted by using hplc. ten phenolic acids were identified in celery extract, among of them phydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in relatively high amounts. in line of these results, yang et al. (2010) identified caffeic acid, p-coumaric acid, and ferulic acid in celery extract. presence of high concentration of total phenolic and phenolic acids in areal parts of celery was previously reported by many investigators (yang et al., 2010; sbai et al., 2017). in greenhouse trial, we evaluated the effects of foliar s pray of aqueous extract of celery and its fractions, hexane, mecl2, etoac and h2o-residue on growth of two-weeks-old c. olitorius, or e. crusgalli or p. oleracea weeds. celery extract at 30, 60 and 90 g l-1 as well as the obtained fractions did not produce any significant effect on growth of three examined weeds. in general, the growth of two-weeks-old weeds tended to be less sensitive to the test fractions than the weed germination process. these results are in agreement of those obtained by inderjit and weston (2000), they found that greenhouse bioassays do not adequately predict the responses observed in laboratory bioassay. hence, weed germination might be the most sensitive index with which to judge allelopathy of plant extracts and its fractions under natural conditions (corrêa et al., 2008). in conclusion, in this study aqueous extract of celery was evaluated for its phytotoxicity against three weed species under laboratory and greenhouse conditions. the extract displayed a great inhibition on germination percentage and seedling growth of target seeds. root growth of c. olitorius and p. oleracea was completely inhibited by extract at 7.5 g l-1, corresponded with 15 g l-1 for e. crusgalli. celery aqueous extract was fractionated by using three less polar sol510 adv. hort. sci., 2018 32(4): 503-510 vents; however, water extract displayed the strongest inhibition effect on germination of three target seeds. celery extract had a relatively high amount of phenolics and phenolic acids. high concentrations of water celery extract 30, 60, 90 g l-1) and the obtained less polar fractions did not produce any significant effect on growth of two weeks-old of tested weeds. water extract of celery may be a useful source for the future development of pre-emergence bioherbicide. references al-humaid a., el-mergawi r., 2014 herbicidal activities of seven native plants on the germination and growth of phalaris minor, echinochloa crusgalli, portulaca oleracea and lactuca sativa. j. agric. sci. tech. a, 4: 843-852. bajwa a.a., mahajan g., chauhan b.s., 2015 nonconventional weed management strategies for modern agriculture. weed sci., 63(4): 723-747. chaves n., escudero j.c., 1997allelopathic effect of cistus ladanifer on seed germination. functional ecology, 11: 432-440. cheema z.a., khaliq a., 2000 use of sorghum allelopathic properties to control weeds in irrigated wheat in a semi-arid region of punjab. agric. ecosyst. environ., 79: 105-112. cheema z.a., khaliq a., ali k., 2002 efficacy of sorgaab for weed control in wheat grown at different fertility levels. pak. j. weed sci. res., 8: 33-38. chevallier a., 1998 the encyclopedia of medicinal 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graveolens l.). j. agric. food chem., 38: 1937-1940. trezzi m.m., vidal r.a., balbinot j.r., von hertwig bittencourt h., da silva souza filho a.p., 2016 allelopathy: driving mechanisms governing its activity in agriculture. j. plant interactions, 11(1): 53-60. turk m.a., tawaha a.m., 2002 inhibitory effects of aqueous extracts of black mustard on germination and growth of lentil. pakistan j. agron., 1(1): 28-30. vyvyan j.r., 2002 allelochemicals as leads for new herbicides and agrochemicals. -tetrahedron, 58: 1631-1646. yang y., sang w., zhou m., ren g., 2010 phenolic composition and antioxidant activities of 11 celery cultivars. j. food sci., 75(1): 9-13. impaginato 13 1. introduction cotton is an economically important plant grown world-wide as a principal source of staple fiber and vegetable oil. a great deal of effort has been made to improve cotton cultivation and characteristics by breeders. cotton is one of the major fiber crops in syria, with a cultivated area of 125,000 ha, a production of 470,000 t of seed cotton, and lint production at 160,000 t. yarn spinning capacity is estimated at 180,000 t (usda, 2011). salinity tolerance is a complex trait that involves physiological, biochemical, cellular, and genetic strategies. at present, out of 1.5 billion ha of cultivated land around the world, about 77 million ha (5%) is affected by excess salt content (moradi et al., 2011). there is evidence that high salt concentrations cause an imbalance of the cellular ions resulting in ion toxicity and osmotic stress, leading to the generation of reactive oxygen species (ros) which alter cellular metabolism causing lipid peroxidation, protein denaturing, and dna mutation (dat et al., 2000; davenport et al., 2003; implay, 2003). moreover, salt stress causes nuclear deformation and subsequent nuclear degradation (katsuhara and kawaski, 1996). structural changes of nuclei caused by salt stress have been previously reported as well (werker et al., 1983). at present, there are several methods (physiological, biochemical, and molecular) available for detecting different kinds of dna damage but with some limitations. recently, molecular markers have been successfully applied to detect dna damage induced by different abiotic stresses, particularly salinity. among others, rapd technique has been well documented as a sensitive means of detecting dna damage and shows potential as a reliable and reproducible assay for the detection of dna fragmentation and chromosomal mutations (citterio et al., 2002). the rapd marker has been extensively applied for salinity tolerance screening in plant breeding programs, such as in date palm (phoenix dactylifera l.) (kurup et al., 2009), aquatic plants hydrilla verticillata and ceratophyllum demersum (gupta and sarin, 2009), in euplotes vannus (protozoa, ciliophora) (zhou et al., 2011), and in acacia senegal (daffalla et al., 2011); in cotton (dojan et al., 2012) and in fish full-sib nile tilapia (oreochromis niloticus), blue tilapia (oreochromis aureus) and their diallel interspecific hybridization (el-zaeem, 2012); and recently, also in soybean (glycine max l.) (khan et al., 2013). rapd bands can be scored for genomic template stability (gts) evaluation to detect various types of dna damage and mutations (rearrangement, point mutations, small insertions or deletions of dna and polyploidy changes) which suggests that rapd bands may potentially form the basis of novel biomarker adv. hort. sci., 2016 30(1): 13-21 doi: 10.13128/ahs-18697 dna changes in cotton (gossypium hirsutum l.) under salt stress as revealed by rapd marker b. saleh department of molecular biology and biotechnology, aecs, p.o. box 6091, damascus, syria. key words: cotton, genomic template stability, rapd, salt stress. abstract: random amplified polymorphic dna (rapd) analysis was applied to evaluate dna changes among four upland cotton (gossypium hirsutum l.) varieties [niab 78 (n78), deir-ezzor 22 (de22), deltapine 50 (dp50) and aleppo 118 (a118)] grown under non-saline conditions (control) and salt stress (200 mm nacl) for seven weeks. changes in rapd profiles were measured as genomic template stability (gts%). the highest estimated gts% value was recorded for the two sensitive varieties, dp50 (79.1%) followed by a118 (58.2%); whereas, the lowest value was recorded for the two other tolerant varieties de22 (36.7%) followed by n78 (26.4%). based upon the data presented, rapd marker could be used as potential tool for early identification of cotton tolerance to salt stress. (*) corresponding author: ascientific@aec.org.sy received for publication 28 may 2015 accepted for publication 20 december 2015 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 13-21 14 assays for the detection of dna damage and mutations in the cells of bacteria, plants, and animals (savva, 1998; atienzar et al., 1999; tanee et al., 2012). it is well documented that genomic template stability ratios (gts) were calculated. gts implies qualitative measure reflecting changes in rapd profiles. changes in rapd and profiles were expressed as reductions in gts in relation to profiles obtained from control samples (gupta and sarin, 2009; aly, 2012; dojan et al., 2012; tanee et al., 2012). therefore, this investigation aimed to detect dna changes induced by nacl application by monitoring the rapd profiles of control and stressed plants in four upland cotton (gossypium hirsutum l.) varieties grown in syria. 2. materials and methods plant materials and growth conditions two local varieties were selected on the basis of their wide-ranging tolerance towards salinity: deirezzor 22 (de22) as salt-tolerant and aleppo 118 (a118) as salt-sensitive variety (saleh, 2011). these two varieties were compared with two introduced cotton varieties, niab 78 (n78) (known as salt-tolerant) and deltapine 50 (dp50) (known as salt-sensitive) under 0 and 200 mm nacl for seven weeks. seeds of upland cotton (g. hirsutum l.) were provided by the general commission for scientific agricultural research of syria (gcsar). seeds were soaked in distilled water for 24 h and then planted in pots filled with a 1:2 (v/v) mixture of perlite:peatmoss. germination was carried out in a greenhouse at 18°c, 12 h photoperiod, and relative humidity of 80%. seedlings were allowed to grow in a greenhouse under controlled conditions (temperature 25°c, 12 h photoperiod, and relative humidity 80%). seedlings were irrigated with tap water for one week before the initiation of nacl treatments. salt stress application was carried out by adding nacl (200 mm) to the water. plants were irrigated twice a week with water with or without salt. all solutions were changed twice a week. the same environmental conditions were maintained during the experiment. the experiment (five replicates/treatment) was carried out in the greenhouse for seven weeks. genomic dna extraction plant genomic dna was extracted from young leaves (bulk of five plants/variety) including the control and stressed plants (200 mm nacl) using ctab (cetyltrimethylammonium bromide) protocols described by doyle and doyle (1987) with minor modifications. leaf tissue (150 mg) was ground in liquid nitrogen and the powder was transferred to a 2 ml eppendorf tube, mixed with 900 μl of extraction buffer (100 mm tris-hcl, ph 8.0, 1.4 m nacl, 20 mm edta, 0.0018 ml β-mercaptoethanol, 2% ctab), and incubated at 65°c for 20 min. one volume of a chloroform:isoamyl alcohol mix (24:1, v/v) was added and centrifuged at 12,000 g for 10 min at 4°c. the aqueous phase was transferred to a fresh tube, and the dna was precipitated with an equal volume of cold isopropanol and kept at -20°c for 10 min. it was then centrifuged at 12,000 g for 10 min at 4°c, the supernatant was discarded, and dna was spooled out and washed with 1 m ammonium acetate and 100% ethanol. the cleaned dna pellet was air dried and dissolved in 100 μl of 0.1x te buffer (1 mm tris-hcl, 0.1 mm edta, ph 8.0). finally 5 μl of rnase (10 mg ml-1) were added and incubation for 30 min at 37°c was applied. dna concentration was quantified by dna spectrophotometer at 260/280 nm and adjusted to final concentration of 10 ng μl-1. dna was stored at -80°c until needed. rapd marker twenty-three rapd primers from operon technologies inc. (usa) and three primers from the university of british columbia were tested to detect dna changes in stressed plants, and their respective controls, for four cotton varieties. rapd marker was performed as described by williams et al. (1990) with a minor modification. pcr amplification reaction was carried out in 25 μl reaction volume containing 1xpcr buffer, 2 mm mgcl2, 0.25 mm dntps, 25 pmol primer, 1.5 u of taq dna polymerase and 30 ng template dna. pcr amplification was performed in a t-gradient thermal cycler (bio-rad; t gradient) programmed to fulfill 42 cycles after an initial denaturation cycle for 4 min at 94°c. each cycle consisted of a denaturation step (1 min at 94°c), an annealing step (2 min at 35°c), and an extension step (for 2 min at 72°c). a final extension cycle was performed for 7 min at 72°c. the pcr products were separated on a 1.5% ethidium bromidestained agarose gel (bio-rad) in 0.5xtbe buffer. electrophoresis was performed for 3 h at 85v and visualized with a uv transilluminator. band sizes were determined by comparison with a 1 kb dna ladder mix, ready for use (fermentas). saleh dna changes in cotton under salt stress 15 rapd data analysis and genomic template stability (gts) estimations dna changes induced in treated plants compared to their respective controls were screened by rapd assay. the polymorphism was calculated in relation to the appearance of new bands and disappearance of bands in treated plants, compared to control band patterns. genomic template stability (gts%) was calculated as follows: gst% = (1a/n) x 100 where (a) is the average number of changes in the dna profile and (n) the number of total bands in the control. polymorphism observed in rapd profiles included disappearance of a normal band and appearance of a new band in comparison to the cotrol rapd profiles (atienzar et al., 2002). 3. results a set of 26 random10-mer primers was used to detect the dna changes among four cotton varieties under salt stress application compared to their respective controls. rapd fragment sizes ranged from 200 to 3000 bp. the generated band characteristics for the four varieties (including control and stressed plants) are summarized in table 1. the total number of characteristic bands (common observed bands in control and stressed plants for the four examined varieties) was 29. the amplification products produced from 26 rapd primers are listed in table 2 in terms of loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately. the rapd analysis carried out on the four cotton varieties produced a number of distinct fragments which varied according to each tested primer. twelve of the 26 rapd primers (opa02, opb05, opc08, opd08, opd20, opj07, opk13, opk17, opr12, opy10, ubc132 and ubc159) produced polymorphic bands under saline conditions for the four tested varieties (table 2). figure 1 shows the amplification products using opa02, opb17 and opy10 rapd primers with template dna from the four varieties under control and saline conditions (200 mm nacl). changes in dna pattern induced by nacl treatment in the four tested cotton varieties were detected based on estimated genomic template stability (gts%) (table 3). in this respect, it was found that the highest gts% was recorded in salt-sensitive cotton, whereas the lowest was found among salt-tolerant varieties (table 3). 4. discussion and conclusions detection of dna changes in cotton via salt stress was assessed using rapd marker system. as shown in table 1, characteristic bands ranged between 0 (opb17, opd20, ope15, opj01, opj07, opk12, opk13, opk17 and ubc132) and 2 (opc08 and opt18), whereas the highest number (three) was yielded by opa04, ope07, opg11, opq01 and opq18 rapd primers (table 1). our findings reveal that nine out of the 26 tested rapd primers generattable 1 characteristic bands identified for the four tested cotton varieties using 26 rapd primers primer name sequence (5' 3') characteristic bands (number and size) opa02 tgccgagctg (1) 950 opa04 aatcgggctg (3) 250, 550 & 1600 opb05 tgcgcccttc (1) 1500 opb17 agggaacgag 0 opc08 tggaccggtg (2) 500 & 800 opc13 aagcctcgtc (1) 450 opd08 gtgtgcccca (1) 1200 opd20 ggtctacacc 0 ope07 agatgcagcc (3) 550, 650 & 800 ope15 acgcacaacc 0 opg11 tgcccgtcgt (3) 700, 1200 & 2100 opj01 cccggcataa 0 opj07 cctctcgaca 0 opk12 tggccctcac 0 opk13 ggttgtaccc 0 opk17 cccagctgtg 0 opq01 gggacgatgg (3) 450, 600 & 1000 opq18 aggctgggtg (3) 650, 750 & 1200 opr09 tgagcacgag (1) 250 opr12 acaggtgcgt (1) 300 opt18 gatgccagac (2) 1200 & 1350 opw17 gtcctgggtt (1) 2000 opy10 caaacgtggg (1) 650 ubc132 agggatctcc 0 ubc159 gagcccgtag (1) 1600 ubc702 gggagaaggg (1) 400 total 29 adv. hort. sci., 2016 30(1): 13-21 16 table 2 markers identified by 26 rapd primers for the four tested cotton varieties under salt stress compared to their respective controls. dna changes induced by nacl treatment using rapd marker as described by loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately primer name n78 de22 dp50 a118 total polymorphic bandsc t c t c t c t opa02 9 8 9 9 ₋ (4) 400, 650, 1800 & 2500 (3) 650, 800 & 2500 (2) 1800 & 2500 (2) 1800 & 2500 16 ₊ (2) 600 & 1500 (3) 500, 600 & 900 nd nd opa04 3 3 3 3 ₋ nd nd nd nd 5 ₊ (3) 400, 500 & 1000 (2) 400 & 500 nd nd opb05 5 5 4 5 ₋ (4) 450, 650, 1000 & 2000 (3) 450, 1000 & 2000 (1) 900 (1) 2000 14 ₊ (3) 500, 700 & 1100 (2) 500 & 1100 nd nd opb17 7 9 8 8 ₋ (5) 600, 700, 1100, 1200 & 1800 (4) 350, 800, 1500 & 1800 nd (5) 400, 600, 800, 1000 & 1800 20 ₊ (2) 400 & 650 (2) 700 & 1100 nd (2) 450 & 750 opc08 4 5 5 4 ₋ (2) 1850 & 1900 (1) 1200 (1) 1850 nd 10 ₊ (2) 300 & 900 (2) 1350 & 1900 nd (2) 1850 & 1900 opc13 6 6 5 5 ₋ (1) 1000 (2) 500 & 1000 nd nd 12 ₊ (5) 300, 800, 900, 1100 & 1350 (4) 300, 1100, 1350 & 1500 nd nd opd08 4 3 4 3 ₋ (2) 450 & 700 (2) 450 & 750 (1) 900 (2) 450 & 800 15 ₊ (2) 650 & 1350 (4) 500, 600, 800 & 1350 (1) 800 (1) 600 opd20 6 4 2 2 ₋ (4) 650, 850, 1100 & 1850 (3) 700, 900 & 1350 (1) 800 (1) 300 13 ₊ (1) 1350 (1) 800 (1) 300 (1) 200 ope07 8 8 8 8 ₋ (5) 1000, 1300, 1800, 2300 & 3000 (1) 1800 nd nd 7 ₊ (1) 1100 nd nd nd ope15 5 4 6 6 ₋ (1) 200 (2) 800 & 1800 nd nd 8 ₊ (4) 800,1300, 1500 & 1800 (1)1500 nd nd opg11 3 3 3 3 ₋ nd nd nd nd 2 ₊ (1) 600 (1) 600 nd nd opj01 7 6 7 7 ₋ (5) 400, 600, 850, 1200 & 2000 (3) 400, 600 & 850 nd (2) 600 & 2000 20 ₊ (4) 350, 500, 950 & 1800 (4) 350, 500, 950 & 1800 nd (2) 500 & 800 opj07 3 4 2 3 ₋ (3) 500, 800 & 1600 (4) 300, 500, 700 & 1600 (2) 550 & 1350 (1) 1100 21 ₊ (3) 700, 900 & 1200 (3) 550, 1000 & 1200 (2) 450 & 1600 (3) 450, 550 & 650 to be continuedt(-) loss bands, (+) gains bands, (nd) no differences. saleh dna changes in cotton under salt stress 17 primer name n78 de22 dp50 a118 total polymorphic bands c t c t c t c t opk12 3 3 2 2 ₋ (3) 700, 900 & 1350 (3) 800, 900 & 1350 nd nd 11 ₊ (3) 850, 950 & 1100 (2) 500 & 1000 nd nd opk13 4 4 3 3 ₋ (2) 650 & 1100 (2) 350 & 1100 (1) 200 (1) 490 20 ₊ (4) 450, 750, 1200 & 1350 (4) 500, 750, 1200 & 1350 (3) 300, 500 & 1100 (3) 700, 1200 & 1350 opk17 8 4 4 6 ₋ (4) 400, 1500, 1850 & 2500 (1) 1850 (2) 700 & 1000 (4) 300, 400, 1000 & 1850 19 ₊ (3) 300, 350 & 800 (2) 350 & 1200 (2) 500 & 1500 (1) 1500 opq01 9 4 4 4 ₋ (5) 300, 800, 1100, 1200 & 1800 (1) 1100 nd nd 10 ₊ (3) 200, 700 & 900 (1) 300 nd nd opq18 7 3 6 6 ₋ (4) 650, 900, 1350 & 1500 nd nd nd 9 ₊ (2) 550 & 1600 (2) 1500 & 1600 nd (1) 1600 opr09 6 3 4 4 ₋ (4) 400, 550, 1100 & 3000 (1) 600 nd (1) 600 9 ₊ (2) 600 & 900 (1) 1000 nd nd opr12 11 10 7 7 ₋ (6) 200, 450, 600, 1100, 2100 & 2250 (6) 250, 500, 800, 900, 1350 & 2250 (1) 900 (3) 200, 1200 & 1350 28 ₊ (4) 250, 500, 900 & 1800 (4) 700, 1000, 1100 & 1800 (2) 1350 & 2250 (2) 450 & 1000 opt18 5 8 10 10 ₋ (1) 650 (2) 1800 & 2000 nd nd 9 ₊ (4) 400, 500, 1000 & 2250 (1) 950 nd (1) 2000 opw17 5 4 3 3 ₋ (2) 600 & 1200 (3) 300, 900 & 1500 nd nd 17 ₊ (5) 450, 500, 800, 1350 & 1500 (4) 300, 450, 500 & 800 nd (3) 250, 450 & 800 opy10 4 5 4 4 ₋ (1) 3000 (2) 1500 & 3000 (1) 400 (3) 400, 900 & 2500 12 ₊ (3) 400, 850 & 2500 (2) 400 & 2500 nd nd ubc132 5 5 4 5 ₋ (4) 1000, 1350, 1500 & 3000 (3) 550, 600 & 3000 (1) 1200 (3) 400, 1000 & 1200 17 ₊ (3) 600, 1200 & 1800 (3) 1000, 1350 & 1500 nd nd ubc159 3 6 3 3 ₋ (1) 500 (2) 1350 & 2000 (2) 500 & 700 (2) 650 & 2000 19 ₊ (5) 550, 700, 950, 1100 & 1350 (3) 300, 700 & 900 (2) 450 & 600 (2) 500 & 700 ubc702 3 3 3 3 ₋ (1) 800 (2) 750 & 800 nd nd 7 ₊ (2) 600 & 2500 (1) 2500 nd (1) 2500 table 2 (continued) adv. hort. sci., 2016 30(1): 13-21 18 ed no characteristic bands for the for tested cotton varieties (table 1). it is worth noting that primer opr12 identified more polymorphisms (28) than any other primer tested (ranging between two for primer opg11 and 21 for primer opj07) (table 2). whereas, the banding patterns produced by primers opa04, opc13, ope07, ope15, opg11, opk12 and opq01 were not polymorphic for varieties dp50 and a118 (table 2). another investigation demonstrated varietal variation in salt tolerance among these cotton varieties based on various examined physiological indices (saleh, 2011). according to the study, the de22 variety could relatively be classified as salt tolerant variety to other tested varieties. dojan et al. (2012) reported the potential of rapd markers for the detection of dna damage induced by nacl in cotton. likewise, the rapd marker has the potential to be applied in environmental pollution detection, e.g. gupta and sarin (2009) applied the same marker to detect pollution by cadmium (cd) in two aquatic plants. zhou et al. (2011) also used rapd bands to indicate dna damage in euplotes vannus (protozoa, ciliophora) induced by nitrofurazone in marine ciliates. previously, aly (2012) used the same marker for genotoxic effect detection of cd stress on egyptian clover and sudan grass plants. changes in rapd profiles were also measured as genomic template stability (gts) and the data suggest noticeable genomic template instability (table 3). reduction in gts values was observed under salt stress, compared to their respective controls for the four tested varieties (table 3). similarly, genetic instability induced by nacl treatment of cotton was reflected by changes in rapd profiles: disappearance fig. 1 rapd banding profiles generated by opa02, opb17 and opy10 primers in the four tested cotton varieties showing dna changes induced by nacl application for seven weeks, c: control, t: treated plants. m: 1 kb dna ladder mix, ready for use. primer name n78 de22 dp50 a118 control 200 mm nacl 200 mm nacl 200 mm nacl 200 mm nacl opa02 100 33.3 25 77.8 77.8 opa04 100 0 33.3 100 100 opb05 100 40 0 75 80 opb17 100 0 33.3 100 12.5 opc08 100 0 40 80 50 opc13 100 0 0 100 100 opd08 100 0 100 50 0 opd20 100 16.7 0 0 0 ope07 100 25 87.5 100 100 ope15 100 0 25 100 100 opg11 100 66.7 66.7 100 100 opj01 100 28.6 16.7 100 42.9 opj07 100 100 75 100 33.3 opk12 100 100 66.7 100 100 opk13 100 50 50 33.3 33.3 opk17 100 12.5 25 0 16.7 opq01 100 11.1 50 100 100 opq18 100 14.3 33.3 100 83.3 opr09 100 0 33.3 100 75 opr12 100 9.1 0 57.2 28.6 opt18 100 0 62.5 100 90 opw17 100 40 75 100 0 opy10 100 0 20 75 50 ubc132 100 40 20 75 40 ubc159 100 100 16.7 33.3 33.3 ubc702 100 0 0 100 66.7 mean 100 26.4 36.7 79.1 58.2 table 3 genomic template stability (gts%) estimated by 26 rapd primers for the four tested cotton varieties under salt stress compared to their respective controls saleh dna changes in cotton under salt stresss 19 of bands and appearance of new bands occurred in the profiles in comparison to those of the controls (fig. 1, table 3). our data supports the suggestion by dojan et al. (2012) that detected dna changes, induced by nacl, using rapd marker could be explained as previously reported by atienzar et al. (1999). it has been demonstrated that dna damage levels could be reflected in gts (atienzar et al., 1999). the later investigation suggested that the loss of bands may be attributed to genomic rearrangements or to point mutations causing alterations in oligonucleotide priming sites, while appearance of new bands could be related to the presence of oligonucleotide priming sites which become accessible to oligonucleotide primers after structural changes (dna mutation, deletions or homologous recombination). table 3 reveals that gts% values decreased with salt application for the four tested varieties. our data show that the highest estimated gts value was recorded for the two sensitive varieties, dp5 (79.1%) followed by a118 (58.2%); whereas, the lowest was recorded for the two tolerant varieties, n78 (26.4%) followed by de22 (36.7%) (table 3). gupta and sarin (2009) reported that the genomic template stability test was significantly affected by heavy metal stress, while aly (2012) reported that gts values decreased obviously with an increase in cadmium (cd) concentration in egyptian clover and sudan grass seedlings. on the other hand, tanee et al. (2012) used gts to identify the vanda species (orchidaceae) of thailand. our results are in accordance with dojan et al. (2012) who reported that there is positive correlation between gts and other parameters (stem and leaf growth and stem length) under nacl stress in cotton. in this respect, the estimated gts values in the current investigation were positively correlated with various physiological indices (biomass and leaf k+/na+ ratio) tested under nacl application in cotton (saleh, 2011). moreover, a positive relationship was also determined between gts values and recent findings (saleh, 2013) based on physiological indices (relative water content, osmotic potential and salt tolerance index) among the same tested varieties (dojan et al., 2012). overall, the lowest estimated gts values combined with the highest polymorphism level recorded for the tolerant varieties (n78 and de22) (where, % polymorphic level was 68.5, 60.9, 21.3 and 36.4% for n78, de22, dp50 and a118, respectively exposed to 200 mm nacl for 7 weeks, using the same marker) could explain their salinity tolerance compared to the other tested varieties. however, the lowest estimated gts value recorded for n78 and de22 varieties could be attributed to genetic variation, inducing new protein in relation to salinity tolerance. it has been successfully demonstrated that environmental constraint induced variation in dna methylation pattern as a developed epigenetic mechanism after exposure to abiotic stress (zhong and wang, 2007; peng and zhang, 2009). our findings could be supported by the data provided in zhong and wang (2007), where genotyping variation in wheat (triticum aestivum l.) cultivar salinity tolerance was reported. in this respect, the study mentioned that the salt-sensitive wheat variety had a lower methylation rate compared to salt-tolerant ones. recently, saleh (2013) reported that n78 and de22 varieties showed a better protection mechanism against salinity damage than the other tested varieties, demonstrating variation in salt tolerance among cotton varieties based on physiological indices. likewise, in the same investigation comparing the protein profiles between control plants and those salts treated using sds-page showed protein changes under salt treatment compared to their respective control. in this respect, the expression of ~19, ~21 and ~26 kda for n78 and ~21 kda protein for de22, was highly increased by salt treatment, indicating that it could play a role in salt stress response. on the other hand, newly synthesized protein of ~30 kda was recorded for both de22 and n78 varieties under saline treatment which was not observed in their respective controls. the other two tested varieties (dp50 and a118) showed decreases in the same protein bands (~19, 26 and 34 kda) under saline conditions, with respect to their respective controls, reflecting their sensitivity to salt stress. salinity promotes the synthesis of salt stress-specific proteins; many of these proteins were suggested to protect the cell against the adverse effect of salt stress. accumulation of these proteins is a common response to salt stress (kong-ngern et al., 2005; metwali et al., 2011). it is worth noting that identified bands in de22 and n78 (salt-tolerant varieties), which were not amplified in salt-sensitive varieties (dp50 and a118), could be related to gene(s) involved in salinity tolerance. these findings are in accordance with kurup et al. (2009). adv. hort. sci., 2016 30(1): 13-21 20 dna variation could be exploited in plant breeding programs to improve salinity tolerance in germplasm. overall, the lowest estimated gts values for nb78 and de22 varieties (known as salt-tolerant) reflect their highest polymorphic values. based on the current results, the rapd marker was useful to establish specific dna markers associated with nacl stress. therefore, the rapd marker could be used as useful tool in plant breeding programs for early identification of cotton tolerance to salt stress. acknowledgements the author would like to thank prof. othman i, director general of aecs and prof. mirali n, head of molecular biology and biotechnology department for their support, and also to the plant biotechnology group for technical assistance. references aly a.a., 2012 application of dna (rapd) and ultrastructure to detect the effect of cadmium stress in egyptian clover and sudan grass plantlets. j. stress physiol. & biochem., 8: 241-257. 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rapd to detect dna damage induced by nitrofurazone in marine ciliate euplotes vannus (protozoa, ciliophora). aquat. toxicol., 103: 225-232. 170 1. apricot in apricot, growth habit and fruiting behavior are strongly inter-related. accordingly, the varieties of apricot can be classified into five groups (guerriero and xiloyannis, 1975 a; bassi et al., 2003). since some changes in shoot morphogenetic gradient during rest period were evident in response to dormancy and chilling (guerriero and scalabrelli 1982), any classification of branch habit should be strictly associated with a specific environment. depending on the type of bud (floral or vegetative) and shoot (sylleptic, long brindle, brindle or spur) the chilling requirement may be very different and, eventually, can be a factor in regulating branch habit and fruiting behavior (guerriero and xiloyannis 1975 b; guerriero and viti, 1997). to decide how to manage pruning, the shoot should be identified by its specific growth rate after bud break. for this purpose, its physiological behavior should be constantly monitored. there is some evidence that the emergence of sylleptic shoots is highly probable when a threshold level of growth rate is exceeded (zucconi, 2003). this means that in some conditions apical dominance is not able to inhibit the growth of lateral meristems, which thus originate sylleptic (anticipated) shoots rather than buds (fig. 1). generally, the buds (once formed) become rapidly dormant (fig. 2) and will only grow in the following spring after a specific amount of chilling, and as a result they will originate proleptic shoots. it is also possible that, depending on growing conditions, sylleptic shoot formation can take place more than once (fig. 1) along the shoot growth. thus buds that are formed on sylleptic shoots at different times compared to proleptic ones may have a different fruiting performance and time of flowering: this is frequently reported by growers. consequently the date of pruning, whether during the vegetative period (summer pruning, early or late) or during the winter before bud break, can have a strong influence in controlling the fruiting of different varieties. moreover, the knowledge of how the shoots grow is very important spring and summer pruning in apricot and peach orchards d. neri, f. massetani dipartimento di scienze agrarie, alimentari e ambientali, università politecnica delle marche, via brecce bianche, 60131 ancona, italy. key words: catalonian open vase, early and late pruning, plant architecture, shoot heading back, shoot thinning. abstract: spring and summer pruning are based on the possibility to manipulate the physiological control of axillary sylleptic growth and carbon allocation in the shoot through alteration of the apical dominance and light distribution in the canopy. the practical result in modern orchards is a higher flower bud differentiation for apricot and an easier training system maintenance for peach with more efficient use of labor. cultivated apricot varieties show diverse tree architectures, habit and fruiting branches. the effect of pruning intensity at different times during spring and summer seasons is specific for the singular growth habits. differences among the peach varieties are less evident than in the apricot. the ease with which peach water sprouts produce axillary sylleptic shoots makes the use of mechanical topping possible in the first two years of intense growth in order to train the tree as a bush and then to open it as a vase with manual pruning (catalonian open vase). in all modern peach orchards, pruning in late summer results very useful to obtain a better light distribution in the canopy and a more efficient carbon allocation to fruiting shoots, preventing and reducing the need for winter pruning. adv. hort. sci., 2011 25(3): 170-178 received for publication 9 june 2011 accepted for publication 17 september 2011 fig. 1 shoot growth rate and sylleptic shoot formation in apricot. this model assumes that the growth rate threshold for inducing sylleptic growth decreases during the season. left: the threshold was overcome only at the end of the season. right: the critical growth rate was overcome twice in the season. 171 for effective pruning, improving the chance of acclimatizing a cultivar that may be productive in a specific environment. pruning for apricot should be modulated both in intensity and timing according to the interaction between the variety and its environment. finally, the specific shoot physiology and architectures of fruiting branches of apricot varieties will determine very dissimilar regimes for pruning. for this reason there should be in each growing area a classification of the varieties, according to their precise branch habit, fruiting behavior and need for pruning. the most common classification in northern italy is arranged into three groups: a with very vigorous and spreading habit, and a tendency to fruit on spurs, brindles and sylleptic shoots; b with less vigorous, assurgent, or semi-spreading habit, fruiting on spurs and vigorous shoots; c with very vigorous, assurgent, or mixed spreading habit, and ability to fruit on all kinds of shoot (neri, 2003; pirazzini, 2004; neri et al., 2010). therefore, for each apricot variety, it is important to predict the response (in terms of the number and type of lateral shoots) to head back pruning of shoots and branches in different periods of the spring and summer seasons. pruning intensity and cultural techniques (fertilization, irrigation, soil management, and eventually forcing and protection conditions) play an extraordinary role in determining the final result and the possible optimal training system (neri et al., 2011). spring shoot heading back and thinning the intensity of head back pruning of growing shoots can be performed within these two extremes: short pruning (leaving half of the shoot or only the basal portion of it with three to five buds, as a spur) and long pruning (which reduces the apical portion of the shoot by pinching or cutting a few centimeters below the tip). generally, these pruning techniques are limited to the spring with fast growing shoots. the time of pruning can be intended as early or late spring pruning, in which the early pruning induces the formation of long sylleptic shoots, while the latter induces nil growth or the formation of few, short, sylleptic brindles with, likely, a higher flower differentiation aptitude. after spring heading back, shoot vigour is strongly reduced and the number of sylleptic shoots is generally increased. short head back pruning was generally less effective than long heading back in inducing flowering brindles, with the exception of weak varieties which need to improve shoot growth. the response to pruning is always higher in fertile and irrigated soil. apparentely the pruning in late spring induces a better response if it is limited to the terminal part of the long shoot (long pruning) (fig. 3). delaying spring pruning (late spring pruning) reduces the number of sylleptic shoots per single cut and also the fig. 2 left: apical dominance intensity along the growing shoot in early spring. centre: apical dominance and bud dormancy intensity along the growing shoot in late spring. right: apical dominance and bud dormancy intensity along the shoot in late summer. fig. 3 number of sylleptic shoots per single cut in response to short and long pruning delaying the operations from early to late spring. 172 flowering intensity. flowering intensity is higher when pruning is applied in early spring (may) in different varieties (fig. 4), probably because more sylleptic brindles were produced. apparently late pruning in june induces a better response if it is performed on the terminal part of the shoot (long pruning). shoot thinning is generally carried out a few weeks before harvest to improve fruit quality in very vigorous trees. it is devoted to eliminating overcrowded and malpositioned shoots. the final goal is to have better light distribution inside the canopy and less carbon directed to water sprouts and suckers which cannot be used for fruit production in the future management of the branches. summer shoot heading back and thinning summer shoot head back pruning aims to increase flower differentiation but in apricot this is possible only if there is new shoot growth, which can be induced by water supply after summer drought or by heavy cuts such as late summer heading back. summer shoot thinning can be performed with the aim of improving the quality of shoots as a consequence of better light penetration and carbon allocation. this practice reduces the need for winter pruning and can be useful in areas where frost damage may challenge flowering and therefore winter pruning must be delayed until after fruit set. different pruning intensity is meant to stimulate more vegetative vigour when the shoot is suddenly cut very short, but to induce the formation of flowering brindles when it remains quite long (fig. 5). under northern italian continental climate, the vegetative response to pruning is always greater in fertile and irrigated soils, resulting in greater shoot vigour. finally, sylleptic shoots tended to bloom later than the rest of the plant (a very important advantage in climates where late frost is common) but to bear fruits of small size, at least in some varieties (pirazzini, personal communication). varietal differences in response to spring heading back pinkcot. early short heading back induces numerous, equally balanced and productive sylleptic shoots. sylleptic shoot growth is very active when it is stimulated on vigorous shoots (water sprouts), which by the end of the season are well ramified. at blooming the number of flowers on sylleptic shoots is higher with long heading back than with short. short heading back resulted in more uniform distribution of brindles and spurs along the original branch. sweetcot. growth is greater after early shoot heading back: nevertheless only few sylleptic shoots were formed, they are not too long and the flower number is increased. in non-irrigated soils late pruning does not improve shoot ramification, while growth is short and rich in flowers. robada. in fertile soil, early pruning generally induces a few sylleptic shoots, often only one as an extension growth from the terminal bud, even though the number of flowers on all type of shoots is high. with late pruning there is a certain number of sylleptic shoots (brindles and fig. 4 flowering estimated entity in 8 varieties in response to pruning applied in may (early pruning), or in june. fig. 5 orange rubis forms shoots after early long-heading back (left): a high number of sylleptic shoot with good flower differentiation is formed. shoot after late short-heading back (right): sylleptic growth and flower differentiation is visible. 173 spurs) but fewer flowers per shoot. in non-irrigated soil, short heading back does not induce any good growth, neither for shoots nor flowers. orange rubis. long early shoot heading back is very effective in stimulating sylleptic growth with high flower differentiation, while short heading back is useful only with weak shoots. heading back of water sprouts, especially when late, does not favor flowering formation. kioto. the number of brindles and spurs is dramatically increased by early pruning, with very high flower differentiation along the old wood. late pruning is positive only on vigorous lateral shoots. flower differentiation is good in all the shoots after early pruning, and intermediate in sylleptic brindles in vigorous shoots after late pruning. flavorcot. both short and long early pruning induce weak growth in comparison with the vigorous cultivars; the sylleptic shoots are limited in number and growth even in water sprouts. late pruning does not induce good sylleptic ramification, but the flower induction is enhanced in brindles and spurs in all the plants. after early pruning, flowers are scarce in all the sylleptic shoots; after late pruning, flower production is much better in the old wood and in sylleptic brindles. zebra. early long head back pruning induces positive sylleptic shoot growth; short heading back is less effective even in water sprouts; flower differentiation confirmed this result. late heading back was negative and induced only few flowers. pieve. early heading back is generally positive and, especially with long pruning, the number of sylleptic brindles is higher and flower differentiation is good. late pruning reduces the branching of shoots, and induces a very limited number of flowers. pisana. for this low fertile variety, it is worth noting that the terminal shoot on the intact branch showed less growth than the second one below, as opposed to the heading back causing the terminal shoot to become the most vigorous of the branch. this means that in low fertility conditions the varieties of this group need to be stimulated by winter pruning instead of weakened by summer pruning. bella di imola. this variety shows very high productivity on the one-year shoot, the terminal portion being the most productive. growth was greater at the terminal position of the branch as well, revealing a much stronger acrotony than pisana, and greater vigor. for this reason it is important to avoid any pruning which induces a vegetative response which is too strong. in fact in orchards with low fertilization, growth was not excessive even with winter pruning; flower differentiation was high in any case. it can be hypothesized that in more fertile soils vigor can be too strong, and so late summer pruning can be widely utilized (neri et al., 2010). pruning in different training systems for apricot actual training systems for apricot are specific for each production area. the two most widely diffused training systems in the northern part of italy are free open vase (with several variations, from delayed open vase to bush) for low density, hilly orchards and spindle for high density systems in flat fertile lands with low vigorous rootstocks. the date and intensity of pruning effectively determines the branch architecture and fruiting potential of each cultivar. these observations lead us to conclude that for apricot, summer pruning is a basic practice in modern orchards but it must be adapted to local conditions and genetic material. shoot physiology, theoretically modeled on the basis of growth rate, can help in choosing the best period and most effective intensity for the pruning of each new cultivar in the different training systems of a particular growing area. we can generalize that summer pruning reduces vigor and induces greater flower production. early long shoot heading back is more effective with high vigor varieties and fertile soil conditions, whereas short shoot and branch head back pruning is favorable for weak and spreading varieties, although the latter habit could be more easily controlled by winter pruning than the former. varieties of group a, such as some of the new varieties, benefit from early summer pruning (early heavy shoot heading back) in order to induce the formation of sylleptic shoots; and summer pruning (without heading back but possibly with shoot thinning) to encourage shoots to be more lignified. group b performs best after winter pruning (shoot thinning and heading back of twoto three-year-old branches). group c may be pruned in late summer or at the end of winter (shoot thinning, heading back the branches), depending on local growing conditions. in order to limit the development of sylleptic shoots, which only bear small fruits, it is better to carry out heading back in late summer instead of in the winter. in any case, every pruning strategy must be tested on each variety before it is adopted throughout commercial orchards. this is due to the possible very specific influence of varietal differences in chilling requirements, and specific shoot and flower differentiation physiology. 2. peach in modern peach orchards, application of spring and summer pruning is increasing (from 20% up to 60% of the total amount of pruning), depending on the training system, production area and farm management (giovannini et al., 2010). peach shows good ability to form sylleptic shoots and strong epinastic control that makes the sylleptic shoot insertion angles wider moving from the top to the basal part of the shoot. these features require an appropriate shoot pruning technique and finally, if well managed, lead to dwarf the tree with the open habit of mature peach plants. during training of modern intensive orchards, spring pruning is therefore applied more than summer pruning (and obviously of winter pruning), in order to address the inclination of vigorous growing shoots and to anticipate formation of the skeleton structure of the canopy. the re174 moval of mal-positioned water sprouts and stimulation of a higher number of well positioned shoots (ferree et al., 1984; lanzellotti et al., 1998) finally dwarf the trees (kappel and bouthillier, 1995; hossain and mizutani, 2008). the second goal of a greater use of spring and summer pruning is to reduce the vegetative unproductive phase and enhance early bearing in all new training systems (giovannini et al., 2010; neri et al., 2010). summer pruning is applied to shorten the not-fruiting initial phase, improve light distribution on fruiting shoots and strengthen the future scaffold branches with a higher number of fruiting shoots (miller, 1987). nevertheless, pruning time in less intensive orchards is often determined by farm organization, depending on the availability of labor and arrangement of the working schedule, rather than on the plant physiology (chalmers et al., 1981; marini and barden, 1987; sansavini and neri, 2005). pruning can be also applied in the spring for biological reasons. in fact for some cultivars pruning intensity can be adjusted near blooming time, when flower buds enlarge and become more visible, depending on the quantity of buds that were damaged by frost during the winter. this kind of spring pruning can thus augment fruit set per tree. if the risk of frost damage is extended to blooming time, pruning can be carried out precisely during the fruit set period. in this case “winter pruning” is completely substituted by an early spring pruning which eliminates the excess shoots, based on the rate of fruit set, by heading back two-year-old branches. late spring pruning is commonly used for training, but in modern orchards it is not very common to control production if trees are mature and equilibrated. also early summer pruning, before harvest time, is used only to thin the water sprouts and to improve light distribution in the canopy, whenever the vigor is too high, to increase fruit color and quality and to prevent diseases. however when this pruning is too heavy or too early it can negatively affect fruit development. summer pruning after harvest can better manage excess vegetative growth and change the distribution of assimilates (rom and ferree, 1984; marini, 1985; mizutani et al., 1997; hossain et al., 2004). it improves bud differentiation and branch hierarchy organization. during summer, shortening branches results in a more regular sprouting in the following spring, with less vigorous shoots and high quality flower buds. in some cultivars, when it is necessary to cut big branches, the summer period is useful also because it induces a more rapid and efficient wound healing. this sort of pruning can also be considered when it is necessary to mitigate severe water stress (lopez et al., 2006; marsal et al., 2006). late summer pruning in august-september is important and widespread in all environments because it contributes to reduce canopy volume and allows shoot hardening. it partially prevents winter pruning (therefore it is called pre-winter pruning) and strongly reduces the need for it. furthermore, it is more selective than winter pruning as the best buds for fruit set are chosen in advance. summer pruning for peach training systems in mediterranean climate depending on the training system, both spring and summer pruning are applied to favor the branch inclination of shoots through pinching or cutting the upper part of the shoot to induce sylleptic ramification (fig. 6), also more than once per season as is done for the catalonian vase (monserrat and iglesias, 2011). removal of excess and mal-positioned shoots is also practiced to give a regular shape to the spindle and to the small open vase (neri et al., 2010). in any case, the pruning intensity is minimal and eventually some spring interventions are postponed from the first to the second year, and/or continued in the third if tree vigor is too weak. in mediterranean areas, with long growing season and early ripening cultivars, also vase training systems (i.e. low open vase) are commonly managed with the application of spring-summer pruning. in fact small vase formation can be improved using summer shoot cuts to direct vegetative growth to the well displaced lateral sylleptic and proleptic shoots. modern systems derived from the vase are characterized by a low scaffold (0.5 m above ground), low tree height (2.5 m), and free growth during the first years (bush type to enhance early bearing). fig. 6 catalonian vase during the first growing season. left: first manual topping when the shoot exceeds 100 cm from the soil. centre: second topping (manual or mechanical) when the shoots exceeds 150 cm from the soil. right: the final growth at the end of the first year (redrawn from monserrat and iglesias, 2011). 175 the catalonian vase, which originated in spain, is the most recent and wide-spreading vase system. spring-summer pruning is repeatedly employed to form and dwarf the trees during the first two years of training. mechanical topping is applied every 50 cm till the final size of the plant is reached (2.5 m) at the end of second year (figs. 6 and 7). topping involves removing a few apical buds per shoot, thus inducing suppression of apical dominance and increasing the possibility for growth of the external sylleptic shoots. this sets off a temporary strong competition among all growing shoots, but in peach the external shoots are privileged, and thus the main shoots are naturally oriented toward the external direction. finally, the whole plant height is lower but several shoots are well positioned to become the future branches of the vase. during the second year, or third in the case of low fruiting cultivars, the vase shape can be completed by thinning the primary branches and cleaning the central part, mainly in late summer. in mature orchards, pruning labor in this system can require less than 100 hr/ha (60% during vegetative season), and the fruiting winter pruning (the remaining 40%) completes the late summer pre-winter pruning (fig. 8). specific summer pruning is required also in the “y” trellis system. inclined branches with angles wider than 40-45° promote the growth of vertical shoots in the internal portion of the canopy and late spring and early summer pruning are necessary to remove them during the initial years. the high density planting of this system, the possibility to use long pruning, without eliminating shoot apex of primary branches, and the reliability of pruning during the vegetative season provide early fruiting. nevertheless, the “y” system is well performing only where climate conditions provide a high level of light, allowing the reduction of branch angles close to 30°, and thus reducing water sprout formation. this angle promotes a balanced vegetative growth and fruit production in all the lateral and basal portions of the two fruiting walls. “v” systems with double tree density and less vigorous rootstocks can be used to reduce further the need for spring and summer pruning. in this case, defining the two oblique productive planes is easier and more rapid but the cost of planting is very high and there is risk for overcrowding in the internal part of the canopy, which could induce an exponential increase in the need for pruning. spring and summer pruning in peach training systems for continental climate in the northern part of italy, where there are short growing seasons and high risk of frost in the spring, with midseason varieties high hedgerow systems (palmette and central leader, 4 m high) are still popular. this is because late spring frost may dramatically damage the production in the bottom part of the tree (first 2 m from the ground). in this condition pruning may be delayed after blooming (when fruit set is already complete). thus winter pruning becomes an early spring pruning, while early summer fig. 7 catalonian vase during the second growing season. left: first mechanical topping when the shoots exceed 200 cm from the soil. right: second mechanical topping when the shoots exceed 250 cm from the soil, and manual pruning to thin the main branches down to four to five in number, opening the centre in very late summer. this last pruning is done only if the variety has a very high productivity (redrawn from monserrat and iglesias, 2011). fig. 8 catalonian vase during the fourth growing season at blooming in the sibari area. the skeleton is completed and the plant is dwarf and equilibrated. 176 pruning is not frequent and limited to lightening the upper part of the canopy (if necessary). during the training period to reduce shoot vigor of the mal-positioned water sprouts, turning and partially crashing them can improve their fruiting aptitude. late summer pruning is used to maintain the shape of the trees and to increase lignification of the shoots that otherwise would be too shaded. the hedgerow made by trees trained as “u” or candelabras along the row requires less green pruning than palmette or central leaders because of less vigor of each vertical branch. for training high density peach orchards, well feathered scions from the nursery must be used to obtain fruit production in the second year. in mature orchards, green pruning is mandatory in two specific seasons: i) in early spring before harvest to eliminate water sprouts and to induce a higher number of productive shoots in well defined positions; after fruit set it is also possible to thin the shoots proportionally to the amount of fruit set; and ii) in late summer after harvest to anticipate the winter pruning (pre-pruning). in this case the water sprouts are eliminated and the vigorous shoots on the main branches are cut only if there is excess flower differentiation; the top part of the branches can be reduced to better permit light distribution in the canopy. if the prepruning in late summer is well executed with light shoot thinning, winter pruning can be avoided. in this way labor can be saved and/or better organized. in low bush open vase (delayed open vase) (sansavini and neri, 2005), for the first three-four years the training of the trees is free with only a few pruning cuts; green pruning is not important. late summer pruning becomes important in the third to fourth year to cut the central leader and to open the centre of the vase. in the fourth to fifth year, the main branches are headed back and the tree is completely formed as a vase. finally, pre-winter pruning is necessary to manage fruit shoot quality and quantity when production becomes important (starting from the third year). production pruning when the tree is well mature and fully formed, spring pruning is less important and must be carried out only in very specific cases when excess vigor of the growing shoots can compete with the growing fruits, interfering with the fruit quality and flower induction for the next year. peach production is located on one-year shoots (brindles, fruiting shoots and in some varieties also in water sprouts) and in a very limited quantity on the spurs (these are important only in clingstone peaches and in some nectarines with low fertile shoots). because of this specific fruiting behavior, it is very important to control shoot growth to form highly specific shoots in each variety (day et al., 1989). if the vigor is very high (generally in early ripening varieties) it is necessary to execute the first pruning before harvest in late spring in order to eliminate mal-positioned water sprouts and to improve light distribution in the canopy. whereas pruning in late summer is very helpful both in early and late ripening varieties to improve the quality of the fruiting shoots, favoring shoot hardening and carbon allocation. to improve light distribution in the canopy it is important to thin the shoots and to head back the branches. this pruning in late summer anticipates winter pruning, which consequently can be delayed at blooming to determine the final number of flowers per plant. winter pruning can even be eliminated and early spring pruning after fruit set can be applied to determine more precisely the number of fruits per plant. it is important to remember that avoiding heavy shading is important to obtain homogeneous distribution of the shoots along a branch. in fact when the shoots are shaded they can be damaged during winter and necrotize. because peach trees do not produce adventitious meristems and do not maintain latent buds for long, winter pruning is not able to recover new adventitious shoot growth and finally the shaded area of the canopy is lost. to avoid this dramatic loss of efficiency of the internal part of the canopy, springsummer pruning is mandatory in modern orchards. this problem is even more accentuated in high density planting systems in which early spring pruning can be associated with fruit thinning to reduce the impact of self shading and inter shading on shoot and fruit quality. traditional low density orchards under continental conditions are mainly pruned using precise winter pruning because once the open vase is well formed it provides high quality and constant fruit production. 3. conclusions in apricot each group of varieties has its own optimum season and intensity for pruning, according to fruiting aptitude and branch habit. for high density orchards, heavy late spring pruning may be used to reduce vigor and improve flower differentiation during the summer. for free open vase, use of late summer pruning only can be suggested to obtain a better carbon partitioning towards the fruiting shoots and a more uniform light distribution in the canopy. the higher the flower differentiation aptitude, the greater the possibility to use different seasons for pruning. with low aptitude, manipulation of spring growth of the shoots and light distribution in the canopy through springsummer pruning is mandatory. apricot varieties can be classified into groups characterized for different vegetative and reproductive habits, accordingly and depending on the fertility of the soil, they can be pruned in summer (if soil fertility is high) or in winter (if soil fertility is low). some of the new low productive varieties benefit from early summer pruning (early or late spring shoot heading back) to induce the formation of sylleptic shoots and to obtain good flower differentiation. weak varieties perform the best after winter pruning (branch heading back). other, very productive varieties may be pruned in late summer or at the end of winter (shoot or branch heading back) depending on local growing conditions (pirazzini, 2004). 177 in peach, habit is mainly influenced by assurgent growth in low chilling varieties that are well adapted to the mild mediterranean climate with low chill in winter, and by the widespread habit of the varieties, suitable for more continental climatic conditions with cold winters and tolerant to late frost in the spring. among these groups of varieties, flower aptitude is another factor which influences the type and intensity of spring and summer pruning. in modern peach orchards, late summer pruning is widely diffused as a common practice to manage light distribution and carbon allocation and finally shoot quality. this technique is applied in substitution or to reduce the amount of the winter pruning. late spring pruning is applied only if necessary when tree vigor is too high, while early spring pruning is used less and limited only to cases of unpredictable fruit set due to erratic climatic conditions. in any case, the labor for all kinds of pruning (spring, summer, and winter pruning) takes not more than 100 hours per hectare per year. with some very productive varieties and appropriate training systems (delayed open vase and free spindle) it is possible to reach an amount of labor for all the manual operations during the season of about 15 hours per ton of fruit (giovannini et al., 2010; neri et al., 2010). during training of catalonian vase in the first two years, spring and summer pruning can be mechanized with a moving machine to further reduce labor. late summer pruning may start in the second year for heavy producing varieties and in the third year for the less productive ones. in conclusion, spring and summer pruning increase the efficiency of labor (both for the ease and speed of the work and for the capability of the tree to rapidly compensate for errors and incorrect interventions) and improve fruit quality. late summer pruning can particularly improve modern orchard management efficiency. acknowledgements the authors wish to acknowledge the regione emilia romagna for financing part of the work (crpv 2008 impiantistica e gestione dei fruttiferi). references bassi d., guerriero r., guerrieri f., monteleone p., rizzo m., viti r., 2003 apricot, pp. 24-49. in: bassi d. (ed.) growth habit in stone-fruit trees. il divulgatore, bologna, italy. chalmers d.j., mitchell p.d., van heek l., 1981 control of peach tree growth and productivity by regulated water supply, tree density and summer pruning. j. am. soc. hortic. sci., 106: 307-312. day k.r., dejong t.m., hewitt a., 1989 postharvest and preharvest summer pruning of ‘firebrite’ nectarine trees. hortscience, 24: 238-240. ferree d.c., myers s.c., rom c.r., taylor b.h., 1984 physiological aspects of summer pruning. acta horticulturae, 146: 243-252. giovannini d., neri d., di vaio c., sansavini s., del vecchio g., guarino f., pennone c., abeti d., colombo r., 2010 efficienza gestionale degli impianti di pesco in un confronto nord-sud. frutticoltura, 7/8: 16-26. guerriero r., scalabrelli g., 1982 relationship between bud dormancy and growing and fruiting behaviour on different apricot varieties along the tuscan coast line. a) changes of one year shoot morphogenetic gradient during rest period. acta horticulturae, 121: 85-92. guerriero r., viti r., 1997 problemi relativi alla biologia fiorale e di fruttificazione dell’albicocco. italus hortus, 4: 29-36. guerriero r., xiloyannis c., 1975 a comportamento produttivo di alcune cultivar di albicocco nel litorale toscano. proceeding of “problemi e prospettive della coltura dell’albicocco”, imola, italy, pp. 83-92. guerriero r., xiloyannis c., 1975 b rapporti tra andamento climatico, dormienza habitus vegetativo e produttivo nell’albicocco. proceeding of “problemi e prospettive della coltura dell’albicocco”, imola, italy, pp. 35-44. hossain a.b.m.s., mizutani f., 2008 dwarfing peach trees and fruit quality development by using summer pruning as physiological changed dwarfing component. austr. j. of basic and appl. sci., 2: 844-849. hossain a.b.m.s., mizutani f., onguso j.m., 2004 effects of summer pruning on maintaining the shape of slender spindle bush of peach tree grafted on vigorous rootstock. j. of the jap. soc, of agric. tech. manag., 11: 55-62. kappel f., bouthillier m., 1995 rootstock, severity of dormant pruning, and summer pruning influences on peach tree size, yield, and fruit quality. canadian j. of plant sci., pp. 491-496. lanzelotti j., gonzalez b.a., boragno o.a., 1998 different dates of pruning in peach tree. acta horticulturae, 465: 629-635. lopez g., mata m., arbones a., solans j.r., girona j., marsal j., 2006 mitigation of effects of extreme drought during stage iii of peach fruit development by summer pruning and fruit thinning. tree physiology, 26: 469477. marini r.p., 1985 vegetative growth, yield and fruit quality of peach as influenced by dormant pruning, summer pruning and summer topping. j. am. soc. hortic. sci., 110: 133139. marini r.p., barden j.a., 1987 summer pruning of apple and peach trees. horticultural reviews, 9: 351-375. marsal j., lopez g., mata m., girona j., 2006 branch removal and defruiting for the amelioration of water stress effects on fruit growth during stage iii of peach fruit development. scientia horticulturae, 108: 55-60. miller s.s., 1987 summer pruning affects fruit quality and light penetration in young peach trees. hortscience, 22: 390-393. mizutani f., rabbany a.b.m.g., amano s., hino a., akiyoshi h., kadoya k., 1997 -effect of summer pruning and gibberellin application on reduction of flower bud formation in “saotome” peach (prunus persica batsch.) 178 trees grafted on p. japonica rootstocks. bulletin of the experimental farm college of agriculture, ehime university, japan, 18: 1-8. monserrat r., iglesias i., 2011 i sistemi di allevamento adottati in spagna: l’esempio del vaso catalano. frutticoltura, 7/8: 18-26. neri d., 2003 ipertesto potatura. crpv, cesena, italy. neri d., giovannini d., massai r., di vaio c., sansavini s., del vecchio g., guarino f., mennone c., abeti d., colombo r., 2010 efficienza produttiva e gestionale degli impianti di pesco in un confronto nord-sud. italus hortus, 17(3): 46-62. neri d., massetani f., giorgi v., 2011 la potatura. edagricole, bologna, italy, pp. 370. neri d., morini f., massetani f. pirazzini p., 2010 pruning: how to manage shoot growth. acta horticulturae, 862: 355-363. pirazzini p., 2004 osservazioni sulla potatura di produzione di alcune varietà di albicocco. frutticoltura, 1: 36-38. rom c.r., ferree d.c., 1984 the influence of summer pruning current season shoots on growth, floral bud development and winter injury of mature peach trees. hortscience, 19: 543-545. sansavini s., neri d., 2005 forme di allevamento e potatura, pp. 115-143. in: fideghelli c., and s. sansavini (eds.) pesco. edagricole, bologna, italy, pp. 259. zucconi f., 2003 nuove tecniche per i fruttiferi. edagricole, bologna, pp. 246. 245 1. introduction the potential impact of atmospheric deposition of pollutants such as sulphur dioxide on vegetation has been the subject of many papers (tingey and olszyk, 1985; darral, 1989; mesanza et al., 1996; johnson et al., 1999). plant injury caused by air pollution is most common near large cities and industrial enterprises, and damage in isolated areas occurs when pollutants are spread over long distances by wind currents (cicek, 2003). some studies have also begun to surface from developing countries (hassan et al., 1995; wahid et al., 1995), where yield losses of cereals and legumes have been attributed to both so 2 and ozone. exposure to so 2 has been reported to decrease or inhibit photosynthesis (silvius et al., 1975; ziegler, 1975; carlson, 1983; rao et al., 1983; katainen et al., 1987; kropff, 1987; sheu, 1994; lorenc-plucińska, 1998; ranieri et al., 1999) and pollen tube growth (karnosky and stairs, 1974; varshney and varshney, 1981). even if the so 2 sensitivity of grapevine is poorly documented (ishikawa, 1972; weinstein, 1984; garciahuidobro et al., 2001), the species is considered sensitive to chronic so 2 exposure, and adverse effects on growth or yield were presumed possible in the field (daines, 1968; fujiwara, 1970). development of black and brown lesions was observed especially on leaves (cicek, 2003); shoot growth reduction and, in some cases, leaf abscission were reported (shertz et al., 1980). the interaction of so 2 with ozone is evident. the combination of the two gases led to an increase in oxidant stipple in leaves (forsline et al., 1983). in other species this interaction has not occurred (kreess et al., 1986), suggesting the role of the genotype in resistance to the pollutant. interaction so 2 /carbon black was also suggested by ionescu et al. (1971) in the industrialized copsa mica zone of romania, where the damaging effects of sulphur-containing effluents were enhanced by the presence of carbon black. fujiwara (1970) found, with increasing so 2 concentration, that leaf abscission, in cv. fredonia, began earlier and progressed at a greater rate. effects of sulphur dioxide were also found on the stomatal apparate of vitis labrusca l. cv. ives, where so 2 induced both stomatal closure and a higher stomatal resistance (rosen et al., 1978). unlike other species (lorenc-plucińska and ziegler, 1989; maurousset and bonnemain, 1990), there is not, for the vine, specific knowledge on the effects of so 2 on shortand long-term effect of sulphite on sucrose transport in grapevine (vitis vinifera l.) leaves. an electrophysiological study e. rinaldelli, r. bandinelli, m. pagano dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione coltivazioni arboree, laboratorio di elettrofisiologia, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: membrane potential, sucrose transport, sulphite, sulphur dioxide, vitis vinifera l. abstract: sucrose is the main carbohydrate translocated in grapevine and its transport may be restricted or inhibited by a number of factors such as the pollutant sulphur dioxide. the present study investigated, for the first time in grapevine, the effects of sulphite on membrane electrical response of sucrose transport in the mesophyll cells. co-transport of sucrose across the membrane is linked to the free energy in a electrochemical proton gradient. without the pollutant, electrophysiological traces displayed a metabolic-dependent sucrose electrical response in which an initial depolarization was followed by complete repolarization. in the presence of sulphite, instead, there were different trends depending on time of contact with the tissue of the pollutant. in the short-term, a slower repolarization was observed and in the long-term (after 6 and 12 h) the extent of depolarization (δ mv) was also reduced. transmembrane electrical potentials, measured in the presence of sulphite, became significantly (p < 0.01) more positive with increasing time of incubation of the tissue. on the whole, electrophysiological results highlight a direct or indirect effect of the pollutant on the activity of proton pump h+/atp ase. since carbohydrate translocation has a central role in the balance between source and sinks in the plant, the results of the research suggest that sulphite can modify the above balance with negative implications for the export of carbohydrate from the leaves. adv. hort. sci., 2011 25(4): 245-252 received for publication 4 november 2011 accepted for publication 24 november 2011 246 sugar transport in leaves. in phaseolus vulgaris and ricinus communis so 2 inhibits assimilate translocation and this inhibition seems to depend on damage to the sucrose transport system or proton pump (noyes, 1980; teh and swanson, 1982; lorenc-plucińska and ziegler, 1987 a, b, 1988). according to a more recent and detailed study on purified plasma membrane vesicles of ricinus communis, the decreased uptake of sucrose may be attributed to a dissipation in the transmembrane ph gradient (russell et al., 1999). sucrose transport in leaves is a secondary active transport (co-transport) in which solute (sucrose) translocation across the membrane is linked to the free energy available in a proton electrochemical potential difference (bush, 1993). it is widely known in the leaves of many plants (giaquinta, 1979; delrot, 1981; delrot and bonnemain, 1981; huber and moreland, 1981), and also in the vine is assumed to be so (mullins et al., 1992). since sucrose transport involves the entry of protons into the cell, it gives rise to changes in transmembrane potential. thus electrophysiological techniques are very useful for following, in real time, the transport of solutes. regarding the effect of sulphite on sucrose transport, the problem has been studied mainly on a biochemical level, while there is little information on the electrophysiological aspects. to our knowledge, only one paper (maurousset and bonnemain, 1990) reports some information about these aspects, however it does not include combined electrophysiological tests with sucrose and sulphite. therefore we felt it was interesting to investigate, for the first time in grapevine, how sucrose membrane electrical response was influenced by so 2 supplied as sulphite. 2. materials and methods plant material electrophysiological tests were carried out on leaves of vitis vinifera l., cv. sangiovese clone ss-f9-a5-48, removed from two-year-old plants grown in a container and grafted on rootstock 420 a. preliminary tests were performed on whole leaves, while the subsequent tests were on leaf segments (3 x 7 mm), cut with a scalpel under b.s. solution. chemicals for electrophysiological experiments, the substance to be tested has to be dissolved in the solution bathing the tissue (treatment solution). sulphur dioxide is highly soluble in water but, being gaseous, it is not easy to dose the exact amount to be dissolved. therefore, in this research, na 2 so 3 was employed, which at ph 5.5, is found mainly in the form of bisulfite ion (hso3-). previous research conducted on isolated chloroplasts of spinacia oleracea l. showed that so 2 and bisulphite ions, at the same equimolecular concentration, have a parallel mode of action in the inhibition of photosynthetic oxygen evolution (silvius et al., 1975). moreover the effects of so 2 on chloroplast enzyme systems were studied by means of hydration products of bisulphite and sulphite (ziegler, 1975). the components of the various treatments were added to the basal solution (b.s.) for the electrophysiological experiments (table 1). in all treatments in which na 2 so 3 alone was employed, even the b.s. contained the same amount of sodium supplied as na 2 so 4 . carbonyl cyanide m-chloro phenyl hydrazone (cccp), a protonophore and uncoupler of oxidative phosphorilation (marrè et al., 1973) was used in 0.05% ethanol, starting from a stock solution of 0.5 mol m-3. controls, in the different trials, also contained the same percentage of ethanol in the treatments. the salts present in the basal solution were only in sulphate form. the presence of clanions was avoided since their entry by symport with h+ can influence cytoplasmic ph (bellando et al., 1995). table 1 composition of the basal solution (b.s.) and concentration of the components in the treatments basal solution treatment components* k2so4 2.5 sucrose 20.0 caso4 0.5 na2so3 1.0 mes 5.0 cccp 10-2 all solutions were adjusted to ph 5.5 by way of tris or dilute h 2 so 4 when na 2 so 3 was present. mes: 2-n-morpholinoethane-sulphonic acid (sigma); cccp: carbonyl cyanide m-chlorophenylhydrazone (sigma); tris: 2-amino-2-hydroxymethyl-1,3-propanediol (fluka). *concentrations are in mol m-3. electrophysiology before beginning the electrophysiological experiments, whole leaves and/or leaf segments were incubated in basal solution in the light for 2 h (short-term). during this period, the solution was renewed twice and constantly aerated. subsequent tests were performed after incubation of leaf segments in sulphite for 6 and 12 h (long-term). even in this case the solution was constantly aerated and renewed every hour. membrane potential (em) was measured according to standard electrophysiological technique as previously adopted (rinaldelli and bandinelli, 1999; rinaldelli, 2005) with some modification. in brief, whole leaves and/or leaf segments were mounted on a poly(methyl methacrylate) chamber secured to a microscope stage. the chamber for whole leaves was slightly inclined and wide enough to accommodate a grapevine leaf, while the one for the segments was small (3 ml) and placed horizontally. continuously aerated b.s. or treatments were permitted to perfuse through the chamber at a flow rate of 10 ml min-1. they reached the tissue by gravity, each through its own adductor channel controlled by a manual valve. in the case of whole leaf, only the stomatal area around the insertion point and the petiole were perfused. a small glass thermometer placed inside the chamber allowed verification of the temperature of the solution. heating or cooling of the solution was obtained by way of a peltier-effect heat pump located along the solution conduit before the poly(methyl methacrylate) chamber; it was electronically controlled. 247 the measuring electrodes used were micropipettes (tip diameter <1 µm) obtained from single-barrelled borosilicate capillaries (w.p.i., usa) by way of a vertical homebuilt puller. the micropipettes and the reference electrode were filled with 500 mol m-3 kcl. the electrodes were connected, by ag/agcl wires, to a high input impedence electrometer (ad 549, 1015 ω). the output signals from the electrometer, before being transmitted to a chart recorder, were passed through a low-pass filter (10 hz) in order to eliminate possible noise. the insertion of microelectrodes into the cells of the central part of the mesophyll took place under two different magnifications. in the case of whole leaves the electrode passed through a stoma and a magnification of 500x was necessary, obtained with a 50x long working distance objective (mitutoyo, japan). in the case of leaf segments, instead, a magnification of 250x, obtained with a 25 x long working distance objective (wpi, usa), was sufficient. the insertion of the microelectrodes was, in both cases, at an angle of about 45°, and took place by way of a very stable, manual, three-axis, homebuilt micromanipulator. treatments started after em stabilized for 5 min. all experiments were performed under faraday cage. the complete electrophysiological set-up is presented in figure 1. except where indicated otherwise, preincubation and electrophysiological tests were carried out at +22°c (± 0.5) in the light (30 watt/m2). fig. 1 schematic electrophysiological set-up for membrane potential measurements. 1. faraday cage. 2. digital cameras. 3. microscope tube. 4. measuring microelectrode. 5. reference electrode. 6. probe. 7. microscope stage. 8. vibration-free table. 9. lamp. 10. micromanipulator. statistical analysis the data relating to transmembrane potentials, measured at different times, in bs and sulphite, were subjected to statistical analysis of variance (anova). comparisons were carried out using duncan’s test. statistical significance of differences were accepted when p <0.01. in the electrophysiological traces, depolarizations (δ mv) are presented as mean ± se. 3. results preliminary experiments on whole leaves and leaf segments since the cuticle is often a barrier to organic molecules (rinaldelli and bandinelli, 1999; rinaldelli, 2005), before working on a tissue it is necessary to verify its permeability to the solutions that will be employed. thus, preliminary tests were performed on whole leaves and leaf segments. they showed that both were permeable to sulphite (fig. 2, a, c) but not to sucrose. in whole leaves, in fact, sucrose depolarization was almost one-quarter that in leaf segments (fig. 2, b, d). based on this evaluation, all subsequent tests were performed on leaf segments. fig. 2 effects of treatments with sucrose and sulphite on whole leaves and leaf segments. all measurements were carried out in light. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. 30 min 248 sucrose transport and its metabolic support as co-transport of sucrose in grapevine leaves was assumed (mullins et al., 1992) but not electrophysiologically investigated, initial tests were carried out to verify this assumption. in the light, in the presence of the uncoupler cccp, sucrose depolarization was less than half, compared to the same test without the inhibitor (fig. 3 a, b). subsequent repolarization was also very slow. in the dark, instead, sucrose depolarization was lower than in the light, and it was completely abolished by the uncoupler (fig. 3 c, d). the different extent of depolarization, in light and dark, reflects metabolic support to the operation sucrose/ h+ symport. as discussed later, cccp only inhibited oxidative phosphorylation and had incomplete or no effect on photophosphorylation. at 5°c sucrose depolarization was nearly annulled (fig. 3 e) and a similar response occurred when the ph of the treatment solution was brought to 8.0 (fig. 3 f). shortand long-term experiments under sulphite in the short-term tests, sucrose depolarization, in the light, was about 20 mv (fig. 3 b) whereas in the dark it was much less (fig. 3 c). also, in the presence of sulphite, differences in extent of depolarization, in light and dark, remained approximately the same as previous tests without pollutant, but a slower repolarization was observed (fig. 4 a, b). based on this evidence, in the short-term sulphite affected only in part the membrane electrical response of sucrose transport. figure 5 shows the electrical responses to sucrose after leaf segments were preincubated in b.s. (control) or sulphite, for 6 and 12 h. since shortand long-term treatments in the dark showed the same trends, only the first are reported. sulphite already reduced the sucrose depolarization after 6 h of incubation (fig. 5, b), but the result was more evident after 12 h where the extent of depolarization defig. 3 effects of chemical and physical treatments to verify energetic support of sucrose transport. except where indicated otherwise, measurements were carried out in light. solution at ph 8.0 was buffered with 2 mol m-3 hepes [n-(2-hydroxyethyl)piperazine-n’-(2-ethanesulfonic acid] -tris. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se 249 creased by almost half as compared to the control (fig. 5d). also the repolarization, after 6 h, and even more after 12 h, was strongly slowed. it should be noted that even with increasing incubation time there is a progressive decrease in the extent of depolarization. also the transmembrane potentials decreased (they became more positive) with increasing incubation time (table 2). these effects were evident under both, b.s. and sulphite. under sulphite, however, they were more marked. table 2 membrane potentials (mv) recorded at different times of incubation, in b.s. and sulphite incubation time (h) membrane potentials b.s. (no sulphite) 1 mol m-3 na2so3 0 -160.2 ± 2.61 a -153.0 ± 2.05 b 6 -152.2 ± 2.04 b -139.7 ± 2.45 d 12 -149.0 ± 2.44 c -125.1 ± 2.33 e each value is the mean ± sd of ten experiments. different letters denote statistically significant differences between membrane potentials (p<0.01). 4. discussion and conclusions energetic support to sucrose transport cccp is an uncoupler of oxidative phosphorilation which, in agreement with the chemiosmotic hypothesis (mitchell, 1961), dissipates the electrochemical gradient fig. 4 short-term treatments in light and dark. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. fig. 5 long-term treatments, after 6 h and 12 h of incubation in b.s. and/or sulphite. all measurements were carried out in light. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. 30 min 30 min 250 of h+ ions preventing atp synthesis. consequently, active transport systems (primary and also, indirectly, secondary), lacking energy, are deactived. under cccp, membrane electrical response of sucrose showed, in light and dark, two different results (fig. 3 a, d). this finding suggests that the metabolic support of the operation of sucrose/h+ symport comes from both respiration and photosynthesis. in dark, since there is no photosynthesis, atp production is only of respiratory origin and it is blocked by the uncoupler cccp. in light, instead, some energetic support appears because cccp does not inhibit, or only partially inhibits, the photophosphorylation. this may be the case, taking into account the variable effects of cccp on photophosphorylation as a function of leaf greening (oelze-karow and butler, 1971; butler et al., 1972) or light intensity (saha et al., 1970; prins et al., 1980). since chemical inhibitors may simultaneously affect many different processes of cells (khalilov et al., 2002), we also tested the effect of low temperature. treatment with sucrose at 5°c nearly led to the annulment of the depolarization. this suggests a metabolic dependence of sucrose transport on the activity of a plasmalemma atpdriven proton pump. at 5°c, since the metabolic energetic support is highly limited (mengel and shubert, 1985; rinaldelli and bandinelli, 1999; rinaldelli, 2000; 2004), the activity of the pump is inhibited. the use of sucrose solution buffered at ph 8.0 strongly reduced depolarization. under these conditions, the lack of protons in the apoplast would explain the annulment of h+ co-transport. this response has also been observed for nitrate (ullrich and novacky, 1981; mcclure et al., 1990) and urea co-transports (rinaldelli, 2004). all these results are considered sufficient to support a metabolic and ph-dependent sucrose co-transport. membrane electrical responses under sulphite sucrose is the main carbohydrate translocated in grapevine. from the leaves, where it is produced, it moves to the various sinks according to the specific needs of the plant (coombe and mccarthy, 2000; hunter and ruffner, 2001). during the annual vegetative and reproductive cycle, it plays a preeminent role in regulation of the carbon/nitrogen ratio (rodriguez-lovelle and gaudillère, 2002). sucrose, being a non-polar molecule, does not have direct effects on membrane potential when it enters the cell, but only indirect, because its transport is related to the flow of protons that move along an electrochemical gradient. this co-transport results in an initial depolarization observed under both b.s. and sulphite (figs. 2 b, d; 3, 4, 5). under sulphite, it has to be excluded that the depolarization could be due to na+ or the accompanying anion, since sucrose and perfusing solutions contained an equal amount of na 2 so 3 . repolarization of the membrane, which sometimes exceeded the starting level (overshoot), is proposed to depend on stimulation of the h+-atpase, caused by either changes in the cytoplasmic ph or the membrane potential itself. the h+-atpase has a ph optimum at 6.6, i.e. well below the physiological ph of the cell cytoplasm (usually around 7.2-7.5). thus, whenever protons start accumulating in the cytoplasm, the activity of the pump increases to remove excess protons from the cell (michelet and boutry, 1995). this response is comparable, as cause and effect, to the one caused by permeant weak acids (marrè et al., 1983; rinaldelli and bandinelli, 1999). sulphite alone, at the concentration used, induced a slight depolarization followed by partial or complete recovery. this is visible in both whole leaves (fig. 2 a) and leaf segments (fig. 2 c). maurosset and bonnemain (1990) found that the depolarization by sulphite, does not depend on the concentration used. effect of the pollutant on the membrane would be, initially, that of “exciting agent”. instead, the repolarization would depend, according to the same authors, on the concentration of sulphite in the treatment. in our study we tested sulphite concentrations below and above 1 mol m-3 (data not shown) finding, however, less marked differences compared to the aforementioned authors. however the comparison is difficult because the study of maurosset and bonnemain (1990) did not include our same electrophysiological tests. their tests, in addition, were conducted on a different species (vicia faba l.) and this may also affect the reported results. when the treatment with sucrose was under sulphite, however, repolarization slowed and decreased gradually moving from shortto long-term. this is the first and most important effect of sulphite on sucrose transport. since repolarization is dependent on proton pump activity, the direct or indirect effect of sulphite should be placed at this level. in the long-term, under b.s. and sulphite, sucrose depolarization was lower (fig. 5) and the transmembrane electropotentials became significantly more positive (table 2). since these effects are evident even in b.s., they should be attributed, at least in part, to phenomena of senescence which start when leaves are detached or segments are cut (thimann et al., 1977; gepstein, 1982; malik, 1982). however, since the variations under sulphite were greater, there is clear evidence, in addition to senescence, of a direct or indirect effect of pollutant on proton pump activity. proton pumps, in fact, are considered the primary motors that build up transmembrane electrochemical proton gradients (felle, 2001). the complex of electrophysiological tests performed in this research highlights a clear membrane electrical response of sucrose transport to sulphite. two points stand out clearly enough: a) in grapevine leaves sucrose membrane electrical response is supported by metabolic energy; b) sulphite alters the above response by acting on the proton pump and/or energetic metabolism, from which the pump draws energy to extrude h+ ions outside the cell. an effect of sulphite on respiration and photosynthesis cannot be excluded, especially in the long-term. the link may be there because both slowed repolarization and more positive transmembrane potentials under sulphite reflect a partial inhibition of the proton pump, whose activity is supported by respiratory and photosynthetic atp. 251 the results obtained constitute an interesting acquisition in grapevine physiology, but it should be taken in account that conditions for the so 2 effect, in the vineyard ecosystem, may differ from those adopted in the laboratory due to the high number of cultural, climatic and pedological variables. acknowledgements this study was supported by university funds, ex 60%. references bellando m., marrè m.t., sacco s., talarico a., venegoni a., marrè e., 1995 transmembrane potential-mediated coupling between h+ pump operation and k+ fluxes in elodea densa leaves 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commonly used cool-season turfgrass in the temperate and subarctic regions of north america, and it is also recognized for its ability to create a high-quality turf (fry and huang, 2004). saline soils reduce growth due to osmotic and ion stresses (marschner and part, 1995; munns, 2002). salinity causes stress in plants in two ways, and plants respond in two distinct phases through time: a rapid response to the increase in external osmotic pressure, and a slower response due to the accumulation of na+ in leaves (munns and tester, 2008). it is generally accepted that silicon can positively affect growth and health status of plants under biotic (adatia and besford, 1986; ma, 2004) and abiotic (barceló et al., 1993; ranganathan et al., 2006) stresses. acceptable results of silicon application against nacl stress have been shown in rice (matoh and kairusmee, 1986; yeo et al., 1999), wheat (ahmad et al., 1992; tuna et al., 2008; tahir et al., 2010; chen et al., 2014), and barley (liang et al., 1996; liang, 1999). possible mechanisms for salt tolerance with the utilization of silicon have been proposed. these include accumulation of silicon in leaves resulting in reduced transpiration (matoh and kairusmee, 1986), turgor enhancement (romeroarandaet al., 2006), formation of na complexes in roots (ahmad et al., 1992), increased photosynthetic activity and protection of plasmatic membranes and chloroplast ultrastructures (liang et al., 1996; liang, 1998; shu and liu, 2001), protection of plant tissues from free radicals through increasing the activity of antioxidative enzymes (liang, 1999; liang et al., 2003; zhu et al., 2004), and alleviation of specific ionic effects (rafiq, 1990) by reducing na uptake (liang, 1999; epstein, 2001; gong et al., 2003). gong et al. (2003) also observed improved water economy and dry matter yield of plants with si application. silicon application is reported to enhance leaf water potential in wheat under drought stress (liang, 1999). the authors suggested that a double layer, comprised of silica and cuticle on leaf epidermal tissue, is responsible for this higher water potential. silicon application enhances water use efficiency, heat/salt tolerance, and resistance to pathogens and adv. hort. sci., 2016 30(2): 87-94 doi: 10.13128/ahs-19134 kentucky bluegrass (poa pratensis l.) silicon-treated turfgrass tolerance to shortand long-term salinity condition s. esmaeili, h. salehi (*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: chlorophyll content, proline content, salt stress, si, turfgrass. abstract: the effects of shortand long-term salinity condition were investigated on silicon-treated and control plants of kentucky bluegrass (kbg) (poa pratensis l.) in a greenhouse study. salt stress solely affected visual quality at ≥15 ds m-1 concentrations while si application increased salt tolerance of kbg after 45 days. in long-term salinity stress, si had no effect on salt tolerance of kbg at ≥15 ds m-1 concentration. si increased morphological parameters including height and number of shoots, and physiological parameters including relative water content (rwc) and chlorophyll content of leaves. in addition, fresh and dry weights of roots and shoots in response to high salt concentrations declined, but showed an increase with si treatment. proline content and electrolyte leakage (el) increased under high salinity levels. in response to the si treatment, na concentration in the shoots significantly decreased at the 5 ds m-1 salinity level. with increasing salinity levels, the concentration of k in roots and shoots decreased while the amount of k in both si-treated roots and shoots reduced. overall, si alleviative effects were more pronounced in 45 days after turfgrasses being salinity treated. (*) corresponding author: hsalehi@shirazu.ac.ir received for publication 23 march 2015 accepted for publication 24 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 87-94 88 heavy metals (yeo et al., 1999; zwieniecki et al., 2001; gao et al., 2004; liang et al., 2005 a, b; wang et al., 2005; guo et al., 2007; liang et al., 2007). it has been highly recommended to use si in turfgrass management (datnoff and rutherford, 2003; datnoff and rutherfood, 2004). in another study on poa pratensis l. ‘baron’, chai et al. (2010) reported that si application under salinity condition raised the transfer of k+ from roots to shoots, but inhibited the absorption and transfer of na+, which may contribute to better turf quality and growth with si treatment under saline conditions. chen et al. (2014) also found that si can enhance plant salt tolerance by alleviating the salt-induced osmotic stress. mateos-naranjo et al. (2013) showed that in wheat, the alleviative effects were more pronounced in the osmotic stress phase than the ion toxicity phase. these results clearly showed that si can enhance plant salt tolerance by alleviating the salt-induced osmotic stress. the purpose of the present study was to evaluate the shortand long-term effects of salinity on growth and physiological parameters of kentucky bluegrass with and without silicon treatment. 2. materials and methods plant materials and growth conditions the experiment was conducted for a period of 90 days in the greenhouse of the department of horticultural science, college of agriculture, shiraz university, shiraz, iran. plastic pots (20 cm in diameter and 10 cm in depth) were filled with 1.3 kg of a mixture of 1:1 sand and perlite, then seeds were cultured. the field capacity and permanent wilting point for the potting mixture were 10% and 4%, respectively. during the germination stage, turfgrasses were irrigated daily with 150 ml deionized water until plants were adequately established. then, irrigation was carried out every two days with 200 ml deionized water. before treatments began, the turfgrass was clipped to 5-7 cm every two weeks. the nutrient solution of a commercial whole fertilizer (cristalone) was used weekly with a concentration of 0.1%. the ec of the nutrient solution was 1 ds m-1. mean relative humidity, daily temperature and light condition of the greenhouse were categorized as 45±5%, 24±4°c and 29 wm-2 (16/8 h day/night), respectively. treatments silicon was applied in the form of potassium silicate (k2sio3) at a concentration of 1 mm as foliar application weekly from 1 september to 1 december 2009. salinity treatment began two weeks after the silicate treatment. different salt concentrations were prepared by adding 1 nacl: 1 cacl2 (w/w) to deionized water to obtain desired ec values; saline water of 5, 10, 15 and 20 ds m-1 along with deionized water as the control was applied (200 ml per pot) every two days. to prevent salinity shock, salinity levels were increased stepwise by 5 ds m-1. measurements physiological parameters were measured twice: at 45 and 90 days following commencement of salinity treatments. chlorophyll content, relative water content (rwc), electrolyte leakage and proline content were measured. chlorophyll content was measured according to the method of saini et al. (2001). half a gram of fresh leaf material, taken from the youngest fully expanded leaf, was extracted with 80% acetone and read using a spectrophotometer at 645 and 663 nm wavelengths. chlorophyll content was calculated using the following formula: mgchl/g f.w. = [(20.2(od 645 nm) + (8.02(od663nm)]*v/ (f.w.*1000) where v is the final solution volume in ml and f.w. is tissue fresh weight in mg. relative water content was measured using the methods of nepomuceno et al. (1998) and sairam et al. (2002). the value of rwc was determined by the following equation: rwc (%) = [(f.w.-d.w.)/(t.w.-d.w.)]×100 where f.w. is the fresh weight, d.w. is the dry weight and t.w. is the turgid weight. electrolyte leakage was determined according to the methods described by saadalla et al. (1990). samples of 0.1 g of fresh leaves were weighed and washed three to four times with deionized water and immersed in a test tube containing 15 ml deionized water, then maintained at 25°c for 24 h. the tubes were then shaken for 15 min and the conductivity of the solution was measured (ec1) using an electrical conductivity meter (metrohm 644, swiss). the test tubes were then placed in an autoclave at 0.1 mpa for 10 min to kill the plant tissue and release all of the electrolytes. the tubes were cooled to 25°c, shaken, and their solution conductance measured again (ec2). the electrolyte leakage was calculated as ec1/ec2 and expressed as percent. proline content was calculated according to the esmaeili and salehi kentucky bluegrass silicon-treated turfgrass tolerance to shortand long-term salinity condition 89 bates et al. (1973) method. a half gram of fresh leaves was homogenized with 10 ml of 3% aqueous sulfosalicylic acid and filtered through whatmans no. 2 filter paper. two ml of filtrate was mixed with 2 ml of acid-ninhydrin and 2 ml of glacial acetic acid in a test tube. the mixture was placed in a water bath at 100°c for 1 h. the reaction mixture was extracted with 4 ml toluene, and the absorbance was measured at 520 nm with a spectrometer (uv-120-20, japan). standard curves of proline were used for the calculation of proline amount in the samples. for fresh and dry weight determination of shoot and root systems and further chemical analysis, leaves and roots were washed three to four rinses in distilled water and then dried at 70°c for 48 h. the dried leaves and roots were ground to powder using an electric mill (ar 10, molinex, china) and subsequently stored in polyethylene bottles at room temperature. one gram of leaf sample was ashed in a furnace at 550°c for 5 h. the ash was then dissolved in 10 ml 2n hcl and diluted to the volume of 100 ml with distilled water. potassium and sodium contents were determined using a flame photometer (pfp7, jenway, england) (champan and pratt, 1982). statistical analysis the experiment was conducted in a complete randomized design with three replications. data were analyzed using mstat-c software. means were compared using the least significant difference (lsd) test at (p<0.05) level. 3. results the turfs treated with silicon, even at high concentration of salinity, maintained their visual quality and turf performance while those without silicon at 20 dsm-1 concentration died after 45 days. during long-term salinity exposure, either with or without si, kentucky bluegrass lost turf performance at 15 and 20 ds m-1 concentrations (data not shown). salt stress reduced chlorophyll content during short-term salinity. silicon treatment increased chlorophyll content extensively both under non saline irrigation and various concentrations of salty solutions (fig. 1). under different salinity exposures, rwc increased through si treatment. results were significantly different in the presence and absence of silicon under non saline conditions. significant differences were observed between salinity control groups, treated with si of otherwise (fig. 2). as shown in figure 3, increased salinity resulted in remarkably enhanced electrolyte leakage. silicon reduced electrolyte leakage after 45 days of salinity. in lower concentrations of salinity, silicon reduced fig. 1 effects of different salinity and si levels on chlorophyll content of poa pratensis after 45 days. 0: control, 5, 10, 15 and 20 ds m-1 as salinity levels; si: k2sio3 1 mm. data are mean ± se at p<0.05 using lsd test. fig. 2 effects of different salinity and si levels on relative water content in of poa pratensis after 45 days. 0: control, 5, 10, 15 and 20 ds m-1 as salinity levels; si: k2sio3 1mm. data are mean ± se. at p<0.05 using lsd test. fig. 3 effects of different salinity and si levels on electrolyte leakage of poa pratensis after 45 days. 0: control, 5, 10, 15 and 20 ds m-1 as salinity levels; si: k2sio3 1 mm. data are mean ± se. at p<0.05 using lsd test. adv. hort. sci., 2016 30(2): 87-94 90 electrolyte leakage even after 90 days (fig. 4). proline concentration increased dramatically after 45 and 90 days of salt stress, although plants with si application had less proline content (figs. 5 and 6). higher concentrations of saline irrigation showed more reduction in shoot number of p. pratensis. silicon markedly increased shoot numbers at lower concentrations (table 1). a dramatic reduction in visual quality based on shoot density and percentage of green leaf canopy area (glca) was observed after 45 days when p. pratensis was cultured at different table 1 interaction effects of different salinity and si levels on growth parameters, shoot and root na and k concentrations of poa pratensis fig. 4 interaction effects of different salinity and si levels on electrolyte leakage of p. pratensis after 90 days. data are mean ± se at p ≤0.05 using lsd test. fig. 5 effects of different salinity and si levels on proline content of poa pratensis after 45 days. 0: control, 5, 10, 15 and 20 ds m-1 as salinity levels; si: k2sio3 1 mm. data are mean ± se. at p<0.05 using lsd test. fig. 6 interaction effects of different salinity and si levels on proline content of p. pratensis after 90 days. data are mean ± se at p ≤0.05 using lsd test. indicator treatments salinity (ds m-1) 0 5 10 shoot length (cm) -si 24.57 ab 20.93 bc 17.37 c +si 29.27 a 20.53 bc 16.4 c shoot number in pot -si 337 ab 121.3 c 62.67 c +si 378.7 a 340.7 ab 258.7 b shoot fresh weight (g) -si 22.57 ab 10.28 cd 3.413 d +si 29.43 a 16.90 bc 8.313 cd root fresh weight (g) -si 46.67 ab 23.57 cd 20.61 d +si 50.20 a 34.96 bc 18.58 d shoot dry weight (g) -si 6.116 ab 3.506 bc 0.947 c +si 9.791 a 6.925 ab 2.033 c root dry weight (g) -si 14.28 ab 6.372 c 6.109 c +si 21.28 a 12.45 bc 5.744 c shoot na concentration (g) -si 360.6 c 948.0 a 826.2 ab +si 234.3 c 562.1 bc 554.6 bc root na concentration (g) -si 40.22 cd 102 ab 118.3 a +si 29.06 d 71.48 bc 81.15 ab shoot k concentration (g) -si 542.2 ab 347.2 bc 175.6 c +si 861.4 a 374.3 bc 216.3 c root k concentration (g) -si 12.16 c 3.966 d 2.242 d +si 26.15 a 18.37 b 13.41 c fig. 7 comparison of different concentrations of salts (a: 5 ds m-1, b: 10 ds m-1, c: 15 and d: 20 ds m-1 with si (+si) and without si (-si) in p. pratensis 45 days after beginning the treatments. esmaeili and salehi kentucky bluegrass silicon-treated turfgrass tolerance to shortand long-term salinity condition. 91 levels of salinity (fig. 7). shoot fresh and dry weight of p. pratensis showed the most prominent decrease at 5 ds m-1 salt concentration (table 1). also, a significant decline in root fresh and dry weight was observed at 5 ds m-1 (table 1). si application partially enhanced shoot fresh and dry weight. in addition, si had a greater impact on root fresh weight with non-saline irrigation (table 1). in the leaves and roots, na+ content significantly increased at low concentrations of salty irrigations after 90 days. in contrast, shoot k+ content was significantly less when salinity level reached 10 ds m-1. si treatment increased shoot k+ content in comparison to saline irrigations without si application. root k+ content reduced markedly at 5 ds m-1, compared to the control. turfs treated with si had a higher concentration of k+ in the roots (table 1). 4. discussion and conclusions kentucky bluegrass (kbg) is generally considered to be a salt sensitive turf. in our research, kbg had no tolerance at periodically extended concentrations higher than 15 ds m-1 salinity, regardless of silicon application; the salinity tolerance threshold of kbg resulted to be 10ds m-1. silicon could increase salinity tolerance at higher concentrations. the silicon remedy was more pronounced during short-term saline conditions, findings that are consistent with previous reports. silicon could increase the amount of chlorophyll and photosynthesis and consequently, growth. si protects plasmatic membranes and chloroplast ultrastructures (liang et al., 1996; liang, 1998; shu and liu, 2001), stimulates h+-atpase activity, and increases k+ in shoots (liang et al., 1996; liang et al., 2003; liang et al., 2005 a). furthermore, it improves the activity of antioxidant enzymes, reducing the damage of reactive oxygen species (ros) (liang, 1999; liang et al., 2003; zhu et al., 2004) and reduces na+ root uptake, alleviating specific ion effects (epstein, 2001; gong et al., 2003; liang et al., 2003). it has been reported that the accumulation of si in plants enhances the strength and rigidity of the tissues (ma and yamaji, 2006; neethirajan et al., 2009). an increased si supply improves the structural integrity of crops and may also improve plant tolerance to disease, drought, and metal toxicities (yeo et al., 1999; richmond and sussman, 2003; ma et al., 2004). these findings are in agreement with our study. some researchers hypothesized that si deposition in the cell wall of root endoderm may contribute to the maintenance of the apoplastic barrier and thereby improve plant tolerance to disease and drought stress (lux et al., 2002; lux et al., 2003; hattori et al., 2005). electrolyte leakage was influenced more in the short-term salinity stress than long-term salinity stress. it appears that plant adaptability to lower salinity levels in the short term increases resistance to salt. as shown in figures 5 and 6, a significant increase in proline content was observed at 15 ds m1. however, silicon only slightly affected proline levels compared to the turfgrasses treated with saline and non-saline waters. proline often accumulated in grasses under salinity stress, however this amount of content was insufficient for osmotic adjustment in grasses (marcum, 2002). foliar application of si increased the unsaturated fatty acid ratios [(18:2+18:3)/18:1] in glycolipids and phospholipids and also proliferated the amount of membrane lipids in strawberries (wang and galletta, 1998). agarie et al. (1998) noted that si increased membrane stability of rice under drought and heat stresses, which prevented the structural and functional deterioration of cell membranes. in concordance, it appears that si plays an important role in maintaining the integrity, stability and function of cell membranes in kentucky bluegrass under salt stress. ashraf and foolad (2007) reported an increase in the amount of proline primarily in cytosols under salinity stress. they found that plant tolerance to salinity stress and proline accumulation are positively related. however, the relationship is not universal and might be cultivar dependent. moreover, bartels and sunkar (2005) and ashraf and foolad (2007) reported other possible roles attributable to proline besides osmotic adjustment in stressed plants, such as acting as a hydroxyl scavenger, the stabilization of membrane and protein structure, serving as a sink for carbon and nitrogen during stress recovery, and the buffering of cellular redox potential under stressful conditions. as shown in figure 2 rwc reduced significantly under higher salinity levels in short-term salt stress. a probable explanation for an increase in rwc under si application may be the prevention of transpiration. marcum and murdoch (1990) reported that shoot water content of zoysia matrella (l.) merrill, z. japonica l., paspalum vaginatum swarts and c. dactylon decreased during a one month salinity stress in solution culture, and suggested that osmotic adjustment was not achieved exclusively by solute accumulation. adv. hort. sci., 2016 30(2): 87-94 92 si is known to decrease na uptake (2004). the present results clearly show that na uptake could be reduced through si treatment. similar results have been achieved in investigations by epstein (2001), liang et al. (2003), gong et al. (2003, chai et al. (2010), and bae et al. (2012). shoot growth and leaf firing decreased as salinity levels increased (fig. 7 and table 1). horst and taylor (1983) stated that growth declines to 50% when the concentration of salt reaches 11 ds m-1 in 44 cultivars of kentucky bluegrass, which was approximately similar to the value obtained in this study. an adverse result was reported by alshammary et al. (2004) who stated that shoot growth decreased by 50% at 5.5 ds m-1 concentration of salinity (a mixture of nacl and cacl2) in kentucky bluegrass. under high salinity levels, cell expansion could be reduced by accumulation of salts in cell walls which would effectively reduce cell turgor and consequently retard growth (oertli, 1968; flower and yeo, 1986). a decrease in growth at higher sodium concentrations due to a decrease in the uptake of k+ and ca2+ has also been reported (sairam and tyagi, 2004). si increased chlorophyll content, rwc, and visual quality under non saline water and low concentrations of salinity in both shortand long-term exposures. proline content and electrolyte leakage increased in response to increasing salinity levels. k+ concentration in shoots and roots increased as a result of si application. shoot number and shoot length dramatically decreased under higher salinity levels while si application increased shoot number and shoot length under low concentration of salt stress. in general, silicate fertilizer is recommended for turfgrass management against environmental stresses such as salinity, high or low temperatures, drought and heavy metals. references adatia m.h., besford r.t., 1986 the effects of silicon on cucumber plants grown in recirculating nutrient solution. ann. bot., 58: 343-351. agarie s., hanaoka n., ueno o., miyazaki a., kubota f., agata w., kaufman p.b., 1998 effects of silicon on tolerance to water deficit and heat stress in rice plants (oryza sativa l.) monitored by electrolyte leakage. plant prod. sci., 1: 96-103. ahmad r., zaheer s.h., ismail s., 1992 role of silicon in salt tolerance of wheat (triticum aestivum l.). plant sci., 85: 43-50. alshammary s.f., qian y.l., wallner s.j., 2004 growth response of four turf grass species to salinity. agric. water manag., 66: 97-111. ashraf m., foolad m.r., 2007 roles of glycine betaine and proline in improving plant abiotic stress resistance. environ. exp. bot., 59: 207-216. bae e.j., lee k.s., huh m.r., lim c.s., 2012 silicon significantly alleviates the growth inhibitory effects of nacl in salt-sensitive ‘perfection’ and ‘midnight’ kentucky bluegrass (poa pratensis l.). hort. environ. biotechnol., 53: 477-483. barceló j., guevara p., poschenriedr c., 1993 silicon amelioration of aluminium toxicity in teosinte (zea mays l. ssp. mexicana). plant soil, 154: 249-255. bartles d., sunkar r., 2005 drought and salt tolerance in plants. crit. rev. plant sci., 24: 23-58. bates l.s., 1973 rapid determination of free proline for water-stress studies. plant soil, 39: 205-207. bradbury m., ahmad r., 1990 the effect of silicon on the growth of prosopis juliflora growing in saline soil. plant soil, 125: 71-74. chai q., shao x., zhang j., 2010 silicon effects on poa pratensis responses to salinity. hort. science, 45: 1876-1881. champan h.d., pratt p.f., 1982 methods of plant analysis, pp. 60-193. in: champan h.d., and p.f. pratt d.f. 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(bangalore), 86: 407-421. shu l.z., liu y.h., 2001 effects of silicon on growth of maize seedlings under salt stress. agro-environ. prot., 20: 8-40. tahir m.a., ramatullah a., aziz t., ashraf m., 2010 wheat genotypes differed significantly in their response to si nutrition under salinity stress. j. plant nutr., 33: 1658-1671. tuna a.l. kaya c., higgs c.d., murillo-amador b., aydemir s., girgin a.r., 2008 silicon improves salinity tolerance in wheat plants. environ. exp. bot., 62: 10-16. wang l., qing n., li m., zhang f., zhuang j., yang w., li t., wang y., 2005 biosilicified structures for cooling plant leaves: a mechanism of highly efficient mid infrared thermal emission. appl. phys. lett., 87: 194105. wang s.y., galletta g.j., 1998 foliar application of potassium silicate induces metabolic changes in strawberry plants. j. plant nutr., 21: 157-167. yeo a.r, flowers s.a., rao g., welfare k., 1999 silicon reduces sodium uptake in rice (oryza sativa l.) in saline condition and this is accounted for by a reduction in the transpirational by pass flow. plant cell environ, 22: 559-565. zhu z., wei g., li j., qiang q., yu j., 2004 silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of salt-stressed cucumber (cucumis sativus l.). plant sci., 167: 527-533. zwieniecki m.a., melcher p.j., holbrook n.m., 2001 hydrogel control of xylem hydraulic resistance in plants. science, 291: 1059-1062. impaginato 249 abbaszade k. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 abdely c. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 adam a. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 adamipour n. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 ahkbari a. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 akbari n. salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato, 151 ahmad a. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 ahmad j. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 amad a. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 alizadeh m. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 al-safadi b. ultrastructural changes in potato (solanum tuberosum) under nacl mediated salinity stress in vitro, 95 arbaoui m. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 arena m. characterization and evaluation of berberis microphylla g. forst pollen grains, 31 aslmoshtaghi e. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 azizi f. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 bagheri f. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 bahadoran m. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 baninaiem e. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 ben hamed k. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 berti m. phenotypic characterisation of almond accessions collected in afghanistan, 207 berti m. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 bettaieb t. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 caboni e. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 carbone k. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 ceccarelli d. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 chand r.r. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling whiteflies (aleurodius dispersus russell), 165 chiechi a. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 ciappellano s. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 comparini d. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 67 creti p. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 cullen g.j. implications of investigating pollination and cross compatibil ity in the almond varieties of afghanistan, 217 das k. influence of organics, inorganic and biofertilizers on growth, fruit quality, and soil characters of himsagar mango grown in new alluvial zone of west bengal, india, 81 dutta p. influence of organics, inorganic and biofertilizers on growth, fruit quality, and soil characters of himsagar mango grown in new alluvial zone of west bengal, india, 81 elbey n. effect of h2o2 pretreatment on the response of index volume 30, 2016 author index 250 two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 enayat n. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 esmaeili s. kentucky bluegrass (poa pratensis l.) silicontreated turfgrass tolerance to shortand long-term salinity condition, 87 fakhar z. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 ferrante a. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 ghous m. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 giordani e. from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan, 197 giordani e. phenotypic characterisation of almond accessions collected in afghanistan, 207 giordani e. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 giro a. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 gopalan r.d. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling whiteflies (aleurodius dispersus russell), 165 hara a. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 hara y. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 hosseini m.s. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 houssian k. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 ibrahim m. arbuscular mycorrhizal isolate and phosphogypsum effects on growth and nutrients acquisition of cotton (gossypium hirsutum l.), 121 idris i. induced resistance in potato plants by a non-pathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 iwase j. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 javanmardi j. salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato, 151 jokhan a.d. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling, whiteflies (aleurodius dispersus russell), 165 kawano t. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 khalil n. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 khorsha s. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 khosh-khui m. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 laera a.m. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 lanzoni c. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 licciulli a. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 luvisi a. insight on trans-plasma membrane behavior of virus-infected plant cells, 135 luvisi a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 mashayekhi k. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 masini g. from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan, 197 materazzi a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 mirzaaliandastjerdi a. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 mortazavi s.n. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 murata y. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 nakar m. ultrastructural changes in potato (solanum tuberosum) under nacl mediated salinity stress in vitro, 95 nisi r. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 pal s. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 panattoni a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 patel a. influence of organics, inorganic and biofertilizers on growth, fruit quality, and soil characters of himsagar mango grown in new alluvial zone of west bengal, india, 81 radice s. characterization and evaluation of berberis microphylla g. forst pollen grains, 31 rastegar s. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during 251 cold storage, 183 ratti c. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 refahi a. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 rehman s. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 rezgui s. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 rinaldelli e. insight on trans-plasma membrane behavior of virus-infected plant cells, 135 rizzo d. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 rubies autonell c. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 saeedi a. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 sakata r. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 saleh b. dna changes in cotton (gossypium hirsutum l.) under salt stress as revealed by rapd marker, 13 salehi h. kentucky bluegrass (poa pratensis l.) silicontreated turfgrass tolerance to shortand long-term salinity condition, 87 salehi h. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 samadi g.r. implications of investigating pollination and cross compatibil ity in the almond varieties of afghanistan, 217 sartori a. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 shahcheraghi s.t. micropropagation of three endemic and endangered fig (ficus carica l.) genotypes, 129 shahsavar a.r. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 shekafandeh a. micropropagation of three endemic and endangered fig (ficus carica l.) genotypes, 129 sivakumar r. impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.), 3 soufi s. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 srividhya s. impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.), 3 stanikzai s. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 stefani l. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 suzuki t. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 takaichi h. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 talento c. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 terlizzi m. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 valori f. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 wali adel m. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 yaqubi m.r. implications of investigating pollination and cross compatibil ity in the almond varieties of afghanistan, 217 yaqubi m.r. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 yaqubi m.r. phenotypic characterisation of almond accessions collected in afghanistan, 207 yaqubi m.r. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 zadran b. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 zahedi s.m. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 zarei m. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 zarghon a.h. implications of investigating pollination and cross compatibil ity in the almond varieties of afghanistan, 217 impaginato 90 adv. hort. sci., 2011 25(2): 90-98 received for publication 8 november 2011. accepted for publication 28 march 2011. the status of chestnut cultivation and utilization in the canary islands d. ríos-mesa*, s. pereira-lorenzo**, a.j.gonzález-díaz***, j.z. hernández-gonzález*, e. gonzález-díaz****, v. galán saúco**** * centro de conservación de la biodiversidad agrícola de tenerife (ccbat), servicio técnico de agricultura y desarrollo rural, cabildo, insular de tenerife. ** escuela politécnica superior, universidad de santiago de compostela. *** servizio de agricultura, cabildo insular de la palma. **** instituto canario de investigaciones agrarias (icia). key words: castanea sativa, food consumption, timber. abstract: chestnut was introduced to the canary islands at the beginning of the 16th century during the time of the spanish conquest. it was utilised by the conquerors as a means of claiming property for communal lands. from that time until today, chestnut has been an important crop in the canary islands. it is important both as a source of food and timber and has contributed to the subsistence of the population, particularly at times when both resources were scarce. nowadays it is mainly cultivated on the islands of tenerife (around 1300 ha) and la palma. on other islands, such as gran canaria, la gomera and el hierro, semi-wild chestnut forests and small plots where farmers collect fruit still exist. morphological and molecular marker (ssrs) studies have shown a great variability within the local population of chestnut trees in the canaries. the main use for chestnut is fruit consumption, but it was also utilised in the past as an exchange commodity to obtain fish and other food. the fruit is consumed in many different ways, mainly toasted or roasted, but also cooked in soups, fish or meat dishes and even as an ingredient for the typical canary islands’ sweet ‘morcillas’ (a type of sausage). the wood of the tree has been used for furniture, with some shoots being utilised for basket making, and also as cattle food. the trunk of the chestnut has also been used to obtain cork or as a bee hive. recent efforts to add value to chestnut cultivation in the canary islands have included the creation of a chestnut farmers association in tenerife that commercializes their products under a brand name. 1. historical background according to recent studies, chestnut was first introduced to the canary islands on el hierro and gomera and then on gran canaria, la palma and finally the island of tenerife, shortly after the process of conquest and colonization of the islands. in the case of la palma, it was most likely introduced around 1493 and quickly expanded and used both for its fruit and wood (ríos mesa, 2004). in fact, there is evidence that 5 ‘arrobas’ (a measure of weight, 1 arroba = 11.502 kg) of fruits were sent from la palma to the island of el hierro in 1546 (hernández-martín, 1999), which seems to indicate the presence of numerous chestnut trees to allow for surplus production. by 1590 frutuoso (2004) had mentioned the existence of chestnuts in the region of puntallana, and there are also many other reports about the presence of chestnut early after the conquest of the islands (hernández-rodríguez, 1983; browne, 2005). many ancient trees are still alive on la palma at present. two are especially important: one located in puntallana with circumference of more than 7 m, and another even larger one with a perimeter of 10 m in the municipality of breña alta. according to the information gathered from the local population, both of them seem to be of the denomination known as “temprano”, one of the few varietal denominations identical to those of spain’s mainland cultivars, which might be a clear indication of its early introduction. the great relevance of the chestnut tree on la palma is reflected in the information given by bandini (1816) in 1813, indicating that 46% of the total production of chestnuts in the canary islands (ci) came from that island. information provided by von fritsch (2006) on exports of chestnut fruits in 1862 from different areas of la palma also confirms the importance of this crop for the island. the role of this tree as part of the landscape of la palma was also highlighted in school texts used at the end of the 19th century (de las casas pestana, 1894). it is also important to mention that the first reference to grafting of chestnuts on la palma was 91 made during the last quarter of the 18th century (rodríguez-benítez, 2004). there are also historical references to the presence of chestnut trees on tenerife as early as 1517 (pereiralorenzo et al., 2007), soon after the conquest. many historical chestnut trees reported previously still survive on this island, such as the ‘castaño de las siete pernadas’, in the area of aguamansa (fig. 1), la orotava county (mendez pérez, 2002), which may be around 500 years old. there is an interesting description of this tree given by rodríguez (2001) in which he indicates that the trunk had a circumference of more than 12 m and that the seven branches of its trunk (siete pernadas) had been reduced to five by wind action. according to ríos-mesa (2004) it is possible that this tree was one of the chestnut trees planted by el adelantado (name given to the first governor of the island), d. alonso fernández de lugo, in the valley of la orotava in the first decades of the 16th century. many well known visitors and naturalists, among them mac-gregor (2005), verneau (2003), von humboldt (2005), du cane (1993), glass (1982), von fritsch (2006) and berthelot (2005), mentioned the presence of chestnut forests as part of the natural landscape of tenerife in their canary island trip reports during 18th and 19th centuries. on gran canaria: galdar, moya, san mateo, telde, teror. on la palma: barlovento, punta gorda, punta llana, san andres y sauces, santa cruz de la palma. on lanzarote: tinajo. on la gomera: vallehermoso. there are no updated figures on the economic importance of chestnut cultivation at a regional level at present. a thorough sampling done by pereira-lorenzo et al. (2001) indicated that chestnut could be introduced in the archipelago as a crop with a double use (fruit and wood) following the example of the spanish mainland from where it was introduced. this study also revealed the presence of isolated, testimonial chestnut trees on the drier eastern islands of lanzarote and fuerteventura. chestnut fruit is nowadays almost exclusively commercialized in a few areas on tenerife and la palma. recent efforts were made to increase the value of chestnut in the canary islands by the creation of a chestnut farmers association on tenerife to market all their products through a brand name. they even differentiate chestnuts harvested in the south one month earlier than those in the north due to microclimate differences, and which usually obtain the highest prices of the year. elorrieta (1949) indicated the presence of chestnut on all the western islands of the canaries but he did not report it as a crop. a recent detailed study carried out in 2006 and 2007 by hernández et al. (2010) localised chestnut trees in 28 counties on the island of tenerife, 23 of them with chestnut orchards. according to this study, the cultivated area of chestnut on tenerife is 1374 ha, with the biggest cultivated area (280 ha) located in la orotava county. in addition 2,567 isolated trees were also found on the island of tenerife. chestnut trees on tenerife are found between 400 and 1,800 m altitude in the southern part of the island (granadilla county). most of the area (647 ha) is located between 400 and 1000 m above sea level, and diminishes progressively after this altitude. only some isolated chestnut trees can be found below 400 m above sea level in the northern county of la laguna. chestnut distribution is different in north and south portions of tenerife. the higher rainfall and humidity on the north slopes due to trade winds, in contrast with a lack of the beneficial influence of them on the south side, explains why chestnut cultivation has been developed at higher altitudes in this latter area than on the north side (ríos-mesa, 2004; hernández et al., 2010). in northern tenerife the area covered by chestnut totals 1,121.72 ha (81.6% of total) and increases constantly from 400 m to 1,000 m above sea level, with a minimum area between 1,000 and 1,200 m. chestnut orchards are located mainly in the orotava valley and tacoronte-acentejo county. most of the 112 ha cultivated in the south are located in the guimar valley, more favoured by trade winds than other southern fig. 1 ancient chestnut tree with 7 main branches named castaño del las 7 pernada. 2. economic importance, cultivated area and geographical distribution chestnut seems to have been an important crop in the middle of the 19th century according to the great number of localities in the canary islands producing chestnuts by 1852 (de leon y falcón, 2005). on tenerife: arafo, candelaria, la matanza, la orotava, el rosario, santa ursula. 92 counties, between 800 and 1,000 m and only 5.6 ha are cultivated between 600 and 800 m. another 118 ha are located between 1,000 and 1,400 m altitude, most of them also in the guimar valley, being a merely testimonial presence of chestnuts at higher altitude (fig. 2). cultivation of chestnut on gran canaria was important years ago but is now only testimonial with isolated plots in the counties of arucas, firgas, valleseco and teror (naranjo-rodríguez and escobio-garcía, 2002). most chestnut trees on the island of el hierro are located in the most humid and cooler areas of el golfo, tiñor, asofa and honduras. chestnut trees are also found on la gomera in the higher parts of the valleys of the north where they are cultivated on mountain slopes. scarce, isolated trees on lanzarote are found in the area affected by the timanfaya volcanic eruptions (1730-1736) which may suggest a later introduction on this island and, as in the case of the other dry island of fuerteventura, a lack of appropriate climatic conditions for chestnut development (pereira-lorenzo et al., 2007). 3. horticultural aspects around 1852, de león y falcón (2005) indicated that chestnuts showed excellent growth on the ci at middle and higher altitudes in soils with a predominant clay, propagated either by seeds or from shoots emerging from their roots, with grafting seldom being practiced. in the case of tenerife, chestnuts are located in highly fertile acid soils, in most cases andisoles and alfisoles, and cultivated, whenever orography and planting density allow, together with other crops such as potato, rye, corn or others (ríos-mesa, 2004). only around 430 ha (31%) of the total surface covered by chestnut on the island of tenerife can be considered to be under good conditions of cultivation and 138 of these, distributed in different counties of the island, can be considered well managed. about 670 ha (48.7% of the total), concentrated mostly on the south side, are in a clear state of abandonment (hernández et al., 2010). in tenerife, extensive areas previously devoted to chestnut cultivation have been abandoned in recent years, although in many cases new trees have grown from seeds among the old grafted trees. these new plants are called chestnut machos or ladrones (males or thieves). the only cultivation practice carried out in these places consists of possible cleaning of the soil surface to facilitate harvesting of early chestnut fruits, perhaps being the only regular horticultural practice undertaken for chestnut cultivation in the different cultivated areas of the islands. plant spacing differs greatly from place to place but it is possible to consider three main systems (ríosmesa, 2004; hernandez et al., 2010). 1 trees planted at high density (30-50 trees/ha) in places where chestnuts were planted for fruit and that correspond with the areas now in a state of abandonment. 2 planted at lower density (15-30 trees/ha) allowfig. 2 chestnut trees in the south of tenerife. three different systems of chestnut cultivation can be found on tenerife. there is a first band at lower altitudes with low density plantings where chestnuts are associated with mixed plantings of vegetables and other fruit trees; a second band with more dense plantings where the lack of light penetration does not allow association with other crops; and the last band occurs at higher altitudes where chestnuts are largely associated with endemic fayal-brezal (myrica faya and erica arborea) or even pinus (pinus canariensis) populations that, in many instances, constitute a part of the native forest and cannot be considered cultivated trees (ríosmesa, 2004). chestnut is more uniformly distributed on the island of la palma with the maximum concentration of chestnut trees being on the west side of the cumbre nueva area in santa cruz de la palma and breña alta counties, where there is more humidity, also in this case, due to an influence of trade wind; on the north and east sides of the island chestnut is more important at middle altitudes. on the drier south and west sides of the island, chestnut as well as other temperate fruit crops is less present and located at higher altitudes. as in the case of tenerife, chestnut is associated with the native vegetation at higher elevations, with laurisilva and fayal-brezal in the north and east and pine forest in the west. frequent fires, the most serious of which occurred in august 2009, have caused the death of many chestnut trees or, on occasion, destruction of the grafted portion of the tree and regrowth of the rootstock (pereira-lorenzo et al., 2007). 93 ing intercropping with associated crops. 3 chestnut trees (10-15 trees/ha) planted only along the edges of plots used for cultivation of other crops such as potatoes, grapes, corn, cabbage, lupines or other vegetables (figs. 3 and. 4). all chestnuts are cultivated without irrigation and no fertilisers are applied, but in cases 2 and 3 above they benefit from the fertilisers applied to the main associated crop. grafting on tenerife (fig. 5) was done in a similar way to that on the spanish mainland, where seedling rootstocks were grafted with two scions placed at 0.5 m height. however peculiarly shaped shrubby chestnuts found in the volcanic south of the island with large pendulous branches touching the soil indicate that this practice was not always carried out. maintenance pruning is seldom practiced except in the case of planting system 3 indicated above. even in this case pruning is scarce and rudimentary, mostly limited to the removal of dead wood or wood that fig. 3 chestnut-corn association. fig. 4 chestnut-grape association. fig. 5 scionwood for grafting. makes the cultivation of the rest of the plot more difficult. there is, however, an important exception in the case of the trees cultivated on volcanic lapilli soils in the counties of arafo (tenerife) and el paso (la palma). in both places, grafting is practiced closer to the soil with a training process orientated at keeping branches as close to the soil as possible to facilitate harvest and to obtain wind protection. trees are also protected against wind on the island of el hierro where a circular wall of rocks, locally called gorona, is built to protect the tree from wind and animals. chestnuts on the ci are in good phytosanitary condition, with no reports of the serious diseases which usually affect this plant on the spanish mainland, such as phytophthora cambivora and phytophthora cinnamomi. this is most likely due to the isolation of the islands from other chestnut areas and no introduction of new plant material for new plantings. several arthropods, such as cydia splendana and balaninus elephas that attack chestnut fruits in the archipelago, are being studied (pereira-lorenzo et al., 2007). 4. plant material efforts made to localise and identify the plant material of chestnuts on the canary islands have only 94 recently been made through extensive samplings and collecting of material during field visits to interview farmers in the producing areas: on tenerife this was carried out between 2000 and 2004; on la palma between 2001 and 2004; on el hierro in 2001; and on la gomera and gran canaria in 2003. the main objectives of these surveys were to finalise the chestnut cultivar spanish inventory, realise the morphological and molecular characterization of ci chestnut plant material, to propose the material to be kept in a germplasm bank and to define the most interesting cultivars for commercial cultivation (pereira-lorenzo et al., 2007). a total of 47 different varietal nominations, according to the names given by farmers, with one or several accessions, were found on the ci (21 on tenerife, 17 on la palma, six on gran canaria, two on el hierro and one on la gomera) coinciding on many occasions with the same names on different islands like ‘blanco’ (white) on la palma and el hierro, ‘mollar’ (freestone) on tenerife, la palma, gran canaria, gomera and el hierro, and ‘negro’ (white), ‘picudo’ (pointed), ‘manso’ (tame) and ‘temprano’ (early) on tenerife and la palma. some new plant materials have been found in recent investigations carried out by the same authors (unpublished data): ‘piñuda’ (with the shape of a pine-cone), ‘menuda’ (small) and ‘merina’ (as the sheep race) on tenerife; ‘colorado’ (red) and ‘arrancado’ (pulled out) on la palma; and ‘chabetudo’ (unknown meaning) on gran canaria. the 39 accessions found on tenerife correspond to 21 varietal nominations (fig. 6), ‘mulato’ (mulatto) with seven accessions being the most common. the 34 accessions of la palma correspond to 17 varietal nominations, ‘jabudo’ (unknown meaning) with six accessions being the most common. from all the varietal nominations of these two islands, only ‘redondo’ (round) and ‘temprano’ coincided with names of cultivars from the spanish mainland, the former in galicia and the latter in andalucía and extremadura. only the plant material from tenerife and la palma has been characterised morphologically and phonologically, using the same methodology as for previous studies of the cultivars of galicia on mainland spain and allowing a comparison among them (pereiralorenzo et al., 1996 a, b, c; pereira-lorenzo and fernández-lópez, 1997 b, c; pereira-lorenzo et al., 2006). analogous molecular characterizations of plant material from tenerife and la palma have also been made following the same methodology as for the cultivars of galicia, comprising the results obtained from 10 microsatellites being utilised in the research project “evaluation, analyses and biodiversity management of castanea sativa mill. (european chestnut) in the atlantic regions (castaneareg)”, interreg iiib, espacio atlantico, feder, 2004-2006. to further allow comparison among cultivars, the four most discriminating morphological characteristics previously studied for the cultivars of galicia (pereiralorenzo, 1994 and pereira-lorenzo et al., 1996 a), favourably tested later for cultivars of different origin (pereira-lorenzo et al., 2006 and 2007) and also utilised under the framework of this same project, have been employed: i) fruit size; ii) fruit shape; iii) type of male flowering; and iv) length of burr spines. from the results obtained through this molecular characterization, it has been possible to group the 74 accessions of tenerife and la palma into 57 different genotypes. it is worth mentioning that, as in the case of the cultivars of galicia (pereira-lorenzo et al., 1996 b), andalucia (pereira-lorenzo and ramos-cabrer, 2003), león (ramos-cabrer et al., 2003) and asturias (pereira-lorenzo et al., 2005; 2006), the cultivars from the ci are all polyclones. the few cases in which more that one genotype was found under the same name seem to indicate that more than one clone of a cultivar might have been propagated. most of the genotypes found were singulars and only 10 groups of coincidence by microsatellites have been detected. when the ci genotypes were compared with those of the spanish mainland (data unpublished), no synonyms were found. all the cases of varietal denominations with only one accession found in the ci have been found to be of a single genotype different from all the others (pereira-lorenzo et al., 2007). 5. tradition and uses chestnuts were traditionally used as an important source of food in times of scarcity and also for wood of important value (pereira-lorenzo et al., 2007). among the different uses of this plant on tenerife, ríos-mesa (2004) specifies the following: the fruits larger ones used for fresh food, for family consumption, exchange or sale, the smaller ones as cattlefig. 6 fruits of castagrande cultivar. 95 feed. chestnut fruits are important for traditional cooking on the islands. they are consumed either boiled or roasted, or even fried in oil alone or accompanying salty fish (a very traditional dish on the islands), in purees or, more recently, in desserts. the wood used for house building, furniture, wine presses, staves for wine barrels and for the bottom of boats, it is also used for making boxes to store various local units of weight, cuartillas, and sowing quantities, almudes (fig. 7). the wood has also served to build small tools such as those used to blow cereals or to fan the grain. ungrafted trees have been preferred, generally, for wood purposes as they are straighter and present fewer nodes than grafted plants. it should be mentioned that the wood has always been cut when the moon is in “its last quarter”. despite early introduction of the tree to the ci chestnut wood was not used, at least on tenerife, for building wine barrels until the middle of the 17th century, as there are reports of imports of wood for that purpose from galicia and portugal (méndez pérez, 2002). chestnut suckers were also utilised to make baskets (fig. 8) of different sizes, such as hand baskets or bigger ones for transporting rocks and even those known as raposas placed on the sides of donkeys to carry various items. suckers also served to make horquetas, long wood sticks ending in a ‘v’ shape which first served to raise and hold the grapevines away from the soil and later in the 20th century for banana cultivation, both to keep the bunch separated from the pseudostem (small horquetas) or to give support to the whole plant in case of wind (bigger horquetas called horquetones). the dry leaves have been used as cattle beds and the green ones as cattle food. ríos-mesa (2004) indicates that chestnut is closely related to cultural aspects of ci life. chestnuts are an important element of the gastronomy linked to popular celebrations. special mention should be given to: the “all the saints feast” when the early chestnut fruits are roasted and consumed together with the “aguapié” (the first liquid extracted from the grapes after the men performed the traditional stomping of the grapes). the “saint andrews feast”, when chestnuts are eaten together with the new wine of the year when the cellars are first opened. at christmas time when chestnuts are considered important christmas symbols, like the spanish turrones (hard almond cakes). it is tradition in some villages to give a small basket full of chestnuts to children as a present. during carnival in some localities in the north of tenerife dry chestnuts are also consumed accompanying wine. for the “saint anthony feast” in the county of acentejo, in the north of tenerife, it is customary to use chestnuts as rosaries to be placed over domestic animals. chestnut wood has been used on the island of la palma for wine barrels and also for making receptacles to export fruits preserved in syrup, made with the sugar coming from the sugar cane industry, or to keep salty meat. they were also used as support poles for grape cultivation (pereira-lorenzo et al., 2007). peeled chestnut wood skin bands were used for building the structure and handles of baskets. several types of baskets, including bread baskets are also made, like those on tenerife (santos-cabrera, 2002). the wood is also appreciated for smoking cheese, a very traditional and delicious dish of the gastronomy of this island. the tender young shoots coming from the main trunks have been traditionally used as cattle feed. unifloral chestnut honey is produced in tenerife and la palma where sometimes bee hives are made of chestnut wood. in the past chestnuts were preserved fig. 7 box for sowing the grain made of chestnut. fig. 8 traditional harvesting basket made with chestnut wood. 96 buried in volcanic lapilli, but it was more common to cover them with sand or to dry them in the sun (pereiralorenzo et al., 2007). it is worth mentioning the historical use of chestnut trees as a means of claiming property for communal lands. it is indicated by rodríguez-brito (1982) that the planting of a chestnut tree in a deforested plot constituted the first step toward claiming it. the “feast of saint martín” (11 november) is the day chosen to drink the new wine and on tenerife it is accompanied by the consumption of chestnut fruits. the night before this feast, children dragged empty oil tins, “cacharros”, in the air which are open at the top and filled with chestnuts and coals. with an evident sense of humour, the old people’s association in the village of san juan de puntallana organise a contest a few days after “saint martín”, coinciding with the time of the chestnut harvest, to nominate the queen of chestnuts, the big chestnut, the small chestnut and the nice chestnut. women participate in the contest by bringing chestnuts collected by themselves or by their husbands. the woman that presents the biggest one is nominated as the queen of chestnuts, and similarly for the smaller or nicer fruits. in addition, the chestnut has erotic relevance on this island as it is used to refer to the female sexual organ (pereira-lorenzo et al., 2007). on the island of el hierro chestnuts have lost their traditional importance as food during autumn and winter except at the “feast of all saints”, when chestnut fruits are used as tafeñas (local chestnut grill) and consumed with new wine. in the past chestnuts were dried in the sun and used as ingredients for the preparation of “morcillas” (blood sausages), a local product still prepared on other islands. these morcillas were used by the inhabitants of the “el golfo” county as exchange for dry figs or potatoes coming from other counties of the island (gil-gonzález 1998). the use of chestnuts on the island of la gomera must have been similar to that of other islands, but in addition chestnut wood is used for making chácaras, a musical percussion instrument similar to the spanish castañuelas (perera-lópez, 2005) (note that chestnut in spanish is castaño.) as indicated above, the consumption of chestnut fruits is very common on the ci where they have been an important source of calories since soon after the spanish conquest of the islands. they are not normally consumed raw, except occasionally when they are very soft, but generally boiled or roasted (fig. 9). boiling is generally done after making a cut in the external shell or after eliminating it. they are cooked for half an hour in plenty of water to which anise, vanilla sticks or cinnamon is added, particularly when used for desserts (iglesias garcía, 2004). they are usually roasted in homes by putting them (after making a cut in the external shell) in a perforated biscuit tin which is then put directly on the kitchen fire. chestnuts are traditionally cooked in small metal boilers placed above charcoal fires on many street corners. in both cases, coarse salt is added during cooking. while roasted chestnuts are directly consumed, boiled chestnuts may be incorporated into typical dishes of the canary island gastronomy, such as roasted sardines, meat compositions or salty fish dressed with oil and vinegar or with the typical “mojo” (a sauce made with capsicum annun) to which they give a sweeter taste. they are also smashed into a puree, either to accompany a dish or as a filling for dishes prepared in the oven mixed with dry fruits (crushed pine nuts or almonds), spices and herbs or even previously fried giblets, adding a special flavour. in addition, they are a typical side dish for wild game (iglesias garcía, 2004), or as an ingredient in sweet ci bloody sausages. chestnuts may be consumed in a broth in the typical “caldo de castañas” (chestnut soup), cooked with potatoes or sweet potatoes, offering consistency and nutritive value which is much appreciated on cold winter days (ríos-mesa, 2004; pereira-lorenzo et al., 2007). fig. 9 traditional chestnut roaster. 97 chestnuts boiled with vanilla sticks are mixed, after removing all the skin, with syrup and eaten or ground as a filling for various sweets and cakes. one of the older recipes for these fillings includes ground chestnuts in addition to sugar, milk and beaten egg yolks. the mixture is cooked on a very slow fire for a few minutes, without allowing it boil. chestnuts are also used as ingredients in stewed fruit preserves. for this use chestnuts are boiled in abundant water, well cleaned to eliminate any remaining skin and then plenty of sugar is added along with anise, mint or hortelan (menta spitaca) and a glass of orange liquor or brandy. when the chestnuts become soft and thick, a syrup is formed; they are kept well covered in the syrup in sealed jars in a dry place (iglesias, 2004). 6. future prospects technical meetings have been organised since 2003 at la matanza municipality on the island of tenerife with the assistance of the centre for conservation of crop biodiversity of tenerife (ccbat) and the extension service. lectures by national and international experts have been given to include horticultural aspects such as pruning and training, marketing and preparation of chestnut products as well as economical and structural aspects and many other subjects dealing with chestnut cultivation on tenerife. special mention should be made of the process of recovery and adding of value to chestnut cultivation initiated in 2004 by the cabildo (local government) of the island of tenerife. this project was developed by the extension service and the ccbat have initiated “ex situ” and “in situ” conservation programmes and aims to create, in connection with the chestnut farmers association of tenerife, a quality label for chestnuts produced on tenerife. this initiative, if continued and extended to other islands, may make a great contribution toward the recovery of commercial cultivation of chestnuts on the canary islands. references 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canarias. 6ª edición. edición j.a. delgado luis y excmo. ayuntamiento de la orotava. la orotava. santa cruz de tenerife, spain. viera y clavijo j., 2004 diccionario de historia natural de las islas canarias. nivaria ediciones. tenerife, spain. von fritsch k., 2006 las islas canarias, cuadros de viaje. taller de historia, 40. von humboldt a., 2005 viaje a las regiones equinocciales del nuevo mundo y otros escritos. nivaria ediciones, santa cruz de tenerife, spain. impaginato 77 1. introduction in these last years, more consumers are interesting in alternative food systems which have grown in tandem with a proliferation of diverse food attribute labels, such as organic, local, humane, fair trade, etc. (berlin et al., 2009). in particular, locally products, such as fruits and vegetables, not offered by conventional grocery stores, are looking for niche markets, in country and urban areas. studies indicate that many consumers associate the local foods with higher perceived quality as well as increased freshness and naturalness of the products (denver and jensen, 2014). the appeal for local varieties, represented by ancient genotypes, is due to the major environmental hardiness (negri, 2005) able to tolerate extreme climate events such as an increase in temperatures and frequency or intensity of precipitation. this peculiarity makes ancient genotypes suitable for sustainable cultivations as support for farmers and consumers addressed to choose healthy products. fruits and vegetables are well recognized to be important source of vitamins, minerals, fibers and many hundreds of compounds with potential antioxidant activity which, acting against cellular oxidation reactions, may have beneficial effects on human health (halliwell, 1996). the antioxidant activity of fruits is strongly affected by the species and, within species, by the variety which is the main factor in determining fruit nutritional quality (scalzo et al., 2005). among other preand post-harvest factors influencing the antioxidant properties, such as cultivation techniques, ripening season, shelf-life and processing, the environmental conditions have an important role. in particular, it has been found that light intensity, temperature together with water availability are related to the antioxidant activity in different fruit species (lee and kader, 2000). the fruit nutritional quality of many commercially important varieties has been characterized, but still scanty is knowledge on the antioxidant properties of ancient genotypes that can show high quality performances, in terms of flavor and nutritional properties (donno et al., 2012; jakobek et al., 2013). recently, a trend to discover and to reintroduce into marketplace genotypes native of a restricted geographical adv. hort. sci., 2017 31(2): 77-84 doi: 10.13128/ahs-21086 chemical characterization and sensory analysis by blind and visually impaired people of local peach varieties s. bartolini 1 (*), r. viti 2, e. ducci 1 1 istituto scienza della vita, scuola superiore sant’anna, piazza martiri della libertà, 33, 56127 pisa, italy. 2 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124, pisa, italy. key words: ancient varieties, physicochemical traits, prunus persica (l.) batsch, total antioxidant capacity, total phenols. abstract: the interest in locally produced foods by the reintroduction of old varieties is due to their environmental hardiness and suitability for low-input agricultural systems. these managements can often produce fruits with imperfections, preventing the consumer acceptance. we have settled a new sensory evaluation going beyond the appearances, involving blind and visually impaired people to provide a quality evaluation of fruits linked to intrinsic rather than exterior characteristics. the research was conducted over two consecutive harvest seasons on three peach old local varieties (‘alberta’, ‘mora di dolfo’ and ‘regina di weinberger’ called in loco ‘regina di bember’) grown in central italy. on fruits, physicochemical traits (fruit weight, peel and flesh color, flesh firmness, ph, total soluble solids, titratable acidity), antioxidant content (total antioxidant capacity and total phenols) and sensory analysis were assessed. the three local peach varieties showed interesting fruit attributes in both studied growth-ripening seasons. the white-fleshed ‘mora di dolfo’, characterized by the highest antioxidant contents, was particularly appreciated by panellists for its aroma. the new sensory analysis, providing an evaluation based on judgment of intrinsic characteristics of peaches, emerges as a valid tool to assess the interest and appreciation of fruits for a conscious consumer’s choice. (*) corresponding author: susanna.bartolini@santannapisa.it received for publication 13 december 2016 accepted for publication 11 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 77-84 78 area took place (krawitzkyab et al., 2014). the local varieties, mainly managed by low-input agricultural systems, often produce fruits with defects, preventing the consumer acceptance (donno et al., 2012). it has been showed that the appearance of fresh fruits is the primary criterion in making purchasing decisions (kays, 1991). to overcome this approach we have settled a new sensory evaluation going beyond the appearances, involving blind and visually impaired people (bartolini et al., 2015 a), who having a higher sense of taste and smell (luers et al., 2014), can provide a quality assessment of fruits linked to intrinsic rather than exterior characteristics. the aim of this research was to determine some fruit quality traits of three peach local varieties originated from lucca province (central italy). physicochemical and antioxidant parameters were studied, and the innovative sensory analysis involving people with visual disability was introduced. 2. materials and methods plant material and cultivation site the research was conducted over two consecutive harvesting seasons (2014-15) on full bearing peach trees of old local varieties: ‘alberta’, ‘mora di dolfo’ and ‘regina di bember’. this latter variety is an example of synonymy; its name was modified by local citizen from the original ‘regina di weinberger’ (bartolini et al., 2015 a), a californian variety widespread at the end of fifties which nowadays has lost importance at national level. the orchards were located in a traditional area at high agricultural vocation (tuscany, lucca province, lat. 44.02 n, long. 10.27 e) and trees were grown in similar soil conditions, using low input farming practices. the main climatic parameters, minimum-maximum temperatures and rainfall data were provided by the regional agro-meteorological service of florence (sir) (http://www.sir.toscana.it). at physiological maturity stage, samples of peaches were collected and analyzed to determine the main physicochemical, antioxidant and sensory traits. physicochemical analysis from each fruit (n = 30 per variety), measurements of weight, peel and flesh color, flesh firmness (ff), total soluble solids (tss), ph and titratable acidity (ta) were determined. fruit weight was expressed according to the following size categories (della strada et al., 1984): very small (≤90 g), small (91-125 g), medium (126-160 g), large (161-195 g), very large (>195 g). peel and flesh color was evaluated using color charts according to upov code (international union for the protection of new varieties of plants, geneva) for peach. the red cover color area of peel (cc) was visually evaluated and expressed as percentage. firmness was assessed with a manual penetrometer on two peeled opposite areas at the equatorial region of peach, using an 11-mm-wide plunger (model 53200sp tr, tr-turoni & c. inc forlì, italy). tss were measured using a refractometer (model 53015c tr, tr-turoni & c. inc forlì, italy) and expressed in °brix at room temperature. the fruit ph was recorded with the help of an electronic ph meter, and ta was determined on fruit juice by titrating a known volume of juice with 0.1n sodium hydroxide (naoh) to ph 8.1. ta was expressed as milliequivalents of malic acid per 100 grams of fresh weight (meq malic ac. 100 g fw-1). total antioxidant capacity (tac) and total phenol (tp) analysis total antioxidant capacity (tac) and total phenols (tp) analyses were carried out on the same fruits that had been previously subjected to the physicochemical determinations. fresh fruit samples (flesh with peel) of 3 g (in triplicate) were immediately frozen and stored at -20°c until extraction. the samples were homogenized using an ultra-turrax blender at 4°c to avoid oxidation. the extraction was performed in 80% ethanol for 1 h in a shaker in the dark and subsequently centrifuged at 4°c for 10 min at 2600 g. the supernatant was used for tac (total antioxidant capacity) and tp (total phenol) analysis. tac was evaluated using the improved trolox equivalent antioxidant capacity (teac) method (arts et al., 2004). the teac value was calculated in relation to the reactivity of trolox, a water-soluble vitamin e analogue that was used as an antioxidant standard. in the assay, 40 μl of the diluted samples, controls or blanks added to 1,9690 μl abts•+ solution, resulted in a 20-80% inhibition of the absorbance. the decrease in absorbance at 734 nm was recorded 6 min after an initial mixing, and plotted against a dose-response curve calculated for trolox (0-30 μm). antioxidant activity was expressed as micromoles of trolox equivalents per gram of fresh weight (μmol te g fw −1). trolox was purchased from sigma chemical co. (st. louis, mo). tp content was determined according to the improved folin-ciocalteu (f-c) method (waterhouse, 2001). the assay provides a rapid indication of the bartolini et al. chemical characterization and sensory analysis of local peach varieties 79 antioxidant status of the studied material and is valuable for different food samples. the standard compound for the calibration curve was gallic acid (ga, sigma chemical co, st. louis, mo). total phenol content was calculated as milligrams of ga equivalent (gae) per gram of fresh fruit weight (mg gae g fw−1). the absorbance of the blue colored solutions was read at 765 nm after incubation for 2 h at room temperature. sensory evaluation the sensory evaluation was carried out on fruits of the varieties ‘alberta’ and ‘mora di dolfo’ as they have the same harvest time (end of july) but different characteristics, such as taste, ground peel (yellow and light-green) and flesh color (yellow and white), respectively. the test was performed without visual inspection, in collaboration with blind and visually impaired persons of the ‘blind and visually impaired people italian association’ of lucca (italy). this is an original experiment on stone-fruits realized for ‘going beyond the appearances’ that, to our knowledge, is the only one after an experience carried out in sicily (http://www.consorzioparsifal.it). taking into account the disability of the assessors-consumers, a tentative of protocol was established using an acceptability test, as a pertinent option for specific applications (varela and ares, 2012). in both years, the same 10 blind and visually impaired persons, aged between 26 and 65, performed the evaluations on the selected varieties. in order to make the evaluation phase easier, prior the acceptability test a short training stage (5 hours) was provided outlining methodology and procedure of the sensory method. a room equipped with individual sites was used. to each panellist, room-tempered and washed halfunpeeled anonymous peaches were served in randomized order, and samples were evaluated in one session. the panellist were invited to rinse the mouth with mineral water and eat saltless bread between samples, in order to avoid tiring effects. the fruit attributes evaluated were the following: shape, size, texture, aroma, sweetness, acid taste, juiciness (shinya et al., 2014). moreover, participants were asked to assess the fruit global appreciation. a continuous non-structured 9-point hedonic scale was utilized for evaluation: 1= dislike extremely; 2= dislike very much; 3= dislike moderately; 4= dislike slightly; 5= neither like nor dislike; 6= like slightly; 7= like moderately; 8= like very much; 9= like extremely (porretta, 2000). the blind judges were requested to indicate the score for each attribute and they were supported by sighted persons helping them to fill the data form. judges were not informed about the characteristic of fruits such as peel and flesh color. statistical analysis physicochemical data of each variety were compared using a student’s t-test analysis with two treatments (2014 and 2015 harvest seasons); for each season, 30 repetitions/parameter were used, excluding ta and ph, where three composite samples containing ten fruit per sample were considered. a two-way anova analysis were performed to test the effects of year and variety on the main physicochemical and antioxidant parameters. differences on the content of antioxidants and phenolics between the three varieties were investigated with the analysis of variance (anova), using tukey’s post hoc test at p≤ 0.05. a correlation analysis of antioxidant capacity versus total phenol content was calculated and correlation coefficient was reported in terms of goodness of fit. for the sensory evaluation, taking judges as factors, results were compared through the student’s t-test analysis between harvest seasons. spearman’s correlations were performed to estimate relationships among sensory and physicochemical traits of fruits. data are reported as means ± standard errors of the means (sem), and the analysis were all performed using the statistical package graphpad prism 5 (graphpad software, inc.). 3. results and discussion weather conditions during the fruit growth, from may to july, the considered harvest seasons were characterized by different weather conditions, in terms of temperatures and rainfall events (table 1). in particular, at the final stages of fruit ripening the strongest differences were observed: the amount of precipitation was consistently higher in 2014 when, only on july, more than 300 mm of rainfall occurred. this condition was unusual for lucca area in comparison to table 1 monthly minimum and maximum temperatures (°c) and cumulative rainfall (mm) from may to july, over a 2-year period (2014-2015) month 2014 2015 t. min. t. max. average rainfall t. min. t. max. average rainfall may 11.8 23.7 17.8 39.8 13.0 24.5 18.8 34.4 june 16.0 29.4 22.7 48.0 16.8 30.2 23.5 29.8 july 17.4 28.7 23.2 314.6 20.7 33.9 27.3 7.4 average 15.1 27.3 21.2 16.8 29.5 23.2 total rainfall 40.4 71.6 adv. hort. sci., 2017 31(2): 77-84 80 average values of the last 10 years, ranging from 8.6 to 60.2 mm (sir toscana). the cumulative rainfall of the early summer seasons (may-july) was about 400 and 72 mm in 2014 and 2015, respectively. the second year was particularly drought and warm by mean temperatures of 4-5°c more than 2014. physicochemical and antioxidant fruit parameters harvest time and mean values of the main physicochemical traits of the peach varieties, recorded over a 2-year period, are presented in table 2. under the environment of lucca area, the harvest time occurred between the first (‘regina di bember’) and third decade of july (‘alberta’ and ‘mora di dolfo’). considering the physical traits of fruit, all varieties showed attractiveness: ‘alberta’ was characterized by fruit of medium size with yellow ground peel (1050% red cover color) and deep yellow flesh; ‘mora di dolfo’ had a large fruit size, light-green peel (3050% red cover color) and white flesh; ‘regina di bember’ had a medium-large fruit size, yellow ground peel, (50-90% extensive red cover color) and deep yellow flesh (fig. 1). within each variety, values of chemical parameters showed variations between years, particularly for soluble sugars (tss), titratable acidity (ta) and ph. ‘regina di bember’ had the greatest differences in relation to the weather conditions occurred during the ripening period of the considered years. tss ranged from about 9 to 12 °brix, and ta changed between 8 and 6 meq malic ac. 100 g fw-1 in the wettest (2014) and driest (2015) year, respectively. as a consequence, the tss/ta ratio varied greatly with the lowest values in the wettest year. a similar trend was recorded in ‘alberta’ where ta changed while tss did not differ as much. no changes in chemical parameters were observed in ‘mora di dolfo’, performing well under the rainy climatic conditions of 2014. in both years, fruits exhibited similar values in tss (11.8-13.9 °brix), ta (9.6-9.8 meq malic ac. 100 g fw-1) and, as a consequence, in their ratio. the anova results comparing harvest year and variety effect showed significant interactions ‘year x variety’ for ta and tss/ta (table 3). during the latter stages of fruit development, the influence of weather *, **, ***: significant at p≤0.05, 0.01, 0.001, respectively. ns= not significant. table 2 harvest time, peel and flesh color, red cover color (cc, %), and main physicochemical parameters recorded on three local peach varieties, over a 2-year period: fruit size, flesh firmness (ff, kg/0.5 cm2), total soluble sugars (tss, °brix), ph, titratable acidity (ta, meq malic ac. 100 g fw-1), sugars/acids ratio (tss/ta) harvest time ‘alberta’ ‘mora di dolfo’ ‘regina di bember’ end july end july early july peel color yellow, 10-50% cc light green, 30-50% cc yellow, 50-90% cc flesh color deep yellow white deep yellow fruit size medium large medium-large 2014 2015 2014 2015 2014 2015 ff 1.2±0.3 1.7±0.1 3.9±0.5 5.0±0.4 2.8±0.5 3.3±0.4 tss 10.7±0.4 11.6±0.2 11.8±0.9 13.9±0.4 9.2±0.4 12.2±0.7 * ph 3.80±0.1 4.15±0.1* 3.71±0.0 3.75±0.0 3.81±0.0 3.76±0.0 ta 10.2±0.1 6.1±0.1 * 9.6±0.1 9.8±0.1 8.3±0.1 6.2±0.1 * tss/ta 1 1.9 * 1.2 1.4 1.1 2.0 * fig. 1 the peach old local varieties ‘alberta’, ‘mora di dolfo’ and ‘regina di bember’ (originally ‘regina di weinberger’). table 3 two-way anova results. variables: tss (°brix), ta (meq malic ac. 100 g fw-1), tss/ta, tac (μmol te g fw-1), and tp (mg gae g fw-1) main effects tss ta tss/ta tac tp p year 0.0001 *** 0.0015 ** < 0.0001 *** 0.6980 ns 0.2792 ns variety 0.0015 ** 0.0001 *** <0.0001 *** <0.0001 *** <0.0001 *** interaction year x variety 0.5636 ns 0.0389 * 0.0016 ** 0.0113 * 0.2500 ns mean±sem. within each variety, asterisk indicates significant differences between years by student t-test (p≤0.05). bartolini et al. chemical characterization and sensory analysis of local peach varieties 81 conditions on pomological and quality properties has been proved: among fruit quality parameters, soluble solids content can vary substantially from year to year, and rainfall and summer sunshine have been identified as the main affecting factors (choi et al., 2003). in peach, extensive rain may reduce the sweetness, while severe water stress or regulated irrigation deficit techniques caused a soluble sugar increased, as a recognized physiological response to stress (kwon et al., 2008). as regards tac (fig. 2a), related to several bioactive compounds such as phenolics, flavonoids, anthocyanins and vitamin c (gil et al., 2002), the considered local varieties showed higher values than those reported in literature for commercial varieties of which, generally, only the flesh was analysed (scalzo et al., 2005). by the contrary, our investigation assessed samples constituted by flesh with peel, fraction of fruit with a key role in determining the antioxidant properties of the whole fruit (remorini et al., 2008; zhao et al., 2015). in both years, significant differences were observed: ‘mora di dolfo’ was characterized by the highest tac levels, on an average of about 14 μmol te g fw–1; ‘alberta’ and ‘regina di bember’ had similar average values (8.5-9.5 μmol te g fw–1). the tp content (fig. 2b), likewise tac results, was the highest in ‘mora di dolfo’ (about 2 gae mg fw–1), while the other varieties showed values from 0.7 to 0.9 gae mg fw–1. the relevant antioxidant content of the white-fleshed variety ‘mora di dolfo’ is in agreement with works reporting the tendency of white-fleshed cultivars to have significantly higher antioxidant content than yellow-fleshed peaches and nectarines (cantìn et al., 2009). a significant relationship between tac and tp content was found (fig. 3), confirming phenolic compounds as important contributors to the antioxidant activity of peach fruits (karav and eksi, 2012). the similar tac and tp values observed between years suggest that different rainfall amounts, occurred during the last growth-ripening period of fruit in 2014 (wet year) and 2015 (dry year), did not impact on the antioxidant power of varieties. these interesting results are not in agreement with studies carried out on several fruit species, where a variability in antioxidant levels in relation to climatic conditions has been found. in particular, the water availability markedly determined changes in fruit nutritional properties, and a linear significant inverse relationship between water status and antioxidant content was reported by several authors (leccese et al., 2010; laribi et al., 2013; bartolini et al., 2015 b). also in peach (cv. suncrest) the irrigation stress induced a fig. 3 linear regression between total antioxidant capacity (tac) and total phenols (tp) of the studied varieties (2year period). fig. 2 (a) total antioxidant capacity (tac) and (b) total phenols (tp) in peach fruits of local varieties recorded over a 2year period. values (means ± sem) are expressed for tac as μmol of trolox equivalent per g of fresh weight (μmol te g fw-1), and for tp as mg of gallic acid equivalent per g of fresh weight (mg gae g fw-1). different letters indicate significant differences at p≤0.01. adv. hort. sci., 2017 31(2): 77-84 82 higher biosynthesis of phytochemical compounds (tavarini et al., 2011). analysis of variance (table 3) showed that variation in tac and tp between varieties was much greater than between harvest years, indicating that genotype plays a more important role than growing season in influencing antioxidant content in peach, as found in other fruit species (howard et al., 2003; leccese et al., 2012). the significant interaction ‘variety x year’ for tac revealed that environmental growing conditions may impact the capacity of genotype to synthesise antioxidants which are influenced by biotic and abiotic factors (biesiada and tomczak, 2012). although investigation on the behavior of these varieties has to be continued, the high antioxidant capacity of peaches can be considered as a part of cultivar value (dalla valle et al., 2007). this peculiarity would be crucial for breeding strategies selecting genotypes with enhanced antioxidant levels which may provide health benefits to consumers. moreover, the availability of peach hardy genotypes will be a key factor for future, in view of the climate variability responsible of extreme event increases, such as drought or intense rainfall. sensory evaluation the sensory evaluation, carried out by blind and visually impaired panellists over a 2-year period, was conducted on varieties ‘alberta’ and ‘mora di dolfo’, characterized by yellow and white flesh color, respectively. considering that color and exterior feature of fruits are nowadays important commercial sensorial traits to attract consumers (kays, 1991), the judgment carried out by sightless assessors allowed to attain a real intrinsic quality evaluation of fruits. ‘going beyond the appearances’ revealed its importance particularly under the wet growing season of 2014; in fact, this condition, favoring the presence of defects on peach peel, could have prevented the consumer’s acceptance. on the basis of panellist’s appreciation, expressed as average degree of linking (1-9), the morphological parameters of fruits (shape and size) were similarly evaluated in both 2014 and 2015 (data not shown), while the organoleptic descriptors (acid taste, aroma, texture, juiciness, sweetness) were differently assessed depending on the harvest season (table 4). concerning the global appreciation, the lowest score was attributed in the year (2014) characterized by exceptional rainy events over the ripening period. these conditions could have negatively influenced components linked to the sensory profile, in agreement with authors who reported that high levels of water before harvest can reduce organoleptic quality and consumer liking degree (navarro et al., 2010). in fact, blind and visually impaired panellists expressed the best agreement for peaches sampled in 2015 when summer drought conditions occurred. most of descriptors showed higher scores and, particularly in ‘mora di dolfo’, the global appreciation was associated with an increase of aroma and juiciness, as observed in other peach varieties (di miceli et al., 2010). moreover, this variety could seem less susceptible to unfavorable climatic conditions since positively judged also in 2014 (score 6.2), although chemical attributes (tss, ta) were similar to ‘alberta’ that, instead, had a lower score (5.2). it has been established that a different sensory perception of peaches could be linked not only to basic organic components (sugars, organic acids, fibers, micro and macro elements) but in great part to the volatile compounds, which define the flavor impact (bononi et al., 2012). the correlation coefficient (table 5) between global appreciation and some sensory and physicochemical traits of peaches showed that panel’s acceptability was highly and positively correlated with aroma, sweetness and tss. moreover, these attributes were significantly correlated each other scores were based on a nine-point hedonic scale: 1= extremely dislike; 5= neither like nor dislike; and 9= extremely likewithin years asterisk indicates significant differences between varieties by student t-test (p≤0.05). table 4 mean scores (acid taste, aroma, texture, juiciness, sweetness and global appreciation) recorded for peach varieties ‘alberta’ and ‘mora di dolfo’, over a 2-year period variety/year ac. taste aroma texture juiciness sweetness glob. appr. 2014 ‘alberta’ 4.0±0.5 5.4±0.7 5.8±0.4 5.8±0.6 5.4±0.7 5.2±0.6 ‘mora di dolfo’ 4.8±1.1 6.2±0.8 5.4±0.9 6.8±0.5 6.2±0.8 6.2±1.0 2015 ‘alberta’ 2.8±0.5 5.8±0.6 5.0±0.5 7.0±0.4 5.7±0.7 6.0±0.4 ‘mora di dolfo’ 3.3±0.6 7.7±0.2 * 5.3±0.5 8.0±0.1* 7.3±0.5* 7.3±0.3 * table 5 coefficients of correlation between scores of global appreciation and some sensory and physicochemical traits of peach fruits attributes r spearman acid taste 0.1 aroma 0.99* juiciness 0.8 sweetness 0.99* texture -0.4 tss 0.99* tss/ta 0.4 significant coefficients are denoted by an asterisk at p≤0.05. bartolini et al. chemical characterization and sensory analysis of local peach varieties 83 (table 6), confirming tss as one of the most important quality indicators in determining acceptance judgments for peaches, due to its influence on the perceived sweetness (crisosto et al., 2006). on the contrary, texture appeared inversely related to global appreciation. the comparison between sensory and the main chemical attributes showed significant relationships: sweetness and aroma and total soluble sugars content were directly related each other. 4. conclusions the three local peach varieties showed interesting quality traits as well as a very high antioxidant content in both studied growth-ripening seasons. the capacity to maintain the bioactive components of fruits also under unfavorable weather events confirms the importance of old genotypes to be more stable in comparison to modern ones (donno et al., 2012). the white-fleshed ‘mora di dolfo’, characterized by the highest tac and tp content, was particularly appreciated by panellists for its excellent eating quality denoted by aroma, sweetness and well balanced sugar/acid ratio. although fruits of this variety are perishable, like the most white-fleshed peach varieties, it could represent a valid hardiness genotype for farmers who are focused to establish local markets where consumers are obviously willing to pay a premium price for niche products. this variety, as source of appreciable attributes, could be useful for specific breeding programs to develop new peach cultivars combining quality traits, hedonic and enhanced nutritional value, actually of high relevance for the consumer. the new sensory analysis, carried out by blind and visually impaired assessors, provided a quality evaluation linked to intrinsic rather than exterior characteristics of peaches that could prevent consumers if defects in appearance are present. this type of test could represent a valid tool to assess the interest and appreciation of fruits for a conscious consumer’s choice, going beyond the appearances, for a reintroduction of interesting ancient peach varieties characterized by unconventional fruit quality traits. acknowledgements research partially supported by ‘comune di capannori (lucca)’. thanks to ‘unione italiana ciechi e ipovedenti di lucca’ for kindly participation to the panel test. references arts m.j.t.j., dallinga j.s., voss h.p., haenen g.r.m.m., bast a, 2004 a new approach to assess the total antioxidant capacity using the teac assay. food chem., 88: 567-570. bartolini s., leccese a., viti r., 2015 b quality and antioxidant properties of apricot fruits at ready-to-eat: influence of the weather conditions under mediterranean coastal area. j. food process technol., 7(1): 1-6. bartolini s., 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china. int. j. mol. sci., 16: 5762-5778. 100 1. introduction ozone is a major gaseous pollutant in the troposphere and ozone concentrations have in recent years increased rapidly in developing asian countries. indeed, the emission of anthropogenic nitrogen oxides (ozone precursors) in asia under a no-further-control scenario was predicted to increase by 350% between 1990 and 2020 (aunan et al., 2000). an elevated ozone concentration will reduce the growth and yield of crop plants including rice, the most important food crop in asia (kobayashi et al., 1995; yonekura et al., 2005). many researchers have described the mechanisms responsible for visible injury on plant leaves by acute ozone exposure (reviewed by kangasjarvi et al., 2005). the primary mechanism is oxidative damage caused by an increase in levels of reactive oxygen species (ros). however, the cause for yield reductions under chronic ozone stress remains unclear. in a previous report we described how ozone sensitivity in evaluated rice cultivars, in terms of visible injury (chlorotic or necrotic lesions), did not coincide with that indicated by the grain yield reduction (sawada and kohno, 2009). in addition, conventional evaluation of chronic ozone effects relies on measurements such as growth and yield reductions, which require large-scale studies (e.g. in a field or greenhouse) and long time periods (e.g. about six months). a rapid and small-scale method for early evaluation of chronic ozone effects, such as the use of molecular markers, would make it faster, easier, and less expensive to select ozone-tolerant cultivars. kubo et al. (2011) reported that during ozone stress, sakuranetin, a flavonone in the phytoalexin family, appears to serve as a molecular marker of the stress response. sakuranetin contents in rice leaves exposed simultaneously to ozone and high temperature increased only in the three cultivars whose grain yield was unaffected by ozone stress. however, their experiment was performed under both elevated ozone and elevated temperature, making it difficult to determine the separate effect of each factor. moreover, the ozone concentration was 150 nl l-1 (ppb), much higher than ambient ozone levels. therefore, more practical markers are needed. proteomic studies are useful to reveal protein markers associated with various stress tolerance (reviewed by kosova et al., 2011). in a previous study, we conducted differential proteome analysis using three rice cultivars that showed different levels of ozone sensitivity (indicated by the reduction in grain yield) when exposed to elevated ozone during the cultivation season in open-top chambers (sawada et al., 2012). in these cultivars, we observed significant changes in the size of spot that contained three proteins: a 60-kda chaperonin (cpn-60), chloroplastic atp synthase, and enolase 1. the change in size of this spot was proportional to their ozone sensitivity, measured as the reduction in grain yield. these results suggest that these proteins are closely involved in the mechanisms that underlie the yield reduction potential marker proteins for ozone-induced yield reduction in rice h. sawada*(1), s. komatsu**, m. tamaoki***, y. kohno* * environmental science research laboratory, central research institute of electric power industry (criepi), 1646 abiko, abiko-shi, chiba 270-1194, japan. ** national institute of crop scinence, 2-1-18 kannondai, tsukuba, ibaraki 305-8518, japan. *** center for environmental biology and ecosystem studies, national institute for environmental studies, onogawa 16-2, tsukuba, ibaraki 305-8506, japan. key words: grain yield, ozone stress, protein markers, 60-kda chaperonin. abstract: three proteins a 60-kda chaperonin (cpn-60), chloroplastic atp synthase, and enolase 1 were evaluated as potential markers of ozone-induced yield responses in six rice (oryza sativa l.) cultivars (‘kirara 397’, ‘koshihikari’, ‘nipponbare’, ‘takanari’, ‘kasalath’, ‘suphanburi 90’) under ozone stress in laboratory-scale tests. the levels of all three proteins decreased after ozone exposure in cultivars identified as ozone-sensitive while they increased or remained constant after ozone exposure in tolerant cultivars, although atp synthase tended to decrease. furthermore, the protein level and grain yield in each cultivar exposed to ozone were significantly positively correlated for all three proteins. thus, cpn-60 and enolase 1 are potential markers for chronic ozone stress in rice. adv. hort. sci., 2014 28(2): 100-104 (1) corresponding author: sawada.hiroko@nies.go.jp received for publication 12 march 2014 accepted for publication 16 june 2014 101 that occurs under elevated ozone levels, and therefore they have potential as molecular markers that can predict the ozone-induced yield loss. to clarify the usefulness of these proteins for use in laboratory-scale tests, we investigated the levels of these candidate proteins in the seedlings of six rice cultivars under short-term ozone exposure in growth chambers, and tested for a significant correlation between the protein levels and relative grain yield. 2. materials and methods chronic ozone exposure six rice (oryza sativa l.) cultivars were used in this study: ‘kirara 397’, ‘koshihikari’, ‘nipponbare’ (japonica cultivars), ‘takanari’ (a hybrid indica cultivar), ‘kasalath’, ‘suphanburi 90’ (indica cultivars). seedlings (n = 40) of each cultivar were grown in seedling boxes for three weeks in a glasshouse under ambient atmospheric conditions, then transplanted into pots (at four plants per pot with a 0.05-m2 surface area and a 0.015m3 volume) in open-top chambers (otcs; 3.6 × 3.6 m) at an experimental field of the akagi testing center of the criepi (maebashi, japan) in the late spring of 2007, 2008, and 2009. fertilizer was supplied at a rate of np 2 o 5 -k 2 o=15-15-15 g m-2. the otc fumigation system has been described previously (frei et al., 2011). ozone was added in the chambers using a mass-flow controller combined with a pid controlling system to maintain the designated concentrations. three ozone-level treatments were established, from transplanting of rice plants into the pots to harvest, for three years with a regular diurnal pattern: charcoal-filtered air (cf), ambient ozone (ozone ×1), and twice ambient ozone (ozone ×2). concentrations of ozone were continuously monitored in each chamber at 3-min intervals using a uv absorption ozone analyzer (ml9810, monitor labs, englewood, co, usa). mean ozone concentration, air temperature and relative humidity in the different treatments are summarized in table 1. measurement of the yield the rice cultivars were harvested between september and november in 2007, 2008, and 2009. harvesting of each cultivar was conducted when about 80% of the grains had turned yellow. after harvesting, grains were separated from the panicles and categorized into two groups (filled and unfilled grains) using an automatic seed-sorting machine (fv-459a, fujiwara seisakusho kk, tokyo, japan). the filled grains (rough rice) were weighed to determine the grain yield. short-term ozone exposure rice seedlings were grown in indoor growth chambers at 28/23°c (day/night), photosynthetic photon-flux density of 400 μmol m-2 s-1, with a 12-h photoperiod, and a relative humidity of 60±5%. after two weeks, the ‘kirara 397’, ‘koshihikari’, and ‘takanari’ seedlings were exposed to three levels of ozone (12 h/day) for three days in three individual replicates: cf, ambient ozone (40 ppb), and twice ambient ozone (80 ppb). similarly, ‘nipponbare’, ‘kasalath’, and ‘suphanburi 90’ seedlings were exposed to cf and 40 ppb of ozone. ozone was generated with a silent electrical discharge in dry oxygen. the concentration of ozone in the chambers was monitored continuously during exposure with a uv absorption ozone detector (model 1150, dylec inc., tokyo, japan). at the end of the exposure, we removed the third leaves, immediately froze them in liquid nitrogen, and stored them in -80°c until the immunoblot analysis was performed. immunoblot analysis leaves (100 mg) were homogenized in sodium dodecyl sulfate (sds) buffer (10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 2.3% (w/v) sds, and 62.5 mm trishcl, ph 6.8). equal amounts of protein samples were separated using 15% sds-polyacrylamide gel electrophoresis (page). after the sds-page, the protein samples were transferred onto a polyvinylidene fluoride membrane or they were stained by coomassie brilliant blue (cbb). table 1 ozone concentrations and environmental conditions in the open-top chambers during the cropping seasons of rice ozone concentration (ppb) temperature (ºc) 24 h mean relative humidity (%) 24 h mean12 h mean 24 h mean mean daily maximum 2007 cf 3.1 2.1 4.1 – – ozone x1 37.6 31.1 61.3 – – ozone x2 68.6 56.3 101.7 – – 2008 cf 4.7 3.9 6.5 21.1 83.4 ozone x1 40.4 27.5 57.6 21.3 83.5 ozone x2 82.7 57.0 118.3 21.3 81.7 2009 cf 5.1 5.0 9.7 20.6 78.7 ozone x1 35.1 27.9 56.9 20.7 78.9 ozone x2 73.5 54.7 110.2 20.9 77.4 measurements of environmental conditions and ozone concentrations were recorded at 3 and 10-minute interval throughout the experiment, respectively. average values of the two replicate chambers per treatment are shown. 12 h means were calculated for the period from 6:00 to 17:59 hours. the temperature and relative humidity were not measured in 2007. 102 the blotted membrane was blocked for 1 h in tbs-t (20 mm tris-hcl, ph 7.6, 150 mm nacl and 0.1% v/v tween-20) containing 5% (w/v) nonfat milk (skim milk; difco, sparks, md, usa). the membrane was subsequently incubated with the monoclonal antibody anti-heatshock protein 60 (acris antibodies gmbh, herford, germany), with the polyclonal antibodies anti-atp synthase β-subunit (antiprot, pullach i. isartal, germany), and antienolase (aviva system biology, san diego, ca, usa) at 1:5000 dilutions for 1 h at room temperature. as secondary antibodies, we used anti-mouse or anti-rabbit igg with conjugated hrp (bio-rad laboratories inc., hercules, ca, usa). after incubation for 1 h with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibodies, we detected the immunoblot signals using the ecl plus western blotting detection kit (ge healthcare, piscataway, nj, usa) following the manufacturer’s protocols and the results were visualized using an las-3000 luminescent image analyzer (fujifilm, tokyo, japan). the relative intensities of the bands were calculated using pdquest software (version 8.0.1, bio-rad). 3. results and discussion after chronic ozone exposure during three years of growing seasons (from 2007 to 2009) each cultivar showed a similar yield response to ozone in all years of the experiment. the grain yields of ‘kirara 397’, ‘takanari’ and ‘kasalath’ decreased significantly by 15 to 36%, 10 to 21%, and 12 to 19%, respectively, under twice the ambient ozone level (about 80 ppb treatment, daily 12-h mean concentration), although the grain yields did not differ significantly from cf under ambient ozone level (about 40 ppb treatment), except for ‘kirara 397’ and ‘takanari’ in 2007 (fig. 1, p <0.05). the grain yields of ‘koshihikari’, ‘nipponbare’, and ‘suphanburi 90’ did not decrease significantly with ozone stress. on this basis, we defined ‘kirara 397’, ‘takanari’, and ‘kasalath’ as ozone-sensitive cultivars, and ‘koshihikari’, ‘nipponbare’, and ‘suphanburi 90’ as ozone-tolerant cultivars. to confirm whether cpn-60, atp synthase, and enolase 1 can be used as markers for ozone-induced rice yield loss in laboratory-scale tests, we analyzed the levels of these proteins (fig. 2a). levels of cpn-60 decreased significantly after three days of exposure to 40 (the ambient concentration) and 80 ppb (twice the ambient concentration) of ozone in ‘kirara 397’ and 80 ppb of ozone in ‘takanari’ (fig. 2b, p<0.05). levels of atp synthase and enolase 1 tended to decrease after ozone exposure, although not significantly (except for ‘takanari’ exposed to 80 ppb of ozone), in both ‘kirara 397’ and ‘takanari’. these cultivars also showed lower grain yield under ozone exposure (fig. 1). in contrast, levels of cpn-60 and enolase 1 in ‘koshihikari’ exposed to 40 ppb of ozone increased significantly and remained the same compared with the levels in cf (p <0.05). moreover, enolase 1 production also remained constant in ‘koshihikari’ at 80 ppb ozone exposure. the level of atp synthase tended to decrease after ozone exposure in ‘koshihikari’. because the levels of cpn-60 and enolase 1 decreased and increased at 40 ppb ozone exposure in ozone-sensitive and ozonetolerant cultivars, respectively, ‘kasalath’, ‘nipponbare’, and ‘suphanburi 90’ seedlings were exposed to cf and fig. 1 effects of chronic ozone exposure on the grain yields of six rice cultivars in 2007, 2008, and 2009. values are mean ± se (n= 40). asterisk indicates a significant difference compared with cf according to dunnett’s test (p<0.05). ‘kasalath’ and ‘suphanburi 90’ were not cultivated in 2007 and 2009, respectively, and yields are shown as “nc”. 103 40 ppb of ozone. the levels of all three proteins increased significantly or were maintained under 40 ppb of ozone in ‘nipponbare’ and ‘suphanburi 90’, but tended to decrease in ‘kasalath’. therefore, the levels of cpn-60 and enolase 1 differed between the ozone-sensitive and ozone-tolerant cultivars: they decreased and increased, respectively at least at 40 ppb. cpn-60 is a molecular chaperone. many molecular chaperones were originally identified as heat-shock proteins (hsps), which function in protein folding, assembly, fig. 2 (a) immunoblot analysis of cpn-60, atp synthase, and enolase 1 in leaves of rice seedlings exposed to charcoal filtered air (cf), or to 40 or 80 ppb of ozone. immunodetection was performed with antibodies specific to these proteins. (bottom panel) cbb-stained sds-page showing the quality and loading quantity of the protein samples. (b) relative intensity for each protein estimated from the immunoblot analysis in panel (a). values are mean ± se (n = 3). asterisk indicates a significant difference compared with cf according to dunnett’s test or t-test (p<0.05). nb, nipponbare; sp90, suphanburi 90. translocation, and degradation during many normal cellular processes, and can assist in protein refolding under stress (wang et al., 2004). cpn-60 (hsp60) appears to be involved in the defense response that mitigates oxidative stresses (wang et al., 2011). enolase 1 is an enzyme involved in glycolysis in the cytosol. bohler et al. (2007) suggested that the enzymes involved in glycolysis increase to produce more energy and to increase the reduction capacity for detoxification of ros and repair oxidative damage in response to ozone stress in the leaves of poplar (populus). in arabidopsis thaliana, cpn60b (at1g55490), encoding homologous protein to cpn-60 in rice, was upregulated in response to drought, uv-b, heat, wounding and oxidative (methyl viologen) stress within 30 min (winter et al., 2007). similarly, eno2 (at2g36530) in a. thaliana, encoding homologous protein to enolase 1 in rice, was upregulated in response to cold, drought, uvb, wounding and heat stress (winter et al., 2007). these studies suggest that cpn-60 and enolase 1 are induced by stresses involved in the production of ros. therefore, the alterations of these protein levels with ozone exposure might result in ozone-derived ros rather than ozone itself. however, there has been no report describing whether cpn-60 and enolase 1 influence grain production in crops under environmental stress, although these proteins might not be specific markers to ozone. further studies will be needed to clarify the relationship between the reduction in grain yield and decreased production of these proteins by ozone-sensitive cultivars. in order to compare the relative levels of each protein upon short-term ozone exposure with the relative grain yields under chronic ozone exposure we performed a linear regression analysis (fig. 3). we found significant positive correlations between the levels of cpn-60, atp synthase, and enolase 1 and the relative grain yield (i.e. yield decreased as the protein concentrations decrease). therefore, the three proteins may serve as potential markers for chronic ozone stress in rice, although further experiments will be required for atp synthase that also tended to decrease in ‘koshihikari’ at 40 ppb ozone exposure (fig. 2b). the level of cpn-60 had the highest goodness of fit (r2 = 0.786) with the grain yield. this suggests that the potential ozone-induced yield reduction can be evalu104 ated using the level of cpn-60 at the seedling stage in laboratory-scale tests. moreover, the protein markers that we identified in this study may be useful in crop breeding to quickly select ozone-tolerant rice varieties. vincent et al. (2007) indicated that the inhibition of shoot growth was best correlated with the level of cpn-60 in two wine grape cultivars exposed to salinity and water deficit stress, suggesting that the protein marker is also applicable to other plant or crops. acknowledgements this work was supported by the environmental research and technology development fund (a0806) of the ministry of the environment, japan. references aunan k., berntsen t.k., seip h.m., 2000 surface ozone in china and its possible impact on agricultural crop yields. ambio, 29:294-301. bohler s., bagard m., oufir m., planchon s., hoffmann l., jolivet y., hausman j.f., dizengremel p., renaut j., 2007 a dige analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism. proteomics, 7: 1584-1599. frei m., kohno y., wissuwa m., makkar h.p.s., becker k., 2011 negative effects of tropospheric ozone on the feed value of rice straw are mitigated by an ozone tolerance qtl. global change biol., 17: 2319-2329. kangasjarvi j., jaspers p., kollist h., 2005 signalling and cell death in ozone-exposed plants. plant cell environ., 28: 1021-1036. kobayashi k., okada m., nouchi i., 1995 effects of ozone on dry matter partitioning and yield of japanese cultivars of rice (oryza sativa l.). agric. ecosyst. and environ., 53: 109-122. kosova k., vitamvas p., prasil i.t., renaut j., 2011 plant proteome changes under abiotic stress contribution of proteomics studies to understanding plant stress response. j. proteomics, 74: 1301-1322. kubo a., cho k., rakwal r., kohno y., shibato j., 2011 method for evaluating influence of ozone on rice using sakuranetin. japan patent kokai, 2011-33533. sawada h., kohno y., 2009 differential ozone sensitivity of rice cultivars as indicated by visible injury and grain yield. plant biol., 11(suppl. 1): 70-75. sawada h., komatsu s., nanjo y., khan n.a., kohno y., 2012 proteomic analysis of rice response involved in reduction of grain yield under elevated ozone stress. environ. exp. bot., 77: 108-116. vincent d., ergul a., bohlman m.c., tattersall e.a.r., tillett r.l., wheatley m.d., woolsey r., quilici d.r., joets j., schlauch k., schooley d.a., cushman j.c., cramer g.r., 2007 proteomic analysis reveals differences between vitis vinifera l. cv. chardonnay and cv. cabernet sauvignon and their responses to water deficit and salinity. j. exp. bot., 58: 1873-1892. wang c.r., gu x.y., wang x.r., guo h.y., geng j.j., yu h.x., sun j.a., 2011 stress response and potential biomarkers in spinach (spinacia oleracea l.) seedlings exposed to soil lead. ecotoxicol. environ. saf., 74: 41-47. wang w., vinocur b., shoseyov o., altman a., 2004 role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. trends plant sci., 9: 244-252. winter d., vinegar b., nahal h., ammar r., wilson. g.v., provert n.j., 2007 an “electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. plos one, 2: e718. yonekura t., shimada t., miwa m., arzate a., ogawa k., 2005 impacts of tropospheric ozone on growth and yield of rice (oryza sativa l.). j. agric. meteorol., 60: 1045-1048. fig. 3 regression analysis for the relative levels of cpn-60, atp synthase, and enolase 1 in rice seedlings exposed to 40 and 80 ppb ozone, plotted as a function of relative grain yield. the grain yields are relative to the values for six cultivars grown in opentop chambers. significance levels: **, p< 0.01; *, p< 0.05. impaginato 45 1. introduction the genus hydrangea l. includes 23 species, mainly distributed in the american and asiatic continents (mcclintock, 1957). it is a very popular ornamental plant for both garden and interior design and has recently been commercialized as a high value fresh or dried cut flower. interest in hydrangeas is mainly due to the striking coloration of its inflorescences (corymbs or panicles), that range from pink, blue, white, to light purple or dark purple. flowers are produced from early spring to late autumn and have two inflorescence morphologies: ‘mophead’ with large flowers forming spherical flower heads; and ‘lacecap’ that resemble round, flat flower heads with a center core of subdued, fertile flowers surrounded by outer rings of showy, sterile flowers. to further increase their popularity, new hybrids and cultivars should be developed. in flowering plants, the main objective of selective breeding is to increase genetic variability in ornamental traits such as flower color, flower shape and plant form. to achieve this objective, intraand inter-specific hybridizations have been widely used in breeding programs. hybrids between h. macrophylla (thunb.) ser. and h. paniculata sieb. were produced using embryo rescue, but the resulting plants were sterile and lacked vigor (reed et al., 2001; reed, 2004). in vitro embryo rescue procedures have been used to facilitate the recovery of interspecific hybrids of many genera (bridgen, 1994; sharma et al., 1996), and have recently been used to recover a putative h. macrophylla (thunb.) ser. x h. arborescens l. hybrid (kudo and niimi, 1999 a, b). hybrid embryos often resume growth and develop into normal plants when removed from ovules and placed on an aseptic nutrient media. this procedure is known as in ovule adv. hort. sci., 2017 31(1): 45-51 doi: 10.13128/ahs-20725 development of pollination and in vitro germination techniques to improve the hybridization in hydrangea spp. g.a. venturieri 1, b. nesi 2, s. lazzereschi 2, s. pecchioli 2, g. burchi 2 1 ufsc/cca-fit, federal university of santa catarina, campus itacorubi, rod. admar gonzaga, 1346 itacorubi, cep 88034-000, florianopolis (sc), brazil. 2 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, unità di ricerca per il vivaismo e la gestione del verde ambientale ed ornamentale (crea-viv), via dei fiori, 8, 51017 pescia (pt), italy. key words: breeding, fertilization barriers, fruit cut system, hortense, seed disinfection, sowing system. abstract: hydrangea is a genus of ornamental plants which is gaining new markets mainly as a fresh or dried cut flower, but it is also important as a pot plant and for landscaping. to expand its market, new hybrids should be developed. to increase the hybridization efficiency, some techniques were developed and tested: i) evaluation of two pollination systems; ii) comparison among fruit-cut systems before in vitro cultivation to develop embryos and to allow the growth of new genotypes; iii) evaluation of seed disinfection systems for in vitro germination; iv) sowing systems using seeds and fruits from stocks cultivated in two environments. to increase interand intra-specific hybridization, pollination by dispersion of previously collected pollen on the top of a corymb by a brush was more effective than pollination using the corymb itself as a brush. a longitudinal cut system can be considered the best treatment to be applied on fruits before in vitro cultivation to allow growth of seedlings. sterilization of seeds can be done by immersion in a solution of commercial bleach for 5 minutes on ms culture medium with ppm®. when stocks are cultivated in greenhouses, in vitro contamination is lower and seeds have a better rate of germination. the results of these experiments were applied in a breeding program on hydrangea using sexual crosses. (*) corresponding author: giorgini.venturieri@ufsc.br received for publication 5 october 2016 accepted for publication 15 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 45-51 46 embryo culture (reed, 2000). in vitro germination of seeds is a common technique used to overcome incompatibility barriers in ornamental plant hybrids (eeckhaut et al., 2006 a, b; lazzereschi et al., 2012; nesi et al., 2012). since most hydrangea seeds are so small (diameter of 0.5 mm) and hybrid seeds production is usually low (0-5 seeds/fruit), so, the development of a successful in vitro method for hydrangea seed germination would be an important tool for breeding programs. to increase seed germination, greer and rinehart (2010) have developed an in vitro method for cultivation and assay of h. macrophylla (thunb.) ser. and h. paniculata sieb. seeds, through germination on solid media in conjunction with plant preservative mixture (ppm®, plant cell technology, inc., washington, dc, usa), and by sterilizing seed with trichloro-s-triazinetrione (trichlor). the objective of the present study was to develop and test several techniques for the generation and recovery of hybrids in hydrangea. these included: two pollination systems (using a brush or an inflorescence on the top of the corymb); different ovary cut systems, aimed at embryo and seed rescue; different seed disinfection systems for in vitro germination (evaluating the time of immersion in a bleach solution and the addition of plant preservative mixture ppm® in media); comparison among three sowing systems using seeds and immature fruits in aseptic conditions, and in compost in climatized beds, from stocks cultivated in two environments (in a greenhouse and under shading net). the outcomes of these experiments were used to determine best practices for the hybridization of hydrangea species using sexual propagation. 2. materials and methods the experiments were carried out at the council for agricultural research and economics landscaping plants and nursery research unit (creaviv) in pescia (pt) (43° 49’ 00” n; 10° 48’ 00” e), italy. at crea-viv, a germplasm collection of hydrangea, composed of 66 genotypes belonging to h. macrophylla ssp. macrophylla (hortensia and lacecap group), h. paniculata, h. serrata, h. villosa, h. quercifolia, h. anomala ssp. petiolaris, h. arborescens, h. heteromalla, h. involucrata, h. aspera and three genotypes of schizophragma hydrangeoides, was maintained. some trials were also arranged in sanremo (im) (43°49′ n; 7°47′ e) with some genotypes replicated from the crea-viv collection. selected cultivars of this collection were used for hybridizations. during the summer of 2014, crosses among different genotypes, belonging to h. macrophylla ssp. macrophylla (38 cultivars), h. macrophylla ssp. serrata and h. paniculata (5 cultivars each); h. arborescens, h. aspera, h. quercifolia and h. involucrata (one cultivar for each species) were made (table 1). then, the capsules obtained from controlled pollinations were collected and used as starting material in the experiments, as described below. pollination systems twelve crosses, involving h. macrophylla, h. arborescens and h. quercifolia, randomly distributed in a germplasm collection, were subjected to two pollination systems (treatments). before pollination, sterile flowers and all extremely immature fertile were removed from inflorescences to be used as females prior to opening of the fertile flowers. after, the petals and anthers of all remaining fertile flowers were removed and the inflorescence was covered with a breathable plastic bag. inflorescences to be used as males were also bagged prior to flower opening. pollination experiments were performed 1 to 4 days following emasculation. the two pollination treatments included: a) pollination by the dispersion of previously collected pollen on the top of a corymb, aided by a brush; b) a simple dispersion of pollen using the corymb as a brush, where anthers presenting freshly-dehisced pollen were touched with the exposed stigmas of the table 1 plant material used for intraand inter-specific crosses between different genotypes of hydrangea spp. species cultivars h. macrophylla ssp. macrophylla ‘alberta’, ‘alpen gluhen’, ‘ayesha’, ‘benxi’, ‘bianca ceriana’, ‘dienemann’, ‘elbatal’, ‘endless summer’, ‘etoille violette’, ‘europa’, ‘first red’, ‘grattino’, ‘green shadow’, ‘hanaby’, ‘harlequin’, ‘intermezzo’, ‘lake san markos’, ‘lanarth white’, ‘lemon wave’, ‘libelle’, ‘magical coral’, ‘magical garnet’, ‘magical jade’, ‘magical noblesse’, ‘masja’, ‘myharayama yae’, ‘nymphe’, ‘paris’, ‘red beauty’, ‘renate wate’, ‘san baronto’, ‘schnball’, ‘seour therese’, ‘sibilla’, ‘tricolor’, ‘white first’, ‘zorro’ and ‘kardinal’ h. macrophylla ssp. serrata ‘acuminata’, ‘blue bird’, ‘miranda’, ‘preziosa’ and ‘yae-no-amacha’ h. paniculata limelight’, ‘phanthom’, ‘pink diamond’, ‘unique’, and ‘vanilla fraise’ h. arborescens ‘annabelle’ h. aspera ‘rowellane’ h. involucrata ‘yorakutama’ h. quercifolia ‘snow queen’ venturieri et al. pollination and in vitro germination techniques applied to hydangea hybridizations 47 emasculated flowers. after pollination, inflorescences were covered again with the bags which remained on the plants until fruit collection. effect of treatment was evaluated by the number of developing fruits on bagged female flowers. a t test was used to compare averages between treatments (sokal and rohlf, 1981). in total, one hundred and thirty-five hand pollinations were carried out in pescia, and 33 in sanremo, using a different subset of hydrangea species. each species cross was repeated 3 times. embryo and seed rescue from immature fruits approximately 90 days after pollination (dap), well-developed fruits (n = 444), obtained from the breeding program were used to investigate rates of embryo and seed rescue from immature fruits. fruits were sterilized in a solution of commercial bleach (5% of active chlorine), in distilled water (1:2, v/v) plus 2 μl/100 ml tween20® (sigma, st. louise, mo, usa) for 10 minutes. subsequently four ovary cut systems (treatments) were applied: a) stigma off stigmatic branches were removed; b) longitudinal cut stigmatic branches were removed and a longitudinal cut, from the top of the fruit up to approximately to peduncle insertion, was made; c) equatorial cut stigmatic branches were removed and a transversal cut at the larger diameter of the fruit was made (both sections were cultivated); d) top cut 1/3 of the distal part of the fruit was removed (fig. 1 a-d). fruits were cultured in a one-half strength murashige and skoog (ms) medium (murashige and skoog, 1962) supplemented with 1 mg/l of naphthalene acetic acid (naa), 1 ml/l of ppm®, 30% sucrose, 6 g/l of agar and ph was adjusted to 5.8. fruits were individually placed in test tubes. six replication were used by cross. all vials were cultivated in a chamber maintained at 23±1°c under 16 h/day photoperiod provided by fluorescent tubes at 35 µmol/m∙s. ovaries that were considered as contaminated or dead were counted 7 days after, and their proportion (p) was a square root of transformed arcsine (ayres et al., 2007) and evaluated using analysis of variance. averages were compared by fisher lsd test of significance for α= 0.05 (ayres et al., 2007). values were expressed as a percentage. physical attributes of germinated seedlings were described for each treatment. disinfection of seeds for in vitro germination fruits considered as mature (130±10 dap) were collected and left to dry on a laboratory bench, and then grinded carefully using a mortar and pestle to liberate seeds from capsules. the obtained mass was passed through 0.71 mm mesh of a soil gradation sieve (grade 25, giuliani tecnologie, scientific instrument, torino, it) to remove large debris, and used in two disinfection experiments as described below. sterilization as a function of time immersion in bleach solution. about 0.025 g of the mass of seeds and debris was placed in a piece of tnt envelope and submerged for the disinfection process (fig. 2 a). the envelopes were dipped in 70% ethanol for 30 sec, followed by sterilization in a commercial bleach solution, as already as described above, for 5, 10, 20, and 30 minutes and rinsed in sterilized distilled water twice (fig. 2 b). then the envelopes were opened (fig. 2 c) and their contents were laid on a one-half ms culture medium supplemented with 1 mg/l of naa, 1 ml/l of ppm®, and 30% sucrose, and adjusting ph to 5.8. tnt was used to cover petri dishes (fig. 2 d), and then cultures were placed in the chamber as described above. germinated seeds and contamination of petri dishes were evaluated after 15 days. frequency of contaminated and uncontaminated petri dishes that contained germinated seeds was used to evaluate the effect of contamination on gerfig. 1 graphic representation of the different cuts applied to the immature fruits of hydrangea: a) stigma off; b) longitudinal cut; c) equatorial cut; d) top cut (fig. 1 a-d); e) development of new plantlet from immature fruits in vitro cultured. adv. hort. sci., 2017 31(1): 45-51 48 mination by fisher’s test. spearman rank order correlation was calculated between number of germinated seeds and disinfection time (ayres et al., 2007). sterilization as a function of duration in bleach solution and ppm® concentration in the media. in this experiment, culture medium and in vitro sowing system were the same as described above, but using a factorial design of two variables: ppm® in the medium at 3 different concentrations (2, 4 and 6 ml/l) and four disinfection times (5, 10, 20 and 30 minutes). about 0.025 g of the mass of seeds was utilized to the disinfection process. each treatment was repeated 3 times. the number of germinated seeds per petri dish and the proportion of contaminated petri dishes were used as parameters. a factorial analysis of variance was performed and averages were compared by fisher lsd test of significance for α=0.05. sowing systems using seeds and fruits from stocks cultivated in two environments seeds were sown in pescia, using material from two environments (pescia and sanremo), following three different systems. from 130 different crosses made in pescia, seeds from 33 crosses were sown in vitro, well developed but still immature fruits, from 22 crosses were cultivated in vitro and seeds from 130 crosses were sown in organic compost (peat and perlite, 1:2, v/v) on artificially climatized beds, inside a greenhouse. from 34 different crosses made in sanremo, seeds from 19 crosses were sown in vitro, well developed but still immature fruits from 24 crosses were cultivated in vitro and seeds from 34 crosses were sown in the same compost and conditions as described above. about 0.025 g of the mass of seeds was utilized to in vitro sowing for each combination of cross. three ovaries of each cross were in vitro cultivated, and of the total mass of seeds were sown in organic compost. each sowing system was repeated 3 times. all in vitro germinations were carried out in fallwinter of 2013 and compost germination in summer of 2014. due to the difficulty in counting the number of seeds sown inside a fruit or inside a mass of sieved seeds and debris, the comparison was based in number of germination events that happened per treatment. a nonparametric χ2 statistical analysis was applied, and where significant intergroup differences were found, multiple comparisons were conducted using the partitioning χ2 test to differentiate between treatments, both for α=0.05 (ayres et al., 2007). 3. results pollination systems according to the t-test, pollination using a brush with pollen collected from the corymb (average of 7.6 fruits per inflorescence cross), showed a mean significantly higher than using a corymb as a brush (average of 4.8 fruits per inflorescence cross) (p= 0.001). although, the time needed for pollination using a brush is considerably greater than that using only a corymb. embryo and seed rescue from immature fruits no statistical significance (p=0.07) was observed in the rate of contamination between different cut treatments. a lower percentage of contamination was observed in the treatment “stigmas off” (fig. 1 a), probably due to less damage in the fruit tissues compared to the other cutting systems (table 2). the number of dead fruits was not affected by the different fruit cut systems applied (p=0.50). cut system were further differentiated based on variation in germination behavior. in the treatments “stigmas off” and “top cut”, fruits swelled but seedlings did not emerge from fruit, suggesting poor germination. furthermore, some seedlings were confined inside the fruit and did not develop and grow outside of the fruit. in the “equatorial cut” treatment, two portions of the fruit were grown: seeds were able to germifig. 2 disinfection process of seeds obtained from mature capsules harvested: (a) tnt envelops with seeds plus debris inside; (b) envelops dipped in the disinfection solution; (c) staples were taken off and envelop opened; (d) seeds plus debris were layered on medium and tnt was left to cover seeds. venturieri et al. pollination and in vitro germination techniques applied to hydangea hybridizations 49 nate and develop new seedlings from the lower portion, but not from the upper portion. this suggests a potential loss of seedlings. conversely, the treatment “longitudinal cut” (fig. 1 b) allowed the seeds to germinate and seedlings promptly grew and developed upright (fig. 1 e). disinfection of seeds for in vitro germination sterilization as a function of time immersion in bleach solution. no statistical differences were observed in the number of contaminated petri dishes between genotypes (p=0.37) or duration of disinfection period (p=0.22). seed germination was lower in contaminated petri dishes only, where sterilization time was positively correlated with the number of germinating seeds (r s of spearman=0.51; p=0.01) (table 3). sterilization as a function of duration in bleach solution and ppm® concentration in the media. seed immersion duration in bleach solution did not significantly affect the rate of contamination (p=1.00); however, a significant difference was observed for ppm® concentration (p≈0.00). no interaction between these two variables was observed (p=1.00). therefore, seeds surface sterilization with bleach solution was not enough to prevent contamination, but sterilization was achieved only with the addition of ppm®. contamination occurred only when 2 ml/l of ppm® were added to the culture medium; when a higher concentration of ppm® was used, no contamination was observed at any time of immersion in the solution of bleach (table 4). so, to sterilize seeds for in vitro germination, the use of a culture media with 4 ml/l ppm® and a sterilization of 5 minutes is recommended. sowing systems using seeds and fruits from stocks cultivated in two environments the average percentage of germination in pescia was 30.5%; and in sanremo, 55.8%. inside the environment “pescia”, no statistical differences were observed between sowing systems (p=0.8, d.f.=2), but in “sanremo” there were differences (p≈0.00 d.f.=2). partitioning χ2 tests revealed that treatments in the sanremo environment were all statistically different from each other (p<0.007, d.f.= 1), with immature fruits cultivated in vitro demonstrating the highest percentage of germination among treatments (fig. 3). 4. discussion and conclusion in the present paper, we use several experiments to define work strategies and priorities to hybridize table 2 proportions of contaminated and dead fruits, by ovary cut system, and behaviour of eventual germinated seeds from survived fruits (z) values followed by the same letter in each column do not differ statistically for α=0.05. table 3 effect of application of bleach solution in function of duration of disinfection by different hydrangea genotypes (z) symbols: ○ uncontaminated petri dishes; ● contaminated petri dishes; ♣ number of germinated seed. table 4 evaluation of ppm® concentration on the number of hydrangea spp. germinated seeds/per petri dishes, and proportion of contaminated petri dishes (z) averages and percentage (by column) followed by the same letter do not differ at α>0.001. cutting systems sample size (n) contamination (%) death (%) behaviors of germinated seedlings stigma off 150 31 a z 31.3 a fruits swelled but seedlings could not emerge from fruit and develop longitudinal cut 150 34 a 38.0 a seeds germinated promptly, grew up and develop upright e q u a t o r i a l cut 72 38 a 29.2 a germinated seed developed but none from the top slices, suggesting potential seedlings loses top cut 72 49 a 31.9 a some seedlings were confined inside the fruit and did not emerged genotype duration of disinfection (minutes) contaminated petri dishes/ genotype5 10 20 30 h. macrophylla spp. macrophylla proc. izu ohoshima ○z ○ ○ ○ 0 h. macrophylla spp. macrophylla proc.takeoka chiba ○ ○ ○ ○ 0 h. involucrata proc. yamamae yoko tama ○ ○ ○ ○ 0 h. macrophylla ‘libelle’ x h. paniculata ‘limelight’ ● ● ●(♣) ●(♣♣♣♣♣) 4 h. involucrata ‘myharayama kokonoe tama’ x h. macrophylla ‘alberta’ ● ● ● ●(♣♣♣) 4 h. macrophylla ‘libelle’ x h. macrophylla ‘europa’ ●(♣) ●(♣) ● ● 4 total of contaminated petri dishes/duration of disinfection 3 3 3 3 ppm® concentration (ml/l) germinated seeds/petri dishes (n) contaminated petri dishes (%) 2 5.5 a z 66.7 a 4 3.8 a 0 b 6 4.0 a 0 b adv. hort. sci., 2017 31(1): 45-51 50 hydrangea species using sexual propagation. the aim of the project was to hybridize all species involved in a breeding program, therefore pollination and in vitro germination techniques had to be developed to maximize efficiency. to efficiently produce fruits of interand intraspecific hybrids, pollination by the dispersion of previously collected pollen on the top of a corymb aided by a brush resulted in better pollination than using the corymb itself as a brush. nevertheless, pollination using only the corymb was used for the purpose of the present project because it was faster and more convenient. however, the hybrid origin should be certified using molecular markers when seedlings are established or at flowering, based on morphological characters. based on the germination behaviour, the “longitudinal cut” system applied to fruits prior to their transfer to the culture medium is recommended to promote vigorous growth of seedlings. it is considered as the best system because seeds readily germinated and developing seedlings promptly grew upright. similar responses were also observed in the production of interspecific hybrids of lilium longiflorum thunb. and l. × elegans (roh et al., 1996). seed surface sterilization for in vitro germination with bleach solution was not enough to prevent contamination, but sterilization was achieved with the addition of ppm®. in some plant species, germination is favoured by the presence of microorganisms, usually attributed to rhizobacteria (saharan and nehra, 2011), but seldom adapted to in vitro germination of ornamental plants except in orchids (tsavkelova et al., 2007); this probably could be an explanation of what happened in the case of hydrangea seeds. for the objectives of the present study, however, it would not be an advantage because almost all culture media were completely covered by fungal mycelia causing seedlings to collapse within the first week following germination. germination in fruits showed a higher number of successful germinations than the other two systems. the highest proportion of germination success, observed for “in vitro inside fruit with a longitudinal cut for embryo and seed rescue” system, could be due to supplementary nutrients, given by the medium, that ensured development of hybrid seeds without endosperm (eeckhaut et al., 2006 a and b). nevertheless, specific studies of endosperm lacking in seeds from hydrangea hybrid crosses are unknown. the number of germinated seeds in pescia was lower than in sanremo: in pescia the stock plants were cultivated under shading net while in sanremo in greenhouse conditions. in fact, the contamination of explants cultivated in vitro can be associated to management of stock plants. pollination system and in vitro germination techniques have allowed us to obtain a number of new individuals with potential ornamental traits. currently this material is under selection, providing the basis for the development of a hydrangea breeding program in course at crea-viv at pescia italy. acknowledgements the authors thank to drs. francesco ferrini and laura mugnai, throughout the international cooperation agreement between the federal university of santa catarina brasil and the university of florence italy; to the council for agricultural research and agricultural economics analysis/ landscaping and nursery plants research unit, at pescia italy, for the laboratories and support. we also thanks for the brazilian government, by the program science without border from capes/cnpq, for the grant given for the first author. and we also acknowledge dr. alistair campbell for linguistic advice. references ayres m., ayres j.m., ayres d.l., santos a.a.s., 2007 bioestat 5.0.: aplicações estatísticas nas áreas das fig. 3 events with successful germination of potential hydrangea spp. hybrids by different sowing systems, in two environments. columns, by environment, followed by the same letter do not differ statistically for α=0.05. venturieri et al. pollination and in vitro germination techniques applied to hydangea hybridizations 51 ciências biomédicas. sociedade civil mamirauá, belém, pará-brasil, pp. 324. bridgen m.p., 1994 a review of plant embryo culture. hortscience, 29: 1243-1245. eeckhaut t., van huylenbroeck j., van laere k., van bockstaele e., 2006 b interspecific hybridization in woody ornamentals: how to deal with barriers? acta horticulturae, 725: 117-126. eeckhaut t., van laere k., de riek j., van huylenbroeck j., 2006 a overcoming interspecific barriers in ornamental plant breeding, pp. 540-551. in: da silva j.a.t. (ed.) floriculture, ornamental and plant biotechnology. advances and tropical issues. vol. i. global science books, uk, pp. 646. greer s.p., rinehart t.a., 2010 dormancy and germination in vitro response of hydrangea macrophylla and hydrangea paniculata seed to light, cold-treatment and gibberellic acid. jeh, 28(1): 41-47. kudo n., niimi y., 1999 a production of interspecific hybrids between hydrangea macrophylla f. hortensia (lam.) rehd. and h. arborescens l. j. jpn. soc. hort. sci., 68(2): 428-439. kudo n., niimi y., 1999 b production of interspecific hybrid plants through cotyledonary segment culture of embryos derived from crosses between hydrangea macrophylla f. hortensia (lam.) rehd. and h . arborescens l. j. jpn. soc. hort. sci., 68(4): 803-809. lazzereschi s., nesi b., pecchioli s., grassotti a., 2012 costituzione, selezione e propagazione di ibridi interspecifici di lilium. acta italus hortus, 6: 188. mcclintock e., 1957 a monograph of the genus hydrangea. proc. calif. acad. sci., 29: 147-255. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue culture. physiologia plantarum, 15(3): 473-497. nesi b., lazzereschi s., pecchioli s., cardona-suarez c.m., colla g., cardarelli m.t., grassotti a., 2012 costituzione, selezione e propagazione di ibridi interspecifici di lilium. acta italus hortus 4: 85-94. reed s.m., 2000 development of an in ovule embryo culture procedure for hydrangea. j. environ. hort., 18(1): 34-39. reed s.m., 2004 floral characteristics of a hydrangea macrophylla × h. paniculata hybrid. proc. southern nursery assn. res. conf., 49: 580-582. reed s.m., riedel g.l., pooler m.r., 2001 verification and establishment of hydrangea macrophylla ‘kardinal’ × h. paniculata ‘brussels lace’ interspecific hybrids. j. environ. hort., 19(2): 85-88. roh m.s., griesbach r.j., gross k.c., lawson r.h., 1996 production and identification of interspecific hybrids of lilium longiflorum and l. × elegans. acta horticulturae, 397: 93-100. saharan b.s., nehra v., 2011 plant growth promoting rhizobacteria: a critical review. life sci. medicine res. lsmr., 21: 1-30. sharma d.r., kaur r., kumar k., 1996 embryo rescue in plants a review. euphytica, 89: 325-337. sokal r.r., rohlf f.j., 1981 the principles and practice of statistics in biological research. 2nd ed. state university of new york at stony brook, san francisco, ca, usa, pp. 776. tsavkelova e.a., cherdyntseva t.a., klimova s.y., shestakov a.i., botina s.g., netrusov a.i., 2007 orchid-associated bacteria produce indole-3-acetic acid, promote seed germination, and increase their microbial yield in response to exogenous auxin. arch. microbiol., 188(6): 655-664. impaginato 3 adv. hort. sci., 2011 25(1): 3-13 received for publication 21 september 2010. accepted for publication 28 december 2010. pollen characteristics, pollination behaviour and pollinizer compatibility of some exotic and indigenous almond [prunus dulcis (miller) d.a. webb] genotypes g. sharma, n. sharma department of fruit science, dr. y.s. parmar university of horticulture and forestry, nauni, solan, hp 173230, himachal pradesh, india. key words: almond, compatibility, pollen, pollination behaviour, pollinizer, abstract: pollination is the most critical and complex part of fruit production, particularly in cross pollinated crops like almond, and it is affected by pollen characteristics. in the present study 100% pollen viability was observed when stained by acetocarmine for all considered genotypes, except gp-17 (99%). optimum stigma receptivity was observed for two days before anthesis, on the day of anthesis and one day afterwards all the stigmas remained receptive. in open pollination, maximum fruit set was noted in primorskij (32.37%) with minimum in makhdoom (17.96%). no fruit set was observed in any of the genotypes by self-pollination, confirming the self-incompatible nature of all tested genotypes. in cross pollination, fruit set was found between 0.00 and 42.84% for different cross combinations. makhdoom, pranyaj, shalimar, waris, nonpareil, waris, waris and shalimar yielded maximum fruits when cross-pollinated with ixl, merced, drake, primorskij, pranyaj, nonpareil, shalimar, makhdoom and waris, respectively. no pollen tube growth was observed in the style when genotypes were crossed with their own pollen. in crosses between ixl and nonpareil, pollen tube growth was arrested in the styles. in compatible crosses, the pollen tube reached the base of the style after different times following pollination. 1. introduction almond [prunus dulcis (miller) d.a. webb], which belongs to the family rosaceae, is an important edible nut with widespread popularity. nuts are a rich source of energy and contain high amounts of fat, protein, minerals and vitamins. presently, the united states of america is the leading producer of almonds and india ranks third as an importer of almonds from the usa after spain and germany. in india, almond cultivation is confined mainly to northern areas including the regions of jammu and kashmir and high hills of himachal pradesh. during 2006-2007, the area under almond cultivation was 16,404 ha with annual production of 15207 mt in jammu and kashmir (directorate of horticulture, personal communication), whereas in himachal pradesh the area was 5766 ha and the production was 1303 mt (directorate of horticulture, personal communication). pollination is the most critical and complex part of fruit production, particularly in cross pollinated crops like almond. it involves a complex and sensitive sequence of events and interactions on a morphological, physiological and biochemical level. climatic conditions and genetic factors of the cultivars have an important impact on pollination. the number of flowers which develop, their interaction and their position within the inflorescence as well as pollen compatibility or incompatibility are some of the determinants for fruiting. the self-incompatibility system in almond limits in-breeding and therefore fails to produce an adequate crop, as a consequence, almond requires pollen from some other compatible cultivar(s) for cross-pollination. trees do produce abundant bloom but fail to set adequately due to lack of pollination and unfavourable weather conditions. sometimes cultivars are self unfruitful, and require pollen from other compatible cultivars for fruit production. according to socias i company (1992) most almond cultivars are self incompatible and nearly 30% pollinizers are required to have an economic crop (kester and griggs, 1959) which necessitates the planting of more than one 4 cultivar with sufficient overlapping of flowering periods to ensure adequate cross pollination and fruit set. some cultivar combinations also exhibit cross incompatibility (talaie and imani, 1998). knowledge of the pollen characteristics and pollination behaviour of different cultivars is therefore an important prerequisite for the successful cultivation of almonds. therefore, studies on pollen characters and pollination behaviour of almond genotypes were undertaken to find the compatibility groups between some exotic and indigenous selections of almond. 2. materials and methods the study of pollen characteristics, pollination behaviour and pollinizer compatibility of almond genotypes was conducted on four introductions from the usa (ixl, merced, drake and nonpareil), two from ukraine (primorskij and pranyaj) and seven indigenous selections from india (shalimar, makhdoom, waris, gp 10, gp 14, gp 17 and gp 19) at the central institute of temperate horticulture, srinagar, jammu and kashmir during 2005-06 and 2006-07. the experimental site was located at a latitude of 34o 05’ north and longitude 74o 50’ east, and at an altitude of 1640 m above mean sea level. the plants were five to six years old and were laid out at a spacing of 4 × 4 m. meteorological data are reported in appendix 1. pollen studies pollen viability was studied by staining with acetocarmine (2% solution) as per the method suggested by das (1995). in vitro pollen germination in sucrose solution was studied in 5, 10, 12.5, 15 and 20% sucrose concentrations. pollen tube growth was assessed for each genotype under a microscope after 24 hr of incubation at 22±2°c. the pollen grains with a pollen tube at least two times longer than pollen size were considered germinated. pollination studies stigma receptivity was studied in unopened, aboutto-open buds and opened flowers with the help of a magnifying lens to visualize the presence of exudates (watery fluid) on the stigmatic surface indicating the stigma to be receptive. to monitor fruit set under open pollination, four shoots in all the directions were selected for each genotype and the number of flowers on each shoot was counted. shoots were left open for natural pollination to occur. percent fruit set was calculated at harvest. under natural self pollination (by bagging) branches with flower buds were selected and all the opened flowers and shrivelled buds were removed. numbers of buds at popcorn stage left on each shoot were counted. these shoots were covered with muslin cloth bags, tied at the lower end and properly labelled. after 35-40 days the bags were removed and percent fruit set was determined at harvest. for hand self-pollination flowers were emasculated at balloon or popcorn stage by removing the entire calyx and corolla, leaving only the pistil. whole branches with emasculated buds were then covered with muslin cloth bags and properly tied and labelled. pollination of the emasculated buds was done on the following or next day with the freshly collected pollen of the same genotype. the pollen was applied to the receptive stigma with the help of a camel hair brush. after pollination the bags were again placed onto the branches. for cross pollination studies, crosses were made in the nine genotypes (exotic and indigenous cultivars) in all the possible combinations. the procedure followed to determine the extent of cross pollination was the same as discussed for self pollination studies, except that the pollen used for crossing belonged to different cultivars. fruit set was counted one month after pollination and percent fruit set was calculated as fruit harvested/flower pollinated × 100. in-vivo pollen tube growth was studied according to the procedure given by ortega et al. (2004). a sample of five pistils was collected at 24, 48, 72, 96 and 120 hr after hand self-pollination and immediately fixed in faa: ethanol 500 ml; formaldehyde 100 ml; acetic acid 50 ml; distilled water 350 ml. pollen tube growth in the style was viewed under fluorescent microscope (olympus bx-40 model) and the extent of pollen tube growth penetration into the style at different intervals of time after pollination was recorded. the data recorded were analysed statistically as per the methods described by panse and sukhatme (1978). 3. results and discussion almond has very low chilling requirements, so trees blossom in early spring, hence pollination and fertilization are negatively affected by the low temperature and rain which may prevail at that time. low temperatures during the flowering period can prevent germination of pollen on the stigma or prevent development of pollen tubes in style. further pollen can be washed away by rains and bees are not active at low temperatures. these factors can create some hindrance in the pollination and fertilization of almond, and subsequently affect fruit set percentage and final yield. therefore, fruit set under open pollination is influenced by a number of factors such as genetic makeup of cultivars, nearness or distance from a compatible pollen source, prevailing weather conditions, bee activity, stigma receptivity, pollen germination, pollen tube growth and fertilization process. pollen viability pollen viability tested with acetocarmine (2%) in different almond genotypes revealed that all the genotypes had 100% pollen viability, except for gp 17 in which 99.00% pollen viability was recorded differing 5 significantly from the others. similar higher values for pollen viability accessed through acetocarmine (2%) were observed previously by das (1995). under invitro pollen germination the percent germination varied significantly in different concentrations of sucrose (table 1). a significant increase in pollen germination percentage was recorded up to 15% sucrose solution and thereafter it decreased significantly in 20% solution. peak pollen germination was recorded in either 12.5 or 15% solution for most of the genotypes. in 5% sucrose solution pollen germination ranged from 12.67% in gp-19 to 76.90% in waris. in 10% solution it varied from 40.40 to 87.83% for gp-14 and ‘nonpareil’, respectively. this latter genotype gave values of 94.05, 96.45 and 90.05% germination in 12.5, 15 and 20% sucrose solution, respectively, whereas, gp 19 demonstrated low pollen germination of 12.67, table 1 in vitro pollen germination of different cultivars of prunus dulcis (miller) d.a. webb genotypes in different sucrose concentrations. values are expressed in percentage ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp-10 gp-17 gp-19 gp-14 cd0.05 genotypes 37.88 e 19.84 d 18.18 e 41.13 e 45.19 d 34.68 e 28.80 d 16.90 e 76.90 c 39.92 e 31.92 e 12.67 d 23.98 d sucrose solution (%) 5 10 12.5 15 20 55.91 d 65.72 b 52.86 d 72.77 d 64.73 b 87.83 d 77.62 c 55.41 d 78.13 c 48.49 d 53.21 d 47.59 c 40.40 c 86.03 a 83.89 a 85.72 b 85.95 c 88.01 a 94.05 b 86.85 a 81.05 b 91.30 a 81.44 b 80.05 b 72.54 b 75.81 b 83.10 b 85.88 a 92.44 a 96.44 a 87.17 a 96.45 a 80.47 b 84.00 a 92.71 a 85.99 a 82.66 a 79.80 a 79.75 a 77.31 c 56.99 c 80.57 b 93.50 c 59.10 c 90.05 c 78.52 c 64.53 c 86.08 b 73.98 c 70.99 c 72.30 b 75.32 b genotypes (g) concentration (c) genotypes x concentration (gxc) 3.27 2.03 7.33 47.59, 72.54, 79.80 and 72.30% respectively at similar levels of sucrose concentrations. the variation in pollen germination in different genotypes under the same sucrose concentration may be attributed to their varied genetic constitution. dhillon et al. (1982) in ‘california papershell’ almond found the highest (80.36%) pollen germination in 20% sucrose solution. eti (1994) found 10 to 15% sucrose concentrations quite suitable for almond pollen germination. stigma receptivity in almond, the importance of the length of the period of flower receptivity to obtain good yield was first described by griggs and iwakiri (1964). the percent of flowers showing stigma receptivity at different durations before and after anthesis in various genotypes is presented in table 2. two days prior to anthesis stigma table 2 stigma receptivity in different almond genotypes ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp 10 gp 17 gp 19 gp 14 genotypes 52 68 72 32 44 56 36 32 44 36 40 28 36 sucrose solution (%) -2 days -1 day 0 day +1 day +2 day 76 72 88 64 84 92 60 56 84 60 84 72 76 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 82 76 68 76 80 72 88 76 80 72 76 72 72 (-) before anthesis, (0) on anthesis, (+) after anthesis. 6 receptivity varied from 28 to 72%; maximum receptivity was observed in drake (72%) and the minimum value (28%) was observed for seedling selection gp19. one day before anthesis, stigma receptivity varied from 60% in shalimar and gp-10 to 92% in nonpareil. in addition, it was observed that all the stigmas were receptive on the day of anthesis and remained so for the second day after anthesis; receptivity decreased thereafter for all genotypes. the work of ortega et al. (2007) is in accordance with the present findings. pollination studies fruit set data under open pollination and self pollination for the years 2006 and 2007 is presented in table 3 and revealed that it varied according to the genotype and the year. the analysis of variance showed that there were significant differences for all the genotypes as well as for the interaction among the genotypes under open pollination. the average effect of the year was observed to be significant as average set for 2006 (26.42%) was higher than that of second year (22.41%). the principal reason for this difference is that the average temperature was higher in march 2006 than it was in march 2007: in 2006 the minimum temperature was above 1°c for all the days while it was 0°c or less in ten out of first 17 days of march 2007. low temperature along with snowfall on 12 and 13 march damaged the blossom of early flowering varieties like makhdoom, shalimar and gp-10. maximum fruit set was recorded for ‘primorskij’ (32.37%) which was at par with ‘pranyaj’ (30.96%) and ‘drake’ (30.51%) and significantly higher than ‘ixl’ (26.27%), ‘nonpareil’ (26.53%), ‘waris’ (25.17%) and ‘shalimar’ (25.02%). minimum fruit set was observed for cultivar makhdoom (17.96%). maximum fruit set in 2006 was recorded for cultivar shalimar (32.99%) and was at par with ‘pranyaj’ (32.25%) and ‘makhdoom’ (30.15%) whereas, the minimum value was observed for merced (21%). in 2007, ‘primorskij’ had the highest fruit set (36.57%) and the minimum recorded was 5.77% for ‘makhdoom’; both these values differed significantly from all other genotypes. low fruit set in early blooming cultivars, due to spring frost, was reported previously by connell (2000). for commercial fruit production fruit set in almond must range between 25 and 40% of the initial number of flowers (kester and griggs, 1959). low fruit set values for both the years of the present study can be further attributed to the non availability of supplemented pollinators (bee hives) during bloom for adequate pollination. variation in fruit setting behaviour under open pollination was reported by talaie and imani (1998), ak et al. (2001) and socias i company et al. (2005). the degree of self compatibility in almond genotypes, assessed by observing fruit set following unassisted self pollination (bagging), revealed that there was no fruit set following bagging in any of the genotypes, thus indicating total self-incompatibility. almond shows a gametophytic self incompatibility system (socias i company, 1992) controlled by a multiallelic locus, known as locus ‘s’ (gagnard, 1954). this implies that the pollen tube of a flower of the same tree, the same cultivar and sometimes of certain other cultivars, will not grow down the style (kester, 1969). in this regard, most almond breeding programmes have fostered the development of self-compatible cultivars to overcome the problems related to cross-pollination of a mostly self incompatible species such as almond table 3 fruit set of prunus dulcis (miller) d.a. webb genotypes calculated for open and self pollination ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp-10 gp-17 gp-19 gp-14 mean cd(0.05) pooled cd 0.05 g y gxy genotypes fruit set (%) open pollination selfing by bagging 2006 2007 pooled 2006 2007 pooled 27.20 21.00 26.93 28.16 32.25 24.23 32.99 30.15 28.70 22.89 24.28 21.97 22.67 26.42 2.07 25.33 21.28 34.09 36.57 29.67 28.83 17.05 5.77 21.65 15.04 19.42 16.99 19.63 22.41 3.24 1.85 0.72 2.63 26.27 c 21.14 de 30.51 b 32.37 a 30.96 ab 26.53 c 25.02 c 17.96 f 25.17 c 18.97 f 21.85 d 19.48 ef 21.15 de 24.41 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7 (social i company, 2002). our results are in accordance with a previous report of kester et al. (1994) wherein a low level of fruit set was recorded following hand pollination in otherwise self-incompatible cultivars. cross pollination cross pollination is essential in almond orchards as most of the cultivars are self-incompatible. in order to guarantee good pollination, at least two cultivars must be inter planted which not only coincide in flowering time, but are also cross-compatible. as the commercial part of the fruit is the seed, a decrease in the number of pollinated flowers often results in crop reduction (kester and griggs, 1959). thus rainy, windy or cold weather interferes with pollination by inhibiting bee foraging (socias i company et al., 1996). in the current work, nine almond cultivars were pollinated with one another in all possible combinations. the data pertaining to fruit set following cross pollination is presented in table 4. the maximum fruit set value was recorded for cross-combination ixl x makhdoom (42.84%) and was at par with nonpareil x pranyaj (39.69%), pranyaj x nonpareil (38.55 %), primorskij x waris (36.29%) and waris x shalimar (35.88%). minimum fruit set was observed when ixl was crossed with its own pollen (0.95%). no fruit set was recorded in merced, makhdoom and shalimar when they were pollinated with their own pollen. the data further revealed that when ixl was used as pollinizer, maximum fruit set was recorded in the cultivar pranyaj (34.47%) followed by waris (25.82%) and primorskij (23.87%). when merced was used as a pollinizer, maximum fruit set was observed with pranyaj (34.71%) followed by drake, waris, ixl, primorskij and ‘nonpareil’ as 28.37, 21.88, 21.29, 20.94 and 14.09%, respectively when merced was used as pollinizer. no fruit set was observed when merced was pollinated by its own pollen. fruit set ranged between 0.88 and 32.67% in different genotypes when drake was used as pollinizer: the maximum was with pranyaj (32.67%) which was statistically at par with fruit set in nonpareil (29.10%) but differed significantly from ixl (25.19%), waris (20.59%), merced (19.69%) and primorskij (18.79%); minimum fruit set (0.88%) was observed when drake was pollinated by its own pollen. in addition, when primorskij was used as a pollinizer, the maximum fruit set was observed with ixl (29.29%), at par with nonpareil (27.44%). fruit set ranged between 2.44 and 38.55% in different genotypes when nonpareil was used as pollinizer. maximum fruit set was noted in pranyaj (38.55%) followed by primorskij (28.12%), drake (27.28%), waris (21.48%) and merced (14.01%). minimum fruit set was observed when nonpareil was pollinated by its own pollen (2.44%), statistically at par with fruit set in ixl (3.05%) when pollinated with nonpareil pollen. when shalimar was used as pollinizer the highest ta ble 4fr uit set (% )i np ru nu sd ulc is (m ille r) d. a. w eb bg en oty pe so bta ine di nd iff ere nt int erva rie tal cro sse s fe ma le ix l m erc ed dr ak e pr im ors kij pr an ya j no np are il sh ali ma r m ak hd oo m w ari s m ea n cd 0.0 5 1x l m erc ed dr ak e pr im ors kij pr an ya j no np are il sh ali ma r m ak hd oo m w ari s m ea n cd 0.0 5 cd 0.0 5 ge no typ es (g ) 0.9 5 21 .29 25 .19 29 .29 13 .72 3.0 5 21 .64 42 .84 24 .14 20 .24 2.2 2 = 16 .16 0.0 0 19 .69 22 .32 32 .24 14 .01 28 .37 23 .95 18 .76 19 .42 1.3 2 6.2 4 18 .16 28 .37 0.8 8 13 .75 12 .64 27 .28 29 .37 28 .55 20 .70 19 .97 2.3 8 23 .87 20 .94 18 .79 1.2 7 19 .37 28 .12 28 .29 31 .60 36 .29 23 .17 2.1 9 34 .47 34 .71 32 .67 21 .56 1.5 7 38 .55 28 .89 28 .44 35 .91 28 .53 5.1 6 2.8 1 14 .09 29 .10 27 .44 39 .69 2.4 4 29 .52 26 .06 31 .90 22 .56 4.9 0 x x x x x x 0.0 0 23 .33 28 .97 17 .43 2.2 9 x x x x x x 14 .23 0.0 0 15 .85 10 .03 2.0 9 25 .82 21 .88 20 .59 24 .50 24 .35 21 .48 35 .88 25 .13 0.9 1 22 .28 1.7 7 17 .46 19 .94 20 .99 20 .02 20 .50 19 .27 24 .06 25 .46 23 .71 1.7 0 2.0 5 2.7 5 2.1 1 3.3 0 3.1 1 3.1 0 2.8 1 3.5 9 m ale x =c ros ses no ta tte mp ted . 8 value of fruit set was observed in waris (35.88%), which differed significantly from other cultivars. the data also showed that no fruit set was observed in cultivar shalimar when pollinated by its own pollen. when makhdoom was used as a pollinizer, the highest fruit set was recorded in ixl (42.84%) followed by primorskij (31.60%). drake, pranyaj, nonpareil, waris, merced and shalimar had fruit set of 28.55, 28.44, 26.06, 25.13, 23.95 and 23.33%, respectively, which were at par with each other when pollinated with makhdoom. no fruit set was observed when makhdoom was pollinated by its own pollen, as was also the case when waris was pollinated by its own pollen. the mean fruit set induced by different pollinizers ranged from 17.46 to 25.46%. makhdoom, as a pollinizer, affected the highest average fruit set (25.46%), followed by shalimar (24.06%), waris (23.71%) and drake (20.99%). the minimum fruit set value was observed when ixl (17.46%) was used as pollinizer. among female parents, the maximum fruit set was observed in pranyaj (28.53%), followed by primorskij (23.17%) and waris (22.28%). cultivar makhdoom (10.03%) had the lowest fruit set value as female parent when pollinated with different pollinizers. cross-incompatibility of ixl with nonpareil had been previously established (gagnard, 1954) and the present study revealed low fruit set in ixl and nonpareil crosses, thus supporting the findings. the rest of the cultivars showed optimum fruit set with crossing (13.72 to 42.84%) thus indicating cross-compatibility between the cultivars. the differences in fruit set may be due to various factors such as genotypic differences of the cultivars under study, response of genotypes to different pollen sources, ovary degeneration, unfavourable climatic conditions during flowering, flower sterility and heterostyly. other workers (talaie and imani, 1998; dalal et al., 2004) reported similar results. in vivo pollen tube growth in temperate tree crops the rate of pollen tube growth to the base of the style is quite low (sedgley, 1982). the present studies, pertaining to in-vivo pollen tube growth, have revealed that in all the cultivars which were pollinated by their own pollen, pollen tube growth was arrested in the style. observations regarding in vivo pollen tube growth are presented in table 5 a and 5 b. the findings indicate that in all crosses where pollen of the same cultivar was used for pollination, the pollen tube failed to reach up to the base of the style. the observations revealed that in ixl the pollen tube reached the base of the style after 120 hr when crossed with pollen from merced and shalimar, whereas it took 96 hr with pranyaj and makhdoom. the pollen tube reached the style after 72 hr when ixl was pollinated by drake, primorskij and waris, while the pollen tube failed to grow in the style of ixl when nonpareil was used as pollen source. in merced it was observed that the pollen tube reached the base of the style after 72 hr when crossed with shalimar and makhdoom; with ixl, drake, nonpareil and waris it reached the same point after 96 hr. primorskij and pranyaj pollen tubes reached the base after 120 hr. likewise, the study of drake styles revealed that pollen tubes reached the base after 72 hr of pollination with merced and waris whereas with other cultivars it reached after 96 hr. the pollen tube of ixl, nonpareil and waris reached up to the base of styles of primorskij after 72 hr of pollination; with merced, drake, shalimar, makhdoom and pranyaj, it reached after 96 hr. it was also revealed that in pranyaj the pollen tube reached up to the base of the style after 72 hr when pollinated with ixl, merced, drake and shalimar while with others it took 96 hr. in vivo pollen tube growth in nonpareil revealed that pollen of drake primorskij, pranyaj and makhdoom reached the earliest (i.e. 72 hr) after pollination, whereas with other pollinzers it took 96 hr to reach the base. in cultivar shalimar it was observed that the pollen tube reached up to the base of the style after 96 hr when crossed with makhdoom and with waris it reached after 120 hr. similarly, in makhdoom the pollen tube reached up to the base after 120 hr and 96 hr when crossed with shalimar and waris, respectively. the pollen tube reached the base of the style at different durations in waris. it took 72 hr for ixl, drake, and pranyaj pollen whereas, with merced, primorskij, nonpareil, shalimar and makhdoom it took 96 hr after pollination. these findings, along with fruit set data, confirm the self incompatibility of cultivars under study. similar results were observed by ak et al. (2001). these authors found that the rejection of incompatible male gametophyte occurred on the stigma, as well as in the style. similarly in pistils of nonpareil and ixl none of the pollen tubes reached the base of the pistil when they were inter-pollinated, thus confirming the cross incompatibility between these two cultivars. however, in the compatible pollination crosses, the pollen tube reached the base of the style after different durations of pollination. the difference did not affect the compatibility relationship of the pollinations. the observed differences must be mostly attributed to the interaction of weather conditions at the time of pollination and thereafter. temperature is an important component for pollen tube growth and the most suitable temperature for pollen tube growth in almond is 12-13°c, and under these temperatures the pollen tube can reach the ovarium within three to four days (loreti and viti, 1984). moreover, pistils may react differently to different pollen sources. overall, it generally took three to five days for pollen tubes to reach the base of the pistil in otherwise compatible pollination. the present findings are in consonance with those of ak et al. (2001) and das and kumar (2004) who reported that pollen tubes reached the base of pistils after four or five days of pollination in almond. other in vivo pollen tube 9 ta ble 5a -i nv ivo po lle nt ub eg row th in sty les of pr un us du lci s( m ille r) d. a. w eb bg en oty pe so bta ine di nd iff ere nt int erva rie tal cro sse s fe ma le po llin ize r m erc ed dr ak e pr im ors kij pr an ya j ix l 24 hrpo lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr ix l m erc ed dr ak e pr im ors kij pr an ya j no np are il sh ali ma r m ak hd oo m w ari s x x 1/4 1/4 1/4 x 1/4 1/4 1/4 x 1/4 2/4 3/4 2/4 1/4 2/4 1/4 2/4 x 2/4 at the ba se at the ba se 3/4 x 3/4 2/4 at the ba se x 3/4 at the ba se x 3/4 at the ba se x at the ba se x at the ba se at the ba se inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 2/4 x 2/4 1/4 2/4 2/4 3/4 3/4 2/4 3/4 x 3/4 2/4 2/4 3/4 at the ba se at the ba se 3/4 at the ba se x at the ba se 3/4 3/4 at the ba se at the ba se x at the ba se at the ba se co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble 1/4 1/4 1/4 1/4 1/4 2/4 1/4 1/4 2/4 2/4 3/4 x 2/4 2/4 3/4 2/4 2/4 3/4 3/4 at the ba se x 3/4 3/4 3/4 3/4 3/4 at the ba se at the ba se x at the ba se at the ba se at the ba se at the ba se at the ba se x co mp ati ble co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 3/4 2/4 2/4 x 2/4 3/4 2/4 2/4 3/4 at the ba se 3/4 3/4 x 3/4 at the ba se 3/4 3/4 at the ba se at the ba se at the ba se x at the ba se at the ba se at the ba se x co mp ati ble co mp ati ble co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble 1/4 1/4 1/4 1/4 x 1/4 1/4 1/4 1/4 2/4 2/4 2/4 1/4 x 1/4 3/4 2/4 2/4 at the ba se at the ba se at the ba se 2/4 x 3/4 at the ba se 3/4 3/4 at the ba se x at the ba se at the ba se at the ba se co mp ati ble co mp ati ble co mp ati ble co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble 10 ta ble 5b -i nv ivo po lle nt ub eg row th in sty les of pr un us du lci s( m ille r) d. a. w eb bg en oty pe so bta ine di nd iff ere nt int erva rie tal cro sse s fe ma le po llin ize r no np are il po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus 24 hr 48 hr 72 hr 96 hr 12 0 hr sh ali ma r po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus 24 hr 48 hr 72 hr 96 hr 12 0 hr m ak hd oo m po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus 24 hr 48 hr 72 hr 96 hr 12 0 hr w ari s po lle nt ub ep en etr ati on (s tyl el en gth ) co mp ati bil ity sta tus 24 hr 48 hr 72 hr 96 hr 12 0 hr ix l m erc ed dr ak e pr im ors kij pr an ya j no np are il sh ali ma r m ak hd oo m wa ris 1/4 1/4 2/4 1/4 1/4 x 1/4 1/4 1/4 1/4 2/4 3/4 3/4 3/4 1/4 2/4 3/4 2/4 x 3/4 at the ba se at the ba se 3/4 x 3/4 at the ba se 3/4 x at the ba se at the ba se x at the ba se at the ba se x x inc om pa tib le co mp ati ble co mp ati ble co mp ati ble co mp ati ble inc om pa tib le co mp ati ble co mp ati ble co mp ati ble x 1/4 1/4 x 2/4 2/4 x 3/4 3/4 x at the ba se 3/4 x at the ba se inc om pa tib le co mp ati ble co mp ati ble 1/4 x 1/4 1/4 x 2/4 2/4 x 3/4 3/4 x at the ba se at the ba se x co mp ati ble inc om pa tib le co mp ati ble 1/4 1/4 1/4 1/4 1/4 1/4 1/4 1/4 x 2/4 2/4 2/4 2/4 3/4 2/4 2/4 2/4 1/4 at the ba se 3/4 at the ba se 3/4 at the ba se 3/4 3/4 3/4 x at the ba se at the ba se at the ba se at the ba se at the ba se x x co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble co mp ati ble inc om pa tib le (-) no ob ser va tio nr ec ord ed . (x )n op oll en tub eg row th ob ser ve d. 11 growth studies in almond indicate that pollen tubes require two to four days or more to reach the ovule (pimienta and polito, 1983; polito et al., 1996). 4. conclusions the present study has shown that all the considered genotypes had optimum pollen viability and confirmed their self-incompatible nature. furthermore, examination of cross pollination has indicated that there is a potential to renew the declining almond industry of india by exploiting the existing diverse gene pool. exotic varieties can be used for commercial cultivation or in future breeding programs to develop varieties suited to local conditions. references ak b.e., acar i., sakar e., 2001 an investigation on the determination of pomological and morphological traits of wild almond grown at sanlurfa province. options mediterraneennes, 56: 139-144. connell j.hr., 2000 pollination of almonds: practices and problems. hort. technology, 10(1): 116-119. dalal m.a., farooqui k.d., das b., 2004 studies on varietal diversity in blooming, productivity and quality characteristics of almond germplasm in kashmir valley. acta horticulturae, 662: 151-156. das b., 1995 studies on compatibility and xenia in almond (prunus amygdalus batsch). m. sc. thesis, dr. y.s. parmar university of horticulture and forestry, solan, himachal preadesh, india. das b., kumar k., 2004 in-vivo pollen germination on stigma and pollen tube growth in relation to inter-varietal cross compatibility in almond. app. biol. res., 6: 44-47. dhillon d.s., dhatt a.s., gill r.p.s., 1982 pollination studies in almond (prunus amygdalus batsch) growing under subtropical conditions. indian j. of hort., 39: 190-195. eti s., paydas v., kuden a.b., kasks n., kurnaz s., ilgin m., 1994 investigations on the pollen viability, germination capability and the growth of pollen tubes on some selected almond types under cukurova conditions. acta horticulturae, 373: 225-233. gagnard j.m., 1954 research on systematic of almond varieties grown in algeria and on the phenomena of sterility. ann. inst. agric. alger., 8(2): 163. griggs w.h., iwakiri b., 1964 timing is critical for effective cross pollination of almond flowers. californian agric., 18(1): 6-7. kester d.e., 1969 almonds, pp. 302-314. in: richard a.j. (ed.) handbook of north american nut trees. northern nut grower association, knoxville, tennessee, usa, pp. 421. kester d.e., gradziel t.m., micke w.c., 1994 identifying pollen incompatibility groups in california almond cultivars. proc. of the amer. soc. of hort. sci., 119(1): 106109. kester d.e., griggs w.hr., 1959 fruit setting in the almond: the effect of cross pollinating various percentages of flowers. proc. of the amer. soc. of hort. sci., 74: 206-213. loreti f., viti r., 1984 recherches sur la pollinisation de certaines varieties d’amandier dans les conditions climatiques du littoral toscan. ciheam options mediterraneennes, 84(ii): 177-183. ortega e., dicenta f., egea j., 2007 rain effect on pollen stigma adhesion and fertilization in almond. scientia hort., 112(3): 345-348. ortega e., egea j., dicenta f., 2004 effective pollination period in almond cultivars. hort. science, 39(1): 19-22. panse v.g., sukhatme p.v., 1978 statistical methods for agricultural workers. indian council of agricultural research, new delhi, india, pp. 347. pimienta e., polito v.s., 1983 embryo sac development in almond [prunus dulcis (mill.) d.a. webb] as affected by cross, self and non pollination. annals of botany, 51: 469-479. polito v.s., micke w.c., kester d.e., 1996 bud development, pollination and fertilization, pp. 98-102. in: micke w.c. (ed.) almond production manual. division of agriculture and natural resources, university of california, usa. sedgley m., 1982 floral development, anthesis and pollination. acta horticulturae, 240: 177-183. socias i company r., 1992 breeding self fertile almonds. plant breeding reviews, 8: 313-338. socias i company r., 2002 latest advances in almond self compatibility. acta horticulturae, 591: 205-212. socias i company r., alonso j.m., gomez j.a., 2005 evaluation of almond selection for fruit set under field conditions. ciheam options mediterraneennes, 63: 133-139. socias i company r., felipe a.j., aparisi j.g., 1996 genetics of late blooming in almond. acta horticulturae, 484: 261-266. talaie a.r., imani a., 1998 flowering, pollination and fruit set patterns in some new iranian almond genotypes. acta horticulturae, 470: 123-130. 12 appendix i meteorological data march, 2006 days temperature maximum minimum i ii rh (%) rh (%) i ii rain (mm) weather 7:30 hr 14:30 hr i ii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 15.00 16.00 17.50 16.00 15.80 15.60 13.00 14.00 16.00 16.00 19.00 20.00 16.00 11.00 13.00 11.00 14.00 16.50 9.00 6.00 14.00 17.50 18.50 11.50 11.40 15.00 14.50 16.50 20.00 20.50 21.60 15.20 1.00 2.50 2.00 3.00 2.60 3.80 2.50 0.50 3.00 3.30 1.50 0.80 3.00 6.40 4.40 0.50 1.20 0.40 5.20 5.00 5.00 3.40 4.60 4.50 5.50 6.50 6.50 5.60 3.00 4.00 9.50 3.57 85.00 94.00 85.00 86.00 94.00 81.00 89.00 94.00 85.00 75.00 88.00 89.00 75.00 90.00 87.00 86.00 81.00 78.00 78.00 94.00 94.00 87.00 73.00 75.00 95.00 75.00 92.00 85.00 95.00 75.00 60.00 84.51 78.00 69.00 53.00 56.00 63.00 77.00 77.00 56.00 60.00 45.00 46.00 42.00 73.00 72.00 87.00 74.00 60.00 67.00 74.00 92.00 58.00 53.00 46.00 69.00 66.00 68.00 68.00 56.00 39.00 39.00 38.00 61.96 0.00 0.00 0.00 0.00 1.00 0.00 5.80 0.00 0.00 0.00 0.00 0.00 0.00 1.80 12.20 7.40 2.20 0.00 0.00 13.00 4.80 0.00 0.00 0.00 8.20 1.60 4.20 4.00 0.00 0.00 0.00 66.20 clear clear clear clear clear cloudy cloudy clear cloudy cloudy clear clear clear cloudy p. cloudy cloudy cloudy clear cloudy rain cloudy clear clear cloudy rain cloudy cloudy cloudy clear clear cloudy clear clear clear clear clear rain cloudy cloudy clear clear clear clear rain rain rain rain clear cloudy cloudy cloudy clear clear clear rain rain cloudy cloudy clear clear clear clear year 2006 month temperature maximum minimum rh% rh% rain (mm) january february march april may june july august september october november december 4.07 12.59 15.20 20.94 28.27 27.96 31.12 28.31 25.05 22.13 14.17 7.47 -1.82 2.38 3.57 5.69 11.45 13.41 17.91 17.26 11.23 6.78 2.79 -0.54 92.19 90.71 84.52 71.30 74.26 79.43 77.35 86.80 88.86 92.16 92.00 93.06 84.06 72.75 61.97 45.17 53.06 65.00 63.32 64.35 69.53 63.94 67.67 76.23 168.10 53.20 66.20 55.50 38.60 35.80 151.60 149.20 108.00 19.00 82.50 94.90 13 march, 2007 days temperature maximum minimum i ii rh (%) rh (%) i ii rain (mm) weather 7:30 hr 14:30 hr i ii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 11.40 12.00 10.00 10.50 13.50 14.50 16.00 17.00 13.50 15.20 9.00 2.00 7.50 9.00 11.50 11.50 10.00 11.40 9.50 5.50 6.00 11.50 15.50 16.50 18.00 19.40 22.50 22.50 23.50 25.00 25.00 13.73 2.70 -1.20 2.50 1.00 0.20 -1.00 -1.00 -0.50 1.50 3.40 4.00 0.00 0.00 -1.50 -2.00 -2.60 -0.50 3.80 4.50 4.50 3.20 3.80 4.00 3.40 1.80 2.40 3.50 3.50 5.00 6.60 8.50 2.04 88.00 100.00 73.00 97.00 94.00 96.00 90.00 87.00 72.00 82.00 71.00 93.00 100.00 93.00 90.00 69.00 85.00 92.00 90.00 97.00 92.00 94.00 89.00 75.00 73.00 62.00 79.00 61.00 65.00 73.00 80.00 83.93 59.00 43.00 64.00 75.00 47.00 35.00 35.00 39.00 53.00 46.00 78.00 90.00 77.00 64.00 50.00 55.00 54.00 76.00 87.00 91.00 97.00 66.00 52.00 53.00 46.00 49.00 45.00 42.00 38.00 42.00 38.00 57.61 5.40 0.00 0.00 3.60 3.00 0.00 0.00 0.00 0.00 0.00 0.00 35.00 165.00 5.40 0.00 0.00 0.00 2.40 0.00 25.40 29.80 6.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 281.8 cloudy clear cloudy cloudy cloudy clear clear clear clear cloudy cloudy snow snow cloudy clear clear cloudy cloudy cloudy rain cloudy cloudy p. cloudy clear clear clear clear clear clear clear p. cloudy cloudy clear rain cloudy clear clear clear clear cloudy clear rain snow clear clear clear clear cloudy cloudy rain rain cloudy clear clear cloudy clear clear clear clear clear clear clear year 2007 month temperature maximum minimum rh% rh% rain (mm) january february march april may june july august september 9.36 11.11 13.74 24.99 25.13 28.54 29.90 29.79 26.88 -2.95 1.8 2.04 6.87 10.52 14.55 16.86 16.53 12.35 88.8 90.03 83.94 70.90 77.32 75.80 82.00 81.84 85.43 53.80 64.43 57.61 39.23 53.29 55.27 56.58 57.39 58.97 8.90 50.50 281.8 1.40 44.50 49.70 57.60 46.40 23.20 source (sasa). 212 1. introduction black cumin, nigella sativa (ranunculaceae), is an annual herbaceous plant. the genus nigella is represented in 20 species of mediterranean-western asian origin (dantuono et al., 2002). only n. sativa, n. damascene and n. arvensis are of interest in jordan; n. sativa is the only species planted by farmers. there is no accurate data about planted area, but the annual production for the year 2005/2006 was 3-5 tons (personal communication). n. sativa is a hermaphroditic species with determined flowering patterns, starting with the flower terminating the main shoot and ending with the flowers on the lowermost branches. in the natural forms, flowers are delicate, and usually colored pale blue and white, with 5-10 petals and characterized by the presence of nectaries. the androecium comprises a large number of stamens, which shed their pollen as the filaments curve outward during the male phase. the gynoecium consists of up to five completely united follicles, each with a long, indehiscent style and composed of a variable number of multi ovule carpels, developing into a follicle after pollination, with single fruit partially connected to form a capsule-like structure. seeds are generally small in size (1-5 mg) dark grey or black (filippo et al., 2002). the fruit is large and its inflated capsule contains numerous seeds. n. sativa is extensively used in traditional medicine for healing various respiratory disorders from morocco to pakistan and in southern europe (filippo et al., 2002). the seeds have been widely added as a spice to a variety of foods such as bread, yoghurt, pickles, sauces, and salads for flavoring. they are also used in jordanian traditional folk medicine for some respiratory, gastrointestinal, rheumatic and inflammatory disorders (nafisy, 1989; zargari, 1990; amin, 1991). n. sativa seeds have been reported to contain essential oil, fixed oil, flavonoids, saponins, alkaloids, and proteins (zargari, 1990; burits and bucar, 2000; al-ghamdi, 2001). pollination studies of n. sativa are very limited in the literature. lloyd (1979) showed that n. sativa is self pollinated without mentioning the mechanism; zohary (1983) showed that n. sativa is capable of setting seed without being cross pollinated. the flowers of n. sativa are visited by honeybees (ricciardelli and oddo, 1981). 2. materials and methods the research considered specific plant species (landraces) of n. sativa, which were planted on-site at different elevations: location a, 150 m below sea level; and location b, 200 m above sea level. n. sativa was obtained from botanical gardens in jordan (ncartt). the seeds were planted in hills 30 cm apart on 5 november 2005. the rows were 20 m long, with 1 m between rows. water was supplied daily by drip irrigation and extra fertilizers (n p k) were applied. black plastic mulch was used. each plant was represented by three rows per pollination of nigella sativa l. (ranunculaceae) in jordan valley to improve seed set k.a. abu-hammour*, d. wittmann** * college of pharmacy, al-isra university, p.o. box 22, 23 isra, university, 11622 amman, jordan. ** institute of natural resource, faculty of agriculture, bonn university, germany. key words: black cumin, jordan, style movement. abstract: in jordan, pollination is one of the problems faced by plants under plastic houses, in open fields and in offseason planting. therefore this study was conducted in jordan to investigate the role of pollinators and to investigate the systems of pollination in nigella sativa species grown at two different altitudes, 150 m under sea level and 200 m above sea level. up to now little attention has been paid to the events associated with pollination such as seed set, and to address this deficit, we examined six pollination treatments of the selected plant species. field work was conducted, repeated and recorded from 2005 to 2007 in jordan. controlled pollinations were carried out in selected individual’s plant at the time of maximum stigma receptivity and anthesis. n. sativa flowers had anthesis intervals which last for five days, then followed by stigmatic receptivity which last for few hours. plants are pollinated trough outcrossing and complete selfing to insure the reproductive assurance. however, self-pollination was occurred due to style movement. the observations confirmed that a mixing mating including a combination of out-crossing and selfing is a better strategy than selfing alone. adv. hort. sci., 2011 25(4): 212-222 received for publication 22 february 2011 accepted for publication 13 november 2011 213 location. missing hills were replanted when necessary. the plants were thinned to two plants per hill when they were at twoto three-leaf stages. the two locations were kept weed-free by cultivation and hand weeding. the time of stigmatic receptivity was determined with the aid of a dissecting microscope. the direct test of receptivity was an assay that detects the presence of stigmatic peroxides. to determine receptivity, the stigmas were treated with hydrogen peroxide 3%: small air bubbles that form by maturation of the stigma indicate that the flower is in the female phase (dafni and maues, 1998). to determine receptive periods, 50 flower buds per plant of each species were marked, 10 flower buds of the same age were bagged a day before the opening of flowers during the anthesis period. on the following day, 10 flower buds were taken to the laboratory in order to check for stigma receptivity. the timing of anthesis was checked in the field using a hand magnifier. after bending, anther capsules were observed with the naked eye. the mechanism of pollen release is described based on direct observations in the field. any rupturing of the capsule causes pollen to release where it is verified by anther dehiscence. in order to observe pollinator visitation tour, the number of visits per bee was estimated by counting the number of visits with anther or stigma contact from the beginning of pollination to fertilization. the counts were conducted every 15 minutes for a period of eight hours on a daily basis during flowering period. controlled pollinations were carried out on selected individual plants at the time of maximum stigma receptivity and anthesis. pollinated flowers were observed periodically for fruit set. the reproductive success of the studied species was assessed by performing a spontaneous self pollination, manual self and cross pollination treatments. following the initiation of the first flower bud, flowers were selected randomly and tagged: 180 flower buds of n. sativa in each location. thirty flower buds were marked for each pollination treatment. pollination treatments were performed from february to march 2006 to determine the best pollination treatment in each locations. in order to conduct geitongamy and xenogamy pollinations, all stamen organs of each flower were removed using special scissors (emasculation). the flowers were pollinated using pollen from freshly dehisced anthers from male flowers (of the same plant) by using a fine brush for geitongamy pollination treatment and from another plant for xenogamy pollination treatment (cross pollination). the flowers were left exposed to any insect as occurs in nature for open pollination treatment. to test the bagged self pollination, flower buds, bagged till the end of pollination stage, were left untreated and uncovered again in order to avoid any negative impact on their germination. in order to check forced self pollination on the same hermaphrodite flower, flowers were bagged till the last day of the male stage. the flowers were pollinated using pollen from freshly dehisced anthers from male to female flowers on the same hermaphrodite flower by using a fine brush. with regard to emasculation, flower buds of nearly the same age were selected in order to remove male flowers to investigate the differences between the role of pollinator and the role of plant, by numbering of fruit set. the anthers were removed with a pair of tweezers and were left to pollinate by pollinator. if an emasculated flower sets fruit, then it must have received pollen from a pollinator. however, if an emasculated flower fails to set fruit, a pollinator will have had no role in fertilization. changes in the relative positions of anthers releasing pollen and the styles was also documented. a total of 30 flower buds were monitored during the study period using a hand magnifier. a single flower from this group was monitored from the morning to the end of the day. each flower was scored for the number of anthers on the flower, the number of anthers dehisced, the position of the dehisced anthers and the positions of anthers relative to the stigma. the length of anther and style were measured using a special caliber. representative photographs were taken of flowers at each stage. thirty marked flower buds were selected to count the number of ovules in order to determine the standard number of ovules in the stigma. the number of ovules per capsule were counted, averaged for both locations and the average number was used as a reference in the calculation. data were analyzed as complete randomized design with three replicates. comparisons between means were made using least significant difference (lsd) at 0.05 probabilities lend (spss). for statistical data, standard descriptive statistics were performed for each of the following quantitative parameters: the number of produced fruits, the number of seed for each stigma, the number of ovules, the number of chambers per capsule, the number of non fecundated seeds and the total number of fecundated seeds. mean number of buds and stigmas of plants, standard deviation, and differences between pollination treatments in terms of seed set per fruit were calculated. the statistical program package spss was used. insect visits were standardized by calculating the number of visits per flower per plant. these data were summarized over the season by taking an average of the observations. minimum and maximum value was observed and graphical analyses were applied. 3. results anthesis and receptivity styles are the first floral organ to emerge and extend, followed by extension of the stamens. when the style has almost straightened, the anthers began to dehisce. after the dehiscence of anthers about half an hour when it is considered as the first day for pollen shedding till fifth day, the male stage activated between 8:30 am to end of the day and anthers were sink down. the male 214 phase is initiated a few days before the stigmas become receptive and male stage lasted for five days. by the fifth day of the male stage, female stage started during this day, stigmatic peroxides tests indicate that receptivity occurred between 8:00-13:00 pm and for one day only. male and female stages synchronized in the last day of the flowering period (fig. 1). the weight of pollen was 0.064 mg/flower, whereas the volume of nectar was 0.13 µl. affluent floral rewards (both nectar and pollen) during the male phase of the flowers. fig. 1 blooming stages of nigella sativa, a) flowers opening, b) anthesis and c) receptivity stage. movements of stigma and anthers the male and female organs at bud stage are presented in (fig. 2 a) at onset of the male stage, all the stamens stand erect (fig. 2 b). they curve outwards one by one, roughly in whorls and strictly reflecting the order of initiation (fig. 2 c). when the anthers reach a horizontal position, the pollen is released (fig. 2 d). then, the stamens sink down. an anther takes 4-7 hours to empty its contents. the stamen movement is not continuous, but it is divided into three phases. in the first phase (12-14 hours) the lower part of the filament inclines slightly, while the upper part curves more strongly, so that the anther is brought into a horizontal position. after reaching this position, movement comes to a standstill. the second phase towards the ends of the male stage, the styles of the five carpels usually curve down (fig. 2 e) and twist (fig. 2 f). this ensures that in the female phase the stigmatic crests, whose bends were making an angle of 45°, continue to make a right angle with ovary to run down nearly the whole length of the style to touch the top of the anther at several points (fig. 2 f). the third stage, in which the stamen sinks down, is much shorter than the previous ones (4-6 hours). finally, the empty anthers curve up. this is a purely passive movement, apparently without any function. after uptaking the pollen, the stigma is pollinated (fig. 2 g), and then the stigma inclined upwardly erect as the order of initiation and makes an angle of 180° with the ovary (fig. 2 h). the maximum style length reached 1.73 cm, whereas the maximum anther length was 1.72 cm. this indicates the equal length of style and anther. pollination season one. location a. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 96±0.5, as shown in (table 1). generally, all flowers under the different treatments produced seeds (table 1). open pollinated flowers produced significantly higher seeds as compared with other treatments 74.9±1.4. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a nonsignificant differences between them with a seed set average of 82.9±1.6, 73.5±1.5 and 78.9±1.6 respectively. a non-fecundated seed production is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination occupied the lowest average of non-fecundated seeds all over other treatments (table 1). hand cross, hand geitongamy and hand forced self ranked secondly in producing a non-significant fecundated seeds with an average of 12±1.7, 21.6±1.6 and 18±1.4 respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (table 1). seed set percentage after open pollination (86.8% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (79.8%, 75.4% and 81% respectively). location b. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 91.1±0.5, as shown in (table 2). generally, all flowers under the different treatments produced seeds (table 2). open pollinated flowers produced significantly higher seeds as compared with other treatments 82.9±1.5. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a nonsignificant differences between them with a seed set average of 72.4±1.4, 70±1.3, and 77.5±1.1 respectively for the first location. characteristics of producing fecundated seeds in the second location were fairly constant in value and regulated mainly by treatments conditions. a non-fecundated seed production table 1 seeds set after different pollination treatments in nigella sativa, location a. season one treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/ capsule open 93.3±0.5 a 74.9±1.4 a 19.0±1.5 a 86.8±1.2 a hand cross 95.6±0.7 b 82.9±1.6 b 12.0±1.7 b 79.8±1.3 b hand geitonogamy 95.1±0.6 b 73.5±1.5 b 21.6±1.6 b 75.4±1.2 b hand forced self 96.0±0.5 b 78.9±1.6 b 18.0±1.4 b 81.0±1.4 b a and b are symbols related to difference in comparison. (a) (b) (c) 7:00 8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 215 fig. 2 a) plant at bud stage; b) the stamens stand erect; c) first phase of stames movement: the stamens curve outwardly in whorls; d) pollens releasing; e) first phase of style movement: the styles of the usually five carpels curve down; f) twisting point of style with anther; g) the stigma is pollinated; h) the stigma inclined upwardly erect. a b c d e f g h 216 is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination occupied the lowest average of non-fecundated seeds (table 2) all over other treatments. hand cross, hand geitongamy and hand forced self ranked secondly in producing a non-significant fecundated seeds with an average of 18.7±1.3, 25±1.5 and 18.1±1, respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (fig. 3). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when cross, hand geitonogamy and hand forced self was used on flowers (79%, 73% and 80% respectively). fig. 3 nigella sativa seed set percentage upon pollination treatments in location a and b season one. season two. location a n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 92.3±1.42, as shown in (table 3). generally, all flowers under the different treatments produced seeds (table 3). open pollinated flowers produced significantly higher seeds as compared with other treatments in both locations 83.4±0.67. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a non-significant differences between them with a seed set average of 74.6±0.68, 73.6±0.67, and 79.7±0.32 respectively for the first location. bagged self pollinated flowers ranked thirdly and produced 44.1±0.75 seeds. the lowest seed set was recorded in the case of emasculated flowers with an average seed production of 12.4±0.33. hand cross, hand geitongamy and hand forced self ranked second. a nonfecundated seed production is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination produced non-significant fecundated seeds with an average of 12±1.7, 21.6±1.6 and 18 ±1.4, respectively. there were significant differences (p≤ 0.05) in the percentage of seed set between treatments (fig. 4). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤ 0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (79%, 78% and 83%, respectively). nearly half of the produced set seed in bagged flowers with an average of 47%. emasculated flowers (13%) recorded the lowest seed set from other treatments with significant difference. location b. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 97.2±1.67, as shown in (table 4). generally, table 2 seeds set after different pollination treatment in nigella sativa location b. season one treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/capsule open 95.6±0.48 a 82.9±1.5 a 12.8± 1.5 a 87.0±1.3 a hand cross 91.1±0.50 b 72.4±1.3 b 18.7± 1.3 b 79.0±1.2 b hand geitonogamy 95.0±0.55 b 70.0±1.3 b 25.0±1.5 b 73.6±1.0 b hand forced self 96.0±0.60 b 77.5±1.1 b 18.1±1.0 b 80.0±0.99 b a and b are symbols related to difference in comparison. table 3 seeds set after different pollination treatment in nigella sativa location a. season two treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/capsule open 96.8±2.19 a 83.4±0.67 a 13.4±1.93 d 87±1.67 a hand cross 96.0±2.31 a 74.6±0.68 b 21.4±2.39 c 79±1.98 b hand geitonogamy 93.0±2.81 a 73.6±0.67 b 21.1±1.81 c 78±1.46 b hand forced self 97.2±1.67 a 79.7±0.32 b 17.6±1.77 c 83±1.54 b bagged self 94.2±1.50 a 44.1±0.75 c 51.9±0.73 b 47±1.10 c emasculation 97.6±1.67 a 12.2±0.33 d 85.4±0.33 a 13±0.46 d a, b, c, and d are symbols related to difference in comparison. 217 all flowers under the different treatments produced seeds (table 4). open pollinated flowers produced significantly higher seeds as compared with other treatments in both locations 82.4±0.57. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced nonsignificant differences between them with a seed set average of 71.8±0.57, 67.9±0.62, and 78.6±0.5, respectively. bagged self pollinated flowers ranked third and produced 43±0.74 seeds. the lowest seed set was recorded in the case of emasculated flowers with an average seed production of 12.4±0.5. open pollination occupied the lowest average of non-fecundated seeds 13.7±1.76 (table 4) all over other treatments. hand cross, hand geitongamy and hand forced self ranked second, a non fecundated seed production is also a common feature of n. sativa’ flowers under the different treatments in producing a non-significant fecundated seeds with an average of 20.2±1.57, 25.1±1.62 and 14.5±1.6, respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (fig. 5). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (78%, 73% and 85% respectively). nearly half of the produced set seed in bagged flowers with an average of 46%. emasculated flowers (13%) recorded the lowest seed set from other treatments with significant difference. behavior of honey bee visitors during our observation, honey bees were the only visitor and pollinator that visited n. sativa in the morning around 7:00 a.m. every flower had one bee at least. each bee spent different time with an average of 12.5 s for nectar collecting, 8 s for pollen collectors. the only diurnal visitor and pollinator were honey bees. honey bees were frequent visitors to n. sativa in the jordan valley. the honey bee had same behavior in the two locations. in the evening, no pollinators were found in the flowers in both sites. the major pollinator was honey bees. n. sativa’ flowers’ mean visit rates for the three replicates in both locations were 14.9 and 14.6 daily visiting tours, respectively. the ultimate activity during the three replicates was approximately from 9:30 to 12:30 in both locations. 33% of the total bees observed were pollen collectors, while the rest 67% were nectar collectors (table 5). honey bees fig. 4 nigella sativa seed set percentage upon pollination treatments in both locations, season two. table 4 seeds set after different pollination treatments in nigella sativa location b. season two treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/ capsule open 96.1±1.76 a 82.4±0.57 a 13.7±1.76 d 87±1.56 a hand cross 92.3±1.42 a 71.8±0.57 b 20.2±1.57 c 78±1.28 b hand geitonogamy 93.1±1.68 a 67.9±0.62 b 25.1±1.62 c 73±1.22 b hand forced self 93.1±1.53 a 78.6±0.50 b 14.5±1.60 c 85±1.39 b bagged self 93.6±1.50 a 43.0±0.74 c 50.6±1.71 b 46±1.10 c emasculation 94.9±1.9 0a 12.4±0.50 d 82.5±2.01 a 13±0.62 d a, b, c, and d; are symbols related to difference in comparison. table 5 behavior of honey bees and their bearings for nigella sativa behaviors of bees average spending time/flower (second) landing on departure of percent of bees according to their bearingspetals anthers twisting petals anthers twisting nectar collector were observed 12.5 • • 67% pollen collector were observed 8.0 • • 33% fig. 5 nigella sativa seed set percentage upon pollination treatments in both locations, season two. 218 visiting tours were conducted in two stages during five days. anthesis period took place in the first four days, and anthesis and receptivity periods were in the fifth day. visiting tours were for functional nectar collecting. on the first day, honey bees landed on petals and then collected nectar during circular stepping upon petals, without getting directly exposed to anthers. on the second till the fourth day, the same behavior occurred. the pollen grains fell down upon bees back from the horizontal anthers during circular motion. on the fifth day, receptivity period began, in which the styles inclined towards anthers, and then the styles twisted themselves around the anthers. honey bees were landing directly on this synapse (not on petals). after that, they left and flew to another flower. 4. conclusions the male phase is initiated a few days before the stigmas become receptive, where the anthesis duration remains for five days full flowering started with the appearance of bright blue petals. male stage started as the anthers started to shed their pollen, since the first day till fifth day, the male stage activated between 8:30 a.m. to the end of day. the viability of one anther remained during one day then started to sink down. it is interesting to point out that anthers remain active for five days, which leads to synchronize the receptivity period in the fifth day. because the flowering period for n. sativa coincides with good temperature in april in jordan, this may lead to an increase of the interval of anthesis since the pollen responds to temperature. it is surprising for pollen of n. sativa to continue for five days. another reason for this long period of anthesis is the large number of anthers in staminate. climatic factors affect the anthesis intervals in n. sativa, there is evidence that high temperature had a direct effect on pollen performance since the pollen responds to temperature. however, at the same time they are advantageous for the pollen by hastening its tube growth rate. on the other hand, low temperatures may act against the pollen by reducing its germination and growth rate, which could limit the fertilization success (thompson and liu, 1973; jakobsen and martens, 1994) the duration of stigmatic receptivity in nigella sativa was approximately hours in angiosperms, the stigma is the first female structure, the pollen grains and pollen tubes have to face on their way to the female gametophyte. the stigma provides an adequate environment for pollen grain germination (knox, 1984; helsop-harrison and shivanna, 1997). one of the most important features of stigmas is stigmatic receptivity, defined as the ability of the stigma to support pollen germination, which is a decisive stage in fertilization success and has a large variability among plant species (helsopharrison, 2000). at the end of the fifth day on the male stage, the female stage started to be active during 8:00 a.m. to 13:00 p.m. and then ended up. it is interesting to point out that the stigma of n. sativa is receptive throughout anthesis. inspite of the flowering period in april when we don’t have high temperature which may hurt the plant; the stigma receptive only for hours. the explanation for that is that the stigma is exposed in direct way to the sun which may increase the exposed area. in addition, the receptivity of stigma occurred after the stigma lost most of the anthers that surrounded the stigma so that the whole stigma is exposed to the sun which may also increase the exposed area to sun. that means high temperature affects stigma receptivity and reduces receptivity interval. there is evidence that ensures stigma responds to high temperature. high temperatures are detrimental for the female part by reducing the length of stigmatic receptivity and accelerating ovule degeneration (postweiler et al., 1985). it is well documented that the reproductive phase, especially from pollination to fertilization, is highly vulnerable to the prevailing environmental conditions including temperature (hall, 1992; stephenson et al., 1992). the duration of stigmatic receptivity is variable depending on the species, and it is also variable within genus. there is evidence that indicates duration of stigmatic receptivity is variable, that the duration of stigmatic receptivity is variable depending on the species and is usually greater in wind-pollinated than in insect-pollinated species (khadari et al., 1995). thus, the stigma can be receptive for not much more than an hour or so, as in avena or dactylis, to as long as several days, as in other grass species (pennisetum or zea) or eucalyptus in which it can remain receptive for more than a week, particularly in hostile environments (helsop-harrison, 2000). from an agricultural perspective, stigmatic receptivity has also a clear practical implication as it limits floral receptivity, the effective pollination period (guerrero-prieto et al., 1985) and hence fruit set (reviewed in sanzol and herrero, 2001). moreover, in an ecologist context, by altering stigmatic receptivity, flowering plants may influence the likelihood of fertilization by indirectly controlling the number and the quality of mating through the control of the number of pollen grains deposited and the time of germination (cruden et al., 1984; primack, 1985; galen et al., 1986). autonomous pollination first of all, i would like to define the autonomous phrase for the reader to understand. as lloyd, 1992 defines it: prior self pollination within-flower: self-pollination that occurs before the opportunity for outcross-pollen receipt for that flower has occurred, competing self pollination within-flower; self-pollination that occurs during the opportunity for outcross-pollen receipt for that flower has occurred, and delayed selfing pollination within-flower; self-pollination that occurs after the opportunity for outcross-pollen receipt for that flower has occurred. one of these three types of self pollination occurred in our research in n. sativa, which is delayed selfing pollination. automonous delayed selfing late in n. sativa flower’s life 219 is favored when honey bees service and thus outcross-pollen receipt is unpredictable. n. sativa flowers attract honey bees but they can also autonomously perform delayed self pollination, which provides reproductive assurance if pollinators fail to visit. the delayed self pollination occurred in our research because the synchronization between male and female occurred in the end of flowering period. i agreed with darwin (1877), muller (1883), baker (1955, 1965) and lloyd (1979, 1992) that pollinator absence or low pollinator abundance during some periods within or among flowering seasons favor shifts from outcrossing to autonomous self-fertilization because self-pollinated seeds provide reproductive assurance. some authors support the research result that absence of pollinators can shift to delayed self pollination; the extinction of pollinators or range expansion in a plant lineage can favor shifts to biotic modes of pollination, including wind pollination and autonomous self fertilization (baker, 1955; stebbins, 1957; regal, 1982; cox, 1991; weller et al., 1998). the results agreed with barrett and harder (1996), and ramsey and vaughton (1996) that pollinator scarcity and reduced pollinator services may result in high selfing rates. cross pollination and bagged self pollinations occur; approved by seed set achieved by all treatments applied on the research where bagged selfing and outcrossing boosted seed production means of 45% and 77% respectively. the results agreed with zohary (1983) as he found that n. sativa are capable of setting seed without being crosspollinated, but he didn’t mention the mechanism for such a result. the results also agreed with faegri and van der pijl (1971) who reported: there are a few flowers that can selfpollinate by their own, but this limits them to in breeding. the results agreed with goodwillie (1999) in believing the ability of self pollination to provide some insurance against pollination failure. in addition to the reproductive assurance benefits, prior selfing could be favored since it reduces the costs associated with the longer floral maintenance time required for outcrossing, and sets the stage for the evolution of reduced investment in cues for pollinators and the amount of pollen per flower. in contrast with early selfing, later-selfing species will retain floral traits and costs associated with outcrossing (i.e., cues to attract pollinators, pollinator rewards, and prolonging floral maintenance relative to prior selfing species. at one extreme, selfing early in a flower’s life (prior) is favored when a population requires pollinators are chronically absent (lloyd, 1992), or when population size is so low as to be undetectable by pollinators (lloyd, 1992; fausto et al., 2001; goodwillie, 2001), or when a population experiences high levels of interspecific pollen flow (fishman and wyatt, 1999). many authors are interested in common type of pollination as cross, open and self pollination, but through our research i have been devoted all our efforts to point out some thing out of traditional efforts such as delayed self pollination. thus, delayed selfing may be achieved by either a partial overlap in timing of male phase with female phase or changes in the relative position of anther and stigma during development. for example, delayed selfing in hibiscus laevis (klips and snow, 1979) and campanula species (faegri and van der pijl, 1979) is characterized by a progressive downward curling of the stigmatic area towards the style where anthers or pollen are located. conversely, in the protogynous aquilegia canadensis (eckhert and schaeffer, 1998) the stamens progressively elongate towards the exerted stigma. in kalmia latifolia (lyon, 1992), anthers collapse into the stigma on the final day of floral development; thereby achieving self pollination. others have found in self pollination late in floral life without changes in morphology. the breakdown of self incompatibility as the flower ages in both lilium and longifolium (ascher and peloquin, 1966) is attributed to degradation of the proteins that control self incompatibility and can be viewed as another form of delayed selfing. faegri and van der pijl (1971) used the term ‘’self pollination’’ or “autogamy” when pollination takes place within one flower (idiogamy), and “allogamy” or “cross pollination” when pollen from one flower is carried out to the stigma of another one. allogamy may further be divided into “geitonogamy” if the flowers are on the same plant and “xenogamy” if they are from different plants. however, it is that geitonogamy that has the ecological properties of cross-fertilizer but the genetic properties of self fertilization. thus, geitonogamy appears to be equivalent to autogamy (lloyd and schoen, 1992). style movement acts towards promoting self-pollination and leads nigella sativa to delayed self-pollination weber (1995) has produced a presentation film showing the pollination mechanism for nigella arvensis. he concisely presented the mechanism in written steps. the mechanism was demonstrating style movement in n. arvensis which exactly resembled our observations on style movement of n. sativa; through pictures shown above. i used his written description has quotation for its meaningful. i have measured the style and anther length and style twisting angle. hence the equal length of anther and stamen demonstrate the style twisting, whereas weber (1995) did not mention the length. weber (1995) mentioned that insects bear pollen on their thorax after touching the horizontal anthers. our observations showed that honey bees are landing on the horizontal anthers and twisting point of style and anther to bear pollen grains on their legs. so, how could insect carry pollen to another flower if the pollen is on their thorax. the beginning of receptivity caused a strong twist for stamens and style that leads to self pollination. this was observed as the end of male stage and the beginning of the receptivity stage. style movement acts towards promoting self-pollination as in n. sativa. in another plant, style movement leads to avoiding self-pollination and promoting cross-pollination as verma and magotra (2004) reported for eremurus himalaicus where they observed the mechanism of the stigma movement away from the dehiscing anthers, hence, it avoided receiving any left over pollen, and so self pollination is impossible. it is interesting to point out that n. sativa plant relies solely on animal vectors to move pollen among individuals, and if 220 pollinators are absent or in low numbers at certain times or years, individuals of n. sativa, that can self pollinate if not previously out crossed, will be at a selective advantage. this reproductive assurance process has been termed delayed selfing. n. sativa mixed mating is a better strategy than selfing alone mixed mating is a better strategy; that means open pollination system is better to seed setting than other pollination treatment. this open system leaves the plant exposed to biotic and abiotic factor. the plant will be without any restriction which may cause any reduction in seed setting. the open system includes the role of honey bees and role of plant to pollinate itself by delayed self pollinated flowers. the manual pollination, which included: hand cross, hand geitongamy and hand forced self, ranked second after open pollination, and this significant difference is attributed to human performance which is not like natural performance. excluding the biotic and abiotic factor from the plant by bagged self, that means plant will be restricted without honey bees, and the plant depends on itself to develop its style to reach the maximum length to catch the anthers in order to twist. in spite of the style movement towards the anthers, it gained half of the seed setting from open pollination, and this attributed to the fact that the style’s movement occurred once the stigma was receptive and at the final stage of anthesis when there is small number of anthers, and then sink down, this may not be enough to get high percent of seed setting as there isn’t enough quantity of pollen. honey bees are pollinator to n. sativa which is considered unattractive to wild bees the only diurnal visitor and pollinator were honey bees. honey bees frequently visited n. sativa in the jordan valley. the honey bee had similar behavior in the two locations. in the evening no pollinators were found in the flowers in both sites and seasons. flower visitors can only be considered pollinators if four pollination conditions have been met: pollen transfer to the vector is observed; pollen transport by the vector is observed, pollen transfer from vector to stigma is observed; and pollen deposited by the vector is shown to result in fertilization of the value (cox and knox, 1988). the flowers of n. sativa were unattractive to wild bees’ visitors. an important aspect used in many pollination studies is the number of visits made by a pollinator (proctor et al., 1996). apis mellifera engaged in pollen and nectar collection as a pollinator of n. sativa flowers with low frequency. the unattractively of n. sativa flowers to wilds bees may be attributed to several factors such as the presence of other floral resources. during our research, n. sativa flowering coincided with that of other species such as centurea syriaca and s. arevensis which are important for apiculture in jordan due to their abundant nectar and the large floral patches through out the area. the attractiveness of any species is a function such as favor, color, nectar volume, sugar concentration (frisch, 1967), and the bees fly to plant species that yield the greatest nectar and pollen (gary, 1979). the role of honey bees in the pollination of n. sativa is too small honey bees’ role as pollinator in fertilizing n. sativa flower buds was very small compared to the role of plant itself and the role of open natural conditions in pollination. the emasculated buds were let exposed to the pollinators in order to fulfill the pollination where it sets up 12% of seed formation percent, while the natural conditions and self pollination conditions gave 87% and 45%, respectively. it is necessary to ask whether the removal of stamens affected subsequent flower development, e.g. the growth of the perianths, a factor that would make it difficult to distinguish between the costs of stamens or pistils and the costs of structures associated with display and reward (andersson, 2003). such effects seem likely considering the work of andersson (2000), who detected a cost of producing and maintaining sepals and petals in a related species (n. degenii), and plack (1957), who found a negative effect of emasculation on corolla size in hermaphroditic plants of the gynodioecious glechoma hederacea (lamiaceae). in the present study of n. sativa, stamen removal caused significant reduction in the mean of seed set. the results agreed with andersson’s study (2003) where he observed the stamen removal produced reduction in total seed number. as a furthermore for n. sativa, andersson (2003) carried out removal of styles from n. sativa flowers and he found that; style-less plants initiated almost three times more flowers and invested 57% more biomass in stamens, than plants whose flowers were permitted to set fruit. he found also stamen-less plants produced significantly heavier seeds after hand-pollination. these observations indicate that stamens draw upon the same pool of resources as the other floral organs and that the removal of immature stamens therefore influences patterns of resource allocation. furthermore, andersson and jorgensen (2005) carried out removal of perianth from n. sativa flowers and found that; perianth removal produced 12.5% heavier seeds and allocated 15.8% more biomass to seed production than plants on which all perianths were left intact, whereas differences in flower production and total seed number were not significant. perianth removal did not significantly affect the proportion of seeds that germinated, but caused a shift toward earlier germination dates. the ultimate visitation rates for n. sativa flower in both locations was diurnal visitation type especially at early morning the ultimate visitation rates for flower in both locations were during 9:30 a.m. to 12:30 p.m., because the bees’ activity is limited by environmental factors; the radiation rate and the daily temperature. visitation rate was estimated by counting the number of visiting tours, those with anther or stigma contact. counts were made for one hour periods during (8) hours a day, while plant species flowers were open. pollinators may accidentally take place without any relationship existing between blossom and agent. even 221 with concept of definite relationship in mind, it is not always easy to draw the line between pollinators and accidental visitors. the quantity of pollen transferred from anthers to stigmas, visit frequency to flower, pollinator forage pattern during anthesis, and floral rewards availability are parameters that can adequately explain the pollination efficiency of floral visitors (primack and silander, 1975; herrera, 1987; 1989). it is generally thought the more visits made, the more efficient is the pollinator, though this also depends on the per visit pollen contribution to the pistillate flower part (primack and silander, 1975; herrera, 1989). references al-ghamdi m.s., 2001 the anti-inflammatory, analgestic and anti-pyretic activity of nigella sativa. j. ethnopharmacol., 76: 45-48. amin g.r., 1991 popular medicinal plants of iran. ministry of 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(eds.) ecology and evolution of plant reproduction. chapman and hall, new york, usa. thompson m.m., liu l.j., 1973 temperature, fruit set, and embryo sac development in italian prune. j. of amer. soc. for hortic. sci., 98: 193-197. verma s., magotra r., 2004 stylar movement avoids self pollination and promotes cross-pollination in eremurus himalaicus. current science, 87(7): 872-873. weber a., 1995 pollination of nigella arvensis (ranunculaceae) (film presentation). pl. syst. evol. (suppl.), 9: 325-326. weller s.g., sakawi a.k., rankin a.e., golonka a., kutchure b., ashby k.e., 1998 dioecy and the evolution of pollination systems in schiedea and alsinidendron (caryophyllaceae: alsinoideae) in the hawaniian islands. american journal of botany, 85: 1377-1388. zargari a., 1990 medicinal plants. tehran university publications, tehran, fifth edition, vol. 1, pp. 43-44. zohary m., 1983 the genus nigella (ranunculaceae). a taxonomic revision. plant systematics and evoluation, 142: 71-107. impaginato 159 1. introduction banana (musa spp.) is a climacteric fruit; therefore, ripening process is induced by ethylene production via acc (1-aminocyclopropane 1-carboxylic acid) biosynthesis. the rate of respiration is followed by reaching a threshold level of ethylene within the cells of fruit then rises rapidly to a peak and subsequently falls as ripening progress. during fruit softening, starch is turned to sugars, the peel color changes to yellow and fruit flavor develop by losing its astringency (pathak et al., 2003). polyamines as natural compounds suppress ethylene synthesis by inhibition of ethylene biosynthesis enzymes activities (lee et al., 1997). they are present ubiquitously in plant organs. the main polyamines are putrescine (1, 4-diaminobutane), spermidine (n3-aminopropyl-1, 4-diaminobutane), and spermine [bis (n-3-aminopropyl)-1, 4-diaminobutane] which are essential in plant growth, differentiation and stress responses (valero and serrano, 2010). they are known to improve the storage life of fruits by inhibiting ethylene production and delaying the ripening process, respectively. polyamines and ethylene have opposite impacts on fruit ripening and senescence. thus, a balance between them is crucial to enhance and retard the fruit ripening process. in general, polyamines level declines throughout fruit senescence along with accelerating ethylene synthesis (valero et al., 2002). much researches have indicated the positive effects of pre and postharvest polyamines application on retarding fruit softening in mango (malik et al., 2003) and pear (franco-mora et al., 2005), reducing weight loss in apricot (martinez-romero et al., 2002), inhibition of ethylene production in peach (zokaee khosroshahi and esna-ashari, 2008), delaying ripening process in nectarine (torrigiani et al., 2004) and peach (bregoli et al., 2002), and maintaining ta at higher levels, diminishing the increase in tss, and declining color change in plum (khan et al., 2008). thus, the present study was carried out to evaluate the application of putrescine for extending quality and storage life of musa acuminata l. adv. hort. sci., 2016 30(3): 159-164 doi: 10.13128/ahs-20278 pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l. m.s. hosseini 1, s.m. zahedi 2, z. fakhar 3 (*) 1 department of horticultural science, hormozgan university, bandar abbas, iran. 2 department of horticultural science, faculty of agriculture, university of maragheh, 551181-8311 maragheh, iran. 3 department of horticultural science, college of agriculture and natural resources, university of tehran, 31587 karaj, iran. key words: firmness, polyphenol oxidase, postharvest, skin color, weight loss. abstract: the study was carried out to determine the effect of putrescine on quality and postharvest life of musa acuminata l. during storage. the fruits were dipped at different concentrations of putrescine (0.5, 1 and 2 mm for 30 min) and distilled water as ‘control’. changes in fruit quality attributes such as weight loss, firmness, skin color (l*, hue angle), total soluble solids (tss), titratable acidity (ta), ph, ascorbic acid, polyphenol oxidase (ppo) and polygalacturonase (pg) enzymatic activity were calculated at harvest and after 5, 10, 15 and 20 days of storage at 0±1°c, 80-85% relative humidity. weight loss, fruit softening, skin color changes, tss, ph, the activity of ppo and pg increased during fruit ripening but the rate of changes was significantly slowed in putrescine treated fruits. moreover, putrescine application maintained higher levels of ta, ascorbic acid and reduced the loss of sensory acceptability and decay incidence compared to control. in conclusion, the postharvest dip treatment of putrescine could be an effective means for extending the storage life of musa acuminata l. (*) corresponding author: za.fakhar@gmail.com received for publication 21 july 2016 accepted for publication 20 september 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 159-164 160 2. materials and methods mature green bananas (musa acuminata l.) were harvested from a commercial orchard in minab, iran, and then transported to the laboratory for experiments. fruits uniform in size and color, without any noticeable defects, were selected and dipped in putrescine solution at different concentrations (0.5, 1 and 2 mm for 30 min) and distilled water as ‘control’. then all of treated and untreated fruits were stored at 13°c and 80-85% relative humidity. then, some physico-chemical attributes were measured at harvest and after 5, 10, 15 and 20 days of cold storage. quality parameters evaluation fruit weight was recorded just after harvest and after the different sampling dates and then expressed as percentage of weight loss relative to the initial weight (soto-zamora et al., 2005). fruit firmness was measured using a fg-5020 penetrometer (lutron electronic enterprise co.) of 5 mm in diameter at 2 equatorial points and was expressed as newton (n). color was determined at opposite sides of each fruit from each replicate with a minolta chromameter cr400; the following parameters were considered: l* (0= black; 100= white), a* (green to red) and b* (blue to yellow) then expressed as l* and hue angle (h°)= arctan (b* a*-1) (ozdemir, 2016). total soluble solids (tss) content was assessed by a digital refractometer (atago n1, japan) at 20˚c and expressed as a percent. titratable acidity (ta) was estimated by titrating 5 ml of diluted juice against 0.1 n naoh using phenolphthalein as an indicator and was expressed as percent malic acid (%). the ph of fruit juice was measured using a mtt65 (japan) ph meter calibrated by ph 4 and 7 buffer solutions. ascorbic acid assessment ascorbic acid content was estimated using the methods of marisa and wall (2006). polyphenol oxidase (ppo) and polygalactronase (pg) activities measurement ppo and pg were assessed using the procedure of marquez cardozo et al. (2015) and zhu et al. (2015) respectively. decay incidence and sensory acceptability determination fruit deteriorations were measured on individual fruit by visual observations. from each fruit 5 slices were obtained and fruits decay was recorded using the following formula: a/b ×100 in which a is the number of decayed fruit slices and b the initial number of all fruit slices. the fruits were rated by a panel of 10 judges on the basis of color, texture, taste and flavor and overall acceptability (as 1-2 unusable, 3-4 unsalable, 5-6 salable, 7-8 good, 9-10 very good). statistical analysis to estimate storability of fruit, a factorial design completely randomized was carried out in three replications. all data were analyzed using sas software package 9.4 for windows and mean comparisons were conducted using duncan’s multiple range tests. 3. results and discussion weight loss and firmness weight loss percentage increased in all the treatments along the storage. however, all putrescine concentrations demonstrated significantly lower weight loss than control. fruits treated with 2 mm putrescine exhibited the lowest weight loss amongst the putrescine concentrations during storage while highest weight loss was registered by control (fig. 1 a). the effect of putrescine on reducing weight loss fig. 1 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on weight loss (a) and firmness (b) of musa acuminata l. during storage. hosseini et al. prolonging storage life of banana by using putrescine 161 may be ascribed to conjugation of polyamines to the cell membrane phospholipids that result in cell membrane integrity (mirdehghan and rahimi, 2016). similar results have been reported in apricot (enas et al., 2010). as shown in figure 1b irrespective of treatments, fruit firmness decreased significantly over storage but putrescine treated fruits were observed firmer, and especially 2 mm putrescine treatments was more effective than others in keeping the firmness. it is suggested that polyamines maintain fruit firmness by their cross-linkage to the pectin substances carboxyl groups in the cell wall and lead to rigidification of cell wall; consequently cell wall degrading enzymes activities of pectin methyl esterase (pme), pectin esterase (pe) and polygalactouronase (pg) are decreased (valero et al., 2002). the results are in line with peach (bregoli et al., 2002). color changes skin color alteration from green to yellow is a predominant index used for evaluating the stage of ripening in banana (gomes et al., 2013). as the storage time progressed, fruit color changed as a result of chlorophyll degradation along with carotenoid synthesis. however, putrescine treated fruits showed higher l* and hue angle than control (fig. 2 a and b). delayed color changes can be associated to the effect of putrescine as anti-senescence by reducing ethylene production and subsequently delaying fruit ripening as well as senescence (drake and chen, 2000). similar results have been observed in apricot (martinez-romero et al., 2002). total soluble solids (tss), titratable acidity (ta) and ph tss content and ta increased along the storage period while ph demonstrated reverse trend in all treated and untreated fruits (fig. 3 a, b and c). lower fig. 2 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on l* (a) and hue angle (b) of musa acuminata l. during storage. fig. 3 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on tss (a), ta (b) and ph (c) of musa acuminata l. during storage. adv. hort. sci., 2016 30(3): 159-164 162 values of tss, ta and higher value of ph content were observed in putrescine treated fruits compared to control (fig. 3). that is ascribed to the role of putrescine on delaying fruit ripening process by reducing ethylene production and respiration rate in fruit (valero et al., 2002). the results are in agreement with those observed in mango (malik and singh, 2006). ascorbic acid the content of ascorbic acid was significantly influenced by putrescine. the value of ascorbic acid was higher in treated fruits than control throughout the storage (fig. 4). it is possible that putrescine inhibits ascorbic acid oxidation by decreasing ascorbate oxidase activity and consequently maintaining ascorbic acid (ishaq et al., 2009). this result is in line with the finding of davarynejad et al. (2013). enzymatic activity of polyphenol oxidase (ppo) irrespective of treatments, the activity of ppo increased during ripening process and it was significantly higher in control than treated fruits (figure 5 a). this trend may be attributed to the role of putrescine on reducing polyphenol oxidase activity (koushesh saba et al., 2012). previously, it has been observed in kiwifruit (jhalegari et al., 2012). polygalacturonase (pg) activity pg is known as an important enzyme on fruit softening, whereas, a reduction in pg activity results in a delay in fruit softening and consequently an increase in the storage life (jhalegari et al., 2012). in this study, the activity of pg increased during the storage. as shown in figure 5 b, untreated fruits demonstrated the highest values of pg activity (1.74 mmol kg-1 s-1 on the 20th day of storage). fruits treated with 2 mm exhibited the lowest pg activity, followed by putrescine at 1 and 0.5 mm respectively. this trend is associated to declining fruit firmness and increasing fruit softening by the loss of membrane integrity (sitrit and bennett, 1998). decay incidence and sensory acceptability the highest rate of fruit decay percent was observed in control while all three concentrations of putrescine reduced the decay development significantly during storage; in particular, the fruits dipped in 2 mm putrescine showed the lowest decay incidence in comparison to others (fig. 6 a). while time passed, sensor acceptability declined. however, fruit treated by putrescine exhibited higher scores of sensor acceptability compared to control at the end storage (fig. 6 b). fig. 4 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on ascorbic acid of musa acuminata l. during storage. fig. 5 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) ppo (a) and pg (b) activities of musa acuminata l. during storage. hosseini et al. prolonging storage life of banana by using putrescine 163 4. conclusions the effect of putrescine treatment at different concentrations (0.5, 1 and 2 mm for 30 min) was investigated to improve and extend storage life of banana (musa acuminata l.). application of putrescine maintained fruit quality attributes such as firmness, color, tss, ta, ph and sensory acceptability. in addition, the reduction of weight loss, ppo, pg, and decay incidence were observed in putrescin treated fruits compared to control. thus, the postharvest dip treatment of putrescine may be an effective tool for prolonging the storage life of musa acuminata l. references bregoli a.m., scaramagli s., costa g., sabatini e., ziosi v., biondi s., torrigiani p., 2002 peach (prunus persica) fruit ripening: aminoethoxyvinylglycine (avg) and exogenous polyamines affect ethylene emission and flesh firmness. physiol. plant., 114(3): 472481. davarynejad g.h., zarei m., ardakani e., nasrabadi m.e., 2013 influence of putrescine application on storability, postharvest quality and antioxidant activity of 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constitute the “orchard system”. this system must be carefully assembled in all its components in order to respond to the diverse environmental, social and economic constraints. designing the system means to define the physical arrangement of the trees, their shape and all the agronomic practices to apply during the entire life span of the orchards. all the system components must be implemented at the time of the orchard design determining its basic traits such as planting density, training system, and scion and rootstock combination. in addition to these physical traits it is necessary to define the proper pruning strategy to be adjusted year by year to the chosen training system taking into account also the agronomic operations direct to the management of the soil root relationship. pruning is the basic tool to manipulate fruit tree architecture and behaviour in order to achieve economically sound crop yield and fruit quality. “pruning” includes a very large and diversified number of operations involving the aerial organs and the root system, performed both during rest and vegetative season (for a glossary of pruning terms see: baldini and scaramuzzi, 1962; liebster and pesserl, 1982; ingels et al., 2002). once the major traits of the orchard have been defined, the pruning strategy may then enter into discussion. the main objective in training fruit trees is the modulation of competition among the vegetative and reproductive parts of the tree, which essentially means the manipulation of the source-and-sink relationships occurring in the plant taking into account other basic objectives such as light interception and distribution in the canopy, and biomass partition properly directed toward the fruits. the relationships between pruning and processes of growth, fruit bearing and senescence assume a strategic relevance as almost every pruning operation affects the growth of a tree or its organs on either a shortor longterm scale. pruning is a very powerful tool but it cannot overcome the effects of basic mistakes in design and management of the orchard. the significance of the architecture and growth traits of trees and their relationships with reproductive activities are essential to understanding how pruning may affect the functioning of the orchard system. in this paper the principal aspects of plant architecture and growth relevant to pruning are examined; since “pruning” includes many diverse operations, a large range of combinations of winter and summer pruning manipulations is possible and therefore their potential effects on tree behaviour are considered and explained in terms of the sourceand-sink relationship and competition among sinks. a number of the references cited in this paper may seem dated, but it must be said that studies and experiments on pruning have recently become rare whereas during the 1980s and ‘90s scientists, researchers and field experts paid great attention to this inalienable orchard management practice. italian contributions to the knowledge of the physiological and practical basis of pruning have been particularly important, as shown by fundamental papers and reference books on this topic (dotti, 1949; morettini, 1963; bargioni, 1992; sansavini and errani, 1998; branzanti and ricci, 2001). it seems, however, useful to reasbasic considerations about pruning deciduous fruit trees c. giulivo dipartimento agronomia ambientale e produzioni vegetali agripolis, università degli studi di padova, via romea, 16, 35020 legnaro (pd), italy. key words: architecture, correlate function, fruit trees, growth, pruning. abstract: crop yield and fruit quality performance determine the economic value of the orchard system. once the basic traits of the orchard design are defined, pruning may represent a powerful tool to modulate the vegetative and productive behaviour of the trees. the various pruning manipulations involve variations in growth, size and geometry of the plant and their effects are explained in terms of correlate functions among the tree organs. in spite of its potential, pruning can not overcome the effects of some basic mistakes made in design and management of the orchard. basic concepts about pruning developed in the past are reassessed in light of recent knowledge on architecture and growth of the tree. adv. hort. sci., 2011 25(3): 129-142 received for publication 13 may 2011 accepted for publication 27 july 2011 130 sess these contributions in light of recent knowledge on the architecture and growth of trees. 2. tree architecture the identification of tree architecture traits is relevant to the pruning strategy. according to costes et al. (2006) plant architecture includes two independent notions: branching and connection between plant units (topology), and the spatial location, orientation, size and shape of the vegetation elements (geometry). tree architecture includes physical structures and physiological functions of fruit trees, and is therefore related to space and time aspects. the multilevel approach of the tree (whole tree, branch, leaf and fruit) should be stated and tree architecture should be described by canopy height, width and shape, by fruiting and vegetative shoot types and their relative proportions, spatial distribution, branching, and growth dynamics. canopy porosity or density should also be considered as light interception and distribution, and pest occurrence are concerned (simon et al., 2007). the orchard structure, function and production may be understood if, in addition to tree architecture, phenotypic plasticity and phenology are considered. trees are modular organisms which develop by reiteration of elementary botanical elements whose anatomical, morphological, dimensional and functional traits change during ontogeny and accordingly to various life stages (bathélémy and caraglio, 2007). during ontogeny plant appearance varies with form and/or structure and with temporal and/or topological changes and, therefore, it is of high practical relevance to orchard design and management (fig. 1). tree architecture depends on the nature and relative arrangement of each of the tree’s parts. at any given time, the architecture is a result of equilibrium between internal growth events and external environmental constraints. pruning must take into account this natural fact and hence it must follow the natural behaviour of the fruit tree and direct it toward the desired economic goals. the main morphological traits used in describing tree architecture are the growth processes, branching modalities, morphological differentiation of axes and location of the reproductive structures (barthélémy and caraglio, 2007). growth processes. organogenesis and extension are two distinct but co-ordinated morphogenetic events that result in the primary growth of a plant. these events basically determine the stem of a plant, which can be considered a succession of internodes and nodes, on which a leaf (or leaves) and axillary bud (or buds) are located; the node and the subtending internode represent the basic structural unit of the plant body (metamer or phytomer). a tree, whatever its final size, is initially formed by the activity of at least two primary meristems, one to develop the aerial part and one the root system, but the subsequent increment in diameter of woody axes (aerial and subterranean) is determined by the activity of secondary meristems. in many species, the apex of the aerial axes may abscise or abort after a time of functioning or it may shift into reproductive structure or other organs: this behaviour is defined as determinate growth. on the other hand, when the apical meristem of the axes maintains indefinitely its growth potential, an indeterminate growth (or extension of the axis) occurs. continuous growth occurs when a shoot does not show a marked endogenous cessation of extension, a case usually occurring in uniform equatorial climates or environments. when a shoot shows marked endogenous extension periodicity and cessation, rhythmic growth occurs. rhythmic extension of leafy shoots is the typical pattern of deciduous fruit trees in which meristem activity shows an alternation of periods of rest and of active extension. the rest period marks on an axis portion a zone of short internodes and/or cataphylls corresponding to the protective organs of the bud from which the axis derives. rhythmic growth of the stem may combined with continuous or more frequently with a rhythmic organogenesis pattern. preformation and neoformation. when metamers and organs of a shoot are already present in the bud before the elongation of the axis deriving from it, the shoot is called preformed. in other cases, more metamers and organs than those included in the bud appear on the shoot and are neoformed by the apical meristem. fruit trees show a strong polymorphism in axis development (costes et al., 2006) (fig. 2). preformed shoots are usually short axes (brachyblast) which after bud burst elongate slightly due to the extension of the preformed metamers. in horticulture short shoots are referred to different names according to the species and to their reproductive or vegetative structures. in stone fruit fig. 1 evolution of the tree crown during plant life (from hilkenbäumer, 1953). 131 species, they are called ‘leaf rosettes’ or ‘clusters’ if they bear, respectively, only leaves or an apical vegetative bud and a variable number of lateral flower buds. in pome fruit species, the short preformed shoots are called ‘dards’ when they are vegetative or ‘spurs’ when they bear also flowers (fig. 2). in many fruit species (cherry, apple and pear trees in particular) the spurs can be one-year-old brachyblasts or consist of a perennial set of branched shoots, which have all remained brachyblasts. longer shoots may be also preformed (mesoblasts) and in this case their limited length derives only from the elongation of internodes; mesoblasts can carry only vegetative buds or carry some lateral flower buds (stone fruits) or an apical fruit bud (pome fruits) and, in any case, are called ‘brindles’(fig. 2). preformation and neoformation can also be combined to produce much longer shoots (auxiblasts) in fruit trees; they are usually vegetative axes in pome fruits (fig. 2), whereas in stone fruits they can bear numerous lateral flower buds as in peach tree. branching process. the complex architecture of a tree consists of several axes, one derived from another by repetitive processes (fig. 3). the branching process is based on axillary meristems located just beside the initiated leaf at a node. more than one axillary bud may be found at the axil of a leaf as in the case of mixed shoots of peach trees or other stone fruit species. the branching pattern of an axis can be monopodial or sympodial according to its indeterminate or determinate growth pattern, respectively. in the case of a sympodial pattern, one, two or more branches may develop after the death, abscission or transformation of the apex. in fruit trees one or two branches arise more commonly. rectilinear stems may be composed of a succession of metamers or growth units or annual shoots, all produced by a single meristem or by a linear succession of sympodial modules (barthélémy and caraglio, 2007). continuous branching occurs when all the axillary meristems of a stem develop into lateral shoots; rhythmic branching occurs when lateral axes are grouped as distinct tiers with an obvious regular alternation of unbranched and branched nodes on the parent stem. branching may be diffuse if only some nodes of the parent axis bear a lateral axis or if a regular distribution of branches in tiers is no evident. acrotony, basitony and mesotomy. the positional preferential development of lateral branches on a vertical parent axis may be classified into three categories. acrotony is the prevalent development of lateral axes in the distal part of the parent axis or shoot, whereas basitony consists in the prevalent development of branches on the proximal part; mesotony is used to denote a privileged development of branches in the middle part of the axis. the topological arrangement of lateral branches along a parent axis is often associated with an increasing or decreasing gradient in length and/or vigour of the branches. in fruit trees, all three categories of lateral axis development are present with some variations even in the same species. the topological arrangement of branches in the tree crown can be strongly modified by pruning. hypotomy, epitomy and amphitomy. considering that an axis may diverge from the vertical, the privileged arrangement of lateral axes on a parental axis is very different if this is horizontal, curved or slanted. the privileged zone may be the upper, lateral or basal position of the parent axis and the terms are respectively epitony, amphitony and hypotomy (fig. 4). in many fruit trees epitony is fig. 2 polymorphism of the axes on an apple branch. left: s (brachyblast) and bs (brachyblast on a bourse) and br (mesoblast) are preformed shoots, ls (auxiblast) are preformed and neoformed shoots. right: long shoot, brindles and spurs on a bourse. fig. 3 evolution of a one-year axis (a) into two-year (b) and threeyear (c) branches. fig. 4 development of lateral axes on an axis. slanted parent axis of peach tree (left) and curved parent axis of apple tree (right). 132 a very common feature (costes et al., 2006), it is often associated with the survival of old branches in old trees (fig. 5). hypotomy is characterized by a privileged development of lateral axes in the curvature zone of a branch. amphitony occurs frequently on rectilinear horizontal or slightly slanted branches. the latter two features may be combined in slanted and curved branches and their incidence in the expansion of lateral branch complexes is of the outmost importance in the aerial architecture of many woody plants. amphitony is a frequent behaviour in rectilinear branches while epitony and hypotony are characterized by the predominant development of lateral axes on the convex side of the curved, downwardlyor upwardlyoriented branches. as they are highly influenced by axis orientation, these branching features are frequently combined with topological arrangement along axes (acrotony, basitony, mesotony) and these combinations can strongly influence the bud fate according to their topological position and space orientation within the tree canopy. polymorphism of axes. the orientation of an axis and the spatial disposition of its leaves are of major importance in the growth strategy of a tree. within a single tree, some of these axes are essential in plant skeleton edification; some are involved in space exploration whereas others are more directed toward reproductive function or light interception and photosynthesis. the differentiation of axes and bud fate may be highly specialized and very different structures (i.e. flowers, inflorescences, spine, shoot, etc.) may be found in a single leaf axil and in a precise position, but the differentiation of an axis may not be an irreversible process. depending on modifications of internal or external conditions or after architectural traumatism or manipulation (pruning), reversion of axis differentiation may frequently occur, indicating that shoot differentiation and bud fate are controlled by a whole plant network of correlated and environmental conditions. the polymorphism of axes is common in fruit trees and represents a morphological differentiation determined by meristem expression and activity. it is common that several types of axis coexist on the same individual tree. however axis specialization is very significant when a tree is trained and pruned for fruit production since in this tree a balanced distribution among skeleton axes, reproductive axes and vegetative axes used for renewal fruiting shoots (fig. 6) must be found. orthotropy, plagiotropy, and mixed axes. orthotropic axes are generally erect to vertical with a radial symmetry, bear leaves in spiral, opposite or verticillate disposition, and lateral axes in all spatial directions; orthotropy is generally associated with plant skeleton edification and the colonization or exploration of vertical space. by contrast, horizontal to slanted oriented axes (plagiotropy) show a bilateral symmetry with distichous phyllotaxis and lateral axes arranged in one plane; plagiotropy is generally concerned with exploration and exploitation of the horizontal space and reproductive functions. in many trees an axis may present an orthotropic proximal portion and a plagiotropic distal end or vice versa. the superimposition of such “mixed axes” is a distinctive feature of trunk edification in trees, but it can be strongly manipulated by pruning (fig. 7). the position of sexuality and reproductive organs can be terminal or lateral and the onset of these organs involves dramatic changes in the architecture of the trees because its impact on plant growth and branching. the lateral or terminal position of reproductive organs is of great relevance for the orientation of the axis that supports the organs (fig. 8). fig. 5 evident epitomic behaviour in an old apple tree (from liebster and pesserl, 1982). fig. 6 in the tree canopy the polymorphims of axes is differently located. in some parts vegetative structures prevail and in others reproductive structures are predominant (redrawn from hilkenbäumer, 1953). 133 architectural model. the growth pattern of a fruit tree species which determines the successive architectural phases must be taken into account if a proper and specific training and pruning is to apply to the trees. the architectural model derives from an inherent strategy that defines both the manner, in which the plant elaborates its form, and the resulting structure. the model results from the nature and the sequence of activity of endogenous morphogenetic processes as determined by the basic growth program on which the entire architecture is established and realized under the environmental and cultivation constraints. a particular combination of simple morphological features may identify typical architectural models: a) the growth pattern (determinate vs. indeterminate growth; rhythmic vs. continuous growth); b) the branching pattern (terminal vs. lateral branching vs. no branching; monopodial vs. sympodial branching; rhythmic vs. continuous vs. diffuse branching; immediate vs. delayed branching; c) the morphological differentiation of axes (orthotropic vs. plagiotropic vs. axes with orthotropic and plagiotropic portions); d) the position of reproductive structures (lateral vs. terminal fruiting) (barthélémy and caraglio, 2007). the tree architecture can be considered a hierarchical branched system in which the axes are grouped into categories according to their morphological, anatomical or distinctive functional features. this branch system, even if very complex, is composed of a simple sequence of axes characterizing its basic architecture. the simple architectural unit lasts during the whole life span of some tree species while in most trees it is possible to recognize repeated architectural units during their development, late in ontogeny, or under particular conditions. this process is called “reiteration”; more precisely it is a morphogenetic process through which the tree duplicates its own elementary architecture. this process may be demonstrated in several structures such as water shoots, root suckers, etc. these reiterated structures may derive from dormant meristems and in this case are called proleptic or delayed. in other cases, reiteration may be a consequence of a shift in the functioning of the apical meristem of a growing shoot that will finally produce a less differentiated structure; in this case, the reiteration is described as sylleptic or better immediate. the trees develop by the repetition of elementary construction units conforming to their model and a differentiation sequence in the activity of their whole set of meristems. the specific and exact structure of a particular organ in a given location within the architecture of the tree may be considered as the result of a complex of several ontogenetic and morphogenetic factors that influences all plant organization levels, at each stage of plant development and during its whole life span. although environmental factors may modulate these sequences of differentiation, they almost never modify the inherent morphogenetic and ontogenetic constructional project of the plant organization. this is also true for horticultural manipulations such as training and pruning, confirming that it is advisable to follow the fruit tree’s development without radically changing its vegetative and reproductive behaviour. in other words, the branching order of an axis must be respected since the higher the order, the higher its degree of differentiation. when the architecture of a branched tree must be manipulated, the modification must be carefully considered according to the specific branching pattern, gradients (acrotony, basitony, mesotony) and the axis spatial orientation and/or geometry (hypotony, epitony, amphitony). in a given plant species, or even in a fig. 7 the relative location of vegetative and reproductive activities may be changed by variations of growth direction of axes imposed by pruning. fig. 8 natural variation in growth direction of a pear brindle with an apical fruit bud (from grisvard, 1957). 134 single tree, very different situations may be observed: at a given stage of development, homologous botanical structures with different features coexist on the same individual (e.g. short vs. long shoots or reproductive vs. vegetative shoots) whereas, by contrast, similar elementary structures with the same morphological features (short shoots with vegetative or reproductive features) may be observed in very different plant ages or stages of development. this may be defined as the physiological age of the meristem, which may generally be characterized by a particular combination of several morphological, anatomical and/or functional attributes of a given plant structure derived from this meristem. the physiological age of the meristem depends on its location in the plant architecture and on the stage of development of the plant; its expression may be modulated by environmental factors and obviously by cultural practices, particularly by pruning (fig. 9). the physiological age of a meristem relates to the degree of differentiation of the structure it produces. typical features of the physiologically aged structures are, for example, the short axes of many trees: growth units are short, bear flowers and may have a short lifetime. these highly differentiated axes may be considered physiologically old whatever their moment of appearance. by contrast, main axes consisting of vigorous growth units and/or annual shoots may be considered physiologically young products and generally appear only in young trees or in strongly pruned fruit trees. identification of meristem age of is very important in order to understand the comprehensive architecture of a plant or even its plasticity, i.e. the effects of the environment and/or of cultivation practices on its development and structure (phenotypic plasticity). 3. tree growth the growth of a living being means the irreversible increase of dry matter, and more currently the term is used for any positive variation in shape, size or fresh weight of an individual or a part of it. trees are characterized by secondary growth processes and by a continuous formation of woody tissue, part of which is non-living biomass. the woody part has the essential functions of tree support, sap transport, and storage of water, nutrients and carbohydrates. from the stand point of modern horticulture, trees of large size are not desirable because they invest a large part of energy and resources in building up and maintain their woody frame. the growth rate of the whole tree is of great economic meaning since it determines, in addition to dry matter accumulation, other important events such as the overcoming of juvenility (if the tree develops from a seed), the duration of the initial low productivity stage, and the acquisition of high fruiting and competing capability. practically speaking, in a grafted fruit tree a fast initial growth rate is an economic advantage for rapidly attaining its definitive size, which in modern fruit trees should be as small as possible in order to realize a high ratio between fruit load and woody frame (bargioni, 1988). tree growth (i.e. permanent increase of dry matter) is balanced between the development of new leaves and new roots. the new leaves exert a positive feedback on the assimilate production and similarly the new roots a positive feedback on absorption of more water and nutrients. at the beginning of a tree’s life, the major part of assimilates produced by the leaves is invested in the construction of the root system. as long as the root system does not achieve an adequate size to supply a sufficient amount of water and nutrients, the reproductive potential of the tree remains zero or extremely low. during maturity the canopy-root ratio is relatively stable and fruiting is kept more or less constant. when the tree attains a given size, the costs of maintaining the structure become very high and fewer and fewer resources are available to sustain root system growth, since its competition potential is weak in relation to other plant organs, particularly fruits. less water and nutrients are then available to the crown, reducing net photosynthesis and assimilate potential and, as a consequence, senescence of the tree is triggered. the net investment of the various resources which enter in and exit out of the tree and partition of the biosynthesis products are two important determinants of the balanced growth of a tree. water, nutrients, carbohydrates, organic acids, lipids, proteins, hormones etc. may be utilized in different fig. 9 formation of lateral axes of different types and ages on an apple tree branch pruned according to the various pruning criteria used in four training systems, which diverge from the natural behaviour very little (a) to very much (d) (redrawn from hilkenbäumer, 1953). 135 ways and times in relation to the physiological balance of the tree. the amount and partition of photosyntates gives a good indication of the growth potential of the tree or of its organs because they are the source of energy which can be used at the time or stored. source-sink interactions, that is the transitory destination of assimilates and their final partition, determine the rate and expression of the relative growth of the plant organs and therefore the architecture and size of the tree. the aim of tree and orchard management is to channel the major part of assimilates toward the reproductive structures, limiting that directed toward other plant organs without affecting the functioning of the whole system. individual tree architecture can vary greatly based on the growth strategy of the plant but can be modified by the environment and cultivation conditions (phenotypic plasticity). an example related to orchard design is the competitive stress induced by planting density (fig. 10). in comparison to a widely spaced population, closely spaced tree populations have a more limited volume of soil available for the individual root systems and thus fewer resources are supplied to the tree canopy. with close spacing, individual trees are smaller and less branched, but the ratio between fruits and woody structure (or volume of the crown) is higher than in individuals in a widely spaced population. not all plant characters are affected equally by the competitive stress, and some effects tend to be speciesand environmentspecific (cannel, 1983). light interception and distribution in the canopy may play a concomitant role in plant growth; in the tree canopy the behaviour at high incident irradiance depends on the degree of light saturation of the leaves, which in turns depends on their geometrical arrangement and the geometry of the tree, which determine the ratio between direct and diffuse radiation (connor, 1983). from outside to inside the canopy the quantity of light decreases sharply, as does the light quality (proctor, 1978). taking into account the variation of light quality inside the canopy (visible light, red/far red, uv), plant form and reproductive performance may be different in dense or open canopies because of the photomorphogenic and photoperiodic effects (connor, 1983). in stands composed of genetically identical trees, like orchards, the effects of competitive stress can be even more emphasized if appropriate pruning and management practices are not applied. the contribution of pruning to the shape, growth and functioning of the tree can be relevant but it does not introduce dramatic variations in the natural vegetative growth habit of the fruit tree. 4. correlative functions the correlative functions within an individual tree (i.e. competition and cooperation among the plant organs) deserve particular attention because they offer some basic concepts for pruning, which l.h. bailey illustrated very well a long time ago in “the pruning manual” published as first edition in 1898. it is worthwhile to report exactly what he wrote. “a tree is essentially a collection or a colony of individual parts. every branch, even every joint of the branches, may do what another branch does it may bear leaves, flowers and seed. every branch competes with other branches; and there are more germs of branches that is, more buds than there can be branches on any tree, or in any other plant that by its nature produces many branches. no two branches of a tree are exactly alike, but are what their position or condition or heredity makes them to be. some are strong and some are weak. that is, there is no definite or proper size or shape for any branch, as there is for different members of an animal or of a flower. the limbs and organs of an animal are not competitors but copartners, each performing some functions or office, that another does not, and they all attain a definite maturity of size and shape. but a branch in a tree-top never attains its full size until it ceases to grow and thereby begins to die. branches are not so much organs as competing individuals. if all these statements are true, then three conclusions follow: there is a contest among the branches of a plant, and some of the contestants perish; the destruction of these branches may conduce to the betterment of those remaining; all the branches of a tree are not necessary to it, but some of them may be superfluous or detrimental to it. in other words, pruning may follow as natural course.” in this context the manipulation of trees by pruning is amply justified in an orchard. citing again bailey, “of course there is a kind of partnership between the branches of a tree, for we assume that each strong branch makes a contribution to the development of the root-system and trunk-system, and there is not the same separateness as between wholly different plants; yet the contest between these branches is apparent, and it has special significance to the present discussion.” of course these considerations may be extended to the other organs of the tree: buds, twigs, shoots, flowers, fruits and roots. fig. 10 effect of planting density on tree structure. closely planted trees bear more fruits per unit canopy volume than widely spaced trees because of phenotypic plasticity (cannel, 1983). 136 taking into account the functional equilibrium between the size and activity of the shoots (carbon fixation) and size and activity of the fine roots (absorption of water and nutrients), that in a constant environment, favouring continuous growth, tends to maintain a constant ratio of root/shoot relative growth rates (cannel, 1985), it is evident that removing a part of a tree by pruning represents a loss of a given amount of tissue and in replacing the lost part the tree expends a certain amount of assimilates. this may seem nonsense, but it is necessary to keep in mind the goals of the orchard, which are the economics and adjustment of treeenvironment interactions. tree vitality is not impaired by removing a part of it unless the removal is so great that it interferes with nutrition (growth) of the remaining parts, for example a great removal of photosynthetic leaf surface or of adsorbing roots. taking into account plant homeostasis, it is obvious that if part of the shoots or branches are removed, assimilates are devoted to rebuilding the lost part and fewer are directed toward the root system, and vice versa. if part of the roots are suppressed, the shoots resume their growth only when the pruned part of the root system is rebuilt and the root/shoot equilibrium is re-established, other aspects remaining constant (richards and rowe, 1971). therefore, shoot pruning temporarily checks root growth, while root pruning temporarily checks shoot growth: the more pruned off, the greater the check in growth, and the longer it takes for the plant to recover the root/shoot relationship that existed before pruning. thus, shoot pruning is a means of promoting new shoot growth, and root pruning is a means of promoting new root growth (cannel, 1985). therefore when part of the treetop is cut away, if not too severely, the tree resources are directed toward the remaining growing points and the tree develops, for example, more vigorous shoots. the consequence is that a correctly pruned tree appears more vigorous and also, as reported by dotti (1949), more productive and longliving than an unpruned tree. for the purpose of this paper, the genetic and metabolic processes causing and governing these correlative functions will not be considered but instead some of the most relevant organ and function competitions for the tree resources are addressed from a phenotypic point of view. hierarchic but not univocal relationships are often established among tree organs and they contribute to the correlative functions that may involve organs of the same or different type. 5. correlative relationships between organs of the same type in this section relationships between meristems, buds, shoots, branches, flowers and fruits are considered. the most important correlative functions are those among the meristems present on a shoot since they originate all the organ typologies. the role of apical dominance, that varies amply according to genotype, development stage and environmental conditions, is known. apical dominance also affects the functional relationships of the buds, determining different vegetative gradients along the shoot. these phenomena offer an important basis for pruning practices. in peach the number of sylleptic shoots appeared to be related to the growth rate displayed by the parent shoot during the early part of the growing season (four to five weeks after bud break). parent shoots with high growth rate formed sylleptic shoots in a greater number than the pinched back ones in which apical dominance was completely abolished. therefore growth rate in the early growing season may play a stronger role than pinching in feather formation. apex removal by pinching (summer pinching) changes the physiological status of a growing shoot, whereas bending affects only the shoot growth rate, thus its feathering (giulivo and ramina, 1974). an interesting case, reported by hilkenbäumer (1953), regards the number and distribution of buds on a tree. three cases are considered and are shown in figure 11: a) when a tree carries a very high number of buds, on each axis a large number of weak shoots are produced and a part of buds remain silent; b) if all the axes are heavily and evenly pruned back, the number of buds is reduced and fewer but vigorous shoots originate in the same manner on each branch; c) if one axis is heavily pruned back and the other two lightly, weak shoots are produced by the former and strong shoots by the latter axes in contrast with case b. cases a and b may be explained by the assimilate partition among many or few buds (hilkenbäumer, 1953). for case c, which is more difficult to explain, it is suggested that the less pruned axes dominate the heavy pruned axis because they bear more buds and sprout earlier, becoming thus stronger sinks. this may be considered an example of the independence-competition among axes of a tree. the fewer the shoots are allowed to grow with a severe pruning, the longer they are (fig. 12). when only very few shoots are present on the tree, not only are they thicker with larger leaves and have longer internodes, but some of the lateral buds grow out to form short shoots or feathers (abbot, 1984). the leading shoot (dominant position) is however always longer than those below it (fig. 13). whichever parent axis is manipulated (fig. 14), there is fig. 11 vegetative responses of branches headed back with different intensities (a and b) or uneven intensity (c) (redrawn from hilkenbäumer, 1953). 137 always one lateral axis that assumes dominance over the other axes. these factors clearly indicate the competition between homologous vegetative organs. a typical case of competition among homologous organs is the high abscission potential or reduced development and growth of fruits that are located, respectively, below or above the fruit that sets first in the corymbs of apple and pear trees (fig. 15). the effect of fruit set priority on competition was also reported in peach tree where the fruitlets which set first and start to grow rapidly have the lowest probability of abscission (ramina 1981; giulivo et al., 1981 b). 6. correlative relationships between organs of different types the strength of relationships among organs are dynamic and change over the growing season and year. usually the organs that have the larger mass (size) and grow more actively dominate the other organs of the plant. the relationship between vegetative and reproductive structures is a typical case. when shoot growth is very intense, fruit growth is limited and vice versa (fig. 16) (giulivo et al., 1981 b; pitacco and giulivo, 1992). in an unpruned tree parts of canopy exist where vegetation dominates and parts where fruiting is prevalent. the position of a vegetative axis in space determines the vegetative-reproductive relationship (fig. 6); in a vertical axis the vegetative activity is strongly favoured and fruiting is repressed. if an axis occupies a lower position in fig. 13 elongation in apical (a), sub-apical (b, i) long shoots and in brindle (br) shoots of three cultivars of peach tree with different ripening time (giulivo et al., 1981 a). fig. 14 position of the dominant shoot in headed-back vertical (a), curved (b) and slanted (c) parent axes (da grisward, 1953). fig. 12 effects of light, medium and severe pruning (one, two, three shoots on a headed-back one-year-old apple tree) (abbott, 1984). 138 the crown and tends toward a plagiotropic position, the relationship is reverted. this phenomenon obviously offers a great opportunity to manipulate a tree with winter and summer pruning. it can be assumed that every manipulation limiting growth can increase the reproductive activity, i.e. fruit bud differentiation and fruit development and growth. two typical cases may be reported: a) by bending, the elongation rate of a growing shoot is decreased but bud differentiation is favoured; b) with limited nitrogen availability the vigour of the tree is depressed but the fruiting capability is increased (hilkenbäumer, 1953). the relationship between fruitlets and shoot carried by a bourse of apple tree can be an interesting case of transient competition between a leafy shoot and a reproductive organ carried by the same plant structure (brachyblast). usually the fruitlets carried by the bourse corymb present a steady abscission for some weeks after full bloom but, if the shoot of the bourse is removed early, fruitlet abscission is delayed and reduced; the fruits on bourse deprived of the shoot growth less and at their final size results much smaller than that of fruits on a normal bourse carrying a shoot (abbot, 1984). this is a consequence of fruit abscission in the corymb, but it may also be an effect of the transport of assimilates to the fruit which takes place when the leaves of the bourse shoot become active exporting organs. as the season progresses, the developing fruits have an increasing demand for assimilates: after some weeks from fruit set they are diverted from shoot and root growth, bud differentiation and later from reserve storage. increasing the fruit load has a dramatic effect: fruit bud differentiation is progressively reduced to a certain threshold, beyond which it is completely abolished (fig. 17). the presence of fruits on the tree slows down the formation of fruit buds as long as the fruits remain on the tree (fig. 18); the later the harvest, the stronger the effect (giulivo et al., 1981 a). a heavy crop load, induced by a very low fruit load during the previous year, strongly reduces shoot growth, flower differentiation for the next year and storage of reserve fig. 16 seasonal growth rate of shoots and fruit of peach tree, cv. andross. when the former is high, the latter is low, and vice versa (pitacco and giulivo, 1992). fig. 17 effects of increasing fruit load of apple tree on shoot and fruit bud formation and on elongation of the longest shoot. (abbott, 1984) fig. 15 competition of the first setting fruitlet over the other fruitlets of the same corymb of pear (left) and apple tree (right). 139 materials (giulivo, 1990). this can explain the succession of onand off-years. a large number of developing fruits requires a great amount of assimilates which are diverted from vegetative growth and bud differentiation and this is a clear example of competition between different plant organs or functions. flower or fruit thinning in the on-year is a powerful tool for modulation of the competition exerted by the fruits (ramina, 1981; abbot, 1984). over-cropping and undercropping, subsequent to offand on-years, can be to some extent overcome by winter pruning, decreasing or increasing the number of fruiting structures of the tree. the onset of fruiting is a dramatic event in the behaviour of a tree and the relative growth of plant parts is radically changed (chalmers and van den ende, 1975): as the crop potential of the tree increases with time (fig. 19), fewer and fewer resources are allocated in the permanent structures of the tree (trunk, branches and root system). this means that cropping of the tree is over time an aging factor and its regulation is a way to delay senescence of the plant. the regulation of cropping by summer and/or winter pruning represents a powerful tool for the maintenance of long-lasting economic performance of fruit trees. during the growing season a heavy fruit load reduces root growth; very strong vegetation acts in a similar way, limiting fine root formation (willianson and coston, 1989). the partitioning of assimilates between shoots and roots is strongly affected by the water status of the tree (schultze, 1982). in many cases and in some stages of development the competition between tree organs is often connected with cooperation. the dynamic relationship between shoots and fruit within the whole tree, and also within a single leafy shoot, is particularly relevant. in fact, in an early stage of fruit growth (fig. 20) the relationship shoot/fruit depends on the number of sinks and their relative position along the mixed shoot, as shown by an experiment on one-year-old peach shoots (giulivo and ramina, 1975). complex relationships among sinks occur, even within a single node of a shoot as observed in peach tree. it is fig. 18 fruit growth and percent of fruit bud formed on shoots of three cultivars of peach tree with different ripening times (giulivo et al., 1981 a). fig. 19 dry matter partition among fruits (points), tree crown (crosses) and root system (asterisks). at increasing tree age represented by the trunk circumference less and less dry matter is allocated in the permanent structure of the tree (redrawn from chalmers and van den ende, 1975). fig. 20 fruitshoot correlative relationships on peach one-year-old mixed shoots 0.2 m long. (circles = fruit; arrows = shoots; square = basal leaf of mixed shoot treated with 14c, all other organs were removed). the organ (or organs) at the higher position was always the strongest sink. the fruit was a very strong sink (situation a); when the fruit was in between two shoots its strength was strongly reduced (situation b); when the fruit was above two shoots it was dominant (situation c) and therefore the relative organ position changed the sink relationship; the number of shoots above and below the fruit change the relationship between the organs (situations b and d). (giulivo and ramina, 1975). 140 known that different combinations of vegetative and flower buds occur at the nodes of peach mixed shoots; if a single fruit originates at a node initially it grows faster than the one or two fruits which are associated with a node with a growing shoot, but after some time, when the shoot rate decreases, these fruits attain a larger size (casella, 1949; giulivo, data not published). this may be explained by the contribution of the shoot leaves to the assimilate requirement by the fruits, in agreement with the results reported by abbot (1984) on the shoot/fruit relationship carried by an apple bourse. it would be possible to mention many other correlative functions, but the cases mentioned here should be adequate to stress the significance of these relationships in pruning management. 7. conclusions the efficiency of the orchard system is a primary requirement for economic success in fruit growing and therefore the architecture of the stand, with an appropriated use of the space available, can permit an efficient use of light, water and nutrients to sustain the basic physical and physiological processes involved in the functioning of the system. sometimes space utilization (planting density) is too low or too high; in the former case light interception by the canopies becomes too scarce and soil evaporation too high; in the later, reciprocal shading of the trees assumes considerable weight. sometimes the structure of the canopy is not optimal, as it may be too dense or too sparse and consequently the conditions inside the canopy are such that leaf and fruit functioning may be modified, pest proliferation may be promoted or light interception may be too scarce to maintain the various functions and processes of the canopy. in designing tree architecture, three basic indexes must be considered: the ratio between the tree parts above and below the ground, the ratio between leaf area surface and canopy volume, and the leaf area surface per unit of fruit weight. these indexes are satisfied differently in the various orchard models in relation to the applied training system and pruning criteria. different training systems with a similar pruning criterion tend to have more or less the same production behaviour while different pruning criteria applied to the same training system tend to induce different crop performance (sansavini and musacchi, 1994). pruning practices are thus a powerful means to modify tree functioning: pruning generally reduces the primary production of the orchard but may induce a great improvement of the performance of the system. the details of pruning for maximum fruit bud production and adequate fruit size and ripening differ for different species but it is possible to identify some general principles. the various pruning techniques have very different effects on the growth and fruiting of a tree; heading-back or removal of organs, variations of spatial position and/or orientation of vegetative axes and transient modification of sap transport induce very different reactions. interventions directed to shortening or removing an axis generally involve vegetative responses which are more or less strong in relation to location, intensity and when they are performed. particular attention is required because strong negative or positive effects on the vegetative and reproductive equilibrium of the tree may arise. the negative effects regard mainly an excessive vegetative reaction but, if the pruning is carefully performed, the tree develops a species-specific fruiting structure more adequate to the economic goal of the orchard. the detachment of fruits (thinning) performs a particular role in realizing a correct fruit-to-leaf ratio in the tree where the crop load is too heavy in relation to the leaves of the canopy. changing the position or the growth direction of the axes in the space (rectilinear, slanted, curved) generally decreases the growth rate and vigour of shoots and branches, improving the formation of reproductive structures. therefore this type of manipulation assume a great importance in training the fruit trees. some pruning techniques, which tend to modify sap transport (incision above or below a bud, bark ringing, strangulation or girdling, shoot twisting and crashing, etc.), influence the relationships among sinks and the source-sink and thus they may stimulate or limit the development of an organ. whatever the manipulations of the above-ground part of the tree, root functioning is modified, but taking into account tree homeostasis, any modification to the root system affects the performance of the canopy. therefore root pruning may be very useful for controlling tree vigour or improving root growth in adult trees. all pruning manipulations mentioned involve variations in plant growth, size and geometry, thus modifying the architecture and functioning of the tree and affecting its productive life. the timing of pruning manipulations is of great importance since the effects on the tree may be very different. in recent years summer pruning tends to prevail over winter pruning in most fruit tree species (bargioni, 1988; sansavini and corelli, 1990; costa, 1997; sansavini et al., 1999; neri and sansavini, 2004). this tendency is mainly based on two reasons: to form small-sized trees and to follow the natural species-specific behaviour of the tree. under the same cultivation conditions winter pruning tends to stimulate the vegetative reaction, whereas summer pruning generally has positive effects on reproductive activity. this may be due to the more powerful action of summer manipulations on source-sink relationships, and among sinks. summer pruning, however, may reduce leaf area surface or the ratio between young, mature and old leaves and this may have some consequence on the photosynthetic performance of the canopy, which can be of some importance when light availability is limited. the timing of summer manipulation of the canopy is critical: if performed when vegetation is growing very fast. in this case the responses of tree may be too strong and consequently a consistent amount of resources are invested in the edification of new 141 vegetative structures or the density of the canopy may be increased. the timing of root pruning is as important as the time of canopy pruning since it has positive effects in the spring when it controls tree vigour, whereas it acts negatively if performed in late summer when storage of reserve materials is needed for the next year. in the last few decades summer pruning (mainly pinching) has been extended to nurseries to train trees of some fruit species (apple, peach, etc.) carrying some feathers which speed up the construction of the tree crown in the orchard. in this way is possible the shortening of the unproductive period or to have young trees more suited for high density orchards or some particular training system (sansavini and corelli, 1990; vigl, 1999; neri and sansavini, 2004). in the 1990s some attempts were made to reduce production costs by omitting pruning during the training of trees and thereafter applying very simplified and superficial pruning, relying on thinning to equilibrate fruit load and leaf area surface. the performance of this technique, called ‘no-pruning’, was quite disappointing because of the serious drawbacks on fruit quality, such as variability in size and ripening of the fruits. even in high density planting, without an appropriate adaptation of the training system, pruning was not able to overcome the excessive tree competition that determines premature aging of the fruiting structures, excessive shading, crop alternation and poor fruit quality (sansavini, 1999). therefore it may be concluded that pruning will always be an unavoidable operation, taking into account the economical goal of the orchards. in conclusion, the best pruning derives from a clear production target and from precise planning while considering tree general physiology, the peculiar characteristics of the fruit species, cultivars, clones, scion-rootstock combinations, and the local environmental and cultivation conditions. finally, it is important to remember that pruning represents only one of the many manipulations which can be performed in the orchard yet it cannot overcome basic mistakes made in designing the system. references abbot d.l., 1984 the apple tree physiology and management. grower books, london, uk, pp. 90. bailey l.h., 1916 the pruning manual. being the eighteenth edition, revised and reset, of the pruning-book, which was first published in 1898 macmillian company, new york, usa, pp. 407. baldini e., scaramuzzi f., 1962 glossario dei termini usati nella potatura degli alberi da frutto a foglia caduca. tipocolor, firenze, italy, pp. 24. bargioni g., 1988 forme di allevamento e tecniche di potatura: come cambieranno? frutticoltura, l(1-2): 97-103. bargioni g., 1992 potatura degli alberi da frutto, pp. 393-494. in: baldoni l., g. bargioni, m. devreux, o. failla, c. fideghelli, g. fontanzza, f. gorini, r. jona, f. lalatta, g. maracchi, r. paglietta, g. rapparini, c. xiloyannis, and a. zocca. frutticoltura generale. reda, roma, italy, pp. 644. barthélémy d., caraglio y., 2007 plant architecture: a dynamic, multilevel and comprehensive approach to plant form, structure and ontogeny. ann. bot., 99: 375-407. branzanti c.e., ricci a., 2001 manuale di frutticoltura. edagricole, bologna, pp. 103-141. cannel m.g.r., 1983 plant management in agroforestry: manipulation of tree, population densities and mixture of trees and herbaceous crops, pp. 455. in: huxley p.a. (ed.) plant research and agroforestry. international council for research in agroforestry, nairobi, kenya, pp. 617. cannel m.g.r., 1985 dry matter partitioning in tree crops, pp. 160. in: cannel m.g.r., and j.e. jackson (eds.) attributes of trees as crop plants. institute of terrestrial ecology publisher, pp. 592. casella d., 1949 mezzi per attenuare la cascola delle gemme, dei fiori e dei frutticini nelle specie legnose fruttifere. atti iii conv. naz. di frutticoltura, ferrara, italy, pp. 200-212. chalmers d.j., van den ende b., 1975 productivity of peach trees: factors affecting dry-weight distribution during tree growth. ann. bot., 39: 423-432. connor d.j., 1983 plant stress factors and their influence on production of agroforestry plant associations, pp. 401. in: huxley p.a. (ed.) plant research and agroforestry. international council for research in agroforestry, nairobi, kenya, pp. 617. costa g., 1997 potatura di produzione e diradamento dei frutti negli impianti di pianura. proceeding symposium melicoltura di pianura: quale futuro, verona, italy, 12 april. costes e., lauri p.e., regnard j.l., 2006 analyzing fruit tree architecture: implications for tree management and fruit production. horticultural reviews, 32: 1-61. dotti f., 1949 recenti vedute sulla potatura dei fruttiferi. atti iii conv. naz. di frutticoltura, ferrara, pp. 213-230. giulivo c., 1990, basi fisiologiche della potatura degli alberi da frutto. proceedings “la potatura degli alberi da frutto negli anni 90”, verona, italy, 27 april. giulivo c., ramina a., 1974 osservazioni sulla formazione dei germogli anticipati nel pesco. ii contributo. riv. ortoflorofrutt. it., 3: 170-176. giulivo c., ramina a., 1975 competizione tra germogli e giovani frutti di pesco. riv. ortoflorofrutt. it., 4: 273-280. giulivo c., ramina a., costa g., 1981 b il contributo del sottoprogetto 5 alle conoscenze dei processi di abscissione e maturazione dei frutti proceedings i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, italy, 26-27 march. giulivo c., ramina a., scrittore g., 1981 a accrescimento e sviluppo riproduttivo in cultivar di pesco con diversa epoca di maturazione dei frutti. proceedings i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, italy, 26-27 march. grisvard p., 1957 la taille des arbres fruitiers. la maison rustique, paris, frances, pp. 160. hilkenbäumer f., 1953 obstbau. paul parey, berlin und hamburg. 142 ingles c., geisel p.m., unruh c.l., 2002 fruit trees: training and pruning deciduous trees. anr, publication 8057, ucd, davis, ca, usa, pp. 7. liebster g., pesserl g., 1982 fachausdrücke des obstbaumschnittes lexikon in deutscher, wörterbuch in englischer, französicher, italienischer und niederländischer sprache. mitteilungen klosterneuburg, 32: 50-100. morettini a., 1963 frutticoltura generale e speciale. reda, roma, italy, pp. 612. neri d., sansavini s., 2004 attualità della potatura nella frutticoltura intensiva. frutticoltura, lxvi(1): 14-23. pitacco a., giulivo c., 1992 dinamica della crescita dei germogli e dei frutti in cultivar di pesco a diversa epoca di maturazione. giornate scientifiche s.o.i., ravello, italy, 8-10 april. proctor j.t.a., 1978 apple photosynthesis: microclimate of the tree and orchard. hort. sci., 6: 641-643. ramina a., 1981 la dinamica della cascola ed alcuni aspetti fisiologici dell’abscissione nel diradamento chimico dei frutti di pesco (prunus persica, l. batsch). conv. i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, 26-27 march. richards d., rowe r.n., 1971 effects of root restriction, root pruning and 6-benzilaminopurine on the growth of peach seedlings. ann. bot., 41: 729-740. sansavini s., 1999 potatura in libertà. frutticoltura, lxi(3). 5-6. sansavini s., corelli grappadelli l., costa g., lugli s., marangoni b., tagliavini m., ventura m., 1999 ricostituzione degli impianti e revisione degli indirizzi produttivi della peschicoltura romagnola. frutticoltura, lxi(3): 8-20. sansavini s., corelli l., 1990 la potatura e le forme di allevamento del melo. proceedings la potatura degli alberi da frutto negli anni ’90. verona, italy, 27 april, pp. 65-99. sansavini s., errani a., 1998 frutticoltura ad alta densità, impianti, forme di allevamento e tecniche di potatura. edagricole, bologna, italy, pp. 593. sansavini s., musacchi s., 1992 modelli di impianto, architettura e potatura nei moderni pereti. frutticoltura, lvi(2): 7-18. schultze e.d., 1982 plant life forms and their carbon, water and nutrient relations, pp. 615-676. in: lange o.l., p.s. nobel, c.b. osmond, and h. ziegler (eds.) physiological plant ecology, ii. encyclopedia of plant physiology. springer verlag, berlin, germany. simon s., sauphanor b., lauri p.e., 2007 control of fruit tree pests through manipulation of tree architecture. pest technology, 1(1): 33-37. vigl j., 1999 l’allevamento del melo a “spindle”. frutticoltura, lxi(3): 29-32. williamson j.g., coston d.c., 1989 the relationship among root growth, shoot growth, and fruit growth of peach. j. amer. soc. hort. sci., 2: 180-183. impaginato 141 1. introduction bermudagrass is a warm-season, c4, perennial grass. it has short, grey-green blades with rough edges, stems of 1 to 30 cm in length and a deep root system that can penetrate 2 m into the ground; however, most of the root mass is less than 60 cm deep (xu et al., 2011). among the many advantages of turfgrass areas are erosion and dust control, aquifer recharge and protection from pollutants, heat reduction in urban environments, reduction of noise and pollution, and providing human health and aesthetic benefits (stier et al., 2013). water scarcity is an increasing challenge to the turfgrass industry and may result in irrigation restrictions being imposed without regard for damage to turfgrass (beard and kenna, 2008). for turf managers, thriving in an industry where turf quality is of utmost importance is difficult when water is limiting. therefore, researches investigating turfgrass resistance to drought stress have become increasingly important (fry and huang, 2004). fu and huang (2001) investigated the effects of drought stress on two cool-season turfgrasses and found that moderate drought stress had not effects on morphological and physiological characteristics, however in intensive drought stress, antioxidant enzyme activities, chlorophyll content, relative water content and shoot dry weight were decreased. in addition to limited amounts of water, turfgrasses are impacted by low-light environments. shade is more problematic for warm-season turfgrasses to maintain quality given their higher light saturation point compared to cool-season turfgrasses (fry and huang, 2004). turfgrasses perform poorly in reduced light environments due to high traffic rate, daily mowing, and reduced photosynthesis. in shade, increased disease presence adversely affects cool-season turfgrass development, while morphological limitations, such adv. hort. sci., 2016 30(3): 141-149 doi: 10.13128/ahs-20250 morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition n. adamipour (*), h. salehi, m. khosh-khui department of horticulture science, college of agriculture, university of shiraz, shiraz, iran. key words: antioxidative enzymes, irrigation, photoperiod, turfgrass. abstract: bermudagrass (cynodon spp.) is the most popular warm-season turfgrass used in warm climatic regions of the world due to its recuperative ability, high traffic tolerance, heat tolerance, and relative drought and salt tolerance. however, shade is a microenvironment in which bermudagrass performs poorly. in order to evaluate the interaction of photoperiod and irrigation on [cynodon dactylon (l.) pers. california origin], a greenhouse experiment was conducted at the research greenhouse of the department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. the experiment was conducted with four field capacity regimes (25%, 50%, 75% and 100%) and three light durations (8, 12 and 16 h) in a completely randomized design factorial arrangements with four replications. results showed that decreasing field capacity and photoperiod decreased fresh and dry weights shoot and root, chlorophyll and starch contents and superoxide dismutase, catalase and ascorbate peroxidase activities. decreasing the field capacity and light duration increased proline content. reducing sugars and peroxidase enzyme in leaves increased with decreasing field capacity. shoot height and leaf area increased by shortening the photoperiod. in overall, results showed that, the increase in irrigation alleviates the destructive effects of reduced day lengths and vice versa. further studies are needed to clarify more the interaction between irrigation and light treatments at structural and ultrastructural levels, in common bermudagrass. (*) corresponding author: adamipournader@yahoo.com received for publication 9 may 2016 accepted for publication 27 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 141-149 142 as reduced lateral stem growth, inhibits warm-season turfgrass development (beard, 1972). variations of shade responses among species and cultivars (jiang et al., 2004; trenholm and nagata, 2005; sladek et al., 2009) make it possible to select turfgrasses with superior shade tolerance. identifying morphological characteristics that are associated with superior shade performance based on genetic variation would add value to germplasm screening for shade tolerant species and cultivars. esmaili and salehi (2012) noted in bermudagrass that were treated with short photoperiod duration, verdure fresh and dry weight, shoot height, tiller density, leaf area and chlorophyll and relative water contents were decreased, however electrolyte leakage and proline content were increased. although bermudagrass, the most widely grown c4 turfgrass on an international basis (shearman, 2006), has been extensively studied, many challenges and questions still remain when light is a limiting growth factor. the main objective of the present study was to investigate the effects of both irrigation interval and light duration on growth and quality of common bermudagrass. 2. materials and methods plant material and experimental conditions this experiment was conducted at the research greenhouse of the department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran (52◦32’e and 29◦36’n, 1810 m asl). seeds of common bermudagrass (cynodon dactylon [l.] pers. california origin) were weighed and cultured in plastic pots with 19 cm in diameter and 25 cm in height, without drainage (0.25 g pot−1) filled with 4 kg clay-loam soil with permanent wilting point (pwp) of 19% and field capacity (fc) 29%. watering was carried out daily prior to beginning of treatments. plants were kept in a greenhouse with 31/25°c (day/night) temperature and 35% relative humidity for one month before the beginning of treatments. treatments were conducted at four irrigation levels (25%, 50%, 75% and 100% fc) and three photoperiod duration [8, 12 and 16 h as short day length (sdl), intermediate day length (idl) and long day length (ldl)]. watering was carried out daily before seed germination and after turf establishment. then, the turves were watered equally when required. established turves were clipped from 3 cm above soil by a hand mower and were transferred to a covered frame which temperature, light (intensity and length) and relative humidity were controlled with digital sensors. the environmental condition of covered frame was 31°c, white and creamy fluorescent lamps one m above the pots with a constant light intensity of 3000 lux, and 35% relative humidity for applying simultaneous irrigation and photoperiod treatments. pots were weighed daily and set to different irrigation treatments (25, 50, 75 and 100% fc), during the whole of experiment. after three months, plants were harvested in order to measure morphological and biochemical traits. growth parameters growth parameters including, shoot height (cm), leaf area (cm2) and fresh and dry weights of shoot and root (g) were measured. dry weights were measured when the materials dried at 60°c for 48 h. chlorophyll content chlorophyll content was measured according to the method of saini et al. (2001) using the following formula: chlorophyll (mg/g f.w.) = [20.2(od 645 nm) + 8.02(od 663 nm) × v/ (f.w.×1000)] where: od is optical density, v is the final solution volume in ml and f.w. is tissue fresh weight in mg. proline content proline was determined according to the method described by bates et al. (1973). using spectrophotometer (biowave ii, england) at 520 nm wavelength, appropriate proline standards were included in calculation of its content in samples. total soluble sugars and starch analysis the total soluble sugars were measured using the method as previously described by dubois et al. (1956). the total soluble sugar content of samples was measured at 490 nm of absorbance and glucose solution was used at different concentrations for standard curve drawing. the starch content was quantified using the bradford method (mccready et al., 1950). the starch content was measured at absorbance of 630 nm and calculated using the standard curve of glucose and multiplying it by 0.92. antioxidant analysis fresh samples were homogenized in extraction buffer (0.1 m phosphate buffer ph 6.8) with mortar and pestle on ice. the homogenate was then centrifuged at 12,000 g for 15 min at 4°c and the supernatant was used as the crude extract for the superoxadamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 143 ide dismutase (sod), guaiacol peroxidase (pod), ascorbate peroxidase (apx) and catalase (cat). the sod, pod, apx and cat enzymes were estimated using the methods previously described by beauchamp and fridovich (1971), chance and maehly (1995), nakano and asada (1981) and dhindsa et al. (1981), respectively. experimental design and data analysis this study was conducted in a completely randomized design with factorial arrangements and two factors: field capacity and photoperiod with four replicates. data were analyzed using statistical software (sas software) and mean comparisons were performed using lsd test at 5% level. 3. results and discussion results of analysis of variance (tables 1 and 2) showed that photoperiod (except for soluble sugar) and irrigation had significantly influenced the measured traits and also the interaction of photoperiod and irrigation had a significant effect on fresh and dry weights of shoot, proline content and the level of activity of superoxide dismutase. shoot height and leaf area shoot height and leaf area significantly declined by decreasing field capacity from 100% to 25% (table 3). shoot height and leaf area decreased (47.09% and 27.77%, respectively) at 25% fc compared to 100% fc (table 3). ryan (2011) reported that growth can be reduced through impairment of cell division and cell expansion which occurs at a lower water stress threshold rather than photosynthetic inhibition. fu and huang (2001) reported that shoot growth of both table 1 analysis of variance of photoperiod, field capacity and interaction between photoperiod and field capacity measured traits source of variability df shoot height (cm) leaf area (cm2) shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) sugars of shoot (mg g-1 d.w.) photoperiod 2 38.45 ** 0.04** 212.24** 20.59** 36.59** 4.87** 5.77 ns field capacity 3 314.82** 0.33** 141.40** 141.40** 1337.54** 324.86** 79732.82** photoperiod * field capacity 6 0.0 ns 0.0 ns 1.25** 1.25** 0.0 ns 0.0 ns 0.0 ns error 33 0.17 0.002 0.24 0.24 0.00 0.08 7.01 cv 2.18 4.32 3.58 6.57 0.00 2.02 1.96 ** and ns significant at the 0.01 level and not significant respectively. table 2 analysis of variance of photoperiod, field capacity and interaction between photoperiod and field capacity measured traits ** and ns significant at the 0.01 level and not significant respectively. source of variability df superoxide dismutase (ug-1 fw) catalase (ug-1 fw) peroxidase (ug-1 fw) ascorbate peroxidase (ug-1 fw) chlorophyll (mg g-1 fw) proline (µmol g-1 fw) starch content (mg g-1 dw) photoperiod 2 4044.08** 97.06** 969.12** 28933.33** 0.59** 4.99** 3.33** field capacity 3 55360.44** 234.71** 5248.15** 257973.85** 1.74** 7238.96** 105235.35** photoperiod * field capacity 6 2.52 ns 0.0 ns 0.0 ns 0.0 ns 0.00 ns 2.27** 0.00 ns error 33 235,20 3.26 27.65 260.03 0.00 0.12 0.00 cv 9.91 5.65 6.67 1.79 0.0 2.68 0.00 variables photoperiod field capacity (%) mean 100% 75% 50% 25% shoot length ldl 22.25 d* 21.27 e 16.67 h 11.05 k 17.81 c (cm) idl 23.75 c 22.77 d 18.17 g 12.55 j 19.31 b sdl 25.35 a 24.37 b 19.77 f 14.15 i 20.91 a mean 23.78 a 22.80 b 18.20 c 12.58 d leaf area ldl 1.22 bc 1.22 bc 1.17 c 0.87 e 1.12 b (cm2) idl 1.23 bc 1.23 bc 1.18 c 0.88 e 1.13 b sdl 1.32 a 1.32 a 1.27 ab 0.97 d 1.22 a mean 1.26 a 1.25 a 1.20 b 0.91 c shoot fresh ldl 20.30 a 20.26 a 16.16 b 14.06 c 17.69 a weight (g) idl 16.32 b 16.26 b 12.81 d 9.13 e 13.63 b sdl 13.85 c 12.83 d 9.69 e 5.34 f 10.42 c mean 16.82 a 16.45 a 12.88 b 9.51 c shoot dry ldl 11.30 a 11.26 a 7.16 d 5.06 e 8.69 a weight (g) idl 10.32 b 10.26 b 6.81 d 3.13 f 7.63 b sdl 9.85 b 8.83 c 5.69 e 1.34 g 6.42 c mean 10.49 a 10.11 a 6.55 b 3.17 c root fresh ldl 39.78 a 38.69 b 29.54 g 16.99 j 31.25 a weight (g) idl 37.80 c 36.71 e 27.56 h 15.01 k 29.27 b sdl 36.81 d 35.72 f 26.57 i 14.02 l 28.28 c mean 38.13 a 37.04 b 27.89 c 15.34 d root dry ldl 19.78 a 18.69 b 13.46 d 8.54 f 15.12 a weight (g) idl 19.69 a 18.60 b 13.37 d 8.45 f 15.03 a sdl 18.82 b 17.73 c 12.50 e 7.43 g 14.12 b mean 19.43 a 18.34 b 13.11 c 8.14 d proline content ldl 5.57 i* 7.06 g 14.35 f 23.25 c 12.56 c (mol g−1 f.w.) idl 5.63 i 6.51 h 15.19 e 24.20 b 12.88 b sdl 5.66 i 6.89 gh 17.29 d 24.74 a 13.65 a mean 5.62 d 6.82 c 15.61 b 24.07 a *in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. ldl= long day length. idl= intermediate day length sdl= short day length. table 3 effect of field capacity and photoperiod and their interaction on shoot length, leaf area, shoot fresh and dry weight, root fresh and dry weight and chlorophyll content adv. hort. sci., 2016 30(3): 141-149 144 kentucky bluegrass and tall fescue generally were not affected by surface soil drying but under full drying, shoot growth declined for both species. the reduced leaf area is a modification to avoid evopo-transpiration loss and to increase water use efficiency in grasses which helps to tolerate water stress. low leaf surface area would reduce transpiration rate also by lowering stomatal activity (riaz et al., 2010). turf shoot height showed considerable difference in ldl treatments compared to sdl treatments. reducing photoperiod significantly increased the shoot height and leaf area (table 3). shoot height and leaf area increased significantly with shortening day length that it’s maximum and minimum decreased (14.82% and 8.19%, respectively) was observed at sld compared to ldl (table 3). similar results have reported on bermudagrass (tegg and lane, 2004) and zoysiagrass (qian and engelke, 1999). shoot fresh weight reducing field capacity from 100% fc to 25% fc significantly decreased the shoot fresh weight to 43.46% at 25% fc compared to 100% fc (table 3). riaz et al. (2010) demonstrated that, water deficit conditions had a significant inhibitory effect on shoot fresh and dry weights of three bermudagrass cultivars. the extended photoperiod (16 h) significantly increased fresh weight compared to shorter photoperiods (12 h and 8 h). shoot fresh weight increased 41.09% under ldl compared to the sdl condition (table 3). sinclair et al. (2004) demonstrated that the extended photoperiod increased biomass accumulation of four grasses (‘pensacola’ bahiagrass, paspalum notatum flugge var. saurde parodi; ‘tifton 85’ bermudagrass, cynodon spp. l. pers.; ‘florakirk’ bermudagrass; and ‘florona’ stargrass, cynodon nlemfuensis vanderyst var. nlemfuensis) compared to short day condition. interaction between field capacity and photoperiod resulted in the highest and lowest fresh weight in 100% fc-ldl and 25% fc-sdl treatments (table 3). shoot dry weight different percentages of field capacity and photoperiod had significant effects on dry weight (table 3). reducing field capacity and photoperiod significantly decreased the dry weight. the shoot dry weight in 100% fc conditions decreased 69.78% compared to 25% fc condition (table 3). similar results have been reported on creeping bentgrass (agrostis stolonifera l.), rough bluegrass (poa trivialis l.), and perennial ryegrass (lolium perenne l.) (pessarakli and kopec, 2008), bermudagrass (cynodon dactylon l.) (riaz et al., 2010). the highest and lowest dry weight was observed in 100% fc-ldl and 25% fc-sdl treatments, respectively (table 3). burton et al. (1988) stated that day length was highly correlated with yield of ‘coastal’ bermudagrass, with yield reduction occurring in day lengths under 13 h. therefore, photoperiod influenced dry matter production of forage grasses. extended photoperiod throughout the cool-season in short-day length conditions substantially decreased forage yield (sinclair et al., 1997, 2001, 2003). root fresh weight root fresh weight significantly declined by decreasing field capacity from 100% to 25%. root fresh weight decreased (59.76%) at 25% fc compared to 100% fc (table 3). the impact of partially closing stomata limits co2 availability and reduces photosynthesis, which is vital to produce and translocate carbohydrates to roots to explore deeper moisture (huang, 2006). huang and gao (2000) found that severe leakage of organic solutes from roots in drying soil gives evidence that root death of tall fescue cultivars during drought stress may correlate with root desiccation. there was a significant difference between ldl, idl and sdl treatments and the highest and lowest root fresh weights were obtained in ldl and sdl treatments, respectively. root fresh weight decreased 9.50% at sdl compared to ldl (table 3). this is in agreement with wang et al. (2004) who reported that an increase in root growth is associated with extended light duration and is related to increase in internal cytokinin concentration and its increased activity in root tips. root dry weight as shown in table 3, reduction in field capacity decreased root dry weight of plants. the highest and lowest root dry weights were observed in 100% fc and 25% fc treatments, respectively and in 25% fc decreased 58.10% compared to 100% fc. pessarakli and kopec (2008) demonstrated that, water deficit conditions showed a significant decrease in root dry weight of three turfgrass species. the highest and lowest root dry weight was obtained in ldl and sdl treatments, respectively (table 3). root dry weight decreased 6.61% at sdl compared to ldl (table 3). beard (1972) reviewed the morphological responses of turfgrasses under shade based on the research conducted before 1995, and found alterations such as: reduced tillering and shoot density, longer internodes with a reduced stem diameter, increased leaf length, decreased leaf width, thinner leaves, more adamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 145 vertical leaf orientation, and fewer roots (mcbee and holt, 1966; almodares, 1980; dudeck and peacock, 1992). a shift in allocation of dry matter occurs in response to shade, resulting in more dry matter partitioning into shoots rather than roots (allard et al., 1991; dias-filho, 2000). in response to lower irradiance, accelerated leaf elongation and decrease in partitioning to root dry matter are adaptive strategies to enhance light capture (semchenko et al., 2012). proline content reducing field capacity and photoperiod significantly increased proline content in all plants. the highest amount of proline content was obtained in 25% fc and the lowest one was obtained in 100% fc treatment (table 3). this is in agreement with (etemadi et al., 2005) who demonstrated that the increase in drought increased proline content in bermudagrass (cynodon dactylon l.). during drought stress, plants respond to different stresses with changes they create in their physiological features. accumulation of soluble material in response to drought is a way to maintain turger. it seems that the accumulation of free proline in plants is the general reaction to the stress. however several other amino acids increase under drought and salinity stress. but the degree of changes is not comparable with proline accumulation (gzik, 1996). in a comparative study between perennial ryegrass and red fescue for the amount of resistance to the drought, it was seen that the amount of proline in red fescue was more than perennial ryegrass (bandurska and jozwiak, 2010). the highest and lowest proline content was obtained in sdl and ldl treatments, respectively (table 3). this is in agreement to the findings reported on the effects of decreased photoperiod on bermodagrass (esmaili and salehi, 2012). interaction between field capacity and photoperiod resulted in the highest and lowest proline content in 25% fc-sdl and 100% fcldl treatments (table 3). chlorophyll content field capacity and light durations had significant effects on leaf chlorophyll content. the highest and lowest chlorophyll content, were observed in 100% fc and 25% fc treatments, respectively (table 4). induction of drought has caused a reduction of electron carrier in photosynthesis and a reduction in chlorophyll content which has been reported by (zuily et al., 1990; moran et al., 1994). prolonged drought, heat, and the combined stresses could lead to loss of chlorophyll and lipid peroxidation, resulting in further turf quality decline (jiang and huang, 2001). water is required to facilitate photosynthesis in plants. low energy electrons are extracted from water and are energized through light energy captured by chlorophyll. these energized electrons enable the production of nadph and atp which are then used to reduce co2. co2 is taken up from the atmosphere through stomata. stomata are very sensitive to external environmental factors such as light, co2, water status, and temperature (hopkins and hüner, 2004). the loss of chlorophyll by the plant in an intense stress can be associated with photo oxidation and consequently oxidative stress (kato and shimizu, 1985). kaiser (1987) indicated that an irreversible decrease in plant photosynthetic capacity occurs as rwc declines below 30%, leading to cell death from membrane damage in chloroplasts. table 4 effect of field capacity and photoperiod and their interaction on proline, sugars and starch contents, activity of superoxidase dismutase, catalase, peroxidase, and ascorbate peroxidase enzymes variables photoperiod field capacity (%) mean 100% 75% 50% 25% chlorophyll ldl 1.81 a 1.78 b 1.48 e 0.99 j 1.52 a content idl 1.74 c 1.71 d 1.41 h 0.92 k 1.44 b (mg chl g−1f.w.) sdl 1.45 f 1.42 g 1.12 i 0.63 l 1.15 c mean 1.67 a 1.64 b 1.33 c 0.85 d sugars of shoot ldl 62.63 de 67.19 c 199.39 b 212.15 a 135.34 a (mg g−1d.w.) idl 61.81 e 66.37 cd 198.22 b 211.33 a 134.52 a sdl 61.45 e 66.02 cd 198.22 b 210.98 a 134.17 a mean 61.96 d 66.53 c 198.72 b 211.49 a starch content ldl 225.30 a 224.10 d 91.50 g 41.30 j 145.50 a (mg g−1 d.w.) idl 224.90 b 223.70 e 91.10 h 40.90 k 145.10 b sdl 224.30 c 223.20 f 90.60 i 40.40 l 144.60 c mean 224.80 a 223.70 b 91.10 c 40.90 d superoxide ldl 136.00 cd 148.50 c 266.00 a 116.00 def 166.62 a dismutase idl 128.50 cde 143.50 c 261.50 a 109.50 ef 160.75 a (ug-1 fw) sdl 106.00 fg 118.50 def 236.00 b 86.00 g 136.62 b mean 123.50 c 136.83 b 254.50 a 103.83 d catalase ldl 31.48 ef 34.45 cd 40.71 a 31.18 efg 34.45 a (ug-1 f.w.) idl 28.89 fgh 31.86 de 38.12 ab 28.59 ghi 31.86 b sdl 29.53 efg 29.53 efg 35.78 bc 26.25 i 29.53 c mean 28.98 c 31.95 b 38.20 a 28.67 c peroxidase ldl 68.40 fg 70.75 f 94.58 c 112.40 a 86.53 a (ug-1 f.w.) idl 60.80 hi 63.15 hi 86.99 d 104.81 b 78.94 b sdl 52.83 j 55.18 ji 79.02 e 96.84 c 70.97 c mean 60.68 c 63.03 c 86.86 b 104.68 a ascorbate ldl 869.64 de 879.64 d 1160.36 a 854.29 ef 940.98 a peroxidase idl 829.64 gh 839.64 fg 1120.36 b 814.29 hi 900.98 b (ug-1 f.w.) sdl 784.64 kj 794.64 ij 1075.36 c 769.29 k 855.98 c mean 827.97 b 837.97 b 1118.69 a 812.61 c *in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. ldl= long day length. idl= intermediate day length sdl= short day length. adv. hort. sci., 2016 30(3): 141-149 146 detrimental effects on chloroplast biochemistry or chlorophyll fluorescence occur when rwc drops below 60% in tall fescue (huang et al., 1998). surface drying had no effects on chlorophyll content in kentucky bluegrass (poa pratensis l.) and tall fescue (festuca arundinacea schreb.) while under full drying, chlorophyll content decreased in both grasses (fu and huang, 2001). our findings were in agreement are (fu and huang, 2001) who reported that amount of chlorophyll in bermudagrass under moderate stress is not reduced, but it will be reduced in the severe drought. chlorophyll content decreased with decreasing day length and the highest and lowest ones were observed in ldl and sdl treatments, respectively (table 4). shorting photoperiod caused decrease in chlorophyll content. in a research, the resistances to low light stress in both bermudagrass and paspalum have been examined and it was concluded that resistance to low light stress in the paspalum is more than bermudagrass (jiang et al., 2004). baldwin et al. (2008) reported that bermudagrass showed significant decrease in chlorophyll content in response to short day length condition. total soluble sugars and starch content regardless of photoperiod, decrease in field capacity significantly increased total soluble sugars in the shoot (table 4). starch content declined by decreasing field capacity from 100% to 25% (table 4). shoot starch content, were highest and lowest in 100% and 25% fc treatments, respectively (table 4). on the other hand, total soluble sugars during the drought can increase making these compounds nonphotosynthetic routes and growth stopping due to the destruction of in soluble sugars and their change to soluble sugars (hissao, 1973). although some researchers have suggested that the destruction of starch can also increase monosaccharaides (düring, 1992). the researchers stated that an increase of amylase in water stress causes starch degradation and the conversion of this large molecule into smaller units (movahhedi-dehnavi et al., 2004). different photoperiod had no significant effects on total soluble sugars (table 4). shoot starch content decreased by different light durations and the highest and lowest one was observed in ldl and sdl treatments, respectively (table 4). starch content decreased in response to shortening the photoperiod. some researchers have reported that prolonging photoperiod increases carbohydrates (hay and pederson, 1986; solhoug, 1991; wang et al., 1998). other researchers reported that the photoperiod had no effect on carbohydrates production (sicher et al., 1982; logendra and janes, 1992). antioxidant enzyme activities apx, pod, cat and sod enzymes activities showed significant differences among field capacity and photoperiod treatments. the activities of apx were not significantly different between 100% fc and 75% fc treatments while were significantly increased in 50% fc treatment and minimum apx activity was observed at 25% fc treatment (table 4). bian and jiang (2009) investigated the accumulation of reactive species of oxygen and antioxidants activity and the pattern of gene expression of antioxidant enzymes in the kentuchy bluegrass in the drought condition. they observed that drought stress increased the activity of apx and cat and decreased sod and they stated that antioxidant enzymes and their gene expression might be different or occur in the immune system of kentuchy bluegrass roots and leaves. pod enzyme activities increased with decrease in field capacity levels. differences in leaf pod enzyme activities were not detected between 100% fc and 75% fc treatments. the maximum and minimum pod activity was obtained in 25% fc and 100% fc treatments, respectively (table 4). in a research on drought tolerance of three cultivars of creeping bentgrass, it was observed that long-term drought stress reduced the activity of antioxidants such as pod and increased lipid peroxidation and the ‘greenwich’ showed high resistance to drought (dacosta and huang, 2007). cat and sod enzymes activities significantly increased with decreasing field capacity from 100% to 50% then, declined in 25% fc treatment (table 4). shao et al. (2005) reported that in the of drought stress, the production amount of three enzymes, cat, sod and pod in resistant bermudagrass varieties have been significantly more than drought-sensitive ones. general declines in antioxidants, including cat were reported in the response of three species of creeping bentgrass to drought stress. moreover, they found that the species agrostis canina l. was the most resistant species to drought (dacosta and huang, 2007). liu et al. (2008) in a research, physiologically and morphologically investigated the five cultivars of kentuchy bluegrass under drought and heat stress and observed that drought and heat stress simultaneously reduces sod enzyme in all cultivars and stated that an increase of sod enzyme activity cannot inhibit stress and would only delay free radicals accumulation. results of present study indicated that regardadamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 147 less of field capacity treatments, apx, pod, cat and sod enzymes activities significantly decreased in response to decreasing day length therefore, the maximum and minimum enzymes activity was observed in ldl and sdl treatments (table 4). similar findings have been previously reported by (burritt and mackenzie, 2003) who stated that when the begonia plant is transferred from low light to bright light, cat activity increases. also, they stated that when the (picea abies l.) seedlings are transferred from low light to high light, the activity of cat enzyme decreases. xu et al. (2010) investigated the effect of nitric oxide and sodium nitroprusside in tall fescue under high light stress and concluded that using sodium nitroprusside reduces enzyme activity of sod, cat and apx, but using nitric oxide increases the activity of mentioned enzymes. jiang et al. (2005) demonstrated that, low light conditions showed a significant decrease in activity apx and cat of bermudagrss and paspalum. grace and logan (1996) reported that the cat enzyme activity varies depending on light intensity. the cat enzyme activity in schefflera [schefflera arboricola (hayata) merrill] and vinca (vinca major l.) plants did not change with a change in light intensity, but in mahonia (mahonia repens (lindley) don.), cat enzyme activity increased with an increase of light intensity. interaction between field capacity and photoperiod resulted in the highest and lowest sod enzyme activities in 50% fc-ldl and 25% fc-sdl treatments (table 4). 4. conclusions the results proved that the reduction in photoperiod led to a progressive increase in shoot height and leaf area, however, the increase in irrigation inhibited their progressive growths. additionally, the reduction in photoperiod caused a decrease in fresh and dry weight of root and shoot. however, the increase in irrigation led to alleviation of these negative effects during the day-time and thus increased the fresh and dry weight of root and shoot. therefore, it appears as though the increased irrigation might have contributed to the enlargement and flexibility of cells, which, in turn helped increasing the dry and fresh weight of root and shoot. the reduced photoperiod led to a reduction in chlorophyll and starch contents and enzymes activities, and the increased irrigation compensated this reduction to some extent. this phenomenon might be, at least in part, explained by the fact that irrigation reduced aba production, inhibited ros production and thus inhibited the closure of stomata. in overall, the increase in irrigation caused the destructive effects of reduced photoperiod to diminish, and vice versa. it seems that the interaction of photoperiod and irrigation treatments has superior effects on alleviating of the symptoms of stressed plants, than their separate. further studies are 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element both in food and horticultural industries. few ppm of ethylene in the storage atmosphere induce very fast ripening in postharvest which causes undesirable product losses (keller et al., 2013). thus it is desired to remove or degrade ethylene from the storage environment in order to preserve the postharvest products and keep them fresh for a longer time period. the important role of ethylene as growth regulator of climacteric products was intensively investigated in the last 50 years and now the ripening process associated to high ethylene production is well known. various methods to remove ethylene have been developed: ventilation, or controlled atmosphere, ethylene oxidation using potassium permanganate. the photocatalytic ethylene oxidation thus represents an innovative way to extend postharvest life of the climacteric fruits and vegetables (park et al., 2009; keller et al., 2013; ye et al., 2013). among the catalysts able to induce ethylene photooxidation, tio2 is promising and attractive due to low cost, non-toxicity and high efficiency. the photocatalytic properties of nanostructured tio2 have already been highlighted in several applications, such as photocatalytic degradation of both air and water organic pollutants, and self-cleaning surface protection (pal et al., 2014). anchoring tio2 nanocrystals on suitable mesoporous substrate brings relevant practical advantages. the immobilization on support materials slows or totally inhibits nanoparticles coarsening, preserving their higher surface area. secondly, the substrate greatly improves handling, processing and recover of tio2 nanopowder. mesoporous silica, as a catalyst support over nano-sized tio2, represent a good choice due to high specific surface area and improved thermal stability of the tio2 anatase crystalline phase. some recent works also demonstrated higher photocatalytic activity of tio2/sio2 mixed oxide composites (fu et al., 1996; zhan et al., 2014). adv. hort. sci., 2016 30(2): 75-80 doi: 10.13128/ahs-19132 photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts a. licciulli 1 (*), r. nisi 1, s. pal 1, a. m. laera 1, p. creti 2, a. chiechi 3 1 dipartimento di ingegneria dell’innovazione, università di salento, via arnesano, 73100 lecce, italy. 2 cnr istituto microelettronica e microsistemi, sezione di lecce, 73100 lecce, italy. 3 salente srl, via monteroni 147, 73100 lecce, italy. key words: gaseous phase photocatalysis, liquid-crystal template, modified storage environment, mixed oxides. abstract: mesoporous tio2/sio2 catalysts were prepared in order to increase the post-harvest life of climacteric fruits and vegetables reducing ethylene concentration by photo-oxidation. tio2/sio2 powders were synthesized by sol-gel method using titanium isopropoxide ti(oipr)4 and tetraethoxysilane si(oc2h5)4 as source of metal oxides. mesoporous sio2 framework was used as catalyst support of nanostructured tio2 to enhance the photocatalytic efficiency. different tio2/sio2 molar ratios were prepared through sol-gel process. a liquid-crystal template route allowed to obtain the mesoporous silica structure, and contemporary tio2 insertion in the silica framework. the x-ray diffraction (xrd) analysis demonstrated that silica insertion in tio2 framework inhibits the anatase to rutile phase transformation at higher sintering temperature. the photocatalytic efficiency of the catalysts was measured by the photo-oxidation of ethylene gas under uv light irradiation. complete photo-oxidation of ethylene was observed after 24 h of reaction time. results show that the silica framework increases the surface area of the composites and make crystalline anatase phase more stable at higher temperature. (*) corresponding author: antonio.licciulli@unisalento.it received for publication 16 february 2016 accepted for publication 5 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 75-80 76 the mesoporous sio2 is able to reduce the recombination of photogenerated electron-hole pair, while higher surface area increases the efficiency in the heterogeneous photocatalysis. over the past thirty years a successful synthetic strategy, named liquid-crystal template, leading to mesoporous materials has been developed allowing to finely tune the porosity (corma et al., 2006; xiao et al., 2008). this methodology involves supramolecular aggregates of ionic surfactants that act as templating agents, which are able to direct and control the crystallization of the inorganic nanoparticles (beck et al., 1992). after the surfactant removal by thermal annealing, mesoporous solids with high surface area can be obtained. the morphological and structural properties of the products can be finely tuned by varying process parameters such as ph, temperature, reagent concentrations, surfactants and silica sources. in this work, tio2/sio2 binary composites obtained through a liquid-crystal template methodology have been tested as catalyst in uv light assisted ethylene oxidation at room temperature for the application in modified atmosphere packaging of climacteric fruits and vegetables. a titania precursor was added in the reaction medium containing the surfactant and the silica source allowing the one-pot formation of tio2 and sio2. structure and morphology of the obtained tio2/sio2 binary composites with several weight ratios were investigated by using brunauer-emmett-teller (bet) surface area, x-ray diffraction (xrd), fourier transform infrared spectroscopy (ftir) and field emission scanning electron microscopy (fesem) measurements. 2. materials and methods synthesis of tio2/sio2 binary oxides all the reagents involved in the catalyst synthesis were purchased from sigma aldrich and were used without any further modification. the starting colloidal solution was obtained by dissolving 28 g of pluronic f127 (eo106 po70 eo106) in 150 g of distilled water through vigorous stirring. the complete polymer dissolution was reached after addition of a solution of 2m hcl (600 g). then the required amount of tetraethoxysilane [si(oc2h5)4, teos] was added drop wise and continued to stirring for an hour. at this stage, titanium tetraisopropoxide [ti(oipr)4, ttip] was slowly added and stirred for overnight. the obtained reaction mixture was heated at 100°c for 24 h and after that, a xerogel was obtained by centrifuging. the xerogel was then dried and calcined at 550°c for 6 h with the heating and cooling rate of 1°c/min to remove the organic content. following this procedure, binary oxide with the tio2/sio2 weight ratio of 9/1, 8/2, and 7/3 were prepared. for comparison, a sample of pure tio2 was also prepared with the same experimental condition. throughout the text, these samples are represented as tsba-10, tsba-91, tsba82, and tsba-73, respectively. characterization crystalline phases of the tio2/sio2 powder samples were characterized by x-ray diffraction (xrd) performed on a rigaku ultima x-ray diffratometer using cukα radiation (λ=1.5406 å) operating at 40 kv/30 ma with the step size of 0.02°. ftir spectra of the obtained composites were carried out with a jasco ftir-6300 over the range 4000-400 cm-1 with a resolution of 4 cm-1 and accumulating 256 scans for each measurement adopting kbr disc method. specific surface area of the samples was measured by bet method using a quantachrome nova 2200e surface analyzer. fesem measurements were performed with a zeiss scanning electron microscope. photocatalytic experimental set up photo-oxidation of ethylene gas under uv irradiation in the presence of tio2/sio2 composite powder samples was performed inside a photocatalytic reactor consisting of a quartz tube of 3l irradiated by 4 uv light sources (osram puritec hns15wg13, 15w, emitting at 254 nm). the pictures of the catalytic reactor have been reported in figure 1. the experimental set up was equipped with flowmeters, vacuum pump, diaphragm pump for gas recirculation, and thermocouples for the temperature control inside the reactor. the tio2/sio2 powder samples (3 g) were placed in the quartz tube on a rectangular quartz vessel. before filling the reactor with ethylene gas/air atmosphere, it was kept in vacuum to ensure the purity inside the chamber. then a gas mixture of air and ethylene (1 equivalent %) was introduced in the reactor with a controlled flow system. all the experiments were performed at atmospheric pressure and the purged gas was continuously circulated in a closed circuit while maintaining room temperature inside the reactor. ethylene photo-oxidation was monitored at regular intervals by using an agilent gas chromatograph 7820 a equipped with a capillary column (hp-plot/q). licciulli et al. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts 77 3. results and discussion to investigate the effect of silica framework and mesoporosity in the catalyst nanostructure, bet specific surface area measurements were performed on the powder samples. bet measurements showed an increase in specific surface area with the increase in silica content. the sample sba of pure silica represents pores with an average diameters of 3.4 nm and a specific surface area of ~553 m2/g, while the pure tio2 (tsba-10) has pores with average diameter of 16.7 nm and a specific surface area of ~59 m2/g. a great ratio of surface/volume is a crucial parameter to optimize photocatalytic efficiency of nanostructered semiconductors. intermediate values of specific surface area and average diameters between those recorded for pure silica and tio2, have been obtained on the binary tio2/sio2 oxides (fig. 2). the phase transformation of the tio2/sio2 catalysts was investigated by xrd and the spectra of each sample are presented in figure 3. the average crystalline size was estimated according to the scherrer’s equation, accounting the most intense diffraction peaks of the corresponding anatase and rutile phases. d= kλ/βcosθ (1) where d is the average crystallite size, k is the shape factor (0.9), λ is the wavelength of x-ray radiation, β is the full line width at half-maxima (fwhm) of the main diffraction peak and θ is the bragg angle of the corresponding diffraction peak (pal et al., 2014). the weight fraction of anatase and rutile contents in the samples were calculated according to the following equations: fig. 1 pictures of the experimental set-up reactor for evaluating photocatalysis efficiency. fig. 2 pore size distribution curve of (a) the mesoporous silica (sba) and (b) tio2 (tsba-10). fig. 3 xrd spectra of the tio2/sio2 samples with different tio2/sio2 weight ratio. adv. hort. sci., 2016 30(2): 75-80 78 wa=1/[1+1.26(ir110/ia101)] (2) wr=1/[1+0.8(ia101/ir110)] (3) where wa and wr are the weighted fraction of anatase and rutile in the mixed phase, and ia101 and ir110 are the integrated intensity of corresponding anatase (101) and rutile (110) diffraction peaks, respectively (pal et al., 2014). phase composition and average crystallite size of the corresponding crystals are summarized in table 1, where it is observed that tsba-73, tsb-82 and tsba-91 samples consist of pure anatase phase whereas in case of tsba-10, there is a small amount of rutile phase formation (~11 wt%). these data clearly indicate that the mesoporous silica framework helps to stabilize the anatase crystalline phase and prevent the thermodynamically favoured phase transformation in to the rutile phase, as reported by fu et al. (1996). ftir measurements were carried out to investigate the bonding of sio2 and tio2 network and they are showed in figure 4. as a reference of pure silica, an ftir spectrum of sba is also presented. all the spectra, except for the sample tsba-10 (100 wt% tio2), show intense and distinct vibrational bands at 1092, 805 and 472 cm-1 which are assigned to the asymmetric stretching of si-o-si (1092 cm-1), si-o-si symmetric stretching (805 cm-1), si-o-si bending vibration mode (474 cm-1). the broad band around 3300-3600 cm-1 and the band at 976 cm-1 are attributed to the stretching vibration of h-bonded silanols (si-oh) with hydroxyl groups of the adsorbed water molecules. another peak of the adsorbed water is located at the wavenumber of 1634 cm-1 due to bending vibration of o-h groups. furthermore, from the spectra it can be observed that the band at 665 cm-1 is absent in sba sample, whereas it is present in all samples containing tio2 and it is therefore attribuited to the ti-o bond. the appearance of broad band at 450-800 cm-1 range is also attributed to the presence of tio2 it is also interesting to see that the vibrational band at 1092 cm-1, due to asymmetric stretching of si-o-si, is gradually decreasing with increasing tio2 content in the mixed composite that confirms the variation of tio2/sio2 ratio. fesem measurements on two different samples, one without silica support (tsba-10) and the other with silica support (tsba-82) were carried out to investigate the effect of silica insertion in the composite catalyst samples. fesem images of these two samples are showed in figure 5. the lower magnification images (fig. 5 a, c) of tsba-10 and tsba-82 samples represent the overall structure of these powder samples. whereas from the images in figure 5 (b, d), it is clearly visible the mesoporous structures. the crystallite size as estimated from the xrd spectra (table 1) of the corresponding samples matches well with the size as observed in the fesem images (fig. 5 b, d). the photocatalytic efficiency of the powder samples was evaluated by the photo-oxidation of ethylene under uv light illumination. the percentage of ethylene photo-oxidation with reaction time under uv light corresponding to four catalysts is summarized in table 2. for a better comparison, data reported in table 2 have been presented as histogram in figure 6. from table 2 and figure 6, it is clear that the overall photo-oxidation efficiency of tsba-82 sample is higher respect to the others. it also shows a superior catalytic activity in early reaction times. the higher catalytic activity of tsba-82 sample can be explained owing to stable anatase crystalline phase, higher specific surface area and having optimum amount of mesoporous silica support. table 1 anatase (wa), and rutile (wr) phase content in different tio2/sio2 samples and their corresponding crystalline sizes calculated from xrd data sample phase content crystalline size (nm) wa wr <da> <dr> tsba-73 1 0 15.749 tsba-82 1 0 15.998 tsba-91 1 0 17.663 tsba-10 0.887 0.111 17.553 13.866 fig. 4 ftir spectra of pure mesoporous silica and of the tio2/sio2 binary systems. licciulli et al. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts 79 4. conclusions tio2/sio2 catalysts, with different tio2/sio2 weight ratios, were synthesized by liquid crystal template technique, being sio2 the mesoporous support fig. 5 fesem images of (a, b) tsba-10 and (c, d) tsba-82 samples. fig. 6 histogram showing the ethylene oxidation efficiency of four catalyst samples with the reaction time under uv light exposure. table 2 percentage of oxidized ethylene for the catalysts tsba-10, tsba-91, tsba-82, and tsba-73 as a function of the uv light irradiation time (h) oxidized ethylene (%) tsba-10 tsba-91 tsba-82 tsba-73 0 0 0% 0% 0% 1 2% 0% 6% 8% 2 8% 5% 15% 6% 3 16% 15% 20% 8% 4 22% 22% 25% 11% 5 26% 31% 30% 13% 24 98% 97% 99% 89% adv. hort. sci., 2016 30(2): 75-80 80 of tio2 catalyst. xrd results prove that a more stable anatase phase with silica insertion even at higher calcination temperature is obtained. increasing the silica content also leads to a higher specific surface area. fesem images showed the well-defined structure of the catalyst samples. the photocatalysis experiments revealed that, after 24 hours of reaction under uv light, total oxidation of ethylene was achieved in case of tsba-82 catalyst. the other catalysts, such as, tsba-0 and tsba-91 also showed interesting results but the highest activity was observed with the tsba-82 sample. this higher efficiency was explained with surface area, phase stability and optimum mesoporous support. overall it can be stated that the tio2/sio2, rather than pure tio2 is a promising candidate in photocatalytic degradation of ethylene. it is therefore expected that tio2/sio2 can be fruitfully used in several applications requiring the control of ethylene to prevent ripening of fruits and vegetables. acknowledgements support by miur in the framework “programma operativo nazionale r&c 2007-2013” project pon01_01435”, “prodotti ortofrutticoli ad alto contenuto in servizio: tecnologie per la qualità e nuovi prodotti” is gratefully acknowledged by the authors. references beck j.s., vartulli j.c., roth w.j., leonowicz m.e., kresge c.t., schmitt k.d., chu c.t.w., olson d.h., sheppard e.w., mccullen s.b., higgins j.b., schlenker j.l., 1992 a new family of mesoporous molecular sieve prepared with liquid crystal templates. j. am. chem. soc., 114: 10834-10843. corma a., diaz-cabanes m.j., moliner m., rodriguez g., 2006 synthesis of microand mesoporous molecular sieves at room temperature and neutral ph catalysed by functional analogues of silicatein. chem. comm., 29: 3127-3139. fu x., clark l.a., yang q., anderson a., 1996 enhanced photocatalytic performance of titania-based binary metal oxide: tio2/sio2 and tio2/zrio2. environ. sci. technol., 30: 647-653. hussain m., bensaid s., geobaldo f., saracco g., russo n., 2011 photocatalytic 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decomposition of ethylene in cold storage by plasmaassisted photocatalyst process with tio2/acf-based photocatalyst prepared by gamma irradiation. chem. eng. j., 225: 499-508. zhan c., chen f., yang j., dai d., cao x., zhong m., 2014 visible light responsive sulphated rare earth doped tio2@fumed sio2 composites with mesoporosity: enhanced photocatalytic activity for methyl orange degradation. j. hazard. mater., 267: 88-97. impaginato 25 1. introduction the increasing demand for basil resulted in cropping area extension by 66% since 2001 in italy (www.istat.it), with the frequent use of local ecotypes (zecchinelli, 1999; tesi and lenzi, 2002). basil (ocimum basilicum l.) is a high marketable value vegetable, which is consumed both as a fresh aromatic ingredient and combined with pasta in a cooked dish (pesto). as today’s consumer choices are oriented towards high quality produce, not only from a sensorial point of view but also in terms of nutritional properties, a particular emphasis is given to this product. in this direction, soilless growing could represent an effective crop management in order to enhance product quality attributes (sgherri et al., 2010), which mainly depend on variety (tesi et al., 1991) but they are also affected by crop system (tesi et al., 1997). plant growing in pots sown with several seeds per pot, to be sold when the plants set reaches a scheduled size, is one of the current farm strategies and interesting market perspectives mainly arise from the winter crop cycle. in this season, light intensity is sufficient for carrying out efficient crop cycles (beaman et al., 2009), but it is necessary ensuring the adequate minimum temperature, which is also positively correlated with basil flavour (chang et al., 2007). moreover, the nutritive solution supplied to plants plays a crucial role as it significantly affects yield (bekhradi et al., 2015) and plant features, such as stem height and dry weight (adler et al., 1989; bione et al., 2014); in this respect, basil is considered a moderately tolerant species (herrera, 2005). the plants density is also of primary importance for the adv. hort. sci., 2017 31(1): 25-30 doi: 10.13128/ahs-20722 effects of nutritive solution electrical conductivity and plant density on growth, yield and quality of sweet basil grown in gullies by subirrigation morano g. 1, amalfitano c. 1, sellitto m. 2, cuciniello a. 1, maiello r. 1, caruso g. 1 (*) 1 dipartimento di scienze agrarie, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. 2 microspore s.p.a., 86035 larino, cb, italy. key words: leaves quality, nutrient uptake, ocimum basilicum l., plants number per pot, production, soilless. abstract: the increasing demand for basil in the last decade has arisen from consumer tendency towards high nourishing produce. soilless growing of this crop is a current farm strategy and the quality targets are affected by nutritive solution as well as by plants density per pot. research was carried out with the aim of assessing plant growth, yield and leaves quality of basil (ocimum basilicum l., cv. gecom ft) grown in pots (peat-lapil) and fed by subirrigation inside plastic gullies, under a heated greenhouse. comparisons were made of four electrical conductivities (ec: 2.2, 2.5, 2.8, 3.1 ms·cm-1) in factorial combination with four plant densities (9, 12, 15, 18 plants per pot) and a split plot design was arranged with three replicates. the 2.8 ms.cm-1 ec resulted in the best yield, growth indexes and biometrical parameters values. water absorption was highest under the 2.8 ms.cm-1 ec, whereas the highest nutrient consumptions as well as the best quality indicators and chemical composition corresponded to the 2.8 to 3.1 ms.cm-1 ec range. the 12 plants per pot density gave the best results, in terms of yield, growth indexes and biometrical parameters, also showing the highest plant water and nutrient uptakes. the leaves quality attributes and chemical composition always displayed decreasing trends as a function of the plant density increase, the highest values corresponding to 9 and 12 plants per pot; only the nitrates concentration showed an opposite trend compared to the other nutrients. in conclusion, the 2.8 ms.cm-1 nutritive solution and the 12 plants per pot density resulted in the best yield and leaves quality. further enhancement of both experimental factors level even caused the reduction of water and nutrient efficiency use. (*) corresponding author: gcaruso@unina.it received for publication 11 september 2016 accepted for publication 23 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 25-30 26 produce quality (bohme and pinker, 2014), as the individual space available has a major impact on the balance among the different plant parts. due to literature shortage on the above mentioned topics, research was carried out on soilless pot-grown basil in naples (southern italy), with the aim of evaluating the effects of nutritive solution electrical conductivity and pot plants density on crop growth, yield and leaves quality. 2. materials and methods research was carried out on basil (ocimum basilicum l. cv. gecom ft) at the experimental site of naples university federico ii in portici (naples, southern italy, 40°49’ n, 14°20’ e, 63 m a.s.l.), in a mediterranean or csa climate (peel et al., 2007), during winter season in 2007 and 2008. the crops were soilless grown in pots, placed in gullies and fed by sub-irrigation, under plastic (ir-pe) tunnels equipped with an air heating system set to 16°c. comparisons were made of four electrical conductivities (ec: 2.2, 2.5, 2.8, 3.1 ms·cm-1) in factorial combination with four plant densities (9, 12, 15, 18 plants per pot) and a split plot design was arranged with three replications; each treatment included 30 pots. the hydroponic equipment consisted of: a) 48 rigid pvc gullies (each 12 cm wide and deep, 300 cm long) supported by plastic elements at 70 cm above ground level, according to 1% slope; b) 12 plastic tanks holding 220 l; c) 12 submerged pumps of 90 watt unit power; d) 24 delivery and return overhead lines. the sowing was performed on 15 january in pots (ø = 10 cm) filled with peat and lapil (1:1 in volume), placed in the gullies through a pierced white pe film and spaced 10 cm along and between the rows (18 pots per m2). they were fed by nutritive solutions (table 1), with 1-6 interventions per day of 5 minutes each, which were never adjusted but they were completely changed at the end of each weekly cycle. besides, during the crop, an insecticide application against aphids was practiced. the crop cycles ended when each plants set reached the scheduled size for pot commercialization, i.e. the plants had four fully expanded leaves couples, and at that time the following determinations were made on plant samples obtained by 8 pots per plot: fresh and dry weight of whole plants and of leaves; leaf area; stems thickness and height. moreover, water consumption was calculated assessing the volume of nutritive solution in each tank at the beginning and at the end of each weekly cycle. concurrently, nutritive solution samples were collected in order to assess nutrient consumption through laboratory analyses of nitrogen, phosphorus, potassium, calcium, magnesium and iron, using the same methods as described below for leaf cation and anion determinations. at the crop cycles end, leaves samples were also collected from 8 pots per plot, in order to perform laboratory analyses. in this respect, two hundred grams of leaves per plot were homogenized in a 1.0 l waring blender (waring laboratory, torrington, ct, usa) and aliquots of this raw homogenate were used for the analyses of cations. the raw homogenate was centrifuged at 10,000 x g for 30 min at 6°c in an 5810r eppendorf refrigerated centrifuge (eppendorf hq, hamburg, germany). the resulting supernatant was passed through a 0.45 µm acrodisc fi lter (gelman sciences, mi, usa). samples of this filtered leaves extract were used for assessing anion, sugar and ascorbic acid contents. the laboratory determinations were performed as follows: the dry residue was assessed in an oven at 70°c with a vacuum; the soluble solids content or ssc (in °brix) was measured at 20°c with a bellingham & stanley, model rfm 81 digital refractometer on the supernatant obtained from raw homogenate centrifugation; anions, sugars and ascorbic acid were determined by high performance liquid chromatography (hplc) as previously described (caruso et al., 2011); table 1 chemical composition of the soilless nutrient solutions for all treatments ph was adjusted to 6.0 and nh4/no3 ratio was 1/9. nutritive solution ec (ms·cm-1) macronutrients (mmol·l-1) micronutrients (m mol·l-1) n p k ca mg s cl fe cu mn zn b mo 2.2 12.3 1.6 5.3 3.8 2.2 1.9 1 35 1 15 5 35 1 2.5 13.8 1.7 5.9 4.3 2.5 2.5 1 35 1 15 5 35 1 2.8 15.7 2.0 6.7 4.8 2.8 2.8 1 35 1 15 5 35 1 3.1 17.5 2.2 7.6 5.4 3.0 3.3 1 35 1 15 5 35 1 morano et al. effects of nutritive solution electrical conductivity on sweet basil grown in subirrigated gullies 27 titratable acidity of the leaves homogenate was determined as previously described (caruso et al., 2014) and it was expressed as grams of anhydrous citric acid per 100 g of leaf fresh weight; cations (ca, mg, k) content in the leaves homogenate was determined by atomic adsorption spectrophotometry as previously described (caruso et al., 2011). data statistical processing was performed by analysis of variance using the spss software version 21, referring to 0.05 probability level, and duncan multiple test was used for mean separation. 3. results and discussion from the data statistical processing, the year of research resulted to have no significant effect either as a main effect or as an interaction with the two experimental factors, therefore in the following tables the mean values of the experimental data of the years 2007 and 2008 are reported. as for yield results relevant to the comparison among the nutritive solution electrical conductivities (table 2), the 2.8 ms.cm-1 ec resulted in the highest basil yield, both as whole plants and of leaves, and in the shortest crop cycle; the lowest nutrient solution strength (2.2 ms.cm-1) showed the worst performances. among the plant densities, the 12 plants per pot treatment resulted in the highest yields and both the 9 and 12 plants per pot led to the shortest crop cycle (table 2). in terms of growth indexes and biometrical parameters (table 3), the 2.8 ms.cm-1 ec also produced the highest values of plant dry matter, leaf area and stem thickness, though the latter was not statistically different from that obtained with the highest ec level (3.1 ms.cm-1); conversely, nutritive solution dilution caused the internodes extension and, indeed, the two lowest electrical conductivities enhanced plant height. with regard to plant density (table 3), the 12 plants per pot treatment resulted in the highest dry weight and lai, whereas the lowest density produced the thickest stems; the 15 and 18 plants per pot densities proved excessive and, in particular, the highest one caused the most unbalanced growth of plants. in fact, the plants grown under the 18 plants per pot treatment showed thinner stems and smaller leaves compared to the other experimental treatments, and they were also taller than the most spaced ones. in contrast with our findings, in previous research (tesi et al., 1995) the 1.6 ms·cm-1 ec showed the best effect on basil yield. moreover, other authors reported that doubling the nutrient availability did not affect basil yield (raimondi et al., 2006), but it led to the increase of leaf dry matter percentage and lai (chen et al., 2004). in our research, the depressing effects of salt stress caused by the 3.1 ms.cm-1 ec on plant vegetative growth and in particular on leaf area corresponds to the rapid plant adaptation to water deficit (munns, 2002). moreover, in our investigation, the density increase presumably caused the light conditions worsening within the canopy and, accordingly, the reduction of plant photosynthetic efficiency. chang et al. (2007) also reported that basil plant weight is adversely correlated with canopy shading. consistently, tesi et al. (1995) recorded the plant weight increase per soil unit area up to a critical density value, over which a decrease occurred; however, they also found that a doubled density, compared to our best treatment of 12 plants per pot provided with the best results using 10 cm diameter pots. moreover, raimondi et al. (2006) found that plant density increase from 66 to 100 plants per m2 results in total yield increase. further, in studies on canopy dynamics simulation (van oosteron et al., 2001), leaf table 2 basil yield results table 3 basil growth and biometrical parameters nutritive solution ec (ms·cm-1) yield crop cycle duration (days) whole plants (g·m-2) leaves (g·m-2) leaves/ plant (%) 2.2 579.1 d 418.7 d 72.3 c 64.0 a 2.5 733.9 c 538.7 c 73.4 b 62.7 b 2.8 958.9 a 713.4 a 74.4 a 61.3 c 3.1 845.5 b 629.9 b 74.5 a 61.0 c no. plants per pot 9 761.9 b 570.7 b 74.9 a 61.2 c 12 931.1 a 695.5 a 74.7 a 61.4 c 15 776.3 b 569.8 b 73.4 a 62.6 b 18 648.5 c 465.6 c 71.8 c 63.9 a nutritive solution ec (ms·cm-1) plant dry matter (g·m-2) lai (m2·m-2) leaf area (cm2·pt-1) plant height (cm) stem thickness (mm) 2.2 45.0 d 0.66 c 28.5 c 17.2 a 2.76 b 2.5 64.8 c 0.92 b 39.3 b 17.0 a 2.90 b 2.8 90.0 a 1.11 a 47.1 a 16.7 b 3.11 a 3.1 82.8 b 0.98 b 41.6 b 16.6 b 3.16 a no. plants per pot 9 73.6 b 0.86 c 51.9 a 16.8 b 3.33 a 12 88.2 a 1.06 a 47.6 b 16.9 ab 3.12 b 15 67.9 b 0.93 b 33.1 c 16.9 ab 2.85 c 18 52.7 c 0.83 c 24.3 d 17.1 a 2.60 d adv. hort. sci., 2017 31(1): 25-30 28 area index showed increasing trend with the plant density enhancement. the highest water and nutrient absorptions were recorded in the last crops week, when plant leaf area reached the highest expansion and the greenhouse temperature showed the highest value of 26.4°c (as an average of the two research years). as reported in table 4, plant water consumption showed a similar trend to the yield one, with the highest values corresponding to 2.8 ms.cm-1 ec, whereas the highest nutrients consumption was assessed under the 2.83.1 ms.cm-1 ec range; the most diluted nutritive solution always resulted in the lowest absorption rates. as for the comparison among the plant densities (table 4), the highest daily values of both water and nutrient absorption occurred in the 12 plants per pot treatment, which also resulted in the best yield (table 2); the highest plant density (18 plants per pot) always showed the lowest consumptions. compared to our research findings, a similar plant response to water deficit was recorded in previous investigations (savvas et al., 2007), where the increase of the nutrient solution strength caused the reduction of plant water absorption. the latter represents a salinity adaptation mechanism, consisting of leaf area and stomata decrease which in turn contributes to reducing transpiration and increasing water use efficiency (chartzoulakis and klapaki, 2000). the quality indicators were significantly affected by the nutritive solution strength (table 5), as their trends were always increasing with the electrical conductivity raise from 2.2 to 2.8 ms.cm-1, whereas no further increases were recorded in the last 0.3 ms.cm-1 rise. the quality parameters showed decreasing trends as a function of the plant density increase (table 5), with the 9 and 12 plants per pot treatments generally attaining the highest values and the 18 plants per pot treatment displaying the worst performances. in previous investigation (adams and ho, 1989), an increase in sugar content and titratable acidity was reported as a consequence of salinity increase or water deficit. moreover, raimondi et al. (2006) found that nutrient solution ec interacted with the cultivars in modifying leaf antioxidant content: i.e. napoletano leaves showed an ascorbate increase with the ec enhancement, whereas genovese displayed opposite trend. the same authors also recorded that the plant density increase from 66 to 100 plants per m2 did not table 4 basil water and nutrient absorptions nutritive solution ec (ms·cm-1) maximum daily absorptions water (l·m-2) nitrogen (g·m-2) phosphorus (g·m-2) potassium (g·m-2) calcium (g·m-2) magnesium (g·m-2) iron (mg·m-2) 2.2 1.6 d 0.35 c 0.11 c 0.42 c 0.29 c 0.11 c 5.76 c 2.5 2.0 c 0.51 b 0.15 b 0.61 b 0.42 b 0.16 b 7.20 b 2.8 2.6 a 0.76 a 0.21 a 0.92 a 0.64 a 0.23 a 9.00 a 3.1 2.4 b 0.78 a 0.22 a 0.93 a 0.65 a 0.24 a 8.46 a no. plants per pot 9 2.2 b 0.62 b 0.19 b 0.74 b 0.53 b 0.19 b 7.56 b 12 2.6 a 0.73 a 0.21 a 0.90 a 0.58 a 0.23 a 9.00 a 15 2.1 b 0.58 b 0.17 c 0.70 b 0.49 c 0.17 c 7.38 b 18 1.7 c 0.47 c 0.13 d 0.54 c 0.40 d 0.15 d 5.94 c table 5 basil leaves quality indicators nutritive solution ec (ms·cm-1) dry residue (%) soluble solids (°brix) titratable acidity (g · 100 g-1 d.w.) glucose (g · 100 g-1 d.w.) fructose (g · 100 g-1 d.w.) sucrose (g · 100 g-1 d.w.) ascorbic acid (mg·100 g-1 d.w.) 2.2 9.5 c 3.2 c 0.76 c 1.80 c 2.25 c 0.40 c 508.4 c 2.5 9.7 bc 3.4 bc 0.84 b 2.12 b 2.52 b 0.52 b 585.7 b 2.8 10.0 ab 3.6 ab 0.96 a 2.34 a 2.83 a 0.63 a 703.8 a 3.1 10.0 a 3.7 a 1.02 a 2.47 a 2.98 a 0.67 a 744.6 a no. plants per pot 9 10.0 a 3.6 a 0.98 a 2.30 a 2.86 a 0.64 a 708 a 12 10.0 a 3.6 a 0.96 a 2.24 a 2.78 a 0.62 a 689.5 a 15 9.8 ab 3.4 ab 0.88 b 2.14 ab 2.54 b 0.52 b 617.3 b 18 9.6 b 3.3 b 0.78 c 2.02 b 2.40 b 0.45 c 527.4 c morano et al. effects of nutritive solution electrical conductivity on sweet basil grown in subirrigated gullies 29 affect fresh produce quality, but it just lowered the soluble solids content. as reported in table 6, mineral nutrient concentrations were significantly affected by the nutritive solution strength, as their trends were always increasing with the electrical conductivity raise from 2.2 to 2.8 ms.cm-1, whereas no further increases were recorded in the last 0.3 ms.cm-1 rise. with regard to plant density (table 6), the 9 and 12 plants per pot treatments always resulted in the highest nutrient accumulation in the leaves, except for the nitrates which showed an opposite trend, increasing from the lowest to the highest density. the decreasing trend of the leaves mineral ion concentrations as a function of the pot plant density increase resulted in relation with the plant nutrient absorptions (table 4). notably, the increase of mineral cations concentration in the plant tissues is caused by salt ion accumulation in the rizhosphere (sonneveld, 2002) as a consequence of the plant active exclusion in response to salt occurrence in the external solution (bethke and drew, 1992). moreover, the nitrate concentration increase in response to nutritive solution strength raise recorded in our research is consistent with the reports of previous investigations (tesi et al., 1997; raimondi et al., 2006). as for plant density, the increasing nitrate accumulation corresponding to the enhancement of the plants number per pot was presumably caused by the gradual light conditions worsening. contrastingly, in previous research (tesi et al., 1995) a decreasing trend of nitrate accumulation as a function of plant density increase was reported. interestingly, in our research the nitrate concentration was always very low and, accordingly, basil leaves consumption not exceeding 563 g per day complies with the acceptable daily intake for nitrate (222 mg·d-1 for 60 kg adult) (authority efs, 2008). 4. conclusions from research carried out in southern italy on soilless pot-grown basil, it can be inferred that the 2.8 ms.cm-1 nutritive solution resulted in the best product yield and quality, whereas a further increase to 3.1 ms.cm-1 caused the reduction of the water and nutrient efficiency use. moreover, the 12 plants per pot density showed the optimal compromise between the individual plants and the pot plants set performances, providing with the highest production and leaves quality. indeed, density intensification to 15 and further to 18 plants per pot caused the reduced efficiency use of water and nutrients and accordingly the plant growth worsening, as well as the crop cycle extension up to 2.7 days. acknowledgements the authors wish to thank mr. rosario nocerino and mr. luigi sannino for their valuable assistance with the greenhouse equipments and farming practices. references adams p., ho l.c., 1989 effects of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. j. hortic. sci., 64: 725-732. adler p.r., simon j.e., wilcox g.e., 1989 nitrogen form alters basil growth and essential oil content and composition. hortscience, 24: 789-790. authority efs, 2008 nitrate in vegetables: scientific opinion of the panel on contaminants in the food chain. the efsa journal, 689: 1-79. beaman a.r., gladon r.j., schrader j.a., 2009 sweet basil requires an irradiance of 500 µmol·m-2·s-1 for greatest edible biomass production. hortscience, 44: 64-67. bekhradi f., delshad m., marin a., luna m.c., garritable 6 basil leaves chemical composition nutritive solution ec (ms·cm-1) nitrates (g·kg-1 d.w.) phosphates (g·kg-1 d.w.) sulphates (g·kg-1 d.w.) calcium (g·kg-1 d.w.) magnesium (g·kg-1 d.w.) potassium (g·kg-1 d.w.) 2.2 3.2 c 3.6 c 1.5 c 5.9 c 3.3 c 45.3 c 2.5 3.7 b 4.1 b 1.8 b 6.4 b 3.8 b 48.8 b 2.8 4.4 a 5.0 a 2.2 a 7.1 a 4.3 a 53.4 a 3.1 4.8 a 5.3 a 2.3 a 7.3 a 4.4 a 55.0 a no. plants per pot 9 3.4 d 4.8 a 2.1 a 7.1 a 4.2 a 54.2 a 12 3.8 c 4.7 ab 2.1 a 7.0 ab 4.1 ab 52.4 ab 15 4.2 b 4.4 bc 1.9 ab 6.5 bc 3.8 bc 49.7 bc 18 4.7 a 4.1 c 1.7 b 6.1 c 3.6 c 46.2 c adv. hort. sci., 2017 31(1): 25-30 30 do y., kashi a., babalar m., gil m.i., 2015 effects of salt stress on physiological and postharvest quality characteristics of different iranian genotypes of basil. hortic. environ. biotechnol., 56 (6): 777-785. bethke p.c., drew m.c., 1992 stomatal and nonstomatal components to inhibition of photosynthesis in leaves of capsicum annuum during progressive exposure to nacl salinity. plant physiol., 99: 219-226. bione m.a.a., paz v.p.d., da silva f., ribas r.f., soares t.m., 2014 growth and production of basil in nft hydroponic system under salinity. rev. bras. eng. agr. amb., 18 (12): 1228-1234. bohme m., pinker i., 2014 asian leafy vegetables and herbs cultivated in substrate culture and aeroponics in greenhouse. acta horticulturae, 1034: 155-162. caruso g., conti s., villari g., borrelli c., minutolo m., russo g., amalfitano c., 2014 effects of transplanting time and plant density on yield, quality and antioxidant content of onion (allium cepa l.) in southern italy. sci. hortic., 166: 111-120. caruso g., villari g., melchionna g., conti s., 2011 effects of cultural cycles and nutrient solutions on plant growth, yield and fruit quality of alpine strawberry (fragaria vesca l.) grown in hydroponics. sci. hortic., 129: 479-485. chang x.m., alderson p.g., hollowood t.a., hewson l., wright c.j., 2007 flavour and aroma of fresh basil are affected by temperature. j. sci. food agric., 87: 1381-1385. chartzoulakis k., klapaki g., 2000 response of two greenhouse pepper hybrids to nacl salinity during different growth stages. sci. hortic., 86: 247-260. chen b.m., wang z.h., li s.x, wang g.x., song h.x., wang x.n., 2004 effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. plant sci., 167: 635-643. herrera e., 2005 soil test interpretation, guide a-122. new mexico state university. http://aces.nmsu.edu /pubs/_a/a-122.html. munns r., 2002 comparative physiology of salt and water stress. plant cell environ., 25: 239-250. peel m.c., finlayson b.l., mcmahon t.a., 2007 updated world map of the kӧppen-geiger climate classification. hydrol. earth syst. sc., 11: 1633-1644. raimondi g., orsini f., maggio a., de pascale s., barbieri g., 2006 yield and quality of hydroponically grown sweet basil cultivars. acta horticulturae, 723: 357-363. savvas d., stamati e., tsirogiannis i.l., mantzos n., barouchas p.e., katsoulas n., kittas c., 2007 interactions between salinity and irrigation frequency in greenhouse pepper grown in closed-cycle hydroponic systems. agric. water manag., 91: 102-111. sgherri c., cecconami s., pinzino c., navari-izzo f., izzo r., 2010 levels of antioxidants and nutraceuticals in basil grown in hydroponics and soil. food chem., 123(2): 416-422. sonneveld c., 2002 composition of nutrient solutions, pp. 179-210. in: savvas d., and h.c. passam (eds.) hydroponic production of vegetables and ornamentals. embryo publications, athens, greece, pp. 483. tesi r., frabotta a., nencini a., tallarico r. 1997 effect of foliar application of fertilizers on yield and nitrate content of sweet basil. colture protette, 3: 95100. tesi r., ghiselli l., tallarico r., 1995 research on pot basil cultivation. colture protette, 24(12): 61-66. tesi r., lenzi a., 2002 evaluation of basil (ocimum basilicum l.) dwarf genotypes. sementi elette, 48(4): 3840. tesi r., paolucci b., tosi d., vidrich v. 1991 basil (ocimum basilicum l.) genetics and breeding ii. cultivars characteristics. sementi elette, 37(5): 7-13. van oosteron e.j., carberry p.s., hargreaves j.n.g., o’leary g.j., 2001 simulating growth, development, and yield of tillering millet ii. simulation of canopy development. field crop res., 72:67-91. www.istat.it, 2011 annual crop data. istituto nazionale di statistica, roma, italy. zecchinelli r., 1999 basil varietal characterization. sementi elette, 45(6): 37-42. 24 1. introduction fresh-cut artichokes suffer several degradative reactions which limit their marketability. enzymatic browning is caused by the oxidation of phenols, catalysed by polyphenol oxidase enzymes (ppo), with the subsequent formation of dark compounds (lattanzio et al., 1994; tomásbarberán and espín, 2001). non-enzymatic browning reactions are caused by iron-polyphenol complexes: chlorogenic acid, the most representative phenolic compound of artichoke heads, in presence of o 2 forms dark-coloured complexes with fe3+, while the same substrate in anoxic conditions forms colourless complexes with fe2+, but after exposure to air the fe2+ complex is quickly oxidized to fe3+ to give coloured compounds (lattanzio, 2003). also, mechanical wounding enhances a different array of enzymatic pathways, many of which are associated with volatile accumulation, such as ammonia, ethanol and acetaldehyde, which leads to the darkening of tissues and onset of off-flavors (salunkhe and do, 1976; rolle and chism, 1987). the use of modified atmospheres can promote or, otherwise, inhibit these degradative reactions, and differences between beneficial and harmful effects of gas mixtures may be small. increased levels of co 2 are used in combination with low o 2 concentrations to maintain the visual quality of several types of fresh-cut produce (beaudry, 1999). carbon dioxide is considered a competitive inhibitor of ppo but increases in ammonia were observed in leafy tissues stored in high co 2 (cantwell et al., 2010); similarly, if the o 2 level in the package decreases below the fermentation threshold, anaerobic respiration is triggered leading to the accumulation of anaerobic metabolites (i.e. ethanol and acetaldehyde) and stimulating the growth of some anaerobic pathogens (oms-oliu et al., 2009). the presence of a very high co 2 concentration (25%) in the storage atmosphere has been proved to be deleterious for fresh-cut artichokes (la zazzera et al., 2012), while only slight beneficial effects were observed at lower concentrations (5 and 15%). other authors, combining a soy protein isolate enriched with cysteine (cys) and different modified atmospheres (active with 5 kpa o 2 +15 kpa co 2 ; active with 80 kpa o 2 and passive), found that the ma did not increase shelf-life compared to coated samples obtaining only 4 days of shelf-life, but the same authors refer an accumulation up to 30% of co 2 , which can be the cause of received for publication 5 september 2014 accepted for publication 27 march 2015 designing a modified atmosphere packaging (map) for fresh-cut artichokes m. la zazzera, m.l. amodio, g. colelli dipartimento di scienze agrarie, degli alimenti e dell’ambiente, università di foggia, via napoli, 25, 71121 foggia, italy. key words: cynara scolymus l., enzimatic browning, gas transmission rate, high co 2 , low o 2 , polylactic acid, visual quality, abstract: ‘catanese’ artichoke quarters were packaged in active modified atmosphere (5% o2+10% co2) in four different materials or in air in macro-perforated bags used as control (ctrl), and stored at 4°c. materials used for modified atmosphere packaging (map) were: polylactic acid (pla), polylactid acid with one line of micro-perforation (pla mf1), polypropilene with two lines of micro-perforations (pp mf2), and polypropylene + polyamide with two lines of microperforations (pp+pa mf2). initially and after 2, 4, and 9 days, overall artichoke appearance, color and weight loss were monitored. o2 and co2 concentrations within the packages were detected initially and after 1, 2, 17, 24.5, 41.5, 49.5, 120 and 216 h (end of the experiment). also, at the last sampling date ethanol and acetaldehyde accumulations in artichoke tissue were measured. all the micro-perforated films maintained gas levels within the range of o2>3% and co2<15%, defined as “safe”, with a positive effect on quality: all samples remained above the limit of marketability until the end of the experiment, without significant differences among them, but showed a slight better overall appearance and, accordingly, a better retention of color parameters when compared with ctrl samples. complete anaerobic condition (16% co2 and 0% o2) developed in pla bags where blackening of cut bracts and receptacle was observed, while black spots appeared on outer bracts, causing drastic quality reduction; samples fell below the limit of marketability after just 2 days. also a significant accumulation of ethanol and acetaldehyde was found in these samples. optimizing map made it possible to maintain the desired gas condition, with positive effects on quality of the produce in absence of any stabilizing treatment, for 9 days. adv. hort. sci., 2015 29(1): 24-29 25 la zazzera et al., a modified atmosphere packaging for fresh-cut artichokes low shelf-life (ghidelli et al., 2015). therefore, the avoidance of extreme conditions in terms of co 2 and o 2 concentrations within the package should be the main objective when designing a modified atmosphere packaging (map) system for fresh-cut artichokes. in this work an active map was designed to maintain inside the package, at a steady state, a gas concentration of 5% o 2 +10% co 2 (target atmosphere) or at least within a “safety range” (o 2 ≥3%+co 2 ≤15%) as defined in previous experiments. the use of micro-perforated films, allowing a greater permeability to o 2 and co 2 than non micro-perforated films, should be recommended for fresh-cut produce with a very high respiration rate, like artichoke (kader, 2002), since an accumulation of co 2 can still be reached within the package but avoids an extreme concentration and total o 2 depletion. a simple recyclable film (polypropylene), a two-layer non recyclable film (polypropylene and polyamide), and a bio-based compostable film (polylactic acid), with different levels of micro-perforations, were used in this work as map materials. 2. materials and methods freshly harvested artichokes (cynara scolymus l. ‘catanese’) were collected in the area of brindisi (italy). in previous studies (cabezas-serrano et al., 2009), ‘catanese’ showed the best attitude for processing as a fresh-cut produce compared to other cultivars, as it is less susceptible to post-cutting degradation phenomena. raw heads were immersed for 2 min in a 100 mg l-1 sodium hypochloride solution in order to reduce the microbiological contamination and eliminate residual soil particles. artichokes were then hand-trimmed using sharp stainless steel knives in order to remove external bracts, leaves and stalks; heads were washed in a naocl solution (100 mg l-1 of free chlorine). after washing, head trimming was completed by further removal of external greener and tougher bracts (inedible fraction) so as to keep just the innermost tender bracts, and by cutting about 2 cm from the top. finally, artichokes were cut into quarters and closed in active modified atmosphere packaging (map) with the initial gas composition of 5% o 2 +10% co 2 using a tecnovac packaging machine (mod. t520, grassobbio, bg; italy). four different packaging materials were used (table 1): polylactic acid (pla), polylactid acid with one line of micro-perforation (pla mf1), polypropilene with two lines of micro-perforations (pp mf2), and polypropylene + polyamide with two lines of micro-perforations (pp+pa mf2). control samples were passively packaged in macro-perforated pp mf2 bags in which additional macro holes (four per side) were manually made with a needle, insuring the complete gas exchange through the film and no atmosphere modification inside the packaging, while protecting the product from excessive weight loss (ctrl). evolution of gas composition inside the package, once it has been sealed, is due to the gas permeability of the plastic material used, to the respiration rate of the packaged commodity and, consequently, to the ratio between the package dimension and product weight. in order to reach and maintain the target atmosphere within the bags, packaging dimensions for a produce weight of 150 g were optimized knowing the film properties (table 1), and the desired gas transmission rate for o 2 (otr) and co 2 (co 2 tr) being respectively 221 ml m-2 day-1 for o 2 and 354 ml m-2 day-1 for co 2, using the following formula: w * rr a = gtr * (%g atm – %g pkg ) where a = packaging surface (m2); w = product weight (kg); rr = respiration rate (ml kg-1 day-1); gtr= gas transmission rate (ml m-2 day-1) % g atm = percentage of the gas in the atmosphere (bar) ; % g pkg = percentage of the gas in the packaging (bar). only for pla mf1 was a produce weight of 120 g used, and the optimal dimension could not be optimized since, based on its low gtr, too-large and not feasible bag dimensions would have been needed. all samples were stored at 4°c. initially and after 2, 4, and 9 days, artichoke colour, overall appearance and weight loss, were monitored. o 2 and co 2 concentrations table 1 film material and packaging characteristics film material packaging dimension (cm) thickness (µm) diameter of holes (µm) number of holes/m2 otr (ml m-2 day-1 bar-1) ß (co 2 tr/otr) wvtr (gm-2 day-1) pla 20x25 30 570 4.2 317 pla mf1 10.6x17 30 60 138 570 4.2 317 pp mf2 24.6x13 30 60 160 1100* 2.4* 5* pp+pa mf2 22.5x25 67 60 222 29* 2.75* 2.01* * refers to the film without micro perforation. pla= polylactic acid without micro-perforation; pla mf1= polylactid acid with one line of micro-perforation; pp mf2= polypropilene with two lines of micro-perforations; pp+pa mf2= polypropylene + polyamide with two lines of micro-perforations; otr= oxygen transmission rate; β = co 2 tr/otr; wvtr = water vapor transmission rate. 26 adv. hort. sci., 2015 29(1): 24-29 within the packages were detected initially and after 1, 2, 17, 24.5, 41.5, 49.5, 120 and 216 h (end of experiment). on the last sampling date, also ethanol and acetaldehyde accumulations in artichoke tissue were measured. o 2 and co 2 concentrations within the packages were measured using a witt gascontrol 100-model mapy 4.0 (wittgasetechnik gmbh & co kg, germany). overall appearance was evaluated by assigning a score to each artichoke quarter, from 5 (excellent) to 1 (very bad) using as a reference a photographic scale associated to brief descriptions (amodio et al., 2007). samples in the bags were weighed at each storage period and the weight loss was calculated as percent of the initial fresh weight. colour in cie l*a* b* scale was measured on the external surface of the outer bract and on the cutting surface, elaborating the images acquired with a spectral scanner (dv srl, italia); hue angle (h°) and chroma were calculated from the primary l*, a*, and b* values. ethanol and acetaldehyde accumulation in artichoke tissues were measured only on the last sampling date with the method of mateos et al. (1993) using a gas chromatograph shimadzu gc-14a equipped with a fid detector (temperature 150°c); separation was carried out isothermally at 80°c on a capillary column 5% cbwx 20m on carbograph 1aw20 80/120, 6’x 1/8’’ x 0.0085 (alltech). ethanol and acetaldehyde were identified and quantified by comparison with standard curves, and expressed respectively as umol ethanol g-1 and nmol acetaldehyde g-1. the experiment was organized in a split-plot design with the packaging condition as the main factor and the time of storage as the secondary factor. the most conservative degrees of freedom were used to determine the effect of time, and packaging x time interaction. at each storage time the effect of packaging treatments was tested performing a one-way anova. means were separated using the tukey test. for the o 2 and co 2 concentrations within the packages and ethanol and acetaldehyde contents, standard deviations (std) were calculated. 3. results and discussion packaging treatment and storage time significantly influenced all the quality parameters with few exceptions (a* value of bract cut surface was not influenced by packaging and b* value of the receptacle was not affected by time of storage). also interactions were found to be significant for all attributes except for a* value in bract cut surface (table 2). the evolution of gas composition within the packages is shown in figure 1. all the micro-perforated films maintained gas levels within the safety range (o 2 higher than 3% and co 2 lower than 15%). for samples stored in pp+pa mf2 and pp mf2 gas evolution showed the same trend: the o 2 level decreased in the first hours, due to the high respiration rate of cut artichokes, reaching a steady state at 12% and 10% respectively for pp+pa mf2 and pp mf2; co 2 concentration increased slightly for both films, reaching a steady state at 11.5% for pp+pa mf2 and 12.5 for pp mf2. in pla mf1 bags, o 2 concentration increased progressively, reaching a maximum of 14.5% after 216 h, while co 2 concentration decreased sharply just after sealing, falling from 10 to 6%, and then rapidly increased, reaching at equilibrium a concentration of about 7.5%. in non-micro-perforated pla packages, anaerobic conditions (0% o 2 and 16% co 2 ) developed after 17 h; then the co 2 level increased further, reaching a maximum of 29% after 120 h of storage. all the samples stored in micro-perforated bags showed a better or comparable visual quality than ctrl samples, allowing the produce to keep marketability until the end of the experiment without significant differences among them (fig. 2). on the contrary, artichokes stored in nonperforated pla resulted heavily damaged: black spots appeared on outer bracts, a blackening of cut bracts and receptacle was observed and off-odours were perceived. after 2 and 4 days of storage, the injuries did not show up until removal from low o 2 atmosphere and exposure to table 2 results of anova test for treatment and time of storage on quality attributes of fresh-cut artichoke quarters during storage in different atmospheres parameter packaging time packaging x time appearance score bract cut surface and receptacle cut surface **** **** **** external bracts **** **** **** color external surface l* **** **** **** a* **** **** **** b* *** **** **** chroma *** **** **** hue angle ** *** ** bract cut surface l* **** **** **** a* ns **** ns b* **** **** **** chroma **** **** **** hue angle * **** ** receptacle cut surface l* *** **** * a* **** **** **** b* *** ns **** chroma **** * **** hue angle **** **** *** weight loss **** **** **** within each row, each factor (treatment and time) and their interaction are significantly different for p≤0,05 (*); p≤0,01 (**); p≤0,001 (***); p≤0,0001 (****), or not significant (ns). 27 la zazzera et al., a modified atmosphere packaging for fresh-cut artichokes fig. 1 o 2 and co 2 concentrations within different packages used for artichoke quarters stored at 4°c: pp+pamf2 = polypropylene + polyamide with two lines of micro-perforations; pp mf2 = polypropilene with two lines of micro-perforations; pla mf1= polylactid acid with one line of micro-perforation; pla= polylactic acid without micro-perforation. mean values of three replicates ± std. fig. 2 changes over time of overall appearance in fresh-cut artichoke quarters packaged in active modified atmosphere (5% o 2 +10% co 2 ) and stored at 4°c: pp+pamf2= polypropylene + polyamide with two lines of micro-perforations; pp mf2= polypropilene with two lines of micro-perforations; pla mf1= polylactid acid with one line of micro-perforation; pla= polylactic acid without micro-perforation. ctrl represents samples stored in air in macro-perforated bags. at each storage sampling different letters indicate significant differences (p<0.05). 28 adv. hort. sci., 2015 29(1): 24-29 air, when in few minutes they became completely inedible, with the onset of off-odours. black spots and blackening could be due to non-enzymatic browning, as suggested by lattanzio (2003) on artichokes: chlorogenic acid, the most representative phenolic compound of artichoke heads, in the absence of o 2 forms colourless complexes with fe2+, but after exposure to air the complexed fe2+ is quickly oxidized to fe3+ to give coloured compounds. in the same way, also enzymatic reactions could have taken place after exposure to o 2 . at the last sampling, after 9 days of storage in extreme atmosphere condition, black spots and blackening began to appear in the produce when still inside the pla package, suggesting a possible necrosis of the tissues. colour data (data not shown) confirmed the deleterious effect of the extreme gas conditions reached within the pla bags, indicating a severe browning (drop on l and b*, increase of a* and higher variation of hue angle) and a better retention on colour parameters for samples stored in micro-perforated films, also compared to ctrl samples. weight loss (fig. 3) was greater in ctrl samples and in samples stored in pla bags (7.5, 5.7 and 3.7% for ctrl, pla and pla mf1 respectively), than in samples stored in pp mf2 and pp+pa mf2 (below 1%). weight loss in non micro-perforated pla bags was significantly higher than in bags of the same material but with microperforations. most probably the deleterious atmosphere condition within these bags played a more important role on weight loss than the possible higher transpiration which the presence of micro-perforations might have generated. a clear accumulation of ethanol and acetaldehyde was found in artichokes stored for 9 days in pla (fig. 4), where complete anaerobic conditions developed. the synthesis of these volatile compounds caused by anaerobic respiration was demonstrated by several authors (shaw, 1970; woodward and topping, 1972; prasad and stadelbacher, 1974). ke et al. (1991) found strong correlations between off-flavour development and ethanol content, and to a lesser extent, acetaldehyde. severe off-odours were produced by broccoli under anaerobic conditions developed after few days in map (forney et al., 1991). nichols and patterson (1987) observed that in apples the injury due to ethanol and acetaldehyde accumulation in anaerobic conditions or high co 2 atmospheres is post-anoxic: it occurs after removal from low o 2 exposures, when ethanol is oxidized to acetaldehyde. 4. conclusions all the tested micro-perforated materials made it possible to reach and maintain the atmosphere composition within a safety range (o 2 >3% and co 2 <15%), with a posifig. 3 weight loss over time of cut bract and external surfaces of freshcut artichoke quarters packaged in active modified atmosphere (5% o 2 + 10% co 2 ) and stored at 4°c: pp+pamf2 = polypropylene + polyamide with two lines of micro-perforations; pp mf2= polypropilene with two lines of micro-perforations; pla mf1= polylactid acid with one line of micro-perforation; pla= polylactic acid without micro-perforation. ctrl represents samples stored in air in macro-perforated bags. at each storage sampling different letters indicate significant differences (p<0.05). fig. 4 ethanol and acetaldehyde accumulation, after 9 days of storage, in fresh-cut artichoke quarters packaged at 4°c in active modified atmosphere (5% o 2 +10% co 2 ), in different packaging conditions: pp+pamf2 = polypropylene + polyamide with two lines of micro-perforations; pp mf2 = polypropilene with two lines of micro-perforations; pla mf1 = polylactid acid with one line of micro-perforation; pla = polylactic acid without micro-perforation. ctrl represents samples stored in air in macro-perforated bags. mean values of three replicates ± std. 29 la zazzera et al., a modified atmosphere packaging for fresh-cut artichokes tive effect on quality of stored fresh-cut artichokes. artichokes packaged in pla mf1 suffered a higher weight loss than produce stored with other packaging materials, which however did not seem to influence its overall appearance. on the contrary, the use of non micro-perforated films was not suitable for cut artichoke, probably due to its high transpiration rate: serious damage occurred to the produce in pla bags, causing a drastic reduction in its quality. extreme conditions in terms of co 2 and o 2 concentrations may be very deleterious for packaged vegetables and the avoidance of these conditions within the packages must be the main objective when designing a map system for fresh-cut artichokes. map optimization for fresh-cut artichokes was possible with the auxilium of micro-perforation, which permitted establishment and maintenance of the desired gas conditions, regardless of the plastic material. the designed map allowed storage of cut artichokes in the absence of any stabilizing treatment for 9 days. acknowledgements this work was carried out within the project “pon02_00657_00186_3417392/1 (infopack -p.o.n. ricerca e competitività” 2007-2013 per le regioni della convergenza codice progetto) co-funded by the italian ministery of high education, university and research ‘ministero dell’istruzione, dell’università e della ricerca’. references amodio m.l., cabezas a., rinaldi r., colelli g., 2007 implementation of rating scales for visual quality evaluation of various vegetable crops. in: kader a.a., and m. cantwell (eds.) produce quality rating scales and color charts. postharvest horticultural series no. 23, university of california, davis, ca, usa, pp. 151. beaudry r.m., 1999 effect of o 2 and co 2 partial pressure on selected phenomena affecting fruit and vegetable quality. postharvest biol. tec., 15: 293-303. cabezas-serrano a.b., amodio m.l., cornacchia r., rinaldi r., colelli g., 2009 screening quality and browning susceptibility of 5 artichoke cultivars for fresh-cut processing. j. sci. food agric., 89(15): 2588-2594. cantwell m., hong g., nie x., 2010 using tissue ammonia and fermentative volatile concentrations as indicators of beneficial and stressful modified atmospheres for leafy and floral vegetables. acta horticulturae, 876: 165-172. forney c.f., mattheis j.p., austin r.k., 1991 volatile compounds produced by broccoli under anaerobic conditions. j. agric. food chem., 39: 2257-2259. ghidelli c., mateos m., rojas-argudo c., pérezgago m.b., 2015 novel approaches to control browning of fresh-cut artichoke: effect of a soy protein-based coating and modified atmosphere packaging. postharvest biol. tec., 99: 105-113. kader a.a., 2002 postharvest biology and technology: an overview, pp. 40. in: kader a.a. (ed.) postharvest technology of horticultural crops. 3rd edition. university of california, anr publication, usa, pp. 535. ke d., goldstein l., o’mahony m., kader a.a., 1991 effects of short-term exposure to low o 2 and high co 2 atmospheres on quality attributes of strawberries. j. food sci., 56: 50-54. la zazzera m., rinaldi r., amodio m.l., colelli g., 2012 influence of high co 2 atmosphere composition on fresh-cut artichoke quality attributes. acta horticulturae, 934: 633-640. lattanzio v., 2003 the role of plant phenolics in the postharvest physiology and quality of fruit and vegetables, pp. 49-83. in: imperato f. (eds) advances in phytochemistry. research signpost, trivandrum, kerala, india. lattanzio v., cardinali a., di venere d., linsalata v., palmieri s., 1994 browning phenomena in stored artichoke (cynara scolymus l.) heads: enzymic or chemical reactions? food chem. , 50: 1-7. mateos m., ke d., cantwell m., kader a.a., 1993 phenolic metabolism and ethanolic fermentation of intact and cut lettuce exposed to co 2 -enriched atmospheres. postharvest biol. tec., 3: 225-233. nichols w.c, patterson m.e., 1987 ethanol accumulation and poststorage quality of ‘delicious’ apples during short-term, low-o 2 , ca storage. hortscience, 22: 89-92. oms-oliu g., hertog m.l.a.t.m., soliva-fortuny r., martín-belloso o., nicolaї b.m., 2009 recent developments in the use of modified atmosphere packaging for fresh-cut fruits and vegetable. stewart posthar. rev. 5(4):1-11. prasad k., stadelbacher g.j., 1974 effect of acetaldehyde vapor on postharvest decay and market quality of fresh strawberries. phytopathology, 64: 948-951. rolle r.s., chism iii g.w., 1987 physiological consequences of minimally processed fruits and vegetables. j. food qual., 10: 157-177. salunkhe d.k., do j.y., 1976 biogenesis of aroma constituents of fruits and vegetables. crit. rev. food sci. nutr., 8: 161-189. shaw g.w., 1970 the effects of controlled atmosphere storage on the quality and shelf-life of fresh strawberries with special reference to botrytis cinerea and rhizopus nigricans. diss. abstr. int. sect. b, sci. eng., 30: 1343. tomás-barberán f.a., espín j.c., 2001 phenolics compounds and related enzymes as determinants of quality in fruits and vegetables. j. sci. food agric., 81: 853-876. woodward j.r., topping a.j., 1972 the influence of controlled atmospheres on the respiration rates and storage behaviour of strawberry fruits. j. hortic. sci., 47: 547-553. impaginato 39 1. introduction berberis microphylla g. forst. is a patagonian native shrub commonly named “calafate”, with a large distribution from neuquén (37° sl) to tierra del fuego (54° 8´ sl) (orsi, 1984). this species has a growing economic potential due to the production of fruits as a non-timber forest product (tacón clavaín, 2004). in fact, its dark blue berries are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. moreover, the fruits have a high content of carbohydrates, phenols and antioxidants (arena and curvetto, 2008; arena et al., 2011, 2012, 2013 b). the genetic and morphological analysis of spontaneous accessions in natural populations of b. microphylla grown on tierra del fuego (giordani et al., 2016), as well as the changes in form and leaf anatomy due to weather conditions (radice and arena, 2015) were recently studied. the study of flower anatomy related to blooming is an important step for programs of genetic resource conservation and improvement, complementing basic studies of floral biology (de castro nunes et al., 2012; wetzstein et al., 2014). flower structure and floral biology was well described by arena et al. (2011), like the phenological stages (arena et al., 2013 a) and flower bud differentiation (arena and radice, 2014). more recently, a comprehensive study of pollen grain was published (radice and arena, 2016 a). nevertheless, pollination was not clear until now. fertilization of patagonian berberis has been classified as cross-pollination by orsi (1984). however, hegi (1958) and romeo et al. (2005) referred the berberis species as autogamous due to the absence of visiting insects together with extreme climatic conditions prevalent in most areas of patagonia. however, during flowering, the activity of different syrphidae was observed (radice et al., 2016). on the other hand, floral movements have been appointed as mechanisms to facilitate self-pollination (darwin, 1862). stamen movement has been documented in a few plant families, among them berberidaceae (lechowski and bialczyk, 1992). however, results of controlled treatments of selfand cross-pollination compared with those of open-pollination performed during three different periods in b. microphylla do adv. hort. sci., 2017 31(1): 39-44 doi: 10.13128/ahs-20724 flower anatomy related to blooming development of berberis microphylla g. forst (berberidaceae) s. radice, m. arena department of plant physiology, facultad de agronomía y ciencias agroalimentarias um-conicet, machado 914, lab. 501, b1708eoh, morón. prov. de buenos aires, argentina. key words: anthesis, barberry, nectar, patagonia, pollen grain, stigma. abstract: berberis microphylla g. forst. is a patagonian native shrub commonly named “calafate”, which has a growing economic potential due to its dark blue berries that are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. moreover, the fruits have a high content of carbohydrates, phenols and antioxidants. the objective of this work was to show the changes observed in the flower from the emergence in relation to the floral phases and the importance that they have on pollination and fertilization. during the anthesis, the nectar is excreted inside and outside of the petal through the epidermis of the secretory tissue. the epidermis of the stigma is papillae with cells of greater length in the periphery of this structure simulating an additional ring. secretory tissue is also present on the area of the fusion carpel. during anthesis, the epidermis glands of the stigma showed active secretion and these conditions favor pollen grain germination. germinated pollen grains were observed after 12 hours of pollination and ten days later the pollen tube reached the ovule area. pollen tube grew surrounded the ovules and probably some of them already accomplished the fertilization. (*) corresponding author: siradice@yahoo.com received for publication 19 november 2016 accepted for publication 15 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 39-44 40 not support this hypothesis (radice and arena, 2016 b). thus, self-pollination resulted only in pollen germination on the stigmas but the pollen tubes were not able to reach the ovules. according to these antecedents, anatomical studies are necessary for a better understanding of the physiological processes during floral biology and pollination that interact with syrphidae activity. the objective of this work was to show the changes observed in the flower from its emergence in relation to the floral phases and the importance that they have on pollination and fertilization. 2. materials and methods plant material flowers of berberis microphylla g. forst. (n = 20) on growth stages ranging from 53 to 68 on the bbch scale proposed for b. buxifolia (arena et al., 2013 a) were collected on plants grown near ushuaia city, tierra del fuego (54° 48’ sl, 68° 19’ wl and 30 m asl), and were fixed in faa (formaldehyde, 100 ml; ethyl alcohol, 500 ml; acetic acid, 50 ml; distilled water, 350 ml). light microscopy button flowers were dehydrated in an ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. fluorescent microscopy flowers fixed in faa were washed with distilled water and softened with naoh (8n) as described by martin (1959). then, they were stained with aniline blue to study pollen tube growth. squash material was observed by a leica microscope (dm 2500) with dapi filter. scanning electron microscopy (sem) button flowers fixed in faa were dehydrated in an ethanol series and critical point drying technique was employed. samples were sputter coated with 20 nm gold and observed with a philips xl 30 sem. ovules and seeds relation the number of ovules on button flowers (n= 130) and the number of seeds on formed fruits (n=100) were counted. 3. results pistil of b. microphylla is similar to a bottle (fig. 1a). flowers before anthesis (growth stage 54) showed anthers with microspores at an advanced stage of development and underdeveloped ovules (figs. 1a-d). in effect, microsporangia contain tapetal cells metabolized, i.e. a thick portion was deposited on the wall of the microspore and inside it is possible to observe vegetative and generative cells that are surrounded by a thin delicate wall (fig. 1d). on the other hand, ovules are rudimentary with an active cellular proliferation on the nucellus and integuments (fig. 1c). the epidermis of the stigma is covered by secretory cells (figs. 2 a, c, e) and the short style is also recovered by glands (fig. 2b). petals have two thick nectaries on the basal position (fig. 3a). both petal epidermises are also formed by glandular cells (figs. 3b, d, e, f). on a later flower phase (growth stage 59), the epidermis and nectariferous cells of the nectar have dense cytoplasm and conspicuous nucleus (figs. 3c). nectar is abundant in the intercellular spaces (fig. 3c). flowers on the anthesis phase (growth stage 60) showed nectariferous cells with a gradual diminution of staining density (fig. 3g). total production of nectar per flower is poor; it is secreted through the gland cells that are present on the epidermis of the nectaries (figs. 3c, g) and two epiderfig. 1 bottom flower of b. microphylla g. forst (light micrograph). a, longitudinal section of a flower on stage 54. b, anther with mature pollen grains; c, rudimentary ovule with nucellus (n) and integuments (i) in development; d, detail of pollen grain with exine (e), vacuole (v) and generative cell (gc) contained into the vegetative cell; n= nucleus. bars: a = 200 µm; b, d = 10 µm; c = 50 µm. radice and arena flower anatomy related to blooming development of berberi microphylla g. forst 41 mal layers of petals (figs. 3e,f). nectar is exuded through the cuticle with rupture of its outer layer. greater presence of vacuoles is observed in both nectar tissues (fig. 3g). flowers on growth stage 59 showed a monocarpellary pistil with a clavate shaped stigma (fig. 4a). epidermis of the stigma is papillae (figs. 4b, c, e) with cells of greater length in the periphery of this structure simulating an additional ring (fig. 4e). these cells secrete a sticky substance that keeps always the stigma hydrated during the anthesis phase (fig. 2f). secretory tissue is also present on the area of the fusion carpel (figs. 4b, d). on the other hand, stigmas on growth stage 59 are receptive, i.e. they can promote pollen germination while stigma on less developed growth phases is not receptive (fig. 2e). flowers on anthesis phase (growth stage 60) fig. 2 sem micrograph of the pistil of b. microphylla g. forst. a, stigma of a flower on stage 59; b, detail of box in the picture a, periphery of the upper end of the ovary with glands; c, stigma front view shown in a; d, stigma front view shown of a flower on stage 60; e, detail of box in the picture d, long hairs peripheral stigma with pollen grains attached; f, detail of circle in the picture c, epidermal cells of stigma; g, detail of circle in the picture d, active secretions glands; h, mixture of pollen grains wrapped in stigmatic mucilage, arrow points to a grain of foreign pollen. bars: a-e =100 µm; f-h= 50 µm. fig. 3 nectary of b. microphylla g. forst. a-d sem micrograph. bc, e-g light micrograph. a, nectary view at the base of petal; b, section of a petal and a nectariferous area; c, detail of tissue of nectariferous area of a flower on stage 59; intercellular space with nectar (arrows); d, detail of circle in the picture a, epidermis with glands, arrows show secretory glands. e, detail of circle in the picture b, petal outer epidermis; f, detail of circle in the picture b, petal inner epidermis; g, detail of tissue of nectariferous area of a flower on stage 60, arrow indicates an epidermal cell in active secretion. bars: a-b= 100 µm; c= 10 µm; d= 50 µm; e-g= 10 µm. fig. 4 light micrograph of the pistil of b. microphylla g. forst. a, longitudinal section of a flower on stage 59.b, view of stigma and the upper end of the ovary; c, glandular cells of the flat part of stigma; d, detail of glandular cells of fusion area of the carpel; e, detail of hairs surrounding the stigma. bars: a= 100 µm; b= 200 µm; c-e= 50 µm. adv. hort. sci., 2017 31(1): 39-44 42 showed mature pollen grain with a well-formed external wall and the cytoplasm of the vegetative cell rich of starch (fig. 5e). in this phase anthers are dehiscent. on the contrary, ovules are externally coated by the integuments and attached to the ovary by the funiculus on basal placentation (fig. 5d). internally, the ovules present some delay in comparison to the pollen grain development. pollinated flowers show ovules with the embryo sac with egg cell, synergists, antipodes and polar nuclei cells developed (figs. 5 a-c); nevertheless, no pollinated pistils show ovules with megaspore mother cells without development. pollen is deposited on the stigma mainly between the secretory cells that surround this structure (figs. 2d, g). germinated pollen grains were observed after 12 hours from pollination (fig. 6b). after 24 hours pollen tube crosses the stigma (fig. 6a) and ten days later the pollen tube reached the ovule area. pollen tube grew surrounded the ovules and probably some of them accomplished the fertilization (figs. 7a, b). subsequently seed growth is observed (fig. 7c) but this process is not given in all the ovules. in effect, on a selected natural population the ovules and seeds were counted, and an average of 8.95 (ranged from 6.9 to 10.0) and 5.28 (ranged from 3.3 to 7.2), respectively, were registered, i.e. 40.97% of all ovules produced aborted. 4. discussion and conclusions flowering plants are associated with a broad spectrum of animal pollinators, among these bees constitute an important but not exclusive one (dötterl and vereecken, 2010). in effect, it has already been demonstrated that b. microphylla is not self-fertile so it depends on insect pollination (radice and arena, 2016 b). tierra del fuego (argentina) offers an extreme climatic situation where the bees cannot prosper; accordingly calafate flowers are visited by different syrphids (radice et al., 2016). pollination by insects, including flies, is commonly a mutualistic interaction, in which both the plant and the insect benefit; thus, anatomical organization and pollination strategies developed on the flowers must be adapted to the environmental conditions. calafate shrubs bloom with abundant yellow flowers that produce aromatic nectar (radice et al., 2016), that exudates inside the flower as well as the outside through the petals mainly in their insertion fig. 5 details of the ovary and pollen grains of a flower on anthesis stage of b. g. forst. a-c, embryo sac with egg cell (a, arrow), synergids (b, arrow) and antipodes (c, arrow) and polar nuclei (c, arrows head); d, sem micrograph of ovules on the basal insertion of ovary; e, mature pollen grains with cytoplasm rich in starch grains. bars: a-c= 200 µm; d= 100 µm; e= 10 µm. fig. 6 fluorescent light micrograph of pollen tube germinated on pistils of b. microphylla g. forst. a, view of stigma with pollen grain germinated and pollen tubes inserted in the ovary; b, pollen tubes on the first stage of growth; c, pollen tube penetrating the ovule. bars: a-c= 100 µm. radice and arena flower anatomy related to blooming development of berberi microphylla g. forst 43 area. this particularity is very important because the fluorescent emission of nectar attracts pollinating insects. b. microphylla have perigonial nectaries type “1a” according to the topographic classification of fahn (1982). unlike what was found on the nectar tissue of berberis corymbosa by bernardello et al. (2000), no stomata were found on this species either on histological sections or through sem observations. in effect, nectar is exuded through the epidermis as cited by bernardello (2007). berberis produce small amounts of nectar per flower; effectively it was registered less than 1 µl on b. corymbosa (bernardello et al., 2000) and 1.57 µl on b. microphylla (radice al., 2016). nectar concentration was considered as intermediate for b. buxifolia (31.2±14.8%) (chalcoff et al., 2006). this result is in coincidence with radice et al. (2016) who measured 36.28 °brix in nectar of the berberis population studied. stigma epidermis is covered by hairs that secrete a stigmatic fluid to promote pollen germination. surface hairs on the stigma can be seen in others species like papaver rhoeas, or lupinus luteus (fahn, 1982). once overcome the stigma, the pollen tubes grow through the carpel wall. in effect the margin of the carpel is covered by glands that nourish the germinated pollen. the berberidaceae are generally considered to have originated in some part of the ranalian complex (chapman, 1936), i.e., it is belonging to the group of the oldest dicotyledons which is confirmed by its structure devoid of carpelar style (fahn, 1982). there are three important elements to attract the pollinating insect on b. microphylla such as color, scent and nectar. it has long been known that bees utilize not only visual but also olfactory flower cues for finding suitable host plants (dötterl and vereecken, 2010). this pollination strategies present in calafate could be useful in other growth areas of the species. on the other hand, young flies in the presence of generic floral scent respond more strongly to a uniformly yellow cue than to any other uniform color cue (green, white, black, blue, red) except for ultraviolet (brodie et al., 2015). nectar is the most commonly sought reward by flower-visiting flies (woodcock et al., 2014) because carbohydrates contained in the nectar provide short-term energy supply. so the abundant number of yellow flowers plus the fluorescent emitted cue by nectar must constitute a strong attraction for syrphids. acknowledgements authors acknowledge to the prefectura naval argentina, the technical assistance of isabel farías. this research was supported by grants pip 0314 subsidized by conicet. fig. 7 fertility of b. microphylla g. forst. a-b, time of discharge of sperm nuclei (arrows) into the egg cell (ec) and polar nuclei (pn), c, growing seed. bars: a-b= 200 µm; c= 100 µm. adv. hort. sci., 2017 31(1): 39-44 44 references arena m.e., curvetto n., 2008 berberis buxifolia fruiting: kinetic growth behavior and evolution of chemical properties during the fruiting period and different growing seasons. sci. hortic., 118(2): 120-127. arena m.e., giordani e., radice s., 2011 flowering, fruiting and leaf and seed variability in berberis buxifolia, a patagonian native fruit species, pp. 117-136. in: marin l., and d. kovaè (eds.) native species: identification, conservation and restoration. nova science publishers, new york, usa, pp. 176. arena m.e., giordani e., radice s., 2013 a phenological growth and development stages of the native patagonian fruit species berberis buxifolia lam. j. food, agric. environ., 11(3-4): 1323-1327. arena m.e., postemsky p., curvetto n.r., 2012 accumulation patterns of phenolic compounds during fruit growth and ripening of berberis buxifolia, a native patagonian species. new zeal. j. bot., 50(1): 15-28. arena m.e., radice s., 2014 shoot growth and development of berberis buxifolia lam. in tierra del fuego (patagonia). sci. hortic., 165: 5-12. arena m.e., zuleta a., dyner l., constenla d., ceci m., curvetto n.r., 2013 b berberis buxifolia fruit growth and ripening: evolution in carbohydrate and organic acid contents. sci. hortic., 158: 52-58. bernardello g., 2007 a systematic survey of floral nectarines, pp. 19-128. in: nicolson s.w., m. nepi, and e. pacini (eds.) nectaries and nectar. springer, dordrecht, the netherlands, pp. 396. bernardello g., galetto l., anderson g.j., 2000 floral nectary structure and nectar chemical composition of some species from robinson crusoe island (chile). can. j. bot., 78(7): 862-871. brodie b.s., smith m.a., lawrence j., gries g., 2015 effects of floral scent, color and pollen on foraging decisions and oocyte development of common green bottle flies. plosone, 10(12): 1-15. chalcoff v.r., aizen m.a., galetto l., 2006 nectar concentration and composition of 26 species from the temperate forest of south america. ann bot., 97(3): 413-421. chapman m., 1936 carpel anatomy of the berberidaceae. amer j. bot., 23: 340-348. darwin c., 1862 on the various contrivances by which british and foreign orchids are fertilised by insects, and on the good effects of intercrossing. john murray, london, uk. de castro nunes r., oliveira bustamante f., techio v.h., mittelman a., 2012 morphology and pollen viability of lolium multiflorum lam. ciên. agrotec., 36(2): 180-188. dötterl s., vereecken n.j., 2010 the chemical ecology and evolution of bee-flower interactions: a review and perspectives. the present review is one in the special series of reviews on animal-plant interactions. can. j. zool., 88(7): 668-697. fahn a., 1982 plant anatomy. nature, pergamon press, usa, pp. 544. giordani e., müller m., gambineri f., paffetti d., arena m., radice s., 2016 genetic and morphological analysis of berberis microphylla g. forst. accessions in southern tierra del fuego. plant biosyst., pp. 1-14. hegi c., 1958 illustrierte flora. von mittel-europa, 4(1): 1-11. lechowski z., bialczyk j., 1992 effect of external calcium on the control of stamen movement in berberis vulgaris l. biol. plant., 34: 121-130. martin f.w., 1959 staining and observing pollen tubes in the style by means of fluorescence. stain technol., 34: 125-128. orsi m.c., 1984 berberidaceae, pp. 325-348. in: correa m.n. (ed). flora patagónica sección 4ª. inta, buenos aires, argentina. radice s., arena m., 2015 environmental effect on the leaf morphology and anatomy of berberis microphylla g. forst. int. j. plant biol., 6(5677): 1-7. radice s., arena m.e., 2016 a characterization and evaluation of berberis microphylla g. forst pollen grains. adv. hort. sci., 30(1): 31-37. radice s., arena m.e., 2016 b effect of different pollination treatments of berberis microphylla g. forst, a patagonian barberry. proceedings of ii international workshop on floral biology and s-incompatibility in fruit species, murcia, spain, may, pp. 23-26. radice s., arena m.e., suárez f.j., landi l.i., caló j.f., 2016 pollination strategies of berberis microphylla g. forst, a patagonian barberry. proceedings of ii international workshop on floral biology and sincompatibility in fruit species, murcia, spain, may, pp. 23-26. romeo r.a., sánchez a.c., novara l., 2005 berberidaceae. aportes botanicos de salta-serie flora, 7(9): 1-10. tacón clavaín a., 2004 manual de productos forestales no madereros. cipma, valdivia, chile, pp. 22. wetzstein h.y., porter j.a., janick j., ferreira j.f.s., 2014 flower morphology and floral sequence in artemisia annua (asteraceae). amer. j. bot., 101(5): 875-885. woodcock t.s., larson b.m., kevan p.g., inouye d.w., lunau k., 2014 flies and flowers ii: floral attractants and rewards. j. pollinat ecol., 12(8): 63-94. 111 1. introduction recent studies have focused on the physiological relaxing effects of the natural environment (park et al., 2009; 2012). it has been reported that staying in a forest environment enhances parasympathetic nervous activity (park et al., 2012; tsunetsugu et al., 2013), suppresses sympathetic nervous activity (park et al., 2012; tsunetsugu et al., 2013; lee et al., 2014), decreases blood pressure and pulse rate (park and mattson, 2009; park et al., 2012), and decreases cortisol concentration (park et al., 2012). studies by li et al. (2007, 2008 a, b) demonstrated that staying in a forest environment for three days and two nights improved the immune function of office workers (li et al., 2007), and this effect was sustained for approximately one month (li et al., 2008 a, b). another study reported that walking in an urban park enhances parasympathetic nervous activity and decreases heart rate (song et al., 2013). in addition, spending time in rooftop gardens enhances parasympathetic nervous activity and suppresses sympathetic nervous activity in elderly people requiring care (matsunaga et al., 2011). evidence-based medicine has been attracting attention globally, with physiological data from field tests making a significant contribution. we expect that accumulating physiological data from field experiments will continue to demonstrate the preventive medical effects of nature therapy in the future (lee et al., 2012). in modern society, many individuals spend the majority of their time in intensely stressful states, and they have no time to make contact with nature outside of their immediate surroundings. high school students, who spend most of their everyday life at school, are typical examples. previous studies have evaluated the psychological stress levels in high school students (anda et al., 2000; takakura and sakihara, 2001). moreover, many high school students have stressful relationships with friends or teachers (miura and kawada, 2008). in a document by the japanese ministry of education, the percentage of students who progressed to universities or junior colleges in 2010 was 56.9%, which was 18.5% higher than the rate in 1975 (statistics bureau, ministry of internal affairs and communications, 2012), and the pressure from entrance examinations is extremely high among high school students (equal employment, children and families bureau, ministry of health, labour and welfare, 2009). physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students h. ikei*, c. song*, m. igarashi*, t. namekawa**, y. miyazaki*(1) * center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa, chiba 277-0882, japan. ** chiba prefectural kashiwanoha senior high school, 6-1 kashiwanoha, kashiwa, chiba 277-0882, japan. key words: heart rate variability, minors, house plant, stress reduction, visual stimuli. abstract: with lifestyles in modern society becoming increasingly stressful, there is growing interest in the physiological relaxing effects of the natural environment. particular interest has been paid to the physiological effects of indoor plants, however no studies have revealed the effects of such visual stimulation on minors. in this study 85 (41 male and 44 female; 16.5±0.9 years; mean±sd) students were exposed, or not as control, to a typical foliage plant, dracaena (dracaena deremensis; lemon lime), for 3 min. physiological indices included heart rate variability (hrv) and pulse rate, using an accelerated plethysmography at the fingertip, were collected continuously during the experiments. the results indicated that the high frequency component (hf), a general index of parasympathetic nervous activity, was significantly higher; the low frequency component [lf/(lf+hf)], a general index of sympathetic nervous activity, was significantly lower; and the pulse rate was significantly lower. after exposure, or not, the subjects completed a questionnaire as psychological evaluation. a 13-point rating scale was used for following parameters: “comfortable-uncomfortable,” “relaxed-awakening,” and “natural-artificial.” results of the study showed that subjects felt more comfortable, relaxed and natural after visualizing the dracaena plants. overall, the physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students is confirmed. adv. hort. sci., 2014 28(2): 111-116 (1) corresponding author: ymiyazaki@faculty.chiba-u.jp received for publication 31 march 2014 accepted for publication 29 july 2014 112 flowers and foliage plants are common natural surroundings that can be incorporated into the school and home. we previously conducted surveys to evaluate the physiological effects of visual stimulation with fresh rose flowers in high school students (ikei et al., 2013), middle-aged and elderly medical staff (komatsu et al., 2013), and office workers (ikei et al., 2014). the results showed enhanced parasympathetic nervous activity (ikei et al., 2013; komatsu et al., 2013; ikei et al., 2014), suppressed sympathetic nervous activity (ikei et al., 2013), and decreased pulse rates (komatsu et al., 2013) during visual stimulation. it was reported in a previous study that natural views from hospital windows or the presence of indoor plants hasten the recovery of patients after surgery and decrease systolic blood pressure (park and mattson, 2009). these effects have also been studied in a classroom, demonstrating that the ambience of indoor space can be improved by including foliage plants (doxey et al., 2009), as reflected by enhanced feelings of comfort among the students (han, 2009). however, there have been no reports on the influence of visual stimulation with foliage plants on heart rate variability (hrv) and subjective feelings in minors. therefore, this study was conducted to examine the effects of exposure to the foliage plant dracaena (ministry of agriculture, forestry and fisheries, 2008) on physiological and psychological variables (hrv, pulse rate, and subjective responses) in high school students. 2. materials and methods the experiments were conducted in a classroom of the chiba prefectural kashiwanoha senior high school in october 2012. the room temperature was approximately 25.9°c, relative humidity approximately 52.6%, and illumination approximately 900  lux. eighty-five high school students (41 male and 44 female; 16.5±0.9 years; mean±sd) participated in the experiment. the study was conducted with the approval of the ethics committee of the center for environment, health and field sciences, chiba university. all subjects provided written informed consent. three dracaena plants (dracaena deremensis, lemon lime), 55-60 cm high, were placed at intervals of 8 cm on a desk in front of each subject (test situation). the distance from the subject’s eyes to the plants was approximately 55 cm, and they were adjusted according to the height of the subjects. no exposure to foliage plants was used as the control condition. before visual stimulation, the plants and the control were covered by a corrugated cardboard box (rest condition). figure 1 shows the study protocol, figure 2 the rest condition, and figure 3 the visual stimuli (the dracaena fig. 1 study protocol for testing the physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students. fig. 2 the rest condition. fig. 3 -the visual stimulation condition. 113 plants or control). after viewing the cardboard box at rest in a sitting posture for 1 min (fig. 2), the subject was exposed to the plants or control for 3 min (fig. 3). after the experiments, each subject completed a questionnaire. the order of stimuli was counterbalanced among subjects. hrv and pulse rate were measured as physiological indices. hrv was calculated by spectral analysis of the coefficient of variation of the a-a interval on an accelerated plethysmograph (apg; artett, u-medica inc., osaka, japan). previous studies have reported that the a-a interval on an apg and r-r interval on an electrocardiogram are strongly correlated (takada and okino, 2004; takada et al., 2008). the sampling frequency was set at 1000 hz. the maximum entropy method was used for frequency analysis, and variance of the low frequency (lf; 0.04-0.15 hz) and high frequency (hf; 0.15-0.40 hz) components were calculated. the lf/(lf+hf) ratio for r-r interval variability was also assessed. the hf component was used as an index of parasympathetic nervous activity and the lf/(lf+hf) ratio was used as an index of sympathetic nervous activity (weise and heydenreich, 1989; cacioppo et al., 1994; sawada et al., 1997). generally, parasympathetic nervous activity is enhanced during relaxation and sympathetic nervous activity is enhanced at the time of awakening and stress (ackerknecht, 1974). therefore, the pulse rate was converted by dividing 60 by the a-a interval on apg. the hrv and pulse rate data were collected continuously during the 3-min experiments and averaged. in addition, the subjects subjectively evaluated the emotional effects of the dracaena plants and control using the modified semantic differential (sd) method (osgood et al., 1957), which uses three pairs of adjectives on 13 scales, including “comfortable-uncomfortable,” “relaxedawakening,” and “natural-artificial.” the statistical package for social sciences software (v20.0, ibm corp., armonk, ny, usa) was used for all statistical analyses. a paired t-test was used to compare the physiological responses to visual stimulation with dracaena plants or control, followed by holm correction of the changes in each 1-min average, while the wilcoxon signed rank test was used to compare the psychological responses to visual stimulation with dracaena plants or control. for both conditions, one-sided tests were used because of the hypothesis that humans are relaxed by visual stimulation with foliage plants. statistical differences were considered significant at p<0.05. 3. results significant differences were found in the values of the hf component and the lf/(lf+hf) ratio of hrv between dracaena and the control. figure 4 shows the hf component of hrv, an estimate of parasympathetic nervous activity. hf between 0 and 1 min was 1210.7±120.3 (mean±se) ms2 during the test condition and 1032.9±95.2 ms2 during the control condition, showing a significant increase of 17.2% (p<0.05) (fig. 4a) during the test condition. similarly, hf between 1 and 2 min was 1052.0±109.3 ms2 during the test condition and 893.1±87.1 ms2 during the control condition, showing a significant increase of 17.8% (p<0.05) (fig. 4a) during the test condition. there was no significant difference in hf between 2 and 3 min. the overall hf during the 3-min experiment was 1083.9 ± 101.5 ms2 in the test condition and 954.8±83.5 ms2 in the control condition, showing a significant increase of 13.5% (p<0.01) (fig. 4b) with the test condition, indicating that parasympathetic nervous activity was significantly higher during dracaena plant exposure. the results of the lf/(lf+hf) ratio, a marker of sympathetic nervous activity, are shown in figure 5. for 1-min segment analysis, the lf/(lf+hf) ratio between 0 and 1 min was 0.47±0.02 during the test condition and 0.52±0.02 during the control condition, showing a significant decrease of 9.6% (p<0.05) (fig. 5a) with the former. fig. 4 the 1-min averages and overall mean high frequency component (hf) of heart rate variability (hrv) during visual stimulation with dracaena plants and control. (a) changes in each 1-min average hf value over 3 min. (b) overall mean hf values. n=85. mean±se. *p < 0.05, **p < 0.01 as determined by the paired t-test, holm correction. fig. 5 the 1-min averages and the overall mean lf/(lf+hf) ratio of heart rate variability. (hrv) during visual stimulation with dracaena plants and control. (a) changes in each 1-min average lf/(lf+hf) value over the 3 min. (b) overall mean lf/(lf+hf) values. n=85. mean±se. *p<0.05 as determined by the paired t-test, holm correction. 114 similarly, between 1 and 2 min, the ratio was 0.48±0.02 during the test condition and 0.54±0.02 during the control condition, showing a significant decrease of 11.1% (p<0.05) (fig. 5a) during the test. no significant difference was observed between 2 and 3 min. for the entire 3-min duration, lf/(lf+hf) was 5.6% lower during the test condition than during the control condition (dracaena: 0.51±0.02, control: 0.54±0.02; p<0.05) (fig. 5b), indicating that sympathetic nervous activity was significantly lower during dracaena plant exposure. clear differences in pulse rate were observed between dracaena and control exposure. the pulse rate between 0 and 1 min was 71.9±1.2 beats/min during the test condition and 72.7±1.2 beats/min during the control condition, showing a significant decrease of 1.1% (p<0.05) (fig. 6a) during the test condition. similarly, between 1 and 2 min, the pulse rate was 72.6±1.2 beats/min during the test condition and 73.3±1.2 beats/min during the control condition, showing a significant decrease of 1.0% (p<0.05) (fig. 6a) during dracaena plant exposure. no significant difference was observed between 2 and 3 min. the mean pulse rate was 0.1% lower during the test condition than during the control condition (dracaena: 72.4±1.2 beats/min, control: 73.0±1.2 beats/min; p<0.05) (fig. 6b). the subjective evaluation data clearly showed the effect of the two different visual stimuli on the psychological states of participants. participants felt significantly more comfortable (dracaena: “slightly comfortable”; control: “indifferent”; p<0.01) (fig. 7, left), relaxed (dracaena: “slightly relaxed”; control: “indifferent”; p<0.01) (fig. 7, center), and natural (dracaena: “slightly natural”; control: “indifferent”; p<0.01) (fig. 7, right) in the dracaena condition than in the control condition after stimuli. 4. discussion and conclusions an improved ambience resulting from the placement of foliage plants in a classroom may significantly enhance physiological relaxation and mental health in high school students, as shown in the present investigation. relatively brief (3 min) visualization of foliage plants resulted in significantly enhanced parasympathetic nervous activity (13.5%), suppressed sympathetic nervous activity (-5.6%), and decreased pulse rate (-0.8%), results which are consistent with previous studies involving the visualization of a forest scene (park et al., 2010; tsunetsugu et al., 2010; park et al., 2011). furthermore, our findings are consistent with those of our previous report on the calming effects of roses in high school students (ikei et al., 2013), where similar physiological responses were found. also in line with our findings, visual stimulation with fresh roses enhanced parasympathetic nervous activity and significantly decreased the heart rate in middle-aged and elderly medical staff (komatsu et al., 2013), while it enhanced parasympathetic nervous activity in office workers (ikei et al., 2014). according to our analysis of the three questionnaires, the subjects in the present study felt more comfortable, relaxed, and natural after visualizing the dracaena plants. this result is consistent with that of our previous report on the calming effects of roses (ikei et al., 2013; komatsu et al., 2013; ikei et al., 2014). the results of this study support the hypothesis that placement of foliage plants in classrooms can induce a relaxing effect, improve physiological activity, and improve the psychological state in high school students. because of the growing interest in mental health in modern times (murray and lopez, 1996), the psychological benefits of indoor plants are expected to play an important role in the promotion of mental health in the future. however this study had limitations. first, we only evaluated hrv. thus, the results cannot be interpreted in terms of a complete physiological evaluation. other experimental indices such as brain activity and stress hormone levels should be assessed to determine the effects of visual stimulation with natural objects, such as foliage plants on human response. second, only dracaena plants were used. fig. 6 the 1-min averages and overall mean pulse rate during visual stimulation with dracaena plants and control. (a) changes in each 1-min average pulse rate over the 3 min. 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“shinrin-yoku” (taking in the forest atmosphere or forest bathing). environ. health prev. med., 15: 27-37. weise f., heydenreich f., 1989 effects of modified respiratory rhythm on heart rate variability during active orthostatic load. biomed. biochim. acta, 48: 549-556. 187 1. introduction an increase in fruit quality is a key objective of fruit tree cultivation, while keeping in mind at the same time the relevance of production cost reduction and environmental issues. the citrus industry is increasingly oriented toward upgrading groves, discarding obsolete plantings and introducing new rootstock/scion combinations which are more tailored to local environmental conditions. moreover, increases in production costs, without a proportionate profit increase, and innovation at the technological level have led to many changes in citrus orchard management and the updating of cultural techniques. new plantings have been realized with regular planting distances for a fully mechanised approach to all cultural practices. in this context, technical evolution also includes pruning, essential for healthy and fruitful orchard management, but without the clear push towards mechanisation as in other countries. this cultural technique as well as all other practices, even though respecting plant physiology, has to be evaluated according to the economic impact. furthermore, it is wise to consider the manifold factors that affect the final result, such as rootstock/scion combination, tree age and development, planting distance, soil and climate conditions. citrus groves in their former conception, although still present in many citrus cultivation areas in italy, were highdensity based (more than 800 plants ha-1) with narrow planting distances. citrus growers were forced to carry out frequent pruning on bearing plants, repeated in spring and at the end of summer, as the only available way for both high plant density and light penetration between and within plant canopies to coexist (rebour, 1971). this situation led to the development of trees with high scaffold (frequently more than 1 m high), usually lacking in skirt and with poor yield. the transition to modern citrus production, based on greater planting distance and average densities of 416 plants ha-1, at least for standard grafting combinations, has led to a new concept of pruning, which in turn has meant substantial changes in the management of this technique, with sometimes substantial negative effects on citrus production (intrigliolo, 1998). similarly to other fruit tree cultivations (giacalone et al., 2004; neri and sansavini, 2004; peano and giacalone, 2004; ventura and sansavini, 2005), pruning practices in citriculture are important to support plant health and reduce stress in order to reach an acceptable balance between vegetative and reproductive activities, a key factor in many stages of citrus grove development. citrus bearing trees in semi arid environments have main shoot growth flushes during the year (spring, summer and autumn flush), with growth stasis periods overlapped with periods of higher and lower temperatures. only for lemon (c. limon (l.) bern) does flowering occur during all growth flushes, whereas for other citrus species flowering is mainly bound to spring flush. flowers, solitary or in inflorescence, can be terminal or axillary and are normally produced on one-year modern trends of citrus pruning in italy f. intrigliolo, g. roccuzzo* centro di ricerca per l’agrumicoltura e colture mediterranee, cra-acm, corso savoia, 190, 95024 acireale (ct), italy. key words: fruit quality, mechanical pruning, rootstock, tree habit. abstract: the citrus industry is at present increasingly oriented toward upgrading groves, discarding obsolete plantings and introducing new rootstock/scion combinations which are more tailored to local environmental conditions. a rise in production costs (but without a proportionate increase in profit) together with technological innovation have both led to many changes in citrus orchard management, and consequently there have been changes in cultural techniques. many cultural and edaphic factors influence the choice of pruning frequency and intensity. the main goal of pruning is to reach a useful balance between yield and growth, and to reduce cultivation costs. drastic pruning in young citrus trees with a vigorous vegetative habit causes an excess of shoot growth, thereby extending the juvenile stage and delaying fruiting. adaptation of the technique to local conditions in bearing groves (species, cultivars, planting distances, etc.) is mandatory for pruning optimization. a fully mechanised approach to pruning leads to substantial changes in citrus grove management and sometimes to negative effects on yield. functional integration of mechanical and assisted pruning seems to be the right choice for the italian citrus industry. adv. hort. sci., 2011 25(3): 187-192 * present address: fruit tree research centre (cra-fru), via di fioranello, 52, 00134 rome, italy. received for publication 20 may 2011 accepted for publication 9 september 2011 188 shoots. in young citrus trees shoots are normally vegetative, since the productive stage begins with flower emission mainly on lateral drooping shoots. in citrus species, like in other tree fruit species, bud differentiation occurs in response to chilling temperatures and mainly depends on interactions at physiological and nutritional level (garcia-luis et al., 1995). drastic pruning in young citrus trees with a vigorous vegetative habit causes an excess of shoot growth, thereby extending the juvenile stage and delaying the beginning of fruiting. in adult trees heavy pruning of branches, twigs and leaves means the removal of reserve substances (i.e. carbohydrates, nitrogen), thus leading to serious reductions in plant growth and overall development. furthermore, in this way pruning in adult trees favors excessive sucker production especially in upright-growing species. on the contrary, in senescent or declined trees light or even hard thinning may be useful to promote growth and healthy fruitwood. 2. pruning of young trees it is essential to take care of citrus trees during the juvenile stage to obtain a balanced scaffold with three to four principal branches developing at 30-50 cm from the collar (fig. 1). if citrus trees are correctly managed in the nursery (cut back or headed) they require little pruning once in the field. trees will grow naturally and they will take the growth habit typical of the species or cultivar. in this case trees assume a drooping shape, ranging from spheroid to ellipsoid (fig. 2). at this stage the most common and severe mistakes are the removal of apical or more drooping branches. in such cases the development of both upper and lower part (the first to produce) of the tree are limited. however, pruning during the first period should be limited to removing an occasionally unwanted branch or buds on the rootstock, to regulate the final scaffold and reduce future severe cuts. 3. pruning mature trees for many years after transplanting citrus trees require no relevant pruning. it is not easy to determine a general rule for the beginning and frequency of regular pruning since this practice depends on many factors: species, cultivar, planting distance, soil and climate conditions and, more relevant, growth status (crowding, presence of deadwood in the internal part, upright shoots exhaustion) and its balance with fruitfulness. pruning frequency can be annual or long-standing, with frequency and severity closely linked. longer time intervals imply more drastic pruning, with wider wounds and a subsequent massive influence on plant growth. in a field trial on full-bearing trees of tarocco orange (citrus sinensis osbeck) the effects of hand pruning with annual, biennial and quadrennial frequency were evaluated (calabretta et al., 2008). quadrennial frequency of pruning showed a decline of fruit quality parameters (above all for average fruit weight), even though they were linked to higher yield and shortening of work time. biennial frequency of pruning showed the best balance fig. 1 young ‘tarocco’ orange tree. fig. 2 ‘navelate’ orange tree with drooping canopy and full skirt. 189 as far as work time (costs), yield and fruit quality were concerned. in the case of aged trees, low in vigor or presenting other problems, it is wise to increase the frequency (intrigliolo, 1984; intrigliolo, 1998). for wider planting distances progressive exhaustion of the internal part of the canopy can be balanced by lateral and vertical expansion, without any influence on yield. therefore in these conditions pruning initially plays a minor role and it could be delayed. on the contrary if pruning is carried out at maturity stage of the orchard, in conjunction with the right fulfillment of all other cultural practices, it becomes necessary to sustain growth vigor, high yield and fruit quality standards. it is not easy to choose the right time to start regular pruning. in the case of a delayed start, the citrus orchard could grow old prematurely, while the opposite case could cause an increase in costs and severe yield reduction (intrigliolo, 1998). another important factor is the timing of pruning. intervention is often undertaken without distinction from january to june, and sometimes is repeated at the end of august or beginning of september to eliminate vigorous upright suckers, due to severe spring cuts. in any case, the right pruning time is extremely variable, depending on species, variety, climate conditions and severity of the previous treatment. early varieties are usually pruned before late ones, either because of earlier harvest times or because of an absence of frost risk. frosts are the main deterrent to early pruning (fisher, 1977; phillips, 1980 b; cutuli, 1985; intrigliolo, 1986 b). as a matter of fact, the removal of the outer part of foliage makes trees more subject to frost injuries since it stimulates the emission of new soft shoots that can be easily damaged by low temperatures. severe treatments should be properly scheduled and deferred until after the juvenile stage: moderation is still the key word. light pruning is advisable to provide deadwood removal and to increase light interception in the internal part of the canopy and between rows. this improves yield and fruit quality, especially fruit size, thus making other cultural techniques easier and less expensive (intrigliolo, 1984; calabretta et al., 2008). studies on pruning timing showed the positive effects of early treatments (end of winter to early spring) in comparison with late summer ones (cameron and hogdson, 1943; milella, 1967; turpin, 1973; fisher, 1977; fucik, 1979). if pruning takes place before spring flush, removal of new vegetation is prevented (turpin, 1973). early pruning is thus highly recommended for skeletonisation and as a general rule for trees low in vigor or weakened by biotic or environmental stresses (cameron and hogdson, 1943; bevington and bacon, 1976; phillips, 1980 a, 1980 b). vigorous trees, on the other hand, will react improperly with extra shooting and will lose fruit bearing surface, as reported by bevington and bacon (1976). phillips (1980 a) reported that light pruning in july and august implied useful fruit thinning, with the consequence of increased fruit size, especially in case of top dressing. as far as yield is concerned, fucick (1979) reported higher levels in texas with grapefruit (c. paradisi macfadyen) resulting from december treatment, whereas in australia bevington and bacon (1976) and bevington (1980), working with valencia late oranges, found similar levels comparing summer and autumn treatments. tree phenological stage as well as scion/rootstock combination and species/variety habit are decisive factors in determining pruning effects. ‘moro’ in the bloody group and many accessions in the navel group represent low vigor orange cultivars; low vigor trees are found among a few lemon cultivars, most bergamot (c. bergamia rissi) and citron (c. medica l.) (fig. 3) cultivars. given the vegetative habit of these genotypes, pruning has the main function of increasing air and light penetration in the internal part of the canopy, avoiding backcuts that would thicken the tree and preferring a balanced thinning. in the case of mandarin (c. reticolata blanco) (fig. 4) and its hybrids these treatments are essential, the canopy being extremely dense due to huge branch production. fig. 3 ‘navelina’ orange tree. fig. 4 ‘avana’ mandarin tree. 190 satsumas (c. unshiu marcovitch) and many clementines (c. clementine hort.) (fig. 5) show an intermediate growth habit. expanded growth habit is normally shown by bloody orange ‘sanguinello’ and ‘tarocco’ clones, with a tendency to upright growth habit in nucellar lines (fig. 6) and in triploids, such as ‘tacle’ and ‘alcantara’. with the aim of obtaining virus-free and viruslikefree accessions, nucellar progeny strategy has been largely adopted in breeding projects in italy. these lines are characterized by their large size (although with some exceptions) thus conditioning plant spacing and orchard management. in this situation concerns arise about the extremely reduced density and about pruning practices, that should be reduced in number and intensity. branches shortening cuts should be preferred mainly in the first years after planting, even though this practice induces very vigorous growth reactions and delay of productive stage. these effects are linked to the increase of costs for future pruning, harvest and other cultural practices. in order to increase yield and reduce the costs of cultural practices (especially pruning), recovery strategies based on micrografting technique should be encouraged, as well as the adoption of new rootstocks able to reduce tree vigor (russo et al., 2011). ‘femmiminello’ lemon trees are characterised by upright irregular shoots (fig. 7), with pronounced apex dominance; young trees show long, thin shoots which are weak and easily prone to breakage. for these trees it is necessary to shorten the branches to reduce plant height, favoring their strength and thus their stability. in late summer, pruning treatments play a prominent role in the eradication of phoma tracheiphila (kanc et ghik) infections, making clear the necessity for tree removal in extreme situations. more extensive pruning (i.e. skeletonisation) is essential in cases of old, decadent trees or in case of damage due to environmental or biotic stresses. in these situations removal of deadwood is useful or absolutely necessary for tree rejuvenate, thus leading to normal growth and production conditions. 4. mechanical pruning in citrus orchard management, pruning is increasingly oriented toward greater levels of mechanization with the aim of combining cost reduction with a proper balance between yield and plant growth. research activity in this field started in italy at the end of 1970s with several experiences of mechanical pruning (giuffrida et al., 1979; blandini et al., 1981; raciti et al., 1981; intrigliolo et al., 1986) with the integration of internal thinning of deadwood and upright shoot removal, and aided pruning by means of pneumatic saws and clippers (intrigliolo and barbagallo, 1987; schillaci, 1988). fig. 5 clementine tree. fig. 6 ‘tarocco’ clone nl 57-e-1 orange tree. fig. 7 ‘femminello’ lemon tree. 191 experiments carried out in italy on mechanic pruning in citrus (giuffrida et al., 1979; blandini et al., 1981; raciti et al., 1981; giametta, 1983; spina et al., 1984; intrigliolo, 1986 a; intrigliolo et al., 1986; intrigliolo et al., 1988; intrigliolo and giuffrida, 1990; raciti et al., 1991) gave largely positive results. trials were carried out on several species and cultivars under different environmental and cultivation conditions, utilizing different equipment and operating systems mainly associated with a traction engine. up till now, however, hand pruning seems to be the most widespread approach in italy and in other advanced citrus cultivation areas like spain (agustì, 2003). frequency and intensity of mechanical pruning represent the key choices to attain high yields and delayed tree senescence (zaragoza-adriaensens and alonso cabo, 1981; intrigliolo, 1986 b; raciti and intrigliolo, 1989). results of a two-year trial on ‘tarocco’ orange trees mechanically pruned with the same intensity in april, june and august, showed that full summer treatments were useful to control plant growth, whereas early treatments stimulated the spring flush (intrigliolo and giuffrida, 1990). yield and fruit quality were only slightly influenced by treatment time. the main purposes of fully mechanized pruning are the fulfillment of the tree’s physiologic demand and the massive reduction of production costs. mechanical pruning is not a selective or thinning practice, but it follows rigid patterns by cutting trees back vertically (hedging) (fig. 8) or removing their tops (topping) (fig. 9) and it is adapted to wide planting distances. thus, the grove is sufficiently open for the passage of equipment for spraying and other cultural practices, reducing shady areas and removing dead or decadent wood (intrigliolo, 1986 b). pneumatic tools reduce the physical effort of workers, amplifying their performance both by replacing hand pruning with traditional tools and complementing mechanical pruning. the economic convenience of pneumatic tool utilization increases as the time needed for the intervention increases. assisted pruning loses its economic convenience in comparison to traditional pruning (intrigliolo and barbagallo, 1987). the reduction of working time accounts for up to 30-40% for assisted pruning, 90% for mechanical pruning and an average of 60-70% when integrated with the latter (fig. 10) (intrigliolo, 1986 a, 1998). the functional integration of mechanical and assisted pruning could be the best way to achieve useful results, hopefully in economic and agronomic terms, at least until further profit loss forces growers toward full mechanizaten. even though experimental results with fully mechanized pruning have to date shown limited effects, it seems that in the near future it will spread to largeand medium-sized citrus orchards. in the traditional italian citrus industry, with terrace cultivation and small-sized farms, for many years pruning has been carried out using pneumatic tools permitting workers to use their own judgment in terms of frequency and limiting costs.fig. 8 mechanical hedging and topping done at the same time. fig. 9 mechanical topping. fig. 10 work time in different kinds of pruning. 192 references agustì m., 2003 citricultura. mundi-prensa, madrid, spain, pp. 422. bevington k.b., 1980 response of valencia orange trees in australia to hedging and topping. proc. fla. state hort. soc., 93: 65-66. bevington k.b., bacon p.e., 1976 response of valencia orange trees to hedging. australian citrus news, 52: 2. blandini g., petrone f., sisinna s., 1981 trials of mechanical pruning machines in italian citriculture. proc. int. soc. citric., vol. i: 180-185. calabretta m.l., giuffrida a., intrigliolo f., 2008 la periodicità della potatura, fattore determinante per la qualità delle arance. rivista di frutticoltura e di ortofrutticoltura, 70(1): 2-5. cameron s.h., hodgson r.w., 1943 effect of time of pruning on the rate of top regeneration of valencia orange trees. proc. am. soc. hort. sci., 42: 280-282. cutuli g., 1985 potatura, pp. 423-451. in: spina p. (ed.) trattato di agrumicoltura. edagricole, bologna, italy, pp. 552. fisher i.e., 1977 hedging and topping. the citrus industry, 57: 6-12. fucik i.e., 1979 long term responses of texas grapefruit trees to hedging and topping. the citrus industry, 59: 2527. garcia-luis a., fornes f., guardiola j.l., 1995 leaf carbohydrates and flower formation in citrus. j. amer. soc. hort. sci., 120(2): 222-227. giacalone g., la iacona t., vittone g., 2004 valutazione di parametri produttivi e qualitativi in impianti di melo condotti con differenti metodi di potatura. proc. vii giornate scientifiche soi, pp. 763-765. giametta g., 1983 prove sperimentali di potatura meccanica degli agrumi. macchine e motori agricoli, 7: 7-20. giuffrida a., melita e., romeo m., 1979 prove di potatura meccanica degli agrumi. rivista di frutticoltura, 41: 23-28. intrigliolo f., 1984 la potatura degli agrumi. l’informatore agrario, 40(22): 49-53. intrigliolo f., 1986 a la potatura meccanica e agevolata in agrumicoltura. l’informatore agrario, 42(9): 93-105. intrigliolo f., 1986 b potatura meccanica e tradizionale a seguito di danni da gelo su arancio cv. ‘sanguinello moscato’. proc. ‘il recente contributo della ricerca allo sviluppo dell’agrumicoltura italiana’, cagliari 29 april-3 may, pp. 267-277. intrigliolo f., 1998 differenti frequenze di potatura meccanica su piante di arancio. atti giornate scientifiche soi, 1: 157-158. intrigliolo f., barbagallo a., 1987 confronto in diverse condizioni di campo fra potatura tradizionale ed agevolata in agrumicoltura. l’informatore agrario, 43(25): 69-72. intrigliolo f., giuffrida a., 1990 interventi di potatura meccanica sull’arancio cv. ‘tarocco’ effettuati in diverse epoche. frutticoltura, 52(5): 91-95. intrigliolo f., raciti g., scuderi a., 1988 mechanical and aided pruning combined with tree removal of nucellar ‘tarocco’ orange. proc. sixth int. citrus congress, vol. ii: 947-952. intrigliolo f., raciti g., scuderi a., giuffrida a., 1986 prove quadriennali di potatura meccanica sul mandarino cv. ‘avana’. proc. ‘il recente contributo della ricerca allo sviluppo dell’agrumicoltura italiana’, cagliari 29 april-3 may, pp. 297-307. milella a., 1967 un quadriennio di osservazioni sull’epoca di potatura dell’arancio. studi sassaresi, sez. iii, ann. fac. agraria, 1: 1-15. milella a., 1980 potatura. in: gli agrumi, reda, pp. 165. neri d., sansavini s., 2004 attualità della potatura nella frutticoltura intensiva. rivista di frutticoltura, 1: 14-23. peano c., giacalone g., 2004 potatura del melo: dalla taille longue all’extinction. proc. vii giornate scientifiche soi, pp. 196-198. phillips r.l., 1980 a hedging and topping practices for florida citrus. the citrus industry, 55: 5-10. phillips r.l., 1980 b rejuvenation pruning of citrus. citrus and vegetable magazine, 44: 6-22. raciti g., cutuli g., intrigliolo f., giuffrida a., 1991 indagine sull’influenza delle tecniche colturali sul mal secco degli agrumi. 2a nota: conduzione del terreno e potatura. l’informatore agrario, 47(22): 47-49. raciti g., intrigliolo f., 1989 nuovi orientamenti sulla potatura degli agrumi. rivista di frutticoltura, 51(1): 2530. raciti g., spina p., scuderi a., intrigliolo f., 1981 three years of mechanical pruning of citrus in italy. proc. int. soc. citriculture, vol i: 175-180. rebour h., 1971 gli agrumi. edagricole, bologna, italy, pp. 371. russo g., reforgiato recupero g, recupero s., intrigliolo f., 2011 la scelta dei nuovi portinnesti i risultati delle ricerche. rivista di frutticoltura, 73(1-2): 6264. schillaci g., 1988 la potatura agevolata in agrumicoltura. macchine e motori agricoli, 4: 55-64. spina p., giuffrida a., melita e., 1984 comparative trials of mechanical and aided pruning. proc. int. soc. citriculture, vol. i: 106-109. turpin i.w., 1973 the place of hedging in the management of citrus orchards. australian citrus news, 49: 6-7. ventura m., sansavini s., 2005 tipologia di rami a frutto e qualità delle mele cvs braeburn, golden delicious e jonagold. rivista di frutticoltura, 67(11): 52-60. zaragoza-adriaensens s., alonso-cabo e., 1981 la frecuencia de la poda manual de los agrios. primeros resultados. anales del instituto nacional de investigaciones agrarias. serie: agrìcola, nùm. 15, separata nùm. 11. 103 1. introduction cut foliage represents an important part of the floricultural industry. they are used as fillers in floral bouquets especially for cut flowers without leaves such as gerberas. the vase life of cut foliage is usually longer than cut flowers, but it may become shorter when they are stored for long periods. storage methods, wet or dry, affect the vase life of cut foliage (ferrante et al., 2002 a) and play a crucial role for preserving quality: it is important, in particular, to delay the symptoms of leaf senescence considering that the intensity of leaf color is the most important quality parameter (pacifici et al., 2007) and closely associated with the marketability of ornamental cut foliage. during postharvest, quality losses of cut foliage are essentially represented by leaf senescence. the most common symptoms are leaf yellowing, leaf desiccation and/or leaf abscission, and weight losses (pacifici et al., 2007). leaf yellowing is common in many cut flowers such as alstroemeria, chrysantemum, lilies, tulips, but also in some cut foliage such as cut danae racemosa foliage (van doorn et al., 1992; ferrante et al., 2002 b, 2003, 2005). postharvest treatments with cytokinins are able to delay leaf yellowing in many cut flowers, but a substituted phenylurea, the thidiazuron (n-phenyln’-1,2,3-thiadiazol-5-yl urea, tdz), with cytokinin-like activity, has been found to be very effective in many cut flowers sensitive to leaf yellowing (ferrante et al., 2002 b); application of low concentrations of tdz delayed leaf yellowing (ferrante et al., 2002 b, 2003; mutui et al., 2005; jiang et al., 2008). during the postharvest chain, another important problem for cut foliage is weight loss. since these ornamental items are sold by weight, any weight loss is directly translated into economic loss. cut foliage is essentially composed of branches with leaves, therefore, weight losses are due to water losses by transpiration. during postharvest, the hydraulic conductance of the branches or cut stems progressively declines due to vessel blockages from bacteria growth, embolism, etc. (van doorn, 1997). cut flowers and branches treated with glycerol are able to enhance osmotic potential and absorb great amounts of water, avoiding reduction of the water potential. glycerol has been used to preserve ornamental plant materials (dubois and joyce, 1992) as it causes a greater water removal due to an increment of osmotic potential. in this way, glycerol treatment reduces leaf weight loss, as well as water loss, which reflects on extending vase life (shanan and shalaby, 2011). in this study, the possibility to extend as much as possible the storage of cut foliage in a cold room at 4°c for 60 days was evaluated with the aim of maximizing cut danae availability on the market. this outlook has practical value for some cooperatives and commercialization companies as extention of the storage period could cover periods when production is lacking (pacifici et al., 2013). evaluation of postharvest storage and treatments in cut ruscus foliage r. bulgari(*), m. negri, a. ferrante dipartimento di scienze agrarie e ambientali, produzione, territorio, agroenergia, università degli studi di milano, via celoria 2, 20133 milano, italy. key words: danae, glycerol, leaf senescence, thidiazuron. abstract: danae racemosa (l.) moench is an important cut foliage in the ornamental market. during postharvest, leaf senescence symptoms are leaf yellowing, weight loss and/or abscission of leaves. the aim of this work was to evaluate the effect of pre-treatments for 24 h with glycerol and thidiazuron (tdz) on danae vase life before and after storage. treatments were applied in vase water containing glycerol 0.1, 1 or 10 mm, 10 µm tdz and a combined treatment of 10 µm tdz plus 10 mm glycerol. the effect of treatments was evaluated through the determination of vase life, chlorophyll content, chlorophyll a fluorescence parameters, sucrose and total sugars content. the cut foliage were stored in sealed plastic bags and placed in a dark room at 4°c for two months. the vase life, before and after storage, was determined in a controlled chamber set to 20°c and rh 50-60% with a light intensity of 20 µmol m-2 s-1. results demonstrated that 10 µm tdz plus 10 mm glycerol was the most effective treatment in maintaining quality during vase life of stored and not stored cut foliage. adv. hort. sci., 2015 29(2-3): 103-108 (*) corresponding author: roberta.bulgari@unimi.it received for publication 26 september 2014 accepted for publication 26 march 2015 104 adv. hort. sci., 2015 29(2-3): 103-108 2. materials and methods plant materials cut branches of danae racemosa (l.) moench. [=ruscus racemosus l.] were provided by a commercial company (floratoscana, pescia, pt, italy) and transported to the postharvest laboratory of the department of agricultural and environmental sciences of the university of milan. cut branches were trimmed to a length of 70 cm to provide homogenous samples for the experiments. chemical treatments and storage cut foliage was pre-treated for 24 h with a solution containing 0.1, 1 or 10 mm glycerol, 10 µm tdz and a combined pre-treatment of 10 µm tdz plus 10 mm glycerol, while control samples were held in distilled water. after pretreatments, the plant material was stored in vases containing distilled water at 20°c and 50-60% relative humidity. each treatment was composed of eight replicates. a part of the cut branches treated with 10 mm glycerol, 10 µm tdz and combined treatment 10 µm tdz plus 10 mm glycerol were stored for 60 days in polyethylene bags at 4°c. the gas composition in terms of oxygen and carbon dioxide percentages was monitored by means of a “binder combigas ga-m3” (from binder, d) portable gas analyzer equipped with an electrochemical cell for oxygen and co 2 measurement and an infrared dispersion cell for nh 3 determination. vase life evaluation after storage, cut branches were transferred to a growth chamber with controlled temperature (20°c), relative humidity (60-70%), and light intensity (10-15 μmol m-2 s-1 ppfd for 12 h per day). non-stored cut branches (control) were used for vase life assessment immediately after harvest. ten branches for each storage period were placed in individual bottles for vase life analysis. vase life was determined as the number of days from when branches were placed in the water to the onset of leaf senescence, yellowing or abscission. chlorophyll determination and chlorophyll a fluorescence chlorophyll content was measured using a chlorophyll meter (cl-01, hansatech, uk) that provides an indicator of green color of leaves. this device determines relative chlorophyll content in vivo using dual wavelength optical absorbance (620 and 940 nm wavelength). chlorophyll a fluorescence was measured with a portable handy plant efficiency analyzer (pea, hansatech, uk). leaves were dark-adapted for 30 min. using a leaf clip (4 mm diameter), a rapid pulse of high intensity light of 3000 μmol m−2 s−1 (600 w m−2) was absorbed by the leaf inducing fluorescence, which was measured by the sensor. the fluorescence parameters were calculated automatically. jip analysis was performed to determine the performance index (pi). sugars determination in order to quantify sucrose and total sugars content, about 0.5 g of leaves were ground in 10 ml of distilled water. the homogenate was centrifuged at 10000 rpm for 5 min. for sucrose determination, 0.2 ml of extract were added to 0.2 ml naoh 2n and incubated at 100°c for 10 min; then 1.5 ml of resorcinol were added and incubated at 80°c for 10 min. a resorcinol solution was prepared by adding 35 mg of resorcinol and 90 mg of thiourea in 250 ml hcl 30%, mixed with 25 ml of acetic acid and 10 ml of distilled water. samples were cooled at room temperature and spectrophotometer readings were performed at 500 nm. a calibration curve was built with sucrose standards at 0, 0.5, 1, 1.5, 2 mm. total sugars were calculated by anthrone method: 0.2 g of anthrone were melted in 100 ml of h 2 so 4 and shacked for 30-40 min; 0.2 ml of diluted extract was added to 1 ml of anthrone solution, cooled in ice for 5 min and mixed thoroughly. samples were incubated at 95°c for 5 min and then cooled on ice. absorbance readings were measured at 620 nm and a calibration curve was built with glucose standards at 0, 1, 2, 3 and 4 mm. leaf extracts were diluted 1:10 for total sugars assay and used pure for sucrose assay. statistical analysis experiments were performed in a completely randomized experimental design with eight replicates for each treatment. the data are reported as means with standard errors. data presented in table 2 were subjected to oneway anova. 3. results gas compositions inside plastic bags the gas composition was determined after 30 and 60 days of storage. results showed that oxygen was lower in the plastic bags containing cut branches treated with tdz plus glycerol. at the same time, also higher values of nh 3 were found in the tdz+gly treatment (table 1). vase life and chlorophyll content the vase life was significantly improved by the combined treatment tdz plus glycerol (33.6 days in average) in comparison with the control (average 21.2 days) and the other treatments (21.8 to 23.6 days) (table 2). vase life was significantly reduced in cut foliage stored for 60 days, but treatment with tdz plus glycerol gave the longest vase life, 22.6 days on average. vase life of the control was 13.8 days, while in the treatment with glycerol and tdz alone it was 10.2 and 15.2 days, respectively (table 2). the chlorophyll content was higher in all treatments compared to control during the first 15 days of vase life, then declined in 10 mm glycerol (this treatment had the best efficiency among the glycerol treatments) while cut foliage treated with tdz showed chlorophyll reduction after 20 days. the combined tdz plus glycerol treatment did not show any chlorophyll reduction until 22 days (fig. 1). after storage the best results were obtained with tdz and tdz plus glycerol treatment. tdz retained the 105 bulgari et al., evaluation of postharvest storage and treatments in cut ruscus foliage chlorophyll content in the treated cut foliage until 10 days, then chlorophyll started to decline. in the combined treatment tdz plus glycerol, the chlorophyll values remained unchanged and similar to the fresh cut foliage (80 a.u.) until 20 days of vase life (fig. 2). chlorophyll a fluorescence measurements chlorophyll a fluorescence was measured during storage by way of a non-destructive method to evaluate the leaf health status of danae leaves during vase life, before and after storage. among the different parameters and indexes calculated, the performance index (pi) is reported in figure table 1 gas compositions inside the plastic bags during storage days gases blank treatments control 10 mm gly tdz tdz+gly 30 co 2 (%) 0 0.30 0.60 0.10 0.3 o 2 (%) 20 12.70 19.90 20.90 13 nh 3 (µl l-1) 2 15.00 0.00 12.00 17 60 co 2 (%) 0.00 0.50 0.40 0.40 0.3 o 2 (%) 20.90 20.30 19.50 20.00 13 nh 3 (µl l-1) 0.00 8.00 8.00 8.00 17 table 2 vase life before and after storage of cut foliage treated with glycerol (0.1, 1 or 10 mm), 10 µm tdz or a combination 10 µm tdz plus 10 mm glycerol vase life of fresh harvest cut foliage control glycerol tdz tdz + glycerol 0.1 mm 1 mm 10 mm 10 µm 10 µm + 10 mm vase life (d) 21.2±1.09 b 21.8±0.45 b 22.2±0.45 b 22±0 b 23.6±1.34 b 33.6±1.14 a storage and vase life post-storage control glycerol tdz tdz + glycerol 10 mm 10 µm 10 µm + 10 mm storage (d) 60 60 60 60 post-storage vase life (d) 13.8±1.09 b 10.2±1.12 b 15.2±0.45 b 22.6±2.61 a storage + vase life (d) 73.8±1.09 b 70.2±1.12 b 75.2±0.45 b 82.6±2.61 b values are means with standard errors (n=5). data were subjected to one-way anova analysis and different letters indicate significant differences for p<0.05. fig. 1 chlorophyll content measured from cut danae branches during vase life with a chlorophyll meter and expressed as relative units. values are means with standard errors (n=8). fig. 2 chlorophyll content during vase life of cut danae branches stored for 60 days. chlorophyll was measured using a chlorophyll meter and expressed as relative units. values are means with standard errors (n=8). 106 adv. hort. sci., 2015 29(2-3): 103-108 3 and 4. in particular, the pi measured during the vase life of non-stored cut foliage did not change in the combined treatment, while it declined in the control and glycerol treatments. instead, in the tdz treated cut branches, the pi declined after eight days of vase life (fig. 3). after two months of storage, the pi was lower in all treatments and dropped faster in the control and tdz, while in the tdz plus glycerol it slightly declined after eight days and remained unchanged until 20 days of vase life (fig. 4). sugars content the sucrose content of cut foliage at harvest was 15 mg/g fw on average and declined during vase life. in glycerol treatments, and in particular in the 10 mm treatment, the reduction was faster and showed lower values. after 14 days of vase life, higher values were found in tdz and tdz plus glycerol. after 19 days only the cut branches treated with tdz and glycerol were alive and the sucrose content was 4.5 mg/g fw on average (fig. 5). in stored cut branches the sucrose content was five fold lower compared to the fresh harvested branches. the sucrose content declined in tdz after eight days of storage and ranged from 1.5 to 2.2 mg/g fw until 15 days of vase life. in tdz plus glycerol treatment the cut branches did not show reduction of sucrose content until 15 days of vase life and a significant decline was observed after 20 days of vase life (fig. 6). total sugars showed a similar trend of sucrose content during vase life before and after storage. the higher values were found in tdz plus glycerol treatment, especially at the end of vase life (data not shown). 4. discussion and conclusions the visual appearance of cut foliage is the most important quality parameter, and preservative treatments for fig. 3 performance index (pi) measured on cut foliage during vase life determination. values are means with standard errors (n=5). fig. 4 performance index (pi) measured on cut foliage stored for 60 days and then transferred to 20 °c for vase life determination. values are means with standard errors (n=5). fig. 5 sucrose content measured in leaves of cut danae branches treated with distilled water (control), 10 mm glycerol, 10 µm tdz or 10 µm tdz plus 10 mm glycerol during vase life. values are means with standard errors (n=3). fig. 6 sucrose content measured in leaves of stored cut danae branches treated with distilled water (control), 10 µm tdz or 10 µm tdz plus 10 mm glycerol during vase life. values are means with standard errors (n=3). 107 bulgari et al., evaluation of postharvest storage and treatments in cut ruscus foliage these ornamental items are aimed at delaying leaf senescence or yellowing, which can occur during long-distance transportation or long storage periods. in addition to external quality, another important parameter to control in cut foliage postharvest is weight loss. since cut foliage is sold on the basis of weight, any weight losses translate into economic losses. danae plants are in active vegetative growth from september to may, thus there are three months without harvest and a lack of cut branches on the market. flower markets and cooperatives are interested in storing as much cut foliage as possible. cut danae branches can be stored under mild vacuum packages or in water for two months while maintaining satisfactory vase life (pacifici et al., 2014). however, preservative treatments are needed to delay leaf yellowing during post-storage vase life. the positive effect of cytokinin treatments on chlorophyll retention was observed in cut eucalyptus branches (ferrante et al., 2002 b) and tdz has been used in cut flowers to inhibit leaf yellowing in sensitive species such as alstroemeria, chrysanthemum and tulips (ferrante et al., 2002 a, 2003, 2005). our results indicate that cut branches treated with tdz plus glycerol provide the best post-storage performance. tdz alone reduced chlorophyll decline but tdz with glycerol showed a synergistic effect; the positive effect of the combination was observed in all parameters measured. chlorophyll a fluorescence measurements can be used during postharvest to evaluate the health status of leaves. in the ornamental field, chlorophyll a fluorescence and relative derived parameters have been used to evaluate quality losses in cut eucalyptus and danae branches (pacifici et al., 2008, 2013). in potted bougainvillea plants, the use of chlorophyll a fluorescence parameters were used to evaluate the efficiency of ethylene inhibitors during the postproduction stage (ferrante et al., 2012). in our experiments, chlorophyll a fluorescence showed that tdz plus glycerol maintained leaf functionality as demonstrated by the fv/fm ratio (data not shown) and pi. storage of cut danae branches in polyethylene bags at 4°c for 60 days gave good results since the cut foliage was available on the market for more than 80 days from harvest. the reduced gas exchanges avoided excessive water loss by transpiration and probably also conditioned respiration, as demonstrated by the gas compositions at the end of storage, especially in the bags containing cut branches treated with tdz plus glycerol. cut e. parvifolia branches treated with glycerol showed longer life compared with control (ferrante et al., 2001) and analogous results were found in e. cinerea (campbell et al., 2000). glycerol uploaded in the cell increases osmotic capacity and induces an initial stress which can be observed by measuring ethylene production (ferrante et al., 2001), but it also helps maintain water balance. sugars represent the energy source for stored products and are usually associated with vase life. among the sugars, sucrose represents one of the most important energy reserves and its degradation provides a direct substrate for respiration (reid, 1991; ferrante and reid, 2006). in the present study, the cut foliage treated with tdz showed higher sugars content. further studies are needed to understand sugar metabolism in tdz treated cut foliage after storage. these results can be further evaluated in combination with postharvest treatments and the use of passive refrigeration systems, which have been applied to different perishable foods (costa et al., 2013). in conclusion, the results obtained from this investigation suggest that pulse treatment for 24 h with tdz and glycerol can be used to extend the vase life of stored and non-stored cut danae branches. however, further studies are required at physiological and biochemical level to elucidate the biological pathways affected by tdz and glycerol. acknowledgements this work was funded by the italian ministry of agricultural, food and forestry policies (mipaaf) under the project traceflor. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references campbell s.j., ogle h.j., joyce d.c., 2000 glycerol uptake preserves cut juvenile foliage of eucalyptus cinerea. australian journal of experimental agriculture, 40(3): 483-492. costa c., antonucci f., menesatti p., pallottino f., boglione c., cataudella s., 2013 an advanced colour calibration method for fish freshness assessment: a comparison between standard and passive refrigeration modalities. food and bioprocess technology, 6(8): 2190-2195. dubois p., joyce d., 1992 preservation of fresh cut ornamental plant material with glycerol. postharvest biology and technology, 2(2): 145-153. ferrante a., hunter d. a., hackett w. p., reid m. s., 2002 a thidiazuron a potent inhibitor of leaf senescence in alstroemeria. postharvest biology and technology, 25(3): 333-338. ferrante a., mensuali-sodi a., serra g., tognoni f., 2001 water consuption, chlorophyll contents and vase life of cut eucalyptus parvifolia cambage foliage. workshop on green cut foliage. ace international flortecnica, santa flavia 4 may 2001 pa, 5: 197-203. ferrante a., mensuali-sodi a., serra g., tognoni f., 2002 b effects of ethylene and cytokinins on vase life of cut eucalyptus parvifolia cambage branches. plant growth regulation, 38: 119-125. ferrante a., mensuali-sodi a., tognoni f., serra g., 2005 postharvest studies on leaf yellowing of chrysanthemum cut flowers. adv. hort. sci., 19(2): 81-85. 108 adv. hort. sci., 2015 29(2-3): 103-108 ferrante a., reid m.s., 2006 postharvest physiology of cut flowers. italus hortus, 13(4): 29-41. ferrante a., tognoni f., mensuali-sodi a., serra g., 2003 treatment with thidiazuron for preventing leaf yellowing in cut tulips and chrysanthemum. acta horticulturae, 624: 357-363. ferrante a., trivellini a., borghesi e., vernieri p., 2012 chlorophyll a fluorescence as a tool in evaluating the effects of aba content and ethylene inhibitors on quality of flowering potted bougainvillea. the scientific world journal, id 684747: 1-11. jiang c.z., wu l., macnish a.j., king a., yi m., reid m. s., 2008 thidiazuron, a non-metabolized cytokinin, shows promise in extending the life of potted plants. acta horticulturae, 847: 59-66. mutui t.m., mibus h., serek m. 2005 effects of thidiazuron, ethylene, abscisic acid and dark storage on leaf yellowing and rooting of pelargonium cutting. j. hort. sci. biotech., 80: 543-550. pacifici s., burchi g., del carlo a., ferrante a., 2013 effect of storage temperature and duration on vase life of cut ruscus racemosus l. foliage. acta horticulturae, 970: 69-74. pacifici s., burchi g., del carlo a., ferrante a., 2014 mild vacuum packaging for long storage of cut danae racemosa (l.) moench foliage. acta horticulturae, 1071:143-148. pacifici s., ferrante a., mensuali-sodi a., serra g., 2007 postharvest physiology and technology of cut eucalyptus branches: a review. agr. med., 137: 124-131. pacifici s., mensuali-sodi a., ferrante a., serra g., 2008 comparison between conventional and vacuum storage system in cut foliage. acta horticulturae, 801: 1197-1204. reid m.s., 1991 effects of low temperatures on ornamental plants. acta horticulturae, 298: 215-224. shanan n.t., shalaby e.a., 2011 influence of some chemical compounds as antitranspirant agents on vase life of monstera deliciosa leaves. afr. j. agric. res., 6(1): 132139. van doorn w.g., 1997 water relations of cut flowers. horticultural rev., 18: 1-85. van doorn w.g., hibma j., de-wit j., 1992 effect of exogenous hormones on leaf yellowing in cut flowering branches of alstroemeria pelegrina l. plant growth regul., 11: 445-448. impaginato 13 adv. hort. sci., 2018 32(1): 13-17 doi: 10.13128/ahs-21241 storage conditions of soft x-ray irradiated pollen for producing seedless watermelons m. akutsu1, 2 (*) 1 national agricultural research center for hokkaido region, hokkaido, japan. 2 department of electrical engineering and computer science, tokai university, kumamoto, japan. key words: fruit quality, n2 storage, seedless watermelon, soft x-ray, storage temperature, vacuum storage. abstract: a safe and efficient method for preserving viable soft x-ray-irradiated pollen for the production of seedless watermelon (citrullus lanatus l.) from diploid plants was tested by packing the pollen under vacuum, o2, co2 or n2 gas at 25°c, 4°c or -25°c. pollen germination rates decreased most rapidly with storage at 25°c and slowest with storage at -25°c. oxygen as a storage gas was not good for storage of pollen, but pollen stored in n2 or co2 gave good germination. pollen stored at -25°c for 90 days germinated, but pollen stored at 4°c for 90 days did not, and n2 storage tended to result in higher fruit set than vacuum storage. fruit set was significantly affected by pollen storage conditions, with n2 storage being more effective than vacuum storage at 4°c. storage at -25°c produced little difference in fruit set between vacuum and n2 storage. thus, temperature was the major factor for maintaining viable and effective pollen, and the use of n2 gas was an effective adjunct. fruit quality was not significantly affected by storage parameters in this experiment. 1. introduction generally, seedless watermelons are produced from triploid plants that are the product of crosses between tetraploid and diploid plants (terada and masuda, 1943; kihara and nishiyama, 1947; kihara, 1951). diploid seedless watermelons have also been produced by pollination with soft x-ray irradiated pollen (sugiyama and morishita, 2000; sugiyama et al., 2002 a). using irradiated pollen is advantageous because seedless watermelons can be produced with ordinary cultivation methods due to the use of diploid plants. however, mass production of seedless watermelon seed by this method will require the production, irradiation, preservation and storage of a lot of pollen. pollen viability after freezing and low temperature storage at low relative humidity has been reported (*) corresponding author: akutsu@tsc.u-tokai.ac.jp citation: akutsu m., 2018 storage conditions of soft xray irradiated pollen for producing seedless watermelons. adv. hort. sci., 32(1): 13-17 copyright: © 2018 akutsu m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 september 2017 accepted for publication 8 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 13-17 14 in many plant species (holman and brubaker, 1926; mcguire, 1952; king, 1961; fatmer and barnett, 1974; nath and anderson, 1975; anderson et al., 1978). miyaji and shirazawa (1977) reported that the best storage conditions for watermelon pollen are 5°c at 20 to 40% humidity, which extended the viability of pollen to 60 days on germination medium, but did not investigate fruit set. watermelon pollen had fruit setting ability for 13 days when stored at 5°c under dry conditions (silica gel) (araki et al., 1987). pollen stored under temperature and humidity control, tend to have rapid germination rate decreases (miyaji and shirazawa, 1977; araki et al., 1987; sugiyama et al., 1998). watermelon pollen storage in organic solvents has also been investigated (kodani and omura, 1981; shimizu, 1983; sugiyama et al., 2002 b). among the organic solvents, ethyl acetate and ethyl ether were the most suitable for watermelon pollen storage (shimizu, 1983). pollen stored in ethyl acetate at -20°c had a germination rate of over 40% for one month on germination medium (morishita et al., 2000), and fruit set was observed up to 79 days of storage in ethyl acetate at -20°c (sugiyama et al., 2002 b). these methods are effective for watermelon pollen, but are not suitable for long-term storage because of a decrease in vigor. also, it is very difficult for farmers to recover and use pollen that has been stored in organic solvents. n2, co2 and/or o2 gas have been used as a general storage medium in the food industry, but there are few reports using gas for pollen storage. in this study, we describe effective new pollen storage method using atmospheric gases. 2. materials and methods storage conditions for watermelon pollen pollen from watermelon cultivar green seeded was collected in october 2005 by cutting the anthers from the flowers and shaking the contents through a stainless steel filter into a stainless steel cup. collected pollen was packed in paraffin paper and irradiated with 600 gy soft x-ray (soft x-ray unit om60r, ohmic ltd.) at 14.5 gy·min-1. irradiated pollen aliquots of about 1 g each were packed with a vacuum packaging machine (‘tospack’ v-380g, tosei, shizuoka, japan), in air or under vacuum storage at 25°c, 4°c or -25°c for storage over 35 days. a second similar storage test was initiated in may 2007 to investigate effect of atmospheric gases on storage. irradiated pollen aliquots of about 3 g each were packed with a vacuum packaging machine under n2, o2, co2, air or vacuum for storage at 25°c for 1-6 days. viability of the stored pollen was judged on the basis of germination on an artificial medium consisting of 14% sucrose, 0.1% boric acid, and 1.5% agar (wako, ltd, osaka, japan). germination percentages were determined after 3 hour incubation at 25°c by counting random samples of 100 pollen or more for each storage condition. the first test was replicated 8 times, and the second test was replicated 5 times. tukey’s multiplerange test (p<0.05) on statistical software (statcel, oms-publishing, saitama, japan) was used to test differences between treatment means. fruit set ability of stored pollen, and fruits quality using stored pollen experiments were performed at the hokkaido research center, sapporo, japan. during cultivation, greenhouse temperatures were recorded (‘ondotori’, t&d corporation, nagano, japan), and monthly mean temperatures were determined. the average temperatures during cultivation in the greenhouse were 25°c in july, 30.4°c in august, and 25.1°c in september. watermelon cultivar fujihikari tr seeds were sown in pots on june 15, 2006 in a greenhouse. seedlings with 5 to 6 leaves were transplanted 50 cm apart in a bed (2.3 x 35 m) in a greenhouse on july 10, 2006. the bed was covered with black and gray polyethylene mulch and fertilized with 9n-9p-9k (in kg·ha-1) before transplanting. plants were topped at the five leaf stage and three lateral vines were allowed to grow. male flowers were pollinated with a paint brash at about the 15th node of lateral branches. all treatments were arranged in a randomized complete-block design with four single plant replications. four fruits were harvested for each treatment. values were compared by tukey’s multiple-range test (p<0.05). source of pollen watermelon cultivar green seeded seeds were sown as a source of pollen for the storage experiments in february 2006, before ‘fujihikari tr’ was sown. male flowers of ‘green seeded’ were harvested in the morning to obtain pollen that would be stored for 14, 28 and 90 days, from april through july. collected pollen was irradiated with 600 gy soft x-ray and packed with a vacuum packaging machine either under n2 gas or vacuum storage at 4 or -25°c for 14, 28 or 90 days. soft x-ray irradiated pollen processed on the morning of the experiment was akutsu pollen preservation for watermelons 15 used as a non-storage control. the stored pollen was used to pollinate female flowers of ‘fujihikari tr’. female flowers of ‘fujihikari tr’ were bagged with cellophane before anthesis. prepared irradiated pollen was applied with a brush. after pollination, female flowers were covered again with cellophane bags to prevent contact with insect-borne pollen for about 3 days. pollination occurred during the period of august 2-6. one or two female flowers on each of the 3 lateral shoots were pollinated from each storage treatment, and fruit set was confirmed about 7 days later. the fruit set rate for each plant was calculated. fruits were selectively thinned, leaving one fruit per plant to mature for each treatment. mature fruits were harvested after about 42 days. after harvesting, fruit weight, shape, rind thickness, flesh color, sugar content (brix) and number of empty seeds exceeding 6 mm in length were recorded for each fruit. fruit shape index is expressed as the ratio of length from peduncle to blossom end, to equatorial diameter. mature harvested watermelons were cut in half, and flesh color was measured with a colorimeter [a*= hue relates to red (+60) green (-60) color axes, nihondenshokukogyo, tokyo, japan]. the germination ability of stored pollen was assayed as in experiment 1 before pollination (the period of august 2-6). fruit set and pollen germination rates were compared by tukey’s multiple-range test (p<0.05). 3. results and discussion storage condition for watermelon pollen pollen germination rates decreased rapidly at 25°c to nearly zero after 7 days (fig. 1). when stored in atmospheric air at 4°c, the germination rate was significantly lower than with other treatments (except at 25°c) after 21 days. after 35 days of storage under vacuum at 4°c, pollen viability was significantly lower than either treatment at -25°c (p<0.05). thus, high temperature is not suitable for storage of pollen, and packing pollen under vacuum at 4°c prolonged viability. the viability of watermelon pollen stored under vacuum or o2 at 25°c decreased during storage to levels that make o2 and vacuum conditions unacceptable as atmospheric media for pollen storage (table 1). pollen stored for 7 days under n2 and co2 showed good germination, indicating that storage of soft x-ray irradiated watermelon pollen under n2 or co2 would enhance its viability. fruit set and quality produced from stored pollen the ability of stored polled to germinate, and to produce agronomically acceptable fruit are two distinct qualities, both of which are necessary for the adoption of soft x-ray irradiated pollen on a commercial scale. there were observable differences in fruit set between pollen stored at 4°c and -25°c (table 2). lower temperatures resulted in good germination rates, and pollen which had been stored at -25°c had a longer shelf-life than at 4°c. pollen stored at 4°c for table 1 the differences of germination ability for cultured periods and treatments at 25°c treatments periods (days) 1 2 3 4 5 n2 25.1 a z 21.2 a 16.5 a 13.8 a 7.9 a o2 11.2 b 4.7 b 0.0 d 0.0 c 0.0 b co2 19.4 ab 17.6 ab 16.3 a 15.1 a 6.9 a vacuum 16.9 ab 10.9 b 2.0 c 1.7 b 0.0 b air 20.3 ab 19.5 ab 9.3 b 3.3 b 0.2 b (z) means followed by the same letter are not significantly different at the 5% level by tukey's multiple-range test. table 2 relationship between storage condition and fruit set treatment storage pollination fruiting fruit set (%) temperature (°c) period (days) control 0 9 8 88.9 nitrogen 4 14 17 17 100.0 nitrogen 4 28 18 7 38.9 nitrogen 4 90 16 0 0.0 nitrogen -25 14 11 10 90.9 nitrogen -25 28 16 14 87.5 nitrogen -25 90 16 15 93.8 vacuum 4 14 17 11 64.7 vacuum 4 28 7 0 0.0 vacuum 4 90 13 0 0.0 vacuum -25 14 15 15 100.0 vacuum -25 28 15 14 93.3 vacuum -25 90 15 13 86.7 fig. 1 germination of pollen after storage at different temperature and treatments. bars indicate ± standard errors (n=8). adv. hort. sci., 2018 32(1): 13-17 16 90 days did not set fruit whether or not it was stored under n2 or vacuum. although germination rates under n2 and vacuum storage were almost the same, fruit set with pollen stored at 4°c for 14 or 28 days was more effective under n2 than under vacuum. pollen stored at 4°c for 28 days under n2 had the ability to set fruit, but pollen stored at 4°c under vacuum did not. snope and ellison (1963) also reported that germination from freeze-dried pollen that had been stored under n2 was got good. these results indicate that pollen vigor is maintained more effectively under n2 than under vacuum. the percentage of germinating pollen stored at -25°c for 28 days was 24.8% and there was fruit set following hand pollination. however, the percentage of germinated pollen stored at 4°c for 28 days was 22.8%, but no fruit was set with this treatment. n2 gas was an effective atmospheric medium for storage at 4°c in both tests. there were many reports that low temperature provides a good environment for stored pollen (araki et al., 1987; sugiyama et al., 1998; morishita et al., 2000). low temperature provides the major benefit, with storage in n2 gas as an adjunct for pollen storage. inert gases may be effective for the maintenance of pollen vitality because they may suppress metabolism of the pollen by displacing oxygen. the additive effect of n2 with low temperature suggests that low metabolic rates are the key factor in maintaining pollen viability over time. there was no significant difference in weight or shape between control fruit and fruit produced by pollen under any of the storage conditions (table 3). rind thickness and flesh color were also the same, except for fruits produced with pollen stored at -25°c for 90 or 14 days, respectively. although the brix of fruit from pollen stored at -25°c for 14 days was the lowest, there was no consistent relationship between storage conditions and brix. sugiyama and morishita (2000) has observed that the number of empty seeds differs widely in individual fruits. in this experiment there were significant differences in the number of empty seeds, but there was no apparent relationship between empty seed numbers and storage conditions. thus, there is no consistent relationship between pollen storage conditions and fruit quality. further statistical analysis of the relationships between treatment, temperature and periods of pollen storage, was done for the survival of pollen, but there were no statistically significant differences. organic solvents are useful as a pollen storage method (kodani and omura, 1981). however, organic solvents carry the risk of environmental pollution and are difficult to use outside the laboratory. storage of pollen at low temperatures and under n2 eliminates the need for organic solvents, thus providing an ecofriendly storage solution which should be applicable for pollen export or import. furthermore, pollen stored under n2 at 25°c for 5 days had germination ability (table 1) and then the temperature in the greenhouse was around 25-30°c, so it might be also no problem to bring the pollen from freezer to the greenhouse during pollination. table 3 fruit quality of seedless watermelon pollination with irradiated pollen in several conditions treatment storage fruit weight (kg) flesh shape (z) thickness of rind (mm) flesh color brix (%) no. of empty seeds temperature (°c) period (days) control 0 5.5±1.1(y) 1.14 13.9±0.0 a (x) 21.4±1.1 a 10.3 a 96.5±12.1 b nitrogen 4 14 4.6±0.6 1.11 13.2±1.0 a 20.4±1.7 a 9.9 a 102.5±86.3 b nitrogen 4 28 4.9±0.5 1.12 13.5±0.2 a 23.9±2.8 a 9.7 b 104.5±17.6 b nitrogen -25 14 5.1±1.0 1.11 12.2±1.0 a 18.4±3.6 b 9.6 bc 150.0±88.3 a nitrogen -25 28 5.5±0.9 1.10 13.5±0.6 a 23.9±1.5 a 9.9 a 104.8±25.2 b nitrogen -25 90 5.2±0.5 1.14 11.9±0.4 b 22.5±1.0 a 9.7 b 222.8±71.7 a vacuum 4 14 4.9±0.3 1.13 12.2±1.4 a 24.0±3.9 a 9.7 b 103.0±44.9 b vacuum -25 14 5.4±0.5 1.10 12.7±1.9 a 20.6±2.5 a 10.2 a 134.8±21.1a vacuum -25 28 5.1±1.5 1.15 12.3±1.6 a 21.0±1.9 a 10.1 a 158.5±81.0a vacuum -25 90 5.7±0.4 1.17 12.5±0.6 a 22.5±0.6 a 10.0 a 127.0±24.5b (z) flesh shape is expressed as the ratio of height to width. (y) mean ± se. (x) value within columns with the same letter are not significantly different (5% level). means followed by the same letter are not significantly different at the 5% level by tukey's multiple-range test. akutsu pollen preservation for watermelons 17 plant breeders may also be able to overcome differences in crop or varietal flowering times by storing pollen with this method. references anderson j.o., nath j., harner e.j., 1978 effects of freeze-preservation on some enzyme. ii. freezing and freeze-drying stresses. cryobiology, 15: 469-477. araki m., hashizume t., hagihara t., 1987 effects of stored watermelon pollen on fruit set. j. jpn. soc. hort. sci., 56(2): 344-345. fatmer r.e. jr., barnett p.e., 1974 low-temperature storage of black walnut pollen. cryobiolgy, 11: 366367. holman r.m., brubaker f., 1926 on the longevity of pollen. univ. cal. publ. bot., 13: 179-204. kihara h., 1951 triploid watermelon. proc. amer. soc. hort. sci., 58: 217-230. kihara h., nishiyama i., 1947 an application of sterility of autotriploids to the breeding of seedless watermelons. seiken ziho, 3: 93-103. in japanese with english summary. king j.r., 1961 the freez-drying of pollens. econ. bot., 15(1): 91-98. kodani t., omura s., 1981 on the collection and storage of pollens of watermelon by the utilization organic solvents. bulletin of tottori agri. exp. stn., 2: 35-46. in japanese with english summary. mcguire dc., 1952 storage of tomato pollen. proc. amer. soc. hort. sci., 60: 419-424. miyaji r., shirazawa m., 1977 influence of temperature and the humidity for melon’s and watermelon’s pollens on their storage. bulletin of the kagoshima agric. exp. stn., 5: 203-206. morishita m., hori t., sakata y., sugiyama m., 2000 storage of pollen irradiated with soft x-ray for producing seedless watermelon. j. jpn. soc. hort. sci., 69(2): 346. nath j., anderson j.o., 1975 effect of freezing and freeze-drying on the viability and storage of lilium longiflorum l. and zea mays l. pollen. cryobiology, 12: 81-88. shimizu t., 1983 studies on the artificial pollination of watermelon. 1. 0n the storage of pollens of watermelon by the utilization of organic solvents. bulletin of the tottori agric. exp. stn., 4: 9-17. snope j.a., ellison j.h., 1963 storage of asparagus pollen under various conditions of temperature, humidity and pressure. amer. soc. hort. sci., 83: 447-452. sugiyama k., morishita m., 2000 production of seedless watermelon using soft-x-irradiated pollen. scientia horticulturae, 84: 255-264. sugiyama k., morishita m., nishino e., 2002 a seedless watermelons produced via soft-x-irradiated pollen. hortscience, 37: 292-295. sugiyama k., morishita m., sakata y., 1998 storage of soft-x-irradiated pollen and effects of stored pollen on fruit quality of seedless watermelon. j. jpn. soc. hort. sci., 67(2): 283. sugiyama m., sakata y., kitadani e., morishita m., sugiyama k., 2002 b pollen storage for production of seedless watermelon (citrullus lanatus) using soft-xirradiated pollen. acta horticulturae, 588: 269-272. terada j., masuda k., 1943 parthenocarpy of triploid watermelon. agric. hort., 18: 15-16. 207 1. introduction coriander (coriandrum sativum) is a culinary and medicinal plant of the umbelliferae, and is an annual herb originally from the mediterranean area; several authors have indicated coriandrum sativum as a wild plant. linnaeus (1780) reported that c. sativum also occurs as a weed in cereals. alefeld (1866) mentioned that c. sativum was a common weed spread from southeastern europe to southern russia. stoletova (1930) reported on wild c. sativum from armenia. all parts of the plant have a strong odor, from which the plant takes its name. cultivation of c. sativum is widespread, but it is planted on a small scale only; it is cultivated as a summer or winter crop. in jordan, it is found in gardens rather than in large fields, while in germany there are many landraces of coriandrum sativum (diederichsen, 1996). description of the plant the plant can reach heights of 20 to 80 cm. the stem is more or less erect, branched sometimes with several side branches at the basal node. each branch finishes with an inflorescence. the color of the more or less ribbed stem is green and sometimes turns to red or violet during the flowering period. the leaves alternate, and the first ones are often gathered in a rosette. the leaves are of two types: lower ones with leaflets and upper ones divided into narrow linear segments (diederichsen, 1996). the coriandrum sativum flower has five irregular-shaped petals, five stamens, five sepals, and two styles. flowering starts with the primary umbel. the first umbels to bloom have hermaphrodite flowers, with possibly a few staminate ones (diederichsen, 1996). the inner flowers of umbellets are staminate. the central flowers are circular, with small inflexed petals. the color of the petals is pale pink or sometimes white. the umbels of higher order usually contain more staminate flowers than the first ones, and their flowering period is shorter (diederichsen, 1996). in a single flower, the five filaments of the staminate are located between the five petals. after the flower opens, the white filaments are visible between the petals. under optimum conditions, many different insect species are pollinators or visitors of c. sativum umbels (diederichsen, 1996), and the species that pollinate the plants depend on the area of cultivation. flowering and pollination biology of c. sativum is typical of that for umbelliferous plants, according to bell (1971). depending on the weather conditions, two to three days after opening of the first flower, the pollen sacs open and spread the pollen. mcgregor (1976) showed that selfing of c. sativum is impossible but glukhov (1955) showed that it is partially self-fertile. he suggested that geitongamy is common and cross is possible. bees are beneficial to c. sativum: glukhov (1955) reported that when they were excluded only 49.4% of the seeds set, but when they were present 68.3% of the seeds set. bogoyavlenseii and akimenko (1966) associated seed yields with greater insect visitation. use of coriandrum sativum this plant is of economic importance since it has been used as a flavoring agent in food products, perfumes and seed contents of coriandrum sativum in jordan valley k.a. abu-hammour*, d. wittmann** * college of pharmacy, al-isra university, p.o. box 22, 23 isra, 11622 amman, jordan. ** institute of crop science and resource conservation (inres), department of ecology of the cultural landscape-animal ecology, university of bonn, d-53127 bonn, germany. key words: coriandrum sativum, fertilized seeds and global warming, petroselinic acid. abstract: the aim of this research was to determine seed contents of coriander (coriandrum sativum). research was conducted in the jordan valley at 150 m below sea level and in northern jordan at 200 m above. analyses of fertilized seeds showed that they contain 14.9% protein while non fertilized seeds had a significantly lower content (4.7%). the seeds contain 7.4% oil, which can vary according to strain. the fatty acid composition varied significantly among the seeds from the selected locations. petroselinic acid was significantly the most concentrated fatty acid (80.10%). this acid (c18:1) can be split to produce c6 (adipic acid) and c12:0 (lauric acid) molecules. adipic acid is used for the manufacture of a wide range of polymers including high grade engineering plastics; at present it is derived from mineral oil by a process that damages the ozone layer and contributes to the releases of gasses such as n2, which affect global warming. petroselinic acid is abundant in c. sativum and could be an alternative, more environmentally friendly raw material for use in industry. adv. hort. sci., 2011 25(4): 207-211 received for publication 18 february 2011 accepted for publication 18 july 2011 208 cosmetics. moreover, the essential oils of the fruits and various extracts from c. sativum have been shown to possess antibacterial (burt, 2004; cantore et al., 2004; kubo et al., 2004), anticancerous and antimtagenic (chithra and leelamma, 2000) properties and the plant has been used in medicine for thousands of years. in jordan, the primary product is the fresh green herb of c. sativum used for its specific flavor, which is completely different from that of the ripe fruits. in other countries the fruits are used as a spice and vegetable. oleum (1993) stated that russia produces high quality c. sativum oil, with a linalool content of 55%. bauer (1942) found that coriandrum sativum attains its greatest yield of volatile oil (0.9%). the fatty oil of coriandrum sativum is of interest because of the high level of petroselinic acid. 2. materials and methods research sites the research was conducted in jordan at two different locations. the first is located 150 m below sea level. this area is humid with warm temperatures in winter, and dry and hot in summer. the other location is 200 m above sea level. it is characterized by rainy, cold winters and dry, mild summers (fig. 1). both locations have produced relatively high biodiversity in wild plants and bees. the jordan valley, latitude 22° 40’ 0’’ and longitude of 35° 30’ 0’’, is located in jordan, a part of the middle east (fig. 2) and extends down the entire flank of jordan 50 km from amman; it is the country’s most distinctive natural feature. the northern segment of the jordan valley, known in arabic as the ghor, is the nation’s most fertile region. it contains the jordan river and extends from the northern border down to the dead sea. several degrees warmer than the rest of the country, its year-round agricultural climate, fertile soils, high winter rainfall and extensive summer irrigation have made the ghor the food bowl of jordan. according to md (2002), the mean maximum and minimum temperatures are 29.9°c and 16.98°c, respectively, with rainfall of around 77-392 mm over 44.84 rainy days yearly. over the last 30 years, mean relative humidity has been 72.45% in winter and 48% in summer. the jordan valley is subjected to ground frost nearly 2.5 days yearly. coriandrum sativum plantation coriandrum sativum seeds, obtained from local markets (landraces), were planted at locations a and b on 5 november 2007. the rows were 20 m long with 1 m between rows. water was supplied daily by drip irrigation, and extra fertilizers (n p k) were applied. both locations were kept weed-free by cultivation and hand weeding. determination of seed content to determine the seed content of c. sativum, 400 g of seeds were collected. oil extraction and preparation of seeds involved the following: drying of seeds, crushing, and extraction. solvents such as carbon dioxide and propane were used to facilitate oil extraction. gas liquid chromatography was used to determine the seed content. seed content analyses were performed at the national center for agricultural research and technology transfer (ncartt) chemist’s lab, amman, jordan. the protein content of c. sativum was analyzed in fecundated and nonfecundated seeds using the international standard method (sop: 130m01-006) (table 1). table 1 analyzed constituents for coriandrum sativum standard method used seed constituent ash sop: 130m01-009 moisture sop: 130m01-010 oil sop: 130m01-001 proteins sop: 130m01-006 trace elements mg mn cu ca k sop no :131m02-005 sop no :131m02-002 oil constituent fatty acids coi/t.20/doc. no. 24 (2001) – aocs ch 2-91 (1997) the seed contains the essential oils based on the analysis applied (table 2). 3. results seed content seeds for testing (for fatty acid composition, essential oils and mineral contents) were chosen from both locations. moisture content was not significantly different among treatments. protein values were significantly different among pollination treatments and ranged from 13.01 to 15.78%. oil content was low in all seeds and varied from 5.61 to 7.40%. ash values ranged between 6.26 and 6.51% (table 2). oleic acid was significantly the most concentrated 80.10% (table 3). all mineral contents fig. 1 jordan valley overview of vegetation covers. 209 location b: 200 m (above sea level).location a: 150 m (below sea level). fig. 2 research sites (53, 15, 30, 30, are the main international highway). table 2 essential oil component and percentage in seed of c. sativum main component total essential oil (%) 1. linalool 66.7 2. alpha-pinene 9.8 3. gamma-terpinene 8.3 4. geranylacetate 3.3 5. camphor 3.0 6. geraniol 1.9 table 3 main components and mineral contents in seed of c. sativum seed content moisture 8.30 % ash 6.31 % protein 15.78 % oil 7.40 % magnesium 0.34 % calcium 0.40 % potassium 1.46 % manganese 18.80 mg/kg cupper 11.60 mg/kg 210 varied significantly among seed samples; manganese and copper were the most prevalent minerals. linalool acid is the common essential oil in coriander and was determined to be 66.7%. fatty acid composition varied significantly among the seeds from the selected locations. (table 4) the fatty oil of coriander is of considerable interest because of the high level of petroselinic acid, which has potential non-food applications in oleo chemistry. protein percentage in coriandrum sativum seeds the protein percent in treated fecundated seeds in coriandrum sativum was 14.9% of the total dry mass, whereas in non-fecundated seeds it was 4.7%. 4. conclusions coriander is an annual herb and is common in the middle eastern and mediterranean cuisine, in which the fresh leaves and dried seeds are the most commonly used parts of the plant. chemicals extracted from coriander have also been used as a traditional treatment for diabetes and hyperlipidemia (chithra and leelamma, 1997; gary and flat, 1999). little is known about the metabolic origin of petroselinic acid (18:1), which is an unusual fatty acid that occurs primarily in seeds of the umbelliferae plant families (tsevegsuren et al., 2004). petroselinic acid is of potential industrial significance because of unsaturation at c-6. through chemical cleavage at its double bond, petroselinic acid can be used as a precursor of both lauric acid, which is a component of detergents and surfactants, and adipic acid, which is the monomeric component of nylon (avato et al., 2001). adipic acid is used for the manufacture of a wide range of polymers including high grade engineering plastics and it has a global market in excess of 2.5 million tons worth over £1 billion. many previous studies have shown that petroselinic acid is the major constituent of the seed oils of many species of umbelliferae such as parsley or coriander and ranges from 15% up to 85% (cahoon et al., 1992; tsevegsuren et al., 2004). keeping with this, the present study illustrated that the major component of the seed oil of coriander tested was petroselinic acid (18:1) and it represented to 80% of the total fatty acid content. recently, derivatives or polymers from under-utilized fatty acids such as petroselinic acid have been regarded as a new raw material, representing an important oleochemical material for the food, cosmetics, chemistry and pharmaceutical industries (avato et al., 2001). additional recent uses include the use as a green vegetable by some ethnic groups and flavoring for dishes and foods such as pickles and sauces. although many studies have focused on the seed content in coriandrum sativum, the percent of protein of the total dry mass in fecundated and non fecundated seeds has not been pointed out. in the present study, the protein percent in treated fecundated and non fecundated seeds for coriandrum sativum was low. protein content in fecundated seeds is more than in non-fecundated seeds, which can be explained by the enzymatic activity of protein synthesis in complete fertilized ovary (embryo) as compared to empty ovary (gamete). in fact, cell division in the fecundated seeds needs more protein in order to complete division. finally, further studies are needed to fully determine the seed contents of coriandrum sativum, and to explore the feasibility of growing coriander on a large scale in jordan. we also believe that the application of advanced plant breeding, together with extensive biochemical studies, could result in more environmentally friendly, high oil, and high petroselinic acid coriander varieties. references alefeld f., 1866 landwirthschaftliche flora. wiegandt and hempel, berline, germany, pp. 165. avato p., fanizzi f.p., rosito i., 2001 the genus thapsia as a source of petroselinic acid. lipids, 36(8), 845-850. table 4 fatty acid content in seed of c. sativum fatty acid trivial name systematic name percentage c 14:0 myristic acid tetradecanoic acid 0.10 c 16:0 palmitic acid hexadecanoic acid 3.33 c 16:1 palmitoleic acid cis-9-hexadecenoic acid 0.42 c 17:0 hexadecanoic acid 0.03 c 17:1 desaturation of cis-9-hexadecenoic acid 0.04 c 18:0 stearic acid octadecanoic acid 0.88 c 18:1, n-7 vaccenic acid cis-11-octadecenoic acid 80.10 c 18:2 linoleic acid cis-9, 12-octadecadienoic acid 14.63 c 18:3 α –linolenic acid cis-9, 12, 15-octadecatrienoic acid 0.29 c 20:0 arachidic acid eicosanoic acid 0.09 c 22:0 behenic acid docosanoic acid 0.03 c 20:1 gadoleic acid cis-9-eicosenoic acid 0.04 c 24:0 lignoceric acid tetracosanoic acid 0.02 211 bauer 1942 landw. jahresber. 92. in: guenther (ed.) the essential oil. vol. 3. robert e. krieger publishing company, huntington, new york, usa. bell c.r., 1971 breeding systems and floral biology of the umbelliferae or evidence for specialization in unspecialized flowers, pp. 93-107. in: heywood v.h. (ed.) the biology and chemistry of the umbelliferae. academic press, new york, pp. 438. bogoyavlenseii s.h., akimenko a.l., 1966 coriander as nectiferous and entomophillous culture. achievements of science and advanced experiment in beekeeping, pp. 119-125. burt s., 2004 essential oils. their antibacterial properties and potential applications in foods a review. int. j. of food microbiol., 94: 223-253. cahoon e.b., shanklintt j., ohlrogge j.b., 1992 expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. proc. natl. acad. sci. usa, 89: 11184-11188. cantore p.l., iacobellis n.s., de marco a., capasso f., senatore f., 2004 antibacterial activity of coriandrum sativum l. and foeniculum vulgar miller var. vulgare (miller) essential oils. j. of agric. and food chem., 52: 7862-7866. chithra v., leelamma s., 1997 hypolipidemic effect of coriander seeds (coriandrum sativum): mechanism of action. plant foods for human nutriton, 51(2): 167-172. chithra v., leelamma s., 2000 coriandrum sativum effect on lipid metabolism in 1, 2,-dimethyl hydrazine induced colon cancer. j. of ethnophamacology, 71: 457-463. diederichsen a., 1996 coriandrum sativum l. promoting the conservation and use of underutilized neglected crops. 3. institute of plant genetics and crop plant research, gatersleben, germany. gary m.a., flat p.r., 1999 insulin-releasing and insulin-like activity of the traditional anti-diabetic plant coriandrum sativum (coriander). british journal of nutrition, 81: 203-209. glukhov m.m., 1955 honey plants. izd. 6, perer. i dop. moskva, gos. izd-vo selkhov litry, pp. 512. in russian. kubo i., fujita k.i., kubo a., nihei k.i., ogura t., 2004 antibacterial activity of coriandrum sativum volatile compounds against salmonella choleraesuis. j. of agric. and food chem., 52: 3329-3332. linnaeus c., 1780 ded ritters carl von linnaeus, koniglich schwedischen leibarztes u.u. vollstandiges pflanzensystem nach der dreyzehnten lateinischen ausgabe und nach anleitung des hollanddischen houttuynischen werks ubersetzt und mit einer ausfuhrlichen erklarung ausgefertiget. von den krautern, gabriel nicolaus raspe, nurnberg 6: 152-154. mcgregor s.f., 1976 insect pollination of cultivated crops plants. usda agricultural handbook, no. 496, pp. 411. md, 2002 metrological report of jordan. metrological departement of jordan. oleum, 1993 the essential oil catalogue. stroud, glouchestershire, uk. stoletova e.a., 1930 polevye i ogorodnye kul’tury armenni. tr. po prikl. bot., gen. i sel. 23: 290-291. tsevegsuren n., aitzetmuller k., vosmann k., 2004 geranium sanguineum (geraniaceae) seed oil: a new source of petroselinic and vernolic acid. lipids, 39(6): 571-576. 187 1. introduction ampelography is a traditional morphological method used for the identification and discrimination of varieties of grapevine (vitis vinifera l.). it has also been found to be very useful in detecting and describing variability among chosen potential clone candidates (poljuha et al., 2006). nowadays, a link between historic descriptions and the molecular fingerprints is also very important, especially in cases of possible homonymy and synonymy of autochthonous varieties (cervera et al., 2001). official descriptors have been published to be used together with the analysis of germplasm material (dettweiler, 1993; ortiz et al., 2004). all the organs used in ampelography (leaves, grapes, shoots etc.) usually change their aspect during phenologic phases, hence it is important to find the characteristics able to discriminate between varieties according to these phases: shoots in the early stage, leaves in the moment when they are mature, and later also the grapes and mature berries (cancellier, 2007). the use of fractal-based measurements of digitallyacquired images eliminates problems with different phenologic phases as well as problems with subjectivity. this allows defining the good shape measure that can be effectively applied to leaf shapes, so they can be compared and analysed by meaningful and objective criteria (mancuso, 1999). using fractal parameters and phyllometric outputs, an artificial neural network can be constructed and effectively used to differentiate varieties and accessions. it is an easy method that requires low-cost equipment such as an optical scanner, personal computer and free software (mugnai et al., 2008). st. claire’s vineyard is an historical area located close to the centre of prague, and where a long tradition of grapevine cultivation dates back to the 13th century. following a period of decline, the vineyard is today regaining its former importance. new varieties are being planted and no ampelographic observations have yet been carried out. the main aim of the present work was to discriminate the grapevine varieties cultivated under the climatic conditions of prague (czech republic) using either the subjective method according to the international descriptors published by ipgri (1997), or an objective computing method which consists of analysis using an artificial neural network. these two approaches are also compared in this paper to assess differences between subjective and objective analysis. discrimination of grapevine varieties cultivated in the czech republic by artificial neural networks e. svobodová*(1), c. pandolfi**, p. hlásná čepková*, s. mancuso** * department of crop sciences and agroforestry in tropics and subtropics, faculty of tropical agriscience, czech university of life sciences prague, czech republic. ** dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: ampelography, phyllometry, vitis vinifera, variety identification. abstract: an artificial neural network approach, based on fractal leaf parameters, and classical ampelography were used to identify nine grapevine varieties cultivated at the st. claire’s vineyard, prague botanic garden. fifty healthy, fully-expanded leaves were collected for each variety, scanned using an optical scanner and then elaborated by computer programs. fourteen phyllometric parameters were qualitatively and quantitatively analysed by the digital image analysis. comparative frames were constructed for each variety and the relationships among varieties were assessed using artificial neural networks. results were then compared with the outcome from traditional ampelographic analysis. the artificial neural network technique appears to be a complementary approach to the traditional ampelography methods commonly used for cultivar discrimination, since the equipment necessary for this analysis is very inexpensive and available. application of the technique led to the distinction of nine selected varieties of vitis vinifera. adv. hort. sci., 2012 26(3-4): 187-192 (1) corresponding author: evasvobodova@its.czu.cz. received for publication 3 september 2012 accepted for publication 6 march 2013 188 2. materials and methods plant material leaf samples for the analysis of nine varieties were collected at the st. claire’s vineyard, prague botanic garden. nine varieties (table 1), cultivated in a sufficient quantity to provide a sufficient source of samples, were chosen for analysis. berry and grape samples were collected at the time of the harvest, which was done according to the further utilization of the grapes. table 1 list of selected varieties variety 1 ‘müller thurgau’ 2 ‘gewürztraminer’ 3 ‘rhine riesling’ 4 ‘italian riesling’ 5 ‘moravian muscat’ 6 ‘sauvignon’ 7 ‘blue portugal’ 8 ‘white chasselas’ 9 ‘red chasselas’ morphological and phenological characterization for the classical ampelography, the following method was applied. in the period 2006-2008, 22 morphological and six phenological traits were evaluated in 10 randomly chosen plants for each variety, according to the descriptors for grapevine (iprgi, 1997). all parameters chosen for the study are listed in table 2 with the codes of updated descriptors (oiv, 2009). digital image analysis of leaves, phyllometric parameters and fractal analysis for each variety, 50 healthy, fully-expanded leaves were collected from ten randomly selected plants in late spring 2008. leaf images were acquired (200 dpi, 256 greyscale) using an optical scanner. based on the method described by mancuso and nicese (1999), 14 phyllometric parameters (table 3) were determined for each leaf using image-analysis software (uthscsa image tool program 3.0). table 2 list of characteristics selected for the description of observed cultivars. the codes are in line with oiv guidelines (oiv, 2009) character code description 001 young shoot: aperture of tip 003 young shoot: intensity of anthocyanin colouration on prostrate hairs of tip 006 shoot: attitude 007 shoot: colour of dorsal side of internode 008 shoot: colour of ventral side of internode 051 young leaf: colour of the upper side of blade (4th leaf) 065 mature leaf: size of blade 067 mature leaf: shape of blade 068 mature leaf: number of lobes 070 mature leaf: area of anthocyanin colouration of main veins on the upper side of blade 076 mature leaf: shape of teeth 079 mature leaf: degree of opening/ overlapping of petiole sinus 102 woody shoot: structure of surface 103 woody shoot: main colour 202 bunch: length (peduncle excluded) 204 bunch: density 220 berry: length 223 berry: shape 241 berry: formation of seeds 225 berry: colour of skin 236 berry: particularity of flavour 303 time of beginning of berry ripening (véraison) 233 berry: must yield 502 bunch: weight of a single bunch 503 single berry weight 505 sugar content of must the codes in bold letters indicate the characteristics which were evaluated by anova. table 3 fourteen phyllometric parameters measured by image analysis software parameter definition 1 area the area of the leaf 2 perimeter the perimeter of the leaf 3 major axis length the length of the longest line that can be drawn through the leaf 4 minor axis length the length of the longest line that can be drawn through the leaf perpendicular to the major axis 5 roundness computed as: (4*π*area) / perimeter2 6 elongation the ratio of the length of the major axis to the length of the minor axis 7 feret diameter the diameter of a circle having the same area as the leaf 8 compactness computed as: sqrt(4*area/π) / major axis length 9 integrated density computed as the product of the mean grey level and the number of pixels in the image of the leaf 10 minimum grey level minimum grey level of the leaf 11 mean grey level mean grey level of the leaf 12 median grey level median grey level of the leaf 13 mode grey level mode grey level of the leaf 14 maximum grey level maximum grey level of the leaf 189 the fractal spectrum of the leaves was obtained using fractal image analysis software (harfa, harmonic and fractal image analyzer 4.9.1,), according to the method described by mancuso (2002). briefly, greyscale image of each leaf was thresholded for a grey value between 0 and 255 and the fractal dimension for each grey value was then assessed using the box counting method. the implementation of these methods has been described in detail by mancuso et al. (1999). after drawing the baseline (fractal dimension = 1) which separates the fractal (>1) from the non-fractal (<1) zone of the spectrum, five fractal parameters (first x, peak x, last x, peak y and total peak area) were calculated (fig.1 a). fig. 1 fractal spectrum of one leaf and identification of five parameters to be implemented in the ann. graphical representation of fractal parameters: theoretical situation (a); and a real value (b). artificial neural network analysis an artificial neural network (ann) was constructed as previously described in pandolfi et al. (2009 a). fourteen phyllometric parameters and five fractal parameters were used as input layers, and the nine grapevine accessions represented the output. to optimize the neural network activity, the number of hidden neurons and the number of iterations was modified. with regard to the hidden layer, many factors (such as learning scheme, numbers of nodes of the output and input and connections between them) play an important role for the determination of the best configuration (zurada and malinowsji, 1994). the ann outputs are represented by a xy-graph for each accession, with the accession names on the x-axis, and the yaxis representing the output. each graph aims to show how the ann was able to discriminate the selected accession in comparison with the others. the level of similarity is expressed by number, which ranges between 0 (false) and 1 (true) (fig. 1 b). due to the natural variability among leaves, the output of the expected class tends to report a value close to 1, but less than 1, while the others should be close to 0 (mugnai et al., 2008). statistical analysis statistical analysis of the selected characteristics was performed by analysis of variance (anova) and a comparision was done using the turkey hsd test (program statistica 7.0 cz). the graphical presentation of variability of the tested varieties was carried out using principal component analysis (pca) (software statistica 7.0 cz). ntsys 2.1 was used to investigate neural network outputs performing a cluster analysis by unweighted pair group method analysis (upgma) based on the similarity matrix calculated using the cosine function (eq. 1). equation 1: 3. results morphological data seven characteristics (table 2), as evaluated by anova, showed differences between the varieties during the three consecutive years of sampling, as well as variability within the variety. the number of lobes observed in the mature leaves varied during the study period in three varieties (‘müller thurgau’, ‘rhine riesling’, and ‘blue portugal’). a significant variation was observed both in the weigh of a bunch and the weight of berries, as well as in the size of berries within one variety. with regard to must yield and its sugar content, there was no significant variance among the varieties, however all the varieties demonstrated a significant correlation between the sugar content of the must and the time of berry ripening (r=0.74). the highest sugar content was measured in 2006, when the mean temperatures remained quite high during whole vegetative period (in july as high as 25˚c), whilst the total precipitation was about 5 mm in july. for most of the 19 characteristics that were not included in the anova analysis, there was considerable stability over the course of the years. the greatest variability was shown in the size of a blade of a mature leaf; it remained stable only in ‘rhine riesling’. in the other eight varieties, the size of the leaves varied significantly. correlations between morphological and phenological characters were also assessed. the output obtained from both morphologic and phenological characteristics after three years of observations (fig. 2) was a score plot crefig. 2 pca representation. 190 ated by pca analysis. according to the graph, factor 1 explained 29.02% of the total variation, whereas factor 2 expressed 22.67% of the total. evaluating both approaches (morphological and phenological), ‘red chasselas’ and ‘white chasselas’ were identified as being quite close, which indicated their relationship to one another. the second group was formed in the very centre of the graph and includes ‘blue portugal’, ‘italian riesling’ and ‘sauvignon’. more distant from the centre group and also from each other were ‘müller thurgau’ and ‘rhine riesling’. the only variety which remained completely separate was ‘gewürztraminer’. neural network analysis in all eight cases, the artificial neural network was able to recognise all the accessions presented in the learning phase. throughout there was a clearly defined peak for the selected accession; therefore the varieties were well-separated one from another. the highest level of similarity was present in ‘müller thurgau’ (0.810). however, all other accessions showed quite high average output in a range from 0.541 (‘rhine riesling’) to 0.657 (‘moravian muscat’) (fig. 3). fig. 3 neural networks of eight chosen varieties. ‘müller thurgau’ also showed the lowest degree of similarity with the other varieties and this was confirmed by the upgma diagram, where ‘müller thurgau’ remained completely separated from all other varieties. another interesting result was the position of both ‘chasselas’ in the neural network diagram and the final dendrogram. in the dendrogram, ‘red chasselas’ was more closely related to ‘italian riesling’ than to ‘white chasselas’. using the data from the neural network, euclidean distances were calculated and a dendrogram based on the distance matrix data, by applying an unweighted pair group method with average mean (upgma) cluster analysis, was constructed (fig. 4). fig. 4 upgma dendrogram. the dendrogram formed two clusters, and left two varieties more (‘müller thurgau’) or less (‘moravian muscat’) separated from the others. two other varieties, ‘rhine riesling’ and ‘sauvignon’, were indicated at the same level. even though they were the two most related samples, they still remained quite distant, with the similarity coefficient at 4.43. the results showed a closer relationship between ‘italian riesling’ and ‘red chasselas’ than the relationship between both ‘chasselas’, even though they are considered to be colour variations of the same variety. 4. discussion and conclusions in the present investigation all general characteristics which are common to vitis vinifera, without regarding the differences between the varieties (ipgri, 1997), were confirmed with regard to morphology for the varieties under study. there was only a slight variation in the number of lobes observed in the mature leaves, which is due to the genotype fixation of this trait, as proposed by poljuha et al. (2006). in three varieties (‘müller thurgau’, ‘rhine riesling’ and ‘blue portugal’) the number of lobes varied during the observation period. this way have occurred due to the unisindentification of the depth of the status. (cancellier, 2007). the size of a berry was strongly correlated either with the weight of a berry, or with the weight of a bunch. the smallest berries were observed in an important must variety 191 ‘rhine riesling’. this result corresponded with the findings of fregoni (2005), who indicated that varieties which produce wines of excellent quality generally have smaller berries with higher amount of skin and lower amount of pulp, in comparision with the table varieties. ojeda et al. (2001) pointed out that the size of a berry is an environment-dependent trait. water deficit that occurs in the early phase from flowering to véraison causes an irreversible decrease in cell volume. due to changes in water import by xylem, this may lead to a decrease of mesocarp cell turgor (thomas et al., 2006). under water stress, especially in the early growth stages, the division and elongation of cells decreases (williams, 2000), thus berries remain smaller. in 2008, when precipitation events were regularly distributed throughout the year, the berries of all nine varieties were larger compared to other observation years. sugar content, and thus the quality of the wine, is significantly influenced by the weather, as proven by grifoni et al. (2006) from observations of italian wines. a surprisingly low sugar content was found in ‘moravian muscat’, despite it being a variety bred in former czechoslovakia (pavloušek 1999), and thus adapted to its climatic conditions. however, in this case the sugar content was influenced by the early harvest date, since the grapes were used for the production of federweisser (a freshly fermented must with low alcohol content). the must yield was also strongly correlated to the size of the berry. according to fregoni (2005) table varieties have lower must yields than must varieties. however, ‘white chasselas’, despite being a table variety, gave the highest must yield during the three study years, while the must varieties, like for instance ‘blue portugal’, produced significantly lower quantities the must (57.00±6.25 ml). this may be the influence of rains right before harvest. in all eight cases, the artificial neural network was able to recognise all the accessions presented in the learning phase, as published for example by mancuso (2002) or mugnai et al. (2008). an interesting result was the position of ‘müller thurgau’, which remained separated from all other accessions. dettweiller et al. (2000) confirmed that ‘müller thurgau’ is the descendent of a crossing between ‘madeleine royal’ and ‘rhine riesling’. nevertheless, in our results ‘rhine riesling’ remained distant from ‘müller thurgau’, and was assessed as close to ‘sauvignon’, corresponding with the data published by moravcovà et al. (2004). as reported in their work (moravcová et al., 2004), ‘red chasselas’ is only a mutation of berry colour of ‘white chasselas’. in the dendrogram, however, ‘red chasselas’ was more closely related to ‘italian riesling’ than to ‘white chasselas’. comparing the dendrogram with the pca score plot, the results were slightly different. both ‘chasselas’ varieties were the closest, which corresponded with the results of moravcovà et al. (2004). but also in this case, there was no evident relationship between ‘rhine riesling’ and ‘müller thurgau’, since they occurred in different quadrants. unfortunately, the position of ‘gewürztraminer’ in the pca graph could not be compared with the ann output. however, the score plot put this variety completely separate from other varieties, e.g. ‘rhine riesling’, as already described by imazio et al. (2002). the great distance of ‘gewürztraminer’ from other varieties might also be due to its closer relationship to vitis silvestris (regner et al., 2000; lacombe et al., 2003). when using the ann approach it is very important to choose well-developed, healthy leaves (mugnai et al., 2008). an ann needs a suitable set of sample data to achieve excellent quality in the training set, as this operation can be usually defined as the key point of all the ann building process (pandolfi et al., 2009 a). unfortunately, the euclidean distance did not permit a clear clustering and thus in our case a pca graph could be considered a more precise tool, even though the data were mostly obtained from subjective observations. however, the possibility of a primary misidentification while still in the vineyard must always be considered: in early spring, when the plants do not have mature leaves or even grapes, an identification error may occur, especially when the varieties are not clearly divided. both approaches ampelography or artificial neural networks – are important methods based on morphological traits, which complement each other to give clear classification of the varieties. the ann technique represents an more economic alternative to genetic methods commonly used for cultivar discrimination since the equipment necessary for this analysis is inexpensive and commonly available (pandolfi et al., 2009 a). in this study, application of anns led to the distinction of nine selected varieties of vitis vinifera. for the further uses, the ann profiles obtained from our analysis may be kept in databases for breeding programs, as well as for the description of new cultivars. the data obtained might also be helpful for rapid and reliable identification and description of still unknown varieties, as shown by pandolfi et al. (2009 b). acknowledgements the authors would like to thank st. clair’s vineyard (prague botanic garden), especially martin beránek, ph.d., for his support and assistance during the experiment. we would also like to acknowledge an unknown reviewer for his/her kind and useful suggestions. references cancellier s., 2007 proposta di una guida per il riconoscimento dei vitigni. esempio: i vitigni di veneto. rivista di viticoltura e di enologia, 60(1): 3-16. cervera m.t., rodriguez i., cabezas j.a., chavez j., martínez-zapater j.m., cabello f., 2001 morphological and molecular characterization of grapevine accessions known as ‘albillo’. amer. j. of enol. and vitic., 52(2): 127-135. 192 dettweiler e., 1993 evaluation of breeding characteristics in vitis.vitis, 32: 249-253. dettweiler e., jung a., zyprian e., töpfer r., 2000 grapevine variety ‘müller-thurgau’ and its true-to-type descent. vitis, 39(2): 63-65. fregoni m., 2005 viticoltura di qualità. 2nd edition, phytoline s.r.l., affi, pp. 819. grifoni d., mancini m., maracchi g., orlandini s., zipoli g., 2006 analysis of italian wine quality using freely available meteorological information. amer. j. of enology and viticulture, 57(3): 339-346. imazio s., labra m., grassi f., winfield m., bardini m., scienza a., 2002 molecular tools for clone identification: the case study of the grapevine variety ‘traminer’. plant breeding, 121: 531-535. ipgri, 1997 descriptors for grapevine (vitis spp.). international union for the protection of new varieties of plants, geneva, switzerland; office international de la vigne et du vin, paris, france and international plant genetic resources institute, rome, italy. lacombe t., laucou v., di vecchi m., bordenave l., bourse t., siret r., david j., boursiquot j.m., bronner a., merdonoglu d., this p., 2003 contribution à la caractérisation et à la protection in situ des populations de vitis vinifera l. ssp. silvestris (gmelin) hegi, en france. les actes du brg, 4: 383-404. mancuso s., 1999 fractal geometry-based image analysis of grapevine leaves using the box-counting algorithm. vitis, 38(3): 97-100. mancuso s., 2002 discrimination of grapevine (vitis vinifera l.) leaf shape by fractal spectrum. vitis, 41(3): 137-142. mancuso s., nicese f.p., 1999 identifying olive (olea europaea) varieties using artificial neural networks. j. amer. soc. hort. sci., 124(5): 527-531. moravcová k., baránek m., pidra m., 2004 the use of rapd markers for differentiation of grapevine varieties registered in the czech republic. horticultural science (prague), 31 (3): 96-101. mugnai s., pandolfi c., azzarello e., masi e., mancuso s., 2008 camelia japonica l. genotypes identified by an artificial neural network based on phyllometric and fractal parameters. plant systematics and evolution, 270: 95-108. oiv, 2009 the 2nd edition of the oiv descriptors list for grape varieties and vitis species. office international de la vigne et du vin, paris, france. ojeda h., deloire a., carbonneau a., 2001 influence of water deficits on grape berry growth. vitis, 40(3): 141-145. ortiz j.m., martín j.p., borrego j., chávez j., rodríguez i., muñoz g., cabello f., 2004 molecular and morphological characterization of a vitis gene bank for the establishment of a base collection. genetic resources and crop evolution, 51: 403-409. pandolfi c., messina g., mugnai s., azzarello e., masi e., dixon k, mancuso s., 2009 b discrimination and identification of morphotypes of banksia integrifolia (proteaceae) by an artificial neural network (ann), based on morphological and fractal parameters of leaves and flowers. taxon, 58(3): 925-933. pandolfi c., mugnai s., azzarello e., bergamasco s., masi e., mancuso s., 2009 a artificial neural networks as a tool for plant identification: a case study on vietnamese tea accessions. euphytica, 166: 411-421. pavloušek p., 1999 vinohradnictví-odrůdy révy vinné. 1. vydání. mendelova lesnická a zemědělská univerzita v brně, brno, pp. 122. poljuha d., sladonja b., peršurić d., 2006 evaluation of intravarietal variability of cv. ‘istrian malvasia’ by the ampelographic parameters. rivista di viticoltura e di enologia, 2(3): 13-18. regner f., stadlbauer a., eisenheld c., kaserer h., 2000 considerations about the evolution of grapevine and the role of traminer. acta horticulturae, 528: 179-184. thomas t.r., matthews m.a., shackel k.a., 2006 direct in situ measurement of cell turgor in grape (vitis vinifera l.) berries during development and in response to plant water deficits. plant, cell and environment, 29: 993-1001. williams l.e., 2000 grapevine water relations, pp. 121126. in: christensen l.p. (ed.), raisin production manual. university of california, division of agriculture and natural resources, publication 3393, oakland, ca. zurada j.m., malinowsji a., 1994 multilayer perceptron networks: selected aspects of training optimization. international journal of applied mathematics and computer science, 4: 281-307. 75 1. introduction grape phylloxera (daktulosphaira vitifoliae), aphidlike gall-forming parasite, is an economically important homopteran pest of grape vines vitis vinifera l. world wide. in syria, there are more than 70,000 ha planted with grapevine and the annual production is about 540,000 t. grape phylloxera annually causes millions of dollars in losses in grape production. grape phylloxera causes direct damage to grapevine by forming damaging root galls. the fleshy galls formed on immature roots, by swelling of the root cortex, are called “nodosities”, while on mature roots they are called “tuberosities”; these latter are considered more damaging to the vine. these galls are metabolically active organs suited to match the nutritional requirements of phylloxera and can support populations with high reproductive rates, making this pest capable of destroying the root system of v. vinifera vines (granett et al., 2001). in most cases the swelling stops rootlet growth, and the affected portion dies. root injuries reduce the vines’ ability to absorb nutrients and water, causing decline in vigor and productivity. weakened plants probably become more susceptible to secondary infections from fungal diseases and other insects, and to environmental stresses. since there is not an effective control method for grape phylloxera, sanitation and quarantine can be considered required procedures to prevent the spread of this insect pest. insecticides and hot water dips are used as quarantine treatments (granett et al., 2001). makee et al. (2010) proposed that gamma irradiation could be economically very useful in quarantine treatments against phylloxera. however, once phylloxera is in a vineyard, the use of resistant rootstocks is the most common and effective means of managing phylloxera. it should be mentioned that some rootstocks were more resistant than others to grape phylloxera (makee et al., 2003). moreover, for unknown reasons, the resistance of some rootstocks may break down and farmers must replant vineyards (granett et al., 1983; song and granett, 1990; de benedictis and granett, 1993). because replanting is costly in money, time, and labor, additional ways to control this pest should be considered. all plants have active defense mechanisms against pathogen attacks. some plant growth-promoting rhizobacteria (pgpr) are able to reduce disease through the stimulation of inducible plant defense mechanisms that render the host plant more resistant to further pathogen ingress. this induced systemic resistance (isr) (pieterse et al., 2002) can be the basis of integrated plant disease management strategies (ramamoorthy et al., 2001; zehnder et al., 2001; saravanakumar et al., 2007). many studies in plants on pgpr against pathogens have been performed. however, only a few of them determined the protective effect of pgpr against insects (zehnder et al., 1997 a, b; zehnder et al., 2001; kloepper et al., 2004; vijayasamundeeswari et al., 2009; valenzuela-soto et al., 2010). a non-pathogenic pseudomonas putida strain (btp1) was shown to enhance the level the influence of a non-pathogenic pseudomonas putida strain btp1 on reproduction and development of grape phylloxera a. adam(1), h. makee, i. idris department of biotechnology, atomic energy commission of syria. p.o. box 6091, damascus, syria. key words: developmental time, fecundity, grape phylloxera, oviposition period, pgpr, pseudomonas putida btp1. abstract: some non-pathogenic rhizobacteria called plant growth promoting rhizobacteria (pgpr) possess the capacity to induce defense mechanisms effective in plant against pathogens. the effect of pseudomonas putida btp1 on reproduction and development of phylloxera, which infested the roots of our local grape variety “balady”, was evaluated. our results showed that the life table of grape phylloxera was different between treated and control plants. the percentage of matured females, developmental time, fecundity and oviposition period were reduced when plants were treated with bacteria. the results showed that the phylloxera resistance was influenced by root soaking duration in p. putida btp1 suspension. the present study provides good information on the possibility of using pseudomonas putida btp1 to increase the resistance of grape to phylloxera. adv. hort. sci., 2012 26(2): 75-80 (1) corresponding author: ascientific@aec.org.sy received for publication 1 march 2012. accepted for publication 7 june 2012. 76 of resistance in cucumber, bean and tomato against the fungal pathogens pythium aphanidermatum and botrytis cinerea, respectively (ongena et al., 1999; adam et al., 2008). these studies revealed that the disease-protective effect was associated with stimulation of defense mechanisms in host plant (ongena et al., 2000, 2004; adam et al., 2008). the main objective of the present study was to evaluate the ability of strain p. putida btp1 to protect grape roots against grape phylloxera. thus, the effect of concentrations and root soaking duration in p. putida btp1 suspension on percentage of matured females, developmental time, fecundity and oviposition period of local phylloxera strain were determined. 2. materials and methods establishment of the phylloxera colony grape phylloxera was originally collected from fieldinfested roots of the local grape varieties in southern parts of syria. the phylloxera colony was established following similar procedures to those mentioned by makee et al. (2003). fresh and healthy pieces of roots (4-7 mm in diameter and 5-7 cm long) of local grape cultivar helwani (v. vinifera) were taken and washed with tap water. each piece was wrapped with moist cotton wool around one end, and then 10 to 15 phylloxera eggs were placed on each piece. the infested root pieces were then placed on a wet filter paper disk inside a plastic petri dish (12 cm diameter). each dish had three to four root pieces. for ventilation purposes the petri dish lid was modified with a 1-1.5 cm cloth-screened hole. the edges of the dishes were sealed with parafilm and they were kept in plastic boxes with tightly fitting lids and incubated at 25±1°c, 70±5% rh and 24 hr darkness. the root pieces were replaced when they desiccated, rotted or the phylloxera became crowded. microbial strains and inoculum preparation p. putida strain btp1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron transport (jacques et al., 1995; ongena et al., 2002); it was maintained and prepared for use in the isr assays as previously described by ongena et al. (2002). for bioassays, two different concentrations (108 and 2x108 cfu/ml) of bacterial suspension were prepared. effect of bacteria-treated roots on phylloxera fresh root pieces were soaked for 3 hr in solutions with various p. putida concentrations: 0 (roots were soaked in distilled water as a control), 108, and 2x108 cfu/ml. all root pieces were then left to air-dry. for each concentration five root pieces were taken. each root piece was infested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°c, 70±5% rh and 24 hr darkness. evaluation procedure a daily microscope inspection of all phylloxera stages on all root pieces was carried out. the number of eggs hatched, feeding nymphs and adults were detected to calculate the percentage of emerged mature females on each root piece at each concentration. also, the mean developmental time (egg to egg) was determined. fecundity (total number of eggs) of phylloxera was evaluated by randomly choosing five individuals of root-feeding phylloxera females on each root piece at each concentration. thus, at each tested concentration 20 females were examined. all eggs laid by each female were observed and counted till the female’s death. additionally, the oviposition period (the time from the first laid egg to the natural death of individual ovipositing females) was recorded for the females chosen for the fecundity measurement. effect of root soaking duration in bacterial suspension on phylloxera fresh root pieces were soaked in solutions with various p. putida concentrations: 0, 108, and 2x108 cfu/ml. at each tested concentration the root pieces were soaked for various periods: 0, 3, 5 and 15 hr. all root pieces were then left to air-dry. at each concentration and soaking duration, five root pieces were taken. each root piece, at each concentration and soaking duration, was infested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°c, 70±5% rh and 24 hr darkness. the same evaluation procedure as described above was followed to determine the percentage of emerged mature females, mean developmental time, fecundity and the oviposition period of phylloxera. statistical analysis all statistical analyses were performed using statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). data were subjected to analysis of variance (anova) for the determination of differences in means between tested plants at each dose. differences between means were tested for significance using tukey hsd test. 3. results effect of bacterial treated roots on phylloxera table 1 shows that when root pieces were treated with p. putida, the percentage of emerged matured females was significantly affected compared to the control (f=84.69; df=15, 64; p<0.05). however, the percentage emerged of matured females was not significantly increased by increasing p. putida concentrations. the result illustrates that the developmental time of phylloxera was significantly decreased by the application of p. putida (f= 15; df = 2, 42; p < 0.05). there was a significant reduction in developmental time as the concentration of p. putida was increased (table 1). there were significant differences in the mean number of laid eggs between all tested root pieces, regardless of concen77 tration (f = 19.9; df = 2, 42; p< 0.05). on untreated root pieces, the mean number of eggs was significantly higher than that on treated ones (table 1). when phylloxera females were reared on p. putida-treated root pieces, the mean number of eggs was markedly reduced by increasing p. putida concentration. table 1 shows that the oviposition period of phylloxera on untreated root pieces was significantly longer than that on treated ones, irrespective of the concentration of p. putida (f = 25.5; df = 2, 42; p < 0.05). there was a significant difference in the oviposition period between p. putida-treated root pieces. the oviposition period on 108 cfu/ml-treated root pieces was significantly longer than that on 2x108 cfu/ml-treated ones. effect of root soaking duration in bacterial suspension on phylloxera there was a significant effect of the soaking duration in p. putida suspension on the percentage emerged of matured females, regardless of the concentration (f= 620.87; df= 11, 24; p<0.05) (fig. 1). at each concentration, the percentage of emerged matured females on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. when the root pieces were soaked, the percentage of emerged matured females with 3 hr soaking duration significantly differed from that on 5 and 15 hr, whatever the p. putida concentration. at each p. putida concentration, there was no significant difference in the percentage of emerged matured females between 5 and 15 hr soaking duration (fig. 1). there was a significant effect of soaking in p. putida suspension on the mean developmental time, regardless of the soaking duration and concentration (f= 16; df= 11, 168; p< 0.05) (fig. 2). at each concentration, the mean developmental time on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. the result illustrates that at 2x108 cfu/ml the observed differences in mean development between soaking durations were not significant. there was a significant effect of the soaking duration in p. putida suspension on mean number of eggs, regardless of the concentration (f= 112; df = 11, 168; p<0.05) (fig. 3). at each concentration, mean number of eggs on soaked root pieces was significantly higher than that on un-soaked ones, regardless of soaking duration. at concentration 0 (root pieces soaked with distilled water), there was no significant difference in the mean number of eggs between 3 and 5 hr soaking duration. however, the mean number of eggs was significantly reduced with 15 hr soaking duration. at 108 and 2x108 cfu/ml concentrations, the mean number of eggs on 3 hr soaking duration was significantly higher than that on 5 and 15 hr (fig. 3). table 1 mean percentage of matured females, developmental time, fecundity and oviposition when phylloxera was reared on p.putida-treated grape root pieces oviposition period/d (mean±se) fecundity (mean±se) developmental time/d (mean±se) % matured female (mean±se) bacterial concentration cfu/ml 14.2±0.54 a 44.73±0.85 a 28.27±0.57 a 78.16±1.48 a 0 12.07±0.27 b 40.07±1.3 b 26.53±0.27 b 36.33±0.88 b 108 9.53±.53 c 34.2±1.3 c 24.53±0.54 c 32.03±1.49 b 2x108 means, in a column, followed by the same letter are not significantly different at the p < 0.05 (tukey hsd test). fig. 1 effect of root soaking duration in bacterial suspension of percentage emerged matured females of phylloxera. fig. 2 effect of root soaking duration in bacterial suspension on mean developmental time of phylloxera. fig. 3 effect of root soaking duration in bacterial suspension on mean number of eggs of phylloxera. 78 a significant effect of the soaking duration in p. putida suspension on mean oviposition period was found, regardless of the concentration (f= 46; df = 11, 168; p<0.05) (fig. 4). at each concentration, the mean oviposition period on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. at both 0 and 108 cfu/ml, the mean oviposition period with 3 hr soaking duration was significantly higher than that with 15 hr. at 0 concentration, there was no significant difference in the mean oviposition period between on 3 and 5 hr soaking duration, while at 108 cfu/ml there was. at 2x108 cfu/ml, there was no significant difference in the mean oviposition period between 3, 5 and 15 hr soaking duration. 4. discussion and conclusions several studies reported the use of rhizobacteria as a biological control of pests (racke and sikora, 1992; zehnder et al., 1997 a, b). to our knowledge, the application of rhizobacteria against phylloxera has not been investigated. our study indicates that when p. putida btp1-treated roots were infested by phylloxera eggs, the percentage of matured females was negatively influenced (table 1). the result illustrates that the percentage of matured females was significantly decreased when the concentration and the soaking duration were increased (fig. 1). the reduction of the number of matured females on the treated roots could be attributed to the inability of phylloxera to feed. thus, nymphs fed on p. putida btp1-treated roots showed antifeeding behaviour. when heliothis zeae (boddie) diet was contaminated with the bacterium pseudomonas maltophila, a 60% reduction in adult emergence and high pupal and adult malformations were observed (bong and sikorowski, 1991). moreover, when chestnut was treated with pseudomonas fluorescens, 20% chestnut weevil mortality was recorded (yaman et al., 1999). therefore, the antifeeding behaviour of phylloxera nymphs on the pgpr treatments could be related to the reduction in the feeding stimulant. consequently, the developmental time was altered leading to early emergence of matured females (table 1). a comparison between p. putida btp1-treated and untreated roots showed that the phylloxera development on treated roots was faster than that on untreated ones. similar results were reported when cucumber beetles and american bollworm were fed on pgpr-treated cucumber plants and cotton bolls, respectively (zehnder et al., 1997 a, b; vijayasamundeeswari, 2009). our current study indicates that the developmental time was significantly shorter on treated root pieces soaked for 15 hr (fig. 2). qingwen et al. (1988) detected a reduction of relative growth rate, consumption rate and digestibility of feed when heliothis armigera fed on p. gladioli-treated cotton plants. it is known that fecundity could be considered an essential factor in assessing the effect of p. putida btp1on phylloxera. phylloxera fecundity on p. putida btp1-treated root pieces was distinctly lower than that on untreated ones. when phylloxera females were reared on treated roots they were unable to produce a normal number of eggs compared to the control, especially at high concentration (table 1). nevertheless, when soaking duration in p. putida btp1 suspension was prolonged, a defective reproductive capacity was obtained (fig. 3). the average number of eggs was 65.3, 40, 32 and 21.6 eggs on 108 cfu/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, respectively. while on 2x108 cfu/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, the average number of eggs was 63.7, 34.2, 23.6 and 17.5 eggs, respectively. inadequate nutrition and inability to establish good feeding sites could directly affect the number of eggs laid. therefore, phylloxera resistance could be reflected in a strong relationship between poor feeding and the reduction of insect reproduction (granett et al., 1983). thus, phylloxera produced more eggs on untreated roots compared with treated ones. correspondingly, the oviposition period of phylloxera on treated roots was markedly shorter than that on untreated ones (table 1). when the concentration of p. putida btp1 was increased a great reduction in the oviposition period was obtained. such reduction was noticeably increased by prolonging the root soaking duration in p. putida btp1 (fig. 4). it is known that the resistance mechanisms of phylloxera could be related to several factors including: 1) reduction of phylloxera fitness (antibiosis); 2) decrease in plant attractiveness to phylloxera (antixenosis) (granett et al., 2001). zehnder et al. (2001) mentioned that the pgpr treatment led to alteration in the plant metabolic pathway which elicited the induction of plant defense compounds. qingwen et al. (1998) reported that polyphenol and terpenoid content were increased with cotton treated with p. gladioli. the synthesis and formation of such materials in p. putida btp1-treated roots would have a negative influence on phylloxera feeding and development. the results illustrate that the soaking of grape roots with p. putida btp1 suspension for 5 hr and 15 hr, regardless of concentration, led to a great reduction in matured females, developmental time, fecundity and oviposition fig. 4 effect of root soaking duration in bacterial suspension on mean oviposition period of phylloxera. 79 period. such reduction might be attributed to increased bacterial cell concentration by prolonging the soaking duration. therefore, roots pieces soaked with 2x108 cfu/ml for 15 hr showed more resistance to phylloxera than lower concentrations and a shorter soaking duration. these results provide essential information about the relationship between p. putida btp1 and phylloxera resistance of grape plants. phylloxera encounters serious difficulties in surviving, feeding and reproducing on p. putida btp1-treated roots. p. putida btp1 reduced the susceptibility of roots to phylloxera. nevertheless, in future it is essential to determine for how long such effects could persist. therefore, we acknowledge that these laboratory studies cannot simulate all conditions that exist in nature. therefore, care must be taken when laboratory-based results are applied in nature. acknowledgements the authors thank prof. i. othman (atomic energy commission of syria) and dr. n. mirali (department of biotechnology) for their help. we thank prof. p. thonart and dr. m. ongena of the university of liège, who gave us the p. putida btp1 strain. references adam a., ongena m., duby f., dommes j., thonart p., 2008 systemic resistance and lipoxygenase-related defence response induced in tomato by pseudomonas putida strain btp1. bmc plant biology, 8: 113. bong c.f.j., sikorowski p.p., 1991 effects of cytoplasmic polyhedrosis virus and bacterial contamination on growth and development of corn earworm, heliothis zeae (boddie). j. invertebr. pathol. 57: 406-412. de benedictis j., granett j., 1993 laboratory evaluation of grape roots as host of california grape phylloxera biotypes. am. j. enol. vitic., 44: 285-291. granett j., bisabri-ershadi b., carey j., 1983 life tables of phylloxera on resistant and susceptible grape rootstocks. ent. exp. & appl., 34: 13-19. granett j., walker m., kocsis l., omer a., 2001 biology and management of grape phylloxera. annual review entomology, 46: 387-412. jacques p., ongena m., gwose i., seinsche d., schroder hr., delfosse p., thonart p., taraz k., budzikiewicz hr., 1995 structure and characterization of isopyoverdin from pseudomonas putida btp1 and its relation to the biogenetic pathway leading to pyoverdines. z. naturforsch., c, j. biosci., 50: 622-629. kloepper j.w., ryu c.m., zhang s.a., 2004 induced systemic resistance and promotion of plant growth by bacillus spp. phytopathology, 94: 1259-1266. makee hr., charbaji t., ayyoubi z., idris i., 2003 evaluating resistance of some rootstocks to grape phylloxera with an in vitro and excised root testing systems. in vitro cell. dev. biol.-plant, 40: 225-229. makee hr., charbaji t., idris i., taher n., 2010 an in vitro assessment of interaction between grape phylloxera and indol acetic acid-treated grape plants. adv. hort. sci., 24: 99-103. ongena m., daayf f., jacques p., thonart p., benhamou n., paulitz t.c., belanger r.r., 2000 systemic induction of phytoalexins in cucumber in response to treatments with fluorescent pseudomonads. plant pathol., 49: 523-530. ongena m., daayf f., jacques p., thonart p., benhamou n., paulitz t.c., cornelis p., koedam n., bélanger r.r., 1999 protection of cucumber against pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and antibiosis. plant pathol., 48: 66-76. ongena m., duby f., rossignol f., fauconnier m.l., dommes j., thonart p., 2004 stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic pseudomonas strain. mol. plant microbe interact., 17: 1009-1018. ongena m., giger a., jacques p., dommes j., thonart p., 2002 study of bacterial determinants involved in the induction of systemic resistance in bean by pseudomonas putida btp1 eur. j. plant pathol., 108: 187-196. pieterse c.m.j., van wees s.c.m., ton j., van pelt j.a., van loon l.c., 2002 signaling in rhizobacteria-induced systemic resistance in arabidopsis thaliana. plant biol. 4: 535-544. qingwen z., ping l., gang w., qingnian c., 1998 on the biochemical mechanism of induced resistance of cotton to cotton bollworm by cutting off young seedling at plumular axis. acta phytolacca sinica, 25: 209-212. racke j., sikora r.a., 1992 isolation, formulation and antagonistic activity of rhizobacteria towards the potato cyst nematode, globodera pallida. soil biol. biochem., 24: 521-526. ramamoorthy v., viswanathan r., raguchander t., prakasam v., samiyappan r., 2001 induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and diseases. crop prot., 20: 1-11 saravanakumar d., vijayakumar c., kumar n., samiyappan r., 2007 pgpr-induced defense responses in the tea plant against blister blight disease. crop prot., 26: 556-565. song g.c., granett j., 1990 grape phylloxera (homoptera: phylloxeridae) biotypes in france. j. econ. entomol., 83: 489-493 valenzuela-soto j.hr., estrada-hernandez m.g., ibarra-laclette e., delano-frier j.p., 2010 inoculation of tomato plants (solanum lycopersicum) with growth-promoting bacillus subtilis retards whitefly bemisia tabaci development. planta, 231: 397410. vijayasamundeeswari a., ladhalakshmi d., sankaralingam a., samiyappan r., 2009 plant growth promoting rhizobacteria of cotton affecting the developmental stages of helicoverpa armigera. j. plant protection res., 49: 239-243. 80 yaman m., demirbag z., belduz a.o., 1999 investigations on the bacterial flora as a potential biocontol agent of chestnut weevil, curculio elephas (coleoptera: curculionidae) in turkey. afr. j. mycol biotechnol., 54: 625-630. zehnder g., kloepper j., tuzun s., yao c., wei g., chambliss o., shelby r., 1997b insect feeding on cucumber mediated by rhizobacteria-induced plant resistance. entomologia experimentalis et applicata, 83: 81-85. zehnder g., kloepper j., yao c., wei g., 1997 a induction of systemic resistance in cucumber against cucumber beetles (coleoptera: chrysomelidae) by plant growth-promoting rhizobacteria. journal of economic entomology, 90: 391-396. zehnder g.w., murphy j.f., sikora e.j., kloepper j.w., 2001 application of rhizobacteria for induced resistance. eur. j. plant pathol., 107: 39-50. 20 1. introduction the genus festuca includes more than 360 species that differ widely in appearance. less than ten species are used as turf, all in cool climates. festuca arundinacea schreb., tall fescue, is a deep-rooted, cool-season perennial grass. it shows vigorous growth in spring and autumn, and its extensive root system helps it to withstand drought conditions. the species is adapted to a wide range of soil and climatic conditions, but performs best where winter is rather mild. its requirement for relatively high mowing times limits its use as lawns in parks, golf course roughs and other areas mowed at 40 mm or more (wiecko, 2006). zeolites are crystalline, hydrated alumino silicates of alkali and earth metals that possess infinite, threedimensional crystal structures. they are further characterized by an ability to lose and gain water reversibly and exchange some of their constituent elements without major changes in structure. nearly 50 natural species of zeoiltes have been recognized and more than 100 species without natural counterparts have been synthesized in the laboratory (breck, 1974; meier and olsen, 1987; mumpton, 1999).the great effectiveness of zeolites as natural sources of trace elements supplementing npk and their high adsorption ability have been reported (kolyagin and kucherenko, 2003). therefore, natural zeolites, due to their structure and properties (inert and non-toxic), can be used as slow-release carriers of fertilizers (ramesh et al., 2011). a 10% addition of clinoptilolite to sand used in the construction of golf-course greens substantially reduced no 3 -leaching and increased fertilizer-n uptake by creeping bent-grass without disturbing the drainage, compaction, or playability of greens (ferguson et al., 1986; nus and brauen, 1991; hung, 1992). the addition of nh 4 + exchanged clinoptilolite in greenhouse experiments resulted in 59 and 53% increase in root weight of radishes in medium with light clay soil (lewis et al., 1984). the water efficiency of surface irrigation systems in iran is low and nitrogenous fertilizers should be added to soil at a higher level. therefore leaching of nitrogen fertilizers occurs resulting in underground water pollution particularly in soils with light texture. clinoptilolite, a naturally occurring zeolite with high exchange capacity, may be used to absorb ammonium and retard excess leaching of nitrate. these facts dictate improving water and fertilization management to decrease the pollution of underground water resources (abdi et al., 2004). the object of the present study was to investigate the effects on tall fescue growth of applying different amounts of natural zeolite and nitrogen to growth medium. 2. materials and methods a greenhouse pot trial was conducted in 2009 at the faculty of agriculture, shiraz university, shiraz, iran, at badjgah, 1810 m above mean sea level, 29° 36´n and 52° growth of tall fescue (festuca arundinacea schreb.) seedlings sown in soil mixed with nitrogen and natural zeolite s. eshghi (1), m. bahadoran, h. salehi department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: clipping, soil mixture, vegetative variables. abstract: to determine the interaction of nitrogen and natural zeolite in culture medium on the vegetative growth of tall fescue (festuca arundinacea schreb.) ‘starlet’ a greenhouse experiment was conducted. a complete randomized design with factorial arrangements including two factors (nitrogen and zeolite) was employed for each treatment with four replications. treatments of nitrogen were 0, 0.06 and 0.12 g kg-1 in the soil mixture and treatments of zeolite were 0, 10, 20 and 30 g kg-1 in the soil mixture. application of zeolite and nitrogen had different effects on seedling height, fresh and dry weights of clippings before first, second and third mowings, chlorophyll and nitrogen content of clippings, and dry weights of roots. adding zeolite at the rate of 30 g kg-1 and nitrogen at the rate of 0.12 g kg-1 to culture medium significantly increased the height of turf seedlings. it is concluded that zeolite could absorb and slowly release nitrogen to the culture medium. adv. hort. sci., 2014 28(1): 20-24 (1) corresponding author: eshghi@shirazu.ac.ir received for publication 4 january 2014 accepted for publication 21 march 2014 21 32´e on tall fescue (festuca arundinacea schreb.) ‘starlet’ to evaluate n uptake from soil mixture with different amounts of zeolite. the soil samples were collected for soil nutrition analysis before the seeds were planted (table 1). seeds of tall fescue cultivar of starlet were planted in 3 kg pots with soil mixture (1:1 v/v field soil and sand). the soil mixture of pots contained 0, 10, 20 and 30 g kg-1 zeolite and 0, 0.06 and 0.12 g kg-1 nitrogen (in the form of urea) in different treatments (4 levels of zeolite × 3 levels of nitrogen × 4 replication= 48 pots). plants were maintained in a greenhouse under natural light (>850 µ mol m-2 s-1) with diurnal temperature 26±3°c and nocturnal temperature 20±3°c, and rh of 56±4%. turf grasses were irrigated every four days in spring and every two days in summer. one month after seed germination the first mowing was carried out; second and third mowings occurred 2 and 3 months after germination of seeds, respectively. seedling height before first, second and third mowings, fresh and dry clipping weights after each mowing, chlorophyll and nitrogen content of clipping, and dry weights of roots were measured. seedling heights were measured. chlorophyll content was determined by spectrophotometric method (saini and buvalda, 1998). the total amount of nitrogen (n) was measured using the kjeldahl digestion method. clippings and roots were weighed for fresh weight and then oven dried at 70oc (karl kolb 112sl) for 48 h and weighed for dry weight. this research was carried out in a complete randomized design with factorial arrangements including two factors (nitrogen and zeolite) for each treatment with four replications from april to august 2009. data were analyzed by mstatc software and mean values were compared using the lsd test at 5% level. 3. results seedling height before first, second, and third mowings (cm) increasing the content of zeolite in the culture medium to 0.12 g kg-1nitrogen, seedling height increased before all mowing times. the effect of zeolite alone on seedling height had shown increase, this increase was significant at 30 g kg-1 zeolite in soil mixture before first and second times of mowing. increasing the nitrogen concentration in the soil mixture had no regular and significant effect on seedling height before the three mowing times when compared to untreated control (table 2). fresh and dry clipping weights after first, second, and third mowings (g) as indicated in figures 1, 2, and 3, an increase in zeolite and nitrogen together in the soil mixture let to greater fresh table 1 some physical, chemical and nutritional characteristics of the soil used in the study cec ph n % p (mg kg-1) k (mg kg-1) oc (%) s.p. clay (%) silt (%) sand (%) 1.3 7.53 0.26 27.5 400 2.72 26 12 34 54 table 2 effect of nitrogen and zeolite on seedling height before first, second and third mowings of festuca arundinaceae schreb ‘starlet’ seedling height before first mowing (cm) means of zeolite seedling height before second mowing (cm) means of zeolite seedling height before third mowing (cm) means of zeolitezeolite g·kg-1 soil mixture zeolite g·kg-1 soil mixture zeolite g·kg-1 soil mixture 0 0.06 0.12 0 0.06 0.12 0 0.06 0.12 nitrogen g kg-1 in soil mixture 0 20.50 ab* 18.25 abc 13.75 cd 17.50 b 17.50 bcd 18.00 bcd 13.50 d 16.33 b 15.25 a 13.50 abc 11.25 bc 13.33 ab 10 22.50 ab 13.25 d 22.75 ab 19.50 ab 19.00 abc 13.50 d 18.75 abcd 17.08 b 14.25 abc 11.00 c 13.75 abc 13.00 b 20 16.50 bcd 20.0 abcd 22.00 ab 19.50 ab 16.00 cd 17.75 bd 22.00 ab 18.58 b 13.50 abc 14.00 abc 13.50 abc 13.67 ab 30 21.50 ab 23.00 ab 24.25 a 22.92 a 20.25 abc 22.00 ab 24.00 a 22.08 a 15.00 ab 15.25 a 15.50 a 15.25 a mean of nitrogen 20.25 a 18.63 a 20.69 a 18.19 a 17.81 a 19.56 a 14.50 a 13.44 a 13.50 a * in each column and row means followed by the same letter(s) (small letters for means and capital letters for means of rows and columns) are not significantly different using lsd test at 5% level. fig. 1 effect of nitrogen and zeolite on clipping fresh weight after first mowing (g) of festuca arundinaceae schreb‘starlet’.. 22 and dry weights of the clippings from the first, second, and third mowings. the means of zeolite had shown increase with increasing in zeolite content of soil mixture and this increase was significant only at 30 g kg-1 zeolite in soil mixture at fresh weight of clipping after all times of mowings. also 30 g kg-1 zeolite in soil mixture had shown significant increase at dry weight of clipping after first and second times of mowings. the mean nitrogen content in the soil mixture had no significant effect on the fresh and dry weights of clippings for all mowing times (figs. 1, 2, 3, 4, 5 and 6). chlorophyll and nitrogen content of clippings with increasing nitrogen and zeolite in the soil mixture, the chlorophyll content of clippings decreased in most treatments but it was not significant when compared with control. maximum chlorophyll content of clippings was found at 10 g/kg zeolite with 0.12 g/kg nitrogen in the soil mixture. the nitrogen content of clippings increased when zeolite and nitrogen increased in most treatments but this increase was not significant compared to control. maximum nitrogen content of clippings was noted at 0 g/ kg of zeolite and 0.12 g/kg of nitrogen in the soil mixture (table 3). dry weight of roots results of this study indicate that by increasing the zeolite content alone in culture medium, the mean dry weight of roots (compared with control) increased but not significantly. an increase in the culture medium of nitrogen alone did not significantly effect the mean of root dry weight; with increasing nitrogen only, dry weight of roots decreased. however, adding nitrogen and zeolite together to the culture medium increased the dry weight of roots (fig. 7). fig. 2 effect of nitrogen and zeolite on clipping dry weight after first mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 3 effect of nitrogen and zeolite on clipping fresh weight after second mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 4 effect of nitrogen and zeolite on clipping dry weight after second mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 5 effect of nitrogen and zeolite on clipping fresh weight after third mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 6 effect of nitrogen and zeolite on clipping dry weight after third mowing (g) of festuca arundinaceae schreb‘starlet’ 23 4. discussion and conclusions application of zeolite and nitrogen to the culture medium increased the seedling height before all mowing times. zeolite absorbs available n (nh 4 +) and releases it gradually to the medium for use by plants. results of our study indicate that nitrogen alone did not increase seedling height but combined treatment with zeolite and nitrogen increased seedling height at most levels. the findings of the present study are in agreement with those of hung and petrovic (1995) who reported that the application of zeolite improved nitrogen efficiency in soil about 16 to 22%. furthermore, zeolite reduced the leaching of ammonium and nitrate 86 to 99% from the soil. zeolite, with a high cation exchange capacity (cec), causes easy storage and release of nitrogen. omar et al. (2011) reported that the application of peat soil water and zeolite with urea significantly increased dry matter, n, p, k uptake and use efficiency in maize plants compared with urea without additives. our results are in accordance with those of sepaskhah and barzegar (2010) who showed that nitrogen and zeolite application resulted in higher grain protein contents in rice and nitrogen recovery efficiency. also aghaalikhani et al., (2011) reported that amending soil with zeolite, reduced nitrogen leaching, increased canola yield and nitrogen useefficiency by avoiding nitrogen leaching and improving soil physical properties so it may be a beneficial approach to decrease chemical fertilizer application rates and develop sustainable agriculture. in the present study, where zeolite and nitrogen were applied to the soil mixture, the chlorophyll content of clippings decreased while the nitrogen content increased. perhaps increased nitrogen resulted in a decrease of some elements and the amount of chlorophyll. our results are in disagreement with abdi et al. (2004) and nazari et al. (2007) who reported that zeolite increases the chlorophyll content of strawberry and african marigold. since the application of zeolite and nitrogen, compared to nitrogen application alone, increased the fresh and dry weights of clippings, it can be concluded that natural zeolite with its high exchange capacity may absorb ammonium and release it slowly. these results are in agreement with abdi et al. (2004) who reported that zeolite application increased shoot dry weight in strawberry. therefore, zeolite balances the amount of nitrogen availability for plants and retards excess leaching of nitrate. in conclusion, natural zeolite as a slow-release compound may be recommended along with nitrogen for use in growth media to improve growth of tall fescue. acknowledgements authors wish to thank mr. pourkhaloie, mr.jozi, miss kakoie and mr. zadnour for their help with the experiment. references abdi g.h., khosh-khui m., eshghi s., 2004 effects of natural zeoilte on growth and flowering of strawberry (fragaria × ananassa duch.). intern. j. agric. res., 1: 384-389. aghaalikhani m., gholamhoseini m., dolatabadian a., khodaei-joghan a., sadat asilan k., 2011 zeolite influences on nitrate leaching, nitrogen-use efficiency, yield and yield components of canola in sandy soil. archives agron. soil sci. doi., 10.1080/03650340.2011.572876. fig. 7 effect of nitrogen and zeolite on clipping dry weight of roots (g) of festuca arundinaceae schreb‘starlet table 3 effect of nitrogen and zeolite on chlorophyll and nitrogen content of clippings of festuca arundinaceae schreb ‘starlet’ chlorophyll content of clipping means of zeolite nitrogen content of clipping means of zeolitezeolite g·kg-1 soil mixture zeolite g·kg-1 soil mixture 0 0.06 0.12 0 0.06 0.12 nitrogen g kg-1 in soil mixture 0 20.40 a* 17.47 a 19.60 a 19.16 a 01.12 ab 01.22 ab 03.36 a 01.90 a 10 13.85 a 14.78 a 20.86 a 16.50 a 01.49 ab 01.37 ab 01.45 ab 01.43 a 20 19.98 a 15.04 a 17.25 a 17.42 a 01.01 b 01.40 ab 00.925 b 01.11 a 30 14.89 a 16.55 a 13.22 a 14.89 a 01.12 ab 01.41 ab 01.79 ab 01.44 a means of nitrogen 17.28 a 15.96 a 17.73 a 01.18 a 01.35 a 01.88 a * in each column and row means followed by the same letter(s) (small letters for means and capital letters for means of rows and columns) are not significantly different using lsd test at 5% level. 24 breck d.w., 1974 zeolite molecular sieves: structure, chemistry and use. wiley j., and sons inc., london, uk, pp. 771. ferguson g.a., ppper i.a., kneebone w.r., 1986 growth of creeping bentgrass on a new medium for turfgrass growth: clinoptilolite zeolite-amended sand. agron. j., 78: 1095-1098. hung z.t., 1992 clinoptilolite zeolite as amendment of sand for golf green root zones. ph.d. thesis, cornell univ., ithaca, ny, usa. hung z.t., petrovic a.m., 1995 clinoptilolite zeolite effect on evaporation rate and shoot growth rate of bentgrass on sand base grass. j. turfgrass manag., 25: 35-39. kolyagin y.s., kucherenko s.p., 2003 yield and longterm effect fertilizers. sakharnaya-svekla., 3:17-18. lewis m.d., moore f.d., goldsberry k.l., 1984 clinoptilolite a fertilizer n exchanger. hortscience, 18: 235-239. meier w.m., olsen d.h., 1987 atlas of zeolite structure types. butterworths, london, uk, 152. mupton f.a., 1999 la roca majica: use of natural zeolites in agriculture and industry. proc. natl. acad. sci. usa, 96: 3463-3470. nazari f., khosh-khui m., eshghi s., salehi h., 2007the effect of natural zeolite on vegetative growth, flower and physiological characteristics of african marigold (tagets erecta l. “queen”. hort. environ. biotechnol., 48(8): 241-245. nus j.l., brauen s.e., 1991 clinoptilolitic zeolite as an amendement for establishment of creeping bentgrass on sandy media. hortscience, 26: 117-119. omar l., ahmed o.h., majid n.m.a., 2011 enhancing nutrient use efficiency of maize (zea mays l.) from mixing urea with zeolite and peat soil water. intern. j. physic. sci., 6(14): 3330-3335. ramesh k., biswas a.k., somasundaram j., rao a.s., 2011 nanoporous zeolites in farming: current status and issues ahead. curr. sci., 99(6): 760-764. saini g.s., buvalda j.b., 1998 kiwifruit, pp. 135-156. in: schaffer b., and p.c. anderson (eds.) handbook of environmental physiology of fruit crops: temperate crops. crc press. inc. boca raton, florida, usa, pp. 112. sepaskhah a.r., barzegar m., 2010 -yield, water and nitrogen-use response of rice to zeolite and nitrogen fertilization in a semi-arid environment. agric. water manage., 98: 38-44. wiecko g., 2006 fundamentals of tropical turf management. biddles ltd, king’s lynn, uk, pp. 205. impaginato 103 1. introduction seashore paspalum is among compatible warmseason turfgrasses used for recreational sites, such as golf courses, which requires low insecticide and fertilizer applications and is tolerant to salt (duncan, 1997). seashore paspalum is found between 35° n-s latitudes in the americas and expands to several islands of the caribbean-atlantic-pacific rim and the mediterranean-african coastal areas. this species lacks winter hardiness (duncan, 1997); it is sensitive to freezing or chilling due to its tropical and subtropical origins (allen and ort, 2001). a remarkable physiological distruption known as chilling injury is exihibited when plants of this species are exposed to temperatures below about 10 to 12°c (lyons, 1973). low temperature may disrupt major components of photosynthesis including thylakoid electron transport, the carbon reduction cycle and controls stomatal conductance (allen and ort, 2001). it constitutes one main issue for the management of warm-season turfgrasses in tropical areas, usually causing yellowing and withering during the winter season (xia et al., 2000). exposure of plants to unfavorable growing conditions such as high temperature, heavy metals, drought, water availability, air pollutants, nutrient deficiency, or salt stress increases the production of reactive oxygen species (ros) such as superoxide (o2.-), hydrogen peroxide (h2o2), hydroxyl radicals (oh) and singlet oxygen (1o2). usually ros production can be coupled with development of oxidative injury and disruption of metabolic functions in plants (mittler, 2002). the major generation site of reactive oxygen species (ros) are reaction centers of psi and psii in chloroplast thylakoids (asada et al., 1999). ros may affect several cellular functions by damaging nucleic acids, oxidizing proteins, and causing lipid adv. hort. sci., 2016 30(2): 103-109 doi: 10.13128/ahs-19136 effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress s. soufi 1 (*), k. ben hamed 2, m. arbaoui 1, n. elbey 1, s. rezgui 1, c. abdely 2, t. bettaeib 1 1 laboratoire des sciences horticoles, département de production végétale, institut national agronomique de tunisie, 43, avenue charles nicolle, 1082 tunis, mahrajène, tunisie. 2 laboratoire des plantes extremophiles, centre de biotechnologie de borj cedria, bp 901, 2050 hammam lif, tunisia. key words: cold stress, hydrogene peroxide, paspalum vaginatum. abstract: seashore paspalum is a warm season grass that requires few maintenance inputs. expanded use of seashore paspalum could play a key role in making recreational sites more sustainable and environmentally. however, one key barrier to widespread seashore paspalum use is a relative lack of winter hardiness. under severe stress conditions, the antioxidant capacity may not be sufficient to minimize the harmful effect of oxidative injury. the search for signal molecules that mediate the stress tolerance is an important step in better understanding how plants acclimate to the adverse environment. this study aims to screen the responses of two paspalum vaginatum cultivars (salam and seaspray) to local weather conditions and to study how to enhance its cold tolerance by a foliar pretreatment by hydrogen peroxide at low concentrations of 10 mm under controlled conditions. the current study provides evidence that exogenous h2o2 decreases the endogenous content of h2o2 and malondialdehyde in the first three days of exposure to cold stress in pretreated ‘seaspray’ plants. in comparison to their control and pretreated ‘salam’. these results indicate that pretreatment with 10 mm h2o2 could improve the tolerance of seashore paspalum to cold stress, especially seaspray cultivar which showed better response to cold stress compared to ‘salam’. exogenous h2o2 could constitute a signaling molecule that significantly increases peroxidase relative density, and decreases mda and h2o2 content. (*) corresponding author: sihemsoufi@yahoo.fr received for publication 22 december 2015 accepted for publication 18 may 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 103-109 104 peroxidation (lpo) (foyer and noctor, 2005). depending on the delicate equilibrium between ros production and scavenging at the proper site and time, ros will act as damaging, or as signaling molecules (gratão et al., 2005). oxidative damage occurs when production of ros exceeds the capacity of these scavenging systems. components of the antioxidant defense system can be divided into enzymatic and non-enzymatic antioxidants. enzymatic antioxidants include superoxide dismutase (sod), catalase (cat), ascorbate peroxidase (apx), guaiacol peroxidase (gd), monodehydroascobate reductase (mdhar), glutathione reductase (gr), and dehydroascorbate reductase (dhar). non-enzymatic antioxidants are ascorbate (asa), glutathione (gsh) (both water soluble), carotenoids and tocopherols (lipid soluble) (munné-bosch and falk, 2004). among ros, h2o2 is considered to be the best suited signaling molecule due to its stability and longer half-life. it is able to cross biological membranes and diffuses from cell to cell or can be transported long distances from its sites of origin in plants depending on the availability of environmental stimuli. prasad et al. (1994) and wahid et al. (2007) found that pre-treated h2o2 at the appropriate concentrations improved salt-tolerance and chilling-tolerance in wheat and maize seedlings respectively (neto et al., 2005). these authors suggest that h2o2 signals the activation of antioxidants in seed. hydrogen peroxide is thus hypothesized to be implicated in enhancing the chilling resistance of plants associated with the function of ros scavenging systems. the aim of the present study is to investigate whether h2o2 supply enhances low temperature resistance of seashore paspalum turfgrass. for this purpose, we used two cultivars of paspalum vaginatum, salam and seaspray, the latter having better response to cold stress than the former (arbaoui et al., 2010). 2. materials and methods field study the study was carried out in tunisia at inat tunisia (national agronomy institute) (36°82’ n; 10°17’ e; 13 m) in 2011-2012 on two mature turfs of seashore paspalum (paspalum vaginatum swartz cv. salam and seaspray). the swards were established in a semiarid area on slit-loam soil (40% clay, 30% silt and 25% sand) with 40 g of p2o5 45%, 40 g of k2so4 as basic mineral fertilizer and ph 7.9. during the trial period, regular mowing and irrigation were applied to maintain a healthy turf. the experimental design was randomized block for each species with four replicates each of 1 m2 area (fig. 1). the conditions of relative humidity, total rainfall, and air temperature are reported in table 1 from the beginning of the vegetative period to the end of the trial. each plot was photographed in winter on minimum green coverage and after winter on spring green up with a digital camera; the images were taken at a height of 1.20 m with an angle of 90°. spring green up was the date when new stolon growth started to occur (recovery) in ecotypes. paspalum coverage was determined using environment for visualizing images (envi version 4.7, itt corporation, ny) (fig. 2). photochemical efficiency (fv/fm) was evaluated during winter, subsequent low temperature, and recovery. leaf water content (lwc) and dry weight (dw) were also measured at the end of the trial. fig. 1 schematic representation of one of the three experimental units with four replicates per species. period total rain fall (mm) average of rh (%) temperature max (°c) temperature min. (°c) average of air temperature (°c) septembre 11.5 64.2 26.0 16.6 20.6 octobre 3.80 62.8 21.5 12.1 16.8 novembre 6.50 67.4 16.7 9.8 12.3 decembre 3.80 54.5 13.3 6.3 10.1 janury 9.20 58.7 10.9 3.8 6.3 february 8.40 68.7 10.7 3.8 7.3 march 3.20 65.6 13.4 4.6 9.0 april 11.2 64.8 17.4 8.7 13.0 table 1 total rainfall, relative humidity, and air temperature from the beginning of the vegetative period to the end of the trial (centre for water resources management, inat tunisia) soufi et al. effects of h2o2 pretreatment on the response of two seashore paspalum cultivars to cold stress 105 laboratory study plant materials and growth conditions. seashore paspalum (paspalum vaginatum cv. salam and seaspray) were collected from a two-year field plot at inat, tunisia. plants were transplanted into plastic pots (0.5 l volume) filled with peat and positioned to grow for one and a half months in a greenhouse at 32°c/26°c (day/night). before the beginning of the experiment, paspalum vaginatum salam and seaspray were sprayed with 30 ml of 0, 10 mm h2o2 three times daily for two days and left in a normal chamber for 12 h for full absorption of the h2o2 solution (fig. 3). these plants were then transferred to a growth chamber with temperature 18°c/8°c (day/night), irradiance of 2000 lux, 11 h photoperiod, and relative humidity 70%. chlorophyll fluorescence was measured before exposure of plants to low temperature. leaves were sampled before initiation (0 days) and after 3 and 6 days of exposure to low temperature stress. samples from each treatment were immediately frozen in liquid nitrogen and stored at 80°c until biochemical analysis. the final harvest occurred after four months of exposure to low temperature to determine dry weight (dw), fresh weight (fw), and water content (wc). growth and water status in seashore paspalum leaves of seashore paspalum samples were excised after four months of chilling treatment and used for fresh weight (fw) and dry weight (dw) determinations. dry weight was obtained after drying leaves in an oven at 75 c until constant weight. these data were used to calculate water content (wc) as follows: wc= (fw-dw) ×100 ×dw-1. chlorophyll fluorescence chlorophyll fluorescence was measured on fully expanded young leaves by using an fim 1500 fluorescence induction monitor (analytical development, hoddesdon, england). each leaf was excised, immediately clipped into a leaf clip (32 mm wide × 80 mm long), and the shutter plate closed to induce darkadaptation for 30 min at room temperature. an array of six high-intensity light emitting diodes (leds) provided red light with a peak wavelength of 650 nm to illuminate the exposed leaf surface (4 mm diameter). maximum intensity of the illuminating light was approximately 3000 µmol m−2 s−1 at leaf surface. measured fluorescence parameters included f0 (initial fluorescence measured at the onset of illumination), fm (maximum fluorescence), fv (variable fluorescence = fm−f0), and fv/fm (ratio of variable to maximum fluorescence indicating the quantum yield). measurements of three leaves from the same pot were averaged to obtain a mean. physiological assays to determine oxidative damage h2o2 determination. the concentrations of h2o2 were estimated following the method of ferguson (ferguson et al., 1983): 100 mg fresh leaf was ground with 3 ml acetone for 30 min at 4°c, and the sample was then filtered through eight layers of gauze cloth. after the addition of 0.15 g active carbon, the sample was centrifuged twice at 3000 g for 20 min at 4°c. next, 0.2 ml 20% ticl4 in hcl and 0.2 ml ammonia were added to 1 ml of the supernatant. the postreacted compound was centrifuged at 3000 g for 10 min; the supernatant was discarded and the pellet was dissolved in 3 ml 1 mol l-1 h2so4 and the absorbance value was determined at 410 nm. the standard curve was made using h2o2, and the h2o2 content in leaf was calculated from the absorbance at 410 nm compared with the standard curve. lipid peroxidation. membrane lipid peroxidation was assessed by measuring the content of malonyldialdehyde in tissue. fresh leaf samples were homogenized in 0.1% (w/v) tca (trichloroacetic acid) solution. the homogenate was centrifuged at 15000 g for 10 min. an aliquot of the supernatant was added to fig. 2 paspalum grass coverage during and after winter. data on minimum green coverage were collected in winter in december and spring green up data were collected in march. fig. 3 schematic representation of the experimental design procedure. paspalum cultivars were first sprayed then left 48 h for full absorption of different concentrations of h2o2 (0 mm (distilled water) and 10 mm h2o2). plants appertaining to each treatment were subsequently exposed to cold stress. adv. hort. sci., 2016 30(2): 103-109 106 0.5% tba (thiobarbituric acid) in 20% tca (trichloroacetic acid). the mixture was heated at 90°c for 30 min in a shaking water bath, and then cooled in an ice bath. the samples were centrifuged at 10 000 g for 5 min, and the absorbance of the supernatant was read at 532 and 600 nm (hernandez et al., 2000). the mda concentration was calculated as the difference of absorbance at 600 nm and 532 nm. protein extraction. aliquots of frozen fresh leaves were ground to a fine powder with liquid nitrogen and extracted (100 mg fw) at 4°c in 100 mm tris-hcl buffer (ph 8.0) containing 10 mm edta, 50 mm kcl, 20 mm mgcl2, 0.5 mm pmsf, 1 mm dtt, 0.1% (v/v) triton x-100, and 10% (w/w) pvp. the homogenate was centrifuged at 14000 g for 30 min at 4°c, and the supernatant was utilized for protein content and the determination of antioxidative enzyme activities. three replicates per treatment were used. protein concentration was determined according to bradford (1976), using bovine serum albumin as standard. native gel electrophoresis and enzyme activity staining. samples of crude paspalum vagintum leaf extracts were separated by gel electrophoresis in 10% (pod) polyacrylamide slab gel, at ph 8.9 under native conditions according to davis (1964). pod isoforms were visualized on gels according to vallejos (1983). the gel was first incubated in a 0.1 m sodium acetate buffer (ph 4.0) containing 1% (v/v) guaiacol for 30 min. then, in a solution containing a final concentration of 4.7 mm 3-amino-9-ethylcarbazole, 38 mm n,n-dimethyl formamide, 0.1 m sodium acetate buffer (ph 5.0), 0.1 m cacl2, 3 mm and h2o2 the revelation was achieved. statistical analysis statistical differences were assessed using anova of sas (9.0) by t-test. the experimental design was a randomized complete block design with three replications. all data were statistically analyzed using least significant difference (lsd) to separate entry means. when the interaction was significant, a subroutine (pdmix800, sas) was used to compare means at (p<0.05%). 3. results local weather conditions in field induced changes in turfgrass coverage, in photochemical efficiency of psii, and growth parameters data on minimum green coverage collected in winter (december) indicated that turfgrass coverage was significantly (p<0.05) altered by local weather conditions (fig. 4, table 1). with spring green up (march) turfgrass coverage of paspalum vaginatum ‘seaspray’ and ‘salam’ exhibited an increase of 44.12% and 75%, respectively (fig. 2, 4). photochemical efficiency of phtosystem ii (fv/fm) followed a significant (p<0.05) similar pattern of decrease then increase through the winter and spring green up. it decreased significantly (p<0.05) and was most pronounced in the second measurement time (table 2). the percentages of decrease were, respectively, 15 and 6.5%. by the end of the trial, dw and wc were slightly different in both paspalum cultivars and were not significantly affected (table 2). effect of pretreatment with h2o2 and long-term cold stress on growth and photosytem ii efficiency of paspalum vaginatum ‘salam’ and ‘seaspray’ the effect of foliar pretrement using low concentrations of h2o2 on biomass production (dw) and water content (wc) of both seashore cultivars is fig. 4 turfgrass coverage on minimum green coverage (in winter) and on maximum green coverage (spring green up) in paspalum vaginatum salam (pvs) and seaspray (pvp). data are the mean ± se of three plants. date 'salam' 'seaspray' fv/fm d1 0.71 b 0.72 b d2 0.70 b 0.71 b d3 0.79 a 0.80 a dw (g) 21.30 a 20.28 b wc (%) 247.73 a 238.61 b data are the mean ± se of twelve replications (n=12). different letters indicate significant differences at a p<0.05 probability level. measurements were taken three times during the trial d1= december; d2= january; d3= march). table 2 changes in photochemical efficiency of psii (fv/fm ratio), dry weight (dw), and water content (wc) in paspalum vaginatum ‘salam’ and paspalum vaginatum ‘seaspray’ under local weather conditions soufi et al. effects of h2o2 pretreatment on the response of two seashore paspalum cultivars to cold stress 107 shown in figure 5. cold stress exposure reduced the dw of pretreated cultivar salam compared to the control with no significant difference observed between pretreated and untreated ‘seaspray’ (p>0.05). biomass production (dw) in pretreated cultivars and their controls were above 300% and leaves did not wilt during the cold stress process however not significant levels were noted (p>0.05). these results seem to be independent of stress exposure and chemical pretreatment of these cultivars. the photochemical efficiency of psii (fv/fm) was monitored three times throughout the cold exposure (table 3) and the fv/fm ratio remained above 0.80 in both control and pretreated seashore cultivars at 10 mm (p<0.05). changes in h2o2, mda and protein content h2o2 pretreatment, exposure duration to cold stress, and the types of paspalum cultivars significantly affected the production of h2o2 and mda content (p<0.05). in cultivar salam, h2o2 content in control and pretreated plants did not differ significantly during cold stress, whereas mda content decreased by 54% in pretreated plants when compared to control ones. in ‘seaspray’ prior to cold stress (0 day), h2o2 content was higher in pretreated plants than in controls (fig. 6). during the third day of cold stress, h2o2 content decreased by 83.77% compared to their controls, before it increased again after 6 days of cold stress. h2o2 pretreated plants lowered (77%) significantly (p<0.05) the mda content in ‘seaspray’, reaching the minimum levels during the third day of cold exposure as compared to the controls (fig. 6). after six days of stress exposure, mda content increased slightly but was lower than mda content recorded on the first day (0 d). protein content in the leaves of ‘salam’ changed slightly and remained at constant concentrations during the stress period. on the other hand, protein content in ‘seaspray’ pretreated with 10 mm h2o2 varied significantly (p<0.05) compared to their controls (fig. 7). chemical pretreatment and cold stress exposure altered relative density of peroxydase isoforms peroxidase activity was assessed on polyacrylamide gel to distinguish the different pod isoforms, and to quantify their activities using image j software. paspalum cultivars show different pod activity reactions under cold stress conditions. pod relative density in all pretreated paspalum vaginatum ‘salam’ (fig. 8) plants increased with increased levels of cold exposure, compared to controls. however, pod relative density in pretreated paspalum vaginatum seafig. 5 dry weight and water content of paspalum vaginatum ‘salam’ (pvs) and paspalum vaginatum ‘seaspray’ (pvp). values expressed as mean ± se (n=3). different letters indicate significant differences at probability level p<0.05. genotype date 0 mm 10 mm paspalum vaginatum ‘salam’ d1 0.78 b 0.80 ab d2 d2 0.81 a 0.80 a d3 d3 0.81 a 0.80 ab paspalum vaginatum ‘seaspray d1 0.80 ab 0.80 ab d2 0.81 a 0.80 a table 3 changes in fv/fm ratio in pretreated plants with 10 mm h2o2 and in control leaves of paspalum vaginatum ‘salam’ and paspalum vaginatum ‘seaspray’ during cold stress exposure data are the mean ± se of six replications (n=6). different letters indicate significant differences at a probability level p<0.05. measurements were taken three times during stress time (dates 1, 2, and 3) under controlled conditions (see materials and methods). fig. 6 protein content of paspalum vaginatum ‘salam’ and paspalum vaginatum ‘seaspray’ leaves in control plants (0 mm) and plants pretreated with 10 mm h2o2 after 0, 3, and 6 days of cold stress exposure. values expressed as mean ± se (n=3). treatment (p<0.001), species (p<0.001), time (p<0.001) and interaction (p<0.001). different letters indicate significant differences at probability level p<0.05. adv. hort. sci., 2016 30(2): 103-109 108 spray was much higher compared to control plants and then decreased during the third day of stress (fig. 8). 4. discussion and conclusions results showed that both cultivars were affected by environmental conditions and could have an impact on turfgrass coverage and on fv/fm. lower fv/fm ratio values were close to 0.7 (date 1), suggesting an altered performance of psii. remarkably, both paspalum cultivars required longer to recover, reaching 0.8 (fv/fm) (date 3). a close relationship has been reported between low temperatures and photosynthetic rates. the recovery of photosynthetic activity under cold stress was faster in other species such as sorghum, maize and pennisetum, while it was slightly slower in soybean, and ryegrass (taylor and rowley, 1971). some of the excessive energy is quenched into chlorophyll fluorescence to minimize damage to photosynthetic systems, particularly in photosystem ii (psii) and subsequent electron carriers (krause and weis, 1991; araboui et al., 2010). changes in membrane structure were considered as the primary lesion of stress injury and may lead to a loss of membrane permeability and metabolic disfunction (lyons, 1973; montillet et al., 2005). the peroxidation of lipids is also a damaging process that can be taken as a single parameter to determine the level of lipid destruction under various stresses. during lipid peroxidation, products are formed from polyunsaturated precursors that include hydrocarbon fragments; such as ketones, mda, and compounds related to them (grag and machanda, 2009). the increase of mda indicated the deterioration of peroxidation and membrane injury induced by ros under cold stress. our findings suggest that within three days of cold stress exposure, 10 mm of h2o2 remarkably decreased mda in cultivar seaspray. he et al. (2009) shown that h2o2 pretreatment enhanced the membrane stability of wheat seedlings, as revealed by a greatly reduced membrane damage rate (mda) and malondialdehyde (mda) content. this was associated with a slight increase in protein content. improving tolerance of both paspalum cultivars to chilling stress also leads to the over production of ros such as o2.and h2o2 in plant tissues (desikan et al., 2003). ros are extremely active molecules that may damage membranes and other cellular components to avoid cold stress or other stress-induced injuries. however, ros could be considered as a signal for molecules that mediate responses to various stimuli. compared to the other ros, h2o2 can be the most suited to act as a signaling molecule due to its higher stability and longer half-life. the fluctuation of h2o2 level in plants should spatially and temporally reflect changes in the environment (desikan et al., 2003, 2004). our study provides evidence that exogenous h2o2 resulted in a significant decrease (p<0.05) of the endogenous content of h2o2 and mda in the first three days of exposure to cold stress in pretreated ‘seaspray’ plants. the higher h2o2 content (0 d) in pretreated ‘seaspray’ was followed by a systematic fig. 7 h2o2 and mda contents of paspalum vaginatum ‘salam’ (a) and paspalum vaginatum ‘seaspray’ (b) leaves in control plants (0 mm) and plants pretreated with 10 mm h2o2 after 0, 3, and 6 days of cold stress exposure. values expressed as mean ± se (n=3). treatment (p<0.001), species (p<0.001), time (p<0.001) and interaction (p<0.001). different letters indicate significant differences at probability level p<0.05. fig. 8 pod isoforms in gel, relative density in control plants (0 mm) and plants pretreated with 10 mm h2o2 under cold stress condition. soufi et al. effects of h2o2 pretreatment on the response of two seashore paspalum cultivars to cold stress 109 high relative density of pod isoforms. with further cold stress, relative densities of pod decreased but remained higher than control plants. this type of response would permit pretreated seaspary cv. plants to still upright to overcome the wilting and mechanical weakness imparted by cold stress. in conclusion, our results have demonstrated that pretreatment with 10 mm of h2o2 could improve the tolerance of seashore paspalum to cold stress, especially cultivar seaspray which showed a better response to cold stress than ‘salam’. exogenous h2o2 could constitute a signaling molecule that significantly increases h2o2 detoxifying and pod activity, and decreases mda and h2o2 content. the meaning of presented results can be important for fundamental and practical sciences. these results would help to understand what are plant reactions to cold stress. i will be also possible to apply low concentrations of hydrogen peroxide in golf course and sport fields, making these fields more viable and sustainable. references allen d.j., ort d.r., 2001 impacts of chilling temperatures on 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introduction historical, cultural and institutional aspects afghanistan, a variegated and multiethnic country, is the result of 5000 years of continuous interaction between different cultures and civilizations, and conflicts with neighbouring powers. more recently the social, educational, and economic situation of this country has been shaped by a conflict dating back more than 30 years. the effects of war have had an overwhelming negative impact on governmental structures (e.g. ministry of agriculture and universities), causing an almost complete absence of guidance and regulations for promoting agriculture, the leading sector of the afghan economy. notwithstanding the destruction generated by the conflict, the rural cultural heritage and social structure has coped with these events better than urban areas where human resources were deeply hit, hence increasing the country’s isolation from the international community. at present, material rehabilitation and human capacity building are still a priority in afghanistan. afghan universities in particular were greatly affected by the conflicts to the point that their level of education and research is still below adequate standards. this is mainly due to a lack of financial support (even for physical re-building and the payment of salaries) and a generalized very poor human capacity (with a few outstanding exceptions). similarly, public institutions such as the ministry of agriculture need international support in order to reassume their expected role in society. over 75% of the afghan people live in rural areas adv. hort. sci., 2016 30(4): 197-205 doi: 10.13128/ahs-20349 from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan g. masini 1, e. giordani 2 (*) 1 perennial horticulture development project, afghanistan, kabul, afghanistan. 2 dipartimento di scienze delle produzioni agroforestali e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: fruit, germplasm, plant nursery. abstract: the afghan economy is based essentially on the primary sector and, namely, on fruit production. a cross road of trade along the “silk route”, local traditional fruitculture based on the bagh (home garden) is widely variegated in terms of species and varieties. afghan fresh and dried fruits (namely raisins) and nuts, sweet and rich in flavors, are demanded by domestic and foreign consumers. the lack of traceability and quality of the propagation material has been considered one of the basic bottle necks hindering development of an advanced horticulture. during the period 20062015 the perennial horticulture development project (phdp), funded by the ec-europeaid program, has been supporting the ministry of agriculture, irrigation and livestock of afghanistan through a process of collection and selection of local fruit varieties in order to improve the private nursery system, as well as perennial horticulture. about 850 accessions of different fruit tree species have been collected and entered into the national collections at six phd centres, where they undergo a standardised characterisation and identification procedure in order to be formally registered in a national list, hence contributing to their safeguard and protection. the best accessions were used as source for a traced propagation system after phytosanitary indexing and also in numerous adaptive experimentations. the phdp contributed in capacity building at technical and institutional level, fostering the establishment of nursery sector associations, providing expertise and developing the afghanistan national nursery growers organization (anngo) and the apex afghanistan national horticulture development organization (anhdo), mainly concerned with value chains. (*) corresponding author: edgardo.giordani@unifi.it received for publication 4 february 2016 accepted for publication 5 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 197-205 198 and the agriculture sector contributes about half of the gdp of the country. afghan agriculture needs to grow at least 5% per year over the next decade (world bank, 2014). this is a big challenge as technology, communication and transport, irrigation, and education are substantially deteriorated due to the conflicts and lack of maintenance. both public and private institutions lack physical infrastructure, necessary regulatory frameworks, and skilled staff to build a modern and competitive agricultural sector. environment two-thirds of total surface of the country (652230 km2) is made up of mountainous terrain and dry plains with little or no vegetation. only 12% of the land is ever cultivated due to high declivity and low rainfall, and out of approximately 8 million ha of cultivated land, only about 2 million can be irrigated. although there are areas rich in high quality groundwater (from melting snow), the irrigation capacity decreases during the summer. a limited number of dams and irrigation schemes were built during the 1950s and the 1960s and efforts are currently on going to rehabilitate water resource infrastructures and extend the irrigated areas. water resources are the main limiting factor for agriculture in afghanistan. precipitation is limited, ranging between 200 and 500 mm/year between december and march in agricultural areas. rain-fed agriculture does not exist, except in limited areas where “spring wheat” cultivation is possible. though arid and often a semi-desert, the afghan landscape also forms a gigantic watershed close to the himalayan and central asian region, with five major river basins and tremendous underground water resources yet to be fully studied and developed. fruit cultivation and crops in general are possible only on irrigated land, hence the typical afghan landscape of dry, deserted mountains and greenery on the foothills, around aquifers and canals. in the irrigated areas, there is high potential for species diversification. the climate is harsh in central and northern afghanistan, with very cold winters and very hot, dry and, in many areas, windy summers. nevertheless, the most common temperate fruit species are grown from 250 m up to 2-3,000 m above sea level. citrus and similar evergreen species can be grown in parts of eastern afghanistan; southern areas have generally mild winters. high mountain chains divide the country into hundreds of deep, narrow valleys with specific microclimates, each one with different typical fruit varieties, which are essentially determined by the interaction between altitude and orography. a good indicator of this is the yearly mean number of frost-free days which ranges from 137 in the salang area (elevation of 3172 m), 188 in gazni (2183 m) to 315 in jalalabad (566 m) (college of agricultural and environmental sciences, 2003). the land tenure the land tenure in afghanistan is extremely diversified, but characterized by fragmentation and a prevalence of smallholdings and subsistence farming (see table below). almost 30% of the irrigated land is owned by 70% of the farms with less than 5 ha. these include 11% of the land and 45% of the owners with less than 2 ha. however, farm owners of more than 10 ha own 70% of the irrigated land (table 1) (maletta and favre, 2003). the generally small size of the farms is making it problematic to achieve an economy of scale. nevertheless, as we have seen in many parts of europe, it could be possible to mitigate this constraint by promoting producers’ associations. that is, whenever safety and the social and economic conditions make it possible as the path towards farmers’ associations is extremely difficult in the present context. commercial horticulture is currently taking place, prevalently in small size orchards often with mixed production, which is a handicap as the market requires high quality and standardization of productions. however, the conditions exist for even small, commercially oriented farmers to plant orchards of commercial size (i.e. 1 ha or more) with the right commercial varieties on suitable rootstocks, now provided by the afghanistan national nursery growers organization (anngo) certified nurseries. these factors, along with the promotion of producers’ associations could render afghan fruit growers much stronger and competitive in the domestic and international market. table 1 distribution of irrigated land in afghanistan farm size (ha) farms (%) land (%) >50 ha 1 15 20-49.9 5 19 10-19.9 9 18 5-9.99 14 18 2-4.99 26 19 <2 ha 45 11 masini and giordani from traditional orchards to advanced fruitculture 199 the development of horticulture in the general agricultural context of afghanistan for many centuries, the livelihood of the afghan people has been associated with the cultivation of fruits and nuts and this has become an intimate part of their life, food, culture, and identity. what is today’s afghanistan developed at a cross road of the trade along the “silk route”, having a comparative climatic advantage over its neighboring countries in hot and humid south asia and very cold central asia. these became and still are today the natural outlets for afghan fruits and nuts. afghanistan’s strategic position explains the broad diversification with many temperate fruit species which originated in the middle east, and central and eastern asia, as confirmed by old records showing a strong traditional linkage between the afghan people and fruits (fresh and dried fruits and nuts). for instance, qasem ebne yousof abunasre herawi, in his “guide to agriculture” of the xiv century, mentioned over 100 types of grapes for the area of herat. bagh is the word meaning “home garden” an area usually protected by clay walls, forming a special micro-climate where a wide range of species and varieties are grown (often from seed) and from which most local varieties originate. bagh also mean specialized mono-specific orchards that supply most of the fruits. scattered trees disseminated in community areas also consistently contribute to fruit production (e.g. nuts like pistachios and seldom “wild” pomegranates). the main perennial fruit species cultivated in the country are grapes (fresh and raisin), almond, pomegranate, apricot, plum, pistachio, citrus (potential “niche”), olive (potential “niche”). the main annuals (vegetables, excluding pulses and cereals) cultivated are onions, potatoes, cumin, and melon, and other cultivated vegetables (including tomato, cauliflower, broccoli, spinach, lettuce, chilli, pepper, okra, cucumber, watermelon, etc.) (college of agricultural and environmental sciences, 2003). afghan consumers are eager for very sweet, extremely low acidic and tasty fruits; the sunny, dry weather contributes to the necessary requirements and facilitates fruit drying and conservation. fruits represent a valuable product both for subsistence and profit. fruit and nut production in 2014 reached 1567000 t (fig. 1), representing more than 16% of the entire primary production of this country, which confirms afghanistan’s vocation for fruitculture, nevertheless yields are very low if compared to those of countries with advanced fruit-growing systems (even if hardly comparable because of inconsistency of statistical data). for instance, afghan yield is about 38% of that of italy for peaches and nectarines. these figures point to problems in terms of fertility, production systems and cultural practices. the adoption of innovative strategies to increase yield and to reduce looses during post-harvest management and marketing, two issues far for being exhaustively afforded and solved, is a must in order to face the high growth rate of the population (average of 3.2%, against 1.2% of the world in the last ten years). in the 1970s the production of fruit and nuts was estimated as 34% of the total export of afghanistan and agriculture as 51% of the total gdp (guimbert, 2004), while exports were only 5.2% of the total gdp (underlining that most of the agricultural production was consumed in the country, thus the importance of the internal market). over the last 35 years, following the russian invasion and internal conflicts with foreign interventions, the production and export of fruits and nuts has continued, but afghan exports lost ground and the important segment of the international market (i.e. eu and usa) was taken by competing production from iran and turkey, which attained higher quality standards. in the last ten years, efforts to revive horticulture have been ongoing with the support of various donors including the eu, wb and usaid. between 2006 and 2015, afghan trade regained some of the lost ground and afghanistan became the seventh largest exporter of raisins (us$ 150 million) and the eleventh largest exporter of almonds (us$ 110 million) (world bank, 2011). fruits and nuts constitute 35% of afghanistan’s total exports. this sector still has great potential for further growth and increased fig. 1 fruit production of afghanistan in 2014 (1,000 metric tons). fao, 2016; other sources. adv. hort. sci., 2016 30(4): 197-205 200 income for rural households. today, the estimated area invested in horticulture is 340,000 ha, representing 14% of the total irrigated land. around 2 million people are involved in horticulture to varying degrees and the contribution to the gdp is estimated at 1.4 billion usd (34% of the agriculture gdp contribution and 6.7% of total) (pain and jensen, 2014; world bank, 2014). brief description and observations regarding the present cropping pattern although afghanistan is in the midst of a major shift towards commercial agriculture, the general cropping pattern is still wheat-based. for a long time, government and international donors have subsidized wheat seed, based on concerns for food security. the very idea of investing part of the land in fodder production is not even an option for most of the rural families, which own or manage a small farm and are mostly focused on subsistence needs. the needs for animal feed are fulfilled through grazing, crop residuals or agro-processing by products (such as cotton and flax), kitchen waste or imported industrial feed (for stall fed animals). wheat straw is largely used as a fodder (often mixed with urea or seed cakes) and it has a market of its own. most of the limited areas invested in fodder crops are intercropped with fruit trees. because of the importance of livestock in the rural economy, the demand for fodder is extremely high and its production can be an income generating activity, as we can see in various parts of the country were fodder like fresh alfaalfa is sold in bundles in bazaars. there is an important economic consideration that concerns the commercial farms. afghanistan has entered into the global market and could import wheat from regions that produce at a lower cost and have a surplus (namely the pakistani punjab, one of the largest grain producing areas in the world). the economic return of an orchard in afghanistan can be four to five times higher than one of the same area invested in wheat production. moreover, the cost of wheat production in afghanistan is much higher than in punjab or other traditional wheat exporting areas. therefore, the investment in temperate fruit production is a natural choice for commercial farms in afghanistan, rather than trying to compete in areas where they are at a disadvantage. it is interesting to note that this is a very fortunate circumstance in which economic, climatic and environmental considerations join hands. as cropping patterns are the result of economic and social factors that have operated for centuries, changes are not easy. however, a shift towards commercial agriculture is in progress and in the near future we can expect that economic factors will drive the most progressive farms towards a greater investment in horticulture, with change and rationalization of the cropping pattern. yet the process may be very slow or limited to a few farms or the private sector may adopt a short-term vision and neglect the important environmental factors. here lies the important role of the public sector to accelerate and incentivize the process. the ministry of agriculture and various donor agencies and development projects could consider introducing in development packages promotion and incentives for changes that rationalize the cropping pattern in order to fully exploit the commercial potential of afghanistan’s agriculture and at the same time mitigate, halt, and reverse ongoing soil fertility degradation. opportunities and constraints of afghanistan fruitculture the main opportunities and positive factors for development are: comparative climatic advantages in the region (dry and hot spring/summer), long tradition of horticulture and reputation of afghan varieties, opportunity to replace growing imports in domestic market (world bank, 2014); opportunity to increase yield and quality through adoption of suitable rootstocks and varieties, intensive cropping systems and suitable orchard management practices; high demand for quality fruit saplings; rich genetic diversity. furthermore the development of horticulture is part of the national development strategy and supported by international donors, few private agribusinesses are starting to invest in quality control, and more farmers associations are under formation. notwithstanding security issues, the presence of consistent international donors developing projects in the primary sector guarantees an emergent agriculture. the main constraints of the afghan fruit-culture can be summarized as follows: small land holdings, small commercial orchards, lack of standardized product, poor orchard management, low yield, lack of infrastructure and substandard storage, sorting, packaging, marketing, transport facilities, high cost of quality inputs, low quality standards, inadequate public services (extension, applied research, metereological services), growing imports to fill higher standards demand in main cities, poor range of varieties masini and giordani from traditional orchards to advanced fruitculture 201 in vegetables and lack of applied research to extend marketing opportunities and marketing season, define cropping calendars, etc. 2. the perennial horticulture development project (phdp i and ii) origin and objectives of phdp during 2002-2003, the italian cooperation funded fao through a grant for the restoration of the afghan germplasm. shortly thereafter, the fao staff approached the european commission for financial support and in 2004 the european commission delegation organised a feasibility study focused on the development of the ministry of agriculture, restoration of the old mother stock nursery systems, and some germplasm collection. actually the subjects involved were: a) germplasm collection and nursery development; b) clean germplasm systems; c) provision of facilities for the development and testing of new varieties and new growing systems of horticultural crops; d) training programmes for implementing agencies; training for senior extensionists/mail staff/farmers/traders; linkages between mail and universities; and e) development of an integrated horticultural research and technology transfer system. the objectives of the perennial horticulture development project were defined in the overall scheme of a number of horticultural development projects taking into account the agriculture master plan (7), following a design aimed at emphasizing the involvement of the ministry of agriculture, irrigation and livestock (mail). the included objectives were the maintenance of reference collections, research and extension production methods and regulation of the industry, backed up by appropriate analytical laboratories and an inspection and quarantine system, and minimizing mail involvement in the production of fruit trees and in the development of mother nurseries. as stated in the project web pages “the specific objective of the project is to develop a demand oriented and export led perennial horticulture industry”. a major purpose of the project was the development of the nursery sector to strengthen and to qualify fruit production, by establishing a traced nursery system based on the propagation of true-to-type local varieties. funding and partnerships in 2006 the european commission-europeaid program funded, through a public/private consortium formed by iak agrar consulting gmbh, aht gmbh and the university of florence-italy, the perennial horticulture development project (phdp) (www.afghanhorticulture.org) in support of mail. a second phase was supported by the ec for the period 2010-2015 to a wider consortium (agriconsulting spa, department of agri-food and environmental science university of florence, department of agricultural sciences university of bologna, centro attività vivaistiche, landell mills uk) with the contract “technical assistance to mail to strengthen the planting material and horticulture industry in afghanistan (europeaid/129-320/c/ser/af/2)”. activities related to the germplasm collection the framework comprised the following steps: a) individuation of superior trees in productive orchards; b) cataloguing and definition of the in situ national collection; c) propagation from the in situ original mother plants; d) establishment of the ex situ national collection; e) characterisation and evaluation; f) foundation of traced and clean mother stock nurseries (msn) (fig. 2). the concept underlyfig. 2 scheme of phdp germplasm activity: from the collection in the traditional mixed bag to the establishment of new specialized orchards. source: phdp. adv. hort. sci., 2016 30(4): 197-205 202 ing all the activities can be summarized with the call for a meeting with local nurseries and fruit-growers: it is the intention of the phdp to undertake various initiatives that create and develop a system that ensures the availability of the best possible planting material for the perennial horticulture industry in afghanistan, and to ensure the long term sustainability of such a system owned by the private sector. support activities, capacity building and sustainability the workplan included the establishment of six centres (the main one located at badam bagh kabul, the others in herat, jalalabad, kandahar, kunduz and mazar-e-sharif) and a laboratory of biotechnology for plant indexing and micropropagation. long term sustainability involved essentially handing over the system built up by phdp to the local staff of mail and of two organizations, the “afghan national horticulture development organization” (anhdo) and the “afghan national nursery growers organization” (anngo)” (giordani et al., 2014). apart from the described objectives, phdp activities were related also to public and private sector institutional and regulatory reform and training and extension. phdp main results a previous publication described the main goals achieved by phdp up until 2013 (giordani et al., 2014). in the present paper, the authors summarise the achievements of the project once it was officially completed (december 31, 2015). more detailed information is available on the web (afghanhorticulture.org). the national collection of fruits and nuts of afghanistan after the surveys of 2006-2008 in the most important areas of fruit production when the best and most “profitable” varieties were chosen together with fruit growers (who were recognized as “custodial” of the in situ selected accessions), an in situ collection formed of over 850 accessions was defined. ex situ collection orchards were established in the six phdp centers in plots with six to ten replicates; they constituted the national collection (nc) (table 2). among fruit species, only citrus, loquat and persimmon accessions, donated by cra-centro di ricerca per l’agrumicoltura e le colture mediterranee acireale (italy) and ivia-valencia (spain) have been almost completely imported and placed in nangarhar valley (jalalabad province) because of its warm climatic conditions. some accessions of cherry, peach, plum, apple, and pear were recovered from plants derived from previously imported propagation material. on the other hand, apricot, almond, grape, and pomegranate varieties were mostly collected locally. the standardised procedures of characterisation (e.g. adoption of internationally recognized specific descriptor lists for the different species) started in 2009 and up to now around 80% of all collectable data have been inserted in a database (table 3). after completion of the description and individuation of true-to-type and unique identified local varieties for each species, the accessions, as previously table 2 number of accessions belonging to the ex situ national collection of fruits and nuts of afghanistan located in the six phdp centers species location of phdp centers number of accessions almond mazar and kunduz 106 apricot kabul and mazar 128 apple kabul and kunduz 81 pear kabul and kunduz 53 sweet and sour cherry kabul and herat 28 japanese and european plum, myrobalan herat and kandahar 77 peach herat and kandahar 116 grape herat and kandahar 139 pomegranate kandahar and jalalabad 79 fig kandahar and jalalabad 16 citrus jalalabad 66 date palm jalalabad 6 loquat jalalabad and kandahar 12 persimmon jalalabad 24 olive jalalabad 10 total 935 species progress in description and characterization almond 56 varieties fully described. register printed and distributed apricot 72 varieties fully described. register printed and distribute pomegranate 78 varieties fully described. register under translation plums 50 varieties fully described. register in preparation peach 82 varieties (description almost completed) cherries 24 varieties fully described. register under translation grape 60% of description completed apple 50% of description done; started description of fruits pear 50% of description done; started description of fruits citrus fruit description in process (only for first evaluation purposes) fig in progress loquat in progress persimmon in progress date in progress pistachio not started yet, accessions recently introduced olive not started yet, accessions recently introduced table 3 status of characterisation of the accessions collected in the national collection of fruits and nuts of afghanistan source: phdpii report agriconsulting. source: phdpii report agriconsulting. masini and giordani from traditional orchards to advanced fruitculture 203 done for almond and apricot (ministry of agriculture, irrigation and livestock, 2014, 2015), will be registered by the ministry of agriculture, irrigation and livestock of afghanistan. actually, the nc represents both a repository of local germplasm and the officially guaranteed source of material to establish the mother stock nurseries (msns) in different parts of the country. capacity building an operative laboratory of plant biotechnology (lbp) has been established in kabul through scientific, educational and technical collaboration of the department of agri-food and environmental science of the university of florence, the department of agriculture of the university of bologna, and the company centro attività vivaistiche (cav) of tebano (ra) italy. the lbp verifies the health status of national and exotic germplasm of fruit trees, applying international standards (e.g. european plant protection office protocols) and, being equipped with the facilities for plant tissue culture, it is in charge of micropropagation of the most valuable rootstocks for peach and cherry. another result focused on sustainability and based on local human resources was the establishment of two organizations. the first, the afghan national nursery growers association (anngo), represents 30 nursery growers associations spread throughout the country. all the involved private nurseries (about 1000) have accepted the anngo regulatory system for the production of true-to-type and traced saplings, deriving from msns with clean material from the nc, to be sold to fruit-growers. anngo also provides services related to business improvement and marketing promotion, sanitary controls, monitoring of the quality of planting material, improvement of nursery techniques and production of clonal rootstocks, certification and inspection services including labeling of certified saplings, technical training and dissemination of innovations, and publication of the catalogue. the production of certified saplings has increased from 340000 in 2012, to 1500000 in the last selling season (2015). the second organization, the afghanistan national horticulture development organization (anhdo), is actually an ngo devoted to the development of a modern horticulture in partnership with mail and the private sector, and it is promoted and tutored by phdp. its aim is to provide continuity to phdp main activities and to enable horticulture stakeholders to improve the industry by providing technical services, upgrading capacities, and promoting private-public coordination. adaptive research following a bottom-up procedure, adaptive research has been integrated within the phdp framework from the very beginning (fig. 3). there were 25 scheduled trials in 2015, most of them related to the topics considered to be at the base of improvement of fruit growing productivity and quality, such as selfand inter-compatibility in almond, plum and apricot varieties; grafting compatibility with vigour reducing fig. 3 the circle of adaptive research within phdp: from the definition of priorities to the impact of the achieved results. source: phdpii report agriconsulting. 204 adv. hort. sci., 2016 30(4): 197-205 rootstocks (pear, cherry); chilling requirement; potential value for breeding and breeding activities (almond and apricot); micropropagation of rootstocks; pruning (grape) and training systems (apple). a plan to develop adaptive research has also been defined for upcoming years. 3. conclusions the pivotal role of the perennial horticulture development project the eu-funded phdp (2006-2015) has set the foundation for modern and sustainable development of commercial fruitculture in afghanistan. the phdp constituted the national collection of fruit and nuts of afghanistan and promoted a traceability system from the national collection to the private nurseries organized in provincial associations and represented nationally by the anngo. for the first time in history, the orchard growers in afghanistan could purchase labeled fruit saplings that certified the origin of the variety and rootstock and phytosanitary control of the mother stock material. as a result, thousands of new orchards are being planted with good marketable varieties, which will result in higher yield, better quality, and more income for the fruit-growers. the phdp also conducted focused adaptive research, identifying the pollination interactions among plum and almond varieties and started a long term breeding program for almond and apricot. at this writing, all the above services have already been handed over to the ministry of agriculture of afghanistan through a process of transition supported by the eu. at the same time, in the second phase, the phdp promoted and developed the anhdo which has become an independent and reputed organization, acting as a catalyst in the private sector, implementing projects to improve the value chain of the main fruit productions and work on post-harvest, quality standards and marketing. by addressing the foundation of horticultural development and having the strength of a strategic vision and the patience to work consistently for nine years, the phdp took a pivotal role in the development of fruitculture in afghanistan. the outputs of phdp will have a relevant impact on development of the sector and on the afghan economy as a whole for many years to come. future projections due to a lack of proper data and statistical treatment, it is extremely difficult to quote figures and attempt projections about future development of agriculture and horticulture in afghanistan. however, based on the latest assessments (altai consulting, 2014) there has been a tremendous expansion in terms of the land invested in horticulture in the last 44 years, passing from 220000 ha in 1960 to the circa 340000 ha of today. but for the last 10 years yields had remained the same, indicating that the increase of horticultural production has been achieved almost exclusively at the expense of investing more irrigated land. taking into account an estimated potential of 750000 ha of land invested in horticulture, a further 73% increase is expected in the next 10 years. this increased investment of land should be combined with extended irrigation schemes, and an upgrading and modernization of the extension and quality input and technical services provided to the farmers. as a result it is hoped that yields, quality standards, and marketability will also improve, thus generating income and jobs. this scenario is certainly possible, but will also require a modernization of the obsolete government institutions so that they become service oriented, providing needed policies and regulations and fostering partnerships with the private sector. this is perhaps the greatest challenge for the entire horticulture sector in afghanistan. acknowledgements a special thanks to all the persons who, directly or indirectly, afghan or foreigners, contributed to develop phdp. particular appreciation is due to the ec delegation in kabul and to the mail for the long term strategic vision and guidance that made the program possible. thanks to the afghan farmers of the remote villages for having kept alive all the accessions collected by phdp. funded by europeaid program european commission. references altai consulting, 2014 agricultural data collection and utilization program final report: roadmap for success. altai consulting, kabul, afghanistan, pp. 110. college of agricultural and environmental sciences, 2003 rehabilitating agriculture in afghanistan. horticultural market survey report for icarda. college of agricultural and environmental sciences, university of california, davis, usa. fao, 2016 faostat www.fao.org. masini and giordani from traditional orchards to advanced fruitculture 205 giordani e., cullen g., degl’innocenti p., masini g., 2014 local fruits and nuts as a tool for the development of afghanistan. junco. journal of universities and international development cooperation, 1: 627635. guimbert s., 2004 structure and performance of the afghan economy. world bank, pp. 40. ministry of agriculture, irrigation and livestock, 2014 the national collection of varieties of fruits and nuts of afghanistan. volume i. almond. ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 194. ministry of agriculture, irrigation and livestock, 2015 the national collection of varieties of fruits and nuts of afghanistan. volume ii. apricot, ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 236. maletta h., favre r., 2003 agriculture and food production in post-war afghanistan. winter agricultural surveys (http://www.fao.org/docrep/007/ae407e/ ae407e00.htm). pain a., jensen p., 2014 review of afghanistan reconstruction trust fund, artf, internal and external studies and evaluations in afghanistan. sida, pp. 54. world bank, 2011 afghanistan understanding gender in agricultural value chains: the cases of grapes/raisins, almonds and saffron in afghanistan. world bank, washington, dc, usa. world bank, 2014 afghanistan agriculture sector review. world bank, washington, dc, usa. 193 1. introduction in western europe plums are grafted on myrobalan b, st. julien a, marianna and other vegetatively propagated rootstocks (nicotra and moser, 1995). in eastern europe it is difficult to propagate dwarfing and semi dwarfing rootstocks for plums and prunes because of long and hard winters. stool beds are often damaged by winter frost and hard-wood cuttings are not properly rooted because of low temperatures in autumn and spring. mother plants in stool beds are also threatened by plum pox. very few nurseries produce trees grafted on st. julien a and gf 655/2. most plum and prune trees are grafted on seedlings of selected types of prunus divaricata. such trees are planted at 4x3 to 5x5 m and trained to open centre form. in poland about 20% of trees are grafted on seedlings of ‘prune wangenheim’ (prunus domestica). this cultivar is self-pollinated, so seed trees grown in isolation produce seedlings with uniform grow habit (sitarek et al., 2001). ‘prune wangenheim’ is compatible with all european plums and prunes. cultivars grafted on ‘prune wangenheim’ are semi dwarf, tolerant to arid soils common in poland and very productive (sitarek et al., 2001). such trees should be suitable for dense plantings in intensive orchards. in germany, zahn (1986, 1994) and brunner (1990) introduced central leader spindle and free spindle (without shoot bending) for dwarf and semi dwarf, densely planted plums and prunes. these systems were tested in belgium (wustenberghs and keulemans, 1996) with 825 and 1250 trees per ha and appeared to be very economical in yielding and harvesting. the goal of the present work was to elaborate an intensive plum orchard suitable for both hand and mechanical fruit harvesting with combined self propelled straddle harvester working in continuous motion, as is used in poland to harvest sour cherries. the harvester requires densely planted trees with central leader up to 3 m high and young flexible shoots coming out of the leader. for this purpose new methods of summer training and pruning were introduced to plum trees. studies were also undertaken on the intensity of tree growth of six cultivars grafted on ‘prune wangenheim’, their growth habit, canopy structure, fruit bud formation in relation to wood age, quality of flower clusters, fruit set, sun irradiation and distribution and yield. these studies enabled to precise the pruning method of trees in full bearing age. 2. materials and methods one-year-old feathered trees of ‘cacanska rana’, ‘cacanska lepotica’, ‘cacanska najbolja’, ‘diana’, ‘katinka’, and ‘silvia’ grafted on semi dwarf seedling rootstock ‘prune wangenheim’ were planted in autumn 2004 on a 0.5 ha plot, on sandy-loam soil at the research institute of pomology, skierniewice, poland. to estimate optimum planting density, trees were spaced 4 m between rows and at various densities in the row: 1.5; 2.0 and 2.5 m (1666; 1250 and 1000 trees/ha). each cultivar was planted in two rows (each consisting of 36 trees): one for hand harvesting, the other for mechanical harvesting. in each row the trees were arranged in three randomized blocks with four trees per plot. in the second year after planting the interrows were grassed down with frequent grass mowing in intensive plum orchard with summer training and pruning a. mika, z. buler research institute of pomology and floriculture, 96-100 skierniewice, poland. key words: canopy structure, fruiting biology, mechanical harvesting, plums, pruning, prunus domestica, training. abstract: an intensive plum orchard model was created for two types of fruit harvesting: by hand and with a self-propelled straddle harvester working in continuous motion. six plum cultivars grafted on semi dwarfing rootstock ‘prune wangenheim’ (prunus domestica) were planted at three densities (1000, 1250 and 1666 trees/ha). a new training system central leader spindle was applied. the leader was not headed after planting and summer training treatments were performed. from the third year onward renewal pruning was done after fruit harvesting. the new training and pruning system resulted in very rapid tree growth, much young wood, fruit bud formation on young wood and early bearing. trees appeared to be suitable for hand and mechanical harvesting within four years from planting. adv. hort. sci., 2011 25(3): 193-198 received for publication 13 april 2011 accepted for publication 1 august 2011 194 conjunction with the maintenance of 1.5-m-wide herbicide strips along the row. a drip irrigation system was installed from the first year. this was necessary because yearly precipitation at skierniewice is around 500 mm whereas plums grown in central poland require 700 mm of rainfall. fertilizers were applied according to the standard recommendation for commercial plum orchards. eight to ten sprayings were essential to control pests and diseases. a new training system with summer pruning was introduced to obtain central leader trees suitable for mechanical harvesting and hand picking (fig. 1). trees having central leaders 1.7 m high at planting time were not headed after planting and side shoots were shortened lightly. subordination of the side branches to the central leader was obtained by summer pruning. at the end of may or beginning of june (depending on the growing season) new shoots that appeared at the top of the central leader were thinned leaving only one to extend the leader. this treatment resulted in numerous short side shoots along the leader most of them setting flower buds in the first growing season. any side shoots growing upright were bent to horizontal position by fixing (clips) pinches to the leader above shoots. these treatments were repeated in the second year. in spring of the third year, trees were nearly 3 m high with at least 1.5 m of canopy diameter and they were able to give the first crop. further training was not necessary. from the fourth year onward, renewal pruning introduced in poland by czynczyk et al. (1976) was performed after fruit harvesting. each branch over three years of age was cut off near the central leader leaving a 10-30 cm stub to ensure new shoot growth (fig. 2). light shoot thinning was carried out as necessary. effect of cultivars and planting distance on tree growth, canopy structure, relation between shoot age and fruit bud formation, and fruit setting was studfig. 1 plum tree training with summer pruning: a) planted feathered tree is left with unheaded central leader and lightly headed side shoots; b) in may side shoots appearing at top of the leader are cut off except for one left for leader prolongation, some lower shoots are bent with clips; c) treatments at top of the leader are repeated in may of the second year; d) tree with fruiting ability in spring of the third year; e) shoot bending with a clip. fig. 2 plum tree pruning by renewal method done after harvesting. old branches are removed to stumps and mainly one-, and twoyear-old twigs are left. 195 ied. to estimate light interception, the irradiation intensity was measured at ground level in rows and interrows with a delta t tube solarimeter tsl and with a light sensor mounted above trees (anderson, 1964). light interception was calculated by subtracting the light intensity at ground level from light intensity above trees and was expressed as percentage of light intercepted by the canopy. light distribution was measured across tree rows on three levels: 0.8; 1.6 and 2.4 m with sun scan probe type ss -1 (delta-t devices ltd, burwell, cambridge, england). harvesting efficiency with a combine harvester was recorded. fruit quality of hand-picked fruit against mechanically harvested fruit was compared. 3. results and discussion in the sixth year after planting (2010) the growth of trees, expressed by trunk cross sectional area (table 1), showed significant differences between cultivars and also significant differences between the most dense tree spacing in the row compared to the two larger spacing treatments. the large differences in growth intensity among cultivars indicate that this factor should be considered in designing intensive plum orchards. treatment in the most densely spaced rows resulted in a dwarfing effect on tree growth. when the orchard is managed over a long period, such dwarfing effect leads to a smaller crop from smaller trees, as is often observed in intensive apple orchards (mika and piskor, 1996). the new applied methods of tree training resulted in rapid vertical growth. all the trees (except for cv. ‘katinka’) reached the required height (3.0-3.5 m) in the fourth year after planting (table 2). in the subsequent years tree height had to be restricted by pruning to coincide with gap required by the harvester. for this reason strong shoots appearing on the top of trees were removed from the fourth year on trees for both machine and hand harvesting. canopy spread (table 3) increased until the sixth year from planting. trees of cv. ‘silvia’ created the most spread whereas ‘katinka’ formed compact trees which were significantly smaller than the other cultivars. some influence of planting density on canopy spread was evident but the differences were not significant. in renewal pruning method, old branches are cut out and replaced by young shoots. due to that, trees are kept in a constant spread. canopy structure is favourable for fruiting because most of new shoots are short, in range 5-10 cm. (table 4). such shoots are able to form fruit buds on one-yearold wood (table 4). there were no significant differences between cultivars and planting density treatments. spur number per tree was also estimated in the fourth year after planting. in spite of renewal pruning, which forced young wood to grow, numerous spurs were present in the tree table 1 influence of cultivars and spacing on tree growth expressed by trunk cross sectional area in the sixth year from planting (2010) influence of cultivars trunk cross sectional area (tcsa) (cm2) ‘cacanska rana’ 46.6 bc ‘cacanska najbolja’ 52.9 d ‘cacanska lepotica’ 35.0 a ‘diana’ 42.8 b ‘katinka’ 33.3 a ‘silvia’ 51.7 cd influence of spacing (m) 4 x 1.5 40.7 a 4 x 2.0 45.6 b 4 x 2.5 44.8 b different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 2 influence of cultivars and spacing on tree growth expressed by tree height in the fourth year from planting (2008) influence of cultivars tree height (m) ‘cacanska rana’ 3.08 b ‘cacanska najbolja’ 3.26 b ‘cacanska lepotica’ 3.20 b ‘diana’ 3.44 b ‘katinka’ 2.50 a ‘silvia’ 3.62 c influence of spacing (m) 4 x 1.5 3.20 a 4 x 2.0 3.17 a 4 x 2.5 3.20 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 3 influence of cultivars and spacing on tree growth expressed by tree spread in the fourth year from planting (2008) influence of cultivars tree spread (m) ‘cacanska rana’ 2.47 b ‘cacanska najbolja’ 2.33 b ‘cacanska lepotica’ 2.50 b ‘diana’ 2.44 b ‘katinka’ 1.74 a ‘silvia’ 2.67 c influence of spacing (m) 4 x 1.5 2.49 a 4 x 2.0 2.38 a 4 x 2.5 2.40 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. 196 canopy (table 5). significant differences among cultivars suggests that some variation in pruning methods may be needed in future for cultivars producing a small number of spurs. bare wood, typical for plums under traditional pruning, did not appear. the significant differences between cultivars show the variation in the growth habit of the chosen cultivars. in the fifth year from planting, trees of three cultivars planted at distances of 1.5 and 2.0 m created dense structures in the bottom part of the canopy; for this reason leaf area index (lai), light interception and light distribution at three planting distances were estimated. this very laborious study was performed only on vigorously growing silvia cultivar having the most regular canopy structure (table 6). the results reveal that lai, expressed as ratio of total canopy leaf area to ground area under tree (m2/m2), achieved a value of 2.5 at planting distance 4 x 1.5 m. this was close to the optimum value (2-3) suggested by jackson (1996) for intensive apple orchards. trees spaced 4 x 2.5 m were far from that value. light interception (table 7) was at an acceptable level at planting distance 4 x 1.5 and 4 x 2.0 m but still insufficient as required for an intensively planted orchard; according to jackson (1996) the value should be 60-70%. light interception at planting distance 4 x 2.5 m was very poor. in a number of studies it has been found that light interception is correlated with fruit production per ha (jackson, 1980). in apple orchards, production increases with light interception up to about 70% available light. light distribution within the fruit tree canopy was acceptable in the upper and middle parts of the trees. in the bottom part (0.7 m above ground) it was critical at the 1.5 x 4 m planting distance, low at 4 x 2 m and sufficient at 4 x 2.5 m planting distances. these results show that the most densely planted trees (1.5 m) require more heavy pruning in the fifth year from planting than trees spaced 2 and 2.5 m in the row. the relationship between the age of wood and percent of cluster fruit bud setting was studied in three years table 4 influence of cultivars and spacing on canopy structure (% of shoots in three classes of length) in the fourth year from planting (2008) influence of cultivars class 5-50 cm class 51-90 cm class 91-150 cm ‘cacanska rana’ 73.5 ij 18.7 cde 6.6 ab ‘cacanska najbolja’ 62.1 hi 30.2 ef 4.8 a ‘cacanska lepotica’ 60.4 gh 13.8 bcd 24.7 def ‘diana’ 48.3 g 34.2 f 14.0 bcd ‘katinka’ 74.6 j 14.6 bcd 10.2 abc ‘silvia’ 54.1 gh 22.7 def 21.7 def influence of spacing (m) 4 x 1.5 61.1 c 21.2 b 15.0 ab 4 x 2.0 62.1 c 22.5 b 12.4 a 4 x 2.5 64.1 c 22.1 b 11.0 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 5 influence of cultivars and spacing on fruiting spurs per tree in the fourth year from planting (2008) influence of cultivars number of fruiting spurs ‘cacanska rana’ 238.8 b ‘cacanska najbolja’ 260.8 b ‘cacanska lepotica’ 136.6 a ‘diana’ 285.7 c ‘katinka’ 146.1 a ‘silvia’ 265.8 b influence of spacing (m) 4 x 1.5 216.8 a 4 x 2.0 4 x 2.5 219.6 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 6 influence of spacing on leaf area, leaf area index (lai), and light interception of ‘silvia’ cultivar in the fifth year from planting (2009) spacing (m) leaf area (m2) lai insolation on ground level (watt/m2)* % of light interception 4 x 1.5 15.16 a 2.52 c 168.0 44.6 b 4 x 2.0 14.20 a 1.78 b 176.3 46.8 b 4 x 2.5 14.75 a 1.48 a 105.5 28.0 a mean insolation above trees 376.7 watt. table 7 influence of spacing on % light distribution in three canopy levels of ‘silvia’ cultivar in the fifth year from planting (2009) spacing (m) canopy level from the ground 0.8 m 1.6 m 2.4 m 4 x 1.5 7.7 a 18.8 bc 48.0 d 4 x 2.0 19.5 bc 29.2 c 48.1 d 4 x 2.5 10.5 ab 32.4 c 49.5 d 197 (2008-2010). there were no significant differences between years. table 8 shows the results in the sixth year from planting on fully-grown trees. many differences were found in the ability of the cultivars to set fruit buds on young wood. in spite of this, most cultivars were able to set 60 to 80% of cluster fruit buds on one-year-old and two-year-old wood. the rest (20-40%) was set on threeyear-old wood. these results indicate that trees treated with renewal pruning produce enough fruiting wood and the pruning method does not have adverse effects on tree yielding. bare wood observed on trees with traditional regulated pruning was not observed in this experiment. detailed studies on flower bud formation revealed that cluster fruit buds on one-year-old wood consist of, on average, fewer flowers than clusters on older wood (table 9). for this reason one can expect that young wood may set less fruit than older wood. however this was not proven. most of the cultivars set 5 to 10% fruit out of 100 flowers (table 10). as the result, the percent of fruit on oneand two-year-old wood was similar to the percent of cluster flower buds (60-80). trees came to blossom in the second year after planting and produced about 0.3 kg of plums per tree. in the third year, blooming was very abundant but spring frost in the first days of may killed all the flowers. for this reason the first yield was obtained in the fourth year after planting. most of the trees produced from 8 to 30 kg per tree. the most productive was ‘cacanska najbolja’, the poorest was ‘katinka’. there were many differences within cultivar and little differences within planting distances. only ‘cacanska najbolja’ and ‘diana’ gave a greater crop when planted at the greater distance. in the fifth year again spring frost in may reduced the crop to less than one kg per tree. acceptable yield was obtained in the sixth year after planting (2010) (table 11) when most trees yielded 15-18 kg per tree. there were again very large differences in tree productivity among cultivars but none in terms of planting distance. this suggests that when renewal pruning is performed trees can be spaced at even 1.5 m in the row. the good yield, calculated per hectare, varied from 13.9 tons at the largest planting distance to 23 tons at the closest spacing. in the fourth and sixth years after planting fruits were harvested by hand and by self-propelled straddle harvester. harvesting was begun when the forces between fruit and stem were 6-8 n, fruit firmness 5 kg/cm, and tss 12-14%. table 8 influence of cultivars and spacing on % of spur fruit buds on young wood in the fourth year from planting (2008) influence of cultivars 1-year-old 2-year-old 3-year-old ‘cacanska rana’ 34.4 efg 42.4 ghi 21.4 bcd ‘cacanska najbolja’ 50.0 i 30.1 def 19.5 bc ‘cacanska lepotica’ 37.5 fgh 25.9 cde 39.4 fghi ‘diana’ 11.9 a 48.2 hi 39.2 fghi ‘katinka’ 40.8 fghi 37.5 fgh 20.6 k ‘silvia’ 16.5 ab 44.5 ghi 38.2 fgh influence of spacing (m) 4 x 1.5 28.2 ab 40.4 e 31.2 abcd 4 x 2.0 29.0 abc 36.7 de 32.0 bcd 4 x 2.5 35.6 cde 36.7 de 24.8 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 9 influence of cultivars and shoot age on number of flowers in one spur fruit bud in the fourth year from planting (2008) influence of cultivars number of flowers in one spur fruit bud ‘cacanska rana’ 4.3 d ‘cacanska najbolja’ 1.9 b ‘cacanska lepotica’ 1.9 b ‘diana’ 2.9 c ‘katinka’ 1.3 a ‘silvia’ 1.3 a influence of shoot age 1-year-old 1.9 a 2-year-old 2.1 b 3-year-old 2.5 c different letters indicate significant differences separately for cultivars and shoot age at p=0.05. table 10 influence of cultivars and spacing on yield (kg/tree) in the fourth and sixth years from planting (2008 and 2010 z) influence of cultivars 2008 kg/tree 2010 kg/tree ‘cacanska rana’ 9.7 a 16.5 c ‘cacanska najbolja’ 28.6 c 4.9 a ‘cacanska lepotica’ 11.4 ab 16.1 c ‘diana’ 14.5 b 17.1 c ‘katinka’ 15.0 b 11.7 b ‘silvia’ 9.0 a 11.1 b influence of spacing (m) 4 x 1.5 13.8 a 12.7 a 4 x 2.0 14.3 b 12.2 a 4 x 2.5 16.0 c 13.9 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. (z) yields in 2007 and 2009 were affected by spring frosts. table 11 calculated yield in 2008 and 2010 (t/ha) influence of spacing (m) 2008 2010 4 x 1.5 23.0 21.2 4 x 2.0 17.9 15.3 4 x 2.5 16.0 13.9 198 the effectiveness of mechanical harvesting was about 95%, with 5% fruit left on the tree or lost on the ground. labour efficiency was 150 kg/8-hour-day at hand picking and 15 ton/day with mechanical harvesting. after grading fruit harvested by machine did not differ visually from that picked by hand. when put in cold storage at temperatures close to 0°c the fruit remained in good condition for five to seven days. the fruits were excellent for processing, but rather poor quality as dessert fruit. references anderson m.c., 1964 light relations of terrestrial plant communities and their measurement. biol. rev., 39: 425486. brunner t., 1990 pruning with bending effect on the basis of inducing hypotonic ramification. physiological fruit tree training for intensive culture. akademiai kiado, budapest, pp. 81-97. czynczyk a., mika a., chlebowska d., 1976 progress report on 12 years of experimentation with established spur, renewal and regulated pruning of apple trees. fruit sci. rep., 3(2): 25-28. jackson j.e., 1980 light interception and utilization by orchard systems. hortic. rev., 2: 208-267. jackson j.e., 1996 light interception and canopy characteristics at low latitudes in relation to orchard system design. acta horticulturae, 451: 417-425. mika a., piskor e., 1996 growth and cropping of dwarf ‘jonagold’ (‘jonica’) apple trees planted at the density ranged from 2000 to 10000 per ha and trained as slender spindle, super spindle and v system. acta horticulturae, 451: 473-477. nicotra a., moser l., 1995 orientamenti per la scelta dei portinnesti del susino. l’informatore agrario, 32: 43-45. sitarek m., grzyb z.s., kołodziejczak p., 2001 effect of rootstocks on growth and yield of plum trees. j. of fruit and ornam. plant res., ix(1-4): 19-24. wustenberghs h., keulemans j., 1996 a comparison of 3 plum tree training systems during the first five years of growth. acta horticulturae, 451: 625-631. zahn f.g., 1986 intensivierung von steinobstanlagen durch stärkenbezogene schnittbehandlung. erwerbsobstbau, 28: 124-140. zahn f.g., 1994 hohengerechter pflanzabstand durch stärkenbezogene baumbehandlung. erwerbsobstbau, 36: 213-220. impaginato 95 1. introduction plants frequently face adverse environmental conditions, such as drought, salinity, chilling, freezing, and high temperatures which may delay growth and development, reduce productivity and, in severe instances, cause plant death (krasensky and jonak, 2012). salt shock is an extreme form of salt stress resulting from the sudden exposure of plants to high levels of salinity (shavrukov, 2013). cellular responses to stress include adjustments in the membrane system, modifications in the cell wall architecture, and changes in cell cycle and cell division. these responsive mechanisms help plants cope with their surrounding environment and tolerate these stresses (sairam and tyagi, 2004; de oliveira et al., 2013). plant defense mechanisms against biotic and abiotic stresses can be either constitutive (continuous) or inducible under stress conditions (freeman and beattie, 2008). in the case of low level salinity stress, for example, the cells undergo osmotic phase due to the none lethal salinity. as a result, they accumulate organic non-toxic solutes such as sugars, proline, mannitol, sorbitol, and amino acids in the vacuoles of the cytoplasm, even if cells do not produce such compounds in normal conditions (läuchli and grattan, 2007; de oliveira et al., 2013). these solutes are called osmolytes, and the reason cells amass them is to achieve osmotic balance and to protect enzyme activity. furthermore, they can play a role as scavenger of oxygen-free radicals produced by salinity (shannon, 1997; sairam and tyagi, 2004; zhu, 2007; etehadnia, 2009; de oliveira et al., 2013). other compounds can also play a role in plant tolerance to stresses. the flavonoid compounds, for example, perform as free radical scavengers and antioxidants against oxidative damage during exposure to various biotic and abiotic stresses such as heavy metals, drought, salinity, excess solar radiation etc. (tattini et al., 2000; ali and abbas, 2003; brown, 2005; zhu, 2007; chutipaijit et al., 2009; samantal et al., 2011). trichomes comprise another type of plant defense response and they play mechanical and chemical roles in controlling water loss by transpiration, increasing tolerance to extreme temperatures, protecting plants against attacks by herbivores, and adv. hort. sci., 2016 30(2): 95-102 doi: 10.13128/ahs-19135 ultrastructural changes in potato (solanum tuberosum) under nacl mediated salinity stress in vitro b. al-safadi (*), m. nakar department of molecular biology and biotechnology, atomic energy commission of syria, po box 6091, damascus, syria. key words: flavanone glycoside, nacl, solanum tuberosum, trichomes, ultrastructural changes. abstract: histological analysis was employed to investigate the way potato plants (solanum tuberosum cv. draga and spunta) face salinity stress. different concentrations of nacl (50, 100, 150, 200 and 250 mm) were used on potato plantlets growing in vitro to simulate salinity stress condition. potato plants treated with 50 and 100 mm concentrations of nacl went into the osmotic stage, and responded with changes: the flavone naringin was created and accumulated in the cells of the aerial parts, and a different type of trichome was observed, in addition to the original types, in potato plants treated with concentration 100 mm. this new type of trichome appeared similar to type b trichomes therefore they were called “type b-like trichomes”. while no substance was exudated from these trichomes in cv. draga, the trichomes, in cv. spunta, green droplets were noted on the glandular vesicle. furthermore, the non glandular trichomes had some swollen stem cells, and branched ones were also observed. thanks to these new trichomes, the plants had increased leaf pubescence. (*) corresponding author: ascientific@aec.org.sy received for publication 10 september 2015 accepted for publication 13 may 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 95-102 96 reducing excessive radiation (gonzales et al., 2008; kang et al., 2010; adebooye et al., 2012). solanum species are well-known for employing their trichomes (especially the glandular types) against insects, for example, the resistance of wild potato cultivars against colorado potato beetle, potato tuber moths, the parasitoid copidosoma koehleri (blanchard) (pelletier et al., 2013), green peach aphid (myzus persicae sulzer) (vallejo et al., 1994), and potato leafhopper (medeiros and tingey, 2006). the role of potato trichomes in abiotic stress tolerance has not been reported. however, tattini et al. (2002) found an integrated role of glandular trichomes and flavonoid glycosides in the mechanisms of acclimation of phillyrea latifolia to excess solar radiation. tuber-bearing solanum species have two major types of trichomes: glandular and non glandular. glandular trichomes are recognized as type a or type b and have been extensively studied (pelletier et al., 2013). type a has a short stalk and a four-lobed glandular head which contains phenolic compounds, while type b is characterized by its long stalk and small glandular vesicle which continuously exudate acyl sugar secretions. wild potato solanum berthaultii bears all types of trichomes, some, or none (pelletier et al., 2013), but some commercial solanum tuberosum cultivars, for example elba and allegeny, have non-glandular and type a glandular trichomes only (medeiros and tingey, 2006). the aim of the present study was to use histological analysis to investigate how potato (solanum tuberosum) responds to salinity shock. 2. materials and methods plant material tubers from the potato cultivars draga and spunta were obtained from general organization for seed multiplication (aleppo, syria). draga variety, a cross svp 50-2017 x mpi 19268 (hzpc, netherlands) is an early variety, consistent performer, with medium yield, medium to large uniform tubers, round shape, cream-coloured skinned with creamy-white flesh and medium depth eyes (eurogrow, 2011). spunta variety, a cross béa x usda 96-56 (higgins agriculture ltd ) is a medium-early ripening variety, with very high yield, long, slightly kidney-shaped, bulky tubers and shallow eyes (ahdb, 2011). explants (nodal segments containing one or two buds) were prepared as previously described (alsafadi and arabi, 2003, 2007). salinity stress in order to simulate salinity stress in vitro, the explants were grown in tubes containing ms medium (murashige and skoog, 1962) with different concentrations of nacl (50, 100, 150, 200, 250 mm) and grown as described by al-safadi and arabi (2007). histo-anatomical study six plants from each salinity treatment were examined. the whole potato plant (shoot, mature leaves, root) and peels of orange fruit were prepared as follows: the samples were fixed in carnoy’s solution (3 ethyl alcohol: 1 acetic acid) for 2 h, then transferred to 70% alcohol and stored at 4°c until analyses. cross-sections were prepared manually by using a blade razor. some sections were stained with safranin o for 5 min (al-khatib et al., 1995; tiţă et al., 2010); for sample examination, a bright field microscope (nikon eclipse 80i) was used and photos were captured by a digital camera (nikon ds-ri1). identification of the crystals solubility. five different plants from each salinity treatment were used. two leaves per plant were taken and immersed in hot water (90°c) for 10 min, and grapefruit juice was diluted with water (50%) and heated to (90°c) for 10 min. the crystals were then studied as cross-sections under nikon eclipse 80i microscope to observe whether any dissolution occurred. reacting to ferric chloride. since physiological parameters showed no significant differences (data not shown) between plants treated with 50 mm and those treated with 100 mm nacl, and for simplicity purposes, only leaves and cross-sections of plants treated with 100 mm of nacl, plus the control, were soaked in a solution of 1% ferric chloride and heated at 90°c for 3-4 min, then left at room temperature for 10 min, to observe the change in colors. crystallization. to obtain and compare the sugar crystals under a nikon eclipse 80i microscope, carnoy’s solution was added for 2 h to the samples for comparison: potato leaves and cross-sections of control and those treated with 100 mm nacl, the pericarp of orange fruit and grapefruit juice. 3. results our first observation when we studied the tissues of plants treated with 150, 200 and 250 mm of nacl al-safadi and nakar ultrastructural changes in potato under nacl mediated salinity stress in vitro 97 was that most of the cells in studied plant parts (roots, shoots, leaves) suffered plasmolysis. this means that the cells were already dysfunctional and the plants were no longer in the tolerance stage but had shifted to the toxic stage, when ions accumulate up to fatal levels in the cytoplasm and cells cannot overcome the damage from accumulating solutes (lauchli and grattan, 2007; de oliveira et al., 2013). on the contrary, at concentrations of 50 and 100 mm, plasmolysis was limited, therefore we focused on these two treatments to detect changes in the cytoplasm. consequently, when referring to treated plants we intend those treated with concentrations of 50 and 100 mm only. studying cross-sections of the stems and leaves of the treated plants from both cultivars (draga and spunta) revealed some interesting and curious structures which did not exist in the tissues of control plants. scattered cells, with rosette-shaped crystals inside, were observed in the cortex layer of the stems and in the spongy mesophyll layer of the leaves (fig. 1 a, b). the crystals resembled the shape of flavanone glycosides found in citrus (hesperidin and naringin) when crystallized. hesperidin is a sweet tasting glycoside usually found in the mesocarp layer (the white pith, albedo) of the pericarp (the peel) of orange fruit citrus auratium (hendrickson and kesterson, 1956; al-khatib et al., 1995), while naringin is a principal flavonoid in grapefruit and gives it its bitter taste and can be found in the juice (kesterson and hendrickson, 1953). both glucosides are very similar in appearance to crystals under the microscope, as the crystal needles agglomerate in a rosette pattern and have very close chemical structures (kesterson and hendrickson, 1953; hendrickson and kesterson, 1956). to confirm our analysis, we took cross-sections of the pericarp of orange fruit and grapefruit juice treated with carnoy’s solution. comparing the results to those in the treated potato, we found the two shapes to be similar (fig. 1 c, d). these crystals were not seen in tissues not fixed with carnoy’s solution. this is considered to be further evidence that these were crystals of a flavanone glycoside dissolved in the cytoplasm and only could be seen under microscope when crystallized by adding alcohol, acetic acid or cooling (kesterson and hendrickson, 1953; hendrickson and kesterson, 1956; al-khatib et al., 1995). moreover, in the control plants, these crystals were not found in the tissues before or after treating with carnoy’s solution or cooling. the crystals, in addition to their rosette shape, had specific naringin’s crystal features: i) brownish tan color while hesperidin crystals are colorless or pale yellow as seen in the mesocarp layer of the peel of orange fruit (fig. 1 a-d) (kesterson and hendrickson, 1953; hendrickson and kesterson, 1956); ii) when interacted with ferric chloride, the color of the leaves of treated plants changed from pale grey to light caramel. on the contrary, when the leaves of the control plants were immersed in ferric acid no difference in color could be seen (fig. 2 a, b). the color of the crystals in the cross sections of the leaves changed from brownish caramel to a very dark red wine color (fig. 3 a, b). the interaction of ferric chloride with naringin, especially at a sufficient level of concentration, caused the change in color which gets darker with the increase in concentration, becoming black at very high concentrations (kesterson and hendrickson, 1953; sinclair, 1972; radhakrishnan et al., 2013); iii) when the leaves were immersed in hot water for 10 min, the crystals in the cross sections and grapefruit juice started diffusing (fig. 3 c-e); it is known that naringin’s crystals are much more soluble in hot than cold water, unlike hesperidin which does not dissolve in hot water (kesterson and hendrickson, 1953; hendrickson et al . , 1954; hendrickson and kesterson, 1956). therefore, we assume that the crystals were fig. 1 rosette-shapedcrystals with caramel color inside cells of the stem's cortex of treated plants with salt concentration 100 mm. a) in draga b) in spunta c) pale hesperidin crystal in the mesocarp layer of the peel of orange fruit, d) tan naringin crystals in the juice of grapefruit; the resemblance between the two structures is obvious. adv. hort. sci., 2016 30(2): 95-102 98 naringin. the control plants and those treated with the 50 mm concentration had only straight non-glandular and type a trichomes on the aerial parts. however, surprisingly, type b-like trichomes were observed in addition to the other type of trichomes in plants treated with the 100 mm concentration. while they had type b trichomes, there was no evidence that they secreted any substance in ‘draga’ plants, although green droplets were noticeable on glandular vesicles in ‘spunta’ plants (fig. 4). in control plants and those treated with 50 mm nacl, non-glandular trichomes were straight, linear with conical shape, and the size of the stem cells were gradually reduced from the base cell to the top. however, in the 100 mm nacl treatment, the trichomes were very long and branched with swollen stem cells on very large base cells (fig. 5). fig. 3 dark red crystals from the leaves of treated plants after ferric acid reaction: a) from cultivar draga; b) from cultivar spunta; c-d) naringin crystals with melted edges after boiling in water: c) from cultivar draga; d) from cultivar spunta, e) from grapefruit juice. fig. 4 photos of trichomes colored with safranin o leaves of: ab) control plants; a. conical straight non-glandular, b) type a. c-d) ‘draga’ plants treated with 100 mm salt concentration; c) a photo of whole type b-like trichomes, d) empty glandular vesicle of the type b-like trichomes without any secrets . e-f) ‘spunta’ plants treated with 100 mm salt secretions; e) a photo of whole type blike trichomes; f) the glandular vesicle of type b-like trichomes secreting green droplets (arrows). bars=100 µm. fig. 2 leaves of treated plants, the leaf immersed in ferric acid (left) and not immersed (right) (differences in color are noticeable): a) draga, b) spunta c) draga control plant leaf: immersed in ferric acid (left) and not immersed (right) (no changes in color could be seen). d) microscope photo showing parts of ‘spunta’ control plant leaf (left) and treated plant leaf (right) , both were immersed in ferric acid and dark red dots (naringin crystals) could be seen only in the cells of treated plant leaves. fig. 5 photos of non-glandular trichomes on the adaxil surface of safranin o.colored leaves of plants treated with 100 mm salt concentration; a) branched trichomes (draga) (arrows), b) two non-glandular trichomes with swollen stem cells (draga) (arrows), c-d) a nonglandular trichome with very large base cell (arrow) compared to the adjacent cells: c) from draga, d) from spunta. (arrows). bars=100 µm. al-safadi and nakar ultrastructural changes in potato under nacl mediated salinity stress in vitro 99 4. discussion and conclusions in this study we found that potato plants treated with low concentrations of salinity (50 and 100 mm) went into the osmotic stage. consequently, cells in the aerial parts created a new substance, which was identified as ‘naringin’ (the rosette-shaped crystals we found in the cortex) (fig. 2 a, b). naringin is a flavanone-7-o-glycoside between the flavanone naringenin and the disaccharide neohesperidose. it is mainly found in grapefruit to which it gives its typical bitter flavor (belajova and suhaj, 2004). flavonoids, such as catechin, epi-catechin, erodictyol, kaempeferol, and naringenin in different amounts, are very common in the cultivated potato solanum tuberosum (brown, 2005). it has been reported for rice (chutipaijit et al., 2009), barley (ali and abbas, 2003) and many other plants (samantal et al., 2011) that flavonoids accumulate in the tissues of stressed-plants. furthermore, gupta and huang (2014) reported that anthocyanin (also a flavonoid) is accumulated in plants exposed to salt stress. flavonoids become more soluble in the form of flavonoid glycoside and thus more effective (chutipaijit et al., 2009; samantal et al., 2011). therefore, we assume that the flavonone naringenin in treated potato became available in larger quantity than normal, as a result of the salinity stress. in potato, naringenin is a byproduct of the pathway of anthocyanin and flavonoles biosynthesis (gramene, 2016). it is unclear, from our work, how naringin was synthesized and accumulated in the tissues of potato plants treated with nacl. however, it is likely that under stress conditions, potato plants produced naringin through the naringenin glycoside biosynthesis pathway (caspi et al., 2016), thus making it possible to visualize naringin under the microscope as crystals. our study also revealed that potato plants treated with 100 mm concentration resulted in an additional type of trichomes, to the original types (non-glandular and type a) (fig. 4 c-f). these new trichomes were similar in shape (long stalk with glandular vesicle) to those in the wild type potato solanume berthaultii (vallejo et al., 1994), hence, we called them “type blike trichomes”. here, we suggest several hypotheses to explain why potato plants formed these trichomes. with regard to the genetics of type b trichomedensity inheritance, we found that most of the studies in the literature were carried out on diploid potatoes. however, some researchers (gibson, 1979; mehlenbacher et al., 1983; mehlenbacher et al., 1984; vallejo et al., 1994; jansky et al., 1999) have convergent conclusions, for example: a) the absence of type b trichomes is controlled by a few genes, at least one of them being recessive due to structural genomic differentiation; b) most of the variation among individuals of offspring of back-crosses studied were not due to heritable genetic differences; c) the inheritance of type b trichome density could be influenced by non nuclear genetic factors such as maternal cytoplasmic dna, as suggested by vallejo et al. (1994), and jansky et al. (1999) who presumed that the cytoplasm in the somatic tetraploid hybrids they studied could repress the gene expression; d) in contrast, the droplet size of type b trichomes was highly heritable. therefore, we wonder if the genes responsible for the presence of type b trichomes were silenced in one way or another in cultivated potatoes, due to structural genomic differentiation during the hybridization, segregation and recombination processes. especially in our case, ‘draga’ and ‘spunta’ are tetraploids with 2n = 4x = 48 chromosomes (caprutoi et al., 2000, 2003). additionally, the expression of these genes could be affected by changes in the cytoplasm (vallejo et al., 1994; jansky et al. 1999; hanson and bentolila, 2004). therefore, we hypothesize (although more genetic studies are called for on this subject) that at least one of the genes that control the presence of type b trichomes was silenced or changed epigenetically, which affected its expression and, subsequently, the other genes could not be expressed during the evolution of the potato cultivars. in the current study we did not investigate the genes involved in formation or activation of trichomes under salinity stress. however, other studies have looked into the wrky transcription factors (tfs) family in plants which comprises numerous members that regulate genes involved in seed germination, seed dormancy, trichomes development, lignin biosynthesis, and both biotic and abiotic stress responses (pnueli et al., 2002; guillaumie et al., 2010; wang et al., 2010). some of the wrky genes have been reported to be activated in response to various abiotic stresses including high salinity (jianchao et al., 2015). furthermore, it has been documented that salinity could induce modifications in the genome, like other abiotic stresses, by making changes in the histone modification pattern, thus activating some genes and/or silencing others (kapazoglou and adv. hort. sci., 2016 30(2): 95-102 100 tsaftarism, 2011; kim et al., 2012; pecinka and scheid, 2012). it could also induce irregularities in the mitotic division and aberrations in the mitotic chromosomes (barakat, 2003). salinity may thereby change the genome epigenetically (changes in the chromatin) and genetically (changes in dna). it is also possible that salinity affected the resultant proteins of those genes; it has been reported that salinity can make changes in the protein patterns (barakat, 2003; kim et al., 2012). on the other hand, without regard to genetic reasons, gonzales et al. (2008) referred to some reports which presumed that plants under salt stress could use the glandular trichomes as facultative salt glands and to eliminate excessive salt (gonzales et al., 2008). in our case, it is possible that a conversion from non-glandular to glandular trichomes under salt stress occurred. however, as we indicated in the results section of this work, we did not observe any substance exudates from the type b-like trichomes in ‘draga’, while in ‘spunta’, these trichomes exuded some kind of green droplets. if the droplets had the same chemical makeup as the exudate from the glandular vesicle of type b trichomes (sucrose esters viscous droplets) (pelletier et al., 2013), it would mean that they may form an irregular local layer on the surface of the leaves; it is known that these trichomes secrete droplets continuously (pelletier et al., 2013; wollenweber et al., 2005). this layer would play a role of additional covering to coat and reduce the absorbance of radiation and water loss. furthermore, we detected some non glandular trichomes with swollen stem cells (fig. 5 a, b, c). the same results were reported by kang et al. (2010) when they studied trichome distortion caused by hairless mutation of tomato (solanum lycopersicum). they found that type i trichomes (equivalent to type b trichomes in potato) on the hl mutant were crooked and had highly swollen stem cells (kang et al., 2012). in contrast, gomes et al. (2011), after treating salvinia auriculata aubl. with different doses of nacl salt, found that the trichomes became more slender as the dose was increased. we also noticed branched trichomes (fig. 5 d) which are probably caused by the extensions in the swell of stem cells, giving the appearance of branching. kang et al. (2010) reported the same observation in tomato affected by hairless mutation (kang et al., 2012). also, irregularity in trichome shape was reported in some plants which were exposed to salt stress (adebooye et al., 2012). nevertheless, the branched trichomes are more effective in protecting the plant from losing additional amounts of water by forming a shield against sunlight, thus maintaining a good quantity of water to adjust the turgor pressure in cells. somehow, the branched trichomes and type b-like trichomes contributed to increasing leaf pubescence, which is a familiar phenomenon among some plants subjected to drought or salinity stress (gonzales et al., 2008; makbul et al., 2011; adebooye et al., 2012). in our study, potato gained new traits: naringin (the flavanone glycoside), branched non-glandular trichomes and type b-like trichomes. the new trichomes allow these plants to have increased leaf pubescence and perhaps different chemicals on the leaf surface, making them more resistant to insects. we believe more research must be carried out on how salinity and drought could be exploited to produce more resistant plants. after all, as some research has pointed out, salinity does not always cause negative effects in plants (shannon and grieve, 1999). in conclusion, potato plants (solanum tuberosum l.) have mechanisms to help them tolerate salinity stress at the cellular, biochemical, and physiological levels. in the present study, some changes were observed in potato plants growing in vitro, including the creation of the “flavanone glycoside” naringin in the cells of the aerial parts (playing the role of antioxidant) and the formation of a new type of trichome. further research is needed to study the role of these cellular changes in potato plants reacting to salinity stress. acknowledgements the authors would like to thank the director general of aecs and the head of molecular biology and biotechnology department for their support. the assistance of n. tafesh and i. karajoli in conducting the experiments is much appreciated. references adebooye o.c., hunsche m., noga g., lankes c., 2012 morphology and density of trichomes and stomata of trichosanthes cucumerina (cucurbitaceae) as affected by leaf age and salinity. turk j. bot., 36: 328-335. ahdb, 2011 spunta potato variety . http://varieties.ahdb.org.uk/varieties/view/spunta. ali r.m., abbas h.m., 2003 response of salt stressed al-safadi and nakar ultrastructural changes in potato under nacl mediated salinity stress in vitro 101 barley seedlings to phenylurea. plant soil environ., 49: 158-162. al-khatib a., bagdadi w., hamad i., soliman m., 1995 botany for freshmen. university of damascus’ press, damascus, syria. al-safadi b., arabi m.i.e., 2003 in vitro induction, isolation and selection of potato mutants resistant to late blight. j. genet. breed., 57: 359-364. al-safadi b., arabi m.i.e., 2007 in vitro induction, isolation, and selection of potato mutants tolerant to salinity. adv. hort. sci., 21(3): 127-132. barakat h., 2003 interactive effects of salinity and certain vitamins on gene, expression and cell division. int. j. agri. biol., 5: 219-225. belajova e., suhaj m., 2004 determination of phenolic constituents in citrus juices: method of high performance liquid chromatography. food chemistry, 86: 339-343. brown c.r., 2005 antioxidants in potato. amer. j. of potato res., 82: 163-172. carputo d., basile b., cardi t., fruciante l., 2000 erwinia resistance in backcross progenies of solanum tuberosum x s. tarijense and s. tuberosum (+) s. commersonii hybrids. potato res., 43: 135-142. carputo d., parisi m., consiglio f., iovene m., caruso g., monti l., frusciante l., 2003 aneuploid hybrids from 5x-4x crosses in potato: chromosome number, fertility, morphology and yield. amer . j. of potato res., 80(2): 93-101. caspi r., billington r., ferrer l., foerster h., fulcher ca., keseler i.m., kothari a., krummenacker m., latendresse m., mueller l.a., ong q., paley s., subhraveti p., weaver d.s., karp pd., 2016 the metacyc database of metabolic pathways and enzymes and the biocyc collection of pathway/genome databases. nucleic acids res., 42: d471-480. chutipaijit s., cha-um s., sompornpailin k., 2009 differential accumulations of proline and flavonoids in indica rice varieties against salinity. pak. j. bot., 41: 2497-2506. de oliveira a.b., alencar n.l.m., gomes-filho e., 2013 comparison between the water and salt stress effects on plant growth and development, pp. 67-94. in: akinci ș. 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(lc50 = 0.004%), cananga odorata (lam.) hook f. and thoms (lc50 = 0.050%), murraya koenigii (l.) spreng. (lc50 = 0.113%), and euodia hortensis forma hortensis (lc50 = 0.114%). the essential oil of m. koenigii (ri=52%) and c. citratus (ri=52%) at 5% (v/v) concentration were found to have a higher repellent toxicity against the spiralling whiteflies. the chemical composition of the selected essential oils was also determined using gc-ms. the trend in the chemical constituent of essential oils revealed that the phenolic and alcoholic compounds were the major groups of contributors to the tested activities. thus, these data suggested that essential oils from the selected medicinal plants found in the south pacific (fiji) have the potential to be employed in the pesticidal activities. (*) corresponding author: chand_rv@usp.ac.fj received for publication 10 august 2016 accepted for publication 18 october 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 165-174 166 diverse use of essential oil can represent a good alternative due to its novel, safe and eco-friendly substitute for its effective insecticidal properties (li et al., 2014; palanisami et al., 2014). several essential oils from medicinal plants have been screened for the repellence and toxicity against grain storage insects, fleas, ticks and lice (leal and uchida, 1998; gandhi et al., 2010; olivero-verbel et al., 2010; caballero-gallardo et al., 2011; cheng et al., 2012; seo et al., 2012; vera et al., 2014). however, very little information on the fumigant and repellent toxicity of essential oils from medicinal plants found in the south pacific (fiji) was available against the spiralling whiteflies. the aim of this study was to assess if essential oils of determined medicinal plants could serve as bio-pesticides for the control of the whitefly pest. the chemical profile of selected essential oils was also studied in order to provide justification for the presence of active compounds in the tested activities. 2. materials and methods essential oils extraction and analysis the plant materials from cananga odorata (lam.) hook f. and thoms (makosoi flowers), cymbopogon citratus (dc.) stapf. (lemongrass leaves), murraya koenigii (l.) spreng. (curry leaves), ocimum tenuiflorum l. (tulsi leaves) and eudioa hortensis forma hortensis (uci leaves) were collected from fiji islands in april to november, 2015. the selected plant materials were verified with the voucher specimens placed at university of the south pacific herbarium and koronivia research station, suva, fiji islands. the plant materials from the medicinal plants were hydro-distilled using clevenger apparatus for 5-7 hours. a meniscus layer (essential oils) was formed in the collecting tube which was then collected in a vial. the samples were dried over anhydrous sodium sulphate (na2so4) and stored at 4°c. the analysis of essential oils using gas chromatography equipped with mass spectrometry (agilent technologies 6890) was performed using an hp-5ms non polar fused silica capillary column (0.25 mm, 30 m, 0.25 μm film thickness; model number: 19091s-433) with the following conditions: the oven temperature was programmed from 50°c to 325°c over 5 min, at equilibration time of 0.50 min. the transfer source and quadrupole temperatures were 150°c, 200°c, 230°c and 250°c respectively, operating at 71 ev ionization energy. for the front inlet the mode used was split with an initial temperature of 250°c at 42.5 kpa at a split ratio of 50:1 and split flow of 43.8 ml/min. helium was used as a carrier gas at a constant linear velocity of 35 cm/sec, flow rate of 0.9 ml/min; the injected sample volume was 1.0 μl which was diluted in hexane (1000 μl). the analysis was carried at the southern cross university, australia. the constituents of essential oils were identified based on mass spectra comparison of retention indices (ri) with standard compounds. for the reference purpose, the database search was done using essoils and adams library. for the purpose of semi-quantification, the normalized peak areas of reported compounds were used without any correction factors for establishing abundance. retention indices (ri) and abundance were calculated using the mean values of 3 injections (el bouzidi et al., 2011). breeding of spiralling whiteflies the adult spiralling whiteflies were brought from a nearby farm (rewa province) without any insecticidal exposure. the collected spiralling whiteflies were brought to the green house where they were introduced to the cassava plants [manihot esculenta (crantz)] in order for them to grow and multiply. the plants were maintained in the greenhouse for appropriately 6-7 months without any pesticide contact before carrying out the actual experiment. the adult spiralling whiteflies were collected in petri dish using a small paintbrush. the conditions that were set in the laboratory were similar to the environment that they were found, that is, under the condition of 28±2°c, 75±5% rh and light regime of 14:10 h (l:d). the spiralling whiteflies (aleurodicus dispersus russell) bred in the greenhouse were brought into the laboratory when required to carry out the fumigant and repellent test. fumigant toxicity assessment the leaves of the cassava pot plants were enclosed with a clear pocket plastic bag (16 cm in length) with 50 whiteflies in each bag irrespective of their sex. the treatments [0.25%, 0.5% and 5% (v/v)] were introduced into each plastic bag using a filter paper (~2 cm in diameter) based on the randomisation. the filter paper discs (~2 cm in diameter) were impregnated on the side of the plastic bag. the control filter disc had tween 20 (5%) (purchased from sigma-aldrich, australia) mixed with the distilled water. the mortality count results after 3, 6, 9, 12 chand et al. bioactivity of essential oils from medicinal plants in fiji 167 and 24 hours were calculated. repellent toxicity assessment a t-shaped olfactometer set was constructed in order to test the repellency on the adult spiralling whiteflies. the setup consisted of a long glass tube (diameter of 50 cm). the external light source was placed between site 1 and site 2. site 1 had the control leaf disc (2 cm in diameter) dipped in tween 20 (5%) solution, while site 2 had the leaf disc with selected concentration of the essential oil. the essential oil concentration for all the five plants tested were 0.25%, 0.5% and 5% (v/v). the test was performed on 50 adult whiteflies with 4 replicates for each concentration. after 6-8 hours the number of whiteflies were counted using a hand lens for each site (chamber). the repellency index (ri %) was calculated using the formula (abdellaoui et al., 2009): ri % = (c-t/ c+t) x 100 where [c= whitefly counts on the control side of the olfactometer] and [t = whitefly counts on the treatment side of the olfactometer] . if the repellency index calculated (ri %) is positive, it means that the whiteflies were repelled with the tested concentration of essential oils and vice versa if the repellency index (ri %) calculated is negative. statistical analysis fumigant test assessment. a factorial anova (5x4x5 split plot design) using tukey’s hsd test was performed. prior to performing anova (significant at p=0.05), the percentage mortalities were transformed by the arcsine of the square root. the total mortalities were converted to percentage mortality. the lethal concentration (lc50) values for the mortality after 24 hours were assessed using probit in xlstat software (version 2015.1) (kabir et al., 2007; postelnicu, 2011). the morality was corrected using abbott’s formula for those that exceed 10% by natural mortality (abbott, 1925). repellent test assessment. to evaluate the statistical difference at 5% level of significance between each essential oil with its respective control, an independent sample t-test was performed. the probit analysis in xlstat software (version 2015.1) was also used to calculate the ec50 for the repelling effect of each essential oil (padhy and panigrahi, 2016, olufayo and alade, 2012). 3. result and discussion chemical analysis of the essential oils a total of 88 compounds were detected in the selected essential oils from the medicinal plants, accounting for 92.76-97.88% of total composition as summarised in table 1 and 2. the main chemical compounds identified in the essential oil of c. odorata were trans, trans-farnesol (29.71%), benzyl benzoate (21.69%), linalool (16.65%) and trans, trans-farnesyl acetate (6.93%). while for m. koenigii the major compounds identified were sabinene (43.80%), βcaryophyllene (16.52%), terpinen-4-ol (7.20%) and αpinene (5.67%). in case of e. hortensis, menthofuran (55.17%) and evodone (25.91%) were the main compounds. the essential oil from o. tenuiflorum revealed the presence of eugenol (58.20%), germacrene d (11.68%) and cis-β-ocimene (10.79%) as the major compounds. the major compounds identified in the c. citratus essential oil were citronellal (45.09%), citronellol (19.11%), geraniol (13.57%) and elemol (6.15%). table 1 composition of essential oils (%) from c. odorata (makasoi), m. koenigii (curry leaves), e. hortenis (uci), o. tenuiflorum (tulsi) and c. citratus (lemon grass) chemical compounds ocimum tenuiflorum (%) cymbopogon citratus (%) cananga odorata (%) euodia hortensis forma hortensis (%) murraya koenigii (%) α-thujene 0.61 0.31# 1.79 linalool 0.21 0.27 16.65 0.10# myrcene 0.38 0.11 0.37 1.84 sabinene 0.43 0.58# 43.80 iso-pulegol 1:17 α-pinene 0.32 5.67 limonene 4.64 1-octen-3-ol 0.19# citronellal 45.09 0.20 iso iso-pulegol 0.46# β-pinene 1.55 α-terpinene 0.23# 2.64 decanal 0.14# methyl benzoate 1.64 menthofuran 55.17 p-cymene 0:23 0.67 citronellol 19:11 0.13# cis-β-ocimene 10.79 0.11 neral 0:55 ethyl benzoate 0.14 limonene-10-ol 0.60 trans-β-ocimene 0.43# 0.39 geraniol 13:57 0.74 terpinen-4-ol 1:01 0.15 7.20 evodone 25.97 β-phellandrene 0.69 ϒ-terpinene 0:37 4.82 geranial 0.74 methyl salicylate 3.15 α-copaene 1.98# 0.79 citronellic acid 0.37# to be continued adv. hort. sci., 2016 30(3): 165-174 168 variability in the essential oils the results obtained also showed variability in terms of the quality, quantity and composition of essential oils in all the selected plants when compared to the available literature, that is, o. tenuiflorum (pino et al., 1998; naquvi et al., 2012), c. citratus (negrelle and gomes, 2007; olivero-verbel et al., 2010; matasyoh et al., 2011; tyagi et al., 2014), c. odorata (katague and kirch, 1963; gaydou et al., 1986; murbach teles andrade et al., 2013), e. hortensis (brophy et al., 1985) and m. koenigii (raina et al., 2002; chowdhury et al., 2008) (table 1). the variability in the composition of essential oil is mainly due to the genetic variations, climatic, ecological locations, soil composition, plant organs, age and vegetative cycle stages of the plant (pietschmann et al., 1998; masotti et al., 2003; stewart, 2005; tchoumbougnang et al., 2005; angioni et al., 2006; koba et al., 2007; nascimento et al., 2008; katoch et al., 2013; erbil et al., 2015; ríos, 2016). fumigant toxicity of selected essential oils among the five tested essential oils (fig. 1 a-c), o. tenuiflorum essential oil showed the most robust fumigant effect against the spiralling whiteflies with lc50 value of 0.003% followed by the essential oils of c. citratus (lc50 = 0.004%), c. odorata (lc50 = 0.050%), m. koenigii (lc50 = 0.113%), and e. hortensis (lc50 = 0.114%) (table 3). statistically, the fumigant activity of o. tenuiflorum and c. citratus essential oils at 0.5% and 5% (v/v) concentrations were significantly higher than the other species (p=0.00). the significant threshold was set at p<0.05. the mortality count of the spiralling whiteflies were also higher at 5% (v/v) concentration for c. odorata, e. hortensis and m. koenigii essential oils as compared to 0.25% and 0.5% (v/v) concentrations. generally the increasing concentrations of the tested essential oils led to increased mortality of whiteflies. the robust effect of o. tenuiflorum essential oil chemical compounds (z) ocimum tenuiflorum (%) cymbopogon citratus (%) cananga odorata (%) euodia hortensis forma hortensis (%) murraya koenigii (%) methyl chavicol 0.45# β-cubebene 0.26 allo-ocimene 0.17# citronellyl acetate 1.05# limonene-10-yl acetate 0.60 geranyl acetate 0.44 trans-anethole 0.27# α-(2) gurjunene 0.59# trans-sabinene hydrate 0.59 α-cubebene 0.18# β-elemene 0.59# δ-elemene 0.24# βcaryophyllene 0.54 isoterpinolene 0.95# eugenol 58.20 1.38 0.33# germacrene d 11.68 0.79# 2.74 0.27# 0.14 trans-α-bergamotene 0.18# trans-p-menth-2-en-1-ol 0.47# δ-cadinene 1.44# 0.88 methyl eugenol 1.77 trans-β-farnesene 0.20# β-bourbonene 0.93 elemol 6.15 β-caryophyllene 4.31 0.49 16.52 β-funebrene 0.23# α-terpineol 0.28 4-α-hydroxyl germacral (10), 5-diene 1.15# humulene 0.33# 0.29# cis-piperitol 0.12# β-copaene 0.35 ϒ-eudesmol 0.72# β-selinene 0.31# 0.40# trans-piperitol 0.17# δ-cardinol 0.27# α-germacrene 0.35# 0.18# ar-curcumene 0.60 ϒ-muurolene 0.40# α-cardinol 3.70 βelemene 1.50 trans, trans-farnesol 29.71 cis, trans-farnesol 0.46# bicyclogermacrene 0.41# trans, trans-farnesal 0.43# benzyl benzoate 0.21# 21.69 β-curcumene 0.56# α-cardinene 0.55# ϒ-cardinene 0.22# trans, trans-farnesyl acetate 6.93 δ-cardinene 0.46# α-selinene 0.78# benzyl salicyate 2.21 caryophyllene oxide 0.24 0.75# epi-1-cubenol 0.13# trans-nerolidol 0.24# α-cadinol 0.87# intermedeol 0.27# ϒ-curcumene 3.79# (z) compounds listed in order of elution from a hp-5ms non polar fused silica capillary column. # indicate that the compounds were detected for the first time as compared to the literature. chemical groups cananga odorata (%) murraya koenigii (%) euodia hortensis forma hortensis (%) ocimum tenuiflorum (%) cymbopogon citratus (%) monoterpenes 1.32 65.51 60.18 13.64 ester 35.76 0.60 1.70 alcohol and phenol 50.85 9.08 0.83 60.61 45.88 sesquiterpenes 4.13 20:27 9.17 22.61 3.41 aldehyde 0:43 0.20 46.52 ketones 25.97 acid 0.37 miscellaneous 0.27 total (%) 92.76 94.86 96.95 96.86 97.88 table 1 (continued) table 2 the major chemical groups present in the essential oils of c. odorata, m. koenigii, e. hortensis, o. tenuiflorum, and c. citratus chand et al. bioactivity of essential oils from medicinal plants in fiji 169 could be attributed to the chemical constituents present in the oil. in this study, o. tenuiflorum essential oil had 60.61% of alcoholic and phenolic compounds as compared to c. citratus (45.88%), c. odorata (50.85%), e. hortensis (0.83%) and m. koenigii (9.08%). according to isman (2000), eugenol compounds were found to be 7-9 times more toxic than terpenes and terpinene-4-ol. this confirms that o. tenuiflorum essential oils from the present study showed a strong fumigant effect due to eugenol (58.20%) compound. in previous studies, eugenols were also reported as a major cause of toxicity against the adult beetle (callosobruchus maculatus) (ajayi et al., 2014), bean weevil (acanthoscelides obrectus) (regnault-roger and hamraoui, 1995), yellow fever mosquito (aedes aegypti) (sosan et al., 2001) and rice weevil (sitophilus oryzae) (lee et al., 2003). cymbopogon citratus essential oil also showed strong fumigant effect (fig. 1 a-c). such effect can be attributed to the major chemical compounds, alcohols and phenols (45.88%), especially citronellol (19.11%) and geraniol (13.57%). these major chemical compounds have showed toxicity and repellent effects on different pests (fradin and day, 2002; ansari et al., 2005; choochote et al., 2007; paluch et al., 2009; sakulku et al., 2009; maia and moore, 2011). similarly, the interaction of different chemical compounds could have played a major role in the repression of the fumigant effect. according to chang et al. (2009), when linalool compound from the basil oil (ocimum family) was mixed with cuelure compounds, the level of toxicity on the tested insect (melon fly, bactrocera cucurbitae) decreased. the above scenario could explain why c. odorata essential oil had the second highest percentage of alcohol and phenol compounds (50.85%) while it was not able to produce a greater fumigant effect as compared to c. citratus (45.88%) essential oil. fig. 1 fumigant effect (mean±se) of selected essential oils on the spiralling whiteflies over different time intervals using different solution concentrations: (a) 0.25%; (b) 0.5%; (c) 5% (v/v). the alphabetical letters represent the respective essential oils and the asterisks indicate results statistically different from the control at p<0.05 (*), p<0.01 (**), p<0.001 (***) using tukey’s test. essential oils time (hours) equation r2 lc 50 /ec 50 (%) χ2 statistic p-value df cananga odorata 24 y = 4.998 + 4.086x 0.750 0.050 118.149 <0.0001 1 murraya koenigii 24 y = 3.408+ 3.933x 0.316 0.113 76.080 <0.0001 1 euodia hortensis forma hortensis 24 y = 3.349+3.887x 0.586 0.114 78.574 <0.0001 1 cymbopogon citratus 24 y = 8.725+3.764x 0.902 0.004 279.950 <0.0001 1 ocimum tenuiflorum 24 y = 12.286+5.020x 0.651 0.003 253.512 <0.0001 1 table 3 dose-effect analysis of the fumigant properties of essential oils on the spiralling whiteflies after 24 hours at 0.25%, 0.5% and 5% (v/v) concentrations the χ² probability ≤0.0001, indicated that the significant difference was brought by the log (concentration) variable and the repellency. each test represents the mean of four replicates of 50 whiteflies. adv. hort. sci., 2016 30(3): 165-174 170 repellent toxicity of selected essential oils the table 4 revealed that none of the essential oils showed a very strong repelling effect on the spirall ing whiteflies. in order, based on the repellency index (ri %) of selected essential oils at the highest concentration [5%( v/v)], we found c. citratus (52%), m. koenigii (52%), o. tenuiflorum (12%), e. hortensis (10%) and c. odorata (9%). a direct relationship was seen between the repellent effect and the concentration (table 4). statistically, it was found that only c. citratus had a strong significant difference (r2 =0.611, p=0.00) at tested concentrations. the ec50 values in ascending order of the repellent effect of selected essential oils were 3.05% (c. odorata), 2.73% (o. tenuiflorum), 0.96% (e. hortensis), 0.43% (c. citratus) and 0.41% (m. koenigii). the chemical analysis in this study revealed the presence of α-pinene (5.67%), β-pinene (1.55%) and myrcene (1.84%) only in the essential oil of m. koenigii. the other active compounds that might have contributed towards the repellent effect can be terpinene-4-ol (7.20%) and eugenol (0.33%). in previous report, these compounds were found to repel yellow fever mosquito (aedes aegypti) (coats et al., 1991; debboun et al. , 2014), bean weevil (callosobruchus chinensis) (haidri et al., 2014) and two-spotted spider mites (lee et al., 1997). the repellent activity of c. citratus and m. koenigii essential oils at the highest concentration [5% (v/v)] were similar. in agreement with nerio et al. (2010), essential oil from c. citratus family were found to have promising repellent properties. the active compounds from previous studies such as α-pinene, limonene, citronellol, citronellal, camphor and thymol have shown higher repellent activity against ticks (amblyomma americanum) and yellow fever mosquito (aedes aegypti) (nerio et al., 2010; debboun et al., 2014). this study also reported the presence of citronellal (45.09%), citronellol (19.11%) and geraniol (13.57%) that may have caused the repellent effect (table 1). interestingly, the essential oil activity of o. tenuiflorum showed a weak repellency against the spiralling whiteflies, despite the fact that the mode of action of essential oils against the spiralling whiteflies in both fumigant and repellent test are known to be similar. in fact, the mode of action of essential oils against the spiralling whiteflies was via neurotoxicity and respiratory toxicity (tanada and kaya, 1993; isman and machial, 2006; satar et al., 2008; li et al., 2014; tehri and singh, 2015). the weak repellent activity of o. tenuiflorum essential oil could be due the eugenol content (58.20%) which could have attracted the spiralling whiteflies rather than repelling. in previous study, eugenol caused attractancy to the japanese beetle (popillia japonica) (isman and machial, 2006). the other chemical compounds from literature that were found to attract the insects were cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and α-terpineol (hammack, 1996; petroski and hammack, 1998). the overall trend of repellent effect of selected essential oils on the spiralling whiteflies can be ranked as c. citratus and m. koenigii followed by e. hortensis, o. tenuiflorum and c. odorata. 4. conclusions all the five essential oils from medicinal plants essential oils conc (v/v) (%) ri (%) equation r2 ec 50 (%) χ2 statistic p-value df cananga odorata 0.25 -29 y = -0.140+0.290x 0.0795 3.046 5.93 0.015 1 0.05 -17 5 9 murraya koenigii 0.25 -13 y = 0.260+0.663x 0.3232 0.406 38.214 < 0.0001 1 0.05 8 5 52 euodia hortensis forma hortensis 0.25 -10 y=0.003+0.188x 0.028 0.964 3.277 0.070 1 0.05 -3 5 10 cymbopogon citratus 0.25 -9 y = -0.285+0.953x 0.6111 0.434 27.474 < 0.0001 1 0.05 3 5 52 ocimum tenuiflorum 0.25 -18 y = -0.163+0.374x 0.1582 2.728 13.928 0.000 1 0.05 -11 5 12 table 4 summary of repellent effect (6-8 hours) on the adult whiteflies at different concentrations (using probit analysis) the χ² probability ≤0.0001, indicated that the significant difference was brought by the log (concentration) variable and the repellency. each test represents the mean of four replicates of 50 whiteflies. chand et al. bioactivity of essential oils from medicinal plants in fiji 171 tested against the spiralling whiteflies showed fumigant and repellent effects. the strongest fumigant effect was shown by o. tenuiflorum essential oils, while for repellent test none of the essential oils showed strong effect. however the m. koenigii and c. citratus showed higher repellency when compared with other tested essential oils. in addition, the results presented in this study are the first given information on the chemical composition of essential oils from the south pacific on the selected plant species. so far only e. hortensis essential oil composition data from fiji is reported (brophy et al., 1985). the selected essential oils from medicinal plants showed potential for the development of possible natural form of controlling the whitefly but needs to be further evaluated to enhance their activity and safety to the humans. acknowledgements a warm thanks to mr ashley dowell and the team from southern cross university, queensland, australia for assisting in the identification of compounds in the selected essential oils. a special thanks to the chief scientist dr rajeswara rao, dr. karuna shanker and the team from the central institute of medicinal and aromatic plants, india, for sharing their thoughts and ideas 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liu, 2003; casacchia and sofo, 2013). this is due to the presence of high added value bioactive compounds, named phytochemicals (iriti and faoro, 2006). these compounds have strong antioxidant properties that enable them to scavenge free radicals, donate hydrogen, chelate metals, break radical chain reactions, and quench singlet oxygen in vitro and in vivo (dai and mumper, 2010). all these properties enable them to act in the prevention of oxidative stress-related diseases (pandey and rizvi, 2009). among phytochemicals, the most abundant class present in fruit is that of polyphenols (manach et al., 2004). phenolic rings have the capacity to scavenge free radicals, first of all hydroxyl ones, by virtue adv. hort. sci., 2016 30(3): 175-181 doi: 10.13128/ahs-20280 comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches d. ceccarelli, c. talento, a. sartori, m. terlizzi, e. caboni, k. carbone (*) consiglio per la ricerca in agricoltura e analisi dell’economia agraria, crea, centro di ricerca per la frutticoltura, via di fioranello, 52, 00134 roma, italy. key words: antioxidant capacity, depigmented peaches, phenolic compounds, phytochemicals, prunus persica. abstract: quality traits and nutraceutical potential of new de-pigmented peaches (“ghiaccio”) were investigated and compared with those of four white flesh peaches. total soluble solids (tss) were measured by digital refractometer, and titratable acidity (ta) by volumetric titration. total phenolic (tpc) and anthocyanin content (tac) were analysed spectrophotometrically and the antioxidant capacity (ac) evaluated by dpph• assay. a strong influence of genotype on quality traits and phytochemical profile of “ghiaccio” peaches was observed. “ghiaccio” series showed, on average, a higher tpc content than that of white flesh peaches, both in flesh and peels (+129% and +14%, respectively). the peels of all peaches analysed were significantly richer in tpc than the flesh. tac was not detectable in de-pigmented genotypes; on the contrary, in the white flesh peaches, it was higher in the peels than in the flesh. ac correlated well with tpc. data confirm, for all peaches analysed, the influence of genotype and fruit tissue on the nutraceutical properties. among genotypes, the best candidates for “ghiaccio” peaches with enriched nutraceutical properties are the advanced selections gø and gx. observed differences in the nutraceutical potential among “ghiaccio” series may open new opportunities for breeding de-pigmented peach varieties with a higher nutritional value. (*) corresponding author: katya.carbone@crea.gov.it received for publication 14 july 2016 accepted for publication 16 november 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 175-181 176 of the aromatic hydroxylation at the ortho-position (xia et al., 2010). these compounds are distributed in every part of the fruit in different ratios, focusing more in the peels (rich in anthocyanins, hydroxycinnamic acids and flavans) and in the seeds (rich in proanthocyanidins and flavans) (lachman et al., 2009). peaches, even though having a lower antiradical capacity than other fruits, are ones of the most important commodities consumed worldwide, both as fresh and processed product (i.e. fruit juice, jam or canned) (cantin et al., 2009). as a consequence, breeding programs aimed to increase peach nutraceutical value is desirable. “ghiaccio” peach series is a new type of peach variety, resulting from a breeding program conducted in the past years at the fruit tree research centre of rome with the aim to obtain varieties with enhanced postharvest fruit characteristics and an improved resistance to disease and pests (nicotra et al., 2001). their progenitor is a stony hard-type peach cultivar, the korean “yumyeong” (kim et al., 1978), from which “ghiaccio” selections have been obtained by self-pollination (nicotra et al., 2001). peaches of “ghiaccio” series have different ripening times but similar pomological traits (nicotra et al., 2001). to the best of our knowledge, there is no phytochemical and nutritional characterisation of “ghiaccio” peaches in literature, making this study quite relevant, providing breeders and consumers with experimental data on this emerging varieties. hence, the aim of the present study was to characterise the quality and nutraceutical properties of these new genotypes by measuring their total polyphenolic and total anthocyanin content, and their relative antioxidant capacity. a comparison with four commercial white flesh peach cultivars was also performed. the ultimate goal was to select “ghiaccio” peach genotypes with enhanced nutraceutical traits, to provide breeders with new varieties having more healthful properties, making them competitive with other fruits known for their healthy properties. 2. materials and methods chemicals all used reagents were of analytical spectrophotometric grade (carlo erba, rome, italy). cyanidin chloride was purchased from extrasyntese (genay cedex, france). folin-ciocalteu reagent, malic acid, chlorogenic acid, 2,2-diphenyl-1-picrylhydrazyl radical (dpph•), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased from sigma-aldrich (milan, italy). plant materials peach fruits from five early-to-late ripening “ghiaccio” genotypes (table 1) and four white flesh peach cultivars were collected in the experimental orchards of the fruit tree research centre of rome (crea-fru, italy), at fully ripening phase. all plants (six years old) were grafted on the same rootstock (gf677), spaced at 4.5x2.5 m and standard pruning, drip irrigation and cultural practices were performed. for each genotype, 15 undamaged and disease-free fruits were collected in order to have three replications of five fruits each for the analysis. traits cultivar/selection ghiaccio ø (g ø) ghiaccio 1® (g1) ghiaccio x (gx) ghiaccio 2® (g2) ghiaccio 3® (g3) size (g) large (168) very large (178) very large (217) very large (205) very large (200) shape oblate oblate oblate oblate oblate shape of pistil end weakly depressed weakly depressed weakly depressed weakly depressed weakly depressed symmetry symmetric symmetric asymmetric symmetric symmetric prominence of suture weak weak weak weak weak ground colour cream white cream white cream white cream white cream white over colour present absent absent present absent hue of over colour pink pink density of pubescence medium sparse medium very sparse very sparse firmness of flesh very firm very firm very firm very firm very firm ground colour of flesh cream white cream white cream white cream white cream white sweetness high high high high high acidity low low low low low ripening time (y) -20 -8 -5 11 25 table 1 pomological and phenological traits of “ghiaccio” series (z) (z) data were detected as community of plant varity office (cpvo) descriptors. (y) the reference cv. rome star ripens in center italy between 25th-30th of july. ceccarelli et al. comparative characterization of de-pigmented “ghiaccio” and white flesh peaches 177 fruit quality attributes after harvesting, fruits were washed, stoned and homogenized, and the homogenate samples were analysed for total soluble solid (tss) content using a digital refractometer (refracto 30 px, mettler toledo, milan, italy); data are given as °brix. the method for analysis of titratable acidity (ta) was based on titration of the acids present in the fruit juice with sodium hydroxide (0.1 n). data are given as g malic acid l-1, since this is the dominant organic acid in peach (david et al., 1956). the ph value was measured using a digital ph-meter (785 dmp, methrom, milan, italy). every analysis was replicated three times. extraction of bioactive compounds fruits from the different genotypes were carefully separated in two different tissues: peel and flesh. the peel fraction was removed from the whole fruit with a sharp knife and immediately frozen with liquid nitrogen, placed in a plastic freezer bag, and stored at -80°c until evaluation. the flesh fraction consisted of a peeled wedge, which was chopped into small pieces, frozen in liquid nitrogen, and also stored at 80°c. samples (5 g) of the different fractions were extracted with a 25 ml hydro alcoholic solution (methanol:water= 70:30, v/v) acidified with hcl (0.005 n) and homogenized with an ultra-turrax blender (ultra turrax t25, ika, milan, italy) at 9000 rpm. then, the homogenates were allowed to stand for 2 hours at 37°c under magnetic stirring to rich a complete solvent extraction. extracts were centrifuged at 8400 rpm for 15 min at 5°c and then the obtained supernatants were analysed as follows. extractions were repeated on three independent samples of the initial homogenate to give triplicate readings. determination of total polyphenol content (tpc) tpc of both flesh and peel samples was determined using the folin-ciocalteu (f-c) method (waterhouse, 2002). tpc was calculated from a calibration curve, using chlorogenic acid as a standard. results were expressed as milligrams of chlorogenic acid equivalents (cae) per 100 g fresh weight (fw). every analysis was replicated three times. determination of total anthocyanin content (tac) tac of both flesh and peel fractions was estimated according to the method of mondello et al. (2000). tac was calculated from a calibration curve, using cyanidin chloride (cc) as a standard. results were expressed on a fresh weight basis as milligrams of cc equivalents (cce) x 100 g fw. every analysis was replicated three times. measurement of antioxidant capacity (ac) ac was assessed by measuring the effect of the bioactive extracts on the content of 2,2-diphenyl-1, picrylhydrazyl radical (dpph•) according to brandwilliams et al. (1995). all the measurements were made in triplicate. ac was expressed as micrograms of trolox equivalents (te) x mg fw. statistical analysis data analysis was performed with spss 17.0 software (spss, inc., chicago, illinois). all measurements were performed at least in triplicate and data were reported, where not specified differently, as means ± standard error of the mean (se). an exploratory data analysis was made to check the data normal distribution (shapiro-wilkinson test) and the equality of variances (levene’s test). when these conditions were met (tss, ta, ac), data were subjected to one-way analysis of variance (anova) and comparisons between means were determined according to tukey’s hsd test. significant differences were accepted at p<0.05 and represented by different letters. when anova assumptions were violated (tpc), even after mathematical transformation of data, a non-parametric data analysis was carried out (kruskal-wallis non parametric test) and significant mean differences were established using the mannwhitney test for independent and non-parametric procedures and a bonferroni’s correction to set the critical value for significance for each test. box-plots were used to display the range, median and distribution density of phytochemicals and ac in the peel and flesh of genotypes analysed. spearman’s correlation coefficient (ρ) was used to determine the correlation among variables in the non-parametric analysis (p<0.01). 3. results and discussion fruit quality attributes table 1 shows the main pomological and phenological traits of “ghiaccio” series analysed. ghiaccio means ice in italian to remind consumers that the colour of the fruit is white or pale cream. they are very different from the common peach type grown in europe and the u.s. these peaches are characterized by totally white cream skin and flesh, very firm flesh, high sugar content (up to 17°brix), 2025 days longevity on the tree, a great productivity and a noteworthy shelf life. moreover, this variety adv. hort. sci., 2016 30(3): 175-181 178 shows a great resistance against diseases, rottenness and pathogenic agents making it particularly suitable for organic practice (nicotra et al., 2001). the “ghiaccio” series includes “ghiaccio 1” (g1), “ghiaccio 2” (g2) and “ghiaccio 3” (g3) genotypes, which have been already licensed, while two advanced selections, “ghiaccio ø” (gø) and “ghiaccio x” (gx) are still under evaluation. all genotypes analysed were harvested between early july and late august and, generally, significant differences in quality traits were detected (table 2). in “ghiaccio” series, the highest tss level (15.2 °brix) was observed in g1 samples, while the lowest one in gø ones (9.1°brix). all samples analysed showed a tss content greater than 8 °brix, which represents the minimum tss content established by the eu to market peaches and nectarines [commission regulation (ec) no. 1861/2004, 10/28/2004]. moreover, “ghiaccio” genotypes showed tss similar to the white flesh peaches analysed. ta values ranged from 3.18 (g3) to 4.23 (g1) g malic acid l-1, with significant differences among selections (table 2). on average, these values are lower than that of the progenitor and “ghiaccio” series can be collocated in sub acidic peach varieties (crisosto et al., 2001). these results are relevant since the acceptance of new cultivars by consumers, which is the ultimate goal of breeders, is linked to the fruit quality. in fact, acid levels, expressed in terms of ph and ta, and sugar concentrations, reported as tss, affect flavour perception of the fruit influencing peach sensory profile and consumer acceptance of peach fruits (crisosto et al., 2001; crisosto and crisosto, 2005). in the present study, we also reported the tss/ta ratio as the relationship between these parameters has an important role in fruit consumer acceptance (diaz-mula et al., 2009). among “ghiaccio” peaches, tss/ta values ranged from 3.62 for g3 to 3.16 for g2, being the latter significant different from the other genotypes of the series. in the fresh market, consumers desire large shaped and flavourful fruit with a high sugar content and low to moderate acidity and these new genotypes appear to respond adequately to such requests. in this regard, “ghiaccio” series showed, on average, a ta content lower than that of the white flesh cultivars analysed, which is reflected in a higher value of the tss/ta ratio. bioactive compounds distribution within the peach genotypes and relative antioxidant capacity fruit antioxidant potential varies in relation to the phytochemical moieties present, and variations can occur among genotypes within a single species (van der sluis et al., 2001; cantin et al., 2009). moreover, it is well known that the content of phytochemicals can vary within different tissues (carbone et al., 2011). in figure 1, box-plots showed the distribution of polyphenols, and the antiradical activity between the peel and the flesh of “ghiaccio” peaches analysed, independently from the genotype. tpc was significantly higher in the peach peel extracts than in the flesh ones (+103%). these data are consistent with those reported in the literature about the influence of the type of fruit tissue on the accumulation of nutraceutical substances (carbone et al., 2011; table 2 fruit quality attributes of genotype analysed (mean±se) tss= total soluble solids. ta= titratable acidity. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). genotype harvest date tss (°brix) ta (g l-1 malic acid) tss/ta ratio ghiaccio 1 july, 22nd 15.20±0.03 e 4.23±0.06 b 3.58±0.04 d ghiaccio 2 august, 11th 13.00±0.03 d 4.13±0.01 b 3.16±0.02 c ghiaccio 3 august, 25th 11.5±0.1 b 3.18±0.02 a 3.62±0.05 d ghiaccio ø july, 7th 9.10±0.07 a nd nd ghiaccio x july, 21st 12.10±0.07 c 3.43±0.04 a 3.54±0.06 d crizia june, 28th 9.6±0.4 a 7.96±0.03 c 1.21±0.05 a maria anna august, 4th 13.60±0.03 d 12.0±0.2 e 1.13±0.02 a redhaven bianca july, 21st 12.2±0.1 c 8.74±0.01 d 1.39±0.01 b silver late september, 8th 13.20±0.06 d 12.61±0.02 f 1.05±0.00 a "ghiaccio" series 12.2±0.5 a 3.7±0.1a 3.47±0.06 b white flesh cvs. 12.1±0.5 a 10.3±0.6 b 1.19±0.04 a ceccarelli et al. comparative characterization of de-pigmented “ghiaccio” and white flesh peaches 179 tomás-barberán et al., 2001). tables 3 and 4 show the flesh and peel phytochemical content and ac of different cultivars analysed. among “ghiaccio” genotypes, gø showed the highest tpc both in the peel and flesh (260 and 134 mg cae 100 g-1 fw, respectively), while g2 the lowest one (70 and 28 mg cae 100 g-1 fw, respectively) (tables 3 and 4). these findings highlight that tpc of “ghiaccio” series is related to the genotype, in agreement with those reported in literature, not only for other peaches but also for other fruit species (tomás-barberán et al., 2001; ceccarelli et al., 2016). interestingly, the average peel polyphenol content of “ghiaccio” series (175 mg cae 100 g-1 fw) was not significantly different from that of the white flesh cultivars analysed (table 3), while the average tpc of the flesh was significantly higher (table 4). obtained results emphasize the healthy properties of the flesh of these new de-pigmented peaches. as regards total anthocyanins, their content was not detectable in “ghiaccio” peaches both in the peel and the flesh. besides, white flesh peaches revealed the presence of these compounds in all tissue analysed. in agreement with the phytochemical data, genotype also influenced the ac of peaches analysed. among “ghiaccio” genotypes, the highest value of ac was found in gø (1.3 µg te mg-1 fw) and gx (1.5 µg table 3 phytochemicals and antioxidant capacity of the peel of genotypes analyzed (mean±se) genotype ac tpc tac ghiaccio 1 1.0±0.1 bc 232±6 e nd ghiaccio 2 0.53±0.08 a 70±2 a nd ghiaccio 3 0.7±0.2 ab 197±4 d nd ghiaccio ø 1.3 ±0.1 cd 260±4 f nd ghiaccio x 1.5±0.1 d 144±4 c nd crizia 1.34±0.03 cd 102±3 b 26.53±0.03 c maria anna 1.42±0.06 cd 260.9±0.7 f 34.55±0.06 d redhaven bianca 1.13±0.04 cd 100±3 b 19.70±0.05 a silver late 1.50±0.08 d 151±2 c 23.40±0.03 b “ghiaccio” series 1.00±0.08 a 175±14 a nd white flesh cvs. 1.35±0.04 b 154±14 a 26±2 ac= antioxidant capacity. tpc= total polyphenol content. tac= total anthocyanin content. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). table 4 phytochemicals and antioxidant capacity of the flesh of genotypes analyzed (mean±se) ac= antioxidant capacity. tpc= total polyphenol content. tac= total anthocyanin content. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). genotype ac tpc tac ghiaccio 1 0.286±0.007 cd 59±8 c nd ghiaccio 2 0.16±0.01 a 28±2 ab nd ghiaccio 3 0.325±0.007 d 126±2 d nd ghiaccio ø 0.308±0.007 d 134±4 d nd ghiaccio x 0.20±0.02 b 71±3 c nd crizia 0.256±0.005 c 23±2 a 7.54±0.01 c maria anna 0.157±0.003 a 38±2 b nd redhaven bianca 0.263±0.004 c 28±2 ab 1.67±0.01 a silver late 0.412±0.005 e 62±2 c 6.45±0.01 b “ghiaccio” series 0.25±0.01 a 86±8 b nd white flesh cvs. 0.27±0.02 a 38±3 a 4.1±0.9 fig. 1 box-plots for the phytochemicals and antioxidant capacity of different peach tissues. the line in the box indicates the median value of the data; the right and the left edges of the box respectively indicates the 75th and the 25th percentiles of the data set, the ends of the horizontal lines indicate the minimum and maximun data values; the point outside the box are outliers or suspected outliers. a) tpc= total polyphenol content (mg cae 100 g-1 fw). b) ac = antioxidant capacity (µg te mg-1 fw). outlier: value more than 1.5 and less than 3 box-lengths from end of box. adv. hort. sci., 2016 30(3): 175-181 180 te mg-1 fw) for what concern the peel and in gø (0.308 µg te mg-1 fw) and g3 (0.325 µg te mg-1 fw) for the flesh. on average, the antioxidant capacity of the peel was lower in “ghiaccio” series than in white flesh cultivars (table 3), probably due to the absence of anthocyanins in the peel of “ghiaccio” series. moreover, correlation analysis pointed out that the scavenging capacity against dpph• of peach extracts and tpc were significantly and positively correlated (ρ= 0.796; p<0.01). this result confirms previous reports on commercial white and yellow flesh cultivars, showing that phenolic compounds can be considered the main phytochemicals contributing to ac in peaches (vizzotto et al., 2007). 4. conclusions peach breeding has supplied a large number of improved cultivars each year to satisfy different market demands. nevertheless, there are some critical issues not yet fully addressed through breeding such as the increasing competition between peach and a broad range of other fruits, as well as the consumer eating habits, which are changed in the last years, with a growing emphasis on nutrition and health properties of food. to meet these challenges, it is necessary to explore new germplasm for the production of new cultivars with improved quality and nutritional characteristics as well as to provide fruits to the market for long periods of time. in this context, the “ghiaccio” series could be a right answer for breeders. the present study describes for the first time the quality traits, phytochemical composition and ac of five genotypes (three cultivars and two advanced selections) belonging to the “ghiaccio” peaches. data point out the key role played by the genotype also within the “ghiaccio” series, underling the importance of the varietal selection. among the genotypes analysed, the best candidates for a “ghiaccio” peach with enriched nutraceutical properties are the advanced selections gø and gx. in addition, data pointed out a higher nutraceutical potential of “ghiaccio” series than that of the commercial white flesh peaches analysed. finally, taking into account that the fruit characteristics (i.e. shape, colour and size) are very similar to each other, but with different ripening periods, “ghiaccio” genotypes, if grown together, would allow the producers to supply the markets with the same type of fruit for a long period of time (i.e. 75 days). references abbott j.a., 1999 quality measurement of fruits and vegetables. postharvest biol. technol., 15: 207-225. bazzano l.a., he j., ogden l.g., loria c.m., vupputuri s., myers l., whelton p.k., 2002 fruit and vegetable intake and risk of cardiovascular disease in us adults: the first national health and nutrition examination survey epidemiologic follow-up study. am. j. clin. nutr., 76: 93-99. brand-willams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. lebensm. wiss.technol., 28: 25-30. cantin c.m., moreno m.a., gogorcena y., 2009 evaluation of the antioxidant capacity, phenolic compounds, and vitamin c content of different peach and nectarine (prunus persica (l.) batsch) breeding progenies. j. agric. food chem., 57: 4586-4592. carbone k., giannini b., picchi v., lo scalzo r., cecchini f., 2011 phenolic composition and free radical scavenging activity of different apple varieties in relation to the cultivar, tissue type and storage. food chem., 127(2): 493-500. casacchia t., sofo a., 2013 antioxidant compounds and nutraceutical benefits of mediterranean red fruit, pp. 107-136. in: carbone k. (ed.) cultivars: chemical properties, antioxidant activities and health benefits. nova science publisher, hauppagem, ny, usa, pp. 281. ceccarelli d., simeone a.m., nota p., piazza m.g., fideghelli c., caboni e., 2016 phenolic compounds (hydroxycinnamic acids, flavan-3-ols, flavonols) profile in fruit of italian peach varieties. plant biosystems, 150(6): 1370-1375. crisosto c.h., crisosto g.m., 2005 relationship between ripe soluble solids concentration (rssc) and consumer acceptance of high and low acid melting flesh peach and nectarine (prunus persica (l.) batsch) cultivars. postharvest biol. technol., 38: 239-246. crisosto c.h., day k.r., crisosto g.m., garner d., 2001 quality attributes of white flesh peaches and nectarines grown under california conditions. j. am. pomol. soc., 55: 45-51. dai j., mumper r.j., 2010 plant phenolics: extraction, analysis and their antioxidant and anticancer properties. molecules, 15: 7313-7352. david j.j., luh b.s., marsh g.l., 1956 organic acids in peaches. j. food sci., 21: 184-194. diaz-mula h.m., zapata p.j., guillén f., martínezromero d., castello s., serrano m., valero d., 2009 changes in hydrophilic and lipophilic antioxidant activity and related bioactive compounds during postharvest storage of yellow and purple plum cultivars. postharvest biol. technol., 51: 354-363. faostat, 2015 http://faostat3.fao.org/home/e. iglesias i., echeverria g., 2009 differential effect of cultivar and harvest date on nectarine colour, quality and consumer acceptance. sci. hortic., 120: 41-50. ceccarelli et al. comparative characterization of de-pigmented “ghiaccio” and white flesh peaches 181 iriti m., faoro f., 2006 grape phytochemicals: a bouquet of old and new nutraceuticals for human health. med. hypotheses, 67: 833-838. kim y.h., cho m.d., lee o.k., lee c.s., yu y.s., 1978 the new peach cultivar, yumyeong. res. rep. off. rural dev., 20: 1-15. lachman j., sulc m., faitovà k., pivec v., 2009 major factors influencing antioxidant contents and antioxidant activity in grapes and wines. int. j. wine res., 1: 101-121. liu r.h., 2003 health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. am. j. clin. nutr., 78: 517s-520s. manach c., scalbert a., morand c., remesy c., jimenez l., 2004 polyphenols: food sources and bioavailability. am. j. clin. nutr., 79: 727-747. mondello l., cotroneo a., errante g., dugo g., dugo p., 2000 determination of anthocyanins in blood orange juices by hplc analysis. j. pharm. biomed. an., 23: 191-195. nicotra a., conte l., moser l., fantechi p., 2001 new types of high quality peaches: flat peaches (p. persica var. platicarpa) and “ghiaccio‟ peach series with long on tree fruit life. acta horticulturae, 592: 131-135. pandey k.b., rizvi s.i., 2009 current understanding of dietary polyphenols and their role in health and disease. curr. nutr. food sci., 5: 249-263. tomás-barberán f.a., gil m.i., cremin p., waterhouse a.l., hess-pierce b., kader a.a., 2001 hplcdad-esims analysis of phenolic compounds in nectarines, peaches and plums. j. agr. food chem., 49: 4748-4760. van der sluis a.a., dekker m., de jager a., wim jongen m.f., 2001 activity and concentration of polyphenolic antioxidants in: apple: effect of cultivar, harvest year, and storage conditions. j. agr. food chem., 49: 3606-3613. vizzotto m., cisneros-zevallos l., byrne d.h., 2007 large variation found in the phytochemical and antioxidant activity of peach and plum germplasm. j. am. soc. hortic. sci., 132: 344-340. waterhouse a.l., 2002 determination of total phenolics, pp. 1-8. in: wrolstad r.e., t.e. acree, e.a. decker, m.h. penner, d.s. reid, s.j. schwartz, c.f. shoemaker, d.m. smith, and p. sporns (eds.) current protocols in food analytical chemistry. john wiley & sons, inc., oxford, uk. xia e., deng g.f., guo y.j., li h.b., 2010 biological activities of polyphenols from grapes. int. j. mol. sci., 11(2): 622-646. impaginato 239 1. introduction a variety of horticultural products is grown in afghanistan with the potential to be (re)developed into valuable export commodities and brands. the primary engine of the afghan economy is its agriculture industry, which engages approximately 80% of the working population. fruit trees, such as peach, plum, apricot or almond and grape are therefore of significant economic importance to the country, with importing or local exchange of plant material for propagation or commercial growing of these fruits likely. the absence of certain viruses is an essential pre-requisite for virus-free certification of plant material in general in many parts of the world (golino and savino, 2005; barba et al., 2015). in afghanistan, there is a tremendous increase in fruit tree nursery production and regulation of phytosanitary certification of plant propagation material is therefore of major importance. in 2005 the ministry of agriculture, irrigation and livestock (mail) stated in the agriculture master plan that the rejuvenation of the horticulture sector would be a government priority. thriving in the 1970s, this sector has become incapable of competing in the international market. three interconnected problems have been identified for the industry: 1) the existing orchards are run-down and production is adv. hort. sci., 2016 30(4): 239-248 doi: 10.13128/ahs-20350 the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey s. rehman 1, j. ahmad 1, c. lanzoni 2, c. rubies autonell 2, c. ratti 2 (*) 1 plant biotecnology laboratory, seed and planting material certification directorate, ministry of agriculture, irrigation and livestock, kabul, afghanistan. 2 dipartimento di scienze agrarie, università di bologna, via g. fanin, 40, 40127 bologna, italy. key words: germplasm, plant pathology, virus disease. abstract: the horticultural industry is a vital component of the agriculture sector of afghanistan, the primary engine of the country’s recovering economy which engages approximately 80% of the working population. this sector was thriving in the 1970s, but is today incapable of competing in the international market. to recover and develop the horticulture of the country, the european community (ec) supports the phdp (perennial horticulture development project), to provide true to type/ecotype and healthy planting materials, and the plant biotechnology laboratory, to ensure the health status of local germplasm. this laboratory started screening the health status of the afghan germplasm national collections in order to ensure the multiplication of not only the best-selected varieties or ecotype, but also to avoid production and distribution of virus-infected trees. inspection for symptoms and sample collection for viral diseases was carried out in all the national collection fields, including cherry, pear, peach, plum, apricot, almond, apple, grape and citrus plants, located in different areas of the country. stone fruit plants infected by apple chlorotic leaf spot virus or prunus necrotic ringspot virus have been identified in the national collection experimental farms located in different provinces of afghanistan. moreover, many grape plants included in the national collection located in herat and kandahar resulted infected by grapevine fanleaf virus, but only few imported plants by grapevine leafroll associated virus 1, grapevine leafroll associated virus 3 or grapevine virus a. finally, in jalalabad (nangarhar province) citrus plants showing vein flecking, yellowing and plant decline symptoms were found to be infected by citrus tristeza virus. some of the identified viral isolates have been characterized molecularly, amplifying a fragment corresponding to the coat protein gene from a selection of positive samples. the presence of those viruses in different accessions of the national collection is of concern for afghan horticulture. implementation of the certification schemes is therefore necessary to quarantine the production and for the employment of virus-free propagating material. (*) corresponding author: claudio.ratti@unibo.it received for publication 4 february 2016 accepted for publication 16 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 239-248 240 dwindling in quantity and quality; 2) nursery owners lack access to quality inputs and services; and 3) the regulatory framework to develop and support a competitive horticulture industry is minimal. to support and develop the horticultural industry of the country, a nursery certification system was started in 2006 from the complete collection afghanistan’s fruit and nut trees crop varieties by the perennial horticulture development project (phdp) funded by european union (eu). one of the main causes for the decrease in production is deleterious pests and diseases, especially plant viruses and viruslike diseases present in afghanistan. virus infection in plants is particularly difficult to detect as viral infection may be sectorial or latent, with symptoms masked or even absent under certain conditions. virus-free certification is currently implemented using conventional techniques for virus detection in vegetal material such as biological indexing, serological or molecular assays. biological indexing requires, in many instances, grafting in greenhouse facilities which is expensive and time consuming. serological assays and molecular techniques, particularly elisa and rt-pcr, have been tested in several laboratories and shown to be efficient for detection and quantification of virus infection in pome and stone fruits (gambino and gribaudo, 2006; faggioli et al., 2013). in a country such as afghanistan, the establishment of a laboratory where sensitive, reliable and specific techniques for routine detection of selected viruses of fruit trees can performed is therefore essential for virus-free certification. many viral diseases can affect fruit trees, resulting in latent or symptomatic infection. according to their effect on the cultivated host, presence of the most important viruses in propagation plant material is regulated at international level. in particular, plum pox virus (ppv) is the causal agent of sharka, the most damaging virus disease of stonefruits. some strains of apple chlorotic leafspot virus (aclsv) can also induce serious diseases in stone fruits. prune dwarf virus (pdv) and prunus necrotic ringspot virus (pnrsv) are of considerable economic significance in stonefruits as well, while citrus tristeza virus (ctv) is considered to be the most destructive virus of citrus crops. apple mosaic virus (apmv), apple stem grooving virus (asgv) and apple stem pitting virus (aspv) are spread worldwide and able to causes significant losses in mixed infections (hadidi et al., 2011; barba et al., 2015). arabis mosaic virus (armv), grapevine fleck virus (gfkv). grapevine fanleaf virus (gflv), grapevine virus a (gva) and “grapevine leafroll-associated viruses” (glravs) such as grapevine leafroll associated virus 1, 2 and 3 (glrav-1, glrav-2 and glrav-3) are of great economic impact in grape and their absence in propagation material is an essential requirement in the certification schemes of most countries (martelli, 2014). national collection centres were established in six agro-ecological zones in afghanistan to collect germplasm. after registration and cataloguing, the mother stock nurseries were established from the same planting material to ensure the availability of high quality and healthy planting material to nursery growers and farmers. theoretical and practical knowledge are essential to develop a complete profile for viral diseases in order to improve the quality of planting material within the national fruit tree germplasm (rizzo et al., 2012, 2015). we therefore developed an in-depth study regarding the detection and identification of viral pathogens through the use of advance and sensitive methodologies and techniques to characterize identified plant viral pathogens and to develop control strategies for the management of diseases. these efforts are aimed at improving the quality of planting material and will ensure access to quality and certified planting material for nursery growers and orchard owners for the establishment of new orchard/motherstock nurseries, as well as for the rehabilitation of existing orchards. screening agricultural products according to the protocols and regulations of global plant quarantine will also positively affect the import and export of afghan agricultural entities. following the standards of international sanitary and phytosanitary rules and regulations is the only way to compete in the international market. 2. materials and methods plant material plant material was collected from the national collection centres (nccs) and mother stock nurseries (msns) from 2009 to 2015. in total, more than 12,000 samples were collected from different species and tested for different viruses. in particular, almond, apricot, cherry, peach and plum samples were tested for the presence of aclsv, apmv, pdv, pnrsv and ppv; apple and pear samples for aclsv, apmv, asgv and aspv; citrus samples for ctv; and grape samples for armv, gva, gfkv, gflv, glrav-1, glrav-2 and glrav-3. rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 241 about 32% of the fruit tree samples were collected from the six nccs located in kabul (apricot and plum), balkh (apricot and almond), kunduz (almond), herat (grape and plum), kandahar (grape and pomegranate) and nangarhar (citrus and pomegranate). moreover, samples were also collected from msns (26%) and from private orchards (nearly 40%) (fig. 1). each year stone fruit samples were collected during the vegetative stage in spring or summer, between march and september, according to the climatic conditions of each location. similarly, apple and pear samples were collected in may, june, july or september; citrus in march, may or november; and grape in dormant stage in january, october or december. the collecting method consisted of sampling leaves (in the case of stone and pome fruits), shoots or canes (grape) or leaves and flowers (citrus) homogeneously distributed around the canopy of the plant. all collected material was maintained under refrigeration (below 10°c) during transport from the field to the laboratory, then kept at -20°c until analyses. serological and molecular analyses all samples were ground in “universal” extraction bags (12x15 cm) using the homex 6 homogenizer (bioreba) then analysed by das-elisa using reagent kits from bioreba (reinach, switzerland) specific for each virus assayed and following the manufacturer’s instructions. samples that resulted positive by das-elisa test were subsequently analysed by rt-pcr reaction using specific primer pairs (table 1). fig. 1 location of the six national collection centres and of the 57 mother stock nurseries in afghanistan. virus primers primers sequences (5’ 3’) size of amplif. (bp) cycling (temp./time)* n° of cycles reference denat. anneal. exten. aclsv sense ttcatggaaagacaggggcaa 677 94°c/20 s 58°c/20 s 72°c/20 s 35 menzel et al., 2002 antisense aagtctacaggctatttattataagtctaa pnrsv mg1 atggtttgccgaatttgc 675 94°c/60 s 55°c/45 s 72°c/60 s 35 glasa et al., 2002 mg2 actctagatctcaagcaggtc gflv m3 atgctggatatcgtgaccctgt 118 94°c/10 s 54°c/10 s 72°c/45 s 35 gambino and gribaudo, 2006 m4 gaaggtatgcctgcttcagtgg ctv ctvf taatggacgacgaacaaaga 655 94°c/40 s 56°c/40 s 72°c/60 s 30 rehman et al., 2012 ctvr ccaagctgcctgacattagt table 1 list of primer pairs, size of amplicons, and rt-pcr conditions used to confirm viral infection in all samples resulting positive to the das-elisa test * pcr amplifications were performed at 94°c for 5 min for initial denaturation and 72°c for 5 min for final extension. adv. hort. sci., 2016 30(4): 239-248 242 total rnas were extracted from each sample using ctab rna extraction method, as previous reported (ratti et al., 2004). complementary dna (cdna) was synthesized using m-mlv reverse transcriptase (promega, usa) in a final volume of 5.0 µl. rna (1.0 µl), mixed with m-mlv 5x buffer, 0.5 µl 10 mm dntps, 1.0 µl 10nmol/ml specific reverse primer, 0.25 µl of 200 u/µl m-mlv and 2.25 µl of nucleasefree water, then incubated at 4 2°c (or 37°c for aclsv and pnrsv) for 1 h. for pcr amplification, 5.0 µl of cdna template, 5.0 µl of 5x green gotaq buffer, 1.5 µl of 25 mm mgcl2, 1.0 µl 10 nmol/ml of specific forward primer, 0.5 µl of 10 mm dntps and 0.2 µl of 5 u/µl gotaq polymerase (promega, usa) were mixed in a total volume of 25 µl. the number of cycles and cycling conditions for each primer set are given in table 1. the amplified pcr products were analysed in 1% agarose gel stained by ethidium-bromide and visualized under uv light after electrophoresis. 3. results das-elisa analyses all apple and pear plants analysed (180 samples) resulted free from aclsv, apmv, asgv and aspv. symptoms such as yellowing, leaf distortion and puckering were observed in some stone fruit plants during collection of the 5521 samples that were tested by das-elisa (fig. 2). among them, 23 (0.4%) samples collected from several msns in kabul, khandahar, parwan, herat and baghlan provinces resulted positive to aclsv. in particular, there were two plants of european plum (clone 364/local name alu bokharashalili), one cross of myrobalan and japanese plum (4031/grangej zard), 17 plants of peach (five from 804/turki sorkh, five from 812/dir ras and seven from 811/turkizard), one plant of almond (159/sattarbai bakhmali) and two plants of apricot (373/shakarpara and 4037/aqa banu). two (0.03%) almond samples, both collected from the ncc in balkh province, resulted infected by apmv; in was interesting that one of them also tested positive to pnrsv. analyses against pnrsv evidenced 38 samples (0.6%), collected from nccs or msns located in balkh, herat, kabul, khandaharand paktya as infected by the virus (13 almond, five plum, nine peach, and 11 cherry plants). finally, one plum sample (cv. alu bokhara shalili), from a msn in kabul, resulted infected by pdv while none of the stone fruit samples tested resulted positive to ppv (tables 2-6 and fig. 3). among the 895 grape samples analysed, one plant from the ncc in kandahar (cv. shir ahmadi herat) resulted infected by gva and only one sample of the cv. pizzutello bianco, imported from italy and collected in the ncc in herat, tested positive to glrav-1 (it also resulted as infected by glrav-3). moreover, glrav-3 was detected in two samples (cv. pizzutello bianco and uva palieri both imported from italy), collected from the nnc in herat. regarding gflv, all the 62 samples which resulted infected by the virus (6.9%) belong to afghan cultivars, with the exception of the cv. regina dei vigneti imported from italy. all grape samples tested resulted negative to armv, gfkv and glrav-2 (table 7) (fig. 3). in addition, the plant biotechnology laboratory analyzed 5400 citrus plants and 318 of them (5.9%) resulted infected by ctv. in particular, among the positive samples, eight plants, showing vein clearing and flecking, yellowing and plant decline symptoms (fig. 4), were collected from the ncc in jalalabad and belong to six different accessions: kumquat cv. fig. 2 yellowing observed in an apricot accession during sample collection in a national collection centre. rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 243 table 3 apricot samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 pdv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 pnrsv north 174 6 (balkh) 0 0 205 5 (balkh) 174 0 53 0 82 0 78 0 766 11 central 2 1 (herat) 0 0 24 0 136 0 103 0 69 0 42 1 (herat) 376 2 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 apmv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 aclsv north 0 0 0 0 205 0 174 0 53 0 82 0 78 0 592 0 central 0 0 0 0 24 0 136 0 103 0 69 0 42 1 (herat) 374 1 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 total 176 7 0 0 229 5 310 0 203 0 151 0 120 2 1189 14 table 2 almond samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul northern regions: badakhshan, baghlan, balkh, faryab, jowzjan, kunduz, samangan, sar-e pol and takhar. central regions: badghis, bamiyan, farah, ghor, herat, kabul, kapisa, kunar, laghman, logar, nangarhar, nurestan, panjshir, parwan and wardak. southern regions: daykundi, ghazni, helmand, kandahar, khost, nimruz, orūzgān, paktia, paktika, zabul. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 pdv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 pnrsv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 apmv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 aclsv north 0 0 0 0 0 0 68 0 0 0 40 0 80 2 (baghlan) 188 2 central 0 0 0 0 318 0 221 0 141 0 69 0 48 0 797 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 total 278 0 833 0 318 0 289 0 170 0 109 0 128 2 2125 2 see legend of table 2 for provinces included in northern, central and southern regions. table 4 cherry samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul see legend of table 2 for provinces included in northern, central and southern regions. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 pdv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 pnrsv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 6 (kabul) 58 1 (kabul) 35 3 (herat) 187 10 south 0 0 0 0 0 0 0 0 18 1 (paktya) 0 0 0 0 18 1 apmv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 aclsv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 total 0 0 0 0 0 0 8 0 111 7 58 1 35 3 212 11 adv. hort. sci., 2016 30(4): 239-248 244 table 6 plum samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul see legend of table 2 for provinces included in northern, central and southern regions. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pdv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pnrsv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 5 (herat) 0 0 190 0 168 0 140 0 31 0 15 0 704 5 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 apmv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 aclsv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 0 0 0 0 190 0 168 0 140 2 (kabul) 31 0 15 0 544 2 south 0 0 0 0 0 0 0 0 33 1 (kandahar) 0 0 0 0 33 1 total 160 5 0 0 190 0 216 0 204 3 31 0 60 0 861 8 fig. 3 distribution, among afghan provinces, of samples resulting positive to aclsv, apmv, ctv, gflv, glrav-1, glrav-3, gva, pdv or pnrsv. see legend of table 2 for provinces included in northern, central and southern regions. table 5 peach samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 pdv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 pnrsvnorth 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 7 (herat) 186 1 (kabul) 21 0 13 0 865 8 south 0 0 0 0 0 0 0 0 75 1 (kandahar) 0 0 0 0 75 1 apmv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 aclsv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 0 0 0 0 267 0 374 0 186 17 (kabul) 21 0 13 0 861 17 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 total 4 0 0 0 267 0 449 7 302 19 30 0 78 0 1130 26 rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 245 margarita (isolates j4 and j8), orange cv. mahali (j61), rough lemon cv. mahali (j101) and mandarin group cvs. fruter (j76), tangelo mapo and clementine di nules. the other 312 plants positive to ctv were collected from private orchards located in the provinces of kunar, laghman, and nangarhar (table 8, fig. 3). rt-pcr and molecular characterization samples from stone fruit plants which resulted infected by aclsv, or pnrsv following das-elisa technique were analysed also by rt-pcr reaction. the results obtained confirmed the presence of each viral agent assayed in the samples tested. in particular, regarding aclsv isolates, a 677-long nucleotide fragment, corresponding to partial coat protein gene, was amplified from all elisa-positive samples by rt-pcr using aclsv sense and antisense primers (menzel et al., 2002). eleven of the identified isolates of aclsv were then characterized molecularly. sequence analysis revealed similarity ranging from 83.6 to 100.0% within aclsv isolates detected in afghanistan. blast analysis showed that sequences of two peach isolates, 812/dir ras, shared the highest nucleotide similarity (95.8 and 96.2%) with the genbank aclsv isolates apr 3 from jordan (aj586631). moreover, the same afghan isolates showed nucleotide identity between 95.0 and 95.7% with isolates s4, pp23, pp63, and hl2 (jn849008, gu327991, gu328003 and gq334211, respectively) from china. sequence analysis of isolate 364/alu table 7 grape samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive gflv herat 0 0 0 0 156 19 117 13 271 22 0 0 0 0 544 54 kandahar 0 0 0 0 0 0 0 0 268 0 0 0 0 268 0 armv herat 0 0 0 0 156 0 117 0 271 0 0 0 0 0 544 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 gva herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 gfkv herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-1 herat 83 0 0 0 156 0 117 1 271 0 0 0 0 0 627 1 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-2 herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-3 herat 83 0 0 0 156 0 117 2 271 0 0 0 0 0 627 2 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 total 83 0 0 0 156 19 117 16 539 23 0 0 0 0 895 58 fig. 4 vein clearing and yellowing symptoms observed in citrus accessions collected from the national collection centre in jalalabad (nangarhar province). table 8 citrus samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ctv nangarhar 0 0 193 6 263 2 1276 153 1554 110 131 0 492 0 3909 271 laghman 0 0 0 0 0 0 180 9 200 4 0 0 296 3 676 16 kunar 0 0 0 0 0 0 318 21 246 9 0 0 249 1 813 31 total 0 0 193 6 263 2 1774 183 2000 123 131 0 1037 4 5398 318 adv. hort. sci., 2016 30(4): 239-248 246 bokhara shalili, 804/turki sorkh and 811/turki zard proved that they are 99.6 to 100.0% identical with each other and share high (94.3 to 94.7%) nucleotide identity with the iranian isolate t12ja (km586376). lower nucleotide similarity (86.2%) was shown by one 804/turki sorkh isolate with the isolate 274-chal (fn391009) from greece. finally, analysis of the sequence of isolates 804/turki sorkh, 811/turki zard and 812/dir ras revealed a high degree of identity (86.2 to 88.4%) with the corresponding nucleotide sequences of the isolate sk-92-pl (hq398253) from slovakia. rt-pcr reaction performed on samples infected by pnrsv, according to das-elisa results, amplified a fragment of 675 nucleotides in length when pnrsv specific primer pair mg1/mg2 was used (table 1). regarding grape samples, specific pcr products of 118 nucleotides where also observed on das-elisa positive samples, using specific primers pair m3/m4 against gflv as reported in table 1. some of the citrus samples tested positive by das-elisa were also tested using primers ctvf and ctvr allowing amplification of specific pcr products 655 nucleotides in length (table 1). moreover, six ctv isolates collected at the jalalabad station were characterized. sequence analysis revealed high similarity, ranging from 91.1 to 99.8%, within the ctv isolates detected. in accordance with the previously defined phylogenetic groups (zemzami et al., 2002), nucleotide sequences of afghan ctv isolates cluster in group 1 (j4 and j8), group 4 (j61 and j76) and group 5 (j101). in particular, j4 and j8 isolates show, respectively, 99.4 and 99.2% identity with reference isolate t36 (m76485) from the usa (florida). furthermore, in group 4, isolate j61 and j76 were more similar to ano-1 isolate (dq211658) from egypt (98.5 and 98.0% identity, respectively) than to isolate 443-4 (ay791844) from croatia (97.4 and 97.5%, respectively). finally, isolate j101, in group 5, shows 95.6% identity with isolates c268-2 (ay750770) and c269-6 (ay750775) from argentina. 4. discussion and conclusions to improve afghanistan’s agricultural sector, the ec supports the phdp (perennial horticulture development project), for true to type/ecotype and healthy planting materials, and the plant biotechnology laboratory, to ensure the health status of local germplasm. the laboratory started screening the health status of the afghan germplasm national collection in order to ensure the multiplication of not only the best-selected varieties or ecotype, but also to avoid production and distribution of virus-infected fruit trees. symptom inspection and sample collection for viral diseases was carried out in all the national collection fields, including peach, plum, apricot, almond, apple, grape and citrus plants, located in different areas of the country. during the first seven years of its activity, the plant biotechnology laboratory based in kabul, played a key role by satisfying the increasing need for certified propagation plant material to establish new plantations in afghanistan. moreover, it contributed to protecting the country from undesirable introduction of pathogens due to increasing exchanges of propagation plant material among different countries and between different continents. until now, the plant biotechnology laboratory has verified the absence of ppv (the most devastating virus for stone fruit) in the afghan germplasm and detected and verified the presence of several important plant viruses in stone fruits, citrus and grapevines. further activity will be addressed to confirming, by rt-pcr, infection by apmv, pdv, gva, glrav-1 and glrav-3 in almond, plum and grape accessions which resulted positive by das-elisa, using specific primer pairs (parakh et al., 1995; menzel et al., 2002; goszczynski and jooste, 2003; osman et al., 2008). the plant biotechnology laboratory first reported aclsv occurrence in peach, plum, almond and apricot plants in afghanistan. according to the preliminary results of sequence analysis, due to the relative low identity with other aclsv isolates present in genbank, our mother stock nurseries appear to be infected by several afghan isolates of the virus, but further studies will be addressed to understanding if aclsv has been introduced into the country through infected propagation material. in any case, the presence of aclsv in important cultivars of peach and plum is a worrying aspect of the afghan certification program. similarly, our results identified, for the first time, pnrsv, apmv, and pdv infected plants in afghanistan. even if the latter two viruses were detected by das-elisa test, the presence of the viruses in accessions of the national collection field is cause for worry for afghan horticulture as all three viruses spread through infected propagating material and nursery productions (mink, 1992). moreover, pnrsv and pdv are also seedand pollen-transmitrehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 247 ted and can be spread by pollinating insects (kelley and cameron, 1986; aparicio et al., 1999; glasa et al., 2002; amari et al., 2007) while seed transmission of apmv is known only in hazelnuts (cameron and thompson, 1985). a comparative sequence and phylogenetic analysis will be performed (zindović et al., 2015) to show if clustering of various isolates is associated or not with geographic and host origin. regarding the detection of viruses in grape, according to our results the presence in the ncc in kandahar of gva and glrav-1, and glrav-3 in herat seems to be restricted to only a few accessions. early detection of these viruses by the plant biotechnology laboratory avoided multiplication and therefore the spread of infected grape material in afghanistan. in contrast, the noticeable presence of gflv (nearly 7%) in local cultivars maintained at the ncc in herat suggests the presence of the virus in afghan germplasm of grape for some time. this hypothesis should be confirmed by studying the genetic variability of afghan isolates in order to investigate the relationship among their geographical origin, sequence variability, and grapevine cultivar (meng et al., 2006; vigne et al., 2009; terlizzi et al., 2015). finally, our analyses identified, for the first time, ctv infected plants in afghanistan (rehman et al., 2012). the presence of a dangerous viral disease such as ctv in many citrus trees (nearly 6% of the assayed plants) from nccs, msns and private orchards represents a serious problem for the afghan citrus industry. high sequence identity with many isolates from usa, egypt, croatia, and argentina together with the large spread of the virus in afghan citrus-cultivating areas suggests the introduction of the virus into the country a long time ago. the presence of this virus seems to be, therefore, endemic; further investigations showed that infected plants did not exhibit quite as dramatic symptoms as those usually found in other citrus cultivation areas (e.g. spain, brazil, argentina etc.) where the virus is extremely dangerous and causes in some cases plants death (rochapeña et al., 1995). a specific research program is in progress to investigate if a mild strain of ctv is present in the eastern region (nangarhar, kunar, laghman) environment, if tolerant/resistant sour orange rootstock clones exist, or if the environmental (climate and soil conditions) influences the infectivity of ctv. due to the economic importance of these crops, implementation of the certification schemes is therefore necessary in afghanistan in order to guarantee the production and employment of virus-free propagating material. it is well known that data regarding the presence and spread of different plant viruses are important for individual countries, their neighbours, and for the whole agricultural community where different viruses can be spread by vegetative propagation via plant material. a continuous survey is therefore necessary to protect afghanistan from the introduction and/or spread of dangerous pests. as previously experienced, most effective management programs require prompt removal of infected trees and replacement with healthy planting material from certified nurseries (hadidi et al., 2011; pallas et al., 2012). establishment of rigid phytosanitary controls related to the importation of propagation material, as well as the eradication of infected trees, is therefore of great importance in afghanistan. references amari k., burgos l., pallás v., sánchez-pina m.a., 2007 prunus necrotic ringspot virus early invasion and its effects on apricot pollen grain performance. phytopathology, 97: 892-899. aparicio 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a.r., fisher h., 2006 genetic diversity analyses of grapevine rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties. j. gen.virol., 87: 1725-1733. menzel w., jelkmann w., maiss e., 2002 detection of four apple viruses by multiplex rt-pcr assays with coamplification of plant mrna as internal control. j. virol. methods, 99: 81-92. mink g.i., 199 ilarvirus vectors. adv. dis. vector res., 9: 261-281. osman f., leutenegger c., golino d., rowhani a., 2008 comparison of low-density arrays, rt-pcr and real-time taqman® rt-pcr in detection of grapevine viruses. j. virol. methods, 149(2): 292-299. pallas v., aparicio f., herranz k., amari k., sanchezpina m.a., myrta a., sanchez-navarro 2012 ilarviruses of prunus spp.: a continued concern for fruit trees. phytopathology, 102, 1108-1120. parakh d.r., shamloul a.m., hadidi a., scott s.w., waterworth h.e., howell w.e., mink g.i., 1995 detection of prune dwarf ilarvirus from infected stone 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1. introduction the japanese apricot, mume (prunus mume sieb. et zucc.) fruit, is widely distributed among the different climate regions of east asia, and is harvested at the yellowpeel stage for pickles called “umeboshi” or at green-peel stage for its juice. mume is known to be a climacteric fruit that produces large amounts of ethylene as it ripens (inaba and nakamura, 1981; koyakumaru, 1997; mita et al,. 1999), making it difficult to preserve for long periods of time as the characteristic taste and firmness is quickly lost. the reason for the degradation of the fruits and measures to prevent this degradation have never been thoroughly investigated. investigating harvest timing and improving conditions for fruit quality preservation during commercial processes may make the fruit more common. nmr spectroscopy is a promising tool for exploring new aspects of plant science, especially given that there are various types of plant materials in which the physical states of cell-associated water change naturally occur, e.g. in the maturation process of red raspberry (williamson et al., 1992), gooseberry (williamson et al., 1993), olives (gussoni et al., 1993), kiwifruit (callaghan et al., 1994), coconuts (jagannathan et al., 1995), cherry tomatoes (ishida et al., 1994), cherry fruits (ishida et al., 1997), in floral malformation in mangos (usha et al., 1994) and maturation in apples (wang et al., 1988). in addition, the detection of water in different subcellular organelles is often used to understand the changing water distribution among plant components. it has been suggested that multi-exponential 1h-nmr relaxation times (t1 and t2) in plant tissues reflect water in different plant cell compartments (burke et al., 1974; stout et al., 1978; gusta et al., 1979; bacic and ratkovic, 1984; hills and duce, 1990; isobe et al., 1999), and can be ascribed to several different causes: cellular heterogeneity and subcellular compartmentation (belton ontogenetic changes of the water status and accumulated soluble compounds in developing and ripening mume (prunus mume) fruit measured by 1h-nmr analysis g. watanabe(1), y. ishibashi(2)*, m. iwaya-inoue(2) (1) agricultural development total center, kagoshima prefecture, ibusuki, 810-0013 kagoshima, japan. (2) laboratory of crop science, department of plant resources, faculty of agriculture, kyushu university, hakozaki, 812-8581 fukuoka, japan. abbreviations: cpmg= carr-percellmeiboom-gill; faa= formalin acetic acid alcohol; nmr= nuclear magnetic resonance; t1= spin-lattice relaxation times; t2= spin-spin relaxation times. key words: histological observation, membrane integrity, mobility of water, nmr relaxation times (t1, t2), water content. abstract: the physiological changes of intact mume (prunus mume sieb. et zucc. cv. rinshu) fruit tissues were examined by measuring the physical states of cell-associated water in the fruit tissues with developing and ripening using 1h-nmr spectroscopy. we found that the water molecules in mume fruit tissues existed in several different compartments with different mobilities. additionally, spectral recovery in the water proton indicated reverse relationships between the pericarps and seeds at the immature and mature stages. in the pericarp tissues, the longest t1 and longer t2 markedly increased, while those in the seeds decreased. from these results, the change in the water status with growth stage had reverse trajectories in the pericarp and seed of the fruit. in the pericarp tissues, both water uptake and dry weight prominently increased with ripening. the epidermis and inner parenchymal cells of the pericarp tissues remarkably enlarged as a sigmoidal growth curve. membrane permeability, indicating a loss of membrane integrity, increased in the pericarp tissues. the elongation in the fully vacuolated cells and changes in the membrane permeability in the pericarp tissues with ripening correlated to the longest t1. in contrast, the high mobility of water in the seeds began to decrease with maturation, while oil began to accumulate. thus, the mobility of water, as analyzed in this study, is considered to reflect the results of physiological changes such as cellular heterogeneity and spatial arrangements both in the pericarp and in seed tissues for mume fruit with development and ripening. adv. hort. sci., 2015 29(1): 3-12 * corresponding author: yushi@agr.kyush-u.ac.jp received for publication 17 september 2014 accepted for publication 28 december 2014 4 adv. hort. sci., 2015 29(1): 3-12 and ratcliffe, 1985; snaar and van, 1992; kumamoto et al., 1998; iwaya-inoue et al., 2004 a, b). the movement of water is controlled by cellular organization, such as compartmentalization by membrane structures (tanner, 1978, 1983), and water is ordered by macromolecules, such as proteins and polymers of organic compounds (hazlewood, 1995). thus, the dynamic states of water in cells closely correlate with the organic properties of macromolecular structures. the objectives of the present research were (1) to examine the ontogenetic changes in the state of water by 1h-nmr spectroscopy, water content, membrane permeability and histochemical observation during development and ripening of japanese apricot fruit, and (2) to study the interrelationships among them. 2. materials and methods plant materials japanese apricot (prunus mume sieb. et zucc, cv. rinshu) fruits cultivated in the orchard at the university farm of kyushu university were harvested at each stage (a total of four times) from april to june. the fruits were defined as belonging to four ripening stages as follows (fig. 1 and table 1): stage 1, fruits at approximately 2.0 g in fresh weight and 1.5 cm in transverse diameter, peels green, and seeds immature and 0.1 g in fresh weight; stage 2, fruits at 15.0 g in fresh weight and 3.0 cm in transverse diameter, and seeds 0.5 g in fresh weight; stage 3, fruit at their maximum size, seeds 0.7 g in fresh weight; stage 4, fruit at maximum size and 45.0 g in fresh weight, 4.0 cm in transverse diameter, the firmness in the pericarp softening, the peels yellow, and seeds hard with a fresh weight of 0.8 g. fruits were separated into pericarp (including epicarps) and seed, and both tissues were used for experiments. tissue water contents were determined by obtaining the weight loss after drying in an oven at 90°c for 20 h. eight to ten fruits were used in each stage in the following experiments. 1h-nmr analysis an nmr spectroscope with a magnet operating at 89.5 mhz for 1h (jeol ex 90a) was used for the measurement of spin-lattice relaxation times (t1), 1h-nmr spinspin relaxation times (t2) and 1h-nmr spectra. a piece of pericarp or seed of an intact japanese apricot fruit was placed in an nmr tube (8 mm in diameter) which was then placed in an outer glass tube (10 cm in diameter) containing 99.8% d 2 o as an internal lock signal; spectroscopic measurement at 25±1°c was then undertaken. the repetition time was 15 s with four accumulation transients for each tissue. the decay between scans was always greater than five times t1. table 1 characteristics of mume fruit tested characteristics stage 1 2 3 4 harvested day april 9 ∼ 24 may 6 ∼ 12 may 29 ∼ june 4 june 13 ∼ 30 skin color green green green yellow endocarp hard hard soft soft size (cm) longitudinal diameter 1.6±0.1 3.2±0.4 3.9±0.1 4.2±0.1 transverse diameter 1.3±0.1 2.9±0.1 3.5±0.9 4.2±0.1 weight (g) whole (z) 1.7±0.2 14.8±1.1 32.8±3.0 45.8±2.4 pericarp 1.1±0.3 11.2±1.1 27.7±2.4 35.6±2.6 seed 0.1±0.0 0.5±0.1 0.8±0.1 0.7±0.1 values represent the mean of eight to ten fruits±se. (z) the weight of the stone is not included. fig.1 mume (prunus mume sieb.) fruit during four ripening stages. p=pericarp; s=seed. s p p s s p p s stage 1 stage 2 stage 3 stage 4 fig. 1 mume (prunus mume sieb.) fruit during four ripening stages. p= pericarp; s= seed. 5 watanabe et al., characteristic of developing and ripening of mume fruit measured by 1h-nmr analysis t1 measurements were determined by the inversion recovery (180°τ -90°pulse sequence) method. τ is the time between the radio-frequency pulses in the sequence, and the indicated angles are between the average direction of the original proton spins and that induced by the radio-frequency pulse (farrar and becker, 1971). twenty-two values of pulse interval (τ) ranging from 0.001 s to 15 s were used to acquire a t1 data set. t1 was determined from the slope of ln (m0 m) /2m0 versus τ, where m is the amplitude of the fid of the water proton signal following the 90° pulse at τ, and m0 is the limiting value of m. the existence of water components with different t1 values was revealed from spectral recovery and semi-log plots of signal intensity of 1h-nmr according to ishida et al. (1994). a graphical method was used to determine the relaxation time of water and the percentage of the fraction (hazlewood and nichlos, 1969; belton and packer, 1974). short t1 values including those below 0.1 s were considered from spectral recovery ranging in the pulse intervals of 0.03 s and 0.05 s. t2s measurements were determined by the cpmg (carr-percell-meiboom-gill) method from the slope of ln m/m0 versus t, where m0 is the magnetization amplitude of the water proton signal occurring at time τ after the initial 90° pulse in the cpmg (90°-τ-180°-2τ-180°2τ---) pulse sequence. t = 2nτ, where n is the number of refocusing pulses (25 points from 2 to 4000 loops) and τ was 0.001s. t2 was also determined from a semi-log plot of signal intensity as in the case of t1. histological observations materials were fixed in faa (formalin acetic acid alcohol; 80% ethanol: 100% acetic acid: formalin = 90:5:5), and 20-μm sections were cut using a microtome (cryostat hm500-om, microm co. ltd) at -20°c. the sections were stained with 0.01% ruthenium red for pectic substances in the middle lamella (fig. 2a) and by sudan iii for lipid substances (fig. 2b), respectively. they were then subjected to microscopic observations (axiphot, carl zeiss co. ltd). leakage of electrolytes pericarp tissues of japanese apricot fruit (about 3.0 g) were cut into pieces of about 2-mm square. these small pieces were immersed in distilled water (50 ml) and stirred at 180 cycles/min. the extent of leakage of electrolytes was determined with an electrolytes conductivity meter (toa conductivity meter, model cm-20e, toa electronics ltd.) and expressed as the percentage of the total electrolytes in each sample measured after samples were killed by a cycle of freezing and thawing (iwaya-inoue et al., 2004 a, b). 3. results histochemical characteristics of fruit tissues with ripening mume fruit are characterized by four stages (fig. 1 and table 1). the histochemical changes of the mume fruit tissues with ripening are shown in figure 2. the individual pericarps and seeds are mainly comprised of the parenchymal tissue, beneath the epidermal tissue and around the vascular tissue. pectic substances stained by ruthenium red are abundant in the middle lamella of cell walls in the pericarp parenchymal tissues from the small green fruit to the large green fruit (fig. 2a, stages 3 and 4). the components in the cell wall of pericarp tissues began to change with maturafig. 2a photomicrographs of cross-sections of the pericarp tissues in mume fruit with ripening (a～d). parenchyma tissues beneath epidermis. a: stage 1; b: stage 2; c: stage 3; d: stage 4. (e～ h) vascular bundles. e: stage 1; f: stage 2; g: stage 3; h: stage 4. epi, epidermis; v.b., vasucular bundle. tissues were stained with ruthenium photomicrographs of cross-sections of the pericarp tissues in mume. bars indicate 50μm. a b c d e f g h epi. v.b stage 1 2 3 4 v.b. v.b v.b epi. epi. epi. fig. 2a) photomicrographs of cross-sections of the pericarp tissues in mume fruit with ripening (a ∼ d). parenchymal tissues beneath epidermis. a= stage 1; b= stage 2; c= stage 3; d= stage 4. (e ∼ h) vascular bundles. e= stage 1; f= stage 2; g= stage 3; h= stage 4. epi= epidermis; v.b.= vascular bundle. tissues were stained with ruthenium. photomicrographs of cross-sections of the pericarp tissues in mume. bars indicate 50 μm. 6 adv. hort. sci., 2015 29(1): 3-12 tion of the fruit (stage 3). as the peel of the fruit assumes a yellow color, pectic substances in the cell wall structures in the parenchymal tissue collapse, except for the epidermis and vascular bundles (fig. 2a, stage 4). on the other hand, lipids in the seeds stained with sudan iii were observed in the epidermis of seed tissues, indicating that the cells of the epidermis are abundant in suberin (fig. 2b). moreover, oil bodies were also observed in the parenchyma of the cotyledons in the matured and ripened fruit seeds (stages 3 and 4). water uptake in relation to cell enlargement in the pericarp tissues, both water uptake and dry weight prominently increased during ripening, especially from the large green-fruit stage to the matured-fruit stage (fig. 3). vacuoles occupy a large part of individual cells in the tissues at these stages (data not shown). the epidermis and inner parenchymal cells of the pericarp tissues remarkably enlarge during ripening in a sigmoidal growth curve (table 2). the enlargement of cells in the pericarp region closely correlated with that for fruit diameter, accompanied by a marked increase in water uptake and dry matter accumulation (tables 1 and 2). in seed tissues, water uptake did not change, while dry weight remarkably increased in both the matured and ripened fruit (fig. 2). therefore, the seed water content remarkably decreased in the mature stages (fig. 4, stage 3). membrane integrity in the pericarp tissues changes in the membrane permeability as well as the cell wall integrity of mume fruit with ripening were considered. leakage of electrolytes from the pericarp tissues increased during ripening, and 90% of the total electrolytes were found to have leaked at fruit maturation (fig. 4, stage 3). thus, membrane permeability, indicating a loss of membrane integrity, arises in the pericarp tissues. tissue specificity of 1h-nmr spectra with various pulse intervals between 180  and 90° the physiological changes of intact tissues were examined by measuring the physical states of cell-associated table 2 changes in cell size of pericarp during ripening of mume fruit cell size (μm) stage 1 2 3 4 epidermis long side×short side 25.1× 9.9 39.0×12.3 38.9×22.9 49.5×49.5 parenchyma small cells long side×short side 44.7×18.6 97.9×54.4 119.7×68.9 142.9×102.9 larage cells diameter 61.4 145.5 181.2 243.9 values represent the mean of ten cells of the pericarp tissues in three fruits. a b c d fig. 2b photomicrographs of cross-sections of the seed tissues in mume fruit with ripening (a～d). parenchyma tissues beneath epidermis. a: stage 1; b: stage 2; c: stage 3; d: stage 4. s.c, seed coat; co., cotyledon. v.b; vasucular bundle. tissues were stained with sudan ⅲ. bars indicate 50μm. s.c co. v.b. s.c co.co. s.c co. s.c s.c 1 2 3 4 stage fig. 2b photomicrographs of cross-sections of the seed tissues in mume fruit with ripening (a ∼ d). parenchymal tissues beneath epidermis. a= stage 1; b= stage 2; c= stage 3; d= stage 4. s.c.= seed coat; co.= cotyledon. v.b= vascular bundle. tissues were stained with sudan iii. bars indicate 50 μm. 7 watanabe et al., characteristic of developing and ripening of mume fruit measured by 1h-nmr analysis water in fruit tissues with ripening using 1h-nmr spectroscopy. 1h-nmr spectra of both intact pericarp and seed in the fruit tissues were examined by spectral recovery of 1h-nmr with the inversion recovery method. the pulse interval was varied between 180o and τ90o pulses. an 1hnmr spectral peak was observed at 4.8 ppm of chemical shift, which corresponds to the 1h nuclei of water. 1hnmr spectra of both the pericarp and seed tissues were not symmetrical, indicating that the peak consists of components with different chemical shifts and various recovery times (fig. 5). in pericarp tissues of the small green fruit, major spectral recoveries were observed in the pulse intervals of 0.3 s and 0.5 s, while in the ripened fruit, these were 0.9 s and 1.5 s (fig. 5a and b, stage 1). on the other hand, in seeds of the small green fruit, several peaks of spectral recovery were mainly observed at pulse intervals of 0.8 s and 1.0 s, fig.3. changes in electrolyte leakage of pericarp during ripening of mume fruit. values represent the mean of three fruit ± se. 0 10 20 30 40 50 60 70 80 90 100 1 stage e le c tr o ly te l e a ka ge ( % ) 1 2 3 4 fig. 4 changes in electrolyte leakage of pericarp during ripening of mume fruit. values represent the means of three fruits ± se. fig. 4a changes in the water content of pericarp (□）and seed (■） for mume fruit with ripening. values represent the mean of eight to ten fruit ± se. w at e r c o n te n t (g h 2 o g 1 d w ) 0 2 4 6 8 10 12 14 16 18 11 2 3 4 stage fig. 3a changes in the water content of pericarp ()and seed () for mume fruit with ripening. values represent the mean of eight to ten fruits ± se. r el at iv e va lu e fig. 4b relative value in the dry weight of pericarp (□）and seed (■） for mume fruit with ripening. relative value of pericarp and seed at stage 1 , respectively, is 1.0. 0 5 10 15 20 25 30 35 1 stage 1 2 3 4 fig. 3b changes in the dry weight of pericarp ()and seed () for mume fruit with ripening. values represent the mean of eight to ten fruits ± se. fig. 5 spectral recovery of 1h-nmr of mume fruit with inversion recovery method. τ: pulse interval. a= spectra of pericarp tissues at stage 1; b= spectra of pericarp tissues at stage 4; c= spectra of seed tissues at stage 1; d= spectra of seed tissues at stage 4. 1; d= spectra of seed tissues at stage 4. 8 adv. hort. sci., 2015 29(1): 3-12 while they occurred between 0.05 s and 0.2 s in ripened fruit (fig. 5c and d, stage 4). the individual values of the t1 components can be obtained by pulse interval (τ) at null point, t1 = null / ln2. thus t1 values that are due to major spectral recovery times in pericarp tissues became longer in the ripened fruit, while the tendency was reversed in the seed tissues. changes of t1 components in the fruit tissues the relaxation times and estimated amounts of the individual water fractions calculated from semi-logarithmic plots of 1h-nmr signal intensities in the fruit tissues with ripening processes are listed in table 3. the spinlattice nmr relaxation time (t1) describes the process of realigning the magnetic moment with the external magnetic field. cellular water exists in two to three components, which are shown by nmr relaxation times; in plant tissues, these water components consist of three states of water: i.e. free water, loosely bound water, and tightly bound water (iwaya-inoue and nonami, 2003). the three compartmentalizations of water originally identified as the vacuole, cytoplasm, and cell wall/extracellular space (apoplast) are reflected by the different relaxation times in the parenchymal tissues of apples (snaar and van as, 1992; hills and remigereau, 1997). thus, differences in the relaxation times (t1, t2) of biological tissues can be interpreted as differences between the ratio of free water to bound water. semi-logarithmic plots of 1h-nmr signal intensity with the inversion recovery method of mume fruit were multiexponential. the long t1 fraction could be understood as highly mobile water (free water) derived from the vacuoles, while the short t1 fraction with restricted mobility represents loosely bound and bound water from the cytoplasm and the apoplastic region, respectively. in the pericarp tissues, t1 values of the longest water component markedly increased from 0.8 to 2.0 s from the small green fruit to the large green fruit (stages 1 and 2). the major fraction of the longest t1 was about 80% and the ratio was constant during the ripening stage. the fraction of the shortest component of t1, at 0.4 s was about 10% and was also constant through all stages. in addition, vacuoles stained by neutral red were observed in a large proportion of individual cells in the pericarp tissues with ripening (data not shown). thus, water in the pericarps was considered to increase the amount of free water with ripening. on the other hand, in the seeds of the small green and large green fruits, t1 values and the fractions of the longest component were about 2.0 s and 75%, respectively (table 3, stages 1 and 2). t1 values of the longest water component in the seeds markedly decreased from 2.0 to 0.4 s from the large green-fruit to matured-fruit stages (stage 2 and 3). the t1 values did not change further for the ripened fruit. however, the fraction ratio of the longest water component in the seed decreased from 45 to 30 % during that stage (stage 4). table 4 changes in components of 1h-nmr spin-spin relaxation times (t2)s during ripening of japanese apricot fruit stage 1 2 3 4 t2 values (ms) fraction (%) t2 values (ms) fraction (%) t2 values (ms) fraction (%) t2 values (ms) fraction (%) pericarp 151±12 28.1±6.0 289±17 26.4±1.2 394±34 25.9±0.9 404±38 26.9±6.7 211±10 71.9±6.0 442±10 73.6±1.2 731±26 74.1±0.9 713±14 73.1±6.7 seed 143±11 12.8±2.8 205±19 16.7±6.3 36± 2 16.7±2.8 27± 2 44.8±2.8 323±24 87.2±2.8 317±14 83.3±6.3 56± 2 83.3±2.8 47± 6 55.2±2.8 values represent the means of eight to ten fruit±se. table 3 changes in components of 1h-nmr spin-lattice relaxation times (t1)s during ripening of mume fruit stage 1 2 3 4 t1 values (ms) fraction (%) t1 values (ms) fraction (%) t1 values (ms) fraction (%) t1 values (ms) fraction (%) pericarp 412±13 12.1±3.2 416± 1 12.1±0.5 413± 5 12.3±0.8 424±13 11.3±1.7 603±55 7.4±3.4 1171±44 9.2±2.4 1332±63 11.9±1.1 1723±28 10.2±2.1 758±54 80.5±1.6 1367±34 78.7±1.7 1619±91 75.8±2.1 2057±94 78.5±1.1 seed 349±11 13.5±4.5 785± 6 12.2±1.1 181± 3 20.2±1.1 138± 2 18.5±1.2 1414±28 13.4±4.3 1443±25 15.9±1.3 321±49 33.7±3.6 193± 3 50.6±1.2 2216±15 73.0±0.4 2040±19 75.6±0.6 443±66 46.1±2.6 370± 2 30.9±0.8 values represent the means of eight to ten samples±se.. 9 watanabe et al., characteristic of developing and ripening of mume fruit measured by 1h-nmr analysis changes of the t2 components in the fruit tissues the spin-spin nmr relaxation time (t2) describes the time-dependent decay of nmr signal due to the dephasing process of the individual spins with respect to each other. the water component estimated by t2 was divided into two fractions (table 4). the regions with long t2 values in glycine max seed tissues had high concentrations of free water, while the regions with short t2 values had high concentrations of loosely bound and bound water (ishida et al., 1987). in the pericarps of the mume fruit, the fractions in t2 comprised about 70% through the four stages. t2 values of both long and short components increased until fruit maturation (stage 3); however, they did not prolong further at the ripened-fruit stage (stage 4). by contrast, t2 values of the longer water component in seeds markedly decreased from about 300 ms to 60 ms from the large green fruit to the matured fruit (stage 2 to stage 3). 4. discussion and conclusions relationship between nmr relaxation time t1 and the characteristics in mume fruits during development and ripening nuclear magnetic resonance (nmr) spectroscopy is a useful technique to follow physiological changes with respect to the state of water in developing japanese apricot fruits. water in living tissues is known to consist of several components with regard to the relaxation of the magnetized protons (hazlewood et al., 1969; hazlewood, 1995; isobe et al., 1999; iwaya-inoue, 2004 a, b). in developing japanese apricot fruits, three components with different t1 values were distinguished on the semi-logarithmic plots of the recovery of the 1h-nmr signal (fig. 5 and table 3). multicomponent water fractions are similar to those reported for other plant tissues (stout et al., 1978; gusta et al., 1979; isobe et al., 1999). referring to the line width of 31p-nmr signals (kano et al., 1990; takagishi et al., 1991), the longest component is ascribed to that of vacuoles containing small molecules, such as metabolic intermediates, secondary products and inorganic ions, and the middle component to that of cytoplasm in plant tissues. the water with the shortest t1 is considered to be exchangeable water around macromolecules, such as starches, proteins and strings of macromolecules in vesicles or between cell walls (rorschach and hazlewood, 1986). large amounts of highly mobile water were detected in the seeds of small green and large green fruits. the mobility of exchangeable water in the seed tissues became higher as fruit developed. however, the mobility became low at fruit maturation; thereafter, it continued to be low. on the other hand, the mobile water in the pericarp of small green fruit was low. this suggests that concentrations of cell components that bind water were high in these tissues. thereafter, the mobile water in the pericarp increased with enlargement and even more so upon coloring of the fruit (figs. 1 and 5, tables 1 and 3). these trends agree with previous reports (ishida et al., 1989; 1994; 1997). changes in the mobility of water are considered to be the result of physiological changes in fruit growth including seed development and maturation, which is the most important natural function of the fruits (crane, 1964). relaxation times are strongly influenced by the availability of the water and the presence of macromolecules to which water molecules can be “bound”. it was shown that t1 closely correlated with water content in developing and maturing rice grains (funaba et al., 2006), in azalea buds subjected to low-temperature stress (kaku et al., 1984), and in the heat-tolerant and heat-sensitive cultivars rice grains subjected to high-temperature stress (tanaka et al., 2009). in the pericarp tissues, the changes in water contents were not correlated with the values of the longest t1 component with ripening (fig. 3a, tables 3). however, our results suggest that other factors may contribute to the motional restriction of water. a similar tendency was observed in sweet potato tubers exposed to cold stress (iwaya-inoue et al., 2004 b). we found that membrane permeability, indicating a loss of membrane integrity, increased in mume pericarp tissues during ripening. an increase in membrane permeability also has been reported in ripening wild-type tomato fruit, but not in the ripening-inhibited (rin) mutant (poovaiah et al., 1975). moreover, the permeability of the plasma membrane increased and the membrane-lipid composition changed in ripening malus domestica fruit (lurie et al., 1987) and during the development and senescence of cucumis melo fruit (lester and stein, 1993). this coincided with our results from the electrolyte leakage study for mume fruit. in wood plants (i.e. galled leaves invaded by insects), a higher ratio of ion leakage was strongly associated with prolongation of the nmr relaxation times (kaku and iwaya-inoue, 1990). in addition, it was demonstrated that the relaxation times strongly related to the size and geometry of the vacuolated cells in mushroom tissues during postharvest senescence (donker et al., 1997). in our experiment, elongation in fully vacuolated cells and changes in membrane permeability in the pericarp tissues may have contributed to the correlation of the longest t1 components with fruit ripening. however, in seed tissues, a considerable decrease in the longest t1 components in seeds of mume fruit was accompanied with markedly decreased water content, from 14 to 1 g h 2 o/g dry weight from stage 2 to stage 4 (fig. 3a and table 3). phenomena such as cold acclimation in plant tissues correlated with the decrease both in t1 and in water content (burke et al., 1974; kaku et al., 1984; fennell et al., 1996; yoshida et al., 1997). the decrease in water content of the seed tissues during maturation is considered to be associated with a reduction in free water, perhaps due to an increase of dry matter, that is, an increase in the accumulation of cellular substances (fig. 2a and b and table 3). 10 adv. hort. sci., 2015 29(1): 3-12 relationship between nmr relaxation time t2 and characteristics in mume fruits during development and ripening the larger fraction of the two, with the long t2, can still be assigned to vacuolar water, whereas the small fraction with short t2 represents water from the cytoplasm and perhaps the contribution of water inside the cell wall and extracellular water (scheenen et al., 2002). the amount of water or t2 in the pericarp and seed changed inversely according to the progression of growth stages. mobile water in the pericarp of small green fruit was low (table 4). thereafter, the mobile water in the pericarp increased with enlargement, but did not change further at the coloring fruit stage. thus, the changes in water contents were not correlated with the values of a longer t2 component with ripening (fig. 3a and table 4). during the stage at which the fruit peel became yellow, however, pectic substances in the cell wall structures in the parenchymal tissue collapsed except for in the epidermis and vascular bundles (figs. 1 and 2a, stage 4). kaneko et al. (1989) reported changes in the pectic substance components during mume fruit ripening. the cellular changes, such as the changes of polysaccharide components or pectic substances, specific to the pericarp tissue ripening characteristics, may contribute to a more ordered state of water, thereby resulting in unchanged relaxation time. in previous reports, mccarthy et al., (1995) measured a decrease in t2 in bruised regions of apples. in addition, marked shortening of t2 values compared with t1 values in the fruit tissues may reflect a correlation between the relaxation times and compartment size of crosslinked polymer gels (murase and watanabe, 1989). on the other hand, in seed tissues, a considerable decrease in the longer t2 components in seeds of mume fruit is accompanied with markedly decreased water content from stage 2 to stage 4 (fig. 3a and table 4). water status can provide useful information about the characteristics of mume fruit development and ripening when the seed of a small green mume fruit was gellified, the mobility of water was high. thereafter, when the color inside the seed of a small green mume fruit turned milky white, the mobility of water, water derived from the vacuole and exchangeable water around macromolecules, became low (fig. 1 and tables 3 and 4). the decrease in water content of the seed tissues during maturation is associated with a reduction in free water, perhaps due to an increase of dry matter, that is, an increase in the accumulation of cellular substances such as oil bodies (fig. 2b and fig. 3b). japanese apricot fruit pericarp tissues may act as a sort of storehouse where photosynthates are temporarily accumulated for further transport into the seed during seed formation. therefore, seed formation, which is the primary object of fruit growth, may require larger amounts of energy than are available in the pericarp. the parenchymal tissues of the pericarps in matured mume fruit began to collapse in the yellow-peel stage (figs. 1 and 2a), probably because the pericarp tissues are no longer necessary to physiological functions. these are considered to be the result of a loss of membrane integrity and efflux of pectic substances in the cell wall structures (figs. 2a and 4). regardless of the constant high water content, the level of high-mobility water, i.e. water derived from vacuoles and exchangeable water, was high (fig. 3a, tables 3 and 4). this increase is considered to result from the fact that the increase of the mobile water by cell wall breakdown is greater than the decrease of the free water by cellular accumulation. in addition, the enlargement of cells in the pericarp region closely correlates with increases in fruit diameter, water uptake, and dry matter accumulation. changes in the fully-vacuolated-cell volume were closely correlated with changes in the values of the longest t1 component during ripening (tables 2, 3 and 4). thus, the water compartments and the mobility of water analyzed in this study are considered to reflect the result of physiological changes such as cellular heterogeneity and spatial arrangements both in the pericarp and in seed tissues for mume fruit with development and ripening. in conclusion, the analysis of water components derived from 1h-nmr spectroscopy provides useful information about the characteristics of fruit ripening. acknowledgements this work was supported in part by a grant-in-aid (no.11460016) for scientific research from the ministry of education, science, sports and culture of japan to m. i. i. we are grateful to drs. h. kano and m. koizumi of the national institute of agrobiological resources and dr. ishida of the national food research institute for their advice regarding nmr techniques and for their helpful suggestions. we also thank kyushu matsushita denki co. ltd. for allowing us the use of their nmr spectroscope, and dr. n. tamura of fukuoka women’s university for the analysis of the nmr spectra. we wish to express our special 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williamson b., goodman b.a., chudek j.a., 1993 the structure of mature gooseberry fruits revealed noninvasively by nmr microscopy. micron., 24(4): 377-383. yoshida m., abe j., moriyama m., shimokawa s., nakamura y., 1997 seasonal changes in the physical state of crown water associated with freezing tolerance in winter wheat. physiol. plant., 99: 363-370. 151 1. introduction the genus ocimum (lamiaceae family), collectively called basil, comprises between 50 and 150 species of herbs and shrubs (darrah, 1980). basil is native to asia (india, pakistan, iran, thailand, and other countries) and can be observed growing wild in tropical and sub-tropical regions (makri and kintzios, 2008). the essential oil profiles of this group of plants are extremely variable, such that several aroma compounds can be found in chemotypes of basil such as citral, eugenol, linalool, methylchavicol, and methylcinnamate that are traded in the international essential oil market (simon et al., 1999). the diversity within basil species has been accentuated by centuries of cultivation and cross compatibility, which has lead to great variation in morphology and chemical profile (javanmardi et al., 2002). basil species have antioxidant, antimicrobial and antitumor activities that are due to the presence of phenolic acids and aromatic compounds (hussain et al., 2008). lemon basil (ocimum × citriodorum vis.), a hybrid of sweet basil (ocimum basilicum) and american basil (ocimum americanum), is a herb grown primarily in northeastern africa and southern asia (fisher and phillips, 2006). it is naturalized in asia and cultivated for its lemon-scented leaves due to the essential oils citral and neral as predominant compounds (grayer et al., 1996). lemon basil is characterized by its small stature, early flowering, and small, narrow leaves. application of organic sources of nutrients, with no or very little use of inorganic fertilizers, is rapidly gaining favor (anwar et al., 2005). compost tea is a highly concentrated microbial solution produced by extracting beneficial microbes from compost. compost tea is produced by mixing compost with water and incubating it for a defined period, either actively aerating (aerated compost tea, act) or not (non-aerated compost tea, nct) and with or without additives that are intended to increase microbial population densities during production (scheuerell and mahaffee, 2002; ingham, 2005). it is a source of foliar and soil nutrients, contains chelated micronutrients for easy plant absorption and the nutrients are in biologically available forms for both plant and microbial uptake (hendawy, 2008). many researchers have pointed out the efficacy of organic manures, compost and compost teas in increasing vegetative growth, biomass and essential oil yield of sweet marjoram (gharib et al., 2008), cumin (safwat and badran, 2002), fennel (azzaz et al., 2009) and sweet basil (khalid et al., 2006). improvements in yield and quality following application of these organic-based substances has been attributed to an enhancement of the beneficial microbial communities in soil, an improvement of mineral absorption conditions for plants, and a stimulation of defense compounds, growth regulators or phytohormones in effects of chicken manure and vermicompost teas on herb yield, secondary metabolites and antioxidant activity of lemon basil (ocimum × citriodorum vis.) j. javanmardi(1), e. ghorbani department of horticultural sciences, faculty of agriculture, shiraz university, shiraz, iran. key words: essential oil, flavonoids, organic agriculture, sustainable agriculture, total phenolics. abstract: effects of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on vegetative growth, herb yield, essential oil content, total phenolics, total flavonoids and antioxidant activity of lemon basil (ocimum × citriodorum vis.) was evaluated in a two-year field experiment. the greatest plant height, number of leaves and flowers, shoot fresh and dry weight and leaf chlorophyll content were obtained using cmt at either 1:5 or 1:10 dilutions with no significant differences. the highest number of lateral branches and flavonoid content were obtained when cmt at 1:5 dilution was applied. essential oil content was at its highest level (0.618%) when cmt or vct were used at 1:10 dilution, while the greatest total phenolic content and total antioxidant activity were obtained at 1:5 dilution of vct. the results emphasize the possibility of using organic-based compost teas for enhancing herbal yield and important secondary metabolites in aromatic medicinal plants. adv. hort. sci., 2012 26(3-4): 151-157 (1) corresponding author: javann@shirazu.ac.ir. received for publication 27 june 2012 accepted for publication 31 january 2013 152 plants (pant et al., 2009). various liquid manures or their extracts are known to serve primarily as a source of soluble plant nutrients, growth stimulants and disease suppressors (khalid et al., 2006). several studies have reported the effects of compost tea on suppression of certain plant diseases such as damping– off caused by phytium ultimum (scheuerell and mahaffee, 2004), gray mold (botrytis cinerea) (scheuerell and mahaffee, 2006), alternaria solani and phytophthora infestans (koné et al., 2010). however, relatively little work has been done to investigate the effect of vermicompost or manure teas on yield, nutritional and quality factors, secondary metabolites and antioxidant activity of vegetable herbs. in the present work the effects of chicken manure and vermicompost teas as soil drench on growth characteristics, chlorophyll content, essential oil yield, total phenolics, total flavonoids and antioxidant activity of lemon basil were evaluated. the significance of this study lies in the possibility of applying compost teas as soil amendments to improve yield components and secondary metabolites in organic agriculture. 2. materials and methods site description, plant material and experimental design the experiment was carried out in two subsequent years, 2010 and 2011, in a field 1810 m above sea level in a silty-loam soil. three soil samples from 0-30 cm depth were collected and sent to a certified local soil laboratory for analysis. the chemical properties of the soil are shown in table 1. the local maximum-minimum mean temperatures and relative humidity during the growing period were 28.7-12.6°c and 47.3%, respectively. seeds of lemon basil (ocimum × citriodorum vis.) were sown in double rows (20 cm apart) with 50 cm spacing. seedlings were thinned three weeks after sowing to 15 cm between plants within the rows. plants were harvested at ground level when they were at full bloom stage (90 days after sowing) for further analyses. irrigation (using a drip-tube system), hand weeding, and other management practices were performed when required throughout the growing period. the experiment was carried out in a complete randomized block design with three replicates per treatment, each of which consisted of 20 plants. five fertilization treatments were applied as soil drench of de-ionized water (control), chicken manure tea (cmt) at 1:5 and 1:10 water dilution (v/v) and vermicompost tea (vct) at 1:5 and 1:10 water dilution (v/v). tea preparation and application method vermicompost and chicken manure teas were prepared as described by javanmardi (2010). briefly, vermicompost and chicken manure were separately mixed with tap water at ratios of 1:5 and 1:10 (v/v) in loosely covered 14 l plastic containers. water was allowed to stand for 24 h for passive chlorine removal before mixing. the mixtures were aerated using an aquarium pump for 72 h brewing time in a shaded area. solutions were filtered through cheesecloth before application. chemical properties for teas are presented in table 2. treatments were started four weeks after sowing and applied five times as soil drench with 600 ml of solution per plant at weekly intervals. fresh solutions were prepared for each application interval. vegetative growth parameters at full bloom stage ten central plants from each replicate (to avoid marginal effect) were cut at ground level and plant height (cm), number of branches per plant, number of flowers, number of leaves, and fresh and dry weight of herb (g per plant) were recorded. chemical analysis chlorophyll content. chlorophyll content was determined as described by saini et al. (2001). randomly selected samples of fully expanded leaves (0.5 g) were used. samples were homogenized with 5 ml of acetone (80% v/v) using a pestle and mortar and filtered through filter paper (whatman no. 2). the process was conducted in the dark to avoid photo bleaching. absorbance was measured with a uv-visible spectrophotometer (camspec m108, spectronic instruments, leeds, uk) at 652 nm and total chlorophyll content calculated using: total chlorophyll (mg·g-1 fw) = [d 652 ×v]×v/w where: v is the total volume of acetone extract (ml) and w, the fresh sample weight (g). essential oil content. quantitative determination of the essential oil obtained from lemon basil subjected to the different treatments was achieved by placing the air-dried herbage in a 2 l flask with distilled water (1:15 w/v) and using a clevenger apparatus, as described by charles and table 1 chemical properties of soil organic matter (%) total n (%) available phosphorus as p (mg·kg-1) bray method available potassium as k (mg·kg-1) fe (mg·kg-1) cu (mg·kg-1) mn (mg·kg-1) zn (mg·kg-1) ph ec (ds·m-1) 1.21 0.05 13.50 540 4.88 1.19 0.39 0.23 7.73 1.50 153 simon (1990). the average essential oil content of aerial parts is reported as percent of plant dry matter. sample preparation for total phenolic and flavonoid content determination. samples were prepared using the method described previously by javanmardi et al. (2003). briefly, 250 mg of dried plant material from each replicate were ground and dissolved in 10 ml of 80% acetone. sample extracts were rotated for 1 h in the dark and centrifuged at 5400 g for 10 min. one ml of supernatant was dried under vacuum at 45°c and kept at -18°c for further use. each sample was dissolved in 1 ml acetone prior to analysis for total phenolic and flavonoid determination. total phenolic compound analysis. the amount of total phenolics in extracts was determined with the folin-ciocalteau reagent using the method of spanos and wrolstad (1990), as described by javanmardi et al. (2003). to 50 ml of each sample, 2.5 ml of 1/10 dilution of folin-ciocalteau reagent and 2 ml of na 2 co 3 (7.5%, w/v) were added and incubated at 45°c for 15 min. the absorbance of all samples was measured at 765 nm using a uv-visible spectrophotometer (camspec m108, spectronic instruments, leeds, uk). gallic acid was used as standard and results are expressed as mg of gallic acid equivalent per g of dry weight (mg gae/g dw). total flavonoid analysis. the method described by adom and liu (2002) was adopted for total flavonoid content analysis. to 0.5 ml of extract, 2.5 ml distilled water were added followed by 0.15 ml of 5% nano 2 solution. the mixture was left to stand for 6 min at room temperature before adding 0.3 ml 10% alcl 3 .6h 2 o solution. the mixture was left for an additional 5 min, then 1 ml of 1 m naoh added and made up to 5 ml with distilled water. the solution was vortexed and the uv absorbance at 510 nm was recorded against catechin as reference. the result is expressed as µg/g dw. total antioxidant activity assay. the antioxidant activity of samples was determined by free radical scavenging activity assay using 1,1-diphenyl-2-picryl-hydrazil (ddph) reagent according to brand-williams et al. (1995). the ground leaves (1 g) were extracted with 50% methanol, 50% water. to 0.75 ml of the extract sample, 1.5 ml of freshly prepared methanolic dpph solution (20 µg·ml-1) were added and stirred. the decolorizing process was recorded after 5 min of reaction at 517 nm and compared with a blank control. the total antioxidant activity is expressed in % calculated as (control absorbance sample absorbance /control absorbance) × 100. statistical analysis the experiment was carried out for two years in a randomized complete block design (rcbd) with three replicates, each of which consisted of 10 plants. data were analyzed using one-way analysis of variance (one-way anova) and means of two years for each trait were compared with least significant difference (lsd) at p≤0.05 by spss12 (spss inc., chicago, il) computer software for windows. the data presented in tables and figures are mean values ± standard errors of two years of data for three replicates. pearson correlation analysis using spss12 (spss inc., chicago, il) was performed to assess the relationship between total phenolics, total flavonoids and essential oils with total antioxidant activity. 3. results and discussion the combined analysis of data from the experiment did not show any significant differences for all traits (data not shown). growth parameters analyses of variance showed that the growth parameters of lemon basil including plant height, number of lateral branches, leaves and flowers, shoot fresh and dry table 2 chemical properties of vermicompost tea (vct) and chicken manure tea (cmt) vermicompost tea (1:5) vermicompost tea (1:10) chicken manure tea (1:5) chicken manure tea (1:10) organic matter (%) total n (%) 6.30 3.14 2.21 1.10 phosphorus as p (%) 1.01 0.50 1.31 0.65 potassium as k (%) 6.18 3.08 5.61 2.81 fe (mg·l-1) 9.20 4.51 9.00 4.48 cu (mg·l-1) 0.07 0.03 5.00 2.48 zn (mg·l-1) 25.0 12.0 3.20 1.60 mn (mg·l-1) 0.18 0.89 0.08 0.04 ph 8.22 8.22 8.29 8.29 ec (ds·m-1) 4.41 2.20 2.82 1.41 154 weight at full bloom stage were significantly affected by vct and cmt (table 3). plant height and number of leaves the greatest plant height and leaf number were observed at 1:5 cmt, which was not significantly different from 1:10 dilution. other treatments did not show significant differences for plant height and leaf number (table 3). the same result has been reported in plantago plants under 300 ml·l-1 compost tea application (hendawy, 2008). the increasing effects of organic manure and bio-fertilizers on plant height on fennel (azzaz et al., 2009) and peppermint (swaefy et al., 2007) have been previously reported. number of lateral branches the highest lateral branch number was observed at 1:5 cmt dilution, however it was not significantly different from 1:10 cmt. the differences among other treatments (1:5 and 1:10 vmt and control) with regard to plant lateral branches were not significant (table 3). a promoting effect of organic fertilizers on the number of branches was observed also by azzaz et al. (2009) in fennel plants, especially when organic fertilizers were used in combination with bio-fertilizers. number of flowers the highest flower numbers were observed in lemon basil plants treated with cmt (1:5 and 1:10 dilutions) and vct (1:5 dilution) with no significant differences. control plants and vct-treated plants at 1:10 dilution showed lower flower numbers (table 3). an increased number of flowers in plantago plants under 300 ml·l-1 compost tea application has previously been reported (hendawy, 2008). shoot fresh and dry weight the highest shoot fresh and dry weights were observed at 1:5 cmt, which was not significantly different from 1:10 dilution. other treatments did not show significant differences for these parameters (table 3). the promoting effect of chicken manure tea on herb yield may be attributed to the micronutrient content and to the action of living micro-organisms and microbial metabolites which stimulate plant growth (diver, 2002; carpenter, 2005). higher fresh and dry weights have also been attributed to the availability of macronutrients, especially nitrogen, and/or to the improvement of soil water-holding capacity (el-sherbeny et al., 2005). furthermore, it has been stated that organic manure activates many species of living organisms which release phytohormones and may stimulate plant growth and absorption of nutrients (naguib and aziz, 2003). the same increased vegetative growth characters (including shoot fresh and dry weights) of basil plants under organic farming has been previously reported (khalid et al., 2006). chemical parameters leaf chlorophyll content. the highest leaf chlorophyll content was observed at 1:5 cmt which was not different from 1:10 cmt dilution. other treatments did not show significant differences for this parameter (table 3). the promoting effect of highly n-containing chicken manure tea on chlo rophyll contents might be attributed to the fact that n is a constituent of chlorophyll molecule. moreover, nitrogen is the main constituent of all amino acids and lipids that act as structural compounds of the chloroplast (al-tarwneh, 2005). in sweet basil, chlorophyll content was significantly higher when organic manure compost was applied than in non-fertilized control plants (taie et al., 2010). essential oil content. the essential oil content of lemon basil was affected by dilution levels of organic compost teas. the 1:10 dilution of vct and cmt was higher in essential oil content compared to 1:5 dilutions (fig. 1). the highest essential oil content was found in the 1:10 vct treatment and it was about 3.12 times higher than in control plants. previously, the highest essential oil content in ocimum basilicum was obtained following soil application of vermicompost at 10 t·ha-1 level compared to application of 10 t·ha-1 farmyard manure and control treatments (anwar et al., 2005). in marjoram plants, aqueous extract of compost increased essential oil percentage and yield (gharib et al., 2008). in basil as a source of essential oils and aroma compounds (simon et al., 1990), the increase in essential oil yield has been attributed to increase in vegetative growth or changes in leaf oil gland population (gharib et al., 2008). our data are in agreement with a previous work that found a higher essential oil percentage in batable 3 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) on plant height, number of leaves, lateral branches and flowers, shoot fresh and dry weight and leaf chlorophyll content of lemon basil plants treatment plant height (cm) no. of leaves no. of lateral branches no. of flowers shoot fresh weight (g) shoot dry weight (g) chlorophyll (mg·g-1 fw) control 27.30±0.89 130.03±12.15 6.80±0.84 6.57±0.90 8.10±1.01 1.45±0.18 0.71±0.016 cmt (1:5) 30.47±1.27 225.93±22.80 10.23±0.92 11.80±1.62 17.63±2.44 2.64±0.36 0.82±0.012 cmt (1:10) 29.23±1.09 197.30±22.50 8.07±0.85 10.27±1.48 14.27±2.41 2.51±0.48 0.80±0.013 vmt (1:5) 27.57±0.64 136.70±13.58 6.77±0.93 10.47±1.80 8.37±0.92 1.60±0.18 0.74±0.016 vmt (1:10) 27.30±1.01 131.13±12.88 5.20±0.72 6.84±0.91 7.87±1.07 1.53±0.17 0.74±0.015 lsd value (p=0.05) 2.65 47.11 2.34 3.85 4.58 0.79 0.039 data (per plant) are mean values of 3 replicates ± standard errors. 155 sil due to application of organic fertilizers as compared to control plants (taie et al., 2010). fig. 1 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on essential oil percent of lemon basil. vertical bars show standard errors of the means (n=3). total phenolic content. application of vct at 1:5 dilution gave the highest total phenolic content. the differences between cmt and 1:10 dilution of vct were not significant (fig. 2). previously, sousa et al. (2005) and taie et al. (2010) reported that total phenolic contents achieved by organic culture were higher than those from conventional practice in tronchuda cabbage (brassica oleracea l. var. costata dc) and sweet basil. asami et al. (2003) and wang and lin (2002) also observed consistently higher levels of total phenolics in organically-grown crops compared with those produced by conventional agricultural practices. the amount of total phenolic content of lemon basil in this experiment is in the range of previously reported total phenolic content of different sweet basil accessions (javanmardi et al., 2003). plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators (lukmanul-hakkim et al., 2008). total flavonoids. the highest total flavonoid content was observed in 1:5 cmt-treated lemon basil plants and it was over 1.4 times higher than that produced in control plants. the differences between the two dilutions of vct were not significant (fig. 3). in previous studies, the amounts of surface flavonoids in ocimum x citriodorum specimens were reported in a range of 0.2 to 5.7 mg g-1 (grayer et al., 2004). our finding is in agreement with a previous work that reported a significant increase in flavonoid content of ocimum basilicum due to compost or compost tea application in comparison with control plants (khalid et al., 2006). it has been stated that the antioxidant activity in basil is largely due to the presence of phenolic components, including flavonoids and phenylpropanoids (juliani and simon, 2002). fig. 3 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total flavonoids of lemon basil. vertical bars show standard errors of the means (n=3). total antioxidant activity. the antioxidant activity of lemon basil extracts is shown in figure 4. the total antioxidant activity (taa) ranged from 48.28% (control) to 58.42% (vct at 1:5 dilution). there were no statistically significant differences between cmt dilutions and 1:10 dilution of vct. pearson correlation analysis between secondary metabolites (total phenolics, total flavonoids and essential oils) fig. 4 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total antioxidant activity of lemon basil. vertical bars show standard errors of the means (n=3). fig. 2 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total phenolic content of lemon basil. vertical bars show standard errors of the means (n=3). 156 with total antioxidant activity showed a significant positive correlation coefficient of r2=0.91 between the amount of total phenolics and total antioxidant activity (table 4). this means that about 91% of total antioxidant activity in lemon basil plants was due to phenolic compounds. other secondary metabolites did not show significant contributions to the antioxidant activity. the same correlation was previously reported in sweet basil (ocimum basilicum l.) accessions (javanmardi et al., 2003). the antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agent, hydrogen donors, single oxygen quenchers and having possible metal chelating activity (rice-evans et al., 1995). table 4 pearson’s correlation coefficients between total phenolics, total antioxidant activity, total flavonoids and essential oil percent of treated lemon basil plants total phenolics total flavonoids essential oil content total antioxidant activity 0.919 0.071 ns 0.320 ns data (per plant) are mean values of 3 replicates ± standard errors. 4. conclusions the findings of this study indicate that fertilization with chicken manure and vermicompost organic teas improves herbal and essential oil yields as well as antioxidative agents such as total phenolics in lemon basil plants. the results point to the beneficial effects of compost tea as possible nutrition sources on growth characteristics and essential oil yields of basil. organic nutrition systems have environmental advantages such as a beneficial impact on soil properties and the production of safe plants. 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(2000). influence of arbuscular mycorrhizal fungi on physiology and fruit quality of pepino (solanum muricatum ait.) in vermicompost amended medium j. javanmardi* (1), m. zarei**, m. saei* * department of horticulture, college of agriculture, shiraz university, shiraz, iran. ** department of soil sciences, college of agriculture, shiraz university, shiraz, iran. key words: fruit quality characteristics, mycorrhizal symbiosis, organic fertilizer, pepino. abstract: the association level of pepino (solanum muricatum ait.) with two arbuscular mycorrhizal fungi (amf) species (glomus etunicatum and g. versiforme) was evaluated for the first time. the first part of experiment showed 30 and 50% root colonization for the two amf species, respectively, while the second part of study was a pot experiment under greenhouse conditions. the effects of vermicompost and root inoculation with g. etunicatum and g. versiforme on reproductive stage, yield and fruit quality of pepino were investigated. treatments included two levels of vermicompost (0 and 20% v/v) and inoculation with the two fungi species along with a non inoculated control. application of vermicompost increased the number of flowers, fruits and fruit weight, but decreased the number of days from plant setting to first flower and fruit set, fruit dry matter percent, fruit titratable acidity and vitamin c content. inoculation with g. versiforme increased fruit dry matter percent, fruit titratable acidity and fruit vitamin c content compared with the non inoculated control (nic) plants. plants inoculated with g. etunicatum showed greater fruit weight and juice ph compared to nic plants. amf inoculation in vermicompost amended pots led to 14 and 10 days earlier flowering for g. versiforme and g. etunicatum, respectively compared to those not amended with vermicompost. g. etunicatum in vermicompost supplemented medium hastened fruit set by 5.5 days compared to those without vermicompost application. fruit quality characteristics were affected differently for the two amf-inoculated plants in presence of vermicompost. adv. hort. sci., 2014 28(1): 35-42 (1) corresponding author: javanm@shirazu.ac.ir received for publication 12 april 2014 accepted for publication 30 may 2014 36 amf inocula amf spores were obtained from the department of soil science, faculty of agriculture, shiraz university, iran. glomus versiforme was isolated from a non-contaminated area of anguran mine, zanjan, iran (zarei et al., 2008) and g. etunicatum was provided from tabriz university, iran; these fungi are abundant in iranian soils (aliasgharzadeh et al., 2001; kariman et al., 2005). mycorrhizal inocula were prepared through the trap culture of maize (zea mays l.) on culture medium composed of autoclaved soil/quartz-sand (<1 mm) (1:4, v/v). simultaneously, some pots containing non autoclaved soil were kept without any spore inoculation to preserve the naturally-occurring microbial association and used for non-inoculated control (nic) treatments. after four and a half months, at the beginning of the maize reproductive period, shoots were removed and the contents of pots (mycorrhizal roots plus soil possessing fungal spores and mycelia) were maintained in polyethylene bags at 4°c. the potential of inoculants (spore numbers of 10-12 g-1 substrate and root colonization of 80-85%) for spore extraction, number, and evaluation of root colonization were measured based on the method described by zarei et al. (2008). examination of arbuscular mycorrhizal symbiosis the association level of pepino plant with two amf species along with a nic was evaluated in a completely randomized design with three replications. the presence of am propagules and the percentage of root colonization was determined after eight weeks of inoculation. the grid-line intersect method was used after cleaning washed roots in 10% koh and staining with 0.01% fuchsin acid in lactoglycerol according to the method described by kormanik and mcgraw (1982). the amf species colonizing roots of pepino plants were used for the main experiment. main experiment the experiment was carried out in a polyethylene greenhouse using 10 l pots. the experimental design was 2×3 factorial with four replications (four plants each) in a completely randomized arrangement. the first factor consisted of control (v 0 : no vermicompost) and v 1 : vermicompost application at 20% (v/v) to soil. the second factor was soil inoculation with g. etunicatum and g. versiforme. non-inoculated pots were considered as control (nic). four-week-old rooted cuttings were planted in pots which contained the described media for treatments. the physical and chemical properties of soil and vermicompost are presented in table 1. for the mycorrhizal treatments 50 g of each amf inoculum was used as a thin layer near the roots of cuttings. nic treatments consisted of adding 50 g of media from control maize trap culture pots (contained nonautoclaved soil with no spore inoculation) as described earlier (see section 2.1). pots containing pepino plants were arranged in rows, 1 m apart and 0.4 m between pots. plants were trained into three main branches. a total amount of 35 g·m-2 of organic soluble fertilizer biomin464-sp (jh biotech, inc. ventura, ca) was applied to fertigated plants during the experimental period. irrigation frequency from transplanting to harvest varied from 2 to 4 days with 0.4 1 l per irrigation based on plant requirements. plant reproductive phase measurements to evaluate the effects of colonization with amf and vermicompost application on the pepino reproductive stage, the following characteristics were assayed: number of days to first flower formation, number of flowers in the first and second truss, number of days from transplanting to first fruit formation, fruit number and fruit set percentage, and fruit fresh weight. fruit quality factors including vitamin c content at maturity [using the method described by association of official agricultural chemists (aoac, 1984)], titratable acidity (gutiérrez-miceli et al., 2007), fruit juice acidity (using ph meter), soluble solid content (using refractometer), and dry matter percent were assayed after harvest. statistical analyses the experiment was arranged in a completely randomized design. four replicates per treatment were used, each with four plants. data were analyzed using jmp statistical software, version 5.1 (sas institute inc., cary, nc, usa). if the interaction was significant, it was used to explain results; if it was not significant, means were separated with least significant differences (lsd) test at p≤0.05. 3. results and discussions the anova revealed significant main and interaction effects of vermicompost and amf for most measured characteristics (table 2). determination of plant association with amf for the first time in literature, our results report the association of pepino with two amf. the results indicate that g. etunicatum and g. versiforme can colonize pepino table 1 physical and chemical properties of soil and vermicompost used for the experiment sand (%) silt (%) clay (%) ec (ds/m) ph n (%) p k soil 34 46 20 1.63 7.82 0.031 5.4 mg/kg 135 mg/kg vermicompost 5 8.25 1.45 1.75% 1.2% 37 roots up to 30 and 50 percent, respectively. it has been stated that the difference between root colonization percentages of glomus strains might be due to the fact that amf have a wide host range, yet certain combinations of hosts and fungi are more efficient than others for either the fungus or the host (douds and millner, 1999; gutierrezmiceli et al., 2008). van der heijden et al. (1998) showed that plant species differed in their dependency on amf. some results suggest that amf has some degree of hostspecificity (eom et al., 2000). root colonization the main and interaction effects of vermicompost and amf on root colonization percent were significant (table 2). root colonization percentages in vermicompost amended soils were about 25% greater than the v 0 treatment regardless of amf (table 3). the greater percentage of mycorrhization in vermicompost amended soils has been attributed to the humic substances found in vermicompost, resulting in an increased metabolism of soil microorganisms, and the nutrient uptake (atiyeh et al., 2002). there was no significant difference between colonized root length of g. versiforme and g. etunicatum in vermicompost amended soils, but compared to nic plants, a greater root colonization was observed (2.46 and 2.80 times, respectively) (table 4). mycorrhizal plants colonized well with introduced amf species. a 15% mean root colonization in nic plants shows that the soil used contained native amf populations. more than 100% greater root colonization was observed in the presence of vermicompost when compared with v 0 treatment (table 5). this could be due to greater organic matter available for growth and development of amf hyphae; it has been reported that amf mycelia can mineralize and enhance utilization of organic materials (feng et al., 2003). days to first flower formation the main and interaction effects of vermicompost and amf on the number of days to first flower formation were significant (table 2). the number of days from planting to first flower formation was much lower in pots amended with vermicompost than in v 0 treatments (table 3). previous studies showing earlier flowering due to vermicompost application on german chamomile, begonia, and coleus (tomati et al., 1983; tomati et al., 1987; azizi et al., 2008) are in agreement with our results. the reason for this has been attributed to the development of efficient photosynthetic structure, higher dry matter production, early initiation, and greater development of the reproductive system (krishna et al., 2008). inoculation with g. etunicatum gave earlier flowering than g. versiforme-inoculated plants (table 4). in agreement with our results, chrysanthemum cuttings inoculated with amf had a significantly shorter flowering time compared with non-inoculated plants (sohn et al., 2003). it is reported that in amf-inoculated tomato plants, the time between emergence and completion of fruit set (the duration of purely vegetative growth) decreased, while the duration of the reproduction period increased (bryla and koide, 1998). this is consistent with the idea that plant resource status serves as a partial control of the switch table 2 analyses of variance for vermicompost application and arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics source of variation df mean squares root colonization days to first flower flower number in truss days from flowering to fruit set fruit number fruit fresh weight fruit dry matter percent fruit juice ph fruit titratable acidity total soluble solids fruit vitamin c content vermicompost 1 67** 1056.25** 28.44** 42.25** 27.39** 2321.47** 14.06** 0.06 ns 0.15** 0.13 ns 264.23** amf 2 26** 44.62** 0.16 ns 45.42** 4.64 ns 513.87* 90.97** 0.08** 0.21** 2.98 ns 165.20** vermicompost × amf 2 12* 32.92 ** 0.75 ns 28.15** 4.99 ns 18.16 ns 28.72** 0.11** 0.12** 5.92* 147.53** error 30 0.004 5.08 0.87 1.58 3.46 130.21 0.58 0.02 0.01 1.00 1.56 total 35 ns, *, *= non-significant, significant at 0.05 and 0.01, respectively. table 3 the main effects of vermicompost application on some pepino plant characteristics treatment root colonization percent days to first flower flower number in truss days from flowering to fruit set fruit number fruit weight (g) fruit dry matter percent fruit titratable acidity (ml/100 ml) fruit vitamin c (mg/100 ml) control 18 b 74.11 a 7.36 b 16.14 a 1.81 b 29.69 b 11.10 a 0.78 a 19.83 a vermicompost added 45 a 63.28 b 9.14 a 13.97 b 3.56 a 45.75 a 9.85 b 0.65 b 14.41 b lsd value at p≤0.05 0.04 1.53 0.63 0.70 1.07 7.77 0.52 0.07 0.85 values in each column with the same letter are not significantly different using lsd test at p≤0.05. 38 from vegetative to reproductive growth (marschner, 1995). in mycorrhizal plants, greater root development leads to more phosphorus in vegetative and reproductive tissues, which eventually leads to early flowering (bryla and koide, 1998). this might explain the early flowering in this experiment due to inoculation with g. etunicatum. plants inoculated with g. versiforme and g. etunicatum in vermicompost amended pots had 15 and 10 days earlier flowering, respectively, compared to non vermicompost amended pots (table 5). the organic material provided by vermicompost can improve growth and development of amf inoculum (bending et al., 2004), which enhances nutrient uptake by the plant and hastens plant growth and development (mahmood and rizvi, 2010). flower number in truss a 24% increase in the number of flowers per truss was observed in vermicompost amended pots compared with v 0 treatments, however the effects of amf and coapplication of amf and vermicompost were not significant (tables 2, 3). previously, a 40% increase in flower number in strawberry was related to the increase in plant biological activity due to a vermicompost application rate of over 10 t/ha (arancon et al., 2004 b). a 20% (v/v) vermicompost application in the present study led to a very significant increase in flower number in trusses (table 2). some possible factors that improve flowering after vermicompost application have been attributed to the improvement in physical structure of growth medium, increased biological enzymatic activities, increased populations of beneficial microorganisms, or the presence of biologically active plant growth-influencing substances (plant growth regulators) in the vermicompost (arancon et al., 2008). our results on increased flower number due to vermicompost are in agreement with previous reports on eggplant and tomato (gajalakshmi and abbasi, 2002). days to first fruit set the main and interaction effects of vermicompost and amf on the number of days from flowering to first fruit set were significant (table 2). vermicompost amended pots showed fruit set occurring an average of 2.17 days earlier compared with v 0 treatments (table 3). non-inoculated control plants and plants inoculated with g. versiforme showed fruit set to be three days earlier than in plants inoculated with g. etunicatum (table 4). the two amf species showed different interactions with regard to the presence of vermicompost in the medium. those inoculated with g. etunicatum in the presence of vermicompost set fruit 5.58 days earlier compared to non vermicompost amended soil (table 5). it seems that the potential efficiency of g. etunicatum for earlier pepino fruit set is greater in the presence of vermicompost than with g. versiforme. nic plants and plants inoculated with g. versiforme with vermicompost added and non vermicompost added media showed no differences. fruit number our results show that vermicompost application was the primary contributing factor in increasing pepino fruit table 4 the main effects of arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics treatment root colonization percent days to first flower days from flowering to fruit set fruit weight (g) fruit dry matter percent fruit juice ph fruit titratable acidity (ml/100 ml) fruit vitamin c (mg/100 ml) nic 15 b 68.08 ab 13.75 b 32.64 b 8.79 b 5.02 b 0.67 b 16.54 b glomus versiforme 37 a 70.85 a 14.12 b 35.41 b 13.65 a 4.99 b 0.87 a 21.08 a glomus etunicatum 42 a 67.15 b 17.29 a 45.10 a 8.98 b 5.15 a 0.62 b 13.73 c lsd value at p≤0.05 5.01 1.88 0.86 9.51 0.63 0.12 0.08 1.04 nic= non inoculated control. values in each column with the same letter are not significantly different using lsd test at p≤0.05. table 5 the interaction effects of vermicompost application and arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics root colonization percent days to first flower days from flowering to fruit set fruit dry matter percent fruit juice ph fruit titratable acidity (ml/100 ml) total soluble solids (brix) vitamin c (mg/100 ml) no-vermicompost added non mycorrhizal control 5 c 72.17 b 13.58 b 8.06 d 5.09 ab 0.68 b 7.13 c 22.39 b glomus versiforme 22 b 78.12 a 14.75 b 15.96 a 4.88 c 1.04 a 9.38 a 24.42 a glomus etunicatum 27 b 72.04 b 20.08 a 9.28 c 5.06 ab 0.61 b 7.68 bc 12.66 e vermicompost added non mycorrhizal control 25 b 64.00 c 13.92 b 9.53 c 4.95 bc 0.65 b 8.15 bc 10.68 f glomus versiforme 53 a 63.58 c 13.50 b 11.35 b 5.10 ab 0.69 b 7.88 bc 17.74 c glomus etunicatum 58 a 62.25 c 14.50 b 8.68 cd 5.23 a 0.61 b 8.52 ab 14.80 d lsd value at p≤0.05 7.0 2.66 1.21 0.90 0.17 0.12 1.18 1.47 values in each column with the same letter are not significantly different using lsd test at p≤0.05 39 number (table 2). fruit number in vermicompost amended soils (an average of 3.56) was about 96% greater than v 0 treatments (1.81 fruits) (table 3). previously, increased yields of strawberry (arancon et al., 2004 b) and pepper (arancon et al., 2005) in vermicompost amended soils in field conditions were attributed to increased fruit number due to the availability of plant growth regulators and humic acids, which produced by the greatly increased microbial populations resulting from earthworm activity (arancon et al., 2004 a; b). according to our results, the simultaneous increased flower number and fruit set percentage considerably increased total yield. fruit fresh weight the main effects of vermicompost and amf on fruit fresh weight showed significant differences, but the interaction of vermicompost and amf was not significant (table 2). comparing fruit weight in vermicompost amended soils with v 0 treatments showed a 54% increase (table 3). this result is in agreement with previous studies on the application of vermicompost for eggplant (moraditochaee et al., 2011), greenhouse pepper (arancon et al., 2004 a), and tomato (arancon et al., 2003). it has been stated that the great microbial activity and populations in vermicompost are probably responsible for a considerable buildup of microbial populations and activity in soils. these improve the soil structure and have an indirect influence on root environment, nutrient absorption, plant growth (arancon et al., 2005), and yield (goswami et al., 2001). no significant differences were found between plants inoculated with g. versiforme and nic treatments. plants inoculated with g. etunicatum produced fruits with an average weight of 45.1 g, which was about 32% greater than nic and g. versiforme-treated plants (table 4). it has been reported that individual tomato fruit weight significantly increased when the plants were colonized with amf (bryla and koide, 1998). such evidence, which is in agreement with our results, also showed different increased levels of fruit fresh weight for other inoculated solanaceous plants, i.e. tomato plants inoculated with g. mosseae (abdel latef and chaoxing, 2010), chili pepper plants inoculated with g. intraradices (castillo et al., 2009), chileancho pepper inoculated with g. fasciculatum (mena-violante et al., 2006), and non-solanaceous cucumber (trimble and knowles, 1995). increased yields have been attributed to the increased yield components due to the positive effects of mycorrhiza including facilitated water and nutrient uptake through extension of root surfaces and increased photosynthesis (ortas et al., 1996; raman and mahadevan, 1996; tarkalson et al., 1998). fruit dry matter percent the main and interaction effects of vermicompost and amf on fruit dry matter percent showed significant differences (table 2). the v 0 treatments produced 12% greater fruit dry matter than pots amended with vermicompost, regardless of amf inoculation (table 3). results of fruit fresh weight and fruit dry matter percentage showed that vermicompost improved water uptake and partitioning in fruits, which had greater fresh weight (due to vermicompost) and lower dry matter percentages (table 3). inoculating pepino plants with g. versiforme increased the fruit dry matter percent to over 50% compared to those inoculated with g. etunicatum and plants not inoculated with amf (table 4). the differences could be related to the developmental pattern of amf species. an increased fruit dry matter percentage in amf plants has been attributed to improved water and nutrient uptake/translocation, higher photosynthesis (vamerali et al., 2003), and also to the pattern of dry matter distribution in inoculated plants, which pointed to a role of amf in carbon partitioning (mena-violante et al., 2006). co-application of vermicompost and amf showed different patterns for fruit dry matter percentage. g. versiforme in v 0 treatment produced about 40% greater fruit dry matter than vermicompost amended media, but no differences were observed between vermicompost and non vermicompost amended soils inoculated with g. etunicatum (table 5). fruit juice ph with regard to fruit juice ph, the main effects of amf and its interaction with vermicompost were highly significant (table 2). the ph of pepino plants inoculated with g. etunicatum was 0.13 higher than in nic plants. the difference between g. versiforme inoculated plants and nic plants was not significant for fruit juice ph (table 4). previously, inoculation of cucumber plants with g. intraradices gave no changes in fruit ph (rouphael et al., 2010). plants inoculated with g. etunicatum exhibited a higher fruit juice ph than g. versiforme inoculated plants in v 0 treatment, but the difference was not significant when vermicompost was used (table 5). fruit titratable acidity the main and interaction effects of vermicompost and inoculation with amf showed significant differences with nic plants for fruit juice titratable acidity (table 2). the plants grown in vermicompost amended soils (0.65 ml/100 ml fruit juice) had a 20% decrease in titratable acidity compared with plants in non-amended soils (0.78 ml/100 ml fruit juice) (table 3). in other studies, the effect of vermicompost on tomato fruit titratable acidity did not show significant differences (gutiérrez-miceli et al., 2007). the effect of inoculation of plants with g. versiforme showed a 40% increase in fruit titratable acidity compared with g. etunicatum inoculated plants and the control (table 4). our result is in agreement with a previous paper that reports a significant increase in fruit titratable acidity of amf inoculated tomato plants (regvar et al., 2003). a different reaction to vermicompost application was observed for inoculated plants with two different amfs (table 5). fruits of plants inoculated with g. versiforme in the v 0 treatment had a 0.5ml/100ml greater titratable acidity than fruits from g. etunicatum-inoculated plants. 40 it seems that the effect of amf on pepino fruit juice titratable acidity is amf-species dependent. total soluble solids the main effects of vermicompost and amf on total soluble solids were not significant but the interaction was significant (table 2). fruits of g. versiforme-inoculated plants in the v 0 treatment had 1.5 brix greater soluble solid content than those grown in vermicompost amended media. the differences between amf-inoculated and nic plants in vermicompost amended treatments were not significant (table 5). greater fruit total soluble solids in amf inoculated tomato plants, compared to non-inoculated plants, has been previously reported (subramanian et al., 2006). different microorganisms have been reported as involved in breaking down (mineralize) and releasing mineral nutrients of organic materials, to then be taken up by plant roots (linderman and davis, 2004). it seems that g. etunicatum has the ability to improve vermicompost utilization by pepino plant roots, which leads to more efficient photosynthetic activity and therefore greater total soluble solids in fruits. fruit vitamin c the main and interaction effects of vermicompost and inoculation with amf showed significant differences for fruit vitamin c content (table 2). fruits produced in vermicompost amended soils had 37% less vitamin c than fruits in the v 0 treatments (table 3). this could be related to a 1.5 times greater fresh fruit weight with constant vitamin c content in a unit volume. it seems that the level of vitamin c in pepino fruits is not affected by vermicompost application. different reports are available on the effect of vermicompost application on fruit ascorbic acid content: some show increased fruit vitamin c in tomato (sable et al., 2007), while others report no significant effect (roberts et al., 2007). the fruit vitamin c content was 53 and 27% greater in fruits from plants inoculated with g. versiforme as compared to fruits from plants inoculated with g. etunicatum and non mycorrhizal treatments, respectively (table 3). higher quantities of ascorbic acid in amf-inoculated tomato plants compared to non-inoculated plants has been previously reported (subramanian et al., 2006). the amf used in this experiment showed different reactions to vermicompost application. fruits from the v 0 treatment inoculated with g. versiforme and those inoculated with g. etunicatum in vermicompost amended soils had greater vitamin c than other treatments (table 5). it seems that the effect of amf on pepino fruit vitamin c is species dependent. those plants treated with g. etunicatum had higher vitamin c content when vermicompost was applied, while the same trend was not observed when vermicompost was applied to g. versiforme-inoculated plants. 4. conclusions despite the influence of amf on crop yield as documented in many reports on solanaceous plants, little is known about 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lead mining region. environ. pollut., 156: 1277-1283. 41 1. introduction it is widely known that traditional japanese gardening requires moss species as key botanical components for covering natural rocks, wooden and stone walls, tree trunks and sidewalk surfaces. the use of mosses is frequently described in the world of japanese literatures, chiefly in haiku, as symbolized by a mythical conversation between legendary haiku poet basho and zen master buccho in which they discuss the buddhist philosophy on growing green mosses (suzuki, 1975). in typical japanese gardens, natural rocks partially covered by a layer of thick moss and ponds surrounded by mossy stones represent miniature-sized rocky mountains with forestry slopes and lakes located deep in the forest. these approaches for miniaturizing natural landscapes are well in line with the buddhist-affected and shintoist-mixed nature-respecting philosophical preferences in japan which encourage people to be surrounded by pieces of nature. in japan, second to mosses, many pteridophytes (ferns and fern allies) have also been traditionally used in japanese garden designing. interestingly, some ground-covering ferns such as members of selaginellaceae have been considered as mosses by gardening practitioners lacking taxonomical background, largely due to their moss-like appearance (fig. 1). in place of mossy mats, two selagipteridophytes as active components in gardening, agricultural and horticultural ecosystems in japan t. kawano (*) laboratory of chemical biology and bioengineering, faculty and graduate school of environmental engineering, the university of kitakyushu, kitakyushu, japan. université paris diderot, sorbonne paris cité, paris 7 interdisciplinary energy research institute (pieri), paris, france. key words: environment, fern, gardening, insects, japan, mycorrhizae. abstract: many members of pteridophytes have been traditionally used in the designs of japanese gardens and a large variety of ferns attract gardeners as greening pieces in the garden designs. however, details or examples of practical uses of ferns in traditional gardening in japan have only rarely been introduced in non-japanese literatures to date, despite the importance of ferns in japanese gardening traditions. in addition to the discussion of ferns in gardening, the use and association of these plants in japanese agricultural and horticultural sceneries are addressed. the presence and importance of 40 familiar fern species in local gardening, agricultural and horticultural ecosystems are also discussed, as well as the roles of introduced ferns as key elements of ecosystems and their interaction with neighboring biota. finally, some examples of uses of fern species in environmental science and engineering are also reviewed. adv. hort. sci., 2015 29(1): 41-47 (*) corresponding author: kawanotom@kitakyu-u.ac.jp received for publication 22 september 2014 accepted for publication 24 march 2015 mini-review fig. 1 living selaginella remotifolia found in a private garden (kiyotake-cho, miyazaki, japan). (a) sheet of growing selaginella remotifolia covering the ground. (b) blue-green colored leaflets. 42 adv. hort. sci., 2015 29(1): 41-47 nellaceae members, such as selaginella remotifolia spring (japanese name: kuramagoke) and selaginella unicinata (desv.) spring (japanese name: konterikuramagoke), can be installed in japanese gardens. japanese names ending with -goke or -koke are conventionally given to moss species, despite the fact that these species are taxonomical members of ferns. furthermore, a large variety of ferns (including standing types) also attract the gardeners as greening pieces in gardening designs. not only in japan but also in other east asian regions such as taiwan and korea, some fern species such as lycopodium fordii bak. (lycopodiaceae) are grown as gardening plants and reflect the local flora (huang et al., 2000). in addition to the lycopsida members mentioned above (eg. lycopodium among subclass aglossopsidae and selaginella among subclass glossopsidae, within the class lycopsida), psilotum nudum (l.) p. beauv. (psilotaceae; japanese name: matsubaran) belonging to the class psilotopsida has long been utilized in japanese gardening landscapes. recently it was suggested that this plant communicates with neighboring plants through a arbuscular mycorrhizal network (winther and friedman, 2009). accordingly, psilotum nudum, with its non-photosynthetic life cycle, can thrive underground mostly by depending on the transport of photosynthetically fixed carbon from the green plants growing nearby. in the gardens where this plant has been introduced, the micro-ecosystem which allows such ecophysiological interspecies communication should be developed. the above fern species have conventionally been considered as members of the phylum microphyllophyta. as might be expected, other pteridophytes belonging to two other conventional phyla, namely spherophyta and pterophyta, are also commonly found in japanese gardening and agricultural landscapes. despite the importance of pteridophytes in japanese gardening traditions, details or examples of practical uses of ferns have only rarely been introduced in non-japanese literatures to date. as most ferns favor a humid subtropical climate, this article aims to briefly describe and list the pteridophytes found in the local gardening and agricultural landscapes in the southwestern half of japan. 2. gardening and ferns historically, the inclusion of ferns in gardens has been popular among people of various classes such as farmers, warriors, monks and aristocrats, thus ferns were often found in the gardens and backyards of imperial and aristocratic palaces, shintoist shrines, buddhist temples, and ordinary houses. today, it has become much harder to find fern culturing in ordinary houses in the large cities in japan, yet in rural areas surrounded by semi-wild forests and far from urban areas, the use of ferns can still be found readily. in traditional japanese gardens, the following fern species can be found. matteuccia struthiopteris (l.) todaro (athyriaceae; japanese name: kusasotetsu also known as kogomi in the northern japan) often welcomes the visitor to the gardens. cyrtomium falcatum (l. fil.) presl. (dryopteridaceae; japanese name: oniyabusotetsu) and equisetum hyemale l. (equisetaceae; japanese name: tokusa) are likely planted surrounding ponds and/or waterfalls. beneath the bushes, polystichum polyblepharum (roem. ex kunze) presl. (dryopteridaceae; japanese name: inode) can be frequently found. equisetum hyemale and dryopteris erythrosora (eaton) o. ktze (dryopteridaceae; japanese name: benishida) can be found in the shade of structures such as a chashitsu (a tea-ceremony house). gaps among the rocks or stones can be naturally filled by cyrtomium falcatum and pteris multifida poir (pteridaceae; japanese name: inomotosō). pteris multifida is a specific host plant that supports the life of caterpillars of a domestic moth of the noctuidae family, callopistria japonibia (fig. 2). inoue and sugi (1958) suggested that geographical distribution (size of habitat) of callopistria japonibia largely depends on the distribution of this fern. this could be a model for studying the contribution of garden environments to harbor specific ferns for insect biodiversity. moreover, some fern species have been passively but very frequently installed in japanese gardens, thus their presence strongly reflects the nature of original wild flora. such species include pteris cretica l. (pteridaceae; japanese name: ōbanoinomotosō), osumunda japonica thunb., botrychium japonicum (prantl) underw. (ophioglossaceae; japanese name: fuyunohanawarabi), cyrtomium falcatum, athyrium niponicum (mett.) hance (athyriaceae; japanese name: inuwarabi), deparia japonica (thunberg) m. kato (woodsiaceae; japanese name: shikeshida) (fig. 3a, d) and pteridium aqualinum (l.) kuhn var. latiusculum (desv.) underw. ex hell (dennstaedtiaceae; japanese name: warabi) (fig. 3e, f). fig. 2 pteris multifida growing on the stone wall in a private garden (kiyotake-cho, miyazaki, japan). (a) top view of growing plants. (b) spore-bearing adaxial side of the leaves. (c, d) plants being fed on by caterpillars of callopistria japonibia. arrows indicate the presence of caterpillars. 43 kawano, pteridophytes in gardening, agricultural and horticultural ecosystems in japan among passively introduced ferns, members of polypodiaceae such as lemmaphyllum microphyllum pres (japanese name: mamezuta), lepisorus thunbergianus (kaulf.) ching (japanese name: nokishinobu), and pyrrosia lingua (thunb.) farw (japanese name: hitotsuba) are epiphytic species which are often attached to trees and rocks (fig. 4). on the surface of rocks and walls, lemmaphyllum microphyllum is often exposed to competition with higher epiphytic plants such as ficus pumila l. (moraceae; japanese name: ōitabi). in addition to epiphytic plants, a climbing species, lygodium japonicum (thunb.) sw. (lygodiaceae; japanese name: kanikusa) is of great interest. historically, suntracking movements associated with a the climbing growth habit were described by charles darwin (1875) in two members of lygodium (l. articulatum and l. scandens). darwin concluded that “as ferns differ so much in structure from phanerogamic plants, it may be worthwhile here to show that twining ferns do not differ in their habits from other twining plants.” the climbing fern native to japan, lygodium japonicum, is commonly known as “japanese climbing fern”. this fern grows very fast and thus often covers nearby living trees, rocks and walls (fig. 5). lygodium japonicum has been exported for ornamental purposes. for instance, this plant was introduced in florida, usa, in 1932 (gordon and thomas, 1997). 3. ferns and japanese people even before ferns were introduced into man-made gardens, people in japan traditionally enjoyed going out fig. 3 deparia japonica and pteridium aqualinum sampled in a private garden (kiyotake-cho, miyazaki, japan). (a) adaxial side (top side) and (b) abaxial side (bottom side) of mature and young bladed of deparia japonica. (c) mature and (d) immature sporangia on leaflets of deparia japonica. (e) living pteridium aqualinum. (f) roots and young emerging frond of pteridium aqualinum. fig. 4 epiphytic ferns found in a countryside private garden (kiyotakecho, miyazaki, japan). (a) lepisorus thunbergianus grown on the trunk of a loquat tree (eriobotrya japonica (thunb.) lindl.). (bd) lemmaphyllum microphyllum growing on the tree and rocks. fig. 5 a climbing fern, lygodium japonicum, found in a countryside private garden (kiyotake-cho, miyazaki, japan). (a) climbing growth of lygodium japonicum on the wooden wall by competing with other climbing plants such as ficus pumila. (b, c) aggressive growth of lygodium japonicum winning the competition with other standing plants. (d) vegetative leaflets. (e, f) development of sporangia on dorsal side of the leaflets. 44 adv. hort. sci., 2015 29(1): 41-47 to the fields and forests to gather edible young fronds of wild ferns. the relationships between japanese people and ferns, especially bracken ferns, are historically, socially and culturally so tight that recreational fern gathering was hardly weakened even among americans of japanese origin, as is revealed by the various recipes for cooking ferns found among traditional japanese communities in the united states (anderson et al., 2000). edible wild ferns in japan include equisetum arvense l. (equisetaceae; japanese name: sugina), pteridium aqualinum (popularly known as warabi), osumunda japonica thunb. (osmundaceae; japanese name: zenmai), and matteuccia struthiopteris. historically, among the japanese, equisetum arvense has been used as an herbal medicine with diuretic, antitussive, antipyretic, hemostatic, and roundworm-eliminating actions. therefore, this herbal medicine has been given to patients with gonorrhea, cystitis, edema, bronchitis, and bleeding hemorrhoids (ikegami, 2013). furthermore, search for lymphangiogenesis-inhibiting agents in the crude extracts from equisetum arvense was recently attempted (jeong et al., 2013). figure 6 shows classical illustrations of rhizome anatomy of equisetum arvense l. these illustrations remind us that, like higher plants, ferns, including equisetum arvense, finely develop rhizomes under the soil, suggesting that ferns are one of the key players in the soil ecosystem. apart from edible and medicinal species, japanese people are also familiar with some fern species for ornamental purposes and crafts. the mature fronds of gleichenia japonica spr. (gleicheniaceae; japanese name: urajiro) are important for traditional new year’s decorations. in summer, outside doors or windows of houses, one can find hanging fern-bearing, peat moss-based green balls called “shinobudama”, which are usually about 10 cm in diameter, as one of the favored customs in japan. a shinobudama literally means a ball of shinobu which is the japanese common name for davallia mariesii moore ex baker (davalliaceae). this ornamental arrangement of shinobu fern is often combined with a wind chime hung outside. use of equisetum hyemale l. in traditional craft works should be also noted. the silicate-rich stems of equisetum hyemale l. have long been used for grinding and honing the surface of wooden table wares and furniture. 4. agriculturally associated flora of ferns in japan paddy fields for culturing rice plants (oryza sativa l.) are man-made wetlands which cover about 50% of the agricultural land in japan. some fern allies bloom out in the paddy field after the harvest of rice. typical examples of dominating aquatic and semi-aquatic ferns in the postrice paddy include ceratopteris thalictroides (l.) brongn. (adiantaceae; japanese name: mizuwarabi), salvinia natans (l.) all (salviniaceae; japanese name: sanshōmo), azolla japonica fr. et sav. (azollaceae; japanese name: ōakaukikusa), azolla imbricata (roxb.) nakai (azollaceae; japanese name: akaukikusa), and marsilea quadrifolia l. (marsileaceae; japanese name: denjisō). marsilea quadrifolia is most likely to be very sensitive to rice field herbicides and thus this plant species is rarely found nowadays (luo and ikeda, 2007). fig. 6 anatomical illustrations of underground structures of equisetum arvense l. found in a classical book by j. duval-jouve (1864). (a) horizontal rhizomes. (b) vertical rhizomes. (c) section of large tuber attached to rhizome. (d) cells of a tuber with starch granules (left) and vessel (right). (e, f) starch granules. this book was originally collected and preserved through collaborative research between université paris diderot and the university of kitakyushu (kawano and bouteau, 2007; kawano et al., 2008). original images were digitally scanned and slightly modified (illustrations of other plant species were masked). 45 kawano, pteridophytes in gardening, agricultural and horticultural ecosystems in japan 5. breeding for horticultural purposes some pteridophytes have been positively subjected to breeding efforts, especially during the era of tokugawa japan known as the edo period (1603-1868). among the ferns listed in this section, the breeding history for psilotum nudum (known as matsubaran) is estimated to have a long history and thus a number of varieties are available (murata and yashiro, 2006). also some ornamental species with attractive leaf orientation, such as onychium japonicum (l.) (thunb.) kunze (parkeriaceae/adiantaceae; japanese name: tachishinobu) and nephrolepis cordifolia (l.) presl (lomariopsidaceae; japanese name: tamashida) are popularly bred and cultivated for both garden use and for ornamental pots. note that, nowadays, nephrolepis cordifolia often requires protection under a glass house especially during the winter season. some fern species are also favored by gardeners and plant growers and are nowadays bred and available on the market. such ferns considered as gardening plants include selaginella unicinata (fig. 1), selaginella tamariscina (beauv.) spring (selaginellaceae; japanese name: iwahiba), selaginella involvens (sw.) spring (selaginellaceae; japanese name: katahiba), adiantum capillus-veneris l. (adiantaceae; japanese name: hōraishida), sphenomeris chinensis (l.) maxon. (lindsaeaceae; japanese name: horashinobu), pteris nipponica w. c. shieh (pteridaceae; japanese name: matsuzakashida) and asplenium antiquum makino (aspleniaceae; japanese name: ōtaniwatari; this plant often requires green house or similar facilities). selaginella unicinata is believed to have been brought from southern regions in china several centuries ago. adiantum capillus-veneris is naturally found in the western half of japan, but there are still some discussions on the origins of this fern (murata and yashiro, 2006). among adiantum species, adiantum monochlamys eaton (adiantaceae; japanese name: hakoneshida) is hardly cultivated in gardens (murata and yashiro, 2006). among the varieties of pteris nipponica, lines or individuals with white spots on the leaves are likely favored on the market (murata and yashiro, 2006). it should be noted that even today, many efforts to develop and/or introduce new horticultural fern varieties of east asian origin are being made (xu et al., 2006). 6. uses of ferns in environmental science and engineering unlike mosses, pteridophytes possess a highly developed root system (rhisomes) by which minerals and metals are effectively taken up by growing plants. from an environmental point of view, some ferns are receiving more attention than ever, after pioneering works by japanese fern specialists, since some ferns might be useful indicators and/or tools to assess and remediate contaminated soils. most metallic ions present at high concentrations are toxic to living plants by targeting and damaging the root cells exposed to the soil (kawano et al., 2005). athyrium yokoscense (fr. rt sav.) christ (athyriaceae; japanese name: hebinonegoza), indigenous to east and north east asia including japan and korea, is known to thrive in and around the sites of mining by rooting in soils contaminated with high concentrations of heavy metals (such as zn, cd, pb, and cu) and as (morishita and boratynski, 1992; van et al., 2006). therefore, the presence or distribution of this plant strongly indicates the history of the soil use or contaminations (morishita and boratynski, 1992). it has been well documented that athyrium yokoscense not only tolerates but also effectively takes up the contaminating elements from the surrounding environments, as examined both at cellular level (yoshihara et al., 2005) and fieldgrown plant level (morishita and boratynski, 1992; kamachi et al., 2005). similar approaches are now being attempted with regard to other fern species. use of a hyper-accumulator fern for arsenic and metals, pteris vittata l. (pteridaceae; japanese name: moejimashida) is one successful example of fern-based phytoremediation of metal-contaminated and arsenic-poisoned soils (an et al., 2006; xie et al., 2009). interestingly, a recent proteomic approach by bona et al. (2011) suggested that the hyper-accumulating nature of pteris vittata requires the presence of symbiotically formed arbuscular mycorrhizae. furthermore, unpublished results presented by chien et al. (2012) during an oral presentation at the 64th annual meeting of the society for bioscience and bioengineering, japan, suggested a possible relationship between the mechanism of arsenic accumulation by pteris vittata l. and bacterial flora in the rhizosphere. these studies consistently support the view that composition of the micro-flora in the soil inevitably affects the environmental responses performed by rhizome-developed fern species. 7. preservation of ferns and related flora and fauna in the gardens based on statistical techniques, namely two-way indicator species analysis (twinspan) and detrended correspondence analysis (dca), murakami et al. (2003) reported that there is a relationship between micro-landforms and the fern species composition in forest islands. this is the case of surveys on the ecological status of fragmented forests within the urbanized area of kyoto city since fern members are largely considered to form a group of environmental indicators in the urban matrix. the authors concluded that the difference in species composition of ferns was shown to reflect differences in micro-landforms. for example, polystichum polyblepharum and many other species are favored in valley bottoms in the small forest islands while some species such as dryopteris erythrosora and gleichenia japonica are favored on the slopes. japanese gardens can be considered as a local ecosystem that allows semi-wild propagation of fern species. this is largely due to the diversified micro-landforms in 46 adv. hort. sci., 2015 29(1): 41-47 japanese garden designs which allocate rocks, mounds, valleys, falls and ponds within small areas. indeed, japanese gardens can be considered models for botanical display and/or preservation of semi-wild fern species and interacting flora, fauna and microbiota. 8. conclusions in this article, the use and associations of ferns in japanese agricultural and horticultural sceneries are reviewed and discussed. through listing 40 key japanese fern species in gardening, horticulture and environmental studies in japan, the author has shown that these ferns behave as key building blocks for the semi-wild and half man-made local ecosystems in gardens and agricultural fields. the author has also noted that these ferns are highly and actively communicating and competing with other organisms such as weeds, trees and insects, and also cope with environmental stress through rhizomes. human-fern interaction may be one such interspecies communication. acknowledgements this work was supported by a grant of regional innovation strategy support program implemented by ministry of education, culture, sports, science and technology (mext), japan. live fern samples were collected from the private garden of mr. munenori kawano and neighboring roads in kiyotakecho, miyazaki prefecture, japan. for determination of ferns and general description of the structures and floral characteristics of ferns species, two handbooks written in japanese, namely, abe (1996) and murata and yashiro (2006) were consulted. references abe m., 1996 ha niyoru shida no kensaku zukan (text in japanese). seibundo shinko sh, tokyo, japan. an z.-z., huang z.-c., lei m., liao x.-y., zheng y.-m., chen t.-b., 2006 zinc tolerance and accumulation in pteris vittata l. and its potential for phytoremediation of zn and as-contaminated soil. chemosphere, 62(5): 796-802. anderson j.a., blahna d.j., chavez d. j., 2000 fern gathering on the san bernardino national forest: cultural versus commercial values among korean and japanese participants. society and natural resources, 13(8): 747-762. bona e., marsano f., massa n., cattaneo c., cesaro p., argese e., sanità di toppi l., cavaletto m., berta g., 2011 proteomic analysis as a tool for investigating arsenic stress in pteris vittata roots colonized or not by arbuscular mycorrhizal symbiosis. j. proteomics, 74(8): 1338-1350. chien m.-f., miyauchi k., endo g., 2012 study on the bacterial flora in rhizosphere of an arsenic hyper-accumulator plant and its arsenic accumulation mechanism. abstract book. 64th sbj society for bioscience and bioengineering, japanannual meeting, 2h, pp. 17. darwin c., 1875 the movements and habits of climbing plants. 2nd edition. john murray, london, uk. duval-jouve j., 1864 histoire naturelle des equisetum de france. j.b. baillière et fils, paris, france. gordon d.r., thomas k.p., 1997 florida’s invasion by non indigenous plants: history, screening, and regulation, pp. 21-37. in: gordon d.r., and thomas k.p. strangers in paradise, impact and management of non indigenous species in florida. island press, washington dc, usa. huang y.-w., chiang t.-y., chiou w.-l., 2008 isolation and characterization of microsatellite loci of lycopodium fordii bak. (lycopodiaceae, pteridophyta). conservation genetics, 9(3): 775-777. ikegami f., 2013 equiseti herba (horsetail herb). wakanyaku, vol. 6, 719: 5-6. jeong d., watari k., shirouzu t., ono m., koizumi k., saiki i., kim y.c., tanaka c., higuchi r., miyamoto t., 2013 studies on lymphangiogenesis inhibitors from korean and japanese crude drugs. biol. pharm. bull., 36(1): 152-157. kamachi h., komori i., tamura h., sawa y., karahara i., honma y., wada n., kawabata t., matsuda k., ikeno s., noguchi m., inoue h., 2005 lead tolerance and accumulation in the gametophytes of the fern athyrium yokoscense. j. plant res., 118(2): 137-145. kawano n., kawano t., lapeyrie f., 2005 environmental metal cation stress and oxidative burst in plants. a review, pp. 631-643 in: lichtfouse e., schwarzbauer j., and d. robert (eds.) environmental chemistry: green chemistry and pollutants in ecosystems. springer, berlin, heidelberg, germany, pp. 780. kawano t., bouteau f., 2007 our current activities: collection, preservation, classification, digitalization, translation and re-evaluation of the classical science literatures from sorbonne libraries. bull. cent. fran. jpn. hist. sci., 1(1): 3-9. kawano t., yokawa k., hiramatsu t., rona j.-p., bouteau f., 2008 mining and revitalization of classical literatures on botanical science derived from sorbonne libraries through collaboration between université paris diderot and the university of kitakyushu. ciee journal, the university of kitakyushu, 6: 13-21. luo x.-y., ikeda h., 2007 effects of four rice herbicides on the growth of an aquatic fern, marsilea quadrifolia l. weed biol. manag., 7(4): 237-241. morishita t., boratynski j.k., 1992 accumulation of cadmium and other metals in organs of plants growing around metal smelters in japan. soil sci. plant nutr., 38(4): 781-785. murakami k., matsui r., maenaka h., morimoto h., 2003 relationship between the micro-landforms and fern species composition in forest islands in kyoto city. annual meeting of japanese institute of landscape architecture, pp. 100. murata t., yashiro k., 2006 handbook for field watching: ferns. zenkoku noson kyoiku kyokai, japan. 47 kawano, pteridophytes in gardening, agricultural and horticultural ecosystems in japan suzuki d.t., 1975 the essentials of zen buddhism, selected from the writings of daisetz t. suzuki. greenwood press, westport, conn., usa. van t.k., kang y., fukui t., sakurai k., iwasaki k., aikawa y., phuong n. m., 2006 arsenic and heavy metal accumulation by athyrium yokoscense from contaminated soils. soil sci. plant nutr., 52(6): 701-710. winther j.l., friedman w.e., 2009 phylogenetic affinity of arbuscular mycorrhizal symbionts in psilotum nudum. j. plant res., 122(5): 485-496. xie q.-e., yan x.-l., liao x.y., li x., 2009 the arsenic hyperaccumulator fern pteris vittata l. environ. sci. technol., 43(22): 8488-8495. xu b., jin s.-h., ding b-y., 2006 selection for shade-tolerance ferns and horticultural utilization. j. zhejiang univ. agric. life sci., 32(3): 329-333. yoshihara t., tsunokawa k., miyano y., arashima y., hodoshima h., shoji k., shimada h., goto f., 2005 induction of callus from a metal hypertolerant fern, athyrium yokoscense, and evaluation of its cadmium tolerance and accumulation capacity. plant cell rep., 23(8): 579-585. 213 olive is one of the most alternating tree species among the commercially grown fruit trees and it is known as such worldwide. fruiting alternation of olive is considered to be dependent both on environmental abiotic and endogenous biotic genetic factors. the degree of orchard fruiting alternation, even of the same cultivar, differs considerably between different areas and regions, thus an initial or independent genetic involvement is questionable. fruit bearing alternation is particularly recognized in regions with climatic conditions that vary annually, particularly winter temperatures such as in the eastern mediterranean basin (fig. 1). in such regions alternate bearing is usually expressed and synchronized within the entire orchard, area or even region. still, alternate bearing, although to a lesser degree, develops also in regions with a stable annual climate favorable for the olive tree’s developmental cycle. the requirement of low, particularly varying temperatures between day and night in the winter to induce reproductive bud differentiation was established many years ago (hartmann and prolingis, 1957; hartmann and whisler, 1975). the buds of olea europaea develop uniformly as they are of undefined nature and need to be induced to become either vegetative or reproductive (fig. 2). various studies describing the anatomical changes occurring in buds have been published and have mainly emphasized the changes leading to the reproductive state, though in some studies also leaf buds (fabbri and alerci, 1999). it is still controversial whether low temperature is required for the induction leading to differentiation, as during the process of vernalization (lavee, 1989; 1996; troncoso alternate bearing in olive initiated by abiotic induction leading to biotic responses s. lavee (*) institutes of plant sciences, faculty of agriculture, huj, rehovot and volcani center, aro, bet-dagan, israel. key words: alternate bearing, environmental control, fruiting metabolism, olive. abstract: alternate bearing of olive trees is one of the most troublesome characteristics of this commodity, impacting its economy due to labor distribution, fruit and oil availability, oil mill capacity and marketing. the metabolic changes leading to alteration in fruit production are generally considered of direct genetic nature. in the present review this approach is challenged, showing that all the biotic-metabolic changes in olive leading to ‘on’ and ‘off’ years are the results of initial abiotic effects on the trees. the nature of the metabolic changes induced by the abiotic regional and annual conditions described are, no doubt, genetically controlled but initiated only as a result of adverse environmental abiotic conditions such as seasonal temperatures, water stress, and soil nutrition conditions. adv. hort. sci., 2015 29(4): 213-219 (*) corresponding author: shimon.lavee@mail.huji.ac.il received for publication 23 february 2015 accepted for publication 3 september 2015 fig. 1 alternate bearing over a period of six years in rain-fed and irrigated olive orchards (lavee, 2006). fig. 2 differentiation of an undefined olive bud to a vegetative and reproductive state (lavee, 1996). 214 adv. hort. sci., 2015 29(4): 213-219 et al., 2012). on the other hand, it was suggested that low temperature is required mainly for predetermined reproductive bud opening, similar to the dormancy process in deciduous trees (rallo and martin, 1991; rallo et al., 1994). some bridging ideas related to that gap of the two approaches have recently been considered, though the need for a chilling period, or comparable conditions causing temporarily seasonal growth retardation for reproductive bud differentiation during the winter, has been well established. various growth and metabolic changes between fruiting (‘on’) trees and low or non fruiting (‘off’) olive trees have been described, as reviewed some years ago for “olea” (lavee, 2006) and fruit crops in general (goldschmidt, 2005). the overall affect of olive alternate bearing is expressed by antagonism between the developing fruit and vegetative growth (fig. 3). as olive fruit is initiated and develops from buds on shoots grown during the previous year, a reduction of vegetative shoot elongation and inhibition of lateral shoot out growth due to fruit development has a major effect on the general number of buds and thus on the potential reproductive buds in particular. as olive is a sectorial tree, the antagonism between developing fruit and vegetative growth might appear on a single scaffold, a tree section or a whole tree depending on the amount and distribution of the fruit in the ‘on’ year. it should be noted that the greater the amount of fruit in the ‘on’ year, the greater the chilling required for a return crop in the following year. still, once the yield in the ‘on’ year exceeded a level specific for the growing conditions and the cultivar additional chilling will not be effective any more (fig. 4a). furthermore, if inflorescences are formed, the amount of male flowers (fig. 5) usually increases and the fruit set potential of complete flowers is markedly reduced (cuevas et al., 1994). a similar effect is induced by harvest time during current yield. harvesting the current fruit late in the season will significantly reduce the level of return flowering in the following year with the degree of chilling having only a minor effect (fig. 4b). all these phenomena are accompanied, and probably controlled by basic metabolic changes within the different organs of the tree. while in leaves during the ‘off’ year the level of proteins was considerable lower than in leaves during the ‘on’ year, the opposite trend was found in the bark of young shoots in which the protein level was significantly higher than in the ‘on’ year (table 1). recently, the molecular origin of some of those proteins, such as ft which is involved in flower induction in arabidopsis, were identified and their possible function in the process leading to flowering of olive was determined (haberman, 2012; samach and smith, 2013). table 1 the protein content (mg/fw) of mature olive leaves of 4 cultivars in “on” and “off” years. sampled in late summer koroneiki uovo di piccione barnea manzanillo tree phase leaves 475 bc 370 a 310 a 295 a “off“ 530 b 510 b 405 c 510 b “on” bark 490 b 490 b 500 b 440 b “off“ 370 a 360 a 370 a 360 a “on” from lavee and avidan, 1994. fig. 3 comparison of the annual vegetative shoot growth of an ‘on’ year (left) and ‘off’ year (right) branch. fig. 4 the effect of olive fruit yield level (a) and harvest time (b) in a current year on their fruiting potential in the following year (lavee, 1989). fig. 5 open olive flower types. on the left a male flower, on the right a complete normal (open perfect) androgynous flower (goor et al., 1960). 215 lavee, alternate bearing in olive initiated by abiotic induction leading to biotic responses another major metabolic change in the leaves of olive trees in their ‘on’ and ‘off’ cycle was identified as a dynamic change of some phenolic compounds, particularly chlorogenic acid (cha). this acid, which olive tissue responds to as a growth promoting auxin (fig. 6), increases in the leaves during fruit-set and remains high throughout the ‘on’ year, inhibiting the differentiation of flower buds for the following year. the resulting low fruit set causes the level of cha to drop again and remains low during the whole ‘off’ year (fig. 7). the negating effect of cha on reproductive bud differentiation was directly demonstrated by injecting cha into scaffolds of cv. manzanillo trees during the winter (fig. 8) which resulted in a reduction of flower bud development for the following spring (lavee et al., 1986). the amount of cha developing in the leaves during the ‘on’ year fruit set is proportional to the amount of fruit developing on the trees (fig. 9). other biotic changes in the metabolism of olive trees leading to alternate bearing are the level or depletion of minerals, activity of endogenous and exogenous gibberellins, and the level of carbohydrates which, although controversial, were also reported to be involved in controlling alternate bearing. various schemes of biotic metabolic changes which lead to or inhibit floral induction were suggested and that indicate the sequences of events controlling vegetative or fig. 6 the effect of auxin (naa) and chlorogenic acid (cha) on the growth of olive callus tissue in vitro (lavee, 1996). fig. 7 the change in content of cha during the year in the leaves of ‘on’ and ‘off’ olive scaffolds and after inflorescences removal of cv. manzanillo olive trees (lavee and avidan, 1994). fig. 8 pressurized winter injection of cha in a scaffold of cv. manzanillo causing a reduction in reproductive differentiation of buds. fig. 9 the relationship between the cha level in leaves and fruit yield per tree of cv. manzanillo (lavee, 1989). 216 adv. hort. sci., 2015 29(4): 213-219 reproductive development during the tree growth cycle (fig. 10). the thermal effect on the biotic processes at the different developmental stages was schematically presented (fig. 11). extreme abiotic conditions, particularly temperature, might change the developmental pattern of the reproductive bud, leading to abnormal organs. juvenal seedlings one to two years old, when submitted to relatively extreme low temperatures, will induce metabolic changes that lead to semi differentiated abnormal reproductive buds (fig. 12). a oneto two-day period of high temperature in mid winter, after the induction of the biotic metabolism leading to flower bud differentiation but prior to initial morphological changes of the buds, will lead to vegetative opening of all the buds along the shoots similar to that of the reproductive fully differentiated ones (fig. 13). thus, the abiotic pulse stopped and changed part of the normal biotic pathway of bud development. long winter periods with insufficient chilling temperature to induce the biotic processes leading to bud differentiation will result in a lack of inflorescence development and therefore cause a fully synchronized alternant bearing, as is common in relatively warmer climates with variable winter temperatures (fig. 14). however, in extreme high winter temperatures, rather uniform day length and no other abiotic factors to induce a winter period with growth cessation the abiotic environment will induce biotic conditions changing the entire reproductive development of the olive by developing single terminal flowers instead of the normal lateral inflorescences (fig. 15). such flowers are usufig. 10 a short schematic presentation of the vegetative-reproductive growth cycle of olive (lavee, 2007). fig. 11 description of temperature involvement in significant metabolic stages during olive vegetative and reproductive development (lavee, 2006). fig. 12 abnormal semi-reproductive development of juvenile buds due to unusual strong abiotic thermal induction. fig. 13 illustration of a fully reproductive olive shoot (left) and a reproductive shoot reversed to vegetative development of all buds due to a period of high temperature during an early stage of winter reproductive differentiation (right) (lavee, 1996). 217 lavee, alternate bearing in olive initiated by abiotic induction leading to biotic responses ally malformed and those which set fruit, found to present, were all parthenocarpic. still, alternate bearing develops in olive also under the most suitable and annually repetitive climates. the alternate bearing under such climatic conditions is less spectacular as it is based on non synchronized alteration of each individual tree. this slowly developing alteration in fruiting is also not due to a genetic property. various slight abiotic stimuli cause the initiation of the biotic processes leading in receptive buds to differentiation and small changes in fruit load which, accordingly, gradually amplify (fig. 16). various abiotic environmental events such as rain during the flowering period wash off the pollen and receptive compounds from the stigma, hot dry winds reduce the respectability of the stigma by drying it, lack of suitable pollen for the required cross pollination, as well as insufficient illumination might lead to the development of shot berries (fig. 17), which are also, in part, instrumental in inducing alternate bearing in olive (stutte and martin, 1986 a, b). however these factors leading to alternate bearing cannot be considered genetic control of alternate bearing as they are all governed by abiotic environmental conditions inducing the onset of specific biotic activities. furthermore, suitable exposure of the trees to light and radiation might create a period of retarded growth compensating for insufficient chilling starting the biotic endogenous processes which lead to fruit development as occurring in some semi tropical regions. fig. 14 sequence of synchronized alternate bearing development due to extreme high or low winter thermal conditions occurring particularly in regions with varying winter temperatures (lavee, 1989). fig. 15 abnormal terminal bud differentiation developing a single flower due to high temperatures under semitropical environments. fig. 16 scheme of alternate bearing development in individual trees under relatively stable low annual winter thermal conditions (lavee, 1989). fig. 17 parthenocarpic fruit (shot berries) development with aborted embryos due to unfavorable reproductive differentiation or mal conditions effecting fruit set in the spring. 218 adv. hort. sci., 2015 29(4): 213-219 to eliminate alternate bearing in olive, or at least reduce it, various horticultural techniques are applied such as fruit thinning, girdling, control of harvest time and, to a lesser extent, pruning. these interventions are selectively used for table olives, but in part also in olives for oil extraction. controlled irrigation and mineral nutrition are helpful tools as well to reduce the biannual bearing, although it does not eliminate it. significant metabolic changes were found in various tree parts between ‘on’ and ‘off’ years during the development of the annual olive life cycle. these biotic changes, and their degree, are strongly affected by the environmental abiotic conditions. this close interaction between the endogenous metabolic processes and the environmental conditions at various olive growing sites led to the assumption that alternate bearing of olive is a basic genetic characteristic of this commodity which exists in close interaction with the surrounding abiotic conditions. there is no doubt that the biotic endogenous processes leading to alternate bearing are strongly affected, and in many cases even controlled, by local abiotic environmental conditions of olive growing regions. thus, all the schemes describing alternate bearing clearly indicate the biotic-abiotic interaction (fig. 18). this can be rather misleading as it is based on the nature and degree of the processes involved during the ‘on’ or ‘off’ phase of an already induced developmental cycle. however this approach deals with the level of alternate bearing and does not take into account its initial induction. based on the various studies dealing with alternate bearing of olive, it should be concluded that the initiation of it is solely abiotic. without an external, environmental and thus abiotic stimulus, alternate bearing of the olive tree is not initiated. analysis of the currently available data clearly indicates that without an abiotic induction alternate fruiting of the olive tree would not occur. conclusions alternate bearing in olive could result from an array of metabolic changes involving tree growth, fruit load, flower and pollen viability, etc., however these biotic changes were shown to occur only as a result of abiotic inductions. thus, biannual bearing, at least in the case of olive, should not be considered a genetic trait developing as a stage of the growth cycle of the tree. on the other hand, the nature and degree of reproductive alternation and the metabolic changes involved are clearly based on an abiotic-biotic interaction. references cuevas j., rallo l., rapoport h.f., 1994 crop load effects on floral quality in olive. scientia hortic., 59: 123-130. fabbri a., alerci l., 1999 anatomical aspects of flower and leaf bud differentiation in olive olea europaea l. acta horticulturae, 474: 245-249. goldschmidt e.e., 2005 regulatory aspects of alternate bearing in fruit trees. italus hortus, 12: 11-17. goor a., spiegel p., gratch h., 1960 the olive. state of israel ministry of agriculture ministry of education, tel aviv, israel, p. 163 haberman a., 2012 study of the endogenic system for flowering control in olive (olea europaea) and characterization of the effect of fruit load on the transition to flowering. msc thesis sub. to the faculty of agriculture, hebrew university of jerusalem, rehovot, israel. hartmann h.t., prolingis i., 1957 effect of different amount of winter-chilling on fruitfulness of several olive varieties. bot. gaz., 119: 102-104. hartmann h.t., whisler j.e., 1975 flower production in olive as influenced by various temperature regimes. j. amer. soc. hort. sci., 100: 670-674. lavee s., 1989 involvement of growth regulators and endogenous substances in the control of alternate bearing. acta horticulturae, 239: 311-322. lavee s., 1996 biology and physiology of the olive, pp. 59106. in: consejo oleicolo internationale (coi) world olive encyclopaedia. plazay jane s.a. barcelona, spain. lavee s., 2006 biennial bearing in olive (olea europaea l). olea, 25: 5-13. lavee s., 2007 biennial bearing in olive (olea europaea). annales: series historia naturalis, 17: 101-112. lavee s., avidan n., 1994 protein content and composition of leaves and shoot bark in relation to alternate bearing of olive trees (olea europaea l.). acta horticulturae, 356: 143-147. fig. 18 a general scheme showing initiation of the abiotic conditions on fruiting induction and subsequent biotic-abiotic interactions on the level of fruit differentiation and development in sequential ‘on’ and ‘off’ years (lavee, 2007). 219 lavee, alternate bearing in olive initiated by abiotic induction leading to biotic responses lavee s., harshemesh h., avidan n., 1986 phenolic acid possible involvement in regulating growth and alternate fruiting in olive trees. acta horticulturae, 179: 317-328. rallo l., martin g.c., 1991 the role of chilling in releasing olive floral buds from dormancy. j. am. soc. hort. sci., 116(6): 1058-1062. rallo l., torreno p., vargas a., alvarado j., 1994 dormancy and alternate bearing in olive. acta horticulturae, 356: 209-210. samach a., smith h.m., 2013 constraints to obtaining consistent annual yields in perennials. ii. environment and fruit load affect induction of flowering. plant sci., 207: 168-176. stutte g., martin g.c., 1986 a effect of light intensity and carbohydrate reserves on flowering in olive. j. am. soc. hort. sci., 111: 27-31. stutte g., martin g.c., 1986 b effect of killing seeds on return bloom of olives. scientia hortic., 29: 107-113. troncoso a., garcia j-l., lavee s., 2012 evaluation of the present information on the mechanisms leading to flower bud induction and differentiation in olea europaea. acta horticulturae, 949: 93-98. 25 1. introduction cherry fruit size and quality is an important factor in production and sales of sweet cherry fruit (proebsting and mills, 1981). sweet cherry trees are typically upright, vigorous and non-precocious (lang et al., 2004) so orchard management practices focus on achieving high yields of premium quality fruit through balancing reproductive and vegetative growth. manipulation of the number of fruits (crop load) on the tree, and leaf area, can be used to encourage larger and sweeter fruit through balanced carbohydrate supply and demand (lang et al., 2004; spayd et al., 1986; whiting and lang, 2004). however, in many of these studies, yield losses due to cracking have not been presented (proebsting and mills, 1981) even when the economic losses due to cracking can be significant (hanson and proebsting, 1996). given that cracking can be induced by internal vascular flow (measham et al., 2009), it is posited that higher crop loads will reduce the incidence of cracking through increased competition between fruit for assimilate supply. it has been previously hypothesised that higher crop loads increase competition between fruit for carbohydrates and that lower crop loads result in higher assimilate supply for individual fruit (spayd et al., 1986), and that there can be a resultant increase in size (spayd et al., 1986) and concentration of sugars (proebsting and mills, 1981). it has also been noted however, that lower crop loads are associated with increased vegetative growth (kappel, 1991) and that current season’s vegetative growth is a strong sink for carbohydrates. diurnal translocation of sugars from leaves to fruit can be variable (richardson, 1998), and therefore it is difficult to assess relationships between sugars and cracking as a result of internally supplied excess water. cherry fruits are strong sinks (ayala and lang, 2008) and it has been noted that removal of spur leaves had little effect on fruit quality because alternative supplies of carbohydrates were sourced (whiting and lang, 2004). fruit and leaf ratio can be manipulated for optimum quality. two flower buds per spur has been suggested as the ideal (whiting and lang, 2004). an interaction between fruit and leaves was also implicated in the development of cracking, in a study by measham et al. (2010), which showed that leaf removal decreased the development of side cracks in cherry fruit during the few weeks prior to harvest. furthermore, diurnal water potential gradients and evaporative demands on the leaf influenced vascular flow to the fruit demonstrating a local fruit and leaf interaction. thus, given that fruit size (simon, 2006) and sugar levels (christensen, 1996) have been associated with the development of cracking, and simon (2006) cites two studies (bullock, 1952; way, 1967) that found trees with high loads that showed little cracking within variety, the potential for crop load manipulation to influence fruit cracking warrants investigation. the aim of this present study is to further examine this relationship between crop load and cracking. crop load manipulation and fruit cracking in sweet cherry (prunus avium l.) p.f. measham*, s.a. bound**, a.j. gracie*, s.j. wilson* * tasmanian institute of agriculture, university of tasmania, private bag 98, hobart, tasmania, 7001 australia. ** tasmanian institute of agriculture, 13 st johns avenue, new town, tasmania, 7008, australia. key words: fruit crop load, incidence of cracking, sweet cherry. abstract: yield loss from rain-induced fruit cracking is a perpetual risk associated with the production of sweet cherries, and is difficult to manage due to the unpredictability of fruit responses to late season rainfall. the aim of this five-year study was to investigate the relationship between fruit crop load and incidence of cracking. the results showed a negative correlation between crop load and incidence of fruit cracking, and it was found in both natural and manipulated crop load trials for all varieties studied and in all seasons assessed. the effect of crop load on final cracking levels are determined post cell division. results from this study showed that fruit width was positively correlated with cuticular cracking but, contrary to what has been purported in literature, no relationship between concentration of soluble sugars or firmness with the incidence of cracking was found. this study has confirmed that crop load should be a major consideration in orchard practices in developing strategies to manage fruit cracking. adv. hort. sci., 2012 26(1): 25-31 received for publication 23 october 2011 accepted for publication 3 february 2012 26 2. materials and methods plant material mature trees, grown on f12/1 rootstock, were used in all field trials. trials were undertaken from late october to late january during seasons 2005/06, 2006/07, 2007/08 and 2010/11 in two commercial orchards in huonville and old beach, tasmania (australia). all orchards were subjected to standard industry management practices. to investigate the effect of crop load on fruit cracking and type, five manipulated crop load trials were undertaken in years with late summer rainfall; trials 1 and 2 in seasons 2005/06 and 2006/07 respectively, trials 3, 4 and 5 in season 2010/11. a study of fruit properties from trial 5 was undertaken. in addition, a survey of natural crop load and fruit properties over three years (2005/06, 2006/07, 2007/08) was performed. the relationship between levels of cracking in situ and the cracking potential using the cracking index (christensen, 1972) was also evaluated. manipulated crop load trials to assess the impact of crop load on crack development, manipulated crop load trials were undertaken on one variety ‘simone’ at one site (huonville) in two seasons: 2005/06 (trial 1) and 2006/07 (trial 2). three further manipulated crop load trials were undertaken on different varieties and sites in one season, 2010/11; on variety ‘sweetheart’ at huonville and old beach (trial 3 and 4 respectively), and on variety ‘regina’ at huonville (trial 5). these varieties were chosen due to the variety of crack types they had previously displayed in earlier studies (measham et al., 2009) in tasmania; ‘simone’ showed a tendency for cuticular cracks, ‘regina’ for side cracks’ and ‘sweetheart’ for both. in trials 1 and 2, treatments included a low, medium and high crop load, which aimed for 2, 5 or 8 fruit per cm2 trunk cross sectional area (tcsa) respectively in a randomised complete block design with three replicates (whole tree plots). treatments were applied at pit-hardening during stage ii of fruit growth and development which occurred post bloom at 4 weeks after full bloom (4wafb). where the high crop load specified could not be reached, natural crop load was determined and used. in trials 3, 4 and 5, treatments included a low, medium and high crop load, applied at three different growth stages in a factorial design with five replicates (whole tree plots). crop load was achieved by thinning each bud to 1, 2 or 4 floral buds per spur and applied pre bloom (prb) at dormant bud stage, full bloom (fb) and post bloom (pob) at four weeks after full bloom. in addition, for trial 3, a sub sample of 30 non-cracked blemish-free fruits were randomly selected from each replicate tree for individual fruit assessments for size, total soluble solids, and firmness. mean fruit property values were used to assess relationships with crop load, and with the incidence of cracking in situ. in all trials cracking incidence was determined at harvest. fruits from each treatment were additionally assessed for cracking index (using 50 non cracked fruits per variety). in all manipulated crop load trials, the actual crop load achieved for all trial trees was recorded. natural crop load and fruit properties survey natural crop load was recorded at harvest over three seasons on three randomly selected whole trees of available varieties which included ‘kordia’, ‘lapin’, ‘regina’, ‘simone’, ‘sweetheart’, ‘sylvia’ and ‘van’. all fruits were harvested and cracking levels recorded. cracking incidence recorded at harvest was assessed in relation to natural crop load. in addition, for each season non-cracked blemish-free fruit from each variety was grouped, and a sub sample of 30 non-cracked fruits were selected for individual fruit assessments for size, weight, total soluble solids, stem length and skin thickness. mean fruit property values were used to assess the relationship with the incidence of cracking in situ for each variety. also in 2007/08, fruits from available varieties were assessed for cracking with the cracking index immersion method (christensen, 1972). the relationship between the cracking index and the level of cracking recorded in the field was investigated. given that this immersion method depends on water uptake across the skin, the impact of stem removal on uptake and the subsequent index value was also investigated using additional sub samples from variety ‘simone’ with stems either removed or left intact. measurements cracking incidence was determined as per measham et al. (2009), but with apical-end cracks and stem-end cracks combined to give a level of cuticular cracks. measham et al. (2010) concluded that these crack types were likely to be induced through the same mode of water uptake. cracking index was determined using the immersion method developed by verner and blodget (1931) as cited in and refined by christensen (1972). all fruit were harvested between 7 a.m. and 12 noon and cracking assessments, morphological measurements and laboratory-based measurements were undertaken on the same day as harvest. climate data for the months preceding and during harvest was obtained from the australian bureau of meteorology stations at huonville (situated less than 5 km from the trial site) and at old beach using a pm-k208 pm-11 phytomonitor weather station. determination of crop load prior to treatment application in manipulated crop load trials, tree girth was measured in centimetres at a point 5 cm above the graft union. tcsa was calculated for each tree for the area (a) of a circle using the formula (a = c2/4π), where c = circumference (cm) as described in measham et al. (2009). crop load was determined as total fruit number per tcsa. to determine natural crop load, all fruits from each tree were counted and crop load expressed as number of fruit per tcsa. 27 fruit property tests fruit size, weight and total soluble solid (tss) concentration (brixo) were measured as described in measham et al. (2009). fruit firmness was measured using a bioworks inc. firmtech 2 with values recorded using controlsoft software. stem length (mm) was measured using vernier callipers and skin thickness was recorded microscopically using a nikon smz800 dissecting microscope. statistical analyses to assess the relationship between crop load and cracking incidence, and crop load with mean fruit properties, data were subject to linear regression tests and anova. interactions between crop load and timing of thinning to desired load were determined prior to assessing main effects. analysis of proportion data was performed on transformed data in order to meet the assumptions of analysis. to assess the effect of natural crop load on fruit properties after accounting for variety, mean fruit property data were subject to anova using variety as a fixed factor, and then to ancova (crop load as the covariate) using proc glm (spss version 17). unless specified, all results identified as ‘significant’ are at probability level of 0.05. 3. results manipulated crop load trials all manipulated crop load trials received rainfall in the three weeks prior to harvest. in 2005/06 and 2006/07 there was a similar amount of rainfall (37 mm and 41 mm respectively). trials 3 and 4 in 2010/11 experienced 49 mm and 50 mm rainfall respectively and trial 5 received 42 mm rainfall (differing due to harvest dates). a negative linear relationship between actual crop load and total cracking incidence was recorded for variety ‘simone’ over both seasons (2005/06 and 2006/07) (fig. 1). the effect was greater in season 2005/06 than 2006/07, as indicated by the significantly greater magnitude of the slope for each crack type (slope (b) = -4.69 and -0.77 for total and side cracks and -0.69 and -0.30 for cuticular cracks). in 2010/11 a significant interaction (p = 0.045) between level and timing of crop load on total cracking in variety ‘sweetheart’ at huonville (trial 3) (fig. 2), but not at old beach (trial 4). at huonville (trial 3), within thinning times, no significant effect of crop load at the dormant (prb) or full bloom (fb) thinning times was seen, but there was a significant effect on total (p = 0.025) and side (p = 0.029) cracks (but not on cuticular cracks) when fig. 1 percentage of total cracked fruit (a), cuticular-cracked fruit (b) and side-cracked fruit (c) with actual crop load (tcsa) for variety ‘simone’. each point is for an individual tree. a significant relationship was found between actual crop load and total cracking incidence in 2005/06 (r2 = 0.903, p<0.001) and 2006/07 (r2 = 0.511, p = 0.03), cuticular cracking 2005/06 (r2 = 0.907, p<0.001) and 2006/07 (r2 = 0.540, p = 0.02) and side cracking in 2006/07 (r2 = 0.575, p = 0.02). a significant difference between seasons was found for total cracking and cuticular cracking. slope (b) of the linear regressions calculated for crop load and cracking incidence were significantly different between years. fig. 2 incidence of total cracking at huonville for variety ‘sweetheart’ under low, medium and high crop loads applied at dormant bud stage (pb), full bloom (fb) and four weeks after full bloom (pob). a significant interaction between crop load level and application timing was evident; low crop load resulted in a significantly higher level of cracking within the post bloom application time only. 28 thinning was applied post bloom (pob) (table 1). high and medium crop load levels set by post bloom thinning resulted in significantly fewer cracked fruit than low crop loads. at old beach (trial 4), there was no interaction between crop load level and timing of thinning; there was, however, a significant main effect of crop load on both total (p = 0.01) and side (p = 0.01) cracks, but not on cuticular cracks. a significant main effect of crop load on total (p < 0.001) and side (p < 0.001) cracks was also seen in variety ‘regina’ at huonville (trial 5) (fig. 3). there was an interaction of crop load level and timing of thinning on fruit size in ‘regina’ (fig. 4) where low crop loads resulted in smaller fruit when thinned pre bloom or post bloom. thinning at full bloom gave smaller fruit with medium loads. no interaction was found for fruit soluble solids or firmness; furthermore, no main effect of crop load or timing was found for fruit firmness. there was a significant main effect of crop load (p = 0.008) and timing of thinning (p = 0.013) on soluble solids with fruit from medium crop loads, and post bloom thinning displaying the highest soluble solids. no fruit properties were correlated with cracking levels except for fruit size, which was positively correlated (p = 0.01) with cuticular cracks only. cracking indices determined for all treatments in trials 3, 4 and 5 are given in table 2. there was a significant relationship (p = 0.017) between index and total cracking fig. 3 percentage of total cracked fruit (a) (r2 = 0.69) and side-cracked fruit (b) (r2 = 0.59) with actual crop load (tcsa) for variety ‘regina’ and percentage of total cracked fruit (c) (r2 = 0.33) and side-cracked fruit (d) (r2 = 0.32) with actual crop load (tcsa) for variety ‘sweetheart’. each point is for an individual tree. significant relationships were found between crop load and total cracking incidence and between crop load and side cracking. table 1 incidence of total and side cracking in ‘sweetheart’ at huonville under high, medium and low crop loads applied post bloom crack type crop load cracking incidence total high 15.22 a medium 16.62 a low 27.40 b side high 11.30 a medium 12.44 a low 22.89 b for each crack type, values followed by different letters indicate a significant difference (p<0.05). 29 recorded in situ for trail 3 only; no relationship was evident between cracking index and cracking in situ for trial 4 or 5. natural crop load and fruit property trials lower natural crop loads had higher levels of cracking incidence (fig. 5). cracking incidence remained low (less than 5%) for crop loads higher than ten fruit per cm2 tcsa in all years and for all crack types (fig. 5). when only using data points of less than 10 fruit per cm2 tcsa, relationships between cracking and crop load were found to be significant for all crack types in 2005/06 (total, r2 = 0.893, p < 0.001; cuticular, r2 = 0.853, p < 0.001; side, r2 = 0.540, p = 0.006) and for total and cuticular cracks in 2006/07 (total, r2 = 0.576, p = 0.005, cuticular, r2 = 0.463, p = 0.03). across all varieties and seasons no significant relationship was found between any of the fruit property values with total cracking incidence or individual crack type incidence. little difference can be observed between either weight or total soluble solids and changes in crop load, except perhaps a slight trend in variety ‘sylvia’ where a decrease in weight, but not in total soluble solids, occurs with a dramatic increase (three fold to 24 fruit per tcsa) table 2 cracking index (n = 50) determined for fruit from the three manipulated crop load trials (trials 3, 4 and 5) time of application crop load cracking in situ (%) cracking index trial 3 trial 4 trial 5 trial 3 trial 4 trial 5 dormant high 18 38 7 5 4 24 medium 18 32 11 10 6 16 low 14 59 28 11 15 21 full bloom high 14 31 6 8 8 3 medium 18 39 11 9 7 7 low 16 69 26 8 14 7 4wafb high 15 28 7 6 9 4 medium 17 32 11 9 4 7 low 27 50 26 18 24 12 mean incidence of total cracking is also given. a significant correlation was found between cracking index and total cracking in trial 3 only. fig. 4 fruit size (mm) determined for fruit from variety ‘regina’ under low, medium and high crop loads applied at dormant bud stage (prb), full bloom (fb) and four weeks after full bloom (pob). a significant interaction between crop load and thinning time was found. fig. 5 the percentage of total cracked fruit (a), cuticular-cracked fruit (b) and side-cracked fruit (c) with natural crop load (tcsa). each point is for an individual tree. a significant relationship was found between crop load and total cracking incidence in 2005/06 and 2006/07, and between crop load and cuticular cracking in 2005/06, and between crop load and side cracking in 2005/06 and 2006/07. 30 in crop load in the third season, 2007/08 (fig. 6). a significant variety effect was found in fruit weight (p = 0.02), total soluble solids (p = 0.001) and stem length (p < 0.001). after accounting for variety, and analysing data using crop load as a covariate, a significant effect of crop load was found for fruit weight only (p = 0.03). fig. 6 mean fruit weight and total soluble solids for seven varieties over three seasons (error bars represent sem, n = 30) with mean crop load levels (n = 3) and cracking incidence for the same varieties and seasons. for cracking incidence, expressed as percentage of total yield, columns represent total incidence, where light areas represent incidence of cuticular-cracked fruit, and dark areas represent incidence of side-cracked fruit. horizontal axis labels represent variety by letter (k kordia, l lapin, r regina, si simone, sw sweetheart, sy sylvia, v van) and season by number (1 2005/06, 2 -2006/07, 3 2007/08). a significant relationship between cracking index with total cracking incidence (p = 0.011) and incidence of sidecracked fruit (p<0.001) was found. no significant relationship was found between cracking index and incidence of cuticular-cracked fruit. no differences were found between values for stemless fruit and fruit with stems attached (both had index values of 25). 4. discussion and conclusions lower crop loads resulted in a greater incidence of fruit cracking in sweet cherry. this was seen in both manipulated crop loads and in the natural crop load survey. responses to rainfall and the difference in magnitude of cracking between 2005/06 and 2006/07 confirm a strong seasonal impact on crack development, consistent with the findings of measham et al. (2009). however, rainfall per se did not account for the differences in cracking between seasons, suggesting that other environmental parameters and fruit growth patterns are also important in the development of cracks. fruit development is important in crack susceptibility as all post bloom thinning showed increased cracking with lower crop loads. this implies that the effect of crop load on final cracking levels are determined post cell division, and cracking is therefore more likely to be attributable to cell expansion during the later stages of growth. cell expansion is a function of internal water entry, which has been linked to increased rates of side-cracked fruit (measham et al., 2010). this also supports the findings of yamaguchi et al. (2002) who linked cracking susceptibility at harvest to cell length. pre bloom thinning would therefore be the preferable option for manipulating crop load for size whilst minimising the risk of cracking. in addition, the development of the cuticle during early growth stages should be investigated with regard to cuticle integrity during the later periods of development. the number of cuticular-cracked fruits in low fruit load trees increased significantly in ‘simone’. during cell expansion, relative canopy cover on a whole tree basis in low fruit load trees, compared to high load trees, may prevent moisture loss from the fruit surface through reduced airflow around fruit bunches in a timely and effective manner, confirming the importance of leaf:fruit ratio in quality management decisions (whiting and lang, 2004). in contrast to other studies (spayd et al., 1986; whiting and lang, 2004) cracking susceptibility in this study did not seem to be related to fruit quality properties, nor did increased crop load limit fruit size or sugar accumulation, or enhance firmness (christensen, 1996) in any of the manipulated crop load trails. in the natural crop load survey, variation in fruit properties was mostly influenced by variety, with crop load only further influencing fruit weight, but not size, sugar level, stem length or skin thickness. cracking incidence was also not significantly correlated with the fruit properties recorded. this is in contrast with accepted views that both fruit size (simon, 2006) and sugar levels (christensen, 1996) are closely linked with cracking. studies suggesting vegetative growth provides a strong photoassimilate sink in apricots (costes et al., 2000) support these results whereby fruit crop load may not strongly influence source:sink relationships. results from both the manipulated and natural crop load trials do confirm studies that report no differences in sugar levels between varieties of varying cracking susceptibility (moing et al., 2004). 31 furthermore, the results of the present study can be explained by the level of crop load achieved under normal orchard practice. fruit loads were relatively low in trees from this study with the majority being lower than 15 fruits per cm2 tcsa; the highest value reached was about 27 fruits per cm2 tcsa, or the equivalent of just over 2000 fruits on a tree with a trunk circumference of 30 cm. it is possible that fruit quality (size and sugars) was not diminishing under this scenario as there were still available resources within the tree from which to draw. this finding highlights the strong potential for encouraging good fruit set, and subsequent crop load, as a practical and viable management tool in mitigating yield losses from rain-induced cherry fruit cracking, given the significant reduction in cracked fruit with increased crop loads. the incidence of cracking recorded in situ was correlated with the cracking index for varieties in the natural crop load survey but for only one of the manipulated crop load trials. the cracking index procedure may not necessarily be reliable for predicting cracking susceptibility given the differences found in cracking incidence with crop load, with seasons (measham et al., 2009), and when compared to other growing regions (christensen, 1996; greco et al., 2008). the strong correlation between cracking index and the incidence of side cracks recorded in situ supports the build up of turgor within the fruit as a likely driver of side cracking (sekse, 1995), which can be somewhat mitigated by skin and cuticular properties. this could be due to differences in shape; curvature of the skin has been related to cracking susceptibility (sawada, 1934), and could also explain why size was the only fruit parameter positively correlated with cuticular cracking. this study has confirmed that crop load management can be successfully used to mitigate cracking without compromising fruit size. the results from this study did not confirm the relationship between fruit size, or sugar, and the incidence or cracking, but highlight the importance of skin properties in crack development. references ayala m., lang g.a., 2008 c-13-photoassimilate partitioning in sweet cherry on dwarfing rootstocks during fruit development, pp. 625-632. in: eris a., and m. burak (eds.) proceedings of the v international cherry symposium. international society for horticultural science, vols. 1 and 2. christensen j.v., 1972 cracking in cherries. iii. determination of cracking susceptibility. acta agriculturae scandinavica, 22: 128-136. christensen j.v., 1996 rain-induced cracking of sweet cherries: its causes and prevention, pp. 297-330. in: webster a.d., and n.e. looney (eds.) cherries: crop physiology, production and uses. cab international, pp. 513. costes e., fournier d., salles j.c., 2000 changes in primary and secondary growth as influenced by crop load in ‘fantasme®’ apricot trees. j. of hortic. sci. & biotec., 75: 510-519. greco p., palasciano m., mariani r., pacifico a., godini a., 2008 susceptibility to cracking of thirty sweet cherry cultivars. acta horticulturae, 795: 379-382. hanson e.j., proebsting e.l., 1996 cherry nutrient requirements and water relations, pp. 243-258. in: webster a.d., and n.e. looney (eds.) cherries: crop physiology, production and uses. cab international, wallingford, uk, pp. 513. kappel f., 1991 partitioning of above-ground dry matter in ‘lambert’ sweet cherry trees with or without fruit. j. amer. soc. for hortic. sci., 116: 201-205. lang g.a., olmstead j.w., whiting m.d., 2004 sweet cherry fruit distribution and leaf populations: modeling canopy dynamics and management strategies, pp. 591-599. in: webster a.d. (ed.) key processes in the growth and cropping of deciduous fruit and nut trees. international society for horticultural science, pp. 715. measham p.f., bound s.a., gracie a.j., wilson s.j., 2009 incidence and type of cracking in sweet cherry (prunus avium l.) are affected by genotype and season. crop and pasture science, 60: 1002-1008. measham p.f., gracie a.j., wilson s.j., bound s.a., 2010 vascular flow of water induces side cracking in sweet cherry (prunus avium l.). adv. hort. sci., 24(4): 243-248. moing a., renaud c., christmann h., fouilhaux l., tauzin y., zanetto a., gaudillere m., laigret f., claverie j., 2004 is there a relation between changes in osmolarity of cherry fruit flesh or skin and fruit cracking susceptibility? j. amer. soc. for hortic. sci., 129: 635-641. proebsting e.l., mills h.h., 1981 effects of season and crop load on maturity characteristics of ‘bing’ cherry. j. amer. soc. for hortic. sci., 106: 144-146. richardson d.g., 1998 rain-cracking of ‘royal ann’ sweet cherries: fruit physiological relationships, water temperature, orchard treatments, and cracking index, pp. 677682. in: ystaas j., o. callesen (eds.) third international cherry symposium, vols. 1 and 2. sawada e., 1934 a physical consideration of the mechanism of the cracking of sweet cherries. transactions of the sapporo natural history society, xii: 365-376. sekse l., 1995 fruit cracking in sweet cherries (prunus avium l.). some physiological aspects a mini review. scientia horticulturae, 63: 135-141. simon g., 2006 review on rain induced fruit cracking of sweet cherries (prunus avium l.), its causes and the possibilities of prevention. international journal of horticultural science, 12: 27-35. spayd s.e., proebsting e.l., hayrynen l.d., 1986 influence of crop load and maturity on quality and susceptibility to bruising of ‘bing’ sweet cherries. j. amer. soc. for hortic. sci., 111: 678-682. whiting m.d., lang g.a., 2004 effects of leaf area removal on sweet cherry vegetative growth and fruit quality, pp. 467-472. in: webster a.d. (ed.) key processes in the growth and cropping of deciduous fruit and nut trees. international society for horticultural science, pp. 715. yamaguchi m., sato i., ishaguro m., 2002 influences of epidermal cell sizes and flesh firmness on cracking susceptibility in sweet cherry (prunus avium l.) cultivars and selections. j. of the japanese soci. for hortic. sci., 71: 738-746. 180 1. introduction apple is a very important fruit tree in syria with total apple acreage of about 41 000 ha. codling moth cydia pomonella l. (lepidoptera: tortricidae) is considered the most important insect pest of apple in syria and insecticides are widely used to control it. such control methods are costly, nonselective, environmentally unsafe and effective for only a short period in the treated area. moreover, c. pomonella has already developed resistance to various insecticides (varela et al., 1993). therefore, a more reliable and environmentally safe control method is required. knipling (1970) and myers et al. (1998) reported that the sterile insect technique (sit) can be considered an important component of an area-wide approach to insect control programs. the possibility of applying sit as an alternative control method to suppress c. pomonella populations has been determined by many researchers (bloem et al., 1999, 2001). this approach relies on mass-rearing and release of both sexes of irradiated moths into wild pest populations. the success of this technique against codling moth depends largely on the release of sexually competitive insects capable of locating and carrying out mating with several feral individuals (knipling, 1981). it is widely agreed that insects with a long lifespan, good dispersal pattern, high incidence of mating, ability to transfer sperm successfully, and a noticeable fecundity would exhibit an acceptable level of mating competitiveness (carpentar et al., 1989). mating in lepidoptera involves the following sequence of events: copulation, spermatophore transfer, insemination and egg fertilization. bues et al. (1992) observed that most lepidopteran females tend to mate within 24 h of emergence. the effect of adult age on mating varies considerably between species as well as sex. previous studies have stated that younger females of pectinophora gossypiella saunders (lingren et al., 1988), eoreuma loftini dyar (spurgeon et al., 1995), lymantria dispar l. (proshold, 1996), and phthorimaea operculella zeller (makee and saour, 2001) had a higher occurrence of successful matings compared to older individuals. on the contrary, young females of ephestia kuehniella zeller showed a low mating incidence. however, the newly emerged males of e. kuehniella were more likely to transfer spermatophores than older males (calvert and corbet, 1973). the effect of adult age on reproductive capacity is well known in several lepidopteran species (ellis and steele, 1982). it has been shown that the reproduction of plutella xylostella (l.) females was significantly reduced by age (nemoto et al., 1992). insect weight plays an important role in mating and reproduction. large males of ephestia elutella hübner were more likely to mate than smaller ones (phelan and barker 1986). similarly, a significant relationship between body size and successful mating has also been reported in the dipteran drosophila melanogaster meigen (partridge and farquhor, 1983). studies have shown that in hemipteran podisus macufactors influencing mating incidence and reproduction in codling moth cydia pomonella l. (lepidoptera: tortricidae) h. makee*(1), i. idris, k. hussian atomic energy commision of syria (aecs), syrian arab republic, p.o. box 6091, damascus, syria. key words: cydia pomonella, fecundity, fertility, mating ability, mating frequency. abstract: the codling moth cydia pomonella l. is a primary pest of apple and various studies have been performed to assess the possibility of applying sterile insect technique as a control method against this pest. in support of this technique, the present work aims to examine the effects of adult age and weight on mating ability, number of matings, fecundity and fertility in c. pomonella. the relationship between number of matings, fecundity, and fertility of females was also studied. female and male weights were found to have an effect on the number of times individuals mate, but male weight only influenced mating success. unlike male weight, female weight affected fecundity and fertility. negative correlations were found between mating success, fecundity and fertility and adult age. multiply-mated females and those which did not accept a second mating showed higher fecundity and fertility than their counterparts that were not given the opportunity to remate. our results provide essential information necessary to increase the effectiveness of sterile insect technique as a control method against c. pomonella. adv. hort. sci., 2012 26(3-4): 180-186 (1) corresponding author: hmakee@aec.org.sy. received for publication 29 february 2012 accepted for publication 15 february 2013 181 liventris and p. connexivus the heavier females presented a better reproductive rate (evans, 1982; zanuncio et al., 1992). to maximize the mating competitiveness of released insects, the influence of adult age and weight on mating incidence and reproduction capacity of codling moth were examined. in addition, the effect of the number of matings on fecundity and fertility of this species was tested. 2. materials and methods a c. pomonella colony has been maintained in our laboratory for several years and was used for the present study. newly emerged adults were crossed in petri dishes (12 cm dim), five pairs in each. a wet cotton wool was placed in each petri dish as drinking source. after 3-4 days, the dishes which had eggs were collected and soaked with 2% sodium hypochlorite solution for 2 min for egg sterilization. then the dishes were washed with tap water and left to dry. eggs were checked daily for hatching. newly hatched larvae were placed on artificial media consisted of the following ingredients: agar-agar, maize, wheat germ, casein, yeast, wesson salts, benzoic acid, fumidil, ascorbic acid, vitamins and nipagine (anisimov, personal communication). all insect stages were kept under constant temperature at 25±1°c with 70±5% rh, and a photoperiod of 16:8 h (l:d). in all the experiments, pupae were sexed and individually placed in small plastic tubes until eclosion. for oviposition, newly emerged females and males were paired in petri dishes (12 cm dim) having a feeding source (a wet cotton wool). “mating incidence” in this study is used to indicate successful spermatophore transfer and/or spermatophore presence in the bursa copulatrix of the female. the number of matings is reflected by the number of spermatophores in the bursa copulatrix. the effect of adult age ten different groups of virgin females (n= 20 in each group) aged 1-10 d were employed. females of each group were individually paired with newly emerged males (<18 h). after 24 h, males were removed and females were kept for oviposition until death. all eggs were collected, counted and allowed to hatch. after death, the females were dissected and examined for the presence of spermatophores in the bursa copulatrix. ten different groups of virgin males (n=20 in each group) aged 1-10 d were examined to determine the effect of male age on successful spermatophore transfer. males of each group were individually paired with newly emerged virgin females (<18 h). after 24 h, males were removed and females were kept for oviposition until death. all eggs were collected, counted and allowed to hatch. after death, the females were dissected and examined for the presence of spermatophores. the effect of adult weight to determine the influence of male and female weight on incidence of mating, number of matings, fecundity and fertility pupae were weighed, sexed, and divided into three groups based on weight: light, medium and heavy. female pupal weights were, respectively, 34-37, 39-42 and 44-47 mg, while male pupal weights were, respectively, 25-31, 36-45 and 47-49 mg. emerged females and males in each group were individually paired with newly emerged adults of the opposite sex. in male and female groups, males were paired with females weighing 36.2±0.5 mg, while females were paired with males weighing 30.6±1.3 mg. in each group, the females and males were kept together until death. all eggs were collected, counted and left to hatch. the females were dissected and the number of spermatophores in the bursa copulatrix were counted. to determine the relationship between adult weight and fecundity and fertility, only mated females were considered. effect of sex ratio on mating ability two experiments were carried out. in the first experiment, 1-d-old males (n = 20) were individually confined with three newly emerged females (1 male: 3 females). males and females were kept together for 24 h, after which time the females were dissected and the number of spermatophores was determined. in the second experiment, 1-d-old females (n= 20) were singly confined with three newly emerged males (1 female: 3 males). after 24 h, females were removed, dissected and the number of spermatophores was determined. a ratio of 1 female: 1 male was used as a control group for the two experiments. effect of female multiple mating on fecundity and fertility two groups of virgin females were used. females of the first group (n= 20) were individually paired with newly emerged males. after 24 h, males were removed and females were kept for oviposition until death. in the second group, females (n= 45) were individually paired with 1-dold males. males were removed after 24 h and replaced with new 1-d-old males. the same procedure was followed for seven successive days. in both groups, all eggs were collected, counted and allowed to hatch. after death, the females were dissected to determine the presence and number of spermatophores. statistical analysis was carried out using the statistic program version 6 (statsoft, inc. 2003) at 5% level (p= 0.05). a simple linear regression analysis was done to study the relationship between adult age and incidence of mating and fertility. data were subjected to analysis of variance for determination of differences between means, which were tested for significance using tukey hsd test. the percentages were analyzed by applying normal approximation test (analysis of proportion). 3. results effect of adult age figure 1 illustrates the effect of male and female mating ability of c. pomonella with regard to age. a regres182 sion line was fitted to present the relationship between incidence of mating and adult age. the percentage of mating ability was significantly correlated with adult age (y= -7.2303x + 95.667, r2 = 0.77, p<0.05; y = -7.8788x + 90.133 r2 = 0.86, p<0.05 for females and males, respectively). a significant increase in mating ability was recorded when males and females became 2 d old. after that, a significant reduction in the mating ability was noticed 4 d and 5 d after male and female emergence (fig. 1). the mating ability of 1-d-old males and females was similar, and afterwards the mating ability of females was higher than that of males, regardless of adult age. fig. 1 effect of adult age on mating ability of codling moth. to determine the effect of adult age on fecundity and fertility only mated females were used in the analysis. figure 2 reveals that the number of eggs increased when the adults became 2-d-old; the number of eggs then significantly declined (f=13.19; d.f=9,190; p<0.05 for female and f=12.9; doff=9,190; p<0.05 for male). significant differences were noticed between males and females at each tested age, except when both sexes were 1-d-old (fig. 2). fig. 2 effect of adult age on mean fecundity of codling moth female. figure 3 illustrates that as females and males got older their fertility decreased. there was a strong relationship between adult age and fertility (y= -5.3455x + 87.8 r2 = 0.90 and y= -8.5939x + 91.867 r2= 0.93 for females and males, respectively). regardless of adult age, female fertility was significantly higher than that of males except at age 1 d. fig. 3 effect of adult age on adult fertility of codling moth. effect of body weight the percentage of mating ability of the females was not affected by their weight. no differences in mating ability were found among the three tested female weight groups (fig. 4). in contrast, the number of female matings was influenced by their weight. the mean number of matings of group 3 females (the heaviest) was significantly higher than that of groups 1 and 2 (f=8.6; d.f=2,74; p<0.05). however, the mean number of matings of group 2 females did not significantly differ from that of group 1 females (fig. 5). fig. 4 effect of body weight on mating ability of codling moth adults. fig. 5 effect of body weight on number of mating of codling moth adults. 183 a significant difference in the percentage of mating incidence was observed among male weight groups 1 and 3 (fig. 4). all males of group 3 (the heaviest) were able to transfer spermatophores, whereas only half of group 1 males (the lightest) was able to do so. the mating incidence of group 2 males did not significantly differ from that of group 3 (fig. 4). the mean number of matings of group 1 males was significantly lower than that of groups 2 and 3 (f=5.19; d.f=2,72; p<0.05). no differences in the number of matings were detected between males in groups 2 and 3 (fig. 5). results from the study show that female body weight significantly affects fecundity (f=6.24; d.f=2,74; p<0.05) and fertility (f=7.09; d.f=2,74; p<0.05) of the codling moth. the mean number of eggs laid and mean number of eggs hatch increased significantly when female weight increased (table 1). table 1 effect of female body weight on fecundity and fertility of c. pomonella female weight (mg) mean fecundity mean fertility 34-37 66.24±11.35 b 50.6±9.37 b 39-42 99.80±12.70 ab 68.9±11.74 b 44-47 134.11±16.03 a 112.5±14.14 a means followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). unlike female weight, male weight did not affect fecundity and fertility. there were no significant differences in fecundity and fertility between heavy and light males (table 2). table 2 effect of male body weight on fecundity and fertility of c. pomonella male weight (mg) mean fecundity fertility 25-31 63.16±12.85 a 52.36±10.8 a 36-45 85.04±9.88 a 57.80±8.6 a 47-49 95.32±12.2a 67.04±9.8 a means followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). effect of sex ratio on mating ability when c. pomonella females were confined with one or three males for 24 h, they mated only once. the incidence of female mating did not differ significantly when confined with one or three males (table 3). c. pomonella males mated only once when paired with a single virgin female for 24 h. when a male was confined with three females, it mated more than once (table 3). effect of female multiple mating on fecundity and fertility when c. pomonella females were exposed to newly emerged males for seven successive days (group 2 females), 18% of them mated once, more than half mated twice or three times and 18% mated four or five times (table 4). table 4 effects of repeated mating on the mean number of eggs and egg hatch percentage of c. pomonella females female group no. of matings females (%) mean no. of eggs/female±se mean fertility/female± se 1 1 60 93.6±19.9 b 93.6±19.9 b 2 0 7 134.7±25.6 b 0 1 18 237.1±32.1 a 237.1±32.1 a 2 35 225.8±20.5 a 225.8±20.5 a 3 22 260.9±27.9 a 260.9±27.9 a 4 11 238.6±8.5 a 238.6±8.5 a 5 7 270.0±25.8 a 270.0±25.8 a means and percentages followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). group 1= females were paired with the males only for 24 h (n = 20). group 2= females were paired with new virgin male every 24 h for 7 successive days (n = 45). the mean number of eggs and fertility of group 1 females, in which the females were not given an opportunity to remate, were significantly lower than those of group 2 females, in which the females were given a chance to remate (f=6.7; d.f=6,59; p<0.05 for fecundity, f=6.7; d.f=6,59; p<0.05, respectively). regardless of the number of matings of group 2 females, the mean number of eggs and fertility did not differ significantly. 4. discussion and conclusions the tendency and number of matings of c. pomonella may be affected by various factors. our results indicate that old males and females of this species were less likely to mate than young individuals. however, differences appeared when the patterns of mating ability of females and males were compared. the female mating ability was greater than that of males. however, the mating ability of females declined less rapidly than that of males with age (fig. 1). in lepidoptera, the ability to release adequate sex pheromone and/or to respond to the sex pheromone of the table 3 effect of number of females and males on number of mating of c. pomonella during a 24-h period sex sex ratio f:m % mated adult per no. of mating 0 1 2 female 1: 1 b35 ab a65 a 0 1: 3 b40 a a60 a 0 male 1: 1 b30 ab a70 a 0 3: 1 b20 b a60 a b20 percentages preceded by different capital letters (raws) and followed by different small letters (columns) are significantly different at p< 0.05 (normal approximation test). 184 opposite sex generally reduces with age (spurgeon et al., 1995; proshold, 1996; makee and saour, 2001). our result confirms that senescence might influence male moths to a greater degree than females. in the current study, male age greatly influenced female fecundity and fertility of c. pomonella (figs. 1 and 2). conflicting results have been reported on the impact of male age on female fecundity and fertility of c. pomonella. vickers (1997) reported that male age had no effect on female fecundity and fertility, whereas knight (2007) showed that female fecundity after mating with 1-d-old males was significantly lower than after mating with 3-d-old males. similarly, female fecundity and fertility of plutella xylostella l. decreased when the females mated with old males (nemoto et al., 1992; wang et al., 2011). female discrimination against older males has been demonstrated in several species (ritchie et al., 1995; jones and elgar, 2004). thus, c. pomonella females preferred mating with younger males since mating with older males diminishes female fecundity and fertility. such reduction in reproductively could be due to an age-correlated reduction in sperm quality (crow, 1997; hansen and price, 1999). the results from the present work clearly indicate both fecundity and fertility were affected by female age at mating, both of which decreased with an increase in age (figs. 1 and 2). similar effects have been observed previously in several species such as spodoptera exigua (hübner), (rogers and marti, 1996), lobesia botrana (dennis & schiffermüller) (torres-vila et al., 2002) and p. xylostella (wang et al., 2011). the reduction of egg production and viability when mating of c. pomenella was delayed after emergence could be related to utilization of the fat body, which is essential source of vitellogenins and lipids for oocyte maturation, for non reproduction metabolism by older females (barrer, 1976). unlike male weight, female weight did not play a role in mating ability in c. pomonella (fig. 4); both light and heavy females had similar mating tendencies. however, female weight did affect the number of matings (fig. 5). similar results were reported in p. operculella (makee and saour, 2001). male weight had important impact on mating ability and number of matings of c. pomonella (figs. 4 and 5). there may be two main reasons for the relationship between male weight, ability to produce and transfer spermatophore, and number of matings: (1) heavy males may be able to produce sufficient quantity of sex pheromone to attract females (thornhill and alcock, 1983; phelan and barker, 1986); (2) c. pomonella females tend to mate with heavier males, as confirmed by our data (i.e. they mated with 100 and 50% of heavy and light males, respectively) (fig. 4). our results show that reproductively of heavy females was greater than for light females (table 1). strong correlation between adult weight and fecundity has been noted in various insect species (evans, 1982). honek (1993) reported that genetic and environmental factors could influence insect weight. there are several environmental factors including food type and temperature (mohaghegh et al., 1999). generally, female weight partly reflects the size of fat-body. this organ is essential for oocyte maturation since it is a site of lipid and yolk protein synthesis (chapman, 1982). therefore, heavy females are able to produce more eggs since they have a larger fat-body. in contrast to female weight, male weight did not influence the fecundity and fertility of c. pomonella (tables 1 and 2). like most lepidopteran, when a c. pomonella male was paired with one female for 24 h, it was able to produce and transfer only one spermatophore (makee and saour, 2001). nevertheless, if several virgin females were available, c. pomonella males were able to transfer more than one spermatophore during one scotophase (table 3). a comparable result has been noted in males of grapholitha molesta busck, spodoptera frugiperda (j.e. smith) and p. operculella (george and howard, 1968; simmons and marti, 1992; makee and saour, 2001). nevertheless, c. pomonella females mated once in 24 h even when they were confined with three newly emerged males (table 3). thus, females needed a lapse of time to remate, regardless of the number of males available. a similar result was reported in p. operculella (makee and saour, 2001). after mating, females released special volatile materials that reduced their receptivity (tompkins and hall, 1981). when c. pomonella females were exposed to newly emerged males for seven successive days, only 18% of them mated once, 35% twice and 40% several times (table 4). c. pomonella females were capable of mating more than five times when they were paired with virgin males for seven successive days. conversely, p. operculella females were unable to mate more than three times when they were paired with virgin males for seven successive days (makee and saour, 2001). whatever the number of matings, the mean number of eggs and fertility of females were similar in both species. this may imply that once-mated females would not seek additional matings since they received sufficient effective sperm during their first mating. several studies reported that p. operculella, heliothis virescens f. and l. dispar females that did not receive an adequate quantity and quality of sperm during the first mating needed to remate (lingren et al., 1988; proshold, 1995; makee and saour, 2001). on the contrary, knight (2007) stated that c. pomonella females that had mated three times had a significantly higher fecundity than singly-mated moths. fecundity and fertility of females with an opportunity to remate were higher than those of once-mated females that were not allowed to remate (table 4). the relationship between the number of matings and the female’s reproductivity could be attributed to: (1) sperm replenishment which is required for egg fertilization; and (2) nutrients derived from spermatophores that are utilized by the female in egg production (greenfield, 1983). the present study provides useful information for situations where sterile insect technique could be considered against c. pomonella: (1) repeated releases of young sterile insects should be executed rather than one major release; (2) production of heavy insects is preferable in mass-rearing procedures; (3) the effectiveness of sterile in185 sect technique against c. pomonella would not restrain by the release of both sterile males and females, since during one scotophase c. pomonella males could remate whereas females were unable to do that. references barrer p.m., 1976 the influence of delayed mating on the reproduction of ephestia cautella walker (lepidoptera: phycitidae). j. stored prod. res., 12: 165-169. bloem s., bloem k.a., carpenter j.e., calkins c.o., 1999 inherited sterility in codling moth (lepidoptera: tortricidae): effect of sub-sterilising 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(lepidoptera: tortricidae). australian journal of entomology, 36: 179-182. wang x.-p., fang y.-l., zhang z.-n., 2011 effects of delayed mating on the fecundity, fertility and longevity of females of diamondback moth, plutella xylostella. insect science, 18: 305-310. zanuncio j.c., bragança m.a.l., díaz j.l.s., sartório r.c., 1992 avaliação dos parâmetros de fecundidade de fêmeas de podisus connexivus (hemiptera: pentatomidae) de diferentes pesos. rev. ceres., 39: 591-596. impaginato 217 1. introduction it is known that most almond [prunus dulcis (mill.) d.a. webb, syn. prunus amygdalus batch] varieties in the world, and until relatively recently, all the major commercial varieties, are self incompatible. this means that each variety needs to be grown with another variety to ensure pollination. not only that, each variety has its own specific incompatibility genes, and has to be pollinated by another variety with a different set of incompatibility genes (micke, 1996). in 2007, the perennial horticulture development project (phdp), funded by the european commission (ec) for the rehabilitation and development of the horticulture industry in afghanistan, began the collection of varieties of fruits and nuts in afghanistan, with a view to characterisation of those varieties and promotion of the best varieties for the development of commercial horticulture. the programme envisaged the distribution of varieties through a system of certified tree production with information provided as to how to plant and grow superior orchards. since every almond orchard that would be planted in afghanistan would need to be planted with a combination of two or more inter compatible varieties, it seemed essential that work be done on identifying suitable combinations of varieties. the phdp programme also included the development of the production of the superior afghan almond types, which include a range of paper shell types that are very much appreciated in the indian market, and which command very high prices. these are exemplified by the sattarbai and qambari types, which are elongated crescent shaped nuts with a paper shell that in the most prized varieties opens along a lateral fissure to expose the kernel inside. since there was no way of predicting the composition of the afghan germplasm in respect of incompatibility genes, a start was made in 2008 on testing the various combinations of varieties. this paper reports on the performed activities and adv. hort. sci., 2016 30(4): 217-223 doi: 10.13128/ahs-20347 short note implications of investigating pollination and cross compatibility in the almond varieties of afghanistan g.j. cullen 1, g.r. samadi 2, a.h. zarghon 3, m.r. yaqubi 3 1 independent consultant afghanistan national horticulture development organisation (anhdo), kabul, afghanistan. 2 kabul university, kabul, afghanistan. 3 afghanistan national horticulture development organisation (anhdo), kabul , afghanistan. key words: breeding, incompatibility groups, national collection, prunus amygdalus, prunus dulcis. abstract: survey and collection of almond accessions for a national collection of fruit and nuts of afghanistan began in 2007. investigations into cross compatibility of almond accessions began in 2008 on in situ collected exemplar trees, and in 2010 on trees in the ex situ collections. the methods varied in relation to specific trials, nevertheless as an average 150 flower buds were isolated ahead of flowering on 7 one-year-old shoots per tree, and used as pollen donors or receptors. the initial trials on in situ accessions were performed on one single tree per variety, while six trees were used for the tests carried on in the ex situ collections. fruit set percentage from self-pollination, cross-pollination and open pollination was calculated. all native afghan varieties tested were shown to be self incompatible. various problems related to weather and other conditions worked against a comprehensive testing of all combinations of varieties, although many useful and surprising conclusions were reached. (*) corresponding author: samadigr@gmail.com received for publication 11 january 2016 accepted for publication 13 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 217-223 218 the first results achieved related to selfand intercompatibility of almond varieties of afghanistan, together with the some notes on the gained experience and the implications of these findings in the almond industry of afghanistan. as illustrated in the following paragraph, different trials have been defined and set up in different locations taking into account farmer knowledge and tradition, local operative conditions and environmental aspects. 2. in situ tree pollination trials in the kunduz, samangan and balkh provinces during the 2007 season, the phdp had collected budwood from some 84 separate accessions from around afghanistan, and budded these onto almond seedling rootstocks at six different centres with a view to planting out duplicate collections in the northern afghanistan centres of mazar-e sharif and kunduz in spring 2009. it had already been ascertained by phdp, in particular through a comprehensive nursery survey, that the main centre of the important almond varieties was in the north of afghanistan, especially in the provinces of kunduz, samangan and balkh provinces. while it was obvious that testing almond pollination attributes would be much easier when the germplasm was collected into the variety collections, it was considered an urgent priority for phdp to find out about the pollination compatibilities of the main varieties. many stories were being reported from the field by the phdp staff and by associated projects, such as roots of peace almond industry development project, concerning the lack of knowledge among many afghan farmers about pollination requirements of almonds. in general, the need for pollination was unknown, and some farmers were insecticide spraying bees that were in the almond blossom, on the assumption that bees were “eating the flowers”. while traditionally many orchards included a range of varieties, they did not necessarily overlap in flowering time, nor were they necessarily compatible. one grower was supposedly so impressed with the results of the nonpareil variety that he planted a whole large block to that single variety, and then wondered why results were disappointing. not only was solving the almond pollination problem considered an urgent priority for phdp because of the lack of knowledge of most small farmers, but as a donor funded project working within the ministry of agriculture, phdp had to demonstrate meaningful results ahead of planting out the national collections of the different species of fruits and nuts. so in 2008, a simple trial at three sites was designed, using the original in situ trees from which the budwood for the germplasm collection was collected. materials and methods the first experiment to study the self and cross incompatibility of almond accessions in the northern part of afghanistan took place from february 2008 (prior to flowering) to august, 2008 (harvesting of fruit). three groups of six in situ trees were selected for their proximity in single orchards or in closely nearby orchards, in khulm (balkh province), in aybak (samangan province) and chardara (kunduz province). each tree was selected before flowering and lengths of branch with 100-200 flower buds were selected and marked. branches used to donate or receive pollen were bagged ahead of flowering using cotton muslin cloth to avoid ingress of bees or other pollinating insects. all the trial was done using single in situ trees, so on each tree there were bags for ten sites (branch) to donate pollen of uncontaminated flowers, bags for ten sites to receive pollen from five other trees (including two replicates for each cross), bags for two sites for self pollination (two replicates), and two sites marked for natural pollination (open pollination). so there were 7 treatments on each tree. pollination, either self or cross pollination, was done on three successive days, to cover the period of flowering, by bringing flowers from the reserved branches to the receptor branches. protective bagging was removed only to allow the hand pollination, until a few days after flower fall, when the protection was removed. the structure of the trial was based on the hypothesis that all the varieties were self incompatible, so that any fruit set was due to the pollen transferred by the hand crossing. for each tree there was also two self pollination replicates, where the tree’s own pollen was used to pollinate flowers. if there had been any self pollination, this would have invalidated the results of the crossing between different trees. results and discussion it was noted that the general level of fruit set on the control sections of branch (open pollination) was generally quite low. hand pollination as practised in this trial would increase the setting of fruit up to ten fold. this indicated a lot of problems with the current practices, probably mostly to do with the absence of cullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 219 pollinating insects. the condition of the flower buds after what had been a very hard winter, the fertility status, the temperatures and humidity at flowering could all have had an effect. presentation of data to farmers and discussion with them indicates a lack of knowledge of the need for pollination which results in the deliberate killing of bees, as they think the bees eat the flowers, and the planting of large areas to single varieties, with resulting nil crop. the data obtained is reported in table 1. comparing the fruit set percentage obtained by selfing and intercrossed pairs or the average of all crosscombinations. it was shown that all six almond successions were self incompatible. on the other hand, the basic criteria for a successful crosscombination was fruit set with hand pollination equal to or higher than fruit set with natural background pollination (open pollination). the criteria for a non-successful combination was fruit set equal or lower than that with self pollination. the low fruit set with natural pollination brought the boundary line between successful/unsuccessful cross to be too near, so there were a lot of inconclusive results. hence, the information from this trial is generally disappointing. fruit set was low in the orchards, but occasionally there were some interesting results. the carmel flowered much later than other varieties, including nonpareil. the variety carmel 167 was too late flowering to be of use with the local varieties and nonpareil 171. nonpareil 171 was pollinated by abdul wahidi 1003, but the reverse cross had nil pollination. 3. pollination trials in the ex situ collections the almond pollination trials in phdp were done in 2008 with in situ trees at three locations in kunduz, samangan, and khulm. for the 2010 and 2011 trials, the same principles were followed, with cross pollination in all combinations of varieties in sets of six varieties. in the national collections, which for almonds are situated in the centres in balkh and kunduz, there are six identical trees of each almond accession, and this layout was exploited to greatly simplify the trials. out of the six trees, one tree was used to provide pollen to the other five varieties, and for self pollination, and each of the other received pollen from one of the other five trees in that trial set. in 2010, the numbers of flowers available for the trials were in many cases too few to permit valid results to be drawn, and most of the varieties in balkh and kunduz suffered damage from a late frost. in 2011, many varieties set reasonable amounts of fruit for drawing conclusions, but other varieties female (receptor) male (pollinizer)/fruit set % location: khulm, balkh province qambari 143 bellabai 144 sattarbai sufi 145 zang kaftar 148 sattarbai bakhmali 149 sattarbai no.4 154 qambari 143 0.2 12 10 4 13 2 bellabai 144 30 1 17 8:05 0 4 sattarbai sufi 145 13 10 0 8 11 2 zang kaftar 148 9 3 0 2 3 2 sattarbai bakhmali 149 14 0 3 16 0 0 sattarbai no.4 154 2 10 3 7 2 0.4 cross-pollination average 13.6 7 6.6 8.75 5.8 2 location: kunduz marawaja kaghazi 166 carmel 167 sattarbai 168 qaharbai 170 nonpareil 171 abdul wahidi 1003 marawaja kaghazi 166 0 0 0 1 0 1 carmel 167 0 1 11 3 0 0 sattarbai 168 2 0 0 0 2 0 qaharbai 170 1 0 1 0 0 6 nonpareil 171 2 2 7 4 1 8 abdul wahidi 1003 1 0 0 15 0 0.3 cross-pollination average 1.2 0.4 3.8 4.6 0.4 3 location: aybak, samangan province sattarbai 156 sattarbai guldar 157 sattarbai bakhmali 159 qaharbai 160 khairodini 161 shokorbai 162 sattarbai sais 156 3 42 42 35 57 26 sattarbai guldar 157 1 0 0 2 1 0 sattarbai bakhmali 159 5 3 6 3 24 9 qaharbai 160 22 15 27 0 20 6 khairodini 161 16 32 8 11 2 6 shokorbai 162 12 1 3 8 8 1 cross-pollination average 11.2 18.6 16 11.8 22 9.4 table 1 pollination data from mature in situ exemplar trees of the national collection of almond varieties, khulm, balkh province, 2008 summery results, based on final percentage. adv. hort. sci., 2016 30(4): 217-223 220 seemed to still remain in a juvenile phase. this did not allow for maximum values to be extracted from the trials. additional pollination trials were undertaken in the badam bagh kabul centre, in the plots of the almond variety demonstration, which included a limited number of accessions. it was considered important that information was collected about the condition of the trees for setting fruit. this is done by allowing a number of flowers to be open to natural pollination from bees and other insects. bees were introduced to the national collections to maximise natural pollination. the proposition was that if any variety was not showing good fruit set with this natural open pollination, then any cross pollination efforts by hand would also not show good fruit set. results from those varieties could clearly not then be used to indicate incompatibility in that cross. after the mostly rather inconclusive pollination trials in 2010 and 2011, by 2012 the trees were quite large, and only individual branches needed to be marked before flowers opened. materials and methods a straight length of branch with an estimated 200+ flowers were chosen for the trial. the beginning and end of the selected length of branch were marked and any flower buds above and below the marked length of branch could be stripped off to allow easy counting. for each variety, the enumerators were asked to pick two trees at either end of the plot, to get a good average of the open pollinated count. the layout of the pollination trial is shown for blocks of six trees, which is the standard layout in the national collections. by using all six trees available in the accession, it can be seen that a set of six varieties can easily be managed (fig. 1). the varieties for the pollination trials were every year chosen based on the relative importance of their nuts in the commercial market. a meeting was held with almond traders in october 2008, where the traders were asked to assign monetary values to a range of samples of almonds in shell collected from the exemplar trees of the national collection of almonds. this selection of the most important varieties was important, as the number of possible combinations of almond accessions to be tested ran into many thousands. results and discussion the 2012 trials repeated some combinations which did not give clear results in 2010 or 2011, and quite a lot of useful information was obtained. the larger number of combinations tested also allowed for progress in identifying varieties that did not combine with each other, and the start of working out some incompatibility groups. once a variety could be allocated to an incompatibility group, predictions can be made on what would be suitable combinations, without actually doing a field test. ahead of the 2010 trials it was suggested that two replicates should be made. however, for each combination there is the reverse cross, which makes a second replication. to obtain reasonable numbers for estimation of pollination compatibility, it was arranged that at least 100 flowers should be pollinated in each combination, but it was not considered necessary to pollinate more than 200 flowers for each combination. after 2010, the flowers were protected from bee pollination by nylon netting rather than cotton muslin. the nylon netting did not absorb moisture during rain, thus avoiding damage to flowers in wet and windy conditions. a substantial number of results were obtained from the 2012 trials. unfortunately in 2013, there were severe late frosts in the north of afghanistan, fig. 1 layouts of almond national collection blocks for cross pollination. each box in the diagram represents one tree in each variety block. each variety is coded by a letter, in order to show the systematic layout of the trial. the code sheets would represent a different accession in each year's trials. cullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 221 which meant that no results were obtained that year. late frosts also affected pollination trials in the north of afghanistan in 2014, and results were limited to the almonds in demonstration plots in kabul, which is a much later flowering site and has not lost a crop due to frost since planting out almond varieties in 2009. 4. successful combinations when tested for cross pollination compatibility 2008-2012 some 93 different combinations of afghan almond accessions (186 combinations when the reverse cross is taken into account) have been shown to be successful. this is calculated before the accession names have since been updated/rationalised in the national register of almond varieties published in 2014. no further information can be deduced from a list of compatible varieties. what is more useful for making further deductions about the possibilities of successful combinations is the classification of pollination groups (kester et al., 1994), which is derived from information about combinations of varieties which do not successfully cross pollinate, as below. from the information about incompatible combinations above, the following attempt was made to classify the different varieties in incompatibility groups, based on results up to 2012. only later was it decided to work on the assumption that sattarbai accessions 168, 771 and 1001 were all the same variety. it was also decided without the incompatibility data that sattarbai 142 should be reclassified as qambari, thus the incompatibility information would indicate that accessions 142 and 143 can be treated as different clones of the same variety. the results are reported in table 2 and 3, where the confirmed incompatible combinations and the individuated incompatibility groups are indicated. at this regard, it is worth noticing that group a can’t be said to be definitively separated from the others, due to non sufficient data at the end of the 2012 trials. conversely, groups b, c and d are considered different. 5. cross pollination for variety verification a range of similar sattarbai accessions, collected under accession number 168, 771 and 1001 from different growers had proven to be very similar when characterised across a range of leaf, flower and fruit characters per the upov standards (upov, 2011). accession numbers 168 and 1001 were also included in the set of nut samples to be assessed by the almond traders in october 2008, and were given almost identical market values ahead of all the other almond varieties. it seemed too much of a coincidence when such similar and high value varieties proved to be incompatible with each other. it is difficult to imagine a scenario whereby one of those three accessions is the parent of the other, because table 2 list of incompatible combinations of almond accessions belonging to the national collection of aghanistan site year accessions khulm 2008 sattarbai bakhmali 149 x bellabai 144 khulm 2008 sattarbai bakhmali 149 x zang kaftar 148 khulm 2008 qambari 143 x sattarbai no. 4 -154 aybak 2008 shokurbai 162 x sattarbai guldar 157 kunduz 2011 qambari 143 x sattarbai 142 kunduz 2011 qambari 143 x qambari 2009 kunduz 2011 sattarbai 142 x qambari 143 kunduz 2011 sattarbai 142 x qambari 2009 kunduz 2011 qambari 2009 x sattarbai 142 kunduz 2011 sattarbai bakhmali 2008 x sattarbai no.4 -154 kunduz 2011 sattarbai 168 x sattarbai 771 mazar 2012 sattarbai 168 x sattarbai 1001 mazar 2012 sattarbai 771 x sattarbai sufi 145 mazar 2012 sattarbai 771 x sattarbai bakhmali 159 mazar 2012 sattarbai sais 777 x sattarbai guldar 157 kunduz 2012 khairodini 846 x abdulwahidi 153 a b c d e sattarbai bakhmali 149 qambari 143 shokorbai 162 sattarbai 168 khairodini 846 bellabai 144 sattarbai no.4 -154 sattarbai guldar 157 sattarbai 771 abdulwahidi 153 zang kaftar 148 sattarbai bakhmali 2008 sattarbai sais 777 sattarbai 1001 sattarbai 142 sattarbai sufi 145 qambari 2009 sattarbai bakhmali 159 table 3 incompatibility groups defined for some almond accessions of the national collection of almond of afghanistan this designation of incompatibility group does not refer to any other classification of almond incompatibility groups that might have been used by other authors. adv. hort. sci., 2016 30(4): 217-223 222 of the intrinsic nature of the incompatibility genes. the most logical explanation, because it is also the simplest, is that the three accession numbers 168, 771, and 1001 were just three different samples (clones) of the one variety that had spread across the north of afghanistan by vegetative propagation by different nursery growers. the three different clones had already been distributed to registered mother stock growers, and all three continue to be maintained under their accession numbers, but the final saplings are all marketed under the new name of “sattarbai mumtaz” (=”superior sattarbai”)(ministry of agriculture, irrigation and livestrock, 2014). following this rationalisation of the sattarbai accessions, it was decided to use this approach to try to identify other duplicates in the national collections. the accession cardinal seemed to be phenotypically very similar to the accession of carmel in the collection. since no other record of an almond variety called cardinal could be found, and because it was known that variety names gradually changed after introduction into afghanistan with unmanaged distribution of varieties, the hypothesis that cardinal was actually a misnamed carmel was tested by attempting cross pollination of the two accessions in the demonstration orchard in kabul. using the same protocol as with the other pollination testing, it was shown that cardinal and carmel would not cross with each other, with pollination in either direction. this was taken as confirmation that cardinal was the same as carmel and was therefore removed from the national collection. other pollination tests were done with the same objective in mind. an accession collected as nonpareil 6041 appeared to be very similar to carmel, but cross pollination with carmel set seed, indicating that 6041 was possibly an offspring of carmel, perhaps from a cross between nonpareil and carmel, which two varieties would have been introduced together into orchards in afghanistan in the 1990s. this accession, 6041, was also included in the sets of six varieties as was normally used in the cross pollination trials, and some of its offspring from crosses with superior afghan almond types continue to be grown and tested in the almond breeding programme. another accession, collected as sattarbai 6038, was considered to be very similar with the varieties ferragnes and ferraduel. again, cross pollination trials with all combinations of ferragnes, ferraduel and 6038 showed that accession 6038 was not either ferragnes or ferraduel. again, ferragnes and ferraduel were also introduced into afghanistan in the 1990s as a combination to be planted together, so accession 6038 could be from a seedling resulting from a cross of those two varieties. 6. incompatibility in almond varieties of afghanistan: final remarks all the trials on pollination in afghanistan have been done within the context of a development project, the perennial horticulture development project (phdp) and its successor project phdpii. the projects have been successful in developing perennial horticulture from the very foundations of the industry, that is from deriving knowledge about how to use the native and imported germplasm to develop sustainable economic production systems. further development continues with continuing private and public sector programmes financed by the european union, within a programme framework that is now attracting other donors. the adaptive research programmes have been based on very simple and basic concepts that are implemented in the context of a country devastated by more than 35 years of almost continuous warfare and internal strife. almonds have been a traditional export for many years, and if a connection can be shown with the fergana valley to the north in tajikistan, then the afghans can claim an almond export industry to india and the persian gulf region for at least five hundred years (hiro, 2006). in terms of planting almond orchards, it is now possible to plant just two varieties in pairs that cross pollinate each other, in the same way there are the famous combinations of nonpareil and carmel in california, and ferragnes and ferraduel in france. a good combination for afghanistan could be nonpareil planted with sattarbai mumtaz. what is clear however, is that there must be quite a few different incompatibility genotypes among the afghan almond germplasm, so the growers can be quite confident, that in the absence of any specific data on varietal combinations, the planting of four different varieties will almost certainly give good fruit set. it should also be noted that there is no reason to think that these four different varieties could not all be different sattarbai types. future work is provide information on the recommended combinations, to demonstrate the imporcullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 223 tance of pollination with bees, to ensure orchard growers understand the importance of planting mixtures of varieties, and taking remedial measures where they have single variety orchards. more combinations of varieties need to be tested for pollination compatibility, until recommendations can be made for all required commercial varieties. however, the testing of further combinations can probably wait until capacity in the plant biotechnology laboratory in kabul is increased to allow for direct examination of the incompatibility factors within the dna of each accession. the testing of almond pollination within the context of a development project has been simplified because of necessity, such that emasculation was not practised (kester et al., 1994). the theory of incompatibility has been extended to encompass pollination testing as an extra verification method in the identification of varieties. the results of the pollination trials, that are the seeds from the inter variety crosses, have been planted out and formed the beginnings of a selection process for almond crosses which is expected to result in the release of several improved lines to growers by 2017. the breeding programme has taken on a life of its own, and is now focused on achieving late flowering, productive afghan almond types. innovative methods of cross pollinating early and very late flowering almond lines have been developed, to ingress late flowering characters into the local germplasm. references hiro d., 2006 babur nama. journal of babur emperor. penguin classics, penguin books, london, england, pp. 384. kester c.e., gradziel t.m., micke w.c., 1994 identifying pollen incompatibility groups in california almond cultivars. j. amer. soc. hort. sci., 119(1): 106109. ministry of agriculture, irrigation and livestock, 2014 national collection of varieties of fruits and nuts of afghanistan. almond. ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 194. micke w.c., 1996 almond production manual . university of california anr, pp. 289. upov 2011 almond. guidelines for the conduct of tests for distinctness, uniformity and stability. ipov, geneva, switzerland. 192 1. introduction carnation (dianthus caryophyllus l.) flowers are one of the most important cut flowers cultivated commercially in the world and they are also used as ornamental plants for decorative purposes in orchards and landscapes (singh et al., 2005). vase life of cut flowers is an important characteristics in crop quality evaluation and it depends on genetic and environmental factors (seglie et al., 2011). ethylene, as a gaseous plant hormone, plays a prominent role in accelerating senescence phenomena of most plant organs such as fruits, flowers, and floral buds (yang and hoffman, 1984). when plants produce ethylene, or are exposed to an external source of ethylene, receptors perceive its presence and the signal is transferred by downstream active genes. ethylene, by inducing expression of senescence-related genes, affects the physiological characteristics vegetative and reproductive organs resulting in organ abscission and yellowing (ahmadi et al., 2008). it has been found that exogenous ethylene in some plants like miniature roses, geranium, and begonia has undesirable effects on flower quality and results in accelerating senescence and decreasing flower life (ahmadi et al., 2009; seglie et al., 2011). application of external ethylene in rose petals causes a considerable increase in ethylene production and activity of acc-synthase and acc-oxidase genes (ma et al., 2006; ahmadi et al., 2009), although ethylene receptor genes are not affected by external ethylene (ahmadi et al., 2009). senescence is an oxidative process in which reactive oxygen species and antioxidants play an important role. in general, reactive oxygen species accelerate flower senescence by increasing cell membrane permeability due to decreasing proteins and nucleic acids resulting from different protease and nuclease enzyme activities (barth et al., 2006). decreasing antioxidant enzyme activity and increasing peroxidation of cell membrane lipids have been indicated as possible reasons of senescence in different plant species (buchanan-wollaston, 1997). in addition, cell death is accelerated in this phase because of a boost in ethylene production during the senescence period (ebeles et al., 1992). plants gain from antioxidant mechanisms to effects of 1-mcp and ethylene on antioxidant enzyme activity and postharvest physio-biochemical characteristics of cut carnation flower cv. fortune a. ranjbar, n. ahmadi (*) department of horticultural science, faculty of agriculture, tarbiat modares university, po box 14115-336 tehran, iran. key word: ethylene, peroxidase enzyme, postharvest longevity, senescence, superoxide dismutase. abstract: carnation (dianthus caryophyllus l.) flowers are one of the most important cut flowers in the world. the majority of carnation cultivars are sensitive to ethylene which affects the physiological and biochemical postharvest characteristics of these flowers. applying inhibitors of biosynthesis and action of ethylene is important factor to protect the display quality and extend postharvest life. in order to evaluate the effects of 1-methylcyclopropene (1-mcp) and ethylene on the antioxidant enzyme activity of cut carnation cv. fortune and subsequently on the extension of vase-life, this experiment was designed as a completely randomized design (crd) with three replications. carnation cut flowers were treated with 1-mcp at concentrations of 0, 0.5, 1 and 1.5 µl/l for 24 h and subsequently exposed to ethylene (1 µl/l) for 16 h. data were analyzed using mstat-c statistical software and means were compared based on least significant differences (lsd) test (p< 0.01). our results showed that 1-mcp treatment had significant effects on vase life and biochemical characteristics like contents of leaf chlorophyll, petal anthocyanin, petal cell membrane stability and antioxidant enzymes activity such as catalase, peroxidase, and superoxide dismutase. the highest vase life and cell membrane stability were appeared in samples treated with 1.5 µl/l 1-mcp which was significantly higher than 0 and 0.5 treatments, although there was no significant difference with 1 µl treatment. the highest chlorophyll and anthocyanin contents were also measured under 1.5 µl/l 1-mcp which was significantly higher than other treatments. the highest and lowest catalase and peroxidase activity were related to 1 and 0 µl/l 1-mcp, respectively. the highest and lowest superoxide dismutase activity was observed in 1.5 and 0 µl/l 1-mcp. in conclusion, application of 1-mcp improved and delayed the onset of senescence symptoms resulted in extending the vase life of cut carnation cv. fortune. adv. hort. sci., 2015 29(4): 192-198 (*) corresponding author: ahmadin@madares.ac.ir received for publication 30 june 2015 accepted for publication 15 august 2015 193 ahmadi, effects of 1-mcp and ethylene on antioxidant enzyme activity of cut carnation flower cv. fortune alleviate the effects of free radicals. these mechanisms include changes in content of defense-related enzymes such as peroxidase, catalase, polyphenol oxidase and other compounds like phenols (staskawicz et al., 1995). since postharvest senescence is an important restricting factor in crop presentation and marketability of many cut-flowers, using high confidence methods to delay crop senescence is of great importance. 1-mcp as an anti-ethylene compound has been proved to be effective in inhibiting ethylene response by competing with ethylene for placing on the site of ethylene receptors (seglie et al., 2011; daneshi nergi and ahmadi, 2014). studies have shown that 1-mcp has inhibited the phenomena of petal fall in geranium, considering that its effectiveness depends on transport conditions, storage temperature, and application times (cameron and reid, 2001). studies on cut carnation showed that all concentrations of 1-mcp decreased ethylene production and chlorophyll destruction was delayed in comparison with control plants (asil et al., 2013). black tulip flowers treated with 1-mcp for 8 h showed maximum anthocyanin till the twelfth day of vase life (chutichudet et al., 2010). application of 1-mcp on soya plants decreased hydrogen peroxide in comparison with untreated plants as well as production of ethylene and free radicals but increased the activity of antioxidant enzymes (djanaguiraman et al., 2011). considering the role of 1-mcp as an ethylene inhibitor, the purpose of the current study was to evaluate 1-mcp efficacy in extending display-quality ‘fortune’ carnation flowers. to gain a deep understanding of biochemical characteristics of cut-flowers, enzyme assays were evaluated in this experiment. 2. materials and methods carnation cut-flowers of ‘fortune’ cultivar were harvested from commercial greenhouses in pakdasht (iran) according to standard indexes. flowers were immediately transferred to the laboratory of post harvest physiology of the horticulture department, faculty of agriculture, tarbiat modares university. healthy and uniform flowers were selected for the considered treatments. cut-flowers were placed in the vase solution and treated with 1-mcp (0, 0.5, 1 and 1.5 µl/l) for 24 h in 200 l glass aquarium chambers. after 1 h of ventilation, the aquarium lids were re-sealed and ethylene was injected inside each chamber using a hamilton syringe to expose cut flowers to 1 µl/l ethylene for 16 h (daneshi nergi and ahmadi, 2014). after termination of the ethylene treatment, the lids of the glass chambers were removed and the vases were placed on the lab bench. experiments were run under the following conditions: temperature 20±2°c, relative humidity of 60-65%, light intensity of 15 µmol/m2s-1 with 12 h light and 12 h darkness (daneshi nergi and ahmadi, 2014). experimentation was conducted in a completely randomized design with three replications and four treatments. sampling was carried out to evaluate physiological and biochemical characteristics at desired times and data were analysed using mstat-c statistical software; means were compared according to the least significant differences (lsd) test (p<0.01) and graphs were designed using excel software. vase life in this study, vase life was considered the time during which cut-flowers can keep their market quality and before senescence symptoms, including bending of petal margins and wilting, appear (singh, 1994). cut-flower durability was based on day distance after the end of cut-flower treatment till their ornamental value disappeared. leaf chlorophyll analysis to measure chlorophyll, leaf samples of 0.5 g were ground using a mortar and pestle with liquid nitrogen, dipped in 15 ml 80% acetone in test tubes and centrifuged at 6000 rpm and 4°c. the absorbance of the solutions was read against blank (solvent) at 663 and 646 nm using a spectrophotometer (bio-rad) (richardson et al., 2002). chlorophyll content was calculated as follows: chlorophyll a (µg.ml) = 12/5a 663 -2/79a 646 chlorophyll b (µg.ml) = 21/51a 646 -5/1a 663 total chlorophyll (µg.ml) = chlorophyll a + chlorophyll b petal anthocyanin petal samples of 200 mg were pulverized in 3 ml 99:1 (v/v) methanol and hydrochloric acid and the obtained extracts were centrifuged at 12000 rpm for 20 min at 4°c. supernatants were kept at 4°c under darkness for 24 h. light absorption was then estimated by spectrophotometer at 550 nm wavelength and using silence coefficient (=33000 mol2 cm-1) (krizek et al., 1993). petal membrane stability index to determine petal membrane stability, two petal samples, each including 200 mg of each replication, were weighted and dipped in 10 ml double distilled water. one sample was placed in 40°c benmary for 30 min and the other 100°c benmary for 15 min. after cooling the sample of 40°c benmary and reaching room temperature, electrical conductivity of the solutions was measured with a conductivity meter and the stability percent of the membrane was determined according ezhilmathi et al. (2007) as follows: membrane stability index (percent) = [1-(c1/c2)] × 100 enzyme assays peroxidase (pod) enzyme. peroxidase (pod) was extracted from 200 mg homogenized samples in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 28 mm guaiacol, 5 mm hydrogen peroxide and crude extract was prepared and its absorbance at 470 nm was detected for 1 min, using spectrophotometer (bio-rad). enzyme activ194 adv. hort. sci., 2015 29(4): 192-198 ity was expressed as absorption delta of 470 nm per mg protein (chance and maehly, 1955). catalase (cat) enzyme. catalase (cat) was extracted from 200 mg samples homogenized in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. the supernatant was transferred to 15 ml tubes and referred to enzyme extract. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 10 mm hydrogen peroxide and crude extract was prepared and its absorbance at 240 nm was detected using a spectrophotometer (bio-rad). enzyme activity was described by measuring the conversion rate of hydrogen peroxide to water and oxygen molecules, as the decrease of absorbance per time per mg of protein (cakmak and horst, 1991). enzyme activity was expressed as absorption delta of 240 nm per mg protein. all steps of enzyme extraction were performed on ice. superoxide dismutase (sod) enzyme. 200 mg of plant tissues were extracted in 50 mm hepes-koh buffer (ph 7.8) containing 0.1 mm edta. the homogenate was transferred to centrifuge tubes and was centrifuged at 12000 rpm for 30 min at 4°c. the supernatant was transferred to 15 ml tubes and referred to enzyme extract. for assay, a mixture consisting of 50 mm hepes-koh (ph 7.8) containing 0.1 mm na-edta, 50 mm na 2 co 3 (ph 10.2), 12 mm l-methionine, 75 µm nitro blue tetrazolium (nbt), 1µm riboflavin and crude extract was prepared and enzymatic extract as a unit of sod activity was considered as enzymatic amount which resulted in 50% inhibition of nbt in 560 nm (chance and maehly, 1955). reaction mix absorption was measured by spectrophotometer. total soluble protein was measured using the bradford (1976) method. absorption of 1 ml bradford reagent along with 100 µl enzymatic extract were mixed completely and registered in 595 nm. protein content was estimated using calibration curve of cow albumin serum (bsa) (bradford, 1976). 3. results and discussion vase life vase life is one of the important post harvest characteristics of ornamental plants especially for cut carnation, which is highly affected by ethylene. reduction of unfavorable effects of ethylene is an appropriate method for enhancing postharvest durability of plants or plant organs and compounds like 1-mcp are used extensively in order to alleviate the undesired effects of ethylene in horticultural crops. results showed that the effect of 1-mcp on characteristics of cut carnation cv. fortune was significant (p<0.01). increasing 1-mcp concentration enhanced vase life of cut flowers so that the longest vase life (11.8 days) was related to 1.5 µl/l 1-mcp which was significantly (p<0.01) higher than control and 0.5 µl/l 1-mcp, while there was no significant (p<0.01) difference with 1 µl/l 1-mcp. by preventing external ethylene action, treatment with 1-mcp increased vase life (fig. 1). in accordance with our results, yamane et al. (2004) showed that 1-mcp increased vase life of cattleya flowers. it seems that 1-mcp prevents acc-oxidase and acc synthase expression, subsequently decreasing ethylene production in primary days of treatment with 1-mcp, resulting in increased vase life (yamane et al., 2004; in et al., 2013; yang et al., 2013). this is also in agreement with the results of chutichudet et al. (2010) who reported that 1-mcp protects tulip cut-flower quality by preventing ethylene production (chutichudet et al., 2010). hence, increasing vase life of cut-flowers treated with 1-mcp is related to inhibiting ethylene action and ethylene biosynthesis (serek et al., 1994; serek and sisler, 2001). membrane stability index 1-mcp treatment preserved membrane stability in carnation cut flowers. the greatest membrane stability on the seventh day after treatment was related to 1.5 µl/l 1-mcp with no significant (p<0.01) difference toward 1 µl/l, but it was significantly (p<0.01) higher than control and 0.5 µl/l 1-mcp. 1-mcp can prevent membrane degradation by decreasing the lipid peroxidation which is regulated by ethylene (yuan et al., 2010) (fig. 2). since ethylene is the main factor for increasing respiration rate in climacteric crops and causes an acceleration of physical characteristic changes and cell membrane phospholipids degradation, it seems that 1-mcp treatment protects membrane stability by preventing ethylene action. chlorophyll content based on the results, the highest chlorophyll content on the seventh day after treatment measured in 1.5 µl/l 1-mcp showed a significant (p<0.01) difference with the other treatments. the effect of 1-mcp on protecting chlorophyll content is a result of ethylene action and consequently inhibition of ethylene biosynthesis, which is considered the fig. 1 effect of 1-mcp on vase life of carnation cv. fortune. data are means with standard errors (n=3). 195 ahmadi, effects of 1-mcp and ethylene on antioxidant enzyme activity of cut carnation flower cv. fortune most important factor of leaf chlorosis in ornamental plants. in accordance with the present results, 1-mcp treatment at all concentrations decreased ethylene biosynthesis which was followed by reduction of chlorophyll destruction compared to control plants (asil et al., 2013) (fig. 3). according to serek et al. (1998) 1-mcp inhibited leaf chlorosis in chrysanthemum and geranium cv. isable (serek et al., 1998). in this case, the effect of 1-mcp was attributed to binding ethylene receptors. a recent study showed that 1mcp can keep carbon assimilation in a condition of good efficiency and prevent tricarboxylic acid cycle. under these conditions, by provoking the biosynthesis of gibberellins, the postharvest senescence process is retarded which appeared as preserving the postharvest quality (wang et al., 2014). anthocyanin content our results showed that the highest anthocyanin content on the seventh day after treatment was related to 1.5 µl/l 1-mcp with significant (p<0.01) differences with the other treatments (fig. 4). positive effects of 1-mcp on inhibition of external ethylene action, delaying senescence and accordingly protecting suitable cell ph were the factors of anthocyanin photosynthesis pigments. chutichudet et al. (2010) found that black tulip cut flowers treated with 300 ppb 1-mcp for 8 h had the highest anthocyanin content till the twelfth day of vase life (chutichudet et al., 2010). usually, postharvest destruction of anthocyanin pigments is as a result of bracteoles’ membrane function destruction (jiang and chen 1995; jiang et al., 2004). the stability of anthocyanin may be due to the role of 1-mcp, which can decrease membrane destruction of fresh crops (hershkovitz et al., 2005). vacuole ph is enhanced during senescence and anthocyanin gets free of color before destruction (zhang et al., 2001). furthermore, anthocyanin destruction occurs as a result of polyphenol oxidase activity (francis, 1989). the preventing from anthocyanins accumulation in tissues could be ascribed to ethylene action, based on research on arabidopsis thaliana showed negative self-regulation in relationship of ethylene, carbohydrates and anthocyanins. stress induced by ethylene resulted in reduced absorption of carbohydrates and consequently decreased accumulation of pigments (das et al., 2011). antioxidant enzymes the highest activity of catalase was found in flowers treated with 1 µl/l 1-mcp, however it was not significantly (p<0.01) higher than 1.5 µl/l but was significantly (p< 0.01) higher than control and 0.5 µl/l 1-mcp treatments (fig. 5). the highest activity of peroxidase was detected in 1 µl/l 1-mcp with significant (p<0.01) differences toward the other treatments (fig. 6). 1-mcp protected antioxidant enzyme activity by inhibiting ethylene action and biosynthefig. 2 effect of 1-mcp on petal membrane stability of carnation cv. fortune. data are means with standard errors (n=3). fig. 3 effect of 1-mcp on petal chlorophyll content of carnation cv. fortune. data are means with standard errors (n=3). fig. 4 effect of 1-mcp on petal anthocyanin content of carnation cv. fortune. data are means with standard errors (n=3). 196 adv. hort. sci., 2015 29(4): 192-198 sis, and subsequently decreased respiration rate. the petal senescence process causes metabolic and physiological changes, which result in the petal death. senescence begins with the expression of a set of genes related to senescence and it emerges at metabolic level as oxidative processes and often the catabolic processes in senescence increase are irreversible (buchanan-wollaston, 1997). in microsomal membranes of carnation, many superoxides are generated during senescence (mayak et al., 1983). here also, increasing free radicals from ethylene stress in plants untreated with (0 µl/1) 1-mcp damaged to immunity system of antioxidant and reduction of immunity enzymes activity. when treated with different concentrations of 1-mcp, there was more antioxidant enzyme activity because of decreasing effects of external ethylene. peroxidase has different biological functions, such as detoxification of hydrogen peroxide, lignin biosynthesis, hormonal signaling and response to stress (gao et al., 2010). catalase is considered an important biological factor and its major function is in the process of superoxide metabolism by playing role in releasing oxygen and hydrogen peroxide free radicals and preventing creation of hydroxyl radicals (spanou et al., 2012). superoxide dismutase like cu-zn superoxide dismutase, mn superoxide dismutase and outside cell superoxide dismutase play a critical role in inhibition of superoxide (miao and st clair, 2009). in fact, peroxidase, catalase and superoxide dismutase play roles in protecting the metabolism balance of oxygen in plant tissues (xie et al., 2003). superoxide causes lipid peroxidation, cell membrane damage and finally senescence; 1-mcp can affect enzyme activities, which remove superoxide (li et al., 2007). in accordance with our obtained results, increasing antioxidant enzyme (catalase, superoxide dismutase and peroxidase) activity of gladiola florets treated with 1-mcp has been reported (fig. 7). it seems that this treatment decreases oxidative stresses in cut-flowers (hassan and ali, 2014). in other words, activity of these enzymes is a factor for the protection of cells against oxidative stresses (zhou et al., 2014). it should be mentioned that even if ethylene decreases in response to 1-mcp, the activity of antioxidant enzymes will increase. in addition, a considerable increase in peroxidase, catalase and superoxide dismutase activity was observed in petals of carnation flower cv. lilacon purple treated with 0.5 µl/l 1-mcp. the 1-mcp treatment fig. 5 effect of 1-mcp on petal catalase enzyme activity of carnation cv. fortune. fig. 6 effect of 1-mcp on petal peroxidase enzyme of carnation cv. fortune. fig. 7 effect of 1-mcp on petal superoxide dismutase enzyme of carnation cv. fortune. 197 ahmadi, effects of 1-mcp and ethylene on antioxidant enzyme activity of cut carnation flower cv. fortune decreased hydrogen peroxide and superoxide (peroxide anion) compared to control plants (karimi, 2014). this decreasing may be a result 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macfad.) are seedless (reuther, 1988). although a major early-maturing citrus, satsuma mandarin (c. unshiu marcow.), is seedless, almost all mid and late-maturing citruses, such as natsudaidai (c. natsudaidai hayata), iyo (c. iyo hort. ex tanaka), and hassaku (c. hassaku hort. ex tanaka), are seedy in japan (iwamasa, 1988). thus, breeding new seedless cultivars is an urgent issue to develop the japanese citrus industry. to develop seedless citrus at the diploid (2x=18) level, utilization of sterility is essential (iwamasa, 1966). sterility can be divided into three types: male sterility, female sterility, and self-incompatibility. in addition, selection of triploid (3x=27) individuals is useful for breeding seedless cultivars (ollitrault et al., 2007). therefore, various kinds of investigations on the mechanism and genetic factors underlying seedlessness in citrus have been conducted and many breeding works have been carried out to develop new seedless cultivars, many of them carried out in japan over the last few decades. in this review, the progress on studies for seedless breeding of citrus in japan, with regard to 1) seedlessness at the diploid level and 2) ploidy manipulation for seedless breeding, is discussed. 2. seedlessness at the diploid level male sterility the degree of male (pollen) sterility is variable in citrus, and usually pollen sterile accessions produce seedless or low-seedy fruits when cultivated in solid blocks. male sterility couples with parthenocarpy to produce seedless fruits when cross-pollination is prevented. even in mixed planting with pollen fertile accessions, male sterility reduces seed production and increases the percentage of seedless fruits because those accessions have a smaller chance of fertilization than male fertile ones (yamamoto et al., 1993, 1995). iwamasa (1966) and ollitrault et al. (2007) summarized the various levels of male sterility at the diploid level in citrus (table 1). chromosome aberration was one of the most important phenomena causing pollen sterility. asynapsis in ‘mukaku yuzu’ (c. junos sirbold ex tanaka) is genetically controlled, while that in ‘eureka’ lemon (c. limon (l.) burm. f.) and ‘mexican’ lime (c. aurantifolia (cristm.) swingle) is induced by low temperature (nakamura, 1943; iwamasa and iwasaki, 1962; iwamasa, 1966). reciprocal translocation is found to cause pollen sterility of ‘valencia’ orange (c. sinensis) (iwamasa, 1966). inversion is the cause of partial pollen sterility of ‘mexican’ lime (c. aurantifolia) (iwamasa, 1966). male sterility that is not caused by chromosome aberration is also well known. anther aborprogress on studies for seedless breeding of citrus in japan m. yamamoto(*) faculty of agriculture, kagoshima university, korimoto, kagoshima 890-0065, japan. key words: female sterility, male sterility, protoplast fusion, self-incompatibility, triploid. abstract: seedlessness is a desirable characteristic for both fresh and processed citrus markets and one of the most important breeding objectives. in this paper, progress on studies for seedless breeding of citrus in japan is reviewed. among the several types of male sterility, anther abortion is the strictest male sterility in citrus and was shown to be controlled by both nuclear and cytoplasmic genes. several seedless cultivars with male sterility have been developed. the mechanism and inheritance of the strictest female sterility derived from ‘mukaku kishu’ (citrus kinokuni hort. ex tanaka) were clarified and seedless cultivar and parental lines with this female sterility have been released. some self-incompatible cultivars show seedlessness when coupled with parthenocarpy. s (self-incompatibility) genotypes of several cultivars have been estimated. tetraploid plants, as parents of triploid offspring, were obtained from nucellar seedlings, and by ploidy mutation, colchicine treatment, and protoplast fusion. triploid plants were produced from the combination of not only tetraploid and diploid crosses but also diploid and diploid crosses. new triploid seedless cultivars were bred by programmed cross-breeding and protoplast fusion. adv. hort. sci., 2014 28(2): 64-72 (*) corresponding author: yamasa@agri.kagoshima-u.ac.jp received for publication 31 march 2014 accepted for publication 17 june 2014 review paper 65 tion in satsuma mandarin (c. unshiu) hybrids is the strictest male sterility in citrus (iwamasa, 1966). the sterile stamen appears only as the filament, and no pollen grains are produced (fig. 1). male sterility of ‘washington’ navel (c. sinensis), ‘tahiti’ lime (c. latifolia tanaka), and some other hybrids is due to early degeneration of pollen mother cells (pmcs) (osawa, 1912; uphof, 1931; frost, 1948; iwamasa, 1966). pollen sterility of satsuma mandarin (c. unshiu) is caused by plural sterility such as abnormal behavior and degeneration of pollen grains (nakamura, 1943; yang and nakagawa, 1969, 1970). to develop new seedless cultivars efficiently, genetic analysis of male sterility has been conducted. among these studies, genetic analysis of anther abortion has progressed remarkably. this male sterility is due to genecytoplasmic interaction [satsuma mandarin (c. unshiu), ‘encore’ mandarin (c. nobilis lour. × c. deliciosa ten.), yuzu (c. junos), and lemon (c. limon) possess sterile cytoplasm] and is probably controlled by more than one major gene (iwamasa, 1966; yamamoto et al., 1992 a, b, 1997; nakano et al., 2001; dewi et al., 2013 a). dewi et al. (2013 a) postulated that a dominant nuclear fertilityrestoring gene system comprising one epistatic gene and two complementary genes controls the restoration of male table 1 diagramatic representation of various kinds of the male-sterility in citrus, according to the sequential order of development (modified from iwamasa, 1966) developmental stage nature of sterility cultivar or hybrid reference initiation of anther development anther abortion satsuma mandarin × sweet orange, etc. iwamasa, 1966 archesporial stage resting stage degeneration of pmcs washington navel tahiti lime lemon × valencia, etc. satsuma mandarin × trifoliate orange osawa, 1912 uphof, 1931 frost, 1948 iwamasa, 1966 meiosis i-division asynapsis (genic) asynapsis (by low temp.) translocation inversion mukaku yuzu eureka lemon mexican lime c. assamensis valencia orange c. assamensis, etc mexican lime iwamasa, 1966 nakamura, 1943 iwamasa and iwasaki., 1962 naithani and raghuvanshi, 1958 iwamasa, 1966 raghuvanshi, 1962 a iwamasa, 1966 ii-division failure of spindle marsh grapefruit raghuvanshi, 1962 b liberation from tetrad degeneration jaffa orange oppenheim and frankel, 1929 mitotic division degeneration satsuma mandarin nakamura, 1943 mature pollen grain fig. 1 flowers of male-sterile (aborted anthers, ms) and male-fertile (normal anthers, mf) citrus. 66 fertility and male-sterile anther size in citrus plants with sterile cytoplasm. nakano et al. (2000) found dna markers linked to aborted anther for juvenile screening of malesterile plants. male-sterile (aborted anther) progenies were also determined at the early period of seedling growth to exhibit precocious flowering, a phenomenon in which very young seedlings have flowers (dewi et al., 2013 b). another type of male sterility, inheritance of pollen fertility/sterility, was also studied (ueno, 1986). some pollensterile progenies arose from two pollen fertile parents. in citrus, not only somatic hybrids but also cybrids were produced by symmetric protoplast fusion (saito et al., 1993; moriguchi et al., 1996; 1997; tokunaga et al., 1999). yamamoto and kobayashi (1995) produced a cybrid having the sweet orange (c. sinensis) nuclear genome and satsuma mandarin (c. unshiu) cytoplasmic genome by fusion between satsuma mandarin protoplasts isolated from embryogenic callus and sweet orange mesophyll protoplasts. the cybrid is useful for seedless breeding because it has sterile cytoplasm derived from satsuma mandarin. several new seedless cultivars with male sterility were released in japan (nishiura et al., 1983; okudai et al., 1991; matsumoto et al., 1991, 2003; yoshida et al., 2005 c) (fig. 2, table 2). all cultivars with aborted anther possess cytoplasm derived from satsuma mandarin (c. unshiu). female sterility female sterility is a very important trait which is closely related to seedlessness. yamamoto et al. (1995) reported that the degree of female fertility/sterility is rated on the basis of the average number of seeds per fruit obtained through hand pollination. a high positive correlation (r = 0.93**) was found between the number of seeds of handpollinated fruits and that of open-pollinated fruit. this result indicated that female sterility is directly related to seediness. female sterility estimated by the above-mentioned method was revealed to be a heritable characteristic (yamamoto et al., 2001). table 2 seedless cultivars and parental lines of citrus released in japan cultivar or parental line cross combination note male sterility kiyomi miyagawa wase (citrus unshiu) × trovita (c. sinensis) seiho kiyomi × minneola (c. paradisi × c. tangerina) tsunokaori kiyomi × okitsu wase (c. unshiu) amaka kiyomi × encore (c. nobilis × c. deliciosa) setoka (kiyomi × encore) no. 2 × murcott (probably tangor) harehime e647 (kiyomi × osceola) × miyagawa wase (c. unshiu) tsunokagayaki (kiyomi × okitsu wase) no. 14 × encore (c. nobilis × c. deliciosa) female sterility southern yellow tanikawa buntan (c. maxima) × mukaku kishu (c. kinokuni) citrus parental line norin no. 5 lee (clementine × orlando) × mukaku kishu (c. kinokuni) citrus parental line norin no. 6 king mandarin (c. nobilis) × mukaku kishu (c. kinokuni) self-incompatibility ariake seike navel (c. sinensis) × clementine (c. clementina) triploid puchimaru oval kumquat (fortunella margarita) × tetraploid meiwa kumquat (c. crassifolia) white love cybrid having lemon (c. limon) somatic hybrid nuclear and sudachi (c. sudachi) cytoplasmic genome + haploid clementine (c. clementina) tokushima 3x no. 1 tetraploid sudachi hs4 (c. sudachi) × sudachi ryokuko-kei (c. sudachi) yellow bell open-pollinated seedling of diploid michitani-line villafranca (c. limon) fig. 2 male-sterile seedless citrus ‘setoka’. 67 ‘mukaku kishu’, a bud variant of the seedy kinokuni mandarin (citrus kinokuni hort. ex tanaka), is completely seedless and considered to have the strictest female sterility in citrus. yamasaki et al. (2007, 2009) studied the mechanism of expression of seedlessness derived from ‘mukaku kishu’. in fruits of ‘mukaku kishu’, specific very small and swollen seeds called “type a seeds” were observed. the expression of ‘mukaku kishu’-type seedlessness is characterized by formation of “type a seed” with an immature seed coat and an embryo arrested at an early stage. however, arrested embryo development in the “ type a seed” is not caused by endosperm abortion. this female sterility is controlled by two major genes: sterility and fertility are dominant and recessive, respectively (nesumi et al., 2001). new seedless cultivar and parental lines with this sterility were bred in japan (yoshida et al., 2005 a, b) (fig. 3, table 2). navel orange (c. sinensis) and satsuma mandarin (c. unshiu) have strong female sterility; only a few seeds were developed when they were hand-pollinated (miki, 1921; nagai and tanikawa, 1926; nishiura and iwasaki, 1963; yamamoto et al., 1995). osawa (1912) observed degeneration of the embryo sac in both navel orange and satsuma mandarin. nesumi et al. (2000) assumed that the female sterility of satsuma mandarin is controlled by two major genes: sterility and fertility are recessive and dominant, respectively, and they were mapped on a linkage map (omura et al., 2000). self-incompatibility self-incompatibility is a genetically controlled phenomenon preventing seed set in self-pollinated plants producing functional gametes. self-incompatibility in citrus is a very important trait for fruit production. without parthenocarpy, it requires cross pollination to achieve stable fruit production (nagai and tanikawa, 1926; miwa, 1951). however, its coupling with parthenocarpy could produce seedless fruit (iwamasa and oba, 1980; yamamoto et al., 1995; yamamoto and tominaga, 2002). thus, much research has been conducted to determine self-incompatibility of many accessions (nagai and tanikawa, 1926; miwa, 1951; nishiura and iwasaki, 1963; iwamasa and oba, 1980; yamamoto and tominaga, 2002; yamamoto et al., 2006, 2012). the incompatibility system of citrus is of the gametophytic type and soost (1965, 1969) proposed s (self-incompatibility) genotypes of some accessions. since then, the source of s genotypes has been less well elucidated. recently, however, research has progressed remarkably via certain methods. pollen tube growth was strongly inhibited in incompatible pollination; those pollen tubes exhibited abnormal behaviors, namely twisted and heavy and irregular callose deposition. on the other hand, in compatible pollination, many normal pollen tubes penetrated into the style (ngo et al., 2001). self-incompatibility s genotypes of several citrus cultivars were estimated by the observation of pollen tube behavior in the styles after controlled pollination with a restricted number of pollen grains on their stigmas (ngo et al., 2010). cross-incompatible, cross-semi-compatible, and cross-full-compatible relationships were clarified based on the results of the number of pollen tubes that reached the base of the style. from these results, s genotypes of several cultivars were estimated. the s genotype could be estimated with the aid of allozymes produced by the glutamate oxaloacetate transaminase isozyme gene (got-3), which appeared to be linked to the s gene (ngo et al., 2011). the most efficient way to determine the s genotype is considered to be pollination with pollen homozygous for the s genotype. kim et al. (2010, 2011) revealed the s genotype of some cultivars by pollination of homozygous s 1 seedlings of ‘hirado buntan’ [c. maxima (burm.) merr.] and ‘banpeiyu’ (c. maxima). there are no differences in estimated s genotypes among the three above-mentioned methods. table 3 shows the estimated s genotypes of several accessions. incompatibility s alleles are distributed widely, not only in self-incompatible accessions but also self-compatible ones such as satsuma mandarin (c. unshiu), grapefruit (c. paradisi), and ‘dancy’ (c. tangerina hort. ex tanaka) (soost, 1965, 1969; vardi et al., 2000). thus, self-incompatible individuals can be produced from cross combinations between two selffig. 3 female-sterile seedless citrus ‘southern yellow’. table 3 estimated s genotype of citrus accessions (kim et al., 2011; ngo et al., 2010, 2011) accession latin name estimated s genotype (z) banpeiyu citrus maxima (burm.) merr. s 1 s 2 tosa buntan c. maxima (burm.) merr. s 1 s 3 iriki buntan c. maxima (burm.) merr. s 1 s 2 kaopang c. maxima (burm.) merr. s 1 s 2 soyu c. maxima (burm.) merr. s 1 s 2 hassaku c. hassaku hort. ex tanaka s 4 s 5 yuge-hyokan c. yuge-hyohan hort. ex yu. tanaka s 6 s 7 shishiyuzu c. pseudogulgul hort. ex shirai s 1 s 6 hyuganatsu c. tamurana hort. ex tanaka s 1 s 8 tachibana no. 1 c. tachibana (makino) tanaka sfs 8 rough lemon c. jambhiri lush. sfs 1 zadaida c. aurantium l. sfs 1 kinukawa c. glaberima hort. ex tanaka sfs 2 kawano natsudaidai c. natsudaidai hort. ex tanaka sfs 2 (z) sf: self-compatible. 68 compatible parents, for example, ‘orlando’ and ‘minneola’ arose from ‘duncan’ grapefruit and ‘dancy’ combination (swingle et al., 1931). in japan as well, self-incompatible seedless ‘ariake’ was bred by crossing self-compatible ‘seike’ navel orange (c. sinensis (l.) osbeck) and self-incompatible clementine (c. clementina hort. tanaka) (yamada et al., 1995; yamamoto et al., 2006) (fig. 4, table 2). 3. ploidy manipulation for seedless cultivar breeding although spontaneous triploid (3x=27) accessions were very rare in citrus (krug, 1943; krug and bacchi, 1943; noro and kajimoto, 1955), many triploid hybrids have been produced by artificial hybridization. since these triploids are seedless, it could be considered that producing triploids is a useful way to promote seedless breeding in citrus efficiently. tachikawa et al. (1961) conducted one of the earliest programmed triploid breeding projects. first, they produced tetraploid materials (4x=36) by colchicine treatment and then triploid (2x=18) hybrids were obtained from these tetraploid and diploid cross combinations. as they showed, since triploids arise from tetraploid and diploid crossing, tetraploid plants are important for triploid breeding. therefore, various tetraploid accessions were obtained by certain methods. oiyama et al. (1980) selected spontaneous autotetraploids from nucellar seedlings of polyembryonic cultivars. they revealed the leaf morphological characteristics of tetraploids: thick and broad leaves and reduced number of stomata per area. kawase et al. (2005) obtained meiwa kumquat (fortunella crassifolia swingle) autotetraploid from 500 seedlings. an autotetraploid also arose as a bud sport (yamao et al., 1993). colchicine treatment is useful for the production of autotetraploids in many higher plants. in citrus in particular, various tetraploids were produced by this treatment. colchicine treatment of seeds was effective in polyembryonic cultivars (yahata et al., 2004). however, this treatment of monoembryonic seeds is a problem because the tetraploids obtained by this treatment are not true-to-type. oiyama and okudai (1986) resolved this problem through a combination of colchicine treatment of isolated small buds and their micrografting. they successfully produced autotetraploids from three monoembryonic cultivars. moreover, in another eight autotetraploids, monoembryonic citrus was produced using the same method (kaneyoshi et al., 2008). it has been revealed that although tetraploids arise from both diploid × tetraploid and tetraploid × diploid crosses, the latter combination is more effective (cameron and burnett, 1978; kaneyoshi et al., 2008) and an unbalanced ploidy ratio between embryo and endosperm is considered to cause this phenomenon (esen and soost, 1973). thus, artificially produced monoembryonic autotetraploids were important as seed parents for triploid breeding. on the other hand, triploid hybrids sometimes appeared from diploid-diploid crosses (esen and soost, 1971; oiyama and okudai, 1983; yasuda et al., 2010); the appearance of triploids is due to the unreduced gametophyte of one parent (esen and soost, 1971). biotechnological methods such as protoplast fusion have contributed to the progress of citrus triploid breeding. since somatic hybrids are tetraploids in general (ohgawara et al., 1985), they are important parents for triploid breeding. somatic hybrids derived from crosses between navel orange (c. sinensis) + satsuma mandarin (c. unshiu), grapefruit (c. paradisi), yuzu (c. junos) and ‘murcott’ (artificial hybrid) were registered as parental lines (kobayashi et al., 1995). triploid hybrids could be produced directly by means of protoplast fusion. somatic hybrids produced from diploid and haploid fusion became triploids (kobayashi et al., 1997). the haploid parents were obtained by diploid × triploid cross (oiyama and kobayashi, 1993). table 2 shows the triploid cultivars released in japan. among the four cultivars, ‘puchimaru’ (yoshida et al., 2003) (fig. 5), ‘white love’, tokushima 3x no. 1’ (tokufig. 5 triploid seedless kumquat ‘puchimaru’ and diploid seedy meiwa kumquat and oval kumquat. p= puchimaru, m= meiwa kumquat, and o= oval kumquat. fig. 4 self-incompatible seedless citrus ‘ariake’. 69 naga et al., 2005), and ‘yellow bell’ (kaneyoshi et al., 2014) were derived from diploid × tetraploid cross, protoplast fusion between diploid and haploid, tetraploid × diploid cross, and diploid × diploid cross, respectively. 4. conclusions seedless breeding of citrus has progressed rapidly in japan over the last few decades. the production of new seedless cultivars is increasing in contrast to the decrease in production of seedy conventional cultivars such as ‘kawano natsudaidai’ (c. natusdaidai) and ‘miyauchi iyokan’ (c. iyo). various kinds of cross combinations using cultivars or parental lines with sterility have been conducted actively and further polyploid breeding is being carried out to produce new triploid plants. in addition, methods to shorten the long juvenile period of citrus have developed in japan (okudai et al., 1980; mitani et al., 2008), making it easier to breed, compared to a few decades ago, various types of new seedless cultivars. understanding the mechanism and hereditary mode of each type of sterility will contribute to produce seedless individuals efficiently and effectively. biotechnological techniques are also very useful for producing seedless materials. the results of genome analysis have provided useful information for breeding new seedless citrus with female sterility (garcia et al., 2000). moreover, owing to the progress of recent dna analysis technologies, the draft whole genome of sweet orange (c. sinensis) has already been reported (xu et al., 2013). the combination of conventional breeding study, biotechnology, and genome analysis is considered to be essential to breed new superior seedless cultivars in citrus. references cameron j.w., burnett r.h., 1978 use of sexual tetraploid seed parents for production of triploid citrus hybrids. hortscience, 13: 167-169. dewi p.t., wakana a., tanimoto y., fujiwara y., sakai k., kajiwara k., 2013 a morphology of sterile anthers and inheritance of cytoplasmic-genetic male sterility in zygotic seedlings of polyembryonic acid citrus. j. japan. soc. hort. sci., 82: 203-214. dewi p.t., wakana a., tanimoto y., sakai k., kajiwara k., 2013 b precocious flowering of citrus seedlings and its use for determination of cultivars generating male sterile progenies. sci. hort., 160: 1-11. esen a., soost r.k., 1971 unexpected triploids in citrus: their origin, identification, and possible use. j. hered., 62: 329-333. esen a., soost r.k., 1973 seed development in citrus with special reference to 2x × 4x crosses. amer. j. bot., 60: 448462. frost h.b., 1948 seed reproduction: development of gametes and embryos, pp. 767-816. in: webber h.j., and l.d. batchelor (eds.) the citrus industry. vol. i. university of california press, barkley and los angeles, usa. garcia m.r., asins m.j., carbonell e.a., 2000 qtl analysis of yield and seed number in citrus. theor. appl. genet., 101: 487-493. iwamasa m., 1966 studies on the sterility in genus citrus with special reference to the seedlessness. bull. hort. res. sta., b6: 1-81. iwamasa m., 1988 citrus cultivars in japan. hortscience, 23: 687-690. iwamasa m., iwasaki t., 1962 on the sterility phenomenon caused by low temperatures in the mexican lime (citrus aurantifolia swingle). bull. hort. res. sta., b2: 25-46. iwamasa m., oba y., 1980 seedlessness due to self-incompatibility in egami-buntan, a japanese pummelo cultivar. bull. fac. agr., saga univ., 49: 39-45. kaneyoshi j., furuta t., kurao m., yamaguchi s., 2008 induced tetraploid by colchicine treatment in some monoembryonic citrus cultivars and triploidy production by using the tetraploid as seed parents. hort. res. 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takishita f., hidaka t., kawai s., 2005 72 b ‘kankitsu chukanbohon nou 6 gou’ (‘citrus parental line norin no. 6’) is useful for breeding seedless and functional component-rich cultivars. bull. natl. inst. fruit tree sci., 4: 53-59. yoshida t., nesumi h., yoshioka t., nakano m., ito y., murase s., takishita f., 2005 c new citrus cultivar ‘harehime’. bull. natl. inst. fruit tree sci., 4: 3745. 239 1. introduction cucumber (cucumis sativus l.), a member of the cucurbitaceae family, is grown as a summer and rainy season crop in the low and mid hills of the northwestern himalaya from april to august and fruits are available from june to october to the plains of northern india. the crop raised in the hills, being of high quality and off-season, brings good returns to the growers. f 1 hybrids in cucumber, as in many vegetable crops, have several well known advantages over open-pollinated varieties (dogra and kanwar, 2011) and hence provide a scope for the breeder to find more appropriate combinations to develop superior hybrids. f 1 hybrids are early, vigorous, high yielding, tolerant to diseases and insectpests and more efficient in the use of water and fertilizers. currently, farmers are purchasing hybrid seeds from private firms who charge exorbitant prices for seed. to tide over the situation, there is a need to develop f 1 hybrids and make their seed available to farmers at a reasonable price. for the development of superior hybrids, estimates of general combining ability of parents and specific combining ability of the crosses help to properly select parents for hybridization. moreover, use of gynoecious lines for developing cucumber hybrids makes the production of f 1 seed more cost effective. furthermore, there is urgent need to develop stable hybrids adapted to a wide range of climatic conditions. 2. materials and methods the present investigations were carried out at two locations: experimental farm nauni (l1) and experimental farm chambaghat (l2) of the department of vegetable crops, dr y s parmar university of horticulture and forestry, solan (himachal pradesh), india, which are 1276 m a.m.s.l. and 1300 m a.m.s.l., respectively. both locations fall in the mid-hill sub-temperate zone of the state of himachal pradesh; nauni lies at latitude and longitude of 300 52’n and 77° 11’ and chambaghat, 30° 55’ n and 77° 06’. all the parents except two gynoecious lines were of monoecious type. crosses among eight parents were attempted in a half-diallel fashion. the material comprising eight parents, 28 f 1 s and one check (pusa sanyog) was sown in randomized block design with three replications. spacing was 1.25x1.00 m. data were recorded on randomly selected plants for yield and horticultural characters at both the locations. griffing’s (1956) method ii model i was used to derive general and specific combining ability estimates. the analysis of variance for combining ability was based on following mathematical model: p ijk = m + g ii + g jj + s ij + b k + e ijk selecting parents for developing superior hybrids in cucumber (cucumis sativus l.) b.s. dogra* , m.s.kanwar** * regional horticultural and forestry research station, dr y s parmar university of horticulture and forestry, bhota-176041, hamirpur (hp), india. ** high mountain arid agriculture research institute (skuast-k), leh, 194101 jammu and kashmir, india. key words: cucumber, cucumis sativus, gca, hybrids, sca. abstract: estimates of general combining ability of parents and specific combining ability of the crosses help to select desidered parents for hybridization and development of superior hybrids. crosses among eight parents were attempted in a half-diallel fashion. the material comprising eight parents, 28 f1s and one check (pusa sanyog) was sown at two locations in randomized block design with three replications. the highest estimates of general combining ability (gca) were exhibited by g2 and gyn1 for most of the characters at both the locations. in general, there was close agreement between gca effects and per se performance, but in some cases it did not hold good, which may be due to a higher degree of gene action involved. the superior cross combinations which recorded high specific combining ability (sca) estimates and per se performance for yield and number of fruits were k-90 x g2 and k-90 x gyn1 and hence may be exploited for the development of f1 hybrid (s) after testing their performance at multi-locations for two to three years. adv. hort. sci., 2011 25(4): 239-244 received for publication 1 june 2011 accepted for publication 21 october 2011 240 where, p ijk = phenotypes of the hybrids between ith and jth parents in kth plots m = population mean g ii = gca effects of ith parent g jj = gca effects of jth parent s ij = sca of the crosses between ith and jth parents b k = block effects e ijk = environmental effect associated with ijkth observation 3. results and discussion analysis of variance (table 1) for combining ability revealed that the importance of gca (σ2g) was more than sca (σ2s), indicating the preponderance of additive gene action for days to first female flower appearance (dfffa) at location 1 and days to marketable maturity (dmm) at both locations. however, in all the other traits, the sca component was higher in magnitude than gca’s, indicating the preponderance of non-additive gene effects. however, mean sum of squares for gca and sca were highly significant for all the characters except tss, suggesting the importance of both additive and non-additive genetic variance in agreement with the findings of om et al. (1978). similar trends at both the locations proved that the conclusions on gene actions are authentic. the parents g 2 , gyn 1 and poinsette had negative estimates for dfffa and node at which first female flower appears (nfff) at both the locations (table 2) showing earliness in fruit bearing and were good general combiners for these characters. among f 1 ’s, the sca effects were significantly negative in 12 and 15 crosses, respectively, for these two traits at l1 (table 3) whereas significantly negative in 15 crosses for each of these two traits at l2 (table 4). the crosses lc-11 x gyn 1 (poor x high) and ec 173934 x lc-40 (poor x poor), respectively, had the highest sca effect at l1 and the crosses lc-11 x lc-40 (poor x poor) and ec 173934 x lc-40 (poor x poor), respectively, had the highest sca effects at l2 for these traits. the parents g 2 and gyn 1 (l1) and g 2 , gyn 1 and poinsette (l2) with significantly high gca estimates (with negative value) were good general combiners for dmm. crosses lc-11 x gyn 1, ec 173934 x lc-40, k-90 x g 2 and k-90 x ec 173934 had high sca estimates at both the locations for dmm. el-shawaf and baker (1978), om et al. (1978), and wang and wang (1980) also reported greater additive genetic variance for dmm. the parents g 2 and gyn 1 may be used in the hybridisation programme for developing early hybrids adapted to a wide range of climate. lc-11 x gyn 1 and ec 173934 x lc-40 may be exploited as early hybrids after further multi-locational testing. these crosses may also be exploited to produce transgressive segregants in advanced generations. with regard to fruit length, the parents gyn 1, lc-11 and k-90 were good general combiners as is evident from their high gca estimates at both locations. fourteen crosses exhibited significant sca effects. the sca effects were high in crosses poinsette x lc-40 and g 2 x poinstte involving poor x poor general combiners. k-90, k-75 and ec 173934 had the highest gca with respect to fruit width and hence were good general combiners. the sca effect was maximum in g 2 x gyn 1 involving poor x poor general combining parental lines (at l1) and in g 2 x k-75 involving poor x high general combining parental lines (at l2). in india, slicing cucumbers are preferred, therefore lengthy fruits are desirable. kupper and staub (1988) and hormuzdi and more (1989) reported contrasting results for fruit length and width due to different experimental material and environment. table 1 analysis of variance for combining ability for different characters in f 1 cucumber source of variation df character days to first female flower appearance node of first female flower days to marketable maturity fruit length fruit width tss flesh to seed cavity ratio fruit weight no. of fruits per plant yield per plant internodal length location 1 nauni gca 7 678.818 * 27.997 * 705.436 * 6.425 * 1.087 * 0.005 0.001 * 3787.657 * 9.898 * 0.735 * 9.512 * sca 28 42.264 * 3.049 * 45.029 * 3.237 * 0.243 * 0.021 0.0015 * 693.149 * 1.159 * 0.193 * 2.183 * error 70 0.557 0.228 0.562 0.004 0.002 0.0013 0.00004 62.357 0.112 0.0013 0.272 σ2g 67.826 2.777 70.487 0.642 0.108 0.0004 0.0001 372.53 0.979 0.073 0.924 σ2s 41.707 2.821 44.467 3.0233 0.240 0.020 0.002 630.79 1.047 0.191 1.911 σ2g/ σ2s 1.626 0.984 1.585 0.199 0.451 0.021 0.068 0.591 0.934 0.383 0.483 location 2 chambaghat gca 7 390.457 * 35.726 * 577.811 * 7.820 * 0.993 * 0.012 * 0.0016 * 3515.486 * 14.247 * 0.786 * 7.800 * sca 28 67.477 * 4.551 * 37.300 * 3.895 * 0.268 * 0.028 * 0.0009 * 612.551 * 1.582 * 0.181 * 1.510 * error 70 0.431 0.205 0.442 0.089 0.023 0.006 0.000035 49.232 0.148 0.0096 0.358 σ2g 39.003 3.552 57.737 0.773 0.097 0.0006 0.000159 346.630 1.409 0.078 0.744 σ2s 67.046 4.346 36.859 3.806 0.245 0.022 0.00088 563.320 1.434 0.171 1.153 σ2g/ σ2s 0.582 0.817 1.566 0.203 0.395 0.029 0.081 0.615 0.983 0.452 0.646 * significant at 5% level of significance. 241 the best general combiners for tss at both locations in order of merit were ec 173934 and lc-40. among 28 specific combinations, 16 (at l1) and 14 (at l2) crosses exhibited positive sca effects being maximum in k-90 x poinsette and poinsette x k-75 at l1 and lc-40 x gyn 1 , k-90 x poinsette and k-75 x lc-40 at l2. for flesh to seed cavity ratio (fsr), the best general combiners were poinsette, ec 173934 and gyn 1 , irrespective of locations. cross combination k-90 x k-75 at l1 and poinsette x ec 173934 at l2 had maximum sca among seven significant and positive specific combinations. in contradiction to the present results, importance of additive gene action for fsr has been reported (dogra, 1995). the parents lc-11, k-90 and k-75 depicted high per se performance with respect to fruit weight at both locations as is evident from their high gca effect (table 2). these parents had maximum concentration of favourable genes for increasing fruit weight. eleven (at l1) and 12 (at l2) specific cross combinations had significantly positive sca effects (tables 3 and 4), being maximum in k-90 x lc-11 (high x high) and k-90 x ec 173934 (high x poor). non-additive gene action for fruit weight was also obtained by ghaderi and lower (1979) in consonance with the present findings. however, gyn 1 and g 2 were identified as good general combiners for number of fruits per plant. the top specific combinations in order of merit were k-90 x g 2 , k-90 x gyn 1 and k-75 x gyn 1 involving medium high, medium x high and poor x high general combiners, respectively. the situation holds good for both the locations with respect to number of fruits. importance of non additive gene action for number of fruits per plant was also reported (om et al., 1978; ghaderi and lower, 1979; dogra, 1995). however, the present results with regard to fruit weight and number of fruits are in disagreement with el hafeez et al. (1997). this may be due to differences in the parental material used for making diallel crosses. for yield per plant, k-90 was the best general combiner in addition to gyn 1 and g 2 irrespective of location (table 2). the sca effects (tables 3 and 4) were high for k-90 x g 2 (high x high), k-90 x gyn 1 (high x high) and lc-11 x gyn 1 (poor x high). the present results on yield per plant were similar to earlier findings of om et al. (1978), ghaderi and lower (1979), wang and wang (1980) and doligibh and sidorova (1983) but in contradiction to the work of gu et al. (2004). parents such as g 2 , gyn 1 and lc-40 had negative gca effects and were considered good general combiners for internodal length. nine (at l1) and 10 (at l2) specific combinations had significant negative values with the maximum in k-90 x poinsette and poinsette x ec 173934, poor x poor general combiners at each location. as is evident from the data in tables 2, 3 and 4, environmental effect was observed as non-significant on genotable 2 estimates of general combining ability of parents for different characters in cucumber source of variation character days to first female flower appearance node of first female flower days to market-able maturity fruit length fruit width tss flesh to seed cavity ratio fruit weight no. of fruits per plant yield per plant internodal length location 1 k-90 0.000 0.367* -0.550* 0.361* 0.364* -0.016* -0.0002 20.083* 0.017 0.276* 0.021 g2 -12.133* -2.567* -12.217* -1.404* -0.041* 0.004 0.004* -25.250* 1.317* 0.302* -1.856* poinsette -2.433* -0.767* -2.0183* -0.105 -0.531* -0.031 0.014* -4.917* -0.217* -0.055* 1.048* ec173934 8.167* 1.633* 8.517* -0.390* 0.191* 0.037* 0.011* 7.417* -0.617* -0.346* 0.144 k-75 0.733* 0.733* 1.017* -0.050* 0.320* -0.004 0.017* 10.083* -0.017 0.024* 1.084* lc-11 6.600* 0.633* 6.583* 0.388* 0.136* -0.022 0.007* 32.750* -0.783* -0.089* 0.604* lc-40 9.800* 2.067* 9.950* -0.225* -0.008* 0.029* 0.005* -8.417* -1.283* -0.379* -0.593* gyn1 -10.733* -2.100* -11.117* 1.425* -0.433* 0.002 0.008* -16.917* 1.583* 0.268* -0.453 se (gi) 0.221 0.141 0.222 0.019 0.013 0.011 0.0019 2.336 0.099 0.011 0.154 cd0.05 (gi) 0.441 0.281 0.443 0.037 0.026 0.021 0.0038 4.658 0.197 0.022 0.307 location 2 k-90 0.075 0.258* -0.267* 0.208* 0.269* -0.021 -0.013* 20.492* 0.508* 0.301* 0.116 g2 -10.092* -2.908* -11.600* -1.355* 0.016 -0.015 -0.016* -23.341* 1.842* 0.285* -1.828* poinsette -1.158* -0.375* -1.133* -0.285* -0.574* -0.008 0.018* -7.141* -0.325* -0.053* 0.693* ec173934 7.642* 1.192* 7.867* -0.592* 0.196* 0.065* 0.014* -5.342* -0.858* -0.384* 0.489* k-75 2.908* 0.792* 1.100* -0.025 0.309* -0.013 -0.010* 8.825* -0.258* 0.058* 0.869* lc-11 2.875* 1.325* 6.300* 0.495* 0.083* -0.028* -0.0001 30.825* -1.092* -0.125* 0.513 lc-40 5.675* 2.358* 7.900* -0.148* 0.083* 0.045* -0.0007 -5.342* -1.358* -0.363* -0.364* gyn1 -7.925* -2.642* -10.167* 1.702* -0.381* -0.026* 0.009* -18.375* 1.542* 0.279* -0.488* se (gi) 0.194 0.134 0.197 0.088 0.044 0.023 0.0018 2.076 0.114 0.029 0.177 cd0.05 (gi) 0.387 0.267 0.393 0.175 0.088 0.046 0.0036 4.139 0.227 0.058 0.353 * significant at 5% level of significance. 242 ta bl e 3 e st im at es o f sp ec if ic c om bi ni ng a bi lit y of f 1 f or d if fe re nt c ha ra ct er s in c uc um be r at n au ni ( l 1) c ro ss es c ha ra ct er s d ay s t o fir st fe m al e flo w er ap pe ar an ce n od e of fi rs t fe m al e flo w er d ay s t o m ar ke ta bl e m at ur ity fr ui t l en gt h fr ui t w id th ts s fl es h to se ed ca vi ty ra tio fr ui t w ei gh t n o. o f f ru its pe r p la nt y ie ld p er pl an t in te r-n od al le ng th k -9 0x g 2 -7 .4 22 * -1 .0 59 * -7 .2 52 * -2 .4 81 * -0 .4 11 * -0 .0 82 * 0. 12 2* -1 3. 25 9* 2. 68 5* 1. 02 3* -0 .6 27 * k -9 0x p oi ns et te -4 .7 89 * -0 .5 26 * -4 .6 18 * -0 .4 40 * 0. 08 6* 0. 27 5* -0 .0 46 * -3 8. 59 3* 0. 84 8* -0 .3 24 * -2 .5 30 * k -9 0x e c 17 39 34 -7 .0 56 * -1 .2 59 * -7 .6 12 * -0 .4 45 * -0 .5 73 * -0 .0 73 * -0 .0 40 * 40 .5 74 * -0 .7 52 * -0 .0 59 * -1 .8 94 * k -9 0x k -7 5 10 .3 78 * 1. 97 4* 9. 48 4* 1. 38 2* 0. 45 1* 0. 01 1 0. 01 2* -3 5. 25 9* -0 .3 52 * -0 .5 43 * 0. 46 6 k -9 0x l c -1 1 9. 17 8* -0 .2 59 9. 94 8* -0 .4 06 * 0. 51 6* -0 .0 61 * -0 .0 31 * 55 .4 07 * -0 .2 52 -0 .3 09 * 2. 24 6* k -9 0x l c -4 0 -5 .3 56 * -1 .6 93 * -5 .0 85 * 1. 32 4* -0 .7 94 * 0. 00 6 0. 00 6* -1 .7 59 -0 .0 85 -0 .3 03 * 2. 04 3 k -9 0x g yn 1 -4 .4 89 * -0 .5 26 * -5 .3 52 * -1 .3 43 * -0 .1 82 * -0 .0 51 * -0 .0 13 * -2 3. 25 9* 2. 38 2* 0. 50 9* -0 .0 30 g 2 xp oi ns et te 2. 01 1* 1. 07 4* 3. 71 5* 2. 33 4* -0 .3 93 * 0. 00 2* -0 .0 45 * 0. 07 4 -0 .1 19 -0 .3 16 * 0. 68 0* g 2 x ec 17 39 34 3. 41 1* 0. 00 7 3. 34 8* 1. 81 9* 0. 48 2* -0 .0 32 -0 .0 29 -4 .0 93 -1 .0 52 * -0 .3 98 * 0. 88 3* g 2x k -7 5 0. 17 8 -1 .0 93 * 0. 51 5 2. 11 3* 0. 30 6* 0. 11 2* -0 .0 09 * 21 .0 74 * 1. 34 8* 0. 49 4* 1. 14 3* g 2 x lc -1 1 -3 .3 56 * 0. 67 4* -4 .0 52 * -1 .1 42 * -0 .3 67 * 0. 04 3 -0 .0 14 * 24 .0 74 * 0. 11 5 0. 26 8* -0 .0 44 g 2 x lc -4 0 9. 44 4* 0. 57 4* 9. 24 8* 0. 63 8* -0 .2 40 * 0. 16 0* 0. 00 3 -2 6. 42 6* -1 .3 85 * -0 .6 59 * 0. 45 3 g 2 x g yn 1 -0 .3 56 * 0. 74 1* 0. 98 2* 1. 42 1* 0. 75 6* 0. 09 3* -0 .0 23 * -1 4. 59 3* 0. 08 2 -0 .1 40 * 1. 01 3* po in se tte x e c 17 39 34 1. 04 4* -1 .1 26 * 1. 31 5* 2. 07 0* 0. 19 2* 0. 08 5* 0. 08 0* 5. 57 4 0. 48 2* 0. 06 5* -2 .3 87 * po in se tte x k -7 5 -0 .5 22 * 0. 10 7 -1 .1 85 * 2. 00 2* 0. 34 3* 0. 22 3* -0 .0 39 * 16 .4 07 * -0 .1 18 0. 20 1* 2. 00 6* po in se tte x lc -1 1 -5 .0 56 * -1 .1 26 * -5 .4 18 * -0 .3 25 * -0 .5 27 * -0 .0 23 * -0 .0 35 * 10 .4 07 * 0. 64 8* 0. 47 8* 1. 41 9* po in se tte x lc -4 0 -4 .9 22 * -1 .2 26 * -5 .4 52 * 2. 62 2* 0. 65 4* -0 .2 56 * 0. 03 2* -2 8. 42 6* 0. 48 1* 0. 08 8* -1 .5 17 * po in se tte x g yn 1 14 .9 44 * 0. 94 1* 15 .6 15 * -1 .9 95 * 0. 12 6* 0. 12 0* 0. 03 2* 18 .4 07 * -0 .3 85 * 0. 26 0* 1. 10 9* ec 17 39 34 x k -7 5 -1 .4 56 * 1. 04 1* -2 .8 85 * 0. 03 3 -0 .2 76 * -0 .2 05 * 0. 00 06 -7 .7 59 * -0 .7 18 * -0 .3 14 * 1. 00 9* ec 17 39 34 x lc -1 1 -0 .3 22 * -2 .5 26 * -0 .7 85 * 1. 16 1 0. 13 9* -0 .0 74 * 0. 04 4* 9. 57 4* 0. 04 8 0. 18 9* 0. 48 9* ec 17 39 34 x lc -4 0 -9 .1 89 * -3 .2 93 * -8 .8 18 * 1. 17 4* -0 .5 25 * -0 .1 07 * -0 .0 02 -5 .9 26 1. 54 8* 0. 44 6* 0. 35 3 ec 17 39 34 x g yn 1 9. 67 8* 3. 54 1* 8. 91 5* -0 .0 92 * -0 .0 59 * 0. 03 6* -0 .0 18 * -1 4. 09 3* -1 .9 85 * -0 .6 02 * 0. 17 9 k -7 5x l c -1 1 3. 77 8* 0. 37 4 3. 71 5* 1. 23 8* 0. 43 6* 0. 02 0 -0 .0 14 -3 6. 25 9* 0. 44 8* -0 .2 58 * -2 .0 84 * k -7 5x l c -4 0 4. 24 4* -2 .0 59 * 4. 68 2* 0. 90 1* -0 .6 77 * 0. 13 4* -0 .0 13 * -0 .0 93 -0 .0 52 -0 .0 28 * -1 .9 54 * k -7 5x g yn 1 -6 .5 56 * -0 .8 93 * -6 .5 85 * -1 .1 16 -0 .3 79 * -0 .2 13 * -0 .0 20 * 13 .4 07 * 1. 41 5* 0. 53 8* 0. 00 6 lc -1 1x l c -4 0 4. 04 4* 4. 04 1* 4. 11 5* 0. 49 6* -0 .5 93 * -0 .0 05 -0 .0 03 22 .2 41 * -0 .2 85 0. 04 9* 1. 25 9* lc -1 1x g yn 1 -9 .4 22 * -1 .4 59 * -9 .8 18 * -0 .4 54 * -0 .1 58 * 0. 12 8* -0 .0 30 * 25 .7 41 * 0. 18 1 0. 51 8* -0 .6 14 * lc -4 0x g yn 1 3. 37 8* -0 .2 26 2. 48 2* -1 .7 07 * 0. 17 3* 0. 21 1 -0 .0 19 * -1 6. 42 6* -1 .3 18 * -0 .3 52 * -2 .0 84 * se (i j)± 0. 67 6 0. 43 3 0. 68 0 0. 05 8 0. 04 4 0. 03 2 0. 00 57 7. 16 0 0. 30 3 0. 03 3 0. 47 2 c d 0. 05 1. 99 4 0. 88 3 1. 35 6 0. 11 6 0. 08 8 0. 06 4 0. 01 1 14 .2 80 06 04 0. 06 6 0. 94 1 *s ig ni fi ca nt a t 5 % le ve l o f si gn if ic an ce . 243 ta bl e 4 e st im at es o f sp ec if ic c om bi ni ng a bi lit y of f 1 f or d if fe re nt c ha ra ct er s in c uc um be r at c ha m ba gh at ( l 2) c ro ss es c ha ra ct er s d ay s t o fir st fe m al e flo w er ap pe ar an ce n od e of fi rs t fe m al e flo w er d ay s t o m ar ke ta bl e m at ur ity fr ui t l en gt h fr ui t w id th ts s fl es h to se ed ca vi ty ra tio fr ui t w ei gh t n o. o f f ru its pe r p la nt y ie ld p er p la nt in te r-n od al le ng th k -9 0x g 2 -7 .8 26 * -0 .9 15 * -7 .2 07 * -2 .2 55 * -0 .7 45 * -0 .0 95 * 0. 00 9* -8 .6 41 * 3. 69 6* 1. 05 8* -1 .0 97 * k -9 0x p oi ns et te -3 .7 59 * 0. 88 5* -3 .6 74 * -0 .0 25 0. 01 2 0. 26 5* -0 .3 90 * -2 8. 17 4* -0 .1 37 -0 .3 61 * -0 .9 17 * k -9 0x e c 17 39 34 -6 .8 93 * -2 .0 15 * -6 .6 74 * -0 .1 52 * -0 .6 25 * -0 .1 42 * -0 .0 28 * 43 .3 59 * -1 .2 70 * -0 .1 40 * -1 .5 80 * k -9 0x k -7 5 9. 17 4* 1. 71 9* 11 .4 26 * 1. 08 1 0. 29 5* 0. 07 0 0. 04 2* -2 7. 47 4* -0 .5 37 * -0 .5 65 * 1. 14 0* k -9 0x l c -1 1 11 .2 07 * -0 .1 48 7. 89 3* -0 .1 72 0. 08 8 -0 .0 49 -0 .0 14 * 33 .8 59 * -0 .3 70 * -0 .2 52 * 2. 26 3* k -9 0x l c -4 0 -1 .5 93 * -1 .5 15 * -3 .3 74 * 1. 70 5* -0 .4 12 * -0 .0 22 -0 .0 04 -1 .6 41 -0 .4 37 * -0 .2 88 * -0 .1 93 k -9 0x g yn 1 -7 .6 59 * -1 .1 82 * -5 .9 74 * -1 .5 78 * -0 .0 82 * -0 .0 50 -0 .0 07 * -1 9. 67 4* 1. 66 3* 0. 43 1* -0 .7 37 * g 2 xp oi ns et te 3. 74 1* 1. 38 5* 4. 65 9* 2. 83 8* -0 .5 02 * -0 .0 09 -0 .0 36 * -1 7. 67 4* -0 .4 70 * -0 .2 55 * 0. 02 7 g 2 x ec 17 39 34 0. 60 7* 0. 81 9* 2. 32 6* 2. 84 5* 0. 42 8* 0. 01 8 -0 .0 22 * -1 4. 47 4* -1 .9 37 * -0 .4 34 * 1. 36 3* g 2x k -7 5 -5 .6 59 * -1 .4 48 * -2 .9 07 * 2. 27 8* 0. 78 2* 0. 19 6* -0 .0 01 23 .0 26 * 0. 79 6* 0. 33 4* -0 .5 83 * g 2 x lc -1 1 -1 .2 93 * 0. 35 2 -3 .7 74 * -1 .2 75 * -0 .3 92 * 0. 04 5 -0 .0 08 * 24 .3 59 * -0 .7 04 * 0. 18 4* -0 .3 27 g 2 x lc -4 0 18 .5 74 * 1. 31 9* 7. 29 3* 0. 70 2* 0. 00 8 -0 .0 95 * 0. 02 3* -1 2. 80 7* -1 .4 37 * -0 .5 15 * 0. 71 7* g 2 x g yn 1 -0 .8 26 * 0. 65 2* 2. 02 6* 1. 01 8* 0. 73 8* 0. 14 3* -0 .0 14 * -7 .5 07 * -0 .6 70 * -0 .0 87 0. 80 7* po in se tte x e c 17 39 34 2. 67 4* -1 .0 48 * 2. 85 9* 0. 90 8* 0. 31 8* 0. 14 5* 0. 07 7* 14 .3 26 * 0. 89 6* 0. 14 7* -1 .9 57 * po in se tte x k -7 5 -3 .5 93 * -0 .3 15 -1 .3 74 * -1 .7 92 * 0. 07 2 0. 19 0* -0 .0 42 * 30 .1 59 * 0. 29 6 0. 25 5* 1. 33 0* po in se tte x lc -1 1 -2 .8 93 * 0. 48 5* -6 .5 74 * -0 .4 78 * -0 .3 02 * 0. 00 5 -0 .0 12 * 11 .4 93 * 0. 79 6* 0. 49 2* 1. 58 7* po in se tte x lc -4 0 -0 .6 93 * -1 .5 48 * -2 .8 41 * 2. 66 5* 1. 06 5* -0 .1 70 * 0. 02 9* -2 9. 00 7* 0. 72 9* 0. 06 6 -1 .9 03 * po in se tte x g yn 1 11 .5 74 * 1. 11 8* 13 .8 93 * -2 .2 52 * -0 .3 72 * 0. 06 9 0. 01 5* 9. 95 9* -0 .1 70 0. 34 4* 0. 75 3* ec 17 39 34 x k -7 5 -1 .7 26 * 3. 45 2* -0 .0 41 -0 .0 85 -0 .1 65 * -0 .2 50 * 0. 00 2 -3 .3 07 -0 .1 70 -0 .2 47 * -0 .2 00 ec 17 39 34 x lc -1 1 4. 30 7* -2 .0 82 0. 75 9* 1. 09 5* 0. 19 5* -0 .0 69 0. 04 2* 19 .6 93 * 0. 33 0* 0. 23 6* -0 .3 43 ec 17 39 34 x lc -4 0 -6 .1 59 * -4 .1 15 * -8 .5 07 * 1. 10 5* -0 .2 05 * -0 .1 75 * -0 .0 04 -1 9. 14 1* 1. 26 3* 0. 34 0* 1. 60 0* ec 17 39 34 x g yn 1 2. 77 4* 0. 21 9 4. 89 3* -0 .4 12 * -0 .1 09 * -0 .0 70 * -0 .0 14 * -1 8. 84 1* -1 .9 70 * -0 .6 18 * 1. 42 3* k -7 5x l c -1 1 5. 70 7* -1 .0 15 * 4. 19 3* 1. 06 2* 0. 58 2* -0 .1 57 * -0 .0 14 * -4 1. 14 1* 0. 39 6* -0 .3 09 * -0 .8 57 * k -7 5x l c -4 0 4. 90 7* -3 .7 15 * 4. 25 9* 0. 93 8* -0 .8 85 * 0. 20 3* -0 .0 17 * -6 .0 41 -0 .0 04 -0 .1 31 * -1 .6 13 * k -7 5x g yn 1 8. 50 7* -1 .0 48 * -5 .6 74 * -1 .5 78 * -0 .2 55 * -0 .2 42 * -0 .0 20 * 10 .3 26 * 1. 42 9* 0. 30 7* 0. 07 7 lc -1 1x l c -4 0 -2 2. 72 6* 3. 08 5* 6. 39 3* 0. 61 8* -0 .8 25 * -0 .0 15 -0 .0 14 * 23 .0 26 * 0. 16 3 0. 02 5* 0. 07 7 lc -1 1x g yn 1 -5 .7 93 * -2 .2 48 * -7 .5 41 * -0 .8 65 * 0. 23 8* 0. 12 3* -0 .0 30 * 28 .3 26 * -0 .4 04 * 0. 57 4* -1 .4 67 * lc -4 0x g yn 1 2. 07 4* 5. 05 2* 1. 85 9* -2 .3 22 * 0. 10 5 0. 31 7* -0 .0 23 * -1 2. 17 4* -1 .1 37 * -0 .3 02 * -1 .3 22 * se (i j)± 0. 59 5 0. 41 1 0. 60 3 0. 27 1 0. 13 4 0. 07 0 0. 00 54 6. 36 2 0. 34 9 0. 08 9 0. 54 2 c d 0. 05 1. 18 6 0. 81 9 1. 20 2 0. 54 0 0. 26 7 01 39 0. 01 1 12 .6 85 0. 69 6 0. 17 7 1. 08 1 *s ig ni fi ca nt a t 5 % le ve l o f si gn if ic an ce . 244 types and hybrid combinations for most of the characters. the results are similar at both locations with developed hybrid combinations and hence hybrids k-90 x g 2 and k-90 x gyn 1 can be exploited in similar types of climates. k-90, g 2 and gyn 1 may be used in hybridisation for developing high yielding hybrids with higher number of fruits per vine, long fruits and high tss on the basis of results from location 1, whereas g 2 and gyn 1 are promising for developing high yielding hybrids with higher number of fruits per vine and short inter-nodal length on the basis of results from location 2. it can be concluded that g 2 and gyn 1 may be used in hybridisation for developing high yielding hybrids with more fruits per vine and wider adaptability. the crosses k-90 x g 2 and k-90 x gyn 1 can be released as hybrids after further testing. references dogra b.s., 1995 heterosis and combining ability studies in cucumber (cucumis sativus l.) m.sc. thesis, dr y s parmar university of horticulture and forestry, solan, india, pp. 591. dogra b.s., kanwar m.s., 2011 exploiting heterosis for yield and horticultural traits in cucumber (cucumis sativus l.). indian j. plant genet. resour., 24(3): 332-339. doligibh s.t., sidorova a.m., 1983 combining ability of induced mutants and partially dioecious forms of cucumber. genetika, 19(8): 1292-1300. el hafeez a.a., el sayed s.f., gharib a.a., 1997 genetic analysis of cucumber yield and its components by diallel crossing. egyptian j. hort. sci., 24(2): 141-159. el-shawaf i.i.s., baker l.r., 1978 inheritance of yield in parthenocarpic hybrid pickling cucumber (abstract). hort. science, 13(3): 355. ghaderi l.r., lower r.l., 1979 heterosis and inbreeding depression for yield in populations derived from six crosses of cucumber. j. amer. soc. hort. sci., 104(4): 564-567. griffing j.b., 1956 concept of general and specific combining ability in relation to diallel crossing system. aust. j. biol. sci., 9: 463-494. gu x.f., zhang s.p., xu c.q., 2004 analysis of combining ability of early yield and total yield character of cucumber cultivated in open field in spring. china vegetables, 6: 1315. hormuzdi s.g., more t.a., 1989 studies on combining ability in cucumber (cucumis sativus l.). indian j.genet., 49(2): 161-165. kupper r.s., staub j.e., 1988 combining ability between lines of cucumis sativus l. and cucumis sativus var. hardwickii (r.) alef. euphytica, 38: 197-210. om y.h., choi k.s., lee c.h., choi c.i., 1978 diallel analysis of several characters in cucumber (cucumis sativus l.). korean j. breeding, 10(1): 44-50. wang y.j., wang y.s., 1980 preliminary analysis of combining ability in autumn cucumber. scientia agricultura sinica, 3: 52-57. impaginato 111 1. introduction the degree of success in any technology employing plant cell, tissue or organ culture is related to quite a few major factors. a significant factor is the choice of nutritional components and growth regulators (gamborg, 1991). a plant tissue culture medium is composed of necessary and optional components required for plant growth, which vary according to the plant species, cultivar, or explant type that is used and must be experimentally defined for each particular case. moreover, all the nutrients in a medium should be present in optimum concentrations to ensure the best possible growth of explants (george, 1993). under in vitro conditions, an intact plant requires macronutrients, micronutrients, plant growth regulators, vitamins, amino acids and other nitrogen supplements and sugars (gamborg, 1991). another important component in plant tissue culture media is the carbon source because it supplies energy to the plants, especially when they are not ready to photosynthesize their own food during the early stage of tissue culture (al-khateeb, 2008 b). carbon source can be in the form of simple or complex sugars (akter et al., 2007). normally, sucrose is used as the carbon source in plant tissue culture. a range of other organic additives have been used in plant tissue culture to promote the growth of the plants, including coconut milk, banana pulp, potato homogenate and juice, honey, date palm syrup, corn extract, papaya extract, guar gum, and isubgol (islam et al., 2003; jain and babbar, 2005; george et al., 2008; murdad et al., 2010; nambiar et al., 2012). such additives are commonly known as organics with undefined compositions (torres, 1989). the advantages of adding such organic materials to medium have already been reported by some researchers, for example, the organic additives help to produce more plbs, shoots and leaves in orchid (akter et al., 2007), increase the size of date palm somatic embryos (alkhateeb, 2008 a), and also promote growth and development of asymbiotic seeds and regeneration adv. hort. sci., 2016 30(2): 111-118 doi: 10.13128/ahs-19137 the usefulness of apricot gum as an organic additive in grapevine tissue culture media s. khorsha, m. alizadeh (*), k. mashayekhi horticulture department, faculty of plant production, gorgan university of agricultural sciences and natural resources, golestan, p.o. box 386, gorgan, iran. key words: apricot gum, callus culture, carrot, grapevine, rooting, stevia, tissue culture. abstract: the growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. in addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. the proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. the growth parameters of treated samples were measured and compared to gum-free medium. earlier callus initiations with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. the usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot multiplication and rooting parameters. due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. overall, the gum at the rate of 4.0 g/l was found to be a logical concentration with respect to encouraging response in all three species. owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. however, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media. (*) corresponding author: mahdializadeh@gau.ac.ir received for publication 9 may 2016 accepted for publication 25 may 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 111-118 112 of cymbidium plantlets (tawaro et al., 2008). the reasons for application of organic additives into culture medium, in addition to being a natural source of carbon, are because they contain natural vitamins, phenols, fiber, hormones, and also proteins (gnasekaran et al., 2010). it was mentioned in a report by al-khateeb (2008 a) that organic additives contained not only sugar but also other nutrients such as proteins, lipids and minerals. in recent years, plant-derived polymers have evoked remarkable attention in various industries due to their diverse applications as food emulsifiers, stabilizers and thickeners, pharmaceuticals, cosmetics, textiles, and in art. they are also used as gelling agents in gels and bases in suppository (nussinovitch, 1996). these polymers are biocompatible, biodegradable and are preferred to semi synthetic and synthetic excipients because of their lack of toxicity, low cost, soothing action, and non irritant nature (deogade et al., 2012). environmental-friendly processing and local availability, especially in developing countries, are considered additional advantages for application of these natural products (jain and babbar, 2005). the rosaceae family, prunus genus, consists of peach, plum, apricot, cherry, and almond trees, all of which can produce exudate gums. herbal exudate gums normally secrete from bark, branch and fruit of trees due to their protection impact against mechanical damage or microbial attacks. gum secretion may also occur due to adaptation to climate of some trees, called physiological gummosis (simas et al., 2008; simas-tosin et al., 2009). a large number of complex natural additives can be very effective in providing an undefined mixture of organic nutrients and growth factors. in the context of carbohydrates, gums are usually considered to be nonstarch, water-soluble polysaccharides with commercial importance. gums are typically more or less sticky in nature and are translucent and amorphous substances which are degradation products of the cell wall of woody species which exude from trees. natural gums (gums obtained from plants) are hydrophilic carbohydrate polymers having high molecular weights, generally composed of monosaccharide units joined by glucosidic bonds (khorsha, 2014). while thorough reports on gum structure (saniewski et al., 2001; lluveras-tenorio et al., 2012), its rheological properties (wang et al., 2008), and its applications in different industries (verbeken et al., 2003) were previously reported, the influence of such natural products as organic addenda in plant tissue culture media has yet to be studied. therefore, the present investigation was conducted to evaluate the effectiveness of apricot gum as an organic additive on growth and in vitro regeneration of three different plant species: carrot (as a model plant), stevia (as herbaceous species) and grapevine (as woody plant). the results of the current study should be applicable to other plant species following minor modifications. 2. materials and methods the present research work was conducted in the plant tissue culture laboratory of the horticulture department, faculty of plant production, gorgan university of agricultural sciences and natural resources, gorgan, iran. the physiological effects of apricot gum on regeneration capacity and growth rate of three different plant species i.e. carrot (as model plant), stevia (as herbaceous plant) and grapevine (as woody plant) were examined. the apricot gum was obtained dried on bark and shoots of apricot trees immediately after secretion, collected from a commercial apricot orchard (gonabad, khorasan, eastern iran) through scraping it from bark surfaces. the bark residues were removed from the collected mass and the clean, pure gum was powdered prior to addition to the culture medium. plant materials and in vitro culture establishement carrot. healthy, undamaged roots of carrot (daucus carrota cv. nantes), 3-4 cm diameters long, were selected and washed thoroughly with normal tap water (30 min) and surface sterilized with ethanol (70% v/v for 40 s) followed by sodium hypochlorite solution (35% v/v plus two drops of tween-20 for 15 min). the clean, sterilized roots were cut transversely, employing a scalpel, to prepare root explants as slices (10×10 mm and 1mm thickness). each explant was prepared in such a way to consist of xylem, secondary phloem and a small part of cambium tissues (hall, 1991). stevia. stevia (stevia rebaudiana) mother plants were procured from golsaran-e-shomal corporation, as tissue cultured pot plants and were transferred to our laboratory. single node explants were disinfected with hgcl2 (0.1% for 6 min) and washed three times with sterilized distilled water inside a laminar hood cabinet. the single node explants were cultured and in vitro culture was established following an already standardized protocol (taherian, 2012). grapevine. single node explants of grapevine (vitis vinifera cv. laal) were utilized following the prekhorsha et al. apricot gum as an organic additive in grapevine tissue culture media 113 viously standardized micropropagation protocol developed by alizadeh et al. (2010). laal is an iranian, commercially grown variety of grapevine. single node explants (2-4 cm length) were pre-washed in 0.10.2% commercial detergent (jam dish-washing liquid, iran) followed by stirring in mancozeb (2 g/l) solution for 45 min. the explants were surface disinfected using 60% (v/v) naoc1 solution (5% available chlorine) for 30 min. after four to five rinses in sterile distilled water, single node cuttings (2-4 cm) were inoculated for in vitro culture establishement. culture media and incubation conditions basal ms (murashige and skoog, 1962) medium was used during the whole experiment for all three plant species. the various media along with their growth regulators for each plant species are reported in table 1. the ph was adjusted to 5.8 prior to the addition of 0.8% agar, and the media were autoclaved at 121°c and 15 psi for 15 min. carrot root cultures were kept in growth chamber in darkness and low light intensity for 7-14 days and the explants were investigated for callus formation. the callus mass was sub-cultured on fresh medium supplemented with apricot gum. the stevia and grapevine single node cultures were incubated at 25±2°c under continuous light (50 μmol m-2 s-1). the established and sprouted cultures were sub-cultured as doublenode explants on proliferation media (table 1) supplemented with different concentrations of apricot gum. measured parameters due to the utilization of explants with different origins, dissimilar parameters were recorded in each species. in the case of carrot callus cultures, days to callus initiation, callus color, firmness, volume and fresh/dry weight were recorded 30 days after caulogenesis. in the case of stevia and grapevine, initially the in vitro cultures were established as explained in above. then, the double-node explants procured from in vitro proliferated cultures were inoculated on their respective proliferation media supplemented with different concentrations of apricot gum. the parameters such as days taken to bud sprouting, number of leaves, leaf area, total chlorophylls and cartenoides, intermodal length, shoot length, days to root initiation, number of roots, root length, rooting percentage and root/shoot fresh and dry weights were measured in both stevia and grapevine regenerated cultures. experimental design and data analysis the present experiment was conducted as a complete randomized design with four replications. the percentage data were transformed using root square method (√ % + 0.5) prior to analysis. the results were analyzed using sas software (sas institute inc., 2003) and the mean values were compared by least significant difference (lsd) test in p<0.01 probability. 3. results the present research was undertaken to ascertain the likely positive effects of apricot gum as an organic additive to plant tissue culture media. the gum was primarily supplemented to carrot callus cultures and, owing to its positive response, was tested in two other plant species, stevia and grapevine, for which in vitro culture propagation protocols were already standardized in our laboratory (alizadeh et al., 2010; taherian, 2012). iran is the world’s second producer of apricot (faostat, 2012) and apricot gum is readily available throughout the country at a very low price. in order to optimize the concentrations of the apricot gum, some preliminary tests were performed and the rate of 2.0-6.0 g/l was found to be a logical range because the higher concentration formed a dense solution that was difficult to dispense in culture vessels. furthermore, in high concentration, it negatively interacted with agar solidification (khorsha, 2014). carrot callus growth the application of gum surprisingly enhanced the performance of in vitro callus initiation and further growth of carrot explants. furthermore, it significantly reduced the time taken to callus initiation and among the treatments caulogenesis occurred even less than two weeks following inoculation of the explants on medium supplemented with 6.0 g/l apricot gum. the callus initiation in control explants was found to start normally after 5 weeks, therefore, precocious callus formation (nearly 20 days earlier than control) is considered a promising result for in vitro application of apricot gum. in addition, callus morphological traits such as callus volume, color, and tissue firmness were also affected by gum treatments table 1 different culture media and growth regulators for each plant species plant species in vitro establishment shoot proliferation rooting carrot 2,4-d (1.0 mg/l) stevia hormone-free medium iba (2.0 mg/l) iba (2.0 mg/l) grapevine ba (2.0 mg/l) + naa (0.2 mg/l) iba (2.0 mg/l) iba (2.0 mg/l) adv. hort. sci., 2016 30(2): 111-118 114 (table 2). the callus mass produced on control medium (30 days after inoculation) were light brown in color and had intermediate firmness. however, with gum application the color was greenish and the tissue mass volume was increased corresponding to gum concentration (fig. 1). the callus fresh weight was also improved in all the treatments, however the dry weight was the same and was determined to be lower than control media (table 3). it is worth noting that the treatment with 6.0 g/l apricot gum may be toxic for carrot callus; however callus fresh weight was not statistically significant compared to 2 g/l level (table 3). due to the positive and promising results with carrot callus cultures, the effect of apricot gum was evaluated on in vitro proliferation of an herbaceous medicinal plant, stevia, for which its proliferated cultures were already established in our laboratory. stevia shoot multiplication and rooting stevia is an herbaceous plant with rapid in vitro proliferation. in our laboratory, stevia cultures were normally sub-cultured at threeto four-week intervals. the growth parameters of stevia shoot proliferation and rooting four weeks after inoculation are shown in table 4. it is clear that apricot gum added to stevia medium was particularly beneficial. the gum significantly reduced the time taken to root initiation and the micro-cuttings inoculated on medium containing gum (6.0 g/l) achieved root initiation in less than a week (6.5 days only), while the control plantlets came into rooting after at least 10 days. root length also increased in the presence of gum, however, the number of roots were not considerably different among treatments. in vitro plantlet length and leaves were reduced on gum-supplemented media, but instead the leaf area and lateral shoots were enhanced (table 4). it seems the lower concentrations of apricot gum (2.0 and 4.0 g/l) led to better vegetative response in stevia cultures. a similar trend was also recorded with respect to stevia leaf pigments. thus, at the rate of 2.0 g/l, gum was more effective in pigmentation and production of dark green leaves. overall, it may be stated that apricot gum had a positive influence on most of the in vitro traits measured in stevia tissue-cultured plants. grapevines shoot multiplication and rooting it has previously been reported that shoot proliferation and rooting occur simultaneously in grapevine (alizadeh et al., 2010), hence, in the present experiment, it was possible to measure related fig. 1 the effect of apricot gum on in vitro growth and proliferation of carrot callus cultures 30 days after inoculation (left to right: control, 2.0, 4.0, 6.0 g/l apricot gum). means in the same row followed by different letters are significantly different at p<0.01 using lsd-test. qualitative traits apricot gum concentration (g/l) control 2.00 4.00 6.00 callus volume <30% 30-50% 50-100% ≥100% callus color light brown beige to light green beige to greenish beige to pistachio green tissue firmness +++ +++ ++++ ++++ table 2 the qualitative traits of carrot callus as affected by gum application 30 days after inoculation table 3 the growth parameters of carrot callus as affected by gum application 30 days after inoculation qualitative traits apricot gum concentration (g/l) control 2.00 4.00 6.00 days to callus initiation 35.75 a 29.80 b 19.60 c 13.70 d callus fresh weight (g) 0.121 c 0.269 cb 1.01 a 0.364 b callus dry weight (g) 21.54 a 10.08 b 11.32 b 13.66 b means in the same row followed by different letters are significantly different at p<0.01 using lsd-test. growth trait apricot gum concentration (g/l) control 2.00 4.00 6.00 plantlet length (cm) 20.00 ab 10.95 bc 22.72 a 8.60 c number of shoots 2.25 b 3.75 ba 3.25 ba 5.25 a number of leaves 68.25 a 27.00 b 36.75 b 47.5 ab average leaf area (cm3) 0.06 c 1.75 a 1.16 ab 0.68 bc days to root initiation 10.75 a 10.25 a 7.25 a 6.50 a number of roots 35.00 a 29.50 a 35.00 a 17.50 a root length (cm) 0.56 b 2.21 a 1.86 a 0.85 b chlorophyll a (mg.g f.w.) 7.50 c 14.45 a 10.96 b 10.68 b chlorophyll b (mg.g f.w.) 4.33 ab 5.43 a 2.77 b 3.87 ba total chlorophyll (mg.g f.w.) 11.91 b 19.97 a 13.76 b 14.72 b carotenoid (mg.g f.w.) 3.15 b 5.95 a 4.25 b 4.53 ba table 4 the growth parameters of stevia shoot proliferation and rooting 28 days after inoculation +++ indicates medium hadness, ++++ indicates medium hardness and fragile callus tissue. khorsha et al. apricot gum as an organic additive in grapevine tissue culture media 115 parameters collectively in a single medium (shoot proliferation cum rooting medium supplemented with apricot gum). furthermore, the results of our grapevine experiment were more obvious and noticeable than for the other species. these distinct effects on grapevine shoot proliferation and rooting are reported in figure 2 and table 5. in the case of rooting parameters, the gum considerably reduced the time taken to root initiation (6 days against 14 days in control explants) and the rooting percentage was also improved, reaching nearly 100 % in the case of 6.0 g/l gum-supplemented media. root length also increased: the greatest length was found in medium containing 4.0 g/l gum. however, all gum treatments showed longer roots compared to control explants. a similar trend was also observed in stevia samples (table 4). shoot proliferation was vigorously enhanced following application of gum to the media (fig. 2 and 3). thanks to the positive effects of apricot gum, longer vines with more lateral shoots, internodes and greater leaf area were achieved (fig. 3). although all three concentrations of gum were found to be effective with regard to the aforementioned vegetative traits as compared to gum-free medium, the efficiency of 4.0 g/l was evident. 4. discussion and conclusions cultured plant tissues need a continuous supply of carbohydrates from the medium to encourage growth and survival in vitro (kozai, 1991). therefore, sugars such as sucrose, glucose, and sorbitol are generally added as a carbon source (kadota and niimi, 2004). furthermore, reliable callus proliferation and subsequent plant regeneration are important for efficient micropropagation and genetic manipulation of plant tissues (khorsha, 2014). carbohydrates play an important role in in vitro cultures as an energy and carbon source, as well as an osmotic agent (george et al., 2008). there are numerous reports on the effects of various carbon sources on in vitro callus growth and regeneration. for example, huang and huang (1999) demonstrated that sorbitol acts as a carbon source, providing energy for callus growth and plantlet regeneration. other studies suggest that it acts only as an osmotic regulator to adjust osmotic pressure in calli (al-khayri and al-bahrany, 2002; george et al., 2008). the capacity of various carbohyfig. 2 the effect of apricot gum on in vitro growth performance of grapevine explants. fig. 3 the effect of apricot gum on in vitro shoot proliferation (above) and rooting (below) of grapevine. table 5 the effect of apricot gum on certain growth parameters of grapevine 30 days after inoculation growth trait apricot gum concentration (g/l) control 2.00 4.00 6.00 appearance of the first leaf 17.25 a 7.00 b 5.00 c 4.75 c number of shoots 1.00 c 1.00 c 2.5 a 2.00 b number of leaves 3.00 b 4.75 b 9.5 a 9.50 a number of roots 20.25 b 32.5 b 77.25 a 65.75 a internode length (cm) 0.94 c 1.02 bc 1.41 a 1.29 ab means in the same row followed by different letters are significantly different at p<0.01 using lsd-test. adv. hort. sci., 2016 30(2): 111-118 116 drates to support growth of japanese morning glory callus was examined and it was found that sucrose was the most effective compound but glucose, fructose, trehalose, maltose, cellobiose, raffinose and soluble starch were also found to be significant (hisajima and thorpe, 1985). apricot gum and callus growth and proliferation gums are typically more or less sticky in nature and translucent and amorphous. chemical analysis of apricot gum has already been performed by lluverastenorio et al. (2012). they found total sugars (60%), galactose (43%), mannose (4%), arabinose (44%), xylose (7%) and ramnose (1%) in apricot gum. it is clear that such composition may be different in each lot, from one tree to another, and the results may not be reproducible in different tissue culture laboratories. however, the present study was performed in five replications and positive responses were found with carrot callus growth. the callus volume produced on media fortified with 4.0-6.0 g/l apricot gum was at least three to four times that of gum-free medium (fig. 1). the enhanced callus growth induced by apricot gum may be considered a significant logical reason for application of gum in plant tissue culture media, especially in species for which indirect regeneration (plant regeneration from callus) is to follow. apricot gum and shoot/root proliferation different types of basal media used in plant tissue culture vary with regard to the concentration of macro and microelements, greatly affecting the in vitro growth and multiplication of shoots. apart from this, type and concentration of carbon sources in the medium affect the physiology and differentiation of tissues (lipavska and konradova, 2004) while serving as osmotic and energy source (george et al., 2008). in our experiment, the utilization of apricot gum caused reasonable shoot proliferation both in stevia and grapevine (table 4, fig. 3), not to mention its very low cost and economic advantages, making it useful in commercial tissue culture laboratories seeking economical and feasible protocols for in vitro propagation of horticultural crops. there are some reports on the utilization of such low-priced and organic materials as a potential plant source with a high amount of sucrose and other sugars which could possibly be used as an alternative carbon source, for example molasses (dhamankar, 1992), sugar cane juice (buah et al., 2011), and date palm syrup (al-khateeb, 2008 a). furthermore, in a review presented by yaseen et al. (2012), factors determining the efficacy of a carbon source including its type, concentration, and their mutual interaction are reported. in our study, apricot gum was exploited, although the application of almond gum or sweet cherry gum would also be possible, since these types of gum are also readily available throughout the country at similar price. however, there may be some differences in in vitro responses of gums from different origins. with regard to concentration, for tissue culture media, 4.0-6.0 g/l may be considered as a feasible range, because beyond this dose the solubility of gum would be difficult even with heat treatments. moreover, in high concentrations the culture media cannot be solidified properly (khorsha, 2014). the mutual interaction between gum and explants is also an important matter. although, in the present experiment all three plant species showed positive responses for most of the measured parameters, however grapevine in vitro shoot proliferation was greatly enhanced in gum-supplemented media (fig. 3). rooting is considered a difficult step in micropropagation of many woody plants (george, 1993) and it is regulated by a number of physiological, biochemical, and genetic factors (pawlicki and welander (1995). generally, rooting occurs in an auxinenriched medium. on the other hand, hassan et al. (2009) mentioned that sucrose at the highest level considerably increased rooting of palm tissue cultures. the results of the present research on rooting parameters of stevia and grapevine correspond with those of al-khateeb (2008 b) on date palm. he observed an enhanced root formation as the sugar concentration increased (60 g/l and above). in conclusion, the present study considered addition of apricot gum as a complex organic addendum in tissue culture of carrot callus, stevia and grapevine. it was found that addition of gum not only was without negative effects, but its usefulness was definitely confirmed. apricot gum significantly increased in vitro growth and proliferation of carrot callus tissues. furthermore, shoot/root vegetative parameters of gum-fortified media were actually promising in stevia and grapevine. overall, gum at the rate of 4.0 g/l was found to be a logical concentration with respect to the positive response in all three species. owing to the promising results found with the present research, the application of gum in commercial tissue culture protocols is highly recommended. however, further studies would be required to fully exploit plant-derived gums as an alternative carbon source. khorsha et al. apricot gum as an organic additive in grapevine tissue culture media 117 acknowledgements the authors are grateful to golsaran-e-shomal tissue culture company for providing stevia mother plants. the help and cooperation of mr. f. salahi for his assistance in grapevine sample collection is fully acknowledged. references akter s., nasiruddin k.m., khaldun a.b.m., 2007 organogenesis of dendrobium orchid using traditional media and organic extracts. j. agric. rural dev., 5(1&2): 30-35. alizadeh m., singh s.k., patel v.b., 2010 comparative performance of in vitro multiplication in four grape (vitis spp.) rootstock genotypes . int. j. plant production, 4: 41-50. al-khateeb a.a., 2008 a comparison effects of sucrose and date palm syrup on somatic embryogenesis of date palm (phoenix dactylifera l.). am. j. biochem. and biotech., 4(1): 19-23. al-khateeb a.a., 2008 b regulation of in vitro bud formation of date palm (phoenix dactylifera l.) cv. khanezi by different carbon sources. bioresource technology, 99 (14): 6550-6555. al-khayri j.m., al-bahrany a.m., 2002 callus growth and proline accumulation in response to sorbitol and sucrose-induced osmotic stress in rice. biologia plantarum, 45: 609-611. buah j.n., tachie-menson j.w., addae g., asare p., 2011 sugarcane juice as an alternative carbon source for in vitro culture of plantains and bananas. am. j. food techn., 6: 685-694. deogade u.m., deshmuk v.n., sakarkar d.m., 2012 natural gums and mucilage’s in ndds: applications and recent approaches. intern. j. pharmaceutical techn. res., 4(2): 799-814. dhamankar v.s., 1992 molasses, a source of nutrients for in vitro sugar cane culture. sugar cane, 4: 14-15. faostat, 2012 http://faostat.fao.org/site/567/ desktopdefault.aspx?pageid=567#ancor. gamborg o.l., 1991 media preparation, pp. 1-24. lindsey k. 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stress states, and much data on the physiological and psychological relaxing effects of forest environments have been accumulated. previous studies have reported that viewing forest scenery or walking in forests can: increase parasympathetic nervous activity, which is enhanced in relaxing situations and suppresses sympathetic nervous activity which is increased in stress states (tsunetsugu et al., 2007; park et al., 2008; lee et al., 2009; park et al., 2009; park et al., 2010; lee et al., 2011; park et al., 2012; tsunetsugu et al., 2013; lee et al., 2014); decrease cerebral blood flow in the prefrontal cortex (park et al., 2007); and decrease salivary cortisol concentration of stress hormone (tsunetsugu et al., 2007; park et al., 2007; park et al., 2008; lee et al., 2009; park et al., 2010). in addition, visiting a forest enhanced natural killer-cell activity and improved immune function (li et al., 2007; li et al., 2008 a, b, c) and the effect lasted 30 days (li et al., 2008 b). in subjective evaluations, it was reported that people feel more “comfortable,” “soothed,” and “natural” when experiencing a forest environment (park et al., 2007; tsunetsugu et al., 2007; park et al., 2008; lee et al., 2009; park et al., 2009; lee et al., 2011; park et al., 2011; tsunetsugu et al., 2013; lee et al., 2014), and that the “tension-anxiety,” “depression,” “anger-hostility,” “fatigue,” “confusion,” and “vigor” of the mood state profile (mcnair and lorr, 1964; mcnair et al., 1992; yokoyama, 2005) improved (li et al., 2008 a, b, c; park et al., 2010; lee et al., 2011; park et al., 2011; tsunetsugu et al., 2013; lee et al., 2014). unfortunately, many people living in cities find it difficult to access forest environments. thus, much attention has been focused on nature-based stimuli, such as walking in an urban park (song et al., 2013), viewing rooftop forests (matsunaga et al., 2011), the presence of plants, including dracaena (igarashi et al., 2014) or roses (ikei et al., 2014), and physical contact with wood (sakuragawa et al., 2008), and the relaxing effects of these stimuli have been reported. nature-based stimuli are intuitively perceived through the five senses. of these five senses, the physiological effects of olfactory stimulation have been characterized physiological and psychological effects of olfactory stimulation with d-limonene d. joung (1) * , c. song(1) **, h. ikei**, t. okuda**, m. igarashi**, h. koizumi**, b.j. park*, t. yamaguchi**, m. takagaki**, y. miyazaki (2) ** * department of environment and forest resources, chungnam national university, 99 daehak-ro, yuseong-gu, daejeon 305-764, republic of korea. ** center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa, chiba 277-0882, japan. key words: heart rate, heart rate variability, limonene, physiological relaxation, semantic differential method. abstract: although d-limonene can be considered an important component of nature-based stimuli, the physiological effects of olfactory stimulation with d-limonene have not been completely clarified by scientific studies. the physiological and psychological effects of olfactory stimulation with d-limonene were studied measuring heart rate variability (hrv), heart rate, and subjective evaluation using a modified semantic differential method; thirteen japanese female university students (mean age±sd, 21.5±1.0 years) participated in the study. a concentration of 60 µl of d-limonene was used as olfactory stimulant and room air as control. subjects were exposed for 90 s while sitting with eyes closed. during d-limonene inhalation: (1) the high-frequency (hf) value of hrv, a marker of parasympathetic nervous activity that is enhanced in relaxing situations, was significantly higher; (2) the heart rate was significantly lower; and (3) subjects reported feeling significantly more comfortable during d-limonene administration than control. the results obtained clearly indicate that olfactory stimulation with d-limonene induced physiological and psychological relaxation, providing important scientific evidence of the health benefits of d-limonene. adv. hort. sci., 2014 28(2): 90-94 (1) these authors equally contributed to the manuscript. (2) corresponding author: ymiyazaki@faculty.chiba-u.jp received for publication 31 march 2014 accepted for publication 30 june 2014 91 in detail. miyazaki et al. (1992) conducted a pioneering study which revealed that olfactory stimulation with chamaecyparis taiwanensis essential oil significantly decreased blood pressure. furthermore, inhalation of rose oil odor was shown to suppress sympathetic nervous activity and decrease adrenaline concentration (haze et al., 2002). lavender oil has been shown to induce deep sleep (goel et al., 2005) and improve concentration (sakamoto et al., 2005). however, evidence-based research using the indices of autonomic nervous activity to clarify the effect of components of these essential oils is lacking. the essential oil components of cryptomeria japonica and pinus densiflora, representative forest trees, have been reported (cimanga et al., 2002; hong et al., 2004; cheng et al., 2009). these oils are composed of various volatile organic compounds, including d-limonene, α-pinene, β-pinene. d-limonene is the main component of citrus peel oil (bernhard, 1960; attaway et al., 1968; shaw, 1979; chiralts et al., 2002; yoo et al., 2004). the purpose of the present study was to investigate the physiological effect of olfactory stimulation with dlimonene on autonomic nervous activity by measuring its effect on heart rate variability (hrv) (camm et al., 1996; kobayashi et al., 1999) and the heart rate. 2. materials and methods subjects thirteen japanese female university students (age range, 21.5±1.0 years; mean±sd) participated in the study. before beginning the experiment, a full explanation about the research aim, the experimental procedure, and all measured indices was provided. informed consent was obtained from all subjects. this study was conducted in accordance with the regulations of the ethics committee of the center for environment, health, and field sciences, chiba university, japan. study protocol physiological and psychological measurements were carried out in a chamber with an artificial climate maintained at 25°c with 50% relative humidity and 230lux illumination. d-limonene (>95.0% purity, tokyo chemical industry co., ltd., japan) was used as an olfactory stimulant, and room air was used as a control. a total of 60 µl d-limonene was injected into a 24-l odor bag (polyethylene terephthalate film heat seal bag; ns-koen co., ltd., kyoto, japan) and the odors were presented to each subject by means of a device fitted on the chest and situated approximately 10 cm under the nose (fig. 1). the flow rate of the odor was set at 3 l/ min. subjective sensitivity to the odor was determined in a preliminary investigation. the subjects were exposed to the odor for 90 s while sitting with their eyes closed. the order of presentation of d-limonene and control was counterbalanced. heart rate variability and heart rate hrv was measured as the periods between consecutive r waves (r-r intervals) in an electrocardiogram recorded with a portable electrocardiograph (activtracer ac-301a, gms, japan). in this study, two major spectral components of hrv, the low-frequency (lf; 0.04–0.15 hz) band and the high-frequency (hf; 0.15-0.40 hz) band were obtained by the maximum-entropy method (memcalc/win, gms, japan). the hf power was considered to reflect parasympathetic nervous activity, and the lf/hf power ratio was considered to reflect the sympathetic nervous activity (camm et al., 1996; kobayashi et al., 1999). heart rate was also investigated using r-r interval data. semantic differential method the subjects provided a subjective evaluation of the emotional impact of the odors according to a modified semantic differential (sd) method (osgood et al., 1957). this method allowed the subject to assess a pair of adjectives, such as “comfortable-uncomfortable,” using a 13-point scale. the sd method was performed after administration of each odor. statistical analysis all statistical analyses were performed using statistical package for social sciences software version 20.0 (ibm corp., armonk, ny, usa). a paired t-test was used to compare differences in the physiological responses over the 90 s of exposure to d-limonene and air. wilcoxon signed-rank test was applied to analyze differences in psychological response between d-limonene and air. a onesided test was used in this study. in all cases, the significance level was set at p < 0.05. 3. results the results of the hrv data after exposure to d-limonene and control were compared, and a significant differfig. 1 olfactory stimulation setup. 92 ence was found in the hf value, which is a marker of parasympathetic nervous activity, as shown in figure 2. the hf value increased 26.4% during d-limonene administration (827.2±191.3 ms2; mean±se) compared with control (654.4 ±163.6 ms2), indicating that parasympathetic nervous activity was significantly higher during d-limonene administration (p<0.05). however, no significant difference was found in the lf/hf power ratio for the two stimuli. figure 3 shows a comparison of the heart rate during the administration of d-limonene and control. heart rate decreased during d-limonene administration (72.8±2.3 bpm) compared with control (74.1±2.5 bpm), and this difference was significant (p<0.05). figure 4 shows the results for a “comfortable” feeling according to the subjective evaluation. subjects reported significantly more comfortable ratings during d-limonene administration than control (p<0.01). 4. discussion and conclusions d-limonene is one of the most common volatile organic compounds in nature (sun, 2007). it is a major component of various citrus oils, such as lemon, orange, grapefruit, and lime (attaway et al., 1968; bernhard, 1960; chiralts et al., 2002; shaw, 1979; yoo et al., 2004), as well as essential oils from coniferous trees, such as pinus densiflora, pinus koraiensis, chamaecyparis obtusa, and cryptomeria japonica (cimanga et al., 2002; hong et al., 2004; cheng et al., 2009). in addition, because of its citrus fragrance, d-limonene is commonly added to perfumes, soaps, and cosmetics (bakkali et al., 2008). although d-limonene is an important component of nature-based stimuli, the physiological effect of olfactory stimulation with d-limonene has not been completely clarified. previously, tsunetsugu et al. (2012) investigated the physiological effect of olfactory simulation with dlimonene on blood pressure and showed that olfactory simulation with a concentration of 10 μl d-limonene decreases subjects’ systolic blood pressure. however, to our knowledge, no previous study has examined the physiological effect of olfactory stimulation with d-limonene on hrv and heart rate. the present study shows that olfactory stimulation with d-limonene induced (1) a significant increase in parasympathetic nervous activities, (2) a significant decrease in the fig. 2 comparison of high-frequency power levels of heart rate variability during olfactory stimulation with d-limonene or control (air). data are expressed as mean ± se; n = 13. *p < 0.05 by paired t-test. fig. 3 comparison of the heart rate during olfactory stimulation with d-limonene or control (air). data are expressed as mean ± se; n = 13. *p < 0.05 by paired t-test. fig. 4 subjective evaluation of “comfortable” measured by a modified semantic differential questionnaire after olfactory stimulation with d-limonene or control (air). data are expressed as mean ± se; n = 13. **p < 0.01 by wilcoxon signed-rank test. 93 heart rate, and (3) a significant increase in a “comfortable” feeling. these results agree with previous studies of other nature-based stimuli (tsunetsugu et al., 2007; park et al., 2008; park et al., 2009; park et al., 2010; lee et al., 2011; park et al., 2012; song et al., 2013; tsunetsugu et al., 2013; ikei et al., 2014, lee et al., 2014). park et al. (2012) showed that the hf value of hrv was significantly increased while viewing scenery of forests using the results of field experiments at 35 forests in japan. ikei et al. (2014) reported that the hf component was significantly increased by viewing roses. song et al. 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(ed.) forest medicine. series public health in the 21st century environmental health. physical, chemical and biological factors. nova science publishers, new york, pp. 316. yokoyama k., 2005 poms shortened version-manual and commentary on cases. kaneko syoboh, tokyo, japan. yoo z.w., kim n.s., lee d.s., 2004 comparative analyses of the flavors from hallabong (citrus sphaerocarpa) with lemon, orange and grapefruit by spte and hs-spme combined with gc-ms. bull. korean. chem. soc., 25: 271-279. impaginato 19 1. introduction milk thistle (silybum marianum l.) is one of the most important medicinal plants in the pharmaceutical industry worldwide, and is used in the production of flavonoids of the silymarin group (silybin, silidianin and silychristine) which are important in the modern pharmaceutical industry (ghavami and ramin, 2007). the origin of this plant has been reported to be the east mediterranean region (keville, 1991). water availability may influence physiological and biochemical properties and seed yield of this medicinal plant. water stress severely limits growth and yield of plants by reducing ground green cover (ghassemigolezani and ghasssemi, 2013), chlorophyll content of leaves, photochemical efficiency of photosystem ii (ghassemi-golezani and lotfi, 2012) and photosynthesis (munns et al., 2006). water stress during vegetative stages largely reduces plant height and biomass, while during reproductive stages it has the greatest negative impact on seed yield (ghassemigolezani et al., 2008). reports in oil crops indicated that water stress decreases oil and increases protein percentages of seeds. however, both oil and protein yields per unit area are decreased as a result of large reduction in seed yield per unit area due to water limitation (ghassemi-golezani and lotfi, 2013; ghassemi-golezani et al., 2015 b). some of the deleterious effects of environmental stresses on plant performance could be alleviated by foliar application of growth regulators such as salicylic acid (sa) (ghassemi-golezani et al., 2015 a). it has been reported that the exogenous application of sa induces plant tolerance to several abiotic stresses including drought tolerance in wheat (singh and usha, 2003), salinity tolerance in safflower (ghassemi-golezani and hosseinzadeh-mahootchi, 2015) and mung bean (ghassemi-golezani et al., 2015 a), heat tolerance in mustard (dat et al., 1998) and chilling tolerance in maize (janda et al., 1999). these studies suggest that sa may enhance the multiple types of stress tolerance in plants by interactive effects on several functional molecules. hayat et al. (2008) found that there was a significant increase in photosynthetic parameters, chlorophyll and proline contents, and antioxidant enzyme activities in sa treated tomato plants. loutfy et al. (2012) reported that sa induced drought tolerance and increased plant biomass, leaf relative water content, and the solute contents in four wheat cultivars. moreover, adv. hort. sci., 2017 31(1): 19-23 doi: 10.13128/ahs-20721 improving oil and flavonoid contents of milk thistle under water stress by salicylic acid k. ghassemi-golezani*, s. ghassemi, i. yaghoubian department of plant eco-physiology, faculty of agriculture, university of tabriz, tabriz, iran. key words: foliar application, plant biomass, seed yield, silybum marianum l., water deficit. abstract: adverse environmental conditions such as water deficit can limit production. however, some of these adverse effects may be overcome by application of plant growth regulators including salicylic acid (sa). thus, a field experiment was conducted in 2015 to evaluate the effects of sa (0 and 1 mm l-1) on yield components, seed yield and oil and flavonoid contents of milk thistle (silybum marianum l.) under different irrigation treatments (i1, i2, i3 and i4: irrigation after 70, 110, 150 and 190 mm evaporation from class a pan, respectively). the experiment was arranged as split-plot based on randomized complete block (rcb) design in three replicates. irrigation treatments and sa levels were located in the main and sub plots, respectively. the results indicated that plant biomass, seeds per plant, 1000 seed weight, seed yield per unit area and harvest index of milk thistle decreased as a consequence of water stress. oil percentage and yield were also reduced, but flavonoid content enhanced with increasing water deficit. all these traits were considerably augmented by foliar application of sa under non-stress and stressful conditions. therefore, it was conclude that sa can be used to improve field performance of milk thistle under different environmental conditions. (*) corresponding author: golezani@gmail.com received for publication 11 october 2016 accepted for publication 20 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 19-23 20 foliar spray of sa decreased the inhibitory effects of drought on phillyrea angustifolia (munné-bosch and penuelas, 2003). abd el-lateef gharib (2006) stated that oil content of basil and marjoram significantly increased with application of sa. however, the responses of some medicinal plants to sa treatment in stressful conditions were not documented so far. thus, this research was undertaken to evaluate the effects of foliar application of sa on seed yield and oil and flavonoid contents of milk thistle under different irrigation intervals. 2. materials and methods seeds of milk thistle (silybum marianum l.) were obtained from pakan bazr, isfahan, iran. the experiment was conducted in 2015 at the research farm of the faculty of agriculture, university of tabriz, iran (latitude 38° 05′ n, longitude 46° 17′ e, altitude 1360 m above sea level). the climate is characterized by mean annual precipitation of 245.75 mm per year, mean annual maximum temperature of 16.6°c and mean annual minimum temperature of 4.2°c. the field experiment was arranged as split-plot based on randomized complete block design in three replications, with irrigation intervals (i1, i2, i3, i4: irrigation after 70, 110, 150 and 190 mm evaporation from class a pan, respectively) in main plots and two levels of salicylic acid (sa; 0 and 1 mm l-1) in sub-plots. seeds of milk thistle were treated with 3.3 g/kg benomyl and then were sown by hand on 28 may 2015 in 3 cm depth of a sandy loam soil. each plot consisted of 6 rows of 3 m length, spaced 25 cm apart. all plots were regularly irrigated up to seedling establishment, but thereafter irrigations were carried out according to treatments. weeds were frequently controlled by hand during crop growth and development. salicylic acid (sa; 0 and 1 mm) was sprayed at vegetative and flowering stages. plant biomass and seed yield at maturity, plants in 1 m2 (8 plants) of the middle part of each plot were harvested and seeds per plant, 1000 seed weight and seed yield per unit area were determined. then above ground biomass was ovendried at 80°c for 48 hours and weighed and subsequently harvest index was calculated. oil extraction oil was extracted from 3 g mature seeds of each plot in petroleum ether for 5 hours using a soxhlet system according to the aocs method (aocs, 1993). oil content was determined as a percentage for each sample and then oil yield per unit area was calculated as: oil yield = seed yield × oil percentage flavonoid extraction powdered air-dried mature seeds (1 g) were extracted in a soxhelt extractor with 100 ml ethanol for an hour and the extract filtered. three ml of the extract was placed in a 15 ml volumetric flask. then 0.3 ml nano2 (1:20) and after 5 minutes 3 ml alcl3 (1:10) and 6 minutes later 2 ml of 1 mol litre-1 naoh were added and the total was made up to 10 ml with distilled water. the solution was mixed well again and the absorbance was measured against a blank at 510 nm with a halo db-20 spectrophotometer (zhuang et al., 1992). the flavonoid content was calculated using the following linear equation: a = 0.01069c 0.001163 where a is the absorbance and c is the flavonoid content in µg/g. analysis of variance analysis of variance of the data appropriate to the experimental design and comparison of means at p≤0.05 were carried out, using genstat 12 and mstatc softwares. excel software was used to draw figures. 3. results analysis of variance (table 1) showed that plant biomass, seeds per plant, 1000 seeds weight, seed yield per unit area and harvest index were significantly affected by water limitation and sa, but the interaction of irrigation × sa was only significant for 1000 seed weight and harvest index. source of variation df mean square plant biomass seeds per plant 1000 seeds weight seed yield harvest index replication 2 1198 2162 0.1163 61.7 0.0129 irrigation (i) 3 38104 ** 56241 ** 4.6906 * 3121.7 ** 4.1715 ** error 6 2795 4424 0.2785 140 0.1257 sa 1 837851 ** 1718420 ** 15.0417 ** 71195.6 ** 48.4504 ** i × sa 3 2633 ns 7585 ns 0.9128 ** 262 ns 3.3993 ** error 8 3987 7553 0.1212 259.2 0.1058 cv (%) 8.8 9.2 1.5 9.2 1.4 table 1 analysis of variance of the data for plant biomass, yield components and seed yield of milk thistle affected by irrigation treatments and salicylic acid (sa) treatments ns, * and ** no significant and significant at p≤0.05 and p≤0.01, respectively. ghassemi-golezani et al. oil and flavonoid of milk thistle affected by watering and salicylic acid 21 plant biomass, seeds per plant and seed yield per unit area decreased with decreasing water availability, but all these traits were considerably enhanced by foliar application of sa. reduction in seeds per plant was only significant under severe water stress (i4), with no significant difference among i1, i2 and i3 treatments. differences in plant biomass and seed yield between i1 and i2 and also between i2 and i3 were not statistically significant. application of sa improved plant biomass, seeds per plant and seed yield by about 71%, 79% and 91%, respectively (table 2). one thousand seeds weight (fig. 1a) and harvest index (fig. 1b) of untreated plants with sa gradually decreased as water stress increased. but, these reductions were only significant under severe water deficit. in contrast, sa treated plants did not show significant reduction in seed weight and harvest index due to water limitation. oil percentage, oil yield and flavonoid content were significantly affected by irrigation and sa treatments, but the interaction of irrigation × sa was not significant for these traits (table 3). oil percentage and yield of milk thistle decreased, but flavonoid content increased as a result of water stress. seed oil percentage and yield and flavonoid content were significantly enhanced by foliar spray of sa. this superiority was more pronounced for oil yield per unit area (table 4). 4. discussion and conclusions reduction in plant biomass due to water stress (table 2) was associated with diminishing leaf area expansion and plant growth during vegetative stages (ghassemi-golezani et al., 2009) and also with early leaf senescence (hugh and richard, 2003). drought fig. 1 mean seed weight (a) and harvest index (b) of milk thistle affected by irrigation and sa treatments. i1, i2, i3, i4= irrigation after 70, 110, 150 and 190 mm evaporation, respectively. sa0, sa1= 0 and 1 mm salicylic acid, respectively. different letters in each column indicate significant difference at p≤0.05. treatments plant biomas (g/m2) seeds per plant seed yield (g/m2) irrigation i1 797.2 a 1034.8 a 197.0 a i2 746.2 ab 984.0 a 185.2 ab i3 705.0 b 939.7 a 172.5 b i4 609.0 c 808.8 b 143.9 c salicylic acid irrigation 528 b 674 b 120.2 b sa1 901 a 1209 a 229.1 a table 2 means of plant biomass, seeds per plant and seed yield of milk thistle for irrigation and salicylic acid (sa) treatments different letters in each column indicate significant difference at p≤ 0.05. source of variation df oil content oil yield flavonoid content replication 2 2.2604 23.12 1.0208 irrigation (i) 3 24.2749 ** 367.88 ** 20.0397 ** error 6 0.6199 5.49 15046 salicylic acid (sa) 1 5.9004 ** 3294.13 ** 26.8182 ** i × sa 3 0.0849 ns 0.59 ns 0.2903 ns error 8 0.1625 10.8 0.3488 cv (%) 2 9.2 1.2 table 3 analysis of variance of oil percentage and yield and flavonoid content of milk thistle affected by irrigation and sa treatments ns, * and ** no significant and significant at p≤0.05 and p≤0.01, respectively. adv. hort. sci., 2017 31(1): 19-23 22 stress decreases water potential of plant, leading to stomata closure and reduction in photosynthesis rate and leaf growth (ozturk, 1999), which ultimately decreases plant biomass. this reduction in plant biomass resulted in decreasing the number of seeds per plant (table 2), 1000 seeds weight (fig. 1a) and consequently seed yield (table 2) and harvest index (fig. 1b). the losses in plant biomass and seed yield due to water deficit have also been reported for sesame (kim et al., 2007), dill (ghassemi-golezani et al., 2008), maize (ghassemi-golezani and dalil, 2011) and safflower (ghassemi-golezani et al., 2016). application of sa largely improved seed yield of milk thistle by enhancing plant biomass, seeds per plant (table 2), 1000 seeds weight and harvest index (fig. 1). sa influences a wide variety of plant processes, including stomatal regulation, chlorophyll content and photosynthesis (yildirim et al., 2008). ghassemigolezani and lotfi (2015) found that exogenous application of sa enhances maximum quantum efficiency of psii (fv/fm) and performance index (pi) in mung bean plants. in another report, ghassemigolezani and hosseinzadeh-mahootchi (2015) stated that chlorophyll content index (cci), photosystem ii efficiency (fv/fm), relative water content (rwc), leaf area index (lai) and finally seed yield of safflower were augmented by foliar application of sa. the low oil percentage due to water deficit (table 4) may be resulted from the short seed filling duration (ghassemi-golezani and lotfi, 2013) and low seed weight (fig. 1a). adequate irrigation during plant growth and development can likely increase seed weight and oil storage. decreasing oil yield per unit area as a consequence of water limitation (table 4) strongly related with reduction in seed yield under stressful condition (table 2). it was similarly reported that water limitation significantly decreases seed and oil yields of sunflower (soleimanzadeh et al., 2010) and maize (ghassemi-golezani et al., 2015 b). increasing seed yield (table 2) and oil percentage by application of sa resulted in considerably higher oil yield of milk thistle (table 4). similar pattern of oil yield improvement by foliar spray of sa was observed in ocimum basilicum and origanium hortensis plants (abd el-lateef gharib, 2006). with decreasing photosynthesis rate under water stress, carbons from the photosynthesis cycle shift to the shikimic acid pathway in order to produce higher flavonoid content (table 4). it was found that phenolics and flavonoids are able to regulate plant growth and improve the physiological efficiency and can enhance effective partitioning of accumulates from the sources to the sinks in plants (ghasemzadeh et al., 2010). stimulation of flavonoid accumulation by sa treatment (table 4) may protect plants from certain biotic and abiotic stresses (dučaiová et al., 2013). an increase in secondary metabolites content was also detected in chamomile (matricaria chamomilla l.) plants as a result of sa spray (dučaiová et al., 2013). this result suggests that flavonoid accumulation is a biochemical response to water stress, and sa can induce flavonoid synthesis, providing an effective protection of milk thistle plants from stress. water stress reduced plant biomass, seeds per plant, 1000 seeds weight, seed yield, harvest index, oil percentage and consequently oil yield of milk thistle. however, flavonoid content of seeds increased with decreasing water availability. all these traits were considerably enhanced by foliar spray of sa under different irrigation intervals. this suggests that exogenous application of sa could be an effective way for improving field production of milk thistle under different environmental conditions. acknowledgements we appreciate the financial support of this work by the university of tabriz. references abd el-lateef gharib f., 2006 effect of salicylic acid on the growth, metabolic activities and oil content of basil and marjoram. int. j. agric. biol., 8: 485-492. aocs, 1993 official methods and recommended practices of the american oil chemists’ society. 4th edn. (methods ag 1-65 and ce 1-62). american oil chemists’ society press, champaign, il, usa. dat j.f., lopez-delgado h., foyer c.h., scott i.m., table 4 means of oil percentage and yield and flavonoid content of milk thistle for irrigation and sa treatments different letters in each column indicate significant difference at p≤0.05. treatments oil content (%) oil yield (g/m2) flavonoid content (µg/g) irrigation i1 22.13 a 43.60 a 48.05 c i2 21.33 a 39.50 b 49.12 bc i3 19.35 b 33.38 c 50.04 b i4 17.67 c 25.43 d 52.34 a salicylic acid sa0 19.63 b 23.60 b 48.83 b sa1 20.62 a 47.24 a 50.95 a ghassemi-golezani et al. oil and flavonoid of milk thistle affected by watering and salicylic acid 23 1998 parallel changes in h2o2 and catalase during thermo-tolerance induced by salicylic acid or heat acclimation in mustard seedlings. amer. soc. plant biol., 116: 1351-1357. dučaiová z., petruľová v., repčák m., 2013 salicylic acid regulates secondary metabolites content in leaves of matricaria chamomilla. biologia, 68: 904909. ghasemzadeh a., jaafar h.z.e., rahmat a., 2010 synthesis of phenolics and flavonoids in ginger (zingiber officinale roscoe) and their effects on photosynthesis rate. int. j. mol. sci., 11: 4539-4555. ghassemi-golezani k., andalibi b., zehtab-salmasi s., saba j., 2008 effects of water stress during vegetative and reproductive stages on seed yield and essential oil content of dill (anethum graveolens l.). j. food, agric. environ., 6: 282-284. ghassemi-golezani k., dalil b., 2011 seed ageing and field performance of maize under water stress. afr. j. biotechnol., 10: 18377-18380. ghassemi-golezani k., ghanehpoor s., dabbagh mohammadi-nasab a., 2009 effects of water limitation on growth and grain filling of faba bean cultivars. j. food, agric. environ., 7: 442-447. ghassemi-golezani k., ghassemi s., 2013 effects of water stress on some physiological traits and grain yield of chickpea (cicer arietinum l.) cultivars. int. j. biosci., 3: 62-70. ghassemi-golezani k., hosseinzadeh-mahootchi a., 2015 improving physiological performance of safflower under salt stress by application of salicylic acid and jasmonic acid. walia j., 31: 104-109. ghassemi-golezani k., lotfi r., 2012 responses of soybean leaves and grain yield to water stress at reproductive stages. int. j. plant, animal environ. sci., 2: 63-68. ghassemi-golezani k., lotfi r., 2013 influence of water stress and pod position on oil and protein accumulation in soybean grains. intl. j. agron. plant. prod., 4: 2341-2345. ghassemi-golezani k., lotfi r., 2015 the impact of salicylic acid and silicon on chlorophyll a fluorescence in mung bean under salt stress. russ. j. plant physiol., 62: 611-616. ghassemi-golezani k., lotfi r., najafi n., 2015 a some physiological responses of mung bean to salicylic acid and silicon under salt stress. adv. biores., 6: 7-13. ghassemi-golezani k., maghferati r., zehtabsalmasi s., dastborhan s., 2016 influence of water deficit and nitrogen supply on grain yield and yield components of safflower. adv. biores., 7: 132136. ghassemi-golezani k., raei n., raei y., 2015 b effects of water deficit and nitrogen levels on grain yield and oil and protein contents of maize. azarian j. agric., 2: 46-50. ghavami n., ramin a.a., 2007 salinity and temperature effects on seed germination of milk thistle. commun. soil sci. plant anal., 38: 2681-2691. hayat s., hasan s.a., fariduddin q., ahmad a., 2008 growth of tomato (lycopersicon esculentum) in response to salicylic acid under water stress. j. plant interact., 3: 297-304. hugh j.e., richard f.d., 2003 effect of drought stress on leaf and whole canopy radiation use efficiency and yield of maize. agron. j., 95: 688-696. janda t., szalai g., tari i., paldi e., 1999 hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize (zea mays l.) plants. planta, 208: 175-180. keville k., 1991 illustrated herb encyclopedia; a complete culinary, cosmetic, medicinal, and ornamental guide to hers. simon & schuster, east roseville, new south wales, australia, pp. 224. kim k.s., park s.h., jenks m.a., 2007 changes in leaf cuticular waxes of sesame (sesamum indicum l.) plants exposed to water deficit. j. plant physiol., 164: 1134-1143. loutfy n., el-tayeb m.a., hassanen a.m., moustafa m.f.m., sakuma y., inouhe m., 2012 changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat (triticum aestivum l.). j. plant res., 125: 173-184. munné-bosch s., penuelas j., 2003 photo and antioxidative protection, and a role for salicylic acid during drought and recovery in field grown phillyrea angustifolia plants. planta, 217: 758-766. munns r., james r.a., läuchli a., 2006 approaches to increasing the salt tolerance of wheat and other cereals. journal of experimental botany, 57: 1025-1043. ozturk a., 1999 the effect of drought on the growth and yield of winter wheat. turk. j. agric. for., 23: 531-540. singh b., usha k., 2003 salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress. plant growth regul., 39: 137-141. soleimanzadeh h., habibi d., ardakani m.r., paknejad f., rejali f., 2010 response of sunflower (helianthus annuus l.) to drought stress under different potassium levels. world applied sciences journal, 8: 443-448. yildirim e., turan m., guvenc i., 2008 effect of foliar salicylic acid applications on growth, chlorophyll and mineral content of cucumber (cucumis sativus l.) grown under salt stress. j. plant nutrit., 31: 593-612. zhuang x.p., lu y.y., yang g.s., 1992 extraction and determination of flavonoid in ginkgo. chinese herbal medicine, 23: 122-124. impaginato 53 1. introduction salinity stress is dependent on environmental condition (kozlowski and pallardy, 1997), farming, water management and genotype (kozlowski and pallardy, 1997). olive (olea europea l.) is one of the most valuable and widespread fruit trees in the mediterranean area. its cultivation is continuously being extended to irrigated land. furthermore, in mediterranean area salinity is becoming a major problem due to high rates of evaporation (kozlowski and pallardy, 1997). olive is considered a moderately salt tolerant plant (ayers and westcot, 1976; aragues et al., 2005; weissbein et al., 2008). in comparison with other mediterranean-grown tree crops, olive is more tolerant than citrus but less tolerant than date palm (ayers and westcot, 1976). the tolerance of olive cultivars are different to salinity stress (therios and misopolinos, 1988; perica et al., 2004; chartzoulakis, 2005). the relationship between saline water and olive cultivation has been intensively studied for many years and significant progress has been made in the understanding of this topic (ayers and westcot, 1976; wiesman et al., 2004). it is generally well established that saline conditions limit the vegetative and reproductive development of olives mainly as a result of interference with the osmotic balance in the root system zone and detrimental effects caused by specific toxic accumulation of chloride and sodium ions in the leaves (weissbein et al., 2008). salt stress reduces water availability in soil solution as a result of an increased osmotic potential, inducing the generation of reactive oxygen species (ros) (zhu, 2001; melloni et al., 2003), the reduction of hormonal signals generated by the roots (munns, 2002), altered carbohydrate metabolism (gao et al., 1998), reduced the activity of certain enzymes (munns, 1993; chartzoulakis, 2005) and ultimately impaired photosynthesis (chartzoulakis, 2005). therefore, these physiological changes result reduced growth in either reduced cell division, expansion or promoting cell death (hasegawa et al., 2000). furthermore these criteria make plant reduce growth rate and yield, chlorophyll destruction which lead to leaf senescence. the plant response to salinity stress is depenadv. hort. sci., 2017 31(1): 53-59 doi: 10.13128/ahs-20726 physiological responses of olive cultivars to salinity stress m. rahemi 1 (*), s. karimi 2, s. sedaghat 1, a. ali rostami 1 1 department of horticultural science, college of agriculture, shiraz university, shiraz, iran. 2 department of horticultural science, college of abouraihan, university of tehran, tehran, iran. key words: electrolyte conductivity, osmoregulation, proline, soluble carbohydrates. abstract: the aim of this study was to evaluate the tolerance of seven promising olive cultivars for southern parts of iran (‘amigdalilolia’, ‘dakal’, ‘zard’, ‘dezful’, ‘tokhm-e-kabki’, ‘shiraz’, and ‘conservalia’) against salinity stress. biochemical and physiological responses of the cultivars irrigated with saline water application (control, 4, 8, and 12 ds m-1) were evaluated and the tolerant cultivars were identified. in contrast to the tolerant cultivars, the sensitive ones continue to grow with lower rate and died under salinity stress. in general, growth indices of olive cultivars were reduced with increasing salinity stress and the lowest growth indices were obtained under 12 ds m-1 treatment. results indicated that the accumulation of higher levels of soluble carbohydrates and proline in the leaves of the tolerant cultivars helps them to deal with salinity stress. the results showed that saline waters up to 4 ds m-1 for irrigation can be used for olive cultivars, however, based on the result of this study, it is not recommended to use water sources with higher electric conductivities to irrigate sensitive olive cultivars. we concluded that the tolerant cultivars stopped growth and used their energy to defend against the salinity stress. (*) corresponding author: rahemi@shirazu.ac.ir received for publication 9 february 2016 accepted for publication 17 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 53-59 54 dent on environmental condition, farming, water management and plant genotype. the aim of this study was to screen for the tolerance of seven olive cultivars from the southern parts of iran, against salinity stress. tolerance was evaluated over several biochemical (proline content, stored carbohydrate, total chlorophyll and starch concentration), and physiological (cell membrane injury) responses of these cultivars under salinity stress. 2. materials and methods experiment was carried out in the department of horticultural sciences, shiraz university during the growing season in 2012, using one year old cuttings of seven olive cultivars: ‘dakal’, ‘zard’, ‘shiraz’, ‘tokhm-e-kabki’, ‘dezful’, ‘amigdalilolia’, and ‘konservalia’ with four replicates for each cultivar. the cuttings were transplanted into 15 kg pots containing soil mixture (1:1:1) of soil (pure soil), sand and leaf mould. the physicochemical characteristics of the soil are shown in table 1. during the establishment phase in greenhouse, olive cultivars were pruned uniformly in order to produce a single stem. the salinity stress treatments were applied by sub irrigation with different salinity levels [control (1.1), 4,8,12 ds/m]. in order to prevent salinity shock, the concentration of salts was gradually increased to reach a given level. the day and night temperature of the greenhouse was 35°c and 25°c, respectively. the saline water was prepared by dissolving sodium chloride (control, 4, 8, 12 ds/m) in the water. the pots were irrigated with saline water for 90 days. they were irrigated with saline water to the field capacity (fc) level, which was equivalent 20% of the dry weight of the soil of pot. the total shoot length of olive cultivars was measured at the beginning and at the end of the experiment. additionally, the number of fully expanded leaves and branches of each cultivar were recorded. at the end of the experiment, the average length of new shoot was measured. using the data collected at the start and the end of salinity stress treatments, the rate of these changes was calculated. total chlorophyll measurement total chlorophyll content was determined by spectrophotometer (saini et al., 2001). briefly, chlorophyll a and b contents were obtained by extraction in 85% acetone solution and measuring their absorbances using camp spec m501 single beam uv/vis spectrophotometer at λ= 663 nm and λ = 645 nm. the concentration of chlorophylls and carotenoids were calculated according to the following formula: total chlorophylls (mg /g fw) = [(20.2×od 645 nm + 8.02×od 663 nm ) × v] /(fw × 1000) where od is optical density, v is the final solution volume in ml and fw is tissue fresh weight in mg. v is the final solution volume in ml and fw is tissue fresh weight in mg. proline measurement free proline was extracted from 0.5 g samples of fully expanded and young leaves with 3%, sulfuric acid and estimated by using ninhydrin reagent, according to the protocol described by bates et al. (1973). the absorbance of the fraction with toluene was determined at 520 nm, using a spectrophotometer (model uv-120-20, japan). cell membrane injury (cmi) cell membrane injury was calculated according to the method of blum and ebercon (1981). for the cmi, 20 samples of stressed and unstressed young leaves were washed with distilled water to remove the dust and injured cells from samples. the samples were then immersed in 20 ml distilled water at room temperature. after 24 h the conductivity of the solutions was read. the samples were autoclaved for 15 min, cooled to room temperature and the conductivity of the solutions was read again. the electrolyte leakage was measured with a conduct meter (644 conduct meter, metrohm, herisau, switzerland). cmi was estimated from the formula: id (drought injury index) = 1 −(1 −t1/t2)/(1 -c1/c2) × 100 where t1 and t2 are the first and second measurement of the conductivity of the solutions in which the table 1 the physiochemical characteristics of the soil used characteristics zn (ppm) 1.5 fe (ppm) 7.6 mn (ppm) 21.14 cu (ppm) 1.76 k (ppm) 400 p (ppm) 23.8 total nitrogen (%) 0.094 oc (%) 1.54 ph (%) 7.9 ec (%) 1.93 clay (%) 34.4 silt (%) 44.2 sand (%) 21.4 rahemi et al. physiological responses of olive cultivars to salinity stress 55 treated samples were immersed and c1 and c2 are the respective values for the conductivity of the solutions. soluble carbohydrate extraction to determine soluble carbohydrate concentration, 150 mg of dried leaf samples was extracted twice with 80% ethanol. the sample was centrifuged at 3500 rpm for 10 min and the volume of the supernatant was adjusted to 25 ml soluble carbohydrate concentration was measured according to the method of buysee and merckx (1993). in summary, 1 ml of supernatant was transferred to a test tube and 1 ml phenol 18% and 5 ml sulfuric acid were added. the mixture was shaken immediately and its absorption was recorded at 490 nm using a spectrophotometer (model uv-120-20, japan). starch concentration starch concentration in the leaf samples was measured using anthron reagent (mccready, 1950). in this method, 5 ml of water (0°c) and 6.5 ml perchloric acid (52%) were added to the pellet used for sugar analysis and mixed for 15 min. about 20 ml water was then added and the sample was centrifuged. the supernatant was separated and the same procedure was repeated with the pellet for each leaf samples. the supernatants were combined and left for 30 min at 0°c. after filtration, the supernatant volume was adjusted to 100 ml. about 2.5 ml of cold 2% anthron solution was added, and the sample was heated at 100°c for 7.5 min. it was then transferred immediately to an ice bath and cooled to room temperature. absorption at 630 nm was recorded using a spectrophotometer (model uv-120-20, japan). statistical analysis the experiment was conducted as a complete randomized design with factorial arrangements. analysis of variance was performed using the spss software package and significant differences among mean values were compared by duncan multiple range test (dmrt) (p<0.05). 3. results effect of salinity on plant growth after 90 days of salinity treatment plant growth (total shoot length, number of branches and leaf number) significantly reduced in all cultivars (table 2, 3 and 4). the effect of salinity on these parameters showed a significant genotypic variation. as expected, the highest reduction of shoot length was found in 12 dsm-1 treatment; considering the mean over all treatments, conservalia and shiraz cultivars had the highest shoot length while ‘dakal’ showed the lowest one under each salinity stress conditions (table 2). the highest reduction in number of branches was found in 8 and 12 dsm-1 treatments on average, the lowest branch number was found in ‘conservalia’ whereas the ‘amigdalilolia’, ‘dakal’ and ‘tokhm-e* mean separation within columns and rows by dmrt, at 5% level. table 2 effect of interaction between salinity treatment and cultivar on total shoot length (in comparison to the beginning of the experiment table 3 effect of interaction between salinity treatment and cultivar on branch number of olive cultivars table 4 effect of interaction between salinity treatment and cultivar on leaf number of olive cultivar (calculated as: leaf number at the beginning of the stress-the leaf number at ending the stress) cultivar total shoot lenght (cm) ec (dsm-1 ) of irrigating water 0 4 8 12 mean ‘conservaliaʼ 158.4 ab* 158.7 a 131.2 a-e 106.3 def 138.6 a ‘shirazʼ 149.1 ab 137.7 a-d 137.7 a-d 124.1 a-f 138.8 a ‘tokhm-e-kabkiʼ 149.1 a-d 119.6 b-f 114.7 def 112.7 def 121.0 bc ‘dezfulʼ 159.9 abc 136.0 a-d 119.2 b-f 121.4 b-f 134.1 ab ‘zard’ 156.3 ab 124.7 a-f 127.3 a-f 118.0 c-f 131.6 ab ‘dakalʼ 154.4 ab 102.7 f 104.8 ef 103.9 ef 116.4 c ‘amigdaliloliaʼ 142.4 a-d 121.8 b-f 106.4 ef 115.7 c-f 120.2 bc mean 151.4 a 128.5 b 120.2 bc 141.6 c * mean separation within columns and rows by dmrt, at 5% level. cultivar branch number ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 21.0 h-k * 24.3 f-j 8.3 l 8.0 l 15.4 d ‘shirazʼ 28.3 c-h 18.6 ijk 18.4 ijk 17.6 ijk 20.7 c ‘tokhm-e-kabkiʼ 40.0 a 35.6 a-d 25.2 e-i 30.2 b-g 32.7 a ‘dezfulʼ 29.8 b-h 28.5 c-h 22.9 g-k 20.9 h-k 25.3 b ‘zard’ 27.6 d-h 17.6 ijk 16.0 k 17.0 h-k 19.3 c ‘dakalʼ 29.3 c-g 31.0 b-f 32.0 b-f 37.6 ab 32.7 a ‘amigdaliloliaʼ 36.0 abc 34.6 a-d 33.0 a-e 22.6 g-k 31.4 a mean 30.3 a 28.0 b 21.8 c 22 c * mean separation within columns and rows by dmrt, at 5% level. cultivar leaf number ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 151.0 a-f * 138.6 c-g 21.0 jk -56.0 l 66.3 d ‘shirazʼ 114.6 b-f 113.6 e-i 74.3 g-j -11.3 kl 80.3 c ‘tokhm-e-kabkiʼ 190.6 a-d 207.1 ab 100.0 f-i 73.6 g-j 142.8 b ‘dezfulʼ 224.6 a 217.6 a 135.0 c-g 113.3 e-i 172.6 ab ‘zard’ 160.0 a-f 96.0 f-i 65.6 g-j 62.6 hij 96.8 c ‘dakalʼ 218.3 a 218.6 a 200.6 abc 130 d-h 191.9 a ‘amigdaliloliaʼ 185.3 a-d 177.3 a-e 174.0 a-e 98 f-i 158.6 b mean 177.8 a 167.0 a 110.1 b 60.0 c adv. hort. sci., 2017 31(1): 53-59 56 kabki’ obtained the most one (table 3). leaf number was also significantly reduced by salinity treatments. at 12 dsm-1, the reduction in leaf number ranged from -56.0 for ‘conservalia’ to 130 for ‘dakal’ with respect to control plants. the greatest number of leaves was found in controls and 4 dsm-1 treatments (table 4). effect of salinity on cell membrane injury (cmi) cell membrane injury (cmi) was significantly increased in all studied cultivars from the treatment of 12 dsm-1 except in ‘amigdalilolia’, ‘zard’ and ‘dakal’. the highest percentage of cmi was obtained in ‘conservalia’ with respect to the control treatment (table 5). furthermore, the maximum and minimum cmi belonged to 4dsm-1 and control, respectively. therefore, cmi was influenced by both salinity treatment and type of cultivar (table 5). proline content leaf proline content of olive cultivars was significantly influenced by salinity treatment and type of cultivar (table 6). maximum proline content was obtained in the leaf tissue of ‘dezful’ while the minimum proline content was determined in the leaves of ‘conservalia’. in general, the highest proline content was observed in 8 dsm-1 treatments and the lowest one was observed in 4 dsm-1 treatments (table 6). soluble carbohydrate content the concentration of soluble carbohydrates of leaves under salinity stress was significantly changed in all studied cultivars and the highest content of soluble carbohydrates was found in shiraz and zard cultivars and the lowest was observed in the leaves of ‘amigdalilolia’ (table 7). the maximum soluble carbohydrate content was found in 4 dsm-1 treatment and the minimum content was observed in control and 12 dsm-1 treatments, respectively. starch concentration content the stored carbohydrate content of leaves was significantly affected by salinity treatments and type of cultivar. ‘conservalia’ had the highest stored carbohydrate content whereas ‘shiraz’ showed the lowest (table 8). plants grown at 12 dsm-1 had the highest stored leaf carbohydrate content and the lowest one was observed in 8 dsm-1 treatments (table 8). table 5 effect of interaction between salinity treatment and cultivar on cmi of olive leaf (%) * mean separation within columns and rows by dmrt, at 5% level. cultivar cell membrane injury (%) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 74.7 abc* 72.8 abc 67.9 b-f 49.1 g 66.1 c ‘shiraz’ 66.6 c-f 60.4 f 52.7 g 52.9 g 58.2 d ‘tokhm-e-kabki’ 72.4 abc 71.6 a-d 63.6 def 63.1 ef 67.7 bc ‘dezful’ 69.7 a-e 66.2 c-f 68.7 a-e 53.3 g 64.5 c ‘zard’ 69.6 a-e 73.0 abc 68.3 b-f 69.4 a-e 70.1 b ‘dakal’ 70.3 a-e 77.1 a 68.9 a-e 68.6 a-e 71.2 b ‘amigdalilolia’ 76.7 ab 75.2 ab 76.3 ab 76.0 ab 75.6 a mean 71.4 a 70.9 a 66.6 b 61.6 c table 6 effect of interaction between salinity treatment and cultivar on proline content of olive cultivar leaf (µm g dw-1) * mean separation within columns and rows by dmrt, at 5% level. cultivar proline content (µm g dw-1) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 29.9 b-e* 21.1 g 24.0 efg 22.1 fg 24.3 c ‘shirazʼ 36.7 ab 22.7 fg 26.8 d-g 23.0 efg 27.3 bc ‘tokhm-e-kabkiʼ 28.9 b-f 28.9 b-f 33.2 a-d 26.6 d-g 29.4 b ‘dezfulʼ 35.4 abc 32.7 a-d 40.7 a 37.8 ab 36.7 a ‘zard’ 26.6 d-g 26.5 d-g 35.7 abc 28.6 b-f 29.3 b ‘dakalʼ 24.1 e-g 29.9 b-e 33.8 a-d 29.9 c-g 28.7 bc ‘amigdaliloliaʼ 22.4 fg 25.9 d-g 26.5 d-g 34.0 a-d 27.2 bc mean 29.1 b 26.8 c 31.5 a 29.5 ab table 7 effect of interaction between salinity treatment and cultivar on soluble carbohydrate content of olive leaf ( %) * mean separation within columns and rows by dmrt, at 5% level. cultivar soluble carbohydrate content (%) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 30.6 abc* 28.3 c-g 26.9 d-h 27.2 d-h 28.3 ab ‘shirazʼ 31.5 a 28.7 a-f 29.9 a-e 28.7 b-f 29.7 a ‘tokhm-e-kabkiʼ 28.0 c-h 31.3 ab 29.0 a-f 26.8 e-h 29.0 ab ‘dezfulʼ 26.7 d-h 29.1 a-f 26.8 e-h 28.3 c-g 27.6 bc ‘zard’ 30.1 a-d 30.0 a-e 28.9 a-f 29.3 a-f 29.6 a ‘dakalʼ 23.5 h 31.2 ab 30.6 abc 28.6 b-f 28.7 ab ‘amigdaliloliaʼ 24.5 gh 25.7 fgh 28.4 c-g 27.3 d-h 26.5 c mean 27.9 b 29.4 a 28.6 ab 28.1 b table 8 effect of interaction between salinity treatment and cultivar on stored carbohydrate content of olive cultivar leaf (mg g dw-1) * mean separation within columns and rows by dmrt, at 5% level. cultivar stored carbohydrate content (mg g dw-1) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 279.6 a-e* 280.6 a-d 251.3 b-g 339.1 a 290.7 a ‘shirazʼ 221.5 d-g 164.0 h 191.0 gh 296.9 abc 220.2 d ‘tokhm-e-kabkiʼ 238.7 c-g 242.8 c-g 239.0 c-g 284.2 a-d 249.7 bc ‘dezfulʼ 221.6 d-g 277.0 a-d 244.9 c-g 261.8 b-e 251.3 b ‘zard’ 251.9 b-f 233.4 c-g 244.2 c-g 237.3 c-g 241.7 bcd ‘dakalʼ 254.6 b-f 229.4 c-g 211.5 e-h 198.6 fgh 221.6 cd ‘amigdaliloliaʼ 288.9 a-d 319.7 ab 233.4 c-g 229.6 c-g 269.1 ab mean 249.4 ab 249.1 ab 230.0 b 264.5 a rahemi et al. physiological responses of olive cultivars to salinity stress 57 chlorophyll content the highest chlorophyll content was observed in the ‘dakal’, ‘dezful’ and ‘amygdalolelia’ while the lowest chlorophyll content was determined in the leaves of ‘shiraz’. in general, the highest chlorophyll content was measured in plants grown with the 4dsm-1 treatment and the lowest was observed for 12dsm-1 treatment (table 9). 4. discussion and conclusions the purpose of this study was to evaluate the salinity tolerance of seven olive cultivars (‘conservalia’, ‘amigdalilolia’, ‘dakal’, ‘tokhm-ekabki’, ‘shiraz’, ‘dezful′ and ‘zard) based on the effect of salinity on growth characteristics, and on physiological and biochemical response of different cultivars, grown in south of iran. salt stress has been reported to have genotypic-linked response (chartozoulakis, 2005). different parameters were used to indicate the response of seven cultivars to nacl stress. the main result concerns the reduced number of leaves in salt treated olive plant respect to control ones. such decrease was not only connected with the growth inhibition effects of salt but also to plants defoliation (karimi et al., 2009). the impact of salinity stress on reducing growth indices was clear and confirmed the response shown in previous research on different species and cultivars of olives (perica et al., 2004). it was clear that the decline in growth rate of olive trees under salinity stress was dependent on the duration of salt exposure, the concentration of salt and the potential of tolerance of cultivars. in the current study, olive cultivars showed different reactions to salinity stress. in general, vigorous cultivars were more susceptible to salinity stress than low growth cultivars. partial growth reduction of olive tree growth can be related to the osmotic stress resulting of elevated level of ions in soil solution and irrigation water (weissbein et al., 2008). because no visual signs of salt toxicity in the plants e.g. tip burn, necrosis and/or shoot die back (chartozoulakis, 2005) under low salinity level; in addition, monitoring leaf number changes indicated no sign leaf abscission due to salt toxicity or oxidative damages under low salt stress level. low osmotic potential of soil solution under salinity stress limits growth of plant by reducing water uptake, transpiration and stomatal conductance, which is associated with the reduced photosynthesis (benasher et al., 2006). hence it can be concluded that, osmotic stress is the primary reason of olive growth limitation under salinity stress. the cmi of leaf is the quantitative index which shows either health of plasma membrane or the rate of membrane disruption in plant leaf tissue under salinity stress. the reduction in cmi of olive cultivar was parallel to intensity of sodium chloride concentration in the irrigation water. under the severe salinity stress, the highest cmi rate was observed in the ‘amigdalilolia’, ‘dakal’ and ‘zard’ (101.8%, 99.4% and 97.6%), respectively and the lowest one belonged to ‘conservalia’ (65.6%). furthermore, leaf tip burning and leaf abscission were observed that might be due to accumulation of specific ions in olive leaf under salinity stress. though the leaf proline content in ‘shiraz’ and ‘conservalia’ declined with the increase of salinity stress, it increased in the other cultivars. during the salinity treatment, proline content was induced to accumulate in plant leaf tissue by the accumulation of the sodium and chloride ions and water stress as a result of enhancing of salt in soil solution (delauney and verma, 1993). in this study, despite salinity stress injuries, the increase in proline content may have played an important role in protection of olive cultivars in stress situations. the data presented here agreed with previous studies which show the direct relationship between tolerance of salinity stress and proline accumulation. it has been demonstrated that proline can protect protein from denaturation and maintain cytoplasmic membrane in salinity stress (khedr et al., 2003; karimi et al., 2009). data were shown that soluble sugar content of leaves of tolerant olive cultivars (‘amigdalilolia’, ‘dakal’) was enhanced during salinity period. table 9 effect of interaction between salinity treatment and cultivar on chlorophyll of olive cultivar * mean separation within columns and rows by dmrt, at 5% level. cultivar chlorophyll ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 1.84 efg* 2.56 a 2.31 a-e 0.74 i 1.86 b ‘shirazʼ 2.10 a-e 1.61 fg 1.55 g 0.20 i 1.36 c ‘tokhm-e-kabkiʼ 2.23 a-e 2.30 a-e 2.12 a-e 1.02 h 1.97 b ‘dezfulʼ 2.52 a 2.47 abc 1.95 d-g 2.05 a-f 2.25 a ‘zard� 1.92 d-g 2.24 a-e 1.98 c-g 1.59 fg 1.93 b ‘dakalʼ 2.25 a-e 2.48 abc 2.42 a-d 2.01 b-g 2.29 a ‘amigdaliloliaʼ 2.51 ab 2.25 a-e 2.27 a-e 1.80 efg 2.21 a mean 2.19 ab 2.27 a 2.09 b 1.36 c adv. hort. sci., 2017 31(1): 53-59 58 however in semi-tolerant cultivars (‘dezful’, ‘zard’ and ‘tokhm-e-kabki’) the sugar content was not significantly changed and in sensitive cultivars (‘shiraz’ and ‘conservalia’), this factor in comparison to control treatment was reduced. soluble sugars play an important role in maintaining turgor pressure in osmotic stress. furthermore, they can protect the plasma membrane in the stress situation (sanchez et al., 1998). this study showed that among tolerant olive cultivars, the accumulation of sugars could be due to the reduced consumption of stored carbohydrate contents (chartzoulakis, 2002). among the sensitive olive cultivars in which the starch and soluble sugar content were limited, it can be concluded that this phenomenon was related to photosynthesis disruption and the subsequent consumption of stored carbohydrate. under severe salinity stress condition, starch accumulation in the tissue of sensitive cultivars, may be due to disruption of enzyme activity and plant metabolism which led to stored carbohydrate in the tissue. salinity stress decreased chlorophyll concentration in the olive cultivar leaf. tolerant cultivars were shown more chlorophyll concentration than susceptible ones. therefore, it can be concluded that leaf chlorophyll concentration might be the best index for evaluating of olive cultivars to tolerate salinity stress as reported by noble and rogers (1994). the same authors suggested that chloroplast dysfunctions and decrease in the number and volume of chloroplast were influenced by salinity stress, and as a result, a reduction in chlorophyll content was a main reaction of olive trees to salinity stress. winicov and seemann (1991) observed that a salt tolerant alfalfa cell line exhibited an 11-fold increase in chlorophyll content compared to the unadapted cell line. the increase in chlorophyll content in alfalfa was associated with large increase in the activity of ribulose-1, 5bisphosphate carboxylase (winicove and seemnann, 1991). in conclusion, the relative tolerance of olive cultivars to salinity stress was in the following order: tolerant cultivars were ‘amigdaliloliaʼ and ‘dakalʼ, semitolerant cultivars included ‘dezful’, ‘zardʼ and ‘tokhm-e-kabkiʼ and finally sensitive cultivars were ‘shirazʼ and ‘conservaliaʼ. acknowledgments authors gratefully thank fars organization of jahad agriculture for their financial support of this project. references aragues r., puy j., royo a., espada j.l., 2005 three year field response of young olive trees (olea europea l., cv. arbequina) to soil salinity: trunk growth and leaf ion accumulation. plant soil, 271: 265-273. ayers r.s., westcot d.w., 1976 water quality for agriculture. fao irrigation and drainage, fao, rome. bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress. plant and soil, 39: 205-207. ben-asher j., tsuyki i., bravdo b.a., sagih m., 2006 irrigation of grapevines with saline water, i. leaf area index, stomatal conductance, transpiration and photosynthesis. agric. water manag., 83: 13-20. blum a., ebercon e., 1981 cell 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kanwar and kumar, 2009; (*) corresponding author: nnari26@gmail.com citation: niyokuri a.n., nyalala s., mwangi m., 2018 residual effects of bioslurry and amino acids plant biostimulant on carnation (dianthus caryophyllus l.) flower quality. adv. hort. sci., 32(1): 137-142 copyright: © 2018 niyokuri a.n., nyalala s., mwangi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 august 2017 accepted for publication 23 october 2017 ahs advances in horticultural science short note adv. hort. sci., 2018 32(1): 137-142 138 renukaradya et al., 2011). although it is indigenous to the mediterranean region, carnation can be grown in almost every climate in glasshouses, plastic houses, and shade nets as well as in open field (aydinsakir et al., 2011). it was fourth among the top ten imported cut flower to the netherlands after roses, st john’s wort (hypericum spp.) and gypsophila with a turnover of €18 million (floraholland, 2017). it was also one of the main cut flowers exported by kenya in 2012 (cbi, 2013) and among the leading cut flowers locally used in flower arrangements and in value addition of flowers, in form of bouquets. in 2014, carnations contributed 4% of the domestic value of floriculture in kenya (hcd, 2015). the quality of carnations is currently affected by many problems such as calyx splitting and short stem length for some varieties. to meet the required quality parameters such as stem length and girth, flowers size and number, farmers resort to heavy application of inorganic fertilizers and synthetic plant growth regulators. although this results in increased production and quality, it adversely affects soil productivity and the environment. this is because pesticides, phosphorus and nitrate used in carnation production represent the major agricultural pollutants that threaten the environment (nardi et al., 2016). with the increasing relevance of social and environmental standards, current research is focusing on developing alternative systems in crop production through development of unconventional and nonpollutant solutions. currently, there are limited organic alternatives to meet plant nutrients. moreover, organic alternatives for plant growth regulators are lacking in many crops and limited in other crops such as carnations. use of compost derived from plants and/or animal wastes as soil amendment or fertilizer additive has been reported as an alternative in the production of several ornamental plants. moreover, bioslurry, the residual manure generated through anaerobic decomposition of various organic materials is considered a quality organic fertilizer (islam, 2006). similarly, the use of biostimulants in sustainable agriculture has been growing particularly for their capacity of enhancing nutrition efficiency and stress response (du jardin, 2012). biostimulants can be obtained from different organic materials and include complex organic materials, humic substances, beneficial chemical elements, peptides and amino acids (protein hydrolysates), seaweed extracts, inorganic salts, chitin and chitosan derivatives, antitranspirants, amino acids and other n-containing substances (du jardin, 2015; nardi et al., 2016). many studies (karki, 2001; islam, 2006; shahbaz, 2011; jeptoo et al., 2013; shahariar et al., 2013) reported yield increases and quality improvement on many crops such as okra (hibiscus esculentus l.), maize (zea mays), cabbage (brassica oleracea var. capitata) and carrot (daucus carota) following bioslurry application. studies by nahed et al. (2009 a, b), paradiković et al. (2011) and mondal et al. (2015) reported that plant biostimulants could be successfully used in the production of ornamental and other horticultural crops such as antirrhinum majus, gladiolus (gladiolus grandflorum l.) sweet yellow pepper (capsicum annuum l.) and eustoma grandiflorum. however, many studies only evaluated the bioslurry and biostimulant direct effects, whereas there are only a few studies which focused on their residual effects on the quality and yield of many crops including carnations. this study was therefore aiming at evaluating residual effects of bioslurry and plant biostimulant on flower quality in carnation plants which previously received applications of bioslurry and plant biostimulant. 2. materials and methods experiment location the study was conducted in a greenhouse at lemotit flower farm of finlays horticulture kenya ltd. situated in kenya at latitude 0o 22’ south and longitude 35o 18’ east, from march to september 2015. experimental design and treatments application carnation ‘walker’ plants planted on soil media at a density of 36 plants per m2 were used. these plants had received drench applications of bioslurry and plant biostimulant, four times at bi-weekly intervals after pinching (three weeks after transplanting) during the period september 2014 to february 2015. the experimental design was a split-plot design with three replications. the main plot measured 5.5 x 1 m (5.5 m2) while the sub-plot was 1 x 1 m (1 m2). buffer zone of 0.5 m and 1 m separated inter-plots and individual main blocks respectively. cow dung bioslurry was applied in main plots at the rate of 0.125, 0.25, 0.5 l m-2 and control diluted in one litre of water prior to application. rates of plant biostimulant used were 2.0, 2.5 and 3.0 l ha-1 and control thoroughly mixed with water at the rate of 5000 l ha-1 and they niyokuri et al. residual effects of bioslurry and amino-acids on carnation flower quality 139 were applied to the sub-plots during the period of september 2014 to february 2015. bioslurry used had at wet basis a ph of 7.44, 0.23% of n, 4.58 ppm of p, 89.3 ppm of k, 4.31 ppm of ca, 19.91 ppm of mg and density of 1.0195 kg l-1. the plant biostimulant used was hicure®, an amino acids based plant biostimulant. this plant biostimulant contains a balanced mixture of free amino acids (with higher proline and glycine contents) and peptides (hydrolysed protein) of natural origin. it is composed of amino acids and peptides (62.5%), total nitrogen (10.9%) and organic carbon (29.4%). after harvest in february 2015, carnation plants were allowed to grow for subsequent season in order to study the residual effects of bioslurry and plant biostimulant. maintenance practices all treatments benefited from a weekly application of mineral fertilizers through fertigation using: 3.06 g n, 3.51 g p205, 5.19 g k20, 1.71 g ca and 0.74 g mg, plus trace elements per square metre. routine crop management practices included irrigation, supporting, weeding, training, disbudding and pest management. harvesting was done at the paint brush stage when petals started to elongate outside the calyx. data collection and analysis data were collected from 10 tagged sample plants per sub-plot on three flower quality parameters namely the length of flower stalk, diameter of flower stalk and flower head size (diameter and head length). the length of flower stalk was measured in centimetres from the point just below the bud to the point of origin of branch on the main stem at harvest; diameter of flower stem was measured in millimetres using digital vernier callipers. the flower head length was recorded in millimetres from the point just below the calyx to the upper point of the flower while the flower head diameter was recorded in millimetres at harvesting from each harvested cut flower at paint brush stage using digital vernier calliper. all data were subjected to analysis of variance (anova) using genstat 14th edition. separation of means was performed using the tukey’s honest significant difference (hsd) test at p≤0.05. 3. results there was no significant residual effect of bioslurry on measured parameters (table 1). however, a significant residual effect of plant biostimulant on plants which had received any level of plant biostimulant was observed on flower stalk length and flower stalk diameter (table 2). carnations plants that had received 2.0, 2.5 and 3.0 l of plant biostimulant had significantly longer flower stalks and significantly thinner flower stalks compared to the control (table 2). the application of different rates of plant biostimulant did not show a significant residual effect on flower head size (diameter and head height) as presented in table 2. the interaction between different levels of bioslurry and those levels of plant biostimulant had no significant residual effects on measured parameters (table 3). 4. discussion and conclusions results of this study showed a significant increase of flower stalk length as a result of the residual effect of plant biostimulant. this increase of flower stalk length may be as a result of enhancement of macro nutrient uptake by plant biostimulant (calvo et al., 2014; rose et al., 2014) which rapidly improves the growth compared to treatments without plant biostimulant. the other probable reason would be the direct uptake of amino acids which are immediately used by carnation plants for their growth and develtable 1 residual effect of bioslurry on carnation flower quality bioslurry levels (l m-2) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 81.01 5.52 21.77 40.58 0.125 81.18 5.56 22.04 40.44 0.25 81.02 5.55 21.86 40.56 0.5 81.15 5.55 21.81 40.48 table 2 residual effect of plant biostimulant on carnation flower quality levels of plant biostimulant (l ha-1) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 80.12 b* 5.628 a 21.97 40.53 2 81.20 a 5.512 b 21.83 40.47 2.5 81.20 a 5.515 b 21.74 40.58 3 81.84 a 5.528 b 21.93 40.48 * means in the same column with the same letter are not significantly different at p≤0.05 using tukey’s hsd test. adv. hort. sci., 2018 32(1): 137-142 140 opment (calvo et al., 2014). there are strong evidences that the increase in flower stalk length may be attributed to residual auxins and gibberellins like activities of the plant biostimulant (brown and saa, 2015). in fact, both auxins and gibberellins are important in plant cell division and elongation (ertani et al., 2009; calvo et al., 2014 and colla et al., 2014). results of this study are in agreement with previous findings by ertani et al. (2009) and colla et al. (2014). colla et al. (2014) reported an increase in coleoptile elongation rate when compared to the control, in a dose-dependent fashion, comparable with the effects of indole-3-acetic acid following the treatment of maize with the protein hydrolysate. the same study provided additional evidences of a gibberellin-like activity of protein hydrolysate when application of plant-derived protein hydrolysate “trainer” at all doses significantly increased the shoot length of the gibberellins deficient dwarf pea plants by an average value of 33% in comparison with the control treatment. similar results previously reported by ertani et al. (2009) showed that the treatment of lettuce with both protein hydrolysate based fertilizers resulted in an increase in the epicotyl length comparable with the effects of exogenous gibberellic acid. the occurrence of this residual effect was perhaps the result of previous down-regulation mechanisms which affected the effect of plant biostimulant in the previous season. ammonium, which was used as a source of nitrogen, has been reported to down-regulate amino acids (henry and jefferies, 2003 cited by gioseffi et al., 2012; thornton and robinson, 2005 cited by gioseffi et al., 2012). the observed reduction in flower stalk diameter is suspected to be a gibberellin-like activity which promoted the flower stalk length (ertani et al., 2009; colla et al., 2014) at the expense of flower stalk diameter. the absence of residual effect of plant biostimulant on flower head size was possibly due to constant supply of nutrients through fertigation. the flower head size is usually a result of carbohydrates stored for subsequent growth and reproductive processes (islam et al., 2010). although there are evidences that biostimulants can enhance macro nutrient uptake (calvo et al., 2014; rose et al., 2014), it is possible that the residual effect of plant biostimulant was limited to stimulating the elongation of flower stalk. hence, the residual effect may have contributed much during early stages when carnation plants started re-growing after the harvest by improving growth and nutrients assimilation as previously revealed by colla et al. (2014). bioslurry did not show any significant residual effect on the flower stalk length, flower stalk diameter and the flower head size. generally, nutrients in cow dung slurry and poultry manure slurry are released in higher amounts compared to their original state (haque et al., 2015). this is because bioslurry, with its narrower c:n than farmyard manure, shows better results on soil nutrient availability at early stages of its application while, farm yard manure affects the nutrient uptake to the plant in more consistent manner because its mineralization occurs at later stages (muhmood et al., 2014). however, results of our study are not in agreement with shahzad et al. (2015) who reported that the application of bioslurry and composted poultry manure as a bio-fertilizer improves soil organic matter contents and availability of soil nutrients (n, p and k) to the subsequent crop, resulting in increased crop productivity and reducing the cost of fertilizer to subsequent crop. in fact, the residual effect of bioslurry may depend on its initial content, characteristics and the quantity applied and for these reasons, the residual effect of bioslurry on carnation grown in the following season was very limited. based on results of this study, we can conclude that the application of plant biostimulant on carnations plants supplied with a full dose of inorganic fertable 3 residual effects of the interaction of bioslurry and plant biostimulant on carnation flower quality bioslurry levels (l m-2) level of plant biostimulant (l ha-1) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 0 79.94 5.57 21.81 40.61 2 81.07 5.48 21.74 40.57 2.5 80.83 5.51 21.71 40.45 3 82.21 5.51 21.8 40.70 0.125 0 79.75 5.65 22.13 40.55 2 81.30 5.53 21.81 40.46 2.5 81.68 5.51 21.79 40.67 3 81.97 5.56 22.44 40.08 0.25 0 80.28 5.67 22.06 40.49 2 81.14 5.52 21.95 40.50 2.5 80.85 5.50 21.76 40.60 3 81.82 5.51 21.69 40.64 0.5 l 0 80.52 5.61 21.88 40.47 2 81.29 5.52 21.84 40.35 2.5 81.44 5.55 21.7 40.58 3 81.35 5.53 21.81 40.41 niyokuri et al. residual effects of bioslurry and amino-acids on carnation flower quality 141 tilizers may have residual effect on the subsequent production flush. however, the residual effect of bioslurry on carnations grown in the same conditions may depend on its nutrient content and physicochemical characteristics. further works would be necessary to study the application of both products under lower rates of inorganic fertilizers and extend over many productions flushes to find out the extent of their residual effects. acknowledgements the authors are thankful to james finlays company for providing the research plant materials and allowing us to conduct experiments in their lemotit farm. references aydinsakir k., tuzel i.h., buyuktas d., 2011 the effects of different irrigation levels on flowering and flower quality of carnation (dianthus caryophyllus l.) irrigated by drip irrigation. afr. j. biotechnol., 10(66): 14826-14835. brown p., saa s., 2015 biostimulants in agriculture. front. plant sci., 6: 671. calvo p., nelson l., kloepper j.w., 2014 agricultural uses of plant biostimulants. plant soil, 383: 3-41. cbi, 2013 cbi tradewatch cut flowers and foliage. survey report. cbi, ministry of foreign affairs, www.cbi.eu. colla g., rouphael y., canaguier r., svecova e., cardarelli m., 2014 biostimulant action of a plantderived protein hydrolysate produced through enzymatic hydrolysis. front. plant sci., 5: 448. du jardin p., 2012 the science of plant biostimulants. a bibliographical analysis. final report “ad hoc study report on bio-stimulants products”, pp. 1-37. du jardin p., 2015 plant biostimulants: definition, concept, main categories and regulation. sci. hort., 196: 3-14. ertani a., cavani l., pizzeghello d., brandellero e., altissimo a., ciavatta c., nardi s., 2009 biostimulant activity of two protein hydrolyzates in the growth and nitrogen metabolism of maize seedlings. j. plant nutr. soil sci., 172: 237-244. floraholland, 2017 facts and figures 2016 . https://www.royalfloraholland.com/en. gioseffi e., de neergaard a., schjoerring j.k., 2012 interactions between uptake of amino acids and inorganic nitrogen in wheat plants. biogeosciences, 9: 1509-1518. haque m.a, jahiruddin m., rahman m.m., saleque m.a., 2015 phosphorus mineralization of bioslurry and other manures in soil. j. environ. waste manag., 2(2): 79-83. hcd, 2015 horticulture. validated report 2014. horticulture crops directorate, pp. 1-68. islam m.r., rahman s.m.e., rahman m., oh d.h., ra c.s., 2010 the effects of biogas slurry on the production and quality of maize fodder. turk. j. agric. for., 34: 91-99. islam m.s., 2006 use of bioslurry as organic fertilizer in bangladesh agriculture. international workshop on the use of bioslurry, domestic biogas programmes, 2728 september, bangkok, thailand. jeptoo a., aguyoh j.n., saidi m., 2013 improving carrot yield and quality through the use of bio-slurry manure. sustainable agric. res., 2(1): 164-172. kanwar j.k., kumar s., 2009 influence of growth regulators and explants on shoot regeneration in carnation. hort. sci. 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humic substances: practical implications for agriculture. adv. agron., 124: 37-89. roychowdhury r., tah j., 2011 evaluation of genetic parameters for agro-metrical characters in carnation genotypes. afr. crop sci. j., 19(3): 183-188. salunkhe d.k., bhat n.r., desai b.b., 1990 postharvest biotechnology of flowers and ornamental plants. springer verlag, new york, usa, pp. 28-46. shahabz m., 2011 potential of bio-slurry and compost at different levels of inorganic nitrogen to improve growth and yield of okra (hibiscus esculentus l.). m.s. thesis, university of agriculture faisalabad, pakistan. shahariar m.s., moniruzzaman m., saha b., chakraborty g., islam m., tahsin s., 2013 effects of fresh and digested cowdung and poultry litter on the growth and yield of cabbage (brassica oleracea). bangladesh j. sci. ind. res., 48(1): 1-6. shahzad k., khan a., smith j.u., saeed m., khan s.a., khan s.m., 2015 residual effects of different tillage systems, bioslurry and poultry manure on soil properties and subsequent wheat productivity under humid subtropical conditions of pakistan. intl. j. biosci., 6 (11): 99-108. 84 1. introduction highly systematized greenhouses for plant growth are called plant factories (pf) in japan, and they have been rapidly increasing in recent years (kobayashi, 2010; nonami, 2010). pf are roughly divided into two types: the solar light-type (spf) and artificial light-type (apf) (murase and fukuda, 2012; kozai, 2013). although both types are essentially the same as greenhouse cultivation, more advanced technologies have been adopted in pf to control growth environments. japan’s ministry of economy, trade and industry (meti) defines a pf as “a facility that aids in the steady production of high-quality vegetables all year round by artificially controlling the cultivation environment (e.g. light, temperature, humidity, carbon dioxide concentration, and culture solution), allowing growers to plan production” (meti, 2014). the netherlands is leading the world in the commercial use of spf, and some well-known companies such as hoogendoorn, hortimax, and priva export excellent cultivation systems to many countries, including japan (kozai, 2013). compared to these advanced companies, the practical application of pf in japan has just begun. nevertheless, research papers and the use of pf have greatly expanded over the last few years. one of the triggers for the expansion of pf was the establishment of the “plant factory working group” of the “agriculture, commerce and industry collaboration study group in april 2009 in cooperation with maff (meti, 2009), who compiled a report describing issues to be addressed, as well as the support required for the promotion and diffusion of pf (meti, 2014). the japanese government appropriated approximately €100 million in the fiscal year (fy) 2009 as a supplementary budget to promote pf. of this, approximately €36 million was spent on constructing and promoting advanced pf, and eight research facilities and 18 pilot plants were established in various areas of japan, including seven universities (kobayashi, 2010). from a broad perspective, there are two ways to promote pf: one is to enhance the productivity of horticultural crops without relying on the experience of farmers, and the other is to allow the private sector to enter agro-industries. 2. present economic status and agricultural production in japan due to the impact of the financial crisis precipitated by the lehman brothers bankruptcy in 2008, the gross domestic product (gdp) of japan decreased by approximately 7% during the five years from 2008 to 2012 (approximately € 3.7 and € 3.4 trillion, respectively) (fig. 1) (imf, 2013). however, japan has been suffering from a long-term economic slowdown for an even longer period, often dubbed “the lost two decades.” this long-term recession heavily impacted the private sector, for example present status and future outlook of plant factories in japan t. nishizawa (1) faculty of agriculture, yamagata university, tsuruoka 997-8555, japan. key words: environment control, fluorescent light, led, lettuce, organic electroluminescence, plant factory, strawberry. abstract: plant factories for the steady production of high-quality vegetables year round, and which can be divided into solar and artificial-light types, have recently been expanding in japan as trigged by a report by a collaboration study group in 2009 and the great east japan earthquake in 2011. common solar-type plant factories with mulch-span roofs are often difficult to construct in the northern areas of japan, especially along the japan sea coast, because of limited sunshine duration and heavy winter snowfall, while artificial light-type factories are more promising in this region although high running costs due to electricity bills for irradiating plants and cooling the room often hinder the promotion of such facilities. the use of leds has recently increased in artificial light-type plant factories, but fluorescent lights are still predominant for economic reasons. generally only small plants can be grown commercially in artificial light-type factories and the light intensity reaching the lower leaves decreases continuously as the stem of the plant elongates, deteriorating light use efficiency. flexible organic electroluminescent devices able to cover the whole plant when irradiation is required and that can easily be applied/removed like a plastic film are expected to be introduced in both types of plant factories. adv. hort. sci., 2014 28(2): 84-89 (1) corresponding author: nisizawa@tds1.tr.yamagata-u.ac.jp received for publication 31 march 2014 accepted for publication 30 june 2014 85 construction industries, resulting in many unemployed workers. although agricultural production in japan accounts for less than 2% of the gdp, production was sustained at a constant level during this period (approximately € 6.1 billion), showing that agriculture in japan was not hit hard by the recession (fig. 1). nevertheless, agriculture in japan faces some difficult problems, such as a decline in the labor force due to a decreasing birthrate and the aging of farmers, limited farmland, high cost of production, and low productivity. however, such difficulties can also provide great opportunities for the private sector to enter into new business. because of this, many companies in the private sector are now interested in entering agriculture, but it is also difficult for them to start new businesses due to legal restrictions hampering their entry into agriculture. 3. laws regulating the entry of the private sector into agriculture until 70 years ago, villages in japan consisted of a few “zinushi” (landowners) who owned most of the farmland and a large number of “kosakunin” (tenant farmers) who worked on these lands (council for regulatory reform, 2002). “zinushi” lent the farmland to “kosakunin” to cultivate the land, and took some farm products, such as rice and beans, as “kosakuryo” (land rent). however, after world war ii, farmlands in japan were divided and distributed to each “kosakunin”. this new policy allowed “kosakunin” to become land owners and saved japan from food shortages during the postwar period. however, the area of distributed farmlands was too small for children to inherit. as the japanese economy developed rapidly during the postwar period, many of these workers left the villages to find jobs in the cities, resulting in the aging of the farming population since the 1960’s. according to statistics from japan’s ministry of agriculture, forestry and fisheries (maff), the total number of japanese farmers in 2013 was 5.62 million, and 2.03 million of them (36.1%) were older than 65 years, which is higher than the percentage of over 65 years of the total population (24.1%) (maff, 2014). aging of the farming population rapidly decreases the production of agricultural products. therefore, the japanese government is now aiming to expand farm management by consolidating farmlands to develop efficient and stable large-scale farm management to improve agricultural productivity and stabilize production. however, such an integration of farmland has not yet given results because many farmers are still eager to possess their farmland as a property or as a means to earn capital gains by using it for alternative purposes (council for regulatory reform, 2002). in addition, the “agricultural land law” and “agriculture promotion law” which were implemented in 1952 and 1969, respectively, work as a barrier when the private sector wishes to enter into agriculture, because they prohibit persons other than farmers from acquiring any farmland. even if a farmer tries to repurpose his farmland, he has to send application forms with opinions from the agricultural committee located in each village, town, or city to the governor of each prefecture who is authorized to give permission through the committee. on the other hand, there is almost no restriction on the sale of horticultural crops, which will enable the private sector to enter agriculture if such companies grow horticultural crops on company-owned land, although the fixed property tax on farmland is considerably lower than that on commercial or industrial areas. another way for the private sector to enter agriculture is to finance agricultural corporations, but it will also be difficult to increase the flow of investment without improving the circumstances of corporate entry into agriculture, such as the further liberalization of corporate entry through farmland acquisition (aritsubo, 2003). for all these reasons, intensive cultivation of vegetables using company-owned factories and lands is possible for the private sector trying to enter agriculture. also, chronic recession affecting the private sector associated with cutbacks in public investment leads to disused equipment, land, and available workers, all of which can also be directed by the private sector toward agriculture. 4. characteristics of greenhouse production in japan in japan, rice production was responsible for the highest sales among agricultural products until 1986, but both horticulture and animal husbandry increased thereafter as the price of rice decreased. at present, the sum of vegetable and flower production accounts for approximately 30% of the gross product, which is greater than rice production and almost the same as animal husbandry (fig. 2). greenhouses have made a marked contribution to increasing the sales of these horticultural products, and the total area of greenhouses in japan is now approximately fig. 1 figures for the gdp (1980-2012) and agricultural production (2008-2012) in japan calculated using the imf world economic outlook database (2013) and statistics released by maff (2013), respectively. 86 50,000 ha (kozai, 2013). however, many of them are still plastic-film greenhouses smaller than 0.1 ha, and only 40% of them have a heating system installed. in addition, computerized environmental control systems have yet to sufficiently circulate commercially, which means that environmental control techniques in greenhouses still largely rely on the experience of farmers. as a result, the average yield of horticultural products from common plastic-film greenhouses still remains far lower compared to that of advanced greenhouses, where environmental management programs have been utilized to automatically optimize plant growth automatically. for example, the average yield of tomatoes in japan is 60 t·ha-1, which is only 13% of that in the netherlands (476 t·ha-1) (fao, 2013). however, the introduction of computerized environmental management programs is not the only answer, cultivation of horticultural crops in japan should be converted from empirical to computer-controlled methods to minimize the decrease in production due to the aging of farmers. 5. introduction of spf as a method to recover from the damage due to the great east japan earthquake (geje) the geje occurred on 11 march 2011, causing considerable damage to the country, including the agricultural sector. according to the data book of maff, the earthquake and subsequent tsunami affected approximately 24,000 ha of farmland in six prefectures located along the pacific coast, which accounted for 2.7% of the total farmland in the region (fig. 3, table 1). among the affected prefectures, the damage to miyagi prefecture was the greatest (10.7% of the total farmland), and it accounted for 60.6% of the total farmland area affected. in 2011, the japanese government established the “law on special geje reconstruction areas” and special support programs to promote early recovery, such as the “geje recovery special loan” program and “geje recovery emergency guarantee” program. according to data from the ministry of finance (maf), the central government spent approximately €137 billion on recovery from the geje during fy 2010-2012 (sato, 2013). miyagi prefecture was a famous area for greenhouse production of horticultural crops such as strawberry, bell-pepper, and tomato, but many facilities were destroyed by the earthquake, and the subsequent tsunami caused serious salt damage to farmland, which resulted in only 33.3% restoration by 11 march 2012 (table 1). therefore, there is much expectation that the affected area will recover as a center for horticultural production table 1 distribution of farmland areas affected by the tsunami following the great east japan earthquake on 11 march 2011 and their restoration prefecture farmland (ha) affected area (ha) % of affected area restored area (ha)(z) % of restored area aomori 156,800 107 0.1 101 94.4 iwate 153,900 1,209 0.8 269 22.2 miyagi 136,300 14,558 10.7 4,855 33.3 fukushima 149,900 5,927 4.0 549 9.3 ibaraki 175,200 1,063 0.6 958 90.1 chiba 128,800 1,162 0.9 1,162 100.0 total 900,900 24,026 2.7 7,894 32.9 (z) restored area on 11 march 2012. table created from databases of maff 2011 and 2012. fig. 2 proportion of each sector for total agricultural production in 2012. the pie chart was created using statistics released by maff (2013). fig. 3 major prefectures in japan. 87 by introducing large-scale spf (ito, 2012), and some facilities have already started running commercially (fig. 4). 6. present and future of spf in japan the productivity of spf largely depends on light conditions. in this regard, weather conditions in japan are often a big barrier to introducing spf. for example, the total duration of sunshine a pacific coast area such as kochi, aichi, and shizuoka prefectures is 2,115-2,158 h per year (5.8-5.9 h per day), while that on the japan sea coast such as akita, aomori, and yamagata prefectures is 1,4901,600 h per year (4.1-4.4 h per day), 69-76% of that on the pacific coast (fig. 3 and table 2). when the duration is divided into summer (april to october) and winter (november to march), the former length does not differ much between the two regions (5.5-5.8 vs. 5.2-5.5 h per day for pacific coast and japan sea coast, respectively), while the latter length on the japan sea coast (2.3-3.3 h per day) is only 36-56% of that on pacific coast (5.8-6.4 ha per day). moreover, the japan sea coast receives far more snowfall compared to the pacific coast (377-669 vs. 0-16 cm per year on the japan sea coast and pacific coast, respectively) (table 2). although spf are usually multi-span type glasshouses with small roofs (fig. 5), such structures oftable 2 comparison of seasonal duration of sunshine and total amount of snowfall between pacific coast and japan sea coast month monthly duration of sunshine (h) pacific coast japan sea coast kochi shizuoka aichi akita aomori yamagata jan 189.9 204.6 169.1 37.7 47.8 80.0 feb 176.9 187.1 174.3 65.5 72.3 99.1 mar 191.9 193.7 199.0 117.6 123.0 136.7 apr 195.4 188.1 198.1 165.9 175.3 170.0 may 185.2 182.1 192.4 175.9 189.7 186.6 jun 134.2 129.3 146.4 171.4 174.6 159.3 jul 174.2 157.5 166.6 146.8 154.1 145.2 aug 206.8 202.6 200.7 184.9 177.2 171.5 sep 168.3 159.9 159.7 153.4 156.0 135.1 oct 180.7 164.2 173.4 145.2 149.9 135.4 nov 166.2 173.0 163.4 81.1 84.2 101.5 dec 188.2 202.5 171.9 44.1 49.1 76.7 jan-dec 2157.9 2144.6 2115.0 1489.5 1553.2 1597.1 daily duration of sunshine (h) apr-oct 5.8 5.5 5.8 5.3 5.5 5.2 nov-mar 6.1 6.4 5.8 2.3 2.5 3.3 jan-dec 5.9 5.9 5.8 4.1 4.3 4.4 total amount of snowfall (cm per year) jan-dec 1 0 16 377 669 426 table created from the database of the japan meteorological agency in 2013. fig. 4 a solar-type plant factory constructed in the area affected by the great east japan earthquake that occurred on 11 march 2011. fig. 5 a multi-span type glasshouse with small roofs constructed in tsukuba city, ibaraki prefecture. 88 ten cannot withstand the weight of heavy snow, especially in northern japan. therefore, apf and not spf are more valuable from a commercial aspect along the japan sea coast of northern japan. 7. present and future of apf in japan pioneering studies of plant growth using artificial light began as early as the 1920’s (harvey, 1922), and in the 1960’s a useful book on artificial light for the production of horticultural crops was published (canham, 1966). in japan, vegetable production using artificial light has been studied since the 1970-80’s, mainly in the research laboratories of maff and universities, and the results have largely contributed to clarifying the mechanisms of flowering, photosynthesis, carbohydrate partitioning, etc., under controlled environments (nishizawa and shishido, 2013). however, the results were not effectively applied commercially (hashimoto, 2012). one of the pioneering examples of aps was the ts-farm of kewpie co., ltd., constructed in ibaraki prefecture (fig. 6), where leafy vegetables have been produced since june 1986 (sekiyama, 1994). on this farm, triangle panels were arranged in an environmentally controlled facility and high-pressure sodium lamps and spraying hydroponics were used for cultivation. another example of a pioneering aps was the “rotary lettuce production facility” of hitachi co., ltd., which was exhibited at the international exposition, tsukuba, japan, 1985 (takatsuji, 2009). although approximately 100 apf are functioning commercially in japan now, many of them have been used for the cultivation of leafy vegetables such as leafy lettuce, while higher valued crops such as strawberry have been grown only in a few facilities (kozai, 2013). moreover, lighting systems of commercial apf still largely rely on fluorescent lights, which have the problem of heat accumulation in the plant during irradiance because of a low and high conversion efficiency from electrical energy to photons and heat, respectively (hoshi et al., 2010; takatsuji, 2010). therefore, the cost for electricity to cool the facility is often more than 50% of that for irradiating the plants (kozai, 2012; 2013). because there is marked potential to overcome the disadvantage of fluorescent lights by using leds (massa et al., 2008), studies for the cultivation of vegetables using this light source have also expanded widely in japan (watanabe, 2011), but the commercial use of leds is still very limited mainly because of the high cost (kozai, 2013). even if the price of leds decreases to that of fluorescent lights, some difficulties still remain regarding the expansion of apf in terms of management. one of the intractable problems is that only small low-valueadded crops are available to grow in such facilities. to solve this problem, studies to add value to the crops have been conducted, for example high-level antioxidative properties (kozai, 2012; 2013), low potassium content (suzuki, 2013), etc. by altering growth environments, or to cultivate more valuable crops such as seedlings (yokoi et al., 2007), medical plants, genetically modified plants (usami, 2011), etc. however, such studies may come up against a limitation in the near future. while almost all horticultural crops can be grown in spf, some which elongate longitudinally are still very difficult to grow in apf because the light intensity is theoretically inversely proportional to the square of the distance. therefore, the light intensity reaching the lower leaves decreases continuously as the stem of the plant elongates. longitudinally arranged fluorescent lights, comprising a line of corded leds in the plant canopy, and the utilization of laser diodes have been tested to solve this problem (takatsuji and mori, 2003), but these methods often aggravate working conditions for growers. one possible solution is to utilize organic light-emitting diodes (oled) which emit light in response to an electric current using an emissive electroluminescent layer with organic compounds (tang and vanslyke, 1987). although the light intensity of oled is still lower than that of other illuminants, a high-performance white-light display of oled has been newly developed at the faculty of engineering, yamagata university in japan (kido et al., 1995), meaning that small horticultural crops such as leafy lettuce and strawberry can be grown (fig. 7). flexible oled devices are theoretically possible to rewind like a negative film. therefore, it may be possible to improve the efficiency of farmers’ work by hanging film-type oled devices from the ceiling of greenhouses and covering the whole plant only when irradiation is required. such an illuminant can be utilized not only in apf but also in spf. references aritsubo t., 2003 structure of agriculture. nippon jitsugyo publishing, tokyo, japan, pp. 172-173. canham a.e., 1966 artificial light in horticulture. contrex publishing, eindhoven, netherland, pp. 1-212.fig. 6 ts-farm constructed by kewpie co., ltd., in ibaraki prefecture. 89 council for regulatory reform, 2002 agricultural, forestry, and fisheries, pp. 1-7 in: second report regarding promotion of regulatory reform. priority regulatory reform measures to promote economic vitalization. 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h., 2011 plant cultivation system in intelligent plant factory. j. pesticide sci., 36(4): 503-509. watanabe h., 2011 light-controlled plant cultivation system in japan. development of a vegetable factory using leds as a light source for plants. acta horticulturae, 907: 37-44. yokoi s., goto e., kozai t., nishimura m., taguchi k., ishigami y., 2007 effects of planting density and air current speed on the growth and that uniformity of tomato plug seedlings in a closed transplant production system. j. shita, 19(4): 159-166. fig. 7 strawberry (a) and leafy lettuce (b) grown under white-light organic electroluminescence display at the faculty of agriculture, yamagata university. impaginato 3 adv. hort. sci., 2018 32(1): 3-12 doi: 10.13128/ahs-21006 24-epibrassinolide improves some physiological disorders in pistachio cultivars f. kamiab (*) department of horticulture, rafsanjan branch, islamic azad university, rafsanjan, iran. key words: abscission, antioxidant enzymes, hormone, physiological disorders, spraying. abstract: the effect of 24-epibrassinolide foliar spray on some physiological disorders of pistachio cultivars was evaluated in two years. this factorial experiment was designed as a randomized complete block design with three replications. factor a involved different pistachio cultivars and factor b involved the application time of hormone with four treatments including t1= bud swelling, t2= after full bloom, t3= t1 + t2, and t4= control. different parameters were recorded. the application of hormone in both different stages caused the maximum fruit set percentage and minimum inflorescence bud and fruit abscission percentage in three sprayed cultivars. despite of the highest percentage of inflorescence bud and fruit abscission in control treatment of cv. kallehghoochi, the application of this hormone decreased these parameters in this cultivar in t3 treatment. chlorophyll, protein, and proline contents of the leaves were increased in all brassinosteroide treatments, especially under two-stage application. also, ion leakage and reduced sugar were decreased in treated leaves after using hormones. the effect of this hormone on antioxidant enzymes showed that all enzymes (cat, sod and apx) except pod were increased by the application of brassinosteroides. the loss of carbohydrate in one-year-old woods indicated the growth stimulating effect of this hormone. thus, 24-epibrassinolide is a good suggestion to increase the quantity and quality of pistachio, especially in cv. kallehghoochi. 1. introduction pistachio (pistacia vera l.) is an important horticultural crop that has high economic value. unfavourable environmental conditions in recent years have resulted in the loss of fruit set and yield in commercial pistachio cultivars. also, physiological disorders such as bud and fruit abscission, and blank and non-split nuts are the most important problems of this produce that have been deteriorated by the biotic and abiotic stresses in most pistachio-growing areas. plant antioxidant defence system consists of such enzymes as superox(*) corresponding author: f.kamiab56@gmail.com citation: kamiab f., 2018 24-epibrassinolide improves some physiological disorders in pistachio cultivars. adv. hort. sci., 32(1): 3-12 copyright: © 2018 kamiab f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 july 2017 accepted for publication 31 october 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 3-12 4 ide dismutase (sod), catalase (cat), peroxidase (pod), ascorbate peroxidase (apx) and also, nonenzymatic components may include osmolytes like proline, glycine betaine, sorbital, and mannitol (anjum et al., 2010, 2012). in these conditions, growth regulators are useful for the prevention of fruit abscission and increased yield. nowadays, foliar spray of growth regulators in fruit orchards has become a usual practice, and their considerable effects are evident on fruit quantitative characteristics. brassinosteroids (brs) are steroid hormones that are widely distributed in the plant kingdom, with a regulatory function in normal plant growth and development (mandava, 1988). exogenous application of brs to roots of young tomato and radish plants induced the hypocotyls and petioles (takatsuto et al., 1983). brassinosteroids are known as hormones with pleiotropic effects that influence different processes like cell elongation, senescence and xylem differentiation, growth, seed germination, rhizogenesis, flowering, stem elongation, pollen tube growth, leaf epinasty, ethylene biosynthesis, proton pump activation, gene expression, and photosynthesis (clouse and sasse, 1998; dhaubhadel et al., 1999; khripach et al., 2000; steber and mccourt, 2001; krishna, 2003; yu et al., 2004; vert et al., 2006). brassinosteroids also could induce resistance to various abiotic stresses (rao et al., 2002). commercial application of this hormone has been started since 1990 and 24-epiprassinolid and 28homobrassinolid are more effective in garden conditions because of their stability (khripach et al., 2000). effects of 28-homobrassinolide have been reported on yields of wheat, rice, groundnut, mustard potato, and cotton (ramraj et al., 1997). foliar spray of brassinoides on watermelon seedling increased female flower, fruit set and yield considerably. in other experiments, effective role of brassinosteroid has been confirmed in vegetative and reproductive growth in strawberries and yellow passion fruit (ramraj et al., 1997; gomes et al., 2006). also, the application of brs could accelerate the ripening of tomato and grape fruits (vardhini and rao, 2002; symons et al., 2006). despite numerous studies on agronomic crops, there are few investigations into the effect of brassinosteroides on fruit trees. hosseinpur et al. (2014) used different concentrations (0.5, 0.75, 1 and 1.5 mg/l) of 24-epibrassinolid before flowering on kallehghuchi pistachio and showed that 1.5 mg/l of epibrassinolid was the best treatment for increasing fruit set of this cultivar. due to the increasing unfavourable environmental conditions during pistachio flowering, this hormone was tested before and after flowering for increasing fruit set. numerous studied has showed that effect of this hormone depends on its application time. thus, we compared different application times on three commercial cultivars to determine the best application time for brassinosteroides on pistachio trees. the objective of the study was to evaluate the effect of 24-epibrassinolid on quantitative and qualitative traits of pistachio fruit. 2. materials and methods plant material and experiments this experiment was done in a commercial orchard (lat. 29°30’ n., long. 56°05’ e., alt. 1605 m.) in the asad abad district, rafsanjan, iran in 2014 and 2015. the results of soil analysis of this orchard are shown in table 1. mineral deficiencies are compensated with the use of fertilizer during the growing season. in 2014, twenty adjacent “on” trees were selected. on each “on” tree, five uniforms “on” shoots were chosen and labeled a week before full bloom. each selected shoot included four clusters with no lateral shoots. because of the alternate bearing habit of pistachio trees, “on” trees in 2014 were naturally “off” trees in 2015, so this experiment was repeated by applying the same treatments on the similar previous year’s trees, naturally going into the “off” year, but the treatments were applied on other one-yearold shoots to avoid any experimental effects of the previous year’s treatments. these selected shoots were also uniform in length and diameter similar to the previous year’s shoots, but each shoot included only one cluster. 24-epibrassinolide (1.5 mg/l, sigma texture soil ph saturation (%) ec (ds/m) depth (cm) k (ppm) p (ppm) n (%) sandy-clay 7.7 28 7 0-30 239 13.8 0.03 table 1 chemical analysis of some macro and micro elements of the soil of the pistachio orchard in asad abad village, rafsanjan kamiab brassinosteroids contribution to pistachio disorders 5 company) was sprayed on crown of the trees. the experiment was carried out on a factorial base with a randomized complete block design with three replications. factor a involved different pistachio cultivars (akbari, kalehghoochi and ohadi). factor b involved the time of the application of 24epibrassinolide in four treatments: t1= bud swelling stage, t2= two weeks after full bloom, t3= application at two stages (t1 and t2) and t4= control (spraying with water). there were 12 treatments with 3 replications in this experiment, and two trees in each experimental unit (totally 72 trees) were compared. inflorescence bud and fruit abscission and fruit set the number of initiated inflorescence buds and the total number of abscised buds on the individual current-year shoots were counted six weeks after full bloom (may) and at the harvest time (september), respectively. the percentage of inflorescence bud abscission was calculated by dividing the number of abscised buds by the total number of buds initiated on each shoot. in order to detect the fruit set percentage and fruit abscission, four branches with almost equal buds in different geographical sides of each tree were selected and marked. these factors were measured by the following formula, respectively: numer of fruit set (30th day) initial fruit set percentage = x 100 number of flower buds numer of fruit set (120th day) harvest fruit set percentage = x 100 number of flower buds fruit characteristics at the harvest time, all clusters were detached from each shoot and were hand-sorted into blank, non-split, and split nuts, fresh and dry weight of fruits were measured on each marked branches. yield factor is reported in results as dry weight of nuts per each branch. biochemical characteristics all biochemical parameters were measured about two weeks after foliar application of brassinosteroides and spectrophotometer was used for all biochemical measurement. assay of protein, apx and pod fresh leaf samples (1 g) from all treatments were ground in 5 ml tris-hcl buffer (0.05 m). the homogenates were centrifuged at 10,000 g for 25 min at 4°c. the protein content in the supernatant was analyzed by the procedure of bradford (1979). the supernatants were also used to analyze the activity of apx and pod enzymes. apx activity was determined by nakano and asada method (1981). assay of sod, cat and h2o2 frozen leaf samples (1 g) were homogenized in 50 mm sodium phosphate buffer (ph 7.8 for sod and ph 7 for cat). then, these materials were centrifuged at 12,000 g for 20 min at 4°c. the supernatant was used to measure the activity of the enzymes. sod activity was measured according to the method described by giannopolitis and ries (1977) and cat activity was determined by the procedure of cakmak and marschner (1992). reducing sugars and proline analysis proline and reducing sugars content were extracted from the fresh leaf samples according to bates et al. (1973) and somogyi (1952), respectively. chlorophyll content of leaves the chlorophyll content was calculated using the method introduced by meidner (1984) as: total chlorophyll (mg/ml-1 g-1): [(17.76 × od646.6) + (7.37× od663.6)] × v/1000w where, od= the read absorbance , v= consumed acetone volume, w= fresh weight of sample (g). ion leakage of leaves the relative permeability of cell membranes was calculated using a slight modification of the method introduced by zhang et al. (2006) as: ec1-ec0relative permeability (%) = x 100 ec2-ec0 total nonstructural carbohydrate of 1-year old wood one-year-old stems were immediately placed on ice and were taken to the laboratory. in the laboratory, the stem tissue were dried at 60°c, weighted, ground to pass a 40-mesh screen and analyzed for total nonstructural carbohydrate (tcn). the concentration of starch was measured by the method of hedge and hofreiter (1962). dissolved sugar was calculated using the phenol and sulphuric acid method introduced by hellebust and craige (1978). the absorbencies were measured at 485 and 630 nm for dissolved sugar and starch, respectively. the concentration of total sugar and starch were summed to adv. hort. sci., 2018 32(1): 3-12 6 give an estimate of total nonstructural carbohydrate. statistical analysis finally, the collected data were analysed using sas software package, the means were compared by duncan test at 5% level, and the diagrams were drawn in ms-excel software package. 3. results interaction of brassinosteroides and cultivars for inflorescence bud, fruit abscission, and fruit set in pistachio the result indicated that the two-stage application of this hormone decreased bud abscission by about 20% in kalleghuchi cultivar but by about 10% in other cultivars (akbari and ohadi) (table 2). in the “on” year, the highest percentage of fruit abscission was observed in control in kalleghuchi cultivar and the application of this hormone especially in two stages decreased fruit abscission by about 30% while fruit abscission was decreased by 20% and 18% in akbari and ohadi cultivars, respectively (table 2) and also the foliar spray of this hormone in two stages increased the fruit set significantly so that it was doubled as compared to control. interaction of brassinosteroides application time and cultivars for blank and split nuts in pistachio the highest blank nut and the lowest split nut were observed in control in ‘kallehghoochi’ in two experimental years and in the “on” year, they were about 6% and 54%, respectively. the lowest blank nut and the highest split nut were observed in kallehghoochi and ohadi cultivars and brassinosteroides spray at two stages in the “on” year at about 4% and 80%, respectively. the percentage of blank nut was decreased in all cultivars in the “off” year by about 50% when the hormone was applied at two stages and by 25% when it was applied at one stage. in the “off” year, the percentage of split nut was significantly increased only in ‘kallehghoochi’ at two stages of brassinosteroides application (table 3). table 2 interaction of stage of brassinosteroides application and cultivar for fruit and bud abscission and fruit set percentage of pistachio in two years ‘ohadi' ‘akbari' ‘kallehghoochi' c t1 t2 t3 c t1 t2 t3 c t1 t2 t3 during 2014 “on year” fruit set (%) 9.5 d 13 c 14 c 16 bc 11 d 14.5 c 18 b 21 a 7.1 f 8.5 e 9.2 d 13.5 c friut abscission (%) 58 b 50 c 47 c 40 d 62 ab 54 bc 55 b 42 c 68 a 47 c 43 c 36 d bud abscission (%) 68 c 63 d 60 d 58 d 88 a 83 b 80 b 78 b 90 a 78 b 72 c 68 c during 2015 “off year” fruit set (%) 11 d 12.5 c 13 c 14 c 15 b 16 b 17 b 19 a 9 d 9.5 d 10 d 12 c fruit abscission (%) 44 c 42.5 cd 40 d 38 d 50 b 46 c 47 bc 42 cd 54 a 50 b 48 bc 45 c bud abscission (%) 50 a 45 ab 43 ab 40 b 30 c 28 c 28 c 22 d 25 cd 24 cd 22 d 22 d table 3 interaction of stage of brassinostroides application and cultivar for blank, split, fresh and dry weight of nut, and yield per shoot of pistachio in two years ‘ohadi' ‘akbari' ‘kallehghoochi' c t1 t2 t3 c t1 t2 t3 c t1 t2 t3 during 2014 “on” year blank nut (%) 4.5 bc 4.2 c 4 c 3.2 d 5.5 ab 5 b 4.5 bc 4 c 6 a 5 b 4.5 bc 4 c fresh weight nut (g) 2.1 e 2.1 e 2.4 d 2.9 c 2.9 c 3 c 3.2 b 3.8 a 2.9 c 3 c 3.2 b 3.9 a dry weight nut (g) 0.67 d 0.7 d 0.8 cd 0.95 c 0.9 c 1 bc 1.1 b 1.2 ab 0.9 c 1 bc 1.1 b 1.3 a split nut (%) 50 d 70 b 65 bc 75 ab 65 bc 70 b 75 ab 79 a 54 d 63 c 70 b 80 a yield per shoot (g) 45 e 75 c 80 c 100 b 60 d 90 b 95 c 120 a 50 ed 65 d 75 c 90 bc yield per tree (kg) 2 f 2.5 ef 3 e 3.5 de 4 d 5 c 6.1 b 7 a 3 e 3 e 4.1 d 5.2 c during 2015 “off” year blank nut (%) 10 b 8.5 c 8 c 5.5 d 12 a 8.5 c 9 bc 6d 11 ab 7 cd 6.5 d 6 d fresh weight nut (g) 2.8 d 3 cd 3 cd 3.8 a 3.2 c 3.7 ab 3.6 b 3.9 a 3.3 c 3.5 b 3.5 b 3.9 a dry weight nut (g) 0.9 e 1 d 1 d 1.2 b 1.1 c 1.2 b 1.2b 1.3 a 1.1 c 1.2 b 1.2 b 1.3 a split nut (%) 75 bc 76 bc 78 b 80 b 8 ab 87 a 87 a 90 a 73 c 80 b 80 b 80 b yield per shoot (g) 14 e 20 b 18 c 22 a 15 ed 20 b 20 b 22 a 16 d 20 b 18 c 22.1 a yield per tree (kg) 1 d 1.1d 1.5 c 1.8 b 1.5 c 1.8 b 2.1 b 2.8 a 1.3 c 1.3 c 1.7 bc 2 b c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. different letters within a row indicate significant differences by duncan’s multiple range tests at p<0.05. c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. different letters within a row indicate significant differences by duncan’s multiple range tests at p<0.05. kamiab brassinosteroids contribution to pistachio disorders 7 interaction of brassinosteroides application time and cultivars for fresh and dry weight of pistachio nut the highest fresh weight and dry weight of fruit were observed in ‘kallehghoochi’ and in brassinosteroides spray at two stages, which were about 3.9 and 1.3 g, respectively. the lowest fresh and dry weights of fruit in the “on” year were observed in control in ‘ohadi’ with no hormone application, which were about 2.1 and 0.67 g, respectively (table 3). interaction of brassinosteroides application time and cultivars for yield per shoot and tree in pistachio table 3 showed that brassinosteroides significantly increased the yield per shoot and tree, especially when they were applied at two stages in the “on” year. the interaction between cultivar and the stage of brassinosteroides spray for the yield per shoot showed that the highest yield of branch and tree were observed in ‘akbari’ and in brassinosteroides spray at two stages, which were about 120 g and 7 kg, respectively. the lowest yield of branch and tree were observed in control of ohadi cultivar and they were about 45 g and 2 kg, respectively in the “on” year (table 3). interaction of brassinosteroides application time and cultivars on proline, reduced sugar, and protein of pistachio leaves table 4 reveals that the highest amount of reduced sugar and the lowest amount of protein and proline was observed in control in ‘kallehghuchi’ in both experimental years. the application of brassinosteroides (at two stages) increased protein and proline contents of the leaves in all pistachio cultivars significantly, especially in the “on” year, and also the lowest amount of reduced sugar was observed in all cultivars in both “on” and “off” years when the hormone was applied at two stages (table 4). interaction of the time of brassinosteroides application and cultivars for chlorophyll and ion linkage of leaves according to table 4, the chlorophyll content was significantly increased in all brassinosteroides treatments as compared to control. maximum chlorophyll content was observed in two-stage application of this hormone in all cultivars. maximum ion leakage was related to the control in all cultivars and all brassinosteroides treatments showed significantly lower ion leakages. minimum ion leakage was observed in twostage application of this hormone in all cultivars (table 4). interaction of the time of brassinosteroides application and cultivars for antioxidant enzyme activities figures 1 and 2 shows that activity of all antioxidant enzymes was significantly higher in the “on” year than in the “off” year. the application of brassinosteroides suppressed the activity of all enzymes (cat, sod and apx) except pod. the lowest activity of all enzymes was observed in control of kallehghuchi cultivar in two trial years so that they were remarkably boosted with application of this hormone as compared to other cultivars. maximum enzyme activity was observed in two-stage application of this hormone in all cultivars. interaction of the time of brassinosteroides application and cultivars on annual changes in total nonstructural carbohydrate (tcn) concentration of oneyear-old wood of pistachio figures 3 and 4 reveal that carbohydrate storage table 4 interaction of stage of brassinostroides application and cultivar for some physiological traits of pistachio in two years c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. different letters within a row indicate significant differences by duncan’s multiple range tests at p<0.05. ‘ohadi' ‘akbari' ‘kallehghuchi' c t1 t2 t3 c t1 t2 t3 c t1 t2 t3 during 2014 “on” year proline (µm/g fw) 20 c 22.6 c 25.7 bc 35 ab 20 c 23 c 28 b 38 a 18 c 20 c 30 b 40 a reducing sugars (mg/g fw) 30 b 25 bc 24 bc 18 c 35 b 30 b 26 bc 20 c 40 a 30 b 25 bc 12 d protein (mg/g fw) 18 bc 19.1 b 20 ab 21 a 19 b 19 b 19 b 21 a 15 d 16.8 c 19 b 2 ab ion leakage (%) 38 b 34 c 35 bc 31 cd 34 c 30 d 30 d 29 d 43 a 40 ab 39 b 34 c chlorophyll (mg/ml fw) 21 c 25 b 28 ab 29.9 a 19.5 c 26 b 27 ab 30.3 a 16.3 d 26.5 b 27.3 ab 30.2 a during 2015 “off” year proline (µm/g fw) 15 c 16 c 16.2 c 20 b 14.5 c 14.5 c 15c 19 b 14.2 c 17.5 bc 20.5 b 25.8 a reducing sugars (mg/g fw) 7.7 ab 7.56 b 7.5 b 6.9 c 6.5 d 6.5 d 6 e 6 e 7. 9 a 7.75 ab 7.75 ab 7.42 b protein (mg/g fw) 17.1 bc 17.1 bc 17.5 b 17.7 b 19 ab 19 ab 19.1 ab 20.1 a 16 c 16.5 c 18.3 b 20 a ion leakage (%) 40 b 37 c 37 c 32 d 36 c 33 d 31 d 29 e 45 a 43 ab 41 b 38 bc chlorophyll (mg/ml fw) 25 c 28 bc 32 ab 34 a 22 d 29 b 30 b 33 a 20 d 29 b 30 b 33 a adv. hort. sci., 2018 32(1): 3-12 8 in shoots of “on” and “off” trees decreased following the spring growth flash. figure 3 shows that, in “on” trees, the lowest amount of carbohydrate was observed in jun and aug in all cultivars. after harvest (sep), amount of carbohydrate increased again. figure 4 shows that, in “off” trees, stored carbohyfig. 1 interaction of stage of brassinostroides application and cultivar for the activities of some antioxidant enzyme of pistachio in the “on” year. c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. different letters within a row indicate significant differences by duncan’s multiple range tests at p<0.05. fig. 2 interaction of stage of brassinostroides application and cultivar on activities of some antioxidant enzyme of pistachio in the “off” year. c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. different letters within a row indicate significant differences by duncan’s multiple range tests at p<0.05. fig. 3 interaction of stage of brassinostroides application and cultivar on annual changes in total non-structural carbohydrate (tcn) concentration of one-year-old wood of pistachio in the “on” year. c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. fig. 4 interaction of stage of brassinostroides application and cultivar on annual changes in total non-structural carbohydrate (tcn) concentration of one-year-old wood of pistachio in the “off” year. c= control, t1= bud swelling phase, t2= two weeks after full bloom, t3= t1 and t2. kamiab brassinosteroids contribution to pistachio disorders 9 drate of control and t1 treatments increased and remained high after initial growth flash but, in t3 and t4 treatments, it decreased in july and then started to increase again. 4. discussion and conclusions the effect of 24-epibrassinolid application on yield and quantitative traits of pistachio in three cultivars was considerable in this experiment. maximum yield was observed in akbari cultivar and brassinosteroides spray at two stages so that it was doubled as compared to the control. also, the application of this hormone at two stages increased fruit set significantly as compared to control. this finding confirms those reported by heidari et al. (2015) who showed that the application of epi-brassinolid increased fruit set in alberta peach. brassinosteroides regulate fruit number and are also able to induce cell division and elongation (rao et al., 2002). gomes et al. (2006) reported that the use of brassinosteroides in yellow passion after flowering has led to an increase in fruit number and yield. foliar application of brassinosteroids resulted in an increase in the number of flowers in strawberry (leubner-metzger, 2001), and also in grape fruits, the foliar application of brassinosteroids in autumn increased the number of flowers (pipattanawong et al., 1996). these results show that the increased yield in this experiment could be due to the increase in pistachio flowers induced by this hormone. another problem in pistachio is fruit abscission whose causes are not clearly known. however, environmental influences and competition for resources such as photo assimilates and plant hormones are the likely causal factors of fruit abscission (thompson, 1996). fruit abscission in this experiment fell by half in kallehghuchi cultivar at two-stage hormone application. foliar spray of epibrassinolid on grape (0.01 mg/l) during flowering reduced fruit abscission (pozo et al., 1994). brassinosteroides can increase carbohydrate content because of the increase in photosynthesis capacity and translate assimilate from source to sink (goetz et al., 2000). evaluation of the carbohydrate storage in oneyear-old wood of pistachio in spring and summer season in this research showed that the amount of carbohydrate decreased in spring (april and may). this reduction of stored carbohydrate could be related to spring growth flash. in “on” year, there were other two steps of reduction of carbohydrate, the first and second steps were due to summer growth flash and period of kernel fill, respectively. this finding confirms the results of timothy et al. (2008). but it should be noted that the application of this hormone, especially in t3 treatment, decreased significantly the amount of stored carbohydrates in first step of reduction as compared to control. this seems to be due to stimulation of growth after application of this hormone. stored carbohydrates, in one-year-old wood, after harvest of pistachio were significantly higher in this treatment. in “off” trees, stored carbohydrate of control and t1 treatments increased and remained high after initial growth flash but stored carbohydrate in t3 and t4 treatments decreased in july and then started to increase again. it seems that decreased carbohydrate in july could be caused by the stimulation of growth by the use of hormone. thus, other reason for higher yield in this experiment is the enhanced growth after application of this hormone caused by the increased number of leaves and shoot and, consequently, more assimilation synthesis for kernel growth and flower bud development. chlorophyll content of leaves was increased in the treated plants. thus, the increased chlorophyll could be the reason for the increase in assimilates in pistachio, resulting in the increased fruit set, fresh and dry weight of nuts and yield. this is consistent with the results of hassan zadeh (2013) who showed that the application of epi-brassinolid increased chlorophyll in cantelop. unfavourable environmental stresses such as drought and high temperature conditions during flowering and after it decrease fruit set and, on the other hand, increase fruit abscission in most pistachio gardens in rafsanjan. plant stress tolerance requires the activation of complex metabolic activities including anti-oxidative pathways, especially ros-scavenging systems within the cells that in turn can contribute to continued plant growth under stress conditions (el-mashad and mohamed, 2012). one of the roles of brassinosteroids is the increased resistance of plants against various abiotic stresses as confirmed by the results of this experiment. so, apx, sod and cat enzymes were increased with the application of this hormone especially its two-stage application. it should also be noted that in ‘kallehghuchi’ as the most sensitive cultivar to physiological disorder, the amount of antioxidant enzymes were lower than other cultivars and that it was increased with the application of brassinosteroides hormone significantly. there are numerous reports in this regard, adv. hort. sci., 2018 32(1): 3-12 10 some of which are reviewed here. brassinosteroidtreated tomato and rice plants grew better than control plants under low-temperature conditions (kamuro and takatsuto, 1991). in rice, 24-epibrassinolide increased the resistance against chilling stress (1-5°c) and the tolerance was associated with the increased atp, proline levels and sod activity, thus indicating brassinosteroid involvement in membrane stability and osmo regulation (wang and zang, 1993). brassinosteroids increased tolerance to high temperature in wheat leaves (kulaeva et al., 1991). results of this experiment showed that the reduction of ion leakage in leaves confirms that this hormone impacts membrane permeability, resulting in resistance to environmental stress. thus, the increased fruit set and decreased fruit abscission in treated trees could be due to the increased resistance to a biotic stress because of hormone application. on the other hand, the application of epi-prassinolid increased proline content and decreased the reduced sugar in pistachio leaves. it seems 24-epi brassinolid could alleviate the adverse effects of abiotic stress on pistachio cultivars, especially on kallehghuchi arguably by increasing the activities of ant oxidative enzymes and the contents of proline. the increase in proline and anti-oxidant enzymes are the natural mechanisms to induce tolerance to abiotic stress in plants. in a similar study, rady (2011) reported that the spray of 5μm 24-epi brassinosteroides to nacl-exposed phaseolus vulgaris improved the elevation of the activities of anti-oxidative enzymes and proline content. aghdam et al. (2012) reported that the treatments with 0.3 and 0.6 μm brs to tomato fruits stored at 1°c for 21 days enhanced proline. foliar application of brassinosteroides was reported to improve cd-tolerance in brassica juncea through the increase in activity of antioxidative enzymes (such as cat, pod, sod) and the content of osmolyte such as proline (hayat et al., 2007). on the other hand, lower amount of reduced sugar in brassinosteroides treatments could reflect the alleviation of the adverse effects of abiotic stress after the application of this hormone. dry and fresh weight of pistachio nuts was increased by about 30% with the application of 24epibrassinolid at two stages. the increase of nut weight may be another reason for the increased yield in this experiment. brassinosteroides have important role in such phenomenon as cell division and elongation, photosynthesis rate, transfer material, regulation of enzyme activity and hormone balance, each of which can increase nut weight and yield in pistachio crop. increased dna and rna polymerase and increased synthesis of protein by the use of brassinosteroides can induce growth (kalinich et al., 1985). vardhini and rao (1998) showed the relationship between increased growth and dna, rna and protein synthesis in peanut. yu (1999) found significant increases in the initial activity of rubisco and in the sucrose, soluble sugars, and starch contents followed by substantial increases in sucrose phosphate synthase, sucrose synthase and acid invertase activities after brs treatment. impaired carbohydrate metabolism and reduced biomass were found in a brassinosteroid-deficient arabidopsis mutant (schluter et al., 2002). increasing of blank nut especially in kallehghoochi and akbari cultivars is one of the most important disorders in pistachio trees. it has been reported that the degeneration of the ovary segments, especially funicle degeneration, is the major cause of blanking in pistachio (shuraki and sedgley, 1996). brassinosteroids are considered as hormones with pleiotropic effects as they influence various developmental processes like growth, flowering, pollen tube growth, tissue differentiation, proton pump activation, gene expression and photosynthesis (khripach et al., 1998, 2000). therefore, brassinosteroide can improve growth and development of reproductive organs and prevent ovary degeneration and, on the other hand, embryo abortion could be decreased by the application of this hormone because of the inducing protein, carbohydrate synthesis and, consequently, the blank nuts can be decreased. in this research the percentage of non-split nuts was decreased with the application of epibrassinolid. a correlation has been found between kernel development and splitting (ferguson et al., 2005). thus, the decrease in the percentage of non-split nuts by the application of this hormone might be attributed to its role in improving the growth and development of pistachio trees and, on the other hand, the application of epibrassinolid increases the production of carbohydrate and assimilation into the plants and, as a result, the growth of kernel increases (crane and iwakiri, 1985). in conclusion, exogenous application of 24-epibrassinolide indicated the possible direct role of this hormone in alleviating the physiological disorders and increasing the quality and yield in pistachio. thus, 24-epibrassinolide is a good suggestion for increasing quantity and quality of pistachio, especially in kallehghoochi cultivar that has the most disorder physiological problems. kamiab brassinosteroids contribution to pistachio disorders 11 references aghdam m.s., asghari m., farmani b., mohayeji m., moradbeygi h., 2012 impact of postharvest brassinosteroids treatment on pal activity in tomato fruit in response to chilling stress. horticultural science, 144: 116-120. anjum n.a., umar s., ahmad a., 2012 oxidative stress in plants: causes, consequences and tolerance. first ed., ik international 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looney (eds.) cherries: crop physiology, production and uses. cabi, wallingford, oxfordshire, uk, pp. 464. timothy m., robert h., dejong m., 2008 seasonal carbohydrate storage and mobilization in bearing and non-bearing of pistachio. tree physiology, 28: 207213. vardhini b.v., rao s.s., 2002 acceleration of ripening of tomato pericarp discs by brassinosteroids . phytochemistry, 61: 843-847. vardhini b.v., rao s.s.r., 1998 effect of brassinosteroids on growth, metabolite content and yield of arachis hypogaea. phytochemistry, 48: 927-930. vert g., nemhauser j.l., geldner n., hong f.x., chory j., 2006 molecular mechanisms of steroid hormone signalling in plants. annual review of cell and developmental biology, 21: 177-201. wang b.k., zang g.w., 1993 effect of epibrassinolide on the resistance of rice seedlings to chilling injury. acta physiologica, 19: 38-42. yu j.q., huang l.f., hu w.h., zhou y.h., mao w.h., ye s.f., nogues s., 2004 a role for brassinosteroids in the regulation of photosynthesis in cucumis sativus. j. exper. bot., 55: 1135-1143. yu j.q., 1999 parthenocarpy induced by n-(2-chloro-4pyridyl)-n-phenylurea (cppu) prevents flower abortion in chinese white-flowered gourd (lagenaria leucantha). environ. exper. bot., 42: 121-128. zhang j., liu y.p., pan q.h., zhan j.c., wang x.q., huang w.d., 2006 changes in membrane-associated h+-atpase activities and amounts in young grape plants during the cross adaptation to temperature stresses. plant sci., 170: 768-777. 21 1. introduction tecomella undulata (roxb.) seem., known as rohida tree, is an ornamental and medicinal shrub species of the bignoniaceae family widespread in tropical regions such as iran, india, and pakistan. this endangered plant species currently grows in arid and semi-arid regions of southern parts of iran. its bark contains lapachol, a naphthoquinone with anticancer, antibacterial, antifungal, antivirus (consolacao et al., 1975; guiraud et al., 1994; hussain et al., 2007) and analgesic (ahmad et al., 1994) activities. it may have a pivotal role in environmental conservation in the arid parts of iran; furthermore it is an accepted tree species in agroforestry systems (tewari and singh, 2009). due to its beautiful flowers and semi-deciduous habit, it can be used in landscaping with some limitations due to its sterility and low potential germination of the few seeds produced in iran. karami and salehi (2010) reported that rooting of stem cuttings is limited from spring to autumn in this species and it is included in the list of hard to root woody plant species. therefore, micropropagation is necessary to protect this endangered species. plant tissue culture is one of the most important steps in genetic transformation and plant biotechnology studies to produce plantlets from stock plants as a rapid and efficient procedure throughout the year (giri et al., 2004; sarmast et al., 2009). there are few reports about micropropagation of t. undulata; in vitro seasonal effect on shoot proliferation was reported (rathore et al., 1991). attempts have been made to multiply t. undulata plants through micropropagation. rathore et al. (1991) reported that about 8-10 shoots were obtained over two to three weeks at 31ºc in ms medium supplemented with bap (2 mg l-1) and iaa (0.05 mg l-1). robinson et al. (2005) claimed that ms basal medium supplemented with 1.5 mg l-1 bap and 0.02 mg l-1 iaa was the most effective medium for maximum (95%) regeneration of nodal explants. they reported that 29 shoots per nodal segment were observed on ms medium supplemented with 0.75 mg l-1 bap and 0.01 mg l-1 iaa within 3 weeks. furthermore, they observed about 27% mortality after transfer of plantlets to soil mixture. aslam et al. (2009) developed a transformation protocol for osmotin gene in t. undulata. the effects of silver nanoparticles (snps) on decontamination in tissue culture systems have been reported (abdi et al., 2008; sarmast et al., 2011). however, the effect of silver-based material on ethylene mode of action is not well acknowledged in plants (taiz and zeiger, 2006). explants grown in media supplemented with silver ions (eapen and george, 1997; zhang et al., 2001) and snps (sarmast et al., 2011) were relatively healthier than control. the most important step of any in vitro propagation system is mass multiplication of plantlets that are genetically homogenous and phenotypically uniform (sarmast et al., 2012). therefore, micropropagation is restricted to direct regeneration. effects of silver nanoparticles on tecomella undulata (roxb.) seem. micropropagation m. aghdaei, h. salehi(1), m.k. sarmast department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: multiplication, rohida tree, snps, tissue culture. abstract: plant tissue culture is a reliable tool for conservation and multiplication of many plants, including medicinal plants. tecomella undulata (roxb.) seem. is a plant native to tropical regions such as iran, india and pakistan; this precious plant which contains lapachol (a strong antiseptic used against jaundice) is an endangered species, therefore its conservation is of prime importance. the aim of this experiment was to evaluate the effects of silver nanoparticles (snps) at concentrations ranging from 5 to 80 mg l-1 alone or combined with 6-benzyl-amino-purine (bap) and indoleacetic acid (iaa) on growth properties of t. undulata in aseptic condition. thidiazuron (tdz) at concentrations from 0.001 to 20 mg l-1 was used in proliferation medium of t. undulata single nodes; combinations of bap (from 0.3 to 1.2 mg l-1), and 2,4-dichloro-phenoxy-acetic acid (2,4-d, 0.2 and 0.4 mg l-1) were also used in callus production and in indirect bud regeneration media. explants were surface sterilized using 10% clorox for 7-8 minutes. results indicated that adding of snps in ms medium increased the mean number of fresh shoots per explants (mnfs/e), the percentage of explants producing shoots (peps) and also plant survival, due to its action on ethylene blockage. tdz at the concentration of 0.1 mg l-1 increased bud proliferation up to two buds per explants, however higher concentration inhibited growth and in some cases caused death of the explants. adv. hort. sci., 2012 26(1): 21-24 (1) corresponding author: hsalei@shirazu.ac.ir received for publication 17 october 2011 accepted for publication 31 january 2012 22 the aim of the present work was to assess snps as ethylene inhibitor for the improvement of micropropagation in t. undulata. a secondary objective was to examine tdz and bap effects on direct and indirect regeneration in t. undulata through single node and callus obtained from the proximal part of the explants. 2. materials and methods more than 15-year-old iranian tecomella undulata plants were chosen for the present study. stem pieces (4 cm long) were cut and prewashed in tap water for 15 min. explants were then treated with 10% clorox (containing 5.25% sodium hypochlorite) plus 0.2% household detergent for 7 min for surface sterilization and then rinsed six times with sterilized distilled water. the stem pieces were finally cut into nearly 10 mm long segments (including a single node) and placed with their proximal ends on ms (murashige and skoog, 1962) basal medium with 3.0% sucrose and 0.8% agar. mean number of fresh shoots per explant (mnfs/e), mean length of shoots per explant (mls/e), mean diameter of callus per explant (mdc/e) and percentage of explants producing shoots (peps) on ms medium supplemented with snps (5 to 80 mg l-1) and combination of bap (2.5 mg l-1) and iaa (0.1 mg l-1) (aslam et al., 2009) along with snps (5 to 80 mg l-1) were evaluated. thidiazuron (tdz) was used at concentrations ranging from 0.001 to 10 mg l-1 for assessment mean number and length of shoots per explant, after two weeks of implementation. the average size of nanoparticles used in this study was 18.5 nm and they were synthesized by nanotechnologies, inc. (nanocid company, tehran, iran) (fig. 1). callus derived from tdz primary treatments was cultured for one week on ms hormone-free medium and then transferred to ms medium supplemented with bap (0.3 to 1.2 mg l-1) in combination with 2,4-d (0.2 and 0.4 mg l-1) for indirect bud formation. the explants were subcultured every two weeks. the ph of the media was adjusted to 5.8 before autoclaving for 15 min at 121°c and 1.5 kg cm-2 pressure. cultures were kept at 25±2°c under cool white fluorescent light (30 µmol m -2 s -1) with 16/8 h photoperiod. the experiment was conducted as a completely randomized design. means were compared using lsd at p≤0.05 with spss software (spss inc., chicago, usa). fig. 1 transmission electron microscopy (tem) micrograph of ag nanoparticles (scale bar of 100 nm). 3. results and discussion completely healthy and disinfected explants were achieved following treatment with 10% clorox for 7 min. explants cultured on ms basal medium supplemented with different concentrations of snps had a higher mnfs/e, mls/e and peps than control (table 1). in other words, snps had positive effects on single node explants of t. undulata, but mdc/e in the control was higher than in other treatments. for this reason, there were not significant differences between 10 mg l-1 snps compared to 80 mg l-1 on mnfs/e and mls/e and, in order to save on cost, the use of 10 mg l-1 snps in tissue culture of t. undulata was proposed. in another experiment (table 1) there were table 1 comparing snps and combination of snps along with bap (2.5 mg l-1) and iaa (0.1 mg l-1) after two weeks on t. undulata (roxb.) single node explants snp (mg l-1) mean number of shoots per explants mean length of shoots per explants mls/e mean diameter of callus per explants percentage of explants producing shoots snp snp+bap+iaa snp snp+bap+iaa snp snp+bap+iaa snp snp+bap+iaa 0 1.06 cd (z) 1.06 cd 2.03 a-c 2.03 a-c 4.27 a 4.27 a 62 ab 62 ab 5 1.00 c 1.24 b-d 1.81 bc 3.01 ab 0.39 c 5.10 a 50 b 67 ab 10 1.65 ab 0.37 e 3.59 ab 1.07 c 0.75 c 2.10 bc 77 a 27 c 20 1.06 cd 1.16 cd 2.33 a-c 2.92 ab 1.19 c 5.02 a 70 ab 70 ab 40 1.82 a 1.41 a-d 3.74 a 1.94 a-c 2.22 bc 5.17 a 80 a 70 ab 80 1.68 a 1.49 a-c 3.02 ab 2.73 a-c 1.27 c 3.81 ab 85 a 70 ab mean 1.37 a 1.12 b 2.75 a 2.28 a 1.68 a 4.24 b 70 a 61 b (z) in each column, means with the same letters are not significantly different at ≤ 0.05 level of probability using lsd. 23 not significant effects between explants grown in medium supplemented with bap (2.5 mg l-1) and iaa (0.1 mg l-1) compared with medium supplemented with different concentrations of snps. when we compared data of snps with snps in combination with plant growth regulators (bap and iaa), there were significant effects between mnfs/e, mdc/e and peps (table 1). our results demonstrate that iranian tecomella undulata (roxb.) seem. had high potential for producing callus from stem segments even in hormone-free medium, but after a couple of days they turned brown and died. even single node explants, after nearly three weeks, had necrotic leaves. the use of common antioxidants such as ascorbic and citric acid or activated charcoal (data not shown), did not have significant effects on survival of single node explants and callus derived from stem segments. leaves of t. undulata produced callus in ms medium but these, when attached to a single node, directly started callus formation. bap at the concentration of 0.9 mg l-1 allowed regeneration of few buds on callus produced from stem segments (data not shown). results indicate that tdz at the concentration of 0.1 mg l-1 increased mns/e and mls/e up to 2 and 3.33 mm respectively from samples taken in autumn (table 2, fig. 2). however tdz at low concentration had a positive effect on callus production, but concentrations of more than 0.01 mg l-1 decreased the dimensions of mdc/e. table 2 regeneration response of t. undulata (roxb.) single node explants on ms medium supplemented with tdz after two weeks snp (mg l-1) number of shoots per explants mean length of shoots per explants (mm) mean diameter of callus per explants (mm) 0.000 0.85 b-d (z) 2.28 a-c 4.23 a-d 0.001 0.90 bc 1.66 a-d 4.80 ab 0.010 1.13 b 2.83 ab 5.33 a 0.100 2.00 a 3.33 a 3.46 a-d 1.000 0.53 b-d 1.66 a-d 4.40 a-c 10.000 0.10 cd 0.40 d 0.16 c-d mean 0.93 1.98 3.63 (z) in each column, means with the same letters are not significantly different at ≤ 0.05 level of probability using lsd. as desert ecosystems currently cover about 35% of the earth’s land surface and also global warming and water deficiency have become worldwide problems, it is urgent to focus on tolerant plant species in these areas (hellen, 1991). utilization of plants for medicinal purposes in iran has been documented in ancient literature. avicenna (abu ali sina) was a persian physician and philosopher whose medical system was for a long time the standard in europe and in the middle east (berman et al., 2009). to protect ecosystems from drought, an option is propagation of tolerant plants such as wild medicinal plants and a rapid and efficient technique for plant propagation is micropropagation (thorpe, 2007). explants used in the present study were collected in october and we believe their low regeneration potential was due to seasonal effects. tdz is one of the most active cytokinin-like substance used successfully for regeneration of recalcitrant woody plants (huetteman and preece, 1993; sarmast et al., 2012). the present study indicates that bap along with iaa did not have a high potential to induce bud regeneration, as previously reported (rathore et al., 1991; robinson et al., 2005). the data presented in this work demonstrated that, at increasing snps concentrations, the dimensions of mdc/e decreased and that buds derived from callus caused somaclonal variation (larkin and scowcroft, 1981; mondal and chand, 2002; sarmast et al., 2012). hence, we conjecture that snps may be helpful in micropropagation to avoid indirect regeneration and the connected somaclonal variability. sarmast et al. (2011) reported that araucaria excelsa explants, grown in ms medium supplemented with snps, were fresher than in ms medium only and that agno 3 in brassica spp. significantly affected bud formation on callus induction medium (akasaka-kennedy et al., 2005) and had positive effects on shoot regeneration in brassica oleracea var. italica (qin et al., 2007). ethylene, the simplest olefin, exists in gaseous state in environmental conditions; it is biologically active in trace amounts and regulates many aspects of the plant life cycle such as senescence (lin et al., 2009; taiz and zeiger, 2006). in tissue culture vessels, plantlet growth and development can be severely influenced by gaseous effects, especially of ethylene at elevated level that may damage explants by suppressing their growth and causing hyperhydration (ziv, 1995). additionally, snps affected ethylene formation and it can be concluded that increasing of durability of explants in culture vessel is due to ethylene blockage. in tissue culture of woody plants, rooting is a severe problem (giri et al., 2004; sarmast et al., 2012), therefore a separate experiment is now in progress to improve rooting of t. undulata with mediating agrobacterium rhizogenes (strain k559). references abdi g.h., salehi h., khosh-khui m., 2008 nano silver: a novel nanomaterial for removal of bacterial contaminants in valerian (valeriana officinalis l.) tisue culture. acta phisial. plant., 30: 709-714. fig. 2 proliferating single node explants of t. undulata in medium supplemented with tdz (0.1 mg l-1). 24 ahmad f., alam khan r., rasheed s., 1994 preliminary screening of methanolic extracts of celastrus paniculatus and tecomella undulata for analgesic and anti-inflammatory activities. j. ethnopharmacology, 42: 193-198. akasaka-kennedy y., toshida h., takahata y., 2005 efficient plant regeneration from leaves of rape seed (brassica napus l.): the influence of agno 3 and genotype. plant cell rep., 24: 649-654. aslam m., singh r., anandhan s., pande v., ahmed z., 2009 development of a transformation protocol for tecomella undulata (smith) seem. from cotyledonary node explants. sci. hort., 121: 119-121. berman p., bianquis t., bosworth c.e., van donzel e., henrichs w.p. brill., 2009 ibn sina (“avicenna”). in: berman p., t. bianquis, c.e. bosworth, e. van donzel, and henrichs w.p. brill (eds.) encyclopedia of islam. 2nd edition. accessed through brill online: www.encislam.brill.nl consolacao m., linardi f., oliveira m.m, sampaio m.r., 1975 a lapachol derivative active against mouse lymphocyte leukemia. j. medicin. chem., 18: 1159-1161. eapen s., george l., 1997 plant regeneration from peduncle segments of oil seed brassica species: influence of silver nitrate and silver thiosulfate. plant cell tiss. org. cult., 51: 229-232. giri c.c., shyamkmar b., anjaneylnu c., 2004 progresses in tissue culture, genetic transformation and application of biotechnology to trees: an overview. trees., 18: 115-135. guiraud p., steiman r., campos-takaki g.m., 1994 comparison of antibacterial and antifungal activities of lapachol and beta-lapachol. planta medica, 60: 373-374. hellen u., 1991 desertification-time for an assessment? ambio, 20: 372-383. huetteman c.a., preece j.e., 1993 thidiazuron: a potent cytokinin for woody plant tissue culture. plant cell tiss. org. cult., 33: 105-119. hussain h., krohn k., ahmad v.u., miana g.a., gree, i.r., 2007 lapachol: an overview. arkivoc., 2: 145-171. karami a., salehi h., 2000 adventitious root formation in rohida (tecomella undulata (am.) seem) cutting. prop. orn. plant., 10: 163-165. larkin p.j., scowcroft w.r., 1981 somaclonal variation a novel source of variability from cell culture for plant improvement. theor. appl. genet., 60: 197-214. lin z., zhong s., grierson d., 2009 recent advances in ethylene research. j. exp. bot., 60: 3311-3336. mondal t.k., chand p.k., 2002 detection of genetic variation among micropropagated tea camellia sinensis (l). by rapd analysis. in vitro cell. dev. biol. plant, 38: 296-299. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-497. qin y., li h.l., guo y.d., 2007 highfrequency embryogenesis, regeneration of broccoli (brassica oleracea var. italica) and analysis of genetic stability by rapd. sci. hort., 111: 203-208. rathore t.s., sigh r.p., shekhavat n.s., 1991 clonal propagation of desert teak (tecomella undulata) through tissue culture. plant sci., 79: 217-222. robinson r., bimlendra k., beniwal s.v., 2005 in vitro shoot multiplication of tecomella undulata (sm.) seem: an endangered tree species. indian j. plant physiol., 10: 372-376. sarmast m.k., salehi h., khosh-khui m., 2011 nano silver treatment is effective in reducing bacterial contaminations of araucaria excelsa r. br. var. glauca explants. acta biol. hung., 62(4): 477-484. sarmast m.k., salehi h., ramzani a., abolimoghadam a., niazi a., khosh-khui m., 2012 rapd fingerprint to appraise the genetic fidelity of in vitro propagated araucaria excelsa r. br. var. glauca plantlets. mol. biotechnol., 50(3): 181-188. sarmast m.k., salehi m., salehi h., 2009 the potential of different parts of sansevieria trifasciata l. leaf for meristemoids production. aust. j. basic appl. sci., 3: 2506-2509. taiz l., zeiger e., 2006 plant physiology. sinauer assoc. inc. 4 ed., pp. 700. tewari v.p., singh b., 2009 site index model for tecomella undulata (sm.) seem. (bignoniaceae) plantations in a hot arid region of india. j. arid environ., 73: 590-593. thorpe t.a., 2007 history of plant tissue culture. mol. biotechnol., 37: 169-180. zhang p., phansiri s., kaerlas j.p., 2001 improvement of cassava shoots organogenesis by the use of silver nitrate in vitro. plant cell tiss. org. cult., 67: 47-54. ziv m., 1995 in vitro acclimatization. automation and environmental control in plant tissue culture. aitkenchristie j., t. kozai, and m.a.l. smith (eds.). klumer academic publisher. netherlands, pp. 577. 181 1. introduction lipase (triacylglycerol acylhidrolases, ec 3.1.1.3) is an extra cellular enzyme which catalyses the hydrolysis of triglycerides to free fatty acids and glycerol (singh and mukhopadhyay, 2012). according to its various industrial applications, biotechnological uses for lipases are steadily increasing (stamatis et al., 1999). although lipases can be obtained from bacteria and yeasts (jaeger et al., 2000; kulkarni et al., 2002), the enzymes from fungi generally meet industrial demand since they are usually excreted extracellularly, facilitating extraction from fermentation media (hiol et al., 2000; abbas et al., 2002). fusarium is a large genus of filamentous fungi, and most of fusarium species are harmless saprobes and relatively abundant members of the soil microbial community (summerell et al., 2001). this ecological habitat of the fungus implies that fusarium would be a useful resource of extracellular enzymes. several different enzymatic activities were investigated in isolates of fusarium species, including lipase (burkert et al., 2004; bakri et al., 2013, 2014). solid-state fermentation (ssf) technique involves the growth and metabolism of microorganisms on moist solids without any free flowing water. ssf has many advantages over submerged fermentation, including an economical use of space that is required for fermentation, simplification of the fermentation media, superior yields, and no requirement for complex machinery (pandey, 1994). however, enzyme production is related to the type and concentrations of nutrients and growth conditions (prazeres et al., 2006). to reach commercial feasibility, enzyme production must be increased by introducing more potent strains, and by optimising culture conditions (singh and mukhopadhyay, 2012). since the effect of carbon sources on lipase production by the fungus f. culmorum has not been investigated so far, a study toward this aim was conducted on the new f. culmorum strain sy6 cultured under solid state fermentation (ssf). 2. materials and methods microorganism the organism used was f. culmorum sy6, isolated in our laboratory and having the ability to produce lipase enzyme (bakri et al., 2014). the strain was grown on petri dishes containing potato dextrose agar (pda, difco, detroit, mi. usa) with 13 mg/l kanamycin sulphate added after autoclaving and incubated at 23°c for 10 days in the dark to allow mycelial growth and sporulation. the cultures were maintained on silica gel at 4°c until needed. cultural conditions the strain f. culmorum sy6 was grown in 250-ml erlenmeyer flasks containing (g/l): na 2 hpo 4 · 2h 2 o 10; kcl 0.5; mgso 4 · 7h 2 o 0.15 and yeast extract 5, as nitrogen source. the mineral salt was added in such a way that the final substrate-to-moisture ratio was 1:5. the ph was adjusted to 6.5 before sterilization. the influences of different carbon sources (wheat bran, corn cobs hulles, beet pulp, tomato pulp, soya cake, cotton seed cake and wheat lipase production by fusarium culmorum in solid state fermentation y. bakri*, y. akeed, r. ouda, th. al-domani, m.i.e. arabi, m. jawhar department of molecular biology and biotechnology, aecs, po box 6091, damascus, syria. key words: fusarium culmorum, lipase activity, solid-state fermentation. abstract: fusarium is a large genus of filamentous fungi which cause some of the most important diseases in agricultural and horticultural crops. this fungus is considered to be a useful producer of enzymes from an industrial point of view. in the present study, lipase production by fusarium culmorum sy6 was investigated under solid-state fermentation (ssf). among the several agronomic wastes, corn cob hulls and tomato pulp supported the highest yield of lipase (170 and 165 u/g of dry substrate, respectively) after five days of incubation. it was determined that ph 9 and 60°c gave optimum enzyme activity. the f. culmorum sy6 strain grown in ssf in a simple medium proved to be a promising microorganism for lipase production. adv. hort. sci., 2015 29(4): 181-184 (*) corresponding author: ascientific@aec.org.sy received for publication 23 february 2015 accepted for publication 9 september 2015 182 adv. hort. sci., 2015 29(4): 181-184 straw) on lipase production were tested (fig. 1). the contents were sterilized by autoclaving at 121°c for 15 min. after cooling, the sterilized medium was inoculated with spores (106/ml) from a seven-day-old culture. the flasks were incubated at 30°c for five days. enzyme assay liplolytic activity was determined titirmetrically on the basis of olive oil hydrolysis (macedo et al., 1997) with some modifications. olive oil emulsion substrate was prepared by mixing 10 ml olive oil and 90 ml from 5% arabic gum solution. the reaction mixture contained 5 ml olive oil emulsion substrate and 1 ml of crude enzyme. the enzyme substrate mixture was incubated on an orbital shaker with a shaking speed of 150 rpm at 50°c for 20 min. to stop the reaction, 10 ml ethanol acetone mixture (1:1) was added to the reaction mixture. liberated fatty acids were titrated with 0.05 mol/l naoh. one lipase unit (u) was defined as the amount of the enzyme that released one µmol fatty acid per min. effect of ph the effect of ph on lipase activity was measured at different ph values ranging from 4 to 10. the ph of the reaction mixture was varied using various buffers (citrate buffer, phosphate buffer, and borate buffer). effect of temperature temperature effect on lipase activity was determined at different temperatures in the range 40-80°c. crude enzyme and substrate were tested by pre-incubating at various reaction temperatures to determine the optimal incubation temperature. statistical analysis all the experiments were repeated twice, and the means were analyzed statistically with the analysis of variance using the stat-itcf program; lsd= least significant differences at p<0.05. 3. results and discussion effect of carbon sources and lipase production the results showed that significant differences (p<0.05) in the mean lipase yield values existed among carbon sources, with values being consistently higher on corn cob hulls and tomato pulp (170 and 165 u/g, respectively) after five days of incubation, whereas, wheat straw exhibited lowest activity. these results might be attributed to the fact that the presence of more available carbon increases both mycelium growth and its activity. moreover, corn cob granulate shows excellent adsorption characteristics (damaso et al., 2008). however, findings these agree with those of abbas et al. (2002), but not with those of burkert et al. (2004). in addition, our results are in agreement with those of gombert et al. (1999) and falcony et al. (2006) in ssf, who reported that lipase enzyme produced by fungi, could be enhanced under ssf. although quantitative comparison of lipase activities reported in literature is not always possible because no standard enzyme substrate has been adopted yet, the lipase productivity from f. culmorum sy6 observed in this work was higher than optimum productivities reported in the literature for some microorganisms grown in ssf (table 1). effect of ph generally, enzymes are sensitive to the concentration of hydrogen ions present in the reaction mixture; therefore, ph is considered an important factor that determines the enzyme activity. the phrelative activity of f. culmorum sy6 lipase was determined in the range 4.0-10.0 ph (fig. 2). the optimum ph was found to be 9. most microbial lipases have their optimum activity at a ph range of 7.0-9.0 (zhang et al., 2005; ulker et al., 2011). an optimum ph of 7.0 for rhizopus oryzae lipase (hiol et al., 2000), ph of table 1 optimum lipase activities produced by filamentous fungi grown in ss microorganism substrate lipase (u/g) reference aspergillus niger j-1 wheat bran 9.14 falcony et al., 2006 penicillium restrictum babassu oil cake 30.3 gombert et al., 1999 fusarium culmorum cobs hulls 170 this work rhizopus oligosporous almond meal 48 ul-ha et al., 2002 rhizomucor pusillus olive oil cake and sugar cane 79.6 cordova, 1998 fig. 1 effect of some agro-industrial wastes as carbon source on lipase production by f. culmorum sy6. 183 bakri et al., lipase production by fusarium culmorum 8.5 for trichoderma harziamum (ulker et al., 2011), and ph 9.0 for penicillium caseicolum lipase (saxena et al., 2003) has been reported. effect of temperature optimization of temperature is vital for enzyme activity. in our work, the activity of f. culmorum sy6 lipase was investigated at different temperatures and the results obtained are shown in figure 3. the data reveal that the optimum temperature for lipase activity was at 60°c, followed by 70 and 80°c however, when the temperature increased or decreased from 60°c, the activity of lipase gradually reduced. prazeres et al. (2006) and (ulker et al., 2011) reported that fusarium oxysporum and trichoderma harzianum presented lipase with maximum activity at 55 and 40°c respectively. 4. conclusions the present study reveals that f. culmorum sy6 strain is a potential and promising microorganism as it produced a high level of lipase under solid state fermentation. adding corn cob hulls and tomato pulp significantly increased the enzyme production (170 and 165 u/g, respectively) after five days of incubation compared to 117 u/g reported by bakri et al. (2014) in which this compound was not used. moreover, the basic parameters such as ph and temperature were found to exert a marked influence on the activity of lipase. acknowledgements authors gratefully acknowledge the director general of aecs and the head of the biotechnology department for their help throughout the period of this research. thanks are also extended to dr. b. alsafadi for critical reading of the manuscript. references abbas h., hiol a., deyris v., comeau l., 2002 isolation and characterization of an extracellular lipase from mucor spp. strain isolated from palm fruit. enzyme micob. technol., 31: 968-975. bakri y., jawhar m., arabi m.i.e., 2013 xylanase production by fusarium solani in solid state fermentation. res. biotech., 4: 31-37. bakri y., jawhar m., arabi m.i.e., 2014 enzymatic activity of the endophytic fusarium species strains isolated from wheat. adv. hort. sci., 28: 129-132. burkert t.f., maugeri f., rodrigues m.i., 2004 optimization of extracellular lipase production by geotrichum sp. using factorial design. biore. technol., 910: 77-84. cordova j., 1998 isolement identification et physiologie des champignons thermophiles en vue de la production de lipases par fermentation en milieu solide montpellier. université de montpellier ii, france. damaso m.c.t., passianoto m.a., freitas s.c., freire d.m.g., lago r.c.a., couri s., 2008 utilization of agroindustrial residues for lipase production by solidstate fermentation. brazillian j. microbiol., 39: 676-681. falcony g., armas j.c., julio c., mendoza d., jose l., hermandez m., 2006 production of extracellular lipase from aspergillus niger by solid-state fermentation. food technol. biotechnol., 44: 235-240. jaeger k.e., dijkstra b.w., reetz m.t., 2000 bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications. annu. rev. microbiol., 53: 315-351. hiol a., jonzo m.d., druet d., comeau l.c., 2000 purification and characterization of an extracellular lipase from a thermophilic rhizopus oryzae strain isolated from palm fruit. enzyme and microbial technol., 26: 421-430. gombert a.k., pinto a.l., castilho l.r., freire d.m.g., 1999 lipase production by penicillium restrictum in solid-state fermentation using babassu oil cake as substrate. process biochem., 35: 85-90. fig. 2 effect of different ph values on the activity of f. culmorum sy6 lipase. fig. 3 effect of temperature on the activity of f. culmorum sy6 lipase. 184 adv. hort. sci., 2015 29(4): 181-184 kulkarni n., gadre r.v., 2002 production and properties of alkaline, thermophilic lipase from pseudomonas fluorescens ns2w. j. ind. microbiol. biotechnol., 28: 344-348. macedo g.a., park y.k., pastore g.m., 1997 partial purification and characterization of an extracellular lipase from a newly isolated strain of geotrichum sp. rev. microbiol., 28: 90-95. pandey a., 1994 solid state fermentation: an overview, in: solid state fermentation. ashok pandey, wiley eastern, new deli, india. prazeres j.n.d., cruz j.a.b., pastore g.m., 2006 characterization of alkaline lipase from fusarium oxysporum and the effect of different surfactants and detergents on the enzyme activity. braz. j. microb., 37: 505-509. saxena r.k, davidson w.s., sheoran a., giri, b., 2003 purification and characterization of an alkaline thermostable lipase from aspergillus carneus. process biochem., 39: 239-247. singh a.k., mukhopadhyay m., 2012 overview of fungal lipase: a review. appl. biochem. biotechnol., 166: 486-520. stamatis h., xenakis a., kolisis f., enantiomeric n., 1999 selectivity of a lipase from penicillium simplicissimum in the esterification of menthol in microemulsions. biotechnol. lett., 15: 471-476. summerell b.a., leslie j.f., backouse d., bryden w.l., burgess l.w., 2001 fusarium: paul e. nelson memorial symposium. 1st ed. american phytopathological society press, st. paul minnesota, usa. ul-ha i., idrees s., rajoka m.i., 2002 production of lipases by rhizopus oligosporous by solid-state fermentation. process biochem., 37: 637-641. ulker s., ozel a., colak a., karaoglu s.a., 2011 isolation, production, and characterization of an extracellular lipase from trichoderma harzianum isolated from soil. turkish j. biol., 35: 543-550. zhang h.y., wang x., ching c.b., wu g.c., 2005 experimental optimization of enzymic kinetic resolution of racemic flurbiprofen. biotechnol. appl. biochem., 42: 67-71. impaginato 121 1. introduction acquisition of mineral nutrients is important to plant growth and productivity. the ability of plants to acquire nutrients may be associated with root colonization with arbuscular mycorrhizal fungi (amf) (clark and zeto, 1996). amf are obligatory biotrophic symbionts occurring in nearly all natural and agricultural soils and commonly colonize roots of many plant species (smith and read, 1997). acquisition of mineral nutrients by plants with amf depends on factors such as soil ph, soil nutrient deficiencies, amf isolate, and plant species (sylvia and williams, 1992). previous studies showed a positive response of cotton to amf (liu et al., 1994; defeng et al., 1998; ibrahim, 2010). in subsistence agriculture systems, it is important to use indigenous amf that are ecotypically adapted to the site (davies et al., 2005). native amf can grow and function better in soils from which they are isolated, e.g. agricultural systems (calvente et al., 2004). phosphogypsum (pg) is the main by-product of the industrial production of phosphoric acid by treatment of rock phosphate with sulfuric acid. calcium sulfate is the dominant component in pg. pg contains the radioactive materials 226ra and 210po, phosphorus, silicon, fe, cu, and f– (al-masri et al., 2004). studies suggest that pg can be used in the improvement of soil structure, plant growth and agricultural production (alcordo and rechcigl, 1993), enhancing seedling emergence (vyshpolsky et al., 2010), and increasing available s and p (al-oudat et al., 1998). the use of pg as a fertilizer in agriculture has been practiced in many parts of the world (enamorado et al., 2009) without constituting environmental hazards to soil and crop tissue (al-oudat et al., 2011). application of pg (a poorly soluble source of p) to soil may become available to plants by solubilization from amf (al-karaki and al-omoush, 2002). solubilization of pg might insure a continuous supply of p without inhibiting root amf colonization (cui et al., 2014). in addition, enhanced acquisition of nutriadv. hort. sci., 2016 30(3): 121-128 doi: 10.13128/ahs-20247 arbuscular mycorrhizal isolate and phosphogypsum effects on growth and nutrients acquisition of cotton (gossypium hirsutum l.) m. ibrahim department of agriculture, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: arbuscular mycorrhizal fungi, gossypium hirsutum, indigenous, phosphogypsum. abstract: cotton was grown in pots with added phosphogypsum (pg) to evaluate the effect of indigenous arbuscular mycorrhizal fungi (amf) and phosphogypsum on cotton growth and acquisition of phosphorus (p), potassium (k), calcium (ca), manganese (mn), iron (fe), copper (cu), and zinc (zn). amf isolate was a mixture of glomus intraradices, glomus viscosum, and glomus mosseae previously isolated from a cotton field. shoot dry biomass was enhanced significantly by both indigenous amf and pg. shoot dry biomass and seed cotton yields were enhanced by the amf and pg combination and even more when pg in compost was added to mycorrhizal plants. p content in amf with pg and in amf with pg/compost treated plants was, respectively, 209.3 and 278.7%, significantly higher than control. acquisition of k, ca, and micronutrients was significantly enhanced by the combination of amf and pg. the treatment of amf with pg/compost induced the highest contents in mn, fe, cu and zn which were found to be, respectively, 287, 201, 192.8, and 171% higher compared to control. results indicate that cotton growth responded to indigenous amf in soils amended with pg. combination of amf with pg added in compost can ensure satisfactory benefits for cotton growth in low input, sustainable cropping systems. (*) corresponding author: ascientific@aec.org.sy received for publication 21 april 2016 accepted for publication 1 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 121-128 122 ents by amf plants in combination with pg has been reported (al-karaki and al-omoush, 2002; bai et al., 2011). compost increases soil organic carbon (el mrabet et al., 2014) and could increase the release of micronutrients in the soil, making them more available to plants (habashy et al., 2008; jan et al., 2014). on the other hand, addition of pg during manure composting decreased the amount of ammonia lost by volatilization (prochnow et al., 1995). therefore, addition of compost and the introduction of mycorrhizal technology may become an effective way of applying pg to soils with p deficiency. the objective of this research was to determine the effects of indigenous amf, in combination with syrian pg alone or integrated in compost, on growth and mineral nutrients acquisition of a syrian cotton variety. 2. materials and methods the experiment was conducted in pots during the summer season (may-september 2013) at deralhajar research station, located southeast of damascus, syria (33°21’ n, 36°28’ e) at 617 m above sea level. the area is located within an arid region in which the total annual precipitation is 120 mm. sandy clay loam soil was air dried, sieved to pass a 3 mm screen, and pasteurized at 5 kgy of gamma ray (gr) with 60co source using a gamma irradiator (robo, russa). phosphogypsum (pg) was previously collected from the area near the phosphoric acid factory in homs (180 km n of damascus). a total of 20 composite samples of pg were obtained from the levels corresponding to pile ages of approximately 1, 3-6 and 7-12 years, from the top, middle and bottom layers, respectively (each sample weighed 1 kg). pg samples were ground, homogenized, and sieving through a 0.5 mm sieve. before planting, soil was mixed with pg alone (at a rate of 30 g kg-1 dry soil) and pg integrated in compost (pg/compost mixture was prepared to be applied at a rate of 30 g of compost plus 30 g of pg per kg dry soil). compost was pasteurized at 120°c for 20 min in the autoclave. apart from pg, no chemical fertilizers were added during the experiment. mycorrhizal inoculum was a mixture of glomus intraradices (schenck & smith), glomus viscosum (nicolson), and glomus mosseae (nicol. & gerd.) gerd. & trappe. amf inoculum was previously isolated (ibrahim, 2010) from a cotton field at der-alhajar research station and multiplied in pot cultures using onion (allium cepa) as a host. the inoculum consisted of fragments of onion root and spores mixed with soil. half of the pots were inoculated with amf inoculum (100 g per pot) at the time of sowing. nonmycorrhizal pots were prepared by mixing the same amount of sterilized amf inoculums. the treatments were: t1, no amf inoculation without pg; t2, amf inoculation without pg; t3, no amf inoculation with pg; t4, amf inoculation with pg; t5, no amf inoculation with pg/compost; and t6, amf inoculation with pg/compost. seeds of cotton (gossypium hirsutum cv. aleppo 33/1) were sterilized in 20% naclo for 1 min and subsequently rinsed with sterilized water. they were then sown five per pot and placed to grow under natural conditions. ten days after emergence, seedlings were thinned to one per pot; the roots of discarded plants were left in the soil to avoid removing the amf inoculum. plants were watered with tap water as needed. growth parameters, such as plant height and fresh weight, were measured at the physiological maturity stage. the total shoot dry weights were measured after oven drying to constant weight at 70°c. boll weight and number of mature bolls per plant at the first handpicking were recorded. the seed cotton yield and percent lint of each plant was determined at one handpicking for all treatments. the vegetative portion of the plants was ground to a fine powder (0.5 mm). nitrogen was determined using the kjeldahl method and phosphorus was determined colorimetrically using a spectrophotometer (thermo spectronic, uk), while determination of ca, k, fe, zn, cu, mn was performed by x-ray fluorescence (xrf). root samples were rinsed free of soil, cut into 1 cm fragments, thoroughly mixed, cleared with koh and stained with acid fuchsin in lactoglycerol. percent root colonization and percent root length colonized by amf were determined microscopically using a gridline intercept method (giovannetti and mosse, 1980). the experimental design was randomized complete blocks with four replications. data were subjected to analysis of variance by the sas program (sas institut inc, 2004) and means were compared using the least significant difference (lsd) test at a probability level of p≤0.05. ibrahim am isolate and phosphogypsum effects on cotton 123 3. results addition of pg to the soil significantly decreased ph (table 1). amf root colonization was between 22.8 and 30.8% regardless of pg addition; the percent was higher for plants grown without added pg than added pg alone. adding pg in compost increased the percent of amf root colonization of cotton plants (table 1). the percentage of mycorrhizal root length was 66.3, 54.5 and 73.3% in the amf plants, amf plus pg, and amf with pg/compost treated plants, respectively (table 1). no amf root colonization was noted for plants grown without amf. significant differences between mycorrhizal and nonmycorrhizal plants were noted for shoot dry biomass regardless of pg addition (table 1). shoot dry biomass was significantly enhanced by indigenous amf and when the mixture of pg/compost was added to soil. application of pg to soil significantly increased shoot dry biomass for both mycorrhizal and nonmycorrhizal plants. shoot fresh weight significantly increased in amf inoculated and pg treated plants in comparison to the control (table 1). plant height at harvest was between 38.3 and 73.8 cm, and it was significantly enhanced by amf inoculation and pg addition compared to control. the maximum plant height was observed for mycorrhizal cotton plants grown with pg/compost mixture. the growth response of cotton plants to amf, pg, and amf plus pg treatments increased by 59.6, 49.4, and 76.3% over control, respectively (table 2). in addition, the growth response of cotton to the combination of amf with pg/compost was higher by 118.9% over control (table 2). yield components of cotton under different treatments are shown in table 2. the number of bolls per plant was significantly increased by both amf and pg in comparison with control. the plants showed the highest number of bolls when pg/compost mixture was added to amf plants (t6). the increase in boll number led to an increase in seed cotton yield, which was improved by both amf and pg. seed cotton yield varied between 50.83 g plant-1 (4574 kg ha-1 on the basis of a density of nine plants m-2) and 18.38 g plant-1 (1654 kg ha-1). the highest seed yield of cotton was observed in amf plants with added pg/compost. boll weight was generally higher in amf plants and pg treated plants than control and it was significantly higher with the amf plus pg/compost treatment compared to other treatments. percent lint varied between 47.3 and 34.3%, and amf inoculation increased it significantly. in addition, lint percentage was significantly increased by pg and this increase was clearly noted when pg was contained in compost. n and p concentrations in the vegetative portions of plants were significantly affected by amf inoculation (table 3, fig. 1). the concentrations of n and p table 1 experimental soil ph, mycorrhizal root colonization, and some growth parameters of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment amf colonization (%) mycorrhizal root length (%) soil ph plant height (cm) fresh biomass (g plant-1) dry biomass (g plant-1) control 0 0 8.1 a 38.3 e 141.72 c 44.40 e amf 24.60 b 66.30 b 8.1 a 55.0 d 256.30 ab 70.38 cd pg 0 0 7.5 b 58.0 cd 240.86 b 65.79 d amf+pg 22.80 bc 54.50 bc 7.5 b 62.0 bc 295.87 a 78.05 b pg/compost 0 0 7.2 c 64.3 b 255.43 ab 72.97 bc amf+pg/compost 30.80 a 73.30 a 7.1 c 73.8 a 316.52 a 96.64 a mean values within columns followed by different letters are significantly different at p<0.05. table 2 growth response and some yield components of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment growth response (%) boll number (per plant) boll weight (g) seed cotton yield (g plant-1) lint (%) control 0 5.3 c 4.9 c 18.4 d 34.3 f amf 59.6 bc 7.8 b 5.1 b 32.9 c 37.9 e pg 49.4 c 7.3 b 5.2 b 29.8 c 42.3 d amf+pg 76.3 b 8.3 b 5.3 b 36.9 b 42.9 c pg/compost 64.7 bc 8.0 b 5.5 b 36.5 b 46.4 b amf+pg/compost 118.9 a 9.5 a 6.1 a 50.8 a 47.3 a mean values within columns followed by different letters are significantly different at p<0.05. values are mean (n= 4). growth response (%)= (dwamf dwcontrol) x100/dwcontrol. lint (%)= (lint weight/seed cotton weight) x 100. seed cotton= seed + lint. table 3 concentrations of k, ca, and micronutrients in vegetative portion of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment k (mg g-1 dm) ca (mg g-1 dm) mn (µg g-1 dm) fe (µg g-1 dm) cu (µg g-1 dm) zn (µg g-1 dm) control 22.28 c 30.42 e 85.05 c 593.8 d 3.63 d 15.23 d amf 28.57 b 33.44 d 127.75 b 676.3 c 4.62 c 18.78 b pg 23.19 c 34.65 d 104.75 bc 688.3 c 3.78 d 16.28 cd amf+pg 29.44 b 40.28 b 154.75 a 797.5 b 4.69 c 20.58 a pg/compost 29.61 b 36.72 c 116.25 b 766.0 b 5.12 bc 16.68 c amf+pg/compost 32.67 a 43.61 a 172.75 a 930.8 a 5.59 a 21.88 a mean values within columns followed by different letters are significantly different at p<0.05. adv. hort. sci., 2016 30(3): 121-128 124 were generally higher for mycorrhizal than for nonmycorrhizal plants. pg significantly increased n concentration only when combined with compost for nonmycorrhizal (t5) and mycorrhizal plants (t6), while it significantly increased p concentration in nonmycorrhizal and mycorrhizal plants. the role of the combination of amf and pg in improving plant p content was noted (table 4). data indicate that maximum plant p contents of 107.8 and 131.9 mg plant-1 were found in amf plus pg and amf with pg/compost treatments, respectively, which was significantly higher (p≤0.05) by 209.3 and 278.7%, respectively, over control (table 5). the concentrations of k and ca were significantly higher in the vegetative portion of mycorrhizal compared to nonmycorrhizal plants regardless of pg (table 3). pg significantly increased ca concentration in nonmycorrhizal and mycorrhizal plants; added alone it had no significant effect on k concentration in either group. the concentrations of both elements increased significantly when pg/compost mixture was added to mycorrhizal plants. higher concentrations of mn, fe, cu, and zn were noted for mycorrhizal than for nonmycorrhizal plants (table 3) and pg significantly increased fe concentration. mn and zn concentrations were increased by pg only in amf plants while pg had no significant effect fig. 1 concentrations of n and p in vegetative portion of cotton plants grown at different treatments (t1= no amf without pg; t2= amf without pg; t3= no amf with pg; t4= amf with pg; t5= no amf with pg/compost; and t6= amf with pg/compost). table 4 contents of nutrients in cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment nutrient content (mg plant-1) n p k ca mn fe cu zn control 708.0 e 35.2 e 465.3 e 634.0 e 1.77 d 12.5 d 0.08 d 0.32 e amf 1165.7 c 86.5 c 884.8 c 1036.8 c 3.97 c 21.0 c 0.14 c 0.58 c pg 890.4 d 45.2 d 595.0 d 887.5 d 2.69 d 17.6 c 0.10 d 0.42 d amf+pg 1331.8 b 107.8 b 1015.0 b 1388.5 b 5.35 b 27.5 b 0.16 c 0.71 b pg/compost 1402.7 b 82.3 c 1013.7 b 1262.0 b 4.02 c 26.5 b 0.18 bc 0.57 c amf+pg/compost 1627.8 a 131.9 a 1276.5 a 1705.1 a 6.76 a 36.4 a 0.22 a 0.85 a mean values within columns followed by different letters are significantly different at p<0.05. table 5 percentage change in nutrient contents (nc) due to pg amendment and amf inoculation of cotton plants treatment nutrient content change (%) n p k ca mn fe cu zn amf 65.4 c 148.5 c 91.8 b 65.2 c 128.6 c 73.3 bc 90.6 b 85.0 c pg 25.9 d 30.0 d 28.2 c 40.6 c 56.2 d 44.4 cd 29.3 c 31.8 d amf+pg 89.1 bc 209.3 b 119.1 b 120.9 b 207.3 b 126.3 b 115.6 b 123.9 b pg/compost 97.7 b 135.6 c 118.5 b 100.0 b 129.2 c 115.3 b 87.4 b 80.4 c amf+ pg/compost 130.9 a 278.7 a 177.5 a 171.2 a 287.1 a 201.1 a 192.8 a 171.0 a data in the same column followed by the same letter are not significantly different (p<0.05). nutrient content (nc) change=(ncamf ncnonamf)x100/ncnonamf. ibrahim am isolate and phosphogypsum effects on cotton 125 on zn and mn concentration in nonmycorrhizal plants. also, pg had no significant effect on cu concentration in either amf or non-amf plants. the highest concentrations of mn, fe, cu, and zn were observed at amf plus pg/compost treated plants compared to other treatments. plant contents of k, ca, mn, fe, cu, and zn were significantly higher for mycorrhizal than for nonmycorrhizal plants (table 4). the data revealed that maximum plant uptake of k, ca, mn, fe, cu, and zn was found in the treatment of indigenous amf with pg/compost, which was significantly (p≤0.05) higher by 177.5, 171.2, 287.1, 201.1, 192.8, and 171%, respectively, over control (table 5). 4. discussion and conclusions the soil p concentration in this study was low (3 mg kg-1), and this nutrient normally has to be added to this soil to provide sufficient p for plant growth. addition of poorly soluble forms of p, such as phosphogypsum, to soil had no negative effect on amf root colonization, as was reported by cui et al. (2014). this may be because amf root colonization often depends on given amounts of soluble p in the soil at the time of root colonization (stribley et al., 1980). in particular, greater root infection was found with amf inoculation plus pg/compost treatment and it was possibly due to the improvement of the rooting zone environment which stimulated better root proliferation (nagahashi et al., 1996; van der heijden and kuyper, 2001). soil ph affects the availability of nutrients and how the nutrients react with each other. application of pg to soil lowered soil ph, a result which is in agreement with literature reports of previous studies (al-karaki and al-omoush, 2002; lee et al., 2009). the lower soil ph caused by pg might be attributed to the release of phosphoric acid and sulfuric acid contained in pg. the increase in cotton plant biomass by pg corroborates reports by zhang et al. (2014) who showed that amendment of pg significantly increased shoot biomass in tobacco, regardless of amf inoculation. according to quintero et al. (2014), increased dry matter of tomato by pg can be ascribed, at least in part, to an increase in water use efficiency. greater fresh biomass and plant height of inoculated cotton compared to control was noted in this study, which is in accordance with other earlier studies on cotton (afek et al., 1991; defeng et al., 1998). enhanced cotton growth with the combination of amf and pg agrees with previous studies conducted with different plant species such as wheat (al-karaki and al-omoush, 2002), maize (bai et al., 2011), tomato (cui et al., 2014), and shallot (gu et al., 2012). improved growth of amf and pg treated plants may have been due to improved soil p availability. the trend noted for n concentration and biomass of amf plus pg/compost treated plants might be due to the compost releasing its nitrogen gradually to the soil/crop to produce a greater number of leaves. strong mycorrhizal effects on cotton were also observed when looking at the nutrients uptake. higher k and ca in mycorrhizal than in nonmycorrhizal cotton is supported by liu et al. (2002) who reported that amf enhanced acquisition of the nutrients that move mainly by mass flow. pg/compost had a positive effect on ca and k concentration in mycorrhizal cotton which could be due to an improvement in soil organic matter and exchangeable ca and k by compost (adeleye et al., 2010). el mrabet et al. (2014) also showed that bio-compost improved soil k-extractable. increasing p uptake of plants due to amf inoculation has been widely reported (deguchi et al. 2007; sharif et al., 2009; ibrahim, 2010). in our study, increased p concentration and uptake by pg addition to mycorrhizal cotton is supported by zhang et al. (2015) who reported that pg amendment significantly increased the concentration and absorption of p in mycorrhizal and nonmycorrhizal tobacco plants. also, gu et al. (2012) found that p concentration in shallot was increased by increasing pg, and the combination of pg and amf colonization can improve p uptake by shallot to different degrees. our results show that indigenous amf increased the concentrations of cu, zn, fe, and mn in cotton; similar results were obtained in cotton inoculated with different species of amf (liu et al., 1994; ibrahim, 2010). our result regarding enhanced acquisition of p and micronutrients in amf cotton grown with pg is supported by al-karaki and alomoush (2002) in their work on mycorrhizal wheat grown with pg. the extension of amf hyphae, beyond the root zone, provides p and other nutrients to plants during growth stages. the ability of the hyphae to extend the root system should be especially beneficial in the case of cotton because its roots have a low density per unit soil volume (mcmichael, 1990). in this case, adv. hort. sci., 2016 30(3): 121-128 126 amf likely contributed p (and other mineral nutrients) from soil and pg particles with which roots would not make contact. high absorption of zn, cu, and fe may be due to greater p uptake by amf plants (clark and zeto, 1996; davies et al., 2005). on the other hand, the positive response of cotton to amf inoculation for nutrient concentration could be due to the effectiveness of the amf isolate in improving soil properties and nutrient availability. previous reports showed that improvement of plant growth and nutrients acquisition by the combination of pg and amf depends on compatibility between plant species, the rate of pg added, and the amf isolate. bai et al. (2011) reported that shoot growth of pg treated maize strain (40 g kg-1) was significantly enhanced when inoculated with diversispora spurcum, but was significantly inhibited when inoculated with glomus aggregatum. gu et al. (2012) reported that the treatment of pg40 addition with glomus mosseae inoculation had a significant effect in improving shallot biomass and p, s uptake. according to zhang et al. (2014), the combination of pg40 and g. aggregatum inoculation had the most desirable effects on tobacco growth. under the conditions in this study, added pg/compost enhanced p and nutrient concentrations of mycorrhizal plants. previous reports have shown that pg application induced changes in soil chemical properties (decreased soil ph and enhanced ece, available p, so4, exchangeable k, ca and mg) (aloudat et al., 1998; lee et al., 2009). in addition, organic fertilizers and amf inoculation could improve soil physico-chemical properties (warnock et al., 2007). amf colonization enhances soil aggregation by exuding the glycoprotein, glomalin, from extraradical hyphae (wright and upadhyaya, 1998). the improved soil structure enhances air and water percolation, improves root system access to soil water and nutrients, and improves crop production (celik et al., 2004). therefore, the increase in nutrients noted in amf and pg/compost treatments could be due to improved soil structure and to increased release of nutrients in the soil, which become more available to the plant (habashy et al., 2008; jan et al., 2014). enhanced acquisition of nutrients and plant growth by amf and pg was reflected by increased yield and yield components of cotton. previous studies showed that pg increased grain yield of barley, wheat, and cotton (al-oudat et al., 2011). cui et al. (2014) reported that tomato yield of amf or amf plus pg seedlings were significantly higher than those of the non-mycorrhizal seedlings. al-karaki and alomoush (2002) reported that grain yield of wheat was enhanced by pg, and even more so when roots were colonized with amf. cotton inoculation with indigenous arbuscular mycorrhizal fungi (amf) and soil amendment with phosphogypsum (pg) enhanced nutrients acquisition from soil and improved growth and yield of cotton. however, mycorrhizal plants grown with pg and compost mixture had greater growth and yield than plants grown with pg alone. therefore, the combination of amf with pg added in compost can ensure satisfactory benefits for cotton growth and yield in low input, sustainable cropping systems. acknowledgements the author would like to thank the atomic energy commission of syria for encouragement and technical support. references adeleye e.o., ayeni l.s., ojeniyi s.o., 2010 effect of poultry manure on soil physic-chemical 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warnock d.d., lehmann j., kuyper t.w., rillig m.c., 2007 mycorrhizal responses to biochar in soil concepts and mechanisms. plant soil, 300: 9-20. wright s.f., upadhyaya a., 1998 a survey of soil for aggregate stability and glomalin, aglycoprotein produced by hyphae of arbuscular mycorrhizal fungi. plant soil, 198: 97-107. zhang l., yue x., gu l., zhang c., shi j., zhang n., xia y., 2014 effects of arbuscular mycorrhizal fungi and phosphogypsum on growth and arsenic accumulation of tobacco (nicotiana tabacum l.). j. agro-environ. sci., 33(7): 1294-1303. zhang l., zhang c., liu y., gu l., zhang n., yue x., xia y., 2015 effect of arbuscular mycorrhizal fungi inoculation on tobacco growth and arsenic pollution risk from phosphogypsum addition. j. plant nutr. fertilizer, 21(2): 475-484. 26 adv. hort. sci., 2011 25(1): 26-31 *corresponding author efallik@volcani.agri.gov.il received for publication 10 february 2011. accepted for publication 18 february 2011. photoselective shade nets reduce postharvest decay development in pepper fruits a. goren, s. alkalia-tuvia, y. perzelan, z. aharon, e. fallik* department of postharvest science of fresh produce, aro, the volcani center, p.o. box 6, 50250 bet dagan, israel. key words: capsicum annuum, postharvest, shelf life, storage. abstract: during two-year studies, we evaluated the influence of photoselective coloured shade nets on the quality of fresh harvested pepper fruits (capsicum annuum) after prolonged storage and shelf life simulation. pepper cultivar ‘romans’ grown in a semi arid region under 35% pearl and yellow shade nets significantly maintained better pepper fruit quality after 16 days at 7°c plus three days at 20°c, mainly by reducing decay incidence during two consecutive years (2008 and 2009), compared to commercial black and red nets. no significant differences were observed in percentage of weight loss, firmness and total soluble solids in fruit harvested under the different coloured shade nets. the skin colour of fruit harvested under pearl net was significantly lighter than that of fruit harvested under red and black shade nets and this fact can be associated with inhibition of fruit ripening during growth. after storability and shelf life simulation however skin colour was red to dark red under all shade nets. pearl and yellow shade nets significantly reduced alternaria spp. population in the field, which was evaluated with alternaria-selective growing medium. the highest alternaria population was found under red shade net. the significant low decay incidence in fruit harvested under pearl and yellow shade nets can be explained by the low inoculum level of alternaria spp. in the field, and inhibition of fungal sporulation, and/or by a slowing of fruit ripening during its growth, reducing fruit susceptibility to fungal infection in the field due to the scattered light, its quality and the ratio between the light spectrum under the two shade nets. 1. introduction sweet bell pepper (capsicum annuum l.) is an important export commodity in israel with more than 120,000 tons per year. fruits may be green (unripe), red, yellow, orange, or brown when ripe. peppers are rich in vitamins, minerals and dietary fibres, and are low in calories (kevers et al., 2007) and they have become popular decorative items (frank et al., 2001). however, to extend the export season with high pepper quality during the summer season, shade netting is necessary to protect agricultural crops from excessive solar radiation and pests (shahak, 2008). in recent years coloured shade netting (photoselective shade nets), designed specifically to manipulate plant development and growth, has become available. these nets can be used outdoors as well as in greenhouses. they can provide physical protection (from birds, hail, insects, excessive radiation), affect environmental modification (humidity, shade, temperature) (perez et al., 2006), and increase the relative proportion of diffuse (scattered) light as well as absorb various spectral bands, thereby affecting light quality (shahak et al., 2004). these effects can influence crops as well as the organisms associated with them. the effect of colour shade nets on plant development in crops, foliage crops, fruit trees and vegetables has been studied in recent years (nissim-levi et al., 2008; shahak et al., 2008). bell peppers are grown commercially in semi-arid regions of israel under black shade nets. netting is frequently used to protect pepper plants from excessive solar radiation (shade-nets), environmental hazards (e.g. hail-nets), or pests (bird, or insect-proof nets) (shahak et al., 2004). shahak (2008) reported that productions of three cultivars of bell pepper were increased by 16 to 32% under pearl and red netting compared with equivalent black shade netting, or with open field production. in addition, elad et al. (2007) reported that blue-silver, green and red nets were associated with lower levels of powdery mildew disease, caused by leveillula taurica in pepper field experiments. other studies have suggested differential effects of photoselective screens and shade nets on pest infestation and vector-borne viral diseases (ben-yakir et al., 2008). in a preliminary research conducted in 2007, 27 fallik et al. (2009) demonstrated the potential use of coloured shade nets to maintain the postharvest quality better of sweet pepper grown under red and yellow nets, during prolonged storage and shelf life. therefore, we undertook the two-year study presented in this work to evaluate, on a commercial growth scale, the influence of different coloured shade nets on the quality of fresh harvested bell pepper after prolonged storage and shelf life in order to understand, in part, the mode-of-action of those shade nets on harvested fresh produce. 2. materials and methods plant materials and coloured net shades red sweet bell pepper (capsicum annuum l.) cv. ‘romans’ was grown at the b’sor experimental station in the south-west of israel (31.271° n; 34.389° e), using commercial cultivation practices (planting seedlings directly into the soil [~ 90% sandy regosol and ~ 10% arid brown soil], automatic drip fertigation), under four different coloured shade-nets, as follows: red, yellow, pearl and black (commercial shade net) with 35% relative shading (in par) (polysack plastics industries, nir-yitzhak, israel under the trade mark chromaticnet), in four random replicates of 18 x 18 m each, all within one large horizontal net-house, 2.5 m high. plants were planted during the third week of may, both in 2008 and 2009, and harvested as described below. light measurements under the nets measurements of light spectra, light scattering and microclimate parameters under the nets were taken as described previously by shahak et al. (2008). spectra of solar radiation outside and under the nets were measured by a spectroradiometer ps-100 (apogee instruments inc. logan ut, usa) which employs a light diffuser of 4 cm diameter above the 300 µm fiber optics. the diffuser was oriented along the sun beams. a round opaque plate of 6 cm diameter was held at 40 cm above the diffuser to block the direct light to measure the scattered light. direct radiation was measured through a 4 x 45 cm tube placed on top of the diffuser. the radiation was measured three times at noon in the middle of august 2009 (fig. 1). quality parameters fruit samples for storability and shelf-life were harvested five times each year, every three weeks, between the beginning of september and the end of november 2008 and 2009. fruits (185±10 g) without defects or diseases were harvested without a calyx, at ~85% light red/red colour (~16% green ‘cheek’), rinsed and brushed in hot water as described by fallik et al. (1999) and packed in four 6.5 kg corrugated cartons. fruit quality parameters were evaluated immediately after each harvest and at the end of 16 days storage at 7°c and relative humidity (rh) of ~ 94%, plus three days at 20°c (rh ~ 70-75%) as follows: weight loss was expressed as percentage of weight loss from the initial weight of ten fruits. fruit firmness was measured on ten fruit as described by ben-yehoshua et al. (1983). each fruit was placed horizontally between two flat surfaces and a 2 kg weight loaded on top of the flat surface. a dial fixed to a graduated plate recorded the deformation of the fruit in millimetres. full deformation was measured 16 s after exerting the force on the fruit, then the weight was removed and the residual deformation was measured 16 s later. fruit was considered very firm with 0-1.5 mm deformation; firm = 1.63 mm deformation; soft = 3.1-4.5 mm deformation; very soft = above 4.6 mm deformation. total soluble solids (tss) were measured on the same ten fruits tested for firmness, by squeezing out juice from fruits onto an atago digital refractometer (atago, tokyo, japan) and taking readings. the development of fruit colour was expressed as colour index (cinx) – on a scale of 1 to 4 with 1 = light red with 10-16% green peel; 2 = light red; 3 = red; and 4 = dark red. the index was calculated as follows: (number of fruits at light red colour x 1 + number of fruits at light red x 2 + number of fruit at red colour x 3 + number of fruits at dark red x 4)/total number of fruits. decay incidence was considered once fungal mycelia appeared on fruit pericarp and/or calyx. decay was expressed as a percentage of the total initial fruit number. evaluation of total microorganism population levels and alternaria spp. levels under coloured shade nets population levels of conidia in the greenhouse envifig. 1 influence of coloured shade nets on the scattered light under the different nets. measurements were taken at noon in august 2009. scattering relate to par (in µmol m-2 s-1). the values represent integrals of the spectra over the following wavelength ranges: par: 400-700 nm; uv: 300-380 nm; blue: 410-470 nm; red: 640-680 nm; far red: 690-750 nm (means of 3 replicates ± se). 28 ronment were evaluated according to elad (1997) by exposing 90 mm petri dishes, under each net, containing pda (potato dextrose agar, difco) supplemented with chloramphenicol (sigma chemicals, 50 µg/ml) at a height of 60 cm above the ground, in five different places (five plates) under each coloured shade net for 60 min. plates were then incubated for four days at ~22°c and colonies were counted. based on preliminary results obtained in 2007, the main decay-causing agent on pepper fruits after harvest was alternaria alternata. population levels of alternaria spp. were evaluated by exposing 90 mm petri dishes containing selective medium for alternaria (soon gyu and pryor, 2004), as described above. colonies were counted after 10 days incubation at ~22°c. the plates were exposed horizontally 60 cm above the ground. each evaluation was repeated three times from the beginning of october to the end of november. statistical analysis all the results were analyzed using a oneor twoway anova statistical analysis at p=0.05 using jmp 6 statistical analysis software program (sas institute inc. cary, nc., usa) (sall et al., 2001). 3. results light quality under different shade nets figure 1 shows the spectra of the scattered light under the different nets. the pearl net significantly reduced uv, while it significantly increased the blue light, thus significantly increasing the ratio between blue/uv compared to all other shade nets. the yellow net significantly increased the scattered red light compared to all other shade nets, while the blue light under this net was significant lower compared to red and pearl nets (fig. 1). the three shade nets significantly increased the far-red scattered light compared to the commercial black net. quality evaluation after 16 days storage at 7°c plus three days at 20°c, the major differences in fruit quality were found between 2008 and 2009 in weight loss and fruit firmness. however, tss was similar in the two years (table 1). in 2008, fruits lost less weight under the red net (2.9%), while under the yellow net weight loss was higher (3.5%). in 2009, percentage of weight loss under the four shade nets was similar (between 3.4 to 3.6%). immediately after harvest, fruits picked from the pearl net treatment were significantly lighter in their red colour index (2.33) than fruit picked from the commercial black or red shades (2.60 and 2.64, respectively) (table 2). no significant differences were observed in fruit colour index between the yellow and pearl treatments. after 16 days at 7°c plus three additional days at 20°c, all fruits turned almost dark red, however fruits picked under the red shade were significantly darker (3.86) than fruits picked under yellow and pearl shades (3.76 and 3.75, respectively) (table 2). decay incidence in both years, decay incidence under yellow and pearl shade nets were significantly lower compared to fruit picked under the commercial black or red net (fig. 2). in 2009, the average decay incidence was higher than in 2008. in 2008, the highest decay incidence was observed on fruit picked under black and red shade nets, which was significantly higher than on fruit picked under the yellow and pearl shade nets. in 2009, black red yellow pearl weight loss (%) z 3.2 abb w 2.9 bb 3.5 aa 3.3 ab firmness (mm) y tss (%) x 3.6 aa 3.4 aa 3.5 aa 3.6 aa 2.1 ab 2.3 ab 2.3 ab 2.0 ab 3.0 aa 3.0 aa 2.7 ba 2.9 aba 6.7 aa 6.4 aa 6.6 aa 6.5 aa 6.6 aa 6.4 aa 6.6 aa 6.4 aa colored shade net 2008 2009 2008 2009 2008 2009 table 1 the influence of coloured shade nets on weight loss, firmness and tss after 16 days at 7°c plus three days at 20°c (means of five experiments each year, with four 6.5 kg boxes per treatment) z percent weight loss from initial. y firmness millimeter deformation (flexibility). x percent total soluble solids. w values followed by the same upper-case letter do not significantly differ between the treatments, while values followed by the same lower-case letter, do not significantly differ between the harvest seasons at p=0.05 according to duncan’s multiple range test. colored shade net immediately after harvest (1-4) z after storage and shelf life (1-4) black red yellow pearl 2.60 a y 2.64 a 2.46 ab 2.33 b 3.80 ab 3.86 a 3.76 b 3.75 b table 2 the influence of coloured shade net on fruit colour index (cinx) development immediately after harvest and after 16 days at 7°c plus three days at 20°c (means of five experiments each year, with four 6.5 kg boxes per treatment) z colour index: 1= light red with 10-16% green peel; 2= light red; 3= red; and 4= dark red. y values within a column followed by the same letter do not significantly differ at p= 0.05 according to duncan’s multiple range test. 29 fig. 2 influence of coloured shade nets on decay incidence in fruit of cv. romans, in 2008 and 2009 after 16 days at 7°c plus three days at 20°c. (means of five experiments with four 6.5 kg boxes per treatment ± se). fig. 3 influence of coloured shade nets on the epiphytic population of fungi detected under the nets (cfu/plate – means of 3 experiments conducted in 2009 ± se). a significantly lower decay incidence was observed on fruit picked under the pearl shade net, whereas the highest one was observed on fruit picked under the red shade net (fig. 2). fig. 4 influence of coloured shade nets on the alternaria spp. population detected under the nets using selective medium (cfu/plate – means of 3 experiments conducted in 2009 ± se). evaluation of total microorganisms population levels and alternaria spp. levels under coloured shade nets the epiphytic population of fungi evaluated on a regular pda medium was lower under the pearl net, compared to the population that was counted under the red or black shade nets. however, no significant difference between treatments was found (fig. 3). using a selective medium for alternaria spp., a significant reduction in its population was observed under pearl and ellow shade nets (fig. 4). the highest alternaria population was found under the commercial black shade net (fig. 4). 4. discussion photoselective coloured shade nets (chromatinets™) have been developed over the last decade to filter selected regions of the spectrum of sunlight, concomitantly with inducing light scattering, and are designed specifically to modify the incident radiation (spectrum, scattering and thermal components) (shahak et al., 2004). depending on the pigmentation of the plastic and the knitting design, the nets provide varying mixtures of natural, unmodified light together with spectrally modified scattered light. they are aimed at optimizing desirable physiological responses, in addition to providing physical protection to the crop, thus improving plant growth, flowering and yield and fruit quality (rajapakse and shahak, 2007; shahak, 2008; shahak et al., 2008). the most prominent effect of the coloured shade nets was found on decay incidence in fruit harvested under pearl and yellow nets, without affecting fruit quality after prolonged storage and shelf life. it is well known that light plays an important role in plant growth. red light induced resistance in broad bean against botrytis cinerea (islam et al., 1998) and alternaria tenuissima (rahman et al., 2003), and in rice against magnaporthe grisea (arase et al., 2000). tabira et al. (1989) reported that continuous irradiation of visible light of 570-680 nm protected apple leaves by inducing insensitivity to alternaria alternata apple pathotype. disease suppression under red light was also observed in glasshouse-grown corynespore cassiicola-inoculated cucumbers, and indicated that delay and suppression of corynespora leaf spot of cucumber were due to induction of resistance in cucumber, and not to differences in environmental conditions or fungal populations between the two greenhouses (rahman et al., 2009). we therefore speculate 30 that under yellow net, significantly more scattered red light penetrates into the plant and fruit (fig. 1), which in turn inhibits fruit ripening as shown by fruit colour index (table 2) and/or, indirectly, induces resistance against alternaria alternata infection after harvest. preliminary results have revealed high amounts of chlorophyll in fruit harvested under pearl and yellow nets and relatively low amounts of carotenoids, which might indicate slow fruit ripening (fallik and goren, unpublished). in parallel, it was found that under the pearl and yellow shade nets the population of alternaria alternata, the main decay-causing agent after harvest, was significantly low compared to commercial black nets. light has profound effects on fungal biology. growth and development of many fungal species are intricately regulated by light (springer, 1993; purschwitz et al., 2006). fungal responsiveness to different wavelengths of light has been well documented, and blue light/uv and red/far-red light are two types of photoresponses primarily observed (mooney and yager, 1990; griffith et al., 1994; purschwitz et al., 2006, 2008; olmedo et al., 2010). blue light or a high ratio of blue to uv radiation was inhibitory to the sporulation of many important phytopathogens (raviv and antignus, 2004; paul et al., 2005). increases in the spectral ratios between transmitted light:blue/near-uv was found to inhibit the sporulation of several isolates of the fungal pathogen botrytis cinerea in tomato (kotzabasis et al., 2008). hence, the ability of the pearl and yellow shade nets to filter uv light and enrich the blue spectrum (shahak et al., 2008) and the blue/uv (fig. 1) may be involved in alternaria alternata inhibition in the field, thus reducing inoculum levels and fruit infection during its growth and thereafter decay development during prolonged storage and shelf life (barkai-golan, 2001). in conclusion, based on our results from two consecutive years of study which have shown a significant reduction in decay development on pepper fruit harvested under pearl and yellow shade nets, it seems that these two coloured shade nets influence both the pathogen in the field and the fruit ripening and its susceptibility to pathological deterioration after harvest. acknowledgements this research was supported by grant no. 430-0184-08/09 from the chief scientist of the ministry of agriculture and rural development, 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apple and japanese pear leaves to am-toxin from alternaria alternata apple pathotype. ann. phytopathol. soc. japan, 55: 567-578. impaginato 61 1. introduction oil pumpkins play a significant role in human nutrition and health. the nutritional value of oil pumpkin seeds is based on high protein and antioxidant content, and high energy potential due to the high percentage of oil (fruhwirth and hermetter, 2007; sari et al., 2008; fokou et al., 2009; lelley et al., 2009; urbanek krajnc et al., 2016). during the last decade, oil pumpkin cultivation declined regarding productivity and quality due to the outbreaks of the zucchini yellow mosaic virus (zymv), extremely high temperatures, radiation stress and prolonged periods of drought (lelley et al., 2009; seebold et al., 2009; gong et al., 2013). years 2013 and 2015 have been excessively hot and we have seen serious problems in fruiting pumpkins related to weather conditions especially high day/night temperatures and drought stress (yavuz et al., 2015). heat stress depends on intensity, duration, and rate of increase in temperature. the extent to which it occurs, in specific climatic zones, depends on duration and level of high temperatures occurring during the day and/or the night. in general, a transient elevation in temperature, 10-15°c above ambient, is considered as heat stress (wahid et al., 2007). it causes an array of morpho-anatomical, physiological and biochemical changes in plants, which affect plant growth and development and may lead to a drastic reduction in economic yield. on the morphological level, high temperature can cause considerable preand post-harvest damages such as scorching of leaves and stems, sunburns on leaves, stems and fruits, leaf senescence and wilting, shoot and root growth inhibition, fruit discoloration and damage, and reduced yield (wahid et al., 2007; ara et al., 2015; johnson, 2015). the physiological changes caused by high temperatures include the negative effect on photosynthesis, respiration, water relations, and modulated levels of hormones and primary and secondary metabolites. on the biochemical and subcellular level the direct injuries due to high temperatures include protein denaturation and aggregation, disorganization of cytoskeleton and increased fluidity of membrane lipids. indirect or slower heat injuries include inactivation of enzymes in chloroplast and mitochondria, inhibition of protein synthesis, protein degradation and loss of membrane integrity. these injuries eventually lead to starvation, inhibition of growth, reduced ion flux, production of toxic comadv. hort. sci., 2017 31(1): 61-73 doi: 10.13128/ahs-20727 the impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes a. urbanek krajnc *, j. rakun, p. berk, a. ivančič faculty of agriculture and life sciences, university of maribor, pivola 10, hoče, slovenia. key words: breeding, cucurbita argyrosperma var. argyrosperma, cucurbita moschata, cucurbita okeechobeensis, cucurbita pepo, fruit temperatures. abstract: fruit temperature is a key parameter for fruit growth and quality which is affected by climate, plant vigorousity, solar exposure and fruit thermal properties. in the present study, the variability in temperature dynamics of styrian oil pumpkin fruits and selected interspecific hybrids involving cucurbita argyrosperma, c. moschata, c. pepo was analysed in two different periods of hot weather. the temperatures were measured with thermistors on (a) attached fruits, (b) detached fruits exposed to the sun and (c) artificially black coloured fruits. the highest average temperatures were determined in the styrian oil pumpkin, whereas the lowest temperatures were determined in genotypes with lighter fruit exteriors suggesting that those are less sensitive to heat stress conditions and may represent a good option for the improvements of adaptability to climatic changes. in order to combine lighter and harder pericarp, the most promising genotypes were crossed with wild cucurbita okeechobeensis. the histological analysis showed that c. okeechobeensis was a good source of genes for obtaining a thicker sclerenchymatic layer within pericarp. (*) corresponding author: andreja.urbanek@um.si received for publication 8 september 2016 accepted for publication 4 march 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 61-73 62 pounds and reactive oxygen species (ros), which coincide with increased synthesis of antioxidants and activity of antioxidant enzymes. furthermore, enhanced expression of a variety of heat shock proteins and other stress-related proteins constitute major plant responses to heat stress (iba, 2002; howarth, 2005; wahid et al., 2007; ara et al., 2013 a, b, 2015). during the ontogenetic development of plants, flowering and fruit set are the most sensitive stages; fruit set of pumpkins is affected at day/night temperatures above 26/20°c and is severeliy affected above 35/26°c (schrader et al., 2004, 2011; saudreau et al., 2011; lei et al., 2014; johnson, 2015). the decrease in yield is due to the rapid decrease of photosynthesis, which reduces the amounts of sugars and other storage products that can go into fruits and developing seeds. on the other hand, hot night temperatures lead to greater cell respiration. high temperatures can also cause increased developmental disorders in fruiting vegetables due to the reduced pollen production leading to a reduction in fruit and seed sets, smaller pods, and split sets. another effect of heat stress in many plant species is induced sterility when heat is imposed immediately before or during anthesis (siddique et al., 1999; wahid et al., 2007). theoretical and experimental evidence shows that fruit temperature can be 10°c higher than the air temperature under sunny conditions (schrader et al., 2004; racskó et al., 2005; schrader, 2011; lei et al., 2014). sunburn of fruits is a surface injury caused by solar radiation which, during the initial phase, results in a light corky layer, golden or bronze discolouration. the damage occurs mainly in the surface and subsurface layers. there are two types of sunburn damage which may have effects on fruits and fruiting vegetables. the first, sunburn necrosis, appears due to the thermal death of cells on the sun exposed side of the fruit; cell membrane integrity is lost and cells start leaking their contents (schrader, 2011; johnson, 2015). the critical fruit tissues’ temperatures for sunburn necrosis vary with the type of fruit. the fruit surface temperature (fst) threshold for sunburn necrosis for cucumbers and pumpkins is between 37 and 42°c (rabinowitch et al., 1983, 1986; ara et al., 2013 a, b; 2015). the second type of sunburn injury is sunburn browning, which is caused by the combination of high fst and high solar radiation. it causes degradation of photosynthetic pigments resulting in yellow spots on the sun-exposed side of the fruit and occurs at a temperature about 5°c lower than sunburn necrosis (schrader, 2011; johnson, 2015). plants have three major ways in which they dissipate excess heat: (1) long-wave radiation, (2) heat convection into the air and (3) transpiration. if transpiration is interrupted by stomatal closure due to water stress, inadequate water uptake or other factors, a major cooling mechanism is not functioning. this will cause internal leaf/fruit temperatures to rise. without transpiration, the only way that plants can reduce heat is by heat radiation back into the air or wind cooling. under high temperatures, radiated heat builds up in the atmosphere around plants, limiting further heat dissipation (wahid et al., 2007; schrader, 2011; johnson, 2015). the adverse effects of high air and soil temperatures, and the high levels of solar radiation can be mitigated by developing plant genotypes with improved thermotolerance. some attempts to develop heat-tolerant genotypes via conventional breeding protocols have been successful (ehlers and hall, 1998; camejo et al., 2005). breeding of cultivated cucurbits was mainly focused in combinig good attributes of c. moschata and c. maxima. (balkaya and karaagac, 2005; balkaya et al., 2009, 2010 a, b, 2011; balkaya and kandemir, 2015). it is well known that c. moschata is best adapted to hot climate and is successfully cultivated in tropical and subtropical regions (balkaya et al., 2010 a, b; balkaya and kandemir, 2015). recently, a study was conducted to determine the extent of heat tolerance of newly developed interspecific squash hybrid named as ‘maxchata’ compared to its parents c. maxima and c. moschata (ara et al., 2013 a, b, 2015) under three different temperature regimes. results showed that various gas exchange and photosynthetic attributes dropped significantly with increasing temperature, while intercellular co2 concentration increased showing the nonstomatal limitations. these trends were more abrupt in c. maxima, reflecting that c. maxima was the most susceptible, while ‘maxchata’ showed intermediate response. c. moschata had the best photosynthetic attributes to sustain the heat regimes (ara et al., 2013 a, b). the ultramorphological, biochemical, and transcriptional analyses gave similar results. the electron microscopy highlighted the maximum degradation of the leaf ultrastructure of c. maxima. c. moschata and ‘maxchata’ exhibited lower degree of subcellular injury upon heat exposure. the antioxidant enzyme activities and their expression were found to be highest in c. moschata, moderate in ‘maxchata’, and lowest in c. maxima (ara et al., 2013 a, b; 2015). the authors concluded that the interspecific hybridization with c. moschata might significanturbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes 63 ly contribute to heat tolerance (ara et al., 2013 a, b, 2015). the presented study is associated with creating heat tolerant pumpkins characterised by lighter exocarp. the work began in 1996 and is based on a modified recurrent selection approach. its aim is to create cultivars characterised by bushy growth, resistance to all major pests and diseases, tolerance to drought and high temperatures, and large and thick seeds having thin seed coats and high concentrations of high quality oil. the basic population (i.e., population of the cycle0) was established by inter-crossing all available sources of genes (i.e., numerous local and commercial cultivars, populations and hybrids of c. pepo, following the semi-diallel scheme). in 1997, the most valuable progenies were planted in new caledonia, at the cirad centre near pouembout (south pacific). due to the favourable semi-tropical climate, it was possible to execute three cycles per year. the problems, however, were seed germination within fruits and rotting fruits due to overheating. as the genetic resources within c. pepo were found to be insufficient for overcoming these problems, it was decided to incorporate interspecific hybridisation and change the exterior fruit colour. the main sources of genes for lighter fruit exterior were c. argyrosperma var. argyrosperma and c. moschata. cucurbita argyrosperma, which was the main source of genes for whitish exocarp and was also used as a genetic bridge between c. pepo and c. moschata. another interesting trait obtained by interspecific crosses was dark yellow fruit exterior which was associated with the same colour of mesocarp. some years later, a wild species c. okeechobeensis (small) l. h. bailey was added to the hybridisation programme, in order to improve the resistance to viruses and harder pericarp. the interspecific hybrids included in this study were indirect progenies developed within the recurrent selection programme which involved intraand inter-population crosses, self-pollinations, and backcrosses. the progress in breeding for heat stress tolerance strongly depends upon understanding the genetic and physiological mechanisms associated with stress tolerance of the whole plants well as at the molecular and cellular levels. our study involved thin-coated seed pumpkins with lighter exocarp because they were considered to have the highest level of tolerance to high air temperatures and high levels of solar radiation during summer. in order to investigate their tolerance to heat stress, pericarp tissue temperature profiles were monitored on the exocarp surface, as well as in the mesoand endocarp of fully developed attached and detached fruits during two different periods of hot weather. 2. materials and methods plant material four different plant materials with thin coated seed were used in the study: (1) styrian oil pumpkin cucurbita pepo subsp. pepo var. styriaca (o), (2) progeny with whitish fruit derived from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma var. argyrosperma (o/a), (3) a progeny derived from crosses involving c. pepo (non-lignified seed type, oil type), c. argyrosperma and tropical c. moschata characterised by yellow fruits (a/mo x o/a) and (4) a three-species hybrid involving c. pepo (nonlignified seed coat, oil type), c. argyrosperma var. argyrosperma and c. okeechobeensis (oke × o/a) (table 1, fig. 1). the temperatures were measured on (a) attached fruits (1st period, 27th july to 5th august 2012), (b) detached fruits exposed to the sun (2nd period, 1st to 11th september 2012) and (c) artificially black coloured fruits (on both periods). the fruits were exposed to sunlight due to loss of foliage caused by drought stress and diseases. for each experiment, three fruits of each studied material were used. fruit temperature measurements the temperatures were measured during two different periods of hot weather, between the 27th july table 1 list of key plant materials with short explanations of abbreviations plant material abbreviations species cucurbita argyrosperma. var. argyrosperma a cucurbita moschata mo cucuribita pepo subsp. pepo var. styriaca o cucurbita okeechobeensis oke interspecific hybrids material obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma. var. argyrosperma, followed by several seasons of intrapopulation recombinations and selection o/a three-species hybrid involving c. argyrosperma (used as a genetic bridge), c. moschata and c. pepo (non-lignified seed coat, oil type), after several seasons of intrapopulation crosses and selection a/mo x o/a a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma var. argyrosperma and c. okeechobeensis oke x o/a adv. hort. sci., 2017 31(1): 61-73 64 to 5th august 2012 and 1st to 11th september 2012. during both periods, three pumpkins of each progeny (o, o/a, a/mo x o/a) were chosen and the thermistors were placed on exocarp the sun-exposed side, as well as inserted in meso(2 cm deep) and endocarp (10 cm deep) (fig. 1a, c, e). the temperatures were recorded every 15 seconds and stored as 10-min averages. the data represented in figures 3-6 show a diurnal and maximum day temperature as average of three fruits out of each progeny. in order to measure temperatures, 32 betatherm 10k3a542i thermistors connected to a campbell’s cr1000 datalogger (campbell scientific inc., logan, ut, usa) were used. the data logger recorded the current times for each iteration supply voltage and analogue input voltage. the thermistors’ readings were performed sequentially as they were all connected to the same adc converter through a multiplexor; so only one of the thermistors was connected to the converter at a time. the selected thermistors had accuracies of 0.2°c and were of negative-temperature-coefficient (ntk) type, which meant that their resistances decreased with the increases of temperature. as the data logger was unable to read the resistance of the thermistor directly, the thermistors were connected in a form of voltage divider, with an additional resistor with 1 kω±0.1% fixed resistance. as the resistive responses of the thermistors were nonlinear, the measured temperatures were calculated according to the temperature table from the datasheet (bethaterm). the results are represented by mean values (n=3), and were statistically evaluated by one-way analysis of variance (anova), using the spss 21 software (spss 21 software, spss inc., chicago, il, usa). significant differences between mean values were determined using the post-hoc duncan test. significant differences (α<0.05) between means were indicated by different letters. air/soil measurements furthermore, air/soil temperatures and relative humidity were measured on the sun-exposed side and within the canopy (5 and 20 cm below soil surface, on soil surface as well as 10 and 20 cm above soil surface using 215 and 107 temperature probe sensors (campbell scientific inc., logan, ut, usa). the temperatures were recorded every 15 seconds and stored as 10-min averages. histochemical evaluation of pericarp from each studied material, 3-6 fruits were taken for histological evaluation. out of each fruit six pieces (diameter 8 mm) of pericarp were sampled on equator of the fruit positioned around the pumpkin in regular intervals. the pieces were cut with a cryotom in order to evaluate the size of the lignified cell layer. an olympus microscope (provis ax 70) with a 100 w mercury arc lamp was used to take analogue images with a 3-chip-colour video camera (sony dxl 950 p, 3 ccd). fluorescence images were obtained through an uplanfi 40x dry objective (n.a., 0.75), a planapo 60x oil immersion objective (n.a., 1.40), and an uplanapo 100 x oil immersion objective (n.a., 1.35). lignified cells were visualised using an olympus filter set (umwu) with 330-385 excitation and 420 nm emission. fig. 1 fruits of selected genotypes with attached sensors, which were selected for lighter and harder fruit exterior and thin coated seeds: a, b) styrian oil pumpkin (o). c, d) three-species hybrid involving c. argyrosperma var. argyrosperma (used as a genetic bridge), c. moschata and c. pepo (non-lignified seed coat, oil type), after several seasons of intrapopulation crosses and selection (a/mo × o/a). e, f) genotype with lighter (i.e., whitish, white-green) fruits material obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma, followed by several seasons of intrapopulation recombinations and selection (o/a). g) c. okeechobeensis. h) a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma and c. okeechobeensis (oke × o/a). urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes 65 3. results and discussion our research was based on the hypothesis that interspecific hybridization aimed in creating lighter exocarp, thicker hypodermal sklerenchyma layer and cuticle would reduce the heat load on the fruit. air/soil measurements during the first period of measurements (jul.aug.), on the sun-exposed sides, the highest temperatures were registered on the ground surface reaching 45°c, whereas 20 cm above ground the maximum daily air temperatures were about 38°c. on the sides shaded by a canopy, the ground temperatures remained cooler and more stable, varying between 18 during the nights and 32°c on the hottest days. twenty cm above ground, the air temperatures reached 43°c. the underground temperatures (20 cm deep) remained stable with 20-22°c day/night variation (fig. 2 a). additionally, the ground temperatures were also measured within the proximities of the studied pumpkins. in the proximity of styrian oil pumpkins, from the 30th of july onwards, they increased from 27°c (daily maximum) to more than 47°c on the 1st august, and remained above 40°c for the next five days (fig. 3 a, b). the maximum day ground temperature in the proximity of white pumpkins were similar, reaching 45°c during the first three days of august (fig. 3 e, f). at the beginning of september, the minimum night temperature was 10°c. one day, on the 7th september, it was particularly hot and the air temperature 20 cm above ground, on the sun-exposed side, reached 45°c, whereas the temperature of the ground surface reached 58°c. a similar situation was observed within the canopy. at 20 cm height, the temperature reached 45°c (fig. 2 b). on attached styrian oil pumpkins (1st period), the maximum day temperatures measured on the exocarp varied between 38 and 50°c. in mesocarp the temperatures ranged between 32 and 51°c. endocarp was more or less 2°c cooler than mesocarp (fig. 3 a, b). on detached styrian oil pumpkins (2nd period) the maximum day temperatures of exocarp varied between 20 and 54°c, whereas mesocarp heated up above the temperature of exocarp to 61°c on the hottest day (7th september), on later days, the maximum day temperatures were around 45°c (fig. 4 a, b). in the ‘yellow fruit’ pumpkin material (a/mo × o/a), the temperatures of exocarp ranged between 42°c and 50°c on attached fruits during the first period and 17°c and 50°c on detached fruits during the second period of hot weather. the temperatures of endocarp varied between 33°c and 42°c during the first period, whereas during the second period the temperatures of mesocarp of detached pumpkins ranged between 16°c and 45°c (fig. 3 c, d, fig. 4 c, d). ‘white fruit’ pumpkin material (o/a) was characterised by the lowest exocarp temperatures, which ranged between 30°c and 41°c during the first period, whereas during the second period the temperatures ranged between 17°c and 44°c. the mesocarp temperatures varied between 35 and 44°c, the endocarp temperatures were more or less 2°c above the mesocarp temperatures. during the second period, where temperatures were measured on detached fruits, the average temperatures of mesocarp were fig. 2 relative humidity and the average air/soil temperatures measured within canopy and on sun exposed side during the period between 27 july-6 august 2012 (a) and between 1-11 september 2012 (b). adv. hort. sci., 2017 31(1): 61-73 66 generally 2°c lower when compared to exocarp but the fruit flesh heated up to 56°c on the extremely hot days of the 6th and 7th september, similarly as was observed in the styrian oil pumpkin (fig. 4 a, b, e, f, f ig. s1 see supplementary material) . transpiration appeared to be vital for maintaining optimal growth temperatures in growing plants. detached fruits, however, lacked the protective effects of transpiration, and direct sources of heat, such as sunlight, can rapidly elevate the internal fruit fig. 3 average and maximum day temperatures measured in exocarp, mesocarp, endocarp of attached pumpkins and ground in the period between 27. july6. august 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 67 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes temperatures to above that of exocarp and towards the thermal death points of their cells. in case of styrian oil pumpkin these lead to localised bleaching and necrosis (sunburn or sunscald). similarly, rabinowitch et al. (1983, 1986) reported that detached cucumbers (cucumis sativus l.) and peppers (capsicum annuum l.) had significantly higher surface temperatures and more serious sunburn injuries than attached fruit. comparing the measured results of the different fig. 4 average and maximum day temperatures measured in exocarp and mesocarp of detached pumpkins in the period between 1.11.september 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 68 adv. hort. sci., 2017 31(1): 61-73 varieties on attached fruits, we can conclude that styrian oil pumpkin was characterised by the highest temperatures within all three tissues (fig. s2), followed by yellow genotype, whereas white genotype had the lowest temperature, confirming the hypothesis that varieties with lighter fruit colour are less heated up. our results of the experiment on detached fruits, however, suggest that the most tolerant is a/mo × o/a which is a progeny derived from the interspecific cross involving the styrian oil pumpkin, c. argyrosperma and c. moschata. it is well known that c. moschata is best adapted to hot climate and is successfully cultivated in the tropical and subtropical regions (balkaya et al., 2010 a, b; balkaya and kandemir, 2015). heat tolerance of c. moschata can be very useful in breeding involving interspecific hybridisation. one of the successful examples, mentioned earlier in the text, is interspecific hybrid of squash named as ‘maxchata’. the authors concluded that the interspecific hybridization involving c. moschata might significantly contribute to heat tolerance (ara et al., 2013 a, b; 2015). in order to test further the resistance of selected genotypes to heat stress, the selected pumpkins were sprayed with black colour and exposed to the sun in order to induce bleaching by the excess heat. the exocarp of the black coloured styrian oil pumpkin fruit heated up to 52°c whereas the mesocarp temperature rose up to 43°c in the afternoon hours between 3 and 4 p.m. during both periods. when analysing the daily temperature curves of attached pumpkins during the july-august period, a depression in the curve was observed at around 12 a.m., reflecting a higher transpiration of fruits in response to heat stress; later, a rapid increase in temperature, may indicate stomata closure (fig. 5a). it is wellfig. 5 average and maximum day temperatures measured in exocarp and endocarp of black coloured attached pumpkins measured in the period between 27 july6 august 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). 69 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes known that if transpiration is interrupted by stomatal closure a major cooling mechanism is lost and the internal fruit temperatures raise (wahid et al., 2007; johnson et al., 2015). the temperatures of coloured fruits were measured for three days since a general collapse and tissue death of the inner pericarp layers was observed later on. the fruits were too much damaged by sunburn and measurements of temperatures no longer made sense. in the september period, when the experiment was performed on detached fruits, the exocarp of styrian oil pumpkin heated up to 51°c, similarly as on attached fruits. mesocarp temperature raised to a higher level on detached fruits (61°c) in comparison to attached fruits (43°c) (fig. 6a, fig. 6b, fig. s2). interestingly, during the 1st experiment on fig. 6 average and maximum day temperatures measured in exocarp and mesocarp of black coloured detached pumpkins measured in the period between 1.-11.september 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 70 adv. hort. sci., 2017 31(1): 61-73 attached pumpkins the exocarp of the black coloured ‘yellow’ pumpkin heated up to 64°c, whereas the endocarp up to 48°c during the july-august period (fig. 5c, d). compared to the styrian oil pumpkin the temperatures were higher. a rapid increase of temperature was determined in the late morning hours until noon. later, an abrupt decrease of temperature followed and maintained at around 38°c between 3 and 6 p.m. after that, during the night hours, the temperature dropped below 20°c (fig. 5c). similarly, in september, during the early afternoon hours of the hottest day (7th september), the maximum day temperatures on exocarp reached 64°c, whereas in mesocarp the temperatures reached 57°c. on later dates the daily maximum temperatures of exocarp constantly reached 42°c, whereas mesocarp was heated up to 57°c (fig. 6 c, d). the ‘white fruit’ genotype (o/a) was characterised by the highest mesocarp temperatures during the hottest days of the 2th period when compared to o and a/mo × o/a progenies. during the first five days, the maximum exocarp temperatures were around 40°c, whereas the mesocarp reached maximum day temperatures between 48°c and 55°c. during the following two days, the exocarp temperatures reached 60°c and 63°c, respectively, whereas the mesocarp 62°c and 71°c. later, the exocarp maximum temperature was comparable to that of the black coloured ‘yellow’ pumpkin, reaching 42°c in the early afternoon hours, whereas the mesocarp was heated up to approx. 55°c (fig. 6 e, f). summarising the results of temperature measurements within the black coloured pumpkins, we can conclude that for the attached pumpkins, the ‘yellow’ genotype was heated up more than the styrian oil pumpkin. by analysing the results of detached pumpkins during the first days of measurements, the highest temperatures of exoand mesocarp were determined in the styrian oil pumpkin, whereas the maximum day temperatures of both ‘yellow’ and ‘white’ genotypes did not differ significantly and were about 5°c lower than those of the styrian oil pumpkin. however, on the hottest days (6th and 7th september), the temperatures of exocarp within ‘yellow’ and ‘white’ genotypes were 8°c higher than those of the styrian oil pumpkin reaching 64°c. consequently, when compared to the styrian oil pumpkin, higher temperatures were also determined in fruit tissues of both studied interspecific hybrids. during the last two centuries, pumpkins have been selected for their softer pericarp in order to ease the laborious and time-consuming hand-harvesting of seeds. however, the resulting pumpkins’ genotypes became more susceptible not only to diseases but also to various types of abiotic stress such as heat stress and drought. in order to create a variety with harder pericarp and also better abscission of the fruit peduncle, those pumpkins with lighter exocarp were crossed with the wild species c. okeechobeensis (fig. 1 g, h), which is characterised by hard perikarp, good abscission of the pedincle and resistance to viruses. the selected hybrids were histologically evaluated and compared to the styrian oil pumpkin and o/a hybrids with lighter pericarps. the styrian oil pumpkin (o) was characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 23 cell layer of more or less isodiametric lignified cells between the chlorenchyma and parenchyma of the mesocarp (fig. 7 a, b). both the ‘yellow’ (a/mo × o/a) as well as the ‘white’ (o/a) genotypes were characterised by a similar 2-3 cell layer of thick lignified cells although the cell walls of this layer as well as the cuticle were thicker (fig. 7 c, f). crosses of o/a genotypes with c. okeechobeensis (oke × o/a) became characterised by a thicker scklerenchymatic layer of 4-5 oblong cells in a radial direction, which were approx. 100 µm long (fig. 7 g, h). this progeny represents a good material for the future breeding for increased heat tolerance. furthermore, c. okeechobeensis has been recognised as promising for interspecific hybridization due to its central position in the genus cucurbita (gong et al., 2013). we may conclude that the genetic breeding of oil pumpkins for heat tolerance is still in its infancy stage and warrants more attention than it has been given in the past. considerable information is presently available regarding the physiological and metabolic aspects of plant heat-stress tolerance (ara et al., 2013 a, b, 2015). furthermore, attempts have been made to include molecular marker technology for genetic characterization and/or development of plants with improved heat tolerance. gong et al. (2013) analysed ssr polymorphisms on a large collection of cucurbita materials in order to obtain an improved insight into the relationships amongst most of the species of the genus. wild species c. foetidissima has been identified as resistant to numerous pathogens and pests but is most distant to other cucurbita species. cucurbita pepo and c. ficifolia were the most outlying of the mesophytic species. the clusters of the six remaining species form three pairs, c. maxima with c. ecuadorensis, c. okeechobeensis with c. lundelliana, and c. moschata with c. argyrosperma. due to the genetic distances 71 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes amongst the cucurbita species, various breeding strategies and biotechnological approaches have been employed (merrick, 1995; lebeda et al., 2007; ortiz-alamillo et al., 2007; lelley et al., 2009; karaağaç and balkaya, 2013) but the success in introgressing desirable traits from one species to another have been limited. cucurbita pepo excels in plant earliness and productivity but lacks genetic resources for disease resistance. cucurbita moschata, on the other hand, carries resistance to various pathogens and is adapted to humid tropics but lacks earliness and productivity (lebeda et al., 2007; lelley et al., 2009; karaağaç and balkaya, 2013). relatively high successes and fertilities have been observed for the cross-combination of c. argyrosperma and c. moschata (montes-hernandez and eguiarte, 2002; ortiz-alamillo et al., 2007) and c. maxima × c. moschata (ara et al., 2013 a, 2015). however, despite all the complexity of heat tolerance and difficulties encountered during the genetic transfer of tolerance, few heat-tolerant inbred lines and hybrid cultivars with commercial acceptability have been developed and released (monteshernandez and eguiarte, 2002; ortiz-alamillo et al., 2007; ara et al., 2013 a, b, 2015). 4. conclusions the presented study suggests that the colour of exocarp is probably one of the key parameters of tolerance to high temperatures. darker colours are generally associated with higher fruit temperatures. in general, for attached pumpkin fruits higher temperatures were measured on exocarp, followed by mesoand endocarp. however, for the detached fruits on extremely hot days in september, the temperatures within mesocarp increased above those of exocarp. it is well known that if transpiration is interrupted by stomatal closure due to water stress and heat stress, a major cooling mechanism is not functioning. this was the reason of rising the internal fruit temperatures in case of detached fruits. one could expect that the genotypes with whitish exocarp (i.e., o/a progenies derived from the cross styrian oil pumpkin × c. argyrosperma) would be the most tolerant to heat stress. the experiment on detached fruits, however, suggest that the most tolerant is the a/mo x o/a progeny derived from the cross involving the styrian oil pumpkin, c. argyrosperma and c. moschata, which is characterized by yellow exocarp. one of the reasons for this could be the introgression of desirable traits of c. moschata, which is more adapted to high temperatures. this parental species was brought from the island of espiritu santo, vanuatu (tropical pacific) and was obviously more tolerant to heat stress than the other two involved pumpkin species. the second important parameter appears to be the histological structure of fig. 7 histochemical analyses of perikarp. a, b) styrian oil pumpkin (o) characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 1-2 cell layer of isodiametric cells and one layer of oblong cells with thick cell walls. c, d) genotype with yellow fruits (a/mo × o/a), with two layers of isodiametric cells with lignified cell walls. e, f) genotype with white fruits (o/a) and three layer of lignified cells below the chlorenchyma. g, h) a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma and c. okeechobeensis (oke × o/a) exhibiting 4-5 cell layers of oblong sklerenchymatic cells below the chlorenchyma. 72 adv. hort. sci., 2017 31(1): 61-73 the pericarp. the genetic improvement of this complex trait should consider both parameters. nevertheless, to accelerate such progresses, major areas of emphasis in the future should be: (1) development of accurate screening procedures at each stage of plant development; (2) identification and characterization of additional genetic resources associated with heat tolerance; (3) discerning the genetic inheritance of heat tolerance; (4) development and efficient screening of large breeding populations to facilitate transfer of genes for heat tolerance to commercial cultivars. acknowledgements this research was funded by the slovenian research agency (arrs, z1-9602, p-0164). the authors would like to express their gratitude to prof. dr. borut bohanec, prof. dr. metka sisko and anja ivanus for their genorous help and cooperation. references ara n., nakkanong k., yang j., hu z., zhang m., 2013 a antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species (“cucurbita maxima” and “cucurbita moschata”) and their interspecific inbred line “maxchata”. int. j. mol. sci., 14(12): 2400824028. ara n., nakkanong k., yang j., hu z., zhang m., 2015 dissecting the heat stress-induced alterations in the leaf ultrastructure and some antioxidant network components in interspecific (cucurbita maxima x cucurbita moschata) inbred line of squash ‘maxchata’ as to its parents possessing variable heat tolerance. plant growth regulation, 76(3): 289-301. ara n., yang j., hu z., zhang m., 2013 b determination of heat tolerance of interspecific (cucurbita maxima x cucurbita moschata) inbred line of squash ‘maxchata’ and its parents through photosynthetic response. j. agric. sci., 19: 188-197. balkaya a., kandemir d., 2015 an overview of winter squash (cucurbita maxima duch.) and pumpkin (cucurbita moschata duch.) growing in turkey. azarian journal of agriculture, 2(3): 57-64. balkaya a., karaagac o., 2005 vegetable genetic resources of turkey. j. veg. sci., 11: 81-102. balkaya a., kurtar e.s., yanmaz r., özbakir m., 2011 the promising winter squash (cucurbita maxima duch.) cultivars candidate developed by selection breeding from the black sea region, turkey (in turkish). the scientific and technical research (tubitak) project, 104: 144. balkaya a., özbakir m., karaagac o., 2010 a evaluation of variation and fruit characterization of pumpkin (cucurbita moschata duch.) populations collected from black sea region. j. agric. sci., 16(1): 17-25. balkaya a., özbakir m., kurtar e. s., 2010 b the phenotypic diversity and fruit characterization of winter squash (cucurbita maxima) populations from the black sea region of turkey. african j. biotechn., 9(2): 152162. balkaya a., yanmaz r., özbakir m., 2009 evaluation of variation in seed characters in turkish winter squash (cucurbita maxima duch.) populations. new zealand j. crop and hortic. sci., 37(3): 167-178. camejo d., rodriguez p, morales m. a., dell’amico j.m., torrecillas a., alarcon j. j., 2005 high temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. j. plant physiol., 162: 281-289. ehlers j.d., hall a.e., 1998 heat tolerance of contrasting cowpea lines in short and long days. field crops res., 55: 11-21. fokou e., achu m.b., kansci g., ponka r., fotso m., tchiegang c., tchouanguep f.m., 2009 chemical properties of some cucurbitaceae oils from cameroon. pak. j. nutr., 8: 1325-1334. fruhwirth g.o., hermetter a., 2007 seeds and oil of the styrian oil pumpkin: components and biological activities. eur. j. lipid. sci. tech., 109: 1128-1140. gong l., paris h.s., stift g., pachner m., vollmann j., lelley t., 2013 genetic relationships and evolution in cucurbita as viewed with simple sequence repeat polymorphisms: the centrality of c. okeechobeensis. genet. resour. crop. ev., 60(4): 1531-1546. howarth c.j., 2005 genetic improvements of tolerance to high temperature, pp. 277-300. in: ashraf m., and p.j.c. harris (eds.) abiotic stresses: plant resistance through breeding and molecular approaches. howarth press inc., new york, usa, pp. 725. iba k., 2002 acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. annu. rev. plant biol., 53: 225-245. johnson g., 2015 fruit set problems and pollination disorders in fruiting disorders in fruiting vegetables. weekly crop update, 25(3): 1-6. karaağaç o., balkaya a., 2013 interspecific hybridization and hybrid seed yield of winter squash (cucurbita maxima duch.) and pumpkin (cucurbita moschata duch.) lines for rootstock breeding. sci. hort., 149: 912. lebeda a., widrlechner m.p., staub j., ezura h., zalapa j., křístková e., 2007 cucurbits (cucurbitaceae, cucumis spp., cucurbita spp., citrullus spp.), pp. 271376. in: singh r.j. (ed.) genetic resources, chromosome engineering, and crop improvement: vegetable 73 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes crops. crc press, boca raton, fl, usa. lei l., troy r.p., zhang q., danfeng h., 2014 modeling apple surface temperature dynamics based on weather data. sensors, 14(11): 20217-20234. lelley t., loy b., murkovic m., 2009 hull-less oil seed pumpkin, pp. 469-492. in: vollmann j., and i. rajcan (eds.) oil crops. handbook of plant breeding. springer, berlin, germany, pp. 548. merrick l.c., 1995 squashes, pumpkins and gourds, cucurbita (cucurbitaceae), pp. 97-105. in: smartt j., and n.w. simmonds (eds.) evolution of crop plants. longman scientific & technical: london, united kingdom. montes-hernandez s., eguiarte l. e., 2002 genetic structure and indirect estimates of gene flow in three taxa of cucurbita (cucurbitaceae) in western mexico. am. j. bot., 89: 1156-1163. ortiz-alamillo o., garza-ortega s., sanchezestrada a., troncoso-rojas r., 2007 yield and quality of the interspecific cross cucurbita argyrosperma ‘c. moschata’. cucurbit. genet. coop. rep., 30: 56-59. rabinowitch h. d., ben-david b., friedmann m., 1986 light is essential for sunscald induction in cucumber and pepper fruits, whereas heat conditioning provides protection. sci. hort., 29: 21-29. rabinowitch h.d., friedmann m., ben-david b., 1983 sunscald damage in attached and detached pepper and cucumber fruits at various stages of maturity. sci. hort., 19: 9-18. racskó j., szabó z., nyéki j., 2005 importance of the supraoptimal radiance supply and sunburn effects on apple fruit quality. acta biologica szegediensis, 49: 111-114. sari n., tan a., yanmaz r., yetisir h., balkaya a., solmaz i., aykas l. 2008 general status of cucurbit genetic resources in turkey . pitrat m. (ed). proceedings of the ixth eucarpia meeting on genetics and breeding of cucurbitaceae. avignon, france, may 21-24th, pp. 21-32. saudreau m., marquier a., adam b., sinoquet h., 2011 modelling fruit temperature dynamics within apple tree crowns using virtual plants ann. botany, 108(6): 1111-1120. schrader l., sun j., zhang j., seo j. h., jedlow l., felicetti d., 2004 fruit skin disorders. proc. wash. tree fruit postharvest conf. schrader l.e., 2011 scientific basis of a unique formulation for reducing sunburn of fruits. hort. sci., 46: 6-11. seebold k., coolong t., jones t., strang j., bessin r., 2009 an ipm scouting guide for common problems of cucurbit crops in kentucky. in: kaiser c (ed.) an ipm scouting guide for common problems of cucurbit crops in kentucky . university of kentucky college of agriculture, lexington, usa. siddique k.h.m., loss s.p., regan k.l., jettner r.l., 1999 adaptation and seed yield of cool season grain legumes in mediterranean environments of south-western australia. aust. j. agric. res., 50: 375-387. urbanek krajnc a., janzekovic i., sober a., ivancic a., 2016 the impact of interspecific hybridization on the chemical compositions of oil pumpkin seeds. phyton, 56: 61-75. wahid a., gelani s., ashraf m, foolad m.r., 2007 heat tolerance in plants: an overview. environ. exper. bot., 61: 199-223. yavuz d., seymen m., yavuz n., turkmen o., 2015 effects of irrigation interval and quantity on the yield and quality of confectionary pumpkin grown under field conditions. agric. water manag., 159: 290-298. sm i adv. hort. sci., 2017 31(1): 61-73, sm i-ii doi: 10.13128/ahs-20727 the impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes a. urbanek krajnc *, j. rakun, p. berk, a. ivančič faculty of agriculture and life sciences, university of maribor, pivola 10, hoče, slovenia. supplementary material copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 61-73, sm i-ii sm ii fig. s1 comparison of the maximum day temperatures measured in the period between 27 july 6 august 2012 in: a) exocarp of styrian oil pumpkin, yellow and white genotype; b) endocarp of styrian oil pumpkin, yellow and white genotype; c) exocarp of black coloured styrian oil pumpkin and yellow genotype; d) endocarp of black coloured styrian oil pumpkin and yellow genotype. fig. s2 comparison of the maximum day temperatures measured in the period between 1-11 september 2012 in: a) exocarp of styrian oil pumpkin, dark yellow and white genotype; b) mesocarp of styrian oil pumpkin, yellow and white genotype; c) exocarp of black coloured styrian oil pumpkin, yellow and yellow genotype; d) mesocarp of black coloured styrian oil pumpkin, yellow and yellow genotype. 37 1. introduction common root rot (crr) caused by cochliobolus sativus (ito & kurib.) drechsler ex dastur (anamorph bipolaris sorokiniana (sacc.) shoemaker), is consistently one of the most damaging diseases of wheat and barley worldwide (gurung et al., 2013; fernandez et al., 2014). crr is considered economically important because it can cause marked reduction in yield and quality of the crop (kumar et al., 2002). this disease produces a brown to black discoloration of the subcrown internode (sci), therefore presence and severity can be determined by pulling up plants and examining sci for disease (kokko et al., 1995; mathre et al., 2003). efforts to minimize the impact of crr have been centered around the use of management strategies such as host resistance, crop rotation, tillage, and fungicide application (fernandez and conner, 2011; burlakoti et al., 2013). from a management perspective, the comparison of crr epidemics across years and locations is necessary to determine the effects of the environment on the efficacy of a given management approach, under similar environmental conditions, and to develop or recommend management strategies or decision thresholds (fernandez et al., 2014). the first step to quantify the effect of crr is to develop a key that clearly defines and standardizes the assessment methods to avoid subjectivity and variability between assessors. therefore, crr evaluation methods need to easily provide objective measurements so that data from different sources are comparable, and provide an adequate sample of the crop for assessment (mathre et al., 2003). reaction of wheat to crr is commonly measured either by incidence (i, proportion of sci units diseased) or severity (s, proportion of sci showing crr symptoms). however, incidence is a binary measurement (madden and hughes, 1999), meaning it is a measure of only one of two possible states, diseased or not diseased. moreover, in spite of the drawback, however, severity is often considered a more important and useful measure of disease intensity than incidence to evaluate yield loss and to deterimine the effectiveness of disease management strategies (fernandez et al., 2009). since measurements of incidence are more easily acquired and more reliable than measurements of severity, and severity is more useful than incidence for certain objectives, a quantitative relationship between incidence and severity would greatly facilitate the evaluation of disease intensity when accurate assessments of severity are not available or possible (seem, 1984; fernandez et al., 2014). therefore, in this study, the i-s relationship of crr was investigated to explore the possibility of simplifying disease assessment. 2. materials and methods disease assessment sites in order to acquire data from crr epidemics of different intensities and to represent a range of environmental, cropping, and management conditions likely to influence a simple approach to assess common root rot severity incidence data in wheat m.i.e. arabi (*), e. al-shehadah, m. jawhar department of molecular biology and biotechnology, aecs, p.o. box 6091, damascus, syria. key words: cochliobolus sativus, common root rot, incidence, severity, wheat. abstract: common root rot (crr) of wheat, caused by cochliobolus sativus, produces discoloration of the subcrown internodes (scis) and is directly related to yield losses. it is critical to clearly define and standardize the crr assessment methods to avoid subjectivity and variability between assessors. therefore, in this study, a comparison between the incidence (i; proportion of diseased scis) and the severity (s; proportion of sci showing crr symptoms) was investigated to explore the possibility of simplifying disease rating. assessments were made visually at multiple sample sites in artificiallyand naturally-inoculated research and production fields for three growing seasons. significant differences (p = 0.001) in mean i and s values were found among cultivars, with values being consistently higher in the susceptible ones. however, crr severity increased linearly as incidence increased in both triticum durum and t. aestivum wheat. their slopes and intercepts of the i–s relationship were consistent over the three growing seasons. this result may be considered a significant contribution for crr assessment in wheat breeding programs. (*) corresponding author: ascientific@aec.org.sy received for publication 17 september 2014 accepted for publication 23 march 2015 adv. hort. sci., 2015 29(1): 37-40 38 adv. hort. sci., 2015 29(1): 37-40 the development of crr, three different locations with several research plots and production fields were selected for crr assessment in three growing seasons (table 1). inoculum preparation the c. sativus isolate (pt4) has been proved to be one of the most virulent isolates to all barley and wheat genotypes available so far (arabi and jawhar., 2002). in the present study, the fungal mycelia were transferred from a stock culture into petri dishes containing potato dextrose agar (pda, difco, detroit, mi, usa) with 13 mg/i kanamycin sulphate and incubated for 10 days at 21±1°c in the dark. host genotypes the ten wheat cultivars (six triticum durum and four t. aestivum) used in this study were chosen for their wide genetic variability for c. sativus reaction from highly susceptible to highly resistant (table 2). the local susceptible landrace salamoni was included in each set as check. experimental design seeds were artificially inoculated with pt4 isolate following the procedure set out by arabi and jawhar (2013). the experimental design was a randomized complete block design with three replicates. the seeding depth was 6 cm (kokko et al., 1995). plot area was 1 x 1 m with a 1 m buffer. each plot consisted of five rows 25 cm apart with 50 seeds sown per row. experimental design, cultural practices, and inoculation methods were performed as described by arabi and jawhar (2002). weeds were controlled by preand post emergence herbicides as appropriate. soil fertilizers were drilled before sowing at a rate of 50 kg/ha urea (46% n) and 27 kg/ha superphosphate (33% p). disease assessment in each field/plot, i and s were estimated visually at several systematically selected sampling sites, 20-25 subsampling from each row were taken at random from each replication. incidence (i) was recorded as the proportion of diseased scis (number of scis with nonzero severity divided by the total number of plants sampled). severity (s) was recorded as infected scis expressed as a proportion of the total area. statistical analysis data for i and s were analyzed by analysis of variance (newman-keuls test), using the stat-itcf program (itcf, 1988). the assumption of coincidence for each year was tested using the anova procedure implemented in the software package statistica 6.1. years were set as the categorical variable and coincidence was tested by simultaneously checking the year’s effect combined with its interaction with the incidence. for all experimental data, each pair of i and s values from each sampling site was considered an observation for data analysis. the experimental data were edited to remove observations with no diseased plants (i.e., i = 0 and s = 0), since the i-s relationship is only defined when disease is present. 3. results significant differences (p = 0.001) in mean i and s values were detected, with values being consistently higher in the susceptible cultivars for the three growing seasons (table 2). the data show that the highest mean i and s were recorded in the t. aestivum landrace salamoni (i and s =100), whereas the lowest was found in the t. durum landrace horani (i and s ≈ 7). in general, the triticum durum genotypes were more resistant than t. aestivum (table 2), in agreement with data presented by bhandari and shrestha (2004). additionally, the data demonstrate that s increased linearly as i increased (fig. 1). there was no difference in the slopes and intercepts of the i-s relationship among the three years, as was shown by the coincidence test (f 3, 32 = 0.309, p = 0.585). in some cases i = s for one or more observations such as in the susceptible landrace salamoni (table 2). this can be explained by the fact that when all plants in the sample are diseased, there is no longer any information on the magnitude of (mean) s in relation to i, other than being larger than the (mean) s when some plants are disease-free. in this extreme situation, i was equal to s for wheat crr reaction. these findings are in agreement with the results of paul et al. (2005) for fusarium head blight on winter wheat. the overall response to crr for the three growing seasons considered in this study differed with the differences table 1 range of magnitude of environmental conditions encountered during three growing seasons (2011, 2012 and 2013) location no. fields temperature (°c) (z) relative humidity (%)(z) average rainfall (mm) (y) altitude (m) directions draa (south) 4 33-39 40-51 256 716.5 36°06’23.86’’ e 33°06’55.71’’ n allepo (north) 4 25-36 41-79 360 297.4 33°55’56.99’’ e 36°01’31.14’’ n hassaka (north east) 5 35-48 35-42 228 313.9 40°40’02.31’’ e 36°31’53.73’’ n (z) average during april, may and june. (y) average from november to april. 39 arabi et al. simple approach to assess common root rot in wheat in susceptibility levels of the cultivars. however, cultivars that are resistant to crr may in fact have different resistance response to the spread of the fungus within the infected plants. hence, for any given i value, a wide range of s values may be observed across cultivars. mcroberts et al. (2003) reported that incidence severity analysis was directly useful in evaluating resistance response. in particular, the i–s relationship could be used to draw conclusions about the relative rate of disease increase among cultivars with different levels of resistance. 4. discussion and conclusions our results show that neither differences in weather conditions for the three growing seasons, nor geographical locations resulted in any different patterns in the i-s relationship. although the locations were up to 50 km apart, it appeared that within a climatologically similar region, i-s relationships did not show distinct differences among sites. moreover, in this study, the number of plants sampled and the small distance among locations did not affect the i-s relationship either. we undertook this study to determine an i-s relationship for crr and then to establish whether that relationship would remain the same for different years, locations and cultivars. the results reveal a positive correlation between crr parameters i and s in wheat which was consistent among seasons and locations. however, characterizing the functional relationship between i and s is still critically important, because through this relationship researchers can identify the cultivars with unusually large or small s for a given i (mcroberts et al., 2003), or through covariance analysis (when there are several pairs of i-s points for each cultivar), identify cultivars with an unusual i-s relationship compared with others. moreover, the estable 2 mean common root rot disease incidence (i) and severity (s) of the most frequently grown wheat cultivars in syria under field conditions for 3 years, combining data for three locations cultivar s i s i s i triticum aestivum sham 2 15.20 e 20.00 e 22.50 de 20.50 e 18.30f 17.60 e bouhouth 4 33.16 d 40.00 d 25.40 d 33.00 d 22.50 e 30.00 d bouhouth 6 42.60 c 50.50 cd 48.20 c 47.60 c 40.30 c 43.00 c salamoni (landrace) 95.50 a 100.00 a 90.07 a 100.00 a 89.30 a 91.00 a doma 4 60.16 bc 73.00 c 52.90 c 50.60 c 40.50 c 39.00 c mexipak 66.50 b 80.00 b 70.30 b 78.30 b 63.20 b 86.20 b t. durum doma 1 31.13 d 33.00 de 27.00 d 30.00 d 35.70 d 39.50 c sham 3 10.30 e 18.00 e 9.10 e 15.10 f 12.20 g 18.30 e horani 7.50e 10.60f 7.80e 8.50fg 5.50h 9.67f bouhouth 7 30.06 d 38.00 d 22.33 de 25.60 de 31.70 d 33.50 d lsd 8.83 6.11 7.42 5.3 4.09 4.01 values followed by different letters columns are significantly different at p= 0.001 according to newman-keuls test. lsd: least significant difference at p<0.05. fig. 1 relationship between incidence (i; proportion of diseased scis) and severity (s; proportion of sci showing crr symptoms) of wheat common root rot for three growing seasons. 40 adv. hort. sci., 2015 29(1): 37-40 timation of mean i from s would substantially reduce the work load in crr quantification in field surveys and treatment comparisons. acknowledgements the authors thank the director general of aecs and the head of the molecular biology and biotechnology department for their continuous support throughout this work. we would like also to thank dr. m. tlas for his assistance to achieve the parallel test analysis. references arabi m.i.e., jawhar m., 2002 virulence spectrum to barley (hordeum vulgare l.) in some isolates of cochliobolus sativus from syria. j. of plant pathol., 84: 35-39. arabi m.i.e., jawhar m., 2013 a simple method for assessing severity of common root rot on barley. the plant pathology journal, 29: 451-453. bhandari d., shrestha s.m., 2004 intensity of common root rot on wheat genotypes. nepal agriculture research journal, 5: 46-48. burlakoti r.r., shrestha s.m., sharma r.c., 2013 impact of seed-borne inoculum irrigation, and cropping pattern on propagation of bipolaris sorokiniana and epidemiology of foliar blight and common root rot in spring wheat. j. of plant pathol., 95: 571-578. fernadez m.r., conner r.l., 2011 root and crown rot of wheat. prairie soils crops journal, 4: 151-157. fernadez m.r., fox s.l., hucl p., singh a.k., stevenson f.c., 2014 root rot severity and fungal populations in spring common, durum and spelt wheat, and kamut grown under organic management in western canada. canadian journal of plant science, 94: 937-946. fernadez m.r., holzgang g., turkington t.k., 2009 common root rot and crown rot of barley crops across saskatchewan and in north-central alberta. canadian journal of plant pathology, 31: 96-102. gurung s., mahto b.n., gyawali s., adhikari t.b., 2013 phenotypic and molecular diversity of cochliobolus sativus populations from wheat. plant disease, 97: 62-73. itcf, 1988 stat-itcf, programme, microsta, realized by ecosoft, 2nd ver. institut technique des cereals et des fourrages paris, france. kokko e.g., conner r.l., kozub g.c., lee b., 1995 effects of common root rot on discoloration and growth of spring wheat root system. phytopathology, 85: 203-208. kumar j., schafer p., huckelhoven r., langen g., baltuschat h., stein e., nagarajan s., kogel h.k., 2002 bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. molecular plant pathology, 3: 185-195. madden l.v., hughes g., 1999 an effective sample size for predicting plant disease incidence in a spatial hierarchy. phytopathology, 89: 770-781. mathre d.e., johanston r.h., grey, w.e., 2003 diagnosis of common root rot of wheat and barley. plant health progress, doi:10.1094/php-2003-0819-01-dg. mcroberts n., hughes g., madden l.v., 2003 the theoretical basis and practical application of relationships between different disease intensity measures in plants. annals of applied biology, 142: 191-211. paul p.a., el-allaf s.m., lipps p.e., maddem l.v., 2005 relationships between incidence and severity of fusarium head blight on winter wheat in ohio. phytopathology, 95: 1049-1060. seem r.c., 1984 disease incidence and severity relationships. annual review of phytopathology, 22: 133-150. 165 1. introduction dracaenas rank second in europe (vonk noordegraaf, 1998) and third in the united states (u.s. department of agriculture, 1999) as popular foliage plants used for interiorscaping. dracaenas are also rich in steroidal sapogenins and saponins (mimaki et al., 1998, 1999; yokoduk et al., 2000), some of which have cytotoxic activities against cultural tumor cells (mimaki et al., 1999), making them an important group of plants for pharmacognosy research. dracaenas as ornamental plants are propagated through stem cuttings, which are predominantly imported from central america, but imported cuttings may carry and spread pathogens and pests (palm and rossman, 2003; prado et al., 2008). for example, an invasive pathotype of ralstonia solanacearum race 1 was identified from eye cuttings of golden pothos imported from costa rica to florida (norman and yuen, 1998). childers and rodrigues (2005) reported some pest mite species on ornamental plants imported to the us from central america. mass propagation through seeds has many limitations like seed dormancy, low rate of germination, and progeny variation in other plant species (venkataramaiah et al., 1980; chand and singh, 2004). to overcome these problems and fulfill the required demand has necessitated restoring the productivity of plants through the use of plant tissue culture techniques (bhattacharjee, 2006) such as in vitro micropropagation which includes the rapid vegetative multiplication of valuable plant material for agriculture and forestry. in addition, the in vitro technique is also widely used in the commercial field for the micropropagation of ornamental plants in large numbers; however, the process is regulated by the biochemical reserve localized in specific organs (thorpe, 1990; bhattacharjee, 2006). the objective of the present investigation was to study different aspects of micropropagation of d. sanderiana (lucky bomboo) and factors affecting its multiplication and rooting, and subsequent acclimatization to propose a commercial protocol for propagation of this plant. 2. materials and methods the experiment was carried out at the tissue culture and biotechnology lab, department of horticultural science, college of agriculture, shiraz university. effects of ms salt strength and number of subcultures green cultivar stem segments, bearing a node, were cultured horizontally on full ms, 3/4 ms and 1/2 ms media supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. the case of subculture times was also assessed. leaf and shoot numbers and the average length of shoots (cm) were recorded. factors affecting in vitro propagation of dracaena sanderiana sander ex mast. cultivars. ii. ms salt strengths, subculturing times, rooting and acclimatization f. kakuei, h. salehi(*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: acclimatization, dracaena, ms salts strength, rooting, subculture. abstract: the interaction effect of ms salt strengths and subculturing times were studied in dracaena sanderiana sander ex mast. (lucky bomboo). stem pieces, each bearing a single node, were cultured horizontally on full, 3/4 and 1/2 strength ms media supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. proliferation rate, shoot length, and number of leaves increased when 1/2 strength ms medium was used. the highest shoot length was recorded after the third subculture. moreover, the greatest number of leaves was obtained at this stage. the highest mean value for parameters such as rooting percentage, the number of roots, and root length were observed on media supplemented with 2 mg l-1 indole-3-butyric acid (iba). plantlets were subsequently planted in a mixture of perlite and vermiculite (1:1) under 95% relative humidity and were then transferred to greenhouse conditions. the proposed protocol can be used in commercial mass production of d. sanderiana. adv. hort. sci., 2015 29(4): 165-170 (*) corresponding author: hsalehi@shirazu.ac.ir received for publication 10 april 2015 accepted for publication 22 june 2015 166 adv. hort. sci., 2015 29(4): 165-170 rooting the experiment was conducted to study the effects of different concentrations of supplementary iba (0, 1, 2, 3 mg l-1) in ms medium on the rooting of both cultivars of d. sanderiana. all treatments contained 30 g l-1 sucrose and 8.0 g l-1 agar in the media used. the ph was adjusted to 5.8 before the addition of agar, then the culture media were poured into 40 ml culture jars and autoclaved at 121ºc at a pressure of 1.5 kg cm-2 for 20 min. shoots were incubated at 25±2oc under 16/8 (light/dark) photoperiod. the light intensity was 30 µm m -2 s-1 provided by cool white fluorescent lamps. transfer and adaptation plantlets were removed from the culture media; roots were washed with distilled water. they were then cultured in a sterilized mixture of 1:1 perlite and vermiculite (v v-1) inside 5 cm pots covered with plastic bags and maintained at a relative humidity of about 95% to later initiate compatibility with field conditions. plastic bags were gradually removed to allow steady acclimatization with lower humidity levels. after two months, they were transferred to the greenhouse where more compatibility was expected to be observed. for better growth, plants were fed with crystalon fertilizer (0.5 mg l-1, once every 15 days). 3. results and discussion effects of ms salt strength, number of subcultures, and number of shoots from the data obtained for the number of shoots (table 1), the greatest mean value (2.00) was recorded when using 1/2 compared to 3/4 and full strength ms media, which resulted in 1.6 and 1.26 shoots, respectively. proliferation rates decreased in subsequent subcultures: 1.8 and 1.2 shoots after the second and third subcultures, respectively. furthermore, the comparison of subcultures derived from full strength ms medium revealed that the first and second subcultures yielded 1.4 shoots, whereas the third subculture yielded one shoot, respectively. probably, the amount of internal tissue hormones have altered with time. shoot length the data presented in table 1 show the effect of ms salt strength, i.e. full, 3/4 and 1/2 strength, on shoot length of cultured d. sanderiana. the longest shoot (3.33 cm) was measured in explants cultured on 1/2 ms; full ms medium gave 2.6 cm and 3/4 ms medium gave 2.5 cm shoot length. with regard to the effect of the interaction between concentrations of ms salts and the number of subcultures on shoot length, it was found that the highest value for shoot length (4.10 cm) was obtained by using 1/2 ms medium after the third subculture, as compared with that obtained from using full ms medium after the first subculture, which gave the lowest value (2.20 cm shoot length). the results reveal that as the salt concentration of ms medium decreases the proliferation and shoot growth rates increase. moreover, as the number of subcultures increased, more positive values for shoot length were obtained which may be attributed to the explants’ freshness and adequate nutrient supply. the presence of vitamins and growth regulators were partly responsible for growth enhancement. number of leaves the data obtained for the number of leaves (table 1) shows that the greatest mean value (4.86) was recorded when using 1/2 ms; 3/4 and full strength ms media produced 3.93 and 4.06 leaves, respectively. the interaction between ms salt strength and the number of subcultures significantly affected the number of leaves produced; i.e. the highest mean value (6.20) was obtained using 1/2 ms after three subcultures, as compared to 3/4 ms medium, regardless of the subculture stages, which gave 3.80 leaves after the first, and 4.00 leaves after the second and third subcultures. these results revealed that the ms salts at full strength were not effective in enhancing the initiation and formation of leaf primordia as compared to the lower concentrations. however, a positive impact was observed when subculturing was done again and again; in other words, the third subculture yielded more new leaves (fig. 1). rooting the data presented in table 2 and figure 2 clearly show the effects of iba concentration on the number of roots table 1 the interaction effect of ms salt strength and subculture number on shoot length, number of leaves, and number of shoots during multiplication stage of dracaena sanderiana sander ex mast. ms strength number of shoots shoot length (cm) number of leaves sub. 1 sub. 2 sub. 3 mean sub. 1 sub. 2 sub. 3 mean sub. 1 sub. 2 sub. 3 mean full ms 1.40 b (z) 1.40 b 1.00b 1.26 b 2.20 c 2.50 bc 3.10 b 2.60 b 3.60 c 4.00 bc 4.60 b 4.06 b ¾ ms 1.80 b 1.50 b 1.50b 1.60 ab 2.00 c 2.50 bc 3.00 b 2.50 b 3.80 bc 4.00 bc 4.00 bc 3.93 b ½ ms 3.00 a 1.80 b 1.20b 2.00 b 2.70 bc 3.20 b 4.10 a 3.33 a 4.00 bc 4.40 bc 6.20 a 4.86 a mean 2.06 a 1.56 b 1.23 b 2.30 b 2.63 b 3.40 a 3.80 b 4.13 b 4.93 a (z) data followed with the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. 167 kakuei and salehi, factors affecting in vitro propagation of dracaena sanderiana. ii and root length of cultured d. sanderiana. it was found that using iba at different concentrations significantly affected percentage of rooting. the highest percentage of rooting was observed at a concentration of 2 mg l-1 iba (81.25%). the highest mean value for root length (3.36 cm) was achieved when using 2 mg l-1 iba as compared with that of untreated explants (1.00 cm).the highest mean value for the number of roots (5.00) was obtained with 2 mg l-1 iba as compared with the control explants which gave the lowest mean number of roots (1.75). these results are in agreement with those obtained by paek et al. (1985) on cordyline. they concluded that using 2.0 or 3.0 mg l-1 iba resulted in more success in the rooting stage. debergh (1975, 1976) succeeded in achieving a 100% rooting percentage in d. deremensis engl. and d. fragrans when using 2 mg l-1 iba. badawy et al. (2005) obtained the greatest root numbers and length in d. fragrans ‘massangeana’ with 1/2 ms medium supplemented with 0.5 mg l-1 iba. transfer and adaptation of ‘green’ plants a month after deployment in the rooting medium, the elongated and well-rooted plantlets were transferred to an equal ratio of sterilized perlite and vermiculite (v v-1). the greatest average length of shoots after transferring to the pots and adaptability to environmental conditions was observed in treatments of 3 mg l-1 ba and 0.25 mg l-1 naa and 2 mg l-1 ba and 0.5 mg l-1 naa, i.e. 6.75 and 5.80 cm, respectively. concentrations of 1 mg l-1 ba and 0.25 mg l-1 naa produced the highest number of leaves (8.80) compared to other treatments. average stem diameter was greatest (5.17 mm) at the concentration of 2 mg l-1 ba and 0.5 mg l-1 naa. chlorophyll content in the treatments with 2 mg l-1 ba and table 2 effect of different concentrations of iba on rooting of dracaena sanderiana iba (mg l-1) rooting percentage average root length (cm) number of roots control 15.00 d (z) 1.00 c 1.75 c 1 31.25 c 1.58 bc 2.25 c 2 81.25 a 3.36 a 5.00 a 3 57.50 b 2.85 ab 3.25 b (z) in each column, means followed by the same letter(s) are not significantly different using lsd test at 5% level. fig. 1 proliferation rate of d. sanderiana ‘green’ explants on 1/2 ms medium at different subcultures. fig. 2 shoots rooted on ½ ms medium containing 2 mg l-1 iba, 30 days after culture. a. first subculture b. second subculture c. third subculture 168 adv. hort. sci., 2015 29(4): 165-170 0.25 and 0.5 mg l-1 naa was 43.94 and 52.16 mg g-1 fw, respectively, which was higher than other treatments (table 3, fig. 3). transfer and adaptation of ‘variegated’ plants in transferred plantlets, the greatest shoot length (3.83 cm) was observed in the treatment with 2 mg l-1 ba and 0.25 mg l-1 naa, which was not significantly different from many other treatments. the highest average number of leaves was obtained in the treatment with 1 mg l-1 ba and 0.5 mg l-1 naa. the stem diameter in transferred plantlets after treatment with 4 mg l-1 ba and 0.25 mg l-1 naa was higher (3.56) than the other treatments. chlorophyll content was the highest with 3 mg l-1 ba and 0.25 and 0.5 mg l-1 naa, i.e. 33 and 29.5 mg g-1 fw, respectively (table 4, fig. 4). most plantlets were adapted, had normal growth, and 70% of them survived. the obtained results agree with the findings of beura et al. (2006) on d. sanderiana, debergh and maene (1981) on d. deremensis engl, and ying et al. (2008) on d. cambodiana pierre ex gagnep. junaid et al. (2013) reported the micropropagation of only one cultivar of d. sanderiana. however, in the present study, high plant regeneration was achieved and this is the first report on mass production of two cultivars of d. sanderiana. the proposed protocol can be used in commercial mass propagation of this plant. 4. conclusions the highest mean value for parameters such as rooting percentage, the number of roots, and root length were observed on media supplemented with 2 mg l-1. plantlets were subsequently planted in a mixture of perlite and vermiculite (1:1) under 95% relative humidity and were then transtable 3 effects of different treatments on some characteristics of acclimatized ‘green’ plantlets, 60 days after transfer ba (mg l-1) naa (mg l-1) mean length of shoots (cm) mean number of leaves mean stem diameter (mm) mean chlorophyll content (mg g-1 fw) 0 0.0 3.40 b (z) 4.50 c 2.70 c 26.40 bc 0.25 3.60 b 8.80 a 2.72 c 33.20 b 1 0.50 3.40 b 5.20 c 2.77 c 26.68 bc 0.25 4.00 b 5.20 c 3.85 b 43.94 a 2 0.50 5.80 a 7.20 b 5.17 a 52.16 a 0.25 6.70 a 5.00 c 3.15 bc 31.82 bc 3 0.50 3.50 b 4.50 c 2.69 c 26.76 bc 0.25 3.90 b 4.40 c 2.76 c 26.58 bc 4 0.50 3.80 b 4.40 c 3.02 bc 22.42 c 0.25 3.80 b 5.40 c 2.79 c 24.24 bc 5 0.50 3.60 b 4.40 c 3.03 bc 29.30 bc (z) in each column, means followed by the same letter(s) are not significantly different using lsd test at 5% level. fig. 3 ‘green’ plantlets transferred to the pots. 169 kakuei and salehi, factors affecting in vitro propagation of dracaena sanderiana. ii ferred to greenhouse conditions. the proposed protocol can be used in commercial mass production of d. sanderiana. references badawy e.m., habib a.m.a., bana a.e., yosry g.m., 2005 propagation of dracaena fragrans plants by tissue culture technique. arab j. biotech., 8: 329-342. beura s., samal p., jagadev p.n., 2006 preliminary studies of in vitro cloning of dracaena (dracaena sanderiana). acta hort. sin., 80: 123-153. bhattacharjee s.k., 2006 advances in ornamental horticulture. pointer publishers, jaipur, india, pp. 2065. chand s., singh a.j., 2004 in vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, pterocarpus marsupium roxb. in vitro cell. dev. biol. plant, 40: 167-170. childers c.c., rodrigues j.c.v., 2005 potential pest mite species collected on ornamental plants from central america at port of entry to the united states. fla. entomol, 88: 408-414. debergh p., 1975 intensified vegetative multiplication of dracaena deremensis. acta hort. sin., 54: 83-92. debergh p., 1976 an in vitro technique for the vegetative multiplication of chimeral plants of dracaena and cordyline. acta horticulturae, 64: 17-19. debergh p., maene l., 1981 “a scheme for commercial propagation of ornamental plants by tissue culture”. scientia hort., 14: 335-345. table 4 effects of different treatments on some characteristics of acclimatized ‘variegated’ plantlets, 60 days after transfer ba (mg l-1) naa (mg l-1) mean length of shoots (cm) mean number of leaves mean stem diameter (mm) mean chlorophyll content (mg g-1 fw) 0 0.0 3.20 a-d (z) 3.35 b 2.50 c 25.53 abc 0.25 2.66 cd 3.66 ab 2.276 c 28.56 a 1 0.50 3.50 abc 5.50 a 2.106 c 27.23 ab 0.25 2.33 d 3.45 b 2.32 c 20.80 bc 2 0.50 3.00 a-d 3.30 b 2.85 abc 27.76 ab 0.25 3.83 a 3.41 b 2.67 bc 33.00 a 3 0.50 3.66 ab 3.66 ab 2.43 ab 29.50 a 0.25 3.00 a-d 3.40 b 3.56 a 26.43 abc 4 0.50 3.16a-d 3.33 b 2.54 c 27.96 ab 0.25 2.83 bcd 3.33 b 2.76 abc 27.63 ab 5 0.50 2.66 cd 3.66 ab 2.72 bc 19.36 c (z) in each column, means followed by the same letter(s) are not significantly different using lsd test at 5% level. fig. 4 acclimated ‘variegated’ plantlets. 170 adv. hort. sci., 2015 29(4): 165-170 junaid a., abdul m., maheshwar p.s., 2013 in vitro micropropagation of dracaena sanderiana sander ex mast: an important indoor ornamental plant. saud. j. biol. sci., 20: 63-68. mimaki y., kuroda m., ide a., kameyama a., yokosuka a., sashida y., 1999 steroidal saponin from the aerial parts of dracaena draco and their cytostatic activity on hl 60 cells. phytochemistry, 50(5): 805-813. mimaki y., kuroda m., takaashi y., sashida y., 1998 steroidal saponins from the stems of dracaena concinna. phytochemistry, 47(7): 1351-1356. norman d.j., yuen j.m.f., 1998 a distinct pathotype of ralstonia (pseudomonas) solanacearum race 1, biovar 1 entering florida in pothos (epipremnum aureum) cuttings. can. j. plant pathol., 20: 171-175. paek k.y., oh m.o., choi j.k., 1985 mass propagation of cordyline and scindapsus in vitro. korean j. hortic. sci., 26: 83-92. palm m.e., rossman a.y., 2003 invasion pathways of terrestrial plant-inhabiting fungi, p. 31-43. in: ruiz g.m., and carlton j.t. (eds). invasive species: vectors and management strategies. island press, washington, dc. prado j., casanoves f., hildago e., benjamin t., sadof c., 2008 effects of production practices on the abundance of quarantine pests in dracaena marginata in costa rican production fields. j. econ. entomol., 101: 1779-1785. thrope t.a., 1990 the current status of plant tissue culture, pp. 1-22. in: bhojwani s.s. (ed.) plant tissue culture: applications and limitation. elsevier sc publishing co., ny, usa, pp. 470. venkataramaiah v., prasad s.v., rajeswara r.g., swamy p.m., 1980 levels of phenolic acids in pterocarpus santalinus l. indian j. exp. biol.,18: 887-889. vonk noordegraaf c., 1998 trends and requirements in floriculture in europe. acta horticulturae, 454: 39-48. ying x., bao-ling h., cheng-qun l., yin-ping z., chuanming z., ying l., 2008 rapid propagation of dracaena cambodiana in vitro. j. anhui agric. sci., 32: 112-120. yokoduk a., mimaki y., sashida y., 2000 steroidal saponins from dracaena surculosa. j. nat. prod., 63: 12391243. 163 1. introduction cultivated olive (olea europaea subsp. europaea var. europaea) (green, 2002) ranks 21st among agricultural species worldwide and first among woody fruit species in terms of surface area (9.5 million ha), producing more than 3 000 000 t of virgin olive oil (fao, 2010) and more than 2 200 000 t of table olives per year (iooc, 2010). virgin olive oil, which makes up slightly more than 2% of total vegetal oil production, accounts for a return in the agricultural sector of more than 22 billion dollars, while the figure for palm oil (30% of vegetal oil production) amounts to a little more than 31 billion dollars (fao, 2010). olive cultivation worldwide has grown over the past 20 years by more than 20% in terms of surface area with increases noted on all continents. there has also been a doubling in production, which is more than proportional to the increase in surface area (fao, 2010). these increases have been due to improvements in cultivation technique in traditional plantings as well as entry into production of new, more rational plantings. the species is in general very efficient in exploiting environmental resources and can produce as much as 2 t ha-1 of healthy oil food. little more than 50 years have passed since olive first expanded beyond its traditional cultivation areas, and the species has shown to be flexible and adaptable in various agronomic, climatic and environmental situations, able to add value to extensive areas and to face the climate changes currently taking place. 2. taxonomy cultivated olive is an evergreen bush that can be, with suitable intervention, grown as a tree and it is considered typical of mediterranean flora. the taxonomic position of the species olea europaea l. has been reviewed (green, 2002), also in light of the data that has emerged from new technologies of molecular identification (besnard et al., 2002). the olea genus is divided into three subgenera and in the subgenus olea, sez. olea, the species olea europaea is grouped as a “complex” with potentially infertile forms, compatible for grafting, and characterized by the presence of flavonoid glucosides in plant tissues and fruit. the olea europaea complex is further divided into six subspecies: cerasiformis (island of madeira), cuspidata (from the south to northeast of africa and from southwest asia to the arid zones of the yunnan and sichuan in china), europaea (mediterranean basin as far as mesopotamia), guanchica (canary islands), laperrinei (hoggar high plain in the south of algeria as far as al jebel marra in the western sudan), and maroccana (southern slope of the atlas mountains in morocco). these subspecies can be considered “geographic entities” with notable molecular differences (for example cerasiformis and maroccana are polyploid) (besnard et al., 2007 a) but their morphologic characteristics are so similar that in certain cases descripoleiculture in progress e. marone*, p. fiorino** * dipartimento di scienze degli alimenti, università degli studi di teramo, piazzale aldo moro, 45, 64100 teramo, italy. ** dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, sezione colture arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. kew words: aging, branching, chilling requirement, cold resistance, flower buds, planting density, triglycerides. abstract: the present work evaluates the limits and possibilities of development with regard to the most recent olivegrowing techniques in light of up-to-date knowledge of species characteristics. after a brief introduction regarding the productive capacity of olive, the new taxonomic position of the cultivated species and a reorganization of the genus olea is presented in the first part of the work. examination follows of the assumed stages of domestication, spread (from the bronze age until decline in the 6-10th centuries a.d.) and then globalization of the species from the 19th century until the present. the second part addresses the spread of olive to the different continents, environmental limitations to its cultivation and the growth model that distinguishes it from most of the other cultivated woody species. the various problems that arise when olive is cultivated outside its areal of origin are considered, from induction processes to effective chilling requirements, as well as the effects of climatic environment on plant growth and product quality following shifts in areal. the paper concludes with a brief analysis of open questions relative to new models of cultivation. adv. hort. sci., 2012 26(3-4): 163-175 received for publication 28 january 2013 accepted for publication 15 february 2013 164 tions can overlap. this fact justifies past imprecision in classifications. many entities which in the past were considered to be separate species, including progenitors of cultivated olive such as o. crysophylla and o. ferruginea (simmonds, 1976), have been placed within the subspecies o. europaea cuspidata. based on this classification, the areal of the olea europaea complex occupies three continents: starting from south africa it crosses the central part of the continent and the horn of africa, from egypt and the red sea it reaches the mediterranean and toward the west into the islands of macaronesia (island of madeira and the canary islands), while in the east it passes through palestine, syria, mesopotamia and western and eastern zones of the himalaya chain as far as southwestern china. 3. origin and domestication of the species due to a scarcity of fossil evidence, it is difficult to determine the geologic period during which this complex taxa became defined and began to evolve. it is believed that both a floristic element of african paleotropical origin and the long evolutionary history of this group spanned a large part of the tertiary (besnard et al., 2009). it seems that the genus olea had its origins in the oligocene and that numerous taxa diversified thanks to catastrophic climatic and tectonic events which were characteristic during the tertiary (from the great glacial period of the oligocene to the raising of mountains in eastern africa and desertification of the sahara). a study on the phylogenesis of the olea genus, conducted by besnard and coworkers (besnard et al., 2007 b) using plastidial dna and nuclear ribosomal dna as biological clocks, positions the principal phylogenetic nodes for determining articulations of the genus from the oligocene (c. 59.2 mya) until the pliocene (c. 4.4 mya), by which time the main taxa of the genus had clearly separated. the desertification of continental africa seems to be correlated to subsequent differentiation of taxa adapted to arid conditions. the new dry environments may have favored establishment of foliar morphotypes in olea. recurrent segregation and hybridization events could very well have been caused by geographic barriers (e.g. the sahara) and land bridges. probably until the end of the last ice age (15 000-12 000 b.c.) the distribution of olive was prevalently in the area of africa where desertification led to isolation of saharan populations (subsp. laperrinei). only later were the southern coasts of the western mediterranean colonized through the spread of glacial refugia. thus, spontaneous olive in the western mediterranean would be of african origin, redistributed as far as the coasts of spain, france and the tyrrhenian portion of italy after the last glaciations (besnard et al., 2001). the subspecies o. europaea europaea is divided into two botanical varieties: europaea, which corresponds to the old denomination olea sativa (weston) and includes the cultivars having seedlings called olivasters; and silvestris (mill.) which corresponds to the old presumed species olea oleaster (hoffman and link), spontaneous olive or oleasters. the wild oleasters forms represent the original postglacial mediterranean population. in the area between ancient palestine (zohary, 1994; zohary and spiegel roy, 1975) and the caucasus, the earliest plants adapted to the needs of proto-cultivators (around the 5th millennium b.c.) were individuated, utilized and multiplied. considering the ease of agamic reproduction and longevity of the plant, it is possible that few generations of crosses divide contemporary cultivars from these not-sodistant progenitors. as colonizers advanced westward, carrying with them plant material and expanding cultivation, they individuated and utilized autochthonous material, progressively diversifying the genetic base of their cultivars and at the same time enriching the local forms. generally speaking, significant molecular differences exist between the oleasters of eastern and western zones of the mediterranean (breton et al., 2006 a) and it is possible to identify separate data groups with a separation line that passes through the adriatic sea and the libyan desert (breton et al., 2006 b). from the cited work, the cultivated genotypes seem rather dispersed which can point to a mixing among the various markers, suggesting repeated attempts at domestication and subsequent crossing among cultivars of different origins. evidence of these events are illustrated by hannachi and coworkers (hannachi et al., 2010): comparing plastidial dna of traditional tunisian cultivars and wild tunisian forms, they demonstrated that seven of the 15 cultivars currently under cultivation in that country are of oriental origin, while the others are linked to wild material of maghrebian origin. even if the center of domestication for olive was in the eastern mediterranean, pre-domestication episodes seem to have occurred more or less at the same time, starting from the 5th millennium b.c., in various areas around the basin, for example on the island of crete and southern spain. in these areas “stratifications” have been found with cultivated olive seeds overlaying oleaster seeds, as well as the contrary with “imported” seeds from cultivated forms supplanted by autochthonous forms (terral et al., 2004). initially olive was used for different purposes compared to its modern use: it was employed for shade, to produce forage and firewood, and to make tools such as poles and staffs; it took thousands of years of cohabitation to arrive at use of the fruit and oil. when cultivation advanced beyond the phase of simply collecting spontaneous fruits, the proto-farmers decided it was more practical, in a preliminary form of cultivation, to group together the best examples from spontaneous forms through agamic propagation by part of stump. this multiplication technique remained in use until the 1970s in many traditional olive-growing countries of the southern mediterranean. 165 4. the spread of cultivation in the beginning, direct use of the fruits must have been limited since the drupes are very bitter, even when extremely ripe, due to the presence of elevated quantities of oleuropein and other phenolic glucosides, and the technology required to remove the bitterness and for brining was too complex for the means available at the time. however, the oil could be extracted through milling using implements similar to those used for grains, making it available for medicinal use, as fuel, for illumination (both sacred and not) and as an unguent. the use of olive oil in the diet came later in this plant’s history, around the first half of the 2nd millennium b.c., when use of this product spread via sea routes first from the eastern mediterranean and aegean sea toward greece, then thanks to the phoenicians along the coast of africa toward the west as far as (and beyond) the pillars of hercules. it is probable that with this progression, attempts at domestication by the inhabitants of the various zones brought advantages. it should be no surprise that ample deposits of knossos oil have been found, which testify to the presence of active olive oil commerce in the minoan period (second half of the 2nd millennium b.c.), nor is transportation of oil from spain toward carthage a surprise as cultivation on the iberian peninsula probably dates back farther than evidence and documentation demonstrate. at the beginning of the 1st millennium b.c. olive cultivation and oil use had reached the various coasts of the mediterranean and the type of “domestication” of the plant is revealed through differentiation: the greeks, as far back as the oldest writings, distinguished the oleaster as κότινος for wood usage from ελαία for oil production. according to pliny, the romans didn’t know olive cultivation until the time of tarquinio prisco (6th century b.c.) but in the 1st century a.d. oil from the italian peninsula was exported to the provinces of the empire. in rome, oil was principally used for external treatment of the body. as the saying went, “wine on the inside, oil on the outside” (intus vini fori olei)1 between the 2nd and 4th centuries the spread of this plant reached its greatest development in the entire mediterranean region. oil was distributed free of charge to the plebs of rome as a food source and sent to the legions in germany. the trade surrounding oil was so important that remains from the jars and amphorae in the port of rome of the time left a hill which is today one of the recognized neighborhoods of the city (testaccio). 5. globalization of cultivation with the fall of the western roman empire in 476 ad and loss of control of the sea routes, trade and use of olive in the western mediterranean declined rapidly. evidence 1 plinio, storia naturale, xiv, 150. of introduction of oil from northern africa in the 6th century can be found (brugnoli and varanini, 2005) but from the 7th century sea transport of oil toward rome ceased, at least in an organized way, to exist. at the same time a period of instability in north africa began, marking a generalized abandonment of olive cultivation in the region; it did not pick up again until after the arab conquest around the 10th century. in europe, olive oil acquired new importance through the christian religion, and for which substitutes were not possible (e.g. for illumination of altars, anointing of the sick, use of plant oils during lent or other periods of abstinence from foods of animal origin). for this reason, cultivation was undertaken in some areas considered marginal in terms of climate for the cultivation of olive (northcentral italy until the alpine valleys) as a way to at least guarantee the needs for churches and monasteries. olive growing zones along the coasts receded with the fall of commerce and only the oldest cultivation areas remained important: palestine, syria, and the island of crete provided oil for venice and constantinople while andalusia furnished muslim areas. the olive oil trade was revived in the 11th and 12th centuries thanks to merchants from genoa and venice. they supplied monasteries, cities in italy, france and constantinople with this precious product needed not only as a food source but also for religious and liturgical purposes, illumination, and soap and wool production. an intense freeze in 1009 killed off the remains of ancient olive growing on the italian peninsula along the entire adriatic and in particular in puglia. subsequently this region experienced a notable increase in olive cultivation with new plantings, at the expense of grain cultivation, thanks to the development of trade by the venetians. in this period apulian markets were open to traffic from venice, genoa and byzantium, thus progressively establishing this area as an important zone for olive cultivation (iorio, 2005). even today puglia accounts for the most extensive and productive portion of italian olive production, with more than 20 million trees of 300-500 years of age. after about 1300, olive oil (for illumination and soap and wool production) became one of the most important products necessary for industrial development in northern italian cities. as it was not possible to rely exclusively on commercial trade, it became necessary for the economies of many zones to provide incentives for cultivation, also in areas with extreme soil and environmental conditions. the 15th century represents a turning point not only in the history of olive but also for humanity. in 1453 constantinople fell, the last remnant of a civilization born with the cultivation of olive, while in 1492 two important events occurred: the fall of granada, the last vestige of muslim dominion in spain, and the landing of colombo on san salvador. the interest of europe shifted westward and with it went the olive, a colonizing plant par excellence. olive was introduced in spanish colonies, first in cuba around 1520 and then in california. the original plant material left the port of seville as seeds or seedlings and so 166 genetically the material can be considered as andalusian in origin. the spanish colonists carried olive with them as they travelled southward from california along the pacific, introducing it in peru and from there crossing to argentina. an olive plant considered to be more than 400 years old exists in the province of la rioja in argentina, which would place its establishment at the time of the first european settlements in that region when the capital, ciudad de todos los santos de le nueva rioja, was founded (1591) (fig. 1). during its trip from california to argentina, the genetic material of olive underwent further selection with identification of cultivars such as ‘mission’ (california) and ‘arauco’ (argentina), the 400-year-old tree belonging to this latter. with the spread of colonialism and the need to transfer plant productions able to satisfy industrial and dietary requirements in the colonies, also olive began to be valued, utilized and cultivated in various areas considered to be suitable. in 1661 dutch merchants carried olive to south africa where spontaneous forms of olea europaea cuspidata were already present. between the end of the 18th and beginning of the 19th centuries cultivated olive, together with cuspidata, landed in southern australia where the two subspecies found a favorable environment, giving rise to a vast phenomenon of spontaneity (breton et al., 2008). the first documented introduction dates back to 1800 when olive officially arrived in sydney (spennemann, 1999). from then until before the second world war, olive from various provenances was introduced (from spain, italy, greece and later israel). starting in 1956 there was interest in olive cultivation in china as well, where the plant was known but its cultivation was difficult due to intense summer rainfall. currently, the cultivation area of olive is spread in both hemispheres between 45° and 30° of latitude with extension toward the warm-temperate zones of our planet. the agronomic success of this species is based on two fundamental characteristics that distinguish olive from all other cultivated fruit species: its particular growth model and flower formation make it easy to manage and the reliability of its production have been determinant for its cultivation since neolithic times. it is adaptable to highly variable climatic conditions and its localities of origin for domestication are characterized by different conditions. 6. environmental limitations the limits for cultivation of olive are attributable to environmental factors and their annual cycles. beyond the degree of latitude (45°) for cultivation for this species, plants are potentially exposed to damage from cold temperatures that can compromise production or even the life of the plant, depending on intensity and timing of the event. cultivated olive is only moderately tolerant of low temperatures and even if it is suitably prepared for winter cold (acclimatization), it can resist only a few degrees below zero due to a protection mechanism (supercooling) that keeps water in cells in the liquid state down to several degrees below the freezing point (table 1). the most resistant tissues are the bud meristems (in ‘ascolana tenera’ the recorded lethal temperature is -19.3°c) (fiorino and mancuso, 2000), so that vegetation severely damaged by fig. 1 old picture (dated around 1950) of the oldest ‘arauco’ plant in argentina. table 1 lethal temperature (°c) as evaluated by differential thermal analysis for various organs in two acclimatized olive cultivars having different resistance to cold: ‘ascolana tenera’, very resistant; ‘coratina’, poorly resistant part of plant lethal temperature (°c) ‘ascolana tenera’ ‘coratina’ leaves -14.5 ± 0.2 -11.8 ± 0.2 shoots -18.6 ± 0.6 -12.6 ± 0.4 buds -19.3 ± 0.6 -13.5 ± 0.4 roots -9.1 ± 0.3 -8.6 ± 0.3 from fiorino and mancuso, 2000. 167 intense winter freeze can copiously resprout in the following spring from latent and adventitious buds, in particular on the stump. differences exist among the cultivars: comparing 21 prevalently italian cultivars for their resistance to cold, employing three different evaluation methods in leaves and sprouts, the authors (azzarello et al., 2009) were able to divide the material into three groups in relation to lethal temperature. with regard to phylogenesis, and considering knowledge of other subspecies such as olea europaea subsp. cuspidata which is poorly resistant to cold stress, it is possible that the varieties cultivated today have reached their maximum result possible through genetic pressure applied by humans in terms of advancement toward colder regions. furthermore, due to its tropical origin, the species is characterized by a relatively high critical temperature (1012°c) (mancuso, 2000), thus pushing cultivation toward northern limits would result in a growing season that is too short. the highest latitude areas with olive cultivation are the alpine lakes in italy, istria (slovenia, croatia), and the olive-growing area of odessa (crimea, ukraine). the factors which limit expansion toward the equator are less evident. olive is a species that can support high temperature: the lethal temperature for leaf tissues varies from 46 to 50°c depending on the cultivar and can reach 52°c in sprouts (mancuso and azzarello, 2002). the greatest limiting factor seems to be the need by flower buds for a period of low temperature in order to pass from an “inductive” phase to that of tissue differentiation and development of the inflorescence. this rest phase is generally divided into two periods with very different thermal needs. in the first phase low temperatures (6-9°c) are needed to remove inhibition for subsequent growth, and in the second warmer temperatures (above 8.5°c) are required to accelerate evolution of the phenomenon. the currently used reference model for olive (de melo-abreu et al., 2004) is based on that of richardson to study periods of rest in peach (richardson et al., 1974). this model considers the period from 1 october to 31 january useful for overcoming chilling need, with 7.3°c being the optimal temperature. temperatures from 0 to 18.5°c can also be considered acceptable with a weighted effect of their distance from the optimal temperature, similar to the richardson model. the authors terminate calculation of the chilling units on 1 february, as they consider that for their study area (cordoba and mas bové reus terragona in spain, elvas and santarém in portugal) by that date the needs of olive for cold should already be satisfied. the authors underline that temperature is not the only factor toward which olive is sensitive and thus the data needed for this calculation should be verified every time there is a change in cultivation zone or variety. interest in the effect of low temperatures for normal development of flower buds is relatively recent: only in the 1950s was it proposed that the amount of flowering is in some way linked to the duration of low winter temperatures (hartmann, 1953; hartmann and porlingis, 1957). in the beginning researchers (hackett and hartmann, 1964) believed that the role of low temperature was more incisive and able, together with other environmental factors, to influence flowering processes from the first phases of induction, but later it was demonstrated (rallo and martin, 1991) that its role is limited to development subsequent to the induction phase. there are however ample areas of research that have only barely been considered. for example, in 1975 it was noted in a study (hartmann and whisler, 1975) that: 1) by applying a suitable period of cold, flowering can be stimulated in any time of the year (this somehow confirms the theory of “aging” of apical meristems that when mature produce buds able to directly develop flowers) (fiorino and marone, 2010); 2) there are ample differences between cultivars in regard to their response to various quantities (constant or variable) of low temperatures (varietal differences represent the weak point of all experimentation in this field); and 3) thermal thresholds of response seem extremely various among cultivars themselves as evidenced by the behavior of local cv. mission which can, by flowering continuously throughout the cool summer typical of the california coast, lead progressively to a modest number of inflorescences. in 1983 a work was published (denney and mceachern, 1983) aimed at improving understanding of responses of this species to various environments. the results were obtained by elaborating temperature data (october-may) from 15 olive-growing stations to determine the effect of the “cold”, measured as the capacity of the olive plant to flower in the subsequent spring, when two conditions have been met: 1) active growth has concluded; 2) daily temperature trend does not exceed an average of 12.5°c. the greatest interest with regard to this study concerns the concept of “effective chilling” which indicates, for regular flowering to occur, the number of days when the average temperature must not drop above 12.5°c. in order to satisfy the thermal needs of olive, 70-80 days of “effective chilling” are necessary, thus setting geographic limits for the species. a comparative analysis of the thermal conditions of some geographical zones at the warmer limits for cultivation (ico, peru and caborca, mexico) and where olive growing is undergoing development (gran chaco, argentina) (ayerza and sibbett, 2001) points out that often in “new”, “warm” olive-growing areas the number of days for wintering are fewer than those considered necessary (70-80 days). for example, at ico (peru) the possibility for wintering does not exist as average daily temperatures are never below 12.5°c, although there are cultivars that produce well enough in the area to allow commercial plantings. the area is characterized by a lack of rainfall despite frequently cloudy skies, making it possible to control growth through drastic reductions in irrigation water which oblige the plant to reduce or interrupt vegetative growth, despite year round temperatures that are relatively high. the authors of the study admit that the reasons for this 168 behavior are not clear and hypothesize that in the autumnwinter period, as the sky is almost constantly overcast, the thermal lows needed for the buds are attained, although perhaps they wouldn’t be in sunny areas. sensitivity to different thermal thresholds or chilling needs to bring about flowering is appearing in new plantings, in areas considered to be homogeneous with consistent seasonality, but where there are specific zones with cold periods of different duration and intensity. for instance, this can be the case in the mediterranean basin where notable differences are recorded for florence (central italy), seville (southern spain) and cairo (egypt). the millennia-long history of olive has made it possible to identify local cultivars perfectly suited to specific environments, thus creating varietal standards able to adapt themselves to the thermal trends of that particular area. in recent olive-growing, cultivars famous in a specific territory have often simply been used elsewhere, although this has led to unsuccessful attempts generally due to a lack of adaptability to the relative high winter temperatures of the new environment. examples of this are the negative results obtained from the transfer of ‘frantoio’ (cultivar from tuscany) to southern coasts of the mediterranean or warm areas of the planet. this sensitivity of the plant to specific thermal requirements for flowering confirms the fundamental role of temperature in all aspects of the life of olive, from the progression of phenological phases to the biochemistry of the oil. there are notable differences among mediterranean germplasm with regard to the effective chilling requirements of autochthonous material which do not necessarily depend on where the material was selected. thus, two varieties selected in similar environments and at approximately the same latitude, for example ‘arbequina’ and ‘frantoio’, have demonstrated opposing behaviors when cultivated in warm areas: the former flowers copiously and early, while the latter rarely flowers much and often not at all. in olive, the effect of low temperature is more complex than in temperate species which are often referred to: for some varieties the “chill” that results from diurnal temperature variation is sufficient to permit normal flowering (e.g. in ‘arbequina’) and the thermal threshold of chilling must be rather elevated since nocturnal temperatures of less than +2 to -1°c markedly diminish flowering under controlled conditions (malik and bradford, 2009). 7. current trends over the course of the millennia of expansion (4th-2nd millennia b.c.) and cultivation (1st millennium b.c. 2nd millennium a.d.) the techniques for growing olive remained essentially unchanged, just as the needs of agriculture to have long-living, hardy, easily managed plants able to produce oil for nutrition, illumination, industries (textile and mechanical) and other purposes did not change over time. it was only in the 19th century that olive-growing began to gain advantage from technological development, in particular with regard to oil extraction, while agronomic techniques remained anchored in acquisitions and requirements from the past. research pertaining to olive-growing was only blandly affected by the innovative spirit that invested agriculture and in the first half of the 20th century knowledge based on millennia-old, empirical observations persisted or models and concepts were adapted that pertained to other cultivated woody species, for example fruit-bearing species from temperate zones. at the turn from the 19th to 20th centuries, in europe olive oil was considered a strategic food but it was only in the middle of the 20th century that the true importance of this product for human nutrition was understood in terms of its nutritional, organoleptic and functional profile. with economic and geographic expansion of olive cultivation in the second half of the last century came awareness of the peculiarity, potentiality and lack of scientific knowledge about this ancient species. olive possesses all the fundamental requisites to become a modern crop: good productivity (more than 2 t ha-1 per year of oil), very early entry into production for plantings that are well positioned in terms of water and nutritional availability (on average the third year after planting), good adaptability to diversified environments and availability of light, soil and other resources, good availability of plant material that can be obtained using new techniques (mist-propagation and micropropagation), and great flexibility of plant material for adaptation to various breeding systems, all depending on the various destinations of the product (for oil or the table) and in relation to the needs of the market. 8. architecture of the plant olive owes its agronomic success to the longevity of plantings, simplicity of its constitution and reconstruction of the canopy, and predisposition for flowering. in nature, the plant grows as a bush with a stump rich in vegetative meristems able to produce suckers that can form trunk-like structures able to survive for hundreds of years; it can grow to have canopy heights and diameters of 10-12 m. the olive model is not greatly described in literature. each vegetative meristem on the stump (or positioned plantlet) can give rise to a sprout with acrotonous, orthotropous attitude and continuous growth that will form the vegetative axis. early on, this sprout is poorly lignified, slender in relation to its height and tends to curve if not supported. at its point of curvature (the new tip of the branch), another vegetative meristem grows and repeats the cycle, while the distal portion of the original branch continues his growth in a lateral direction. with lignification and subsequent radial growth, the insertion angle of the two consecutive, opposing sections attenuates and the structure takes on the form of a single trunk resulting from fusion of the segments (fiorino et al., 2012). the originally-vegetative apical meristems, over time and with growth, mature, change in function, lose their orthotro169 pous characteristics, become plagiotropous and take on reproductive functions (“aging”) (fiorino and marone, 2010) with subsequent formation of fruit-producing vegetation. this particular type of growth and shift to production makes olive a very flexible plant as it can be grown either with a central axis and short branchlets arranged at various heights or with varying vase-like forms, obtained via subsequent vegetative axes, having or not a central trunk. having these characteristics, the parts of the canopy destined to support the vegetation are semi-permanent structures that periodically need to regenerate the growth-agingflowering cycle typical of olive branches. this growing habitus simplifies pruning principles and operations. 9. induction and differentiation growth and lengthening of the branch in subsequent years is almost completely delegated to apical meristem activity, creating a continuous linear structure (branchlimb) with persistent (three years), opposite leaves and lateral flowering that allows further apical growth. the branch-limb complex is made up of a succession of nodes generated by an evolving meristem (vegetative vs. reproductive) that will determine bud functions. the growth-aging process continues until a progressive weakening leads to the loss of the apical meristem or until itself transforms into a flower (fig. 2). two types of buds coexist at each node. the main, most evident ones that can remain on the plant for no more than two vegetative cycles, and at least two accessory buds positioned above the main one (fig. 3). these accessory buds are poorly visible and often covered after their formation by cortical tissues (and therefore also called hidden buds) of the growing branch to the point of not even being considered (lavee, 2007), and are destined to become latent and able to burst many years after their formation. the evolutionary phases of a flower meristem are not distinguishable to the naked eye before the burst of the buds (barone and di marco, 2003; andreini et al., 2008) and sometimes, even under morpho-anatomical analysis, some early manifestations of differentiation are ambiguous and difficult to interpret (troncoso, 1966). histological and histochemical indications point out that: a) there are histochemical differences in the formation and development of lateral buds in different genotypes (e.g. ‘leccino’ and ‘puntino’); b) there are differences in zeatin levels in buds of the same cultivar (e.g. ‘leccino’) taken from plants with different fruit loads and these hormone level differences start early, from the month of july (andreini et al., 2008); c) there are differences in development (timing and forming structures) among homologous buds removed from rising branches or identified in peripheral parts of the canopy and these also start early, from the month of july (fabbri and alerci, 1999). it was believed (lavee, 2007) that in growing sprouts the principal buds of originally mixed function could shift toward flower formation only when a specific sequence of events occurred, which are successive (preinductionconfirmation), temporally separated and controlled by endogenous and exogenous factors. more recent research has indicated the primary role of apical growth in the formation of fruit-bearing vegetation and in the evolution that is determined by growth. in order to reach adequate levels of aging, in relation to the cultivar, buds with defined function flowers for the principal buds, vegetative for the accessory buds form along the fig. 2 terminal bud transformed in floral grape in cv. koroneiki (photo e. marone). fig. 3 node showing the two main buds producing floral grapes and in upper position the two accessory buds (photo e. marone). 170 lengthening sprouts (fiorino and marone, 2010; marone et al., 2013). in this way, and possible due to an adequate intensification of cultivation, the amount of annual growth of mature branch-limbs increases leading directly to an increase in the number of flower clusters and thus fruit load on the branch. the fruits, due to their weight, pull the vegetation downward forming fruit-bearing cascades that are particularly suitable for mechanical harvesting by horizontal shakers (fiorino et al., 2010). 10. adaptability due to olive’s adaptability to xeric environments, currently expansion of cultivation takes place with success mainly in warm-temperate dry zones below 30-35° latitude in both hemispheres: these areas, where land is available, are characterized by short winters, early-spring temperature rise and hot, sunny summers, all factors which affect the quantity of growth, progression of phenological phases and oil characteristics as particularly influenced by temperature, according to results presented in the literature. in a study carried out at one location (montepaldi, tuscany, 43° 40’ n lat., 11° 09’ e long., 266 m a.s.l.) to evaluate the behavior of cultivars selected over time from areas having different climatic conditions (mancuso et al., 2002), analysis of the relationship between variations in annual climatic conditions and development of phenological phases revealed the following roles played by temperature: a) in the determination and evolution of the various phases together with different behavior of cultivars coming from different areas. cultivars ‘coratina’ and ‘carolea’, selected from southern italy where spring temperatures are warmer, have more accelerated bud burst and phenological phases up until fruit set compared to cultivars (‘moraiolo’ and ‘leccino’) selected in the cool, test area; and b) in the appearance of phenological phases in relation to average increase in temperature and duration of insulation. from a data pool pertaining to five cultivars (the four previously mentioned plus ‘picholine languedoc’) it was found that an increase of 0.5°c leads to an anticipation of flower bud burst by more than three days, with notable effects from the time of pit hardening. in addition, an increase in average insulation seems to increase the speed with which these phenological phases progress. the influence of spring temperatures on the date of flowering in different cultivars was confirmed by de melo-abreu and coworkers (de melo-abreu et al., 2004). more recently, orlandi et al. (2012) found that pollen release was generally determined by meteorological factors in the period before flowering, with effects on both pollen amounts and timing of flowering (early or delayed). the role of temperature in oil composition is more extensive. independently in each cultivar, temperature trend during fruit growth and maturation influences the ratio among fatty acids of the triglycerides in the oil (fiorino and ottanelli, 2003; marone et al., 2003) with a reduction of the percentage normally expected for oleic acid in years characterized by prolonged periods of elevated temperature. with increased average seasonal temperatures (expressed as thermal sums, gdh, with 10°c threshold) (software for the calculation of gdh with variable thresholds was elaborated by marone, 2003), the percentage of oleic acid content drops and is substituted by increases in palmitic and linoleic acid percentages. employing a collection of italian germplasm, with plants grown in the same environment (mirto, calabria) and with adequate agronomic techniques, and through analysis of the variations between levels and ratios of the three principal fatty acids (palmitic, oleic, linoleic) and the thermal sums values of gdh for the relative years, it was possible to measure the existing regression between thermal sums and levels of oleic acid percentages in the oils obtained from the cultivated accessions (fig. 4) (lombardo et al., 2008). each cultivar constantly adapts its triglycerides composition, modifying equilibriums and increasing palmitic or linoleic acid (lombardo et al., 2008). the graph shown in figure 5 illustrates the division of a population of cultivars into three clusters separating samples with different characteristics. the first, the most numerous, groups the cultivars that compensate for the reduction in percentage of oleic acid with linoleic acid and subordinately palmitic acid (cv. canino, nera di gonnos, tonda di cagliari and moraiolo). the second group reacts in the opposite manner (cv. raja sabina and moraiolo t. corsini), while only 5% of the tested population is composed of stable triglycerides (cv. nocellara messinese). the intensity of the response to temperature changes can be such that the oil from some cultivars fig. 4 regression between the % content of oleic acid in the tags of different cultivars and gdd (population 188 cultivars in total with at least two analytical data in the five years; p≤ 0.01 and r=0.473). on the left, the coolest year (2005), on the right, 2003 (the warmest, practically superposed with 2001) (from: lombardo et al., 2008). 171 approaches or exceeds the limits established by law in some countries with regard to triglyceride composition. working in the province of rome, italy (41° 53’ n lat., 12° 14’ e long., 30 m a.s.l.) on ‘arbequina’ and ‘arbosana’ plants grown as hedges under superintensive cultivation, the acidic composition of the oil was well balanced and characterized by elevated amounts of oleic acid and low or very low levels of linoleic acid in both cultivars (marone et al., 2009). however, when the same varieties were grown in warm, dry valleys in the northwest of argentina, with a prolonged growing period and high temperatures during the growing season, the acidic composition was modified: there was a drop in oleic acid and a rapid rise in level of linoleic acid (rondanini et al., 2011). due to this sensitivity to high temperatures during fruit growth and maturation, the need to review the limits for some parameters in the oil, as prescribed by international laws intended to verify the genuineness of the product, becomes necessary with the extension of olive growing in warm areas or the marketing of local products that had previously been destined for self-consumption. unadulterated productions can, for example, present not only variations in the ratios among triglycerides (often the amount of linoleic acid exceeds the allowed limit of 1%), but also alterations with regard to the phytosterols, with increases in particular in campesterol levels beyond established limits and which could indicate an addition of seed oils to the olive oil. there have been few observations of the result of transfer of cultivars from warm to cooler zones. in this case, an opposite phenomenon has been noted with an increase in oleic acid percentage (fiorino and marone, unpublished data); it is possible that special characteristics of some productions from cool olive cultivation zones derives precisely from the specific interactions between some cultivars and the various climatic zones. 11. open questions new plantings require the transformation of current cultivation techniques, which are still tied to millenniaold traditions. this shift calls for not only radical cultural changes, but also notable investment for establishment of new or modification of existing plantings as well as for machinery. the objective of new plantings is to produce oil, with a lengthening of the production chain from the fruit to the finished product extra virgin olive oil which remains unique among other vegetal oils on the market for the presence of complex antioxidants (i.e. polyphenols) that derive directly from this plant’s origins. for researchers and technicians who direct their efforts toward globalization of cultivation, the weak point remains the modest amount of basic knowledge available in order to respond to questions that arise regarding varietal adaptability to different environments, regularity of production and canopy management. in particular, with regard to the spread of new intensive or superintensive olive-growing models (terms linked to the elevated number of plants per hectare), there are a series of questions: what is the productive lifespan of orchards? what are the characteristics of the product? what is the varietal platform? the reduced productive lifespan of orchards represents the greatest difficulty to overcome for the spread of this model in countries where olive-growing is deeply rooted in culture and history. today, plantings last only a few years: about 10 for peach, 15 for apple and pear, perhaps 20 or a bit more for olive, with plants having performed their economic duty in this period of time. as for the characteristics of the product, research has pointed out that it depends more on cultivar/environment interaction than on the growth system. initial data seem to confirm that, in any case, the chemical and sensory characteristics of the oil do not change in intensive or superintensive plantings (marone et al., 2009). the varietal platform suitable for superintensive plantings is made up of few cultivars typical of specific areas (‘arbequina’ and ‘arbosana’, spain; ‘koroneiki’, greece). growth and productivity characteristics are known for the new planting models (rallo, 2006; tous et al., 2003) as well as the characteristics of the product (marone et al., 2009). novelties, such as tosca 07® and chiquitita® (redacción olint, 2007; rallo et al., 2008), have been more recently introduced in new plantings and still need to be better defined agronomically. the longevity and vitality of olive, and the marginality of its cultivation and production have penalized genetic improvement of this plant: it was undertaken unconsciously in the past but has been more targeted in the last century. the ideotypes must combine different characteristics (fiorino, 1999). in addition to growth habit, fructification, fig. 5 fuzzy clustering in a ternary plot (fuzziness f= 2.25), utilizing the absolute value’s differences for the two compared years between 69 cultivars for saturated, monounsaturated and polyunsaturated fas (from: lombardo et al., 2008). 172 and resistance to biotic and abiotic stress, they have to respond to precise composite characteristics for oil which indicate the nutritional value for humans and are by now used internationally for marketing of the product. despite the existence of a broad genetic base, with a total of more than 1200 cultivars in germplasm collections (bartolini, 2008), there is little reliable information available about genetic determination of traits and their heredity (bellini et al., 2003) and this lack makes it difficult to choose the genetic material to use for crossing programs. a recent review (rugini et al., 2011) about the major needs for genetic improvement of olive, indicated systems and aim to achieve different goals in this species, but at present it often ends up being based simply on the phenotypic behavior of parental lines. also variability in the triglyceride and unsaponifiable fraction in descendents from crosses with the same parents was wide (tables 2-4) and sometimes with percentages of some fatty acids outside normal values (fiorino, 2001). the table 2 reports the values for fatty acids, polyphenols and tocopherols in oils obtained from eight genotypes of open-pollinated ‘arbequina’ grown in the same environment. the lineage is characterized by great variability in acidic composition, above all for linoleic acid contents. it has only been in the past ten years that indications regarding the technological characteristics of the various germplasm collections are present in terms of the qualitative and organoleptic characteristics of the oil (rotundo and marone, 2002). furthermore, characterization of the oils produced by single cultivars in specific environments has begun in this past decade as well (cimato et al., 2004; di vaio, 2012). new plantings call for control of canopy dimensions, and as most varieties selected over the millennia are very vigorous, new input has come from selection of clonal rootstocks able to influence growth and vigor in olive. by using rootstocks from specific varieties (‘tosca 07® and ‘leccino dwarf’) vigor and architecture have been modified in plants of ‘cerasuola’ highly vigorous and ‘biancotable 2 oil characteristics of genotypes obtained from open-pollinated ‘arbequina’ (ghiza, egypt) genotypes fatty acids (%) 16 52 56 61 67 68 94 105 c 16:0 10.7 14.2 14.2 16.2 19.3 14.9 15.7 18.6 c 16:1 1.0 0.6 1.2 2.2 3.4 2.0 2.1 3.4 c 18:0 2.5 2.1 2.3 2.2 1.8 1.7 2.5 1.7 c 18:1 75.3 66.7 50.7 60.6 42.0 45. 9 61.1 58.5 c 18:2 8.5 14.2 29.3 16.6 31.7 33.3 16.6 15.7 c 18:3 0.7 0.9 1.3 1.2 0.9 1.2 1.0 1.0 tocopherols (mg/kg) 416 168 219 366 286 347 91 420 polyphenols (mg/kg) 34 165 67 51 77 91 50 101 from fiorino, 2001. table 3 values for the most important fatty acids in oil from crosses of ‘arbequina’ fatty acids (%) genotypes 127* 129* b28** c 16:0 13.7 11.7 19.6 c 16:1 0.9 0.8 2.4 c 18:0 2.1 2.3 2.1 c 18:1 66.1 76.6 50.2 c 18:2 14.7 6.3 23.3 c 18:3 1.0 0.8 1.2 * no. 127 and no. 129 = ‘arbequina’ x ‘aggezi shami’. ** b28 = ‘arbequina’ x ‘picholine languedoc’. from fiorino, 2001. table 4 oil characteristics of genotypes obtained from controlled crosses of ‘manzanilla’ x ‘picholine languedoc’ genotypes fatty acids (%) a2 a5 a4 a14 a18 a19 c 16:0 12.0 10.9 13.7 16.6 9.9 16.1 c 16:1 0.9 1.2 1.9 1.9 0.9 2.1 c 18:0 2.5 2.1 2.3 1.9 2.1 2.1 c 18:1 68.6 75.0 64.9 63.9 72.0 58.0 c 18:2 13.7 8.8 15.1 13.7 12.8 19.5 c 18:3 0.8 0.7 0.8 0.9 0.9 1.3 tocopherols (mg/kg) 173 185 215 177 214 318 polyphenols (mg/kg) 169 286 140 305 from fiorino, 2001. 173 lilla’ low vigorous (caruso et al., 2012). these results are very promising, even if they must be verified in the field and in different environments. 12. conclusions as with other woody fruit trees (e.g. citrus spp.) and prevalently herbaceous species (e.g. corn, soy, sugarcane, manioc), olive is rapidly expanding its territory into new areas, which in turn permits the latent capacities of this species to be expressed to an equal or greater extent compared to that possible in the species’ areas of domestication and millennia of cultivation. olive seems able to gain added value in some temperate-warm areas of the planet where otherwise unutilized desert zones could offer ample space. in these areas it would be possible to control growth and fructification through the water stress inherent to the climate as well as temporal space during the annual growth cycle to satisfy the modest chilling requirements of some cultivars. the current geographical limit toward the equator (30° lat.) does not seem insurmountable and, in particular conditions, the margins for maneuvering with regard to enlarging the areal still seem quite wide. the limits of this development are related to a lack of some background knowledge and uncertainties in interpretation of research data, especially with regard to control mechanisms for flowering, flower formation (inductive phase), and flower development (chilling requirements). olive oil has special characteristics that make it not only a food and condiment, but also a product able to protect the human organism from dysfunctions and pathologies, thanks to the presence of a number of components, and thus it plays an important role in a balanced diet. olive has a particular capacity 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vinifera l.) is an economically important fruit crop, served fresh and used for wine production. grape is one of the most studied fruit crops, given that the grape genome sequence is available (jaillon et al., 2007; velasco et al., 2007). grape skin contains several phenolic compounds, such as anthocyanin, resveratrol, and catechin, which are important not only for resistance to biotic and abiotic stresses but also for berry qualities such as color, astringency, and human health benefits (kader, 2002; steyn, 2009). resveratrol, a stilbenoid accumulating in the grape berry, is a key compound in the “french paradox” (renaud and de lorgeril, 1992) and is attracting attention in medicine and food science. atp binding cassette (abc) transporters are wellknown transporters responsible for secondary metabolite accumulation in plants (yazaki, 2006). they form a large gene family and are found in all living organisms (rea, 2007). plants have much larger numbers of abc transporters than animals or microorganisms: arabidopsis and rice have more than 120 abc proteins (rea, 2007; yazaki et al., 2009; kretzschmar et al., 2011). abc transporters have a transmembrane domain (tmd) and a nucleotide-binding domain (nbd) comprising atp-binding walker a and b motifs (martinoia et al., 2002). abc transporters are classified into eight subfamilies (abca-h) according to their structure and sequence similarity. half-size abc transporters contain one tmd and one nbd, whereas full-size abc transporters contain two repeats of the structure of half-size abc transporters, two tmds and two nbds (verrier et al., 2008). the substrate specificity of abc transporters is broad, and plant abc transporters have been reported to transport various compounds, such as secondary metabolites, heavy metals, lipids, chlorophyll catabolites, xenobiotics, and plant hormones (rea, 2007; yazaki et al., 2009). abc transporters show different localizations, such as the plasma membrane, vacuole, er, golgi apparatus, mitochondrion, and peroxisome; subcellular localizations of abc transporters in the same subfamily are not always the same (yazaki et al., 2009; kretzschmar et al., 2011). the abcg subfamily is a major abc transporter subfamily. it contains both half-size transporters, called the molecular cloning and characterization of abcg/pdrtype abc transporter in grape berry skin m. suzuki 1, 2, m. jasinski3,4, e. martinoia5, r. nakabayashi6, m. suzuki6, k. saito6, 7, k. shiratake1 (*) 1 graduate school of bioagricultural sciences, nagoya university, chikusa, nagoya 464-8601, japan. 2 national institute of vegetables and tea science, minaminakane, taketoyo, aichi 470-2351, japan. 3 department of biochemistry and biotechnology, poznań university of life sciences, dojazd, 60-637 poznań, poland. 4 institute of bioorganic chemistry, polish academy of science, noskowskiego 61-704 poznań, poland. 5 university of zurich, institute of plant biology, zollikerstrasse ch-8008 zurich, switzerland. 6 riken center for sustainable resource science, suehiro-cho, tsurumi-ku, yokohama 230-0045, japan. 7 graduate school of pharmaceutical sciences, chiba university, inohana, chuo-ku, chiba 260-8675, japan. key words: full-size abcg transporter, gene expression, grape, resveratrol, uv. abstract: grape (vitis vinifera l.) skin contains the phenolic compound resveratrol which is important not only for resistance to biotic and abiotic stresses but also for human health. however, little is known about resveratrol transport in plant cells. abc (atp binding cassette) transporters are well-known transporters responsible for secondary metabolite accumulation in plants. previous reports speculated that the full-size abcg transporter pleiotropic drug-resistant (pdr) is involved in resveratrol transport in fungi and plants. in this paper, all full-size abcg transporters found in the grape genome database are listed and focus is placed on vvabcg44/vvpdr14 as a candidate resveratrol transporter. the fulllength cdna of vvabcg44 was cloned by rt-pcr using mrnas extracted from grape berry skin. vvabcg44 expression was induced by uv irradiation, and the expression pattern of vvabcg44 in various grape organs was similar to that of stilbene synthase (sts), a key enzyme in resveratrol synthesis. resveratrol content in grape berry skin increased after uv irradiation. these results suggest that vvabcg44 functions as a resveratrol transporter in grape. adv. hort. sci., 2014 28(2): 53-63 (*) corresponding author: shira@agr.nagoya-u.ac.jp received for publication 31 march 2014 accepted for publication 16 june 2014 54 white-brown complex (wbc) subfamily, and full-size transporters, called the pleiotropic drug-resistant (pdr) subfamily. one of the best studied full-size abcg subfamily members is yeast pdr5 (lamping et al., 2010; prasad and goffeau, 2012). pdr5 is an exporter in yeast plasma membrane and is associated with multidrug resistance (decottignies and goffeau, 1997; golin et al., 2007). in plants, full-size abcg transporters have been reported to transport phytoalexins (banasiak et al., 2013), abscisic acid (aba) (kang et al., 2010), strigolactone (kretzschmar et al., 2012), and other compounds. a strain of the plant pathogenic fungus botrytis cinerea, lacking a full-size abcg transporter bcatrb, is sensitive to resveratrol (schoonbeek et al., 2001). on the other hand, after treatment with an elicitor, cyclodextrin, grape culture cells accumulated resveratrol and full-size abcg transporter genes were induced in the cells (zamboni et al., 2009). these results suggest that full-size abcg transporters are associated with resveratrol transport in fungi and plants. in this study, we listed all full-size abcg transporters (pdrs) found in the grape genome database and focused on vvabcg44/vvpdr14 as a candidate resveratrol transporter. we cloned the full-length cdna of vvabcg44 and determined its gene expression in various organs and after uv irradiation. vvabcg44 expression was induced by uv irradiation and the expression pattern of vvabcg44 in various grape organs was similar to that of stilbene synthase (sts), a key enzyme in resveratrol synthesis. to the best of our knowledge, this is the first report of an abcg transporter in grape. 2. materials and methods plant material and treatments vitis vinifera “pinot noir” was harvested in the vineyards of the azumi apple corporation in nagano prefecture and of nagoya university in aichi prefecture, japan. for molecular cloning and gene expression analysis, young leaves, mature leaves, tendrils, stems, seeds, pulp, and berry skin were harvested in june and july. for molecular cloning, the skin of the berries after uv irradiation was used. for uv irradiation and aba treatment analyses, the grape berry clusters were harvested in june and july, before the veraison stage. uv irradiation and aba treatment were performed as described below. berry clusters were irradiated using a uv-c lamp (253.7 nm, gl-15, toshiba, japan) at a 50-cm distance for 1 h. control samples (dark) were covered with a box and placed beside the sample receiving uv irradiation. for rna extraction, the skin of the berries was collected immediately after uv irradiation. for measurement of resveratrol content, after uv irradiation, berry clusters were maintained for 23 h in the dark at room temperature and then the skins of the berries were collected. berry clusters were sprayed with 960 mm aba containing 0.05% (v/v) tween 20 and maintained in the dark at room temperature for 48 h. control samples (water) were sprayed with water containing 0.05% (v/v) tween 20 and placed beside the aba-treated samples. after treatment, the skins of the berries were collected. three biological replicates were assayed for each treatment. identification of full-size abcg transporter genes in the 12× version 1 of vitis vinifera genome full-size abcg transporters in grape were searched with blast (basic local alignment search tool) at ncbi (http://www.ncbi.nlm.nih.gov/) against the predicted protein sequence dataset of the 12× version 1 (v1) of cribi (http://genomes.cribi.unipd.it/grape/) using the nppdr1 protein sequence (cac40990) as a query. because the average full-size abcg protein comprises 1,400 amino acids (rea, 2007), only sequences comprising more than 400 amino acids were taken into account. these nomenclatures were represented according to çakır and kılıçkaya (2013). the sequences corresponding to fullsize abcg transporters confirmed that there was at least one pdr motif. molecular cloning of vvabcg44 the genome sequence corresponding to the partial cdna sequence of a grape full-size abcg transporter [tentative consensus sequencetc76318, the grape gene index database (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain. pl?gudb=grape)], induced by cyclodextrin (zamboni et al., 2009), was searched in the ncbi database (http://www.ncbi. nlm.nih.gov/) by blast. a genome sequence (accession number am449250.2), provided by the iasma research center (http://genomics.research.iasma.it/), was matched. the open reading frame (orf) of the gene was predicted by softberry (http://linux1.softberry.com/berry.phtml) and primers to amplify the entire orf were designed (table 1). total rna was extracted from the berry skin by hot borate method (wan and wilkins, 1994). the full-length cdna of vvabcg44 was amplified using the primescript high fidelity rt-pcr kit (takara, japan) according to the manufacturer’s instructions. three motifs (walker a, walker b, and abc signature) were confirmed according to van den brûle and smart (2002). tmd was predicted by phd (nps@) (rost and sander, 1993, 1994). sequence data of vvabcg44 have been deposited in ddbj under accession number ab910387. gene expression analysis total rna from grape tissues was extracted by the technique described above. total rna was reverse-transcribed using a primescript rt reagent kit with gdna eraser (perfect real-time) (takara) according to the manufacturer’s recommendations. transcript levels were determined by quantitative rtpcr using syber premix ex taq ii (perfect real-time) (takara) and thermal cycler dice real time system tp800 (takara) software ver. 3.00d. primers for vvabcg44, sts, and actin are shown in table 1. reaction conditions for thermal cycling were as follows: after enzyme 55 activation at 95°c for 10 s, amplification was performed in a two-step pcr with 40 cycles of 5 s at 95°c for denaturation and 30 s at 60°c for annealing/extension. transcript levels were calculated using a standard curve, and normalized against actin as described by reid et al. (2006). all reactions were performed in triplicate with three biological replicates. measurement of resveratrol content extraction of resveratrol (cas number 501-36-0) and its analysis using an lc-q-tof/ms system equipped with an esi interface (hplc: waters acquity uplc system; ms: waters xevo g2 q-tof , waters, germany) were performed according to tamura et al. (2014). identification, determination, and semi-quantification were compared with a 100 μm chemical reference standard. 10-camphorsulfonic acid was used as the internal control. three samples of biological replicates were divided into two aliquots and a total of six samples were analyzed for each treatment. 3. results zamboni et al. (2009) reported the partial sequence of a grape full-size abcg gene that was induced by cyclodextrin. to obtain the full-length cdna clone of the gene, we searched the genome sequence corresponding to the gene and successfully amplified a full-length cdna using primers designed from the genome sequence data. the gene was designated vvabcg44 or vvpdr14. vvabcg44 had a 4,350 bp coding region and was predicted to encode a protein of 1,450 amino acids (fig. 1a) with two tmds and two nbds (fig. 1b). a phylogenetic tree of plant full-size abcg transporters including vvabcg44 and all full-size abcg transporters in arabidopsis (fig. 2) shows that ntpdr1 (bad07483), nppdr1 (cac40990), and mtabcg10 (aes68070) are the closest homologues to vvabcg44. ntpdr1 (crouzet et al., 2013) and nppdr1 (jasiński et al., 2001) were reported to transport diterpenes including sclareol, whereas mtabcg10 (banasiak et al., 2013) transported isoflavonoids. a close homologue of vvabcg44, sptur2 (o24367) (van den brûle and smart, 2002), transported sclareol. other close homologues transport different compounds; atabcg40 (aaf71978) (kang et al., 2010) and papdr1 (jq292812) (kretzschmar et al., 2012) transport aba and strigolactone, respectively. the substrate range of vvabcg44 homologues is very broad, and it is not easy to identify the substrate of vvabcg44. to determine the tissues in which vvabcg44 is expressed, quantitative rt-pcr analyses were performed table 1 primers used in this study primer name purpose primer sequence(5’-3’) take2_forward cloning cac cat ggc gac ggc tga aat tta tar ag take2_reverse cloning tcg cct ttg gaa gtt caa tgc vvpdr14_exp_fw gene expression tag gag tgg ttg cag ctg tg vvpdr14_exp_rv gene expression ttt tgc tcc gtg tga ctt ctt vvsts_exp_fw gene expression ggg tca cta aga gcg agc ac vvsts_exp_rv gene expression gct cct caa gca ttt ctt cg vvact_fw gene expression tcc tgt gga caa tgg atg ga vvact_rv gene expression cttgca tcc ctc agc acc tt fig. 2 phylogenetic tree of all full-size abcgs in arabidopsis, vvabcg44, and characterized full-size abcgs from various plant species. nppdr1 and nppdr2 from nicotiana plumbaginifolia, ntpdr1 and ntpdr3 from tobacco, sptur2 from spirodella polyrhiza, osabcg36 and osabcg43 from rice, hvabcg31 from barley, papdr1 from petunia, lr34 from wheat, and gmpdr12 from soybean. the neighbor-joining tree was constructed with mega5 (tamura et al., 2011). 56 (fig. 3). the highest expression of vvabcg44 was observed in mature leaves, which was 9.6 times higher than that in young leaves. vvabcg44 expression in tendril and stem was higher than that in young leaves, but was not as high as that in mature leaves. vvabcg44 expression was relatively low in the grape berryvvabcg44, where it was highest in the skin and lowest in seeds. we also determined the gene expression of stilbene synthase (sts), a key enzyme in resveratrol synthesis. the expression pattern of sts in various grape organs is similar to that of vvabcg44 (fig. 3), suggesting a relationship between vvabcg44 and resveratrol synthesis. a fig. 1 nucleotide sequence, amino acid sequence, and topology of vvabcg44 a: nucleotide sequence of vvabcg44 and the deduced amino acid sequence. walker a motifs are underlined. abc signature motifs are boxed. walker b motifs are double underlined. transmembrane domains are dotted-lined. arrows indicate the primers for quantitative pcr analysis. b: putative topology of vvabcg44. the protein is composed of two halves and each half harbors tmd (gray boxes) and nbd (dashed lines), which contains an abc signature and walker a and b motifs. b 57 later, we determined the induction of vvabcg44 in grape berry skin by uv irradiation and by aba treatment. expression of vvabcg44 was upregulated 2.7 times by uv irradiation, and the sts gene was strongly induced by uv irradiation. furthermore, resveratrol content in the grape berry skin increased 159 times after 23 h of incubation following uv irradiation (fig. 4). these results suggest a relationship between vvabcg44 and resveratrol accumulation. on the other hand, the expression of vvabcg44 was not induced by aba treatment in the grape berry skin (fig. 5). 4. discussion and conclusions there are few reports of grape abc transporters, although comprehensive analyses, such as transcriptomics and proteomics, report the expression of abc transporters in grape. recently, çakır and kılıçkaya (2013) identified all abc proteins using whole genome sequencing with 12× coverage and francisco et al. (2013) identified an abcc transporter of grape as a vacuolar anthocyanin fig. 3 gene expression of sts andvvabcg44 in various grape organs [yl ( young leaves), ml; (mature leaves), stem, tendrils, seeds, pulp, and skin]. mrna levels of sts and vvabcg44 were detected by quantitative pcr. actin was used as an internal control. each value represents mean ± se of three independent experiments. fig. 4 expression of sts,vvabcg44 and resveratrol content in the grape berry skin after uv irradiation. mrna levels of sts and vvabcg44 were detected by quantitative pcr. actin was used as an internal control. each value represents mean ± se of three independent experiments. resveratrol content was assayed by lc-esi-q-tof/ms system in negative ion mode. each value represents mean ± se of six independent measurements. fig. 5 expression of vvabcg44 in the grape berry skin after aba treatment. mrna level of vvabcg44 was detected by quantitative pcr. actin was used as an internal control. each value represents mean ± se of three independent experiments. 58 transporter. to the best of our knowledge, this is the only characterized full-size abc transporter in grape. why have such few full-size abc transporters been studied? this is because of the difficulty in cloning fulllength cdna encoding full-size abc transporters, particularly full-size abcg transporters. one of the reasons for this difficulty is the very large size (ca. 4,000 bp) of full-size abcg transporter cdnas. another reason is the frequently observed low growth rate of escherichia coli harboring full-size abcg transporter cdna. the reason for this low growth rate of e. coli is unclear. therefore, few or no full-length cdna clones encoding full-size abcg transporters are found in public cdna databases or resources, and should be cloned. although 15 full-size abcg transporters are present in arabidopsis (van den brûle and smart, 2002), only five of them, atabcg40, atabcg37, atabcg36, atabcg32, and atabcg30, have been characterized (campbell et al., 2003; lee et al., 2005; ito and gray, 2006; kobae et al., 2006; stein et al., 2006; kim et al., 2007; badri et al., 2009; strader and bartel, 2009; kang et al., 2010; kim et al., 2010; růz̆ic̆ka et al., 2010; bessire et al., 2011; underwood and somerville, 2013; xin et al., 2013). in other plant species, only two full-size abcg transporters (osabcg36, osabcg43) in rice (moons, 2003; oda et al., 2011) and five full-size abcg transporters (nppdr1, nppdr2, ntpdr1, ntpdr3, and abcg5/pdr5) in tobacco family plants have been studied (jasinski et al., 2001; sasabe et al., 2002; schenke et al., 2003; ducos et al., 2005; stukkens et al., 2005; trombik et al., 2008; bultreys et al., 2009; navarre et al., 2011; bienert et al., 2012; seo et al., 2012; crouzet et al., 2013). in this study, we successfully cloned the full-length cdna of vvabcg44 using the primers designed from the grape genome sequence data, using a high-grade enzyme for pcr reactions and optimized e. coli culture conditions (culture at lower temperature and in higher volume). this appears to be the first report of a grape full-size abcg transporter. two different data sets of grape genome sequences have been disclosed to the public. first, the pinot noir clone entav115 was released by an italian group, iasma research center (http://genomics.research.iasma.it/) (velasco et al., 2007). we used this information for cdna cloning of vvabcg44. second, the pinot noir-derived inbred pn40024 was sequenced by the french-italian public consortium (jaillon et al., 2007) (http://www.genoscope.cns. fr/externe/genomebrowser/vitis/). the latter data set was updated from 8× to 12× and is now widely used. recently the 12× version1(v1) has been made available by an italian group, cribi (grimplet et al., 2012) (http://genomes.cribi. unipd.it/grape/). therefore, we used v1 to find all full-size abcg transporters in the grape genome (table 2). fifteen and 23 full-size abcg transporters were found in arabidopsis (van den brûle and smart, 2002) and rice (moons, 2008), respectively. in the grape genome data, we found 34 full-size abcg transporters (table 2, fig. 6). this number is much larger than that in arabidopsis and rice, suggesting a diversity of roles of full-size abcg transporters in grape. as mentioned above, substrate and subcellular localization of full-size abcg transporter cannot be determined from sequence similarity. however, full-size abcg transporters are responsible for transport of secondary metabolites, plant hormones, cutins, and heavy metals (fig. 2) and should have an important role in grape berry. recent reports showed that plant full-size abcg transporters, transport plant hormones or their precursors, such fig. 6 phylogenetic tree of all full-size abcgs in arabidopsis and vvabcg44. a neighbor-joining tree was constructed with mega5 (tamura et al., 2011). 59 ta bl e 2 fu llsi ze a b c g t ra ns po rt er s in g ra pe ( v iti s vi ni fe ra ). c ol um ns c on ta in t he v it is v in if er a 12 × v 1 id , c hr om os om e lo ca tio n, p ro te in l en gt h, p d r s ig na tu re s, a nn ot at ed d es cr ip tio n by t ai r1 0, pr ot ei n ac ro ny m ( n am e) a nd v it is v in if er a 12 × v 0 id f or e ac h ge ne a re g iv en 12 x v 1 id c hr om os om e lo ca tio n pr ot ei n pd r si gn at ur es ** d es cr ip tio n of t ai r1 0 sa nc he zh g n c ** * 1 2x v 0 id c hr st ra nd st ar t en d ll lg pp g ld ss t g ld a r a a g i c od e sh or t d es cr ip tio n su bf am ily n am e su bf am ily n am e v it _1 1s 00 16 g0 45 40 11 + 38 25 50 6 38 37 07 9 14 22 + + + at 2g 26 91 0. 1 pl ei ot ro pi c dr ug re si st an ce 4 v vp d r 1 v va b c g 31 g sv iv t0 10 15 45 60 01 v it _1 1s 00 16 g0 45 90 11 38 91 36 7 38 98 72 7 14 78 + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 2 v va b c g 32 g sv iv t0 10 15 46 10 01 v it _0 9s 00 02 g0 35 50 9 + 32 29 01 2 32 42 58 2 64 9 + + at 1g 15 21 0. 1 pl ei ot ro pi c dr ug re si st an ce 7 v vp d r 3 v va b c g 33 g sv iv t0 10 16 99 10 01 v it _0 9s 00 02 g0 35 60 9 + 32 42 58 3 32 44 57 4 42 7 + at 3g 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 v vp d r 4 v va b c g 34 g sv iv t0 10 16 99 20 01 v it _0 9s 00 02 g0 35 80 9 + 32 46 54 4 32 52 73 4 69 1 + + at 3g 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 v vp d r 5 v va b c g 35 g sv iv t0 10 16 99 30 01 v it _0 9s 00 02 g0 36 30 9 33 18 73 2 33 27 35 4 14 11 + + + at 1g 59 87 0. 1 a b c -2 a nd p la nt p d r a b c -ty pe v vp d r 6 v va b c g 36 g sv iv t0 10 16 99 80 01 v it _0 9s 00 02 g0 36 40 9 33 28 21 2 33 36 62 6 14 94 + + + at 3g 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 v vp d r 7 v va b c g 37 g sv iv t0 10 16 99 90 01 v it _0 9s 00 02 g0 53 60 9 50 99 14 6 51 14 84 9 14 90 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 8 v va b c g 38 g sv iv t0 10 17 18 40 01 v it _0 9s 00 02 g0 53 70 9 51 15 50 5 51 22 76 0 14 22 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 9 v va b c g 39 g sv iv t0 10 17 18 50 01 v it _0 9s 00 02 g0 54 00 9 51 46 16 7 51 60 09 0 15 65 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 10 v va b c g 40 g sv iv t0 10 17 18 70 01 v it _0 9s 00 02 g0 54 10 9 51 69 12 5 51 76 18 9 14 38 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 11 v va b c g 41 g sv iv t0 10 17 18 80 01 v it _0 9s 00 02 g0 54 90 9 52 16 53 6 52 23 50 7 12 80 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 12 v va b c g 42 g sv iv t0 10 17 19 60 01 v it _0 9s 00 02 g0 55 30 9 52 59 17 5 52 66 31 4 14 60 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 13 v va b c g 43 g sv iv t0 10 17 19 80 01 v it _0 9s 00 02 g0 55 60 * 9 52 81 29 6 52 88 25 5 14 55 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 14 v va b c g 44 g sv iv t0 10 17 20 10 01 v it _0 9s 00 02 g0 55 70 9 52 94 43 7 53 01 67 7 14 55 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 15 v va b c g 45 g sv iv t0 10 17 20 20 01 v it _0 9s 00 02 g0 55 90 9 53 16 14 4 53 23 42 0 14 55 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 16 v va b c g 46 g sv iv t0 10 17 20 40 01 v it _0 9s 00 02 g0 56 00 9 53 36 09 0 53 43 69 9 14 51 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 16 v va b c g 46 g sv iv t0 10 17 20 40 01 v it _0 5s 00 20 g0 06 80 5 + 25 48 76 2 25 57 92 1 14 38 + + + at 3g 53 48 0. 1 pl ei ot ro pi c dr ug re si st an ce 9 v vp d r 17 v va b c g 47 g sv iv t0 10 17 67 60 01 v it _0 6s 00 04 g0 65 60 6 + 72 84 90 1 72 97 72 4 12 74 + + + at 2g 29 94 0. 1 pl ei ot ro pi c dr ug re si st an ce 3 v vp d r 18 v va b c g 48 g sv iv t0 10 24 74 30 01 v it _1 4s 00 60 g0 04 70 14 + 43 97 01 44 86 96 14 49 + + at 3g 53 48 0. 1 pl ei ot ro pi c dr ug re si st an ce 9 v vp d r 19 v va b c g 49 g sv iv t0 10 31 31 40 01 v it _0 6s 00 61 g0 14 90 6 1, 9e +0 7 1, 9e +0 7 14 55 + + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 20 v va b c g 50 g sv iv t0 10 31 37 70 01 v it _0 6s 00 61 g0 14 80 6 1, 9e +0 7 1, 9e +0 7 14 61 + + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 21 v va b c g 51 g sv iv t0 10 31 37 80 01 v it _0 6s 00 61 g0 14 70 6 1, 9e +0 7 1, 9e +0 7 11 23 + + + at 1g 66 95 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 1 v vp d r 22 v va b c g 52 g sv iv t0 10 31 38 00 01 v it _0 8s 00 07 g0 37 10 8 + 1, 8e +0 7 1, 8e +0 7 14 52 + + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 23 v va b c g 53 g sv iv t0 10 33 80 40 01 v it _1 3s 00 74 g0 06 60 13 88 18 11 3 88 27 87 4 14 73 + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 24 v va b c g 54 g sv iv t0 10 34 74 10 01 v it _1 3s 00 74 g0 06 80 13 88 59 78 6 88 67 04 7 14 77 + + at 1g 66 95 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 1 v vp d r 25 v va b c g 55 g sv iv t0 10 34 74 50 01 v it _1 3s 00 74 g0 06 90 13 88 76 00 0 88 83 07 8 13 79 + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 26 v va b c g 56 g sv iv t0 10 34 74 60 01 v it _1 3s 00 74 g0 07 00 13 88 97 68 8 89 04 96 5 14 81 + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 27 v va b c g 57 g sv iv t0 10 34 74 80 01 v it _0 4s 00 08 g0 42 30 4 35 96 68 3 36 05 45 2 14 22 + + at 2g 26 91 0. 1 pl ei ot ro pi c dr ug re si st an ce 4 v vp d r 28 v va b c g 58 g sv iv t0 10 35 71 50 01 v it _0 4s 00 08 g0 47 90 4 42 27 01 7 42 34 51 8 14 37 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 29 v va b c g 59 g sv iv t0 10 35 78 00 01 v it _0 4s 00 08 g0 48 20 4 + 42 58 54 1 42 65 24 1 14 20 + + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 30 v va b c g 60 g sv iv t0 10 35 78 40 01 v it _0 4s 00 08 g0 48 30 4 + 42 82 42 5 42 86 09 4 76 4 + + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 31 v va b c g 61 g sv iv t0 10 35 78 50 01 v it _0 4s 00 08 g0 48 40 4 + 42 86 95 4 42 95 63 1 11 20 + at 1g 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 v vp d r 32 v va b c g 62 g sv iv t0 10 35 78 60 01 v it _0 6s 00 80 g0 00 40 6 + 2e +0 7 2e +0 7 15 07 + + at 2g 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 v vp d r 33 v va b c g 63 g sv iv t0 10 36 18 40 01 * v it _0 9s 00 02 g0 55 60 s ho w n w ith b ol d le tte rs is c or re sp on de d to v va b c g 44 . ** pd r s ig na tu re s w er e re po rt ed b y va n de n b rû le a nd s m ar t ( 20 02 ). ** * s an ch ez -f er na nd ez a nd h g n c s ub fa m ily n am es w er e re po rt ed b y ç ak ır a nd k ılı çk ay a (2 01 3) . 60 as aba (kang et al., 2010), strigolactone (kretzschmar et al., 2012), and auxin (ruzicka et al., 2010). one of the closest homologues of vvabcg44, atabcg40, was reported to transport aba (kang et al., 2010) (fig. 2). in grape berry, aba accumulates just before maturation, called “veraison”, and aba works as a trigger of berry maturation (coombe and hale, 1973; davies et al., 1997). after veraison, both sugar and anthocyanin accumulate considerably in the grape berry (coombe, 1992; davies et al., 1997; deluc et al., 2007). in this study, we determined vvabcg44 induction by aba in the berry skin before veraison. however, no induction was observed (fig. 5). many plant full-size abcg transporters have been suggested to be associated with biotic and abiotic stress resistance, particularly resistance against pathogens, and some have been observed to transport secondary metabolites that function as phytoalexins (fig. 2). therefore, vvabcg44 is considered to be associated with biotic and abiotic stress resistance and transports phytoalexins. vvabcg44 was first found as an elicitor-induced gene in grape culture cells and the induction of vvabcg44 corresponded to resveratrol accumulation in the cells (zamboni et al., 2009). it is known that uv irradiation induces resveratrol accumulation in the grape berry skin (douillet-breuil et al., 1999; adrian et al., 2000; versari et al., 2001; takayanagi et al., 2004). therefore, we determined the effect of uv irradiation on vvabcg44 expression together with sts expression, a key enzyme for resveratrol synthesis and resveratrol accumulation. a clear induction of vvabcg44 by uv irradiation, though not large compared with that of sts expression and resveratrol accumulation, vvabcg44 was observed (fig. 4). a similar pattern in the gene expression of vvabcg44 and sts in various grape organs was also observed (fig. 3). these results suggest a relationship between vvabcg44 and resveratrol accumulation. close homologues of vvabcg44, ntpdr1 (crouzet et al., 2013), nppdr1 (jasiński et al., 2001), mtabcg10 (banasiak et al., 2013), sptur2 (van den brûle and smart, 2002), atabcg40 (kang et al., 2010), and papdr1 (kretzschmar et al., 2012), transport diterpenoids, isoflavonoids, aba, and strigolactones (fig. 2). these functions are surprising because their molecular structures are completely different. resveratrol is a compound belonging to the stilbenoids and both stilbenoids and flavonoids belong to the phenylpropanoids. the closest homologue of vvabcg44, mtabcg10, transports isoflavonoids (banasiak et al., 2013). although no direct evidence of stilbenoid transport activity of full-size abcg transporter has been reported, it was observed that b. cinerea, lacking a full-size abcg transporter, bcatrb, was more sensitive to resveratrol than the wild-type strain (schoonbeek et al., 2001). this result suggests that bcatrb is an exporter of resveratrol in b. cinerea. it can be concluded that vvabcg44 may work as a resveratrol transporter in grape. we attempted to express vvabcg44 in yeast lacking eight abc transporters (kang et al., 2010) and measure resveratrol transport activity. however, this attempt was unsuccessful because heterologous expression of plant full-size abcg transporters is difficult not only in e. coli but also in yeast. we identified 34 full-size abcg transporters in the grape genome, including vvabcg44. it is assumed they transport key compounds for plant growth and stress resistance, including secondary metabolites, plant hormones, cutins, and heavy metals, and have important roles in grape. further study on full-size abcg transporters in grape is warranted. acknowledgements we thank mr. hiroya saito and mr. chiharu uchikata at azumi apple corporation for supplying grape berries. we also thank mr. yutaka nishikawa and mr. kenji wada of mie prefecture agricultural research institute and dr. takafumi tezuka of nagoya university for advice on uv treatment of grape berries. we also thank mr. tetsuya mori at riken csrs for technical assistance. this work was supported by the programme for promotion of basic and applied researches for innovations in bio-oriented industry from bio-oriented technology research advancement institution (brain) and by grant-in-aids for scientific research from the japan 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helped: today it is at its best when it comes to pruning and harvesting. in this context, the volume by xavier rius and josé m. lacarte is timely and offers a wealth of information. the title itself suggests the quality of the content: research and experimentation that have radically transformed an obsolete management system into new hope for olive growing in the third millennium. the text is divided into four chapters: 1. el aceite de oliva en el mundo (olive oil in the world); 2. la oportunidad de negocio en las plantaciones de olivar superintensivo (the economic opportunity in the superintensive planting system olive groves). 3. el cultivo superintensivo del olivar: respuesta a las necesidades del sector (the superintensive planting system in olive groves: response to sector needs). 4. el cultivo superintensivo en los principales países oleícolas (españa, italia, grecia, portugal, túnez, marruecos, estados unidos, chile, australia, argentina, resto del mundo) (the superintensive planting system in the major olive-producing countries: spain, italy, greece, portugal, tunisia, morocco, usa, chile, australia, argentina, rest of the world). a wealth of tables, figures and color image aid consultation of the text and add value to its presentation. considering the global interest of the topic, translation of the text into english is desiderable and perhaps predictable in the near future. enrico rinaldelli il paesaggio mantovano. nelle tracce materiali, nelle lettere e nelle arti. iv. il paesaggio mantovano dall’età delle riforme all’unità (1700-1865). atti del convegno di studi (mantova, 19-20 maggio 2005)(the mantuan landscape. from material, literary and artistic traces. the mantuan landscape from the period of reform to unity (1700-1865). prooceedings from the study convenction in mantua, 1920 may 2005). camerlenghi e., v. rebonato, and s. tammaccaro (eds.). leo s. olschki, florence (italy), 2010. pp. x + 454 + 7 figures and 70 plates, 63 of which in color. isbn 978-88-222-5845-8. € 55.00. the proceedings, edited by e. camerlenghi, v. rebonato, and s. tammaccaro, of the study convenction held in mantua, 19-20 may 2005 have recently been published. they are the fourth volume of the work on the mantuan lanscape presented by accademia nazionale virgiliana di scienze, lettere, e arti and edit by leo s. olschki. the various contributions which make up the volume, and include images from the period, have been prepared by outstanding experts in the field of historical italian landscapes and offer stimulation for debate about the causes behind the development of the mantua area during the subject period. ample space is dedicated to hydraulic conditions, which were tied to the control of power of padanian principalities, agronomic aspects and the evolution of crops, as well as to literary sources, from folengo to renaissance novelists. all this, without overlooking the urban landscape, which in this historic period in mantua offered moments of great splendor. some of the chapters clearly highlight the changes which occurred in the landscape, above all with regard to the management of water resources, while others focus on the parks, gardens and urban green spaces of mantua during the period. the text, which includes a generous list of bibliographic references, also contains illustrations (both color and black and white) and color photos. this volume is a valuable addition, under a scientific and technical profile as well as from a historical and cultural point of view, to the vast literature that exists on the evolution of historical-landscape areas. the work thus offers the reader insight and knowledge about numerous and important aspects of the landscape which make up our image of the mantuan territory. francesco ferrini 46 l’architetto sapiente. giardino, teatro, città come schemi mnemonici tra il xvi e il xvii secolo. koji kuwakino. giardini e paesaggio, vol. 28. leo s. olschki, florence (italy), 2011. pp. xxiv-326 + 70 figures and 7 plates in colour. isbn 978-88-222-6046-8. € 29.00. in this work, by the japanese scholar koji kuwakino and published by leo s. olschki of florence, the little-known relationship between architecture, the art of memory and encyclopaedism of the first modern age is examined through examples of the fruitful interaction between words, images and space. the subtitle of the book underlines the investigated subject: garden, theater, and city as mnemonic schemes in the 16th and 17th centuries. this architectonic typology offers the schemes of a complex conceptual construction able to collect, order and internalize the enormous quantity of knowledge acquired and accumulated over the centuries, from the ancient world up to the contemporary age. the author analyzes the ideal gardens of agostino del riccio and giovan battista ferrari, the universal theater of samuel von quiccheberg and the mnemonic city of cosma rosselli. as the author points out in the introduction, it is “an ideal journey” aimed at analyzing “three architectonic typologies often utilized as mental spaces: the garden, the theater, and the city”. these spaces “are not so much concretely real, but rather are ideal plans created with words, and the authors are not professional architects but gifted men and virtuosi who understand architecture”. dr. kuwakino goes on to explain that these examples of spaces “set outside the specialized field of ‘ars aedificatoria’, present only in text and for the most part unknown to historians, reveal, as in filigree, aspects that were previously hidden or scarcely examined despite being of considerable interest and rich in suggestion”. the analysis presented in the book aims to offer a complementary reading and enrichment of the history of 16th century architecture. through this work the author succeeds in shining light on fundamental cognitive aspects of the architectonic culture of the first modernity which previously had not been sufficiently emphasized. “l’architetto sapiente” demonstrates clearly how much architecture is under the influence of the spirit of the times architectonic space become, for example, a metaphor for knowledge and how much architecture has contributed to the building of new structures of modern thought. after the author’s introduction, the text is dived into six chapters: “l’arte della memoria architettonica” (the art of architectonic memory); “il giardino del tardo cinquecento come luogo del pensiero” (the garden in the late 16th century as a place for thought); “la rappresentazione emblematica del sapere enciclopedico nel giardino mnemonico di del riccio” (emblematic representation of encyclopaedic knowledge in the mnemonic garden of del riccio); “il ‘paradiso celeste’ nel giardino italiano” (the ‘paradiso celeste’ in the italian garden); “il grande teatro del pensiero creativo” (the great theater of creative thought); and “città celeste e ‘loca communia’”, (the celestial city and the ‘loca communia’), followed by the conclusions. the text includes a ample list of bibliographic references and is enriched by various illustrations and photos (black and white and color) which highlight the presented material. this work is clearly a precious addition to the existing literature on landscape studies, from both scientific and historical-cultural points of view, and offers, page after page, new and significant knowledge to the reader. cinzia silori territori delle acque. esperienze e teorie in italia e in inghilterra nell’ottocento (territories of water: experiences and theories in italy and england in the 19th century). aquae studi e testi sulle terme, 4. gabriele corsani (ed.). leo s. olschki, florence (italy), 2010. pp. 164. isbn 978-88-222-5988-2. € 20.00. water, promethean and domestic, represents the magnificent destiny of progress as the most radical adherence to the spirit and laws of nature. certainly, it is not easy to reduce down to a single text the myriad aspects of water and its interaction between the earth and the living things which occupy our planet. for example, water as power and energy but also as nourishment for life and the human spirit. in this context, gabriele corsani has deftly succeeded in telling the story of water in italy and england over the course of the 19th century. the volume, which contains a wealth of bibliographic information and illustrations from the period, is a complete work from different points of view: an interesting historical analysis and a precious bibliographic source for historians and researchers. 47 the text, compiled by notable authors, includes the following ten contributions: gabriele corsani, territori delle acque: esperienze e teorie in italia e in inghilterra nell’ottocento (territories of water: experiences and theories in italy and england in the 19th century). antonello boatti, storici e scrittori lombardi dell’ottocento di fronte al paesaggio. corsi d’acqua naturali e artificiali tra agricoltura e industria negli scenari disegnati da carlo cattaneo e cesare cantù (lombard historians and writers in the 19th century in a landscape context. natural and artificial waterways in agriculture and industry in scenes drawn by carlo cattaneo and cesare cantù. carlo cattaneo, prospetto della navigazione interna delle province lombarde con alcune notizie sulla loro irrigazione (“il politecnico”, 1841) (prospectives of navigation within the province of lombardy and notes on irrigation) (“il politecnico”, 1841). cesare cantù, storia di milano, diocesi e provincia di milano, cap. v. acque (history of milan, diocese and province of milan, chapter v. water). marco geddes da filicaia, corrado tommasi-crudeli e il tema dell’acqua ai tempi del colera (corrado tommasi-crudeli and the topic of water during times of cholera). corrado tommasi-crudeli, la canalizzazione delle città (canalization of the city). katia caldari, alfred marshall e l’importanza delle risorse naturali (alfred marshall and the importance of natural resources). alfred marshall, l’acqua come un elemento della ricchezza nazionale (water as an element of national wealth). gabriele corsani, la appendix howardiana: tecnica e retorica comunitaria per le acque ludiche di città giardino (the howardian appendix: technique and community rhetoric for water in the city garden). ebenezer howard, appendice. l’approvvigionamento idrico (appendix. the provisioning of water). enrico rinaldelli filippo de pisis botanico flâneur, un giovane tra erbe, ville, poesia. ricostruita la collezione giovanile di erbe secche. roncarati paola and rossella marcucci. giardini e paesaggio, vol. 30. leo s. olschki, florence (italy), 2011. pp. 208. isbn 978-88-222-6139-7. € 28.00. this attractive volume by paola roncarati and rossella marcucci presents ample and detailed documentation about an interesting but rarely mentioned, multifaceted man: filippo de pisis. de pisis is identified principally as a notable painter of the early 20th century but he was also, from a young age, a talented writer and poet as well as a passionate entomologist and botanist. indeed, it is on these latter aspects of his career that the two authors, through a combination of their areas of expertise, have focused the current work, offering an important contribution to the complex and heterogeneous biography of the painter. the volume begins with an introduction by lucia tongiorni tomasi and luigi zangheri entitled “l’erbario essiccato, un inedito tributo alla personalità di filippo de pisis”, (the dried herbarium, a tribute to filippo de pisis), which is then followed by a carefully written preface by gianni venturi, who admirably comments on the meaning and significance of the work: “it is clear that this book is difficult to define as a study of an herbalist, just as every work of poetry includes a multitude of meanings which render it precious, not only for a specific interest, but above all as an important step toward understanding the life and works of the artist in greater depth.” the text is divided into two parts. the first is dedicated to “le implicazioni culturali di una passione botanica” (the cultural implications of a passion for botany) and the second to “luoghi di erborizzazione, escursioni della mente, passeggiate dello sguardo” (places for herbalism, excursions of the mind, and a stroll for the eyes). finally, there is an analytical comment by the authors on the estrosità nell’erbario: lo sguardo “asistematico” di un botanico originale”. (creativity in the herbarium: an asystematic outlook by an original botanist). the work also includes a series of documents, including an article by giuseppe viviani “a passeggio con de pisis” (a walk with de pisis) and a rich bibliography,. enrico rinaldelli impaginato 3 1. introduction strawberry (fragaria x annanassa duch.) is one of the most important berries produced in the world. in argentina, 33000 tons are produced, mainly cultivar camarosa (gómez riera et al., 2013). it is highly accepted by consumers firstly because of its physical aspect and organoleptic and nutritional properties (shin et al., 2008; garriga et al., 2015). nevertheless, its postharvest quality rapidly declines due to the soft texture, high metabolic activity and susceptibility to bacterial and fungal rots. usually, there is quality decay during transport and commercialization (dotto et al., 2011). cell wall degradation is one of the principal causes of strawberry´s postharvest quality decay. as pectine synthesis occurs along the fruit maturation, they are less firmly attached to the cell wall. additionally, middle lamella´s debilitation and solubilization during fruit ripening diminishes cell cohesion (lara et al., 2004). calcium plays a preferential role on permeability and cell integrity and has a direct influence on fruit firmness and storage time (fernández et al., 2006). calcium functions as an intracellular cement because it forms calcium-pectine complexes that give firmness to vegetable tissues. calcium´s presence also favours pectic material insolubilization and inhibits its degradation by polygalacturonase enzyme (alonso, 1995). an immersion in calcium at harvest could increase calcium´s content in strawberry, increasing fruit firmness (galetto et al., 2010) and thereby storage period. however, the key factor for quality maintenance is temperature optimum for strawberry is 0°c (mitcham et al., 2015). could an immersion in calcium allow an increase in storage temperature, maintaining fruit quality? other factors, such as growing system and substrate, can influence strawberry´s quality. greenhouse production improves organoleptic quality of fruits and vegetables because they are not exposed directly to air conditions as in open field (gruda, 2009). soilless production, moreover, diminishes incidence of diseases and pests (urresterazu, 2004). adv. hort. sci., 2017 31(1): 3-10 doi: 10.13128/ahs-20719 a calcium lactate treatment at harvest, growing system and refrigerated modified atmosphere can affect strawberry’s ´camarosa´ postharvest quality? m. harris (*), m.c. llorens, d. frezza universidad de buenos aires, facultad de agronomía, departamento de producción vegetal, cátedra de horticultura, avenida san martin 4453 (1417) ciudad autónoma de buenos aires, argentina. key words: nutritional quality, organoleptic quality, perlite, postharvest behaviour, soil, storage temperature. abstract: the aim of this work was to evaluate the effect of a calcium lactate treatment on postharvest behaviour, organoleptic and nutritional quality of strawberries (fragaria x ananassa duch., cv. camarosa) grown in different growing systems and stored in refrigerated modified atmosphere. strawberry grown in perlite and soil in greenhouse and soil in open field was harvested and dipped in a calcium lactate 1% solution. fruits were packed in modified atmosphere at 1°c and 8°c. at 1, 3 and 7 days of storage postharvest behaviour, organoleptic and nutritional quality was evaluated. calcium had a positive effect on fruit firmness and no differences were observed between storage temperatures in calcium treated fruits. organoleptic quality (except visual quality) was better in fruits grown in open field soil, regardless calcium treatment and storage temperature. nutritional quality was better in untreated fruits and stored at 1°c. (*) corresponding author: mharris@agro.uba.ar received for publication 6 june 2016 accepted for publication 12 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 3-10 4 the aim of this work was to evaluate the effect of a calcium lactate treatment on postharvest behaviour, organoleptic and nutritional quality of strawberries grown in three growing systems and stored at two temperatures in modified atmosphere during 7 days of storage. 2. materials and methods plant material and growth conditions the experiment was conducted in the horticulture experimental field, school of agriculture, university of buenos aires (latitude 45° s, longitude 58° 31´ w, altitude 26 m asl). strawberry seedlings (commercial variety ‘camarosa’) were planted in three growing systems: soil in open field, soil and perlite in greenhouse. plant density was 7 plants m-2. soil treatments were covered with a black plastic mulching. all treatments were fertirrigated: nutrient solutions were formulated according to strawberry´s requirements (table 1). hourly intervals of temperature (°c), relative humidity (%) and radiation (w·m-2) were measured in open field and greenhouse with datalogger hobo. harvest mature strawberry fruits (at least 75% red colour) were harvested and immediately submerged in cold water to decrease fruit temperature. half of the fruits of each growing system were treated with a calcium lactate 1% solution during one minute. although calcium chloride is most commonly used, calcium lactate was used as a firming agent (codex alimentarius world health organization, 2015) because chloride can give a bitter taste to fruits (oms-oliu et al., 2010). sixty-five grams (g) of fruit were packed in a modified atmosphere (medium density polyethylene semi rigid container). modified atmosphere increases postharvest life in fruits and vegetables by reducing respiratory rate (sandhya, 2010). containers were stored at storage chambers at 1°c and 8°c. postharvest at 1, 3 and 7 days of storage, quality characteristics of fruits were evaluated as follows: postharvest behaviour oxygen and carbon dioxide concentration in the container was measured with dansensor gas analyzer and expressed as percentage. fresh weight loss. fruits were weighted and weight loss was expressed as percentage relative to the initial value. organoleptic quality visual quality. the overall visual and sanitary quality was determined by scoring each strawberry using a 1-10 hedonic scale, being 10 excellent and 6 the commercialization limit. colour was measured with minolta chromameter cr300. l, a, b, c, h parameters were determined in four spots in equatorial zone of three fruits per treatment. firmness was measured in equatorial zone with ludwig penetrometer fitted with a 3 mm diameter round probe. total soluble solids were determined with atago refractometer and expressed as °brix. nutritional quality ascorbic and dehidroascorbic acid was determined by liquid chromatography and expressed as mg of ascorbic acid 100 g-1 of fresh weight. antioxidant capacity. antioxidant capacity was estimated by determining the free-radical scavenging capacity evaluated with the stable radical dpph (adapted from brand williams et al., 1995 and leong and shui, 2002). two g of edible portion of the fruit was homogenized using a blender and inserted into a 50 ml centrifuge tube. twenty ml of 50% aqueous ethanol was added (1:10 w/v) and mixed in a vortex mixer for 15-30 seconds. the extract was centrifuged at 2000 g for 5 min al 4°c. the supernatant was filtered before using. a 25 mg solution of dpph (1,1 diphenyl-2-picrylhydrazyl) was prepared in methanol. for calibration curve, aliquots of ascorbic acid (0, 25, 50, 75, and 100 μl) solved in aqueous ethanol 50% (0.1 ml ml-1) were placed in tubes with in 3 ml dpph. absorbance at 517 was measured at 1, 10, 30, 60, 90 and 120 minutes. an aliquot of 50 ml of an antioxidant/fruit extract solution was added to 3 ml of the dpph solution. the decrease in absorbance at 517 macroelements microelements element concentration (mg l-1) element concentration (mg l-1) nitrogen 64 iron 2.8 phosphorus 31 sodium 1.2 potassium 200 manganese 0.5 sulfur 64 boron 0.5 magnesium 48 copper 0.02 -zinc 0.05 molybdenum 0.01 table 1 nutrient solution formulation harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 5 nm was measured at 0, 1, 5 and then every 10 minutes until the reaction reached a plateau. the decreased absorbance of dpph remaining at the steady-state was calculated and expressed as mg of ascorbic acid (aa) equivalents per 100 g of homogenate (aeac). the aeac was calculated using the following equation: aeac=δ a x f x v x 100 x 1/w where δ a is the change of absorbance after addition of fruit extract, f is the inverse of the calibration curve slope, v is the volume of filtrate (ml) and w is the weight of homogenate used for extraction (g). statistical analysis a completely randomized factorial design with 3 replicates per treatment was used. the results were analyzed by multivariate analysis of variance repeated in time with a 5% significance level. tukey test to compare means was used (kuehl, 2001). infostat software was used (di rienzo et al., 2015). 3. results and discussion oxygen and carbon dioxide concentration in the container temperature, as expected, generated the most significant differences: strawberries stored at 1°c respired less than those stored at 8°c (fig. 1). nevertheless, in all cases, at equal temperatures, strawberries treated with calcium decreased the respiration. on the other hand, an interaction between growing systems and calcium treatment was observed (p<0,0001) both in oxygen and carbon dioxide levels: strawberries grown in soil systems (both open field and greenhouse) and without calcium treatment showed larger differences between storage temperatures: those stored at 8°c showed a high oxygen decrease and carbon dioxide increase, especially at the 7th day of storage. treated fruits, instead, showed similar behaviour during the seven days of storage. thus, calcium treatment could be considered as a regulator because differences between temperatures in treated strawberries were smaller. waghmare and annapure (2013) observed that a combination of modified atmosphere, calcium chloride and nitric acid treatment in chopped papayas stored at 5°c had a significant decrease of oxygen and increase of carbon dioxide compared to fruits only stored in modified atmosphere. equilibrium modified atmosphere was not established, especially those stored at low temperatures. this could be because medium density polyethylene was used and this material has low permeability to gases: 2600 cm3/m2.d.atm for oxygen and 7600 cm3/m2.d.atm for carbon dioxide (sandhya, 2010). additionally, as temperature increases, material permeability increases as well. thus, containers stored at 8°c had a higher permeability (oliveira et al., 2015). fig. 1 oxygen and carbon dioxide concentration (%) in containers at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. adv. hort. sci., 2017 31(1): 3-10 6 fresh weight loss maximum allowable weight loss in strawberry is 6% (laurin et al., 2003). all treatments, except one case, were below those values (fig. 2). the smaller weight loss was observed in open field fruits (p<0.0001), independently storage temperature and calcium treatment. preharvest temperatures can affect postharvest shelf life. for example, fruits grown at high temperatures can exhibit water soaking (benkeblia et al., 2011). in this work, differences between greenhouse and open field temperatures reached 7°c. fruits harvested in greenhouse soil and soilless systems had surely higher temperature, what determined higher weight loss in those systems. visual quality fruits grown in perlite had better quality: 7.7% more than open field soil and 20% compared to greenhouse soil fruits (fig. 3). at the 7th day of postharvest, all the strawberries stored at 1°c prefig. 2 fresh weight loss (%) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. maximum allowable weight loss (6%) is indicated. fig. 3 visual quality at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. each graph represents a combination of growing system and storage temperature. commercialization limit (6) is indicated. harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 7 sented visual and sanitary quality above commercialization level. fruits stored at 8°c, instead, were below that level and in most cases they had botrytis cinerea symptoms. temperature management is a key factor to minimize postharvest deterioration in strawberry. at high storage temperatures, fruits have higher respiration rates, and consequently, shorter postharvest shelf life (shin et al., 2008). colour significant differences were not observed for all colour parameters. shin et al. (2008) did not find colour changes during 7 days of storage of l c and h° values. firmness an interaction was observed between growing system and calcium treatment (p=0,0203): treated strawberries grown in soil systems, both open field and greenhouse had 20% more firmness than others. in perlite, difference between treated and untreated fruit was not significant (fig. 4). firmness increased in fruits stored at 1°c along the storage time. shin et al. (2007) also observed an increase in firmness along 4 days of storage at high (10.5°c ) and low (0.5°c) temperatures. the same authors investigated firmness during 12 days of storage and observed a positive tendency in fruits stored at 3°c and a decrease in those stored at 10°c (shin et al., 2008). firmness increase in low storage temperatures is due to physical changes in cell wall: cold produces an increase in pectin viscosity, which impacts positively in fruit firmness (lara et al., 2004). total soluble solids total soluble solid content (table 2) in all cases was above the minimum content recommended (7°brix) for the postharvest quality maintenance (mitcham et al., 2015). a significant interaction was found between temperature and growing system (p<0,0001): fruits stored at 8°c and grown in open table 2 total soluble solids (°brix) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures growing system storage 1°c storage 8°c 1 day 3 days 7 days 1 day 3 days 7 days ca with ca without ca with ca without ca with ca without ca with ca without ca with ca without ca with ca without soil open field 8 a bc 10 a d 7.63 ab ab 7.83 ab bc 8 a bc 6.33 a a 8.50 a bc 7.17 a a 9.17 ab cd 12 a e 8 a ab 9 ab cd soil greenhouse 8 a bc 7.33 b a 7.75 ab ab 8.67 bc cd 8.17 ab bc 9.33 ab de 8.33 a cd 8.17 bc bc 8.33 b cd 7.33 bc ab 6 b a 8.83 b e perlite greenhouse 8 a a 9.5 a bc 8 a a 9.17 cd ab 8 a a 8.83 bc ab 9 ab cd 6.5 ab a 8 bc ab 8.33 cd bc 10.5 c de 7.67 bc ab different capital letters indicate differences between row and different small letters indicate differences between columns (p<0.05). fig. 4 firmness (kpa) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. adv. hort. sci., 2017 31(1): 3-10 8 field had higher content of soluble solids. it was only in the open field system that a difference between storage temperatures was found: those stored at 8°c had a 12.5% higher content of total soluble solids. calcium lactate immersion affected negatively the total soluble solid content. similar results were found by other authors with different calcium sources. singh et al. (2007) observed that weekly foliar applications of calcium chloride since flowering decreased total soluble solid content in 10% at harvest. dunn and able (2006), as well, found that a calcium deficiency during growth stage increased significantly soluble solids content in fruits. ascorbic acid content in almost all cases, ascorbic acid content (fig. 5) decreased during storage period. phillips et al. (2016) also observed a decrease in strawberries stored at 1.5°c during 7 days. only the fruits stored at freezers (-10 to -20°c) and ultra freezers (-55°c) maintained ascorbic acid contents similar to those observed at harvest time. low temperature storage is a key factor to maintain ascorbic acid content during postharvest (lee and kader, 2000). an interaction was observed between calcium treatment and storage temperature (p=0.0026). untreated fruits stored at 1°c had 26% more ascorbic acid compared to the others. many authors recognize that a calcium treatment at harvest enhances ascorbic acid content in fruit mainly due to an increase in fruit firmness (aghdam et al., 2013). nevertheless, other authors did not find differences between calcium treatment and the control (shaffie et al., 2010). regardless the calcium treatment, temperature was the key factor to maintain ascorbic acid content in strawberries. fruits grown in perlite had a 49% higher content compared to those grown at soil (both open field and greenhouse). these results are in agreement with de data reported by treftz and omaye (2015), who observed that soilless grown strawberries had a 74% higher content of ascorbic acid compared to soil grown fruits. for other fruits, the results were contradictory: isabelle et al. (2010) observed higher ascorbic acid content in pepper and özcelik and akilli (1999) in tomato. however, gruda (2005) didn´t find differences in strawberry. antioxidant capacity several interactions were observed for antioxidant capacity. untreated and greenhouse soil grown fruits presented 13% higher antioxidant capacity (p=0,0003) (table 3). wang and zheng (2001) observed that strawberries grown under higher temperature (day and night) had higher antioxidant capacity, as it was observed in this work, where differences of temperatures between greenhouse and fig. 5 ascorbic acid content (mg 100 g-1) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 9 open field reached 7°c. as well, untreated fruits stored at 1°c had 10% higher antioxidant capacity (p=0.0295). many authors, as described in 3.7, explain that a calcium treatment enhances ascorbic acid content (and consequently antioxidant capacity) due to an increase in cell wall firmness. nevertheless, other authors express that a disruption in cell wall composition increases antioxidant capacity, but this increase is different depending the product (reyes et al., 2006). with respect to storage temperature, shin et al. (2008) found significant differences since the 12th day. different capital letters indicate differences between columns and different small letters indicate differences between rows (p< 0.05). yield in the growing systems although yield was not an objective of this work, we observed that the perlite system had 35% and 17% higher yield than soil in greenhouse and soil in open field systems, respectively. 4. conclusions strawberry is highly accepted by consumers but its postharvest quality rapidly declines. a calcium lactate treatment can increase principally strawberry’s firmness. this, in addition to storage temperature and growing system can increase postharvest shelf life of fruits. in this research, the calcium lactate treatment had a positive effect on fruit firmness, especially in those grown in open field and greenhouse soil. furthermore, treated fruits had less respiration during storage time and there was no significant difference between storage temperatures in fruits treated with calcium lactate. equilibrium modified atmosphere was not established. the other variables were not affected or had a negative response to calcium lactate treatment. weight loss and organoleptic quality (except visual quality) were better in fruits grown in open field soil, regardless calcium treatment and storage temperature. on the other hand, nutritional quality was better in untreated fruits and stored at 1°c. in conclusion, even though temperature is substantial to maintain fruit quality at postharvest, a calcium lactate treatment could be useful to improve strawberry (cv. camarosa) quality during transport and commercialization and decrease incidence of postharvest diseases. acknowledgements the present work was funded by grants from buenos aires university references aghdam m.s., dokhanieh a.y., hassanpour h., rezapour fard j., 2013 enhancement of antioxidant capacity of cornelian cherry (cornus mas) fruit by postharvest calcium treatment. sci. hortic., 161: 160164. alonso j., rodriguez t., canet w., 1995 effect of calcium pretreatments on the texture of frozen cherries. role of pectinesterase in the changes in the pectic materials. j. agric. food chem., 43: 1011-1016. benkeblia n., tennant d.p.f., jawandha s.k., gill p.s., 2011 preharvest and harvest factors influencing the postharvest quality of tropical and subtropical fruits, pp. 112-142. in: yahia e. 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at a time when the study of crops is increasingly refers to the soil characteristics in which they grow, the text by giacomo certini and fiorenzo cesare ugolini is unanimous and enthusiastically welcomed by the scientific world, and agricultural world in general. knowing soil texture, its constitution and origin is the first step to set up and rationally manage each crop. furthermore, in the context of fruit tree species, the optimal balance between vegetation and production depends largely on the relationships between rootstock and soil and, therefore, between plant and peculiar soil characteristics which for many years will host the root system. the work of the aforementioned authors is, therefore, a long-awaited, scientific and technical reference for those who want to deepen their knowledge in the “horizon” of soil. the text is well articulated and the essential themes, accurately and deeply treated, reflect the rigorous expertise of the authors in the field. the volume includes the following eight chapters. 1. che cos’è il suolo, a cosa serve e come si forma (what is soil, what is it for how does it forms); 2. le fasi del suolo (soil phases); 3. il profilo del suolo e la sua descrizione (the soil profile and its description); 4. le forme di humus (the forms of humus); 5. i fattori della formazione del suolo (factors of soil formation); 6. i processi pedogenetici (pedogenic processes); 7. la soil taxonomy. gli orizzonti diagnostici e i regimi pedoclimatici (soil taxonomy. diagnostic horizons and soil and climate regimes); 8. i dodici ordini della soil taxonomy (the twelve orders of soil taxonomy). the volume is completed by an ample and rich list of references and a useful analytic index. furthermore, the wide range of tables, figures in black and white and color make it easy to read and support the display of topics. the text is an indispensable tool for students, to whom is addressed, but there is no doubt about the value it can bring to those working in agriculture, either as scholars or direct operators. cinzia silori oleum. manuale dell’olio da olive. ricci a. (ed.) edagricole, edizioni agricole il sole 24 ore, bologna, 2011. pp. 320. isbn 978-88-506-5276-1. € 45.00. with the release of “oleum”, this interesting and qualified volume adds to those concerning the complex and brood agro-industries topic. the text, written in the specific context of the processing of olives into oil, has makes use of the collaboration, synergistic and additive, of the most talented scholars in the field. the result obtained by the editor, antonio ricci, is that of a harmonic fusion of scientific knowledge that, right from the first chapter, gives the reader a broad and updated view of knowledge and acquisitions. each chapter, cleverly arranged in a crescendo of technical and scientific horizons, deals with the specific issues by providing, in addition to an up-to-date view of the topic, also a large repertoire of bibliographic references. the topics are organized as follows. 1. la chimica dell’olio da olive (chemistry of olive oil) (g. lercker); 2. normativa sui requisiti chimici, fisici e organolettici degli oli di oliva (regulatory requirements on chemical, physical and organoleptic characteristics of olive oil) (l. conte); 3. i contaminanti e la filiera produttiva degli oli di oliva (contaminants and the olive oil production process) (l. conte, s. moret, g. purcaro, t. populin, a. ermacora, and m. marega); 4. caratteristiche chimico-fisiche dell’oliva e dell’olio con riferimenti alle cultivar più diffuse (physical and chemical characteristics of olive and oil with reference to the most cultivated cultivars) (g. panelli); 5. l’olivo e l’olio nell’emisfero sud (olive and oil in the southern hemisphere) (g. panelli); 6. caratterizzazione molecolare delle varietà di olive e degli oli di oliva (molecular characterization of olive varieties and of olive oils (l. baldoni, r. mariotti, and n.g.m. cultrera); 7. tecnologie di estrazione, conservazione, packaging e loro riflessi sulla qualità dell’olio vergine di oliva (extraction, storage and packaging technologies and their impact on virgin olive oil quality) (m. servili, s. esposto, a. taticchi and r. sacchi); 8. utilizzazione dei reflui oleari (use of oil wastewater production) (m. servili, s. esposto, s. urbani, a. taticchi, and m. petruccioli); 9. caratteristiche meccaniche e funzionali degli impianti di estrazione dell’olio di oliva (mechanical and functional characteristics of olive oil extraction machinery and wastewater mills) (p. amirante); 10. modelli strutturali di frantoi e collocazione logistica degli impianti (structural models of mills and logistic location of facilities) (p. amirante); 11. oli aromatizzati (aromatic oils) (l. cerretani); 12. utilizzo dell’olio nella preparazione dei prodotti agroalimentari (using oil in the preparation of foodstuffs) (l. cerretani); 13. valutazione sensoriale degli oli vergini di oliva e loro classificazione (sensory evaluation of virgin olive oils and their classification) (a. giomo); 14. la valutazione sensoriale degli oli vergini di oliva (sensory evaluation of virgin olive oils) (b. alfei); 15. normativa relativa alla commercializzazione dell’olio di oliva (legislation on olive oil marketing) (r. filo della torre). the elegant typographical presentation and the impressive range of tables and images in black and white and color make the work not only attractive but also make reading it a pleasent and fuild experience. for its peculiar characteristics, the text addresses a broad spectrum of readers: from student to scientists of the subject, from the technical crusher to the manufacturer of machinery for olive oil extraction, and from traders to the legislators. enrico rinaldelli 123 book reviews 164 1. introduction persimmon trees, having no dwarf rootstocks of commercial value, tend to grow high. lowering tree height by heavy dormant pruning has been a routine practice for efficient management of many persimmon orchards. in densely planted orchards, heavy pruning is an inevitable practice to restrict tree size. this practice, in turn, causes vigorous shoot growth resulting in an excessive crowding of the canopy. poor fruit set and excessive supply of nitrogen also stimulate the occurrence of vigorous shoots. tree crowding not only hinders orchard operations, but deteriorates fruit quality as the tree interior becomes heavily shaded. shoots of vigorous and succulent growth make the tree susceptible to anthracnose (colletorichum gloeosporioides) when humidity within the canopy is high. summer pruning is one of many options to alleviate the problems of crowding, ensuring adequate light penetration into the canopy and controlling excessive shoot growth. however, removal of shoots during growing season involves the loss of functional leaf surface, which may lead to reduced tree development and fruit growth. loss of leaf area may also reduce reserve accumulation for early growth the next season. the effect of summer pruning on physiological process and tree growth seems well documented in other deciduous fruit crops, but limited studies have been conducted in persimmon. in this paper we evaluate the effects of summer pruning on tree growth, fruit quality, and nutrient composition of perennial organs in persimmon. 2. tree response to summer pruning vegetative growth since removal of active leaf area reduces the production of dry matter during the growing season, summer pruning suppresses vegetative growth in other fruit crops, the effect of which is closely related to pruning severity, timing and cultivars (taylor and ferree, 1984; rom and ferree, 1985; marini and barden, 1987; mediene et al., 2002; zamani et al., 2006). in young ‘fuyu’ and ‘nishimurawase’ persimmons, thinning 26% of total shoot length in late july reduced dry weight of dormant one-year-old twigs, but it did not significantly affect total dry weights of aerial wood and the root (choi et al., 2003 a). the result might be related in part to a high photosynthetic activity of remaining leaves until later in the season as shown in leaf chlorophyll concentrations (fig. 1). although not significant, negative relationships were observed between the increment of trunk cross-sectional area and pruning severity in the same experiment (choi et al., 2003 a) and summer heading-back (song et al., 2001). summer pruning effect on tree growth and fruit production of persimmon s.t. choi*, d.s. park*, k.p. hong*, s.m. kang** * sweet persimmon research institute, gyeongnam agricultural research and extension services, 262-1 woodong, jinyoung, gimhae 621-802, republic of korea. ** department of horticulture, institute of agriculture and life science, gyeongsang national university, 900 gazwa-dong, jinju 660-701, republic of korea. key words: pinching, secondary shoot, topping, tree vigor, water sprout. abstract: this paper reviews the effect of summer pruning in persimmon (diospyros kaki thunb.) with regard to its positive and negative aspects on tree growth and fruit production. in order for this practice to be of a significant value, a clear understanding is needed to appreciate the fact that so many variables are involved and much remains to be substantiated. major reasons for summer pruning of persimmon are to improve fruit quality by enhancing light penetration into the tree canopy and to restrict vegetative shoot growth. summer pruning generally suppresses tree growth even though it elevates leaf activity. positive effects of summer pruning on skin color, soluble solids, and appearance of fruits are observed mainly in those orchards where the trees are heavily dormant-pruned to lower tree height and to secure space in high density plantings. secondary shoot pruning and topping could also improve fruit quality, increasing flower bud formation of remaining shoots. summer-pruning effects are highly dependent on its severity and timing to affect tree growth, shoot regrowth, reserve accumulation, and fruit quality. adv. hort. sci., 2011 25(3): 164-169 received for publication 11 april 2011 accepted for publication 13 may 2011 165 leaf activity summer pruning is known to increase photosynthesis, dark respiration, and transpiration of shoot leaves in other fruit crops (taylor and ferree, 1981; marini and barden, 1982; myers and ferree, 1983). as shoot thinning severity in late july increased in ‘nishimurawase’ persimmon, specific leaf weight tended to become higher (table 1). chlorophyll content per unit area increased by 1.9-fold and 2.3-fold in ‘fuyu’ and about 2-fold in ‘nishimurawase’ trees that had been thinned 17% and 26% of their shoots, respectively (fig. 1). results indicated that photosynthesis can hardly compensate for the leaf area that has been removed, and there would be a sizable reduction in the production of photosynthates depending on pruning time regrowth it is possible that shoot regrowth following summer pruning can have serious negative effects on the supply of photosynthates to fruit and shoot and resistance to diseases and pests (forshey et al., 1992; choi et al., 2003 b). in addition, excessive regrowth may reduce flower bud formation by gibberellins produced during shoot regrowth (forshey et al., 1992). the extent of regrowth following summer pruning was influenced by the time and severity (miller, 1982; ferree et al., 1984). when ‘fuyu’ persimmon was summer-pruned on 20 june or 4 july, the earlier pruning produced more secondary growth more than the later one (fujimura, 1932). summer pruning after late july, when buds enter physiological rest, did not result in a great problem of regrowth in persimmon trees, but heavy summer pruning may stimulate some buds to break into growth (table 1). heading-back cuts in vigorous shoots would easily induce regrowth more than thinning cuts. late july through early august is the appropriate time for thinning out vigorous shoots to avoid regrowth in south korea. reserve accumulation early loss of foliage from pruning may result in reduced carbohydrate levels of the tree, and that could adversely affect cold hardiness (marini and barden, 1987). when the current shoots were thinned up to 26% of their total length in late july in young ‘fuyu’ and ‘nishimurawase’ persimmon, the differences in non-structural carbohydrates and inorganic nutrients in fourto five-year-old wood of aboveground parts and the large root, measured on 2 april the following year, were not consistent with the pruning severity the previous summer (choi et al., 2003 a). this result might be due to the increased activity of the leaves as presented in chlorophyll concentration in figure 1. however, thinning 50% of total shoots decreased carbohydrate concentration in one-year-old shoots of field-grown ‘nishimurawase’ (table 2) but not in those of ‘fuyu’ (choi et al., 2003 b). in the same experiment, the severe summer pruning also reduced flower buds in both cultivars the following year, since low carbohydrates in the shoots (choi et al., 2011) and excessive regrowth (forshey et al., 1992) were negatively related to flower bud formation. 3. fruit quality affected by summer pruning summer pruning has been recommended to improve table 1 effect of summer pruning severity on 29 july on light penetration and tree growth of ‘nishimurawase’ persimmon (choi et al., 2003 b) shoot-thinned (%) light penetration (%) tcsa increment (%) regrowth (cm/tree) slw (mg·cm-2) leaf spad reading 0 11.3 b 6.8 a 0 a 10.14 b 51.6 a 25 24.7 a 7.8 a 63 a 10.92 ab 52.8 a 33 27.9 a 7.4 a 163 a 10.93 ab 51.8 a 50 30.9 a 6.1 a 215 a 11.18 a 54.8 a after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. tcsa= trunk cross-sectional area. slw= specific leaf weight. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤ 0.05. fig. 1 effect of different severities of shoot thinning on 22 july on leaf chlorophyll of five-year-old ‘fuyu’ and ‘nishimurawase’ persimmon on 3 november. chlorophyll content includes chlorophylls a and b. values sharing the same letter are not significantly different from each other by duncan’s multiple range test at p≤ 0.05 (choi et al., 2003 a). µg 166 fruit quality in vigorous persimmon by increasing light interception (mowat, 1987; ullio, 2003; george et al., 2005; yamada, 2008). however, there was no consistent effect of the pruning on fruit characteristics in many studies. table 3 shows that thinning out some water sprouts in late july increased skin color with no reduction in fruit size. this kind of positive result might be possible in vigorous ‘fuyu’ trees in densely-planted orchards, and the leaf-fruit ratio after the pruning was sufficiently high. kim (2010) also found larger size and higher coloration of fruits in vigorous ‘fuyu’ trees after thinning out some water sprouts in late july. lower water consumption and thus improved water status during the growing season after summer pruning could benefit fruit growth and relieve the potential detriment due to carbohydrate shortage in apple (li et al., 2003) and peach (lopez et al., 2006). fruit firmness was not significantly changed by the differences in pruning severity (tables 4 and 5). size, soluble solids, and skin color of persimmon fruits decreased when the leaf-fruit ratio was low (choi et al., 2010). the appropriate leaf-fruit ratio for quality ‘fuyu’ fruits has been set at about 20 in korea (choi et al., 2010) and japan (kishimoto, 1975; yamamura et al., 1989). loss of leaf area by summer pruning results in poor fruit quality table 3 effect of removing water sprouts on 28 july on light penetration and fruit characteristics in vigorous ‘fuyu’ persimmon orchard (choi et al., 2005) treatment light penetration (%) fruits average weight (g) skin color (hunter a) soluble solids (°brix) non-pruning 15 243 27.9 15.0 summer pruning 33 257 29.8 15.2 significance ** ns * ns by summer pruning of 1-year-old trees, water sprouts were removed to maintain leaf-fruit ratio 20 in an orchard planted at 6 x 3 m. ns= not significant; * = significant at p≤ 0.05; ** = significant at p≤0.01. table 4 effect of summer pruning severity on 28 july on fruit characteristics of ‘fuyu’ persimmon harvested on 31 october (choi et al., 2003 b) shoot-thinned (%) average weight (g) skin color (hunter a) fruit firmness (n) soluble solids (°brix) skin damage (%) 0 234 a 27.6 a 20.7 a 15.2 a 13.2 a 10 244 a 28.4 a 21.9 a 15.7 a 9.3 cb 20 249 a 28.3 a 20.1 a 15.1 a 7.8 c 30 233 a 29.5 a 20.3 a 15.3 a 9.8 b after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 39 of 0% thinning to 28 of 30% thinning in an orchard planted at 6 x 3 m. skin damage: blemish or stains on fruit skin. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤0.05. table 5 effect of summer pruning severity on 29 july on fruit characteristics of ‘nishimurawase’ persimmon harvested on 26 september (choi et al., 2003 b) shoot-thinned (%) average weight (g) skin color (hunter a) fruit firmness (n) soluble solids (°brix) skin damage (%) 0 144 a 32.2 a 34.3 a 14.5 a 8.8 a 25 139 a 32.2 a 33.4 a 14.0 ab 3.7 ab 33 137 a 31.2 ab 32.5 a 13.6 ab 4.6 ab 50 141 a 27.7 a 31.9 a 13.0 b 1.6 b after summer pruning of seven-year-old trees on 29 july, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. skin damage: blemish or stains on fruit skin. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤0.05. table 2 effect of summer pruning severity on 29 july on concentrations of carbohydrates and inorganic elements in dormant shoots of ‘nishimurawase’ persimmon on 17 february (choi et al., 2003 b) shoot-thinned (%) carbohydrates (% dw) inorganic elements (% dw) soluble sugars starch n p k ca mg 0 12.2 a 5.4 a 0.60 a 0.21 b 0.60 a 0.36 b 0.17 a 25 12.0 a 4.4 ab 0.62 a 0.24 ab 0.73 a 0.39 ab 0.17 a 33 10.3 a 3.5 ab 0.60 a 0.24 ab 0.74 a 0.43 a 0.17 a 50 9.7 a 3.2 b 0.62 a 0.28 a 0.75 a 0.44 a 0.18 a after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤ 0.05. 167 if the pruning significantly reduces the ratio. when leaffruit ratio decreased to less than 13 with severe removal of the shoots, fruit size, skin color or soluble solids were significantly reduced (table 5) (choi et al., 2003 a). on the other hand, strong winds result in blemish of fruit skin due to rubbing of shoots and leaves against fruits (george et al., 1997 a). skin staining of persimmon fruits is often caused by humid conditions in autumn (george et al., 1997 a), which become aggravated by the shoots growing vigorously. thinning out some of the shoots in summer reduces these damages by improving the micro-environment within the tree canopy (tables 4 and 5). therefore, summer pruning has been recommended as a means to reduce the occurrence of skin staining (george et al., 1997 a, 2005), especially in newly-released japanese cultivars like a crack-susceptible ‘taishu’ (yakushiji and nakatsuka, 2007). the influence of summer pruning on fruit quality varies by the degree of canopy crowding and the timing and its severity, as has been documented in other fruit crops (marini and barden, 1987; forshey et al., 1992; zamani et al., 2006). 4. water sprout management removing all water sprouts of vigorous trees too early in the season may induce severe regrowth (fujimura, 1932), which disturbs light penetration and reserve accumulation in perennial organs (kappel et al., 1983). therefore, instead of removing all water sprouts, thinning some of them combined with bending or twisting the remaining ones down to horizontal in june may be recommended (park and choi, 2000; huh et al., 2003; george et al., 2005). securing leaf area even in water sprouts helps prevent the regrowth that may be related to the action of hormones produced in old leaves (forshey et al., 1992). some of the water sprouts may have some value as mother branches the next season. water sprouts of ‘fuyu’ could form flower buds as long as their terminal buds are set by early august in south korea (choi et al., 2011): they could serve as fruiting branches for the following year. in vigorous trees with poor fruiting, using the sprouts as mother branches to set fruits the next season is extremely important to make up for the yield reduction as well as to control tree vigor. when the sprouts were twisted and bent down under horizontal in midto late june after thinning out some, flower buds were formed in 84 to 97% of them the following year (park and choi, 2000). changes in endogenous hormones might be closely involved in this treatment, particularly low gibberellin and high cytokinin, which play a role in flower bud formation (banno et al., 1985). heading-back pruning to four buds is also practiced in new zealand to ensure future fruiting site from water sprouts (mowat, 1987). 5. secondary-shoot pruning, pinching and topping secondary growth from shoot terminal occurs from mid-june in vigorous persimmon trees (nii, 1980; park et al., 2003). secondary growth often induced fruit drop (george et al., 1997 b). park et al. (2003) studied the heading-back effect of secondary shoots leaving two to three basal leaves of the secondary growth on ‘fuyu’ persimmon. when the heading-back was done on fruiting shoot, they found that the shoots had a lower dry weight which was accompanied by an increase in fruit weight. the result indicated the necessity of such cuts for fruit growth. compared with the heading-backs before 10 july or after 10 august, those on 25 july produced the largest fruits inseason and exhibited the highest percentage of shoots that bore flowers and fruits the following year, indicating that pruning the secondary shoots on 25 july helps to direct the assimilates to fruit growth and flower bud development, not to vegetative growth. ‘nishimurawase’ persimmons tend to bear staminate, not pistillate, flowers when the trees are old and not vigorous enough. chijiwa and hayashi (2007) reported a way to use water shoots to serve as fruiting mother branches for the next season by pinching at 15 cm from the base between may and june. shoots left after the pinching or summer shoots regrown from the cut bore more pistillate and fewer staminate flowers the following year than the un-treated water shoots. takano (1994) also reported a better mother branch formation by pinching adventitious buds of persimmon. the size of trees and yield efficiency are among the most important considerations in high density orchards. when the vigorous shoots of young ‘uenishiwase’ persimmon were topped to a 30cm length in early june, followed by the removal of the regrowth, trunk growth decreased and fruit set increased the following year (song et al., 2001). the occurrence of secondary growth after heading was dependent on the treatment date. topping the shoots at the fourth bud from the terminal on 20 may promoted secondary growth of ‘hiratanenashi’ persimmon, but the same treatment after 22 june did not (hasegawa and nakajima, 1984). the summer topping of long shoots in summer was effective in forming fruiting shoots the following year in their lower parts. pinching and topping the shoot would promote flower bud formation of that shoot if regrowth is not severe. 6. conclusions severe summer pruning could suppress vegetative and fruit growth due to the lowered photosynthetic capacity, reducing the carbohydrate reserves in persimmon tree. however, summer pruning which is appropriate to tree vigor and environmental conditions would increase production of quality fruits by improving light distribution in the tree canopy or restricting vegetative growth. since tree vigor is a result of the complex interactive effects with cultural practices, the pruning alone should not be used as a tool for controlling vegetative growth. it is necessary to incorporate the pruning into a comprehensive program 168 such as tree training, fruiting, irrigation, and fertilization to reduce vigorous growth. growers should employ summer pruning methods while carefully monitoring for potentially negative effects on tree growth and fruit quality. references banno k., hayashi s., tanabe k., 1985 effects of sadh and shoot-bending on flower bud formation, nutrient components and endogenous growth regulators in japanese pear (pyrus serotina rehd.). j. japan. soc. hort. sci., 53: 365-376. chijiwa h., hayashi k., 2007 the formation of fruiting mother shoot from water shoot by pinching in japanese persimmon. hort. res., 6: 265-270. choi s.t., kang s.m., park d.s., song w.d., ahn g.h., 2003 a effect of different severities of summer pruning on fruit characteristics and tree growth in young ‘fuyu’ and ‘nishimurawase’ persimmon. j. korean soc. hort. sci., 44: 569-574. choi s.t., park d.s., ahn g.h., 2003 b studies on efficient production and quality improvement of sweet persimmon effect of summer pruning on fruit qualities of sweet persimmon. rural development administration report. suwon, korea, pp. 5-34. (in korean with english summary). choi s.t., park d.s., kang s.m., 2011 nutrient accumulation and flower bud formation affected by the time of terminal bud set on water sprouts of persimmon. hortscience, 46: 523-526. choi s.t., park d.s., kang s.m., cho y.c., 2010 effect of fruit-load on the growth, absorption, and partitioning of inorganic nutrients in young ‘fuyu’ persimmon trees. sci. hort., 126: 408-412. choi s.t., park d.s., kim s.c., 2005 summer pruning and management for vigorous persimmon trees. 2005 farming technology. rural development administration. suwon, korea. (in korean) ferree d.c., myers s.c., rom c.r., taylor b.h., 1984 physiological aspects of summer pruning. acta horticulturae, 146: 243-252. forshey c.g., elfving d.c., stebbins r.s., 1992 training and pruning apple and pear trees. amer. soc. hort. sci., pp. 60-63. fujimura j., 1932 effects of summer pruning on the secondary growth of one-year-old persimmon and japanese pear trees. j. japan. soc. hort. sci., 3: 9-20. george a.p., mowat a.d., collins r.j., 1997 a factors affecting blemishing of persimmon in new zealand and australia. acta horticulturae, 436: 171-178. george a.p., mowat a.d., collins r.j., morleybunker m., 1997 b the pattern and control of reproductive development in non-astringent persimmon (diospyros kaki l.): a review. sci. hort., 70: 93-122. george a.p., nissen b., broadley r., collins r.j., rigden p., jeffers s., isaacson b., ledger s., 2005 sweet persimmons grower’s handbook. department of primary industries and fisheries, brisbane, queensland, australia. hasegawa k., nakajima y., 1984 effects of topping on the growth and fruit quality of the japanese persimmon. res. rpt. kochi univ. (agr.), 33: 43-53. huh m.s., song i.k., cho d.h., jeong y.s., 2003 bending effect of vigorous shoot on persimmon tree. 2003 annual research report. gyeongsangbuk-do agr. res. ext. serv. daegu, korea. (in korean) kappel f., flore j.a., layne r.e.c., 1983 characterization of the light microclimate in four peach hedgerow canopies. j. amer. soc. hort. sci., 108: 102-105. kim b.s., 2010 development of cultivation techniques for the stable production of high quality sweet persimmon fruits to export a study on quality enhancement of sweet persimmon for export by stable tree vigor. rural development administration, suwon, korea. (in korean). kishimoto o., 1975 estimations of optimum range of degree of fruit thinning and desirable fruit weight in japanese pears and persimmon tree. j. japan. soc. hort. sci., 43: 368-376. li k., lakso a.n., piccioni r., robinson t., 2003 summer pruning reduces whole-canopy carbon fixation and transpiration in apple trees. j. hort. sci. biotechol., 78: 749-754. lopez g., mata m., arbones a., solans j.r., girona j., marsal j., 2006 mitigation of effects of extreme drought during stage iii of peach fruit development by summer pruning and fruit thinning. tree physiol., 26: 469-477. marini r.p., barden j.a., 1982 net photosynthesis, dark respiration, transpiration, and stomatal resistance of young and mature apple trees as influenced by summer or dormant pruning. j. amer. soc. hort. sci., 107: 170-174. marini r.p., barden j.a., 1987 summer pruning of apple and peach trees. hort. rev., 9: 351-375. mediene s., jordan m.o., pagès l., lebot j., adamowicz s., 2002 the influence of severe shoot pruning on growth, carbon and nitrogen status in young peach trees (prunus persica). tree physiol., 22: 1289-1296. miller s.s., 1982 regrowth, flowering, and fruit quality of ‘delicious’ apple trees as influenced by summer pruning. j. amer. soc. hort. sci., 107: 975-978. mowat a., 1987 pruning persimmons. hortresearch publication. new zealand. march 28, 2011. http://www.hortnet. co.nz/publications/science. myers s.c., ferree d.c., 1983 influence of summer pruning and tree orientation on net photosynthesis, transpiration, shoot growth, and dry-weight distribution in young apple trees. j. amer. soc. hort. sci., 108: 4-9. nii n., 1980 current shoot and growth in japanese persimmon, diospyros kaki cv. fuyu, in relation to the development of the tissue system in the leaf. j. japan. soc. hort. sci., 49: 149-159. park d.s., choi s.t., 2000 development of technology for production of high quality fruits in sweet persimmon studies on the method of fruit-load adjustment for high quality of fruit production. rural development administration report, suwon, korea. (in korean with english summary). park d.s., kang s.m., choi s.t., lim c.a., song w.d., 2003 effect of secondary-shoot prunings on fruit growth and following year’s fruit set of ‘fuyu’ persimmon. j. korean soc. hort. sci., 44: 678-682. rom c.r., ferree d.c., 1985 time and severity of summer pruning influences on young peach tree net photosynthesis, 169 transpiration, and dry weight distribution. j. amer. soc. hort. sci., 110: 455-461. song w.d., choi s.t., kim s.c., park d.s., kang s.m., 2001 effect of pinching on fruit set of young sweet persimmon trees during the following years. 2001 annual research report. gyeongsangnam-do agr. res. ext. serv. jinju, korea. (in korean) takano s., 1994 better branch formation by pinching adventive buds of japanese persimmon. bull. nara agr. expt. sta. j., 25: 36-37. taylor b.h., ferree d.c., 1981 the influence of summer pruning on photosynthesis, transpiration, leaf abscission, and dry weight accumulation of young apple trees. j. amer. soc. hort. sci., 106: 389-393. taylor b.h., ferree d.c., 1984 the influence of summer pruning and cropping on growth and fruiting of apple. j. amer. hort. sci., 109: 19-24. ullio l., 2003 persimmon growing in new south wales. agfact h3.1.17, 3rd edition. elizabeth macarthur agricultural research institute, camden, australia. yakushiji h., nakatsuka a., 2007 recent persimmon research in japan. japan. j. plant sci., 1(2): 42-62. yamada m., 2008 persimmon propagation, orchard planting, training and pruning in japan. adv. hort. sci., 22: 269-273. yamamura h., matsui k., matsumoto t., 1989 effects of gibberellins on fruit set and flower-bud formation in unpollinated persimmon (diospyros kaki). hortscience, 38: 77-86. zamani z., saie a., talaie a.-r., fatahi r., 2006 effects of summer pruning on growth indices of two important iranian apple cultivars ‘golab’ and ‘shafi-abadi’. acta horticulturae, 707: 269-274. 95 1. introduction citrus is one of the most important fruit crops in japan and also worldwide. various accessions of citrus species are adapted to the southwest of japan, and although they are cultivated in this region, almost all of them are nonnative, that is, they were introduced from abroad, arose as chance seedlings, were selected from bud sports, and were bred by artificial pollination. only two species, citrus tachibana (makino) tanaka (tachibana) and citrus depressa hayata (shiikuwasha) were present in japan before recorded history. c. tachibana mainly grows indigenously on the pacific side of the southwest of japan’s main islands (kyushu, shikoku, and honshu). c. tachibana was recorded in “kojiki”, the oldest chronicle in japan dating from the early 8th century. its indigenous trees were also found on the ryukyu islands (islands including the okinawa islands, sakishima islands, and amami islands, which were ruled by japan from the 17th to19th centuries) and taiwan (tanaka, 1931; lin and chen, 2006; inafuku-teramoto et al., 2010). c. depressa is indigenous to both the ryukyu islands and taiwan (tanaka, 1936; lin and chen, 2006). compared to c. tachibana, c. depressa is considered to be adapted to a warmer climate; the former is usually used as an ornamental for gardens and its fruit is inedible. on the other hand, fruit of c. depressa is in much demand as an ingredient for food and drinks, to garnish dishes similar to a lemon or lime, to make juice and jam, and as an additive to soy sauce and distilled spirits. recently, this fruit has attracted attention because it contains high levels of polymethoxyflavonoids, one of the most important health-promoting components of citrus (inafuku-teramoto et al., 2010). we have investigated the phylogenetic relationships of citrus and its relatives through the analysis of genes encoded in chloroplast dna (cpdna) (tshering et al., 2010, 2013). in our recent study (tshering et al., 2013) in which various citrus accessions were used as materials, we found that c. tachibana and c. depressa possess a characteristic cpdna genome based on the sequences of the chloroplast matk genes, which encode a maturase involved in splicing type ii introns from rna transcripts (hilu and liang, 1997; hilu et al., 2003; olmstead and palmer, 1994). there are many accessions in both species, and intraspecies diversity is found within each species (hirai et al., 1990; yamamoto et al., 1998; kinjo, 2007; inafuku-teramoto et al., 2010; yamamoto et al., 2011). characterization of chloroplast matk sequences of citrus tachibana and citrus depressa, two indigenous species in japan y. nagano1, s. inafuku-teramoto2,3, m. hashimoto4, t. mimura4, r. matsumoto4, m. yamamoto5(*) 1 analytical research center for experimental sciences, saga university, honjo-machi, saga 840-8502, japan. 2 faculty of agriculture, university of the ryukyus, nishihara, okinawa 903-0213, japan. 3 botswana-jica jatropha project, dar, sebele, gaborone, botswana. 4 faculty of agriculture, saga university, honjo-machi, saga 840-8502, japan. 5 faculty of agriculture, kagoshima university, korimoto, kagoshima 890-0065, japan. key words: cpdna, genetic resources, ryukyu islands, shiikuwasha, tachibana. abstract: citrus tachibana, c. nippokoreana, and c. depressa are indigenous mandarin species in japan. we deduced their phylogenetic relationships from nucleotide sequences of the chloroplast matk gene. the results indicate that c. tachibana, c. nippokoreana, and c. depressa accessions can be classified into two types: type a, all sixteen c. tachibana and six c. depressa; type b, eleven c. depressa and one c. nippokoreana. both type a and type b accessions of c. depressa were found on the okinawa islands, whereas only type b accessions of c. depressa were found on the sakishima and amami islands. this cpdna divergence seemed to indicate a polyphyletic origin of c. depressa. the matk genes of type a were found only in c. tachibana and some c. depressa. from these results, both species probably possess a characteristic chloroplast genome among various citrus species. adv. hort. sci., 2014 28(2): 95-99 (*) corresponding author: yamasa@agri.kagoshima-u.ac.jp received for publication 31 march 2014 accepted for publication 1 july 2014 96 however, a limited number of accessions were used in our previous study (tshering et al., 2013). therefore, for the present work, we analyzed the matk gene sequences of a number of c. tachibana and c. depressa plants grown in various regions in japan to reveal their characteristic profiles of the cpdna genome. c. nippokoreana (korai tachibana), a c. tachibana relative indigenous to hagi city, yamaguchi prefecture, japan, and cheju island, korea (kimura and taninaka, 1995), was also investigated. 2. materials and methods plant materials sixteen c. tachibana, one c. nippokoreana, 17 c. depressa, and 13 control accessions were used in this study. the sources of the materials are shown in table 1 and figure 1. pcr amplification and dna sequencing genomic dna was extracted from leaves using the dneasy plant mini kit (qiagen, valencia, ca, usa). by using this genomic dna as a template, the matk gene was amplified by pcr using proofreading primestar gxl dna polymerase (takara bio, ohtsu, shiga, japan). the primers used for pcr amplification of the matk gene were matk1f (5′-accgtatcgcactatgtatc-3′) and matk1r (5′-gaactagtcggatggagtag-3′). the amplified dna fragments were purified using the nucleospin gel and pcr clean-up kit (macherey-nagel, düren, germany). the primers used for sequencing of the matk gene were matk1f, matk2f (5′-acggttctttctccacgagt-3′), matk3f (5′-ggtccgatttctctgattct-3′), matk1r, matk2r (5′-agaatcagagaaatcggacc-3′), and matk3r (5′-actcgtggagaaagaaccgt-3′). the purified dna fragments were sequenced in both directions in an applied biosystems 3130 genetic analyzer (applied biosystems) with a bigdye terminator cycle sequencing ready reaction kit v. 3.1 (applied biosystems) as described previously (platt et al., 2007). sequence data were submitted to ddbj/genbank/ebi and were assigned accession numbers ranging from ab839905 to ab839932. the sequences of the accessions from no. 29 to no. 34 were deposited in our previous study (tshering et al., 2013). phylogenetic analyses the neighbor-joining (nj) and maximum likelihood (ml) methods from the mega (version 5.2.1) program (tamura et al., 2011) were used to create phylogenetic trees. the reliability of each branch was tested by bootstrap analysis with 1,000 replications. 3. results and discussion we constructed multiple sequence alignments of 1,630-bp fragments containing the matk gene from different citrus accessions. each sequence contained a 1,530-bp protein-coding sequence and 100 bp of the 3′ utr. one exception is the matk gene of trifoliate orange (poncirus trifoliata), which has a 6 bp insertion at the 3′ utr. of these, 23 bases were variable and six bases were phylogenetically informative. we created phylogenetic trees using the nj and ml methods. the topologies of the different trees were identical (data not shown). therefore, we present here only the ml tree (fig. 2). c. tachibana, c. depressa, and c. nippokoreana accessions were classified into two types as follows: type a: all 16 c. tachibana and six c. depressa [shiikuwasha-okinawa#1 (no. 17), shiikuwasha-okinawa#3 (no. 19), shiikuwasha-okinawa#6 (no. 22), and shiikuwashaoku (no. 26), kabishi (no. 29), and fusubuta (no. 31)]. type b: eleven c. depressa [shiikuwasha-taketomi (nohara) (no. 11), shiikuwasha-taketomi (takana) (no. 12), shiikuwasha-iriomote (no. 13), shiikuwasha-iriomote (katoura) (no. 14), shiikuwasha-kohama (ufudake) (no. 15), shiikuwasha-kohama (omori) (no. 16), ishikunibu (no. 28), mikanguwa (no. 30), kaachi (no. 32), shiikunin (no. 33), and shiikurubu (no. 34)] and one c. nippokoreana. none of the control accessions belonged to type a, whereas all seven control mandarin accessions belonged to type b. the other control accessions were clearly distinguished from type a and type b. this finding is consistent with the results of our previous study (tshering et al., 2013). all 16 c. tachibana accessions carried an identical matk sequence. previous studies (hirai et al., 1990; yamamoto and tominaga, 2003) reported that c. tachibana was genetically differentiated from citrus species originating from all fig. 1 collection sites of citrus tachibana, c. nippokoreana, and c. depressa in the present study. 97 table 1 citrus tachibana, c. nipponkoreana, and c. depressa accessions used in the present study no. accession latin name origin note 1 tachibana-dazaifu (uchi) citrus tachibana (makino) tanaka fukuoka, kyushu planted tree 2 tachibana-dazaifu (soto) c. tachibana (makino) tanaka fukuoka, kyushu planted tree 3 tachibana-heian jingu c. tachibana (makino) tanaka kyoto, honshu planted tree 4 tachibana-iwashimizu hachimangu c. tachibana (makino) tanaka kyoto, honshu planted tree 5 tachibana-kitano tenmangu c. tachibana (makino) tanaka kyoto, honshu planted tree 6 tachibana-toshijima (mie) c. tachibana (makino) tanaka mie, honshu native tree 7 tachibana-matsuoyama (kochi) c. tachibana (makino) tanaka kochi, shikoku native tree 8 tachibana-nangoku (kochi) c. tachibana (makino) tanaka kochi, shikoku planted tree 9 korai tachibana c. nippokoreana tanaka kochi, shikoku planted tree 10 tachibana-ishigakijima c. tachibana (makino) tanaka ishigaki-jima, sakishima native tree 11 shiikuwasha-taketomi (nohara) c. depressa hayata taketomi-jima, sakishima native tree 12 shiikuwasha-taketomi (takana) c. depressa hayata taketomi-jima, sakishima native tree 13 shiikuwasha-iriomote c. depressa hayata iriomote-jima, sakishima native tree 14 shiikuwasha-iriomote (katoura) c. depressa hayata iriomote-jima, sakishima native tree 15 shiikuwasha-kohama (ufudake) c. depressa hayata kohama-jima, sakishima native tree 16 shiikuwasha-kohama (omori) c. depressa hayata kohama-jima, sakishima native tree 17 shiikuwasha-okinawa#1 c. depressa hayata okinawa-honto native tree 18 tanibuta-okinawa#2 c. tachibana (makino) tanaka okinawa-honto native tree 19 shiikuwasha-okinawa#3 c. depressa hayata okinawa-honto native tree 20 tanibuta-okinawa#4 c. tachibana (makino) tanaka okinawa-honto native tree 21 tanibuta-okinawa#5 c. tachibana (makino) tanaka okinawa-honto native tree 22 shiikuwasha-okinawa#6 c. depressa hayata okinawa-honto native tree 23 tanibuta-okinawa#7 c. tachibana (makino) tanaka okinawa-honto native tree 24 tanibuta-okinawa#8 c. tachibana (makino) tanaka okinawa-honto native tree 25 garagara c. tachibana (makino) tanaka okinawa-honto native tree 26 shiikuwasha-oku c. depressa hayata okinawa-honto native tree 27 tanibuta c. tachibana (makino) tanaka okinawa-honto native tree 28 ishikunibu c. depressa hayata okinawa-honto native tree 29 kabishi c. depressa hayata okinawa-honto native tree 30 mikanguwa c. depressa hayata okinawa-honto native tree 31 fusubuta c. depressa hayata okinawa-honto native tree 32 kaachi c. depressa hayata okinawa-honto native tree 33 shiikunin c. depressa hayata tokuno-shima, amami native tree 34 shiikuribu c. depressa hayata okinoerabu-jima, amami native tree control accessions satsuma mandarin ‘aoshima’ c. unshiu marcow. ponkan ‘yoshida ponkan’ c. reticulata blanco mediterranean mandarin c. deliciosa ten. dancy c. tangerina hort. ex tanaka kinokuni ‘hirakishu’ c. kinokuni hort. ex tanaka sunki c. sunki (hayata) hort. ex tanaka cleopatra c. reshni hort. ex tanaka yuzu ‘yamane’ c. junos siebold ex tanaka sweet orange ‘fukuhara’ c. sinensis (l.) osbeck lemon ‘eureka’ c. limon (l.) burm. f. pummelo ‘mato buntan’ c. maxima (burm.) merr. citron ‘maru busshukan’ c. medica l.   trifoliate orange ‘standard’ poncirus trifoliata (l.) raf.     98 other countries except japan. the present study also confirmed that the matk sequence of c. tachibana was not identical to those of studied accessions originating from all other countries except japan. however, we found that the matk sequence of c. tachibana was identical to those of some investigated c. depressa accessions that are indigenous to the ryukyu islands, japan. this suggests that c. tachibana has been isolated from the mandarins elsewhere, and evolved in japan in unique ways. we found no diversity within species. however, further study considering more accessions is needed since the materials used here did not cover the entire area where c. tachibana grows. the matk sequence of c. nippokoreana was not identical to that of c. tachibana, indicting genetic differentiation between the two species. because it is considered that c. nippokoreana is related to c. tachibana (the japanese name “korai tachibana” means “tachibana from korea”), this finding is interesting. c. depressa accessions were divided into two types according to matk sequences. one was the same type as c. tachibana and the other was the same type as several mandarins such as c. reticulata and c. sunki. this result completely agrees with the results of our previous study (tshering et al., 2013). differentiation of the cpdna genome in c. depressa was also reported by urasaki et al. (2005) and yamamoto et al. (2013), who analyzed the trnl-trnf and trnf-trnvr regions, respectively. these results strongly suggest a polyphyletic origin of c. depressa. this divergence of matk genes was found in c. depressa accessions grown on okinawa-honto (the main island of okinawa islands) but not in those grown on the sakishima and amami islands. c. depressa possessing c. tachibana-type cpdna (a type) was found only on okinawa-honto. similar results were reported by yamamoto et al. (2013) who studied c. depressa on okinawa-honto and the amami islands. however, urasaki et al. (2005) found that c. depressa accessions possessed c. tachibana-type cpdna (trnl-trnf sequence) on the sakishima islands. thus, further study using many c. depressa accessions grown on various islands is necessary to resolve the distribution of each type. there is a possibility that type a c. depressa is genetically closer to c. tachibana than type b. however, this hypothesis is not supported since the proportion of common bands from random amplified polymorphic dna (rapd) analysis between c. depressa of type a and c. tachibana was not so different from that of type b and c. tachibana (yamamoto et al., 1998). since the origin and/or relationship of c. depressa to c. tachibana cannot be elucidated only by cpdna analysis, cpdna analysis combined with nuclear genome analysis such as simple sequence repeat (ssr), sequencerelated amplified polymorphism markers (sraps) (barkley et al., 2006; uzun et al., 2009), and restriction site-associated dna sequences (rad-seq) (baird et al., 2008) is considered to be necessary. for this purpose, structural analysis (barkley et al., 2006) seems to be informative. the present work demonstrates the characteristic profiles of the chloroplast genome of citrus tachibana and citrus depressa, two indigenous species in japan, using a number of accessions grown in various regions based on the results of matk sequencing. furthermore, the divergence of the cpdna genome of c. depressa seems to indicate a polyphyletic origin of this species. these findings are a contribution to progress in the study of the genetic resources in citrus and related genera. acknowledgements we would like to express our deep appreciation to dr. tetsuo koyama, kochi prefectural botanical garden for help in obtaining tachibana samples. we also acknowledge the help of staff of the following prestigious shrines, dazaifu tenmangu, kitano tenmangu, heian jingu, and iwashimizu hachimangu, for supplying tachibana samples from their sacred trees. references baird n.a., etter 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s., kinjyo h., kawano s., 2005 single nucleotide polymorphism in shiikuwasha (citrus depressa hayata) chloroplast dna, trnl-trnf. jpn. j. trop. agr., 49: 246-251. uzun a., yesiloglu t., aka-kacar y., tuzcu o., gulsen o., 2009 genetic diversity and relationships within citrus and related genera based on sequence related amplified polymorphism markers (sraps). sci. hortic., 121: 306-312. yamamoto m., kouno r., nakagawa t., usui t., kubo t., tominaga s., 2011 isozyme and dna analyses of local citrus germplasm on amami islands, japan. j. japan. soc. hort. sci., 80: 268-273. yamamoto m., matsuo y., kuniga t., matsumoto r., yamada y., 1998 isozyme and rapd analyses of shiikuwashas (citrus depressa hayata). bull. natl. inst. fruit tree sci., 30/31: 39-51. yamamoto m., tominaga s., 2003 high chromosomal variability of mandarin (citrus spp.) revealed by cma banding. euphytica, 129: 267-274. yamamoto m., tsuchimochi y., ninomiya t., koga t., kitajima a., yamasaki a., inafukuteramoto s., yang x., yang x., zhong g., nasir n., kubo t., tominaga s., 2013 diversity of chloroplast dna in various mandarins (citrus spp.) and other citrus demonstrated by caps analysis. j. japan. soc. hort. sci., 82: 106-113. 265 1. introduction potato (solanum tuberosum l.) is one of the major crops contributing to the world’s food requirement because it is a rich source of starch, having protein of high biological value (eppendorfer and eggum, 1994). in lebanon, potato occupies an area of 19,700 ha with a total production of 460,000 t, but average yield is only 23 t ha-1, which is much below the crop’s potential productivity. such a low yield seems to be due to the imbalance in nutrients applied for the agricultural production of this crop. owing to the current trend of intensive cropping in lebanon, soils have developed multi-nutrient deficiencies. farmers usually diagnose and correct the deficiencies of nitrogen (n) and phosphorus (p), but often neglect the effect of deficiencies of other essential macronutrients such as potassium (k). in addition to n and p, potato is a heavy remover of soil potassium and its response to potassium varies with variety, source and method of potassium fertilizer application (sharma and sud, 2001; kumar et al., 2007; abd el-latif et al., 2011). since biomass and bulking rate of potato tubers are positively affected by synthesis and accumulation of starch, k plays a key role in this regard as it is the most efficient monovalant cation that stimulates the activity of the starch synthase enzyme, catalyzing the incorporation of simple glucose molecules into complex molecules of starch (moinuddin et al., 2004). starch accumulation is coupled with cell and tissue growth of the tubers as k enhances the overall growth of the plants (singh and singh, 1996), and facilitates the translocation of assimilates to the sinks/tubers (moinuddin et al., 2005), which could ultimately increase the tuber bulking capacity and, thereby, its biomass and yield. thus, potato removes large quantities of k and other soil nutrients, particularly n and p, in a short period coupled with a high rate of dry matter production (perrenoud, 1993; singh and trehan, 1998). an optimum k level, along with optimum levels of n and p would, therefore, be required to exploit the full genetic potential of the crop and achieve an improved level of tuber yield and quality. in spite the efforts aimed at optimizing potato response to k fertilization, little has been done on the time of k application for potato farming. this study, conducted in the central bekaa valley of lebanon, aims at assessing potato response to increasing in-season potassium rates (four progressive rates of k) applied at different times of tuber potato response to potassium application rates and timing under semi-arid conditions f. karam*, r. massaad**, s. skaf**, j. breidy**, y. rouphael*** * international center for agricultural research in dry areas, po box 5466, aleppo, syria. ** lebanese agricultural research institute, po box 287, zahleh, lebanon. *** department of crop production, faculty of agricultural engineering and veterinary medicine, lebanese university, dekwaneh-el maten, beirut, lebanon. key words: aggregate tuber yield, potassium application rates, potassium application timing, solanum tuberosum l., specific gravity, tuber dry matter. abstract: a two-year experiment (2004-2005) was conducted at tal amara research station in the bekaa valley of lebanon to evaluate the influence of progressive application of k rates and application timing on yield, yield components and tuber quality of potato (solanum tuberosum l. cv. agria). four levels of potassium (0 (k0), 75 (k75), 150 (k150), and 225 (k225) kg k2o ha-1) and two application timings (tuber initiation and tuber bulking stages) were used in a split-plot design. the progressive application of potassium fertilizer from 0 to 225 kg k2o ha-1 significantly affected the yield and yield components of potato. in both years, small grade tubers and aggregate tuber yield increased quadratically with increasing k application rates up to 150 kg k2o ha-1, reaching a plateau thereafter, showing luxury consumption of the nutrient at 225 kg k2o ha-1. in 2004 when averaged over k application rates, large and medium grade tubers and aggregated tuber yield were 120%, 22%, and 12% greater, respectively, with k application at tuber bulking than at tuber initiation. a similar trend was also observed in 2005, when the small grade tubers and aggregate tuber yield were 20% and 12% higher, respectively, with k application at tuber bulking than at tuber initiation stage. finally, no significant difference among treatments was observed for tuber dry matter (avg. 19.8%) and specific gravity (1.08 g cm-3). adv. hort. sci., 2011 25(4): 265-268 received for publication 2 august 2011 accepted for publication 20 october 2011 short note 266 growth (tuber initiation and tuber bulking stages) and to depict the optimal rate to achieve target yield. 2. materials and methods experimental site field experiments were conducted from april to august during the 2004 and 2005 growing years at tal amara research station in the central bekaa valley of lebanon (33º 51’ 44’’ n lat., 35º 59’ 32’’ n long., 905 m a.s.l). the details of the experimental site have been described elsewhere (karam et al., 2003, 2005, 2006, 2007, 2009 a,b, 2011). tal amara has a well-defined hot and dry season from may to october and very cold conditions for the remainder of the year. average seasonal rainfall is 592 mm, with 95% of the rain occurring between november and march, and a maximum of 145 mm in january. historical data indicate no rain occurrence at tal amara from june to september. rainfall amounts during the growing period were 35 and 25 mm during 2004 and 2005, respectively. soils of the experimental site were deep, non-calcareous, clay eutric cambisols with an average bulk density of 1.2 g cm-3. soil chemical and physical properties were: available n content 45.5 g kg-1, available p content 17.0 g kg-1, and available k content 11.5 g kg-1, organic matter content 1.2% and ph 7.9. crop management, k-treatments and experimental design potato (solanum tuberosum, l.) seeds of cultivar agria were sown under field conditions on 5 april 2004 and 11 april 2005. the soil was plowed and disked each year in anticipation for bed preparation. in both years, seeds were planted in a conventional “hill” system, where single soil beds were separated by relatively deep furrows spaced 70 cm apart, giving a theoretical plant density of 70000 plants ha-1. the experiments were conducted under optimum irrigation conditions in both years. at planting, the soil surface was thoroughly moistened using a sprinkler irrigation system at the application rate of 4.5 mm h-1. when plants reached 8 to 10 cm in height (two weeks after emergence), a drip irrigation system was installed along the furrows. the drip system consisted in polyethylene (pe) distribution lines, 16 mm in diameter, 40 cm spaced drippers, delivering each 4 l h-1 at 1 bar of head pressure. experiments were set up in a split plot design (main plot: potassium application rate; sub-plot: application timing). the trial covered four levels of potassium (0 (k 0 ), 75 (k 75 ), 150 (k 150 ), and 225 (k 225 ) kg k 2 o ha-1) and two application times (tuber initiation and tuber bulking stages), with five replications. each experimental unit consisted of six rows, 5 m in length. in both years, preplant fertilizer was broadcast (150 kg·ha-1; 17n 17p -17k) and incorporated into the soil. moreover, a fertilizer dose of 144 kg n and 96 kg p 2 o 5 ha-1 was applied in two splits after planting (35 days after planting, dap) and at tuber initiation (60 dap) uniformly to all the plots to assure rigorous shoot development. potassium was applied as k 2 o (0-0-46) in one split at tuber initiation (60 dap) and tuber bulking (80 dap) stages in four application rates with irrigation water. the experiments were concluded on 3 august 2004 (120 days after planting) and on 8 august 2005 (119 days after planting). data collection after harvest, the tuber yield was grouped into three grades, grade 1 (200-400 g), grade 2 (85-200 g) and grade 3 < 85 g. the grade-wise and aggregate tuber yields were recorded. tubers were dried in a forced-air oven at 80°c for 72 h and weighed to determine the tuber dry matter (dm). tuber specific gravity (tuber weight in air/tuber weight in water) (dunn and nylund, 1945) was determined on subsamples of acceptable tubers. statistical analysis all data were statistically analyzed by anova using the spss software package (spss 10 for windows, 2001). duncan’s multiple range test was performed at p=0.05 on each of the significant variables measured. 3. results and discussion in experiment 1 (2004), small grade tubers and aggregate tuber yield were significantly affected by k application rates, whereas large and medium grade tubers and aggregate tuber yield were highly influenced by k application timing, with no ‘k rates x k timing interaction’ (table 1). while in experiment 2 (2005), small grade tubers and aggregated tuber yield were significantly influenced by k application rates, k application timing, with no significant ‘k rates x k timing interaction’ (table1). in both years, no significant difference among treatments was observed for tuber dry matter (avg. 19.8%) and specific gravity (1.08 g cm-3) (table 1). these results on tuber quality (i.e. dry matter and specific gravity) are consistent with the findings of davenport and bentley (2001) who observed no response in tuber quality, mainly specific gravity, in response to increasing k rates. in contrast, others have reported that excess k fertilizer reduces dry matter content and specific gravity of tubers (westermann et al., 1994 a, b). explanations for this disagreement could be the different environments in which the plants were grown, and variations between potato genotypes in response to potassium application rates. in 2004 when averaged over k application rates, large and medium grade tubers and aggregated tuber yield were 120%, 22%, and 12% greater, respectively, with k application at tuber bulking than at tuber initiation. a similar trend was also observed in 2005, when the small grade tubers and aggregate tuber yield were 20% and 12% higher, respectively, with k application at tuber bulking than at tuber initiation stage (table 1). in both years, irrespective of k application timing, the highest small grade tubers yield was recorded with k application rates of 150 kg k 2 o ha-1 (avg. 30 and 33 t ha-1, in 2004 and 2005 respectively), 267 whereas the highest aggregate tuber yield was observed at both k 150 and k 225 with no significant difference observed between the two k application rates followed by k 75 and finally k 0 treatment. in both experiments, aggregate tuber yield increased quadratically with increasing k application rates up to 150 kg k 2 o ha-1, reaching a plateau thereafter, indicating the luxury consumption of the nutrient at 225 kg k 2 o ha-1 (table 1). significant increase in tuber yield of potato as a result of k application is well documented (cordova and valverede, 2001; singh et al., 2001; tawfik, 2001; umar and moinuddin, 2001; moinuddin et al., 2004, 2005). in fact, potato has a higher potassium requirement for optimum production compared to cereals, pulses, oilseeds, and other commercial crops and produces much more dry matter in short growth duration. it produces large amounts of starch due to k-mediated carbohydrate metabolism (perrenoud, 1993; singh and trehan, 1998). in addition, it helps in efficient translocation of photoassimilates to the developing sinks/tubers (beringer, 1978) and enabling the plants to fully utilize applied n and p fertilizers (mengel and kirkby, 1987). thus, k helps the potato tubers to attaine large size and heavier weight. this was evident in the current study, as we observed a progressive increase in aggregate tuber yield. these results are consistent with the findings of moinuddin et al. (2004, 2005) and abd ellatif et al. (2011) who showed an increase in tuber yield with a progressive application of k fertilizer from 0 to 225 kg k 2 o ha-1 (moinuddin et al., 2004, 2005) and from 72 to 120 kg k 2 o fed.-1 (abd el-latif et al. 2011). moreover, in line with our results, singh et al. (1997) reported that an increase in k application rates resulted in an increase in the yield of small-grade tubers. to summarize, we can conclude that the progressive application of potassium fertilizer from 0 to 225 kg k 2 o ha-1 significantly affected the yield and yield components of potato. in both experimentation years, small grade tubers and aggregate tuber yield increased quadratically with intable 1 effects of potassium application rates and k application timing on grade-wise and aggregate tuber yield, tuber dry matter and specific gravity of potato plants grown in 2004 and 2005 year k timing k rate grade-wise tuber yield (t ha-1) aggregate yield tuber dry matter specific gravity kg k2o ha-1 grade 1 grade 2 grade 3 t ha-1 % g cm-3 2004 tuber initiation stage 0 0.6 30.7 19.9 51.2 20.0 1.078 75 0.3 23.4 28.1 51.8 19.8 1.079 150 0.9 24.2 28.9 54.0 19.5 1.077 225 0.2 26.0 29.4 55.6 20.2 1.081 tuber bulking stage 0 1.0 27.4 25.5 53.9 19.5 1.077 75 1.7 30.8 24.3 56.8 19.2 1.075 150 0.4 33.0 31.1 a 64.5 20.0 1.079 225 1.3 36.5 25.2 63.0 20.0 1.080 significance (z) k rate ns ns ** ** ns ns k timing * * ns * ns ns k rate x k timing ns ns ns ns ns ns 2005 tuber initiation stage 0 0.8 29.1 22.7 52.6 19.8 1.078 75 1 27.1 26.2 54.3 19.5 1.077 150 0.6 28.0 30 58.6 19.8 1.078 225 0.8 31.2 27.3 59.3 20.1 1.081 tuber bulking stage 0 0.9 28.2 27.3 56.4 19.7 1.077 75 1.3 29 31.4 61.7 19.3 1.076 150 0.5 31.0 36 66.5 19.9 1.079 225 1 33.9 32.8 67.7 20.0 1.080 significance (z) k rate ns ns * ** ns ns k timing ns ns * * ns ns k rate x k timing ns ns ns ns ns ns (z) ns * **. ***non significant or significant at p < 0.05, or 0.01 respectively. creasing k application rates up to 150 kg k 2 o ha-1, reaching a plateau thereafter, showing luxury consumption of the nutrient at 225 kg k 2 o ha-1, indicating the detrimental effect of over fertilization. the results also demonstrated that k application during tuber bulking stage was more effective in terms of yield and yield components, and not in terms of quality, than at tuber initiation stage. references abd el-latif k.m., osman e.a.m., abdullah r., abd el kader n., 2011 response of potato plants to potassium fertilizer rates and soil moisture deficit. advances in applied science research, 2: 388-397. beringer h., 1978 functions of potassium in plant metabolism with particular reference to yield, pp. 185-202. in: sekhon g.s. (ed.) potassium in soils and crops. potash research institute of india, new delhi, india. cordova j., valverede f., 2001 potato response to potassium application in volcanic soil. better crops, 15: 1617. davenport j.r., bently e.m., 2001 does potassium fertilizer form, source, and time of application influence potato yield and quality in columbia basin? american journal of potato research, 78: 311-318. dunn l.e., nylund r.e., 1945 the influence of fertilizers on the specific gravity of potatoes grown in minnesota. american potato journal, 22: 275-288. eppendorfer w.h., eggum b.o., 1994 dietary fiber, starch, amino acids and nutritional value of potato as affected by sulphur, nitrogen, phosphorus, calcium and water stress. acta agric. scand. section b, soil plant sci., 44: 107-115. karam f., breidy j., stephan c., rouphael y., 2003 evapotranspiration, yield and water use efficiency of drip irrigated corn in the bekaa valley of lebanon. agricultural water management, 63: 125-137. karam f., kabalan r., breidi j., rouphael y., oweis t., 2009 a yield and water-production functions of two durum wheat cultivars grown under different irrigation and nitrogen regimes. agricultural water management, 96: 603-615. karam f., lahoud r., masaad r., daccache a., mounzer o., rouphael y., 2006 water use and lint yield response of drip irrigated cotton to the length of irrigation season. agricultural water management, 85: 287-295. karam f., lahoud r., masaad r., kabalan r., breidi j., chalita c., rouphael y., 2007 evapotranspiration, seed yield and water use efficiency of drip irrigated sunflower under full and deficit irrigation conditions. agricultural water management, 90: 213-223. karam f., masaad r., bachour r., rhayem c., rouphael y., 2009 b water and radiation use efficiencies in drip-irrigated pepper (capsicum annuum l.): response to full and deficit irrigation regimes. european journal of horticultural science, 74: 79-85. karam f., masaad r., sfeir th., mounzer o., rouphael y., 2005 evapotranspiration and seed yield of field grown soybean under deficit irrigation conditions. agricultural water management, 75: 226-244. karam f., saliba r., skaf s., breidy j., rouphael y., balendonck j., 2011 yield and water use of eggplants (solanum melongena l.) under full and deficit irrigation regimes. agricultural water management, 98: 1307-1316. kumar p., pandey s.k., singh b.p., singh s.v., kumar d., 2007 influence of source and time of potassium application on potato growth, yield, economics and crisp quality. potato research, 50: 1-13. mengel k., kirkby e.a., 1987 principles of plant nutrition. international, potash institute, 4th ed., bern, switzerland. moinuddin, singh k., bansal s.k., 2005 growth, yield, and economics of potato in relation to progressive application of potassium fertilizer. journal of plant nutrition, 28: 183-200. moinuddin, singh k., bansal s.k., pasricha n.s., 2004 influence of graded levels of potassium fertilizer on growth, yield and economic parameters of potato. journal of plant nutrition, 27: 239-259. perrenoud s., 1993 fertilizing for higher yield potato. international potash institute, 2nd ed., ipi bull. 8. bern, switzerland. sharma r.c., sud k.c., 2001 potassium and management for yield and quality of potato. central potato research institute, pp. 363-381. singh d., singh d., marwaha s.k., 1997 response of potato to nitrogen and potassium application under faizabad condition. journal indian potato association, 24: 40-43. singh j.p., trehan s.p., 1998 balanced fertilization to increase the yield of potato, pp. 129-139. in: barar m.s. and s.k. bansal (eds.) proceedings of the ipi-prii-pau workshop on balanced fertilization in punjab agriculture. international potash institute, bern, switzerland. singh m.v., jaiswal r.c., singh a.p., 2001 effect of phosphorus and potash on yield of potato crop raised from t.p.s. seedlings. journal indian potato association, 28: 267-269. singh v.n., singh s.p., 1996 influence of split application of potato. journal indian potato association, 23: 72-74. tawfik a.a., 2001 potassium and calcium nutrition improves potato production in drip-irrigated sandy soil. african crop science journal, 9: 147-155. umar s., moinuddin, 2001 effect of sources and rates of potassium application on potato yield and economic returns. better crops, 15: 13-15. westermann d.t., james d.w., tindall t.a., hurst r.l., 1994 a nitrogen and potassium fertilization of potatoes: sugars and starch. american potato journal, 71: 433-452. westermann d.t., tindall t.a., james d.w., hurst r.l., 1994 b nitrogen and potassium fertilization of potatoes: yield and specific gravity. american potato journal, 71: 417-432. 268 88 1. introduction strawberry (fragaria x annanasa dutch) has been grown commercially in various parts of the world for many years but in india it was only introduced in the early 1960’s (sharma and sharma, 2004) and it has now acclimatized well in different parts of india. this is essentially a temperate fruit crop hence its expansion in the kashmir valley has been gaining popularity in the last decade. it is not only consumed as fresh fruit but is also used in processed foods such as jam, ice cream, biscuits and so on. the demand for strawberry fruits for domestic as well as export markets has been increasing steadily. the standard planting time in the valley ranges from late october to the first fortnight of november, and harvesting from 15 march to late april in controlled conditions and from the second fortnight of april to late may in open conditions. climatic conditions under various production methods during the growing season may affect fruit quality. soluble solids concentration, acidity and colour of strawberry fruit have all been reported to be affected by environmental factors (sacks and shaw, 1994; vlachonasios et al., 1995), as well as harvest date (shaw, 1988). temperature is an important factor for floral initiation under short day conditions. the optimum temperature for short day floral initiation is 1518oc, while below 10°c and above 25°c short day induction is rather ineffective (manakasem and goodwin, 2001; sonsteby and heide, 2006; verheul et al., 2007). although both diurnal and nocturnal temperatures are important, strawberry requires an optimum daytime temperature of 22°c and nighttime temperature of 13°c for maximum growth and yield (shoemaker, 1977). growing strawberries under polyhouse decreases the dependence of fruit quality on climate and soil conditions. such cultivation system also enables better water, light and temperature control to a certain extent. in kashmir valley, cultivation is generally carried out under open field conditions and takes advantage of the local climatic conditions, but due to the long duration of winters the availability of fruit is very late. there is no information regarding the protected cultivation and management practices on the performance of commercialized cultivars of strawberry in the kashmir valley. hence, the present study was conducted to study the feasibility of growing strawberry under polyhouse to obtain an early crop and to evaluate the cultivars which may be suitable for commercial exploitation. 2. materials and methods experimental site and material the experiment was laid out under polyhouse during 2008-09 and 2009-10 at krishi vigyan kendra, pulwama, jammu and kashmir. the kvk is located at 33° north and 74° east at an altitude of 1601 m amsl. the mean annual rainfall ranges from 500 to 850 mm. the minimum and maximum temperatures of the station during summers range between 10 and 30oc and between -4 and 10°c durgrowth, yield and fruit quality of strawberry under protected cultivation in south kashmir a. kumar, i. ahad krishi vigyan kendra, extension training centre, sher-e-kashmir university of agricultural sciences and technology kashmir, malangpora (pulwama) jammu & kashmir, india. key words: fruit quality, growth, protected cultivation, strawberry, yield. abstract: field experiments were conducted at krishi vigyan kendra, pulwama, jammu and kashmir at an altitude of 1601 m amsl to identify the suitable strawberry cultivars for higher production of good quality fruits. eight strawberry cultivars were evaluated for two consecutive years (2008-09 and 2009-10) under polyhouse conditions. maximum plant spread (27.43 cm) along with maximum number of runners (8.54) was produced by ‘chandler’. ‘tioga’ produced first flower in 97 days after planting, while runners of ‘chandler’ (56.79) flowered for maximum number of days. ‘chandler’ produced maximum number of flowers per plant (27.23) and set maximum berries (86.01%), however final yield of berries was more in ‘tioga’ which recorded maximum yield per plot (2.26 kg), closely followed by ‘chandler’ (2.19 kg). berry weight (12.24 g) and berry size (5.10 cm length and 4.73 cm) was maximum in ‘tioga’. ‘catskill’ registered maximum for all the biochemical characters, closely followed by ‘tioga’. overall, ‘chandler’, ‘catskill’ and ‘tioga’ performed well under polyhouse conditions in kashmir valley. adv. hort. sci., 2012 26(2): 88-91 received for publication 17 october 2011. accepted for publication 21 may 2012. 89 ing winter under open conditions. a polyhouse with steel pipe framework clad with twin layer uv stabilized 200 µm plastic sheet of was used to create a modified environment. the polyhouse was additionally fitted with a high pressure fan on each west side. under the polyhouse the temperature was maintained up to 25°c. the soil of the location is silty clay, loam neutral in reaction (ph 7.07) having organic c 10.02 g/kg, available n 248.6 kg/ha, available p 14.7 kg/ha and available k 250.3 kg/ha. the experimental materials were comprised of eight commercial cultivars (‘catskill’, ‘chandler’, ‘confutura’, ‘gorella’, ‘pajaro’, ‘selva’, ‘tioga’ and ‘fern’) collected from skuast-k and the department of horticulture, ramban, jammu and kashmir. the experiment was laid out in a completely randomized block design (crbd) with three replications. the spacing between the runners was 30 x 30 cm on 1 x 1 m2 raised beds of 15 cm height with 55 cm spacing between the beds. uniform runners were planted in the first week of november 2008 in three rows on each bed accommodating nine runners. for the second year crop, the emerged runners were removed in the last week of october 2009 in order to maintain the proper spacing for the next year’s crop. usual irrigations, manures and fertilizers, weeding and hoeing were applied equally to the experimental plots during the study years. observations recorded data were recorded for different growth, flowering and fruiting characters for three years. plant spread (cm) and length of the runners (cm) was measured with the help of a measuring tape. number of runners per plant, number of flowers per plant and number of berries per plant were counted from five randomly selected plants. days to first flower was recorded from the date of planting of runners to initiation of first flower. flower duration was counted by subtracting the date of initiation of first flower from the date of last flowering. percentage of berry set was calculated by dividing the number of berries by the number of flowers. yield per plant (g) was calculated by weighing whole fruits from a single plant. ten fruits were randomly selected for all the physio-chemical characters. berry weight was determined with the help of a weighing scale; berry length and width were determined using a vernier calliper. tss, acidity and tss/acid ratio were estimated using standard procedures. total sugar and reducing sugar were determined by shaffer somogy, micro method (ranganna, 1991). data on temperature and humidity under polyhouse were recorded with a portable thermohygrometer. data analysis the pooled data of two years were statistically analyzed following panse and sukhatme (1985). the mean of attributes was compared by paired ‘t’ test and the least significant difference was calculated at 5% level. 3. results and discussion the average monthly data on minimum and maximum temperature and relative humidity inside the polyhouse from transplanting to harvesting are presented in figure 1. the pooled data of two consecutive years shown in table 1 reveal that ‘chandler’ had maximum plant spread (27.43 cm) which was statistically at par with ‘catskill’ (25.49 table 1 growth and flowering characters of strawberry cultivars under polyhouse cultivar plant spread (cm) no. of runners/ plant runners length (cm) days taken first flower to produce duration of flowering number of flower/ plant catskill 25.49 ef 7.09 ef 81.94 f 106 def 50.86 ab 26.92 e chandler 27.43 f 8.54 h 82.59 fg 101 bc 56.79 d 27.23 f confutura 24.18 cde 6.11 d 89.78 h 99 ab 53.92 bcd 20.93 ab gorella 25.44 def 5.67 bc 75.40 de 103 cd 51.12 abc 21.90 bc pajaro 21.75 ab 4.89 a 67.58 b 105 de 49.39 a 20.48 a selva 23.08 bcd 5.44 b 73.95 cd 116 h 47.82 a 21.84 bc tioga 20.19 a 7.35 fg 70.42 bc 97 a 55.49 cd 25.64 d fern 22.35 abc 6.93 e 60.92 a 110 g 48.91 a 21.45 bc mean 23.74 6.50 75.32 104.6 51.79 23.31 cd 0.05 2.40 0.29 3.70 3.84 4.43 0.99 fig. 1 average maximum, minimum temperature (oc) and relative humidity (%) month wise from transplanting to harvesting time. 90 cm) and ‘gorella’ (25.44 cm), while minimum plant spread was recorded for ‘tioga’ (20.19 cm). maximum number of runners per plant in pooled data was found in ‘chandler’ (8.54) which differed significantly from all other cultivars, whereas minimum number of runners per plant was recorded in ‘pajaro’ (4.89). the pooled data of two years shows that ‘confutura’ (89.78 cm) produced maximum runner length which differed significantly from the other cultivars; minimum length of runners was observed in ‘selva’ (67.58 cm). two years of data relative to runner length shows that in the first year runner length was greater than in the second year: the material may have degenerated with the passage of time (childers, 1975). ‘tioga’ (97) produced flowers earlier among the considered cultivars and was closely followed by ‘confutura’ (99) and ‘chandler’ (101); ‘selva’ ranked last and took 116 days to produce first flower (table 1). runners of ‘chandler’ (56.79) flowered for the maximum number of days followed by ‘tioga’ (55.49) and ‘confutura’ (53.92) which was statistically at par with both the cultivars, whereas ‘selva’ (47.82) produced flowers for the fewest number of days. kaska et al. (1997) cultivate nine cultivars of strawberry under high tunnels in adana (turkey) observed that ‘selva’ took maximum days to produce first flower and flowered for least number of days while ‘chandler’ flowered for maximum number of days. maximum number of flowers per plant (27.73) (table 1) and maximum number of berries per plant (23.42) (table 2) were both produced by ‘chandler’ which was statistically at par with ‘catskill’ (26.92) with respect to the number of flowers per plant, while for the number of berries per plant the former was significantly higher than all the cultivars. this indicates that the number of flowers per plant certainly has a bearing on the number of fruits per plant to be harvested, i.e. greater the number of flowers/plant, greater the number of fruits to be harvested but the total yield per plant may vary due to berry weight. ‘pajaro’ (20.48) produced the fewest flowers per plant, which was statistically at par with ‘fern’ (21.45), however the lowest number of berries per plant was produced by the latter (14.49) which was statistically at par with ‘pajaro’ (14.74). paraskevopoulou-paroussi et al. (1990) also recorded a minimum number of flowers per plant and number of berries per plant in ‘pajaro’ and ‘fern’ while growing these cultivars under greenhouse in northern greece. the data in table 2 reveal that ‘chandler’ (86.01%) gave the maximum berry set, which was statistically at par with ‘tioga’ (83.48 %); the minimum berry set (71.84%) was recorded for ‘pajaro’. maximum yield per plot of berries was recorded in ‘tioga’ (2.26 kg) which was statistically at par with ‘chandler’ (2.19 kg) and ‘catskill’ (2.17 kg), and minimum yield per plot was recorded in ‘selva’ (1.08 kg). paraskevopoulou-paroussi et al. (1990) also noted that ‘pajaro’ (24%) and ‘fern’ (38%) produced marketable yield under greenhouse conditions in northern greece, however, kaska et al. (1997) reported that ‘chandler’ and ‘selva’ produced the highest and lowest yield, respectively under high tunnel conditions in adana (turkey). observations from two years of data on yield per plant shows that yield was greater in the second year than the first which might be due to substantial annual variation in fruit set in strawberry (smolarz et al., 1968; misic et al., 1976). ‘tioga’ scored as having maximum berry weight (12.24 g) along with maximum berry length (5.10 cm) and berry breadth (4.73 cm), closely followed by ‘chandler’. ‘selva’ had minimum berry weight (8.71 g), however minimum size [i.e. berry length (3.70 cm) and berry breadth (3.62 cm)] was recorded for ‘pajaro’. pathak et al. (2006) observed similar results with respect to weight, length and breadth of berries while growing strawberry cultivars under cover. maximum tss was scored by ‘catskill’ (9.85%), followed by ‘tioga’ (9.32%), however both these cultivars significantly differed from each other (table 3); ‘selva’ had minimum tss (6.72%). minimum acidity was observed in ‘catskill’ (0.88%), closely followed by ‘tioga’ (0.89%), yet the pooled data of two years showed non significant results. ‘catskill’ (10.51) showed maximum tss/ acid ratio which was statistically at par with tioga (10.47) while minimum tss/acid ratio was recorded for ‘selva’ (6.25). maximum reducing sugar (6.27 %) and total sugar (8.09%) were observed in ‘catskill’ which was the highest table 2 yield and fruiting characters of strawberry cultivars under polyhouse cultivar number of berries/ plant berry set (%) yield/plot (kg) berry weight (g) berry length (cm) berry breadth (cm) catskill 22.32 e 82.90 e 2.17 d 11.11 e 4.98 de 4.55 de chandler 23.42 f 86.01 f 2.19 d 12.11 f 4.79 d 4.31 c confutura 16.93 c 80.86 d 1.44 d 10.54 d 4.78 d 4.48 cd gorella 16.62 c 75.81 c 1.28 c 9.76 c 4.25 bc 3.76 a pajaro 14.74 a 71.84 b 1.18 b 9.03 b 3.70 a 3.62 a selva 15.94 b 72.73 b 1.08 a 8.71 a 4.39 c 3.72 a tioga 21.32 d 83.48 ef 2.26 d 12.24 f 5.10 e 4.73 e fern 14.49 a 66.95 a 1.13 ab 9.22 b 4.03 b 4.01 b mean 18.22 77.57 1.59 10.34 4.50 4.14 cd 0.05 0.64 2.73 0.09 0.27 0.22 0.23 91 among all the studied cultivars, and ‘pajaro’ scored minimum reducing sugar (3.71%) and total sugar (5.55%). our investigation showed much variation in the various cultivars for all the characters and this could be attributed to the genetic make up of the cultivars (dhaliwal and singh, 1983; chandel and badiyala, 1996). factors which may significantly influence strawberry composition include mineral and organic fertilization but weather conditions and variety are also important. it is concluded from the present study that ‘chandler’ for growth and yield characteristics, ‘catskill’ for biochemical characters and ‘tioga’ for yield and physical characters of strawberry fruits are profitable for cultivation under polyhouse conditions in the kashmir valley. references chandel j.s., badiyala s.d., 1996 performance of some strawberry cultivars in foothills of himachal pradesh. annals of agricultural research, 17(4): 375-378. childers n.f., 1975 modern fruit science. horticulture publication, rutgers university nichol avenue, new brunswick, n.j., usa, pp. 976. dhaliwal g.s., singh k., 1983 evaluation of strawberry cultivars under ludhiana conditions. haryana journal of horticultural science, 12(1/2): 36-40. kaska n., turemis n., kafkas s., comlekcioglu n., 1997 the performance of some strawberry cultivars grown under high tunnel in the climatic condition of adana (turkey). acta horticulturae, 439: 297-300. manakasem y., goodwin p.b., 2001 responses of day neutral and june bearing strawberries to temperature and day length. j. of hortic. sci. and biotech., 76: 629-635. misic p.d., tdodrovic r.r., lakie n.k., 1976 self fertility in strawberry. jugoslovensko vocarstovo, 10(37/38): 355-360. panse v.g., sukhatme p.v., 1985 statistical methods for agricultural workers. indian council of agricultural research (icar), new delhi, india. paraskevopoulou-paroussi g., vassilakakis m., dogras c., 1991 performance of five strawberry cultivars under plastic greenhouse or field conditions in northern greece. acta horticulturae, 287: 273-280. pathak p.k., gurung m.r., mitra s.k., 2006 performance of strawberry cultivars under cover in the plains of west bengal. the orissa journal of horticulture, 34(2): 35-37. ranganna s., 1991 manual of analysis of fruits and vegetable products. tata mcgraw hill publishing co., new delhi, india, pp. 77. sacks e.j., shaw d.v., 1994 optimum allocation of objective colour measurements for evaluating fresh strawberries. j. of americ. soc. for hortic. sc., 119: 330-334. sharma v.p., sharma r.r., 2004 the strawberry. indian council of agricultural research (icar), new delhi, india. shaw d.v., 1988 genotypic variation and genotypic correlations for sugars and organic acids of strawberries. j. of americ. soc. for hortic.l sc., 113: 770-774. shoemaker j.s., 1977 small fruitculture. the avi publishing company, westport, connecticut, usa. smolarz k.d., sobezy k.w., kostrzena z., michalski w., bustydzienski w., 1968 yield of four strawberry cultivars in different regions of the country. prace. instytutu sadownictwa, 12: 21-26. sonsteby a., heide o.m., 2006 dormancy relations and flowering of the strawberry cultivars korona and elsanta as influenced by photoperiod and temperature. scientia horticulturae, 110: 57-67. verheul m.j., sonsteby a., grimstad s.o., 2007 influences of day and night temperatures on flowering of fragaria x ananassa duch., cvs. korona and elsanta. scientia horticulturae, 112: 200-206. vlachonasios c., vasilakakis m., dogras c., mastrokostas m., 1995 out of season glasshouse strawberry production in north greece. acta horticulturae, 379: 305-312. table 3 biochemical characters of strawberry cultivars under polyhouse cultivar tss (%) acidity (%) tss/acid ratio reducing sugar (%) total sugar (%) catskill 9.85 h 0.88 10.51 g 6.27 f 8.09 e chandler 9.06 ef 0.94 9.64 d 5.45 d 7.18 d confutura 9.24 fg 0.97 10.11 e 5.85 e 7.14 d gorella 8.03 d 1.02 7.85 c 4.63 c 6.65 c pajaro 7.81 b 1.00 7.78 c 3.65 a 5.55 a selva 6.72 a 1.04 6.25 a 4.04 b 5.86 b tioga 9.32 g 0.89 10.47 fg 5.88 e 7.19 d fern 7.83 cd 1.02 7.64 bc 3.76 a 5.70 a mean 8.48 0.97 8.78 4.94 6.67 cd 0.05 0.20 ns 0.35 0.19 0.22 impaginato 85 1. introduction sunflower (helianthus annuus l.) is one of the most important oilseed crops. besides soy and rapeseed oil, sunflower oil is ranking third with a worldwide production of about 44 million tons each year from 2012 to 2016, 28 mill ion tons in the eu (committee for the common organization of agricultural markets, 2016). sunflowers prove to be a protein source of great interest for human nutrition; moreover, the residues originating from oil extraction are rich in phenolic antioxidants, which account for 1-4% of the total mass with chlorogenic acid being the predominant component (weisz et al., 2009). polyphenols in sunflower seeds were identified and quantified after hplc analysis (aramendia et al., 2000; pedrosa et al., 2000). the main phenolic compounds present in both the kernel and hull besides chlorogenic acid are caffeic acid and caffeoylquinic derivatives. phenolic compounds in sunflower seeds have been shown to exert a high antioxidative potential, which might be beneficial from a biofunctional point of view and may be used as effective antioxidants for sunflower oils (de leonardis et al., 2003, 2005; anjum et al., 2012). organic agriculture is most widely used and its benefits concern overall the environment and the health of food, which is not contaminated with pesticides and synthetic fertilizers. however organic fertilizer of biological origin may lead in the long term to the “conventionalization of organic farming” (darnhofer et al., 2010). in order to go deeper in the organic management, the montepaldi long term experiment (molte) trial in central italy has been set up to compare three agro-ecosystems with different management: two organic (old organic since 1992 and young organic since 2001) and one conventional (migliorini, 2014). the aim of this research is to follow the fate of polyphenols in teguments, kernels and oil from sunflower seeds grown under the above-mentioned three different conditions (one conventional and two organic) in order to assess whether such condition may affect the polyphenols profile and which compounds may be regarded as innovative parameters in order to deeper investigate the relation among soil condition, agricultural management and seed characteristics. 2. materials and methods the montepaldi long term experiment (molte) has been active since 1991 in the farm of the university of florence (location montepaldi, san adv. hort. sci., 2017 31(2): 85-88 doi: 10.13128/ahs-20608 seeds and oil polyphenol content of sunflower (helianthus annuus l.) grown with different agricultural management a. romani 1, p. pinelli 1, v. moschini 2, d. heimler 2 (*) 1 dipartimento di statistica, informatica, applicazioni “g. parenti”, università degli studi di firenze, viale morgagni, 40, 50134 firenze, italy. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: organic management, polyphenol content, sunflower. abstract: using a long term experiment (20 and 11 years of organic cultivation on the same soil), sunflower was cultivated under organic management and in a different part of the same farm under conventional management. kernels, teguments and oils were analyzed for their polyphenols content. five caffeoylquinic acids were identified. no qualitative differences were found in the three cases, while quantitative differences have been pointed out and discussed. (*) corresponding author: daniela.heimler@unifi.it received for publication 2 may 2017 accepted for publication 15 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 85-88 86 casciano, val di pesa, long. 11°09’08’’ e, lat. 43°40’16’’ n) covering a slightly sloping surface of about 15 ha at 90 m a.s.l. the molte experiment has a system approach and includes the following three different micro agro-ecosystem managements: a) “old organic” (oldo) with an area of 5.2 ha, divided into 4 fields under organic since 1992; b) “youngorganic” (yngo) with an area of 5.2 ha, divided into 4 fields (integrated farming) from 1992 to 2000 and converted into organic management since 2001; c) “conventional” (conv) area of 2.6 ha divided into 2 conventional fields, where farming techniques used were those normally used in the territory of the study area for conventional management. on october 28, 2013, after harvesting the whole sunflower plant, seeds were put in an oven at 60°c for 48 hours and then preserved in paper bags. seeds were manually dehulled in order to obtain kernel and tegument. phenolic compounds were extracted for 30 min from kernels (about 2 g) and from teguments (about 1 g) with 70:30 ethanol/water (25 and 15 ml, respectively). the extracts were evaporated under vacuum at room temperature and finally dissolved in 10 ml ethanol/water (70:30). cold-pressed seeds oils (25 ml) were extracted with 3x25 ml of 70:30 ethanol/water, adjusted to ph 2.0 with formic acid; each step involved an extraction for 30 min at room temperature. the extracts were combined and defatted with 3x50 ml hexane. the defatted extracts were evaporated to dryness under vacuum at room temperature and finally dissolved in ethanol/water (70:30) to a final volume of 4 ml. solvents and reagents all the solvents (hplc grade) and formic acid (acs reagent) were purchased from aldrich chemical company inc. (milwaukee, wisconsin, usa). chlorogenic acid was obtained from extrasynthese s.a. (lyon, nord-genay, france). the hplc-grade water was obtained via double-distillation and purification with a labconco water pro ps polishing station (labconco corporation, kansas city, usa). hplc analysis hplc/dad analysis. the hplc/dad analyses were performed with an hp 1100l liquid chromatograph equipped with hp dad (agilent technologies, palo alto, ca, usa). a kinetex c18 column 100×2.1 mm, 5 μm (phenomenex) operating at 30°c was used. the eluents were h2o adjusted to ph 3.2 by formic acid and acetonitrile. a four-step linear solvent gradient was performed starting from 100% water up to 100% acetonitrile, with a flow rate of 0.2 ml/min for a 30min period (table 1). hplc/esi-ms analysis. the hplc-ms analyses were performed using an hp 1100l liquid chromatograph equipped with a dad and 1100 ms detectors. the interface was an hp 1100 msd api-electrospray (agilent technologies). mass spectrometer operating conditions were the following: gas temperature 350°c at a flow rate of 10.0 l/min, nebulizer pressure 30 psi, quadrupole temperature 30°c and capillary voltage 3500 v. the mass spectrometer operated in positive and negative ionization mode at 80-120 ev, for both ionization modes. 3. results and discussion in figure 1, the chromatographic profile of organic sunflower kernel, registered at 330 nm, maximum wavelength of absorbance of caffeic acid and its derivatives, is reported. eight compounds have been identified: 3-o-caffeoylquinic acid, chlorogenic acid, 4-o-caffeoylquinic acid, three derivatives of caffeic acid, 3,5-o-dicaffeoylquinic acid, and 4,5-o-dicaftable 1 linear solvent gradient system used in hplc-dad and hplc-ms analysis time (min) a b 0.10 100.0 0.0 5.00 80.0 20.0 7.00 80.0 20.0 13.00 70.0 30.0 18.00 70.0 30.0 22.00 30.0 70.0 26.00 30.0 70.0 30.00 0.0 100.0 fig. 1 hplc chromatogram of sunflower kernel extract recorded at 330 nm. peaks= 1. 3-o-caffeoylquinic acid; 2. chlorogenic acid; 3. 4-o-caffeoylquinic acid; 4-6. caffeic acid derivatives; 7. 3,5o-dicaffeoylquinic acid; 8. ac. 4,5o-dicaffeoylquinic acid. solvent a= h2o adjusted to ph 3.2 by hcooh. solvent b= ch3cn. romani et al. seeds and oil polyphenol content of sunflower 87 feoylquinic acid. in the shells the same compounds with the exception of 4-o-caffeoylquinic acid and the two dicaffeoylquinic acids were identified. the same caffeoyl and dicaffeoylquinic acid derivatives were already found by aramendia et al. (2000), pedrosa et al. (2000), and weisz et al. (2009). the qualitative pattern does not change along with the agricultural management. as concerns quantitative data, table 2 reports the quantitative data of kernel and teguments. total polyphenols amount is the lowest, in the case of kernel, when the plant grows on the old organic field, while it is almost the same with the other two managements; in the case of teguments, on the contrary, the lowest amount is observed in the case of conventional management. it should however be noted that in the case of teguments, polyphenols content is very low changing from about 10% to 2% with respect to kernel content. chlorogenic acid is the main compound and it accounts for about 90% in tegument. chlorogenic acid in our samples, both kernel and tegument, is about ten times more abundant with respect to the quantitative data reported by aramedia et al. (2000) and pedrosa et al. (2000). the comparison of quantitative data is however difficult since different techniques and sample treatments are involved (weisz et al., 2009). polyphenols content along with agriculture management has recently reviewed and in most vegetables grown under organic conditions, a higher content of polyphenols has been found; on the other hand a higher soil nitrogen availability decreases polyphenols content (heimler et al., 2017). our data support previous findings that indicate how old managed organic soil was the most efficient in term of c and n storage (migliorini et al., 2014). furthermore, when the individual compounds are taken into account the relative percentages of compounds from new organic and conventional managed soils are similar, while in the case of the old managed organic soil, notwithstanding the lowest total polyphenol content, a higher dicaffeoylquinic acids content was found (17% with respect to 12%). chlorogenic acid and dicaffeoylquinic acids derive from the phenylpropanoid pathway. generally, this pathway is induced by biotic and abiotic stress such as wounding, uv irradiation, or pathogen attack (moglia et al., 2008). no information is available on the regulation of dicaffeoylquinic acids in any plant species, even if the same regulation of chlorogenic acid synthesis could be foreseen (moglia et al., 2008). in table 3, the data of sunflower oil are reported. oil has been obtained by means of a mechanical oldo= old organic; youngo= young organic; co= conventional (see experimental section). nd= not determined. standard deviation within brackets. table 2 polyphenols (mg/g, fresh weight) in sunflower kernel and tegument compound agricultural management kernel tegument oldo youngo co oldo youngo co 3-o-caffeoylquinic acid 2.51(0.37) 4.57(0.82) 4.74(0.76) 0.08(0.01) 0.08(0.01) 0.05(0.01) chlorogenic acid 6.25(1.12) 11.39(1.94) 11.82(1.42) 0.95(0.18) 0.96(0.18) 0.41(0.07) 4-o-caffeoylquinic acid 0.44(0.08) 0.8(0.11) 0.83(0.13) 0.03(0.005) traces traces caffeic acid derivatives 0.21(0.04) 0.38(0.04) 0.39(0.06) traces n.d. traces 3,5-o-dicaffeoylquinic acid 1.3(0.24) n.d. n.d. n.d. n.d. n.d. 4,5-o-dicaffeoylquinic acid 0.77(0.11) 2.36(0.34) 2.45(0.27) n.d. n.d. n.d. total polyphenols 11.48 19.5 20.23 1.06 1.04 0.46 oldo= old organic; youngo= young organic; co= conventional (see experimental section). nd= not determined. standard deviation within brackets. table 3 polyphenols (mg/l) in sunflower oil compound agricultural management oldo youngo co 3-o-caffeoylquinic acid 0.232(0.192) 0.35(0.09) 0.176(0.057) chlorogenic acid 1.96(0.393) 2.43(0.11) 1.936(0.12) p-coumaroylquinic acid 2.36(0.318) 0.112(0.022) 0.592(0.079) 4-o-caffeoylquinic acid 0.048(0.005) 0.104(0.011) 0.08(0.02) caffeic acid derivatives 2.28(0.644) 0.328(0.079) 3.216(0.415) not identified polar compounds 0.504(0.011) 1.2(0.045) 1.456(0.429) total polyphenols 7.384 4.368 7.456 adv. hort. sci., 2017 31(2): 85-88 88 press, which is the only technology that allows the maintenance of polyphenols high content (bendini et al., 2011). oil yields are different (22.2% for old organic soil, 29.8% for young organic soil and 24.7% for conventional management) and, the young organic soil, with the highest yield, exhibits the lowest total polyphenol content. sunflower oil-amount of 10 mg kg-1 has been reported (de leonardis et al., 2005). polyphenols content of the three seeds oils (table 3) are almost in accordance with such amount. young managed organic soil gave rise to an oil with the smallest polyphenols content notwithstanding the high polyphenols content of almond and teguments. acknowledgements the authors acknowledge the regione toscana ‘‘praf 2012-2015 misura 1.2 e)’’ program (project volatosca). references anjum f.m., nadeem m., khan m.i., hussain s., 2012 nutritional and therapeutic potential of sunflower seeds: a review. brit. food j., 114: 544-552. aramendia m.a., garcia i.m., lafont f., lizaso j., marinas j.m., urbano f.j., 2000 rapid determination of chlorogenic acid and related compounds in sunflower seeds by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. rapid commun. mass spectrom., 14: 1019-1022. bendini a., barbieri s., valli e., buchecker k., canavani m., gallina toschi t., 2011 quality evaluation of cold pressed sunflower oils by sensory and chemical analysis. eur. j. lipid sci. technol., 113: 1375-1384. committee for the common organization of agricultural markets, 2016 oilseeds and protein crops, market situation. http://ec.europa.eu/agricult u r e / s i t e s / a g r i c u l t u r e / f i l e s / c e r e a l s / presentations/cereals-oilseeds/market-situationoilseeds_en.pdf darnhofer i., lindenthal t., bartel-kratochvil r., zollitsh w., 2010 conventionalization of organic farming practices: from structural criteria towards an assessment based on organic principles. a review. agron. sust. dev., 30: 67-81. de leonardis a., macciola v., di domenico n., 2005 a first pilot study to produce a food antioxidant from sunflower seed shells (helianthus annuus). eur. j. lipid sci. technol., 107: 220-227. de leonardis a., macciola v., di rocco a., 2003 oxidative stabilization of cold pressed sunflower oil using phenolic compound of the same seed. j. sci. food agric., 83: 523-528. heimler d., romani a., ieri f., 2017 plant polyphenol content, soil fertilization and agricultural management: a review. eur. food res. technol., 243(7): 1107-1115. miglioriini p., moschini v., tittarelli f., ciaccia c., benedettelli s., vazzana c., canaliu s., 2014 agronomic performance, carbon storage and nitrogen utilization of long-term organic and conventional stockless arable systems in mediterranean area. europ. j. agron., 52: 138-145. moglia a., lanteri s., comino c., acquadro a., de vos r., beekwilder j., 2008 stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke. j. agric. food chem., 56: 8641-8649. pedrosa m.m., muzquiz m., vallejo c.g., burbano c., cuadraro c., ayet g., robredo l.m., 2000 determination of caffeic and chlorogenic acids and their derivatives in different sunflower seeds. j. sci. food agric., 80: 459-464. weisz g.m., kammerer d.r., carle r., 2009 – identification and quantification of phenolic compounds from sunflower (helianthus annuus l.) kernels and shells by hplc-dad/esi-ms. food chem. 115: 758-765. impaginato 14 adv. hort. sci., 2011 25(1): 14-20 received for publication 15 september 2010. accepted for publication 14 january 2011. radiation-induced chromosomal aberrations in grape phylloxera h. makee, n. tafesh, i. idris department of biotechnology, atomic energy commission of syria, po box 6091, damascus, syria. key words: chromosome aberrations, grape phylloxera, irradiation, reproduction. abstract: chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation were detected. the results showed that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. when phylloxera nymphs were irradiated, the chromosomal number on the metaphase plate of some embryos’ cells was increased. the result indicated that the chromosomal aberrations influenced the mortality, longevity and reproduction of phylloxera. eight autosomal chromosomes were identified according to their length. additionally, the karyotype of irradiated and unirradiated populations of local phylloxera strain was defined. 1. introduction grape phylloxera, daktulosphaira vitifoliae fitch (homoptera: phylloxeridae), an aphid-like gall-forming parasite, is one of the most destructive insect pests of cultivated grape vitis vinifera l. world wide. grape phylloxera causes direct damage to grapevine by forming damaging root galls. these galls are metabolically active organs suited to match the nutritional requirements of phylloxera and can support populations with high reproductive rates. granett et al. (1985) reported that there was frequent decline of commercial vineyards as result of this pest, and consequently losses of quality and yield of grapes. the use of resistant rootstocks is the most common and effective means of managing phylloxera. our previous studies showed that some rootstocks were more resistance than others to grape phylloxera (makee et al., 2003). however, for unknown reasons, the resistance of some rootstocks may break down and farmers must replant vineyards (granett et al., 1983; song and granett, 1990; de benedictis and granett, 1993). therefore, additional ways to control this pest should be considered. sanitation and quarantine can be considered as required procedures to prevent the movement of this serious pest. insecticides and hot water dip treatments are used as quarantine treatments (granett et al., 2001). ionizing radiation has been recognized as an alternative method for treating agricultural products to overcome quarantine barriers in trade (fao, 2003). irradiation treatment does not influence the quality of many commodities; it is reasonably safe to the consumers and environment. previously, several authors have presented reviews of this subject (hallman, 1998; johnson and marcotte, 1999; hallman, 2000, 2001). irradiation has been successfully used for the control of many insect pests such as codling moth cydia pomonella (l.), beetle prionolplus reticularis white (lester et al., 2000), apple maggot, rhagoletis pomonella (walsh), the borer eucosma notanthes meyrick (lin et al., 2003), coconut scale aspidiotus destructor signoret (follett, 2006), the weevil sternochetus mangiferae (f.) (follett, 2001), cigarette beetle, lasioderma serricorne (f.) (hu et al., 2002), and the rice weevil sitophilus zeamais motschulsky (hu et al., 2003). makee et al. (2008) proposed that gamma irradiation could be economically very useful in quarantine treatments against phylloxera. the results showed that the percentage of matured phylloxera females and fecundity were markedly reduced when higher doses of gamma irradiation were used (makee et al., 2008). however, to our knowledge efforts of associate performance parameters of irradiated phylloxera with chromosomal rearrangements have not yet been studied. therefore, the purpose of the present research was to detect chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation. the influence of such chromosomal aberrations on longevity and reproduction of phylloxera was examined. moreover, determination of the number and length of autosomal and sex chromosomes was undertaken. such 15 study will allow definition of the karyotypes of irradiated and unirradiated populations of the local phylloxera strain. 2. materials and methods establishment of phylloxera colony grape phylloxera was originally collected from field-infested roots of the local grape varieties in southern parts of syria. all insects were reared on fresh and healthy pieces of local grape variety balady roots, 4-7 mm in diameter and 5-7 cm long as outlined in makee et al. (2003). the experiments were conducted at 25±1°c with 70±5% rh, and 24 hr darkness. egg sterilization was carried out as described by makee et al. (2003). a co60 source (issledov gamma irradiator, techsnabexport co. ltd., moscow, russia) delivering a dose rate of 60 gy/min was used to treat the insects. effect of irradiation on matured females, fecundity, and oviposition period new phylloxera eggs were placed on fresh root pieces and left until hatching. a group of three-weekold feeding phylloxera nymphs was taken. nymphs were irradiated at different doses: 0-10-20 and 30 gy (n= 25 nymphs at each dose). irradiated and unirradiated nymphs were kept at 25±1°c with 70±5% rh, and 24 hr darkness. a daily microscopic inspection of all phylloxera stages at each applied dose was carried out. the number of feeding nymphs, which were able to develop to adult stage, was observed to determine the percentage mortality at each dose. female fecundity and longevity was determined at each tested dose. chromosome preparations mitotic metaphase chromosomes were obtained from 24 to 36-hr-old embryos. at each dose, 10 eggs were taken and placed in a 1.5 ml tube. the eggs in each tube were fixed in carnoy’s fixative (ethanol:chloroform:acetic acid 6:3:1) and shaken for 10 min. then a drop was taken and transferred onto a clean slide. shortly before drying, a drop of 60% acetic acid was added and macerated for 2-3 min with fine tungsten needles. then the specimen was spread on the slide using a heating plate at 45°c to allow evaporation of the acetic acid. the preparation was then stained and mounted in lactic acetic orcein for 5 min; redundant stain was removed with a piece of filter paper. the cover glass was sealed with nail polish. chromosome preparations were examined in phase contrast micrographs. chromosome measurement the lengths of the chromosomes from 12 well spread orcein-stained metaphase chromosomes were measured in digital images using digitizier software, version 1 (developed by the mathematics department, atomic energy commission of syria). chromosome lengths were ranked for each cell nucleus and means and standard deviations (sd) were calculated. relative chromosome lengths were calculated as percentages of the total length of all chromosomes in the diploid set. 3. results effect of irradiation on mortality, longevity and fecundity our results show that the percentage mortality of irradiated phylloxera nymphs was significantly higher than that of unirradiated ones. a regression line was fitted to present the relationship between gamma irradiation and percentage mortality of phylloxera (fig. 1), showing that the percentage mortality was positively correlated with dose (t= 7; p=0.05). the lowest percentages of mortality were recorded at 10 gy, after which the percentages started to increase. only about 16% of phylloxera nymphs were able to reach matured female stage at 30 gy. % mo rta lit y fig. 1 effect of gamma irradiation on percentage mortality of grape phylloxera. table 1 effect of different doses of gamma irradiation on mean number of eggs and longevity of phylloxera dose (gy) mean no. eggs (±se) mean longevity (d) (±se) 0 10 20 30 60.0±4.0 a 24.5±2.7 b 10.7±1.0 c 0.7±0.2 d 14.6±0.75 a 10.0±0.88 b 7.0±0.69 c 0.6±0.01 d means followed by different letters (columns) are significantly different at p< 0.05 (tukey hsd test). table 1 illustrates that phylloxera longevity and number of eggs were considerably influenced by the applied dose (table 1). the mean value for longevity and the mean number of eggs were significantly reduced by increasing the dose (f=75.67; df=3, 96; p=0.05 and f=106.78; df=3, 96; p=0.05, respectively). 16 chromosomal analysis under a light microscope from matured phylloxera females, 24 to 36-hr-old embryos, which contain a higher proportion of dividing cells, were taken to analyze the metaphase chromosomes. it was revealed that the wild-type metaphase karyotype of phylloxera consists of 10 chromosomes. there were eight autosomal chromosomes and sex chromosomes (xx) (fig. 2 a). a karyotype of 2n=9 was also found (fig. 2 b). based on our observations, phylloxera metaphase chromosomes appeared like thick condensed rods. in some cells, an additional very small chromosome was detected (fig. 3). a b 1 µm 1 µm fig. 2 normal metaphase kryotype from embryonic cells of grape phylloxera, daktulosphaira vitifoliae fitch: a) 2n= 10; b) 2n=9 + small chromosome (the arrow). 1 µm ý fig. 3 normal metaphase kryotype from embryonic cells of grape phylloxera: 2n= 10 + small chromosome (the arrow). to study the effect of different doses of gamma irradiation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose, and it was found that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. at all different doses, sticky chromosomes were observed in the embryo cells (fig. 4). when 20 gy was applied, inter-chromosome translocations were clearly visible (fig. 5). however, increasing in the chromosomal number on the metaphase plate in some cells was noticed in embryos when phylloxera nymphs were irradiated (fig. 6). a b 1 µm 1 µm c d 1 µm 1 µm e 1 µm fig. 4 mitotic metaphase chromosomes from embryos of treated grape phylloxera with gamma ray, showing sticky chromosomes at deferent doses: a-b at dose 10 gy; c) at dose 20 gy, d-e) at dose 30 gy. fig. 5 orcein-stained preparations from embryos of irradiated phylloxera at dose 20 gy showing mitotic metaphase chromosomes with a translocation on the large chromosome (the arrow). ý 1 µm 17 chromosomes measurement it was possible to identify eight autosomal chromosomes according to their length when different metaphase chromosomes were investigated. table 2 illustrates the mean length and relative length for each identified chromosome. the total mean length of complete metaphase chromosomes was 12.17±1.97 µm. chromosome pair no. 1 is an extra large chromosome with an average total length of 2.31±0.3 µm and relative length of 18.18%; chromosome pair no. 2 is a large chromosome with an average total length of 1.33±0.3 µm and relative length of 10.5%; chromosome pair no. 3 is a medium chromosome (1.12±0.65 µm); and chromosome pair no. 4 is a short chromosome (0.90±0.160 µm) (table 2). the sex chromosome (xx) was clearly recognized. the sex chromosome is the shortest chromosome in the metaphase complements with an average total length of 0.69±0.1 µm and relative length of 5.51%. the mean length of the additional chromosome, which was observed in several cells, was only 0.5±0.1 µm. considerable variation was noted among the species tested within these orders (makee and saour, 1999; bakri et al., 2005; follett et al., 2007). in fact, a few studies were carried out to determine the radioresistance of hemiptera (scales, mealy bugs, aphids, and whiteflies). a previous study showed that the egg hatch of phylloxera decreased when eggs were subjected to high doses of gamma irradiation and the percentage of matured phylloxera females significantly increased as older nymphs and lower doses were used (makee et al., 2008). on the contrary, fecundity was markedly reduced when older nymphs and higher doses were employed. however, a relationship between chromosomal aberrations induced by irradiation and phylloxera biology was not determined in the study. phylloxera can be considered a cytogenetically exciting insect species because of abnormal features related to its cyclical parthenogenesis, and because it has holocentric chromosomes (chromosomes that lack a localized centromere). phylloxera populations can consist totally of parthenogenetic (thelytokous) females. several studies showed that grape phylloxera mainly reproduces asexually (parthenogenesis): an egg cell can develop into offspring without fertilization by a sperm. thus, the offspring and its siblings are assumed to be genetically identical to the mother (vorwerk and forneck, 2006). parthenogenetic reproduction of phylloxera has been observed in the field and can be easily maintained under constant conditions in the greenhouse or in vitro. this type of reproduction allows phylloxera populations to be replicated several fold, thus several asexual generations can be analyzed within a short period. once a year, xx parthenogenetic phylloxera females, like aphids, produce one egg that develops as an xo male, having lost half its x chromatin during the single maturation division (blackman and hales, 1986; blackman, 1987). the current study has shown that phylloxera nymph mortality increased with irradiation (fig. 1), confirming the results reported by makee et al. (2008). correspondingly, dohino et al. (1998) found that the survival of aphids was significantly decreased when they were treated with doses of 400-600 gy. the high death rate, especially when phylloxera nymphs were exposed to higher doses of gamma irradiation, can be attributed to the effects of the dominant lethal mutations induced in phylloxera nymphs’ chromosomes by irradiation (lachance, 1967). when low doses were applied, a small portion of irradiated nymphs successfully completed development and produced matured females, survival which was due to the holokinetic nature of phylloxera chromosomes. irradiation can cause fragmentation but the resulting fragments are still able to move on the mitotic spindle so that chromosome breakage does not lead automatically to the loss of genetic material (hughes-schrad and ris, 1941). our results reveal that the fecundity and longevity fig 6 orcein-stained preparations from embryos of irradiated phylloxera showing: a) at dose 20 gy: a cell with about 15 chromosomes which some of them are stuck together; b) at dose 30 gy: a cell with 14 chromosomes. a b 1 µm 1 µm table 2 the mean length and relative length (%) of phylloxera chromosomes no. chromosome mean length (µm±sd) relative length (%) 1 1 2 2 3 3 4 4 x x total mean length 2.31±0.3 2.31±0.3 1.33±0.3 1.33±0.3 1.12±0.65 1.12±0.66 0.90±0.16 0.90±0.16 0.69±0.1 0.69±0.1 12.17±1.97 18.18 18.18 10.5 10.5 8.81 8.81 7.1 7.1 5.41 5.41 100 4. discussion and conclusions several studies demonstrated that dipteran, coleopteran and hemipteran species tend to be more radiosensitive than lepidopteran species. however, 18 of surviving matured females, irradiated as nymphs, were greatly impacted by irradiation (table 1). comparable results were reported when crawls and nymphs of mealybug, maconellicoccus hirsutus (green), were irradiated (jacobsen and hara, 2003). therefore, at low doses some matured phylloxera females were recorded, but they laid only few eggs and lived for a short period of time. it could be that the induced chromosomal aberrations in irradiated nymphs prevent the normal process of mitotic division, which leads to egg production. therefore, the matured females were unable to produce a normal number of eggs. to study the effect of different doses of gamma irradiation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose. it was noted that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. we noticed sticky chromosomes, inter-chromosome translocations, and increases in the chromosomal number on the metaphase plate in some cells. all these chromosomal aberrations in the embryos were expected as the phylloxera, like lepidptera species, has holocentric chromosomes. it is reported that irradiation causes fragmentations and translocations in many species of lepidptera (traut et al., 1986; makee and tafesh, 2006, 2007). and because the chromosomes are holocentric when a break occurs, the fragments are usually not lost and can still be attached on the spindle. in the present study on phylloxera, a lot of chromosomal breaks occurred during the formation of eggs in the ova of the nymphs which gave sticky chromosomes and inter-chromosome translocations in the cells of the embryos. it can also be said that spindles were affected by the irradiation which caused an increase in the chromosomal number on the metaphase plate in some embryo cells. in this work, the embryos of laid eggs varied greatly in their chromosomal rearrangements (figs. 2, 3, 4 and 5). however, such rearrangements allow the formation of embryos but it is unknown if they will permit the development of embryos until egg hatch. in mealybug only embryos with an approximately normal amount of paternal chromosomal material were able to survive (nelson-rees,1962). the current study has shown that the metaphase complement of the syrian strain of the phylloxera female consists of 10 chromosomes, representing eight autosomal and two sex chromosomes, confirming the results presented by forneck et al. (1999) and maillet (1957). we found also normal karyotype with 2n= 9 in the embryonic cells coincidently. forneck et al. (1999) found one karyotype containing 2n = 9 in the somatic cells of phylloxera and they interpreted it as a male sexual phylloxera, although they did not find any spermatides during their study, which leads us to think that maybe this deficiency in chromosome number is a kind of variety of the karyotype. when defining the karyotype of six phylloxera populations from germany, forneck et al. (1999) noticed extra chromosomes. similarly, in our study an additional very small chromosome was observed in some examined cells of the syrian phylloxera strain. the detection of supernumerary chromosomes was reported in aphids as well (blackman, 1976; wilson et al., 2003). such supernumerary or accessory chromosomes are not essential for the life of a species and are lacking in most of the individuals; they do not carry genes necessary for basic growth, but may have some functional significance such as to increase asymmetry chiasma distribution or increase variation by increasing crossing over and recombination frequencies. in aphid, blackman (1980, 1981) suggested that the differences in chromosome numbers might be due to dissociations or fusions involving elements of the normal diploid set, or to the presence of supernumerary b chromosomes. the centromeric activity of holocentric chromosomes, dispersed along its full length, allows the broken chromosomal fragments to segregate during mitosis (ris, 1942). moreover, blackman (1980) proposed that thelytokous reproduction of aphids is a factor that permits karyotype variation within populations of the same species. the phylloxera karyotype consists of 10 chromosomes. the total complement length is about 12ìm and the chromosomes range in length from 0.7 to 2 ìm. when mitosis of agallia constricta (leafhopper) was examined, the metaphase chromatin appeared to be a 2-3 ìm wide (rieder et al., 1990). based on embryo metaphase, the chromosomes of phylloxera females could be sorted into five different size-dependent groups: extra long, long, medium, short and extra short (table 2). however, in some cells a dot-like chromosome, that represents the additional chromosome, was observed. forneck et al. (1999) classified phylloxera chromosomes into two classes: one pair of large chromosomes and four pairs of shorter chromosomes. nevertheless, in their study they did not mention the exact length of each pair. the x chromosome is the shortest one in phylloxera karyotype forneck et al. (1999). however, blackman et al. (2003) reported that in most aphids species the x chromosomes could be identified as the longest or second longest pair. on the contrary, in some aphids the x chromosome was the shortest pair (blackman, 1986; blackman et al., 2003). the present study confirms the efficiency of cytogenetic techniques in analyzing the karyotype and chromosomal length of phylloxera, as well as tracing chromosome aberrations in irradiated phylloxera populations. this investigation is a contribution to the search for genetic variation of phylloxera behaviour and development from different populations and provides useful information that can be taken into account in pest management and quarantine measurements against phylloxera. however to apply irradiation technology more comprehensive studies are still needed. 19 references bakri a., heather n., hendrichs j., ferris i., 2005 50 years of radiation biology in entomology: lessons learned from ididas. ann. entomol. soc. am., 98: 1-12. blackman r.l., 1976 cytogenetics of two species of eureraphis (homoptera, aphididae). chromosoma, 56: 393-408. blackman r.l., 1980 chromosome numbers in the aphididae and their taxonomic significance. syst. entomol. 5: 7-25. blackman r.l., 1981species, sex and parthenogenesis in aphids, pp. 75-85. in: forey p.l. 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makee h., tafesh n., 2007 sex chromatin body as a cytogenetic marker of w chromosome aberrations in cydia pomonella females. in: vreysen m.j.b., a.s. robinson, and j. hendrichs (eds.) area-wide control of insect pests. from research to field implementation. springer, dordrecht, the netherlands, pp. 792. makee h., tafesh n., marec f., 2008 analysis of radiation-induced w chromosome aberrations in codling moth cydia pomonella (l.) by fluorescence in situ hybridization techniques. j. of pest science, 81: 143-151. nelson-rees w.a., 1962 the effects of radiation damaged heterochromatic chromosomes on male fertility in the mealy bug, planococcus citri (risso). genetics, 47(6): 661-683. rieder c.l., bowser s.s., cole r., rupp g., peterson a., alexander s.p., 1990 diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran agallia constricta (leafhopper) cell line ac-20. cell motility and the cytoskeleton, 15: 245-259. ris h., 1942 a cytological and experimental analysis of the meiotic behavior of the univalent x chromosome in the bearberry aphid tamalia (= phyllaphis) coweni (ckll.). j. exp. zool., 90: 267-330. song g.c., granett j., 1990 grape phylloxera (homoptera: phylloxeridae) biotypes in france. j. econ. entomol., 83: 489-493. traut w., weith a., traut g., 1986 structural mutants of the w chromosome in ephestia (insecta, lepidoptera). genet20 ica, 70: 69-79. vorwerk s., forneck a., 2006 reproduction mode of grape phylloxera (daktulosphaira vitifoliae, homoptera: phylloxeridae) in europe: molecular evidence for predominantly asexual populations and a lack of gene flow between them. genome, 49: 678-687. vorwerk s., sonntag k., blaich r., forneck a., 2008 application of current in situ hybridization techniques for grape phylloxera (daktulosphaira vitifoliae, fitch) and grapevine (vitis spp. l.). vitis, 47(2): 113-116. wilson a.c.c., sunnucks p., hales d.f., 2003 heritable genetic variation and potential for adaptive evolution in asexual aphids (aphidoidea). biological journal of the linnean society, 79: 115-135. impaginato 106 adv. hort. sci., 2011 25(2): 106-111 (1) corresponding author: skuhf07@yahoo.co.in received for publication 4 april 2011. accepted for publication 10 may 2011. in vitro mutagenesis and detection of variability among radiomutants of chrysanthemum using rapd a. kaul, s. kumar*(1), m. ghani department of biotechnology, university of horticulture and forestry, solan 173 230 (h.p.), india. key words: dendranthema grandiflora, in vitro mutagenesis, radiomutants, somaclones. abstract: the present study was undertaken to induce mutations in dendranthema grandiflora cv. snow ball through in vitro mutagenesis by exposing the in vitro shoots to 5, 10, 20 and 30 gy gamma radiation. rapd analysis was used to detect genetic polymorphism among the variants and the control. morphological variations were not observed with the 5 gy gamma dose during the first season. shoot regeneration, rooting and survival of shoots were affected by gamma ray doses. 10 gy of gamma irradiation was the most effective in inducing mutations in flower colour through direct in vitro mutagenesis. the shoots irradiated with 20 and 30 gy gamma radiation did not root and died. twenty rapd primers were used to amplify dna segments from the genomic dna of the control and its 10 variants, and the genetic similarity among them ranged from 0.06 to 0.79 revealing high genetic diversity. 1. introduction chrysanthemum is a major horticultural crop and it is the second largest in terms of cut flowers after rose, among the ornamental plants traded in the global flower market (kumar et al., 2006). the common garden chrysanthemum is hexaploid with 54 chromosomes (wolff, 1996). it is propagated vegetatively and has a strong self incompatibility system (richard, 1986), hence new cultivars are difficult to obtain by crossing. traditionally, new cultivars have been obtained from spontaneous mutations in vegetative reproduction, sports, being some variations more stable than others (miñano et al., 2009). in the last few years, induced mutations and somaclonal variations derived from the tissue culture process have been employed as a new source of variability (schum, 2003; datta et al., 2005; jain and spencer, 2006; zalewska et al., 2007; jain, 2010; barakat et al., 2010). although extensive work has been carried out to develop novelties in chrysanthemum through induced mutations using physical and chemical mutagens (broetjes and van harten, 1978), there is always a need to explore the possibility of new variety for floriculture trade. mutation breeding by radiation has been widely used to upgrade well-adapted plant varieties and also to develop new variations within improved agricultural characteristics. since most cultivated chrysanthemum cultivars are polyploids with high genetic heterogeneity, mutants with allied flower colour, shape, floral size and shape are often recovered. allied flower colours with chimeric tissue can be easily induced by radiation and can be isolated using in vitro tools (kumar et al., 2006). identification and characterization of cultivars is extremely important in horticultural crops in order to protect the plant breeder’s rights. traditionally, identification has been based on morphological characters; however the development of new technologies has made it possible to base this analysis on dna information. one approach is a pcr-based technique rapd (william et al., 1990) that has been widely used for plant germplasm characterization (wolff et al., 1994; huang et al., 2000; martin et al., 2002; martin and gonzalez-benito, 2005). the aim of the present study was to induce mutation in chrysanthemum cv. snow ball through in vitro mutagenesis by treating the in vitro shoots with gamma radiation and to apply rapd analysis for the detection of genetic polymorphism among the mutants and control. 2. materials and methods plant material and in vitro mutagenesis nodal segments (2-3 cm) of chrysanthemum grandiflora (tzelev) cv. snow ball, collected from one107 year-old mother plants maintained in the glasshouse of the department of biotechnology, university of horticulture and forestry solan (himachal pradesh), india, were used as explants. the explants were surface sterilized with 5% sodium hypochlorite (naocl2) for 10 min, washed thoroughly three to four times with sterilized water and cultured on ms (murashige and skoog, 1962) medium supplemented with 2 mg/l benzyladenine (ba), 0.5 mg/l α-naphthalene acetic acid (naa), 30 g/l sucrose (w/v) and 8 g/l agar (w/v; sigmaaldrich, bangalore). the culture without growth regulators served as control. the ph of the medium was adjusted to 5.8 before autoclaving at 121oc at a pressure of 1.1 kg cm-2 for 15 min. the cultures were maintained under 16-hr photoperiod with a light intensity of 50-60 µmol cm-2 s-1 provided by cool, white fluorescent lamps at 24±2oc. after four weeks of culture, the shoots were subcultured on shoot multiplication medium consisting of ms salts supplemented with 0.5 mg/l ba, 0.1 mg/l indole acetic acid (iaa) and 1 mg/l gibberellic acid (ga3). shoots about 2.5-3.0 cm long were irradiated in a gamma cell (60 cobalt source) with 5, 10, 20 and 30 gy doses. after four weeks, the shoots were multiplied and 3-4 cm long shoots were transferred to 1/2 strength ms medium supplemented with 0.3 mg/l indolebutyric acid (iba) and 0.2% activated charcoal for rooting. the shoots with well developed roots were removed from the culture vessel, washed with running tap water to remove the adhering agar and transferred to plastic pots (25 cm diameter) filled with soil:sand:fym in 1:1:1 ratio and kept in the glasshouse with 80-90% relative humidity. after acclimatization for four weeks, the plants were observed for any variations in morphological characters from the control and pcr analysis was carried out using the genomic dna. statistical analysis the data recorded for different parameters were subjected to completely randomized design (gomez and gomez, 1984). the statistical analysis based on mean value per treatment was made using analysis of variance (anova). the lsd multiple range test (p ≤ 0.05) was used to determine differences between treatments. polymerase chain reaction genomic dna was isolated from fresh, young green leaves of the control and gamma ray-irradiated plants following the method of doyle and doyle (1987) with some modifications. for amplification, reaction mixtures (21.8 µl) contained 1 µl oligonucleotide, 2.5 µl 10x taq polymerase buffer, 1.5 nm mgcl2, 2 µl each of dntps, 3 µl genomic dna and 3 µl taq polymerase (3 units/µl). random oligonucleotide primers were used for rapd amplification (bangalore genei, bangalore, india). amplification was performed in a thermocycler (mj research, usa) programmed for a first denaturation step of 3 min at 94oc followed by 36 cycles of 94oc for 30 s for denaturation, 50oc for 30 s for annealing, and 72oc for 2 min (extension). twenty operon random primers (operon technologies, inc., usa), ten of the opa series (1-10) and ten of the opb series (1-10), were employed for amplification using the cycling condition mentioned above. all samples were given 10 min at 73oc for postamplification. pcr products were separated on a 1.4% agarose gel using 1 x tae buffer and were stained with ethidium bromide. in all amplification reactions, a reaction mixture with water instead of genomic dna was used as negative control. 1 kb dna ladder (fermantas, lithuania) was used as the size marker. the amplified products were visualized using a uv transilluminator and photographed using gel documentation system (alpha imager, usa). amplified dna was scored as either presence (1) or absence (0) of band. pairwise comparison between the control and variants was performed to calculate similarity (j) between the samples (jaccard, 1908) using simqual programme of ntsys-pc (version 2). a dendrogram was produced from the resulting similarity using upgma method. 3. results effect of gamma radiation on in vitro cultures the data presented in table 1 reveal that the survival of shoots was affected by gamma ray doses. a decrease in survival was observed with an increase of irradiation dose; a significantly higher survival percentage was observed in the control shoots. the variation in survival percentage between 5 and 10 gy was statistically insignificant. a decrease was also observed on shoots forming roots. a lethal effect of higher doses (20 and 30 gy) was noticed on rooting of shoots. the shoots did not root, hence they turned yellowish brown and died. no variation was observed in shoots forming roots with 5 and 10 gy irradiation. a significant difference in percent root initiation was observed between the control and irradiated shoots. the rooted plants were acclimatized as explained above and allowed to grow in the glasshouse till flowering. table 1 effect of radiation doses on survival of in vitro shoots in d. grandiflora dose strength survival (%) number of shoots producing roots (%) 0 5 10 20 30 lsd0.05 90.47 (79.93) 66.60 (59.25) 52.38 (46.39) 42.85 (40.83) 19.05 (20.15) 25.11 (21.89) 100.00 (90.90) 82.60 (65.48) 82.35 (65.40) 0(0) 0(0) 6.25 (4.88) figures within parentheses are arc sine transformed values. 108 effect of gamma radiation on morphological characters morphological variations were not observed in the acclimatized plants exposed to 5 gy gamma radiation. all the plants flowered true to the mother floret colour/shape. therefore, selections were made among the plants exposed to 10 gy gamma radiation for agronomic and molecular characterization of mutants. at flowering stage, plants in the glasshouse were observed for any variations in morphological characters. results revealed that there were 10 variants with different morphological characters compared to the control. the data presented in table 2 reveal that morphological characters of d. grandiflorum cv. snow ball and its mutant were statistically, highly significantly affected by gamma ray doses. plant height plant height was significantly reduced in the variants compared to control (table 2). a significant variation in plant height was also observed among the variants (table 3). variant v10 gave the significantly highest value for plant height (69.00 cm), compared with the control (62.10 cm). variant v8 showed the lowest value for plant height, followed by variant v2. the plant height was significantly lower in most of the variants as compared to the control. leaf number and leaf area (cm2) the number of leaves was significantly reduced in the variants compared to control (table 2). there was a significant increase in the number of leaves in variant v4. in general, the number of leaves in the variants was lower compared to the control. variant v2 had the fewest number of leaves, followed by variants v7 and v8. statistically leaf area did not differ between the variants and control (table 2), however leaf area differed among the variants (table 3). variant v7 had significantly higher mean values of leaf area, followed by variants v10, v2 and v9 in comparison to the control. number of flower buds and flowers table 2 reports that the number of flower buds was significantly different between the variants, whereas the difference in the number of flowers was not significant. the number of flower buds and flowers varied among the variants (table 3) with variants v3 and v8 having a significantly lower number of flower buds and flowers, followed by variant v7. it was observed that in most of the variants about 82-83% of buds opened into flowers while in variant v3 only 50% of the buds opened into flowers. flower diameter and flower colour the variation in flower diameter between the control and the variants was statistically not significant (tables 2 and 3). the original flower colour of the cv. snow ball was white with incurve ray florets (fig. 1a), whereas one branch of variant v9 produced yellowcoloured flowers with incurve ray florets (fig. 1b). the results indicate that the irradiation dose of 10 gy was an effective dose in inducing mutations in flower colour, but no changes were observed in flower shape and size. table 2 analysis of variance of the morphological characters of control and variants of chrysanthemum treatment plant height (cm) number of leaves leaf area (cm2) number of buds number of flower flowers diameter (cm2) control variants lsd0.05 62.10 54.80 2.97 38.70 31.60 4.18 8.60 10.93 2.35 15.10 11.70 4.05 11.40 8.10 3.44 6.60 6.50 1.58 table 3 effect of gamma radiation (10 gy) on morphological characters in d. grandiflora treatment plant height (cm) number of leaves leaf area (cm2) number of buds number of flower diameter flowers (cm2) control v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 62.10 56.00 40.00 60.00 58.00 60.00 55.00 58.00 37.00 55.00 69.00 38.70 23.00 16.00 40.00 49.00 38.00 35.00 19.00 19.00 40.00 37.00 8.61 7.39 13.00 8.48 7.87 10.85 10.48 15.16 9.32 12.56 14.15 15.10 15.00 5.00 14.00 16.00 14.00 10.00 7.00 5.00 13.00 18.00 11.40 10.00 4.00 7.00 9.00 11.00 7.00 5.00 3.00 10.00 15.00 6.60 5.50 6.00 8.00 5.50 6.60 7.30 6.00 7.50 6.80 6.60 109 characterization twenty decamer primers (table 4) were used for the amplification of genomic dna of control and mutants of dendranthema grandiflora snow ball. the number of dna fragments amplified ranged from one to five depending upon the primer and the dna sample with a mean value of 2.90 bands per primer (table 4). the amplification product ranged from 250 to 3000 bp. a total number of 58 markers were produced by the 20 primers. a total of 100% of the 58 scored bands were polymorphic in 11 genotypes (one control and its ten fig. 1 induced somatic mutations in chrysanthemum cv. snow ball after 10 gy gamma radiation treatment. (a) control, whitecoloured flowers; (b) mutated, yellow-coloured flower. table 4 nucleotide sequences and rapd amplification results of the primer used in the pcr amplification primer sequence (5’-3’) scored bands polymorphic bands polymorphism (%) opa-1 opa-2 opa-3 opa-4 opa-5 opa-6 opa-7 opa-8 opa-9 opa-10 opb-1 opb-2 opb-3 opb-4 opb-5 opb-6 opb-7 opb-8 opb-9 opb-10 total gtttcgctcc tgatccctgg catccccctg ggactggagt tgcgcccttc tgctctgccc ggtgacgcag gtccacacgg tgggggactc ctgctgggac caggcccttc tgccgagctg agtcagccac aatcgggctg aggggtcttg ggtccctgac gaaacgggtg gtgacgtagg gggtaacgcc gtgatcgcag 3 4 3 1 4 5 4 4 0 2 4 1 1 3 2 2 4 3 3 5 58 3 4 3 1 4 5 4 4 0 2 4 1 1 3 2 2 4 3 3 5 100 100 100 100 100 100 100 100 0 100 100 100 100 100 100 100 100 100 100 100 58 fig. 2 rapd profile with primer opb-4 showing polymorphism in control and its variants in chrysanthemum cv. snow ball. lanes 110: variants (v1 to v10); lane 11: control; lane 12: dna ladder (1kb). variants). figure 2 shows the amplification profiles, generated by primer opb-4 across the chrysanthemum genotypes. the rapd markers produced by the 20 primers were used to construct a similarity matrix (table 5). five clusters can be observed. the first cluster includes only table 5 jaccard’s similarity matrix in control (c) and mutated (v) plants based on rapd analysis c v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 c 1.00 0.32 0.31 0.31 0.28 0.37 0.20 0.29 0.20 0.06 0.28 v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 1.00 0.79 0.67 0.76 0.37 0.30 0.65 0.50 0.27 0.62 1.00 0.54 0.65 0.37 0.26 0.68 0.54 0.26 0.61 1.00 0.67 0.37 0.32 0.53 0.37 0.28 0.44 1.00 0.42 0.32 0.72 0.50 0.29 0.51 1.00 0.37 0.40 0.26 0.24 0.31 1.00 0.45 0.37 0.26 0.32 1.00 0.62 0.33 0.62 1.00 0.25 0.59 1.00 0.35 1.00 110 yellow-coloured flowers (group 5c, fan 1) (british colour council, 1938). in order to check the stability of flower colour, the mutated branch was propagated vegetatively. the plants were grown in a glasshouse, where they expressed the same colour/shape. over recent years, rapd analysis has been used in ornamental breeding to characterize genotypes and to identify genes controlling important traits (huang et al., 2000; rumińska et al., 2004; kumar et al., 2006; miñano et al., 2009). in the present study, a high level of polymorphism was observed in 10 radiomutants of chrysanthemum. wolff and van rijn (1993) also noticed a high degree of polymorphism in chrysanthemum cultivars using rapd markers. this high level of polymorphism may be due to the strict out-crossing, resulting in a higher level of heterozygosity in chrysanthemum (wolff et al., 1994). rapd markers were used to construct a similarity matrix and the results of characterization analysis revealed a high diversity between the control and its somaclones. the greatest genetic distance between control and variant 9 (v9) may be due to the fact that v9 is a somaclone where one branch was mutated and produced yellow-coloured flower heads. although the plants differed in flower colour, bands specific for flower colour could not be distinguished due to the resolution capacity of the tested primers. wolff (1996) reported that the choice of the primers may be an important factor in obtaining a rapid discrimination between samples. barakat et al. (2010) reported that mutants with different flower colour could be identified at the molecular level using rapd technique, holding promise to identify unique genes as scar markers. bhattacharya and teixeira da silva (2006) attempted to understand the molecular systematic and genetic difference between 10 original chrysanthemum cultivars and 11 mutants and reported that similarity ranged from 0.17 to 0.90 using rapid analysis. kumar et al. (2006) reported genetic distances from 0.43 to 0.96 between 13 chrysanthemum cultivars. a great genetic distance among the different cultivars showed the existence of introgressing new and novel genes from the chrysanthemum gene pool. it may be suggested, with regard to the present study, that by using rapd markers, it is possible to differentiate newly evolved chrysanthemum cultivars from their parents which can be a useful tool to supplement the distinctness, uniformity and stability analysis for plant variety protection in future. our results are in accordance with those of other workers who reported rapd as a powerful tool in the assessment of genetic variability as well as for genetic characterization, allowing differentiation of chrysanthemum mutants/cultivars (huang et al., 2000; rumińska et al., 2004; kumar et al., 2006; chatterjee et al., 2006; miñano et. al., 2009). therefore, it may be concluded that 10 gy gamma irradiation was most effective in inducing variations in flower colour and other morphological characters. morphological variations were not observed with the 5fig. 3 dendrogram of control and mutants of chrysanthemum cv. snow ball variant 9 (v9); the second includes the control (c); the third includes variants 5 and 6 (v5 and v6); the fourth includes variants 8 and 10 (v8 and v10), and the last cluster includes variants 3, 4, 7, 2 and 1 (v3, v4, v7, v2, v1). it can be seen from figure 3 that the shortest genetic distance was found between variant 1 (v1) and variant 2 (v2), whereas the greatest distance was observed between the control and variant 9 (v9). 4. discussion and conclusions analysis of explant sensitivity is one of the basic requirements for an effective use of mutation induction in plant breeding programmes (walther and sauer, 1986). in the present study, the radio sensitivity of shoots was assessed by survival and rooting of shoots after irradiation in order to select a suitable dose of gamma irradiation. predieri (2001) also reported the necessity to identify an appropriate dose to apply in mutagenic treatments. the effect of gamma radiation on in vitro cultures in crop breeding has been studied by many workers (shen et al., 1990; charbaj and nabul, 1999; predieri and gatti, 2000; datta et al., 2005; barakat et al., 2010). reductions in survival, plant height, number of leaves and flowers were recorded after gamma irradiation. mutation in flower colour was detected as chimera in one branch of the plant, which produced 111 gy gamma dose and a lethal effect of 20 and 30 gy doses was observed on rooting and survival of in vitro shoots. one hundred percent polymorphism was observed among the radiomutants using pcr technique. the present results also indicate the applicability of gamma radiation in crop breeding, and the assessment of genetic variability and characterization of radiomutants at genomic level by rapd can be a useful tool in breeding programs aimed to improve ornamental characters of chrysanthemum cultivars. references british colour council, 1938 horticultural colour chart i and ii. the british colour council, london, pp. 1-200. barakat m.n., abdel-fattah r.s., badr m., eltorky g., 2010 “in vitro” mutagenesis and identification of new variants via rapd markers for improving chrysanthemum morifolium. african j. agric. res., 5: 748-757. bhattacharya a., teixeira da silva j.a., 2006 molecular systematics in chrysanthemum x grandiflorum (ramat) kitamura. sci. hort., 109: 379-385. broetjes c., van harten a.m., 1978 application of mutation breeding methods in the improvement of vegetatively propagated crops. an interpretative literature review. elsevier, amsterdam, the netherlands, pp. 324. charbaj i., nabul s., 1999 mutational analysis of racial differentiation in oryza sativa. curr. sci., 32: 451-453. chatterjee j., mandal a.k.a., ranade s.a., teixeira da silva j.a., datta s.k., 2006 molecular systematics in chrysanthemum x grandiflorum (ramat) kitamura. sci. hort., 119: 373-378. datta s.k., mishra p., mandal a.k.a., 2005 “in vitro” mutagenesis a quick method for establishment of solid mutant in chrysanthemum. curr. sci., 88: 155-158. doyle j.j., doyle j.l., 1987 a rapid dna isolation procedure for small amounts of fresh leaf tissue. phytochem. bull., 19: 11-15. gomez k.a., gomez a.a., 1984 statistical procedure for agricultural research. john wiley and sons, new york, usa. huang s.c., tsai c.c., sheu c.s., 2000 genetic analysis of chrysanthemum hybrids based on rapd molecular markers. bot. bull. acad. sin., 41: 257-262. jaccard p., 1908 nouvelles recherches sur la distribution florale. bull. soc. vaud. sci. natl., 44: 223-270. jain s.m., 2010 mutagenesis in crop improvement under the climate change. romanian biotechnol. lett., 15: 88-106. jain s.m., spencer m.m., 2006 biotechnology and mutagenesis in improving ornamental plants, pp. 589-600. in: teixeira da silva j.a. (ed.) floriculture, ornamental and plant biotechnology: advances and topical issues, vol. i. global science books, isleworth, uk, pp. 2481. kumar s., prasad k.v., choudhary m.l., 2006 detection of genetic variability among chrysanthemum radiomutants using rapd markers. curr. sci., 90: 1108-1113. martin c., gonzález-benito m.e., 2005 survival and genetic stability of dendranthema grandiflora tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. cryobiology, 51: 281-289. martin c., uberhuaga s., perez c., 2002 application of rapid markers in the characterization of chrysanthemum varieties and assessment of somaclonal variation. euphytica, 127: 247-253. miñano h.s., gonzález-benito m.e., martin c., 2009 molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using rapd markers. sci. hort., 122: 238-243. murashige t., skoog f., 1962 revised medium for the rapid growth and bioassay with tobacco tissue culture. physiol. plant., 15: 473-479. predieri s., 2001 “in vitro” propagation and breeding of ornamental plants: advantages and disadvantages of variability. euphytica, 115: 12-14. predieri s., gatti j., 2000 pear mutagenesis: “in vitro” treatment with gamma-rays and field selection for vegetative form trials. euphytica, 114: 79-90. richard a.j., 1986 plant breeding systems. george allen and unwin, london, uk. rumińska j.l., zalewska m., sadoch z., 2004 radiomutants of chrysanthemum (dendranthema grandiflora tzvelev) of the lady group rapd analysis of the genetic diversity. plant breeding, 123: 290-293. schum a.r., 2003 mutation breeding in ornamentals: an efficient breeding method? acta horticulturae, 612: 47-60. shen t., li w., dai c., chen j.y., 1990 the induction of sports in chrysanthemum by gamma radiation. theor. appl. genet., 88: 129-136. walther f., sauer a., 1986 “in vitro” mutagenesis in roses. acta horticulturae, 189: 37-46. william g.k., kubelik a.r., livak k.l., rafalski j.a., tingey s.v., 1990 dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acid res., 18: 159-164. wolff k., 1996 rapd analysis of sporting and chimerism in chrysanthemum. euphytica, 88: 159-164. wolff k., van peter r.j., hofstra h., 1994 rflp analysis in chrysanthemum l. probe and primer development. theor. appl. genet., 88: 472-478. wolff k., van rijn p.j., 1993 rapid detection of genetic variability in chrysanthemum (dendranthema grandiflora tzvelev) using random primers. heredity, 71: 336-341. zalewska m., rumińska l.j., miller n., 2007 “in vitro” propagation using adventitious bud techniques as a source of new variability in chrysanthemum. sci. hort., 113: 70-73. 109 1. introduction peaches and nectarines are widely-consumed summer fruit and, in the last few years, there has been an increasing interest in their nutritional value (ramina et al., 2008; wolfe et al., 2008). peach fruit contains a wide range of chemical compounds but, from a dietary point of view, the most important fruit constituents are carotenoids, phenolics and fibre (ramina et al., 2008). yellow-fleshed peaches are considered a good source of β-carotene and β-cryptoxanthin (gross, 1987) while flavonols, that are glycosilated forms of quercetin and kampferol, are the most abundant phenolics in peaches and other stone fruit (young et al., 1989). all these compounds are reported to have antioxidant activity (fu et al., 2011; haminiuk et al., 2012) and, when added to the human diet, have a protective action against cancer and cardiovascular diseases (steinmetz and potter, 1996). after harvest, firmness, acidity and other quality parameters of peaches and nectarines are subjected to important changes (crisosto, 2006; ramina et al., 2008). fruit nutritional quality varies greatly among cultivars (gil et al., 2002) and often decreases after refrigerated storage (di vaio et al., 2001; tsantili et al., 2010). after storage at low temperatures, sensory characteristics, and especially aroma, of peach decreases (infante et al., 2008) while in fruit ripened at 18°c, the level of volatile compounds was found to be similar to tree-ripened fruit (aubert et al., 2003). the aim of this work was to study the evolution of qualitative characteristics and the concurrent change in bioactive compound concentrations in eight peach and nectarine cultivars during postharvest ripening at 20°c. 2. materials and methods a white(‘honora’) and two yellow-fleshed (‘dr. davis’, fairtime) peach cultivars and a white(‘maria anna’) and four yellow-fleshed (‘diamond ray’, ‘fairline’, ‘nectaross’, ‘sweet red’) nectarines were harvested in the experimental field of the cra-fruit tree culture of rome. immediately after harvest, all fruits were sent to the crafood technology research unit of milan where 30 fruits postharvest changes in quality characteristics, antioxidant activity and bioactive compounds of peach and nectarine cultivars [prunus persica (l.) batsch] m. nuzzi 1, m. grassi 1, a. sartori 2, m. terlizzi 2, m. buccheri 1(*) 1 consiglio per la ricerca e la sperimentazione in agricoltura, unità di ricerca per i processi dell’industria agroalimentare, via venezian, 26, 20131 milano, italy. 2 consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la frutticoltura, via di fioranello, 52, 00134 roma, italy. key words: acidity, carotenoids, color, dpph, firmness, folin-ciocalteau, hue, phenolics, shelf-life. abstract: three peach (‘honora’, ‘dr. davis’ and ‘fairtime’) and five nectarine (‘maria anna’, ‘diamond ray’, ‘fairline’, ‘nectaross’, ‘sweet red’) cultivars were analyzed at harvest and after a postharvest ripening period. physicochemical characteristics [peel ground color (l*,c*, h°), soluble solids content (ssc), flesh firmness and titratable acidity (ta)], the concentration of some bioactive compounds [total phenol content (tpc) and total carotenoids (tc)] and the total antioxidant activity (taa) were evaluated at harvest and after a shelf-life period of five days at 20 °c. phenolic compounds and antioxidant activity were assayed on two different extracts of each sample: ethanol/hcl and ethanol/acetone. after shelf-life, all the cultivars showed a decrease in firmness and an increase in the ratio ssc/ta. the h° parameter of the peel background color had a good correlation with firmness, ssc, ta and the ratio ssc/ta in some of the cultivars, but no relationships were found in the white-fleshed varieties and in two of the nectarines evaluated. the trend of the carotenoids content after postharvest ripening was found to be cultivar-dependent, while taa or tpc showed an increase in nectarines and remained unchanged in peaches. the ethanol/acetone mix was able to extract almost the double of antioxidant compounds with respect to the ethanol/hcl extract. adv. hort. sci., 2015 29(2-3): 109-115 (*) corresponding author: marina.buccheri@entecra.it received for publication 13 september 2014 accepted for publication 26 march 2015 110 adv. hort. sci., 2015 29(2-3): 109-115 per cultivar were selected for size uniformity and absence of damage. fruits of each cultivar were randomly divided into two sets of 15 fruits each and analyzed immediately [harvest (hr)] or after five days of shelf-life at 20°c (7580% rh) in a temperature-controlled ripening room (sl). quality characteristics color (l*, a*, b*, cie values), soluble solids content (ssc) and titratable acidity (ta) were analyzed on each fruit. skin background color was measured by a spectrophotometer (cm-2600d, konica minolta, japan) on the two cheeks of each fruit (15 fruit/sampling); flesh color was assessed on two opposite sides of each fruit after removing 2.5 mm of peel and flesh. hue value (h°) was calculated as arctangent of b*/a* and expressed in degrees, while the color saturation index (c*) was calculated as √a*2+b*2. ssc was measured by a digital refractometer (rfm 81, bellingham+stanley, uk). ta was measured by titrating 10 ml of fruit juice with 0.1 n naoh to ph 8.1 and calculating ta as g of malic acid/100  g fresh weight. the maturity index (artés and salmerón, 1996; crisosto et al., 2001; crisosto, 2006) was calculated as the ratio ssc/ta. bioactive compounds and antioxidant activity three replicates (five fruits/replicate), were analyzed at each sampling. each extract was prepared in duplicate. total carotenoids (tc) were assessed by the method of picchi et al., (2012) with some modification. briefly, 5 g of homogenized flesh were added to 150µl of butylated hydroxytoluene (bht) (1% in methanol w/v), 0.05 g of ammonium sulphate and 10 ml of extracting solution (hexane: ethyl acetate: ethanol, 2:1:1 v/v). samples were vortexed for 10 s and then centrifuged (15 min, 4°c, 15000xg) and the supernatant was filtered through cheesecloth and stored at -20°c until analysis. absorbance was recorded at 450 nm (uv-uvidec 320 spectrophotometer, jasco, japan) and total carotenoids were estimated by comparison with a standard curve obtained with different amounts of β-carotene. the results were expressed as µg β-carotene equivalent (β-carotene eq) /100 g f.w. total phenolic content (tpc) and total antioxidant activity (taa) were analyzed preparing two different extracts: 5g of homogenized flesh to 20 ml of ethanol (96%): hcl 0.04n (1:1 v/v) (e extract) or 20 ml of ethanol (96%): acetone (1:1 v/v) (e/a extract). samples were vortexed for 10 s and centrifuged (15 min, 4°c, 10000xg), and the supernatant was filtered through cheesecloth and stored at -20°c until analysis. tpc was measured using the folin-ciocalteu method (singleton et al., 1999) with some modifications: 150 µl of sample extract, 5 ml of deionized water and 1 ml of folin-ciocalteu reagent were put in 10 ml test tubes and, after 5 min, 2 ml of 20% sodium carbonate solution were added. samples were kept 120 min in the dark and the absorbance at 730 nm was read against a blank (the same reaction mix but without the sample extract). tpc was calculated from a calibration curve, using gallic acid as standard. results were expressed as mg of gallic acid equivalent (gae)/100 g f.w. taa was measured using the dpph assay. the effect of peach extracts on the content of 2.2-diphenyl-1.picrylhydrazyl radical (dpph•) was estimated according to the method of lo scalzo et al., (2004) with some modifications: 100 µl of sample extract or trolox standard solution (0.01 to 0.5 mg/ml) were added to 2 ml of ethanol and 500 µl of dpph• (0.5mm in ethanol) and the decrease in absorbance at 517 nm was recorded after 3 min. each reading was done against its blank (2.5 ml ethanol, 100 µl of sample extract). the dpph scavenging capacity of the samples was calculated using a standard curve of trolox, and expressed as mg trolox eq/100 g f.w. 3. results and discussion quality characteristics based on ‘redhaven’ peach maturity (july 10 in central italy) the evaluated varieties were considered (table 1) as “middle-late” (honora, maria anna, nectaross), “late” (dr. davis, diamond ray, sweet red) or “very late” (fairline, fairtime) maturity cultivars. firmness at harvest differed considerably from cultivar to cultivar but, as reported by other authors (gil et al., 2002), peach cultivars have, on average, lower flesh firmness than nectarines (40.6 n and 51.1 n respectively, p<0.01). after five days of shelf-life, firmness of all the cultivars was comparable, with the exception of ‘dr. davis’ (yellow peach) and ‘fairtime’ (yellow nectarine) which showed the highest values (13.7 and 18.8 n, respectively). flesh firmness alone is not considered a satisfactory maturity index because it can vary among varieties, fruit size or climatic conditions (crisosto, 1994). in general, mature fruit of early-season peach or nectarine is less firm than late season varieties (crisosto, 1994). at harvest all cultivars reached the ssc (10%) proposed by kader (1997) as the minimum quality standard. all the nectarines and, above all, the “very late” cultivar ‘fairline’, had higher ssc than peaches, both at harvest and after shelf-life. after shelf-life the ssc showed a slight increase in ‘fairline’ while it remained unchanged in all the other cultivars. ta was higher in nectarines than in peaches (1.3 and 0.7 g/100 g fresh weight respectively, p<0.01) and decreased in all the cultivars after shelf-life. in peaches, ssc was shown to correlate well with consumer acceptance (crisosto and crisosto, 2005), but ssc and ta can be determined by several factors (crisosto, 2006) and large differences are reported among peach varieties (crisosto, 1994). the ratio ssc/ta (maturity index) was judged by some authors (lill et al., 1989; artés and salmerón, 1996) to be a more reliable quality index. in the present work, the maturity index increased in all cultivars after shelf-life and, on average, was higher in peaches than in nectarines (17.7 and 10.8% respectively, p<0.01). color changes that are associated with ripening strongly influence visual and eating quality of peaches (ramina et 111 nuzzi et al., postharvest changes in peach and nectarine cultivars al., 2008). in our experiment, peel background color was affected by postharvest ripening. almost all the cultivars had a lower (more yellow) h° value after shelf-life (table 2), except for the white peach ‘honora’ and for the nectarine ‘diamond ray’. in this latter, the red color covered the whole fruit surface and it was very difficult to measure the background yellow color. l* and, above all, c* values of the peel background color markedly differed from cultivar to cultivar but seemed not to be affected by shelf-life. hue of the flesh decreased slightly after shelf-life in all the cultivars but not in white-fleshed peaches ‘honora’ and ‘maria anna’ and in the nectarine ‘diamond ray’. h° and l* values of the flesh were similar between whiteand yellow-fleshed cultivars. the color of the white-fleshed peach (‘honora’) and nectarine (‘maria anna’) differed from the yellow cultivars only for a lower c* value, which indicates a lower saturation of the color, rather than a real difference in the hue. table 1 physical and chemical characteristics (means ±standard error) of different peach and nectarine cultivars at harvest (hr) or after five days of shelf-life (sl) at 20°c fruit type flesh color cultivar harvest date time firmness (n) ssc ta (g/100 g f.w.) ssc/ta peach white honora aug. 4 hr 41.2±5.2 10.8±0.3 1.1±0.04 9.8±0.2 sl 4.30±0.1 11.2±0.5 0.9±0.03 12.4±0.2 yellow dr. davis aug. 18 hr 38.8±1.4 13.1±0.1 0.7±0.00 19.9±0.1 sl 13.7±0.5 13.1±0.2 0.5±0.01 25.6±0.2 fairtime sept. 23 hr 41.8±2.8 11.2±0.2 0.7±0.02 16.6±0.2 sl 7.20±0.3 10.7±0.6 0.5±0.02 20.4±0.3 nectarine white maria anna aug. 4 hr 25.6±3.1 14.1±0.3 1.7±0.02 8.5±0.2 sl 2.30±0.2 14.9±0.2 1.0±0.06 15.3±1.1 yellow diamond ray aug. 10 hr 43.4±2.5 11.8±0.5 1.2±0.05 9.9±0.3 sl 4.00±0.3 12.4±1.1 1.0±0.02 13.0±0.8 fairline sept. 16 hr 72.0±2.3 17.3±0.3 1.3±0.02 13.7±0.3 sl 18.8±2.6 19.8±0.8 1.3±0.03 14.7±0.2 nectaross aug. 4 hr 56.8±4.8 13.7±0.2 1.6±0.02 8.7±0.0 sl 4.60±0.4 14.5±0.5 1.5±0.05 9.9±0.6 sweet red aug. 18 hr 57.6±3.6 11.1±0.4 1.3±0.05 8.4±0.1 sl 7.00±0.7 11.4±0.4 1.2±0.04 9.2±0.2 table 2 peel and flesh color parameters (means ±standard error) of peach and nectarine cultivars at harvest (hr) or after five days of shelf-life (sl) at 20°c fruit type flesh color cultivar time peel background color flesh color l* h° c* l* h° c* peach white honora hr 62.1±3.0 55.5±5.5 33.3±0.9 77.9±1.6 77.7±6.2 23.7±0.3 sl 60.2±0.8 52.9±3.5 36.9±1.2 74.8±2.7 76.6±2.0 26.8±0.8 yellow dr. davis hr 75.1±0.2 83.5±0.4 60.3±1.3 82.2±0.4 85.4±0.5 50.3±1.6 sl 74.7±0.6 79.3±0.7 62.6±0.3 81.6±0.3 83.8±0.3 51.5±0.4 fairtime hr 76.6±0.4 89.7±0.3 51.8±1.4 82.6±0.2 87.4±0.3 47.8±0.9 sl 75.4±0.7 84.8±0.5 56.3±1.2 80.1±0.4 84.1±0.3 51.3±1.2 nectarine white maria anna hr 70.3±1.7 65.2±3.9 31.3±1.0 78.4±0.7 64.7±3.5 19.5±0.8 sl 64.8±2.2 54.7±3.6 37.5±1.2 77.2±1.3 73.2±6.1 20.8±0.7 yellow diamond ray hr 41.7±0.7 31.9±0.8 47.6±1.7 69.4±3.3 68.3±4.6 49.8±1.7 sl 44.5±2.2 35.8±2.3 47.7±1.8 66.8±4.5 68.0±5.8 48.1±2.5 fairline hr 75.3±0.1 87.6±0.3 58.7±0.2 80.5±0.6 86.4±0.3 51.3±0.2 sl 71.9±0.3 81.2±1.0 61.9±0.6 78.2±0.5 83.0±0.7 55.5±0.6 nectaross hr 71.2±1.2 79.7±1.7 51.8±0.4 79.3±0.7 85.4±0.9 50.4±0.9 sl 70.4±1.2 75.1±1.9 58.3±1.0 75.0±1.2 79.8±2.1 50.8±0.6 sweet red hr 72.3±1.5 82.7±2.5 50.6±1.0 79.2±0.5 85.7±0.2 52.3±0.9 sl 73.2±1.2 77.3±1.0 53.9±0.9 77.8±0.7 81.9±0.7 51.1±0.5 112 adv. hort. sci., 2015 29(2-3): 109-115 flesh or peel background color are reported by different authors (delwiche and baumgardner, 1985; byrne et al., 1991; crisosto, 1994; lewallen and marini, 2003) to be highly correlated with firmness and other quality parameters of peaches and nectarines, so that background color is often used as maturity index (kader, 1997). peel background color or flesh color are not affected by sunlight and, thus, are more dependable indices of maturity than red color (crisosto, 1994). in this work we found good correlations between peel background color or flesh color (h°) and different quality parameters in some of the evaluated cultivars (table 3). in particular, peel background color of the yellow peaches ‘dr. davis’ and ‘fairtime’ showed good correlations with firmness, ta or ssc/ta and, in ‘fairtime’, these parameters were also related with flesh color. with regard to the yellow nectarines, peel and flesh color of ‘fairline’ and ‘nectaross’ were related with different quality parameters but without showing very high r values. good correlations were found only in ‘nectaross’ between h° of the flesh and ssc or ssc/ta. ‘sweet red’ nectarine had a high correlation coefficient between flesh color and firmness, without showing any significant correlation coefficient with the other parameters. similar, but slightly lower, r values were found for a* color parameter (data not shown). no relationships were found between flesh or peel color and any of the quality characteristics in the white peach (‘honora’) and nectarine (‘maria anna’) while ‘diamond ray’ showed only a low correlation coefficient between flesh color and ta and between flesh color and ssc. bioactive compounds and antioxidant activity peaches and nectarines are rich in bioactive compounds such as carotenoids and phenolics (gil et al., 2002). the major carotenoids in peaches are β-carotene and β-cryptoxanthin (ramina et al., 2008). white and yellow peaches show different levels of carotenoids production, especially in the last phase of maturity (brandi et al., 2011). in our experiment we found a lower level of total carotenoids in the two white-fleshed cultivars (fig. 1). carotenoid content increased, after shelf-life, in the two yellow peaches (‘dr. davis’ and ‘fairtime’) and in the nectarine ‘diamond ray’, while it remained constant in the other nectarine cultivars (‘fairtime’, ‘nectaross’ and ‘sweet red’). carotenoid content showed a rather good correlation coefficient with the chroma (c*) of the flesh (r=0.64, p<0.01) and of the peel (r=0.62, p<0.01) but not with the h° values (r=0.29 and r=0.33 with the flesh and peel hue values, respectively, p<0.05). the literature is inconsistent regarding the trend of tc after harvest. a decrease is reported by ramina et al. (2008) while other authors (caprioli et al., 2009, bianchi et al., 2015) described an increase in carotenoids after shelflife at 20°c. as is shown also by our results, the evolution of carotenoids after harvest could be cultivar-dependent. phenolic compounds were measured on two different extracts of each sample. in general, the extraction of phenolic compounds in alcoholic solution provides satisfactory results (perva-uzunalić et al., 2006); on pomegranate, some authors reported that the extraction in a mixture with methanol, ethanol, acetone and water had better results (li et al., 2006). for this reason we decided to perform the classic ethanol/hcl (e) extraction plus an ethanol/acetone (e/a) extraction. total phenolic content (fig. 2), evaluated by the two extraction methods, remained almost unchanged after shelf-life in the peach cultivars while it increased in nectarines, with the only exception being ‘diamond ray’ that table 3 correlations among peel background color or flesh color and different quality parameters in yellow or white-fleshed peaches and nectarines fruit type flesh color cultivar peel background color flesh color firmness ssc ac ssc/ac firmness ssc ac ssc/ac peach white honora ns ns ns ns ns ns ns ns yellow dr. davis 0.73** -0.59** 0.76** -0.82** ns ns 0.38* -0.39* fairtime 0.70** ns 0.76** -0.81** 0.77** ns 0.83** -0.82** nectarine white maria anna ns ns ns ns ns ns ns ns yellow diamond ray ns ns -0.48* ns ns -0.52* ns ns fairline 0.58** -0.68** ns -0.56** 0.56** -0.77** ns -0.66** nectaross 0.42* -0.38* 0.58** -0.61** 0.52** -0.45* 0.53** -0.82** sweet red ns ns ns ns 0.82** ns ns ns significance of r= p<0.05 (*), p<0.01(**), ns= not significant. fig. 1 total carotenoids content of the flesh of peach and nectarine cultivars at harvest and after five days of shelf-life at 20°c. bars refer to standard error. 113 nuzzi et al., postharvest changes in peach and nectarine cultivars showed a very low tpc content. between the two methods used, the ethanol/acetone mix (31.4 mg gae/100g f.w, on average, against 26.4 mg of the ethanol extract, p<0.01) was able to extract a higher quantity of phenolics. phenolic compounds are a class of compounds that is very broad and complex. in stone fruit the most abundant phenolics are flavonols and cinnamic acids, including chlorogenic and neochlorogenic acids (ramina et al., 2008), but phenolic extracts of plants are always a mixture of different classes of compounds which are selectively soluble in the solvents (koffi et al., 2010). furthermore, solvent polarity plays a key role in increasing phenolic solubility (naczk and shahidi, 2004). since there were differences in tpc extraction between ethanol/hcl and ethanol/acetone solution, total antioxidant activity was also assayed on both extracts. as for phenolic compounds, peaches and nectarines showed different behaviors after shelf-life (fig. 3): in peach fruit, total antioxidant activity, measured on e or e/a extracts, remained unchanged, while in nectarines it showed an increase after five days at 20°c. other authors also showed a significant increase in taa in several nectarine cultivars after refrigerated storage (seven days at 2°c) while the increase was not significant or there was a decrease in peaches (di vaio et al., 2001, 2008). antioxidant activity of peaches and nectarines measured on e/a extract was more than double that measured on ethanol/hcl extract (19 mg trolox eq/100 g f.w., on average, with respect to 9.4 of the e extract, p<0.01). this fact indicates that probably more antioxidant compounds can be extracted by the combined action of the solvent mix ethanol/acetone. acetone is a polar aprotic solvent that solvates ions without making bonds. having also a lipophilic portion [-c-(ch 2 ) 2 ], it probably allows a better extraction of non-polar compounds like lipophilic phenols or carotenoids with respect to the ethanol/hcl extract. the correlation coefficient between the difference in taa values measured on the two extracts (taa ethanol/acetone-taa ethanol/hcl) showed a slight correlation with total carotenoids (r=0.34, p<0.05), while there was no relationship between taa measured on e extract and total carotenoids. as shown by gil et al. (2002), total antioxidant activity was highly correlated with tpc (r=0.81 between taa and tpc measured on e extract and r=0.87 between taa and tpc measured on e/a extract, p<0.01). principal component analysis to obtain a global picture of the difference in quality and nutritional characteristics of the different cultivars, all the data were subjected to pca. four functions were extracted, explaining 84.7% of total variance. considering the first two principal components (fig. 4) pc1 (39.2% of total variance) was positively related to all the evaluated fig. 2 total phenol content (tpc) of ethanol/hcl (e) or ethanol/acetone (e/a) extracts from flesh of different peach and nectarine cultivars at harvest and after five days of shelf-life at 20°c. bars refer to standard error. fig. 3 total antioxidant activity (taa) of ethanol/hcl (e) or ethanol/ acetone (e/a) extracts from flesh of different peach and nectarine cultivars at harvest and after five days of shelf-life at 20°c. bars refer to standard error. fig. 4 principal component analysis of quality characteristics and bioactive compounds of eight peach and nectarine cultivars at harvest (hr) and after shelf-life (sl). cultivars: hon= honora; dr.d= dr. davis; f.tim=fairtime; m.an= maria anna; d.ray= diamond ray; f.lin=fairline; nec=nectaross; sw.r=sweet red. factors: ssc= soluble solids content; h°, c*, l*= color parameters; bg= peel background; tc= total carotenoids content; ta= titratable acidity; taa= total antioxidant activity; tpc= total phenol content; (e) ethanol/hcl extract; (e/a)=ethanol/acetone extract. 114 adv. hort. sci., 2015 29(2-3): 109-115 factors, except for firmness and ta; pc2 (21.6%) grouped c* and h° color parameters, firmness and total carotenoids, opposite to ssc and ta. the biplot of pc1 versus pc2 (fig. 4) revealed four distinct groups. the first group was formed by the two white-fleshed cultivars that showed negative values for both pc1 and pc2 and, hence, were negatively related to c* and total carotenoids. the second group was made up of the two ‘diamond ray’ samples, which showed very negative scores on pc1, probably because of their low values of bioactive compounds. the third group is composed of the two ripe samples (after shelf-life) of ‘nectaross’ and ‘fairline’ that had positive values on pc1 and negative on pc2 and, hence, linked mainly with a high content of antioxidant compounds. the last group was formed by the remaining yellow peaches and nectarine samples that showed positive values on pc1 and pc2, which are linked with high values in color parameters and high carotenoids content. the scores of the all the samples on pc1 and pc2 showed important differences from cultivar to cultivar (fig. 5). after shelf-life pc1 scores increased in all the nectarines but remained unchanged in the peach cultivars, while pc2 scores did not show important changes, except for ‘nectaross’ and ‘fairline’. 4. conclusions this study has shown a high variability in peach and nectarine characteristics after postharvest ripening. peel background color, that is often used as maturity index (kader, 1997) had, after shelf-life, a good correlation with firmness, ssc, ta and ssc/ta in some of the cultivars, but no relationships were found in the white-fleshed varieties and in two of the nectarines evaluated. for these cultivars it could be desirable to evaluate other nondestructive parameters such as near infrared spectrometry (nir) or time-resolved reflectance spectroscopy (trs) (carlomagno et al., 2004; zerbini et al., 2006), which might be better related with the ripening stage. the trend of carotenoids content after postharvest ripening was found to be cultivar-dependent, while taa and tpc measured on two different extracts (ethanol/hcl and ethanol/acetone) showed an increase in nectarines and remained unchanged in peaches. the e/a mix was able to extract almost double the antioxidant compounds with respect to the simple ethanol/hcl extract, probably because of the higher extraction of non-polar compounds due to the presence of acetone. with principal component analysis, the nectarine ‘diamond ray’ was grouped differently from the other cultivars, probably because of its low content in bioactive compounds. in general, all the evaluated cultivars did not show, after postharvest ripening, a significant decrease in quality parameters other than firmness. on the contrary, all the cultivars maintained or increased their antioxidant activity and their initial content in bioactive compounds. acknowledgements this work was funded by the project “validazione di cultivar e selezioni avanzate di actinidia pesco e melo per uso in coltura biologica (biofru)” of the italian ministry of agriculture. we acknowledge our colleagues dr. roberto lo scalzo for his suggestions and help on the extraction methods and dr. anna rizzolo for her help with statistic analysis and manuscript revision. references artés f., salmerón m.c., 1996 quality attributes, 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identification and comparison of flavonoid compounds in the fruit skins and leaves of peach cultivars. acta horticulturae, 254: 35-40. zerbini p.e., vanoli m., grassi m., rizzolo a., fibiani m., cubeddu r., pifferi a., spinelli l., torricelli a., 2006 a model for the softening of nectarines based on sorting fruit at harvest by time-resolved reflectance spectroscopy. postharvest biol. technol., 39: 223-232. impaginato foreword when i was asked to prepare a brief preface to this special issue of the journal advances in horticultural sciences, i gladly accepted because i thought that the topic of the recovery and enhancement of indigenous germplasm of a country so far away and so problematic as afghanistan was fascinating and full of great potential in various fields of agricultural science and beyond. while i was reading the articles included in this issue, i heard news of a further attack by isis on the architectural masterpieces of the city of palmyra, syria, on 20 january. the facade of the roman theater and the spectacular tetrapylon were destroyed, irretrievably. although there is no clear link between the two events (the recovery of the fruit germplasm of afghanistan and the destruction of historical monuments) it set me thinking that these two events are actually part of a single negative picture of modern times that man, consciously or not, is perpetrating against the biological, cultural and artistic gifts that our ancestors have been handing down to us for centuries. the systematic deletion of the vestiges of the past carried out by isis is not so different from what decades of uninterrupted war have caused to a country like afghanistan, which for historical and cultural reasons, is one of the most important points of contact and exchange between cultures and the biodiversity of the east, the middle east, africa and europe. a country which through commercial and military exchanges along the silk road, has witnessed a merging of genotypes of many species of agricultural interest, as well, of course, of traditions and cultures belonging to people who had never met before. it is easy to imagine what kind of genetic variability has been able to generate through these exchanges which allowed the spread, throughout the rest of the globe, of fruit species which today are some of the most cultivated worldwide. in the temperate climates where these crops have since been established, the strong selection imposed by man in favour of genetic or phenotypes traits of production and commercial interest has meant that, over the centuries, the gene pool of these species has been increasingly shrinking. this became particularly acute when specialized vegetative propagation began to be used through grafting techniques in order to obtain almost total uniformity in the reproduced trees and the fixing of those positive characters that are essential from an economic point of view. however in an area like afghanistan, these species have survived, as witnessed by the authors of the articles in this special issue, in an environment in which the technological evolution of agricultural practices has remained almost stationary since the beginning of the last century. in this environment selective processes have taken place that are much closer to the natural processes than to those imposed by man for cultivation purposes. given that afghanistan is a country rich not only in contradictions, but also in geomorphological variability, soil and climate, the strategic importance of the conservation of its biodiversity is clear. this country, which is considered by many as ‘the land of dust’, with extreme climatic conditions, both in terms of drought and flooding, high and low temperatures, and a topography with considerable altimetric changes, has led to an evolutionary process of adaptation to these conditions on the part of both cultivated and non­cultivated species. these conditions thus suggest the presence of genes that can resist extreme climatic conditions, which may be a key to the future adaptation of fruit tree species grown in temperate climates to the progressive and unstoppable changes that climate changes are causing. unfortunately, genetists from around the world have established that the fruit tree species grown in temperate areas no longer have, and maybe have never even had, a gene pool that would lead to improvements in the resistance abiotic stress that has become increasingly frequent. hence the vital importance of participatory research programs such as those described in this special issue. and here again lies the analogy between the destruction of the archaeological sites and the gradual loss of irreplaceable biodiversity that has been handed down for generations and generations: the loss of this heritage permanently eliminates the future possibility of re­establishing a balance between us and our environment and achieving harmony between the people, nature and history of a country. it is difficult, however, to assume that the preservation of this heritage can be obtained through protective measures aimed merely at conservation, without any organized and responsible participation by the local populations. just as the statues of the bamiyan buddhas and ancient books and statues of the mosul museum were not saved from the devastating fury of the taliban and isis, so too the fragile afghan germplasm of the fruit tree species would have little chance of survival if its conservation was confined to collection orchards designed like an open air museum but subjected to some nefarious purpose in a country where the emergency of war, combined with the food and economic emergency, leaves little room ahs vol. 30 no. 4 (2016): special issue on "afghanistan" for investments in an unproductive genetic heritage that cannot actually be exploited by the local people. the papers in this special issue thus represent an effective model of approach to the problem which, in my opinion, is the only way to reach such a high objective ­ the protection of biodiversity threatened by genetic erosion. a fundamental part of this approach is also the participation of local people who can then gain a possible means of livelihood and income, providing that they are properly trained and supported both technically and culturally. this is an enormously difficult process, most importantly due to the need to recover all that knowledge that enabled the older generation to pass on, until a few decades ago, the cultivation and planting techniques of suitable plants for the particular geographical area in question. the gradual abandoning of agricultural practices for food in order to concentrate on the almost universal production of opium poppies has led to the disappearance of several generations of farmers and the almost irretrievable loss of know­how in agriculture. introducing new and effective cultivation and propagation techniques for fruit species can play a vital role in the reconstruction of an agricultural micro­entrepreneurship, which is both self­ sufficient and motivated to succeed. the challenge for researchers who are attempting this enterprise is very great. we can only hope that their efforts will be rewarded by the expected results so that they can guarantee for tomorrow the survival of our agricultural production if we can keep that genetic heritage that will represent the basis for future breeding programs. for now we can only wish the greatest success to the researchers in this volume who, with great personal sacrifice, have started working on the challenge with so much enthusiasm and so much hard work. good luck and thank you! rossano massai impaginato 31 1. introduction apple (malus domestica borkh) is one of the most important horticultural crops considered as the third major fruit in the world (garming, 2014). the main proportion (½ million tons) of apple production in iran is used for fresh consumption (iranian ministry of agriculture, 2016), while some problems such as tissue softening during storage, tissue browning due to physical damages at post-harvest handling, high water loss and some physiological disorders such as bitter pit, water core, scald and internal browning are the most dominant post-harvest restriction factors (esna-ashari and zokaee khosroshahi, 2011). generally, quality apple must be mature, firm, crispy and juicy with a good flavor composition and free from mechanical damage, physiological disorders, and pathological diseases (baldwin, 2002). however, reducing consumer acceptance or nutritional value usually happens in the period after harvesting. apples contain several health-promoting compounds functioning as antioxidants, or modulators of enzyme activity. peel of red apples contains higher antioxidant than their flesh. meanwhile, drougoudi et al. (2008) discovered a positive correlation between phenolic content and antioxidant capacity in both flesh and peel of apple. color is also another important factor regarding fruit evaluation. there is a close correlation between color and overall quality of fruits (ritenour and khemira, 2007). salicylic acid (sa) is a natural compound that functions as plant growth regulator. sa carries a high potential of controlling post-harvest losses of horticultural crops. it has been discovered that the sa is associated with a delay in fruit ripening, (srivastava and dwivedi, 2000; zhang et al., 2003; mo et al., 2008), induction of disease resistance (shafiee et al., 2010), increasing antioxidant and phenolic compounds (peng and jiang, 2006; geransayeh et al., 2015), maintenance of post-harvest quality (srivastava and dwivedi, 2000; zhang et al., 2003; wang et al., 2006; mo et al., 2008; harindra et al., adv. hort. sci., 2017 31(1): 31-38 doi: 10.13128/ahs-20723 effect of preand post-harvest salicylic acid treatments on quality and antioxidant properties of ‘red delicious’ apples during cold storage m. hadian-deljou, m. esna-ashari*, h. sarikhani department of horticultural sciences, bu-ali sina university, hamedan, iran. key words: anthocyanin, firmness, phenolic compound, soluble solid content, weight loss. abstract: salicylic acid is a natural phenolic compound known as a plant hormone having positive effect on storage life and quality of fruits. this study aimed to investigate the effects of preand post-harvest application of salicylic acid on antioxidant properties and quality of ‘red delicious’ apples during 193 days cold (0±0.5°c) storage. both preand postharvest salicylic acid treatments did not affect soluble solid content, titratable acidity and fruit firmness, with the exception of 1 mm at pre-harvest application for titratable acidity. fruit juice ph was reduced in all fruits at the end of storage, while it was not quite uniform during storage. although there was no significant difference between the concentrations of salicylic acid in terms of fruit weight loss, but the highest amount of weight loss was observed in post-harvest treatments. salicylic acid application increased total phenolics and antioxidant activity at the earlier stages of storage showing the highest capacity with 2 mm followed by 1 and 4 mm salicylic acid concentrations, while 1 mm concentration belonged to the highest antioxidant capacity at the end of storage. anthocyanin content showed a gradual increase during storage until day 60, then decreased right afterwards. the highest amounts of anthocyanin were obtained from the concentrations of 1 and 2 mm salicylic acid in pre-harvest treatments, while 4 mm treatment was not encouraging. overall, salicylic acid treatments could increase apple storage life and quality for a short period of time only. (*) corresponding author: m.esnaashari@basu.ac.ir received for publication 6 december 2016 accepted for publication 7 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 31-38 32 2015) and increasing storage life of horticultural crops. hence, sa is suggested to be utilized in postharvest handling of fruits and vegetables (peng and jiang, 2006) which necessitates further investigation into know-how of its application. wen et al. (2008) found that treatment of grape berries with sa could induce an increase in phenylpropanoid and phenylalanine ammonia-lyase (pal). qin et al. (2003) reported a significant increase in polyphenoloxidase, pal and β-1, 3-glucanase activity in cherries fruit through sa treatment. exogenous application of sa on tomato green mature fruits has shown a delay in biosynthesis of some biochemicals including carotenoids, lycopene, ascorbic acid, total phenolics, free amino acids and γ-amino butyric acid that led to increase in the keeping quality of fruits (kant et al., 2016). pre-treatment of sa combined with lower storage temperature could provide a useful means of maintaining beneficial antioxidant activity during storage of navel orange (huang et al . , 2008). exogenous application of sa enabled grape leaves to maintain relatively higher activities of antioxidant enzymes under normal temperature, heat, or cold stress. the effect of sa on alleviating chilling injury of peaches during cold storage may be attributed to its ability to induce antioxidant systems (wang et al., 2006). the aim of this study was to maintain apple quality through the application of sa controlling physiological damages as well as investigating content of phenolic compounds, anthocyanin and antioxidant activity of fruits during cold storage. 2. materials and methods plant materials and salicylic acid treatments apples (malus domestica borkh cv. red delicious) were provided from a 20 years old orchard in horticultural research center, bu-ali sina university, hamedan, iran. this study was conducted as a factorial experiment based on a complete randomized block design with three replications. first stage of experiment launched on september when apples’ green skin had just started to turn reddish. four concentrations of sa including 0 (control), 1, 2 and 4 mm were sprayed on previously-selected trees, branches and fruits. sa concentrations were made by dissolving powdered sa (merck, germany) in hot water. in the second stage of experiment, non-treated fruits were first harvested according to the maturity index (starch test) using cornell starch-iodine chart in late september. starch test is a standard method of determining apple maturity to estimate optimum harvest dates well before picking fruit (blanpide and silsby, 1992). the harvested fruits then dipped in the same concentrations of sa solution for approximately three minutes at room temperature (25±1ºc). previously-treated apples were also harvested at this time. in this experiment, 15 apple fruit were used in each replicate for a totally of 45 fruit for each treatment. two apples per each replicate were used for the measurments of all parameters (except for the weight loss) at any time of determination (totally 6 times). three remaining apples from each replicate were kept to weigh at any time of determination for the evaluation of weight loss. for the packaging of the samples, each group of five apples was packed in a cubic plastic container with two small pores (2.5 mm in diameter) in each side, and stored at 0±0.5°c with 90% relative humidity and kept up to 193 days. measurements of all parameters started at the beginning of the storage and then continued until the day of 90 with 30 days intervals. two other measurements were taken 157 and 193 days of storage. soluble solid content, titratable acidity and juice ph apple juice was first prepared using a domestic electric apple juice maker available in the local shops. soluble solid content (ssc) was determined by measuring refractive index of the juice with a handhold refractometer (n1, atago co., tokyo, japan) at room temperature (25±1°c) and the results were expressed as °brix. titratable acidity (ta) was measured by titration with a calibrated titrator using a solution of the juice and water (2/10 ratio) with 0.1 n naoh to ph 8.2±0.1 and converted to malic acid percentage. fruit juice ph was determined using an aqualitic digital ph meter (model al10 ph) with a gel electrode. fruit firmness and weight loss firmness values of each individual apple were measured at three points of their equatorial region after which the peel was removed by using a manual penetrometer (fdk; wagner instruments, greenwich, ct, usa) with a 2 mm diameter flat probe (zhang et al., 2009). total phenolic content total phenolic content (tpc) of treated fruits were determined through a slightly modified version of folin-ciocalteu’s method as suggested by slinkard and singleton (1977). chlorogenic acid was used as standard phenolic compound. extraction was performed by homogenizing 0.5 g flesh tissue powder in 3 ml of 85% meoh and the extract was filtered with hadian-deljou et al. effect of salicylic acid on quality and antioxidant properties of ‘red delicious’ during cold storage 33 no.1 whatman filter paper. sample extract (300 µl) was mixed with 1500 µl of folin-ciocalteu’s reagent. after 5 min, 1200 µl of sodium carbonate solution (7.0%, w/v) was added, and the mixture vortexed and allowed to stand at room temperature (25±1°c) in the dark for 90 min. the absorbance was read at 765 nm in a uv/vis spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa), and the total phenolic concentration was calculated from a calibration curve, using chlorogenic acid as the standard. results were expressed as mg l-1 chlorogenic acid equivalents. total anthocyanin content for the assessment of anthocyanin contents of the apples, only their peels were used through applying a ph differential protocol (giusti and wrolstad, 2003). the absorbance was measured at 510 and 700 nm with a uv/vis spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa). anthocyanin content was then calculated using the following equation and expressed as mg cyanidin 3-galactoside equivalent per gram of fresh weight. mg cya-3-gal /g fw = [(a 510 a 700 ) ph1 (a 510a 700 ) ph4.5 ]×mw×f×1000/ε×d where: a= absorbance, mw= molecular weight of cyanidin 3-galactoside = 445.2 [g/mol], f= dilution factor = 10, d= cell pathlengths [cm], ε= molar absorbance of cyanidin 3-galactoside = 34300 [l/ mol×cm] and 1000= factor for mg. total antioxidant activity antioxidant activity was estimated using the stable 1,1-diphenyl-2-picryl hydrazyl (dpph) free radical (arnous et al., 2001) with slight modifications. the absorbance was read at t = 0 and t = 30 min with a spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa). for all the above assessments (except the anthocyanin measurement), the apple flesh was used. statistical analysis statistical analysis (analysis of variance) of the data was performed with sas software (version 9.1, 2002-2003, sas institute inc., cary, nc, usa). means were compared with duncan’s multiple range test. differences at p=0.05 were considered as significant. 3. results and discussion soluble solids content, titratable acidity and juice ph soluble solids content increased gradually during storage with no significant differences between the treatments (fig. 1). with pre-harvest application of 1 mm sa, ta slightly increased until the 60th day of storage, when reached its maximum value being significant with the other treatments, but decreased gradually afterwards (fig. 2). fruit juice ph was reduced in all fruits at the end of storage, while it was not quite uniform during storage (table 1). ssc may increase during fruit ripening due to the action of sucrose-phosphate synthase, a key enzyme in sucrose biosynthesis. this enzyme is activated by ethylene and the ripening process itself during storage. because organic acids are substrates of respiration, their levels decrease during ripening. utilization of these compounds over post-harvest period is the main reason of increasing sweetness in originally fig. 1 effect of preand post-harvest salicylic acid treatments on ssc of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. fig. 2 effect of preand post-harvest salicylic acid treatments on ta of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. adv. hort. sci., 2017 31(1): 31-38 34 high-sugar, high-acid apples or insipidity and blandness of fruit when sugar and acid concentrations are initially low (jackson, 2003). similar to our founding have been reported by sayyari et al. (2011) in which ssc increased during storage in both control and treated fruits. as previously reported, in climacteric fruits such as apples, starch turns into sucrose during post-harvest storage. mo et al. (2008) studies showed ssc in treated fruits to be lower than the control, suggesting that sa slowed starch degradation. this could be a possible reason why ssc increased in this study. however, dehydration of fruits during storage could be mentioned as another reason. organic acids are consumed in the process of respiration during post-harvest storage. it could be concluded that, the apples’ respiration rate in this study was possibly higher at the first two months of storage, and for this reason, the ta values were increased over this period, then lowered afterwards. srivastava and dwivedi (2000) stated that sa treatment has made ta fixed in banana. similar results have been reported while investigating chestnut by peng and jiang (2006). as the contents of organic acids in fruit are mainly dependent to the activities of their synthetic and hydrolytic enzymes, sa treatment was probably regulated the activities of related enzymes (ding et al., 2007). effects of salicylic acid on fruit softening fruits showed signs of softening during storage (fig. 3). firmness of apples was initially decreased in both control and sa-treated fruits, and then appeared rapidly. similar results was found by ding et al. (2007) and sayyari et al. (2011) who reported that fruit firmness decreased during storage. a close relationship between the change at endogenous sa level and the rate of fruit ripening and softening in kiwifruit was observed by zhang et al. (2003). softening could be the result of turgor loss, starch degradation, and most importantly cell wall degradation associated with weakening intercellular cohesive forces (jackson, 2003). sa decreases production of ethylene and inhibits activation of cell wall and membrane degrading enzymes such as polygalacturonase, lipoxygenase, cellulose and pectinemethylesterase resulting in the reduction of softening (srivastava and dwivedi, 2000; zhang et al., 2003). adding sa to the nutrient solution could induce firmness and delay the softening process in strawberry and apple fruits (shafiee et al., 2010; kazemi et al., 2011). effects of sa on weight loss sa-treated fruits demonstrated controlled weight loss during initial stages of post-harvest. the lowest amount of weight loss was observed in 2 mm concentration of sapre-harvest-treated fruits. higher concentrations of sa could induce fruit weight loss, but did not control it (fig. 4). it was observed that the apples kept their quality until the 157 days of storage, so that they were still suitable to be transferred to the market, but they gradually started to show visible defects on the peel losing their quality afterwards. therefore, we recommend our method to be suitable for the storage of red delicious apples for the period of five months. weight loss is caused by both dehydration and consumption of soluble solids during respiration. sa table 1 effect of preand post-harvest salicylic acid treatments on ph of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 4.55 a 4.34 b 4.36 a 4.39 a 4.28 a 4.18 a po 4.56 a 4.54 a 4.34 a 4.43 a 4.31 a 4.10 b t0 4.52 a 4.39 c 4.35 a 4.41 a 4.27 a 4.13 a t1 4.54 a 4.42 bc 4.35 a 4.42 a 4.31 a 4.14 a t2 4.58 a 4.49 a 4.33 a 4.39 a 4.28 a 4.13 a t3 4.55 a 4.46 ab 4.37 a 4.42 a 4.33 a 4.15 a prt 0 4.53 a 4.30 d 4.34 a 4.36 a 4.26 a 4.15 abc prt 1 4.57 a 4.34 cd 4.36 a 4.41 a 4.30 a 4.25 a prt 2 4.52 a 4.36 cd 4.34 a 4.38 a 4.26 a 4.19 ab prt 3 4.59 a 4.37 c 4.40 a 4.40 a 4.32 a 4.15 abc pot 0 4.40 a 4.49 b 4.36 a 4.46 a 4.28 a 4.12 bc pot 1 4.53 a 4.50 b 4.34 a 4.43 a 4.32 a 4.03 c pot 2 4.46 a 4.62 a 4.32 a 4.40 a 4.31 a 4.08 bc pot 3 4.51 a 4.55 b 4.34 a 4.43 a 4.34 a 4.16 ab fig. 3 effect of preand post-harvest salicylic acid treatments on firmness of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. hadian-deljou et al. effect of salicylic acid on quality and antioxidant properties of ‘red delicious’ during cold storage 35 could decrease water loss and respiration rate through controlling degradation of cell wall and reducing ethylene biosynthesis (srivastava and dwivedi, 2000), respectively. jonagold apples immersed in sa have shown a significant decrease in weight loss during cold storage (kazemi et al., 2011). in pear fruits, ratio of weight loss has been decreased at 0.5 mm sa after 48 h of treatment (imran et al., 2007). strawberries dipped in sa solution demonstrated less weight loss as well (shafiee et al., 2010). it seems that high concentrations of sa could induce aggregation of intoxicated materials such as certain polyphenols in later stages of storage, which is probably why plant tissues degrade followed by increasing weight loss. further investigations are needed to clarify this. effects of salicylic acid on accumulation of phenolic compounds application of sa partially induced accumulation of phenolic compounds and these effects were significant only from the 60th day until the end of storage (fig. 5). since then, sa-treated fruits contained higher quantities of phenols than the control. these results are in agreement with the findings of harindra et al. (2015) in grapes and geransayeh et al. (2015) in strawberries. in contrast with the results of this work, kant et al. (2016) reported a delay in the biosynthesis of total phenolics in tomato green mature fruits dipped in sa solution resulting in an increase in fruit quality that is possibly related to the different physiological status of tomato green mature fruits during storage. apples contain many bioactive compounds including phenols. sa may act as modulator of phenylpropanoid metabolism leading probably to the accumulation of phenolics correlated with the induction of enzymes which are involved in general phenylpropanoid metabolism, e.g. pal (godoyhernandez and loyola-vargas, 1997). it has shown that pal activity could be induced by sa elicitation in citrus (lafuente et al., 2001) and grapes (chen et al., 2006), which would result in the accumulation of plant secondary metabolites. effects of salicylic acid on anthocyanin content anthocyanin content of apples revealed an increasing trend until the 60th day of storage while decreased right afterwards (table 2). initially, effects fig. 4 effect of preand post-harvest salicylic acid treatments on weight loss of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. fig. 5 effect of preand post-harvest salicylic acid treatments on tpc of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. table 2 effect of preand post-harvest salicylic acid treatments on anthocyanin content (mg/g fw) of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 5.57 a 7.39 a 12.41 a 9.15 a 4.91 a 2.54 a po 3.74 a 5.53 b 10.92 a 9.10 a 5.13 a 2.22 a t0 5.75 a 6.32 ab 12.08 a 7.84 b 4.59 a 2.83 a t 1 7.37 a 8.29 a 13.08 a 11.62 a 5.07 a 1.54 a t 2 4.33 a 6.46 ab 11.63 a 10.02 ab 5.44 a 2.22 a t 3 3.29 a 4.68 b 10.06 a 7.29 b 4.85 a 2.91 a prt 0 5.26 a 5.66 bc 12.50 a 7.65 b 4.04 a 2.88 ab prt 1 5.68 a 8.54 ab 15.39 a 12.98 a 5.24 a 1.35 b prt 2 7.56 a 10.13 a 12.02 a 9.43 ab 5.94 a 2.47 ab prt 3 4.46 a 5.38 bc 9.69 a 6.46 b 3.93 a 3.64 a pot 0 5.17 a 7.14 abc 11.53 a 8.00 b 5.14 a 2.79 ab pot 1 5.67 a 7.98 ab 10.78 a 10.27 ab 4.96 a 1.70 ab pot 2 2.75 a 3.52 c 11.23 a 10.74 ab 5.02 a 2.03 ab pot 3 2.10 a 3.97 c 10.43 a 7.96 b 5.54 a 2.36 ab adv. hort. sci., 2017 31(1): 31-38 36 of sa on anthocyanin content were significant. the highest contents of anthocyanin were obtained in the concentrations of 1 and 2 mm at pre-harvest treatments with no significant differences between one and with 1mm of post-harvest treatment. similar result has been reported by jamali et al. (2013) who sprayed strawberry plants by sa together with nickel sulfate and found higher amounts of anthocyanin in strawberry fruits. anthocyanins degrade by polyphenol oxidase during post-harvest, and this might be the main reason behind the reduction of anthocyanin compounds. the role of sa on anthocyanin production is unknown, one may hypothesize that sa could activate the key enzyme (chalcone synthase) in the anthocyanin biosynthetic pathway (godoyhernandez and loyola-vargas, 1997). obinata et al. (2003) reported that sa could markedly increase the production of procyanidin in grape. total anthocyanin has also been increased with storage period in both control and treated pomegranates (sayyari et al., 2011). our results agree with findings of obinata et al. (2003) and sayyari et al. (2011). effects of salicylic acid on antioxidant activity antioxidant activity increased in all treatments during storage (table 3). antioxidant activity was increased in sa-treated fruits, but this process was not uniform, i.e. changes antioxidant activity was not followed from fixed pattern during storage. antioxidative potential of apple, however, is known to depend on the concentration and composition of phenolics. numerous researchers have indicated that sa and its functional analogs have inhibitory effects on cat and pod activities or serve as substrates for pod. knorzer et al. (1999) reported, when applied exogenously at suitable concentrations, sa was found to enhance the efficiency of antioxidant system in plants. imran et al. (2007) and sayyari et al. (2011) found that sa have the capacity of increasing antioxidant, the result that confirms the present study. kazemi et al. (2011) also observed the higher ascorbic acid (vitamin c) content as well as peroxidase and superoxide dismutase activities in apples treated with sa exogenously. sa is possibly effective through the activation of responsible genes for producing antioxidant compounds (wang et al., 2006) and increasing activity of the antioxidant enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase (mo et al., 2008). 4. conclusions this study showed the effectiveness of preand post-harvest treatment of sa on the quality of ‘red delicious’ apples. application of sa affected on juice ph and anthocyanin content until the middle of storage, maintaining phenolic compounds and increasing antioxidant activity. no significant difference between sa treatments was seen in terms of ssc, ta and fruit firmness. the lowest weight loss was observed on pre-harvest-sa treatment when sprayed, and the highest concentration of phenolic compounds was observed in post-harvest treatment when dipped. anthocyanin content was also increased during storage up to 60 days, but by the day 90, it was gradually decreased until the end of storage when reached to the lower level. as a conclusion, treatment of ‘red delicious’ apples with sa could properly maintain the quality of fruits and increase their post-harvest life and antioxidant capacity for at least 2 months. references arnous a., makris d.p., kefalas p., 2001 effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. j. agric. food chem., 49: 5736-5742. baldwin e., 2002 fruit flavour, volatile metabolism and consumer perceptions, pp. 89-106. in: kneen m. (ed.) fruit quality and its biological basis. crc press, boca table 3 effect of preand post-harvest salicylic acid treatments on antioxidant activity (% of dpph inhibition) of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 60.20 a 74.34 a 62.00 b 73.67 b 78.67 a 82.18 a po 47.40 a 67.35 b 84.25 a 81.53 a 76.79 a 82.49 a t 0 59.29 a 62.43 c 55.62 b 68.22 b 72.50 c 78.76 b t 1 50.14 a 69.88 b 79.16 a 78.26 a 83.93 a 83.67 ab t 2 61.03 a 81.16 a 81.52 a 81.93 a 79.84 ab 80.36 b t 3 58.24 a 70.27 b 79.68 a 81.42 a 74.55 bc 86.33 a prt 0 58.46 a 70.85 cd 50.37 d 70.33 cd 82.55 a 63.54 c prt 1 61.94 a 68.59 d 68.09 b 73.09 bcd 84.57 a 77.94 b prt 2 56.35 a 78.56 b 60.33 c 75.35 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to high temperature stress. j. plant growth regu., 55: 1-10. zhang h., wang l., ma l., dong y., jiang s., xu b., zheng x., 2009 biocontrol of major post-harvest pathogens on apple using rhodotorula glutinis and its effects on post-harvest quality parameters. biol. control., 48: 79-83. zhang y., chen k., chen q., zhang s., ren y., 2003 effects of acetylsalicylic acid (asa) and ethylene treatments on ripening and softening of post-harvest kiwifruit. acta botan. sinica, 45(12): 1447-1452. zhang y., chen k., zhang s., ferguson i., 2003 the role of salicylic acid in post-harvest ripening of kiwifruit. post-harvest biol. technol., 28: 67-74. 100 1. introduction open-field elevation cultivation of strawberries (fragaria x ananassa duch.) is being developed for fruit production in tropical regions (lópez et al., 2002; pirlak et al., 2002; riyaphan et al., 2005; pádua et al., 2009) and also in temperate areas (faedi, 2010; gambardella, 2010; rowley et al., 2010). nevertheless, little information is available on the quality of strawberries obtained from plants grown in mountain areas. a strong variability of horticultural characteristics and antioxidant profiles were observed among 12 cultivars grown at 730 m asl in the trentino region (italian alps), the most relevant attributes limiting quality being represented by poor taste and low flesh firmness (giongo et al., 2006). in the same area, a study by faedi et al. (2009) reported that ‘elsanta’ showed the best qualitative results in the first season crop among the tested cultivars. two commercial, ordinary june-bearing strawberry cultivars, ‘elsanta’ and ‘marmolada®onebor’, were selected for an elevation cultivation trial in the area of abetone mountain (apennine) in italy. the present paper reports the results observed on the quality of berries of these cultivars grown in open field mountain area. two cultivation strategies are also comparatively discussed one based on “first season crop” (fsc), carried out in 2008, and the subsequent year (2009) “second season crop” (ssc) as well as some aspects related to the ecological environment. 2. materials and methods location and environmental characteristics three experimental fields (ef1, ef2 and ef3) were established in three different locations around the abetone pass (apennine mountains) in tuscany (italy); details of the sites are reported in table 1. the three experimental fields had good exposure to light; all of them laid on humic umbrisols soils (lamma, 2012) with similar chemico-physical characteristics. the yearly average rainfall for the area is 2,000 mm; snow is present from late november until late spring (may). historical average of air temperature and humidity data and rainfall for the period 2008-2009 are reported in figure 1. from an ecological point of view, the selected sites for the experiment are represented by scarcely anthropized natural grasslands, highly biodiverse in terms of flora being constituted mainly by festuca pucinellii, trifolium thalii, plantago alpina, poa alpina, brachypodium genuense and nardus stricta, and surrounded by forests of european beech (fagus silvatica), european silver fir (abies alba mill.) and elder (sambucus racemosa l.) often consociated with blueberry (vaccinium myrtillus l.) (dondini and vergari, 2009). production of strawberries (fragaria x ananassa duch.) in mountain areas: a comparative evaluation of berries from two june-bearing cultivars e. giordani, w.a. petrucci, d. morelli, a. ferri dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: elevation cultivation, ‘elsanta’, garden strawberry, ‘marmolada®onebor’. abstract: two uniferous strawberry cultivars, ‘marmolada®onebor’ and ‘elsanta’, were tested in three experimental fields located at 1,200 m asl; phenological phases and fruit quality of first and second season crops were compared. harvesting time in the first year crop occurred 40 days later than in the second season crop. cultivar significantly affected fruit skin brightness and chroma index, flesh firmness, titratable acidity and yield in the first season crop; the experimental field location exerted a significant effect also on fruit weight and diameter, but not on total acidity. fruit weight was higher in the second season crop, and ‘marmolada®onebor’ fruits (22.7 g) resulted heavier than those of ‘elsanta’ (19.7 g). total solid soluble content ranged from 7.4 to 8°brix in ‘elsanta’, compared to 6.2 to 7.4°brix in ‘marmolada®onebor’. neither diseases nor arthropod attacks were noticed on plants and fruits. adv. hort. sci., 2012 26(2): 100-109 received for publication 17 february 2012. accepted for publication 4 june 2012. 101 soil management and plant spacing in may 2008 the soil area destined for the three experimental fields (500 m2 each) was ploughed 30 cm deep and mature manure was distributed uniformly at the rate of 80 t/ha; soil was not fumigated. the strawberries were planted in parallel hill rows 110 cm apart with a distance of 30 cm between plants within the row, with a density of 4 plants/m2; the plants of each cultivar were distributed in an alternate four-row scheme for a total of eight rows for each experimental field. a black polyethylene film was used along the rows for mulching. each plot was surrounded by guard rows. plant material, planting and management one-crown homogeneous-size certified refrigerated plants of ‘elsanta’ and ‘marmolada®onebor’ were supplied by a qualified commercial nursery. in early may 2008 strawberries were planted in the experimental fields, from which the fsc was obtained; the same open field unprotected, overwintered and not de-crowned strawberry plants generated the ssc in 2009. plants were drip irrigated with 1,000 l/d average per experimental field, from flowering time up to the end of the productive season; no treatment was adopted for pest and disease control. weeds were manually removed. data collection during the two years of study (2008 and 2009), observations regarded phenological, morphological and chemical characteristics. flowering and ripening time were recorded on one sample every five plants and reported as days from the beginning of the year. morphological data were measured on four replicated sets of 25 ripened fruits each for every experimental unit (cultivar/location) during the period of maximum productive peak. fruits were harvested and promptly characterized for weight and maximum diameter, skin colour, flesh firmness (two sides), fruit shape and presence of internal cavity. weight (g) was measured with a precision balance (sartorius te 150/2s sartorius) and diameter (mm) with hand calliper; colour (l, a, b coordinates) was determined with a minolta chromameter cr200 konica minolta electronic colorimeter and the a and b coordinates were transformed in the chroma index (a2 + b2)1/2; flesh firmness (g) was measured with a 6 mm diameter plunger hand penetrometer tr 53200. fruit shape and presence of an internal cavity were visually assessed following the upov descriptor for strawberry (upov, 2008). chemical parameters such as ph, titratable acidity and total solid soluble (tss) content were assessed on samples (four replicates per experimental unit) of juice extracted from 10 fruits. titratable acidity (meq malic acid/100 g fresh weight) was determined at ph 8 with a 0.1 n solution of naoh, adopting a basic 20 crison ph meter; total soluble solid content (°brix) was quantified with a hand refractometer (atago n1 atago co., ltd). fruits were tasted by five experts to assess sensorial quality and flavour quality was rated on a five-score scale (1 very weak; 2 medium weak; 3 medium; 4 good; 5 very good), while for persistence of taste 3 min after ingestion and scent intensity, a three-score scale (1 weak; 2 medium; 3 strong) was adopted. experimental design and statistical analysis each of the three experimental fields was split into 16 rows (eight alternate rows per cultivar); every row held 125 plants. a two-way analysis of variance was employed to test the significance of the effects due to cultivar (‘eltable 1 description of experimental fields (ef): altitude, coordinates, soil texture, n concentration (‰), c concentration (%), ph, field capacity (f.c.) and wilting point (w.p.) experimental fields altitude (m a.s.l.) coordinates (n/w) soil texture n‰ c% ph f.c. (-0,33 bar) w.p. (-15 bar) ef 1 1,213 n 44°08.650’ sandy-loam 0.3 3.5 5.2 20.1 11.6 w 10°41.412’ ef 2 1,250 n 44°08.517’ loam 0.1 3.6 6.2 18.4 11.1 w 10°42.043’ ef 3 1,250 n 44°07.810’ sandy-loam 0.1 3.7 5.9 23.6 16.5 w 10°45.336’ fig. 1 mean air temperature (°c), relative humidity (%) and rainfall (mm) of the trial location of years 2008, 2009 and historical series (data elaborated from lamma). 102 santa’ and ‘marmolada®onebor’) and experimental field location (efl1, efl2 and efl3), both considered as independent factors, and their interaction. duncan’s test was applied for mean separation; the averages were reported with standard errors. two-tail chi2 test was applied to qualitative and scale-scored parameters exposed as frequencies. spss statistics 17.0 software was used for analyses; differences at p < 0.05 and p < 0.01 were considered significant and very significant, respectively. 3. results the anova statistical significance of the different factors on the studied parameters are reported in table 2; each of them will be discussed in the following sections. first season crop (fsc) both elsanta and marmolada®onebor cultivars showed the same pattern of phenological stages in the three locations; in detail the full blooming started on 8 july 2008 (day 190) and portrayed until day 203 for a total of 13 days. the first fruits of both cultivars ripened 24 days later (day 214), and harvest time closed on day 234; the period of maximum peak of ripened strawberries fell in the period 4-11 august 2008 (days 217-224) (fig. 2). average fruit weight resulted identical for both cultivars (15.0±0.4 g)(anova; p = 0.69) (table 2). field location and its interaction with cultivar exerted a statistically significant effect on this parameter (anova; p < 0.01), with the highest and the lowest weights of 19.1±0.7 g and 11.8±0.7 for ‘marmolada®onebor’ obtained in ef1 and ef3, respectively; for ‘elsanta’ the maximum and minimum values were 15.9±0.7 g in ef1 and 13.8±0.7 g in ef3 (fig. 3a). as expected, the fruit maximum diameter (average 32.9±6.1 mm in ‘elsanta’ and 32.04±6.2 mm in ‘marmolada®onebor’) followed a behaviour similar to that observed for fruit weight, and it was affected significantly by the experimental field and its interaction with cultivar (fig. 3a). skin colour was analysed taking into account brightness (l) and chroma index. l values ranged from 30.9 to 47.6 for ‘elsanta’ and 24.7 to 47.8 for ‘marmolada®onebor’, with averages of 38±0.3 and 35.2±0.3, respectively (anova; p <0.01). fruit skin brightness showed differences of 2 units (≈ 35 against ≈ 37) due to the effect of the experimental field location (anova; p < 0.01). analogous results were observed for chroma index, with average values of 40.3±0.4 and 37.1±0.4 for ‘elsanta’ and ‘marmolada®onebor’ (anova; p < 0.01); similarly to skin brightness, ef exerted a very significant influence on chroma index, with a variation of ≈ 2 units. flesh firmness resulted statistically different (anova; p<0.01) between the cultivars; the highest average value was observed in ‘marmolada®onebor’ (473.1±9 g), which is about 33% higher than the firmness of ‘elsanta’ fruits (355.6±7.9 g). the experimental field location exerted a significant effect on fruit firmness (p<0.05); a 5% difference between the maximum and the minimum average values was observed. no relevant differences were found in ph of juice, showing a value of 3.6 for both cultivars, and no effect was attributable to the location of the experimental fields. different results were obtained for titratable acidity, which was higher in ‘elsanta’ fruits (12.6±0.8 meq/100 g fw) than in ‘marmolada®onebor’ (9.6±0.4 meq/100 g fw) (anova; p<0.05); location did not affect this parameter. taking into account the total solid soluble content of strawberry juice, the cultivar elsanta showed an average value of 7.4±0.3 °brix, against 6.2±0.3 °brix of fig. 2 blooming and fruit ripening time in years 2008 (fsc) and 2009 (ssc). table 2 significance of cultivar, experimental field location and their interactions on the quantitative parameters analyzed on strawberry fruits resulted from analysis of variance (anova) attribute crop cultivar cv experimental field ef cv*ef replicates fruit weight fsc ns ** ** ns ssc ** ** ** ns fruit diameter fsc ns ** * ns ssc ns ** ns ns fruit brightness (l) fsc ** ** * ns ssc ns ** ** ns chroma index fsc ** ** ** ns ssc ns ** ** ns flesh firmness fsc ** * ** ns ssc ** ** ** ns fruit ph fsc ns ns ns ns ssc ns ns ns ns titratable acidity fsc * ns ns ns ssc ** ** ns ns tss content fsc ns ns ns ns ssc * ns ns ns ns= non significant; * = 0.05<p<0.01; **= p<0.01. 103 ‘marmolada®onebor’; such differences were not statistically significant (anova; p>0.05). the distribution of fruit shape resulted similar among cultivars; no statistical differences were observed between cultivars for conical and globose-conic classes, the most frequent fruit shapes. significant differences (chi2; p< 0.01) were found in the percentages (40% and 27%) of globose-conic fruits of ‘elsanta’ and ‘marmolada®onebor’, respectively (fig. 4). the internal cavity was present in approximately 81% of fruits of both cultivars; a significant statistical difference (chi2; p<0.01) was observed taking into account the experimental field (ef factor), since almost 100% of fruits from ef2 showed internal cavity, against about 75% of those obtained in the other two sites. the distribution of fruits for each cultivar taking into account the quality of taste, resulted as an average value of five taste assessors, is reported in figure 5a. ‘elsanta’ showed a percentage (86%) of fruits of acceptable quality (namely the sum of the “medium”, “good” and “very good” classes) which was higher than the one observed in ‘marmolada®onebor’ (63%); such difference resulted non significant (chi2; p>0.05). on the other hand, no relevant effect was exerted by the experimental field location. the fruits of ‘elsanta’ showed a higher percentage of fruits with medium (69%) and strong (21%) persistence of taste 3 min after ingestion, against 61% and 3% observed on ‘marmolada®onebor’ fruits. the effect of the location of the experimental field was not statistically significant for this parameter, but the best results were obtained in ef2. a similar situation was observed for scent intensity, which resulted superior in ‘elsanta’ fruits compared to those of ‘marmolada®onebor’, with a higher percentage of strongly scented fruits (26%) against (8%)(chi2; p <0.01)(fig. 6a); the location producing more fruits with strong scent was ef2. second season crop (ssc) full blooming of ‘elsanta’ and ‘marmolada®onebor’ was observed in the three plots on 29 may (day 150) and it finished 14 days later (day 164). the first fruits ripened at day 177 (26 june 2009) and the last ripened on day 209 (28 july 2009), with the maximum peak of ripened fruits between days 186 and 192 (fig. 2). the highest fruit weight was observed for ‘marmolada®onebor’ (average 22.4±0.5 g) while a lower mean value was found in ‘elsanta’ fruits (19.7±0.5 g) fig. 3 fruit weight and diameter for ‘elsanta’ and ‘marmolada®onebor’ in the first (a) and second (b) season crop observed in each experimental field (ef). bars with different letters are statistically different (duncan’s test; p<0.05). fig. 4 fruit shape distribution (%) of ‘elsanta’ and ‘marmolda® onebor’ for years 2008 (fsc) and 2009 (ssc). chi2 statistical differences within columns: ns= not significant; * significant (p<0.05). ns ns ns 104 (anova; p< 0.01). also the experimental field location affected fruit weight very significantly, with the highest mean values observed in ef3 (28.7±0.7 g); a reduction of about 40% of this parameter was observed in ef1 and ef2. the effect of the interaction cultivar by location on fruit weight was also very significant (anova; p<0.01) with the highest mean values found in fruits of ‘marmolada®onebor’ grown in ef3 (31.5±0.9 g) and the lowest for those of ‘elsanta’ grown in ef1 (15.3±1.0 g). fruit diameter resulted practically identical between the cultivars (37.2±0.4 mm and 36.1±0.4 mm for ‘marmolada®onebor’ and ‘elsanta’, respectively); in acfig. 5 fruit taste: distribution (%) of the first (a) and second (b) season crop strawberries for different quality classes of ‘elsanta’ and ‘marmolada®onebor’. acceptable fruit is the sum of medium, good and very good classes. bars with different letters are statistically different (chi2; p<0.05). fig. 6 fruit scent intensity: distribution (%) of strawberries of elsanta and marmolada®onebor cultivars for the first season crop (a) and the second season crop (b). bars with different letters are statistically different (chi2; p<0.05). 105 cordance with fruit weight, the greatest diameters were observed in ef3 for fruits of ‘marmolada®onebor’ (fig. 3b). skin brightness (l coordinate) was similar between the cultivars, ranging from 29.3 to 47.8 for ‘elsanta’ and 29.2 to 47.1 for ‘marmolada®onebor’, with averages of 37.7±0.3 and 37.3±0.3 respectively. only field location exerted a strong effect on this parameter (anova; p<0.01); skin brightness reached the highest value (39.7±0.4) in ef2, followed by ef3 (36.8±0.4) and ef1 (35.9±0.3). similarly, chroma index of skin colour was influenced by the location of experimental field (anova; p<0.01) with the highest values in fruits from ef2, decreasing then in those obtained in ef3 and ef1. the chroma index average value for fruits of ‘elsanta’ was 34.3±0.5 while for ‘marmolada®onebor’ it was 33.8±0.5. the interaction of cultivar and location affected very significantly these two colour parameters (table 2). taking into account fruit flesh firmness, ‘elsanta’ showed the highest average (490±0.5 g) compared to that of ‘marmolada®onebor’ fruits (460.2±4.7 g)(p<0.01). the experimental field location and its interaction with cultivar exerted a very significant effect on fruit firmness (p<0.01), with differences of around 13% between the maximum and the minimum average value found in ef3 and ef1, respectively. the juice of ‘elsanta’ and ‘marmolada®onebor’ fruits showed the same ph value (3.6), while titratable acidity was affected by the cultivar and location of cultivation. ‘elsanta’ showed a higher titratable acidity (average 12.8±0.6 meq/100 g fw) than that observed in ‘marmolada®onebor’ fruits (10.7±0.5 meq/100 g fw) (anova; p< 0.01). analogously, the location of cultivation exerted a very significant effect on this parameter; the highest mean value (13.5 meq/100 g fw) was found in berries obtained in ef2, against 10.9 meq/100 g fw of those harvested in ef1. the cultivar elsanta showed an average value of 8.1±0.7 °brix for total solid soluble content of juice, against 7.4±0.8 °brix of ‘marmolada®onebor’ (anova; p <0.05). no significant differences were observed due to the location of the experimental field; in any case, the highest value was observed in ‘elsanta’ cultivated in ef2 (8.6±0.3 °brix); ‘marmolada®onebor’ showed the maximum value of total solid soluble content in ef1 (8±0.3°brix). the most frequent fruit shapes were conical and globose-conic (fig. 4). the differences between the cultivars resulted statistically very significant (p<0.01) for both shapes; ‘elsanta’ presented 68% and 26% of fruits belonging to the globose-conic and conical shapes, while in ‘marmolada®onebor’ the frequencies were of 36% and 58%, respectively (fig. 4). the difference between cultivars for the presence of internal cavity was non significant, with values of 35% and 45% of cases in ‘elsanta’ and ‘marmolada®onebor’ respectively. the location of cultivation exerted a significant effect (chi2; p<0.05) on this characteristic; the lower percentage of fruits showing internal cavity was observed in ef1. the distribution of cases for fruit taste is illustrated for both cultivars in figure 5b. taking into account the percentage of acceptable fruits in terms of quality of taste, 59% and 46% for ‘elsanta’ and ‘marmolada®onebor’ respectively, the cultivar effect resulted statistically not significant. on the contrary, the effect of the experimental field location on fruit taste was very significant: ef1 showed 72% of fruits with acceptable taste, while these values were of 44% and 18% for ef2 and ef3, respectively (chi2; p<0.01). the persistence of taste after ingestion resulted identical for both cultivars and the location did not exert a relevant effect on this parameter. none of the cultivars produced fruits with strong scent; ‘elsanta’ showed higher percentages of fruits belonging to the medium class (59%) against 22% of ‘marmolada®onebor’ (chi2; p<0.01) (fig. 6b). taking into account the location of the experimental field, ef3 showed the worst result, with the highest percentage of fruits with weak scent (80.7%), against ≈ 55% observed in the other two fields. 4. discussion and conclusions no differences were observed for flowering and fruit ripening time between cultivars and experimental fields. in 2008, the interval between planting date and flowering time corresponded to 24 days for both cultivars, which is almost half the time indicated by perez de camacaro et al. (2004) for ‘elsanta’ plants grown in the uk; conversely the interval flowering-ripening time (36 days) coincided with that found by perez de camacaro et al. (2004). in the second season crop, this interval was eight days shorter for the strawberries grown in tuscany (28 days), and the same (36 days) as in the trial reported by perez de camacaro et al. (2004). the fsc ripening time for ‘elsanta’ and ‘marmolada®onebor’ cultivars showed a delay of 40 days in relation to the second year crop. this finding confirms the possibility of scheduling the strawberry harvesting time offered by elevation cultivation, as indicated by faedi et al. (2009). the average weight of fsc fruits was about 15 g for both cultivars, but experimental field and its interaction with the cultivar exerted a strong effect on this parameter. the heaviest fruits (average 19.1 g) were obtained from the plants of ‘marmolada®onebor’ grown in ef1; they weighed about 7 g more than the fruits collected in ef3. a lower magnitude difference was observed in ‘elsanta’ fruits. in the second season, crop fruit weight was on average 25% higher than the value for 2008; furthermore the cultivar factor was more relevant, since ‘marmolada®onebor’ fruits (average 22.7 g) were about 3 g heavier than those of ‘elsanta’ (19.7 g). a similar situation was observed by coman et al. (2002) for the same cultivars grown on the north-eastern coast of the usa (19 and 14 g/fruit for ‘marmolada®onebor’ and ‘elsanta’, respectively). the values obtained for fruit weight of ‘elsan106 ta’ in the second season crop are similar to those indicated by fitogest (2011) and much higher than those observed by giongo et al. (2006) in similar growing conditions and by palha et al. (2009) in soilless culture. on the other hand, the fruit weight of ‘marmolada®onebor’ fruits was very close to the value indicated by fitogest (2011). slightly lower average weights (17.2 g) were found in a set of cultivars grown in brazil at 1,370 m asl by pádua et al. (2009), while a higher value (26.3 g/fruit) was observed for ‘marmolada®onebor’ in cesena (italy) on oneyear-old planted strawberries (tagliavini et al., 2005). the observations confirm that average fruit weight is lower in the first season crop than in the second, which is in accordance with the results of previous studies on ‘elsanta’ and ‘bolero’ (perez de camacaro et al., 2004). also the experimental field influenced fruit weight with heavier fruits obtained in ef3. the opposite influence of ef3 on fruit weight in the second year crop can be attributed, more than to the intrinsic characteristics of the location (e.g. soil chemico-physical properties), to a predominant effect of the cultivar due to cumulate effects associated to the changes in vegetative growth of the second year. in strawberry, the predominant sink is represented by fruits (olsen et al., 1985); fruiting affects dry-matter partitioning, hence the changes in vegetative development may significantly interfere with fruit production as suggested by perez de camacaro et al. (2004). fsc strawberries were underweight by a few grams when taking into account the optimal market values indicated by lovati (2010). conversely this parameter was acceptable for the second year crop. fruit diameter resulted highly correlated with fruit weight (r2= 0.87; p<0.01), nevertheless it was less influenced than fruit weight by cultivar effect. again, the observed values of ‘elsanta’ fruit diameter are in accordance to those indicated by giongo et al. (2006). taking into account fruit colour, brightness resulted close or superior to the acceptable threshold of 37, as indicated by lovati (2010) for both cultivars and for the two crops; on the other hand, chroma index was lower than the limit value (40). colour parameters were less affected by cultivar in the second season crop; conversely experimental field was found to be a more relevant factor. during 2008 the fruits obtained in f1 showed the highest values of brightness and chroma index, while in 2009 the most attractive fruits in terms of brightness and chroma index were grown in ef2. brightness values in both years were almost the same: chroma index was 4 points higher in 2008 than in 2009. flesh firmness of fruits was significantly affected by the studied factors and by their interactions. the optimal range of flesh firmness for harvesting and handling strawberries is indicated as 300-400 g (lovati, 2010), about 20% lower than that observed for first and second season crops for both cultivars under study. the average firmness found for ‘elsanta’, regardless of year and experimental field, was 423 g, approximately 25% higher than that found by giongo et al. (2006) in plants grown at 730 m asl, demonstrating a good texture of the fruits at ripening time, and hence better suitability to handling and transport. fruit juice ph was not affected by any studied factor, resulting substantially stable with a value of 3.6 in both fsc and ssc. titratable acidity was significantly influenced by the cultivar in both fsc and ssc, with average values of about 12.7 and 10.2 meq/100 g fw for ‘elsanta’ and ‘marmolada®onebor’ respectively. such values are within the range of 10-15 meq/100 g fw indicated as optimal for strawberries by roudellac and trajkovsky (2003) and lovati (2010). giongo et al. (2006), observed a higher value (13.6 meq/100 g fw) in ‘elsanta’ fruits obtained in similar growing conditions; in a trial conducted in slovenia, ‘elsanta’ showed a total acid content higher than the one found for ‘marmolada®onebor’ (sturm et al., 2003), which is in accordance with the results of this research. titratable acidity was lower (7 meq/100 g fw) in the juice of ‘marmolada®onebor’ fruits collected in cesena (italy) from one-year-old plants (tagliavini et al., 2005); location seems to have a strong effect on this parameter, as confirmed in our experiment for the second season crop where location exerted a significant influence with a maximum variation of about 30% of the amount of titratable acidity. taking into account the total solid soluble content of strawberry juice, the cultivar ‘elsanta’ showed an average value of 7.4 and 8°brix, against 6.2 and 7.4°brix of ‘marmolada®onebor’, in 2008 and 2009 respectively. these results, even if higher in value, show a similar trend to those found for ‘elsanta’ (about 5.8°brix) and ‘marmolada®onebor’ (about 5.2°brix) grown under identical conditions in slovenia by sturm et al. (2003). similar results were obtained by radajewska and dejworborowiak (2002) on both cultivars grown in poland. nevertheless, different values of total solid soluble content of ‘elsanta’ fruits have been found by different authors and for plants growing in diverse areas and cultivation systems. kovačević et al. (2008) noted a value of 7.2°brix in fruits grown under organic and conventional cultivation. palha et al. (2009) found values ranging from around 7.7 up to 10 °brix on strawberries in soil less cultivation, depending on planting date, tray and bare-rooted plant type, and similar values were observed by voća et al. (2007) in croatia for the same cultivar. giongo et al. (2006) recorded an average value of 8.4°brix in fruits obtained from cultivation in buckets at 730 m asl. lower mean values of solid soluble content (7.2°brix) were observed by pádua et al. (2009) on a different set of cultivars grown in brazil at 1,370 m asl. the values observed for ‘marmolada®onebor’ were higher than those shown by the same cultivar grown in flat areas (4.7°brix) (tagliavini et al., 2005), thus confirming the positive influence of environmental conditions of mountain areas on fruit quality. no statistical differences were observed taking into account the experimental fields and their interaction with the cultivar, nevertheless it is worth noting that ‘elsanta’ fruits obtained in ef1 reached 8.1°brix, which is in accordance with the values found by giongo et al. (2006). however the results obtained in the present study are higher than the threshold of 7°brix, 107 adopted as a standard quality parameter for strawberries (roudeillac and trajkosky, 2003). the results here obtained for ‘marmolada®onebor’ are higher than those reported by kovačević et al. (2008) and plantgest (2012) of 5.7-6.0 and 5.7 °brix respectively. in this study total solid soluble content was associated with taste quality, even if this sensorial parameter is influenced also by many other factors such as acidity, flavours, flesh texture, etc. ‘elsanta’ fruits resulted better in taste quality than those of ‘marmolada®onebor’ for the two years of observation (fig. 5) and in taste persistence in the fsc. similarly ‘elsanta’ fruits showed a higher percentage of fruits in the superior class of scent intensity (fig. 6). these results are in accordance with those found by coman et al. (2002) in fruits of ‘marmolada®onebor’ and ‘elsanta’ grown on the north-eastern coast of the usa and with those of radajewska and dejwor-borowiak (2002) in a trial conducted in poland. fruits of the fsc resulted better than those obtained as ssc for all the analysed sensorial attributes (taste, taste persistence and scent intensity); this result may be associated to the about 15% higher total soluble content and slightly lower titratable acidity observed in the juice of strawberries derived from the fsc, with respect to those of the ssc. most of consumers prefer conical, or slightly roundconical strawberries (tirelli, 2010). the predominant fruit shapes found in the present study were conical and globose-conic; a cultivar effect was noted, but location did not seem to exert any influence on this parameter. the most frequent class of fruit shape in the fsc was conical while conversely globose-conic was predominant for the second season crop. the variation from one class to the other was more evident in ‘elsanta’ fruits (fig. 4). the internal cavity is a morphological attribute that does not have a relevant effect on quality but may modify the shape and volume of fruits. this parameter was not cultivar-dependent but location had a significant influence on the percentage of fruits with an evident internal cavity. furthermore, the growing system (fsc or ssc) seemed to exert a similar effect, since during the fsc around 80% of fruits showed this attribute compared to 40% in the second year crop. ‘elsanta’ is considered highly susceptible to soil-born diseases and powdery mildew, while ‘marmolada®onebor’ is deemed to be less susceptible to fungal diseases (baruzzi et al., 2009). nevertheless, no disease or arthropod attacks were observed in this trial on either of the cultivars in both years of experimentation. conversely łabanowska et al. (2004) observed heavy symptoms caused by powdery mildew and strong attacks of mites (tetranychus urticae and phytonemus pallidus ssp. fragariae) on ‘elsanta’ plants, while ‘marmolada®onebor’ was strongly infected by leaf spot (mycosphaerella fragariae) and attacked by strawberry blossom weevil (anthonomus rubi). analogously, hietaranta et al. (2004) evaluated ‘elsanta’ as insufficient for resistance to pest and diseases in nordic european countries. this means that the environmental characteristics of the area chosen for the trial exert a positive effect on plant health. this is not a secondary aspect in strawberry production, since the practices adopted to reach high yields in flat area cultivation (including a massive use of pesticides, soil fumigants and fertilizers) are in contrast with the market demand oriented towards healthy, organic and high quality strawberries (gengotti et al., 2008; mennone et al., 2008). furthermore, the demand by consumers for healthy fresh fruits, rich in antioxidants, produced locally and in unpolluted environments is noticeably increasing (tulipani et al., 2008). strawberry high-altitude cultivation opens a new scenario since the ecological conditions (climate, soil, and spontaneous flora and fauna, associated with a generally low anthropized environment), seem to positively influence the nutritional properties of berries, for example increasing their polyphenol content as reported by szajdek and borowska (2008), and above all exert a strong influence on the amount and spectrum of harmful pests and diseases and hence on the cultural practices needed for production. guerena and born (2007) found that spontaneous flora alongside strawberry fields represented a shelter and a source of pollen and nectar to predators and parasites of insect pests, thus reducing the amount of damaged plants and strawberries. furthermore, in almost natural conditions and sustainable farming systems the soils are rich in arbuscular mycorrhizal fungi which strongly contribute to counter-balance the appraisal and diffusion of soil-born diseases, such as fungi like fusarium, phytophthora, pythium, rhizoctonia and verticillium, and nematodes (branzanti et al., 2002; harrier and watson, 2004). additionally, naturally fertile soils, namely in terms of organic matter content, are typical of many mountain areas. in this regard, gonzalez and acuña (2009) found that in strawberry cultivation a soil rich in organic matter may supply fertilization at least for the first year of establishment, hence reducing the chemical inputs which are a source of pollution to adjacent ecosystems (tagliavini et al., 2005). strawberry high-elevation cultivation is feasible also in temperate areas as shown by this study. the main factors affecting fruit quality and productivity are related to cultivar and to location, this latter being a relevant aspect even under highly homogeneous environmental conditions. the first season crop, showing higher fruit quality than the second season crop, is particularly adequate for scheduling ripening time, hence filling the gaps in product offer by flat-area cultivation during specific periods. furthermore, mountain cultivation in marginal and low anthropized areas allows production of high quality marketable strawberries. further comparative tests are needed to study in detail the effect of climate, soil, and especially of the ecological background of mountain areas on strawberry cultivation. acknowledgements research funded by arsia tuscany region and comunità montana appennino pistoiese. 108 references baruzzi g., capriolo g., lucchi p., rocco m., sbrighi p., faedi w., 2009 liste varietali fragola. terra e vita, 49: 24-35. branzanti m.b., gentili m., perini r., neri d., cozzolino e., 2002 the micorrhizal status of strawberry plants under different pre-planting and cultivation techniques in the field. acta horticulturae, 567: 495-497. coman m., faedi w., nourse t., 2002 field performance of some italian strawberry genotypes under conditions of the northeastern coast of usa. acta horticulturae, 567: 191-193. dondini g., vergari s., 2009 natura sull’appennino pistoiese (toscana settentrionale). felici editore, pisa, italy, pp. 190. faedi w., 2010 fragola nel mondo, pp. 358-361. in: angelini r. 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(ed.) la fragola. art servizi editoriali spa, bologna, tulipani s., mezzetti b., capocasa f., bompadre s., beekwilder j., ric de vos c.h., capanoglu e., bovy a., battino m., 2008 phenolic compound and nutritional quality of different strawberry genotypes. j. agric. food chem., 56: 696-704. upov, 2008 guidelines for the conduct of tests for distinctness, uniformity and stability. strawberry. http://www. upov.int/en/publications/tg_rom/tg_index.html. voća s., dobričević n., skendrović babojelić m., družić j., duralija b., levačić j., 2007 differences in fruit quality of strawberry cv. elsanta depending on cultivation system and harvest time. agriculturae conspectus scientificus, 72(4): 285-288. 17 1. introduction although in italy lawns are traditionally established with cool season grasses, the use of warm season species has been increasing over the last two decades (croce et al., 2004; volterrani et al., 2010). warm season grasses show several advantages over cool season species. they tolerate high temperatures and drought, show lower water consumption, tolerate higher salinity concentrations in the soil and in irrigation water, establish rapidly and present excellent recovery properties due to the abundant production of stolons and rhizomes (biran et al., 1981; carrow and duncan, 1988; beard, 1989; dudeck and peacock, 1993; beard and sifers, 1997; volterrani et al., 1997; croce et al., 2006; harivandi and marcum, 2008). bermudagrasses (cynodon spp.) are the warm season turfgrass species most widely used in the transition and warm regions of the world due to their aggressive growth habit, tolerance to a wide range of mowing heights, marked resistance to many abiotic stresses, and the high quality of the cultivars recently developed by breeding programs (wu et al., 2009). a technique recently developed for turf establishment of warm season species is based on the transplant of single plug plantlets pre-rooted in the nursery (volterrani et al., 2008). nursery activity consists of the pot cultivation of donor plants from which stolons are collected, divided into one-node sprigs about 2 cm long, then cultivated in alveolate trays until the formation of mature plantlets. the crucial point of this practice is stolon fragmentation. this operation is very time consuming and consequently labour-costly. in fact, stolons must be cut several times to obtain sprigs of the right length due to long internodes. the effectiveness of trinexapac-ethyl (te) in controlling bermudagrass growth is reported by many authors (johnson, 1994, 1997; fagerness and yelverton, 1999; lowe and whitwell, 1999; fagerness and yelverton, 2000; fagerness et al., 2002; bunnel et al., 2005; mccullough et al., 2005; baldwin et al., 2006; mccullough et al., 2007; baldi et al., 2010). in these reports, te is applied to bermudagrass turfs in order to facilitate their management (mainly by reducing mowing requirements), ameliorate their quality or enhance stress tolerance (e.g. salinity tolerance or cold tolerance). the rates of te normally adopted in bermudagrass turf management are around 0.11 kg a.i. ha-1, which is the recommended rate for bermudagrass maintained at golf fairway height (cooper, 2003). plant height, texture, density, clipping weight and root mass are the growth parameters related to plant response to te to be considered. on the contrary, since turf establishment by means of pre-rooted plantlets propagated by stolon in the nursery has been recently developed, the effects of plant growth regulators (pgrs) on grass plants grown to produce propagating material have not been investigated. furthermore, parameters such as stolon length, internode length and stolon number, important for nursery activity, have been seldom considered. traditionally, pgrs are classified into two main groups, type i and type ii, according to their biological mode of effects of trinexapac-ethyl on stolon development in potted patriot bermudagrass a. lenzi, a. baldi, m. nannicini, a. pardini, r. tesi dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: internode length, nursery production, plant growth regulators, sprigs, warm season turfgrasses. abstract: a recent technique developed for establishment of warm season turfgrasses is based on the transplant of single plug plantlets pre-rooted in the nursery. plantlets are obtained from one-node sprigs about 2 cm long derived from stolon fragmentation. usually, stolons must be cut several times to obtain sprigs of the right length because of overly long internodes. in the present study, potted plants of patriot bermudagrass grown in the nursery were treated with trinexapac-ethyl (te) at the rates 0.1, 0.2, 0.4 and 0.8 kg a.i. ha-1. te application was aimed at obtaining internode shortening in order to facilitate the stolon division practice. in fact, te-treated plants showed a decrease in the average length of internodes with respect to control at any applied rate. nevertheless, the lowest rate applied (0.1 kg a.i. ha-1) did not assure a prolonged effect while the highest rate (0.8 kg a.i. ha-1) caused a decrease in the yield of sprigs. therefore, our results suggest that te may be advantageously used and at rates of 0.2-0.4 kg a.i. ha-1 to control stolon development of patriot bermudagrass for nursery purposes. adv. hort. sci., 2012 26(1): 17-20 received for publication 20 september 2011 accepted for publication 24 january 2012 18 action. type i pgrs inhibit cell division and differentiation in the plant meristems and are excellent seedhead inhibitors; type ii compounds suppress growth by inhibiting the biosynthesis of gibberellic acid, which is needed for cell elongation (watschke et al., 1992). in turfgrass, three of the most commonly used pgrs [flurprimidol (fl), trinexapac-ethyl (te) and paclobutrazol (pbz)] are type ii chemicals (lowe and whitwell, 1999). turf plants treated with type ii pgrs usually show an altered morphology of leaves and a more compact growth habit due to a reduced internode length (rossi, 1993). therefore, for their mode of action, type ii pgrs may presumably be useful to reduce stolon length by controlling internode elongation. in this paper, the effects of te on potted plants of bermudagrass grown in the nursery are described. the aim of the te treatment was to obtain a stolon shortening to facilitate the stolon division practice and, hopefully, without reducing the overall production in sprigs, that result from the number of stolons per plant and the number of nodes per stolon. 2. materials and methods plant material, growing conditions and experimental treatments the experiment was carried out between april and june 2010 at the pacini horticultural nursery located in rigoli (pisa), central italy (45°45’ n, 10°26’ e, 6 m a.s.l.). on 7 april 2010, plantlets derived from one-node sprigs of bermudagrass [cynodon dactylon (l.) pers. x c. transvaalensis burtt-davy] cv. patriot were transplanted in 20 cm-diameter, 3.80 l volume polyethylene pots (three plantlets per pot) filled with peat (baltikum-s132; 92% organic matter, ph 5.5-6.5, electrical conductivity 0.6-1.0 ds m-1) fertilized with 2 g l-1 of osmocote-pro (scotts) 1810-11 + 2 mgo. on 12 may 2010, after stolons had started to develop, plants were trimmed down to the pot rim height and five treatment rates [0 (= control), 0.1, 0.2, 0.4 and 0.8 kg a.i. ha-1] of te [(4-cyclopropyl-α-hydroxy-methylene)-3,5dioxocyclohexanecarboxylic acid methyl ester] were applied by foliar spraying (1250 l ha-1) of the commercial product primo maxx (syngenta). during the experiment, plants were irrigated as needed by an overhead sprinkler system. during the period, average maximum and minimum temperatures were 35°c and 20°c, respectively, while relative humidity (r.h.) reached 55% and 70% as minimum and maximum values, respectively. a randomized block design was adopted with five replicates per treatment (1 replicate = 1 pot). data collection after treatment, stolon length and the number of nodes per stolon were recorded weekly for seven weeks on three stolons per pot, and average internode length was calculated. on 30 june 2010, seven weeks after treatment, fresh weight and dry weight of aerial biomass (after oven-drying at 80°c for 48 hr) and the number of stolons of each pot were determined. in addition, the total number of sprigs per pot was calculated as the product of the number of stolons per pot and the final number of nodes per stolon. statistics data were subjected to one-way analysis of variance (anova) and means were compared using the lsd test at p≤0.05 level of significance. 3. results te induced a statistically significant shortening of stolons compared to the control one and two weeks after application at the rates 0.2, 0.4 and 0.8 kg a.i. ha-1 (fig. 1). three weeks after treatment, the effect of te treatments on stolon length began to wane (fig. 1). during the entire monitoring period (seven weeks), no statistically significant differences were observed between te-treated plants and control plants in the number of nodes per stolon (fig. 2). fig. 1 stolon length in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. fig. 2 number of nodes per stolon in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. 19 te-treated plants showed a decrease in the average length of internodes (fig. 3). in control plants internode length varied between 2.7 and 3.0 cm; in te-treated plants stolons showed an average internode length varying from 1.8 (first week) to 2.5 cm (seventh week) at the rate 0.1 kg a.i. ha-1, from 1.4 (first week) to 2.1 cm (seventh week) at the rate 0.2 kg a.i. ha-1, from 1.2 (first week) to 1.8 cm (seventh week) at 0.4 kg a.i. ha-1, and from 1.2 (third week) to 1.5 cm (seventh week) at 0.8 kg a.i. ha-1 (fig. 3). the differences between control and te-plants were statistically significant at all the applied rates one week after treatment and afterwards at the rates 0.2, 0.4 and 0.8 kg a.i. ha-1. at the end of the monitoring period, no statistically significant differences were observed between te-treated plants and control plants in fresh matter production, while dry matter decreased significantly compared to control at 0.2 and 0.8 kg a.i. ha-1 but, surprisingly, not at 0.4 kg a.i. ha-1 (table 1). table 1 aerial biomass production in patriot bermudagrass treated with different doses of te (kg a.i. ha-1) seven weeks after treatment treatment te (kg a.i. ha-1) fresh matter (g per pot) dry matter (g per pot) number of stolons per pot number of sprigs per pot 0 128.5±5.1 ab (z) 38.3± 4.6 a 110±7.4 a 915±83.3 a 0.1 136.7±5.8 a 37.9±1.3 a 104±6.4 ab 912±138.3 a 0.2 113.2±5.2 b 31.8±1.5 b 86±2.6 b 673±48.0 ab 0.4 121.0±4.7 b 34.5±2.6 ab 89±6.3 b 786±80.6 ab 0.8 115.0±6.6 b 31.3±2.0 b 62±5.6 c 597±72.9 b (z) values in the same column followed by different letters are significantly different for p≤0.05 (lsd test). values are means (± se) of five replicates. te treatment had a detrimental effect on stolon number per pot starting from the rate 0.2 kg a.i. ha-1 upwards, but the average number of nodes derived from one pot decreased significantly only at the highest rate applied (0.8 kg a.i. ha-1) (table 1). 4. discussion and conclusions the effect of te on the number of bermudagrass stolons was previously investigated by fagerness et al. (2002). these authors found that te (0.11 kg a.i. ha-1) did not affect the number of stolons in tifway bermudagrass when plants were grown in a 22/17°c day/night temperature regime, while at 36/31°c te-treated plants showed about twice as many stolons as control plants four weeks after te application. in our experiment, temperature ranged between 20 to 35°c and 0.1 kg te ha-1 did not cause any effect on stolon number in patriot bermudagrass seven weeks after its application. on the contrary, higher rates of te (0.2, 0.4 and 0.8 kg a.i. ha-1) reduced significantly the number of stolons compared to control. an indirect evaluation of the effect of te on stolon lengthening in turfgrass species may come from percentage lateral regrowth, a parameter used to estimate the horizontal growth or recovery of turfs as described by bunnell et al. (2005). these authors applied te at 0.039 kg a.i. ha-1 to tifeagle bermudagrass every three weeks and measured percentage lateral regrowth weekly for eight weeks. averaged weekly data of te-treated plants did not show any differences compared to untreated plants. totten et al. (2006) reported that te did not affect the percentage lateral regrowth in tifway bermudagrass when used at the rate 0.052 kg a.i. ha-1, while at 0.104 kg a.i. ha-1 percentage lateral regrowth was significantly reduced compared to control two weeks after te application. in our experiment, a te rate of at least 0.2 kg a.i. ha-1 was needed for a significant decrease of stolon length in patriot bermudagrass (fig. 1). that effect lasted for two weeks after te treatment. internode length after one, two or three te applications at 0.153 kg a.i. ha-1 to a mature tifway bermudagrass turf was measured by richardson (2002). the measurements were taken ten weeks after the last te application and no effect of the pgr was detected. in our experiment internode length was significantly shortened in te-treated plants at any applied te rate one week after one application; from two to seven weeks after treatment stolon showed average internode length with statistically significant differences between control and te-treated plants at the rate 0.2 kg a.i. ha-1 and higher. in conclusion, our results suggest that te may be advantageously used to control stolon development of patriot bermudagrass for nursery purposes (shortening of the internodes in plants grown in pot to facilitate stolon division practice). in order to obtain prolonged effect, higher rates than those normally adopted in bermudagrass turf management seemed to be required (at least 0.2 kg a.i. ha-1). the highest rate applied (0.8 kg a.i. ha-1) was not advisable as it caused also almost a halving in the number of stolons per pot and a statistically significant decrease in the yield of sprigs. under the conditions in which we operated, the best result was achieved at the rates 0.2 and 0.4 kg a.i. ha-1. with such rates, although the number of stolons per pot was significantly reduced compared to control, the yield in nodes remained unvaried. fig. 3 average internode length in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. 20 acknowledgements this research was funded by the ministry of agricultural, food and forestry policies of italy. project: “sistemi avanzati per la produzione vivaistica di tappeti erbosi di specie macroterme ad uso multifunzionale a basso consumo idrico ed energetico”. the authors wish to thank the pacini horticultural nursery for the use of the greenhouses and other facilities. references baldi a., lenzi a., nannicini m., pardini a., tesi r., 2010 plant growth regulator treatments for the management of patriot bermudagrass. proceedings of the second conference of the european turfgrass society. angers, france, april 11-14, pp. 32-34. baldwin c.m., liu h., mccarthy l.b., bauerle w.l., toler j.e., 2006 effect of trinexapac-ethyl on the salinity tolerance of two ultradwarf bermudagrass cultivars. hortscience, 41(3): 808-814. beard j.b., 1989 turfgrass water stress: drought resistance components, physiological mechanisms, and species-genotype diversity. proceedings of the sixth international turfgrass research conference, tokio, japan, pp. 23-28. beard j.b., sifers s.i., 1997 genetic diversity in dehydration avoidance and drought resistance within the cynodon and zoysia species. int. turfgrass soc. res. j., 8: 603-610. biran i., bravdo b., bushkin-harav i., rawitz e., 1981 water consumption and growth rate of 11 turfgrasses as affected by mowing height. agron. j., 79: 85-90. bunnell b.t., mccarty l.b., bridget jr. w.c., 2005 ‘tifeagle’ bermudagrass response to growth factors and mowing height when grown at various hours of sunlight. crop sci., 45: 575-581. carrow r.n., duncan r.r., 1988 salt-affected turfgrass sites. assessment and management. john wiley & sons, hoboken, new jersey, usa, pp. 185. cooper r.b., 2003 summary of 2003 cutless 50wp turfgrass growth regulator research on 419 bermudagrass fairways. sepro corp. www.sepro.com/documents/cutlesscooper.pdf. croce p., de luca a., falcinelli m., 2006 tappeti erbosi. edagricole, bologna, pp. 340. croce p., de luca a., mocioni m., volterrani m., beard j.b., 2004 adaptability of warmseason turfgrass species and cultivars in a mediterranean climate. acta horticulturae, 661: 365-368. dudeck a.e., peacock c.h., 1993 salinity effects on growth and nutrient uptake of selected warm-season turf. int. turfgrass soc. res. j., 7: 680-686. fagerness m.j., yelverton f.h., 1999 effects of trinexapac-ethyl on late season development and cold hardiness of ‘tifway’ bermudagrass. proc. northeast. weed sci. soc., 53: 63. fagerness m.j., yelverton f.h., 2000 tissue production and quality of ‘tifway’ bermudagrass as affected by seasonal application patterns of trinexapac-ethyl. crop sci., 40: 493-497. fagerness m.j., yelverton f.h., livingston d.p. iii, rufty jr. t.w., 2002 temperature and trinexapac-ethyl effects on bermudagrass growth, dormancy, and freezing tolerance. crop sci., 42: 853-858. harivandi m.a., marcum k.b., 2008 a review of salt tolerance among sports field turfgrasses. acta horticulturae, 783: 159-162. johnson b.j., 1994 influence of plant growth regulators and mowing on two bermudagrasses. agron. j., 86: 805-810. johnson b.j., 1997 growth of ‘tifway’ bermudagrass following application of nitrogen and iron with trinexapac-ethyl. hortscience, 32(2): 241-242. lowe d.b., whitwell t., 1999 plant growth regulators alter the growth of ‘tifway’ bermudagrass (cynodon transvaalensis x c. dactylon) and selected turfgrass weeds. weed technol., 13(1): 132-138. mccullough p.e., liu h., mccarty l.b., toler j.e., 2007 trinexapac-ethyl application regimens influence growth, quality, and performance of bermudagrass and creeping bentgrass putting greens. crop sci., 47: 21382144. mccullough p.e., mccarty l.b., liu h., whitwell t., 2005 response of ‘tifeagle’ bermudagrass (cynodon dactylon x cynodon transvaalensis) to ethephon and trinexapac-ethyl. weed technol., 19: 251-254. richardson m.d., 2002 turf quality and freezing tolerance of ‘tifway’ bermudagrass as affected by late-season nitrogen and trinexapac-ethyl. crop sci., 42: 1621-1626. rossi f., 1993 what is it with plant growth regulators? the grass roots, 21(4): 27. totten f.w., toler j.e., mccarty l.b., 2006 ‘tifway’ bermudagrass growth regulation with the use of trinexapacethyl and flurprimidol. weed technol., 20: 702-705. volterrani m., grossi n., lulli f., gaetani m., 2008 establishment of warm season turfgrass species by transplant of single potted plants. acta horticulturae, 783: 77-84. volterrani m., grossi n., pardini g., miele s., gaetani m., magni s., 1997 warm season turfgrass adaptation in italy. int. turfgrass soc. res. j., 8(2): 13441354. volterrani m., magni s., gaetani m., de luca a., croce p., mocioni m., 2010 bermudagrass evaluation trial in italy. proceedings of the second conference of the european turfgrass society. angers, france, april 11-14, pp. 223-225. watschke t.l., prinster m.g., brenninger j.m., 1992 plant growth regulators and turfgrass management, pp. 557-588. in: waddington d.v., r.n. carrow, and r.c. shearman (eds.) turfgrass. american society of agronomy, agronomy monograph, n. 32. madison, wi, usa, pp. 805. wu y., martin d.l., anderson j.a., bell g.e., anderson m.p., walker n.r., moss j.q., 2009 recent progress in turf bermudagrass breeding research at oklahoma state university. usga turfgrass and environmental research online, 8(16): 1-11. impaginato 112 adv. hort. sci., 2011 25(2): 112-122 received for publication 24 march 2011. accepted for publication 3 june 2011. root distribution in young chétoui olive trees (olea europaea l.) and agronomic applications c. masmoudi-charfi *, m. masmoudi ** n. ben mechlia** * institut de l’olivier, tunisie. **institut national agronomique de tunisie. key words: irrigation, overall root length, root-canopy ratio, root density, root volume, water requirement. abbreviations: etc (mm)= crop evapotranspiration as determined by the fao method (allen et al., 1998). et* (m3)= evapotranspiration volume of an individual tree relative to its root area. kc =crop coefficient. kr = minorative coefficient introduced in the formulae of etc fao to take into account the soil coverage. ksupply = a supply ratio determined in order to link the water supplied to trees to the evaporative demand, it takes into account only the tree-related quantities. eto (mm)= reference evapotranspiration determined following to penman-monteith equation. pe (mm)= effective rainfall determined following the usda-scs method (fao, 1976). i (mm)= irrigation amount supplied during the irrigation period. i*(m3)= irrigation amount supplied by localized system or in small basins around the trunk. p (mm)= total rainfall. p* (m3)= effective rainfall for a single tree received around the trunk. sc (m2)= the maximum projected canopy area determined for each of the six trees assuming a circular shape. sr (m2) = area concerned by tree transpiration i.e. where roots are active. (t)= the number of years from planting. lo, lx= dimension of interest respectively at planting and at maximum growth. α, β = adjustment parameters within the logistic root and canopy growth curve. abstract: the study was carried out to have a comprehensive view of the root system behavior of young olive trees cultivated under field conditions. the experiment involved irrigated trees (olea europaea l., cv., chétoui) cultivated at 6x6 m2 spacing in mornag (36.5°n, 10.2°e), northern tunisia. the way in which roots explore the soil volume during the first years after planting was explored through ‘in situ’ root system drawings and estimation of root densities. the relationship between canopy and root growth parameters was also investigated. the last section of this paper proposes a methodological approach for determining irrigation requirements of young olive trees and how water supply could be linked to the development of canopy and root system during the first years of cultivation when ground cover and the root system are not completely developed. some agronomic applications were then deduced concerning water and fertilizers for such orchards. results show that the main development of the olive root system occurs during the two to four first years of cultivation confining most roots (70%) to the top soil layers (20-40 cm). maximum root densities were observed at this depth at a distance of 0.4 m from trunks. for young trees, water and fertilizers should be supplied at these depths and distances from trunk to allow easy and efficient root absorption. obtained results also show a significant relationship between canopy and root areas which can be approximated by a linear model (r = 0.94). the root-canopy ratio estimated from their areas decreased rapidly beginning from the second year after planting, resulting from the establishment of competition between vegetative growth and fruiting. the optimum ratio root length/leaf canopy area of 2.3 km m-2 was found for the six-year-old tree indicating good equilibrium between the above and underground parts. the mathematical model developed on the basis of canopy cover and root extension allows precise estimation of water needs taking into account the actual root surface. however, while the canopy cover measurement was relatively easy to carry out, it was much more difficult to determine the surface covered by the root system. results obtained in the present work also show an over-estimation of water needs when the fao method is adopted to estimate the evapotranspiration of young trees. 113 1. introduction the primary function of the root system, i.e. water absorption and acquisition of soil nutrients, has a great influence on many of the physiological processes in the tree (doussan et al., 2003). however, despite of this importance, the root system is possibly the least explored area in crop physiology because of the difficulty involved in reaching it, in addition to the highly spatial-temporal variability which can generate many constraints to root extension. amongst the first papers dealing with this area, are those of yankovictch and berthelot (1947), vernet and mousset (1963) and abdel-rahman et al. (1966) which were carried out in north africa, mainly on cultivars chemlali and picholine marocaine. research conducted a few years later in spain and italy investigated the relationship between water and root extension (pisanu and corrias, 1971; bohm, 1979; nunuez-aguilar et al., 1980; martin-aranda et al., 1982; michelakis and vougioucalou 1988; pastor et al., 1998; smit et al., 1999; palease et al., 2000). it was shown that apart from genetics and the origin of the plant (ayachi-mezghani, 2009) root distribution and extension can be markedly influenced by neighboring trees and soil texture and depth (ben rouina et al., 1996). also, roots proliferate within the potential root zone regardless of irrigation application and method (fernandez et al., 1991, 1992, 2003; fernandez and moreno, 1999; connor and fereres, 2005). these last authors noted that localized irrigation increased root length density of manzanilla olive trees but it decreased their spread, largely confining them within the wetted volume and nearby trunks. they reported also that except under the canopy, roots were less frequent in the top layers than in deeper strata. root extension is also dependent on the available carbohydrate resources (dichio et al., 2002) and growth stage (michelakis, 2000). rapid growth is observed in spring and autumn; it depends on water supply. root growth precedes shoot growth and may be drastically limited by the previous year’s fruit load. in fact, when no competition for carbohydrates occurred with other organs, for example for young olive trees or/and for vigorous canopy growth trees, important root extension and greater root densities were reported (palease et al., 2000). in contrast, limited carbohydrate resources led plants to reduce their canopy growth and root length and even could deteriorate the root-canopy ratio as a result of competition between shoots, flowers, fruits and roots (dichio et al., 2002). this relationship between root growth and the aboveground development is complex because it integrates many other factors and physiological processes like temperature, radiation, hormones, variety and alternate bearing. reduction of the root-canopy ratio implies systematic reduction of the capacity of the rooting system to absorb water. in terms of root balance, the importance of the water collecting system resides in its capacity to obtain water to support the transpiring leaf area (connor and fereres, 2005; connor, 2006) and it can be determined via an estimation of total root length through monitoring of root density. these techniques are reported by tennant (1975) and fernandez and moreno (1999). such measurements could provide reliable estimates of comparative activities. for olive trees, connor and fereres (2005) reported root densities ranging between 0.1 and 1.0 cm cm-3. these values are lower than those provided for herbaceous crops and some deciduous orchards, although olive root systems can be extensive and deep. it appears from this short review that fundamental research on this subject is of prime interest: when and where the roots grow is crucial to understanding the functioning of the root system and its relationship with the above-ground organs. in fact, without precise information on root distribution, we cannot expect to efficiently manage the irrigation of the orchard. for these purposes, we have carried out the present study in order to have a comprehensive view of the root system behavior of young olive trees cultivated under field conditions. in this work, we examine how roots explore the soil volume during the first years after plantation. the relationship between root and canopy development was also investigated. the last section of this paper proposes a methodological approach to determine irrigation requirements of young olive trees and considers how water supply could be linked to the development of canopy and root system during the first years of cultivation when ground cover and the root system are not yet completely developed. 2. materials and methods olive orchard the study was carried out during the period 19982003 at the experimental farm of the institut national agronomique de tunisie, located 15 km south of the capital tunis (36.5ºn, 10.2ºe), northern tunisia. in this region, climate is mediterranean with yearly averages of 450 mm rainfall and 1200 mm reference evapotranspiration. it is dry and hot from may to september. the orchard, of 1.6 ha, was planted in 1998 at 6x6 m2 spacing on a textural clay soil (29%c, 49%l, 23%s) of about 2 m depth. the volumetric soil water content was measured in the laboratory at field capacity (50%) and at the wilting point (26%). crop management practices carried out in the orchard, i.e. pruning, fertilizer (masmoudi-charfi and ben mechlia, 2009) and pest management practices, were similar to those applied in intensive orchards (masmoudi-charfi, 2006; masmoudi-charfi et al., 2006). the trial concerned trees of cultivar chétoui, which is the main oil variety of northern tunisia. 114 climatic data and irrigation management daily crop evapotranspiration (etc) was determined according to allen et al. (1998) for the non-standard conditions such as: etc = eto ×kc ×kr, kc ranging between 0.3 and 0.5 according to age, while kr values were determined experimentally and varied between 0.69 and 0.75. for this purpose, a large white grilled (10 cm/10 cm) sheet was used. it was placed below the tree and the shade squares were counted and compared to the total number of squares (those lighted by sun and those shaded by leaves). this percentage represents the kr value. daily reference evapotranspiration (eto) was computed according to the penman-monteith equation, with maximum and minimum yearly values of 1320 mm (1999) and 1212 mm (2003), respectively. data relative to rainfall, eto and temperature are reported in table 1. all climatic data were recorded continuously with an automatic weather station located about 150 m from the young olive orchard. during the six years of the study, rainfall amounts varied from 327 mm (2001) to 790 mm (2003), while effective rainfall amounts ranged between 226 mm and 546 mm. these values were determined according to the usda-scs method (fao, 1976). accounting for these conditions, olive trees were irrigated every year during the spring-summer season. water flows were programmed four times per season regardless of the critical stages and water availability. irrigation was supplied by furrows (basin and drain) during the four first years and then by a drip system (2002 and 2003). two parallel drip lines were fixed on the soil surface at about 0.5 m from trunks. there were four emitters per tree, two at each side of the tree trunk, separated 1 m from each other; each having a 4-l h-1 flow rate. the area wetted by irrigation application varied between 1 m2 (1st year) and 6 m2 (6th year). watering conditions for the whole period are given in table 2. water requirements were covered at levels varying between 0.3 etc and 1.1 etc according to year and water availability. measurements soil water content. the volumetric water content of the soil was measured with a neutron probe (solo 25) which was previously calibrated for the soil in question (masmoudi-charfi, 2008). twenty-eight access tubes, 1.5 m long, were placed at the corners of a square of 2 m2 below the canopy but also within the tree line and between tree lines. the soil moisture in each of these tubes was recorded frequently during the irrigation period every 0.3 m to 1.2 m depth, and the mean calculated separately for each position: below the canopy, far from the emitters, along and between the lines of tree (unpublished data). for the top 0.2 m soil layer, soil water content was determined by gravimetry. more details are given in masmoudi-charfi (2008). table 1 climatic data recorded during experimentation (1998-2003) annual rainfall (mm) effective annual rainfall (mm) absolute tmax (°c) absolute tmin (°c) average tmax (°c) average tmin (°c) annual eto (mm) 1998 1999 2000 2001 2002 2003 376 260 47.0 3.0 25.0 13.3 1313 440 304 41.0 1.0 23.7 15.0 1320 410 283 44.0 4.0 25.2 14.8 1293 327 226 42.0 3.0 25.8 15.8 1282 345 238 43.0 3.0 25.6 15.5 1231 790 546 46.0 3.0 24.9 14.9 1212 table 2 water requirement and irrigation application for young olive trees of cultivar chétoui during the experimental period irrigation system first irrigation last irrigation dose (m3/tree) irrigation amount (m3/tree/year) i + pe (mm)* etc (mm)* i+pe / etc 1998 1999 2000 2001 2002 2003 basin march august 0.12 0.84 140 243 0.6 basin may september 0.18 0.72 61 241 0.3 drain april september 0.22 0.88 180 291 0.6 drain april september 0.44 1.76 141 287 0.5 drip march august 0.7-1.7 4.98 248 273 0.9 drip may september 0.3-1.0 5.41 389 368 1.1 (*) indicates that values are determined for the irrigation period. pe is the effective rainfall determined according to the usda-scs method (fao, 1976) and i is the irrigation amount. the ratio i+pe / etc was calculated for the irrigation period. the tree downward projection canopy flat area varied between 2% (first year) and 33% (sixth year). 115 root distribution. distribution of the root system was studied during the rest period (november-december) on the same chétoui olive trees by extensive observations of their root system. the trench method was used as described by fernandez et al. (1991). for this purpose, a large pit was opened at 0.4 m from the trunks and roots were counted on the internal trench wall, which was divided into five layers of 0.2 m width each and down to 1.0 1.2 m depth. root diameter was measured by means of a caliper 1/100. maximum distance of roots from trunk was determined at each soil layer in order to estimate lateral root extension. total volume of soil and the area explored by the root system were determined assuming central symmetry to the trunk. root density. root densities were determined on the same chétoui olive trees by using the cylinder method as described by fernandez et al. (1991). soil samples were taken during the rest period by a conventional auger at 0.4 m, 0.8 m and 1.2 m from trunks in order to quantitatively assess the importance of the root system through an estimation of root densities as described by tennant (1975). samples were taken within layers of 0.2 m width, down to 1.0 1.2 m depth, following east and south directions, along the line of drippers (south) as well as perpendicular to this. they were then washed out abundantly and sieved through a 0.5 mm screen. extracted roots were counted by adopting a reference scale (tennant, 1975). root length was then derived from the average root density value for each of the six trees. figure 1 presents details on both protocols. with this scheme, it was possible to obtain information on root distribution in the zones affected and not affected by irrigation. canopy measurements. canopy diameter measurements were monitored at the same time as the study of the root system and on the same experimented trees. the maximum projected canopy area (sc) was determined for each of the six trees assuming a circular shape. these measurements were used to set a typical model of growth and to examine the relationship between root and canopy development. canopy leaf area was determined for the six-yearold olive tree by computing the number of leaves on representative shoots and estimating its specific leaf area. it reached 14 m2 on may 2003. this value was adopted to calculate the root length/leaf canopy ratio. methodological approach to determine irrigation requirements of young olive trees. this section proposes a methodological approach to determine irrigation requirements of young olive trees and how the water supply can be linked to the development of the canopy and root system during the first years of cultivation when ground cover and root system are incompletely developed. determination of water requirements according to the fao method (allen et al., 1998) is adequate for standard conditions, i.e. when soil coverage reaches 60% or more. however, when the coverage area is less, a reductive coefficient kr is introduced (coi, 1997; allen et al., 1998). in some cases, particularly for young and new orchards (low tree canopy cover), this coefficient may not be precise enough to allow good estimation of water needs. in addition to problems estimating kr values, the kc is strongly affected by conditions that influence evaporation from the soil surface (orgaz et al., 2006). recently, testi et al. (2004) proposed a simple linear relationship between the olive ground cover (and leaf area index) and the average kc of the summer months, valid for ground cover fractions up to 0.25, along with its variation when wet surface soil spots are present. these authors indicate that this relationship does not apply outside a rainless summer, and the contribution to soil evaporation from the drip system depends on the surface area and location of the wet spots and is not scalable. thus, we developed the following approach which is designed to determine the consumptive use of olive trees in relation to their canopy growth and root development during the first six years after planting. before full development of the root system, only a fraction of rainfall water is accessible to trees. thus, the water balance equation should consider the area concerned by tree transpiration i.e. where roots are active (sr); sr is assumed to be circular and to increase following a logistic-shaped curve. root extension, as well as canopy increase, seems to coincide with a logistic growth curve as given by the following equation: l(t)=lo+ lx-lo 1+exp[α(t-β)] where (t) is the number of years from planting; lo, lx dimensions of interest, respectively, at planting and at maximum growth; α, β are adjustment parameters. in order to link the water supplied to trees to the evaporative demand, a supply ratio (ksupply) that takes into account only the tree-related quantities is defined by this equation: ksupply = (p* + i*) / et* fig. 1 scheme of sampling to determine root distribution and root densities for young olive trees aged one to six years. root profiles were mode following to nw direction while samples of root density determination were taken at 0.4 m, 0.8 m and 1.2 m from trunks to 1.2 depth following to se direction. : emitter. 116 considering that irrigation (i*, m3) is supplied by a localized system or in small basins around the trunk, only a small surface is wetted and affected by soil evaporation and transpiration. irrigation water is therefore assumed to be fully accessible to the root system of the tree. on the other hand, effective rainfall for a single tree (p*) is taken as the volume of rainfall water available to the root system which could be approximated by the following equation: p* (m3) = p (m) x sr (m2) p is rainfall, considered here as total rainfall. the evapotranspiration volume of an individual tree (et*) can be estimated from the root area of the tree as: et* (m3) = kc x eto (m) x sr (m2). different water supply ratios are determined as kc fao, i/eto, p*+i*/et*and i*/et*. the ratio i/eto is the irrigation supply, p*+i*/et* is the volumetric total supply and i*/et* is the volumetric irrigation supply. these ratios are for the period april-august over the first six years of olive tree cultivation. values are represented in the same figure to compare results. 3. results soil water status simultaneous monitoring of soil moisture carried out during the 2003 campaign at the canopy limit and near the emitters showed that soil water contents vary from 15 to 39% according to depth and distance to trunk (fig. 2). low values of soil water content were observed in the upper layers, while minimums were recorded within the superficial strata (0-20 cm) as a result of soil water evaporation and root absorption. this result confirms the concordance between root development and soil water depletion. the results showed large variation between measurements at the limit of the canopy, while low variation of soil moisture was observed near the emitters with values ranging between 32 and 38% according to depth (fig. 2). root system drawings root profiles for the tagged trees show two or three types of roots according to age (fig. 3). during the first years after planting, trees developed fine roots in the upper 0.2 m of the soil layer, which then extended fig. 2 soil water content (%) measured at two sites: on the left at 10 cm from the emitters and on the right at the limit of the canopy during the 2003 campaign. table 3 maximum number of roots and root diameter emerging from the trench face for each soil layer for olive trees aged one to six years 0-20 20-40 40-60 60-80 80-100 total number of roots maximum root diameter (mm) 2 3 4 5 6 6 2 0 0 0 8 2 2 3 8 2 0 15 6 16 6 3 4 0 29 23 10 5 1 4 0 20 32 9 5 3 5 3 25 24 51 91 116 97 81 472 27 1soil layer (cm) age (year) fig. 3 drawings of the root system of young olive trees of cultivar chétoui aged one to six years. roots were counted on the internal trench wall, down to 1.0 1.2 m depth depending on age. rapidly in lateral and vertical directions with inclinations varying from 30° to 60° depending on their size and position. for older plants, larger roots were observed beyond the first 0.3 m and they developed horizontally with numerous fine roots. the number and diameter of roots which emerged from the lateral face of the trench are summarized in table 3. results indicate that most roots (70%) are localized in the first 0.6 m of soil. the maximum number is found in the top layers, with diameters ranging between 2 mm (one-year-old tree) and 32 mm (four-year-old tree). some roots developed in deeper strata, reaching 1.0 m depth. very few roots were found below this depth even for the oldest tree. 117 extension of the root system results presented in table 4 show that the main development of the root system occurred during the first two to four years of cultivation, horizontally and within the top layers (0.2-0.3m). during this period, the soil volume explored by roots increased at a regular rate of about 1.0 m3 yearly. for the three-year-old tree, roots explored a volume of 3.65 m3. the soil volume explored by the root system of the five-year-old-tree represents 47% of that reached by the older tree (sixyear-old tree). root density results relative to root density estimation are reported in figure 4. a noticeable root concentration is observed for both east and south directions and close to trunk in the top layers around each of the six trees. average values varied between 0.001 cm cm-3 and 0.670 cm cm-3 depending on depth, distance to trunk, direction and tree age. greater values, by up to 0.5 cm cm-3, were recorded in the first 60 cm and at 0.4 m from trunk. these values decreased significantly as the distance to trunk increased (except some measurements for twoand threeyear-old plants). roots were less frequent at all depths outside the canopy limit and particularly for the deeper layers. at these depths, however, it should be mentioned that root densities rarely exceed 0.4 cm cm-3 for both directions, while average values ranged between 0.067 cm cm-3 and 0.303 cm cm-3 (table 5). root system length the overall length of the root system varied from 1.0 km to 33.9 km depending on age (table 6). a significant increase of the overall length of the root system was observed for the six-year-old tree. it was 4.8 times greater than that recorded the previous year. the lowest value was recorded for the four-yearold tree. there was no apparent cause which could explain this result. root development and canopy growth results presented in figure 5 showed for tree aged one to four years that roots grew at higher rates than table 4 maximum distance of roots to trunk (m) and volume of soil explored by the root system (m3) for olive trees aged one to six years 0-20 20-40 40-60 60-80 80-100 explored soil volume (m3) 2 3 4 5 6 1.05 1.15 0 0 0 1.45 1.05 1.10 1.00 0.80 0 2.55 1.25 1.30 1.25 1.00 0 3.65 1.45 1.45 1.25 1.25 0 4.60 1.50 1.45 1.25 1.25 1.00 5.30 2.12 1.95 1.80 1.65 1.55 11.2 1 depth (cm) age (year) fig. 4 root densities (cm cm-3) recorded for olive trees of cultivar chétoui aged one to six years based on direction and depth. for each tree, three measurements were carried out for both directions at different distances from trunk; the first observation was made at 0.4 m, the second at 0.8 m and the third at 1.2 m. table 5 average root densities (dr, cm cm-3) determined for trees aged one to six years dr (cm cm-3) 2 3 4 5 6 0.067 0.079 0.196 0.075 0.133 0.303 1 table 6 the overall length of root system (lr, km) for trees aged one to six years lr 2 3 4 5 6 1.005 1.975 7.056 3.450 7.049 33.936 1 fig. 5 maximum root distance from the trunk (m) and maximum canopy radius (m) following to age for olive trees chétoui ages one to six years. di sta nc eo rr ad ius (m ) 118 canopy radius. then, differences between the canopy radius and the root-to-trunk-distance decreased. roots reached for the six-year-old tree a maximum distance to trunk of 2.10 m, while the canopy limit was observed at 1.95 m. the projected canopy area (sc) increased slowly after planting to reach 0.21 m2 for the oneyear-old tree and 11.94 m2 for the six-year-old-tree (table 7), while the root area progressed at a constant rate of 1.2 m2 per year to reach 13.8 m2 for the six-year-old-tree. a significant relationship was found between canopy (sc, m2) and root (sr, m2) areas, which can be approximated by a linear model with a correlation coefficient r of 0.94, as illustrated by figure 6, where sc = 1.183 sr 3.602 (r2 = 0.876) the sr/sc ratio derived from both canopy and root areas decreased significantly from 20 to 0.9 depending on tree age. for the four-, fiveand six-year-old trees, this ratio approximated the unit. a decrease of the sr/sc ratio implies a tendency to equilibrium between the under-ground and aboveground organs beginning from the fourth year after planting, which apparently results from the establishment of competition between shoots, roots and fruits (and explains the decrease of this ratio). in fact, trees began to produce olives within the second year after planting and the first commercial crop arrived in year four (6.5 kg / tree). results indicate also that plants seem to be able to adjust their root systems to the larger above-ground development during the winter rest. this feature is well represented by the root length/leaf canopy area ratio. a value of 2.3 km m-2 of leaves for the six-year-old tree was found in the present study, a value which is considered optimum for such conditions. irrigation supply as a function of canopy and root development in order to link the water supplied to trees to the evaporative demand, a supply ratio (ksupply) that takes into account only the tree-related quantities is defined as developed in section ‘measurements. methodological approach to determine irrigation requirements of young olive trees’. this ratio could be considered as a crop coefficient for young trees when reference evapotranspiration, rainfall and irrigation amounts are computed according to the previous equations and expressed in m3/tree. adoption of such a ratio allows estimation of irrigation requirements for different rainfall and evapotranspiration regimes. the different water supply ratios, kc fao, i/eto, p*+i*/et* and i*/et*, determined for each of the six olive trees are given in figure 7 for comparative purposes. results show that the ratio of applied irrigation (i, mm) to reference evapotranspiration (eto, mm) during the dry season from april to august was very low. it increased from 0.02 to 0.14 when trees grew from one to six years. when using the volume method to calculate the irrigation and precipitation falling on the area covered by roots, ksupply comes very close to the kc-fao. estimation of effective precipitation remains however big challenge for using the proposed method. table 7 canopy and root area estimations (m2) of olive trees aged one to six years root area (sr) canopy area (sc) sr / sc 4.20 0.21 20.00 1 3.80 0.82 4.60 2 5.30 1.86 2.80 3 6.60 3.79 1.70 4 7.10 8.04 0.90 5 13.80 11.94 1.20 6 fig. 6 relationship between canopy and root areas for young olive trees aged one to six years. fig. 7 variation of kc-fao, irrigation supply (i/eto), volumetric total supply (p*+i*/et*) and volumetric irrigation supply (i*/et*) ratios calculated for the period april-august over the first six years of olive tree cultivation, 1998-2003, mornag tunisia. 119 4. discussion and conclusions this study provides preliminary results on root distribution of young olive trees of cultivar chétoui, which could be exploited to manage young olive orchards efficiently. root profiles for trees aged one to six years show rapid extension of the root system during the first two to four years of cultivation following to horizontal direction. most roots (70%) are localized in the first 0.6 m of soil with a maximum number developed in the top layers. some roots developed in deeper strata, reaching 1.0 m depth, but at this age very few roots were found below this depth. the largest roots were observed beyond the first 0.3 m with maximum diameters between 2 mm and 32 mm according to age. results indicate also that lateral fine roots are abundant and give rise to a fibrous root system which represents the main absorbing surface as it was reported by palease et al. (2000). these roots originate from the branching of a parent root and constitute their ramifications, generally at right angles, as indicated by doussan et al. (2003), who classified the roots into three main categories according to their ontogenesis: primary, adventitious and lateral. in our case, the primary root constitutes the main root of the cutting. it was not dominated at the outset by a principal axis as it occurs in trees grown from seedlings. rather, many adventitious roots are produced from the base of the cutting. similar results were found in the literature for young olive trees, although studies were carried out under different conditions. abd-el-rahman et al. (1966) reported for young trees, aged seven years and grown under 150 mm of rainfall, that roots are contained in the shallow tillage (0.15-0.30 m) to approximately 0.3 m from trunk. in sardinia, pisanu and corrias (1971) observed a very shallow root system in the roots of excavated trees. their photographs and drawings clearly illustrate the horizontal development of the roots and the fact that roots of contiguous trees avoid competition by developing outwards from the tree row. in spain, nunuez-aguilar et al. (1980) observed for 12-year-old ‘manzanilla’ olive trees, that most roots are localized in the outer layers at 0.45 m from the trunk with diameter less than 0.5 mm. mickelakis and vougioucalou (1988) observed for five-year-old ‘kalamon’ olive trees cultivated in create, a maximum number of roots at a depth of 0.4 m. later, bongi and palliotti (1994) indicated that the root system of young olive trees is mainly confined to the top meter of soil, growing at depths between 0.15 and 0.40 m at a maximum distance of 0.30-0.40 m from the trunk. results relative to soil volume exploration showed a regular increase of about 1.0 m3 yearly but this rate is apparently lower than that reported in other studies. for three-year-old trees cultivated on loamy soil in southern italy, dichio et al. (2002) found volumes of about 8.6 m3 for the irrigated trees and 5.1 m3 for those cultivated under rain-fed conditions (670 mm/year of rainfall). in our case and for trees of the same age, roots explored a volume of 3.65 m3 only. this extension represents, according to fernandez and moreno (1999), doussan et al. (2003), fernandez et al. (2003) and connor and fereres (2005), the plants’ evolutionary response to the spatiotemporal variability. it explains, in our case, the lateral spread of roots and the depths they achieve; soil characteristics (clay) and its mechanical resistance may adversely affected root exploration. increases in soil strength during the summer months, as a consequence of occasional water shortage (interval between irrigations varying between 20 and 50 days), may have reduced the average number of laterals developed on the primary axes. during the following years (fifth and sixth years) the application of drip irrigation led trees to limit their root development, confining most roots to the upper layers with a noticeable root concentration observed for both east and south directions close to the trunk. an increase of root density is however observed with average values varying between 0.001 and 0.670 cm cm-3 depending on depth, distance to trunk, direction and tree age. similar values ranging between 0.1 and 1.0 cm cm-3 were reported by connor and fereres (2005). greater values of up to 0.5 cm cm-3 were recorded in the first 60 cm and at 0.4 m from the trunk. values of root density then decreased, significantly as distance to trunk increased (except some measurements for twoand three-year-old trees). roots were less frequent at all depths outside the canopy limit and particularly for the deeper layers. nunez-aguilar et al. (1980) observed similar results for 12-year-old ‘manzanilla’ olive trees with highest values of about 0.7 cm cm-3 at 0.45 m from the trunk. for seven-year-old olive trees growing with only 150 mm mean annual rainfall, abd-el-rahman et al. (1966) also found maximum root densities in the top layers at 0.15 0.30 m and up to 0.3 m from the trunk. these results show good concordance between soil profiles made for the six experimental trees and their root density distribution, having agronomic applications, since they could be used to manage more efficiently irrigation and also fertilization. water and fertilizer supplies should be given at these distances from trunks for young trees to guarantee their efficacy. many factors are cited to explain root density distribution (fernandez and moreno, 1999) amongst the cultural practices are reported in most papers. in our case, the six-year-old tree provided the highest values with average density of 0.303 cm cm-3, however the root densities recorded for the three-year-old plant were greater than those observed for the older trees. genetic factors inherent to the potentialities of that tree may be involved (michelakis and vougioucalou, 1988). however, it seems that the most influential factor that affected root density is the heterogeneous distribution of water in the orchard. results showed spatial variability of soil moisture with lower differences between measurements near the emitters (values ranging between 32 120 and 38% depending on depth) and larger variation between measurements at the limit of the canopy. this result was unexpected, but it may indicate lower rates of root absorption around the emitter despite the high densities observed at this distance from trunk (0.4 m). for such a situation, fernandez and moreno (1999) indicated that sites of maximum root density may coincide with low root activity as a compensation mechanism; thus root activity may be higher in zones of low root density than in zones of high density. the influence of soil water content on root distribution is reported by fernandez et al. (1991), who observed that adequate watering makes roots continue to grow during the dry season, thus, increasing the period of their activity and preventing their shrinking during this period. palease et al. (2000) and bongi and palliotti, (1994) indicated that root extension depends largely on the distributed water amounts and the irrigation frequency. larger volumes of water would favor the existence of wider wet bulbs and could increase root length density. in opposite, low water availability can slow down root growth because roots are able to sense the soil dryness and order stomata to close; thereby reducing water losses and preventing excessive water stress. water shortage may also increase mortality of fine roots even in the irrigated orchards; roots developed outside the wetted area during the rainy period may die. root distribution and densities are also highly dependent on leaf area and canopy development. in fact, this trial shows that olive tree establishes equilibrium between root and canopy development rapidly, around the fourth year after planting despite the larger extension of roots observed during the first two years in comparison to canopy growth. such increases in root area could be explained as a need to adequate the root system to a more vigorous canopy development. inversely, greater leaf area could provide greater total carbohydrates reserve for root activity. this relationship between leaf and root is very important to consider because dry soil conditions determine a cumulative effect over the years which indirectly affected root activity through an integrated chemical and hydraulic signaling mechanism controlling leaf water relationships, as stated by fernandez and moreno (1999). it could be represented by the under-/above-ground ratio. our results show high values of this ratio during the first year after planting, indicating a greater availability of water per unit of leaf area. however, beginning from the second year after planting this ratio decreased rapidly to attain a minimum value of 0.9. dichio et al. (2002) explains that a decrease of root-canopy ratio is a consequence of lack of water during the growing phase, which led plants to several physiological modifications; thus it can be used as an indicator of tree adaptation to water shortage. other reasons could be evoked to explain the decrease of this ratio such as the establishment of competition for nutrients between shoots, roots and fruits, which are considered the strongest sinks. the establishment of such competition is important to insure a balanced development of the tree, once it begins to set fruits. during this period of youth and first-fruit-set, the tree re-orientates the mobilization of carbohydrates (proietti and tombesi, 1996; palease et al., 2002) and high amounts of assimilates are drain to growing olives against the competing demand of the growing roots and shoots. as a results, the number of roots and their length could be reduced because their growth remain highlydependent of the available assimilates. under adequate watering conditions, olive trees seem to be able to adjust their root systems to the larger above-ground development during the winter rest essentially when no (or low) competition with other organs occurs. such result was reported by palease et al. (2002) and connor and fereres (2005) who indicate that this feature is well represented by the root length /leaf canopy area ratio. in our experiment we found a value of 2.3 km m-2 of leaves for the six-year-old tree. this ratio is concordant with the optimum values of 2.2 2.9 km m-2 which were determined for intensive plantations (connor and fereres, 2005), and this result is very important for the current work because it indicates that olive trees were adequately irrigated. such management of water ensured good development of the root system and the canopy despite the difficulties involved in fixing the irrigation amounts for such young trees with regard to the incomplete soil coverage and root development. for such young orchards, it is known that only a fraction of rainfall water is accessible to trees. thus, the water balance equation should consider only the area concerned by tree transpiration i.e. where roots are active. this area is assumed to be circular and results show that it increases following a logistic-shaped curve (masmoudi et al., 2007). this factor was taken into account to develop a mathematical model which allows estimation of irrigation needs of young trees, based on the study of the root/canopy over a long period of time. in this model a supply ratio was determined as shown previously in order to link the water supplied to the evaporative demand that takes into account only the tree-related quantities. results show that the ratio of applied irrigation to reference evapotranspiration during the dry season from april to august was very low. it increased from 0.02 to 0.14 when trees grew from one to six years. when using the volume method to calculate the irrigation and precipitation falling on the area covered by roots, the supply ratio comes very close to kc-fao. estimation of effective precipitation remains, however, a big challenge for using the proposed method. the present study provides preliminary results on root distribution of young olive trees of cultivar chétoui, which could give insight into the efficient management of intensive orchards. however, these results 121 should be enhanced by root activity observations. furthermore, development of a more detailed study on young trees would be useful to get more information on the relationship between root activity and root distribution because sites of heavy root density may present lower root activity, even in young trees. in such study, the root system should be viewed as a population of roots with varying, although coordinated, morphological and physiological properties. measurements of carbohydrate status at different stages of development at both root and canopy levels would also improve these results and give us more valuable information on the relationship between the rooting system distribution and canopy development, essential for irrigation requirement estimation as they determine evapotranspiration and water available for the root system. more knowledge is needed on root growth in young trees because they are more vulnerable to water shortages. for such trees, and particularly plants obtained from rooted cuttings, water uptake remains highly dependent on the effective areas of transpiration and water absorption. thus, it is probably more convenient to consider evapotranspiration, rainfall and irrigation in terms of volume of water/tree instead of mm. our progress in the future will be measured by our capacity to integrate knowledge on water supply, evaporative demand and the soil volume explored by the root system for different locations and planting densities. references abd-el-rahmane a.a., el-sharkawi h.m., 1974 response of olive and almond orchards to partial irrigation under dry farming practices in semi-arid regions. water relations in olive during the growing season. plant and soil, 41: 13-31. allen r.g., pereira l.s., raes d., smith m., 1998 crop evapotranspiration. guidelines for computing crop water requirements. fao irrigation 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meteorol., 121(1-2): 1-18 therios i., 2008 irrigation of the olive. in: therios l. (ed.) olives. crop production science in horticulture series. cabi, oxon, wallingston, uk, pp. 166. vernet a., mousset p., 1963 ecologie de l’olivier: alimentation en eaux. rapport de l’ecole nationale supérieure d’agriculture de tunis. yankovictch l., berthelot p., 1947 rapport sur l’enracinement de l’olivier et des autres arbres fruitiers en tunisie. annales du service botanique et agronomique de tunisie. 20: 109-176. 105 1. introduction in recent years, malignant neoplasm (cancer), cerebrovascular disease and heart disease have become major causes of death among japanese people, with approximately 75% of deaths in japan caused by these diseases (japanese ministry of health, labour and welfare, 2009 a). such diseases are generally called lifestyle diseases because they are thought to be strongly related to a lack of exercise and high intake of fat and salt. therefore, emphasis has shifted from early diagnosis (secondary prevention) to lifestyle improvement (primary prevention) (japanese ministry of health, labour and welfare, 2009 b). in this context, research on foods that have pharmacological effects or physiological functions, such as disease prevention and health maintenance, has become more important (namba, 1999; tokui et al., 2003). regarding the pharmacological effects of food, there is a form of medicinal cooking called “yakuzen” which is based on the philosophy of oriental medicine and is intended to maintain good health and improve physical condition. the preparation of yakuzen dishes draws from the theory of “yaku-shoku dou-gen”, which means that the same principle underlies the daily diet and medical treatment, and on the yin-yang theory, the five-phase theory in oriental medicine. as a result, yakuzen has attracted considerable attention for the prevention of lifestyle diseases. to prepare yakuzen dishes, in-season materials with appropriate properties and tastes are selected according to the health status and constitution of each person (namba, 1995; lan et al., 2002; tokui et al., 2003). these properties and tastes are called “sei-mi” in yakuzen theory. sei-mi consists of four properties (making the human body hot, warm, cool, or cold) and five tastes (salty, bitter, sweet, pungent, and sour), and each is considered to have its own function in the human body (namba, 1999; tokui et al., 2003). if the concept of sei-mi can be applied to vegetables, a cultivar that has a strong flavor and a high content of functional constituents related to the properties and taste is considered to have strong sei-mi and is suitable for yakuzen dishes. however, the inherent flavor of vegetable cultivars has been weakened by breeding because priority has been given to ease of consumption for consumers or ease of production for growers. in the case of eggplant (solanum melongena l.), popular cultivars in japan today are f 1 (first filial generation) cultivars derived from a parental line with oval fruit, a deep purple pericarp, and high yield; these cultivars have improved fruit quality with less unpleasant or harsh taste. evaluation of local eggplant cultivars in terms of the suitability as materials for “yakuzen” dishes s. tsukagoshi* (1), k. kuroda**, m. hohjo*, f. ikegami*, n. kunisaki***, t. hanamura***, k. yamada***, t. hagiwara*** * center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa, chiba 277-0882, japan. ** faculty of horticulture, chiba university, 648 matsudo, matsudo, chiba 271-8510, japan. *** research and development department, nichirei foods inc., 9 shinminato, mihama-ku, chiba 2610002, japan. key words: functionality, local cultivars, property and taste, solanum melongena. abstract: “yakuzen” is a form of medicinal cooking based on the theories of oriental medicine. to prepare yakuzen dishes, in-season materials with appropriate properties and tastes, “sei-mi,” are selected according to the health status and constitution of each person. in this study, the suitability of eggplant (solanum melongena l.) cultivars for yakuzen was evaluated by sensory tests and by analysis of the functional constituents considered to be closely related to the taste and functionality of yakuzen dishes. twenty-two eggplant cultivars including 21 japanese and 1 italian cultivar, and a thai species (solanum xanthocarpum schrad & wendl.) were evaluated. principal component analysis (pca) was used for comprehensive evaluation among the cultivars. from the pca, many of the cultivars with round or oval fruit were characterized as juicy and sweet and considered easy to eat; most of the long-fruit cultivars were characterized as having higher specific amino acid contents. the small and round fruit cultivar dewako and the thai species (makhuea pro) were considered to contain many functional ingredients, such as ash, polyphenols, and specific amino acids, and to have higher suitability for yakuzen dishes. adv. hort. sci., 2014 28(2): 105-110 (1) corresponding author: tsukag@faculty.chiba-u.jp received for publication 31 march 2014 accepted for publication 07 july 2014 106 however, eggplant was introduced into japan more than 1,260 years ago, and it is mentioned in nara-period documents (the shõsõin documents) edited in 750 a.d. (yoshida, 2010). therefore, many cultivars have been developed over the long history of cultivation, and even today there are many cultivars with local origins. the chemical composition, flavor, and texture of the edible parts of plants vary among cultivars. local cultivars grown for many years may have retained more flavor, and some of them may be more suitable for yakuzen dishes than the currently popular cultivars. regarding the functionality of foods, many studies have focused on certain ingredients and have discussed the relationship between the amounts of those ingredients and the functionality of the food. however, it is important to consider the functionality of foods comprehensively in yakuzen theory. in previous studies, we used principal component analysis (pca) to comprehensively evaluate the functionality of local cultivars, and we demonstrated that two local cultivars of japanese radish (raphanus sativus l.), two of carrot (daucus carota l.), and one of bitter melon (momordica charantia l.) were more suitable for yakuzen dishes than the widely used f 1 cultivar (saito et al., 2010; tsukagoshi, et al., 2011 a, b). in this study, two f 1 cultivars and 21 local cultivars of eggplant were evaluated for their suitability as materials for yakuzen dishes according to their taste and content of functional constituents. 2. materials and methods plant materials and growing condition twenty-one japanese eggplant cultivars with different fruit shapes were selected for this study (table 1). most of them were purebred cultivars, but two f 1 cultivars were included. in addition, an italian cultivar and a thai species (solanum xanthocarpum schrad & wendl.) were used. all cultivars were grown at the center for environment, health and field sciences, chiba university. seeds of all cultivars were sown in 9-cm plastic pots filled with upland soil on 17 april 2012, and the seedlings were raised in a glasshouse. on 5 june seedlings were transplanted to the open field at a spacing of 50 cm between plants and 100 cm between rows. fruits were harvested when they reached the regular size for each cultivar. harvest began on 31 july and ended on 14 september. all other management was carried out according to the conventional methods in japan (chino, 2001). sensory test fruits were harvested on 7 and 9 august and the sensory test was conducted the following day. the fruits were washed, cut to a size of 1.5×1.5×3.0 cm, and steamed at 100°c for 5 min. the characteristics of steamed samples were evaluated by six panelists. the panelists passed recognition tests for five tastes (sweetness, umami, saltiness, table 1 eggplant cultivars used for the experiment fruit shape (country) cultivar abbreviation fruit length, weight and color remark small, round (japan) dewako de 3-8 cm, 10-15 g, deep purple minden min 3-8 cm, 10-15 g, deep purple round (japan) aizu maru am 8-10 cm, 200-300g, deep purple   kamo nasu kn 12-15 cm, 200-350 g, deep purple tonosama to approx. 15 cm, 300-450 g, deep purple yamatoyo maru ym 10-12 cm, 250-350g, deep purple money pouch (japan) nagaoka kinchaku nk 8-10 cm, 300-350g, deep purple   (swelling toward the bottom) saitama ao daimaru sa approx. 15 cm, 300-450 g, green oval (japan) heta murasaki hm approx. 5 cm, 30 g, deep purple   senryo 2 gou se2 10-12 cm, 80-90 g, deep purple f 1 , control cultivar in this experiment se2 (control) sk 10-11 cm, 150-180 g, deep purple wase shinkuro ws 10-12cm, 80-120 g, deep purple yamashina ya 10-12cm, 80-120 g, deep purple long (japan) chikuyou chi 20-25 cm, 120-150 g, deep purple f 1 hakata naga hak 40-45 cm, 200-300 g, deep purple hhogo naga hho 17-18 cm, 85-90 g, deep purple himo nasu hn 25-30 cm, 150-200 g, pale purple kitta chunaga kc 10-12cm, 80-120 g, deep purple kurume oh naga ko 30-35 cm, 250-300 g, deep purple shikon sendai naga ss 8-10 cm, 20-30 g, bluish purple shin nagasaki naga sn 35-40 cm, approx. 250 g, deep purple big, oval (italy) zebra ze 20-25 cm, 300-400 g, purple and white stripes small, round (thailand) makhuea pro mp 4-5 cm, approx.10 g, green solanum xanthocarpum 107 bitterness, and sourness) and the discrimination tests for four solutes (sucrose, sodium chloride, tartaric acid, and sodium glutamate). in addition, they had more than one year experience in evaluating vegetables and were classified as expert assessors (japanese society for sensory evaluation, 2009). the characteristics listed in table 2 were evaluated on a scale of -5 (weaker) to 5 (stronger) compared to se2, the control cultivar in this study. taste sensor analysis and amino acid content approximately 50 g of fresh fruit was homogenized in 100 ml of water in an ice bath, then filtered through cotton cloth. although filtrates were prepared separately from three or four fruit samples, the filtrates were mixed to obtain the quantity necessary for measurements. bitterness, astringency, acridity, and pungency of the filtrates were measured using a taste sensor system (sa402b, intelligent sensor technology, kanagawa, japan). each value was expressed relative to the control cultivar (se2), which was set at zero. a portion of each filtrate was filtered again through a 0.45μm filter (dismic-25cs, advantec, tokyo, japan) and the amino acid content was measured using an amino acid analyzer (jlc-500/v, jeol, tokyo, japan). soluble solids content fresh fruit was cut into small pieces and pressed in gauze to extract the juice. the soluble solids content of the juice was measured using a refractometer (pal-1, atago, tokyo, japan) and expressed as percent brix. ash and polyphenol contents the harvested fruit was stored at -30°c until use. fresh-frozen fruit samples were freeze-dried and ground into a fine powder. ash content was determined using the dry ashing method. briefly, 0.3 g of the powder was put in a crucible and ashed at 550°c for 24-48 hr. after cooling, the weight of the residue was measured. polyphenol content was determined using iron tartrate spectrophotometry. first, 0.2 g of the powder was mixed with 10 ml of distilled water, and shaken for 10 min at 80°c. after cooling, the sample was centrifuged at 3,000 rpm for 15 min. then, 3.2 ml of the supernatant was mixed with 1.6 ml of iron tartrate reagent (0.1% (w/v) ferrous sulfate and 0.5% (w/v) potassium sodium tartrate) and 3.2 ml of phosphate buffer (0.1 m, ph7.5). the absorbance at 540 nm was then measured using a spectrophotometer (u-2000, hitachi, tokyo, japan). the polyphenol content was calculated from a table 2 evaluation of the taste of eggplant cultivars by sensory test (z) fruit shape cultivar aroma   softness juiciness sweetness bitterness astringency     good grassy   pericarp flesh       & acridity small, round de (y) 0 (x) 0 -1 0 0 0 1 1 min 1 1   0 -1 0 -1 2 1 round am 0 1 0 -1 0 0 0 1 kn 1 1 0 -1 0 0 0 0 to 0 0 -1 0 0 0 1 0 ym 0 0   0 1 1 1 -1 0 money pouch nk 0 2 -3 -2 -1 -1 0 0   sa 0 2   -4 0 -1 0 0 1 oval hm 0 0 0 1 0 0 0 0 se2 (control) 0 0 0 0 0 0 0 0 sk 0 2 0 1 2 1 0 0 ws 0 0 -1 0 -1 -1 0 0 ya 1 0   -1 0 0 0 0 0 long chi 0 0 0 1 0 -1 0 0 hak 0 0 1 1 -1 0 -1 0 hho 0 0 0 0 0 0 0 0 hn 0 1 -1 1 -1 -1 0 0 kc 0 0 -2 1 0 -1 0 1 ko 0 0 -2 1 -1 -1 0 0 ss 0 1 -1 0 0 -1 0 1 sn 0 2 0 1 -2 0 -1 0 big, oval ze 2 2   0 -2 0 -1 0 2 small, round mp 0 3   0 -1 0 -1 0 1 (z) eggplant fruit was cut to the size of 1.5 x 1.5 x 3.0 cm, then steamed at 100 degree c for 5 min before the test. (y) amino acid which is considered to be important for the functionality of eggplant in yakuzen theory. (x) tastes were evaluated on a scale of -5 (weaker) to 5 (stronger) as compared to senryo 2 gou (se2). 108 standard curve of ethyl gallate. principal component analysis and characterization of cultivars data were analyzed by principal component analysis (spss for windows version 13), and the characteristics of cultivars were comprehensively evaluated to determine the suitability of the cultivars as materials for yakuzen dishes. 3. results and discussion cultivars nk and sa (with money-pouch fruit shape) tended to have harder fruits, and long-fruit cultivars tended to be less sweet (table 2). the aroma of the italian cultivar, ze was characterized as both “good” and “grassy”. however, most cultivars were very similar to se2 (the control cultivar in this study) in the sensory test. taste sensor analysis showed that the local cultivars tended to have a less unpleasant taste than se2 (table 3). this result did not correspond to the results of the sensory test, and the difference may be due to the heating of samples before the sensory test but not before the taste sensor analysis. nevertheless, we can conclude that the local cultivars were not unpalatable compared with the commonly used cultivar. min, ym and some other cultivars tended to have higher soluble solids contents, but there was no significant difference between cultivars (table 4). “mi” (the taste) of eggplant is “kan” (sweet). in yakuzen theory, mi means not only the taste on the tongue but also specific functions in the human body (tokui et al., 2003). in this study, we could not discern differences of kan characteristics from the results of sensory test and soluble solids contents among cultivars; therefore, the suitability of cultivars was evaluated on the basis of other characteristics. ash content was higher in cultivars de and min (both of which have small, round fruit) and lower in cultivar ws, and tended to be lower in cultivars with oval fruit. “sei” (the property) of eggplant is to cool the human body. potassium accounts for most of the ash of eggplant fruit (usda, 2013), and the function of this mineral is to release heat inducing diuresis. this function is closely related to the property of eggplant, and higher ash content may be related to greater suitability of cultivars for yakuzen dishes. polyphenol content was also higher in de and min. eggplant contains polyphenols such as chlorogenic acid and nasunin, which are considered to have antioxidant activity, and to suppress lipid peroxidation, aging, various lifestyle diseases, and cancer (kimura et al., 1999; noda et al., 2000; kitsuda et al., 2005; singh et al. 2009). das et al. (2011) reported that grilled eggplant had a higher polyphenol content, though the cardioprotective ability was not different. the high polyphenol content in de and min may increase the pharmacological value of these cultivars. as mentioned above, mi (the taste) also encompasses specific functions in the human body, and mi of eggplant is kan (sweet). kan is considered to have functions such as supplying nutrition and energy, promoting relaxation, etc. some amino acids are considered to have kan functions. for example, glutamine is an energy source for digestion and plays an important role in the maintenance and improvement of immunity and the repair of organs (ajinomoto co. inc., 2003 a, b), and this may correspond to a kan function. alanine has a sweet taste and supplies sugars to the body, and it is also considered to have a kan function. the amino acids strongly related to the kan of eggplant include alanine, citrulline, glycine, glutamine, proline and serine. therefore, these amino acid contents were summed to give specific amino acid content (table 4): it was highest in se2 and sn and tended to be higher in the long-fruit cultivars and lower in the round-fruit cultivars. total amino acid content also tended to be higher in the long-fruit cultivars, especially in sn. however, no other trends in amino acid content were observed. the higher content of specific and total amino acids in sn would indicate greater suitability of this cultivar for yakuzen dishes. table 3 evaluation of the taste of eggplant cultivars by taste sensor (z) fruit shape cultivar bitterness astringency acridity pungency small, round de (y) -0.27 (x) 0.06 -1.74 -0.69 min -0.38 0.04 -2.27 -0.96 round am -0.23 0.20 -1.14 -0.48 kn -0.11 0.21 -2.11 -0.94 to -0.11 0.13 -2.18 -1.18 ym -0.16 -0.02 -1.33 -1.30 money pouch nk -0.26 0.27 -1.78 -0.64   sa -0.07 0.30 -2.30 -1.07 oval hm 0.22 0.13 -1.83 -0.99 se2 (control) 0.00 0.00 0.00 0.00 sk -0.28 0.05 -1.51 -0.76 ws -0.06 0.23 -2.26 -1.17 ya -0.09 0.08 -2.57 -1.30 long chi -0.33 0.12 -1.39 -0.61 hak -0.28 -0.09 -2.55 -1.40 hho -0.15 0.05 -1.61 -0.86 hn -0.01 0.15 -2.87 -1.45 kc -0.12 0.11 -2.24 -1.28 ko -0.43 0.07 -0.52 -0.15 ss -0.26 0.04 -2.03 -1.00 sn -0.47 -0.05 -2.27 -1.09 big. oval ze -0.32 -0.02 -2.73 -1.23 small. round mp -0.34 0.19 -2.15 -0.75 (z) taste sensor was prepared to express the value of se2 was zero. (y) amino acid which is considered to be important for the functionality of eggplant in yakuzen theory. (x) positive and negative value means the taste was stronger and weaker than se2, respectively. 109 from the pca, 23 principal components (pcs) were derived and 14 pcs were considered to be meaningful (contribution rate>1). these 14 meaningful pcs accounted for 96.6% of the total rate (data not shown). although the first two pcs accounted for only 41.3% of the total rate, a twodimensional scatter diagram of factor loading was constructed. specific and total amino acid contents were in the positive direction along the x-axis, and juiciness, sweetness, percent brix and some unpleasant taste were in the negative direction. ash, polyphenol content, and bitterness were in the positive direction along the y-axis, and softness was in the negative direction (data not shown). a two-dimensional scatter diagram constructed from the pc1 and pc2 scores of each cultivar enabled classification of the cultivars (fig. 1). many of the common f 1 cultivars of eggplant used for commercial production in recent years have been developed to improve ease of consumption and cultivation. se2 is one of the common cultivars in japan. it was characterized as juicy and sweet by the pca and is considered easy to eat. the other cultivars with round or oval fruit were also characterized as juicy and sweet. on the other hand, most of the long-fruit cultivars were distinct from the round-fruit and oval-fruit table 4 soluble solid, ash, polyphenol and amino acid content of eggplant cultivars fruit shape cultivar soluble solids (% brix) ash (g 100 g-1 fw) polyphenol (z) (mg 100 g-1 fw) specific amino acid (y) (mg 100 g-1 fw) total amino acid (mg 100 g-1 fw) small. round de (x) 5.1 a (w) 0.60 a (w) 673.4 a (w) 120.6 284.9 min 5.2 a 0.60 a 434.0 a 71.1 186.0 round am 4.8 a 0.45 ab 169.0 b 37.2 151.2 kn 5.4v 0.53 (v) 150.0v 48.8 203.1 to 4.8 a 0.39 b 253.7 ab 52.5 196.6 ym 5.3 a 0.50 ab 255.0 ab 36.3 144.3 money pouch nk 5.0 a 0.45 ab 172.9 b 63.1 197.8 sa 4.6 a 0.50 ab 247.3 ab 39.5 167.3 oval hm 4.7 a 0.50 ab 300.6 ab 64.5 203.1 se2 (control) 4.6 a 0.44 ab 129.1 b 145.6 302.0 sk 4.3 a 0.39 b 158.4 b 48.0 170.6 ws 5.1 a 0.37 b 181.9 b 52.8 199.1 ya 4.3 a 0.40 b 248.5 ab 58.1 185.4 long chi 4.5 a 0.42 ab 188.1 b 34.1 161.2 hak 4.3 a 0.38 b 127.9 b 116.3 286.5 hho 4.8 a 0.52 ab 166.2 b 76.8 253.3 hn 4.5 a 0.52 ab 144.1 b 83.6 288.6 kc 4.4 a 0.48 ab 236.6 ab 50.8 211.0 ko 5.1 a 0.45 ab 135.1 b 93.6 314.2 ss 4.1 a 0.38 b 250.6 ab 106.1 281.8 sn 4.5 a 0.41 ab 119.2 b 139.2 342.0 big. oval ze 3.8 a 0.46 ab 124.0 b 95.1 308.9 small. round mp 5.1 a 0.56 ab 245.9 ab 63.9 196.7 (z) polyphenol content was expressed as ethyl gallate equivalent. (y) amino acid strongly related to kan of eggplant. (x) abbreviations as in table 1. (w) different letter within the row indicates significant difference by tukey’s multiple range test at 5% level (n=5). (v) number of harvested fruit was not enough for statistical analysis. fig. 1 two dimensional scatter diagram of the principal component score of eggplant cultivars ■: small, round ◆: round ●: monry pouch ▲: oval : long : italy : thailand. abbreviations as in table 1. 110 cultivars. they were characterized as having higher specific amino acid contents and little unpleasant taste or sweetness. the small and round fruit cultivar de and the thai species (mp) were considered to contain many functional ingredients, such as ash, polyphenols, and specific amino acids, and to have greater suitability for yakuzen dishes. 4. conclusions among the local japanese cultivars used in this study, cv. de is highly suitable for yakuzen dishes because it contains many ingredients associated with the properties and taste (sei-mi) of eggplant in yakuzen theory. in addition, the italian cultivar (ze) and the thai species were highly distinct. different results may have been obtained had we grown the cultivars in another area or under different conditions. nevertheless, we have demonstrated that some local eggplant cultivars have stronger sei-mi than current f 1 cultivars. these characteristics could add value to the local cultivars and lead to regional development. references ajinomoto co. inc., 2003 a amino san no hataraki (function of amino acid). amino acid handbook. ajinomoto co. inc., kogyo chousa-kai publishing, tokyo, japan, pp. 20-63. ajinomoto co. inc., 2003 b hinmoku betsu kakuron (detailed explanation of each amino acid). amino acid handbook. ajinomoto co. inc., kogyo chousa-kai publishing, tokyo, japan, pp. 147-174. chino k., 2001 nasu no roji saibai (cultivation method of eggplant in open field). yasai tsukuri no jissai -ka sai 1(practical method of vegetable cultivation fruit vegetables 1). rural culture association japan, tokyo, japan, pp. 80-90. das s., raychaudhuri u., falchi m., bertelli a., braga p.c., das d.k., 2011 cardioprotective properties of raw and cooked eggplant. food funct., 2: 395-399. japanese ministry of health, labour and welfare, 2009 a annual changes in death rate for leading causes of death. health, labour and welfare report 20082009 part 1. japanese ministry of health, labour and welfare, no. 10. japanese ministry of health, labour and welfare, 2009 b changes in national health promotion measures, health, labour and welfare report 2008-2009 part 2. japanese ministry of health, labour and welfare, no. 62. japanese society for sensory evaluation, 2009 panel. kannou hyouka shi text (textbook for sensory evaluator). japanese society for sensory evaluation, kenpakush, tokyo, japan, pp. 49-53. kimura y., araki y., takenaka a., igarashi k., 1999 protective effects of dietary nasunin on paraquat-induced oxidative stress in rats. biosci. biotechnol. biochem., 63: 799-804. kitsuda k., nakamura t., morita n., imahori y., suzuki t., ikeda h., 2005 antioxidative activity in eggplant ‘mizu-nasu’ fruit and its enhancement after injury. hort. res. (japan), 4: 229-232. lan s., sakai e., tanaka t., 2002 the role of “ishokudougen” in japan. a study of “yaku-zen” which is both old and new meal science. a. proc. gifu pharm. univ., 51: 47-53. namba t., 1995 yakuzen to kampo (yakuzen and oriental medicine). farumashia, 31: 19-21. namba t., 1999 yakuzen no genri to shoku/yaku zai no kouyou (1) [principes of yakuzen, and the effect of food and medicine (1)]. j. cookery sci. japan, 32: 374-379. noda y., kaneyuki t., igarashi k., mori a., packer l., 2000 antioxidant activity of nasunin, an anthocyanin in eggplant peels. toxicology, 148: 119-123. saito y., hohjo m., tsukagoshi s., ikegami f., nakao c., hanamura t., yamada k., hagiwara t., 2010 study on the adequacy of vegetables as materials for medicinal foods. 5. comprehensive evaluation of pre-selected momordica charantia l. cultivars by principal component analysis. hort. res., 10 (suppl.1): 481. singh a.p., luthria d., wilson t., vorsa n., singh v., banuelos g.s., pasakdee s., 2009 polyphenols content and antioxidant capacity of eggplant pulp. food chemistry, 114: 955-961. tokui n., minari y., zhang z.l., guo x., 2003 yakuzen no gaiyou (outline of yakuzen). yakuzen to chu-igaku (yakuzen and chinese medicine). kenpakusha, tokyo, japam, pp. 2-32. tsukagoshi s., inuzuka s., hohjo m., ikegami f., takenaga s., nakao c., uryu n., hagiwara t., aoki h., hanamura t., 2011 a evaluation of japanese radish cultivars in terms of medicinal properties for “yakuzen” dishes. hortresearch, 65: 81-86. tsukagoshi s., takano a., hohjo m., ikegami f., hagiwara t., nakao c., yamada k, hanamura t., takenaga s., aoki h., 2011 b evaluation of some local cultivars of carrot in terms of the suitability as materials for “yakuzen” dishes. j. trad. med., 28: 106-114. united states department of agriculture, 2013 national nutrient database for standard reference, release 26. united states department of agriculture, http://ndb.nal. usda.gov/ndb/search/list. yoshida t., 2010 gensan to raireki (origin and history of eggplant). nougyo gijutsu taikei yasai hen (compendium of agriculture, vegetables edition). vol. 5. kisohen (basis). rural culture association japan, tokyo, japan, pp. 3-7. 199 1. introduction summer pruning of apples (malus x domestica) is a practice used primarily to enhance fruit quality through the manipulation of tree physiology and alteration of the canopy environment, particularly light. the majority of studies of summer pruning in apple have focused on physiological impacts such as photosynthate partitioning, winter hardiness, return bloom, fruit coloring and post-harvest quality. however, summer pruning can have impacts on apple diseases, because it alters canopy microclimate, can remove diseased tissue, improves deposition of fungicides and other chemicals, and in altering tree physiology may change resistance to disease. summer pruning may enhance disease management, but in some cases may increase the risk of disease. of the few studies that have investigated interactions between summer pruning and apple diseases, most were done on semi-dwarf trees, and very few have looked at high-density systems. in semi-dwarf trees, summer pruning offered both horticultural and disease management benefits. this is illustrated by an example from the mid-1980’s in the northeastern us, where summer pruning was used to compensate for the loss of daminozide in commercial apple production (autio and greene, 1990). the predominant cultivar produced in the region at that time was ‘mcintosh’, which frequently drops fruit to the ground before they are sufficiently colored for harvest. daminozide (alar®) prevented this premature fruit drop, allowing development of full fruit color. when the manufacturers of daminozide removed the registration for apple use, it created potentially devastating crop loss through premature drop in ‘mcintosh’. as a response, growers were encouraged to summer prune, primarily to accelerate development of fruit color through increased light penetration in the canopy. the practice was successful, to a large extent compensating for daminozide treatments. it also had a side-benefit, in that incidence of the summer disease complex sooty blotch and flyspeck (sbfs) decreased in summer-pruned trees, primarily as the result of reduced humidity and improved fungicide penetration in the canopy (cooley et al., 1997). at this time, the most recent major review of summer pruning was written 25 years before this review, focusing exclusively on effects on tree growth, yield, flowering, and fruit development (saure, 1987). since then, commercial apple production has seen wide-spread adoption of highdensity planting systems in which the methods and impacts of summer pruning would be expected to differ substantially from those used in semi-dwarf (e.g. m.7, ca. 5 m tall) trees, but few studies have examined the impacts of summer pruning on apple diseases in high-density trees. in larger, semi-dwarf trees, summer pruning does not have an impact on the tree scaffold, but focuses on small branches and is largely intended to increase light penetration and summer pruning of apple: impacts on disease management d.r. cooley, w.r. autio department of plant, soil and insect sciences, university of massachusetts amherst, amherst, ma, usa. key words: apple scab, black rot, canopy management, cultural control, fire blight, malus x domestica, microclimate, nectria canker, powdery mildew, sanitation, sooty blotch and flyspeck, white rot. abstract: pruning, including summer pruning, of apples can have a positive impact on disease management in two basic ways: through removal of dead tissue and inoculum, and through alteration of the canopy microclimate. summer pruning can also increase diseases if it is done when disease risk is high. however summer pruning is used almost exclusively as a horticultural tool to improve fruit quantity and quality. as orchard planting and training systems have moved from semi-dwarf trees to high-density, fully-dwarf trees, very few summer pruning studies have looked at impacts related to disease, yet summer pruning in high-density systems may have important disease management effects. growers should avoid summer pruning practices which will increase disease risks, and use those that offer both horticultural and disease management benefits. more research in this area is needed, as cultural components of apple disease management will become increasingly important in sustainable production systems. this review looks at important apple diseases, including apple scab, fire blight, sooty blotch and flyspeck, black rot, white rot, nectria canker and powdery mildew, and uses dormant pruning studies plus knowledge of the epidemiology of the diseases to suggest ways that summer pruning would be expected to impact disease management. adv. hort. sci., 2011 25(3): 199-204 received for publication 20 june 2011 accepted for publication 27 july 2011 200 air circulation, beneficial to both fruit quality and disease management. however, in modern high-density production systems a primary goal is light penetration, and overall tree training maintains a relatively small, open canopy. in such trees, summer pruning may not significantly increase air circulation, improve drying in the canopy, or improve pesticide deposition, and it is unclear whether it has benefits in terms of disease management. instead, pruning cuts during the growing season may increase the risk of infection. alternatively, pruning and removing diseased tissue, sanitation, may reduce disease impacts and future risks, but such cuts may conflict with desired tree architecture in a high-density system. yet as restrictions on chemical use in plant disease management increase, cultural controls such as sanitation and inoculum destruction become more important. much of this review must extrapolate from studies on semi-dwarf trees, dormant pruning, and the epidemiology of apple diseases to identify potential benefits and risks of summer pruning related to disease. it outlines types of summer pruning used in high-density systems, and then looks at important apple diseases that may be impacted by summer pruning and how pruning for sanitation may be useful. 2. impacts on tree growth, yield, flowering and fruit development summer pruning can take on various forms from simple watersprout removal only to significant reductions in canopy density. much study of summer pruning came from an interest in enhanced light penetration into the summer canopy, thus improving fruit color development. vincent (1917), preston and perring (1974), stiles (1980), lord and greene (1982), marini and barden (1982), morgan et al. (1984), autio and greene (1990), schupp (1992), and ystaas (1992) all showed increased fruit redness as a result of summer pruning. decreases in fruit size, however, have also been reported in some studies (stiles, 1981; marini and barden, 1982; greene and lord, 1983; myers and ferree, 1983) but not all and not consistently. li et al. (2003) modeled tree physiology as a result of summer pruning and found reductions in carbohydrates, potentially leading to a carbohydrate shortage after summer pruning. the potential for shortage was greater as the intensity of summer pruning increased. fruit size impacts of that shortage can be mediated by improved water status resulting from reduced transpiration. additionally, varying responses may be partially explained by the location of the summerpruning treatment. greene and lord (1983) suggested that, as the severity of pruning increased or as the distance between the cut and the fruit decreased, the potential for a size reduction is enhanced. timing of summer pruning also is an important consideration. in general, regrowth during the period following summer pruning is greater the earlier the summer pruning is performed. autio and greene (1990) showed a linear decrease in the amount of regrowth as the pruning was performed from early (~45 days after full bloom) to late summer (~105 days after full bloom). zamani et al. (2006) described a similar response from summer pruning from 30 to 90 days after full bloom. such data indicate that summer pruning acts as a stress on apples. if a stress is severe enough it can predispose woody plants to disease particularly from canker pathogens, but plants will recover from light to moderate stress (schoeneweiss, 1981). at the same time, abiotic stresses can induce disease resistance in plants, including apples, though the physiological mechanisms behind induced resistance are not well understood (hammerschmidt, 1999; poupard et al., 2003). developing a better understanding of the detrimental and positive impacts of pruning stress at the physiological level is an area in need of more research. 3. impacts on disease pruning apple trees, including summer pruning, can impact disease in several ways: by altering microclimate and architecture of the canopy, by removing inoculum and infected tissue, and by creating wounds that pathogens can invade. in their experiment in an organic orchard, simon et al. (2006) observed that centrifugal training decreased apple scab and key insect pests in an organic orchard, and listed five explanatory hypotheses: 1) removal of inoculum and arthropods with the removal of fruiting spurs; 2) change in canopy microclimate, particularly better aeration; 3) decreased shoot density and increased distance between growing shoots slowing transmission; 4) changes in tree physiology inducing resistance or otherwise changing tissue susceptibility; 5) decreased canopy density improving pesticide penetration and deposition. pruning for sanitation is specifically designed to remove inoculum, thereby delaying or slowing epidemics and decreasing disease incidence and severity. for example, pruning removes primary inoculum of apple powdery mildew and fire blight, and is routinely recommended as part of the management programs for these diseases (covey and fischer, 1990; xu, 1999; steiner, 2000; holb, 2005). however, pruning for sanitation often requires pruning cuts that do not conform to horticultural goals, and in most cases is performed in winter or early spring rather than summer. summer pruning may remove inoculum, though it is not the primary purpose of the practice. changing the canopy density alters microclimate thereby impacting infection and disease development. microclimate factors, particularly those related to moisture, have a major effect on plant diseases (huber and gillespie, 1992), and altering canopy density, as summer pruning does, influences canopy drying and disease (gubler et al., 1987; cooley et al., 1997; sentelhas et al., 2005; batzer et al., 2008). leaf wetness duration (lwd) is a particularly important factor in plant disease epidemics and is often used in disease forecast models (gleason et al., 2008). for example, lwd has a major impact on whether or not apple scab (venturia inaequalis) infections occur (mills, 1944; 201 machardy and gadoury, 1989; stensvand et al., 2005; xu and robinson, 2005). high humidity and lwd also increase disease incidence and severity of sbfs (cooley et al., 2011), cedar-apple rust (gymnosporangium juniperivirginianae) (aldwinckle et al., 1980), black rot (botryosphaeria obtuse) (arauz and sutton, 1989), white rot or bot rot (botryosphaeria dothidea) (sutton and arauz, 1991), nectria canker (nectria galigena) (krahmer, 1981; xu et al., 1998), and fire blight (erwinia amylovora) (steiner, 2000). of these diseases, apple scab, sbfs, black rot, white rot and bitter rot drive most of the fungicide applications made in the eastern us, and account for substantial fungicide use in many apple production areas around the world (cooley, 2009). by maintaining an apple canopy that dries relatively quickly, lwd periods are shortened, which may allow decreases in fungicide use. the relationship between plant growth and the rate of an epidemic is complex, depending on tissue susceptibility, existing infections and the density of susceptible tissue, among other factors (ferrandino, 2008). for apple scab, the relative risk of infection is affected by the amount of leaf tissue available for infection interacting with increasing ontogenic resistance and inoculum availability, which all change over time (ficke et al., 2002). summer pruning removes target tissue as well as sources of inoculum, and hence should slow development of apple scab epidemics. the timing of tissue removal should affect disease, and in general removal early in the growing season should reduce disease more than midor late-season removal. still the interaction between pruning and the different epidemiological factors is complex. in the scab example, if early pruning stimulates vegetative growth, the rapid development of relatively large amounts of young, susceptible tissue may erase any earlyseason disease suppression. holb et al. (2004) observed that heavy winter pruning suppressed foliar scab, but the impact on fruit at harvest was not significant. for fire blight, it is recommended that flower clusters be removed from non-bearing trees before bloom, because the flowers are an important infection court for e. amylovora (steiner, 2000). pruning relatively non-productive flower clusters, as in centrifugal training, may reduce risk of fire blight. pruning also usually improves penetration of fungicides and other disease controlling chemicals (sutton and unrath, 1984; travis et al., 1987; cooley et al., 1997; cross et al., 2003). both summer (cooley et al., 1997) and winter pruning (ocamb-basu et al., 1988; holb, 2005) have been shown to improve spray penetration relative to non-pruned trees, though a comparison between two high density pruning methods in which centrifugal training reduced scab relative to original solaxe pruning did not show a difference in spray deposition (simon et al., 2006). apple scab scab is probably the most important diseases threatening apples in humid production regions, but only one published study has been done on the impacts of summer pruning on the disease. disease incidence in apple scab is strongly related to the duration of wetting periods and the amount of inoculum available in an orchard, with longer wetting periods and more inoculum increasing disease incidence and severity (machardy, 1996). simon et al. (2006) showed a decrease in scab in high-density plantings pruned using centrifugal pruning relative to original solaxe pruning. they suggested that centrifugal pruning shortened wetting periods in the canopy thereby reducing the number and/or intensity of infection periods, though they do not present data on canopy microclimate. a study by holb (2005) looked at different levels of winter pruning on scab in high-density organic orchards, and concluded that heavy pruning significantly reduced the area under the disease progress curve for foliar and fruit scab on susceptible cultivars. they hypothesized that suppression of scab epidemics were caused by a reduction of inoculum overwintering in apple buds, improved fungicide deposition in pruned trees, and modification of the in-canopy microclimate, though microclimate factors did not consistently vary among pruning regimens. fire blight fire blight is an increasingly serious disease of apples worldwide. the disease affects all apple tissues, but is most damaging when it migrates from primary infections, commonly in blossoms and young shoots, to limbs and trunks. scaffold limb and trunk infections are particularly damaging, cutting production over several seasons and often killing trees (van der zwet and beer, 1995; steiner, 2000; thomson, 2000). primary infections may also occur when trees are damaged mechanically by hailstones or other means, allowing the bacterial pathogen to enter the plant. in the case of fire blight, pruning for horticulture purposes also may provide entry points for the pathogen. an early study of effects of summer pruning on fire blight in apple showed that it markedly increased infections (lake et al., 1975). hence pruning when fire blight models (e.g. (steiner and lightner, 1996; smith, 1999) indicate risk of infection is high should be avoided if possible, or a treatment of streptomycin or other chemical prior to pruning be made if it is not. for example, mechanical hedging as practiced in fruiting walls would be expected to open multiple sites to fire blight infection for several days, and would be analogous to a hailstorm in terms of generating risk of infection from fire blight. fire blight epidemics often force growers to prune in an attempt to stop the progress of infections and remove inoculum. recommendations for such pruning generally suggest cutting back to a healthy branch union approximately 25 cm below visible infections; disinfection of pruning tools with alcohol or bleach between cuts may also be recommended (van der zwet and beer, 1995; steiner, 2000; toussaint and philion, 2008). in apples it has generally been recommended diseased tissue be pruned out as soon as symptoms are observed, and pruning continued at frequent intervals thereafter in order to slow and stop epidemics (covey and fischer, 1990; steiner, 2000; toussaint 202 and philion, 2008). shtienberg et al. (2003) found that factors related to the host, pathogen and environment should all be taken into account when determining whether and how to prune fire blight in pears, and elements of this approach may be useful in apples. sooty blotch and flyspeck summer pruning has been shown to decrease sbfs in apples, primarily because it reduces relative humidity and improves fungicide penetration and coverage in the canopy (cooley et al., 1997). this study was conducted on freestanding semi-dwarf apple trees approximately 5 m tall by 3 m diameter. it is not known whether similar results would be obtained in systems using fully dwarf trees in dense plantings. however a trial in the us showed that it took over 450 hours lwd for sbfs symptoms to develop in fully-dwarf, well-maintained trees while large trees with dense canopies developed signs at 225 hours lwd (ellis et al., 1999). the extent to which fully-dwarf trees in high-density systems may benefit from summer pruning has not be studied. mummified fruit have been shown to harbor inoculum for sbfs pathogens, and removing these mummies can reduce disease incidence (gleason et al., 2011). black rot and white rot black rot and white rot are fungal diseases that can attack fruit, foliage and woody tissue of apples. sutton (1981) showed that much of the inoculum for these diseases comes from prunings in or near production blocks, and it is recommended that prunings be removed or chopped so that they rapidly disintegrate so as to remove inoculum from the orchard. removing mummified fruit is also recommended as a cultural control. in that rates of these diseases on fruit is related to wetness duration and can be controlled with fungicides, pruning that reduces canopy humidity and improves fungicide coverage would be expected to enhance their management (sutton, 1981; arauz and sutton, 1989; arauz and sutton, 1990; sutton and arauz, 1991; parker and sutton, 1993). nectria canker nectria can cause infections on fruit and woody tissue in apple. pruning wounds are susceptible to the disease, and hence summer pruning can have an impact on canker incidence (krahmer, 1981; xu et al., 1998). studies have consistently shown that new wounds, including pruning cuts, are readily colonized by n. galigena. while this is another example of a disease that can be exacerbated by summer pruning, unlike fire blight, there are no forecast models to predict when summer pruning is less risky. there are fungicidal chemicals that can be very effective in reducing canker incidence if applied right after pruning, including the organically accepted slaked lime (heijne et al., 2005). powdery mildew since the early 20th century, dormant pruning has been recommended as a control measure against powdery mildew (fisher, 1920). more recent studies from eastern europe suggest that summer pruning that targets infected shoots can significantly reduce the disease, and even eliminate the disease (e.g. berbekov et al., 2006; holb and abonyl, 2007). disease reduction is probably primarily the result of inoculum removal, though reduction in canopy humidity may play some role as well (xu, 1999). 4. conclusions while the role of summer pruning in modern highdensity apple orchards is not disease management, the practice does alter the canopy architecture in ways that may reduce moisture levels and wetting period duration. for some diseases, notably fire blight and nectria canker, summer pruning can increase the risk of infection by causing wounds. while some recent studies show that summer pruning can reduce risk for major apple diseases, such as scab and powdery mildew, virtually no studies have been done outside europe. there is a need to assess the horticultural and disease management benefits and costs of summer pruning across a broader range of climates and highdensity production systems, in order to determine whether summer pruning can be an element in economically and environmentally sustainable apple production. 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(ed.) fire blight: the disease and its causative agent, erwinia amylovora. cabi, oxon, wallingford, uk, pp. 370. toussaint v., philion v., 2008 natural epidemic of fire blight in a newly planted orchard and effect of pruning on disease development, pp. 313-320. in: johnson k.b., and v.o. stockwell (eds.) proceedings of the eleventh international workshop on fire blight. international society horticultural science, leuven 1, belgium. travis j.w., skroch w.a., sutton t.b., 1987 effect of canopy density on pesticide deposition and distribution in apple trees. plant disease, 71: 613-615. van der zwet t., beer s.v., 1995 fire blight its nature, prevention, and control: a practical guide to integrated disease management. cornell university, usda agricultural information bull. no. 631, pp. 91. vincent c.c., 1917 winter versus summer pruning of apple trees. idaho agric. expt. sta. bul. university of idaho, usa. xu x.m., 1999 modelling and forecasting epidemics of apple powdery mildew (podosphaera leucotricha). plant pathology, 48: 462-471. xu x.m., butt d.j., ridout m.s., 1998 the effects of inoculum dose, duration of wet period, temperature and wound age on infection by nectria galligena of pruning wounds on apple. eur. j. plant pathol., 104: 511-519. xu x.m., robinson j., 2005 modelling the effects of wetness duration and fruit maturity on infection of apple fruits of cox’s orange pippin and two clones of gala by venturia inaequalis. plant pathology, 54: 347-356. ystaas j., 1992 effects of summer pruning on yield, fruit size, and fruit quality of the apple cultivar ‘summerred’. acta horticulturae, 322: 277-282. zamani z., saie a., talaie a., fatahi, r., 2006 effects of summer pruning on growth indices of two important iranian apple cultivars ‘golab’ and ‘shafi-abadi’. acta horticulturae, 707: 269-274. impaginato 89 1. introduction recently, aromatic plants have received great attention in several fields such as agroalimentary, food, perfumes, pharmaceutical and natural cosmetic products (baatour et al., 2010). among aromatic plants, mint is an herbaceous rhizomatous perennial plant belonging to the lamiaceae family, widely used since ancient times for its characteristic flavor and aroma. belonging to the mentha genus, spearmint (mentha spicata l.), cornmint (m. arvensis l.) and peppermint (m. piperita l.) are the species commonly used as spices in food preparation and nowadays represent one of the most important spices throughout the world marketed fresh or dried. the drying process is used to increase shelf-life and to prevent some biochemical reactions that may alter the organoleptic characteristics. moreover, mint essential oil is commonly used as flavoring food agent, perfumery, cosmetic and pharmaceutical products (díazmaroto et al., 2003). the chemical composition of spearmint essential oil depends on plant genetic structure, growth conditions and agronomic practices (sangwan et al., 2001; figueiredo et al., 2008). in the past, the spearmint essential oil has been intensively investigated using gas chromatography (gc) technique and the mainly compounds identified are carvone, monoterpene synthesized and stored in glandular thricomes (gershenzon et al., 1989), followed by limonene and 1,8 cineole (díaz-maroto et al., 2003; antal et al., 2011; orio et al., 2012) while the monoterpene menthol is one of the main conadv. hort. sci., 2017 31(2): 89-95 doi: 10.13128/ahs-20588 short note characterization of volatile compounds in mentha spicata l. dried leaves e. masi 1 (*), s. caparrotta 1, c. taiti 1, f. ieri 2, p. fiume 3, n. moselhy 4, s. mancuso 1, a. romani 2 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente dispaa, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 dipartimento di statistica, informatica, applicazioni “g. parenti” disia, università degli studi di firenze, viale morgagni, 59, 50134 firenze, italy. 3 ciheam, istituto di agronomia mediterranea, via ceglie, 9, 70010 valenzano (ba), italy. 4 desert research center (dcr), cairo, egypt. key words: aroma compounds, gas chromatography mass spectrometry (gc-ms), headspace solid phase microextraction (hs-spme), proton transfer reaction time-of-flight mass spectrometry (ptr-tof-ms), spearmint. abstract: gas chromatography-mass spectrometry (gc-ms) is one of the most common techniques used to measure and to characterize volatile organic compounds (vocs). at the same time, the proton transfer reaction time-of-flight mass spectrometry (ptr-tof-ms) represents a recent innovative tool that allows the on-line monitoring of vocs providing the whole mass spectra with short response time, high mass resolution and without sample preparation. we reported as major constituents of headspace in spearmint dried leaves the monoterpenes carvone, followed by dihydrocarvone, limonene and 1,8-cineole (eucalyptol) although other monoterpenes such as a-pinene, 3-carene, terpineol and neodihydrocarveol, alcohols (3-octanol, 1-octen-3-ol and 3-methyl-1-butanol), esters and ketones (3-octanone) were detected in low concentrations. in this study, the analytical gc-ms and ptr-tof-ms techniques allowed to characterize the entire volatile profile of the sample bypassing the limitations of each tool. (*) corresponding author: elisa.masi@unifi.it received for publication 13 april 2017 accepted for publication 19 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 89-95 90 stituents of m. piperita essential oil (orio et al., 2012). surprisingly, few studies have been carried out to assess the aroma composition of fresh or dried spearmint leaves (díaz-maroto et al., 2003; antal et al., 2011; silva and câmara, 2013). from several years the conventional sample technique used to analyze the aroma of plant material was the headspace solid phase microextraction (hsspme) (arthur and pawliszyn, 1990) coupled to gcms technique. this procedure has been introduced in early 90s, as a powerful solvent-free sample preparation technique where the volatile fraction is collected by exposing a fiber, coated with single or multiple polymers, directly to the headspace above the sample. subsequently, the fiber is introduced into the injection chamber of a gas chromatograph and high temperatures allow the release of the analytes into the chromatographic column. recently, a new instrument has been developed such as proton transfer reaction mass spectrometry (ptr-tof-ms), which allows the on-line monitoring of volatile compounds and the achievement of the whole mass spectra of complex volatile matrices with short response times, high mass resolution and without sample preparation (vita et al., 2015). from one side, the ptr-tof-ms advantages include high sensitive with a rapid detection system (response time of about 100 ms) and very poor fragmentation of the volatile molecules (taiti et al., 2017). by the other side, the main disadvantage is represented by the difficulty to separate volatile compounds with same chemical formula, being the unique identification of the vocs not always possible (taiti et al., 2017). for the first time, in the present study two mass spectrometry techniques (hs-spme/gc-ms and ptrtof-ms) are reported with the aim to evaluate and to characterize the aroma compounds of spearmint dried leaves. 2. materials and methods plant material mentha spicata seeds were germinated in fine peat in a growth chamber at 24°c, 68% humidity with a 10/14 h day/night cycle. subsequently, seedlings were transplanted into pots (40 cm head diameter × 20 cm height), filled with sandy loam soil and transferred in greenhouse (university of florence, dispaa). after planting, plants were grown and maintained for 50 days in a greenhouse at day/night temperatures 26-30°c and 18-21°c, respectively and 80% humidity. the plants were irrigated every 2 days and fertilized after 10-25-40 days of growth. forty five days after planting, midlife leaves were collected from each experimental unit and dried at 45°c for 24 h using a common heater. subsequently, the dried leaves were finely chopped with a grinder (mulinex ar 11, groupe seb, france) with a particle size about 1.0 mm. determination of volatile composition ptr-tof-ms analysis. the volatile profile of spearmint dried leaves was assess by ptr-tof-ms (ptr tof 8000 model, ionicon analytik gmbh innsbruck, austria) using h3o+ as reagent ion for the proton transfer reaction. for a detailed explanation of the ptr-tof-ms technology see blake et al. (2009). for sample preparation and experimental setup has been followed the procedure previously used by taiti et al. (2016). briefly, one-gram aliquots of chopped spearmint dried leaves were transferred into a glass jar (2/3 l) suitable for volatile analysis and provided to screw caps with two holes respectively connected with teflon pipes to zero air generator (peak scientific) and ptr-tof-ms. the employed setup allows the formation of a dynamic headspace sampling system with a constant air flow of 0.3 l min-1 and a constant humidity and temperature (75% ur and 24°c), which are critical parameters for vocs determination (mancuso et al., 2015). mass spectra were recorded in the mass-to-charge (m/z) range of m/z 20-220 with a time resolution of 1 s. the tool worked with the following parameters in the drift tube: pressure 2.3 mbar, voltage 594 v, temperature 110°c, extraction voltage at the end of the drift tube (udx) 35 v and was operated at an e/n value of 140 (1 td = 10217 v cm2). the inlet line, consisted of a peek capillary tube (internal diameter 0.40 mm), was maintained at 110°c in order to minimize the adsorption of chemicals onto surface (lanza et al., 2015). subsequently, the raw data and peak extraction were acquired and processed by the tofdaq software (tofwerk ag, switzerland), using a procedure previously reported by taiti et al. (2016). in order to guarantee high mass accuracy for the post processing and for a precise conversion of time-of-flight into m/z values, the internal calibration was performed off-line, using together with known low mass ions (no+ peak m/z = 29.997 and c3h7o+ peak m/z = 59.049) a known compound with high mass ion as the carvone (c10h15o+ peak m/z = 151.075). in this way the exact masses have been assigned and determined the sum formula of all vocs recorded with the ptr-tof-ms. masi et al. characterization of volatile compounds in mentha spicata l. dried leaves 91 finally, once determined the sum formula, each peak has been tentatively assigned to a specific compound, with the help of the results obtained by gcms analysis, and of the available literature on volatile compounds emitted by mint products. raw data acquired by the ptr-tof-ms were processed and analyzed using a tofdaq software (tofwerk ag, switzerland) and the methodology used for data processing is reported elsewhere (taiti et al., 2017). hs-spme/gc-ms procedure. as reported by silva and câmara (2013), the trivalent fiber divinylbenzene-carboxen-polydimethylsiloxane (dvb/car/ pdms) was the most effective spme fiber able to isolate the volatile compounds from m. piperita and m. spicata fresh leaves. in our analysis, a 2 cm trivalent spme fiber (dvb/car/pdms) was used to extract the volatile fraction from the m. spicata dried leaves. spme sampling device was manually inserted into the sealed vial and the fiber was exposed to headspace above the sample for 20 min at 25°c. after extraction the fiber was insert into the injector port of the gcms system, an agilent 7890 a gc equipped with a 5975c msd, where the volatile compounds where thermally desorbed at 280°c and then transferred to the analytical capillary column. the analytes separation was achieved with a agilent db innowax, length 50 m, 0.20 μm id, 0.40 μm df column. chromatographic conditions were: initial temperature 40°c for 0.5 min, then 6°c min-1 up to 260°c. this temperature was maintained for 1 min. only the compounds with higher intensity were identified in order to support the ptr-tof-ms analysis; the identification was based on mass spectra matching with the standard nist08/wiley98 libraries. 3. results and discussion the aim of our research was a comparison among ptr-tof-ms and conventional hs-spme/gc-ms techniques performed simultaneously on spearmint dried leaves. as reported in recent literature surveys, gcms can be considered the main method for the analysis of volatile fractions. the hs-spme/gc-ms analysis led to the identification of the most representative compounds from a quantitative point of view, and the typical gas chromatogram obtained is reported in figure 1a. consequently, over fifty volatile compounds were identified (fig. 1a). among these, the most abundant compounds were, in order of abundance: terpenes (with carvone, dihydro-carvone, d-limonene and eucalyptol or 1,8-cineole as the most represented); alcohol (mainly 1-octen-3-ol and 3-methyl-1-butanol); ketones (e.g. 3-octanone); esters (e.g. 2-methyl-ethyl ester-butanoic acid) (fig. 2). the same compounds were reported by silva and câmara (2013) in the volatile fraction of spearmint fresh leaves analyzed using the same method. the data obtained by ptr-tof-ms analysis were filtered following the procedure previously described by taiti et al. (2017); firstly all peaks imputable to water chemistry and to interfering ions (e.g. oxygen, nitrogen monoxide) and secondly the peaks whose average concentrations were lower than 1 ppbv (parts per billion by volume) were eliminated. after filtering, a total of 54 masses were revealed and could be tentatively attributed to compounds such as aldehydes, ketones, esters, terpenes and other unidentified compounds in the range of measured masses m/z from 20 to 220 (fig. 1b, table 1). as fig. 1 a) typical gas chromatogram obtained by volatile fraction of m. spicata dried leaves obtained with hs-spme/gc-ms using a dvb/car/pdms fiber. b) snapshot of the mass spectra of headspace of spearmint dried leaves obtained with ptr-tof-ms. adv. hort. sci., 2017 31(2): 89-95 92 reported in materials and methods section, the detected vocs were integrated with the fragmentation patterns of each compound (maleknia et al., 2007; kim et al., 2009; brilli et al., 2011) and with the results of the gc-ms-based profile. indeed, previous researches performed on different matrices demonstrated the good agreements with gas-chromatographic peaks and ptr-ms mass spectral data (king et al., 2010; aprea et al., 2015). generally, the correlation between gc-ms and ptr-tof-ms data allows the characterization of the sample entire volatile profile bypassing the limitations of each tool. all compounds identified and their signal intensity are listed in table 1, together with their m/z ratio, chemical name, molecular formula, percentage on the total, and additional reference for peak identification. the most abundant signals detected with ptr-tof-ms (>3% of the total) were: alkyl fragment (m/z = 41.038), isoprene (m/z = 69.069), 3-methyl-1butanol (m/z = 71.085), dimethyl-furan (m/z = 97.065), p-cymene fragments (m/z = 119.085), pcymenene (m/z = 133.101), carvone (m/z = 151.075), dihydrocarvone (m/z = 153.126), neodihydrocarveol together with menthol (m/z = 157.155) (table 1). most terpenes volatile compounds were reported in previous articles and identified as the main constituents of spearmint aroma (kokkini et al., 1995; díaz-maroto et al., 2003; orio et al., 2012). alcohols, esters and aldehydes were also detected in spearmint dried leaves even though in low content. interestingly, 3-methyl-1-butanol (m/z 71.085) was detected using both technique while some compounds such as 3-octanone, 1-octen-3-ol and 3octanol were identified only with hs-spme/gc-ms (fig. 2, table 1). as reported by kokkini et al. (1995) spearmint aroma is mainly formed by a complex mixture of various vocs, mainly terpenes such as carvone, limonene, and 1,8-cineole that are the most important aroma constituents due to their high percentage in the volatile fraction. indeed, terpenes (monoterpenes and sesquiterpenes) and terpenoids are the common constituents of flavor and fragrance of some plants and in particular of spearmint aroma (telci et al., 2010). terpenes are hydrocarbons (carbon and hydrogen are the only elements present) whereas terpenoids have been denatured by oxidation and thus contain additional functional groups. regarding monoterpenes (m/z 137 and 135) and sesquiterpenes (m/z 205) hydrocarbons, their fig. 2 mass spectra enlargement of widest class compounds identified by hs-spme/gc-ms. masi et al. characterization of volatile compounds in mentha spicata l. dried leaves 93 table 1 volatile compounds identified via ptr-tof-ms m/z tentative identification protonated chemical formula signal intensity (ppbv) compound (% on the total) references 27.022 acetylene c 2 h 3 + 22.9 ± 6.4 1.11 vita et al. (2015) 31.018 formaldehyde ch 3 o+ 56.3 ± 5.2 2.79 kushch et al. (2008) 39.022 isoprene fragment c 3 h 3 + 15.7 ± 2.2 0.74 maleknia et al. (2007) 41.038 alkyl fragment c 3 h 5 + 111.3 ± 12.7 5.81 vita et al. (2015) 43.018 alkyl fragment c 2 h 3 o+ 58.7 ± 21.5 2.9 vita et al. (2015) 43.054 alkyl fragment (propene) c 3 h 7 + 56.3 ± 5.2 2.66 taiti et al. (2017) 45.033 acetaldehyde c 2 h 5 o+ 13.5 ± 6.8 0.77 taiti et al. (2016) 53.04 cyclobutadiene c 4 h 5 + 37.3 ± 0.1 1.83 vita et al. (2015) 55.055 c 4 aldehydes fragment c 4 h 7 + 40.3 ± 5.1 1.98 taiti et al. (2017) 57.033 alkyl fragment (2-hexenal) c 3 h 5 o+ 8.0 ± 2.4 0.38 brilli et al. (2011) 57.07 alkyl fragment (hexanol/valeric acid) c 4 h 9 + 19.8 ± 0.2 0.97 aprea et al. (2015) 59.049 propanal, acetone c 3 h 7 o+ 57.4 ± 17.2 2.7 taiti et al. (2016) 61.028 acetates c 2 h 5 o+ 12.7 ± 4.6 0.62 taiti et al. (2016) 65.039 alkyl fragment c 5 h 5 + 6.1 ± 0.2 0.3 goacher et al. (2010) 67.055 terpene fragment c 5 h 7 + 33.0 ± 3.0 1.62 yener et al. (2015) 69.033 furan c 4 h 5 o+ 29.9 ± 6.4 1.47 taiti et al. (2017) 69.069 isoprene c 5 h 9 + 118.8 ± 24.4 6.06 taiti et al. (2015) 71.049 2-butenal c 4 h 7 o+ 17.7 ± 2.2 0.87 vita et al. (2015) 71.085 3-methyl-1-butanol/pentenal fragment c 5 h 11 + 117.3 ± 4.6 5.81 aprea et al. (2015) 77.04 alkyl fragment c 6 h 5 + 55. 4 ± 36.8 2.7 goacher et al. (2010) 79.054 benzene c 6 h 7 + 56.3 ± 5.2 2.66 vita et al. (2015) 81.069 1,3-cyclohexadiene/terpenes fragment c 6 h 9 + 26.5 ± 6.8 1.35 taiti et al. (2016) 83.086 c 6 compounds/hexenol fragment c 6 h 11 + 37.3 ± 0.1 1.83 maleknia et al. (2007) 91.054 alkyl fragment c 7 h 7 + 46.3 ± 5.1 2.27 yener et al. (2015) 93.068 terpene fragments c 7 h 9 + 8.6 ± 2.4 0.42 maleknia et al. (2007) 95.085 terpene fragments c 7 h 11 + 19.8 ± 0.2 0.97 aprea et al. (2015) 97.065 dimethyl-furan c 6 h 9 o+ 69.8 ± 17.2 3.91 yener et al. (2015) 97.101 alkyl fragments c 7 h 13 + 12.7 ± 4.6 0.62 aprea et al. (2015) 99.08 cyclohexanone/hexenal c 6 h 11 o+ 6.1 ± 0.2 0.3 buhr et al. (2002) 101.096 3-hexen-1-ol c 6 h 13 o+ 33.0 ±3.0 1.7 buhr et al. (2002) 103.101 2-hexanol c 6 h 15 o+ 6.5 ± 1.0 0.32 buhr et al. (2002) 105.069 styrene c 8 h 9 + 32.7 ± 1.6 1.6 yener et al. (2015) 107.049 benzaldehyde c 7 h 7 o+ 4.9 ± 1.2 0.24 yener et al. (2015) 107.086 ethylbenzene c 8 h 11 + 3.9 ± 0.7 0.19 de gouw et al. (2003) 109.065 phenylmethanol/benzenemethanol/p-cresol c 7 h 9 o+ 17.6 ± 0.3 0.86 leskinen et al. (2015) 109.101 terpene fragments c 8 h 13 + 40.6 ± 0.6 1.99 maleknia et al. (2007) 119.085 p-cymene fragments c 8 h 11 + 153.2 ± 10 7.68 maleknia et al. (2007) 121.101 sesquiterpene fragments c 9 h 13 + 6.1 ± 1.0 0.3 demarcke et al. (2009) 123.08 4-ethylphenol c 8 h 11 o+ 17.5 ± 2.7 0.86 chatonnet et al. (2009) 123.116 sesquiterpene fragments c 9 h 13 + 19.8 ± 2.1 0.97 demarcke et al. (2009) 133.101 p-cymenene c 10 h 13 + 85.2 ± 10.7 4.18 kus and van ruth (2015) 135.135 p-cymene c 10 h 15 + 4.2 ± 0.8 0.21 maleknia et al. (2007) 137.137 monoterpenes compounds (limonene, 3-carene) c 10 h 17 + 27.1 ± 9.3 1.18 maleknia et al. (2007) 139.111 2-pentylfuran/nopinone c 9 h 15 o+ 1.4 ± 0.2 0.07 wisthaler et al. (2001) 145.135 octanoic acid c 8 h 17 o 2 + 1.4 ± 0.1 0.07 aprea et al. (2015) 149.135 sesquiterpene fragments c 11 h 17 + 4.7 ± 1.0 0.23 kim et al. (2009) 151.075 monoterpenes oxygenate (carvone, myrtenal, carvacrol) c 10 h 15 o+ 79 ± 13.1 3.74 kim et al. (2009) 153.126 monoterpenes oxygenate (carveol, dihydrocarvone) c 10 h 17 o+ 71.3 ± 1.3 3.45 maleknia et al. (2007) 155.145 alcohol monoterpenes (neodihydrocarveol/terpineol) c 10 h 19 o+ 21.5 ± 8.8 3.07 bourtsoukidis et al. (2014) 157.155 decanol/menthol c 10 h 21 o+ 90.6 ± 26.8 4.55 vita et al. (2015) 159.135 nonanoic acid/c9 ester c 9 h 19 o 2 + 3.6 ± 1.5 0.18 aprea et al. (2015) 163.148 sesquiterpene fragments c 12 h 19 + 18.2 ± 3.9 0.38 demarcke et al. (2009) 203.18 sesquiterpene compounds (trans-calamenene) c 15 h 23 + 21.5 ± 4.9 0.89 taiti et al. (2017) 205.195 sesquiterpenes compounds c 15 h 25 + 29.3 ± 7.9 1.15 taiti et al. (2015) data are expressed in ppbv as mean ± deviation standard, n=3. adv. hort. sci., 2017 31(2): 89-95 94 amount was similar and represented about 2.5% of total compounds detected by ptr-tof-ms, while terpenoids (m/z 151, 153, 155, 157) were the most abundant (about 15%) (table 1). finally, considering the entire dataset and following the odor descriptor (www.thegoodscentscompany.com), the spearmint aroma might be due to the interaction between compounds that give a different odors such as: minty (e.g. carveol, 1,8-cineole, carvone), citrus (e.g. limonene), spicy (e.g. carvacrol, menthol, p-cymene, terpineol), green or herbaceous (e.g. 3-hexen-1-ol, hexenal, hexenol), fruity (e.g. 3methylbutanoate, ethyl 2-methyl butyrate) and floral notes (e.g. phenylmethanol). 4. conclusions ptr-tof-ms has been shown to be a very useful tool to detect vocs emitted by dried herbs. the results obtained in this study demonstrated that ptrtof-ms technique coupled with the gc-ms for the identifications of isomers, can be a usefully screening tool in order to (1) assess the volatile compounds of very complex samples such as aromatic herbs and (2) achieve a fast and simple fingerprinting. this technique is able to provide the whole mass spectra of complex trace gas mixtures with short response times, high mass resolution and without sample preparation. moreover, this technique, when coupled with hs-spme/gc-ms, allows the overcoming of the disadvantage related to the identification of vocs. in summary, the spearmint aroma might be due to the interaction between compounds with minty (e.g. carveol, 1,8-cineole, carvone), citrus (e.g. limonene), spicy (e.g. carvacrol, menthol, p-cymene, terpineol), green or herbaceous (e.g. 3-hexen-1-ol, hexenal, hexenol), fruity (e.g. 3-methylbutanoate, ethyl 2-methyl butyrate) and floral notes (e.g. phenylmethanol). acknowledgements the marsadev project is financed by italian ministry of foreign affairs and international cooperation implemented by ciheam 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f., sangwan r.s., 2001 regulation of essential oil production in plants. plant growth regul., 34(1): 3-21. silva c.l., câmara j.s., 2013 profiling of volatiles in the leaves of lamiaceae species based on headspace solid phase microextraction and mass spectrometry. food res. int., 51(1): 378-387. taiti c., costa c., guidi nissim w., bibbiani s., azzarello e., masi e., pandolfi c., pallottino f., menesatti p., mancuso s., 2017 assessing voc emission by different wood cores using the ptr-tof-ms technology. wood sci. technol., 51(2): 273-295. taiti c., costa c., menesatti p., caparrotta s., bazihizina n., azzarello e., petrucci w.a., masi e., giordani e., 2015 use of volatile organic compounds and physicochemical parameters for monitoring the post-harvest ripening of imported tropical fruits. eur. food res. technol., 241(1): 91-102. taiti c., marone e., bazihizina n., caparrotta s., azzarello e., petrucci a.w., pandolfi c., giordani e., 2016 sometimes a little mango goes a long way: a rapid approach to assess how different shipping systems affect fruit commercial quality. food anal. methods, 9(3): 691-698. telci i., demirtas i., bayram e., arabaci o., kacar o., 2010 environmental variation on aroma components of pulegone/piperitone rich spearmint (mentha spicata l.). ind. crop. prod., 32(3): 588-592. vita f., taiti c., pompeiano a., bazihizina n., lucarotti v., mancuso s., alpi a., 2015 volatile organic compounds in truffle (tuber magnatum pico): comparison of samples from different regions of italy and from different seasons. scientific reports, 5: 12629. wisthaler a., jensen n.r., winterhalter r., lindinger w., hjorth j., 2001 measurements of acetone and other gas phase product yields from the oh-initiated oxidation of terpenes by proton-transferreaction mass spectrometry (ptr-ms). atmos. environ., 35(35): 6181-6191. yener s., romano a., cappellin l., granitto p.m., aprea e., navarini l., mark t.d., gasperi f., biasioli f., 2015 tracing coffee origin by direct injection headspace analysis with ptr/sri-ms. food res. int., 69: 235-243. impaginato 81 1. introduction mango (mangifera indica l.) is the most important tropical fruit and considered king of all the fruits in india. the fruits are a rich source of iron and various anti-oxidants. in addition, mango is a rich source of vitamin a, e and selenium, which help to protect against heart disease and other such related aliments. nowadays, continuous use of chemical fertilizer without organic manure causes problems of soil health and fruit quality. fruit qualities are being deteriorated through the use of chemical fertilizers (huyskens-keil and schreiner, 2003). organic farming is currently gaining gradual momentum worldwide with growing awareness of health and environmental issues in agriculture and consumers demanding the production of organic fruit, thus offering an attractive source of rural income. organic farming in india has attracted many farmers throughout the country and different fruit crops like banana, papaya, pineapple and sapota have been successfully tested. according to neuhoff et al. (2011) working with oranges and ilic et al. (2013) tomato, organically grown fruits are rich in various minerals such as p, k, ca and mg. soil microbes were found to increase in organic systems compared to the conventional system of planting (dutta and kundu, 2011). however, the influence of organic, inorganic, and biofertilizer on growth characters and fruit quality of mango and soil properties are not well documented. hence, a study was initiated to evaluate their effect on fruit quality and soil properties. 2. materials and methods the study was conducted at the university research station, gayeshpur, bidhan chandra krishi viswavidyalaya, west bengal, india on 11 years old tree of mango cv. himsagar with 10×10 m spacing. the following eight treatments were imposed: vermicompost at 5 kg/plant/year, fym at 10 kg/plant/year, inorganic fertil izer (npk at 1000:500:1000 g/plant/year), 50% vermicompost + adv. hort. sci., 2016 30(2): 81-85 doi: 10.13128/ahs-19133 influence of organics, inorganic and biofertilizers on growth, fruit quality, and soil characters of himsagar mango grown in new alluvial zone of west bengal, india p. dutta (*), k. das, a. patel department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 dt. nadia, west bengal, india. key words: growth characters, mangifera indica, quality, shelf life, soil microbial population. abstract: a field study with organic manures, inorganic manures and biofertilizers alone and in combination was carried out on 11-year-old mango (mangifera indica l.) cv. himsagar spaced 10 m apart in a randomized block design at the university research station, gayeshpur, bckv, west bengal, india. the objective was to determine their effect on growth, soil characters, and fruit quality of mango grown in a new alluvial zone of west bengal. respiration, physiological loss in weight (plw) and shelf life at ambient room temperatures, and soil characters were also assessed. results revealed that among the eight treatments, biofertiliser (azotobacter + psm) along with 50% inorganic fertilizer significantly increased the growth characters of mango trees. this treatment also increased the physico-chemical character of fruit while biofertilizers alone improved the fruit quality, viz. total soluble solids, total sugar and β-carotene. fruits treated with biofertilizers also had increased shelf life with lower plw and respiration rate. soil microbial population and other soil characters were improved by application of biofertilizer as well. it is concluded that biofertilizer application in mango gives better growth, fruit quality, and soil health. (*) corresponding author: pallab_bckv@rediffmail.com received for publication 9 august 2015 accepted for publication 11 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 81-85 82 50% inorganic fertilizer, 50% fym+50% inorganic fertilizer, biofertilizer (azotobacter at 150 g/plant) + psm at 100 g/plant), biofertilizer + 50% inorganic fertilizer, and control (no treatment). treatments were applied separately in split doses, once at fruit set and another at harvest the experiment was laid out in a randomized block design with three replications. mature fruits were harvested and brought to the laboratory for physico-chemical analyses following all standard procedures as described by ranganna (2000). total soluble solids were determined using a hand refractometer (0-32°brix). total soluble sugar content was analysed using fehlings’ a and b solution, according to the method described by ranganna (2000) and expressed as percentage. in this method, for inversion of room temperature an aliquot of claried and diluted solution was transferred for a flask. 10 ml of hcl (1:1) was added and allowed to stand at room temperature for 24 hrs. the solution was then neutralized with concentrated naoh solution and made to volume. an aliquot was taken and the total soluble sugars were determined as invert sugars using fehling’s a and b solution. titratable acidity (% malic acid) was estimated by titrating fruit juice (5 ml) to ph 8 against 0.1 m naoh using phenolphthalene as indicator. total carotenoids were estimated by the method described by ranganna (2000). five grams of fresh sample were taken, a few crystals of anhydrous sodium sulphate were added, and then crushed in 10 ml acetone with the help of a mortar and pestle. the supernatant was decanted into a beaker. the process was repeated twice or thrice and the combined supernatant was transferred to a separating funnel out on standing. petroleum ether (10 to 15 ml) was added in the separating funnel and rinsed, the pigment was then transferred to the petroleum ether phase by diluting the acetone with water or water containing 5% sodium sulphate. the extraction of the acetone phase with a small volume of petroleum ether was repeated, if necessary, until no more colour was extracted. the lower layer was discarded and the upper layer was collected in a 100 ml volumetric flask. the petroleum ether extract was filtered through anhydrous na2so4 and the volume was made up to 100 ml with petroleum ether. the optical density was recorded at 452 nm using petroleum ether as blank containing 3 ml acetone per 100 ml and expressed as µg 100 g-1 pulp. as carotenoids are light sensitive, all steps were performed under subdued light. shelf life was determined at ambient room temperature (34±1°c). the co2 evolution of fruits was determined by titration of residual ba(oh)2 in the solution with standardized n/10 hcl as described by mitra et al. (1971). growth characters such as plant height, plant spread, and trunk girth were measured after fruit harvest. soil properties were recorded as per the standard procedure given by black (1965). soil microbial population was counted using the method described by collin and lyne (1985). statistical analysis was carried out according to the standard procedures. 3. results and discussion growth parameters table 1 reveals that different nutrient treatments significantly increased plant height, canopy, spread, and trunk girth. biofertilizer + half inorganic fertilizer gave maximum (6.72 m) plant height, canopy spread (6.37×6.92 m), and trunk girth (79.32 cm), followed by vermicompost (2.5 kg/plant/year) + half chemical fertilizer (rdf-npk at 1000:500:1000 g/plant/year); treatments plant height (m) canopy spread trunk girth (cm) (m) e-w n-s 1. vermicompost (5 kg/plant) 5.99 5.49 5.12 69.72 2. fym (10 kg/plant) 6.10 5.97 6.14 72.11 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 6.00 5.97 6.11 73.72 4. 50% vermicompost + 50% inorganic fertilizer 6.24 6.12 6.31 70.47 5. 50% fym + 50% inorganic fertilizer 6.11 6.00 6.09 73.15 6. biofertilizer (azotobacter @ 150g/plant + psm @ 100 g/plant) 5.99 5.84 5.91 70.37 7. biofertilizer + 50% inorganic fertilizer 6.72 6.37 6.92 79.32 8. control 5.97 5.82 5.90 71.41 sem± 1.01 0.72 1.11 2.40 cd (p=0.05) 3.11 1.92 3.31 3.14 table 1 growth characters of mango cv. himsagar as influenced by inorganic and organic manures dutta et al. organics, inorganic and biofertilizer influence on growth, quality and soil characters of himsangar mango. 83 the lowest measurements for these three parameters were found in untreated control plants. korwar et al. (2006) in aonla and shukla et al. (2009) in guava obtained similar results. these findings may be due to a better nutritional environment: application of organic matter improves soil health by improving the physico-chemical and biological activities (schnitizer, 1991) and biofertilizer was found to enhance the rate of mineralization and availability of the nutrients, further enhancing plant growth (sahoo and singh, 2005). physico-chemical composition of fruit different nutrient treatments significantly improved the physico-chemical composition of fruits (table 2). biofertilizer + half of inorganic fertilizer produced maximum fruit weight (285.15 g), yield (57.20 kg/plant), fruit length/breath (9.14/8.19 cm), followed by biofertlizer alone. also with regard to fruits, control trees gave minimum results. unlike the physical characters, the bio-chemical composition of fruits was more effected by biofertilizer alone. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant/year) gave maximum total soluble solids (19.80°brix, total sugar (16.00%) and β-carotene (6123 µg/100 g) with minimum (0.16%) acidity. fruits treated with inorganic fertilizers showed minimum total solids (18.20°brix) and β-carotene (4792 µg/100 g) with maximum (0.31%) acidity. results revealed that fruits grown under organic manure/biofertilizer had better fruit quality. the increase in physicochemical parameters in fruits due to bio-fertilizer might be because of their role in nitrogen fixation, production of phytohormone-like substances and increased uptake of nitrogen as reported by dutta and kundu (2012). furthermore, micro-organisms are an important component of soil environment (arshad and frankemberger, 1992). thus, utilization of biofertilizer could be a better preposition for improving biological attributes of soil, which in turn may increase quality and productivity potential of various crops as reported by allen et al. (2002). soil nutrient status and soil bacterial population different nutrient treatments significantly increased the soil ph and soil organic carbon (table 3). biofertilizer alone gave maximum (6.70) soil ph while inorganic fertilizer-treated soil the minimum table 2 fruit quality of himsagar mango as influenced by inorganic and organic manures treatments fruit weight (g) yield (kg/plant) fruit length/breadth (cm) tss (°brix) total sugar (%) acidity (%) β carotene (µg/100g) 1. vermicompost (5 kg/plant) 250.00 50.95 8.40/7.29 18.70 16.12 0.14 5720 2. fym (10 kg/plant) 151.42 50.35 8.31/7.31 18.90 15.91 0.17 5824 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr) 248.43 51.75 8.00/7.44 18.20 15.33 0.31 4792 4. 50% vermicompost + 50% inorganic fertilizer 265.14 53.92 9.11/8.12 19.00 15.11 0.25 5012 5. 50% fym + 50% inorganic fertilizer 270.00 54.98 9.00/8.15 19.10 15.23 0.26 5170 6. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant) 270.40 54.12 8.99/85.16 19.80 16.00 0.16 6123 7. biofertilizer + 50% inorganic fertilizer 285.15 57.20 9.14/8.19 19.60 15.92 0.18 5814 8. control 240.40 48.00 8.10/7.60 18.00 14.94 0.19 4914 sem± 1.15 0.70 1.01/0.72 0.51 0.07 0.01 4.31 cd (p=0.05) 3.72 2.10 3.11/1.97 1.37 0.21 0.03 12.39 treatments ph ec (1:2.5) (dsm-1) bulk density (g/cc) oc (%) soil microbial population (bacteria) (cfug-1 soil) 1. vermicompost (5 kg/plant) 6.57 0.263 1.21 0.90 1.6 × 106 2. fym (10 kg/plant) 6.59 0.137 1.29 0.84 5.9× 106 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 6.00 0.171 1.49 0.61 4.3× 105 4. 50% vermicompost + 50% inorganic fertilizer 6.45 0.214 1.12 0.72 5.1× 106 5. 50% fym + 50% inorganic fertilizer 6.42 0.219 1.41 0.74 5.4× 106 6. biofertilizer (azotobacter @ 50 g/plant + psm @ 100 g/plant) 6.70 0.198 1.31 0.78 8.3× 106 7. biofertilizer + 50% inorganic fertilizer 6.42 0.197 1.27 0.74 6.0× 106 8. control 6.10 0.111 1.69 0.60 6.9× 105 sem± 0.13 0.70 0.03 0.02 4.72 cd (p=0.05) 0.39 2.10 0.09 0.07 14.43 table 3 soil characters as influenced by inorganic and organic manures adv. hort. sci., 2016 30(2): 81-85 84 (6.00). organic carbon content of soil also varied significantly. vermicompost or fym-treated soil gave maximum (0.90/0.84%) content of organic carbon, followed by biofertilizer-treated soil (0.78%). soil organic carbon was at the lowest level in untreated control. the increase in organic carbon of soil may be due to the addition of organic matter through organic manure or microbes and recycling of organic materials in the form of crop residue, which brings the soil ph nearer to neutral and increases the nutrient availability. our results are in close conformity with earlier findings (dutta and kundu, 2012). the effective conductivity (ec) of soil solution depends on the presence of soluble salts. the concentrations of these salts affect the growth and absorption of water. different treatments significantly influenced the ec of soil. application of vermicompost led to the highest ec (0.263 dsm-1) in this study, whereas the control plot gave the lowest. this could be due to the fact that vermicompost contains salts, mostly cl and so4, but not at toxic levels (masciandaro et al., 1998). bulk density varied due to different treatments, with the untreated control giving maximum bulk density (1.69 g/cc) of soil. the soil microbial population varied with the different treatments: the maximum (8.3x106 cfug-1 soil) was found with biofertilizer (azotobacter at 50 g/plant + psm at 100 g/plant). soil applied with biofertilizer showed more soil bacteria, whereas least was formed by vermicompost (table 3). similar results were obtained by dutta et al. (2010) in litchi. plw, shelf life and respiration of fruit physiological loss in weight (plw) of fruit varied significantly among the different treatments (table 4). untreated fruit had the highest (18.95%) plw while the lowest was found with fruit grown in biofertilizer (11.31%). fruits grown in biofertilizer showed maximum (10 days) shelf life with minimum respiration rate (109.72 mg/hr/kg fruit) during storage. untreated control fruit recorded maximum respiration and plw with minimum shelf life (5 days). improvement of shelf life due to application of biofertilizer was previously reported in mango (dutta and kundu, 2012). 4. conclusions the present study reveals that application of organic and biofertilizer are more beneficial for quality mango production and increase soil health. therefore, this approach can be spread among growers to improve the quality in mango orchards. references allen m.f., jasper d.a., zak j.c., 2002 microorganism, pp. 257-278. in: perrow m.r., and a.j. davy (eds.) handbook of ecological restoration. volune i. principles of restoration. part 4. manupulation of the biota. cambridge university press, cambridge, uk, pp. 445. arshad m., frankemberger jr. w.t., 1992 microbial production of plant growth regulators, pp. 307-348. in: meeting f.b. jr. (ed.) soil microbial ecology. application in agricultural and environmental management. marcel dekker, inc. new york, usa. black c.a., 1965 method of soil analysis. amer. soc. of agron. inc. publisher, madison, wisconsin, usa, vol. 9, parts 1 & 2, pp. 1572. collin c.h., lyne p.m., 1985 microbiological methods. butterworth co., london, uk, pp. 450. dutta p., kundu s., 2011 effect of organic manure and biofertilizers on organic production of mango. treatment plw (%) shelf life (days) respiration co 2 evolution (mg/hr/kg fruit) 1. vermicompost (5 kg/plant) 12.62 8 120.41 2. fym (10 kg/plant) 12.44 9 118.32 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 17.92 6 170.41 4. 50% vermicompost + 50 % inorganic fertilizer 14.31 7 150.37 5. 50 % fym + 50 % inorganic fertilizer 14.45 2 159.37 6. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant) 11.31 10 109.72 7. biofertilizer + 50% inorganic fertilizer 13.00 8 122.14 8. control 18.95 5 163.14 sem± 1.10 0.61 4.11 cd (p=0.05) 3.42 1.82 12.12 table 4 plw, shelf life and respiration of fruit at the end of storage life as affected by inorganic and organic manures dutta et al. organics, inorganic and biofertilizer influence on growth, quality and soil characters of himsangar mango. 85 proceedings of the third scientific conference of isofar, 1: 475-478. dutta p., kundu s., 2012 effect of biofertilizers on nutrient status and fruit quality of himsagar mango grown in new alluvial zones of west bengal. j. crop weed sci., 8(1): 72-74. dutta p., kundu s., biswas s., 2010 integrated nutrient management in litchi cv. bombai in new alluvgial zone of west bengal. indian j. hort., 67: 181-184. huyskens-keil s., schreiner m., 2003 quality of fruit and vegetable. j. appl. bot., 77: 147-151. ilic z.s., kapoulas n., milenkovic l., 2013 micronutrient composition and quality characteristics of tomato (lycopersicon esculentum) from conventional and organic production. indian j. agric. sci., 83: 651-655. korwar g.r., pratibha g., ravi v., palanikumar d., 2006 influence of organics and inorganics on growth, yield of aonla (emblica officinalis) and soil quality in semi-arid tropics. indian j. agric. sci., 76: 457-461. masciandaro g., ceccanti b., garcia c., 1998 changes in soil biochemical and cracting properties induced by living much system. canadian j. soil sci., 77: 579-589. mitra d., guha j., chaudhuri s.k., 1971 studies in botany. vol, ii, moulik library, kolkata, india. neuhoff d., vlatschkov v., raigon d., 2011 comparison of the quality of conventionally and organically grown oranges in spain. proceedings of the third scientific conference of isofar, pp. 487-490. ranganna s., 2000 manual of analysis of fruits and vegetables products. tata mcgrow hill publishing co. ltd. 3rd ed. new delhi, india. sahoo s.k., singh d.b., 2005 effect of different level of biofertilizers on growth, yield and quality of strawberry (fragania ananassa duch) cv. sweet charley. the orissa j. hort., 33: 82-85. schnitizer m., 1991 soil organic matter in the next 75 years. soil sci., 151: 41-59. shukla a.k., sarolia d.k., bhavana k., kaushik r.a., mahawer l.n., bairwa h.l., 2009 evaluation of substrate dynamics for integrated nutrient management under high density planting of guava cv. sardar. indian j. hort., 66: 461-464. 13 received for publication 26 september 2014 accepted for publication 4 december 2014 1. introduction pollen incompatibility is a recognition mechanism that enables plants to prevent inbreeding. many flowering plants have these systems and prevent self-fertilization and subsequently prevent inbreeding depression (hiscock, 2002). pollen compatibility is an essential factor in breeding programs of fruit trees and for selection of the best pollenizers in orchard establishment for all fruit and nut species, including the european hazelnut (corylus avellana l.) (mehlenbacher, 1997). most hazelnut cultivars are self-incompatible; selfand cross-incompatibility in hazelnut cultivars are widespread phenomena which are of the sporophytic type (germain, 1994). dominance relations may lead to reciprocal differences in pollen incompatibility between cultivars, a result that makes pollinizer selection a complicated decision (hampson et al., 1993). cross-pollination is crucial for efficient nut set in hazelnuts. at least two different pollinizers are recommended for commercial yields to ensure sufficient amounts of viable, compatible pollen when needed, since flowers continue to emerge for several weeks (hampson, et al. 1993). therefore, in commercial orchards there should be several verified compatible cultivars to achieve appropriate nut production. to do so, it is suggested to have 6 to 15% of orchard trees as pollenizers (mehlenbacher and miller, 1988). several studies have been carried out to determine the level of selfand cross-compatibility in different hazelnut cultivars from different geographical regions (mehlenbacher and smith, 1991; mehlenbacher, 1997; erdogan and mehlenbacher, 2000; mehlenbacher and smith, 2006; vicol, et al., 2009; mehlenbacher, 2014). there is a serious need to examine selfand cross-compatibility of hazelnut cultivars in different climates and regions to determine the most compatible pollenizers for main hazelnut cultivars in the region. accurate information on this aspect could also enhance breeding efficiency and contribute to knowledge about hazelnut pollen-stigma incompatibility as well as helping to improve further efforts to study interspecific and intraspecific crosses (molnar, 2011). notwithstanding the importance of this requirement, there is little information on pollen compatibility of hazelnut cultivars grown in iran. the present investigation was undertaken as a preliminary study to determine the level of selfand cross-compatibility of three iranian and four imported hazelnut cultivars. 2. materials and methods the study was carried out in winter 2010 at the astara hazelnut research station in astara, province of guilan, iran. three iranian cultivars including shastak, pashmine, and tabestane, and four imported cultivars barcelona, daviana, segorbe, and merville (= merveille de bollwiller) were selected. three trees of each cultivar and some a preliminary study on pollen compatibility of some hazelnut cultivars in iran hosseinpour a. (1), seifi e. (1), javadi d. (2), ramezanpour s.s. (3) (1) department of horticulture, faculty of plant production, gorgan university of agricultural sciences and natural resources, gorgan, iran. (2) guilan research center of agriculture and natural resources, rasht, iran. (3) department of plant breeding and biotechnology, faculty of plant production, gorgan university of agricultural sciences and natural resources, gorgan, iran. key words: corylus avellana l., filbert, nut set, pollen incompatibility, pollination. abstract: pollen incompatibility is a major problem among hazelnut cultivars and can result in considerable crop loss in hazelnut orchards. thus, identifying the level of self-compatibility of a cultivar and compatibility between cultivars are crucial aspects to selecting the proper pollenizers. controlled selfand cross-pollinations were carried out in three local and four imported hazelnut cultivars. based on cluster set, partial self-compatibility was found in cultivars pashmine and shastak, and complete self-compatibility in ‘tabestane’. the best pollenizers for the cultivars pashmine, tabestane, shastak, barcelona, segorbe, daviana and merveille were segorbe, barcelona, segorbe, shastak, shastak, pashmine and, pashmine, respectively. adv. hort. sci., 2015 29(1): 13-16 14 adv. hort. sci., 2015 29(1): 13-16 shoots of the trees were selected: for each tree of iranian cultivars six shoots were selected (for self-pollination, open-pollination and pollination with four imported cultivars); and also for each tree of imported cultivars four shoots were selected (for open pollination and pollination with three iranian cultivars). the experimental design was completely randomized blocks with each tree considered as one block. based on the method described by mehlenbacher (1997), after removing the catkins, the selected shoots were isolated with long paper bags. the selected shoots for open-pollination were left unbagged to receive airborne pollen. at the same time pollination and pollen receptivity period of all cultivars were monitored and recorded. since the life of pollen and the receptivity of stigma are short and there is a gap between the periods of female and male flowers bloom, it is essential to know the duration of their activity for all cultivars in the region. pollen was collected in january, before the beginning of pollen shedding, and kept in vials at -18°c. controlled pollinations were performed by hand when styles appeared and became receptive. the number of pollinated pistillate inflorescences was recorded after pollination. produced nut clusters were picked and counted in early september. the percentage of nut cluster set was determined as the ratio of the produced nut clusters to the pollinated inflorescences. to evaluate the level of selfand cross-incompatibility, the index of self-incompatibility (isi) was applied (zapata and arroyo, 1978). isi is the ratio of nut set after selfor cross-pollination to nut set after open-pollination, as a potential compatible cross. when the ratio is ≤ 0.2, the cross is incompatible, 0.2-1 is partially compatible, and ≥ 1 is completely compatible. since this ratio was applied to evaluate both selfand cross-incompatibility, the index of pollen-incompatibility (ipi) was used instead of isi, as suggested by seifi et al. (2011). the obtained data were analyzed by analysis of variance (anova) using sas software version 9.1 for windows (sas institute, 2001). 3. results and discussion pollination and pollen receptivity of all cultivars are illustrated in figure 1. dichogamy was predominant in all cultivars. all seven cultivars were protanderous, which was expected according to the literature (germain, 1994). the most distinct dichogamy was obvious in the cultivar tabestane. although there was complete self-compatibility through hand pollination in this cultivar, its pollen will not touch its stigma when such dichogamy exists. however, it is noticeable that dichogamy is influenced by climatic conditions and may differ from year to year. the shortest and longest period of pollen release were recorded in ‘shastak’ (20 days) and ‘merville’ (35 days), respectively. further, the shortest and longest period of pollen receptivity by stigma of pistillate flowers were recorded in ‘shastak’ (10 days) and ‘tabestane’ (45 days), respectively (fig. 1). the activity of male inflorescence of ‘merville’ had an appropriate overlap with receptivity of the female inflorescence of all studied cultivars. pashmine ‘pashmine’ is a local iranian cultivar that is widespread throughout iran. little information is available about this cultivar’s characteristics. results showed that there was a significant difference between pollenizers in terms of nut set in ‘pashmine’. as can be seen in table 1, there was no significant difference between self-pollination and open-pollination in ‘pashmine’ which produced 39.4% nut set after self-pollination, indicating its partial self-compatibility. this result is roughly similar to nut set after self-pollination in the cultivar tombul (44%) (mehlenbacher and smith, 1991). this level of self-compatibility is noticeable in this species and is reported here for the first time in iran. the pollen of ‘barcelona’, ‘daviana’ and ‘merville’ was completely incompatible with ‘pashmine’ (ipi < 0.2) and produced no nuts. pollen of ‘segorbe’ was partially compatible with ‘pashmine’ (ipi = 0.21) (table 2). tabestane this cultivar has a small nut and kernel and is widespread throughout traditional orchards in iran. statistical analysis revealed a highly significant difference between treatments (pollenizers) (p < 0.01) (table 1). there was no significant difference between selfand open-pollination, and self-pollination resulted in 59.82% nut set, indcating complete compatibility (ipi = 1.22) (table 2). this is a rare and interesting phenomenon in hazelnut. this level of self-compatibility was higher than the two other local pollenizers ‘pashmine’ (39.4%) and ‘shastak’ (19.33%). nevertheless, this nut set percentage was lower than that after self-pollination in some genotypes reported by mehlenbacher and smith (2006). fig. 1 period of bud break and female and male bloom of 10 hazelnut cultivars. male bloom; female bloom. 15 hosseinpour et al., a preliminary study on pollen compatibility of some hazelnut cultivars in iran shastak the size of nut and kernel of this local cultivar is medium. according to analysis of variance, pollenizers had a significant difference in terms of nut set. the highest nut set was obtained through open pollination (63.14%). ‘shastak’ showed partial self-compatibility (ipi = 0.31) (table 2). ‘barcelona’, ‘daviana’ and ‘merville’ with ipi lower than 0.2 were considered as completely incompatible pollenizers (table 2). barcelona in the literature, the pollenizers ‘casina’, ‘daviana’ and ‘merville’ have been suggested for ‘barcelona’ (wilkinson, 2005). pollenizers for this cultivar were significantly different. open pollination resulted in 50% nut set which was statistically different from other pollenizers (p < 0.01) (table 1). pollen of ‘pashmine’ was completely incompatible with ‘barcelona’ (ipi = 0) and produced no nut and consequently is not suggested for ‘barcelona’. more research is needed to ensure this finding. segorbe this cultivar originated from france and has upright growth with medium to large nuts. its pollen grain is shed in early winter and pistillate flowers are observable in mid-winter (wilkinson, 2005). observations demonstrated significant differences between treatments. the highest nut set in ‘segorbe’ was obtained after open pollination (82.62%). this could be as a result of the effect of mixing various cultivars’ pollen grains from more than 40 genotypes at the station. based on calculated ipi, ‘tabestane’ (0.22) and ‘shastak’ (0.24) were partially compatible with ‘segorbe’; but ‘pashmine’ was incompatible (table 2). daviana ‘daviana’ originated from england and is an upright growing cultivar with few root suckers and medium and oblong nuts. its pollen grain is shed in mid-winter and pistillate flowers are observed in late winter. this cultivar is considered an appropriate pollenizer for ‘barcelona’ and ‘butler’ (wilkinson, 2005). based on analysis of variance, there was a significant difference between all pollenizers. the highest nut set resulted from open pollination (26.78%) which actually was not that much in such an orchard containing almost 40 different cultivars. ‘pashmine’ was partially compatible (ipi = 0.35); however, the amount of nut set was low (tables 1, 2). the pollenizers ‘tabestane’ and ‘shastak’ showed complete incompatibility. therefore, according to the preliminary findings, the three mentioned pollen sources are not suggested for ‘daviana’. table 1 cluster set in controlled selfand cross-pollinations of some hazelnut cultivars (%) main cultivar pollenizer pashmine tabestane shastak barcelona segorbe daviana merville pashmine 39.4±20.8 ab 0.00±0.00 c 2.38±2.38 b 9.48±1.97 a 25.16±3.41 b tabestane 59.82±15.61 a 17.26±11.28 b 18.35±6.33 b 1.59±1.59 b 18.38±6.62 b shastak 19.33±8.51 bc 17.76±5.88 b 19.44±15.47 b 1.19±1.19 b 20.73±1.49 b barcelona 8.4±4.55 bc 13.63±9.77 b 9.42±8.14 c segorbe 12.3±6.32 bc 10±3.71 b 30.47±4.38 b daviana 7.5±1.5 bc 6.72±4.15 b 5.84±2.95 c merville 5.61±1.98 c 5.75±3.02 b 2.3±1.15 c open 57.38±6.81 a 49.02±8.22 a 63.14±6.31 a 50±1.92 a 82.62±7.98 a 26.78±9.71 a 76.18±6.17 a mean±se. means within a column followed by the same letter are not significantly different (p<0.01). within the table, hyphen (-) means that the cluster set was not determined where it was not aimed. table 2 index of pollen incompatibility (ipi) in different crosses of hazelnut cultivars main cultivar pollenizer pashmine tabestane shastak barcelona segorbe daviana merville pashmine 0.69 0.00 0.03 0.35 0.33 tabestane 1.22 0.35 0.22 0.059 0.24 shastak 0.31 0.36 0.24 0.044 0.27 barcelona 0.15 0.28 0.15 segorbe 0.21 0.20 0.48 daviana 0.13 0.14 0.09 merville 0.0017 0.12 0.04 open within the table, hyphen (-) means that the cluster set was not determined where it was not aimed. 16 adv. hort. sci., 2015 29(1): 13-16 merville this cultivar is known as hall’s giant in the united states and australia and has been created from a seedling selection in germany in 1788 (usda, 2010). its pollen is shed in late winter, after other cultivars, for a short time. its pistillate flowers also appear in late winter (wilkinson, 2005). results showed that open pollination led to the highest nut set (76.18%) which was significantly different from the other pollenizers. ‘pashmine’, ‘tabestane’ and ‘shastak’ showed partial compatibility with ‘merville’, without any significant difference between them (table 1). 4. conclusions in conclusion, based on cluster set observations, partial self-compatibility was found in the cultivars pashmine and shastak, and interestingly complete selfcompatibility in tabestane. the best pollenizers for the cultivars pashmine, tabestane, shastak, barcelona, segorbe, daviana and merveille were segorbe, barcelona, segorbe, shastak, shastak, pashmine, and pashmine, respectively. since the observed compatibility in hazelnut cultivars may not be consistent over the years, complementary pollination studies should be carried out to verify the previous compatibility results and therefore document more reliable data. references erdogan v., mehlenbacher s.a., 2000 interspecific hybridization in hazelnut (corylus). j. amer. soc. hortic. sci., 125(4): 489-497. germain e., 1994 the reproduction of hazelnut (corylus avellana l.): a review. acta horticulturae, 351: 195-209. hampson c.r., azarenko a.n., soeldner a., 1993 pollen-stigma interactions following compatible and incompatible pollinations in hazelnut. j. amer. soc. hortic. sci., 118(6): 814-819. hiscock s.j., 2002 pollen recognition during the self-incompatibility response in plants. genome biol., 3(2): 1004.11004.6. mehlenbacher s.a., 1997 testing compatibility of hazelnut crosses using fluorescence microscopy. acta horticultural, 1052: 107-116. mehlenbacher s.a., 2014 incompatibility alleles of hazelnut cultivars. hortscience, 41(2): 482-483 mehlenbacher s.a., miller a.n., 1988 pollenizer management in a hazelnut orchard. proc. nut growers soc. oregon, washington and british columbia, 73: 68-81. mehlenbacher s.a., smith d.c., 1991 partial selfcompatibility in ‘tombul’ and ‘montebello’ hazelnuts. euphytica, 56(3): 231-236. mehlenbacher s.a., smith d.c., 2006 self-compatible seedlings of the cutleaf hazelnut. hortscience, 41(2): 482-483. molnar t.j., 2011 corylus l., pp. 15-48. in: kole c. (ed.) wild crop relatives: genomic and breeding resources of forest tree. springer-verlag, berlin, heidelberg, germany, pp. 166. sas institute, 2001 sas/stat user’s guide. sas institute inc., cary, nc, usa. seifi e., guerin j., kaiser b., sedgley m., 2011 sexual compatibility and floral biology of some olive cultivars. n.z.j. crop and hortic. sci., 39(2): 141-151. usda, 2010 national nutrient database for standard reference. release 23. united states department of agriculture, agricultural research service, usa. vicol a., botu i., botu m., giorgota a., 2009 preliminary study of incompatibility alleles expressed in pollen of romanian hazelnut cultivars. bulletin uasvm horticulture, 66(1): 480-483. wilkinson j., 2005 nut grower’s guide landlinks press, collingwood, vic, australia, pp. 240. zapata t.r., arroyo m.t.k., 1978 plant reproductive ecology of a secondary deciduous tropical forest in venezuela. biotropica, 10(3): 221-230. 125 1. introduction in the past century, the use of synthetic agro-chemicals has contributed to the exponential increase of food production, particularly in industrialized countries. however, it is more and more evident that safeguarding human health and the environment depends upon an appropriate use of synthetic chemicals in food and ecological chains (frangenberg, 2000). as a result, new and more restrictive regulations have become mandatory, significantly limiting the use of agro-chemicals. among fungicides, only a few are authorized in postharvest and the levels of residues are strongly restricted. in addition, european union (eu) regulation establishes for some crops (e.g. stone fruits) that postharvest treatments with conventional agro-chemicals is not allowed (european commission, 2008). furthermore, the control of postharvest diseases may be jeopardised by the outbreak of resistant biotypes of postharvest fungal pathogens to fungicides (holmes and eckert, 1999; ma et al., 2003). this is a real challenge for highly perishable crops such as stone fruits (peach, nectarine, plum and prune) where postharvest physio-pathological disorders can inflict severe economic losses. in recent years, alternative control of postharvest diseases of fresh commodities has become a chief field for research (gonzález-león and valenzuela, 2007; palou et al., 2008). among the investigated alternatives, having hardly any restriction, are some food additives, preservatives and generally recognized as safe (gras) compounds that provide an acceptable control of decay, although lower than synthetic fungicides (palou et al., 2009; molinu et al., 2010). the efficacy of these agents was shown to depend upon the commodity and the pathogen. indeed, carbonic acid salts, especially sodium carbonate and bicarbonate, proved to be fungistatic and to control in vivo penicillium digitatum sacc., the agent of citrus green mould, whereas p. italicum, the agent of citrus blue mould, was much lesser affected, especially with mandarin fruit (smilanick et al., 1999; palou et al., 2002). palou et al. (2009) evaluated, in a primary in vivo screening, the efficacy of several safe compounds against the major postharvest pathogens of stone fruits and found that sodium carbonate exerted an inhibitory activity on most pathogens. however, when applied at 20°c for 1 m in a small-scale in vivo trial, the efficacy and persistence resulted unacceptable, whereas when solutions were heated to 55 or 60°c an increased efficacy was attained, but it was not superior to that of the heated water alone. brief hot water treatment was employed by karabulut et al. (2010) to control monilinia fructicola on artificially inoculated californian plums and they observed that hot water applied at 60°c for 60 s completely inhibited the disease after five days of incubation at 20°c ad 90% rh. effect of short heat treatments with a sodium bicarbonate solution on storability of the yellow germoplasm plum ‘meloni’ m.g. molinu (*), a. dore, g. d’hallewin istituto di scienze delle produzioni alimentari (ispa), cnr, traversa la crucca, 3, loc. baldinca, 07040 sassari, italy. key words: decay control, gras compounds, penicillium expansum, quality. abstract: the behavior of a sardinian plum, cv. meloni, was investigated with regard to short-heat treatments at 20, 50, 55 or 60°c in water with 0 or 2% nahco3 (sbc) for 0, 15, 30, 45 or 60 seconds. fruits were stored for one month at 5°c and 95% rh followed by a simulated marketing period (smp) at 20°c and 80% rh for six days. quality and decay percentage were monitored. in addition, fruits were artificially inoculated with p. expansum and stored for 10 days at 25°c and 95% rh. compared to the control, all short-heat dip treatments lowered the degree of decay, and the efficacy was positively correlated with temperature and treatment duration. the use of sbc increased the efficacy of decay control and the best results were attained at 55 and 60°c. heat treatments increased levels of total flavonoids and antioxidant activity after smp. none of the heat treatments induced rind damage (browning or discoloration), but the overall appearance decreased significantly when fruit was treated at 55 or 60°c for 60 s after smp. scanning electron microscopy (sem) observations showed that treatments at 55 and 60°c with sbc cause damage and loss of cuticular wax on fruit surface. adv. hort. sci., 2015 29(2-3): 125-132 (*) corresponding author: mariagiovanna.molinu@ispa.cnr.it received for publication 26 september 2014 accepted for publication 10 june 2015 126 adv. hort. sci., 2015 29(2-3): 125-132 conducting a comparative study between local prune and international plum varieties, molinu et al. (2010) found that decay was significantly reduced on prunes but not plums when adding 2% sbc to water heated at 55°c. the results attained with plums, nectarines and peaches are in contrast with those attained with citrus fruits where a synergic interaction increased the efficacy of heated salt solutions. in citrus fruits, in addition to the direct fungistatic activity of carbonic acids, a clear induction of natural resistance contributed to increase decay control efficacy (venditti et al., 2005). this aspect has received slight attention in pome and stone fruits treated with gras compounds. in addition, limited data are available on the effect of postharvest treatments with gras compounds on the quality of prune fruit and no literature is available concerning changes in bioactive components during storage. in order to improve our understanding on the effect of postharvest treatments with heated gras solutions, a factorial design experiment was carried out employing 2% sbc with immersion duration and water temperature as quantitative variables. 2. materials and methods plant material and processing fruit. european plum or prune fruit (prunus domestica l. cv. meloni) was harvested at commercial maturity in an ex situ germplasm conservation orchard belonging to the ispa-cnr located in oristano (sardinia italy). after harvest, fruits were selected and randomized in order to obtain homogeneous sets. the fruit surface was disinfected by 2 min. immersion in a 1% (v/v) commercial sodium hypochlorite solution, followed by a deionised water rinse and finally air drying. treatment and storage. the sets of fruit were arranged in a 3-factorial randomized complete block design (rcbd) (2x5x4) with two treatments (with 0 or 2% sbc), five immersion times (0, 15, 30, 45 or 60 s) and four solution temperatures (20, 50, 55 or 60°c). each set was made up of three replicates of 30 fruits. after treatment, fruit was left to dry and then stored at 5°c and 95% rh for one month, followed by a six-day simulated marketing period (smp) at 20°c and 80% rh. inoculation treatments. artificial inoculation was performed with a wild isolate of p. expansum link, obtained from a decayed plum (‘stanley’) subjected to a two-month storage at 2°c. the inoculum was obtained from a 12-dayold sporulating culture by adding 5 ml of sterile water with 0.05% (v/v) tween 80 and gently scrubbing the agar. after filtering, the spore concentration was determined using a haemacytometer and concentration adjusted to 1x104 conidia/ml with sterile water. the artificial inoculation experiment was performed by wounding, once, 1200 fruits at the equatorial area with a sterile stainless rod (2 mm wide by 2 mm deep) and by introducing, 1 h later, 15 ml (~ 150 conidia) of the inoculum into half of the wounds (20 sets of 30 fruits), and into the remaining fruits (20 sets of 30 fruits) 15 ml of distilled water (controls). one hour later, fruit was treated according to the experimental plan. when dry, each set was placed into a plastic container, covered and stored at 25°c and 95% rh in the dark. the percentage of decay was monitored after 10 days and treatment efficacy was expressed as inhibition percentage compared to control. weight loss and appearance. mass change of prunes, expressed as % of weight loss compared to harvest weight, was determined on 30 fruits after storage and smp. visual quality was evaluated by employing a rating scale composed of five categories (9, excellent; 7, very good; 5, good, limit of marketability; 3 fair, limit of usability; 1, very poor, unusable) (chena and zhub, 2011). the extent of quality loss was described as an index, which was determined by summing the products of prunes in each category by the value of each category, and then dividing this sum by the total number of prunes assessed. chemical analysis ph, titratable acidity and total soluble solid content. all chemical analyses were performed on centrifuged and filtered juice obtained by homogenizing de-stoned prunes using a blender. ph, titrable acidity (ta) and total soluble solid content (tss) were carried out in three replicates of ten fruits each at harvest, after cold storage, and after smp. a 5 ml sample of juice was titrated with 0.1 n sodium hydroxide to an end point of ph 8.2 using a digital ph meter (orion, model 420a) and the acidity was calculated as % of malic acid content (g/100 ml of juice). soluble solids concentration was measured using an atago 0-32°brix temperature compensating refractometer (atago co., japan). after storage and smp fruit was rinsed in deionised water before analysis. antioxidant activity, total flavonoids and phenolics. the total antioxidant activity, total flavonoids and phenolics were measured at harvest, after storage, and smp in fruit immersed for 60 s in water at 20 and 55°c, with or without sbc. antioxidant activity (aa) was measured using two different spectrophotometer methods: abts and dpph assay according to surveswaran et al. (2007). for each assay, 0.1 ml of diluted juice (1:10 in water) was used, a calibrated standard curve with trolox (3-15 μm; r 2 =0.992 for dpph assay and r 2 =0.998 for abts assay) was made and results expressed as teac units (mmol trolox equivalents per 100 g of fruit). for both assays, absorbance was recorded with an agilent spectrophotometer (8453 uvvisible spectrophotometer, agilent technologies, palo alto, ca, usa). samples were analysed in triplicate. total flavonoids (tf) were determined according to the colorimetric assay described by kim et al. (2003). an aliquot of diluted juice was used for the assay and the tf in samples were quantified by catechin calibration curve (2.5-20 µg/ml, r 2 =0.999). the absorbance was measured at 510 nm with an agilent spectrophotometer (8453 uv127 molinu et al., short heat treatments on storability of the yellow germoplasm plum ‘meloni’ visible spectrophotometer, agilent technologies, palo alto, ca, usa). the results were expressed as mg of catechin equivalent (ce) per 100 g of fruit. analyses were performed in triplicate on each sample. total phenolics (tp) levels were measured in juice using the folin-ciocalteu assay (singleton and rossi, 1965). aliquots of diluited samples were mixed with folin-ciocalteu reagent (1:1) and 10 ml of 7.5% sodium carbonate in a 25-ml volumetric flask. reaction mixture was incubated for 120 min at room temperature and the absorbance measured at 750 nm with an agilent spectrophotometer (8453 uv-visible spectrophotometer, agilent technologies, palo alto, ca, usa). results were expressed as mg of gallic acid equivalents (gae ) per 100 g of fruit by means of a calibration curve of gallic acid (10-100 mg/l, r 2 = 0.989). samples were analysed in triplicate. scanning electron microscopy (sem) prunes subjected to the different treatments were used to study the effect of the treatment on the cuticle and epicuticular waxes by means of sem. observations were performed, on replica of the fruit rind, adapting the methodology of dore et al. (2010). replicas of the same marked area were made on dry fruit before and after the treatment (1 and 30 d post-treatment). samples were observed with a dsm 962 sem (zeiss, oberkochen, germany) at 20 kv. statistical analysis a one-way or three-way analysis of variance was applied to data using openstat (2007). data from disease incidence were transformed to the arcsine of the square root of the proportion of decayed fruit. when appropriate, means were separated by fisher’s protected least significant difference test with a significance level of p = 0.05. synergy testing of the combined treatments was performed according to plascencia-jatomea et al. (2003), calculating the expected and registered efficacy by limpel’s equation: e e = x + y (xy/100), where e e was the expected additive response to hot water treatment and bcs immersion, and x and y were the percentages of inhibition relative to each factor (heat or sbc) used alone. 3. results decays concerning the development of molds during the storage trial (table 1), over 90% of natural infections were caused mainly by p. expansum, while the remaining 10% consisted of m. fruticola, botrytis cinerea, and rhizopus stolonifer. it is possible to observe that salt, temperatures, and immersion times significantly influenced decay control. as a general rule, decay control was improved by increasing the immersion duration and by heating the sbc solution. after storage, the lowest degree of decayed fruit was attained in the sets dipped in the sbc solution at 55 or 60°c for 45 s and at 60°c for 60 s. as a result, compared to untreated fruit (28.5% decay) those treatments significantly reduced the natural infection to 4.1, 3.8 and 7.1%, respectively. after the smp decay augmented in all sets of fruit, however, the best control remained that attained by dipping the fruit for 45 s in the heated salt solution at 55 or 60°c (7 and 6.7% of rots) with about 80% reduction of decay compared to control (35.4% of rots). table 1 ‘meloni’ prunes with decay (%) after 1 month of storage at 5°c and 95% rh and 6 days at 20°c and 80% rh (smp) when treated with (+) or without (-) a 2% nahco 3 solution at 20, 50, 55 or 60°c for 15, 30, 45 or 60 s or left untreated (control) (z) temperature (°c) immersion duration (s) storage 15 15 30 30 45 45 60 60 control (-) (+) (-) (+) (-) (+) (-) (+) 28.5 aa 20 21.0 bb 20.8 bb 22.1 bb 19.1 bb 22.1 bb 18.1 bb 21.1 bb 23.4 bb 50 20.4 bb 19.1 bb 22.4 bb 19.4 bb 18.8 bb 11.4 cc 15.1 cb 13.4 cc 55 22.4 bb 18.1 bb 19.4 bb 17.1 bb 10.4 cc 4.1 dd 15.8 cb 12.4 cc 60 19.4 bb 16.1 bcc 15.4 cc 10.4 cd 7.4 cd 3.8 de 8.8 dc 7.1 dd smp 15 15 30 30 45 45 60 60 control (-) (+) (-) (+) (-) (+) (-) (+) 35.4 aa 20 26.7 bb 26.7 bb 26.0 bb 22.0 bc 26.0 bb 21.3 bc 25.0 bb 20.3 bc 50 26.3 bb 23.0 bb 25.3 bb 24.3 bb 22.7 bb 15.3 cc 20.0 db 16.3 cc 55 26.3 bb 22.0 bb 23.3 bb 23.0 bb 15.3 cc 7.0d d 19.7 db 15.3 cc 60 26.3 bb 19.0 bb 19.3 bb 15.3 cc 11.3 cc 6.7 dd 11.7 cc 10.0 cc (z) values are means with n=90 each time. capital letters relate to comparisons within rows, lower case letters to comparisons within columns, different letters indicate differences at p≤0.05 according to newman-keuls test. 128 adv. hort. sci., 2015 29(2-3): 125-132 inoculation treatments the in vivo experiment with artificially inoculated fruit (fig. 1) evidenced that the immersion for 15, 30 or 45 s in a 2% sbc solution at 20 or 50°c did not improve decay control compared to water dips. the same occurred when fruit was treated for 15 s at 55 or 60°c, indicating that with immersions up to 45 s no additive effects took place by heating the solution up to 50°c; we observed the same behavior at higher temperatures with 15 sec dips. it is interesting to note that synergistic interactions took place at 55°c for 30, 45, 60 s while no such interaction occurred at 60°c, except for the 30-s dip. the best result was obtained when fruits were immersed in sbc solutions at 55°c for 45 and 60 s with an increase of decay inhibition by 49 and 53% respectively compared to the heat treatment only (20 and 25% respectively). weight loss and appearance weight loss during storage and smp ranged between 2 and 4% for control fruit. the immersion of fruit at 20°c, with or without sbc, had no effect on weight loss, while it increased when fruit was immersed in heated water, reaching a maximum of 9.5% after smp with 60 °c for 60 s. a significant increase of weight loss took place when fruit was immersed in the sbc solution at 55 and 60°c for 45 or 60 s (8.7, 9.7, 11.3 and 13%, respectively). the visual quality of fruit in the storage experiment evidenced a harmful effect on fruit when immersed in water at 55 and 60°c for 60 s (visual quality score between 2 and 3). the reason for this result was related to fruit shrivelling, which was particularly evident after smp. the sbc solution was not the cause of shrivelling but contributed to increasing the impairment when employed at 55 or 60°c. indeed, at 50°c no shrivelling was observed, while at 55°c it appeared when fruit was immersed for more than 30 s, and it resulted slightly more visible with the sbc solution. ph, titratable acidity and total soluble solid content at harvest the ph, ta and tss (°brix) resulted 3.69, 0.69 and 19.2, respectively. during storage the values changed slightly in untreated fruit, while after smp the ph and tss increased (3.84 and 21.6 °brix, respectively) and ta decreased (0.56). with respect to untreated fruit (control), the different treatments did not affect the ph value, both during storage and smp. total acidity decreased during the smp except for a notable increase observed in fruit of the set treated at 55 and 60°c for 60 s. consistent increases of tss occurred during smp in fruit from sets immersed at 55°c for 45 or 60 s without sbc (24.2 and 23.1, respectively) or with sbc (22.3 and 23.7, respectively). on the other hand, fruit treated at 60°c, with or without sbc (20.2 and 20.1, respectively), had lower tss compared to untreated fruit (control) or that treated at 55°c. antioxidant activity, total flavonoids and phenolics figure 2 shows the data regarding total antioxidant activity measured by the dpph assay after storage and smp in fruit immersed for 60 s in water at 20 and 55°c, with or without sbc. with respect to harvest, the antioxidant activity decreased during storage and treatment temperature did not affect this trend nor did the sbc solution. compared to the end of storage, an increase of the aa occurred in all sets of fruit after the smp. the increase in heat treated fruit with or without sbc reached values higher than the aa at harvest. data relating to abts assay were well correlated with dpph (r2=0.70). fig. 1 p. expansum inhibition (%) in ‘meloni’ plum artificially inoculated 24 h before immersion at 15, 30, 45 or 60 s in water at 20, 50, 55, or 60°c with (sbc) or without (no sbc) 2% nahco 3 after 10 days at 25°c and 95% rh. mean decay control= 90.5%. 129 molinu et al., short heat treatments on storability of the yellow germoplasm plum ‘meloni’ the correlation between total polyphenols and total flavonoids resulted good (r2= 0.70) and showed a similar trend with the aa values, thus increasing during the smp and reporting higher values for heat treated fruit (data not shown). following smp the tf of fruit dipped at 55°c for 60 s, with or without sbc, resulted significantly higher (81.9 and 76.2 ce/100 g, respectively) compared to cold-water treated fruit (59.4 ce/100 g). heat treatment increased both the tf and the aa values, measured by dpph assay, attaining a high correlation (r2= 0.80) between them, while the tp and dpph values resulted less correlated (r2= 0.63). scanning electron microscopy (sem) sem observations of the fruit surface at harvest evidenced a constant layer of epicuticular wax with several wax plates (fig. 3, micrograph a). after immersion for 45 or 60 s in water, at 50 and 55°c, the epicuticular wax layer was melted (b), while when immersed under the same conditions in the 2% sbc solution, new wax with a different crystalline structure appeared on the surface (micrographs c-d). when the immersion was performed for 45 or 60 s at 60 °c the cuticle was damaged by evident cracks near the stomata (e). when those conditions were applied with the 2% sbc solution, an erosion of the epicuticular wax was observed in most samples (micrograph f). 4. discussion and conclusions the hot water treatments at 55 and 60°c with 2% of sbc were effective in controlling p. expansum decay during one month of storage at 5°c and the subsequent smp in both experiments. the most effective treatments remained those of dipping the fruit for 45 s at 55 or 60°c in sbc solution; this result confirms the findings reported by molinu et al. (2010) for other cvs of sardinian germplasm. karabulut et al. (2010) indicated 55 and 60°c as effective temperatures for decay control of “casselman” plums inoculed with m. fructicola 12-16 h before treatments. the authors observed a total rot control at 60°c for 1 min, while with ‘meloni’ prunes, inoculated 24 h pre-treatment, we obtained under the same conditions an inhibition percentage of about 53%, which rose to 55% with the sbc solution. also palou et al. (2009) reported a significant increase of treatment efficacy by heating the solutions to 55 or 60°c with several gras compounds in stone fruits, inoculated in vivo with seven major postharvest pathogens. the trends at 55 °c with sbc are similar to those attained with citrus fruit, but with a much lower efficacy (smilanick et al., 1999). this difference was also observed by palou et al. (2009) in stone fruits. the observed diversities between species is probably related to notable differences in fruit tissue texture, rind structure, cuticle thickness and chemical composition within the potential infection courts. indeed, in wounded citrus fruits, alkaline hydrolysis of pectins is promoted by carbonic acid salts along with an increased ph of the albedo and a remarkable induction of phytoalexin biosynthesis (dore et al., 2010). these additional factors affecting pathogenicity have not yet been investigated in stone fruits and may contribute to explaining the higher or lower efficacy according to species. concerning overall appearance, a significantly lower score was attributed to fruit immersed in water at 55 and fig. 2 total antioxidant activity in ‘meloni’ plum immersed for 60 s in water at 20 and 55°c, with (+) or without (-) bcs, expressed as teac units (mmol trolox equivalents per 100 g of fruit) by dpph assay, after 1 month of storage at 5°c and 95% rh and 6 days at 20°c and 80% rh (smp). 130 adv. hort. sci., 2015 29(2-3): 125-132 60°c for 60 s. this as the result of fruit shriveling, particularly evident after smp in heat treatments with the sbc solution. also fruit weight loss was affected by the heatsbc treatments and the same trend was reported for other crops even though to a lesser degree. in citrus fruit, the greater weight loss was attributed to an increase of fruit transpiration following alterations on the cuticular permeability (dore et al., 2010). peelimpairments, especially at the cuticle level near stomata, were evidenced by sem on ‘meloni’ fruit immersed in the sbc solution at 60°c for 45 and 60 s. as reported for fig. 3 micrographs a-f. scanning electron microscopy micrographs of ‘meloni’ prune epicuticular wax at harvest (a), following immersion at 55°c for 60 s in water (b) and in a 2% nahco 3 solution (c) with arrows indicating crystalline wax (c), and a magnification of the new wax layer (d). in micrograph ‘e’ damage to the cuticle and wax erosion (f) following immersion at 60°c for 60 s in the 2% sbc solution are evidenced (arrows). 131 molinu et al., short heat treatments on storability of the yellow germoplasm plum ‘meloni’ citrus fruits, it is likely that also for stone fruits harming of the cuticle produces the observed greater fruit weight loss and wilting. with regard to chemical analysis, the variations occuring during storage and smp in untreated and in 20°c treated fruit are consistent with literature. in fact, díazmula et al. (2009) reported a 40-45% decrease of total acidity detected in eight plum cultivars after 35 days of storage at 2°c and a significant increase of tss in almost all the cultivars studied. compared to treatments at 20°c, fruit dipped at 55°c had higher tss and ta contents. according to serrano et al. (2004) this trend could be related to a reduction of the respiration rate caused by the heat treatment. somewhat less clear is the significant drop of tss in fruit dipped at 60°c. a similar phenomenon was reported for citrus fruit when the treatment temperature reached the fruit-damage threshold, and this drop was attributed to fruit physiology connected to stress-induced metabolic responses (schirra and d’hallewin, 1997). with respect to harvest, aa, tp and tf decreased during cold storage, while an increase was observed after smp and, in heat treated fruit, values were higher compared to harvest. díaz-mula et al. (2009) monitored the bioactive compounds in several plum cultivars during cold storage and reported rising and dropping trends according to cv. and ripening stage at harvest. the increase of aa, tp and tf following heat treatments has been reported also for pomegranate and strawberry (mirdehghan et al., 2006; vicente et al., 2006), supporting our findings that heat affects nutraceutical properties in a positive way. the results attained with the heated sbc solution suggest that only heat and not sbc plays a role inducing ac, tp and tf. as far as we are aware, no literature is available on the changes of bioactive compounds and antioxidant activity during cold storage and subsequent smp of stone fruits according to treatment with sbc solution. from the results attained with the european plum ‘meloni’ it appears clearly that decay is furthermore reduced, compared to water at 55°c, when 2% of sbc is added and immersion is performed for 45 or 60 s. in addition, as for citrus fruits, we observed that sbc induces the production of crystalline wax and that treatments at 60°c induce cuticular damage and loss of epicuticular wax from the fruit surface. these effects on the cuticle and epicuticular waxes can increase fruit transpiration, as demonstrated by the greater weight loss and shrivelling. after smp, postharvest immersion of ‘meloni’ prunes in hot water resulted in an increase of bioactive compounds and sbc did not contribute to this increase. thus, heating a sbc solution seems not to affect nutraceutical properties but from our results it significantly improves the efficacy of this gras compound in containing p. expansum infection during storage. it is also important to evidence that a synergic anti-penicillium effect took place by employing the heated sbc solution. epicuticular damage highlighted by sem undermines or hide the effects on fruit quality and probably further research is needed in order to establish the most appropriate concentration of the salt aimed at reducing the epicuticular damage and at clarifing the salt influence on the nutraceutical properties of fruit. acknowledgements research granted by miur “caratterizzazione di composti con proprietà nutraceutiche in cultivar di susino (prunus domestica) del germoplasma autoctono della sardegna” (cisia) “conoscenze integrate per la sostenibilità e l’innovazione del made in italy agroalimentare” legge 191/2009. the authors wish to thank mrs gavina serra and mr salvatore marceddu for assistance in the laboratory and sem observations, respectively. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references chena z., zhub c., 2011 combined effects of aqueous chlorine dioxide and ultrasonic treatments on postharvest storage quality of plum fruit (prunus salicina l.). postharvest biol. tech., 61: 117-123. díaz-mula h.m., zapata p.j., guillén f., martínezromero d., castillo s., errano m., valero d., 2009 changes in hydrophilic and lipophilic antioxidant activity and related bioactive compounds during postharvest storage of yellow and purple plum cultivar. postharvest biol. tech., 51: 354-363. dore a., molinu m.g., venditti t., d’hallewin g., 2010 sodium bicarbonate induces crystalline wax generation, activates host-resistance and increases imazalil level in rind wounds of oranges, improving the control of green mould during storage. j. agric. food chem., 58: 72977304. european commission, 2008 food safety from the farm to the fork. chemical safety. food additives. available at: http:// ec.europa.eu/food/chemicalsafety/additives/index-en.htm. frangenberg a., 2000 integrated crop management as fundamental basis for sustainable production. pflanzenschutz-nachrichten bayer, 53: 131-153. gonzález-león a., valenzuela a.i., 2007 the postharvest use of synthetic fungicides, implications on human health, pp. 1-20. in: troncoso-rojas r., m.e. tiznado-hernández, and a. gonzález-león (eds.) recent advances in alternative postharvest technologies to control fungal diseases in fruits and vegetables. transworld research network, trivandrum, kerala, india. holmes g.j., eckert, j.w., 1999 sensitivity of penicillium digitatum and p. italicum to postharvest citrus fungicides in california. phytopathology, 89: 716-721. karabulut o.a., smilanick j.l., crisosto c.h., palou l., 2010 control of brown rot of stone fruits by 131 1. introduction despite considerable advances in technology, agriculture is exposed to climate changes in all parts of the world. among climatic factors, rainfall is the most critical because 70% of the main areas under cultivation are still without irrigation (wilhite, 2001). water shortage is the main characteristic of agriculture in mediterranean regions, inducing water stress during spring and summer. fruit trees survive in this situation because they are prone to physiological or morphological changes which enable them to avoid drying damage, cast it back, or tolerate it (torrecillas et al., 1999). drought tolerance is observed with different rates in almost all plant species. understanding plant responses to the external environment is an essential component for selection stress tolerance (reddy et al., 2004). in recent years, in southern iran, low and poor distribution of rainfall has caused great damage to plants. water stress, particularly in rain-fed fig production areas (e.g. estahban) has become a big issue. in the estahban region not only the annual production has decreased, but also the highly productive trees are in danger of destruction. one strategy to confront this problem is the identification and selection of more tolerant genotypes. traditional breeding approaches for selection of tolerant plants are time consuming and complex processes. imposing water stress in field-grown crops is difficult because of the unpredictability of rainfall and the possibility of seepage from adjoining plots and drought escape of deep roots from osmotic stress. since field evaluation of drought effects is highly correlated with environmental conditions, in vitro screening techniques allow for a better control of culture conditions. tissue culture also offers the possibility of screening many plants in limited space and time, assuming that there is a correlation between cellular, tissue, organ and in vivo plant responses (mohammad et al., 2000). polyethylene glycol (peg) has been used to simulate water stress in plants. peg of high molecular weight is a non-ionic osmoticum lowering the water potential of nutrient solution without being taken up or being phytotoxic (hassan et al., 2004). it has been shown that the shoot length decreases with increasing water stress via in vitro culture in cherry (sivritepe et al., 2008) and mulberry (tewary et al., 2000). in a study on mulberry, leaf relative water content (rwc) decreased with increasing water stress (ramanjulu et al., 1998). accumulation of proline is the most common plant response to decreasing water potential (helal ragab and samir moustafa, 2008). it has been reported that with increasing peg in date palm seedling culture, the rate of proline was increased in tolerant cultivar (djibril et al., 2005). carbohydrates have an important role in osmotic regulation in different plant parts (masoudi-sadaghiani et al., 2011). simple sugars such as glucose and fructose were increased and the rate of sucrose and starch decreased under water stress (sharp and davies, 1979; munns and weir, 1981; wang and stutte, 1992; nawar and ezz, 1993; clifin vitro drought effects on morphological and physiological indices of two fig (ficus carica l.) cultivars a. shekafandeh(*), s. hojati department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: drought, growth parameters, polyethylene glycol, proline. abstract: in vitro responses of two fig cultivars, ‘sabz’ and ‘siah’, were evaluated in ms media containing four levels of polyethylene glycol (peg) (0, 2, 4, 6%) as a simulation of water stress. the results showed that in sabz cultivar, shoot length, shoot fresh and dry weights were 43, 36 and 25%, respectively, lower than control in drought treatments caused by 4% peg, while the leaf area and specific leaf area were not significantly (p>0.05) affected. in siah cultivar, shoot length, fresh and dry weights were 57, 58 and 40%, respectively, lower in stressed media in comparison to control. in contrast to sabz cultivar, leaf area and specific leaf area of siah cultivar were significantly reduced by addition of 6% peg (56 and 21.5%, respectively). naturally, the amount of proline in ‘sabz’ was higher than in ‘siah’ (81.8 μmole g-1 versus 16.7 μmole g-1). however, in both cultivars, with addition of peg in culture media, leaf proline content was increased, in comparison to control. with increasing peg% in culture media, the amount of leaf soluble sugars content increased and the amount of starch decreased. the result show that ‘siah’ is more sensitive to drought than ‘sabz’ and that in vitro culture can be used to evaluate drought tolerance of cultivars. adv. hort. sci., 2012 26(3-4): 131-137 (*) corresponding author: shekafan@shirazu.ac.ir. received for publication 26 april 2012 accepted for publication 12 september 2012 132 ford et al., 1998; perez-perez et al., 2007; mafakheri et al., 2010). in iran, fars province with about 36,000 hectares under fig cultivation is the primary fig production area. estahban district with 20,000 hectares under fig cultivation is the center of fig cultivation in fars province, as well as in iran. as mentioned above, due to drought in recent years, highly productive trees are in danger of destruction. the cultivars sabz and siah are the most desirable cultivars in iran; there has not been report on water stress tolerance of these cultivars. selection of more drought tolerant cultivars can be used to establish of new orchards and a promising rootstock for other interested cultivars. the aim of this investigation was to evaluate the effects of drought created by polyethylene glycol (peg) on morpho-physiological changes of two important fig cultivars (‘sabz’ and ‘siah’) and leading to selection of more drought tolerant varieties using in vitro culture. 2. materials and methods plant material and explant decontamination in this research, the in vitro-derived micro-shoots of two cultivars of ficus carica l. ‘sabz’ and ‘siah’ were used. the branches (20~30 cm long) were cut from rain-fed, mature mother trees established in estanban fig research station located at 29° 07’ n latitude and 54° 02’ e , 197 km southeast of shiraz. freshly grown stems with 2~6 nodes were cut from the above branches and washed in a solution of water-detergent (three drops of detergent in 100 ml of water) for 30 min, then placed in a solution of benomyl (3%) for 1 h (torres, 1998). to prevent the secretion of phenolic compounds into the culture media, explants were treated in a solution of 2% ascorbic and citric acid for 45 min. at this point, the stems were transferred to a laminar air flow cabinet. they were first dipped in 70% (v/v) ethanol for 45 s, then treated with 15% (v/v) chlorox solution (a household bleach containing 5.25% sodium hypochlorite) for 15 min and rinsed three times with sterilized distilled water. nodal segments of 1~1.5 cm long were cut and used as explants. the basal medium was ms (murashige and skoog, 1962), containing 3% sucrose (merck, lgaa 64271darmstadt, germany) and 1mg l-1 (4.4 µm) benzyl adenine (ba) and solidified by 0.8% agar-agar (merck, lgaa 64271darmstadt, germany). the ph was adjusted to 5.7±0.05 prior to autoclaving at 1.2 atm pressure and 121˚c for 20 min. cultures were maintained in a growth chamber at 25±2˚c, 58% relative humidity and a photon flux density of 40 μmol m-2 s-1 was provided by white fluorescent tubes, with a 16 h photoperiod. peg treatments after four weeks, the micro-shoots (2 cm length) from the above cultures were transferred into 500 ml culture vessels containing 100 ml ms basal media supplemented with different levels of peg (0, 2, 4 and 6%) as selective agent and maintained under the environmental conditions described above. growth measurements seven weeks after the beginning of experiments, shoot length and shoot fresh and dry weight were evaluated. leaf area was measured by leaf area meter (deltatdevices england) and specific leaf area was calculated as follows: specific leaf area = leaf area (cm2)/leaf dry weight (g) relative water content leaf relative water content (rwc) was estimated according to the method described by whetherley (1950). twenty healthy leaf discs of 0.7 cm diameter were cut from plants, using a leaf punch and washed three times with double distilled water. leaf discs were weighed (fw), then placed into a 10 ml conical flask, immersed in 10 ml distilled water for 4 h in dark. turgid weight (tw) of leaf discs were then measured and samples were dried in an oven (80ºc) until constant weight (dw) was achieved. rwc was calculated from the following equation: relative water content % = fw dw/tw dw x 100 chlorophyll content for chlorophyll determination, prior to extraction fresh leaf samples were washed with deionized water to remove any surface contamination. one g leaf samples were ground in 80% acetone using a pestle and mortar. the mixture was centrifuged at 4800 rpm for 20 min. the optical density of the supernant was measured at 663 and 645 nm wavelengths and chlorophyll content was calculated using the following equation: mg chl g fw = [20.2 (od645 nm) + 8.02 (od663 nm)] x v/fw x 1000 where v is final volume of solution (ml) and fw, leaf fresh weight (mg). proline contents to determine proline content of the leaves, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous of sulfosalcylic acid and the homogenate filtered through whatman # 2 filter paper. two ml of filterate was reacted with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a test tube for 1 h at 100°c and the reaction terminated in an ice bath. the reaction mixture was extracted with 4 ml toluene, mixed for 15~20 seconds. chromophore containing toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm. the proline concentration was determined based on standard curve and calculated as follows: μ moles proline/g fw = (μg proline/ml x ml toluene) / g sample/5 115.5 μg / μmole electrolyte leakage electrolyte leakage was measured as an assessment of cell wall permeability. electrolyte leakage was measured using an electrical conductivity meter. two mature leaves per plant were taken and cut into 1 cm segments. after three washes to remove surface contamination, leaf samples were placed in individual vials containing 10 ml of 133 distilled water. the samples were incubated at room temperature on a shaker (100 rpm) for 24 h. electrical conductivity (ec) of bathing solution (ec1) was read after incubation. samples were then placed in an autoclave at 120°c for 20 min and the second reading (ec2) was determined after cooling the solution to room temperature. the electrolyte leakage was calculated as ec1/ec2 and expressed as percent. soluble sugars content soluble sugars were extracted from 0.1 g fresh leaves by heating with 5 ml of 80% ethanol in a water bath at 70°c for 30 min. the insoluble residue was removed by centrifuging at 5000 g for 10 min. one ml of the resulting extract was mixed with 1 ml of 5% phenol solution and 5 ml of sulfuric acid. the mixed solution was permitted to cool to room temperature, then vortexed. the absorbance was read at 490 nm using a spectrophotometer. the soluble sugar content of each sample was determined using standard curve for glucose and expressed as mg glucose g-1 fw (mccready et al., 1950; dubois et al., 1956). starch content the solid residue remaining in the centrifuge tube after removal of all soluble sugars in the previous section was washed, re-extracted and re-centrifuged four times using 80% (v/v) ethanol. starch content in the samples was determined colorimetrically using anthrone method (mccready et al., 1950). the absorbance was determined at 630 nm in a digital spectrophotometer (spectronic 20 d+; spectronic instruments inc., new york, usa.) as described by lópez et al. (2002). statistical analysis the experiment was arranged as a 2×4 factorial experiment in completely randomized design (crd) with 10 replicates, each consisting of three plants. thus, there were 30 plants in each treatment and a total of 240 plants in the experiment. data were subjected to analysis of variance using the spss software (ver. 13.0) spss inc. mean differences were determined by duncan’s multiple range tests at p≤0.05. 3. results and discussion water stress caused a reduction in micro-shoot growth of the two fig cultivars (table 1). in ‘sabz’, shoot fresh and dry weights and shoot length were 36, 25, and 43% lower, respectively, in drought treatments caused by 4% peg compared with the control, while in ‘siah’, these traits were 58, 40 and 57% lower respectively, in the same stressed media (4% peg), than the control. in this latter cultivar, in contrast to ‘sabz’, leaf area and specific leaf area were significantly affected by water deficit (56 and 21.5%, respectively) (table 2). the findings of this study showed that the growth rate of 'siah' in optimum conditions (control) was the same as sabz cultivar, but with increasing intensity of water stress, the growth reduction rate in ‘siah’ was more than in ‘sabz’. the main effect of cultivars showed that without respect to different levels of peg, ‘sabz’ had a significantly higher leaf area (8.2 cm) than ‘siah’ (5.51) (table 2). water stress significantly reduced vegetative growth indices of fig micro-shoots. this phenomenon has been previously reported (oukabli et al., 2008). the long-term use of peg in vitro on growth reduction and shoot regeneration in other plants has been well documented (bressan et al., 1982; handa et al., 1982; handa et al., 1983; dami and hughes, 1995; al-khayri and al-bahrany, 2004). in most cases, peg has been used to stimulate table 1 interaction of water stress and cultivar on micro-shoot length, fresh and dry weight drought stress (peg%)  0 2 4 6 cultivar average fresh weight (g) mean siah 3.80 a (z) 2.1 bc 1.6 c (58) 2.10 bc 2.4 a sabz 3.00 ab 2.3 bc 1.9 bc (36) 2.10 bc 2.3 a mean 3.41 a 2.2 b 1.8 b 2.08 b  average dry weight (g) siah 0.50 a 0.30 bc 0.3 bc (40) 0.30 bc 0.34 a sabz 0.40 ab 0.40 ab 0.3 bc (25) 0.30 bc 0.36 a mean 0.46 a 0.34 b 0.3 b 0.31 b shoot length (cm) siah 2.60 a (z) 1.4 b 1.1 b (57) 1.20 b 1.57 a sabz 2.33 a 1.4 b 1.3 b (43) 1.30 b 1.59 a mean 2.47 a 1.4 b 1.2 b 1.25 b (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. table 2 interaction of water stress and cultivar on leaf area (cm2) and specific leaf area (g cm-2) of micro shoots drought stress (peg%) 0 2 4 6 cultivar leaf area (cm2) mean siah 8.2 a (z) 6.9 ab 3.4 b 3.6 b (56) 5.51 b sabz 12.4 a 8.2 a 6.9 ab 6.6 ab 8.52 a mean 10.0 a 7.6 ab 5.4 b 5.4 b specific leaf area (g cm-2) siah 14.4 a 14.6 a 13.4 a 11.3 b (21.5) 13.4 a sabz 16.8 a 15.3 a 15.1 a 13.1 a 15.1 a mean 15.6 a 14.9 a 14.4 a 12.2 a (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. 134 water stress in plants. peg with high molecular weight is a non-penetrating inert osmoticum which lowers the water potential of nutrient solutions without being taken up or being phytotoxic (hassan et al., 2004). it has been shown that in vitro growth reduction of apple (molassiotis et al., 2006) and cherry (sivritepe et al., 2008) was due to the decrease in water potential created by peg. under water stress conditions, the reduction of mineral absorption has resulted in limited leaf growth and development and decreased plant water transpiration. therefore, producing smaller leaves can be the first plant defense mechanism against water deficit. hsiao (1973) reported that a decrease in leaf area results in lower light absorption and photosynthetic capacity; thereby reducing photosynthetate and plant growth. this can be true with plants in vivo, but probably this is less important in vitro, because in vitro plants are more heterotrophic, so decreases in their growth cannot be due to a deficiency of carbohydrates. in the present work it was clearly shown that the decrease in water availability is the main cause of decreasing growth. specific leaf area (sla) is a function of leaf area. the reduction of sla in water deficit conditions is due to the fact that leaf area development is more affected than deposition of dry matter (blum and pneul, 1990). in both cultivars with increasing peg% in culture media, leaf relative water content (rwc) decreased and an increase in ion leakage was recorded (table 3, fig. 1). these findings are in agreement with the results obtained by other researchers in vitro (sawwan et al., 2000; al-khayri and al-bahrany, 2004; chai et al., 2005). leaf yellowing and chlorosis were the consequence of structural damage to cell membranes in explants. interestingly, in 6% peg, although the rate of relative water content reduction in leaves of sabz cultivar (50%) was more than ‘siah’ (20%), the difference in their ion leakage was not significant. leaves accumulated significant quantities of proline and, in contrast, the amount of chlorophyll was decreased with increasing peg percentage in culture media (fig. 2). table 3 interaction of water stress and cultivar on leaf relative water content (%) and leaf ion leakage (%) drought stress (peg%) leaf relative water content (%) cultivar 0 2 4 6 mean siah 69.2 a (z) 68.6 a 59.3 ab 54.9 ab (20) 63.0 a sabz 73.6 a 51.4 ab 61.7 a 36.7 b (50) 55.8 a mean  71.4 a 60.6 ab 58.8 ab 45.7 b leaf ion leakage (%) siah 50.6 d 57.1 bcd 55.5 bcd 64.2 abc 56.9 b sabz 54.1 cd 57.0 bcd 68.7 a 66.2 a 61.5 a mean 52.3 c 57.0 bc 62.1 ab 65.2 a (z) in each row and column, means with similar letters are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. fig. 1 changes in leaf water content (rwc) and ion leakage in two fig cultivars: ‘sabz’ (a) and ‘siah’ (b). fig. 2 changes in proline and total chlorophyll in two fig cultivars: sabz (a) and siah (b). 135 naturally, the amount of proline in ‘sabz’ was higher than in ‘siah’ (81.8 µmole g-1 versus 16.7 µmole g-1) (table 4). in both cultivars, with increasing peg % in media, proline content increased. however, the results indicated that, in all levels of peg treatments, the amount of proline in ‘sabz’ was higher than in ‘siah’. plants produce and accumulate such compounds compatible to their metabolism to overcome adverse effects of drought stress (zhu, 2001). accumulation of such substances would cause more negative water potential in plants, a necessary condition to absorb and keep the water in plant tissues (sivritepe et al., 2008). table 4 interaction of water stress and cultivars on leaf proline content (µm g-1 fresh weight) drought stress (peg%) cultivar 0 2 4 6 mean siah 16.8 c (z) 50.5 b 73.7 b 121.0 b 65.5 b sabz 81.8 b 320.7 a 293.6 a 289.5 a 246.4 a mean 42.8 b 170.6 a 199.4 a 205.2 a (z) in each row and column, means with similar letters are not significantly different using duncan’s multiple rang test p≥0.05 with increasing the severity of water stress, proline and soluble sugars (as compatible substances) accumulated significantly in explant leaves of both cultivars. the accumulation of these substances has been well documented in field (clifford et al., 1998; zamani et al., 2002; mafakheri et al., 2010) and in vitro conditions (handa et al., 1982; brito et al., 2002; al-khayri and al-bahrany, 2004; molassiotis et al., 2006; sivritepe et al., 2008). a notable point in our results was the significant increase in proline accumulation in leaves of both cultivars. it has been reported that there is a direct positive relationship between drought tolerance and proline concentration in plant tissues (al-khayri and albahrany, 2004; sivritepe et al., 2008). the role of proline in destroying reactive oxygen species (ros) in water stress conditions has been reported in different plant species (turkan et al., 2005; verslues et al., 2006). therefore, it can be expected that plants which accumulate more proline under water stress are more tolerant. thus, it may be concluded that ‘sabz’ is more tolerant than ‘siah’, although in some plants a relationship was not found between proline accumulation and drought tolerance. the changes in leaf sugar and starch contents under different levels of peg are shown in table 5 and figure 3. in both cultivars, with increasing peg%, the amount of soluble sugar content increased and the amount of starch deceased. in drought conditions, soluble sugar can act as osmoticum and also osmoprotectant. in addition, accumulation of sugar may partly protect protein against the oxidative damage created by free radicals (bohnert and shen, 1999). in the present work, soluble sugars content in ‘sabz’ was greater than in ‘siah’, but it did not have significant effect on water absorption. accumulating soluble sugars can be due to starch hydrolysis, which is in agreement with the results obtained by shawky et al. (1997). taylor et al. (1982) also reported that carbohydrates (reducing and non-reducing sugars) are the most abundant component in osmotic adjustment in tomato seedlings. table 5 interaction of water stress and cultivar on leaf tss and starch (mg g-1 dry weight) drought stress (peg%) 0 2 4 6 cultivar tss (mg g-1 dry weight) mean siah 170.3 c 168.9 c 256.6 bc 287.0 b 220.69 b sabz 276.4 bc 344.5 b 357.5 b 522.2 a 375.2 a mean  215.8 c 256.7 bc 311.6 b 404.6 a starch (mg g-1 dry weight) siah 133.5 ab 103.8 bc 73.7 bc 65.3c 94.1 a sabz 167.0 a 124.2 abc 76.3 bc 80.3 bc 111.9 a mean  152.56 a 112.83 b 75.4 b 73.9 b (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05 in this study, with increasing peg in culture media, the amount of total chlorophyll declined in leaves of the two fig cultivars. effect of drought stress on reduction of chlorophyll content of other plants in vitro has previously fig. 3 changes in leaf sugar and starch content in two fig cultivars: ‘sabz’ (a) and ‘siah’ (b). 136 been reported (hernández-sebastià et al., 2000; brito et al., 2002; molassiotis et al., 2006). under field conditions, also chlorophyll content was reduced with increasing drought (munne-bosch and penuelas, 2004). in the present experiment, with increasing water deficit (with addition of peg), chlorophyll content was decreased along with accumulation of leaf proline (fig. 3). this may be due to the fact that chlorophyll and proline are synthesized through glutamate pathway, causing an increase in synthesis of proline under drought conditions, which resulted in reduction of chlorophyll synthesis (aspinall and paleg, 1981). 4. conclusions the assessment of drought tolerance of two fig cultivars showed that under water stress conditions in both cultivars, leaf fresh and dry weight, total chlorophyll and starch contents decreased, but proline and soluble sugar 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(gupta et al., 2010), enabling clean and high-efficiency combustion with reduced emission of no x , so x , and particulate matter (semelsberger et al., 2006). furthermore, dme can be efficiently reformed to h 2 at low temperatures, and most importantly, dme does not have large issues with toxicity, production, infrastructure, or transportation as is the case with various other fuels (semelsberger et al., 2006). apart from its use as a fuel or a precursor to other organic chemicals, dme has attracted the attention of scientists and engineers since it behaves as a subcritical solvent. for instance, performance of subcritical dme as an effective media for the extraction of medicinal, flavoring, and pungent agents from some spices (ginger, black pepper, and chili powder) has been demonstrated (catchpole et al., 2003). in addition to the subcritical approach, dme can be used as a low-temperature solvent or extraction agent, and thus, it is applicable to laboratory procedures for the extraction of organic molecules from bio-materials (kanda and makino, 2010; kanda and li, 2011). recently, an industrial use of liquefied dme was reported for rapid removal of water from sub-bituminous coal without any heating process (kanda and makino, 2010). this approach encourages us to testify the performance of liquefied dme in de-watering of biomaterials including watery horticultural crops. one may believe that the usefulness of dme as a solvent is limited by its low boiling point (-23°c). however, this property could be highly beneficial as it may facilitate the removal of the solvent from the extracted samples. in biochemical exercises at ambient temperature, a variety of conventional solvents such as alcohols, acetone, hexane, etc. are used for extraction of organic substances of interest including flavoring agents and pigments from various biological samples. however, extraction of some reactive or highly degradable chemicals requires handling at use of liquefied cold temperature dimethyl ether for extraction of pigments from fresh vegetable tissues a. noriyasu(1), h. furukawa(1), a. kikuchi(1), h. takaichi(1), f. bouteau(2), x. li(1), s. nishihama(1), k. yoshizuka(1), t. kawano(1,3)* 1 faculty and graduate school of environmental engineering, the university of kitakyushu, itakyushu, japan. 2 université paris diderot, sorbonne paris cité, institut des energies de demain (umr8236), paris, france. 3 université paris diderot, sorbonne paris cité, paris 7 interdisciplinary energy research institute (pieri), paris, france. key words: carotenoids, carrot, chlorophyll, dme, japanese squash, spinach. abstract: dimethyl ether (dme) is known as a useful precursor to other organic compounds and is a promising alternative fuel without issues of toxicity, production, infrastructure, and transportation as is the case with various other fuels. recently, dme has attracted the attention of scientists and engineers since it behaves as a subcritical solvent or a lowtemperature solvent applicable for the extraction of organic molecules from bio-materials. this paper presents the extraction of chlorophylls and carotenoids from green peel and yellow cortex of japanese squash, spinach leaves and carrot roots using low-temperature liquefied dme. spectroscopic and fluorescence analyses of the extracted pigments revealed that chlorophylls were successfully extracted by liquefied dme from green materials (squash peel and spinach leaves). hplc analysis further confirmed that chlorophylls extracted include both chlorophylls a and b. by using liquefied dme, carotenoids were extracted from all vegetable samples examined. the performance of dme as a novel pigment extracting agent is confirmed in this work and its use as a “green” solvent, as opposed to conventional solvents, for the preparation and extraction of various plant pigments is highly encouraged from an environmental point of view. adv. hort. sci., 2015 29(1): 48-52 * corresponding author: kawanotom@kitakyu-u.ac.jp received for publication 9 january 2015 accepted for publication 24 march 2015 49 noriyasu et al., dimethyl ether for extraction of pigments from fresh vegetable tissues low temperature. due to the nature of dme in liquefied form at ambient pressure (or relatively low pressure) at low temperature, it may allow us to perform extraction of pigments at low temperature, preferably below 0ºc. in the present study, we examine the application of liquefied dme for the extraction of model plant pigments such as chlorophylls and carotenoids from fresh vegetable tissues by passing the low-temperature solvent through the layer of homogenates of the tissues. 2. materials and methods plant materials the pericarp (fruits) of japanese squash (cucurbita moschata, cv. ebisu, cultivated in hokkaido, japan), leaves of spinach (spinacia oleracea l., cv. hunter, cultivated in fukuoka prefecture, japan), and roots of carrot (daucus carota l., cv. koyo-2, cultivated in hokkaido, japan) were obtained from a local market and used for the extraction of pigments. squash pericarp was dissected into green peel (1 mm from the surface) and yellow tissue while spinach leaves and carrot roots were used without such separation. following homogenization of the vegetable tissues in a mixer, the obtained pastes (1-2 g fresh weight, g fw) were layered in the sample holder in the extraction chamber. chemicals reagents for separation and analysis of pigments such as hplc grade acetone and methanol (wako pure chemical industries, ltd., osaka, japan), and ammonium acetate (sigma-aldrich japan, tokyo, japan) were obtained through local vendors. apparatus for cryo-liquefaction of dme and extraction of pigments the dme gas cylinder was obtained from a local gas vendor (air-gases kitakyushu inc.) and supply of gaseous dme was made through 0.5 mpa gauge. the newly-designed apparatus consisted of (1) dme gas cylinder connected to a flow meter, (2) cooling unit, (3) extraction chamber, and (4) liquid trapping port (fig. 1a). each of the units was connected with aluminum capillaries. within the extraction chamber, there was a disk-shaped sample holder for vegetable paste layering. since the extraction chamber of acryl resin was transparent, the amount of cryo-liquefied dme could be monitored and manually controlled (fig. 1b, c). treatment of plant materials with liquefied dme for extraction of model pigments from the homogenized vegetable tissues, the apparatus shown in figure 1 was used. dme gas, supplied directly from the gas cylinder at 0.4 mpa, was rapidly cryo-liquefied in the cooling unit. by handling (opening and closing) of a stopcock between the cooling unit and extraction chamber, collection and release of liquid dme precipitated in the cooling chamber was manually controlled. after loading ca. 15 ml liquid dme into the extraction chamber, the vegetable sample was allowed to have contact with liquefied dme only for a few seconds. the mixture of passed dme and accompanying liquid was collected in a test tube by opening the stopcock between the extraction chamber and the collection unit (fig. 1d, e). in turn, dry matter was left in the extraction chamber. following passive dme removal under ambient pressure, mixtures of vegetable sap (water) and vegetable-derived oily materials containing pigments were recovered. spectroscopic analysis pigments recovered after extraction with dme were diluted with acetone and used for spectroscopic scanning and fluorescence analyses using a spectrophotometer (u3310, hitachi high-technologies, tokyo, japan) and a fluorescence spectrophotometer (f-4500, hitachi hightechnologies, tokyo, japan), respectively. for fluorometric analysis of pigments, a three-dimensional (3d) contour plot was performed as described elsewhere (kawano et al., 1999). the fluorescence contour profile was obtained with an excitation range between 250 nm and 700 nm (slit size, 5 nm; sampling interval, 5 nm) and an emission range between 300 nm and 750 nm (slit size, 5 nm; sampling interval, 5 nm), at scanning speed of 30000 nm/min. recovery of chlorophyll a in the extracts was spectroscopically quantified according to the formula proposed by wellburn (1994) as follows: [chlorophyll a] (µg/ml) = 12.25 x a 663.2 nm 2.79 x a 646.8 nm . similarly, concentration of total carotenoids in the extracts was spectroscopically quantified using the following formula proposed by the national agriculture and food research organization of japan: [total carotenoids] (mg/l) = 4.143 x a 475 nm 0.561. fig. 1 apparatus for pigment extraction with liquefied dme. (a) composition of the system. arrows indicate the positions of two stopcocks designed for fine controls of gaseous and liquefied dme flows. (b, c) extraction chamber. (d, e) trapping port with a plastic tube. 50 adv. hort. sci., 2015 29(1): 48-52 high-performance liquid chromatography (hplc) aliquots of the extracts containing pigments were analyzed by the reverse-phase hplc system according to maeda et al. (1998). hplc (waters 2690 separations module, waters, usa) equipped with inertsil ods-80a column (gl science, tokyo, japan) and two types of detectors, namely, a detector with photodiode arrays (water alliance pda system, waters, usa) and a fluorescence detector (waters 474 scanning fluorescence detector, waters, usa), for the separation and detection of pigments. analysis of chlorophylls was carried out using the solvent system which consisted of a (1:4 mixture of 1 m ammonium acetate and 80% methanol) and b (1:4 mixture of acetone and methanol). the program started with a linear gradient from a to b (15 min), continued with an isocratic run with b (5 min), and returned to a (2 min), and continued isocratically for 8 min at a flow rate of 1 ml/ min. peaks corresponding to pigments were detected with a fluorescence detector (excitation at 405 nm, emission at 660 nm). analysis of carotenoids was carried out using the solvent system consisting of 9:1 mixture of methanol and ethanol at flow rate of 1 ml/min. peaks corresponding to pigments, chiefly carotenoids, were detected by monitoring absorbance at 455 nm using a detector with pda. 3. results and discussion preparation of samples and pigment extraction fresh tissues of japanese squash (green peel and yellow cortex), spinach (green leaves) and carrot (roots) were homogenized and the resultant pastes were prepared for extraction with liquid dme (fig. 2a-k). after passing liquid dme through the plant sample layer (homogenates) packed in the column of the extraction chamber, a dry powder resembling acetone powder was left in the apparatus (fig 2l-o). in turn, liquid samples mostly the mixtures consisted of extracted water (due to de-watering action of dme), oil (due to solvent action of dme), and the carrier liquid dme, were collected in the tubes placed beneath the extraction apparatus. as dme can be readily evaporated out under conditions of ambient pressure and temperature, solvent removal from the collected liquid was passively allowed by leaving the tubes with liquid samples at ambient temperature for at least 30 min. as a consequence, dense colored aqueous samples remained in the tubes (fig. 2p-s) which were a mixture of oil and water. this indicated that both water and oils were extracted from the plant samples by liquid dme. note that pigments were separately extracted from the green peel (fig. 2a, d, h, l, p) and yellow cortex (pericarp; fig. 2a, e, i, m, q) of japanese squash. the above extraction processes were repeated three times. the total yield of oil/water mixture extracted with dme flow largely depends on the water content in the vegetable samples; the highest yield was found with carrot roots, the most watery sample among those tested (fig. 3). since a drastic change in color after dmebased extraction was observed in squash peel (fig. 2h, l) and carrot root tissues (fig. 2k, o), we assumed that extraction of pigments was successfully carried out. in fig. 2 fresh tissues, homogenized paste, dry powder, and pigment mixture of vegetables. peel and cortex of japanese squash (a, d, e, h, i, l, m, p, q), leaves of spinach (b, f, j, n, r) and carrot roots (c, g, k, o, s) were used for extraction of pigments using dme. whole vegetables (a-c), fresh tissues prior to homogenization (d-e), paste of homogenates (h-k), dry powder remaining after extraction with dme (l-o), and pigment mixtures obtained after evaporation of dme (p-s) are shown. fig. 3 yield of oil/water mixture extracted from vegetables using dme as solvent. mixture of oil and water containing pigments from vegetables are compared. 51 noriyasu et al., dimethyl ether for extraction of pigments from fresh vegetable tissues contrast, pigment extraction from sticky pastes of yellow squash pericarp and spinach leaves required further modifications (fig. 2i, j, m, n). quantitative and qualitative analysis of extracted pigments spectroscopic analyses of the pigments extracted from vegetable samples were performed (fig. 4 left) and the resultant data was used for quantification (fig. 5). carotenoids were observed in all samples. as expected, the presence of chlorophylls was detected only in the samples derived from green tissues. the chlorophyll content in squash peel was found to be higher than that in spinach leaves (fig. 5 top), possibly due to the loss of chlorophyll during extraction from the leafy sample as previously suggested (fig. 2n). the total carotenoid contents in vegetable extracts were also compared and revealed that the green peel of squash and carrot roots had the highest values (fig. 5 bottom). the presence of chlorophylls (chiefly chlorophyll a) in the extracts from squash peel and spinach leaves was further confirmed by fluorescence spectroscopy (fig. 4 right) and hplc (fig. 6). while it was difficult to dissect the spectroscopic and fluorescent signals for chlorophyll a from those of concomitantly present chlorophyll b, hplc chromatograms showed clearly separated peaks of both chlorophylls a and b in the extracts from squash peel and spinach leaves (fig. 6). with the aid of hplc, the presence of both αand β-carotenes were also confirmed in carrot roots while the other three samples showed the presence of β-carotene only (fig. 7). 4. conclusions in the present study, the extraction of chlorophylls and carotenoids from green peel and yellow cortex of japanese squash, spinach leaves and carrot roots using low-temperature liquefied dme has been demonstrated. spectroscopic and fluorescence analysis of the extracted pigments revealed that chlorophylls were successfully extracted by dme from green materials (squash peel and spinach leaves). hplc analysis further confirmed that the extracted chlorophylls included both chlorophyll a and b. carotenoids were shown to be extracted by dme from all vegetable samples examined as confirmed by spectroscopic and hplc analyses. the performance of dme as a novel pigment extracting agent is confirmed in this work fig. 4 spectroscopic and fluorescent profiles of dme-extracted pigments from vegetable samples. comparison of absorption spectra (left) and fluorescence spectral contour (right) recorded in extracts from squash peel (top), yellow squash cortex (second line from the top), spinach leaves (third line from the top), and carrot roots (bottom). prior to assays, extracts (oil/water mixtures) obtained after evaporation of dme were dissolved in acetone. fig. 5 quantification of pigments extracted from vegetable samples. chlorophyll contents (top) and total carotenoid contents (bottom) in the extracts from green squash peel, yellow squash cortex, spinach leaves, and carrot roots are compared. bars, s. d. (n = 3). 52 adv. hort. sci., 2015 29(1): 48-52 and its use as a “green” solvent, as opposed to conventional solvents, for the preparation and extraction of various plant pigments is highly encouraged from an environmental point of view. acknowledgements this work was supported by a grant of regional innovation strategy support program implemented by ministry of education, culture, sports, science and technology (mext), japan. references catchpole o.j., grey j.b., perry n.b., burgess e.j., redmond w.a., porter n.g., 2003 extraction of chili, black pepper, and ginger with near-critical co 2 , propane, and dimethyl ether: analysis of the extracts by quantitative nuclear magnetic resonance. j. agric. food chem., 51: 4853-4860. gupta k.k., rehman a., sarviya r.m., 2010 bio-fuels for the gas turbine: a review. renew. sustain. ener. rev., 14: 2946-2955. kanda h., li p., 2011 simple extraction method of green crude from natural blue-green microalgae by dimethyl ether. fuel, 90: 1264-1266. kanda h., makino h., 2010 energy-efficient coal dewatering using liquefied dimethyl ether. fuel, 89: 2104-2109. kawano t., adachi m., kurata h., azuma r., shimokawa k., 1999 calcium-dependent catabolism of phaeophorbide a in tomato fruit. j. japan. soc. hort. sci., 68: 810-816. maeda y., kurata h., adachi m., shimokawa k., 1998 chlorophyll catabolism in ethylene-treated citrus unshiu fruits. j. japan. soc. hort. sci., 67: 497-502. semelsberger t.a., borup r.l., greene h.l., 2006 dimethyl ether (dme) as an alternative fuel. j. power sources, 156: 497-511. wellburn a.r., 1994 the spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. j. plant. phyiol., 144: 307-313. zhu w., li x., kaneko h., fujimoto k., 2007 lpg synthesis from dme with semi-direct method. stud. surface sci. catal., 167: 355-360. zhu w., li x., kaneko h., fujimoto k., 2008 selective transformation of dimethyl ether into small molecular hydrocarbons over large-pore beta zeolite. catal. lett., 120: 95-99. fig. 7 detection of αand β-carotenes with hplc. hplc chromatograms show the peaks of carotenoids extracted with dme from squash peel, squash pericarp, spinach leaves and carrot roots. pigments eluted from inertsil ods-80a column were detected by monitoring the changes in absorption at 455 nm. fig. 6 detection of chlorophylls with hplc. hplc chromatograms showing the peaks of chlorophylls a and b extracted from squash peel and spinach leaves using dme. pigments eluted from inertsil ods-80a column were detected by intrinsic fluorescence intensity (excitation, 405 nm; emission, 660 nm). impaginato 53 1. introduction calla and peruvian lily are commonly cultivated in tuscany (italy) and they represent one of the main cut flower productions. calla lily [zantedeschia aethiopica (l.) spreng] is known to be susceptible to at least 13 virus species, mainly belonging to potyviridae, bunyaviridae, and tombusviridae (huang et al., 2007). peruvian lily (alstroemeria spp.) has become one of the most popular cut flowers worldwide. it has been reported as the natural host of various plant viruses, including members of potyviridae, betaflexiviridae or bunyaviridae (park et al., 2010). in tuscany, virus surveys were carried out on various woody plants such as grapevine (rizzo et al., 2012; 2015 a) but to our knowledge no reports are available for calla and peruvian lily in italy. in order to evaluate the health status of these plants in tuscan nurseries, various viruses were assayed, belonging to the following families: betaflexiviridae [alstroemeria carla virus (alcv), lily symptomless virus (lsv)], bromoviridae [cucumber mosaic virus (cmv)], bunyaviridae [impatiens necrotic spot virus (insv), iris yellow spot virus (iysv), tomato spotted wilt virus (tswv)], comoviridae [arabis mosaic virus (armv), broad bean wilt virus 1 (bbwv-1), broad bean wilt virus 2 (bbwv-2)], potyviridae [alstroemeria mosaic virus (almv), bean yellow mosaic virus (bymv), dasheen mosaic virus (dsmv), konjac mosaic virus (komv), lily mottle virus (lmov), turnip mosaic virus (tumv), zantedeschia mosaic virus (zamv), zantedeschia mild mosaic virus (zammv)], tombusviridae [carnation mottle virus (carnmv)] and unassigned [tobacco rattle virus (trv)]. an additional aim of this survey was to evidence new viral records in italy for these widespread cultivations, due to the intense international exchanges that characterize the commercialization of lily. 2. materials and methods tests were carried out on 90 z. aethiopica plants and 48 alstroemeria spp. plants collected from 12 tuscan nurseries in two years. plants showed sympadv. hort. sci., 2016 30(1): 53-56 doi: 10.13128/ahs-18702 occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy a. luvisi 1,2 (*), d. rizzo 3, l. stefani 3, a. panattoni 2, a. materazzi 2 1 dipartimento di scienze e tecnologie biologiche ed ambientali, centro ecotekne università del salento, via provinciale lecce monteroni, 73100 lecce, italy. 2 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. 3 servizio fitosanitario regionale, regione toscana, via ciliegiole, 99, 51100 pistoia, italy. key words: alstroemeria, rt-pcr, zantedeschia. abstract: in order to evaluate the health status of calla and peruvian lily in tuscan nurseries, 18 viruses belonging to six families and one unassigned virus were assayed. tests were carried out on 90 zantedeschia aethiopica plants and 48 alstroemeria spp. plants collected from 12 tuscan nurseries in two years, via rt-pcr tests. z. aethiopica was mainly affected by viruses belonging to the potyviridae family, with the main infection caused by dasheen mosaic virus (dsmv) and zantedeschia mild mosaic virus (zammv). even if alstroemeria spp. plants were affected by potyviridae family viruses too, higher infection rates were recorded for betaflexiviridae, where lily symptomless virus infected more than half of plants. this is the first known report of lily mottle virus (lmov) in alstroemeria spp. and z. aethiopica or zammv in alstroemeria spp. in italy. (*) corresponding author: andrea.luvisi@unisalento.it received for publication 24 november 2015 accepted for publication 22 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 53-56 54 toms such as foliar chlorosis, yellow spot and stripes. total rna was extracted from foliar tissue (2 g) using rneasy plant mini kit (qiagen, netherlands) protocol, modified according to mackenzie et al. (1997). tissues (2 g) were ground using a tissue lyser (qiagen) adding 5 ml of grinding buffer (4.0 m guanidine isothiocyanate, 0.2 m sodium acetate ph 5.0, 25 mm edta, 2.5% pvp-40 and 2.0% sodium bisulfate) just before use. the homogenate (1 ml) was transferred to a 1.5 ml tube and 100 μl of 20% sarkosyl were added. after 2 min centrifugation, 600 μl were transferred to a qiashredder spin column (qiagen) placed in a 2 ml collection tube. the subsequent steps of rna extraction were according to the manufacturer’s protocol. the extracted rna was then retro-transcribed into cdna using the iscript cdna synthesis kit (biorad, usa). for each sample, 2 µl of cdna were amplified in a total volume of 20 μl containing 1x hotmaster buffer, 0.5 μg/μl bsa and 1 u of hotmaster taq dna polymerase (eppendorf, germany). primers and rt-pcr parameters were chosen following the protocols reported in table 1. 18s rrna was used as internal control (osman and rowhani, 2006). finally, 10 μl of the amplification mix was electrophoresed in a 1.5% agarose gel in tae buffer [40 mm tris base, 20 mm sodium acetate, 1 mm edta ph (8.0)]. the amplified cdna fragments were visualized on a uv transilluminator. data were analyzed using sigma-plot software (version 11; systat software, san jose, ca). the software was used to perform analysis of variance (anova). data expressed in percent were converted to arcsin values. p <0.05 was considered to be significant. 3. results the health status of calla and peruvian lily as determined by the present survey is reported in table 2. z. aethiopica was mainly affected by viruses belonging to the potyviridae family. more than two plants out of three were infected by dsmv and 50% table 1 list of references for primers and rt-pcr conditions target rt-pcr assay comoviridae armv faggioli et al., 2005 bbwv-1 ferrer et al., 2008 bbwv-2 ferrer et al., 2008 betaflexiviridae alcv spence et al., 2000 lsv lim et al., 2009 bromoviridae cmv faggioli et al., 2005 bunyaviridae iysv kritzman et al., 2000 insv liu et al., 2009 tswv mumford et al., 1994 potyviridae almv spence et al., 2000 bymv ganesh selvaraj et al., 2009 dsmv wen-chi et al., 2010 komv wen-chi et al., 2010 lmov lim et al., 2009 tumv wen-chi et al., 2010 zamv kwon et al., 2003 zammv wen-chi et al., 2010 tombusviridae carnmv cevik et al., 2010 unassigned trv wei et al., 2009 table 2 health status of calla lily (zantedeschia aethiopica) and peruvian lily (alstroemeria spp.) expressed as percentage of infected plants target zantedeschia aethiopica alstroemeria spp. comoviridae armv bbwv-1 bbwv-2 betaflexiviridae alcv lsv 56.3 a bromoviridae cmv 12.5 b bunyaviridae iysv insv 3.3 c * tswv 3.3 c potyviridae almv dsmv 66.7 a 13.5 b komv lmov 16.7 b 12.5 b tumv zammv 50.0 a 6.3 c tombusviridae carnmv unassigned trv * values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). luvisi et al. occurrence of viruses in calla and peruvian lily in tuscan nurseries 55 of tested plants were infected by zammv. lower infection rates were reported for viruses belonging to bunyaviridae. even if alstroemeria spp. plants were affected by potyviridae viruses too, higher infection rates were recorded for betaflexiviridae, where lsv infected more than half of plants. further infections were caused by bromoviridae viruses. comoviridae, tombusviridae or trv infections were not found in both plants. in z. aethiopica, mixed infections were set at 40% for dsmv/zammv, 6.7% for dsmv/lmov/zammv and 3.3% for dsmv/lmov(data not shown). in alstroemeria spp., mixed infections were set at 6.3% for lsv/zammv, lsv/lmov, dsmv/zammv, cmv/lsv or cmv/dsmv/lsv. with regard to alstroemeria spp., the sequence obtained from a zammv amplicon (genbank accession no. kf156666 (table 3) had 99% nucleotide identity with the corresponding fragment of a reference zammv isolate (genbank accession no. ay626825). further isolates of lmov were detected (genbank accessions no. kf156667, kf156668, kf156669). each further isolate had 99% nucleotide identity with the corresponding fragment of a reference zammv isolate. all four isolates are different but had 99% nucleotide identity. the sequence obtained from lmov amplicon (genbank accession no. kf156662) (table 3) had 94% nucleotide identity with the corresponding fragment of a reference lmov isolate (genbank accession no. jn703466). the same lmov amplicon was obtained from a z. aethiopica sample as well. further isolates of lmov were detected in both species with 100% nucleotide identity with kf156662 (genbank accessions no. kf156663, kf156664, kf156665). 4. conclusions various viral infections, mainly due to viruses belonging to two families, betaflexiviridae and potyviridae, seem to affect calla and peruvian lily in tuscan nurseries. most detected viruses are frequently reported for both plants and mixed infection of virus belonging to potyviridae are quite common in z. aethiopica (huang et al., 2007). however, some evidence is rarer. to our knowledge this is the first report of lmov in alstroemeria spp. and z. aethiopica or zammv in alstroemeria spp. in italy, while zammv was recently identified in taiwan (huang and chang, 2005) and in italy (rizzo et al., 2015 b). this report puts in evidence how widespread the virus is within these common plants and the need for constant monitoring of the health status for flower production. even if some of viruses that affect calla and peruvian lily may be eradicated by thermotherapy (panattoni et al., 2013) and heat treatment may help in bromoviridae control (luvisi et al., 2015), prevention represents the preferred method of virus control. references cevik b., bakir t., koca g., 2010 first report of carnation mottle virus in turkey. plant pathol., 59: 394. faggioli f., ferretti l., albanese g., sciarroni r., pasquini g., lumia v., barba m., 2005 distribution of olive tree viruses in italy as revealed by one-step rtpcr. j. plant pathol., 87: 49-55. table 3 sequence of isolates of zantedeschia mild mosaic virus and lily mottle virus isolate detected in italy zantedeschia mild mosaic virus isolate 2079 polyprotein gene, partial cds (genbank: kf156666) tcattgagtaccaaccccaacagtccgatctgtttaatactcgcgcgtcacaaacccaattcaataattggtatgatgcgatcaaaaatgagtatggggttgatgatagtcagatgcagagaatcatgaatggcttcatggtgtggtgtctcgagaatgggacatcaccaaacataaatggcgtgtgggttatgatggatggggatgaacaagtagaatttccactaaaaccaatggtggagaatgccaagcctacgctgcgtcaaataatgcaccacttttcagacgcagccgaggcttacattgaacttaggaatgccgctgccccatatatgcctagatatgggttgctgcggaacttaagagacagaggtctagcacgcttcgcattcgacttctatgaagtcacttcaaagacaccagatcgtgctagagaagctgtagcgcagatgaaggcagcagcgctaaacaatgtttccacaaggatgtttggattggatggaaatattgcaactgccacggagaacactgaaaggcacactgctaaggatgtaagtccgagcatgcactcgctactcgggatctcagccttgcagtaaaggagctggaaacagcccacagttattgtcttgcgatagggtttaaatagccgtactattgtcgttgctagatgttgcagtgtgggcctcccaccttaaggtttatcagtgtggctttccacctagttccttacattgcgcatagtatgtgt lily mottle virus isolate 2409 coat protein gene, partial cds (genbank: kf156662.1) tgctggggcctctagctccacacaaacgagtcgcccaacacgtccagagattgccgcggtcgatgtagcaccacaacagagctctgaggctagagtgcgtgatcgtgatgttgatgctggcaccgtgggaacataccaaatccctcgactgaaagcactggcaacaaagattaacgtacccaaggtcaaggggcgaacaatagtgaacactgggcaccttgtgaactacaacccagaccaaacagatatttcaaatacaaggtcaacccagaagcaatttgagacctggcataacgctgtgaaagatgagtatggtctcaacgacgagagtatggctctcgcaatgaatggtctgatggtttggtgcatagagaatggcacctcaccaaacataaatggcgtgtggctcatgatggacggagatcagcaagttgaatttcctttacgtcctatacttgaacacgcaaaaccgacgctgcgccaaattatggcgcatttctcaaacctcgctgaagcttatattgagaagcaaaatttggagaaaccgtacatgcctaggtacggccttcagcgaaatctcaccgatttcaatctagcacgatttgcttttgatttctatga adv. hort. sci., 2016 30(1): 53-56 56 ferrer r., escriu f., luis-arteaga m., guerri j., moreno p., rubio l., 2008 new molecular methods for identification of broad bean wilt virus 1. mol. cell. probes, 22: 223-227. ganesh selvaraj d., pokorny r., holkova l., 2009 comparative analysis of elisa, one step rt-pcr and icrt-pcr for the detection of bean yellow mosaic virus in gladiolus. commun. agric. appl. biol. sci., 74: 853859. huang c.h., chang y.c., 2005 identification and molecular characterization of zantedeschia mild mosaic virus, a new calla-lilly-infecting potyvirus. arch. virol., 150: 1221-1230. huang c.h., hu w.c., yang t.c., chang y.c., 2007 zantedeschia mild mosaic virus, a new widespread virus in calla lily, detected by elisa, dot-blot hybridization and ic-rt-pcr. plant pathol., 56: 183-189. kritzman a., beckelman h., alexandrov s., cohen j., lampel m., zeidan m., raccah b., gera a., 2000 lisianthus leaf necrosis: a new disease of lisianthus caused by iris yellow spot virus. plan dis., 84: 11851189. kwon s.b., ha j.h., yoon j.y., ryu k.h., 2003 zantedeschia mosaic virus causing leaf mosaic symptom in calla lily is a new potyvirus. arch. virol., 147: 2281-2289. lim h.j., bae e.h., lee y.j., park s.h., lee k.j., kim s.r., 2009 detection of lily symptomless virus, lily mottle virus, and cucumber mosaic virus from lilium grown in korea by rt-pcr. korean journal of microbiology, 45: 251-256. liu h.y., sears j.l., mou b., 2009 spinach (spinacia oleracea) is a new natural host of impatiens necrotic spot virus in california. plant dis., 93: 673. luvisi a., panattoni a., materazzi a., 2015 heat treatments for sustainable control of soil viruses. agron. sustain. dev., 35: 657-666. mackenzie d.j., mclean m.a., mukerji s., green m., 1997 improved rna extraction from woody plants for the detection of viral pathogens by reverse transcriptionpolymerase chain reaction. plant dis., 81: 222-226. mumford r.a., barker i., wood k.r., 1994 the detection of tomato spotted wilt virus using the polymerase chain reaction. j. virol. methods, 46: 303-311. osman f., rowhani a., 2006 application of a spotting sample preparation technique for the detection of pathogens in woody plants by rt-pcr and real-time pcr (taqman). j. virol. methods, 133: 130-136. panattoni a., luvisi a., triolo e., 2013 elimination of viruses in plants: twenty years of progress. span. j agric. res., 11: 173-188. park t.h., han i.s., kim j.b., 2010 review on the development of virus resistant plants in alstroemeria. j. plant biotechnol., 37: 370-378. rizzo d., materazzi a., stefani l., farina p., vanarelli s., panattoni a., luvisi a., 2015 a distribution of regulated viruses in cv. sangiovese vineyards in tuscany. j. plant pathol., 97: 131-135. rizzo d., panattoni a., stefani l., paoli m., nesi b., lazzereschi s., vanarelli s., farina p., della bartola m., materazzi a., luvisi a., 2015 b first report of zantedeschia mild mosaic virus on zantedeschia aethiopica (l) spreng in italy. j. plant pathol., 92: 399. rizzo d., stefani l., paoli m., triolo e., panattoni a., luvisi a., 2012 the sustainability of old grapevine mother plants in relation to new mandatory diagnostic tests for virus control. adv. hort. sci., 26(3-4): 148150. spence n.j., mills p.r., barbara d.j., 2000 a surbey of viruses of alstroemeria in the uk and the characterization of carlaviruses infecting alstroemeria. eur. j. plant pathol., 106: 843-847. wei t., lu g., clover g.r.g., 2009 a multiplex rt-pcr for the detection of potato yellow vein virus, tobacco rattle virus and tomato infectious chlorosis virus in potato with a plant internal amplification control. plant pathol., 58: 203-209. wen-chi h., chin-hsing h., shu-chuan l., chun-i w., ya-chun c., 2010 detection of four calla potyviruses by multiplex rt-pcr using nad5 mrna as an internal control. eur. j. plant pathol., 126: 43-52. 171 1. introduction in agriculture, seeds are considered as starting points of plant growth. assuming seeds are non-dormant, germination is the key component of seedling emergence (forcella et al., 2000). germination may be defined as the process associated with the initiation and completion of embryo emergence; it refers to the progress of a seed from imbibition through radicle emergence (gniazdowska et al., 2010). germination parameters are useful for estimating the conversion of seeds to seedlings and, thus, the suitability of a seed lot for commercial seedling production. germination parameters are also useful in determining the type of seed pre-treatment as well as nursery management practices needed to attain a high level of germination (kolotelo et al., 2001). abscisic acid (aba) induces dormancy during maturation, and gibberellins (gas) play a key role in dormancy release and in the promotion of germination (kaur et al., 2006). in many cases, endogenous levels of these hormones (ga, aba) are associated with the ability of the seeds to germinate (seo et al., 2006); seed dormancy must be broken to induce germination. in morphophysiological dormancy, seeds must be exposed to cold, heat, gibberellic acid, or chemical materials for dormancy breaking (hilton, 1984; dehghanpour et al., 2011). the use of potassium nitrate has been an important seed treatment in seed-testing laboratories for many years without a good explanation for its action mechanism (çetinbas and koyuncu, 2006). hydrogen cyanamide (hcn , dormex®) has been identified as one of the most effective dormancybreaking agents in many deciduous plant species (sillercepeda et al., 1992) and has been exploited mainly as an artificial rest-breaking agent to stimulate budbreak in mild winter climates (ben mohamed et al., 2012). however, the emission of hcn has been detected during the pre-germination period of many seeds (gniazdowska et al,. 2010). pistachio (pistacia vera l.), a member of anacardiaceae family (khan et al., 1999), is a dioecious species and for this reason it has acquired large genetic diversity (behboodi, 2002). seed dormancy, oily nature of cotyledons leading to early spoilage, risk of heterozygosis, and production of male/female plantlets are major problems associated with pistachio sexual propagation (jangali baygi, 2012). therefore, under natural conditions, an erratic and irregular germination over a quite long period. on the other hand, pistachio plants cannot be readily propagated from cuttings taken from mature trees and are currently propagated by grafting on seedling rootstocks (behboodi, 2002). furtherdormancy removal in pistachio nut: influences of hydrogen cyanamid (dormex®) as compared to ordinary seed chemical pre-treatments m. jangali baygi 1, m. alizadeh 1 (*), f. ghaderifar 2, m. sharifani 1 1 horticulture department, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2 agronomy department, gorgan university of agricultural sciences and natural resources, gorgan, iran. key words: dormancy, germination, gibberellin, kno 3 , hydrogen cyanamid, pistachio. abstract: seed germination is considered an important phenological stage of plant growth during which the viable, nondormant embryos develop into seedlings under favorable conditions. currently, various seed priming treatments may be used to enhance germination values (germination percentage, lag phase, time to reach 50% germination) and breaking dormancy; gibberellins and kno3 are applied as common treatments in this regard. in the present study, efficacy of hydrogen cyanamid (hcn, dormex®) on seed germination of two cultivated pistachio varieties (abasali, shahpasand) were studied and the results were compared to ordinary ingredients, namely gibberellic acid (ga3) and kno3. the extraction of parameters was carried out using four-parameter hill function. the results reveal that hcn may significantly improve critical parameters involved in pistachio seed germination. in shahpasand variety, seeds treated with hcn germinated prior to other treatments (150.53 hours after treatment) and achieved 50% germination earlier than other treatments (199.00 hours only). according to the results, utilization of hcn in seed testing experiments may be encouraged. adv. hort. sci., 2015 29(4): 171-175 (*) corresponding author: mahdializadeh@gau.ac.ir received for publication 19 may 2015 accepted for publication 23 june 2015 172 adv. hort. sci., 2015 29(4): 171-175 more breeding programs and development of hybrid seedlings/superior rootstocks require a rapid and reliable method for seed germination (ghazaeian et al., 2012). hence, in the present study, effects of hcn on dormancy removal and seed germination of two pistachio varieties were studied and the results were compared to widespread chemical treatments such as ga 3 and kno 3 as well as cold stratification. 2. materials and methods the present study was undertaken in the physiology laboratory at the horticulture department, gorgan university of agricultural sciences and natural resources, golestan, gorgan, iran. two iranian cultivated pistachio varieties, (‘abbasali’ and ‘shahpasand’) were procured from a commercial orchard located in damghan, semnan province. initially, the healthy and bold seeds were selected and the extracted kernels were treated for 24 h with different chemicals, i.e. ga 3 (50, 100 mg/l), kno 3 (1.0, 1.5 %), and dormex® (50, 100 mg/l), all procured from sigma. dormex is a mineral component with chemical formula h 2 cn 2 (siller-cepeda et al., 1992). the chemical component utilized in the present study was aqueous hydrogen cyanamid (h 2 cn 2, 187364 sigma, f.w.= 42.04 g/mole). initially the required amount of hcn was dissolved in a few drops of ethanol to prepare the stock solution from which the concentrations of 50-100 mg/l hcn were prepared with distilled water. the control seeds were also soaked in distilled water only and some seeds of the same lot were kept in a refrigerator (5°c) as cold stratification treatment. in order to prevent fungal contamination, the treated kernels were surface sterilized with sodium hypochlorite 10% and then rinsed at least three times with autoclaved water. these were then cultured in plastic containers filled with autoclaved sand (diameter 2 mm). the samples were observed daily for a period of 54 days and the number of germinated seeds was recorded. radicle emergence (5 mm in length) was taken as germinated seed. the cumulative germination count of each pistachio variety seed lot was fitted to a four-parameter hill function (4-phf; equation 1) already reported by elkassaby et al. (2008) as a mathematical representation and parameter extraction of seed germination: (1) y = y 0 + axb cb + xb in the equation 1, y is considered as the percentage of cumulative germination at time x, y 0 is the intercept on the y axis (≤0), a is the asymptote, or maximum cumulative germination percentage, which is equivalent to germination capacity, b is a mathematical parameter controlling the shape and steepness of the germination curve and c is the “half-maximal activation level” measured in days and represents the time required for 50% of viable seeds to germinate (g 50% ). following extraction of a, b, c and y 0, from 4-phf, the time at germination onset (lag) and the final germination percentage (g max ) were also computed using the following equations: (2) lag = b -y 0 cb a + y 0 √ (3) g max = y 0 + a the entire research work was designed as a completely randomized design (crd) with factorial arrangement including seven treatments and three replications. the data were analyzed using sas software. 3. results and discussion pistachio plants cannot be readily propagated from cuttings procured from mature trees and are currently propagated through budding/grafting on seedling rootstocks. though tissue culture techniques were proposed as new and advanced methods for multiplication of this species (behboodi, 2002) seedling development followed by budding is still considered a superior method for pistachio orchard establishment. however, seed dormancy and erratic germination cause significant trouble for growers, nurserymen, and fruit breeders (jangali baygi, 2012). dormancy release is accomplished by diverse mechanisms that include complex interactions with the environment mediated by plant hormones and other small molecules, and is supposed to select conditions for germination that are most favorable for plant survival (finkelstein et al., 2008). application of ga 3 . thiourea, and kno 3 as common priming agents were formerly reported in dormancy removal of some nut species such as wild pistachio (khan et al., 1999), walnut (kaur et al., 2006), and pecan (ghazaeian et al., 2012). hence, the present study was also undertaken on two cultivated pistachio varieties and the following results were found. the results of analysis of variance of the effects of chemical treatments on dormancy removal and germination parameters of pistachio varieties are shown in table 1. the data demonstrate a sharp and considerable effect of chemical priming on germination percentage, the time at germination onset (lag), and time taken to 50% germination in each seed lot. furthermore, the results demonstrate the effectiveness of dormex® to release pistachio seeds from dormancy. the lag phase is an important parameter to determine quality of seed lots. in abbasali variety the control seeds germinated 731.28 h after culture, while the chemical priming considerably reduced this period. for example, seeds treated with 1.5% kno 3 germinated at 263.26 h only. as shown in figure 1, application of dormex® at the rate of 100 mg/l had the similar effect on lag phase and the germination at onset was recorded at 385 h following treatment. in the case of shahpasand variety, dormex® was found to be the best treatment to remove seed dormancy and the lag period from 150.35 h in control seeds was reduced to 52.78 h only. this result indicates that the priming treatment with dormex® may lead to precocious and early seed 173 jangali baygi et al., dormancy removal in pistachio nut: influences of hydrogen cyanamid (dormex®) germination. an application of calcium cyanamide was previously proposed to overcome a lack of winter chilling in many plants (walton et al., 1991). ben-mohamed et al., (2012) investigated the effect of hcn on dormancy release and carbohydrate metabolism in the buds of grapevine. their results showed that application of hcn to dormant grape buds advanced budbreak to 10 days after treatment while the untreated buds opened within 14 days after treatment. furthermore, hcn caused significant differences in the rate and percentage of budbreak (85% as compared to 42% budbreak in the controls). however, there are quite a few reports on the effect of this compound on seed dormancy. gniazdowska et al. (2010) studied the role of hcn on breaking dormancy of apple embryos. interestingly, they found that the deep dormancy of apple embryos may be removed by a quite short pre-treatment (3-6 h) of hcn. furthermore, the development of abnormal seedlings was also lower than control, non treated seeds. such effect of hcn in apple embryos is comparable with the response of shahpasand pistachio variety in the present study (fig. 2). the time taken to reach 50% seed germination is also considered as a remarkable parameter in seed germination experiments. according to figures 3 and 4, application of chemical treatments significantly reduced this table 1 the results of analysis of variance of the effects of chemical treatments on dormancy removal and germination parameters of two cultivated pistachio varieties sorces of variations d.f. mean square d 50 g max lag variety 1 2508961.5** 1138.38** 1328137.5** treatments 6 66477.16** 911.36** 49107.3** variety x treatment 6 33053.44* 528.47** 21142.09* error 28 12569.26 87.34 17639.87 cv 19.68 12.28 52.83 g max= germination percentage. d 50 = time to reach 50% germination. lag= time to germination onset. fig. 1 effect of different priming agents on time to germination at onset (lag phase) of abbasali pistachio variety. fig. 2 effect of different priming agents on time to germination at onset (lag phase) of shahpasand pistachio variety. fig. 3 effect of different priming agents on time to reach 50% germination (g 50% ) of abbasali pistachio variety. co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% 174 adv. hort. sci., 2015 29(4): 171-175 period. in shahpasnd variety, the time taken to g 50% in control seeds was recorded as 562 h while in the case of priming treatments it was effectively reduced to 199 h (dormex® 100 mg/l), 234 h (ga 3 100 mg/l) and 286 h (kno 3 1.5%). in the case of abbasali, the same trend was observed and kno 3 1.5% was found to be the most effective treatment. in both varieties, cumulative germination in control seeds was low but it was enhanced following chemical priming (fig. 5 and 6). this means that g max in ‘abbasali’ increased from 46.23% in control seeds to 86.30% in seeds treated with kno 3 . the same variety had 81.91% of g max when treated with dormex®. application of kno 3 in ‘shahpasand’ seeds led to 99.25% germination. hence, the results clearly reveal that dormex® was effective in dormancy removal and germination of pistachio nuts and that in some cases it was comparable or even better than ga 3 and kno 3 treatments. the effectiveness of gibberellins in seed germination has been reported in numerous studies (bewley and black, 1994; seo et al., 2006). in vitro germination of walnut (juglans regia l.) embryos was undertaken by kaur et al. (2006) and they used components such as ga 3, benzyl adenine, and kinetin to improve embryo growth and development. they reported ga 3, along with low temperature, as the best treatment for walnut in vitro embryo germination. in another experiment for dormancy breaking of tulip seeds, rouhi et al. (2010) also applied ga 3 at rates from 250 to 500 mg/l. they observed an enhancement of seed germination as ga 3 levels were increased. in general, two main functions have been proposed for gibberellins during seed germination process. first, ga increases the growth potential of the embryo. secondly, it is necessary to overcome the mechanical restraint conferred by the seed-covering layers, by weakening the tissues surrounding the radicle (kucera et al., 2005). gibberellins stimulate germination by inducing hydrolytic enzymes that weaken the barrier tissues such as the endosperm or seed coat, inducing mobilization of seed storage reserves, and stimulating expansion of the embryo (finkelstein et al., 2008). potassium nitrate is considered to be one of the most effective and widely used components with respect to breaking seed dormancy (çetinbas and koyuncu, 2006). as far as pistachio seed germination is concerned, khan et al. (1999) applied kno 3 at the rate of 1% to wild pistachio and they observed 26% seed germination as compared to control with only 2%. in an experiment carried out on prunus avium l., the stratified seeds were treated with ga 3, kno 3 and thiourea (çetinbas and koyuncu, 2006). treatments with 7,500 mg/l kno 3 after 120 days of stratification were more effective, yielding 64.54% germination of seeds with coat. however, in case of seeds without coat (kernels), ga 3 was found to be more effective than other chemicals. the role of hcn in dormancy removal may be attributed to its stimulatory effects on endogenous ethylene production. ethylene increases respiration as well as starch degradation of seed reserves and the embryo then starts germination. a similar result was also reported for tobacco in which ethylene was involved in endosperm rupture and fig. 4 effect of different priming agents on time to reach 50% germination (g 50% ) of shapasand pistachio variety. fig. 6 effect of different priming agents on cumulative germination percentage (g max ) of shahpasand pistachio variety. fig. 5 effect of different priming agents on cumulative germination percentage (g max ) of abbasali pistachio variety. co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% co ntro l ga 50 m g/l ga 10 0 m g/l hcn 50 m g/l hcn 10 0 m g/l kno 3 1% kno 3 1. 5% 175 jangali baygi et al., dormancy removal in pistachio nut: influences of hydrogen cyanamid (dormex®) high bglu i expression during seed germination (kucera et al., 2005). ben mohamed et al. (2012) also stated that starch concentration was high in dormant buds of grapevine and declined rapidly to reach its lowest level following hcn application. the same process may occur in treated pistachio seeds leading to 99.25% germination in shahpasand variety (fig. 6). another reason for the role of hcn in improvement of seed germination can be the activation of the pentose-phosphate pathway which is thought to be necessary for the breaking of dormancy in buds and seeds (walton et al., 1991). they demonstrated that application of hcn to kiwifruit dormant buds led to production of high levels of proline amino acid. they stated that elevated levels of proline in hcn-treated plants could be associated with a greater stimulation of the pentose-phosphate pathway, resulting in a greater percentage of budbreak and increased bud fruitfulness. in conclusion, it can be stated that hcn is an effective compound for pistachio seed germination. moreover, the role of ordinary chemical seed pre-treatments such as ga 3 and kno 3 to improve pistachio seed germination was reconfirmed. application of hcn also leads to precocious germination and early seedling development. this parameter may be useful for nurseries or fruit breeders as a useful measure to develop hybrid seedlings in a shorter time. overall, although more experiments with other species should be carried out to confirm the results of the present research work, it can be said that utilization of hcn in seed testing experiments should be encouraged as an effective pre-treatment and dormancy breaking agent. references behboodi b. sh., 2002 tissue culture results of all wild pistachio species and some cultivars in iran. acta horticulturae, 591: 399-409. ben mohamed h., vadel a.m., geuns j.m.c., khemira h., 2012 carbohydrate changes during dormancy release in superior seedless grapevine cuttings following hydrogen cyanamide treatment. scientia horticulturae, 140: 19-25. bewley j.d., black m., 1994 seeds: physiology of development and germination. plenum, ny, usa, pp. 367. çetinbas m., koyuncu f., 2006 improving germination of prunus avium l. seeds by gibberellic acid, potassium nitrate and thiourea. hortscience., 33(3): 119-123. dehghanpour faradhah h., tavakkol afshari r., sharifzadeh f., chavoshinasab s., 2011 germination improvement and α-amylase and β-1,3-glucanase activity in dormant and non-dormant seeds of oregano (origanum vulgare). australian journal of crop science, 5(4): 421-427. el-kassaby y.a., moss i., kolotelo d., stoehr m., 2008 seed germination: mathematical representation and parameters extraction. forest science, 54(2): 220-227. finkelstein r., reeves w., ariizumi t., steber c., 2008 molecular aspects of seed dormancy. annu. rev. plant biol., 59: 387-415. forcella f., benech arnold r.l., sanchez r., ghersa c.m., 2000 modeling seedling emergence. field crop res., 67: 123-139. ghazaeian m., zamani s., safarnejad a., kheradmand g., 2012 effects of stratification treatments on germination of pecan seeds (carya illinoensis). international journal of agriscience, 2(7): 588-591. gniazdowska a., krasuska u., bogatek r., 2010 dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway. planta., 232: 1397-1407. hilton j.r., 1984 the influence of light and potassium nitrate on the dormancy and germination of avena fatua l. (wild oat) seed and its ecological significance. new phytol., 96: 31-34. jangali baygi m., 2012 analysis of seed germination using hill function: a case study of pistachio. msc thesis, horticulture dep., gorgan university of agricultural sciences and natural resources, gorgan, golestan, iran. kaur r., sharma n., kumar k., sharma d.r., sharma s.d., 2006 in vitro germination of walnut (juglans regia l.) embryos. scientia horticulturae, 109: 385-388. khan j., rauf m.a., harun-ur-rashid a.z., khattack m.s., 1999 different stratification techniques on seed germination of pistachio cv. wild. pakistan journal of biological sciences, 2(4): 1412-1414. kolotelo d., van steenis e., peterson m., bennett r., trotter d., dennis j., 2001 seed handling guidebook. bc ministry of forests, tree improvement branch, victoria, bc, canada, pp. 106. kucera b., cohn m.a., leubner-metzger g., 2005 plant hormone interactions during seed dormancy release and germination. seed science research, 15: 281-307. rouhi h.r., shakarami k., tavakkol afshari r., 2010 seed treatments to overcome dormancy of waterlily tulip (tulipa kaufmanniana regel.). australian journal of crop science, 4(9): 718-721. seo m., hanada a., kuwahara a., endo a., okamoto m., yamauchi y., north h., marion-poll a., sun t., koshiba t., kamiya y., yamaguchi s., nambara a., 2006 regulation of hormone metabolism in arabidopsis seeds:phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism. the plant journal, 48: 354-366. siller-cepeda j.h., fuchigami l.h., chen t.h.h., 1992 hydrogen cyanamide-induced budbreak and phytotoxicity in ‘redhaven’ peach buds. hortscience., 27(8): 874-876. walton e.f., clark c.j., boldingh a.l., 1991 effect of hydrogen cyanamide on amino acid profiles in kiwifruit buds during budbreak. plant physiol., 97: 1256-1259. 179 1. introduction slender spindle bush types are commonly used for compact-sized fruit trees such as apple cultivars grafted on dwarfing rootstocks. peach trees can be dwarfed and trained as slender spindle types when they are grafted on prunus tomentosa and p. japonica ( mizutani et al., 1985; yaegaki et al., 2008 ). however, these rootstocks often show graft-incompatibility for some peach scion cultivars several years after grafting (nakano and shimamura, 1983; yaegaki et al., 2008). it is difficult to maintain trees grafted on vigorous rootstocks as slender spindle types by winter pruning. their shoot growth is so great that the inside parts of the tree are shaded, resulting in poor growth and even death of shoots near the trunk. in relation to shading, neri et al. (2003) reported that shading caused leaf wilting, necrosis and abscission earlier under water-stressed conditions. it is important to maintain shoots and buds alive near the trunk to maintain the slender spindle types. otherwise, shoots extend outward resulting in the crown type of tree. however, when the tree vigor is so great, severe annual winter pruning only repeats imbalanced vegetative-oriented growth cycles each year. commercial fruit production is difficult under such conditions. many reasons have been given to support the practice of summer pruning in peach trees. it has been reported that summer pruning reduces vegetative growth, improves light penetration, enhances fruit quality, concentrates fruit maturation and increases the number of flower buds. in general, it is considered that the time of flower bud formation in deciduous fruit trees is around late july and august in the temperate zones of the northern hemisphere. thus, the time of summer pruning is very important in relation to flower bud formation, especially when heading back pruning is conducted. after summer heading back pruning, new shoots are regenerated from remaining twig parts. even thinning out pruning sometimes enhances branching and burst of buds which otherwise remain quiescent. in relation to shoot regrowth after summer pruning, neri et al. (1992) reported that it was induced only when the whole root system was well irrigated. after summer pruning the regenerated shoots are considered physiologically young compared with the spring flushes. in apple trees, the earlier the pruning time, the greater the number of flower buds (mizutani et al., 2000). apple flower buds tend to bear in the apical buds of shoots. when summer pruning is carried out earlier, new plural shoots come out and bear flower summer pruning to maintain slender spindle bush type of peach trees grafted on vigorous rootstocks f. mizutani faculty of agriculture, ehime university, matsuyama, ehime prefecture, 790-8566 japan. key words: peach, pruning, shading, shoot heading back, summer shoot thinning. abstract: the slender spindle bush type system is commonly used for compact-sized trees, especially grafted on dwarfing rootstocks. it is difficult to apply this system to trees grafted on vigorous rootstocks by winter pruning. such practices only cause the trees to repeat imbalanced vegetative-oriented growth every year. therefore, in the current work summer pruning was applied to slender spindle bush type of early maturing peach trees grafted on vigorous rootstocks. three trials were conducted: summer shoot thinning, shoot heading back trials in the field and a shoot heading back and shading trial in the pot experiment. summer shoot thinning reduced tree growth and recovered proper balance between vegetative and reproductive growth. the following season such shoot thinning enhanced bud burst and initial growth of new shoots but the final tree growth was less in the summer-pruned trees compared with winter-pruned trees. the fruit matured earlier and soluble solids content was greater and titratable acidity was lower in the summer-pruned trees. the summer shoot heading back trial revealed that it regenerates shoots, although they bear fewer flower buds compared with winter-pruned trees. such heading back is effective to keep alive shoots and buds near the trunk in slender spindle bush type systems. summer shoot heading back and shading experiments in the pot showed that shading reduced the number of regenerated shoots and flower bud formation and delayed flower blooming in the following year. thus summer shoot thinning and heading back are applicable to early maturing peach cultivars grafted on vigorous rootstocks to maintain the slender spindle bush type because thinning favors reducing tree vigor and light penetration near the trunk, and heading back keeps alive shoots and buds near the trunk which otherwise weaken or die back due to apical dominance and/or shading. adv. hort. sci., 2011 25(3): 179-186 received for publication 31 march 2011 accepted for publication 31 august 2011 180 buds in each shoot apex resulting in greater flower bud numbers (mizutani et al., 2000). however, later summer pruning diminishes such effects. it seems likely that a certain period is required for regenerated shoots to bear flower buds. erez (1982) also reported that in the meadow orchard system of peach trees, four to five months are required for sufficient shoot regeneration and flower bud formation. therefore he recommends that such systems are only applicable to early maturing cultivars, with a long enough growing season after fruit harvest. three trials (summer shoot thinning, heading back in the field, and heading back and shading in the pot) were conducted in the present work to develop new methods to maintain slender spindle bush type peach trees grafted on vigorous rootstocks. 2. maintaining tree shapes as slender spindle bush type in peach trees grafted on vigorous rootstocks by summer shoot thinning a. objectives at first we tried to maintain tree shapes as slender spindle bush type with ‘ab-1’ (‘akatsuki’x unknown peentao) peach trees grafted on vigorous rootstock (prunus persica batsch, wild form) by winter pruning. the trees grew well in the orchard (previously vineyard) in spite of the fact that chemical fertilizers were not applied. it was difficult to maintain tree shape as slender spindle type while producing quality fruit annually. to keep shoots and buds near the leader trunk, excessive severe winter pruning was practiced. such practices resulted in an improper balance between vegetative and reproductive growth. the vegetative-oriented growth is represented by vigorous shoot growth, poor fruit set, much june drop and delayed fruit maturation. when the peach trees are vigorous, severe winter pruning only repeats such a tree cycle every year. therefore, the objective of the first trial in the field was to determine whether summer shoot thinning can alter such imbalanced tree growth to the proper balance in the slender spindle type of peach trees grafted on vigorous rootstocks. b. materials and methods the orchard used in the experiment was formerly a vineyard to which chemical fertilizers had been applied according to a standard instruction in the experimental farm, faculty of agriculture, ehime university. for the purpose of dwarfing peach trees grafted on vigorous rootstocks, we planned no application of chemical fertilizers during the course of experiments. by using nine-year-old ‘ab-1’ peach trees which had so far received winter pruning, we tried summer pruning to maintain the tree as slender spindle bush type. the harvest time of ‘ab-1’ peach is mid july. summer pruning was conducted just after fruit harvest; most of it was conducted as thinning out methods not heading back. the weight of pruned shoots and leaves was measured. the following year new shoot growth, flowering, leaf mineral content, fruit growth and quality of harvested fruit were determined. c. results figure 1 shows summerand winter-pruned trees just after summer pruning on 24 july. the weight of shoots removed by summer pruning was less than that removed by winter pruning (fig. 2). however, in the case of winter-pruned trees, there were no leaves at pruning time so that the actual biomass removed from the winter-pruned trees was much fig. 1 peach trees just after summer pruning on 24 july (left: tree after summer pruning; right: tree without summer pruning) fig. 2 weight of shoots removed by summer and winter pruning from peach trees. data are presented as mean ± standard error (se). s ho ot w ei gh t (k g f. w . / t re e) summer pruning winter pruning 181 greater than the shoot weight pruned. three or four days earlier bud break and flowering were observed in summer-pruned trees compared with winter-pruned trees in the following year (fig. 3). the ovary size was greater in summer-pruned trees (fig. 4). the number of flowers however was less in summerpruned trees (fig. 3), while mineral and carbohydrate content in the new leaves and shoots was greater in the summer-pruned trees (figs. 5 and 6). this indicates that the shoots remaining after summer pruning received enough fig. 3 effect of summer and winter pruning on the formation of new shoots, leaves and flowers of peach trees in the following season on april 4. fig. 4 effect of summer and winter pruning on ovary size of peach flowers on 4 april. data are presented as mean ± standard error (se). o va ry le ng th w id th (m m ) 8 6 4 2 0 length width fig. 5 effect of summer and winter pruning on mineral content of stems and leaves in peach trees on 4 april. data are presented as mean ± standard error (se). n ut rie nt c on te nt (% ) 5 4 3 2 1 0 summer pruning n ut rie nt c on te nt (% ) winter pruning 5 4 3 2 1 0 fig. 6 effect of summer and winter pruning on sugar and starch content in stems and leaves in peach trees on 4 april. data are presented as mean ± standard error (se). summer pruning c on te nt (m g/ g d w ) winter pruning 120 100 80 60 40 20 0 c on te nt (m g/ g d w ) 120 100 80 60 40 20 0 182 solar radiation to accumulate carbohydrate as reserves and mineral nutrient from the roots. on the other hand, in the winter-pruned trees, the shoots remaining near the trunk are supposed to have been shaded in the previous summer and fall. although the initial tree growth was slightly enhanced in the summer-pruned trees, the shoot growth was accelerated in the winter-pruned trees in the middle growing season and final tree size became greater in the latter group (fig. 7). fruit number and yield per tree were greater in the summer-pruned trees (tables 1 and 2). fruit weight was similar in both treatments but fruit seemed to mature earlier in summer-pruned trees. in this regard, the total soluble solids content in the juice was greater and titratable acidity was lower in summer-pruned trees. thus, summer shoot thinning seems to be applicable to vigorous peach trees grafted on vigorous rootstocks in slender spindle bush type system to recover balanced vegetative and reproductive growth. 3. shoot regeneration and flower bud formation after summer shoot heading back a. objectives because of apical dominant nature of shoots, the terminal shoot grows well, which retards the growth of lateral shoots. whereas the apical part of buds on shoots burst and extend, the lower part of buds mostly remain quiescent. in the slender spindle bush type of tree it is very important to keep alive buds or shoots near the trunk. without shoot heading back, terminal shoots extend outward, while the inside of the crown becomes shaded and shoots and buds near the trunk die back. in the second trial in the field, we examined the effects of summer shoot heading back on shoot regeneration, leaf chlorophyll content (spad value), leaf drop and flower bud formation by using peach tree grafted on vigorous rootstocks. b. materials and methods five-year-old ‘hikawahakuho’ peach trees grafted on vigorous rootstock (prunus persica batsch, wild form) which were trained as slender spindle type were used. the harvest time of this cultivar is early july. trees had been trained as slender spindle bush type by winter pruning before the experiment started. no chemical fertilizers were applied, as mentioned above, because the orchard was formerly a vineyard where the recommended amount of fertilizers had been applied according to the standard instruction. tree vigor of ‘hikawahakuho’ peach grafted on vigorous rootstocks used in this experiment was less compared with ‘ab-1’ peach described above in the previous section. summer pruning consisted of heading back of current shoots to about 10 cm and removal of vigorous shoots, which was conducted after fruit harvest on 15 july. the number of regenerated shoots, shoot growth, flower bud formation, spad values and leaf drop were detertable 1 effect of summer pruning on peach fruit yield and quality in the following season (2001) treatment fruit/tree (no.) yield (kg/tree) fruit weight (g) fruit length (mm) fruit diameter (mm) ssc (%) titratable acidity (%) summer pruning 136.0±36.7 13.0±3.1 102.2±5.3 59.4±0.7 62.2±0.7 9.27±0.26 0.27±0.01 winter pruning 96.2±5.3 8.7±0.8 102.2±4.0 58.4±0.5 60.7±0.4 8.45±0.24 0.52±0.04 data are presented as mean ± standard error (se). table 2 effect of two-successive-year summer pruning on peach fruit yield and quality in the following season (2002) treatment fruit/tree (no.) yield (kg/tree) fruit weight (g) maturity degree (z) ssc (%) titratable acidity (%) summer pruning 76.8±15.0 8.3±2.1 108.9±4.4 3.7±0.4 12.10±0.32 0.20±0.01 winter pruning 66.4±8.0 6.6±1.0 103.5±3.5 2.4±0.3 9.31±0.29 0.45±0.08 (z) for maturity degree, the score was given to green fruit=1 and ripen fruit=5. data are presented as mean ± standard error (se). fig. 7 the photos show tree sizes on 24 july in the following year. 183 mined. the following year, tree growth and fruit yield and quality were determined. c. results figures 8 and 9 show the shoot regeneration after summer pruning. there was no regrowth of shoots in winterpruned trees. spad values of leaves were greater in summer-pruned trees than winter-pruned trees (fig. 10). leaf retention was prolonged by summer pruning (fig. 11). mierowska et al., (2002) also reported that in apple spur leaf total chlorophyll content was higher in summer-pruned than non-pruned trees. these facts indicate that regenerated shoots are physiologically young compared with spring flushes. the percent of flower buds were lower in summerpruned trees than winter-pruned trees (table 3); total shoot length in the following year was less in summer-pruned trees (table 3). fig. 8 effect of summer pruning on regenerated shoot numbers of peach trees. data are presented as mean ± standard error (se). fig. 9 effect of summer pruning on the regenerated shoot length of peach trees. data are presented as mean ± standard error (se). table 3 effect of summer pruning on flower bud formation and total shoot length of peach trees in the following year treatment flower buds (%) total shoot length in the following year (m) summer pruning 17.2±4.5 21.8±4.9 winter pruning 65.7±8.7 45.0±9.0 data are presented as mean ± standard error (se). fig. 10 effect of summer pruning on spad (chlorophyll content) in the leaves of peach trees in september and november. data are presented as mean ± standard error (se). fig. 11 effect of summer pruning on leaf drop in peach trees from september to late january. data are presented as mean ± standard error (se). 184 therefore it can be said that the tree size of summerpruned trees was reduced compared with winter-pruned trees. figure 12 shows single year and two-successiveyear summer pruning on the weight of shoots pruned. two-successive-year summer pruning reduced the weight of pruned shoots. fruit quality of harvested fruit as affected by single year and two-successive year summer pruning is presented in table 4. summer pruning enhanced maturation and increased soluble solids contents and reduced titratable acidity. 4. shoot regeneration and flower bud formation after summer shoot heading back under shaded conditions a. objectives it is important to keep alive shoots and buds near the trunk to maintain peach trees as a slender spindle type. since shoots have apical dominance, terminal shoots extend outward so that inner parts of the crown become shaded without pruning. we further examined the effects of shading and summer shoot heading back on shoot regeneration and flower bud formation by using peach trees grafted on vigorous rootstocks in the pot trial. b. materials and methods potted (30 cm diameter) one-year-old ‘hikawahakuho’ peach grafted on vigorous rootstock (wild form) were used in the trial. fertilizers (n, p2o5, k2o=15%, 15%,15%) were applied at the rate of 30 g and 15 g per pot in mid march and early september, respectively. treatments consisted of shading with white and black cheesecloth, shoot heading back (to 5 cm length from shoot base) and their combinations. summer shoot heading back was conducted on 1 september. the number and length of regenerated shoots, leaf drop, spad values, flower bud formation and flowering time were determined. c. results no shoot regeneration was found without summer shoot heading back under either non-shaded or shaded conditions (fig. 13). summer shoot heading back regenerated shoots but shading reduced their number (fig. 13). heavy shading (black cheesecloth) accelerated shoot growth as compared to light shading (white cheesecloth) and non-shading (fig. 14). the number of flower buds was reduced by summer pruning and the tendency was accelerated by shading (fig. 15). shading tended to delay bloom in the following spring (fig. 16). this indicates that the inner side shoots of the tree crown delayed bud burst and initial shoot growth as described in winter-pruned ‘ab-1’ peach in the previous section (fig. 3). fig. 13 effect of late summer pruning and shading on the number of regenerated shoots of peach trees. sp = summer pruning; wp = winter pruning, wc = white cheesecloth; bc = black cheesecloth. data are presented as mean ± standard error (se). fig. 12 effect of summer and winter pruning on pruned shoot weight of peach trees. summer-pruned shoots include leaves. spⅰ, wpⅰ and spⅱ, wpⅱ indicate single-year and two-successiveyear pruning, respectively. data are presented as mean ± standard error (se). table 4 effect of summer and winter pruning on fruit yield and quality in peach trees treatment fruit no./tree fruit weight (g) yield (kg/tree) maturity degree (z) ssc (%) titratable acidity (%) summer pruning i (y) 28.1±4.1 131.0±0.5 3.7±0.7 3.5±0.4 12.8±0.2 0.27±0.03 winter pruning i(y) 41.1±5.8 128.1±0.4 5.3±1.0 2.7±0.2 11.8±0.2 0.39±0.03 summer pruning ii 22.2±3.6 136.2±6.0 3.0±0.6 3.9±0.3 13.7±0.3 0.24±0.02 winter pruning ii 30.4±4.3 128.6±0.4 3.9±0.8 2.9±0.3 12.7±0.2 0.38±0.03 (z) for maturity degree, the score was given to green fruit=1 and ripen fruit=5. (y) indicate single-year and two-successive-year pruning, respectively. data are presented as mean ± standard error (se). 185 5. discussion and conclusions slender spindle bush type training systems have been easily adopted in compact-sized trees grafted on dwarfing rootstocks (mizutani et al., 1985). however, when this system is applied to trees grafted on vigorous rootstocks, imbalanced vegetative-oriented cycle between vegetative and reproductive growth occurs. for commercial quality fruit production, a good balance between vegetative and reproductive growth is necessary. as suggested in the present work, application of summer shoot thinning can reduce vegetative growth in such vegetative-oriented trees of spender spindle type trees grafted on vigorous rootstocks. neri et al. (2010) also reported that summer pruning reduced vigor in apricot trees. to maintain the slender spindle type, it is crucial to keep alive shoots and buds near the trunk. however, shoots exhibit apical dominance by nature so that they extend outward, thus the space near the trunk becomes shaded. shading accelerates dieback of shoots and buds inside the crown. with regard to shading, neri et al. (2003) reported that shading caused leaf wilting, necrosis and abscission earlier under water-stressed conditions. thinning of shoots is effective for the penetration of solar radiation near the trunk in the crown. furthermore, heading back of shoots near the trunk induces shoot regeneration from the buds at the base, which are otherwise quiescent or died back. unless heading back is conducted, the base parts of shoots become bare without alive shoots or buds. summer shoot heading back is efficient to keep such shoots and buds near the trunk, even under shaded conditions. readers interested in further detailed information for current work are also referred to hossain et al. (2004, 2005, 2006). in conclusion, summer thinning is effective for reducing tree vigor and light penetration near the trunk, whereas summer heading back is essential for keeping alive shoots and buds near the trunk in slender spindle bush type peach trees grafted on vigorous rootstocks. references erez a., 1982 peach meadow orchard: two feasible systems. hortsci., 17: 138-142. hossain s.a.b.m., mizutani f., onguso j.m., 2004 effects of summer pruning on maintaining the shape of slender spindle bush of peach tree grafted on vigorous rootstock. j. jap. soc. agr. tech. man., 11: 55-62. hossain s.a.b.m., mizutani f., onguso j.m., elshereif a.r., 2005 effects of late summer pruning and cheesecloth shades on shoot regeneration, flower bud formation and blooming time of peach trees. bull. exp. farm fac. agr. ehime univ., 27: 11-17. hossain s.a.b.m., mizutani f., onguso j.m., el-shereif a.r., rutto k.l., 2006 effects of summer pruning on shoot growth and fruit quality in peach trees trained as slender spindle bush type. mem. fac. agr. ehime univ., 51: 9-13. fig. 14 effect of late summer pruning and shading on regenerated shoot growth of peach trees. data are presented as mean ± standard error (se). fig. 15 effect of late summer pruning and shading on flower bud formation of peach trees. sp=summer pruning; wp= winter pruning; wc=white cheesecloth; bc=black cheesecloth. data are presented as mean ± standard error (se). fig. 16 effect of late summer pruning and shading on flower blooming of peach trees in the following year. data are presented as mean ± standard error (se). 186 mierowska a., keutgen n, huysamer m., smith v., 2002 photosynthetic acclimation of apple spur leaves to summer pruning. sci. hort., 92: 9-27. mizutani f., kogami t., moon d.g., bhusal r.c., rutto k.l., akiyoshi h., 2000 effects of summer pruning on the number of apical buds near the trunk in slender-spindle-trained apple trees grafted on semi-dwarfing rootstocks. bull. exp. far. coll. agr., ehime. univ., 22: 1-10. mizutani f., yamada m., taniguchi, t., koizumi k., sugiura a., tomana t., kaddya k., 1985 dwarfing effect of p. japonica and p. tomentosa rootstocks on ‘ohkubo’ peach trees. j. jap. soc. hort. sci., 54 : 327-335. nakano m., shimamura k., 1983 growth and yield of peaches on rootstocks of prunus tomentosa and prunus persica. sci. rep. fac. agr. okayama univ., 61: 67-75. neri d., castagnoli m., poni s., corelli l., 2003 diversified response to drought of light exposed and shaded leaves of potted grapevine, peach and pear trees. j. fruit orn. plant res., 11: 5-15. neri d., morini f., massetani f., 2010 pruning: how to manage shoot growth. acta horticulturae, 862: 355-363. neri d., sansavini s., sugiyama n., 1992 summer and root pruning of split-root potted peach trees. acta horticulturae, 322: 177-189. yaegaki h., inomata y., murase s., ito a., yoshioka h., takatsuji t., suzuki k., 2008 influence of the combination of scion peach cultivars and prunus japonica thumb. and p. tomentosa thumb. as a dwarfing rootstock on growth and fruit production. bull. natl. inst. fruit tree sci., 7: 1-12. 14 1. introduction grapevine phylloxera (daktulosphaira vitifoliae fitch) is considered the most destructive grapevine insect pest in syria where more than 70,000 ha of this crop, annually producing approximately 540,000 t of grapes, are planted (makee et al., 2010). grape phylloxera invaded syria on nursery stock from bordering countries during the 1920s where it quickly spread in syrian vineyards among the non-resistant local vitis vinifera l. grape (makee et al., 2003). in syria, the most commonly used resistant rootstocks are ru140 (v. rupestris x v. berlandieri), r99 (v. rupestris x v. berlandieri), 3309c (v. riparia michaux x v. rupestris) and 41 b (v. vinifera x v. berlandieri planchon). however, local phylloxera demonstrate an ability to develop and reproduce on all american rootstocks (makee et al., 2010). in addition, the susceptibility of ru140 rootstock was found to be higher than that of r99 and 3309c rootstocks (makee et al., 2003). the rootstocks often allow limited growth and reproduction of phylloxera, but they may also reduce the growth vigor of grafted varieties because of incompatibility phenomena. moreover, most syrian grapevines are planted in areas of 300-500 mm rainfall, thus they are unirrigated and such conditions are inappropriate for resistant rootstocks (makee et al., 2010). granett et al. (1996) reported that once a vineyard is infested with grape phylloxera it is expected to cease its production in about two to five years. generally, spring phylloxera is found primarily on the small feeder roots that proliferate during root flush. however, during summer when the climate is dry and the top of the plant is growing, phylloxera is found extensively on the infected mature older roots. in winter, phylloxera is found on mature roots due to the disappearance of feeder roots (omer et al., 1997). once the phylloxera’s proboscis is inserted into root bark cells, the parenchyma cells below the suberized outer layer, they inject saliva that, in turn, trigger the root cells to increase in size and number. these galls provide a feeding site from which stored nutrients such as sugars and amino acids can be extracted by developing phylloxera, which leads to a decrease in vine vigor and eventually destroys the roots causing vine death (omer et al., 2002). the injured grapevines are usually attacked by secondary soil-borne pathogens at the insect feeding site (omer and granett, 2000). granett et al. (1998) and lotter et al. (1999) revealed that fungal infections associated with pylloxera galls are pathogenic and cause root death. in addition, fungal invasion of the roots at grape phylloxera feeding sites cause severe infections in grapevines. fusarium is a large genus of filamentous fungi, and most fusarium species are harmless saprobes and relatively abundant members of the soil microbial community (domsch et at., 1980; nwanma et al.,1993). this ecological habitat of the fungus implies that fusarium could be a useful resource of extra cellular enzymes. the fungal isolate f. solani sy7 was the best xylanase producer among tested isolates (arabi et al., 2011; bakri et al., 2012). vine varieties susceptible to grape phylloxera were also highly susceptible to f. oxysporum (omer et al., 1995; 1999). granett et al. the relationship between grape phylloxera and fusarium root infection i. idris,(1) m.i.e. arabi department of biotechnology, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: fusarium ssp., grape phylloxera, grape root. abstract: phyloxera seems to have key role in the fungal pathogen infection ratio while the fungal spread reduces the ability of pheloxera to reproduce. intact roots of four-month-old grape plants were inoculated with phylloxera eggs in presence or absence of fungal pathogens. fusarium solani sy7 infection was detected in all plant parts when grapevine roots were infested with phylloxera. the spread ratio of fusarium solani sy7 increased from 74 to 100% of the infested plants with phylloxera. on the other hand, the phylloxera on f. solani sy7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly decreased 49% and 31% respectively. the total plant biomass decreased to 29% in the presence of both f. solani sy 7 and phylloxera as compared to 9 and 17% in the presence of f. solani sy7 or phylloxera, respectively. this study sheds light on the correlation between fungi, phylloxera and grapevine and could help in the application of integrated pest management (ipm) programs against grape phylloxera. adv. hort. sci., 2014 28(1): 14-19 (1) corresponding author: ascientific@aec.org.sy received for publication 26 march 2014 accepted for publication 29 april 2014 15 (1998) also demonstrated that the two root types with v. vinifera parentage, carignane and axr#1, which are most susceptible to phylloxera feeding, are also susceptible to infection by f. oxysporum pathogen. the present study therefore aims to investigate whether the presence or absence of phylloxera could enhance f. solani sy7 fungal infection in the local balady v. vinifera variety vine roots and the consequences of the interaction between the grape phylloxera and f. solan sy7 on the vine. in this context, the interaction between root infection and the three fusarium species root and rootfeeding grape phylloxera were first investigated under controlled conditions, then the effects of fungal infection with and without the presence of phylloxera on grape vine vigor (plant biomass, roots, branches and leaves, internodes weights) were determined. the process included identification of the fusarium species that invade the roots from grape phylloxera feeding sites. 2. materials and methods establishment of the phylloxera colony grape phylloxera was originally collected from fieldinfested roots of the local grape varieties in southern parts of syria. the phylloxera colony was established according to the procedures mentioned by de benedictis and granett (1993). fresh and healthy pieces of local grape variety (makee et al., 2008) “balady” roots, 4-7 mm in diameter and 5-7 cm long, were taken and washed with tap water. each piece was wrapped with moist cotton wool at one end, and then 10 to 15 phylloxera eggs were placed on each piece. the infested root pieces were placed on a wet filter paper disk inside plastic petri dishes (12 cm diameter and about 1 cm deep, three to four root pieces per dish). for ventilation purposes, the petri dish lid was modified with a 1-1.5 cm cloth–screened hole. dish edges were sealed with parafilm, kept in plastic boxes with tightly fitting lids and incubated at 25°c in the dark with 75% relative humidity. the root pieces were replaced when they desiccated rotted or the phylloxera became crowded. potted inoculation procedure before inoculation, three-day-old eggs (n =100) were removed from the colony and placed in 1.5 ml plastic tubes for surface sterilization. one ml of formaldehyde was added to the eggs and the tubes were gently shaken for 10 min. the sterilizing solution was removed from the mix and the eggs were extracted and placed on a sterile filter paper and dried for 5 min. they were then kept in a petri dish, sealed with parafilm to prevent contamination and the escape of the phylloxera crawlers. egg sterilization was carried out under sterile conditions (makee et al., 2003). fungal isolates the fungal isolates were obtained from the plant pathology laboratory of the atomic energy commission of syria (arabi et al., 2011) (table 1). host plant root samples infested with fusarium were collected from different locations in syria. roots were sterilized in 5% sodium hypochlorite (naocl) for 5 min. after three washings with sterile distilled water, roots were dipped in 70% ethanol for 1 min and then washed once with distilled water. roots were cut into small slides under sterile conditions and transferred to petri dishes containing potato dextrose agar (38g/l) (pda, difco, detroit, mi, usa) (alazem, 2007). thirteen mg/1 kanamycin sulphyate were added after autoclaving and 10 days incubation at 23 ± 1°c in the dark to allow mycelia growth. all isolates were identified morphologically, according to nelson et al. (1993). emphasis was placed on selecting isolates that induced differential reactions on specific genotype, pathogencity and in vitro xylanase activity (alazem, 2007; arabi et al., 2011; bakri et al., 2012). the above mentioned parameters lead to select of three monosporic isolates f. culnorum sy3, f. solani sy7 and f. equisesti sy24. the fusarium isolates used in this study, their location, year of collection and xylanase production are listed in table 1 (arabi et al., 2011). the cultures were maintained on silica gel at 4°c until needed. eighty “balady” grape stem pieces were dipped in a solution of 2000 ppm iaa (indol butric acid) for 2 min, then planted in plastic pots contacting sterile moistened soil. finally, they transferred to 10-l plastics pots, after four months. the experiment was conducted in greenhouse, using a randomized complete block design with four replicates of four plants for each of the following treatments in each treatment. 1 infection with phylloxera: roots of vines were infected with phylloxera eggs (50 eggs/root). 2 infection with fusarium spp: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (f. culnorum sy3, f. solani sy7, f. equiseti sy24) for 15 s, then dried for 30 min. 3 infection with fusarium spp and phylloxera: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (f. culnorum sy3 , f. solani sy7, f. equiseti sy24) for 15 s, dried for 30 min, and then infected randomly by phylloxera eggs. 4 plant control (free of fusarium spp. and phylloxera): plants were transplanted into 10-l pots. each experimental unit consisted of two plants. pots were filled with sterile soil. the pots were all placed in a greenhouse at 25 ± 1°c ( day) and 23±1°c (night) with 16-h table 1 fusarium isolates, location, year of collection and extra cellular xylanase production in solid state fermentation after five days of inoculation at 30°c isolate location of syria year of collection xylanase (u/g) f. culnorum sy3 north-west 2005 163.69 f. solani sy7 middle-region 2003 908.2 f. equiseti sy24 north-east 2005 122.43 16 daylight and 85-95% relative humidity. plants were irrigated with water as needed. a year later, the following parameters were measured: plant biomass, root weight, root number, internode weight. five root pieces of each tested plant were sampled. microscopic inspection was performed to determine the number of tuberosities and feeding nymphs for each tested plant. fungal inspection plant samples (roots, leaves and branches) were collected from each tested plant, surface sterilized for 3 min in 5% naocl, and rinsed twice in distilled water. six disks to each roots, leaves and branches were transferred to petri dishes containing pda with 13 mg/l kanamycin sulphyate added after autoclaving and incubated for 4 weeks at 23 + 1°c in the dark to allow mycelia growth. mycelia edges of f. culnorum sy3, f. solani sy7, and f. equiseti sy24 were identified morphologically as describe by nelson et al. (1993). the infection percentage of fungal ratio was calculated using the formula r = f e / ft x 100, where r = the percentage of fungal ratio, f e = number of disks that contain the fungi from roots, leaves and branches, and ft = number of the total disks with or without fungi from roots, leaves and branches. statistical analysis was performed using the statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). means were subjected to analysis of variance tested for significance using tukey hsd test. 3. results incidence of fusarium spp infection in different parts of plant with or without phylloxera the results demonstrated that fungal infection ratio in the whole plant was 74, 1.7 and 0% of f. solani sy7, f. equiseti sy24 and f. culmorum sy3, respectively. furthermore, the ratio of infection with f. solani sy7 in treatment 4 increased 26% to reach 100% (table 2). effect of different treatments on plant the plant biomass (fig. 1, i) decreased significantly in the presence of phylloxera and fusarium solani sy7 (9 and 17%, respectively). this effect is more evidenced in the presence of fungi and phylloxera 29% (f=2; df=1, 3; p<0.05). vine roots infection with fusarium spp. had no effect on root weight (fig. 1, ii) compared with the control, while infection with phylloxera increased 36% significantly comparing to control (f=2; df=1, 3; p<0.05). branch and leaf weight (fig. 1, iii) decreased significantly (f=2; df=1, 3; p<0.05 ) compared to the control in the presence of phylloxera and f. solani sy7 (53% and 31%, respectively). this effect is more evidenced in the presence of phylloxera with infection of f. solani sy7 58%. the average internode weight (fig.1, iv) in treated plants with both phylloxera and fungi increased considerably (50%) as compared to the control. however, no obvious correlation between phylloxera and fungi concerning the internode weights was shown in the experiment. in addition, the root number of vines exposed to the fungus was notably decreased but to a lesser degree compared to plants infested with phylloxera or both fungi and phylloxera (f=3.6; df=1, 3; p<0.05) (fig. 2). effects of f. solani sy7 infection on nymph and tuberosity numbers the numbers of nymphs and tuberosities in vines infested with phylloxera decreased significantly (f=85.3; df=1.1; p<0.05), (f20=; df=1.1; p<0.05) in comparison with vines infested with both phylloxera and fungi. in addition, nymphs and tuberosities were reduced by 49 and 31%, respectively, compared to the control plants (table 3). 4. discussion and conclusions phylloxera seems to have key a role in the fungal pathogen infection ratio and, in contrast, the fungal spread reduces the ability of pheloxera to reproduce. the high infection ratio of f. solani sy7 (74%) may attribute to its ability to spread in the phloem parenchyma through a special mechanism which allows the fungi to infect the roots (omer et al., 1999). phylloxera can serve as a vector and transport fungal propagates from infected to healthy roots (omer et al., 2000). therefore, our data suggest that the injury caused by phylloxera may give benefit to f. solani table 2 incidence of fusarium spp. infection in different parts of plant with or without phylloxera treatment f. solani sy7 % f. equiseti sy24 % f. culnorum sy3 % roots branches leaves roots branches leaves roots branches leaves phylloxera 0 0 0 0 0 0 0 0 0 fusarium mix 72 77 75 0 0 0 0 0 0 phylloxera + fusarium mix 100 100 100 5 0 0 0 0 0 control 0 0 0 0 0 0 0 0 0 data represents the infection percentage of fusarium spp. ratio in roots, branches and leaves in each treatment. 17 sy7 to spread throughout the entire plant 100%. however, the other two fungi (f. culmorum sy24 and f. equiseti sy3) species may not possess this mechanism to spread in the phloem parenchyma and eventually they are not able to infect the vine despite the presence of phylloxera which may be attributed either to the lack of proper growth condition in vine or to the presence of immune response in the plant which prevents the growing of the later fungi species (omer et al., 1999; fossen, 2002). to confirm the effect of f. solani sy7 and phylloxera on vines, we established an experiment to evaluate five parameters (the weights of plant biomass, root internodes, roots, branches and leaves and root numbers in presence or absence of phylloxera compared to control plants). from these biological parameters, we can estimate the relationship between f. solani sy7 and phylloxera. omer et al. table 3 effects of f. solani sy7 infection on numbers of nymphs and tuberosities treatments nymphs number (se) tuberosities number (se) phylloxera 25 ± 1 a 4.9 ± 0.9 a f. solani sy7 0 0 phylloxera + f. solani sy7 12.9 ± 0.9 b 3.4 ± 0.5 b control 0 0 data represents nymphs number mean in tested roots of 16 plants in each treatment. data were subjected to anova analysis and the differences between means were tested for significance using tukey hsd test. means followed by different letters (columns) are significantly different at p <0.05. b a b b 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi r oo ts w ei gh ts gr am treatments ii a b c d 0 20 40 60 80 100 control phylloxera fungi phylloxera + fungi p la n ts b io m a ss g ra m treatments i b a a a 0 5 10 15 20 25 control phylloxera fungi phylloxera+fungi in te rn od es w ei gh ts gr am treatments iv a c b c 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi b ra nc he s an d le av es w ei gh ts gr am treatments iii fig. 1 effect of different treatments on plant biomass (i), root (ii), branches + leaves weights (iii) and internode weights (iv). a b b b 0 5 10 15 20 25 30 35 40 45 50 control phylloxera fungi phylloxera+fungi r oo ts n um be rs treatments fig . 2. effects presents of phylloxera and fungi on roots numbers fig. 2 effects presents of phylloxera and fungi on roots numbers i ii iii iv 18 (1995) demonstrated that greenhouse experiments with effects of fungal infection on grapevine vigor after pruning at week 13 showed that damage was significantly greater in vines infested by phylloxera, f. solani and pythium ultimum than the damage in vines infested with phylloxera alone. total biomass was reduced by 16% in vines infested with phylloxera and by 24% in vines infested by phylloxera and f. solani (omer et al., 1995). in our study it was found that the total biomass was reduced by 9% in vines infested with phylloxera and by 29% in vines infested with phylloxera and f. solani sy7, compared to the controls. the differences between f. solani sy7 and f. solani (omer et al., 1995) may be attributed to the disparity of aggressiveness of the isolates. therefore, the infection with phylloxera and fungi together caused a synergetic effect where the plant biomass tend to decrease in presence of phylollxera or fungi. this can be attributed to the damage in the vine roots caused by either phylloxera or fungi which cause dysfunction of the root as they should be in the normal conditions. as both phylloxera and fungi affected the roots directly therefore, more work focused on the roots. the root weight and internodes increased significantly by 36 and 63% comparing to the control, in the presence of phylloxera and fungi respectively. this increase is normal in the presence of phylloxera due to the tuberosities and nodosities formed by the phylloxera. however, fungi alone did not cause changes in the root weight (omer et al., 1995). our results demonstrated that the significantly growth of infested vines with phylloxera alone or with a combination of phylloxera and f. solani sy7 may attribute to the rapid growth observed after potting. moreover phylloxera may require more time necessary to establish feeding sites (omer et al., 1995). additionally, a reduction of 50%, 30% respectively was observed in branch and leave weight and in the number of roots in the presence of phylloxera or both phylloxera and fungi compared to control, is attributed to roots death.therefore, f. solani sy7 infection can spread radially causing necrosis in the parenchyma and phloem. ultimately the infection kills the roots and eventually the plant (omer et al., 1999). similarly fossen (2002) found that the virulence isolates in present of v. vinifera on vine roots led to proportion of the root circumference that became necrotic in a 5-week period. some isolates were highly virulent, causing up to 80% necrosis while other isolates were negligibly virulent (fossen, 2002). it has been shown in other plant-herbivore-pathogen systems that co-occurrence can, through direct interaction or changes in host’s susceptibility, affect the performance of the pest or the pathogen (karban et al., 1987; hatcher., 1995). omer et al. (2002) reported that the ability of grape phylloxera to exploit grape roots increased in the absence of fungal pathogens and indicated that phylloxera on infected roots developed more slowly, and had substantially reduced survival and reproduction rates. therefore our results revealed the presence of fungi reduced the ability of phyloxera to form tuberosities by 31%. our data also demonstrated that the total root weight decreased in presence of fungi with the phylloxera-infested vine roots which were also reflected by a decrease in the number of nymphs. therefore our assays demonstrated that phylloxera on f. solani sy7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly decreased by 49% and 31% respectively. the reproduction and feeding activities of phylloxera were significantly decreased in the presence of fungal infection, consequently this result is in agreement with omer et al.( 2000). furthermore the ability of f. solani sy7 to spread within the plant parts increased when grapevine roots were infested with phylloxera grape. these results provide interesting piece of information about the relationship between f. solani sy7 pathogens and phylloxera. however these results are to be proved in the field. the present study provides preliminary information that could help in application of integrating pest management (ipm ) program against grape phylloxera. acknowledgements the authors thank the general director of aecs and the head of the molecular biology and biotechnology department for their continuous support throughout this work . we also wish to thank dr h. makee, and dr t. charbaji for helping us in this work. references alazem m., 2007 characterization of syrian fusarium species by cultural characteristics and aggressiveness. ph.d. thesis, faculty of agriculture, university of damascus, damascus, syria. arabi m.i.e., bakri y., jawhar m., 2011 extracellular xylanase production by fusarium species in solid state fermentation. polish. j.m., 60(3): 209-212. bakri y., jawhar m., arabi m.i.e., 2012 correlative analysis of fusarium species pathogenicity and in vitro xylanase activity. j. plant bio. research, 1(2): 86-92. de benedictis j., granett j., 1993 laboratory evaluation of grape roots as host of california grape phylloxera biotypes. am. j. enol. vitic., 44(3): 285-291. domsch k.h., gams w., anderson t.h., 1980 compendium of soil fungi. academic press, london. fossen m., 2002 resistance of grapevine rootstocks to rootpathogenic fungi vectored by grape phylloxera (daktulosphaira vitifoliae fitch). ms thesis, university of california, davis, pp. 28. granett j., omer. d.a., pesserequ p., walker m., 1998 fungal infections of grapevine roots in phylloxerainfested vineyards. vitis, 37: 39-42. granett j., walker m., de benedictis j.a., fong g., lin h., weber e., 1996 california grape philloxera more variable than expected. calif. agric., 50(4): 9-13. hatcher p.e., 1995 three-way interactions between plant pathogenic fungi, herbivorous insect and their host plants. 19 bio. rev. cambridge philosoph. soc., 70: 639-694 karban r., adamchak r., schnathorst w.c., 1987 induced resistance and interspecific competition between spider mites and a vascular wilt fungus. science, 235: 678-680 lotter d.w., granett j., omer a.d., 1999 differences in grape phylloxera-related grapevine damage in organically and conventionally managed vineyards in california. hort sci., 34: 1108-1111. makee h., ammounha h., idris i., 2010 development and reproduction of phylloxera on some local grape varieties in syria. adv. hort. sci., 24(3): 169-175. makee h., charbaj i.t., ayyou b.z., idris i., 2003 evaluating resistance of some rootstocks to grape phylloxera with an in vitro and excised root testing systems. in vitro cell. dev. biol. plant., 40(2): 225-229. makee h., charbaj i.t., idris i., ayyoubi z., 2008 effects of gamma irradiation on survival and reproduction of grape phylloxera. adv. hort. sci., 22(3): 1-5. nelson p.e., toussoun t.a., marasas w.f.o., 1983 fusarium species: an illustrated maual for identification. the pennsylvania state university press, university park, usa, pp. 203. nwanma b., onyike n., nelson p.e., 1993 the distribution of fusarium species in soils planted of millet and sorghum in lesotho, nigeria and zimbabwe. mycopathologia, 121: 105-114 omer d.a., granett j., 2000 relationship between grape phylloxera and fungal infections in grapevine roots. j. plant diseases and protection, 107: 285-294. omer d.a., granett j., de benedictis j.,walker m.a., 1995 effects of fungal root infections on the vigor of grapevines infested by root-feeding grape phyloxera. vitis, 34(3): 165. omer d.a., granett j., downie d.a., walker m.a., 1997 population dynamics of grape philloxera in california vineyards. vitis, 36(4): 199-205. omer d.a., granett j., walker m.a., 1999 pathogeniticy of fusarium oxysparum on different vitis rootstocks. phytopathology, 147: 433-436. omer d.a., granett j., walker m.a., 2002 influence of plant growth stage on grape phylloxera (homoptera: phylloxeridae) populations. environ. entomol., 31(1): 120-126. 151 1. introduction summer pruning is a fairly broad term comprising a set of practices performed on the canopy during the growing season with an array of aims, including regulation of size, vigour and crop and reduction of the susceptibility to biotic and abiotic stress. if it is considered that at least two such operations, e.g. selective shoot and cluster thinning, still require manual execution, the total amount of necessary seasonal labour, calculated as man × hr/ha, readily exceeds the demand for winter pruning and becomes a primary determinant of vineyard economics (intrieri and poni, 1995). while it is commonly heard that the ‘perfect’ vineyard needs no summer pruning, perfect in reality has proved to be a very rare occurrence. yet, we should certainly like to see vineyards of the future moving towards a more focused application of summer pruning operations. the major change is that a given summer cut is not solely or exclusively seen as something the grower “has to do”, say, to accommodate adjustments for excessive shoot growth or canopy density. rather it should also be viewed as something that the grower may ‘use’ to head vine and cluster growth towards better grape composition or to specific features consonant with adjustments needed because of climate change. along with traditional summer pruning operations, which define the grapevine canopy management strategy and include cluster and shoot thinning, shoot positioning and hedging, elimination of lateral shoots and late season basal leaf removal, over the last few years innovative summer techniques such as pre-flowering leaf removal (poni et al., 2006; intrieri et al., 2008; poni et al., 2008; diago et al., 2010 a; palliotti et al., 2011 b) or early and late season anti-transpirant sprays (palliotti et al., 2010 and 2011 a) have been introduced. these latter management practices are useful in any situation where the main aims are to reduce the vine yield and improve both technological and phenolic maturation. moreover, global warming is leading to a progressive shift toward sub-tropicalization of several viticulture areas, shorter time intervals between phenological stages (schultz, 2000; jones et al., 2005) as well as increased probability for berry sunburn (spayd et al., traditional and innovative summer pruning techniques for vineyard management a. palliotti*, s. poni** * dipartimento di scienze agrarie e ambientali, università degli studi di perugia, borgo xx giugno, 74, 06121 perugia, italy. ** istituto di frutti-viticoltura, università cattolica del sacro cuore, via emilia parmense, 84, 29100 piacenza, italy. key words: cluster thinning, grape composition, leaf removal, shoot hedging, shoot thinning, vegetative growth. abstract: this review paper highlights physiological and vine performance effects of widely adopted summer pruning operations such as leaf removal, shoot trimming and positioning and cluster thinning. leaf removal is addressed either under its traditional configuration, i.e. removing in dense canopies some or all leaves around clusters usually pre-veraison to improve fruit microclimate and facilitate spraying and early (pre-flowering) defoliation primarily aimed at inducing looser clusters via a concurrent reduction of fruit-set and berry size. time consuming and still non mechanisable cluster thinning is evaluated primarily in terms of response variability vs. season and intensity with emphasis on lack of significant reduction of final yield per vine in thinned treatments when large crop compensation occurs. variability of expected final grape composition improvements in thinned vines is also discussed based on the actual vine balance when the operation is performed. although fully mechanisable, shoot trimming is still a debated choice in terms of timing and severity. while severe (i.e. fewer than six or seven main leaves retained) and late (i.e. several weeks after bloom) cuts should possibly be avoided, the effects of shoot trimming on final grape composition is discussed as a function of seasonal changes in leaf area development, demography, fraction of lateral leaves from the total and leaf to fruit ratio. it is indicated that, for vertically shoot-positioned trellises, if the support trellising is correctly designed and vine vigour is balanced, timing and severity of trimming are dictated by the vine “itself” rather than by grower choices. overall, this review underscores the importance of leading the vineyard to a “natural” control of vegetative growth, which would minimise the need for an extensive use of summer pruning. in other words, such vineyard operations should be viewed not just as something the growers “have to do”, instead as specific tools used to achieve targeted final grape composition. adv. hort. sci., 2011 25(3): 151-163 received for publication 9 may 2011 accepted for publication 6 june 2011 152 2002; tarara and spayd, 2005; greer et al., 2006). finally, clear evidence does exist for faster ripening leading to significant increases in grape sugar concentration at harvest (dokoozlian, 2009). 2. leaf removal this operation has been historically defined as “the removal of some leaves from the fruiting area between fruit set and veraison” (smart, 1973) with the prevailing aim to ameliorate bunch microclimate and reduce rot incidence in canopies that are too dense (gubler et al., 1991). ongoing research has provided knowledge to distinguish two types of leaf removal aimed at quite distinct goals. traditional leaf removal although this practice may have different purposes, it is usually employed from fruit set to veraison on high-density canopies to improve light exposure and air circulation around the clusters, with substantial benefits in terms of pigmentation and tolerance to rot (smart, 1985; bledsoe et al., 1988; gubler et al., 1991; percival et al., 1994; reynolds et al., 1996). this operation can be done manually, requiring up to about 60 hr/ha, although increasing labour costs nowadays strongly advise a mechanical approach which can be easily performed in less than 2 hr/ha. the best timing for machine use is about one to two weeks prior to veraison when berries are still hard while specific bunch weight is already much higher than that of leaves. yield may not change (bledsoe et al., 1988; smith et al., 1988; hunter et al., 1995) or might even occasionally increase as compared with non-defoliated vines (zoecklein et al., 1992). the variability of the impact that leaf removal has on yield and their components is likely dependent upon the negative effects on fruit set and berry growth in the current year and positive effects on bud induction and differentiation for the next year’s crop via an improvement in canopy microclimate. although this type of leaf removal usually leads to undeniable improvements in fruit composition, which more frequently are a slight increase in sugars and ripe fruit characters and a decreased malic acid content and attenuated herbaceous and grassy wine characters (smart, 1985; reynolds et al., 1996; zoechlein et al., 1992; scheiner et al., 2010), its popularity has probably decreased over the last two decades due to either advancement in leaf and whole-canopy physiology and new pressure from global warming. a study from petrie et al. (2003) found that leaf removal from the lower quarter of the canopy during the lag phase of berry growth caused a significant decrease of whole-vine photosynthesis, even on a per-unit leaf area basis, thus suggesting that the lower portion of the canopy contributed more than the upper portion to the whole-vine carbon budget. a possible explanation of this finding is that although basal, and hence older leaves are removed by defoliation, they are also the largest leaves along the shoot and their size can offset lower photosynthetic rates (poni et al., 1994). therefore, lowering shoot photosynthesis might not be negligible especially for leaf removals performed after fruit set. removal of all the leaves from the fruiting area, which thereby exposes the clusters to full sun, might lead in warm climates to compromised fruit composition because of excessive berry temperatures, which can hinder colour formation and cause a sharp drop in malic acid concentrations (spayd et al., 2002; tarara et al., 2008). for such reasons and in association with increasing concern for berry sunburn, criteria for applying leaf removal have become more restrictive and more often conceive retaining some leaf cover around the fruiting area. differentiation in the actual need and/or severity of leaf removal also depends upon specific planting choices. for instance, no or very light defoliation is usually applied on the south facing side of an east-west oriented row, whereas more severe leaf stripping might be required on the north facing row side; basically the same applies for westand east facing sides of north-south oriented rows, respectively. more physiological insights have also been provided about “why” a traditional leaf removal might become mandatory. backward to the still shareable rule indicated from dr. shaulis in that “no leaf removal is needed if while standing in front of a canopy at veraison about 50-60% of the clusters are visible”, other more recent findings have shown that in a significant number of cases, excessive canopy crowding in the bunch zone leading, in turn, to the need of stripping leaves, is caused by other wrong or rushed vineyard management choices (fig. 1). one example is worthwhile above all: spur pruned vertically shoot positioned (vsp) cordontrained canopies are usually prone to leaf removal due to too high shoot density per meter of canopy length. yet, this often happens as vines burst many either secondary or base bud originated shoots casting additional shade in the bunch area. more equilibrated vines would better comply with the shared requirement that, on average, one shoot is expected from each single retained node and, if so, the subsequent leaf removal would become quite likely unnecessary. fig. 1 interrelationships of excessive shoot vigour stimulated by a too narrow within-row vine spacing and related consequences on summer pruning needs (drawn by authors). 153 early leaf removal this practice has mainly been inspired from long-standing knowledge according to which carbohydrate supply at flowering is a primary determinant of fruit set (coombe, 1959; may et al., 1969). the temporary source limitation induced by removing an average of six main basal leaves before flowering has led, under a broad array of genotypes and growing conditions, to a significant decrease in fruit-set, which in turn increases cluster looseness and tolerance to rot (gubler et al., 1991; poni et al., 2006; intrieri et al., 2008; poni et al., 2008; diago et al., 2010 a). yet, the most important outcome is that, irrespective of genotype, this early leaf removal markedly improves grape composition and wine sensory properties as compared to non-defoliated shoots (poni et al., 2006; diago et al., 2010 a; palliotti et al., 2011 b). there are multiple mechanisms involved in such a positive response. defoliated shoots generally have a higher final leaf-to-fruit ratio than control, thus implying that the yield reduction induced by defoliation was more than proportional to the leaf removal constraint due to a fruit-set and berry-size effect (poni et. al., 2006). furthermore, it is known that a precocious source limitation carried out in the form of defoliation or darkening the basal shoot zone hastens translocation of assimilates towards the cluster (quinlan and weaver, 1970). improved grape composition in the defoliated shoots also relates to the ‘quality’ of the source. for example, it is indeed true that removing the main six basal leaves at pre-bloom causes an abrupt and severe decrease in vine photosynthesis [75% less than with not-defoliated (nd) according to poni et al., 2008]. however, removing source leaves around bloom also triggers a series of dynamic changes in canopy growth, age and photosynthesis. defoliated vines have a ‘younger’ canopy at veraison since median and apical shoot leaves at this time are now mature and more lateral leaves may be present as a compensating reaction to early main leaf removal, while some, albeit temporary, photosynthetic compensation usually occurs in both main and lateral leaves of defoliated plants. poni et al. (2008) have recently shown that whole canopy net co2 exchange rates (ncer) monitored uninterruptedly for three months in defoliated (d) vs. non-defoliated sangiovese vines indicated no differences in data expressed on a per-vine basis. yet when the same data were given on a per-unit leaf area basis, defoliated vines showed higher rates than nd vines (4.75 µmol m-2 s-1 vs. 4.16 µmol m-2 s-1) and, most importantly, ncer/ yield increased by 38% in d vines, thus resulting in enhanced carbohydrate supply for ripening (table 1). however, the most intriguing outcome from these early-season defoliation tests is that a significant increase in relative skin mass has consistently been found in separate field studies conducted on a three-year basis in cv. barbera (poni and bernizzoni, 2010), regardless of absolute berry mass (fig. 2). it is reasonable to think that such an early table 1 effects of early defoliation on yield components and whole shoot net co2 exchange rate (ncer)/fresh fruit mass treatment flowers/cluster (no.) fruit set (%) total berries/ cluster (no.) cluster weight (g) berry weight (g) ncer shoot/yield (nmol/s x g) cluster compactness (rating) control 435 38.8 169 334 1.98 2.43 6.60 defoliated 487 21.0 103 207 2.01 3.31 4.25 significance ns ** ** ** ns ** ** **, ns= significant at p ≤ 0.05 or not significant, respectively. fig. 2 correlation between relative skin and berry mass in 2006, 2007 and 2008 for non defoliated and defoliated barbera grapevines (from poni and bernizzoni, 2010). 154 basal leaf removal, besides favouring berry hardening in the long run, would also impose more favourable microclimate conditions for cell division and berry skin deposition, which typically takes place within four to five weeks after flowering. mescalchin et al. (2008) have shown in pinot gris that the earlier the defoliation, the lesser the incidence of skin burning on vsp and pergola-trained varieties due to both more time allowed for cluster cover after treatment and adaptation towards the formation of a thicker skin. mechanization is feasible by preferably using at preflowering (i.e. closed-flower stage) an air pressure blowing machine which can run two passages per row in about 5-7 hr/ha (intrieri et al., 2008). best performance is obtained on canopies characterized by vertical and well positioned shoots and on cultivars having mostly erect inflorescences. it has to be kept in mind that early leaf removal is specifically recommended in highly productive vineyards which often present heavy, thick bunches very susceptible to rot. based on the constancy of the results obtained under the above circumstances, this practice is nowadays an interesting alternative to traditional methods of crop control such as bunch thinning. advantages are feasibility of mechanization, hence cost saving, and different mechanisms by which the crop level on the vine is adjusted. if early leaf removal is chosen, the primary regulation for crop restriction is via a decrease in fruit set with or without a significant reduction in berry size. therefore, cluster number is unchanged, yet each bunch is smaller and looser. conversely, hand bunchthinning, besides being time consuming, drastically lowers bunch number per vine and favours undesirable yield compensation mechanisms such as larger berries and heavier clusters (ough and nagaoka, 1984; keller et al., 2005). anti-transpirant applications a very recent development of the above work investigated whether the precocious, albeit temporary, source limitation sought with early leaf removal can be induced through the non-invasive and easy-to-do application of anti-transpirants (palliotti et al., 2010). their use could sort out the inherent limitations of high labour demand for manual work while eliminating the risks of direct damage to the inflorescences linked to the use of a leaf plucker. results reported for cvs. sangiovese and ciliegiolo subjected to pre-bloom treatment of anti-transpirant vapor gard® (a.i. di-1-p-menthene at 3% concentration, intrachem bio italia, grassobbio, bg, italy) show similar reductions of net photosynthesis (from 30% to 70%) over several weeks after spraying as compared to control vines (fig. 3). the treated sangiovese vines showed reduced yield, berry weight, cluster compactness and, on a two-year basis, lower vigour and unchanged vine capacity per year. at harvest, the treated vines showed higher °brix in all seasons and higher anthocyanin concentration two years out of three. overall, early-season applications of a filmforming anti-transpirant caused a leaf function limitation strong enough to reduce yield and cluster compactness through smaller final berry size. over the last decade, climate change along with improvements in vineyard management and clonal selection have exerted a strong impact on vine yield and grape and wine composition. among the most important effects, the increase in grape sugar concentration at harvest, is to be considered, which resulted in wines with high alcohol config. 3 seasonal trends of air vapour pressure deficit (vpd) and total photosynthetic active radiation (par) (a), assimilation rate (b), transpiration rate (c) and intrinsic water use efficiency (d) recorded on fully expanded, median sangiovese (top image) and ciliegiolo (bottom image) leaves sprayed twice with anti-transpirant vapor gard® at 3% (t) or left unsprayed (c). bold arrows indicate the time of application. data are means ± se (from palliotti et al., 2010). 155 tent (vierra, 2004; duchêne and schneider, 2005; godden and gishen, 2005). there is a surge of interest from the wine industry in tools suitable to lower wine alcohol content such as the de-alcoholisation process which also agrees with the eu legislative measure no 606/2009. conversely, it would thus be helpful to find strategies able to reduce grape sugar concentration in the vineyard, thus limiting the need to operate in the winery without detrimental effects on wine characteristics. in association with traditional management practices which can be used to slow down the accumulation of sugars in the grape berry, interest is growing in late season applications of anti-transpirants. in a recent contribution by palliotti et al. (2011 a), the anti-transpirant vapor gard® sprayed about one month before harvest significantly delayed sugar accumulation in sangiovese, tocai rosso and trebbiano toscano berries which, at harvest, had -1.2 to -2.7 less °brix than the un-sprayed control according to genotype and crop load. the temporary reduction of photosynthesis, due to the film formed by the anti-transpirant, limited the amount of assimilates translocated into the ripening berry, thus lowering must sugar concentration with a potential effect on wine alcohol content. 3. cluster thinning the achievement of an adequate balance between growth and fruiting can be obtained by the regulation of crop level through cluster thinning treatments. despite additional labour costs, cluster thinning might play an important role in all cases where over cropping occurs (e.g. excess of vigour due to cultivar and rootstock, high soil fertility, low planting density, use of drip fertigation, etc.) and in cases where winter pruning severity has not overcome cropping due to high bud fertility. the negative effects of over cropping include delay in grape maturation, worsening of overall grape quality, increased susceptibility to biotic disease and poor wood maturity (winkler et al., 1974). furthermore, different environmental parameters, particularly air temperature, light intensity, photoperiod and soil water content, together with phyto-hormones and the availability of mineral ions are known to influence bud fertility and fruit-set (srinivasan and mullins, 1981). therefore, it is not always possible to regulate the yield level by solely adjusting bud load, especially in vineyards with low planting density and in years and areas characterized by unfavourable environmental conditions. however, the results regarding the effects of high yield levels on fruit composition (sugar, acidity, colour, etc.) and wine quality (taste, flavours, colour and potential for aging) are quite contradictory. for example, some authors found an increase in anthocyanin concentration upon cluster thinning (bravdo et al., 1984 a, reynolds, 1989; guidoni et al., 2002), whereas no improvement in anthocyanin content or wine colour in cluster-thinned vines were found by bravdo et al. (1984 b) and ough and nagaoka (1984). location, application time and intensity of cluster thinning treatment significantly affected the results and can therefore justify, at least in part, the discrepancy of the experimental results in literature. the results of a three-year trial on the effects of three levels of cropping (0%, 20% and 40% cluster thinning treatments) applied just before veraison in sangiovese, merlot and cabernet sauvignon showed that this management practice caused a significant reduction of yield only at the 40% severity and in two out of the three seasons studied (table 2) (palliotti and cartechini, 1988). in each cultivar, in 1995 and 1996, yield was linearly correlated with cluster thinning intensity. cluster thinning treatment at the 40% level caused a reduction of vine yield that ranged from 22% to 47%. the reduction of yield observed was, in general, not proportional to the cluster thinning intensity due to a significant increase of berry and clustable 2 effects of cluster thinning on yield and cluster characteristics in sangiovese, merlot and cabernet sauvignon grapevine cultivars cultivar thinning yield (kg/vine) cluster/vine (n°) cluster weight (g) berry weight (g) 1995 1996 1997 1995 1996 1997 1995 1996 1997 1995 1996 1997 sangiovese 0% 12.4 11.3 10.1 40.6 46.4 39.9 306 245 251 2.30 2.36 2.33 20% 11.8 9.5 10.0 35.1 34.3 32.9 340 271 300 2.47 2.60 2.68 40% 9.5 6.9 9.5 25.2 22.6 24.3 381 308 387 2.70 2.82 3.38 significance ** *** ns ** *** ** *** *** *** *** *** *** r2 0.76 0.92 --0.76 0.94 0.75 0.90 0.89 0.92 0.87 0.94 0.96 merlot 0% 8.1 8.7 8.7 57.8 64.1 60.5 147 137 149 1.70 1.82 2.03 20% 7.7 8.0 8.2 49.5 52.1 50.5 159 154 160 1.76 1.83 2.12 40% 6.1 6.6 7.9 35.4 38.8 37.5 172 170 212 1.92 1.94 2.53 significance * *** ns *** *** *** *** *** *** *** * ** r2 0.47 0.87 --0.84 0.91 0.83 0.89 0.91 0.84 0.85 0.46 0.73 cabernet s. 0% 7.2 7.9 6.2 56.1 58.9 51.6 131 135 123 1.35 1.94 1.39 20% 7.4 7.0 6.1 44.2 47.6 42.2 167 146 146 1.60 2.02 1.57 40% 5.6 4.2 6.0 32.2 27.9 30.5 176 154 198 1.70 2.06 1.89 significance * *** ns *** *** *** *** *** *** ** ** *** r2 0.42 0.84 --0.90 0.93 0.92 0.85 0.87 0.84 0.72 0.70 0.84 *,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. 156 ter weight. at the 20% intensity of cluster thinning, vine self-regulation warranted full yield compensation through significantly increased berry size and cluster weight. in 1997, due to quite favourable environmental conditions for ripening, +156 and +143 degree-days, base 10°c, as compared to 1995 and 1996, respectively, and lower rainfall during the two months prior to harvest, the impact of the 40% cluster thinning on vine yield was negligible. total soluble solids, anthocyanins and phenolics increased linearly with thinning severity in two out of the three seasons (tables 3 and 4). juice ph and titratable acidity (ta) were rather variable, although cluster thinning tended to reduce ta and increase ph (table 3). in 1995 and 1996, improvements in soluble solids content in cluster-thinned vines were consistent with lower yield levels (table 3) whereas the reduction of titratable acidity and the slight increase of juice ph were probably attributable to an earlier ripening. similar results have also been reported by looney (1981), bravdo et al. (1984 a) and reynolds (1989). table 4 effects of cluster thinning on anthocyanins, polyphenols and total nitrogen content at harvest in sangiovese, merlot and cabernet s. grapevine cultivars cultivar thinning anthocyanins (mg/cm2 berry skin) polyphenols (mg/cm2 berry skin) total nitrogen (% s.s.) 1995 1996 1997 1995 1996 1997 1996 1997 sangiovese 0% 0.412 0.453 0.602 1.42 1.95 1.34 0.35 0.56 20% 0.580 0.596 0.652 1.89 2.37 1.84 0.56 0.56 40% 0.610 0.692 0.639 1.94 2.42 1.87 0.49 0.70 significance *** *** ns ** * ns ns ** r2 0.83 0.96 --0.80 0.57 ----0.65 merlot 0% 0.491 0.487 0.576 1.51 1.63 1.24 0.42 0.49 20% 0.571 0.554 0.641 1.73 2.00 1.46 0.63 0.49 40% 0.824 0.742 0.653 2.10 2.37 1.56 0.49 0.53 significance *** *** * ** ** ns ns ns r2 0.90 0.91 0.49 0.68 0.77 ------cabernet s. 0% 0.652 0.786 0.691 1.80 1.91 2.08 0.38 0.29 20% 0.670 0.772 1.021 2.10 2.60 2.52 0.70 0.42 40% 1.024 0.942 1.073 2.70 2.84 2.55 0.56 0.56 significance ** * *** *** ** ns ns *** r2 0.78 0.62 0.83 0.81 0.79 ----0.95 *,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. table 3 effects of cluster thinning on soluble solids, titratable acidity and ph at harvest in sangiovese, merlot and cabernet sauvignon grapevine cultivars cultivar thinning soluble solids (°brix) titratable acidity (g/l) juice ph 1995 1996 1997 1995 1996 1997 1995 1996 1997 sangiovese 0% 17.3 17.1 21.4 8.5 8.2 6.3 3.01 3.08 3.26 20% 18.0 18.9 21.8 8.8 7.5 6.1 3.04 3.11 3.22 40% 18.4 21.1 22.0 8.0 7.2 5.9 3.04 3.12 3.21 significance * *** ns ns ** ns ns ns ns r2 0.54 0.94 ----0.71 --------merlot 0% 20.6 21.0 21.4 9.7 6.8 6.5 3.13 3.29 3.32 20% 21.4 21.2 22.8 9.5 6.7 6.3 3.15 3.27 3.28 40% 22.6 22.8 22.6 8.8 6.5 6.4 3.18 3.44 3.36 significance ** ** ns ** ns ns *** ns ns r2 0.79 0.67 --0.64 ----0.85 ----cabernet s. 0% 20.2 21.0 21.6 9.7 8.0 7.6 3.05 3.21 3.22 20% 20.0 21.4 22.2 9.5 7.9 7.1 3.09 3.19 3.23 40% 22.0 23.2 22.0 8.8 7.5 7.2 3.13 3.25 3.27 significance * ** ns * ns ns * ns ns r2 0.55 0.74 --0.55 ----0.46 ----*,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. 157 data pooled from cultivars and years resulted in negative correlations between total soluble solids and yield level, while positive linear relationships were found between anthocyanins in berry skin and soluble solids in berry juice (fig. 4). overall, regulation of yield through cluster thinning is strictly dependent on year; the grape composition is generally improved and this assumes particular importance in seasons marked by unfavourable environmental conditions or in very productive vineyards due to either high fertility cultivars (i.e. sangiovese) or soils. the increase of polyphenols and anthocyanin content recorded in both 20% and 40% cluster-thinned vines is of great significance for the production of high quality red wine, especially when targeted to aging. since manual cluster thinning is a very expensive operation due to large labour requirements, its mechanization is a very needed, yet largely unresolved issue. in grenache and tempranillo grapevines trained to vertical, shoot-positioned mechanical berry thinning performed with a grape harvester was effective to reduce yield while achieving more ripened grapes and wines with higher alcohol and ph values, more intense colour and increased phenolic compounds (diago et al., 2010 b). 4. shoot hedging practices aimed at manipulating vegetative growth during late-spring and summer, particularly in vigorous vineyards, can substantially influence yield and grape composition (intrieri et al., 1983; kliewer and bledsoe, 1987; reynolds and wardle, 1989). hedging is a common management practice used to maintain canopy shape, reduce vine vigour, improve the microclimate in the fruiting zone, increase the efficiency of disease treatments and facilitate harvest and access of machines to the vineyard rows. compared with other summer management practices used for similar purposes, such as leaf removal and pulling of lateral shoots, hedging is commonly used because it can fig. 4 relationship between yield per vine and total soluble solids (left) and total soluble solids and anthocyanins content in the berry skin at harvest (right). 158 be done completely mechanically and therefore is easy, fast and cheap. the effects of hedging on yield and fruit quality, considering the variables of timing and severity of application, are strictly associated to the ability of the cultivar to develop lateral shoots and their photosynthetic capacity from veraison to harvest (cartechini et al., 1998). the impact of hedging severity on vine performance is well known; severe hedging, i.e. less than six main leaves retained per shoot, generally reduces grape quality (kliewer and bledsoe, 1987; reynolds and wardle, 1989; palliotti, 1992), whereas the time of application is rather controversial because other factors may also influence these effects such as bud load, shoot orientation, training system, environmental conditions, soil characteristics, water availability, and so on (intrieri et al., 1983; reynolds and wardle, 1989). vertical shoot positioned (vsp) training systems are normally trimmed when their shoots exceed the wires placed at the top of the canopy. therefore, the timing is poorly dependent on grower’s decisions and it is instead a function of intrinsic shoot vigour and vine balance. a balanced vineyard would reach the height suitable for trimming around fruit set, whereas an excessively vigorous one would get to the same growth stage much earlier, therefore making shoot trimming more likely to be repeated again later in the season. timing of trimming follows different rules when performed on sprawl canopies (i.e. a single high wire trellis) where an early (pre-flowering) shoot trimming might be made necessary by the need to induce mostly upright shoot growth habits. a two-year trial, aimed at assessing the effect of timing of hedging (one and five weeks after full bloom, afb) on yield and grape composition in different red and white grapevine cultivars grown on fertile clay soil and trained to a single high wire trellis, showed that hedging at the 9-10th node on primary shoots, carried out one week afb, markedly changed canopy characteristics, yield and grape composition (fig. 5 and tables 5, 6, 7 and 8) (cartechini et al., 1998). in untrimmed sangiovese, cabernet sauvignon and verdello vines, leaf area build up progressed rapidly from about 30 to 120 days after bud burst (fig. 5). the development of laterals and relative leaf area occurred from 60 to 110 days after bud burst in sangiovese and from 60 to 140 days after bud burst in cabernet sauvignon and verdello. fig. 5 development of primary and lateral leaves in sangiovese, cabernet sauvignon and verdello grapevine cultivars hedged one and five weeks after full bloom (afb) as compared to the untrimmed control (n = 3 ± se). 159 in all the cultivars, from flowering to veraison, the total leaf area increased more than three-fold. at the end of canopy growth, the sangiovese had less total leaf area than cabernet sauvignon and verdello (-1.5 and -2.0 m2/vine, respectively) and the laterals represented 18, 32 and 22% of the total leaf area in sangiovese, cabernet sauvignon and verdello, respectively. up to the end of canopy growth, sangiovese, cabernet sauvignon and verdello hedging one and five weeks afb produced about 1.1, 3.9 and 3.5 and 0.9, 3.4 and 3.1 m2 of new leaves per vine, respectively, derived mainly from lateral development. in all cultivars, early-hedging, one week afb, generally increased the contents of soluble solids, total nitrogen and total polyphenols (tables 6, 7 and 8) as well as anthocyanins content in the red cultivars (table 8). early-hedging significantly reduced the titratable acidity and juice ph in all the cultivars (table 6 and 7). late-hedging, five weeks afb, instead significantly reduced yield in sangiovese and, except for sauvignon blanc, the soluble solid content was significantly reduced as well as anthocyanins content in both red cultivars. the positive outcomes of the early-hedging were likely dependent upon a cultivar’s ability to develop lateral shoots after trimming (fig. 5). all the cultivars with a good capacity to produce laterals, such as cabernet sauvignon, verdello, drupeggio and sauvignon blanc, responded better to early summer pruning as shown by the increased cluster weight and yield and improved contents of soluble solids, total polyphenols and nitrogen content. trimming vines increased lateral growth and table 5 yield and average cluster weight at harvest in vines of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control (n= 60) cultivar yield (kg/vine) cluster weight (g) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 7.4 b 7.3 b 6.0 a 279.8 b 292.4 b 253.5 a cabernet s. 6.0 a 7.8 b 5.5 a 122.9 a 143.7 b 110.5 a verdello 7.0 a 8.2 b 6.9 a 215.6 a 276.6 b 218.7 a drupeggio 7.4 a 9.1 b 7.2 a 238.7 a 275.7 b 235.4 a sauvignon b. 4.0 a 5.2 b 3.9 a 106.5 a 129.3 b 103.8 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. table 6 soluble solids content and titratable acidity at harvest in different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar soluble solids (°brix) titratable acidity (g/l) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 23.2 b 23.9 b 21.8 a 6.6 b 6.1 a 6.8 b cabernet s. 23.4 b 23.7 b 22.9 a 7.1 b 6.6 a 7.3 b verdello 19.4 b 21.0 c 17.8 a 8.5 b 8.0 a 8.6 b drupeggio 20.3 b 21.9 c 18.1 a 8.4 b 7.8 a 8.3 b sauvignon b. 20.5 a 23.1 b 20.4 a 8.8 b 8.2 a 9.0 b for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. table 7 juice ph and berry nitrogen content at harvest in vines of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar juice ph total nitrogen (% d.w.) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 3.42 b 3.35 a 3.36 a 0.48 a 0.63 b 0.45 a cabernet s. 3.40 b 3.22 a 3.29 a 0.63 a 0.98 b 0.55 a verdello 3.06 b 3.00 a 2.99 a 0.42 a 0.59 b 0.41 a drupeggio 3.08 b 3.03 a 3.04 a 0.44 a 0.68 b 0.38 a sauvignon b. 3.07 b 3.01 a 3.02 a 0.51 a 0.66 b 0.45 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. 160 the total final leaf area was always less than that recorded in control vines (from 15 to 49% less). at harvest, in all the grapevines tested, early-hedging reduced the leaf/ fruit ratio from 33 to 45% in comparison to the control vines and improved the soluble solids content (from 0.3 to 1.6°brix), whereas late-hedging caused a reduction of both leaf/fruit ratio and soluble solid accumulation in the berries (fig. 6). the rejuvenation of leaf area in the canopy following early-hedging and their high photosynthetic efficiency from veraison to harvest of the newly formed lateral leaves (fig. 7) likely reduced the leaf area per gram of fruit required to achieve adequate ripeness. these laterals also translocate assimilates to the subtending clusters very efficiently (candolfi-vasconcelos and koblet, 1990). negative results found on late-hedged vines, also reported by other authors (intrieri et al., 1983; palliotti, 1992), are probably linked to the fact that lateral shoots compete with the developing grapes for carbohydrates, causing delayed berry growth and sugar accumulation. early-trimming reduced titratable acidity as compared to control vines due to greater cluster exposure to sunlight table 8 anthocyanins and total polyphenol content at harvest in the berry skin of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar anthocyanins (mg/cm2 berry skin) polyphenols (mg/cm2 berry skin) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 0.754 b 0.958 c 0.412 a 1.65 b 2.24 c 1.09 a cabernet s. 1.095 b 0.998 b 0.773 a 2.07 a 2.96 b 1.90 a verdello ------0.88 a 1.25 b 0.80 a drupeggio ------0.91 a 1.19 b 0.81 a sauvignon b. ------0.82 a 1.12 b 0.75 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. fig. 6 relationship between must total soluble solids and leaf/fruit ratio at harvest in vines of sangiovese, cabernet sauvignon and verdello either untrimmed or trimmed one (a) and five (b) weeks after full bloom (afb). fig. 7 evolution of net photosynthesis of primary and lateral leaves from veraison to leaf fall in sangiovese, cabernet sauvignon and verdello grapevine cultivars (n = 8 ±se). 161 and a consequent decrease of malic acid content due to respiration activity. in addition, the reduced must ph with early-hedging is probably linked to the reduction of both the malic acid and potassium contents in the must in association with lower total leaf area. bledsoe et al. (1988) found a significant positive correlation between these two parameters and juice ph. in all the grapevine cultivars that develop many laterals after hedging, the greater transpiration rate (from +15 to 35%, data not shown) assessed in these leaves, compared with primary ones, particularly in august and september, may aggravate susceptibility to vine water stress especially in hot environments and in particularly dry years. during the first two weeks of november, the laterals on the vines had net photosynthesis values that ranged from 0.7 to 1.6 µmol co2 m -2 s-1 (fig. 6), in a period when all the carbohydrates fixed are very useful for the reserve accumulation, and therefore for increased cold hardiness (wample and bary, 1992) and even for budbreak and initial shoot growth the following season. thus, at the end of the season care must be taken to maintain the integrity of these leaves until total abscission occurs. early winter pruning, practiced in some viticulture areas, should be avoided. 5. shoot positioning in vsp canopy trellis systems, shoot positioning is performed to maintain canopy form and shoot separation, to create a uniform distribution of leaves that minimizes cluster shading as well as to optimize canopy light interception and allowing the transit of mechanical equipment between rows. shoot positioning also exerts a positive effect on disease incidence and severity; usually disease pressure is lessened due to increased air flow and sunlight penetration inside the vine canopy. another important effect of this canopy management technique is that it has a positive impact on the development of fruitful buds and therefore for the vine yield in the following year. the way shoot positioning is performed depends mainly on the training systems. in a vsp system the process consists of directing the shoots growing up between a set of catch wires as they develop. the vertically positioning of shoots can be done manually or using movable wires and done several times during the growing season. mechanical shoot positioning on vsp trellis systems with specialized equipment has undergone a notable increase in recent years. on geneva double curtain (gdc) training system the shoots are positioned downward and separated out from the permanent cordon in order to reduce the vigour of shoots and attain optimal canopy density. in the gdc trellis, shoot positioning is performed on the interior part of the canopy to maintain two distinct canopies avoiding excessive shading in the central part of the canopy. usually, in most training systems, shoot positioning is performed one or two weeks after bloom, before tendrils have become firmly attached. for best results, however, two or three shoot positioning runs during the season are needed. 6. conclusions vineyard management should aim to achieve and maintain high efficiency over time, which is closely dependent on the ability to control the competition both between-and intra-vine. this approach would warrant a fair and fruitful balance between vegetative and productive activity of the vines and the best expression of grape quality (smart and robinson, 1991) without costly additional inputs. since the “perfect” vineyard able to reach and maintain this equilibrium in a natural way during the season is generally utopia, summer pruning often plays a crucial role. in light of the climate change in progress, an important challenge for old and new vineyards will be the matching of tradition and innovation. this raises the question of new techniques of canopy management, availability of rootstocks of low-to-moderate vigour, new cultivars better adapted to higher temperatures and water shortage and more intense mechanization. the latter assumes particular importance especially when the wines produced must be sold in un-bottled form or within large organized distribution (lod) chains, like supermarkets, hypermarkets and discount markets. currently, at least in italy, lod commercialize about 70% of the entire italian wine production (which corresponds to about 48-50 million hl per year) (ismea, 2007), where the binomial “adequate quality”“moderate selling price” is still dominant. global warming requires rapid adaptation and poses the crucial question of ripening modulation. in white grape varieties, the major challenge is the preservation of organic acids and primary grape flavours; whereas in black-berried cultivars the priority is producing wines with moderate alcohol content without modifying colour intensity and wine sensory. some traditional and innovative canopy management practices can help to achieve these results, such as light pruning (petrie et al., 2003), early leaf removal (poni et al., 2006; palliotti et al., 2011 b), late defoliation and severe summer pruning (stoll et al., 2010), use of anti-transpirants (palliotti et al., 2010, 2011 a), canopy treatment of exogenous auxins (böttcher et al., 2010) and brassinosteroid and brassinazole steroidal 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kliewer w.m., lider l.a., 1974 general viticulture. univ. calif. press. berkeley, usa. zoecklein p.w., wolf t.k., duncan n.w., judge j.m., cooke m.k., 1992 effects of fruit zone leaf removal on yield, fruit composition and fruit rot incidence of chardonnay and white riesling (vitis vinifera l.) grapes. am. j. enol. vitic., 43: 139-148. impaginato 21 adv. hort. sci., 2011 25(1): 21-25 received for publication 30 november 2010. accepted for publication 3 march 2011. growing spinach in a floating system with different volumes of aerated or non aerated nutrient solution a. lenzi, a. baldi, r. tesi dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: hydroponics, hypoxia, leafy vegetables, nitrate accumulation, oxygen, spinacia oleracea l. abstract: vegetables grown in a floating system may encounter problems of hypoxia at root level, especially in the summer when temperature is high. depending on the species, oxygen deficiency may cause a lower yield due to a reduction in water and mineral uptake by the plants. on the other hand, plants under oxygen stress may reduce nitrate accumulation, thus ameliorating produce quality. in the present work spinach was grown in summer and autumn in a floating system in different volumes (252, 126 and 60 l per m2 of cultivated area) of aerated or non aerated nutrient solution. aeration kept oxygen concentration at 7-8 mg l-1 while in the non aerated solution oxygen decreased gradually reaching at harvest, on average, values of 1.92 mg l-1 and 2.83 mg l 1 in summer and autumn respectively. such levels of hypoxia did not affect yield and did not reduce nitrate accumulation either. on the contrary, in the summer cycle leaf nitrate content was significantly lower when the nutrient solution was aerated. reduction of the volume of the solution to 60 l m-2 of cultivated area induced a decrease in nitrate accumulation without negative effect on yield. no significant aeration x volume interaction was observed. 1. introduction a floating system is a soilless cultivation technique where plants are grown on alveolate or fissured polystyrene panels floating in tanks containing the nutrient solution (lazzarin et al., 2001; tesi, 2002). the system is particularly suitable for growing small-size, shortcycle species like leafy vegetables, especially if aimed at producing ready-to-use salads (nicola et al., 2007). as in other hydroponic systems, plants grown in a floating system may encounter problems of oxygen deficiency (hypoxia) at root level, as roots themselves gradually consume the oxygen dissolved in the nutrient solution. oxygen deficiency may cause a reduction in water and mineral uptake by the plants, with consequences on the development of their aerial part and therefore on yield (morard and silvestre, 1996). the problem is surely more urgent in the summer, since with higher temperatures the quantity of oxygen dissolved in a solution decreases and root respiration rate increases (boisseau et al., 1988). in a floating system the stillness of the solution, that may favour the occurrence of hypoxia, is compensated for by the large volumes normally adopted (150-250 l per m2 of cultivated area) and the shortness of the growing cycles. in any case, in order to avoid any risk, growers aerate the nutrient solution to enrich it with oxygen, thus incurring additional costs. on the other hand, plants under oxygen stress are known to increase the activity of nitrate reductase (garcia-novo and crawford, 1973; lambers et al., 1978; veen, 1988), the key enzyme for nitrate utilization by plants (campbell, 1988). therefore, the reduction of oxygen concentration in the nutrient solution could actually control nitrate accumulation in hydroponically-grown vegetables, as suggested by ferrante et al. (2003). in the present work oxygen trend during summer and autumn cultivation of spinach in a floating system equipped or not with a device to aerate the nutrient solution was studied and the possible repercussions on spinach yield and quality (namely nitrate content) were investigated. the aim of the work was to verify if, when the nutrient solution was not aerated (thus achieving a cost reduction), oxygen level was kept high enough to ensure spinach yield but at the same time decreased enough to control its nitrate accumulation. furthermore, different volumes of solution were tested in order to verify if a cost saving may be achieved in the floating system by reducing water and nutrient consumption without negative effects on yield and produce quality, at least in spinach, and if such eventuality may be limited by oxygen concentration when the nutrient solution is not aerated. 22 2. materials and methods growing conditions spinach (spinacia oleracea l.) cv. seven r was cultivated in a floating system in a glasshouse in sesto fiorentino (fi, italy). two cultivation cycles were carried out, one in the summer (sowing on 1 july 2008) and the other in autumn (sowing on 30 september 2008). plants were grown in polystyrene 32.5x51.4x5 cm 160-alveoli trays (three seeds per alveolus). trays floated in polypropylene 36x56x29 cm tanks, lined with a black (inside)/white (outside) polyethylene sheet, on 42, 21 or 10 l of continuously aerated or non aerated nutrient solution (252, 126 or 60 l of nutrient solution per m2 of cultivated area, respectively). the bottom of the tanks containing 10 and 21 l of nutrient solution was raised in order to bring the cultivated panels at the same height as panels floating on 252 l m-2 of nutrient solution. aeration was provided by aquarium aerators (air flux: about 2 l hr-1 per l of nutrient solution). the nutrient solution was composed as follows (macronutrients in mmol l-1, micronutrients in µmol l1): 12 n-no3, 3.8 n-nh4, 2.8 p, 8.4 k, 3.5 ca, 1.4 mg, 40 fe, 10 mn, 40 b, 5 zn, 1 cu, 1 mo. during the cultivation cycles the nutrient solution consumed by the plants was restored once (after 10 days and 14 days of cultivation in the floating system for the summer and autumn cycle, respectively) by adding tap water. tap water was composed as follows (macronutrients in mmol l-1, micronutrients in µmol l1): 0.08 n-no3, <0.0027 n-nh4, traces of p, 0.9 k, 1.8 ca, 0.57 mg, 0.14 fe, <0.04 mn, 10.17 b, 0.09 zn, 0.36 cu, 0 mo. at harvest, the overall consumption was on average 79.9 l m-2 in summer and 74.0 l m-2 in autumn, without differences due to the imposed treatments (aeration/volume). plants were harvested on 23 july and 30 october 2008 for the summer and autumn cycle, respectively, when their developmental stage was suitable for ready-to-use salads (about 12 cm height). during the cultivation period, maximum, minimum and mean air temperature were 38.3, 19.9 and 27.5°c, and 34.3, 15.6 and 21.6°c, respectively in the summer and autumn. data collection during the cultivation period, every two-three days oxygen concentration and temperature of the nutrient solution were measured using a portable dissolved oxygen meter hi 9146 (hanna instruments, padova, italy). at the same time, ph and electric conductivity (ec) were also checked by the portable ph and conductivity meter hi 991300 (hanna instruments, padova, italy). at harvest, yield and produce quality were evaluated by collecting the following data: leaf and root fresh weight (fw, determined by weighing the whole production from each tank), leaf dry weight (dw, by ovendrying one sample of 100 g of fresh leaves per tank at 80°c until constant weight), leaf area (measured on five plants per tank using the leaf area meter li-3000 by li-cor, lincoln, ne, usa), leaf colour (estimated on 10 plants per tank using the chlorophyll meter spad-502, konica minolta, tokyo, japan) and nitrate content, which was measured spectrophotometrically on two samples per tank using the salicylic-sulphuric acid method (cataldo et al., 1975). statistics the treatments were arranged in a two-way randomized block design with three replications (one replication=one tank). data were subjected to analysis of variance (anova) and means were compared using the snk test at p=0.05 level of significance. 3. results and discussion nutrient solution aeration of the nutrient solution affected ph and oxygen concentration, while it did not show any effect on ec and temperature (fig. 1 and 2). in the aerated nutrient solution ph increased more than in the non aerated one, although without exceeding well-tolerated values for the species. aeration kept oxygen concentration at 7-8 mg l-1 while in the non aerated solution oxygen decreased gradually reaching values below 5 mg l-1 after 17 days in summer and after 21 days in autumn, fig. 1 effect of aeration (left) and volume (l per m2 of cultivated area) (right) of the nutrient solution on its o2 concentration, temperature, ph and ec during the summer cultivation cycle of spinach. error bars (±se) are shown when larger than symbols. arrows indicate the date when the consumed solution was restored with tap water. 23 that is five and nine days before harvest, respectively. therefore, when the nutrient solution was not aerated, plants were subjected to sub-optimal oxygen concentrations only for a short period of their cultural cycle. at harvest oxygen level in non aerated tanks was on average 1.92 mg l-1 in summer and 2.83 mg l-1 in autumn. no significant differences were noticed in temperature and oxygen concentration due to the volumes of the solution, while ph and ec showed variations that were greater when the volume was lower (fig. 1 and 2). those variations (a rise in ph and a decrease in ec) were due to the water added to the tanks to restore the solution consumed by the plants (about 8 l per tank in summer and 7 l in autumn), whose effect was inversely proportional to the starting volume of the solution. spinach yield and quality in summer, temperature conditions unfavourable to germination in the first week after sowing (29.5°c as mean temperature) led to a lower plant density, justifying the lower yield in the summer cycle compared to the autumn one (on average 1.6 kg m-2 and 2.7 kg m-2 of fresh leaves, respectively). on the other hand, in summer the single plants showed a larger leaf area and more developed roots (table 1 and 2). leaf and root production and leaf area did not show any significant differences due to the treatments in both the summer and the autumn cultivation cycles (table 1 and 2). fig. 2 effect of aeration (left) and volume (l per m2 of cultivated area) (right) of the nutrient solution on its o2 concentration, temperature, ph and ec during the autumn cultivation cycle of spinach. error bars (±se) are shown when larger than symbols. arrows indicate the date when the consumed solution was restored with tap water. table 1 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on leaf production (leaf fw and leaf dw), leaf area and root production (root fw) in spinach grown in a floating system in the summer season treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume leaf fw (kg m-2) leaf dw (g m-2) leaf area (cm2 plant-1) root fw (g m-2) 1.6 a 1.7 a 1.5 a 1.8 a 1.6 a ns 85.9 a 91.6 a 94.5 a 91.7 a 80.0 a ns 55.3 a 53.7 a 50.5 a 59.5 a 53.6 a ns 162.7 a 138.0 a 134.0 a 169.0 a 148.0 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. table 2 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on leaf production (leaf fw and leaf dw), leaf area and root production (root fw) in spinach grown in a floating system in the autumn season treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume leaf fw (kg m-2) leaf dw (g m-2) leaf area (cm2 plant-1) root fw (g m-2) 2.5 a 2.8 a 2.5 a 2.9 a 2.6 a ns 112.0 a 127.7 a 109.7 a 130.2 a 119.7 a ns 53.2 a 45.2 a 47.1 a 50.9 a 49.6 a ns 93.3 a 84.0 a 85.0 a 93.0 a 88.0 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. 24 spinach tolerance to hypoxia in a floating system was previously observed in spring (lenzi et al., 2008; baldi et al., 2009), in summer (lenzi et al., 2008) and in autumn (tesi et al., 2003 b); on the contrary, when cultivated in summer, spinach showed a reduction in yield due to oxygen depletion (tesi et al., 2003 b) but in this case the length of the cycle was 40 days and plants were exposed to hypoxia conditions for almost twice as long compared to the present work, as well as with respect to the work described in lenzi et al. (2008). no effect on crop yield of root hypoxia in a floating system was detected in rocket (ferrante et al., 2003; lenzi et al., 2008; baldi et al., 2009) and lamb lettuce (ferrante et al., 2005). in head lettuce, roots were exposed to oxygen concentration of 2.1 mg l-1 without any significant effect on plant growth (goto et al., 1996), while oxygen levels close to 0 mg l-1 during the last two weeks of cultivation caused an evident decrease in yield (tesi et al., 2003 a). as far as the quality aspect is concerned, the imposed treatments did not cause any differences in the colour of the product (spad values), while they showed some effects on nitrate accumulation in the summer cycle (table 3). in previous experiments cultivation in a floating system without a device to oxygenate the nutrient solution reduced nitrate accumulation in lamb lettuce (ferrante et al., 2005) and head lettuce (tesi et al., 2003 a) but did not show any effect in spinach and rocket (lenzi et al., 2008; baldi et al., 2009). in the experiment of ferrante et al. (2003) rocket showed a reduction in nitrate content when plants were subjected to anoxia conditions by bubbling nitrogen gas through the nutrient solution one week before harvesting. in the present experiment spinach nitrate accumulation was even higher when the nutrient solution was not aerated, with statistically significant differences compared to the aerated nutrient solution in the summer season (table 3). probably, to obtain a decreasing effect on nitrate accumulation by the onset of oxygen stress according to the mechanism suggested by gracia-novo and crowford (1973) (nitrate used as an electron acceptor alternative to free oxygen), prolonged conditions of anoxia or severe hypoxia are necessary. in experiment presented in this work, a decrease in leaf nitrate content was obtained also by reducing the volume of the nutrient solution, with statistically significant differences again in the summer cycle (table 3). such decrease, already observed both in spinach and rocket (baldi et al., 2009), was not correlated to oxygen, as demonstrated by the non-significant interaction aeration x volume and by the fact that oxygen concentration in the solution did not change due to its volume (fig. 1 and 2). instead, since the lower volume underwent a stronger diluting effect from the water added to restore plant consumption, the nitrate decrease was probably simply due to a lower availability of nitric ions for plants. a similar result was obtained in spinach and other fresh-cut vegetables grown in a floating system by using low-concentrated nutrient solutions (alberici et al., 2008; de pascale et al., 2008). in autumn, when environmental conditions are more favourable to plant nitrate accumulation (maynard et al., 1976), nitrate content was on average higher than in the summer. as in the summer, nitrate was lower with aeration and with the lower volume, although the differences with respect to non aerated conditions and higher volumes, respectively, were not statistically significant (table 3). 4. conclusions from the literature it appears that when vegetables are grown in a floating system without devices to enrich the nutrient solution with oxygen, root hypoxia may have or not a negative effect on yield depending on the species, the season and the length of the growing cycle. the season and duration of the cycle result in different hypoxia levels and more or less prolonged exposure to it. in spinach, in the case of short cycles (20-30 days), both in summer and autumn, hypoxia was not severe enough to influence yield even with low volumes of nutrient solution (up to 60 l per m2 of cultivated area). extent and duration of oxygen deficiency are probtable 3 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on spad and leaf nitrate content in spinach grown in a floating system in the summer and autumn seasons treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume spad leaf nitrate (mg kg-1 fw) spad leaf nitrate (mg kg-1 fw) 24.0 a 26.2 a 24.6 a 25.1 a 25.7 a ns 3610 b 4085 a 4090 a 3901 ab 3553 b ns 23.6 a 24.9 a 23.9 a 23.7 a 25.2 a ns 4495 a 4814 a 4822 a 4780 a 4361 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. summer cycle autumn cycle 25 ably the most important factors influencing also nitrate accumulation. while anoxia or severe hypoxia may reduce vegetable nitrate accumulation (ferrante et al., 2003; tesi et al., 2003 a), we found that in the case of short exposure to oxygen depletion up to 2-3 mg l-1 not aerating the nutrient solution may result in an even higher nitrate content in spinach leaves. therefore, although the necessity to aerate or not the nutrient solution in a floating system depends on the aim (yield/quality) and the specific situation, we support the use of aeration. furthermore, reduction of the volume of the nutrient solution per m2 of cultivated area, which did not cause any effect on spinach yield and reduced nitrate accumulation up to 60 l, appears an interesting strategy in order to save water and fertilizers and at the same time ameliorate produce quality. acknowledgements this research was funded by the italian ministry for education, university and research, research project prin 2006 “nitrate uptake regulation in leafy vegetables grown under limited oxygen conditions”. references alberici a., quattrini e., penati m., martinetti l., gallina p.m., ferrante a., schiavi m., 2008 effect of the reduction of nutrient solution concentration on leafy vegetables quality grown in floating system. acta horticulturae, 801: 1167-1176. baldi a., lenzi a., cillerai g., tesi r., 2009 controllo del tenore in nitrato in rucola e spinacio coltivati in floating system. proceedings “xxviii convegno sia”, florence, italy, 21-23 september, pp. 421-422. boisseau y., maertens c., morard p., 1988 influence de la température sur la respiration du blé. 7th. colloquium international association optimization plant nutrition, copenhagen, denmark. campbell w.h., 1988 nitrate reductase and its role in nitrate assimilation in plants. physiol. plant., 74: 214-219. cataldo d.a., haroon m., sehrader l.e., youngs v.l., 1975 rapid colorimetric determination of nitrate in plant tissue by titration of salicylic acid. commun soil sci. plant. anal., 6: 71-80. de pascale s., maggio a., orsini f., bottino a., barbieri g., 2008 sulphur fertilisation affects yield and quality in friarielli (brassica rapa l. subsp. sylvestris l. janch. var. esculenta hort.) grown in a floating system. j. hort. sci. biotechnol., 83: 743-748. ferrante a., incrocci l., maggini r., tognoni f., 2003 preharvest and postharvest strategies for reducing nitrate content in rocket (eruca sativa). acta horticulturae, 628: 153-159. ferrante a., quattrini e., martinetti l., schiavi m., maggiore t., 2005 per la quarta gamma. colture protette, 12: 72. garcia-novo f., crawford r.m.m., 1973 soil aeration, nitrate reduction and flooding tolerance in higher plants. new phytol., 72: 1031-1039. goto e., both a.j., albright l.d., leed a.r., 1996 effect of dissolved oxygen concentration on lettuce growth in floating hydroponics. acta horticulturae, 440: 205-210. lambers h., steingröver e., smakman g., 1978 the significance of oxygen transport and metabolic adaptations in flood-tolerance in senecio species. physiol. plant., 43: 277-281. lazzarin r., enzo m., pimpini f., 2001 floating system. in: pimpini f. (eds.) principi tecnico-agronomici della fertirrigazione e del fuori suolo. veneto agricoltura, padova, italy, pp. 204. lenzi a., baldi a., tesi r., 2008 effect of hypoxia on yield and quality of leafy vegetables grown in floating system. abstracts “first symposium on horticulture in europe”, vienna, 17-20 february, pp. 212-213. maynard d.n., barker a.v., minotti p.l., peck n.h., 1976 nitrate accumulation in vegetables. adv. agron., 28: 71-118. morard p., silvestre j., 1996 plant injury to oxygen deficiency in the root environment of soilless culture: a review. plant and soil, 184: 243-254. nicola s., hoeberechts j., fontana e., 2007 ebb-andflow and floating systems to grow leafy vegetables: a review for rocket, corn salad, garden cress and purslane. acta horticulturae, 747: 585-593. tesi r., 2002 colture fuori suolo in orticoltura e floricoltura. edagricole, bologna, italy, pp. 112. tesi r., lenzi a., lombardi p., 2003 a effect of different o2levels on spinach (spinacia oleracea l.) grown in a floating system. acta horticulturae, 614: 631-637. tesi r., lenzi a., lombardi p., 2003 b effect of salinity and oxygen level on lettuce grown in a floating system. acta horticulturae, 609: 383-387. veen b.w., 1988 influence of oxygen deficiency on growth and function of plant roots. plant and soil, 111: 259-266. 232 1. introduction a key to success for the planting of new trees, both in open-field nursery and in the urban environment, is the protection of young plants from non-crop plant species (including some hardwoods, shrubs, grasses, and forbs). these fast-growing plants often kill or greatly suppress desired trees by competing with them for light, water, and nutrients. as a result, nurserymen, arborists and urban forest managers generally use herbicides to suppress noncrop vegetation. however, the use of herbicides in the urban environment may be limited or even banned in certain countries and/or municipalities. as a consequence, to protect young trees, environmentally sound, effective, cost-efficient, and socially acceptable techniques for managing non-crop vegetation are needed. in this scenario, we focused on the need for the establishment of environmentally friendly and low cost management methods for nurseries and urban green areas. mulching and its skilled use can contribute to such a development by improving organic matter content in the soils and by affecting other soil characteristics (harris et al., 2004). for tree management in the urban landscape, especially in the first years after planting, mulching with organic materials can be advantageous. organic mulching with different materials (mainly shredded wood, chipped wood, pine bark and composted materials), when skillfully applied, is an environmentally friendly way of establishing, protecting and managing young trees at a low cost in a new plantation. a recent review compared the costs and benefits of landscape mulches as reported in the technical and scientific literature, underlining how plants and soil can both benefit in terms of weed suppression, evaporation reduction and other environmental modifications (chalker-scott, 2007). even if mulching is a world-wide practise in urban green areas and different materials (as noted above) can be used for this purpose (rakow, 1989), little research has been done in italy to determine its effectiveness. positive effects following organic mulch application have been obtained in previous research, demonstrating beneficial effects on soil physical and chemical properties (fraedrich and ham, 1982; litzow and pellett, 1983; watson, 1988; appleton et al., 1990; himelick and watson, 1990; smith and rakow, 1992; iles and dosmann, 1999; dahiya et al., 2007; tiquina et al., 2007) and on plant growth and physiology (watson, 1988; green and watson, 1989; appleton et al., 1990; himelick and wateffects of mulching with compost on growth and physiology of acer campestre l. and carpinus betulus l. a. fini, f. ferrini dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: chlorophyll fluorescence, leaf gas exchange, mulching, soil respiration, soil temperature, spad. abstract: the aim of this work was to evaluate the effects of mulching with compost on growth and leaf gas exchange of two widely-used ornamental trees in comparison to local nursery management standards. in addition, effects on soil respiration, soil temperature and water evaporation from soil were determined. an equal number (180 each) of uniform hedge maple (acer campestre l.) and 180 hornbeam (carpinus betulus l.) were planted in an experimental plot located in pistoia. treatments compared were: 1) chemical weeding by herbicides; 2) natural grass cover, mowed twice per year; 3) harrowing once a year; and 4) mulching with mixed compost (50% green+50% from household waste, 5 to 10 cm thick). over a two-year period, stem diameter, shoot extension and leaf gas exchange were measured. in the second year leaf chlorophyll content, chlorophyll fluorescence, soil respiration, soil evaporation content and soil temperature were also recorded. mulching with compost influenced shoot extension and stem diameter growth of field maple and white hornbeam. plants grown with natural grass cover had, generally, smaller stem diameters and shoot growth than the other treatments. leaf gas exchange, chlorophyll content and chlorophyll fluorescence were influenced by the different treatments. soil respiration was unaffected by the different treatments while soil temperature was significantly lower in mulched plots. adv. hort. sci., 2011 25(4): 232-238 received for publication 5 august 2011 accepted for publication 9 november 2011 233 son, 1990). however, sometimes the results from mulching are variable as they are affected by different environmental conditions and different tree species (whitcomb, 1979; iles and dosmann, 1999). moreover, if the quality of the mulching materials supplied by the producers is not satisfactory, tree performances can be affected in a negative way. this fact can be related either to quality or misuse, i.e adding too much material which can negatively affect soil oxygen content (gilman and grabosky, 2004; hanslin et al., 2005), although watson and kupkowski (1991) found no detrimental effect from the application of 0.45 m of wood chip mulch over soil in which the roots of trees were growing. the application of bark mulch can sometimes decrease growth in the first year, but the effects on plant growth are positive when examined in the long term (samyn and de vos, 2002). this can be due to a temporary nitrogen depression until the microorganisms are able to decompose a sufficient amount of organic material to provide the needed nitrogen (craul, 1992). in fact, although this temporary depression mainly affects the interface between mulch and soil, it has been shown that fine roots tend to grow into the organic mulch layer (watson, 1988; watson and kupkowski, 1991), where nconcentration is temporary depressed. as far the composted material as concerned, it has to be remarked that it needs to be well characterised for nutrient values, stability and other properties for the support of tree growth and effect against weeds. in a review of the use of composts for mulching and soil amendments, sæbø and ferrini (2006) suggest designing the composts to fit the specific effects that are wanted. for example composts for mulching should consist of layers of compost of different particle sizes, so that nutrients can be supplied and weeds are not given good germination conditions. the purpose of the present study was to investigate the use of mulching materials and their possible influence on growth and physiology of two shade tree species widely grown in the urban environment. in addition, effects on soil respiration, soil temperature and water evaporation from soil were assessed in the second year of the experiments. 2. materials and methods one hundred eighty uniform acer campestre l. and 180 carpinus betulus l. two-year-old seedlings 1.20 m in height from container were planted in an experimental plot located in pistoia (43°56’ n; 10°54’ e). planting density was 1.80 m in-row and 2.50 m between rows. mean temperature and rainfall over the last 50 years are 14.3°c and 1257 mm/year respectively. however, during the years of the current experiment, a decrease in rainfall and an increase in mean temperatures were recorded: rainfall in the first and second year was 971 and 903 mm, respectively; while in both the first and second year, an extended dry period with no rainfall was recorded from early june to the end of july. plants were irrigated the first year after planting to help establishment. from the second year no irrigation or fertilization were applied to plants. after two years from planting, when trees had overcome transplant phase, the experiment was started and the following thesis were compared: 1) weeding by herbicides using glyphosate twice a year (w); 2) natural grass cover, which was mechanically cut twice per year (g); 3) tillage by harrowing at a depth of 15 cm, once per year (t); and 4) in-row mulching with green compost (layer 5-10 cm thick and 1.5 m wide) and natural grass cover soil between the rows (m). the experimental design was a randomized complete block with six blocks and 30 plants per block. the following year (three years after planting and one year after treatments), biometric and physiological analyses were performed. stem diameter was measured 5 cm above the root flare on all plants at the end of the growing season. at the same time shoot extension was determined on 10 shoots per plant on three plants per block (18 plants per treatment). during the growing season, leaf gas exchange was measured with an infrared gas analyzer (ciras-2, pp-system, new hertfordshire, uk) on six leaves per block (36 leaves per treatment x species) twice during the first growing season and four times during the second. measured parameters were net assimilation (a, µmol*m2*s-1) and transpiration (e, mmol*m-2*s-1). measurements were taken between the hours 8.00 and 13.00 on the first fully expanded leaf from the shoot apex. measurements were taken under fixed co 2 concentration (360 ppm) and saturating irradiance (1300 µmol*m-2*s-1) provided with a built-in red light emitting diode radiation source. water use efficiency (wue) was calculated as the ratio between a and e as described in a previous work (ferrini et al., 2008). leaf greenness index was determined in the second growing season with a minolta spadmeter (spectrum technologies, plainfield, il, usa) on six leaves per block (36 leaves per treatment x species). for each leaf, the spad value was obtained averaging three different measurements made in different points of the leaf blade. this parameter is a good indicator of leaf chlorophyll, n and carotenoid content (percival et al., 2008). chlorophyll fluorescence was measured three times in the second year with a portable plant efficiency analyzer handypea (hansatech ins., king’s lynn, uk) on six leaves per block (36 leaves per treatment x species). fv/fm values were obtained by placing leaves in darkness for 30 min by attaching light-exclusion clips to the leaf surface. fv/fm is the maximum quantum yield of the psii and it is a reliable indicator of the occurrence of environmental stress (maxwell and johnson, 2000). soil respiration and evaporation were measured with a soil respiration chamber (src, pp-system, new hertfordshire, uk) at a depth of 5 cm below soil surface. measurements were taken twice in july around midday on soil exposed to full sunlight (not shaded by the trees). at least 30 days had passed since the last remarkable rainfall 234 (fig. 1). four measurements per treatment per species and per replicate were made. before measuring respiration and evaporation, the mulch was removed from an area of about 25 cm2 and the chamber was placed on the soil beneath. after taking the measurements, mulch was spread again. soil temperature was measured with a temperature probe at a depth of 10 cm below soil surface four times per treatment per species and per replicate. all data were analyzed with glm using the spss statistical package for windows (spss inc., chicago, il, usa). effects of soil management technique and species were analyzed with a random model two-way anova. when no significant interaction between factors was found, differences among soil management techniques were tested with duncan’s multiple range test (p≤0.05 and p≤0.01). parameters which showed significant interaction between factors were plotted separately in order to compare each level of factor a (soil management) for each level of factor b (species) (chew, 1976). data on leaf gas exchange, fv/fm and soil parameters were analyzed per single sampling date, merged together and processed again to obtain an average value on annual basis. 3. results and discussion mulching with compost (m) affected shoot growth of acer campestre and carpinus betulus (table 1). significant interaction between species and soil management technique was found for shoot growth (table 1). in the first year of measurements, mulched plants of both species had higher shoot growth than the other treatments (table 2). in maple, no difference was found among treatments w (herbicide), t (tilling) and g (ground cover). in hornbeam, shoot growth was lower in g than in t and w. in the second year, m carpinus plants had greater shoot growth than w and t which, in turn, had longer shoots than g. in the second year, m and t acer had greater shoot growth than w. again, the shortest shoots were found in g maples. in the first year and second years, g plants had lower stem ditable 1 summary table of two-way anova for the investigated parameters parameter measurement unit management technique species management x species shoot growth (1 year) cm ** ** ** shoot growth (2 year) (g) ** ** ** stem diameter (1 year) (g) ** ns ns stem diameter (2 year) (g) ** ns ns leaf greenness index spad unit ** ** ns fv/fm ** ns ns a (1 year) μmol m-2 s-1 ** ** ns a (2 year) μmol m-2 s-1 ** ** ns e (1 year) mmol m-2 s-1 ns ** ns e (2 year) mmol m-2 s-1 ** ** ns wue (1 year) μmol co2/mmol h2o ns ns ns wue (2 year) μmol co2/mmol h2o ** ** ns soil temperature (2nd year) ° c ** ns ns soil respiration (2nd year) μmol m-2 s-1 ns ns ns soil evaporation (2nd year) mmol m-2 s-1 ** ns ns * and ** indicate significant differences between treatments at p<0.05 and p<0.01, respectively. table 2 effect of soil management technique on shoot extension in acer campestre and carpinus betulus in the first and second years shoot growth (cm) acer campestre carpinus betulus treatment 1 year 2 year 1 year 2 year chem. weeding 70.2 b 58.3 b 70.0 b 44.7 b tillage 70.0 b 57.5 b 67.0 b 53.4 a grass cover 69.6 b 45.5 c 57.1 c 35.6 c mulching 86.0 a 72.0 a 74.0 a 54.4 a different letters within the same column indicate significant differences at p≤ 0.01. fig. 1 rainfall from 1 june (day 1) to 31 july (day 61) in 2006 and 2007 measured in the experimental centre where the research was carried out. 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 1st year 2nd year weeks m m o f ra in 235 ameter than the other treatments (table 3); no differences in stem diameter were found between species (table 3). there are several reports on how turf or natural grass cover decrease plant growth (garrity and mercado, 1994; stork and jerie, 2003; yao et al., 2005; chalker-scott, 2007). in the present experiment, natural grass cover decreased shoot growth and stem diameter in both the species studied. also, carpinus betulus, whose growth was affected both in the first year and second year, is probably a worse competitor with turf than acer campestre. mulch increased growth of both species, and mulched plants had shoot growth and stem diameter similar or higher than plants grown in tilled soil. greater plant growth in response to mulching has been observed by many authors (sæbø and ferrini, 2006; chalker-scott, 2007; ferrini et al., 2008). some authors found that mulching decreased growth in the first year after application and most of the authors attributed this reduced growth to temporary immobilization of soil n due to the high c/n ratio of the mulch (ferrini et al., 2009). the mulch applied in this experiment had relatively low c/n ratio (about 30), so no nitrogen immobilization occurred and growth was enhanced even in the first year after application, in agreement with that reported in previous works (tilander and bonzi, 1997; erhart and hartl, 2003; sonsteby et al., 2004; granatstein and mullinix, 2008). leaf greenness index was affected by the different treatments and species (table 4). regardless of the species, mulched plants showed higher values than the other treatments. the lowest readings were found in w and g plants, whereas t plants performed intermediately. higher spad readings following low c/n mulch application were also found by granatstein and mullinix (2008), who found a soil n-enrichment due to mulch mineralization. we did not consider the effect of management technique on soil n, however according to percival et al. (2008), the higher spad-reading of m plants may reflect a better nutritional status generated by mulch application and its decomposition. leaf greenness index was also affected by species, with field maple having higher values than hornbeam (table 4). fv/fm was affected by soil management technique but not by species. fv/fm measurements of healthy, unstressed plants are associated with values ranging from 0.76 to 0.85 (percival, 2004; percival et al., 2006). regardless of the treatment, all plants in this experiment were subjected to some degree of stress because of the very low rainfall during the summer (fig. 1), since fv/fm values were lower than 0.75. in any case, m and t plants had a significantly higher fv/fm than g plants (table 4). fv/fm is very sensitive to oxidative stresses, and to drought stress in particular (angelopulos et al., 1996; maxwell and johnson, 2000; rong-hua et al., 2006). thus, the lower spad and fv/fm observed in g plants must be attributed to grass competition for nutrients and water. leaf gas exchange was affected by soil management technique and species (table 5). in the first year, net assimilation was higher in m plants than in the other treatments. in the second year, m and t had greater assimilation than g and w. transpiration was not affected by management technique in the first year, while in the second year t plants had greater transpiration than the other treatments. water use efficiency was not affected by soil management technique in the first year. in the second year, m and w had greater wue when compared to t and g. leaf gas exchange was also affected by species: hedge maple always showed higher values than hornbeam (table 5). a significant quadratic relation was found for acer (p<0.01; r2=0.855) and carpinus (p<0.01; r2=0.298) between leaf greenness index and net assimilation (fig. 2). leaf greenness index has shown to be accurate in predicting leaf n content (follett et al., 1992; wood et al., 1992). in our experiment, the relationship between spad and a was much stronger in hedge maple than in hornbeam, suggesting a n limitation to photosynthesis in the case of maple and a less n-dependant limitation in hornbeam. soil temperature was affected by management technique: in july (second growing season) plots mulched with compost were 13°c, 10.8°c and 7.2°c cooler than bare soil, tilled and turf plots respectively (table 6). this is consistent with previous works, which found that mulching is more effective than cover crops and tillage to reduce extreme summer temperatures and that, in dry climates, mulching can lead to a reduction in soil temperature of up to 10°c (martin and poultney, 1992; zhang et al, 2009). table 3 effects of soil management technique and species on stem diameter growth in the first and second years stem diameter (cm) 1 year 2 year effect of different treatments chem. weeding 6.5 a 7.5 a tillage 6.1 a 7.6 a grass cover 5.6 b 6.7 b mulching 6.2 a 7.9 a effect of species a. campestre 6.1 7.4 c. betulus 6.1 7. 4 different letters within the same column indicate significant differences at p≤0.01. table 4 effects of soil management technique and species on leaf greenness index and fv/fm in the second year greenness index (spad) fv/fm effect of different treatments chem. weeding 38.7 c 0.721 ab tillage 40.2 b 0.731 a grass cover 38.8 c 0.701 b mulching 42.3 a 0.743 a effect of species a. campestre 43.0 a 0.728 c. betulus 40.0 b 0.719 data are the average of two measurement campaigns made in the second year. different letters within the same column indicate significant differences at p≤ 0.01. 236 the significant reduction in soil temperature contributed to create a more favourable environment for root growth: soil temperature in mulched plots did not exceed 35°c, which is considered the threshold temperature above which root growth is hampered and root mortality increases (coder, 1996; fini and ferrini, 2007). the reduction of soil temperature under mulch is due to the low albedo and thermal conductivity of woody mulches (montague and kjelgren, 2004). this finding means that the radiation reaching the mulch was not reflected, but the mulch acted as insulation and prevented energy from being conducted to the soil. soil temperature under natural grass cover was lower than tilled and bare soil, but higher than mulch. the cover crop acted as a barrier which absorbed solar energy and shaded soil surface, but it also transpired soil water, reducing soil water content and, by consequence, its buffering capacity (zhang et al., 2007, 2009). this is confirmed by water evaporation from soil (table 6). evaporation was measured 30 days after the last rainfall. in the mulched plot, the mulch layer was temporary removed and the measurement taken on the soil below. the lowest evaporation was found in g plots and the highest value was found in m plots. in absence of irrigation and natural rainfall, the higher value measured in mulched soil provides further confirmation of the effectiveness of mulching to reduce evaporation: after 30 days of drought, soil was very dry fig. 2 relationship between leaf greenness index (chl, spad-units) and net assimilation (a, mmol*m-2*s-1) in acer campestre and carpinus betulus. table 5 effect of soil management technique and species on net assimilation (a, µmol*m-2*s-1), transpiration (e, mmol*m-2*s-1) and water use efficiency (wue, µmol co 2 /mmol h 2 o). data are the average of two (the first year) and four (the second year) measurement campaigns a (μmol m-2 s-1) e (mmol m-2 s-1) wue (μmol co2/ mmol h2o) 1 year 2 year 1 year 2 year 1 year 2 year effect of different treatments chem. weeding 8.2 b 6.3 b 2.5 2.0 b 3.6 3.5 a tillage 8.4 b 7.5 a 2.5 2.6 a 3.8 2.9 b grass cover 7.7 b 6.3 b 2.2 2.1 b 3.8 3.1 b mulching 9.3 a 7.5 a 2.5 2.0 b 4.0 3.7 a effect of species a. campestre 10.3 a 8.3 a 2.9 a 2.4 a 3.8 3.6 a c. betulus 6.5 b 5.5 b 1.9 b 1.9 b 3.7 2.9 b different letters within the same column and factor indicate significant differences at p<0.01. table 6 effect of the different treatments on soil temperature (measured 10 cm below soil level), soil evaporation (measured 5 cm below soil level), and soil respiration (measured 5 cm below soil level). data are the average of two measurement campaigns made in the second year treatment soil temperature (°c) soil evaporation (mmol m-2 s-1) soil respiration (µmol m-2 s-1) chem. weeding 43.4 a 28.0 b 0.3 tillage 41.2 a 29.7 b 0.4 grass cover 37.6 b 18.3 c 0.3 mulching 30.4 c 94.2 a 0.5 different letters within the same column indicate significant differences at p≤ 0.01. 237 in treatments g, w and t and no further evaporation was possible. on the contrary, moisture was still available under the mulch so that, after mulch removal, evaporation was higher than in the other treatments. soil respiration was somewhat higher in m and t plots, but differences were not significant (p=0.172). most of the parameters measured showed that plants performed better when mulching was used and there are probably multiple causes which determined these results. the effect of mulching in reducing soil temperature which can allow a higher root growth is probably the most important under the conditions of this study, also because it might have prevented dehydration. compost mineralization might also have increased soil nutrient content. these effects could have allowed a greater root growth and, as a consequence, greater water and nutrients absorption. unfortunately we did not analyze the soil and this might be a potential shortcoming of this research. 4. conclusions the use of compost as mulching material had great impact on plant growth and physiology. in agreement with the results obtained in a previous work (ferrini et al., 2008), the present study confirms that mulching with compost is a useful practice to improve plant growth, leaf gas exchange and leaf chlorophyll content. positive effects of organic mulching on soil organic matter, soil water holding capacity and weed suppression have already been revealed in previous works (sæbø and ferrini, 2006; chalker-scott, 2007; granatstein and mullinix, 2008; mulumba and lal, 2008). the present investigation provides the evidence that mulching also has positive effects on plant growth and physiology, comparable or even superior to tillage. despite being inexpensive and very effective in weed control, the use of chemical herbicides reduced shoot growth and gas exchange when compared to mulching. an even greater reduction in growth and carbon assimilation and a significant increase of oxidative stress on psii was found in plants growing with natural grass cover, mechanically cut twice per year. considering that mulching is cheaper than tillage and mechanical weeding (zhang et al., 2009), it can be considered an environmentally-friendly and sustainable alternative for managing plants in the forest, in the nursery and in the urban environment. acknowledgements we 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bird g.w., abawi g.s., thies j.e., 2005 orchard floor management practices that maintain vegetation or biomass groundcover, stimulate soil microbial activity and alter soil microbial community composition. plant & soil, 271: 377-389. zhang s., lövdahl l., grip h., jansson p.e., tong y., 2007 modelling the effects of mulching and fallow cropping on water balance in the chinese loess plateau. soil till. res., 93: 283-298. zhang s., lövdahl l., grip h., tong y., yang x., wang q., 2009 effects of mulching and catch cropping on soil temperature, soil moisture and wheat yield on the loess plateau of china. soil till. res., 102: 78-86. 75 1. introduction peach (prunus persica l.), a member of the rosaceae family, is one of the most economically important fruit species in the world, with italy being the second world producer after china and the first in europe (faostat, 2012). the flesh color of the fruit, white or yellow, is an important characteristic driving consumer choice and thus represents a key breeding trait for this crop. from a genetic point of view, it is controlled by a single locus and the white phenotype is fully dominant over the yellow (connors, 1920; falchi et al., 2013). from a biochemical point of view, yellow flesh color is determined by the accumulation of carotenoids in chromoplasts. in particular, in the early stages of the fruit developmental cycle, both whiteand yellow-fleshed peaches have very high concentrations of total carotenoids, although masked by the green due to the presence of chlorophyll (ma et al., 2014). in the later stages of the fruit cycle, carotenoids are massively degraded in the white-fleshed, but not in the yellowfleshed fruits, which keep accumulating these compounds and whose content peaks at full fruit ripening (ma et al., 2014). recent studies carried out on peach chimeric mutants for flesh color showed a strongly reduced expression of ccd4, the gene codifying for dioxygenase in the yellowfleshed genotype compared to its white-fleshed mutant, assessment of antioxidant activity of carotenoidenriched extracts from peach fruits using the new lox/rno method m.n. laus 1(*), m. soccio1, d. giovannini 2, e. caboni 3, i. quacquarelli 2, m.l. maltoni 2, f. scossa 3, e. condello 3, d. pastore 1 1 dipartimento di scienze agrarie, degli alimenti e dell’ambiente, università degli studi di foggia, via napoli 25 71122 foggia, italy. 2 consiglio per la ricerca e la sperimentazione in agricoltura, unità di ricerca per la frutticoltura, cra-frf, via la canapona 1 bis 47121 forlì, italy. 3 consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la frutticoltura, cra-fru, via fioranello, 52, 00134 roma, italy. abbreviations: aa, antioxidant activity; aaph, 2,2’-azobis(2-amidinopropane); abts, 2,2’-azino-bis-(3-ethylbenzothiazoline6-sulfonate); auc, area under curve; dpph, 2,2-diphenyl-1-picrylhydrazyl radical; frap, ferric reducing antioxidant power; fluorescein, 3’,6’-dihydroxyspiro[isobenzofuran-1[3h], 9’[9h]-xanthen]-3-one; f.w., fresh weight; lox, lipoxygenase (linoleate: oxygen oxidoreductase, ec 1.13.11.12); orac, oxygen radical absorbance capacity; rno, 4-nitroso-n,n-dimethylaniline; teac, trolox equivalent antioxidant capacity; trolox, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid. key words: antioxidant activity, carotenoids, lox/rno method, peach. abstract: peach (prunus persica l.) fruits contain several health-promoting phytochemicals. among these, carotenoids, in addition to being involved in determining flesh color, play a relevant role in cell protection against oxidative stress. nevertheless, antioxidant activity (aa) of peach carotenoids so far has not been investigated in as much detail as phenols. in the present study, for the first time, aa of peach carotenoid extracts was evaluated using the innovative lipoxygenase/4nitroso-n,n-dimethylaniline (lox/rno) method, able to simultaneously detect different antioxidant mechanisms and synergistic antioxidant interactions, as well as using the well-known orac and teac assays. in particular, extracts were obtained from fruits collected in s4 development stage from two yellow-fleshed (armking and redhaven) and three white-fleshed (silverking, caldesi 2000, iff331) varieties. the lox/rno method gave high aa values (10-150 µmol eq. trolox/g f.w.), about 85-1900-fold higher than orac and teac methods. moreover, the ratio between aa values, measured by the lox/rno method, of yellowand white-fleshed peaches resulted equal to 14, but only 2.6 and 3.6 for orac and teac, respectively. results of this study indicate that the lox/rno method, measuring high aa values and easily discriminating among samples, is an advisable tool to assess the aa of the carotenoid component in peach. adv. hort. sci., 2015 29(2-3): 75-83 (*) corresponding author: maura.laus@unifg.it received for publication 26 september 2014 accepted for publication 9 march 2015 76 adv. hort. sci., 2015 29(2-3): 75-83 suggesting a key role of this gene in controlling carotenoid accumulation, hence flesh color in peach (brandi et al., 2011; adami et al., 2013). in addition to their role in peach flesh color, carotenoids also represent relevant biologically active compounds, responsible for some beneficial health properties associated to increased consumption of fruit and vegetables (liu, 2003 and references therein; schreiner and huyskens-keil, 2006). in particular, β-carotene, α-carotene and β-cryptoxanthin have an essential function in human nutrition as they are a primary dietary source of provitamin a, which humans are unable to synthesize de novo. moreover, the antioxidant properties of carotenoids, that may act as free-radical scavengers, singlet oxygen quenchers, and lipid antioxidants, have been reported to be highly involved in reducing the risk of cardiovascular disease, cancer, diabetes and other degenerative diseases associated with oxidative stress (rao and rao, 2007; jomova and valko, 2013). in recent years several studies have been performed to investigate the antioxidant properties of peach fruit due to carotenoids, as well as anthocyanins and other phenolic compounds; nevertheless, the role of carotenoids so far has not been completely clarified. peach carotenoid content has been quantified in these reports, as well as antioxidant activity (aa) evaluated using assays measuring i) the peroxyl radical scavenging capacity (oxygen radical absorbance capacity, orac) (campbell and padilla-zakour, 2013), ii) the 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (abts) radical cation scavenging capacity (trolox equivalent antioxidant capacity, teac) (dalla valle et al., 2007; di vaio et al., 2008; legua et al., 2011; garcía-parra et al., 2011; oliveira et al., 2012), iii) the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity (gil et al., 2002; lavelli et al., 2009; puerta-gomez and cisneros-zevallos, 2011; zielinski et al., 2014), and iv) the fe3+ ion reducing capacity (ferric reducing antioxidant power, frap) (gil et al., 2002; remorini et al., 2008; tavarini et al., 2008; durst and weaver, 2013; zielinski et al., 2014). in most of these studies, aa was evaluated by studying methanol extracts. only in very few reports, the aa of both waterand fat-soluble antioxidant fractions (obtained from fruit extraction using water and acetone or thf or ethyl acetate, respectively) was investigated separately (dalla valle et al., 2007; di vaio et al., 2008; legua et al., 2011). this was made possible by using only the teac assay, measuring mainly the antioxidant reducing capacity; interestingly, generally lower aa values of the lipophilic antioxidant fraction compared to those of the hydrophilic component were obtained, positively correlated to total carotenoid content (legua et al., 2011). in the present study, aa of extracts highly enriched in carotenoids obtained from the flesh of peach fruits was evaluated for the first time using the advanced and innovative lipoxygenase/4-nitroso-n,n-dimethylaniline (lox/rno) method (pastore et al., 2009). this is a recently developed aa assay based on the rno bleaching reaction due to some radical species generated by secondary anaerobic reactions catalyzed by soybean lox-1 isoenzyme, occurring when the main aerobic cycle of linoleic acid (lh) hydroperoxidation has consumed oxygen (pastore et al., 2000 a) (fig. 1). with respect to the majority of aa assays, the lox/rno method is able to detect the scavenging capacity of antioxidants towards different physiological and biologically relevant radical species, such as alkoxyl (lo•), peroxyl (loo•), hydroxyl (•oh) and perhaps alkylic (l•) radicals, as well as singlet oxygen (1o 2 ), but this latter only in the presence of imidazole (pastore et al., 2000 a; 2009). moreover, other important antioxidant functions may be simultaneously detected, including chelating or reducing activities of iron ions essential for the catalysis and generation of radical species, as well as any possible inhibition of the apo-enzyme (pastore et al., 2009) (fig. 1). consistently, the lox/rno method has been found to highlight very well synergistic effects among antioxidants (pastore et al., 2009; laus et al., 2012 b; laus et al., 2013 b). to date, the lox/rno method has been applied to assess aa of food-grade antioxidants (laus et al., 2013 b) and of whole flour of some cereals (flagella et al., 2006; pastore et al., 2009; laus et al., 2012 b), pseudocereals (laus et al., 2012 a), and grain-derived products (laus et al., 2013 a), showing a much higher performance with respect to other commonly used aa assays. fig. 1 schematic representation of the aerobic and anaerobic cycles catalyzed by the soybean lipoxygenase (lox)-1 isoenzyme involved in the bleaching of the 4-nitroso-n,n-dimethylaniline (rno). the soybean lox-1 apoenzyme is represented as eshaped and fe3+ indicates non-heme iron atom essential for the lox-1 catalysis. the aerobic cycle of soybean lox-1-mediated hydroperoxidation of linoleic acid (lh) is schematized, as well as secondary anaerobic reactions involving generation of radical species, able to induce the rno bleaching. the short black arrows indicate some different mechanisms by which antioxidant compounds may inhibit the lox-1-dependent rno bleaching. antiox, antioxidant compounds; lo•, loo•, l•, alkoxyl, peroxyl and alkylic radicals of lh; looh, 13-hydroperoxy derivative of lh; 1o 2 , singlet oxygen; •oh, hydroxyl radical. 77 laus et al., antioxidant activity of peach carotenoid extracts and the lox/rno method the objective of this study was to evaluate the performance of the lox/rno method with respect to aa assessment of the carotenoid fraction extracted from the flesh of peach fruits of some yellow and white varieties. to do this, the lox/rno method was compared with two well-established methodologies for aa measurement: the teac (re et al., 1999) and orac (ou et al., 2001) assays. 2. materials and methods chemicals all reagents at the highest commercially available purity were purchased from sigma-aldrich corp. (st. louis, mo., u.s.a.). plant material fruit from yellow-fleshed (armking and redhaven) and white-fleshed (silverking, caldesi 2000 and iff331) cultivars/breeding selections was harvested from trees grown in the experimental farm of cra-fru (roma, italy) and cra-frf (forlì, italy). replicates of three representative fruits at stage s4 (full ripening phase) of fruit development were sampled, peeled, cut into 0.5-cm slices and the mesocarp was immediately frozen in liquid nitrogen and stored at -80°c. preparation of aqueous solutions of linoleate and ß-carotene the sodium linoleate solution was prepared as described in pastore et al. (2000 b; 2009) and the exact linoleate concentration was determined by means of the lox assay (pastore et al., 2000 b), by using a perkin-elmer λ45 uv-vis spectrophotometer (perkin-elmer, wellesley, ma), managed by uv winlab software (perkin elmer version 2.85.04). the β-carotene solution was prepared according to the method reported by pastore et al. (2000 b; 2009), and the concentration was spectrophotometrically determined as described in pastore et al. (2000 b). extraction of carotenoids from peach fruit extraction was carried out according to the procedure described in fraser et al. (2000) with some modifications. all manipulations were carried out on ice and shielded from strong light. freeze-dried material (about 3.5 and 5 g for whiteand yellow-fleshed fruits, respectively) was finely ground into a powder with a mortar and pestle using liquid nitrogen. methanol was added according to a (v/w) ratio equal to 7.5 ml/g of ground tissue; the suspension was mixed by inversion and incubated for 5 min in an icewater bath. then, 50 mm tris-hcl buffer ph 7.5 (containing 1 m nacl) was added to the mixture according to a (v/w) ratio equal to 7.5 ml/g of homogenized tissue and incubated in an ice-water bath for 10 min. chloroform at a (v/w) ratio equal to 2 ml/g of tissue was added to the mixture, incubated on ice for 10 min and then centrifuged at 5000xg for 5 min at 4°c. the aqueous phase was removed and the chloroform hypophase was pooled. determination of total carotenoid content was performed on chloroform extract as described in the next section. then, the chloroform extract was partitioned in two volumes. for aa measurements with the teac and orac methods, an aliquot was evaporated to dryness under vacuum at 40°c using a buchi evaporator and the dry residue was reconstituted in absolute ethanol. as for the lox/rno assay, the second aliquot of the chloroform extract was added with tween 80 according to a ratio equal to 0.4 µl tween 80/µg of carotenoids; the mixture was dried under vacuum at 40°c and the dry residue was reconstituted in 100 mm sodium borate buffer ph 9.0. spectrophotometric determination of total carotenoids of extracts from peach fruits quantification of total carotenoids was carried out according to the procedure described by lichtenthaler (1987) and by lichtenthaler and wellburn (1983). briefly, the absorbance spectra of appropriate dilutions in 80% (v/v) acetone of the chloroform extract (see previous section) was recorded in the vis region. carotenoid concentration was calculated by means of a proper equation using a specific absorption coefficient at 470 nm of 198 ml·mg-1 ·cm-1, and involving a correction for chlorophyll a and b content based on absorbance measurements at 663 and 647 nm. determination of antioxidant activity (aa) by the lox/ rno, orac and teac methods the lox/rno reaction was spectrophotometrically monitored, as described in pastore et al. (2000 a; 2009), by measuring the rno absorbance decrease at 440 nm and 25°c in a reaction mixture (2 ml) containing 100 mm sodium borate buffer ph 9.0, 1 mm sodium linoleate, 1.5 µl tween 20/µmol linoleate and 15 µm rno; the reaction was started by adding 0.3 enzymatic units (eu) of soybean lox-1. since carotenoid extracts reconstituted in sodium borate buffer containing 0.4 µl tween 80/µg carotenoids were analyzed, all lox/rno measurements were carried out in the presence of a constant volume (0.5 µl/ml) of tween 80 in the assay mixture. the lox/rno reaction was measured both in the absence (control) and presence of carotenoid extract (or ±-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid, trolox, used as a standard antioxidant). the (%) decrease of the rate of rno bleaching measured in the presence of extract (or trolox) with respect to the rate of the control reaction was used to quantify aa. this was done by means of a dose-response curve derived for trolox by plotting the (%) decrease of the rate of rno bleaching as a function of standard antioxidant concentration. in particular, to calculate the rate of the lox/rno reaction in the presence of extract, the lox-1-depedent bleaching of carotenoids was also monitored by measuring the absorbance decrease at 440 nm in the above reported assay mixture lacking rno. then, a new trace was built, representing the difference between the trace of the lox/rno reaction in the presence of carotenoid extract and that relative to carotenoid bleaching 78 adv. hort. sci., 2015 29(2-3): 75-83 (for details see results). the orac protocol, described in ou et al. (2001) and modified as in pastore et al. (2009) and laus et al. (2012 b), was applied. fluorescence intensity decay due to 3’,6’-dihydroxyspiro[isobenzofuran-1[3h], 9’[9h]xanthen]-3-one (fluorescein) oxidation by peroxyl radicals generated by 2,2’-azobis(2-amidinopropane) (aaph) thermal decomposition was continuously monitored at 37°c at excitation and emission wavelengths of 485 and 515 nm, respectively. to quantify aa, the area under the fluorescence decay kinetic curve (area under curve, auc) was used and, in particular, the net auc (auc net ), obtained by subtracting auc of the blank from that of the sample. aa was calculated by means of a proper dose-response curve prepared with trolox by plotting the auc net as a function of standard antioxidant concentration. since carotenoid extracts reconstituted in ethanol were used, all orac measurements were carried out in the presence of a constant volume of ethanol in the assay mixture. the teac protocol, reported in re et al. (1999) and modified as in pastore et al. (2009) and laus et al. (2012 b), was used. the coloured radical cation abts•+ was produced by abts oxidation with potassium persulfate solution. absorbance at 734 nm and 25°c (a 734 ) was measured after a fixed time of incubation of carotenoid extract (or trolox) with the abts•+ solution (diluted in absolute ethanol). the (%) decrease of a 734 measured after 3 min incubation of extract (or trolox), with respect to a 734 of the uninhibited radical cation solution, was calculated; aa was quantified by means of a proper concentration-response curve prepared with trolox by plotting the (%) decrease of a 734 as a function of standard antioxidant concentration. for all three methods, determinations were carried out in triplicate by analyzing at least three different amounts of extract. a linear dependence of the inhibition on the amount of extract was verified by linear regression analysis of data. then, aa was obtained by comparing the slope derived by linear regression analysis with that of the calibration curve prepared with trolox. statistical analysis distribution of data from figure 4 was evaluated using the shapiro-wilk and jarque-bera tests, and homogeneity of variances was verified by the bartlett’s test. figure 4 data were submitted to a “one-factor” analysis of variance (anova) and the mean separation was tested by the duncan’s test at 0.01 p level of significance. statistical analysis was performed using statistica (data analysis software system), version 7 (statsoft, tulsa, oklahoma, usa). 3. results application of the lox/rno method to aa assessment of extracts showing high ß-carotene concentration is complicated both by the high absorbance of ß-carotene in the visible light region and by the ability of lox to cause carotenoid bleaching. thus, a preliminary investigation regarding this point was carried out. in figure 2 the absorbance spectra of both rno and β-carotene aqueous solutions are reported, as recorded in the visible region of the electromagnetic spectrum: a large overlapping of both spectra in the entire investigated visible region is clearly evident, as well as a high absorbance value of ß-carotene at 440 nm, representing the wavelength of the rno absorption maximum. in light of these absorbance properties of carotenoids, the lox-1-catalyzed rno bleaching cannot be easily monitored at 440 nm. to overcome these problems, in this study a new simple lox/rno protocol was developed for aa assessment of carotenoid-enriched extracts. figure 3 shows evaluation according to the new protocol of the lox-1-dependent rno bleaching in the presence of carotenoids extracted from the yellow-fleshed peach fruits of cv. armking. in particular, in figure 3a a typical experimental trace of the lox/rno control (in the absence of extract) reaction is reported: it was spectrophotometrically monitored by continuously measuring the rno absorbance decrease at 440 nm and it consists of a lag phase (representing the time occurring to consume oxygen in the reaction mixture due to the primary lox-1 reaction of linoleate hydroperoxidation), and a true rno bleaching phase (due to the radical species generated by lox-1 when anaerobiosis is approached in the assay mixture), occurring, in this experiment, at a rate equal to about 0.15 δa 440 nm · min-1. in figure 3b the trace relative to the rno bleaching in the presence of 1.13 µg carotenoids from armking (rno plus carotenoid bleaching, trace a) is shown. in the same figure, the trace b is reported, representing the experimental curve relative to carotenoid bleaching reaction, obtained fig. 2 rno (a) and ß-carotene (b) absorbance spectra. aqueous solution of ß-carotene was prepared as reported in methods. absorbance spectra of 15 µm rno (trace a) and 8 µm ß-carotene (trace b) were recorded in 2 ml of 100 mm sodium borate buffer ph 9.0. 79 laus et al., antioxidant activity of peach carotenoid extracts and the lox/rno method by measuring the absorbance decrease at 440 nm in the absence of rno: it clearly shows carotenoid absorbance decrease due to oxidation by radicals produced during secondary anaerobic reactions associated to the lox-1-mediated linoleate hydroperoxidation (barimalaa and gordon, 1988; pastore et al., 2000 a). moreover, the curve representing the difference between traces a and b, obtained by mathematical processing of these curves using a specific software, is also reported (trace indicated as “a minus b”), showing a rate equal to about 0.12 δa 440 nm ·min-1. this newly built curve only reflects the effect of carotenoids on rno bleaching, excluding any absorbance decrease due to lox-1-dependent carotenoid bleaching; thus, its rate may be compared to that of the lox/rno control reaction. the comparison of the slopes of the trace “a minus b” and the control one shows that the amount of extract from cv. armking containing 1.13 µg of carotenoids is able to induce an about 25% decrease of the rate of rno bleaching. in the same experiment, an increasing inhibition of the reaction rate with increasing amount of armking carotenoid extract, evaluated according to the same protocol, was also observed. in particular, a linear dependence between inhibition and amount of extract was found in the studied 0.75-2.25 µg carotenoid range (fig. 3d). in figure 3c the trolox-dependent inhibition of the rate of rno bleaching as a function of the standard antioxidant concentration is reported, showing a linear dependence of the inhibition in the 20-50% range on trolox concentration ranging from 2 to 7.5 mm, described by the equation: y(inhibition) = 6.059x(concentration millimolar of fig. 3 the lox-1-dependent rno bleaching in the absence (a, control) and presence (b) of carotenoid-enriched extract from peach fruit and linear dependence of the inhibition on trolox concentration (c) and extract amount (d). the lox/rno reaction was monitored as described in methods in the absence (a, control) and in the presence of 1.13 µg carotenoids extracted from the yellow-fleshed cv. armking (b, trace a). in (b) the lox-1-dependent bleaching of the same amount (1.13 µg) of carotenoid extract was also reported (trace b), as well as the curve obtained from the difference between traces a and b (trace “a minus b”). in (c) the calibration curve obtained with trolox is reported; in (d) the (%) inhibition of the rate of the lox/rno reaction is reported as a function of extract amount, expressed as carotenoid content. the rates of the rno bleaching, calculated as the highest slopes (dotted lines) to the experimental curves, are reported, expressed as δa 440 nm · min-1. for aa calculation see the text. 80 adv. hort. sci., 2015 29(2-3): 75-83 trolox) + 8.505 (r=0.999, p≤ 0.001). by means of this trolox-based calibration curve, the aa value for armking extract was calculated, resulting, in this experiment, equal to 120±6 µmol eq. trolox/g fresh weight. the new developed protocol was also applied to measure the aa of carotenoid extract obtained from the white-fleshed cv. silverking. in this case, a linear dependence between inhibition and amount of extract was obtained in the studied 0.075-0.2 µg carotenoid range; the inhibition corresponded, in that experiment, to an aa value equal to 7.5±2 µmol eq. trolox/g fresh weight. interestingly, the ratio between aa values of the yellow-fleshed armking and the whitefleshed silverking extracts resulted equal to 16, very similar to the ratio (about 17) between carotenoid content of the extracts (13.5 and 0.8 µg/g fresh weight, respectively, in these experiments). the study was extended to three other peach varieties, one yellow-fleshed (redhaven) and two white-fleshed (caldesi 2000 and iff331). in table 1, aa values of carotenoid extracts obtained from all tested genotypes, measured by means of the lox/rno, are reported and compared with those obtained by using the orac and teac assays. the lox/rno method provided aa values ranging from 10 (white-fleshed genotypes) to 150 µmol eq. trolox/g fresh weight (yellow-fleshed genotypes). in table 1 the carotenoid content of extracts is also reported for all genotypes under study, ranging from 0.25 (white-fleshed genotypes) to 18.5 µg/g fresh weight (yellow-fleshed genotypes). interestingly, a highly statistically significant positive correlation was obtained between aa values provided by the lox/ rno method and carotenoid content (r = 0.996, p≤0.001). aa values obtained by the teac assay also resulted significantly correlated to carotenoid content (r = 0.997, p≤ 0.001); a lower correlation was obtained for the orac protocol (r = 0.824, p≤0.001); high correlations were also obtained between aa values measured by the lox/rno method and that obtained using the other assays (lox/rno-teac r = 0.992, p≤0.001; lox/rno-orac r = 0.837, p≤0.001; orac-teac r = 0.839, p≤0.001). figure 4 presents the ratio between the yellow-fleshed cultivars and white-fleshed genotypes under study in terms of carotenoid content, as well as the ratio between aa mean values of yellowand white-fleshed peach fruits, as evaluated by means of the lox/rno, orac and teac methods. the yellow-fleshed genotypes have a carotenoid content on average 36 times higher than that of white-fleshed varieties. interestingly, the aa mean value obtained by the lox/rno method for the yellow-fleshed genotypes resulted about 14 times higher than those measured for the white-fleshed varieties. the same ratios aa yellow-fleshed fruits / aa white-fleshed fruits were only about 2.6 and 3.6 in the case of the orac and teac assays, respectively. 4. discussion and conclusions in this study, the lox/rno method was applied for the first time to assess antioxidant properties of peach fruits, in table 1 antioxidant activity (aa), evaluated by means of the lox/rno, orac and teac methods, and carotenoid content of extracts from yellowand white-fleshed peach genotypes genotype carotenoid content (µg/g f.w.) aa (µmol eq. trolox/g fresh weight) lox/rno orac teac yellow-fleshed redhaven 18.33±0.75 155.3 ± 10.3 0.325 ± 0.042 0.082 ± 0.002 armking 14.11±0.71 125.3 ± 9.5 0.444 ± 0.063 0.068 ± 0.001 white-fleshed silverking 0.75 ± 0.08 9.6 ± 3.5 0.211 ± 0.039 0.025 ± 0.001 caldesi 2000 0.34 ± 0.02 10.5 ± 1.2 0.112 ± 0.007 0.018 ± 0.0001 iff331 0.24 ± 0.01 9.8 ± 1.5 0.129 ± 0.006 0.020 ± 0.0001 all data refer to fresh weight and are reported as mean value ± standard deviation (n=3 different experiments). fig. 4 ratio between yellowand white-fleshed peach fruits in terms of carotenoid content and antioxidant activity, evaluated by means of the lox/rno, orac and teac methods. data are reported as mean value (n=3 different experiments). different capital letters indicate significant differences at 0.01 p level, according to the duncan’s test. 81 laus et al., antioxidant activity of peach carotenoid extracts and the lox/rno method comparison with the widely used orac and teac assays. in particular, the carotenoid component was studied by comparing some yellowand white-fleshed genotypes. unfortunately, application of the lox/rno method to aa determination of carotenoid extracts from peach fruits is strongly affected by absorbance properties of carotenoids, whose absorbance spectra largely overlap the one of rno. moreover, it should be considered that carotenoids are also subjected to absorbance bleaching due to their co-oxidation associated to the lox-mediated hydroperoxidation of polyunsaturated fatty acids (barimalaa and gordon, 1988; pastore et al., 2000 a). in a previous study, in order to evaluate the effect of ß-carotene on the lox/rno reaction, these problems were overcome by measuring the rno absorbance changes as absorbance difference (δ 394nm-512nm ) using a double-wavelength spectrophotometer, an expensive and not widespread laboratory instrumentation (pastore et al., 2009). interestingly, using that experimental approach, a very high sensitivity to ß-carotene of the lox/rno reaction was found, with an ic 50 value in the micromolar range (pastore et al., 2009), consistent with the inhibition by ß-carotene of loxes from different sources (lomnitski et al., 1993). in this paper, to evaluate antioxidant properties of carotenoids by means of the lox/rno reaction, a new experimental approach was developed involving the use of a simple spectrophotometer, which is generally found in research laboratories. this is a multi-step protocol involving the following spectrophotometric measurements: i) the lox-1-depedent rno bleaching in the absence of carotenoid-enriched extract (control reaction); ii) the rno bleaching in the presence of carotenoids (rno plus carotenoid bleaching reaction); iii) the carotenoid bleaching (at 440 nm) in the absence of rno (carotenoid bleaching reaction); iv) mathematical processing by specific software of the traces obtained at points ii) and iii) so as to build a new curve in which the absorbance value at each time is represented by the difference between the respective values of the traces ii) and iii). excluding absorbance decrease due to lox-1-dependent carotenoid bleaching, the rate of the new curve reflects only the effect of carotenoids on rno bleaching, making it possible to compare the rate of the lox/rno control reaction (point i), and allowing calculation of the (%) decrease of rate of rno bleaching and, finally, aa quantification by means of a dose-response curve prepared with trolox. to validate the newly developed protocol, measurement of the aa of carotenoid extract obtained from the yellow-fleshed peach fruits of cv. armking was undertaken, as well as from the white-fleshed cv. silverking. in both cases, the inhibition of the reaction rate was found to be linearly dependent on the amount of carotenoid extract; the inhibitions measured in these experiments allowed calculation of an aa value of the yellow-fleshed armking fruits about 15-fold higher than that obtained for the white-fleshed silverking extract. interestingly, extract from armking fruits showed a carotenoid content about 16-fold higher than that measured for silverking. these data confirm the suitability of the modified lox/rno protocol to measure aa of carotenoid compounds. the study was extended to carotenoid extracts obtained from other yellowand white-fleshed peach varieties. as already observed in previous studies (pastore et al., 2009; laus et al., 2012 a, b; laus et al., 2013 a, b), the method lox/rno provided very high aa values (10-150 µmol eq. trolox/g fresh weight) which were much higher than those measured by orac and teac assays (about 85350-fold and 530-1900-fold higher, respectively) and they showed a statistically significant positive correlation with carotenoid content, higher than that obtained with the other assays in comparison. moreover, the ratio between aa values measured by the lox/rno method of yellowand white-fleshed peaches resulted much higher than that obtained using the orac and teac assays, thus indicating the capability of the newly developed lox/rno protocol to highlight much higher differences among the different tested samples with respect to the other compared aa assays. on the whole, the results of this paper indicate that the lox/rno method, applied according to the new protocol developed in this study, is able to measure high aa values of carotenoid-enriched extracts from peach fruits, highly related to carotenoid content, and to easily discriminate among samples. although teac and orac values also show correlation with carotenoid content, they measure low aa and few differences among yellowand whitefleshed varieties. in conclusion, the lox/rno method may represent a recommended tool to assess aa of the carotenoid component in peach fruits. therefore, the use of this assay in studies regarding aa measurements of other fruits and vegetables is worthwhile. acknowledgements this work was supported by the research project of the ministry of agriculture: “miglioramento delle proprietà igienico-sanitarie, salutistiche e funzionali di commodity per l’alimentazione dell’uomo e/o degli animali (alisal)”. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 2223 may 2014. references adami m., de franceschi p., brandi f., liverani a., giovannini d., rosati c., dondini l., tartarini s., 2013 identifying a carotenoid cleavage dioxygenase (ccd4) gene controlling yellow/white fruit flesh color of peach. plant mol. biol. rep., 31: 1166-1175. barimalaa i.s., gordon m.h., 1988 cooxidation of β-carotene by soybean lipoxygenase. j. agric. food chem., 36(4): 685-687. 82 adv. hort. sci., 2015 29(2-3): 75-83 brandi f., bar e., mourgues f., horváth g., turcsi e., giuliano g., liverani a., tartarini s., lewinsohn e., rosati c., 2011 study of “redhaven” peach and its white-fleshed mutant suggests a key role of ccd4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism. bmc plant biol., 11:24 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pastore d., 2012 b dissection of antioxidant activity of durum wheat (triticum durum desf.) grains as evaluated by the new lox/rno method. j. cereal sci., 56: 214-222. lavelli v., pompei c., casadei m.a., 2009 quality of nectarine and peach nectars as affected by lye-peeling and storage. food chem., 115(4): 1291-1298. legua p., hernández f., díaz-mula h.m., valero d., serrano m., 2011 quality, bioactive compounds, and antioxidant activity of new flat-type peach and nectarine cultivars: a comparative study. j. food sci., 76(5): c729-c735. lichtenthaler h.k., 1987 chlorophylls and carotenoids: pigments of photosynthetic biomembranes. method enzymol., 148: 350-382. lichtenthaler h.k., wellburn a.r., 1983 determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. biochem. soc. trans., 11: 591-592. liu r.h., 2003 health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. am. j. clin. nutr., 78: 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carotenoids and human health. pharm. res., 55: 208-216. re r., pellegrini n., proteggente a., pannala a., yang m., rice-evans c., 1999 antioxidant activity applying an improved abts radical cation decolorization assay. free radical bio. med., 26: 1231-1237. remorini d., tavarini s., degl’innocenti e., loreti f., massai r., guidi l., 2008 effect of rootstocks and harvesting time on the nutritional quality of peel and flesh of peach fruits. food chem., 110(2): 361-367. schreiner m., huyskens-keil s., 2006 phytochemicals in fruit and vegetables: health promotion and postharvest elicitors. crit. rev. plant sci., 25: 267-278. tavarini s., degl’innocenti e., remorini d., massai r., guidi l., 2008 preliminary characterization of peach cultivars for their antioxidant capacity. int. j. food sci. tech., 43(5): 810-815. zielinski a.a.f., ávila s., ito v., nogueira a., wosiacki g., haminiuk c.w.i., 2014 the association between chromaticity, phenolics, carotenoids, and in vitro antioxidant activity of frozen fruit pulp in brazil: an application of chemometrics. j. food sci., 79(4): c510-c516. 97 1. introduction clementines (citrus clementina hort. ex tan.), due to their high quality, are one of the most important cultivated citrus mandarins in southern italy. production in the last decade has increased considerably thanks to remarkable consumer preference. these fruits are very perishable and the occurrence of various fruit diseases and physiological disorders affect their marketing value. the major postharvest diseases of citrus fruit, including clementines, can be separated into two categories based on their initial infections: preharvest infections including brown rot (phytophthora spp.), alternaria rot (alternaria citri ellis et pierce, a. alternata (fr.) keissl), stem-end rot (diplodia natalensis pole-evan, phomopsis citri fawcett), grey mould (botrytis cinerea pers.), anthracnose (colletotrichum gloeosporioides penz.); and postharvest infections including green mould (penicillium digitatum sacc.), blue mould (p. italicum weh.) and sour rot (geotrichum candidum link) (ohr and eckert, 1985; brown and miller, 1999; schena et al., 2011). the most common and serious diseases, which occur in italy, during storage and marketing of clementine fruit are green and blue moulds. infection takes place only through wounds, where nutrients are available to stimulate spore germination and fruit decay begins at these infected injury sites (eckert and eaks, 1989; smilanick et al., 1997; 2006; ismail and zhang, 2004). the incidence of other pathogens is generally low, but can be a serious problem in warm, wet years. these diseases, however, can cause significant economic losses during storage, transport and marketing. chilling injury (ci) represents the major disorder of citrus fruit occurring during low non-freezing temperature storage (0-10°c), and it depends on species and cultivars; mandarin hybrids are sensitive to ci. the severity of ci is related to the temperature and the duration of exposure (chalutz et al., 1985; eckert and eaks, 1989; lafuente and zacarias, 2006). aging is indicated by the shrivelling and collapse of the stem-end button tissue (porat et al., 2004). the use of synthetic fungicides in packinghouses, before fruit storage, remains the major means of control for managing citrus postharvest diseases (eckert and ogawa, 1988; ismail and zhang, 2004; smilanick et al., 2006). however, the development of resistance in fungal pathogens to fungicides (schwinn et al., 1982; viñas et al., 1993; holmes and eckert, 1999) and the growing public concern regarding the potential impact on human health and environmental hazdecay control of cold stored citrus clementina hort. ex tan. fruit by preand postharvest application of potassium phosphite m.c. strano1 (*), s. di silvestro1, m. coniglione2, r. magnano san lio1 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca per l’agrumicoltura e le colture mediterranee, cra-acm, corso savoia, 190, 95024 acireale (ct), italy. 2 decco italia, bivio aspro zona industriale piano tavola, 95032 belpasso (ct), italy. key words: aging, chilling injury, citrus fruit, defence stimulators, preand postharvest decay. abstract: the effectiveness of preand postharvest application of potassium phosphite against naturally occuring postharvest decay and storage disorders on clementine monreal fruit (citrus clementina hort. ex tan.) was investigated. phosphite solutions were applied according to the following experimental protocol: by spraying fruit on the trees (2.5 g/l), at fruit colour breaking and 15 days before harvest; by the combination of preharvest (2.5 g/l) and postharvest dipping application (4 g of a.i./l). preharvest applications were compared to phosethyl-al solution (2.5 g/l) and water control. decay and disorders were assessed after 30 days of cold storage at 6±1°c and 90-95% rh, followed by 7 days of shelf life at 20±2°c. the combination of preand postharvest application of phosphite was more effective in reducing green (penicillium digitatum sacc.) and blue mould (p. italicum weh.) incidence, as compared to water control, but it was not so effective in reducing the incidence of minor decay. potassium phosphite treatments, before harvest and in pre-postharvest combination, significantly reduced chilling injury and aging with respect to water control. based on these results, pre and postharvest application of potassium phosphite can be considered a useful strategy to be included in an integrated approach for controlling green and blue mould of citrus fruit in storage. adv. hort. sci., 2015 29(2-3): 97-102 (*) corresponding author: mariaconcetta.strano@entecra.it received for publication 26 september 2014 accepted for publication 9 march 2015 98 adv. hort. sci., 2015 29(2-3): 97-102 ards, have resulted in a significant interest in the development of alternative methods of disease control. the development of treatments to enhance plant defences is an attractive area to seek further improvements in postharvest disease control. among preharvest treatments, phosphite products, which elicit biochemical defences against invading fungi, can offer an alternative means of decay control. in italy, phosphite products (potassium, calcium and copper phosphite salts) are registered as fertilizers but not yet authorized as disease control agents; they require oxidation to phosphate prior to use by plants and this process is mediated by microbes (adams and conrad, 1953; landschoot and cook, 2005). although foliar phosphite applications increased flower numbers and yields on ‘valencia’ orange, their benefits may result from the control of fungal pathogens, as well as mitigating abiotic stresses, among other mechanisms, such as defence stimulators (albrigo, 1999). product activity is carried out primarily through two mechanisms: direct inhibition of the pathogen, with modification of the phosphate metabolism, and induction of host defence responses (induced systemic resistance mechanisms), such as the phytoalexins scoparone, scopoletin and umbelliferone (smillie et al., 1989; guest and bompeix, 1990; guest and grant, 1991). many growers of citrus fruit and other crops often apply phosphites before harvest to protect fruit from postharvest decay from fungal pathogens (cerioni et al., 2013 a). in fact, they are effective for the control of diseases caused by oomycetes (phytophthora and related fungi), particularly susceptible to inhibition by phosphite (gaulliard and pelossier, 1983; cohen et al., 1987; guest and grant, 1991; martin et al., 1998; mcdonald et al., 2001; adaskaveg, 2009). on the other hand, few investigations, instead, describe control of penicillium spp. by phosphites and also report the major efficacy of phosphites when applied in heated solution (amiri and bompeix, 2011; bassay blum et al., 2007; cerioni et al., 2013 a). in the united states phosphites are exempt from residue tolerances (us epa, 2006), and two commercial potassium phosphite formulations are registered for postharvest use. the objective of the present research was to investigate the effectiveness of preand postharvest application of potassium phosphite against postharvest decay (in particular green and blue moulds), and physiological disorders (chilling injury and aging) on cold stored clementine fruits. the efficacy of the product was compared to phosethyl-al, a phosphategenerating fungicide. in order to simulate actual commercial conditions, experiments were conducted on naturally-infected fruit instead of on artificially inoculated specimens. 2. materials and methods plant material field trials were conducted in the fall 2013, on 20-yearold clementine trees (citrus clementina hort. ex tan.) cv. monreal, located in the “palazzelli” experimental orchard (sicily region, southern italy) belonging to ‘consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria centro di ricerca per l’agrumicoltura e le colture mediterranee (cra-acm)’. solution preparation commercial formulations of potassium phosphite (deccophosk, decco italia s.r.l., belpasso, catania, italy) and phosethyl-al (aliette, bayer cropscience) were dissolved manually in water to achieve a final concentration of 2.5 g l-1. treatments and storage scheduled treatments are reported in table 1. for preharvest treatments, trials were arranged in a completely randomized block design with three replicates of four plants each. plants were selected for uniformity of fruit development, absence of evident symptoms of diseases and disorders, and sprayed with potassium phosphite, phosethyl-al and tap water. treatments were carried out at fruit colour breaking and 15 days before harvest using a commercial motor-driven back sprayer (approximately 5 l plant-1 of solution). at commercial maturity, fruits were harvested from treated plants and placed into plastic boxes (one box per plant), each containing 50 fruits, with the exception of potassium phosphite treatments (two boxes per plant), in order to use the extra fruit for the postharvest treatment. for the combination of preand postharvest treatments, a group of 600 fruits from plants a, already treated in the field, were immersed in a solution of potassium phosphite (4 g of a.i./l) at 40°c (±0.5°c) for 120 s. the fruits were not rinsed after treatment and were allowed to dry for 2 h at room temperature. all fruits, placed in three plastic boxes per treatment (each containing 200 fruits), were stored for 30 days at 6±1°c and 90-95% rh, followed by 7 days of shelf life at 20±2°c. these storage conditions were used to simulate actual commercial conditions. at the end of cold storage and after shelf life, decay incidence, chilling injury and aging were assessed. decay incidence was expressed as the percentage of fruit infected by fungal pathogens. diseases were visually identified and classified as green mould (p. digitatum), blue mould (p. italicum), mix of green and blue mould (p. digitatum and italicum present on the same fruit), and minor decay (phytophthora, alternaria, rhizopus, botrytis, phomopsis, diplodia, etc.). severity of chilling injury (ci) was evaluated table 1 scheduled treatments on clementine fruits treatment dose period of treatment potassium phosphite (a) 2.5 g/l two preharvest treatments phosethyl-al (b) 2.5 g/l two preharvest treatments water control (w) two preharvest treatments potassium phosphite (ap+p) 2.5 g/l 4 g of a.i./l two preharvest treatments and a postharvest treatment 99 strano et al., decay control of cold stored citrus clementina by preand postharvest application of potassiuim phosphite using a four-grade scoring system. a subjective rating of 0 (none), 1 (light), 2 (moderate), and 3 (severe) was used to estimate damage of the rind. a light rating indicated damage <10% of peel area, not perceived to be objectionable to the discerning consumer, moderate (10-30%) was injury estimated to be objectionable, and severe (>30%) indicated damage that would cause consumers to reject the product. aging was expressed as percentage of fruit damaged. in order to evaluate the effect of treatments on fruit weight loss, 30 fruits per treatment were regularly weighed at the beginning, at the end of cold storage and after one week of shelf life. the percentage of weight reduction was recorded. statistical analysis data were analysed using one-way analysis of variance (anova) procedures, using statistica 6.0 software. percentage data were arcsine transformed to normalize variance. mean values of treatments were compared by using tukey’s test at p=0.05 level. data in the figures are actual percentages of decayed fruit. 3. results postharvest rots on clementines at the end of storage were mainly due to p. italicum (blue mould) and p. digitatum (green mould) alone and present in the same fruit (mix of green and blue mould). minor decay was caused by geotrichum spp., alternaria spp., botrytis spp., phytophthora spp., etc. in all cases preharvest application of potassium phosphite and the combination of preand postharvest applications, showed variable effects in reducing decay incidence, depending on the pathogens involved. since the trials were conducted on naturally occurring infections, disease incidence in the control treatments was not very high. after 30 days of storage at 6±1°c followed by a week of shelf life at 20±2°c, preharvest application of potassium phosphite on clementines significantly reduced the percent infection of blue mould, the mix of green-blue mould, and minor decay as compared to the water control (fig. 1b-1c-1d); no significant reduction was observed on the green mould incidence as compared to the water control (fig. 1a). the combination of preand postharvest applications of potassium phosphite was, instead, more effective in reducing the incidence of green and blue mould, as compared to water control (fig. 1a-1b). the improved control of blue mould, known for its greater ability to grow at low temperature, was of particular interest. conversely, its efficacy in reducing the incidence of minor decay, on preharvest treatments was not improved by postharvest application (fig. 1d). fig. 1 incidence of green mold (p. digitatum) (a), blue mold (p. italicum) (b), mix of green-blue mold on the same fruit (c) and minor decay (d), on clementine, after 30 days of storage at 6±1°c followed by one week at 20±2°c. each treatment was applied to three replicates of 200 fruit each. water treatment was used as control. columns marked with the same letters are not statistically different according to tukey’s test (p = 0.05). w= water control, preharvest treatments; a= potassium phosphite, preharvest treatments (2.5 g/l); ap+p= potassium phosphite, pre(2.5 g/l) and postharvest treatments (4 g a.i./l); b= phosetyl-al, preharvest treatments (2.5 g/l). 100 adv. hort. sci., 2015 29(2-3): 97-102 concerning ci, potassium phosphite treatments before harvest (a) and in pre-postharvest combination (ap+p) significantly reduced light and moderate values, as compared to water control and severe values as compared to phosetyl-al (fig. 2). all treatments (a, ap+p and b) were significantly effective in reducing aging with respect to water control (fig. 3). postharvest treatment with potassium phosphite had no phytotoxic effect on clementines. in addition, after 30 days of storage and one week of shelf life, the general external appearance of fruit was not affected by different treatments. no statistically significant differences were found for weight loss, among all treatments, both at the end of cold storage and after a week of shelf life (data not shown). 4. discussion and conclusions the main objective of the present study was to evaluate the efficacy of preand postharvest application of potassium phosphite, in controlling postharvest decay, particularly green and blue moulds of clementine, in order to extend its application for disease control of citrus fruits in italy. reports describing the preand postharvest use of phosphite to control diseases caused by true fungi are few. gutter (1983) reported that the posphite-generating compound phosetyl-al, in vitro and in vivo, had modest activity on the control of p. digitatum; bassay blum et al. (2007) reported that immersion of apple fruit in potassium phosphite solutions controlled blue mould caused by p. expansum. cerioni et al. (2013 a) reported that improved control of green and blue mould, in postharvest treatments, was influenced by heating the solution (50°c), and by increasing the phosphite concentration (15 g/l). regarding post-treatment storage temperature, 10°c were able to control green mould on citrus fruit, but had less effect on blue mould, even when the phosphite solution was heated to 50°c. our data showed that treatment with potassium phosphite was more effective against green and blue mould when applied before and after harvest, whereas, when applied only before harvest, it did not influence green mould incidence as compared to the water control. this different result is probably due to defence stimulation that treatment activates on the tree in field trials followed by the defence stimulation activated on fruit, in postharvest treatment (4 g of a.i./l), at the temperature of 40°c. the reduced efficacy of potassium posphite, on minor decay, in preand postharvest applications, was unexpected since its field application is effective against different pathogens. although not evaluated for the single control of phytophthora brown rot, phosphites have long been known to control this fruit decay (gaulliard and pelossier, 1983; cohen and coffey, 1986; graham and timmer, 2011), which causes significant losses in wet years. adaskaveg (2009) reported the excellent results obtained for the pre and postinfection control of phythophthora citrophthora on orange fruit dipped in 0.27 g/l of potassium phosphite. thus, the phosphite treatments that controlled green and blue mould would be expected to control brown rot. phosphite is more costly than other alternatives used in packinghouses (sbc), but are compatible with sbc and with all of the fungicides currently registered for postharvest use such as imazalil (imz) and thiabendazole (tbz), improving their performance (cerioni et al., 2013 a, 2013 fig. 2 effect of treatments on the severity of chilling injury on clementine, after 30 days of storage at 6±1°c followed by one week at 20±2°c. each treatment was applied to three replicates of 200 fruits each. water treatment was used as control. columns marked with the same letters are not statistically different according to tukey’s test (p = 0.05). w= water control, preharvest treatments; a= potassium phosphite, preharvest treatments (2.5 g/l); ap+p= potassium phosphite, pre(2.5 g/l) and postharvest treatments (4 g a.i./l); b= phosetyl-al, preharvest treatments (2.5 g/l). fig. 3 effect of treatments on aging percentage on clementine, after 30 days of storage at 6±1°c followed by one week at 20±2°c. each treatment was applied to three replicates of 200 fruits each. water treatment was used as control. columns marked with the same letters are not statistically different according to tukey’s test (p = 0.05). w= water control, preharvest treatments; a= potassium phosphite, preharvest treatments (2.5 g/l); ap+p= potassium phosphite, pre(2.5 g/l) and postharvest treatments (4 g a.i./l); b= phosetyl-al, preharvest treatments (2.5 g/l). 101 strano et al., decay control of cold stored citrus clementina by preand postharvest application of potassiuim phosphite b; palou et al., 2001; 2002). thus, the combination of potassium phosphite with sbc could be used to reduce costs, and in combination with imz could improve effectiveness for the control of imz-resistant isolates of p. digitatum (kinay et al., 2007). in conclusion, our results have demonstrated that the incidence of green and blue mould on clementine fruit can be reduced by applying potassium phosphite twice before harvest and in postharvest treatments. preand postharvest application of potassium phosphite can be considered a useful strategy to be included in an integrated approach for controlling postharvest diseases of citrus fruit. in any case, less infected fruit on packing lines should also reduce the demand for sanitizers during washing procedures (lanza and strano, 2009). practical application of potassium phosphite on citrus fruit needs to be further optimized as the obtainable level of protection is affected by various factors, first of all citrus variety, timing and number of applications. additional research is in progress on different citrus varieties to improve the application strategy. acknowledgements we thank michele scirè for technical assistance. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references adams f., conrad j. p., 1953 transition of phosphite to phosphate in soils. soil sci., 75: 361-371. adaskaveg j.e., 2009 management of citrus brown rot. http://www.calcitrusquality.org/wp-content/uploads/2009/05/citrus-brown-rot-ja-9-29-11.pdf. albrigo l.g., 1999 effects of foliar applications of urea or nutriphite on flowering and yields of valencia orange trees. proc. fla. state hortic. soc., 112: 1-4. amiri a., bompeix g., 2011 control of penicillium expansum with potassium phosphite and heat treatment. crop prot. 30: 222-227. bassay blum l.e., vidal tamarini do amarante c., dezanet a., barreto de lima e., hack neto p., duarte avila r., siega v., 2007 fosfitos aplicados empós-colheita reduzem o mofo-azul em maçãs ‘fuji’ e ‘gala’. rev. bras. frutic., 2: 265-268. brown g.e., miller w.r., 1999 maintaining fruit health after harvest, pp. 175-192. in: timmer l.w., and l.w. duncan (eds.) citrus healthy management. aps press, st. paul, mn, usa, pp. 197. cerioni l., rapisarda v.a., doctor j., fikkert s., ruiz t., fassel r., smilanick j., 2013 a use of phosphite salts in laboratory and semicommercial tests to control citrus postharvest decay. plant dis., 97: 201-211. cerioni l., sepulveda m., rubio-ames z., volentini s.i., rodriguez-montelongo l., 2013 b control of lemon postharvest diseases by low-toxicity salts combined with hydrogen peroxide and heat. postharvest biol. technol., 83: 17-21. chalutz e., waks j., schiffmann-nadel m., 1985 a comparison of the response of different citrus fruit cultivars to storage temperature. sci. hortic., 25: 271-277. cohen e., shalom y., axelrod y., adato i., rosenberger i., 1987 control and prevention of contact infection of brown rot disease with fosetyl-aluminum, and residue levels in post-harvest-treated citrus fruit. pestic. sci., 20: 83-91. cohen y., coffey m.d., 1986 systemic fungicides and the control of oomycetes. annu. rev. phytopathol., 24: 311338. eckert j.w., eaks i.l., 1989 postharvest disorders and diseases of citrus fruits, pp. 179-260. in: reuter w., e. calavan, and g.e. carman (eds.) the citrus industry, vol. v. university of california press, riverside, usa, pp. 383. eckert j.w., ogawa j.m., 1988 the chemical control of postharvest diseases, deciduous fruits, berries, vegetables and root/tuber crops. annu. rev. phytopathol., 26: 433-469. gaulliard j.m., pelossier r., 1983 efficacite de phosethyl al en trempage des agrumes (fruits) contre phytophthora parasitica agent de la pourriture bruneet contre penicillium digitatum. fruits, 38: 693-697. graham j.h., timmer l.w., 2011 florida citrus pest management guide: brown rot of fruit. fla. coop. ext. serv. doc., pp. 148. guest d.i., bompeix g., 1990 the complex mode of action of phosphonates. aust. plant pathol., 19: 113-115. guest d.i., grant b.r., 1991 the complex action of phosphonates as antifungal agents. biol. rev., 66: 159-187. gutter y., 1983 supplementary antimold activity of phosethyl al, a new brown rot fungicide for citrus fruits. phytopathol. z., 107: 301-308. holmes g.j., eckert j.w., 1999 sensitivity of penicillium digitatum and p. italicum to postharvest citrus fungicides in california. phytopathology, 89: 716-721. ismail m., zhang j., 2004 post-harvest citrus diseases and their control. outlooks pest manage., 15: 29-35. kinay p., mansour m.f., gabler f.m., margosan d.a., smilanick j.l., 2007 characterization of fungicide-resistant isolates of penicillium 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starvation response. j. plant nutr., 24: 1505-1519. ohr h.d., eckert j.w., 1985 postharvest diseases of citrus fruits in california. leaflet no. 21407. university of california, cooperative extension service, riverside, california. palou l., smilanick j.l., usall j., viñas i., 2001 control of postharvest blue and green molds of oranges by hot water, sodium carbonate and sodium bicarbonate. plant dis., 85: 371-376. palou l., usall j., munoz j.a., smilanick j.l., viñas i., 2002 hot water, sodium carbonate, and sodium bicarbonate for the control of postharvest green and blue molds of clementine mandarins. postharvest biol. technol., 24: 93-96. porat r., weiss b., cohen l., daus a., aharoni n., 2004 reduction of postharvest rind disorders in citrus fruit by modified atmosphere packaging. posharvest biol. technol., 33(1): 35-43. schena l., strano m.c., sanzani s.m., ippolito a., 2011 le malattie degli agrumi in postraccolta. protezione delle colture, anno iv, 9: 30-41. schwinn f.j., sijpesteijn a.k., davidse l.c., de waard m.a., fuchs a., beever r.e., byrde r.j.w., wolfe m.s., zadoks j.c., bollen g.j., gilpatrick j.d., uesugi y., brent k.j., eckert j.w., 1982 fungicide resistance in crop protection. in: dekker j., and s.g. georgopoulos (eds.) fungicide resistance in crop protection. pudoc, wageningen, netherlands, pp. 265. smilanick j.l., brown g.e., eckert j.w., 2006 postharvest citrus diseases and their control, pp. 339-396. in: wardowski w.f., w.m. miller, d.j. hall, and w. grierson (eds.) fresh citrus fruits. second ed. florida science source, inc., longboat key, fl, usa. smilanick j.l., michael i.f., mansour m.f., mackey b.e., margosan d.a., flores d., weist c.f., 1997 improved control of green mold of citrus with imazalil in warm water compared with its use in wax. plant dis., 81: 1299-1304. smillie r., grant b.r., guest d., 1989 the mode of action of phosphite: evidence for both direct and indirect modes of action of three phytophthora spp. in plants. phytopathol., 79(9): 921-925. us epa, 2006 title 40: protection of environment. us epa, part 180. code of federal reg., 71: 49373. viñas i., vallverdú n., monllao s., usall j., sanchis v., 1993 -. imazalil resistant penicillium isolated from spanish apple packinghouses. mycopathol., 123: 27-33. 39 1. introduction supply-chain traceability systems are suitable tools for controlling plant disease diffusion and they can be implemented by means of integrated computer-based information systems (icbiss) which incorporate data from different production centers (porto et al., 2011). with regard to propagative material for grapevine in the european union (2005/43/ce), there are four main categories, labeled with a color coding system. the primary source, derived from selected grapevine by a conservative breeder and is grown under his/her responsibility: this source produces pre-basic material, as well as the derived basic material. this material is intended for delivery only to nurseries having the necessary qualification; certified grapevines are derived from this source. italian regulations (dm 8 february 2005, dm 7 july 2006 and dm 24 june 2008) define which propagative material must be periodically assayed for pests: each material stage is intrinsically characterized by increasing risks of re-infection, and rapid and safe retrieval of mother plant data can be useful for prompt intervention when it is necessary to limit spread of the pathogen. surely an efficient and durable traceability system can provide for information retrieval regarding propagated material, particularly if it is supported by an information technology (it) solution: radiofrequency (rfid)-based technologies can be implemented in platforms to share and manage data in agriculture, providing a safe and durable link between items such as plants and information (sørensen et al., 2010), with positive effects on traceability. sørensen et al. (2011) stated that communication and automated processing of data require a digital form and a machine-readable format which can be interpreted unambiguously by all entities involved in the information flows. increasingly, common data sources and sensor systems in agriculture produce digital, machine-readable data which can be used for decision making. with regard to grapevine propagative material, rfid technology has been successfully used to identify all plants during propagative material of grapevine: rfid technology for supporting traceability of “basic” and “certified” material along the wine production chain a. luvisi*, a. panattoni*, r. bandinelli**, e. rinaldelli***, m. pagano***, e. triolo* * dipartimento di coltivazione e difesa specie legnose, sezione di patologia vegetale, università degli studi di pisa, via del borghetto, 80, 56124 pisa, italy. ** associazione toscana costitutori viticoli, tos.co.vit, via vecchia di marina, 6, 56010 san piero a grado (pi), italy. *** dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione di coltivazioni arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: conservative breeder, foundation block, electronic identification. abstract: four main categories of propagative material in the european union (eu) have been indentified for grapevine: the primary source, pre-basic, basic and certified material. each type of material has to be periodically assayed for pathogens and each material stage is intrinsically characterized by increasing risks. radiofrequency (rfid) can be considered an efficient and durable traceability system to provide retrieval of propagated material or check producer identity. rfid tagging of grapevines of different categories along the production line could establish a durable link between stakeholder and products. to evaluate this approach, histological observations and growth parameters of basic or certified rfidtagged grapevine were performed, as well as requirement analysis for management of sanitary checks and for traceability of the wine production chain. basic material can be safety tagged with rfid tags to establish mother plant vineyards; derived certified material can also safely be tagged. no detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first year of production life. requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as the rfid-tagging steps and methods to collect sanitary and production data that are useful for traceability purposes. adv. hort. sci., 2012 26(1): 39-43 received for publication 2 february 2012 accepted for publication 5 march 2012 40 sanitary, ampelographic and genetic checks during selection, with no losses in retrieving information from plants (pagano et al., 2010). grapevine tagging with a rfid microchip (tag) can be performed internally (bandinelli et al., 2009) in order to guarantee a durable identification of plants: a microchip identification code can also be associated to an electronic identification datasheet (eid) to store virtually unlimited data for each microchip/plant (luvisi et al., 2010 a). in this perspective, rfid tagging of grapevines of different categories belonging to a production line could establish a durable link between stakeholder and products: interestingly, this application could be combined with corks containing a rfid inlay for highvalue wines (collins, 2005; launois, 2008). histological observations and growth parameters were performed in four-year-old rfid-tagged basic plants to evaluate plant response to implanting method at production stage: tests were also performed on one-year-old derived plants, the information from which was linked to mother plants. requirement analyses for plant management were also performed, while the wine production chain was described in order to define a traceability scheme based on rfid microchips linked to each production stage, from primary source registration to bottle tagging. 2. materials and methods plant materials in order to evaluate the traceability procedures from foundation block to grower, pre-basic vitis vinifera cv. sangiovese were externally tagged with a rfid wristband. then, derivate v. vinifera cv. sangiovese cuttings was grafted on rootstock 1103 paulsen (vitis berlandieri x vitis rupestris) by a foundation block in 2007, in order to obtain two rows of basic material. hardwood cuttings were obtained from these plants in 2009 and 2010, and used by a nursery to graft on rootstock 1103 paulsen in 2010 and 2011 respectively, in order to obtain two rows of plants of certified material. plants belonging to basic or certified categories were tagged with rfid tags, following the luvisi et al. (2010 b) procedure: microchips were inserted inside pith of rootstock after 4-cm depth direct drilling of pith from a distal cut of rootstock just before omega grafting. the microchip was positioned 3 cm below grafting point. untagged plants were used as control in order to evaluate production performances. in order to evaluate the tagging system along the entire grapevine production chain, conservative breeder activities were included in the trial. to minimize experimental times, v. vinifera cv. sangiovese plants involved in a clonal selection procedure started in 2007 were used as primary source of plant used for the foundation block. similarly, barrels and wine bottles were marked with external tagging to simulate data matching with plants, thus completing the traceability system of the wine production chain using rfid. electronic materials and requirement analysis transponder glass rfid tags were used (diameter 2.1 mm, length 12 mm), working at a frequency of 125 khz (intermedia sas, forlì, italy, www.rfid360.net). they were used for internal implanting or attached to plastic wristbands for the external tagging procedure. tags were electronically read using a card flash reader connected by sd slot to a palm-pc (dell axim x51) able to identify the microchips from a distance of 100 mm. software for managing data was java™ and adobe® flex®. tag reliability, as number of readable microchips out of the total, and accuracy, as number of readable microchips within 30 sec, were evaluated in implanted basic (four-year-old) or certified (one-year-old) grapevine plants. requirement analysis was performed by stakeholder identification, interviews, collecting data relative to regulations, farm rules and existing software for managing rfid-tagged plants. according to porto et al. (2011), the requirement analysis step regarded the a) identification of traceability objectives; b) analysis of the regulatory requirements concerning grapevine production, c) identification of plant propagating materials produced along the grapevine production chain; d) definition of rfid-tagged materials; e) analysis of the material flows within the plant supply chain; and f) analysis of the information to be managed in the traceability system. plant assay in order to evaluate the productivity of rfid-tagged basic plants, growth of branches expressed mean relative growth rate (mrgr, mg d-1) with one sampling period of 45 or 90 days was calculated from when shoots started growing in 2009 and 2010. in addition, mean number of buds recovered for grafting and mean diameter of buds were evaluated at the same time. considering these parameters, mean data from 20 plants per treatment are reported. to estimate long-term damage or losses in productivity, effects of implanting procedures on functional vascular tissue area (%) were measured at three heights in rfid-tagged basic (four-year-old) or certified (one-yearold) grapevine. measurements were performed on fresh trunk sections in proximity of the microchip location, at approximately mid-length of the microchip (“height 0”), 3 mm higher (“height 3”) and 3 mm lower (“height -3”). in unmarked plants, sections were taken at the same height as in marked plants. vascular tissue area was calculated using software for image analysis (cerri et al., 1993), measuring total vascular tissue area and non-necrotic vascular tissue area. for histological observations, fresh transversal sections (20 μm thick) were made with a rotary microtome (reichert-jung, autocut 2040, austria) and stained with toluidine blue o (sigma-aldrich corporation, usa); sections were immediately observed with a light microscope (leica, wetzlar, germany). data analysis the effects of treatments were compared by analysis of variance in a random design. duncan’s multiple range test 41 at 5% level was calculated in order to compare treatments for functional vascular area, characterized by undamaged vessels, and in which xylem rays are developed as control, and for growth parameters as well. data in percentage were normalized by arc sin square root transformation. 3. results requirement analysis and microchip tests the objectives for traceability of requirement analysis were: to record genetic links among plant categories; to record mandatory or voluntary assays; to identify structures or people involved in plant production and resulting wine; and to use an identification system which relies on rfid codes associated with electronic identity cards (luvisi et al., 2010 a). regulations concerning non standard grapevine production require mandatory assay for conservative breeders or foundation stock (2005/43/ce). data relative to assays, in particular periodical health assays, can be associated to tags through the use of a database. in any case, other checks, for example those performed by phytosanitary services or those for voluntary certification, can be implemented within the system. identification of plant propagation materials produced along the grapevine production chain led to the creation of six plant categories to be included in the plant management system (fig. 1): plants belonging to these categories can be tagged diffig. 1 data flow within rfid-tagged grapevine production chain. 42 ferently in relation to their morphological characteristics. external tagging is for grapevines selected by conservative breeders, primary sources and non grafted pre-basic material: these plants cannot be tagged using methods proposed thus far. taking into account trunk dimension (which in the case of older plants may be adequate), transversal drilling of trunk and microchip implanting may be possible. on the other hand, tags can be implanted in basic and certified material by foundation blocks and nurseries, respectively. furthermore, to establish a link with the final product, barrels and bottles can be externally tagged. figure 1 offers a scheme to analyze the material flows within the plant supply chain. analysis of the information to be managed in the traceability system identifies the information as: information from grapevine cultivar registries (i.e. the italian registro nazionale delle varietà di vite); information useful for grapevine purchasers, such as botanical name and cultivar, plant category, clone and rootstock specifications, name of producer, rfid-related codes; product history, such as the conservative breeder, mandatory or voluntary assays, information about stakeholders; a digital map accessible via mobile or desktop systems, providing a “virtual vineyard” in which grapevine plants marked by rfid can be viewed (luvisi et al., 2011); and details of wine production, wine producer and cellar. with regard to tests of microchip reliability, tags were fully functional in both plant categories and the implanting procedures did not compromise readability. tags were readable within 30 s in more than 98% of the reading tests, without significant differences between plant categories (table 1). table 1 microchip reliability (as number of readable microchips out of the total) and accuracy (as number of readable microchips within 30 s), implanted in basic (four-year-old) or certified (one-year-old) grapevine plants grapevine category microchip reliability microchip accuracy basic 100.0 a (z) 98.8 a certified 100.0 a 98.1 a (z) within each parameter, values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). plant assay rfid-tagged grapevines belonging to the basic category did not show detrimental effects for parameters affecting their productivities such as growth and bud production. in fact, after two years from tagging, no differences in mrgr at 45 or 90 days were measured compared to control (table 2). rfid-tagged plants develop a comparable number of buds with adequate diameters compared to control (table 3). the absence of detrimental effects of rfid implanting in both categories was confirmed by measurement of the functional vascular tissue area, not affected by tag presence (table 4). table 2 growth of branches of rfid-tagged basic grapevine expressed as mean relative growth rate (mrgr, mg d-1) with one sampling period of 45 or 90 days, calculated from when shoots started growing year mrgr (mg d-1) 45 days mrgr (mg d-1) 90 days control rfid-tagged control rfid-tagged 2009 0.095 a (z) 0.100 a 0.121 a 0.125 a 2010 0.0101 a 0.099 a 0.128 a 0.126 a (z) within each parameter, values in the same line followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. table 3 mean number of buds for rfid-tagged basic grapevine recovered for grafting and mean diameter of buds year mean no. of buds/plant mean diameter of buds (mm) control rfid-tagged control rfid-tagged 2009 27.3 a (z) 25.1 a 8.5 a 9.1 a 2010 29.4 a 32.5 a 10.8 a 10.0 a (z) within each parameter, values in the same line followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. table 4 effects of implantation procedures on functional vascular tissue area (%), measured at three heights, in rfid-tagged basic (four-year-old) or certified (one-year-old) grapevine procedure functional vascular tissue area (%) height -3 height 0 height 3 basic certified basic certified basic certified control 99.1 a (z) 98.7 a 99.9 a 99.4 a 99.8 a 99.0 a rfidtagged 99.2 a 99.1 a 99.8 a 98.7 a 99.9 a 98.8 a (z) values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. 4. discussion and conclusions plant labeling represents a key step in the certification scheme, with labels supplied by certifying authorities such as a government agency or an officially recognized private organization. for various woody plants, labeling is strictly regulated by law, and currently electronic identification is not an viable option for substituting plastic labels. however, interest in these tools by organizations such as the european commission cannot be ignored, as reported for bovines, already in use in several eu member states on a private basis mainly for farm management purposes. this approach seems to be crucial for localizing and tracing individual animals for veterinary purposes as a tool to control infectious diseases (european commission, 2011). 43 pets can also be legally subjected to microchip implanting, as reported for dogs in italy (ordinanza 6 agosto 2008). even if plants cause less concern about human health when compared to the meat production chain, implications do exist in terms of worldwide spread of plant pathogens (in particular viruses) and chemical residuals. plant traceability by rfid can be a useful tool when it comes to risk management. considering the grapevine production line, basic material can be safely tagged with rfid tags to establish mother plant vineyards, the same for derived certified material. no detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first phase of production life. tags were readable to check identities after four years from implanting. data associated to basic material can be linked to previous categories such as pre-basic and primary sources that can be externally tagged, completing the traceability of the grapevine production chain. requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as rfid-tagging steps and methods to collect information and match data, from the plant to the wine bottle. even if the availability of user-friendly details about wine production may seem sufficient for the final user (i.e. details about the cellar or viewing of virtual vineyards), the system guarantees retrieval of detailed, exhaustive data for inside users, thus supporting a virtuous circle of trust. references bandinelli r., triolo e., luvisi a., pagano m., gini b., rinaldelli e., 2009 employment of radiofrequency technology (rfid) in grapevine nursery traceability. adv. hort. sci., 23(2): 75-80. cerri s., panattoni a., triolo e. 1993 studio, progetto e sperimentazione di una procedura semiautomatica per l’analisi di alterazioni del legno in barbatelle di vite. consiglio nazionale ricerche, nota interna, b4-25: 1-24. collins j., 2005 wine bottles get corked with rfid. http://www.rfidjournal.com/article/articleview/2117/1/1/ european commission, 2011 electronic identification of bovines to further strengthen food safety and animal health in the eu. http://europa.eu/rapid/pressreleasesaction.do?reference=ip/11/991&format=html&aged=0&lan guage=en launois a., 2008 rfid tracking system stores wine bottle data. http://www.foodproductiondaily.com/packaging/rfidtracking-system-stores-wine-bottle-data luvisi a., pagano m., bandinelli r., rinaldelli e., gini b., scartòn m., manzoni g., triolo e., 2011 virtual vineyard for grapevine management purposes: a rfid/gps application computers and electronics in agriculture, 75: 368-371. luvisi a., panattoni a., bandinelli r., rinaldelli e., pagano m., gini b., triolo e., 2010 b rfid microchip internal implants: effects on grapevine histology. scientia horticulturae, 124: 349-353. luvisi a., triolo e., rinaldelli e., bandinelli r., pagano m., gini b., 2010 a radiofrequency applications in grapevine: from vineyard to web. computers and electronics in agriculture, 70: 256-259. pagano m., bandinelli r., rinaldelli e., panattoni a., triolo e., luvisi a., 2010 rfid technology for clonal selection purposes. adv. hort. sci., 24(4): 282-284. porto s.m.c., arcidiacono c., cascone g., 2011 developing integrated computer-based information systems for certified plant traceability: case study of italian citrus-plant nursery chain. biosystem engineering, 109(2): 120-129. sørensen c.g., fountas s., nash e., pesonen l., bochtis d., pedersen s.m., basso b., blackmore s.b., 2010 conceptual model of a future farm management information system. computers and electronics in agriculture, 72: 37-47. sørensen c.g., pesonen l., bochtis d.d., vougioukas s.g., suomi p., 2011 functional requirements for a future farm management information system. computers and electronics in agriculture, 76: 266-276. 53 1. introduction turfs and lawns are an important part of the landscape: they enhance its beauty (geren et al., 2009) and provide important ecological benefits (beard, 1973; linse et al., 2001; busey, 2003; argenti and ferrari, 2009) especially in areas modified by human intervention. recently, the need for larger spaces for individual, family environments and common areas for socialization has increased the concept that green spaces represent a higher standard of living in urban areas (hull, 1990; roberts, 1990). this awareness has increased interest in turf, as a surface capable of fulfilling technical, aesthetic and recreational goals, ensuring high quality standards, practicability and durability of the turf (volterrani and magni, 2007). in turn, this has led to the development of precise management practices (gaetani et al., 2013) through which to minimize the negative impact on the environment. the main problem connected to turfgrass is the amount of input necessary to obtain a high quality surface (easton and petrovic, 2005). the predominant challenge is the utilization of water for irrigation (youngner et al., 1981; sevostianova and leinauer, 2014). this aspect may reach critical levels especially in mediterranean regions, characterized by low rainfall and hot summer temperatures. these climatic characteristics suggest the use of a low water rate and drought tolerant species such as warm-season turfgrasses (marchione, 2008) that can provide high quality turf with suitable water consumption and low fertilizer and pesticide input (turgeon, 2002; schiavon et al., 2013). the main objection to the use of these grasses is their lack of green color during the winter period, when they enter in dormancy and loose chlorophyll (bernardini, 2007). this occurs especially in central italy, an area characterized by high summer and cold winter temperatures, where cold tolerance and rapidity of recovery from winter dormancy are also important qualities (magni et al., 2014). the objective of this investigation was to assess performance and adaptation, especially in relation to cold temperature, of three species (seven cultivars) of c4 warm-season turfgrass species and a cool-season grass as a comparison, in an internal area of the tuscany region. 2. materials and methods the experimental site was located in a borderline area of mediterranean climate in the centre of tuscany, characterized by hot summer and low winter temperatures. the experimental trial started in the late spring of 2011 in antria (tuscany, central italy) on natural soil, containperformance of warm-season turfgrasses in an area of central italy i. seppoloni (1) *, n. staglianò (2), s. cecchi (2), g. argenti (2) (1) c.n.r., ibimet, via giovanni caproni 8, 50145 firenze, italy. (2) dispaa, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: bermudagrass, ground cover, growing season, turf quality, weeds. abstract: traditionally, in italy, the c3 cool-season grasses have been the dominant species used for turfs, even though they do not appear to be the most suitable for the mediterranean climate. however, recent limited water availability and the need to reduce energy inputs have placed drought tolerant warm-season turfgrasses under the spotlight. these species combine aesthetics with performance advantages in terms of water consumption, and with regard to the reduction of fertilizer and pesticides use. the present research was aimed to test the performance of warm-season turfgrass species (three cultivars of cynodon dactylon, two cultivars of paspalum vaginatum and two of zoysia japonica) in a climatic transition zone in tuscany, to evaluate their potential for use in this environment. the assessment of several parameters, which were estimated periodically, permitted performance evaluation of each species/cultivar, thereby enhancing the existing knowledge of these species and their potentiality in this environment. results showed that the species with the best adaptation to the environment was cynodon dactylon, which had higher performances compared to the other species. paspalum vaginatum reported good quality in terms of color and density, but was damaged by low temperatures during winter. zoysia japonica displayed a poor performance during the first year, but quality increased during the second year, yielding satisfactory results. adv. hort. sci., 2015 29(1): 53-58 * corresponding author: i.seppoloni@ibimet.cnr.it received for publication 28 january 2015 accepted for publication 1 may 2015 54 adv. hort. sci., 2015 29(1): 53-58 ing 16.4% sand, 54.1% silt and 29.5% clay. the study was conducted over two growing seasons (2011 and 2012), starting after full establishment of the turf, up to the period of dormancy. due to the presence of a meteorological station nearby, weather parameters were recorded in detail for the entire experimental period. the species under comparison included cynodon dactylon (‘black jack’, ‘casino royale’ and ‘la paloma’), paspalum vaginatum (‘marina’ and ‘sea spray’), and zoysia japonica (‘compadre’ and ‘zenith’) and a cool-season grass as control (lolium perenne ‘kokomo’). the eight different accessions tested were replicated three times for a total of 24 plots (2x3 m with a surface area of 6 m2) according to a complete randomized block design. sowing took place on the 10 june 2011. seed rate was 25 g m-2, as an average of the suggested dose for warm-season species (panella et al., 2000). seeding was performed manually and followed by rolling. cultural practices for the growing season included four or five applications of standard fertilizers (yearly total amount 318-85-81 kg ha-1 npk). irrigation was performed to restore 100% of crop evapotranspiration (etc) and it was applied with a sprinkler system. periodically, mowing was carried out with a mowing height of 30 mm performed by a rotary mower. a glyphosate treatment was performed during the dormancy period to reduce the presence of weeds (1 l ha-1). the following parameters were monitored during the trial and evaluated every two weeks (pardini et al., 2002; reyneri and bruno, 2008): aesthetic quality of the turf: scores were visually assigned on a scale ranging from 1 to 9 (1= poorest quality, 9= highest quality) (piano, 2005; bigelow and walker, 2008). quality was also compared to additional parameters measured during the trial (color, ground cover and weeds) by multiple regression analysis to establish which aspects are the most related to quality; turf color: scores were visually assigned on a scale ranging from 1 to 9 (1= light green, 9= dark green) (bullitta et al., 2005; kir et al., 2010); ground cover: estimated visually as a percentage of soil cover; weed infestation: estimated visually as percentage of soil cover. all the data were grouped to obtain seasonal aggregation, where s1 was the first season of trials corresponding to summer 2011, s2 was autumn 2011, s3 spring 2012, s4 summer 2012 and s5 was the end of the study in autumn 2012. the mean length of winter dormancy was estimated for each species, and plants were considered dormant when color scores means reached a value of 2. statistical analysis was carried out by means of anova and tukey test to discriminate differences among averages species values. moreover, through multiple regression analysis, it was possible to evaluate which of the tested parameters (color, ground cover and presence of weeds) was strongly related to the quality on a global scale. all analysis were performed utilizing ibm spss statistics software (release 20). 3. results meteorological trends during experimental period both years were characterized by very hot and dry summer periods. in particular, 2011 was exceptionally dry until late autumn and had a total rainfall of 500 mm compared to the average of 800 mm (2002-2010). the highest recorded temperature occurred in august (38°c). during winter the coldest month was february 2012 with severe temperatures below 0°c (-9°c minimum recorded) and, even in full dormancy, warm-season grasses can be damage by the cold. damage is also proportional to the duration of low temperatures. in 2012 there was a total rainfall of approximately 900 mm but it was concentrated at the end of the year. for this reason, during the summer, there was a severe water deficit replaced by irrigation. spring was exceptionally warm, whereas the summer was one of the driest in recent decades. turf quality figure 1 shows the interaction between quality and season (s1= summer 2011, s5= autumn 2012) for the eight species or cultivars. the species that showed the best performance over time was cynodon dactylon, with good values and a constant quality trend. all three cultivars belonging to this species performed well: in particular, ‘black jack’, obtained higher scores at the beginning of the trial. the highest value (7.1) was attained during summer 2012 (s4), even though the presence of thatch probably influenced the estimation. paspalum vaginatum, after a good start and an improvement in autumn, proved to be the most sensitive species to low winter temperatures, having slower vegetative growth during the spring 2012 (s3) and the worst score, due to damage that occurred during winter. ‘sea spray’ showed better results in comparison to ‘marina’. zoysia japonica performed badly during the year of establishment until the second growing season. thereafter, fig. 1 global quality of the species/cultivars under investigation during the trial period (assessment with 1-9 scale). bars = standard error. l.p. = lolium perenne, c.d. = cynodon dactylon, p.v. = paspalum vaginatum, z.j. = zoysia japonica. 55 seppoloni et al., performance of warm-season turfgrasses in an area of central italy it gradually improved and gave very good scores at the end of the second year. ‘zenith’ performed better than ‘compadre’ on an overall scale. lolium perenne was the most unsuitable species, showing a rapid initial establishment, after which the quality began to decrease from s2, attaining the worst score (2) in s4. color the best performance regarding color was obtained with lolium perenne: it was clearly superior with a darker and brighter color in each period (fig. 2). only p. vaginatum, in s3, showed equivalent scores and it resulted to be the species with the best color among the tested warmseason grasses. cynodon dactylon gave unsatisfactory results, showing a decrease in quality in both years (in late summer and in the beginning of autumn), due to a rapid entrance into dormancy. moreover, the light green color, typical of these seeded cultivars of cynodon and the high production of thatch, influenced the scores. the trend of the two cultivars of zoysia japonica appears to be very similar to that of cynodon. ground cover and weed infestation table 1 shows the average ground cover values for each species during the different seasons of the trial. it is clear that the warm-season grasses increased the soil cover over time, while the percentage of l. perenne remained almost unchanged compared to the initial one (approximately 5560%). p. vaginatum, due to the winter cold, had a poor cover percentage in the spring of the second year but it was able to regenerate an efficient soil cover thanks to its strong stolons, and this trend is evident for both cultivars. c. dactylon demonstrated good and constant soil coverage and it was more satisfactory than other species, whereas z. japonica displayed the typical behavior of this species, attaining optimal values at the end of the growing season of the second year (s5). as regards the presence of weeds (table 1), infestation spread rapidly during the first year on l. perenne and z. japonica. the high presence of weeds on lolium is also due to the fact that this species is characterized by a bunch type table 1 average value (±se) of ground cover and infestation of the tested cultivars over the 5 seasons species/ cultivar ground cover (%) s1 s2 s3 s4 s5 lp 59.0±7.5 a 58.3±2.1 bcd 58.3±4.6 ab 51.7±4.8 d 61.7±2.5 c cd1 73.7±5.2 ab 86.3±5.1 a 76.7±8.8 a 92.8±1.5 a 95.0±0.1 a cd2 63.7±5.8 ab 81.3±4.4 a 78.3±2.2 a 92.2±1.1 a 94.4±0.6 a cd3 57.0±5.6 ab 79.6±4.2 ab 73.3±3.6 a 92.2±1.1 a 93.9±1.1 a pv1 67.0±3.2 ab 78.3±2.1 ab 40.0±6.3 b 58.1±9.3 cd 83.3±5.9 b pv2 61.7±2.6 ab 76.7±6.9 ab 42.5±14.4 b 71.9±12.3 bc 88.9±5.3 ab zj1 24.7±2.4 c 40.8±3.4 d 52.5±6.3 b 78.9±1.5 ab 87.2±2.0 ab zj2 39.7±7.6 bc 56.3±6.3 cd 60.0±3.8 ab 83.3±2.5 ab 89.4±0.6 ab species/ cultivar infestation (%) s1 s2 s3 s4 s5 lp 4.0±2.3 ab 16.7±4.0 bc 30.0±5.8 c 23.3±4.4 b 30.6±4.0 b cd1 0.3±0.3 a 5.8±0.8 ab 8.3±2.2 a 6.7±1.0 a 16.1±2.9 ab cd2 1.7±0.9 a 10.8±1.1 abc 8.3±2.2 a 9.4±2.8 a 18.9±5.3 ab cd3 1.7±1.2 a 5.4±1.1 a 7.5±2.5 a 9.4±4.4 a 13.9±5.6 a pv1 1.3±0.9 a 3.3±1.1 a 19.2±5.8 bc 14.7±6.8 b 20.0±3.3 ab pv2 1.0±1.0 a 4.2±0.4 a 15.0±2.5 ab 12.5±2.7 ab 19.4±7.2 ab zj1 4.3±1.3 ab 17.9±4.2 c 19.2±4.4 bc 11.7±3.5 ab 18.9±4.5 ab zj2 8.7±1.3 b 19.6±1.8 c 23.3±2.2 bc 13.9±2.4 ab 9.4±0.6 a lp= lolium perenne kokomo, cd1= cynodon dactylon black jack, cd2= c. dactylon casinò royale, cd3= c. dactylon la paloma, pv1= paspalum vaginatum marina, pv2= p. vaginatum sea spray, zj1= zoysia japonica compadre, zj2= z. japonica zenith. values in a column with the same letter are not significantly different (p<0.05) according to tukey test. fig. 2 average color of the species under investigation during the trial period (assessment with 1-9 scale). bars = standard error. l.p. = lolium perenne, c.d. = cynodon dactylon, p.v. = paspalum vaginatum, z.j. = zoysia japonica. 56 adv. hort. sci., 2015 29(1): 53-58 habitus that makes it less competitive with opportunistic weed species in comparison to warm-season grasses. in addition, lolium, after being stressed by the high summer temperatures, was also the only species not treated with glyphosate, taking into account its vegetative activity during the winter. for zoysia, the infestation was easily attributable to a slow establishment that left plenty of space for the development of invasive species at the beginning of the trial. nevertheless, for all the species, despite the winter control with glyphosate, a manual control against weeds in the second year was necessary. after these operations, the presence of weeds was shown to be reduced, thanks to the ability of these highly-competitive species to suppress weed growth. multiple regression analysis this model permits the calculation of turf quality value (considered the dependent variable) through the other observed parameters. the model applied (a stepwise model) involves a mechanism of removal of the independent variables provided that they do not cause a decline in the power of the model. the elimination of the variables from the model is performed automatically by comparison with a threshold value of significance, which in this case was set to be equal to 0.05. from the analysis the following formula was obtained: global quality= -1.447+0.067*cover-0, 050*weeds+0.443*color (r2 = 0.884) the parameters were ordered starting from the one most related to overall quality to the one with the lowest influence on the formula. no variable was removed from the model, demonstrating the importance of all investigated parameters in describing the aesthetic value of a turf. the presence of weeds, as expected, produced a negative value, as it was inversely related to the aesthetic appearance of the turf. the very high determination coefficient indicated the good correlation between the independent variables and the dependant variable (turf quality). this type of analysis could be used in future investigations to predict the overall performance of a turf. winter dormancy table 2 shows the dates at the beginning and the end of the growing season, and the length of vegetation and dormancy periods for the species during the trial years. at the end of the first year of experimentation, dormancy took place in november for all the species, with differences of about two weeks between the earliest (zoysia) and latest (paspalum) entrance into dormancy. the period of dormancy during winter 2011-2012 was particularly long for paspalum, also because the growing season in the spring 2012 started at the end of april. cynodon and zoysia showed early vegetation activity at the end of march. the growing season for 2012 was longer for z. japonica (236 days), due to its early vegetative growth and the entrance into dormancy in mid-november, which was exceeded only by paspalum vaginatum. nearly the same duration was found for c. dactylon (233 days), with greater precocity in vegetative growth, but with an earlier loss of green color than with other species. the shortest growing season, as expected, was found in p. vaginatum (208 days), despite its late entrance into dormancy. the period of dormancy during winter 2012-2013 showed a substantial analogy to the previous year for zoysia (137 days in 2011/2012 and 142 in 2012/2013) and paspalum (151 days compared to 159) but a considerable lengthening for cynodon (124 days compare to 160 in the second year). this behavior could be connected to the fact that mean and maximum spring temperatures in 2013 were lower compared with the previous year. this trend definitely affected the vegetative growth of the earliest species (cynodon), highlighting the important role temperatures play in the vegetative activity of warm-season grasses. 4. discussion and conclusions in this study, cynodon dactylon emerged as the species with the best overall behavior and the best potential adaptation to the environment. it displayed good quality standards, rapid establishment, good overall appearance, high ground cover and a competitive behavior against weed table 2 dates of the beginning and the end of the growing season and length of vegetation and dormancy periods for the species during the years of trial species beginning end vegetation (days) dormancy (days) cynodon dactylon 16/11/2011 124 paspalum vaginatum 26/11/2011 151 zoysia japonica 08/11/2011 137 cynodon dactylon 20/03/2012 08/11/2012 233 paspalum vaginatum 26/04/2012 20/11/2012 208 zoysia japonica 25/03/2012 16/11/2012 236 cynodon dactylon 17/04/2013 160 paspalum vaginatum 28/04/2013 159 zoysia japonica 07/04/2013 142 57 seppoloni et al., performance of warm-season turfgrasses in an area of central italy infestation. the constancy of studied parameters along experimental trial are consistent with volterrani et al. (1997). the worst performance concerned color of this species that probably resulted remarkably affected by presence of thatch, confirming the observations of holm et al. (1991). paspalum vaginatum presented an overall satisfactory appearance. however, the species was highly susceptible to cold winters, in agreement with de luca et al. (2008), and tesi (2012) reported cold damages on paspalum with temperature around -7°c. anyway this species performed in a very good manner concerning other variables, as it displayed a dark green-blue color, as well as a good color retention during autumn, as reported previously by volterrani et al. (1996) and geren et al. (2009). given that the stability of the turf was compromised, this behavior establishes a limit to the use of this species in areas that border the mediterranean climate or with severe risks of low temperatures during winter. zoysia japonica exhibited a typical growing pattern, confirming that observed by patton and reicher (2007), and it showed very poor performance in the first year, slow development and not competitive attitude against weeds. similar results regarding this species were found by geren et al. (2009) in a comparative study among warm-season grasses in turkey. on the other hand, starting from the beginning of the second year, a gradual increase in quality, color and coverage was observed, and the species achieved remarkable results at the end of second growing season, in agreement with sladek et al., (2011). through multiple regression analysis it was possible to evaluate which of the tested parameters influenced turf quality the most. the results showed that the ground cover and the presence of weeds are strongly related with the overall aesthetic quality. the winter season revealed important differences in relation to the length of the period of dormancy among species. p. vaginatum had the longest period of dormancy (about 155 days as the average of the two years of experimentation). a delay in the vegetative activity in paspalum, in comparison to other warm-season grasses, was also reported by other authors in different climatic areas (duncan, 1996; miele et al., 2000; volterrani et al., 2001), as well as in both coastal and internal areas of tuscany (volterrani et al., 2000; pardini et al., 2002) and in the province of rome (croce et al., 2001). c. dactylon showed a rapid recovery from winter dormancy in warm springs, while a longer length of dormancy was observed during the cold spring 2013. z. japonica remained dormant for approximately 140 days during both years of the experiment. in general, in the interior zones of this study, the duration of dormancy was found to be longer than those verified by other experiments along the coastal areas of central italy (croce et al., 2004). for this reason, in this environment, it is necessary to plan and analyze costs and benefits associated with the use of warm-season grasses. where it is not possible to accept the absence of green color during winter, it is evident that the use of warm-season species can be extended to these areas only by adopting appropriate overseeding programs, but these interventions seem possible only in high standard turf such as those related to sport pitches. acknowledgements the authors acknowledge prof. marco volterrani for his valuable and constructive suggestions during the planning and development of this research work and green grass s.r.l. for its technical support for this project. research was funded by the italian ministry of agricultural and forest policies, “bando oiga 2009”. references argenti g., ferrari l., 2009 plant cover evolution and naturalisation of revegetated ski runs in an apennine ski resort (italy). iforest, biogeosciences and forestry, 2: 178182. beard j.b., 1973 turfgrass: science and culture. prenticehall, inc., englewood cliffs, nj, usa. bernardini v., 2007 i manti erbosi sportivi ad alto livello: l’esperienza dell’uso delle macroterme allo stadio olimpico di roma. tappeti erbosi, aspetti tecnici, ambientali e paesaggistici. i quaderni del c.i.r.a.a., felici editore, pp. 65-70. bigelow c.a., walker k.s., 2008 annual nitrogen 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cultivars in a mediterranean climate. acta horticulturae, 661: 365-368. de luca a., volterrani m., gaetani m., grossi n., croce p., mocioni m., lulli f., magni s., 2008 warmseason turgrass adaptation in northern italy. proceedings of the 1st european turfgrass society conference, pisa, italy, 2008, pp. 75-76. duncan r.r., 1996 the environmentally sound turfgrass of the future. seashore paspalum can withstand the test. a usga-sponsored research project, pp. 17-19. 58 adv. hort. sci., 2015 29(1): 53-58 easton z.m., petrovic a.m., 2005 impact of organic and mineral fertilizers on run-off from turf. better crops, 89: 16-17. gaetani m., lulli f., andreucci a., masini a., vittori g., volterrani m. 2013 sprouting and plant regeneration capability in saline conditions of seashore paspalum, manilagrass, and hybrid bermudagrass stolons. propagation of ornamental plants, 13: 57-64. geren h., avcioglu r., curaoglu m., 2009 performances of some warm-season turfgrasses under mediterranean conditions. african journal of biotechnology, 8: 4469-4474. holm l.g., plunknett d.l., pancho j.v., herberger j.p., 1991 the world’s worst weeds: distribution and biology. krieger publishing company, malabar, florida, usa. hull r.j., 1990 the psychological value of turf and ornamental plants. agronomy abstracts, 175. kir b., avcioglu r., demiroglu g., simic a., 2010 performance of some cool season turfgrass species in mediterranean environment: i. lolium perenne l., festuca arundinacea schreb., poa pratensis l., and agrostis tenuis sibth. turkish journal of field crops, 12: 174-179. linse s.j., mergen d.e., smith j.l., trlica m.j., 2001 upland erosion under a simulated most damaging storm. journal of range management, 54: 356-361. magni s., gaetani m., grossi n., caturegli l., la bella s., leto c., virga g., tuttolomondo t., lulli f., volterrani m., 2014 bermudagrass adaptation in the mediterranean climate: phenotypic traits of 44 accessions. adv. hort. sci., 28(1): 29-34. marchione v., 2008 performance of several cynodon dactylon and zoysia japonica cultivars in southern italy. proceedings of the 1st european turfgrass society conference, pisa, italy, 2008, pp. 125-126. miele s., volterrani m., grossi n., 2000 warm-season turfgrasses: results of a five-year study in tuscany. agricoltura mediterranea, 130: 196-202. panella a., croce p., de luca a., falcinelli m., modestini f.s., veronesi f., 2000 tappeti erbosi. calderini edagricole, bologna, italy. pardini a., tallarico r., parrini d., 2002 comportamento di cultivar di specie graminacee micro e macroterme da tappeto erboso in una località del mugello. rivista di agronomia, 36: 257-263. patton a., reicher z., 2007 zoysiagrass establishment rates. golf course management, march, pp. 98-101. piano e., 2005 inerbimenti e tappeti erbosi nella realtà italiana: motivazioni e finalità per lo sviluppo della ricerca. atti del convegno “inerbimenti e tappeti erbosi per l’agricoltura, l’ambiente e la società”. volume 1. comunicazioni poster. istituto sperimentale per le colture foraggere, lodi, italy, pp. 5-20. reyneri a., bruno g., 2008 effects of clipping management on the quality of a low maintenance tall fescue turf. proceedings of the 1st european turfgrass society conference, pisa, italy, pp. 159-160. roberts e.c., 1990 the significance of turf benefits. an overview. agronomy abstract, 174. schiavon m., barnes b., shaw d., henry j., baird j., 2013 strategies for converting tall fescue to warm-season turf in a mediterranean climate. horttechnology, 23: 442-448. sevostianova e., leinauer b., 2014 subsurface-applied tailored water: combining nutrient benefits with efficient turfgrass irrigation. crop science, 54: 1926-1938. sladek b.s., henry g.m., auld d.l., 2011 effect of genotype, planting date, and spacing on zoysiagrass establishment from vegetative plugs. hortscience, 46: 1194-1197. tesi r., 2012 importanza dei tappeti erbosi e ruolo delle poaceae macroterme. atti del convegno accademia dei georgofili, firenze, italy. turgeon a.j., 2002 turfgrass management. prentice hall inc., upper saddle river, nj, usa. volterrani m., gaetani m., miele s., 2000 la trasemina autunnale di specie microterme su tappeti erbosi realizzati con paspalum vaginatum swartz e cynodon dactylon x transvaalensis burtt. davy. rivista di agronomia, 34: 28-34. volterrani m., grossi n., pardini g., miele s., gaetani m., magni s., 1997 warm season turfgrass adaptation in italy. international turfgrass society research journal, 8: 1344 -1354. volterrani m., magni s., 2007 il tappeto erboso ieri, oggi e domani. tappeti erbosi, aspetti tecnici, ambientali e paesaggistici. i quaderni del c.i.r.a.a., felici editore, pp. 29-36. volterrani m., miele s., magni s., gaetani m., pardini g., 2001 bermudagrass and seashore paspalum winter overseeded with seven cool-season turfgrasses. international turfgrass society research journal, 9: 957-961. volterrani m., pardini g., grossi n., gaetani m., miele s., pietrini e., 1996 valutazione dell’adattabilità di specie graminacee macroterme da tappeti erbosi alle condizioni ambientali dell’italia centrale. italus hortus, 3: 1016. youngner v.b., marsh a.w., strohman r.a., gibeault v.a., spaulding s., 1981 water use and turf quality of warm-season and cool-season turfgrasses. proceedings of the fourth international turf research conference, 4: 251-257. impaginato 31 1. introduction in spite of the well-known importance of wild flora as a source of food and medicinal substances, more studies on the diversity and agronomic value of these plant species are still needed (arena and vater, 2005). areas with indigenous flora offer non-domesticated plants (monge et al., 2000), like berberis genus in patagonia (orsi, 1984) for these purposes. berberidaceae in the widest sense is a small family, consisting of 10 to 12 genera and about 600 species, with as many as 500 of these belonging to berberis l., widely distributed in both the old world and new world (nowicke and skvarla, 1981). in patagonia, berberis genus is well represented by 16 species of native shrubs and they are distributed from neuquén to tierra del fuego (arena and curvetto, 2008). however, according to a later classification of the genus (landrum, 1999), the number of species is less than previous studies cited by orsi (1984), as landrum groups the species b. buxifolia, b. microphylla and b. heterophylla under b. microphylla g. forst, postulating that the differences among them may fluctuate to retain its range of species. in particular, b. microphylla (ex b. buxifolia lam.), commonly known as “calafate”, is an evergreen shrub that is present throughout the region mentioned, prevalent on the island of tierra del fuego over other species of berberis. b. microphylla has growing economic potential due to the production of fruits as a non-timber forest product (tacón clavaín, 2004). in fact, its dark blue berries are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. moreover, the fruits have a high content of phenols and antioxidants (arena and curvetto, 2008; arena et al., 2012). some characteristics of its phenological phases (arena et al., 2013 a; arena and radice, 2014), fruit composition and production (arena and curvetto, 2008; arena et al., 2003; 2011; 2013 b) have already been studied in natural populations of this species. characterization of pollen grains is an important step for programs of genetic resource conservation and improvement, complementing basic studies of biological data that characterize genotypes (de castro nunes et al., 2012). selection on male gameadv. hort. sci., 2016 30(1): 31-37 doi: 10.13128/ahs-18699 characterization and evaluation of berberis microphylla g. forst pollen grains s. radice (*), m. arena conicet, facultad de agronomía y ciencias agroalimentarias, universidad de morón, machado 914, b17108eoh morón, buenos aires, argentina. key words: barberry, germination, patagonia, size, viability. abstract: berberis microphylla, commonly known as “calafate”, is a non-timber forest product native from patagonia, and its berries possess highlighted nutraceutical value. the objective of this research was to describe the morphology and anatomy of pollen grains of berberis microphylla g. forst genotypes growing spontaneously on the island of tierra del fuego (argentina), and evaluate their vitality and germination. pollen grain diameter varied from 40 to 47.26 µm, the pollen grains of 124 and 201 genotypes being significantly smaller than the others. vitality measured by dapi methodology was also variable among genotypes, although always about 50%. in vitro germination of pollen grains measured one day after the flowers were collected was very high for some genotypes (near 80%), and then decreased after 21 days of storage, except for genotype 123 whose germination value increased from 44.34 to 69%. the significant variability found in pollen performance (size, viability and germination) among b. microphylla genotypes from a natural population could be interpreted as an enhanced survival strategy to maximize reproduction fitness, with a marked capacity of response to environmental changes. high viable pollen frequency together with germination percentages observed in all the genotypes tested could indicate a good fertilization process. the correlation observed between size and germination percentage could be used as markers of pollen grain performance, paving the way for possible b. microphylla breeding. (*) corresponding author: siradice@yahoo.com received for publication 31 october 2015 accepted for publication 4 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 31-37 32 tophytes (pollen) to alter the genetic constitution of the subsequent sporophytic generation has been suggested as an interesting tool in plant breeding programs (hormaza and herrero, 1992). pollen competitive ability describes the reproductive success of a pollen grain and can therefore be considered as equivalent to pollen fitness (sari-gorla and frova, 2005). the variability in pollen performance (size, viability and germination) among genotypes of a natural population could be interpreted as a survival strategy to maximize reproduction fitness (tejaswini, 2002), while enabling a capacity of response to environmental changes (hedly et al., 2005). nevertheless, aspects related to fertility and reproductive organs have not yet been studied on berberis microphylla. the objective of this research was to describe the morphology and anatomy of pollen grains of berberis microphylla g. forst genotypes growing spontaneously on the island of tierra del fuego (argentina), and evaluate their vitality and germination. 2. materials and methods plant material flowers (n=20) in phase e (before anthesis according to arena et al., 2011) were collected from each b. microphylla genotype grown near ushuaia city, tierra del fuego (54° 48’ sl, 68° 19’ wl and 30 m asl) (table 1), in october 2013. the flowers were immediately placed in petri dishes with wet paper at 5°c for viability and germination studies. pollen grain description and size equatorial and polar diameters of the pollen grains (n=50, randomly selected) were measured for each studied genotype using a leica dm 2500 microscope. the average of the two parameters for each pollen grain was then calculated. light microscopy button flowers were dehydrated in an ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. scanning electron microscopy button flowers were dehydrated in an ethanol series and a critical point drying technique was employed. samples were sputter coated with 20 nm gold and observed with a philips xl 30 sem. transmission electron microscopy anthers were pre-fixed in 2.5% glutaraldehyde in phosphate buffer (ph 7.2) for 2 h and then post-fixed in oso4 at 2 oc in the same buffer for 3 h. they were then dehydrated in ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were made on a sorval ultramicrotome, stained with uranyl acetate and lead citrate (o’brien and mc cully, 1981). sections were observed with a jeol-jem 1200 exii tem at 85.0 kv. pollen grain viability pollen grains were hydrated with sucrose solution (15%) and treated with fluorescein diacetate (10%) and propidium iodine (2%) (greissl, 1989). the number of viable and not viable pollen grains was recorded under optic microscope, with a minimum of 300 pollen grains per genotype. pollen grain germination pollen grains were put on micro drops of a saline solution composed of 2 x 10 -3 m h3bo3 and 6 x10 -3 m ca (no3)2 added with sucrose 30% (dafni, 1992). micro drops were placed on the inside of the lid of a petri dish in which 3 ml of water were added in the base to create a humid chamber. incubation was at 21±2°c. the number of germinated and aborted pollen grains was recorded under optic microscope 24 h after the test started and performed with anthers conserved for 1, 10 and 21 days at 5°c. data analysis measurements were analyzed by anova and tukey’s test and chi-square test was employed to evaluate pollen vitality. table 1 berberis microphylla genotype number and its satelital position in tierra del fuego (argentina) genotype number sl wl 107 54 49 43 0 68 19 01 7 108 54 49 42 9 68 19 02 1 111 54 49 43 5 68 19 00 1 122 54 49 40 9 68 19 04 1 123 54 49 42 4 68 19 07 1 124 54 49 42 8 68 19 04 2 125 54 49 46 1 68 19 00 6 126 54 49 45 4 68 18 58 7 177 54 49 42 9 68 19 27 9 201 54 49 50 7 68 19 21 7 202 54 49 51 2 68 19 20 1 radice and arena characterization and evaluation of berberis microphylla pollen grains 33 3. results pollen grain description and size flowers collected in phase e had five stamens; the anthers were not yet dehiscent (fig. 1a) although the pollen grains were already mature (fig. 1b, 2a-b). a mature pollen grain is formed of a vegetative cell with a dense cytoplasm with numerous starch grains (fig. 1b). b. microphylla pollen grains are spherical with a psilate punctate surface interrupted by mild cracks (fig 2. a-b), resembling a tennis ball. the pollen wall is formed by an exine and intine of considerable thickness (fig. 3a). transmission electron micrographs make it possible to identify an exine with two different layers, the ektexine and the endexine (fig. 3a). ektexine is nearly amorphous and not organized into typical foot layer, columellae, and tectum units. conversely, ektexine appears as an external irregular cover with channels and small enclosed areas which are electron translucent (fig. 3). endexine has greater electro-density than ektexine. immediately below this, the intine is present, at least four times thinner than the exine and with a very low electro-density. pollen grain wall appears different on the apertures or pore zone (fig. 3b). ektexine is less structured and is represented by nodules above the endexine. the pollen grain diameters varied significantly among genotypes, from 40 to 47.26 µm (p≤0.001). the maximum values were observed for genotype 107, which was significantly higher than genotypes 124, 126, 177, and 201 (fig. 4). pollen grains of genotypes 124 and 201 were significantly smaller than genotypes 107, 111, 123 and 202 (fig. 4). pollen grain viability pollen grains stained with fluorescein diacetate and propidium iodine showed very different colors depending on whether they were vital or non-vital. fig. 1 androecium and pollen grain of b. microphylla flower in phase e. a) view of androecium with anther (a) no dehiscent; b-c) microphotograph of cross-section of anther and mature pollen grain with starch grains (arrowhead). bars: a = 1mm; b = 10 µm; c = 100 µm. fig. 2 sem micrograph of mature pollen grain of b. microphylla. a) view of microsporangium with pollen grain; b) detail of a pollen grain. bars: a = 10,000 nm; b = 50 µm. fig. 3 tem micrograph of pollen grain wall of b. microphylla. a) detail of different parts of exine: endexine (end), ektexine (ekt) and intine (i); b) detail of pore zone with ektexine fragmented (arrows). barras = a-b = 1 µm. fig. 4 mean diameter of b. microphylla pollen grains of the studied genotypes. adv. hort. sci., 2016 30(1): 31-37 34 vital pollen grains were bright green while non vital ones stained red (fig. 5). in this latter case, another category was evaluated, the sub-vital pollen grains, those that can germinate but it is uncertain whether they can be efficient in fertilization. pollen viability of different genotypes gave very different results (p≤0.001). for genotypes 111, 123, 124 and 202, values of vital pollen grains were above 70%, while for genotype 108 the value was 51.47% (fig. 6). in coincidence, the 108 genotype shows a 36.80% of sub-vital pollen grains, value significantly greater than that observed for the 111, 123, 124, 126, and 202 genotypes. pollen grain germination the pollen grain germination of b. microphylla was significantly different among genotypes (p≤0.001) and days of conservation (p≤0.001), and the interaction between the two factors studied (p≤0.001) was significant (fig. 7). germination of the pollen grain was at maximal level after one day of collection in most genotypes, except for genotype 123 which showed a maximum value after 21 days of storage, although without significant differences between 10 and 21 days (fig. 7). genotypes 124 and 125 showed percentages of pollen grain germination up to 70%, while genotype 201 presented a maximum value of 87.03% after one day of the collected flowers (fig. 7). the pollen germination rate remained unchanged among the three tested dates, except for genotypes 122, 126 and 177. in addition, the values were significantly lower after 21 days of conservation for genotypes 122, 126 and 177 (fig. 7). this decrease in the percentage of pollen germination is in accordance with the percentage of aborted pollen grains (data not shown). anova values were 0.001 for both date and genotype factor and their interaction. furthermore, the genotypes 108 and 202 showed an increase in the percentage of germinated pollen grains between the second and third test, i.e. the values obtained after 21 days of flower conservation were higher than those obtained with 10 days of storage but these differences were not significant (fig. 7). finally, a significant negative correlation between the pollen grain germination and the pollen grain size was found (r= -0.252; p≤0.001). 4. discussion and conclusions it was observed that flower differentiation on b. microphylla started 12 weeks after bud break in coincidence with the end of the first fruit growth phase fig. 5 viable (v), and non-viable (nv) pollen grains observed by fluorescence microscopy. fig. 6 viability of b. microphylla pollen grains from the assessed genotypes. columns with different letters indicate significant differences between genotypes and date assessed (2 p<0.05). fig. 7 germination percentage of b. microphylla pollen grains from the assessed genotypes. columns with different letters indicate significant differences between genotypes and date assessed (tukey p<0.05). radice and arena characterization and evaluation of berberis microphylla pollen grains 35 (arena and radice, 2014). nevertheless, final development of male gametes occurs the following spring when the flower bud elongates (data not shown). mature pollen grains can be found in the stage of lower emergence code 59 according to the bbch scale (arena et al., 2013 a). there are few published studies on the pollen of berberis species. erdtman (1952) and heusser (1971) described the pollen grains of the berberis genus and they determined that the pollen grains measured an average of 30 to 65 µm with an exine 2-3 µm thick. nowicke and skvarla (1981) emphasized the presence of irregular apertures, a psilate surface and an unstratified exine. in adittion, the exine of b. micropylla shows an endexine as a prominent fibrous-granular layer and the ektexine with cavities and channels which suggest the endexine. pollen performance traits are often genetically based (hedly et al., 2005; hove and mazer, 2013), however they could be also affected by differences in the nutritive status of the developing pollen grains as well as by the environmental conditions (hedly et al., 2005). the size of pollen grains is considered to be one indicator of their viability (i.e. germinability and pollen tube growth rate), while the proportion of large pollen grains has been used to estimate pollen performance. variation in pollen grain size among plants has been documented for several species (varis et al., 2011). larger pollen grains are thought to contain more resources for germination and, thus, have greater viability than smaller grains (dufaÿ et al., 2008). however, in b. microphylla a negative correlation between pollen size and germination percentage was observed, as was also found in pinus sylvestris (varis et al., 2011), as the growth of the pollen tube may be more dependent on pollen storage than pollen size. good fertilization is directly related to very good pollen viability. it is estimated that the value of viability should be above 70% for fruit production (urquieta, 2010). on the other hand, pollen grains which exceed 50% viability would be the only one that can be selected for use as male parents (urquieta, 2010). all genotypes of b. microphylla tested presented viability values above 50%, in other words having very good prospects for fruit production. urquieta (2010) found similar results when pollen grains of b. bidentata, b. darwinii, b. parodii and b. trigona were tested. pollen grain viability of these species was variable between 59.6 and 74.1%. it has been found that high percentages of pollen viability are due to high degrees of adaptability of the species to different environmental conditions (kelly et al., 2002). in effect, high frequency of viable pollen reflects the adaptability of the species, since environmental plasticity submitted by pollen allows the genotype a satisfactory performance to different environmental conditions (paupière et al., 2014). on the other hand, pollen viability was influenced by relative humidity, temperature, atmospheric composition, and oxygen pressure after release from the anthers (bots and mariani, 2005), so it is expected that the experimental values obtained for b. microphylla are less than true values. pollen germination under in vitro conditions always produces lower values than those obtained with the viability test (ontivero et al., 2006). in the present study, this premise is true for genotypes 107, 111, 123, 124, and 202 (figs. 6 and 7). on the contrary, all other genotypes showed viability values higher than those obtained by germination viability test. note that after 21 days of harvested flowers, pollen germination values obtained were only 10% lower than viability values for genotypes 108 and 123. these results could be due to the protective effect that antioxidants have on pollen. in effect, it is well known that secondary metabolites produced in the tapetum, such as phenolic compounds, can spread to the pollen and play a role in pollen colour, in the attraction of pollinators, in pollen tube germination, and in protection against abiotic stress of pollen (paupière et al., 2014). pollen germination and tube growth is largely due to the presence of flavonols in mature pollen grains (yistra et al., 1992). although the content of flavonols was not measured in the anthers of b. microphylla, it is well known that flavonols are present in berberis species (končić et al., 2010). in effect, accumulation patterns of phenolic compounds during fruit growth and ripening in b. buxifolia (b. microphylla) was studied by arena et al. (2012). it is likely that high values obtained from in vitro germination of pollen grains could be explained by the protective effect that these compounds perform. the variability found in pollen performance (size, viability and germination) among genotypes of the natural population of b. microphylla, and its correlations, suggest the existence of pollen competition leading to unequal reproductive success in this species, as was observed for camellia sinensis by muoki et al. (2007). the significant variability found in pollen performance (size, viability and germination) among b. microphylla genotypes from a natural population could be interpreted as a highlighted survival strategy adv. hort. sci., 2016 30(1): 31-37 36 to maximize reproduction fitness, with a marked capacity of response to environmental changes. high viable pollen frequency, together with germination percentages observed in all the genotypes tested, could indicate a good fertilization process. the correlation observed between size and germination percentage could be used as a marker of pollen grain performance, with these findings representing the first antecedents useful for b. microphylla breeding. acknowledgements the authors thank the prefectura naval argentina and mrs. isabel farías for her assistance with the histology. this research was supported by grants pip 314 subsidized by conicet. references arena m.e., curvetto n., 2008 berberis buxifolia fruiting: kinetic growth behavior and evolution of chemical properties during the fruiting period and different growing seasons. scientia horticulturae, 118(2): 120127. arena m.e., giordani e., radice s., 2011 flowering, fruiting and leaf and seed variability in berberis buxifolia, a patagonian native fruit species, pp. 117-136. marin l., and d. kovaè (eds.) native species: identification, conservation and restoration, nova sciencia publisher inc., hauppauge, ny, usa, pp. 176. arena m.e., giordani e., radice s., 2013 a phenological growth and development stages of the native patagonian fruit species berberis buxifolia lam. j. food, agric. environ., 11(3-4): 1323-1327. arena m.e., postemsky p., curvetto n.r., 2012 accumulation patterns of phenolic compounds during fruit growth and ripening of berberis buxifolia, a native patagonian species. new zealand journal of botany, 50(1): 15-28. arena m.e., radice s., 2014 shoot growth and development of berberis buxifolia lam. in tierra del fuego (patagonia). scientia horticulturae, 165: 5-12. arena m.e., vater g., 2005 native and introduced species of small fruits in austral patagonia, tierra del fuego, pp. 96-110. in: dris r. (ed.) fruits: growth, nutrition, and quality . wfl publisher, helsinki, finlandia, pp. 220. arena m.e., vater g., peri p., 2003 fruit production of berberis buxifolia lam. in tierra del fuego. hortscience, 38(2): 200-202. arena m.e., zuleta a., dyner l., constenla d., ceci m., curvetto n.r., 2013 b berberis buxifolia fruit growth and ripening: evolution in carbohydrate and organic acid contents. scientia horticulturae, 158: 5258. bots m., mariani c., 2005 pollen viability in the field. research reports no. cgm5. dafni a., 1992 pollination ecology: a practical approach. oxford university press, oxford, uk, pp. 250. de castro nunes r., oliveira bustamante f., techio v.h., mittelman a., 2012 morphology and pollen viability of lolium multiflorum lam. ciência e agrotecnologia, 36(2): 180-188. dufaÿ m., vaudey v., de cauwer i . , touzet p., cuguen j., arnaud j.f., 2008 variation in pollen production and pollen viability in natural populations of gynodioecious beta vulgaris ssp. maritima: evidence for a cost of restoration of male function? j. evol. biol., 21: 202-212. erdtman g., 1952 pollen morphology and plant taxonomy. gff, 74(4): 526-527. greissl r., 1989 vitality analysis of monadic and polyadic pollen grains using optical contrast-fluorescence microscopy. scientific and technical information, ix(5): 180-184. hedly a., hormaza j.i., herrero m., 2005 influence of genotype-temperature interaction on pollen performance. j. evol. biol., 1(8): 1494-1502. heusser c.j., 1971 pollen and spores of chile: modern types of the pteridophyta , gymnospermae, angiospermae. university of arizona press, tucson, arizona, usa, pp. 167. hormaza j.i., herrero m., 1992 pollen selection. theoretical and applied genetics, 83: 663-672. hove a.a., mazer s.j., 2013 pollen performance in clarkia taxa with contrasting mating systems: implications for male gametophytic evolution in selfers and outcrossers. plants, 2: 248-278. kelly j.k., rasch a., kalisz s., 2002 a method to estimate pollen viability from pollen size variation. am. j. bot., 89(6): 1021-1023. končić m.z., kremer d., karlović k., kosalec i., 2010 evaluation of antioxidant activities and phenolic content of berberis vulgaris l. and berberis croatica horvat. food and chemical toxicology, 48(8): 21762180. landrum l.r., 1999 revision of berberis (berberidaceae) in chile and adjacent southern argentina. ann. missouri bot. gard., 86(4): 793-834. monge a., chorgade m., erhardt p.w., genellin c.r., koga n., lindberg p., perun t.j., topliss j.g., trivedi b.k., wermuth c.g., 2000 medicinal chemistry in the development of societies: biodiversity and natural products. eur. j. med. chem., 35(12): 1121-1125. muoki r.c., wachira f.n., pathak r., kamunya s.m., 2007 potential male gametophyte competition among camellia sinensis genotypes in isolated biclonal seed orchards. african crop science journal, 15(2): 59-66. nowicke j.w., skvarla j.j., 1981 pollen morphology and phylogenetic relationships of the berberidaceae. radice and arena characterization and evaluation of berberis microphylla pollen grains 37 smithsonian contributions to botany, 50: 1-83. o’brien t.p., mc cully m.e., 1981 the study of plant structure: principles and selected methods . termarcarphy pty, melburne, australia, pp. 357. ontivero m., radice s., giordani e., bellini e., 2006 effects of different pollination treatments in genotypes of prunus salicina lindl. international journal of horticultural science, 12(2): 141-146. orsi m.c., 1984 berberidaceae, pp. 325-348. in: correa m.n. (ed.) flora patagónica sección 4ª. inta, buenos aires, argentina. paupière m.j., van heusden a.w., bovy a.g., 2014 the metabolic basis of pollen thermo-tolerance: perspectives for breeding. metabolites, 4(4): 889-920. sari-gorla m., frova c., 2005 pollen tube growth and pollen selection, pp. 333-351. in: shivana k.r., and v.k. sawhney (eds.) pollen biotechnology for crop production and improvement. cambridge university press, ny, usa, pp. 468. tacón clavaín a., 2004 manual de productos forestales no madereros. cipma, valdivia, chile, pp. 22. tejaswini p., 2002 variability of pollen grain features: a plant strategy to maximize reproductive fitness in two species of dianthus? sexual plant reproduction, 14: 347-353. urquieta c.a.p., 2010 evaluación de la viabilidad polínica de cuatro especies pertenecientes al género berberis l. (berberidaceae). thesis, chile university, pp. 85. varis s., reiniharju j., santanen a., ranta h., pulkkinen p., 2011 the size and germinability of scots pine pollen in different temperatures in vitro. grana, 50(2): 129-135. yistra b., touraev a., moreno r.m.b., stöger e., van tunen a.j., vicente o., heberle-bors e., 1992 flavonols stimulate development, germination, and tube growth of tobacco pollen. plant physiology, 100(2): 902-907. impaginato 23 1. introduction food security is a central theme of the new millennium and it must be faced at national and international levels, taking into consideration the multidisciplinary nature of the field which involves socio-cultural, political and environmental, as well as agronomic and economic aspects (deaton and paxson, 1998). from an economic and environmental point of view, food security is defined as a situation in which people have safe and appropriate food with nutritional requirements, for an active and healthy life (wfs plan of action, 1996). food security is based on three pillars: food availability, food access, and food uses as reported in the fao guidelines (matushke, 2009). food security is a priority in all developing countries, in particular in the urban areas of africa with the egyptian situation and the conditions of its capital city, cairo, being critical. despite the underestimation of risks, the population of the slum areas is affected by malnutrition. in 2005 the egyptian demographic and health survey stated that 18% of egyptian youth and 16% of the residents of urban areas were affected by malnutrition. in a city like cairo, the improvement of agricultural hydroponic systems might be a possible solution to this problem, in particular for informal urban settlements. the development of an efficient and productive growing protocol such as simplified hydroponic systems could be used to grow horticultural produce and increase the availability of natural and safe food for the poorest classes of the population (dresher, 2004). food insecurity is a global problem because it is caused by growth of the demand by the world population for secondary food like meat, making, basic resources, such as vegetables and cereals, less available for the poor parts of population (godfray et al., 2010). the world’s urban population is expected to double in the next 30 years, meaning there will be a growing number of urban poor people. according to the united nations human settlement programme (un-habitat), urban population expansion will be more pronounced in developing countries as a result adv. hort. sci., 2016 30(1): 23-29 doi: 10.13128/ahs-18698 vegetable production using a simplified hydroponics system inside city of dead (cairo) a. giro 1 (*), s. ciappellano 2, a. ferrante 1 1 dipartimento di scienze agrarie e ambientali produzione, territorio, agroenergia, università degli studi di milano, via celoria, 2, 20133 milano, italy. 2 dipartimento di scienze per gli alimenti, la nutrizione e l'ambiente, università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: food security, heavy metals, middle east, sustainable agriculture. abstract: this research work was performed in the poorest urban area of cairo (egypt) in a slum area called al-quarafa. the aim was to develop and evaluate a simplified hydroponics system (hs) to grow vegetables for the local inhabitants who live in an extreme status of food unsafety. in the hydroponic growing system the tomato plants were cultivated and two substrates were compared: peat:perlite (70/30 w/w) and sand:coir (50:50 w/w). this technique guarantees high levels of production and low contamination by avoiding the use of polluted urban soils which are often common in slum areas. macro and microelements in tomato fruits were analysed by icp-ms. results showed low concentrations of heavy metals (cd, sr, as, cr, mo) with all heavy metals under the levels set by european community laws. lack of food safety in slums is more closely linked with malnutrition than starvation, so it is also important to understand mineral availability (both micro and macro) and nutritional significance for health. for this reason micro and macro elements (k, ca, na, fe, mn, mg, cu, zn) were analysed in the harvested fruits. this study showed that hs is a valid method to grow vegetables in urban areas to improve food security in middle east cities. (*) corresponding author: andrea.giro@unimi.it received for publication 19 october 2015 accepted for publication 28 january 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 23-29 24 of high birth rates and immigration from rural areas as people flock to cities in search of food, employment and security. population growth will lead to an increase in urban slum areas, with high levels of unemployment, lack of food safety, and malnutrition. the increase of urbanization in developing countries enhances food insecurity of large cities. the poorest people move from rural to urban areas in order to improve their life with hopes of finding a job. unfortunately, in many cases people do not find employment and are obliged to live in the slum areas, increasing the food demand. for this reason, they become more vulnerable in their new position as citizens and consumers. by 2030, it is estimated that approximately 800 million people in developing countries will live in big cities, instead of in rural areas. this prospective is shocking for many nations, in particular for the social and economic relationships among citizens; this phenomenon is unescapable due to economic development and it could create a negative impact on the food security of populations of cities and, in particular, megacities (cohen and garrett, 2010) (fig. 1). in order to counteract the reduction of food availability in african cities, urban people started growing vegetables in urban and peri-urban areas. studies showed a considerable degree of self-sufficiency in the production of fresh vegetables, poultry production and the raising of other animals in many large cities of developing countries (armar-klemesu, 2000). for example, dakar produces 60% of its vegetable consumption and produces poultry for 65-70% of the national demand (mubvami and mushamba, 2004). accra produces 90% of the fresh vegetables consumed in the city (cencosad, 1994; maxwell et al., 2000). in dar es salaam, more than 90% of leafy vegetables in the markets come from the open spaces adjacent to the city or in garden houses (lee-smith, 2010). urban agriculture improves the nutritional state of vulnerable communities as evidenced by studies carried out by mwangi (1995) in nairobi. however, one of the main problems of urban agriculture is the competition for spaces within the city itself between people, vehicles, and animals. another important issue is the agricultural knowledge of the people. many of them do not know how to grow plants, therefore very simple growing systems must be developed. simplified hydroponic (sh) systems allow cost-effective agricultural production in confined spaces, such as internal urban spaces, particularly in the cities of developing areas (seikh, 2006). the sh technique allows higher yield, safer food, and at lower costs. the costs of the entire sh system are fundamental for sustainability of the production of goods; for this reason local materials, best if recycled, were used to construct low budget hydroponic systems. horticulture works well in urban and peri-urban zones because it is highly labour-intensive, involving perishable products and short-cycle, productive, high-value crops, which require less land and water per unit of product than other food crops. hydroponic systems are growing techniques that do not use soil, making them suitable in urban areas to cultivate horticultural commodities (seikh, 2006). it is a relatively young technique used in commercial fields over the past 40 years (grewal et al., 2011). the potential of sh is underestimated: it can be a solution for marginal areas, in developing countries with malnutrition problems. sh technique has the objective of reducing the costs of traditional hydroponic techniques, thus making it available and easy to manage for the poor and people without agriculture skills living in slums, like al-quarafa. for the aims of this study, we used local materials to construct sh greenhouses in order to diminish the cost and to make the technique reproducible for the local population. one of the main costs of sh is the substrate, so it is important to understand which substrate is best in which environment. standardisation of an efficient protocol of sh is important in order to cultivate horticultural products within the urban context (santos and ocampo, 2005). nowadays, many examples of hydroponics are available in developing countries but they are not usually well set up and tend to be expensive. hence, the new challenge is represented by the inception fig. 1 rural and urban index of food energy. international food policy research institute (ifpri). giro et al. vegetable production using a simplified hydroponics system 25 and enhancement of a hydroponic system sustainable in all aspects, from production to management. 2. materials and methods simplified hydroponic systems and plant cultivation boxes made of wood (100x50x20 cm) ensure the best conditions for plant growth and for transportation which are fundamental aspects for the socioenvironmental context (iwasa and roughgarden, 1984). tomato plants (solanum lycopersicum l.) require a minimum depth of 20-30 cm for root system development (pardossi et al., 2005). for the present study, plant density was 3 plants/m2. individual modules of locally manufactured palm wood boxes (50x50x25 cm) were used to reduce the cost of materials, planting one tomato plant per box. the ratio between the surface and the plant was chosen following the standard for tomato crops in open field. all boxes were plastic-coated with a black polyethylene film in order to maintain the moisture of the substrate, preventing excessive evaporation (hassanwassef, 2004). during the summer season, high temperatures in cairo cause rapid water evaporation, therefore to reduce this affect, the cultivation boxes were covered with white mulching. evaluation of substrate performance was carried out by comparing the production of 24 tomato plants. for each substrate, plant growth and fruit quality were determined. substrates comparison perlite, peat or coconut fibre, and sand were compared. the substrates were mixed and the final composition was 50% sand + 50% coir (s+c) and 70% peat + 30% perlite (p+p). three steps followed: fertilization and irrigation up to saturation, mulching and planting, and covering with net-shading to reduce sun intensity. the fertigation strategy used was based on an initial fertilization with 25 g of 20-20-20 npk fertilizer containing fe 5 g/kg, zn 5 g/kg, and mg 1 g/kg during the preparation of substrate with the addition of 1 g microelements (table 1). the peat used had the following characteristics: ph 3.5; ec 10 ms/m; hydric retention 70% vol. peat ph was adjusted to 5.5 by adding 200 g caco3. the simplified weekly fertigation schedule provided irrigation with 20 l/week of water containing 3 g ca(no3)2 for each plant. nitrogen was provided as 13.7% n-no3 and 1.5% n-nh4, with a total of 15.2%. the addition of 25 g npk and 1 g of micronutrients was carried monthly for each box. assessment of tomato growth and fruit quality plant growth was evaluated monthly and the following parameters were recorded: height, time of flowering, and number of flowers for plants in the different substrate conditions. fruit quality evaluations were carried out on dried fruits and comprised the determination of macro (k, na, ca) and microelements, which are important for a balanced human diet. the concentrations of heavy metals (cd, pb, sr, cr, mo, as), as possible contaminants, were also measured. analytical determinations were carried out using an icp-ms (inductivity coupled plasmamass spectroscopy). to better understand the potential of the cultivation technique related to the lack of food safety in the slum community, we also analysed the quality of tomatoes bought in local markets of the cemetery area of al-quarafa. to evaluate the quality, comparisons were made with standard dried tomato analysed by the fda (food and drug administration). dry materials were ground and digested with nitric acid (7:0.1). mineral compounds (fe, mn, zn, and cu) were measured using inductively coupled plasma mass spectrometry (icp-ms). values were reported as means and standard errors (n=3). data were subjected to anova analysis and differences among means were determined using bonferroni’s post-test. 3. results plants growth and productivity plant growth was monitored in sand and coir or peat and perlite in the two growth containers during cultivation. higher values were found in plants grown in the s+c substrate in both palm boxes (fig. 2a) and woody boxes (fig. 2b). however, statistical differtable 1 eu heavy metal security limits (ec 420/2011) elements concentration cr 0.050 mg/kg pb 0.100 mg/kg mo 0.025 mg/kg as 3.500 mg/kg cd 0.050 mg/kg adv. hort. sci., 2016 30(1): 23-29 26 ences were found only in woody boxes. in the s+c substrate the plants reached an average height of 50 cm after three months. in the p+p substrate the plants ranged from 35 to 38 cm. in june, in both substrates, plants reached a plateau (fig. 2). food quality, nutrients and heavy metals content the results revealed that simplified hydroponics guarantee the safety of food produced, even in heavy metals-polluted environments. the main mineral nutrient contents were determined in ripe tomato fruits and compared with those sold in the local markets. the sodium (na) content was lower in fruits harvested from plants grown in the s+c substrate (1.7 mg/kg dw) compared with the other samples. the fruits purchased from local markets had a higher na amount: 7.5 mg/kg dw on average (fig. 3a). the magnesium (mg) content in fruits grown in sh was similar without significant differences between the two substrates and ranged from 2.7 to 3.4 mg/kg dw (fig. 3a). in fruits obtained from local markets, the mg content was almost double (fig. 3a). analogous results were observed for potassium (k) and calcium (ca). the k in fruits harvested from plants grown in the two different substrates ranged from 65.5 to 68.0 mg/kg dw, while for fruits purchased from the markets the values were higher, 110.0 mg/kg dw on average (fig. 3). the ca content was similar to mg, double the content was found in the commercial tomato fruits compared with those harvested from the sh system (fig. 3a). considering the heavy metals microelements, different concentrations were found in the tomato fruits. copper (cu) in the analysed fruits ranged from 0.6 to 0.8 mg/100 g dw. the highest values were found in the p+p substrate (fig. 3). manganese (mn) was similar in distribution in the different treatments, but the concentration was higher: from 0.7 to 4.4. mg/100 g dw. the highest value was found in the fruits harvested from plants grown in the p+p substrate (fig. 3). on the contrary, zn content was higher in the sh cultivated fruits compared with those obtained from local markets. the values ranged from 1.3 to 2.0 mg/100 g dw. iron (fe) was fig. 3 nutritional elements expressed as mg/100 g dried weight. a) na, mg, k and ca. b) cu, mn, zn and fe in tomato fruits harvested from plants grown in sand and coir (s+c), peat and perlite (p+p) or purchased at the local market (m values are means with standard errors. data were subjected to two-way anova analysis and differences among means were determined using bonferroni’s post-test. different letters indicate statistical differences for <0.05. the heavy metals in fruits were under the safety thresholds established by the european union. fig. 2 plant height of tomato plants grown in palm boxes (a) or woody boxes (b). values are means with standard errors. data were subjected to two-way anova. differences among means were determined using bonferroni’s post-test. giro et al. vegetable production using a simplified hydroponics system 27 higher in fruits obtained from plants grown in the p+p substrates and the mean was 13.0 mg/100 g dw. half the amount was found in fruits harvested from the s+c grown plants and purchased from local markets (fig. 3). heavy metals were found in trace amounts and the values were below those set by eu regulations. the as content was higher in local market fruits (6.03 µg/kg dw), while in the sh system the as content was 0.63 µg/kg dw in fruits harvested from plants grown on the s+c substrate and 2.58 µg/kg dw in those obtained from plants grown on p+p substrate. strontium (sr) in the sh system ranged from 17.97 to 20.7 µg/kg dw, while in tomatoes purchased from the local market the concentration was 44.6 µg/kg dw on average (fig. 4). molybdenum (mo) was 10fold higher in fruits harvested from plants grown in the p+p substrate compared with s+c and local market tomato samples. cadmium (cd) was higher in fruits sold in local markets compared with those obtained from the sh system. the lowest value, 1.6 µg/kg dw, was found in fruits harvested from plants grown on the p+p substrate. analogous results were observed for lead (pb); the highest value found in fruits purchased from the market was 28.97 µg/kg dw (fig. 4). on the contrary, higher cr values were found in the fruits harvested from the sh system (21.03-45.67 µg/kg dw) compared with 16.83 µg/kg dw measured in the fruits purchased at the market (fig. 4). 4. discussion and conclusions the cultivation of vegetables in highly polluted environments where there is poor soil quality can only be achieved using soilless systems. most people living in the slum areas do not have skills or knowledge about agricultural practises, therefore urban agricultural systems must be simple and easy to manage. unfortunately, high density urban and periurban areas can also be subjected to heavy metal pollution. contamination can also come from airborne trace elements such as cd and mo (francini et al., 2010) that are deposited on the surface of fruits. heavy metal accumulation in vegetables is closely correlated with the pollution level of the area (antisari et al., 2015). sh is a good strategy to improve food security for urban slum populations and, in particular, it is linked to an increase of food availability (orsini et al., 2013). however, the food situation could also improve through direct sale of the products if the cultivated surface were enough to have a surplus of fresh produce. for these reasons it is important when choosing the cultivar to consider the most profitable ones in order to improve saleability of the products. the sh technique makes it possible to cultivate safe vegetables, in small spaces, and with low-tech instruments. the results of the current study reveal that local materials for substrate and palm boxes were a good compromise between cost and production capability. the community of al-quarafa follows a typical mediterranean diet based on cereals and legumes, however they do not consume fresh vegetables because they are very expensive and perishable (arenas and vavrina, 2002; hamza and mason, 2004). thus, the application of sh represents an interesting method to combat food insecurity in al-quarafa, a symbol of all cairo slums. moreover, the use of the substrates in the study avoided the accumulation of heavy metals in vegetables and allowed enrichment of some of the micronutrients that improve human health. sh have been used in africa for 10 years to cultivate fresh products in urban and peri-urban settlements. for example in dakar, in 1999 a fao pilot project for family farming employed sh. the dakar models, however, were adapted to tropical temperature and climate. the substrates were 40% peanut shells, 40% rice husks, and 20% perlite, suitable for leafy vegetable production because they are harvested at a young stage and do not have vertical growth. fig. 4 heavy metals expressed as µg/kg dw determined in tomato fruits harvested from plants grown in sand and coir (s+c), peat+perlite (p+p) or purchased from the local market (m). values are means with standard errors. data were subjected to two-way anova analysis and differences among means were determined using bonferroni’s post-test. different letters indicate statistical differences for <0.05. adv. hort. sci., 2016 30(1): 23-29 28 unfortunately, they are not able to support the growth of plants such as tomatoes. in fact, tomatoes prefer more stable substrates, from a mechanical point of view, such as peat/perlite or coir/sand (esposito, 2010). also the depth (14-20 cm) of the recycled-pallet boxes used in dakar was less than that required by cultivated varieties that need greater volume for adequate root development to favour production (ghehsareh et al., 2011). the irrigation schedule in the dakar project provided 2 l/m2 two to three times a day (ghehsareh et al., 2011). the nutritional elements were recollected in a small container under the boxes. this daily schedule means that plants must be managed three times a day and this is one of the differences between the dakar project and the method applied in the current study tested in cairo in which an irrigation schedule was followed of once a week, thanks to the water capacity (wc) of the peat and coir. the combination of 50% coir and 50% sand provided the best growth performance for two reasons: 1) because using a weekly irrigation schedule the coir had greater wc than peat (abad, 2002), and 2) because the weight of sand simplified the growth of the vegetative part of the plants, offering mechanical stability. it is also important to consider the ph of the substrate used: the combination between coir and sand naturally had a sub acid ph (arenas and vavrina, 2002), with is the naturally ideal ph for tomato growth. this aspect favoured the availability of the micro and macro nutrients (ghehsareh et al., 2011). from a productive point of view, the tomato plants produced on average 0.4-0.5 kg per plant in the current investigation; the best results were achieved with tomatoes grown in sand/coir. considering two plants per box, these values translate to a productive capability of 0.8-1 kg/m2 of tomatoes on average. these productivity ranges consider the stressed conditions of the climate in cairo which allows two growing cycles per year (september to november, march to june). in terms of nutrition, considering the nutritional level of macro and micro elements of tomatoes analysed by the fda, the tomatoes grown in sh in the current study had similar nutritional levels and sometimes better than the tomatoes the fda considered as standard. social agriculture and future strategy this sh growing system has been developed for poor urban communities with very limited agricultural knowledge. these cultivation systems are suitable for urban and social agriculture as reported in fao guidelines. moreover, for production in highly populated areas, sh systems also play a social role as they can be meeting points for the families within the community. therefore, these growing systems should not be considered only as vegetable production tools. these systems must be easy to manage especially for water and nutrients supply. the management of the cultivation is often carried out by women and children, who are the most susceptible to food insecurity. in slum areas like al-quarafa, people are depressed, marginalized and unemployed. hence, the growing areas improve food production, as well as the physic and psychological health of the inhabitants. collaborations among the people living the community improve relationships between families and create opportunities to establish networks. these networks are essential to sharing information on crop cultivation in sh systems and to organising selling points in local market. in the future, the “cairo model” of the sh system may be exported to other depressed urban areas in africa and in the middle east. this new, low-cost and low-tech growing system for vegetable production provides a great opportunity for poor populations who want to redeem themselves within society. acknowledgements this study was logistically supported by ngo “liveinslums” and financially supported by municipality of milan. references abad m., 2002 physico-chemical and chemical properties of some coconuts dust for use as a peat substrate for containerised ornamental plants. bio-resource technology, 82: 241-245. antisari l.v., carbone s., gatti a., vianello g., nannipieri p., 2015 uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide (ceo2, fe3o4, sno2, tio2) or metallic (ag, co, ni) engineered nanoparticles.environ. sci. pollut. res., 22(3): 1841-1853. arenas m., vavrina c.s., 2002 coir as an alternative to peat in media for tomato trasplant production. hortscience, 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(ed.) crops: quality, growth and biotechnology. wfl publisher science & tecnology, helsinki, pp. 1356. santos p.j., ocampo e.t., 2005 snap hydroponics: development & potential for urban vegetable production. philippine journal of crop science, 30(2): 3-11. seikh b.a., 2006 hydroponics: key to sustain agriculture in water stressed and urban environment. pak. j. agric., agril. eng., vet. sci., 22: 53-57. impaginato 371 adv. hort. sci., 2018 32(3): 371-378 doi: 10.13128/ahs-23266 postharvest application of hydrogen peroxide and salicylic acid differently affects the quality and vase life of cut rose (rosa hybrida l.) petals and leaves g. cocetta, a. ferrante dipartimento di scienze agrarie e ambientali, università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: chlorophyll fluorescence, cut flowers, elicitors, oxidative stress, phenolic compounds, senescence. abstract: rose represents an important product in the market of cut flowers. its quality is related to visual appearance and is affected by oxidative stress and senescence. to maintain the quality and extend the product vase life, innovative technical solutions to be applied in postharvest are needed. in this work the effectiveness of hydrogen peroxide (h2o2) and salicylic acid (sa), was assessed on cut rose leaves and petals. several physiological indicators of quality were studied, including chlorophyll content, chlorophyll a fluorescence, color, lipid peroxidation, phenolic compounds and anthocyanins. after 7 days of vase life 73.33% of untreated roses were not marketable anymore, while the percentage was lower in response to treatments (47.06% in sa and 25.53% in h2o2). the application of h2o2 induced a reduction in leaf chlorophyll and in the performance index which, at the end of the vase life decrease by 76% and 49% respectively. consistently, the lipid peroxidation in leaves treated with h2o2 increased by a 53%. after 4 days of vase life, sa allowed maintaining higher levels of anthocyanins in petals compared to h2o2 and to controls. the results obtained allowed individuating different responses, depending on the treatment applied as well as on the plant organ. 1. introduction the market of cut flowers is characterized by the high perishability of the commercialized products. the pipeline from growers to the final consumers, is often long and at the same time, the item must have very highquality standards. flowers showing discoloration, shriveling, bending or any other visible sign of damage can no longer be commercialized, and product losses represents a crucial problem for this sector. rose (rosa hybrida l.) is one of the most important species in the flower market and it is particularly appreciated by the consumers as cut flower. the quality of cut roses is strictly related to the integrity and quality of petals, but also to the color of leaves, which contributes to their visual appearance. chlorophyll degradation and leaf yellowing reduce the (*) corresponding author: giacomo.cocetta@unimi.it citation: cocetta g., ferrante a., 2018 postharvest application of hydrogen peroxide and salicylic acid differently affects the quality and vase life of cut rose (rosa hybrida l.) petals and leaves. adv. hort. sci., 32(3): 371-378 copyright: © 2018 cocetta g., ferrante a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 may 2018 accepted for publication 6 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 371-378 372 ornamental value of cut flowers and compromise their commercialization. during transportation and handling, leaves and petals can undergo mechanical damage, which can lead to tissue browning with negative effect consequences on quality. on the physiological point of view, the loss of quality is a direct consequence of oxidative stress which in turn can lead to senescence (kumar et al., 2008). these phenomena are characterized by the activation of several physiological responses at cellular level and involve signaling molecules, enzymes, and transcriptional regulators such as genes and transcription factors (gregersen et al., 2013; thomas, 2013). in the recent years, several efforts have been made to maintain the quality of cut roses and extend their vase life. these efforts include breeding programs the improvement of growing conditions, and the optimization of transportation. moreover, the effectiveness of several treatments applied during the vase life has been assessed (reid and jiang, 2012). salicylic acid (sa) is a plant hormone involved in several plants functions, including stress responses, development, and plant signaling (hayat et al., 2010; an and mou, 2011; rivas-san vicente and plasencia, 2011). spray application of sa has been reported to prolong vase-life in cut rose flowers by increasing the activity of the ros-scavenging enzyme catalase and improving the water balance. interestingly, the lowest dosage applied (50 µm) was the most effective (alaey et al., 2007). also, in other studies, low concentrations of sa (0.01 mm) have been reported as the most effective in inducing plants responses to stress (horváth et al., 2007). hydrogen peroxide (h2o2) is an important molecule produced and accumulated in plant cells because of the aerobic metabolism. it belongs to the reactive oxygen species (ros) and can play a key role in stress metabolism and senescence. the increment in ros levels is, in fact, a typical feature of senescence and can lead to cellular damage and finally to cell death (panieri et al., 2013). however, slight alterations of the ros balance, including h2o2 accumulation, can be considered as important signals in plant cells and can trigger a series of defense responses, which in some case can also involve sa (kalachova et al., 2013; herrera-vásquez et al., 2015). while high concentrations of h2o2, can directly induce oxidative stress in plant tissue, low doses can lead to the activation of positive physiological responses. it has been suggested that h2o2 priming can help in modulating ros-mediated stress tolerance in plants (hossain et al., 2005). non-toxic levels of this molecule must be maintained to have beneficial effects and to improve the quality and vase life of cut flowers, as reported for lily (liao et al., 2012). the aim of this work was to evaluate the effect of low concentrations of h2o2 and sa when applied to cut roses during the vase life. attention has been given to the individuation of specific and common responses induced by these two molecules. to achieve this goal, several indices in both petals and leaves were considered, including chlorophyll content, chlorophyll a fluorescence, color, lipid peroxidation, phenolic compounds and total anthocyanins. 2. materials and methods plant material cut rose stems (rosa hybrida l. ‘tacazzi’) at the commercial developmental stage were purchased on july 20th, 2017, from the milan flowers market and brought to the laboratory within one hour. each stem was around 60 cm-long. once in the laboratory, 20 stems were randomly selected from a pool of 60 and placed in a beaker filled with 2 l of distilled water (control). similarly, two other groups of 20 stems each, were placed in a sa (0.01 mm) and h2o2 (0.1 mm) solution respectively. the concentrations of the treatments were chosen among the lowest effective dosage found in the literature to reduce the risk of toxicity. after 24 hours, all the solutions were substituted with 2 l of fresh distilled water and the volume was maintained until the end of the trial. the vase life conditions were: 20±2°c, 55% relative humidity (rh), 18 µmol m-2 s-1 light intensity, with a photoperiod of 12 hours. samples of petals and leaves were collected at the beginning of the trial as well as after 4 and 7 days of vase life. after 7 days the remaining stems were maintained until all of them were considered unmarketable. vase life and damaged flowers (%) at each time point, the number of damaged flowers were estimated based on the visual appearance. those flowers showing wilting, shriveling or desiccation were considered as not marketable anymore and thus counted as damaged. this information was used to calculate the percentage of damaged flowers and the average vase life (reported as days). water content (%) at harvest and after 7 days three roses for each treatment (the whole stem) were weighed and cocetta and ferrante effect of h 2 o 2 and salicylic acid on cut rose vase life 373 immediately placed in an oven at 115°c until the complete drying. the water content was calculated as percentage of water respect to fresh weight. chlorophyll and chlorophyll a fluorescence-related parameters during vase life in vivo determination of leaves chlorophyll content was performed using a cl-01 chlorophyll mater (hansatech, united kingdom). similarly, chlorophyll a fluorescence was measured on dark adapted leaves using a portable fluorimeter (handy pea, hansatech, united kingdom). the parameters measured were: the quantum maximum efficiency of psii (fv/fm), the performance index (pi), the number of reactive centers per cross section (rc/csm), and the amount of energy dissipated per reaction center (dio/rc). color measurement changes in petal coloration in response to treatments and to vase life were evaluated at each time point by using a minolta cr-400 colorimeter (konica minolta, inc., japan). the color parameters were described using the lab* and the munsell (l*c*h) color spaces. briefly, in the lab*color space the lightness is represented by l* (with 0 indicating black and 100 white). the colors in the red green region are represented by a* (red with positive values and green with negative values) while b* describes the interval from yellow (positive values) to blue (negative values). in the munsell color space h represents the hue while c* is the chroma (or color purity). phenolic index and total anthocyanins for the extraction of phenolic compounds and anthocyanins, around 1 g of petals and leaves, collected at each time point, were homogenized in 8 ml of acidified methanol (1% hcl v/v) and extracted overnight in the dark. the phenolic index was calculated as the absorbance at 320 nm of the diluted extracts, normalized to fresh weight (ke and saltveit, 1989). total anthocyanins were determined spectrophotometrically at 535 nm using an extinction coefficient (ε) of 29,600 mm-1 cm-1 and expressed as cyanidin-3-glucoside equivalents and (klein and hagen, 1961). lipid peroxidation the level of lipid peroxidation was measured with the thiobarbituric acid reactive substances (tbars) assay (heath and packer, 1968). briefly, around 1 g of petals and leaves was homogenized in 5 ml 0.1% trichloroacetic acid (tca) solution. the extract was mixed with 4 ml of 20% (w/v) tca, 25 µl of 0.5% thiobarbituric acid (tba) and distilled water. after vortexing, the mixture was heated at 95°c for 30 minutes in a water bath and then cooled on ice. absorbance at 600 nm was subtracted from the absorbance at 532 nm (as an index of non-specific turbidity) and the concentration of tbars was expressed as malondialdehyde (mda) equivalents (nmol g-1 fw), calculated using an extinction coefficient (ε) of 155 mm-1 cm-1. statistical analysis all data were subjected to two-way anova followed by bonferroni’s multiple comparisons test, except for vase life data which were subjected to a one-way anova. statistics were performed using graphpad prism version 6 for windows, graphpad software, la jolla, california, usa (www.graphpad. com). 3. results vase life and water content after 4 days of vase life all the flowers treated with h2o2 and sa were considered marketable, while 10% of untreated flowers showed a marked loss of quality. after 7 days, the higher rate of damage was recorded in the controls (73.33%), followed by sa (47.06%) and h2o2 (25.53%) (fig. 1 a). after 8 days all fig. 1 percentage of damaged flowers (a) and average vase life (b) measured in cut ‘tacazzi’ rose. data are means ± se (b: n=14; c: n=3). different letters indicate significant differences (p<0.05). adv. hort. sci., 2018 32(3): 371-378 374 the control flowers were already damaged, while 6 flowers treated with sa were still marketable until 10 days, and 6 among those treated with h2o2 lasted until 12 days. the treatment with h2o2 determined a significant increment in the average vase life compared to sa and control (fig. 1 b). the initial water content was 77.86% and showed a slight progressive decline without any significant difference among the treatments. after 7 days the values were: 70.01%, 70.61%, 70.64%, in control, sa and h2o2 respectively. the flowers treated with h2o2 showed a reduced opening compared to those treated with sa or to controls (fig. 2 a, c). color after 7 days of vase life, all the petal samples looked paler compared with to the beginning of the trial and an increment in the l* value was recorded despite the treatments applied (table 1). h2o2-treated flowers did not show any change in the colorrelated parameters compared to controls. on the other hand, after 7 days, sa induced a decrement in all the color attributes compared to the beginning of the trial, including a*, b*, hue (h) and chroma (c*). this variation was consistent with the visual observation of the petals which appeared darker and turned from red to a more violet coloration (fig. 2 c). chlorophyll content and chlorophyll a fluorescencerelated indexes treatment and vase life duration significantly affected the chlorophyll content of cut rose leaves. after 4 days of vase life, the chlorophyll content was fig. 2 visual appearance of cut roses (cv. tacazzi) as affected by h 2 o 2 or sa application after 4 (a, b) and 7 (c) days of vase life. cocetta and ferrante effect of h 2 o 2 and salicylic acid on cut rose vase life 375 tionality after 7 days of vase life, evidenced by a significant decrement in the pi value (fig. 3 c). no significant changes were observed in fv/fm, rc/csm and in dio/rc (fig. 3 b, d, e), even though these indexes showed a trend similar to the one observed in the pi. lipid peroxidation the level of lipid peroxidation in leaves was significantly affected by treatment and by vase life duration. in leaves treatments induced higher oxidative damage compared to control, already after 4 days fig. 3 chlorophyll content (a) and chlorophyll a fluorescence-related indexes: maximum quantum efficiency of psii (fv/fm) (b), performance index (pi) (c), number of reaction centers per cross section (rc/csm) (d), energy dissipated per reaction center (dio/rc) (e). measurements were conducted on leaves of cut ‘tacazzi’ rose. data are means ± se (n=6). different letters indicate significant differences (p<0.05). significantly lower in the controls and in sa-treated leaves compared to h2o2-treated ones. however, h2o2 induced a pronounced discoloration of leaves, particularly at the end of the trial (fig. 2 b, c). this observation is confirmed by the significant reduction in chlorophyll content which has been recorded after 7 days of vase life (fig. 3 a). all the parameters related to chlorophyll a fluorescence showed a trend consistent with the one observed in chlorophyll content. in particular, stems treated with h2o2 showed a loss in the leaf psii funcdata are means ± se (n=6). different letters indicate significant differences (p<0.05). table 1 changes in petal color parameters of cut ‘tacazzi’ rose during vase life and in response to treatments treatment days of vase life l* a* b* h c* 0 41.17±0.64 a 55.93±0.31 a 14.10±.01 a 14.14±0.98 a 57.70±0.37 a control 4 34.13±1.93 a 48.57±1.89 ab 9.75±1.12 ab 11.29±1.05 ab 49.56±2.00 ab h 2 o 2 0.1 mm 4 39.00±2.16 a 49.08±1.69 ab 10.46±2.13 ab 11.81±2.09 ab 50.77±2.30 ab sa 0.01 mm 4 36.09±1.18 a 50.09±1.78 ab 10.12±1.94 ab 11.21±1.77 ab 51.17±2.11 ab control 7 47.73±3.02 b 48.12±2.83 ab 9.45±0.56 ab 11.16±0.63 ab 49.05±2.84 ab h 2 o 2 0.1 mm 7 51.12±1.22 b 50.13±1.67 ab 9.80±0.99 ab 11.00±0.81 ab 50.97±1.78 ab sa 0.01 mm 7 50.19±2.67 b 44.57±3.14 b 6.49±1.02 b 8.52±1.64 b 45.10±3.04 b 376 adv. hort. sci., 2018 32(3): 371-378 and lipid peroxidation reached the maximum level in h2o2-treated leaves after 7 days of vase life (fig. 4 a). the amount of mda equivalents was markedly higher in flowers (fig. 4 b) compared to leaves, but no significant changes were observed in petals in response to vase life or to treatments. phenolic index and total anthocyanins the levels of total phenolic compounds, estimated with the phenolic index, were stable, with similar values between leaves and flowers and without showing any significant change in response to treatment or to vase life (fig. 5 a and b). on the other hand, total anthocyanins, which were highly accumulated in petals, progressively declined during the vase life (fig. 5 c and d), with a significant effect of vase life time in both leaves and petals. this trend was more pronounced in petals, which showed a significant decrement already after 4 days in controls and in response to h2o2 treatment. fig. 5 changes in the phenolic index (a: leaves; b: petals) and in total anthocyanins levels (c: leaves; d: petals) of cut ‘tacazzi’ rose during vase life and in response to treatments. data are means ± se (n=3). different letters indicate significant differences (p<0.05). fig. 4 changes in lipid peroxidation in leaves (a) or petals (b) of cut ‘tacazzi’ rose during vase life and in response to treatments. data are means ± se (n=3). different letters indicate significant differences (p<0.05). 377 cocetta and ferrante effect of h 2 o 2 and salicylic acid on cut rose vase life then again, after 4 days sa-treated petals showed significantly higher levels of anthocyanins compared to h2o2 and to control. 4. discussion and conclusions the results obtained in this work allowed individuating independent ways of action for the two treatments and evidenced positive and negative aspects of both molecules. as a first consideration is important to observe that both treatments determined a reduction in the product losses compared to control and in case of h2o2, a significant extension of the vase life. this positive result is consistent with other previously published works (gerailoo and ghasemnezhad, 2011; liao et al., 2012). it is also interesting to point out that the two treatments applied, induced different responses in rose leaves and petals. the application of h2o2 determined a delay in the flower opening compared to control and to sa, this is consistent with what already observed in cut lily (liao et al., 2012) and in tree peony (shi et al., 2015), and can have positive implications in the postharvest management of cut roses. in fact, the possibility of modulating or delaying the flower opening could be successfully be used to extent the commercial life of cut flowers and to better manage the storage and transportation. moreover, h2o2 helped in maintaining low levels of mda in petals during the first days of vase life. as a drawback, h2o2 induced leaves yellowing which in turn can affect the visual appearance of the stem. the detrimental effect of h2o2 on leaves has been confirmed by the marked loss of chlorophyll and by the higher level of peroxidation recorded at the end of the vase life. the parallel decrement in the pi, suggests a possible positive association between chlorophyll a fluorescence and plant stress, as previously reported (kalaji et al., 2006). considering the results obtained in this trial, it appears that h2o2 is not affecting the accumulation of phenolic compounds and pigments in cut ‘tacazzi’ rose. it is known that the effect of h2o2 depends on the dosage (liao et al., 2012), thus it is possible that the concentration of h2o2 applied in this trial was enough to induce oxidative stress and damage in the leaves while having a positive effect on flowers. there are not many reports about the effect of sa in ornamental leaves, however recently, a role for sa in extending the vase life of acacia holosericea foliage has been suggested (chen and joyce, 2017). the positive effect of sa on the leaves of cut roses has been confirmed in the present work, since at the end of the trial, sa-treated leaves showed a better maintenance of chlorophyll and leaf functionality indexes compared to controls and to h2o2-treated stems. the positive effect of sa could be due to the activation of plants defenses against ros, including the increment in antioxidant compounds and the activation of antioxidant enzymes. treatments with sa have been reported to reduce lipid peroxidation in gladiolus cut flower spikes (rahmani et al., 2015). also, sa alone or in combination with sucrose, was shown to affect the rate of lipid peroxidation (mda) and chlorophyll content in carnation petals (kazemi et al., 2011), but different results were observed depending on the dosage applied. unexpectedly, in the present work, the application of 0.01 mm of sa was not effective in preventing lipid peroxidation of cut roses, perhaps because of the too low concentration applied. the degradation of anthocyanins is a typical phenomenon which negatively affects the quality of ornamental flowers, including rose (luo et al., 2017). the role of exogenous sa in stimulating the biosynthesis of anthocyanin pigments has been documented (ram et al., 2013), thus it can be hypothesized that sa treatment could delay the degradation of pigments in petals. the increase of phenolic compounds as well as the anthocyanins content could help in counteracting the senescence and extend the flower life as observed in model plants (mcnish et al., 2010). despite the low dose applied, sa helped in maintaining higher levels of anthocyanins in cut rose petals after 4 days of vase life. also, the petal coloration was affected by sa after 7 days. in conclusion, the negative effect of h2o2 in leaves compared to the beneficial effect observed in flowers is controversial and it would surely worth to be investigated further. perhaps the negative effects could be circumvented by testing other forms of application, such as direct spraying on the petals avoiding contact with leaves. the application of sa did not induce any damage, but at the same time, the positive effects were not as evident as expected compared to untreated control. this suggests that the conditions of treatment with sa can be further perfectioned. in future experiments the application of higher doses of sa could intensify the positive effects observed in this trial. given the results obtained, the assessment of the pi, based on the chlorophyll fluorescence mea378 surement, could represent an innovative approach in the evaluation of the vase life of cut flowers, as it followed a trend consistent with the decline observed in chlorophyll content and with the increment in mda. in fact, the non-destructive estimation of leaf functionality through this index could be further studied, implemented and successfully used for the rapid quality control of cut flowers and ornamentals. references alaey m., babalar m., naderi r., kafi m., 2011 effect of pre-and postharvest salicylic acid treatment on physio-chemical attributes in relation to vase-life of rose cut flowers. postharvest biol. technol., 61(1): 9194. an c., mou z., 2011 salicylic acid and its function in plant immunity. j. integr. plant biol., 53(6): 412-428. chen y., joyce d.c., 2017 salicylic acid and jasmonic acid treatments for potentially extending the 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role in plant growth and development. j. exp. bot., 62(10): 3321-3338. shi j., shi g., tian z., 2015 effect of exogenous hydrogen peroxide or ascorbic acid on senescence in cut flowers of tree peony (paeonia suffruticosa andr.). j. hortic. sci. biotechnol., 90(6): 689-694. thomas h., 2013 senescence, ageing and death of the whole plant. new phytol., 197(3): 696-711. impaginato 3 1. introduction drought stress can affect plant growth, development and yield. it has been estimated that up to 45% of world agricultural lands are subjected to drought (bot et al., 2000). water deficit leads to the perturbation of most of the physiological and biochemical processes and consequently reduces plant growth and yield (boutraa, 2010). abscission of reproductive organs like flower buds and flowers is a major yieldlimiting factor in vegetable crops (wien et al., 1989). the abscission of floral organs during stresses has been associated with changes in physiological processes (aloni et al., 1996). it has been reported that, in tomato, the abscission of flowers and flower buds and the reduction in photosynthesis were higher in susceptible cultivars compared to tolerant cultivars (bhatt et al., 2009). in soybean, flower retention and fruit set are highly sensitive to environmental stresses (kokubun et al., 2001). etsushi et al. (2009) reported that soil plant analytical development (spad) method readings significantly correlated with chlorophyll content, rubisco content, photosynthetic rate, and fv/fm ratio. a major portion of soluble protein (50%) in leaves is occupied by rubisco, a prime enzyme for carbon fixation in photosynthesis (noggle and fritz, 1986). daniel and triboi (2002) showed that heat stress decreased the duration of soluble protein accumulation in terms of days after anthesis but not in terms of thermal time. drought decreased leaf n, whereas heat stress did not influence it and, however, the total soluble protein content was decreased during drought, heat, and a combination of drought and heat. heat and drought stress induced suppression of photosynthesis by mainly decreasing the proportion of soluble protein to total leaf n, adversely affecting the rubisco protein and activity (xu and zhou, 2006). bhatt et al. (2009) reported there was a considerable decrease in sps activity in flowers under water deficit condition; a relatively higher decrease was observed in the susceptible genotypes. sps activity was shown to decrease during leaf desiccation (foyer et al., 1998), or to remain constant (zrenner and stitt, 1991). however, yang et al. (2002) and niedzwiedzsiegen et al. (2004) observed an increase in sps activity in rice and wheat leaves, respectively, under adv. hort. sci., 2016 30(1): 3-11 doi: 10.13128/ahs-18696 impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.) r. sivakumar (*), s. srividhya department of crop physiology, tamil nadu agricultural university, coimbatore 641 003, tamil nadu, india. key words: ascorbic acid, drought, flower abscission, lycopene, soluble protein, sps, yield. abstract: the effect of drought stress on flowering, yield and quality of tomato (solanum lycopersicum) genotypes was investigated under field conditions in rainout shelter. the drought condition was imposed on the first day after transplanting based on field capacity of soil. experimentation was undertaken with ten genotypes adopting factorial randomized block design with three replications and two treatments viz., 1.0 iw/cpe and 0.5 iw/cpe field capacity. as the stress increased from 100% field capacity to 50% field capacity, reductions in chlorophyll index, soluble protein content, days to flower initiation, sucrose phosphate synthase (sps) activity, fruit volume, fruit diameter, yield and increased flower abscission percentage were noted. significant increases in tss and lycopene were observed under drought. the genotypes le 118, le 57 and le 114 showed significantly less reduction in soluble protein content; sps activity and fruit yield during drought were considered as drought tolerant. genotypes le 1 and le 125, which gave the lowest soluble protein content, sps activity and ultimately poor yield, were considered as drought susceptible. (*) corresponding author: sivatnau5@gmail.com received for publication 26 january 2015 accepted for publication 6 november 2015 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 3-11 4 mild drought. tomato (solanum lycopersicum) is one of the most popular and widely grown vegetables in the world. considering the potentiality of this crop, there is plenty of scope for its improvement, especially under the drought situation. water is a scarce resource for irrigation. although the concept of drought tolerance has been viewed differently by molecular biologists, biochemists, physiologists and agronomists, the major concern is to enhance the biomass and yield under limited water input, which is a characteristic feature of rainfed agriculture. there are several physiological and biochemical traits contributing to the drought tolerance of horticultural crops. however, a large number of tomato genotypes have not been screened for drought tolerance or exploited for their cultivation under drought. to breed drought tolerant genotypes, it is necessary to identify physiological traits of plants which contribute to drought tolerance. chlorophyll pigments and soluble protein content in leaves which have a greater correlation to crop yield show reduced levels under drought. the reproductive stage of the crop is highly sensitive to any abiotic stress. hence, reduction in crop yield under drought is mainly attributed to the effect on flowering characters and a decrease in sps enzyme activity. therefore, the present investigation was carried out to study the physiological traits such as spad value, soluble protein content, sps activity, flowering characters, and yield to facilitate the screening and selection of tomato genotypes for drought tolerance. 2. materials and methods plant materials and cultivation the study was undertaken to determine the effect of drought on flowering and yield of tomato genotypes in the field experiment at rainout shelter of crop physiology department, tamil nadu agricultural university, coimbatore, tamil nadu during 2012-13. the experiment was conducted with 10 tomato genotypes (le 1, le 27, le 57, le 114, le 118, le 125, co 3, pkm 1, tnau thco 3 and coth 2) and two treatments (1.0 and 0.5 iw/cpe (irrigation water/ cumulative pan evaporation) with three replications following a factorial randomized block design. seeds of selected genotypes were sown in trays filled with vermicompost for nursery use. twenty-five day old seedlings were transplanted in the rainout shelter; plot size 1.35 x 1.5 m. drought was imposed on the first day after transplanting to all genotypes in both iw/cpe ratio treatments. furrow irrigation was applied when the cumulative pan evaporation reading reached 50 mm (1.0 iw/cpe ratio) and 75 mm (0.50 iw/cpe ratio). crop was supplied with fym (25 t/ha rate), npk fertilizers (75:100:50 kg/ha), borax 10 kg and zinc sulphate 50 kg/ha as basal dose and 75 kg n/ha on the 30th day after planting during earthing up. other cultivation operations including weed control (applied pendimethalin 1.0 kg a.i./ha as preemergent herbicide followed by one hand-weeding at 30 days after planting) and plant protection measures were carried out as per the recommended package of practices of tamil nadu agricultural university, coimbatore. physiological parameters spad readings were recorded using chlorophyll meter (spad 502) designed by the soil plant analytical development (spad) section, minolta, japan. data were recorded as described by peng et al. (1996). soluble protein content of the leaf was estimated as per the method of lowry et al. (1951) and expressed as mg g-1 fw. sucrose phosphate synthase (sps) activity was determined as described by pavlinova et al. (2002). sucrose was measured using anthrone reagent as modified by ashwell (1957). absorbance was measured at 630 nm and the activity was expressed in mg sucrose mg-1 protein h-1. flowering parameters days to flowering (the number of days from seedling emergence to opening of the first flower) were recorded for each plant in the three replications and the average was taken. abscission study was conducted on single flower basis. flower number of tagged plants and dropped flowers per plant were counted every three days. these records were used to calculate the flower abscission and expressed in terms of percentage. yield parameters average fruit weight was calculated by adding the weight of five fruits from each plant at second harvest and dividing it by the total number of fruits and expressed in g fruit-1. the fruit volume was estimated by water displacement method. individual fruits were immersed in 1 l of water; amount of water displaced was measured and volume was worked out per fruit and expressed as cc. polar diameter was measured from stalk end to blossom end of fruit by using vernier calipers and the average of five fruits sivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 5 was worked out and expressed in cm. the fruit weight per plant was recorded in control and stressed plants for each picking and fruit yield (kg per plant) was calculated as fresh weight of fruits in all the pickings. quality parameters juice extracted from cut fruit was used to determine tss with the help of a hand refractometer (0 to 32°brix) at room temperature and the value was noted in °brix. ripened fruit samples were analyzed for ascorbic acid content, using 2,6-dichlorophenol indophenol dye titrimetrically as per sadasivam and manickam (1996). lycopene content of fruit was extracted using petroleum ether and od of the extract was measured at 503 nm in uv-vis-spectrophotometer using petroleum ether as a blank (ranganna, 1986). lycopene content of the sample was calculated using the following formula and expressed in mg 100 g-1. 3.1206 x od of sample x volume made up x dilution lycopene = x 100 weight of sample x 1000 statistical analysis data from the various parameters were analyzed statistically as per the procedure of gomez and gomez (1984). 3. results decreased spad value under drought control (1.0 iw/cpe) plants showed a higher mean chlorophyll index value (47.57) than treated (0.5 iw/cpe) plants (44.04). among the genotypes, co 3 and pkm 1 recorded significantly higher chlorophyll index values of 50.1 and 46.1, and 49.5 and 46.7 at 1.0 iw/cpe and 0.5 iw/cpe conditions, respectively. during drought, the genotypes le 114 (45.8), le 57 (45.7), coth 2 (45.5) and le 118 (45.0) were found to be on par with each other. decreased soluble protein due to drought control (1.0 iw/cpe ratio) plants showed a higher mean soluble protein content (13.26) than the drought (0.5 iw/cpe ratio) imposed plants (8.98). among the genotypes, coth 2 (15.63) and thco 3 (15.18) registered the highest soluble protein content under 1.0 iw/cpe ratio level and le 57 (11.99) and le 118 (11.74) under drought conditions (0.5 iw/cpe). the lowest soluble protein content was found for le 125 (8.16) and le 1 (8.68) (table 1). altered flowering characters from drought our data on number of days to flower initiation revealed that genotypes, treatments and interactions attained statistical significance (table 2). in the case of treatments, the plants under drought initiated flowers earlier (26) than control plants (30). at 0.5 table 2 effect of drought on flowering characters of tomato genotypes during flowering stage genotypes days to flower initiation (dat) flower abscission (%) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 29 25 27 10.90 20.10 15.50 le 27 30 27 29 13.20 15.80 14.50 le 57 30 25 28 12.60 15.10 13.80 le 114 29 26 28 11.90 15.40 13.70 le 118 32 28 30 10.90 13.90 12.40 le 125 31 25 28 11.90 17.10 14.50 co 3 26 23 25 10.20 15.90 13.10 pkm 1 28 24 26 9.90 16.40 13.20 thco 3 32 28 30 10.90 17.40 14.20 coth 2 31 27 29 11.20 18.10 14.70 mean 30 26 28 11.40 16.50 13.90 g t g x t g t g x t sed 0:18 0:08 0:26 0.08 0.03 0.11 cd (0.05) 0:37 0:17 0:52 0.16 0.07 0.22 table 1 effect of drought on chlorophyll index spad value and soluble protein content of tomato genotypes at 60 dat genotypes spad value soluble protein (mg g-1) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 42.00 38.40 40.20 10.85 6.51 8.68 le 27 48.20 44.40 46.30 13.98 10.72 12.35 le 57 47.60 45.70 46.70 15.03 11.99 13.51 le 114 48.20 45.80 47.00 13.43 10.19 11.81 le 118 48.40 45.00 46.70 14.58 11.74 13.16 le 125 45.00 39.50 42.30 11.07 5.24 8.16 co 3 50.10 46.10 48.10 11.55 8.69 10.12 pkm 1 49.50 46.70 48.10 11.33 7.69 9.51 thco 3 48.40 43.30 45.90 15.18 8.46 11.82 coth 2 48.30 45.50 46.90 15.63 8.58 12.11 mean 47.57 44.04 45.82 13.26 8.98 11.12 g t g x t g t g x t sed 1.15 0.38 1.62 0.137 0.061 0.194 cd (0.05) 2.28 0.76 ns 0.278 0.124 0.393 adv. hort. sci., 2016 30(1): 3-11 6 iw/cpe ratio level, le 118, thco 3 (28), le 27 and coth 2 (27) registered a delay in flowering compared to other genotypes. the variety co 3 showed its supremacy for earlier flowering, both under control (26) and drought (23) conditions, compared to the other genotypes; pkm 1 (28 and 24) ranked next. the differences due to genotypes, treatments and interactions attained significance for flower abscission (table 2). among the treatments, the plants imposed with 1.0 iw/cpe ratio recorded lesser mean percentage of flower abscission (11.4) than 0.5 iw/cpe ratio plants (16.5). among the genotypes, le 1 recorded greater mean percentage of flower abscission (15.5) than the other genotypes considered. interestingly, among the control plants, le 27 and le 57 recorded higher flower abscission (13.2, 12.6) while, under drought, le 1 and coth 2 recorded higher flower abscission of 20.1 and 18.1, respectively. at 0.5 iw/cpe ratio level, le 118 showed its supremacy with lower abscission of 13.9 followed by le 57 (15.1), le 114 (15.4) and le 27 (15.8). sps is the plant enzyme thought to play a major role in sucrose biosynthesis. it is considered to play a major role in the re-synthesis of sucrose (wardlaw and willenbrink, 1994) and sustain the assimilatory carbon flux from source to developing sink (isopp et al., 2000). among the genotypes, le 57 recorded significantly higher enzyme activity of 2.75 under control followed by the genotypes le 27 (2.61), le 118 (2.53) and le 114 (2.46). at 0.5 iw/cpe ratio level, the highest activity of 1.97 was registered by le 118 followed by le 57 (1.90) while the lowest was recorded by le 1 (0.57) and le 125 (0.69) (fig. 1). the data on yield components such as average fruit weight, fruit volume, fruit diameter, fruit number and fruit yield attained statistical significance except fruit diameter. comparing the treatments, control (1.0 iw/cpe ratio) plants recorded higher fruit weight (20.25) than under drought condition (13.75) (table 3). relating the genotypes, coth 2 recorded higher fruit weight of 45.40 which was on par with thco 3 (44.45) at 1.0 iw/cpe ratio level while the lowest was registered by le 125 (9.06) followed by le 57 (9.48). at 0.5 iw/cpe ratio level, relatively higher fruit weight was recorded by coth 2 (29.87) followed by thco 3 (28.78), co 3 (19.93), fig. 1 effect of drought on sps activity of tomato genotypes at 60 dat. genotypes average fruit weight (g) fruit volume (cc) fruit diameter (cm) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 11.18 5.72 8.45 12.020 6.48 9.25 3.41 2.43 2.920 le 27 10.68 7.23 8.96 11.52 7.95 9.74 3.34 3.05 3.20 le 57 9.48 7.89 8.69 10.09 8.62 9.36 3.11 3.48 3.30 le 114 9.55 7.49 8.52 10.31 8.25 9.28 3.53 3.25 3.39 le 118 10.01 7.95 8.98 10.92 8.61 9.77 4.12 3.76 3.94 le 125 9.06 3.89 6.48 9.65 4.64 7.15 3.30 2.72 3.01 co 3 26.38 19.93 23.16 28.39 22.69 25.54 5.40 4.84 5.12 pkm 1 26.31 18.75 22.53 28.34 22.49 25.42 5.51 4.71 5.11 thco 3 44.45 28.78 36.62 48.02 29.64 98.33 6.57 5.59 6.08 coth 2 45.40 29.87 37.64 48.95 30.69 39.82 6.31 5.73 6.02 mean 20.25 13.75 17.00 21.82 15.01 18.41 4.46 3.96 4.21 g t g x t g t g x t g t g x t sed 0.582 0.260 0.823 0.594 0.266 0.841 0.053 0.024 0.075 cd (0.05) 1.178 0.527 1.667 1.203 0.538 1.702 0.108 0.048 0.152 table 3 effect of water deficit on the yield parameters of tomato genotypes sivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 7 pkm 1 (18.75), le 118 (7.95) and le 57 (7.89). the data on fruit volume recorded similar trend of fruit weight. regarding the treatments, the plants imposed with 1.0 iw/cpe ratio recorded the fruit volume of 21.82 than 0.5 iw/cpe ratio (15.01) (table 3). under control, higher volume of fruit was recorded in the genotype coth 2 (48.95) which was on par with thco 3 (48.02). under 0.5 iw/cpe ratio level, the lowest fruit volume was recorded by le 125 (4.64) followed by le 1 (6.48). other than hybrids and varieties, le 57 showed higher fruit volume of 8.62 followed by le 118 (8.61), le 114 (8.25) and le 27 (7.95) at 0.5 iw/cpe ratio level. with regard to fruit diameter, thco 3 (6.08) and coth 2 (6.02) recorded higher average diameter of fruits which was on par with each other. for treatments, plants imposed with 1.0 iw/cpe ratio recorded higher fruit diameter (4.46) than 0.5 iw/cpe ratio (3.96) (table 3). among the genotypes, coth 2 registered higher fruit diameter of 5.73 followed by thco 3 (5.59), co 3 (4.84) and pkm 1 (4.71) at 0.5 iw/cpe ratio level. other than hybrids and varieties, le 118 showed higher fruit diameter of 3.76 followed by le 57 (3.48), le 114 (3.25) and le 27 (3.05) at 0.5 iw/cpe ratio condition while the lowest was recorded by le 1 (2.43) and le 125 (2.72). comparing the irrigation treatments, plants that received 1.0 iw/cpe ratio recorded higher fruit yield than 0.5 iw/cpe ratio (fig. 2). among the genotypes, le 57 recorded significantly superior fruit yield of 16.64 followed by coth 2 (15.89), le 118 (15.02), thco 3 (14.67) and le 27 (14.42) with 1.0 iw/cpe ratio level. but, at 0.5 iw/cpe ratio condition, le 57 documented higher fruit yield of 11.12 followed by le 118 (10.14), le 114 (8.54) and le 27 (8.13) while the lowest yield of 2.22 was recorded by le 125 followed by le 1 (2.57). quality parameters were altered under drought the data on tss content of the fruits revealed that the genotypes, treatments and interactions attained statistical significance (table 4). among the treatments, plants imposed with 0.5 iw/cpe ratio recorded higher brix value (3.01) than 1.0 iw/cpe ratio (2.89). among the genotypes, thco 3 recorded higher average brix value of 4.00 than the rest of the genotypes. at 0.5 iw/cpe ratio condition, the highest tss value was recorded by thco 3 (4.1) followed by coth 2 (3.9), pkm 1 (3.6) and co 3 (3.4) while the lowest was registered by le 125 (2.2). plants imposed with 0.5 iw/cpe ratio recorded fig. 2 effect of drought on the yield of tomato genotypes. table 4 effect of water deficit on fruit quality of tomato genotypes genotypes tss (° brix) lycopene (mg 100 g-1) vitamin c (mg 100 g-1) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 2.50 2.70 2.60 2.21 2.39 2.30 14.45 14.42 14.44 le 27 2.50 2.60 2.55 2.52 2.73 2.63 14.76 14.96 14.86 le 57 2.40 2.60 2.50 2.46 2.68 2.57 14.95 15.30 15.13 le 114 2.40 2.50 2.45 2.82 2.88 2.85 13.97 14.05 14.01 le 118 2.40 2.50 2.45 2.85 2.95 2.90 14.36 14.36 14.36 le 125 2.20 2.20 2.20 2.13 2.67 2.40 13.46 13.43 13.45 co 3 3.30 3.40 3.35 4.54 4.84 4.69 24.05 24.17 24.11 pkm 1 3.50 3.60 3.55 3.78 4.05 3.92 23.06 23.21 23.14 thco 3 3.90 4.10 4.00 3.35 3.53 3.44 15.42 15.64 15.53 coth 2 3.80 3.90 3.85 3.54 3.55 3.55 16.19 16.30 16.25 mean 2.89 3.01 2.95 3.02 3.23 3.12 16.47 16.58 16.53 g t g x t g t g x t g t g x t sed 0.03 0.01 0.04 0.048 0.022 0.068 0.147 0.066 0.208 cd (0.05) 0.05 0.02 0.07 0.097 0.044 0.138 0.297 ns ns adv. hort. sci., 2016 30(1): 3-11 8 higher lycopene content (3.23) than 1.0 iw/cpe ratio (3.02). with respect to the genotypes, co 3 recorded significantly higher average lycopene content (4.69). at 0.5 iw/cpe ratio level, lowest lycopene content was recorded by le 1 (2.39) and le 125 (2.67). the data on vitamin c content indicated that a narrow increment under drought compared to control (table 4). 0.5 iw/cpe ratio recorded higher ascorbic acid (16.58) than 1.0 iw/cpe ratio (16.47). among the genotypes, co 3 recorded higher average vitamin content of 24.11 followed by pkm 1 (23.14). at 0.5 iw/cpe ratio level, the higher value was registered by co 3 (24.17) followed by pkm 1 (23.21), coth 2 (16.30) and thco 3 (15.64) while the lowest values were recorded by the genotypes le 125 (13.43) and le 114 (14.05). interestingly, only genotypes attained significant difference not the treatments or interactions in the case of ascorbic acid. 4. discussion and conclusions in the present study, spad value, an index for total chlorophyll content in plants, showed a reduction under drought stress. hawkins et al. (2009) reported that spad values can be used to evaluate the response of plant species to drought and heat stresses in the field. the adverse effect of drought on greenness of the leaf in the current investigation could be observed in the susceptible genotypes le 125 and tnau thco 3 which depicted the highest reduction of spad values at the time of reproductive development stage. on the contrary, the tolerant genotype le 57 showed only a very low reduction in spad value. hence, the intensity of greenness in terms of spad values of the plant influenced the photosynthetic rate and thereby plant efficiency for increased biomass production. ma et al. (1995) also reported a highly significant correlation of spad readings with photosynthetic rate in soybean. the ability of the genotypes le 57 and le 114 to maintain high spad values under field conditions in response to water deficit has been revealed. therefore, these genotypes were able to endure drought injury better than the sensitive lines. the soluble protein content of the leaf, a measurement of rubp carboxylase activity, was considered an index for photosynthetic efficiency. rubisco enzyme makes up nearly 80% of the soluble proteins in leaves of many plants (joseph et al., 1981). diethelm and shibles (1989) opinioned that, the rubisco content per unit leaf area was positively correlated with that of soluble protein content of the leaf. several studies have reported that drought stress in tomato (bartholomew et al., 1991), arabidopsis (williams et al., 1994), and rice (vu et al., 1999) leads to a rapid decrease in the abundance of rubisco small subunit (rbcs) transcripts, which may indicate the decreased synthesis of soluble protein. the present study also confirms the above findings with a 32.3% reduction of soluble protein content under drought compared to control. drought stress induces degradation of soluble proteins and this effect could be revealed through a reduction in leaf soluble protein content of various genotypes. in the present study, the reduction was, however, low in le 118 and high in coth 2 and thco 3 under drought stress. maintenance by the genotypes of soluble protein content could be attributed to higher rubisco activity, leading to more carbon fixation and ultimately to higher photosynthetic efficiency under drought, which is one of the important traits for drought tolerance. reproduction is the crucial stage to be affected by any abiotic stress in any crop. an increase in the frequency of water stress days during flower development affects plant reproduction with immediate and long-term effects (srivastava et al., 2012). drought stress, in general, induces early flowering and in the present study as well flower initiation occurred three days earlier than the control. this early flowering under drought might be due to rapid phenological development in order to complete the life cycle under an unfavorable environmental condition. differences due to genotypes, treatments and interactions attained significance for flower abscission (table 2). among the treatments, the plants imposed with 1.0 iw/cpe ratio recorded a lower mean percentage of flower abscission than plants under 0.5 iw/cpe. among the genotypes, le 1 recorded the highest percentage of flower abscission. interestingly, under control conditions, genotypes le 27 and le 57 gave high flower abscission, while le 1 and coth 2 recorded higher flower abscission under drought. an earlier finding by bhatt et al. (2009) in tomato strongly supports the results of the present study. a lower rate of flower abscission in the tolerant genotypes might be due to the maintenance of photosynthesis and efficient translocation of photosynthates to the reproductive parts under drought. the reduction in photosynthesis during stress may decrease the availability of assimilates to the develsivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 9 oping floral organs and leads to the abscission of flowers and flower buds in susceptible cultivars. however, some workers are of the opinion that the abortion of reproductive organs is not solely due to a poor assimilate supply but also due to other factors such as assimilate utilization (ruiz and guardiola, 1994; aloni et al., 1996). in the present study, there was a reduction in sps activity under drought conditions compared to control. the highest percent reduction (74.3) was observed in the genotype le 1, however the lowest reduction was noted for le 118. as observed also by huber and huber (1996), there was a significant elevation in sps activity in response to water stress. in contrast to this finding, the present study revealed a decreased activity of sps under drought conditions. bhatt et al. (2009) observed a considerable reduction in sps activity in susceptible cultivars during stress. the present study corroborates these findings. the reduced photosynthesis during water stress may also lead to a reduction in the capacity for both starch and sucrose synthesis and cause a decline in the sps activity (vassey and sharkey, 1989). our results reveal that drought stress caused the reduction in fruit weight up to 30% under field conditions. among the genotypes, le 57, le 118, and le 114 had a lower reduction in fruit weight. all the genotypes exhibited a similar trend in fruit volume and fruit diameter in response to drought stress, which also caused remarkable changes in fruit number and an overall reduction up to 31% was observed compared to control. the reduction in fruit weight, in response to drought stress, had a direct influence on fruit yield of the various genotypes of tomato. drought stress resulted in an overall yield loss of tomato fruits up to 55%. the greatest yield loss (70 to 80%) was exhibited by le 1 and le 125. the varieties and hybrids showed a reduction of fruit yield from 40.5 to 50.4% compared to control. significantly less reduction (35 to 40%) was exhibited by le 118, le 57, le 114, and le 27 showing their somewhat tolerant nature toward drought stress (fig. 2). therefore, it can be clearly stated that water deficit, as a result of soil drying, caused a major adverse effect on yield and yield components even in tolerant genotypes. the present study confirms previous findings by farooq et al. (2009) and manjunatha et al. (2004). doorenbos and kassam (1979) indicated that the highest demand for water supply in tomato plants occurs at the flowering phase. water deficit during this stage would have reduced the number of flowers produced and, as suggested by mahendran and bandara (2000), limitation of water at flowering stage not only reduces flower formation but also increases flower shedding. purseglove et al. (1981) stated that, although the cultivar has a dominant influence over quality determinant properties, the environment in which it grows also has a significant role in the quality characters. fruit quality, mainly total soluble solids, vitamin c, and acid contents have been reported to change under moisture stress (kozlowski, 1972). however, in the present study, a slight enhancement in ascorbic acid content was noticed in all the genotypes in response to drought stress. furthermore, tss, lycopene and citric acid content of the fruit also increased slightly. our work corroborates earlier findings by ali et al. (1980) in tomato. also nahar et al. (2011) explained that the fruit quality improvement under water deficit conditions in tomato might be due to the synthesis of ascorbic, citric, and malic acid. in the present study, le 118, le 57, and le 27 showed their primacy with the highest ascorbic acid content, as well as higher tss and lycopene content. this finding is strongly supported by tambussi et al. (2000), who also reported that the increase in ascorbic acid might be an effective strategy to protect membranes from oxidative damage in water stressed condition. from perusal of the results obtained for spad value, soluble protein, sps activity, fruit characters, lycopene, ascorbic acid, tss and yield, it can be inferred that genotypes le 114, le 57, le 118, and le 27 performed better under drought conditions and can be categorized as drought tolerant genotypes compared to genotypes le 1 and le 125, drought susceptible ones. however, further studies are required to confirm the results by molecular evidence. the tolerant genotypes could be utilized for further breeding programmes to evolve new tomato genotypes for better drought tolerance with higher yield. acknowledgements the authors wish to express their sincere thanks to the professor and head, department of vegetable science, for providing seed materials for this research and all the faculty members of the department of crop 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14(9): 939-946. 159 1. introduction dracaena sanderiana sander ex mast. (lucky bomboo) belongs to the family agavaceae. it is distributed in tropical and subtropical open lands of africa and india. despite its medicinal and ornamental importance, not much work has been undertaken with regard to its in vitro propagation; conventional vegetative propagation is the most prevalent method (junaid et al., 2008). vegetatively propagated plants accumulate several bacterial, fungal, viral, and mycoplasma diseases however the in vitro condition can overcome these problems and offers rapid vegetative multiplication of plants (predieri, 2001; muthusamy et al., 2007). usually this plant is propagated by means of seeds or cuttings however micropropagation is a proposed technique to produce healthy plants. to cover the needs for such plants, it is necessary to study the different factors affecting productivity of the explants to standardize the technique used for enhancing the multiplication of this important plant for indoor and outdoor uses. paek et al. (1985) found that cordyline terminalis electra and scindapsus aureum marble queen shoot tips were successfully multiplied on a solid ms medium supplemented with indole-3-acetic acid (iaa) at 1.0 mg l-1, kinetin at 3.0 mg l-1 and adenine sulphate at 100 mg l-1 and na 2 h 2 po 4 .h 2 o at 150 mg l-1. atta-alla et al. (1996) stated that the highest rate of shoot proliferation in dracaena marginata lam. ‘tricolor’ was achieved on ms medium supplemented with ba at 4.0 mg l-1 plus naa at 0.05 mg l-1. furthermore, tian-lang et al. (1999) reported that using ms medium supplemented with ba (3-3.5 mg l-1) and naa (0.02 mg l-1) was suitable for multiplication of d. sanderiana cv. virescens. el-sawy et al. (2000) produced the largest number of shoots per explant on ms medium supplemented with ba at 4.0 mg l-1. kobza and vachunova (1989) reported that ms medium enriched with ba and iaa or ba and naa was the best. stem explants of d. deremensis warneckii ‘lemon lime’ with a dormant bud provided the most suitable propagation material. leffring et al. (1985) found that, as both growth regulators (iaa and aba) increased over a few month culture period, the downward movement of iaa resulted in basal callus formation of cordyline cultivars and prevented shoot development as it antagonized the effects of kinetin in the culture medium. kobza and vachunova (1991) found that ms medium supplemented with 1.0 mg l-1 kinetin and 0.8 mg factors affecting in vitro propagation of dracaena sanderiana sander ex mast. cultivars. i. sterilization, explant browning, and shoot proliferation f. kakuei, h. salehi(*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: dracaena, growth regulators, proliferation rate, propagation, sterilization. abstract: dracaena sanderiana sander ex mast. (lucky bomboo) is an ornamental plant belonging to the family agavaceae. the factors affecting sterilization, explant browning, and shoot proliferation of two cultivars green and variegated of d. sanderiana were studied. micropropagation of d. sanderiana is very important because of the limitations in its conventional propagation by classical vegetative propagation methods which give rise to several bacterial, fungal, viral and mycoplasma diseases. the in vitro condition can overcome these problems. half-strength murashige and skoog’s (ms) media supplemented with different concentrations of 6benzyl-amino-purine (ba mg l-1) and naphthalene acetic acid (naa) (0.25 and 0.5 mg l-1) were used for shoot proliferation and plant regeneration studies. the effect of explant positioning on the culture media, whether horizontal or vertical, was also assessed on proliferation and growth of shoots produced. explants of the ‘green’ cultivar, cultured horizontally on media, were successful in yielding proliferated shoots. the highest mean value (4.8) was recorded on the medium supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. explants of the ‘variegated’ cultivar, cultured horizontally on media, were also successful in yielding proliferated shoots. the highest mean value (3.66) was recorded on the medium supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. adv. hort. sci., 2015 29(4): 159-164 (*) corresponding author: hsalehi@shirazu.ac.it received for publication 10 april 2015 accepted for publication 22 june 2015 160 adv. hort. sci., 2015 29(4): 159-164 l-1 iaa was the best medium for d. concina kunth shoot production. the objective of the present investigation was to study different aspects of micropropagation of d. sanderiana (lucky bomboo) and factors affecting its sterilization, browning decrease in explants, and proliferation of shoots to propose a commercial protocol for propagating this plant. 2. materials and methods this experiment was carried out at the tissue culture and biotechnology lab, department of horticultural science, college of agriculture, shiraz university. sterilization stage and effects of antioxidants numerous shoot explants of d. sanderiana (both cultivars) measuring 3-4 cm long were removed from the mother plants and placed in a weak detergent solution (about 0.2%) for 20 min. to prevent the formation of polyphenols and the occurrence of browning, the explants were exposed to citric acid and ascorbic acid at 50 and 100 mg l-1, respectively, and an integrated combination, for 30 min. they were then washed with running tap water for 20 min, followed by surface sterilization with 70% ethanol for 3 min plus 10% or 15% clorox (a commercial bleach, containing 5.25% sodium hypochlorite) with 3-5 drops of tween-20 for 10, 15, and 20 min. they were then rinsed three times with sterilized distilled water. culture media murashige and skoog’s (1962) (ms) medium was used for the establishment stage supplemented with 30 g l-1 sucrose and 8 g l-1 agar. the ph was adjusted to 5.8 before the addition of agar, then 40 ml of the culture medium were poured into 40 ml culture jars and autoclaved at 121ºc at a pressure of 1.5 kg cm-2 for 20 min using the following media: full strength ms medium and 1/2 strength ms medium. all the chemicals were purchased from sigmaaldrich authorized distributer, kimia gostar pooyesh co., ltd., tehran, iran. shoot proliferation and plant regeneration half strengths ms media supplemented with different concentrations of ba (1, 2, 3, 4 and 5 mg l-1) and naa (0.25 and 0.5 mg l-1) were used for shoot proliferation and plant regeneration of both dracaena cultivars. also, the effect of the direction of explant positioning on media was studied to evaluate the proliferation and growth rate of the produced shoots. data recording and analysis all the main experiments were conducted in a completely randomized design with eight replicates; the experiments were repeated at least two times. data were statistically analyzed and the means were calculated using spss (version 15) software whereby means were compared using lsd test at 5% level. 3. results and discussion sterilization stage results indicated that the use of clorox significantly affected explant contamination (table 1). increasing clorox concentration increased the decontaminated explants in d. sanderiana. the highest mean value of explant decontamination (0%) was obtained when applying 15% (v v-1) of clorox for 20 min. an optimum value of healthy explants, free of contamination, could be obtained by using clorox at 15%, whereas the higher concentration had a negative effect on this characteristic. our results are consistent with those obtained by kunisaki (1975) on cordylin terminalis (l.) kunth, chua et al. (1981) on d. marginata link, sagawa and kunisaki (1990) on dracaena sp., and badawy et al. (2005) on d. fragrans (l.) ker gawl. cv. ‘massangeana’. effects of antioxidants antioxidants were used to reduce browning in explants. after placing the explants in a weak detergent solution (about 0.2%), they were placed in a solution containing the concentrations of 0, 50 and 100 mg l-1 ascorbic acid and citric acid separately and in combination for at least 30 min. the data presented in table 2 clearly show that both antioxidants significantly prevented browning in cultured explants of d. sanderiana. the highest mean of explants saved from browning was recorded when 100 mg l-1 of both ascorbic acid and citric acid were mixed and used in combined form. the highest browning frequency and intensity belonged to the control. the use of 100 mg l-1 ascorbic acid, significantly reduced the browning of explants compared with utilization of the same amount of citric acid. in a study conducted by huang et al. (2002) it was observed that ppo (polyphenol oxidase) enzyme activity retable 1 effect of different concentrations of clorox on surface sterilization of explants of dracaena sanderiana sander ex mast. clorox concentrations (%) time (min) contamination (%) control (0) 0 100.00 a (z) 10 43.75 b 10 15 37.50 bc 20 18.75 bcd 10 18.75bcd 15 15 6.25 cd 20 0.00 d (z) means followed by the same letter(s) are not significantly different using lsd test at 5% level. 161 kakuei and salehi, factors affecting in vitro propagation of dracaena sanderiana. i mains stable at ph 10 and strongly acts against browning; the use of ascorbic acid was to prevent the occurrence of browning. elmore et al. (1990) also used ascorbic acid as an antioxidant and anti-browning substance in the culture medium for plant cells, tissues, and organs. shoot proliferation rate in horizontal and vertical explants: green cultivar table 3 shows the difference between the proliferation rate of horizontal and vertical explants of d. sanderiana ‘green’ after 60 days of deployment following treatment with concentrations of 1, 2, 3, 4 and 5 mg l-1 ba, and 0.25 and 0.5 mg l-1 naa. according to the results, the direction of explant positioning in the culture medium, either horizontal or vertical, had significant effects on successful establishment and proliferation rates. the highest mean value (4.8) was recorded when using 2 mg l-1 ba and 0.25 mg l-1 naa. generally, explants showed a good response to this ba concentration to the extent that some cultures produced up to 10 shoots. however, regardless of growth regulator concentration, proliferation failed to occur on vertical explants (table 3, fig. 1). the highest average length of shoots in horizontally cultured explants of ‘green’ was observed when ba and naa were utilized at concentrations of 1 mg l-1 and 0.5 mg l-1, respectively. this resulted in the production of shoots measuring 3.25 cm long. furthermore, shoots were successfully produced measuring 3.05 cm long when ba and naa were used at 3 mg l-1 and 0.5 mg l-1, respectively. the greatest average length of shoots achieved in vertically cultured explants of ‘green’ was 4.12 cm when 2 mg l-1 ba and 0.5 mg l-1 naa were used (table 4). table 2 effect of different antioxidants on browning rate of dracaena sanderiana sander ex mast. antioxidants concentrations (mg l-1) browning (z) control (0) 8.60 a (y) as50 5.20 bc ci50 5.80 b as100 3.80 cd ci100 5.20 bc as50+ci50 3.60 d as100+ci100 2.20 e (z) 10 the highest rate of browning and 1 the lowest rate of browning. (y) means followed by the same letter(s) are not significantly different using lsd test at 5% level. as= ascorbic acid; ci= citric acid. table 3 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the proliferation rate of ‘green’ treatments (mg l-1) average number of shoots meanexposure position of explant ba naa horizontal vertical 0 0 1.00 c (z) 1.00 c 1.00 b 1 0.25 1.20 bc 1.00 c 1.11 b 0.50 1.40 bc 1.00 c 1.22 b 2 0.25 4.80 a 1.00 c 3.11 a 0.50 1.60 bc 1.00 c 1.33 b 3 0.25 2.20 b 1.00 c 1.66 b 0.50 1.60 bc 1.00 c 1.33 b 4 0.25 1.00 c 1.00 c 1.00 b 0.50 1.40 bc 1.00 c 1.22 b 5 0.25 1.20 bc 1.00 c 1.11 b 0.50 1.60 bc 1.00 c 1.33 b mean 1.72 a 1.00 b (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. table 4 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the length of shoots in ‘green’ treatments (mg l-1) average length of shoots (cm) meanexposure position of explant ba naa horizontal vertical 0 0 2.40 b (z) 1.88 b 2.16 ab 1 0.25 1.95 b 2.30 ab 1.91 b 0.50 3.25 ab 2.62 b 2.40 ab 2 0.25 2.30 b 2.25 b 2.80 ab 0.50 2.35 b 4.12 a 3.14 a 3 0.25 2.96 ab 2.88 ab 2.92 ab 0.50 3.05 ab 2.00 b 2.27 ab 4 0.25 2.70 b 2.12 b 2.40 ab 0.50 2.70 b 3.00 ab 2.83 ab 5 0.25 2.50 b 2.25 b 2.7 ab 0.50 2.40 b 2.62 b 2.50 ab mean 2.59 a 2.51 a (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. fig. 1 rate of shoot proliferation in dracaena ‘green’ cultured horizontally on 1/2 ms medium with mg l-1 ba and 0.25 mg l-1 naa40 days after culture (left) and 60 days after culture (right). 162 adv. hort. sci., 2015 29(4): 159-164 average leaf number in horizontally cultured explants of ‘green’ showed no significant difference in any of the treatments used, but in vertically cultured explants the greatest number of leaves (5.75) was obtained with 2 mg l-1 ba and 0.5 mg l-1 naa (table 5). with increasing concentration of growth regulators, deformity was observed in most of the shoots produced. shoots created from horizontally cultured explants of ‘green’, at a concentration of 4 mg l-1 ba, showed the greatest deformity and deviated from their natural state. the least deformity was observed in the control and 2 mg l-1 ba treatments. the level of deformity in shoots obtained from vertically cultured explants of ‘green’ was also higher at 5 mg l-1 ba. deformity was not detected at low growth regulator concentrations and in the control explants (table 6). shoot proliferation rate in horizontal and vertical explants: variegated cultivar table 7 shows the difference between the proliferation rate of horizontally and vertically cultured explants of d. sanderiana ‘variegated’ after 60 days of deployment with the concentrations of 1, 2, 3, 4 and 5 mg l-1 ba, and 0.25 and 0.5 mg l-1 naa. according to the results, the direction of explant position in culture medium had a significant effect on proliferation rate: horizontally cultured explants showed a greater shoot proliferation rate. the highest mean value (3.66) was recorded when using 2 mg l-1 ba and 0.25 mg l-1 naa (table 7, fig. 2). when explants were treated with 1 mg l-1 ba and 0.5 mg l-1 naa, the average length of shoots in vertically cultured explants of ‘variegated’ measured 3.00 cm more than other treatments. the average length of shoots in vertically cultured explants of ‘variegated’ was the highest with 2 mg l-1 ba and 0.5 mg l-1 naa. the average length was 2.83 cm (table 8). five leaves were obtained on average with the use of 1 mg l-1 ba and 0.5 mg l-1 naa. this average was higher table 5 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the average leaf number in ‘green’ treatments (mg l-1) average number of leaves (cm) meanexposure position of explant ba naa horizontal vertical 0 0 3.80 b (z) 4.00 b 3.88 ab 1 0.25 3.70 b 4.75 ab 4.16 ab 0.50 4.30 ab 3.50 b 3.94 ab 2 0.25 4.45 a 4.25 ab 5.02 a 0.50 3.90 b 5.75 a 4.05 ab 3 0.25 4.46 ab 4.75 ab 4.60 ab 0.50 4.30 ab 3.25 b 3.83 ab 4 0.25 4.00 b 3.50 b 3.77 b 0.50 4.50 ab 3.75 b 4.16 ab 5 0.25 4.20 b 4.00 b 4.11 ab 0.50 3.80 b 4.25 ab 4.00 ab mean 4.12 a 4.15 a (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. table 6 the interaction effect of different concentrations of ba and naa, and exposure position of explants on deformity in ‘green’ treatments (mg l-1) deformity (z) meanexposure position of explant ba naa horizontal vertical 0 0 0 g (y) 0 g 0 e 1 0.25 1.00 d-g 0 g 0.55 de 0.50 1.00d-g 0.50 fg 0.77 de 2 0.25 1.20 d-g 0.75efg 1.00 cd 0.50 0.80 efg 0.62 fg 0.72 de 3 0.25 1.60 def 1.50 def 1.55 c 0.50 3.00 abc 1.87cde 2.50 b 4 0.25 2.20 bcd 2.75abc 2.40 b 0.50 3.70 a 3.40 a 3.61 a 5 0.25 3.20 ab 3.50 a 3.33 a 0.50 3.50 a 3.50 a 3.50 a mean 1.96 a 1.67 b (z) 5= highest deformity and 1= lowest deformity. (y) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. table 7 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the shoot proliferation rate in ‘variegated’ treatments (mg l-1) average number of shoots meanexposure position of explant ba naa horizontal vertical 0 0 1.00 c (z) 1.00 c 1.00 b 1 0.25 1.00 c 1.00 c 1.00 b 0.50 1.00 c 1.00 c 1.00 b 2 0.25 3.66 a 1.00 c 2.33 a 0.50 1.00 c 1.00 c 1.00 b 3 0.25 2.00 b 1.00 c 1.5 ab 0.50 2.00 b 1.00 c 1.5 ab 4 0.25 1.00 c 1.00 c 1.00 b 0.50 1.00 c 1.00 c 1.00 b 5 0.25 1.00 c 1.00 c 1.00 b 0.50 1.00 c 1.00 c 1.00 b mean 1.42 a 1.00 b (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. 163 kakuei and salehi, factors affecting in vitro propagation of dracaena sanderiana. i than other treatments. the average leaf number in vertically cultured explants of ‘variegated’ was 5.66 when 3 mg l-1 ba and 0.25 mg l-1 naa were used (table 9). with increasing concentration of growth regulators, deformity was observed in most shoots produced. shoots created from horizontally cultured explants of ‘variegated’, resulting from 5 mg l-1 ba with 0.25 and 0.5 mg l-1 naa, showed the greatest deformity and were deviated from their natural state. deformity in shoots obtained from vertical explants of ‘variegated’ was also higher at concentrations of 5 mg l-1 ba and 0.25 or 0.5 mg l-1 naa; deformity was absent in low growth regulator concentrations and in control explants (table 10). successful growth of explants under controlled conditions is a function of the proper combination of nutrients, especially compounds controlling growth. because of the small size of the explants used, synthesis and production of these compounds is greatly needed for organ growth and development. therefore, in order to achieve maximum growth rate, it is necessary to use plant hormones in approtable 8 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the length of shoots in ‘variegated’ treatments (mg l-1) average lenght of shoots (cm) meanexposure position of explant ba naa horizontal vertical 0 0 1.00 f (z) 1.33 def 1.16 cd 1 0.25 1.00 f 1.00 f 1.00 d 0.50 3.00 a 1.33 def 2.16 ab 2 0.25 1.50 c-f 2.16 a-e 1.83 a-d 0.50 2.33 a-d 2.83 ab 2.58 a 3 0.25 1.63 c-f 2.33 a-d 1.98 ab 0.50 2.00 a-f 1.33 def 1.66 bcd 4 0.25 1.16 ef 2.00 a-f 1.58 bcd 0.50 1.83 b-f 1.16 ef 1.50 bcd 5 0.25 1.33 def 1.66 c-f 1.50 bcd 0.50 1.16 ef 2.50 abc 1.83 a-d mean 1.63 a 1.70 a (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. fig. 2 rate of shoot proliferation in dracaena ‘variegated’ cultured horizontally on 1/2 ms medium with 2 mg l-1 ba and 0.25 mg l-1 naa55 days after culture (left) and on 1/2 ms medium with 3 mg l-1 ba and 0.25 mg l-1 naa 70 days after culture (right). table 9 the interaction effect of different concentrations of ba and naa, and exposure position of explants on the average leaf number in ‘variegated’ treatments (mg l-1) average number of leaves meanexposure position of explant ba naa horizontal vertical 0 0 3.25 c (z) 3.00 c 3.12 b 1 0.25 3.66 bc 3.00 c 3.33 b 0.50 5.00 ab 3.00 c 4.00 ab 2 0.25 3.25 c 3.50 bc 3.45 ab 0.50 3.00 c 3.66 bc 3.33 b 3 0.25 3.41 bc 5.66 a 4.53a 0.50 3.66 bc 3.00 c 3.33 b 4 0.25 3.33 bc 3.50 bc 3.50 ab 0.50 3.33 bc 3.00 c 3.16 b 5 0.25 3.33 bc 3.00 bc 3.33 b 0.50 3.66 bc 4.00 bc 3.83 ab mean 3.53 a 3.42 a   (z) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level. table 10 the interaction effect of different concentrations of ba and naa, and exposure position of explants on deformity in ‘variegated’ treatments (mg l-1) deformity (z) meanexposure position of explant ba naa horizontal vertical 0 0 0 e (y) 0 e 0.00 e 1 0.25 0.33 de 1.00 cde 0.66 de 0.50 1.00 cde 1.25 cd 1.16 cd 2 0.25 1.33 cd 1.50 bc 1.41 bcd 0.50 1.33 cd 1.50 bc 1.41 bcd 3 0.25 1.40 cd 1.66 bc 1.53 bcd 0.50 1.33 cd 1.66 bc 1.50 bcd 4 0.25 2.00 bc 1.80 bc 1.90 bc 0.50 1.66 bc 2.50 abc 2.08 bc 5 0.25 3.33 a 3.25 a 3.29 a 0.50 3.40 a 3.66 a 3.53 a mean 1.52 b 1.90 a (z) 5 highest deformity and 1 lowest deformity. (y) data followed by the same letter(s) (lower letters for interactions and capital letters for factor means) are not significantly different using lsd test at 5% level 164 adv. hort. sci., 2015 29(4): 159-164 priate concentrations. having this goal necessitates the use of different hormones responsible for growth stimulation (hu and wang, 1983). the increase in shoot proliferation rate is partly due to the increase in ba concentration. this can be interpreted through the rate of cytokinins role in stimulating cell division and growth of lateral buds. furthermore, shoot proliferation culture media that is rich in cytokinins often results in lateral buds that have been released from terminal bud dominance (taji et al., 1997). explants that were taken from young stems of d. sanderiana, as compared to older explants, showed greater shoot production in the media used. the results of this experiment were in agreement with those of tian lang et al. (1999) on d. sanderiana cv. virescens. they found that the use of 3 to 3.5 mg l-1 ba with 0.02 mg l-1 naa yields the highest shoot proliferation rate in this species. attaalla et al. (1996) and el-sawy et al. (2000) on d. marginata cv. tricolor showed the highest shoot proliferation rate on ms medium containing 4 mg l-1 ba and 0.05 mg l-1 naa. moreover, our results were more or less the same as those of maheran et al. (1996) on d. fragrans ‘massangeana’ and ying et al. (2008) on d. cambodiana pierre ex gagnep. 4. conclusions explants of the ‘green’ cultivar, cultured horizontally on media, were successful in yielding proliferated shoots. the highest mean value (4.8) was recorded on the medium supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. explants of the ‘variegated’ cultivar, cultured horizontally on media, were also successful in yielding proliferated shoots. the highest mean value (3.66) was recorded on the medium supplemented with 2 mg l-1 ba and 0.25 mg l-1 naa. references atta-alla h., zaghloul m., waly a.k., khattab s.h., 1996 micropropagation of some ornamental plants. 3. in vitro culture and establishment of dracaena marginata var. ‘tricolor’. j. agric. sci., 34: 1153-1162. badawy e.m., habib a.m.a., bana a.e., yosry g.m., 2005 propagation of dracaena fragrans plants by tissue culture technique. arab j. biotech., 8: 329-342. chua b.u., kunisaki j.t., sagawa y., 1981 in vitro propagation of dracaena marginata ‘tricolory’. hort. sci., 16: 494-498. elmore h., samples b., sharma s., harrison m., 1990 influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media. plant cell tissue organ culture, 20: 131-135. el-sawy a., bekhee s.a., hossny y.a., 2000 a protocol for micropropagtion of dracaena marginata. egypt. j. hort., 27: 29-40. hu c.y., wang p.j., 1983 meristem shoot tip and bud culture, pp. 177-227. in: evans d.a., w.r. sharp, p.v. ammirato, and y. yamada (eds.) handbook of plant cell culture. macmillan publishing company, ny, usa, pp. 833. huang l.c., lee y.l., huang b.l., kou c.i., shaw j.f., 2002 high polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture. in vitro cell dev. biol. plant, 38: 358-365. junaid a., mujib a., sharma m.p., 2008 effect of growth regulators and ethylmethane sulphonate on growth, and cholorophyll, sugar and proline contents in dracaena sanderiana cultured in vitro. j. plant biol., 52: 569-572. kobza f., vachunova j., 1989 propagation of dracaena in vitro. i. propagation of dracaena deremensis. acta university agric. faculty of hort., 4: 33-37. kobza f., vachunova j., 1991 propagation of dracaena in vitro. ii. propagation of dracaena concina kunth. acta university agric. faculty of hort., 6: 51-55. kunisaki j.t., 1975 in vitro propagation of cordyline terminalis l. kunth. hort. sci., 10: 601-602. leffring l., vonkk c.r., ribot s.a., 1985 hormonal effects on shoot formation of cordyline cultivars. acta botanica neerlandica, 34: 238-245. maheran a.a., ooi h.l., rashid a.a., 1996 in vitro responses of dracaena fragrans massangeana to growth regulators. pertanika j. trop. agric. sci., 19: 123-127 murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant, 15: 473-497. muthusamy a., vasanth k., sivasankari d., chandrasekar b.r., jayabalan n., 2007 effects of mutagens on somatic embryogenesis and plant regeneration in groundnut. j. plant biol., 51: 430-435. paek ky., oh m.o., choi j.k., 1985 mass propagation of cordyline and scindapsus in vitro. korean j. hortic. sci., 26: 83-92. predieri s., 2001 mutation induction and tissue culture in improving fruits. plant cell tissue organ culture, 64: 185210. sagawa y., kunisaki j.t., 1990 micropropagation of floricultural crops, pp. 25-34. in: evans d.a., w.r. sharp, p.v. ammirato, and y. yamada (eds.) handbook of plant cell culture. macmillan publishing company, ny, usa, pp. 833. taji a.m., dodd w.a., willims r.r., 1997 plant tissue culture practices. une press, australia, 3rd edition, pp. 71. tian-lang t.h., zhang-lin t.l., tan h.y., zhang l., 1999 stem segment culture and tube propagation of dracaena sanderiana cv. virscens. acta hort. sin., 26: 133-134. ying x., bao-ling h., cheng-qun l., yin-ping z., chuanming z., ying l., 2008 rapid propagation of dracaena cambodiana in vitro. j. anhui agric. sci., 32: 112-120. impaginato 115 1. introduction over the last 20 years, low-input and organic agriculture has increased worldwide to preserve agroecosystem functionality (postma-blaauw et al., 2010). the main point of such an agriculture is a systemic approach to integrate sustainable yield and crop quality together with high-energy efficiency and low environmental impact (pimentel et al., 2005; moonen and bàrberi, 2008). in the framework of this view, the natural roles of microorganisms, such as arbuscular mycorrhizas in improving soil fertility have gained a growing interest for the use of such microrganisms as ecosystem engineers and biofertilizers (fitter et al., 2011). although arbuscolar mycorrhizal fungi normally infect most species of plants, some plants taxa do not usually form generally recognisable mycorrhizas. among them, the family of brassicaceae have been considered to be nonmicorrhyzal plants (lambers and teste, 2013), probably because their roots released anti-fungal metabolites such as isothiocyanates in the surrounding environment (tester et al., 1987). isothiocyanates are produced by hydrolization of glucosinolates that are a group of secondary metabolites present in brassicaceae (halkier and gershenzon, 2006). the endophyte fungus piriformospora indica (p. indica), a basidiomycete of the order sebacinales, was isolated from the indian thar desert in 1997 (varma et al., 1999). p. indica has received a great attention over the last few decades due to its ability to promote plant growth, protection and stress tolerance in colonized plants (verma et al., 1998; banhara et al., 2015). p. indica is similar to arbuscular mycorrhizal fungi, but it is a facultative symbiont and can be easily grown on various synthetic media. likewise, p. indica has a wide host range, colonizes the host roots, grows inter and intracellularly, and forms pearshaped chlamydospores within the cortex, improving the growth of many plant species, enhancing nutrient uptake, enabling plants to cope with environmental conditions, and to survive under abiotic stresses. it also confers resistance to toxins, pathogenic microorganisms, and increases seed biomass yield (oelmuller et al., 2009). among others, p. indica is able to coloadv. hort. sci., 2017 31(2): 115-119 doi: 10.13128/ahs-20664 fungal colonization improved growth and modulated the expression of myrosinases in black cabbage r. del carratore 1 (*), a. podda 2, b.e. maserti 2 1 istituto di fisiologia clinica, consiglio nazionale delle ricerche, area della ricerca cnr, via moruzzi, 1, 56124 pisa, italy. 2 istituto per la protezione sostenibile delle piante, consiglio nazionale delle ricerche, area della ricerca cnr, via madonna del piano, 10, 50019 sesto fiorentino (fi), italy. key words: brassica oleracea, colonization, piriformospora indica. abstract: the role of beneficial microorganisms, such as mycorrhizas, in improving the resistance to environmental stress of colonized plants is well-known. plants of brassicaceae family are of large economic importance, especially for the synthesis of anticarcinogenic compounds such as glucosinolates and their derivatives isothiocyanates. the endophyte fungus piriformospora indica is able to colonize them and improves their growth and response to environmental stress. however, no information are available on the impact of colonization on glucosinolate metabolism. in this work, colonization of black cabbage (brassica oleracea cv. acephala sabellica) is reported as well as the effects on plant growth and on the expression of myrosinase encoding genes, the isothyocianate producing enzymes. results indicate that p. indica successfully colonized black cabbage as validated by the expression of the marker gene ptef1. colonized plants showed increase of biomass weights and shoot length respect to the uncolonized plants and a decrease of myrosinase gene expression. this last finding indicates that p. indica might affect the resistance against biotic stress of black cabbage. (*) corresponding author: rdc@ifc.cnr.it received for publication 3 may 2017 accepted for publication 21 june 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 115-119 116 nize plants of brassicaceae family (sherameti et al., 2005) and improves their growth and response to environmental stimuli. p. indica triggered local and systemic root responses in arabidopsis thaliana (pedrotti et al., 2013). in chinese cabbage (brassica rapa), it has been reported that p. indica colonization confers drought tolerance stimulating antioxidant enzymes, the expression of drought-related genes and the plastid-localized ca(2+)-sensing receptor (cas) protein in the leaves (sun et al., 2010). black cabbage, (brassica oleracea cv. acephala sabellia) a variety of kale largely used in italian cuisine, especially in tuscany, where has been grown for centuries (appleman et al., 2008), is generally considered a nonmicorrhyzal plants (lambers and teste, 2013). in this work, with purpose to assess whether p. indica colonizes black cabbage and to study the colonization effects on this cultivar, seedlings were inoculated with p. indica; morphological parameters and the expression of myrosinase encoding genes were studied. 2. materials and methods growth conditions of plants and fungus, and estimation of plant growth seeds of brassica oleracea l. ssp. oleracea convar acephala (dc.) alef. var. sabellica l. were surfacesterilized with 75% alcohol three times for 10 min, and then placed on a petri dish containing sterilized water. plates were incubated at 22°c under continuous illumination (for seed germination). after 7 days, seedlings were transferred in petri dish plates with solid (1.5% agar) complete medium (cm) (pham et al., 2004). six seedlings were used per plate. piriformospora indica growth conditions p. indica cultures, dsm11827, leibniz institute dsmz-german collection of microorganisms and cell cultures, braunschweig, germany (lahrmanna et al., 2013) were propagated at 28°c in liquid cm for two days than plated in agar cm (fig. 1a). the amount of 200 mg of fungal mycelium were used to colonize the seeds. 0.1 ml of cm medium containing fungal mycelium were positioned 1 cm away from each seedling. the same amount of autoclaved mycelium was used as control. plant growth was monitored day by day. histograms report biomass weight and shoot length as mean±sd. the statistical significance of differential findings between samples was determined by anova using nia software; p<0.05 was considered statistically significant. rna extraction and genes expression pitef1 expression brassica leaves were disrupted by liquid nitrogen and then suspended in the double volume of pbs. total rna extraction and cdna synthesis were performed from 50 mg of lised leaves samples, modifying the protocol of the taqman gene expression cells-to-ct tm kit (applied biosystems) as reported in podda et al. (2014). two μl of the cdna were used for sqrt-pcr amplification performed with gotaq green mastermix (promega, usa). the following standard thermal profile was used for all pcrs: 94°c for 3 min; 35 cycles of 90°c for 30 s, 59°c for 40 s, and 72°c for 40 s; 72°c for 7 min as final extension. pcr products were separated by 1% agarose gel electrophoresis and stained with gelred (biotium). cdna fragments were purified from gels and sequenced by bmr-genomics (italy). transcript levels were measured by scion image program and normalized with the constitutive reference actin gene (wang et al., 2016). three independent biological replicates were used. in order to verify the colonization level, the presence of p. indica transcription elongation factor pitef (butehorn et al., 2000) was tested in the p. indica leaves before or after fungal colonization. the following primers have been used: fig. 1 p. indica grown in liquid (left panel) or agar medium (right panel) (a); control (p. indica) or colonized (+ p. indica) black cabbage seedlings grown on 1.5% agar (b); pitef1 expression in -p.indica (left panel) or + p.indica plants (right panel) (c). del carratore et al. black cabbage colonization by p. indica 117 3. results and discussion p. indica growth and black cabbage colonization in order to evaluate the effects of p. indica colonization in black cabbage, the protocol used by dolatabadi and goltapeh, (2013) has been optimized for this kale variety. the fungus p. indica was grown in liquid medium (fig 1 a, left panel) and then transferred on agar complete medium (fig 1 a, right panel). then 7 week-old black cabbage seedlings were inoculated with p. indica mycelium in sterilized conditions in tubes. to validate the successfully colonization, the expression of pitef1 was assessed as the gene has been demonstrated to be useful for estimating the amount of active mycelium introduced in seedlings (butehorn et al., 2000). a strong expression of pitef1 was observed in leaves of colonized seedlings of black cabbage one and three weeks after fungal inoculation whereas no transcript was observed in not-colonized seedlings (fig. 1c). evaluation of black cabbage growth parameters the effects of colonization on growth parameters, biomass weights and shoot lengths, were measured in the inoculated plants in the first three weeks of growth. colonization by p. indica resulted in a rapid enhancement of about 30% of root and shoot biomass respect to the not colonized plants, just after one week from the inoculation (fig. 2 a, b). results are in agreement with those reported by dolatabadi and goltapeh, (2013) who found that p. indica and sebacina vermifera improved the growth of b. oleracea and other brassicaceae plants. satheesan et al., (2012) reported improved growth of centella asiatica after inoculation by p. indica. expression of tgg1, tgg2, pen2 in the leaves an increase of glucosinolates was found within ten days from germination in black cabbage. glucosinolates are secondary metabolites present in brassicaceae (yi et al., 2015). when plants are damaged due to insect herbivore attack, glucosinolates are hydrolyzed quickly with myrosinase (β-thioglucoside glucohydrolase or thioglucosidase) resulting in production of isothiocyanates, thiocyanates, nitriles and others compounds (bones and rossiter, 2006; hopkins et al., 2009). no information are available in the literature on the modulation of glucosinolate by products in the leaves during fungal colonization. fig. 2 influence of p. indica on black cabbage growth parameters during three weeks from the inoculation. picture of the plants after one week from the inoculation (a); biomass weight (mg) or shoot length (mm), at 0, 1 and 3 weeks after p. indica colonization (b).values are the mean of ten independent experiments for each condition (control or inoculated) ±sd. asterisk means significant difference at p≤0.05. f 5’ rew 5’ pitef1 attgcctgcaagttctccga cttcgtaaccttgccaccct tgg1 tcttaacgtgtgggatggct cctcctttgttcactcccct tgg2 agatgtgctggacgaactca cggcgtaacaggtaggatca pen2 gcatcatcatccaacagcgt acgccttgatcagttctcca actin aatggtaccggaatggtcaa agttgctcacaacaccatgc adv. hort. sci., 2017 31(2): 115-119 118 thus, in this work the expression of tgg1 and tgg2, which encode myrosinases hydrolysing aliphatic glucosinolates or pen2, encoding the enzymes hydrolysing indole glucosinolates was evaluated. intriguingly, a decrease of tgg1, tgg2 and pen2 expression was observed at three weeks of colonization (fig. 3). similar results have been reported by witzel et al. (2015) in arabidopsis thaliana infected by verticillium longisporum. as the glucosinolatemyrosinase system is relevant for defence against insect-herbivore (winde and wittstock, 2011), the decrease of the expression of the genes relative to this pathway, is of particular importance and should be further investigated for extensive periods. although p. indica colonization improves plant growth and resistance to abiotic stress (sun et al., 2010), a negative impact on defence response pathway might increase the susceptibility of colonized black cabbage against biotic stress. acknowledgements this study was supported by funds of the regione toscana ‘‘praf 2012–2015 misura 1.2 e’’ program (call “agrifood”, project volatosca)”. references appleman n., lindegren s., leahy k., 2008 a16: food and wine. random house, ten speed press, pp. 288. banhara a., ding y., kühner r., zuccaro a., parniske m., 2015 colonization of root cells and plant growth promotion by piriformospora indica occurs 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matured on the tree develop most of the above characteristics but such fruits have been associated with environmental hazards such as insect attack, sunburn, wind abrasion, sap spurt at harvest, damage due to hail and pre-harvest disease infection (oosthuyse, 1997). bagging may be useful as a means of preventing such problems in mango and can reduce disease and physical damage as well as improve colour at harvest in a number of fruits (bentley and viveros, 1992; byers and carbaugh, 1995). this approach has been tested to produce high quality unblemished mango fruits in queensland (hofman et al., 1997), south africa (oosthuyse, 1997) and the philippines (bugante et al., 1997). however, different bagging materials behave in different ways, as has been reported by ann et al. (1998). according to these authors fruit bagging at an early stage was the most effective method to control mango anthracnose disease. the use of different bagging materials did not affect disease controls, although it did affect fruit maturation, colour and °brix. while some benefits (e.g. reduction in physical damage) could be expected, there may also be negative effects on quality as different days of bagging can delay the development of ripening characteristics of fruits. the present paper describes the results of experiments on bagging in fruit quality and mineral element content of mango fruit cv. himsagar, an important commercial cultivar of west bengal, india. influence of bagging on fruit quality and mineral composition of himsagar mango grown in new alluvial zones of west bengal p. dutta*, d. majumder** * department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 west bengal, india. ** department of agricultural statistics, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 west bengal, india. key words: fruit bagging, fruit quality mineral composition, himsagar mango. abstract: the effect of polybagging of mango (mangifera indica l.) fruits was evaluated at different stages of fruit development (35, 45, 55 and 65 days after fruit set). fruits were harvested at different stages of maturity (75, 85 and 90 days after fruit set) and allowed to ripen at room temperature (34-36°c, rh 85-90%). the use of bagging at different stages of fruit development improved the appearance of fruit, fruit weight and size through other effects such as increased relative humidity and a consequently reduced fruit water loss. the maturity of fruits, at all stages of fruit harvest, was delayed with increasing bagging duration. early bagging of fruit (35 days after fruit set) delayed the development of ripening characteristics in comparison to delayed bagging and unbagged control fruit, which ripened earliest. this was clearly evident from the carotene content in the mango flesh, at the different stages of harvest and of ripening fruit, which was the result of higher temperature inside the bags. in bagged fruits usually day/night temperature fluctuations were reduced and there was a cut off in the temperature curve inside the bag. the total soluble solids and sugar content were higher and titratable acid content was always less in unbagged fruit at all stages of fruit harvest and fruit ripening. mineral elements were also affected by the number of days of bagging. the reduced ca concentration in long-duration bagging (early bagging) might be due to increased rh around the fruits. fruits bagged for 55 days recorded an increased content of n, p, zn, mn and fe while fruit calcium concentration was reduced by bagging for 55 days. anthracnose and stemend-rot (ser) caused by colletotrichum and diplodia spp. respectively were reduced by bagging in both years through a reduction in contact between disease propagules and fruits. these results indicate that bagging can improve fruit quality by reducing disease, lead to a better appearance of fruit and increase fruit weight and size. adv. hort. sci., 2012 26(3-4): 158-162 received for publication 16 january 2012 accepted for publication 24 september 2012 159 2. materials and methods the experiment was conducted at the mondouri horticultural research station and the post harvest technology laboratory of the faculty of horticulture, bidhan chandra krishi viswavidyalay, nadia, west bengal, india during 2006-2008. study trees of mango cv. himsager were selected from the orchard at mondouri (23.5° n latitude and 89° e longitude). the trees were healthy, uniform in size and more than 15 years old. about 250 developing mangoes were tagged and bagged with transparent polyethylene bags at different stages of fruit development: 35, 45, 55 and 65 days after fruit set and a control without any bagging. then on each sampling date (75, 85, 90 days after fruit set), 10 mangoes were harvested at random from each bagging-date lot of polyethylene-bagged fruits. the fruits were washed and dried at ambient temperature (32°c±1°c), and kept in the laboratory for ripening. ripe fruits were analyzed for physico-chemical characteristics; bagged fruits were analysed 55 days after fruit set for mineral elements and disease incidence. physico-chemical analysis a. physical characters. weight, length and diameter. the weight of ripe fruit was determined using a digital balance and expressed in grams. length of fruit was measured from the base to the apex and diameter at its widest part, near the shoulder of the fruit, with the help of a vernier caliper. both were expressed in centimeters. b. bio-chemical constituents. total soluble sugars, total soluble solids (tss), titratable acidity. total soluble sugar content was analysed using fehlings’ a and b solution, according to the methods of the aoac (1996) and expressed as percentage. in this method, for inversion at room temperature an aliquot of clarified and diluted solution was transferred to a flask. 10 ml of hcl (1:1) was added and allowed to stand at room temperature for 24 h. the solution was then neutralized with concentrated naoh solution and made to volume. an aliquot was taken and the total soluble sugars were determined as invert sugars using fehling’s a and b solution. in determining reducing sugar, acid hydrolysis was not done. total soluble solids (tss) content of juice was determined using a hand refractometer and expressed as °brix at 20°c. titratable acidity (% malic acid) was estimated by titrating fruit juice (5 ml) to ph 8 against 0.1 m naoh using phenolphthale as an indicator. total carotenoids total carotenoids were estimated by the method of ranganna (1977). five grams of fresh sample were taken, a few crystals of anhydrous sodium sulphate were added, and then crushed in 10 ml acetone with the help of a pestle and mortar. the supernatant was decanted into a beaker. the process was repeated twice or thrice and the combined supernatant was transferred to a separating funnel out on standing. petroleum ether (10 to 15 ml) was added in the separating funnel and rinsed; the pigment was then transferred to the petroleum ether phase by diluting the acetone with water or water containing 5% sodium sulphate. the extraction of the acetone phase with a small volume of petroleum ether was repeated, if necessary, until no more colour was extracted. the lower layer was discarded and the upper layer was collected in a 100 ml volumetric flask. the petroleum ether extract was filtered through anhydrous na2 so 4 and the volume was made up to 100 ml with petroleum ether. the optical density was recorded at 452 nm using petroleum ether as blank containing 3 ml acetone per 100 ml and expressed as µg 100 g-1 pulp. as carotenoids are light sensitive, all steps were performed under subdued light. flesh minerals dried flesh samples from the ripe fruit (unbagged and bagged 55 days after fruit set) were collected and ground with a mortar and pestle, further dried at 70°c for 2 days, then finely ground in a shatter box. a 0.3-0.5 g sub-sample was digested in 15 ml nitric acid/perchloric acid and analysed by atomic absorption spectroscopy against standards prepared in the same matrix. nitrogen was determined using kjeldahl digestion. disease incidence disease incidence was measured 10 days after harvest. anthracnose lesions (caused by colletotrichum spp.) on the side of the fruit, and stem end rot lesions (ser : caused by diplodia spp.) at the stem end of the fruit were rated for incidence (percentage of the fruit affected). statistical analyses data regarding observed characters were statistically analysed by complete randomised design and test of significance was carried out following the method described by panse and sukhatme (1967). 3. results and discussion fruit weight, length and diameter fruit bagging with polyethylene bags significantly increased the weight of fruit as compared with the control at all stages of fruit harvest (75, 85 and 90 days after fruit set). early bagging (35 days after fruit set) proved most effective in increasing fruit weight compared to those bagged later (table 1). it is evident from table 2 that fruit length table 1 effect of bagging on fruit weight (g) at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 303.50 322.75 345.00 45 287.50 296.75 319.60 55 244.75 291.20 310.17 65 229.72 259.86 301.87 control 205.25 229.50 276.02 sem ± 21.22 28.52 24.12 lsd (p= 0.05) 63.62 85.11 72.42 160 increased along with the increase in duration of bagging and also with delayed harvesting. fruits bagged early (35 and 45 days after fruit set) were almost always longer than those bagged later (55 days after fruit set). however, the increase in fruit length due to polybagging was not significant for fruits sampled 85 days after fruit set. like fruit length, the diameter of fruit also increased due to polybagging; the increase in diameter was significant at all stages of fruit harvest (table 3). total soluble solids (tss), total soluble sugar and titratable acidity (ta) total soluble solids and sugar contents of fruit were significantly affected by treatment with polybagging (tables 4 and 5). tss content of ripened fruit was always higher in those bagged later or not bagged at all. at fruit harvest 90 days after fruit set, tss content was higher in fruits that were bagged later (at or after 55 days of fruit set) than the rest of bagged or not bagged fruits, however tss content was greatest in fruits bagged earliest (35 day after fruit set). as tss, total sugar content of fruits was always greater than those that were bagged later (65 days after fruit set) or those that were not bagged at all (control). fruit acidity decreased with a decrease in bagging period (table 6). however titratable acid content of ripened fruits did not show any significant variations due to bagging at different stages of fruit development. carotenoid contents analysis at the eating ripe stage showed that the carotenoid content of fruit increased as harvesting was delayed from 75 to 90 days (table 7). early bagged fruits usually developed less carotenoid upon ripening as compared to those bagged later or to the control. the unbagged fruits table 2 effect of fruit bagging with polyethylene on length (cm) of fruit at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 10.75 12.75 13.91 45 10.30 11.50 12.28 55 10.06 11.35 11.94 65 9.95 11.12 11.82 control 9.40 10.92 11.32 sem ± 0.425 0.414 0.380 lsd (p = 0.05) 1.241 ns 1.144 table 3 effect of fruit bagging with polyethylene on diameter (cm) of fruit at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 7.77 8.00 8.43 45 7.33 7.71 8.25 55 7.19 7.70 7.95 65 7.05 7.44 7.94 control 6.91 7.25 7.68 sem ± 0.172 0.210 0.211 lsd (p = 0.05) 0.521 0.591 0.610 table 4 effect of fruit bagging on total soluble solids (°brix) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 45 9.90 12.20 15.40 55 10.05 13.50 17.30 65 10.40 14.30 17.85 control 11.90 15.30 16.25 sem ± 0.161 0.072 0.051 lsd (p = 0.05) 0.483 0.219 0.153 table 5 effect of fruit bagging on total soluble sugar (% of fresh weight) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 4.42 6.85 11.81 45 4.77 6.85 12.57 55 5.02 7.52 13.69 65 5.50 7.81 13.82 control 6.20 7.97 12.83 sem ± 0.129 0.061 0.051 lsd (p = 0.05) 0.391 0.180 0.158 table 6 effect of fruit bagging on titratable acid (% malic acid) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 0.250 0.210 0.200 45 0.240 0.220 0.190 55 0.210 0.190 0.180 65 0.180 0.180 0.150 control 0.170 0.160 0.140 sem ± 0.012 0.014 0.015 lsd (p = 0.05) ns ns ns table 7 effect of fruit bagging on carotene (µg/100 g pulp) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 2247.50 4345.50 7782.50 45 2380.00 4773.50 8276.00 55 3565.00 6566.00 8958.50 65 3955.00 7318.50 9168.00 control 4354.00 8485.50 9768.00 sem ± 12.95 17.25 19.00 lsd (p = 0.05) 36.79 52.12 57.12 161 (control) at all stages of harvest developed maximum content of carotene while early bagged fruits (35 days after fruit set) contained minimum carotene upon ripening at all different stages. fruit minerals and disease incidence mineral element contents of fruits varied due to different days of bagging. table 8 shows that n, p, zn, mn and fe content of fruits were higher in bagged fruit (55 days bagged) while k and ca contents were higher in control fruit (0 day bagged) as compared to 55 days of bagging. bagging did not significantly reduce the incidence of anthracnose and stem-rot (ser) (table 9) of ripe fruit. reductions of only 5.5 and 6.0% of anthracnose and stemend-rot, respectively, were noted in fruits bagged 55 days after fruit set. table 8 effect of fruit bagging on mineral elements contents of ripe mango fruit mineral elements days bagged significance 0 55 n (%) dry weight 0.57 0.74 * p (%) dry weight 0.12 0.14 * k (%) dry weight 0.71 0.64 * ca (%) dry weight 0.07 0.03 * zn (ppm) 22.50 23.33 ns mn (ppm) 11.75 12.18 ns fe (ppm) 49.00 49.75 ns assessment were made at eating soft stage. ns indicates p>0.1. * represent effects significant at p<0.05. the results of the present investigation clearly demonstrate the benefits of polybagging on the development of mango fruit and fruit quality. bagging of fruits improved fruit weight and size of litchi (tyas et al., 1998), banana (johns and scott, 1989) and pomegranate (hussein et al. 1994). bagging can affect fruit size through other effects such as increased relative humidity and therefore reduced fruit water loss (tombesi et al., 1993). according to hofman et al. (1997), in the present study fruit mass and size may be due to the use of polybag instead of paper bag. the polybagging of fruits delayed the development of ripening characteristics of fruits. however, the extent of the fruit surface coloured yellow was greater with polybagged fruits, which was due to maintenance of a higher temperature inside the bags. in bagged fruits usually day/night temperature fluctuations were reduced, hence the range of minimum temperatures was between 18 and 27°c, which is very close to the optimal temperature range (24-30°c) for the development of himsagar mango (mukherjee, 1953; whiley et al., 1989). the total soluble solids and total sugar content of ripe mango were almost always greater in control fruits and those with a shorter bagging period as compared to early-bagged fruits. titratable acid content of ripe fruits was greater in early-bagged fruits and acid content of ripe fruits declined as the period of bagging was reduced; minimum acid levels were noted in the control. this is only due to bagging which delayed the development of ripening characteristics of fruits. similarly, the carotene content in the pulp of ripe mango fruit was at maximum in control and minimum in early-bagged fruits. this is mainly due to fact that polybagging of mango fruits delayed the development of ripening characteristics and therefore there was less development of carotene in fruits that were bagged early in comparison to control. these results are in close conformity with the previous findings of singha (2002). however, hofman (1997) opined that fruit quality of mango was not affected by bagging. fruit mineral (n, p, k and ca) contents were significantly influenced by bagging. bagged fruit in the 55-day group had a amount of calcium in comparison to unbagged fruits. calcium is transported mainly in the transpiration stream (grange and hand, 1987), thus tissues with greater transpiration generally have higher tissue ca concentrations (witney et al., 1990 b). increased rh around the fruit can reduce fruit ca accumulation (grange and hand, 1987, combrink et al., 1995) and bagging of apple fruit has been associated with reducing fruit ca concentrations and increasing bitter pit incidence (witney et al., 1991). similar effects were observed in mangoes bagged for 55 days, although the higher fruit ca concentration with longer bagging times may suggest a capacity of the fruit to import ca by a mechanism other than transpiration. longer bagging times may have caused changes in the surface of fruits that allowed higher transpiration rates and thereby the higher ca accumulation noted with increased bagging times as described by hofman et al. (1997). reduction in fruit disease as a consequence of bagging in a number of different fruits has been noted by kitagawa et al. (1992). in mango, colletotrichum infection occurs during fruit development and remains quiescent until fruit ripening (dodd and jeffries, 1989). the reduction in anthracnose incidence with bagging (55 days) could be partly due to a reduction in contact between disease propagules and fruit as mentioned by hofman et al. (1997). in conclusion, bagging of mango can have important commercial benefits as a means to prevent problems like table 9 effect of bagging on fruit disease incidence (percent of fruit affected) parameter incidence (%) days bagged anthracnose stem-end-rot 0 82.5 77.0 55 56.5 50.5 significance ns ns all assessment were made at 10 days after harvest. ns indicates p>0.1. 162 insect attack, sunburn, sap spurt at harvest and damage due to small hail storms by reducing physical damage of fruit, as well as delaying the development of ripening characteristics of fruits. acknowledgements we thank the university grant commission, india for financial support through minor research project. references ann p.j., lu l.s., chuang t.y., kao c.w., 1998 effect of fruit bagging and mulching on control of mango fruit anthracnose disease. plant pathology bull., 7: 19-26. aoac, 1996 aoac methods. association of official analytical chemists, washington, dc., usa. bentley w.j., viveros m., 1992 brown bagging granny smith apples trees stops coding moth damage. california agriculture, 46 : 30-32. bugante r.d. jr., lizada m.c.c., de ramos m.b., 1997 disease control in philippine ‘carabao’ mango with preharvest bagging and post harvest hot water treatment. acta horticulturae, 455: 797-804. byers r.e., carbaugh d.h., 1995 chemical, cultural and physiological factors influencing ‘stayman’ fruit cracking. bulletin virginia agricultural experiment station, 95: 1-33. combrink n.j.j., jacobs g., maree p.c.j., maras e.m., 1995 effect of relative humidity during fruit development of muskmelon fruit quality. journal of south africa society for horticultural science, 5 : 43-46. dodd j.c., jeffries p., 1989 management strategies to control latent infection in tropical fruit. aspects applied biology, 20: 49-56. grange r.i., hand d.w., 1987 a review of the effects of atmospheric humidity on the growth of horticultural crops. journal of horticultural science, 62(2): 125-134. hofman p.j., smith l.g., joyce d.c., johnson g.i., meiburg g.f., 1997 bagging of mango (mangifera indica cv. keitt) fruit influence fruit quality and mineral composition. postharvest biology and technology, 12: 83-91. hussein a.a., abd-el-rahaman a.g., ahmed r.b., 1994 effectiveness of fruit bagging on yield and fruit quality of pomegranate (punica granatum l.). annals of agricultural science, 32(4): 943-957. johns g.g., scott k. j. 1989 delayed harvesting of banana with ‘sealed’ covers on bunches. effect on fruit yield and quality. australian journal of experimental agriculture, 29: 727-733. kitagawa h., manabe k., esguerra e.b., 1992 bagging of fruit on the trees to control disease. acta horticulturae, 321: 870-875. mukherjee s.k., 1953 the mango its botany, cultivation, uses and future improvement, especially as observed in india. economic botany, 7(2): 130-162. oosthuyse s.a., 1997 a method of eliminating many of the problems which reduce the value of mangoes. south african mango grower’s association yearbook, 17: 41-44. panse v.g., sukhatme p.v., 1967 statistical method for agricultural workers. icar, new delhi. ranganna s., 1977 manual of analysis of fruit and vegetable products. tata mc graw hill publishing company limited, new delhi, 3rd edition. singha s., 2002 studies on the effect of bagging on fruit maturity and ripening behaviour of himsagar mango. m.sc. thesis submitted to bidhan chandra krishi viswavidyalaya, west bengal. tombesi a., antognozzi e., palliotti a., 1993 influence of light exposure on characteristics and storage life of kiwifruit. new zealand journal of crop and horticultural science, 21: 85-90. tyas j.a., hofman p.j., underhill s.j.r., bell k.l., 1998 fruit canopy position and panicle bagging affects yield and quality of “tai so” lychee. scientia horticulturae, 72: 203-213. whiley a.w., rasmussen t.s., saranah j.b., wolstenholem b.n., 1989 effect of temperature of growth, dry matter production and starch accumulation in the mango (mangifera indica l.) cultivars. journal of horticultural science, 64: 753-765. witney g.w., hofman p.j., wolstenholme b.n., 1990 b mineral distribution in avocado trees with reference to calcium cycling and fruit quality. scientia horticulturae, 44: 279-291. witney g.w., kushad m.m., barden j.a., 1991 induction of bitterpit in apple. j. of amer. soc. of hortic. sci., 47: 173-176. 79 1. introduction perilla (perilla frutescens l. britton) belongs to the lamiaceae family and is cultivated in china and japan (pandey and bhatt, 2008). the seeds yield oil and the leaves are used for medicine or garnish for fish (nitta et al., 2005; hossain et al., 2010). dried red perilla leaves are also used as ‘soyou’ in kampo medicine and it is one of the components of ‘saibokuto,’ which is used to treat bronchial asthma (homma et al., 1992; ueda et al., 2002). the seeds of perilla have been used for food for birds or humans, oil as a fuel or a cooking oil, and also the leaves are used as a potherb for medicine or food coloring and the foliage to produce an essential oil for flavoring (brenner, 1993). perilla has a variable chromosome complement (brenner, 1993); a haploid chromosome count of fourteen plus zero to two beta chromosomes (vij and kashyap, 1976) and chromosome counts of both n=20 and 2n=38, and three distinguished chromosome sizes (yamane, 1950). as the traditional crop landraces are facing danger of complete extinction in some areas, some of perilla frutescens species are at the verge of extinction in the central himalaya (negi et al., 2011). usually perilla species are conserved in genebanks as plant genetic resources (arora, 1997). biotechnological tools are important to conserve the critical genotypes of medicinal plants like perilla species. however, previous reports of plant regeneration for perilla used seedlings segments such as the cotyledon and hypocotyl as material (hou and jia, 2005; zhang et al., 2005; hossain et al., 2010). moreover, there are few reports about effective clonal propagation of perilla. cryopreservation techniques are now used for plant germplasm storage at several institutes around the world (niino, 2006) and this method has become an important tool for the long-term storage of plant germplasm and of experimental materials that possess unique attributes, minimizing space and maintenance requirements without causing genetic alterations (sakai, 1997; matsumoto et al., 2013). recently, a vitrification protocol using the aluminum cryo-plate method (v-cryo-plate method) has been reported (sekizawa et al., 2011; yamamoto et al., 2011 a, b; 2012). niino et al. (2013) reported that the v-cryo-plate method has two main advantages: a user-friendly procedure and very high cooling and warming rates of treated samples. as a result, very high regrowth was obtained after cryopreservation of the tested materials (niino et al., 2013). in this study, we have developed effective clonal propagation by node culture and cryopreservation of in vitro grown shoot tips using the v-cryo-plate method for perilla. development of in vitro propagation by node culture and cryopreservation by v-cryo-plate method for perilla frutescens t. matsumoto*(1), k. yoshimatsu**, n. kawahara**, s.-i. yamamoto***, t. niino**** * faculty of life and environmental sciences, shimane university, matsue 690-1102, japan. ** tsukuba division, research center for medicinal plant resources, national institution of biomedical innovation, tsukuba 305-0843, japan. *** national institute of agrobiological sciences (nias), tsukuba 305-8602, japan. **** gene research center, university of tsukuba, tsukuba 305-8572, japan. key words: aluminum plate, cryopreservation, perilla, v-cryo-plate method. abstract: a clonal propagation method by node culture of perilla (perilla frutescens l. britton) was investigated. nodes were plated on solidified 1/2 ms medium with ba and optimum shoot elongation and propagation was obtained at ba 0.05 and 0.1 mg l-1. cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown perilla shoot tips. excised shoot tips from nodes were precultured on 1/2 ms medium with 0.3 m sucrose and embedded on an aluminum cryo-plate with alginate gel. the cryo-plate with shoot tips was osmo-protected with ls solution and dehydrated in pvs2 for 20 min at 25°c prior to immersion into liquid nitrogen. the recovery growth after cryopreservation was found to be about 80%. this new v-cryo-plate method has many advantages and may facilitate the cryo-storage of other medicinal plants. adv. hort. sci., 2014 28(2): 79-83 (1) corresponding author: tmatsumoto@life.shimane-u.ac.jp received for publication 31 march 2014 accepted for publication 18 june 2014 80 2. materials and methods in vitro propagation by node culture the material, seeds of perilla frutescens l. britton, was obtained from tsukuba division, research center for medicinal plant resources, national institution of biomedical innovation (tsukuba, japan). the seeds were surface sterilized in 70% ethyl alcohol for 30 sec then in 1% naocl for 15 min. after rinsing three times in sterile distilled water, they were placed on hormone-free halfstrength solidified ms medium (murashige and skoog, 1962; termed 1/2 ms) and incubated at 25°c with 16 h light/8 h dark photoperiod under a light intensity of 50 µmol m-2 s-1 for germination. after one month, the axillary buds were placed on solidified 1/2 ms medium with 0, 0.01, 0.05, 0.1 and 0.5 mg l-1 ba and incubated. the shoot length, number of leaves, number of shoots, number of roots and longest root length were measured. ten buds were used for each treatment with three replicates. cryopreservation by v-cryo-plate method axillary shoot tips (1 mm size) were excised from in vitro grown plants of perilla and used for cryopreservation by vcryo-plate method (yamamoto et al., 2011 a, b) with some modification. figures 1 and 2 show the schematic diagram of the aluminum cryo-plate used and the v-cryo-plate procedure. cryo-plates used in this study were obtained from the national institute of agrobiological sciences (tsukuba, japan). the following steps were performed: 1) excised shoot tips were precultured on solidified 1/2 ms medium with 0.3 m sucrose for 1 day at 25°c to induce the osmo-protection. 2) an aluminum cryo-plate (fig. 3) was placed in a petri-dish and 2.0-2.5 µl of 2% (w/w) na-alginate solution with 0.4 m sucrose in 1/2 ms medium was poured in a well. 3) the precultured shoot tips were positioned in each well and 100 mm cacl 2 solution with 0.4 m sucrose in 1/2 ms were added to the aluminum plate for 15 min for polymerization (fig. 3). 4) after removing cacl 2 solution, the cryo-plate with shoot tips was treated with ls solution (2 m glycerol + 0.6 m sucrose) (matsumoto et al., 1994; yamamoto et al., 2011 b) in a petri dish (8 cm in diameter) for 20 min at 25°c for osmo-protection. fig. 1 aluminum cryo-plate with embedded shoot tips. size: 7 mm × 37 mm × 0.5 mm with ten wells (diameter 1.5 mm. depth 0.75 mm). fig. 2 procedure of v-cryo-plate for cryopreservation of perilla shoot tips. fig. 3 shoot formation by node culture after 2 months 1/2ms + ba 0.1 mg l-1. bar = 10 mm. 81 5) after ls treatment, the cryo-plate with shoot tips was dehydrated with pvs2 solution (sakai et al., 1990) for 0 to 40 min at 0 c and for 0 to 20 min at 25°c. 6) the cryo-plate was then transferred to an uncapped 2 ml cryotube held on a cryo-cane and directly plunged into liquid nitrogen (ln) for at least 30 min. 7) for plant regeneration, the cryo-plate with shoot tips in ln was transferred to 1.2 m sucrose solution with 1/2 ms in a petri dish (8 cm in diameter) for 15 min at 25°c for rapid warming and unloading. shoot tips were then plated on solidified 1/2 ms medium with 3% sucrose and incubated at 25°c under standard conditions. three replicates of 10 shoot tips were tested in each experiment. statistical analyses were performed using tukey’s test and significant differences (p<0.05) were determined. 3. result and discussions in vitro propagation by node culture after incubation for 30 days on 1/2ms medium with different concentrations of ba, all nodes of perilla formed shoots without callus formation. the optimum shoot elongation and propagation was obtained at ba 0.05 and 0.1 mg l-1 (table 1). in previous reports, plant regeneration of perilla was obtained from hypocotyl segment including apical bud (hossain et al., 2010), cotyledon, and hypocotyl (zhang et al., 2005). the shoot recovery rate of this node culture (100%) is higher than that of the previous reports (65 to 91%). moreover, regenerated plants from cotyledon and hypocotyl segments are not genetically the same and the propagation systems are not suitable for clonal propagation. in this experiment, 1.6 shoots with 5.8 leaves (average of ba 0.05 and 0.1 mg l-1) were obtained from one node after 30 days of incubation. nodes were located basal respect to each petiole, and we found that one node produced 9.28 nodes (5.8 × 1.6) after 30 days incubation. this propagation efficiency (9.28) is considered to be as high as clonal propagation. furthermore, no abnormalities were observed in the regenerated shoots (fig. 3) indicating that this method can be considered a suitable propagation method for the material of cryopreservation. cryopreservation by v-cryo-plate method in our previous reports of cryopreservation using vitrification (matsumoto et al., 1994; 1995), we demonstrated that the osmo-protection treatments (preculture of high sucrose medium, ls treatment of 2 m glycerol + 0.4 m sucrose solution) were necessary to produce high regrowth before pvs2 dehydration for shoot tips of most plant species. the v-cryo-plate procedure was carried out according to yamamoto et al. (2011 a, b) with these two osmo-protection treatments. figure 4 shows the regrowth rates with different exposure times to pvs2 at 0 and 25°c. the highest regrowth rates obtained were about 55% for 30 min at 0°c and about 70% for 15 min at 25°c. in the vitrification protocol, direct exposure of less tolerant cells and meristems to highly concentrated pvs2 at 25°c was found to possibly be harmful due to osmotic stress or chemical toxicity (matsumoto and sakai, 2003). in this experiment, the regrowth rate at 0°c was 15% lower than that at 25°c. the reason of this result was not clear, but might be related to problems associated with sensitivity to pvs2 and/or dehydration. the high regrowth rate after cryopreservation by v-cryoplate method is due to rapid cooling (4,000-5,000°c min-1) and warming (3,000-4,500°c min-1) (niino et al., 2013). in vitrification-based procedures, damage can be caused by chemical toxicity of pvs2 and osmotic stress by excessive duration of pvs2 treatment (engelmann, 1997; sakai et al., 2008). our results suggest that this high regrowth rate with exposure to pvs2 at 25°c may be due to the rapid cooling and warming. for practical use for cryopreservation, the time for the cryogenic procedure should be short and the regrowth rate after cooling should be high (at least 70%). it is worth noting that no abnormalities were found in shoots developed from table 1 effect of ba concentration on shoot regrowth from nodes in perilla ba (mg l-1) shoot length (mm) no of leaves no of shoots no of roots longest root (mm) regrowth (%) 0 6.7 ab 5.4 a 1.0 b 1.9 b 26.5 ab 100 0.01 10.1 a 5.1 ab 1.0 b 1.4 ab 30.7 a 100 0.05 11.3 a 6.0 a 1.5 a 2.2 b 16.3 ab 100 0.1 8.2 a 5.6 a 1.7 a 1.3 ab 10.8 ab 100 0.5 5.1 b 3.7 b 1.0 b 0.7 a 6.2 b 100 fig. 4 effect of different exposure times and temperatures of pvs2 solution on regrowth of cryopreserved perilla shoot tips using v-cryo-pate method. exposure time to pvs2 (min) re gr ow th ij 82 cryopreserved shoot tips using v-cryo-plate method treated at 25°c. thus, the pvs2 dehydration at 25°c in this v-cryo-plate method is suitable for use with perilla. in conclusion, an efficient clonal propagation method for perilla using node culture was successfully established. in addition, we demonstrated that the v-cryoplate procedure led to about 70% regrowth and this procedure was thus a very efficient and practical method for cryopreservation of perilla germplasm. this protocol appears promising for cryopreservation of other medicinal plants, as well as other plant species and/or cultivars of horticultural interest after marginal modifications to the procedure. acknowledgements this study was supported by the research grants from genebank, national institute of agrobiological sciences (nias) and health labor sciences research grant, japan. references arora p.k., 1997 proc. indian natn. sci. acad. b63(3): 175186. brenner d.m., 1993 perilla: botany, uses and genetic resources, pp. 322-328. in: janick j., and j.e. simon (eds.) new crops. john wiley & sons ltd, new york, usa, pp. 710. engelmann f., 1997 in vitro conservation methods, pp. 119-162. in: ford-lloyd b.v., j.h. newbury, and j.a. callow (eds.) biotechnology and plant genetic resources: conservation and use. cab international, wallingford, uk, pp. 320. homma m., oka k., yamada t., niitsuma t., ihto h., takahashi n., 1992 a strategy for discovering biologically active compounds with high probability in traditional chinese herb remedies: an application of saiboku-to in bronchial asthma. anal. biochem., 202: 179-187. hossain h.m.m.t., kim y.h., lee y.s., 2010 the apical bud as a novel explant for high-frequency in vitro plantlet regeneration of perilla frutescens l. britton. plant biotechnol. rep., 4: 229-235. hou s.w., jia j.f., 2005 in vitro regeneration of perilla frutescens from hypocotyl and cotyledon explants. biologia plantarum, 49: 129-132. matsumoto t., akihiro t., maki s., mochida k., kitagawa m., tanaka d., yamamoto s., niino t., 2013 genetic stability assessment of wasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis. cryoletters, 34(2): 128-136. matsumoto t., sakai a., 2003 cryopreservation of axillary shoot tips of in vitro-grown grape (vitis) by a two-step vitrification protocol. euphytica, 131: 299-304. matsumoto t., sakai a., yamada k., 1994 cryopreservation of in vitro-grown apical meristems of wasabi (wasbia japonica) by vitrification and subsequent high plant regeneration. plant cell reports, 13: 442-446. matsumoto t., sakai a., yamada k., 1995 cryopreservation of in vitro-grown apical meristems of lily by vitrification. plant cell, tissue and organ culture, 41: 237241. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-487. negi v.s., rawat l.s., phondani p.c., chandra a., 2011 perilla frutescens in transition: a medicinal and oil yielding plant need instant conservation, a case study from central himalaya, india. environ. we int. j. sci. tech., 6: 193-200. niino t., 2006 developments in plant genetic resources cryopreservation technologies, pp. 197-217. in: kang j.-h. (ed.) effective genebank management in apec member economies. niab, korea. niino t., yamamoto s., fukui k., castillo martinez c.r., arizaga m.v., matsumoto t., engelmann f., 2013 dehydration improves cryopreservation of mat rush (juncus decipiens nakai) basal stem buds on cryoplates cryoletters, 34: 549-560. nitta m., lee j.k., kang c.w., katsuta m., yasumoto s., liu d., nagamine t., ohnishi o., 2005 the distribution of perilla species. genet. resour. crop evol., 52: 797-804. pandey a., bhatt k.c., 2008 diversity distribution and collection of genetic resources of cultivated and weedy type in perilla frutescens (l.) britton var. frutescens and their uses in indian himalaya. genet. resour. crop evol., 55: 883-892. sakai a., 1997 potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems, pp. 53-66. in: razdan m.k., and e.c. cocking (eds.) conservation of plant genetic resources in vitro. science publishers, enfield, new hampshire, usa. sakai a., hirai d., niino t., 2008 development of pvsbased vitrification and encapsulation-vitrification protocols, pp. 33-58. in: reed b.m. (ed.) plant cryopreservation: a practical guide. springer, new york, pp. 513. sakai a., kobayashi s., oiyama i., 1990 cryopreservation of nucellar cells of navel orange (citrus sinensis osb. var. brasiliensis tanaka) by vitrification. plant cell reports, 9: 30-33. sekizawa k., yamamoto s., rafique t., fukui k., niino t., 2011 cryopreservation of in vitro-grown shoot tips of carnation (dianthus caryophyllus l.) by vitrification method using aluminium cryo-plates. plant biotechnology, 28: 401-405. ueda h., yamazaki c., yamazaki m., 2002 luteolin as an anti-inflammatory and anti-allergic constituent of perilla frutescens. biol. pharm. bull., 25: 1197-1202. vij s.p., kashyap s.k., 1976 cytological studies in some north indian labiatae. cytologia, 41: 713-719 yamamoto s., fukui k., rafique t., khan n.i., castillo martinez c.r., sekizawa k., matsumoto t., niino t., 2011 a cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminium cryo-plates. plant genetic resources: characterization and utilization, 10: 14-19. 83 yamamoto s., rafique t., fukui k., sekizawa k., niino t., 2012 v-cryo-plate procedure as an effective protocol for cryobanks: case study of mint cryopreservation. cryoletters, 33: 12-23. yamamoto s., rafique t., priyantha w.s., fukui k., matsumoto t., niino t., 2011 b development of a cryopreservation procedure using aluminium cryo-plates. cryoletters, 32: 256-265. yamane y., 1950 cytogenetic studies on the genus perilla and coleus i. chromosome numbers. jpn. genet., 25: 220. zhang t., wang x.y., cao z.y., 2005 plant regeneration in vitro directly from cotyledon and hypocotyl explants of perilla frutescens and their morphological aspects. biologia plantarum, 49: 423-426. anthocyanins. biosynthesis, functions, and applications. gould k., k. davies, and c. winefield (eds.) springer science+business media, llc, new york, ny, usa, 2009. pp. i-xviii + 329. isbn 978-0-387-77334-6. usd 149.00. gbp 79.00. € 99.95. plant colours are attracting not only because they provide spectacular views of nature, but also for their functional roles. it is well known that nothing in nature is made by chance. anthocyanins, molecules of plant secondary metabolism, are those natural pigments giving predominant contributions to the painted word. their appearance on external portions of plant organs seems to be a kind of language to communicate with the animal kingdom, with the aim of attracting or repelling in order to facilitate plant reproduction and diffusion or plant defence, respectively. learning about the beneficial effects of anthocyanins in plants, humans have discovered how beneficial anthocyanins can be to their health as well. considerable literature in plant sciences is devoted to understanding anthocyanin-involved mechanisms, however much remains as yet undisclosed. the book anthocyanins. biosynthesis, functions, and applications edited by kevin gould, kevin davies and chris winefield is certainly a useful reference for all researchers involved in the many multidisciplinary studies of these natural pigments and represents a valuable collection of research results and needed future work for this rapidly expanding field. the different aspects of anthocyanin properties covered by this book include antioxidant activity and photoprotection, the role in plant defence mechanisms, the function in fruits and flowers. extensive chapters are dedicated to plant cell cultures for the biosynthesis of anthocyanins and their biotransformation by microorganisms. in addition, the biochemical pathways of reactions occurring in vivo for the stabilization of anthocyanins and the characterization of new anthocyanin-derived compounds as food colorants are also considered. finally, the last chapter deals with the phytochemical role of anthocyanins to promote human health. being an outsider to the plant science academic world, but working in spectroscopic monitoring of vegetation for about 20 years, i thoroughly enjoyed reading this volume and warmly recommend finding a place for this book on everyone’s desk. giovanni agati la civiltà delle acque tra medioevo e rinascimento. the culture of water between the middle ages and the renaissance. calzona a. and d. lamberin (eds.). collana ingenium, vol. 14. edizioni leo s. olschki, firenze, 2010. volume i and ii pp. xviii + 718, 11 figures, and 83 plates, 22 of which in colour. isbn 978 88 222 5969 1. € 78.00. these two volumes contain the proceedings of the international meeting of the same name held in mantua 1-4 october 2008 and include presentations by 37 authors of national and international level. the works address the literary, philosophic, historic, political-economic and social, historical-artistic and architectural, and scientific and technical-engineering issues relative to the management and significance of freshwater in italy from the middle ages to the renaissance. the works are grouped into five sections: literary and imaginary; political and economic management; art, architecture, landscape and territory; engineering, infrastructures, science and criticism; gardens, parties and spectacles. a final section deals with the magical, thermal, and nutritive aspects of the waters. the overall complex provides a true treatise on the subject matter, making these volumes valuable not only as a guide for those directly involved in the sector but they can also be a source for consultation for others who have personal interest in the topic. various illustrations and tables enrich each paper and there are numerous bibliographic citations, offering the reader the possibility to go into greater depth. this subject is of considerable historical-cultural importance in italy and both the meticulous work and the efforts by the various contributors – some of whom present their life work – attest to just how much there is to know about fresh waters in our country. these two volumes are therefore of great interest and usefulness and are an important contribution to the literature available on the topic and will surely be helpful for not only technical workers and students but also an increasing number of enthusiasts. francesco ferrini 69 book reviews 70 san rossore nella storia: un paesaggio naturale e costruito. san rossore in history: both a natural and built landscape. panattoni r. collana giardini e paesaggio, vol. 27. edizioni leo s. olschki, firenze, 2010. pp. xxxii + 230. 2 figures and 32 plates in colours. isbn 978-88-222-6023-9. € 27.00. this work by rita panattoni was awarded the premio verbania, editoria e giardini in 2009. the title of the volume is, in itself, significant: the subject is not the history but rather the park of san rossore. this difference may not seem important but the title of any publication should define the whole, which in this case is the role played by this park in the history of the region. the book opens with an essay on the evolution of the botanical context by fabio garbari who underlines the extraordinary environmental value of this area which lies somewhere between being a garden and a landscape, and contains a total of 335 pages divided into five parts: the ancient san rossore; san rossore of the medici family; san rossore of the lorena family; san rossore of the house of savoy; the post-world war ii period, the 1950s. overall the work effectively illustrates the story of the transformation of the original selva dei tomboli pisani into the current san rossore estate. the earliest information about the san rossore area – covering an area of 4800 hectares between the serchio and arno rivers, composed of woodlands and pine forests, beaches, dunes, fields and marshlands and approximately 12 km of coast – dates back to about 1000 ad. up until the beginning of the 16th century this vast area was utilized for hunting and fishing or for the cultivation and harvesting of wood. it was the medici family who began the work of transforming the estate, first as renters (in the 16th century) and later as owners. subsequently ownership passed to the lorena family, who used the estate frequently and added improvements. in the mid-1800s, when tuscany became a part of the new kingdom of italy, the house of savoy came to san rossore and they occupied the estate for long periods of the year. after the war and the fall of the monarchy, san rossore became the property of the president of the republic. the volume, which contains an ample list of bibliographic references and numerous illustrations in black and white and in color, is an important addition to the vast literature on the evolution of historical landscapes not only from a scientific and technical profile but also from an historical and cultural point of view. this approach thus offers the reader meaning and knowledge about the important and multiple values of the park of san rossore which has a role in creating an image of tuscany. francesco ferrini paesaggio rurale: strumenti per la pianificazione. strategica. the rural landscape: tools for strategic planning. agnoletti m. edagricole, bologna, 2010. figures 220. pp. xvi + 348. isbn 978-88-506-5226-6. € 39.00. almost always legislation regarding a particular topic is distributed among various laws and decrees, which generally are based on or are handed down from previous periods and are more or less modified as needed. as a consequence, consultation of the regulations pertaining to a subject of interest can be often difficult and laborious and sometimes one has the feeling of not having investigated fully or having interpreted incorrectly, especially when the regulations are apparently contradictory (a not uncommon occurrence). fortunately, prof. agnoletti has prepared this volume which represents a clear and precise compendium of the piano nazionale di sviluppo rurale 2007-2013 (psn). the text is divided into six sections: 1) the evolution of the rural landscape from unification until today; 2) the evolution of forest landscapes from unification until today; 3) spatial characteristics and structural dynamics: the system of tuscan monitoring; 4) the current structure of the italian landscape; 5) the rural landscape in territorial politics; and 6) strategies and actions in order to add value. the appendix contains a section dedicated to analysis methods and management processes. the piano nazionale di sviluppo rurale 2007-2013 (psn) has included landscape amongst its strategic objectives, underlining its role in the new agricultural policy and the transformations which have occurred in terms of understanding the importance of this resource as well as that of the grower and the rural territory. this initiative is in line with the activities set out by the european landscape convention and with landscape planning promoted by the new codice dei beni culturali involving workers in the sector, public decision-makers and universities. the meaning of landscape and the perception of it has, in fact, changed over time. it is the essential element in defining a development model that is particularly suitable to the rural context, representing not only the cultural identity of our country but also economic and environmental values. however it is necessary to elaborate a cultural and scientific proposal that is appropriate for the rural setting, which takes into account the fact that landscape is the result of a combinationof social, economic and environmental factors in space and time where man plays a central, not marginal role. the current volume proposes and adds to the materials utilized to develop a picture and definition of the strategies for landscape by the ministry of agricultural, alimentary and forestry policy, illustrating approaches to analysis and providing additional material for integrated planning. the text includes illustrations, tables, graphs and an ample list of bibliographic references which offer the reader a key to acquiring greater depth of knowledge in the particular aspects of the subject matter. this work is of considerable scientific importance and the book can be of great interest for researchers and students in the field of environmental studies, as well as anyone who deals with landscape on a number of different levels. francesco ferrini 71 handbook of plant nutrition. barker a.v. and d.j. pilbeam (eds.) taylor and francis group. crc press, new york, ny, usa, 2007. pp. xvi + 614 + cd-rom. isbn 0-8247-5904-4. us$ 157.95. the increasing demand on the world food supply, coupled with concern over the use of chemical fertilizers, has determined an interest towards the practice of precision agriculture, has led to a better control and monitoring of plant nutrition to maximize the rate of growth, the yield of crops as well as their nutritional value. this handbook covers principles of plant nutrition from a historical standpoint to current knowledge of the requirements of crops for certain elements and the beneficial effects of others. the book consists of twenty chapters, each one dedicated to an essential macro or micronutrient or beneficial element. more in details, each chapter, written by eminent researchers from across the world, gives historical information on the specific nutrient, explaining why it is either essential or beneficial for the plants; moreover an explanation of how appearance and composition of plants can be used to assess nutritional status is given, as well as recommendations on fertilizers that can be applied to remedy nutritional deficiencies. this handbook, including a cd-rom containing more than 40 illustrations in full colour, can be considered of great value, and for this reason recommended, to growers, agricultural consultants, agronomist and plant scientist, providing a practical easy-to-use reference for determining, monitoring, improving the nutritional needs of plants. the graphical presentations of plant interactions with nutrients and beneficial elements, and the straight-forward explanations of how nutrient deficiencies arise are especially useful to those seeking knowledge of plant nutrition. francesco paolo nicese orticoltura mediterranea sostenibile. tesi r. pàtron editore, bologna, 2010. pp. 504. isbn 978-88-555-3062-0. € 42.00. the original work of romano tesi is inspired and basically derives from the interesting connection among some important facts. first of all, the mediterranean basin is the area of origin and varietal differentiation of many important vegetables, but it also allowed the selection of many others imported from asia and america during the colonization era. to this is added the fact that the countries bordering the mediterranean basin are the most important producers and consumers of those products. in the context of this mixture of cultures and traditions the well-known “mediterranean diet” has developed, which is considered worldwide as a bright example of the highest dietary level. “orticoltura mediterranea” is a new and updated book which gives particular attention to the changing market needs. thus, in addition to general and specific aspects of the traditional horticulture, the book focuses into the composition and nutritional properties of vegetables, together with a outlook on the principal aspects of integrated production and organic farming. the book is divided into two parts. the first part covers the horticulture in the mediterranean basin, which is deeply described in the following eight chapters: 1) l’orticoltura mediterranea (mediterranean horticulture); 2) classificazione degli ortaggi (classification of vegetables); 3) tipi di orticoltura e sostenibilità (sustainability of the different kind of horticulture); 4) i sistemi colturali (cultivation systems); 5) qualità dei prodotti orticoli (quality of horticultural products); 6) mezzi di protezione (protection devices); 7) sementi e vivaismo orticolo (seeds and horticultural nursery); 8) gestione dell’azoto e dell’acqua di irrigazione nel pieno campo (nitrogen and irrigation water management in the field horticulture). the second parts is dedicated to the monographic description of more than one hundred horticultural crops, with broad and updated insights. due to its modern setting, the rich collection of tables, the presence of numerous coloured or black and white images in which the scientific knowledge of the author can be easily recognized, the book may serve to a wide group of readers, from the student to the teacher, from the expert to the manager. enrico rinaldelli impaginato 47 1. introduction the potato (solanum tuberosum l.) has been considered one of the most important food crops, along with rice, wheat, and maize (ross, 1986; douches et al., 2004). potatoes grow in a variety of geo-environmental conditions. developing countries cultivate potato to add nutritional balance to their food basket (douches et al., 2004; navarre et al., 2009). in syria, more than 29,000 ha were planted with potato, producing about 609,000 t of tubers in 2005 (alammouri, 2008). however, severe damage may occur to potato crops at storage periods particularly in developing countries. within the lepidoptera order, potato tuber moth phthorimaea operculella (zeller) belongs to the gelechiidae family and it has been reported in more than 90 countries, making it a cosmopolitan pest (visser, 2005; golizadeh and esmaeili, 2012). it damages potato throughout the growing season by mining stems, petioles, leaves and tubers by larvae, with the latter considered the typical damage. the procedure of potato damage begins when larvae penetrate the foliage, including leaves and stems. this insect can infest potato tubers stored and in field or it may develop on plants remaining in the field including tomatoes, aubergine or other solanaceous plants (gilboa and podoler, 1995; coll et al., 2000; alvarez et al., 2005). farmers depend broadly on the use of insecticides and other varieties of farming practices (clough et al., 2008); insecticides are widely used to control this pest. however, insecticides are costly, nonselective, unfriendly to the environment, and affective for only a short period of time (simmons et al., 2006). additionally, the phenomenon of resistance to insecticides in lepidoptera has increased significantly (gonzalez and trevathan, 2001). plant resistance, together with appropriate biological and farming practices in combination with insecticides may provide the best management options (rondon, 2010). plants defeat pathogens through their active defense mechanisms that can be stimulated in some cases by plant growth-promoting rhizobacteria (pgpr) which ultimately reduce disease and render the host plant more resistant to any foreseeable pathogen attacks (pieterse et al., 2002). induction of such enhanced defensive capacity is systemic as root adv. hort. sci., 2016 30(1): 47-52 doi: 10.13128/ahs-18701 induced resistance in potato plants by a non-pathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller) a. adam (*), i. idris, n. khalil, k. houssian department of molecular biology and biotechnology, atomic energy commission of syria (aecs), p.o. box 6091, damascus, syria. key words: biocontrol, plant resistance, potato tuber moth, p. putida btp1, rhizobacteria. abstract: pseudomonas putida strain btp1 is able to promote induced systemic resistance (isr) in a wide spectrum of pathosystems. in this study, we investigated induced resistance in potato plants against potato tuber moth (phthorimaea operculella zeller) by non-pathogenic p. putida btp1. several physiological indicators in the life cycle of the potato tuber moth, such as survival rate, mean weight of pupae, and sex ratio were studied to assess the protective effect of p. putida btp1. our results showed that treatment of potato tubers by bacterial suspension of p. putida btp1 caused evident disturbance to the development of p. operculella in potato plants. survival rate of larvae feeding on treated plant leaves and mean weight of pupae decreased significantly. in addition, a clear deviation in the sex ratio in moths, in favor of males, resulted from larvae fed on bacteria-treated plants. this study preliminarily reports the ability of btp1 to induce resistance in potato plants against potato tuber moth. consequently, p. putida strain btp1 could be a promising approach for potato tuber moth biocontrol. (*) corresponding author: ascientific@aec.org.sy received for publication 9 august 2015 accepted for publication 8 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 47-52 48 treatment with a pgpr was shown to trigger protective effects on above-ground plant parts. these reactions are thought to typically result from the activation of latent defense mechanisms that are overexpressed upon subsequent pathogen challenge (van loon et al., 1998; ongena et al., 2002; bakker et al., 2007). this induced systemic resistance (isr) can be the basis for integrated plant disease management strategies (ramamoorthy et al., 2001; zehnder et al., 2001; saravanakumar et al., 2007). induced resistance in plants by non-pathogenic rhizobacteria against pests is a very important additional factor for the protection of agricultural crops. several studies have indicated the ability of many strains of rhizobacteria (pgpr) to induce systemic resistance against a large number of insect pests (racke and sikora, 1992; zehnder et al., 1997). for example, in cucumber against striped cucumber beetle acalymm avittatum (zehnder et al., 1997, 2001), in cotton against american boll worm helicoverpa armigera (vijayasamundeeswari et al., 2009), in tomato against whitefly bemisia tabaci (valenzuela-soto et al., 2010) and also in cucumber against spider mites tetranychus urticae (tomczyk, 2006). a non-pathogenic btp1 showed enhancement of resistance level in many plants including bean, cucumber, and tomato against fungal pathogens (ongena et al., 2002, 2004; adam et al., 2008). in a previous study performed in vitro on grapevine rootstocks, we demonstrated the influence of p. putida btp1 on reproduction and development of grapevine phylloxera (adam et al., 2013). however, to our knowledge no studies have been performed yet to assess the effects of pgpr on p. operculella in potato plants. therefore, the present work aims to demonstrate the protective effect triggered by p. putida strain btp1 against p. operculella in potato plants. the larvae survival rate, the mean weight of pupae and the sex ratio were studied as a biometers to detect the induced resistance. 2. materials and methods establishment of the potato tuber moth colony insects used in the experiments were reared on waxed potato slices as described by rahalakar et al. (1985). the experiments were conducted at a constant temperature of 25±1˚c with 70±5% rh, and a photoperiod of 12:12 (l:d) h. microbial strain and inoculum preparation psudomonas putida strain btp1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron transport (jacques et al., 1995; ongena et al., 2002). it was maintained and prepared for use in the isr assays as previously described by ongena et al. (2002). for the bioassays, btp1 strain was grown in erlenmeyer flasks (250 ml) containing 100 ml of casamino acids medium (caa) for 24 h on a rotary shaker (150 rpm) at 28 oc. cells were removed by centrifugation at 16500 g for 15 min at 4°c and washed in sterile nacl (5g l–1). the final pellet was resuspended in an adequate volume of sterile distilled water to obtain a bacterial suspension at 108 cfu ml-1. assays for induced resistance “draja” potato tubers were washed in sterile water, dipped separately in a suspension of p. putida strain btp1 for 30 min, and air-dried, while control tubers were treated with sterile water. the tubers were then planted in 10 l plastic pots containing autoclaved, moistened soil (three tubers/pot) to exclude any microorganism could affect btp1. the pots were placed in a greenhouse at 25±1°c (day) and 23±1°c (night) with daylight of 16 h and relative humidity of 85-95%. both control and treated plants were under the same watering and fertilizing conditions during the planting period. fresh leaves excised from potato plants (six to seven weeks old) were used for feeding the newly hatched larvae (24 h). for each treatment, 120 larvae in 10 (18 x 12 x 8 cm) plastic boxes (12 larvae/box) were fed on leaves until they reached the pupal stage. the boxes were resealed with parafilm to keep the larvae from escaping, and were then incubated at 25±1°c with daylight of 12 h and relative humidity of 70%. each four-day-old pupae was weighed and placed separately within a small plastic tube. the pupae were classified into three groups according to their weights: small pupae (<5 mg), medium pupae (6-7 mg) and large pupae (>8 mg) to determine the larger sex. the number of pupae and the number of emerging moths (males or females) were recorded in order to calculate the survival rate of larvae and the sex ratio (the number of male/the number of female). the experiment was repeated three times. isolation of bacteria from potato plant leaves small leaf samples were taken from different parts of the potato plants treated with p. putida adam et al. potato plant resistant to potato tuber moth by a non-pathogenic pseudomonas putida 49 btp1. the samples were sterilized with sodium hypochlorite solution (5%) for 3 min and washed three times for 3 min. samples were left to dry on sterile paper. they were then grown on petri dishes containing the casamino acid (caa) medium. the dishes were incubated at 30±1°c for 72 h. statistical analysis statistical analyses were performed using statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). data were subjected to analysis of variance (anova) for the determination of differences between means. differences between means of pupal weight were tested for significance using tukey hsd test. ratio analysis test (z-test) was used to compare the percentages of larval survival rate. 3. results isolation of bacteria from potato plant leaves the isolation of bacteria test on treated-potato plant leaves showed no bacterial colonies grew in the petri dishes, indicating that p. putida btp1 did not migrate through the plant (from the tubers to the leaves). there was no direct contact between bacteria and larvae. effect p. putida btp1 on potato plants against potato tuber moth larvae survival rate. induced resistance experiments showed the death of large numbers of larvae of p. operculella in different ages of development, particularly in p. putida btp1-treated potato plants (fig. 1.1). the larval survival rate decreased significantly (35%) when the larvae were fed on the excised leaves from p. putida btp1-treated plants compared with the control plants (fig. 1.2). significant differences between btp1 and control were observed in all experiments. effect on pupal weight. the results of three independent experiments showed that there was a negative impact on mean pupal weight in p. putida btp1treated potato plants. where mean of pupal weight was 7.77±0.12 mg in the control potato plants, it decreased significantly in p. putida btp1-treated plants to 6.24±0.15 mg (fig. 2). this implies an approximately 20% weight reduction of pupae in potato plants pre-inoculated with p. putida btp1 as compared with the control. significant differences between btp1 and control were observed in all experiments. with regard to pupae weight, we observed that the pupae, which were classified into three groups according to their weight (small, <5 mg; medium, pupae 6-7 mg; large >8 mg), in control plants were mostly large pupae (approximately 61%) while the fig. 1 example of potato leaves infested by potato tuber moth showing larvae feeding on p. putida btp1-treated plant leaves (1a), and control plant leaves (1b). (1c): profile of the dead larvae in different stages because of malnutrition. (2): influence of potato tuber treatment by the bacterial suspension of p. putida btp1 on the survival rate of larvae of the potato tuber moth. three separate experiments were carried out (120 larvae per treatment and per experiment were used). data were subjected to anova and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). adv. hort. sci., 2016 30(1): 47-52 50 small pupae was almost absent (3%) (fig. 3). in contrast, in btp1-treated plants, the percentage of the large pupae decreased significantly to reach (36%), while the percentage of the small pupae increased significantly to reach (27%) (fig. 3). sex ratio of p. operculella the sex ratio of moths (number of males/number of females) emerged from control (180 moths) and p. putida btp1-treated potato plants (91 moths) was calculated. a clear deviation was observed in the sex ratio in favor of males from feeding on btp1-treated plants: 1.84:1, compared to 1:1 in control plants. 4. discussion and conclusions our study has shown that the larval survival rate and the mean of pupal weight were significantly decreased in btp1-treated potato plants compared to control. these results are consistent with previous studies conducted on whitefly, which showed a significant decrease in survival rate (number of nymphs which are able to develop and reach the adult stage) in tomato plants treated by rhizobacteria (valenzuela-soto et al., 2010). in addition, similar results were found in pgpr-treated cotton bolls with mortality of larval, malformation of pupal and adult with decreased adult emergence of american bollworm h. armigera (vijayasamundeeswari et al., 2009). moreover, changes in dietary behavior of the rice leaf roller (cnaphalocrocis medinalis) was observed, and there was a decrease in the larval and pupal weight in treated rice leaves by rhizobacteria (radjacommare, 2002). the reduction of larval survival rate and pupal weight in btp1-treated potato could be attributed to the inability of larvae to feed on treated plant leaves. it is well known that the growth of phytophagous larvae is affected indirectly by chemical or physical conditions or even both which characterize their host plants. for instance, pgpr-treated plants may have a decrease in essential nutrients or have compounds that inhibit growth, or both (reese and field, 1986; bong and sikorowski, 1991; yaman et al., 1999). on the other hand, treatment of potato tubers with p. putida btp1 also caused a clear deviation in sex ratio in favor of males. quezada-garcia et al. (2014) proved that nutritional variation causes differential mortality to the larger sex and the most sensitive to nutritional stress (female) in spruce budworm (choristo neural fumiferana (clemens); lepidoptera). in contrast, house et al. (2011) demonstrated that offspring mortality in the dung beetle (onthopha gustaurus; coleoptera) vitally depends on the amount of resources that females have provisionally. in addition, they also showed that males have greater nutritional demands than females during development, which ultimately leads to higher mortality in the male population (the larger sex and the fig. 2 influence of potato tuber treatment with bacterial suspension of p. putida btp1 on mean of pupal weight of the potato tuber moth. three separate experiments were carried out. each column represents the weight mean of 25 pupae. data were subjected to anova and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). fig. 3 influence of potato tuber treatment with bacterial suspension of p. putida btp1 on percentages of pupal weight of the potato tuber moth (75 pupae/treatment). data were subjected to test ratio analysis (z-test) to determine the significant differences at p<0.05 (values with different letters are significantly different). adam et al. potato plant resistant to potato tuber moth by a non-pathogenic pseudomonas putida 51 most sensitive to nutritional stress) (house et al., 2011; quezada-garcia et al., 2014). these findings are consistent with our results which showed that females were larger (16.74%) than males. in conclusion, understanding the mechanisms of induced defense by p. putida btp1 is very important to enhance the resistance in potato plants. the current study provides evidence that p. putida strain btp1 has a protective effect in potato plants against potato tuber moth. based on similar studies that illustrated that the accumulation of some toxic phenolic compounds in the cells of resistant plants led to an increase in the death rate in insects, we believe that the treatment of potato tubers with p. putida btp1 leads to secondary metabolic changes in treated plant cells which elicit the production of defense compounds (lattanzio et al., 2000; zehnder et al., 2001; arimura et al., 2005; melvin and muthukumaran, 2008). this study preliminarily reports the ability of p. putida strain btp1 to induce resistance in potato plants against potato tuber moth. this bacterial strain could be a promising agent for potato tuber moth biocontrol. however, the controlled environment (plastic pots, sterile soil, and humidity) may lead to different results compared to farm applications due to competitors, t/hr condition, dispersion of inoculum, etc. consequently, more research is needed to determine the mechanisms of defense induced in potato plants. acknowledgements the authors thank prof. i. othman (atomic energy commission of syria) and dr. n. mirali (department of biotechnology) for their help. we thank prof. p. thonart and dr. m. ongena of the university of liege, who provided us with the p. putida btp1 strain. references adam a., idris i . , ayyoubi z., 2013 in vitro 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visser d., 2005 guide to potato pests and their natural enemies in south africa. arcroodeplaat vegetable and ornamental plant institute, pretoria, south africa. yaman m., demirbag z., belduz a.o., 1999 investigations on the bacterial flora as a potential biocontol agent of chestnut weevil, curculio elephas (coleoptera: curculionidae) in turkey. african journal of mycology biotechnology, 54: 625-630. zehnder g., kloepper j., yao c., wei g., 1997 induction of systemic resistance in cucumber against cucumber beetles (coleoptera: chrysomelidae) by plant growthpromoting rhizobacteria. j. econ. entomol., 90(2): 391-396. zehnder g.w., murphy j.f., sikora e.j., kloepper j.w., 2001 application of rhizobacteria for induced resistance. eur. j. plant pathol., 107(1): 39-50. impaginato 231 1. introduction during the last decades, after over ten years of reconstruction projects, afghanistan horticulture has grown to the pre-war level. in 2014, the estimated horticulture crops extended over 340,000 ha that represents the 14% of the total irrigated land. the overall contribution of horticulture is valued at 1.4 billion (6.7%, to the total gdp) and the 34% of the agriculture gdp contribution with a crops employment generated estimated in 2 million people involved to various degrees. principal crops are grapes ($330m), almonds ($120m), pomegranates ($100m), all of which are largely exported to the neighbour countries. by comparison, illicit crops (opium poppy, cannabis) in the same period are grown on 220,000 ha with an estimated value of about $1 billion. many important fruit and vegetables are origin from afghanistan (e.g. pistachio, walnut and pomegranate) and afghanistan has favourable climate condition suitable for high quality horticulture. the hypothetically development of the horticulture sector is huge as well as the potential international interest for their quality (pomegranates, apricots, almonds, raisins). despite the improvements, especially the road network, the horticulture sector claims a lack of massive interventions. as a consequence, the overall quality is still low due to a wellknown list of constraints: small land holdings, small commercial orchards, lack of standardized product, poor orchard management, lack of infrastructure and substandard storage, sorting, packaging, marketing, transport facilities. afghan quality control system in general, the quality of the agriculture products is the final result of a long list of interventions, controls and standards adopted by a nation, generically define as quality control system. the quality control system has the primary function of protecting its citizen by selecting active controls of the imported commodities at the borders and promote the safety of the exported goods. the lack adv. hort. sci., 2016 30(4): 231-237 doi: 10.13128/ahs-20351 introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study f. valori 1 (*), a. ahkbari 2, m.r. yaqubi 2 , n. enayat 2, f. azizi 3, m. wali adel 2 1 independent consultant, florence, italy. 2 afghanistan national horticulture development organization, kabul, afghanistan. 3 ministry of agriculture, irrigation and livestock, kabul, afghanistan. key words: harvest date, maturity index, sweet cherry. abstract: in the last decade, afghanistan has been under the political attention by several institution and organizations whose invested millions of dollars for the reconstruction of the country. part of such efforts were focused on the agricultural sector, as the principal source of income of afghanistan. mainly, perennial horticulture represents the one of the most challenging opportunity for the developing economies of afghanistan, and the high contribute of eu in such direction, shown significant results. however, the level of the export is still neglected and the reputation of afghan’ products has lost the ancient brittleness. the generic low hygienic conditions and poor quality of the products are the primary cause of such situation. in this scenario, phdp (perennial horticulture development project) first and anhdo (afghanistan national horticulture organization) then, tried to improve the quality of perennial horticulture crops. phdp has, for the first time created a certification scheme for the production of certified perennial plants, anhdo has the main goal of improving the quality of the fruit value chain of the principal crops in afghanistan. in this study the first protocol for the determination of the optimum harvest date (ohd) for cherry var. burlat is given, as a case study of the new course of horticulture in afghanistan. (*) corresponding author: federico.valori@gmail.com received for publication 10 march 2016 accepted for publication 5 september 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 231-237 232 of an efficient quality control system, affects the horticulture sector in many ways. the general situation in afghanistan is critical in both the import/export directions: international requirements (rules and consumers education) on food are extremely rigid and difficult to overcome for afghan traders. from the import side, the situation is without control. the absence of an explicit laws with a specific phytosanitary standards regulation, allows to the neighbour countries to freely provide goods and services without any reliable control. the consequence is a risk for the food safety and human health aspects (e.g. ogm) and besides that also the religious point of view is involved. the respect of halal rules is in fact often not certified and the customs authority has technical difficulties to check the provided certificates. for the above mentioned reasons, the national quality control system of afghanistan is now under a deep revision: afghanistan has started the world trade organization (wto) membership path. this is a very important step forward for the quality control of the country, especially for the agricultural commodities: join the wto will allow to afghanistan to implement non-tariff barriers useful to protect his local market from the massive import from neighbours countries i.e china, pakistan and india on top (wto, 1995 a). beyond wto, the gap between the afghan food industries and the international competitors, is consistent. afghan industries may require large investment to make them efficient and competitive in a reasonable period of time. applying a protection policy is probably, the best way for afghanistan to fill the gap with its competitors. protection would act as incentive for such industries, the introduction of technical barriers agreements (tbt) may establish a more transparent trade with the neighbour countries and allow to afghanistan a better control of the imported commodities based on harmonised rules. application of tbt required the wto membership and the main obstacle to this is the lack of a food law that will clarify the different responsibilities among the ministries and will produce the conditions to the creation of the national quality control system. the responsibilities on the food safety are now divided between the ministry of public health (moph) and the ministry of agriculture irrigation and livestock (mail) but such division is not clear, with the consequence of an inefficient sanitary and phytosanitary system, with respect to the requested sps agreement (wto, 1995 b). at the best of our knowledge, the food law is still stuck in the parliament and afghanistan doesn’t adopts any procedures and a clear set of harmonized standards. such a law represents a necessary milestone for the future of agriculture sectors, but it is not sufficient to promote a real food quality control in afghanistan. the first goal of such law must be the protection of human health from risks created by uncontrolled trading, tools as quarantine measures, certification, borders checks, inspections network and laboratory tests are the pillars of the quality control. in the last ten years, the phdp has successfully developed a certification scheme for the horticulture crops and keep the horticulture to an upper level of quality in the whole afghanistan. moreover, phdp developed the national collection orchards (nc) where the local and international varieties were tested for their adaptive level in the specific afghanistan climate. within its activities, phdp has also set-up six pomology laboratories where, for the first time, local and introduced horticulture varieties were characterized under many aspects (e.g. upov, grafting, etc.). the phdp project ends up with a great numbers of varieties, however, not all these varieties were fully characterized and some basic information are still missing. specifically, the harvesting date and maturity index were not included as tested parameters because out of the scope of the project. horticulture private sector development project (hps) has, in one specific subjective (so3), as primary goal, the evaluation and the promotion of the fruit commercial varieties. anhdo is the implementing partner of hps and in close collaboration with mail (that has inherited the ownership of the pomology laboratories) has succeeded to the phdp cultural heritage and is developing the missing horticulture parameters: in the next two years at least 60 varieties of the major commercial horticulture crops will be fully characterized and studied for the pomology basic tests, including post-harvest tests, maturity indexes and harvesting date by hps specific sub objective (so2 adaptive research). anhdo/hps ambitions are high and even if the pomology laboratories are well equipped and the technical staff has been trained, the overall technical level is still critical due to the general environmental situations and only basic tests are possible. fruit quality in afghanistan: cherries as least development country, afghanistan has a total potential catchment not depending on the quality of the products and the domestic market has the peculiarity of accepting every type of fruits. the wide range of the existing social classes creates consumers valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 233 with different tolerance and allow traders to sell all the product without any formal losses. the wellknown effect of the extended cherry shelf-life over the 7-10 days (loss of firmness, color and flavour, stem discoloration, desiccation and mould growth) will not affect the trade. however, such condition is changing with the increase of the consumer awareness. soon, farmers and traders will face, for the first time, the necessity of an improvement in the cherries quality. to be prepared, for the very first time in afghanistan, hps has set-up a formal protocol to start the characterization of the main commercial fruit varieties, with the final aim of helping farmers to harvest the crops in the optimum period for domestic consumption or international export. aim of the work the aim of the study is to provide a simple and pragmatic in-field tool for the anhdo/hps beneficiaries farmers, to identify the appropriate harvesting date and suitable maturity index for cherry cv. burlat. the proposed approach is a compromise between scientific research (that requires at least twenty single observations) and the real condition of afghanistan that forced to reduce such observations to five. phdpii project provided initial data on the harvesting period of many varieties, based on visual observations. this study will confirm the hypothetical harvesting date and will provide (among different existing parameters) one or two easy and quick maturity indexes for the afghan farmers. determination of optimum harvest date of sweet cherry cv. burlat grown in kabul optimum harvest date (ohd) is defined as the date in which harvesting results in optimum fruit quality of the produce after long-term storage (baumann, 1998). typically, ohd is predicted using calculation models or through direct measurement of certain physiological fruit parameters and quality characteristics during a given period until harvest (çalhan et al., 2014). the first method required historical series of meteorological and phenological data that are not available in afghanistan. on the contrary, direct measurements of fruit characteristics, in specific intervals before the estimated harvest date, are more reliable for specific cultivars and applicable in afghanistan. this study was conducted to evaluate ohd through physical and chemical measurements of fruit quality as a maturity index for estimating proper harvest time of burlat cherries, in kabul. among the exiting maturity indexes, sugar content (ssc), titratable acidity (ta), ssc/ta ratio, skin color, and firmness have all been used as indices of cherry fruit maturity. however, skin color has long been accepted as the best indicator for the appropriate harvest maturity of sweet cherries (drake and elfving, 2002). 2. materials and methods cherry cv. burlat has leaves sawed, average green, slightly lengthy and pointed. burlat fruit has a dark red or purple skin, slightly flat and mean sized. the flesh is dark red, with sweet taste, juicy and firm. it is defined as early ripening, it is self-incompatible and it needs a pollinizer. fruit from the sweet cherry cv. burlat was harvested during 5 different dates (between 18.05.2015 and 27.05.2015) from the national collection in badam bagh, kabul, afghanistan. full bloom date of burlat was at 19.04.2015. the harvesting period was concentrated in 10 days due to existing data obtained from previous tests on the same station. in the national collection each varieties is replied 6 times in a row. fruits were collected early in the morning from branches with the same orientation (south-east) and all the fruit from the selected branches were immediately transferred to the pomology laboratory. in the laboratory, samples with defects (split, diseased or damaged fruit) were annotated and then rejected. the collected cherries were then divided into 5 maturity class according to their visual skin color: a) yellowish immature; b) light red nearly mature; c) red mature; d) dark red ripe; e) overripe. the number of cherries for each class was annotated. in this study only red and dark groups were considered. fruits parameters were collected by 24 fruits per class for a total of 48 samples. weight was measured by an electronic balance (kern emb-200) to an accuracy of 0.01 g; results were expressed as gram (g). fruit width and length was measured using manual calliper (verinier caliper 0-150 mm). results were expressed as millimeter (mm). total soluble solids concentration (tss) was determined with a temperature compensated digital refractometer (atago pal-1) previously calibrated with distilled water and results expressed as °brix. titratable acidity (ta), was estimated by juice titration with 0.1 n naoh to the titration end point of ph 8.1, monitored adv. hort. sci., 2016 30(4): 231-237 234 with a ph meter (hanna instruments hi3221) and expressed as malic acid content (mg/100 ml). flesh firmness was defined as the maximum load required to push the 6 mm diameter probe into the fruit firmness one side of each fruit, to a depth of 6mm with fruit texture analyser (fruit pressure tester ft 327). the results were expressed in newton (n). skin color was measured on 24 fruits per class using a minolta chromometer model cr 400 and average readings at six pre-determined points on the circumference of the fruits were recorded. the instrument was calibrated against a standard white color plate (y=93,9, x=0,313, y=0,321) (konika minolta, 2013), color space (xyz) (batu, 2004; žnidarčič and požrl et al., 2006). all the above mentioned parameters were measured between 18.05.2015 and 27.05.2015. the final dataset was statistically analysed by pearson correlation test, anova test and pca using systat 12 software and minitab 17. part of the graphs do not shown red group values at the latest observation. this happened because during the division in maturity classes immediately done after the harvest in the last part of the maturity process, the red group was without samples. such a situation is obviously due to the ripening of cherries and the lack of non-mature fruits. 3. results as reported in figure 1, red groups weight increase at maximum rate to 24/05 and then starting decreasing. on the contrary, the dark group results do not shown significant variation of weight. this suggest that the maturity weight was reached before the start of the observations, and is confirmed by the red group trend that increases as confirmation of the maturity development. as reported in figure 2, height increase during the observation period. however, as expected, the height/diameter ratio is not changing (table 1). the difference between the first and the last observations reports an increase of 7.56%. this result is clearly explained by the shorter observation period where the contiguous observations are not significant. firmness (fig. 3), as expected, decrease during the observations period and drastically dropped between 24/05 and 26/05. considering the dark red and red groups separately (fig. 3), the decrease is 31.89% and 50.23% respectively. sugar content (fig. 4) is constantly increasing for both dark and red groups. the most significant increase is visible between 24/05 and 27/05, in which the sugar content passes from 20.27 °brix to 24.73 °brix for the dark group. in the last day of observation (27/05), none of the sampled cherries was classified as red group, as consequence of the ripening process. the ph values, in this study, do not provide a reliable maturity index, as the variation observed during the experimental period were significant but ranged within the instrument fig. 2 effects of different picked dates on 48 fruit height (mm) of sweet cherry cv. burlat. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). means with different letter are significant different (tukey’s hsd p<0.05). fig. 1 effects of different picked dates on 48 fruit weight (g) of sweet cherry cv. burlat fruits. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). table 1 effects of different picked date of ratio between height and diameters (h/d) on 48 fruits of sweet cherry cv. burlat date height-diameter ratio dark red group red group error d.r. error r. 5/18/15 0.852 a 0.893 a 0.017 0.018 5/20/15 0.859 a 0.879 a 0.018 0.018 5/24/15 0.833 a 0.839 a 0.018 0.019 5/26/15 0.834 a 0.861 a 0.018 0.018 5/27/15 0.984 b 0.018 valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 235 error (fig. 5). the graph reported in figure 6, perfectly, explain and reflects the burlat maturity pattern of ta: the total acidity starts decreasing from the 26/05 in the dark group. such a result, compared with sugar content clearly states that cherry acidity is decreasing, while the sugar content is still growing (fig. 4). considering the path of the sugar content and the ta result, is possible to set the maturity days between 26/05 and 27/05. the same conclusion is obtained by looking the color xyz-cie results (fig. 7), in which the variation of color is stable in the same period. the identification of the most suitable parameter used as easy maturity index, passed through a first analysis of the data set using a pca analysis. however, the interpretation of a pca with more than 8 factors and at least three principal components, is not easy. to overcome such problem the pearson correlation matrix was used to reduce the number of parameters analysed with pca. the main correlations among x, y, z parameters are reported in table 2. the color components are very well correlated, and such strong correlations justify to use only one of the fig. 3 effects of different picked dates on 48 fruit firmness of sweet cherry cv. burlat compared with red and dark red group. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 4 effects of different picked dates on 48 fruit sugar content (brix°) of sweet cherry cv. burlat for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 5 effects of different picked dates on 48 fruit ph of sweet cherry cv. burlat. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 6 effects of different picked dates on 48 fruit sugar content and tritatable acidity (ta) of sweet cherry cv. burlat fruits for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 7 effects of different picked dates on 48 fruit xyz cie color of sweet cherry cv. burlat for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). table 2 pearson correlation for the xyz-cie parameters x y z x 1.000 y 0.993 1.000 z 0.834 0.883 1.000 adv. hort. sci., 2016 30(4): 231-237 236 three components in the pca (tabachnick and fidell, 2006). therefore, only x factor has been considered in the simplified pca test, because from the statistical analysis (fig. 7) such variable appears more sensible to the ripening process compared to y and z. table 3 reported all the other correlations. between diameter and weight the correlation is sufficient to justify the usage of the height and diameter ratio (h/d) to get an unique shape factor that will reduce the variability in the data set (tabachnick and fidell, 2006). sugar content and ph are well positively correlated (0.694), as expected. applying the above mentioned consideration, the simplified pca is reported in figure 8. the first three components explain 71.8% of the variability. table 4 reports loadings values for the first three components. ssc and ph have the highest loadings values and are positively correlated, thus able to explain almost all the variance of the dataset. also color has a high loading value (table 3) that suggests it as one of the possibly maturity index suitable for the afghan farmers. the parameter weight explains the remaining variance. weight, as very easy and cheap measure can be used as additional maturity index with sugar content, ph and color. 4. conclusions the cherry cv. burlat ohd is set for the year 2015 at 26th of may. such result must be verified in the next coming years and correlated with the meteorological situation. based on the whole results dataset, the suggested ‘burlat’ characteristic at the ohd are: height: 19.167±0.254 mm, weight: 6.705±0.126 mm, ph: 3.246±0.009, sugar content: 22.488±0.5°brix, ta: 15.433±0.450 g/ml, firmness 0.653±0.037 n, x: 6.391, y: 5.229, z: 5.368 (rgb=6b3540 html color). as explained, the aim of the study was providing a simple tool applicable in the field as maturity index to the afghan farmers. as reported, sugar content, ph, color and weight have very good chances to be used as maturity indexes. however, for the color determination, the required instrument is very expensive and not suitable for the afghan condition, in which the gross national income per capita is 640 usd (world bank, 2004). a similar conclusion is valid for the ph. sugar content is, thus, the suggested solution for farmers. it is relatively cheap and extremely easy to use. however, based on the laboratory results, hps may provide color gauge specific for each single varieties to the hps beneficiaries and confirm with ssc, weight, ph and color tests the validity of sugar content as maturity index. the same methodology applied for ‘burlat’, and explained in this study, was used with four other table 4 variance explained by components table 3 pearson correlation for all paramenters fig. 8 simplified pca using the following parameters: weight, ssc, ta, ph, h/d, firmness, x. weight height diameter grade firmness ssc x ph ta weight 1 height 0.274 1 diameter 0.599 0.485 1 grade 0.540 0.121 0.423 1 firmness -0.207 0.053 0.119 -0.098 1 ssc -0.134 -0.259 -0.339 0.035 -0.251 1 x -0.302 0.073 0.023 -0.382 0.417 -0.704 1 ph -0.199 -0.207 -0.315 -0.037 -0.260 0.694 -0.481 1 ta 0.129 0.045 0.153 0.262 -0.193 0.152 -0.306 -0.126 1 variable pc1 pc2 pc3 weight 0.027 -0.609 0.166 h/d -0.133 0.367 -0.724 firmness 0.317 0:35 -0.001 ssc -0.568 0.069 0.003 ph -0.532 0.733 0.166 ta 0.072 -0.477 -0.645 x 0.520 0.291 0.070 valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 237 cherry cultivars: balck star, cherry pie, santina and stella compact, following the estimated ohd: ‘black star’: 6 june, ‘cherry pie’: 30 may-6 june, ‘santina’: 4 june, ‘stella compact’: 6-11 june. acknowledgements this study was funded by phdpii (europe aid/129320/c/ser/af/2) and hps (europe aid/133872/l/af). in memory of dr. monica berti. references batu a., 2004 determination of acceptable firmness and color values of tomatoes. j. food engineering, 61(3): 471-475. baumann h., 1998 decrease of starch to predict the ‘optimum harvest date’ for apple storage. acta horticiculturae, 466: 41-46. çalhan ö., ebru o.c., güneyli a.,·eren i., demirtaş i., 2014 optimum harvest date of sweet cherry cv. lapins grown in isparta. türk tarım ve doğa bilimleri, 7(7): 1905-1910. drake s.r., elfving d.c., 2002 indicators of maturity and storage quality of lapins’ sweet cherry . horttechnology, 12(4): 687-690. konika minolta, 2013 precise color communication. color control from perception to instrumentation. konika minolta camera co. ltd. japan, pp. 62. tabachnick b.g., fidell l.s., 2006 using multivariate statistics. 5th ed. allyn & bacon, boston, ma, usa, pp. 1008. world bank, 2014 data indicators. country afghanistan available at http://data.worldbank.org/country/ afghanistan. wto, 1995 a agreement on agriculture (aoa). is an international treaty of the world trade organization. it was negotiated during the uruguay round of the general agreement on tariffs and trade, and entered into force with the establishment of the wto on january 1. world trade organization. wto, 1995 b agreement on the application of sanitary and phytosanitary measures (sps agreement). is an international treaty of the world trade organization. it was negotiated during the uruguay round of the general agreement on tariffs and trade, and entered into force with the establishment of the wto at the beginning of january. world trade organization. žnidarčič d., požrl t., 2006 comparative study of quality changes in tomato cv. ‘malike’ lycopersicon esculentum mill.) whilst stored at different temperatures. acta agriculturae slovenica, 87: 2. impaginato 37 adv. hort. sci., 2011 25(1): 37-43 received for publication 4 march 2011. accepted for publication 15 march 2011. foliar application of molybdenum: effects on yield quality of the grapevine sangiovese (vitis vinifera l.) e. masi*, m. boselli** * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 26, 50019 sesto fiorentino (fi), italy. ** dipartimento di biotecnologie, università degli studi di verona, villa lebrecht, via della pieve, 70, 37029 san floriano (vr), italy. key words: grapevine, foliar spray, molybdenum, yanc. abstract: field experiments with sangiovese vines were carried out in the chianti classico region over a period of two years to examine the effect of molybdenum (mo) foliar sprays on nutrient composition of leaves, petioles and berries, leaf gas exchanges, must composition, total yield, bunch size and pruning weight. two mo foliar doses and time sprays (mox1: one application in early flowering; mox3: three applications in early flowering, early fruit set and veraison) were applied. basal sample of petioles, leaves and berries collected at fruit set (except berries), veraison and harvest for mineral analyses showed not relevant interactions between mo and the main macro and micronutrients. leaf gas exchanges monitored after the applications, as well as spad units, showed a higher activity in the mox3-treated vines. increased vigour was also confirmed by the slightly higher total yield, bunch size and pruning weight, as well as the delay in fruit maturation (lower sugar and polyphenol contents at harvest). no relevant discrepancy between mox1 and the control was found, except for higher soluble solid and yeast-assimilable nitrogen contents (yanc) in the treated vines. yanc was positively influenced also in the mox3 vines, however with no significant differences towards the mox1 treatment. the application of mo as a useful tool to stimulate nitrogen metabolism, as well as indications about dose and time of mo application, are discussed. 1. introduction the transition element molybdenum (mo) is a very rare but essential micronutrient for all organisms (bortels, 1930; fortescue, 1992), and understanding of its role and function in plants is progressing rapidly. its importance for plants has been known for a long time (coughlan, 1980), even though mo itself seems to be catalytically inactive in biological systems until it is complexed by a special cofactor, the pterin (mendel and hänsch, 2002) which binds to diverse apoproteins. this latter compound is a unique pterin named molybdopterin or metal-containing pterin. in this form, it occurs in more than 40 enzymes catalysing many redox reactions, four of which have been found in plants (hille, 1996; kappl et al., 2002). one of these is nitrate reductase (nr) that catalyses the first step in nitrate assimilation, a pathway of key importance for plant nutrition. nitrate reductase is the key-enzyme for nitrate assimilation while nitrogenase is found in nitrogen-fixing bacteria inside nodules of symbiotically growing species. the last step of abscisic acid biosynthesis is catalyzed by the molybdenum-enzyme aldehyde oxidase, and sulfite oxidase protects the plant against toxic levels of sulfite (hänsch and mendel, 2009). nitrogen (n) is assimilated into the cell as a fully reduced form, ammonia (nh3). nh3 may be obtained in free form (e.g. by fertilizers) and from the degradation of amino acids. if these sources are not available, then ammonia must be produced via nitrate (no3)reduction. when nr is a limiting factor, plant growth, development and protein synthesis by plants are reduced (solomonson and barber, 1990). hence a shortage of mo in the soil, even though plants’ requirement for it is very low, or a mutational block of the cellular ability to use mo leads to the loss of essential metabolic functions and can cause the death of the plant. mo deficiency has been reported for many plant species including herbs, crops and trees (gupta, 1997) and could determine poor nr activity (hewitt, 1983), causing an inability to utilize n, with visible symptoms of chlorosis or yellowing of the leaves. 38 in grapevine, n is essential in overall vine establishment and maintenance, fruit quality, and the conversion of grape juice to wine. in grape berries, n is found primarily as ammonium cations and organic compounds such as amino acids (proline, arginine, glutamine, glutamate, etc.), hexose amines, peptides, nucleic acids and proteins. there are two phases of intense n incorporation in the fruit: the first takes place during the two weeks before the “pea-size” stage of the berries; the second starts one month later at veraison and lasts an additional two weeks (löhnertz, 1991). the amount of n in the clusters at harvest is approximately 40-44% of total available n of the entire plant (alexander, 1957; conradie, 1991; weinbaum et al., 1984). furthermore, n compounds are required by yeast for the production of cell biomass and the synthesis of proteins and enzymes necessary for the biochemical process of fermentation. the readily (easily assimilable) fermentable n compounds in juice and must consist primarily of nh3 and n available from the alpha amino acids present at harvest, particularly arginine (bisson, 1991). low levels of yeast-assimilable n content (yanc) in grape juice and must at harvest have been associated with sluggish and stuck fermentation and consequent undesirable levels of residual sugar in wines (kunkee, 1991; jiranek et al., 1995). slow and stuck fermentations are sometimes associated with grapes from vineyards composed of not vigorous plants or with high planting density, clearly characterized by scarce availability of n (masi and boselli, 2007). in some cases, despite plants having a sufficient level of n in their tissues, plant growth and crop yield show a clear n deficiency making it possible to hypothesize that most of the n in the plant is stored in non available forms. however, fertilizations with n are considered to be difficult, especially concerning choice of the right dose for optimum plant and yeast growth: even a low dose of n frequently causes excessive canopy growth and an unacceptable delay in fruit maturation being, at the same time, not sufficient in order to assure an optimal assimilable n concentration of the juice (spayd et al., 1995). assuming the inadequacy to reach optimal levels of available forms of n by chemical fertilization, mo supply could be a possible solution since its key role in the activation of n metabolism could be compared to a n fertilization itself. moreover, mo application has been shown to increase fruit quality, sugar content (rus’-ko, 1979; strakhov, 1988) and qualitative and quantitative composition of free amino acids (veliksar, 1977) in grapevine. mo is important in nitrogen nutrition of vines and has been suggested as a primary cause of millerandage in merlot vines (longbottom et al., 2004; williams et al., 2004). it is thought that mo directly affects the development of reproductive structures. molybdenum is necessary for successful pollen tube growth, ovule penetration, and fertilization (longbottom et al., 2004). williams et al. (2004) showed that mo increased the percent of coloured berries with one or more functional seeds and decreased the proportion of green berries, thus suggesting that mo application affected pollination and/or fertilization, and thereafter berry development. the present study, comprising two different levels and time of mo supply, was undertaken to determine the effect of mo on n metabolism by studying fruit and vegetative development in the grapevine sangiovese (vitis vinifera l.). 2. materials and methods plant material the study was carried out in a six-year-old vineyard of sangiovese vitis vinifera (l.) cultivar, grafted onto berlandieri x riparia 420a, located in the chianti classico docg region (elevation about 380 m asl; weather was characterized by mild winter, hot and dry season during the summer and average yearly rainfall of 700 mm, with the highest levels in november and march). the vines were spaced 0.8 m within the row and 2.4 m between rows and pruned to a single cordon system with four spurs, with an average of eight buds growing off the cordon. soil was rich in clay and “skeletal” material. pre-planting studies showed that the soil chemical fertility was good for most of the elements except for n (table 1). for that reason, every year, soil fertilizations rich in n (400 kg of npk-fertilizer 12+10+20) had been executed since the vineyard was established; although tissue analysis did not show any mineral deficiency, neither for microelements nor macroelements (fig. 1), light symptoms of n-deficiency were present on the vines at the moment the experiment was performed, consisting in low vigour and poor yanc must content in the previous vintages. treatments during two consecutive years (2002-2003), the vineyard was divided into three homogeneous areas, of six rows each, for two mo treatments and a control table 1 soil chemical analysis soil parameter ph (h2o) electrical conductivity total lime (caco3) active lime (caco3) cation exchange capacity organic matter n ca mg k (k2o) p (p2o5) fe mn b 8.2 126.5 µs ·cm-1 7.6% 0.7% 18.5 me·100g-1 0.9% 0.4% 1157 mg·kg-1 134 mg·kg-1 146 mg·kg-1 12.5 mg·kg-1 78.3 mg·kg-1 85.7 mg·kg-1 2.8 mg·kg-1 39 (untreated vines). foliar applications were sprayed using 350 g·ha-1 of a custom-made mo fertilizer (12% w/w) dissolved in 200 l of water. the first treatments provided a single application in early flowering (mox1); the second provided three applications, respectively in early flowering, early fruit set and early veraison (mox3). recommended cultural practices of the vineyard were applied. data collection three basal leaf and petiole samples were collected (at fruit set, veraison and harvest); two berry samples were also collected from veraison to harvest. each sample was the result of 50 to 60 organs harvested from random positions among the six rows of each treatment area. the material was rinsed with distilled water in order to take away any pesticide or fertilizer traces, then oven dried at about 80°c until thoroughly dry. each dried sample was then ground and sent to the laboratory for chemical analysis of macro and microelements; the analyses were performed three times and results averaged. gas exchange measurements were taken after veraison on 25 replications obtained by selecting five leaves on five plants per treatment: the leaves were of the same age, spatial orientation and light exposure. net photosynthetic rate (pn) and transpiration rate (e) were recorded with a portable photosynthesis system (ciras-i, ppsystems, hertfordshire, uk). leaves were enclosed in a ventilated leaf cuvette and exposed to saturating irradiance (par≥1200 µmol·m-2); concentration of co2 used for the measurements was 350 mg·l-1. in addition, water user efficiency (wue) was calculated as the rate of net photosynthesis per unit of transpired water. according to the same statistical design, after veraison (and 30 days later) on the same plants and leaves leaf chlorophyll and nitrate content (westerveld et al., 2003) was estimated non-destructively using a portable chlorophyll meter (spad-502, minolta corp., ramsey, n.j., u.s.). at fruit set, veraison and harvest, berry samples were picked randomly from 10 plants per treatment, crushed, pressed and the juice analyzed for the yeast fermentable nitrogen content. the yanc is expressed as mg·l-1 of n as the sum of the assimilable nitrogen from ammonia plus the assimilable nitrogen from alpha amino acids. fig. 1 primary macronutrient (n, p, k) concentrations in basal leaf, petiole and berry at fruit set, veraison and harvest in mo-treated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. 40 at harvest, 300 berries were collected, weighed and then squeezed by hand and filtered through a strainer. samples were harvested randomly from 10 plants (30 berries per plant) per treatment. the expressed juice was measured to determine total soluble solids (brix) with a hand refractometer and titratable acidity by titration with 0.1 n naoh to ph 7. titratable acidity was expressed by g·l-1 of tartaric acid. anthocyanin content of the must was performed via glories indices (glories, 1984). indices evaluated were: total anthocyanins a1 (mg·l-1); extractable anthocyanins a3.2 (mg·l-1); extractability assay ea (based on the ratio of the above two anthocyanin values); and extractability of the tannins contained in the seed mp. during the season, five plants per treatment were labelled; they were harvested separately in order to calculate average yield per vine and their pruning materials were weighed as an indicator of the vegetative vigour. the ratio of total yield to total pruning weight per vine, referred to as the ravaz index (champagnol, 1984) was used as an indicator of balance between fruit and vegetative growth. statistical analysis of variance on the obtained data was performed with separation of the means by lsd test at 5%. no year effect was observed, thus data shown are the average of the measurements of two years. 3. results tissue analysis showed in general no deficiency or excess in the levels. the effect of applied mo on the concentration of the nutrients in basal petioles, leaves and berries was small and of little practical importance. nitrogen (n) content of leaf, petiole and berry compared with control treatment was not influenced by mo (fig. 1 a). leaf and petiole phosphorous (p) content tended to decrease in leaf and petiole after mo supply, especially in the mox3 treatment (fig. 1 b), while berry content was not influenced. on the contrary, potassium (k) level was significantly higher in berries of motreated plants (fig. 1 c) at harvest. calcium (ca) content was not affected by any kind of treatment in all analyzed organs (fig. 2 a), while petiolar and leaf manganese (mn) tended to increase (fig. 2 b), especially at harvest time and for mox3 treatment. moreover, for the same treatment, a higher and significant level of mn was found in berries during both measurements (at veraison and harvest time). no significant differences were detected for the level of iron (fe) (fig. 2 c). co2 assimilation rate of mox3-treated vines was significantly higher than those of the mox1-treated and untreated vines (fig. 3 top). mox3 showed also relevant difference towards the other treatments in transpifig. 2 secondary macro (ca) and micronutrient (mn, fe) concentrations in basal leaf, petiole and berry at fruit set, veraison and harvest in motreated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. 41 ration rate (fig. 3 middle); as a result of the higher transpiration rate, however, the mox3 vines showed more critical water use efficiency values (fig. 3 middle). accordingly, the spad unit measurements were significantly influenced by mo treatments (fig. 3 bottom). mox3 showed the highest values in both sampling data; the values decreased for mox1 treated vines after veraison, although remained higher than those of the control plants. fig. 3 top: assimilation rates measured after veraison in mo-treated and untreated grapevine plants. middle: transpiration rates and water use efficiency measured after veraison in mo-treated and untreated grapevine plants. bottom: spad units measured after veraison and a month later in mo-treated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. different letters correspond to significant differences (p ≤ 0.05). mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. the yanc content increased significantly after the applications showing the positive role of mo on nr activity to provide an available form of n for the plant (table 2). furthermore, at veraison, after more than a month from the first (and single, for mox1) application, both mox1 and mox3 berries had higher levels of yanc compared to the control ones. surprisingly, no significant difference was found between the two mo treatments, not showing a linear correlation between mo dose and nr activity. in any case at harvest time, no effect was found for either of the mo treatments: the difference tended to decrease during berry ripening, disappearing at harvest when both the treated and untreated vines had quite the same amount of available n. significant differences in must soluble solids (ss) analysis at harvest were observed between mox1 treatment (higher value) and both mox3 and control treatments (lower values). titratable acidity (ta) and ph levels were similar for treated and untreated vines (table 3). total anthocyanin content was significantly greater in the untreated vines compared to the treated ones while extractable anthocyanins in mox1-treated and control plants were similar and higher than in mox3-treated vines (table 3). mox1-treated vines must extractability assay and mp value suggest a better polyphenolic evolution during ripening. mox3 treatment negatively influenced anthocyanin extractability and polyphenol evolution as confirmed by the highest value of mp. table 2 yeast assimilable nitrogen content (yanc) yanc (mg·l-1 n) mox1 mox3 untreated 96 a 92 a 74 b fruit set veraison harvest 66 a 62 a 34 b 68 ns 64 ns 55 ns different letters within the same column correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. table 3 berry soluble solids (ss), ph, titratable acidity (ta), berry glories indices (a1: total anthocyanins; a3.2: extractable anthocyanins; ea: extractability assay; mp: extractability of the tannins contained in the seed analysis results) technol. analysis mox1 mox3 untreated 23.9 a 22.1 b 22.6 b ss (brix) ph ta (g·l-1) 3.1 ns 3.0 ns 3.0 ns 7.3 ns 7.5 ns 7.3 ns different letters within the same column correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. polyphenol. analysis a1 (mg·l-1) a3.2 (mg·l-1) ea (%) mp (%) mox1 mox3 untreated 1535 b 1437 b 2038 a 1007 a 706 b 1005 a 34.4 b 50.9 a 50.7 a 29.6 b 40.5 a 32.0 b 42 the different ripening development between mox3 and untreated vines probably has to be linked to the influence of mo on the fruit and vegetative activity of the plant, suggested also by the average bunch weights (fig. 4 right) and the total pruning weight, which were higher in the mox3 treatment (fig. 4 left): plants treated with higher dose of mo had greater difficulty completing the ripening process within the same time as the other vines. no significant differences were however revealed with regard to total yield per vine (data not shown) and ravaz index (fig. 4 left). 4. discussion and conclusions the present study supports earlier suggestions that mo affects the n metabolism in grape, whose unquestionable evidence is in the yanc analysis of the berry. the fact that the reaction of the plants to mo applications was measurable, in terms of yanc, as early as a few days after the first treatments performed before fruit set gives interesting indications about its rate of speed. the absence of differences in yanc content between the two mo treatments suggests that when the general plant availability of n is good (like in this case), higher doses of mo result to be useless. this hypothesis, therefore, is not confirmed by the analysis of the bunch weight and total yield per vine nor in the development of the ripening process, when relevant differences were found between the two mo treatments, as well as a clearly positive correlation between the quantity of mo supplied and the appearance of such phenomena, well known to be connected with an increased n-availability for the plant. in fact, mo, especially when supplied at high rate, is able to increase plant activity (see results about gas exchange analysis) as the result of rendering the n of the plant more available for the metabolisms and, consequently, plants tend to be generally more vigorous. finally, important information about fertilization management can be underlined: a high dose treatment (mox3) seems to be inadequate and sometimes contrary to the quality of production. mox1-treated vines, on the other hand, did not show an influence of the treatment on vegetative and fruit balance nor on phenolic evolution and, interestingly, differentiated positively from the control regarding the sugar content of the berry and the yanc, even though its level at harvest was not optimal. assuming the mox1 dose and time optimal for quality, further analysis should be aimed at correlating the role of mo on nr activity towards n availability in order to understand how to increase must quality. references alexander d., 1957 seasonal changes in the nitrogen content of the sultana vine. aust. j. agr. res., 8: 162-178. bisson l.f., 1991 influence of nitrogen on yeast and fermentation of grapes, 128-131. in: rantz j.m. (ed.). proceedings of the international symposium on nitrogen in grapes and wines. am. soc. enol. vitic., davis, ca, usa. bortels h., 1930 molybdan als katalysator bei der biologischen stickstoffbindung. archiv für mikrobiologie, 1: 333342. champagnol f., 1984 élements de physiologie de la vigne et de viticulture générale. saint-gély-du-fesc, france, pp. 351. conradie w.j., 1991 distribution and translocation of nitrogen absorbed during early summer by two-year-old grapevines grown in sand culture. am. j. enol. viticult., 42(3): 180-190. coughlan m.p., 1980 aldehyde oxidase, xanthine oxidase and xanthine dehydrogenase: hydroxylases containing molybdenum, iron-sulfur and flavin, 119-185. in: coughan m.p. (ed.) molybdenum and molybdenum-containing enzymes. pergamon press, oxford, uk. fortescue j.a.c., 1992 landscape geochemistry: retrospect and prospect 1990. appl. geochem., 7: 1-53. glories y., 1984 la couleur des vins rouges. 2e. partie: mesure, origine et interpretation. conn. vigne vin, 18(4): 253-271. gupta u.c., 1997 soil and plant factors affecting molybdenum uptake by plants, 71-91. in: gupta u.c. (ed.) molybdenum in agriculture. cambridge university press, cambridge, uk. hansch r., mendel r.r., 2009 physiological functions of mineral micronutrients (cu, zn, mn, fe, ni, mo, b., cl). curr. fig. 4 bunch weight (left), total pruning weight and ravaz index (right) measured in mo-treated and untreated grapevine plants. right: error bars reflect the lsd at p ≤ 0.05. different letters correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. 43 opin. plant biol., 12(3): 259-266. hewitt e.j., 1983 molybdenum requirements of plants, pp. 277-330. in: robb d.a., and w.f. pierpoint (eds.) metals and micronutrients. uptake and utilisation by plants. pergamon press, oxford, uk. hille r., 1996 the mononuclear molybdenum enzymes. chem. rev., 96: 2757-2816. jiranek v., langridge p., henschke p.a., 1995 amino acid and ammonium utilization by saccharomyces cerevisiae wine yeast from a chemically defined medium. am. j. enol. viticult., 46(1): 75-83. kappl r., hüttermann j., fetzner s., 2002 the molybdenum-containing hydroxylases of quinoline, isoquinoline, and quinaldine, 481-538. in: sigel a., and h. sigel (eds.) molybdenum and tungsten. their roles in biological processes. marcel dekker, new york, usa. kunkee r.e., 1991 relationship between nitrogen content of must and sluggish fermentation, pp. 148-155. in: rantz j.m. (ed.) proceedings of the international symposium on nitrogen in grapes and wines. am. soc. enol. vitic., davis, ca, usa. löhnertz o., 1991 soil nitrogen and the uptake of nitrogen in grapevines. international symposium on nitrogen in grapes and wine, seattle, washington, pp. 1-11. longbottom m., dry p., sedgley m., 2004 foliar application of molybdenum pre-flowering: effects on yield of merlot. aust. grapegrower, 491: 36-39. masi e., boselli m., 2007 molibdeno si, ma con misura. vq, 1: 54-58. mendel r.r., hänsch r., 2002 molybdoenzymes and molybdenum cofactor in plants. j. exp. bot., 53(375): 16891698. rus’-ko e.a., 1979 the effect of soil and foliar nutrition with minor element on grapevine growth and productivity. sadovodstvo vinogradarstvo i vinodelie moldavii, 1: 30-32. solomonson l.p., barber m.j., 1990 assimilatory nitrate reductase: functional properties and regulation. annu. rev. plant physiol. plant mol. biol., 41: 225-253. spayd s.e., nagel c.v., edwards c.g., 1995 yeast growth in riesling juice as affected by vineyard nitrogen fertilization. am. j. enol. viticult., 46(1): 49-55. strakhov v.g., 1988 trace elements fertilizer application in vineyards. vliyanie udobrenii na obmen veshchestv i produktivnost’ rastenii, pp. 153-160. veliksar s.g., 1997 the effect of molybdenum on the changes in the aminoacid composition of grapevine shoots. sadovodostvo, vinogradarstvo i vinodelie moldavi, 5: 25-27. weinbaum s.a., klein i., broadent f.e., micke w.c., muraoka t.t., 1984 effects of time of nitrogen application and soil texture on the availability of isotopically labelled fertilizer nitrogen to reproductive and vegetative tissue of mature almond trees. j. am. soc. hortic. sci., 109: 339-343. westerveld s.m., mckeown a.w., mcdonald m.r., scott-dupree c.d., 2003 chlorophyll and nitrate meters as nitrogen monitoring tools for selected vegetables in southern ontario. acta horticulturae, 627: 259-266. williams c.m.j., maier n.a., bartlett l., 2004 effect of molybdenum foliar sprays on yield, berry size, seed formation, and petiolar nutrient composition of ‘‘merlot’’ grapevines. j. plant nutr., 27: 1891-1916. impaginato 151 1. introduction tomato as one of the most widely produced and consumed ‘vegetable’ in the world (heuvelink, 2005) contains high levels of antioxidant active compounds such as vitamin c, polyphenlos and carotenoids (tommonaro et al., 2012). salicylic acid (sa) has been the focus of intensive research due to its role in plant defense mechanisms and response to abiotic stresses (rivas-san vicente and plasencia, 2011). besides, it is stated that sa plays a crucial role in physiological and biochemical processes during the entire lifespan of the plant (rivas-san vicente and plasencia, 2011). sa as an endogenous plant growth regulator controls a large variety of physiological processes: from regulatory signal in plants mediating defense against pathogens, to ethylene biosynthesis, action and inhibition. it is also involved in plant responses to abiotic stress conditions such as salt and osmotic stresses (khalil, 2014). exogenous application of sa also results in many different changes in plant physiological processes and reactions such as prevention of ethylene production (khan et al., 2003); increases in plant height, number of branches, number of leaves (saharkhiz et al., 2011) and antioxidant activity (ananieva et al., 2004). most of the researches on sa have focused on mediating local and systemic plant defense and resistance to biotic and abiotic stresses (atkinson and urwin, 2012), while studies on its effect on physiological, biochemical and quality features of fruit are limited (ali et al., 2014). since plant growth, development and the level of bioactive compounds especially antioxidant active substances depend on the cultivar, and by agronomic and environmental conditions (tommonaro et al., 2012), the aim of this work was to study the influences of sa on growth, fruit quality attributes including fruit firmness and storability, vitamin c, antioxidant activity, total phenolic, flavonoids and yield of tomato under greenhouse conditions. adv. hort. sci., 2016 30(3): 151-157 doi: 10.13128/ahs-20277 salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato j. javanmardi, n. akbari department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. key words: antioxidant activity, flavonoids, solanum lycopersicum, total phenolics, yield components. abstract: most of the researches on salicylic acid (sa) have focused on postharvest application or acquiring stress resistance, while studies on its effect on plant growth, secondary metabolites and fruit quality are limited. sa as foliar application (0, 150, 300 and 450 mg/l) at different plant growth stages on fruit yield, secondary metabolites and quality features of tomato (solanum lycopersicum l. cv. kardelen) under greenhouse conditions were evaluated. the highest fruit yield per plant (about 1.3-fold greater than control) was obtained from 300 mg/l sa when applied three weeks after fruit set. comparing to control plants, the highest fruit firmness, 10 days prolonged storability, highest total phenolics (22.6 mg gallic acid equivalent per 100 g fw); and highest antioxidant activity (65.11) were observed when 450 mg/l sa applied at fruiting stage and 3 weeks later. an increasing pattern in ascorbic acid content was observed with increasing sa concentration irrespective to application time. the same concentration effect was observed in flavonoid content when plants treated at 3 weeks after fruiting. the highest effect of flavonoids on antioxidant activity was calculated using pearson correlation (r=0.82). sa concentrations greater than 450 mg/l showed significant adverse effects on all measured traits. the effect of exogenous sa on tomato plant depends on the developmental stage and sa concentrations tested. improved fruit quality factors may happen in a certain concentration range, while over that may have negative or adverse effect. (*) corresponding author: javanm@shirazu.ac.ir received for publication 1 may 2016 accepted for publication 9 august 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 151-157 152 2. materials and methods plant material and experimental design the tomato (solanum lycopersicum cv. kardelen) seeds were obtained from gento seeds® co., turkey. six weeks old seedlings having 4-5 leaves were transplanted into a 500 m2 polycarbonate greenhouse siltloam soil (greenhouse research center, college of agriculture, shiraz university, shiraz, iran) at 50 cm double rows and 150 cm between rows spacing (density of 2 plants/m2). the greenhouse conditions were set to temperature of 25±2°c and relative humidity of 60-70% during the entire study. soil was already fertilized according to the soil test results and certified lab recommendations for growing greenhouse tomato (papadopoulos, 1991). treatments consisted of sa (merck millipore corporation, germany) foliar application at four concentrations (0, 150, 300, and 450 mg/l) at different growth stages (transplant establishment, onset of flowering, fruit set, 3 weeks after fruit set) and their all possible combinations (table 1). the experiment was arranged in a completely randomized design with three replications. each sample for analyses consisted of three fruits (per plant) of four plants for each replicate per treatment. measurement methods fruits were harvested at mature red stage based on the “color classification requirement in united states standards for grades of fresh tomatoes” chart, published by usda. tomato produces fruits on clusters. fruits of two first clusters were considered as yield and expressed as kg/plant. fruit firmness was measured as penetration force on the fruit flesh (over the fruit locules) using force gauge, fg-5005 (lutron electronic enterprise co. taipei, taiwan) with a probe diameter of 8 mm. the average values obtained for each fruit was calculated and expressed as newton. fruit storability was measured as the number of days from keeping red fruits in a storage at 12±1°c and 80% relative humidity to starting fruit shrinkage or lose their shiny appearance. vitamin c quantification was performed according to the method described by the aoac (1984) and results were expressed as mg ascorbic acid per 100 grams of fruit dw. total phenolic content (tpc) was determined with the folin-ciocalteau reagent using the method of spanos and wrolstad (1990) and the results of three replicates were expressed as mg gallic acid equivalent per 100 grams of fruit dw (mg gae/100g dw). measured trait salicylic acid (mg/l) application time establishment flowering fruiting 3 weeks after fruiting establishment + flowering establishment + fruiting establishment + 3 weeks after fruiting flowering + fruiting flowering + 3 weeks after fruiting fruiting + 3 weeks after fruiting all 4 times fruit firmness (n) 150 9.97±0.47 no 10.76±0.59 mn 12.59±0.13 kl 12.20±0.21 lm 12.33±0.14 lm 12.62±0.24 kl 12.27±0.05 lm 12.67±0.36 kl 10.89±0.64 mn 13.49±0.99 j-l 13.94±0.20 i-k 300 13.96±0.35 i-k 14.69±0.68 g-j 16.14±0.82 e-g 14.51±0.39 g-i 15.07±0.24 f-j 15.56±0.12 e-h 14±0.50 h-k 15.33±0.28 f-i 14.38±0.07 g-i 16.11±0.52 e-g 16.10±0.65 e-g 450 16.19±0.83 e-g 16.28±0.87 e-g 19.92±0.78 a 16.95±0.21 c-e 16.23±0.40 e-g 18.28±0.31 bc 16.45±0.37 ef 18.86±0.12 ab 16.60±0.32 d-f 18.89±1.17 ab 18.10±0.54 b-d control 9.07±0.51 o shelf life (days at 12°c) 150 21.00±0.57 d 21.66±0.33 b-d 21.66±0.33 b-d 22.00±1.0 b-d 21.66±0.23 b-d 21.66±0.66 b-d 21.66±0.31 b-d 21.33±0.33 cd 21.66±0.41 b-d 22±0.57 b-d 22±0 b-d 300 21.66±0.33 b-d 22±0.57 b-d 22±0.51 b-d 22.33±0.33 b-d 22.00±0.54 b-d 22.33±0.33 b-d 22.33±0.13 b-d 22.33±0.34 b-d 22±0.02 b-d 22.33±0.33 b-d 22.33±0.33 b-d 450 22.33±0.66 b-d 22±0.57 b-d 22.33±0.88 b-d 24.66±1.33 a 21.83±0.44 b-d 22.33±0.33 b-d 22.66±0.88 bc 22.66±0.23 bc 23±0.57 b 23±0.57 b 22.33±0.33 b-d control 17.00±0.00 e vitamin c (mg/100 g dw) 150 13.88±0.35 pq 13.99±0.35 pq 14 39±0.42 o-q 18.28±0.26 l-o 15.42±0.81 n-q 16.92±0.62 m-p 19.12±0.51 l-n 14.74±0.39 o-q 20.37±0.92 k-m 21.03±1.24 j-l 21.87±1.90 i-l 300 23.56±0.22 h-k 24.91±1.63 h-j 27.15±1.25 e-h 28.47±1.63 b-g 25.10±0.68 h-g 25.13±0.13 h-g 26.01±0.48 f-h 27.30±0.51 d-h 27.52±1.53 d-h 27.81±1.06 c-g 29.35±2.28 b-f 450 31.11±0.79 a-e 31.62±0.89 a-c 32.47±0.12 ab 32.50±0.09 ab 31.18±0.73 a-e 31.26±0.72 a-d 31.95±0.92 ab 31.73±0.92 a-c 32.25±0.38 ab 34.34±2.07 a 32.50±0.84 ab control 11.82±0.24 q total phenolics (mg gae/100 g dw) 150 14.19±0.09 l 14.51±0.16 k 15.60±0.25 i 16.14±0.14 gh 15.12±0.10 j 15.18±0.10 j 15.71±0.05 i 14.59±0.23 k 15.70±0.20 i 15.84±0.11 hi 16.26±0.22 g 300 19.66±0.17 f 19.73±0.14 f 20.13±0.12 e 20.24±0.06 e 20.04±0.06 ef 20.12±0.065 e 20.13±0.06 e 19.99±0.12 ef 20.22±0.03 e 20.26±0.05 e 21.45±0.06 d 450 21.47±0.03 d 21.71±0.29 b-d 22.64±0.09 a 22.67±0.12 a 21.54±0.07 cd 21.51±0.09 cd 21.59±0.11 cd 21.49±0.02 cd 21.88±0.18 bc 21.73±0.19 b-d 22.01±0.06 b control 10.84±0.10 m flavonoids (mg gae/100 g dw) 150 1.05±0.08 n 1.06±0.08 n 1.06±0.01 n 1.09±0.03 kl 1.09±0.08 kl 1.07±0.03 mn 1.09±0.08 lm 1.08±0.03 lm 1.08±0.01 lm 1.08±0.09 lm 1.09±0.06 j-l 300 1.09±0.06 j-l 1.13±0.04 i-k 1.11±0.07 h-j 1.14±0.01 b-e 1.09±0.06 j-l 1.09±0.09 j-l 1.12±0.06 g-i 1.10±0.05 j-l 1.12±0.03 f-h 1.14±0.09 d-g 1.09±0.10 j-l 450 1.14±0.06 d-g 1.14±0.06 c-f 1.13±0.01 e-g 1.18±0.05 a 1.14±0.07 c-f 1.14±0.05 c-f 1.16±0.06 bc 1.14±0.01 d-g 1.15±0.07 b-d 1.16±0.08 ab 1.14±0.08 c-f control 0.99±0.07 o table 1 effect of salicylic acid foliar application at different tomato plant phonological stages on some fruit characteristics averages±sd for each measured trait with the same letters showing no significant differences using lsd test at p<0.05. control plants did not receive any sa at any time. javanmardi and akbari salicylic acid effects on greenhouse tomato 153 total antioxidant activity (taa) was measured using dpph (2,2-diphenil-1-picrylhydrazyl) (merck millipore corporation, germany) assay as described by patras et al. (2009) at the absorbance of 517 nm using micro plate reader (epoch, germany). taa was calculated according to the following equation: taa = [1(a sample at 517nm /a control at 517nm )]×100 total flavonoid content (tfc) was determined by the aluminum chloride colorimetric assay based on the formation of a complex flavonoid-aluminum, having a maximum absorbance at 510 nm (toor and savage, 2005) and results expressed as mg gallic acid equivalent per 100 gram of fruit dw (mg gae/100 g dw). statistics data were analyzed using sas 9.1 statistical software (sas institute inc., cary, nc, usa). means were compared using lsd test at p ≤ 0.05. pearson correlation statistical method was used to determine the correlation between secondary metabolites and antioxidant activity. 3. results and discussion our preliminary tests on different concentrations of sa application showed significant adverse effects on all measured traits when concentration was higher than 450 mg/l (data not shown). this could be due to the nature of sa that acts as a plant growth regulator. it is stated that the responses to sa are highly concentration dependent, so that moderate doses of sa improve features such as antioxidant status and induce stress resistance, while higher concentrations trigger a hypersensitive cell death pathway (tounekti et al., 2013). fruit yield fruits of the first two clusters were evaluated as yield. the greatest yield was obtained from 300 mg/l sa applied at three weeks after fruit set (fig. 1). this was 1.26 fold greater than control. the stimulatory effect of sa on flowering regulation which has been well known for a long time (raskin, 1992; rivas-san vicente and plasencia, 2011) would eventually affect total number of fruits (ondrašek et al., 2007) and enhance efficiency in fruit production. previously, increased yield of strawberry (aghaeifard et al., 2015) and tomato (javaheri et al., 2012) has been related to promoted cell division and cell enlargement due to sa (hayat et al., 2010) through its influence on other plant hormones such as auxin, cytokinin and aba balances (shakirova, 2007) and enhanced net photosynthetic rate, internal co2 concentration and water use efficiency (fariduddin et al., 2003). it is reported that in non-thermogenic plants such as tobacco, sa levels increase 5and 2-fold in their leaves at the initiation of or during transition to flowering, respectively (abreu and munné-bosch, 2009). fruit firmness firmness is an important physical parameter for postharvest storage, transportation and monitoring the fruit ripening process. sa and its derivatives are widely in use to enhance fruits postharvest life by enhancing fruit firmness during storage (wang et al., 2006). in our experiment, sa significantly affected fruit firmness. the firmness became 2.9 times higher than the control group when 450 mg/l sa was applied at fruiting stage. plants treated with 150 mg/l sa at establishment stage showed the lowest fruit firmness as equal to control plants (table 1). a 2-fold greater tomato fruit skin thickness due to 0.01m sa application comparing to control has been previously reported (javaheri et al., 2012). metabolic reactions respiration, ethylene production (aktas et al., 2012) breakdown of cell wall and activation of enzymes involved therein are key events in fruit ripening and softening (prasanna et al., 2007). these metabolic activities can be harmful to maintain fruit quality. there are reports that sa could prevent the activity of such enzymes, while affect the swelling of cells in a manner that results in firmer fruit (zhang et al., 2003; prasanna et al., 2007). sa prevents fruit softening. shafiee et al. (2010) have cited several reports indicating that rapid softening of fruits during ripening was simultaneous with rapid decrease in endogenous sa of fruits. increased firmness in climacteric fruits due to pre-harvest sa spray has been attributed to the role of sa in preventing cell wall and membrane degrading enzymes (polygalacturonase, fig. 1 effect of salicylic acid (sa) foliar application at different tomato plant phenological stages on yield (two first clusters) at greenhouse condition. transplant establishment (est), flowering (fl), fruiting (fr), three weeks after fruiting (3wfr). adv. hort. sci., 2016 30(3): 151-157 154 lipoxygenase, cellulase, pectinemethylesterase), ethylene production (zhang et al., 2003) and reduced hydrolysis of soluble starch and therefore higher firmness (tareen et al., 2012 b), while in non-climacteric fruits such as “flame seedless” grape it has been attributed to the role of sa in preventing decay (khalil, 2014). fruit storability the greatest fruit storability period was observed in 450 mg/l sa when applied at 3 weeks after fruiting (table 1). this was over 10 days more than control plants. aghdam et al. (2014) attributed longer storability and higher chilling resistance of detached tomato fruits treated with sa to increased endogenous proline content. lowered ethylene biosynthesis has been also considered to be the main cause of prolonged storability due to regulatory potential of exogenous sa on fruit ripening of green mature tomato fruits (kant et al., 2013). parallel to this, sa can activate the alternative respiration pathway in many plant tissues which results in a lower respiratory rate and delay in the climacteric peak (raskin, 1992). srivastava and dwivedi (2000) stated that the concentration of sa determines the extent to which these effects actualize. reduced the quality loss during storage due to sa were previously reported for tomato (ding et al., 2001) and sweet peppers (fung et al., 2004). vitamin c human diet consists of about 91% of ascorbic acid coming from fruits and vegetables (tareen et al., 2012 a). an increasing pattern of ascorbic acid content was observed with increasing sa concentration irrespective to application time (table 1). the greatest ascorbic acid content was obtained from 450 mg/l sa. in most cases, this increase was about 3 times than control plants. some researches indicated that the treatment of tomatoes (javaheri et al., 2012; kalarani et al., 2002) and strawberry (aghaeifard et al., 2015) with sa caused them to acquire higher levels of ascorbic acid comparing to control plants. sa can activate ascorbate peroxidase, which is the precursor to ascorbic acid in fruits and prevents vitamin c from being destroyed in cells and therefore causes the accumulation of ascorbic acid in the fruit (wiśniewska and chełkowski, 1999). total phenolics phenolic compounds as secondary plant metabolites are synthesized by all plants and responsible for the flavor and color of fruit products (jeong et al., 2008). the highest amount of phenolic compounds was observed in plants treated with 450 mg/l sa during the fruiting stage and three weeks after the fruiting (table 1). in those treatments, a 2.9 times higher phenolic compounds content than control plants was observed. although aghdam et al. (2012) reported no significant effect of sa application on total phenolics content of mature green tomato fruits, our results were similar to reports on sweet cherry (valero et al., 2011) and grapes (ranjbaran et al., 2011; khalil, 2014), which all concluded sa application induced greater total phenolics and other secondary metabolites with antioxidant properties (ranjbaran et al., 2011). previously, the application time of sa on sweet cherry at three fruit developmental stages (pit hardening, initial color changes and onset of ripening) increased fruit weight and led to higher concentration of total phenolics and total anthocyanins, as well as higher antioxidant activity (giménez et al., 2014). antioxidant activity it is well known that the positive effect on health associated with tomato consumption is exerted by the pool of antioxidants, with noticeable synergistic effects (tommonaro et al., 2012). the highest antioxidant activity was observed in 450 mg/l sa treatment when applied on fruiting stage plus three weeks after fruiting. this was 1.84 times greater than the control, which had the least antioxidant activity. an increasing pattern in antioxidant activity was observed with increasing sa concentration, irrespective to application time (fig. 2). the same pattern was previously found in orange (huang et al., 2008 b) and pears (cao et al., 2006). increased total antioxidant activity of strawberry due to sa has been previously reported (aghaeifard et al., 2015). regular applications of salicylic acid at different stages of plant growth and fruit development can increase the antioxidant activity fig. 2 effect of salicylic acid (sa) foliar application at different tomato plant phenological stages on fruit total antioxidant activity at greenhouse condition. transplant establishment (est), flowering (fl), fruiting (fr), three weeks after fruiting (3wfr). javanmardi and akbari salicylic acid effects on greenhouse tomato 155 (shakirova, 2007). it is frequently hypothesized that sa has direct physiological effects on the activity of antioxidant enzymes which promote the synthesis of metabolites existing in fruits and vegetables, especially those with nutritional value in the product (huang et al., 2008 a). flavonoids flavonoids comprise a diverse group of natural compounds and are among the best-known natural phenols, exhibiting an array of chemical and biological pathways such as radical scavenging and antimicrobial activities. an increasing pattern in the amount of flavonoids was found by increasing sa concentration irrespective to application time. the highest amount of flavonoids was observed in 450 mg/l sa treatment when plants were treated either in three weeks after fruiting or fruiting plus three weeks after fruiting stages with about 1.18 times greater than control treatment (table 1). reports have been cited stating that exogenous sa applications boost the accumulation of flavonoids in several plant species (tounekti et al., 2013). on the other hand, research has proved that flavonoids possess antibiotic activities (al-matani et al., 2015). this can generate debate as to whether higher amounts of flavonoids can contribute to a longer shelf life against the rot of perishable fruits like tomato. when vegetables are heated for special purposes in food industries, excess heat can cause degradations in flavonoids and thus can reduce its overall content (sharma et al., 2015). correlation between secondary metabolites and antioxidant activity a pearson correlation analysis was performed to determine relationships between the individual parameters phenolic compounds, flavonoids and ascorbic acid which contribute in antioxidant activity. significant correlations were found for all measured traits with the highest effect of flavonoids on antioxidant activity; however, it was not a simple sum of their contribution (table 2). this has been related to synergistic effect among all antioxidants and their interactions with other constituents of the fraction (jimenez et al., 2002; lenucci et al., 2006). similar to our results, ilahy et al. (2011) found a good significant correlation between antioxidant activity and main antioxidants (vitamin c, flavonoids and total phenols). given the key role of sa in increasing ascorbic acid (dat et al., 1998), total phenolics and other secondary metabolites with antioxidant properties (ranjbaran et al., 2011), the rise in antioxidant activity can thus be explained. 4. conclusions treatments with sa could be a promising tool to improve tomato yield, fruit quality attributes and health beneficial compounds (including phenolic compounds, vitamin c and flavonoids having antioxidant activity) because of its diverse regulatory roles in plant metabolism. the effect of exogenous sa on plant depends on the plant species, developmental stage, and the sa concentrations tested. fruit setting stage and 3 weeks later are the best two important stages for sa application. a concentration of 300 mg/l sa for increased yield and 450 mg/l sa for improved fruit quality attributes are recommended. it is 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ricerche, via madonna del piano, 10, 50019 sesto fiorentino (fi), italy. ** centro sperimentale orticolo “po di tramontana”, veneto agricoltura, via moceniga, 7, 45010 rosolina (ro), italy. key words: cryopreservation, genetic resources, pvs2, rapd, red chicory, vitrification. abstract: shoot tips from in vitro stock plants of red chicory ‘rosso di chioggia’ line were cryopreserved by one-step vitrification. after two days of cold-hardening on hormone-free ms medium and loading for 30 min in a mixture of 2 m glycerol and 0.4 m sucrose at 25°c, shoot tips were dehydrated with pvs2 vitrification solution at 0°c for 60 min and plunged directly into liquid nitrogen. the post-thaw survival of shoot tips was achieved 79% when was cultured on recovery medium containing 0.5 mm ba. observed regrowth, after six weeks of culture in the same medium composition, was 100%. rooted cryopreserved microshoots showed good quality when transferred to the greenhouse. preliminary results proved that the genetic fidelity of the cryopreserved line was maintained. the same vitrification protocol was then applied to three other red chicory lines, ‘rosso di treviso precoce’, ‘rosso di treviso tardivo’ and ‘castelfranco’. a simple and effective protocol for the cryopreservation of red chicory shoot tips has been successfully developed as a result of this study. 1. introduction in italy, the major production area of red chicory (cichorium intybus l. var. intybus), covering about 9 thousand hectares, is located in the veneto region, one of the most economically important areas for vegetable production. an ancient typology, ‘rosso di treviso tardivo’, that was progenitor of the present varieties of red chicory, was introduced into italy in the 15th century. over time, growers selected those plants having good production, while in recent years this leafy vegetable has undergone intense selection and breeding work, and several improved typologies have been produced (such as ‘rosso di treviso precoce’, ‘rosso di verona’, ‘rosso di chioggia’, ‘variegato di castelfranco’, and others) (veneto agricoltura, 2002), which are highly appreciated for their quality and productivity. at the “po di tramontana” experimental farm in rosolina (veneto agricoltura, rovigo, italy) a specific breeding program to select high-performance lines has been continuing for many years. every year after infield evaluation, the most valuable lines are introduced and maintained in vitro by subculturing every three weeks. the stock plants obtained from these lines are transferred to the greenhouse to produce high quality seeds to be used for the production of high quality red chicory. the costs of stock culture maintenance and the risks of contamination and decay of lines can be reduced by introducing cryopreservation as a tool for a long-term preservation. cryopreservation involves the maintenance of plant propagules at ultra-low temperatures (-196°c, ln): under these conditions, biochemical and most physical processes are completely arrested and as such, plant material can be stored for unlimited periods. cryopreservation studies have been reported for belgian endive (cichorium intybus l. var. foliosum, cvs. flash, rumba and carolus) by controlled-rate freezing (demeulemeester et al., 1992; 1993) and encapsulation-dehydration techniques (vandenbussche et al., 1993). controlled-rate freezing is regarded as the traditional approach to plant cryopreservation. although controlled-rate freezing has been effective for cryopreserving differentiated tissues (reed and uchendu, 2008), reports on cryostorage using this technique are limited. one of the practical limitations of the controlled-rate freezing approach is the need for an expensive programmable freezer (engelmann, 2000). however, applying traditional (controlled-rate freezing) and new procedures (one-step freezing technique), the first examples of “cryogenic banks” are available today in several countries (reed, 2001; sakai and engelmann, 2007). at the same time, the development 45 of a vitrification system has made it possible to increase the number of plant species that can be cryopreserved (towill and bajaj, 2002; lambardi and de carlo, 2003, 2009; sakai et al., 2008). in the present study, a vitrification/one-step freezing procedure was developed using ‘rosso di chioggia’ shoot tips, with exposure of explants to the vitrification solution and rapid cooling by directly immersing in liquid nitrogen (ln). this procedure was then applied to three different red chicory typologies. in addition, validation of this method using molecular markers is presented with regard to maintenance of genetic stability. 2. materials and methods plant material microshoots of red chicory ‘rosso di chioggia (mp c7 4/212) were adventitiously induced from leaf portions through direct organogenesis (fig. 1 a). the lines were obtained by isolating each adventitious shoot and transferring them onto a semi-solid ms (murashige and skoog, 1962) medium, supplemented with 30 g l-1 sucrose, 1.0 µm 6-benzyladenine (ba) and 7 g l-1 agar (proliferation medium). the ph of the medium was adjusted to 5.8 before autoclaving for 20 min at 121°c. the shoot cultures were placed at 21±1°c under fig. 1 cryopreservation of red chicory “rosso di chioggia” line (a-f). a microshoots induced adventitiously from a leaf portion; b a shoot tip just after excision from a shoot bud; c shoot tip survival after three weeks and a well-formed shoot tip after cryopreservation with 60 min of pvs2 treatment (detail); d regrowth in plastic cylinder on ms medium with 0.5 µm ba; e cryopreserved rooted microshoot; f plantlets from cryopreservation, after potting and acclimation in the greenhouse. 46 12-hr photoperiod conditions (60 µmol m-2 s-1 photosynthetically active radiation) and subcultured every three weeks (standard culture conditions). short-day conditions avoided floral initiation. vitrification procedure before bud excision, stock plants were exposed to 4°c with an 8-hr photoperiod for a three-week coldhardening period. shoot tips (2-3 mm long), consisting of the apical meristem and 4-5 leaflets, were then excised from microshoots (fig. 1b) under a stereo microscope in aseptic conditions, and were precultured on hormone-free ms medium supplemented with 0.09, 0.3 or 0.7 m sucrose for two days at 4°c under an 8-hr photoperiod to determine the effect of sucrose pretreatment on cryopreserved explant survival. to induce dehydration, the shoot tips were loaded with a cryoprotectant (loading solution, ls; 2 m glycerol and 0.4 m sucrose) (matsumoto et al., 1994) for 30 min at 25°c in 2-ml nalgene® cryovials (10 shoot-tips per cryovial) and subsequently incubated at 0°c with the pvs2 (plant vitrification solution 2; 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dmso in ms medium containing 0.4 m sucrose) (sakai et al., 1990). in order to choose the longest possible exposure time of shoot tips to the vitrification solution while avoiding toxic effects, shoot tips were exposed to pvs2 for 30, 60, 90 or 120 min and evaluated for shoot tip survival. after dehydration, the samples were suspended in 0.6 ml of fresh pvs2 solution and rapidly frozen to –196°c by direct immersion in ln, where they were stored for at least 2 hr. for recovery, they were quickly rewarmed by plunging the cryovials into a 40°c waterbath (warming rate: about 150°c min-1), unloaded from the pvs2 solution, washed for 20 min at 25°c in a liquid ms medium containing 1.2 m sucrose and finally plated onto the proliferation medium and maintained under standard culture conditions. cryopreserved shoot tips were assessed for survival after three weeks. in addition to proliferation medium, five different recovery media were tested to improve the regrowth of cryopreserved shoot tips: ms added with 0.5, 1.0 or 5.0 µm ba, ms with 1 µm thidiazuron (tdz) and ms with 0.5 g l-1 activated charcoal (ac). the role of ac has been discussed in many reports on plant tissue culture with different effects (thomas, 2008). survival was defined as a percentage of green shoot tips after three weeks from thawing. explants were transferred to plastic tubes (one shoot for each tube) and six weeks after ln treatment the percentage of plants demonstrating regrowth was assessed. explants treated with pvs2 but not exposed to ln were used as controls. in vitro rooting of cryopreserved shoots cryopreserved shoots were transferred to jars containing 100 ml of ms rooting medium with 20 g l-1 sucrose, 7 g l-1 agar and supplemented with 2.5 µm indole butyric acid (iba). the jars were kept in a climatic chamber, at 21°c, under a light intensity of 40 µmol m-2 s-1 and a 12-hr photoperiod. after four weeks, the rooted microplantlets were transferred to ex vitro conditions. the plantlets were transferred to the greenhouse into pots with substrate containing both sterilised peat and sand in a ratio of 2:1. after five weeks, the plants were transferred to the field and their morphological stability was evaluated. cryopreserved chicory lines the cryopreservation procedure, optimized for ‘rosso di chioggia’ shoot tips, was then tested on shoot tips from in vitro plants of three red chicory lines, ‘rosso di treviso precoce’ (tvp s5), ‘rosso di treviso tardivo’ (tvt) and ‘variegato di castelfranco’ (c90 s6’). all three lines are included in a program to safeguard and conserve red chicory in the veneto region. encapsulation-vitrification procedure ‘rosso di chioggia’ shoot tips, cold-hardened at 4°c for two days, were encapsulated into 2% na-alginate beads and treated with ls solution for 1 hr on a rotary shaker at 25°c. when this solution was removed, the beads were dehydrated by exposure to pvs2 for 2, 3 or 4 hr at 0°c, then placed in cryovials (five beads per cryovial) and plunged immediately into ln. encapsulated shoot tips were thawed in a waterbath at 40°c for 3 min and placed on ms proliferation medium. survival was assessed after three weeks. data analyses a minimum of 30 samples were used for each treatment and each experiment was repeated twice. statistical analysis of percentages was carried out by the χ2 test or non-parametric statistical test, the post hoc multiple comparison test (marascuilo and mcsweeney, 1977), both at p ≤ 0.05. genetic stability assessments of cryopreserved plantlets random amplified polymorphic dna (rapd) analysis was applied on red chicory in vitro shoots to evaluate the genetic stability of cryopreserved and non-cryopreserved shoots (untreated control) collected from the same stock cultures. genomic dna was extracted from approximately 100 mg of leaves with a ‘dneasy plant mini kit’ (quiagen). dna concentration was determined using a spectrophotometer at 260 nm, and an aliquot of dna was diluted to a working concentration of 20 ng µl-1. rapd profiles were generated using 24 arbitrary 10-mers as primers, of which 10 primers were then selected for the reproducibility, the legibility and the 47 stability of the rapd pattern: 1253, 1247, m2, m3, m10, m13, a1, a5, a9 and c7 (table 1). the amplification of dna was performed according to vettvori et al. (1996). fragments were separated on 2% agarose gels by electrophoresis and visualized by ethidium bromide staining under uv light. for each primer, amplification reactions were repeated at least twice and only those having reproducible band partners were used. minor fragments, which tend to be unstable in staining intensity, and therefore not reliable, were not considered. 3. results influence of pvs2 times and preculture treatments prolonged exposure at 0°c to the vitrification solution appeared to be harmful for explants. indeed, shoottip survival decreased after exposures of 90 min or more, while 30-min treatment was not enough to protect the explants during ultra rapid freezing (table 2). when the incubation time was limited to 60 min, more than 72% of cryopreserved (+ ln) shoot tips survived after plating onto the proliferation medium (fig. 1c). incubation for 90 and 120 min in the pvs2 solution showed even a slight decrease in percentage survival of shoot tips without freezing (ln). this result highlights the importance of incubation time with vitrification solution for shoot tip survival. to enhance the shoot tips’ osmotolerance to the vitrification solution, preculture treatments with different sucrose concentrations for two days at 4°c was applied before the ls and pvs2 treatments. however, frozen explant survival exhibited a significant decline when the shoot tips were pre-cultured on media containing high sucrose concentrations (table 3), indicating that sucrose treatments are not appropriate to improve red chicory shoot tip survival. all three red chicory lines can be successfully cryopreserved by loading shoot tips in pvs2 for 60 or 90 min, prior to freezing in ln. among the lines, maximum explant survival ranged between 65% in ‘rosso di treviso precoce’ and 76% in ‘rossso di treviso tardivo’ and ‘variegato di castelfranco’, respectively with 60 and 90 min of pvs2 treatment (table 4). recovery media the addition of ba to the recovery medium was found to be beneficial for post-thaw recovery of the shoot tips, even if with an increase of the concentration callus formation is stimulated. the highest post-thaw survival of ‘rosso di chioggia’ shoot tips was obtained with 0.5 µm ba (79%) (table 5). when 1 µm tdz was used, 33% of shoot tips survived, but after the first subculture (21 days), they stopped growing, after which no shoots developed. there was no survival of shoot tips on hormone-free medium supplemented with activated charcoal. table 1 nucleotide sequences of dna primers used for rapd analysis primer primer sequence 1253 1247 m2 m3 m10 m13 a1 a5 a9 c7 gtt tcc gcc c aag agc ccg t aca acg cct c ggg gga tga g tct ggc gca c ggt ggt caa g cag gcc ctt c agg ggt ctt g ggg taa cgc c gtc ccg acg a table 2 survival of cryopreserved shoot tips of ‘rosso di chioggia’ after exposure to pvs2 for different time periods. data were recorded three weeks after thawing (ln, liquid nitrogen) pvs2 exposure time (min) shoot tip survival (%)(z) 30 60 90 120 100 a 100 a 80 b 80 b ln + ln 33.5 b 72.5 a 40.0 b 30.0 b (z) in each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. table 3 effect of sucrose concentration in preculture medium (two days at 4°c) on ‘rosso di chioggia’ shoot tips cryopreserved following 60 min pvs2 treatment sucrose concentration (m) shoot tip survival (%)(z) 0.09 0.3 0.7 68.0 a 47.5 a 17.5 b (z) percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. table 4 shoot tip survival percentage of three selected red chicory lines, following incubation and immersion in ln red chicory line loading time with pvs2 (min) (z) for each select line and each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the χ2 test. shoot tip survival (%)(z) ‘rosso di treviso precoce’ ‘rosso di treviso tardivo’ ‘variegato di castelfranco’ ln + ln 60 90 60 90 60 90 80.0 a 70.0 a 80.0 a 90.0 a 90.0 a 100 a 65.0 a 55.0 a 66.7 a 76.7 a 50.0 a 76.0 b table 5 effect of recovery media on ‘rosso di chioggia’ shoot tip survival (three weeks) and regrowth (six weeks) after thawing recovery medium 0.5 µm ba 1.0 µm ba 5.0 µm ba 1.0 µm tdz 0.5 g/l ac 79.1 a 47.6 b 47.8 b 33.3 c 0 shoot tip survival (%) shoot tip regrowth (%)(z) 100 a 95.0 a 95.4 a 0 -(z) in each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. 48 a further increase of regrowth was easily achieved by transferring the explants in tubes containing proliferation medium with ba. the best concentration for the development of ‘rosso di chioggia’ chicory shoots was again 0.5 µm. indeed, the plantlets obtained from this cytokinin treatment were generally taller and produced more shoot apices (fig. 1d). after in vitro rooting (fig. 1e), the microplantlets were transplanted into pots and successfully acclimated in vivo (fig. 1f). field observations of the plantlets confirmed the morphological stability of acclimatized plants with the original mother plants (fig. 2). vitrification vs encapsulation-vitrification cryopreservation by shoot-tip vitrification, using the pvs2 solution, was compared with the encapsulation-vitrification procedure in ‘rosso di chioggia’ line. the highest survival percentage of cryopreserved shoot tips was obtained by vitrification procedure (table 6). fig. 2 experimental field, the arrow indicates the plot with cryopreserved ‘rosso di chioggia’ plants. cryogenic procedure shoot tip survival (%)(z) vitrification encapsulation vitrification 76.0 a 35.2 b (z) percentages followed by different letters are significant at p ≤ 0.05 by the post hoc multiple comparison test. the encapsulation-vitrification procedure proved to be less effective for the cryopreservation of the selected red chicory line. however, only a maximum of 35% explant survival was achieved when the longest treatment of the beads with pvs2 was applied; this percentage is markedly lower than that reported in the literature using an encapsulation-dehydration procedure in chicory (vandenbussche et al., 1993). assessment of molecular stability by rapd to assess the genetic fidelity of plantlets regrown from cryopreserved shoot-tips, the rapd patterns were compared with untreated samples of the same red chicory lines. out of 24 primers screened, 10 selected primers produced clear and reproducible bands. the number of bands for each primer varied from five to twelve. each primer generated a set of amplification products of a size ranging between 350 bp and 3000 bp. preliminary results proved that the genetic fidelity of the cryopreserved lines was maintained. for all 10 primers tested, rapd fragment patterns of plantlets from cryopreserved shoot-tips did not show differences with respect to untreated shoots. figure 3 represents amplified band patterns produced by two primers (1253 and 1247). genetic fidelity was confirmed also by the other primers. 4. discussion and conclusions ‘rosso di chioggia’ shoot-tips cryopreserved using ls treatment for 30 min, pvs2 for 60 min at 0°c, and recovery on medium containing 0.5 µm ba resulted with a high survival rate (79%). this result is slightly inferior to that obtained in belgian endive, cv. flash (83%) where demeulemeester et al. (1992) used a more complex procedure, consisting of cooling the explants to -40°c at a rate of 0.5°c min-1 prior to immersion in ln. in the present study, the vitrification protocol applied to different red chicory lines showed high survival percentages, even if tolerance to the pvs2 varied in different lines. differences among lines can be considered to be genotypedependent; these results are consistent with other experiences on cryopreservation in other plant species (de boucaud et al., 2002; kim et al., 2006; benelli et al., 2009). table 6 shoot tip survival percentage of ‘rosso di chioggia’ shoottips after two different cryogenic procedures fig. 3 rapd profiles generated with primers 1253 (left) and 1247 (right) from untreated and cryopreserved plantlets of ‘rosso di chioggia’ line. in both images, lanes m correspond to the 100-bp ladder; lanes 1-9 correspond to the amplification products from untreated samples, lanes 10-17 correspond to the amplification products from cryopreserved plantlets. 49 in many cases, preculture with sucrose can be very efficient to improve cryopreservation (matsumoto et al., 1998), but such treatments applied on apical buds of several species did not induce increases in recovery after vitrification (sakai, 2000). sugar treatments can influence the membrane and protein composition (ramon et al., 2002; carpentier et al., 2005), influencing flexibility and permeability of the membrane. in red chicory shoot tips, preculture treatment on ms with 0.09 m sucrose for two days before pvs2 loading resulted in the best survival with respect to 0.3 and 0.7 m sucrose, but it did not improve the post-thaw survival percentage in the cryogenic procedure. the type and concentration of growth regulators in the recovery medium is important for survival and regrowth of cryopreserved explants (turner et al., 2001). red chicory shoot tip survival (79%) was improved when the recovery medium contained 0.5 µm ba; callus formation was induced when the ba concentration was increased, in particular with 5 µm ba. callus is not desired and represents a limitation in development of the cryopreserved explants. this phenomenon is observed in several species when a high concentration of ba was used (wang et al., 2003). demeulemeester et al. (1993) held the explants for one week on medium supplemented with plant growth regulators and then transferred them to hormone-free medium to avoid a callus phase in three varieties of chicory (flash, rumba and carolus) after cryopreservation. this protocol was effective for restricting callus formation, but led to a decrease in survival percentage. in the present study, with the ‘rosso di chioggia’ line, we obtained the highest survival rate without callus formation by applying the lowest ba concentration and the best regrowth when the single cryopreserved shoot was transferred in tube. addition of activated charcoal in post-thaw recovery medium resulted deleterious for shoot tip survival and regrowth; whereas thidiazuron gave a low survival of explants after three weeks but no subsequent growth. rooted microshoots of ‘rosso di chioggia’, obtained from cryopreserved shoot-tips, exhibited high survival and vigour in the greenhouse. application of the encapsulation-vitrification procedure showed a limited survival of cryopreserved explants and further experimentation is needed to optimize the protocol for these red chicory lines. a few genetic studies using molecular markers have been carried out on red chicory, mainly for the genetic characterization of commercial varieties (barcaccia et al., 2003), and particularly rapd markers were used to construct the genetic map of c. intybus (de simone et al., 1997) to identify and to evaluate the phylogenetic relationships among cultivars of chicory (koch and jung, 1997; van stallen et al., 2000, 2001; barcaccia et al., 2003). assessment of the genetic stability of cryopreserved shoots can be performed with different techniques (harding, 2004). in this study, rapd markers were adopted because of their simplicity, rapidity and ability to screen a randomly large part of the genome. genetic stability was reported after rapd analysis of cryopreserved plantlets and the untreated shoots. comparison of the dna patterns of control and frozen material did not reveal any variations caused by the cryoprotectant treatments or cryostorage. the results obtained are confirmed by numerous studies that demonstrated that cryopreservation did not affect the genetic stability in various species, such as potato (harding and benson, 2000), grape and kiwi (zhai et al., 2003), chrysanthemum (martin and gonzalez-benito, 2005), citrus spp. (lambardi et al, 2007), and apple (liu et al., 2008). moreover, the morphological analysis carried out in the field confirmed that there were no significant differences between plants derived from control and cryopreserved shoots. acknowledgements the authors are grateful to mr. francesco da re (veneto agricoltura) for his technical assistance in this research. references barcaccia g., pallottini l., soattin m., lazzarin r., parrini p., lucchin m., 2003 genomic dna fingerprints as a tool for identifying cultivated types of radicchio (cichorium intybus l.) from veneto, italy. plant breed., 122: 178-183. benelli c., de carlo a., giordani e., pecchioli s., bellini e., kochanova z., 2009 vitrification/one-step freezing procedure for cryopreservation of persimmon dormant buds. acta horticulturae, 833: 163-168. carpentier s., witter e., laukens k., deckers p., swennen r., panis b., 2005 preparation of protein from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. proteomics, 5: 2497-2507. de boucaud m.t., brison m., helliot b., herve’paulus v., 2002 cryopreservation of prunus. in: towill l.e. and y.p.s. bajaj (eds.) cryopreservation of plant germplasm ii. vol. 50. spring-verlag, berlin, heidelberg. de simone m., morgante m., lucchin m., parrin p., marocco a., 1997 a first linkage map of cichorium intybus l. using a one-way pseudo-testcross and pcr-derived markers. mol. breed., 3: 415-425. demeulemeester m.a.c., panis b.j., de proft m.p., 1992 cryopreservation of in vitro shoot tips of chicory (cichorium intybus l.). cryoletters, 13: 165-174. demeulemeester m.a.c., vandenbussche b., de proft m.p., 1993 regeneration of chicory plants from cryopreserved in vitro shoot tips. cryoletters, 14 : 57-64. engelmann f., 2000 importance of cryopreservation for the conservation of plant genetic resources, pp. 8-20. in: engelmann f., and h. takagi (eds.) cryopreservation of tropical plant germplasm. ipgri, rome, pp. 496. harding k., 2004 genetic integrity of cryopreserved plant cells: a review. cryoletters, 25: 3-22. harding k., benson e.e., 2000 analysis of nuclear and 50 chloroplast dna in plants regenerated from cryopreserved shoot-tips of potato. cryoletters, 21: 279-288. kim h.h, lee j.k., yoon j.w., ji j.j., nam s.s., hwang h.s., cho e.g., engelmann f., 2006 cryopreservation of garlic bulbil primordial by the droplet-vitrification procedure. cryoletters, 27: 143-153. koch g., jung c., 1997 phylogenetic relationships of industrial chicory varieties revealed by rapds and aflps. agronomie, 17: 323-333. lambardi m., de carlo a., 2003 application of tissue culture to the germplasm conservation of temperate broad-leaf trees, pp. 815-840. in: jain s.m., and k. ishii (eds.) micropropagation of woody trees and fruits. kluwer ac. pub., dordrecht. lambardi m., de carlo a., 2009 tecniche ed applicazione della criogenia alla conservazione ed al risanamento del germoplasma vegetale. italus hortus, 16: 79-98. lambardi m., halmagyi a., benelli c., de carlo a., vettori c., 2007 seed cryopreservation for conservation of ancient citrus germplasm. adv. hort. sci., 21: 198-202. liu y.g., liu l.x., wang l., gao a.y., 2008 determination of genetic stability in surviving apple shoots following cryopreservation by vitrification. cryoletters, 29: 7-14. marascuilo l.a., mcsweeney m., 1977 post hoc multiple comparison in sample proportions for test of homogeneity, pp. 141-147. in: marascuilo l.a., and m. mcsweeney (eds.) non parametric and distribution-free methods for the social science, brooke/cole publishing company, california, usa. martin c., gonzalez-benito m.e., 2005 survival and genetic stability of dendranthema grandiflora tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. cryobiology, 51: 281-289. matsumoto t., sakai a., yamada k., 1994 cryopreservation of in vitro-grown apical meristems of wasabi (wasabia japonica) by vitrification and subsequent high plant regeneration. plant cell rep., 13: 442-446. matsumoto t.a., sakai a., nako y., 1998 a novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (wasabia japonica) cooled to –196°c by vitrification. cryoletters, 19: 27-36. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-497. ramon m., geuns j., swennen r., panis b., 2002 polyamines and fatty acids in sucrose precultured banana meristems and correlation with survival rate after cryopreservation. cryoletters, 23: 345-352. reed b.m., 2001 implementing cryogenic storage of clonally propagated plants. cryoletters, 22: 97-104. reed b.m., uchendu e., 2008 controlled rate cooling, pp. 77-92. in: b.m. reed (ed) plant cryopreservation: a practical guide. springer verlag, berlin. sakai a., 2000 development of cryopreservation techniques, pp. 1-7. in: f. engelmann and h. takagi (eds) cryopreservation of tropical plant germplasm. ipgri, rome, pp. 496. sakai a., engelmann f., 2007 vitrification, encapsulationvitrification and droplet-vitrification: a review. cryoletters, 28: 151-172. sakai a., kobayashi s., oiyama i., 1990 cryopreservation of nucellar cells of navel orange (citrus sinensis var. brasiliensis tanaka) by vitrification. plant cell rep., 9: 3033. sakai a., hirai d., niino t., 2008 development of pvsbased vitrification and encapsulation-vitrification protocols, pp. 33-58. in: b.m. reed (ed.) plant cryopreservation: a practical guide. springer verlag, berlin. thomas t.d., 2008 the role of activated charcoal in plant tissue culture. biotech. adv., 26: 618-631. towill l.e., bajaj y.p.s., 2002 biotechnology in agriculture and forestry. cryopreservation of plant germplasm ii. springer-verlag, berlin, heidelberg. turner s.r., touchell d.h., senaratna t., bunn e., tan b., dixon k.w., 2001 effects of plant growth regulators on survival and recovery growth following cryopreservation. cryoletters, 22: 163-174. van stallen n., noten v., demeulemeester m., de proft m., 2000 identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic dnas and amplified fragment length polymorphisms. plant breed., 119: 265270. van stallen n., noten v., neefs v., de proft m., 2001 the phylogenetic relationship between different cichorium intybus cultivars and cultivar groups, as revealed by rapds. plant breed., 120: 425-428. vandenbussche b., demeulemeester m.a.c., de proft m.p., 1993 cryopreservation of alginate-coated in vitro-grown shoo -tips of chicory (cichorium intybus l.) using rapid freezing. cryoletters, 14: 259-266. veneto agricoltura, 2002 il radicchio rosso di chioggia, di verona, di castelfranco. veneto agricoltura, vol. 3. legnaro (padova), italy. vettori c., paffetti d., pietramellara g., stotzky g., gallori e., 1996 amplification of bacterial dna bound on clay minerals by the random amplified polymorphic dna (rapd) technique. fems microbiology ecology, 20: 251-260. wang q., li p., batuman o., ron g., mawassi m., 2003 effects of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro. cryoletters, 24: 293-302. zhai z., wu y., engelmann f., chen r., zhao y., 2003 genetic stability assessments of plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-tips using rapd. cryoletters, 24: 315-322. 61 1. introduction it is well known that disinfection represents one of the most critical processing steps influencing quality, safety and shelf-life of fresh-cut fruits and vegetables. in the fresh-cut industry, chlorine is generally used as a product sanitizer; however, there is a trend in eliminating this substance due to the environmental and health risks associated with the formation of carcinogenic halogenated disinfection by-products (gil et al., 2009). ozonation represents a sustainable technology able to improve the quality of the product; ozone is useful in diminishing the microbial load and the level of toxic organic compounds. furthermore, this gas has a stronger and more rapid antimicrobial action against spores, faecal and pathogenic microorganisms and viruses with respect to chlorine (artés and allende, 2005). the antimicrobial effect of ozone and its relatively simple application, in addition to its characteristic of not leaving any residue or forming carcinogenic trihalomethanes (such as chlorination), make this treatment suitable for various applications in the food industry (gil et al., 2009; alexopoulos et al., 2013). moreover, products treated with ozone do not modify their sensory characteristics. after it gained gras (generally recognized as safe) status in 1997, the use of this gas has been approved as a disinfectant or sanitizer in foods and food processing in europe and in the us (ölmez and kretzschmar, 2009). the primary systems for ozone application include gaseous phase storage and ozonated dips (artés et al., 2009). when ozone is used as a gas, the exposure time is longer (1-4 h) than ozone dissolved in water (1-10 min) (carletti et al., 2013). in recent years the use of this gas as a pretreatment to preserve fruit and vegetable quality has received great attention for its antimicrobial activity against bacteria, fungi, viruses, and bacterial and fungal spores. several studies have focused on the effect of ozone treatment on the safety and quality of iceberg lettuce (rico et al., 2006; yuk et al., 2006; hassenberg et al., 2007). in addition, various applications have been proposed concerned the sanitizing effect of ozone dissolved in water used for washing on the quality and safety of minimally processed fruits and vegetables (seydim et al., 2004; zhang et al., 2004; selma et al., 2008; artes et al., 2009; ölmez and kretzschmar, 2009), but there is scant literature about ozone applied to fresh-cut iceberg lettuce (beltran et al., 2005; ölmez and akbas, 2009). the aim of this work was to evaluate the effect of refrigeration at 4°c, associated with ozonization treatment application of ozone in fresh-cut iceberg lettuce refrigeration f. galgano1(*), m.c. caruso1, n. condelli1, s. stassano1, f. favati 2 1 school of agricultural, forestry, food and environmental sciences, university of basilicata, viale dell’ateneo lucano 10, 85100 potenza, italy. 2 department of biotechnology, university of verona, strada le grazie 15, 37134verona, italy. key words: environmental sustainability, fresh-cut vegetables, fresh vegetable quality, minimally fresh processed leaf vegetables, shelf-life. abstract: recently, technological innovations have been geared to supporting environmental sustainability, also in the fruit and vegetable industry. the application of ozone in the cold storage of fruits and vegetables is a sustainable technology used to improve product quality and its antimicrobial effect, simple use, and characteristic of not leaving any residue, makes this treatment suitable for many applications in this field. the aim of this work was to evaluate the effect of refrigeration at 4°c, associated with ozonization treatment at a concentration of 0.2 ppm on the shelf life of fresh-cut lettuce, compared to a lettuce control stored only at 4°c. lettuce quality throughout the storage period (7 days) was determined by means of color and microbiological indexes, such as total bacterial count, total coliforms, escherichia coli, pseudomonadaceae, yeasts and molds. the lettuce used in the experiment was found to have a low microbiological load. microbiological results obtained at different storage times have shown that the use of ozone is effective in containing microbial growth during chilling storage of the raw material compared to the refrigerated control. in particular, the positive effects of ozonation were appreciable after the third day of storage. furthermore, the ozone treatment did not affect the color of the product. (*) corresponding author: fernanda.galgano@unibas.it received for publication 26 september 2014 accepted for publication 9 march 2015 adv. hort. sci., 2015 29(2-3): 61-64 62 adv. hort. sci., 2015 29(2-3) 61-64: at a concentration of 0.2 ppm on the shelf life of freshcut lettuce, compared to a lettuce control stored only for 7 days at 4°c. 2. materials and methods sampling and ozone treatment iceberg lettuce (lactuca sativa l.) was obtained from società agricola ”rago” (battipaglia, sa, italy) and, immediately after harvesting, it was stored at 4°c and submitted to a continuous ozone exposure of 0.2 ppm. a control sample was stored at 4°c without ozone treatment. ozone gas was generated using a corona discharge ozone generator (model oxy met twin, met, san lazzaro di savena, bo, italy) from purified oxygen gas. functioning of this equipment takes place through sensors placed inside the storage compartment/chamber that reveal the exact concentration present in the refrigeration cell and it is reported on the display of the instrument. the tests lasted up to 7 days and the ozonated and non-ozonated product was sampled at 1, 2, 3, 5 and 7 days for analyses. colorimetric analysis color was measured on the lettuce surface using a minolta chroma meter cr-300 (minolta camera co. ltd., osaka, japan), with a d 65 illuminant, using cielab l*, a*, b* values. the data reported are the average of a set of four determinations carried out on the lower and upper surface of two leaves of lettuce. microbiological analysis at each treatment time, 9 g of ozonated or control product were placed in a stomacher bag containing 81 ml of tryptone water (liofilchem, teramo, italia) and homogenized for 2 min. samples were then serially diluted in tripton water and surface plated in duplicate on compactdry ec (pbi international, milano, italy) for the recovery of surviving total coliforms and escherichia coli, on compact-dry ym (pbi international, milano, italy) for yeasts and moulds and on cetrimide agar (liofilchem, teramo, italy) for the enumeration of pseudomonas aeruginosa and pseudomonas spp. total mesophilic bacteria were enumerated on plate count agar (liofilchem, teramo, italy) using the pour plate technique. all the inoculated media were incubated at 30°c for 48 h. statistical analysis in order to study the effect of ozone treatment and storage time on fresh-cut iceberg lettuce quality characteristics, data were processed by two-way analysis of variance (anova). moreover, a student test was performed to compare the mean values between the samples treated with ozone and control samples for each time of storage. all statistical procedures were computed using the statistical package systat for windows (ver. 10, 2003) (systat software, chicago, usa). 3. results and discussion ozone can be used to disinfect fruit and vegetables by dissolving it in the water used for washing (gil et al., 2009), or by keeping a constant ozone concentration, through the use of an appropriate generator, during storage of the product in a cooling room at 4°c. in this paper we evaluated the effect of cold storage at 4°c, accompanied by ozonization at a concentration of 0.2 ppm on the shelf life of the fresh-cut lettuce, compared to a control subjected only to cold storage. preliminary tests showed that the use of ozone at low concentrations, such as 0.2 ppm, did not determine sensory alterations; for this reason, the effect of treatment on both perceptible sensory parameters, such as color stability, and microbiological parameters, for safety of the product, has been evaluated. a two-way analysis (time, treatment) showed a significant effect of both factors on the colorimetric parameters (p= 0.000). in particular, total bacterial count and coliforms increased throughout the storage period, being significantly lower for ozonated samples than control samples. therefore, the microbiological results obtained at different storage times show that the use of ozone is effective in containing microbial growth during storage in a cooling room of raw material compared to the untreated control. in particular, the positive effects of ozonation were appreciable after the third day of storage, as shown by the trend of the total bacterial count and total coliforms of the chilled and ozonized product compared to the refrigerated only product (fig. 1 and 2). in particular, ozone was able to inhibit the proliferation of total coliforms, which were not detectable even after 7 days of storage in the ozonized samples, while in control samples these microorganisms reached 1.6 x 103 cfu/g at the same storage time. the growth of colonies on the plates inoculated for the detection of other microorganisms was sporadic and not significant for both the experimental theses considered. in the literature a reduction of 3-4 log units of escherichia coli o157: h7 inoculated in lettuce and carrots, by sequential washes with aqueous solutions of cl 2 o/ozonated water, has been reported (singh et al., 2002). various experiments carried out by treating vegetables with ozonated water have shown the ability of ozone to reduce the growth of different microorganisms, including pathogens, and also to reduce enzymatic activities, including enzymatic browning (artes et al., 2009). alexopoulos et al. (2013) have highlighted the importance of keeping the concentration of ozone constant during treatment by continuous generation. in fact, the immersion of vegetables in water presaturated with ozone determined the reduction of 0.5 log of cbt after 15 min, whereas the immersion for the same time in continuously ozonated water involved the reduction of 2 log of cbt, showing in this latter case a greater effectiveness of ozonization with respect to chlorination. in fact, the reduction from 1.5 to 2.5 log units of the microflora of the lettuce was obtained with ozonated water with 1.5-3 ppm; this effect was comparable to the use of 100 ppm of chlorine 63 galgano et al., application of ozone in fresh-cut iceberg lettuce refrigeration (ölmez and kretzschma, 2009). furthermore, ozone treatment did not adversely affect product color (fig. 3 and 4), as also confirmed by twoway analysis of variance; in fact, a non significant effect of ozone treatment and storage time was recorded for the colorimetric indexes. it is well known that ozone is a strong oxidizing agent, which can induce damage to the structure and may influence the color of the products depending on the time of contact, the concentrations applied, and the type of product (horvitz and cantalejo, 2014; olmez et al., 2009). however, in the present study, at concentrations of 0.2 ppm of gaseous ozone on lettuce stored at 4°c, no difference was detected relative to colorimetric parameters l *, a *, and b * between the control and the product treated with ozone has (fig. 5 and 6). 4. conclusions the microbiological results obtained at different storage times have shown that the use of ozone is effective in containing microbial growth during storage of the raw material compared to the untreated control. in particular, the positive effects of ozonation were appreciable after the third day of storage. moreover, ozone treatment did not affect the color of the product. in order to better assess the fig. 1 evolution of total mesophilic bacteria during storage at 4°c. for each storage time, data followed by different letters are significantly different (tstudent test at p<0.05). fig. 2 evolution of total coliforms during storage at 4°c. for each storage time, data followed by different letters are significantly different (tstudent test at p<0.05). fig. 3 lettuce not treated with ozone after 3 days of storage at 4°c. fig. 4 lettuce treated with ozone after 3 days of storage at 4°c. fig. 5 l*, a* and b* values determined on the upper leaf surface. 64 adv. hort. sci., 2015 29(2-3) 61-64: treatment effect of ozonation on lettuce, studies considering material with a higher degree of microbial contamination, or inoculated with microorganisms that have not been detected even in the thesis used as a control in this experiment, should be undertaken. acknowledgements the authors would like to thank società agricola ”rago” for supplying the lettuce samples. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references alexopoulos a., plessas s., ceciu s., lazar, v., mantzouranii., voidarou c., stavropoulou e., bezirtzoglou e, 2013 evaluation of ozone efficacy on the reduction of microbial population of fresh-cut lettuce (lactuca sativa) and green bell pepper (capsicum annuum). food control, 30: 491-496. artés f., allende a., 2005 processing lines and alternative preservation techniques to prolong the shelf-life of minimally fresh processed leafy vegetables. europ. j. hort. sci., 70: 1-15. artés f., gómez p., aguayo e., escalona v., artés hernández f., 2009 sustainable sanitation techniques for keeping quality and safety of fresh-cut plant commodities. postharv. biol. technol., 51: 287-296. beltran d., selma m.v., marin a., gil m.i., 2005 ozonated water extends the shelf-life of fresh-cut lettuce. j. agric. food chem. 53(14): 5654-5663. carletti l., botondi r., moscetti r., stella e., monarca d., cecchini m., massantini r., 2013 use of ozone in sanitation and storage of fresh fruits and vegetables. j. food agric. environm., 11: 585-589. gil m.i., selma m.v., lópez-gálvez f., allende a. 2009 fresh-cut product sanitation and wash disinfection: problems and solutions. int. j. food microbiol., 134: 37-45. hassenberg k., idler c., molloy e., geyer m., plöchl m., barnes j., 2007 use of ozone in a lettucewashing process: an industrial trial. j. sci. food agric., 87: 914-919. horvitz s., cantalejo m.j., 2014 application of ozone for the postharvest treatment of fruits and vegetables. crit. rev. food sci. technol., 54: 312-339. ölmez h., akbas m.y., 2009 optimization of ozone treatment of fresh-cut green leaf lettuce. j. food. eng., 90(4): 487-494. ölmez h., kretzschmar u., 2009 potential alternative methods for organic fresh-cut industry for minimizing water consumption and environmental impact. lwt food sci. technol., 42: 686-693. rico d., martin-diana a.b., frias j.m., henehan g.t.m., barry-ryan c., 2006 effects of ozone and calcium lactate treatments on browning and texture properties of fresh-cut lettuce. j. sci. food agric., 86: 2179-2188. selma m., allende a., lópez-gálvez f., conesa m.a., gil m.i., 2008 disinfection potential of ozone, ultraviolet-c and their combination in wash water for the fresh-cut vegetables industry. food microbiol., 25: 809814. seydim z.g., bever p.i., greene a.k., 2004 efficacy of ozone to reduce bacterial populations in the presence of food components. food microbiol., 21: 475-479. singh n., singh r.k., bhunia a.h., stroshine r.l., 2002efficacy of chlorine dioxide, ozone, and thyme essential or a sequential washing in killing escherichia coli o157:h7 on lettuce and baby carrots. lebensm. wiss. u. technol., 35: 720-729. yuk h.g., yoo m.y., yoon j.w., moon k.d., marshall d.l., oh d.h., 2006 effect of combined ozone and organic acid treatment for control of escherichia coli o157: h7 and listeria monocytogenes on lettuce. j. food sci., 71(3): 83-87. zhang l., zhaoxin l., zhifang y., xiang g., 2004 preservation of fresh-cut celery by treatment of ozonated water. food control, 16: 279-283. fig. 6 l*, a* and b* values determined on the lower leaf surface. 176 1. introduction harvesting is one of the most important operations of the whole cultivating cycle in olive production, both in order to obtain high quality oils and to reduce costs (tombesi, 1990; famiani et al., 1998; cicek, et al., 2010; ferguson et al., 2010). the aim of this trial was to evaluate the efficiency of a trunk shaker with a reversed umbrella interceptor for the mechanical harvesting (visco et al., 2008; farinelli et al., 2012 a, b) of two autochthonous cultivars, ‘ortice’ and ‘ortolana’, in southern italy. 2. materials and methods the experiment was carried out in 2006 in a commercial olive grove, belonging to the “uliveto” farm, located in southern italy (41°15’ n, 14°38’ e) (province of benevento). adult trees of two autochthonous cultivars, ‘ortice’ and ‘ortolana’ (di vaio et al., 2013), trained to the vase system and planted at a spacing of 6 x 6 m were studied. the olive grove had a slope of less than 3% and was drip irrigated. pruning was carried out annually and fertilization and pest management were carried out according to local standard practices. for the mechanical harvesting, carried out on 30 november, a self-propelled machine, “f3” model with three traction wheels and a reversed umbrella interceptor manufactured by sicma (catanzaro, italy), was used (fig. 1). the main characteristics of the trunk shaker were: an engine power of 77 kw (105 cv), a very-highfrequency vibrating head (1800-2000 vibrations/min), a self-braking system and a 6-m diameter umbrella opening. the work force consisted of two workers, one for maneuvering the harvesting machine, the other for handling the olives. during drupe maturation, periodically on three samples of 100 olives per date, the following parameters were determined: olive detachment force, coloration (jaén index between 0 and 7), fresh weight, pulp firmness (with a manual penetrometer with a 1.0 mm diameter plunger on the equatorial zone of fruit) and oil content (determined using a soxlhet extractor). on four trees per cultivar, at the mechanical harvesting of oil olives by trunk shaker with a reversed umbrella interceptor c. di vaio*, n. marallo*, s. nocerino*, f. famiani** * dipartimento di arboricoltura, botanica e patologia vegetale, università di napoli federico ii, via università, 100, 80055 portici (na), italy. ** dipartimento di scienze agrarie e ambientali, università degli studi di perugia, borgo xx giugno, 74, 06121 perugia, italy. key words: mechanical harvesting, olea europaea l., ‘ortice’, ‘ortolana’, oil quality, trunk shaker with reversed umbrella. abstract: trunk shakers are primarily used for the mechanical harvesting of oil olives in intensive orchards. the objective of this trial was to determine the efficiency of mechanical harvesting of olives with a self-propelled trunk shaker with a reversed umbrella interceptor (model f3, sicma, catanzaro, italy), from adult trees of two autochthonous cultivars, ‘ortice’ and ‘ortolana’, growing in southern italy with 6 × 6 m spacing and trained to the vase system. the main characteristics of the trunk shaker were: an engine power of 77 kw (105 cv), a very-high-frequency vibrating head (1800-2000 vibrations/min), a self-braking system and a 6-meter diameter umbrella opening. the worksite consisted of two workers one for maneuvering the harvesting machine and the other for handling the olives. mechanical harvesting was carried on 30 november 2006 when the fruits of ‘ortice’ and ‘ortolana’ had a weight and detachment force around 2.8 g and 3.1 n and 3.8 g and 4.6 n, respectively, and the fruit drop was around 14% and 10%, respectively. both cultivars had a good production (26.06 and 21.18 kg/tree). the mechanical harvesting yield (percentage of mechanically harvested olives) was very high, reaching values around 97% in both cultivars. moreover, the low number of workers, the reduced time for the operation (2.5 min/tree), the good yield/tree and the high quantity of harvested fruit allowed a very high work productivity to be obtained: around 302 kg/h/worker for ‘ortice’ and 246 kg/h/worker for ‘ortolana’. the quality of the oils extracted from the harvested olives met the requirements set by european law for extra virgin olive oils. the results indicate that the use of a trunk shaker with a reversed umbrella can be an efficient solution for mechanical harvesting of the ‘ortice’ and ‘ortolana’ cultivars in southern italy. adv. hort. sci., 2012 26(3-4): 176-179 received for publication 6 february 2013 accepted for publication 14 february 2013 177 beginning of ripening, eight small branches were labeled (two per cardinal point) and the fruit was periodically counted up to harvesting time in order to estimate fruit drop. mechanical harvesting was carried out on 10 trees/ cultivar and the drupes were weighed. after harvesting, undetached olives were harvested by hand and weighed. the ratio between mechanically harvested olives/total olives on the canopy, expressed as percentage, was used to determine the mechanical harvesting yield (%). the trunk cross-sectional area (at about 0.5 m above the ground) and canopy width (w), height (h) and volume [volume = ((w/2)2 x 3.14 x h) x 2/3] were measured/calculated on each of the harvested trees. work productivity was calculated and expressed as the amount of harvested olives/h/ worker. in both cultivars, after mechanical harvesting, 100 kg of olives were collected and micro-milled to obtain two samples of mono-variety oils, on which free acidity, peroxide number, spectrophotometric indices and sensorial characteristics (by panel test) were determined. data were submitted to analysis of variance (anova) using msta-c software and mean separation was performed by the multiple range duncan test at the 5% significance level. moreover, the standard errors (se) of the means were also calculated. 3. results and discussion during maturation, the cv. ortice was resistant to drupe detachment, which decreased at the beginning of november until reaching 309.17 g at harvest time. the cv ‘ortolana’, instead, followed a rather constant course, and at harvest time reached 455.83 g (fig. 2 and table 1). pulp firmness decreased constantly during maturation, reaching fig. 1 mechanical harvesting of olives with a trunk shaker with a reversed umbrella interceptor. fig. 2 evolution of olive detachment force during maturation (mean ± standard error). table 1 characteristics of trees and fruit at harvest, machine efficiency and work productivity (mean ± standard error) cv. ortice cv. ortolana olive weight (g) 2.79 a±0.06 3.77 b±0.01 olive detachment force (g) 309.17 a±19.30 455.83 b±18.20 olive detachment force/olive weight (n/g) 1.11 a±0.08 1.21 a±0.09 pulp firmness (g/mm2) 267.00 a±5.11 231.58 b±0.45 drupe coloration jaèn index (0-7) 2.10 a±0.05 4.35 b±0.17 fruit drop (%) 13.95 a±2.27 10.36 a±2.49 canopy volume (m3) 47.34 a±2.35 56.31 b±1.56 trunk cross section area (cm2) 1587.31 a±337.96 613.43 b±172.04 total olive yield per tree (kg) 26.06 a±7.72 21.18 a±5.02 olives mechanically harvested per tree (kg) 25.17 a±7.21 20.49 a±5.03 productive efficiency of tree (kg/m3) 0.37 a±0.17 0.25 a±0.13 mechanical harvesting yield (%) 96.58 a±1.15 96.74 a±0.88 work productivity (kg/h/worker) 302.04 a±18.01 245.88 a±14.10 in each row, means with the same letter are not significantly different by duncan multiple range test (p<0.05). 178 values of 267.00 g/mm2 and 231.58 g/mm2 for ‘ortice’ and ‘ortolana’, respectively (fig. 3 and table 1). at harvest, the accumulation of oil in the drupe was 26.09% in ‘ortice’ and 16.51% in ‘ortolana’ (fig. 4). olive pigmentation, scored according to the jaén index, differed for the two cultivars: 2.10 in ‘ortice’ and 4.35 in ‘ortolana’ (table 1). both cultivars had good production (26.06 and 21.18 kg/ tree) (table 1), and the productive efficiency was 0.37 and 0.25 kg of olives/m3, respectively (table 1). the canopy volume of the trees was 47.34 m3 for ‘ortice’ and 56.31 m3 for ‘ortolana’. pre-harvest fruit drop increased during the entire period, reaching values of 13.95% for ‘ortice’ and 10.36% for ‘ortolana’ at harvest time (fig. 5 and table 1). at harvest fruit weight was 2.79 g for ‘ortice’ and 3.77 g for ‘ortolana’ (table 1). the ratio between the olive detachment force and weight decreased throughout the ripening period and was 1.11 and 1.21 n/g at harvest (fig. 6 and table 1). in general, fruit characteristics and ripening patterns were similar to those reported by di vaio et al. (2013) for the same cultivars. despite the high canopy volume of the trees, the mechanical harvesting yield (percentage of mechanically harvested olives) was very high: 96.58% and 96.74% for the ‘ortice’ and ‘ortolana’ cultivars, respectively (table 1). these high values are likely due to the relatively low olive detachment/weight ratios at the time of harvesting, which were close to one. indeed, farinelli et al. (2012 a) reported a significant negative relationship between the olive detachment/weight ratio and the mechanical harvesting yield obtained with a trunk shaker. the same authors observed that high mechanical harvesting yields are obtained when the ratio is less than 2. in this regard, it can be noted that in the present work, for both cultivars, the olive detachment/weight ratio, as a result of the medium weight and the medium/low detachment force of the olives, was less than 2 for the entire ripening period. this indicates that mechanical harvesting could be carried out efficiently fig. 3 evolution of pulp firmness during maturation (mean ± standard error). fig. 6 evolution of the ratio between olive detachment force and drupe weight during maturation (mean ± standard error). fig. 5 evolution of pre-harvest fruit drop during maturation. fig. 4 evolution of olive oil content (% f.w.) during maturation. 1.8 1.6 1.4 1.2 1.0 0.8 179 during the entire ripening period. at the farm level, this allows for flexibility, also considering that oil quality changes during olive ripening and so oils with different characteristics can be obtained by modulating harvesting time (inglese et al., 2011). moreover, the low labour requirement (only two workers), the reduced time for operation (about 2.50 min/tree, which included the approach to the tree and attachment of the shaker to the trunk, opening of the reversed umbrella, shaking and closing the reversed umbrella), the good yield/tree and high quantity of harvested fruit allowed very high values of work productivity to be obtained: about 302.04 kg/h/worker for ‘ortice’ and 245.88 kg/h/worker for ‘ortolana’ (table 1). this result highlights the importance of a good yield/tree in determining high work productivity and therefore the economic convenience of using machines for olive harvesting (famiani et al., 1998). the oil quality indices (acidity, peroxide number, spectrophotometric indices and sensorial characteristics by panel test) reported in table 2 show that all analytical and sensory evaluations of the oils from the two cultivars met the requirements set by law for extra virgin olive oils (ec regulation n. 2568, 1991). therefore, the use of the trunk shaker with interceptor allowed high quality oils to be obtained. in conclusion, the results of the present study show that the trunk shaker with the reversed umbrella interceptor, which allows for high harvesting yields and high labour productivity, can be considered an interesting solution for mechanical harvesting of the autochthonous cultivars ‘ortice’ and ‘ortolana’ in southern italy. acknowledgements this study was financed by the mi.p.a.a.f. within the riom project “research and innovation for the olive growing of southern italy”. the authors wish to thank dr. mark walters for editing the manuscript. references cicek g., sumer s.k., kocabiyik h., 2010 effect of different harvest methods on olive yield and work capacity. african journal of agricultural research, 5(23): 3246-3250. di vaio c., nocerino, s., paduano a., sacchi r., 2013 characterization and evaluation of olive germplasm in southern italy. journal of the science of food and agriculture. doi: 10.1002/jsfa.6057 ec regulation 2568, 1991 on the characteristics of olive oil and olive residue oil and on the relevant methods of analysis. off. j. eur. commun, nl 248/1 famiani f., proietti p., palliotti a., guelfi p., nottiani g. 1998 possibilità di meccanizzazione della raccolta delle olive in diverse tipologie di oliveto. rivista di frutticoltura, 7/8: 33-39. farinelli d., ruffolo m., boco m., tombesi a., 2012 a yield efficiency and mechanical harvesting with trunk shaker of some international olive cultivars. acta horticulturae, 949: 379-384. farinelli d., tombesi s., famiani f., tombesi a., 2012 b the fruit detachment force/fruit weight ratio can be used to predict the harvesting yield and efficiency of trunk shakers on mechanically harvested olives. acta horticulturae, 965: 61-64. ferguson l., rosa u., castro-garcia s., lee s.m., guinard j.x., burns j., krueger w.h., o’connell n.v., glozer k., 2010 mechanical harvesting of california table and oil olives. adv. hort. sci., 24(1): 53-63. inglese p., famiani f., galvano f., servili m., esposto s., urbani s., 2011 factors affecting extra-virgin olive oil composition. horticultural reviews, 38: 83-147. tombesi a., 1990 physiological and mechanical advances in olive harvesting. acta horticulturae, 286: 399-412. visco t., molfese m., cipolletti m., corradetti r., tombesi a., 2008 the influence of training system, variety and fruit ripening on the efficiency of mechanical harvesting of young olive trees in abruzzo, italy. acta horticulturae, 791: 425-429. table 2 quality indices of oils obtained from cv. ortice and ortolana olives mechanically harvested with a trunk shaker (mean±standard error) cultivar acidity % oleic acid peroxide value meq o 2 kg-1 uv defects at panel 0-5k232 k270 ∆k ortice 0.52±0.04 8.2 b±0.13 1.967±0.065 0.145±0.014 -0.003±0.000 0±0.00 ortolana 0.46±0.02 6.5 b±0.14 1.824±0.032 0.155±0.036 -0.005±0.000 0±0.00 29 1. introduction bermudagrass is still the dominant warm-season turfgrass in warm to temperate climatic regions of the world. it is well adapted to a wide range of soil types, and its drought tolerance, recuperative ability, salt tolerance, wear tolerance, aggressive stoloniferous and rhizomatous growth habit, and overall appearance make bermudagrass an ideal turfgrass in many environments (taliaferro, 2003; shearman, 2006). bermudagrass includes several taxa of the genus cynodon (l.) rich. but the two species that represent the genetic pool from which the present cultivars descend are cynodon dactylon (l.) pers. var. dactylon and cynodon transvaalensis (burtt-davy) also known as african bermudagrass (taliaferro, 2003). c. transvaalensis is morphologically distinct from c. dactylon due to narrow erect pale leaves producing a fine textured turf with a yellowish-green colour (de wet and harlan, 1970; taliaferro, 1992). c. dactylon can be found as far north as 53° n latitude and from sea level to 3000 m altitude (taliaferro, 2003). asexual reproduction has played a role in bermudagrass enhancement as well. remarkable breeding progress has been obtained from inter-specific hybridization and mutation breeding. the inter-specific hybridization of c. dactylon and c. transvaalensis has been extensively used to obtain sterile cultivars for which clonal propagation is necessary due to a lack of viable seeds. among hybrid genotypes, a number of cultivars have been selected for plant size and morphology in response to the lower cutting height adopted over the years on golf greens. the more recently released “ultradwarf” cultivars have become routinely adopted thanks to an improved density, a slower vertical leaf extension and an increased dominance of stoloniferous growth relative to rhizomes at low mowing heights (beard and sifers, 1996). parameters used to evaluate cynodon turf typically include turfgrass quality, colour, percent spring green-up, establishment rates, leaf texture, and density. as the turfgrass industry moves towards more sustainable management practices, the types of parameters potentially related to better stress tolerance are increasingly important (baldwin and liu, 2013). one of the most important parameters is cold tolerance and rapidity of recovery from winter dormancy in the spring (anderson et al., 2007; patton et al., 2008). lowtemperature tolerance depends on a combination of sevbermudagrass adaptation in the mediterranean climate: phenotypic traits of 44 accessions s. magni*, m. gaetani*(1), n. grossi*, l. caturegli*, s. la bella**, c. leto**, g. virga**, t. tuttolomondo**, f. lulli***, m. volterrani* * dipartimento di scienze, agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56127 pisa, italy. ** dipartimento di scienze agrarie e forestali, università di palermo, viale delle scienze, 208, 90128 palermo, italy. *** turf europe r&d, pisa, italy. key words: colour, cynodon dactylon, green-up, node density, quality, shoot density. abstract: the use of bermudagrass in the mediterranean area is increasing for its outstanding tolerance to heat and drought, and its aggressive growth and high recuperative potential make it particularly suited to heavily worn areas and appreciated for sports turfs. however, the overall performance of a given genetic type can be affected by the adaptation to a specific environment. the objective of this research was to determine the variability of a number of phenotypic traits that can affect bermudagrass turf performance on a wide range of bermudagrass accessions grown in two locations in italy. in may 2010, 44 accessions of bermudagrasses, grouped in “wild”, “improved” “hybrid” and “dwarf” types were transplanted in the center of field plots in pisa and palermo. in 2011, when the turf was completely established, the following traits were determined: shoot density, horizontal stem density, node density, leaf width, colour, quality, spring green-up, and fall colour retention. dwarf and hybrid types yielded the best aesthetic characteristics. with respect to colour retention and spring green-up, great variability was recorded within the groups. dwarf types presented the earliest dormancy, while the hybrid types were in general the ones to green-up first in spring. adv. hort. sci., 2014 28(1): 29-34 1 corresponding author: monica.gaetani@unipi.it received for publication 14 april 2014 accepted for publication 19 may 2014 30 eral factors, including environmental conditions, cultural practices, and especially genetic factors (blum, 1988; anderson and taliaferro, 2002). bermudagrass survives the dormancy period using its reserves of nonstructural carbohydrates and nitrogen compounds accumulated during the previous growing season in storage organs such as stolons and rhizomes (macolino et al., 2010; volterrani et al., 2012; giolo et al., 2013; pompeiano et al., 2013). in the last two decades several southern european universities have developed research programs to study warm-season turfgrass species, including bermudagrass (volterrani et al., 2008; lulli et al., 2011; lulli et al., 2012; nikolopoulou et al., 2012; agati et al., 2013; gómez de barreda et al., 2013), and in particular their adaptability to the mediterranean environment (volterrani and magni, 2004). the aim of our research was to determine the variability of a number of phenotypic traits and aesthetical characteristics that can affect bermudagrass turf performance in a wide range of bermudagrass accessions grown in two locations in italy. this information can provide further insight into bermudagrass adaptability in the mediterranean climate. 2. materials and methods plant material with the objective of expanding morphological diversity of the plant material, 44 accessions of bermudagrass [cynodon (l.) rich.], representative of both wild populations and cultivars, were included in the present study. group one included 13 entries that were called “wild types”, naturally occurring populations of c. dactylon (l.) pers. collected from contrasting environments supposed to generate a selective pressure. collection sites were located in italy (certes-1= warm temperate, salt affected soil; certes-2= warm temperate, fertile soil; certes-3= warm temperate, polluted soil; certes-13= warm temperate, fertile soil), france (certes-4= cool humid, fertile soil), greece (certes-5, -6 and -7= warm temperate, salt affected soils), croatia (certes-8= warm temperate, salt affected soil), argentina (certes-9= warm temperate, salt affected pastureland), united arab emirates (certes-10 and -11= warm arid, desert sand), and maldives (certes-12= tropical humid, salt affected soil). group two included 13 entries that were called “improved types”. these were experimental or commercial vegetative and seeded improved c. dactylon cultivars. group 3 included 11 entries called “hybrid types”, commercial or experimental inter-specific hybrids (cynodon dactylon x transvaalensis burtt.-davy) of which those labelled tifwere kindly provided by dr. w. hanna (university of georgia, usa). group four included seven entries called “dwarf types”, commercial interspecific dwarf and ultradwarf hybrid cultivars and two cynodon transvaalensis burtt.-davy accessions, one a commercial cultivar (uganda) and the other (roma) a line of african bermudagrass that was collected in a turf nursery in rome (italy) where the species was first introduced presumably as a weed. african bermudagrasses were included in the “dwarf types” due to their similarity in leaf texture, density and growth habit with the well-known hybrid dwarf bermudagrasses. 2010 turf establishment on 15 april 2010 at the university of pisa, italy, all the accessions were propagated in the greenhouse (24±5°c) in peat-filled honeycomb seed trays (7 cm2 area and 25 cm3 volume each cell). vegetatively propagated genotypes were planted as single stolon and seeded cultivars were seeded as single seed. on 13 may 2010 plants in the greenhouse were fertilized (30 kg ha-1 n, 10 kg ha-1 p, and 10 kg ha-1 k) using a soluble fertilizer (grow more inc., gardena, ca, usa). on 31 may 2010, plants were mown to 5 cm and transplanted into field plots in two locations in italy: the research station of the university of pisa (43°40’n, 10°19’e, 6 m a.s.l.) and the research station of the university of palermo (38°06’n, 13°20’e, 50 m a.s.l.). experimental plots were 1.5 by 1.5 m with 0.5 m bare soil pathways arranged in a randomized complete-block design with four replications. one plant of bermudagrass was transplanted in the centre of each plot. soil type at pisa was silt-loam (28% sand, 55% silt and 17% clay) with a ph of 7.8 and 18 g kg-1 organic matter while at palermo soil type was sandy clay loam (54% sand, 23% silt and 23% clay) with a ph of 7.6 and 14 g kg-1 organic matter. irrigation was applied as needed to encourage establishment. plots received 50 kg ha-1 n, 10 kg ha-1 p, and 40 kg ha-1 k per month from june to september 2010. in order to minimize weed competition, from two years before establishment, the experimental areas were treated twice a year with glyphosate [n-(phosphonomethyl) glycine] at 2.88 kg ha-1 a.i. the day before planting, oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4oxadiazol-2(3h)-one] was applied at 3.36 kg ha-1 a.i. plots were not mowed during the year of establishment to avoid genotype x mowing interaction. weeds occurring during the trial period were manually removed, even if it is not a standard cultivation technique, as the accessions could be differently injured by chemical removal. in the trial, no pesticides were applied. encroachment of stolons into adjacent plots was avoided by using toothpicks that redirected the growing tip back toward the plot centre. 2011 in 2011, in the first and second weeks of march at palermo and pisa, respectively, scalping was carried out. from the end of april (the end of green-up) to october 2011, the turf was mowed weekly with a reel mower (john deere 20sr7) at a mowing height of 2.5 cm. the irrigation program was adjusted according to soil temperature and evapotranspiration rate, with supplement irrigations applied as needed to prevent visual wilt of the turf (croce et al., 2004). plots received 50 kg ha-1 n, 20 kg ha-1 p, and 40 kg ha-1 k per month from may to august 2011. 31 weeds were manually removed inside the plots during the trial period as the accessions could be differently injured by chemical treatments. in the trial, no pesticides were applied. to avoid the encroachment of stolons into adjacent plots, the corridors were treated with glyphosate at 2.88 kg ha-1 a.i. every other week. no turf cultivation, or verticutting or phytosanitary treatment was practiced on the plots. monthly mean maximum and minimum temperatures recorded at the two trial sites are reported in table 1. there were 13 days in pisa with air temperatures below 0°c from november 2010 to march 2011; zero days were recorded in palermo for the same period. assessments spring green-up (15 march-15 may 2011) and fall colour retention (15 november 2011-15 january 2012) were estimated and expressed as percentage of green ground cover. in the second week of october at both testing sites a 50 cm2 core sample per plot was collected and the following parameters determined: leaf width (20 fully expanded leaves per plot measured with a precision vernier caliper and data reported in millimeters), shoot density (direct counting with data reported as shoot no cm-2), horizontal stem density (stolons and rhizomes collected after soil washing measured with a ruler and data reported as cm cm-2) and node density (nodes of stolons and rhizomes collected from the core samples counted and reported as no cm-2) (roche and loch, 2005; volterrani et al., 2008; volterrani et al., 2010; pompeiano et al., 2012). at the pisa location two additional parameters were determined: at 30-day intervals throughout the growing season (may-october) colour, with a rating scale of 1=light green and 9=dark green, and quality with a rating scale of 9 = best and 1 = poorest (morris and shearman, 2007; patton et al., 2009 ) were estimated. data were subjected to analysis of variance using costat software (monterey, ca, usa). to test the effects of location, accession and their interaction, a factorial combination was used. significantly different means were separated using fisher’s least significant difference (lsd) at the t-probability level of 0.05. 3. results the interaction of treatments was not statistically significant for any of the parameters recorded in pisa or palermo. for all the parameters, the location and accession mean effects were statistically significant. location effect is reported as average across accessions and accession effect is reported as average across locations. location mean effect for the location effect, shoot density and node density were on average higher in pisa (3.1 shoots cm-2 and 2.4 nodes cm-2 respectively) compared to palermo (0.8 and 1.0) (table 2). also horizontal stem density was higher in pisa (4.2 cm cm-2) while leaf width recorded in pisa was on average less (1.4 mm) compared to palermo (2.4 mm). spring green-up evaluations as average across locations showed green cover percentages (april 14) higher in palermo (81%) with respect to pisa (60%), while fall colour retention (december 17) showed higher green cover percentages in palermo (77%). accession mean effect shoot density all wild type entries, with the exception of certes 12 (2.4 shoot cm-2) which produced a density comparable to hybrid and improved types, had a similar shoot dentable 1 monthly mean air temperatures (°c) during the trial period (2011) at pisa and palermo month air temperature (°c) pisa palermo mean maximum mean minimum mean maximum mean minimum january 10.6 3.5 14.7 9.3 february 11.8 3.3 13.9 9.3 march 13.9 5.2 16.2 10.4 april 19.2 9.0 19.6 13.1 may 23.8 11.8 21.4 15.9 june 26.2 17.0 25.2 20.2 july 27.6 18.3 28.6 22.9 august 30.2 18.5 29.0 23.0 september 26.7 16.0 26.9 21.0 october 21.8 10.5 22.6 16.5 november 17.5 6.6 20.2 14.5 december 13.3 4.7 16.9 12.6 table 2 bermudagrass [cynodon (l.) rich.] shoot density, horizontal stem density, node density, leaf width. location effect averaged across accessions shoot density (n° cm-2) horizontal stem density (cm cm-2) node density (n° cm-2) leaf width (mm) spring green-up (%) fall colour retention (%) pisa 3.1 4.2 2.4 1.4 60 33 palermo 0.8 1.9 1.0 2.4 81 77 means are significantly different at the 0.05 level of probability as determined by fisher’s protected lsd. 32 sity with values ranging from 0.5 to 1.0 shoots cm-2 (table 3). the most dense improved type was wintergreen (2.3 shoots cm-2), hybrid type values ranged from 1.7 shoots cm-2 (patriot) to 3.8 shoots cm-2 (tif 00-1), while the most dense dwarf type was miniverde with 5.1 shoots cm-2. horizontal stem density the highest value was recorded for miniverde with 5.5 cm cm-2 while certes 12 had a slightly lower value (5.1 cm cm-2) (table 3). the variability within the different groups was high for this parameter with values ranging from 1.5 to 5.1 cm cm-2 (respectively for certes 1 and certes 3 versus certes 12) for the wild types, from 2.1 to 4.9 cm cm-2 (respectively for scotts r6la and yukon versus bull’s eye) for the improved types, from 2.7 to 4.3 cm cm-2 (for tif 00-18 compared to santa ana and tif 00-1) for the hybrid types, and from 1.6 to 5.1 cm cm-2 (respectively for tifdwarf and miniverde) for the dwarf types. node density certes 3 and miniverde were the entries with the lowest and the highest node density with 0.6 and 5.5 nodes cm-2, respectively (table 3). the variability within the groups was high for this parameter, with the exception of the hybrid types that had values ranging from 1.6 to 2.5 nodes cm-2 (respectively for tif 00-18 and santa ana). leaf width coarser leaves were found in the wild types with values ranging from 2.1 mm (certes 10) to 3.0 mm (certes 5, 6, 7) with certes 12 (1.1 mm) the exception (table 3). improved cd types had a leaf width ranging from 1.6 mm (wintergreen and yukon) to 2.2 mm (scotts r6la and sovereign). for hybrid types values ranged from 1.3 mm (tif 00-10) to 1.9 mm (tif 00-27), while for dwarf types values ranged from 1.0 mm (roma) to 1.5 mm (uganda). spring green-up green cover percentage evaluated in mid-april showed accessions scoring values above 80% and not statistically different from each other in each group (table 4). in more detail, the accession with the best score was certes 5 (93%); the lowest score was found in sovereign (24%). fall colour retention green colour retention evaluated in mid-december showed a great variability within the groups (table 4). the highest value was recorded for tif 00-2, with the lowest for three dwarf types, miniverde, tifdwarf and tifeagle (11%). colour in pisa, the highest and lowest values, barazur (score 8.4) and riviera (score 6.0) respectively, were recorded within the improved type group. in the wild type group, the values ranged from 6.1 (certes 1) to 7.4 (certes 4 and 9) (table 4). with the exception of barazur, the highest values were recorded within the hybrid type group with values ranging from 7.1 (tifsport) to 8.2 (patriot), while table 3 bermudagrass [cynodon (l.) rich.] accessions. shoot density, horizontal stem density, node density and leaf width. accession effect averaged across locations accessions shoot density (n° cm-2) horizontal stem density (cm cm-2) node density (n° cm-2) leaf width (mm) wild types (cd) certes-1 0.8 1.5 0.7 2.8 certes-2 0.7 1.7 0.8 2.8 certes-3 0.9 1.5 0.6 2.8 certes-4 0.8 2.9 1.8 2.5 certes-5 0.9 1.9 0.9 3.0 certes-6 1.0 3.1 1.7 3.0 certes-7 0.6 2.1 0.9 3.0 certes-8 0.5 1.8 0.8 2.9 certes-9 0.9 2.3 0.9 2.8 certes-10 0.9 2.1 0.9 2.1 certes-11 1.0 2.5 1.1 2.7 certes-12 2.4 5.1 3.0 1.1 certes-13 0.7 1.9 1.1 2.8 improved types (cd) argentina 1.0 4.0 1.8 2.1 barazur 2.0 3.1 2.1 1.7 bull’s eye 1.9 4.9 2.4 1.9 celebration 1.3 2.5 1.3 1.9 grand prix 2.2 2.9 1.6 1.8 princess 77 2.1 3.6 1.9 1.8 riviera 1.1 2.3 0.9 1.9 scotts r6la 1.3 2.1 1.0 2.2 sovereign 0.9 3.3 1.4 2.2 sr 9554 1.3 2.2 0.7 2.0 veracruz 1.3 3.4 1.6 1.8 wintergreen 2.3 2.9 1.5 1.6 yukon 1.2 2.1 1.1 1.6 hybrid types (cdxt) patriot 1.7 3.9 2.0 1.8 santa ana 3.1 4.3 2.5 1.4 tifsport 2.3 3.4 1.7 1.8 tifway 2.6 3.8 2.0 1.4 tif 00-1 3.8 4.3 2.3 1.5 tif 00-2 3.2 3.3 2.1 1.5 tif 00-7 3.2 3.2 2.2 1.5 tif 00-10 3.5 3.7 2.0 1.3 tif 00-18 2.7 2.7 1.6 1.4 tif 00-24 2.0 3.7 1.9 1.4 tif 00-27 2.3 3.6 2.1 1.9 dwarf types (cdxt/ct) champion 2.2 4.0 2.0 1.2 miniverde 5.1 5.5 5.5 1.3 tifdwarf 2.6 1.6 1.0 1.3 tifeagle 3.1 4.4 4.2 1.1 tifgreen 2.7 1.9 1.5 1.2 roma 4.1 3.7 2.2 1.0 uganda 3.0 4.7 2.6 1.5 lsd 0.05 0.7 1.6 1.2 0.3 33 for the dwarf types the values ranged from 7.0 (tifgreen) to 7.9 (miniverde). quality in pisa, the highest quality was recorded for the dwarf type miniverde with a score of 8.4, however no significant differences were recorded within this group (table 4). wild types ranged in quality from 4.3 (certes 1) to 7.0 (certes 12), while improved types ranged from 5.8 (riviera) to 7.7 (barazur and bull’s eye); the variability of ratings within both these groups is worthy of note. the hybrid types scored from 6.8 (tif 00-7) to 8.1 (patriot and tifway). 4. discussion and conclusions the study carried out on a pool of genetically and morphologically different entries belonging to the genus cynodon has highlighted a wide variability of aesthetic and morphological traits. morphological characteristics such as shoot density, node density, and horizontal stem density highlighted the better quality of the majority of dwarf and hybrid type cultivars, with improved types showing performances similar to those of wild types; certes 12 was the exception. the genetic differences among groups are reflected more clearly with regard to leaf width, with values getting lower going from wild to dwarf types, with the exception of certes 12. recovery from winter dormancy in the spring, expressed as spring green up, showed a great variability within and among the groups. this parameter is associated with carbohydrate reserves accumulated in storage organs as observed by macolino et al. (2010). other studies (volterrani et al., 2012) focused on carbohydrates in stolons in the first year of establishment and the relationship with growth and establishment rate. cold tolerance, expressed as fall colour retention, highlighted the better performances of the hybrid types with the dwarf types being the first cultivars in which dormancy begins. the parameters representing turf aesthetic quality (colour and turf quality), although they indicate a great variability within groups, showed improving mean values from wild to dwarf types and confirmed what patton et al. (2009) observed concerning the differences between improved and hybrid types. acknowledgements the authors wish to acknowledge dr. wayne hanna, the university of georgia, for providing experimental clones of hybrid bermudagrass. this trial was carried out within the project “sistemi avanzati per la produzione vivaistica di tappeti erbosi di specie macroterme ad uso multifunzionale a basso consutable 4 bermudagrass [cynodon (l.) rich.] accessions. spring greenup (april 14 2011) and fall colour retention (percentage of green colour) (17 december 2011). accession effect averaged across locations. colour (visual estimation based on a 1-9 scale) and quality (visual estimation based on a 1-9 scale) refer only to the pisa location (mean values may-october 2011) accessions spring green-up (%) fall colour retention (%) colour (1-9) quality (1-9) wild types (cd) certes-1 55 16 6.1 4.3 certes-2 75 32 7.0 5.7 certes-3 66 38 6.3 5.8 certes-4 65 38 7.4 6.8 certes-5 93 76 6.4 6.0 certes-6 73 49 7.2 6.4 certes-7 74 37 7.1 6.2 certes-8 59 33 6.6 6.2 certes-9 85 63 7.4 6.7 certes-10 51 53 6.7 5.9 certes-11 72 68 6.2 5.6 certes-12 79 54 6.8 7.0 certes-13 60 41 6.7 5.9 improved types (cd) argentina 66 45 6.4 6.1 barazur 55 43 8.4 7.7 bull’s eye 76 59 7.9 7.7 celebration 49 59 7.5 6.4 grand prix 77 78 6.8 6.9 princess 77 72 73 6.9 6.9 riviera 57 50 6.0 5.8 scotts r6la 73 57 6.9 6.4 sovereign 24 56 6.4 6.3 sr 9554 67 56 6.8 6.5 veracruz 71 61 6.8 6.9 wintergreen 83 69 7.1 6.5 yukon 70 35 7.3 7.1 hybrid types (cdxt) patriot 75 33 8.2 8.1 santa ana 77 73 7.4 7.9 tifsport 79 68 7.1 7.2 tifway 87 83 7.9 8.1 tif 00-1 86 77 7.9 7.5 tif 00-2 89 86 7.2 7.6 tif 00-7 62 75 7.9 6.8 tif 00-10 82 81 8.1 7.8 tif 00-18 84 80 8.0 8.0 tif 00-24 74 79 7.7 7.0 tif 00-27 87 81 7.4 7.6 dwarf types (cdxt/ct) champion 86 76 7.7 8.0 miniverde 53 11 7.9 8.4 tifdwarf 63 11 7.8 7.9 tifeagle 52 11 7.4 8.1 tifgreen 72 31 7.0 7.9 roma 75 56 7.4 8.0 uganda 84 78 7.8 7.9 lsd 0.05 17 7 0.5 0.7 34 mo idrico ed 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green section record, 30(4): 10-12. taliaferro c.m., 2003 bermudagrass [cynodon (l.) rich.], pp. 235-256. in: casler m.d., and duncan r.r. (eds.) turfgrass biology, genetics, and breeding. wiley & sons, hoboken, new jersey, usa, pp. 384. volterrani m., grossi n., gaetani m., pompeiano a., 2010 zoysiagrass cultivar establishment rate and turf quality in central italy. acta horticulturae, 881: 313-316. volterrani m., grossi n., lulli f., gaetani m., 2008 establishment of warm season turfgrass species by transplant of single potted plants. acta horticulturae, 783: 77-84. volterrani m., magni s., 2004 species and growing media for sports turfs in mediterranean area. acta horticulturae, 661: 359-364. volterrani m., pompeiano a., magni s., guglielminetti l., 2012 carbohydrate content, characterization and localization in bermudagrass stolons during establishment. acta agriculturae scandinavica, 62(1): 62-69. impaginato 225 adv. hort. sci., 2017 31(4): 225-233 doi: 10.13128/ahs-20814 detection of not allowed food-coloring additives (copper chlorophyllin, coppersulphate) in green table olives sold on the italian market c. negro, l. de bellis, e. sabella, e. nutricati, a. luvisi, a. miceli dipartimento di scienze e tecnologie biologiche ed ambientali, università di salento, via monteroni, 165, 73100 lecce, italy. key words: adulteration, cu-chlorophyllin, e141ii, high performance liquid chromatography, table green olives. abstract: table olives are a common and well-known food in the whole mediterranean area, produced and consumed in great quantities. many deepgreen olives can be found on sale in the south of italy. sometimes a deep color could be the result of the fraudulent addition of a coloring agent (e141ii, copper chlorophyllins) during the pickling process, in spite of the european union legislation that does not allow the addition of any colorant to fruits included table olives. the objectives of this study were to use a relatively simple method of detection of e141ii added to table olives, to verify the presence on the italian market of artificially colored table olives, and to show that also cuso4 can be employed for table olive re-greening. compounds with chromatographic and spectral characteristics similar to the ones from the e141ii (cu chlorin e6, cu isochlorin e4, cu pyropheophorbide a) were found in 8 samples out of 16. these results show that the fraudulent addition of colorant to table olives is a quite common practice. more pressing controls and analysis are required to ensure the complete food safety and the compliance with the current law. 1. introduction table olives are a common and well-known food in the whole mediterranean area, produced and consumed in great quantities especially in southern italy. manufacturing techniques have been refined along the years, in order to optimize the quality and attractiveness of the final product and to cover different market niches. table olives found on market’s shelves exhibit different colors and shapes, depending on cultivar type, ripening stage or processing method. there are green olives, spotted and fully ripened ones. in southern italy the greek method and the spanish method are commonly used to produce green table olives, while the castelvetrano method covers a smaller but increasing market. with the greek method, olives are washed with water and then stored in brine (5-8%). the addition of salt promotes the product preservation (*) corresponding author: carmine.negro@unisalento.it citation: negro c., de bellis l., sabella e., nutricati e., luvisi a., miceli a., 2017 detection of not allowed food-coloring additives (copper chlorophyllin, copper-sulphate) in green table olives sold on the italian market. adv. hort. sci., 31(4): 225-233 copyright: © 2017 negro c., de bellis l., sabella e., nutricati e., luvisi a., miceli a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 june 2017 accepted for publication 22 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 225-233 226 and the development of fermentation-capable microorganisms. during this stage of treatment, olives slowly lose their bitterness (due to oleuropein’s enzymatic degradation) and acquires their final taste and properties. the development of lactic fermentation is favoured (olives average final ph of 5.2), in order to improve both the food safety and its organoleptic characteristics (piga et al., 2001). the spanish method, the most used worldwide, requires the olives to be treated with a solution of sodium-hydroxide and water in order to hydrolyze most of the oleuropeinic glucosides. afterwards, olives are washed repeatedly with water and then stored in brine (5-6%) in which they naturally undergo a complete lactic fermentation, reaching an average ph value of 4 (garrido-fernandez et al., 1997). olives treated with the castelvetrano method are plunged for 10 to 15 days in a sodium-hydroxide solution which concentration depends on olive caliber and their ripening stage. later on, marine salt (nacl) will be slowly added to the solution. after such treatment drupes will undergo several washings with water (salvo et al., 1995) so that, in absence of a lactic fermentation, ph never goes below 6.5 (fodale et al., 2007). anyway, castelvetrano type olives appears brilliant-green and with more compact pulp (owen et al., 2003), so that they are preferred to green table olives prepared with the greek or spanish methods. as a matter of fact, treatment and storage in acid solutions are known to be the main responsables of chlorophylls degradation in table olives (minguez mosquera et al., 1989). the loss of the magnesium from the chlorophylls causes to turn into the corresponding pheophytins, shifting their color from green to brown (scotter et al., 2005). at the end of the various pickling processes, therefore, olives had lost most part of their original chlorophylls (and of their original dye) becoming yellowish-green; in spite of that, many deep-green olives can be found on sale in the south of italy, especially in the bari province. their sometimes unnatural colour could be the result of the fraudulent addition of a colouring agent during the pickling process, in spite of the european union legislation does not allow the addition of any colorant to fruit and table olives (eu, reg ce 94/36/ce, 1994). among the most used green food colorants, there are the chlorophyll-derived ones; copper complexes of chlorophyillins, particularly, play a major role within this group. copper chlorophyllins are manufactured by chlorophyll saponification which, in turn, is extracted by means of organic solvents from edible plant, such as alfalfa (medicago sativa l.) and nettle (urtica dioica l.) (mortensen, 2006). the resulting chlorophyll-based salts are marketed as e141 colorant and are available in two forms: e141i (liposoluble) and e141ii (hydrophilic). these salts are widely used as food colorants (e.g. ice creams, snacks, food decorations) but their addition to table olives and other fruits is expressly forbidden. in spite of that, e141ii (due to its hydrophilic properties and color stability) is sometimes fraudulently added to table olives during the pickling process as re-greening agent. the objectives of this study were to confirm the presence on the market of artificially colored table olives through the detect fraudulent color adulteration with e141ii, and to verify if the addition of copper-sulphate during the pickling process could allow a re-greening of table olives as result of its interaction with chlorophylls, leading to the formation of cu-chlorophyllins. nowadays, copper-sulphate is not allowed and not even mentioned in food legislation, but it is known that it has been widely utilized by food manufacturers because of its re-greening properties on preserved vegetables (cerutti, 2006) but its use is very dangerous because copper is toxic to the liver. continuous consumption of olives treated with this compound could result in damage to the consumer due to its accumulation in the body (stern, 2010). all this to generate a warning for consumers and public authorities. 2. materials and methods chemicals all reagents were analytical or hplc grade: hexane and water were supplied from romil ltd. (cambrdge, uk); methanol from carlo erba reagents (rodano, italy); acetic acid and tert-butyl metyl ether from j.t. baker (deventer, netherlands). chlorophyll a and b, pheophorbide a and copper sulfate were purchased from sigma-aldrich co. (saint louis, usa). sample of e-141ii colorant were supplied by chimica d’agostino (bari, italy). plant material obviously deep-green table olives may have been fraudulently colored, while table olives appearing from pale-green to mustard-yellow are to be considered not colored. therefore, 16 table olives samples were purchased from local markets (bari, brindisi and lecce): 9 olives were with a brilliant-green or deepnegro et al. detection of not allowed food-coloring additives in green table olives in italian market 227 green color, while drupes from the remaining 7 samples were pale-green or mustard-yellow. ph values of the brine were recorded with a pc 650 probe (eutech instruments, singapore). due to the lack of information reported on the labels, we could not assess the cultivar and origin of each olive sample (table 1). pigment extraction all procedures were performed under dimmed green light to avoid any photo-oxidation of chlorophylls. 40 g of olive pulp were collected and homogenized with 25 ml of methanol-water solution (80/20, v/v) using an ultra-turrax t25 (janke e kunkle, ikalabortechnik, germany); the resulting paste was filtered by means of a buckner’s funnel with a paper filter (perfecte 2 extra rapida, superfiltro milano, italy) and a suction flask connected to a vacuum pump. the solid residue was collected, added to 50 ml of methanol and stirred for 1 hour; the resulting solution underwent a second filtration step with the same procedure previously used. for same samples appearing still green, 50 ml of methanol were added and a third stirring-filtering cycle has been performed. the filtrate was then mixed with an identical amount of hexane in a separating funnel in order to separate the lipophilic substances from the hydrophilic ones. the latter phase was recovered and evaporated under vacuum in a re 111 rotavapor (büchi, flawil, switzerland) at room temperature. the evaporation remnant was finally diluted up to 2 ml with methanol and utilized for the hplc/dad analysis. this procedure was made up for extraction hydrophilic pigments preferably. cuso4 addition tests in order to verify the possibility of re-greening using copper sulphate during the olive production process or the marketing, samples of olives were treated with cuso4. it is known, in fact, that the addition of copper stabilizes the tetrapyrrolic ring of pheophytin, resulting a re-greening of olive drupes (niir, 2004). a local food company provided us a sample of olives processed with the castelvetrano method and stored in a sodium-hydroxide/salt/water solution (ph 11.5). four groups of 10 olives were taken from the sample: the first one was washed with water for 24 hours, stored in brine (6% nacl, 0.6% citric acid, 0.05% ascorbic acid, ph: 2.3) for 48 hours (ph stabilized at 7.3) and then analyzed; the remaining 3 subsamples were placed in beakers with 150 ml of their original packing solution, 1.5 g (1%), 7.5 g (5%) and 30 g (20%) of cuso4 were then added. after 3 hours of stirring, the coloring solution was discarded and the samples were washed for 24 hours with water. the samples were then stored in brine solution for 48 hours until ph stabilization (ph 5.4). after the brine storage, all the sub-samples went through the before mentioned pigments extraction procedure. another trial was run to test the effect of cuso4 addition on olives treated with the classical spanish method. 10 olives were selected from a sample which analysis already excluded any fraudulent colouration (s16). drupes were first put in an alkaline solution (150 ml of 1% naoh-water solution, ph: 12.8) until ph stabilization was reached (ph: 8.3), 30 g (20%) of cuso4 were then added to this medium. the drupes were then immersed in brine solution; after 48 hours the ph reached a stable value of 4.0. the sample was then treated until pigment extraction following the already cited procedures. analysis of chlorophylls compounds by hplc/dad the determination of pigment products were carried out by hplc using a agilent 1100 liquid chromatograph fitted with a manual injector. a stainless steel column, alltech prontosil c30, 200 å, 5 µ, 250× 4.6 mm i.d. was used. the column was protected by precolumn packed with the same material. separation was performed using an elution gradient (flow rate 1 ml min-1) with the mobile phases (a) methanol: distilled water: acetic acid (90:10:0.5 v/v/v) and (b) tert-butyl metyl ether: methanol: acetic acid (100:10:0.5 v/v). the gradient scheme was 0-50% b in 30 minutes, 50-100% b in 10 minutes, 100% b for 5 minutes and 100-0% b in 5 minutes. sample ph colour cultivar origin s1 5.4 g n.r. n.r. s2 5.9 g n.r. greece s3 7.1 g n.r. greece s4 5.7 g nocellara del belice italy (sicily) s5 6.1 g n.r. n.r. s6 5.9 g n.r. italy s7 5.1 g n.r. italy s8 5.7 y n.r. n.r. s9 6.4 g n.r. n.r. s10 4.2 y bella di cerignola n.r. s11 3.8 y n.r. n.r. s12 4.1 y n.r. n.r. s13 5.0 y n.r. n.r. s14 3.3 y n.r. n.r. s15 4.0 y n.r. n.r. s16 4.4 g n.r. n.r. table 1 main characteristics of the olives samples analyzed g= green; y= pale-green or mustard-yellow. n.r.= not reported. adv. hort. sci., 2017 31(4): 225-233 228 sequential detection was performed with a photodiode array detector (dad) at 430 nm and 650 nm also the online uv-vis spectra were recorded from 250 to 800 nm. 100 µl of 1 mg/ml e141ii in methanol or 100 µl of extract was utilized for the analysis. to analyze the samples treated with cuso4 the flow rate was lowered at 0.7 ml/min and the elution of b was set to 0-45% in 30 minutes, 45-100% in 10 minutes, 100% for 5 minutes and 100-0% in 5 minutes. data were collected and processed with a lc agilent chemstation (revision software b.04.02). pigments were identified by co-chromatography with authentic samples and/or by comparison their spectral characteristics with literature or compound standard when available. all analysis were made in duplicate. 3. results and discussion in order to identify and recognize the e141ii components likely to be present in olive samples, industrial copper chlorophyllin was analyzed by hplc/dad, injected and monitored at 650 nm. the resulting chromatogram shows 8 major peaks indicates with a to h in figure 1. the analysis of the elution times and absorption spectra of the peaks (fig. 2) compared with literature (inoue et al., 1994; chernomorsky et al., 1997; mortensen and geppel, 2007; roca et al., 2010; aparicio-ruiz et al., 2011; gandul-rojas et al., 2012) allowed the tentative identification of 7 of the 8 peaks: (a) cu rhodin g7, (b) cu chlorin e6, (c) cu chlorin p6, (d) cu isochlorin e4, (e) cu 151-oh-lactone-pheophytin a (g) cu rhodochlorin, (h) cu pyropheophorbide a. we were unable to identify without any doubt peak f. the main components of our e141ii standard eluted according to montensen and geppel (2007) procedure which utilize a chromatographic method similar, were: cu rhodin g7 < cu chlorin e6 < cu isochlorin e4 < cu pyropheophorbide a. figures 3 and 4 show chromatograms resulting from the hplc/dad analysis of the studied samples; they have been recorded at 650 nm to facilitate the detection of chlorophylls and their derivatives, avoiding in the same time interferences from carotenoids. in 7 samples out of 16 (fig. 3) we assessed the presence of compounds with chromatographic characteristics similar to the ones from the industrial chlorophyllin sample: cu isochlorin e4 (peak d) and cu pyropheophorbide a (peak h). this finding agrees with bibliographical data, since cu isochlorin e4 is referred as one of the main component of commercial copper chlorophyllins (inoue et al., 1994; chernomorsky et al., 1997; ferruzzi et al., 2002). cu chlorin e6 (peak b) were found in 2 samples. finally, the presence of cu rhodin g7, cu chlorin p6, cu 151oh-lactone-pheophytin a and cu rhodochlorin was never observed. this result is supported by the work of gandul-rojas et al. (2012) which demonstrates fig. 1 hplc/dad analysis of food colorant e141ii, recorded at 650 nm, showing the peaks of the main identified components: a) cu rhodin g7; b) cu chlorin e6; c) cu chlorin p6; d) cu isochlorin e4; e) 151 oh lactone pheophytin a; f) unknown; g) cu rhodochlorin; h) cu pyropheophorbide a. fig. 2 spectra uv/vis of the copper chlorophyllins pigments found in the colorant e141ii and in some samples of green table olives. negro et al. detection of not allowed food-coloring additives in green table olives in italian market 229 that the addition of e141ii to table olives led to the chromatographic identification of cu-chlorin type compounds (mainly cu chlorine e6 and cu isochlorin e4) very different from chlorophyll derivatives usually found in green table olives. moreover, minguezmosquera et al. (1995) showed that under certain (still unexplained) circumstances, small amounts of cu-chlorophyll compounds can be spontaneously synthesized within the olives, leading to localized pigment alteration on their surface (green staining alteration). the visual analysis of our samples led us to notice no traces of green staining on the drupes: cucompounds identified in our samples must therefore be the result of a colorant addition. in those samples we were also able to identify several other pigments, marked with numbers, such as chlorophyll b (peak 4; fig. 3-s1); pheophitin a (peak 5; fig. 3-s6 to 3-s8) and isochlorin e4 (peak 7; fig. 3-s5 to 3-s8). cu-compounds were never found in 8 of the analyzed samples (fig. 4). on the other hand, those samples contained pigments identified as chlorophylls (e.g chlorophyll a; peak 6; fig. 4-s9, 4s11, 4-s13, 4s16) and chlorophyll derivatives (e.g. pheophorbide a; peak 2; fig. 4-s13, 4-s15, 4-s16, pheophytin a; peak 5; fig. 4-s10 to 4-s16). fig. 3 hplc/dad chromatograms recorded at 650 nm of the samples (s1-s8) containing cu-components related to ones from e141ii colorant. peaks described in table 2. adv. hort. sci., 2017 31(4): 225-233 230 gandul-rojas et al . (2012) show that chromatograms of olives treated with the spanish method exhibit a series of major peaks belonging to mg-free chlorophyll derivatives (mainly phaeophytins), while chromatograms of samples treated with the castelvetrano method show peaks from chlorophylls or degraded chlorophylls with mg (e.g. glycoxylic acid chlorophylls; formyl chlorophylls) in addition to the first ones. table 2 indicates the chromatographic, spectral characteristics and origin of the chlorophyll derivates pigments identified in the commercial e141ii and in the olive samples. the results obtained are in agreement with the literature (hynninen 1973; inoue et al., 1994; chernomorsky et al., 1997; mortensen and geppel, 2007; roca et al., 2010; aparicio-ruiz et al., 2011; gandul-rojas et al., 2012). the pigments identified are cu chlorophyllins (peaks a-h), chlorophylls and derivatives (peaks 1-7). the analysis of the 9 samples initially suspected of have been fraudulently colored (indicated as g in table 1), largely confirmed our hypothesys: 8 samples contains cu compounds related to the e141ii colorant (fig. 3). on the other hand, 6 out of the 7 samples chosen accordingly to their pale-green or mustad-yellow dyes, do not contain compounds related to the food coloring agent, underlining that the addition of e141ii during the pickling process could be the fig. 4 hplc/dad chromatograms recorded at 650 nm of the samples (s9-s16) do not contain cu-components related to ones from e141ii colorant. peaks described in table 2. negro et al. detection of not allowed food-coloring additives in green table olives in italian market 231 main responsible of the bright or deep-green dye of the analyzed olives. concerning the addition of copper-sulphate, the re-greening made at alkaline ph was more pronounced in samples treated with the addition of 5 and 20% cuso4 in comparison respect to olives treated with 1% cuso4. in fact, untreated samples do not show peaks referable to cu-chlorophyll compounds (fig. 5-s17), while samples treated with 1, 5 and 20% of cuso4 show well defined peaks in their chromatograms: 2 of them were identified as cu chlorin e6, (peak b) and cu pyropheophorbide a (peak h) (fig. 5-s18; 5-s19; 5-s20). the sample previously processed with spanish method (therefore at acid ph) and treated with cuso4, shows a lighter colour change with respect to the other three samples. this is supported by the very low adsorbance values of cu chlorin e6 (peak b) pointed out in its chromatogram (fig. 5-s21). the copper of cuso4 seems therefore to react only at alkaline ph (before the final acidification) with the degraded chlorophylls within the olives the same way it reacts with saponificated chlorophylls during industrial colorant production, leading to the synthesis of cu-chlorophyll derivatives similar to the ones identified in the e141ii reference sample. pigment peak (z) kc soret spectral data in hplc eluent sample (and/or e141ii dye) where the pigment is present referencesi ii iii iv v vi m r m r m r m r m r m r chlorine e6 1 8.83 402 500 18.02 528 25.66 (642) 9.45 662 5.47 s5 to s7, s10, s11, s13 to s16 [4] cu rhodin g7 a 11.21 436 366 3.27 577 6.85 623 3.11 e141ii [3] cu chlorin e6 b 12.42 408 (395) 1.21 502 8.02 (588) 4.80 634 2.19 e141ii, s4, s8, s18 to s21 [2] [3] [4] [6] cu chlorin p6 c 13.48 407 388 1.29 500 19.52 (597) 9.76 640 2.22 e141ii [4] pheophorbide a 2 17.77 408 (380) 1.53 (400) 1.10 507 9.33 537 9.34 609 9.76 666 2.01 s13, s15, s16 [2] [7] std cu isochlorin e4 d 17.96 406 (394) 1.16 501 18.71 (585) 11.65 627 2.20 e141ii, s1 to s7, s18 to s20 [2] [4] cu 151-oh-lactonepheophytin a e 19.07 412 392 1.27 498 19.33 540 23.22 598 7.96 644 1.53 e141ii [5] pheophytin b 3 20.75 436 416 2.58 526 8.03 560 19.84 600 16.01 656 4.43 s6 [2] unknown f 21.93 417 (397) 1.86 505 19.50 545 14.31 623 6.78 671 1.67 e141ii chlorophyll b 4 22.62 466 (550) 17.46 600 8.45 650 2.49 s1, s9 [2] std pheophytin a 5 23.31 410 (380) 1.52 (400) 1.11 507 9.44 538 10.11 610 10.62 667 2.12 s5 to s7, s11, s13 to s16 [2] cu rhodochlorin g 25.04 408 393 1.29 498 21.50 (593) 9.67 636 1.83 e141ii [4] chlorophyll a 6 26.16 430 (386) 1.70 (416) 1.12 (580) 9.80 618 5.10 665 1.13 s9, s11, s16 [2] std cu pyropheophorbide a h 26.31 424 (366) 1.78 403 1.15 510 19.60 554 606 5.44 652 1.13 e141ii, s1 to s4, s6, to s8, s18 to s20 [1] [4] [5] isochlorin e4 7 28.55 400 500 11.87 530 31.62 560 74.80 609 31.60 665 3.06 s5 to s8, s13, s16 [1] table 2 the chromatographic and spectroscopic characteristics of the chlorophyll derivates pigments present in the e141ii dye and in the analyzed green table olives compounds derived from copper-free chlorophylls are indicated by numbers; the other, containing copper, present in the e141ii dye and in some samples, with letters. retention factor. kc = (tr tm)/tm where tr is the retention time of the pigment peak and tm is the retention time of an unretained component. m = maximun absorbance (nm); r = quotient of absorbance at soret band divided by absorbance at wavelength indicated. the values in parentheses indicate inflection points in the absorption spectrum. [1] aparicio et al., 2011, [2] gangul-rojas et al., 2012; [3] inoue et al., 1994; [4] mortensen et al., 2007; [5] roca et al., 2010, [6] chernomorsky et al., 1997, [7] hynninen, 1973, std = chemical standard. adv. hort. sci., 2017 31(4): 225-233 232 4. conclusions the use of a relatively simple method of detection of e141ii added to table olives shows that the fraudulent addition of colorant (or copper sulfate) to table olives is a quite common practice. in fact, the 50% of the analyzed samples possess copper chlorophyll pigments, most likely due to the addition of colorant e141ii, or, worse still, of copper sulphate during the production process. our work has shown that in these samples there is often the presence of cu chlorin e6 , cu isochlorin e4 and cu pyropheophorbide to which can therefore be considered markers for these adulterations. more controls and analysis are required to ensure the complete food safety and the compliance with the current law. the finding that even copper-sulphate can be used to modify olive dye before marketing them, makes this need even more pressing, especially in relation to the severe liver damages it may cause. references aparicio-ruiz r., riedl k.m., schwartz s.j., 2011 identification and quantification of metallo-chlorophyll complexes in bright green table olives by highperformance liquid chromatography-mass spectrometry quadrupole/time-of-flight. j. agr. food chem., 59: 11100-11108. cerutti g., 2006 residui, additivi e contaminanti degli alimenti. tecniche nuove, milano, italy, pp. 128-129. chernomorsky s., rancourt r., sahai d., poretz r., 1997 evaluation of commercial chlorophyllin copper complex preparations by liquid chromatography with photodiode array detection. journal of association of analytical communities international, 80: 433-435. european union, 1994 official journal of the commission of the european communities. eu regulation no. 94/36/ce, l237. ferruzzi m.g., failla m.l., schwartz j., 2002 sodium copper chlorophyllin: in vitro digestive stability and accumulation by caco-2 human intestinal cells. j. agr. food chem., 50: 2173-2179. fig. 5 hplc/dad chromatograms, recorded at 650 nm, of samples treated with various percentages of cuso4: (s17) without cuso4 addition; (s18) cuso4 1%; (s19) cuso4 5%; (s20) cuso4 20%. (s21) classical spanish method and cuso4 20%. peaks described in table 2. negro et al. detection of not allowed food-coloring additives in green table olives in italian market 233 fodale a.s., mule r., tagliavia m., tucci a., 2007 the processing of green olives using the “castelvetrano” method. industrie alimentari, 46(742): 895-900. gandul-rojas b., roca m., gallardo-guerrero l., 2012 detection of the colour adulteration of green table olive with copper chlorophyllin complexes (e141ii colorant). lwt-food sci. technol., 46: 311-318. garrido-fernandez a., fernandez-diez m.j., adams m.r., 1997 table olives. production and processing. chapman & hall, london, uk, pp. 496 hynninen p., 1973 preparation and purification of some derivatives of chlorophylls a and b. acta chemica scandinavica, 5: 1771-1780. inoue h., yamashita h., furuya k., nonomura y., yoshioka n., li s., 1994 determination of copper (ii) chlorophyllin by reversed-phase high-performance liquid chromatography. j. chromatogr. a, 679: 99-104. minguez-mosquera m.i., gallardo-guerrero l., hornero-mendez d., garrido-fernandez j., 1995 involvement of copper and zinc ions in green staining of table olives of the variety gordal. j. food protect., 58: 564-569. minguez-mosquera m.i., garrido-fernandez j., gandul-rojas b., 1989 pigment changes in olives during fermentation and brine storage. j. agr. food chem., 37: 8-11. mortensen a., 2006 carotenoids and other pigments as natural colorants. pure appl. chem., 78: 1477-1491. mortensen a., geppel a., 2007 hplc-ms analysis of the green food colorant sodium copper chlorophyllin. innov. food sci. emerg., 8: 419-425. niir, 2004 food colours, flavours and additives technology handbook. chapter 9 natural colours in food. niir national institute of industrial research, pp. 626. owen r.w., haubner r., mier w., giacosa a., hull w.e., spiegelhalder b., bartsch, h., 2003 isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes. food chem. toxicol., 41: 703717. piga a., gambella f., vacca v., agabbio m., 2001 response of three sardinian olive cultivars to greekstyle processing. ital. j. food sci., 1: 29-40. roca m., gallardo-guerrero l., minguezmosquera m.i., gandul rojas b., 2010 control of olive oil adulteration with copper-chlorophyll derivates. j. agr. food chem., 58: 51-56. salvo f., cappello a., giacalone l., 1995 destinazione della produzione olivicola. l’olivicoltura nella valle del belice, istituto nazionale di economia agraria, italy, pp. 155-187. scotter m.j., castle l., roberts d., 2005 method development and hplc analysis of retail foods and beverages for copper chlorophyll (e141[i]) and chlorophyllin (e141[ii]) food colouring materials. food addit. contam., 22: 1163-1175. stern b.r., 2010 essentiality and toxicity in copper health risk assessment: overview, update and regulatory considerations. j. toxicol. env. health, part a, 73: 114-127. impaginato 235 adv. hort. sci., 2017 31(4): 235-240 doi: 10.13128/ahs-20517 the influence of synthetic strigolactones and plant extracts on the morphological parameters of onion (allium cepa) t. kopta 1 (*), m. antal 2, m. jurica 1, j. volková 2, r. pokluda 1 1 department of vegetable growing and floriculture, faculty of horticulture, mendel university brno, brno, czech republic. 2 research institute for organic synthesis (vuos), rybitivi, czech republic. key words: leaf length, leaf weight, number of leaves, stability, strigolactones, vegetables. abstract: in recent years there has been frequent reference to the significance of strigolactones as a new group of hormones which might have a significant effect on horticultural production. the aim of this work was to find an ideal combination of stable synthetic strigolactones and plant extracts with potential effects on onion plants. the synthetic strigolactone fenyl 7 (dihydro-3-[[2,5dihydro-4-methyl-5-oxo-2-furanyl)oxy]-methylene]-5-phenyl-2(3h)-furanone) was tested in a carrot macerate, with citric acid and with salicylic acid. from the results it was confirmed that increasing the ph of the preparation leads to improving the stability of fenyl 7. evaluation has repeatedly confirmed the effect of the preparation, combining synthetic strigolactone and a macerate of carrot in a mixture of surfactants with added citric acid. in all the experiments this combination showed a statistically demonstrable influence on leaf weight (increased by 12-31%) and length (increased by 6-13%) in comparison with the controls. 1. introduction in several countries fertiliser use on agricultural soil results in serious problems associated with the accumulation of residues in the food chain and groundwater pollution (hegazi et al., 2010). the increased costs of chemical fertilisers and the massive input of non-renewable resources have led to promote the research in the field of alternative preparations (board, 2004). several technological innovations have been proposed in order to enhance the sustainability of production systems through a significant reduction in the use of chemicals. an effective tool would be the use of biostimulants (hamza and suggars, 2001) a group of compounds that act neither as fertilisers nor as pesticides, but have a positive impact on plant performance when applied in small quantities (calvo et al., 2014). carotenoids are essential photosynthetic pigments also identified as (*) corresponding author: tomas.kopta@mendelu.cz citation: kopta t., antal m., jurica m., volková j., pokluda r., 2017 the influence of synthetic strigolactones and plant extracts on the morphological parameters of onion (allium cepa). adv. hort. sci., 31(4): 235-240 copyright: © 2017 kopta t., antal m., jurica m., volková j., pokluda r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 april 2017 accepted for publication 27 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 235-240 236 precursors of different signalling molecules and hormones such as strigolactones. strigolactones, recently recognised as a new class of plant hormones, play a key role in different developmental processes such as plant architecture, the number of shoot branches and nutrient availability (al-babili and bouwmeester, 2015). several studies have reported the ability of these molecules to stimulate root hair elongation (kapulnik et al., 2011) and root growth (arite et al., 2012), but also to inhibit adventitious root formation (rasmussen et al., 2013; urquhart et al., 2015). furthermore, strigolactones increase stem thickness by stimulating secondary growth in interaction with auxin (agusti et al., 2012), positively regulate internode length (de saint germain et al., 2013) and accelerate leaf senescence (yamada et al., 2014). strigolactones are also signalling molecules, which participate in communication with mycorrhizal fungi, initiating the arbuscular mycorrhiza formation (garcía-garrido et al., 2009). through mycorrhiza strigolactones are also involved in plant nutrition increasing the reduction of the phosphates in soils (garcía-garrido et al., 2009). moreover, studies have shown the role of this plant hormone on seed germination (toh et al., 2012) and early seedling development (tsuchiya et al., 2010). strigolactones stimulate the germination of parasitic plants, for example witchweed or broomrape, which frequently survive for years in a dormant state. these seeds can be stimulated by preparations containing strigolactones obtained from plant roots showing effects already in extremely low concentrations: for example, a concentration less than 10-10 mol l-1 is sufficient to stimulate the germination of parasitic plants (humphrey et al., 2006). plant extracts are mostly rather complex mixtures, and therefore individual components can function antagonistically but also synergistically. the assumption that plant extracts stimulate the growth with similar effects to strigolactones was tested in experiments with the germination of the striga hermonthica plants. this test is a known method for ascertaining the presence of strigolactones and is standardised for the effects of the synthetic strigolactone gr24 (sato et al., 2005; yoneyama et al., 2008). strigolactones are now known to modify positively many plant traits important for crop yield and quality (mason, 2013) and many commercial opportunities could arise (wood production, shoot architecture, root development, nutrient acquisition and more). already it is possible to identify certain strigolactones that affect one process and not another. however, one challenge is to identify stable strigolactones or methods of delivery that enhance their stability (beveridge, 2014). the aim of this work was to find a suitable combination of stable synthetic strigolactone and plant extracts with a positive effect on onion growth parameters. in the experiment the stability of the synthetic strigolactone fenyl 7 was evaluated and the application of several types of bio-additive in onion seedlings and the influence on morphological parameters was followed. 2. materials and methods fenyl 7 (dihydro-3-[[2,5-dihydro-4-methyl-5-oxo2-furanyl)oxy]-methylene]-5-phenyl-2(3h)-furanone) is a synthetic strigolactone protected as utility model no. 29064 by industrial property office and produced in vuos (research institute for organic synthesis, rybitvi) by synthesis from 3-methyl-2(5h)-furanone and phenylbutyrolactone. the obtained compound, which occurs in two stereoisomers, was purified with the aid of column chromatography. the compound has a similar structure to other synthetic strigolactones (e.g. gr5, gr7 and gr24), but is simpler and cheaper to produce. fractions of individual isomers were obtained and also a fraction with a mix of isomers in a concentration of 10-2 mol l-1 was used for additive preparation. given the instability of fenyl 7 in water a method was sought to increase the stability of this substance in solution. aside from using surfactants a possible means of increasing stability is to change the ph of the application solution. natural strigolactones arise from the biosynthesis of carotenoids, and so for this purpose carrot macerate (ph = 5.2) was used, which presents its positive effects (lópez-ráez et al., 2010). to further reduce ph and also to conserve the macerate, citric or salicylic acid were added (ph = 3.6). fenyl 7 was dissolved in the mixture of surfactants or in the carrot macerate and converted with the aid of a vacuum evaporator to a 3 g l-1 mixture of surfactant. the resulting concentration for application on plants was 10-5 m. the carrot macerate was prepared by the maceration of carrot roots for 3-5 days in ethyl acetate. for the experiment a 1:1 mixture of mdge (diethylene glycol monomethyl ether) and pegsho (poly(ethylene glycol)sorbitol hexaoleate) was used. first the stability of fenyl 7 in buffers (mdge and pegsho) of ph 4, 7 and 9, according to oecd (2004), kopta et al. stability of the synthetic strigolactones fenyl 7. influence on morphological parameters 237 was followed. these solutions were left at a temperature of 4°c and the decline in fenyl 7 was steadily monitored over a period of 30 days or until the drop was greater than 90%. liquid chromatography diodearray detection (hplc-dad) was used to determine the fenyl 7. the determination was carried out on a lichrospher rp-18 column of length 125 mm and internal diameter of 4 mm. the average particle size in the column packing was 5 μm. the column temperature was set at 30°c. a wavelength of 239 nm was used for the detection of measurements. a mixture of 40% meoh (component a) and 100% meoh (component b) was used as a mobile phase. the flow of the mobile phase was set at 0.9 ml min-1. analysis of one sample with a volume of 10 μl last 15 min. the gradient of the mobile phase was set at: 0 min 0% component b, 10 18 min 50% component b and 21 min 0% component b. the influence of preparations selected according to previous laboratory research (table 1) on the morphological parameters of onion seedlings (allium cepa) was then followed. the experiment was carried out in three periods during two years. the experiments labelled 1, 2 and 3 were evaluated on the dates 12/5/2014, 17/7/2014 and 13/5/2015. the variety lusy (semo, cz) was sown in the substrate klasmann ts3 fine 416, the ph was 6.0 and the electrical conductivity of the substrate was 30 ms m-1. the nutrient content of the substrate was as follows: 100 mg l-1 n, 70-150 mg l-1 p2o5, 140-300 mg l-1 k2o and 60-100 mg l-1 mg (pasic, cz). the experiment took place in a greenhouse in type 96 trays (cell volume 27 ml). 100 ml per tray (dilution of 2 ml l-1 water), which corresponds to a dose of 2 ml per plant, was applied as a foliar spray. only water was used to spray the control treatment in the same period. for treatments, the variants given in table 1 were selected in two applications (the first in the phase of the first true leaves, and the second after 14 days). the determination of fresh leaf weight (g), leaf length (m) and their number (pcs) was carried out 8 weeks from sowing on 25 plants. data were analysed by analysis of variance and evaluated by using the lsd test at a probability of p = 0.05 (statistica 12.0, stafsoft). 3. results and discussion given the instability of fenyl 7 in water, a means was sought for increasing the stability of this compound in water. accordingly, aside from the use of surfactants the ph of the environment was modified. the assumption that making the ph more acidic would lead to greater stability of fenyl 7, as stated by (babiker et al., 1988) in the case of gr7 in soil, was confirmed in this experiment. in fact, as clearly reported in figure 1, in the case of a solution with ph 9 there was a degradation of fenyl 7 by more than 90% in three days. with reduced ph there was a reduction in the degradation of fenyl 7. the lowest level of degradation was determined in the variant with a ph 4, where the reduction in the concentration was only by 8.5%. the ascertained state corresponds to the summary from zwanenburg and pospíšil (2013), that a lower ph does not lead to the hydrolysis of the tested synthetic strigolactones (gr7, gr24). in the tested bio-additives (table 1) it is possible to conclude the presence of an influence on the evaluation of morphological parameters as it is shown in table 1 composition of bio-additives used and labelling of experiments label composition of mixture used bioa-1 fenyl 7 (isomer 1) in mixture of surfactants bioa-3 fenyl 7 (mix of isomers) in mixture of surfactants bioa-7 fenyl 7 (mix of isomers) in mixture of surfactants with added salicylic acid (20 g l-1) bioa-10 fenyl 7 (isomer 1) and carrot macerate in mixture of surfactants bioa-11 fenyl 7 (isomer 1) and carrot macerate in mixture of surfactants with added citric acid (20 g l-1) fig. 1 degradation of fenyl 7 in dependence on ph. adv. hort. sci., 2017 31(4): 235-240 238 table 2. all the treatments had a positive effect which however was not manifested in repeated experiments with all variants. the number of leaves in onion was positively influenced only in experiment no. 2 (summer 2014) in variants bioa-1, 3, 7 and 10 while a reduction in number of leaves was found in experiment no. 1 (spring 2014) only in variant bioa-3. on the contrary, in the experiment no. 3 (spring 2015) the number of leaves was not influenced in comparison to control conditions. from the results obtained it is not possible to conclude that there was a positive influence of the bio-additive used on the number of leaves in onion plants. from the evaluation over more replicates however it is possible to confirm the positive effect of preparation bioa-11. in all the experiments this combination showed a statistically demonstrable influence on the leaf weight and length, which attained higher values compared to the controls, as is apparent from table 2. the recorded increase in the leaf weight was of 12-31% and in the leaf length was of 613% within all the evaluations. from the overall evaluation of the all tested bio-additives there is an evident tendency for the more frequent manifestation of a positive influence in the later (summer) sowings compared to the spring sowing. the recorded increase in all the evaluated parameters in the spring period of 2014 in comparison with the same period in 2015 can be assigned to the higher sunlight and higher average temperature recorded, which could have had the effect in accelerating the plant growth. according to the meteorological data from the station it follows that the total sunlight hours for march to may was 7% higher in 2014 than in 2015. similarly, the average temperature for the same period was 11% higher in 2014 than in 2015. the positive effect of the external application of strigolactones on leaf length might be related to the report of (pasare et al., 2013) who stated that plants with significantly reduced strigolactone content showed, among others, decreased in plant height. although the pea plants supplied with 3 µm of synthetic strigolactone (gr24) via hydroponics for 16 days did not significantly increase the internode length (de saint germain et al., 2013) the effect on onion leaf weight and length has been proven with 10 µm of fenyl 7. from the experiments carried out we obtained the following findings. essentially the use of one single isomer or a mixture (bioa-1 vs. bioa-3) does not have an influence on the evaluation of the morphological parameters. from the results it is not possible to conclude that there is an unequivocal difference between these variants. also in itself the use of the carrot macerate does not have a fundamental influence on some of the morphological parameters (bioa-1 vs. bioa-10). against this, clearly better results are achieved when the ph was modified with the aid of one of the acids (bioa-7 and bioa-11). while bioa-7 was positively evaluated in two out of the three experiments, bioa-11 was positively evaluated in all the experiments carried out. it can thus be concluded that acidification with citric acid and the presence of carrot macerate ensures the repeated effectiveness of fenyl 7 on the morphological paratable 2 average values of the monitored morphological parameters experiment number evaluated factor treatment control bioa-1 bioa-3 bioa-7 bioa-10 bioa-11 1 (spring 2014) leaf mass (g) 2.61 a 2.77 a 2.68 a 3.61 b 2.68 a 3.43 b leaf length (cm) 32.92 a 34.27 ab 33.20 ab 36.80 c 32.86 a 34.73 b number of leaves (pcs) 3.80 b 3.73 b 3.25 a 3.52 ab 3.55 ab 3.55 ab 2 (summer 2014) leaf mass (g) 1.08 ab 1.56 d 1.02 a 1.24 bc 1.29 c 1.35 cd leaf length (cm) 23.88 a 28.12 d 25.70 bc 25.20 ab 26.40 bc 27.06 cd number of leaves (pcs) 2.47 a 3.23 d 2.77 bc 3.00 cd 2.85 bc 2.60 ab 3 (spring 2015) leaf mass (g) 1.27 ab 1.19 a 1.39 bc 1.27 ab n/a 1.43 c leaf length (cm) 28.87 a 29.73 ab 30.33 bc 30.60 bc n/a 31.27 c number of leaves (pcs) 3.10 a 3.10 a 3.20 a 3.00 a n/a 3.10 a the letter after the value indicates the demonstrable difference according to the lsd test at p=0.05 (different letters in rows indicate significant differences between treatment variants). n/a= not evaluated. kopta et al. stability of the synthetic strigolactones fenyl 7. influence on morphological parameters 239 meters in onion plants. 4. conclusions from the experiments it was confirmed that changing the ph of the solution into acidic territory leads to higher stability of fenyl 7. in the case of a solution with a ph 9 the degradation of fenyl 7 was more than 90%. with a drop down in ph there was a reduction in the degradation of fenyl 7. the least degradation was recorded in the variant with a ph 4, where there was a reduction in concentration by 8.5%. it is also possible to summarise the positive effect of preparation bioa-11 (fenyl 7 and carrot macerate in a mixture of surfactant with added citric acid) on the growth parameters of onion seedlings. in repeated tests an increase in leaf weight (by 12-31%) and leaf length (by 5-8%) was verified in comparison with the control variant. accelerated production through an appropriate bio-additive, leading to a reduction in the pre-cultivation period could have, especially in the spring period, marked economic benefits for growers. it would be of benefit in future experiments to verify to what extent the effects on seedlings will be manifested in the ensuing vegetation. acknowledgements 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the species is significantly grown in greenhouse (1052 ha) (istat, 2012). in the temperate area of the northern hemisphere, asparagus harvest begins after winter rhizome dormancy, when soil temperature is favourable to hypogeous bud resumption (mccormick and geddes, 1996; heiβner et al., 2006). therefore, the latter is earlier at lower latitudes and gradually delayed towards the north: in fact, it starts in january in california desert valleys, in february in italian southern regions and in south carolina, in may in holland, end of june in michigan and in washington districts (dufault, 1994 a). the spring harvest duration in greenhouse in southern italy is usually extended to ninety days, as it occurs in other areas with similar climatic conditions (shou et al., 2007). indeed, in such cases the favourable season length allows the plants to completely recover rhizome reservoirs, insuring crop vigour and longevity (takatori et al., 1970). however, as in italy the production period is exclusively concentrated in spring, in the other seasons of the year the product demand is satisfied by imports from warmer areas (south africa, chile, mexico, southern california). as an alternative, asparagus production can be achieved by summer forcing, through total aerial biomass cutting and subsequent irrigation a few days before the scheduled harvest. the possibility of annual double harvest has also been proposed in south carolina (dufault, 1991, 1995 and 1996) and its convenience seems to be mainly dependent on the favourable season duration. particularly, the crop forcing achieved at the end of spring or summer could reveal, respectively, too early or late and therefore damage the rhizome reservoir and consequently plant longevity. instead, a second harvest effects of crop method and harvest seasons on yield and quality of green asparagus under tunnel in southern italy g. caruso*1, g. villari**, c. borrelli*, g. russo*** * dipartimento di scienze del suolo, della pianta, dell’ambiente e delle produzioni animali, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. ** stazione sperimentale per l’industria delle conserve alimentari, via nazionale, 121, 84012 angri (sa), italy. *** ph.d., università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: annual double harvest, amino acids, asparagus officinalis l., fiber, organic management, sugars, vitamin c. abstract: a three-year study (2007-2009) was carried out on green asparagus under tunnel in campania (southern italy) with the purpose of verifying both the possibility to practise organic management and annual double harvest in order to extend the eco-compatible production period and to avoid expensive imports. comparisons among eight experimental treatments were made. treatments were obtained by factorial combination of two crop methods (conventional and organic) and four spring and summer harvest periods of 90-day total duration (75 days in spring plus 15 in summer; 60 days in spring plus 30 in summer; 45 days in spring plus 45 in summer; as a control, 90 days in spring), arranging a split plot design with three replicates. the conventional management led to the highest yield, as a consequence of the higher spear number per plant, while the organic management resulted in both spear calibre and mean weight increase. organic spears showed a higher level of residues and sugars but a lower content of nitrate and fibre. the treatment with 75-day harvest in spring and 15 in summer proved the best double harvest combination, leading to the highest comprehensive yield (11.5 t·ha-1), not different from the control harvested only in spring for 90 days. summer spears showed higher values of optical residue, glucose, fructose, vitamin c and some mineral nutrients; instead, spring spears attained lower nitrate and average fibre content. asparagus annual double harvest revealed economically interesting results, but the profits are strictly related to the prices of summer spears, which were evidently higher in summer than in spring in the three years of research. adv. hort. sci., 2012 26(2): 51-58 (1) corresponding author: gcaruso@unina.it received for publication 20 march 2012. accepted for publication 4 may 2012. 52 period managed in july-august could allow the plants to reconstruct the aerial biomass both after the spring production and before the winter quiescence. in comparing the traditional spring three-month yield and the comprehensive production derived from the spring and summer harvest, it should be taken into account that the double harvest period can satisfy the consumer requirement in both seasons. particularly, in summer higher economic profits could be achieved due to both a lack of asparagus spear offer at italian markets and the potential product demand by tourists. also, harvest season influences the quality of asparagus spears, which are considered a good source of essential minerals, vitamins, amino acids and dietary fibers (lopez et al., 1996). in fact, spear nutritional features are highly affected by environmental factors, especially temperature and light (makus, 1994, 1995; papadopoulou et al., 2003). in addition, bhowmik and coworkers (2001) proved that carbohydrate and organic acid contents in asparagus spears were season-dependent. also fibrousness, though being a varietal peculiarity (poll and van kuistrum, 1990; simón, 1990; billau et al., 1990; gast et al., 1991; sanchez, 1996), is influenced by crop environment and seasonal climate. in particular, it was inversely correlated with rainfall (keulder and riedel, 1990) and temperature (sosa coronel et al., 1976; keulder and riedel, 1990; poll, 1996), which regulate the spear growth rate. fibrousness is the consequence of wall lignification of the pericycle cells and vascular bundles (baxter et al., 1987). according to haard et al. (1974), spear removal causes endogenous ethylene development at an amount stimulating the peroxidase activity involved in lignin synthesis. also spear diameter influences fibrousness (clore et al., 1976; billau et al., 1990; poll, 1996) because the fibrous bundle number does not change and thus the fibre has a lower relative incidence in higher calibre spears (herner, 1973). with the purpose of studying the different aspects mentioned above, research was carried out in salerno province (southern italy) on green asparagus under tunnel with the aim of evaluating the effects of crop method and annual double harvest on spear yield and quality. 2. materials and methods research was carried out in the period 2007-2009 in fisciano (salerno province) under tunnel (40°46’ n, 14° 48’ e, 150 m a.s.l.). the asparagus crop was planted in 2004 on clay-loam soil (table 1), using cultivar desto crowns spaced 1.20 m between rows and 0.40 m between the plants along rows. temperature time course of the three research years are shown in figure 1. eight experimental treatments were compared. treatments were obtained by factorial combination of two crop methods (conventional and organic) and four spring and summer harvest periods of 90-day total duration (75 days in spring plus 15 in summer, labelled as 75+15; 60 days in spring plus 30 in summer, labelled as 60+30; 45 days in spring plus 45 in summer, labelled as 45+45; as a control, 90 days in spring). a split plot design was used, with three replicates, assigning the crop methods to plots and the harvest periods to sub-plots; the latter had a 17.3 m2 surface area. polytunnels, covered by thermal polyethylene, were made of three structural units, each of them 20 m long, 8 m wide, 2 m high at wall and 3.5 m at roof. the organic system was managed according to ec regulation 834/2007, using only substances permitted under the law. for the crop, three fertilizations per year were achieved, before and after spring harvest and after summer harvest, each with 60 kg·ha-1 of n, 40 of p 2 o 5 and 70 of k 2 o, using mineral fertilizer for the conventional method and manure for the “organic”. moreover, weed control was undertaken using linuron chemical treatment (0.75 l·ha-1 of a. p.), at spear pre-emergence, in conventional plots and by hand in organic. drip irrigation was activated at 80% soil available water consumption. harvest was carried out twice a year, in spring and in summer, except for the control harvested only in spring. spear emission in summer was urged by cutting the crop aerial biomass and, at the same time, activating irrigation. in particular, these interventions were made at the beginning of july for the 45+45 treatment, at mid-july for 60+30 and at the end of july for 75+15. table 1 soil characteristics constituents coarse sand % 24.27 fine sand % 29.87 silt % 27.27 clay % 18.97 composition organic matter (walkley and black method) % 3.47 total nitrogen (n) kjeldahl method % 0.21 available phosphate (p 2 o 5 ) olsen method ppm 12.57 available potassium (k 2 o) ammonium acetate method ppm 146.97 reaction ph 6.57 fig. 1 trend of monthly temperatures in greenhouse. 53 harvest was practised by hand when the spears emerged 150 mm out of soil and it began on 28 february, 3 march and 2 march, respectively in 2007, 2008 and 2009. at each picking, the plot product was separated in marketable and waste fractions. the latter included the folded, damaged or less than 10 mm thick spears. after harvesting, marketable spears were cut at 30 mm from the base and their calibre was measured on 30-unit random samples. moreover, with the aim of estimating product prices during spring harvests, data published by infomercati (www.infomercati.com) were used. informercati is a consortium of the main italian fruit and vegetable wholesale markets, which records and processes the daily prices of each product. in july and august the prices were detected by local sellers to whom the research spears were given, as in summer no asparagus transactions were recorded. in order to evaluate spear qualitative characteristics, random 30-unit samples were collected in each plot (for three replicates) both in conventional and organic treatments. in the control, this procedure was achieved 45 days after beginning of harvest; in the 75+15, 60+30 and 45+45 treatments sample collection was made 37, 30 or 22 days, respectively after spring harvest and 7, 15 or 22 days after summer harvest. spears were immediately transferred to the experimental station for the food preserving industry, angri (salerno) branch, where the following determinations were made: dry residue: in an oven at 70°c under vacuum until steady weight; optical residue (expressed in °brix): on spear sap after squeezing, at a temperature of 20°c, by means of a digital refractometer, model r.f.m81, from bs (bellingham+stanley); reducing sugars (glucose and fructose) and sucrose: by hplc, using the 600e waters chromatographic system and a column sugar-pak waters at 85°c, edta-ca in water solution as eluent (50 mg·l-1); titratable acidity: expressed as grams of monohydrate citric acid per 100 g of product in agreement with the official analysis methods for vegetable preserves of the italian ministry of agriculture and forestry (mipaf, 1973); proteins: with the kjeldahl method, utilising a foss tecator digestor with a kjeltec 2300 distiller; lipids: measured in accordance with the official analysis methods for vegetable preserves of the italian ministry of agriculture and forestry (mipaf, 1973); vitamin c: by hplc using the model 600e waters chromatographic system, equipped with a 486 waters uv detector set to 410 nm λ and a column biorad mod. hpx87h at 35°c; fibre: the sigma chemical company enzymatic kit was used. the samples were weighed, dried (105°c), gelatinized in the presence of heat-resistant α–amylase and digested enzymatically by proteases and amydoglucosydase, to remove proteins and starch, whereas soluble fibre was precipitated by ethanol. the residue, filtered, washed with ethanol and acetone, dried and weighed, was split into two fractions used to determine, respectively, proteins and ash. fibre content was obtained by the difference between the weights of the residue and the proteins and ash; free amino acids: by means of hplc with a waters 600e chromatographic system, connected to a personal computer using millenium32 software, version 3·05·01, and equipped with a waters 717 autosampler and a fluorescence detector set at a λ of 205 nm-395 nm. the measurements were carried out utilising: a waters accqtag column (spherical c-18, 4 µm 150 x 3.9 mm, at a temperature of 35°c; in condition of gradient with eluent a, or a 140 mm sodium acetate trihydrate buffer, tea, edta-na2, sodium azide, eluent b, consisting of acetonitrile, and eluent c, which consists of water and with an injection volume equal to 5 μl; mineral anions (chlorides, nitrates, phosphates): by hplc with 600e waters system, a mod. 717 autosampler and a dionex column (mod. as11, 4 x 250 mm); mineral cations: (calcium, magnesium, potassium, sodium, iron, copper, zinc): by atomic adsorption spectrophotometry, after sulpho-nitric mineralization, with a model 1100 perkin-elmer spectrophotometer. data were processed by analysis of variance and duncan multiple range test was used for mean separation at 0.05 and 0.01 probability levels (n = 3). 3. results and discussion from the data reported in table 2 it can be stated that there are no significant differences among the research table 2 asparagus yield results as influenced by crop method and harvest seasons treatment marketable spears yield t·ha-1 no. per plant mean weight g calibre mm year 2006 10.3 14.3 35.8 15.6 2007 10.1 14.6 34.4 15.5 2008 10.2 15.1 33.9 15.3 ns ns ns ns crop method conventional 10.7 15.6 34.0 15.1 organic 9.7 13.7 35.3 15.8 * * * * harvest seasons spring 90 days 11.7 a 16.4 a 35.4 a 15.8 a spring 75 days + summer 15 days 11.5 a 16.5 a 35.1 a 15.6 a spring 60 days + summer 30 days 9.5 b 13.7 b 34.7 a 15.5 a spring 45 days + summer 45 days 8.1 c 12.1 c 33.4 b 14.9 b ns= not significant; * significant at p≤ 0.05. means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 54 years in terms of yield. this means that spring harvest did not condition the subsequent summer plant productive behaviour and summer harvest did not condition the crop performances in following year’s spring. moreover, no plant density reductions were recorded. conventional asparagus management caused the highest yield, as a consequence of the higher spear number per plant (table 2). this is consistent with poll and van kruistum (1987) referring to a correlation between spear number and yield. organic management, instead, resulted in spear calibre and mean weight increase. these differences between the two crop methods are the consequence of the yield differences recorded both in spring and summer (table 3), though the conventional method resulted in higher production than the organic one by 14.2% in spring but only by 6% in summer. contrary to our results, in previous research (warman, 1991) no significant difference was detected between the two crop methods. as for the comparison among the annual double production periods (table 2), the 75+15 treatment (75-day spring harvest plus 15 days in summer) caused the highest comprehensive yield (11.5 t·ha-1), owing to the highest spear number, calibre and mean weight. however, it was not different from the control harvested only in spring for 90 days. the lowest production was attained by the combination of 45-day spring harvest plus 45 days in summer (8.1 t·ha-1). notably, the comprehensive yield obtained by the double harvest crops was influenced to a greater extent by spring results than by summer ones, as in the first season more remarkable differences among the treatments were recorded (table 3). in fact, in spring the production increases caused by the 75+15 treatment were, respectively, by 54% and 170% in comparison with 60+30 and 45+45, though correspondingly the harvest period was 25% and 67% longer. in summer, however, an opposite yield trend was shown by the annual double harvest treatments, but unable to balance the spring results. in fact, in summer the 45+45 treatment had a longer harvest period than 60+30 and 75+15, respectively, by 100% and 200%, to which the same percentage yield increases corresponded. the productive results were, therefore, affected by the lower temperatures recorded in march which, in agreement with previous reports (heiβner et al., 2006), conditioned the shooting rate. the latter was, instead, favoured by the gradual temperature increase, which allowed in the 75+15 treatment to get more than 60% of the whole spring production in the last 30 days of harvest. in fact, quick spear emission is promoted by an air temperature of about 24°c (bouwcamp and mccully, 1975) or soil temperature of about 20°c and it is correlated with the total yield (poll and van kruistrum, 1987; keulder and riedel, 1996). therefore, summer temperature was always very favourable to spear immediate and continuous emission, in agreement with the reports of dufault (1994 b), thus differences among the harvest treatments were not so remarkable as those recorded in spring. but in summer, yield results were conditioned by spear calibre, with values showing an inverse relation with temperature, consistent with previous findings (wagenvoort et al., 1980; pill et al., 1993). notably, both in spring and summer spear calibre and mean weight decreased from the shortest to the longest harvest period, confirming the effects of harvest pressure on these variables (mcgrady and tilt, 1990). table 3 asparagus spring and summer yield results as influenced by crop method and harvest seasons marketable spears yield (t ha-1) no. per plant mean weight (g) calibre (mm) spring summer spring summer spring summer spring summer year 2006 8.0 d 3.0 10.6 d 4.9 38.2 31.0 16.5 14.1 2007 7.9 d 3.0 11.0 d 4.9 36.4 30.6 16.3 13.9 2008 8.0 d 2.9 11.5 d 4.8 35.7 30.4 16.2 13.8 ns ns ns ns ns ns ns ns crop method conventional 8.4 d 3.1 11.8 d 5.1 36.0 30.1 15.9 13.7 organic 7.6 d 2.9 10.2 d 4.6 37.6 31.2 16.8 14.1 * * * * * * * * harvest seasons spring 90 days 11.7 a 16.4 a 35.4 c 15.8 c spring 75 days + summer 15 days 10.0 b 1.5 c 14.1 b 2.4 c 35.7 c 31.4 a 15.7 c 14.6 a spring 60 days + summer 30 days 6.5 c 3.0 b 8.8 c 4.9 b 37.1 b 30.6 b 16.5 b 13.8 b spring 45 days + summer 45 days 3.7 d 4.4 a 4.8 d 7.4 a 38.9 a 30.0 c 17.2 a 13.4 c ns = not significant; * significant at p≤ 0.05. means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 55 our research showed that the goal of annual double harvest is pursuable in terms of yield in irrigation regime, if the traditional spring harvest is reduced from 90 to 75 days and integrated with 15 days of summer harvest. alternative double harvest combinations, represented by 60-day spring harvest plus 30-day summer harvest or by 45-day harvest both in spring and summer, caused significantly lower yields than the control. nevertheless, their economic profits are strictly related to the prices of summer spears. in this regard, the mean prices of green asparagus spears recorded in the three years of research were evidently higher in summer (5.5 €·kg-1 in july and 6.0 in august) than in spring (3.45 €·kg-1 in march, 2.68 in april and 2.36 in may). from the statistical processing of spear quality data no significant differences were recorded both between the examined years (2008 and 2009) and among the different harvest durations within the same season. therefore, only the results concerning the comparison between conventional and organic methods and between spring and summer harvest are reported (tables 4, 5, 6 and 7). with regard to quality indicators (table 4), organic management resulted in higher values of dry and optical residues, glucose, fructose and sucrose than conventional cultivation, whereas acidity, proteins and lipids were not affected by crop method. also the harvest season had significant influence on quality, as summer spears showed higher values of optical residue, glucose, fructose and vitamin c than the spring ones; instead, no significant change was detected for dry residue, sucrose, titratable acidity, lipids and proteins. the latter result was similar to that of shou et al. (2007) but, differently from our findings, these authors reported a decreasing trend for dry residue and reducing sugars and an increasing trend for acidity. among the sugars, fructose attained higher incidence than glucose and sucrose, as also it resulted in our research. with respect to ascorbic acid, both esteve et al. (1995) and shou et al. (2007) found a content decrease in summer spears compared with spring ones. fibre content was affected by crop method (table 5), as organic spears attained lower values than conventional ones. also significant differences were recorded between spring and summer spears, as the latter showed higher average fibrousness, consistent with previous reports (shou et al., 2007). presumably, in summer the spear fibre increase caused by calibre reduction prevailed the opposite effect of temperature; in fact, the latter is inversely correlated with fibre content during spear growth, as reported by sanchez (1996). with respect to the fibre content of each 5 cm fraction obtained by spear split, the organic method led to higher values than the conventional in all the comparisons (table 5). regarding harvest season, it was found that summer spears showed higher fibre content than the spring ones only from the basal to the middle fraction. as for fibre content along the spear, the highest value was detected in the white basal fraction and it was as much as 2.6 times higher than the one recorded in the green tip (respectively, 28.1 and 11.0%). haard et al. (1974) stated that fibre reduction towards the spear tip is the consequence of isoperoxidase changes, which start from the cut surface and gradually extend to the tip. table 4 asparagus spear quality as influenced by crop method and harvest seasons treatment dry residue (g·100 g-1 fw) optical residue (°brix) glucose (g·100 g-1 dw) fructose (g·100 g-1 dw) sucrose (g·100 g-1 dw) acidity (g·100 g-1 dw) protein (g·100 g-1 dw) lipids (g·100 g-1 dw) vitamin c (mg·100 g-1 dw) crop method conventional 8.38 6.72 4.54 7.54 1.55 4.93 31.4 6.20 659.9 organic 9.11 7.26 5.40 8.41 1.77 5.02 32.4 6.26 673.0 * * * * * ns ns ns ns harvest seasons spring 8.64 6.81 4.83 7.78 1.63 4.87 31.7 6.13 627.0 summer 8.85 7.18 5.11 8.17 1.69 5.08 32.1 6.33 705.9   ns * * * ns ns ns ns * ns= not significant; * significant at p≤ 0.05 (n=3). table 5 asparagus spear fibrousness as influenced by crop method and harvest seasons treatment spear fibre content fibre content per spear section from tip to base (cm) 0-5 g·100 g-1 dw 5-10 g·100 g-1 dw 10-15 g·100 g-1 dw 15-20 g·100 g-1 dw 20-25 g·100 g-1 dw crop method conventional 18.8 11.7 12.8 14.1 24.8 30.7 organic 15.9 10.3 11.1 11.7 20.8 25.5 * * * * * * harvest seasons spring 16.6 11.0 11.6 12.1 21.6 26.5 summer 18.1 10.9 12.3 13.7 24.0 29.7 * ns ns * * * ns= not significant; * significant at p≤ 0.05 (n=3). ( )( )( )( )( ) 56 concerning the amino acids, we have reported only the mean composition and the statistical differences among the individual compounds (table 6), as both crop method and harvest season did not cause any significant effect on this variable. with regard to composition, glutamine and asparagine showed the highest content in the asparagus spears, whereas cystine, methionine, tyrosine and histidine displayed the lowest. these results are consistent with those reported by other authors (shou et al., 2007; slupski et al., 2010). as for spear chemical composition (table 7), sodium and copper showed significant higher contents in organic crops, whereas the conventional method led to higher nitrate accumulation; the other ions examined were not affected by crop management. in previous research (warman, 1991) no significant differences were recorded between conventional and organic methods with regard to spear mineral nutrients. with respect to harvest season, summer spears showed higher sodium, iron and zinc than the spring ones. in the latter season, contrary to the findings of other authors (shou et al., 2007), a significantly higher nitrate content was detected, which was however lower than the one recorded in other research (shalaby et al., 2004). 4. conclusions a three-year investigation (2007-2009) was carried out on green asparagus under tunnel in salerno province (campania, italy) in order to investigate the effects of crop method and annual double harvest on spear yield and quality. organic crops did not give as high yields as the conventional, but produced better spear quality in terms of higher level of residues and sugars and lower content of nitrate and fibre. asparagus summer forcing, achieved through the plant aerial biomass cut and irrigation, did not limit the crop productivity but revealed interesting economically. compared with the traditional 90-day harvest period, the annual double production practised for 75 day in spring and 15 in summer did not significantly differ in terms of yield. however, production decreased with the other spring-summer combinations, made up of 60-day harvest in spring plus 30 in summer or of 45-day harvest in both seasons. nevertheless, the annual double harvest effectiveness also depends on asparagus market receptivity, which in summer is potentially interesting owing to tourist demand. this perspective is supported by the investigation conducted during our research: higher spear prices in summer than in spring (on average, respectively 5.75 €·kg-1 vs 2.83) due to the lack of italian product offer. from a qualitative point of view, summer spears showed higher table 7 asparagus spear chemical composition as influenced by crop method and harvest seasons treatment calcium mg·100 g-1 dw magnesium mg·100 g-1 dw potassium mg·100 g-1 dw sodium mg·100 g-1 dw iron mg·100 g-1 dw copper mg·100 g-1 dw zinc mg·100 g-1 dw chlorides mg·100 g-1 dw phosphates mg·100 g-1 dw nitrates mg·100 g-1 dw crop method conventional 25.0 22.2 249.6 3.5 1.19 0.12 0.95 57.1 62.4 45.9 organic 23.5 21.7 263.9 4.2 1.16 0.15 0.93 57.7 66.7 14.2 ns ns ns * ns * ns ns ns * harvest seasons spring 24.0 21.7 254.0 3.7 1.11 0.13 0.89 55.2 63.9 43.2 summer 24.5 22.2 259.5 4.0 1.24 0.14 0.99 59.6 65.3 16.9   ns ns ns * * ns * ns ns * ns= not significant; * significant at p≤ 0.05 (n=3). ( ( ( ( ( ( ( ( ( () ) ) ) ) ) ) ) ) ) table 6 asparagus spear amino acid composition (average of crop methods and harvest seasons) amino acids content (g·100 g-1 dw) alanine 1.18 bd arginine 1.24 bc asparagine 3.60 ac cystine 0.16 hc glutamine 3.97 ac glycine 0.76 cf histidine 0.40 fh isoleucine 1.03 ce leucine 1.11 bd lysine 1.23 bd methionine 0.29 gh phenylalanine 0.62 eg proline 1.56 bc serine 1.05 cd threonine 0.74 df tyrosine 0.37 fh valine 0.97 ce means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 57 values of optical residue, glucose, fructose, vitamin c and some mineral nutrients, compared with the spring product. nevertheless, the latter attained lower fibre content, though only from the middle to the basal fraction. acknowledgements the authors thank mr. roberto maiello for his assistance with laboratory analyses. references baxter l., waters l. jr., breenew w., 1987 a quick method for determining fibrousness of 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j.t.k., van kruistrum g., 1987 some aspects of asparagus forcing. asparagus research newsletter, 5(2): 27-34. poll j.t.k., van kuistrum g., 1990 the effect of temperature on yield and quality of forced asparagus. acta horticulturae, 267:151-152. sanchez m.t., 1996 influence of variety and preharvest factors on the texture of white and green asparagus. alimentaria, 34(276): 29-34. shalaby t., sator j., haneklaus s., schnug e., 2004 influence of variety and cultivation on mineral elements and protein content of asparagus (asparagus officinalis l.). acta horticultuare, 629: 313-320. shou s., lu g., huang x., 2007 seasonal variations in nutritional components of green asparagus using the mother fern cultivation. sci. hortic., 112: 251-257. simón a., 1990 fibrousness of white asparagus varieties. investigación agraria, producción y protection vegetales, 5(2): 239-246. 58 slupski j., korus a., lisiewska z., kmiecik w., 2010 content of amino acids and the quality of protein in aseaten green asparagus (asparagus officinalis l.) products. intern. j. food sci. & techn., 45(4): 733-739. sosa coronel j., vest g., herner r.c., 1976 distribution of fiber content in asparagus cultivars. hortscience, 11: 149-150. takatori f.h., stillmann j.i., souther f.d., 1970 asparagus yields and plant vigor as influenced by time and duration of cuttings. calif. agric., 24(4): 8-10. wagenvoort w.a., van os e., simonse l., 1980 forcing asparagus in water in forcing trays. groenten en fruit, 36(23): 64-67. warman p.r., 1991 effects of manures and fertilizers on asparagus yield, fern mineral content and soil fertility. sci. hortic., 47(3-4): 231-237. 260 1. introduction salinity is a widespread problem around the world, especially in arid and semi-arid regions. each year more and more land becomes non-productive due to salt accumulation. at least 25% of currently cultivated land throughout the world suffers from excess salinity (bohnert and jensen, 1996) and all major crop species are intolerant to salt (fairbairn et al., 2000). the most economic and sustained way to overcome the problem of salt-stress is to develop salt-tolerant varieties (frommer et al., 1999). in vitro culture has been widely used for the propagation and conservation of crop genetic resources in both agriculture and horticulture crops (barakat and el-lakany, 1992). mutation breeding programs using gamma irradiation on apricot buds were carried out by several investigators (legave and garcia, 1988; ageeva, 1989; gulcan and aksoy, 1995). legave and garcia (1988) reported that bud sticks of five apricot cultivars were exposed to up to 70 gy gamma rays and scored for bud survival and growth. the effect of gamma irradiation in the range 10-70 gy on variation in the characters of apricot was also investigated (ageeva, 1989). the varieties reacted in different ways to treatment. induced mutations in apricot breeding were also investigated (gulcan and aksoy, 1995). apricot (prunus armeniaca) was treated with 0.3 kr gamma irradiation (source 60co). mutagenesis affected vigour, dry matter and vitamin c content, and the level of carotene in fruit. in vitro cultures of japanese plum (prunus salicina) cv. shiro were also gamma-irradiated by predieri and gatti (2000). in vitro culture may offer potential for quick evaluation of germplasm against salt stress (cano et al., 1998). recently, jain (2001) reported that tissue culture generates a wide range of genetic variation in plant species which can be incorporated into plant breeding programs. the effect of nacl and cacl2 in prunus cerasifera was investigated by lucchesini and vitagliano (1993). the in vitro response of peach cv. redhaven and of the peach/almond hybrid rootstock gf677 to increasing concentrations of nacl in the medium was reported (biricolti and pucci, 1995). the response to increasing rates of nacl or cacl2 and proline on ‘mr.s 2/5’ (prunus cerasifera) peach rootstock cultured in vitro has also been reported (dimassi-theriou, 1998). towards in vitro selection studies for salinity tolerance in canino apricot cultivar. effect of gamma irradiation on in vitro mutation and selection for salt-tolerance a.s. el-sabagh*1, m.n. barakat**, e.a-e. genaidy*** * department of horticulture, faculty of agriculture, damanhour branch, university of alexandria, po box 22516, damanhour, egypt. ** department of crop science, faculty of agriculture, alexandria university, egypt. *** pomology department national research center, dokki, geza, egypt. key words: iba, bap, m3 and ms3 medium, mutation gamma ray, propagation, salt tolerance. abstract: in vitro mutation method was used to obtain salt-tolerant clone in apricot. small propagules of canino apricot cultivar were irradiated with gamma ray at doses of 0, 10, 25, 35, 50, 75 and 100 gy. after 30 days from treatment, both the radio sensitivity and post-irradiation recovery were assessed as the number of proliferated shoots per explants, fresh weight of cultures, shoot length and productivity of irradiated explants. a sudden and sharp decrease in the survival percentage occurred with the dose 75 gy, while the highest dose (100 gy) was lethal for all propagules. a marked decline in the number of regenerated shoots per explant and fresh weight of produced cultures was associated with an increase of irradiation doses. doses in the range of 10-75 gy, which preserved high survival percentage of irradiated explants, seemed to be more suitable for in vitro mutation in canino apricot cultivar. irradiated shoots were exposed to different concentrations of nacl which were added to the multiplication medium at the rates of 25, 50, 75, 100, 125 mm and after 30 days, vigorous shoots were selected from salinity treatments. in conclusion, apricot tissues exposed to different doses of gamma irradiation in the range of 10-75 gy, followed by culturing the plantlets produced in a medium containing additional salts (ranging from 25 to 125 mm) can be considered a good method to identify the most tolerant mutants to salts in apricot cultivars. adv. hort. sci., 2011 25(4): 260-263 1 corresponding author: ahmed_elsabagh67@yahoo.com received for publication 12 january 2011 accepted for publication 12 october 2011 261 recently, increased sodium chloride (nacl) salinity effects on bitter almond (amygdalus communis) (prunus dulcis) growth, cell osmolarity and nutrient acquisition were studied in vitro (shibli et al., 2003) and it was found that elevating salinity from 0.0 (control) to 50, 75, 100 mm nacl resulted in reductions in shoot growth (shoot height, shoot dry weight) and rooting (rooting percentage, root number, root length). the objective of the present work was to obtain salttolerant clone (s) in apricot using an in vitro mutation method. 2. materials and methods the present work was carried out in the biotechnology laboratory, crop science department, faculty of agriculture, alexandria university from 2001 to 2005. small propagules of canino apricot cultivar initiated from in vitro culture by the follwing protocol: two explants shoot tips and single node cutting; of 0.5-1 cm in length was used. shoot tips and single node were soaked in 100 mg/l ascorbic acid +150 mg/l citric acid for 30 min. explants were dried for 30 min. before they were immersed in fungicide ridomil (1 g/l solution) for 30 min. and then washed with distilled water, then soaked in clorox (7% sodium hypochlorite) for 7 min and washed with sterile distilled water three times. the explants were aseptically excised and placed in jar containing 50-60 ml of culture medium. each jar contained one explant, considered as one replication. cultures were incubated at 25±2oc under 16 hour’s illumination (2000 lux, day light fluorescent tubes). in order to check the phenol oxidation and to establish the explants with free from phenol, the explants were cultured on two medium (ms and m3,) supplemented with four pvp concentrations (0.0, 40.0, 80.0, 160.0 mg/l). the effect of five medium protocols was examined: modified woody plant medium (m3) (perez-tornero et al., 2000), ms medium (ms1) and ms medium (murashige and skoog, 1962) modified by reducing kno3 and nh4no3 by 25%, 50% and 75% which were designated ms2 , ms3 and ms4, respectively. all media were supplemented with 3.0% sucrose, 4 mg/l adenine sulfate, 160 mg/l pvp, 0.4 mg/l bap and 0.01 mg/l iba. the ph of the media was adjusted to 5.7 by using 1.0 n hcl or 1.0 n naoh and agar was added after adjusting the ph. the best two media (m3 and ms4) for proliferation were used to test the optimum effect of three types of cytokinins benzyladenine (bap), kiniten and isopentenyladenine (2ip) and their concentration on shoot tip proliferation, four different concentration of cytokinin 0.2, 0.4, 0.6 and 0.8 mg/l for each type using m3. also, four different concentration of cytokinin 0.5, 1.0, 2.0 and 4.0 mg/l for each type using ms4. the proliferation was evaluated six weeks after the beginning of the experiment and the number of shoots, (longer than 5 mm) per explant, their length and productivity (number of shoots x the average shoot length) were recorded. shoots of apricot derived from the shoot tips multiplication were cultured on m3 medium perez-tornero et al. (2000). the medium was supplement with either naa (0.0, 0.5, 1.0, 2.0 mg/l) or iba (0.0, 2.0, 4.0, 6.0 mg/l) were employed. in vitro mutation and selection for salt-tolerance effect of gamma irradiation on in vitro shoot culture. small propagules of canino cultivar initiated from in vitro culture (fig. 1) were irradiated in a gamma cell with a cobalt60 source at the middle-east regional radioisotopes centre for arab countries, el-dokki, giza with 10, 25, 35, 50, 75 and 100 gy doses. the irradiated propagules were removed from the jar and recultured on a fresh proliferation medium. after six weeks incubation, impact of the irradiation was assessed by determinating the number of shoots, the fresh weight of shoot multiplication, shoot length and productivity. 0.2 mg bap 0.4 mg bap 0.8 mg bap 0.6 mg bap fig. 1 in vitro micro propagation of apricot cv. canino on m3 medium derived from shoot tip explants. in vitro selection. small propagules of the cultivar (canino) (fig. 1) were irradiated in a gamma cell with cobalt 60 source gy at a dose of 10, 25, 35, 50 and 75 gy. the propagules were subcultured five times before in vitro selection for salt-tolerance. individual shoots from irradiated cultures were grown on the proliferation 0.6 mg/l bap m3 medium supplemented with different concentrations of nacl (25, 50, 75, 100 and 125 mm). after six weeks of incubation, the vigorous shoots were selected and transferred to fresh medium free from salt. analysis of variance with sas software (sas institute, 1988) was carried out. treatment means were compared using the lsd test at 5% level probability. data were analyzed as a factorial arrangement in randomized complete block design according to steel and torrie (1980). 3. results and discussion effect of gamma irradiation on in vitro apricot culture the basic requirement for effective use of mutation induction in plant breeding programs is the analysis of radio sensitivity of the explant material (walther and sauer, 262 1986). predieri (2001) reported that one of the first steps in mutagenic treatment is the estimation of the most appropriate dose to apply. the aim of the present work was to determine the radio-sensitivity of in vitro apricot culture, as assessed by the number of regenerated shoots, the fresh weight of shoot multiplication, the shoot length and productivity in order to select the suitable dose of gamma irradiation to conduct in vitro mutation for improvement. the collected data, reported in table 1, indicate that a clear decrease in in vitro traits occurred with increasing irradiation dose. complete lethality (100% death) was observed with an irradiation dose higher than 75 gy (fig. 2). several other studies have been conducted on the radio-sensitivity of in vitro cultures of fruits, such as prunus avium (walther and sauer, 1985), kiwifruits (shen et al., 1990), grapevine (lima da silva and doazan, 1995; charbaji and nabulsi, 1999) and prunus salicina (predieri and gatti, 2000). previously, laneri et al. (1990), working with gerbera jamesonii, stated that in a mutation breeding experiment, the dose chosen for the main experiment should result in the highest survival of irradiated explants and that a low inhibition of the rate of production of new shoots gives the highest efficiency in recovering useful mutants. in light of these studies, the results obtained in the present investigation suggest that doses of 10 gy to 75 gy seem to be the most suitable for inducing mutation for apricot improvement. in vitro mutation and selection for salt-tolerance in canino apricot cultivar mutation breeding can be employed as a promising technique that allows diversification of apricot. induced mutations change only one or a few specific traits of an elite cultivar without undesired additional variations (predieri, 2001). in fact predieri concluded that the most suitable method may be mutation treatment and propagation of in vitro axillary shoots without passage through undifferentiated growth, and it can contribute to fruit improvements without upsetting the requirements of the fruit industry nor the consumers. through in vitro selection, mutation with a useful agronomic trait, e.g. salt or drought tolerance or disease resistance, can be isolated in a short time (jain, 2001). the present work was conducted to obtain salt-tolerant clone(s) in apricot cv. canino using in vitro shoot mutation. small propagules were irradiated with 0, 10, 25, 35, 50, or 75 gy and explants were multiplied for five subcultures. the generated irradiated shoots were subjected to a salt (nacl) which was added to the medium with the concentrations 25, 50, 75, 100, or 125 mm. the number of vigorous shoots of cv. canino showed marked differences in their in vitro salinity tolerance (table 2). it is clear that the number of vigorous shoots decreased rapidly with increasing salinity. the highest number of vigorous shoots was obtained in medium supplemented with 25 mm selective agent of salinity when the propagules were exposed to 25 and 50 gy, respectively (table 2). from these results, it can be concluded that apricot cv. canino tissues exposed to different doses of gamma irradiation in the range 10-75 gy, followed by culturing in medium containing a higher concentration of additional salts (ranging from 25 to 100 mm) can be considered a good method to identify mutants in apricot cv. canino which are the most tolerant to salts. fao/iaea (1997) reported that plant biotechnology in combination with mutation induction and conventional breeding might open new frontiers for obtaining salt-tolerance rice varieties. the application of mutation techniques in breeding has increased constantly over the past years. these techniques must be rapid to keep pace with the large quantity of breeding materials generated after mutagenesis. screening under field conditions is difficult due table 1 effect of gamma irradiation six weeks after the treatment on in vitro apricot shoot gamma irradiation (gy) doses weight number of shoots shoot length productivity control 5.85 a 23.40 a 6.15 a 147.36 a 10 3.58 b 14.30 b 2.99 b 42.27 b 25 3.45 b 13.80 b 3.14 b 42.52 b 35 2.40 bc 9.60 bc 3.63 b 41.68 b 50 1.50 cd 6.00 cd 0.77 c 11.05 c 75 0.68 d 2.70 d 0.48 c 4.18 c means within a column or a row followed by the same letter(s) are not significantly different at the 0.05 level of probability. table 2 effect of irradiation doses on the number of vigorous shoots of apricot cv. canino, after six weeks from culturing in media containing different salt concentrations salt concentration mm irradiation dose (gy) 0 10 25 50 75 total 25 0 2 5 4 3 14 50 0 2 3 1 2 8 75 0 0 0 2 2 4 100 0 0 0 1 2 3 125 0 0 0 0 0 0 total 0 4 8 8 9 29 fig. 2 effect of gamma irradiation 100 gy, six weeks after the treatment on in vitro apricot shoot. 263 to stress heterogeneity, presence of salt-related stress and the significant influence of environmental factors such as temperature, relative humidity and solar radiation. genetic modification of crop plants to improve their salt-tolerance is a possible way of increasing production, especially for regions of the world where arable lands must be extended to marginal areas, and sometimes irrigated with saline water (dorion et al., 1999). references ageeva n.g., 1989 effect of gamma irradiation on variation in the characters of apricot. bulletin’ gosudarstvennogo nikitskogo botanicheskogo, 68: 67-70. barakat m.n., el-lakany m.h., 1992 clonal propagation of acacia saligna by shoot tip culture. euphytica, 59: 103-107. biricolti s., pucci s., 1995 effects of increasing nacl rates on ‘redhaven’ peach and ‘gf677’ rootstock cultured in vitro. adv. hort. sci., 9(2): 75-78. bohnert h.j., jensen r.g., 1996 metabolic engineering for increased salt tolerance. the next step. australian journal of plant physiology, 23: 661-667. cano e.a., perez-alfocea f., moreno v., caro m., bolarin m.c., 1998 evaluation of salt tolerance in cultivated and wild tomato species through in vitro shoot apex culture. plant cell and organ culture, 53: 19-26. charbaji t., nabulsi i., 1999 effect of low doses of gamma irradiation on in vitro growth of grapevine. plant cell, tissue and organ culture, 57: 129-132. dimassi-theriou k., 1998 response of increasing rates of nacl or cacl2 and proline on “mr. 2/5” (prunus cerasifera) peach rootstock cultured in vitro. adv. hort. sci., 12(4): 169-174. dorion n., wies n., burteaux a., bigot c., 1999 protoplast and leaf explants culture of lycopersicon cheesmanii and salt to lerance of protoplast-derived calli. plant cell, tissue and organ culture, 56: 9-16. fairbairn d.j., liu w., schachtman d.p., gomezgallego s., day s., teasdale r.d., 2000 characterization of two distinct hkt1like potassium transporters from eucalyptus camaldulensis. plant molecular biology, 43: 515-525. fao/iaea, 1997 develop low cost tissue culture techniques for mutation induction. plant breeding unit. frommer w.b., ludewig u., rentsch d., 1999 taking transgenic plants with a pinch of salt. science, 285: 1222-1223. gulcan r., aksoy u., 1995 utilization of induced mutations in apricot breeding has been investigated. acta horticulturae, 384: 231-236. jain s.m., 2001 tissue culture-derived variation in crop improvement. euphytica, 118: 153-166. laneri u., franconi r., altavista p., 1990 somatic mutagenesis of gerbera jamersonii hybrid: irradiation and in vitro cultures. acta horticulturae, 280: 395-402. legave j.m., garcia g., 1988 radio sensitivity of apricot bud sticks exposed to strong gamma rays and nursery observations on the second vegetative generation from irradiated buds. agronomy, 8(1): 55-59. lima da silva a., doazan j.p., 1995 gamma ray-mutagenesis on grapevine rootstocks cultivated in vitro. j. int. sci. de la vigne et du vin, 29: 1-9. lucchesini m., vitagliano c., 1993 effect of nacl and cacl2 in prunus cerasifera tissue culture. acta horticulturae, 336: 109-113. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco cultures. physiol. plant., 15: 473-497. perez-tornero o., lopez j.m., egea j., burgos l., 2000 effect of basal media and growth regulators on the in vitro propagation of apricot (prunus armenica l.) cv. canino. journal of horticultural science & biotechnology, 75(3): 283-286. predieri s., 2001 mutation induction and tissue culture in improving fruits. plant cell, tissue and organ culture, 64: 185-210. predieri s., gatti e., 2000 effects of gamma radiation on plum (prunus salicina lindl.) ‘shiro’. adv. hort. sci., 14: 215-223. predieri s., gatti e., 2000 effects of gamma radiation on micro cuttings of plum (prunus salicina lindl.) ‘shiro’. istituto di ecofisiologia delle piante arboree da frutto, cnr, 14(1): 7-11. shen x.s., wan j.z., luo w.y., ding x.l., 1990 preliminary results of using in vitro axillary and adventitious buds in mutation breeding of chinese gooseberry. euphytica, 49: 77-82. shibli r.a., shatnawi m.a., swaidat i.q., 2003 growth, osmotic adjustment, and nutrient acquisition of bitter almond under induced sodium chloride salinity in vitro. communications in soil science and plant analysis, 34(13/14): 1969-1979. steel r.g.d. torrie j.h., 1980 principles and procedures of statistics: a biomaterical approach. mcgrow hill book co. inc., new york, usa, pp. 196-201. walther f., sauer a., 1985 analysis of radio-sensitivity a basic requirement for in vitro somatic mutagenesis. i. prunus avium l. acta horticulture, 169: 97-104. walther f., sauer a., 1986 analysis of radio-sensitivity: a basic requirement for in vitro somatic mutagenesis. ii. gerbera jamesonii. proc. int. symp. nuclear techniques and in vitro culture for plant improvement. iaea/fao, vienna, pp. 155-159. impaginato 207 1. introduction almond [prunus dulcis (mill.) d.a. webb] presumably originated in mountain and desert areas of central asia and the middle east (martínez-gómez et al., 2007; gradziel, 2011; zeinalabedini et al., 2012). after its domestication, which possibly occurred during the third millenium bc, it spread towards western areas via seeds carried by caravans along the old silk route (fernandez i martì et al., 2015). in afghanistan, a geographical area criss-crossed by ancient trade routes and a cultural bridge between east and west, almond is called by its persian name ‘‘bādām’’, similarly to iran, india, kashmir, pakistan, tajikistan, tibet, and turkey, or “badam-e-shirin”. almond is considered a species belonging to afghan flora (alam, 2011) and its cultivation has spread widely from ancient times. the presence of bitter tasting seeds is considered a relic of the domestication process (rigoldi et al., 2015), hence the ancient presence of almond in afghanistan can be corroborated by the occurrence of many bitter tasting seed specimens scattered throughout the afghan territory. furthermore, recent studies on almond evolution showed the existence of an unambiguous gene flow of wild almonds from the centre of origin to iran (fernandez i martì et al., 2015), neighbouring to afghanistan. on the other hand, amygdalus species, among which a. zabulica, growing spontaneously in a restricted area of afghanistan, was not considered by ladizinsky (1999) as a possible almond progenitor because of its fleshy fruits, as well as a. kuramica, another species found only in afghanistan and north west pakistan; nevertheless kester et al. (1991) reported that this species may bring sweet and “paper shell” fruit types, the latter trait being quite common in afghan almond germplasm. although it has a cold, harsh continental climate, with hot dry summers and cold winters, afghanistan is an important almond producing and exporting country; about 53,000 t of almonds in shell have been produced yearly in the last five years, representing about 1.8% of total world production (faostat, 2016). almond kernels are an important source of nutritional and nutraceutical compounds for local populations, as well as a significant income for the adv. hort. sci., 2016 30(4): 207-216 doi: 10.13128/ahs-20346 phenotypic characterisation of almond accessions collected in afghanistan e. giordani 1 (*), m. berti 2, m. rauf yaqubi 3 1 dipartimento di scienze delle produzioni agroforestali e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 independent consultant 3 afghanistan national horticulture development organisation (anhdo), kabul, afghanistan. key words: genetic resources, germplasm, kernel, prunus dulcis. abstract: almond [prunus dulcis (mill.) d.a. webb] accessions of afghanistan have been surveyed, propagated, and grown in ex situ collections. trees, leaves, flowers, and fruits were characterised following standardized procedures taking into account 48 phenotypic traits. the national collection of varieties of almonds of afghanistan showed a significant variability in terms of morphological traits, with a predominance of early flowering time accessions. among the 56 accessions, sattarbai, a unique group of cultivar typology characterized by crescent dry fruit, soft and thin “paper shell” and high kernel/dry fruit weight ratio (>0.65) was clearly represented by cluster analysis. other accessions resulted closer to the international cultivars lauranne, carmel, ferraduel and ferragnes, considered as reference. (*) corresponding author: edgardo.giordani@unifi.it received for publication 23 july 2015 accepted for publication 3 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 207-216 208 country since a substantial percentage of almond nuts are exported to india and other countries of the region (kaska et al., 2006; aaido, 2015). the main production areas of export quality almonds are located in the regions of samangan, kunduz, takhar, and balkh (aaido, personal communication), and almonds are also traditionally grown in the “bagh” (home garden) and cultivated in orchards in almost all the valleys and flatlands of the country. the specific orography of the country, rich in narrow and isolated valleys, the multi-ethnic composition of the afghan population, together with a primitive traditional agriculture, based in ancient times mostly on sexual reproduction of trees which is a suitable method of propagation for a species with a short juvenile period like almond, were factors that fostered the occurrence of a rich and differentiated local almond germplasm. furthermore, almond is considered the most diverse species belonging to the genus prunus (socias i company and felipe, 1992) due to its self-incompatibility system (socias i company, 1990), the use of open pollinated seedlings in traditional fruitculture, and its adaptability to more diverse microclimates (fernandez i martì et al., 2015). within the framework of the perennial horticulture development project (phdp phase i), several almond producing areas of the country were surveyed during the period 2007-2008 in order to collect the most valuable varieties (giordani et al., 2014). this was achieved by following the indications of local fruit growers, scientists, and agronomists. the in situ collection was comprehensive of 82 local accessions which were propagated by budding. the obtained saplings were planted in ex situ replicated repositories in kunduz and mazar-i-sharif, representing the national collection of fruits and nut of afghanistan. usually the primary evaluation of germplasm accessions of fruit tree species, including almond, is based on the observation and standardized description of phenotypic traits of the tree and its main organs, with the adoption of specific descriptor lists (gülcan, 1985; talhouk et al., 2000; de giorgio et al., 2007; chalak et al., 2006; upov, 2011; rigoldi et al., 2015) and suitable uniand multivariate statistical analyses to study the morphological variability, the relationships among the studied accessions and the correlation between the described traits (lansari et al., 1994; khadivi-khub and etemadi-khah, 2015). such approach is essential for germplasm characterisation and protection against bio-piracy, and it is a suitable method for the phenotypic distinction of collected varieties, a basic step for the further development of the almond industry given the high occurrence of synonyms and homonyms among the accessions indicated for commercial propagation and production purposes. the present study aims to describe and analyse the morphological variability of a set of 56 accessions of almond described in the national collection of varieties of fruits and nuts of afghanistan almond (ministry of agriculture, irrigation and livestock of afghanistan, 2014), in relation to the results achieved by similar approaches on other almond gene pools. 2. materials and methods plant material flowers (n. 10), fully expanded leaves (n. 10), and fruits (n. 20) were randomly collected for two consecutive years from trees (n. 6) of the same accessions grown following traditional cultural practices in the orchards of the national collection of fruits and nuts located in kunduz (latitude 36.70893 n; longitude 68.86145 e; altitude 405 m a.s.l.) and mazar-elshariff (latitude 36.73232 n; longitude 67.02216 e; altitude 353 m a.s.l.) in afghanistan. trees, grafted on almond seedlings, were four to five years old, productive and free of harmful viral diseases; off-type trees observed in the six planting plots were not included in the trial. data collection morphological and pomological characterisation of genotypes were performed following the perennial horticulture development project procedures, based on dus-upov and ipgri guidelines for almond (gülcan, 1985; upov, 2011). data related to tree characteristics were collected in the field; measurements and descriptions of the other organs were performed in the laboratory. descriptive traits were determined based on rating and coding according to the adopted almond descriptor. data were entered in microsoft excel® sheets, designed for the purpose with multiple choice notes for qualitative traits or open fields depending on the descriptor typology. quantitative variables were measured and weighed adopting a manual calliper and a precision (0.01 g) electronic balance, respectively. colour parameters were visually determined using a specifically designed colour chart; other qualitative characteristics such as organ shape were attributed by using illustrated charts. giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 209 data processing and statistical analysis average and mode were calculated for all quantitative and qualitative parameters respectively; such values were used for the attribution of each accession to one class, regardless of the year and place of data collection. classes and relative notes were defined for each measured quantitative trait. after mean values standardization, multivariate principal component analysis and hierarchical cluster analysis (euclidean distance and ward’s agglomeration method) were performed on xls stat software. 3. results and discussion studied accessions the 56 studied accessions (table 1) were initially collected in stands (orchards or scattered plants in home gardens) located in different areas distributed throughout the country, within latitudes 31° 36’ 42.8400’’ n 36° 43’ 42.9276’’ and longitudes 65° 40’ 51.9600’’ e 71° 9’ 10.0008’’ e. altitudes ranged from 287 m to 1022 m a.s.l. most of the accessions (>70%) were collected in various districts of northern provinces of kunduz, samangan and balkh, considered the typical areas of almond production, although all accessions were susceptible in varying degrees to the risk of frost damage during the flowering time common in these locations. there were no late flowering varieties equivalent in flowering time to cultivars such as lauranne, ferragnes and ferraduel. morphologic and phenological trait variation minimum, maximum, mean and standard deviation values for each analysed trait for the whole set of studied accessions is reported in table 2. among 140 possible notes attributable to 46 different internationally agreed morphological descriptors related to tree, leaf, flower, shell and kernel, only 17 (≈12 %) of them showed no cases, indicating a wide variability for almost all traits. the sole descriptor showing no variation is related to the number of pistils (always 1 in 100% of cases); colour of tip of flower, sepals and petals showed low variability as well. among fruit traits, a special note, not present in the international standardised descriptor lists, was included to describe the “crescent” shape of green fruits and kernels, which showed a frequency of 1% and 16% of cases respectively for the whole collection. the set of described accessions was characterised mainly by vigorous, open trees with dense foliage; medium size, mostly dark green leaves with short petiole and crenate margin. flowers very frequently showed red brown sepals, broad elliptic white petals, stigmas above the anthers, and reddish stamen filaments. the predominant dry fruit traits were elongated and crescent shape, pointed apex, thick but medium-low resistant to cracking endocarps. kernels were characterised by elliptic-narrow elliptic and crescent shapes, red brown colour, medium-weak rugosity and small-medium size. kernel weight (1.1±0.4 g) was lower than the mean value (2.4 g) reported for a germplasm collection of iranian, european and north american accessions, and similar to those observed in different almond collections of the persian and middle-east areas (talhouk et al., 2000; chalak et al., 2006; zeinalabedini et al., 2012; khadivi-khub and etemadi-khah, 2015) and afghanistan (kaska et al., 2006). a distinctive trait of afghan almonds is related to shell cracking resistance, a characteristic with relevant implications in value-added products (ledbetter, 2008), which was found to be very low or low for 30% of accessions, indicating a consistent occurrence of “paper shell” and soft dry fruits. conversely, a similar percentage of accessions with hard and very hard shell (28%) was observed. kernel weight/dry fruit (nut + shell) weight ratio, sometimes indicated as shelling percentage or kernel yield, is another important commercial trait. afghan accessions in the present study showed higher mean value (0.51±0.12) for this characteristic than those found by other authors on almond germplasm of the same geographic area (between 0.31 and 0.35 for the persian area) (sorkheh et al., 2010; zeinalabedini et al., 2012; khadivi-khub and etemadi-khah, 2015), from lebanon (from 0.30 to 0.41)(talhouk et al., 2000; chalak et al., 2006), and similar to the mean value (0.50±0.11) found in a previous study carried out on 17 local varieties of northern afghanistan (kaska et al., 2006). lower mean values of kernel/dry fruit ratio were observed also on moroccan almond germplasm (0.38)(lansari et al., 1994), on european germplasm collections (godini, 1984; cordeiro et al., 2001; de giorgio and polignano, 2001), and on newly released cultivars (vargas et al., 2008). with regard to other fruit morphological traits linked to market value, the percentage of double kernel, a negative trait for international standardised trade but not very influent on domestic and subregional commerce, resulted absent or very low (0-2% adv. hort. sci., 2016 30(4): 207-216 210 national collection code cultivar name area of collection (province/locality) remarks afg0153 abdul wahidi balkh khulm good quality afg0144 belabai balkh khulm slight bitterness afg0167 carmel kunduz char darah (i) international cultivar, later flowering than any native afghan almond afg0527 changaki kandahar kandahar low market value afg1008 du maghza kulula kunduz char darah low market value afg1007 du maghza spin kunduz char darah afg6308 ferraduel kunar asad bad (i) international cultivar afg6206 ferragnes nangarhar jalalabad (i) international cultivar afg0793 kaf samangan aybak medium market value afg0773 kafmal kunduz char darah afg4048 kaghazi du posta herat guzara low market value afg4016 kaghazi gerd herat guzara medium market value afg0739 kaghazi herati herat enjil low market value (exceptionally large kernel) afg0532 kaghazi kalan kandahar kandahar valuable for kernel size (afg0739 has 15% bigger kernels and half the percentage of doubles afg6040 kaghazi maida nangarhar muhmand dara low market value; small kernel afg0535 kaghazi sia dana kandahar kandahar good quality afg0847 kajak samangan samangan aybak good market quality afg0778 kelk arus kunduz char darah afg0146 khairodini balkh khulm moderately hard shellafg6309 lauranne kunar asad bad (i) international cultivar afg0173 mahali kunduz kunduz char darah low market value; hard shell afg0147 majidi balkh balkh khulm small kernel afg2010 majidi samangan samangan aybak small kernel afg0166 marawaja doum kunduz char darah low market value afg0165 marawaja du maghdza kunduz char darah afg0775 marawaja maida dana kunduz char darah low market value afg0534 marawaja safid post kandahar kandahar hard shell afg0151 pista badam balkh khulm low market value afg0160 qaharbai samangan aybak afg1003 qaharbai allah mir kunduz char darah afg1006 qaharbai aykhanum kunduz char darah afg0780 qaharbai hazratan kunduz char darah afg0143 qambari balkh khulm afg0142 qambari 142 kunduz char darah afg0772 qambari kunduz kunduz char darah afg0158 sangak dahum samangan aybak low market value afg0740 sangak haftum herat enjil low market value afg0519 sangak hashtum kandahar kandahar low market value afg0530 sangak nohum kandahar kandahar low market value afg0531 sangak shashum kandahar kandahar low market value afg0380 sangi du maghza kalan kandahar kandahar low market in shell afg0774 satarbai yaqubi kunduz char darah afg0159 sattarbai bakhmali samangan aybak high market value afg1002 sattarbai doum kunduz char darah high market value afg0157 sattarbai guldar samangan aybak high market value afg0168 sattarbai mumtaz kunduz char darah highest market value, intrinsically low yielding afg0154 sattarbai no.4 balkh khulm high market valueafg2011 sattarbai sais aybak samangan aybak high market value afg0164 sattarbai sais kunduz kunduz char darah high market value afg0156 sattarbai sais talkhak samangan aybak afg0777 sattarbai sais zuhrabi samangan aybak high market value afg0145 sattarbai sufi balkh khulm afg1005 shakh-i-buz kunduz char darah afg0155 shakh-i-buz safid balkh khulm afg0162 shokorbai samangan aybak acceptable market value afg0148 zang kafter balkh khulm table 1 list of the described almond [prunus dulcis (mill.) d.a. webb] accessions from afghanistan, area of in situ collection, altitude and remarks on their commercial value giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 211 of fruit with double kernel) and low (3-5% of fruits with double kernel) in 29% and 20.5% of cases, respectively, with a general average of 4% of fruits with double kernels. this value is similar to the average value (4.5%) found by sorkheh and colleagues (2010) in a germplasm collection composed of iranian and european and north american accessions, but much lower than the findings of kaska (kaska et al., 2006) on a set of almond fruit collected mainly in northern provinces of afghanistan (≈ 46 %) and those observed on persian (39%)(khadivi-khub and etemadi-khah, 2015) and lebanese (≈ 43%) germplasm (chalak et al., 2006). time of flowering showed a high variation, with table 2 list of phenotypic traits, codes, states, classes and descriptive statistics adopted to describe the set of 56 accessions from the national collection of almond from afghanistan. in brackets the percentage of cases observed for each note/descriptor no. trait classes (% of cases) min. max. mean sd 1 juvenile tree vigour 1 weak (0); 2 medium (45); 3 strong (55) 2 3 2.55 0.81 2 tree habit 1 up-right (3); 2 slightly open (19); 3 open (52); 4 spreading (24); 5 drooping (2) 1 5 3.00 0.63 3 foliage density 1 loose (4); 2 medium (48); 3 dense (48) 1 3 2.41 0.29 4 leaf blade length 1 short (12); 2 medium (88); 3 long (0) 1 2 1.91 0.42 5 leaf blade width 1 narrow (9); 2 medium (91); 3 broad (0) 1 2 1.84 0.60 6 leaf blade – ratio length/width ratio 1 small (17); 2 medium (81); 3 large (2) 1 2 2.54 0.45 7 leaf blade colour 1 light green (5); 2 medium green (36); 3 dark green (59) 1 3 1.73 0.47 8 leaf blade incisions of margin 1 serrate (26); 2 crenate (74) 1 2 1.32 0.50 9 leaf petiole length 1 short (67); 2 medium (33); 3 long (0) 1 2 1.41 0.36 10 flower buds: shape 1 conical (59); 2 ovoid (41); 3 rounded (0) 1 2 2.02 0.69 11 flower bud colour of tip of petals 1 white (3); 2 pink white (93); 3 pale pink (2); 4 pink (2); 5 carmine (0); 6 white, with carmine tip (0) 1 4 2.82 0.86 12 flower bud colour of sepals 1 green (0); 2 brown green (34); 3 red brown (50); 4 dark red (16) 2 4 2.34 0.64 13 flower bud hairiness of sepals 1 absent or very weak (24); 2 weak (22); 3 medium (52); 4 strong (2); 5 very strong (0) 1 4 1.80 0.82 14 flower size 1 small (31); 2 medium (57); 3 large (12) 1 3 2.27 0.40 15 flower shape of petals 1 narrow elliptic (24); 2 elliptic (26); 3 broad elliptic (50) 1 3 1.20 0.65 16 flower colour of petals 1 white (83); 2 pink white (17); 3 pink (0); 4 dark pink (0) 1 3 1.18 0.39 17 flower number of stamens 1 few (31); 2 medium (55); 3 many (14) 1 3 1.84 0.53 18 flower number of pistils 1 always 1 (100); 2 sometimes 2 (0); 3 frequently two (0); 4 fewmedium (0); 5 medium many (0) 1 1 1.00 0.00 19 flower position of stigmas compared to anthers 1 below (2); 2 same level (36); 3 above (62) 1 3 2.61 0.41 20 stamen anthocyanin colouration of filaments 1 absent (22); 2 present (78) 1 2 1.79 0.81 21 stigma size 1 small (26); 2 medium (65); 3 large (9) 1 3 1.73 0.63 22 green fruit length 1 small (9); 2 medium (72); 3 large (19) 1 3 2.11 0.53 23 green fruit shape 1 rounded (2); 2 ovate (9); 3 elliptic (2); 4 pointed (74); 5 crescent (1) 1 5 3.84 0.78 33 green fruit pubescence 1 slight (29); 2 medium (59); 3 much (12) 1 3 1.82 0.64 34 dry fruit shape 1 type 1 (5); 2 type 2 (0); 3 type 3 (48); 4 type 4 (28); 5 type 5 (19); 1 5 3.59 0.99 35 dry fruit shape of apex 1 flat (7); 2 rounded (9); 3 pointed (84) 1 3 2.77 0.57 36 dry fruit thickness of endocarp 1 thin (28); 2 medium (34); 3 thick (38) 1 3 2.09 0.82 37 dry fruit resistance to cracking 1 very low (9); 2 low (21); 3 medium (42); 4 high (19); 5 very high (9) 1 5 3.00 1.08 38 dry fruit keel development 1 absent or very weak (9); 2 weak (28); 3 medium (37); 4 strong (24); 5 very strong (2) 1 5 2.79 0.97 39 kernel shape 1 crescent (16); 2 narrow elliptic (31); 3 elliptic (34); 4 broad elliptic (17); 5 very broad elliptic (2) 1 5 2.57 1.02 40 kernel size 1 small (64); 2 medium (36); 3 large (0) 1 3 1.36 0.48 41 kernel thickness 1 thin (24); 2 medium (71); 3 thick (5) 1 3 1.82 0.51 42 kernel main colour 1 yellow (2); 2 yellow brown (26); 3 light brown (16); 4 red brown (49); 5 dark chestnut brown (7) 1 5 3.32 1.01 43 kernel intensity of colour 1 light (9); 2 .medium (74); 3 dark (17) 1 3 2.07 0.50 44 kernel rugosity 1 very weak (3); 2 weak (34); 3 medium (58); 4 strong (5) 1 4 2.63 0.65 45 kernel percentage of double kernel 1 absent/very low (23); 2 low (17); 3 medium (30); 4 high (22); 5 very high (8) 1 4 2.66 1.21 46 kernel/dry fruit weight percentage 1 very low (0); 2 low (8); 3 medium-low (15); 4 medium (23); 5 medium-high (33); 6 high (21); 7 very high (0) 1 6 4.48 1.19 47 time of flowering 1 very early (48); 2 early (31); 3 medium (14); 4 late (5); 5 very late (2) 1 5 1.88 1.03 48 ripening time (harvesting) 1 very early (0); 2 early (62); 3 medium (33); 4 late (5); 5 very late (0) 1 4 2.38 0.49 adv. hort. sci., 2016 30(4): 207-216 212 cultivars flowering from very early (before february 26; 49% of cases, among which many “sattarbai” and “sangak” type accessions) to very late (after march 12; ≈ 2% of cases, represented by the international cultivar lauranne), which clearly indicates a prevalence of early flowering accessions in the almond germplasm from afghanistan. conversely, the national collection of almond lacks very early (before july 14) and very late (after 29 august) ripening accessions. the range of phenological and measured morphologic traits are shown in table 3. relationship between the described accessions individual descriptive sheets were published in 2014 as the national collection of varieties of fruits and nuts of afghanistan almond and are available online on the internet (http://afghanistanhorticulture.org/pages/germplasm.aspx), while the results of multivariate pca and cluster analysis are reported in table 4 and figure 1. principal component analysis traits scales leaf blade length (mm): short (< 58); medium (58-83); long (>83) leaf blade width (mm): narrow (<20); medium (20-29); broad (>29) leaf blade ratio length/width ratio: small (<2.5); medium (2.5-3.5); large (>3.5) leaf petiole length (mm): short (<11); medium short (11-19); medium (>19-28); medium long (>28-36); long (>36) flower size (mm): small (<3); medium (3-4); large (>4) green fruit length (mm): small (<32); medium (32-50); large (>50) kernel size (g): small (<0.7); medium (0.7-1.9); large (>1.9) kernel thickness (mm): thin (<5.5); medium (5.5-10.5); thick (>10.5) double kernel (%) very low (<15); low (15-27); medium-low (28-39; medium (40-51); medium-high (52-63); high (64-75); very high (>75) time of flowering very early (before february 26); early (february 27 march 2); medium (march 3-6); late (march 7-11); very late (after march 11) time of maturity very early (before july 14); early (july 14-29); medium (july 30 august 13); late (august 14-28); very late (after august 28) table 3 scales defined for quantitative and phenological traits observed on the almond accessions from afghanistan principal component f1 f2 f3 f4 f5 f6 f7 eigenvalue 5.3 3.0 2.6 2.4 2.2 2.0 1.7 variability (%) 14.0 7.9 6.8 6.5 5.9 5.3 4.6 cumulative variability % 14.0 21.9 28.8 35.3 41.3 46.6 51.3 variable correlation juvenile tree vigour 0.19 0.13 -0.27 0.05 0.12 0.12 0.25 tree habit -0.28 -0.01 -0.03 0.01 -0.50 -0.11 -0.43 foliage density -0.01 -0.34 0.07 0.04 0.55 0.16 -0.07 leaf blade length -0.01 0.44 0.47 0.19 0.28 0.25 0.09 leaf blade width 0.07 0.30 -0.57 0.03 0.16 -0.31 -0.21 leaf blade ratio length/width 0.07 0.36 0.62 0.19 -0.03 0.19 0.25 leaf blade colour 0.01 0.07 0.14 0.50 -0.23 -0.13 -0.03 leaf blade incisions margin -0.03 0.27 0.41 0.34 -0.15 0.41 -0.13 leaf petiole length 0.03 0.42 0.26 -0.36 0.10 -0.33 -0.17 flower bud shape 0.27 0.21 -0.22 -0.17 0.31 -0.20 0.26 flower bud colour of tip of petals 0.21 0.17 -0.13 0.46 -0.26 -0.25 0.16 flower bud colour of sepals 0.23 0.10 0.36 0.06 0.08 -0.26 -0.13 flower bud hairiness of sepals -0.04 0.51 0.05 -0.37 -0.08 0.06 -0.16 time of beginning of flowering -0.08 0.39 -0.33 0.21 -0.07 0.38 -0.07 flower size 0.19 0.11 0.49 -0.38 0.13 -0.19 -0.10 flower shape of petals 0.24 0.05 0.28 -0.24 0.22 0.11 0.13 flower colour of petals 0.11 -0.23 0.14 0.16 0.34 -0.10 -0.29 flower number of stamens 0.08 0.55 -0.13 -0.30 0.03 -0.11 -0.09 flower position of stigmas compared to anthers -0.31 0.32 -0.23 0.19 0.39 0.06 -0.43 stamen anthocyanin colouration of filaments -0.09 0.09 0.12 -0.38 -0.07 0.48 -0.18 stigma size 0.23 0.36 0.11 -0.14 0.33 -0.18 0.29 green fruit size -0.66 -0.15 -0.10 0.19 0.25 -0.08 0.15 green fruit shape -0.62 -0.01 0.26 0.34 0.07 -0.39 0.08 green fruit pubescence -0.04 0.12 -0.28 0.09 0.23 0.47 0.34 time of maturity 0.26 0.26 0.01 0.20 -0.38 0.09 -0.10 dry fruit shape -0.76 -0.06 0.26 0.09 0.10 -0.31 0.04 dry fruit shape of apex -0.50 0.18 0.01 0.10 -0.17 0.09 0.48 dry fruit thickness of endocarp 0.71 -0.16 0.14 0.23 0.03 -0.03 0.04 dry fruit resistance to cracking 0.81 -0.38 0.08 0.09 0.09 -0.10 0.04 dry fruit keel development -0.27 0.45 -0.06 -0.05 -0.07 -0.14 0.21 fruit percentage of double kernels -0.07 -0.30 -0.01 -0.36 0.19 0.01 -0.12 kernel shape 0.74 0.07 -0.11 0.09 -0.07 0.25 -0.25 kernel size -0.09 0.01 -0.44 0.18 0.28 -0.01 0.06 kernel thickness 0.63 0.24 -0.20 -0.26 -0.06 -0.10 0.26 kernel main colour 0.35 0.31 -0.01 0.32 0.20 -0.27 0.01 kernel intensity of colour 0.28 0.41 -0.03 0.46 0.31 -0.05 -0.35 kernel rugosity -0.16 -0.13 0.11 0.16 0.48 0.32 -0.11 kernel dry/fruit ratio -0.73 0.31 -0.09 -0.21 0.08 0.07 -0.14 table 4 eigenvalues, expressed variability and correlations of the first seven principal components (accounting for 51.3% of variability) obtained from the whole accession/variable data set. the highest absolute correlation of each variable is in bold giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 213 based on the averages of the discrete scores of the 48 morphological and phenological variables for each accession indicated a low correlation between the utilized variables, with 15 pcas with eigenvalue higher than 1, accounting for 75.8% of variability within the whole set. such result can be attributed to the adoption of a multi-state discrete scoring methodology but also to a generalized low correlation between variables. the average euclidean distance of the generated proximity matrix between the studied accessions resulted equal to 8.46±1.21, ranging from 4.94 (minimum value) observed in the pair “kaghazi kalan kaghazi herati” (collected from kandahar dehe kochi village and herat province, enjil district, kahdestan village, respectively) up to 12.59 (maximum value) for the couple “sattarbai n.4 marawaja safid post”. the dendrogram generated by cluster analysis is reported in figure 1, where three main significant clusters (c1, c2 and c3) can be observed. the first cluster c1 (7.35±0.92 average distance; 5.43 minimum distance and 9.53 maximum distance for pairs “sattarbai sufi sattarbai mumtaz” and “sattarbai n.4 sattarbai doum”, respectively) is almost completely composed by “sattarbai” denominated accessions (10 of 14 accessions), while only one “sattarbai” type (“sattarbai sais talkhak”) belongs to a different cluster (c1). table 5 reports the representative characteristics of the “sattarbai” almond type, considered the best in terms of fruit quality and market value, as resulted from the analysis of the 48 morphological and phenological variables of the 10 “sattarbai” accessions included in cluster c1. even if a certain degree of variation within single traits of leaves (colour), flowers (petal shape), dry fruits (shape, thickness of endocarp, keel development and percentage of double kernel) and kernels (shape and main colour) was observed, as a whole, these accessions are very closely related and they constitute a clearly defined special subset within the afghan almond germplasm (fig. 1). the accession sattarbai sais talkhak, the only “sattarbai” named cultivar outside cluster c1, showed six characteristics (namely leaf colour, flower size, shell resistance to cracking, dry fruit shape and kernel main colour) with significant differences and 16 traits slightly differing from the “sattarbai” standard type; this fact substantiates its location in a different cluster. on the other hand, qambari, qambari kunduz, qambari 142 are varieties often confused with “sattarbai” type also by expert traders, which confirms the high phenotypic similitude between “qambari” and “sattarbai” groups and justifies their presence in cluster c1. conversely, belabai, albeit showing many traits of the “qambari/qaharbai” type, differentiates for the distinctive and sometimes bitter flavour of the kernel which detracts from its otherwise medium value. cluster c2 is formed by the four international cultivars (lauranne, carmel, ferragnes and ferraduel) belonging to the national collection and by 21 local accessions, among which all “sangak” (sangak meaning “stone” in local languages) accessions, a part of “kaghazi” types and other unique varieties. broad elliptic sepals, type 3 dry fruit shape, thick endocarp, fig. 1 dendrogram (euclidean distance; ward's agglomerating method) obtained by analyzing 48 multi-state, discrete morphological variables of 56 almond accessions belonging to the national collection of fruits and nuts of afghanistan (bold cursive: international cultivars; bold: accessions belonging to c1 cluster "sattarbai group"). adv. hort. sci., 2016 30(4): 207-216 214 medium resistance to cracking, red brown kernel, medium percentage of double kernels and medium kernel/dry fruit weight ratio (average 0.45, about 30% less that c1 mean value) were the traits differentiating this group of varieties from those of cluster c1. the last cluster (c3) held 17 local accessions mostly belonging to “qaharbai”, “marawaja” and “majidi” types and other unique varieties; such accessions resulted characterised by medium size flowers, dry fruits with weak keel development, high percentage of double kernels, and medium high kernel/dry fruit weight ratio (average 0.53). table 5 morphological and phenological characteristics of "sattarbai" almond type no. trait note (mode) range 1 juvenile tree vigour medium medium-strong 2 tree habit open open-spreading 3 foliage density dense medium-dense 4 leaf blade length medium medium-long 5 leaf blade width medium medium 6 leaf blade ratio length/width medium small medium 7 leaf blade colour medium green medium green dark green 8 leaf blade incisions margin crenate crenate serrate 9 leaf petiole length short short-medium 10 flower bud shape conical conical ovoid 11 flower bud colour of tip of petals pink white pink white 12 flower bud colour of sepals red brown red brown brown green 13 flower bud hairiness of sepals medium medium weak 14 flower size small small medium 15 flower shape of petals narrow elliptic narrow elliptic elliptic 16 flower colour of petals white white 17 flower number of stamens few few medium 18 number of pistils always 1 always 1 19 flower position of stigmas compared to anthers above above same level 20 stamen anthocyanin colouration of filaments present present ansent 21 stigma size small small medium 22 green fruit size large large medium 23 green fruit shape pointed pointed crescent 33 green fruit pubescence medium medium slight 34 dry fruit shape type 5 crescent type 5 type 4 35 dry fruit shape of apex pointed pointed 36 dry fruit thickness of endocarp thin thin medium 37 dry fruit resistance to cracking very low very low low 38 dry fruit keel development strong medium strong 39 kernel shape narrow elliptic narrow elliptic crescent 40 kernel size small small medium 41 kernel thickness thin thin medium 42 kernel main colour yellow brown yellow brown light brown 43 kernel intensity of colour medium medium light 44 kernel rugosity medium medium weak 45 fruit percentage of double kernels medium high absent very lowhigh 46 kernel dry/fruit ratio high (0.64) high (0.58-0.72) 47 time of beginning of flowering very early very early early 48 ripening time (harvesting) early early medium giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 215 4. conclusions this first comprehensive study of morphological and phenological traits of almond accessions from afghanistan clearly reveals an important level of variation among traits of the local germplasm. analogous findings were reported for iranian and lebanese germplasm (talhouk et al., 2000; chalak et al., 2006; zeinalabedini et al., 2012; khadivi-khub and etemadikhan, 2015). furthermore, a special subset of local varieties, to the best of our knowledge not yet reported by scientific international literature and unique in its kind because of dry fruit and kernel crescent shape and very soft “paper shell” endocarp, denominated “sattarbai” type, has been clearly highlighted. the best sattarbai types are extremely crescent shaped, with very narrow, elongated kernels and a very clear fissure in the “paper shell” along its length. the qambari types have less exaggerated length and crescent shape nuts, but clearly have “paper shell”. qaharbai types are less elongated and tend to have more thickening of the semi “paper shells”. shakhe buz (=”goat’s horn”) has an exaggerated “s” shape. other kaghazi or “paper shell” types may be downgraded because of untypical shape, but the accession kaghazi herati afg0739 has kernels some 40-50% heavier than the typical sattarbai or qambari kernel. the sattarbai, qambari, qaharbai and some other types of almond were found exclusively in the north of afghanistan, indicating a probable connection to the central asian germplasm, particularly to the famous almond growing areas of the fergana valley. nevertheless, a very early flowering time represents a limit to the expansion of these cultivars, and breeding programs are already being developed to overcome this obstacle. specific morphological studies associated to dna fingerprinting would be the next step in order to better clarify the identity of individual local accessions, to reveal the relationship of the afghan almond germplasm with prunus dulcis genetic resources of other areas, and to study the role of afghanistan, an intersectionbetween east and west, in the flow of almond around the world. acknowledgements activity founded by the european commission europeaid program. a special appreciation to the staff of the ministry of agriculture, irrigation and livestock of afghanistan for the availability and support. references aaido afghan almond industry development organization, 2015. http://www.aaido.af. personal communication. alam m., 2011 trees and shrubs of afghanistan. a dendrological guide. rossolis, musée botanique cantonal lausanne, switzerland, pp. 512. chalak l., chehade a., kadri a., 2006 morphological characterization of cultivated almonds in lebanon. fruits, 62(3): 177-186. cordeiro v., monteiro a., oliveira m., ventura j., 2001 study of some physical characters and nutritive composition of the portuguese’s (local) almond varieties. xi grempa 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(ed.) fruits and nuts. genome mapping and molecular breeding in plants. springer berlin heidelberg, germany, pp. 370. ministry of agriculture, irrigation and livestock of afghanistan, 2014 national collection of varieties of fruits and nuts of afghanistan. almond. ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 194. rigoldi m.p., rapposelli e., de giorgio d., resta p., porceddu a., 2015 genetic diversity in two italian almond collections . electronic journal of biotechnology, 18: 40-45. socias i company r., 1990 breeding self-compatible almonds. plant breeding reviews, 8: 313-338. socias i company r., felipe a.j., 1992 almond: a diverse germplasm. hortscience, 27(7): 817 and 863. sorkheh k., shiran b., khodambashi m., moradi h., gradziel t.m., martínez p., 2010 correlations between quantitative tree and fruit almond traits and their implications for breeding. scientia horticulturae, 125(3): 323-331. talhouk s.n., lubani r.t., baalbaki r., zurayk r., alkhatib a., parmaksizian l., jaradat a.a., 2000 phenotypic diversity and morphological characterization of amygdalus l. species in lebanon. genetic resources and crop evolution, 47(1): 93-104. upov, 2011 almond. guidelines for the conduct of tests for distinctness, uniformity and stability. upov, geneva, switzerland. vargas f., romero m., clavè j., vergès j.s., battle i., 2008 ‘vayro’, ‘marinada’, ‘constantı´’, and ‘tarraco’ almonds. hortscience, 43(2): 535-537. zeinalabedini m., sohrabi s., nikoumanesh k., imani a., mardi m., 2012 phenotypic and molecular variability and genetic structure of iranian almond cultivars. plant. syst. evol., 298: 1917-1929. 138 1. introduction in tunisia water resources (36 km3 annually) are mostly used to irrigate about 400 000 ha of annual and perennial crops. these amounts fulfill on average 75% of the crop water requirements (hamza, 2009). in the north and center of the country, priority is always given to fruit trees and vegetables, although olive is considered the most important species and it is cultivated over a large area (1 700 000 ha). over the two last decades, the amount of available water has decreased consistently, thus the imbalance between water supply and water demand has intensified. this situation has given rise to much attention from the relevant authorities and the general public in terms of the average and long term water uses. obviously, water should be used judiciously with reasonable amounts to meet water needs and without any wastage. determination of crop water needs, i.e. crop evapotranspiration (etc), is therefore necessary to efficiently manage irrigation at the field level. however, it involves a highly complex set of processes which are influenced by watering conditions and tree and land cover characteristics. we have published in recent years technical papers (masmoudi-charfi, 2006; masmoudi-charfi et al., 2006) presenting the water requirements of olive trees for different cultivation areas, according to age, soil coverage and growth stages based on the climatic method of the fao (allen et al., 1998; habaieb and masmoudi-charfi, 2003; masmoudi-charfi et al., 2004). however, during the calculation procedure, we were confronted with a lack of information about the crop coefficient. in addition, long term climatic data were not available for all sites. the lysimetric measurements give more precise information on water use, but is hard to carry out and expensive (deidda et al., 1990). estimates of actual evapotranspiration for adult olive trees were published in tunisia and elsewhere for different environments (ozyilmaz and ozkara, 1989; cohen, 1991; pastor et al., 1998; michelakis, 2000; musters and bouten, 2000; palomo et al., 2002; bandino and dettori, 2003). these estimates require regular measurements of soil water content, which are essential to calibrate models estimating the vertical distribution of root water uptakes (hazrat et al., 2000; palese et al., 2000). gravimetry is amongst the devices used to reliably measure soil water content (hv) in the field. however, it is more useful for calibrating other devices than for scheduling irrigation because it takes a full day to dry samples and irrigation may be needed before the results of the measurements are obtained. the time domain reflectometer (tdr) is easy to use and reliable but the number of sites for measurements is limited. the neutron-scattering method was extensively used in field studies for measuring soil storage and its changes over quantitative analysis of soil water content in young drip-irrigated olive orchards c. masmoudi charfi olive tree institute, 17 avenue ahmed tlili, 1004 el menzah v (carnoy), tunisie. key words: evapotranspiration, neutron probe calibration, root density, stage of development. abstract: the present work was carried out in northern tunisia (36°40’ n, 10°16’ e) during a single growing season in order to examine how water is distributed within young drip-irrigated olive orchards on the basis of distance to trunk and depth. soil water content was measured by using a time domain reflectometer (tdr) and a neutron probe calibrated by concurrently measuring soil water content gravimetrically. measurements were made below the canopies, along the line of drippers and out of the projected canopy area, at distances of 1.4 m, 2.2 m, 2.8 m and 4.2 m from trunks (compartments g1, g2, g3 and g4), taking into account the heterogeneous distribution of roots. results showed significant and positive correlations between the series of data collected simultaneously with the pvc and aluminium access tubes and those collected using the different methods and apparatus, demonstrating that any device may be employed, depending only on their availability. results showed significant changes in soil water contents and stocks according to the season, depth and distance to trunk. during the rainy period, the stocks of water increased homogeneously within all soil compartments, but varied consistently during the dry season with lower values recorded within the upper soil layers. the area situated at 4.2 m from trunks was the driest in summer but it was the wettest during the rainy period. no roots were found at this distance while maximum root densities were observed at 0.4 m from trunk within the upper layers. the lack of water recorded after june affected tree height and fruit growth rates, although irrigation application was sufficient to meet the seasonal crop water needs. adv. hort. sci., 2012 26(3-4): 138-147 received for publication 22 june 2012 accepted for publication 21 december 2012 139 time (vachaud et al., 1977; evans et al., 1996; tarara and ham, 1997; xiong and guo, 1999). with this apparatus, measurements can be made at different depths and sites, but these ‘measurements’ represent a property of the soil that can be related to soil-water content and are, therefore, indirect estimates (hewlett et al., 1964; rana and katerji, 2000). on the other hand, a survey of literature (hewlett et al., 1964; vachaud et al., 1977; sinclair and williams, 1979; haverkamp et al., 1984; vauclin et al., 1984; villagra et al., 1995) shows that little attention has been paid to the associated errors and uncertainties resulting from the definition of the calibration curve itself, when calibrating the apparatus. instrumentation, timing and location variances are identified as the different components of the total variance of an individual water content estimate. sinclair and williams (1979) reported a comprehensive analysis of the contribution of instrument calibration and location variances to the variance of mean water content values and their changes in time. implicitly they assumed that all the observations were independent of one another. the present work illustrates, with results from a single growing season of a young olive orchard cv. chétoui aged six years and cultivated in northern tunisia, how water is distributed in such orchards taking into account time (stage of development) and root distribution. our approach is based on estimating water content at different distances from trunks. data were analyzed considering both spatial and temporal variability within different soil reservoirs, throughout the campaign and on some typical days. specifically, the aim was to highlight the main difficulties found when measuring soil water content in dripirrigated orchards characterized by discontinuous and low soil coverage. 2. materials and methods experiment site the study was performed during a single growing season (2003) on a young olive orchard located at the experimental farm of the institut national agronomique de tunisie, northern tunisia (36°40’ n, 10°16’ e). the area is characterized by a mediterranean climate with average annual water deficit of 750 mm and reference evapotranspiration (et0) of 1200 mm. weather variables were recorded continuously in a nearby automatic weather station. daily average values were used for et0 calculation (table 1) following the penman monteith equation (allen et al., 1998). table 1 annual and seasonal (march september) weather variables recorded in 2003 weather variables value rainfall (mm/year) 790 seasonal rainfall (mm) 346 et 0 (mm/year) 1211 seasonal et 0 (mm) 982 the year of experiment was rainy and hot with annual and seasonal effective rainfall amounts of 546 mm and 239 mm, respectively. these values were estimated following the usda method (fao, 1976). average maximum and minimum temperatures (24.9°c and 14.9°c, respectively) showed an increase of 3 and 7%, respectively, with regard to the average values recorded during the 25 previous years. rising temperatures were noted during the three first months of the year, resulting in an increase of the growth degree day (gdd) of about 300 days. olive orchard three six-year-old olive trees (cv. chétoui), representative of the whole orchard, were used in this experiment. they were planted at 6 m x 6 m spacing and stand on a textured clay-loamy soil of about 2 m depth. soil characteristics were determined at the beginning of the experiment for each trench of soil to 1 m depth (table 2). average bulk density (d a ), soil water contents at field capacity (θ cc measured at -0.3 mpa) and at wilting point (θ pfp measured at -1.5 mpa) were 1.6 g/cm3, 0.50 m3/m3 (50%) and 0.26 m3/m3 (26%), respectively. table 2 soil characteristics of the olive orchard horizon (cm) 0-20 20-40 40-60 60-80 80-100 average ecartype clay % 39 34 28 22 20 29 7.1 loam % 50 52 48 48 46 49 2.0 sand % 11 14 24 32 34 23 9.3 d a (g/cm3) 1.55 1.64 1.60 1.60 1.68 1.61 0.04 θ cc (%) 48 50 50 51 50 50 0.98 θ pfp (%) 25 26 27 26 25 26 0.75 trees were intentionally chosen of the same variety, with similar shape. leaf area (la) was determined after pruning by computing the number of leaves on representative branches and their specific area by planimetry (fernandez and moreno, 1999); individual average value of la was 14 m2. soil coverage was low, rarely exceeding 35%. at the end of the campaign, mean tree height and canopy diameter reached 4.9 m and 4.0 m, respectively. water requirements and irrigation management average weather variables published in local papers (masmoudi-charfi, 2006) were used to estimate daily et0 values. crop evapotranspiration (etc) was then determined following the fao method for non-standard conditions (allen et al., 1998) as et c = et o x k c x k r , with a crop coefficient k c = 0.5 (six-year-old trees) and kr = 0.75 (coi, 1997) accounting for an average soil coverage of about 33% (masmoudi-charfi, 2008). trees were irrigated from 15 may to 5 september with amounts ranging between 0.333 m3/tree and 1.098 m3/tree according to the stage of growth. the seasonal irrigation amount was 5.4 m3/tree. periods and doses of irrigation are 140 reported in table 3. water was supplied using four emitters per tree with a total discharge of 16 l/h. fresh water was provided alternatively from the ‘medjerda’ canal, the main river of northern tunisia and nearby wells. table 3 irrigation supply periods and amounts (m3/tree) 1 2 3 4 5 6 7 irrigation period 15-20/5 2-3/6 30/6-2/7 10-15/7 21-30/7 5-10/8 28/8-5/9 irrigation amount 0.823 0.333 0.549 0.843 0.902 0.902 1.098 field monitoring experimental protocol and soil water content measurements. this work was carried out in order to highlight the difficulties met at field level when elaborating protocols concerning irrigated olive orchards, characterized by heterogeneous distribution of light, soil coverage, roots and water application. difficulties concerned mainly the choice of measurement sites and the right measuring device, particularly: at which depths, frequency and distances from trunks measurements should be taken? which kind of apparatus and access tubes should be used for easy and precise soil moisture monitoring? is there any relationship between measurements taken with different apparatus? how many repetitions (trees) are necessary to get significant results? what precautions should be taken when preparing and installing the access tubes and when calibrating the neutron probe? taking all these questions in mind, but also the results obtained for this same orchard relative to the root distribution (masmoudi-charfi and ben mechlia, 2011), a specific diagram was built in which the area surrounding the three olive trees was instrumented with access tubes covering all soil occupation cases. figure 1 shows a series of 28 access tubes implemented vertically in the soil at distances from trunks ranging between 1.4 m and 4.2 m. measurements of volumetric water contents (h v ,%) were carried out within this area from april to october at depths ranging from 0.20 to 1.20 m using a neutron probe (solo 25, nardeux, france). two types of tubes were experimented and compared. a correlation was then established between measurements made simultaneously with aluminum and pvc-polyamide tubes, which were locally assembled (4 cm inside diameter and 170 cm long). specific glue was used to seal the components of the pvc-polyamide tubes in order to assure their tightness and impermeability to water. neutron probe countings were coupled with routine observations of h v made at the limit of the canopy (2 m from the trunk) on the eastern side of the medium tree, about 0.60-0.70 m from the emitters and 0.20, 0.40, 0.60, 0.80 and 1.0 m depth by using a time domain reflectometer (tdr) (fig. 1). fig. 1 distribution of access tubes for neutron probe measurements taking into account root distribution, soil humectation and soil coverage. tubes were set around three olive trees of cultivar chétoui at 1.5 m depth at distances of 1.4 m, 2.2 m, 2.8 m and 4.2 m from trunks. the heterogeneous distribution of roots and discontinuity of the soil coverage make the interpretation of our measurements difficult. for this reason, four soil compartments designated g 1 , g 2 , g 3 and g 4 were considered according to the distance to trunk. the groups g 1 and g 2 include measurements of soil water content made below the canopy at 1.4 m and 2.2 m, respectively, with three and two replications. observations made along the line of drippers at 2.8 m and out of the projected canopy area at 4.2 m belong to groups g 3 and g 4 , respectively. probe calibration. calibration of the neutron probe consists in relaying the count ratio (n/n water ) and the soil water content values determined for all depths exceeding 0.20 m by concurrently monitoring the probe countings and the gravimetric soil moisture. measurements were made at the same sites, weekly, in dry and humid conditions to cover all potential values, and more frequently during the irrigation period. before and after sampling, the counting was sampled in a water medium in order to control the possible drift in the electronic device provided by the probe itself. the average value was used to adjust the measurements made on the same day. also, we have considered for each trench of soil a specific value of the bulk density (d a ) instead of using an average value for all soil layers, which may increase the error intervals. finally, the series of data were correlated considering each trench of soil separately. the correlative equations were used to translate the counting values obtained during all the campaign into h v estimates. soil water storage. water stored in the soil was determined for each trench of soil and then for the whole profile to a depth of 1.2 m using the following equation: s (mm) = 10 x h v x d, where d = is the layer depth (0.20 m), assuming a standard error of 0.02 m3m-3 on h v measurements. analyses of results. soil water content values determined for each compartment (groups g 1 , g 2 , g 3 and 141 g 4 ) were analyzed separately considering two temporal scales. the first analysis was made during the campaign and the second concerned some typical days representing the main physiological processes that evolved during the growing season. the first date (29/5/2003), designated (s), coincided with the rapid fruit growth stage and it was dry, without any rain or irrigation supplies. the second date (16/7/2003) was chosen during an irrigation episode, corresponding to the stage of flower induction, designated (i). the third date (23/09/2003) was the period of fruit enlargement, designated (p). it corresponds to a high soil moisture period and was chosen after the first heavy rains (90 mm) of that autumn. through measurements taken on these dates, we analyzed soil behavior under welland low-watered conditions and different climatic demand. 3. results calibration of the neutron probe count ratios (n/n water ) and soil water content values (h v ) obtained gravimetrically were positively correlated with r correlative coefficients (r) ranging between 0.69 and 0.83 for the portion of soil from 0.20 to 1.20 m depth (fig. 2). the curves established for the four medium layers (20-40 cm, 40-60 cm, 60-80 cm, 80-100 cm), drawn with either the same or different values of bulk density, provided the same coefficient of correlation (r= 0.83, r= 0.76, r= 0.70 and r= 0.73 for 0.20-0.40 m, 0.40-0.60 m, 0.600.80 m, 0.80-1.00 m, respectively) for each soil layer. the trench of soil from 1.00 to 1.20 m showed some deviation with r= 0.69 when using a specific da value, and r= 0.66 when using an average value of da. these differences are due to transition between the clay-loamy and clay-sandy soil layers. relationships between tdr, neutron probe and gravimetric soil water content measurements measurements of h v values taken using aluminum and pvc-polyamide access tubes were inter correlated, showing a positive and significant correlation curve with r = 0.73 (fig. 3). the tdr-measurements were also correlated to the neutron probe estimates and to the gravimetric observations with high correlative coefficients of 0.87 and 0.79, respectively (fig. 4). these results are of practical interest as they allow use of any apparatus or method with confidence depending on their availability. however, it is important to take into account the representativety of the measurements which could not be taken simultaneously every time at each location (the case of tdr). fig. 3 relationship between h v (%) measurements made simultanuously with the aluminium and pvc-polyamide access tubes. fig. 2 probe calibration curves determined for the neutron solo 25 for different soil layers. gravimetric measurements (h v ) were correlated to the counting ratio n*= n/n water , where n and n water referred to counting made into the soil and water, respectively. fig. 4 relationships between h v (%) measurements made simultanuously with tdr, neutron probe and gravimetry. 142 spatio-temporal variability of soil water content soil water contents measured with tdr fluctuated during the growing season by 15 to 46% depending on depth, season and watering conditions (fig. 5). during the irrigation period (beginning from 15 may), h v -values ranged from 25 to 39% with maximum and minimum values observed within the medium depths and at the top soil layer, respectively. resumption of irrigation at the end of august provided a significant increase of h v values which decreased rapidly after mid october. the lowest values were recorded at the end of the year under low soil evaporation conditions and root activity, and specially in the upper layer. fig. 5 evolution of the tdr-soil water content measurements according to depth in 2003. soil water content also varied according to the distance to trunk (fig. 6). this was observed through estimates of water stocks determined throughout the campaign for all soil compartments (g 1 , g 2 , g 3 and g 4 ). considerable variability was observed between these reservoirs with regard to soil humectation events (i.e. rainfall and irrigation supplies) with values between 225 and 400 mm. the stock of water recorded at the beginning of the campaign (april) was close to 350 mm with little variability between soil compartments. it then decreased during the first decade of may in response to the increasing climatic demand and plant activity. the beginning of irrigation on 15 may marked the start of a consistent increase of the stocks around the emitters, enhancing the disparities between compartments. values subsequently evolved continuously with a downward trend following water application. from mid july to the end of august, irrigation was interrupted, leading to a significant decline of these stocks, to reach their lowest value of 225 mm in g 4 . rainfall received in the autumn increased and homogenized the soil water status. the highest stocks were recorded in this case for g 1 , while reservoirs g 3 and g 2 provided intermediate values; these areas were not subject to irrigation but they were partially shaded during the diurnal period (reduction of soil evaporation). reservoir g4 showed the lowest stocks in summer, but it gave the highest values during the rainy period. these results indicate that soil water status mainly depends on depth, water application and distance to trunk, but it may vary depending on plant activity. soil water content and tree response root growth and canopy relationship. roots extended rapidly during the growing season to reach in may the limit of the canopy at 2.12 m from trunks. maximum root number was observed at 0.40-0.60 m depth (table 4), i.e. at depths characterized by high soil water contents (fig. 5), while maximum root densities (dr) were recorded in the top soil layers at 0.40 m from the trunks (fig. 7). the highest value of dr (0.67 cm/cm3) was observed in g 1 . then, as distance to trunk increased, root densities decreased, as did the stocks of water. at a greater distance from trunks (80 cm), maximum root densities ranged between 0.15 cm/cm3 (deeper layers) and 0.35 cm/cm3 (upper layers). during this same period, the canopy diameter increased at similar rates leading to equilibrium between the above and underground areas, just a few years after planting. we recorded at the end of the campaign an optimum la/l r (leaf area/root length) value of 2.3 km/m2 while the ratio s r /s c (root area/projected canopy area) approximated the unit (table 5). this result indicates that as leaf area increased, the amount of carbohydrates increased allowfig. 6 soil water stocks (mm) determined for all the profile (0-100 cm depth) at 1.4 m (g 1 ), 2.0 m (g 2 ), 2.8 m (g 3 ) and 4.2 m (g 4 ) from trunks. table 4 root distribution, number and diameter observed during the experimental year for six-year-old chétoui olive trees compared with measurements made on five-year-old tree soil layer (cm)/age 5-year-old tree 6-year-old tree distribution of roots 0-20 9 51 20-40 5 91 40-60 3 116 60-80 5 97 80-100 3 81 100-120 0 36 total number of roots 25 472 maximum root diameter (mm) 24 27 volume of the rooting system (cm3) 5.3 11.2 maximum distance to trunk (cm) 150 212 143 ing good development of roots and fruits. this hypothesis is analyzed in the following section through simultaneous monitoring of fruit growth and tree height. watering conditions, tree growth and fruit development. growth patterns relative to tree height and fruit diameter observed in 2003 were different from those recorded for the previous year (fig. 8). in 2002, tree height and fruit diameter increased with irregular rates, but continuously from april till october, peaking at 1.4 cm/day (105 doy) and 0.22 mm/day (142 doy), respectively. in the following year, we did not record any peak values for tree height but rather a low and constant rate of about 0.100.15 cm/ day. from april to june, fruits grew with increasing rates to peak at 0.29 mm/day on 155 doy. soil water contents recorded during this period of cell division and early fruit growth (10/4-10/6, 100-160 doy) (fig. 9) were apparently sufficient to assure suitable fruit development. fig. 8 growth patterns of olive tree height (cm/day) and fruit diameter (mm/day) recorded in 2003 compared to tree height and fruit development curves recorded in 2002. values are averages of 96 tree height and 480 fruit diameter measurements made on chétoui olive trees. tree height and fruit growth decreased significantly in july-august most likely because of interruption of irrigation and the decrease of soil water content values (fig. 9). after this period of a lack of water, hv-values increased particularly in the deeper depths, enhancing the ultimate fruit development (284 doy, 0.1 mm/day). soil profiles established in may, july and september showed different behavior depending on the watering conditions and the climatic demand. for measurements made at 1.4 m from trunk (g 1 ), soil water content values ranged fig. 9 soil water content curves observed at different stages of fruit development. fig. 7 average root densities (cm/cm3) recorded for fiveand six-year-old olive trees at 0.40, 0.80, and 1.20 m distance from trunks. measurements were made in both east and south directions at different depths (0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm and 80-100 cm). table 5 characteristics of the rooting system of the six-year-old-tree of cultivar chétoui compared with those recorded for a fiveyear-old tree soil layer (cm)/age 5-year-old tree 6-year-old tree root area (m2) 7.10 13.8 projected canopy area (m2) 8.04 11.94 root area (sr, m 2) / projected canopy area (sc, m 2) 0.9 1.2 maximum canopy radius (m) 1.60 1.95 average root density (cm/cm3) 0.13 0.30 length of the rooting system (km) 7.05 33.94 144 between 30 and 42% (fig. 10), providing distinguishable profiles with constant differences between the lowest and the highest values within each trench of soil. however, minimum and maximum soil water contents were not observed during the driest (s) and the wettest period (p), respectively. minimum values of h v were recorded in the first 40 cm in may (s) and at deeper depths (0.40-1.20 m) in july (i), while maximum values were recorded in july in the superficial top layer, in september for the 0.20-0.60 m layer and in may at deeper depths (0.60-1.20 m). fig. 10 soil water content (h v , %) measured within the first reservoir (g 1 ). measurements were made to 1.2 m depth at a distance of 1.4 m from trunks on may (29-05-2003) after a 20-day period of dryness (g 1 -s), in july (16-07-2003) during an irrigation period (g 1 i) and in september (23-09-03) following the first heavy autumn rains (g1-p). groups g 2 , g 3 and g 4 showed important variations between h v values within the top soil layers (0-0.40 m) and small differences at deeper depths despite their distances from trunks (2.0 m, 2.8 m and 4.2 m) (fig. 11). minimum values (20%) were recorded in may and july despite the different climatic conditions, while the highest values of soil water content were about 40 % at all depths. 4. discussion and conclusions this case study demonstrates the potential of using the neutron probe, tdr and gravimetric measurements to determine soil water content in a young olive orchard, taking into account the heterogeneous distribution of roots, localized irrigation and low canopy shade. values of h v were obtained by using the probe calibration curves established specifically for the solo 25 apparatus for each trench of soil. these curves cannot be used in any other situation. data collected with the different methods and apparatus showed positive correlations between soil water content measurements. this result is of practical interest because it indicates that any of these apparatus or methods can be used depending only on their availability. for example, the relationship developed between the two types of access tubes allowed us to use with confidence the pvc-polyamide tubes which were assembled locally with a lower cost. regarding the apparatus, the tdr with probes installed vertically has proven to be fast, accurate and non-destructive, but it allowed measurements in one location only. this is a major disadvantage. on the contrary, gravimetric measurements can be made at any location and the method is suitable for calibrating other methods, although it is destructive and hard to carry out. measurements made with the neutron probe are difficult. the apparatus requires specific calibration and some care should be taken when using such radioactive probes. with regard to these ‘constraints’, uncertainties arise because soil water content monitoring is incomplete as it is impossible to do measurements at all depths and in all directions. for the neutron probe, it is important to know if the observed variance of water content measurements is really due to errors associated with the location, which can be randomly distributed or spatially structured, or to errors arising from the use of a neutron probe itself. indeed, uncertainty intervals are also influenced by the charge of the probe battery, the number of access tubes used, their placement and the depths at which they were installed. the number of access tubes should be determined depending on the heterogeneity of the orfig. 11 soil water content (%) measured within the second (g 2 ), third (g 3 ) and fourth (g 3 ) reservoirs. measurements were made to 1.2 m depth at distances of 2.0, 2.8 and 4.2 m from trunks, respectively, in may (29-05-2003) after a 20-day-long period of dryness (g 1 -s), in july (16-072003) during an irrigation period (g 1 -i) and in september (23-09-2003) following the first heavy autumn rains (g 1 -p). 145 chard in order to avoid situations where sample data size is not sufficiently distributed in the field. about 25 locations would be necessary to obtain a mean value with a relatively high precision; observations are thus considered independent of one another regardless of their location in the field. it is also obvious that when installing soil-water measuring devices in the plant row in the irrigated field, at least one device should be located in each of the major soil types to take into account all soil occupation cases. additional care should be taken with regard to possible drift in the electronic device: (1) counting in a water medium before and after profiles are sampled, (2) using the apparatus with a fully charged battery and (3) the use of a specific d a value for each trench of soil rather than an average value for all soil layers when the soil texture is variable. however in light of recent concepts introduced in soil physics studies, and as previously stated, it is obvious that the auto-correlation between measurements in estimating the variance of the mean must be taken into account. soil water contents varied consistently according to the proximity of measurements to trunk and depth. maximum variations were observed in the first top soil layers at 0.40 m from trunks as a result of the heterogeneous distribution of roots. there was a massive presence of roots near the trunk, mainly confined to the canopy projected area (masmoudi et al., 2007; masmoudi-charfi et al., 2011). these results are concordant with those reported by bonachela et al., (1999), fernandez and moreno (1999), palese et al., (2000), fernandez et al., (2003) and connor and fereres (2005) regarding high root densities in these areas. some roots were also found at greater distances from the trunk, outside the canopies, i.e. within the reservoirs g 2 and g 3 but with lower densities in comparison to values obtained within the first compartment. this indicates that root uptakes are still possible in these areas, and these roots may have a significant role in enhancing root absorption and water transfer. furthermore, results showed that the zones where roots develop behave differently, even below the canopy, involving different processes of water uptake and depletion. unfortunately it was not possible to separate these processes in the present study to determine if these roots are more active than those located near the trunk or not. fernandez and moreno (1999) and connor and fereres (2005) explain that root densities are necessarily higher in the area of irrigation but roots may be less active. on the contrary, roots far from the trunk may be larger with numerous fine roots and thus they are more active. in another study carried out during the same year, abid-karray (2006) reported the presence of lateral water transfers within an olive orchard cultivated in central tunisia under complementary irrigation. high water depletion was observed in that olive orchard and others cultivated under semi-arid and arid climates due to advective transfers of heat in soil (fernandez et al., 1990; fernandez et al., 1991; villagra et al., 1995; bonachela et al., 1999; granier et al., 2000; fernandez et al., 2003). this makes the situation more complex because roots situated outside of the projected canopy limit may contribute significantly to supply other reservoirs. their role is however, highly dependent on the distance from the point of water but also the stage of development. we have published in previous papers (masmoudi-charfi and ben mechlia, 2007 and 2008) that under irrigated conditions young olive trees cultivated in this same location continued to grow even during the winter months, under different watering conditions (extreme rainy and rainless years), however with relatively low rates. variability of soil water content is also dependent on soil coverage, which varied consistently from year to year, following the season, the severity of pruning and measurement site, thus modifying significantly the relative importance of the evaporative processes involved within each soil reservoir (dichio et al., 2002; masmoudi et al., 2004; 2007). this is because the contribution of the different processes of water uptake and water depletion depend on the amount of solar radiation intercepted by the tree canopy, which is the most important factor controlling water losses and extension of the leaf area. water applied during the growing season seemed to be sufficient to meet the overall tree water needs although some water shortage was observed during the fruit set-maturation period. stocks of water recorded at the beginning of the growing season (in may) were apparently insufficient to insure suitable growth of the tree, but high enough to assure early fruit growth. however, the lack of water observed from midjuly to end of august under high evaporative demand, reduced both tree height and fruit size (-10%), fruit weight (-26%) and also fruit number through an important fruit drop observed early september. comparative results between the year of study and the preceding year showed a significant reduction of yield at harvest. six-year-old olive trees yielded 1.9 t/ha (2003 was normally an ‘on’ year), while yield exceeded 2.0 t/ha in 2002 (‘off’ year). these different responses may be inheritent to other exogenous factors like soil type, pruning, and fertilizer schedules (masmoudi-charfi and ben mechlia, 2009) or to some endogenous parameiters which have an impact on how irrigation changes affect the production and growth levels. in september, although water was abundant and trees were loaded with fruits, water uptakes seem to be reduced due to the decrease of climatic demand. these results are supported by observations of sap fluxes recorded during the same period (masmoudi-charfi et al., 2011; 2013), showing that when water is available, sap flux measurements increased in correlation with the increasing climatic demand. this experiment, concerning the choice of devices used to measure soil water content, despite the observed constraints, has practical interest. the positive correlations developed between soil water content measurements made with the different methods and apparatus indicate that any of these devices can be used indifferently, depending only on their availability and ease. particularly, the relationship developed between the two types of access tubes allowed us to use with confidence the pvc-polyamide tubes assembled locally with a lower cost. this study has shown also that soil water content consistently affects tree height 146 and fruit growth rates and varies depending on the depth and distance from the trunk. this spatio-temporal variability makes it difficult to provide proper assessment of the components of the water balance, if it is used for water consumption estimation in young orchards. therefore, associating methods should be used that make it possible to distinguish between soil evaporation and water lost by transpiration like sap flux measurements. additional measurements of root activity are also necessary to confine intervals in simulated uptake distributions. nevertheless, these methods and estimations remain useful tools to decide when and how to irrigate, how much water is stored in the soil for plant use (soil water logging capacity) and to determine allowable water depletion. acknowledgements author gratefully acknowledge prof ben mechlia nétij, dr 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difficulties of estimating evapotranspiration from the water balance equation. agricultural and forest meteorology, 72: 317-325. xiong l., guo s., 1999 a two-parameter monthly water balance model and its application. journal of hydrology, 216: 111-123. impaginato 75 adv. hort. sci., 2011 25(2): 75-80 received for publication 24 february 2011. accepted for publication 18 march 2011. antioxidant capacity and total phenolic content of hydrothermally-treated ‘fuerte’ avocado e.r. daiuto *, j.g.f. fumes**, r.l. vieites***, n.c. cabia**, r.s.d. castro**** * department of horticulture, faculty of agronomic sciences, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. ** agronomic engineering, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. *** department of agribusiness management and technology, faculty of agronomic sciences, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. **** food engineer, unesp, c.p. 237, 18610307 botocatu, são paulo, brazil. key words: antioxidant capacity, bioactive compounds, persea americana mill., refrigeration. abstract: avocados possess high nutritional value with proven effectiveness in preventing cardiovascular diseases, attributed primarily to their unsaturated fatty acids content. this fruit is also rich in carotenoids and vitamins, particularly vitamin e. this work evaluates the antioxidant capacity and total phenolic content of hydrothermally-treated fuerte avocado. fruits were selected and hydrothermally treated at 45oc for 5, 10, 15 and 20 min. they were then stored in a refrigerator (10 ± 1oc and 90±5% relative humidity) and evaluated over a 15-day period. the total phenolic content increased up to the sixth day of storage, and decreased thereafter, without differences between the treatments. the percentage of antioxidant capacity of the control and the hydrothermally-treated samples for 5 and 10 min increased during storage. untreated fruits showed the highest percentage of antioxidant capacity. however, the antioxidant capacity of avocado fruits subjected to these treatments declined starting on the twelfth day of storage, possibly due to the fruits’ senescence. hydrothermal treatments for 15 and 20 min delayed fruit senescence while the antioxidant capacity continued to increase up to the fifteenth day of storage. no significant correlation was found between antioxidant capacity and total phenolic content. the antioxidant capacity of ripe fuerte avocado was higher than that of unripe or overripe avocado. 1. introduction avocado (persea americana mill.) has considerable nutritional quality, with a high content of fibers, proteins and mineral salts, particularly potassium and vitamins, especially vitamin e (usda, 2007). it also contains significant amounts of unsaturated fatty acids, which are beneficial for the prevention of cardiovascular diseases (tango et al., 2004). previous studies have also shown that this fruit contains anticarcinogenic lipophilic compounds such as carotenoids (ding et al., 2007). wang et al. (2010) pointed out the scantiness of studies on the phytochemical composition of avocados and the lack of knowledge about the total phenolic content and antioxidant capacity of different avocado varieties, or cultivars. the aforementioned authors conducted studies to determine the antioxidant capacity of the pulp, seed and peel of different avocado varieties, but not the fuerte variety. ‘fuerte’ avocados are small and are highly valued in european and american markets. antioxidants, which are compounds that inhibit and/or reduce the effects of free radicals (soares et al., 2005), can be defined as compounds that protect the cells against the harmful effects of oxygen and nitrogen free radicals that are formed in oxidative processes. high free radical levels generate an imbalance, triggering oxidative stress, the metabolic process responsible for the onset of several types of chronic degenerative diseases. antioxidants can be obtained by eating food containing vitamins e and c, carotenoids, phenolic compounds, and other compounds (ali et al., 2008). phenolic compounds are responsible for most of the antioxidant activity in fruits, making them a natural source of antioxidants (heim et al., 2002). the phenolic content in food and plants depends on a number of intrinsic factors such as the genus, species and cultivar, and on extrinsic factors such as agronomic and environmental factors, handling and storage (thomas-barberán and espín, 2001). avocado is a climacteric fruit which ripens a few days after harvest (hardenburg et al., 1986; seymour and tucker, 1993) and whose postharvest behavior can be influenced by temperature and storage time. the literature contains several studies about the increase in 76 the conservation period of avocado, involving the evaluation of storage temperature, the use of modified atmosphere with the application of wax, gamma irradiation and thermal treatment to prevent chilling injury (zauberman et al., 1973; castro and bleinroth, 1982; seymour and tucker, 1993; germano et al., 1996; de oliveira et al., 2000; sanches, 2006; morgado, 2007; donadon, 2009). thermal treatment has been applied postharvest to solve the problem of contamination by fungal diseases and insect infestation in fruit (fawcett, 1922 apud couey, 1989) or to reduce problems caused by low storage temperatures (kluge et al., 2006). to this end, thermal treatments are performed prior to refrigeration, in the form of conditioning, or during refrigerated storage, in the form of intermittent warming. thermal conditioning consists of exposing fruits briefly to moderate (15 to 25oc) or high temperatures (37 to 53oc) before putting them in refrigerated storage (kluge et al., 2006). daiuto and vieites (2008) conducted a study on hass avocado to evaluate the polyphenol oxidase (ppo) and peroxidase (pod) content in unripe and ripe fruits hydrothermally treated at 45oc for 10 min and stored at 9oc (±1). the enzyme inactivation in ripe fruits subjected to the treatment was 78 to 94% compared to untreated fruits. daiuto et al. (2010) evaluated the weight loss and respiratory rate of ‘hass’ avocado by subjecting it to different physical treatments (thermal, uv and gamma radiation) and reported a decrease in the intensity of the fruit’s respiratory peaks. the evaluation of antioxidant capacity has become increasingly important to determine the effectiveness of natural antioxidants in protecting vegetable products against oxidative damage and loss of their commercial and nutritional value. therefore, the present research focused on an evaluation of the antioxidant capacity and total phenolic content of ‘fuerte’ avocado subjected to hydrothermal treatment. 2. materials and methods ‘fuerte’ avocados were harvested carefully at the point of physiological maturation and according to their oil content. the fruits, which were selected with a view to uniform size, color and absence of injuries and defects, were hydrothermally treated in a water bath at 45°c for 5, 10, 15 and 20 min (four treatments), after which they were stored under refrigeration (10±1°c and 90±5% relative humidity). fruits not subjected to the hydrothermal treatment were used as control. the fruits of these five treatments were evaluated at threeday intervals for two weeks. fruit extraction the extraction process was performed with a solvent mixture of ethanol:water (80:20 v/v). fruit extracts were obtained in triplicate. aliquots of 3.0 g of pulp were weighed and placed in falcon tubes, to which were added 30 ml of an ethanol:water mixture (80:20 v/v). the tubes containing pulp and solvent were then processed at room temperature in a turrax crushing disperser for several minutes, and then centrifuged at 5000 x g for 15 min. the extracts were filtered and stored in dark vials at 8ºc for no longer than a week prior to analysis. total polyphenol analysis the total phenolic content was determined by the folin-ciocalteu spectrophotometric method, as described by singleton et al. (1999), using gallic acid as standard. an aliquot of 0.5 ml of the resulting extracts was then transferred to a test tube and 2.5 ml folin-ciocalteu reagent diluted in water 1:10 was added. the mixture was allowed to rest for 5 min, after which 2 ml of sodium carbonate 4% was added and the tubes were left to stand for 2 hr in the dark. the absorbance was measured in a spectrophotometer operating at a wavelength of 740 nm. a blank sample was subjected to the same procedure and conditions. the results are expressed in µg gae/100 g-1 of dry weight. dpph radical scavenging activity the radical scavenging activity was determined by dpph method (mensor et al., 2001). tocopherol and bht at a concentration of 90 µg ml-1 were used as standards. the reaction mixture consisted of 500 µl of fruit extract, 3.0 ml of ethanol 99%, and 300 µl of the dpph radical in a solution of ethanol 0.5 mm, which was incubated for 45 min at room temperature in the dark. the negative control was prepared by replacing the volume of extract for an equal volume of the extraction solvent. a processing time of 45 min was defined after determining the half maximal effective concentration, ec50. to determine the stabilization time, readings of the antioxidant in five concentrations (1, 2, 3, 4 and 5 g) were taken at 15-min intervals (sanchesmoreno et al., 1998). according to do rufino et al. (2007), in subsequent experiments with the same fruit, readings can be limited to the previously established time (ec50 time), accompanied by the initial reading of the control. the blank was prepared by substituting the volume of the dpph solution for an equal volume of solvent. the free radical scavenging activity was determined in the form of antioxidant activity (aa), using the equation: aa (%) = 100[(aa – ab) x 100] / ac, where: aa = absorbance of the sample; ab = absorbance of the blank; and ac = absorbance of the negative control. all the analyses were performed in triplicate and accompanied by a control. a variance analysis was performed using tukey’s test for multiple comparisons of the averages, at a significance level of 5%. the data were then subjected to a regression analysis and to pearson’s correlation for the two parameters evaluated, using the sas version 77 9.2 software program. 3. results and discussion table 1 presents the average and standard deviation of total polyphenols identified in ‘fuerte’avocado. although the four treatments produced similar results, a difference was detected as a function of storage time (p=0.007). total polyphenol content was higher in the control treatment. mean values of 45.7, 47.0, 47.1 and 47.2 µg gae/100 g-1, respectively, were obtained in 5, 10, 15 and 20 min hydrothermal treatment, while the control showed 49.8 gae/100g-1. the lowest value obtained was 42.7 µg on the first day of analysis and the highest was 62.1 µg for the control treatment on the sixth day of analysis. the composition of phenolic compounds in fruit may be modified as a function of the environment and postharvest factors, including processing and storage. processing and storage can induce prolonged enzymatic and chemical oxidation of phenolic compounds, contributing to their reduction (kaur and kapoor, 2002). many studies have shown that phenolic compounds generally decrease in climacteric fruit such as tomatoes, bananas, mangos and guavas during ripening (haard and chism, 1996; lakshimnarayana et al., 1970; mitra and baldwin, 1997; selvaraj and kumar, 1989). the total phenolic content increased up to the sixth day of storage, decreasing thereafter due to the onset of senescence (fig. 1). daiuto et al. (2010) found that the average respiratory peak of ‘hass’ avocado subjected to different physical treatments occurred on the ninth day of storage, after which senescence set in. this decrease can be attributed to a series of chemical and enzyme amendments that occur during the accelerated process of maturation of this fruit. these changes may include glycoside hydrolysis by glycosidases, phenol oxidation by phenoloxidases, and polymerization of free phenolic content (robards et al., 1999). the average antioxidant capacity measured in the treatments varied from 21.1% (20 min treatment) to 28.5% (control). the lowest value obtained was 17.6% on the first day of analysis and the highest was 67.6% on the twelfth day of analysis in the control treatment (table 2). the overall average, taking into account the days of storage time, indicated an increase in antioxidant capacity. table 1 average and standard deviation of the total polyphenol content in hydrothermally-treated ‘fuerte’ avocado as a function of treatment and storage day treatments storage days 0 3 6 9 12 15 overall average per treatment control 5 min 10 min 15 min 20 min overall average per storage day 42.7±2.7 42.7±2.7 42.7±2.7 42.7±2.7 42.7±2.7 42.7b±2.7 41.8±2.7 51.8±12.8 58.5±2.7 51.6±5.1 49.3±1.7 50.6ab±8.8 62.1±20.5 42.3±13.5 48.0±4.6 58.4±9.1 54.6±10.1 53.1a±13.0 54.2±1.3 52.9±15.4 48.8±6.5 41.4±15.8 45.3±6.8 48.5ab±10.3 36.5±9.3 42.7±3.7 44.1±7.5 42.8±4.5 45.6±2.9 42.4b±6.1 61.7±3.5 42.2±6.0 39.8±15.1 45.6±6.0 45.4±7.9 46.9ab±10.8 49.85±13.2 45.75±9.9 47.05±9.5 47.15±9.4 47.25±6.5 upper case letters compare overall averages on each storage day table 2 average and standard deviation of antioxidant capacity of hydrothermally-treated ‘fuerte’ avocado as a function of hydrothermal treatment and storage day treatments storage days 0 3 6 9 12 15 overall average per treatment control 5 min 10 min 15 min 20 min overall average per storage day 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6±1.1 9.3ab±2.0 24.7aab±1.0 15.4aba±4.0 13.2aab±4.3 17.7aab±2.5 16.1±5.9 20.6abb±7.2 10.8bb±6.4 15.7aba±7.4 38.4aa±5.4 32.6ab±13.3 23.6±12.9 26.6ab±6.8 25.1aab±9.7 34.3aa±16.9 9.3ab±4.5 10.81b±3.8 21.2±12.9 67.6aa±1.4 43.2aa±33.6 16.9ba±0.7 13.2bab±0.7 7.2bb±4.8 29.6±17.5 29.4aab±3.6 29.6aab±12.6 31.7aa±12.2 33.4aab±8.4 40.9aa±34.6 33.0±15.7 28.5±19.5 25.1±16.6 21.9±11.2 20.9±13.4 21.1±17.8 lower case letters compare averages per treatment per day. upper case letters compare averages of each treatment on each storage day. fig. 1 total phenolic content (µg gae/100g-1) of hydrothermallytreated ‘fuerte’ avocado (overall average of storage days). 78 the highest percentages of antioxidant capacity were obtained in the fruit without hydrothermal treatment. the percentage of antioxidant capacity declined in thermally-treated fruits starting on the twelfth day of storage, possibly due to senescence. the fruits thermally treated for 15 and 20 min showed values of 33.4 and 40.9%, respectively, after 15 days of storage. however, the values declined between the sixth and twelfth day of storage. this tendency for the percentage of antioxidant capacity to decrease may be a result of the thermal treatment, but the profile presented here with low values at nine and 12 days may be a consequence of the heterogeneity of fruit samples. this may explain the results of this research, which indicated that the antioxidant capacity of pulp thermally treated for 15 and 20 min increased up to the fifteenth day of storage. with a less intense respiratory peak, the degradation reactions were also diminished. arancibia-avila et al. (2008) reported that total polyphenols, flavonoids and anthocyanins were significantly higher (p < 0.05) in ripe durian fruit than in unripe or overripe fruit (durio zibethinus murr., cv. mon thong). the overall average antioxidant capacity during the storage period was 17.6, 16.1, 23.6, 21.2, 29.6, and 33.0% for the different treatments, indicating the tendency for antioxidant capacity to increase as the fruits ripened. the total phenolic content is not necessarily involved in the quantification of antioxidant activity (jacóbo-velasquéz and cisneros-zevallos, 2009). the correlation analysis of antioxidant capacity and phenolic compounds in fuerte avocado did not reveal significant results (p=0.992 and r=0.001). arancibia-avila et al. (2008) found a correlation of 0.98 between the total phenolic content and antioxidant capacity of durian fruit. these authors concluded that the high polyphenol content was the main factor responsible for the fruit’s antioxidant capacity. wang et al. (2010) found a significant correlation between the total phenolic content and antioxidant capacity (≥ = 0.79) of avocados of different cultivars. the two parameters evaluated by these authors showed no correlation with the chlorophyll and carotenoids content (r<0.1). furthermore, for these authors the high correlation found between procyanidins and the polyphenol content and antioxidant capacity suggests that this compound is the main polyphenol contributing to the antioxidant capacity of avocado. in the present research, the low correlation found for the evaluated parameters may indicate that another food metabolite is responsible for the antioxidant activity of avocado. it should be noted that vitamin e is a powerful antioxidant which may also contribute to the antioxidant capacity of avocado fruits. in a study of the effect of heat treatment on the antioxidant capacity of vegetables, melo et al. (2009) found that several events that occur during this treatment explain changes in the antioxidant activity of foods, which may be increased, reduced or unaltered. in situations in which the antioxidant activity of food increases, heat treatments favor the partial oxidation of the bioactive compound with the highest ability to donate a hydrogen atom to a radical starting from the hydroxyl group, and/or the aromatic structure of the polyphenol is more able to withstand the displacement of the unpaired electron around the ring. moreover, heat treatments may favor the formation of new compounds such as maillard reaction products (reductones), which exhibit antioxidant activity (nicoli et al., 1999). because refrigeration is the most efficient method for controlling fruit maturation, it may have contributed to maintaining the antioxidant capacity of the fruits during the storage period. the heat treatment had a negative effect on the maintenance of the fruit’s antioxidant capacity compared to that of the control. the longer the fruit is exposed to a hydrothermal treatment, the higher the loss of its antioxidant capacity. 4. conclusions the total phenolic content increased up to the sixth day of storage, decreasing thereafter, without differences between the treatments. the antioxidant capacity of the control fruit and the fruit hydrothermally treated for 5 and 10 min increased throughout the storage period. the highest percentages of antioxidant capacity were obtained for the fruit without heat treatment. the percentage of antioxidant capacity of these treatments declined starting on day 12 of storage, possibly due to senescence. hydrothermal treatments of 15 and 20 min delayed senescence, with antioxidant capacity continufig. 2 dpph antioxidant activity of hydrothermally-treated fuerte avocado. legend: t = thermal treatment considering all the treatments, the highest antioxidant capacity was found in the control treatment, followed by the 5-min thermal treatment. figure 2 shows increasing values of antioxidant capacity over storage time in the control and the 5and 10-min hydrothermal treatments. 79 ing to increase up to the fifth day of storage. no significant correlation was found between the antioxidant capacity and the content of phenolic compounds. the antioxidant capacity of ripe ‘fuerte’ avocado was higher than that of unripe or senescent fruit. acknowledgements the authors gratefully acknowledge the company jaguacy (bauru, sp, brazil) for its support of this study, and the brazilian research funding agencies fapesp (são paulo research foundation) and capes (federal agency for the support and evaluation of postgraduate education) for their financial support. references ali s.s., kasoju n., luthra a., singh a., sharanabasava h., sahuaa., bora u., 2008 indian medicinal herbs as sources of antioxidants. food research international, 41(1): 1-15. arancibia-avila p., toledo f., park y.-s., jung s.-t., kang s.-g., heod b.g., lee s.-h., sajewicz m., kowalska t., gorinstein s., 2008 antioxidant properties of durian fruit as influenced by ripening. food science and technology , 41(10): 2118-2125. castro j.v., bleinroth e.w., 1982 conservação do abacate em atmosfera controlada e à temperatura ambiente. boletim do instituto de tecnologia de alimentos, 19(2): 165182. couey h.m., 1989 heat treatment for control of postharvest diseases and insect pests of fruits. hort science, 24(2): 198202. daiuto e.r, vieites r.l., 2008 atividade da peroxidase e polifenoloxidase em abacate da variedade hass, submetidos ao tratamento térmico. revista iberoamericana de tecnologia postcosecha, 9(2): 106-112. daiuto e.r., vieites r.l., trecomoldi m.a., citadini russo v., 2010 taxa respiratória de abacate ‘hass’ submetido a diferentes tratamentos físicos. rev. iber. tecnología postcosecha, 10(2): 101-109. de oliveira m.a., dos santos c.h., henrique c.m., rodrigues j.d., 2000 ceras para conservação pós colheita de frutos de abacateiro fuerte, armazenados em temperatura ambiente. scientia agricola, 57(4): 777-780. ding h., chin y.w., kinghorn a.d., d’ambrosio s.m., 2007 chemopreventive characteristics of avocado fruit. seminars in cancer biology, 17(5), 386-394. do rufino m.s., alves r.e., de brito e.s., de morais s.m., de sampaio c.g.; pérez-jiménez j., sauracalixto f.d., 2007 metodologia científica:determinação da atividade antioxidante total em frutaspela captura do radical livre dpph. comunicado técnico on line. 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extração de óleo. revista brasileira de fruticultura, 6(1):17-23. tomás-barberán f.a., espín j.c., 2001 phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. j. sci. food agri., 81: 853-879. usda, 2007 national nutrient database for standard reference, release 20. department of agriculture, agricultural research service nutrient data laboratory, http://www.ars.usda.gov/ba/bhnrc/ndl. wang w., bostic t.r., gu l., 2010 antioxidant capacities, procyanidins and pigments in avocados of different strains and cultivars. food chemistry, 122: 1193-1198. zauberman m.s., schiffmann-nadel m., yanko u., 1973 susceptibility to chilling injury of three avocado cultivars stages of ripening. hortscience, 8(4): 511-513. impaginato 32 adv. hort. sci., 2011 25(1): 32-36 received for publication 24 august 2010. accepted for publication 21 february 2011. fibrous root distribution in pineapple orange trees under semi-arid irrigated ecosystem r.p.s. dalal, a. thakur punjab agricultural university, regional station, dabwali road, bathinda, punjab, 151001 india. key words: cleopatra (citrus reshni), depth, radial distance, root excavation, root length density, rootstocks, rough lemon (citrus jambhiri lush), troyer citrange (poncirous trifoliate x citrus sinensis osbeck). abstract: the root distribution pattern of 17-year-old pineapple orange trees budded on rough lemon, cleopatra and troyer citrange rootstocks were studied by root excavation method at four radial distances, 0-75, 75-150, 150-225 and 225-300 cm from tree trunk, and at three depths, 0-15, 15-30 and 30-60 cm. fibrous root length density (frld) and fibrous root length percentage differed significantly at various depths and radial distances among rootstocks. frld was closer to tree trunk on both horizontal and vertical planes. root density decreased from 0.183 to 0.084, 1.051 to 0.238 and 0.238 to 0.095 cm.cm-3 from 0-15 cm to 30-60 cm depth within 0-75 cm radial distances from tree trunk in trees on rough lemon, cleopatra and troyer citrange, respectively. cleopatra contains the highest 0.231 cm.cm-3 frld as compared to 0.051 cm.cm-3 in rough lemon and troyer citrange. troyer citrange has intensive lateral root development with 84% fibrous roots (fr) within 75 cm radial distance, whereas rough lemon and troyer has an appreciable amount up to 225 cm distance (extensive lateral). cleopatra contained 57% fr in upper soil layer (0-15 cm) (intensive vertical). in rough lemon and troyer 54% fr are confined to lower depth 15-60 cm (extensive vertical root development). troyer and rough lemon had the same vertical, whereas rough lemon and cleopatra showed the same horizontal rooting pattern under arid irrigated ecosystem. thus, irrigation depth and fertilizer placement should be critically rootstock specific. 1. introduction citrus production depends not only upon soil, climate and high density planting but also rootstocks play an important role as different rootstocks have different intensities of root proliferation and penetration (castle and krezdorn, 1975; neves et al., 2004; morgan et al., 2007). moreover, roots are the principal organ for absorption of nutrients and water from soil. root system structure determines the volume of the soil accessible to the crop plant and it is important to maintain sufficient water and nutrient concentration within the soil occupied by the crop root system for optimal nutrient and water uptake (kramer and boyer, 1995; scholberg et al., 2002). increasing the density of fibrous root within a crop root system increases the amount of water and nutrients available to the crop (eissenstat et al., 1999; tinker and nye, 2000). the rootstock in turn can be influenced by the scion and soil environment. performance of rootstock in a certain environment is related to total volume, configuration, lateral distribution and depth of the root system (cintra et al., 1999). the root distribution pattern of a tree varies from region to region and from one rootstock-scion combination to another. even a single rootstock-scion combination may differ in root distribution with a change in climatic condition. mikhail and elzefhoui (1979) found that 79% of the total fibrous root of valencia orange occurred in the first 60 cm of soil depth on sandy soil, whereas clay soil contained 94% in the same depth. boman et al. (1999) reported that citrus production in deep sandy soils with a high volume irrigation system tends to cause the upper soil layer to dry out between long irrigation intervals and this condition favours deep rooting. hipondoka et al. (2003) reported that most of the root activities in trees with regard to water uptake are performed near the soil surface in arid ecosystems of africa. these differences in rooting pattern among rootstocks and soil environments are more likely to reflect the adaptation of plants to a given environment. since citrus growth and root distribution system is rootstock-dependent and may be modified as a result of changes in the root environment, a clear understanding of the root system is important to best deal with management practices such as irrigation and nutrient application and fixing the geometry in a particular ecosystem. keeping in mind the above facts, the present investigation was carried out with the objectives to 33 determine the rooting pattern of 17-year-old pineapple orange budded on three rootstocks under an arid ecosystem of punjab (india). 2. materials and methods study sites the trial was conducted at the experimental orchard of the punjab agricultural university, regional station, bathinda located at 211 m above mean sea level, latitude 74° 58’ e and longitude 30o 17’, and average rainfall 400 mm/year. however 80% of the rainfall is received during the southwest monsoon season (first week of july to mid september). the mean maximum temperature is 40-45°c in june with hot winds and minimum temperature is 4-5°c in january. soil characteristics the soil samples collected from the experimental orchard at a depth of 0-30 cm were analysed for their physical and chemical properties. the soil type was loamy sand with clay content 13%, bulk density 1.5g/cc with moisture holding capacity of 40-45%, moisture at field capacity 25-28%. the ph of the site was 8.32 with electrical conductivity (ec) 0.2 dsm-1 and calcium carbonate 5-12%. the available n, p, k contents were 160-182, 13-17 and 320-346 kg/ha, respectively. treatments mature pineapple orange trees budded on three rootstocks, i.e. rough lemon (citrus jambhiri lush), cleopatra (citrus reshni) and troyer citrange (poncirous trifoliate x citrus sinensis osbeck), at a spacing of 6x6 m planted in 1990 were selected for the study. all three sets of five mature 17-year-old trees were grown under uniform cultural practices (i.e. irrigation with flooding); fertilizer application at 880 g nitrogen and 440 g. phosphorus/plant/year and mechanical weeding/hoeing were selected randomly in a randomized block design and examined for the root distribution system. sample collection for each plant a circle with a radius of 3 m from the tree trunk was marked. this radius was further divided into four segments with 0-75, 75-150,150-225 and 225300 cm radius. the circle circumference was divided into eight parts and one-eighth sections were excavated at three depths, viz. 0-15, 15-30 and 30-60 cm (fig. 1). the roots of 15 plants were excavated with a jet of water at a pressure of 10-15 psi. the plants were exposed to a radial distance of 3 m from the trunk and down to a depth of 15 cm from the ground surface; exposed roots were painted red. the roots were then excavated to a depth of 30 cm (i.e. between 15-30 cm) and exposed roots were painted yellow. the roots were further excavated to a depth of 60 cm (i.e. between 3060 cm) and these roots were kept as such to distinguish them from other roots. after the entire root system was exposed, the roots were collected from each segment of depth and radial distance separately and washed. the root diameter was measured with the aid of a vernier caliper and those having diameter < 0.2 cm were categorized as fibrous roots. the fibrous root length of each segment was measured using a meter scale separately. fig. 1 scheme of the root sampling areas around the trunk (radial distances: a = 0-75 cm; b = 75-150 cm; c = 150-225 cm; d = 225-300 cm). data collection we estimated root length per soil volume represented by the volume of soil calculated as the radial distance from the trunk by the depth increment (0-15; 1530; 30-60 cm) for each of the 15 orange trees. the surface area of the ring of radial distance 0-75 cm (a) was determined by calculating the area of a circle with radius of 75 cm. the area of ring between radial distances 75-150 cm (b) was equal to the area of a circle with a radius of 150 cm minus the area of ring a. similarly the areas of the other rings (c and d) were determined by subtracting consecutively the area of the adjacent smaller circle from the larger one. the volume of soil used to determine the estimated root length at each sampling location was the product of the area of each ring determined by the sampling distance and soil depth (0-15, 15-30 and 30-60 cm) then divided by 8 because only one-eighth of each ring was excavated. fibrous root length density (frld) was determined by dividing the sample fibrous root length for each sampling location by their respective sample soil volume and expressed as cm cm-3. the root length percentage at various depth zones and radial distances from tree trunk was determined on the basis of total root length, irrespective of radial distance and depth respectively. th 34 statistical analysis the experiment was set up in randomized block design with three sets and five replications. the frld and root length percentage of the three rootstocks at various depths and radial distances from the trunk were analysed by one-way anova using duncan’s multiple range test (p< 0.05). 3. results fibrous root length density (frld) was significantly different among the rootstocks. therefore the frlds were pooled and analysed for interaction among rootstocks, soil depths and distances from tree trunk. although the average frld to a 60-cm depth was statistically significant for cleopatra (0.231 cm.cm-3) in respect to rough lemon (0.048 cm.cm-3) and troyer citrange (0.051 cm.cm-3) which were otherwise at par (table 1). a significant interaction of rootstock and depth suggests distinctly different root distribution patterns among the three rootstocks. trees on cleopatra had significantly greater frld than trees on rough lemon and troyer citrange, whereas the frld was not statistically significant between rough lemon and troyer citrange at all soil depths. frlds decreased significantly with every increase in soil depth in rough lemon and cleopatra, whereas with troyer citrange frld was at par between 0-15 and 15-30 cm depth. the maximum frlds (0.067, 0.390 and 0.070 cm.cm-3) were observed in the top 15-cm soil layer in rough lemon, cleopatra and troyer citrange, respectively. a high proportion of fibrous root length (frl) was found in the upper 0-15 cm soil containing 46, 57 and 45% of rough lemon, cleopatra and troyer citrange, respectively, which also did not differ significantly. the proportion of frl differed significantly with every increase in soil depth in rough lemon and cleopatra and at par in troyer citrange at 0-15 and 15-30 cm depth. however trees grown on rough lemon and troyer citrange have more frl (57%) deeper than 15 cm compared with trees grown on cleopatra (43%), resulting in only 45% of rough lemon and troyer citrange root length at more than 15 cm depth. frld and percentage root length of cleopatra differed significantly compared to rough lemon and troyer citrange at all depth zones, whereas rough lemon and troyer citrange were not significantly different. unlike soil depth, distance from trunk had more effect on distribution of fibrous roots among rootstocks (table 2). cleopatra had significantly greater frld at all 75-cm increments in radial distances from trunk in respect to rough lemon and troyer citrange rootstocks. troyer citrange showed significantly more frld (0.173 cm.cm-3) compared to rough lemon (0.129 cm.cm-3) at the 0-75 cm radial distance, whereas for greater radial distances, rough lemon contained significantly more frld compared to troyer citrange. frlds differed significantly with every increase in radial distance in rough lemon and cleopatra while in troyer frlds at 75-150 and 150-225 cm radial distances did not differ significantly. the highest proportion of frl was observed close to the trunk (i.e. 0-75 cm radial distance from trunk) in all the rootstocks. however, troyer citrange showed maximum frl (84%) within 75 cm radial distance whereas, trees table 1 pineapple orange tree mean fibrous root length density (frld) and percentage of root length in the radial distance up to 300 cm of the soil for rootstock and soil depth soil depths (cm) rough lemon frld (cm.cm-3) root length 0-60cm (%) cleopatra frld (cm.cm-3) root length 0-60cm (%) troyer citrange frld (cm.cm-3) root length 0-60 cm (%) 0-15 15-30 30-60 average 0.067 a 0.048 b 0.030 c 0.048 46.19 a 33.24 b 20.56 c 0.390 a 0.210 b 0.093 c 0.231 57.02 a 29.39 b 13.59 c 0.070 a 0.057 a 0.028 b 0.052 45.11 a 36.74 a 17.88 b fibrous root length density (frld) and root length (%) separation by duncan’s multiple range tests. values followed by different letter within a column are significantly different (< 0.05) from other values in the same column. mean (n=5). table 2 pineapple orange tree mean fibrous root length density (frld) and percentage of root length in the upper 60 cm of soil for rootstock and distance from the tree trunk radial distances (cm) rough lemon frld (cm.cm-3) root length 0-300 cm (%) cleopatra frld (cm.cm-3) root length 0-300 cm (%) troyer citrange frld (cm.cm-3) root length 0-300 cm (%) 0-75 75-150 150-225 225-300 0.129 a 0.031 b 0.022 c 0.012 d 66.54 a 15.87 b 11.39 c 6.19 d 0.642 a 0.139 b 0.097 c 0.035 d 70.31 a 15.23 b 10.58 c 3.87 d 0.173 a 0.017 b 0.011 bc 0.004 c 84.20 a 8.26 b 5.37 bc 2.15 c fibrous root length density (frld) and root length (%) separation by duncan’s multiple range tests. values followed by different letter within a column are significantly different (< 0.05) from other values in the same column. mean (n=5). 35 grown on rough lemon and cleopatra, showed 82-85% of frl within 150 cm radial distance. the proportion of frl beyond 75 cm radial distance was for rough lemon and cleopatra at par and significantly more than troyer citrange. all rootstocks differed significantly for frld with every increment in radial distance, but cleopatra and rough lemon did not significantly differ in root length percentage and differ significantly compared to troyer citrange. however, the greatest frld in the top 15-cm depth ranged from 0.08 to 1.05 cm.cm-3 soil at a distance of 300 cm or less for trees on cleopatra, whereas frlds ranged from 0.016 to 0.183 and 0.006 to 0.238 cm.cm3 at the same depth and distance from trees on rough lemon and troyer citrange, respectively (fig. 2). the figure illustrates that the fibrous roots are concentrated closer to the tree trunk (i.e. up to 75 cm radial distance and 0-15 cm depth). beyond the radial distance of 75 cm there was a very sharp decrease in frlds in all rootstocks. the effect is more pronounced in troyer citrange at 0-60 cm depth, followed by cleopatra (1560 cm) and rough lemon (30-60 cm). 4. discussions and conclusions fibrous root density was influenced by depth and distance from trunk and rootstock. however, fibrous root length observed was lower than earlier reports (kaufman et al., 1972; castle, 1980; morgan et al., 2007) which may be due to sampling time in late spring because in citrus root growth is periodic; root activity declines during fall/winter with unfavourable environment and moisture stress condition then a spring growth flush takes place. root activity then increases immediately after the cessation of shoot elongation in summer months. there is a gradual decrease in frld with depth and distance from tree trunk. however the frld’s were highest near the surface and closer to trunk in all rootstocks (castle, 1980; kurien et al., 1991; swietlik, 1992; zhang et al., 1996). cleopatra had more overall fibrous root length compared to rough lemon and troyer citrange, the latter which showed the same intensity. this may be due to differences in their rooting pattern or genetic make-up. cleopatra had more roots (57%) in the upper layer (0-15 cm) compared to rough lemon and troyer citrange (45% each), however cleopatra rootstock showed only 42% fibrous roots between 15-60 cm while rough lemon and troyer citrange contained 54%. hence cleopatra may be classified as shallow rooted. thakur et al. (1981) concluded that citrus is basically a surface feeder. similarly, avilan et al. (1985) reported that most cleopatra roots (80%) were located in the top 30 cm of soil under the canopy of the tree. similar results were reported previously by zhang et al. (1996): root density was greater (75%) at 0-15 cm depth when field is flooded and nitrogen is spread and less than 10% at 30-60 cm depth in grapefruit on sour orange. neves et al. (2004) found that 80% of the roots grow under 31 cm for african rough lemon and more root area was observed at lower horizon of the soil in p. trifoliata and c13 citrange as compared to rough lemon and sunki mandarin for tahiti lime. sharma and chauhan (2005) found in apple nearly all fibrous roots above the 50 cm depth with very few roots between 75100 cm. troyer citrange showed 84% fibrous root closer to tree trunk (0-75 cm) and at higher distance there was a very sharp decrease showing less than 10% at 75-150 cm distance, whereas, rough lemon and cleopatra has 82-85% fr within 150 cm radial distance with less than 10% fibrous root length beyond 225 cm. thus troyer citrange has an intensive lateral root development and cleopatra and rough lemon showed an extensive lateral root system. the maximum root growth in citrus takes place during summer months following rainy season, hence this may cause more lateral and less vertical root development due to the availability of water in the upper layer during active root growth period. in arid climates, higher root density are in irrigated compared to non-irrigated zones and effect fig. 2 changes in fibrous root length density as a function of soil depth from the surface and radial distance from the tree trunk for pineapple orange trees on (a) rough lemon (n=5); (b) cleopatra (n=5) and (c) troyer citrange (n=5) rootstock. r o o t le n g th d en si ty (c m .c m -3 ) r o o t le n g th d en si ty (c m .c m -3 ) r o o t le n g th d en si ty (c m .c m -3 ) 36 of irrigation is closer to tree trunk due to shading effect or lower evaporation under the canopy (bielorai, 1985; roth and gardner, 1985; morshet et al., 1989). furthermore, rough lemon and cleopatra have a dense and large canopy in comparison to troyer citrange, hence the dense and large canopy reduced soil water losses by evaporation forming a favourable environment for root development in the upper layer. secondly, the largest part of roots are formed within the 0-15 cm depth, the most important layer for plant nutrient supply specially, phosphorus that stimulates root growth in layer fertilized with nutrients. troyer citrange showed a reduced and somewhat upright growth of canopy hence more moisture loss under the canopy took place which make roots to divert to lower horizon for water uptake. these results are in accordance with those of misra et al. (2003) in grape fruit budded on trifoliate orange. carrizo citrange rootstock has intensive type root system and less lateral development in hamlin rootstock (castle and krezdorn, 1975; morgan et al., 2007). similarly castle (1980) and cintra et al. (1999, 2000) found that rough lemon and cleopatra have large root system and rough lemon extensive lateral and vertical development. kurien et al. (1991) reported that most root activity (75-80%) was confined within a radius of 80 cm and 24 cm in depth in acid lime on karna khatta (citrus karna) in this study, we have observed that frld distribution of pineapple orange trees grown on rough lemon, cleopatra and troyer citrange rootstocks decreased with soil depths and lateral distances. the overall maximum frld was recorded in cleopatra at all the depths and radial distances. the density of feeder roots was concentrated at a depth of 0-15 cm within 75 cm radial distance. trees grown on troyer citrange and rough lemon showed an appreciable amount of fr up to 60 cm in depth and may be classified as plants with an extensive vertical root development, whereas, in cleopatra and rough lemon a noticeable amount of fr is confined up to 225 cm radial distance and hence can be considered as extensive lateral development. trees on troyer citrange have fr very closer to tree trunk (0-75 cm) i.e. intensive lateral roots. cleopatra showed more roots in the upper soil layer (0-15 cm) and it can be considered as upper intensive root development. therefore depth of irrigation and placement of fertilizer based on root distribution should be rootstock specific and deep ploughing should be avoided. references avilan r.l., menesses l., sucre r., 1985 root distribution system in fine textured soil on cleopatra rootstock budded with valencia orange. agronomia tropical, 33: 509-534. bielorai h., 1985 moisture, salinity and root distribution of drip irrigated grapefruit. third intl. drip/trickle irr. congr., 2: 567573. boman b., levy y., parsons l., 1999 water management, pp. 72-81. in: timmer l.w. and l.w. duncan (eds.) citrus health management. aps press, st. paul, mn, usa. castle w.s., 1980 fibrous root distribution of ‘pineapple’ orange trees on rough lemon rootstock at three tree spacing. j. amer. soc. hort. sci., 105: 478-480. castle w.s., krezdorn a.h., 1975 effects of citrus rootstocks on root distribution and leaf mineral contents of ‘orlando’ tangelo tree. j. amer. soc. hort. sci., 100: 1-4 cintra f.l.d., libardi p.l., jorge l.a.c., 1999 distribuicao do sistema radicular de porta-enxertos de ecossistema de tabuleiro costeiro. revista brasileira de fruticulture, 21: 313-317. cintra f.l.d., libardi p.l., saad a.m., 2000 balanco hidrico no solo para porta-enxertos de citros em ecossistema de tabuleiro costeiro. revista brasileira de engenharia agricolae ambiental, 4: 23-28. eissenstat d.m., whaley e.l., volder a., wells c.e., 1999 recovery of citrus surface roots following prolonged exposure to dry soil. j. exp. botany, 50: 1845-1854. hipondoka m.h.t., aranibar j.n., chirara c., lihavha m., macko s.a., 2003 vertical distribution of grass and tree roots in arid ecosystem of southern africa: niche differentiation or competition? j. arid environ., 54: 319-325. kaufmann m.r., boswell s.b., lewis l.n., 1972 effect of tree spacing on root distribution of 9-years old ‘washington’ navel oranges. j. amer. soc. hort. sci., 97: 204-206. kramer p.j., boyer j.s., 1995 water relations of plants and soils. academic press, ny, usa. kurien s., goswami a.m., deb d.l., 1991 the distribution of root activity in acid lime (citrus aurantifolia). exp. agri., 27: 431-434. mikhail e.h., el-zefhoui b.m., 1979 effect of soil types and rootstocks on root distribution, chemical composition of leaves and yield of valencia oranges. australian j. soil res., 17: 335342. misra k.k., singh n., jaiswal h.r., 2003 studies on root distribution pattern of grapefruit (citrus paradisi macf) budded on trifoliate orange rootstock. indian j. hort., 60: 49-52. morgan k.t., obreza t.a., scholberg j.m.s., 2007 orange tree fibrous root length distribution in space and time. j. amer. soc. hort. sci., 132: 262-269. morshet s., cohan y., fuchs m., 1989 water use and yield of a mature ‘shamouti’orange orchard submitted to root volume restriction and intensive canopy pruning. proceeding of international society of citriculture, 2: 739-746. neves c.s.v.j., murata i.m., stenzel n.m.c., medina c.de c., borges a.v., okumoto s.h., lee r.h.c., kanai h.t., 2004 root distribution of rootstocks for ‘tahiti’ lime. scientia agricola, 61: 94-99. roth r.l., gardner b.r., 1985 root distribution of mature orange trees irrigated by pressurized systems. proceeding of third international drip/trickle irrigation congress, 2: 579-586. scholberg j.m.s., parsons l.r., wheaton t.a., mcneal b.l., morgan k.t., 2002 soil temperature, nitrogen concentration and residence time affect nitrogen uptake efficiency of citrus. j. environ. quality, 31: 759-768. sharma d.d., chauhan j.s., 2005 effect of different rootstocks on root distribution of apple. acta horticulturae, 696: 167-171. swietlik d., 1992 yield, growth and mineral nutrition of young ‘ray ruby’ grapefruit trees under trickle or flood irrigation and various nitrogen rates. j. amer. soc. hort. sci., 117: 22-27. thakur r.s., rajput m.s., srivastava k.k., 1981 root distribution studies in some fruit crops with special reference to tracer technique. a review. haryana j. hort. sci., 10: 45-53. tinker p.b., nye p.h., 2000 solution movement in the rhizosphere. oxford university press, ny, usa. zhang m., alva a.k., li l.c., calvert d.v., 1996 root distribution of grapefruit trees under dry granular broadcast vs. fertigation method. plant and soil, 183: 79-84. 3 1. introduction buriti (mauritia flexuosa l.) is a palm tree found from the atlantic forest to the cerrado (a vast tropical savanna ecoregion) of the brazilian north, northeast and mid-west in the state of minas gerais (manzi and coomes, 2009). it also extends to the state of mato grosso, as well as bolivia, colombia, ecuador and peru. there is debate between brazilian and peruvian scientists about its origin (cavalcante, 1991). female buriti palm trees produce four to eight infructescences, and each raceme bears 500-2000 fruits (goulding and smith, 2007). the fruit is a reddish-brown drupe, with a thin oily yellow-orange pulp that surrounds a relatively large seed (manzi and coomes, 2009). the oil contains tocopherols (de frança et al., 1999; albuquerque et al., 2005), carotenoids (mariath et al., 1989; silva et al., 2009) and pro-vitamin a (mariath et al., 1989; klemm et al., 2008). moreover, candy made from buriti is an effective treatment of xerophthalmia in children in northeastern brazil, but the fruit used to make it is not easily preserved. it is good for no more than two to three days under ambient conditions. the buriti fruit has a hard, red shell that covers an oily pulp that contains carotenoids and ascorbic acid (embrapa, 2007; silva et al., 2007). the local population collects the fruits when they are released from the mother plant and most of the fruits on the ground are near the desired state of maturity. a 55-kg bunch produces 40 kg of fruits. the local population collects the fruits that they are going to use from the ground. buriti and products made from it are widespread in the brazilian cerrado. the fruit pulp is used to make a flour for a porridge that helps meet the nutritional requirements of the locals (almeida and silva, 1994). the fruit is also a source of vegetable oil, as described by albuquerque et al. (2003). these authors obtained an ir spectrum of the oil that revealed the presence of triolein, the triglyceride of oleic acid, which could be used to control cholesterol in the blood. the oil has been reported to have a relatively high concentration of so-called monounsaturated fatty acids (de frança et al., 1999), although it really contains fatty acyls that are a part of mono-, diand triglycerides. that is, the oil had to be hydrolyzed to break down the mono-, diand triglycerides, forming glycerol plus non-volatile free fatty acids, which are then converted to volatile fatty acid methyl esters and analyzed by gas chromatography (de frança et al., 1999; aoac, 2003). in order for the fruits to be used, they must be preserved properly. this is discussed in the next paragraph. usually the fruits are collected in a form that is not easily preserved. the palm trees are cut to facilitate the removal of the fruits, drastically reducing the population of buriti trees in the amazonian region of peru. this forces the harvesters to travel long distances to gather a significant quantity. the management of buriti via extractavism is small compared to the demands of the regional market (manzi and coomes, 2009; horn et al., 2012). extractavism refers to natural tropical forest areas that are reserved for the extraction of potentially renewable commercial forest products. so, better storage methods are needed to meet the needs of the market and provide social benefits, which are described next. buriti offers social benefits to the local population as a source of wealth and employment in the manufacture of products such as licorice, wine, candy, juice and sorbets. refrigerated storage of the fruits of buriti (mauritia flexuosa l.) e. fujita*, r.l. vieites*, é.r. daiuto*, r.e. smith**(1) * unesp botucatu, faculty of agronomic sciences, unesp, c.p. 237, botucatu 18610307, sp, brasil. ** fda, 11510 w 80th street, lenexa, ks 66224, kansas, usa. key words: brazilian cerrado, buriti, mauritia flexuosa, post-harvest. abstract: the objective of this study was to evaluate different storage conditions to maximize the shelf-life of buriti fruits; under ambient conditions the fruits last only 2-3 days. buriti fruits were stored refrigerated at 10, 12 and 15oc with 85±5% relative humidity, and at room temperature (23±5°c) and 60±5% relative humidity. fruits were analyzed every three days over a 12-day period for weight loss, respiratory activity, soluble solids, ph, titratable acidity, lipids, protein and fiber. under the considered conditions, refrigeration at 15oc was found to give the best results. adv. hort. sci., 2014 28(1): 3-8 (1) corresponding author: robert.smith@fda.hhs.gov received for publication 16 september 2013 accepted for publication 10 december 2013 4 however, these activities have not been given the technical or scientific support needed to make them sustainable and more profitable. while an adequate storage method has not yet been well defined, it is known that the fruit must be scraped off the seeds and dehydrated at room temperature, followed by refrigeration for an undetermined period of time (almeida and silva, 1994). therefore, the objective of this study was to evaluate different storage conditions for buriti fruits in order to find the best way to maximize shelf-life with attention to the post-harvest behavior of the fruits so as to maintain their quality. 2. materials and methods the fruits used in the study were from the ecological preserve in jalapão, near the city of dianópolis in the state of tocantins at 11°37’40” south latitude and 46°49’14” west longitude at an altitude of 691 m above sea level, where the trees grow in sandy soil. the fruits were picked from the trees when they were 3-4 cm in diameter and dark yellow to brown. the trees were not cut. due to the height of the tree (10 m), a scaffolding was erected so that the bunches of fruit could be removed with the utmost caution, to prevent damage caused by falling. the bunches were placed in a ventilated polystyrene box to maintain a temperature of 16°c. after collection, the fruits were sent to the laboratório de frutas e hortaliças do departamento de gestão e tecnologia agroindustrial da faculdade de ciências agronômicas – unesp, botucatu, são paulo, where they arrived after two days. they were separated randomly into four lots, each containing ten fruits. three were stored under refrigeration at 10, 12 and 15°c and 85±5% relative humidity. the fourth lot (control) was stored under ambient conditions (23±5°c and 60±5% relative humidity). the following analyses were carried out on ten fruits for each storage condition: weight loss, respiratory activity, soluble solids, titratable acidity, and ph. analyses were carried out for fruits that were viable for commercialization. respiratory activity was determined by the release of co 2 in each package according to the method of bleinroth et al. (1976), using a saturated solution of barium hydroxide and 0.1 n koh (0.1 normal koh, which is the same as 1 mol/l koh) and using the formula: tco 2 = 2.2 (vo-v 1 ) 10 m t where: tco 2 is the rate of respiration (ml of co 2 kg-1h-1); vo = ml of hcl needed to titrate the koh as a standard before the absorption of co 2 ; v 1 = ml of hcl needed to titrate the koh after the absorption of co 2 ; m = mass of the fruits; t = respiration time; 2.2 = equivalent weight of co 2 (44/2), multiplied by the concentration of hcl; 10 = adjustment for the total amount of koh used. soluble solids, ph and titratable acidity were determined by the method of the instituto adolf lutz (ial, 2008). soluble solids were measured with a digital palette pr 32 refractometer (atago inc., bellevue, wa), equipped with automatic temperature compensation. results were expressed directly in obrix. the ph was measured with a ph meter and titratable acidity was measured by titrating the acidic fruits with 0.1 mol/l naoh. the amount of total sugars, lipids, proteins and fibers were determined using the method of somogyi (1945) and nelson (1944) by reacting samples with the somogyi reagent and measuring the absorbance at 535 nm using a micronal b382 spectrophotometer (micronal, são paulo, sp, brazil). in detail, samples were diluted sufficiently so that an absorbance between 0.2 and 0.8 was produced after reacting 1 ml of neutralized and filtered sample with 1 ml of somogyi reagent. after putting the samples in a boiling water bath for 10 min, they were cooled to room temperature. then, 1 ml of the nelson reagent and 7 ml of water were added. finally, the absorbance at 535 nm was read. the somogyi reagent is an arsenomolybdate complex formed by the reaction of ammonium molybdate with sodium arsenate. the nelson reagent was made of two parts: part a contained 2.5 g each of na 2 co 3 and potassium sodium tartrate, 2 g each of nahco 3 and na 2 so 4 in 100 ml water. part b contained 7.5 g cuso 4 ·5h 2 o per 100 ml water, acidified with a drop of conc h 2 so 4 . glucose was used to construct a calibration curve. total lipids were determined by performing a 2-h soxhlet extraction on 3 g of sample using 200 ml of petroleum ether, evaporating off the solvent and weighing the residue. protein was determined on 0.1 to 0.2 g of sample using the kjeldahl method using a conversion factor of 6.5 to convert percent nitrogen to percent protein. the experimental data were analyzed as a 4x5 matrix (temperature x time) by the sisvar 4.6 program. averages were evaluated by the tukey test at 5% probability (gomes, 1987). 3. results and discussion beginning on the third day of storage, a weight loss of >10% was found in all storage conditions. the fruits stored at 10 and 23oc had the greatest loss of mass after 12 days. the lowest loss was at 15oc (table 1). all weight losses were calculated by comparing weights to day zero. ten fruits were analyzed in each experiment. according to finger and vieira (2002) the weight loss of most fresh fruits should be 5-10% to avoid withering or wrinkling. thus, buriti fruits examined suffered a weight loss that reduced their commercial value. 5 buriti fruits demonstrated respiratory behavior that is characteristic of climacteric fruits, as shown in table 2. according to chitarra and chitarra (2005) they are characterized by a rapid increase in respiration and ethylene production during ripening. the apparent peak at day three of storage was probably due to an adaptation of the fruits to the storage conditions. the fruits that were kept at ambient temperature had their peak respiration on the sixth day of storage, while the fruits stored under other conditions had their peak respiration on the ninth day of storage. the lowest respiratory activity (lowest production of co 2 ) was found in fruits stored at 10ºc, i.e. with the lowest production of co 2 . therefore, a temperature of 15ºc proved to be the most effective for storing the fruits as it resulted in the lowest loss of weight and the latest peak in respiratory activity. the amounts of soluble solids, ph and titratable acidity are shown in table 3. table 1 weights of buriti when stored refrigerated temperature initial wt. (g) 3 days (g) 6 days (g) 9 days (g) 12 days (g) 10°c 263.03±7.81 a 238.43±5.74 a 214.50±6.78 a 197.95±3.98 a 191.60±2.45 b 12°c 256.32±6.82 a 237.31±4.81 a 222.10 ±4.91 c 212.56±4.89 c 208.59± 3.99 c 15°c 261.23±5.73 a 245.60±5.97 b 233.65±5.60 d 226.13±6.71 d 223.12± 4.58 d ambient 274.75±6.99 b 251.80±6.52 c 229.53±4.57 b 207.65± 5.86 b 194.47±4.98 a averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. table 2 respiratory activity in buriti fruits stored under refrigeration and 80 ± 5% relative humidity for 12 days respiratory activity temperature days of storage 0 3 6 9 12 10°c 24.5±0.64 35.1±1.90 18.5±0.99 35.5±0.42 13.8±0.64 12°c 24.5±0.64 12.1±0.14 17.3±0.42 58.6±2.62 44.2±3.11 15°c 24.5±0.64 31.4±0.35 17.9±0.78 70.9±0.28 36.8±0.64 ambient 24.5±0.64 54.2±0.28 55.6±0.07 50.8±0.35 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. table 3 soluble solids (°brix), ph and titratable acidity (g of acid 100g-1 fresh weight) in buriti fruits under refrigerated storage (80±5% relative humidity) and ambient conditions (23±5ºc and 60±5% relative humidity) temperature days of storage 0 3 6 9 12 soluble solids 10°c 12.37±1.51 ab 14.67±0.58 aab 16.33±2.52 aa 15.00±1.73 aab 16.00±0.00 aa 12°c 12.37 ±1.51 aa 14.67±0,58 aa 14.00±0 aba 13.66±0.58 aa 14.67±1.53 aa 15°c 12.37 ±1.51 ab 14.67±1.53 aab 13.33 ±2.08 bcab 12.67± 0.58 ab 16.00 ±0.0 aa ambient 12.37±1.51 aa 10.33±0,58 bb 10.80±0.00 cb c.v. (%) 9.37 ph 10°c 3.83± 0.06 ac 3.77± 0.58 abc 4.33±0.15 aab 4.57±0.06 aa 4.1±0.00 abb 12°c 3.83 ±0.06ac abc 3.73±0.58 abc 4.07 ±0.15 bab 3.93± 0.23 bbc 4.23±0.25 aa 15°c 3.83± 0.06ac abc 3.67±1.53 bc 4.13±0.06 aba 4.00±0.10 bab 3.97±0.12b ab ambient 3.83±0.06ac ab 3.90±0.58 aab 4.10 ±0.0 ba c.v. (%) 2.63 titratable acidity 10°c 0.68±0.06 ab 0.84±0.02 aa 0.41±0.02 ac 0.47±0.07 bc 0.60±0.00 ab 12°c 0.68 ±0.06 ab 0.84±0.02 aa 0.51 ±0.03 ac 0.60 ±0.04 abc 0.52±0.05 ac 15°c 0.68 ±0.06 aab 0.76 ±0.02 aa 0.49±0.02 ac 0.58±0.04 abbc 0.60 ±0.01 aab ambient 0.68± 0.06 aa 0.63±0.02 ba 0.42±0.00 ab c.v. (%) 9.54 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. 6 the amount of soluble solids did not show a significant difference when stored at 10, 12 or 15°c. the fruits stored under ambient conditions exhibited a decrease in soluble solids on day three, but it dropped no further on day six. on the ninth day, contamination by the fungus monilinia fructicola was observed and this made the fruits unsuitable for consumption. the fruits stored under ambient conditions maintained a relatively low amount of soluble solids during the experimental period, possibly due to the lower respiratory rate compared to refrigerated storage. the small increase in the concentration of soluble solids at the end of the experiment could be related to the weight loss. on the third day of storage, there was a tendency for the ph to increase, as shown in table 3. in the case of ambient storage, this can be due to the process of senescence (chitarra and chitarra, 2005). the increase in ph was seen throughout the experiment. when stored at 10°c, the ph was nearly constant on day three, but increased on days six and nine, followed by a decrease on day 12. when stored under ambient conditions, the ph increased until the last day of analysis. the titratable acidity increased on day three under all storage conditions, followed by a decrease on subsequent days. ambient storage presented the lowest amount of titratable acidity, probably due to infestation by pathogens that consumed acid in their metabolism (chitarra and chitarra, 2005; özcan and haciseferogullari, 2007). the data on total sugars, lipids and protein are presented in table 4. albuquerque et al. (2005) reported levels of total sugars in buriti that varied by about 2.10%, similar to what we found. moreover, hiane et al. (1992) reported values of 11.36% ± 1.81, which are higher than those found in the present experiment. the amount of total sugars increased from day zero to the third day of storage, followed by a decrease on the sixth day and increases on subsequent days. carbohydrates are oxidized by the respiratory process (chitarra and chitarra, 2005; rodriguez-guisado et al., 2009), causing the decrease. the increase in concentration on later days was probably related to the loss of weight. according to cavalcante (1991), buriti fruit contains a relatively large amount of lipids, which are an important source of energy. this was also reported by de frança et al. (1999), albuquerque et al. (2005), and silva et al. (2009), rodrigues et al. (2010). however, all these authors reported finding free fatty acids, when they were most likely fatty acyls as a part of mono-, diand triglycerides. table 4 amounts of total sugars, lipids, protein and fiber (%) in buriti fruits under refrigerated storage (80±5% relative humidity) and ambient conditions (23±5ºc and 60±5% relative humidity) for 12 days temperature storage days 0 3 6 9 12 total sugars 10°c 2.22 ab 3.25 aa 0.91 ac 1.29 bc 2.72 aab 12°c 2.22 ab 2.93 aa 0.80 ac 2.69 aab 2.42 aab 15°c 2.22 ab 3.09 aa 0.83 ac 2.40 ab 2.68 aab ambient 2.22 ab 3.07 aa 0.96 ac c.v. (%) 12.64 lipids 10°c 14.00 ac 18.67 ab 18.53 ab 14.80 cc 21.00 aa 12°c 14.00 ac 18.13 ab 17.30 abb 18.23 ab 21.33 aa 15°c 14.00 ac 15.67 bb 16.93 abb 16.70 bb 20.47 aa ambient 14.00 ab 13.30 cb 18.00 ba c.v. (%) 3.65 protein 10°c 0.26 ad 0.26 ab 0.35 ab 0.37 ac 0.33 aa 12°c 0.26 ac 0.25 ab 0.21 cb 0.29 bb 0.31 ba 15°c 0.26 ab 0.21 bb 0.35 ab 0.27 bb 0.26 ca ambient 0.26 ab 0.22 bb 0.29 ba c.v. (%) 2.75 fiber 10°c 10.43 ac 10.73 abc 10.10 bc 11.30 ab 13.00 aa 12°c 10.43 ac 9.60 bd 10.37 abcd 11.70 ab 12.67 aa 15°c 10.43 ac 8.50 cd 10.90 ac 11.53 aab 11.83 ba ambient 10.43 aa 8.70 cb 10.50 aba c.v. (%) 3.37 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. 7 this is a common mistake which is made when fatty acyl amounts are determined by gas chromatography (gc) only after hydrolyzing the glycerides. this forms free fatty acids, which are then esterified to form fatty acid methyl esters (fames) that are volatile enough to be analyzed by gc. therefore, a clever marketing or sales representative from producers of a competing company could say that all these studies demonstrate that buriti fruits rapidly turn rancid, since free fatty acids were supposedly found. when oils (glycerides) turn rancid, it is due to partial hydrolysis of glycerides to form malodorous free fatty acids. the article by silva et al. (2009) is especially confusing, since it reports actually finding 3.1% free fatty acids in buriti oil, based on their separation by size exclusion chromatography: it is not clear whether the authors really meant free fatty acids or fatty acyls that are part of triglycerides. they go on to report the profile of free fatty acids, but it is based on gc analysis of fames, so it refers to the fatty acyl profile of mono-, diand triglycerides. vegetable oils contain mono-, diand triglycerides, and therefore the analysis method requires that they be hydrolyzed into free fatty acids and glycerol, followed by forming volatile fatty acid methyl esters (fames), which can be analyzed by gas chromatography (mannina et al., 1999; aoac, 2003). it is not clear whether the 3.1% free fatty acids separated by size exclusion chromatography were part of glycerides or if they were truly free fatty acids, caused by the oil turning rancid. however, the present experiment truly measured total lipids and found more than the 2.5 to 5.5% reported by hiane et al. (1992) and donadio et al. (2002). albuquerque et al. (2005) reported finding 11.24% lipids. carneiro and carneiro (2011) found 18.16% lipids in buriti pulp. the amount of lipids increased starting on the third day of storage and stayed almost constant throughout the 12-day experiment (table 4). on the third day, the fruits stored at 10 and 12°c showed no significant differences, but more lipids were found when stored at 15°c. the fruits stored under ambient conditions had fewer lipids that the fruits stored under refrigeration. on day 12 there were no significant differences in lipid content in the refrigerated samples. the increase in lipid concentration with time could be related to the weight loss that occurred. according to donadio et al. (2002) buriti fruits had about 2.3 to 5.5% protein, while hiane et al. (1992) found 2.12%, carneiro and carneiro (2011) found 1.30% and darnet et al. (2011) found 3.7%. in the present study, there were no significant differences in protein concentrations in fruits stored at 10 and 12°c, but these values were higher than those of fruits stored at 15°c and under ambient conditions, which were not statistically different from each other. on the sixth day of storage, there was an increase in protein concentration, with the exception of those stored at 12°c. on the ninth day, the concentration of protein increased in the fruits stored at 10 and 12°c, while those stored at 15°c showed a decrease. donadio et al. (2002) found that the fiber content in buriti fruits varied from 10.4 to 27.5%. hiane et al. (1992) reported about 12.31% fiber. darnet et al. (2011) found 22.8% dietary fiber in buriti fruits from the amazon. the concentrations of fiber found in the current study ranged from 8.5 to 13.0%: on the third day, the fruits stored at 10°c had the highest concentration of fiber, followed by those stored at 12°c; those stored at 15°c and under ambient conditions had the lowest concentrations and were not significantly different from each other. starting on the third day there was an increase in the concentration of fiber which then continued slowly throughout the 12-day experiment. the increase could be simply due to the loss of weight, so the total amounts were about the same. 4. conclusions refrigeration was effective in extending the shelf-life of buriti fruits, increasing it by at least three days. the data presented regarding the decrease in weight and respiratory activity demonstrate that a temperature of 15°c was the most effective in maintaining the quality of buriti fruits. this work should not be taken as reflecting fda policy or regulation. references albuquerque m.l.s., guedes i., alcantara p. jr., moreira s.g.c., 2003 infrared absorption spectra of buriti (mauritia flexuosa l.) oil. vibrat. spectroscopy, 33: 127-131. albuquerque m.l.s., guedes i., alcantara p. jr., moreira s.g.c., barbosa neto n.m., correa d.s., zilio s.c., 2005 characterization of buriti (mauritia flexuosa l.) oil by absorption and emission spectroscopies. j. braz. chem. soc., 16(6): 113-117. almeida s.p., silva j.a., 1994 piqui e buriti importância alimentar para a população dos cerrados. empresa brasileira de pesquisa agropecuária do brasil (embrapa cpac), planaltina, df, brasil, pp. 25-34. aoac, 2003 aoac official method 969.33. fatty acids in oils and fats. aoac international gaitherburg, md, usa. bleinroth e.w., zuchini a.g.r., pompeo r.m., 1976 determinaçâo das características fisícas e mecânism de variedades de abacate e a su conservaçâo pelo frio. coletânea do instituto de tecnologia de alimentos, 7(1): 29-81 carneiro t.b., carneiro j.g.m., 2011 frutos e polpa desidratada buriti (mauritia flexuosa l). rev. verde de agroecol. desenvolv. sustent., 6(2): 105-111. cavalcante p.b., 1991 frutas comestíveis da amazônia 5º ed. ver. ed. cevup, belém, brasil, pp. 168-171. chitarra m.i.f., chitarra a.b., 2005 pós-colheita de frutos e hortaliças: fisiologia e manuseio. esal/faepe, lavras, mg, brasil, pp. 785. darnet s.h., da silva l.h.m., rodrigues a.m.c., lins r.t., 2011 nutritional composition, fatty acid and tocophenol contents of buriti (mauritia flexuosa) and patawa (oenocarpus bataua) fruit pulp from the amazon region. ciênc. tecnolog. alimen., 31(2): 488-491. 8 de frança l.f., reber g., meireles m.a.a., machado n.t., brunner g., 1999 supercritical fluid extraction of carotenoids and lipids from buriti (mauritia flexuosa), a fruit from the amazon region. j. supercrit. fluid., 14: 247-256. donadio l.c., morô f.v., servidone a.a., 2002 frutas brasileiras. editora novos talentos, jaboticabal, sao paulo, 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a.j.a., 2009 characterization of oil extracted from buriti fruit (mauritia flexuosa) grown in the brazilian amazon region. j. amer. oil chem. soc., 86(7): 611-616. somogyi m., 1945 determination of blood sugar. j. biol. chem., 160: 69-73. 115 1. introduction mangosteen (garcinia mangostana l.), the queen of tropical fruits (almeda and martin, 1976; ibpgr, 1986; kusumo and verheij, 1994), is a very important crop of the warm humid tropics. the crop like other polyaxial tropical and sub tropical trees such as rubber, mango, cashew and citrus, exhibits a rhythmic growth habit under the relatively constant environmental conditions of the tropics (alvim, 1964; borchert, 1973). it bears profusely and fits very well as a component of home gardens of kerala. though it is a fruit crop with immense potential, both as a monocrop and as a mixed crop in coconut gardens with very high domestic and foreign demand, its cultivation on a commercial basis is limited by its long gestation phase (wiebel et al., 1992 b). the apomictic mangosteen (richards, 1990; normah et al., 1992) seedlings used for commercial planting are extremely slow growing, both at nursery stage and in orchards (hume, 1947; almeda and martin, 1976; wiebel et al., 1991, 1992 a). the slow growth rate and consequent long pre-bearing phase have been a cause for concern wherever mangosteen is grown. though the long gestation period, ranging from 10-15 years (lim, 1984; richards, 1990; wiebel et al., 1995), can be reduced by resorting to vegetative propagation, the problem of slow growth becomes all the more conspicuous (wiebel et al., 1992 b, 1995). this crop lacks root hair (richards, 1990) and this reduced vital link responsible for absorption of nutrients and water may be one of the prime reasons for slow growth. low carbon acquisition capacity and prolonged dormancy of buds at the apex have also been listed as probable causes of the slow growth rate (downton et al., 1990). a critical review of the literature has revealed that only very few references to the crop exist. selection index studies are available in palms but the aims differ as the thrust of the present work is directed toward the growth rate. this study was undertaken with the objectives of identifying some of the basic reasons for the slow growth rate and, secondly, to address this problem by identifying the mother plant, fruit and seed characters in relation to seedling growth and formulating the selection indices at these three basic levels. accelerating the growth rate of mangosteen trees and thereby reducing the gestation period is one of the pre-requisites for an extensive commercialization of this crop. preliminary studies on selection indices for activating seedling growth in mangosteen (garcinia mangostana l.) l.m. yusuf, s. kurien(*) department of pomology and floriculture, kerala agricultural university, kau post, thrissur, kerala, 680656 india. key words: d2 values, dendrogram, discriminant function analysis, multiple regression analysis, principal component analysis, selection indices, garcinia mangostana. abstract: studies on selection indices for activating seedling growth in mangosteen were conducted in the central orchard at the main campus of kerala agricultural university. the present investigation was undertaken with the main aim of identifying some of the basic reasons for slow growth in mangosteen, and to address this problem by developing and identifying criteria to select the age of the mother plant, fruit, seed and seedling characters or direct selection indices at all four stages with respect to seedling growth. mother plants of four distinct age groups were used in the study. variables were generated using all fruit, seed and seedling characters such as fruit index, seed index and seedling index by principal component analysis (pca). using pca and multiple regression analysis, prediction models was fitted for the three indices. major fruit, seed and seedling characters were identified by stepwise regression. hierarchial analysis was performed based on euclidean distance to find the similarities between the four age groups. discriminant function analysis was performed and six discriminant functions were fitted with corresponding d2 values to discriminate the six pairs involving the four age groups of the mother plants. for practical purposes, selection indices and best age group of mother plants are described in the work. adv. hort. sci., 2012 26(3-4): 115-130 (*) corresponding author: sajanalice@gmail.com. received for publication 21 february 2012 accepted for publication 12 september 2012 116 2. materials and methods the materials and methodologies used in this investigation are presented separately. identification of mother plants belonging to various age groups the first experiment on fixation of a selection index of mangosteen (garcinia mangostana l.) was based on the age of the mother plant and on fruit, seed and seedling characters. hence a preliminary survey was conducted in the kerala mangosteen growing tracts during the bearing phase ending with the onset of southwest monsoon. the main aim was to identify plants belonging to the different age groups as envisaged in the study. the required number of plants belonging to different age groups could have been located in different regions, but the study was restricted to pariyaram village of chalakudi taluk as age groups and numbers were available in a compact area. hence the mother plants and fruit collection were centered on this area. furthermore this approach aided in eliminating possible errors from an ecological point of view to the minimum. location of mother trees (pariyaram village) pariyaram village is located at 10° 20’ n latitude and 76° 26’ e longitude, at an altitude of 3.25 m above mean sea level and 5 km (east) of the chalakudi railway station, thrissur district. climate and soil conditions of the area the area receives an average rainfall of 2150 mm distributed over a period of a year. the mean maximum temperature ranges 28-36°c with a mean of 32°c. the mean minimum temperature ranges 12.8 to 20.6°c with a mean of 16.7°c. the relative humidity ranges from 90 to 98% with a mean of 94%. the soil of the area where the mother trees are located is sandy alluvium with ph 5.5 -5.8 and belonging to the order of entisol. the soil is low in available n and p 2 o 5 and high in available k 2 o. once fruit collection and observations were completed, the studies on seed germination and observations on seedling characters were carried out in the central orchard attached to the department of pomology and floriculture, the college of horticulture in the main campus of kerala agricultural university, vellanikkara, thrissur district, kerala. the experiment site experiences a warm humid tropical monsoon climate. it is situated at 12° 32’n latitude and at 74° 20’e longitude at an altitude of 22.5 m above mean sea level. soil type is a typical sandy clay loam with ph 5.4, ec 1.25 dsm-1, and belonging to the order ultisols. mother plant characters trees belonging to various age groups were identified and categorized into the four age groups as envisaged in this investigation: 1. trees having an age of less than 25 years; 2. trees having an age of 25-50 years; 3. trees having an age of 51-75 years; 4. trees having an age of more than 75 years. within each age group, ten fruits per tree were collected from five mother plants randomly, so that each age group had a total of fifty fruits. care was taken to see that the mother plants under selection fell near the mid value of each age group and so that the five plants in a group were more or less uniform in growth characters (height and spread). fruits were labeled individually and seeds were extracted from the fruits. the seeds were sown separately in black polythene bags (45x30 cm) filled with potting mixture made up of farmyard manure, sand and cow dung in the ratio of 2:2:1. the following observations on various fruit, seed and seedling characters of the four age groups (table 1) were recorded separately as presented below. fruit characters fruit weight, fruit girth, fruit volume, pulp weight, rind weight, number of segments, number of seedless fruits, number of one-seeded fruits, number of two-seeded fruits, number of threeor more seeded fruits, number of seedlings produced per fruit and seed specific gravity were recorded. seed characters observations on seed weight, seed length, seed thickness at centre, seed volume, seed specific gravity, number of seeds per fruit, number of seedlings per seed, number of ungerminated seeds, number of seeds producing one seedling, number of seeds producing two or more than two seedlings (table 2), number of days taken to germination, and germination rate were recorded based on ista guidelines (ista, 2003). seedling characters height, girth at collar, total number of leaves, and survival rate of the seedlings at three months, six months, nine months and one-year stage after germination were recorded. increment in height, girth, and total number of leaves at quarterly intervals up to one year were computed. number of new flushes per year, number of leaves/flush, and total leaf area were limited to a twelve-month stage. shoot and root characters shoot and root weight, dry weight, root to shoot dry weight ratio, length of longest root, number of primary, secondary and tertiary roots, and total number of roots were recorded. based on the fruit index, seed index and seedling index the selection indices were determined. biochemical analysis the following biochemical characters were estimated in seeds and leaves of the mother plants and seedlings of the four age groups. however biochemical analyses were restricted to the treatments showing the growth of best, in117 termediate and least categories, in the experiments of activation of seedling growth using growth regulators and a m fungi separately. 1. nitrogen seeds and leaves 2. phosphorus seeds and leaves 3. potassium seeds and leaves 4. protein seeds and leaves 5. sodium seeds and leaves 6. chlorophyll leaves only 7. total sugar content seeds only 8. total carbohydrates seeds and leaves 9. total phenols seeds and leaves 10. abscisic acid seeds and leaves selection index for this experiment, fully matured (dark purple colour) fruits were harvested randomly and the following observations were taken. fruit characters individual fruits were labeled immediately after harvest. fruit weight was then recorded using an electronic balance (contech precision balance) and the average expressed in grams. girth of the fruit was measured using a thread and its length measured using a metre scale and the average expressed in centimeters. fruit volume was measured by water displacement method and the average volume expressed in milliliters. the fruit hull was carefully removed and the weight of the white-segmented pulp (aril) of each fruit with seeds was measured using an electronic balance. seeds were extracted and their weight recorded and the average expressed in grams. pulp weight of each individual fruit alone was calculated using the formula: pulp weight alone (g) = pulp weight with seed (g) seed weight alone (g) the rind weight of each individual fruit was calculated using the following formula and the average expressed in grams: rind weight (g) = total fruit weight (g) pulp weight with seed (g) numbers of white juicy segments were counted immediately after the fruits were opened and the average table 1 latent vectors and variance of the principal component analysis performed for generating fruit, seed and seedling indices separately in mangosteen (garcinia mangostana l.) sl. no. characters principle component 1 principle component 2 fruit characters 1 fruit weight 0.62 0. 14 2 fruit volume 0.62 0 .14 3 fruit girth 0.02 0.00 4 no. of segments 0.00 0.00 5 pulp weight 0.15 0.16 6 rind weight 0.47 -0.62 7 fruit specific gravity 0.00 0.00 8 number of seedless fruits 0.00 0.00 9 number of one seeded fruits 0.00 -0.01 10 number of two seeded fruits 0.00 0.0 1 11 number of three and multi seeded fruits 0.00 0.01 12 number of seedlings/fruit 0.00 0.04 cumulative variance 96.27 98.68 seed characters 1 seed weight 0.00 -0.07 2 seed length 0.00 -0.04 3 seed thickness at center 0.00 -0.03 4 volume 0.00 -0.08 5 seed specific gravity -0.01 0.1 i 6 number of seeds 0.00 -0.04 7 number of seedling/seed 0.00 0.00 8 number of days to germination 0.02 0.99 9 seed with one seedling 0.00 0.00 10 seed with twin and multiple seedlings 0.00 0.00 11 germination percentage l .00 -0.01 cumulative variance 97.63 99.50 seedling characters 1 height 0.03 -0.01 2 girth at collar 0.00 0.00 3 total number of leaves 0.03 0.00 4 number of flushes/year 0.00 0.00 5 total leaf area 0.98 -0.21 6 survival rate at twelth month (%) 0.04 0.18 7 shoot fresh weight 0.01 0.06 8 root fresh weight 0.01 0.03 9 total fresh weight 0.02 0.09 10 shoot dry weight 0.01 0.03 11 root dry weight 0.00 0.01 12 total dry weight 0.01 0.04 13 root to shoot dry weight ratio 0.00 0.00 14 root length (longest root) 0.03 0.1 8 15 total number of roots 0.20 0.94 cumulative variance 70.05 95.77 table 2 average fruit index, seed index and seedling index values of four age groups in mangosteen (garcinia mangostana l.) age groups (years) fruit index seed index seedling index <25 90.55 80.54 93.69 25-50 88.62 88.36 101.17 51-75 132.90 84.47 65.20 >75 110.42 62.12 67.17 average 102.16 81.58 86.21 118 expressed as a number. numbers of seeds per fruit were counted and categorized as one, two, three and more in three-seeded and seedless fruits and the average expressed as a number. total number of seedlings obtained from individual fruits was counted irrespective of the number of seeds per fruit and the average was expressed numerically. the specific gravity fruits was calculated using the following formula and the average expressed as grams/milliliter: fruit specific gravity = (fruit weight / fruit volume) seed character the seeds were extracted from the pulp and the weight of individual seeds in fruits was measured using an electronic balance; the average seed weight was expressed in grams. seed length and thickness at the centre were also measured, with the aid of a meter scale, and averages expressed in centimeters. seed volume was measured by water displacement method and the average expressed in milliliters. seed specific gravity was calculated using the formula seed specific gravity = (seed weight / seed volume) and the average expressed as grams/milliliter. total number of seeds present in individual fruits, total number of seedlings produced by individual seeds and total number of ungerminated seeds were counted and averages expressed as a number. the number of seedlings produced by an individual age group was calculated and the average expressed as a number. the number of seedlings produced by individual seeds was counted and categorized as seeds producing one, two and more than two seedlings and the average expressed as a number. the number of days from date of sowing to seed germination was counted and the average expressed as number of days. germination rate was calculated for each individual age group and the average expressed as a percentage. germination % = (total number of seeds germinated / total number of seeds sown) x 100 seedling characters seedling height was measured from the collar region to the tip of the main stem using a meter scale and expressed in centimeters. seedling girth was measured at the collar using a thread; thread length was measured using a meter scale and averages expressed in centimeters. total number of leaves produced by an individual seedling was counted and the average expressed as a number. the increments were calculated by computing the difference between two consecutive values of the particular interval and the average expressed in centimeters. the number of flushes and number of leaves/flush produced by an individual seedling were counted and the average expressed as a number. leaf area was calculated by multiplying the length, the breadth and the factor (0.6727) and the average expressed as cm2. the factor was pre-standardized, for this purpose, by measuring the length and breadth of 100 leaves: the leaf area of the corresponding leaf was measured by leaf area meter to work out the factor value. thus, the factor value (0.6727) was derived using the formula: factor = (leaf area / length x breadth) survival rate was determined from the number of established plants as a percentage of the total number of seedlings observed after germination. survival % = (total number of seedings observed at each interval / total number of seedlings observed immediately after germination) x 100 shoot and root characters seedlings were uprooted one year after germination. the plants were immediately cut and separated into shoots and roots. fresh shoot and root weights were recorded separately using an electronic balance and the average expressed in grams. the samples collected for the fresh weight were dried in an oven at 60°c till the weight of the samples remained constant. dry weights were recorded separately and averages expressed in grams. dry weight ratio of root to shoot was calculated by the formula: dry weight ratio = (root dry weight / shoot dry weight) length of the longest root (taproot) was measured from the collar region to the growing tip of the taproot using a meter scale and expressed in centimeters. after carefully removing the potting mixture using water spray, the number of primary, secondary, tertiary roots, and total number of roots were counted and their averages expressed as the number of roots. biochemical studies leaf samples from seedlings were collected one year after germination; leaf samples from mother plants were also collected. the third leaf from the tip was collected and oven dried at 60°c, ground and used to estimate the content of n, p, k and na. the total nitrogen content of leaf samples was determined by microkjeldhal method (jackson, 1973) and the average expressed as percentage. the phosphorus content of the samples were determined using the di-acid extract method (jackson, 1973). a spectrophotometer was used to determine colour intensity developed by vanado-molybdo phosphoric yellow colour method and readings were taken at 420nm wavelength. phosphorous content was cal119 culated using a standard graph and the average expressed as a percentage. the potassium and sodium contents of samples were determined with di-acid extract (jackson, 1973) and read in an eel flame photometer, at 548 nm and 598 nm respectively, and averages expressed as a percentage. nitrogen content was estimated by microkjeldhal method (jackson, 1973) and the value of nitrogen content was multiplied with the factor 6.25 to get the crude protein content and the average expressed as a percentage. chlorophyll content (total chlorophyll, chlorophyll a and chlorophyll b) was estimated in leaf samples by arnon’s acetone method (sadasivam and manickam, 1996) and the average expressed in milligrams. total sugars were estimated using standard procedure (a.o.a.c., 1980), total carbohydrates were estimated using anthrone method (dubois et al., 1951), and averages expressed in milligrams; total phenol content was estimated using the folin-ciocalteau method (sadasivam and manickam, 1996) and expressed in milligrams. procedure adopted for quantification of abscisic acid the procedure adopted for quantification of abscisic acid was a modification of the standard method of little et al. (1972). the modification became imperative as bands were not obtained. the procedure was standardized and bands were obtained corresponding to the standard abscisic acid. further quantification was done using a u-v spectrophotometer and standards of known concentration from which a standard graph was obtained. generation of new varieties as index variability in morphology characters (38 characters) were recorded. principal component analysis (pca) was carried out independently for 12 fruit, 11 seed and 15 seedling characters in order to establish a list of minimum descriptors (a.o.a.c., 1980) making it possible to identify the best age group of mother plants. the first principal component which accounted for maximum possible variance was selected. this is supported by the work of manzano et al. (2001). estimation of correlation associations between the various characters were made using the karl pearsons product movement correlation coefficient (r). correlations between age of mother plant, fruit characters, seed characters, seedling characters, fruit index, seed index and seedling index were calculated according to the method of searle (1961). principal component analysis the observations of 12 fruit, 11 seed and 18 seedling characters of the four age groups and their corresponding index values (i.e. fruit, seed and seedling) of the 135 plants were used for the study. the volume of the data had to be reduced first for the sake of simplicity. this was based on the fact that principal component analysis is one of the variabledirected techniques aimed at reducing dimensionality of the problem and which finds new variables that make the data easier to understand (chatfield and collins, 1980). principal component analysis (pca) was used to determine the relationship among the fruit characters, seed characters, seedling characters and four age groups (meilgard et al., 1991) and was performed in xlstat version 5.1v2 package. it was also used to provide a graphical description of the characteristics. factor scores were calculated for each fruit, seed and seedling characteristics using the formula: factor scores= each attribute factor loading x the original attribute mean score although the characteristic factor scores were calculated from a single analysis, they were plotted on separate figures to facilitate interpretation. these figures then provided a visual representation of the dominant age groups and characters for each fruit, seed and seedling character (dever et al., 1996). step-wise regression to facilitate the prediction of indices reduction in number of variables is imperative. hence the step-wise regression procedure was adopted to identify major characters, which can be used to predict the fruit, seed and seedling indices. furthermore, adoption of step-wise regression has been scientifically established to reduce multi-collinearity among the independent variables and to arrive at the best subset of variables (draper and smith, 1966). cluster analysis cluster analysis was performed to find the similarities (or dissimilarities) between the four age groups based on absolute square euclidean distance (johnson and wichern, 1998). the clusters and square euclidean distances (chatfield and collins, 1980) were graphically represented by dendrograms (manzano et al., 2001). the euclidean distance was calculated using the formula p drs : {∑ (x ri – x si )2 }to ½ and the variable were the principal component co-ordinates. where drs distance from individual r to individual s, x rj value of individual r x sj value of individuals. selection index selection index or total index values were worked out using principal component analysis. principal component analysis was performed on all the fruit, seed and seedling characters collectively and the first principal component was taken as the index value for selection, as in the method described above. regression analysis was performed for each age group separately to find the best age group with equal weightage of all fruit, seed and seedling characters. then the best age group or selection index (total index) 120 was predicted using the regression equation with the fruit, seed and seedling indices as the explanatory variables. the predictor equation was found to be y=a 1 f1 + a 2 s1 + a 3 sdli + c where a 1 , a 2 , a 3 were regression coefficient or weightage of f1, s1 and sdli, which are fruit index, seed index and seedling c the intercept constant (manoj, 1992). 3. results the results of the study have been divided into broad aspects for presentation. fruit, seed and seedling indices the numbers of independent variables were large in the present study. hence, to reduce the number of descriptors, principal component analysis was performed using the deviation squares and products matrix of the 12 fruit, 11 seed, and 15 seedling characters independently in order to establish a list of minimum descriptors (i.e. fruit index, seed index and seedling index), thereby enabling identification of the best age group of mother plants, as well as reducing the complexity of the statistical analysis. the first principal components of fruit, seed and seedling characters, which accounted for the cumulative variance of 96.27, 97.63 and 70.05% respectively, are given in table 1. the computed average fruit, seed and seedling index values of each age group are presented in table 2 and figure 1. fruit index the highest fruit index value was in the age group of 51-75 years of mother plants, followed by the age group of more than 75 years. seed index the highest seed index value was recorded in the 25-50 year age group, followed by 51-75 years. the lowest seed index values were observed in the more than 75 years age group. seedling index the highest seedling index was observed in the age group 25-50 years, followed by the age group of less than 25 years. an important observation was made: the difference between the best group (25-50 years) and the two age groups of more than 50 years was near to one and a half times more. index values observed for fruit, seed and seedling characters differed but the trend was the same for the seed and seedling characters. however, equal importance was given to each of the three index values and to the age group for finally determining the best age group of mother plant. principal component analysis principal component analysis was performed using the deviation squared and product matrix of the 14 fruit characters, 13 seed characters, 20 seedling characters, the age of the mother plants and all the three fruit, seed and seedling indices separately. the first two principal components, which accounted for the cumulative variance of fruit, seed and seedling characters, were found to be 88.78, 92.94 and 92.49% respectively. the cumulative variance, factor scores and contribution of variation of each character are presented in table 3 and the same for each age group in table 4. the fruit index among the fruit characters, seed index among the seed characters and seedling index among the seedling characters were predicted using the regression equation with the first two principle components as the explanatory variables. the predictor equations were found to be y = 0.0695 p 1 – 0.1283 p 2 0.0197 with an r2 of 0.999 where y 1 p 1 and p 2 are the fruit index first and second principal components respectively; y= 0.3198 p 1 + 0.6307 p 2 0.0135 with an r2 of 1.00 where y 1 p 1 and p 2 are the seed index, first and second principal components respectively; y= 0.7003 p 1 0.0703 p 2 + 3.263 with an r2 of 0.999 where y 1 , p 1 and p 2 are the seedling index first and second principal components respectively. also the relationship and dominance of the fruit, seed and seedling characters including four age groups of the mother plants and three (fruit, seed and seedling) indices separately were determined using factor scores. the depiction of the various characters of factor i showed negative scores for characters such as number of seeds per fruit, specific gravity and selection index, whereas factor ii revealed positive scores only for seedlessness and seed specific gravity (table 3). distinct variations in the dominant fig. 1 fruit, seed and seedling index of four age groups in mangosteen (garcinia mangostana l.). 121 characters were observed between the various age groups. in the case of fruit characters, fruit index, fruit weight, fruit girth, fruit volume, pulp weight, number of segments and rind weight characters of age group three (51-75 years) were dominant. on the other hand, the number of seeds per fruit was observed as the dominant characters in the table 3 eigen vectors, factors loadings and contribution of variations by each character in fruit, seed and seedling characters in mangosteen (garcinia mangostana l.) characters eigenvectors factor loadings contributions of variation (%) f1 f2 f1 f2 f1 f2 fruit characters fruit weight 0.30 -0. 17 0.96 -0.28 9.27 3.01 girth 0.21 -0.07 0.66 -0.11 4.44 0.44 volume 0.29 -0.24 0.92 -0.39 8.50 5.84 pulp weight 0.21 -0.46 0.66 -0.73 4.39 20.90 rind weight 0.31 -0.05 0.99 -0.08 9.86 0.25 segments 0.30 -0.14 0.95 -4.22 9.14 1.94 seeds -0.28 -0.20 -0.87 -0.32 7.70 3.97 seedless fruits 0.29 0.27 0.90 0.43 8.20 7.08 one seeded fruits -0.27 -0.24 -0.84 -0.39 7.22 5.98 two seeded fruits -0.21 -0.18 -0.65 -0.29 4,29 3.37 three and >three seeded fruits -0.27 -0.31 -0.84 -0.49 7.16 9.32 specific gravity -0. 11 0.58 -0.34 0.94 1.16 34.01 fruit index 0.31 -0.06 0.97 -0.10 9.54 0.41 selection index -0.30 -0.19 -0.95 -0.30 9.13 3.49 cumulative variance 70.37 88.79 seed characters seed weight 0.29 -0.28 0.82 -0.55 8.29 7.81 seed length 0.34 -0.02 0.98 -0.04 11.68 0.03 thickness at center 0.31 0.06 0.89 0.l l 9.68 0.31 seed volume 0.29 -0.27 0.84 -0.54 8.69 7.40 specific gravity -0.28 0.29 -0.80 0.57 7.81 8.17 number of seedlings / seed 0.32 0.07 0.90 0.14 9.94 0.49 ungerminated seeds -0.30 -0.22 -0.87 -0.44 9.30 5.04 seeds producing one seedlings 0.26 0.27 0.74 0.53 6.69 7.23 seeds producing 2 and >2 seedlings 0.33 0.10 0.95 0.19 11.05 0.94 days to germination -0.24 0.31 -0.68 0.62 5.72 9.81 germination % 0.24 0.37 0.68 0.73 5.59 13.73 seed index 0.20 0.41 0.57 0.82 4.01 17.04 selection index -0.12 0.47 -0.35 0.93 1.54 21.99 cumulative variance 62.79 92.94 seedling characters height 0.26 0.09 0.99 0.15 6.85 0.88 girth 0.26 -0.09 0.97 -0.13 6.62 0.74 total leaves 0.26 0.08 0.97 0.12 6.60 0.59 new flushes / year 0.10 0,55 0.39 0.85 1.09 29.76 total leaf area 0.25 0.19 0.95 0.30 6.35 3.63 survival rate 0.2l -0.36 0.78 -0.55 4.27 12.70 shoot fresh weight 0.23 0.06 0.87 0.09 5.34 0.33 root fresh weight 0.26 0.05 0.99 0.08 6.87 0.23 shoot dry weight 0.24 -0.04 0.90 -0.06 5.64 0. 14 root dry weight 0.26 0.03 0.99 0.04 6.89 0.08 root to shoot dry weight ratio -0.14 0.47 -0.54 0.72 2.08 21.65 root length 0.23 -0.16 0.86 -0.25 5.23 2.68 primary roots 0.25 -0.15 0.94 -0.23 6.18 2.29 secondary roots 0.26 -0.01 1.00 -0.02 7.01 0.02 tertiary roots 0.14 0.49 0.55 0.76 2.09 24.22 total roots 0.26 0.01 1.00 0.02 7.01 0.02 seedling index 0.26 -0.01 0.99 -0.02 6.93 0.02 selection index 0.26 -0.01 0.99 -0.02 6.93 0.02 cumulative variance 79.13 92.49 122 less two age groups (i.e. <25 and 25-50 years age groups). interestingly, in age group four (>75 years), the most dominant character was observed to be seedlessness (fig. 2). the depiction of the various seed characters of factor i showed negative scores for seed specific gravity, ungerminated seeds, days to germination and selection index. in the case of factor ii the negative scores were found for seed weight, seed length, seed volume and ungerminated seeds. all other characters showed positive factor scores. in the case of seed characters, also variations in dominant characters were observed with respect to age groups. the seed index, selection index, germination percentage, seeds producing one seedling and number of seedlings per seed were the dominant characters observed in the second age group (25-50 years). in age group one (<25 years) the number of days taken to germination and seed specific gravity were noted to be the dominant characters. in age group three, seed weight, seed volume and seed length were the dominant characters, whereas in age group four, the dominant character was the number of ungerminated seeds (fig. 3). in the case of seedling characters, negative scores were observed only for the root to shoot dry weight ratio in factor i; in factor ii negative scores were found for girth, survival rate, shoot dry weight, root length and number of roots (primary, secondary and tertiary), seedling index and selection index. all other characters showed positive factor scores. a study of the dominant characters of seedlings revealed that the major characters (seedling index seedling girth, total number of roots, number of primary, secondary roots, survival rate, shoot, fresh weight, dry weight, root dry weight, height of the seedlings and selection index were the dominant characters in age group two. in the case of age group one characters such as the root fresh fig. 2 plot showing the percentage of variation contributed by each fruit character and the relationship between age of the mother plant and fruit characters in mangosteen (garcinia mangostana l.). table 4 factor scores and contribution of variations by each age group based on the fruit, seed and seedling characters in mangosteen (garcinia mangostana l.) characters factor scores contributions of age groups {%} f1 f2 f1 f2 fruit characters age 1 -1.74 -0.62 6.14 2.99 age 2 -4.34 -0.66 38.27 3.39 age 3 4.96 -l .76 49.91 24.02 age 4 1.60 3.00 5.21 69.59 mean -0.48 0.05 0.47 0.02 seed characters age 1 -1.64 1.04 6.59 5.53 age 2 -0.64 2.87 0.99 41 .98 age 3 5.26 -1.13 67.89 6.55 age 4 -3.16 -2.99 24.46 45.70 mean 0.17 0.22 0.07 0.24 seedling characters age 1 3.35 0.92 15.73 7.10 age 2 4.93 -0.91 34.14 6.95 age 3 -4.52 -2.27 28.75 42.95 age 4 -3.90 2.27 21 .35 43.00 mean 0. l5 -0.01 0.03 0.00 123 weight, total number of leaves, number of tertiary roots and new flushes per shoot were the dominant characters. in the higher two age groups, no specific dominance were observed (fig. 4). principal component analysis reduces only the dimensionality, but not the number of variables involved. hence, to reduce the number of variables and to identify the major fig. 3 plot showing the percentage of variation contributed by each seed character and the relationship between age of the mother plant and seed characters in mangosteen (garcinia mangostana l.). fig. 4 plot showing the percentage of variation contributed by each seedling character and the relationship between age of the mother plant and seedling characters in mangosteen (garcinia mangostana l.). 124 variables that contribute greater variation to the fruit, seed and seedling index, a step-wise regression was performed. step-wise regression step-down regression of the fruit index on 13 fruit characters, seed index on the same 13 fruit characters, seed index on the 12 seed characters separately and all the 25 fruit and seed characters collectively, and seedling index on 13 fruit characters, 12 seed characters, 19 seed characters including age separately and also collectively (all the 42 fruit, seed, seedling characters and age) was carried out and the following regression equations with major variable, to each, indices on each group of characters, were observed. fruit index on fruit characters alone y = 1.295 x 7 + 1.7 l5x 8 + 1.3 19 x 14 4.17 with an r2 at 0.993 where y fruit index, x 7 pulp weight, x 8 rind weight, x 14 number of seedlings per fruit. seed index on fruit characters alone y = -a.282 x2 + 0.427 x3 + 14.416 x5 + 22.368 x10+ 126.73 with an r2 at 0.203. where y – seed index, x2 – age of the mother plant, x 3 fruit weight, x 5 fruit girth, x 10 number of seedless fruits. seed index on seed characters alone y= 0.007 x15 0.004 x16 0.012 x17 + 0.009 x180-008 x19 + 0.002 x20 +0.004 x21 + 0.015 x22 + 0.002 x24 + x25 + 0’01 with an r2 at 1.00 where y seed index, x 15 seed weight, x 16 seed length, x 17 seed thickness at centre, x 18 seed volume, x 19 seed specific gravity, x 20 number of seeds per fruit, x 21 number of seedling per seed, x 22 number of days to germination, x 24 number of seeds producing two or more than two seedlings, x 25 – germination percentage. seed index on fruit and seed index collectively y = -0.002 x 11 0.006 x 15 0.006 x 17 + 0.006 x 18 0.007 x 19 + 0.005 x 21 + 0.015 x 22 – 0.002 x 23 + x 25 + 0.01 with an r2 at 1.00 where y seed index, x 11 – number of one seeded fruits, x 15 seed weight, x 17 seed thickness at centre x 13 seed volume, x 19 seed specific gravity, x 21 number of seedling per seed, x 22 number of days to germination, x 23 seed with one seedling x 25 germination percentage. seedling index on fruit characters including age y =-0.481 x2 + 107.15 with an r2 of 0.0388 where, y seedling index, x 2 age of the mother plant. seedling index on seed characters y =136.67 x17 1083 with an r2 of 0.1087 where, y seedling index, x 17 seed thickness at centre. seedling index on seedling characters y= 0.979 x30 + 0.037 x31 + 0.042 x32+ 0.027 x33 + 0.031 x39+ 0.202 x40 + 0 .36 with an r2 of 1.00 where, y seedling index, x 30 total leaf area per seedling, x 3l survival rate at l2-months stage, x 32 – shoot fresh weight, x 33 root fresh weight, x 39 foot length, x 40 total number of roots. seedling index on fruit, seed and seedling characters collectively including age of mother plant y= 0.026 x26 + 0.030 x28+ 0.977 x30 + 0.037 x31 + 0.0034 x320.029 x33+ 0.017 x35 + 0.009 x36 + 0.031 x 39 + 0.031 x 39 + 0.201 x 40 with an r2 of 1.00 where, y seedling index, x 26 height of the seedling, x 28 total number of leaves, x 30 total leaf area, x 31 survival rate at twelve-months stage, x 32 shoot fresh weight, x 33 root fresh weight, x 35 shoot dry weight, root dry weight, x 36 root dry weight, x 39 root length, x 40 total number of roots. cluster analysis cluster analysis was performed to highlight the similarities and differences based on the fruit, seed and seedling characters separately and all characters collectively among the four age groups. absolute euclidean distances between the four age groups were calculated based on the fruit, seed and seedling characters and are presented in table 5. figure 5 illustrates the dendrogram showing the clusters (or similarities) based on the fruit, seed and seedling characters among the four age groups. considering fruit characters, closest distance was observed between age groups of more than 25 years and 2550 years, followed by the age group of 25-50 years and more than 75 years; in all the remaining combinations greater distances were observed, indicating that the age group of more than 25 years and 25-50 years have more similar fruit characters. with regard to seed characters, closest distance was again observed between age groups of more than 25 years and 25-50 years, followed instead by the age group of more than 25 years and more than 75 years. with regard to all the remaining combinations greater distances were observed, indicating that the age group of more than 25 years and 25-50 years have more similar seed characters. for seedling characters, closest distance followed the same trend (i.e. age group of more than 25 years and 25-50 years). all the remaining combinations were observed at greater distances, indicating that also for seedling charac125 ters the age group of more than 25 years and 25-50 years are more similar. when the fruit, seed and seedling characters were taken collectively, the closest distance was observed between age group of more than 25 years and 25-50 years, followed by the age group of more than 25 years and more than 75 years; in all the remaining combinations greater distances were observed. finally it may be confirmed that, based on all four dendrograms, maximum similarity for all the characters was between the age group of less than 25 years and 25-50 years. discriminant function analysis fruit index, seed index and seedling index of the four age groups were discriminated using discriminant functional analysis. discriminant functions were fitted for discrimination pairs of these age groups. the functions derived were as follows. for groups 1 and 2 z = 0.0004 fi 0.0547 si 0.0004 sdli with d2 = 0.4313 for groups 1 and 3 z = -0.0483 fi 0.0313 si + 0.0075 sdli with d2 = 2.381 for groups 1 and 4 z = -0.0183 fi 0.0680 si + 0.0047 sdli with d2 = 1.739 for groups 2 and 3 z = -0.0406 fi + 0. 1264 si + 0.0137 sdli with d2 = 2.782 for groups 2 and 4 z = -0.0265 fi + 0.3508 si + 0.0150 sdli with d2 = 10.29 for groups 3 and 4 z = 0.0024 fi + 0.2152 si + 0.0004 sdli with d2 = 4.064 where fi the fruit index, si the seed index and sdli the seedling index are the explanatory variables. the average values of fruit index, seed index and seedling index for each age group were fitted in the equation and the discriminant values calculated for each pair of age groups. the direction of association of the discriminant coefficients in each age group is given in table 6. in this maximum number of positive directions were observed in the age group of 25-50 years, when discriminated with other three table 5 proximity matrix showing the absolute squared euclidean distance between four age groups based on fruit, seed, seedling characters separately and collectively in mangosteen (garcinia mangostana l.) characters and age groups absolute squared euclidean distance 1 2 3 4 fruit 1 0.00 14.41 39.25 20.57 2 14.41 0.00 3 1.60 15.6 3 39.25 31.60 0.00 22.57 4 24.57 15.60 22.57 0.00 seed 1 0.00 13.14 30.09 13.24 2 13.14 0.00 21.58 20.37 3 30.09 21.58 0.00 33.59 4 13.24 20.37 33.59 0.00 seedling 1 0.00 5.65 29.97 35.44 2 5.65 0.00 26.48 34.63 3 29.97 26.48 0.00 11.83 4 35.44 34.63 11.83 0.00 all characters 1 0.00 39.06 109.58 77.77 2 39.06 0.00 100.31 90.23 3 109.58 100.31 0.00 75.05 4 77.77 90.23 75.05 0.00 fig. 5 dendrogram showing the similarity and proximity of the four age groups based on the fruit, seed and seedling characters individually and collectively in mangosteen (garcinia mangostana l.). table 6 direction of association of six discriminant function coefficients involving the four age groups in mangosteen (garcinia mangostana l.) pairs of compared age groups magnitude of the values fruit index seed index seedling index 1 and 2 + 1 and 3 + 1 and 4 + + 2 and 3 + + 2 and 4 + + 3 and 4 + + 126 age groups (table 7). the corresponding discriminant values of each age group (criteria) and the mid values of each pair of discriminant values were taken to draw the conclusion that, if the values is below the mid values it falls in the group i, if not it falls in the group ii. the criterion and criteria are presented in table 8. selection index principal component analysis was performed on all the fruit, seed and seedling characters collectively and first principal component was taken as the index value for selection (fig. 6). the selection index was predicted using the multiple regression equation constant for each age group independently and also the average of all age groups. the predictor equation was found to be for the less than 25 years group y = -0.0402 fi + 0.0513 si + 0.9960 sdli – 0.4317 (adj. r2= 0.99) table 7 direction of association of six discriminant function coefficients when used to compare each age group with the remaining three age groups in mangosteen (garcinia mangostana l.) discriminating pairs of age groups magnitude of the values total number of positive values age group i age group ii fruit index seed index seedling index 1 2 + 1 1 3 + 1 1 4 + + 2 total positive values for age group 1 4 2 1 + + 2 2 3 + + 2 2 4 + + 2 total positive values for age group 2 6 3 1 + + 2 3 2 + 1 3 4 + + 2 total positive values for age group 3 5 4 1 + 1 4 2 + 1 4 3 + 1 total positive values for age group 4 3 table 8 criteria and criterion for discrimination of age groups in mangosteen (garcinia mangostana l.) sl. no age groups comparisons criteria 1 criteria 2 criterion conclusion 1 <25 and 25-50 -4.41 -4.84 -4.62 if the value is >-4.62 it falls in group i, if not group ii 2 <25 and 51-75 -6. 19 -8.57 -7.38 if the value is >-7.38 it falls in group i, if not group ii 3 <25 and >75 4.26 2.52 3.39 if the value is > 3.39 it falls in group i, if not group ii 4 25-50 and 5 1-75 8.96 6.17 7.57 if the value is > 7.57 it falls in group i, if not group ii 5 25-50 and >7 5 30.17 19.87 25.02 if the value is >25 .02 it falls in group i, if not group ii 6 51-75 and >75 18.47 13.61 16.04 if the value is >16.04 it falls in group i, if not group ii fig. 6 selection index or total index values of all the four age groups in mangosteen (garcinia mangostana l.). 127 for group 25-50 years y= -0.0562 fi + 0.0129 si + 0.9979 sdli + 4.331(adj. r2= 0.99) for the 51-75 years group y= -0.0479 fi + 0.0115 si + 0.9967 sdli + 3.070 (adj. r2 = 0.99) for the more than 75 years group y= -0.0495 fi + 0.0191 si + 0.9955 sdli + 2.247 (adj. r2 = 0.99) for the average of all age groups y= -0.0527 fi + 0.0446 si + 0.9978 sdli + 1.056 (adj. r2 = 0.99) where fifruit index, siseed index and sdliseedling index. the average of fruit index, seed index and seedling index values for each age group were fitted in the equation independently and the selection index values were determined. the averages of all the age groups were recorded and are given in table 9. table 9 mean selection index or total index values of four age groups in mangosteen (garcinia mangostana l.) age groups (years) selection index values less than 25 years 93.38 25 50 years 101 .45 51 75 years 62.66 more than 75 years 64.84 mean 80.64 index values the highest index value was found in the 25-50 years age group of mother plant, which was concluded to be the best age group for selection of the mother plants. this was followed by the age group of less than 25 years. the mean values of the important characters in the best age group (25-50 years) were fixed on the basis of the corresponding character for selection. classification was made as above average if positively correlated and below average, if negatively correlated. 4. discussion and conclusions presentation of the results of the studies are broadly discussed and organized under subheadings to make them more accessible to the reader. association of morphological characters of fruit, seed and seedling the fruit index was positively correlated with most of the fruit and seed characters, except fruit specific gravity, number of one-seeded fruits, seed thickness at centre, seed specific gravity, number of seedlings per fruit, and germination rate; all the seedling characters were negatively correlated. an analysis of the age group variations revealed that the maximum values of all the morphological characters of fruit and seed were recorded in the 51-75 years group and the lowest values were recorded in the 25-50 years group. as fruit index is a function, which is derived from all the morphological characters of fruit, the characters ought to be naturally correlated. however the negative correlations observed were due to the lowest values of fruit characters recorded in the 25-50 years age group, which in turn gave maximum seeded fruits, maximum germination of seeds, maximum survival rate and the best quality seedlings as revealed by the improved morphological characters. seed index was observed to be negatively correlated with all the fruit characters. although the pattern of gradation observed in the values of seed characters in the various age groups was the same as that observed for fruit characters in the same age group, a corresponding size or weight of the seed was not observed. the relative differences observed at seed level are not as explicit as in the case of fruits and this could be the reason for the negative correlation observed between seed index and fruit characters. this is more evidenced if a ratio of the seed to fruit weight is computed. the second age group registered the highest ratio values, which again reveals that seed weight expressed as a fraction of the fruit weight is a more important character. similarly, the differences are most clear in seed thickness to fruit girth ratio, where the second age group showed a very high ratio in spite of the fact that for both of the above characters the first age group had the highest values individually. the positive correlations of seed index with all seedling characters once again confirms that the seedling characters are more governed by both the individual as well as collective characters of the seed. while in the standard methodology that was adopted, equal importance (weightage) was given to seed, fruit, and seedling characters and age of the mother plant, the results confirm that it is the seed characters which play a more determining role for better seedling characters. even though identical reports are not available in mangosteen, the studies of reddy (1997) revealed that the size of the seeds was highly variable, the difference in seed weight brought about the variation in germination and that the ability to germinate and grow successfully is related to the amount of food stored in the seed. another author suggested that it is better to establish new plantings only from the larger seeds weighing 1 g and above in florida (campbell, 1966). seedling index, which was computed as a total variation of all the seedling characters collectively, was negatively correlated with number of seedlings per seed, number of days to germination, and seeds producing more than two seedlings. this is basically due to the fact that as a consequence of an increase in the number of seedlings per seed, the growth characters of the seedling are affected. the number of days taken to germinate was negatively correlated with seedling index because the seed characters 128 were positively correlated with seedling characters and the age groups that recorded the highest values for seed morphological characters also recorded the least number of days to germination. the number of seeds producing more than two seedlings were negatively correlated with the seedling index as the positive aspect of more seedlings produced per seed was negated by the character of comparatively slower growth. another positive aspect in this case is that normally seedlings with exposed food storage or haustorial cotyledons are very vulnerable, and this attractive food source is prone to attack by rodents when it is present above ground. however in the garcinia type, the food reserve is stored in the hypocotyl, which is protected by the envelopments and situated at, or below, soil level making it less vulnerable (vogel, 1980). in general, most of the fruit characters are positively correlated with seed characters and negatively correlated with seedling characters; most seed characters are positively correlated with seedling characters. generally this is because the values of fruit characters increase with mother plant age group, but this increase is not matched by a corresponding increase in seed characters. in the case of seedlings, the age groups with increased fruit characters produce only weaker seedlings, as revealed by the lower seedling index. the increase generally observed in the seed characters leads to increased seedling growth and, hence, over emphasizes the absolute command of seed characters in determining the quality attributes of seedlings. principal component analysis. fruit characters and index a figurative plot of the principal components of the fruit characters is presented in figure 6, showing very narrow angles among the fruit characters such as rind weight, pulp weight, fruit weight, fruit index, number of segments, fruit girth and fruit volume and describing actually the high positive correlation that exist between them. the principal component analysis of fruit characters and age of the mother plant showed that factor i and factor ii account for 88.76% of the cumulative variance. a critical analysis of the plot reveals that for all major characters of the fruit, age group three showed narrow angles, underlining that most of the improved fruit morphological characters are observed in this group. on the other hand, seedlessness showed narrow angles to age groups three and four, confirming that it is a factor that goes hand in hand with increased age. seedlessness in fruits and seediness (one-seeded, two-seeded and more than three-seeded) are in opposite directions, illustrating that they are negatively correlated. a similar positioning is observed in the case of specific gravity and pulp weight, which reconfirms the negative correlation. seed characters and index principal component analysis of the seed characters and age of the mother plant showed that factors i and ii account for 92-94% of the cumulative variance. a critical perusal of the plot (fig. 3) of the seed characters revealed that maximum seed characters (seed index, selection index, germination percentage, seed producing one seedling, number of seedlings per seed and seed thickness at centre) were in close proximity and with very narrow angles to age group two, which clearly establishes the distinct superiority of this age group. the number of ungerminated seeds was in close proximity to age group four and positioned opposite germination percentage, showing the negative correlation. similarly, seed weight and volume are positioned at opposite ends with respect to the days to germination and specific gravity, confirming the highly significant negative correlation observed in the study. seedling characters and index principal component analysis of the fruit characters and age of the mother plant showed that factors i and ii account for 92.49% of the cumulative variance. the most important or major characters of the seedling and selection index are in close proximity to age group two (fig. 4), which clearly establishes that seedlings of this age group are superior in all characters. the narrow angles between the various characters and this age group also establish the high positive correlations of the characters with this age group. the placement of survival rate and root to shoot dry weight ratio at opposite ends not only reveals the negative correlation but, more notably, the importance of roots which are critical in the case of mangosteen. step-wise regression step-wise regression was carried out to identify the variables contributing maximum variations and to reduce the number of variables. there was reduction in the number of variables influencing the fruit characters. with regard to seed characters, the thickness of the seed at the centre was found to be the most important factor. this is supported by the highest seed specific gravity found to be maximum in the best age group. for seedling index, the important characters were height of seedling, total number of leaves, total leaf area, survival rate, shoot and root fresh weight and dry weight, root length and total number of roots. as for seedlings, root characters were logically found to be important as the root system in mangosteen is magnolioid but cannot be recommended as an index for selection as they are underground. mangosteen plants are very sensitive and even removal of bits of leaves for chemical analysis normally resulted in death of the plant. similarities between age groups based on fruit, seed and seedling characters a critical analysis of the various age groups points to a very important conclusion: for most of the fruit characters, age groups one and two gave comparatively lesser values, whereas the older groups gave maximum values and have higher fruit index. likewise in the case of seeds, the older groups had better size but the younger age groups gave more seeded fruits and gave better germination and more 129 seedlings per seed. these characters though were highest in the age group two were equally high in the youngest age group and thus the similarity between these two younger age groups were higher resulting in more closer distance in the dendrogram showing hierarchical clusters based on the euclidean distance. in the case of seedling characters, a comparative study reveals more similarity and also when all fruit, seed and seedling characters were collectively taken. there was more similarity between the younger two age groups and this should be the reason for the very close distance between these age groups (less than 25 years and 25 50 years) and the close proximity in the dendrogram (fig. 5). discriminant function further extrapolation of the coefficients (table 7) used in discrimination of various groups were critically analysed and are presented in table 8. the directions of coefficients were used to prepare the dendrogram presented in figure 7, showing both the positive and negative direction of the coefficients of fruit index, seed index and seedling index. it is again obvious from the table and the dendrogram that maximum coefficients were positively linked in the case of age group two when two specific pairs of age groups were discriminated as envisaged in this study. this was actually a reflection of the higher seed and seedling characters in age group two, compared to the other age groups, which reconfirms that this group is the best age group for selection as mother plants. selection index based upon the equation, the elaborated selection index revealed that the 25-50 years age group was the best. this is actually more a reflection of the improved seedling characters, particularly the germination percentage, survival rate and other morphological characters. the seed characters as revealed by the seed index were also highest in this particular age group. in the selection index equation, the seed characters were given equal weightage with all characters and hence this age group, which showed better seed characters, gave higher index values. although the fruit characters were better in the older groups, seedlessness, low germination percentage and poor survival rate were the main reasons for the lower selection index values. root characters cannot be taken as indices for selection as they are below ground, and hence not visible. furthermore, any attempt to lift the bare seedlings normally results in death of the plants, as they are highly sensitive. as the root characters are positively correlated with shoot characters, selection based on the shoot characters reflects root characters as well. having identified the best age group, the mid value of the dominant characters were chosen as viable indices for the selection. the mid values of seedling characters were fixed so that destructive procedures could be avoided. considering the complex of results obtained in the study, the following characters and mid values were selected as indices for selection. the selection should be such that the fruits from mother plants of the age group of 25-50 years should have fruit weight of not less than 58 g, fruit girth of not less than 5 cm, fruit volume of not less than 51 ml, pulp weight of not less than 16 g, rind weight of not less than 41 g, and number of segments less than 5. seeds obtained from the fruits with the above characters should have a seed weight of not less than 0.66 g, seed length of not less than 1.61 cm, seed thickness at centre of not less than 0.66 cm, seed volume of not less than 0.44 ml, seed specific gravity of not less than 1.50, days to germination not less than 21, germination percentage not less than 87% and preferably have more than one seed per fruit. furthermore, seedlings at the one-year stage from this age group with the identified fruit and seed characters should have a height of not less than 10 cm, seedling girth at collar region of not less than 2 cm, total number of leaves per seedling not less than 9, number of new flushes per year more than one, total leaf area per seedling not less than 72 cm2 and survival rate not less than 85%. in conclusion, it can be said that the present study has generated results of immense practical relevance which will directly aid in selection or act as a powerful tool for the selection of mother plants, fruit, seed and seedling characters for improved seedling growth. acknowledgements the authors acknowledge the assistance provided by the associate dean, college of horticulture, kerala agricultural university during the survey of mother plants, and for the infrastructural support received and lab facilities available for the study. fig. 7 dendrogram showing the directions of discriminant coefficients of fruit, seed and seedling indices of each age group when compared to the remaining three age groups in mangosteen (garcinia mangostana l.). 130 references a.o.a.c., 1980 official methods of analysis of the association of official analytical chemists. l3th ed., washington d.c., usa, pp. 1098. almeda n., martin f.w., 1976 the mangosteen. cultivation of neglected tropical fruits with promise. part i. agricultural research service, us department of agriculture, pp. 5. alvim p.t., 1964 tree growth periodicity in tropical climates, pp. 479-497. in: zimmermann j.h. (ed.). the formation of wood in forest trees. academic press, new york. borchert r., 1973 simulation of rhythmic tree growth under constant conditions. physiol. plant., 29: 173-180. campbell c.w., 1966 growing the mangosteen in southern florida fla. state hortic. soc., 79: 399-400. chatfield c., collins r.j., 1980 introduction to multivariate analysis. chopman and hall, london, uk, pp. 51-79. dever m.c., mcdonald r.a., cliff m.a., lane w.d., 1996 sensory evaluation of sweet cherry. hort. sci., 31(1): 150-153. downton w.j.s., grant w.j.r., chacko e.k., 1990 effect of elevated carbon dioxide on the photosynthesis and early growth of mangosteen (garcinia mangostana l.). scientia horticulturae, 44(34): 215-225. draper n.r., smith h., 1966 applied regression analysis. john wiley and sons, new york, usa, pp. 54-104. dubois m., gilles k., hamilton j.k., robers p.a., smith f., 1951 a colorimetric method for the determination of sugars. nature, 168: 167. hume e.p., 1947 difficulties in mangosteen culture. trop. agric., 14(32): 36. ibpgr, 1986 genetic resources of tropical and sub-tropical fruits and nuts. international board for plant genetic resources, fao, rome, italy, pp. 43-46. ista, 2003 handbook for seedling evaluation. ed. don r, switzerland, pp. 1-50. jackson m.l., 1973 soil chemical analysis. prentice hall inc. engle wood cliffs, usa, pp. 498-504. johnson r.a., wichern d.w., 1998 applied multivariate statistical analysis. prentice hall, fourth edition, new jersey, usa, pp. 1-124. kusumo s., verheij e.w.m., 1994 mangosteen, the queen of tropical fruits: problems and suggested research in indonesia. central research institute for horticulture, indonesian agricultural research and development journal, jakarta, indonesia, 16(3): 33-36. lim a.l., 1984 the embryology of garcinia mangostana l. (clusiaceae). garden’s bullettin, singapore, 37: 93-103. little c.h., strunz g.m., france r.l., bonga j.m., 1972 identification of abscicic acid in abies balsamea. phytochem., 11: 3535-3536. manoj p.s., 1992 biometrical studies in cashew (anacardium occidentale l.) hybrids. m.sc. (hort.) thesis, college of horticulture, kerala agricultural university, vellanikkara, thrissur, india. manzano a.r., nodals a.a.r., gutierez m.i.r., mayor z.f., alfonso l.c., 2001 morphological and isoenzyme variability of taro (colocasia esculenta l. schott) germplasm in cuba. plant genetic resources newsletter, 126(3): 1-40. meilgard m., civille g.v., carr b.t., 1991 sensory evaluation techniques. 2nd ed. crc press, boca raton, fl., usa, pp. 301-305. normah m.n., rosnah h., noor-azza a.b., 1992 multiple shoots and callus formation from seeds of mangosteen (garcinia mangostana l.) cultured in vitro. actahorticulturae, 292: 87-91. reddy b.m.c., 1997 propagation techniques for the tropical fruits. regional training course on the conservation and use of germplasm of tropical fruits in asia, 18-31 may, iihr, bangalore, india. richards a.j., 1990 studies in garcinia dioecious tropical fruit trees: the origin of the mangosteen (garcinia mangostana l.). botanical journal of the linnean society, 103(4): 301-308. sadasivam s., manickam a., 1996 biochemical methods. 2nd ed. new age international pvt. ltd., coimbatore, pp. 193-194. searle s.r., 1961 phenotypic, genotypic and environmental correlations. biometrics, 17: 474-480. vogel e.f.d., 1980 seedlings of dicotyledons seedlings ecology. center for agricultural publishing and documentation, wageningen, the netherlands, pp. 130133. wiebel j., chacko e.k., downton w.j.s., 1991 the mangosteen (garcinia mangostana l.). a potential crop for tropical northern australia. fruits, 46(6): 685-688. wiebel j., chacko e.k., downton w.j.s., loveys b.r., ludders p., 1995 carbohydrates levels and assimilate translocation in mangosteen (garcinia mangostana l.). gartenbatwissenschaft, 60(2): 90. wiebel j., chacko e.k., downton w.j.s., subhadrabandhu s., 1992 a mangosteen (garcinia mangostana l.) a potential crop for tropical northern australia. actahorticulturae, 321: 132-137. wiebel j., downton w.j.s, chacko e.k., 1992 b influence of applied plant growth regulators and bud dormancy and growth of mangosteen (garcinia mangostana l.). sci. hortic (amst.), 52(1-2): 27-35. 92 1. introduction the spread of grapevine varieties through different countries over time has produced many of cases of synonymy that now need clarification. correct identification is very important in order to gain more precise knowledge of the varietal assortment and to better manage grapevine catalogues and germplasm repositories. the italian catalogue of grapevine varieties is one of the most copious in the world. nonetheless, it contains various duplicates or triplicates, such as ‘alicante’/‘cannonao’/‘tocai rosso’, ‘vermentino’/ ‘pigato’/‘favorita’, ‘biancame’/‘trebbiano toscano’, ‘albarola’/‘bianchetta genovese’. also ‘verdicchio’, one of the most prized white grape varieties of the marche region (central italy) and registered in the italian catalogue with code no. 254, has an officially recognized duplicate, ‘trebbiano di soave’ (code no. 239), the name used in several provinces of the veneto region (north-eastern italy). other synonyms in the same region are ‘trebbiano di lugana’ and ‘turbiana’. in the lazio region (central italy) it is known as ‘trebbiano verde’ (bruni, 1962). to our knowledge, the first citation of ‘verdicchio’ was by the botanist costanzo felici as far back as 1569 (in arbizzoni, 1986). ‘verdello’ is considered a minor grapevine variety of the umbria region (central italy). it is registered in the italian catalogue with code no. 253 and is used for the doc wines of orvieto, colli amerini, colli del trasimeno and torgiano. scalabrelli and grasselli (1988) report that ‘verdello’ is also cultivated in the south of tuscany under the synonym ‘duropersico’, mainly in the territory of pitigliano doc, where it has been present for at least two centuries and contributes to the wine of the same name. the two authors refer that in the same territory a variety known as ‘uva angiola’ is also present; they generically link it to the trebbiano group, but this variety more specifically resembles to those analyzed in the present study. furthermore, in the most northern part of tuscany, a variety morphologically similar to ‘verdicchio’ has been grown for a long time. it is called ‘verduschia’ (soderini, 1600; acerbi, 1825), ‘verdella’ or ‘verdarella’, depending on the cultivation area. the diffusion of this variety is limited to the small lunigiana territory (provinces of la spezia and massa carrara) and to a few residual specimens in old vineyards (scalabrelli and dodi, 1998). ‘verduschia’ falls into the category of minor grapevine varieties on the ‘verdello’, ‘verdicchio’ and ‘verduschia’: an example of integrated multidisciplinary study to clarify grapevine cultivar identity m. crespan*, a. armanni**, g. da rold*, b. de nardi*, m. gardiman*, d. migliaro*, s. soligo***, p. storchi** * consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la viticoltura, cra-vit, viale xxviii aprile, 26, 31015 conegliano (tv), italy. ** consiglio per la ricerca e la sperimentazione in agricoltura, unità di ricerca per la viticoltura, cra-vic, via romea, 53, 52100 arezzo, italy. *** centro regionale per la viticoltura, l’enologia e la grappa, cerveg, veneto agricoltura, via zamboni, 31015 conegliano (tv), italy. key words: ampelography, ‘duropersico’, ssr markers, ‘trebbiano di soave’, ‘turbiana’, ‘uva angiola’. abstract: ‘verdello’, ‘verdicchio’ and ‘verduschia’ are registered in the official italian catalogue as three distinct grapevine varieties. twenty-five accessions of these cultivars, encompassing known or presumed synonyms, coming from cra repositories and from vineyards where they are traditionally cultivated, have been genotyped with eleven ssr markers. for morphological comparison, one accession for each variety has been described with 57 characters of oiv 2009 list; phenological and yield traits have also been recorded. in addition, the phenotypic comparison has been extended to the literature descriptions. the same dna profile has been obtained for all 25 accessions; moreover, present and historical ampelographic data showed a very high similarity. all this information leads to the conclusion that these three varieties are, in fact, the same cultivar. adv. hort. sci., 2012 26(2): 92-99 received for publication 16 february 2012. accepted for publication 31 may 2012. 93 verge of extinction; although registered in the italian catalogue since 1971 with code no. 297, it does not seem to have been propagated. dna genotyping allowed us to hypothesise an unrecognized synonymy among ‘verdicchio’, ‘verdello’ and ‘verduschia’. in fact, based on previous results obtained with molecular analysis of some accessions of ‘verdello’, interesting for the clonal selection of materials from lazio, we found that they had the same profile of the ‘verdicchio’ accession held in the centro di ricerca per la viticoltura (cra-vit) repository. given the interest aroused by this preliminary information, we extended the molecular comparison to numerous accessions of ‘verdicchio’, ‘verdello’ and their synonyms, as well as to ‘verduschia’, the tuscan variety morphologically similar to ‘verdicchio’. the samples for genotypic comparison were singled out both in the cra-vit repositories and in the cultivation areas of each variety, for a total of 25 accessions, including a commercial clone of ‘verdello’ (clone vcr1) and another of ‘verdicchio bianco’ (clone r2). this study was integrated with morphological comparison: one accession for each variety was characterized with 57 descriptors of the oiv 2009 list; phenological and yield traits were also recorded. in addition, ampelographic comparison was extended to the literature descriptions of these three varieties: most of the traits reported in literature were retrieved and harmonized according to the oiv 2009 descriptors list used for examining the actual materials. 2. materials and methods plant material twenty-five accessions were sampled, coming from cra-vit repositories in spresiano (tv) and susegana (tv), from unità di ricerca per la viticoltura (cra-vic) repositories in arezzo, from the veneto agricoltura collection and from vineyards in veneto, tuscany, umbria, marche and lazio regions (table 1). ampelographic, phenological and yield data comparison three accessions were described: one ‘verdello’ of umbrian origin (accession no. 4 in table 1) and one ‘verduschia’ (no. 15 in table 1) coming from lunigiana (province of massa carrara), both held in the cra-vic germplasm collection in arezzo, and one ‘verdicchio’ coming from marche (no. 25 in table 1), held at the azienda poggio gagliardo in montescudaio (pisa, italy). morphological descriptions of the three varieties were performed according to 57 descriptors (oiv, 2009); phyllometric analyses were also carried out on samples of 20 leaves per cultivar using superampelo software (soldavini et al., 2009). the comparison was widened to the ‘verdello’, ‘verdicchio’ and ‘verduschia’ descriptions given in the literature: in particular, the umbrian ‘verdello’ was described by cartechini and moretti (1989), the ‘verdicchio’ from marche by bruni (1962) and the tuscan ‘verduschia’ by breviglieri and casini (1965). to facilitate the comparison, all these descriptions were standardized according to the oiv 2009 descriptor list. dna extraction total genomic dna was extracted from young leaves of the 25 accessions using nucleospin® 8 plant kit (macherey-nagel gmbh, düren, germany) automated on the microlab® star liquid handling robot according to the macherey-nagel nucleospin® 8 plant protocol. dna concentration and quality were assessed with a spectrophotometer and by 1% agarose gel electrophoresis. dna samples were then diluted to 10 ng/µl prior to amplification. ssr analysis ssr analysis was performed in order to verify the varietal identity of the studied accessions. eleven microsatellite loci were analyzed: the six core loci selected within genres 081 european project (vvs2, vvmd5, vvmd7, vvmd27, vrzag62 and vrzag79) (this et al., 2004); vvmd28 (bowers et al., 1999); isv2 (vmc6e1), isv3 (vmc6f1) and isv4 (vmc6g1) (crespan, 2003); vmcng4b9 (welter et al., 2007). a multiplex pcr mixture was prepared, amplifying simultaneously all 11 ssr loci. the reaction mixture consisted of: 1 x pcr buffer (promega; pharmacia biotech), 200 µm of each dntps, 1 u of taq dna polymerase (promega; pharmacia biotech), 2.0 mm mgcl 2 and the primer concentrations ranged between 0.09 µm and 0.40 µm, according to signal  intensity; the forward primers were labelled with 6-fam, vic, pet or ned fluorescent table 1 list of the analysed accessions id accession name provenance 1 verdicchio cra-vit repository 2 verdello cra-vit repository 3 verdello clone vcr1 vivai cooperativi of rauscedo (pordenone) italy 4 verdello covio 6 orvieto (terni) italy 5 verdello sugano orvieto (terni) italy 6 verdello fausto 1 porano (terni) italy 7 verdello 553 ercolani farm, capodimonte (viterbo) italy 8 verdello 550 ercolani farm, capodimonte (viterbo) italy 9 verdello 549 ercolani farm, capodimonte (viterbo) italy 10 verdello 514 catercia farm, capodimonte (viterbo) italy 11 verdello 405 bianchi farm, bagnoreggio (viterbo) italy 12 verdello 146 ar pitigliano (grosseto) italy 13 verdello lazio 14 verdello cra-vit repository 15 verduschia aulla (massa carrara) italy 16 verdella aulla (massa carrara) italy 17 verdicchio carr 10 terranuova bracciolini (arezzo) italy 18 turbiana vicenza italy 19 trebbiano verde viterbo italy 20 trebbiano di soave cra-vit repository 21 trebbiano di soave cra-vit repository 22 trebbiano di lugana veneto 23 duropersico 165 ar pitigliano (grosseto) italy 24 uva angiola cra-vit repository 25 verdicchio clone r2 marche 94 dyes; the final volume was 12.5 µl. pcr was carried out in geneamp 9700 (applied biosystems) with the following thermal profile: 2 min at 94°c, followed by 30 cycles at 94°c for 45 sec, 55°c for 1 min and 30 sec, 72°c for 1 min and a final step at 72°c for 30 min. pcr products (1 µl) were added to 0.1 µl liz 500 size standard and 8.9 µl hi-di formamide (applied biosystems) and separated by capillary electrophoresis using an abi prism 3110xl dna analyzer (applied biosystems) and pop-7 polymer (applied biosystems). after data collection, genotyping analysis was performed with abi prism® genemappertm software version 3.0. 3. results the results of the ampelographic comparison between ‘verdello’, ‘verdicchio’ and ‘verduschia’ are reported in table 2. the descriptions from the present study show that the three grapevine varieties share 47 out of 57 traits. ten diverge (in bold in table 2): in particular, ‘verdello’ differs from ‘verdicchio’ and ‘verduschia’ by having a slight bronze tinge on the young leaf and the mature leaf with less pronounced lower lobes and shorter petiole compared to the length of the middle vein. ‘verdicchio’ showed a larger average size of the mature leaf, a longer middle vein n1 and a greater density of prostrate hairs on the lower side of the blade (descriptor no. 84), as well as longer internodes (no. 353). finally, three morphological traits differ in ‘verduschia’ with respect to ‘verdello’ and ‘verdicchio’: the dorsal side of the shoot is green (oiv codes 007 and 009) and berry size is smaller. therefore, comparison of our descriptions shows a substantial analogy among these varieties, which share 82% of the expression levels, with very minor discrepancies. their similarity is also confirmed by the elaboration of the mature leaf measurements with superampelo software: the cluster analysis performed by the program shows that the degree of similarity is even higher and above 93% (fig. 1). fig. 1 cluster analysis of ‘verdello’, ‘verdicchio’ and ‘verduschia’ phyllometric data elaborated with superampelo software. percentage of similarity values are indicated. table 2 list of ampelographic traits of the varieties ‘verdello’, ‘verdicchio’ and ‘verduschia’. there are two types of comparisons: i) among current descriptions: discordant characters observed in the present study are written in bold; ii) among descriptions from other authors: slightly discordant traits inferred from the literature are underlined; clearly discordant characters are underlined in italics. an asterisk indicates the characters defined by the software superampelo. nr: not recorded character. organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 sh oo t 002 young shoot: distribution of anthocyanin coloration on prostrate hairs of the shoot tip absent absent absent absent absent piping 003 young shoot: intensity of anthocyanin coloration on prostrate hairs of the shoot tip none or very low none or very low none or very low none or very low none or very low low 004 young shoot: density of prostrate hairs on the shoot tip medium very high medium very high medium medium or very high 006 attitude semi-erect semi-erect semi-erect semi-erect semi-erect semi-erect 007 colour of the dorsal side of internodes green and red green and red green and red green and red green green 008 colour of the ventral side of internodes green and red green and red green and red green and red green and red green and red 009 colour of the dorsal side of nodes green and red green and red green and red green and red green green 010 colour of the ventral side of nodes green and red or red green green and red or red green and red green and red green and red 013 density of prostrate hairs on nodes low none or very low low none or very low low low 015_2 intensity of anthocyanin coloration on the bud scales weak weak weak nr weak nr 016 number of consecutive tendrils 2 or less 2 or less 2 or less 2 or less 2 or less 2 or less 95 organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 yo un g le af 051 colour of upper side of blade (4th leaf) green / bronze / yellow green green / yellow green / bronze green / yellow green 053 density of prostrate hairs between main veins on lower side of blade (4th leaf) high very high high very high high very high 055 density of prostrate hairs on main veins on lower side of blade (4th leaf) low low low nr low nr m at ur e le af 065 size of blade medium-small* medium medium* medium medium-small* medium-small 067 shape of blade wedge-shaped or pentagonal* circular wedge-shaped or pentagonal* circular or pentagonal wedge-shaped or pentagonal* pentagonal 068 number of lobes five or three five five five or three five five or three 069 colour of the upper side of the blade between medium green and dark green medium green between medium green and dark green between medium green and dark green between medium green and dark green dark green 070 area of anthocyanin coloration of main veins on upper side of blade absent absent absent absent absent absent 071 area of anthocyanin coloration of main veins on lower side of blade absent absent absent absent absent absent 073 undulation of blade between main or lateral veins present present present present present present 074 profile of blade in cross section involute, v-shaped or twisted v-shaped involute, vshaped or twisted flat or twisted involute, vshaped or twisted flat or twisted 075 blistering of upper side of blade medium weak medium medium medium nr 076 shape of teeth mixture between straight and convex sides one side concave, one side convex mixture between straight and convex sides mixture between straight and convex sides mixture between straight and convex sides both sides convex 078 length of teeth compared with their width short short short short short short 079 degree of opening/overlapping petiole sinus overlapped strongly overlapped overlapped closed or overlapped overlapped overlapped 084 density of prostrate hairs between main veins on lower side of blade medium medium high very high medium very high 086 density of prostrate hairs on main veins on lower side of blade low low low high low nr 090 density of prostrate hairs on petiole low none or very low low none or very low low none or very low 093 length of petiole compared to length of middle vein (n1) shorter than n1 longer than n1 equal to n1 nr equal to n1 nr 601 length of vein n1 short* nr medium* nr short* nr 602 length of vein n2 medium* nr medium* nr medium* nr 603 length of vein n3 medium* nr medium* nr medium* nr 604 length of vein n4 very long* nr very long* nr very long* nr w oo dy sh oo t 101 cross section circular or elliptic circular circular or elliptic circular or elliptic circular or elliptic circular or elliptic 103 main colour brownish brownish brownish between brownish and grey brownish brownish in flo re sc en ce 151 flower: sexual organs hermaphrodite hermaphrodite hermaphrodite hermaphrodite hermaphrodite hermaphrodite 152 insertion of 1st inflorescence 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 153 number of inflorescences per shoot 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 96 organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 bu nc h 202 length medium-long medium medium-long medium-long medium-long long 204 density dense very dense dense dense or medium dense medium 206 length of peduncle short very short short medium short short 208 shape conical / funnel shaped / cylindrical conical conical / funnel shaped / cylindrical conical or cylindrical-conical conical / funnel shaped / cylindrical conical 209 number of wings of the primary bunch 1-2/3-4 with wings 1-2/3-4 wings with wings 1-2/3-4 with wings be rr y 220 length medium medium medium medium between medium and short short 222 uniformity of size uniform uniform uniform uniform uniform uniform 223 shape globose globose globose globose globose globose 225 colour of skin yellow yellow yellow yellow yellow yellow 227 bloom medium medium medium medium medium medium 228 thickness of skin thin thick thin thin thin thick 229 hilum visible visible visible visible visible visible 241 formation of seeds complete complete complete complete complete complete ve ge ta tio n 306 autumn coloration of leaves yellow yellow yellow yellow yellow yellow 351 vigour of shoot growth medium-strong medium medium-strong medium-strong medium-strong medium-strong 352 growth of lateral shoots weak weak weak medium weak weak 353 length of internodes medium long long medium-long medium medium 354 diameter of internodes medium small medium medium medium medium with regard to the descriptions from the literature, most of the 57 oiv descriptors we used were retrieved. only the measurements from code 601 to 604 relative to the mature leaf, three traits of ‘verdicchio’ and five of ‘verduschia’ were excluded (indicated as not recorded characters in table 2) and so a total of 50 descriptors for ‘verdello’ and ‘verdicchio’ and 48 for ‘verduschia’ were taken into consideration. as expected, the literature data comparison highlighted greater differences, given that the three descriptions were made by different authors. twenty-eight descriptors, underlined in table 2, showed slightly discordant expression levels, as they are limited to a single interval or with different coexisting or intermediate expression levels. these are mainly characters whose expression may be influenced by environmental factors like the exposure to the light or by cropping factors and vegetative vigour, in addition to the subjectivity of the observer. other descriptors (7 out of 52), underlined in italics in table 2, showed markedly different expression levels. four of them regard the mature leaf and in particular the density of prostrate hairs on the lower side of the blade (no. 84 and 86) and the profile and shape of the teeth (no. 74 and 76); the other three regard the bunch and the berry (no. 204, bunch density; no. 206, peduncle length; no. 228, thickness of skin). comparing this last group of seven morphological features with the set of results from our field measurements, it appears that our descriptions match better with those of ‘verdicchio’ described by bruni (1962); instead, fewer similarities are noted with the descriptions in the literature for ‘verduschia’ and even fewer for ‘verdello’. lastly, the comparison of the phenological and yield data collected in the two environments [arezzo and montescudaio (pisa)] are reported in table 3. the data obtained from the same references for ampelographic descriptions are also shown for comparison and ‘trebbiano toscano’ is proposed as common reference variety to enhance data significance. ‘verduschia’ differs by the longer vegetative cycle and the lower berry weight. instead, there are no relevant differences between the other two varieties. molecular analysis of the 25 accessions listed in table 1 produced the same dna profile, reported in table 4 together with ‘sangiovese’, ‘pinot noir’ and ‘muscat blanc à petits grains’ profiles, proposed as reference varieties for easier data comparison. to our knowledge this is the first time that the synonymy with ‘trebbiano verde’ from lazio, ‘duropersico’, ‘verduschia’ or ‘verdella’ and ‘uva angiola’ from tuscany and ‘turbiana’ from veneto, is confirmed by molecular data. 97 table 3 phenology and yield data for ‘verdello’, ‘verdicchio’ and ‘verduschia’: average of 2007-2010 for the vineyard in arezzo, average of 2004-2008 for the vineyard in montescudaio (pisa). the data from some reference ampelographic descriptions are also shown for comparison. ‘trebbiano toscano’ is proposed as common reference variety to enhance data significance ‘verdello’ ‘verdicchio’ ‘verduschia’ ‘trebbiano toscano’ place of data collection arezzo1 orvieto (terni)2 montescudaio (pisa)3 iesi (ancona)4 arezzo1 firenze5 arezzo1 montescudaio (pisa)3 bud burst 13 april (medium) medium 7 april (medium) medium-late 12 april (medium) 10 20 april (medium) 16 april (medium) 7 april (medium) flowering 3 june (medium early) medium 2 june (medium -early) early 7 june (medium) 1 10 june (medium) 8 june (medium) 3 june (medium) veraison 11 august (medium) medium 5 august (medium) medium 13 august (medium late) 21 31 august (late) 17 august (medium late) 6 august (medium) ripening 25 september (medium) late 20 september (medium) medium-late 27 september (late) 1 10 october (late) 4 october (late) 28 september (late) average weight of the bunch (g ± sd) 320±95 269 455±43 280 352±66 245 357±51 402±60 average weight of the berry (g ± sd) 1.87±0.18 1.80 1.94 2.05 1.60±0.64 0.80 1.71±0.21 1.82±0.19 ° brix (± sd) 22.0±1.2 22.3 21.4±0.6 20.0 20.6±0.7 21.0 18.9±1.2 21.1±0.8 titratable acidity (g/l ± sd) 6.71±1.36 8.90 6.80±0.40 8.25 6.42± 0.75 6.97 7.32±0.48 5.29±0.62 1 cra-vic repository, lon: 11°49’29’’ e, lat: 43°28’30’’ n. 2 data from cartechini and moretti (1989). lon: 12° 12’ e, lat: 42° 42’ n. 3 poggio gagliardo farm, lon: 10°32’53’’ e, lat: 43°18’52’’ n. 4 data from bruni (1962), lon: 13° 11’ e, lat: 43° 38’ n. 5 data from breviglieri and casini (1965), lon: 11°19’21’’ e, lat: 43°45’11’’ n. table 4 ssr profiles of ‘verdicchio’/‘verdello’/‘verduschia’ and three cultivars proposed as reference to favour comparison with other databases ssr loci verdicchio, verdello and verduschia sangiovese pinot noir muscat blanc à petits grains vvs2 133 133 137 133 155 133 151 133 vvmd5 228 226 228 228 240 236 238 236 vvmd7 239 239 239 233 247 263 243 249 vvmd27 179 179 185 179 185 185 189 194 vvmd28 239 237 221 249 261 247 239 271 vrzag62 195 193 187 185 195 195 193 195 vrzag79 248 242 238 250 256 258 244 254 isv2 (vmc6e1) 165 143 151 141 165 165 165 143 isv3 (vmc6f1) 135 139 133 133 139 139 145 139 isv4 (vmc6g1) 169 177 169 169 197 197 177 187 vmcng4b9 164 158 158 158 166 168 162 166 98 4. discussion and conclusions the large number of accessions analyzed, most of which come from traditional cultivation areas, together with the results of the ampelographic comparisons, allow us to affirm that ‘verdicchio’, ‘verdello’ and ‘verduschia’ represent a new group of synonyms. this discovery is important because the three varieties are registered as distinct in the italian catalogue. this is further supported by the results of the molecular analysis on the commercial clone of ‘verdello’ (clone vcr1), which showed to be identical to the other ‘verdello’ accessions analyzed in this study. it emerges from present work that the extent of the cultivation area in italy and the interest in ‘verdicchio’/ ‘verdello’/‘verduschia’ are greater than previously supposed from the information available on the already known synonyms. according to the italian census of agriculture data (istat, 2004) the total area under ‘verdicchio’, ‘verdello’, ‘verduschia’ and ‘trebbiano di soave’ cultivation in italy is about 6000 ha, mainly in marche (53%) and veneto (30%) and, to a much lesser extent, in umbria (8.3%) and lazio (1.6%). cultivation is also authorized in emilia romagna, tuscany, abruzzo, molise and even in sardinia. the success with which this variety has been cultivated for centuries in central and north-eastern italy is obvious; the broad and historic diversification of the denominations, even in very close areas, supports the old age of the variety and also the interest in the locally produced wines, with recognised quality. the synonymy ‘verdicchio’/‘verdello’/‘verduschia’ is added to a growing number of redundancies found in the italian catalogue and highlights the value of molecular analysis to facilitate rapid comparison among varieties. this tool has been very useful in revealing other, never previously suspected cases of synonymy, such as ‘greco di tufo’ and ‘asprinio’ (costantini et al., 2005), ‘malvasia delle lipari’ and ‘malvasia di sardegna’ (crespan et al., 2006), or to definitively clarify long disputed synonymies, such as ‘malvasia nera di brindisi’ and ‘malvasia nera di lecce’ (crespan et al., 2008; gasparro et al., 2008). it is extremely difficult to hypothesize the centre of diffusion of ‘verdicchio’/‘verdello’/‘verduschia’, also because the pedigree of this variety is unknown. one not scientifically supported hypothesis infers that this cultivar arrived in marche from northern italy in the second half of the 15th century, with colonies of farmers from veneto and lombardy, and from there it moved to lazio, to finally turn up in tuscany (pollini, 2006). the discovery of this new group of synonyms has been greatly facilitated by the use of ssr markers, which suggest comparisons among varieties independently from preliminary ampelographic indications. given the wealth of varieties registered in the italian catalogue, molecular analysis, associated with the building of a reliable and complete molecular database of reference, has been shown to be very useful to highlight redundancies. ampelographic comparison is indispensable to confirm the preliminary data acquired via molecular fingerprinting. indeed, the definition of a cultivated variety is tied to the dus criteria (distinctness, uniformity and stability), since the somatic mutants for agronomically important characters are legally registered as separate cultivars, as are mutants for berry colour or earliness of ripening. lastly, genotyping represents a strategic tool in order to avoid the recording of duplicates in the future. acknowledgements this research was financed by the ministry of agricultural, food and forestry policies as part of the rgv-fao and aser-identivit projects, and with funds from the regional agency ‘veneto agricoltura’. the authors thank francesco anaclerio (vcr) for providing the clone of ‘verdello’ vcr1 and marina niero for the information on the clonal selection of ‘verdello’ in the lazio region. references acerbi g., 1825 delle viti italiane. silvestri, milano. arbizzoni g., 1986 costanzo felici, scritti naturalistici. dell’insalata e piante che in qualunque modo vengono per cibo del’homo. quattroventi, urbin, italy, pp. 105. bowers j.e., dangl g.s., meredith c.p., 1999 development and characterisation of additional microsatellite dna markers for grape. am. j. enol. vitic., 50: 243-246. breviglieri n., casini e., 1965 verduschia. in: i principali vitigni da vino coltivati in italia. vol. iv, ministero dell’agricoltura e delle foreste, rome, italy. bruni b., 1962 verdicchio bianco. in: i principali vitigni da vino coltivati in italia. vol. ii, ministero dell’agricoltura e delle foreste, rome, italy. cartechini a., moretti g., 1989 verdello, pp. 679690. in: ministero dell’agricoltura e delle foreste i principali vitigni da vino coltivati in italia, nuova serie. vol. i:. ministero dell’agricoltura e delle foreste, istituto sperimentale di viticoltura di conegliano, grafiche zoppelli, dosson (treviso), italy. costantini l., monaco a., vouillamoz j., forlani m., grando m.s., 2005 genetic relationships among local vitis vinifera cultivars from campania (italy). vitis, 44(1): 25-34. crespan m., 2003 the parentage of muscat of hamburg. vitis, 42(4): 193-197. crespan m., cabello f., giannetto s., ibáñez j., kontić j.k., maletić e., pejić i., rodriguez i., antonacci d., 2006 malvasia delle lipari, malvasia di sardegna, greco di gerace, malvasia de sitges and malvasia dubrovačka synonyms of an old and famous grape cultivar. vitis, 45(2): 69-73. crespan m., coletta a., crupi p., giannetto s., antonacci d., 2008 ‘malvasia nera di brindisi/lecce’ grapevine cultivar (vitis vinifera l.) originated from ‘negroamaro’ and ‘malvasia bianca lunga’. vitis, 47(4): 205-212. 99 gasparro m., caputo a.r., brini m.l., coletta a., crespan m., la penna f., antonacci d., 2008 characterization of malvasia nera accessions with molecular markers. cd-rom, section varieties: knowledge, characterization, valorization. proceedings of the 31th oiv congress held in verona, 15-20 june. istat, 2004 5° censimento generale dell’agricoltura 2000, “la coltivazione della vite in italia”. volume ii. vitigni. istat, rome, italy. oiv, 2009 oiv descriptor list for grape varieties and vitis species (2nd edition). http://www.oiv.int/oiv/info/frplubicationoiv. pollini l., 2006 viaggio attraverso i vitigni autoctoni italiani. alsaba, siena, italy, pp. 481 scalabrelli g., dodi s., 1998 caratteristiche ampelografiche di alcuni vitigni ad uva bianca reperiti in lunigiana. proceedings “il recupero del patrimonio viticolo della lunigiana”, aulla, massa-carrara, italy, pp. 77-130. scalabrelli g., grasselli a., 1988 recupero dei vitigni coltivati nella zona del “bianco di pitigliano”. l’informatore agrario, 41(5): 59-66. soderini g., 1600 trattato della coltivazione delle viti. filippo giunti, firenze. soldavini s., stefanini m., dallaserra m., policarpo m., schneider a., 2009 superampelo: a software for ampelometric and ampelographic descriptions in vitis. acta horticiculturae, 827: 253-258. this p., jung a., boccacci p., borrego j., botta r., costantini l., crespan m., dangl g.s., eisenheld c., ferreira-monteiro f., grando m.s., ibáñez j., lacombe t., laucou v., magalhães m., meredith c.p., milani n., peterlunger e., regner f., zulini l., maul e., 2004 development of a standard set of microsatellite reference alleles for identification of grape cultivars. theor. appl. genetics, 109(7): 1448-1458. welter l.j., göktürk-baydar n., akkurt m., maul e., eibach r., töpfer r., zyprian e.m., 2007 genetic mapping and localization of quantitative trait loci affecting fungal disease resistance and leaf morphology in grapevine (vitis vinifera l.). mol. breed., 20: 359-374. 223 1. introduction celosia, a c3 plant, belongs to the amaranthaceae family and is of tropical origin. in ghana, celosias are not only grown as a cut flower crop, but also as bedding plants, pot plants, and vegetable crops (norman, 2004). in this study, disbudding entailed the removal of axillary buds or bud breaks on a single stem, leaving the terminal flower bud intact and able to develop into a large flower head. this practice is reported to be a standard operation in the cultivation of roses, carnations, chrysanthemums and celosias (machin and scopes, 1978; janick, 1986; dole and wilkins, 1999; norman, 2004). celosia requires one stem as a cut flower; here developing flower buds on a flowering shoot must be disbudded in order to improve the quality of the terminal flower (norman, 2004). reports indicate that disbudding increases plant height in dahlias (parshall, 2007) and in celosias reduces the number and size of undesirable side (or axillary) shoots on the flower stem and thus resulting in increased plant height, flower stem length and flower head size (norman et al., 2009). also, disbudding induces early harvesting and a more concentrated harvesting period (norman, 2004). growth analysis has been widely used to study yieldinfluencing factors and plant development as net photosynthates accumulation over time (gardner et al., 1985). this approach uses simple primary data in the form of weights, areas, volumes and contents of plant components to investigate processes within and involving the whole plant (hunt, 1990). the leaf area index (lai) of a crop at a particular growth stage indicates its photosynthetic potential or the level of its dry matter accumulation. the greater the lai, the higher the dry matter accumulation potential of the crop and vice versa (rasheed et al., 2003). its value can vary with environmental and cultivation conditions (board and harville, 1992). the leaf area and its duration (lad) are measurements of growth of plants and plant physiological processes (miralles et al., 1997). lai and lad control the total production of dry matter and subsequently yield and yield attributes (jirali et al., 1994). crop growth rate (cgr) is a prime factor in determining crop yield because it reflects the capacity of assimilates production and affects dry matter accumulation. there is a close association between maximum dry matter production and maximum cgr (ball et al., 2000). the analysis of cgr has been shown to be important in evaluating treatment differences among crop species or cultivars with species in relation to yield (fageria et al., 2006). relative growth rate (rgr) is described as the rate of increase of total dry weight per plant (hunt, 2003). relative growth rate curves of crops are in opposition to dm accumulation during the life cycle of crops (fageria et al., disbudding effects on growth analysis of celosia (celosia cristata) p. adjei-frimpong, j. ofosu-anim, j.c. norman department of crop science, university of ghana, po lg 44, legon, rupublic of ghana. key words: celosia cristata, disbudding, growth analysis. abstract: experiments to investigate the effects of disbudding on growth analysis of two celosia cultivars, ‘carmine’ and ‘chief gold’, were carried out on the field in 2009 and 2010 at the sinna garden of department of crop science, university of ghana, legon, accra, ghana. the treatments consisted of disbudding once, disbudding twice, and no disbudding, as control, and were arranged in a 3x2 split plot in a randomized complete block design with four replications in 2009 (experiment 1) and three in 2010 (experiment 2). the two cultivars were harvested weekly during the growing period and separated into the various plant parts and oven-dried for dry weights, using appropriate formulae to calculate the various growth parameters. analysis of variance (anova) was used to analyse the data and a correlation coefficient matrix showed relationships among growth parameters. disbudding resulted in increased leaf area index, leaf area ratio, leaf area duration, relative growth rate, and harvest index, but reduced crop growth rate and net assimilation rate. ‘chief gold’ had a higher harvest index than ‘carmine’. disbudding plants once gave the best flower head size and weight result. ‘carmine’ gave the best flower yield and quality results in experiment 1 and ‘chief gold’ in experiment 2. adv. hort. sci., 2011 25(4): 223-231 received for publication 7 june 2011 accepted for publication 12 october 2011 224 2006). results from the studies of medhet et al. (2000) on growth analysis of sunflower, helianthus annuus, under drought conditions indicated a reduction of rgr value from early growth stages to final stages. however, recent reports by fageria et al. (2006) indicate that values of rgr are generally higher during early growth stages of the crop and decrease with age. measurement of net assimilation rate (nar) is important to detertmine the efficiency of plant leaves for dm production. nar values decrease with crop growth due to both the shedding of leaves and reduced photosynthetic efficiency of older leaves (fageria et al., 2006). similarly, law-ogbomo and egharevba (2008) reported that abscission with plant growth of the lower leaves in tomato causes a decrease in nar. however, there is no detailed information on the quantitative growth aspects and growth analysis of celosia cristata grown for cut flower production. the only previous reference to growth characteristics is that of celosia argentea (grown as a leafy vegetable crop) by ojo (2001) who reported a positive relationship between yield and leaf area, which was enhanced by increasing population density and cutting height. the present experiment was therefore undertaken to investigate the effects of disbudding on the growth indices of two cultivars (carmine and chief gold) of celosia cristata and to identify relationships between these indices (parameters) and flower yield. 2. materials and methods experimental site the study was conducted at the sinna garden, department of crop science, university of ghana, legon, between july and september 2009 for experiment 1 and december 2009 to february 2010 for experiment 2. the soil at the experimental site is of the adenta series (brammer, 1960) and classified as ferric acrisol (fao/unesco, 1990). the soil is sandy loam and moderately well drained with moderate levels of organic matter. climatological data during the experimental period are shown in table 1. experimental design a randomized complete block design with split plot arrangement and cultivars as the main plots and disbudding as the subplots were used for the experiment. there were four replications in experiment 1 and three in experiment 2. the disbudding treatments were: disbudding once; disbudding twice; and no disbudding (as control). the cultivars used were ‘carmine’ and ‘chief gold’. the plants were established at a spacing of 15 x 9 cm. there were 90 plants (experiment 1) and 96 plants (experiment 2) per sub-plot in which five plants were sampled weekly for dry weights and 10 plants were tagged for field data collection. cultivation practices in experiment 1, seeds were first sown in seed boxes using sandy soil on 17 july 2009 and the germinated seedlings were planted in the field on 7 august 2009. before planting, each sub-plot (0.9 x 1.5 m) received an application of 15-15-15 npk fertilizer at the rate of 674 kg/ha on 6 august 2009. in experiment 2, cow dung was incorporated into the plots at 25 t/ha on 8 december 2009. seeds were sown in plastic seed trays on 16 december 2009 using peat as the soil mix and the germinated seedlings were pricked out into plastic seed trays on 30 december 2009. the seedlings were planted in the field on 13 january 2010. a day before planting, each sub-plot (0.99 x 1.65 m) received an application of 15-15-15 npk fertilizer at the rate of 600 kg/ha. in each experiment, hand watering was done twice a day. routine weed control was carried out either by handpicking of weeds or by hoeing when necessary. diseases and insect pests were controlled by spraying of insecticide and fungicide. dithane m45 was sprayed on 19 august 2009 and 11 february 2010, in both experiments respectively, to control leaf spot diseases. on 2 september 2009, 28 january and 3 february 2010, in both experiments respectively, cydim super was also sprayed to control grasshoppers and whiteflies. in experiment 1, plants were sidedressed four weeks after planting with potassium nitrate at table 1 climatological data during experimental period month mean maximum temperature (°c) mean minimum temperature (°c) total rainfall (mm) mean maximum relative humidity (%) mean minimum relative humidity (%) experiment 1 2009 july 28.3 23.3 91.5 94 77 august 28.3 23.0 11.5 92 74 september 30.5 23.2 6.3 91 69 experiment 2 2009 december 2010 33.4 24.8 10.3 92 64 january 33.3 25.0 49.6 94 65 february 33.7 25.4 57.2 94 66 source: meteorological services of ghana, mempeasem, accra, ghana. 225 a rate of 100 kg/ha while in experiment 2, side-dressing was done at three weeks after planting at the same rate. disbudding disbudding was carried out as follows: 1. disbudding once: axillary flower heads and side shoots were removed on all the plants in the field except the control plants at 22 days after planting (dap) on 29 august 2009 and at 18 dap on 1 february 2010. 2. disbudding twice: the removal of axillary flower heads and side shoots was undertaken on only the plants designated for this treatment at 27 dap on 3 september 2009 and at 25 dap on 8 february 2010. sampling sampling started two weeks after planting and every week thereafter until the sixth week in experiment 1; in experiment 2 it started a week after planting and every week thereafter until the fifth week. five plants were randomly sampled from each sub-plot, carefully dug up and the roots washed of soil particles. the leaf area was calculated using a leaf area meter (model am 100 by analytical development company limited, england). the plant parts (leaves, flower heads, flower stems, side shoots, axillary flowers and roots) were separated and chopped into pieces and put in different sampling envelopes and ovendried to a constant weight of 80°c for 48 hr to determine their dry matter. two types of measurements are needed for growth analysis: the plant weight, usually the oven dry weight (g); and the size of the assimilating system, usually in terms of leaf area (cm2). the crop growth rate, net assimilation rate (nar), relative growth rate (rgr), leaf area index, leaf area ratio (lar), leaf area duration and harvest index were calculated as follows. leaf area index (lai) leaf area index is defined as leaf area per unit area of land. it is a dimensionless ratio (watson, 1947) and calculated with the formula: leaf area index = total leaf area land area crop growth rate (cgr) crop growth rate is defined as the increase in plant dry matter per unit of time per land area unit (radford, 1967) with the formula: cgr (gm-2 day-1) = w2 – w1 t2 – t1 relative growth rate (rgr) relative growth rate is the increase of plant material per time unit. it was calculated for each interval between sampling with the formula given by radford (1967). the rgr of the first harvest could not be calculated because there was no dry weight before the first harvest. rgr (mgg-1 day-1). = ln w2 – ln w1 t2 – t1 net assimilation rate (nar) the net assimilation (nar) is the increase of plant material per unit of the assimilating material per unit of time. it was calculated for each interval between two samplings with the formula described by watson (1947) and radford (1967). the nar of the first harvest could not be calculated because there was no leaf area value before the first harvest. nar (gm-2 day-1) = (w1)(w2) lad leaf area duration (lad) leaf area duration is the photosynthetic potential of a plant, i.e. a measurement of the entire opportunity for assimilation a plant possesses during a growth period (watson, 1947). this was calculated using the formula: lad = (lai1 + lai2) x (t2 – t1) 2 leaf area ratio (lar) leaf area ratio of a plant at an instant in time (t) is the ratio of the assimilatory material per unit of plant material present. the lar was calculated with the following formula: lar (cm2/g) = (lai1) – (lai2) (w1)(w2) harvest index (hi) the harvest index is the ratio of economic yield (flower head and stem) to biological yield (donald and hamblin, 1976). its computation uses the following formula: harvest index (hi) = economic yield (flower head and stem) biological yield (total dry weight) x 100 where w2 and w1 = dry weight at second and first harvest, t2 and t1= time corresponding to second and first harvest. leaf chlorophyll content leaf chlorophyll content was measured using a chlorophyll meter (model spad, minolta, japan). flower head size index this was calculated as the product of the vertical and horizontal lengths of the flower head divided by 2. number of side shoots this was obtained by stripping off side shoots on the flower stem and counting. 226 harvesting harvesting of the 10 tagged plants of each plot started 60 days after sowing for experiment 1 and 63 days after sowing for experiment 2. ‘carmine’ was harvested two days earlier than ‘chief gold’. statistical analysis the data collected were analysed using analysis of variance (anova) (genstat, ver. 9). significant differences among treatment means were determined using the least significant difference (lsd) test at p = 0.05. correlation analysis correlation analysis for flower quality parameters and other measured growth variables was also determined using spearman’s rank correlation coefficient. 3. results and discussion flower head dry weight and size tables 2 and 3 show the effects of disbudding and cultivar on flower head dry weight and size. disbudding significantly influenced flower head size production. in experiment 1, disbudding twice produced the heaviest flower heads with the control producing the lightest. plants subjected to disbudding once produced the heaviest flower heads with the control producing the lightest in experiment 2. larger flower heads were also produced by disbudding-once plants followed by disbudding-twice, with the control producing the smallest flower heads. ‘carmine’ produced significantly larger flower heads than ‘chief gold’ in experiment 1. however, the opposite was true in experiment 2. flower head size has the potential to increase when the sink-source ratio is reduced, i.e. when the number of competing sinks for assimilates is reduced or the source activity is increased (cockshull, 1982; lee et al., 2001). in the present study, disbudding increased flower head size significantly. similar observations were made by carvalho et al. (2006) in chrysanthemums and norman et al. (2009) in celosia. in a celosia plant, the axillary flower heads, roots, leaves and side shoots compete with the flower stem and head for assimilates. as the number of flower heads per plant increases, the flower head size tends to decrease. reducing the number of flower heads on a flower stem allows the plant to distribute assimilates to the terminal flower that then attains a larger size. competition among the terminal flowers as well as between the flower and the vegetative plant parts for available assimilates explains the smaller and lighter flower heads produced by the control plants. these experienced a high intra-plant competition for photosynthetic radiation, thus influencing the assimilate allocation to the terminal flower. an increased in the number of small flowers has also been reported in chrysanthemum by carvalho et al. (2006) as a result of removal of the terminal flower bud. crop growth rate (cgr) figure 1 shows the effects of disbudding and cultivar on cgr of celosia plants. significant interactions were observed but showed no differences among treatments at 3 wap in experiment 1. however, in experiment 2, disbudding significantly affected cgr with the control plants recording the highest cgr and this was significantly different from the other treatments. significant table 2 effects of disbudding on flower head dry weight and size of two celosia varieties at harvest. experiment 1 treatment flower head dry weight (g) flower head size index (cm) ‘carmine’ ‘chief gold’ mean ‘carmine’ ‘chief gold’ mean disbudding once 2.08 2.20 2.34 21.7 19.7 20.7 disbudding twice 2.48 1.81 1.94 18.9 15.0 17.0 control 1.09 0.84 0.97 12.2 9.5 10.9 mean 1.89 1.62 17.6 14.7 lsd(5%):cultivar ns ns lsd(5%):disb 0.56 7.20 lsd(5%):cultivar x disb 1.22 9.65 table 3 effects of disbudding on flower head dry weight and size of two celosia varieties at harvest. experiment 2 treatment flower head dry weight (g) flower head size index (cm) ‘carmine’ ‘chief gold’ mean ‘carmine’ ‘chief gold’ mean disbudding once 2.21 2.47 2.34 16.9 23.9 20.4 disbudding twice 2.11 2.13 2.12 18.1 20.2 19.1 control 1.43 1.02 1.08 11.9 10.2 11.1 mean 1.82 1.87 15.7 18.1 lsd(5%):cultivar ns ns lsd(5%):disb 0.36 3.50 lsd(5%):cultivar x disb 0.41 4.30 227 interactions were observed with ‘chief gold’ having significantly higher cgr than ‘carmine’ at the 3 wap. crop growth rate was observed to increase with plant development (fig. 1a and b). an increase in cgr was recorded for both experiments at the final sampling (5-6 wap). crop growth rate is a prime factor in determining crop yield because it reflects the capacity of assimilates production and affects dry matter accumulation. there is a close association between maximum dry matter production and maximum cgr (ball et al., 2000). the observed significant and positive correlation between total aboveground biomass and cgr (r = 0.241*) and (r = 0.245*) in both experiments, respectively, supports this hypothesis. celosia plants produced a lot of side shoots and axillary flower heads during growth. therefore, it can be speculated that the dm accumulated in these organs, in addition to the other plant organs, accounted for the higher cgr and also enhanced nar in the control plants (fig. 2a and b). crop growth rate was significantly and positively correlated with flower stem dry weight (r = 0.3*). leaf area index (lai) disbudding did not significantly affect lai at the initial growth stages. leaf area index was significantly different among the various treatments (fig. 3a and b) and at harvesting (5-6 wap), disbudding significantly affected lai in both experiments. in experiment 1, plants disbudded twice had the highest lai (2.47) followed by those disbudded once (2.43); the control plants produced the lowest (1.55). in experiment 2, plants disbudded once had the highest lai (2.58) followed by those disbudded twice (2.52), while the control plants had the lowest lai (1.83). all disbudded treatments had a significantly higher lai than the control treatments. ‘carmine’ produced a lot of leaves in experiment 1, and they were broader than the ones of ‘chief gold’, hence ‘carmine’ had a higher lai. ‘chief gold’ responded earlier to disbudding than ‘carmine’ in lai as the control plants recorded lower values right from the initial stages. fig. 1 crop growth rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief’ gold (b). experiment one experiment two fig. 2 effects of disbudding and cultivar on side shoot production: ‘carmine’ (a) and chief gold’ (b) cultivars of celosia over the growing period in both experiments. experiment one experiment two fig. 3 leaf area index as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two disbudding once disbudding twice control c ro p gr ow th r at e (m g-2 d ay -1 ) c ro p gr ow th r at e (m g-2 d ay -1 ) n um be r of s id e sh oo ts n um be r of s id e sh oo ts l ea f ar ea in de x l ea f ar ea in de x l ea f ar ea in de x l ea f ar ea in de x n um be r of s id e sh oo ts n um be r of s id e sh oo ts disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice controldisbudding once disbudding twice control disbudding once disbudding twice control weeks after planting c ro p gr ow th r at e (m g-2 d ay -1 ) c ro p gr ow th r at e (m g-2 d ay -1 ) weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting disbudding once disbudding twice control 228 the lai determines the photosynthetic capacity of a crop. the higher lai of the disbudded treatments means that there were more (expanded) leaves per plant for higher radiant energy interception for photosynthesis and, therefore, more dry matter partitioning into the economic yield. this assertion is supported by the significant positive correlation between chlorophyll content and lai in experiment 1 (r = 0.040*). a significantly higher number of leaves per plant was produced by ‘carmine’ (17.44) than ‘chief gold’ (12.82) (fig. 4). the lower lai induced by the control treatments might be due to a lower leaf number and area which might have resulted from the competition among the various plant parts for assimilate partitioning. maximum dm production is achieved at an optimal lai. the optimal lai obtained for disbudded plants in celosia is between 2 and 2.5. although the control plants had a lower lai, they had the highest cgr. previous reference to growth characteristics of celosia argentia was made by ojo (2001) who reported a positive relationship between yield and leaf area. the results of experiment 2 confirm what reported above this. a linear relationship was observed between total aboveground biomass and lai (r = 0.040*). however, total aboveground biomass had a significantly negative relationship with lai (r = -0.343*) in experiment 1. lai had a positive and significant association with flower stem dry weight (r = 0.04*) and flower head dry weight (r = 0.07*). relative growth rate (rgr) in both experiments, rgr decreased linearly during the early growth and increased towards maturity (fig. 5a and b). in experiment 1, plants disbudded twice exhibited a higher rgr than control and disbudded-once plants. however, in experiment 2, plants disbudded once had a higher rgr than the other treatments. ‘carmine’ exhibited a higher rgr in experiment 1 than ‘chief gold’ in experiment 2. the observed decrease in rgr may be attributed to the decreasing trend in leaf area ratio (lar) with plant growth as indicated by the linear relationship between lar and rgr (r = 0.343*), (r = 0.168*) in both experiments, respectively. relative growth rate had a positive and significant association with flower stem dry weight (r = 0.12*) and flower head dry weight (r = 0.39*). increased rgr due to disbudding also resulted in increased flower yield. relative growth rate also had a significant negative relationship with cgr (r = -0.04*). net assimilation rate (nar) net assimilation rate showed no significant differences among treatments at 3 wap in experiment 2 (fig. 6a and b). in experiment 1, disbudding did not affect nar significantly; in experiment 2 disbudding lowered nar significantly. correlation analysis shows that nar had a negative and significant correlation with flower head dry weight (r = -0.05*) and flower stem dry weight (r = -0.02*) in experiment 1 but correlated positively and significantly with flower head dry weight (r = 0.13*) in experiment 2. thus, the lower nar observed in experiment 2 was compensated for bigger flower head production. generally, ‘chief gold’ had a higher nar than ‘carmine’. the decline in nar with plant growth observed in experiment 2 after disbudding might be due to both the shedding of leaves and reduced photosynthetic efficiency of older leaves (fafig. 4 leaf growth as influenced by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b) cultivars of celosia. experiment one experiment two fig. 5 relative growth rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two n um be r of le av es n um be r of le av es n um be r of le av es n um be r of le av es disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting r el at iv e gr ow th r at e (m g-2 d ay -1 ) r el at iv e gr ow th r at e (m g-2 d ay -1 ) r el at iv e gr ow th r at e (m g-2 d ay -1 ) r el at iv e g ro w th ra te (m g-2 d ay -1 ) 229 geria et al., 2006). similarly, law-ogbomo and egharevba (2008) reported that the abscission of the lower leaves with plant growth in tomato causes a decline in nar. leaf area ratio (lar) leaf area ratio decreased for both cultivars in both experiments with plant age. from 3-6 wap, disbudding significantly affected lar (fig. 7a and b). the disbudded plants had a significantly higher lar than the control plants. since lar indicates how much leaf area a plant produces per gram of dry matter, a high lar indicates that a plant is efficient at producing leaf area. since leaf area determines light interception, which is also an important parameter affecting plant growth, a high lar would be expected to result in a high growth rate (kang and van iersel, 2004). this further explains the linear relationship between lar and rgr (r = 0.343*), (r = 0.168*) in both experiments, respectively. leaf area ratio correlated negatively and significantly with flower stem dry weight (r = -0.05*) and flower head dry weight (r = -0.02*). leaf area duration (lad) the effect of disbudding and cultivar on lad is shown in figure 8a and b. disbudding affected lad but this was significant. however at harvesting (5-6 wap), significant differences were observed among disbudded treatments. significant interactions were also observed with all disbudded plants of ‘carmine’ producing a higher lad than that of ‘chief gold’ (fig. 8a and b). according to gifford and evans (1981), lad is more important for determining the final yield. however, in the current study, the higher lad of ‘carmine’ in both experiments did not lead to a yield advantage since ‘chief gold’ had a higher yield in terms of both the flower head and flower stem (economic sinks). fig. 6 net assimilation rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two fig. 7 leaf area ratio as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two fig. 8 leaf area duration as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting n et a ss im ila tio n ra te (m g2 d ay -1 ) n et a ss im ila tio n ra te (m g2 d ay -1 ) l ea f a re a ra tio (c m 2 / g) l ea f ar ea d ur at io n l ea f ar ea d ur at io n l ea f a re a du ra ti on l ea f a re a du ra ti on l ea f a re a ra tio (c m 2 / g) l ea f a re a ra ti o (c m 2 / g) l ea f ar ea r at io ( cm 2 / g) n et a ss im ila tio n ra te (m g2 d ay -1 ) n et a ss im ila tio n ra te (m g2 d ay -1 ) 230 the most likely explanation for this disagreement is the inefficiency of ‘carmine’ to use its entire lad for dm production even though flower heads were harvested before they were fully matured (market requirement) for both cultivars. a positive linear association was observed between lad and flower head dry weight (r = 0.02*) and flower stem dry weight (r = 0.04*). harvest index (hi) the effects of cultivar and disbudding on mean harvest index are presented in table 4. disbudding significantly influenced hi. all disbudded plants had a higher hi than the control plants (table 4). cultivars did not differ significantly in mean hi. significant disbudding and cultivar interactions were also observed. the hi for the disbudded ‘chief gold’ plants was relatively higher than that of ‘carmine’, indicating that ‘chief gold’ had a more efficient translocation system compared to ‘carmine’. differences in hi may be related to differences in the pattern of allocation of photosynthate (gent and kiyomoto, 1989). ‘chief gold’ had higher hi than ‘carmine’, which indicates that ‘carmine’ is less efficient in converting dm to flower stem and head yield (flower yield). hi showed linear associations with lai (r = 0.131*), lar (r = 0.154), nar (r = 0.019*) and rgr (r = 0.010*). 4. conclusions the overall result of the present study shows the effect of variations in disbudding on growth and development of the two considered cultivars. disbudding increased leaf area index, leaf area ratio, leaf area duration, relative growth rate, and harvest index, but reduced crop growth rate and net assimilation rate. ‘chief gold’ had a higher harvest index than ‘carmine’. disbudding plants once gave the best flower yield and quality in terms of flower head size and weight. in addition, ‘carmine’ gave the best flower yield and quality results in experiment 1 and ‘chief gold’ in experiment 2. disbudding once is therefore a highly recommended technique for celosia cut flower growers. references ball r.a., purcell l.c., vories e.d., 2000 yield compensation in response to plant population and water regime for short-season soybean production. crop sci., 40: 757-764. board j.e., harville b.g., 1992 explanations for greater light interception in 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both experiments treatment mean harvest index ‘carmine’ ‘chief gold’ mean epx 1 exp 2 epx 1 exp 2 epx 1 exp 2 disbudding once 36.99 29.34 39.26 29.34 38.12 29.34 disbudding twice 37.41 37.03 42.90 28.49 40.15 27.76 control 21.35 15.58 20.10 15.47 20.73 15.53 mean 31.92 23.99 34.09 24.44 lsd(5%):cultivar ns ns lsd(5%):disb 4.35 4.04 lsd(5%):cultivar x disb 5.48 4.87 231 pea (cicer arietinum l.) genotypes under receding soil moisture conditions. indian j. of plant physiol., 37: 275-276. kang j.g., van iersel m.w., 2004 nutrient solution concentration affects shoot:root ratio, leaf area ratio, and growth of sub irrigated salvia (salvia splendens). hortscience, 39: 49-54. law-ogbomo k.e., egharevba r.k.a., 2008 effects of planting density and npk fertilizer on growth and fruit yield of tomato (lycospersicon esculentus mill). res. j. agric. and biol. sci., 4: 265-272. lee b.j., won m.k., lee d.h., shin d.g., 2001 effects of source sink on flowering and growth in chrysanthemum (dendranthema 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scdahlias. org. radford p.j., 1967 growth analysis formula their use and abuse. grop science, 7: 171-175. rasheed m., hussain a., mahmood t., 2003 growth analysis of hybrid maize as influenced by planting techniques and nutrient management. int. j. agric. and biol., 2: 169-171. watson d.j., 1947 comparative physiological studies on the growth of the field crops. i. variation in net assimilation rate and leaf area between species and varieties and within and between years. annals of botany, 11: 41-76. 185 1. introduction norway spruce [picea abies (l.) karst.] is an economically important tree species that plays a key role in natural ecosystems in the boreal zone from norway to siberia, and is one of the most valuable european forest tree species. the natural distribution of p. abies in italy spans the entire alpine range, where it ascends to an altitude of 2,000 m, and it is also found in part of the northern apennines. a large portion of the spruce forests are used commercially and managed with varying levels of intensity. in italy, this species is also the most cultivated for the production of christmas trees for its conical shape, compact, intense green foliage, and rapid growth. the christmas trees present on the italian market are certified because they derived from 90% cultivations in forest nurseries and the trees are usually obtained by seed. seeds are collected from selected stands, and the obtained seedlings are cultivated in a nursery for almost four years: two years as “seedlings” in seedbeds, and two to three years as “transplantations” in fields depending on the desired size. christmas tree cultivation is concentrated mainly in veneto and tuscany (arezzo and pistoia districts) where about 800 hectares are devoted to this crop. nevertheless, a considerable amount of the seedlings are imported from belgium, denmark, holland, and germany for the establishment of christmas tree plantations. recently, increased importation from hungary, romania and moldavia has evidenced problems related to the genetic control of propagation material. thus, to avoid genetic mixing between autochthonous species and those coming from abroad, the imported seedlings carry a special label which certifies their origin from specialized cultivations, nationality, and indication as not intended for reforestation. a sustainable christmas tree market should aim to increase the production of plants from autochthonous species characterized by high levels of germinability and growth, and to produce seedlings of adequate size and quality in a short time for transplantation. the commercial importance of genetic characteristics and origin of the propagation material for plantation quality are well documented. in particular, rates of early growth and morphological and physiological characteristics are among the features that differ between populations. consequently, the provenance of seeds in relation to the choice of planting site is extremely important. furthermore, identification of both the best seed provenance and environmental conditions for germination and growth of seedlings could be the first objective to improve nursery production. the influence of provenance has not been widely reported on seed germination of picea, while it is well documented for seedling growth. at provenance level, strong relationships are generinfluence of seed provenance on the propagation of picea abies (l.) karst l.m.r. rinaldi ¹, a. leva ¹ (*), l.p. d’acqui ² ¹ istituto per la valorizzazione del legno e delle specie arboree, consiglio nazionale delle ricerche, via madonna del piano, 10, 50019 sesto fiorentino (fi), italy. ² istituto per lo studio degli ecosistemi, consiglio nazionale delle ricerche, via madonna del piano, 10, 50019 sesto fiorentino (fi), italy. key words: germination, growth, picea abies, somatic embryogenesis, substrate. abstract: picea abies (l.) karst. is the most common species cultivated in italy for sale as christmas trees. the aim of this study was to identify the best seed provenance to improve plant production. three italian provenances gran bosco di salbertrand, pezzel e fochino, and val di fiemme were examined. a part of seeds were sown on benches in a greenhouse after cold stratification, and another part was used for induction of somatic embryogenesis. in the first experiment, two different amounts of organic matter (manure), as components of growing media, were evaluated, while in the second one different levels and combinations of growth regulators in the medium were tested. the seed provenance and composition of the growing medium influenced seed germination and seedling growth. the best performance on both growing media was achieved by the val di fiemme provenance, and in general seedling emergence and growth were the most favourable in the growing medium with a lower organic matter amount. also, the in vitro cultures evidenced differences in the efficiency of somatic embryogenesis among the provenances. adv. hort. sci., 2015 29(4): 185-191 * corresponding author: leva@ivalsa.cnr.it received for publication 21 july 2015 accepted for publication 1 august 2015 186 adv. hort. sci., 2015 29(4): 185-191 ally observed between traits that characterise the duration of the growth period and the degree of lignification. variation in bud flushing and initiation of shoot growth among plants derived from different norway spruce seed provenances is assumed to be regulated both by differential responses to the accumulated temperature sum in spring and by conditions during acclimation in the preceding year (schmidt-vogt, 1976; dormling, 1982). accordingly, trees from with northern latitude or high altitude provenances, which are adapted to a short growing season, have inferior growth potential compared with those adapted to a longer growing season. variation between plants from different provenances has been demonstrated for a number of other traits, such as nutrient demands, respiratory activity, shade tolerance, and differences in crown shape, and resistance to snow and ice damage (schmidt-vogt, 1976). on the other hand, seedling morphological characteristics before planting were found to highly affect seedling growth during the first years after transplantation (tsakaldimi, 2006). furthermore, fertilizer application during two years improved survival and increased annual height of betula pubescens and larix sibirica (o´skarsson et al., 2006). many studies have reported the effect of manure on increase diameter and height growth of some species of pinus (nourshad and ghorani, 1990), and biomass (root and shoot dry weight) of potted and bare rooted p. taeda seedlings (kiani et al., 2005). moreover, combinations of types of soil in different ratios of nutrients have also influenced seed germination of important forest species (selivanovskaya and latypova, 2006). germination, survival, growth, and biomass of cupressus arizonica and cupressus sempervirens are enhanced using organic matter and in particular, manure has increased the maximum quality index (dickson et al., 1960; ahmadloo et al., 2012). therefore, the aim of this study was to assess the influence of seed provenance on emergence and seedling growth of picea and, as an ancillary purpose, to evaluate if an increase of the amount of organic matter (dust manure) in the growing media compared to that commonly used can improve or affect the emergence and seedling growth of picea seeds of different provenance. further, considering that somatic embryogenesis is potentially the most promising method for vegetative propagation of coniferous genera (dunstan et al., 1995), the influence of seed provenance has also been evaluated on somatic embryo germination starting from embryogenic tissues. 2. materials and methods seed lots the seeds were provided by the national centre for the study and maintenance of forest biodiversity (cnbf) of peri (verona, italy). cnbf is primarily involved in the production, selection, and conservation of forest seeds (of trees and shrubs). specifically, the forest seeds produced by the centre come from forests enrolled in the “libro nazionale boschi da seme” (l.n.b.s.), and traditional and “special” harvesting methods are used in order to preserve the genetic diversity of the species. all the material is certified and traceable through global positioning system (gps) technology (http://www3.corpoforestale.it). in this study, mature seeds of picea abies (l.) karst. harvested in october 2011 from selected populations of the following three italian provenances were evaluated: gran bosco di salbertrand gs (l.n.b.s n. 088, csr n. 034-pie; 45°n; altitude 1030-1900 m); pezzel e fochino pf (l.n.b.s n. 137 so 037; 46°n; 1350-1540 m); and val di fiemme vf (l.n.b.s n. 023 val di fiemme tn; 46°n; 1600-2000 m). certification of seed quality (purity, weight of 1000 seeds, germinability, cut test, viability, value crop) in accordance with the ista (international seed testing association) was carried out by cnbf and is reported in table 1. in vivo experiment the seeds from each provenance were stored separately in plastic bags at 5°c. later, two sowing beds (each 1.60 m × 5.0 m) were prepared in a greenhouse. a common nursery soil, composed of a mixture of 75% sand, 10% perlite, and 15% organic material (mixture of peat, manure) for a total organic c content of 6.26%, was utilized for the first bed (substrate 1). the same substrate with a rate higher than one-and-a-half of organic material (total organic c 9.82% ) was used for the second bed (substrate 2) (table 2). chemical parameters of growing media were determined according to the siss methods (siss, 1985), whereas the ones of organic carbon (oc), nitrogen (n), and inorganic carbon (ic) by using na 1500 chns analyzer, carlo erba (milan, italy) coupled with the procedure reported by santi et al. (2006). in march 2012, an average of 20 and 15 g seeds, respectively, for gs, pf, and vf provenances were sown on each sowing bed. before the trials, the seeds were soaked in water for 12 h. the seeds were sown in seven rows, and a thin layer table 1 certification of seed quality seed provenance purity (%) weight 1000 seeds germinability (%) cut test (%) no. viable seeds/g value crop (%) gran bosco salbertrand (lnbs n.088 (sr n.054 pie.) 94.5 7 71 91 96 67.1 pezzel e fochino (lnbs n.137 so037) 92 6.4 74 88 113 68.1 val di fiemme (lnbs n.023 val di fiemme) 97.2 8.8 92 97 119 89.4 187 rinaldi et al., influence of seed provenance on the propagation of picea abies (l.) karst of sand was applied to cover the seeds. the sowing beds were watered as necessary. after emergence, the seedlings were fertilised twice at intervals of 15 days with 50 ppm flory 4 (agrochimica). emergence rates were recorded in march, april, and may. the final stem height (cm) was determined for all seedlings at 90 days, the time of their transfer to pots. in june 2012 the seedlings were transferred to pots (volume 80 ml) filled with the same growing media. two hundred seedlings per provenance were distributed in five multipot pvc pots of 40 places each. the seedlings were transferred to a shaded (50% light reduction) location outdoors and cultivated until the end of july 2013. growth was evaluated after 4, 12 and 52 weeks. on the final date, 15 seedlings per provenance were randomly selected for root and shoot biomass measurements. the fresh weight (fw) and dry weight (dw), obtained by drying in an oven at 70°c for 48 h, were determined. the root-to-shoot dw ratio was also calculated for each seedling. in vitro experiment for induction of embryogenic tissues, the seeds were sterilized in 2% (v/v) sodium hypochlorite solution for 20 min and rinsed three times with sterile distilled water. the zygotic embryo was dissected from each seed and placed on solid bm1 medium (gupta et al., 1987) supplemented with 1% sucrose, hydrolysed casein (500 mg/l), l-glutamine (450 mg/l), and myo-inositol (1g/l), and solidified with 0.3% phytogel. five or 10 μm 2,4-dichlorophenoxyacetic acid (2,4-d) in combination with 2.5 or 5 μm 6-benzyladenine (ba) were included in the medium. the trial was carried out in petri dishes (100 mm × 15 mm) with 40 embryos per provenance, divided into five replicates of eight embryos each. the embryos were incubated in the dark at 20°c. the cultures were assessed after incubation for four weeks. for culture proliferation, the embryogenic tissues were subcultured three times on fresh medium and kept in the dark for development of early-stage somatic embryos. the fresh weight of embryogenic tissues was measured at the end of each subculture. for somatic embryo maturation, embryogenic tissues from material derived from each provenance were transferred to medium containing abscisic acid (aba) to allow maturation of the somatic embryos. the maturation medium was bm1 medium supplemented with 20 μm aba, 2% sucrose, and 7.5% polyethylene glycol (peg-4000). embryogenic tissue clumps growing vigorously and of about the same size (about 1 cm³ in volume) were selected to monitor the embryo maturation process. the cultures were maintained in the dark at 20°c for about five weeks until the somatic embryos reached cotyledonary stage. thirty randomly selected mature somatic embryos per provenance were transferred to germination bm1 basal medium supplemented with 0.2% activated carbon (ac), 2% sucrose and 0.4% phytogel. the embryos were cultured under photoperiod (16h/8h, light/dark) for eight weeks, after which time the plantlets were transferred to ex vitro conditions. they were placed in multipot pvc pots filled with a mix of pumice, peat, and organic fertilizer, and they were transferred to greenhouse conditions. collection data and statistical analysis with regard to seedling emergence in the greenhouse, the number of seeds that were sown on each seed bed for each provenance was calculated using the proportion: weight in grams of 1000 seeds:1000=weight in grams of seeds sown on each bed:x for each provenance, seedling emergences were normalized with respect to the number of seeds put to germinate on each bed. therefore, the data on the emergence of seedlings from different provenances were normalized to 100 according to this proportion: seedling emergence:total seeds put to germinate x row of seed bed=x:100 seedling data (emergence and height) were arranged in a completely randomized design, and the statistical significance of all differences in emergence and height of the seedlings was tested using factorial analysis of variance (anova), with the substrate and provenance as the main factors. differences among provenances were evaluated using tukey’s multiple-range tests (snedecor and cochran, 1980) at the 5% level of significance. for seedling growth parameters, means and standard errors were calculated for material derived from each provenance. all statistical analyses were performed with statistix software version 8 (analytical software, tallahassee, fl, usa). 3. results in vivo experiment the three provenances significantly (p<0.05) varied in emergence and seedling height (cm) in the two different table 2 chemical characteristics of growing media growing media substrate 1 substrate 2 c(z) (%) 6.26 9.82 organic matter %) 10.91 17.25 n (y) (%) 0.2 0.39 c/n 31.3 25.17 ic (x) (%) 2.83 1.47 cec (w) ( mol c kg-1 ) 14.4±2.7 32.0±3.9 ph 7.3±0.2 7.1±0.2 (z) c= organic carbon; (y) n= nitrogen; (x) ic= inorganic carbon; (w) cec= cationic exchange capacity. 188 adv. hort. sci., 2015 29(4): 185-191 substrates used in the seed beds (table 3). seed provenance significantly affected all these processes. in addition, the substrate effect was also significant except at 15 days after sowing, and the substrate x provenance interaction effect was also significant. more precisely, seedling emergence in the greenhouse occurred 15 to 30 days after sowing, and thereafter it was very low or non-existent (data not shown). with regard to substrate 1, which contained a lower organic matter content, seedling emergence differed significantly among the three provenances at both 15 and 30 days after sowing. at 15 days, vf showed 58% seedling emergence, whereas gs and pf showed 38 and 20% seedling emergence, respectively. after 30 days, the percentage of seedling emergence for pf increased to 25%, whereas gs and vf (fig. 1a) showed the highest seedling emergences of 62 and 91%, respectively. as for substrate 2, which contained the highest organic matter content, the emergence percentage ranged from 34% for gs to 36 and 38% for vf and pf, respectively, after 15 days. subsequently, a large percentage of seedlings for gs and pf appeared flaccid and twisted as if suffering from wilt. these seedlings continued to decline, assumed a dark colour and greasy appearance, and then quickly disintegrated to leave areas of bare soil in the seed bed (fig. 1b). after 30 days, seedling emergence was reduced to 20%, whereas that for vf increased to 47%. seeds from the vf provenance generated the most seedlings on both substrates; seeds from pf displayed the worst performance on the same substrates at the end of culture in the greenhouse (table 3). in contrast, gs provenance produced the most seedlings only on substrate 1. the final stem height was determined for all seedlings at 90 days from sowing. the stem height of seedlings ranged from 2.4 to 4.3 cm on substrate 1, and from 2.1 to 3.2 cm on substrate 2. vf seedlings showed the greatest stem height on substrate 1; pf seedlings exhibited the lowest on both substrates. the stem height of gs seedlings was highest on substrate 1. the growth trends were not different for seedlings of the provenances measured after 4, 12 and 52 weeks cultivation in pots (fig. 2). at the time of initial measurement (4 weeks), pf (7.9 cm) and gs (7.4 cm) seedlings showed superior stem growth compared to vf seedlings (6.4 cm). at the end of the cultivation period (52 weeks), gs and pf seedlings had mean stem heights of 14.7 and 13.8 cm, respectively, whereas vf seedlings attained a mean height of 10 cm (fig. 2). the minimum standard defined by the european economic community for growth of three-yearold seedlings is 14 cm in height (e.e. c. minimum standards) (magini, 1977). therefore, the recorded growth in seedlings of 15 months old from gs and pf provenance exceeded the minimum standard. among the provenances, seedlings grown in pots exhibited changes in early growth, and fresh and dry matter allocation (table 4). gs seedlings showed significantly higher values in fresh shoot biomass. on the other hand, vf provenance, compared with gs, table 3 seedling emergence and early growth of three provenances of p. abies in two different substrates growing medium provenance seedling emergence (%) seedling height (z) (cm)15 days 30 days substrate 1 gran bosco di salbertrand 38 b 62 b 4.3 a pezzel e fochino 20 c 25 cd 2.4 d val di fiemme 58 a 91 a 3.7 b substrate 2 gran bosco di salbertrand 34 bc 20 d 2.6 d pezzel e fochino 38 b 20 d 2.1 d val di fiemme 36 bc 47 bc 3.2 c two-way anova (p values) substrate (s) 0.4152 0.0000 0.0000 provenance (p) 0.0005 0.0000 0.0000 s x p 0.0001 0.0013 0.0000 values for each parameter, column, and factor followed by different letters are significantly different (tukey’s multiple range test, p < 0.05). (z) the final stem height was determined for all seedlings at 90 days from sowing: the average for gran bosco di salbertrand, pezzel e fochino and val di fiemme was calculated on 200, 123 and 331 seedlings for the substrate 1 and on 867, 257 and 801 seedlings for the substrate 2, respectively. fig. 1 emergence and growth of seedlings on different seed beds at 30 days after sowing: (a) seedlings from the provenance val di fiemme on substrate 1; (b) at left, seedlings from the provenance pezzel and fochino, showing areas of bare soil, and at right, seedlings from the provenance val di fiemme on substrate 2. 189 rinaldi et al., influence of seed provenance on the propagation of picea abies (l.) karst showed similar values in dry shoot biomass, root biomass, and root/shoot ratio, but lower values were always found in pf provenance. in vitro experiment explants from all examined provenances give rise to embryogenic tissues (table 5). the mature zygotic embryos from gs seeds yielded a higher frequency (13.5%) of embryogenic tissue induction, compared with those for pf (8.6%) and vf (5.5%). a higher frequency (13.4%) of embryogenic tissue induction, independent of provenance, was achieved on medium supplemented with low concentrations of growth regulators (5 μm 2,4-d and 2.5 μm ba) than on medium with higher concentrations of growth regulators (10 μm 2,4-d and 5μ m ba). with regard to the proliferation of embryogenic tissues, only the provenance gs showed the highest increase in fresh weight of embryogenic tissue and the capacity for embryo maturation in the presence of low concentrations of growth regulators. in contrast, a greater increase in embryogenic tissue fresh weight was achieved for all provenances on medium supplemented with the higher concentrations of 2,4-d and ba. these cultures were whitish to translucent, and were characterised by vigorous growth (fig. 3 a, b); maturation of embryogenic tissues for each provenance is reported in table 5. specifically, gs and pf embryogenic tissues showed the highest maturation efficiency (mean of 52 and 73 somatic embryos per clump, respectively), whereas the vf embryogenic tissues produced an average of only 24 somatic embryos per clump. the maturation process up to the cotyledonary stage required about 5 weeks of culture on the maturation medium (fig. 3 c-e). over 74% of the somatic embryos germinated rapidly after transfer to the germination medium. the development of plantlets with true needles was observed within 1 to 2 weeks (fig. 3f). table 4 morphological characteristics of picea abies seedlings grown in pots outdoors provenance mean number shoot biomass (g) root biomass (g) root to shoot ratio branche roots fresh dry fresh dry g s 3.0±0.46 a 9.7±1.34 a 5.15±0.09 a 2.71±0.08 a 1.23±0.12 a 1.06±0.07 a 0.395 a p f 1.3±0.44 a 7.0±1.29 a 2.15±0.18 c 1.88±0.16 b 0.52±0.12 b 0.44±0.07 b 0.257 a v f 1.8±0.63 a 7.0±1.29 a 4.45±0.10 b 2.66±0.09 a 1.16±0.14 a 1.05±0.09 a 0.390 a all parameters of growth were evaluated after 52 weeks of growth in pots. the data were subjected to analysis of variance. values are the mean of a minimum of 15 seedlings for each provenance. means within a column followed by a different letter are significantly different (tukey’s multiple range test, p< 0.05). gs= gran bosco di salbertrand; pf= pezzel e fochino; vf= val di fiemme. table 5 induction of embryogenic cultures, proliferation, and somatic embryo maturation provenance embryogenic cultures (%) increase of mass embryogenic (g) embryo maturation 5μm 2.4d+ 2.5μm ba 10μm 2.4d+ 5μm ba no. somatic embryos/clump total no. embryos/clump gran bosco salbertrand 13.5±2.35 a 1.09±0.60 a (z) 1.51±0.26 a 52.0±15.0 b 619 pezzel e fochino 8.6±2.72 a 0.22±0.15 a 1.66±0.39 a 73.0±11.5 a 881 val di fiemme 5.5±2.11 a 0.30±0.10 a 1.13±0.48 a 24.0±9.0 c 288 growth regulators 5μm 2.4d+ 2.5μm ba 13.4±1.57 a 10μm 2.4d+ 5μm ba 5.0±2.72 b (z) data represent increase of fresh weight over time. the data were subjected to analysis of variance. values within a column followed by a different letter are significantly different (tukey’s multiple range test, p < 0.05). fig. 2 stem heights of picea abies seedlings grown in pots outdoors measured after 4, 12, and 52 weeks of cultivation in pots. data represent the means (±se) for seedlings from the gran bosco di salbertrand (1), pezzel e fochino (2), and val di fiemme (3) provenances. a minimum of 200 seedlings were measured for each provenance. 190 adv. hort. sci., 2015 29(4): 185-191 4. discussion and conclusions the efficiency of p. abies propagation from seeds and by somatic embryogenesis was closely related to the provenance of the plant material. furthermore, the emergence and growth of seedlings propagated in seed beds, and the capacity for somatic embryogenesis were influenced by the composition of the growing medium. with regard to seed propagation, the best performance in terms of production and seedling growth was achieved by the val di fiemme provenance on both growing media, whereas the pezzel e fochino provenance showed the worst performance. moreover, the positive results of vf provenance on emergence and growth of seedlings in seed beds are also confirmed by the best values regarding seed quality of this provenance. in addition, the organic matter added to growth medium and its content should be taken into account. malakouti and homaei (2004) reported that organic matter improved seed germination and seedling growth, providing suitable conditions for seedling production. on the contrary, this study shows that the highest content of organic matter seems to induce a detrimental effect on growth and seedling survival. probably high amounts of organic matter in the nursery environment promotes favourable conditions for the development of pathogens living in the growing medium. therefore, the mortality of gran bosco di salbertrand and pezzel e fochino seedlings could be due to a disease, likely damping off, as evidenced from areas of bare soil in the seed beds. in contrast, the vf seedlings showed superior growth and survival and seem to be more resistant to pathogens. consequently, control of organic-matter content, humidity level in seed beds, and reduction in the duration of cultivation under greenhouse conditions are important factors to achieve a high percentage of seedling emergence and growth. at the end of the cultivation period in pots, the fifteen-month seedlings of gs provenance showed a height growth in excess of the minimum standard for three-year-old seedlings. however, the vf and gs provenances showed similar values in dry matter, fig. 3 somatic embryogenesis from mature zygotic embryos of picea abies; proliferation of embryogenic tissues (a and b); maturation (c-e) and germination (f) of somatic embryos. 191 rinaldi et al., influence of seed provenance on the propagation of picea abies (l.) karst while lower values were always found in pf. on basis of these results, the production of christmas trees could be increased by the use of val di fiemme seeds, which showed higher germinability and growth of seedlings and by shortening the time for transplanting. to the authors’ knowledge, this is the first report of the application of somatic embryogenesis technology to p. abies seeds from different italian provenances. our results demonstrate a low production of embryogenic tissues, and among the provenances a different efficiency was evident with regard to the proliferation and maturation of somatic embryos. only the gs provenance was able to develop somatic embryos capable of germination on both proliferation media; the other provenances gave rise only to somatic embryos on medium with a high concentration of growth regulators. specifically, gs and pf provenances showed the highest maturation efficiency in terms of the number of embryos per embryogenic clump. these results obtained in vitro are in line with previous studies conducted on conifers. chen et al. (2010) reported low frequency of embryo initiation and genetic specificity of explants as serious problems associated with embryogenesis induction. moreover, the induction of somatic embryogenesis varies greatly among different genotypes under identical culture protocols and proliferation and maturation levels have been identified as major constraints to somatic embryogenesis (tang et al., 2001). in order to improve the efficiency of propagation from seeds and by somatic embryogenesis, further studies will be finalized to manipulate the composition of the growing medium to improve the emergence and growth of seedlings and frequency of embryogenic tissues. acknowledgements the norway spruce seed material was kindly supplied by the national centre for the study and maintenance of the forest biodiversity of peri (verona, italy). the authors are grateful to the” istituto per l’agricoltura ed ambiente” of firenze for providing the planting site for the trials, prof. g. coppola for the helpful suggestions, and p. pestelli for his support in seedling production in greenhouse. references ahmadloo f., tabari m., hamed y., kooch y., ahmad r., 2012 effects of soil nutrient on seedling performance of arizona cypress and medite cypress. annals of biological research, 3(3): 1369-1380. chen s., chen s., chen f., wu t., wang y., shanjun y., 2010 somatic embryogenesis in mature zygotic embryos of picea likiangensis. biologia, 65(5): 853-858. dickson a., leaf a.l., hosner j.f., 1960 quality appraisal of white spruce and white pine seedling stock in nurseries. the forestry chronicle, 36(1): 10-13. dormling i., 1982 frost resistance during bud flushing and shoot elongation in picea abies. silva fennica, 16: 167-177. dunstan d.i., tautorus t.e., thorpe t.a., 1995 somatic embryogenesis in woody plants, pp. 471-538. in: thorpe t.a. (ed.) in vitro embryogenesis in plants. kluwer academic publishers, dordrecht, the netherlands, pp. 566. gupta p.k., durzan d.j., finkle b.j., 1987 somatic polyembryogenesis in embryogenic cell masses of picea abies (norway spruce ) and pinus taeda (loblolly pine) after thawing from liquid nitrogen. canadian journal of forest research, 17: 1130-1134. italian society for soil science, 1985 metodi normalizzati di analisi del suolo. edagricole, bologna, italy. kiani b., sharaji rostamii t., taheri f., 2005 studying ability rhizogenesis pinus taeda seedlings in bare root and potting condition. iranian journal natural resources, 58(2): 333-338. magini e., 1977 appunti di vivaistica forestale. clusf cooperativa editrice universitaria. malakouti m.j., homaei m., 2004 soil fertility of arid and semi-arid regions (difficulties and solutions). tarbiat modares university press, tehran, iran, pp. 482. nourshad m., ghorani m., 1990 research projects selection the best soil mixture in order to container seedlings production. afforestation and park bureau press, mazandaran, pp. 57. o´skarsson h., sigurgeirsson a., raulund-rasmussen k., 2006 survival, growth, and nutrition of tree seedlings fertilized at planting on andisol soils in iceland: six-year results. forest ecology and management, 229(13): 88-97. santi c.a., certini g., d’acqui l.p., 2006 direct determination of organic carbon by dry combustion in soils with carbonates. communications in soil science and plant analysis, 37: 155-162. schmidt-vogt h., 1976 die fichte. band i. taxomomie, verbreitung, morphologie, okologie, waldgesellschaften. verlag paul parey, hamburg and berlin. selivanovskaya s.y.u., latypova v.z., 2006 effects of composted sewage sludge on microbial biomass, activity and pine seedlings in nursery forest. waste management, 26(11): 1253-1258. snedecor g.w., cochran w.g., 1980 statistical methods. iowa university press, ames, iowa, usa. tang w., whetten r., sederoff r., 2001 genotypic control of high-frequency adventitious shoot regeneration via somatic organogenesis in loblolly pine. plant science, 161: 267-272. tsakaldimi m., 2006 kenaf (hibiscus cannabinus l.) core and rice hulls as components of container media for growing (p. halepensis m.) seedlings. bioresource technology, 97(14): 1631-1639. impaginato 252 afghan accessions almond, 207, 217 pomegranate, 225 afghanistan special issue almond, 207, 217 fruitculture, 197 germplasm, 197, 207, 217, 225, 231, 239 phytosanitary status, 239 pomegranate, 225 sweet cherry, 231 aleurodicus dispersus russell see spiralling whiteflies almond afghan accessions, 207, 217 afghanistan special issue, 207, 217 breeding, 217 cross compatibility, 217 genetic resources, 207 germplasm, 207 imcompatibility groups, 217 kernel, 207 phenotipic characterisation, 207 phytosanitary status, 239 pollination, 217 alstroemeria spp. see peruvian lily antioxidant activity peach, 175 tomato, 151 antioxidative bermudagrass, 141 apple phytosanitary status, 239 apricot phytosanitary status, 239 apricot gum carrot, 111 grapevine, 111 stevia, 111 arbuscular mycorrhizal fungi cotton, 121 ascorbate peroxidase tomato, 183 ascorbic acid tomato, 3 carrot apricot gum, 111 callus culture, 111 carbon source, 111 organic additive, 111 tissue culture, 111 chemical composition spiralling whiteflies, 165 chlorophyll content kentucky bluegrass, 87 mexican lime, 39 citrus aurantifolia l. see mexican lime climateric fruits banana, 75, 159 ethylene, 75 gaseous phase photocatalysis, 75 high ethylene production, 75 liquid-crystal template, 75 mixed oxides, 75 modified storage environment, 75 post-harvest, 75 uniform ripening, 75 cold stress seashore paspalum, 103 cold tolerance seashore paspalum, 103 control activities spiralling whiteflies, 165 cotton arbuscular mycorrhizal fungi, 121 dna changes, 13 genome template stability, 13 growth, 121 mineral nutrients, 121 phosphogypsum, 121 rapd markers, 13 salt stress, 13 cross compatibility almond, 217 cut flower tuberose, 69 cymbopogon citratus spiralling whiteflies, 165 cynodon dactylon (l.) pers. see subject index banana climateric fruits, 75, 159 ethylene production, 159 putrescine treatment, 159 quality, 159 ripening process, 159 shelf life, 159 barberry germination, 31 pollen grains pollen size, 31 pollen viability, 31 berberis microphylla g. forst see barberry bermudagrass antioxidative, 141 irrigation, 141 morpho-physiological alteration, 141 photoperiod, 141 turfgrass, 141 biocontrol potato, 47 biofertilizers mango, 81 breeding almond, 217 building thermal control japanese climbing fern, 59 ‘burlat’ sweet cherry, 231 calla lily new viral evidence, 53 rt-pcr tests, 53 tuscan nurseries, 53 viruses occurrence, 53 callus culture carrot, 111 grapevine, 111 stevia, 111 cananga odorata spiralling whiteflies, 165 carbon source carrot, 111 grapevine, 111 stevia, 111 253 bermudagrass daucus carota l. see carrot decay tomato, 183 de-pigmented cultivar peach, 175 dna changes cotton, 13 drought tomato, 3 drought deficit mexican lime, 39 endangered genotypes fig, 129 environmental measurements japanese climbing fern, 59 essential oils spiralling whiteflies, 165 ethylene climateric fruits, 75 tuberose, 69 ethylene production banana, 159 euodia hortensis spiralling whiteflies, 165 explant fig, 129 ficus carica l. see fig fig endangered genotypes, 129 explant, 129 micropropagation, 129 rooting, 129 shoot proliferation, 129 single node, 129 flavanone glycoside potato, 95 flavonoids tomato, 151 flower abscission tomato, 3 food security tomato, 23 growth cotton, 121 growth characters mango, 81 harvest date sweet cherry, 231 heavy metals tomato, 23 high ethylene production climateric fruits, 75 ‘himsagar’ mango, 81 histological analysis potato, 95 hydrogen peroxide treatment seashore paspalum, 103 hydroponic system tomato, 23 imcopatibility groups almond, 217 inorganic manure mango, 81 ion fluxes grapevine, 135 tobacco, 135 irrigation bermudagrass, 141 japanese climbing fern building thermal control, 59 environmental measurements, 59 green roof, 59 green wall, 59 roof top garder, 59 urban agriculture, 59 kentucky bluegrass chlorophyll content, 87 proline content, 87 salt stress, 87 silicon treatments, 87 turfgrass, 87 kernel almond, 207 leaf water potential mexican lime, 39 liquid-crystal template climateric fruits, 75 fruitculture afghanistan special issue, 197 germplasm, 197 plant nursery, 197 traditional orchard, 197 fumigant toxicity spiralling whiteflies, 165 gaseous phase photocatalysis climateric fruits, 75 gc-ms test spiralling whiteflies, 165 genetic resources almond, 207 genome template stability cotton, 13 germination barberry, 31 germplasm aghanistan special issue, 197, 207, 217, 225, 231, 239 almond, 207 fruitculture, 197 phytosanitary status, 239 pomegranate, 225 ‘ghiaccio’ peach, 175 gibberellic acid tuberose, 69 glomus etunicatum mexican lime, 39 gossypium hirsutum l. see cotton grapevine apricot gum, 111 callus culture, 111 carbon source, 111 ion fluxes, 135 organic additive, 111 phytosanitary status, 239 plant-virus interaction, 135 potassium, 135 tissue culture, 111 trans-plasma membrane, 135 green roof japanese climbing fern, 59 green wall japanese climbing fern, 59 greenhouse conditions tomato, 151 254 longevity tuberose, 69 lycopene tomato, 3 lygodium japonicum thunb. sw. see japanese climbing fern macro elements tomato, 23 malus domestica borkh. see apple mangifera indica l. see mango mango biofertilizers, 81 growth characters, 81 ‘himsagar’, 81 inorganic manure, 81 organic manure, 81 quality, 81 shelf life, 81 soil microbial population, 81 maturity index sweet cherry, 231 mexican lime chlorophyll content, 39 drought deficit, 39 glomus etunicatum, 39 leaf water potential, 39 pseudomonas fluorescence, 39 micro elements tomato, 23 micropropagation fig, 129 middle east tomato, 23 mineral nutrients cotton, 121 mixed oxides climateric fruits, 75 modified storage environment climateric fruits, 75 morphological variability pomegranate, 225 morpho-physiological alteration bermudagrass, 141 mother stock nurseries phytosanitary status, 239 phytochemicals peach, 175 phytosanitary status aghanistan special issue, 239 almond, 239 apple, 239 apricot, 239 germplasm, 239 grapevine, 239 mother stock nurseries, 239 peach, 239 pear, 239 plum, 239 sweet cherry, 239 virus disease, 239 plant nursery fruitculture, 197 plant resistance potato, 47 plant-virus interaction grapevine, 135 tobacco, 135 plum phytosanitary status, 239 poa pratensis l. see kentucky bluegrass polianthes tuberose l. see tuberose pollen grains barberry, 31 pollen size barberry, 31 pollen viability barberry, 31 pollination almond, 217 pomegranate afghan accessions, 225 aghanistan special issue, 225 germplasm, 225 morphological variability, 225 nutraceutical properties, 225 relationship, 225 post harvest climateric fruits, 75 tomato, 183 tuberose, 69 potassium grapevine, 135 tobacco, 135 murraya koenigii spiralling whiteflies, 165 musa acuminata l. see banana new viral evidence calla lily, 53 peruvian lily, 53 nicotiana tabacum l. see tobacco nutraceutical properties peach, 175 pomegranate, 225 ocimum tenuiflorum spiralling whiteflies, 165 organic additive carrot, 111 grapevine, 111 stevia, 111 organic manure mango, 81 paspalum vaginatum sw. see seashore paspalum peach antioxidant activity, 175 de-pigmented cultivar, 175 ‘ghiaccio’, 175 nutraceutical properties, 175 phenolic compounds, 175 phytochemicals, 175 phytosanitary status, 239 quality, 175 white flesh, 175 pear phytosanitary status, 239 peruvian lily new viral evidence, 53 rt-pcr tests, 53 tuscan nurseries, 53 viruses occurrence, 53 pesticidal activities spiralling whiteflies, 165 phenolic compounds peach, 175 phenotypic characterisation almond, 207 phosphogypsum cotton, 121 photoperiod bermudagrass, 141 255 potato biocontrol, 47 flavanone glycoside, 95 histological analysis, 95 plant resistance, 47 potato tuber moth, 47 pseudomonas putida btpi, 47 rhizobacteria, 47 salt stress, 95 trichomes, 95 ultrastructural changes, 95 potato tuber moth potato, 47 pre-harvest treatment tomato, 183 tuberose, 69 proline content kentucky bluegrass, 87 prunus amygdalus batch. see almond prunus armeniaca l. see apricot prunus avium l. see sweet cherry prunus domestica l. see plum prunus dulcis mill. see almond prunus persica l. see peach pseudomonas fluorescence mexican lime, 39 pseudomonas putida btpi potato, 47 punica granatum l. see pomegranate putrescine treatment banana, 159 pyrus l. see pear quality banana, 159 mango, 81 peach, 175 tomato, 183 quality control system sweet cherry, 231 rapd markers cotton, 13 relationship pomegranate, 225 repellent test spiralling whiteflies, 165 cymbopogon citratus, 165 essential oils, 165 euodia hortensis, 165 fumigant toxicity, 165 gc-ms test murraya koenigii, 165 ocimum tenuiflorum, 165 pesticidal activities, 165 repellent test, 165 sps tomato, 3 stevia rebaudiana bert. see stevia stevia apricot gum, 111 callus culture, 111 carbon source, 111 organic additive, 111 tissue culture, 111 sustainable agriculture tomato, 23 sweet cherry afghanistan special issue, 231 ‘burlat’, 231 harvest date, 231 maturity index, 231 phytosanitary status, 239 quality control system, 231 tissue culture carrot, 111 grapevine, 111 stevia, 111 tobacco ion fluxes, 135 plant-virus interaction, 135 potassium, 135 trans-plasma membrane, 135 tomato antioxidant activity, 151 ascorbate peroxidase, 183 ascorbic acid, 3 decay, 183 drought, 3 flavonoids, 151 flower abscission, 3 food security, 23 greenhouse conditions, 151 heavy metals, 23 hydroponic system, 23 lycopene, 3 macro elements, 23 micro elements, 23 middle east, 23 quality, 183 post harvest, 183 pre-harvest treatment, 183 salicylic acid, 151, 183 rhizobacteria potato, 47 ripening process banana, 159 roof top garden japanese climbing fern, 59 rooting fig, 129 rt-pcr tests calla lily, 53 peruvian lily, 53 ‘salam’ seashore paspalum, 103 salicylic acid tomato, 151, 183 salt stress cotton, 13 kentucky bluegrass, 87 potato, 95 seashore paspalum cold stress, 103 cold tolerance, 103 hydrogene peroxide treatments, 103 ‘salam’, 103 ‘seaspray’, 103 ‘seaspray’ seashore paspalum, 103 shelf life banana, 159 mango, 81 shoot proliferation fig, 129 silicon treatment kentucky bluegrass, 87 single node fig, 129 soil microbial population mango, 81 solanum lycopersicon l. see tomato solanum tuberosum l. see potato soluble protein tomato, 3 spiralling whiteflies cananga odorata, 165 chemical composition, 165 control activities, 165 256 soluble protein, 3 sps, 3 sustainable agriculture, 23 total phenolics, 151 weight loss, 183 yield, 3 yield components, 151 total phenolics tomato, 151 traditional orchard fruitculture, 197 trans-plasma membrane grapevine, 135 tobacco, 135 trichomes potato, 95 tuberose cut flower, 69 virus disease phytosanitary status, 239 viruses occurrence calla lily, 53 peruvian lily, 53 vitis vinifera l. see grapevine weight loss tomato, 183 white flesh peach, 175 yield tomato, 3 yield components tomato, 151 zantedeschia aethiopica l. spreng. see calla lily ethylene, 69 gibberellic acid, 69 longevity, 69 post harvest, 69 pre-harvest treatment, 69 turfgrass bermudagrass, 141 kentucky bluegrass, 87 tuscan nurseries calla lily, 53 peruvian lily, 53 ultrastructural changes potato, 95 uniform ripening climateric fruits, 75 urban agriculture japanese climbing fern, 59 impaginato 99 adv. hort. sci., 2011 25(2): 99-105 received for publication 6 april 2011. accepted for publication 13 may 2011. growth reduction in root-restricted tomato plants is linked to photosynthetic impairment and starch accumulation in the leaves s. mugnai*, h.s. al-debei** * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione di coltivazioni arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. ** department of horticulture and crop science, faculty of agriculture, university of jordan, 11942 amman, jordan. key words: chlorophyll, leaf gas exchange, solanum lycopersicum l., sugar content, total water potential. abstract: the mechanisms responsible for reduced shoot growth due to restricted root growth is still not fully understood. therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root restriction conditions and to determine the time frame within which these changes occurred. in particular, this research aims to evaluate the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in tomato (solanum lycopersicum l.). our results show that growth reduction by root restriction is mainly linked to a photosynthetic impairment, caused by a concurrent limited stomatal conductance (probably driven by stomatal factors and hormonal substances) together with a strong accumulation of starch in the tissues, which led to a feedback inhibition of the photosynthetic process. 1. introduction the use of root-restricted cultivation for vegetable production has significantly grown in the last decades (shi et al., 2008), as it appears an effective technique for saving resources, controlling root environment, and regulating early yield and quality (marsh and paul, 1988; shi et al., 2008). root restriction may occur wherever pot size or rooting volume is physically limited (tschaplinski and blake, 1985; ismail and noor, 1996; saito et al., 2008; mugnai et al., 2009), mostly with greenhouse-grown horticultural crops (thomas, 1993). root restriction leads to a denser root mass and a reduced root growth (ismail and noor, 1996). besides limiting the volume of the soil available to the root system for water and nutrient uptake, it also suppresses canopy growth (ismail and noor, 1996; shi et al., 2008) via many plant physiological and biochemical processes. the mechanisms responsible for reduced shoot growth due to restricted root growth is still not fully understood. several hypotheses were investigated including water and nutrient stresses (hameed et al., 1987), decrease in root respiration (shi et al., 2007) and photosynthesis (shi et al., 2008), and production of plant hormones (liu and latimer, 1995), but reports indicated that there are contradictory results as to which of these factors play a significant role in the response of aerial plant parts to restricted root growth and indicated differences between species. leaf photosynthesis strongly depends on environmental conditions such as radiation, co2 concentration and temperature. in addition to these environmental conditions, photosynthesis is subjected to internal regulation associated with sink demand for assimilates (marcelis, 1991). the presence of a physical restriction to root growth, a major metabolic sink for photsynthetically fixed carbon at seedling stage (thomas and strain, 1991) resulted in feedback inhibition mechanisms, with lower rates of carbon metabolism and photosynthesis as a result of carbohydrate accumulation (schaffer et al., 1996; shi et al., 2008). therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root restriction conditions and to determine the time frame within which these changes occurred. in particular, this research aims to study the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in tomato (solanum lycopersicum l.). 100 2. materials and methods plant material experiments were carried out at the department of plant biology, university of pisa (italy). seeds of tomato (solanum lycopersicum l.) cv. ‘cal j’ were sown in seedling flats filled with vermiculite and placed in a germinating room at constant temperature (25°c) and light intensity (300 mol m-2 s-1 of par). after germination, seedlings with the first true leaves were selected for uniformity and single plants were transplanted into 7 ml (root restricted, rr) and 230 ml (control) speeding flats filled with vermiculite. flats were placed in a greenhouse and suspended 15 cm above the benches to facilitate air pruning of roots and to induce root restriction treatment through out the experiment period. in each flat 24 seedlings were planted regardless of the original number of cells per flat to minimize the effect of mutual shading, to avoid light competition between plants and to allow for uniform plant density. in order to avoid any water or nutrient stress, a closed fertirrigation system controlled by a timer was established to supply water and nutrients at frequent and regular intervals. the nutrient solution was composed thus: 10 mm no3 -, 1 mm h2po4 -, 8 mm k+, 4 mm ca2+, 1.5 mm mg2+, 1 mm so4 2-, 0.04 mm fe2+ and microelements (ph 6.0, ec=1.2 ms cm1). the nutrient solution was renewed every week. growth measurements five plants per treatment were sampled at weekly intervals. roots were carefully washed, then plants were separated into leaves, stems and roots. leaf area was measured with an area meter (delta t-devices ltd., cambridge, uk), plant height was estimated using a ruler and dry weight for each organ was obtained after oven drying (48 hr at 70°c). leaf gas exchange measurements net co2 assimilation (a), stomatal conductance (g) and transpiration (e) measurements were performed weekly (n=5) on the central sector of the youngest fully-expanded leaf using an open system (cms 400, heinz walz, effeltrich, germany) connected to an assimilation chamber and equipped with a high sensitivity irga (binos, leybold haeraeus, germany) under temperature (24°c) and growing light (400 mmol m-2 s-1 par) conditions provided by a mercury vapour lamp (osram hqi-ts 250 w/ndl). calculation of all the parameters was performed following von cammerer and farquhar (1981) using a specific software (diagas 2.02, walz, effettrich, germany). chlorophyll content five leaf disks (10 mm diameter) were randomly taken from the uppermost fully-expanded leaves at weekly intervals, and extracted in 2 ml of n,ndimethylformamide for 24 hr in the dark. absorbance was then determined for each sample using a spectrophotometer at 647 and 663 nm. chlorophyll a and b contents, and a/b ratio were calculated according to moran (1982). determination of total, osmotic and turgor potentials leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange (n=5). total water potential (ψw) was determined using a pressure chamber (pardossi et al., 1991). osmotic potential (ψs) of the leaf xylem sap was determined using an osmometer (precision system, usa) by determining the freezing point depression of the sample. leaf turgor potential (ψp) was calculated using the following equation (eq. 1): ψp = ψw ψs (eq. 1) measurement of sugar content leaf, stem, and root samples (approx. 50 mg each) were taken at weekly intervals (n=5) and directly freeze-dried in liquid nitrogen. samples were homogenized and extracted with 1 ml hot 80% ethanol, boiled for 5 min, and centrifuged at 12000 rpm for 15 min; the supernatant was then collected. the pellet was extracted again as described above, and the supernatant was collected again. at the end of the procedure, the pellet was evaporated to remove any excess ethanol. particulates including starch were suspended in 1 ml of koh 20 mm, boiled and centrifuged at 8000 rpm for 15 min and the supernatant was collected. the extract from ethanol was used for sucrose, glucose and fructose determinations, and the extract from koh was used for starch determination. for sugar determination, two 200 µl aliquots from the ethanol extract were taken, one incubated for 30 min at 37°c with 100 µl solution containing invertase (1 mg invertase ml-1 na-acetate 50 mm at ph 4.6), the other with 100 µl solution containing na-acetate 50 mm at ph 4.6, then both brought to the final volume (1 ml) with a solution containing 100 mm tris-hcl, ph 7.6, 3 mm mgcl2, 2 mm atp, 0.6 mm nadp, 1 unit hexokinase and 1 unit glucose-6-pdehydrogenase (incubated at 37°c for 30 min). absorbance at 340 nm was then measured using a spectrophotometer. the concentration of glucose in each solution was determined from glucose standard curves according to guglielminetti et al. (1995). the solution without invertase was used to calculate the amount of free glucose in the sample and the difference between the two gave the amount of sucrose (as glucose equivalent). for each of them 10 µl of solution containing 15 µl of phosphoglucoisomerase in 150 µl of tris-hcl 300 mm at ph 7.6 were incubated at 37°c for 15 min, then absorbance at 340 nm was determined. the difference between the one without invertase and treated with phosphoglucoisomerase and the other without invertase at the first determination gave the amount of free fructose (as glucose equivalent). for starch determination, 100 µl of extract was incubated at 101 37°c for 1 hr with 100 µl solution of na-acetate 100 mm ph 5.2/10 α-amylase. this solution was incubated with 100 µl of na-acetate 100 mm ph 4.6/10 u amyloglucosidase at 55°c for 1 hr. finally, the solution was boiled and centrifuged to eliminate denaturated protein from α-amylase and amylogluco-sidase. 100 µl from this solution was taken and brought to 300 µl with distilled water, then starch analysis (as glucose equivalent) was carried out as mentioned above for glucose. statistical analysis data were analyzed by one-way anova, and means (n=5) were separated using duncan’s multiple range test (p≤0.05). statistical analysis was performed using graphpad prism 4.0 (graphpad software). 3. results growth parameters were greatly affected by root restriction treatment (rr), with significant reductions in total dry weight, leaf area and plant height (fig. 1a, b and c) starting from an early stage of seedling development. rr plants also showed a significantly higher root:shoot ratio (fig. 1d), due to a higher allocation of biomass in the root system compared to canopy (stem and leaves). during the first month, no significant differences were noticed in leaf gas exchange parameters. from day 29, however, stomatal conductance (g) started to significantly decrease in rr plants (fig. 2a), leading to a significant reduction from day 36 in both net co2 fig. 1 growth parameters measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total dry weight (a), leaf area (b), plant height (c) and root:shoot ratio (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 2 leaf gas exchange parameters measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: stomatal conductance (a), net co2 assimilation (b) and transpiration (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. assimilation (fig. 2b) and transpiration (fig. 2c) until the end of the experiment. the reduction in net co2assimilation was not related to a decrease in the chloro102 phyll content of rr plants (table 1), as no significant differences were noticed for chlorophyll a, b, and a/b ratio between the two treatments. leaf water status did not affect stomatal closure, as total water potential (fig. 3a) and turgor potential (fig. 3c) did not show any significant difference throughout the entire experiment in both the treatments, even if a slight, but not significant, reduction in total water potential was measured on day 43 in rr plants. this behaviour also confirmed the fact that no water stress symptoms occurred during the experimental period, giving a positive feedback of our experimental system. on the contrary, sugar content determination led to interesting results. while sucrose content trend was not uniform during the experiment, leading to contradictory results (fig. 4a), rr treatment led to a clear increase in glucose content (fig. 4b) and a concurrent decrease in fructose content (fig. 4c) together with a great accumulation of starch (fig. 4d). in particular, starch accumulation in the tissues began early in the developmental process (day 29). starch was mainly compartimentalized in the leaves (fig. 5a) and stems (fig. 5b) of rr plants, whereas no significant differences were noticed in roots between control and rr plants (fig. 5c). 4. discussion and conclusions our growth data are in line with several previous results concerning growth depression induced by root restriction in other horticultural crops (carmi and heuer, 1981; tschaplinski and blake, 1985; thomas and strain, 1991; rieger and marra, 1994; liu and latimer, 1995; van iersel, 1997; kharkina et al., 1999; saito et al., 2008; shi et al., 2008). root restriction generally caused an increase in root:shoot ratio (carmi et al., 1983; mugnai et al., 2000); roots in smaller volume formed a highly branched mat, whereas plants in large volume had long tap roots and showed little branching. the increased root:shoot ratio reported by some researchers for many crop species subjected to table 1 chlorophyll content (a, b and a/b ratio) measured at weekly intervals from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants control plants root-restricted plants (rr) 22 29 36 43 50 chl a (mg cm-2) chl b (mg cm-2) a/b 8.075 7.403 7.414 9.076 10.924 3.221 3.274 3.276 3.686 4.320 0.484 0.703 0.702 0.596 0.624 chl a (mg cm-2) chl b (mg cm-2) a/b 8.423 9.261* 9.949* 9.809 10.873 3.333 3.708 3.500 3.863 4.054 0.483 0.566 0.216* 0.547 0.422* * indicates significantly different values between the two treatments for the same parameters for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 3 leaf water status determined at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total water potential (a), osmotic potential (b) and turgor (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. root restriction might be attributed to an increased substrate temperature in smaller containers in conjunction with a possible temperature dependence of root elongation as suggested by hurley et al. (1998). our results reveal that root restriction significantly reduces stomatal conductance, as previously noted by other authors for different species (carmi et al., 1983; thomas and strain, 1991; ismail and noor, 1996; day 103 fig. 4 sugar content measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total sucrose (a), total glucose (b), total fructose (c) and total starch (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 5 starch content in the different plant organs measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: leaf (a), stem (b) and roots (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. kharkina et al., 1999), and that stomatal conductance was the primary cause of decrease in co2 assimilation in root-restricted plants suggesting a stomatal factor limiting the photosynthetic rate under root-restriction conditions (shi et al., 2008). the decline in stomatal conductance was not correlated to a concurrent decline in total water potential, as leaf tissues were able to maintain a high level of turgor during the whole experiment. this means that other factors are largely involved in the stomatal closure. it has been suggested that root volume restriction induces a reduction in the stomatal conductance via a decrease in the supply of growth substances from roots to shoots and/or an imbalance in root and shoot hormones. for example, shi et al. (2008) reported that shoot growth suppression may be caused by the influence of aba originating from the restricted roots. carmi (1995) found that the higher level of aba in the leaves of root-restricted plants was not a consequence of an enhanced transport from the restricted roots, concluding that root-zone restriction might promote aba accumulation in the root and the shoot, with a possible influence of such accumulation on other processes in root-restricted plants, such as leaf gas exchange. the decline in net co2 assimilation observed in root-restricted conditions was also interpreted as a feedback inhibition by carbohydrate accumulation (pezeshki and santos, 1998). plant growth is strongly affected by leaf photosynthetic activity, since photosynthates are essential either as the source of carbon used for the build-up of organic compounds or as the source of energy needed for biochemical reactions involved in growth and maintenance processes. growth rate may regulate photosynthesis either through effects on the supply of growth substances translocated into leaves or through the effect on the translocation rate of photosynthates from leaves to the growing organs (carmi et al., 1983). the accumulation of photosynthates is influenced by the rate of their translocation to the sink organs (sonnewald and willmitzer, 1992), and sink demand for photosynthates has a marked influence on source leaf photosynthesis, which is greatly dependent on sink strength, considered as a product of sink size and sink activity (sonnewald and willmitzer, 1992). however, sink size is determined by different parameters. roots are recognized as 104 a metabolic sink that influences the partitioning of photosynthetically fixed carbon (gifford and evans, 1981; robbins and pharr, 1988). sink limitation caused by root restriction can greatly reduce leaf photosynthetic rate in many crop species (hameed et al., 1987; ismail and noor, 1996; schaffer et al., 1996; whiley et al., 1999; shi et al., 2008), and reduced translocation of assimilates from leaves (robbins and pharr, 1988; kharkina et al., 1999). root volume restriction often promotes an accumulation of nonstructural carbohydrates in the stem and leaves in response to the lack of the active sinks (nishizawa and saito, 1998), meaning that the difference in the growth rate between root-restricted and control treatments was not due to a decrease in assimilates supply to the organs whose growth was restricted (mandre et al., 1995). our results suggest that the role of the leaves and stem as sink organs may increase when root growth is extremely limited by volume restriction and a relatively larger amount of carbohydrates may accumulate in the canopy. a new shoot to root equilibrium may be established for an increased function of leaves and stem, together with a concurrent diminished function of the roots. therefore, it can be concluded that as a result of reduced vegetative growth an excess of assimilates was produced which could not be used for growth, and thus accumulated in the form of starch, as also indicated by carmi and heuer (1981), robbins and pharr (1988) and shi et al. (2008). accumulation of non-structural carbohydrates in the leaves in response to root restriction could provide a feedback mechanism that reduces carbon metabolism (thomas and strain, 1991). starch accumulation may reduce net photosynthetic rate by avoiding intracellular co2 transport (shi et al., 2008). however, contradictory results were obtained by rieger and marra (1994), who suggested that reduced co2 assimilation cannot always be explained by a feedback inhibition of carbohydrates. the relatively low maximum assimilation (amax) rates for container-grown plants compared to field-grown plants may be attributed to containers restricting the root sink, thus causing the photoassimilate supply to exceed the capacity of demand (i.e. endproduct inhibition of photosynthesis) as indicated by arp and drake (1991) and whiley et al. (1999). in conclusion, our results show that growth reduction by root restriction is mainly linked to a photosynthetic impairment, caused by a limited stomatal conductance (probably driven by both stomatal factors and hormonal substances) and a strong accumulation of starch in the tissues, which probably leads to a feedback inhibition of the photosynthetic process. acknowledgements the authors are particularly indebted to prof. franco tognoni for scientific support. references arp w.j., drake b.g., 1991 increased photosynthetic capacity of scirpus olneyi after 4 years of exposure to elevated co2.plant cell environ, 14: 1003-1006. carmi a., 1995 growth, water transport and transpiration in root-restricted plants of bean, and their relation to abscisic acid accumulation. plant science, 107: 69-76. carmi a., hesketh j.d., enos w.t., peters d.b., 1983 interrelationships between shoot growth and photosynthesis, as affected by root growth restriction. photosynthetica, 17(2): 240-245. carmi a., heuer b., 1981 the role of roots in control of bean shoot growth. ann. bot., 48: 519527. gifford r.m., evans l.t., 1981 photosynthetic carbon partitioning and growth. ann. rev. plant physiol., 32: 485-509. guglielminetti l., perata p., alpi a., 1995 effect of anoxia on carbohydrate metabolism in rice seedlings. plant physiol., 108: 735-741. hameed m.a., reid j.b., rowe r.n., 1987 root confinement and its effects on the water relations, growth and assimilate partitioning of tomato. ann. bot., 59: 685-692. hurley m.b., rowarth j.s., trought m.c.t., barnes m.f., rowe r.n., 1998 variations in water availability and temperature in the root environment during root volume restriction studies. new zeal. j. crop hort. sci., 26: 127-134. ismail m.r., noor k.m., 1996 growth, water relations and physiological processes of starfruit plants under root growth restriction. scientia hort., 66: 51-58. kharkina t.g., ottosen c.o., rosenqvist e., 1999 effects of root restriction on growth and physiology of cucumber plants. physiol. plant., 105: 434-441. liu a., latimer j.g., 1995 water relations and abscisic acid levels of watermelon as affected by rooting volume restriction. j. environ. bot., 289: 1011-1015. mandre o., rieger m., myers s.c., seversen r., regnard j.l., 1995 interaction of root confinement and fruiting in peach. j. amer. soc. hort. sci., 120(2): 228-234. marcelis l.f.m., 1991 effects of sink demand on photosynthesis in cucumber. j. exp. bot., 42: 1387-1392. marsh d.b., paul k.b., 1988 influence of container type and cell size on cabbage transplant development and field performance. hortscience, 23(2): 310-311. moran r., 1982 formula for determination of chlorophyll pigments extracted with n,n-dimethylformamide. plant physiol., 69: 1376-1381. mugnai s., ferrante a., petrognani l., serra g., vernieri p., 2009 stress-induced variation in leaf gas exchange and chlorophyll a fluorescence in callistemon plants. res. j. biol. sci., 4: 913-921. mugnai s., vernieri p., tognoni f., 2000 container volume effects on morphology and physiology of tomato seedlings. acta horticulturae, 516: 49-56. nishizawa t., saito k., 1998 effects of rooting volume restriction on the growth and carbohydrate concentration in tomato plants. j. amer. soc. hort. sci., 123(4): 581-585. pardossi a., vernieri p., tognoni f., 1991 evaluation of the pressure chamber method for the assessment of water status in chilled plants. plant cell environ., 14: 675-682. pezeshki s.r., santos m.i., 1998 relationships among rhizosphere oxygen deficiency, root restriction, photosynthesis, and growth in baldcypress (taxodium distichum l.) seedlings. photosynthetica, 35: 381-390. rieger m., marra f., 1994 response of young peach trees to root confinement. j. amer. soc. hort. sci., 119: 223-228. robbins n.s., pharr d.m., 1988 effect of restricted root growth on carbohydrate metabolism and whole plant growth of cucumis sativa l. plant physiol., 87: 409-413. 105 saito t., fukuda n., iikubo t., inai s., fujii t., konishi c., ezura h., 2008 effects of root-volume restriction and salinity on the fruit yield and quality of processing tomato. j. japan. soc. hort. sci., 77: 165-172. schaffer b., searle c., whiley a.w., nissen r.j., 1996 effects of atmospheric co2 enrichment and root restriction on leaf gas exchange and growth of banana. physiol. plant., 97: 685-693. shi k., fu l.j., ding x.t., dong d.k., zhou y.h., yu j.q., 2008 root restriction-induced limitation to photosynthesis in tomato leaves scientia hort., 117: 197-202. shi k., hu w.h., dong d.k., zhou y.h., yu j.q., 2007 low o2 supply is involved in the poor growth in root-restricted plants of tomato. env. exp. bot., 61: 181-189. sonnewald u., willmitzer l., 1992 molecular approaches to sink-source interactions. plant physiol., 99: 1267-1270. thomas r.b., strain b.r., 1991 root restriction as a factor in photo-synthetic acclimation of cotton seedlings grown in elevated carbon dioxide. plant physiol., 96: 627-634. thomas t.h., 1993 effects of root restriction and growth regulator treatments on the growth of carrot (daucus carota l.) seedlings. plant growth regul., 13: 95-101. tschaplinski t.j., blake t.j., 1985 effects of root restriction on growth correlations, water relations and senescence of alder seedlings. physiol. plant., 64: 167-176. van iersel m., 1997 root restriction effects on growth and development of salvia (salvia splendens). hortscience, 32(7): 1186-1190. von cammerer s., farquhar g.d., 1981 some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. planta, 153: 376-387. whiley a.w., searle c., schaffer b., wolstenholme b.n., 1999 cool orchard temperatures or growing trees in containers can inhibit leaf gas exchange of avocado and mango. j. amer. soc. hort. sci., 124(1): 46-51. 59 1. introduction pear psylla is one of the most important pests affecting production of pears of pyrus communis parentage. a sucking insect that causes severe wilting and defoliation, which reduces yields and weakens the trees. at least seven species of pear (pyrus)-feeding psyllids in the genus cacopsylla (formerly psylla) are recognized, but there are three major species which occur primarily west of china: c. pyricola foerster is the only species found in north america; c. pyri l. and c. pyrisuga foerster are also endemic to europe. c. pyricola probably originated in western eurasia in contact with wild pyrus (bell et al., 1996). all of the major cultivars of the european pear are susceptible to this homopteran insect, varying only slightly in the degree of infestation and tolerance to feeding. major cultivars of p. pyrifolia parentage are slightly less susceptible, while those of p. x p. bretschneideri or p. ussuriensis origin (e.g., ya li and tzu li) appear to be moderately resistant (beutel, 1985). 2. morfology and life of psylla c. pyri is characterized by a seasonal dimorphism, which is strong enough that the two morphotypes were at one time considered to be distinct species (slingerland, 1892). the winter form is a large dark red overwintering adult with wide blackish longitudinal and traverses scratches, that is quite larger (2.6-2.9 mm) than the smaller and light-colored summerform adult (2.1-2.7 mm). pear psylla spends much of the winter in reproductive diapause, characterized by immature ovaries and a lack of mating. dispersal of winterforms from the orchard in autumn begins in early september (civolani and pasqualini, 2003), and peaks during late-october and early-november, coinciding with leaf fall in pear. this winterform adults overwinter both alone or in small groups in bark crevices, branch intersections and at the base of shoots on the pear host plant (priore, 1991) and away from the host plant, in the earth, or under rocks and clods which are exposed to sun irradiance (nguyen, 1962). so, some individuals spend the entire winter on pears, others recolonize pear early in spring before bud break, long before any sign of green foliage, since they would feed on a plant other than pear but not complete develop (fye, 1983). cool, wet autumns result in a reduction in dispersal out of the orchard. as soon as the weather conditions become favourable, winterforms reach the apical twigs and pierce the plant by inflicting their stylets at the bud base. in sicily overwintering nymphs from which adults flutter in february have been noticed as well (nucifora, 1969; tremblay, 1995). the egg maturation is very slow in overwintering females and seems to be accelerated when psylla adults ceases to disperse among orchards (rieux et al., 1992; lyoussoufi et al., 1994). generally diapause terminate in mid december, but in the more precocious females this event may occur starting from the end of november (rieux et al., 1990). at the end of january all females are mature pear resistance to psilla (cacopsylla pyri l.). a review s. nin*, a. ferri*, p. sacchetti**, e. giordani* * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. ** dipartimento di biotecnologie agrarie, università degli studi di firenze, via maragliano, 75-77, 50144 firenze, italy. key words: breeding, germplasm, pest control, p. communis abstract: pear psylla, cacopsylla pyri l., is one of the most important insect pests in european pear production areas. control measures are directed specifically at controlling pear psylla and require accurate and timely information about insect densities in the orchard. thus, there is a widespread interest in the search for suitable biological control agents and in breeding for resistance to pear psylla. modes of host plant resistance to pear psylla damage have been studied extensively by several authors and the susceptibility of many european pear genotypes have been investigated in order to detect cultivars resistant or highly tolerant to this pest useful in breeding programs. this review presents an update of published results and knowledge on psylla life, host finding for feeding and oviposition, type of damages, monitoring and control strategies with renewed and improved efficacies, resistance characterization and breeding, with particular regard to the identified sources of resistance and the screening methods. adv. hort. sci., 2012 26(2): 59-74 received for publication 18 may 2012. accepted for publication 26 june 2012. 60 and inseminated, but for the beginning of egg-laying by overwintered winterforms a temperature over 10°c for 2 consecutive days (thermic quiescence) is required (nguyen, 1975). in italy egg-laying begins in late february in campania (priore, 1991), early march in emilia-romagna (giunchi, 1959) or early april in veneto (terza and pavan, 1988). because of the absence of foliage at this time, the first eggs are deposited directly on wood, generally at the base of unopened buds (spurs), in a number of 300-400 per female. as foliage becomes available in midto latemarch, oviposition shifts to occur primarily on expanding leaves and flowers: eggs are deposited along mid-veins and petioles of developing leaves and on stems and sepals of blossoms. first nymphal instars escape from wintereggs in concurrence with bud opening and leaves sprouting and infest the new vegetation. the first generation of summerform adults appears in april and fecundity of females appears to be quite higher compared to winterforms due to their major longevity (on average about 600 eggs per female) (stratopoulou and kapatos, 1995), although high temperatures can cause a substantial reduction in fecundity. springand summer-deposited eggs require ca. 6-10 days to hatch, depending upon temperature (mcmullen and jong, 1977). there are 5 nymphal instars. nymphs require 3-4 weeks to complete development at moderate (21-27°c) temperatures (georgala, 1956; mcmullen and jong, 1977). male and female progeny are produced in equivalent numbers (burts and fischer, 1967). afterwards (may) feeding nymphs of second generation develop on growing shoots with an aggregate distribution on leaves and internodes (deronzier and atger, 1980; pasqualini et al., 1997) immersed in pools of honeydew, which they produce in extremely large amounts. further generations overlap with all the ontogenetic stages and phases till autumn. in the warmer periods aestivation phenomenons may occur (stratopoulou and kapatos, 1995). c. pyri develops 5-7 generation per year. 3. host finding, probing and feeding behaviour host finding for feeding and oviposition contemplates a sequence of phases of hierarchic nature. the first, out of the three principal phases, consist of a host selection that the insect makes from distance using visual and olfactive impulses. the second phase occurs when the insect takes contact with the plant surface getting information about its physical structure. these impulses may strongly influence female egg-laying. the third phase regards the discrimination between host and non host plant through gustative impulses perceived during the survey of the internal tissues, the so called ‘probing behaviour’. although most of the homopteran insects have been reported to make little use of volatile compounds for the long and mid distance host finding and acceptance, the chemical characteristics are thought to be much more plant specific than the quality of the visual spectrum (prokopy and owens, 1983; dethier, 1982). the role of olfactive and gustative sensilli existing in the antennas and tarsi in not yet known, whereas that of gustative sensilli in the mouth apparatus is evident. psylla rarely initiates oviposition activities immediately upon leaf contact. rather, oviposition activities tend to be preceded by settling-probing activity, as evidence that plant cues received on initial contact are insufficient to release oviposition activity but that plant cues received during settling-probing activity release oviposition activities. thus plant cues received during oviposition activities ultimately affect whether the egg is depositated (horton and krysan, 1991). deprivation or habituation may result in higher number of eggs oviposited on less suitable genotypes than in free-choice tests. indeed, c. pyricola adult has been found to be more selective in oviposition activities than in its settling-probing activities, i.e. probing is not likely to be an indicator of a variety’s acceptability, since c. pyricola is able to colonize and feed nonhosts like pyrus calleriana and malus spp., but without laying eggs (horton and krysan, 1990). as for plant cues that mediate host acceptance it has been reported that pear psylla readily settled on nonhosts to an extent that initially unacceptable species eventually receive eggs, thus suggesting either that plant cues that release settling attivity differ from those that release abdomen bend activity and oviposition, or that thersholds for these activities differ. yet, there is evidence that settling is partially mediated by leaf surface characteristics for winterform psylla. first, settling-probing activity differs between upper and lower leaf surfaces, suggesting that cues received at the leaf surface affect activity. second, at leaf contact, pear psylla scrape the leaf surface with their tarsi (ullman and mclean, 1988; horton and krysan, 1990). finally, despite the tendency to settle readily on apple, the amount of time between initial leaf contact and onset of settling-probing was smaller for psylla encountering bartlett pear than for those encountering apple, suggesting that leaf surface cues affected behaviour. psylla adults ingest more frequently xylematic tissue, while nymphs prefer phloematic tissue or at least that of vascular fasces. a good knowledge on the different c. pyri feeding phases by means of epg may allow to discriminate between susceptible and resistant selections and to locate the mechanisms of resistance within plant tissues (civolani et al., 2010). up to know only little differences have been found in the feeding behavior of psylla adults on the susceptible william and the resistant ny10353 pears. however, lasting of the first and second non-probing is longer on william compared to ny10353, and c. pyri needs less time to reach the phloematic fasces in the susceptible plant, in accordance wih the assumption of horton and krysan (1990, 1991). 4. types of damage pear psylla causes three primary types of damage: fruit russet, psylla shock, and pear decline (burts, 1970; westi61 gard et al., 1979; beers et al., 1993). fruit russet is caused by the feeding activities of nymphs and is of most concern to growers, and control programs are generally directed at preventing this injury, since it can be caused by relatively low population densities (burts, 1988). as in other homoptera, pear psylla ingests excessive quantities of plant juices and other plant products that must be eliminated (as honeydew) during the digestive process. in fact, lymph in the phloematic tissue is rich of carbohydrates and poor of nitrogenous substances, due to this deficiency the insect has to absorb a great quantity of lymph that it afterwards excretes through his digestive apparatus producing honeydew. adult psylla excrete these waste products as small, waxy pellets that cause no harm to the plant or fruit. conversely, the immature form of the insect secretes copious quantities of honeydew, a sugary, sticky substance. if nymphal-produced honeydew is in contact with fruit for a significant period of time it causes dark blotches or streaks on the surface of the fruit (russetting), which in turn results in downgrading of the fruit at harvest (burts, 1970). honeydew allows a black, sooty mold fungus (antennaria, aureobasidium, capnodium, ceratocarpia, cladosporum, torula, ulocladium) to grow on both fruit and leaves, not only reducing the quality of the fruit, but also blocking sunlight from the leaves and decreasing photosynthesis. a second type of injury, also caused by the sucking nymphs at high densities, is of a more indirect nature than that previously mentioned. infected leaves turn brown and often fall and the fruits drop prematurely or are small and of poor quality, thus suppressing root growth and reducing tree vigor and yield. (westigard and zwick, 1972). these symptoms have collectively been termed psylla shock, and are caused by a toxin in the saliva of feeding nymphs (beers et al., 1993). symptoms of the injury can be similar in appearance to those associated with pear decline disease. psylla shock can be particularly damaging because the effects are not always restricted to the year of infestation, but symptoms may carry-over into a second year even if densities are not high the second year (beers et al., 1993). cultivars that are less preferred by psylla, such as some red pears or pears of asian origin, tend less likely to experience this type of damage. finally, adult pear psylla vector the mycoplasma-like organism (hibino and schneider, 1970) that is the causal agent of pear decline disease especially during vegetative growth (carraro et al., 1998; davies et al., 1998; guerrini et al., 2000). the feed and phytoplasma are assumed together from a diseased plant and transmitted to a healthy plant during the salivation of phloematic feedings (carraro et al., 1998). the way of transmission is persistentdispersive, as pear decline phytoplasma propagate in the insect body. disease acquisition and inoculation require at least 1-2 hours of phloematic feeding; thereafter the vector undergoes to a period of latency (about 1-2 weeks) during which the phytoplasma circulates and propagate within its body till he reaches the slave glands. both winterform and summerform pear psylla can be important in the transmission of pear decline (blomquist and kirkpatrick, 2002). this pathogen causes sieve-tube necrosis at or below the graft union (batjer and schneider, 1960; westigard et al., 1979), preventing tree-synthesized nutrients from reaching the roots and resulting in starvation of the roots (wilde and mcintosh, 1964). symptoms of the disease include a slow to abrupt decline or collapse in growth and vigor, causing a reduction in yield and (often) death of the tree. certain affected pear trees may recover if psylla densities are kept low or during winter quiescence thanks to the degeneration of epigeous phloematic tubes (giunchedi and refatti, 1997; davies et al., 1998). severity of the disease depends upon psylla density and type of rootstock (beers et al., 1993). cultivars that have been grafted onto p. communis rootstock are less susceptible than those grafted onto p. pyrifolia or p. ussuriensis rootstock. quince (cydonia oblongata) rootstocks posses a limited aptitude to allow phytoplasma survival between one vegetative cycle and another. resistant rootstock has largely remedied this problem in various pear growing regions. 5. monitoring and control tactics the psyllid c. pyri, along with its natural enemies, needs to be carefully monitored for correct integrate pest management and biological pest control decision making. moreover, timing of spray application against c. pyri is crucial because recommended insecticides are only efficient at certain stages. monitoring should provide starting from spring density of eggs, nymphs, adults and presence of the principal antagonist. a simple method for estimating densities of pear psylla is desirable. monitoring pear psylla is made difficult by the uneven distribution of insects (eggs and nymphs) on the trees, a distribution that may in fact change seasonally. densities of psylla may also vary with height in the tree canopy (and sex) both for c. pyricola (brunner, 1984; horton, 1994) and c. pyri (stratopoulou and kapatos, 1995). currently, sampling of the adult population is necessary to determine the onset of reentry in late winter or the popolation density, and sampling of fruit spurs for eggs is often the easiest way to determine the beginning of egglaying. counts have been obtained in usa using frappage, sticky traps, beat trays and open-ended organdy bags, the last one providing direct estimates of psylla numbers per leaf but being extremely time consuming (horton, 1994; horton and lewis, 1997). effectiveness of yellow stickyboard traps have been examined by several authors and seasonality of the catch and flight activity of pear psylla (c. pyricola) according to weather conditions have been reported (krysan and horton, 1991; horton, 1994; civolani and pasqualini, 2003; erler, 2004), as well as diurnal difference (horton, 1993) and intraorchard changes in distribution associated with leaf fall (horton et al., 1993). laboratory study have shown that males of both the sum62 merform and winterform morphotypes in c. pyricola are attracted to volatiles given off by females, whereas in the field male has shown a clear preference for sticky traps that have been baited with live females compared with traps baited with live males or left unbaited (brown et al., 2009). limb beating or limb jarring to collect arthropod specimens from trees has been known for a long time and in several variations: frappage or beating tray (two-dimensional) and beating umbrella (three-dimensional). the first of this procedure has been applied by several authors and has been reported by jenser et al. (2010) to depend considerably on weather condition, while the second one to be much less temperature and wind-sensitive, due to its vertical extension, and much more suitable for collecting fast moving or flying beneficial organisms than the twodimensional method. in contrast with the hypothesis that any data collected for the adults using a beating umbrella would be influenced more by weather conditions than those gathered using funnels, sanchez and ortìn-angulo (2011) have found a higher efficacy of the net in relation to the funnel. the same authors also have stated a low efficacy of the beating techniques for sampling nymphs that may be due to the fact that they hold tight to the substrate and are not easily removed by the act of beating. both the application of the sticky board traps and beating tray provide accurate information about the changes of pear psylla population density (jenser et al., 2010). in particular the capture of adults using either the funnel or the net may be used to estimate the absolute number of c. pyri nymphs on trees, thanks to the high correlation found by sanchez and ortìn-angulo (2011) between nymphs counted on shoots and the capture of adults using either of the beating techniques. several authors have reported the same relationship for other psyllid species (horton, 1994; jenser et al., 2010). the beating techniques also have the advantage of being less time-consuming than the sampling of leaves and, for beating over a net or tray, samples may be processed directly in the field, although the amount of collected insects using beating umbrella some times makes necessary the laboratory process. in europe the dinamic of c. pyri populations have been studied using frappage by various authors (deronzier, 1984; rieux at al., 1992). according to civolani and pasqualini (2005) frappage is the sampling method which best represents the dinamics of populations of psylla and its predators (antocoridis, coccinellids, lacewings). predators overwintering in bark cervices may be estimated by using corrugated cardboard traps (bogya et al., 1999; horton et al., 2002; civolani and pasqualini, 2003; jenser et al., 2010). alternatively the psylla eggs have been counted on the shoots and leaves using a binocular dissecting microscope by several authors (jenser et al., 2010) and a few of the authors investigated and counted both the eggs and larvae. this method provides real data, but it’s time consuming; the sample must be taken into the laboratory and the counting completed within a short time. berlese funnel is a widespread technique for extracting arthropods mainly from soil and litter samples (stäubli et al., 1992). moreover the mite brushing machine or leaf brushing machine developed by henderson and mcburney (1943) is a technology that can reduce the time required to obtain either absolute counts or estimates of arthropods on leaves from samples. recently developed, the wash-down method described by jenser et al. (2010) offers the advantages of the independence of the weather conditions (temperature, wind, rain) and the daily rhythm of the examined psylla stages. since practically every larvae developing on the flowers and shoots are extracted, it provides suitable data about the pear psylla population density and its changes, as well as about the effectiveness of the insecticides. this method has been suggested to provide also significant data to judge the susceptibility or tolerance of the pear cultivars to pear psylla species. since observing the population development of pear psylla is time-consuming and prone to error, phenological models could assist growers in the timing of monitoring and control measures, as they simulate and predict, by means of driving variables (usually temperature), the timing of natural events. there have been modest attempts to develop degree-day models that predict onset of egglaying and appearance of first generation nymphs (westigard and zwick, 1972; brunner, 1984; beránková and kocourek, 1994) and timing of reentry (horton et al., 1992), with aims toward improving timing of the dormant spray. morgan and solomon (1993) have provided a phenological model for c. pyricola which have been integrated into a multipest forecasting system. further on, a phenological model for c. pyri based on biological mechanisms, in particular the emergence of juvenile instars of the second generation, has been developed by schaub et al. (2005). in italy the defence against c. pyri is mainly based on integrated pest management (ipm), supported by natural control aimed to equilibrate the complex biological relationships of the field community (civolani, 2012). among the basic strategies there are the ‘good agricultural practice’ (gap) techniques that reduce tree suitability for growth and reproduction of pear psylla by avoiding overuse of fertilizers, incorrect or over pruning, and reducing excessive plant vigor (beers et al., 1993; civolani, 2012). suckers or water sprouts should be removed from scaffold limbs (beers et al. 1993), because these are a source of rapidly growing and highly nutritious foliage. also the strategies to control other pest species, such as the technique of mating disruption and the use of granulosis virus (cpgv) employed to control codling moth (cydia pomonella), may influence psylla and assist in chemical control. however, in the last decade commercial pear growers have relied primarily on the use of synthetic products to control pear psylla, and the advantages and disadvantages of the main strategies performed in the last 20 years in integrated and conventional farms have been described by civolani (2012). unfortunately, these methods are not always entirely effective, as their efficiency depends both on the active in63 gredients employed and the weather conditions at the time of treatment and moreover pear psylla has developed resistance to several classes of commonly used insecticides (riedl et al., 1981; follett et al., 1985; burts et al., 1989; croft et al., 1989). current control recommendations emphasize destruction of the overwintered generation, or offspring of the overwintered generation with insecticides. a typical control program for overwintered adults is performed at leaf fall, commonly in france on c. pyri and in north america on c. pyricola, with the application of pesticides belonging to the pyrethroid family (with or without mineral oil added), repeated as necessary in late winter (at bud swelling stage or bud break) to break down the population of females emerging from winter shelters and about to lay eggs. these pyrethtoids are completely non-selective but broad spectrum and therefore dangerous for the beneficial insects. for this reason the treatment must be performed only at complete leaf fall (late november or early december), when a. nemoralis populations have already found shelter in bark crevices while c. pyri adult winter forms are still active on plants. efficiency of treatments may vary considerable upon seasonal conditions. for example most psylla adults take shelter early and survive to the late autumn treatments when an early frost occurs at the beginning of autumn. similarly the efficiency of chemicals is reduced after frost waves at the end of winter which interrupt and delay the emergence of adults, while activity of pesticides is best after a mild winter when almost all adults leave their shelters at the time of treatment (civolani, 2000; civolani and pasqualini, 2003). in italy the dormant sprays are discouraged since the pest population, after an initial sharp decline, soon recovers in spring because the natural control by its predator a. nemoralis is limited, then increases again in may, reaching the economic threshold for spring-summer treatments (civolani, 2012). the main side effect of the use of pyrethroids in late winter is that they sharply reduces the psylla first generation and therefore could starve the anthocorids, interfering with their settlement during early plant growth in spring. various alternative biorational solutions to synthetic pesticides have been tested against the overwintering generations, and among them kaolin and some oily compounds. kaolin, a white, non-abrasive, fine-grained allumosilicate mineral that is purified and sized so that it can be easily dispersed in water, creates a mineral barrier on plants that prevents oviposition and insect feeding (puterka et al., 2000). treatment with kaolin has been reported to hinder egg anchorage on the leaf surface and inhibiting host-plant acceptance. moreover, some insects have been found to be less mobile and unable to reach the laying site (host location) on plants, as their body and wings have became soiled (pasqualini et al., 2003; daniel and wyss, 2006). further on, puterka et al. (2005) investigated the effects of particle film type (hydrophobic versus hydrophilic) and formulation determining that there are a number of biological effects particle films have on pear psylla beyond the deterrence of adult settling and oviposition. alternatively, mineral oils and oily compounds could also be used to interfere with egg deposition by psylla adults. a good reduction of the number of eggs laid has been obtained in northern italy with pure mineral oil alone (‘dormant oil’) (pasqualini et al., 2003) and in turkey with fish-liver oil and summer oil (erler, 2004). some growth regulators have proved to possess a good activity against eggs and nymphs of first and second generation by interfering on the cuticle transport and deposition during larvae development (erler and cetin, 2005). at the beginning of the second generation growers can assess the risk to their orchard and still target specific stages. therefore, treatments target mostly eggs and/or young larvae of the second generation. the treatments against summer generations can be performed towards eggs or nymphs. chitin inhibitors, usually employed against c. pomonella, have shown a secondary effect on second generation eggs, usually laid in the first decade of may, especially when they are applied on newly laid eggs (white eggs) or on eggs laid in a short time after the treatment. however, control strategies against juvenile stages are of most relevance, and were performed in the past with generic organophosphorates, whereas are based in present times on specific synthetic active ingredients, often acaricides. among these, abamectin (produced by the soil bacterium streptomyces avermitilis) is the basic chemical employed today against young nymphs of second generation (usually in may) and included in the italian disciplinary of integrated management. the best results are obtained when yellow eggs are mostly present and when the hatching peak has not yet achieved (pasqualini and civolani, 2006). abamectin is allowed only once in a year or twice in case of young orchards; since it’s not systemic, the addition of mineral oil may improve its penetration within 24 hour time. a new broad spectrum acaricides, namely spirodiclofen (baj2740, trade name: envidor®), belonging to the new chemical class of tetronic acid derivatives, has been discovered by bayer cropscience during the 1990s and is commercially available since 2007. spirodiclofen has a new original mode of action (interference with lipid biosynthesis) and shows no cross-resistance to any resistant mite or whitefly field population, representing an invaluable new tool to manage insecticide resistance in rotation with abamectin. it’s efficiency is best on yellow eggs some days before the hatching of first instar nymphs and is improved by addition of mineral oil, although often lower than that of abamectin (pasqualini and civolani, 2007; boselli and cristiani, 2008; marčić et al., 2009). besides summer mineral oils, whose main action is that of dissolving honeydew, sodium dioctyl sulphosuccinate or other vegetal free fatty acids may be used for washing the trees (briolini et al., 1989). recently some other novel compounds have been used, similar to liquid glue and capable of controlling almost all juvenile instars of c. pyri. these products are synthetic sugar esters (sucrose oc64 tanoate) and represent a relatively new class of insecticidal compounds that are produced by the reaction of sugars with fatty acids, valuable in crop integrated pest management programs (puterka et al., 2003). it’s important to keep in mind that summer psylla infestation depend on the antagonists development in spring, first of all the most important one, a. nemoralis, which has to be protected. for first instar nymphs control threshold is given by the ratio between number of infested shoots and number of shoots with the antagonist antocoride, which have to be ≥ 5 (marani and reggidori, 2007). also the relevant effect of weather conditions on pest populations should not be underestimated. in fact, the development of psylla is strongly reduced by high summer temperatures that cause the death of eggs and the slowdown of juvenile growth. on the other hand, cold and rainy periods during blossoming and petal fall encourage nymph spreading on plants, often clustering in flower calyxes, sometimes causing russet blotches or young fruit drop (civolani, 2012). climate conditions, such as wind, have been demonstrated to have an impact on the clustering of psyllids, wheras spatial factors, such as distance from a mixed hedge have been found to be related to beneficial arthropod community (debras et al., 2008). localized resistance cases to organophosphorates insecticides, pyrethroids and carbamates pesticide families and chitin inhibitors family have been developed and have been largely documented, especially for c. pyricola in north america since 1960 (harries and burts, 1965). resistance rates among the active ingredients has been reported to be very variable in laboratory tests and probably there are different mechanisms involved in the resistance to different pesticide families, as reported by civolani (2012). in italy, cases of loss of efficiency of abamectin have been noticed in some orchards in emilia-romagna region, indicating that there is a high risk of selection for resistance to abamectin, especially if the number of treatments per year is high. up to now, the tests data indicate that no apparent resistance to abamectin has been developed in c. pyri populations of that region, but may rather be related to incorrect pest defence management (civolani et al., 2007). control strategies should be based on a limited use of pesticides, possibly selective ones, in order to foster the development of a. nemoralis populations, which become a relevant factor to control the pest, preying on both eggs and nymphs of psylla. in emilia-romagna a. nemoralis generally shows three generations and may feed also on other insects, for example aphids and the pear sawfly hoplocampa brevis. laboratory tests have shown an average predation of about 300 psylla nymphs during the entire life of an adult, which lasts about 60 days (civolani, 2012). one problem is that the populations of this anthocorid grow rapidly in spring only if there is psylla of first generation in the orchard for feeding, therefore in may-june some amounts of the pest have to be tolerated. a further weakening of the wild a. nemoralis populations may be caused by the large amount of active ingredients used against other pests, having significant toxic effects on a. nemoralis. among these, thiacloprid, the most frequently pesticide used against the codling moth, c. pomonella, as well as the neonicotinoids, not employed in italy and europe as specific psyllicides, but against aphids and the pear sawfly h. brevis. the artificial introduction of the antagonist antocoride at the end of march beginning of april is a very useful mean for controlling eggs and young nymphs of the first generation. the flow is made with about thousand individuals per hectar fractioned in 3 times at weekly intervals. resettlement is much more feasible much wider the cultivated area is (minimum 1 hectar). some authors however retain that 500 individuals would be sufficient for each introduction (beninato and morella, 2000). good natural equilibrium have been obtained in veneto with the introduction of 500-600 psylla adults per ha in a sole time in may (mori and sancassani, 1984). in france the introduction of a. nemoralis has been performed by the distribution of pelargonium stems containing 2.940 eggs of the psylla antagonist (fauvel et al., 1994; rieux et al., 1994). some authors indicated that the efficacy of this predator is not strongly mediated by plant quality, at least at tree scale, thus, for systems where pest population growth is strongly tied to plant vigor or quality, the reduction of fertilizers to the minimum level required for proper fruit set is likely to improve the success of pest biocontrol (daugherty et al., 2007). 6. new pesticides and strategies for integrated pest management in the last years new pesticides have been developed with generally low toxicity towards beneficial insects. aksebio2 is a mixture of various aromatic plant essential oils, edible plant extracts and a bacterium tr 2000 which decreases oviposition and immature stages of the pest (erler et al., 2007). spirotetramat (movento®) is a new, fully systemic and ambimobile active ingredient particularly effective against a broad range of sucking pests, similar to the tetronic acid derivate spirodiclofen. its singularity depends upon its unique translocation property, which allows the protection of new shoots or leaves appearing after foliar application, in fact after foliar uptake the insecticidal activity is translocated within the entire vascular system (nauen et al., 2008). due to the lack of any cross-resistance to existing chemical classes of insecticides, spirotetramat is a very interesting alternative to be used in rotation schedules. natural plant compounds, fungal pathogens and different orchard ground cover all seem promising controls. among nontoxic plant compounds, sugar-ester extracted from wild tobacco has proved to be most successful in psylla control (usa), killing most nymphs within 2 hours. even nymphs that hatched 3 to 5 days after spraying die as soon as they walk on leaves (stanley, 1993). rapeseed 65 oil and petroleum oil as well have showed a total efficacy against eggs laid by winterforms females of c. pyri (marčić et al., 2008, 2009). several naturally occurring fungal pathogens (spores of beauveria, verticillium, and paecilomyces mixed with either oil or water) have given 100-percent control as well within 5 days. the advantage is that fungi can last indefinitely compared to the sugarester that may persist on the plant for about a week. they are host-specific, completing their life cycle on infected insects on the plant, and therefore nontoxic to humans, animals and beneficial insects. after killing their host, the fungi release hundreds of spores, each capable of infecting another pear psylla. since pear psylla also have several predators (table 1), planting ground covers with perennial crops between tree rows to attract them could provide a measure of control (stanley, 1993). new strategies for integrated pest management of psylla may be offered in the future by the optimization of the recently identified sex attractant pheromone, the 13-methylheptacosane, for c. pyricola winterforms males (guédot et al., 2009). 7. resistance to pear psylla all of the main cultivars of the european pear grown commercially (abate fétel, william, conference, doyenne de comice, kaiser, etc.) (bellini and nin, 2002) are susceptible to this arthropod pest and biological controls are becoming of limited effectiveness since resistance to insecticides has developed rapidly. host plant resistance would therefore be a valuable control strategy. resistance to the pear psylla has been demonstrated in the east asian pear species, p. betulifolia bunge, p. calleryana decne., p. fauriei schneid., p. ussuriensis maxim., and p. x bretschneideri redh. (westigard et al., 1970; quamme, 1984; moore and ballington, 1991). hybrids of p. ussuriensis x p. communis have been found to be resistant to c. pyricola (harris, 1973; harris and lamb, 1973; quamme, 1984) as well as to c. pyri (robert et al., 2004). different interspecific hybrids between p. communis and p. longipes or p. pyrifolia have shown high levels of resistance to c. pyri, too (robert et al., 2004). resistance has been reported also for a few genotypes of p. nivalis jacq. and sorbopyrus (westigard et al., 1970; bell, 1992). small fruit size of the pure species and gritty or coarse texture of both the pure species and interspecific hybrids may limit the utility of some of this germplasm for rapid transfer of resistance into cultivars with p. communis type fruit. within p. communis, moderate resistance has been demonstrated in the old italian cultivar spina carpi (quarta and puggioni, 1985), and in eleven ‘primitive’ cultivars from yugoslavia and hungary (bell and stuart, 1990; bell, 1992). all of these genotypes have relatively poor fruit quality but are important sources of resistance within the primary gene pool available for improvement of p. communis cultivars. in many countries ex situ pear collections have been established in some important pear growing areas with a great diversity of national, local and foreign cultivars, mainly for evaluation of resistance to major disease and insects, to be used as potential parents in breeding (quarta and puggioni, 1985; braniste et al., 1994; braniste and militaru, 2008; benedek et al., 2010). more than 200 pear cultivars of tuscan, national and international origin, intable 1 natural enemies associated with pear psylla in europe natural enemy taxonomic group species predators arachnida, araneae unidentified spiders dermaptera forficula auricularia linnaeus heteroptera, anthocoridae anthocoris nemoralis (fabricius) orius spp. heteroptera, nabidae nabis spp. heteroptera, miridae several species heteroptera, lygaeidae several species neuroptera, chrysopidae chrysoperla carnea (stephens) chrysopa formosa brauer chrysopa septempunctata wesmael anisochrysa prasina (burmeister) coleoptera, coccinellidae several species belonging to different genus diptera, syrphidae episyrphus balteatus (de geer) epistrophe spp. parasitoids hymenoptera, encyrtidae trechnites psyllae (ruschka) prionomitus mitratus (dalman) hymenoptera, pteromalidae syrphophagus mamitus (walker) entomopathogenic fungi entomophthora sphaerosperma from: armand et al., 1991; tremblay, 1995; civolani and pasqualini, 2003; erler, 2004. 66 cluding also 25 afghan accessions, are presently being evaluated in ex situ and in situ collections for psylla resistance at the department of plant, soil and environmental science of the florence university (dipsa-unifi) within the ager project ‘innovapero: management and crop innovations for high-quality pear production’. moreover, evaluation of insect preference in tunnel is being in progress on 26 local cultivars showing good pomological traits. taking earlier and present results into account almost 60 european pear cultivars being resistant or highly tolerant to pear psylla infestation and damage can actually be listed (table 2). some of these ancient or local cultivars may be exploited both in organic farming or in breeding, but further investigations are needed to estimate their yield capacity and fruit quality (benedek et al., 2010; szabó et al., 2010). moreover, some varieties considered resistant in field have shown to be susceptible, if isolated and articially infested by adults (westigard et al., 1970; harris, 1975). methods of evaluating host resistance are sufficiently developed and rapid nymphal feeding bioassays have been developed to screen pear germplasm for antibiosis-based resistance by harris (1973, 1975) and butt et al. (1989) for the evaluation of pear germplasm introduced in north america from eastern europe, and then modified by different authors (table 3). the results of tests can vary, depending on the type of assay and host phenological stage, which affects ovipositional preference (bell and puterka, 2003). genetic psylla resistance do not follow a general rule and is supposed to be often polygenically inherited (harris and lamb, 1973). lespinasse et al. (2008) found that psylla resistance was not well transmitted from the p. ussuriensis x p. communis hybrid ny10355 to its progenies, assuming that genetic resistance in ny10355 may result table 2 pear cultivars showing some degree of resistance to psylla as reported by different authors cultivar tolerant resistant moderately resistant low susceptible country reference 20th century x serbia stamenkovic et al., 1993 bartjarka x usa bell, 1992 bókoló körte x hungary benedek et al., 2010; szabó et al., 2010 bötermö kálmán x hungary benedek et al., 2010; szabó et al., 2010 bulgaresti x romania braniste et al., 1994 cantalupesti x romania braniste et al., 1994 cantari x romania sestras et al., 2009 cj16-9-13 x romania straulea et al., 1992 craiesc x romania braniste et al., 1994 cure x romania straulea et al., 1992 cure-6 x hungary benedek et al., 2010 d’aout lamer x france robert and raimbault, 2005 daoyenné de poitiers x france robert and raimbault, 2005 erabasma x usa bell and stuart, 1990 ewerd x romania braniste et al., 1994 füge alakú x hungary benedek et al., 2010 general osmanwill x romania braniste et al., 1994 haydeea x romania sestras et al., 2009 honeysweet x usa quamme, 1984 imperiale x romania braniste et al., 1994 imperiale x romania sestras et al., 2009 jerisbasma x usa bell, 2003 kajzerka x usa bell, 1992 karamanka x usa bell, 2003 karamanka x serbia stamenkovic et al., 1993 karamanlika x usa bell and stuart, 1990 katman x usa bell and stuart, 1990 katman x france robert and raimbault, 2005 kései kálmán x hungary benedek et al., 2010 kieffer seedling x romania braniste et al., 1994 67 either from the combination of several small-effect resistance genes, according to pasqualini et al. (2006), or from a combination of dominance or epistatic effects or from both. a genetic mapping approach should help researchers to understand the genetic mechanism of psylla resistance. the molecular interaction between pear tree and the piercing/sucking psylla has been investigated through the construction and characterization of cdna subtracted libraries. genes expressed upon insect infestation were identified in a susceptible and a resistant pear genotype. the two expression profiles were found to be different: in the resistant plant more genes involved in the response to biotic and abiotic stress were activated than in the susceptible one. the further characterization of the identified genes could lead to the development of molecular markers associated with tolerance/resistance to psylla (salvianti et al., 2006). the quantitative resistance to pear psylla has been analyzed recently in a progeny of the european pear angelys crossed with the resistant genotype ny10355, and by screening parents/seedlings with microsatellite markers a qtl (quantitative trait loci) that explained 15% of the phenotypic variability has been determined and mapped on the linkage group 17 (bouvier et al., 2011). cultivar tolerant resistant moderately resistant low susceptible country reference kieffer x hungary benedek et al., 2010 kieffer éd x hungary benedek et al., 2010 krupen burnusus x usa bell and stuart, 1990 krupen burnusus x usa puterka, 1997 lorencz kovacs x romania sestras et al., 2009 lorenz x romania braniste et al., 1994 lucele x usa bell, 1992 magness x romania braniste et al., 1994 magness x serbia stamenkovic et al., 1993 mednik x usa bell and stuart, 1990 mednik x usa puterka, 1997 monglow x italy quarta and puggioni, 1985 nagyasszony körte x hungary benedek et al., 2010 nyári kálmán x hungary benedek et al., 2010 obican vodenac x usa bell and stuart, 1990 obican vodenac x usa puterka, 1997 pinguoli x serbia stamenkovic et al., 1993 rocha portugheza x romania braniste et al., 1994 rozs nyári körte x hungary benedek et al., 2010 severinka x romania sestras et al., 2009 sierra x usa quamme, 1984 sirrine x usa quamme, 1984 sirrine x italy quarta and puggioni, 1985 smokvarka x usa bell and stuart, 1990 spadona x romania braniste et al., 1994 spina carpi x italy quarta and puggioni, 1985 spina carpi x france robert and raimbault, 2005 steiner x hungary benedek et al., 2010 téli kálmán x hungary benedek et al., 2010 tomnatice x romania braniste et al., 1994 topka x usa bell and stuart, 1990 triomphe de joidogne x romania braniste et al., 1994 triomphe de joidogne x romania sestras et al., 2009 vidovaca x serbia stamenkovic et al., 1993 viki körte x hungary benedek et al., 2010; szabó et al., 2010 william precoce morettini x romania braniste et al., 1994 zelinka x usa bell and stuart, 1990 zelinka x usa puterka, 1997 68 8. resistance characterization resistance is characterized by both ovipositional non-preference (antixenosis = settling and oviposition) and feeding inhibition, delayed development and increased nymphal mortality (antibiosis) (bell and stuart, 1990). while antixenosis influences the size of the initial nymphal population, antibiosis probably exerts the greatest effect on population levels over a season. so, feeding rejection is a major component of resistance and leads directly to a precocious nymphal mortality; the mechanism for feeding acceptance or rejection is probably internal to the leaf as reported by butt et al. (1988). table 3 pear resistance to psylla: assay methods adopted in controlled conditions reference number of insects used for artificial infestation site of infestation replications per cultivar/selection observations (hours or days after infestation) harris, 1975 100 adults plant 1 4 days: removal of adults, growth and development of the resultant progeny butt et al., 1988 1 nymph lower midrib of 10 fully expanded detached-leaves 3-10 2 h, 4 h, 6 h, 24 h: position of nymph and presence of honeydew butt et al., 1988 1 nymph lower midrib of 10 fully expanded leaves of potted trees 2 2 h, 4 h, 6 h, 24 h: position of nymph and presence of honeydew butt et al., 1988 10 first instars lower midrib of the 2 youngest fully expanded leaves of potted trees 2 24 h: position of nymph and presence of honeydew butt et al., 1989 25 first-instar nymphs 2 youngest and fully expanded leaves of a shoot 4 each day: feeding determined by excretion of honeydew puterka et al., 1993 2-6 females excised twig collected at different stages of bud development 8 24 h after infestation at stages of dormant bud, green tip, fully expanded leaf: adults per twig 48 h: adults per twig 72 h: eggs per twig berrada et al., 1995 15 pairs of sexually mature adults 10-16 leaves (≈300 cm2) 4 24 h: removal of adults and egg count each day: survival of eggs and larvae until they developed into adults baldassari et al., 1996 6-10 third-fourth instar nymphs 2 younger and more expanded leaves of a shoot 2-3 5 days: vitality of nymphs, amounts of produced honeydew, possible development of sooty moulds 15 days: number of deaths baldassari et al., 1996 10 first instar nymphs 2 younger and more expanded leaves of a shoot 3 every day: number and age of dead nymphs, days needed for possible development of adults puterka et al., 1997 5 nymphs 4 fully expanded terminal leaves 5 4 days: nymphal survival and development alternating 3rd and 4th day up to day 29: nymphal survival and development robert et. al., 1999 1 female plant 1 2-7 days: female removal after 50 eggs on average per plant had been laid each week: larval mortality and count of different instars bell, 2003 10 second or third instar nymphs underside of the top 2 youngest fully expanded leaves of 5 48 h: number of surviving and actively feeding nymphs robert and raimbault, 2005 4 females and 1-2 males in two times at 8 day-interval plant 7-8 15 days: number of eggs on the 8 upper leaves of shoots 36, 63 and 98 days: number of nymphs 134 days: shoot and leaf state pasqualini et al., 2006 300-400 males and females plant 5-16 10 -25-50 days: number of adults per plant 10-25 days: number of eggs per plant 25-50 days: number of nymphs producing honeydew per plant pasqualini et al., 2006 1 female upper surface of a leaf in a clip-cage 3-11 48-72 h: number of laid eggs per female bouvier et al., 2011 8 insects plant 7 presence of honeydew on the first, second and last third of the plant. the quantity of larvae present on the whole plant 69 volatile substances emitted by the leaves of different varieties are not substantially dissimilar and therefore do not probably play a basilar role in the affinity an repulsion of psylla adults (miller et al., 1989), but bioassays on this topic are still lacking. resistance of genotypes is not directly proportional to leaf cuticle thickness, the resistant genotype ny10355 for instance has a lower content of cutin compared to the susceptible william variety (gérard et al., 1993). pubescence is not a major factor in feeding deterrence according to bell and stuart (1990). however, antixenosis is influenced by both the physiological status (bigre and lefeuvre, 1982) and bud phenological phase (stuart et al., 1989; puterka et al., 1993) of pear tree. thus differences in leaf morphology may influence psylla oviposition, bearing in mind that the insect prefers to lay the eggs on prominent structures such as leaf vein or crevices at the base of fruiting spurs. resistant genotypes express antibiosis with the production of a limited amount of honeydew and a strong nymphal mortality (butt et al., 1988, 1989). however, the quantity of produced honeydew has not been denotive of the infection intensity on selections obtained by induced mutagenesis (baldassari et al., 1996). ingestion of substances belonging to the group of polyphenols (for example tannins) has been suggested to be the cause of this mortality (bell, 1984). challice and williams (1968) underlined the presence of the group of active components flavone glycosides in the asiatic pyrus ussuriensis, which is lacking in pyrus communis. braniste et al. (1994) evidenced a lower total isoperoxidase activity in resistant pear cultivar compared to susceptible ones. also sugar content in leaves differed between resistant and susceptible genotypes, an increase of sugar content due to a reduced level of starch synthesis and also its rapid degradation was noticed in susceptible cultivars. fiori and lamb (1982) found the presence of secretory cells to be much more extensive in the phloem of leaf midveins of pear genotypes with resistance against p. pyricola and suggested that average percentage of the phloem area occupied by secretory cells in may-june may provide a valid method for determining the resistance of pear trees to p. pyricola. antibiosis towards preimmaginal stages is accompanied by a reduction of feeding frequency, which may be linked to the presence of nutritional inhibitors (butt et al., 1989) or to an insufficient plant alimental appetizer (chang and philogène, 1975). later on, the ex novo induction of a phenolic compound (3-o-trans-p-cumaroyltormentic acid) has been demonstrated after 12 hours from the phytophaga attack with a pick after 30 days from infection (scutareanu et al., 1999). this induction has been recently shown to be local (conference) or systemic (william and ny10355), but there are no evidence on whether this induction can modify c. pyri behavior or not (scutareanu et al., 1999). a different effect has been attributed to other volatile substances still originating during the wounding process of the mouth apparatus. some of these essences released from infected pear leaves, i.e. the monoterpene (e,e)-α-farnesene and the phenolic compound methyl salicylate, are primarily responsible of the attraction of the main psylla predators, namely anthocoris nemoralis and anthocoris nemorum (scutareanu et al., 1997, 1999, 2001). the capacity of some plant species to emit mixture of volatile compounds, dominated by terpenes, to attract carnivorous arthropods that prey on or parasitise herbivorous insects or mites, has been well documented as plant defence strategy (degenhardt et al., 2003). finally, antixenosis and antibiosis are often associated in resistant genotypes, but are supposed to be indipendent from a genetic point of view, since only one of this two mechanisms of resistance exist in some genotypes (puterka et al., 1993). 9. breeding fortunately, pear species vary considerably in their resistance to pear psylla and breeding for resistance is possible. for breeding, the use of the larger fruited species (p. ussuriensis and p. x bretschneideri) should prove to be more efficient for combining resistance with europeantype fruit quality. in italy, the experimental institute for fruit crops, rome, forlì section (isf-fo) has been studying genetic improvement of pear for about 35 years, looking with particular regard for fire blight and pear psylla resistant cultivars. the breeding activity for the transfer of pear psylla resistance lists 22 crossing combinations, about 8,200 seedlings and 13 advanced selections, 3 of which are rather tolerant to pear psylla (baldassarri et al., 1996). praiseworthy is the selection isf.68-14-44-11, which is rather tolerant to pear psylla, although the fruit does not have sufficient eating quality (rivalta and dradi, 1998). transfer of resistance traits have been reported more recently in crosses with different ny selections (isf 94-1/174-267, isf 94-4/103-267, isf 94-5/-51-268) and selections of p. pyrifolia (isf 98-5-70-150, isf 90-12/110-149) (pasqualini et al., 2006). the department for tree crops, bologna university (dca-ubo), has been implementing a programme of both intervarietal and interspecific cross breeding, which began about 30 years ago, in order to develop diversified pears for quality and ripening calendar, without disregarding the evaluation of fire blight and psylla resistance (sansavini and rosati, 1986; sansavini, 1999). ny10353 and ny10355 have been used as male parents, while max red bartlett and doyenne de comice as female parents, and eight seedlings have been selected and are under evaluation. among these, dca 92052105-119 (ny10353 x doyenne de comice) has shown a great degree of psylla resistance in controlled growth chamber and is actually under evaluation in open field (musacchi et al., 2005; pasqualini et al., 2006). moreover, a number of 90 aflp primer combination has allowed to indentify, through a bulk segregant analysis, a first step of molecular markers linked to psylla (sansavini, pers. com.). 70 in france, a close collaboration between inra and the national institute of horticulture (inh), angers, has been recently started for the definition of precocious tests for evaluation of pear psylla resistance as well as potential parents to be used in the breeding project. now, some 10,000 resistant hybrids and 60 selections are under study using artificial inoculation tests (le lezéc, 1991; le lezéc, pers. com.). of great importance is also the breeding programme which is undergoing at the fruit research stations of pitesti-maracineni, cluj-napoca and voinesti, romania, whose goals have been focused since many years on resistance improvement to fire blight, pear psylla and scab by means of intra and interspecific hybridization followed by backcross. the initial sources concerning psylla resistance were represented by biotypes derived from p. serotina and subsequent f 1 and f 2 interspecific selections. some foreign and native p. communis cultivars were used as parents (napoca, butirra precoce morettini, butirra hardy, butirra six, doyenné d’hiver, etc.) and the psylla resistant or tolerant cultivars haydeea, euras, getica and ina estival have been promoted and named (andreis, pers. com.; braniste, pers. com.; sestras et al., 2009). in north america the breeding programmes initiated in the 1920’s and 1930’s developed in the 1960’s into two impressive programmes for disease and insect resistance at harrow in canada and at kearneysville (usda) in the united states, based on hybridisation with cultivars and selections from p. ussuriensis and p. pyrifolia (characterised by a higher resistance, probably of monogenic type), with fruit characteristics of p. communis being recovered by backcrossing to selected p. communis cultivars (bellini and nin, 1997). resistance to pear psylla represent an additional breeding objective of the pear programme at harrow (brunner, 1997; hunter, 1994; hunter, pers. com.), while has been added as specific primary objective of the united states department of agriculture (usda) breeding programme. selection methods have been developed from detailed studies of the modes of resistance (a 24-hour nimphal feeding bioassay plus choice and non choice oviposition assays for further resistance characterization), almost 4,400 seedlings have been evaluated and rapd markers associated with resistance to nymphal feeding antixenosis are in progress. among the most recent cultivars coming from united states are elliot, gourmet, potomac and blacke’s pride (bell and van der zwet, 1992; bell et al., 1996). often, parallel studies are carried on in order to support and speed up the attainment of the pursued goals. generally, the future direction of such programs will include a biotechnology component, with the objective of identifying and transferring genes for resistance to fire blight and pear psylla (bellini and nin, 1997; bellini et al., 2000). acknowledgements supported by progetto ager, grant n° 2010-2107. references armand e., lyoussoufi a., rieux r., 1991 évolution du complexe parasitaire des psylles du poirier psylla pyri et psylla pyrisuga [homoptera: psyllidae] en vergers dans le sud-est de la france au cours de la période hivernale, printanière et estivale. entomophaga, 36(2): 287-294. baldassarri n., baronio p., rocchetta g., salvaterra g., 1996 indagine sullo sviluppo di cacopsylla pyri (l.) 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(2014) found that in citrus fruit the application of edible natural biodegradable coatings enhanced fruit gloss, slightly increased fruit firmness, but was mostly not effective in preventing water loss and decreased the flavor acceptability in mandarins due to off-flavor development. plums treated with a coating material based on carbohydrate plus sorbiinfluence of edible coatings on postharvest physiology and quality of honeydew melon fruit (cucumis melo l. inodorus) m. vanoli(*), m. grassi, m. buccheri, a. rizzolo consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, unità di ricerca per i processi dell’industria agroalimentare (cra-iaa), via g. venezian 26, 20133 milano, italy. key words: electronic nose, ethane, ethylene, fermentative metabolites, firmness, internal oxygen, total soluble solids. abstract: several techniques have been developed to preserve the quality of horticultural products throughout the supply chain. edible coatings represent a promising technology as they can improve quality and extend shelf life of fruit and vegetables by changing gases and moisture permeabilties, enhancing fruit appearance, and reducing microbial contamination. the aim of this work was to assess the effectiveness of two kinds of novel coatings on the shelf life extension of honeydew winter melons during retail. sixty melons were used: 24 were uncoated as control; 18 were treated with a cellulose polymer coating (f1) and 18 with a synthetic polymer (f2) coating. upon arrival, and after 6, 9 and 13 days at 13°c, six melons/ treatment were individually analyzed for internal o2, ethylene and ethane concentrations, fermentative metabolites, quality parameters, and aroma pattern. already after six days, internal o2 levels in coated fruit fell to ~1% in f1 and ~3% in f2 melons, triggering fermentative pathways as shown by the increased productions, mainly in f1 fruit, of acetaldehyde, ethanol, ethyl acetate, and ethane. this pattern caused changes in the responses of electronic nose sensors which were able to distinguish the three treatments. coating did not influence fruit firmness and internal ethylene concentration. f1 coating reduced soluble solids content, strongly enhanced skin glossiness, and delayed yellowing, but it was not able to prevent moisture losses. in contrast, f2 coating significantly reduced weight loss and showed a slight positive effect on fruit appearance. (*) corresponding author: maristella.vanoli@entecra.it maristella.vanoli@gmail.com received for publication 26 september 2014 accepted for publication 9 march 2015 adv. hort. sci., 2015 29(2-3): 65-74 66 adv. hort. sci., 2015 29(2-3): 65-74 tol showed an extended shelf life period due to weight loss decrease and delayed changes in firmness, color, ph and acidity (eum et al., 2009). strawberries coated with edible coatings showed significant delays in the changes of weight loss, decay incidence, acidity, ph and soluble solids, and ascorbic acid contents and maintained higher concentration of total phenolics and anthocyanins in comparison to control fruit (gol et al., 2013). positive effects of coating were also observed in mangoes treated with a nanomultilayer coating of pectin and chitosan: coated fruit presented a better external appearance, a less dehydrated surface apparently without fungal growth and lower mass loss after 45 days of storage (medeiros et al., 2012). carboxymethyl cellulose (cmc) coatings alone and in combination with gamma irradiation were tested for maintaining the storage quality and extending shelf life of pears. cmc alone was effective in extending shelf life of pears by six days, following 45 days of refrigeration, while the combinatory treatment maintained the storage quality and delayed pear decay, prolonging the shelf life period up to 12 days (hussain et al., 2013). fisk et al. (2008) found that coatings improved the surface appearance of kiwifruit without impairing ripening, and fruits were well liked by consumers even if no benefit was observed on weight loss. as for melons, the majority of investigations on edible coatings have concerned their applications on fresh-cut rather than on the whole fruit (oms-onliu et al., 2008 a, b; amaro et al., 2012). fallik et al. (2005) evaluated the external, internal and sensory traits of ‘galia’-type melon fruit coated with three polyethylene-based waxes. the best results were obtained by using waxes that contain no, or very low amounts, of shellac as these waxes reduced water loss, improved the general appearance, and maintained pleasant, sweet and fruity aroma notes of the fruit even after prolonged storage, while control fruit suffered from high-decay incidence and soft texture. when shellac was present in high amounts, off-flavor significantly increased in the melon fruit due to high internal levels of co 2 , ethanol, acetaldehyde and ethyl acetate. cong et al. (2007) found that the bilayer coating of chitosan and polyethylene wax microemulsion containing natamycin extended the shelf life of ‘hami’ melon by reducing weight loss, fruit decay, and the decrease of ascorbic acid content during storage at ambient temperature even if some doubts remained regarding the sensory quality of coated fruit. the aim of the present work was to assess the effectiveness of different kinds of novel coatings (under patent) on shelf life extension of winter melon during retail by studying internal gas concentrations coupled to off-flavor development and fruit quality. 2. materials and methods the experiment was carried out in 2014 on winter melons (cucumis melo l. inodorus type honeydew cultivar natal) assigned for a large retail chain. fruits were harvested in a commercial orchard in the rio grande do norte region on 21 january 2014 and arrived in milan on 11 february, when the trial began. natal melons are characterized by a yellow skin and white flesh. sixty melons were selected: 24 uncoated fruits were used as control (t); 36 fruits were treated with two coatings (f1 and f2) currently under patent. f1 coating is a complex based on a cellulose polymer, it is water soluble, with a concentration of 25±2°bx. f2 coating is a synthetic polymer, also water soluble, with a concentration of 15±1°bx; f2 was used after dilution 1:2 (w/w) with tap water. fruits were dipped in the coating solutions for 30 s, dried for 24 h at room temperature and then put at 13°c together with uncoated melons. upon arrival in the laboratory of cra-iaa in milan (d0), and after 6 (d6), 9 (d9) and 13 (d13) days at 13°c, six melons/treatment were individually analyzed for internal o 2 , ethylene and ethane concentrations, fermentative metabolites, skin and pulp color, flesh firmness, soluble solid content (ssc), weight loss and aroma pattern by a commercial electronic nose (e-nose). internal oxygen internal oxygen was measured in the seed cavity by using a fluorescence-based optical sensor system (neofox fosfor-r, ocean optics). this fiber optic oxygen sensor uses the fluorescence of a chemical complex in a sol-gel to measure the partial pressure of oxygen. the pulsed blue led sends light, at ~475 nm, to an optical fiber. the optical fiber carries the light to the probe. the distal end of the probe tip consists of a thin layer of a hydrophobic solgel material. a sensor formulation is trapped in the sol-gel matrix, effectively immobilized and protected from water. the light from the led excites the formulation complex at the probe tip. the excited complex fluoresces, emitting energy at ~600 nm. if the excited complex encounters an oxygen molecule, the excess energy is transferred to the oxygen molecule in a non-radiative transfer, decreasing or quenching the fluorescence signal. the degree of quenching correlates to the level of oxygen concentration or to partial pressure of oxygen on the film, which is in dynamic equilibrium with the oxygen in the sample. the energy is collected by the probe and carried through the optical fiber to the spectrometer and the data are then displayed in the ooisensors software. internal ethylene and ethane internal ethylene and ethane concentrations were measured by withdrawing 1 ml samples of internal gas from the seed cavity of each melon using a syringe equipped with 15 cm long, 15-gauge needle. the sample was injected in a dani gs 86.10 gas chromatograph equipped with a deactivated aluminum oxide f1 (80-100 mesh) column (1/8 in. ×200 cm) and a flame ionization detector according to rizzolo et al. (2005). quantitative data were obtained by relating the peak of each hydrocarbon to that of its external standard and were expressed as ppm. 67 vanoli et al., edible coatings and quality of melon fruit fermentative metabolites fermentative metabolites (ethanol, acetaldehyde and ethyl acetate) were determined on the fruit pulp by means of static hs-gc. for each fruit, 10 g of homogenized pulp (two replications) were put into 25 ml vials tightly closed with an aluminum cap with a silicone-teflon rubber septum; then samples were immediately frozen and kept at -20°c until analysis. after a 60 min thawing at room temperature and e-nose analysis, each vial was heated at 80°c for 30 min, and 0.5 ml of the headspace gas was sampled and injected using the automatic headspace sampler hss 86.50 dani fitted to a gas chromatograph dani 8521, equipped with a ptv injector port operating in splitless mode, a fid detector, and a db-1 column (60 m × 0.53 μm i.d., 1 μm film thickness). the following gc conditions were used: helium carrier gas flow rate, 1.6 ml min−1; hydrogen flow rate, 66 ml min−1; air flow rate, 146 ml min−1; oven temperature program, 10 min at 50°c, 4°c min−1 to 100°c, injector port and detector temperatures, 200 and 250°c, respectively. fermentative metabolites were quantified by relating the peak area of each one to that of external standards and were expressed as mg kg−1. skin and pulp color skin and pulp color were measured on two opposite sides in the equatorial region of the fruit with a spectrophotometer cm-2600d (minolta co, japan), using the primary illuminant d65 and 10° observer in the l*, a*, b* color space. the gloss index was also calculated by the spectramagic acquisition program by using the sci and sce numerical gloss control. color readings were averaged for each fruit. flesh firmness flesh firmness was measured on the fruit flesh after having cut the melon into two parts along the longitudinal axis; measurements were carried out on one part in six opposite areas at the top, mid, and bottom positions of each fruit using an 8 mm diameter plunger mounted on an instron universal testing machine (model 4301, instron ltd, great britain) with the crosshead speed at 200 mm min–1. the six measurements were averaged for each fruit. soluble solids content soluble solids content (ssc) was determined on the juice that came out during plunging at the same positions of firmness measurements; ssc was measured using an automatic refractometer (rfm81, bellingham-stanley ltd., england) and the six readings were averaged for each fruit. aroma pattern aroma pattern was determined by a commercial e-nose on the same samples used for the fermentative metabolite analysis, soon after the 60 min thawing at room temperature. a pen3 portable electronic nose (win muster airsense analytics inc., germany) was used (rizzolo et al., 2013). the pen3 e-nose consists of a sampling section, a detector unit containing the array of sensors, and a pattern recognition software (win muster v. 3.0) for data recording and elaboration. the sensor array is composed of ten metal oxide semiconductor (mos) type chemical sensors: w1c (aromatic), w5s (broad range), w3c (aromatic), w6s (hydrogen), w5c (aromatic aliphatics), w1s (broad), w1w (sulfur organic), w2s (broad alcohol), w2w (sulfur chlorinate), and w3s (methane aliphatics). the sensor response is given by the ratio of the conductivity response of the sensors to the sample gas (g) relative to the carrier gas (g 0 ) over time (g/g 0 ). the headspace gas was pumped over the sensor surfaces for 60 s (injection time) at a flow rate of 45 ml min−1, and during this time the sensor signals were recorded. after sample analysis, the system was purged for 120 s with filtered air prior to the next sample injection to allow reestablishment of the instrument baseline. each sample was evaluated three times. for each e-nose run, the conductivity g/g 0 of the 10 sensors at the time corresponding to the normalized maximum of all signals was taken as the vector of sensors signal. the average of the runs of each replicate was used for statistical analysis. statistical analysis data were submitted to analysis of variance (statgraphics ver.7, manugistic inc., rockville, md, usa) considering coating and day at 13°c as a sources of variation, and means were compared by tukey’s test at p≤0.05%. e-nose data were also submitted to principal component analysis. the principal component (pc) scores were then subjected to anova and means were compared by tukey’s test at p≤0.05% considering as factors coating and day at 13°c. correlations between pcs and internal o 2 , ethylene and ethane concentrations and fermentative metabolites were also analyzed. 3. results internal oxygen, ethylene, and ethane upon arrival in the laboratory, melons showed an internal o 2 amount of about 16.3%. already after 6 days at 13°c o 2 levels fell to 1.3% in f1-coated melons and to 3.2% in f2 ones (fig. 1) and remained at about these percentages up to 13 days. in contrast, in control fruit, internal o 2 levels decreased to about 14% at d6, slightly decreased at d9, falling to about 5% at d13. f2 melons showed higher variability in internal o 2 amounts in comparison to f1 ones, as in f2 fruit the internal o 2 ranged from 1 to 6.8%, and in f1 fruit from 0.8 to 1.8%. ethylene amount was 0.14±0.07 ppm at d0, increased at d6 to about 0.55 ppm and 0.78 ppm in control and f1 melons, respectively, then it decreased up to d9, to a greater extent in control fruit than in f1 ones, and remained constant up to the end of the storage period (fig. 1). in f2 melons, ethylene was constant up to d6 and increased at d9, remaining at this level up to d13 (fig. 1). f2 fruit showed the highest ethylene amounts from d9 to d13, while control fruit developed the least ethylene quantity in the same period. 68 adv. hort. sci., 2015 29(2-3): 65-74 ethane was absent at d0; in control fruit it was produced only at d13 in very low amounts (about 8 ppm), while in f1-coated melons ethane levels steeply increased to about 60 ppm already after 6 days at 13°c, then decreased in the subsequent three days maintaining a high amount (30 ppm) up to d13 (fig. 1). f2-coated melons showed a slight increased in ethane production, reaching about 13 ppm at d13 (fig. 1). fermentative metabolites at d0, fruit had acetaldehyde=12.3±0.6 mg kg-1, ethanol=24.2±3.2 mg kg-1 and ethyl acetate= 1.7±0.5 mg kg-1. fig. 1 internal oxygen, ethylene, and ethane concentrations (left) and fermentative metabolites (acetaldehyde, ethanol, ethyl acetate right) amounts of control and coated (f1, f2) melon fruit during storage at 13°c. bars refer to standard error of the mean (n=6). 69 vanoli et al., edible coatings and quality of melon fruit all three fermentative metabolites dramatically increased in f1 fruit already after 6 days at 13°c (fig. 1), increased further up to d9, showing a slight but not significant increase up to d13, with the exception of acetaldehyde which did not change from d9 to d13. also in f2 fruit, fermentative metabolites significantly increased from d0 to d6 but to a lesser extent than in f1 melons; then they slightly increased up to d9, maintaining this amount up to 13 days. in contrast, in control melons, fermentative metabolites did not show any changes during storage at 13°c. skin and pulp color the application of the coatings significantly affected b* and gloss index in the fruit skin and a* and b* of the pulp (table 1). skin b* was higher in control fruit than in coated fruit, and no difference was found between the two kinds of coating. gloss index showed the highest values in f1 melons, intermediate in f2 ones and the lowest in control fruit. as for pulp color, a* was highest in control fruit and lowest in f2 melons, with f1 fruit showing intermediate values, whereas b* had the lowest values in control fruit at d6, when f1 fruit had the highest values; no difference was found at d9 among the treatments, while at d13 control fruit still had lower b* than both types of coated fruit. no changes were observed in skin and pulp color parameters during storage at 13°c, except for skin l* and gloss index and for pulp b* which significantly decreased during storage at 13°c. firmness, soluble solids and weight loss firmness did not change with coating treatments and days at 13°c, while ssc, on average, were lower in f1 fruit and did not change with days at 13°c (fig. 2). weight loss showed the lowest values in f2 fruit (fig. 3). a different trend in weight loss increase during storage at 13°c was observed according to the treatment; a sharp increase was observed in f1 melon from d6 to d13, while in f2 fruit weight loss increased from d6 to d9 and then remained constant up to d13; in control melons weight loss dramatically increased up to d6, then it continued to increase but to a lesser extent up to d9 and then steeply increased up to d13. aroma pattern the behavior of the signals generated by the sensor array is reported in figure 4. each line represents the average signal variation of replicated samples for one sensor of the array, linking the conductance increase or decrease experienced by the sensors to the evolution of the coating type during the storage time. the responses of the 10 mos sensors significantly changed with coating presence, with the exception of w1w, w2w and w3s sensors. the sensors w1c, w3c and w5c showed the lowest responses in f1 fruit and the highest in control melons; the opposite behavior was observed for w5s, w6s, w1s and w2s sensors. no sensors changed in control fruit with storage days, while the responses of w1c, w3c and w5c sensors decreased with storage time and those of w5s, w1s and table 1 skin and pulp color (mean ± standard error; n=6) of control and coated (f1, f2) melon fruit during storage at 13°c and anova results storage skin color pulp color l* a* b* gloss index l* a* b* day 0 75.1±1.0 3.8±0.8 75.1±0.9 3.7±1.0 73.1±1.1 -3.0±0.3 15.3±0.6 day 6 control 74.5±1.1 5.7±1.4 76.6±1.1 5.0±0.5 74.7±0.9 -1.9±0.4 12.7±0.4 f1 74.6±0.8 3.7±1.3 72.8±1.2 21.5±4.5 76.4±0.6 -2.2±0.2 14.0±0.3 f2 74.0±0.8 4.7±0.9 73.2±1.3 12.8±2.2 72.9±0.4 -3.3±0.2 15.5±0.2 day 9 control 75.7±0.5 4.2±1.2 77.3±0.8 1.7±0.6 73.7±0.8 -1.9±0.8 14.6±1.1 f1 73.6±0.2 7.1±0.7 73.7±0.4 20.5±4.5 74.1±0.8 -2.2±0.2 13.8±0.1 f2 73.5±0.3 6.6±0.5 73.9±0.5 14.7±2.6 75.1±0.7 -2.4±0.3 13.2±0.4 day 13 control 73.9±0.8 5.8±0.6 75.4±1.2 0.0±0.0 73.5±1.2 -1.6±0.2 12.0±0.5 f1 72.3±0.5 6.9±0.9 71.7±0.6 10.5±3.3 74.7±0.3 -2.1±0.1 13.4±0.3 f2 72.9±0.7 6.8±0.8 73.3±0.4 5.8±1.1 72.5±1.2 -2.5±0.2 13.6±0.8 anova coating (a) ns ns *** *** ns ** ns days at 13°c (b) * ns ns ** ns ns * a x b ns ns ns ns ns ns ** ***, p<0.001; **, p<0.01; *, p<0.05. ns= not significant. 70 adv. hort. sci., 2015 29(2-3): 65-74 w2s decreased in coated fruit. in f2 melons, the e-nose sensor responses were always intermediate between control and f1 melons. to see whether the sensor array was able to distinguish the different kinds of coating, pca was applied to the e-nose measurements. two functions were extracted, explaining about 89% of the variability (fig. 5). pc1 grouped w1s, w2s and w5s sensors opposite to w1c, w3c and w5 ones, while in pc2 w1w and w2w sensors were opposed to w3s ones. pc1 scores showed the lowest values for control melons, intermediate for f1 ones and highest for f2 (fig. 6). the pc1 score was also lower at d0 in comparison to the other days at 13°c. pc2 scores didn’t change, neither with coating nor with storage time (fig. 6). to compare the e-nose patterns with composition data, the correlations between pc1 and pc2 scores with internal o 2 , ethylene, ethane, and fermentative metabolites were studied (table 2). high and positive correlations were found between pc1 fermentative metabolites and ethane while a negative correlation was observed between pc1 and internal o 2 levels. no correlation was found for pc1 with ethylene amount and for pc2 with all compounds. 4. discussion and conclusions edible coatings have been used in order to retain quality and to extend shelf life of fresh fruits and vegetables. most fruits and vegetables possess a natural waxy layer on the surface (cuticle) which generally has a low permeability to water vapor. applying an external coating could enhance this natural barrier as a semi-permeable membrane is formed on the fruit surface and thus it will be possible to obtain a better control of gas diffusion and moisture loss, delaying ripening and senescence. fig. 2 firmness (left) and soluble solids content (ssc, right) of control and coated (f1, f2) melon fruit during storage at 13°c. bars refer to standard error of the mean (n=6). table 2 linear correlation coefficients (r) of pc1 and pc2 scores, obtained from principal component analysis, with oxygen level, ethylene, and ethane amounts and fermentative metabolites correlation coefficient pc1 pc2 oxygen 0.668*** 0.040 ethylene 0.211 0.090 ethane 0.702*** 0.034 acetaldehyde 0.844*** 0.034 ethanol 0.836*** 0.059 ethyl acetate 0.882*** 0.101 significance of r: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001) fig. 3 weight loss of control and coated (f1, f2) melon fruit during storage at 13 °c. bars refer to standard error of the mean (n=6). 71 vanoli et al., edible coatings and quality of melon fruit the quality of fruits and vegetables depends on their internal o 2 and co 2 concentrations which in turn are affected by the environmental concentrations of these gases (hagenmaier, 2005). a reduced o 2 level is desirable for slowing down respiration and preventing exchange of food aroma and flavor compounds with the environment. the coatings applied to fruit form barriers to the diffusion of o 2 and co 2 through the fruit peel. when the supply of o 2 needed for respiration or the release of co 2 is blocked, fruit and vegetables quickly become inedible and rotten. such blockage lowers and raises the interior o 2 and co 2 concentrations, respectively, causing off-flavor development due to anaerobic fermentation. thus, the selection of appropriate coating materials and formulations with a proper gas permeability represents a crucial point to obtain successful results. our results showed that already after 6 days at 13 °c, internal o 2 levels in coated fruit fell to about 1% in f1 melons and to 3% in f2 ones. this o 2 drop led to a remarkable increase in ethane, acetaldehyde, ethanol, and ethyl acetate amounts, mainly in f1 fruit. this means that f1 fig. 4 relative conductivity (g/g 0 ) of each sensor in control and coated (f1, f2) melon fruit during storage at 13°c. bars refer to standard error of the mean (n=6). fig. 5 pca of e-nose data: loadings and scores of pc1 versus pc2 according to coating treatment (control; coated=f1, f2) and day at 13°c (d0, d6, d9, d13 refers to arrival, after 6, 9 and 13 days at 13°c, respectively). fig. 6 pca scores of pc1 and pc2 according to coating treatment (control; coated=f1, f2) and days at 13°c (d0, d6, d9, d13 refers to arrival, after 6, 9 and 13 days at 13°c, respectively). bars refer to standard error of the mean (n=18). 72 adv. hort. sci., 2015 29(2-3): 65-74 melons initiated anaerobic respiration, by which glucose is converted to pyruvate by glycolysis and then, pyruvate metabolized to acetaldehyde and acetaldehyde to ethanol. the recommended percentage of o 2 in a modified atmosphere for fruits and vegetables for both safety and quality falls between 1 and 5% (sandhya, 2010). it has been established that at a 2% o 2 level anaerobic respiration may result in the development of off-flavors and offodors. fruits exposed to such low o 2 levels may also lose their ability to attain uniform ripeness upon removal from the modified atmosphere packaging. the minimum o 2 concentration tolerated in controlled atmosphere storage of whole cantaloupe melons is 2% and impaired ripening, off-flavors and odors could develop when o 2 falls to 1% and co 2 goes up to 20% (kader et al., 1989). oms-oliu et al. (2008 a, b), studying low oxygen modified atmospheres on shelf life extension of fresh-cut melons, found that fermentative pathways were triggered under a 2.5 kpa o 2 + 7 kpa co 2 atmosphere. this atmosphere caused a rapid reduction in the o 2 levels below 1 kpa and an accumulation of co 2 so the initial respiratory quotient (rq-ratio of co 2 produced to o 2 consumed) of 1.2 increased above 1.3 after 10-14 days of storage. when rq is higher than 1, anaerobic respiration takes place and fermentative products are developed (fonseca et al., 2002). in fact, fresh-cut melons produced acetaldehyde and ethanol mainly inside package when o 2 concentrations drop below 2 kpa level (oms-oliu et al., 2008 a, b). whole melon fruit coated with waxes characterized by high amounts of shellac developed off-flavors when internal o 2 and co 2 levels reached about 3% and 20%, respectively. these fruits developed higher amounts of acetaldehyde, ethanol, and ethyl acetate than uncoated ones and were characterized by a ‘bad flavor’ as fruity-pleasant notes (due to butyl acetate and 2-methyl-propyl acetate) were very low, while ‘ethyl acetate note’ (which causes a solvent like smell) and the ‘ethanol-like’ note were high. in our work, when melons were cut for analysis, a fermentative smell was perceived for f1-coated fruit, as the amounts of acetaldehyde and ethanol were higher than their detection thresholds of 25 µg l−1 and 990.000 µg l−1, respectively (czerny et al., 2008). flores et al. (2004) considered inedible a melon fruit when ethanol was about 64 µmol kg−1, as found in fruit packed in modified atmospheres due to higher co 2 levels. in contrast, when oxygen levels remained at about 4% and co 2 levels at about 10%, fresh-cut cantaloupe cubes retained salable quality for 9 days at 5 °c and fruit showed better color retention, reduced translucency, respiration rate and microbial populations (bai et al., 2001). hagenmaier (2005) found a rather wide range of internal co 2 and o 2 values when individual coated oranges and apples were considered, in comparison with uncoated fruit showing a rather tight cluster of values. the internal gas values were particularly scattered for fruit with shellac and resin coatings, which caused the greatest reduction in peel permeance. this means that low-permeance coatings result in fruit with higher variation in product quality. on the contrary, in our work a large variation in internal o 2 levels was found for uncoated fruit followed by f2-coated fruit, while f1 melons which had the lowest o 2 levels also had the lowest variability in o 2 levels. f1 melons also exhibited a dramatic increase in ethane levels already after 6 days at 13°c when o 2 fell from 16% to 1%. similarly, rizzolo et al. (2008) found that ethane production was maximum in pears under 0.1 kpa o 2 and absent in fruit stored at o 2 ≥2 kpa. in our work a slight ethane production was also detected in f2-coated fruit where the o 2 level was about 3%. ethane production depends also on co 2 levels, as it was high in pears stored under 5 kpa co 2 whatever the po 2 (rizzolo et al., 2008). ethane is usually considered as a marker of lipid peroxidation in the cell membranes and it is released in pears affected by core browning while it was not detectable in healthy fruit (veltman et al., 1999; larrigaudière et al., 2001). the aroma pattern as revealed by e-nose reflected the different o 2 and ethane levels as well as the different amounts of fermentative metabolites found in melon fruit according to coating treatment. by using an e-nose it was possible to distinguish control fruit from f1and f2-coated ones. w1s, w2s, w5s and w6s sensors showed higher responses for coated melons in comparison with control fruit, which in turn had the highest responses for w1c, w3c and w5c sensors. w1s, w2s, w5s and w6s sensors were grouped in pc1 and were positively related to acetaldehyde, ethanol, ethyl acetate, and ethane and negatively related to o 2 levels. pc1 had the highest scores in f1 fruit which were characterized by the lowest o 2 levels and by the highest production of ethane and fermentative metabolites and showed the lowest values in control fruit at day 0 when no fermentative metabolism occurred. the same relationships between mos sensors and fermentative metabolites were found by riva et al. (2005) in strawberries as a consequence of the formation of a peripheral layer of sugar with reduction of tissue porosity occurring during the osmodehydration process. differently from that found for cold-stored peaches for which a high and significant correlation was found between ethylene production and e-nose pattern (rizzolo et al., 2013), no correlation was found between e-nose pattern and internal ethylene. ethylene production was very low as expected for a honeydew-type melon and slight changes were observed in relation to coating treatments and to days at 13°c in agreement with barreiro et al. (2001). considering quality parameters, coating treatments had no influence on fruit firmness, while reduced ssc content was found when f1 coating was applied on fruit, and this decrease may be due to the fermentative pathway of accumulations of acetaldehyde and ethanol catalyzed by the enzymes pyruvate decarboxylase and alcohol dehydrogenase, respectively (kader, 1995). coating also affected skin and pulp color of melon fruit as f1 coating enhanced skin glossiness and delayed skin yellowing in compari73 vanoli et al., edible coatings and quality of melon fruit son both to control and f2-coated fruit, while f2 melons showed a white-slightly greener pulp than control fruit. in our experiment, weight loss was quite low, reaching the maximum values of about 1.5 % in f1-coated fruit and was lower in f2 fruit than in control and f1 ones. the inability of f1 coating to control weight loss could be due to the fact that this type of coating is based on a cellulose polymer and it is well known that the hydrophilicity of this material does not provide a sufficient moisture barrier (lin and zhao, 2007; falguera et al., 2011; dhall, 2013). application of coating to whole winter melons caused the onset of fermentation processes especially in fruit coated with the cellulose-based polymer (f1) where the o 2 level dropped to 1% which is the threshold value causing anaerobic metabolism and development of off-flavors and off-odors. probably this kind of coating strongly decreased permeance of the skin to gas exchanges while it had no effect on water loss. the coating (f2) based on a water-soluble synthetic polymer showed a higher permeance to o 2 exchanges as o 2 levels were maintained at about 3% and therefore the development of fermentative metabolites was limited; this coating was also able to prevent fruit weight loss but no other positive effect was observed on fruit quality as firmness and soluble solids content were similar to those of uncoated melons. however, the cellulose based polymer coating improved fruit appearance, strongly enhancing fruit gloss and delaying skin yellowing, but these positive effects are of secondary importance in comparison to the anaerobic metabolism induced by this kind of coating. acknowledgements research carried out within the project “utilizzo di prodotti innovativi derivanti da polimeri organici di interesse per il settore agro-industriale (pro-inn)” of the italian ministry of agriculture. the work reported in this paper was presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 22-23 may 2014. references amaro a.l., beaulieu j.c., grimm c.c., stein r.e., almeida d.p.f, 2012 effect of oxygen on aroma volatiles and quality of fresh-cut cantaloupe and honeydew melons. food chem., 130: 49-57. arnon h., zaitsev y., porat r., poverenov e., 2014 effects of carboxymethyl cellulose and chitosan bilayer edible coating on postharvest quality of citrus fruit. postharvest 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valencia-chamorro s., perez-gago m., del rio m., palou l., 2010 effect of antifungal hydroxypropyl methylcellulose-lipid edible composite coatingson penicillium decay development and postharvest quality of coldstored ‘ortanique’ mandarins. j. food sci., 75: s418-s426. veltman r.h., sanders m.g., persijn s.t., peppelenbos h.w., oosterhavenj., 1999 decreased ascorbic acid levels and brown core development in pears (pyrus communis l. cv. conference). physiologia plantarum, 107: 39-45. 65 adv. hort. sci., 2011 25(1): 65-68 received for publication 20 october 2010. accepted for publication 18 february 2011. short note performance of tomato under greenhouse and open field conditions in the trans-himalayan region of india m.s. kanwar sher-e-kashmir university of agricultural sciences and technology of kashmir, regional agricultural research station, leh 194 101, ladakh, jammu and kashmir, india. key words: greenhouse, ladakh, open field, solanum lycopersicon, tomato. abstract: production of tomato is limited by harsh climate and a short growing season in the trans-himalayan ladakh region of india. the performance of five tomato genotypes was compared under polyhouse and open field conditions. the study revealed that the performance of all tested tomato genotypes is far superior in the polyhouse, as compared to open field conditions, for all the considered characters. ‘shivalik’ performed best with respect to yield characters followed by ‘pusa rohini’ under polyhouse conditions. however, in the open field, ‘pusa rohini’ showed the highest values, followed by ‘shivalik’. cultivation of tomato under the polyhouse produced 136.12% more yield per ha and 188.93% more fruits per plant compared to open field cultivation. therefore, tomato cultivation under protected conditions is advised for ladakh growing conditions, employing specific polyhouse-responsive varieties. 1. introduction tomato (solanum lycopersicon l.) is available throughout the year in india. however, in the state of jammu and kashmir, with the exception of the jammu region, it is mostly confined to the summer season. in the trans-himalayan ladakh region, production of tomato is limited by climate and a short growing season. ladakh has a harsh climate and extreme temperature fluctuations ranging from -37°c to +38°c. in ladakh, tomato can be grown in open conditions but yield remains poor with low quality and it remains weather-dependent. therefore, protected cultivation is a feasible answer for successful cultivation of tomato in this region. singh and asrey (2005) also recommended that cultivation of tomato in a greenhouse would help obtain high productivity and better return. therefore, it is useful to study tomato production potential in the ladakh region with respect to yield and horticultural traits under protected conditions (preferably in a zero-energy polyhouse) in comparison to the open field. 2. materials and methods the experiment was conducted under naturally ventilated polyhouse and open field conditions at the experimental farm, stakna (leh) of the regional agricultural research station (skuast-k) located at 3319 m amsl with latitude 33°58.551’ ns and longitude 77°41.995’ew. the climate of the area is typically dry temperate. five genotypes including four hybrids (ph5, shivalik, jaya and naveen 2000+) and one op variety (pusa rohini) were transplanted in a naturally ventilated polyhouse and the open field. planting distance was 60 x 30 cm. the design of the experiment was factorial rbd and material was replicated thrice. individual data of each location were also subjected to statistical analysis in rbd to have more authentic information with regard to tomato genotypes. data recorded on 13 characters were subjected to statistical analysis as per snedecor and cochran (1967). 3. results and discussion there were significant differences among tomatoes grown under polyhouse and open field condition for all the characters, except for locules per fruit, confirming thereby the certain role of polyhouse in the cultivation of tomato in the trans-himalayan region. similar 66 pu sa ro hin i ph -5 sh iva lik jay a na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) pla nt he igh t( cm ) va rie tie s 91 .12 98 .12 12 5.7 0 12 2.9 0 16 9.2 0 12 1.4 0 10 .43 29 .95 ns 77 .50 56 .25 96 .25 95 .50 11 5.0 0 88 .10 12 .63 84 .31 77 .19 11 1.0 10 9.2 14 2.1 ns 10 .25 9.5 0 10 .00 10 .00 10 .00 9.9 5 1.1 4 ns ns 6.2 5 5.5 0 6.0 0 5.7 5 6.5 0 6.0 0 ns 8.2 5 7.5 0 8.0 0 7.8 8 8.2 5 ns 91 .5 89 .5 89 .5 91 .00 88 .50 95 .65 3.3 0 ns ns 95 .25 96 .25 93 .75 99 .00 94 .00 90 .00 ns 93 .37 92 .87 91 .62 95 .00 91 .25 ns 10 .25 9.5 0 10 .00 10 .00 10 .00 9.9 5 0.5 1 ns ns 6.2 5 5.5 0 6.0 0 5.7 5 6.5 0 6.0 0 0.6 7 8.2 5 7.5 0 8.0 0 7.8 8 8.2 5 ns 68 .50 67 .25 68 .75 71 .50 74 .50 70 .05 4.0 7 ns ns 39 .00 35 .50 38 .25 36 .75 39 .50 37 .80 ns 53 .75 51 .37 53 .50 54 .12 56 .88 ns l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n ste m gir th (m m) da ys to fir st ha rve st no .o fh arv est ing ha rve st du rat ion (da ys ) ch ara cte rs lo ca tio n ta ble 1pe rfo rm an ce of tom ato ge no typ es un de rp oly ho us ea nd op en co nd itio ns for pla nt ch ara cte rs l1 =p oly ho us e. l2 =o pe nf iel d. results were obtained by singh and asrey (2005) as they found excellent tomato crops in polyhouses compared to the outside environment. significant differences were observed among tomato genotypes pooled over locations for number of fruits per plant, yield per plant, yield per ha, fruit length, tss and locules per fruit. however, no polyhouse x genotype interaction was noted. comparative performance of tomato genotypes is presented here. plant characters in polyhouse and open conditions, ‘naveen 2000+’ had significantly higher plant length. results are in conformity with those of singh and singh (2000) under open field conditions. ‘naveen 2000+’ also had a significantly greater number of harvests, followed by ‘pusa rohini’ in open field conditions. under the polyhouse, the number of harvests ranged from 9.50 to 10.25. cargnelutti et al. (2004) obtained 11-14 harvests under plastic greenhouse. the polyhouse had a significant effect on all the plant characters under study (table 1). ganesan (2002) also reported better plant height in polyhouses as compared to open field conditions. yield characters yield per plant, yield per ha and number of fruits per plant were highest in ‘shivalik’ followed by ‘pusa rohini’ under greenhouse conditions (table 2). gualberto et al. (2007) also recorded significant differences for yield per ha. however, in open field conditions, ‘pusa rohini’ gave the highest value for all three characters, followed by ‘shivalik’. however, the differences were non-significant for yield per ha. the performance of ‘pusa rohini’ was the best, followed by ‘shivalik’, for all these characters when data was pooled from the different locations. the performance of tomato was statistically superior in polyhouse cultivation compared to open conditions. ganesan (2002) observed similar trends for yields per plant in polyhouses respect to open field conditions. fruit characters the highest recorded value for fruit length under polyhouse and open conditions, as well as for pooled data, came from ‘naveen 2000+’ however this genotype was at par with ‘jaya’ and ‘shivalik’ in the polyhouse (table 3). eklund et al. (2005) recorded fruit weight of 147.35 g, fruit length of 57.67 mm, fruit diameter of 69.75 mm and 5.25 locules per fruit for an elite hybrid in a controlled protected structure. all these values were higher than those found in the present study with the probable reason being fluctuating environment at fruit development as the present experiment was conducted under a naturally ventilated polyhouse. in open field and pooled data, ‘naveen 2000+’ had statistically superior fruit length. tss was statistically the highest in ‘pusa rohini’ under polyhouse conditions and in pooled data, while in open conditions ‘shivalik’ exhibited the highest tss, which was at par 67 pu sa ro hin i ph -5 sh iva lik jay a na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) no .o ff rui ts pe rp lan t yi eld pe rp lan t( kg ) yi eld pe rh a( q) l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n 25 .03 22 .64 26 .81 20 .72 22 .34 25 .31 1.6 1 3.9 5 13 .00 4.4 9 9.9 6 7.3 6 8.9 7 8.7 6 2.7 6 ns 19 .02 13 .56 18 .39 14 .04 15 .65 2.5 3 1.2 43 1.0 66 1.2 82 0.9 94 0.9 83 1.1 13 0.2 37 ns 0.6 57 0.2 13 0.5 37 0.3 54 0.4 62 0.4 44 0.0 87 0.1 48 0.9 5 0.6 4 0.9 1 0.6 7 0.7 2 0.1 37 69 0.4 2 59 2.3 6 71 2.2 2 55 1.2 5 54 5.8 3 61 8.4 0 13 1.4 3 ns 36 5.1 4 11 8.3 4 29 8.2 0 19 6.3 9 25 6.3 9 26 1.9 0 57 .34 ns ns 52 7.8 39 2.8 50 5.2 37 3.8 40 1.1 90 .65 ta ble 2pe rfo rm an ce of tom ato ge no typ es un de rp oly ho us ea nd op en co nd itio ns for yie ld ch ara cte rs l1 =p oly ho us e. l2 =o pe nf iel d. pu sa ro hin i ph -5 sh iva lik jay a na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) fr uit we igh t( g) va rie tie s 78 .00 78 .10 67 .40 71 .05 74 .70 73 .85 11 .49 ns ns 60 .06 54 .09 59 .23 57 .86 57 .22 57 .69 ns 69 .03 66 .09 63 .32 64 .45 65 .96 ns 48 .97 44 .75 49 .61 49 .59 53 .74 49 .33 2.3 5 4.5 2 ns 44 .51 40 .47 46 .49 47 .36 52 .82 46 .33 4.7 0 46 .74 42 .61 48 .05 48 .47 53 .28 3.7 2 52 .23 57 .18 48 .97 51 .89 49 .27 51 .91 3.4 0 ns ns 47 .63 48 .10 48 .43 48 .08 45 .45 47 .56 ns 49 .99 52 .64 48 .70 49 .98 47 .36 ns 6.7 5 5.1 0 5.4 0 5.9 5 5.0 5 5.6 5 0.1 2 0.2 6 ns 5.3 5 5.1 0 5.5 5 5.1 0 5.4 0 5.3 0 0.3 0 6.0 5 5.1 0 5.4 8 5.5 3 5.2 3 0.1 8 3.0 3.5 3.0 3.0 2.2 5 2.9 5 ns 0.5 6 ns 3.0 3.0 2.5 3.0 2.5 2.8 0 ns 3.0 0 3.2 5 2.7 5 3.0 0 2.3 8 0.3 7 l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n fr uit len gth (m m) fr uit dia me ter (m m) ts s( °b ) lo cu les /fr uit ch ara cte rs lo ca tio n ta ble 3pe rfo rm an ce of tom ato ge no typ es un de rp oly ho us ea nd op en co nd itio ns for va rio us fru itc ha rac ter s l1 =p oly ho us e. l2 =o pe nf iel d. ch ara cte rs va rie tie slo ca tio n with ‘naveen 2000+’ and ‘pusa rohini’. the statistically lowest number of locules per fruit in polyhouse conditions was recorded for ‘naveen 2000+’, while in pooled data ‘naveen 2000+’ and ‘shivalik’ were at par. performance improvement perusal of data in table 4 reveals that mean yield per ha, number of fruits per plant, fruit weight, plant height, harvest duration and number of harvests were 136.12, 188.93, 16.16, 37.80, 85.32 and 65.83% more, respectively, under polyhouse conditions compared to the open field. these findings demonstrate the suitability, as well as economic feasibility, of polyhouses in the pusa rohini ph-5 shivalik jaya naveen 2000+ mean percent increase in yield per ha number of fruits per plant 89.08 400.56 138.73 180.79 112.89 136.12 fruit weight plant height harvest duration no. of harvest 52.54 404.23 169.18 181.52 149.05 188.93 29.87 44.39 13.79 22.80 30.55 16.16 17.57 74.44 30.60 28.69 47.13 37.80 75.64 89.44 79.74 94.56 88.61 85.32 64.00 72.73 66.67 73.91 53.85 65.83 genotype table 4 percent improvement in tomato performance under polyhouse versus open conditions for economic characters trans-himalyan ladakh region for tomato cultivation. gualberto et al. (1998) also reported 40-45 % higher marketable yield in greenhouses than with open field conditions. growth and yield attributes were also recorded as poor in the open field condition. therefore, it may be concluded that naturally ventilated polyhouses are a good and less expensive option for tomato cultivation in the trans-himalayan region to obtain higher yield, number of fruits per plant and longer harvest duration. varieties like ‘shivalik’ and ‘pusa rohini’ are responsive to protected cultivation in this region and may be used for cultivation after further testing to increase the return per unit area. references cargnelutti filho a., radin b., matzenauer r., storck l., 2004 number of harvest and comparison of tomato genotypes cultivated under plastic greenhouse. pesquisa agropecuaria brasileira, 39(10): 953-959. eklund c.r.b., caetano l.c.s., shimoya a., ferreira j.m., gomes j.m.r., 2005 performance of tomato genotypes under protected cultivation. horticultura brasileira, 23(4): 1015-1017. ganesan m., 2002 effect of poly-greenhouse models on plant growth and yield of tomato (lycopersicon esculentum) indian journal of agricultural science, 72(10): 586-588. gualberto r., de oliveira p.s.r., de guimaraes a.m., 1998performance of fresh market tomato cultivars under protected cultivation. horticultura brasileira, 25(2): 244-246. gualberto r., resende f.v., de guimaraes a.m., ambrosio c.p., 2007 performance of long-life salad tomato cultivars grown in a protected environment and under field conditions. unimar ciencias, 7(2): 133-138. singh a.k., singh a., 2000 performance of tomato hybrids under sub-montane and low hills subtropical condition of h.p. crop. res., 20(3): 539-540. singh r., asrey r., 2005 performance of tomato and sweet pepper under unheated greenhouse. haryana j. hort. sci., 34(1-2): 174-175. snedecor g.w., cochran w.g., 1967 statistical methods. oxford and ibh publishing co. pvt. ltd., new delhi, india, pp. 593. 68 impaginato 249 adv. hort. sci., 2018 32(2): 249-264 doi: 10.13128/ahs-22261 evaluation of salinity tolerance in fourteen selected pistachio (pistacia vera l.) cultivars a. momenpour 1 (*), a. imani 2 1 national salinity research center, agricultural research, education and extension organization, areeo, yazd, iran. 2 temperate fruit research center, horticultural research institute, agricultural research education and extension organization, areeo, karaj, iran. key words: chlorophyll fluorescence, ghazvini cultivar, growth indices, pistacia vera l., salinity water. abstract: cultivars and rootstocks tolerant to salinity are determinant to increase the salt tolerance of planted fruit trees including pistachio. in this research, the effect of salinity stress on morphological and physiological traits as well as the concentration of nutrition elements in some pistachio cultivars was investigated based on completely randomized design (crd), with two factors cultivars and irrigation water salinity. studied cultivars were ghazvini, shahpasand, akbari, khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi, kaleh ghochi and badami zarand and levels of salinity were 0.5, 4.9, 9.8, 14.75 and 19.8 ds/m. each treatment had nine replicas. the results showed that increasing salinity reduced branch height, branch diameter, number of total leaves, and percentage of green leaves, relative humidity content, chlorophyll a, chlorophyll b and total chlorophylls in all cultivars. but percentage of necrotic leaves, percentage of downfall leaves, relative ionic percentage and cell membrane injury percentage were increased. the results showed that salinity stress affected the young trees through increasing the amount of minimum fluorescence (f0) and decreasing the maximum fluorescence (fm) and reducing variable fluorescence (fv) as well as the ratio of variable fluorescence to maximum fluorescence from 0.83±1 in the control plants to 0.59±0.015 in rezaie zood res cultivar and 0.61±0.009 in mousa abadi cultivar. the results also showed that in the total cultivars studied, the highest amount of na+ in leaves and roots (2.09±0.04% and 3.04±0.06%), and the lowest amount of k+ in leaves and roots (0.40±0.02% and 0.34±0.01%), were observed in treatment 19.75 ds/m. overall, ghazvini was found to be the most tolerant cultivar to salinity stress. this cultivar could well tolerate salinity 14.75 ds/m. 1. introduction pistachio (pistacia vera l.) is one of the important commercial crops in iran. majority of pistachio orchards are located in areas with saline soil and are irrigated with low quality and salty waters. although pistachio trees are classified as tolerant to salinity, researches have demonstrated (*) corresponding author: a.momenpour@areeo.ac.ir citation: momenpour a., imani a., 2018 evaluation of salinity tolerance in fourteen selected pistachio (pistacia vera l.) cultivars. adv. hort. sci., 32(2): 249-264 copyright: © 2018 momenpour a., imani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 249-264 250 that growth rates of pistachio trees decrease with increasing sodium chloride (nacl) concentration in soil and there is a positive correlation between sodium (na+) as well as chloride (cl−) concentration in plant tissue and soil (sepaskhah and maftoun, 1988; noitsakis et al., 1997; munns and tester, 2008; zrig et al., 2015). salinity stress also negatively affects photosynthesis rate, morphology of leaves, and nutrient balance in pistachio trees (picchioni and myamoto, 1990; saadatmand et al., 2007; karimi et al., 2011). walker et al. (1987) and karimi et al. (2009) reported that the highest chloride concentrations were observed in lamina and petiole of pistachio seedlings irrigated with salty water, whereas highest sodium concentration was observed in roots. ferguson et al. (2002) suggested that the decrease of water potential in plant in higher salinity levels is one of the main reason for decrease pistachio yield. it has been reported that salinity stress is one of the most important environmental factors limiting photosynthesis in the majority of worldwide cultivated crops, including pistachio crop (maxwell and johnson, 2000; ranjbarfordoei et al . , 2006). chlorophyll fluorescence (cf) has been used to study plant responses to different kinds of stress (baker and rosenqvist, 2004). chlorophyll (chl) fluorescence yield (chl fy) such as minimal chl fy (f0) and variable chl fy (fv) can be used for evidencing stress and damage of the photosynthetic apparatus, and characterizing the environment where plants grow (herda et al., 1999; deell and toivonen, 2003; kodad et al., 2010). fv/fm ratio has been used in many studies related to stress in plants. in most of plants, when ratio fv/fm is around 0.83 means that stress has not been introduced to the plant. values lower than this will be seen when the plant has been exposed to stress, indicating in particular the phenomenon of photo inhibition (herda et al., 1999; deell and toivonen, 2003; kodad et al., 2010). selecting nutrient sources that do not add harmful ions and salinity to irrigation water to avoid compounding salinity problems would be the best option. in areas affected by soil and water salinity, nevertheless, it is more convenient to use salt-tolerant rootstocks for the species characterized by a certain degree of salt tolerance, i.e. pistacia sp. an important characteristic of pistacia sp. is their ability to store large quantities of na+ in roots, which might make pistachio tolerant to na+ (picchioni and myamoto, 1990; karimi et al., 2011). sepaskhah and maftoun (1988) reported that 50% reduction in shoot growth was observed when the average root-zone salinity was between 7.9 and 10 ds/m (ece). saadatmand et al. (2007) postulated that salinity stress had more negative influence than drought stress on pistachio growth. they reported that sarakhs variety showed higher sensitivity to soil salinity than qazvini variety, but with increasing in irrigation intervals, sarakhs was more-tolerate to salinity than qazvini. although, other researchers have studied the influence of soil and water salinity on the growth indices and chemical composition of pistachio cultivars, but in before researches a low number of cultivars was investigated. therefore, in this research, the effects of five levels of irrigation water salinity on morphological and physiological traits as well as the concentration of nutrition elements in fourteen selected pistachio (pistacia vera l.) cultivars have been investigated in order to find most tolerant cultivars to salinity. 2. materials and methods plant material and natural salt treatments in this research, the effects of salinity stress on morphological and physiological traits and on the concentration of nutrition elements in 14 pistachio cultivars such as ghazvini, shahpasand, akbari, khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi, kaleh ghochi and badami zarand were investigated. the experiment was carried out in the research greenhouse of temperate fruit research center, horticultural research institute in karaj-iran in years of 2013 and 2014 based on completely randomized design (crd), with two factors; cultivars with 14 levels and irrigation water salinity by 5 levels (control= 0.5 ds/m, a= 4.8 ds/m, b= 9.8 ds/m, c= 14.75 ds/m and d=19.8 ds/m) and with nine replications for each treatment, for a total of 630 pots. seeds were germinated according to the method described by karimi et al. (2009). seeds were pretreated with benomyl (wettable powder-50%; dupont, wilmington, de, usa) for 24 h, and then incubated at 30°c within layers of sterile moist crisped cloth. after radicle emergence, seeds were planted in jiffy pots (jiffy group, moerdijk, netherlands) and grown in a greenhouse for three months. seedlings with 10 to 15 cm height were transplanted to pots 2 kg filled with soil series of fine loamy mixed, which its characteristics are listed in table 1. salinity treatment was started and continued for momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 251 two and half months. for salinity treatments, salts were collected of salt lake shore in qom-iran. then, salinity treatments were obtained by solving 0, 2.4, 4.8, 7.2 and 9.6 g of salt in 1 l of water (treatments composition is reported in table 2). also, to avoid sudden shock and plasmolysis, salt treatments were gradually added and reached to the final concentration within a week (2 stages of irrigations). field capacity (fc) of soil in pots was determined before transferring plants to units by a pressure plate (model f1, make usa). irrigation schedule was organized according to pots changes in weight and leaching requirement. electric conductivity and ph rate were regularly measured in drainage water to maintain the electric conductivity of both input and soil solutions in a stable range. at the end of the experiment, the soil of pots in each level salinity was mixed together. then three samples of each treatment (in total 15 samples) were analyzed (table 3). growth parameters at the end of the experiment, growth characteristics including main-shoot length, trunk diameter and number of leaves, were measured as well as percentage of necrotic leaves, downfall leaves and green leaves were calculated (papadakis et al., 2007). fresh weight of leaves, main-shoots and roots were measured immediately after removing, using a digital scale. dry weight of the samples was measured using an oven at 75°c for 48 h (papadakis et al., 2007). physiological parameters for determination of leaf chlorophyll, 0.2 g of leaf was extracted (in total 630 samples, means nine replicas for each cultivar and for each salinity treatment), with ethanol 80% and chlorophyll a, chlorophyll b and total chlorophyll content were calculated with the method described by arnon (1949). leaf greenness (chlorophyll index) was evaluated on the same leaves used for gas exchange and fluorescence using a spad (minolta, 502, made in japan) after 75 days since treatments introduction. leaf relative water content (rwc) was determined with nine replicas (made by four leaves each) for each treatment and for each cultivar, for a total of 630 samples. fresh weight (fw) was recorded and then samples were put into distilled water and kept at 4°c for 24 h in the dark. after the emission of extra humidity, samples were weighed again to obtain the total weight (tw). subsequently, samples were kept in the oven at 105°c for 24 hours and dry weight (dw) was recorded. finally, relative water content was calculated via formulae (yamasaki and title value saturation percentage (%) 39 field capacity (%) 27.33 permanent wiltering point (%) 14.8 ds/m (ec) 1.28 ph 7.5 n (%) 0.15 organic carbon (%) 1.49 p (ppm) 104.9 sand (%) 46 silt (%) 34 clay (%) 20 texture loam ca (ppm) 1230 mg (ppm) 316.2 total neutralizing value (%) 13.8 cu (ppm) 2.12 zn (ppm) 4.86 fe (ppm) 27.34 k (ppm) 690 mn (ppm) 16.26 na (ppm) 93.15 table 1 physical and chemical characteristics of soil mixture table 2 salt solution characteristics treatments electrical conductivity (ds/m) (ph) na (mg/l) cl (mg/l) ca (mg/l) mg (mg/l) hco 3 -(mg/l) control 0.50 7.30 22.1 35.5 62 17.1 98 a 4.90 7.60 809 1386 79 23.01 137 b 9.80 7.78 1653 2836 99 25.7 159 c 14.75 7.87 2443 4199 123 28.5 186 d 19.80 7.95 3276 5610 151 31.9 214 treatments ec (ds/m) ph control 1.2 7.4 a 5.7 7.65 b 10.9 7.87 c 15.95 7.96 d 21.3 8.05 table 3 ec and ph soil treated with different levels of salinity adv. hort. sci., 2018 32(2): 249-264 252 dillenburg, 1999). rwc= [(fw-dw) / (tw-dw)] ´100 for the determination of relative ionic content was determined with nine replicas (made by four leaves each) for each treatment and for each cultivar, for a total of 630 samples. the amount of 0.5 g of each sample was put in tubes with 25 ml of distilled water at 25°c for 24 h on a shaker with speed 120 in/min. electrical conductivity (ec) of the medium was then read using a conductivity meter (conduct meter; radiometer, copenhagen). following the initial reading (lt), samples were autoclaved for 20 min to kill leaf tissues and then kept at 25°c for 2 h on shaker with speed 120 in/min and a final reading (lo) was obtained. finally, relative ionic percentage was calculated via formulae: relative ionic percentage = (lt/lo)/100 as described in lutts et al. (1995). after calculation relative ionic percentage, cell membrane injury in samples’ treatment with natural salt ratio samples control was performed as follows: % injury = 1[1(t1/t2)/ 1(c1/c2)] × 100 where t and c refer to the ec values of stresstreated and control tubes and 1 and 2 refer to the initial and final ec, respectively (lutts et al., 1995). chlorophyll fluorescence parameters chlorophyll fluorescence of leaves was measured using a portable fluorometer pam-2000 (h. walz, effeltrich, germany). before measuring chlorophyll fluorescence parameters, three leaves on mainbranch of each plant were put in dark-adapted state (das) for 30 min using light exclusion clips (maxwell and johnson, 2000). maximum quantum efficiency of photosystem ii (fv/fm) was determined as fv/fm= (fm-f0)/fm; where fm and f0 were maximum and minimum fluorescence of dark-adapted leaves, respectively. concentration of na+ and k+ concentration of na+ and k+ in leaves and roots was determined with nine replicas for each treatment and for each cultivar, for a total of 630 samples. leaves and roots of each plant, oven-dried at 75°c for 48 h, and then milled to a fine powder to pass through a 30-mesh screen. the amount of 0.5 g of each sample was dry-ached for 6h at 550°c, dissolved in 3 ml of 6 mol l−1 hcl and diluted to 50 ml with deionized water. subsequently, concentration of na+ and k+ were determined using atomic absorption spectroscopy (papadakis et al., 2007). statistical analysis this experiment was carried out based on completely randomized design (crd), with factors cultivars in 14 levels and irrigation water salinity in 5 levels and with nine replicas for each treatment in research greenhouse of temperate fruit research center, horticultural research institute in karaj-iran in years 2013 and 2014. finally, data were analyzed using analysis of variance (anova) using sas software. means were also compared by duncan’s multiple range test at 1% level. 3. results as reported in table 4, salinity treatments negatively affected plant height, trunk diameter and number of leaves. with increasing salinity concentration in irrigation water, final height, trunk diameter and number of leaves in all studied cultivars were decreased. the lowest branch height, trunk diameter and leaf number were observed in salinity level d. the rate of decrease branch height, trunk diameter and number of leaves among the cultivars showed a significant difference with each other. the height of khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi and kaleh ghochi cultivars were decreased in salinity level b compared to control plants. while the height of akbari, shahpasand and badami zarand cultivars in salinity level c and in qazvini cultivar only in salinity level d was decreased significantly compared to control plants. as reported in table 4, as the salt concentration increases, the trunk diameter and its growth were decreased during the application of salinity stress in all cultivars. the decrease in trunk diameter in the cultivars showed a significant difference with each other. the trunk diameter of khanjari, fndoghi 48, rezaie zood res, mousa abadi and kaleh ghochi cultivars was decreased in salinity level b compared to control plants. while the trunk diameter of italiyayi, jandaghi, ebrahimi, sabz pesteh tohg, ahmad aghaee, akbari, shahpasand and badami zarand cultivars in salinity level c and in qazvini cultivar only in salinity level d was decreased significantly compared to control plants. the results showed that number of leaves with increasing salinity concentrations were reduced, but the amount of reduction in the number of leaves in different cultivars had significant differences. the maximum number of leaves were observed in control momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 253 table 4 effect of interaction between salinity and cultivar on some of the morphologic traits means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. *= a/ is less than z. given that variety of data was very wide, duncan’s test grouped data between a to z and less than z such as a/, b/, c/, d/ and e/. cultivars treatments no. of green leaves green leaves (%) trunk diameter (mm) branch height (cm) khanjari control 27.33±1.0 h-n 100.00±0.0 a 8.38±0.13 a 32.50±1.56 f-g a 25.00±1.93 n-r 100.00±0.0 a 8.38±0.09 a 31.47±0.37 g-j b 22.55±1.42 s-v 97.02±3.81 a-d 8.07±0.09 b 28.67±1.17 k-n c 20.00±1.22 t-w 76.21±5.82 m-o 7.57±0.09 cd 25.55±0.52 r-s d 16.33±1.32 x-z 51.41±6.87 q 6.94±0.05 f-h 21.31±0.54 w-x akbari control 24.67±1.80 o-s 100.00±0.0 a 7.53±0.10 c-d 25.05±2.78 r-s a 22.89±0.78 s-u 100.00±0.0 a 5.55±0.09 c-d 24.38±2.69 s-u b 22.11±0.70 s-v 100.00±0.0 a 7.31±0.05 d-e 23.55±1.26 s-v c 18.45±0.72 u-y 96.86±4.23 a-d 7.14±0.06 e-g 22.67±1.29 t-y d 16.45±0.88 x-z 89.43±4.92 g-i 6.65±0.08 h-j 20.18±1.79 w-y ghazvini control 27.23±0.77 h-n 100.00±0.0 a 5.82±0.07 p-u 22.57±1.08 t-w a 27.07±0.26 i-m 100.00±0.0 a 5.81±0.07 p-u 22.55±0.57 t-w b 26.52±0.67 j-o 99.55±0.0 a 5.70±0.05 s-w 21.67±0.50 u-x c 25.77±0.67 m-r 97.72±5.63 a-c 5.46±0.04 u-x 20.7±1.0 w-y d 23.81±2.24 q-u 90.88±7.07 e-h 5.02±0.06 x-y 18.11±1.21 y-z italiaie control 31.67±2.34 e 100.00±0.0 a 6.71±0.03 h-j 33.00±2.95 e-h a 31.06±2.0 e-f 100.00±0.0 a 6.74±0.16 h-j 32.30±1.21 f-h b 29.22±1.39 f-i 98.85±2.40 a-b 6.53±0.12 j-m 30.17±0.94 i-k c 25.22±1.71 n-r 90.72±8.49 e-h 6.15±0.29 n-q 26.43±1.19 o-r d 22.33±2.34 q-u 84.88±5.04 i-k 5.60±0.24 t-x 22.71±1.90 t-w kaleh ghochi control 27.00±1.93 i-n 100.00±0.0 a 6.63±0.05 f-h 32.09±0.87 f-h a 26.85±0.86 i-n 10000±0.0 a 6.59±0.04 j-k 31.87±0.86 f-i b 23.44±0.72 q-u 97.66±3.04 a-c 6.28±0.03 l-p 28.87±0.86 k-n c 19.33±1.0 u-w 89.60±5.26 f-i 5.78±0.04 p-u 25.43±0.71 r-s d 15.33±1.0 y-z 72.92±7.64 o 5.25±0.03 w-x 20.28±0.48 x-y jandaghi control 27.89±1.05 g-j 100.00±0.0 a 6.05±1 p-r 23.56±0.73 s-v a 27.11±2.26 i-m 100.00±0.0 a 6.04±1 p-r 23.45± 1.05 s-w b 25.33±0.70 n-r 95.80±3.46 a-e 5.97±1 q-s 21.03±0.53 w-x c 20.67±1.93 t-w 80.96±6.21 k-m 5.33±1 u-x 18.73±0.68 y-z d 15.44±1.42 y-z 64.71±7.56 p 4.81±1 y-z 16.45±0.51 a mousa abadi control 32.00±1.22 e 100.00±0.0 a 5.78±1 p-u 30.00±1.87 j-l a 30.44±1.74 e-f 100.00±0.0 a 5.61±1 s-w 29.48±0.87 k-m b 26.88±1.69 i-n 93.80±3.13 b-e 5.33±1 w-x 26.65±0.75 n-r c 22.11±2.02 s-v 78.71±5.28 l-n 5.12±1 x-y 21.75±0.72 u-x d 15.00±1.22 z 52.47±4.98 q 4.57±1 z 14.90±1.07 b ebrahimi control 29.33±1.93 f-h 100.00±0.0 a 6.07±0.09 p-r 33.67±1.58 e-f a 28.77±1.20 f-i 100.00±0.0 a 6.08±1.29 p-r 33.55±1.26 e-f b 27.44±1.33 g-k 91.85±2.60 d-h 5.75±0.66 p-u 31.26±1.29 h-j c 23.67±1.32 q-u 84.68±4.53 i-k 5.44±0.09 u-x 26.56±1.44 n-q d 19.33±1.32 u-w 62.98±8.62 p 5.17±0.09 w-y 22.15±1.34 s-v badami zarand control 25.00±1.22 n-r 100.00±0.0 a 6.73±0.05 h-j 29.32±1.09 k-m a 24.67±1.32 o-s 100.00±0.0 a 6.69±0.06 h-j 29.35±1.30 k-m b 23.33±1.0 q-u 97.39±0.0 a-c 6.56±0.08 j-l 27.40±0.92 m-q c 21.44±1.23 t-v 93.00±3.37 c-h 6.25±0.06 m-p 24.51±0.81 r-t d 17.78±1.48 v-y 83.71±4.45 j-l 5.80±0.12 p-u 21.19±0.90 w-x fandoghi 48 control 35.00±1.87 d 100.00±0.0 a 6.62±0.19 i-j 36.53±1.11 d a 34.33±1.58 d 100.00±0.0 a 6.53±0.11 j-m 36.23±0.92 d b 31.11±1.69 e-f 94.55±4.30 b-e 6.30±0.10 k-o 33.17±1.26 e-g c 26.77±1.85 j-o 87.96±2.29 h-j 5.90±0.15 q-s 29.34±1.65 k-m d 21.11±1.69 t-w 75.97±6.88 o 5.67±0.09 s-w 23.45±1.02 s-v sabs pesteh togh control 32.00±1.50 e 100.00±0.0 a 6.43±0.09 j-n 28.22±1.21 l-o a 31.67±0.86 e 100.00±0.0 a 6.44±0.05 j-n 27.73±1.10 m-p b 28.00±1.87 g-k 93.78±0.05 b-g 6.13±0.08 n-p 25.70±0.92 p-r c 24.33±1.32 p-t 83.08±3.09 h-j 5.73±0.08 s-w 22.91±0.29 s-v d 20.11±1.45 t-w 67.29±5.39 no 5.33±0.09 u-x 21.01±0.64 w-x ahmad aghaee control 30.00±1.87 e-g 100.00±0.0 a 5.71±0.11 s-w 23.11±1.16 s-w a 27.88±1.16 g-k 100.00±0.0 a 5.67±0.08 s-w 21.33±1.32 w-x b 25.00±1.22 n-s 95.55±3.16 a-e 5.35±0.11 u-x 18.33±0.96 y-z c 22.11±1.26 s-v 82.32±4.41 kl 5.11±0.08 x-y 17.21±0.54 z d 19.04±0.72 u-x 72.01±2.50 o 4.73±0.08 y-z 16.22±0.66 a/* rezaie zodres control 30.67±1.32 e-g 100.00±0.0 a 5.81±0.18 p-u 34.63±2.23 e a 29.22±1.30 f-h 100.00±0.0 a 5.50±0.26 u-x 32.22±1.27 e-h b 25.77±1.71 m-r 91.76±5.73 d-h 5.17±0.16 w 28.11±1.32 m-p c 20.55±1.01 t-w 75.51±6.87 no 5.05±0.33 x-y 23.46±1.48 s-v d 16.00±0.86 y-z 53.48±4.82 q 4.49±0.24 z-a 17.95±1.08 y-z shahpasand control 45.00±1.65 a 100.00±0.0 a 7.77±0.11 c 47.51±1.40 a a 43.67±1.0 a 100.00±0.0 a 7.65±0.10 c 47.07±1.29 a b 41.67±0.70 a-b 98.55±0.0 a-b 7.52±0.10 cd 45.01±0.98 a-b c 39.11±1.16 c 93.89±1.81 b-g 7.21±0.06 e-f 42.19±3.33 c d 35.33±1.58 d 83.01±4.73 j-l 6.90±0.09 g-i 37.01±1.06 d adv. hort. sci., 2018 32(2): 249-264 254 plants of shahpasand cultivar (45±1.65 leaves), and the lowest amount of them were observed in mousa abadi, kaleh ghochi, jandaghi and rezaie zood res cultivars in salinity level d (15±1.22, 15.33±1.00, 15.44±1.42 and 16±0.86 leaves), respectively. the results showed that with increasing salinity of irrigation water, the percentage of green leaves in all cultivars was decreased. in control plants and also plants treated with salinity level a, all leaves of plants were green and were not observed any necrotic leaves. necrosis and fallen leaves in all cultivars were observed in salinity levels b (except for akbari cultivar), c and d. the lowest percentage of green leaves was found in salinity level d and in mousa abadi (52.47±4.98%), and rezaie zood res (53.48±4.82%), cultivars, respectively. as reported in table 5, in all the cultivars as the salinity increases, the percentage of necrosis leaves were increased and the first symptoms of necrosis except for akbari cultivar were observed in salinity level a. in all cultivars, the highest incidence of necrosis leaves was observed in salinity level d. the percentage of fallen leaves also were increased with increasing salinity levels while in all cultivars except for akbari and ghazvini cultivars were observed fallen leaves in salinity levels c and d. the results showed that leaves and shoots fresh and dry weights in all studied cultivars significantly decreased by applying salinity stress and increasing its concentration. shoots and leaves fresh and dry weights in kaleh ghochi, italiyayi, jandaghi, fndoghi 48, ebrahimi, mousa abadi and rezaei zood res cultivars in salinity levels b, c and d, and in khanjari, sabz pesteh tohg, ahmad aghaee and badami zarand cultivars in salinity levels c and d, and in akbari, shahpasand and qazvini cultivars only in salinity level d, were decreased significantly compared to control plants. based on the results of this study, as the salinity increases, the amount of minimum chlorophyll fluorescence (f0) was increased significantly. the highest amount of f0 in all cultivars was observed in salinity level d. the highest amount of f0 was observed in the leaves of khanjari cultivar treated with salinity level d (table 7). also, maximum chlorophyll fluorescence (fm) in all cultivars was decreased significantly as the salinity increased. the highest amount of fm was observed in control plants while the lowest amount of fm was observed in rezaie zood res (365.22± 20.90), jandaghi (380.67±11.69) and mousa abadi (387.67±29.06) cultivars that was treated with salinity level d, respectively (table 7). the results showed that in all studied cultivars, (fv/fm) ratio was reduced significantly by applying salinity stress and increasing its concentration. furthermore, there was a significant difference fv/fm values in different levels of salinity among tested cultivars. in the leaves of the control plants fv/fm was 0.83±1 indicating the existence of ideal and nonstressed environmental conditions for the growth of all cultivars throughout the experimental period. regarding changes in (fv/fm) ratio the stress intensity in rezaie zood res and mousa abadi cultivars was more severe than other cultivars, (0.59±0.015 and 0.61±0.009 respectively). therefore, the susceptibility of these cultivars to salinity stress in levels c and d were higher than other cultivars. on the contrary, gazvini and akbari cultivars were less damaged, (0.76±0.003 and 0.75±0.007, respectively) (table 7). in other words, fv/fm in this cultivars, showed the lowest decrease. results on chlorophyll a, b and total chlorophyll content of the leaves treated in different salinity levels are reported in table 8. chlorophyll a content was reduced significantly in all of the studied cultivars in salinity level d compared to control plants while chlorophyll b content in salinity levels c and d was reduced significantly compared to the control plants. total chlorophyll content was decreased significantly in ghazvini cultivar only in salinity level d, and in akbari and badami zarani cultivars in salinity levels c and d while total chlorophyll content in other cultivars decreased significantly in salinity levels b, c and d (table 8). chlorophyll index was decreased significantly under salinity stress. the lowest chlorophyll index was observed in the leaves of the plants that were irrigated with salinity level d. the highest reduction in chlorophyll index was observed in mousa abadi (22.60±1.50), jandaghi (27.83±0.98) and kaleh ghochi (31.47±3.08) cultivars. the lowest reduction in chlorophyll index was observed in shahpasand (55.35±1.35), akbari (53.70±1.24) and ghazvini (49.78±1.10) cultivars (table 8). according to the results reported in table 9, the content of relative humidity of leaves decreased significantly as the salinity increased. the content of relative humidity in leaves of control plants were higher than 79.83% (of 79.83±0.24% in control plant leaves of shahpasand cultivar to 85.25±0.64% in control plant leaves of khanjari cultivar), while relative humidity content in leaves of mousa abadi, rezaie zood res and sabz pesteh togh plants in salinity level d, were 64.17±0.52%, 66.49±0.57% and 66.95± 0.77%, respectively. in ghazvini and akbari cultivars momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 255 table 5 effect of interaction between salinity and cultivar on the morphologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments leaf dry weight (g) leaf fresh weight (g) downfall leaves (%) necrosis leaves (%) khanjari control 2.77±0.02 d-f 6.28±0.06 e-f 0.00±0.0 l 0.00±0.0 q a 2.77±0.01 d-f 6.30±0.02 e-f 0.00±0.0 l 0.00±0.0 q b 2.67±0.02 f-g 6.05±0.06 f-g 0.00±0.0 l 2.98±3.81 n-q c 2.54±0.01 g-i 5.71±0.03 h 11.72±3.47 d 16.07±4.47 d-e d 2.47±0.03 h-k 5.04±0.06 k-m 19.24±3.22 b 29.35±4.24 a akbari control 1.73±0.12 s-u 3.67±0.26 q-t 0.00±0.0 l 0.00±0.0 q a 1.61±0.05 t-u 3.41±0.11 r-u 0.00±0.0 l 0.00±0.0 q b 1.48±0.04 u-x 3.16±0.10 s-v 0.00±0.0 l 0.00±0.0 q c 1.43±0.06 v-x 2.95±0.14 t-w 0.00±0.0 l 3.14±4.23 m-q d 1.34±0.06 w-y 2.69±0.13 u-x 0.00±0.0 l 10.57±4.34 f-h ghazvini control 2.45±0.06 h-l 5.44±0.15 h-j 0.00±0.0 l 0.00±0.0 q a 2.44±0.02 h-l 5.41±0.05 h-j 0.00±0.0 l 0.00±0.0 q b 2.40±0.06 h-l 5.30±0.13 i-k 0.00±0.0 l 0.45± 0.30 q c 2.31±0.06 k-o 5.02±0.13 j-l 0.00±0.0 l 3.28±5.63 m-q d 2.25±0.21 m-p 4.76±0.44 l-n 0.00±0.0 l 9.12±3.68 g-i italiaie control 2.52±0.18 h-j 5.70±0.42 h 0.00±0.0 l 0.00±0.0 q a 2.54±0.15 h-j 5.75±0.35 g-h 0.00±0.0 l 0.00±0.0 q b 2.30±0.10 k-o 5.14±0.24 j-l 0.00±0.0 l 1.15±2.40 p-q c 1.98±0.13 o-s 4.38± 0.29 o-p 3.78±3.50 g-l 5.50±5.62 i-n d 1.73±0.18 s-u 3.77±0.39 q-t 5.13± 3.05 f-j 8.99±3.09 g-j kaleh ghoch control 2.36±0.16 h-m 5.13±0.36 i-k 0.00±0.0 l 0.00±0.0 q a 2.37±0.07 h-m 5.13±0.16 i-k 0.00±0.0 l 0.00±0.0 q b 2.01±0.06 o-s 4.29±0.13 o-p 1.19±2.50 kl 1.15±3.04 p-q c 1.58±0.08 t-u 3.32±0.17 r-u 4.91±2.70 f-k 5.49±2.78 i-n d 1.18±0.11 y-z 2.43±0.15 v-y 17.10±7.08 b-c 9.98±6.03 f-h jandaghi control 2.92±0.23 c-d 6.41±0.24 e 0.00±0.0 l 0.00±0.0 q a 2.89±0.06 c-e 6.32±0.50 e-f 0.00±0.0 l 0.00±0.0 q b 2.55±0.18 g-h 5.53±0.14 hi 1.87± 1.90 i-l 2.33±2.95 n-q c 1.95±0.12 p-t 4.13±0.38 p-r 11.54± 4.80 d 7.50±4.54 g-l d 1.41±0.04 v-x 2.90±0.26 t-w 17.74± 6.60 b-c 17.55±6.71 cd mousa abadi control 1.83±0.02 r-t 4.07±0.09 p-r 0.00±0.0 l 00.00±0.0 q a 1.91±0.09 p-t 3.99±0.06 p-s 0.00±0.0 l 00.00±0.0 q b 1.51±0.10 u-x 3.22±0.20 s-u 2.65±1.30 i-l 3.55±1.50 m-q c 1.18±0.05 y-z 2.43±0.22 v-y 8.15± 2.50 d-f 13.04±2.74 e-f d 0.72±0.12 z 1.42±0.11 z 27.06± 4.90 a 20.47±4.70b-c ebrahimi control 1.94±0.07 p-s 4.52 ±0.29m-o 0.00±0.0 l 00.00±0.0 q a 1.87±0.10 r-t 4.32±0.18 o-p 0.00±0.0 l 00.00±0.0 q b 1.61±0.04 t-u 3.61±0.23 q-t 3.82±2.33 g-l 5.33±2.11 j-o c 1.22±0.12 x-y 2.70±0.09 u-x 5.02±2.89 f-k 9.29±3.24 f-h d 0.71±0.12 z 1.50±0.25 y-z 15.05±2.95 c 21.96±3.11 b badami zarand control 2.47±0.13 h-k 5.25±0.25 i-k 0.00±0.0 l 0.00±0.0 q a 2.43±0.09 h-k 5.15±0.27 j-k 0.00±0.0 l 0.00±0.0 q b 2.29±0.11 k-o 4.91±0.20 k-m 0.00±0.0 l 2.61±1.11 n-q c 2.08±0.14 o-r 4.28±0.24 o-p 3.50±2.70 h-l 3.50±2.51 m-q d 1.71±0.16 s-u 3.41±0.28 r-u 5.64±2.99 f-i 10.65±3.80 f-h fandoghi 48 control 3.11±0.14 c 7.03±0.37 d 0.00±0.0 l 0.00±0.0 q a 3.06±0.14 c 6.87±0.31 d 0.00±0.0 l 0.00±0.0 q b 2.73±13 e-f 6.07±0.32 f-g 0.00±0.0 l 5.45±2.37 l-p c 2.29±0.15 k-m 4.98±0.34 k-m 3.74±2.50 g-l 8.28±2.30 g-k d 1.73±0.13 s-u 3.67±0.29 q-t 18.15±3.70 b-c 15.88±3.53 de sabs pesteh togh control 2.34±0.04 j-n 4.99±0.09 k-m 0.00±0.0 l 0.00±0.0 a a 2.35±0.05 j-n 4.99±0.12 k-m 0.00±0.0 l 0.00±0.0 a b 2.29±0.06 k-o 4.76±0.13 m-n 1.11±1.17 j-l 5.01±1.39 k-p c 2.13±0.08 o-r 4.33±0.16 o-p 6.88±2.70 e-h 10.04±2.84 f-h d 1.98±0.08 o-s 3.96±0.17 p-s 14.46±3.67 cd 18.25±3.53 cd ahmad aghaee control 1.89±0.05 p-u 3.85±0.11 p-s 0.00±0.0 l 0.00±0.0 q a 1.87±0.03 p-t 3.68±0.06 q-t 0.00±0.0 l 0.00±0.0 q b 1.78±0.04 s-t 3.48±0.08 r-u 0.50±1.10 l 3.95±2.73 l-q c 1.66±0.04 t-v 3.18±0.08 t-v 6.56±2.12 e-h 11.12±2.64 f-g d 1.42±0.05 v-x 2.66±0.09 u-x 11.37±2.50 d 16.62±2.56 de rezaie zodres control 2.27±0.05 k-m 4.52±0.10 m-o 0.00±0.0 l 0.00±0.0 q a 2.25±0.03 k-m 4.45±0.05 m-o 0.00±0.0 l 0.00±0.0 q b 2.15±0.04 m-p 4.21±0.07 p-s 1.31±0.70 j-l 6.93±3.84 h-m c 1.85±0.067 r-t 3.56±0.13 q-t 9.31±3.12 de 15.18±3.82 de d 1.58±0.05 t-u 2.97±0.09 t-w 27.41±4.45 a 29.11±4.80 a shahpasand control 4.34±0.05 a 8.36±0.10 a 0.00±0.0 l 0.00±0.0 q a 4.34±0.02 a 8.32±0.05 a 0.00±0.0 l 0.00±0.0 q b 4.29± 0.03 a 8.17±0.06 a-b 0.00±0.0 l 1.45±0.5 o-q c 4.23±0.05 a 7.93±0.10 b 3.06±1.50 h-l 3.05±1.62 m-q d 4.02±0.08 b 7.41±0.16 c 7.54±3.01 e-g 9.45±3.08 f-h adv. hort. sci., 2018 32(2): 249-264 256 table 6 effect of interaction between salinity and cultivar on the morphologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. *= a/ is less than z. given that variety of data was very wide, duncan’s test grouped data between a to z and less than z such as a/, b/, c/, d/ and e/. cultivar treatments branch fresh weight (g) root fresh weight (g) root dry weight ratio to aerial organ dry weight root fresh weight ratio to aerial organ fresh weight khanjari control 4.42±0.02 g-i 8.12±0.13 i-l 0.65±0.01 m-v 0.76±0.01 o-y a 4.38±0.01 g-j 8.13±0.07 h-l 0.65±0.01 m-v 0.76±0.01 o-y b 4.16±0.05 h-m 7.75±0.28 l-o 0.67±0.02 m-u 0.78±0.02 o-x c 3.69±0.05 j-s 6.69±0.11 r-s 0.69±0.01 m-u 0.81±0.01 m-u d 3.01±0.06 r-z 6.14± 0.06 t-u 0.73±0.01 k-s 0.88±0.01 i-r akbari control 3.25±0.36 o-x 8.58±0.51 f-i 0.99±0.08 c-j 1.24±0.10 c-f a 3.17±0.34 o-x 8.51±0.25 f-j 1.03±0.05 c-h 1.28±0.06 c-e b 2.99±0.16 s-z 8.01±0.14 j-n 1.03±0.04 c-h 1.28±0.05 c-e c 2.76±0.15 u-a/ 7.95±0.05 j-n 1.04±0.04 c-g 1.31±0.06 c-e d 2.28±0.20 a/-c/ 7.79±0.04 l-o 1.11±0.06 c-d 1.42±0.08 a-c ghazvini control 3.44±0.16 n-v 9.46±0.15 a-b 0.91±0.02 d-l 1.06 ±0.02e-n a 3.38±0.08 o-w 9.42±0.35 a-b 0.91±0.03 d-l 1.07±0.04 e-n b 3.18±0.07 o-x 9.35±0.03 a-b 0.94±0.01 d-k 1.10±0.01 e-k c 2.66±0.14 v-a/ 9.29±0.03 a-c 1.03±0.01 c-h 1.21±0.03 c-g d 2.42±0.16 y-b/ 9.15±0.04 a-e 1.09±0.07 c-e 1.28±0.08 c-e italiaie control 4.35±0.39 g-k 9.60±0.07 a 0.82±0.06 g-n 0.96±0.06 g-p a 4.23±0.15 h-l 9.58±0.05 a 0.83±0.03 g-n 0.96±0.03 g-p b 3.89±0.12 h-o 9.41±2.98 a-b 0.85±0.28 f-n 0.99 f±0.33-o c 3.30±0.14 o-x 9.19±0.06 a-d 1.02±0.05 c-i 1.19±0.06 c-h d 2.72±0.22 v-a/ 8.96±0.07 b-f 1.20±0.09 b-c 1.37±0.10 b-d kaleh ghochi control 3.21±0.08 o-x 5.46±0.07 w-y 0.56±0.01 p-w 0.65±0.01 r-y a 3.12±0.08 p-y 5.46±0.03 w-y 0.57±0.01 o-w 0.67±0.01 q-y b 2.74±0.08 v-a/ 5.41±0.04 w-z 0.66±0.01 m-u 0.77±0.02 o-y c 2.23±0.06 a/-c/ 5.20±0.05 w-a/ 0.80±0.03 h-p 0.93±0.04 h-q d 1.62±0.03 c/ 4.90±0.03 z-b/ 1.03±0.04 c-h 1.21±0.05 c-g jandaghi control 3.49±0.10 m-u 5.24±0.06 w-a/ 0.46±0.01 u-w 0.53±0.01 v-y a 3.42±0.15 n-v 5.22±0.04 w-a/ 0.47±0.02 t-w 0.54±0.02 u-y b 2.90±0.07 t-z 5.01±0.07 y-b/ 0.52±0.01 r-w 0.59±0.01 t-y c 2.34± 0.08 z-b/ 4.78±0.05 a/-c/ 0.65±0.03 m-v 0.74±0.04 o-y d 1.81±0.05 b/ 4.28±0.03 d/ 0.79±0.03 i-p 0.91±0.04 i-r mousa abadi control 3.30±0.20 o-w 5.16±0.18 w-a/ 0.63±0.03 n-w 0.70±0.03 p-y a 3.18±0.09 o-w 5.08±0.10 y-a/ 0.63±0.01 n-w 0.71±0.01 p-y b 2.46±0.07 xa/ 4.97±0.07 y-b/ 0.75±0.02 k-r 0.84±0.03 k-t c 1.96±0.06 a/d/ 4.53±0.05 b/-d/ 0.95±0.03 d-k 1.08±0.04 e-l d 1.16±0.08 d/ 4.13±0.07 d/e/ 1.39±0.06 a-b 1.60±0.07 a-b ebrahimi control 5.05±0.23 e-g 7.51±0.09 n-p 0.76±0.03 j-q 0.79±0.03 n-w a 5.04±0.18 e-g 7.49±0.06 n-q 0.77±0.02 j-p 0.80±0.02 n-v b 4.41±0.18 f-j 7.32±0.04 o-q 0.87±0.02 e-m 0.91±0.02 i-r c 3.50±0.19 m-t 7.04±0.07 p-r 1.07±0.03 c-f 1.13±0.03 d-j d 2.68±0.16 w-a/ 6.69±0.06 r-s 1.47±0.13 a 1.61±0.16 a badami zarand control 5.57±0.20 d-e 8.80±0.09 c-g 0.78±0.02 j-p 0.81±0.02 m-u a 5.54±0.24 d-e 8.73±0.13 d-g 0.78±0.03 j-p 0.82±0.03 l-t b 5.10±0.17 e-f 8.65±0.13 e-h 0.84±0.02 g-n 0.87±0.03 j-s c 4.41±0.14 g-j 8.32±0.26 g-k 0.92±0.03 d-l 0.96±0.04 g-p d 3.64±0.15 k-s 8.06±0.17 i-m 1.09±0.05 c-e 1.14±0.06 d-i fandoghi 48 control 7.71±0.23 a-b 7.62±0.11 l-o 0.41±0.01 w 0.51±0.01 x-y a 7.60±0.19 a-b 7.58±0.05 m-o 0.42±0.01 v-w 0.52±0.01 w-y b 6.81±0.25 c 7.30±0.09 o-q 0.46±0.01 u-w 0.57±0.01 t-v c 5.78±0.32 d 6.99±0.08 q-s 0.52±0.02 r-w 0.65±0.033 r-y d 4.36±0.19 g-j 6.50±0.14 s-t 0.65±0.03 m-v 0.82±0.04 l-t sabs pesteh togh control 3.81±0.16 i-p 6.02±0.11 t-v 0.69±0.02 l-u 0.68±0.02 q-y a 3.74±0.14 i-r 5.99±0.07 u-v 0.70±0.01 l-t 0.69±0.01 p-y b 3.21±0.11 o-x 5.71±0.18 u-w 0.73±0.01 k-s 0.71±0.01 p-y c 2.61±0.03 x-a/ 5.38±0.14 w-z 0.78±0.01 j-p 0.78±0.01 o-x d 2.14±0.06 a/-c/ 4.95±0.11 y-a/ 0.82±0.02 g-n 0.81±0.02 m-u ahmad aghaee control 3.83±0.07 i-p 5.72±0.06 u-w 0.76±0.01 j-q 0.75±0.01 o-y a 3.76±0.03 i-q 5.73 ±0.07u-w 0.79±0.01 j-p 0.77±0.01 o-y b 3.55±0.07 l-t 5.61±0.07 v-x 0.82±0.01 g-n 0.80±0.01 m-v c 3.35±0.07 o-w 5.47±0.08 w-y 0.86±0.01 f-n 0.84±0.01 k-t d 2.85±0.09 t-z 5.02±0.07 y-a/ 0.91±0.01 d-l 0.91±0.01 i-r rezaie zodres control 4.56±0.09 f-h 4.56±0.12 b/-d/ 0.49±0.01 t-w 0.50±0.01 y a 4.46±0.12 f-i 4.41±0.11 b/-d/ 0.50±0.02 s-w 0.50±0.02 y b 4.13±0.09 i-n 4.28±0.16 d/ 0.51±0.01 s-w 0.51±0.01 x-y c 3.78±0.09 i-q 3.98±0.15 e/ 0.53±0.02 q-w 0.54±0.02 u-y d 3.05±0.10 q-z 3.53±0.09 f/ 0.58±0.01 o-w 0.59±0.01 t-y shahpasand control 8.01±0.07 a 9.44±0.11 a-b 0.47±0.01 t-w 0.57±0.01 t-y a 7.98±0.08 a 9.43±0.08 a-b 0.48±0.01 t-w 0.58±0.01 t-y b 7.83±0.12 a-b 9.21±0.05 a-d 0.48±0.01 t-w 0.58±0.01 t-y c 7.49±0.07 a-b 8.98±0.13 b-f 0.49±0.01 t-w 0.59±0.01 t-y d 7.18±0.10 b-c 8.53±0.16 f-i 0.50±0.01 s-w 0.60±0.01 s-y momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 257 table 7 effect of interaction between salinity and cultivar on chlorophyll fluorescence parameters means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments (f v /f m ) maximum florescence (f m ) minimum florescence (f o ) khanjari control 0.82±0.003 b-c 603.67±3.46 b-d 107.22±1.71 r-u a 0.82±0.003 b-c 606.44±3.39 b-d 110.67±1.58 o-r b 0.80±0.005 d-e 586.00±8.74 e-f 116.55±1.66 k-m c 0.74±0.015 j-k 540.33±5.50 k-m 139.88±8.52 e d 0.65±0.025 q 467.77±5.65 r 163.11±11.20 a akbari control 0.83±0.002 a-b 625.44±6.72 a 106.11±2.31 s-u a 0.82±0.004 b-c 617.67±5.85 a-c 109.77±2.16 p-s b 0.82±0.007 b-c 614.11±5.01 a-c 110.55±3.77 o-s c 0.78±0.003 f-g 607.11±7.09 b-d 134.22±2.38 f-g d 0.75±0.007 i-j 555.44±11.58 h-k 141.33±1.58 e ghazvini control 0.82±0.003 b-c 602.44±11.54 c-d 106.11±3.51 s-u a 0.82±0.003 b-c 602.55±17.00 c-d 108.67±4.09 q-t b 0.81±0.004 c-d 591.00±2.87 d-f 109.77±2.27 o-s c 0.80±0.003 c-e 578.33±5.61 d-g 118.67±1.93 j-m d 0.76±0.003 h-i 538.11±4.59 l-m 129.33±2.39 h italiaie control 0.83±0.004 a-b 600.77±7.41 c-e 101.22±3.89 v-w a 0.83±0.003 a-b 604.22±9.31 b-d 103.77±3.70 t-v b 0.81±0.004 c-d 576.44±3.71 f-g 112.00±2.39 n-q c 0.78±0.008 f-g 532.11±13.50 m 117.11±2.14 k-m d 0.73±0.009 l 488.67±6.61 o-q 133.55±5.12 f-g kaleh ghochi control 0.83±0.002 a-b 538.44±9.46 l-m 93.22±1.98 y-z a 0.82±0.005 b-c 542.88±5.94 j-m 93.33±2.64 y-z b 0.80±0.005 d-e 529.88±7.18 m 106.44±2.29 s-u c 0.76±0.005 h-i 489.00±8.38 o-q 119.55±3.20 i-k d 0.69±0.009 m 444.11±12.31 s 137.00±3.46 e-f jandaghi control 0.83±0.005 a-b 580.33±18.67 f 100.67±2.64 v-w a 0.82±0.006 b-c 557.00±3.80 h-j 101.44±3.46 v-w b 0.77±0.006 g-h 479.88±6.73 p-r 110.33±2.54 p-s c 0.7±0.0043 l 417.33±9.04 t-u 113.89±3.25 m-p d 0.66±0.004 p 380.67±11.69 w 129.44±3.08 h mousa abadi control 0.84 a±0.005 551.78±7.15 h-l 91.00±2.87 z a 0.82±0.007 b-c 530.00±7.29 m 94.00±3.57 x-z b 0.79±0.002 e-f 495.22±14.77 op 104.55±3.46 t-v c 0.73±0.008 kl 447.22±21.89 s 122.44±4.97 i-j d 0.61±0.009 s 387.67±29.06 w 151.00±10.34 b-c ebrahimi control 0.83±0.003 a-b 608.11±8.08 b-c 104.55±2.50 t-v a 0.83±0.004 a-b 609.67±8.17 a-c 105.44±3.77 t-v b 0.80±0.006 d-e 586.44±10.27 e-f 114.44±3.35 m-p c 0.77±0.006 g-h 539.67±14.41 k-m 123.67±4.24 i d 0.73±0.011 kl 512.44±12.28 n 140.67±4.44 e badami zarand control 0.82±0.003 b-c 602.44±9.83 cd 105.55±2.12 s-u a 0.82±0.002 b-c 606.33±8.81 a-c 106.55±2.45 s-u b 0.81±0.003 c-d 597.11±10.32 d-f 108.55±1.58 q-t c 0.77± 0.007 g-h 542.55±13.92 j-m 124.44±4.92 i d 0.73±0.004 k-l 512.67±12.79 n 137.44±3.16 e-f fandoghi 48 control 0.84±0.006 a 585.44±10.90 f 95.11±2.97 x-z a 0.83±0.004 a-b 585.48±9.48 f 98.11±2.47 w-x b 0.79±0.004 e-f 579.00±8.95 f 118.89±3.33 j-m c 0.73±0.010 k-l 491.44±10.87 o-q 131.55±4.63 g-h d 0.64±0.018 r 404.55±14.39 u-v 147.33±4.74 d sabs pesteh togh control 0.83±0.005 a-b 562.00±14.96 g-h 95.67±1.73 x-y a 0.82±0.007 b-c 539.00±21.68 l-m 97.77±1.78 w-x b 0.76±0.009 h-i 470.89±14.88 r 110.88±2.52 o-r c 0.72±0.007 l 427.11±15.39 t 118.55±4.15 j-l d 0.67±0.009 o 409.11±14.58 u 134.00±3.93 f-g ahmad aghaee control 0.83±0.003 a-b 610.33±12.40 a-c 106.44±2.24 s-u a 0.82±0.004 b-c 603.22±19.07 cd 111.22±3.07 o-r b 0.78±0.011 f-g 543.89±20.01 i-m 117.11±2.61 k-m c 0.75±0.009 i-j 510.33±12.10 n 130.00±3.27 g-h d 0.67±0.022 o 469.89±30.25 r 153.33±4.66 b rezaie zodres control 0.84±0.008 a 562.89±13.27 g-h 92.67±3.04 y-z a 0.82±0.006 b-c 538.55±14.52 k-m 95.67±2.00 x-y b 0.76±0.012 h-i 467.77±16.20 r 105.33±3.60 t-v c 0.68±0.016 n 391.00±10.14 v-u 123.55±5.41 i d 0.59±0.015 t 365.22±20.90 x 147.77±7.52 cd shahpasand control 0.83±0.003 a-b 531.00±9.73 m 86.67±2.00 a/ a 0.83±0.004 a-b 530.88±8.26 m 88.00±1.58 a/ b 0.82±0.007 b-c 527.44±17.25 m-n 95.11±2.52 x-z c 0.79±0.012 e-f 499.67±18.36 o-p 105.77±2.81 s-u d 0.75±0.011 i-j 461.00±13.79 r 115.00±3.42 l-o adv. hort. sci., 2018 32(2): 249-264 258 table 8 effect of interaction between salinity and cultivar on the physiologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments total chlorophyl (mg/g) chlorophyll b (mg/g) chlorophyll a (mg/g) chlorophyll index (spad) khanjari control 1.13±0.03 k-n 0.36±0.02 g-j 0.77±0.02 g 57.58±1.52 f-h a 1.11±0.02 m-n 0.36±0.04 g-j 0.75±0.02 g-i 56.62±1.62 g-j b 1.06±0.02 p-r 0.34±0.02 i-l 0.72±0.05 i-k 53.60±0.82 i-l c 0.88±0.01 w 0.31±0.01 l-o 0.57±0.02 s 48.55±1.50 o-q d 0.59±0.02 a/ 0.22±0.01 t 0.37±0.04 w-x 40.00±1.73 y-z akbari control 1.45±0.007 c 0.51±0.008 a 0.95±0.003 c 61.27±0.98 c-d a 1.45±0.11 c 0.52±0.008 a 0.94±0.009 c 61.40±1.57 c-d b 1.40±0.02 c-d 0.51±0.1 a 0.89±0.03 c-d 60.15±1.55 c-e c 1.23±0.01 f 0.46±0.03 cd 0.77±0.01 g 56.97±1.04 g-i d 1.05±0.009 q-s 0.38±0.02 e-h 0.67±0.02 m-o 53.70±1.24 k-m ghazvini control 1.20±0.01 f-g 0.46±0.007 c-d 0.74±0.01 h-j 55.17±1.27 g-l a 1.21±.005 f-g 0.47±0.01 c-d 0.74±0.008 h-j 54.67±1.25 i-l b 1.17±0.006 g 0.45±0.007 c-d 0.72±0.005 j-k 54.04±1.09 j-m c 1.14±0.02 g-m 0.43±0.008 d 0.71±0.003 j-l 51.98±0.85 l-n d 0.97±0.004 t 0.35± 0.01 h-k 0.62±0.008 q-r 49.78±0.82 n-o italiaie control 1.10±0.01 m-o 0.36±0.004 g-j 0.74±0.003 h-j 49.95±1.10 n-o a 1.09±0.01 n-p 0.36±0.004 g-j 0.73±0.01 i-j 50.02±1.38 n-o b 1.04±0.007 q-s 0.32±0.01 k-n 0.72±0.003 j-k 45.17±1.36 r-u c 0.91±0.008 v 0.26±0.009 q-r 0.6±0.0075 op 43.95±1.43 t-w d 0.70±0.01 z 0.20±0.004 t 0.50±0.005 u 40.41±1.46 x-z kaleh ghochi control 1.07±0.007 o-q 0.38±0.008 e-h 0.69±0.005 l-m 45.17±0.60 r-u a 1.06±0.008 p-r 0.37±0.004 f-i 0.69±0.003 l-m 43.95±0.16 t-w b 1.03±0.01 q-s 0.37±0.004 f-i 0.66±0.009 m-o 41.50±0.28 u-z c 0.80±0.009 x 0.31±0.008 l-o 0.49±0.02 u-v 36.24±0.40 a/ d 0.60±0.003 a/ 0.22±0.01 t 0.38±0.01 w-x 31.47±3.08 b/ jandaghi control 1.04±0.007 q-s 0.36±0.04 g-j 0.69±0.002 l-m 42.77±0.82 u-x a 1.04±0.008 q-s 0.36±0.04 g-j 0.68±0.004 l-n 42.77±0.76 u-x b 0.95±0.01 t-u 0.32±0.03 k-n 0.63±0.006 p-q 38.54±1.37 y-z c 0.74±0.004 y 0.26±0.03 q-r 0.48±0.010 u-v 35.45±0.85 a/ d 0.53±0.004 b/ 0.17±0.004 u 0.36±0.01 x 27.83±0.98 c/ mousa abadi control 1.05±0.007 q-s 0.36±0.008 g-j 0.69±0.002 l-m 42.40±0.95 v-y a 1.04±0.008 q-s 0.36±0.004 g-j 0.68±0.003 l-n 42.65±0.51 u-y b 0.95±0.01 t-u 0.30±0.005 m-p 0.63±0.005 p-q 38.11±1.00 z-a/ c 0.72 ±0.01y-z 0.23±0.01 s-t 0.49±0.009 u-v 32.33±1.10 b/ d 0.48±0.03 c/ 0.15±0.004 u 0.33±0.008 y 22.60±1.50 d/ ebrahimi control 1.20±0.04 f-h 0.45±0.03 d 0.74±0.01 h-j 55.21±1.22 g-l a 1.19±0.008 g-i 0.45±0.02 cd 0.74±0.01 h-j 54.97±1.65 h-l b 1.12±0.02 l-n 0.42±0.01 e-g 0.70±0.008 j-l 49.97±1.98 no c 0.95±0.01 t-u 0.35±0.01 h-k 0.60±0.01 r 45.51±1.88 r-t d 0.74±0.01 y 0.27±0.01 p-r 0.47±0.01 v 40.27±1.54 x-z badami zarand control 1.15±0.02 j-l 0.38±0.008 e-h 0.77±0.008 g 54.86±1.32 h-l a 1.15±0.02 j-l 0.39±0.01 e-g 0.76±0.01 g-h 54.80±1.47 i-l b 1.10±0.02 l-p 0.37±0.01 f-i 0.73±0.01 g-l 52.89±0.70 l-m c 0.93±0.01 u-v 0.30±0.01 m-p 0.63±0.01 p-q 49.39±1.53 n-p d 0.73±0.02 y-z 0.20±0.01 t 0.53±0.01 t 45.55±1.19 r-t fandoghi 48 control 1.23±0.02 f 0.41±0.03 e 0.83±0.01 e 56.88-i±1.16 g a 1.23±0.02 f 0.40±0.02 e-f 0.83±0.01 e 56.00±1.22 g-k b 1.16 ±0.009h-j 0.36±0.01 f-i 0.80±0.01 e-f 51.67±1.32 m-n c 1.02±0.03 s 0.32±0.01 k-n 0.70±0.01 j-l 47.22±1.48 p-r d 0.73±0.02 y-z 0.25±0.01 r-s 0.49±0.01 u-v 40.00±1.87 y-z sabs pesteh togh control 1.19±0.03 g-i 0.44±0.05 d 0.74±0.02 h-j 50.36±1.14 n-o a 1.16±0.02 i-k 0.44±0.02 d 0.73±0.007 i-j 46.93±7.47 o-r b 1.05±0.02 q-s 0.35±0.01 h-k 0.70±0.009 j-l 46.77±1.28 q-s c 0.81±0.008 x 0.28±0.02 o-q 0.53±0.01 t 42.47±0.90 v-y d 0.58±0.01 a/ 0.21±0.01 t 0.36±0.005 x 36.92±1.03 a/ ahmad aghaee control 1.04±0.01 q-s 0.37±0.01 f-i 0.67±0.007 m-o 46.48±3.78 q-t a 1.02±0.02 s 0.35±0.01 h-k 0.67±0.007 m-o 44.15±0.22 s-v b 0.97±0.01 t 0.32±0.009 k-n 0.64±0.006 o-q 41.37±0.44 w-z c 0.83±0.01 x 0.29±0.01 n-q 0.53±0.01 t 36.60±0.30 a/ d 0.62±0.01 a/ 0.23±0.01 s-t 0.39±0.01 w 31.23±0.75 b/ rezaie zodres control 1.12±0.02 l-n 0.36±0.008 g-j 0.76±0.02 g-h 57.72±1.04 e-g a 1.11±0.02 m-n 0.37±0.01 f-i 0.75±0.01 g-i 56.01±0.84 g-k b 1.02±0.02 s 0.32±0.01 k-m 0.70±0.01 j-l 53.25±1.63 lm c 0.86±0.02 w 0.29±0.01 n-q 0.57±0.01 s 48.56±1.41 o-q d 0.58±0.03 a/ 0.21±0.005 t 0.37±0.02 w-x 40.24±1.61 x-z shahpasand control 1.54±0.007 a 0.50±0.008 a-b 1.04±0.007 a 65.52±1.16 a a 1.54±0.01 a 0.50±0.01 a-b 1.03±0.009 a-b 64.52±1.19 a-b b 1.50±0.03 a-b 0.48±0.02 b-c 1.02±0.008 a-b 62.57±0.99 a-c c 1.33±0.03 e 0.40±0.02 e-f 0.93±0.01 c 59.98±0.79 d-f d 1.10±0.02 m-o 0.29±0.02 n-q 0.81±0.01 e-f 55.35±1.35 g-l momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 259 table 9 effect of interaction between salinity and cultivar on the physiologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments cell membrane injury (%) relative ionic leakage (%) relative water content (%) khanjari control 37.70±0.010 m-o 85.25±0.64 a a 2.98±1.28 w-y 39.14±0.008 k-o 84.16±0.46 a-b b 8.09±0.91 t-u 44.34±0.005 i-o 81.31±0.51 b c 23.09±3.10 k-m 51.75±0.019 d-k 77.60±0.62 c d 46.47±0.70 b 64.42±0.004 a-c 70.36±1.04 f akbari control 38.05±0.007 l-o 85.08±0.40 a a 0.83±1.21 x-y 38.41±0.016 l-o 84.52±0.60 a-b b 5.27±1.07 u-w 42.30±0.006 j-o 82.90±0.60 a-b c 16.37±1.78 o-q 45.96±0.011 g-o 82.08±0.68 a-b d 27.83±2.18 h-i 50.09±0.013 d-k 80.22±0.56 b ghazvini control 35.62±0.013 o 83.19±0.57 a-b a 0.42±0.19 y 36.59±0.006 m-o 82.98±0.64 a-b b 2.67±2.28 x-y 38.37±0.014 l-o 81.58±0.42 b c 5.89±2.81 u-w 39.73±0.018 k-o 80.88±0.36 b d 16.17±0.62 o-q 45.50±0.16 g-o 78.95±0.45 b-c italiaie control 40.85±0.012 j-o 84.43±0.35 a-b a 0.75±0.79 x-y 40.88±0.005 j-o 84.21±0.38 a-b b 12.16±3.06 r-s 47.75±0.018 e-n 81.51±0.36 b c 22.24±2.62 l-m 52.75±0.015 c-i 78.40±0.37 c d 33.65±3.51 j-l 60.54±0.020 a-d 74.06±0.50 d-e kaleh ghochi control 39.43±0.015 k-o 83.41±0.32 a-b a 3.89±2.81 v-y 40.81±0.017 k-o 82.41±0.29 a-b b 11.97±0.59 r-s 43.67±0.003 i-o 81.45±0.32 b c 21.88±1.09 l-n 48.89±0.006 d-l 78.35±0.33 c d 38.43±2.46 de 59.29±0.015 b-f 73.59±0.30 e jandaghi control 37.68±0.015 m-o 82.57±0.32 a-b a 3.06±1.36 w-y 38.85±0.008 l-o 81.45±0.34 b b 10.47±1.29 s-t 45.53±0.008 h-o 79.35±0.25 b-c c 18.68±2.28 n-p 55.70±0.014 b-i 74.67±0.29 d-e d 31.21±3.47 g-h 66.61±0.021 a-b 67.38±0.41 g mousa abadi control 37.59±0.007 l-o 80.42±0.27 b a 4.47±1.83 u-x 39.39±0.011 k-o 79.53±0.28 b-c b 18.68±2.77 n-p 49.41±0.017 d-k 75.55±0.35 d c 33.47±2.27 f-g 57.80±0.014 b-g 70.52±0.66 f d 54.83±4.50 a 71.34±0.028 a 64.17±0.52 i ebrahimi control 40.04±0.010 k-o 81.51±0.25 b a 4.88±4.12 u-w 42.77±0.027 j-o 80.40±0.45 b-c b 12.92±4.01 q-s 46.57±0.024 f-o 78.49±0.31 b-c c 18.34±2.62 n-p 49.90±0.016 d-l 76.34±0.34 c-d d 27.70±4.05 h-i 56.65±0.024 b-h 69.95±0.45 f-g badami zarand control 35.90±0.018 n-o 80.56±0.26 b-c a 2.36±3.55 w-y 37.60±0.028 l-o 79.41±0.24 b-c b 6.96±2.50 t-v 41.66±0.016 j-o 78.13±0.30 c c 14.18±2.66 q-r 45.00±0.017 h-o 76.38±0.32 c-d d 21.21±2.04 l-n 49.51±0.013 d-l 73.21±0.47 e fandoghi 48 control 41.44±0.012 j-o 82.51±0.33 a-b a 4.18±3.07 v-y 43.52±0.022 j-o 81.64±0.30 b b 10.24±2.12 s-t 47.33±0.012 f-o 79.22±0.47 b-c c 26.48±4.68 i-k 56.86±0.027 b-h 75.25±0.54 d d 41.04±3.79 c-d 65.40±0.022 a-b 69.12±0.47 f-g sabs pesteh togh control 41.06±0.014 j-o 81.34±0.31 b a 5.35±3.95 u-w 44.64±0.029 h-o 79.60±0.41 b-c b 14.57±6.87 q-r 50.94±0.039 d-k 77.18±0.32 c-d c 28.48±4.47 h-i 57.93±0.025 b-g 73.67±0.59 e d 42.89±6.21 c 66.20±0.035 a-b 66.49±0.77 g-i ahmad aghaee control 39.50±0.013 k-o 85.02±0.32 a a 3.27±1.44 v-y 41.35±0.008 j-o 84.39±0.36 a-b b 9.63±0.93 s-t 44.05±0.005 i-o 83.26±0.42 a-b c 19.71±1.64 m-o 50.30±0.010 d-j 80.1±0.38 b d 35.57±2.85 e-f 60.11±0.017 a-e 75.38±0.38 d rezaie zodres control 40.12±0.006 k-o 81.05±0.42 b a 2.18±1.61 w-y 40.97±0.010 j-o 80.50±0.25 b-c b 10.48±1.51 s-t 48.81±0.009 d-l 77.17±0.51 c-d c 26.78±3.43 i-j 55.85±0.020 b-i 74.34±0.45 d-e d 47.45±2.95 b 68.31±0.017 a-b 66.95±0.57 g-i shahpasand control 39.92±0.006 k-o 79.83±0.47 b-c a 3.61±3.10 v-y 40.43±0.021 k-o 79.49±0.22 b-c b 5.98±1.87 u-w 42.63±0.011 j-o 78.89±0.51 b-c c 15.32±3.43 p-r 46.68±0.020 f-o 77.15±0.53 c d 27.77±2.98 h-i 53.23±0.018 c-j 74.41±0.39 d-e 260 adv. hort. sci., 2018 32(2): 249-264 was observed the least decrease in the relative humidity content of the leaves. relative ion leakage percentage in all studied cultivars was increased by increasing salinity concentration. the increase in the relative ion leakage percentage was significant between the studied cultivars. the highest relative ion leakage percentage was observed in mousa abadi cultivar in salinity level d. after this cultivar, rezai zod res, jandaghi, sabz pesteh togh, fndoghi 48, kanjari and italiyayi cultivars had the highest relative ion leakage percentage. the increase in relative ion leakage percentage was not significant in ghazvini cultivar compared to the control plants (table 9). the results showed that the cultivars had a significant difference in cell membrane injury percentage. the highest cell membrane injury percentage was observed in the leaves of mousa abadi (54.83±4.50%), and the lowest cell membrane injury percentage was observed in the leaves of ghazvini (16.17±0.62%). results reported in table 10 assessed that with increasing salinity concentration in irrigation water, the sodium concentration in the leaves and roots of total cultivars increased. the increase in sodium concentration in the leaves of ghazvini cultivar was only significant when plants were treated with salinity level d, while in akbari, badami zarand and shahpasand cultivars was observed a significant increased when treated with salinity levels c and d, compared to the control plants. while the increase of sodium concentration in the leaves of other cultivars was significant different salinity levels b, c and d, compared to the control plants (table 10). the highest sodium concentration in leaves was observed in the salinity level d and in mousa abadi (2.09± 0.045%), rezaie zood res (2.05±0.030%), khanjari (2.03±0.115%) and jandaghi (1.90±0.035%) cultivars treated. also the highest sodium concentration in roots was also observed in salinity level d, and in mousa abadi (3.04±0.06%) and rezaie zood res (2.99±0.05%) cultivars. with increasing salinity levels (to 14.75 ds/m), potassium concentration increased in leaves and roots of akbari, ghazvini, shahpasand, badami zarand and ebrahimi cultivars while potassium content in the leaves and roots of other cultivars except mousa abadi and rezaie zood res increased to salinity level c. potassium content in the leaves and roots of mousa abadi and rezaie zood res cultivars was increased only in salinity level b. overall, the highest potassium content in leaves and roots was observed in salinity level c and in ghazvini (1.81±0.02%) and akbari (1.38±0.02%) cultivars. 4. discussion and conclusions based on the results of this study, with increasing salinity concentration in irrigation water, final height, trunk diameter and number of leaves in all studied cultivars decreased. plant height is heavily dependent on growth environment. since the growth phenomenon gained vital activities in which condition the plant must be in possession of enough water, reduction in the height occurs in case of failure to provide the required water due to the reduction of cell turgor pressure and length of the cells would be negatively affected (munns, 2002; munns and tester, 2008). the osmotic effects of salinity stress can be observed immediately after salt application and are believed to continue for the duration of exposure, resulting in inhibited cell expansion and cell division (munns 2002; munns and tester, 2008). in this research, trunk diameter and its growth were decreased during the application of salinity stress in all cultivars. these results are consistent with other results (sepaskhah and maftoun, 1988; munns and tester, 2008; zrig et al., 2015). it has been reported that growth rates of pistachio trees decrease with increasing sodium chloride (nacl) concentration in soil. it has been also reported that there is a positive correlation between sodium (na+) as well as chloride (cl-) concentration in plant tissue and soil (sepaskhah and maftoun, 1988; munns and tester, 2008; zrig et al., 2015). based on the results of this study, number of leaves with increasing salinity concentrations reduced. our results are consistent with studies reporting that increasing salinity levels negatively affect morphology and number of leaves in pistachio trees (picchioni and myamoto, 1990; saadatmand et al., 2007; karimi et al., 2011). the results of this research showed that with increasing salinity, percentage of green leaves, leaves and shoots fresh and dry weights in all cultivars decreased but the percentage of necrotic leaves and percentage of downfall leaves increades. the cultivars showed different responses to salinity levels. these results are consistent with the results of karimi et al. (2009 and 2011). in these studies, effect of salinity levels on pistachio cultivars was investigated and was reported that pistachio cultivars showed different responses to salinity levels. although pistachio trees are classified as tolerant to salinity, but amount of their tolerance to salinity is differently (sepaskhah and maftoun, 1988; momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 261 table 10 effect of interaction between salinity and cultivar on root and leaf k+ and na+ contents means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments root na+ (%) leaf na+ (%) root k+ (%) leaf k+ (%) khanjari control 0.55±0.03 s-y 0.43±0.027 q-v 0.60±0.03 z-a/ 1.35±0.03 k-m a 0.59±0.01 q-y 0.46±0.023 q-v 0.76±0.05 p-w 1.44±0.11 d-i b 0.70±0.06 o-x 0.64±0.023 m-r 0.73±0.05 q-y 1.56±0.03 c-f c 1.57±0.11 g-i 1.25±0.055 g-h 0.50±0.04 b/ 1.30±0.06 i-o d 2.59±0.06 c-d 2.03±0.116 a 0.37±0.04 d/ 1.01±0.04 u-x akbari control 0.42±0.01 y 0.37±0.013 t-v 0.80±0.03 m-u 1.08±0.03 r-x a 0.44±0.008 x-y 0.39±0.005 t-v 0.99±0.03 f-i 1.21±0.05 m-r b 0.48±0.03 v-y 0.42±0.007 r-v 1.37±0.03 a 1.58±0.04 c-e c 0.97±0.03 l-n 0.73±0.035 k-n 1.38±0.02 a 1.59±0.02 b-d d 1.86±0.04 f 1.40±0.066 e-g 0.77±0.02 o-v 1.11±0.03 q-v ghazvini control 0.46±0.003 w-y 0.41±0.014 s-v 0.79±0.02 n-v 1.31±0.02 i-n a 0.47±0.004 w-y 0.41±0.007 s-v 0.84±0.02 k-r 1.48±0.02 d-h b 0.49±0.006 v-y 0.43±0.004 q-v 0.95±0.02 g-k 1.55±0.03 c-g c 0.52±0.006 u-y 0.46±0.007 q-v 1.08±0.02 d-f 1.81±0.02 a d 1.05±0.02 k-m 0.82±0.023 k-m 0.85±0.02 j-q 1.35±0.02 h-m italiaie control 0.57±0.02 r-y 0.34±0.006 v 0.93±0.02 h-l 1.08±0.02 r-x a 0.61±0.01 q-y 0.36±0.007 t-v 0.98±0.03 f-i 1.19±0.02 n-t b 0.78±0.03 n-t 0.43±0.007 q-v 1.13±0.02 cd 1.23±0.02 m-p c 1.21±0.02 j-k 0.82±0.027 k-m 0.88±0.02 i-p 0.99±0.02 v-x d 1.81±0.04 f 1.55±0.035 c-e 0.60±0.02 z-a/ 0.67±0.02 a/ kaleh ghochi control 0.67±0.03 p-y 0.45±0.005 q-v 0.89±0.02 h-o 1.10±0.02 r-w a 0.70±0.02 o-x 0.47±0.003 p-v 0.95±0.02 g-k 1.18±0.04 n-t b 0.75±0.009 n-u 0.49± 0.003 p-v 1.14±0.02 b-d 1.57±0.02 c-e c 1.38±0.02 i-j 0.76±0.029 k-n 1.11±0.02 de 1.55±0.02 c-g d 2.55±0.04 d 1.50±0.044 d-f 0.82±0.02 l-t 1.17±0.02 n-t jandaghi control 0.49±0.006 u-y 0.41±0.005 s-v 0.68±0.02 u-z 0.82±0.02 y-z a 0.60±0.005 q-y 0.46±0.004 q-v 0.72±0.03 r-z 0.93±0.02 x-y b 0.82±0.01 m-q 0.57±0.017 n-u 0.75±0.05 q-x 1.08±0.02 r-w c 1.52±0.02 g-i 1.22±0.035 g-h 0.69±0.02 u-z 0.71±0.02 z-a/ d 2.73±0.03 cd 1.90±0.035 a-b 0.35±0.03 d/ 0.46±0.02 b/ mousa abadi control 0.53±0.007 t-y 0.44±0.002 q-v 0.60/±0.01 z-a 1.05±0.02 s-x a 0.65±0.01 q-y 0.47±0.005 p-v 0.85±0.02 j-q 1.13±0.02 p-v b 0.83±0.03 m-p 0.71±0.034 l-o 0.63±0.02 a/ 1.07±0.02 r-x c 1.76±0.02 f-g 1.33±0.027 f-h 0.45±0.02 c/ 0.95±0.02 w-y d 3.04±0.06 a 2.09±0.045 a 0.34±0.01 d/ 0.40±0.02 c/ ebrahimi control 0.46±0.005 w-y 0.39±0.005 t-v 0.68±0.02 u-z 1.26±0.03 k-q a 0.48±0.005 v-y 0.41±0.004 s-v 0.77±0.02 o-v 1.37±0.02 h-l b 0.53±0.007 t-y 0.45±0.006 q-v 0.82±0.02 l-t 1.48±0.02 d-h c 1.14±0.04 k-l 0.87±0.020 j-l 0.83±0.02 k-s 1.50±0.02 d-h d 2.11±0.03 e 1.62±0.027 c-d 0.60±0.02 z-a/ 1.15±0.01 o-u badami zarand control 0.51±0.007 u-y 0.48±0.005 p-v 0.71±0.02 s-z 1.04±0.01 t-x a 0.53±0.003 t-y 0.49±0.004 p-v 0.74±0.02 q-y 1.16±0.02 n-u b 0.56±0.008 r-y 0.51±0.019 o-v 0.97±0.02 f-j 1.43±0.01 e-j c 0.83±0.02 m-q 0.64±0.027 m-r 1.01±0.01 e-h 1.47±0.01 d-h d 1.73±0.04 f-g 1.23±0.027 g-h 0.70±0.02 t-z 1.05±0.01 s-x fandoghi 48 control 0.60±0.006 q-y 0.42±0.002 r-v 0.82±0.02 l-t 1.19±0.02 n-t a 0.64±0.008 q-y 0.44±0.005 q-v 0.91±0.02 h-n 1.28±0.03 j-p b 0.99±0.01 l-n 0.79±1.21 k-m 0.92±0.02 h-m 1.35±0.10 h-m c 1.43±0.02 h-j 0.93±0.01 j-k 0.90±0.02 h-n 1.30±0.02 i-o d 2.74±0.04 c-d 1.73±0.04 b-c 0.64±0.02 x-z 1.01±0.02 u-x sabs pesteh togh control 0.60±0.007 q-y 0.48±0.005 p-v 0.62±0.09 y-z 1.07±0.02 r-x a 0.64±0.01 q-y 0.51±0.005 o-v 0.88±0.01 i-p 1.28±0.02 j-p b 0.79±0.04 n-s 0.57±0.02 n-u 1.01±0.02 e-h 1.44±0.02 d-i c 1.55±0.03 g-i 1.02±0.05 i-j 0.67±0.009 v-z 1.19±0.01 n-t d 2.81±0.07 b-c 1.83±0.06 b 0.44±0.01 c/ 0.83±0.01 y-z ahmad aghaee control 0.62±0.006 q-y 0.55±0.004 n-v 0.95±0.02 g-k 1.37±0.02 h-l a 0.65±0.006 q-y 0.58±0.005 n-t 1.08±0.02 d-f 1.54±0.03 c-g b 0.81±0.02 n-p 0.63±0.02 m-s 1.39±0.02 a 1.65±0.03 b-c c 1.64±0.02 f-h 1.17±0.02 h-i 1.35±0.01 a 1.50±0.05 d-h d 2.60±0.03 c-d 1.83±0.04 b 0.64±0.01 w-z 1.17±0.02 n-t rezaie zodres control 0.55±1.41 s-y 0.42±0.005 r-v 0.82±0.02 l-t 1.22±0.02 l-r a 0.60±0.008 q-y 0.45±0.005 q-v 1.08±0.02 d-f 1.41±0.02 f-k b 0.93±0.02 l-o 0.68±0.03 l-p 0.85±0.01 j-q 1.21±0.02 m-r c 1.74±0.03 f-g 1.31±0.02 f-h 0.62±0.03 y-z 0.98±0.03 v-x d 2.99±0.05 a-b 2.05±0.03 a 0.39±0.02 d/ 0.64±0.02 a/ shahpasand control 0.43±0.004 y 0.35±0.005 u-v 0.97±0.02 f-j 1.40±0.03 g-k a 0.45±0.003 x-y 0.36±0.004 t-v 1.05±0.01 d-g 1.48±0.02 d-h b 0.48±0.006 v-y 0.38±0.006 t-v 1.23±0.01 b-c 1.68±0.03 a-c c 0.91±0.02 l-p 0.65±0.02 m-q 1.24±0.02 b 1.72±0.03 a-b d 1.76±0.02 f-g 1.28±0.07 g-h 0.90±0.03 h-n 1.37±0.03 h-l 262 adv. hort. sci., 2018 32(2): 249-264 munns and tester, 2008). based on the results of this study, fv/fm ratio was 0.83±1 in the leaves of the control plants indicating the existence of ideal and non-stressed environmental conditions for the growth of all cultivars throughout the experimental period. in many plant species, when fv/fm ratio is about 0.83, it means that stress hasn’t been introduced to the plant and, lower levels indicate stress condition in plants (maxwell and johnson, 2000). regarding changes in fv/fm values the stress intensity in rezaie zood res and mousa abadi cultivars were more severe than other cultivars (0.59±0.015 and 0.61±0.09, respectively). on the contrary, gazvini and akbari cultivars were less damaged (0.76±0.003 and 0.75±0.007, respectively). these results are consistent with the results of (herda et al., 1999; starck et al., 2000; deell and toivonen, 2003; kodad et al., 2010). it has been reported that salinity stress is one of the most important environmental factors limiting photosynthesis. symptoms of salinity stress are expressed at both stomatal and non-stomatal levels. at stomatal level, the plant closes its stomata to prevent injuries (maxwell and johnson, 2000, ranjbarfordoei et al., 2006). as a result, net photosynthesis is unavoidably reduced due to a decrease in co2 availability, which potentially damages the photosynthetic apparatus (lawlor and cornic, 2002). most of the decrease in photon flux energy used for photochemistry can be explained as an increase in non-photochemical dissipation of excitation energy (lawlor and cornic, 2002). the results of this research indicated that under salinity stress amount of chlorophyll b was reduced more than amount of chlorophyll a. these results are consistent with the results of dejampour et al. (2012). these researchers investigated the effect of nacl on the amount of chlorophyll a, b and total chlorophyll in some of the prunus genus, and they reported that amount of chlorophyll b and total chlorophyll significantly decreased under salinity stress. however, reduction in amount of chlorophyll a in these plants was not significant. also, total chlorophyll content was decreased significantly in all studied cultivars with increasing salinity that are consistent with the results of karimi et al. (2009 and 2011). researcher reported that salinity stress leads to reduction chlorophyll content and photosynthesis capacity in plants which are the major reasons of decreases growth and yield in plants (levitt, 1980; munns, 2002; munns and tester, 2008). the results showed that content of relative humidity were decreased significantly as the salinity increased. the highest reduction in relative humidity content was observed in leaves mousa abadi, rezaie zood res and sabz pesteh togh cultivars under salinity level of 19.8 ds/m. the results are consistent with the data reported by shibli et al. (2000) and massai et al. (2004). salinity, through the gradual accumulation of sodium ions, reduces the relative water content and osmotic potential of the leaf in full turgor state. relative ion leakage percentage and cell membrane injury percentage in all studied cultivars were increased by increasing salinity concentration. the highest relative ion leakage percentage and cell membrane injury percentage were observed in mousa abadi cultivar under treatment 19.8 ds/m. these results are consistent with the results of other studies. it has been reported that using a relative ionic leak test is one way to find out the extent to which cell membranes are damaged. recording the relative ion leakage rate allow for tissue damage estimation. this method was used for the first time by dexter et al. (1930 and 1932) to investigate the resistance to cold in plants and, over time, was used to measure cell membrane damage in relation to other environmental stresses, including salinity stress (chen et al., 1999). with increasing salinity concentration in irrigation water, the sodium concentration in the leaves and roots of total cultivars studied increased. the highest sodium concentration in leaves and roots were observed in salinity level 19.8 ds/m and in mousa abadi and rezaie zood res cultivars which had the highest percentage of leaves necrosis and loss, and at the end of the experiment, only 52.47±4.98% and 53.48±4.82% of leaves were greens. in researches on various plants under salt stress, it has been reported that the loss of water availability, toxicity of na+ and ion imbalance leads to growth limitation in plants (mahajan and tuteja, 2005; szczerba et al., 2009). it is repeatedly reported that k+ deficiency and na+ toxicity are major restrictors of crop production worldwide (mahajan and tuteja, 2005; szczerba et al., 2008, 2009). the results indicated that the type of cultivar is effective in potassium absorption and its transmission to the aerial part. in this research, ghazvini and akbari cultivars with increasing the amount of potassium in its leaves and roots could reduce the negative and destructive effects of sodium better than other cultivars. potassium plays an important role in vital metabolites in salinity stress conditions, so that the k+ can counteract na+ stresses, thus the potential of plants to tolerate salinity is strongly dependent on their potassium nutrition 263 (aleman et al., 2011; nieves et al., 2016). generally, the results of this study showed that by applying salinity stress and increasing its concentration, growth indices including branch height, branch diameter, number of total leaves, percentage of green leaves, fresh and dry weight of leaves, shoots and roots, relative humidity content, chlorophyll a, chlorophyll b and total chlorophyll content, have been reduced in the all cultivars studied. but the percentage of necrotic leaves, percentage of downfall leaves, relative ionic percentage and cell membrane injury percentage were increased. however, the reduction and increase of measured traits were significantly different among studied cultivars. the results also showed that salinity stress affected the young trees through increasing the amount of minimum fluorescence (f0) and decreasing the maximum fluorescence (fm) and reducing variable fluorescence (fv) as well as fv/fm ratio from 0.83±1 in the control plants to 0.59±0.015 in rezaie zood res and 0.61±0.009 in mousa abadi cultivar. based on the results mentioned above, reducing fv/fm ratio was symptoms of the damaging stress in plants. the results of method chlorophyll fluorescence in this research are consistent with the results of morphological and physiological traits and therefore, it can be said that chlorophyll fluorescence technique (fv/fm indicator) is a rapid, sensitive and non-destructive method to check the intensity of stress that induced to plants. overall, the result showed that type of cultivar and level of salinity was affected on concentration of na+ and k+ in leaves and roots. ghazvini cultivar was recognized as the most tolerant cultivar to salinity. this cultivar could tolerate salinity 14.75 ds/m. after this cultivar, akbari, badami zarand and shahpasand cultivars had more tolerance to salinity, respectively. in contrast, rezaie zood res and mousa abadi cultivars were recognized as the most sensitive cultivars to salinity stress. after these cultivars, khanjari, jandaghi and fndoghi 48 cultivars had more sensitive to salinity. references alemán f., nieves-cordones m., martínez v., rubio f., 2011 root k+ acquisition in plants: the arabidopsis thaliana model. plant cell physiol., 52(9): 1603-1612. arnon d.i., 1949 copper enzymes in isolated chloroplast polyphenol oxidase in beta vulgaris. plant physiol., 24: 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under sun and shade conditions. j. plant sci. res., 2(1): 1-10. adv. hort. sci., 2018 32(2): 249-264 81 1. introduction due to consumer demand for high-convenience foods, fresh-cut peppers may represent an interesting product to add to the existing fresh-cut products. the quality of a fresh-cut product is generally affected by pre-harvest and post-harvest factors, including processing. genotype, growing conditions, cultural practices, and maturity stage at harvest, from a pre-harvest point of view, may greatly influence initial quality of the product to be processed; on the other hand, postharvest handling and storage of raw materials, and processing conditions, including finished product fate throughout the distribution chain, markedly determine its final quality. maturity stage is an important factor conditioning final quality and processability of fresh-cut products, particularly fruits. less mature fruits, in fact, are more suitable for processing due to their greater firmness, compared to more mature fruits, but this can result in lower sensorial quality as observed in melons (watada and qi, 1999) and mangoes (bender et al., 2000). in peppers, the maturity stage was found to have an impact on flavonoids, carotenoids, and ascorbic acid concentration (marin et al., 2004; fox et al., 2005), with carotenoids and ascorbic acid increasing, and flavonoids decreasing, as maturity proceeds, while deepa et al. (2007) observed an increase in the main antioxidant compounds, including phenols ascorbic acid, capsicin, and carotenoids for 10 different genotypes. moreover, in ‘domino’ bell peppers, an increase in firmness was observed with the increasing of fruit size, occurring with ripening, most probably due to the increase of pericarp thickness (tadesse et al., 2002). the same authors reported an increase in soluble solids, and a decrease in respiration rate and ethylene production and suggested as maturity index at harvest for this cultivar firmness values of 35 n and a minimum of 6°brix. molinari et al. (1999) reported greater titrable acidity on full-ripe peppers ripened on the plant compared to those harvested at the color-break stage and ripened in storage. in addition, agricultural practices, soil, and climate conditions may differently affect quality attributes of fresh produce, including external attributes (kays, 1999), firmness (sams, 1999), and nutritional composition (lee and kader, 2000). in particular, nitrogen fertilization seems to decrease the concentration of vitamin c in many fruits and vegetables (lisiewska and kmiecik, 1996). all these factors can consequently affect quality of fresh-cut produce effect of type of fertilization and maturity on quality of fresh-cut red and yellow peppers (capsicum annuum l. ) f. piazzolla*, m.l. amodio*, r. rinaldi*, f. raimo**, g. colelli* * dipartimento di scienze delle produzioni e dell’innovazione dei sistemi agroalimentari mediterranei, università degli studi di foggia, via napoli, 25, 71121 foggia, italy. ** consiglio per la ricerca e sperimentazione in agricoltura, unità di ricerca per le colture alternative al tabacco, cra-cat, via p. vitiello, 108, 84018 scafati (sa), italy. key words: appearance, organic fertilization, phenolics, vitamin c. abstract: the aim of this work was to evaluate the effect of the type of fertilization (mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation) and stage of maturity at harvest (mature-green and full-colored) on post-cutting quality of red and yellow ‘cazzone’ peppers. peppers were cut into strips, and air-stored for 8 days at 5°c. during storage, color, appearance score, firmness, respiration rate, soluble solids, acidity, ph, vitamin c, total phenols, and antioxidant activity were measured. the maturity stage influenced color parameters and soluble solids, acidity and ph for both yellow and red types. full-colored peppers showed a lower respiration rate, and higher ssc than mature-green peppers; for the yellow type, a lower firmness value was observed for full-colored fruits compared to the mature-green ones. a lower antioxidant activity was also observed in the yellow type fertilized with the combined treatment, while phenol content in full-colored peppers was higher than in mature-green ones. fresh-cut yellow peppers showed higher susceptibility to decay compared to red types: after 8 days of storage, the appearance score in mineral fertilized full-colored yellow peppers dramatically decreased below the limit of marketability. the results of this experiment show that the type of fertilization and maturity stage can have varying impact on the quality of yellow and red peppers. adv. hort. sci., 2012 26(2): 81-87 received for publication 26 april 2012. accepted for publication 25 june 2012. 82 and their storability, but very few works have directly investigated the impact of pre-harvest factors on the quality of fresh-cut products, and none of them regard bell peppers. more works are available on the impact of processing, and among them artés-hernández et al. (2010) studied the effect of the cut type and of the modified atmosphere packaging on quality of bell peppers (cv. requena) observing that in general peppers cut in a ‘ring’ suffer greater weight loss than peppers cut in strips or dice, although respiration was not affected by the cutting mode and was not significantly different from the whole product. the objective of the present work was to evaluate the effect of the type of fertilization (mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation) and stage of maturity at harvest (mature-green and full-colored) on post-cutting quality of red and yellow ‘cazzone’ peppers. 2. materials and methods plant material the experiment was carried out in scafati (sa, italy, coordinates 40° 44’n, 14° 30’e, 10 m a.s.l.), on soil of sandy loam texture, basic ph and with organic and mineral content as reported in table 1. table 1 soil parameters soil parameters ph in water (1:2.5) 8.4 ec 25°c (1:2) (ds/m) 0.6 c (g/kg) 15.4 organic matter (g/kg) 26.6 n (g/kg) 1.3 c/n 11.6 assimilated p 2 o 5 (mg/kg) 85.0 exchangeable k (ppm) 917.1 exchangeable na (ppm) 251.8 exchangeable ca (ppm) 2090.9 exchangeable mg (ppm) 497 ecotypes of red and yellow ‘cazzone’ pepper (capsicum annuum l.) (density of 3.3 plants m-2) were subjected to two different tecniques of fertilization: mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation. mineral fertilization was in compliance with the campania region guidelines: 100 kg ha-1 of nitrogen were applied for 1/3 in pre-transplant and 2/3 during plant growth. for the combined fertilization, 20 t ha-1 of dry organic compost obtained from urban organic waste (characteristics reported in table 2) were applied in pre-transplant, and integrated with 50 kg ha-1 of mineral nitrogen during plant growth. transplanting was carried out on 25 may. pepper fruits were harvested on 15 september at two stages of maturity (mature-green and full-colored), and transported to the laboratory of postharvest technology at the university of foggia (italy). table 2 compost parameters compost parameters humidity (%) 31 ph 6-8 c (% dm) 28 humic and fulvic carbon 8 n (% dm) 2 c/n 14 cu (mg/kg dm) 110 zn (mg/kg dm) 250 salinity (meq/100 g) 21 experimental design and protocol for each ecotype/fertilization/stage of maturity combination, six lots (two replicates x three storage sampling) of 15 strips were individually placed in plastic trays closed in pet macroperforated bags, together with wet paper (to maintain high level of rh), and stored at 5°c for eight days. initially, and after four and eight days of storage at 5°c quality attributes (including color, appearance score, firmness, respiration rate, weight loss, soluble solids, titrable acidity, ph, vitamin c, phenols content, and antioxidant activity) were monitored. respiration rate and weight loss respiration rate (ml co 2 kg−1 hr−1) was measured using the static system, measuring the amount of co 2 accumulated in the headspace of sealed pvc containers (5 l). co 2 concentration, determined by a shimadzu gas chromatograph (model 17a) equipped with a tcd detector, was then referred to the weight of the sample, to the volume of the headspace, and to the elapsed time. samples were individually weighed and the weight loss was calculated as % of the initial fresh weight. physical analysis the colour of the strips was measured in two different points of the mesocarp, randomly selected, using a spectrophotometer (cm 2600d konica minolta, osaka, japan) in the reflectance mode using the cie l*a*b* colour scale. hue angle and saturation were then calculated. appearance score evaluation was subjectively assessed using a scale of 5 to 1 where 5= excellent, no defects; 4= very good, few defects; 3= good, moderate defects, limit of marketability; 2= poor, many defects; and 1= inedible. a score of 3 was considered as the limit of marketability and a score of 2 as the limit of edibility (amodio et al., 2007). firmness was measured on two points of the mesocarp, as resistance of the strips to a 2-mm penetration by a probe 83 of 6-mm diameter, using a digital penetometer (tierre s.r.l., torino). chemical analysis ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents were determined as described by zapata and dufour (1992), with some modifications. samples of 20 μl were analysed with an agilent 1200 series hplc (waldbronn, germany) equipped with a binary pump, an autosampler, and a photodiode array detector. separations of dhaa and aa were achieved on a zorbax eclipse xdb c18 column (150 mm × 4.6 mm; 5 μm of particle size; agilent technologies, santa clara, ca, usa). aa and dha contents were expressed as milligrams of ascorbic or dehydroascorbic acid per kilogram of fresh weight (mg kg−1). total phenols were determined according to the method of singleton and rossi (1965). the absorbance was read at 725 nm against a blank using a uv-1700 shimadzu spectrophotometer (jiangsu, china). the content of total phenols was calculated on the basis of the calibration curve of gallic acid, and was expressed as grams of gallic acid per kilogram of fresh weight (g ga kg−1). antioxidant assay was performed following the procedure described by brand-williams et al. (1995), with minor modifications. trolox was used as a standard and the antioxidant activity was reported in milligrams of trolox equivalents per kilogram of fresh weight (g te kg−1). four grams of fresh juice were used to determined total soluble solids (tss) (measured with a digital refractometer, atago pr32), ph and titratable acidity (ta). titratable acidity was determined with an automatic titrator (titromatic crison 1s), using the juice samples and titrating with 0.1 n naoh up to ph 8.1, and the value was expressed as percentage of citric acid. data analysis for mean data at harvest of peppers of both ecotypes, a two-way anova for stage of maturity and fertilization effects was run, while on the whole data set, a three-way anova was performed with stage of maturity, fertilization treatment and time of storage as factors. when interactions among factors were not significant, main effects were analyzed. mean separation among treatments at each time of storage was performed with the tukey test. (p< 0.05, n=10). 3. results and discussion most of the quality attributes were not affected by the type of fertilization, except respiration rate, a* value and ph, whereas, as expected, most of them were affected by the stage of maturity. the interaction between the type of fertilization and the stage of maturity was statistically significant for respiration rate and ph (table 3). pepper fruits treated with mineral fertilization showed a higher respiration rate (9.6 ml co 2 kg-1 hr-1) than fruits treated with the combined fertilization (8.8 ml co 2 kg-1 hr-1 ), a higher a* value (0.6 vs. -1.1) and ph (5.44 vs. 5.35) although the absolute ph difference was very little (table 4). table 4 illustrates the effect of stage of maturity on quality attributes of fresh-cut yellow peppers at harvest. in particular, the stage of maturity affected the respiration rate, l*, a*, and b* values, hue angle, chroma, soluble solids, and ph. full-colored peppers showed lower respiration rate than mature-green peppers (7.6 and 10.7 ml co 2 kg-1 hr-1 respectively), higher soluble solids (6.1 vs. 4.7°brix ) and lower ph (5.75 vs. 5.04). these findings confirmed that reported by tadesse et al. (2002) for ‘domino’ bell peppers, and mainly that soluble solids, respiration rate and firmness (other than color) evolve during fruit ripening, and may be used as maturity index at harvest. the increase of titratable acidity and decrease of ph during bell pepper growth and ripening has also been observed in several studies (fox et al., 2005), during plant ripening (molinari et al., 1999; serrano et al., 2010) and, in particular serrano et al. (2010) observed an increase of citric acid during ripening. for red ‘cazzone’ peppers, no differences were observed for the measured attributes according to the type of fertilization and interaction between the type of fertilization and the stage of maturity, whereas the stage of matutable 3 effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored), and their interaction on quality attributes of fresh-cut yellow ‘cazzone’ peppers at harvest quality attributes fertilization (f) stage of maturity (s) fxs firmness (n) ns ns ns respiration rate (ml co 2 kg-1 hr-1) * *** *** l* ns **** ns a* * **** ns b* ns **** ns hue angle (*) ns **** ns chroma ns **** ns soluble solids (°brix) ns *** ns titrable acidity (% citric acid) ns ns ns ph ** **** *** vitamin c (mg/100 g fw) ns ns ns ascorbic acid (mg/100 g fw) ns ns ns l-dehydroascorbic acid (mg/100 g) ns ns ns antioxidant activity (mg trolox/100 g fw) ns ns ns total phenol content (mg gallic acid/100 g fw) ns ns ns * when p≤ 0.05; ** when p≤ 0.01; *** when p≤ 0.001; **** when p≤ 0.0001; ns when not significant; n= 10. 84 rity affected several quality attributes (table 5). the respiration rate of full-colored peppers was about half that of peppers at a mature green stage; soluble solids increased by about 2°brix with ripening. in addition, an increase of acidity from 0.1% to 0.15% and a decrease of ph were also observed (5.93 and 4.93, respectively) (table 6). most of the differences observed at harvest were maintained during storage for yellow peppers. some differences among chemical constituents were found for yellow table 4 main effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers at harvest quality attributes type of fertilization stage of maturity mineral combined mature green full-colored firmness (n) 19.0 nss 18.3 nss 19.2 nss 18.1 nss respiration rate (ml co 2 kg-1 hr-1) 9.6 as s 8.8 b s 10.7 a s 7.6 b s l* 45.2 nss 46.1 nss 39.3 b s 52.1 a s a* 0.6 a ss -1.1 b s -13.2 b s 12.7 a s b* 37.7 nss 38.3 nss 26.4 b s 49.6 a s hue angle (*) 95.0 ns s 97.3 nss 116.6 a s 75.6 a s chroma 40.3 nss 40.5 nss 29.5 b s 51.3 a s soluble solids (°brix) 5.4 nss 5.5 nss 4.7 b s 61.0 a s titrable acidity (% citric acid) 0.16 ns 0.14 ns 0.14 ns 0.15 ns ph 5.44 a s 5.36 bs 5.75 a s 5.04 bs vitamin c (mg/100 g fw) 59.4 nss 58.8 nss 61.4 nss 56.8 nss ascorbic acid (mg/100 g fw) 58.6 nss 58.0 ns s 60.4 nss 56.2 nss l-dehydroascorbic acid (mg/100 g) 0.8 nss 0.8 nss 1.0 nss 0.7 nss antioxidant activity (mg trolox/100 g fw) 169.8 nss 155.0 nss 153.7 nss 171.1 nss total phenol content (mg gallic acid/100 g fw) 114.1 nss 119.5 nss 108.0 nss 125.5 nss for each row, mean values followed by a different letter are significantly different (n=10 and p≤0.05). table 5 effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored), and their interaction on quality attributes of fresh-cut red ‘cazzone’ peppers at harvest quality attributes fertilization (f) stage of maturity (s) fxs firmness (n) ns ns ns respiration rate (ml co 2 kg-1 hr-1) ns ** ns l* ns **** ns a* ns **** ns b* ns *** ns hue angle (*) ns **** ns chroma ns ** ns soluble solids (°brix) ns *** ns titrable acidity (% citric acid) ns *** ns ph ns *** ns vitamin c (mg/100 g fw) ns ns ns ascorbic acid (mg/100 g fw) ns ns ns l-dehydroascorbic acid (mg/100 g) ns * ns antioxidant activity (mg trolox/100 g fw) ns ns ns total phenol content (mg gallic acid/100 g fw) ns ns ns * when p≤ 0.05; ** when p≤ 0.01; *** when p≤ 0.001; **** when p≤ 0.0001; ns when not significant; n= 10. table 6 main effect of stage of maturity (mature-green and fullcolored) on mean values of quality attributes of fresh-cut red ‘cazzone’ peppers at harvest quality attributes mature green full-colored firmness (n) 18.9 ns 18.9 ns respiration rate (ml co 2 kg-1 hr-1) 9.8 a 4.9 b l* 40.2 b 31.8 b a* -13.1 b 32.4 a b* 27.0 b 16.6 b hue angle (*) 115.9 a 27.2 b chroma 30.0 b 36.4 a soluble solids (°brix) 4.1 b 6.3 a titrable acidity (% citric acid) 0.10 b 0.16 a ph 5.93 a 4.93 b vitamin c (mg/100 g fw) 55.0 ns 52.0 ns ascorbic acid (mg/100 g fw) 53.6 ns 51.4 ns l-dehydroascorbic acid (mg/100 g) 1.4 a 0.6 b antioxidant activity (mg trolox/100 g fw) 149.1 ns 163.9 ns total phenol content (mg gallic acid/100 g fw) 107.2 ns 123.2 ns for each row mean values followed by a different letter are significantly different (n=10; p≤0.05). 85 ‘cazzone’ peppers, depending on the fertilization treatment. for this last ecotype, table 7, the main effect of the fertilization treatment over storage on quality attributes is shown. in particular, a lower antioxidant activity was observed for peppers fertilized with the combined treatment (139 mg trolox/100g fw) compared to mineral fertilization (157 mg trolox/100g fw), whereas phenol content was positively affected by the stage of maturity, showing a value 10% higher for full-colored peppers compared to mature green (table 7). the higher phenol content of fullcoloured peppers compared to mature green ones confirms that phenols and antioxidant compounds increase during ripening, as found by marin et al. (2004) and deepa et al. (2007). in addition, a higher susceptibility to decay of fresh-cut yellow peppers compared to red type was observed. as shown in figure 1, after eight days of storage the appearance score in mineral-fertilized full-colored yellow peppers dramatically decreased below the limit of marketability (score 3) due to the presence of decay and most likely to the high respiration rate, even if it was found not significant among treatments (fig. 1). for red peppers, no differences related to the fertilization treatment were observed during storage; on the other hand, the stage of maturity had effects on many attributes of fresh-cut red peppers after storage (table 8). in particular, the mature-green peppers showed a higher respiration rate (8.6 ml co 2 /kg/h) than full-colored peppers (5.9 ml co 2 /kg/h), probably causing the greater weight loss (0.9% and 0.5%, respectively). full-colored red peppers accumulated 2.4°brix with respect to the mature green fruits, also showing an increase of titrable acidity and lower ph (0.20% citric acid vs. 0.11%) which resulted in a lower ph (4.72 vs. 5.80). table 7 main effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers during storage quality attributes type of fertilization stage of maturity mineral combined mature green full-colored firmness (n) 16.5 ns 17.0 ns 15.9 ns 17.5 ns respiration rate (ml co 2 kg-1 hr-1) 9.7 ns 7.8 ns 9.2 ns 8.3 ns l* 44.6 ns 45.0 ns 39.0 b 50.6 a a* 0.4 a -0.8 b -12.6 b 12.2 a b* 37.5 ns 37.7 ns 26.4 b 48.8 a hue angle (*) 95.0 ns 96.6 a 115.6 a 76.0 b chroma 39.8 ns 39.8 ns 29.3 b 50.3 a appearence score 4.0 b 4.2 a 4.1 ns 4.0 ns soluble solids (°brix) 5.9 ns 5.8 ns 4.8 b 6.9 a titrable acidity (% citric acid) 0.15 ns 0.14 ns 0.11 b 0.18 a ph 5.26 ns 5.26 ns 5.66 a 4.86 b weight loss (%) 0.8 ns 0.7 ns 0.9 ns 0.6 ns vitamin c (mg/100 g fw) 60.9 ns 61.8 ns 58.5 ns 64.2 ns ascorbic acid (mg/100 g fw) 58.6 ns 59.3 ns 55.9 ns 62.1 ns l-dehydroascorbic acid (mg/100 g) 2.3 ns 2.5 ns 2.6 ns 2.2 ns antioxidant activity (mg trolox/100 g fw) 157.0 a 139.0 b 141.7 ns 154.3 ns total phenol content (mg gallic acid/100 g fw) 111.4 ns 106.8 ns 103.6 b 114.6 a for each row mean values followed by a different letter are significantly different (n=10; p ≤0.05). table 8 main effect of type stage of maturity (mature-green and fullcolored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers during storage quality attributes mature green full-colored firmness (n) 16.5 ns 17.2 ns respiration rate (ml co 2 kg-1 hr-1) 8.6 a 5.9 b l* 39.7 a 30.9 a a* -12.6 b 31.4 a b* 26.6 a 16.3 b hue angle (*) 115.3 a 27.4 b chroma 29.5 b 35.4 a appearance score 4.2 ns 4.2 ns soluble solids (°brix) 4.4 b 6.8 a titrable acidity (% citric acid) 0.11 b 0.20 a ph 5.80 a 4.72 b weight loss (%) 0.9 a 0.5 b vitamin c (mg/100 g fw) 54.5 ns 58.8 ns ascorbic acid (mg/100 g fw) 51.8 ns 56.3 ns l-dehydroascorbic acid (mg/100 g) 2.7 ns 2.5 ns antioxidant activity (mg trolox/100 g fw) 136.6 ns 147.9 ns total phenol content (mg gallic acid/100 g fw) 96.1 ns 107.2 ns for each row mean values followed by a different letter are significantly different (n=10; p ≤0.05). 86 fig. 1 effect of storage at 5°c on appearance score, respiration rate and firmness of fresh-cut yellow and red ‘cazzone’ peppers (m=mineral fertilization; c=combined fertilization). at each storage evaluation different letters indicate significant differences among treatments (n=10; p≤0.05). 87 all red peppers received a score higher than 3 at the end of storage, without differences among treatments, whereas a lower firmness was observed for peppers fertilized with the combined treatment (fig. 1). particularly, after four days of storage, the mature-green peppers fertilized with the mineral system showed greater firmness (19.80 n) than mature-green peppers fertilized with the combined system (12.09 n), while intermediate results were observed for full-colored peppers. at eight days of storage, full-colored and mature-green peppers treated with mineral fertilization showed greater firmness than peppers fertilized with the combined treatment. for yellow peppers, no significant differences in firmness were observed. in conclusion, the results of this experiment show that the type of fertilization and the maturity stage had a different impact on quality of yellow and red peppers. in particular, for yellow full-colored peppers, the combined fertilization treatment allowed a longer shelf-life than the mineral treatment, and this should be considered when processing fresh-cut peppers. these results may also encourage further study of the feasibility of using ‘environmentally friendly’ fertilization techniques on bell peppers and eventually to extend these trials to other species. acknowledgements we thank consiglio per la ricerca e sperimentazione in agricoltura, unità di ricerca per le colture alternative al tabacco (cra-cat) for supplying the peppers. references amodio m.l., cabezas-serrano a.b., rinaldi r., colelli g., 2007 implementation of rating scales for visual quality evaluation of various vegetable crops. in: kader a.a., and m. cantwell (eds.) produce quality rating scales and color charts. postharvest horticulture series, no. 23. university of california, davis, ca, usa, pp. 151. artés-hernández f., conesa a., artés f., 2010 minimally fresh processed pepper under different kind of cuts. acta horticulturae, 857: 25-30. bender r.j., brecht j.k., baldwin e.a., maludo t.m.m., 2000 aroma volatiles of mature-green and treeripe ‘tommy atkins’ mangoes after controlled atmosphere versus air storage. hortsci., 35(4): 684-686. brand-williams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. food sci. and technol., 28(1): 25-30. deepa n., charanjit k., binoy g., balraj s., kapoor h.c., 2007 antioxidant constituents in some sweet pepper (capsicum annuum l.) genotypes during maturity. food sci. and technol., 40(1): 121-129. fox a.j, del pozo-insfron d., lee j.h., sargent s.a., talcott s.t., 2005. ripening-induced chemical and antioxidant changes in bell peppers as affected by harvest maturity and postharvest ethylene exposure. hortsci., 40(3): 732-735. kays s.j., 1999 preharvest factors affecting appearance. postharv. biol. and technol., 15: 233-247. lee s.k., kader a.a., 2000 preharvest and postharvest factors influencing vitamin c content of horticultural crops. postharv. biol. and technol., 20: 207-220. lisiewska z., kmiecik w., 1996 effect of level of nitrogen fertilizer, processing conditions and period of storage for frozen broccoli and cauliflower on vitamin c retention. food chem., 57: 267-270. marin a., ferreres f., tomàs-barberan f.a., gil m.i., 2004. characterization and quantification of antioxidant constituents of sweet peppers (capsicum annuum l.). j. agric. food chem., 52: 3861-3869. molinari a.f., de castro l.r., antoniali s., pornchaloempong p., fox a.j., sargent s.a., lamb e.m., 1999 the potential for bell pepper harvest prior to full color development. proc. fla. state hort. soc., 112: 143-146. sams c.e., 1999 preharvest factors affecting postharvest texture. postharv. biol. and technol., 15(3): 249-254. serrano m., zapata p.j., castillo s., guillén f., martínez-romero d., valero d., 2010 antioxidant and nutritive constituents during sweet pepper development and ripening are enhanced by nitrophenolate treatments. food chemistry, 118(3): 497-503. singleton v.l., rossi j.a., 1965. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic., 16(3): 144-158. tadesse t., hewett e.w., nichols m.a., fisher k.j., 2002 changes in physicochemical attributes of sweet pepper cv. domino during fruit growth and development. sci. hort., 93(2): 91-103. watada a.e., qi l., 1999 quality of fresh-cut produce. postharv. biol. and technol., 15: 201-205. zapata s., dufour j.f., 1992 ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse phase ion interaction hplc. j. food sci., 57(2): 506-511. 143 1. introduction summer pruning is a cultural techniques which drives vine vigour to ensure fruit quality and plant vegetative balance. while summer pruning is the most expensive cultural operation 44.2% of total management costs (crescimanno et al., 2011) it helps to improve the microclimate in the canopy, promotes good ripening of the grapes and creates less suitable conditions for the development of pathogens. good results depend on the vegetative-productive behaviour of the vineyard, intensity and age of cultural operation (crescimanno et al., 1986). summer pruning defines the final productivity of plants by modifying the number of shoots per plant with shoot thinning, the number of clusters per shoot with cluster thinning, and the number of berries per bunch with berry thinning. other summer operations include leaf removal, shoot trimming and girdling (di lorenzo, 2003). general indications about summer pruning techniques to enhance quality of production are very difficult to formulate because cultivar behaviour, vigour of the vineyard and environmental conditions must all be considered. 2. leaf removal leaf removal causes a reduction of vine leaf area. if it occurs at or before bloom, it may cause berry drop, a reduction in fruit set or a reduction in bud fertility in the following season (candolfi-vasconceloset and koblet, 1990). the intensity of leaf removal should be based on canopy density and light penetration into the fruit zone. the removal of basal leaves around the clusters is widely adopted to improve grape quality and to reduce the incidence of fungal infection (gubler and marois, 1987; caspari et al., 1998). leaf removal should be performed near berry set or after fruit softening (dokoozlian et al., 2000 a). the leaves immediately above the cluster are the main source for photosynthates translocated to the cluster, particularly during the early stages of its development (hunter and visser, 1988). also at pea-size stage the loss of basal leaves increases fruit abscission, reduces berry size and decreases bud fertility; it has no effect when applied at veraison (caspari et al., 1998). after berry setting, usually all primary leaves and lateral shoots beginning from the base of the shoot to the node opposite the top cluster on each shoot are removed. elimination of apparently superfluous sinks, such as lateral shoots, reduces canopy density and °brix, but it has minor impact on ta and ph (reynolds and wardle, 1989; barbagallo et al., 2007 a). the leaves left on the vines after defoliation increase photosynthetic activity to recover the reduction on total leaf area activity and to supply the photoassimilates demand of sinks (poni et al., 2006; scafidi et al., 2010). on the other hand, candolfi-vasconcelos and co-workers (1994) found that defoliated plants had similar or even slightly lower photosynthetic rates compared to control plants, not only during the stress period but also in the following season. a photosynthesis response to leaf removal may be apparent only if the source-sink ratio is sufficiently limited. under conditions of source deficiency due to leaf removal in the fruit zone, plants promote the activity of apical meristems to replace the missing leaf area (barbagallo et al., 2007 b). basal leaves should not be removed before veraison, especially in varieties susceptible to heat damage or sunburn like ‘red globe’, ‘thompson seedless’. during fruit ripening leaves opposite the clusters have limited importance compared to the younger leaves at the top of the canopy (candolfi-vasconcelos et al., 1994; hunter et al., 1995). younger leaves show a higher transpisummer pruning in table grape r. di lorenzo, c. gambino, p. scafidi dipartimento demetra, università degli studi di palermo, viale delle scienze, 11, 90128 palermo, italy. key words: girdling, leaf removal, thinning, trimming, vitis vinifera l. abstract: this paper reviews cultural practices to improve fruit quality in table grape during vegetative and reproductive seasons. summer pruning in table grape (vitis vinifera l.) has more effects than winter pruning, above all with regard to plant productivity and final number of bunches for harvesting. thinning is one of the most cultural technique and it consists in the elimination of vegetative or reproductive organs in excess. other summer canopy management techniques include leaf removal, fruit shoots positioning, shoot trimming and girdling. adv. hort. sci., 2011 25(3): 143-150 received for publication 9 june 2011 accepted for publication 8 september 2011 144 ration rate, but also higher water use efficiency than those opposite the clusters (candolfi-vasconcelos et al., 1994). some weeks before harvest random defoliation is usually undertaken to fully develop the colour of white, red and some black grape varieties. 3. thinning thinning consists in the elimination of vegetative or reproductive organs in excess. it is very rarely performed before bloom since negative climatic events can lead to the loss of many shoots or irregular fruit set; in some areas and for some cultivars thinning performed before bloom can lead to excessive fruit set and tight bunches. shoot thinning shoot thinning is the elimination of double, weaker and sterile shoots and it is very important to aerate the canopy, improve the growth of remaining shoots and adjust cluster numbers. there may be an advantage with shoot thinning in vigorous vines to reduce shoot crowding and thus increase light exposure of the remaining shoots. shoot thinning should be performed when shoot length reaches 25-30 cm. (dokoozlian et al., 2000 b) when it is possible to define which shoots have bunches in good position and which are well located as pruning material for the next year. on spur-pruned vines two shoots per spur are retained and latent shoots are removed from older wood, arms and cordons, while cane-pruned vines are sometimes shoot thinned, especially when several canes are wrapped together on a single wire. cluster thinning cluster thinning is usually performed after fruit set in order to adjust the crop load, distribute clusters evenly on the vine and canes, select the best clusters (shape, size and position) and eliminate those that are misshaped and weak (figs. 1 and 2). generally the aim is to have an equal number of cluster and shoots on the plant, leaving two clusters on the distal shoots. the number of flowers per inflorescence, berry per cluster and cluster weight (table 1) is positively affected by the node position long the cane (sottile et al., 1996). a cluster/shoot ratio of less than 0.8 usually determines a reduction in terms of yield (tables 2 and 3) without any significant improvement in terms of quality (di lorenzo, 2003). several studies demonstrated that crop removal significantly increases soluble solids (fig. 3) and berry colour (dokoozlian et al., 1995). in a trial conducted on ‘flame seedless’ in fresno california, berry weight, size and fruit composition varied little among vines thinned one week prior to bloom and those thinned four weeks following fruit set (dokoozlian et al., 1995). berry thinning berry thinning is a widely performed technique and involves the removal of a few berries from the cluster (di lorenzo, 2003). this operation is necessary to decrease the compactness of bunches and to give them a more attractive shape with large, uniform-size berries (fig. 4 table 4). berry thinning is performed when berries are at pea-size in order to give more uniform clusters in terms of weight and shape, satisfying packaging and marketing needs. in some cases for some cultivars, the partial removal of inflorescence or flowers with small scissors or small combs is performed in order to avoid an excessive clusters weight and/or closeness (di lorenzo, 2003). how the berries are removed depends on the cultivar. the best results are obfig. 1 effect of cluster thinning on evolution of berry weight (dotted line) and volume (continuous line) in treated (square) and control (triangle) plants. fig. 2 cluster thinning. table 1 influence of node position on number of flowers per inflorescence, number of berries per cluster, and berry and cluster weight (sottile et al., 1996) node position flower (no.) berry (no.) berry weight (g) cluster weight (g) 1-4 258 101 7.0 779 5-9 517 128 7.3 1015 10-12 744 148 6.9 1090 145 tained with the “helicoidal” method, which consists of eliminating shoulders arranged in a spiral around the axis of the rachis. another method is the “fish spine” system where two parallel cuts are made on each side of the axis of the rachis, but without injuring it. the resulting bunch is very flat, but when the berries grow, the respective ramification occupies the space around the rachis. in ‘thompson seedless’ the most common method is to clip the cluster leaving only the upper four to six shoulders (dookolzian et al., 1995); in ‘red globe’ and ‘flame seedless’ usually the upper six to eight shoulders are kept (dookolzian and hirschfelt, 1995); in superior seedless® one-third of the bottom part of the cluster is removed. in cultivars such as ‘italia’, instead, berry thinning requires plucking small seedless or irregularly developed berries by hand, a very expensive operation which may take up to 50-80 labour days/hectare. in seedless varieties the use of giberelic acid (ga3) is widespread; dose and time of application is highly dependent on the variety. the success of treatment is extremely variable, and is greatly influenced by climate during flowtable 2 influence of cluster:bud ratio on production of grape cv. italia. score of grapes from different vineyards and different thesis (crescimanno et al., 1986) vineyards different time i ii iii 0.6 0.8 0.6 0.8 0.6 0.8 1 627.45 509.15 592.20 565.40 581.17 499.62 2 680.17 ab 539.25 b 724.47 a 708.97 a 731.80 a 704.95 a 3 336.27 abab 305.20 abcd 355.67 aab 230.77 bc 393.22 aa 303.65 abbc 4 948.55 816.72 960.07 842.97 846.05 860.92 2a 919.72 890.85 945.70 845.75 851.62 894.35 3a 821.75 ab 871.47 ab 698.35 b 782.85 ab 946.00 a 787.62 ab 4a 832.05 a 633.78 b 714.30 ab 756.52 ab 818.62 ab 774.40 ab values sharing the same letter are not significantly different from each other by duncan’s multiple range test at p≤ 0.01 and p≤ 0.05. table 3 influence of cluster:bud ratio on production of grape cv. italia. score of grapes from different vineyards and different thesis (crescimanno et al., 1986) different time i ii iii vineyards 0.6 0.8 0.6 0.8 0.6 0.8 1 2 9 8 9 8 8 8 3 4 9 8 8 8 7 8 2a 10 9 8 8 9 8 3a 10 9 9 9 9 8 4a 10 7 10 10 8 8 thesis average 9.6 8.4 8.8 8.6 8.2 8 time average 9 8.7 8.1 = poor quality of the product (not packable). fig. 3 effect of cluster thinning on evolution of °brix content (dotted line) and total acidity (continuous line) in berries of treated (square) and control (triangle) plants. fig. 4 berry thinning. 146 ering (especially air temperature). one of the goals of breeding programs is to obtain varieties that do not require berry thinning. 4. girdling and cane-scoring girdling is the removal of a ring of bark (only phloem) around the trunk or bases of the individual canes, while scoring is a simple knife-cut encircling the branch (fig. 5 a and b). the phenological stage at which girdling is carried out is the greatest factor determining the nature and magnitude of the obtained effects (di lorenzo, 2003). both operations stop movement through the phloem, modifying the hormonal balance of the vine after girdling (kriedemann and lenz, 1972) and consequently producing an increase of carbohydrates above the girdle (weaver and mccune, 1959); carbon exchange between the shoot and the rest of the vine is thus eliminated. girdling reduces net co2 assimilation rate and stomatal conductance of leaves until the girdle heals (kriedemann and lenz, 1972; williams and ayars, 2005). water use efficiency decreases following girdling without an application of ga3 at berry set. once the girdle heals, vine water use increases up to harvest (bucks et al., 1985; williams and ayars, 2005). the reduction in stomatal conductance, and concomitant reduction in vine water use in response to girdling is probably due to an accumulation of abscisic acid (aba) in the leaves (loveys and kriedemann, 1974; during, 1978; williams et al., 2000; williams and ayars, 2005). girdling has negative effects on some berry characteristics, such as a decrease of malic acid concentration in the must (orth et al., 1994). the effect of girdling is reduced by leaf removal and declines while the number of leaves decreases (caspari et al., 1998). cane girdling at 12°brix sugar content on cv. vittoria determines a qualitative improvement of grapes (tables 5 and 6): particularly, single girdling increases ratio sugar: acidity, double girdling (first time performed at pea-size stage, second time at veraison) increases the berry weight (fig. 6) (di lorenzo and gambino, 2010). canescoring increases the average berry size of ‘emperatriz’ seedless grape and bunch weight compared to unscored vines, but has no effect in ‘aledo’ seeded grape (casanova et al., 2009). the author supposes that in seeded fruits the availability of carbohydrates is guaranteed by the seed’s ability to synthesize plant growth hormones leading to powerful sink capacity, while seedless fruit has an insufficient sink capacity to grow. trunk girdling is a more rapid technique than cane girdling and all clusters are subjected to treatment. with cane girdling or scoring there may be a few clusters, located below the cut, that remain unaffected. the bark ring removed has to be complete; incomplete cuts result ineffective (jensen et al., 1979). usually the girdle cut heals in approximately four weeks through callus formation that recovers the vascular connections (williams et al., 2000). girdling and cane scoring are carried out seven to 10 days before flowering to improve berry-set, at berry set to increase berry size, and at veraison to advance sugar and colour development in red varieties. table 4 effect of two different thinnings on qualitative traits (di lorenzo, 2003) thinnings cluster weight (g) berry weight (g) berry weight variation coefficient (%) closeness index thinning intensity more than 40% of traditional 626 10.6 23.1 2.59 “traditional” berry thinning 934 8.9 28.1 3.39 fig. 5 cane girdling and trunk girdling. a 147 in a trial of trunk girdles applied at fruit set on ‘crimson seedless’, vines girdled at fruit set produced larger berries compared to vines girdled at berry softening and ungirdled vines. trunk girdles applied at fruit set increased berry weight 38%, berry length 12% and berry diameter 10% compared to the fruit of ungirdled vines. the berry weight and diameter of vines girdled at veraison were significantly lower than those of ungirdled vines, while the berry length of these treatments was similar. in addition, berry firmness of vines girdled at fruit set was significantly greater compared to vines girdled at berry softening and ungirdled vines. due primarily to their larger berry size, the total yield of vines girdled at fruit set was approximately 45% greater than vines girdled at berry softening and ungirdled vines. a fruit quality defect among the girdling treatments was poor colour, and so only a portion of this increase in total yield was packable fruit (dokoozlian et al., 1995; dokoozlian et al., 2000 a). in contrast, in the same variety, brar and coworkers (2008) indicated that girdling at berry set was an effective practice to stimulate berry colour development. in ‘autumn royal’ berry weight can be increased 10 to 15% by girdling at berry set, but also in this variety girdling delays colour development and harvest (dokoozlian et al., 2000 a). trunk girdling at berry set and bunch thinning, in an early-season black seedless table grape variety (sugrathirteen® or midnight beauty®) improved berry size, sugar content and berry firmness (gentilesco et al., 2011). girdling increases the risk of skin burn, and should never be done on the same vine more than once a year. repeated girdling over a number of years may reduce bunch size and the life expectancy of the plant. 5. shoot trimming intensive growth of vines in warm climates requires measures to control vigour in order to ensure fruit quality and vegetative balance of the plants. the main control measure, besides the careful use of water and fertilizers, is shoot trimming which is usually performed after flowering; the exact moment depends on the cultivar and the objective of the culture (camargo, 2005). shoot trimming carried out just before bloom may improve fruit set: in fact in this stage it stops trophic competition of top shoot. in “t”, “y” or open gable trellis, shoot trimming or hedging can be performed to improve cluster exposure to sunlight and to reduce humidity within the fruit zone. early hedging may stimulate lateral shoot growth. hedging should be performed after berry softening to avoid potential problems with fruit sunburn. both sides of the canopy should be trimmed to allow the uniform penetration of sunlight into the canopy interior. care must be taken not to remove too much foliage when hedging as excessive foliage removal may slow fruit maturation and significantly retard fruit colour development (dokoozlian et al., 2000 a). table 5 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on parameters of berries at ripening on cv. vittoria (di lorenzo et al., 2010) average berry weight ±se (g) weight range average p.d. ±se (mm) average e.d ±se (mm) average berry form (dp/de)<6 6-8 > 8 1 8.3 c ±0.20 15 42 43 27.3 bc ±0.39 21.1 b ±0.30 1.30 ±0.02 2 7.4 b ±0.16 21 44 35 26.8 b ±0.23 19.7 a ±0.20 1.47 ±0.01 3 8.7 c ±0.19 10 35 55 27.8 c ±0.33 21.0 b ±0.17 1.32 ±0.01 control 6.8 a ±0.10 38 43 19 25.0 a ±0.21 21.1 b ±0.14 1.18 ±0.01 table 6 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on harvest parameters (cv. vittoria) (di lorenzo et al., 2010) soluble solids °brix total acidity (g/l) 05-07 15-07 05-07 15-07 1 13.2 b 13.3 a 5.8 a 5.5 a 2 12.0 a 14.2 b 6.2 b 5.8 b 3 13.2 b 13.5 a 5.8 a 5.9 b control 12.0 a 13.5 a 6.2 b 5.8 b fig. 6 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on growth rate of the shoot and berry (di lorenzo and gambino, 2010). 148 6. plant growth regulators plant growth regulators play a notable role in current worldwide table grape cultivation. some of these can be included among summer management techniques, in order to reduce berry set, increase berry size and accelerate or improve fruit ripening. before discussing their effects and possible uses, it must be pointed out that in each country there are different rules and regulations for their use (e.g. forchlorfenuron and ethephon are forbidden in many countries). gibberellic acid (ga3) gibberellic acid (ga3) is commonly used to reduce fruit set and increase berry size of seedless table grape cultivars. ga3 rates and timing applications are quite specific and depend on the cultivar, region, and desired effects on berry growth and fruit quality (dokoozlian et al., 1995). ga3 sprays are generally carried out: several weeks before bloom to elongate the cluster rachis. while many studies have reported that pre-bloom ga3 application has no effect on cluster length or compactness at harvest (dokoozlian, 2000), commercially it is still used (about 10 ppm rate). it could have a negative influence on bud fruitfulness in the following year. between 30 and 100% bloom to improve berry thinning. the mechanism by which gibberelic acid works as fruit thinner is still not understood. an initial hypothesis was that ga3 acts as a pollenicide, interfering with pollen germination, however many studies have shown that the ga3 concentration normally applied for thinning does not reduce pollen germination. some authors suggest, instead, that ga3 applied at bloom alters the endogenous hormone balance causing flower or fruit abscission. the most reliable hypothesis is that ga3 induces nutrient competition between flowers and shoots, and among flowers/small fruits within the cluster. in the latter case ga3 stimulates nutrient competition among berries, and so physiologically advanced berries become strong sinks, while weaker berries are unable to compete for nutrients and drop (dokoozlian, 2000). the ga3 rate is closely related to variety and climate conditions, and it can vary from 1 to 20 ppm. a higher rate of ga3 applied at bloom generally does not improve thinning, but can significantly increase the number of shot berries per cluster. single or multiple applications usually result in similar levels of fruit thinning, however it seems that multiple applications produce larger berries at harvest compared to single applications (dokoozlian, 2000). ga3 spray at bloom often produces inadequate levels of berry thinning, which results in a need for manual berry thinning. after fruit set to increase berry size. gibberellic acid applied to growing berries increases cell division and elongation. also in this case the rate depends on the cultivar and prefixed quality target. the timing of application has a big influence on the efficacy of treatment; usually berry size should be in the range 4-6 mm, to a maximum of 10 mm. ga3 treatments can increase berry size at harvest 50% or more, but they delay fruit maturity and reduce berry colour in red varieties (dokoozlian, 2000). also in this stage, high rates might cause a decrease in bud fruitfulness in the following year. ga3 molecules enter in plant tissues better if applied in low ph solution (ph ≈ 4) since at low ph ga3 molecules are neutral and are able to move easily through plant tissues. forchlorfenuron (cppu) forchlorfenuron (cppu) is a synthetic cytokinin that increases cell division and elongation. usually, cppu can be sprayed on grape: immediately before bloom to increase fruit set (≈ 10 20 g/ha) (dokoozlian, 2000); after fruit set to increase berry size (≈ 5 40 g/ha). in different varieties (‘thompson seedless’, ‘ruby seedless’, ‘redglobe’ and ‘melissa’) cppu applied at fruit set increased berry weight, diameter and length, while cppu applied at fruit softening had no significant effect on berry growth. a two-week delay in harvest of most cultivars was obtained when 9-12 mg/l cppu was applied at berry set, while pigment accumulation was either delayed or significantly reduced (dokoolzian, 2001). cppu does not reduce the fruitfulness of either seedless or seeded table grape cultivars, while it increases the rachis size and the force required to remove berry from the capstem (dokoolzian et al., 1995). ethephon ethephon (trade name ethrel®) is commonly applied to red-pigmented table grape cultivars at the beginning of fruit ripening to enhance berry colour. the active ingredient in ethephon, [(2-chloroethyl) phosphonic acid], produces ethylene upon its degradation. ethylene is an endogenous plant hormone that accelerates the ripening of many fruits, including grapes. ethephon, applied on ‘crimson seedless’ when approximately 5 to 10% of the berries were showing red colour, had no effect on fruit soluble solids content, however vines treated with ethephon had lower titratable acidity compared to untreated vines (dokoozlian et al., 1995). ethephon had no significant effect on berry weight, length or diameter, while it significantly improved fruit colour, increasing packable yield (+38%), but significantly reduced berry firmness compared to untreated vines (dokoozlian et al., 1995). abscisic acid (aba) the plant hormone abscisic acid (aba) appears to be one of the factors for anthocyanin accumulation. exogenous applications of aba increased the anthocyanin content of grape skins (peppi et al., 2006; peppi et al., 2007). 149 application of abscisic acid (aba) may improve colour more effectively than ethephon, but it may potentially influence postharvest quality, though in a trial carried out on ‘crimson seedless’ the aba and ethephon treatments did not affect berry firmness or predispose the fruit to postharvest shatter (cantína et al., 2007). in that trial grapes treated with 300 μl l−1 aba coloured quickly and thus were harvestable about 30 days earlier than untreated grapes, and 10 days earlier than grapes treated with ethephon. on average, grapes treated with 150 μl l−1 aba were harvestable at about the same time as grapes treated with 300 μl l−1 aba or ethephon, and grapes treated with either 150 μl l−1 aba or ethephon were harvestable about 15 day before non-treated grapes. however, tss, ta, and the ratio of tss to ta differed among treatments. grapes treated with 300 μl l−1 aba were harvested at the lowest tss, followed by grapes treated with 150 μl l−1 aba, and grapes treated with ethephon or not treated. grapes treated with 300 μl l−1 aba or ethephon had the highest acidity (≈ 5.0 g l−1) and the lowest tss:ta ratio (cantína et al., 2007). in ‘flame seedless’ 300 ml l−1 aba applied at veraison was superior to the other aba concentrations and to ethephon applied at any of the tested times. moreover, any concentration of aba between 75 and 300 mg l−1 applied after veraison improved colour better than ethephon applied at the same time (peppi et al., 2006), although the same rate in ‘red globe’ increased pigmentation and improved colour, it also caused fruit softening (peppi et al., 2007). references barbagallo m.g., grippi f., scafidi p., lino t., 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forchlorfenuron (cppu) on the growth and composition of table grapes. asev 52nd annual meeting, san diego, california, usa. dokoozlian n., hirschfelt d.j., 1995 the influence of cluster thinning at various stages of fruit development on flame seedless table grapes. am. j. enol. vitic., 46(4): 429-436. dokoozlian n., luvisi d., moriyama m., schrader p., 1995 cultural practices improve color, size of ‘crimson seedless’. cal. agric., 49(2): 36-40. dokoozlian n., peacock b., luvisi d., vasquez s., 2000 a cultural practices for crimson seedless table grapes. pub. tb 16-00, university of california, usa. dokoozlian n., peacock b., luvisi d., vasquez s., 2000 b cultural practices for autumn royal table grapes pub. tb 17-00, university of california, usa. during h., 1978. studies on the environmentally controlled stomatal transpiration in grape vines. ii. effects of girdling and temperature. vitis, 17: 1-9. gentilesco g., amendolagine a.m., giannandrea m.a., notarangelo l., roccotelli s., suriano s., savino m., romanazzi v., tarricone l., 2011 effects of crop load and time of girdling on sugrathirteen® table grape: first results. proceedings of giesco xvii international symposium, asti-alba, italy, pp. 419-422. 150 gubler w.d., marois j.j., 1987 control of botrytis bunch rot of grape with canopy management. plant dis., 71: 599-601. hunter j.j., ruffner h.p., volschenk c.g., le roux d.j., 1995 partial defoliation of vitis vinifera l. cv. cabernet sauvignon/99 richter: effect on root growth, canopy efficiency, grape composition, and wine quality. am. j. enol. vitic., 46: 306-314. hunter j.j., visser j.h., 1988 distribution of 14-c photosynthate in the shoot of vitis vinifera l. cv cabernet sauvignon. the effect of leaf position and developmental stage of the vine. s. afr. j. enol. vitic., 9(1): 3-9. jensen f., luvisi d., swanson f., leavitt g., mitchell g., mayer g., 1979 effects of complete and incomplete girdles on thompson seedless and ribier table grapes. am. j. enol. viticult., 27: 65-67. kriedemann p.e., lenz f., 1972 the response of vine leaf photosynthesisto shoot tip excision and stem cincturing. vitis, 11: 193-197. loveys b.r., kriedemann p.e., 1974. internal control of stomatal physiology and photosynthesis i. stomatal regulation and associated changes in endogenous levels of abscisic and phaseic acids. austral. j. plant physiol., 1: 407-415. orth c.h.f., stevens s., van dik b.w., 1994 palatability of dauphine table grape is it affected by different viticoltural practices?. proc. international symposium on table grape production, anaheim, california, usa, 28-29 june, pp. 223-226. peppi m.c., fidelibus m.w., dokoozlian n., 2006 abscisic acid application timing and concentration affect firmness, pigmentation, and color of ‘flame seedless’ grapes. hortic. sci., 41: 1440-1445. peppi m.c., fidelibus m.w., dokoozlian n., 2007 application timing and concentration of abscisic acid affect the quality of ‘redglobe’ grapes. j. hortic. sci. biotechnol., 82: 304-310. poni s., bernizzoni f., canalini l., civardi s., intrieri c., 2006 effects of early leaf removal on shoot photosynthesis, yield components, and grape quality. am. j. enol. vitic., 57: 397-407. reynolds a.g., wardle d.a., 1989 impact of various canopy manipulation techniques on growth, yield, fruit composition, and wine quality of gewürztraminer. am. j. enol. vitic., 40: 121-129. scafidi p., pisciotta a., squadrito m., di lorenzo r., barbagallo m.g., 2010 effetto dell’epoca di sfogliatura sul comportamento vegeto-produttivo della cv cabernet sauvignon in sicilia. italus hortus, 17: 33-41. sottile i., di lorenzo r., barbagallo m.g., giuffrida s., 1996 characteristics of inflorescence and cluster of tablegrape “italia” cultivar as influenced by position on fruit cane and shoot. gesco 9th groupe europèen d’etudes des systèmes de conduite de la vigne, budapest. hungary, pp. 137-142. weaver r.j., mccune s.b., 1959 girdling: its relation to carbohydrate nutrition and development of thompson seedless, red malaga and ribier grapes. hilgardia, 28: 421456. williams l.e., ayars j.e., 2005 -water use of thompson seedless grapevines as affected by the application of gibberellic acid (ga3) and trunk girdling practices to increase berry size. agricultural and forest meteorology, 129: 85-94. williams l.e., retzlaff w.a., yang w., biscay p.j., ebisuda n., 2000 effect of girdling on leaf gas exchange, water status, and non-structural carbohydrates of fieldgrown vitis vinifera l. (cv. flame seedless). am. j. enol. vitic., 51: 49-54. 141 1. introduction decay represents the major cause of postharvest loss of fig (ficus carica), especially in fruit of the second crop when high humidity levels and precipitation cause skin cracking, ostiole splitting, and the growth of pathogens. postharvest life of figs can vary from a few days to one to two weeks (ferguson et al., 1990; crisosto and kader, 2004). more than in other fruits, the ripening process in figs is very rapid; under favourable environmental conditions, flesh tissue changes from a spongy dry state to a juicy, sweet condition in one-two days. these sudden changes have long been the object of controversy, and whether figs should be considered climacteric or non-climacteric fruit. indeed, if on one hand the rapid changes of rheological and compositional features are typical of climacteric fruit, on the other hand figs do not share the ability to continue the ripening process once harvested. normally, unripe harvested figs never rich an optimum eating stage as happens with other climacteric fruits, such as peaches, pears, kaki or apples. surely, the maturing and ripening processes are so close and rapid to overlap, not allowing a clear sequential separation between these two physiological stages. nevertheless, the classification of figs as a climacteric species is generally accepted (marei and crane, 1971; ferguson et al., 1990). susceptibility of figs to decay and physical damage dramatically increases with ripening: as fruit ripens, the defence mechanism of unripe fruit is rapidly lost and various pathogenic microorganisms can develop. infection sites may involve the outer tissue of the fruit, with pathogens starting to develop on the peel or the underneath tissue through wounds or cracks, or from inside, through infections originating in the syconium cavity, in most cases transported by wasps or other insects (crisosto et al., 2011). in parthenocarpic cultivars with closed or partially closed ostiole, which do not need caprification to produce, visits by fig wasps and other insects inside the syconium cannot take place or are markedly reduced. consequently, infections starting from inner tissues like endosepsis (fusarium monilifome), souring or fermentation incited by different types of yeast and bacteria carried by different insects, are easier to control than in cultivars with open ostioles (ferguson et al., 1990; michailides et al., 1996). in all cases, an efficient control of decay can be achieved by field treatments with insecticides and fungicides. azoxystrobin is a strobilurin-like partial-systemic fungicide with broad-spectrum activity against several important pathogens (gullino et al., 2000). it is considered a influence of azoxystrobin dip treatments on postharvest decay of second-crop fig (ficus carica) fruits from sardinian germoplasm s. d’aquino 1(*), a. palma 1, d. satta 2, l. de pau 2, m. schirra 1 1 istituto di scienze delle produzioni alimentari, cnr, traversa la crucca 3, loc. baldinca, 07100 sassari, italy. 2 agris sardegna dipartimento per la ricerca nell’arboricoltura, via mameli 126/d, 09123 cagliari, italy. key words: azoxystrobin, cold storage, fig fruit, fig decay, postharvest treatment. abstract: fig (ficus carica l.) fruits from the second-crop, cultivars verde, san pietro, perdingiana, and carcanzi trota, were harvested on 25 august and 8 september 2005, subjected to 50 or 100 mg/l azoxystrobin (azo) dip treatments for 30 s and stored for seven days at 18°c (simulated marketing conditions, smc) or for seven days at 5°c (cs) plus seven days at smc. after seven days at 5°c, the external decay incidence in control fruit was 3-10%. treatment with azo completely suppressed external decay in ‘san pietro’, and ‘carcanzi trota’ and resulted in 2 and 8% decay in ‘verde’ and ‘perdingiana’, respectively. after cs plus 3 days at 18°c, decay in control fruit was measured as 23-41% and 71-85% of august and september harvests, respectively, while in those treated with azo, average losses were 4-11%. at the end of smc, all fruit in all treatments decayed, although the rotten area was smaller in azo treated fruit. similarly, in fruit stored directly in smc, azo significantly reduced decay during the first three days; after seven days all fruit decayed. internal decay originating from the syconium cavity was higher in fruit harvested in september and was not affected by azo treatments. adv. hort. sci., 2015 29(2-3): 141-144 (*) corresponding author: salvatore.daquino@ispa.cnr.it received for publication 26 september 2014 accepted for publication 22 july 2015 142 adv. hort. sci., 2015 29(2-3): 141-144 reduced-risk-fungicide by the united states environmental protection agency and has been registered for field application or postharvest treatments on several crops. the present study evaluates the efficacy of postharvest treatments with azoxystrobin to control decay on four cultivars of second-crop fig fruits of sardinian germplasm. forniti fruits of these cultivars have an open ostiole, so they are very prone to internal decay. 2. materials and methods the investigation was carried out on four fig cultivars from sardinia germoplasm (chessa and nieddu, 1994): verde, san pietro, perdingiana, and carcanzi trota. fruits of the second crop were picked on 25 august or 8 september from the collection field of the “agris sardegna” in sassari. trees received standard agricultural practices, but no chemical treatment to control pests or diseases had been applied in the previous three years. fruits were harvested early in the morning and immediately transported to the laboratory, which was located about 10 km from the orchard. fruit without defects from each cultivar were selected and divided into three groups. each group was dipped for 30 s, at room temperature, in: water (control) or water with 50 or 100 mg/l of azoxystrobin (azo) (ortiva , syngenta crop protection milan, italy). after dipping and before storage, all figs were dried at room temperature and each treatment group was divided into two subgroups including eight replications of 25 fruits. the first subgroup was stored for three or seven days at 18°c and 90% relative humidity (rh) (simulated marketing conditions (smc), while the remaining subgroup was stored for seven days in cold storage (cs) at 5°c and subsequent three or seven days of smc. afterwards, cs and smc fruit were inspected for external and internal decay (endosepsis and souring). data were subjected to analysis of variance after transformation of average decay-percentage values in √x or arcsin√x depending on the range of variation of decay. separation of the means was accomplished according to fisher’s test of the least significant difference (lsd); actual values are reported. 3. results the development of external and internal decay was greatly influenced by picking date and storage conditions. fruits harvest in august were significantly less prone to decay than those harvested in september, regardless of the cultivars (figs. 1, 2; tables 1-4). ‘perdingiana’ figs (table 3) of both harvest dates were the most susceptible with high percentages of external and internal decay, whereas no relevant differences were detected among other cultivars. after seven days of storage at 5°c, the percentage of fruit showing external decay was low in all treatments and harvest dates. when fruits were transferred to smc, table 1 influence of 30-s-dip treatments with azoxystrobin (azo) at 20°c on external and internal decay incidence in second crop ‘verde’ figs after three days at 18°c and 65% rh, or for seven days at 5°c plus three days at 18°c treatments 3 days at 18°c 7 days at 5°c 7 days at 5°c plus 3 days at 18°c external decay % internal decay % external decay % internal decay % external decay % internal decay % harvested in august control 21 b (z) 23 a 6 b 0 a 31 b 18 a 50 mg/l azo 5 a 35 a 0 a 0 a 7 a 31 b 100 mg/l azo 4 a 27 a 0 a 0 a 8 a 24 ab harvested in september control 70 b 88 a 9 b 10 a 82 b 86 a 50 mg/l azo 9 a 75 a 2 a 18 a 11 a 75 a 100 mg/l azo 9 a 83 a 1 a 16 a 9 a 81 a (z) for each storage period and harvesting time values in columns followed by different letters are significantly different at p≤0.05 according to fisher’s test of the least significant difference. fig. 1 influence of picking date and storage conditions on external decay incidence in second crop production of verde, san pietro, perdingiana and carcanzi trota figs. for each storage period histograms with different letters are significantly different, p≤0.05. fig. 2 influence of picking date and storage conditions on internal decay incidence in second crop production of verde, san pietro, perdingiana and carcanzi trota figs. for each storage period histograms with different letters are significantly different, p≤0.05. 143 d’aquino et al., influence of azoxystrobin dip treatments on postharvest decay of second-crop fig sharp increases in decay development were recorded in all fruit samples especially those of the second harvest date. in particular, after three days at 18°c external decay percentage in control samples ranged between 71 (san pietro) and 85% (perdingiana), whereas after seven days at 18°c all fruit decayed, regardless of the treatments (data not shown). azo treatments significantly reduced external decay in all cultivars. however, the protective activity of azo lasted few days in fruit held at 18°c. after seven days at 18°c all azo-treated fruit showed external decay, although the extent of the diseased area was considerably lower than in untreated fruit (data not shown). no statistical differences were detected between the two concentrations of azo (tables 1-4). various pathogens developed on the same fruit. in fruit harvested in august, alternaria rot (alternaria alternata) and to a lesser extent, cladosporium rot (cladosporium herbarum) accounted for more than 90% of decay, whereas in fruit harvested in september the number of pathogens increased. however, alternaria rot was always the main cause of decay, followed by cladosporium rot, grey mold (botrytis cinerea), and penicillium mold (penicillium spp.). moreover, moulds of these pathogens, which first initiated the infections, in a nested fashion, were often overwhelmed by rhizopus rot (rhizopus stolonifer). internal decay severely developed in all cultivars, especially in ‘perdingiana’. fruit affected by internal decay were significantly more in samples harvested in september. storage at 5°c reduced the development of internal decay; when fruits were moved to smc it dramatically increased (tables 1-4). the influence of azo against internal decay was negligible. 4. discussion and conclusions results of this experiment confirmed the high postharvest perishability of the studied fig cultivars, especially ‘perdingiana’. the susceptibility to microbiological deterioration was highly affected by the harvesting period. fruits harvested in august experienced significantly less decay than those harvested in september. this is because the higher environmental humidity in september is more favourable to field infection than in august, when weathtable 2 influence of 30 s-dip treatments with azoxystrobin (azo) at 20°c on external and internal decay incidence in second crop ‘san pietro’ figs after three days at 18°c and 65% rh, or for seven days at 5°c plus three days at 18°c treatments 3 days at 18°c 7 days at 5°c 7 days at 5°c plus 3 days at 18°c external decay % internal decay % external decay % internal decay % external decay % internal decay % harvested in august control 19 b (z) 32 a 3 a 4 a 23 b 28 a 50 mg/l azo 3 a 25 a 0 a 7 a 10 ab 34 a 100 mg/l azo 0 a 27 a 0 a 9 a 2 a 29 a harvested in september control 35 b 73 a 5 a 41 b 71 b 76 a 50 mg/l azo 8 a 65 a 0 a 37 ab 7 a 71 a 100 mg/l azo 6 a 72 a 0 a 28 a 9 a 67 a (z) for each storage period and harvesting time values in columns followed by different letters are significantly different at p≤0.05 according to fisher’s test of the least significant difference. table 3 influence of 30-s-dip-treatments with azoxystrobin (azo) at 20°c on external and internal decay incidence in second crop ‘perdingiana’ figs after three days at 18°c and 65% rh, or for seven days at 5°c plus three days at 18°c treatments 3 days at 18°c 7 days at 5°c 7 days at 5°c plus 3 days at 18°c external decay % internal decay % external decay % internal decay % external decay % internal decay % harvested in august control 35 b (z) 33 a 9 b 11 a 41 b 44 b 50 mg/l azo 4 a 31 a 0 a 12 a 6 a 41 ab 100 mg/l azo 3 a 35 a 0 a 14 a 4 a 33 a harvested in september control 76 b 91 a 13 b 24 ab 85 b 100 a 50 mg/l azo 10 a 98 a 8 ab 27 b 19 a 95 a 100 mg/l azo 13 a 87 a 5 a 19 a 16 a 100 a (z) for each storage period and harvesting time values in columns followed by different letters are significantly different at p≤0.05 according to fisher’s test of the least significant difference. table 4 influence of 30-s-dip treatments with azoxystrobin (azo) at 20°c on external and internal decay incidence in second crop ‘carcanzi trota’ figs after three days at 18°c and 65% rh, or for seven days at 5 °c plus three days at 18°c treatments 3 days at 18°c 7 days at 5°c 7 days at 5°c plus 3 days at 18°c external decay % internal decay % external decay % internal decay % external decay % internal decay % harvested in august control 26 b1 24 a 0 a 3 a 29 b 18 a 50 mg/l azo 6 a 29 a 0 a 8 a 8 a 21 a 100 mg/l azo 4 a 27 a 0 a 5 a 3 a 24 a harvested in september control 58 b 85 ab 10 b 16 a 77 b 82 a 50 mg/l azo 10 a 77 a 0 a 14 a 6 a 81 a 100 mg/l azo 4 a 91 b 0 a 9 a 2 a 79 a (z) for each storage period and harvesting time values in columns followed by different letters are significantly different at p≤0.05 according to fisher’s test of the least significant difference. 144 adv. hort. sci., 2015 29(2-3): 141-144 er conditions are usually dry. azo was highly effective against all the main pathogens causing external diseases, confirming its broad spectrum of activity (gullino et al., 2000). however, its effectiveness lasted only three days in fruit held in smc. after seven days at 18°c all fruit treated with azo exhibited visible infections, although the extension of the lesions were notably less than in control fruit. azo was ineffective against internal decay. all studied cultivars had open ostiole. figs with open ostiole may be more susceptible to endosepsis caused by fusarium moniliforme and souring, incited by different kinds of yeasts and bacteria (crisosto et al., 2011). infections of both diseases are caused by entrance into the syconia of fig wasps and vinegar flies (drosophyla spp.), dried-fruit beetles (carpopilus spp.), and thrips (thrips spp. and frankliniella spp.) (michailides et al., 1996; crisosto et al., 2011). in figs with close or narrow ostiole, in which only wasps and bees can enter, internal decay is generally lower. proper postharvest technologies, such as precooling, film-wrapping, and conditioning in modified atmosphere environment are shown to extend the keeping quality of fig fruit (turk, 1989; piga et al., 1995, 1998; d’aquino et al., 1998, 2003). however, under shelf-life conditions, the market life of figs decreases dramatically, especially in second crop fruit. the present results reveal that postharvest application of azo, even at very low rates which could leave on fruit reside levels lower than field treatments with higher rates, resulted only in a slight delay of decay development, as most of the infections generally occur in the orchard and remain latent until fruit ripen. thus, postharvest azo treatments associated with low temperatures can give better results if pest management includes a preventive disinfestation program aimed at reducing the insect populations which act as carrier of pathogens’ conidia. acknowledgements the research was supported by italian national council of research. references chessa i., nieddu g., 1994 il fico, pp. 361-404. in: agabbio m. (ed.) patrimonio genetico di specie arboree da frutto le vecchie varietà della sardegna. carlo delfino editore, sassari, pp. 424. crisosto c.h., kader a.a., 2004 fig. in: gross k.c., c.y. wang, and m. saltveit (eds.) the commercial storage of fruits, vegetables, and florist and nursery stock. usda, ars, agricultural hdbk. 66. washington dc. http:// www.ba.ars.usda.gov/hb66/115prickly.pdf crisosto h., ferguson l., bremer v., colelli g., 2011 fig (ficus carica l.), pp. 134-158. in: yahia elhadi m. (ed.) postharvest biology and technology of tropical and subtropical fruits vol. 3. woodhead publishing limited, cambridge, uk, pp. 584. d’aquino s., palma a., dore a., agabbio m., 2003 non conventional treatments to reduce figs decay. acta horticulturae, 604: 817-821. d’aquino s., piga a., molinu m.g., agabbio m., papoff c., 1998 maintaining quality attributes of “craxiou de porcu” fresh fig fruit in simulated marketing conditions by modified atmosphere. acta horticulturae, 480: 289-294. ferguson l., michailides j.t., shorey h.h., 1990 the california fig industry. horticultural review, 11: 409490. gullino m.l., leroux p., smith m., 2000 use and challenges of novel compounds for plant disease control. crop protection, 19: 1-11. marei n., crane c., 1971 growth and respiratory response of fig [(ficus sativa l.) cv. mission] fruits to ethylene. plant physiol., 48: 249-254. michailides t.j., morgan d.p., subbarao k.v., 1996 fig endosepsis. an old disease still a dilemma for california growers. plant dis., 80: 828-841. piga a., d’aquino s., agabbio m., papoff c., 1995 influenza del confezionamento con film plastici sulla conservazione del fico. italus hortus, 2: 3-7. piga a., d’aquino s., agabbio m., papoff c., 1998 short-term nitrogen atmosphere exposure extends shelf-life of fresh “niedda longa” fig fruits. acta horticulturae, 480: 295-299. turk r., 1989 effect of harvest time and precooling on fruit quality and cold storage of figs (ficus carica l., cv. bursa siyahi). acta horticulturae, 258: 279-285. impaginato 107 1. general aspects rocket salad also known as arugula is an annual herbaceous plant whose name encloses several species of the brassicaceae family characterized by leaves with peculiar pungent taste and strong flavour. the crop has been originated in the mediterranean and near east, with a major centre of diversity in the regions of western mediterranean (hall et al., 2015), which represent also the main areas of cultivation thanks to their growing conditions and climate. several species, mainly belonging to the genus eruca and diplotaxis, are widely cultivated and recognized as rocket salad. the most common are eruca vesicaria (l.) cav. and diplotaxis tenuifolia (l.) dc. eruca vesicaria includes four subspecies namely subsp. vesicaria, subsp. sativa (miller), subsp. longirostris (uechtr.) and subsp. pinnatifida (desf.) (gomez-campo, 2003). among these, the subsp sativa, also called eruca sativa, has been spreaded in different part of the world as cultivated rocket and is the most consumed and economically relevant. this species is diploid with eleven chromosomes (2n = 22) (padulosi and pignone, 1996) with annual life cycle flowering at begin of spring and ending with the production of seeds in late spring/early summer. nowadays it is cultivated in all continents in both marginal areas and/or fertile soils. plants of this species are characterized by a height of about 15.0 cm, flowers with calyx caduceus, sepals only two cucullate and corolla cream or whitish (gomezcampo, 2003). diplotaxis genus includes about 33 species (www.theplantlist.org) with great variability related to morphological traits and chromosome number (table 1). the genus includes both annual and perennial plants with leaves of different shape, thickness, adv. hort. sci., 2017 31(2): 107-113 doi: 10.13128/ahs-21087 mini review rocket salad: crop description, bioactive compounds and breeding perspectives p. tripodi (*), g. francese, g. mennella consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca orticoltura e florovivaismo (crea-of), 84098 pontecangnano-faiano (sa), italy. key words: diplotaxis tenuifolia, eruca sativa, flavonols, genetic improvement, glucosinolates, phytochemicals, rocket salad. abstract: rocket salad is a plant member of the brassicaceae family whose name encloses species of the eruca and diplotaxis genera characterized by leaves with peculiar pungent taste and strong flavour. it has been originated in the mediterranean area and nowadays is worldwide cultivated and consumed as food condiment and in ready-to-use mixed salad packages. several other uses are recognized in cosmetics and medicine. this crop represents a valuable source of health benefits due to the presence of a range of health-promoting phytochemicals including carotenoids, vitamin c, fiber, polyphenols, and glucosinolates. these compounds are potentially linked in the prevention of certain diseases and types of cancer. glucosinolates, represent the major class of compounds in rocket, and their hydrolysis products are responsible of the typical pungent aromas and flavours. despite the continuous increase of the global consumption during the recent years, few efforts have been carried out in genetic improvement programs aimed to constitute new varieties due to biological and reproductive barriers. in the present article is provided a brief overview of the principal species of rocket salad used in dietary and discussed the qualitative properties as well as the potentiality and constraints for breeding. (*) corresponding author: pasquale.tripodi@crea.gov.it received for publication 7 march 2017 accepted for publication 21 june 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 107-113 108 indentation, and flower colour (white, yellow and purple). the most common species cultivated across all continents are diplotaxis tenuifolia and diplotaxis muralis. both are perennial being cultivated in the winter and producing new sprouts in the spring. this aspect, combined to the dehiscence of the siliqua and the large number of viable seeds, helps to spread these species as weeds. diplotaxis tenuifolia is the most used for human consumption; plants are characterized by average height of 80 cm, a deep tap root, fleshy leaves, and oblong, lobed with pointed apexes. main differences occurred between eruca and diplotaxis genus in terms of plant architecture, leaf morphology, chromosomal number and phytochemical compound contents. eruca species, being annuals, tends to have a higher growth rate, increased size of leaves and early flowering. these characteristics result in a high production of biomass, which make the system of cultivation different respect to the diplotaxis spp. and requiring a lower seeding density and a lower number of harvests. another trait discriminating the two genera is the larger seed dimension of the eruca species (1.5 mm in length) with respect to diplotaxis spp. (about 0.7 mm in length) (padulosi and pignone, 1996). a wider germination temperature range and greater speed of germination is also observed within the eruca species, which is probably due to their annual nature, requiring greater energy of the plant to produce viable seeds (hall et al., 2015). the consumption of rocket salad dates back since ancient time and included food and non food uses such as oil, deodorant, cosmetic and medical purposes (hall et al., 2012). aphrodisiac properties and medical uses related to anti inflammatory and depurative effects (padulosi and pignone, 1996) were emphasized by ancient poets during greek and roman times. nowadays leaves are eaten fresh in salads or as topping of many dishes (e.g. pizza) or cooked in soups. several recipes provide the preparation of pureed, sauces and pesto. other cosmetic uses concern the production of creams and lotions for body. rocket salad is worldwide cultivated and commercialized in many countries as mix salad packages. in europe, the needing of prepared products ready to use as well as the major attention given to a well balanced and assorted diet, composed of a variety of health-promoting compounds, has facilitated its consumption. in central and northern markets, over half of the rocket comes from italy and spain, which, table 1 list of diplotaxis species recognized and their chromosome number * na = not available. species chromosome number d. acris (forsk.) boiss. var. acris 11 var. duveyrieriana (coss.) coss. na * d. antoniensis rustan na d. assurgens (del.) thell. 9 d. berthautii br.-bl. and maire 9 d. brachycarpa godr. 9 d. brevisiliqua (coss.) mart.-laborde 8 d. catholica (l.) dc. var. catholica 9 var. rivulorum (br.-bl. and maire) maire na d. erucoides (l.) dc. subsp. erucoides 7 subsp. longisiliqua (coss.) gómez-campo 7 d. glauca (j.a. schmidt) o.e. schulz 13 d. gorgadensis rustan subsp. gorgadensis na subsp. brochmanii rustan na d. gracilis (webb) o.e. schulz 13 d. griffitthii (hook.f. and thomps.) boiss. na d. harra (forsk.) boiss. subsp. crassifolia (rafin.) maire 13 subsp. harra 13 subsp. lagascana (dc.) o. bolòs and vigo 13 subsp. confusa mart.-lab. 13 d. hirta (chev.) rustan and borgen 13 d. ibicensis (pau) gómez-campo 8 d. ilorcitana (sennen) aedo et al. 8 d. kohlaanensis a. miller and j. nyberg na d. muralis (l.) dc. subsp. ceratophylla (batt.) mart.-laborde na subsp. muralis 21 d. nepalensis hara na d. ollivierii maire na d. pitardiana maire na d. scaposa dc. 9 d. siettiana maire 8 d. siifolia kunze subsp. bipinnatifida (coss.) mart.-laborde na subsp. siifolia 10 subsp. vicentina (samp.) mart.-laborde 10 d. simplex (viv.) sprengel 11 d. sundingii rustan 13 d. tenuifolia (l.) dc. subsp. cretacea (kotov) sobr. vesp. 11 subsp. tenuifolia 11 d. tenuisiliqua subsp. rupestris (j. ball) mart.-laborde na subsp. tenuisiliqua 9 d. varia rustan na d. villosa boulos and jallad na d. viminea (l.) dc. var. viminea and var. integrifo10 d. virgata (cav. dc.) subsp. sahariensis coss. na subsp. virgata 9 subsp. rivulorum (br.-b1. and maire) mart.na subsp. australis mart.-lab. na d. vogelii (webb) o.e. schulz na tripodi et al. qualitative properties and breeding perspectives in rocket salad 109 thanks to their geographical position and mild climatic conditions, represent the main producers. diplotaxis is much more cultivated, fitting better to the needs of the farmers and being better suited to commercial utilization, thanks to the possibility to perform several harvests per cycle with yield increasing after the first harvest (hall et al., 2015). arugula is recommended in diets, having a very low-calorie vegetables (25 calories per 100 grams of fresh leaves) and being a very good source of vitamins and minerals (table 2). furthermore, it contains a range of vital compounds, with important nutraceutical and anticancer properties, which are discussed in the next paragraph. 2. bioactive compounds many studies associate a highly significant reduction in the risk of cancer as well as a tumorogenesis inhibition and hepatoprotective effects with increasing consumption of cruciferae (lynn et al., 2006; juge et al., 2007; lamy et al., 2008; alqasoumi et al., 2009). rocket contains a range of health-promoting phytochemicals including carotenoids, vitamin c, fiber, polyphenols and glucosinolates (bennett et al., 2006; heimler et al., 2007). glucosinolates (glss) represent the major class of compounds in rocket and their contents in this crop have been well documented in the literature (d’antuono et al., 2008; pasini et al., 2012). when glucosinolates are exposed to myrosinase (ec 3.2.1.147, thioglucoside glucohydrolase) during tissue damage, glucose and an unstable intermediate are formed. this intermediate degrades to produce a sulfate ion, and a variety of products including isothiocyanates, nitriles and, to a lesser extent, thiocyanates, epithionitriles and oxazolidines. the relative proportion of these hydrolysis products depends on the plant species studied, on the glucosinolate itself (as side chain substitution), and reaction conditions like ph, metal ions or epithiospecifier protein (bennett et al., 2007). both eruca and diplotaxis species contain similar profiles of glss within the leaf tissue, the most prominent of which are glucosativin (4-mercaptobutyl-gls), glucoerucin [4-(methylthio)butyl-gls] and glucoraphanin [4-(methylsulfinyl)butyl-gls]. glucosativin and glucoerucin breakdown products are thought to contribute most to pungency and flavour in rocket (pasini et al., 2012). numerous other glss have also been identified within rocket tissue, for example diglucothiobeinin [4-(b-d-glucopyranosyldisulfanyl) butyl-gls] (kim et al., 2007), 4hydroxyglucobrassicin (4-hydroxy-3-indolymethylgls) (cataldi et al., 2007) and 4-methoxyglucobrassicin (4-methoxy-3-indolymethyl-gls) (kim and ishii, 2006). phenolics are the most abundant antioxidants in the human diet. considerable evidence indicates that some of the protective effects of phenols in fruits and vegetables may be due to flavonoids (clifford and brown, 2006). rocket species also contain large concentrations of polyglycosylated flavonol compounds, which are known to infer numerous beneficial health effects in humans and other animals. particularly of note are their effects on the gastrointestinal tract and in cardiovascular health (bjorkman et al., 2011; traka and mithen, 2011). several studies in rocket have identified and quantified polyglycosylated flavonols, which belong to three core aglycones: table 2 rocket salad nutritional values for 100 g of fresh leaves (usda nutrient database *) *https://ndb.nal.usda.gov/ndb/foods/show/3569?manu=&fgcd= &ds= (z) dietary folate equivalents. (y) retinol activity equivalents. nutrient unit value energy kcal 25 water g 91.71 carbohydrate g 3.65 protein g 2.58 sugars g 2.05 fiber g 1.6 lipid g 0.66 vitamins vitamin c mg 15 thiamin (vitamin b 1 ) mg 0.044 riboflavin (vitamin b 2 ) mg 0.086 niacin (vitamin b 3 ) mg 0.305 pyridoxine (vitamin b 6 ) mg 0.073 folate (vitamin b 9 ), dfe (z) µg 97 vitamin a, rae (y) µg 119 vitamin a iu 2373 vitamin e mg 0.43 vitamin k µg 108.6 minerals calcium, ca mg 160 iron, fe mg 1.46 magnesium, mg mg 47 phosphorus, p mg 52 potassium, k mg 369 sodium, na mg 27 zinc, zn mg 0.47 adv. hort. sci., 2017 31(2): 107-113 110 isorhamnetin, kaempferol and quercetin (bennett et al., 2006). pasini et al. (2012) studied the glucosinolate and phenolic profiles of 37 rocket salad accessions (32 eruca sativa and 5 diplotaxis tenuifolia) obtained by liquid chromatography-mass spectrometry. the authors isolated eleven desulpho-glucosinolates (dsglss) and the glucosinolate profiles did not differ between the two species. total ds-gls content, expressed as sinigrin equivalents (se) revealed a certain variability, ranging from 0.76 to 2.46 mg g-1 dry weight (dw) but, again, the quantitative analysis did not discriminate eruca from diplotaxis. moreover, the polyphenol evaluation by hplc-dad-ms allowed the identification of two different classes of compounds in the two rocket salad species. qualitative differences were observed between the polyphenol profiles at specific level: quercetin derivatives were the main phenolics of diplotaxis, whereas kaempferol derivatives characterised eruca samples. the contents of total flavonoids determined as rutin equivalents (re) ranged from 4.68 to 31.39 mg g-1 dw. kaempferol-3,4’-diglucoside (71.4-82.2%) and isorhamnetin-3,4’-di-glucoside (7.8-18.4%) were always isolated as first and second more abundant phenolic compounds in eruca samples. no marker phenolic compounds were isolated in diplotaxis samples. durazzo et al. (2013) reported significant differences in the quality of conventional and integrated cultivation practices on the nutritional properties and benefits of wild rocket [diplotaxis tenuifolia (l.) dc.], while no influence on biological activity was evidenced. the authors also determined the cytotoxicity and antiproliferative activity of rocket polyphenol extract on human colon carcinoma (caco-2) cells, evidencing a significant accumulation of cells in g1 phase and a consequent reduction in the s and g2 + m phases in response to the treatment. regarding antioxidant properties, they found frap (ferric reducing antioxidant power) values ranged from 4.44±0.11 mmol kg-1 fresh weight (fw) to 9.92±0.46 mmol kg-1 fw for conventional rocket and from 4.13±0.17 mmol kg-1 fw to 11.02±0.45 mmol kg-1 fw for integrated rocket. villatoro-pulido et al. (2013) analysing four e. sativa accessions reported the total content of glucosinolates ranged from 6.12 to 12.33 mg g−1 of dw. glucoraphanin represented up to 52% of the total glucosinolates in leaves of one accession. accessions showed differences in the hydrolysis of glucoraphanin to the isothiocyanate sulforaphane. no correlation between these compounds was observed, which insisted differences in the myrosinase activity within accessions. the same authors highlighted that rocket leaves had variable phenolic profiles represented by quercetin-3-glucoside, rutin, myricetin, quercetin and ferulic and p-coumaric acids. a high variability was observed for the total carotenoids ranged from 16.2 to 275 μg g-1 with lutein as the main carotenoid. moreover, they found glucose was the predominant sugar, representing >70% of the total soluble carbohydrates. bell and collaborators (2015) used liquid chromatography mass spectrometry (lc-ms) to obtain glucosinolate and flavonol content for 35 rocket accessions and commercial varieties. they identified 13 glucosinolates and 11 flavonol compounds; semiquantitative methods were used to estimate concentrations of both groups of compounds. minor glucosinolate composition was found to be different between accessions; concentrations varied significantly. according to pasini et al. (2012) they confirmed flavonols differentiation between genera, with diplotaxis accumulating quercetin glucosides and eruca accumulating kaempferol glucosides. the authors detected several compounds in each genus that have only previously been reported in the other. recently, we investigated the qualitative and quantitative profiles of glucosinolates and polyphenols, highlighting flavonoid glycoside compounds (flavonols), in 39 accessions of wild and cultivated rocket (taranto et al., 2016). seven ds-glss were detected in rocket leaves belonging to two chemical classes: five aliphatic compounds (glucoerucin, glucoraphanin, progoitrin, glucoalyssin, and glucosativin) and two structurally related compounds containing one intermolecular disulfide linkage, 4-(β-d-glucopyranosyldisulfanyl)butyl-gls and dimeric 4-mercaptobutyl-gls. the species studied significantly differed for gls content: total average concentrations being 29.61 and 19.41 mg g-1 dw for e. sativa (21 accessions) and d. tenuifolia (16 accessions), respectively. total gls content ranged from 2.10 to 40.96 mg g-1 dw and from 11.61 to 26.96 mg g-1 dw, for eruca and diplotaxis accessions, respectively. additional accessions of d. muralis and erucastrum spp. were evaluated exhibiting an average gls content of 17.39 and 3.63 mg g-1 dw, respectively. fifteen flavonol compounds were tentatively identified in the thirty-nine accessions studied. diplotaxis accessions were characterized by nine diftripodi et al. qualitative properties and breeding perspectives in rocket salad 111 ferent flavonols mainly represented by quercetin derivatives, total average content being 7.17 mg g-1 dw with a range from 4.91 to 8.57 mg g−1 dw. the most abundant flavonol compound in diplotaxis was quercetin 3,4’-diglucoside-3’-(6-sinapoylglucoside). as regards eruca accessions, the more abundant flavonoid group was represented by kaempferol derivatives, in agreement with a previous report (martínez-sánchez et al., 2007). the 21 eruca sativa accessions showed a flavonol total average concentration of 8.13 mg g−1 dw, the lowest and the highest content being 0.82 and 10.16 mg g−1 dw, respectively. the most abundant flavonol was kaempferol 3,4’diglucoside. according to previous research, isorhamnetin 3,4’-diglucoside was the only compound common to diplotaxis and eruca accessions studied (martínezsánchez et al., 2008; pasini et al., 2012; bell et al., 2015). however, some exceptions have been observed. specific compounds mainly detected in eruca such as kaempferol 3-glucoside and kaempferol 3-diglucoside-7-glucoside have been reported also in diplotaxis commercial varieties (bell et al., 2015). other compounds specific for diplotaxis (i.e., quercetin 3,4’-diglucoside-3’-(6-caffeoylglucoside) and quercetin 3,4’-diglucoside-3’-(6-sinapoylglucoside) have been also identified in eruca (bell et al., 2015). these inconsistencies could be related to the genetic material used. overall the results of the analysis of glucosinolates and flavonols evidenced how the eruca sativa gene pool contains potential candidates to use in breeding programs for quality. 3. potentiality and perspectives for breeding despite the global consumption of rocket salad has increased in the recent years, little efforts have been spent by both private and public breeding programs aimed to constitute new varieties. the importance in phytochemicals has been above discussed and novel knowledge as source of resistances have been recently described (pane et al., 2017). nowadays, constraints are mainly caused by pathogens, nitrate accumulation, early flowering and physiological disorders due to intensive culture system. accessions of eruca sativa are reported to be late-bolting (kenigsbuch et al., 2014) and to accumulate less nitrate than diplotaxis tenuifolia (cavaiuolo and ferrante, 2014), being good candidates for the improvement with respect to the latter. however, several limitations for the transfer of these useful traits are linked to the failure of intergeneric crosses between eruca and diplotaxis due to post-zygotic barriers (tripodi unpublished) resulting in the absence of a cost effective hybridization system available for rocket. moreover, interspecific crosses among diplotaxis species are difficult due to their different chromosomes number (table 1). eruca sativa x brassica rapa and diplotaxis tenuifolia x brassica rapa hybrids are instead possible using embryo rescue (agnihotri et al., 1990; jeong et al., 2009) and somatic hybridization (zhang et al., 2008) techniques, making the two rocket salad species a good source to use for the improvement of brassica rapa. the possibility of intercross has been applied in the development of cytoplasmic male sterile (cms) eruca sativa plants transferring a male sterile cytoplasm from brassica oleracea or brassica napus (merete et al., 2012). two approaches have been used: one requiring the application of embryo rescue after the first cross hybridization, subsequent chromosome doubling and backcrossing of the resulting hybrid to eruca sativa, another, using protoplast fusion from cytoplasmic male sterile brassica, subsequent regeneration of allogenic cells and crossing of the regenerated plant with pollen from eruca sativa . the same approach has been used by hosemans and leviell (2012) by transferring cytoplasmic male sterility from raphanus sativus to diplotaxis tenuifolia. raphanus sativus has been also used to transfer cms in eruca sativa (nothangel et al., 2016). despite these achievements, breeding activities are still carried out by means of traditional selection schemes such as mass selection or single seed descent. new possibilities may be obtained by tilling (mccallum et al., 2000) in order to select mutants for gene of interest or genome wide association approaches (gwas) (huang and han, 2014) for the dissection of the genetic basis of complex traits and the development of markers for breeding assisted selection. mutagenesis mediated by ethyl methanesulfonate (ems) is already reported with success in diplotaxis tenuifolia (kenigsbuch et al., 2014), resulting in a mutant showing late flowering and delayed postharvest senescence. these approaches successful in brassica species (stephenson et al., 2010; xu et al., 2016) may be also applied in rocket salad for a better exploitation of the genetic potentiality of this crop, and furthermore, to address the challenges of the modern agriculture that demands major security and quality of foods. adv. hort. sci., 2017 31(2): 107-113 112 acknowledgements the authors wish to acknowledge the project: “innovazione e potenziamento della filiera sementiera della rucola per la iv gamma (fiser)” funded through the f.e.a.s.r. european funding program (p.s.r. campania 207/2013, measure 124). references agnihotri a., gupta v., lakshmikumaran m.s., shivanna k.r., prakash s., jagannathan v., 1990 production of eruca brassica hybrids by embryo rescue. plant breeding, 104: 281-289. alqasoumi s., al-sohaibani m., al-howiriny t., alyahya m., rafatullah s., 2009 rocket “eruca sativa”: a salad herb with potential gastric anti-ulcer activity. world j. gastroenterol., 15: 1958-1965. bell l., oruna concha m.j., wagstaff c., 2015 identification and quantification of glucosinolate and flavonol compounds in rocket salad (eruca sativa, eruca vesicaria and diplotaxis tenuifolia) by lc−ms: highlighting the potential for improving nutritional value of rocket crops. food chem., 172: 852-861. bennett r.n., carvalho r., mellon f.a., eagles j., rosa e.a.s., 2007 identification and quantification of glucosinolates in sprouts derived from seeds of wild eruca sativa l. 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resources. food chem., 133: 1025-1033. stephenson p., baker d., girin t., perez a., amoah s., graham j.k., østergaard l., 2010 a rich tilling resource for studying gene function in brassica rapa. bmc plant. biol., 10(62): 1-10. taranto f., francese g., di dato f., d’alessandro a., greco b., onofaro sanajà v., pentangelo a., mennella g., tripodi, p., 2016 leaf metabolic, genetic, and morphophysiological profiles of cultivated and wild rocket salad (eruca and diplotaxis spp.). j. agric. food chem., 64 (29): 5824-5836. traka m.h., mithen r.f., 2011 plant science and human nutrition: challenges in assessing health-promoting properties of phytochemicals. plant cell., 23(7): 24832497. villatoro-pulido m., priego-capote f., alvarezsnachez b., saha s., philo m., obregon-cano s., de haro-bailón a., font r., del río-celestino m., 2013 an approach to the phytochemical profiling of rocket [eruca sativa (mill.) thell]. j. sci. food agric., 93(15): 3809-3819. xu l., hu k., zhang z., guan c., chen s., hua w., jiana l., jing w., bin y., jinxiong s., chaozhi m., jinxing t., tingdong f., 2016 genome-wide association study reveals the genetic architecture of flowering time in rapeseed (brassica napus l.). dna res., 23: 43-52. zhang c., yang z., gui x., liu y., mao x., xia g., lin l., 2008 somatic hybridization between brassica napus and eruca sativa mill. chin j. biotechnol., 24: 793-802. 32 1. introduction changes in consumers’ social environment represent a constraint to vegetable and fruit consumption and lead to convenience orientation (candel, 2001). a wide assortment of minimally processed vegetables and fruits (chunks, mousse, smoothies) has been developed to meet consumer needs for ‘‘quick’’ and convenient products, and to benefit from the healthy image of fruit and vegetables (ahvenainen, 1996). one of the main factors that influences quality of fresh-cut products is the enzymatic browning that occurs on product surfaces after cutting (garcia and barret, 2002). thus, cultivar study should be aimed at identifying cultivars which are less susceptible to browning, but also have higher nutritional and sensorial quality, covering all the season, and lead to consumer satisfaction and repeat purchase. cultivar survey represents an important step when developing a new product (cabezas-serrano et al., 2009 a, b) allowing identification of genotypes that better respond to postharvest handling and to minimal processing. peaches and nectarines are nutritionally important because they contain meaningful amounts of carotenoids including β-carotene (especially yellow-fleshed fruits), lutein and β-cryptoxanthin (gil et al., 2002). although their availability is limited by season they are one of the most important fruit commodities consumed worldwide (cantin et al., 2009). many studies on minimally processed vegetables and fruits focus on microbiological quality, safety, processing and packaging issues (foley et al., 2002; amodio and colelli, 2008) but still little information is available on varietal susceptibility. in this light, the aim of the present work was to investigate the suitability of peach varieties to be processed as fresh-convenience products throughout the peach season. 2. materials and methods from june to september 2010, 26 varieties of peach fruits from the apulia region (italy) were collected, including peaches (prunus persica l.), nectarines (prunus persica l. var nectarina) and clingstones peaches (prunus persica l. batsch). based on the ‘redhaven’ peach maturing date, peach fruits were divided into three groups: early maturing (group a), middle maturing (group b) and late maturing (group c), as shown in table 1. fruits were harvested at a commercial maturity stage, typical for each cultivar, as established by the growers. peach fruits were then transported to the postharvest laboratory (university of foggia) influence of quality attributes of early, intermediate and late peach varieties on suitability as fresh-convenience products f. colantuono, m.l. amodio, f. piazzolla, g. colelli dipartimento di scienze delle produzioni e dell’innovazione nei sistemi agro-alimentari mediterranei (prime), università degli studi di foggia, via napoli, 25, 71122 foggia, italy. key words: clingstone peaches, fruit puree, harvest season, mechanical damage, nectarines, sensorial analysis, soluble solids. abstract: fresh convenience products represent a category of minimally processed fruit and vegetables (chunks, mousse, smoothies) that respond to the changes in consumer attitudes. thanks to the image of convenience (time-saving, snack sizes, no waste, smart packaging) and healthiness their sales are steadily increasing. in this study 26 varieties (including peach, nectarines, and clingstone peaches) from apulian production were divided into three groups according to harvest dates in early (a), middle (b) and late (c) maturing. physical, chemical and sensorial analyses were performed in order to select high quality fruits for minimal processing according to the harvesting season. a multivariate principal component analysis was applied to discriminate different varieties for quality attributes. within group a, ‘honey kist’ showed the lowest acidity and intermediate susceptibility to mechanical damage. for group b, ‘stark red gold’, ‘zee glo’ and ‘venus’ resulted different in sensorial evaluation, while ‘loadel’ and ‘eolia’ were more susceptible to browning. for group c, ‘tardi belle’ and ‘baby gold7’, although more sensitive to mechanical damage, were differentiated for flavor. results of this work confirm the extreme variability among varieties in terms of sensorial quality, susceptibility to browning and to mechanical damage, and the importance of assessing varietal screening for selection of most suitable varieties for minimal processing. adv. hort. sci., 2012 26(1): 32-38 received for publication 29 september 2011 accepted for publication 22 february 2012 33 and kept under controlled temperature and humidity (5°c and 95% rh) for one day before processing. fruits were washed in a naocl solution (100 ppm), rinsed in clear water and then gently dried with a paper tissue. twenty fruits were used to assess mechanical damage, while the remaining fruits were divided into three replicates of 20 fruits and used to evaluate quality attributes. physical parameters were evaluated mainly on the whole fruit (with the exception of puree viscosity), while chemical and sensorial analysis were performed on the fruit puree. table 1 peach varieties grouped in peach, nectarine and clingstone peach type peach nectarine clingstone royal glory big bang loadel prince diamond big top eolia red elegance honey kist baby gold7 rome star amiga marilyn bigi lara zee lady ambra o’henry laura tardi belle spring bright fire top maria camilla zee glo guerriera venus stark red gold lidy star physical and chemical analysis for each variety the resistance to mechanical damage, in particular the susceptibility to impact bruising, was assessed on 20 fruits. fruits were individually impacted on one side from a fixed height (30 cm) using a free-falling steel ball (40 g and 21.4 mm diameter), and held at room temperature for 48 hr before measuring. the extension and depth of the bruise (in mm) after peeling was measured. in addition, for each variety the incidence of mechanical bruising (%) was calculated as the ratio between the number of fruits damaged and the total number of fruits considered. peach fruit firmness was assessed for each replicate, measuring the force (in n) required by a 8-mm probe to penetrate the peeled surface in two opposite regions of the fruit mesocarp, using a digital penetrometer (tr, italy). flesh fruit color was measured with a colorimeter (cm 2600d, konica minolta, japan) in the cie l*a*b* mode, taking two measurements per fruit after removing the peel. hue angle (h°) was calculated as arctg b*/a*. in order to carry out analysis on the fruit puree, fruits from each cultivar were divided into three groups of 15 fruits each, corresponding to three replicates and from each replicate three subsamples were analyzed. fruits were peeled, de-stoned, cut into big chunks and blended for 90 s. the purees were promptly transferred to sealed glass jars and stored at 5°c. a few drops of peach puree were used to measure the total soluble solids content (tss) with a digital hand refractometer (atago, japan). a small amount of fruit puree was transferred to a falcon tube and centrifuged with a centrifuge (pk 121r, thermo electron corporation, france) at 4°c for 5 min. five grams of surnatant were then used to measure the titratable acidity (ta), with an automatic titrator (titromatic 1s, crison, spain) measuring the volume of naoh 0.1n to reach a final ph of 8.1. results were expressed as percent of citric acid referred to the juice. at the same time, the ph was also measured for each puree sample. to determine the dry matter content, puree samples were desiccated in an oven at 105°c up to constant weight and the dry matter content was calculated as difference from initial weight. peach fruit puree viscosity was determined on samples at 20°c by means of a consistometer (bostwick, usa); the final results were expressed in cm * 30 s-1. sensorial analysis sensorial evaluations were carried out on the puree samples, kept at 5°c, within 3 hr after processing. puree of each variety was distributed into clear plastic cups labeled with a three-digit code. the sensorial test was performed in a sensorial laboratory with 10 trained panelists. to evaluate the intensity of aroma, freshness, sweetness and sourness, an hedonic scale, from 1 to 5, labeled 1=less intense to 5=very intense, was used. for overall quality the scale reference was 1= really poor and 5= excellent. for the level of browning, scale reference was 1= severe browning and 5= not browned. statistical analysis standard deviation was calculated on mean values for each quality attribute. principal component analysis (pca) was performed on the data. the biplot technique was used to display the relative positioning of quality attributes and cultivars according to the first two pcs. 3. results and discussion analysis of fruits in terms of physical, chemical, and sensorial attributes, revealed significant variation among the cultivars. evaluation of physical and chemical attributes the resistance to mechanical damage showed a different incidence among the cultivars. percentage of incidence to mechanical damage is shown in table 2: it was not related to the fruit harvest dates but only to the cultivar, even if cultivars in group b seemed to be more resistant to mechanical damage than the others. within group a, most 34 of the varieties showed a bruise incidence around 55%; ‘amiga’ was the most damaged variety (73.7%) whereas ‘spring bright’ did not show any evidence of damage. within group b only the clingstone peach ‘loadel’ showed a high bruise incidence (80%) whilst for the other varieties the average incidence was less than 30%. in group c all the varieties evaluated were characterized by a high bruise incidence, especially for ‘tardi belle’ (100%). no differences among the cultivars were found in bruise depth (data not shown) and only the results related to bruise extension are reported. cultivars from group a showed a great variability (table 2), while in group b the average value of bruise size was 6 mm with the exception of ‘guerriera’ and ‘marilyn’ that showed the highest (9 mm) and the lowest (2 mm) values, respectively. within group c, ‘baby7’ and ‘tardi belle’ showed the same bruise size, which was significantly higher than that of ‘o’henry’. results confirmed that bruise size and incidence of mechanical damage does not correlate with flesh firmness, as reported by mitchell and kader (1989). in fact, within the range of commercial maturity studied (table 2), varieties with the same firmness (i.e. ‘big bang’ and ‘spring bright’) showed different response to mechanical damages (55 and 0% respectively), confirming the great variability within the same peach type and among cultivars. fruit flesh color, expressed as hue angle value (table 2), was not statistically different among cultivars in groups b and c, while in group a ‘royal glory’ presented a significantly greener value (hue angle greater than 90°, in the second quadrant of l*a*b* color space) than ‘bing bang’, ‘amiga’, ‘ambra’, and ‘laura’, although showing a very low flesh firmness and acidity. fruit puree viscosity was different among the studied varieties; values ranged from 1.5 (in ‘eolia’) to 9 cm * 30 s-1 (in ‘ambra’), with the most frequent values between 4 and 5.5 cm * 30 s-1 (table 2). the high variability among cultivar viscosity was related to the maturity level; indeed in some cases it seemed influenced by fruit firmness. group a presented cultivars with higher values compared table 2 chemical and physical parameters evaluated on peach, nectarine and clingstone peach varieties for group a (early maturing), group b (middle maturing) and group c (late maturing) ripening group cultivar bruise incidence % bruise extension (mm) firmness (n) hue angle (°) viscosity (cm*30 s-1) ph ta (% citric acid) tss (°brix) a big bang 55 6±0.1 37.6±6.7 75.9±6.4 4.4±0.1 3.77±0.04 0.55±0.70 8.7±1.6 big top 50 11±0.5 43.3±9.1 85.5±8.1 4.6±0.1 3.65±0.13 0.75±0.15 11.7±0.9 honey kist 36 8±0.4 28.0±10.1 87.7±8.2 7.4±0.3 4.00±0.09 0.65±0.09 16.3±2.4 amiga 74 10±0.5 19.5±9.0 71.6±11 6.5±0.3 3.37±0.21 1.22±0.13 10.6±0.1 bigi lara 50 12±0.3 25.4±7.7 86.4±5.4 7.5±0.1 3.55±0.03 1.00±0.09 10.9±0.3 ambra 60 7±0.2 21.0±7.6 77.8±7.9 9.1±0.5 3.57±0.15 1.19±0.08 10.9±0.3 laura 29 4±0.4 18.6±8.2 79.8±3.5 8.4±0.3 3.63±0.09 1.03±0.06 10.4±0.4 spring bright 0 0±0.0 37.9±10.5 83.2±4.4 5.5±0.2 3.69±0.16 1.18±0.20 11.1±0.8 royal glory 65 11±0.3 18.8±9.5 91.4±5.0 3.5±0.2 3.85±0.10 0.68±0.11 9.9±1.0 fire top 25 3±0.1 31.2±11.0 82.8±10 3.4±0.2 3.50±0.06 1.41±0.13 11.7±0.5 b maria camilla 11 6±0.2 25.9±9.1 87.7±6.0 4.6±0.3 3.39±0.05 1.21±0.16 10.2±0.9 zee glo 25 6±0.1 16.8±4.3 85.4±3.8 7.1±0.2 3.43±0.03 1.32±0.38 11.4±1.2 guerriera 22 9±0.5 13.2±5.7 82.1±5.7 5.1±0.1 3.49±0.05 1.08±0.10 12.1±1.0 diamond princess 15 8±0.3 16.5±7.2 81.7±4.1 4.8±0.1 3.38±0.13 0.88±0.08 11.7±0.5 red elegance 25 6±0.3 61.9±13.9 84.1±2.7 2.1±0.1 3.44±0.02 0.93±0.08 12.2±0.4 rome star 0 0±0.0 40.7±11.6 81.7±4.2 4.3±0.2 3.52±0.03 0.88±0.03 12.3±0.4 loadel 80 6±0.3 32.7±6.0 83.1±4.5 4.5±0.1 3.59±0.06 0.76±0.08 13.4±0.9 eolia 25 5±0.2 42.1±14.2 79.0±4.3 1.5±0.2 3.81±0.14 0.77±0.06 12.2±0.4 venus 20 6±0.4 37.4±13.2 80.2±5.3 4.1±0.1 3.35±0.07 1.25±0.15 13.3±0.6 stark red gold 20 9±0.8 17.3±4.5 71.4±10 5.5±0.2 3.38±0.04 1.24±0.05 13.5±0.6 lidy star 30 4±0.2 13.0±3.10 78.4±4.8 5.1±0.2 3.76±0.10 0.82±0.06 13.0±0.5 marilyn 6 2±0.0 27.6±12.7 82.7±8.2 4.0±0.3 3.53±0.05 0.96±0.13 13.5±0.6 zee lady 20 4±0.2 25.8±10.6 81.5±2.7 3.5±0.2 3.46±0.07 0.96±0.09 13.1±0.3 c o’herny 64 4±0.2 62.6±8.7 80.3±4.1 7.0±0.3 3.43±0.06 0.78±0.03 13.4±0.7 tardi belle 100 5±0.1 42.9±9.3 81.6±4.3 7.5±0.1 3.59±0.07 0.69±0.08 12.8±0.4 baby gold 7 87 5±0.1 41.5±5.3 78.6±3.5 4.0±0.3 3.67±0.06 0.52±0.03 12.9±1.2 mean values±standard deviation. 35 to the other two groups, in particular ‘ambra’ and ‘laura’ (9.1 and 8.4 cm * 30 s-1 respectively ) that were characterized by a low flesh firmness (21 and 18.6 n), whereas ‘red elegance’ that showed low viscosity (2.1 cm * 30 s-1) had a high flesh firmness (61.9 n). viscosity is an important technological parameter for the formulation of smoothies and fruit purees, due to its influence on product smoothness (or thickness) which may have an effect on the mouth feel of the product. as for chemical attributes (table 2), within group a ‘amiga’ showed the lowest ph value (ph 3.37) that was significantly different from ‘honey kist’ (ph 4), which indeed were characterized by a different ta: higher for ‘amiga’ (1.22% citric acid) and lower for ‘honey kist’ (0.65% citric acid). in terms of total soluble solids (tss), ‘honey kist’ showed the highest soluble solid content (16.3°brix) even though its flesh firmness was similar to the other cultivars. the high tss value confirms its nonacid characteristics, showing the highest tss:ta ratio as well (25); this ratio is commonly used as a quality index because it is related to taste perception (byrne et al., 1991). other researchers (liverani et al., 2003) indicated that the tss:ta ratio at commercial harvest in non-acid cultivars is three to four times higher than in acid cultivars. ‘royal glory’ showed high ph value (ph 3.85) together with a low ta (0.68% citric acid) and low tss (9.9 °brix); this cultivar was significantly different from ‘fire top’ and ‘bigi lara’ that had similar ph values (ph about 3.5) and similar tss contents (11.7 and 10.9°brix respectively) but a different ta content (1.41 and 1.00% citric acid respectively). peach fruit acidity is controlled by several factors such as the cultivar, environmental conditions, canopy position, crop load and fruit maturity (crisosto et al., 1997; castellari et al., 2006). cultivars with the same flesh firmness as ‘spring bright’ and ‘big bang’ showed similar ph values (ph 3.69 and 3.77 respectively) but different ta values (1.18 and 0.55% citric acid) and tss (11.1 and 8.7°brix); this implies that at harvest not only the firmness but also the other chemical attributes must be taken into account to select the right maturity stage for harvesting. within group b, ‘maria camilla’ resulted different from the other cultivars since it showed a lower tss content (10.2°brix) with ph 3.39 and high ta (1.21% citric acid). even if it was in the range of maturity for consumption (firmness 25.9 n) it would have had a low potential impact on consumer preference due to the very low tss:ta ratio (8.4). ‘eolia’ and ‘lidy star’ showed the highest ph values (ph 3.81 and 3.76 respectively) and they were similar in ta (0.77 and 0.82% citric acid) and tss (12.2 and 13°brix). ‘eolia’ was significantly different from ‘venus’ and ‘stark red gold’ that had lower ph together with higher ta (1.25 % citric acid) and same tss (13.3 °brix). with regard to harvest date, it has been reported that medium and late season cultivars have a greater capacity to accumulate sugars compared to early season cultivars, and this is due to the non-interruption of the growing process, sugar accumulation, acid degradation and aroma synthesis (byrne, 2002). among late maturing varieties (group c), ‘o’henry’ showed lower ph value (ph 3.43) than the others, indeed it also had higher ta (0.78) than ‘baby gold7’ and no differences in terms of tss were found among them. the lowest ta value in ‘baby gold7’ gave a higher tss:ta ratio value (24.8, data not shown) with a potential high consumer preference. dry matter includes both soluble (largely sugars) and insoluble solids (mainly the structural carbohydrates and starch). as a large proportion of the dry matter at harvest is starch plus soluble sugars, its value can be related to the soluble sugars that will be contained in the ripe fruit. indeed in accordance with tss values, dry matter contents ranged between 8.6% for ‘big bang’ and 16.4 % for ‘honey kist’. burdon et al. (2004) proposed dry matter in kiwifruit as being both a maturity indicator for timing harvest and also as a predictor of the sensory quality of the fruit once ripe. results obtained in the present study on peach fruits were in accordance with this theory since peach fruit cultivars (‘honey kist’, ‘lidy star’, ‘stark red gold’, ‘baby gold7’) with high dry matter content showed a higher value of tss and were also preferred for sweetness by panelists during sensorial tests. sensorial analysis it is well documented that in peach organic acids and soluble sugars are the major determining factors of fruit taste and, together with the volatiles (responsible for the aroma), have an impact on the overall eating quality of the fruit (iglesias and echeverría, 2009). among the cultivars tested, ‘honey kist’ was the most preferred from group a together with ‘big bang’, ‘laura’ and ‘ambra’ as indicated by the overall evaluation score (table 3). the puree obtained with ‘honey kist’ was described as sweet and fresh, and received a high overall evaluation (score 4.4). varieties in group b showed differences for aroma, freshness, sweetness, and overall evaluation: ‘zee glo’, ‘guerriera’, ‘diamond princess’, ‘stark red gold’, ‘venus’, ‘lidy star’ and ‘loadel’ resulted the most pleasant with a score between 3 (intense or fair) and 4 (good). moreover, ‘maria camilla’, ‘zee glo’, ‘diamond princess’, and ‘stark red gold’ were evaluated positively in terms of color, with a score of 3.5 (slightly browned). in group c ‘tardi belle’ and ‘o’henry’ were evaluated well balanced on freshness and aroma while ‘baby gold7’ was considered the sweetest, most probably because of its high tss:ta ratio. in general, panelists disliked those varieties that were less sweet and more sour, rating them negatively (score 2-2.5) since ta plays an important role at low tss levels (<10%). when tss and ta are low even with a high tss:ta ratio (‘royal glory’), the perception of sweetness is low, as reported by crisosto et al. (2006). moreover, in the selection of new varieties, low acid content (non-acid) and a sweet taste are desirable traits, which give an acceptable flavor and result in better quality for consumers (nicotra and conte, 2003). the nectarine ‘honey kist’, a new variety, was the most appreciated due to its tss content (16.4°brix), higher than 36 the optimum level (11-12%) suggested by hilaire and mathieu, (2004) for consumer satisfaction. principal component analysis each sample from groups a and b was plotted using the first and second pc factors, which retained 69% of the total variance, while in group c the first and second pc factors retained 99% of total variance, but in this case only three cultivars were used. grouping of component loadings separated quality attributes into three groups for all maturing groups (well displayed by the biplot graphs in figure 1). fig. 1 biplot graphs for group a (early maturing) and group b (middle maturing) varieties. grouping of peaches, nectarines and clingstone peaches according to their physical, chemical and sensorial attributes determined by pca. table 3 sensorial parameters evaluated on purees of peach, nectarine and clingstone peach varieties for group a (early maturing), group b (middle maturing) and group c (late maturing) ripening group cultivar color aroma freshness sweetness sourness overall evaluation a big bang 3.7±0.5 4.4±0.8 4.3±1.1 2.6±0.8 2.9±0.9 3.0±0.8 big top 1.4±0.5 2.6±0.8 2.0±0.7 2.8±0.8 1.8±0.4 2.8±0.8 honey kist 2.4±0.9 3.8±0.8 4.2±0.8 4.0±0.0 1.6±0.5 4.4±0.5 amiga 4.2±0.8 3.6±0.5 3.2±0.4 1.6±0.5 3.4±1.5 2.4±1.0 bigi lara 2.0±1.0 3.2±0.8 2.8±0.8 2.4±0.9 1.8±0.8 2.4±0.9 ambra 5.0±0.0 4.4±0.9 3.6±0.9 2.2±0.8 3.2±0.4 3.2±0.8 laura 4.2±0.4 4.2±0.4 3.0±0.7 2.4±0.9 3.4±0.9 3.0±1.2 spring bright 2.2±0.8 2.8±0.4 2.8±1.3 2.4±0.9 3.4±1.5 2.2±0.8 royal glory 2.0±0.0 3.6±0.5 3.6±0.5 2.0±0.7 3.2±0.4 2.4±0.5 fire top 1.5±0.5 2.6±1.0 2.8±1.0 2.6±1.5 2.7±1.2 2.3±0.9 b maria camilla 3.5±1.5 3.3±0.9 3.3±1.0 3.0±1.2 2.8±0.6 3.5±0.8 zee glo 3.5±1.5 3.3±0.9 3.3±1.0 3.0±1.2 2.8±0.6 3.5±0.8 guerriera 1.6±0.5 3.7±1.0 3.2±0.7 3.4±0.9 1.8±0.7 3.3±0.9 diamond princess 3.5±0.4 3.1±1.1 3.5±0.8 2.7±0.5 3.0±1.0 3.1±0.6 red elegance 1.7±0.8 2.1±0.8 2.5±1.4 2.4±0.5 2.8±0.8 2.4±1.1 rome star 1.3±0.5 2.2±0.7 2.8±0.4 2.5±0.8 2.3±1.0 2.8±0.8 loadel 2.2±0.7 3.5±0.5 3.2±1.5 3.3±0.4 1.9±0.7 3.6±0.5 eolia 1.2±0.4 2.5±0.8 2.8±1.2 3.4±0.6 2.0±0.7 3.2±0.7 venus 2.7±1.0 2.7±0.8 2.9±1.0 2.6±0.6 3.2±0.7 2.8±0.8 stark red gold 3.5±1.0 3.0±1.2 3.6±0.9 2.7±1.0 2.7±1.0 2.8±0.7 lidy star 3.0±1.0 3.0±1.0 3.0±1.4 3.5±1.2 2.3±1.1 3.5±1.2 marilyn 1.2±0.4 3.1±1.2 2.6±1.2 2.4±0.6 2.9±0.7 2.1±0.6 zee lady 2.8±0.9 3.2±0.7 2.9±0.6 3.1±0.7 2.6±0.5 3.0±0.7 c o’herny 3.4±0.8 3.7±0.9 3.4±0.5 2.9±0.8 2.7±1.1 3.3±0.5 tardi belle 3.0±1.0 3.3±0.6 3.7±0.6 2.0±0.0 3.0±1.0 2.7±0.6 baby gold 7 2.7±0.4 3.6±0.9 3.7±1.0 4.0±0.7 1.6±0.5 3.7±0.6 intensity of aroma, freshness, sweetness and sourness scored from 1=less intense to 5=very intense; overall quality scored from 1= really poor to 5= excellent; color scored from 1= severe browning to 5= not browned. mean values±standard deviation. 37 in the early maturing cultivar biplot graph, the first group associates acidity and sourness, the second tss, sweetness, dry matter and overall evaluation, and the third viscosity, color, bruising susceptibility, aroma, and freshness. based on this positioning, opposite relationships between acidity and sensorial evaluation, and between sourness and sweetness were found. specifically, pca analysis in this study indicated that in early maturing cultivars, ‘fire top’, ‘spring bright’, ‘royal glory’, ‘big top’ and ‘bigi lara’ were positioned in the upper quadrant along with acidity since ta values were higher than for other varieties. ‘honey kist’ was in the lower right quadrant along with tss, sweetness, dry matter and overall evaluation. this positioning is in accordance with the chemical and sensorial analysis results; ‘honey kist’ showed the highest tss value (16°brix) associated with a high impact on consumer sweetness perception. cultivars ‘laura’, ‘ambra’, ‘amiga’, and ‘big bang’ were spread in the lower left quadrant according to sourness, color, bruising susceptibility, viscosity, and freshness; in particular ‘ambra’ obtained a high sensorial score for aroma and color. it could be concluded that for group a, nectarine ‘honey kist’ showed the highest tss content, the lowest acidity, and an intermediate susceptibility to mechanical damage, with a potential positive impact on consumer preference together with ‘laura’ and ‘ambra’ that retained good color after processing. in the middle maturing cultivar biplot graph the first group was associated by tss, sweetness, sensorial evaluation and bruising, the second by viscosity, freshness, color and ta, the third by sourness. within this group, an opposite relationship between sweetness and sourness, and between dry matter content and ta was observed. ‘eolia’ was in the left upper quadrant along with dry matter in accordance with chemical analysis. ‘lidy star’ and ‘guerriera’ were in the upper right quadrant for sweetness and sensorial evaluation, ‘stark red gold’ was associated with aroma due to the high overall score, while ‘loadel’ differentiated for bruising due to the high incidence of mechanical damage. ‘zee glo’, ‘diamond princess’, ‘venus’ and ‘maria camilla’ were spread around freshness, ta and color. ‘marilyn’, ‘zee lady’, ‘rome star’ and ‘red elegance’ formed a third group positioned in the lower left quadrant between sourness and dry matter, due to their similar dry matter content (13%) and to the high score received for sourness. within this group ‘stark red gold’, ‘zee glo’, ‘venus’, and ‘diamond princess’ resulted different from the others because of a higher overall evaluation and a lower susceptibility to bruising, indicating their suitability to minimal processing. on the contrary, clingstone peaches ‘loadel’ and ‘eolia’ were more susceptible to browning after blending, and although pleasant for sweetness and overall quality, their use for fresh convenience products would not lead to promising results. as for group c, the principal component analysis is not really meaningful due to the low number of cultivars. from the biplot graph (not shown) it was observed that ‘tardi belle’ was in the upper right quadrant associating with bruising susceptibility, due to the very high incidence to mechanical damage (100%). although it was the most sensitive to mechanical damage, ‘tardi belle’ differentiated from the other varieties for the well balanced flavor. ‘o’henry’ positioned in the lower right quadrant between ta and overall evaluation, while ‘baby gold7’ was in the left quadrant along with sweetness, aroma, freshness, and color, according to the sensorial results obtained. in conclusion, this work confirms the extreme variability existing among genotypes in terms of sensorial quality, susceptibility to browning and to mechanical damage, and the importance of assessing screening to select the most suitable varieties for minimal processing. from these preliminary data, the best suited cultivars for minimal processing were ‘honey kist’, ‘laura’ and ‘ambra’ from group a (early maturing), ‘stark red gold’, ‘zee glo’, ‘venus’ and ‘diamond princess’ from group b (middle maturing); cultivars from group c (late maturing) did not show many differences although this may be due to the limited number of varieties evaluated. further studies are needed in order to better understand the biochemical and technological behavior and to extend the screening to other potentially interesting varieties. references ahvenainen r., 1996 new approaches in improving the shelf life of minimally processed fruit and vegetables. trends in food science & technology, 7: 179-187. amodio m.l., colelli g., 2008 effect of thermal treatment and dipping on quality and shelf-life of fresh-cut peaches. adv. hort. sci., 22(1): 21-26. burdon j., mcleod d., lallu n., gamble j., petley m., gunson a., 2004 consumer evaluation of “hayward” kiwifruit of different at harvest dry matter contents. postharvest biology and technology, 34: 245-255. byrne d., 2002 peach breeding trends: a worldwide perspective. acta horticulturae, 592: 49-59. byrne d.h., nikolic a.n., burns e.e., 1991 variability in sugars, acids, firmness, and colour characteristics of 12 peach genotypes. j. am. soc. hort. sci., 116(6): 1004-1006. cabezas-serrano a.b., amodio m.l., cornacchia r., rinaldi r., colelli g., 2009 a suitability of five different potato cultivars (solanum tuberosum l.) to be processed as fresh-cut products. postharvest biol. technol., 53: 138-144. cabezas-serrano a.b., amodio m.l., cornacchia r., rinaldi r., colelli g., 2009 b screening quality and browning susceptibility of 5 artichoke cultivars for freshcut processing. j. sci. food agric., 89(15): 2588-2594. candel m.j.j.m., 2001 consumers’ convenience orientation towards meal preparation: conceptualization and measurement. appetite, 36: 15-28. cantin c.m., moreno m.a., gogorcena y., 2009 evaluation of the antioxidant capacity, phenolic compounds, and vitamin c content of different peach and nectarine [prunus persica (l.) batsch] breeding progenies. j. of agric. and food chemistry, 57: 4586-4592. 38 castellari l., malavolti a., colombo r., rondinelli g.p., 2006 l’impiego dei ‘‘panel test’’ nella valutazione qualitativa di alcune nettarine emiliano-romagnole. rivista di frutticoltura, 7-8: 60-63. crisosto c.h., crisosto g., neri, f., 2006 understanding tree fruit quality based on consumer acceptance. acta horticulturae, 712: 183-189. crisosto c.h., scott johnson r., dejong t., day k.r., 1997 orchard factors affecting postharvest stone fruit quality. hortscience, 32(5): 820-823. foley d.m., dufour a., rodriguez l., caporaso f., prakash a., 2002 reduction of escherichia coli o157:h7 in shredded iceberg lettuce by chlorination and gamma irradiation. radiation physics and chemistry, 63: 391-396. garcia e.l., barrett d.m., 2002 preservative treatments on fresh-cut fruits and vegetables, pp. 276-303. in: lamikanra o. (ed.) fresh-cut fruits and vegetables. science, technology and market, crc press, boca raton, florida, usa, pp. 467. gil m.i., tomas-barberan f.a., hess-pierce b., kader a.a., 2002 antioxidant capacities, phenolic compounds, and vitamin c contents of nectarine, peach and plumcultivars from california. journal of agriculture food chemistry, 50: 4976-4982. hilaire c., mathieu v., 2004 test hédoniques sur varieties de peche. d’abord, satisfaire le consommateur. infosctifl, 162: 32-35. iglesias i., echeverría g., 2009 differential effect of cultivar and harvest date on nectarine colour, quality and consumer acceptance. scientia horticulturae, 120: 41-50. liverani a., giovanini d., brandi f., merli m., 2003 le pesche subacide. l’informatore agrario, 31: 43-49. mitchell g.f., kader a.a.,1989 factor affecting deterioration rate in peaches, plums and nectarines growing and handling for fresh market. division of agriculture and natural resources, university of california, publication no. 3331. nicotra a., conte l., 2003 nuove tipologie di frutto per il mercato delle pesche: nascono la serie ‘‘ufo’’ e ‘‘ghiaccio’’. rivista di frutticoltura, 7-8; 20-25. 17 1. introduction the subgenus cerasus of the genus prunus includes more than 50 species, most of which are distributed in temperate areas in the northern hemisphere, especially in china, where 33 wild species occur (yu and li, 1986). in japan, nine native species are recorded: p. jamasakura sieb. ex koidz., p. sargentii rehder, p. verecunda (koidz,) koehne, p. incisa thumb. ex murray, p. nipponica matsum., p. apetala (sieb. et zucc.) fr. et sav., p. lannesiana (carr.) wilson var. speciosa (koidz.) makino, and p. pendula f. ascendens (makino) ohwi. in addition, three wild species, p. pseudo-cerasus lindl., p. cerasoides d. don and p. campanulata maxim., have been popularly cultivated since their introduction from china, taiwan, and nepal, respectively (kawasaki, 1991). several classifications based on morphological observations have been proposed for japanese flowering cherries (kawasaki, 1991; kobayashi, 1992; ohba, 1992), and they have been classified into five sections: apetalae (p. apetala), incisae (p. incisa and p. nipponica), sargentiella (p. jamasakura, p. sargentii, p. verecunda, and p. lannesiana), phyllomahaleb (p. maximowiczii), and microcalymma (p. pendula). the phylogenetic relationships among these taxa have been investigated using restriction fragment length polymorphism (rflp) analysis of chloroplast dna (kaneko et al., 1986), randomly amplified polymorphic dna (rapd) analysis (shimada et al., 2001), and analyses of rdna its sequences (lee and wen, 2001), ssr markers for nuclear dna (ohta et al., 2005), and plastid subtype identity (psid) sequences (ohta et al., 2006). more than 250 cultivars of flowering cherries, including prunus × yedoensis matsum. ‘somei-yoshino’ (iketani et al., 2006), have been created through repeated natural and artificial hybridizations among wild cerasus species (kawasaki, 1993). ‘somei-yoshino’ was first proposed to have arisen as a hybrid between p. lannesiana var. speciosa and p. pendula f. ascendens (wilson, 1916). takenaka (1962, 1965) produced hybrids between the two species and noted that they had similar morphological characters to ‘somei-yoshino.’ however, these hybrid plants, such as ‘amagi-yoshino’ and ‘izu-yoshino,’ were taller and produced many more flowers with white petals than ‘somei-yoshino.’ based on ssr marker analysis, iketani et al. (2007) pointed out clonal status of ‘someiyoshino,’ which has been propagated by grafting. origin of prunus × yedoensis ‘somei-yoshino’ based on sequence analysis of pola1 gene nakamura i.(1)*, takahashi h.(1), ohta s.(2), moriizumi t.(3), hanashiro y.(4), sato y.-i.(5), mii m.(1) (1) graduate school of horticulture, chiba university, matsudo, matsudo 271-8510, japan. (2) department of agriculture, shizuoka university, ohya, shizuoka 422-8529, japan. (3) bex co. ltd., itabashi, tokyo 173-0004, japan. (4) ocean exposition commemorative national government park management foundation, kunigami, okinawa 905-0206, japan. (5) future center, kyoto sangyo university, kamigamo, kita-ku, kyoto 603-8555, japan. key words: flowering cherry, phylogenetic relationships, pola1 gene, rna polymerase i largest subunit. abstract: prunus × yedoensis ‘somei-yoshino’ is the most popular cultivar of flowering cherry in japan. although the origin of this cultivar has been considered hybrid between p. pendula f. ascendens and p. lannesiana var. speciosa, the paternity of p. lannesiana has not been clearly proven by molecular analysis. to reveal the origin of ‘somei-yoshino,’ we analyzed sequences of intron 19 and exon 20 of pola1, a single-copy nuclear gene encoding the largest subunit of rna polymerase i. one of two exon 20 sequences found in ‘somei-yoshino’ was the same as that of p. pendula, whereas the other sequence was shared with several taxa in seven wild species, including p. jamasakura and p. lannesiana. ‘someiyoshino’ contained two different haplotypes of the intron 19 sequences; one was the same as that of p. lannesiana, which is endemic to the izu and boso peninsula in japan. while another haplotype of ‘somei-yoshino’ was different from that of p. pendula by two snps but identical to one of two haplotypes of p. pendula ‘komatsu-otome,’ which is a cultivar found in the ueno park, tokyo. these results indicated that ‘somei-yoshino’ probably originated by the hybridization of cultivars derived from p. pendula and p. lannesiana. adv. hort. sci., 2015 29(1): 17-23 * corresponding author: inakamur@faculty.chiba-u.jp received for publication 14 november 2014 accepted for publication 13 february 2015 18 adv. hort. sci., 2015 29(1): 17-23 previously, kaneko et al. (1986) showed that p. pendula might be the maternal parent of ‘somei-yoshino’ based on rflp patterns of chloroplast dna. a recent analysis of psid sequences provided additional support that ‘somei-yoshino’ had the 10a-t-4a haplotype specific to p. pendula, but not the 14a haplotype for p. lannesiana and p. jamasakura (ohta et al., 2006). recently, roh et al. (2007) proposed that variations for nuclear issr markers and two plastid dna sequences of the p. yedoensis population on korean jeju island overlapped those of ‘somei-yoshino.’ their data, however, did not prove the paternal origin of ‘somei-yoshino.’ utilizing the associations of nuclear dna markers dispersed over the genome, it is especially difficult to identify the paternal parent in cerasus species. because the cerasus species have complete self-incompatibility, those dna markers recombine every generation. to resolve the paternity of ‘somei-yoshino,’ we decided to compare a relatively short sequence within a singlecopy gene, because a short dna sequence is thought to be a block consisting of many closely linked dna markers, for which recombination is difficult. sang (2002) stated that the sequences of singleor low-copy nuclear genes are particularly helpful for understanding the interand intraspecific relationships of various plant groups. recently, we were interested in pola1 as a candidate single-copy gene, which encoded the largest subunit of the rna polymerase i complex. the dna sequences of intron 19 of the pola1 gene were highly polymorphic whereas the exon 20 sequences showed species-specific variations in the genera petunia (zhang et al., 2008), oryza (takahashi et al., 2009), and triticum (takahashi et al., 2010). the present study was initiated to reveal the origin of ‘somei-yoshino’ through the analysis of intron 19 and exon 20 sequences in pola1 gene. 2. materials and methods plant material most of the dna samples used in this study were provided from the faculty of agriculture, shizuoka university, and some dna samples were extracted from leaves of the clonally propagated plants that were maintained in the tama forest science garden, tokyo, japan. a total of 42 individuals of nine wild species native to japan (table 1) were analyzed; p. apetala (three individuals), p. incisa (four), p. nipponica (four), p. jamasakura (eight), p. sargentii (two), p. verecunda (four), p. lannesiana (three), p. maximowiczii (three), and p. pendula (six), and three alien wild species, p. campanulata (two), p. cerasoides (two), and p. pseudo-cerasus (one) (table 1). two cultivars, ‘somei-yoshino,’ p. pendula f. ascendens ‘komastuotome’ hayashi & nshida (hayashi, 1989), and five edohigan trees were collected in ueno park, tokyo, japan. one individual of apricot (prunus armeniaca l.) was also analyzed as out-group material. genomic dna isolation and pcr amplification genomic dna was extracted from ca. 50 mg of young leaves using a modified ctab method (doyle and doyle, 1987). the forward primer designated as 19ex5p (5’ctcgctggacggggtgagatgaatg-3’) and the reverse primer designated as 21ex3p (5’-atttactggcaatccaagacagat-3’) were designed based on pola1 gene (genbank accession no. nm_125397) of arabidopsis thaliana and est (genbank accession no. bq641151) of almond (prunus dulcis mill.), respectively. dna fragments containing intron 19 and exon 20 sequences of pola1 gene were amplified by pcr using a pair of 19ex5p and 21ex3p primers (fig. 1). the reaction mixture of 25 µl contained 10-50 ng of genomic dna, 1 unit of ex taq dna polymerase (takara co., japan), 2.5 µl of 10× buffer (100 mm tris-cl, 500 mm kcl, and 15 mm mgcl 2 , ph8.0), 2 µl of 2.5mm dntps, 1 µl of 2.0 µm each primer (19ex5p and 21ex3p), and 17.5 µl of distilled water. pcr was performed with a condition of 35 cycles of 94°c for 1 min denaturation, 58°c for 1 min annealing, and 72°c for 2 min elongation in ptc200 thermocycler (mj research co., usa). direct sequencing of pcr products containing the intron 19 and exon 20 the amplified pcr products were subjected to 1.2% agarose gel electrophoresis, purified using qiaquick pcr purification kit (qiagen co., usa), and directly sequenced with 19ex5p or 21ex3p primer used for the pcramplification by abi3100 automated dna sequencer with a bigdye terminator cycle sequencing kit (life technologies co., usa). either 20ex3p (5’-ttgaagatgttcaggtatggggag-3’) or 20ex5p (5’-ataagttgaagaaaatcac tgtgg-3’) primer were also used as an internal sequencing primer. the two internal primers were designed based on the partially determined sequences of pola1 exon 20 of cerasus in this study. the determined sequences of the intron 19 and exon 20 of pola1 gene were analyzed using a ncbi web-based blast server (altschul et al., 1990), and aligned using web server of mafft ver 6.0 (kato and toh, 2008), and then the aligned sequences were subjected to phylogenetic analysis using upgma software, with bootstrap analysis using 1,000 replicates, in the mega 4.0 (tamura et al., 2007). fig. 1 dna fragments containing intron 19 (pi19) and exon 20 (pe20) of pola1 gene were amplified using a pair of 19ex5p (pe19) and 21ex3p (pe21) primers. the sequences were determined by direct sequencing using primers for the initial amplification and internal sequence primers, 20ex5p and 20ex3p. 19 nakamura et al., origin of prunus × yedoensis ‘somei-yoshino’ based on sequence analysis of pola1 gene table 1 samples used in this study species name (z) locality pi19 (y) pe20 (y) prunus apetala (sieb. et zucc.) fr. et sav. tj063 kawaguchiko, yamanashi 507 nd. tj093 chino, nagano 507 847 tj164 hachioji, tokyo (tfsg) 507 847 p. incisa thumb. mm048 gotenba, shizoka 507 847 mm131 fujimi, nagano 507 847 mm160 amatsukominato, chiba (tfsg) 507 847   mm165 fujiyoshida, yamanashi (tfsg) 507 nd. p. nipponica matsum. tk077 shizuoka, shizuoka 507 nd. tk113 ashiyasu, yamanashi 507 nd. tk140 fujimi, nagano 507 847 tk188 kusatsu, gunma 507 847 p. jamazakura sieb. ex koidz. ym001 morimachi, shizuoka 507 nd. ym011 ishikawa forest exper. station 485,507 nd. ym038 amagiugashima, shizuoka 485 823 ym154 hachioji, tokyo (tfsg) 507 nd. ym245 kushikino, kagoshima 507 847 ym256 izumi, kumamoto 507 847 ym272 kinkai, nagazaki 507 nd.   ym277 yayoi, notsu, oita 485,507 nd. p. sargentii rehder oy024 ishikawa forest exper. station 507 nd. oy162 mamurogawa, yamagata (tfsg) 507 847 p. verecunda (koidz.) koehne ks016 ishikawa forest exper. station 507 847 ks136 fujimi, nagano 507 nd. ks183 yahiko, niigata 507 nd. ks211 nishiki, yamaguchi 507 847 p. lannesiana (carr.) wilson var. speciosa (koidz.) makino os017 ishikawa forest exper. station 507 847 os166 miyake, tokyo (tfsg) 507 847 osmtd matsudo, chiba 507 nd. p. maximowiczii rupr. my076 shizuoka, shizuoka 507 823 my139 fujimi, nagano 498 nd.   my158 chichibu, saitama (tfrg) 507 823 p. pendula maxim. f. ascendens (makino) ohwi. eh015 ishikawa forest exper. station 506 nd. eh149 ochiai, okayama (tfrg) 506 nd. eh150 takekawa, yamanashi (tfrg) 506 823 eh155 oya, hyogo (tfrg) 506 nd. eh163 oguchi, kagoshima (tfrg) 506 823 ehfsg a mountain behind tfrg 506 nd. p. pseudo-cerasus lindl. shinami sn009 ishikawa forest exper. station 507 823 p. campanulata maxim. kn014 ishikawa forest exper. station 507 823 kn101 tsukubo botanical garden 507 nd. p. cerasoides d. don. hm001 katomandu, nepal (shizuoka u.) 507 823 hm002 katomandu, nepal (shizuoka u.) 507 nd. p. armeniaca l. anz chiba university 510 823 p. × yedoensis matsum. ‘somei-yoshino’   chiba university 505, 507 823, 847 p. pendula maxim. ‘komatsu-otome’   ueno park, tokyo 505, 506 823 (z) nos. are according to ohta et al. (2006). (y) length (bp). nd= not determined. 20 adv. hort. sci., 2015 29(1): 17-23 3. results polymorphisms of the pola1 intron 19 sequences using total dna extracted from 43 individuals in ten species as template, ca. 2.2-kb-long dna fragments containing intron 19 and exon 20 of the pola1 gene were clearly amplified by pcr (fig. 2). the pola1 intron 19 sequences of most cerasus species were 507 bp in length (table 1). all six individuals of p. pendula contained 506 bp because of a one-base insertion at position 49 and a two-base deletion at position 349-350. one individual (ym038) of p. jamasakura and one individual (my139) of p. maximowiczii had shorter intron 19 lengths of 485 and 498 bp, respectively. two individuals (ym011 and ym277) of p. jamasakura had two intron 19 sequences of different lengths (485 and 507 bp), although these sequences could not be confirmed. the dna sequences described in this paper have been deposited in ddbj dna database (accession nos. lc010372lc010416). polymorphisms of the pola1 exon 20 sequences prunus pendula, p. maximowiczii, p. pseudo-cerasus, p. campanulata, p. cerasoides, and p. armeniaca (outgroup) contained an 823-bp exon 20 (table 1), whereas, seven species (p. apetala, p. incisa, p. nipponica, p. jamasakura, p. sargentii, p. verecunda, and p. lannesiana) showed a long 847-bp-long exon 20 with a 24-bp insertion. one individual (ym038) of p. jamasakura had the short exon 20 (823 bp), and two individuals (ym011 and ym277) possessed both long and short exons 20 (table 1). the dna sequences described in this paper have been deposited in ddbj dna database (accession nos. lc010540 lc010565). phylogenetic tree of the pola1 intron 19 and exon 20 sequences the 41 sequences determined for intron 19 and the 23 sequences for exon 20 were aligned using mafft and subjected to phylogenetic analysis using the upgma method in mega 4.0 with 1,000 bootstap replicates. in the phylogenetic tree for exon 20, the nine japanese wild species were classified into two groups, jamasakura and pendula (fig. 3). prunus campanula belonged to a distantly-related clade. in the jamasakura group, p. jamasakura, p. nipponica, p. incisa, p. lannesiana, and p. apetala shared a long exon 20 and formed a closely-related clade, and ten individuals of these five species contained the same sequence for the exon 20. one individual (ks016) of p. verecunda and one individual (oy162) of p. sargentii also shared an identical exon 20. one individual (ym038) possessed the short exon 20 and was distantly related to the other individuals in the jamasakura group. the remaining two species, p. pendula and p. maximowiczii, formed the pendula fig. 2 pcr products (arrow) of dna fragments containing intron 19 and exon 20 of pola1 gene in the cerasus species, 1: prunus lannesiana var. speciosa, 2: p. pendula f. ascedens, m: marker (λdna/hindiii plus øx144 dna/haeiii). fig. 3 phylogenetic upgma tree of the exon 20 sequences in the pola1 genes from 22 individuals in nine cerasus species. individuals used are listed in table 1. pcr amplification 21 nakamura et al., origin of prunus × yedoensis ‘somei-yoshino’ based on sequence analysis of pola1 gene group together with p. pseudo-cerasus and p. cerasoides. unlike the species in the jamasakura group, the four species in the pendula group were clearly differentiated from one another (fig. 3). in the phylogenetic tree for intron 19, p. pendula was positioned as the most distantly-related clade (fig. 4) because this species contained a unique insertion (position 49) and a unique deletion (positions 349-350) (fig. 5). except for p. pendula, the individuals in the jamasakura and pendula groups formed two independent clades. although the species in the pendula group were clearly differentiated from one another, concurring with the results for exon 20, most individuals of the six species in the jamasakura group, except for ym038, shared similar sequences at intron 19, and 14 individuals of the six species possessed the same intron 19 sequence. three individuals of p. lannesiana had the same sequence and belonged to an independent sub-clade (fig. 4). analysis of the origin of p. × yedoensis ‘somei-yoshino’ when the two allelic sequences of exon 20 of ‘someiyoshino’ were determined, one was identical to that of p. pendula, and the other was the same as that shared by five species in the jamasakura group. for the intron 19 sequence, the (o) haplotype of p. lannesiana was distinguished from the haplotypes of p. jamasakura by three unique singlenucleotide polymorphisms (snps) (positions 25, 101, and 171), which were also found in one of two ‘somei-yoshino’ haplotypes (fig. 5). by contrast, the other (k) haplotype was found to differ from the (e) haplotype of p. pendula by one base deletion at position 49 and one base substitution of c to a at position 392 (fig. 5). consequently, we found that p. pendula f. ascendens ‘komatsu-otome’ possessed two haplotypes (k and e): one was the same haplofig. 4 phylogenetic upgma tree of the intron 19 sequences in the pola1 genes from 40 individuals in nine cerasus species. individuals used are listed in table 1. fig. 5 haplotypes (h) of the intron 19 sequences in the pola1 genes among ‘somei-yoshino’ (ko), p. pendula (ee), ‘komatsu-otome’ (ke), p. lannesiana (oo), and p. jamasakura (yy), a: polymorphic bases and their positions in two haplotypes are shown, b: sequence charts were produced using 21ex3p primer and converted to the complementary charts using the 4peak software. 22 adv. hort. sci., 2015 29(1): 17-23 type of ‘somei-yoshino’ and the other was identical to that of a wild p. pendula individual. except for cultivars derived from ‘somei-yoshino,’ we found that ‘komatsu-otome’ and two other trees (nos. 142, 145) in ueno park had the same haplotype as ‘somei-yoshino’ (fig. 6). 4. discussion speciation of wild cerasus species in japan comparing the sequences of intron 19 and exon 20 (figs. 3 and 4), the nine wild species of japanese cerasus were clearly classified into two groups; the jamasakura group of seven species (p. apetala, p. incisa, p. nipponica, p. jamasakura, p. sargentii, p. verecunda, and p. lannesiana) and the pendula group of two species (p. pendula and p. maximowiczii). although the former group had been grouped into sections, apetalae, incisae, and sargentiella based on morphological differences (kawasaki, 1966, 1991; kobayashi, 1992; ohba, 1992), all seven species shared the same 24-bp insertion within the long exon 20 (847 bp), suggesting that they originated from the same ancestor. the remaining two wild species, p. pendula and p. maximowiczii, contained the short exon 20 (823 bp), in common with three alien wild species (p. pseudo-serasus, p. campanulata, and p. cerasoides), and with p. armeniaca (out-group). the results from the sequence analysis of exon 20 were thought to be more reliable than those for intron 19 for classifying the subgenus cerasus, and polymorphisms found in intron 19 will be useful for discriminating among closely-related taxa and cultivars. although the seven species in the jamasakura group have clearly different phenotypes, such as the apetala flower of p. apetala and dwarf stature of p. incisa and p. nipponica, these species have formed a large hybridizing population because they share the same sequences for intron 19 and exon 20. the short intron 19 found in three individuals (ym011, ym038, and ym277) of p. jamasakura might have been derived from an ancestral cryptic species. these results suggest that the classification of seven species in the jamasakura group remains to be revised based on further molecular information. origin of ‘somei-yoshino’ ‘somei-yoshino’ is the most popular flowering cherry cultivar in japan and the rest of the world. ever since wilson (1916) proposed a hypothesis for the hybrid origin of ‘somei-yoshino,’ the biological and geographical origin of this cultivar has been disputed in japan. in this study, we found that the o haplotype for intron 19 of p. lannesiana contained three unique snps, and these snps were also found in one of the two haplotypes (k and o) in ‘someiyoshino’ (fig. 5). this indicates that the paternal parent of ‘somei-yoshino’ was p. lannesiana or its cultivars. as p. lannesiana is endemic to the izu peninsula and the izu oshima islands, ‘somei-yoshino’ may have originated on the the izu peninsula (takenaka, 1962) or in edo and tokyo (iwasaki, 1989), and not on jeju island, korea (park et al., 1984; roh et al., 2007). the other (k) haplotype of intron 19 in ‘somei-yoshino’ was identical to that (e) of p. pendula, except for two snps (fig. 5). we also found that one of the two haplotypes (k and e) for intron 19 of ‘komatsu-otome’ was the same as that of ‘somei-yoshino.’ the original individual of ‘komatsuotome’ grows inside the ueno park, tokyo (fig. 6) and has a dwarf stature with pinkish flower petals. this implies that the maternal origin of ‘somei-yoshino’ is a cultivar related to ‘komatsu-otome.’ out of five trees grown in the same position with ‘somei-yoshino’ and ‘komatsu-otome’ shown in figure 6, two trees (nos. 142, 145) contained k haplotype and three (nos. 141, 142, 144) were hybrids between p. pendula f. ascendens and p. lannesiana var. speciosa. these results suggest that there were sufficient genetic resources to develop ‘somei-yoshino.’ because p. pendula and ‘komatsu-otome’ bloom two weeks earlier than p. lannesiana, ‘somei-yoshino’ and ‘komatsu-misaki’ were probably produced in tokyo through artificial hybridizations between ‘komatsu-otome’ or a related cultivar, and p. lannesiana or a related cultivar, before the end of the edo period (iwasaki, 1989). acknowledgements we would like to express our sincere thanks to professor dr. toshio ando of the graduate school of horticulture, chiba university and dr. akira kobayashi of the management office of tokyo metropolitan parks for their kind assistance during the course of this research. this work was supported by the ocean exposition commemorative national government park management foundation, okinawa, japan. fig. 6 haplotype of the pola1 intron 19 of trees (nos. 135-145) found around “komatsu no miya” statue (s) in the ueno park. ‘somei-yoshino’ (133, 134, 136, 138) and ‘komatsu-otome’ (135) share a haplotype k, p. pendula (e), p. lannensiana (o). iy, kn and kz are flowering cherry cultivars, ‘ichi-yo,’ ‘kanzan,’ and ‘kanzakura,’ respectively. 23 nakamura et al., origin of prunus × yedoensis ‘somei-yoshino’ based on sequence analysis of pola1 gene references altschul s.f., gish w., miller w., myers e.w., lipman d.j., 1990 basic local alignment search tool. j. mol. biol., 215: 403-410. doyle j.j., doyle j.l., 1987 a rapid dna isolation procedure for small quantities of fresh leaf tissue. phytochem. bull., 19: 11-15. hayashi y., 1989 komatsu-otome. hana no tomo 36: 40-41 (in japanese with latin nomenclature. iketani h., katsuki t., kawahara t., 2006 prunus × 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(eds). flora reipublicae popularis sinicae 38. science press, beijing, rp china (in chinese). zhang x., takahashi h., nakamura i., mii m., 2008 molecular discrimination among taxa of petunia axillaris complex and p. integrifolia complex based on pola1 sequence analysis. breed. sci., 58: 71-75. impaginato 81 adv. hort. sci., 2011 25(2): 81-89 (1) corresponding author: gcaruso@unina.it received for publication 22 march 2011. accepted for publication 5 may 2011. influence of crop cycle and nitrogen fertilizer form on yield and nitrate content in different species of vegetables g. caruso*(1), s. conti**, g. la rocca*** * dipartimento di scienze del suolo, della pianta, dell’ambiente e delle produzioni animali, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. (1) ** dipartimento di arboricoltura, botanica e patologia, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. *** servizi integrati per l’agricoltura, s. felice circeo (lt), italy. key words: diplotaxis tenuifolia (l.) d.c., raphanus sativus l., cucurbita pepo l., cultivation time, fertilization, production, nitric ion accumulation. abstract: research was carried out in latina province (italy) on rocket, radish and zucchini grown under tunnel. ten treatments, obtained by the factorial combination of two crop cycles (autumn-winter and winter-spring) and six nitrogen fertilizer forms (organic, organic-mineral, mineral in three modes, control with no nitrogen fertilization) were compared. the effects of these treatments were evaluated in terms of yield and nitrate content in the edible organs. in rocket, no significant difference in yield was detected between the autumn-winter and winter-spring crop cycles, although the former cycle resulted in a higher leaf nitrate content. the organic fertilizer treatment and the n-unfertilized control gave the lowest yields, but the mineral fertilizers caused the highest leaf nitrate accumulation. radish yield did not vary between the two crop cycles, but the hypocotyl nitrate content was higher in the autumn-winter cycle. the crops fertilized with the two highest mineral supplies produced the highest yields, compared with the organic or organic-mineral treatments. in the autumn-winter crop, the mineral n fertilization resulted in the highest hypocotyl nitrate content, whereas in the winter-spring crop only the highest mineral n dose caused a higher nitrate content compared with the organic fertilizer. the highest zucchini yield was obtained from the winter-spring cycle at the two highest mineral fertilizer supplies. in the autumnwinter crop the highest mineral nitrogen dose resulted in the highest fruit nitrate content, while in winter-spring the two highest supplies caused this effect. 1. introduction an adequate supply of nitrogen fertilizers is generally needed to achieve high yield and quality performance of vegetable crops (hochmuth, 1992), although a positive correlation between nitrogen availability and production has not always been shown (mccall and willumsen, 1998). the ratio between nitric and ammonium nitrogen in n fertilizers also affects yield: in a study on zucchini, chance et al. (1999) reported that a ratio between 1:0 and 1:3 is more effective than 3:1 in terms of production. on the other hand, an excessive supply of n fertilizer may have negative effects both on the quality of vegetables and on the environment (beretta et al., 1990). excessive nitrogen fertilization may also result in nitrate accumulation, especially in leafy vegetables and a high food nitrate content is considered to be potentially dangerous to human health. this occurs because 5-10% of the ingested nitrate is reduced by bacterial enzymes in saliva and the gastrointestinal tract into the more toxic nitrite ion (walters and smith, 1981) which can, in turn, react with amines and amides giving rise to carcinogenic n-nitrose compounds (hill, 1999). a positive correlation between drinking water nitrate levels and diabetes mellitus incidence in northern england was also reported (parslow et al., 1997). however, some beneficial effects of nitrate on human health were also recognized (duncan et al., 1997; addiscott and benjamin, 2000), while the high content in antioxidant compounds of some vegetables, like wild rocket (martinez-sanchez et al., 2008), can inhibit the formation of carcinogenic compounds (steinmetz and potter, 1991). in this respect, a contrasting report was published by vermeer et al. (1998) who found that despite the low 82 nitrate content of cauliflower, peas, beans and carrots, the presence of vitamin c and other antioxidants in the same vegetables could not prevent nitrosamine formation. therefore, the nitrate content alone of vegetable foods cannot be considered as a quality indicator. genetic, environmental and cultural factors affect nitrate uptake and accumulation in plants (bonasia et al., 2002). nitrate in the soil is readily taken up by the plant where it is reduced to nitrite and ultimately converted into organic compounds by the nitrate-reductase enzyme complex, whose presence in the leaves increases in response to light and to nitrate availability in the soil (guerrero et al., 1981). when the rate of nitrate uptake is faster than its assimilation rate, it is mainly accumulated in the plant cell vacuoles where is not toxic, differently from the ammonium ion (crawford and glass, 1998). nitrate accumulation rate depends on the vegetable species and on the plant organs, the highest concentrations being found in leafy/petiole species while lower nitrate contents are found in root/hypocotyl (meah et al., 1994, santamaria, 1999 b) or in fruit crops (quince and dvorak, 1980). the nitrate content also varies among cultivars within the same species: large differences were found in spinach (cantliffe, 1972), celeriac (delorez and vulsteke, 1985), lettuce (reinink et al., 1987), endive (reinink et al., 1994) and carrot (gutezeil and fink, 1999). in rocket, the nitrate content was found to increase under low solar radiation, as did the flavonoid content and antioxidant activity (jin et al., 2009). however, when this crop is grown under high light intensity, more rapid plant growth requires high organic nitrogen availability, which consequently prevents nitrate accumulation. in fact, according to padgett and leonard (1993), organic nitrogen compounds can replace nitrate as osmolyte and adjust the plant nitrate absorption. temperature also affects the uptake, translocation and assimilation of nitrate: nutrient solution heating stimulates nitrate absorption (malorgio et al., 1995) but excessive air temperature favours its accumulation in the plant tissues (behr and wiebe, 1992). as regards fertilization, ammonium and nitrate ions are the main nitrogen sources for plants. even though nitrate assimilation is energetically rather expensive, it is usually the plant-preferred form (salsac et al., 1987). in fact, a rapid ammonium uptake rate, exceeding its assimilation rate, would result in ammonium accumulation and toxicity (maynard and barker, 1969), while nitrate accumulation in plant tissues does not cause negative consequences. however, the preference between the two nitrogen forms depends on several factors: plant species, plant age, cultural method and the ratios between the concentrations of nitrogen/ammonium and other nutrients in the growth medium. in fact, while celery and fennel prove indifferent to the inorganic nitrogen form, chard is inhibited by ammonium nutrition (santamaria et al., 1999 b). in lettuce, nitrate content can be reduced by distributing a part of the whole nitrogen supply as ammonia form, without changing the overall yield results (van der boon et al., 1990) while in endive the exclusively ammonia form allows for no nitrate heads (elia and santamaria, 1997). moreover, rouphael and colla (2005) reported that a greater nitrate content in zucchini was found in soil-grown plants compared to a soilless cultural system. in the latter context, it was found that interrupting the nitrogen supply at an advanced stage of the crop cycle, the fruit nitrate content was significantly reduced (santamaria et al., 1998). this reduction appears to be related to the plant’s ability to use the nitrate previously accumulated in vacuoles for protein synthesis (blom-zandstra and lampe, 1983), in order to ensure growth when the substrate resources decrease (koch et al., 1988). finally, nitrapirine, a nitrification inhibitor, did not improve productive results in radish (mills et al., 1976). the present study was carried out to evaluate the effects of different nitrogen fertilization forms on yield and nitrate content of wild rocket, radish and zucchini in the pontina plain (latina, italy), grown under tunnel in two cultural cycles, autumn-winter and winterspring. 2. materials and methods wild rocket, radish and zucchini were grown on sandy soil (table 1) in fondi (pontina plain, latina province, italy) in 2003-2004. the crops were grown under a thermal pe tunnel equipped with anti-freeze irrigation, activated at a temperature of 5°c. the structural unit was 7.20 m wide, 40.00 m long and 2.00 to 3.50 m tall, respectively, from wall to roof. temperature and solar radiation values of the trial environment are reported in figure 1. the experimental protocol was planned in order to compare 10 treatments, which originated from the factorial combination of two crop cycles (autumn-winter and winter-spring) with six nitrogen fertilizer forms (organic, organic-mineral, mineral in three modes, control not fertilized with nitrogen). for the treatment distribution within each crop cycle, a randomized block table 1 soil characteristics fondi (latina, italy), 2003-2004 soil characteristics sand silt clay organic matter total nitrogen kjeldhal method available phosphorus olsen method available potassium ammonium acetate method total lime ph electrical conductivity (1:5) at 25°c % % % % % ppm ppm % ds·m-1 86.00 4.00 10.00 2.06 0.14 69.00 245.00 not detected 6.40 0.74 83 design with three replicates was followed. for each crop, the local standard agricultural practices were respected: plowing, hoeing, ridging, preplanting chemical weed control, drip irrigation and parasite control. the fertilization plans were scheduled according to the average nutrient requirements of the crops (tesi and lenzi, 2005) and to the expected production levels. crops were harvested at midday in all of the plots and random representative samples of the edible plant parts were collected. the samples were transferred to the laboratory, where nitrogen content was determined by high-performance liquid chromatography (hplc) using a waters 600e chromatographic system, managed through the millennium software version 3.05.01. the analytical column was a dionex anion exchange column (model as11, 4×250 mm, code p/n 044076) with a dionex pre-column (4×50 mm, code p/n 044078). the eluent was 21 mmol l-1 naoh at a flow rate of 1 ml min-1. the detector was a dionex pulsed electrochemical detector together with an anion selfregenerating suppressor. statistical data processing was performed by anova, using the duncan test for mean separation. wild rocket (diplotaxis tenuifolia (l.) d.c.) pre-planting fertilization was applied with 50 kg·ha-1 of p2o5 from mineral superphosphate 18/20 and 150 of k2o from potassium sulphate 48/50. with the exception of the n-unfertilized control, in all plots 90 kg·ha1 of n were supplied as follows: organic n fertilization was applied at pre-planting as roasted leather; mixed organic-mineral n fertilization was supplied with 45 kg·ha-1 at planting from roasted leather and 45 kg·ha-1 at dressing from ammonium nitrate 26/27; mineral n fertilization was supplied as 25 kg·ha-1 at planting from ammonium sulphate 20/21 plus 65 kg·ha-1 from ammonium nitrate during the crop cycle. organic-mineral n fertilization was applied in two phases, at six and three weeks before the expected second harvest, while the three mineral n treatments differed with respect to the application time at one, two or three weeks before the expected second harvest date. alveoli containing 10 plants each were transplanted on 25 september 2003 and on 15 january 2004 in the first and the second crop cycle, respectively. in plots of 12.60 m2 (3.00 x 4.20 m) three ridges were made 0.40 m apart from each other, which included six rows with spacing 0.20 m, with a density of 215 plants per m2. rocket was harvested in two phases, between 13 november and 9 january (2003-2004) in the first cycle and from 14 march to 28 april 2004 in the second cycle, according to the fertilization treatment. this crop was hand harvested by cutting the plants at about 2 cm above the soil surface when they reached 18 cm in height; crop weight was measured. at the second harvest, a random sample of 100 plants was collected in each plot and transferred to the laboratory for nitrate determination. radish (raphanus sativus l. subsp. parvus) radish cv. suprella, a commonly grown variety in the experimental area, was sown on 24 november 2003 in the first crop cycle and on 2 march 2004 in the second. the plots had a 5.20 m2 (2.60 x 2.00 m) surface area including 0.80 m wide beds, with 0.10 x 0.05 m spacing between plants. pre-planting fertilization was applied with 80 kg·ha1 of p2o5 from mineral superphosphate 18/20 and 100 kg·ha-1 of k2o from potassium sulphate 48/50. in addition, 100 kg·ha-1 n, corresponding to the crop requirement, was supplied to all plots with the exception of the n-unfertilized control: organic n fertilization was applied at pre-planting as roasted leather; mixed organic-mineral n fertilization was applied in two phases as 50 kg·ha-1 n as roasted leather at pre-planting plus 50 kg·ha-1 n as ammonium nitrate at 20 days after sowing. the mineral n fertilization treatments supplied 70, 100 or 130 kg·ha-1 n: one-half of the total n supply was given at pre-planting as ammonium sulphate while the remaining 50% was given as ammonium nitrate in two applications, at 12 and 24 days before the expected harvest date. therefore, the range of mineral n fertilization treatments supplied the intermediate amount (100 kg·ha-1) +/a 30% increase or reduction. radish crops were harvested 16-22 january and 611 april in 2004, depending on the crop cycle and fertilization treatment. radish plants were manually harvested when the hypocotyls reached the 30-40 mm caliber: weight, number and average weight data were recorded. moreover, a sample of 100 units was collected in each plot and transferred to the laboratory for nitrate assessment. zucchini (cucurbita pepo l.) zucchini cv. velvia, a commonly grown variety in the experimental area, was used for this study. the plants were transplanted on 23 september 2003 in the fig. 1 trend of temperature and solar radiation. fondi (latina), 20032004. 84 first cultural cycle and on 20 january 2004 in the second. each plot was 35.10 m2 (5.40 x 6.50 m) and plant density was 1.71 plants per m2 (1.80 m between double rows and 0.65 m between plants along the row). the fertilization doses were adjusted to the expected yields, which changed with the crop cycle: 60 kg·ha1 of p2o5 from mineral superphosphate 18/20 and 240 of k2o from potassium sulphate 48/50 were supplied at planting for the autumn-winter cycle; while 120 kg·ha1 of p2o5 and 480 of k2o were supplied at planting for the winter-spring cycle. in addition, nitrogen fertilization supplied 150 or 300 kg·ha-1 n for the autumn-winter or the winter-spring cycle, respectively. n fertilization was supplied as follows: organic n fertilization was applied at pre-planting as roasted leather; organicmineral n fertilization was applied in two phases (50% at planting as roasted leather and 50% at dressing as ammonium nitrate). mineral n fertilization treatments supplied 105, 150 and 195 kg·ha-1 or 210, 300 and 390 kg·ha-1 to the autumn-winter or winter-spring crop, respectively. one-third of the total n dose was applied at planting (35, 50, 65 kg·ha-1 as ammonium sulphate in the autumn-winter cycle or 70, 100, 130 kg·ha-1 in the winter-spring crop) while the remaining two-thirds of the total n dose was applied during the crop cycle (70, 100, 130 or 140, 200, 260 kg·ha-1 as ammonium nitrate). therefore, the range of mineral n fertilization treatments supplied an intermediate rate (150 or 300 kg·ha-1) +/a 30% increase or reduction. the n fertilization at dressing was evenly distributed in three applications with a 21-day interval. fruits were hand harvested when the corolla was open, from 23 october to 22 december 2003, and from 29 march to 27 may 2004, depending on the crop cycle and fertilization treatment. fruit weight, fruit number and average fruit weight were recorded. in addition, 10 days after the last fertilizer application a sample of 20 marketable fruits was collected from each plot and transferred to the laboratory for nitrate assessment. 3. results no significant difference in rocket yield was found between the autumn-winter and winter-spring crop cycles (data not shown). in the first cycle, however, the leaf nitrate content was as much as 64.3% higher than in the winter-spring. table 2 shows that in the autumn-winter cycle there was no yield difference between the mineral and mixed organic-mineral n fertilization forms. however, the mineral and organic-mineral treatments produced better results compared with the organic n fertilization and with the n-unfertilized control. in rocket, mineral n fertilization resulted in the highest leaf nitrate content compared with the other treatments, while the n-unfertilized control resulted in the lowest. in the winter-spring cycle (table 3), the production trend was similar to that observed in the autumn-winter cycle. however, organic-mineral fertilization did not produce significantly different results from the organic n fertilization treatment. the mineral and organic-mintable 2 wild rocket under tunnel: yield results and leaf nitrate content in the autumn-winter cycle, as influenced by nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: one week before the second harvest mineral 2: two weeks before the second harvest mineral 3: three weeks before the second harvest marketable yield (t·ha-1) leaf nitrate content (mg·kg-1 of fresh weight) 10.3 c 13.9 b 15.4 a 16.0 a 16.3 a 16.4 a 2706.7 c 3866.7 b 4240.3 b 5032.0 a 5150.0 a 5317.7 a means followed by different letters are significantly different according to the duncan test at p≤0.05. table 3 wild rocket under tunnel: yield results and leaf nitrate content in the winter-spring cycle as influenced by nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: one week before the second harvest mineral 2: two weeks before the second harvest mineral 3: three weeks before the second harvest marketable yield (t·ha-1) leaf nitrate content (mg·kg-1 of fresh weight) 11.0 c 15.1 b 16.8 ab 17.3 a 17.4 a 17.6 a 1626.0 c 2322.8 b 2838.3 a 2842.2 a 3083.3 a 3300.0 a means followed by different letters are significantly different according to the duncan test at p≤0.05. 85 eral n fertilization treatments resulted in higher leaf nitrate content compared with the organic n fertilizer application or with the n-unfertilized control. radish yield did not vary between the two crop cycles: the hypocotyl number per unit surface area and their average weight were unaffected by the crop cycle factor (table 4). in contrast, n fertilizer form significantly affected the edible organ production: mineral fertilization at the two highest doses was more effective than the organic and the organic-mineral n fertilizer forms. the organic n supply had better effects on yield, compared to n-unfertilized control, but it was less effective than the other treatments. these results were mainly affected by the average hypocotyl weight, which changed significantly in response to the different n fertilization forms while only the unfertilized control resulted in a reduced hypocotyl number. similarly to rocket, also in radish (table 5) nitrate content was higher in the autumn-winter cycle, on average as much as 25.3% compared to the winter-spring cycle. in the autumn-winter cycle, leaf nitrate content was higher in response to organic-mineral and mineral n fertilization. instead, during the winter-spring cycle the highest mineral nitrogen supply caused a nitrate concentration increase only in comparison with the organic form. moreover, in the winter-spring cycle, the maximum mineral fertilizer dose (130 kg·ha-1 equally distributed before and after planting) resulted in a three-times higher leaf nitrate content compared with the unfertiltable 4 radish under tunnel: hypocotyl yield as a function of crop cycle and nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment crop cycle autumn-winter winter-spring nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 70 kg·ha-1 mineral 2: 100 kg·ha-1 mineral 3: 130 kg·ha-1 marketable hypocotyl yield no. per m2 mean weight (g) means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) 26.9 27.4 ns 20.8 d 25.4 c 27.9 b 28.5 ab 30.0 a 30.5 a 139.9 137.0 ns 128.2 b 138.0 a 140.0 a 140.2 a 142.2 a 142.3 a 19.2 20.0 ns 16.2 e 18.4 d 20.0 c 20.4 bc 21.1 ab 21.5 a table 5 radish under tunnel: hypocotyl nitrate content as a function of crop cycle and nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrate content (mg·kg-1 of fresh weight) autumn-winter winter-spring nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 70 kg·ha-1 mineral 2: 100 kg·ha-1 mineral 3: 130 kg·ha-1 1020.3 d 1229.7 c 1431.7 b 1670.0 a 1697.0 a 1743.0 a 478.3 c 1038.3 b 1258.3 ab 1311.7 ab 1357.3 ab 1571.7 a means followed by different letters are significantly different according to the duncan test at p≤0.05. table 6 zucchini under tunnel: yield results and fruit nitrate content in the autumn-winter cycle as a function of nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 105 kg·ha-1 mineral 2: 150 kg·ha-1 mineral 3: 195 kg·ha-1 marketable fruit yield no. per plant fruit nitrate content (mg·kg-1 of fresh weight) 13.8 e 19.9 d 23.1 c 23.9 bc 25.8 ab 26.9 a means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) mean weight (g) 9.3 c 12.0 b 13.0 ab 13.3 ab 14.0 a 14.3 a 87.1 c 97.5 b 103.8 ab 105.7 ab 107.5 a 110.6 a 496.7 e 638.7 d 723.7 d 850.0 c 1022.0 b 1186.0 a in both crop cycles, the highest zucchini production was obtained with the two highest mineral fertilizer supplies (tables 6 and 7). however, in the autumn-winter cycle (table 6) the intermediate dose was not different from the lowest one and the latter was as effective as the organic-mineral treatment in both crop cycles. the control treatment resulted in lower fruit number and average weight while the mineral n fertilization produced the highest values. the winter-spring yield (table 6 and 7) was more than double the autumn-winter yield, as a consequence of the increased fruit number (+100%), while their ized control, whereas in the autumn-winter cycle a 71% increase was recorded between the same two treatments. 86 average weight was not significantly different. in the autumn-winter cycle (table 6), the maximum mineral n rate and the unfertilized control resulted in the highest and lowest fruit nitrate concentrations (1186 vs 497 mg·kg-1), respectively. furthermore, no difference was detected between the organic and organicmineral n fertilization treatments. in the winter-spring cycle (table 7), the mineral n treatments led to a greater fruit nitrate accumulation compared with the control and the organic n fertilization treatment. 4. discussion and conclusions in the present investigation rocket and radish did not show yield differences between the autumn-winter and winter-spring cycles. this is presumably due to the light and temperature requirements of these crops which allow for an equally good production in both seasons. in fact, the two crop periods are quite similar in terms of duration and day-length, though the latter decreases from the beginning to the end of the autumn-winter cycle while the opposite trend occurs in winter-spring. our results are in accordance with those reported by inada and yabumoto (1989) who found that growth of a radish crop was promoted by increasing day-length, but it was less sensitive to variations in temperature regime. differently from rocket and radish, zucchini yield was affected by the cultural cycle, as the winter-spring cycle resulted in a higher fruit production compared with the autumn-winter cycle. presumably, the increasing daylength, light intensity and temperature of the second part of the winter-spring cycle played a crucial role in improving crop productivity. similar results were reported by rouphael and colla (2005). as regards n fertilization, the wild rocket yield was favourably affected by the mineral fertilizer treatment as well as the organic-mineral treatment. a contrasting report was published by cavarianni et al. (2008) who found that an increasing mineral n supply caused the rocket yield to decrease. differently from rocket, radish showed a positive response to nitrogen increase, both in organic-mineral and in mineral form. guven (2002) reported that radish yield benefited from an increase of inorganic nitrogen table 7 zucchini under tunnel: yield results and fruit nitrate content in the winter-spring cycle as a function of nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 210 kg·ha-1 mineral 2: 300 kg·ha-1 mineral 3: 390 kg·ha-1 marketable fruit yield no. per plant fruit nitrate content (mg·kg-1 of fresh weight) 30.3 d 39.9 c 47.0 b 48.3 b 54.5 a 55.0 a means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) mean weight (g) 19.3 c 23.3 b 25.7 ab 25.7 ab 28.0 a 28.0 a 91.3 d 100.3 c 107.6 b 109.7 ab 113.6 ab 115.5 a 331.7 c 426.7 bc 443.0 bc 536.3 ab 606.0 a 678.3 a supply, while fuke et al. (2000) reported a better effect of the mixed organic-mineral fertilization compared with the exclusively mineral treatment. in the present work, the organic n fertilization was less effective on radish production than the other examined treatments, although it produced better results than the unfertilized control. yield was influenced almost exclusively by the hypocotyl average weight, which gradually decreased from the highest nitrogen rate to the unfertilized control. the number of marketable edible organs was conditioned instead only by the absence of n in the control treatment, which resulted in 9% deformed or undersized hypocotyls. the zucchini yield in both crop cycles was better affected by mineral nitrogen fertilization achieved with the intermediate or 30% increased rate, compared to the organic-mineral or organic fertilization forms. nevertheless, in the autumn-winter cycle the intermediate mineral supply did not produce better yield results than the 30% reduced treatment. zotarelli et al. (2008) reported that, in the same cultural cycle, a 50% increased n supply, compared to the crop requirement, did not modify zucchini yield but a 50% decrease was less effective. moreover, the reduced mineral nitrogen application did not result in a better production than the organic-mineral supply while, as recorded also for wild rocket and radish, the organic n treatment accelerated zucchini plant development only compared to the control. however, it should be stressed that the lower yield in the organic n fertilization treatments resulted from a combination of the production factors. in fact, these treatments did not affect the fruit emission rate nor their size. termine et al. (1987) reported that organic n fertilization did not condition the production level of leek and turnip. in this study the tested species showed a different attitude toward accumulation of nitrate in their edible organs. in particular, rocket was the only crop that in the autumn-winter cycle displayed nitrate levels above the highest ec regulation limits (4500 mg of no3 -·kg-1 of fresh weight), confirming also the influence of cultivation time on vegetable nitrate content. a clear tendency to accumulate more nitrates in the autumn than in the spring was shown, the latter being characterized by low cloud cover and increasing photoperiod. this is in agreement with the reports of rouphael and colla 87 (2005) on zucchini and elia et al. (1997) on broccoli rab grown in growth chamber and subjected to additional lighting. in the latter case, the leaf blade nitrate reduction was a result of nitrate-decreased absorption rather than its assimilation increase. in fact, in spinach (steingröver et al., 1986 b), parallel to the leaf blade nitrate reduction, the net nitric ion adsorption was reduced while the nitrate-reductase activity did not change except at the end of the night period. this effect would not result from direct light inhibition, but from an adsorption feedback regulation achieved by the amino acids formed in the leaf blades and then transported to the roots. moreover, it was found that under low solar radiation, typical of the winter season, the nitrate content is high even with a low nitrogen supply, whereas with high solar radiation the ion concentration increases only by supplying nitrogen (roorda van eysinga and van der meijs, 1985; santamaria et al., 1999 a). this indicates that solar radiation intensity and photoperiod length regulate the nitratereductase activity, the enzyme which reduces nitrate to nitrous ion (steingröver et al., 1986 a). in particular, the solar radiation effect is multiple, as it gives input to the nitrate-reductase synthesis and induction, supplying also the reducing power (nadh) through photosynthesis (behr and wiebe, 1992). it is believed that in the vacuole, nitrate acts as the cell turgor osmotic regulation, as an alternative to sugars and organic acids, poorly synthesized under low radiation conditions (blom-zandstra and lampe, 1985). plant nitrate concentration is known to be also subject to the available nitrogen amount and quality (citak and sonmez, 2010), and this explains why the unfertilized control plants always exhibited the lowest tissue nitrate levels. only in the zucchini fruits harvested in spring, values were not different between the control and organic or mixed treatments, confirming this species’ lower attitude. on the contrary, wild rocket displayed a remarkable tendency to accumulate nitrates in the aerial apparatus: in the unfertilized plants, a nearly triple (2.9) and more than five-fold (5.2) content was detected, compared with the corresponding control of radish and zucchini, respectively. according to quinche and dvorak (1980), the latter accumulate less nitrate because they receive organic nitrogen mainly through the phloem, which does not carry inorganic nitrogen forms. in other studies, rocket was reported to accumulate high nitrate levels both under reduced nitrogen availability (bianco et al., 1998; santamaria et al., l999 a) or increased supply (santamaria et al., 2002). organic fertilization, compared to the other supply forms, caused the lowest nitrate content in the edible organs; it was only higher than the unfertilized control (as much as 29% in zucchini and 51% in radish on average). similar results were reported for lettuce (stopes et al., 1989) and radish (ebid et al., 2008) crops fertilized with composted manure. the organic-mineral treatment resulted in nitrate content increases between 41% in zucchini and 80% in radish compared with unfertilized plants. therefore, even in such circumstance zucchini demonstrated to be a refractory species to nitrate accumulation. the mineral nitrogen fertilization caused the highest nitrate content in the edible organs: compared to the non-fertilized control, the increase was +90.2, +108.0 and +96.3% on average, respectively in rocket, radish and zucchini. santamaria et al. (1993) reported that mineral nitrogen caused a higher nitrate accumulation than the organic-mineral form in spinach. nevertheless, in the autumn-winter cycle a 30% reduction of the mineral nitrogen dose resulted in a lower nitrate accumulation, compared to the maximum and to the intermediate supply, respectively in radish and in zucchini. previously, other researchers found a direct relationship between the nitrogen rate supplied and the vegetable nitrate content (maynard et al., 1976; tesi et al., 1995; cavarianni et al., 2008). with regard to the time of fertilizer supply, in rocket, in both crop cycles, the latest n dressing application (one week before harvest) did not cause a higher leaf nitrate accumulation than the earliest one. opposite findings were reported by graifenberg et al. (1990) for lettuce: leaf nitrate content increased with the reduction of the interval between nitrogen fertilizer application and harvest. in conclusion, the studies carried out in the pontina plain (latina, italy) under tunnel, in 2003-2004, showed that the cultivation time produced a significant effect only on zucchini yield, which was better affected by the winter-spring cycle. moreover, the autumn-winter crops exhibited higher nitrate content in the edible organs. as for the fertilization treatments, in rocket n fertilization in organic-mineral form was the most appropriate, as it gave yield values as high as the exclusively inorganic supply. in addition, the organic-mineral fertilizer resulted in a lower leaf nitrate accumulation in the autumnwinter cycle, which did not exceed the ec threshold. in contrast, in radish and zucchini the two mineral supplies corresponding to nitrogen crop requirement or to its 30% increase produced the best yield results. moreover, they also caused the highest radish hypocotyl nitrate content, which was however much lower than the limits set by some european countries for vegetable trade (santamaria, 2006). acknowledgements this research was funded by lazio region in the frame of the project “accumulo dei nitrati nelle principali specie ortive dell’agro pontino”. the authors thank dr. christopher latham for his help with the english language, and mr. roberto maiello for his assistance with laboratory analyses. references addiscott t., benjamin n., 2000 are you taking your nitrate? food sci. techn. today, 14(2): 59-61. 88 behr u., wiebe h.j., 1992 relation between photosynthesis and nitrate content of lettuce cultivars. sci. hortic., 49: 175-179. beretta g.p., bonomo c., nurizzo c., 1990 nitric ion contamination of water table. inquinamento, 1: 54-61. bianco v.v., santamaria p., elia a., 1998 nutritional value and nitrate content in edile wild species used in southern italy. acta horticulturae, 467: 71-87. blom-zandstra m., lampe 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of lettuce as affected by total nitrogen and chloride concentration, nh4/no3 ratio and temperature of the recirculating nutrient solution. j. hort. sci., 65: 309-321. vermeer i.t.m., pachen d.m.f.a., dallinga j.w., kleinjans j.c.s., van maanen j.m.s., 1998 volatile n-nitrosamine formation after intake of nitrate at the adi level in combination with an amine-rich diet. environ. health perspect, 106: 459-463. walters c.l., smith p.l.r., 1981 the effect of water-borne nitrate on salivary nitrite. food. chem. toxicol., 19: 297302. zotarelli l., dukes m.d., scholberg j.m., hanselman t., le femminella k., muñoz-carpena r., 2008 nitrogen and water use efficiency of zucchini squash for a plastic mulch bed system on a sandy soil. sci. hortic., 116(1): 8-16. impaginato 97 1. introduction pomegranate (punica granatum l.) is a nutrient dense and one of the most popular fruits native to iran. with a production of 700,000 tons/year, iran is the world’s leading producer (sarkhosh et al., 2009). historical evidence reveals that the primary origin of pomegranate is iran and that it has been spread from this region to other areas (levin, 1996). a large number of pomegranate varieties can be found in iran, more than 760 original, wild and decorative cultivars (mousavinejad et al., 2009). considering lack of water resources and intensification of abiotic stresses such as drought and salinity, importance of pomegranate has increased in recent years, since this species is a tolerant fruit crop and thrives well under arid and semi-arid climatic conditions (la rue, 1980). previous investigations indicate varied levels of tolerance to abiotic stress conditions such as drought and salinity among different pomegranate cultivars (tabatabaei and sarkhosh, 2006; okhovatianardakani et al., 2010; ibrahim, 2016) because abiotic stress tolerance is a complex parameter and depends on both genetical and physiological properties. differences in adaptability potentials to prevailing environmental conditions and abiotic stresses tolerance among plant species or cultivars including pomegranate can be attributed to their varied ability for nutrients uptake and subsequent different concentration of macro and micronutrients within plant organs and tissues (jamali et al., 2015). nutrients deficiencies or imbalances exert secondary, often unpredicted influences on the growth of plants by changes in growth pattern, chemical composition, and antioxidant defense capacity of plants and particularly decrease the resistance of plants to biotic and abiotic environmental stresses (hajiboland, 2012). macronutrients are mainly important structural components of plants and their optimum concentrations result in improved growth (marschner, 1995). activity of enzymes and/or production of some metabolites involving in the plants response to their surrounding environment or even modulations in the signal transduction pathways are under micronutriadv. hort. sci., 2017 31(2): 97-105 doi: 10.13128/ahs-20488 evaluation of adaptability potential of seven iranian pomegranate cultivars in southern iran, arsenjan region b. jamali (*), a.r. bonyanpour horticulture crops research department, fars agricultural and natural resources research and education center, agricultural research, education and extension organization, shiraz, iran. key words: adaptability, macro and micronutrients, punica granatum, tolerance. abstract: present study was carried out to compare leaf mineral composition and some physiological parameters in seven iranian pomegranate cultivars for evaluation of their adaptability differences in arsenjan region/fars province/southern iran and selecting probable more abiotic tolerant cultivars in this region. uniform and healthy rooted plants of seven commercial pomegranate cultivars were purchased from a commercial nursery and planted in a completely randomized block design in an orchard site in arsenjan region. after full establishment the orchard samples (fresh leaves) were taken and transferred to lab for analysis. cultivars included: malas yousefkhani saveh, naderi badroud, malas daneh ghermez yazd, rabab neiriz fars, shirin shahvar fars, shirin poust daneh ghermez and zard anar arsenjan. significant differences were found among studied pomegranate cultivars for concentrations of leaf potassium, calcium magnesium, sodium and iron concentrations. also parameters such as leaf total chlorophyll and carotenoids content, total sugars, relative water content and electrolyte leakage, α-tocopherol and ascorbic acid concentration were significantly different in studied cultivars. ‘zard anar arsenjan’, an indigenous cultivar of the region, and ‘rabab neiriz fars’ were evaluated as probable more tolerant cultivars in comparison to other cultivars. this can be attributed to their more optimized leaf mineral composition and antioxidant statues. (*) corresponding author: babakjamali@ymail.com received for publication 29 march 2017 accepted for publication 25 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 97-105 98 ents influence (hajiboland, 2012). cultivars with a higher capacity for macro and micronutrients absorption possess a more efficient growth and development cycle, with a better adaptability to environmental conditions (jamali et al., 2015). previous literatures on comparison of differences in leaf mineral composition and also physiological characteristics in various iranian pomegranate cultivars are limited (in comparison to other important crops) and more investigations for evaluation of adaptability potential in different pomegranate cultivars seem necessary. so the goal of the present study was to compare leaf mineral composition and also some physiological and biochemical parameters in seven iranian cultivars for a deeper understanding of their adaptability differences and selecting probable more tolerant cultivars in arsenjan region. 2. materials and methods orchard and plants uniform and healthy rooted plants of seven commercial iranian pomegranate cultivars were purchased from a commercial nursery and planted in a completely randomized block design with 3 replications (each replication had 3 plants) with 3 m distance in rows and 5 m distance between rows in an orchard site in arsenjan region (hub of pomegranate growing and production in fars), fars province, iran. average annual climate parameters in the experimental region were; precipitation: 200 mm, relative humidity (max: 55%, min: 23%), temperature (max: 38, min: 4°c). the soil of the orchard was sampled at two depths and analyzed for soil texture, mineral content, organic matter, ph and ec (table 1). cultivars included: malas yousefkhani saveh (mys), naderi badroud (nb), malas daneh ghermez yazd (mdgy), rabab neiriz fars (rnf), shirin shahvar fars (ssf), shirin poust daneh ghermez (spdg) and zard anar arsenjan (zaa). after 4 years and full establishment of the collection orchard, samples (fresh leaves) were taken from the trees. leaf samples were taken from different orientations of the trees (north, south, west and east); 25 fully expanded mature leaves from each side of all trees (100 leaves per tree as bulk samples), and transported to laboratory. leaves were taken from shoots without terminal fruit. the leaves were of spring bloom, the middle third of the branch, at a height of between 1-1.5 m including the petiole. leaves with abnormal symptoms such as chlorosis and mechanical lesions caused by pests or diseases were avoided. the trees were grown under drip irrigation; water quality parameters are presented in table 2. routine cultural practices suitable for commercial fruit production were carried out during experimental period. the following parameters were measured in studied cultivars for two consecutive years and an average was reported. measurements trunk circumference. circumference of trunks was measured 5 centimeter above ground and expressed as centimeter. leaf dry matter content. three uniform leaves were selected and washed with tap and distilled water, after drying with clean towel they were weighed with digital scale and then were oven dried for 72 hours in 70°c and weighed. leaf dry matters percentage was calculated by the following formula (eshghi and jamali, 2009): leaf dry matters (%) = [leaf dry weight (g)/leaf fresh weight (g)] × 100 leaf water content. leaf relative water content (lrwc) was measured by using ten leaf discs. the leaf discs of each treatment were weighed (fw). they were then hydrated until saturation (constant table 1 analysis of soil samples in the experimental region soil depth (cm) soil texture soil mineral content (mg kg-1) organic carbon (%) ec (ds m-1) ph nitrate ca mg k fe zn mn 0-30 loamy clay 32 1200 150 150 8.4 1 8.7 0.75 0.82 7.6 31-60 loamy sand 40 1150 150 140 7.32 0.95 7.1 0.65 0.71 7.6 table 2 analysis of quality parameters of irrigation water parameters value ec (ds m-1) 0.91 ph 7.1 na (%) 34.75 cl (mg l-1) 45.71 sar 3.5 tds (mg l-1) 590 jamali and bonyanpour adaptability of seven iranian pomegranate cultivars in southern iran 99 weight) for 48 h at 5°c in darkness (tw). leaf discs were dried in an oven (dw). relative water content was calculated according to the following expression (jamali and eshghi, 2015): lrwc% = (fw-dw)/(tw-dw) × 100 leaf electrolyte leakage. leaf electrolyte leakage (el) was determined by recording the electrical conductivity (ec) of leaf leachates in double distilled water at 40 and 100°c. leaf samples were cut into discs of uniform size and taken in test tubes containing 10 ml of double distilled water. the test tubes were kept at 40°c for 30 min and at 100°c in boiling water bath for 15 min and their respective electric conductivities (ec1 and ec2) were measured by conductivity-meter (metrohm conductometer 644, switzerland) (jamali et al., 2015): electrolyte leakage (%) = (ec1/ec2) × 100 leaf total sugars. the total leaf soluble carbohydrates were determined according to irigoyen et al. (1992) and glucose (0-100 mg l-1, from merck) was used as a standard. leaf samples of 0.5 g (dry weight) were homogenized in 5 ml ethanol (95%) and centrifuged at 4500 × g for 15 min, the supernatant was removed from the sample and the residue was resuspended in 5 ml of 70% ethanol. then the supernatant was centrifuged again for final extraction. both supernatants were combined. anthrone-sulfuric acid assay was used for determination. an aliquot of 100 μl was added to 3 ml of anthrone-sulfuric acid solution and the mixture was shaken, heated in a boiling water bath for 10 min and cooled at 4°c. the absorption at 625 nm was determined by spectrophotometer. leaf chlorophyll and carotenoids concentration. leaf discs of 0.5 g were extracted in 5 ml of acetone (80%), then centrifuged for 10 min in 8,000 × g. the supernatant was used to make a final volume of 100 ml of the leaf extract. extraction of leaf tissue with the buffer continued until decoloration. absorbance of the extract was read at 470, 645 and 663 nm with a spectrophotometer and 80% acetone was used as a blank. finally, chlorophyll and carotenoids contents was calculated according to the following equations (lichtenthaler, 1987): chl a (mg. g-1 fresh weight): [(12.25a663 2.79a645) × v / 1000 × w] chl b (mg. g-1 fresh weight): [(21.50a6455.10a663) × v / 1000 × w] chla + chlb (mg. g-1 fresh weight): [(7.15a663 + 18.71a645) × v /1000 × w] carotenoids (mg. g-1 fresh weight): 1000a4701.82chla85.02chlb / 198 where chla = chlorophyll a; chlb = chlorophyll b; chla+b = total chlorophyll; a = absorbance at ƛ (nm). leaf anthocyanins concentration. leaf total anthocyanins were measured spectrophotometrically by ph differential method with two buffer systems: potassium chloride buffer, ph 1.0 (0.025 m) and sodium acetate buffer, ph 4.5 (0.4 m). 0.5 g leaf samples were extracted with 2 ml methanol: water: concentrated hcl solution (80:20:1 v/v/v). 0.4 ml of leaf extract was mixed with 3.6 ml of corresponding buffers and read against water as blank at 510 and 700 nm. absorbance (a) was calculated as a = (a515 a700) ph 1.0 (a510 a700) ph 4.5 then total anthocyanins content was calculated using the equation: anthocyanin (µg. g-1 fresh weight) = (a × mw × df × 1000) / e where a is the absorbance of the diluted sample and df is the dilution factor (10), mw is molecular weight of cyanidin-3-glucoside (449.2) and e= 26,900 l/mol.cm, molar extinction coefficient of cyanidin-3glucoside. leaf total polyphenols. leaf polyphenols was determined with folin-ciocalteu reagent using gallic acid as a standard phenolic compound. in brief, 1 g of lyophilized leaf samples were placed in an eppendorf tube, with 1 ml of methanol (80%), grinded at 4°c and centrifuged at 10000 × g for 15 min. the extract was mixed with 0.5 ml of folin-ciocalteu reagent (diluted 1:1 with water) then 1 ml of a 5% sodium carbonate solution was also added. after 30 min, absorbance was measured at 725 nm and expressed as mg on g fresh weight-1. leaf α-tocopherol concentration. leaf α-tocopherol was extracted according to chong et al. (2004).two hundred mg lyophilized sample was homogenized in 1 ml acetone with a prechilled mortar and pestle at 4°c. following the addition of 0.5 ml hexane, the homogenate was first vortexed for 30 s, then centrifuged at 1000 × g for 10 min. the upper hexane layer was removed while the acetone layer containing vitamin e remained in the vial. a second aliquot of 0.5 ml hexane was added, and the extraction process was repeated at least twice. α-tocopherol was estimated by the method of kanno and yamauchi (1997). adv. hort. sci., 2017 31(2): 97-105 100 a 0.4-ml aliquot of 0.1% (w/v) 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine was added to 0.2 ml of pooled extract. the volume was made up to 3 ml with absolute ethanol, 0.4 ml 0.1% (w/v) ferric chloride (fecl3.6h2o) was added, and the content was gently mixed under dim light in a dark room to avoid photochemical reduction. after a 4 minutes reaction at room temperature, 0.2 ml 0.2 m orthophosphoric acid was added and the mixture left for another 30 min. absorbance was determined at 554 nm spectrophotometrically and reported as µg. g fresh weight-1. the blank was prepared in the same manner except that absolute ethanol was used instead of the sample. α-tocopherol (sigma chemical) was used as a standard. leaf ascorbic acid concentration. ascorbic acid was estimated by the method of omaye et al. (1979). briefly, to 1 g of lyophilized leaf sample, 10% ice-cold tca was added and centrifuged for 20 min at 3500 × g in room temperature. one ml of the supernatant was mixed with 0.2 ml of dtc reagent and incubated for 3 h at 37°c. then 1.5 ml of ice-cold 65% h2so4 was added, mixed well and the solutions were allowed to stand at room temperature for an additional 30 min. the color developed was read at 520 nm spectrophotometrically and reported as µg. g fresh weight-1. macro and micronutrients. oven-dried leaf samples were used for determination of macro and micro-nutrients. dried samples (0.5 g) were ground and ashed at 550°c in a porcelain crucible for 6 h. the white ash was mixed in 2 m hot hcl, filtered and finally made up to 50 ml with distilled water. sodium (na) and potassium (k) concentration of samples were determined using flame emission method using a sherwood scientific ltd model 360 flame photometer. atomic absorption spectrophotometer (aa 6200, double beam atomic absorption spectrophotometer, shimadzu, kyoto, japan) was used to determine ca, mg and micronutrient element including fe, zn, mn concentrations (kalra, 1998). nitrogen (n) concentration was measured using the kjeldahl digestion method (kalra, 1998). phosphorus (p) concentration was determined colorimetrically (kalra, 1998). chlorine (cl) was measured by precipitation titration with silver nitrate (mohr’s method) (kalra, 1998). statistical analysis data were analyzed by sas and means were compared using duncan’s multiple range test at 5% probability level. 3. results leaf macronutrients and na and cl concentrations in studied pomegranate cultivars are indicated in table 3. leaf n, p and cl concentrations were not statistically different in studied cultivars. rnf and zaa had significantly higher leaf k concentration in comparison to spdg and nb, other cultivars were not statistically different. the highest leaf ca concentration was observed in rnf cultivar (2.53±0.06 mg g-1 dry weight), however mdgy, ssf and zaa were not statistically different. this macronutrient was 22% lower in spdg compared to rnf. leaf mg concentration was significantly higher in mys, mdgy, rnf and zaa in comparison to nb, spdg and ssf. leaf na concentration was 41% higher in mys in comparison to zaa. other cultivars were not statistically different. leaf fe, zn and mn concentrations in studied pomegranate cultivars are presented in table 4. leaf fe concentration is 32% higher in zaa in comparison to nb. other cultivars are not statistically different. no significant difference was found among studied cultivars for leaf zn and mn concentrations. leaf dry matters, total sugars, relative water content and electrolyte leakage are shown in table 5. means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated are mean ± standard error (n = 3). table 3 leaf macronutrient and na and cl concentrations in studied pomegranate cultivars cultivars n (mg g-1 dw) p (mg g-1 dw) k (mg g-1 dw) ca (mg g-1 dw) mg (mg g-1 dw) na (mg g-1 dw) cl (mg g-1 dw) malas yousefkhani saveh 20.50±2.07 a 1.59±0.09 a 21.60±0.72 bc 1.99±0.12 cd 0.97±0.02 ab 3.53±0.47 a 1.56±0.23 a naderi badroud 22.80±0.81 a 1.63±0.02 a 20.33±0.41 c 2.11±0.05 bcd 0.89±0.06 b 2.74±0.43 ab 1.48±0.28 a malas daneh ghermez yazd 21.453±1.48 a 1.62±0.11 a 22.60±0.72 abc 2.38±0.09 abc 1.13±0.01 a 2.43±0.36 ab 1.89±0.13 a rabab neiriz fars 20.83±0.89 a 1.60±0.09 a 23.56±0.29 a 2.53±0.06 a 1.06±0.04 a 2.49±0.49 ab 1.96±0.10 a shirin shahvar fars 22.43±0.74 a 1.59±0.02 a 22.80±0.44 abc 2.21±0.08 abcd 0.88±0.06 b 2.76±0.40 ab 1.76±0.14 a shirin poust daneh ghermez 22.50±1.41 a 1.53±0.09 a 20.91±1.21 c 1.97±0.02 d 0.86±0.07 b 2.28±0.35 ab 1.47±0.26 a zard anar arsenjan 21.78±0.87 a 1.65±0.06 a 23.55±0.27 ab 2.50±0.02 ab 1.13±0.3 a 2.07±0.07 b 1.44±0.30 a jamali and bonyanpour adaptability of seven iranian pomegranate cultivars in southern iran 101 rnf had significantly higher leaf dry matter compared to mys, ssf and spdg. zaa, nb and mdgy were not statistically different. the highest leaf total sugars (47.63±4.99 mg g-1 dry weight) were detected in rnf which were significantly higher than spdg, ssf, nb and mys. zaa showed the highest leaf relative water content (83.83±0.74%) however rnf, ssf and mdgy were not statistically different. this parameter was lower in mys, nb and spdg compared to zaa. leaf electrolyte leakage was significantly higher in mys in comparison to all other cultivars. concentrations leaf pigments in studied pomegranate cultivars are indicated in table 6. nb had significantly lower leaf total chlorophyll concentration in comparison to rnf, other cultivars were not statistically different. leaf carotenoids concentration was significantly higher in zaa, spdg, ssf and rnf compared to nb and mys. leaf anthocyanins concentration was not different among studied cultivars. mys showed lower chlorophyll a to b ratio in comparison to rnf, ssf, spdg and zaa. leaf total polyphenols concentration was statistically higher in rnf, spdg and zaa in comparison to mys, nb and ssf (fig. 1). the highest leaf α-tocomeans followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated are mean ± standard error (n = 3). table 4 leaf micronutrient concentrations in studied pomegranate cultivars cultivars fe (mg g-1 dw) zn (mg g-1 dw) mn (mg g-1 dw) malas yousefkhani saveh 58.66±2.02 ab 23.00±4.50 a 53.00±9.70 a naderi badroud 43.00±5.85 b 25.00±3.46 a 49.00±3.51 a malas daneh ghermez yazd 46.33±4.91 ab 29.33±2.84 a 49.33±5.81 a rabab neiriz fars 53.00±8.38 ab 22.33±4.33 a 55.00±9.23 a shirin shahvar fars 53.667±2.33 ab 22.33±3.66 a 50.66±3.38 a shirin poust daneh ghermez 58.00±2.08 ab 23.00±3.05 a 48.66±0.88 a zard anar arsenjan 64.667±3.71 a 26.66±7.35 a 56.00±2.64 a means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated are mean ± standard error (n = 3). table 5 leaf dry matter, total sugars, relative water content and electrolyte leakage in studied pomegranate cultivars cultivars leaf dry matter (%) leaf total sugars (mg g-1 dw) lrwc (%) el (%) malas yousefkhani saveh 24.33±1.20 d 22.44±2.26 d 79.05±0.67 c 24.80±1.77 a naderi badroud 30.33±1.45 ab 29.50±1.45 cd 80.92±1.27 bc 20.08±1.28 b malas daneh ghermez yazd 32.33±0.66 ab 39.63±2.56 ab 82.22±0.63 ab 19.20±1.51 b rabab neiriz fars 33.00±1.52 a 47.63±4.99 a 82.70±0.24 ab 18.66±0.50 b shirin shahvar fars 25.66±1.76 cd 35.72±3.29 bc 81.62±0.89 abc 18.55±1.24 b shirin poust daneh ghermez 28.66±0.66 bc 29.66±1.15 cd 80.55±0.86 bc 17.20±0.49 b zard anar arsenjan 30.66±2.18 ab 42.41±1.22 ab 83.83±0.74 a 17.10±0.10 b means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated are mean ± standard error (n = 3). table 6 leaf pigments concentrations in studied pomegranate cultivars cultivars total chlorophyll carotenoids anthocyanins chlorophyll a/b ratio malas yousefkhani saveh 1.35±0.06 ab 0.15±0.01 c 0.25±0.004 a 0.88±0.04 c naderi badroud 1.16±0.08 b 0.20±0.01 b 0.25±0.007 a 1.01±0.04 bc malas daneh ghermez yazd 1.30±0.17 ab 0.25±0.01 ab 0.24±0.002 a 1.15±0.02 ab rabab neiriz fars 1.46±0.04 a 0.27±0.005 a 0.26±0.01 a 1.19±0.03 a shirin shahvar fars 1.44±0.04 ab 0.28±0.004 a 0.25±0.02 a 1.19±0.01 a shirin poust daneh ghermez 1.40±0.07 ab 0.26±0.02 a 0.25±0.02 a 1.29±0.03 a zard anar arsenjan 1.34±0.10 ab 0.29±0.006 a 0.26±0.01 a 1.26±0.04 a fig. 1 leaf polyphenols concentration in studied cultivars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=3). adv. hort. sci., 2017 31(2): 97-105 102 pherol concentration was obtained from spdg (229 µg. g fresh weight-1), however it was not statistically different in comparison to zaa, rnf and ssf. mys, nb and mdgy had significantly lower leaf α-tocopherol concentration compared to spdg (fig. 2). leaf ascorbic acid concentration in rnf was significantly higher in rnf in comparison to mdgy, other cultivars were not statistically different (fig. 3). rnf had significantly higher trunk circumference in comparison to mys. other cultivars were not statistically different (fig. 4). 4. discussion and conclusions in present study significant differences were found between studied pomegranate cultivars for concentrations of leaf macro and micronutrients. this was in agreement with previous studies. giménez et al. (2000) compared leaf mineral composition (macro and micronutrients) in two pomegranate cultivars; mc1 (one of the most commonly grown cultivars in the south-east of spain) and a well-known israeli early variety. they found significant differences between these two pomegranate cultivars for concentrations of leaf macro and micronutrients. normality ranges for n, p, k, ca and mg were also varied. el-agamy et al. (2010), compared the salt tolerance of two important egyptian pomegranate cultivars, manfalouty and nab el-gamal in vitro. they found significant differences in these two cultivars under saline and non-saline conditions for absorption of n, k and ca. khayyat et al. (2014) evaluated physiological and biochemical responses of malas mommtaz and shishe kab under saline conditions; they reported with increase in salinity (from 4.61 to 7.46 ds m-1) shoot cl concentration augmented in malas mommtaz but decreased in shishe kab, also this cultivar (shishe kab) managed na+ transport into the leaves better than malas mommtaz. sarafi et al. (2017) indicated that wonderful and ermioni cultivars had different ability for p, k, ca, mg and zn uptake under optimum conditions. similar differences were observed between rabab and shishekab fig. 3 leaf ascorbic acid concentration in studied cultivars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=3). fig. 2 leaf α-tocopherol concentration in studied cultivars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=3). fig. 4 trunk circumference in studied cultivars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=3). jamali and bonyanpour adaptability of seven iranian pomegranate cultivars in southern iran 103 by hasanpour et al. (2015). rnf and zaa had higher relative water content and lower electrolyte leakage in comparison to mys. presence of higher k concentration in plant tissues will lead to more efficient water relationships in the leaves (marschner, 1995). photosynthesis, stomatal activity, transport of sugars, protein synthesis are all dependent on k (prajapati and modi, 2012). ca pectates provide stability and mechanical strength to cell walls (pilbeam and morley, 2007; taiz and zeiger, 2010). structural impairments in membrane structure are very common in ca-deficient cells which make cell membranes very leaky and cause extensive loss of organic (e.g., sugars, amino acids) and inorganic electrolytes from root or leaf cells (white and broadley, 2003). leaf total sugars varied among studied cultivars. about half of osmotic potential in glycophte species is related to presence of sugars (cram, 1976). accumulation of higher total sugars has been reported to be associated with higher tolerance to salinity and/or drought in various species; grape, barley, zygophyllum album and soybean (ashraf and harris, 2004). five sunflower cultivars were evaluated for their salinity tolerance, more resistant lines had higher total sugars (ashraf and tufail, 1994). comparison between wild populations of melilotus indica (salt tolerant) and eruca sativa (salt sensitive) showed that the former had higher leaf sugars (ashraf and harris, 2004). significant difference was observed between cultivars for leaf chlorophyll and carotenoids concentration in present study. in various studies the chlorophyll concentration were used as a sensitive indicator of the cellular metabolic state (chutipaijit et al., 2011). higher chlorophyll concentration is related to elevated tolerance against abiotic stresses such as drought and salinity (hasanuzzaman et al., 2013). in addition to harvesting solar energy, carotenoids play protection roles keeping integrity of photosynthesis apparatus against photo oxidative damages by scavenging free radicals (dall’osto et al., 2007; andradesouza et al., 2011). carotenoids are precursor of aba which is an important phyto-hormone regulating plant responses in response to stresses. so presence of higher carotenoid concentration lead to lower photo-oxidative damage and higher potential for regulating plant growth under stress conditions (götz et al., 2002; han et al., 2008). non enzymatic antioxidants (leaf polyphenols, αtocopherol and ascorbic acid concentration) were statistically different among studied cultivars. varied antioxidant profile in different species and cultivars is one of the main reasons responsible for their different adaptability and abiotic tolerance potential (munns and tester, 2008; jamali et al., 2016). polyphenols have strong antioxidant properties and presence of an elevated level of them is associated with increased abiotic stress tolerance (jamali et al., 2016). among vitamin e family, α-tocopherol has the highest antioxidant activity (garg and manchanda, 2009). several lines of evidence indicate that α-tocopherol plays a major role in keeping an adequate redox state in chloroplasts (munne-bosch, 2005). deficiency of this antioxidant leads to a slightly increased susceptibility to photooxidative stress (kanwischer et al., 2005). plants have different capacity of ascorbate metabolism which is due to the variation of ascorbic acid synthesis and regeneration. plant with higher amount of ascorbic acid content demonstrate better protection against oxidative stress. ascorbate influences many enzyme activities, minimizing the oxidative damage through synergic function with other antioxidants (foyer and noctor, 2005 a, b). ascorbic acid plays a role as a co-factor of violaxanthin de-epoxidase, thus sustaining dissipation of excess excitation energy (pourcel et al., 2007). trunk circumference was significantly higher in rnf compared to mys. difference in mineral composition and ability of nutrients uptake in these cultivars could be one of the reasons responsible for this, as discussed above. difference in enzymatic (data not shown) and non-enzymatic antioxidant responses and also growth regulators such as ga3, zeatin and aba (data not shown) in these cultivars are other reasons for varied growth rate observed between pomegranate cultivars in the present study. pomegranate is a tolerant species, however various varieties have significantly different adaptability potential. this can be contributed to varied ability for macro and micronutrients uptake, different enzymatic and non-enzymatic antioxidant profile and endogenous plant growth regulators. however, environmental conditions play an important role in this regard and might alter the final response of plant. zaa is an indigenous variety of arsenjan region/fars province/iran. high level of leaf k, ca, mg and fe, enzymatic and non-enzymatic antioxidants (data not shown) and higher level of zeatin (data not shown) are the reasons responsible for better 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tolerance. improvement in plant-water relations is affected by direct and indirect mechanisms (davies et al., 1993). in general, plants that have mycorrhizal symbiosis grow and perform better as they absorb more nutrients and water from the soil. these plants are also more tolerant towards environmental stresses including biotic and abiotic stresses (porcel and ruiz-lozano, 2004). most varieties of citrus, like orange, trifoliate orange, cleopatra mandarins, swingle citrumelo, and citrange, are very dependent, because of their hairy roots, on glomus species (davies et al., 1993). plant adaptations to arid climate conditions, morphological and physiological changes, and concentration of novel metabolites along with structural variations, increase their efficiencies in stress conditions (wu et al., 2006). when plants are under drought stress, osmotic adjustments occur to reduce potential water loss. this phenomenon leads to good water flow maintenance from the soil to plant roots (porcel and ruizlozano, 2004). g. versiforme fungus increased leaf water potentials of trifoliate orange and mandarin seedlings under both drought stress and enoughwater-supply conditions (wu et al., 2006, 2008). moreover, when trifoliate orange seedlings were under drought stress, the leaf relative water content (rwc) significantly increased compared to plants with no fungus (wu et al., 2006). in mandarin seedlings, plant height, leaf area and number of leaves per plant, decreased under drought stress conditions, while all those factors were improved using g. versiforme fungi (wu and zou, 2009). in citrus plants, g. versiforme fungi increased growth and adv. hort. sci., 2016 30(1): 39-45 doi: 10.13128/ahs-18700 analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions a.r. shahsavar 1 (*), a. refahi 1, m. zarei 2, e. aslmoshtaghi 1 1 department of horticultural science, college of agriculture, shiraz university, shiraz, iran. 2 department of soil science, college of agriculture, shiraz university, shiraz, iran. key words: chlorophyll content, drought deficit, leaf water potential, mexican lime. abstract: to analyze the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria on some morphological and physiological characteristics of mexican lime plant under drought stress conditions, a factorial experiment was conducted. this experiment was based on a completely randomized design with three replicates; each replicate was composed of two pots. the factors used consisted of g. etunicatum fungi and control, pseudomonas fluorescence bacteria and control, and drought stress at three levels (-0.35, -0.47, and -0.6 bars). the analyzed characteristics were leaf chlorophyll content, leaf temperature, rate of net photosynthesis, transpiration, leaf relative water content (rwc), and percentage of root colonization. data analysis revealed that both fungi and bacteria increased leaf chlorophyll content, net photosynthesis rate, transpiration, and leaf rwc. moreover, the presence of fungi reduced leaf temperature while inoculation of bacteria had no effects on that the parameter. in addition, with the increase of irrigation periods, leaf temperature and transpiration were also increased. results showed that root colonization percentage dropped with increased irrigation and the highest root colonization percentage was observed in simultaneous inoculations of fungi and bacteria with a two-day irrigation period. (*) corresponding author: shahsava@shirazu.ac.ir received for publication 31 october 2015 accepted for publication 4 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 39-45 40 biomass while they reduced their colonization percentage. these increases were attributed to the improvement of fungi water absorptions and increases in the length and volume of plant fungal roots (faber et al., 1991; bryla and duniway, 1997; wu et al., 2011). in these plants, root colonization increases with a decrease in drought stress (augé, 2001). under drought stress conditions, g. versiforme fungi increased fresh and dry weight of plant roots and shoots and increased the root colonization percentage (wu and xia, 2006). g. intraradices fungus, under drought stress conditions, increased root growth and respiration rate of rough lemon (leyv and syvestern, 2006). many studies showed that g. etunicatum fungi could affect plant-water relations of host plants including citrus, under both drought stress and enough-water-supply conditions (wu et al., 2006). therefore, they cause higher water use efficiency and this water use efficiency in mycorrhizal plants becomes even more tangible in drought stress conditions (davies et al., 1993). glomus etunicatum fungus increased phosphorus, potassium, zinc, and copper in pistachio trees planted under sufficient water supply conditions and also increased nitrogen and calcium in pistachio trees planted under drought stress conditions. however, this fungus did not change the magnesium concentration (abbaspour et al., 2011). it is reported that pseudomonas bacteria enhances growth and yield of some plants (rodriguez and fraga, 1999). construction of active metabolites such as vitamins, amino acids, and indole acetic bacteria may have a direct effect on the growth and metabolite contents of piriformospora indica and mycorrhizal fungi. as a helpful microorganism, it seems that bacteria supports fungal performances (vivas et al., 2003). plant inoculations with different types of pseudomonas bacteria in drought stress situations increased plant proline contents, thus the plants’ water levels were maintained and their protein contents and membranes remained safe from drought stress damage (yoshiba et al., 1997). inoculation with pseudomonas species, led to moderation of drought stress effects, improvement of plant growth and increase of proline, soluble sugars and amino acids production, explaining their effectiveness in absorbing water and nutrients from the soil (wu et al., 2008). these types of bacteria also help the plant maintain its rwc and lwl (leaf water loss) levels under drought stress conditions. studies have shown that mycorrhizal plants absorb more co2 in the presence of light. hence, their photosynthesis rates are also higher. the increase of co2 absorption in mycorrhizal plants is related to a decrease of liquid-phase resistance of mesophyll cells to co2 transmission (wu and zou, 2009). miller (2000) reported that in mycorrhizal plants, due to the increase of photosynthesis materials and rate, water use efficiency increased per water use unit. mycorrhiza can increase plant weight, leaf area, and plant pigments, and these increases may be attributed to the improvement of fungi water and phosphorus absorptions (bethlenfalvay et al., 1988; davies et al., 1993). glomus etunicatum and pseudomonas bacteria have positive effects on plant growth and employing them, instead of fertilizers, is considered a positive approach to reduce fertilizer use (davies et al., 1993). despite the lack of comprehensive scientific investigations on the horticultural characteristics of mexican lime (citrus aurantifolia swingle cv. mexican lime) as a rootstock, its seed availability for propagation and some its characteristics, such as good crop load and vigorous habit of grafted cultivars as scion, have made it a favorite in fars province, iran. considering the positive effects of fungi and bacteria in symbiosis with some plant roots, the aim of this study was to investigate the effects of g. etunicatum fungi and pseudomonas fluorescence bacteria and their interactions on some morphological and physiological characteristics of mexican lime plant under drought stress conditions. 2. materials and methods preparation and inoculation of plant materials mexican lime seedlings, six months of age and disease-free, were provided in khafr city of fars province, iran. they were transferred to the greenhouse. planting soil mixture in ratio 1:1:1 (sand:soil: leaf compost) was sterilized and 2.7 kg were placed in plastic pots. the arbuscular mycorrhizal fungi isolate used in this study was g. etunicatum supplied from the soil lab of the faculty of shiraz university. the lyophilized fungal inoculum of pseudomonas fluorescence was supplied from tehran university school of soil and water and was prepared as follows. to prepare a solution containing growth-stimulating bacteria, a nutrient broth (nb) medium was applied. first, 0.8 g of nb was dissolved in 100 mg of distilled water and then media were sterilized by autoclaving at 121°c and pressure of 1.1 atm for 25 shahsavar et al. effects of glomus etunicatum and pseudomonas fluorescence on mexican lime under drought stresss 41 min. a lyophilised pre-culture vial was first suspended in 0.3 ml of nutritive medium. one drop (1 ml) of that suspension was added to 5 ml of nutritive medium and incubated on an orbital shaker at 28°c for 24 h. this final preparation of medium was used as the inoculum. after the incubation period, roots were placed in a solution containing bacteria for 30 min. moreover, to ensure its effectiveness, 10 cc of the solution containing bacteria were added to each pot. for drought stress treatments, pots containing 2.7 kg soil without a seedling were selected and their moisture contents were equilibrated with the previously measured field capacity. the wet soils of the pots were weighed daily for 15 days, always at the time. daily water reductions and moisture curves were graphed. using those diagrams, irrigation periods were identified for every 2, 4 and, 6 days. for g. etunicatum fungus inoculation, 70 g of inoculum containing spores, hyphae, and root fragments were introduced 5 cm beneath the soil surface in the pots, and mixed thoroughly. equal to the amount of added inoculum, hyphae, and mycelium to the fungal treatment pots, inoculum without hyphae and mycelium was added to control pots. for bacteria inoculation, seedlings were placed in a solution containing pseudomonas fluorescence bacteria for 30 min and were then planted into pots. for fungi and bacteria treatments, bacteria-inoculated seedlings were planted in pots in which fungus was previously added. one seedling was planted per pot and, two months later, water treatments were applied. after six months, the implants were removed. the study was conducted using a factorial experiment, based on a completely randomized design with three replications in two replicate pots. factors used in the experiment were: 1) g. etunicatum fungus in two levels of g. etunicatum and control; 2) growth stimulating bacteria in two levels of pseudomonas fluorescence and control; 3) drought stress at three levels. the kormanik and mcgraw method (kormanik and mcgraw, 1982) was used to measure colonization percentage. in this method, 2 g of roots previously stored in faa (formaldehyde acetic acid ethanol) were washed with water three or four times and were placed in falcon tubes containing 10% koh solution for 24 h at room temperature. the color of the solution was almost yellow or light yellow. the solution was then poured out and the roots were again washed with water three or four times. the samples were placed in 2% hydrochloric acid for at least 15 min for staining. the acid was poured out and a colored solution was poured over the acidic roots. acid fuchsine stain was used in this study; the ratio of the fuchsine acid colored solution was 14 ml lactic acid, 1 ml glycerin and, 1 ml water. the roots and the solution were kept at room temperature for 24 h. the coloring solution was then removed. besides, due to elimination of extra colors, the coloring solution was poured on the roots. after 6-12 h, fungal organs such as arbuscules, hyphae, and vesicles were observed under a light microscope and colonization was calculated as a percentage. after application of water stress treatments, leaf chlorophyll content was measured with a spad-502 chlorophyllmeter using three fully-expanded leaves to find an average for chlorophyll content. leaf temperature factors, net photosynthesis and transpiration rates were measured by portable photosynthesis meter (lci, adc, england). relative water content was determined by using ten 7 mm-diameter leaf discs. leaf discs for each treatment were weighed (fw). they were hydrated until saturation (constant weight) for 48 h at 5°c in darkness (tw). the leaf discs were then dried in an oven at 105°c for 24 h (dw). relative water content was calculated according to the following expression (filella et al., 1998): rwc% = (fw-dw)/(tw–dw) × 100 statistical analysis the data were analyzed for significance (p<0.050) by anova (analysis of variance) with mean separation by duncan’s multiple range test. 3. results and discussion leaf relative water content (rwc) analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime leaf rwc, at different irrigation periods, identified that the maximum leaf rwc was observed in simultaneous inoculation of fungi and bacteria with the two-day irrigation period (74.7%). the general results indicate that the leaf rwc decreased with the increase in irrigation period, while inoculation with fungi or bacteria significantly increased rwc in all irrigation periods (table 1). osmotic adjustment is one of the most important factors in plant drought tolerance and it is closely related to rwc (haley et al., 1993). when plants are under a drought stress condition, osmotic adjustadv. hort. sci., 2016 30(1): 39-45 42 ment occurs to reduce water potential and maintain a good flow of water from the soil to the plant roots. plants with mycorrhizal fungi have more osmotic adjustment potentials than plants without fungi (porcel and ruiz-lozano, 2004). manette et al. (1988) reported that plants which are under drought stress conditions have specific morphological and physiological characteristics that enable them to store more water. clarke and craig (1982) stated that plants under drought stress conditions loose their water content more slowly. they also indicated that there are significant relationships between water content of the loss of leaves, plant drought tolerance, and leaves ability to retain water content (clarke and craig, 1982). therefore, mycorrhizal plants have higher osmotic adjustment and are more capable of retaining their water content. chlorophyll content chlorophyll content decreased with the increase of irrigation periods. in addition, inoculations of g. etunicatum fungi and pseudomonas fluorescence bacteria increased leaf chlorophyll content. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria leaf chlorophyll content identified that the maximum leaf chlorophyll content was observed when both fungi and bacteria were inoculated and there was a two-day irrigation period (634.7). the lowest chlorophyll content was observed in the treatment without fungi and bacteria inoculations with six-day irrigation periods (table 2). analysis of the effects of g. etunicatum fungi and pseudomonas fluorescence bacteria inoculations on chlorophyll content of the mexican lime leaves in the current study revealed that the chlorophyll content decreased with an increase of drought stress periods. however, inoculations of fungi and bacteria largely reduced the deleterious effects of drought. this can be explained by the fact that in drought stress conditions, the chlorophyllase enzyme becomes activated while its activation results in the loss of chlorophyll content (shaharoona et al., 2008). under drought, oxygen free radicals, which are damaging to various cellular organelles, are formed. one of the most sensitive organelles to drought stress and free radicals is chloroplast (kaya et al., 2003). g. etunicatum fungi and pseudomonas fluorescence bacteria, by increasing antioxidant content and antioxidant enzyme activities, cause a loss of detrimental free radicals and consequently preserve plant chlorophyll content (molinari et al., 2007). they also increase the absorption of elements such as magnesium, iron, and nitrogen that lead to the plant’s production of more chlorophyll (molinari et al., 2007). leaf temperature our results indicate that the increase of irrigation periods led to an increase of leaf temperature. the presence of g. etunicatum fungi decreased leaf temperature while pseudomonas fluorescence bacteria inoculation had no effect on it. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on leaf temperature revealed that the minimum leaf temperature was with simultaneous inoculation of fungi without bacteria and a two-day irrigation period (31.47°c). likewise, the maximum temperature was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation periods (table 3). rate of net photosynthesis analysis of the net photosynthesis rate of mexican lime revealed that it declined with the increase of irrigation periods: the maximum and minimum rates were observed with twoand six-day irriirrigation periods (day) ge + ge pf + pf pf + pf 2 634.7 a 574.3 b 565.4 b 529.6 c 4 578.2 b 512.9 cd 511.7 cd 441.7 f 6 503.8 d 484.6 e 479.3 e 320.5 g table 2 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf chlorophyll content with different irrigation periods (spad value) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 74.7 a 73.5 ab 72.9 ab 72.6 ab 4 71.9 b 70.3 b 69.5 bc 67.6 c 6 70.2 b 69.5 bc 68.4 bc 66.3 c table 1 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf rwc with different irrigation periods (%) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. shahsavar et al. effects of glomus etunicatum and pseudomonas fluorescence on mexican lime under drought stress 43 gation periods, respectively. the results also indicated that the presence of g. etunicatum fungi and pseudomonas fluorescence bacteria increased the plants’ rate of net photosynthesis. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria identified that the maximum rate was observed in simultaneous inoculation of both fungi and bacteria and with a two-day irrigation period (12.3 micromole/m2/s) (table 4). rate of transpiration analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime transpiration rate in plants grown with different irrigation periods identified that the highest rate was observed in simultaneous inoculation of both fungi and bacteria and a two-day irrigation period (10.25 micromole/m2/s). likewise, the minimum transpiration rate was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation period. the overall results showed that the leaf transpiration rate increased with the increase of irrigation period (table 5). wu and xia (2006) specified that under drought stress conditions, g. versiforme fungi increase leaf water potential, photosynthesis rate, respiration rate, rwc, and stomatal conductance of mandarin seedlings; however, leaf temperature is decreased compared to plants without fungi. effects of irrigation period on leaf temperature, photosynthesis rate, and transpiration showed that with the increase of irrigation period, they all declined (figueiredo, 2008). this can be explained by the fact that under drought condition, more stomata are closed; with a loss of evaporation, the leaf surface loses less heat and the leaf temperature increases (dietz and foyer, 1986.). moreover, because of stomata closure, less water is lost and the transpiration rate decreases. it should be noted that stomata closure causes less carbon dioxide to enter into the leaf, resulting in a lower rate of photosynthesis (zhang et al., 2010). the presence of g. etunicatum fungi and inoculation with pseudomonas fluorescence bacteria leads to better water absorption and higher drought stress tolerance, thus increasing the plant’s rate of photosynthesis. many studies have reported the effects of g. etunicatum fungi on increasing photosynthesis rate (johnson et al., 1986), increasing root hydraulic conductivity for water uptake (graham and syvertsen, 1984), and increasing transpiration rate (leyv and syvestern, 2006). root colonization percentage results of the present study showed that root colonization occurred in the presence of g. etunicatum fungi and pseudomonas fluorescence bacteria. moreover, an increase of irrigation period led to a decrease of root colonization percentage. analysis of the effects of interaction between inoculation of g. irrigation periods (day) ge + ge pf + pf pf + pf 2 32.59 de 31.47 e 33.16 d 33.05 d 4 34.25 d 33.87 d 34.92 c 36.50 b 6 35.94 b 36.35 b 36.28 b 38.41 a table 3 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf temperature with different irrigation periods (°c) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. table 4 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime photosynthesis rate with different irrigation periods (micromole/m2/s) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 12.3 a 11.6 ab 11.4 b 10.50 c 4 10.2 c 10.2 c 10.1 c 9.06 d 6 9.51 d 8.52 de 8.37 e 6.48 f table 5 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime transpiration rate with different irrigation periods (micromole/m2/s) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 10.25 a 9.96 b 10.07 ab 9.83 b 4 9.68 c 9.58 c 9.16 d 8.74 e 6 9.17 d 9.72 b 8.91 e 8.65 e adv. hort. sci., 2016 30(1): 39-45 44 etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime percentage of root colonization revealed that the maximum percentage was observed in simultaneous inoculation of both fungi and bacteria with a two-day irrigation period (49.66%) (table 6). as previously mentioned, root colonization occurred only in the presence of g. etunicatum fungi and its percentage dropped with an increase in irrigation period. until now, no specific reason has been proposed for the reduction of colonization in drought stress conditions. probably water is one important element in fungi growth. the formation of secondary metabolites that prevent fungi growth in the plant roots is also a possible explanation. wu et al. (2006) reported that, in the case of citrus roots, the highest colonization percentage of mycorrhizal fungi occurs when the roots are not under drought stress conditions, which is consistent with the present study results. regarding other types of citrus, they found similar results in their subsequent studies (wu et al., 2006, 2008). in order to utilize root colonization of fungi and bacteria capacities in sustainable agriculture, there must be appropriate establishment of both fungi and bacteria on the plant roots. accordingly, observation of mexican lime root colonization percentage in the current investigation was a very important and valuable factor. in addition, specification of the appropriate colonization percentage for effective interaction between fungi and plant is an important issue. 4. conclusions the results of the current study and other research projects in this field have shown the practical and scientific advantages of g. etunicatum fungi and pseudomonas fluorescence bacteria applications in arid or semi-arid areas. the synergistic effect, which was observed between g. etunicatum fungi and pseudomonas fluorescence bacteria, could increase most of the plant characteristics such as leaf chlorophyll content, net photosynthesis and transpiration rates, leaf rwc and root colonization percentage which provide the material energy and information for plant growth, development and reproduction. pseudomonas fluorescence bacteria could reduce the negative effects of drought stress less than g. etunicatum fungi. using their hyphae and extra/intra root mycelia, g. etunicatum fungi expand root evacuation area for better uptakes of water and nutrients. arbuscular mycorrhizal fungi can be integrated in soil management to achieve low-cost sustainable agricultural systems, offering a sustainable and environmentally safe treatment to improve drought tolerance. consequently, using these fungi as well as pseudomonas fluorescence bacteria can be very effective in achieving the goals of sustainable agriculture. references abbaspour h., saeidsarand s., afshar h., 2011 improving drought tolerance of pistacia vera l. seedlings by arbuscular mycorrhiza under greenhouse conditions. j. medicinal plants research, 5: 70657072. augé r.m., 2001 water relations, drought and vesiculararbuscular mycorrhizal symbiosis. micorrhiza, 11: 3-42. bethlenfalvay g.j., brown m.s., ames r.n., thomas r., 1988 effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake. physiol. plant., 72: 565-571. bryla d.r., duniway j.m., 1997 effects of mycorrhizal infection on drought tolerance and recovery in safflower and wheat. plant and soil., 197(1): 95-103. clarke j.m., craig t.n., 1982 excised-leaf water retention capability as an indicator of drought resistance of triticum genotypes. can. j. plant sci., 62: 571-578. davies f.t., potter j.r., linderman r.g., 1993 drought resistance of mycorrhizal pepper plants independent of leaf p concentration response in gas exchange and water relations. physiologia plantarum., 87: 45-53. dietz k.j., foyer c., 1986 the relationship between phosphate status and photosynthesis in leaves. planta, 167(3): 376-381. faber b.a., zasoske r.j., munns d.n., shackel k., 1991 table 6 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime root colonization percentage with different irrigation periods (%) irrigation periods (day) ge + ge pf + pf pf + pf 2 49.66 a 42.36 ab 0 d 0 d 4 38.73 b 36.87 bc 0 d 0 d 6 34.24 c 35.12 c 0 d 0 d in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; 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of causarina equisetifilia seedlings by arbuscular mycorrhizal under glasshouse conditions. new for., 40(3): 261-271. impaginato 59 1. introduction to aid summer-time thermal control in urban areas, the creation of shade above buildings and/or streets is highly important for a number of reasons. first, shade can provide protection for residents and passersby by buffering the changes in housing thermal conditions inside the building and/or local climate on the streets during heat wave attacks, a serious concern as discovered in the case of the historic, heat-related disaster in paris in 2003 (keller, 2013). second, minimizing energy consumption within buildings has become an important goal of architecture and urban planning in recent years. as a result, several guidelines have been developed depending on the climatic zones, aimed at increasing solar exposure for buildings in cold climates and reducing solar exposure for buildings in warm climates (okeil, 2010). adv. hort. sci., 2016 30(2): 59-67 doi: 10.13128/ahs-19130 optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant a. hara 1, h. takaichi 1, y. murata 1, r. sakata 1, y. hara 1, j. iwase 2, 3, d. comparini 1, 2, 4, t. suzuki 1, 4,t. kawano 1, 2, 4 (*) 1 faculty and graduate school of environmental engineering, the university of kitakyushu, kitakyushu, japan. 2 linv kitakyushu research center, kitakyushu, japan. 3 kyushu institute of technology collaboration center, kitakyushu, japan. 4 international photosynthesis industrialization research center, the university of kitakyushu, kitakyushu, japan. key words: building thermal control, environmental measurements, green roof, green wall, rooftop garden, urban agriculture. abstract: recently, thermal properties of the landscaped rooftops and walls have attracted the interest of researchers because of the potential to minimize energy consumption in urban areas and to aid summer-time thermal control. for this reason the creation of a plant-based shade for walls or above buildings is highly important. in this paper we evaluate using lygodium japonicum, one of the many ferns and fern allies traditionally used in japanese gardening, as a component of thermal-buffering green walls. lygodium japonicum, the only climbing fern species in japan, is fast-growing, adheres easily to walls and has a climbing nature. a simple thermal analysis of the sun-shading effect of lygodium canopy suggested that local surface temperature above the ceramic tiles placed on the rooftop of a building can be buffered (lowered in daytime and maintained relatively warm at night) by the presence of leafy climbing ferns covering the tiles, possibly due to the reflection and absorbance of solar radiation. furthermore, the presence of the plants may also slow the night-time release of heat from the building surface. because plants installed on tall walls or on the tops of buildings are not easily accessed for manual care, we performed a real-time routine monitoring and control of plant growth status using various optical sensors that could be automated and monitored remotely for large-scale applications. for this purpose, the optical properties of a l. japonicum canopy under solar incident light have been determined. in order to evaluate the natural shading and growing properties of a green canopy, the incident solar radiation spectrum (j), leaf canopy-filtered light spectrum (transmittance, t) and leaf-reflectivity spectrum (r) were measured. by reading the reflectivity spectrum, concomitant chlorophyll fluorescence signals (f) from lygodium leaves were also detected at 760 nm, which corresponds to the o2-a fraunhofer line. our data suggests that the daily change in photosynthetic status (p) can be traced by monitoring the change in relative f in relation to the estimated heat loss (h) and measured j, r, and t using a series of practical equations designed to roughly estimate the gross photosynthetic response within the plant canopy. using our equations, the photosynthetic capacity in the plant canopy structure could be simply simulated and predictable by optical sensors. (*) corresponding author: kawanotom@kitakyu-u.ac.jp received for publication 23 december 2015 accepted for publication 19 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 59-67 60 the thermal properties of greened rooftops and walls have recently been extensively documented. a study was performed to compare greened and nongreened walls with and without additional covering shade cloths using model buildings with thermal thin walls (yamasaki et al., 2009). by quantitatively evaluating the cooling load reduction effect under/inside the greened roof/walls, the room temperature of the green-covered building was shown to be significantly lower due to the sun-shielding effect of plants covering the building, and electrical power consumption for air conditioning was also lower in the green building. according to this model, around 40 to 45% of energy could be saved in the greened building compared to the non-greened building. it is natural to conclude that lowering total temperature inside the model buildings is the consequence of the local temperature controls on the surface of the walls or roofs. as a lack of or excess water could drastically alter the growth status of the plants installed, water consumption by the plants needs to be understood. in addition, the rate of water consumption and air-cooling properties of plants have a close relationship since transpiration by plant leaves plays a key role in local heat removal. therefore, quantitative evaluation of water consumption by a model wall-greening system is of great importance. toward this purpose, simple models with net-supported vine plants (such as ivy and morning glory) have reportedly been conducted (takayama et al., 2014). like other forms of green infrastructure, green façades in which climbing plants are grown either directly against, or on support structures affixed to external building walls, have recently been gaining the attention of architects as a design feature aimed at reducing internal building temperatures, reducing building energy consumption, and facilitating urban adaptation to a warming climate (hunter et al., 2014). today, such vertical greenery systems (vgs) are viewed as passive tools for energy savings in buildings (pérez et al., 2014). accordingly, not only the lowering of building temperature, but also many economic, environmental and social benefits are associated with the use of vgs (safikhani et al., 2014). furthermore, various new green wall construction methods have been developed to date, although many of these technologies have yet to be evaluated and even to be amended, mainly due to the difficulty of maintaining the active growth and development of plants under stressful conditions such as forced adhesion by artificial supports, exposure to thermal stresses, lack of or excess irrigation and/or fertilization on site and on time (tachibana et al., 2011). reports on approaches for the greening of buildings using self-growing plants are increasing day-byday. for an instance, a canadian team has designed a prefabricated piece to be used on building envelopes, interior partitions, façades or landscape enclosures, in which the vegetation is integrated within the wall construction instead of being adhered to it (ardila et al., 2009). accordingly, the designed system by an incorporated conduit system included the self-supporting, self-irrigating and self-fertilizing performance for the growing plants. an urban geographer, gandy (2010), has explored the work of french botanist patrick blanc, who applies his knowledge of botany and related sciences to urban wall design with inspiration from the mur végetal (green wall) first made in 1988. blanc intended to transform the urban sceneries into ravines or rainforests by covering the streets and buildings with ferns and mosses. it is well known in japan that, second to mosses, many members of pteridophytes (encompassing ferns and fern allies) have been traditionally used in japanese garden design (kawano, 2015). as encouraged by a french botanist, one of the authors (tk) recently propounded that the use of japanese fern species on green walls and/or roofs is worth pursuing (kawano, 2015). for this reason, we would like to discuss the criteria for the plant components of heat buffering green walls which should also be applied to the fern species. the first criterion is the plants’ adhesion to the surfaces of walls or roofs. among the common garden ferns found in japan, members of polypodiaceae such as lemmaphyllum microphyllum presl (japanese name: mamezuta), lepisorus thunbergianus (kaulf.) ching (japanese name: nokishinobu), and pyrrosia lingua (thunb.) farw (japanese name: hitotsuba) are epiphytic species often attached to trees and rocks. therefore, these plants can be effectively used to cover walls. however, lemmaphyllum microphyllum is often exposed to competition on the surface of rocks and walls with neighboring epiphytic higher plants such as ficus pumila l. (moraceae; japanese name: ōitabi). therefore, we would like to emphasize that the second criterion for plant components of thermal buffering green walls is that they should be fast growing. the third criterion for green wall ferns must be an ability to not only adhere, but also climb up the poles and nets, and cover irregular walls of the greening structures. the last criterion is the heat-buffering property of the greenery components. hara et al. optical evaluation of the shading properties of lygodium japonicum 61 however, to date, studies on the influence of climbing plant characteristics are still very limited, and even fewer works have investigated the impact of green façade design components on thermal performance (hunter et al., 2014). as a candidate fern species to be listed as a green wall component, lygodium japonicum (thunb.) sw. (lygodiaceae; japanese name: kanikusa), the only climbing fern species in japan, is of great interest since this plant species has a fast-growing, wall adhesive, and climbing nature. the sun-tracking and rotating movements associated with the climbing growth in two lygodium members (l. articulatum and l. scandens) were briefly described by charles darwin (1875). in his book, he concluded that “as ferns differ so much in structure from phanerogamic plants, it may be worthwhile here to show that twining ferns do not differ in their habits from other twining plants”. finally, the heat buffering property under solar radiation must be determined with l. japonicum. through the minimal model tests described here, we attempt to demonstrate that local surface temperature above ceramic roof tiles of a building can be effectively buffered, thus lowering daytime temperature and maintaining relative warmth during the night, by the presence over the tiles of leafy climbing ferns, possibly due to reflection and absorbance of solar rays and prevention of the release of heat from the building surface. however, once plants are installed on tall walls or the top of buildings which are far from accessible for manual daily care, realtime routine monitoring and controls of plant growth status should be automated using various optical sensors. for this purpose, background data for optical properties of a lygodium canopy under solar incident light, reflecting the thermal and growing status, have been investigated. 2. materials and methods plant materials and experimental set up lygodium japonicum is commonly known as “japanese climbing fern”. this native fern grows very rapidly and thus often covers neighboring living trees, rocks and walls in gardens (fig. 1). for ornamental purposes, l. japonicum has been exported out of the country. for instance, this plant was introduced in 1932 in florida, usa (gordon and thomas, 1997). in the hibikino campus of the university of kitakyushu (wakamatsu-ku, kitakyushu, japan; 33˚ 53’24’’ north latitude, 130˚ 42’ 49’’ east longitude), semi-wildly propagating l. japonicum plants (fig. 1ae) directly exposed to sunlight were sampled, replanted in pots, and kept in the greenhouse for three days under fluorescent light to recover prior to experiments. model set-up on building roof to measure daytime roof tile temperature with and without lygodium leaf canopy the experimental was set up on the rooftop of a building to assess the shading properties of the lygodium canopy (fig. 2a). lygodium plants were potted and placed on the building roof-top, with bunches of leaflets covering ceramic tiles on which pairs of fine thermocouples (thermal sensors) were set. we previously reported the real-time measurement of rapid and accurate temperature changes in the micro-environments within a plant cell culturing system (lin et al., 2006). the real-time thermo-sensing units employed here have similar set-ups. the units consist of fine thermocouples (kft-25-200-100, anbe smt co., japan), an ad/da 8 channel converter (mr-500, keyence, japan), and a pc with a display (fig. 2 b-d). each sensory unit, calibrated immediatefig. 1 the semi-wildly propagating japanese climbing fern, lygodium japonicum, used in this study. (a) vegetative leaflet. (b) reproductive leaflet. (c) semi-wild plants surviving in the gaps between stones (as indicated by arrows). (d) aggressive growth of l. japonicum winning the competition with other standing plants. (e) climbing growth of l. japonicum on the concrete and aluminum walls, thus naturally greening the building. (f) even though it came after, l. japonicum plants are growing on the concrete wall by rapidly covering over the pre-existing vines of ficus pumila l. plants were found on hibikino campus of the university of kitakyushu, wakamatsu-ward, kitakyushu, japan (a-e), and a private garden in miyazaki prefecture, japan (f). adv. hort. sci., 2016 30(2): 59-67 62 ly before the experiment, possesses small heat capacity, thus enabling immediate and accurate measurements. solar spectra, canopy-filtered light spectra and leafreflectivity spectra to measure solar and leaf canopy light spectra, a cl-500a illuminance spectrophotometer (konika minolta, tokyo, japan), which covers the range between 360 nm and 780 nm, was used. spectroscopic analyses of reflectivity on the surface of leaves were carried out using a portable nearinfrared (nir) field spectro-radiometer, fieldspec handheld 2 (asd inc., atlanta, ga, usa), designed for spectral measurements (ranging from 325 nm to 1075 nm) on site (fig. 2e). in figure 2f and g, the fate of incident solar light illuminating plant leaves and generalized modes of energy transfer by short wavelength (blue) light and long wavelength (red) light after illumination of chlorophylls are illustrated. based on the experimental design described here, spectroscopic data on incident light (j), reflection by leaves (r), transmittance through leaves (t), light energy captured by leaves (j) and chlorophyll fluorescence (f) can be non-invasively and remotely monitored. 3. results and discussion effects of lygodium japonicum canopy on ceramic roof tile surface temperature the heat-blocking or buffering action by the leafy canopy of l. japonicum was assessed by monitoring the changes in local temperature on the surface of model ceramic tiles with and without l. japonicum coverage (fig. 3). comparisons were made on a cloudy day (2 october 2014) (fig. 3, top) and a sunny day (3 october 2014) (fig. 3, bottom). in both cases, daytime temperature was higher on the control tiles without plant canopy. data clearly suggest that the fluctuation of temperature due to direct exposure to naturally changing solar light intensity could be buffered. fig. 2 experimental set up to assess the shading properties of the lygodium japonicum canopy on the roof of a building. (a, b) l. japonicum plants set up on the roof. (c) one of the fine thermocouples placed on the ceramic tiles. (d) composition of the monitoring units. (e) measurement of reflection spectra on the surface of plant leaves using a portable nir field spectro-radiometer. (i) pc control booth beneath the solar panels. arrows (ii) and (iii) indicate the positions of the thermocouples. (iv) a note pc, (v) an ad/da 8-channel converter, (vi) connecting cables. (f) fate of incident solar light illuminating plant leaves. j, incident light; r, reflection; t, transmittance; j, captured light energy; h, local heat loss; f, fluorescence; and p, photosynthesis. optically, j, r, t, j and f can be determined. (g) energy transfer by short wavelength (blue) light and long wavelength (red) light after illumination of chlorophylls. fig. 3 assessment of the sun-shading effect of l. japonicum canopy by monitoring of the surface temperature. typical data, recorded on 2 and 3 october 2014, are shown. hara et al. optical evaluation of the shading properties of lygodium japonicum 63 in addition to the action of the plant canopy in the daytime, the nighttime changes in local temperature were also buffered by the presence of the l. japonicum leaves, possibly by minimizing the bulk flow of heat-removing air reaching the tile surface, and also by blocking the heat transfer out of ceramic tiles through the highly reflective nature of the leaves in the nir region. these hypotheses will be the subject of critical experimental examination in future studies. filtering of solar rays through lygodium japonicum leafy canopy solar radiation above and under the l. japonicum leafy canopy were monitored with a hand-held spectrophotometer (fig. 4). by subtracting the level of radiation under the canopy from that recorded above the plants, total light filtering performance by l. japonicum canopy could be calculated (fig. 4, bottom). data from two nearby experimental sites, each with its independent plant, indicated that a majority of solar radiation was filtered by the leaf canopy, possibly through absorption and reflection of light. reflection of solar rays by lygodium japonicum leaves reflectivity of l. japonicum leaflet surface was measured under sunlight (fig. 5) (3 october 2014). due to the presence of chlorophylls, there was always a pair of valleys of reflectivity in the blue and red regions (in the range below 450 nm and around 660 nm), which correspond to the absorbance by chlorophyll a and its related metabolites (kawano et al., 1999). in the range of visible light, green-colored light (peaking at around 550 nm) was most highly reflected as expected by the presence of chlorophylls in the leaves. since green light was also the major light component of the under-canopy radiation (fig. 4), absorption of green light by plant pigments was shown to be negligible. in the nir region, a high rate of solar radiation was reflected upwards (fig. 5), suggesting that the decrease in nir radiation below the plant canopy is largely due to reflection. a steep increase in reflectivity spanning from the red region to nir region represents the so-called “red edge” of reflection. this is a phenomenon commonly observed in various green fig. 4 spectrometric analysis of solar radiation and its filtering by l. japonicum canopy. typical data, recorded on 3 october 2014, are shown. fig. 5 analysis of leaf reflectivity using the leaves of l. japonicum plants covering ceramic roof tiles. (a) typical reflectance spectra from l. japonicum leaves recorded at 9:00, 12:00, 15:00 and 18:00 on 3 october 2014 are shown. (b) an example of processed reflectance spectrum. relative signal intensity was determined after the first order differentiation of the spectral data. (c) changes in the normalized chlorophyll fluorescence signal. signals corresponding to reflectance in the green and red-to-nir regions are labeled as “green reflection” and “red edge”, respectively. chlorophyll-dependent fluorescence signals detected at 760 nm and 679 nm, corresponding to o2-a and o2-b fraunhofer lines, respectively, are labeled. adv. hort. sci., 2016 30(2): 59-67 64 leafy plants, suggesting that most green plant canopies are capable of creating shade, minimizing the nir radiation allowed to reach ground level, thus blocking the heating radiation to reach beneath the leaves. in this context, a l. japonicum canopy fulfills one of the key criteria as a thermal buffering plant canopy. detection of chlorophyll fluorescence signals in the reflectivity spectra at 760 nm, the wavelength corresponding to the o2-a solar fraunhofer line (telluric absorption band) near 760 nm, a spike of signal corresponding to chlorophyll fluorescence was observed especially under morning solar radiation (fig. 5a) (labelled as o2-a). however, chlorophyll signal at o2-b near 687 nm solar fraunhofer line could hardly be detected since 687 nm coincides with the steep increase in “red edge” reflectance by leaves (fig. 5a). after firstorder differentiation of the reflectance spectra, the red edge reflectance signal no longer interferes with the reading of chlorophyll fluorescence at 687 nm (fig. 5b) (labelled as o2-b). in this way, quantification of chlorophylls and estimation of the spread and density of vegetative plant tissue can be non-invasively and even remotely monitored as plant vegetation performance mapped by remote-sensing satellites (meroni et al., 2009; guanter et al., 2010; mazzoni et al., 2012). however, we have to be cautious about the handling of fluorescence data to assess the area of leaf coverage since fluorescence signals can potentially report the status of gross photosynthesis (without considering the rate of respiration) and therefore, it may be altered over the course of the day. the fate of light energy reaching the plant leaf surface can be expressed as follows: j= r+t+h+f+p [1] j= j-r-t [2] where j, r, t, h, f, p, and j stand for incident light, reflection, transmittance, heat loss, fluorescence, photosynthesis, and captured light energy, respectively. then, the fate of j can be traced as follows: j= h+f+p [3] p= j-h-f [4] in this study, we directly and fully monitored j (fig. 4) (solar spectrum), r (fig. 5a), and t (fig. 4) (canopy spectrum), and partial f, the intensity of which is proportional to the total f (fig. 5a, b). by definition, j can be readily estimated from recorded j, r and t. therefore, the rate of gross photosynthesis (p) under constant or known j should be negatively proportional to the rate of heat loss (h) + fluorescence (f). assuming that h is constant (actual changes in local heat loss in the leaves should be determined in future experiments), changes in f indicate the photosynthetic status of the plants. in fact, quantification of fluorescence signal and monitoring of its temporal changes can be readily performed after normalization with green reflection (fig. 5b, c). taken together, the data in figure 5c suggest that l. japonicum plants are fully ready for photosynthesis only after midday. need for the evaluation of photosynthesis we have recently proposed a series of practical equations designed to describe the collective gross photosynthetic response within the plant canopy (okamoto et al., 2016). using our equations, the photosynthetic capacity in the plant canopy structure can be simply simulated based on minimal sampling of a single top-positioned leaf through measurement of (i) pi-curve in a horizontally placed single leaf, (ii) state of dark respiration in a single leaf, and (iii) transmittance through a single leaf. as pointed out by monsi and saeki (2005), t through layer of leaves can be expressed according to the definition by beer-lambert law as follows: t=e-ax [5] where a and x are absorption coefficient and length of the path within the leaf layer, respectively. for simplification of the model, we assume that the canopy structure consists of uniform leaves. by experimentally determining the value for t in a single leaf, we can approximate the total light used for photosynthesis within the canopy structure as follows: [6] where i is the number of leaves (i=0 is initial light intensity above the leaves). today, michaelismenten-type photosynthetic equation proposed by platt and jasby (1976) is widely accepted by the plant research community to describe the nature of gross photosynthesis as below: [7] recently, we proposed that photosynthetic light response curves can be generated based on a limited number of experimental data points through application of platt-jasby equation by determining pmax values and kj values from least-sized experiments (nagasawa et al., 2015). by substituting j in equation [7] with the total hara et al. optical evaluation of the shading properties of lygodium japonicum 65 light used for photosynthesis within the canopy as shown in equation [6], we can obtain the following equation: [8] where pn stands for p in the nth leaf in the canopy. since the collective light yield rapidly converges, k can be replaced with ∞ in a practical sense. this equation can be rewritten to modify the apparent michaelis constant as follows: [9] by accurately determining p or total p in the canopy through a model experiment, we can more accurately estimate the local heat loss (h) on the leaf as: h= j-f-p [10] climbing plants it is well known that climbing plants, as represented by the tendril-bearing plant families, chiefly belong to higher flowering plant families such as vitaceae, bignoniaceae, passifloraceae and cucurbitaceae (fabre, 1855; darwin, 1875; gerrath et al., 2008), many of which are agriculturally and economically important (kawano et al . , 2012). interestingly, only a few climbing species can be found among the seedless vascular plant lineages, including ferns (darwin, 1875). many more climbing fern species may have been lost in the course of evolution, since it is believed that there was a dramatic drop in the diversity and abundance of most fern species, inversely-proportional to the burst of diversification in angiosperms during the cretaceous period (schneider et al., 2004). timing of model experiments we planned to examine the slowing effects of l. japonicum canopy on both local heating during daytime and local cooling during nighttime; for this purpose, early october (2014) was chosen as the timing for model experiments. although the attempt presented here provides preliminary data in support of the thermal buffering capacity of l. japonicum canopy, further model experiments taking place under two extreme conditions, namely in mid-summer and mid-winter, are required in order to fully assess the thermal buffering capacity of this species. finally, there is great interest in assessing the quantitative heat balance and the radiation balance on the surface of walls or roofs based on the larger scale experiment with special reference to the thermal buffering effect of l. japonicum in all seasons in japan. future environmental studies green components covering buildings and walls in urban areas are exposed not only to natural environmental stresses but also to artificial stressful conditions, chiefly exposure to polluted air containing ozone (kadono et al., 2006; tran et al., 2013) and related oxidants (yukihiro et al., 2012). most plants exposed to such oxidative stress readily develop visible symptoms on the leaves reflecting the onset of programmed cell death (kadono et al., 2010). the sensitivity and/or tolerance of l. japonicum to such stressful conditions must be studied prior to its wider application in urban greening projects. 4. conclusions the minimal thermal analysis of the sun-shading effect of l. japonicum canopy was performed by monitoring changes in tile surface temperature. in order to optically monitor the natural shading and growth properties of a green canopy consisting of the leaves of a climbing fern, the following optical approaches have been performed for the first time: here, optical properties of l. japonicum under solar incident light, namely, the natural shading and growing properties of green canopy were studied. the incident solar radiation spectrum (j), leaf transmittance (t) spectrum, and leaf-reflectivity spectrum (r) were measured. in the reflectivity spectrum, concomitant chlorophyll fluorescence signal (f) was detected at 760 nm, corresponding to the o2-a fraunhofer line. data suggests that the daily change in photosynthetic status (p) can potentially be traced by monitoring the change in relative f in relation to the estimated heat loss (h) and measured j, r, and t. acknowledgements this work was supported by a grant from the regional innovation strategy support program implemented by ministry of education, 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adv. hort. sci., 2019 33(1): 39-48 doi: 10.13128/ahs-23606 reflective materials and management practices on the physicochemical and biochemical quality of merlot grapes a.h. porto 1 (*), a. wagner júnıor 1, v. bandeira cabral 1, i. citadin 1, j. zanela 2, i.b. nunes 2, p.c. conceição 1 1 post-graduate programme in agronomy, universidade tecnológica federal do paraná, via do conhecimento, km 1, pato branco, paraná, brasil. 2 universidade tecnológica federal do paraná, estrada para boa esperança, km 4, dois vizinhos, paraná, brasil. key words: fruit growing, irradiation, vine, vitis sp. abstract: the aim of this study was to evaluate the use of reflective materials on the soil surface and management practices on the physicochemical and biochemical quality of the merlot grapes (vitis vinifera l.). the experimental design was of randomized blocks, in factorial 3 x 2 (“material type” x “management practice”), with 4 replications, using 4 plants by plot. the “management practice” factor was divided into two levels, with and without its realization. for “material type”, two reflective films were tested in the soil surface, it being white raffia plastics of polypropylene (reflective film 1), metallic raffia plastic (reflective film 2) and without the use of any reflective material. when they reach the harvesting point in the 2009/2010 and 2010/2011 production cycles, the physicochemical and biochemical evaluations of fruit quality were submitted; physiological characteristics of the plant and microbiological properties of soil were also evaluated. the use of reflective material on the soil surface improved the quality of the grape in some aspects, especially determining the reduction of the berry dropping and, consequently, an increase in the productivity. furthermore, it provided greater microbial activity in the soil, which may be important for grapevine. the cultural practices had no influence on the physicochemical and biochemical quality of the vine fruits, providing only a greater number of berries per bunch. 1. introduction grape is one of the most economically important fruits in the world, with brazil in 2014 occupying 78,765 hectares, while production was 1,454,183 tons (fao, 2014; camargo et al., 2011). approximately 57% of this production was used for winemaking (mello, 2011), with the rest consumed in natura or as dried fruits (conde et al., 2007). viticulture for winemaking is mainly concentrated between the 30th and 50th parallel of north latitude and between 30th and 45th of south lati(*) corresponding author: porto@alunos.utfpr.edu.br citation: porto a.h., wagner junior a., bandei̇ra cabral v., ci̇tadi̇n i., zanela j., nunes i.b., , conceição p.c., 2019 reflective materials and management practices on the physicochemical and biochemical quality of merlot grapes. adv. hort. sci., 33(1): 39-48 copyright: © 2019 porto a.h., wagner junıor a., bandeira cabral v., citadin i., zanela j., nunes i.b., conceição p.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 july 2018 accepted for publication 4 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 39-48 40 tude. the main climates that allow the cultive of vines are those of temperate, mediterranean and arid areas, in different levels (tonietto and mandelli, 2003). however, the state of paraná, brazil, presents potential for the cultivation of grapes destined for the in natura consumption and for the elaboration of juices or wines. in particular, in the southwest region of paraná there are numerous vine orchards in small rural properties even though the grapes produced in this region had sporadically shown inferior quality to what is considered as good for wine and/or juice, especially when related to soluble solids content. fruit quality is linked directly to genetic characteristics, edaphoclimatic factors and cultural practices. the management adopted in the vine orchard usually aims to promote the penetration of solar rays inside the canopy, allowing the leaves located in the lower part of the plant to increase the photosynthetic rate and increase the availability of the photo assimilates for the fruits (mota et al., 2009). the solar radiation is one of the main factors to obtain fruits with high quality, with regard to size, firmness, sugar content, coloration, anthocyanins, starch content and acidity (procton and lougheed, 1976; erez and flore, 1986; jackson, 1989). thus, the increase in the efficiency of its use is fundamental for obtaining more attractive fruits. among the management techniques performed in the pre-harvesting of the grape, which can improve the penetration of light and aeration inside the canopy, favouring the obtaining of quality fruits, are green pruning, defoliation, thinning, removal extra shoots (suckering) in the grape tree leaf (mendonça et al., 2016). however, many winegrowers are resistant to these practices, preferring the adoption of management techniques that allow them to obtain fruits with quality, without the need to increase the demand for labour and the costs in the property. one of the alternatives that can be adopted is the use of reflective materials in the soil, since they allow to increase the total light absorbed and the distribution of the same inside the canopy, improving the quality of the fruits (chavarria and santos, 2009). in ‘fuji’ apple the use of reflective materials in the soil was able to increase fruit quality as the red intensity of the epidermis and increase the size of fruits from the interior of the canopy (andris and crisosto, 1996). the use of reflective plastics, under the canopy of plants, is a very used management technique in some countries of america, europe and asia. the main objective is to reflect the sunlight to the inside of the crown, to intensify the coloration of the fruit epidermis, to improve the flavour, as well as to antecipate maturation (trevisan et al., 2006; meinhold et al., 2011). this additional light is beneficial for photosynthesis and anthocyanin production (layne et al., 2002). the principle of the use of these materials is the ability to reflect the solar radiation to the interior of the canopy and it can provide a 40% increase in the photosynthetically active radiation reflected when the soil is completely covered and, around 24%, when partially covered (green et al., 1995). the present study aimed to evaluate the use of reflective materials and management practices on plant physiology and physicochemical and biochemical quality of merlot grape (vitis vinifera l.). 2. materials and methods the experiment was conducted in a commercial vineyard in the dois vizinhos city pr, brazil, in santa lucia community (25° 51’ 08” s, 53° 06’ 15” w, 594 m of altitude), for two productive cycles (2009/2010 and 2010/2011), indicated as cycles 1 and cycles 2 in the following text. the studied vineyard was planted in 2004 with merlot (vitis vinifera l.) grafted 1103 rootstock paulsen (v. berlandieri x v. rupestris). the spacings between rows are 3.0 m and between plants are 2.0 m in an espalier system, totalling 1.666 plants per hectare. the experimental design was of randomized blocks, in multifactorial 3 x 2 (“material type” x “management practice”), with 4 replications, considering each 4 plants as a plot, the two central ones being useful, disregarding those near the border. the “management practice” factor was divided into two levels, with and without its realization. the treatments with the management practices consisted of the adoption of the shoot topping, withdrawal of tertiary branches, disbudding and defoliation. in the treatments without the management practices, only the disbudding was done. for “material type”, two reflective films were tested in the soil: polypropylene white raffia plastic (reflective film 1) and metallic raffia plastic (reflective film 2), both placed below the projection of the porto et al. reflective materials and management on quality of merlot 41 crown of the plants, in the lines and between the lines. the films were placed in the vineyard 30 days after plants broke their dormancy; as third level, none film was used. upon reaching the harvesting point, in both cycles, the fruits were harvested and taken to the laboratory of plant physiology, (universidade tecnológica federal do paraná campus dois vizinhos) for physicochemical and biochemical evaluations. the evaluated variables were rot incidence (bunch and berry), bunch and berries weight, number of berries and bunches, berry drop, total soluble solids content (°brix) (ss) titratable total acidity (tta) (g tartaric acid 100 ml-1), ss/tta ratio, ph and production per plant (kg). the incidence of rot was determined visually, considering fruits with damage when they presented lesions and typical characteristics of pathogen attack. the berry drop was determined by the percentage of berries detached from the general total of the bunch. the ss contents were determined by refractometry. the titratable acidity was determined by titrating 10 ml of juice in 90 ml of water diluted with 0.1 n naoh solution to ph 8.1; the results being expressed in g of tartaric acid 100 ml-1. analysis of biochemical variables of productive cycle 1 fruits included total proteins, total phenols, total and reducers sugars, phenylalanine ammonialyase enzyme (pal), flavonoids and anthocyanins, constituting a total of four samples per treatment. for total protein dosage, the pulp samples from the fruits of each treatment were macerated in a mortar with 10 ml of 0.2 m phosphate buffer (ph 7.5). then the material was centrifuged (14.000 g for 10 min at 4°c) and the supernatant collected. for the quantification of the total protein content in the samples, the bradford test (1976) was used and the spectrophotometer, model uv-sp2000-spectrum at 630 nm, with bovine serum albumin as standard. the quantification of the total phenolic compounds of the pulp of the fruits of each treatment was carried out in two stages; the first one, following the method adapted from bieleski and turner (1966) and the second one according to jennings (1991). the total soluble sugar concentrations of fruit pulp from each treatment were determined by the phenol-sulfuric method as described by dubois et al. (1956). the activity of pal was evaluated based on the difference in absorbance resulting from the conversion of phenylalanine to trans-cinnamic acid (hyodo et al., 1978). were evaluated the efficiency of water use (%), co2 assimilation rate (μmol co2 m-2 s-1), water conductance (mol h2o m-2 s-1), intracellular co2 concentration (μmol co2 mol-1), transpiration rate (mmol h2o m-2 s-1) and foliar temperature (°c) in the productive cycle 1 and productive cycle 2. these physiological analyses of the plants were performed on three different dates (8th, 15th and 21st of january, 2010) for the productive cycle 1 and a date (14th of december, 2010) during the productive cycle 2. the gas exchange readings always started at 9:30 a.m. using an open gas measurement system equipped with a li-6400xt infrared gas analyser (irga li-cor, lincoln, nebraska usa) and an artificial source of red and blue light, performed on the fully developed and healthy middle third leaves of two plants per plot. the microclimatic conditions in the sample chamber were maintained constant during the readings, being 1100 μmol m-2 s-1 par (photosynthetically active radiation) and ambient co2 concentration (average of 383 μmol co2 mol-1). microbiological analyses of the soil were also performed according to the type of coverage adopted, by means of the quantification of the respiratory activity of the same, according to method proposed by öhlinger (1993). the analyses were carried out in the laboratory after the collection of the same, in the period of seven days after the harvest of each productive cycle to the depth of 10 cm. maximum, average and minimum temperatures of the plant body were obtained after reflection of the solar radiation for the productive cycle 1, captured by infrared images (thermographic) by a therma cam sc500 camera, always installed in the direction of the region of the plant, at 1 m of distance from the projection of the plant, discarding those of the border of each treatment. the temperature was obtained using the therma cam 200 professional software based on the readings of 15th and 22nd of january, 2010 starting at 9:00 a.m. the results of all variables were submitted to analysis of variance and the means were compared by the tukey’s test (α = 0.05), except for the soil microbiological analyses where the duncan’s test (α = 0.05) was applied. the data expressed in percentage was transformed by sine arc √(x/100) and when expressed in numbers by √(x + 1), according to the lilliefors’ normality test. all analyses were performed by the statistical software sanest (zonta and machado, 1984). adv. hort. sci., 2019 33(1): 39-48 42 3. results and discussion in the course of the experiment, excessive rainfall occurred during the two productive cycles (figs. 1a and 1b). in addition to the occurrence of hail in productive cycle 1, which impaired the performance of phytosanitary treatments, creating a favourable environment for disease incidence, it being observed among these, a greater incidence of mildew (plasmopara viticola), close to 25% of bunches or berries. there were no statistically significant differences for the multifactorial interactions between the type of material and the management practices in the physicochemical variables (table 1) during productive cycles 1 and 2 and biochemical (table 2) of productive cycle 1, except for the ss/att ratio of the productive cycle 2. when factors were analyzed separately, significant results were observed, except for the total weight of berries and bunch, berry drop, yield per plant and total sugars concentration for the factor “material type” of production cycle 1 (table 3) and number of berries per bunch for the factor “management practice” of cycle 1 (table 4). for berry drop of the grapes, in the productive cycle 1, it was verified that the use of the reflective film 2 (metallized raffia plastic) presented the lowest values in comparison to the reflective film 1 (white raffia plastic) and without film (table 1). however, in production cycle 2 these values were lower in relation to cycle 1. the highest values of berry drop productive cycle 1 obtained in the reflective film 1 and without film, may be related to the greater attack of fungal diseases in the berries, because the cycle had an average temperature around 25°c and a high incidence of rainfall (280 mm) near the harvest season fig. 1 data obtained from inmet’s meteorological station, from productive cycle 1 (2009/2010) (a) and productive cycle 2 (2010/2011), located at universidade tecnológica federal do paraná campus dois vizinhos pr. table 1 berry drop (%), bunch weight (g), weight of total and individual berries (g), ss (°brix), att (g of tartaric acid 100 ml-1), ph, ss/tta ratio, number of berries per bunch, number of bunches, rot berries and bunches (%), production per plant (kg) of merlot grapes submitted or not to management practices with or without the use of reflexive films on the soil, in productive cycle 1 of (2009/2010) and productive cycle 2 of (2010/2011) ns= non-significant by the f test. ** significant at 5% of probability.t1= with management practices and no reflexive film; t2= with management practices with reflexive polypropylene white raffia plastic (film 1); t3= with management practices with reflexive metallic raffia plastic (film 2); t4= without management practices and no reflexive film; t5= without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). treatment (z) berry drop bunch weight total weight of berries berry weight (g) ss att ph ss/att berries no. per bunch bunches no. berries with mildew bunch with mildew production (kg) productive cycle 1 (2009/2010) t1 12.71 ns 132.4 ns 125.70 ns 1.73 ns 16.64 ns 5.57 ns 3.44 ns 2.99 ns 72.76 ns 25.67 ns 25.24 ns 24.02 ns 3539.04 ns t2 15.29 162.65 155.17 2.43 15.74 4.71 3.47 3.34 63.62 27.69 20.44 25.57 4222.51 t3 4.59 155.96 148.5 1.98 15.89 4.84 3.53 3.28 74.87 26.71 21.96 24.92 4535.66 t4 16.76 117.29 111 1.98 16.64 5.34 3.42 3.12 56.06 23.91 25.24 22.5 2831.5 t5 11.36 148.45 139.96 2.22 15.74 4.77 3.43 3.3 62.95 30.06 20.45 27.48 4503.96 t6 4.68 157.17 150.16 2.48 15.89 4.51 3.45 3.52 60.65 29.12 21.96 25.66 4554.81 cv (%) 18.77 14.06 14.29 18.33 7.98 8.29 2.21 16.52 8.12 8.79 4.54 5.1 24.37 productive cycle 2 (2010/2011) t1 1.59 ns 250.58 ns 237.82 ns 1.75 ns 60.25 ns 4.04 ns 3.67 ns 4.14** 137.2 ns 60.25 ns 4.47 ns 15.61 ns 15321.1 ns t2 4.8 259.4 241.74 1.76 62.63 3.77 3.36 4.44 139.75 62.62 6.25 15.98 16389.38 t3 5.42 283.37 268.07 1.74 70.25 4.58 3.06 3.72 152.97 70.25 4.13 14.13 19997.24 t4 3.72 245.29 233.95 1.78 59.63 4.4 3.37 3.86 131.97 59.62 6.04 11.26 14907.88 t5 3.63 265.81 253.84 1.75 60.88 4.08 3.2 3.93 145.06 60.88 4.85 17.11 16278.66 t6 3.78 246.34 238.1 1.62 70.3 4.43 3.35 4.48 147.22 69.38 8.06 18.99 17504.40 cv (%) 27.72 18.25 17.92 6.6 25.57 10.93 2.99 11.79 16.16 25.57 49.36 26.24 30.64 porto et al. reflective materials and management on quality of merlot 43 (fig. 1), favouring the development of pathogens. although they did not present statistically significant difference in both cycles, it is assumed that the use of metallized raffia plastic as a reflective film beneath the merlot vines had provided greater heat in the area of the crown projection as compared to the use of white raffia plastic without film creating an environment with low levels of moisture, unfavourable to the attack of diseases. the same was visualized in productive cycle 2 whose incidence of fungal diseases attack was reduced, resulting in lower values of berry drop. this hypothesis can be verified by the analysis of plant surface temperatures (minimum, average and maximum) after reflection of the solar radiation, captured by infrared (thermographic). in fact, we found divergent results for the type of cover and management practice, and the use of reflective film allowed an increase of up to 3°c in the average and maximum temperatures, in relation to the non-use of reflective film (tables 3 and 4, respectively). the same significant effect was not obtained for the maximum, average and minimum temperatures of the plants during the second evaluation (table 2). treatment (z) flavonoids (mg/100 g) phenols (mg/g) total sugar (mg/g) anthocyanins (mg/100 g) pal (uabs/min/ mg prot) proteins (mg/g) surface temperature in 2nd evaluation intracellular co2 concentration min (°c) med (°c) max (°c) 1st evaluation 3rd evaluation t1 159.12 ns 1.78 ns 26.92 ns 128.08 ns 0.09 ns 0.92 ns 26.33 ns 30.23 ns 37.90 ns 255.55 ns 242.07 ns t2 153.31 1.01 28.02 119.43 0.02 1.02 25.2 29.98 38.77 251.23 250.12 t3 110.41 1.32 30.76 86.01 0.04 0.79 24.85 31.38 41.65 246.61 241.07 t4 121.79 1.56 26.82 94.87 0.04 0.45 25.92 29.6 35.75 253.19 235.12 t5 151.74 1.63 27.76 118.22 0.03 0.74 26.83 31.13 39.73 255.55 244.82 t6 137.52 1.27 28.16 107.13 0.39 0.54 23.05 31.1 38.6 246.94 242.71 cv (%) 31.83 33.38 5.67 30.82 282.43 71.27 8.13 4.76 7.99 3.1 3.99 table 2 flavonoids (mg/100 g), phenols (mg/g), total and reducing sugar (mg/g), anthocyanins (mg/100 g), pal (uabs/min/mg prot) and proteins (mg/g), minimum, average and maximum surface temperatures of the second evaluation, intracellular co2 concentration (μmol co2 mol-1) of the first and third evaluation, of merlot vine submitted or not to management practices and the use or not of reflexive films on the soil, in productive cycle 1 (2009/2010) ns= non-significant by the f test. ** significant at 5% of probability. (z) t1= with management practices and no reflexive film; t2= with management practices with reflexive polypropylene white raffia plastic (film 1); t3= with management practices with reflexive metallic raffia plastic (film 2); t4= without management practices and no reflexive film; t5= without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). table 3 berry drop (%), total weight of bunches and berries (g), production per plant (kg) and total sugar (mg/g), minimal, medium and maximum surface temperatures during the first evaluation, of merlot vines submitted or not to management practices with the use or not of reflexive films on the soil, in productive cycle 1 (2009/2010) ns= non-significant by the f test. *means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). type of material (z) berry drop total weight of berries bunch weight production per plant total sugar surface temperatures in first evaluation min (°c) med (°c) max (°c) reflexive film 1 13.26 a* 147.57 a 155.55 a 4519.81 a 27.92 ab 20.43 ns 29.84 ab 39.98 ab reflexive film 2 4.63 b 149.33 a 156.57 a 4388.66 ab 29.46 a 18.81 30.45 a 41.55 a no film 14.68 a 118.35 b 124.84 b 3185.27 b 26.87 b 18.26 29.09 b 38.20 b cv (%) 24.62 14.29 14.06 24.37 5.67 14.35 2.75 5.83 table 4 minimum, medium and maximum surface temperatures during the first evaluation, number of berries per bunch in merlot vines submitted or not to management practices, in productive cycle 1 (2009/2010) * means followed by the same lowercase letter in the column do not differ significantly at 5% level of significance by tukey’s test. management practice surface temperature in first evaluation n° of berries per bunch min (°c) med (°c) max (°c) with management 20.75 a * 29.79 a 38.63 b 70.33 a without management 17.58 b 29.79 a 41.8 a 59.86 b cv (%) 14.35 2.75 5.83 5.67 44 adv. hort. sci., 2019 33(1): 39-48 regarding the bunch weight, total berries weight, and production per plant, the highest values were obtained when the reflective films 1 and 2 (white raffia plastic and metallized raffia plastic, respectively) were used, and when observed in the productive cycle 2 these values were even more expressive, but without statistical difference for the effect of the treatments (table 1). observing the productive cycles 1 and 2, we noticed the alternation of productivity, which may be related to the lower pluviosity in the final period of development of the fruits (fig. 1) and lower incidence of fungal diseases, providing best sanitary conditions of the vines resulting in increased production. it is believed that the use of reflective films in productive cycle 1 may have provided an increase in the use of the photosynthetically active radiation by the plant, since when it reaches the films, it is reflected, mainly in the regions below the canopy, usually more shaded, increasing the production of photo assimilates, which allowed a greater weight of bunch and berries and, consequently, greater production. this fact can also be observed for productive cycle 2, however, with no statistical difference. the influence of the radiation on the weight of the berries was also verified by cargnello (1992), kliewer and lider (1968), and todic et al. (2007), who verified that the weight of berries in plants exposed directly to the radiation was superior to those kept under shading conditions. this fact was also verified by morrison (1988), reporting that shading of the leaves undermined the weight of the berries. during fruit development, shading affects the photosynthetic rate, which consequently limits carbohydrate sources for their development (garriz et al., 1998). in part, this hypothesis can be verified by the results obtained regarding the intracellular co2 concentration during the second evaluation in productive cycle 1, since there was a significant effect for the interaction between the factors tested (table 5), demonstrating that when the management practice was performed, there was a lower value with the use of reflective film 2 (metallized raffia plastic), followed by the use of reflective film 1 (white raffia plastic) and during productive cycle 2, the results remained favourable for the use of some kind of material, however, with no statistical difference. it is assumed that these lower values in the intracellular co2 concentration with the use of the reflective films are due to the greater photosynthetic activity by the plant, quickly converting all co2 absorbed into photo assimilates, as previously attributed to the results regarding bunch weight, total of berries weight and production per plant. the same effect of the films was obtained for the contents of total sugars, with a higher mean obtained in fruits, where the plants had reflective films 1 and 2, which is also related to the greater use of the photosynthetically active radiation. in grapevine, kliewer (1980) described that the fruits located inside the canopy, therefore, with lower illumination, presented less accumulation of sugar. this fact also occurred in the fruits of those plants that did not have reflective films below the canopy, which demonstrates that the use of these is important for the greater use of photosynthetically active radiation. when submitted to the management practices the ss/att ratio of the fruits obtained higher results significantly using the reflective film 1 and without the use of reflective films. however, when they observed the non-use of management practices, the use of reflective material did not present statistical difference (table 6). the same phenomenon can be also observed concerning, the management practice. with its realizatable 6 ss/att ratio in merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). table 5 intracellular co2 concentration (μmol co2 mol-1) during the second evaluation in merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 1 (2009/2010) material type (z) ss/att ratio with management without managment reflexive film 1 4.44 aa 3.93 aa reflexive film 2 3.72 ba 4.48 aa no film 4.14 aa 3.86 aa cv (%) 11.8 material type (z) intracellular co2 with management without management reflexive film 1 266.35 aba* 259.50 aa reflexive film 2 261.19 ba 261.53 aa no film 274.13 aa 254.73 ab cv (%) 2.03 * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). porto et al. reflective materials and management on quality of merlot 45 tion, it is believed that there was increase in the photosynthetic rate of the plant, as results in the intracellular co2 concentration (tables 5, 7, and 8), since practices like disbudding and defoliation allow higher incidence of light to enter the plants. in part, the greater light penetration can be proven by obtaining the increase in the minimum temperature of the plants during the first evaluation, when the management practice is carried out (table 4), which is advantageous for greater metabolic activity of the plant, consequently providing increase in energy production and photo assimilates. however, when there was no management practice the plants showed a higher maximum temperature in comparison with those in which it was carried out (table 4). this demonstrates that the accomplishment of the management practice besides increasing the minimum temperature of the plant, provides lower maximum temperature, favouring the photosynthetic activity, since very high temperatures can provide stomatic closure, reducing the entrance of co2, which diminishes the photosynthetic activity in addition to reducing the absorption of water and nutrients. on the other hand, the average surface temperature of the plant presented equal means with or without the management practice (table 4). as for the other physiological variables (water use efficiency, co2 assimilation rate, water conductance, transpiration rate and leaf temperature), there was no significant interaction or for the factors individually during the production cycle 1 in the three evaluations carried out (table 7), also verified for intracellular co2 concentration in the first and third evaluation (table 2), and in the water use efficiency, co2 assimitable 7 water use efficiency (%), co2 assimilation rate (μmol co2 m-2 s-1), water conductance (mol h2o m-2 s-1), transpiration rate (mmol h2o m-2 s-1), leaf temperature (°c), intracellular co2 concentration (μmol co2 mol-1) of merlot vines submitted or not to management practices with or without reflexive films on the soil, during the first, second and third evaluations, in productive cycle 1 (2009/2010) ns= non-significant by the f test. (z) (t1) with management practices and no reflexive film; (t2) with management practices with reflexive polypropylene white raffia plastic (film 1); (t3) with management practices with reflexive metallic raffia plastic (film 2); (t4) without management practices and no reflexive film; (t5) without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). treatment (z) water use efficiency (%) co 2 assimilation rate (μmol co 2 m-2 s-1) water conductance (mol h 2 o m-2 s-1) transpiration rate (mmol h 2 o m-2 s-1) leaf temperature (°c) 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. t1 0.26 ns 0.25 ns 0.38 ns 12.12 ns 10.55 ns 10.9 ns 0.23 ns 0.24 ns 0.18 ns 4.76 ns 4.76 ns 2.94 ns 32.59 ns 31.58 ns 28.3 ns t2 0.27 0.27 0.38 12.57 11.93 11.8 0.26 0.25 0.23 4.69 4.69 3.41 31.82 31.16 28.38 t3 0.31 0.3 0.37 11.92 12.53 10.69 0.22 0.23 0.2 4.57 4.57 3.27 32.45 31.13 28.58 t4 0.28 0.3 0.39 12.44 11.52 10.98 0.23 0.22 0.18 4.5 4.5 2.96 31.84 31.21 28.6 t5 0.24 0.27 0.39 12.96 11.05 11.5 0.26 0.21 0.2 5.34 5.34 3.23 32.94 31.82 28.49 t6 0.26 0.3 0.37 12.9 12.62 11.78 0.23 0.25 0.17 4.34 4.34 2.8 31.78 30.93 28.02 cv (%) 15.03 9.78 10.72 7.22 9.69 10.72 10.85 15.89 20.67 11.69 13.37 13.43 2.99 2.07 1.41 table 8 intracellular co2 concentration (μmol co2 mol-1), water use efficiency (%), co2 assimilation rate (μmol co2 m-2 s-1), water conductance (mol h2o m-2 s-1), transpiration rate (mmol h2o m-2 s-1), leaf temperature (°c) of merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) treatment (z) intracellular co 2 concentration water efficiency (%) co 2 assimilation rate (μmol co 2 m-2 s-1) water conductance (mol h 2 o m-2 s-1) transpiration rate (mmol h 2 o m-2 s-1) leaf temperature (°c) t1 261.31 ns 1.43 ns 14.67 ns 0.23 ns 3.29 ** 24.44 ns t2 263.7 1.46 14.36 0.23 3.2 24.51 t3 263.09 1.46 14.09 0.22 3.36 24.32 t4 260.05 1.41 15.02 0.23 3.23 24.37 t5 263.43 1.44 14.71 0.23 3.67 24.5 t6 261.82 1.44 14.44 0.22 3.04 24.26 cv (%) 2.78 7.84 7.85 8.74 7.48 2.85 ns= non-significant by the f test. (z) (t1) with management practices and no reflexive film; (t2) with management practices with reflexive polypropylene white raffia plastic (film 1); (t3) with management practices with reflexive metallic raffia plastic (film 2); (t4) without management practices and no reflexive film; (t5) without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). adv. hort. sci., 2019 33(1): 39-48 46 lation rate, water conductance for productive cycle 2. the transpiration rate of productive cycle 2 showed significant interaction indicating that the use of reflective film 2 in conjunction with the management practice were superior to the other treatments, which possibly may have contributed to the quantity and quality of the fruits in the cycle productive 2 (table 9). it was verified that the adoption of management practices in spite of not influencing most of the variables analysed in the present study, provided a greater number of berries when carried out on the merlot vines in productive cycle 1 (table 4), demonstrating the importance of its execution for the winegrower. it is believed that the lower number of berries in the plants without management practices can be due to the attack of fungal diseases. champagnol (1984) and fregoni (1998) reported in their studies that mildew, after effective fruiting, may be one of the causes for the lower number of berries per bunch. indeed, the practices of shoot topping, suckering, withdrawal of tertiary branches and defoliation may have improved the penetration of light and air in the area of projection of the plants, not allowing the formation of favourable microclimate for fungal diseases. regarding the microbiological analysis of the soil, a statistical difference was observed for the type of reflexive film used in productive cycle 1, with the highest microbial activity in the soil when white raffia plastic and metallized raffia plastic were used. the latter did not differ statistically from the treatment without reflexive film on the soil (table 10). for production cycle 2, although the values obtained were higher, which makes it favourable for productivity, the same did not present statistical differences. it is assumed that the use of the white or metallic raffia plastic films to reflect the solar radiation allowed lower temperature in the soil, as well as reduced water loss, favouring the full metabolic activity of soil microorganisms, given the maintenance of conditions optimum temperature and humidity, decreasing problems with water stress for the plant. hungria et al. (1997) mentioned that the reduction of soil microbiota impairs the temporary fixation of nutrients, increasing their losses and resulting in soil impoverishment. in general, soils with high microbiological activity indicate low human interference with the environment and are, in principle, desirable for crops, since they contribute to a faster decomposition and re-synthesis of organic matter, to nutrient cycling, to specific biochemical transformations (nitrification, denitrification, oxidation and sulphur reduction) for biological nitrogen fixation (silveira and freitas, 2007). thus, the use of reflective material on the soil presents additional benefits to the crop and the environment, and according to the peculiarities of the area, the effects of the application of the technology on the soil microbial population can become as important or more than the benefits obtained directly on the quality of the fruits. attention must be paid to determining the most appropriate moment for the installation of the reflective film in the soil, since the influence of the radiation on the quality of the fruit varies according to the phenological stage of the crop (weston and barth, 1997; kader, 2002; meinhold et al., 2011). material type (z) transpiration rate (mmol h 2 o m-2 s-1) with practice without practice reflexive film 1 3.20 b a 3.67 a a reflexive film 2 3.36 a a 3.04 a ab no film 3.29 a a 3.23 a b cv (%) 7.48 table 9 transpiration rate (mmol h2o m-2 s-1) of merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). table 10 microbiological respiratory activity of the soil according to the material type addopted for merlot vines, in productive cycle 1 (2009/2010) and productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). material type (z) microbiological resiratory activity (mg of co2 kg of soil-1) productive cycle of 2009/2010 reflexive film 1(z) 45.65 a reflexive film 2 43.45 ab no film 35.75 b cv (%) 11 productive cycle of 2010/2011 reflexive film 1 100.10 ns reflexive film 2 102.3 no film 82.13 cv (%) 30.87 porto et al. reflective materials and management on quality of merlot 47 4. conclusions the use of reflective films on the soil improved the quality of merlot grapes in some aspects as reducing berry drop, consequently increasing productivity. management practices here tested didn’t have influence on the physicochemical and biochemical characteristics of the fruits, resulting only in more berries per bunch. the use of reflective films on the soil provided higher microbiological activity in the soil, which may be important for grapevines. references andris h., crisosto c., 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group. they are stable at high concentrations of this element and are able to accumulate up to 1 mg of se per g of dry weight during selenium uptake without causing growth inhibition (terry et al., 2000). although se is not regarded as an essential trace element for most of agricultural crops, including onion, a moderate selenium concentration is reportedly a powerful natural antioxidant, capable of protecting plants against different types of stress, such as salinity, drought, uv-radiation, heavy metals, and attack by herbivores (germ et al., 2007). notably, the antioxidant role of se in polyunsaturated fatty acid (pufa) stability of seed oil seems to be of special interest. oils are a vital component of the human diet and are readily available in some vegetables species. their accumulation in plant seeds, as well as their fatty acids composition, is affected by genetic, environmental, and farming factors (sidlauskas and bernotas, 2003). in addition, their antioxidant action is of great importance due to pufa disposition against oxidation. an investigation of the germination of capsicum annuum seeds demonstrated that fatty acids content and composition may play a major role on germination and seedling vigour at different temperatures (kaymak, 2014). a significant decrease of seed germination during storage is reportedly connected with lipid auto-oxidation leading to pufa degradation and an increase in cell membrane permeability (harmann and mattick, 1976). in this respect, the inhibitory effect of seed oil content, fatty acids composition and antioxidant properties as affected by genotype in allium cepa l. and perennial onion species n.a. golubkina1 (*), s.m. nadezhkin1, a.f. agafonov1, o.v. kosheleva2, a.v. molchanova1, g. russo3, a. cuciniello3, g. caruso3 1 agrochemical research center, all-russian institute of vegetable breeding and seeds production, selectsionnaya 14, 143080 moscow region, odintsovo district, vniissok, russia. 2 laboratory of vitamins and mineral compounds, institute of nutrition, ustinsky pr. 2/14, 119480 moscow, russia. 3 department of agricultural sciences, naples university federico ii, via università 100, 80055 portici (na), italy. key words: oil fatty acids composition, onion seed oil, proteins, selenium, total phenolics. abstract: the antioxidant content in plant seeds is deemed to affect seed oil protection against auto-oxidation to a large extent, whereas the relationship between a strong antioxidant element such as selenium (se) and either seed oil accumulation or fatty acids composition has not been investigated so far. the aim of the present work was to assess se concentrations in seeds and their relationships with oil content and fatty acids composition in: a) ten allium cepa cultivars, i. e. eight russian and two italian; and b) six perennial onion species (a. schoenoprasum, a. obliquum, a. altaicum, a. fistulosum, a. nutans, a. ramnósum). fatty acids composition of allium seed oil was determined by gas chromatography method, whereas total and water soluble se concentration was assessed by microfluorimetric method. the oil content of allium cepa seeds was 1.7 fold higher (10.7-16.5%) than that recorded in perennial onions (4.0-10.8%) and it was positively correlated with the total se concentration. within a. cepa, the seeds of the two italian cultivars ramata di montoro and rossa di tropea were characterized by the highest oil content (16.5-16.6%) and oleic acid (25-27%). linoleic (c18:2) acid was the main fatty acid, followed by oleic (c18:1) and palmitic acids (c16:0) in all cultivars. among the perennial onion seeds, the highest oil percentage was detected in a. schoenoprasum (10.8%) and the lowest in a. ramnósum (4.0%). compared to a. cepa cultivars, the perennial onion species showed a similar oil fatty acid composition, with the main acids being c18:2, c18:1 and c16:0 in decreasing order, a lower level of c16:0, and enhanced levels of minor sfa, such as c20:0, c22:0 and c24:0. further differences also included decreased levels of c16:1, 11-trance c18:1 and a higher concentration of c22:1. the concentration of water soluble forms of se in seeds was positively correlated with linoleic acid and with total phenolics. conversely, oleic acid was negatively correlated with water soluble forms of se. adv. hort. sci., 2015 29(4): 199-206 (*) corresponding author: segolubkina@rambler.ru received for publication 25 september 2015 accepted for publication 4 november 2015 200 adv. hort. sci., 2015 29(4): 199-206 selenium on lipid peroxidation was described for ryegrass (hartikainen et al., 2000) and germination was enhanced in lettuce seeds stored long-term as a result of se application (xue et al., 2001). moreover, a direct correlation was found between the content of water soluble se forms and germination rate of apiaceae seeds (dobrutskaya et al., 2010). unlike the reports mentioned above, soybean biofortification with selenium did not affect the stability of seed oil pufa against oxidation (fallen et al., 2011). a high oil content is typical in allium species, with allium cepa seeds containing about 26%, with the predominance of highly labile linoleic acid (dini et al., 2008; yalcin and kavuncuoglu, 2014); a gradual germination decrease over time is however frequent in allium species. actual perspectives of allium seed oil utilization in the human diet as well as in phytotherapy of cardiovascular and dermatological diseases (nakamura et al., 2008) also require investigations into the relationship between selenium and either oil content or composition in allium species, also connected to the genotype. therefore, due to the lack of information reported in literature on these topics, research was carried out in order to assess selenium concentration, oil content and fatty acids composition, total phenolics in the seeds, as well as the significant correlations between these four parameters, in: a) ten allium cepa l. cultivars, native of northern europe (eight russian cultivars) and the mediterranean area (two italian cultivars); and b) six perennial allium species. 2. materials and methods research was carried out to compare the seeds obtained from northern europe and mediterranean onion cultivars with regard to selenium concentration, oil content and fatty acids composition, proteins, and total phenolics. in this context, seeds from eight russian allium cepa cultivars (ledocol, cherny prince, alba, sigma, myachkovsky, zolotnichock, zolotie cupola, globus) and six perennial onion species (a. schoenoprasum, cv. medonos; a. obliquum, novichock; a. altaicum, alves; a. fistulosum, troitsa; a. nutans, lider; a. ramnósum, aprior) were produced in the experimental fields of “all-russian institute of vegetable breeding and seeds production” in the moscow region in 2014. in the same year, seeds were obtained from two italian allium cepa cultivars (ramata di montoro and rossa di tropea) grown in an experimental field arranged by the department of agricultural sciences in a private farm sited in pontecagnano (salerno, italy). in both locations, five onion bulbs per square meter of 46-70 mm caliber were planted in march and the infructescences containing physiologically ripe seeds were harvested in july-august. the farming practices before planting were: ploughing at 40 cm depth and hoeing at 15 cm; fertilization with 70 kg ha−1 of n as ammonium sulphate, 80 kg ha−1 of p 2 o 5 as superphosphate and 130 kg ha−1 of k 2 o as potassium sulphate. during the crop, the following practices were performed: hand weeding; fertilization with 130 kg ha−1 of n and 140 kg ha−1 of k 2 o as calcium nitrate and potassium nitrate; drip irrigation, activated when the soil available water capacity decreased to 70%; copper oxychloride application against rust. the seed oil content was gravimetrically assessed after oil extraction with n-hexane (60°c) for 6 h in a soxhlet extractor, according to aoac (1990), and hexane removal from the oil by rotary evaporation under reduced pressure (20 mbar at 30°c). the fatty acids composition of allium seed oil was detected by gas chromatography, according to aocs (1999), via appropriate methyl ethers chromatography on capillary glass column, using an agilent 6890 gas cromatograph (agilent technologies, santa clara, california, us), equipped with a flame ionization detector. the carrier gas was helium and the total gas flow rate was 20 ml·min-1. the oven temperature was initially held at 80°c for 10 min, then sequentially increased to 175°c for 15 min, to 200°c for 5 min, and to 225°c for 70 min. the injector and detector temperatures were 260 and 240°c respectively. the peaks of fatty acids methyl ethers were identified by comparison to the retention times of reference standards (aocs, 1999). the microfluorimetric method was used to determine total and water soluble selenium content in seeds (alfthan, 1984). total phenolics in water extracts were determined using folin-ciocalteu colorimetric method (sagdic et al., 2011). protein content was estimated by kjeldahl method. a randomized complete block design was used for the distribution of the treatments in the fields, with three replicates, and the plot size was 18 m2 (4.0 x 4.0 m). data were statistically processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 17. data expressed as percentage were subjected to angular transformation before processing. standard deviation (sd) was assessed for the three replicate data per experimental treatment and coefficient of variation (cv) for the data set linked to each variable. 3. results as reported in tables 1 and 2, the oil content recorded in allium cepa seeds fell in the 10.7-16.6% range and was significantly higher than that detected in perennial onion species (4.0-10.8%). among allium cepa cultivars, the two italian ones (ramata di montoro and rossa di tropea) showed higher content of seed oil (16.5-16.6%) compared to the russian cultivars (10.7-13.5%). a. ramnosum seeds were characterized by a 1.8-2.7 fold lower oil content than the other perennial onion seeds. among the perennial onion species tested, the highest oil concentration was measured in a. schoenoprasum seeds (10.8%). 201 golubkina et al., seed oil content as affected by genotype in onion selenium concentration in onion seeds varied greatly (108-476 and 112-261 µg·kg-1 in perennial onions and allium cepa respectively), giving the highest coefficient of variation (25% in a. cepa and 37% in perennial onions); the lowest cv was recorded for the oil content (9.2 and 11.2% in a. cepa and in perennial species seeds respectively). moreover, only allium cepa seeds showed a direct correlation between total se concentration and oil percentage (r= 0.82; p < 0.05) (fig. 1). as reported in the tables 3, 4, 5, the predominant fatty acids of seed oil in both allium cepa and perennial onion species are the following in decreasing order: linoleic c18:2 (61.5-73.7%), oleic c18:1 (19.0-27.2%) and palmitic c16:0 (5.1-8.6%). moreover, among the allium cepa cultivars considered, the highest content of oil saturated fatty acids (sfa) was found in ledokol seeds (11.3%), whereas the seed oil of allium cepa italian cultivars ramata di montoro and rossa di tropea showed the highest content of monounsaturated acids (mufa). among perennial onion species, the highest sfa value was detected in a. ramnosum (12.7%), whereas the highest content of oleic acid c18:1 was detected in a. nutans seed oil. moreover, pufa represented as much as 74% of table 1 chemical composition of allium cepa seeds cultivar oil content (%) proteins (%) total phenolics (mg g-1 f.w.) selenium (µg kg-1 f.w.) sigma 10.7 ± 0.9 d 22.4 ± 2.1 ce 3.0 ± 0.2 bc 112 ± 9 e globus 12.0 ± 0.9 c 22.1 ± 2.0 de 2.8 ± 0.2 c 124 ± 7 de alba 12.5 ± 1.0 bc 24.1 ± 2.2 bd 1.9 ± 0.1 f 113 ± 7 e ledocol 12.9 ± 1.0 bc 26.2 ± 2.2 b 2.7 ± 0.2 cd 192 ± 10 bc myachkovsky 13.1 ± 1.1 bc 19.1 ± 1.7 f 3.8 ± 0.3 a 121 ± 9 de zolotie cupola 13.1 ± 1.0 bc 34.7 ± 3.1 a 3.2 ± 0.3 b 159 ± 8 cd cherny prince 13.2 ± 1.0 bc 25.2 ± 2.0 bc 2.1 ± 0.1 ef 112 ± 9 e zolotnichock 13.5 ± 1.1 b 20.5 ± 1.8 e 2.7 ± 0.2 cd 135 ± 39 de ramata di montoro 16.5 ± 1.4 a 22.0 ± 1.9 df 2.4 ± 0.2 de 261 ± 17 a rossa di tropea 16.6 ± 1.4 a 22.3 ± 1.9 cd 3.0 ± 0.3 bc 203 ± 12 b mean ± sd 13.5 ± 1.3 23.9 ± 3.0 2.8 ± 0.4 153 ± 40 cv (%) 9.2 12.4 14.6 25 ± within each column: the data are reported as mean ± standard deviation; cv = coefficient of variation; means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). table 2 chemical composition of perennial onion species seeds cultivar oil content (%) proteins (%) total phenolics (mg g-1 f.w.) selenium (µg kg-1 f.w.) a. ramnósum (cultivar aprior) 4.0±0.3 d 24.6±2.1 bc 1.7±0.1 e 223±25 b a. nutans (lider) 7.0±0.6 c 35.2±3.1 a 3.0±0.3 bc 108±12 c a. fistulosum (troitsa) 7.1±0.6 c 23.8±2.0 c 3.8±0.3 a 476±12 a a. altaicum (alves) 8.6±0.7 bc 26.4±2.2 bc 3.3±0.3 ab 182±7 bc a. obliquum (novichock)   9.1±0.7 b 19.9±1.7 d 2.4±0.2 cd 211±11 b a. schoenoprasum (medonos) 10.8±0.9 a 27.2±2.5 b 2.1±0.2 de 146±8 bc mean ± sd 7.8±1.7 bc 26.2±3.4 2.7±0.7 224±84 cv (%) 11.2 13.0 24.3 37 ± within each column: the data are reported as mean ± standard deviation; cv = coefficient of variation; means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 260 280 y = -145,4 + 22.3x r² = 0.66 *220 240 260 280 g-1 y = -145,4 + 22.3x r² = 0.66 * 200 220 240 260 280 g kg -1 y = -145,4 + 22.3x r² = 0.66 * 140 160 180 200 220 240 260 280 s e, m cg k g-1 y = -145,4 + 22.3x r² = 0.66 * 140 160 180 200 220 240 260 280 s e, m cg k g-1 y = -145,4 + 22.3x r² = 0.66 * 100 120 140 160 180 200 220 240 260 280 s e, m cg k g-1 y = -145,4 + 22.3x r² = 0.66 * 100 120 140 160 180 200 220 240 260 280 10 11 12 13 14 15 16 17 s e, m cg k g-1 y = -145,4 + 22.3x r² = 0.66 * 100 120 140 160 180 200 220 240 260 280 10 11 12 13 14 15 16 17 s e, m cg k g-1 oil content (%) y = -145,4 + 22.3x r² = 0.66 * 100 120 140 160 180 200 220 240 260 280 10 11 12 13 14 15 16 17 s e, m cg k g-1 oil content (%) fig.1 fig. 1 relationship between selenium concentration and oil content in allium cepa seeds (r = 0.82; p<0.05). 202 adv. hort. sci., 2015 29(4): 199-206 fatty acids in a. altaicum seed oil (table 4), while 70.3% was the top value of these compounds in allium cepa cultivars, i.e. in myachkovsky and zolotie cupola (tables 3 and 4). sfa content in seed oil of perennial onions decreased according to the following sequence: a. ramnosum > a. nutans > a. fistulosum = a. obliquum > a. schoenoprasum > a. altaicum. as regards mufa, the ranking sequence was: a. nutans > a. schoenoprasum > a. ramnosum > a. obliquum > a. fistulosum > a. altaicum. compared to allium cepa, the seed oil of perennial species was characterized by a decreased content in c16:0, 11-trans c18:1 and c16:1 fatty acids and high concentrations of c20:0, c22:0, c24:0 and c22:1, c 20:2 acids (table 5). negative correlation was found between water soluble se concentration and oleic acid (r= -0.64, p<0.05) and positive correlation between water soluble se and linoleic acid (r= 0.63; p<0.05) (fig. 2 and 3). the highest content of total phenolics (3.8 mg g-1) was detected in ‘myachrovsky’ seeds among allium cepa cultivars and in a. fistulosum among perennial onion species. moreover, total phenolics were positively correlated with se water soluble forms (r= 0.92; p<0.01), but they showed table 3 fatty acids composition (%) of seed oil in allium cepa cultivars fatty acid onion cultivar a lb a c he rn y pr in ce si gm a g lo bu s z ol ot ni ch oc k m ya ch ko vs ky z ol ot ie k up ol a r am at a di m on to ro r os sa d i t ro pe a l ed ok ol 14:0 0.14 b 0.12 c 0.11 c 0.15 b 0.12 c 0.11 c 0.12 c 0.12 c 0.12 c 0.19 a 16:0 8.62 a 8.44 ab 7.61 bc 7.59 bc 7.58 bc 7.82 ac 5.13d 7.82 ac 7.23 c 8.53 a 17:0 0.04 a 0.01 c 0.03 b 0.04 a 0.03 b 0.03 b 0.03 b 0.04 a 0.04 a 0.04 a 18:0 1.30 cd 1.47 bc 1.39 cd 1.73 a 1.50 b 1.26 d 1.43 bc 1.69 a 1.36 cd 1.58 ab 20:0 0.15e 0.16 de 0.14e 0.22 c 0.27 b 0.17 de 0.17 de 0.22 c 0.19 cd 0.81 a 22:0 0.10 ef 0.10 ef 0.14 c 0.17 b 0.19 a 0.11 e 0.13 d 0.14 c 0.14 c 0.09 f 24:0 0.00 d 0.00 d 0.00 d 0.00 d 0.00 d 0.04 b 0.05 a 0.04 b 0.05 a 0.02 c sfa 10.35 ab 10.30 ab 9.42 b 9.90 b 9.69 b 9.54 b 7.06 c 10.08 ab 9.23 b 11.26 a 16:1 0.10 a 0.06 cd 0.05 d 0.07 bc 0.07 bc 0.05 d 0.08 b 0.06 cd 0.07 bc 0.07 bc 16:1 9 cis 0.22 ab 0.20 bd 0.21 ac 0.23 a 0.19 ce 0.17 e 0.11 f 0.18 de 0.19 ce 0.19 ce 18:1 9 cis 20.77 ce 22.44 cd 20.35 de 21.79 cd 19.18 e 18.97 e 19.06 e 24.96 ab 27.16 a 23.00 bc 18:1 11 trans 1.00 cd 1.05 bc 1.06 bc 1.17 ab 1.11 bc 0.91 de 0.83 e 1.25 a 1.30 a 1.09 bc 20:1 0.20 a 0.20 a 0.20 a 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b 22:1 0.02 d 0.02 d 0.02 d 0.11 a 0.05 c 0.06 bc 0.07 b 0.03 d 0.02 d 0.05 c mufa 22.31ce 23.97 bc 21.89 ce 23.37 cd 20.60 de 20.16 e 20.15 e 26.48 ab 28.74 a 24.40 bc 18::2i 0.03 de 0.06 ab 0.06 ab 0.07 a 0.04 cd 0.02 e 0.05 bc 0.07 a 0.07 a 0.07 a 18:2 66.90 ab 65.24 ab 66.98 ab 66.52 ab 69.43 a 69.91 a 69.81 a 62.99 b 61.50 b 63.77 ab 20:2 0.21 cd 0.21 cd 0.23 bc 0.19 d 0.29 a 0.19 d 0.25 b 0.25 b 0.31 a 0.25 b 18:3 ω-3 0.12 de 0.12 de 1.34 a 0.19 b 0.14 cd 0.14 cd 0.16 bd 0.09 e 0.10 0.17 bc 20:3 0.05 a 0.04 ab 0.03 b 0.04 ab 0.04 ab 0.03 b 0.03 b 0.05 a 0.03 b 0.05 a pufa 67.26 ac 65.67 ac 68.64 ac 67.01 ac 69.94 ab 70.29 a 70.30 a 63.45 c 62.01 c 64.31 bc along each row, means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). sfa= saturated fatty acids; mufa = mono unsaturated fatty acids; pufa = poly unsaturated fatty acids. 29 27 29 ) 25 27 29 % ) y = 31,52 0.35 x r² = 0.46 *23 25 27 29 (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 *23 25 27 29 (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 21 23 25 27 29 ic a ci d (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 19 21 23 25 27 29 o le ic a ci d (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 17 19 21 23 25 27 29 o le ic a ci d (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 5 17 19 21 23 25 27 29 o le ic a ci d (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 15 17 19 21 23 25 27 29 20 25 30 35 40 45 o le ic a ci d (c 18 :1 ), ( % ) y = 31,52 0.35 x r² = 0.46 * 15 17 19 21 23 25 27 29 20 25 30 35 40 45 o le ic a ci d (c 18 :1 ), ( % ) water soluble se, mcg kg-1 y = 31,52 0.35 x r² = 0.46 * 15 17 19 21 23 25 27 29 20 25 30 35 40 45 o le ic a ci d (c 18 :1 ), ( % ) water soluble se, mcg kg-1 fig.2 fig. 2 correlation between water soluble selenium concentration and oleic acid concentration in allium seeds (r = -0.64, p<0.05). 203 golubkina et al., seed oil content as affected by genotype in onion no correlation with total se concentration (fig. 4). finally, no significant correlation was recorded between proteins and se, the latter considered either as total concentration or water soluble forms, in onion seeds. 4. discussion and conclusions seed aging and viability are affected by a number of factors, both during seed production and storage. the seed aging process depends on a seed’s ability to resist degenerative changes as well as on its protection mechanisms, which are species-specific. notably, seeds rich in lipids have limited longevity due to their specific chemical composition and, in fact, during seed storage of oily species, a declining trend of both total oil content and seed germination can be observed. however, fatty acids composition is table 4 fatty acids composition (%) of seed oil in perennial onion species fatty acid a.schoenoprasum medonos a.altaicum alves a.obliquum novichock a.ramnosum aprior a.fistulosum troitsa a.nutans lider 14:0 0.12 b 0.08 d 0.10 c 0.17 a 0.18 a 0.09 cd 16:0 5.13 c 4.35 d 5.89 b 7.83 a 5.25 bc 5.62 bc 17:0 0.02 d 0.04 bc 0.04 bc 0.07 a 0.03 cd 0.05 b 18:0 1.43 bc 1.56 b 1.32 c 3.11 a 1.47 bc 1.61 b 20:0 0.56 c 0.71 b 0.34 d 0.96 a 0.59 c 0.94 a 22:0 0.21 c 0.30 b 0.21 c 0.38 a 0.33 b 0.38 a 24:0 0.07 de 0.09 c 0.06 e 0.16 a 0.08 cd 0.11 b sfa 7.54 c 7.13 c 7.96 bc 12.68 a 7.93 bc 8.80 b 16:1 0.08 b 0.05 c 0.10 a 0.10 a 0.07 b 0.08 b 16:1 9-cis 0.11 a 0.05 c 0.05 c 0.09 b 0.06 c 0.10 ab 18:1 9-cis 23.02 a 18.27 b 19.49 b 22.29 a 18.30 b 24.17 a 18:1 11-trance 0.62 a 0.40 b 0.67 a 0.44 b 0.47 b 0.61 a 22:1 0.09 c 0.10 bc 0.07 d 0.18 a 0.11 b 0.07 d mufa 23.92 a 18.87 b 20.38 b 23.10 a 19.01 b 25.03 a 18::2i 0.09 a 0.05 c 0.07 b 0.05 c 0.06 bc 0.05 c 18:2 67.76 ac 73.68 a 71.00 ab 63.57 c 72.34 a 65.32 bc 20:2 0.38 bc 0.04 e 0.32 d 0.44 a 0.34 cd 0.41 ab 18:3 ω-3 0.26 a 0.20 bc 0.21 bc 0.19 c 0.22 b 0.27 a 20:3 0.04 c 0.02 d 0.06 b 0.02 d 0.09 a 0.10 a pufa 68.53 ac 73.99 a 71.66 ab 64.27 c 73.05 a 66.15 bc along each row, means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). sfa= saturated fatty acids; mufa = mono unsaturated fatty acids; pufa = poly unsaturated fatty acids. 76 72 74 76 nt , % 72 74 76 nt en t, % 68 70 72 74 76 c 18 :2 ) co nt en t, % 68 70 72 74 76 c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0 44 * 66 68 70 72 74 76 c ac id (c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0.44 * 64 66 68 70 72 74 76 le ic a ci d( c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0.44 * 62 64 66 68 70 72 74 76 l in ol ei c ac id (c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0.44 * 62 64 66 68 70 72 74 76 l in ol ei c ac id (c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0.44 * 60 62 64 66 68 70 72 74 76 20 25 30 35 40 45 l in ol ei c ac id (c 18 :2 ) co nt en t, % y = 54 + 0.46 x r² = 0.44 * 60 62 64 66 68 70 72 74 76 20 25 30 35 40 45 l in ol ei c ac id (c 18 :2 ) co nt en t, % water soluble se, mcg kg-1 y = 54 + 0.46 x r² = 0.44 * 60 62 64 66 68 70 72 74 76 20 25 30 35 40 45 l in ol ei c ac id (c 18 :2 ) co nt en t, % water soluble se, mcg kg-1 fig.3 fig. 3 correlation between selenium water soluble forms and linoleic content in allium seeds (r = 0.66, p<0.05). 45 9 98 + 7 18 40 45 -1 y = 9,98 + 7.18 x r² = 0 84 ** 40 45 k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 30 35 40 45 e se , m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 30 35 40 45 e se , m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 25 30 35 40 45 so lu bl e se , m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 25 30 35 40 45 at er s ol ub le s e, m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 20 25 30 35 40 45 w at er s ol ub le s e, m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 20 25 30 35 40 45 w at er s ol ub le s e, m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 15 20 25 30 35 40 45 1 1,5 2 2,5 3 3,5 4 w at er s ol ub le s e, m cg k g-1 y = 9,98 + 7.18 x r² = 0.84 ** 15 20 25 30 35 40 45 1 1,5 2 2,5 3 3,5 4 w at er s ol ub le s e, m cg k g-1 total phenolics content mg g-1 y = 9,98 + 7.18 x r² = 0.84 ** 15 20 25 30 35 40 45 1 1,5 2 2,5 3 3,5 4 w at er s ol ub le s e, m cg k g-1 total phenolics content, mg g-1 fig.4 y = 9,98 + 7.18 x r² = 0.84 ** 15 20 25 30 35 40 45 1 1,5 2 2,5 3 3,5 4 w at er s ol ub le s e, m cg k g-1 total phenolics content, mg g-1 fig.4fig. 4 correlation between water soluble selenium concentration and total phenolics in allium seeds (r = 0.92; p< 0.01). 204 adv. hort. sci., 2015 29(4): 199-206 the main factor responsible for oil susceptibility to oxidation (morello et al., 2004). the results obtained in our research indicate that the oil contained in allium cepa and perennial onion species seeds are similar in fatty acids composition and may be related to a group of natural oils, such as grape, poppy, sunflower and safflower oil, with a high content of linoleic acid (60-80%) (fig. 5). moreover, the high pufa/ sfa ratio detected in perennial onion seed oil is favourable for the treatment of atherosclerosis and cardiovascular diseases (dini et al., 2008; yalcin and kavuncuoglu, 2014) and, in this respect, a. altaicum is a very interesting species with the highest pufa/sfa ratio (10.4) in our trial. in addition, statistically significant differences in the content of oil fatty acids in seeds, both among allium cepa cultivars and perennial onion species, have only been recorded for minor sfa and mufa, such as c20:2, c22:1, c18:1 11-trans and c16:1 9 cis (table 5). notably, fatty acid profiles of these two groups of allium species indicate deeper sfa synthesis in perennial onion seed oil, containing higher concentrations of c20:0, c22:0, c24:0 and a lower level of c18:0. other remarkable differences have been found in oil content, as allium cepa seeds showed a 1.7 fold higher table 5 mean values of fatty acids composition of oil in allium cepa and in perennial onion species seeds fatty acid perennial onions (%) allium cepa (%) difference significance probability (p) myristinic 14:0 0.12±0.03 0.13±0.02 > 0.5 palmitic 16:0 5.68±0.79 7.64±0.70 < 0.001 17:0 0.04±0.01 0.03±0.01 < 0.1 stearic 18:0 1.75±0.45 1.47±0.12 < 0.1 eicosanoic 20:0 0.68±0.19 0.25±0.12 < 0.001 behenic (docosanoic) 22:0 0.30±0.06 0.13±0.03 < 0.001 lignoceric 24:0 0.10±0.03 0.02±0.02 < 0.001 sfa 8.68±1.37 9.68±0.63 < 0.1 16:1 0.08±0.01 0.07±0.01 < 0.5 16:1 9-cis 0.08±0.02 0.19±0.02 < 0.01 palmitoleic ∑16:1 0.16±0.03 0.26±0.02 < 0.002 18:1 9-cis 20.90±2.24 21.77±2.10 < 0.5 18:1 11-trans 0.54±0.10 1.08±011 < 0.001 oleic ∑ 18:1 21.46±2.26 22.95±2.10 < 0.2 eicosaenic 20:1 0.00 0.06±0.08 < 0.05 erucic 22:1 0.10±0.03 0.05±0.02 < 0.001 mufa 21.72±2.30 23.21±2.19 < 0.2 18::2i 0.06±0.01 0.05±0.02 < 0.2 18:2 68.95±3.40 66.30±2.34 < 0.1 linoleic ∑ 18:2 69.01±3.39 66.31±2.33 < 0.1 eicosadienic 20:2 0.32±0.09 0.24±0.03 < 0.05 γ-linolenic 18:3 ω-3 0.23±0.03 0.30±0.26 < 0.5 eicosatetraenic 20:3 0.06±0.03 0.04±0.01 > 0.5 pufa 69.61±3.29 66.89±2.43 < 0.1 ufa (mufa+pufa) 91.33±1.37 91.20±1.70 > 0.5 sfa= saturated fatty acids; mufa = mono unsaturated fatty acids; pufa = poly unsaturated fatty acids; ufa = unsaturated fatty acids. c l flax s apiáceae canola flax s b sesame apiáceae canola flax c wheat germ cotton soybean sesame apiáceae canola flax c wheat germ cotton soybean sesame apiáceae canola flax a hemp corn wheat germ cotton soybean sesame apiáceae canola flax s fl a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax p perennial onion sunflower a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax g d poppy perennial onion sunflower a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax safflower grapeseed poppy perennial onion sunflower a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax 0 20 40 60 80 100 safflower grapeseed poppy perennial onion sunflower a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax 0 20 40 60 80 100 safflower grapeseed poppy perennial onion sunflower a.cepa hemp corn wheat germ cotton soybean sesame apiáceae canola flax fig. 5 content (%) of oleic acid in several natural oils. 205 golubkina et al., seed oil content as affected by genotype in onion level than perennial onions. notably, the higher oil concentration detected in the two italian cultivar seeds may be explained by both genotype attitude and the warmer climate in southern italy. allium seeds showed a significant amount of se (tables 1 and 2), which suggests a possible active participation of this natural antioxidant in pufa protection against peroxidation. a direct correlation between total se concentration and oil content was found only in allium cepa, which contained a higher seed oil percentage compared to perennial onion seeds, indicating a functional role of this element. a similar correlation was reported in previous research on flax seeds (golubkina et al., 2012) where a stable proteinoil complex was identified. in this respect, it may be supposed that linseed proteins contain se-aminoacids, such as selenomethionine or/and selenocystein due to the ability of se to substitute sulphur in organic compounds (pilonsmith, 2015). the existence of a protein-oil complex in allium seeds is unknown and this topic calls for special investigation, as no correlation has been demonstrated between se and proteins nor between proteins and oil content, both in allium cepa and in perennial onion seeds. interesting focus has also arisen from se and pufa positive correlations. oleic acid (c18:2), which predominates in allium seed oil, is known to be 17 times more sensitive to oxidation than linoleic acid (c18:1) and, accordingly, antioxidant protection of their degradation has great importance. this is also true for the italian cultivar seeds, grown in the mediterranean area where high temperatures during vegetation stimulate the synthesis of oleic acid (c18:1) and inhibit linoleic acid biosynthesis (c18:2) (bellaloui et al., 2013). such protection may be achieved by se via protein-oil interaction (golubkina et al., 2012) or via activation of appropriate enzymes. indeed, it is known that se demonstrates antioxidant properties in plants by increasing the activity of glutathione peroxidase (hartikainen et al., 2000) or of superoxide dismutase (xue et al., 2001), thus leading to protection of tocopherol reduction; the activation of these enzymes suggests the involvement of se water soluble forms. previous reports indicated that se water soluble forms show the highest biological activity in the environment (golubkina et al., 2010). moreover, it is known that the concentration of water soluble forms of the element increase significantly during seed germination due to auxine activation of hydrolytic enzymes (golubkina and papazyan, 2006). indeed, the content of water soluble se derivatives is shown to be directly proportional to germination value of apiáceae family seeds (golubkina et al., 2010). these data are in accordance with our research results, where a correlation with pufa content is only concerned with water soluble forms of this element. theoretically, antioxidant defense against lipid peroxidation should be enhanced by se in allium species because of the relatively low content of oleic acid (c18:1) which shows a stabilizing effect on lipid peroxidation (north et al., 1994). in any case, it seems obvious that se in allium seeds does not independently act, but displays strict interaction with other antioxidants, especially with phenolics. in fact, antioxidants are considered to form a cooperative network, using a series of different redox reactions and this is confirmed by the significant positive correlation between se water soluble forms and total phenolics content in allium seeds recorded in our research (fig. 4). although the direct relationship between se and total phenolics has already been noted in various agricultural crops enriched with the element (du et al., 2009; põldma et al., 2013; bystrická et al., 2015), such relationship in untreated plant seeds has been demonstrated for the first time in our research. notably, the highest phenolics content in allium seeds was recorded in a. fistulosum, which also gave the highest total se content (table 1). phenolics concentrations detected in allium seeds in our research are close to the values previously reported for amaranth (vollmannova et al., 2013) and soybean (malencic et al., 2012) and underline the high nutritional value of allium seeds. phenolic antioxidant properties reportedly improve both sunflower seed oil stability during storage (žilić et al., 2010) and, following exogenous application, microencapsulated linseed oil oxidative stability (rubilar et al., 2012). phenolics also play an important role in plant resistance and protection against microbial infections, which are intimately connected with reactive oxygen species (emmons and peterson, 2001; grassmann et al., 2002; malenčić et al., 2008, 2012) and possibly with se content. indeed, the concentration of se water soluble forms was shown to be positively correlated with the resistance of brassica chinensis to bacterial diseases (golubkina et al., 2002). in conclusion, interesting results have arisen from the research carried out on both allium cepa cultivars and perennial onion species seeds, due to the positive correlations found between oil content, se and total phenolics concentrations. notably, se is supposed to favour an increase in seed oil content and to interact with phenolics, essential for building up protection against oxidation and infections. in this respect, fatty acids composition also exerts a crucial role. acknowledgements the authors wish to thank dr. alina kolesnikova for her effective help with the research team arrangement. references alfthan g., 1984 a micromethod for the determination of selenium in tissues and biological fluids by single-test-tube fluorimetry. anal. chim. acta, 65: 187-194. aoac, 1990 official methods of analysis. 1st ed., association of official and analytical chemists. american oil chemists society, washington, usa. aocs, 1999 fatty acid composition by gas chromatography. 206 adv. 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(fi), italy. 2 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, via r. balzarini, 1, 64100 teramo, italy. key words: proton transfer reaction time-of-flight mass spectrometer, protonated masses, temperature, vegetable oils, volatile organic compounds. abstract: in this work the emission of volatile organic compounds (vocs) upon the heating process of an extra virgin olive oil (evoo) and a high oleic sunflower oil (sfo) was evaluated in real time by spectrometry. two tests were carried out, in the first vocs emitted from both kinds of oil were measured at room temperatures (not heated, nh) and at 180°c; in the second test, vocs emission for selected masses were monitored under increasing temperatures over time: at room temperature not heated oils (nh), 60, 90, 120, 150, and 180°c, respectively. the spectra were acquired using a proton transfer reaction time of flight mass spectrometer (ptr-tof-ms). the total vocs emission increased at 180°c, determined both by the rise of the amount of compounds present in the nh samples and by the formation of new masses generated by oxidative chemical reaction from triglycerides and fatty acids. from the set of results it is evident that a good control of the temperatures can be useful in reducing the quantities of masses potentially harmful to health in human food. 1. introduction a common worldwide operation extensively used in food preparation is cooking by immersion in hot oil, as for fried and deep fried operations. types of oils used for fried and deep frying operations vary with different cultures and are widely used both domestically and commercially. when frying and deep frying are used, fats and oils are carried at elevated tem(*) corresponding author: emarone@unite.it citation: sabbatini l., taiti c., redwan m., azzarello e., marone e., mancuso s., 2018 monitoring in real time the changes in vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms. adv. hort. sci., 32(1): 149-153 copyright: © 2018 sabbatini l., taiti c., redwan m., azzarello e., marone e., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 december 2017 accepted for publication 23 march 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(1): 149-153 150 peratures often over 180°c, which in presence of atmospheric oxygen (between 160 and 240°c) produce many flavor compounds (alkanes, fatty acids, aldehydes, ketones, polycyclic aromatic hydrocarbons), pleasant or unpleasant, and change their flavor stability. the flavor compounds released during the heating process are affected by some parameters such as cooking temperature, preparation time, chemical composition of the oils, physical and physicochemical constants and the presence of additives and contaminants (fullana et al., 2004). the volatile compounds and sensory characteristics from different edible oils (brewer et al., 1999; katragadda et al., 2010) were altered after intense heating and the main alterations were in the emission of volatile compounds linked to the oxidation process, compounds which are negatively related with the sensory attributes. in particular, the oxidation process in oils mainly lead to the formation of alkyl radicals, alkylperoxyl radicals and decomposition of hydroperoxide and cause the decay of both their nutritional and sensory quality (velasco and dobarganes, 2002; silvagni et al., 2012). moreover, there are some human health risks both linked to oil oxidations process and to many of the originated compounds released by the heating process (vaclavik et al., 2013). the ptr-tof-ms tool represents a simple, and high-throughput strategy, applicable without any laborious sample preparations and treatments, particularly suited for analysis in real time of dynamic flavor release (both in vitro and in vivo) from diverse food matrices along the food-to-fork production chain (costa et al., 2016). the aims of this study are (1) to test the ptr-tofms as a sampling system for the detection of the released compounds from not heated olive and sunflower oils and to compare with heated oils at their smoking point (180°c); (2) to monitor and evaluate in real-time the vocs emitted from different oils, upon the heating from 25°c to 180°c, to understand the dynamic of oxidation phenomena. 2. materials and methods vegetable oil material two different types of commercial oils were used for this experiment, extra virgin olive oil (evoo) and a high oleic sunflower oil (sfo). they have different heating temperatures, this latter (sfo) considered more resistant to thermal increases (smith et al., 2007). both oils were acquired at the local supermarket in florence city (italy). the analysis of volatile organic compounds (vocs) emitted from both oils virgin has been obtained through a ptr-tof-ms (ionicon analytik gmbh, innsbruck, austria), which is an instrument with high resolution mass and sensibility (5-10 ppt) and makes analysis possible without any pretreatment of samples. h3o+ was used as a reagent ion for the proton transfer reaction. sample preparation and analysis first experiment. for this experiment, 100 ml of each type of oil was placed inside a 250 ml pyrex glass jar, hermetically sealed; the jar’s lid was equipped with two holes that permitted the insertion of teflon tubes, which were respectively connected to a zero-air generator (peak scientific instruments, usa) and to the ptr-tof-ms system. it was essential to avoid the entry of foreign substances inside the jar, and that was obtained by means of a sealing paste placed around the lid and the two entry holes. oil samples were analyzed at two points: room temperature (25°c) and at 180°c. second experiment. the same steps of the first experiment was followed, in addition each oil sample was heated by using a round electric heating plate and analyzed in real time to verify the vocs emitted from both types of oil at different temperatures: room temperature (25°c), 60, 90, 120, 150, 180°c. once reached each of the five thermal steps, the temperature was kept constant for 5 minutes and then passed to the subsequent thermal step. the evaluation of the aromatic profile of each oil examined was carried out in real time in order to monitor the behavior of the various volatile substances during the heating process. for each sample the analysis lasted for a period of about 50 minutes, recording a spectrum per second for a total of 60 spectra per minute. to determine the achievement of the selected temperatures, a trial was performed in parallel; in details, a thermometer was placed into the jar contained 100 ml of different kinds of oil and the times required to reach each temperature, for both samples, were measured as well. the whole analyses were carried out inside a conditioned room, with an internal temperature of 25°c in order not to condition the chemical reactions, as they are strongly sensitive to variations in temperature and humidity. the value of the white (control) represented by an empty sealant jar was recorded before starting with samples analysis, and the volatile profile of the white (control) has always been subtracted from the final value of sabbatini et al. vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms 151 each sample. the vocs in the headspace were observed by direct injection into the ptr-tof-ms and separation of single ions happened accordingly to their mass to charge (m/z) ratio. all the instrumental parameters were settled in the following way as in taiti et al. (2017): drift tube ionization condition at 600 v and a continuous pressure of 2.20±0.02 mbar; while instrument internal calibration was based on: m/z = 29.997 (no+); m/z = 59.049 (c3h7o+) and m/z = 21.022 (h3o+) and was performed off-line. the experiment data was acquired through the tofdaq software (tofwerk ag, switzerland) and all spectra were analyzed and acquired using a dead time of 20 ns for the poisson correction and peak extraction following the methodology described in cappellin et al. (2011), using a modified gaussian peak shape. 3. results the signal intensity (ncps) generally vary among different oils. in figure 1 the signal intensity (ncps) of protonated m/z detected at 25°c and 180°c in sunflower oil (sfo) and extra virgin olive oil (evoo) are clearly indicated. as a general consideration, the intensity of the signals is always higher in evoo than in the sfo at the same temperature. at room temperature (25°c) the sfo spectrum is smoother, characterized by only 16 signals, being a very refined oil compared to evoo. the evoo presents instead 33 signals at room temperature when not heated, which are characteristics of the extra virgin olive oil in general, even if among them there are some masses that could generate off-flavor (m/z = 47.049, m/z = 61.028, m/z = 89.060) (marone et al., 2017; taiti and marone, 2017). at temperatures of 180°c, 43 m/z were measured in evoo and 39 m/z in sfo, with the formation of 23 new masses generated as a result of the high temperature in sfo and 11 new m/z in evoo, of which only 7 common to both matrices. to better verify the intensity of the signals at temperatures below 180°c, some masses of particular relevance have been selected in sfo and evoo, found in the most recent literature (klein et al., 2016). in particular, in the sfo emerge the masses m/z = 57, m/z = 101, and m/z = 113 (fig. 2); these masses are also reported in klein et al. (2016) as particularly relevant emissions by sfo. for olive oil emerge the masses m/z = 57, m/z = 101, m/z = 115, m/z = 143, also reported in klein et al. (2016). moreover, as its peculiar trend, it should be noted the mass m/z = 45 (fig. 2), that sharply increases in evoo starting from 150°c. the emission assessment of the selected masses by ptr-tof-ms allows to directly examine in real time the trend of their quantities in sfo and evoo in relation to the heating temperatures, avoiding alterations due to foods present together with the cooking oil, as generally found in the current scientific literature (ontanón et al., 2013; klein et al., 2016). the increasing temperature determines the increase of the emission of the total vocs produced at each temperature point for both sfo and evoo. the intensity of vocs emission by the refined sfo, always appears lower than that of the evoo. in both oils the increases are progressive, and related to the increasing temperature, in agreement with katragadda et al. (2010). both in sfo and evoo the mass m/z = 57, still absent at 90°c, present at 120°c, showed a dramatic increase at 150°c according to katragadda et al. (2010), and subsequently restart a trend proportional to the increase in temperatures. for each matrix all the other selected masses showed a common trend; fig. 1 signal intensity (ncps) of vocs compounds in sunflower oil (sfo) and extra virgin olive oil (evoo) at 25°c and 180°c. bars represent standard deviations (n = 3). adv. hort. sci., 2018 32(1): 149-153 152 while they were absent, or present in small quantities, up to a temperature of 90°c, they have a relatively modest rise to 120°c, and showed a dramatic increase from 150°c, according to nunes et al. (2013). the mass m/z = 57, always absent in the unheated oils, would derive from the dehydration of glycerol subjected to high temperatures (klein et al., 2016), and this would be a phenomenon common to all vegetable oils: the aldehydes that may be present in virgin olive oils as a result of a natural enzymatic oxidation of polyunsaturated fatty acids (lypoxigenase cascade, lox) increase due to the temperature through a chemical oxidative reaction of the same polyunsaturated fatty acids (muik et al., 2005). 4. discussion and conclusions the ptr-tof-ms tool represents a simple, and high-throughput strategy, applicable without any laborious sample preparations and treatments, for the real-time detection of oxidative changes occurred during the heating process of both vegetable oils. there are numerous studies that examine the quality of the oil, as well as its composition and characteristics during the heating or frying process; nevertheless, a fewer number of studies examine the composition of the fumes generated during this process. in this work, it is pointed out that evoo emissions are always higher than those of the sfo; the effect of the heating causes an increase in the emission of the masses present at the origin and the generation of new compounds, deriving from transformations of triglycerides and above all of the polyunsaturated fatty acids (moreno et al., 1999). in particular, the trend of emissions in relation to the temperature has been highlighted for some selected masses, and it is clear that change due to chemical oxidation occur between 120 and 150°c, temperatures above which the production of volatiles increases enormously for all the examined masses. since the increase in oil temperature proportionally increases the emission of vocs, including some compounds that could be considered as pollutants, it is clear that an accurate control of the temperatures of oils used for frying food is important for the industry and for the home cooking. many vocs depend on the temperature, emissions at temperatures below the smoke point will be actually reduced. some compounds, such as mass m/z = 57, start to form consistently already at relatively low temperatures (150°c). it should be noted that in the case of evoo, masses of naturally occurring compounds are also very high, as they are derived from an oxidative process linked to enzymatic reactions of the polyunsaturated fatty acids (lipoxygenase pathway, lox) (campestre et al., 2017). the increase determined by high temperatures is no longer enzymatic but chemical oxidative. further studies are needed to deepen the knowledge on the effect of heating temperature in the formation of compounds potentially harmful to human health, in order to reduce the effect of pollution not only at commercial but also at the domestic level, for the daily use of fried oils. references brewer m.s., vega j.d., perkins e.g., 1999 volatile compounds and sensory characteristics of frying fats. j. food lipids, 6(1): 47-61. cappellin l., biasioli f., granitto p.b., schuhfried e., soukoulis c., costa f., tillman m.d., gasperi f., 2011 on data analysis in ptr-tof-ms: from raw specfig. 2 trend of signal intensity (ncps) of different protonated m/z in sunflower oil (sfo) and extra virgin olive oil (evoo) at different temperatures (25, 60, 90, 120, 150, and 180°c) upon heating sabbatini et al. vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms 153 tra to data mining. sens. actuators b chem., 155: 183-190. campestre c., angelini g., gasbarri c., angerosa f., 2017 review. the compounds responsible for the sensory profile in monovarietal virgin olive oils . molecules, 22: 1833. costa c., taiti c., strano m.c., morone g., antonucci f., mancuso s., claps s., pallottino f., sepe l., bazihizina n., menesatti p., 2016 multivariate approaches to electronic nose and ptr-tof-ms technologies in agro-food products, pp. 73-82. in: rodrígez mendéz m.l. 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a new precise and simple analytical tool to discriminate virgin olive oils. adv. hort. sci., 31(4): 329-337. taiti c., marone e., lanza m., azzarello e., masi e., pandolfi c., giordani e., mancuso s., 2017 nashi or williams pear fruits? use of volatile organic compounds, physicochemical parameters, and sensory evaluation to understand the consumer’s preference. european food res. techn., 243(11): 1917-1931. vaclavik l., belkova b., reblova z., riddellova k., hajslova j., 2013 rapid monitoring of heat-accelerated reactions in vegetable oils using direct analysis in real time ionization coupled with high resolution mass spectrometry. food chem., 138(4): 2312-2320. velasco j., dobarganes c., 2002 oxidative stability of virgin olive oil . european j. lipid sci. techn., 104(9-10): 661-676. impaginato 129 figs are one of the earliest cultivated fruit-bearing trees. iran is one of the major producing countries of fig (ficus carica l.) and it stands fifth after turkey, egypt, algeria and morocco (fao, 2012). in iran, some of the genotypes, including ‘bargchenari’, ‘runu’ (faghih and sabet-sarvestani, 2001) and ‘dehdez’ (gholami, 2012) are endangered. compatible old native varieties and their wild relatives as genetic resources are the basis of the breeding programs. in addition, the continuing the search for high yielding varieties with resistance to pests and pathogens ensures the availability and maintenance of a large genetic resources that guarantee accessibility of a useful genetic material at any time (chawla, 2009). the number of endangered plant species in the world has increased in recent years due to anthropogenic and environmental pressures. numerous species are described as endangered, and integrated programs are required to protect and preserve current biodiversity (sarasan et al., 2006). special attention must be given to endemic flora, which is a critical target in conservation strategies because it is restricted to small areas (mallon et al., 2008). genetic conservation of plant can be performed in situ or in vitro. field collections (in situ), loose genetic biodiversity because of pests, diseases and adverse weather conditions and their maintenance is laborintensive and expensive. recently, the use of in vitro tools is an important way of safeguarding the germplasm of endangered plants and become increasingly popular for conservation purposes (sarasan et al., 2006; bunn et al., 2007; mallon et al., 2008; piovan et al., 2010). in vitro techniques can be used for not only the production of large number of plantlets in short period of time, but also for conservation of rare and endangered plant species, including genetic resources of recalcitrant seed and vegetatively propagated of elite genotypes (engelmann, 2011). several reports described shoot regeneration and organogenesis from various explants by the use of different culture media, types and concentrations of plant growth regulators in f. carica l. (brum et al., 2001; fráguas et al., 2004). somatic embryogenesis from leaf segment of fig (sultani genotype) was reported on ms medium supplemented with 30 mg l-1 adv. hort. sci., 2016 30(3): 129-134 doi: 10.13128/ahs-20248 micropropagation of three endemic and endangered fig (ficus carica l.) genotypes s.t. shahcheraghi, a. shekafandeh (*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: explant, rooting, single node, shoot proliferation. abstract: in this research, in vitro propagation of three endangered and endemic fig genotypes named ‘bargchenari’, ‘dehdez’ and ‘runu’ were investigated. for shoots proliferation, nodal explants on murashige and skoog (ms) medium were treated with different concentrations of growth regulators. rooting of shoots was initiated by the use of ms and ms/2 media and various concentrations of indole-3-butyric acid (iba) (0, 0.5, 1 or 1.5 mg l-1). the experiments were conducted in a completely randomized design. results showed that shoots proliferation improved as subcultures increased. the highest shoot numbers per explant 13.67 and 8.8 were achieved eight weeks after second subculture in ‘runu’ and ‘bargchenari’ genotypes respectively, when ms medium supplemented with 0.5 mg l-1 benzyl adenine (ba) and 0.2 mg l-1 n6-(δ2-isopentenyl)adenine (2ip). in ‘dehdez’ genotype, the number of shoots was 4.4 per explant, when culture media were supplemented with 6 mg l-1 kinetin (kn) and 0.2 mg l-1 6-α-naphthaleneacetic acid (naa). the highest root numbers (2.23) were obtained in ‘bargchenari’ genotype on ms/2 medium containing 1.5 mg l-1 iba. 1. introduction (*) corresponding author: shekafan@shirazu.ac.ir received for publication 7 december 2015 accepted for publication 8 julyl 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(3): 129-134 130 n6-(δ2-isopentenyl)adenine (2ip) and 7 mg l-1 thidiazuron (tdz) and 0.25 mg l-1 6-α-naphthaleneacetic acid (naa) (soliman et al., 2010). dhage et al. (2012) obtained shoot regeneration from leaf callus initiated of four fig genotypes on ms medium supplemented with 0.5 mg l-1 naa and 7 mg l-1 tdz. for successful micropropagation axillary buds or shoot tip cultures are preferred as preexisting meristem easily develop into shoots while maintaining clonal fidelity and reduce the risk of somaclonal variability (anis et al., 2003; ning et al., 2007). the present study was undertaken with the aim to evaluate the regeneration potential of three endangered genotypes and to establish an efficient in vitro method for their rapid propagation by culture of nodal explants using various plant growth regulators. 2. materials and methods preparation and decontamination of explants shoots with a length of 5-20 cm were collected from mother plants of three different genotypes (‘bargchenari’, ‘dehdez’ and ‘runu’) grown in a greenhouse with 40% relative humidity, day and night average temperatures of 30±2°c and 24±2°c respectively. the shoots were kept for one hour under the running tap water then dipped for 20 min in a solution of 4 g l-1 benomyl. explants were surface sterilized with 70% ethanol for 5 min, then were disinfected with 15% clorox (commercial bleach, 5.25% sodium hypochlorite) for 15 min and they were rinsed 3-4 times with sterile distilled water. then, the single-node segments were isolated and inoculated on a proliferation medium (ms) containing 100 mg l-1 ceftriaxone (darusazy exir, iran) to control bacterial contamination. for control of phenolic substances 2 g l-1 activated charcoal was added in the culture medium. shoot proliferation based on availability of explants different experiments were conducted as follow: in the first experiment the effects of different concentrations of kinetin (kn) (2, 4, 6 and 8 mg l-1) supplemented with 0.2 mg l-1 naa and control (without plant growth regulator) were separately studied for two genotypes ‘bargchenari’ and ‘dehdez’. data were collected after 4 weeks of culture. the second experiment was carried out on three genotypes of ‘bargchenari’, ‘dehdez’ and ‘runu’. the explants were sub cultured two times with the interval of 4 weeks and a little change in growth regulators in culture medium as follows: culture. the single-node explants cultured on ms (murashige and skooge, 1962) medium containing 0.5 mg l-1 benzyl adenine (ba). first subculture. after 4 weeks of culture and data collection, the explants were subcultured into the same medium. second subculture. after another 4 weeks and data collection, the explants were subcultured on a new medium with the same growth regulator composition plus 0.2 mg l-1 2ip. in this step, after 4 weeks, the proliferated shoots were compact and short, thus, they were transferred into new medium containing 1 mg l-1 gibberellic acid (ga), and data were recorded after another 4 weeks. the experiments were conducted in completely randomized design with 4 replications of at least three explants in each. rooting the produced shoots (above 1 cm length) were used for rooting. in this experiment, the effects of different concentrations of indole-3-butyric acid (iba; 0, 0.5, and 1.5 mg l-1) and different media (ms and ms/2) on shoot rooting performance were investigated. a factorial test was conducted in a completely randomized design with 8 replications. after 4 weeks, the rooted shoots were transferred to pots containing soil mixture (field soil, leaf mold and sand; 1v:1v: 1v). data were subjected to analysis of variance by sas software, version 9.1 (sas institude, cary, nc, usa) and means comparison were done by duncan multiple range test at probability of 5%. 3. results and discussion shoot proliferation first experiment. in ‘dehdez’ genotype, the results showed that the highest shoots number per nodal segment (4.4 shoots/explant) was obtained at concentration of 6 mg l-1 kn which was significantly higher than the other treatments (table 1, fig. 1). however, by increasing of kn from 6 to 8 mg l-1 significantly reduced the number of shoots. the positive effect of kn in suitable concentration on shoot proliferation have also been reported previously in fig (fráguas et al., 2004) and strawberry (balakrishnan et al., 2009). shahcheraghi and shekafandeh micropropagation of three endangered fig genotypes 131 with increasing kn in culture medium, the shoot length significantly decreased so that the highest shoot length was observed in control (27.67 mm) and the lowest one in 8 mg l-1 kn (7.56 mm). our results was in agreement with the findings of fráguas et al. (2004), they reported that although the kn is required for the induction of shoot proliferation, in fig ‘roxo de valinhos’, the supra optimal concentrations can be toxic, and reduce the number and length of shoots. nevertheless, it was in disagreement with the results reported by mustafa and taha (2012) in which 2.5 mg l-1 kn encouraged shoot length. the highest number of leaves (26.4) was gained at the concentration of 6 mg l-1 kn and lowest one (5.33) at 2 mg l-1. in ‘bargchenari’ genotype, the results showed different concentration of kn had no significant effect on shoots number (fig. 2 a), although with adding kn in culture media, shoot number increased. with increasing kn in medium culture the shoots length decreased (fig. 2 b). the m2 medium produced the highest numbers of leaf/explant that was significantly higher than control (fig. 2 c). shoot length reduction in m1 and m2 media and increment of number of leaves in the same medium is indicative of internodes length reduction (fig. 2). second experiment. the results showed that after 4 weeks of nodal segments culture, there were not significant differences between three genotypes in relation to shoot number, shoot length and leaf number. in the first subculture, the number of shoot (1.78 per explant) and shoot length (18.11 mm) in genotype ‘bargchenari’ were significantly higher than ‘dehdez’ and ‘runu’ (table 2). in second subculture the maximum shoot numbers was obtained in ‘runu’ (13.67 shoots/explant) (fig. 3, a3) which was not significantly different from ‘bargchenari’ (8.08 shoots/explant), and ‘dehdez’ (2.8 shoots/explant) produced the lowest number of shoots (fig. 3, b3). it seems that in the second subculture, the adding of 0.2 mg l-1 2ip in addition to 0.5 mg l-1 ba into the medium improved shoot proliferation. previous reportes, on ficus shoot proliferation with implication of ba and 2ip in culture media, showed ba achieved better than 2ip on shoot proliferation on ficus benjamina (rzepka-plevnes and kurek, 2000) and ficus anastasia (al malki and elmeer, 2010). it has been also reported that ba (a synthetic fig. 1 effect of naa and kn on nodal explant proliferation in ‘dehdez’, a= control, b= 0.2 mg l-1 naa and 6 mg l-1 kn, 8 weeks after culture. fig. 2 effects of different treatments of kn and naa on shoot number (a), shoot length (b) and leaf number (c) per explant in ‘bargchenari’ genotype. c= control (no growth regulator); m1= kn 2 mg l-1+naa 0.2 mg l-1; m2= kn 4 mg l-1+naa 0.2 mg l-1 ; m3= kn 6 mg l-1+naa 0.2 mg l-1 ; m4= kn 8 mg l-1+naa 0.2 mg l-1. treatments (mg l-1) shoot number shoot length (mm) leaf number control 1.67 c 27.67 a 12.67 c kn 2 + naa 0.2 1.67 c 9.78 c 5.33 e kn 4 + naa 0.2 3.00 b 9.96 c 17.86 b kn 6 + naa 0.2 4.40 a 11.59 b 26.40a kn 8 + naa 0.2 2.22 c 7.56 d 9.89 d significant ** ** ** table 1 effects of different treatments of kn and naa on shoot number, shoot length and leaf number per explant in ‘dehdez’ genotype mean values followed by the same letters are not significantly different according to duncan's multiple range test (dmrt) at p<0.05. adv. hort. sci., 2016 30(3): 129-134 132 cytokinin) effect is stronger than the other cytokinins on shoot regeneration (torres, 2013). however, in this study, simultaneous application of 2ip and ba in culture media highly improved shoot proliferation of the three fig genotypes. this indicates that two kinds of cytokinins had synergistic effect and enhanced shoot proliferation. the positive effect of transferring explants to a new medium on explant growth have also been reported by other researchers on different species of ficus, including carica (mustafa and taha, 2012) and religiosa (hassan et al., 2009; siwach et al., 2011; siwach and gill, 2014). the results showed in ‘dehdez’ and ‘bargchenari’, there were no significant differences in shoot length with increasing subcultures. however, the shoot length of ‘bargchenari’ was significantly higher than ‘dehdez’. in ‘runu’, the shoot length decreased in first subculture and increased in second subculture. as described in materials and methods, 4 weeks after the second subculture explants were transferred to a new medium containing ga. the results showed in spite of high shoot proliferation in second subculture, positive effect of ga on stem elongation. it has been reported ga induces internode elongation, meristem and buds growth in in vitro culture (torres, 2013). fráguas et al. (2004) also reported a positive effect of ga on shoot length elongation on fig ‘roxo de valinhos’. eight weeks after second subculture the highest number of leaves (66.67) was observed in ‘runu’ and the lowest number (10.8) in ‘dehdez’ genotype. the results showed that all measured parameters, especially shoot proliferation are genotype dependent. in other species such as prunu mume (ning et al., 2007), pomegranate (al-wasel, 1999; naik et al., 1999) the effect of genotype on shoot proliferation rate has been reported. rooting the highest rooting percentage (84.61), root number (2.23) and root length (1.51 cm) per explant were observed in ms/2 medium containing of 1.5 mg l-1 iba which were significantly higher than their controls (table 3). on ms/2 medium supplemented with 0.5 mg l-1 iba did not produce any roots (fig. 4). fig. 4 effects of iba on rooting of shoots in ‘bargchenari’ genotype in in vitro condition, a= control, b= 0.5 mg l-1 iba on ms medium, c= 1.5 mg l-1 iba on ms 1/2 medium. medium iba rooting root number root length (mg l-1) (%) (cm) ms 0 18.18 c 0.45 bc 0.11 b 0.5 71.43 ab 1.14 ab 0.30 b 1 16.67 c 0.33 bc 0.08 b 1.5 28.57 bc 0.71 bc 0.09 b ms 1/2 0 30.77 bc 0.92 bc 0.10 b 0.5 0.00 c 0.00 c 0.00 b 1 28.57 bc 0.28 bc 0.43 b 1.5 84.61 a 2.23 a 1.51 a significant * ** ** table 3 effects of different concentrations of iba and media (ms and ms/2) on rooting characteristics in ‘bargchenari’ genotype mean values followed by the same letters are not significantly different according to duncan's multiple range test (dmrt) at p<0.05. fig. 3 compared growth of nodal explants in ‘runu’ (a), ‘dehdez’ (b) and ‘bargchenari’(c) genotypes in different cultures, 1= culture, 2= 4 weeks later, first subculture, 3= 8 weeks later, second subculture. genotype subculture shoot number shoot length (mm) leaf number ‘dehdez’ culture 1.00 c 9.90 bc 2.50 c first subculture 1.00 c 10.30 bc 3.25 c second subculture 2.80 b 6.45 c 10.80 c ‘bargchenari’ culture 1.00 c 14.89 ab 5.44 c first subculture 1.78 b 18.11 a 7.67 c second subculture 8.08 a 17.18 a 38.50 b ‘runu’ culture 1.00 c 14.20 ab 3.60 c first subculture 1.12 c 8.62 c 3.87 c second subculture 13.67 a 13.52 ab 66.67 a table 2 effects of genotype and subculture on shoot numbers, shoot length and leaf numbers per explants on ms medium mean values followed by the same letters are not significantly different according to duncan's multiple range test (dmrt) at p<0.05. shahcheraghi and shekafandeh micropropagation of three endangered fig genotypes 133 the results showed a high interaction between rooting media and different concentrations of iba on rooting percentage and root number, such that in ms/2, the best results was obtained with 1.5 mg l-1 iba, where in ms medium it was 0.5 mg l-1 iba that produced the best result. hepaksoy and aksoy (2006) also reported that iba is necessary for in vitro rooting of fig, sarilop cultivar, that is in consistent with our results. the obtained results are in disagreement with those reported by brum et al. (2001) and fráguas et al. (2004) who found iba are not essential for in vitro rooting of fig cv. roxo de valinhos. in this experiment, the best rooting occurred on ms/2 medium. dhage et al. (2012) also introduced ms half strength medium for in vitro fig rooting, while yakushiji et al. (2003) and kim et al. (2007) reported ms full strength was the best medium for in vitro rooting of fig cultivars. adventitious root formation on proliferated shoots in a commercial propagation is very important. the ability of plant tissue to form adventitious roots depends on the interaction of many different endogenous and exogenous factors. the role of auxins in root development was reviewed, and it is a well-established fact that auxins are the main factors involved in the root formation (németh, 1986). 4. conclusions simultaneous application of 2ip and ba in culture media highly improved shoot proliferation of the three fig genotypes. this indicates that two kinds of cytokinins had synergistic effect and enhanced shoots proliferation. the highest shoot numbers per explant 13.67 was achieved eight weeks after 2nd subculture in ‘runu’ genotype. the highest root number (2.23) was obtained in ‘bargchenari’ genotype on ms/2 medium containing 1.5 mg l-1 iba. acknowledgements we are grateful to the center of excellence in rainfed fig for partially providing cost of this research. references al malki a.a.h., elmeer k.m.s., 2010 influence of auxin and cytokinine on in vitro multiplication of ficus anastasia. afr. j. biotech., 9(5): 635-639. al-wasel a.s., 1999 in vitro colonel propagation of “albelehi” pomegranate (punica granatum l.) journal of king saud university, agri. sci., 11: 3-14. anis m., faisal m., singh s., 2003 micropropagation of mulberry (morus alba l.) through in vitro culture of shoot tip and nodal explants. plant tissue cult., 13: 47-51. balakrishnan v., latha m.r., ravindran k., robinson j.p., 2009 clonal propagation of morus alba l. through nodal and axillary bud explants. bot. res. inter., 2: 42-49. brum g., pasqual m., silva a., chalfun n., 2001 sucrose, culture media, and their interactions during in vitro proliferation of ‘roxo de valinhos’ (ficus carica l.). acta horticulturae, 605: 131-135. bunn e., turner s., panaia m., dixon k.w., 2007 the contribution of in vitro technology and cryogenic storage to conservation of indigenous plants aust. j. bot., 55: 345-355. chawla h.s., 2009 introduction to plant biotechnology. crc press, taylor and francis group, abingdon, uk, pp. 730. dhage s., pawar b., chimote v., jadhav a., kale a., 2012 in vitro callus induction and plantlet regeneration in fig (ficus carica l.). j. cell tiss. res., 12: 33953400. engelmann f., 2011 use of biotechnologies for the conservation of plant biodiversity. in vitro cell. develop. biol. plant., 47: 5-16. faghih h., sabet-sarvestani j., 2001 fig, plant and harvesting. 1st ed rahgosha, shiraz, iran, pp. 292. 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shamshiri 2, h.r. roosta 2, e.u. khan 3 1 department of horticulture, agri-college, vali-e-asr university of rafsanjan, iran. 2 department of horticulture, vali-e-asr university of rafsanjan, iran. 3 nuclear institute for agriculture and biology (niab), faisalabad, pakistan. key words: nutrients, organic matter, pistachios, poultry manure, yield elements. abstract: the effectiveness of poultry manure application time was studied on pistachios (pistacia vera l.) trees. the experiment consisted of seven different poultry manure application time, including poultry manure application as one time in 1) last week of october 2) last week of december 3) last week of january 4) mid-march and dividing into two parts and use in fall or in winter, dividing into four parts and use in dormant seasons (fall and winter). based on the results, there were significant differences among treatments. the highest number of fruit per cluster (27.4) was found in poultry manure applied in last week of october. the highest nut splitting percent (84.3%) and the lowest nut blanking percent (8.6%) were obtained in poultry manure applied by dividing into four parts. half kernel nuts followed a similar trend with blanking percent. weight of 1000 nuts increased and responded positively and number of pistachios nut per ounce decreased by manure application when divided into two parts and used in the winter. application of poultry manure in the mid-march enhanced the nut protein (19.63%). 1. introduction production technologies of horticultural crops including pistachio tree have undergone vast changes recently, and led to the extension of innovative technologies about nutrient management. the critical factor of nutrient management of nut trees like pistachio is to elevate the net yield and improve the quality of nut fruits. pistachio blanking and flower bud abscission can be directly related to nutrition management (mahmoudi meimand and ghanbari odivi, 2013). nowadays, fertilizers play a key role in nutrition of fruit trees. because of harmful side effects of chemical fertilizers, (ljoyah and sophie, 2009) the use of bio fertilizers are increasing recently. organic fertilizers, such as animal manure, have a long history of use by men (baybordi and malakuoti, 2003). different animal manures such as sheep, cow and poultry manures have been used as natural crop (*) corresponding author: mmeimand@ut.ac.ir citation: mahmoudi meimand m.j., shamshiri m.h., roosta h.r., khan e.u, 2018 poultry manure application time on pistachio (pistacia vera l.) trees adv. hort. sci., 32(2): 177-183 copyright: © 2018 mahmoudi meimand m.j., shamshiri m.h., roosta h.r., khan e.u. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 177-183 178 fertilizers for centuries. poultry manure, because of its high level of nitrogen which is absorbable for pistachio trees, has been recognized as one of the main favorable manures. it has been documented that poultry manures also supply other essential pistachio nutrients and act as soil amendant by raising organic matter content, which helps improve the moisture level of soil and nutrient maintenance. according to alimoradi (2011) nearly 5.3 million tons of poultry manure is consumed in rafsanjan and kerman pistachio orchards every year. these bio fertilizers are known as main source of manure for iranian pistachio growers. nutrients provided by poultry manures have been indicated to establish effects on different crops, including fruit crops (mitchell et al., 1993; miller, 1996). it has been reported that poultry manure is composed of essential elements for fruit crops containing about 3% of nitrogen nh4 +, 63.2% phosphorus p2o5 and 4.1% potassium k2o (reddy and reddy, 1995). the main form of nitrogen in poultry manures is nh4 +, which elevate the availability of nitrate to the plants for a longer period (burmester, 1993; crawford and chalk, 1993; touchton and bosewell, 1980). on the other hand, organic manures application to decrease the use of chemical fertilizers in pistachio orchards and other fruit trees is an important goal in fruit production (reganold et al., 2001; forge et al., 2002). organic manures increase the fertility of soil and the crop yield. therefore, they can be helpful to achieve sustainable agriculture. it has been demonstrated earlier that poultry manure improved growth parameters, yield and quality in different crops (ram and rajput, 2002; ingle et al., 2003; arancon et al., 2003). pimpini et al. (1992) revealed higher rate of ex-tractable sucrose by using 4 t ha-1of poultry manure in crops. increased content of total carbohydrate, protein and ascorbic acid were reported by abusaleha and dutta (1988) when poultry manure was used. improving photosynthesis, plant biomass and glycosides content of stevia were also demonstrated before (xiangyang et al., 2010). enhanced starch content, crude fiber, ash, crude protein, phosphorus, calcium and magnesium of d. bulbifera were reported by ezeocha et al. (2014). adekiya and agbede (2017) showed that poultry application increased soil organic matter, leaf n, p, k, ca, mg contents, growth and yield in tomato. they also showed that the application of poultry manure at 3 weeks before transplanting had highest effects on leaf nutrient concentrations, growth and yield in tomato (adekiya and agbede, 2017). now, pistachio growers are frequently using poultry manure as a source of plant nutrient. to our knowledge, there are no reports available to recommend favorable time for poultry manure application in orchard of pistachio trees. the aim of this investigation is to study the effect of poultry manure application time on nut yield and quality in pistachios trees. 2. materials and methods the experimental orchard selected for this study is located at khatam, yazd province, iran. it is located at 39.33°n latitude and 54.40°e longitudes, at an elevation of 1605 m above sea level. the average temperature of the zone is 18.5°c, the annual total chilling hours (≤7.2°c) is about 950; the average annual rainfall is 300 mm. the climate of this area is typically subtropical. just before poultry manures application, soil sample of the experimental orchard were collected and chemically analyzed (table 1). this research was conducted on 12 years old pistachio cv. akbari grafted on ‘badamii zarand’ rootstock. management factors such as irrigation regime, pruning practices, and weed control were followed according to local standards. trees were trained with a modified central leader system and distance of trees was 2x6 m. to achieve better results, uniform trees were selected with uniform vigor and age and three uniform shoots were selected from different sides of the tree for data collection. harvest index was considered when several nuts in the cluster were light colored and the hull was easily separated from shell. characteristics of yield, percentage of splitting, weight of 1000 nuts, blanking, pistachio weight in soil characteristics value ph 7.6 ec ds m-1 4 p ppm 28.1 k ppm 180 o.m. % 1.3 t.n.v. % 22.1 sand % 52 silt % 34 clay % 14 texture loam esp 17.9 table 1 soil fertility analysis report of the experimental orchard mahmoudi meimand et al. poltry manure application on pistachio trees 179 ounce, dry weight and some other characteristics related to kernel and leaf were recorded. the experiment consisted of seven different poultry manure application time, with three replications based on the randomized complete block design (rcbd). the treatments were poultry manure application as one time in 1) last week of october 2) last december 3) last week of january 4) mid-march and dividing into two parts and use in fall or in winter, dividing into four parts and use in dormant seasons (fall and winter). these treatments were conducted beneath the tree canopy and mixed well with surface soil, 10 kg per tree. data collecting was performed in next growth season. statistical analysis was conducted using the sas software (9.2) and means were compared by duncan’s multiple range test (p≤ 0.05). 3. results and discussion effect of poultry manure application time on yield parameters number of fruits per cluster. the study indicated that poultry manure application time had significant effects on number of fruits per cluster (p≤ 0.05) (table 2). the highest number of fruit per cluster was found by poultry manure application in last week of october (27.4) followed by the last week of december (26.9) (table 2). the minimum value (19.2) was observed in control treatment. the higher fruit per cluster with poultry manure application in last week of october (concurrently with tree deciduous) might be related with the positive role of nitrogen and other critical elements on cluster final development. burmester (1993) crawford and chalk (1993) touchton and bosewell (1980) demonstrated the positive role and proportion ratio of elements on reproductive growth parameters. nut splitting percent. also, our results demonstrated that poultry manure application time had significant effects on nut splitting percent (table 2). highest nut splitting percent (84.3%) was obtained in treatment of poultry manures divided into four parts and used in last weeks of october, december, january and mid-march followed by divided into two parts and used in fall (83.5%) and in winter (83.1%) (table 2). the minimum value for nut splitting percent was reported in control treatment. based on our results, the poultry manure application in four different times, thus involving a longer period of the year improved nut splitting percent. it has been demonstrated before that poultry manure was improved yield quality in different crops (arancon et al., 2003; ingle et al., 2003). number of pistachio nuts per ounce. table (2) indicates that the use of poultry manure increased the pistachio ounce index or decreased number of inshell pistachio nuts per ounce significantly (p≤ 0.01). the results showed also that when a portion of poultry manure is allocated at the end of dormant season, increase fruit size is achieved. previous findings showed that poultry manure improved yield component in different crops (ram and rajput, 2002; arancon et al., 2003). blanking percent. based on the results, the lowest value (8.6%) for nut blanking was observed when treatment consisted in dividing poultry manures into four parts and using in last weeks of october, december, january and mid-march; this was followed by treatment divided into two parts and used in winter (11%) (table 2). the maximum amount of blanking (%) was observed in control treatment (13.9%) with no significant difference with fall application. therefore, poultry manure application at over the year in four different times decreased nut blanking percent. yield component improving demonstratlwo= last week of october, lwd= the last week of december, lwj= the last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. means followed by the same letters are not significantly different (duncan test, p≤ 0.05). parameter\treatment lwo lwd lwod lwj mm lwjm lwodjm control number of fruit 27.4 a 26.9 b 22.6 c 21.2 e 21.05 e 22.1d 26.9 b 19.2 f nut splitting % 77.3 b 76.4 b 83.5 a 70 c 69.8 c 83.1 a 84.3 a 64.8 d fruit ounce 22.6 a 22.6 a 22 ab 22.6 a 22.3 a 20.6 b 20.6 b 23 a blanking % 13.3 ab 13.5 ab 13.1 bc 12.5 bc 12.8 bc 11 d 8.6 e 13.9 a half seed fruit % 1.3 c 1.2 d 0.7 e 4.4 b 3.1 c 0.9 e 0.7 e 5.0 a weight of 1000 nuts 1249 b 1249 b 1288 b 1249 b 1267 b 1372.1 a 1370 a 1231 b table 2 effect of poultry manure application time on yield parameters adv. hort. sci., 2018 32(2): 177-183 180 ed by ram and rajput (2002) and arancon et al. (2003). half kernel fruit percent. half kernel nuts followed a trend similar to blanking percent, it was decreased with dividing poultry manure into four parts (0.7%), followed by manure divided into two parts and used in fall (0.7%) or winter (0.9%) (table 2). the highest half kernel nuts percent was obtained in control (table 2). weight of 1000 nuts. weight of 1000 nuts increased and positively and significantly (p>0.05) responded to the treatments. highest weight of 1000 nuts (1372.1 g) were observed in poultry manure divided into two parts and applied in winter followed by treatment consisting in manure divided into four parts and used in last weeks of october, december, january and mid-march (1370 g). however, other treatments exhibited lowest weight of 1000 nuts (table 2). previous findings indicated that poultry manure improved yield component in different crops (ram and rajput, 2002; arancon et al., 2003). effect of poultry manure application time on nut quality parameters fruit stening. fruit stening was influenced by application of poultry manure in different times (table 3). maximum amount for fruit stening (7.23%) was noted at mid-march application (7.23%) and control (7.16%) and the lowest value for this parameter was obtained with treatment divided into two parts and used in fall treatment (table 3). it has been demonstrated earlier that poultry manure improved yield and quality in different crops (ram and rajput, 2002; arancon et al., 2003; ingle et al., 2003). nut protein. nut proteins percent was also enhanced by the application of poultry manure in all treatment versus control (table 3), but maximum value for nut protein percent was observed at midmarch (mm) application (19.63%) followed by the last week of january (19.5%) and in poultry manures divided into four parts and used in last weeks of october, december, january and mid-march (19.2%) with no significant differences (table 3). the minimum value for nut protein percent was observed in control treatment (17.4%). increasing amount of total carbohydrates, proteins and ascorbic acid content reported by abusaleha and dutta (1988) in a similar work that well described poultry manure application on increasing secondary metabolites. enhancing crude protein in d. bulbifera was reported by ezeocha et al. (2014). the results reported by adekiya and agbede (2017) showed that poultry application produced higher percent protein content, more growth and yield in tomato. effect of poultry manure application time on vegetative and reproductive parameters leaf fresh weight. according to obtained results, poultry manure application time had significant effects on leaf fresh weight (p≤ 0.05) (fig. 1). the highest amount of leaf fresh weight was obtained at poultry application in mid-march (2.1 g), followed by fig. 1 changes of leaf fresh (x) and dry weight (■) (gr) of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. parameter\treatment lwo lwd lwod lwj mm lwjm lwodjm control fruit stening (%) 5.6 d 6.56 b 4.2 f 6.8 b 7.23 a 5.9 c 4.96 e 7.16 a nut protein (%) 19.1 bc 19 c 18.9 c 19.5 ab 19.63 a 19.2 ab 19.2 ab 17.4 d table 3 effect of poultry manure application time on nut quality parameters lwo= last week of october, lwd= the last week of december, lwj= the last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. means followed by the same letters are not significantly different (duncan test, p≤ 0.05). mahmoudi meimand et al. poltry manure application on pistachio trees 181 application divided into two parts and used in winter (1.9 g) (fig. 1). the lowest rate of leaf fresh weight was occurred in control (1.5 g) and last week of october (1.5 g), respectively (fig. 1). based on our result, poultry manure application in mid-march at once increased leaf fresh weight. increasing photosynthesis and plant biomass of stevia was also demonstrated before (xiangyang et al., 2010). leaf dry weight. dry weight of leaves was significantly affected by different times of manure application (p≤ 0.05). maximum leaf dry weight was found in poultry application in mid-march (0.8 g) and divided into two parts and used in winter (0.8 g), whereas the minimum was observed in control (0.4 g) and last week of october (0.4 g) (fig. 1). this increase in leaf dry and fresh weights with application of poultry manures at the end of dormant season can be explained as following, this time of application cannot make the expected effect and causes to increase vegetative parameters, similar to reports published by xiangyang et al. (2010). leaf nitrogen content. results revealed that the leaf nitrogen content of pistachio trees fertilized with poultry manure in different times varied significantly (p≤ 0.05). leaf nitrogen content was the highest (3.1%) at poultry application in mid-march once at any time compared to the treatments divided into two parts and applied in winter (3.03%) and in last week of january (2.9%) (fig. 2). the higher leaf nitrogen content in mid-march application of poultry manures could be in relation with releasing more nitrogen nh4 + form which is useful for vegetative parameters and causes nitrogen accumulation in the leaves. previous findings emphasis on high level of nh4 + form in poultry manure. the application of poultry manure with high level of n, have influenced the growth and vegetative factors of tree and production of fruits (reddy and reddy, 1995). adekiya and agbede (2017) indicated that application of poultry manure increased percent of leaf n, p, k, ca, mg content, growth parameters and yield in tomato. poultry manure usage at 3 weeks before transplanting caused higher leaf nutrients concentrations and more growth and yield in tomato (adekiya and agbede, 2017). shoot length. based on our findings, shoot length was significantly affected by different application times of manure (p≤0.05). maximum shoot length was obtained when manure was divided into two parts and used in winter (44 cm) and mid-march application (43.3 cm). our results revealed that minimum value for this parameter was observed in control (26.6 cm) and in manure divided into two parts and used in fall (32.6 cm) (fig. 3). similar to the dry and fresh weights, application of poultry manures at the end of dormant season caused to increase shoot length. our results demonstrated that application of poultry manures during fall stimulates a least vegetative growth with no significant differences between various times in fall (fig. 3). increasing vegetative growth and similar findings by using poultry manure fig. 2 changes of leaf nitrogen content of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. fig. 3 changes of shoot length of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. adv. hort. sci., 2018 32(2): 177-183 182 have been reported before (xiangyang et al., 2010). leafy bud number, floral bud number and leafy to flower bud ratio. results indicated that leafy and floral buds and leafy to flower bud ratio of pistachio trees fertilized with poultry manure was positively affected by treatments (p≤ 0.05). number of floral buds in the next growing season was the highest in last week of october and fall (in last weeks of october and december) (7.33) (fig. 4). minimum value for floral bud number was observed in control (fig. 5). results for leafy bud numbers showed that maximum value was obtained in treatment divided into two parts and applied in winter (5.6) and midmarch (5.3) (the letter two treatments have not any significant difference) (fig. 4). floral bud abscission ratio. it was revealed that floral bud abscission during the grow season by poultry manure usage was significantly less than control, which it might be due to the role of poultry manure in decreasing floral bud abscission totally. any treatment could not show significant effect on floral bud abscission, but minimum amount for this parameter was observed in treatment that was conducted in last week of december (0.32) (fig. 5). previous reports showed that poultry manure improves the yield component (ram and rajput, 2002; arancon et al., 2003), although there are no exact results about reproductive phase and parameters affected by poultry manure treatment, but flower bud abscission can be directly related to nutrition management (mahmoudi meimand and ghanbari odivi, 2013). 4. conclusions it is clear that application of organic fertilizers will improve the nut yield and quality, but application of poultry manure through dormant season can be effective on reproductive and vegetative parameters in pistachio. based on our findings, dividing poultry manures into four parts and applying in last weeks of october, december, january and mid-march, or using a single application in fall showed the best results for optimum nut yield, quality and floral bud emergence in pistachio. references abusaleha, dutta o.p., 1988 inter relationship of yield components in cucumber. veg. science, 15(1): 79-85. adekiya a.o., agbede t.m., 2017 effect of methods and time of poultry manure application on soil and leaf nutrient concentrations, 6 growth and fruit yield of tomato (lycopersicon esculentum mill). j. saudi soc. fig. 4 changes of leafy bud number (■), floral bud number (×) and leafy to flower bud ratio (○) of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. fig. 5 changes of floral bud abscission ratio of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. mahmoudi meimand et al. poltry manure application on pistachio trees 183 of agric. sci., 16: 383-388. alimoradi m., 2011 the value of poultry by-products, livestock specialist. vtyvr blog. arancon n.q., galvis p., edwards c.a., yardim e., 2003 the trophic diversity of nematode communities in soils treated with vermicompost. pedobiología, 47: 736-740. baybordy a., malakouti m.g., 2003 effect of iron, manganese, zinc and copper on the yield and quality of wheat in saline conditions. khosro-shahr agricultural research institute. burmester c.h., 1993 cotton production guide. alabama cotton notes . alabama cooperative extension service, auburn university, usa. crawford d.m., chalk p.m., 1993-sources of n uptake by wheat (triticum aestivum l.) and n transformations in soil treated with nitrification inhibitor. plant soil, 149: 59-72. ezeocha v.c., nwogha j.s., ohuoba a.n., chukwu l.i., 2014 evaluation of poultry manure application rates on the nutrient composition of dioscorea bulbifera (aerial yam). nigerian food journal, 32(2): 92-96. forge t.a., hogur e., neilsen g., neilsen d., 2002effect of organic mulch on soil microfaunce in root zone of apple: implications for nutrient fluxes and functional diversity of the soil foos webappl. soil ecol., 22: 39-54. ingle h.v., athawale r.b., ingle s.h., 2003 effect of organic and inorganic fertilizers on yield and quality of nagpur mandarin. orissa j. hortic, 32(1): 10-13. ljoyah m.o., sophie v.l., 2009 effects of different level of decomposed poultry manure on yield of cabbage (brassica oleraceae l.). j. trop. agric., food, environ. extension, 8(1): 20-23. mahmoudi meimand m.j., ghanbari odivi a., 2013 new approach for cultivation pistachio trees, pub by nushe publisher, isbn:978-600-6376-18-8. miller d., 1996 poultry litter as a soil amendment in southeastern row crops: a feasibility study based on agronomics, environmental and economic factorssustainable agriculture research and education. southern region, as93-10, ls9139. final report. mitchell c.c., burmester c.h., wood c.w., 1993 broiler litter as a source of n for cotton. beltwide cotton conf., new orleans, la, natl, 1357-1359. pimpini f., giardini l., borin m., gianquinto g., 1992 effect of poultry manure and mineral fertilizers on the quality of crops. j. agric. sci. camb., 118: 215-221. ram a.a., rajput m.s., 2002 role of biofertiliser and manures in production of guava (psidium guajava l.) cv. allahabad safeda. haryana j. hortic. sci., 29(3-4): 193194. reddy t.y., reddy g.h., 1995 principles of agronomy. 2nd edition, kalyani publishers, new delhi, india, pp. 223. reganold j.p., glover d., andrews p.k., hinmam h.r., 2001 sustainability of three apple production systems. nature, 410: 926-929. touchton j.t., bosewell f.c., 1980 performance of nitrification inhibitors in the south east in j. j. meisinger (ed.) nitrification inhibitors: potential and limitation. asa spec. publ. asa and sssa, madison, usa, pp. 6374. xiangyang l., guangxi r., yan s., 2010 the effect of organic manure and chemical fertilizer on growth and development of stevia rebaudiana bertoni. energy procedia, 5: 1200-1204. 30 1. introduction it is well known that the vase life of cut flowers depends on water quality in terms of sanitation, nutrients and acidity regulators. water lost during the postharvest period can normally be replaced through the vase solution. nevertheless, desiccation is one of the most important postharvest problems for cut flowers due to the plugging of xylematic vessels (i.e. bent neck of cut roses or stem break of cut gerbera flowers), disorders caused by plugging of the cut surface of the stem due to bacteria, exudations or colloidal materials. thus, non destructive identification of the water status in the stem of cut flowers can be useful to predict postharvest vase life. zantedeschia aethiopica (commercially known as calla lily) has long been an important cut flower, and new green-tinged and different-shaped variants are increasingly important. the flowers are normally pulled from the rhizome, and re-cut to ensure adequate water uptake (reid, 2004). this practice is very important to guarantee a long shelf life if flowers are maintained at the correct, low temperature. the fresh weight of spathe of z. aethiopica decreases with time while that of scape and spadice increases (tjia and funnel, 1986). a standard preservative solution used to prolong the longevity of cut flowers (8-hqc + sucrose) was deleterious to zantedeschia foliage, reducing display life several fold (skutnik et al., 2001). in a recent paper (ahmad et al., 2013 a) it was shown that floral preservative was ineffective to prolong vase life of calla while it was tolerant of high water ph (8.1) and vase life varied from 9.2 d for acidic solutions (ph 3.2) to 10.1 d for solutions with intermediate ph (6.3). all of these results are conditioned by the water quality and behaviour. mri (magnetic resonance imaging) has been used to monitor the developmental change of zantedeschia spreng. tuber (robinson et al., 2000) but no paper has reported, to our knowledge, its application on flower stems. nir (near infrared) spectroscopy is an excellent technique to detect the water content inside the tissue in a non destructive way because water has a very strong signal (cozzolino et al., 2006). it has been applied to measure the water potential in vine leaves (de bei et al., 2011) as well as the water loss of grape berry during dehydration (bellincontro et al., 2011). in the research reported here, a portable nir-aotf spectrometer was used to non destructively measure the water content and weight loss of calla lily, as model cut flower, to predict its vase life; innovative application of nir-aotf and mri to study water behaviour in cut flowers a. bellincontro(1), m. valentini(2), r. forniti(1), f. mencarelli(1)* (1) dibaf, università della tuscia, via de lellis, 01100 viterbo, italy. (2) cra, centro di ricerca per lo studio delle relazioni tra pianta e suolo, azienda sperimentale di tor mancina, strada della neve km 1, 00015 monterotondo (rm), italy. key words: dry matter, flower, image software, mri, nir-aotf, plugging, water content. abstract: in order to study the water status of cut flowers, a comparision study was made between flowers stored in water and flowers stored «dry pack», by using a portable nir (near infrared)-aotf(acousto-optical tunable filter) instrument and mri (magnetic resonance imaging). as model flower, zantedeschia aethiopica (commercially known as calla lily) was used. to predict the weight loss and water content by nir-aotf, cut flowers were dried in a cold room at 10°c (±1°c) and 85% (±5%) relative humidity (rh), and measured for weight loss. for mri application, 4 and 20°c storage temperatures were used for flowers kept in water or dry; two stem sections, basal and middle, were measured. significant correlation results for weight loss and water content ( r2 in calibration = 0.98 for estimated % of water loss and 0.96 for % of water content; r2cv = 0.95 and 0.90) were obtained by nir-aotf spectra acquisitions. mri detected vessel degradation in the stem of the water-stored flowers at 4°c but at 20°c in dry storage no vessel degradation appeared and images were correlated with dry matter values. the use of image software allowed the transformation of images in normalized population and pixel intensity, which gave hints about the potential use of these data to combine with nir-aotf data. nir-aotf, an easy-to-use and non destructive instrument, can be used to predict the vase life of cut flowers by measuring water content or weight loss. mri is a powerful tool to identify the plugging of vessels but it is destructive; the image software used represents a useful tool to correlate mri with nir-aotf to predict vessel plugging. adv. hort. sci., 2015 29(1): 30-36 * corresponding author: mencarel@unitus.it received for publication 10 january 2015 accepted for publication 14 april 2015 31 bellincontro et al., innovative application of nir-aotf and mri to study water behaviour in cut flowers subsequently, mri was applied to study the water traslocation in calla lily stems kept at two storage temperatures (4 and 20°c). image software was used to transform the mr images in pixel intensities for graphical representation and comparative evaluation. in subsequent investigations, the correlation between mri results and nir-aotf spectra will be tested with the aim of performing a calibration model able to predict vessel plugging of flowers. 2. materials and methods material and experimental procedure calla lily (zantedeschia aethiopica) cv. childsiana flowers, at commercial stage of development, were picked in the morning and immediately placed in water, the stem surface was placed in sterilized water after recutting. the average weight of flowers was 80 g; color of spathe l= 75.7±2.9 a= -9.7±1.2 b= 44.5±4.5; color of stem l= 50.1±6.6 a= -8.8±0.5 b= 28.6±1.3 measured by a cm-2600d colorimeter (konica minolta inc., ramsey, ny) set at sce (specular component excluded) measuring cielab coordinates l, a, and b. in order to build a calibration curve for weight loss, in the first experiment, calla flowers (50) were kept dry, horizontally placed in cardboard boxes covered with plastic film to avoid excess air flow, in a cold room at 10°c (±1°c) and 85% (±5%) rh, with coil fans blowing during the cooling intervals. each single flower was weighed on a scale (cubis mod., sartorius-stedim italia spa, florence, italy) every 8 h. weight loss and water content were calculated and expressed as %. nir spectra collection a luminar 5030 miniature, hand-held nir-aotf analyzer (brimrose corporation, baltimore, maryland, usa) was used for the nir spectra acquisitions, obtained by putting the optical sensor in contact with four sections of the flower: basal, middle, upper, and spathe. ten spectral acquisitions were run for each section (along and all around), recorded in transmittance mode (bellincontro et al., 2011) and then averaged. a single measurement, at the speed of 16000 wavelength sec-1, was conducted in the 1100-300 nm range, with 2 nm wavelength increments and 50 spectra per average, which represents a good compromise between acquisition speed and signal quality of the spectrum. near infrared spectroscopy analisys and chemometrics raw spectra were statistically pre-treated for absorbance (log 1/t) transformation using snap 2.03 software (brimrose corporation, baltimore, maryland, usa). the absorbance spectra, obtained as spectral average, were used as x-variables and opposite to the y-variables (water content and water loss) in model calculation, performed by chemometric procedures of filtering and partial least square (pls) calculation. model validation in parameter prediction was obtained by full cross-validation (leaveone-out) method, and no outlier identification and elimination was applied. the statistical r2 indexes (coefficient of multiple determination) in calibration (r2 c ), and in crossvalidation (r2 cv ) were defined to establish the correlation between nir spectra and destructive measurement. root mean standard error of cross validation (rmsecv) and the number of latent variables (lvs), minimizing the error in modeling, were used to determine the significance of the calculations. finally, the rpd values, defined as the ratio between sd and secv, were also calculated in order to define the robustness of predicting responses. this experiment was run until complete wilting of the flowers (10 days). a parallel destructive experiment was carried out: flowers (50) were kept under the same conditions and nir acquisitions were performed in a similar way as described above; after each nir acquisition, sections were cut and used to measure water content by drying them in an electric ventilated oven (tecno-lab srl, brescia, italy) at 70°c for 72 h. ten flowers were used for each sampling time. mri experiment in the second experiment, flowers were collected and in the laboratory the stems were recut in sterilized water and then placed in the same. flowers were divided into two lots: 60 flowers were kept in water (10 flowers per vase, 6 vases); other 60 flowers were placed horizontally, wrapped, not tightly, in plastic film as used commercially. both lots were split and stored in two storage rooms at 4 or 20°c (±1°c) at 70% (±5%) rh. the experiment lasted 12 days. dry matter was measured by using a ventilated oven as described above, at each sampling time. mri measurements were performed on flower sections at time 0, after 3 and 12 days. at each sampling time, mri analyses were performed on two sections (15 cm long) of the stem, cut from 5 cm above the stem cut surface and from 15 cm below the spathe, named basal and middle, respectively. a bruker avance 300 mhz spectrometer (bruker biospin corp, bilerica, ma, usa) equipped with cylindrical birdcage single-tuned nucleus (1h) coil probehead with an inner diameter of 20.0 mm was used (taglienti et al., 2009). the water signal was monitored and used for the image reconstruction. gradient-echo (gefi) and multislice-multi-echo (msme) experiments, m_gefi_ortho and m_msme_ortho, respectively (bruker library), were performed according to standard procedures. in gefi measurements, which generate echoes by applying gradient pulses, the field of view was 20.0 mm x 20.0 mm, the matrix size 128 x 128 pixels and spectral width 100.0 khz. the echo and repetition times were set equal to 2.445 ms and 60.0 ms, respectively. the number of scans was 1; slice thickness was 1.01 mm; and excitation pulse was a sinc3. the data were processed to obtain images 128 x 128 in size and a field of view of 20.0 mm x 20.0 mm. the processing mode was ft_mode, the latter was complex_fft and spikes elimination was allowed. in msme experiments, which produce echoes via a spin-echo-based sequence, the field of view was 20.0 mm x 20.0 mm; the 32 adv. hort. sci., 2015 29(1): 30-36 matrix size 128 x 128; spectral width 100.0 khz; the echo and repetition times were set equal to 17.5 ms and 6000.0 ms, respectively; the number of echoes and images were 196; number of scans and dummy scans was 1; slice thickness was 1.0 mm; and the excitation pulse was a sinc3. the data were processed to obtain images 128 x 128 in size, a field view of 20.0 mm x 20.0 mm, the processing mode ft_mode, the latter complex_ fft and with spikes elimination allowed. the intensity decay of the nmr signal vs. the echo time was calculated by the bruker software paravision 3.0.2 in order to calculate the local transverse relaxation time, i.e. t 2 . gefi and msme images were also analysed by imagej 1.41 (rasband, 2007) which allows for greyscale analysis. for gefi experiments, the axial image was analysed by imagej software, while for msme the intensities of the 24th acquired image were reported to a scale from 0 to 255, the first value corresponding to full black colour and the second to complete white. the whole sample was selected, and parameterised images were used to create curves according to the pixel intensities. 3. results and discussion the absorbance mean spectra of the acquisitions in the floral sections showed characteristic molecular references of the absorbance peaks (data not shown). spectra were characterized by two principal water absorption bands around 1450 nm and 1920-1950 nm (nicolai et al., 2007). these bands are assigned to the first overtone of the symmetric and asymmetric oh stretching and/or combination bands (1450 nm), and to the combination of the oh stretching band and to the oh bending band (19201950 nm), respectively (shenk and westerhaus, 1996). this spectral response is strictly correlated with the floral water content; thus the weight loss estimation, mainly due to water loss, is predictable. in table 1 analytical measurements of weight loss (%) and water content (%) are statistically defined by descriptive indexes. data range (as min and max values), mean, and sd (standard deviation) are reported to describe the variability of the data sets destined to the multivariate calibration models. for regression models, different pretreatments were tested on the spectra sets (data not shown) previously transformed in absorbance (log 1/t). first derivation by savitzky-golay filter (11 points of smoothing, 2nd order) proved to be the best performing and were used in subsequent chemometric applications and regressive pls1 calculation. calibration and cross-validation results for the models obtained from the two tested parameters, are reported, as characteristic scatter plots of multivariate regressions, in figure 1. significant correlation results were achieved for both models: the determination coefficients (r2) in calibration were 0.98 and 0.96 for the estimated percentage of the weight loss and the estimated percentage of the water content, respectively; the coefficients of determination in cross-validation (r2cv) were, respectively, 0.95 and 0.90. for the estimation of the predictive accuracy of the models, an r2cv greater than 0.9 represents a valid quantitative information (maeda et al., 1995). cross validation is a practical method to demonstrate how nirs can predict a qualitative attribute, even if it would be better to estimate the accuracy of the application by using an appropriate, preferably external, test or validation set (dardenne, 2010). in leave-one-out cross validation, one sample is removed from the dataset and a calibration model is built on the basis of the remaining subset, using that samples to calculate the residual prediction (cozzolino et al., 2011). the significant results obtained in terms of correlation on the predictive models can also be attributed to the high degree of accuracy and precision of the reference data (bellincontro et al., 2012). table 1 statistical analyses of sample sets relative to the two analyzed parameters (weight loss and water content) during the first experiment parameter samples mean sd min max weight loss (%) 95 7.95 4.45 1.51 19.60 water content (%) 75 93.11 3.31 93.11 97.11 mean, standard deviation (sd), range (min. and max.) are reported expressed as %. number of sample s (i.e. the number of destructive measurements carried out) is reported fig. 1 scatter plots relative to the prediction models for the percentage of weight loss of flowers (top). scatter plots relative to the prediction models for the percentage of water content of flowers (bottom). measured values are plotted versus predicted values and significant indexes of calibration and validation are reported. 33 bellincontro et al., innovative application of nir-aotf and mri to study water behaviour in cut flowers however, the real and applicative performance of the predictive models is better defined if combined with the estimation indexes referring to potential errors in calibration and prediction or cross-validation (rmsec, rmsep and rmsecv). here we report rmsecv (root mean standard error in cross-validation) which was 0.81% and 1.48% in predicting the flowers’ weight loss and water content, respectively. quite low lvs values were obtained for both pls models (fig. 1), and it is possible to observe that a small number of latent variables could reduce the possible errors in predictive responses of the models. rpd (ratio of performance to deviation) indexes are also reported in figure 1. the rpd ratio is another statistical index useful for evaluating the predictive ability of the nirs and is calculated as the ratio between sd of destructive measurements and the standard errors of prediction. a rpd below 2.53 means that the model has low ability of discrimination from high values of the response variable; values above 5 indicate good discrimination, especially if destined for quality control (williams and sobering, 1996). as regards the second experiment on the use of mri, the dry matter in the basal (cut surface) and middle sections were similar at the start of the experiment (table 2). after 3 days, at 4°c, no differences were observed among samples, whatever the location of the section or the method of storage (dry or water). at 20°c, after 3 days, the dry matter was similar (4.7 and 4.9%) in the middle sections regardless of the storage method and similar to the one at 4°c while, the basal sections had 3.4 and 4.1% of dry matter in flowers kept in water or dry, respectively; after 12 days the values significantly decreased, 3.7 and 2.8%, in the middle and basal sections, respectively, of flowers kept in water. the results for dry matter indicate that storage temperature plays an important role in preserving the integrity of the stem as has been reported recently for lilium (prisa et al., 2013) and also to maintain fresh and dry weight (ahmad et al., 2013 b). dry matter was also affected by storage method: storage in water preserved the integrity of the internal structure of the calla stem. dry matter concentration is mainly due to sugars concentration which are known for their importance in vase life of cut flowers. the significant reduction of dry matter in middle and basal sections of the flowers kept in water at 20°c might be due to the presence of bacteria. bacteria can enter maintenance solutions from the external surface of the flowers (teixeira da silva, 2003). in tap water, acinetobacter sp., bacillus pumilus and pantoea agglomerans cells moved from the outside to inside the flower parts, becoming endophytic bacteria with a role in stem break of gerbera flowers (balestra et al., 2005). xylematic vessel blockage could be due to some amorphous or physiological deposition and rod-shaped bacteria located within the 5 cm stem end of the cut flower as shown by scanning electron microscope (wang et al., 2014). a partial relationship between the lower content of dry matter and the tissue degradation seems to be confirmed by mri. in figure 2, degradation of the cut stem surface (white area indicates that water moves freely) is clear, while the middle section still maintains intact tissue and its vessels are table 2 dry matter content (%) of sections (basal and middle) of calla lily flowers kept at 4 or 20°c, dry or in water water m.s. dry m.s. water b.s. dry b.s. middle section basal section initial time 4.8±0.3 bcd 4.4±0.4 cd 4°c after 3 days 5.2±0.3 ab 5.2±0.2 ab 4.7±0.4 bcd 4.7±0.4 bcd after 12 days 4.9±0.4 abc 4.9±0.5 abc 4.9±0.4 abc 4.9±0.5 abc 20°c after 3 days 4.7±0.4 bcd 4.9±0.4 abc 3.4±0.4 ef 4.1±0.2 de after 12 days 3.7±0.2 e 4.5±0.4 cd 2.8±0.2 f 4.3±0.2 cd data are the mean of 10 flowers at each sampling time ± sd. values with different letters are significantly different (p<0.05) by lsd. (m.s.= medium section; b.s.= basal section). fig. 2 msme mri images of flowers, middle and basal sections. on top, initial samples. ten images were taken for each sample at each sampling time. the reported images are representative of the pool of 10 for each sample, which showed similarity of behaviour. 34 adv. hort. sci., 2015 29(1): 30-36 visible (white dot); this image is very clear in the initial samples. at 4°c in water, the degradation is less diffuse than at 20°c, both in the basal and middle sections. these images do not reflect the data of dry matter probably because the lower temperature reduces the consumption of sugars by respiration. in contrast, similarity of the images between the initial samples and the samples kept dry at 4 or 20°c is evident, in agreement with dry matter data. in water at 20°c, the images show a great degradation which is in line with the data of dry matter loss. elaboration of the images in terms of normalized population vs pixel intensity, in order to have numbers to combine with the nir data, made it possible to emphasize that the basal sections of the flowers kept in water, not only at 20°c but also at 4°c, are contaminated already in the first 3 days of vase life. the gefi image (mr signal prosectional to water content) of the basal section of flowers kept at 20°c, at a pixel intensity of 100, shows how great the difference is (0.2 vs 0.05-0.1 normalized population) between the initial sample (black line) and the lines (red and bleu) of the flowers kept in water, at 3 and 12 days (fig. 3 a). this result pinpoints a greater water content in the basal tissue than in the middle section where the difference is not evident (data not shown). the same images elaborated as msme signal (mr signal prosectional to water mobility) (fig. 3 b) show a shift of the red and blue lines (flowers in water at 3 and 12 days, respectively) toward the left (y axis), compared to the other samples, and a particular peak at 200 pixels (which suggests a change in water mobility) similar to the one observed in the gefi image (water content) but at 100 pixels (fig. 3a). both these results point out that a change in water content and mobility occurs; this event could be attributed to the presence of bacteria. the curve pattern of the middle section is similar but with a greater distance between the two pairs of lines (red and bluu vs green and light blue) and no peak is observed at 200 pixel (fig. 3 c). the peak of the normalized population for red and blue lines is at 25 pixels while for green and light blue lines it is between 50 and 75 pixels, meaning a clearer image, as can be observed in figure 2. the patterns of curves of basal and middle sections are very different, especially for red and blue lines: at 50 pixels the value of (a) (b) (c) (d) fig. 3 normalized population vs pixels (a.u. = auxiliary units) elaboration of gefi (3a) and msme (3b,c,d) images. 35 bellincontro et al., innovative application of nir-aotf and mri to study water behaviour in cut flowers the basal section is 0.4 normalized population while the one of the middle section is 0.2; for the green and light blue lines (dry flowers at 3 and 12 days) the 100-pixel values are similar for the basal and middle sections. thus, for flowers that were kept dry, clearer images and less difference in the images of the sections can be noted. at 4°c, the basal section showed coupled lines on the basis of sampling times, with a shift towards the y axis, compared to the black line (initial sample): the peak of the normalized population of the initial sample is around 75 pixels, while for the rest of the lines it is between 25 and a little bit more than 50 pixels (fig. 3 d). in the range 50 and 100 pixels, the green and red lines, which refer, respectively, to dry and water samples at 3 days, had lower values than the bleu and the light bleu lines, referring to water and dry samples but at 12 days. even in this case, at 150 pixels an increase of the values of normalized population of the red and blue lines was observed as was shown at 20°c, in line with the presence of degraded tissue. in the middle section, the cited increase at 150 pixels is not visible and the pattern of lines is more confused (data not shown). 4. conclusions application of the portable nir-aotf instrument to the stem of cut flowers (calla lily in the present study) to non-destructively measure weight loss and water content, especially for dry shipped flowers, can be very useful to predict vase life. mri is a very powerful tool to study the water status in the xylematic vessels and the use of of imagej software permits transformation of the image into numbers (pixel intensity or normalized population). unfortunately the cost of the instrument, which depends on magnet size, makes commercial use of the instrument impossible. the use of software will permit correlation of the pixel intensity or normalized population data to spectra values of other non destructive instruments, such as the nir-aotf as will be examined in our future research. acknowledgements research supported by the ministry of agricultural policy, traceflor project. we would like to thank floratoscana sag of pescia (pt) italy and dr. maria castellani for the material and the technical assistance. references ahmad i., dole j.m., carlson a.s., blazich f.a., 2013 a water quality effects on postharvest performance of cut calla, hydrangea, and snapdragon. sci. hort., 153: 26-33. ahmad s.s., tahir i., shahri w., 2013 b effect of different storage treatments on physiology and postharvest performance in cut scapes of three iris species. j. agric. sci. technol., 15(2): 323-331. balestra g.m., agostini r., bellincontro a., mencarelli f., varvaro l., 2005 investigation on bacterial populations related to the gerbera (gerbera jamesonii l.) stem break. phytopathologia mediterranea, 44: 291-299. bellincontro a., cozzolino d., mencarelli f., 2011 application of nir-aotf spectroscopy to 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inflorescences. acta horticulturae, 181: 451458. wang r., zheng x., xu x., 2014 evidence for physiological vascular occlusion in stems of cur gerbera cv. hongyan. j. agric. sci. technol., 16: 365-372. williams p.c., sobering d.c., 1996 how do we do it: a brief summary of the methods we use in developing near infrared calibrations, pp. 185-188. in: davies a.m.c., and p.c. williams (eds.) near infrared spectroscopy: the future waves. nir publications, chichester, uk, pp. 742. 84 1. introduction ‘abbé fétel’ is the most important pear cultivar in italy in terms of production (conerpo, 2010) and it can be stored in normal air (na) for three to four months and in controlled atmosphere (ca) for up to six months (bai et al., 2009). however, when stored in na for more than four months ‘abbé fétel’ pears became sensitive to superficial scald (vanoli et al., 2008). ‘conference’ pears in italy are often subjected to superficial scald in cold storage, and it has been reported that under predisposing climatic conditions, up to 70% of fruit developed scald, thus impairing their marketability (folchi and bertolini, 2008). scald is manifested as brown or black patches on the skin; it can take several forms and, along with superficial scald, it is an expression of damage and/or death within the surface layers of cells (lurie and watkins, 2012; whitaker, 2013). scald has been related to the presence of oxidation products [conjugated trienols (ctols), primarily αfarnesene], acting on epidermal cells (whitaker, 2007) and could be prevented or controlled by storage in ca (bertolini et al., 1997; lurie and watkins, 2012). however, the low levels of oxygen used in ca for ‘abbé fétel’ pears can induce soft scald (bertolini et al., 2002; rizzolo et al., 2010; vanoli et al., 2010 a). up to now, the traditional strategy to prevent superficial scald in pears is a pre-storage treatment with ethoxyquin, which recently has been excluded from the list of active ingredients of chemicals used in food production (ec council directive 91/414) (calvo and kupferman, 2012). the most effective alternatives to ethoxyquin are treatment at harvest with 1-methylcyclopropene (1-mcp) or storage under controlled atmosphere with low levels of o 2 such as ultra-low oxygen, initial low oxygen stress, and dynamic controlled atmosphere (calvo and kupferman, 2012; lurie and watkins, 2012). it was found that 1-mcp inhibited superficial scald and prevented or controlled soft scald and internal breakdown in ‘bartlett’ pears (villabolos-acuña et al., 2011 a, b). in ‘conference’ doses ranging from 50 to 1000 µl l–1 did not prevent the formation of superficial scald, both in ca and in na, but either controlled it, keeping the incidence of scald within commercially acceptable rates, or reduced symptom severity (eccher zerbini et al., 2003; rizzolo ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene: physiological and quality implications of initial low oxygen stress and controlled atmosphere storage m. vanoli, m. grassi, g. bianchi, m. buccheri, a. rizzolo* consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, unità di ricerca per i processi dell’industria agroalimentare (crea-iaa), via g. venezian 26, 20133 milano, italy. key words: α-farnesene, ethylene production, fermentative metabolites, pulp mechanical properties, sensory profiles, storage disorders. abstract: superficial scald is a disorder developed in cold storage by ‘conference’ and ‘abbé fétel’ pears and it has been related to the presence of oxidation products, mainly conjugated trienols (ctols), of which α-farnesene is primary, acting on epidermal cells. among tested postharvest methods to control scald, there is treatment at harvest with 1-methylcyclopropene (1-mcp) and initial low oxygen stress (ilos). the investigation presented here studied, in ‘conference’ and ‘abbé fétel’ pears treated with 1-mcp (300 μl l–1), the physiolological and quality implications of storage in controlled atmosphere (ca, 2 kpa o2 + 0.7 kpa co2, -0.5°c) after two 2-weeks ilos (0.3-0.5 kpa o2) periods at three-week intervals after 13 and 21 weeks of storage and shelf life at 20°c up to seven days. results showed that 1-mcp treatment severely reduced α-farnesene, ctol269, ctol281 and ethanol after ilos treatment in both cultivars, and ethyl acetate in ‘abbé fétel’ pears. furthermore, it impaired fruit softening, delayed skin yellowing and reduced ethylene production in shelf life. at sensory analyses, 1-mcp treated ‘conference’ and ‘abbé fétel’ pears were described as being firmer and less juicy, sweet and aromatic than untreated fruit. 1-mcp treated pears did not develop superficial scald and soft scald in ‘abbé fétel’, nor superficial scald and black speck after 21 weeks of storage in ‘conference’. adv. hort. sci., 2015 29(2-3): 84-96 * corresponding author: anna.rizzolo@entecra.it received for publication 26 september 2014 accepted for publication 11 may 2015 85 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene et al., 2005; folchi and bertolini, 2008). the reduction of symptom severity in ‘conference’ fruit was related to lower amounts of α-farnesene and ctols (rizzolo et al., 2005; folchi and bertolini, 2008). in ‘abbé fétel’ pears, after shelf life, ctols and α-farnesene were significantly higher in fruit affected by superficial scald and lower in those affected by senescent scald (vanoli et al., 2010 b). storage under controlled atmosphere with a low level of o 2 was suggested as an alternative to ethoxyquin and to 1-mcp treatment not only in controlling scald development but also in improving overall fruit quality (prange et al., 2011; lurie and watkins, 2012). widespread adoption of low o 2 regimes has not taken place due to concerns of anaerobic damage when fruit is held below the lower oxygen limit (lol), that is the environmental o 2 level at which cell metabolism changes from being predominantly aerobic to fermentative (wright et al., 2012). zerbini and grassi (2010) and rizzolo et al. (2008, 2010) found that lol for ‘conference’ pears was 0.4 kpa o 2 and for ‘abbé fétel’ pears 0.6 kpa o 2 . vanoli et al. (2008, 2010 a) and rizzolo et al. (2010) found that storage of ‘abbé fétel’ pears at 0.7 kpa o 2 at -0.5°c completely prevented superficial scald development and reduced soft scald incidence compared to 2 kpa o 2 , but increased internal browning and internal breakdown. as for the initial low oxygen stress, wang and dilley (2000) found that an ilos with 0.25 kpa o 2 for two weeks, carried out one or two times at two-month intervals, strongly inhibited α-farnesene and its volatile oxidation product (6-methyl-5-hepten-2-one), increased ethanol, and was effective in controlling scald development in several apple cultivars. as for pears, calvo et al. (2002) found that ilos (0.5 kpa o 2 ) followed by low oxygen ca (1.5 kpa o 2 ) significantly inhibited the development of superficial scald after nine months of storage in ‘beurré d’anjou’ cultivar, while rizzolo et al. (2015) reported that in ‘conference’ pears after an ilos (0.2-0.5 kpa o 2 ) period followed by low oxygen ca (2 kpa o 2 ) there were lower amounts of α-farnesene, ctol 258 and acetaldehyde, and higher quantities of ethanol than after ca and na storage, developing less scald than the other atmospheres. the objective of the present research was to evaluate in ‘conference’ and ‘abbé fétel’ pears the effect of 1-mcp application on fruit stored in a low oxygen ca after two two-week ilos periods at three-week intervals. physiological aspects (fermentative metabolites, conjugated trienols) in storage, and ethylene production, quality and sensory characteristic changes with post-storage shelf life and storage disorders are discussed. 2. materials and methods the experiment was carried out in 2012 on ‘conference’ and ‘abbé fétel’ pears (pyrus communis l.) (about 1000 fruit/cv) harvested from commercial orchards in the modena province (italy) on 20 august and 10 september, respectively, at a commercial degree of maturity [mean ± standard error: ‘abbé fétel’: firmness, 62.5±1.3 n; hue, 104±0.6°; starch hydrolysis, 4.0 ± 0.5 (eurofru 1-10 scale); ‘conference’: firmness, 71.0±1.6 n; hue, 108.9±0 .5°; starch hydrolysis, 3.0 ± 0.1 (eurofru 1-10 scale)] and randomized in 14 boxes. for each cultivar, on the day after harvest, half of the fruits were treated with 300 µl l–1 1-mcp (smartfresh™, agrofresh inc., rohm and haas, spring house, pa, usa) and seven boxes of untreated fruit were used as control. ‘conference’ fruits were then put in na at -0.5°c for four weeks before the beginning of ilos periods and ca storage, while for ‘abbé fétel’ pears the ilos experiment began two days after the 1-mcp treatment. for both control and 1-mcp treated ‘conference’ and ‘abbé fétel’ pears, two ilos periods at 0.3-0.5 kpa o 2 for about two weeks were applied with a three-week interval in ca at 2 kpa o 2 + 0.7 kpa co 2 at -0.5°c. four containers were used, each one dedicated to one sample (1-mcp dose and cultivar); the gas composition of each container was controlled and checked with centralized analyzers, supervised by a specific fruit control equipment software; fluorescence firm™ sensors monitoring (harvestwatch™, satlantic, canada) was carried out in each container from the beginning of the first ilos period till the first storage time (13 weeks). the first ilos period was applied from d0 to d17, and the second one from d40 to d53. then pears were stored in ca at -0.5°c in 2 kpa o 2 + 0.7 kpa co 2 up to 21 weeks. samplings α-farnesene, ctols and fermentative metabolites were analyzed (6 fruits/1-mcp dose/cultivar) at the beginning and at the end of the second ilos period, and at the first storage time (13 weeks); hereafter these samplings are referred as d0, d13 and d40. after 13 and 21 weeks of storage, 3 boxes/1-mcp dose/ cultivar were put in shelf life at 20 °c up to 7 days. at 1, 5 and 7 days of shelf life (d1, d5, d7) 20 fruit/1-mcp dose/ cultivar were analyzed for background skin color and pulp mechanical properties (firmness, stiffness and energy-torupture). ethylene production was measured at d1, d5 and d7 on ten fruits of sample d7, while sensory analyses were carried out on ten fruits at d5 and d7. after 7 days at 20 °c the incidence of storage disorders was evaluated on 3 boxes/1-mcp dose/cultivar. α-farnesene and ctols α-farnesene and ctols (ctol 258 , ctol 269 , ctol 281 ) were determined in the skin according to zoffoli et al. (1998), by sampling eight skin disks of 0.8 cm2 area from the equatorial region of two pears (three replications) and extracting overnight at 2°c with 6 ml of hplc-grade hexane with 1 g of anhydrous na 2 so 4 . the absorbance of the extracts at 232, 258, 269, 281 and 290 nm was measured using a jasco (model 7800) spectrophotometer. concentrations of α-farnesene and ctols were calculated according to huelin and coggiola (1970) and du and bramlage (1993). data were expressed as nmol cm–2. 86 adv. hort. sci., 2015 29(2-3): 84-96 fermentative metabolites fermentative metabolites (ethanol, acetaldehyde and ethyl acetate) were determined on the pulp of the same fruit analyzed for α-farnesene and ctols by means of hsspme-gc, by pooling the six fruits of each sample. ten grams of homogenized pulp (three replications) were put into 25 ml vials tightly closed with an aluminum cap with a silicone-teflon rubber septum; samples were then immediately frozen and kept at -20°c until analysis. after 60 min thawing at room temperature, the spme headspace volatile sampling was carried out for 30 min at 40°c using a 50/30µm dvb-car-pdms fiber (supelco), which was desorbed for 5 min in the gc injector port at 250°c. fermentative metabolites were separated on a supelcowax-10 column (60 m ×0.25 mm i.d., 0.25 µm film thickness) using the following conditions: carrier gas, helium at a flow of 1.5 ml min–1; temperature program, 40°c×13 min, 15°c min–1 to 185°c; fid temperature, 250°c. fermentative metabolites were quantified by relating the peak area of each one to that of external standards. background skin color background skin color was measured on the greener side of fruit with a spectrophotometer cm-2600d (minolta co, japan) using the primary illuminant d65 and 10° observer in the l*, a*, b* color space. from a* and b* values, hue (h°) and chroma (c*) were computed according to h°=arctan (b* a*–1) and c*= (a*2 + b*2)−2. pulp mechanical properties the mechanical properties of pear tissue of each fruit were measured on two opposite peeled areas in the equatorial region of the pear using an 8 mm diameter plunger mounted on an instron universal testing machine (model 4301, instron ltd, great britain) with crosshead speed at 200 mm min–1. from the force-displacement curve the following pulp mechanical properties were measured (rizzolo et al., 2014): firmness (n), stiffness (n mm–1) and energy-to-rupture (mj). firmness, stiffness and energy-torupture readings were averaged for each fruit. ethylene production rate the ethylene production rate (ep) was measured by static hs/gc on fruit put in 1.7 l gas-tight glass jars (ten replications, one fruit per jar) for 2 h at 20 °c according to rizzolo et al. (2005). one milliliter of the headspace gas was sampled and analyzed using a deactivated aluminum oxide f1 (80-100 mesh) column (1/8 in 200 cm) at a column temperature of 100°c and fid detection. quantitative data were obtained by relating the ethylene peak area to that of a 10 μl l–1 standard and were expressed as pmol kg–1 s–1. sensory analysis sensory analyses were carried out in a sensory lab using a panel of ten short-term trained judges at d5 and d7 of shelf life at 20°c. for both the cultivars in each session, one peeled slice/1-mcp dose was presented to each panelist. at the beginning of the session, a slice of a fruit not included in the experimental plan was tasted to eliminate the first tasting effect. drinking water was provided as a palate cleaner between samples. each sample was evaluated for the intensity of attributes related to fruit structure (firm, juicy, grainy) and taste and flavor (sweet, sour, aromatic, bitter, astringent) using 120 mm unstructured line scales with anchors at 12 mm from the extremes (low, high). in addition, in order to have a rough idea of sample pleasantness, at the end of the tasting session, panelists were also asked to score samples for overall acceptability using a 120 mm unstructured line scale with “low” and “high” anchors near the extremes. details on panel training and attributes are reported by rizzolo et al. (2014). storage disorders storage disorders were evaluated on three boxes/1− mcp dose/cultivar. for each box the percentages of healthy fruit and of fruit affected by superficial scald, rots (both cultivars), soft scald (‘abbé fétel’), early blackening, black speck and black spot (‘conference’) were computed. statistical analysis data were submitted to analysis of variance (statgraphics ver.7, manugistic inc., rockville, md, usa). prior to statistical analysis the rating scores of each sensory attribute were standardized by panelists in order to remove the variability due to their using different parts of the scale (bianchi et al., 2009). percentage data were submitted to angular transformation before anova. 3. results oxygen levels and chlorophyll fluorescence figure 1 shows the oxygen partial pressure and the corresponding response of chlorophyll fluorescence (fα) for control and 1-mcp treated ‘abbé fétel’ and ‘conference’ pears from the beginning of the first ilos period till the first storage time (13 weeks). by comparing the graphs of o 2 partial pressure of containers of control and 1-mcp treated ‘conference’ pears, it is evident that during the first ilos period the o 2 values ranged from about 0.2 to 0.6 kpa and during the second ilos from 0.15 to 0.55 kpa. the two ilos periods induced a remarkable rise in chlorophyll fluorescence (fα) in untreated fruit and much smaller ones in 1-mcp treated pears, when o 2 concentration decreased below 0.4 kpa. in control ‘abbé fétel’ pears the o 2 concentration decreased below 0.6 kpa only for a short time during the first ilos period, while during the second ilos period o 2 values ranged from 0.3 to 0.6 kpa, causing a very slight rise in fα at the end of the second ilos period. on the other hand, in the container with 1-mcp treated ‘abbé fétel’ pears o 2 values ranged from 0.1-0.4 kpa during the two ilos periods, causing a slight rise in fα in coincidence with the two ilos periods. 87 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene α-farnesene and conjugated trienols on average 1-mcp treated ‘conference’ pears had lower amounts of α-farnesene, ctol 269 and ctol 281 than control fruit, and ctol 281 at the end of the ilos period (d13) was not detectable in 1-mcp treated fruit (table 1). in control fruit α-farnesene, ctol 269 and ctol 281 significantly increased at d40, in correspondence wth the first storage time. likewise, 1-mcp treated ‘abbé fétel’ pears table 1 amounts (mean ± standard error) of α-farnesene (α-farn), ctols (ctol 258 , ctol 269 , ctol 281 ) and fermentative metabolites (ace, acetaldehyde; etoh, ethanol; etac, ethyl acetate) in control (mcp0) and 1-mcp treated (mcp300) ‘conference’ pears at the beginning (d0) and at the end (d13) of the second ilos period and in correspondence with the first storage time (d40) and anova results α-farn nmol cm–2 ctol 258 nmol cm–2 ctol 269 nmol cm–2 ctol 281 nmol cm–2 ace µg kg–1 etoh µg kg–1 et ac µg kg–1 mcp0 d0 11.32±2.97 0.97±0.24 1.16±0.18 0.21±0.08 27.33±5.38 38.77±13.85 0.10±0.05 d13 9.26±2.81 0.80±0.27 0.90±0.22 0.60±0.33 29.15±3.23 69.45±3.71 0.14±0.09 d40 23.04±8.69 1.80±0.60 2.32±0.58 0.91±0.33 35.34±0.29 24.71±1.13 1.77±1.52 mcp300 d0 3.77±0.28 1.01±0.15 1.08±0.12 0.14±0.04 40.26±4.42 55.81±7.39 0.06±0.03 d13 2.96±0.31 0.62±0.15 0.68±0.16 nd 17.31±8.67 21.30±14.83 0.05±0.04 d40 3.90±0.37 0.77±0.15 0.93±0.14 0.04±0.02 33.89±3.82 23.01±2.66 0.87±0.45 anova (z) a: day ns ns * * ns * ns b: 1-mcp ** ns * ** ns ns ns a × b ns ns ns * ns ** ns (z) ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant. fig. 1 oxygen partial pressure (gray) and corresponding response of chlorophyll fluorescence fα (black) for control and 1-mcp treated containers of ‘abbé fétel’ and ‘conference’ fruit. the dotted lines indicate the o 2 partial pressure inducing stress evidenced by fα increase (‘abbé fétel’ 0.6 kpa; ‘conference’ 0.4 kpa) 88 adv. hort. sci., 2015 29(2-3): 84-96 had lower amounts of α-farnesene, ctol 258 , ctol 269 and ctol 281 than control fruit (table 2). the α-farnesene and ctols concentrations did not significantly change from the beginning (t0) to the end of the second ilos period (d13) both in control and 1-mcp treated ‘abbé fétel’ fruit, and then they increased at d40. the concentration of α-farnesene in pear skin was three to five times higher in control than in 1-mcp treated fruit, those of ctol 258 and ctol 269 two (‘conference’) to four-six times (‘abbé fétel’), and that of ctol 281 two to eight times in ‘abbé fétel’ and fifteen to twenty-three times in ‘conference’. in both cultivars the ratios ctol 258 /ctol 281 and ctol 269 / ctol 281 (fig. 2) were higher in 1-mcp treated fruit than in control ones, being, on average, three times higher in ‘conference’ pears, and about 50% higher in ‘abbé fétel’ treated fruit. in ‘conference’ the highest values for both ratios were observed in 1−mcp treated fruit at d 40, while in ‘abbé fétel’ in 1−mcp treated fruit at d0 and d13. control ‘abbé fétel’ and ‘conference’ pears at d40 showed the lowest values for both the ratios. fermentative metabolites in ‘conference’ pears the 1-mcp treatment at harvest did not influence the amounts of fermentative metabolites (table 1). in control ‘conference’ pears ethanol increased with the second ilos period, then it significantly decreased table 2 amounts (mean ± standard error) of α-farnesene (α-farn), ctols (ctol 258 , ctol 269 , ctol 281 ) and fermentative metabolites (ace, acetaldehyde; etoh, ethanol; etac, ethyl acetate) in control (mcp0) and 1-mcp treated (mcp300) ‘abbé fétel’ pears at the beginning (d0) and at the end (d13) of the second ilos period and in correspondence with the first storage time (d40) and anova results α-farn nmol cm–2 ctol 258 nmol cm–2 ctol 269 nmol cm–2 ctol 281 nmol cm–2 ace µg kg–1 etoh µg kg–1 et ac µg kg–1 mcp0 d0 14.80±4.65 0.83±0.14 0.70±0.14 0.42±0.10 10.38±0.31 14.86±5.18 0.92±0.07 d13 15.10±1.41 0.70±0.05 0.63±0.02 0.38±0.01 16.40±3.56 31.10±6.65 0.08±0.06 d40 55.14±9.81 3.83±0.53 6.41±0.91 5.14±0.79 26.78±6.56 39.62±15.98 0.06±0.01 mcp300 d0 2.51±0.08 0.43±0.05 0.34±0.06 0.18±0.03 14.03±2.83 17.89±5.11 0.51±0.03 d13 2.69±0.06 0.47±0.01 0.34±0.01 0.18±0.01 16.21±2.10 34.94±5.34 0.09±0.05 d40 10.91±1.94 1.01±0.30 1.01±0.32 0.62±0.17 27.33±3.04 48.99±11.30 0.04±0.02 anova (z) a: day *** *** *** *** *** * *** b: 1-mcp *** *** *** *** ns ns ** axb ** *** *** ns ns ns *** (z) ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant. fig. 2 ctol 258 /ctol 281 and ctol 269 /ctol 281 ratios of control (mcp0) and 1–mcp treated (mcp300) ‘abbé fétel’ and ‘conference’ pears at the beginning (d0) and the end (d13) of the second ilos period and in correspondence with the first storage time (d40). bars refer to standard error of the mean. 89 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene at d40 (i.e. 13 weeks storage time), while in 1-mcp treated fruit it decreased with the second ilos period and did not change further at d40. the sampling time had no significant influence on acetaldehyde and ethyl acetate amounts both in control and 1-mcp treated fruit, probably due to the high standard errors of data, especially for ethyl acetate. the 1-mcp treated ‘abbé fétel’ pears (table 2) had, on average, lower ethyl acetate amounts at d0 than control fruit. ethyl acetate both in control and 1-mcp treated pears decreased steeply with the ilos period, and afterwards did not change at d 40. both in control and 1-mcp treated ‘abbé fétel’ fruit acetaldehyde concentration did not change from the beginning (d0) to the end of the second ilos period (d13), and then increased at d40, while ethanol increased throughout the sampling time. ethylene production ethylene production in control ‘conference’ pears was lower than in ‘abbè fétel’ (fig. 3). in ‘conference’ pears ep was significantly affected only by the 1-mcp treatment (table 3), whereas in ‘abbé fétel’ fruit both 1-mcp treatment and post storage shelf life significantly influenced ep (table 3). in both cultivars the 1-mcp treatment reduced ep to values lower than 10 pmol kg–1 s–1. in ‘abbé fétel’ control and 1-mcp treated fruit after 13 weeks storage showed a decreasing ep with shelf life, and after 21 weeks a minimum ep at d5. pulp mechanical characteristics upon removal, 1-mcp treated ‘conference’ and ‘abbé fétel’ pears maintained firmness similar to that at harvest (‘conference’: 71.0±1.6 n; ‘abbé fétel’: 62.5±1.3 n), but lower stiffness and higher energy-to-rupture than at harvest (values at harvest: stiffness: ‘conference’, 21.0±0.5 n mm−1; ‘abbé fétel’, 24.5±0.6 n mm−1; energy-to-rupture: ‘conference’, 0.106±0.04 j; ‘abbé fétel’, 0.074±0.003 j). the same scenario was found for control ‘conference’ pears, whereas control ‘abbé fétel’ fruit had lower firmness and energy-to-rupture than at harvest. on average, 1-mcp treated ‘conference’ pears had higher firmness, stiffness and energy-to-rupture than control fruit, without any difference between storage times; firmness and energy-to-rupture did not change with shelf life, whereas stiffness decreased, but to a lesser extent than control fruit (fig. 4). in control ‘conference’ pears the values of all the mechanical properties decreased with shelf life with the main changes at d5. as for ‘abbé fétel’ pears (fig. 4), on average, 1-mcp treated fruit showed higher firmness, stiffness and energy-to-rupture than control fruits, without any difference between the storage times, except for a higher energy-torupture of 1-mcp treated fruit after 21 weeks of storage. fig. 3 ethylene production rate (ep) of control (left) and 1–mcp treated (right) ‘abbé fétel’ and ‘conference’ pears during shelf life at 20°c after 13 and 21 weeks of storage. bars refer to standard error of the mean. results of anova analysis are reported in table 3. table 3 multifactor anova results for pulp mechanical characteristics (firmness, f, stiffness, st and energy-to-rupture, e f ), color parameters (lightness, l*; chroma, c* and hue) and ethylene production rate (ep) for ‘abbé fétel’ and ‘conference’ pears abbé fétel conference ep f st e f l* c* hue ep f st e f l* c* hue a: storage time ns ns ns *** ns ** *** ns ns ns ns *** *** *** b: 1-mcp *** *** *** *** *** *** *** *** *** *** *** *** *** *** c: shelf life *** *** *** *** *** *** *** ns *** *** *** *** *** *** a×b ns ns * ns * ns ns ns ns ns ns ns ns * a×c ns * ns * ns ns ns ns * ns ** ns ns ns b×c *** *** *** *** ns ns * ns *** *** *** ** * * a×b×c ns ** ** * ns ns ns ns ns ns ns ns ns ns ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant) 90 adv. hort. sci., 2015 29(2-3): 84-96 at d1 of shelf life, control ‘abbé fétel’ pears had lower firmness and energy-to-rupture but similar stiffness than d1 1-mcp treated fruit. firmness, stiffness and energy-to-rupture of control ‘abbé fétel’ pears decreased after 5 days and then they did not change further. in 1-mcp treated ‘abbé fétel’ pears, firmness and energy-to-rupture did not change with shelf life, whereas stiffness decreased after 5 days, but to a lesser extent than in control fruit, without any further change with the increase of shelf life time. skin color on average in ‘conference’ pears l* and c* were higher in control fruit after 21 weeks storage (fig. 5) than in 1-mcp treated fruit at both storage times and in control fruits after 13 weeks storage, while the highest h° was found for 1-mcp treated fruit at both storage times and the lowest for control fruit after 21 weeks. hue decreased with storage time only in control fruit. at d1 of shelf life control ‘conference’ pears had l*, c* and h° values not different from those of 1-mcp treated fruit. with shelf life, l* and c* increased and h° decreased both in control and 1-mcp treated fruit, with control fruit at d7 showing the highest values of l* and c* and the least of h°. in ‘abbé fétel’ the highest l* value was found in control fruit after 13 weeks of storage and the lowest in 1-mcp treated fruit at both storage times (fig. 5). chroma on average was higher in control fruit than in 1-mcp treatment without any influence of storage time, whereas h° was higher in 1-mcp treated fruit than in control pears, without any difference between storage times, with control fruits having the lowest h° value after 21 weeks storage. at d1 of shelf life control ‘abbé fétel’ pears had higher l* and lower h° than 1-mcp treated fruit. l* increased with shelf life in control fruit at both storage times and in 1-mcp treated ones after 21 weeks of storage. h° decreased with shelf life only in control fruit, while c* increased both in control and 1-mcp treated fruit, with control fruit having the highest values at d7. sensory analysis with regard to sensory analysis, ‘conference’ 1-mcp treated pears were on average firmer, more grainy, less juicy, sweet, sour and aromatic than control ones (fig. 6), with the average scores being (1-mcp and control, respectively): 84 and 45 for sensory firmness, 52 and 38 for graininess, 25 and 71 for juiciness, 47 and 62 for sweetness, 19 and 24 for sourness, and 33 and 59 for aromatic. no changes in sensory profile with storage time and shelf life were observed for 1-mcp treated ‘conference’ pears. in contrast, in control ‘conference’ pears sensory firmness decreased during shelf life, without any differences between the storage times, while sweetness decreased with shelf life after 13 weeks storage and increased after 21 weeks storage, with d5 pears after 13 weeks being sweeter than fruit after 21 weeks. juiciness in control fruit increased with shelf life only after 13 weeks storage and it fig. 4 firmness, stiffness and energy-to-rupture of control (mcp0) and 1-mcp treated (mcp300) ‘abbé fétel’ and ‘conference’ pears after 1, 5 and 7 days of shelf life at 20°c after 13 and 21 weeks of storage. bars refer to standard error of the mean. results of anova analysis are reported in table 3. fig. 5 lightness (l*), chroma (c*) and hue angle (degree) of control (mcp0) and 1-mcp treated (mcp300) ‘abbé fétel’ and ‘conference’ pears after 1, 5 and 7 days of shelf life at 20°c after 13 and 21 weeks of storage. bars refer to standard error of the mean. results of anova analysis are reported in table 3. 91 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene was not significantly affected by the storage time. overall acceptability was higher in control fruit and it was not influenced by storage time and shelf life both in control and 1-mcp treated ‘conference’ fruit. on average control ‘abbé fétel’ pears were less firm and more juicy, sweet and aromatic than 1-mcp treated fruit, the average scores being (1-mcp and control, respectivley): 93 and 50 for sensory firmness, 33 and 58 for juiciness, 43 and 59 for sweetness, and 33 and 50 for aromatic. similarly to that found for ‘conference’ pears, control ‘abbé fétel’ fruit at d5 of shelf life (fig. 6) were firmer than at d7, without any influence of storage time. juiciness was higher in d7 fruit after 13 weeks storage than in those after 21 weeks. sweetness and aromatic scores were lower in d5 fruit after 21 weeks than in d5 ones after 13 weeks, the latter showing also higher sourness and astringency than fruit stored for 21 weeks at the end of shelf life. as for 1-mcp treated ‘abbé fétel’ pears, sensory firmness did not change with shelf life in fruit stored for 13 weeks, whereas it increased in those stored for 21 weeks, with the d7 fruit after 21 weeks being firmer than fruit at d5 of both storage times. 1-mcp treated ‘abbè fétel’ pears at d5 after 13 weeks of storage were less firm and juicier and sweeter than those of the same storage time at d7 and than those stored for 21 weeks. overall acceptability decreased with storage time and shelf life: control fruit after 13 weeks storage and 5 days of shelf life had the highest overall acceptability, while 1-mcp treated fruit at fig. 6 sensory profiles and overall acceptability of control (mcp0) and 1-mcp treated (mcp300) ‘abbé fétel’ and ‘conference’ pears after 13 and 21 weeks of storage and 5 (d5) and 7 (d7) days of shelf life at 20°c. within each 1-mcp dose, for each attribute the significance of fratio (**, p<0.01; *, p<0.05; (*), p<0.10; no symbol, not significant) is reported. within each 1-mcp dose, bars with different letters refer to statistically different means (tukey’s test, p < 0.05%). 92 adv. hort. sci., 2015 29(2-3): 84-96 d7 after 13 weeks and at d5 and d7 after 21 weeks storage had the least overall acceptability. in addition, 1-mcp treated ‘abbé fétel’ pears stored for 13 weeks at 5 days of shelf life had an overall acceptability score that was not different from that of control fruit stored for 21 weeks. storage disorders in ‘conference’ pears at the end of storage and after 7 days at 20°c the percentage of healthy fruit was very low and decreased with storage time both in control and 1-mcp treated fruit (table 4). three types of peel disorders were detected: blackening, superficial scald and black speck. two forms of blackening were distinguishable, differing for the color and severity of the disorder: early blackening, characterized by a grey net covering part of the peel, without any specific localization, and black spot, characterized by a very tight black net which, in the most severe forms, covered almost all the fruit surface. the percentages of fruit affected by the two forms of blackening were very high (table 4). early blackening was found only after 13 weeks of storage and its incidence was not influenced by 1-mcp treatment; black spot was found only after 21 weeks of storage, with higher incidence in 1-mcp treated fruit than in control ones. superficial scald incidence was low (less than 5%) and developed only in control fruit after 21 weeks of storage. black speck incidence was low (about 5%) and was found both in control and 1-mcp treated pears after 13 weeks of storage. rot incidence was very low and was influenced neither by storage time nor by the 1-mcp treatment. in ‘abbé fétel’ pears the percentage of healthy fruit on average was 93.3% in 1-mcp treated fruit and 77.4% in control ones and slightly decreased with storage time (table 5). two types of peel disorders were detected: soft scald and superficial scald, both of them developed only in control fruit. the incidence of soft scald increased somewhat with the increase of storage time, while superficial scald was detected at percentages lower than 1.5% only after 21 weeks of storage. rot incidence was low, ranging from 4.2% (1-mcp treated after 21 weeks) to 8.6% (control after 21 weeks), and was not significantly influenced by storage time nor by the 1-mcp treatment. 4. discussion in control and 1-mcp treated ‘conference’ pears, the two ilos periods induced a remarkable rise in chlorophyll fluorescence (fα) in untreated fruit and much smaller ones in 1-mcp treated pears, when o 2 concentration decreased below 0.4 kpa, which is the lower o 2 limit (lol) at which metabolism of ‘conference’ pears changes from aerobic to fermentative (zerbini and grassi, 2010). the lesser fα increase found in 1-mcp treated ‘conference’ pears could be due to respiration reduction induced by 1-mcp, which reduces lol, as observed in other pear cultivars (watkins, 2006) and in ‘abbé fétel’ fruit by rizzolo et al. (2008, 2010). in control ‘abbé fétel’ pears the o 2 concentration decreased below 0.6 kpa, which is its lol, only for a short table 4 storage disorders in control (mcp0) and 1-mcp treated (mcp300) ‘conference’ pears after 13 and 21 weeks of storage and 7 days of shelf life at 20°c and anova results (n=3) healthy early blackening black spot superficial scald black speck rot mcp0 13 w 33.2 ±0.3 59.4±0.7 0 0 4.6 ±0.4 0.3±0.1 21 w 22.2±0.4 0 71.3±0.3 4.7±0.4 0 0.3±0.3 mcp300 13 w 29.0±0.01 64.3±0.01 0 0 5.3±0.01 1.3±0.01 21 w 10.8±0.2 0 88.4±0.2 0 0 0.5±0.1 anova (z) a: storage time ** *** *** ** *** ns b: 1-mcp ns ns * ** ns ns a × b ns ns * ** ns ns (z) ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant. table 5 storage disorders in control (mcp0) and 1-mcp treated (mcp300) ‘abbé fétel’ pears after 13 and 21 weeks of storage and 7 days of shelf life at 20°c and anova results (n=3) healthy soft scald superficial scald rot mcp0 13 w 82.5±0.3 10.6±0.1 0 5.6±0.2 21 w 72.8±0.1 15.1±0.2 1.4±0.4 8.6±0.2 mcp300 13 w 95.6±0.03 0 0 4.4±0.03 21 w 91.5±0.2 0 0 4.2±1.2 anova (z) a: storage time * ns ns ns b: 1-mcp *** *** ns ns a × b ns ns ns ns (z) ***, p<0.001; *, p<0.05; ns, not significant. 93 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene period during the first ilos period, while during the second ilos period o 2 values ranged from 0.3 to 0.6 kpa. in contrast, in the container with 1-mcp treated ‘abbé fétel’ pears during the two ilos periods o 2 values ranged from 0.1-0.4 kpa, causing a slight rise in fα in 1-mcp treated ‘abbé fétel’ pears probably due to the fact that in this container the o 2 values were much lower than the lol value. it has been reported that fruit stored in ultralow oxygen pressure, below 2 kpa inducing fermentation, when compared to fruit stored in air develop lower quantities of straight-carbon chain compounds, esters, aldehydes and ketones (mattheis et al., 1991, fellman et al., 1993) and increased amounts of ethanol, acetaldehyde and ethanolderived ethyl esters, mainly ethyl acetate (argenta et al., 2004). furthermore, lumpkin et al. (2014) found for apples that acetaldehyde, ethanol and ethyl esters amounts increased with po 2 , decreasing from 1.5 kpa to 0.3 kpa, mainly during the first weeks of storage, while mattheis et al. (2013) found in ‘d’anjou’ pears a significant correlation between pithy brown core incidence and ethanol, suggesting a relationship between disorder development and abnormal oxidative metabolism due to an hypoxic storage environment. according to these findings we can infer that, in control ‘conference’ pears and in control and 1-mcp treated ‘abbé fétel’ fruit, the stress due to the low po 2 applied during the ilos period, as highlighted by the fluorescence monitoring, impacted on fermentative metabolites. the reduction of respiration induced by 1-mcp significantly influenced fermentative metabolite development, as in ‘conference’ no more ethanol was produced during the low po 2 ilos period and in ‘abbè fètel’ pears it lowered the production of ethyl acetate. as for the relationships between ilos period and αfarnesene and ctols content in the peel, for both cultivars no significant changes in their concentrations following the ilos period were found, with the exception of ctol 281 in 1-mcp treated ‘conference’ pears, which became not detectable at the end of the ilos period. then, after 13 weeks of storage α-farnesene and ctols concentration in the peel increased in control fruit of both cultivars and in 1-mcp treated ‘abbé fétel’ pears. on the other hand, in both cultivars 1-mcp treatment significantly inhibited the production of α-farnesene and ctols not only during the ilos period but also after the ca storage period till 13 weeks storage time, with ‘abbé fétel’ pears showing a more marked reduction in α-farnesene, ctol 258 and ctol 269 concentrations and ‘conference’ fruit in ctol 281 concentration. the α-farnesene and ctols trends observed are in agreement with previous findings on ‘conference’ and ‘abbé fétel’ pears (lo scalzo et al., 2002; folchi and bertolini, 2008; eccher zerbini et al., 2005; vanoli et al., 2010 b) and could be due to the fact that low temperature storage induces, in scald-susceptible cultivars, a high rate of αfarnesene synthesis, which causes its marked accumulation in the skin during the first two to three months of storage. then, the concentration of α-farnesene declines as a consequence of its in vivo oxidation to the highly reactive conjugated trienols (gapper et al., 2006; isidoro and almeida, 2006; whitaker, 2007), which disrupt cell membranes and lead to polyphenoloxidase-mediated browning of the skin (bain and mercer, 1963) and necrosis of the hypodermal cell layers. moreover, it was found that both in apples and pears inhibition of α-farnesene synthesis by 1-mcp was closely correlated with suppression of the α-farnesene synthase gene pcafs1, which encodes the last enzyme in the α-farnesene biosynthetic pathway (lurie et al., 2005; pechous et al., 2005; gapper et al., 2006). in highly scaldsusceptible apple and pear cultivars, inhibition of ethylene production and α-farnesene synthesis by pre-storage 1-mcp treatment is often lost after several months in cold storage, and this coincides with loss of scald control (gapper et al., 2006; tsantili et al., 2007). for ‘d’anjou’ pears zoffoli et al. (1998) reported that ctol 269 was the main peak for conjugated trienols and increased during cold storage, as did the other two ct peaks; also for ‘packham’s triumph’ pears the three ctols increased during storage, but ctol 258 was proportionally higher than ctol 281 and almost the same as ctol 269 , while in ‘bartlett’ pears the main ctol was ctol 258 , which increased with storage, with very low amounts for the other two ctols. whitaker et al. (2001) associated the absorbance measured at 258 nm in the skin hexane extracts to a family of p-cumaryl fatty esters which act as antioxidant rather than to an oxidation product of α-farnesene. in view of these findings, our results suggest that the capacity of ‘conference’ pears stored in ca after ilos periods to generate scald-related antioxidants (ctol 258 ) is higher than the fruit’s ability to produce scaldrelated α-farnesene oxidation products (ctol 281 ), while the opposite scenario was found for control ‘abbè fétel’ fruit. considering the ratio ctol 258 /ctol 281 , a potential marker of superficial scald, du and bramlage (1993) found that values lower than 1.0 were generally associated to high scald susceptibility while values greater than 2.0 were associated to lower scald susceptibility. in this work, higher values of ctol 258 /ctol 281 were found in 1-mcp treated fruit of both cultivars, whereas values below 1.0 and below 2.0 were found in control fruits of ‘abbé fétel’ and ‘conference’, respectively, both developing some superficial scald at the end of storage. du and bramlage (1993) reported that ctol 269 /ctol 281 ratio values generally reflected those of the ctol 258 /ctol 281 ratio, but differences were less distinct when comparing lots of apple fruit with different scald potential. on the other hand, zoffoli (1994), when considering the trends of the ctol 258 /ctol 281 and ctol 269 / ctol 281 ratios with cold storage time in pears, suggested that ctol 258 could be the precursor to ctol 269 , and ctol 269 the precursor to ctol 281 . in the present study, data of the two ratios confirmed zoffoli’s (1994) hypothesis: indeed in ‘abbé fétel’ pears at the beginning and at the end of the second ilos period ctol 258 was predominant, both in control and 1-mcp treated fruit, but in correspondence with the first storage time ctol 269 was predominant in control fruit, while it was almost the same in 1-mcp treated pears. similarly, in control ‘conference’ fruit at the first storage 94 adv. hort. sci., 2015 29(2-3): 84-96 time, ctol 269 was predominant. the low incidence of superficial scald found in this work could be due either to seasonal non predisposing conditions, as low superficial scald incidence was observed in ‘abbé fétel’ fruit stored in na (rizzolo, grassi and vanoli unpublished) or to the beneficial effect of ca in ‘conference’ pears with respect to na stored fruit (rizzolo et al., 2015). both in ‘conference’ and ‘abbé fétel’ pears, 1-mcp treatment prevented the development of scald after storage and shelf life. in ‘conference’ a high proportion of fruit developed blackening in a less severe form after 13 weeks storage, and with higher incidence and severity after 21 weeks storage. blackening has been detected in ‘conference’ pears for about twenty years, but its incidence and severity has been increasing over the last few years. according to previous observations (bertolini, personal communication), blackening development is not related to α-farnesene and ctols development and it is neither controlled nor prevented by 1-mcp treatment, rather it induced higher severity than in control fruit. black speck was developed in ‘conference’ only after 13 weeks storage and, similarly to blackening, it was neither controlled nor prevented by 1-mcp treatment. black speck has been reported for mature-green ‘d’anjou’ pears (lee et al., 1990) and it was suggested that it is provoked by fruit stress related to low temperature in conjunction with low oxygen ca storage, as confirmed by mattheis and rudell (2011), who found that the low o 2 partial pressure set points established by monitoring fruit chlorophyll fluorescence can prevent ‘d’anjou’ scald but may result in black speck development. the pre-storage 1-mcp treatment drastically reduced ethylene production during post-storage shelf life both in ‘conference’ and ‘abbé fétel’ fruit, as found in previous studies on these cultivars (eccher zerbini et al., 2003, 2005; rizzolo et al., 2005, 2008; vanoli et al., 2008, 2010  a) and on other pear cultivars (watkins, 2006 and references herein). control ‘abbé fétel’ pears produced more ethylene than control ‘conference’ pears and were less sensitive to 1-mcp treatment, as shown mainly by the sensory analysis results and, secondly, by quality parameters, as found by eccher zerbini et al. (2003, 2005). control fruit from both cultivars with shelf life underwent skin yellowing and pulp softening. however, control ‘abbé fétel’ pears already upon removal after 21 weeks storage in ca soften to firmness values lower than 40 n, the threshold value corresponding to edible-firm texture for this cultivar (predieri and gatti, 2009). ‘conference’ control fruit softened rapidly with shelf life, reaching the minimal level of acceptable eating quality of 10 n (chiriboga et al., 2013) already after 5 days of shelf life at 20°c. fruit softening in control pears was similar to that found in previous experiments on ca and dca ‘abbé fétel’ fruit stored at -0.5°c (rizzolo et al., 2014; vanoli et al., 2015) and on ‘conference’ fruit stored in na and ca at -1°c (folchi and bertolini, 2008) and in air at -0.5°c (chiriboga et al., 2013). the treatment with 1-mcp at the concentration of 300 nl l–1 prevented ripening during the 7-day shelf life at 20°c, even if slight decreases in firmness, stiffness and energy-to rupture were found for 1-mcp treated ‘abbé fétel’ pears; these slight changes, indeed, influenced the sensory firmness and juiciness. in contrast, in 1-mcp treated ‘conference’ pears, only stiffness slightly decreased with shelf life, and no changes in the sensory profile were found with storage time and shelf life. on the contrary, folchi and bertolini (2008) observed that in ‘conference’ pears harvested at 64.7 n and treated with 300 nl l–1 1-mcp, stored at -1°c up to four months there was a slight softening of 6 n, followed by a further softening to 40 n, prolonging storage up to seven months plus seven days of shelf life at 20°c. this difference could be due to the fact that ‘conference’ pears had been harvested at a less advanced stage of maturity as assessed by the firmness value of 71 n and hue value of 108°, which are similar to the values reported by chiriboga et al. (2013) for early and mid harvests. these authors reported that in two years ‘conference’ pears harvested before or around the commercial harvest date remained firm after treatment with 1-mcp and lost their ability to soften even after several days at 20°c. in contrast 1-mcp applied at more advanced stages of maturity slowed down the softening process without completely blocking it. 5. conclusions the 1-mcp treatment at harvest influenced the physiological and quality changes of ‘conference’ and ‘abbé fétel’ pears stored in ca after ilos periods. 1-mcp dramatically decreased the concentrations of α-farnesene and ctols in the fruit skin at the end of the ilos period both in ‘conference’ and ‘abbé fétel’ pears, and affected the concentrations of fermentative metabolites in the fruit pulp, lowering, after the ilos period, the ethanol concentration in both cultivars and the ethyl acetate amount in ‘abbé fétel’ pears. the 1-mcp treatment drastically reduced the ethylene production during shelf life, impaired fruit softening to edible texture, and delayed fruit yellowing both during storage and shelf life. upon sensory tasting, 1-mcp treated fruit was firmer and less juicy, sweet and aromatic than control fruit, without any changes with shelf life in ‘conference’ fruit and with a slight decrease in sensory firmness and juiciness in ‘abbé fétel’ pears. it was confirmed that 1-mcp prevents superficial scald in both cultivars, and soft scald in ‘abbé fétel’ pears, whereas in ‘conference’ fruit it either has no effect or enhances the incidence and severity of blackening, a disorder which may not be dependent on oxidation products in the skin. acknowledgements research carried out within the project “effects of 1-mcp application on ‘abbé fétel’ and ‘conference’ 95 vanoli et al., ‘conference’ and ‘abbé fétel’ pears treated with 1-methylcyclopropene pears stored in different low oxygen atmospheres” was funded by agrofresh europe. references argenta l.c., mattheis j.p., fan x., finger f.l., 2004 production of volatile compounds by fuji apples following exposure to high co 2 or low o 2 . j. agric. food chem., 52: 5957-5963. bai j., prange r.k., toivonen p.m.a., 2009 pome fruits, pp. 267-286. in: yahia e.m. 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2019 33(3): 381-390 doi: 10.13128/ahs-23830 the physiological responses of four turfgrass species to drought stress z. gholamian jazi (*), n. etemadi, h. aalipour department of horticulture, college of agriculture, isfahan university of technology, isfahan, iran. key words: antioxidant, bermudagrass, cool-season turfgrasses, electrolyte leakage. abstract: drought stress is one of the most important factors which reduce turfgrass growth and quality in the area with restricted rainfall or irrigation water supply. using resistant species and varieties can be a useful management program for reducing irrigation requirement in turfgrass. the present study was carried out to examine the physiological changes of four species turfgrass in response to drought stress conditions. the rhizomes of bermudagrass, and seeds of tall fescue, perennial ryegrass and kentucky bluegrass were cultivated at the greenhouse in pvc pots (20 cm in diam., 20 cm long). after four months, when the seedlings well established, drought stress was applied in 100% field capacity (fc), 75% fc, 50% fc and 25% fc. proline, electrolyte leakage (el), malondialdehyde (mda), relative water content (rwc), chlorophyll, catalase (cat), superoxide dismutase (sod) and peroxides (pod) was measured. all species showed an ability to tolerate drought stress, but tall fescue exhibited more tolerance, with a higher rwc and proline content. tall fescue also revealed higher cat, sod, pod activities and lowest mda, el. this study found that kentucky bluegrass was more vulnerable to severe water stress, and displayed the highest mda and el as compared to the other examined species. 1. introduction water deficit is the main problem for turf management, especially in arid and semi-arid zones. turfgrasses play a significant role in the design of urban green spaces, in most cases other plants can not be utilized instead of turfgrasses; therefore it is necessary to find species and cultivars of turfgrass that require little water and are able to maintain their visual quality in drought conditions (fiorio et al., 2012). increased competition for water has fostered interest in water conservation practices for both warm-season and cool-season turfgrasses. responses of turfgrass to drought can be viewed in a number of ways. drought stress will affect visual quality, growth rate and evapotranspiration (et) (krishnan et al., 2013). some adaptations and mitigation strategies are necessary to dispose of drought stress. grass species and cultivars have been found to respond differently to drought stress (vurukonda et al., 2016). some grass genotype like tall fescue were able to tolerate drought condition in a research it was demonstrated that this kind of grass can be one of the (*) corresponding author: z.gholamianjazi@gmail.com citation: gholamian jazi z., etemadi n., aalipour h., 2019 the physiological responses of four turf‐ grass species to drought stress. adv. hort. sci., 33(3): 381-390 copyright: © 2019 gholamian jazi z., etemadi n., aalipour h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 october 2018 accepted for publication 27 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 381-390 382 most suitable plant species to be used for cultivation under arid, semi-arid regions, and areas with limited water supplies or drought conditions (alam et al., 2018). some traits affected by drought stress include relative water contant, electrolyte leakage and some enzyme activities. rwc and el are indicators for the selection of drought-tolerant plants (salehi lisar et al., 2012). reactive oxyen species cause lipid peroxidatin, which leads to damage of cell membrane. drought stress increased lipid peroxidatin and membrane damage percent in different plants. it is been reported that tall fescue exhibiting a more effective protection mechanism, mitigated oxidative stress and lipid peroxidation by maintaining higher superoxide dismutase (sod) and catalase (cat) activities than kentucky bluegrass (xu et al., 2013). there are many reports about increase, decrease, or no change inoxidative enzymes in plants exposed to stress (fu and huang, 2001; ramachandra reddy et al., 2004; sharma and dubey, 2005). synthesis of compatible solutes such as proline seems to has a central role in osmotic adjustments, preventing or reducing the loss of turgor (vinocur and altman, 2005). in fact, studies on several turfgrass species have shown that the free proline concentration increases in leaves with water stress. this phenomenon has been demonstrated in festuca arundinacea scherb. (salehi and salehi, 2012). many researches evaluated the physiological adaption or the functional and qualitative response of different cool and warm season grasses to increasing water deficits (qian and engelke, 1999; bastug and buyuktas, 2003; fu et al., 2004). althought all of these studies report that during the drought period, warm-season grasses are more tolerant to drought sress than cool-season grasses, different climatic conditions will influence relative drought tolerance of cool-season and warm-season turfgrass, necessitating regional evaluations. the main goals of this study were to compare the physiological and biochemical responses of cool-season turfgrasses (tall fescue, kentucky bluegrass, perennial ryegrass) and warmseason turfgrasses (bermudagrass) under drought stress condition in isfahan, iran. 2. materials and methods plant materials and experimental: this reserch was performed during 2015 to 2016 in department of horticultural at isfahan university of technology, isfahan, iran under greenhouse conditions (32°39’ n, 51°40’ e). polyvinyl chloride (pvc) pots (20 cm in diam., 20 cm long) filled with sterilized silt-loam soil, which collected from the isfahan’s landscape. for this experiment seeds of festuca arundinacea. ʻastrixʼ, lolium perenne. ʻnumanʼ and poa pratensis. ʻmiracleʼ were sown and rooted rhizomes of cynodon dacty‐ lon. ʻtifwayʼ were planted in 48 pvc pots. irrigation was applied as needed to prevent any visible drought stress during grass establishment. in general, turfs were watered three times weekly to maintain plants under well-watered condition and soil moisture at field capacity. plants were maintained at a cutting height of 5 cm and moved once a week using a reeltype mower. a fertilizer (urea) was applied at 5 g.m-2 rates once every two weeks to provide nutrients and to facilitate plant establishment before initiation of treatments. drought stress treatment and experiment design this study was carried out as a factorial experiment based on randomized complete block design (rcbd), with two treatments consisted of four levels of drought stress (100%, 75%, 50%, and 25% field capacity (fc) ), four turfgrass species (tall fescue, bermudagrass, kentucky bluegrass, and perennial ryegrass), with three replications and 16 pots were used for each replication (numbers of pots= 48). the fc was determined by the gravimetric method, which consists on the difference between the wet soil after saturation and free drainage, and the weight of the dry soil (cleide de souza et al., 2000). the soil water content was kept at gravimetric water capacity (measured 100%, 75%, 50% and 25% fc were 22.3%, 16.72%, 11.15% and 5.57% respectively) by adding tap water. drought stress was applied for two months and at the end of the experiment, physiological traits were measured. chlorophyll content leaf samples were selected randomly from the plants and homogenized in a mortar in 10 ml of 100% acetone. the extract was centrifuged at 2000 rpm for 10 min. absorbance of the supernatant was recorded at 663, 645 and 450 nm spectrophotometrically. chlorophyll (chl) content was determined following the method of (lichtenthaler, 1987). electrolyte leakage: leaf electrolyte leakage which is used to assess membrane permeability leakage was assayed base on lu et al. (2008) methods. leaf samples (0.1 g) was placed into a vial with 20 ml of double distilled water. after incubating the samples at room temperature on a shaker (150 g) for 24h, the gholamian jazi et al. ‐ physiological response of four turfgrass species to drought stress 383 electrical conductivity (ec) of the bathing solution (ec 1 ) was determined. the same samples were then placed in water bath at 100°c for 1h and a second reading (ec 2 ) was determined after cooling solution to room temperature. the electrolyte leakage was calculated as ec1/ec2 and expressed as percent. relative water content we determined relative water content (rwc) according to the method developed by ghoulam et al. (2002). about 0.2 g of the fresh leaf sample was cut into smaller pieces and weighed (w1). then the leaf samples were saturated in 100-ml deionized water for 24 h at 4°c and weighed to determine the turgid weight (w2). finally leaf samples were dried at 70°c for 24 h, and the dry weight was recorded (w3). rwc was determined using the following equation: rwc (%) = (fw-dw) / (tw-dw) ×100. where fw, dw, and tw are fresh, dry and turgid weights respectively. proline content proline content measurement was carried out according to a previously described method (bates et al., 1973). leaves were homogenized in 3% aqueous sulphosalicylic acid, then centrifuged 5,000 g for 20 min at 4°c. 2 ml of this homogeny solution react acid-ninhdrin and 2 ml of glacial acetic acid in a tube for 1 hour at 100°c and the reaction is torn up in an ice bath and then extracted with 4 ml of toluene. it was kept at room temperature to stabilize. proline content was measured by spectrophotometer (uv160a, shimadzu, tokyo, japan) at 520 nm (bates et al., 1973). malondialdehyde in order to determine the content of malondialdehyde (mda) in the leaves, 0.1 g of leaf tissues was homogenized in 5 ml of 0.1% (w/v) trichloroacetic acid (tca) for 10 minutes and then was centrifuged at 5,000 g. 1 ml of conventional solution was mixed with 4 ml of thiobarbituric acid (tba) (0.5% of tba in 20%). then the reaction mixture was placed in a hot bath at 100°c for 15 minutes. finally the mixture centrifuged at 5,000 g for 10 minutes and the amount of mda was subsequently read by the spectrophotometer at 450, 532 and 600 nm (wang et al., 2008). enzyme assay for enzyme extraction, 0.1 g leaf powder was extracted with 1 ml of sodium phosphates extraction buffer and triton. the extractions were centrifuged at 12000 × g for 30 min at 4°c, and supernatant was collected for enzyme assay. the supernatant was used as a source of sod enzyme. sod was measured by a photochemical method (giannopolitis and ries, 1977). the reaction mixture (3 ml) contained 0.1 mm edta (ethylenediamine tetra acetate) 0.05 ml hepeskoh buffer (ph=7.8), 50 mm na 2 co 3 , 13 mm methionine, 63 μm nbt (nitro blue tetrazolium) 0.05 ml enzyme extract and 1.3 μm riboflavin. the absorbance was read at 560 nm and one unit of sod activity was defined as the amount of enzyme causing 50% inhibition of photochemical reduction of nbt. cat (ec: 1.11.1.6) activity was assayed in a reaction mixture containing 100 mm phosphate buffer (ph 7.0), 15 mm h 2 o 2 and enzyme 0.05 ml of aliquot. the decomposition of h 2 o 2 was followed at 240 nm (aebi, 1984). the catalase (cat) activity is defined in international unit equals (1 unit) as the amount of catalase necessary to decompose 1 μm of h 2 o 2 per minute. activity of peroxides (pod) was determined in a reaction mixture (2.95 ml), which consisted of 75 mm suitable amount of guaiacol, 15 mm h 2 o 2 , 100 mm phosphate buffer and 0.05 ml of enzyme extract. the absorbance of the supernatant at 470 nm was measured (maehly, 2006). one unit of pod activity was defined as the amount of enzyme necessary to decompose 1 μm of h 2 o 2 per minute. statistical analyses statistical analysis system (sas 9.1) was used for variance analysis and the differences between treatment means were assessed by the least significance difference (lsd) at p= 0.05 probability level. 3. results chlorophyll content results from leaf chlorophyll content measurements showed a significant difference between water stress treatments, species and interaction effects (p≤ 0.01) (table 1). water stress negatively influenced the chl in all species. chl content significantly (p<0.05) decreased under water stress condition compared to the well-watered treatment (fig. 1). as shown in figure 1, lolium perenne in 25% fc showed the lowest chlorophyll content (12.6 mg/g fw) and chlorophyll content was higher at 100% fc in poa pratensis (44.24 mg/g fw) compare to other species (fig. 1). electrolyte leakage el significantly decreased under water stress treatments compared to 100% fc level. the highest el was manifested under 25% fc level (table 2). the adv. hort. sci., 2019 33(3): 381-390 384 el reached the peak (95.4%) in p. pratensis, and the lowest el (89.3%) was recorded in the festuca arun‐ dinacea (table 2). interaction of drought stress and species showed no significant effect on electrolyte leakage (table 1). relative water content the results showed that water stress, species, and their interaction had the significant effect on rwc (p ≤0.01) (table 1). as shown in figure 2, all species showed high value of rwc under 100% fc. percentage reduction of rwc under 25% fc, were 43.2, 43.9, 51.1 and 59.6 for tall fescue, bermudagrass, perennial ryegrass and kentucky bluegrass respectively, as compared with well-watered control plants (fig. 2). proline content water stress and species both showed significant effect on proline content (p≤0.01) and their interaction effects were significant at 5% level (table 1). as shown in figure 3, proline concentrations in four species were all increased under drought stress which indicates osmotic adjustment in turfgrasses. f. arun‐ dinacea at 25% fc level showed the highest proline content (with 0.3 μmol/g fw) whereas lower proline content was shown for well-watered lolium perenne, table 1 analysis of variance of chl (chlorophyll content), el (electrolyte leakage), rwc (relative water content), pr (proline content), mda (malondialdehyde), cat (catalase), pod (peroxidase), and sod (superoxide dismutase) activities of turfgrasses species under drought stress ns= not significant, *p≤0.05, **p≤0.01. fig. 1 interaction effects of water stress and species on chlorophyll content. vertical bars (mean ± 1.37) not connected with the same letter represent significant difference between treatments according to lsd test (p˂0.05). table 2 interaction effects of water stress and species and mean comparison on electrolyte leakage the values represent the mean ± standard error of three replicates. different letters are showing considerable differents at p≤0.05. effect mean df chl el rwc pr mda cat pod sod block 2 15.00 * 54.31 ** 3.93 ns 0.0001 ns 0.01 ns 0.003 ns 0.01 ns 0.34 ns species 3 834.1 ** 16.08 ** 354.15 ** 0.003 ** 2.09 ** 0.07 ** 34.89 ** 21.18 ** drought 3 353.86 ** 29.72 ** 4618.81 ** 0.115 ** 6.70 ** 0.16 ** 60.59 ** 59.101 ** drought × species 9 29.38 ** 2.61 ns 22.33 ** 0.0007 * 0.38 ** 0.01 ** 4.70 ** 8.76 ** error 30 2.84 1.44 889.86 0.0003 0.02 0.002 0.02 0.86 coefficient of variation 7.42 1.28 2.33 10.89 9.4 15.45 3.2 9.94 species drought stress treatment electrolyte leakage mean festuca arundinacea 100% fc 89.31 h 93.08 b 75% fc 92.95 defg 50% fc 94.41 bcde 25% fc 95.64 ab lolium perenne 100% fc 92.10 fg 93.33 b 75% fc 92.59 efg 50% fc 93.74 bcdef 25% fc 94.89 bcd poa pratensis 100% fc 94.66 bcd 95.40 a 75% fc 94.17 bcde 50% fc 95.42 abc 25% fc 97.34 a cynodon dactylon 100% fc 91.48 g 92.92 b 75% fc 92.40 efg 50% fc 93.53 cdef 25% fc 94.26 bcde gholamian jazi et al. ‐ phsyiological response of four turfgrass species to drought stress 385 100% fc (fig. 5). also as shown in figure 6, the highest pod activity was obtained under 50% fc in f. arun‐ dinacea while the lowest activity was obtained for poa pratensis at 100% fc level., on the whole, pod activities of for species followed a similar pattern under drought stress, which was characterized by a gradual increase until 50% fc level followed by a decline after this treatment (fig. 6). moreover, it poa pratensis and cynodon dactylon (fig. 3). malondialdehyde according to the results, the mda content were significantly affected by water stress level, grass species, and their interaction effects (p≤0.01) (table 1). as the water stress increased, a clear increase in the mda content has been seen in all species (fig. 4). as shown in the figure 4, the highest (3.6 μmol g-1 fw) and lowest (0.44 μmol g-1fw) amounts of mda were obtained in p. pratensis grown under severe water stress (25% fc) and f. arundinacea under control treatment, respectively (fig. 4). enzyme the results showed that water stress, species, and interaction effects had significant effect on catalase, peroxidase and superoxide dismutase (p≤0.01) (table 1). as shown in the figure 5, the maximum cat activity belonged to f. arundinacea at 25% fc whereas the minimum cat activity belonged to c. dactylon at fig. 2 interaction effects of water stress and species on relative water content (rwc). vertical bars (mean±1.96) not connected with the same letter represent the significant difference between treatments according to lsd test (p˂0.05). fig. 3 interaction effects of water stress and species on proline. vertical bars (mean ± 0.01) not connected with the same letter represent significant difference between treatments according to lsd test (p˂0.05). fig. 4 interaction effects of water stress and species on malondialdehyde. vertical bars (mean ± 0.11) not connected with the same letter represent significant difference between treatments according to lsd test (p˂0.05). fig. 5 interaction effects of water stress and species on catalase. vertical bars (mean ± 0.04) not connected with the same letter represent significant difference between treatments according to lsd test (p˂0.05). fig. 6 interaction effects of water stress and species on peroxidase. vertical bars (mean ± 0.12) not connected with the same letter represent significant difference between treatments according to lsd test (p˂0.05). 386 adv. hort. sci., 2019 33(3): 381-390 could be obviously seen that sod activities followed the similar pattern as pod; applying 50% fc in f. arundinacea and 100% fc in l. perenne scored the highest (15 umg-1protein) and the lowest (2.5 umg1protein) respectively (fig. 7). traits correlation electrolyte leakage with proline, mda and cat showed the significant positive correlation (p≤0.01). our results showed the significant negative correlation between rwc and el (p≤0.01). proline was positively correlated with mda, cat, sod, and pod (p ≤ 0.01). also, it showed the negative correlation between rwc and chlorophyll at p≤0.01 (table 3). our results showed a positive correlation between mda and cat (p≤0.01) (table 3). sod showed significant positive correlation with cat and pod (p≤0.01). 4. discussion and conclusions in the present study, increasing in water deficit resulted in an decrease in the plant chl content compared with the plants under well-watered conditions. chlorophyll content (chl) in live plants is an important factor in determination of photosynthetic capacity. in the present study, leaf chl content decreased gradually during the stress periods (fig. 1). decreased or unchanged chl content level during drought stress has been reported in other cultivars, depending on drought duration and severity (zhang and kirkham, 1996; jagtap et al., 1998). under the water deficit stress, chloroplast ultra-structures are the first target to be damaged at the cellular levels since it is the major site of reactive oxygen species (ros) production (munné-bosch and peñuelas, 2003). an enriched ros in stressed tissues impairs cellular membrane and organelles which affects the integrity of the cell. in our study, electrolyte leakage gradually increased with increasing water stress in turfgrass species. increase of el with development of drought stress has been reported by many researchers (guo et al., 2006; liu et al., 2008). electrolyte leakage increase is occurred with the increase of cell permeability (blum and ebercon, 1981). results of a study by abraham et al. (2004) showed at higher electrolyte leakage in poa pratensis and its hybrids under drought stress, while low levels of electrolyte leakage (an indicator of cell membrane stability) were observed in the drought-tolerant plants during drought stress. fu and huang (2001) reported that el and mda increased simultaneously in five species of kentucky bluegrass which indicated the positive relationship between these traits in this research, drought stress conditions decresed rwc due to reduced leaf water potential, consistent with results reported by fu and huang (2001), farkhondeh et al. (2012). rwc as an indicator of plant water status is one of the most reliable indicators for defining water retention in plants. in this study, rwc was affected significantly by water stress fig. 7 interaction effects of water stress and species on superoxide dismutase. vertical bars (mean ± 0.75) not connected with the same letter represent significant difference between treatments according to lsd test (p˂ 0.05). table 3 pearson correlation coefficients of electrolyte leakage, proline, mda (malondialdehyde), sod (superoxide dismutase), cat (catalase), chlorophyll, and pod (peroxidase) of turfgrasses species under drought stress traits electrolyte leakage proline mda sod rwc cat chlorophyll pod electrolyte leakage 1 proline 0.454 ** 1 mda 0.59 6** 0.672 ** 1 sod 0.258 ns 0.575 ** 0.354 ** 1 rwc -0.576** -0.855 * -0.890 ** -0.374 ** 1 cat 0.422 ** 0.760 ** 0.510 ** 0.491 ** -0.587 ** 1 chlorophyll -0.029 ns -0.475 ** -0.242 ns -0.308 * 0.455 ** -0.205 ns 1 pod 0.273 ns 0.627 ** 0.311 ** 0.690 ** 0.364 ** 0.694 ** -0.274 ns 1 ns= not significant, *p≤0.05, **p≤0.01. gholamian jazi et al. ‐ physiological response of four turfgrass species to drought stress 387 in all species. however, reduction was less pronounced for f. arundinacea, which maintained a higher rwc than other species. the amount of rwc in plant with high resistance to drought stress is higher than that of susceptible plants. in other words, plant having higher yields under drought stress should have higher rwc (liu et al., 2002). under water deficit, the cell membrane is subjected to changes such as penetrability and decrease in sustainability (blokhina et al., 2003). results of a study by wang and huang (2003) showed a decline in the rwc under drought stress, especially in; susceptible cultivars (wang and huang, 2003). bian and jiang (2009) showed that rwc in p. pratensis decreaed during drought stress. the initial physiological response of plants to drought stress is osmoregulation which decreases water potential and maintains turgor to hold water inside of tissues and absorbing moisture from the environment at the same time, thus finally maintain other physiological activities of the cell (li et al., 2015). the results of the present study clearly showed that proline content increased in all turfgrass species under water deficit compared to the wellwatered conditions. increase of proline under drought stress has been reported by many researchers (turkan et al., 2005; wang et al., 2008). proline content of f. arundinacea under drought stress increased dramatically, which could be a significant factor for maintaining the relative water content. the correlation between proline and antioxidant enzymes had been reported by morot-gaudry et al. (2001). since proline can act as a scavenger or reducer of superoxide production, it is normal to find a significant positive correlation seems between proline and antioxidant enzymes. bian et al. (2009) concluded that proline content increased in creeping bentgrass (agrostis stolonifera l.) under drought stress. lipid peroxidation has been associated with damages provoked by some environmental stresses (jaleel et al., 2008). the rise in mda content under different stress conditions showed that drought could induce membrane lipid peroxidation by means of ros (moussa and abdel-aziz, 2008). in this condition, low concentration of mda has been associated with drought-tolerant plants (dacosta and huang, 2007; hassan et al., 2015). in our experiment, f. arundinacea, with low concentration of mda in different levels of drought stress treatment, showed more tolerance to drought stress. according to other studies (e.g. sharma and dubey, 2005; pan et al., 2006; zlatev et al., 2006), drought stress increased mda concentrations in leaves. positive correlation between mda and cat indicates that the antioxidant enzymes act as a first defensive line to counter oxidative stress in plants. oxidative stress occurs when the antioxidant defense decreases or the formation of free oxygen radicals increases (matés et al., 1999). to cope with detrimental effects of oxidative stresses under extremely adverse conditions, plants have developed an antioxidant defense system which includes the antioxidant enzymes sod, apx, pod, and cat. the levels of antioxidant enzymes are higher in tolerant cultivars than sensitive ones under various environmental stresses (wang et al., 2009). accordingly, we observed higher sod activity in f. arundinacea at 50% fc level compared to other species, which suggest that this drought-tolerant grass possess a better reactive oxygen scavenging ability. however, in the 25% fc this trend changed considerably and the amount of sod decreased. previous studies have shown that responses of sod activity to water deficit have varied with drought severity, duration, and species. zhang and kirkham (1996) suggested that water stress did not influence sod activity under moderate stress in sorghum [sorghum bicolor (l.) moench]. in wheat (triticum aestivum l.), sod activity increased or remained unchanged in the early phase of drought and then decreased with further water stress (zhang et al., 1995). this reduction in sod activity could be associated with reduced synthesis or enhanced degradation of the enzyme. sod converts the toxic o 2 radicals to h 2 o 2 which must be scavenged to o 2 and water by the antioxidant enzyme such as cat, pod, and apx (ozkur et al., 2009). increase in pod activity under various stress conditions has been linked with protection from oxidative damage, lignification, and cross-linking of the cell wall to cope with such adverse conditions (moussa and abdel-aziz, 2008). in our study, drought-induced pod activity in shoot of four species. however, activity of this enzyme in f. arundinacea under both control and stress conditions was higher than others species, suggesting a better antioxidant system for removing h2o2 by pod. in kentucky bluegrasss this enzyme activity initially increased and then decreased with development of drought (fu and huang, 2001), similar to changes of this enzyme,s activity founding p. pratensis in the response to drought stress. the activity of pod increased during initial periods of drought stress and decreased as stress intensity increased. the similar trend of pod activity during water stress has been reported in poa pratensis adv. hort. sci., 2019 33(3): 381-390 388 (farkhondeh et al., 2012). catalase is another antioxidant enzyme that scavenges h2o2 in cells (shao et al., 2007). high activity of cat indicated drought tolerance in chrismas tree (sharma and dubey, 2005) and wheat (simovastoilova et al., 2010). fu and huang (2001) reported that cat activity was decreased in all studied poa pratensis and f. arundinacea cultivars under water stress condition. accordingly, they concluded that the reduction of cat activity was supposedly due to inhibition of enzyme synthesis, change in the inhibition of enzyme precursor, or protein degradation under drought stress. in the present research, cat activity in f. arundinacea was higher than other species in different levels of water stress treatment. the high activity of cat in f. arundinacea during drought stresses demonstrated more ability of these species to decomposition of h2o2 in stress condition. cat showed the positive correlation with pod. the correlation between antioxidant enzymes reported in mature leaves of arabidopsis under drought stress (jung, 2004). mercado et al. (2004) reported the significant positive correlation between pod activity and rwc content which are in agreement with our results. according to the results presented here, it can be concluded that among the evaluated species, tall fescue has good tolerance to drought stress. the higher tolerance induced by tall fescue was associated with more efficient osmotic adjustment, which was reflected by the smaller reduction in rwc and cell membrane stability. the identification of these indices is valuable because they can be rapidly assessed and can be used in the early stages of breeding turfgrasses for screening drought tolerant species; however, for efficient selection and better understanding of the mechanisms involved in drought tolerance, biochemical and molecular markers must also be included. acknowledgements this work was supported by isfahan university of technology. authors would like to thank the research section and laboratory of the university for their help. references abraham 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33(1): 67-75 doi: 10.13128/ahs-23043 influence of post-véraison water deficit on berries yield and quality of three table grape cultivars m. ashori 1, m. ghasemnezhad 2 (*), m.h. biglouei 3 1 department of horticulture, university campus 2, university of guilan, rasht, iran. 2 department of horticulture, faculty of agriculture, university of guilan, rasht, iran. 3 department of water engineering, faculty of agriculture, university of guilan, rasht, iran. key words: antioxidant activity, anthocyanin, drought, total phenol, water requirement. abstract: in order to investigate the effect of regulated deficient irrigation (rdi) on some quantitative and qualitative characteristics of the three commercial grapevine cultivars (i.e. keshmeshi, sahebi and sharabi), an experiment was set in split plot arranged in an rcbd design with three replications. in this experiment, irrigation treatments including 100 (as control), 80%, 60% and 40% crop evapotranspiration (etc) were implemented in a period between onset of berries color change (véraison) to harvest of fruits. at the end of experimental period, some traits such as berry weight, berry length, berry diameter and cluster weight as well as some fruit quality traits such as total soluble solids (tss), titratable acidity (ta), flavonoids, anthocyanin, total phenol, and total antioxidant capacity were measured. the results of this experiment revealed that effect of rdi at different levels on yield, berry and cluster weight, berry length and diameter, tss, ta, anthocyanin, phenol, and flavonoid was significant. also, there was no significant difference between control and 80% etc in terms of yield, berry diameter, tss, and ta; and these treatment enhanced total phenol, anthocyanin and antioxidant activity in the three cultivars. in both years of experiment, rdi remarkably enhanced anthocyanin, flavonoids, antioxidant activity and phenolic concentration. overall, the results indicated that 80% etc might be sufficient to gain adequate yield in ‘keshmeshi’, ‘sahebi’ and ‘sharabi’ without undermining the quality of fruit. 1. introduction water deficiency is one of limiting factors influencing grape production throughout the world (rabiei et al., 2003). in order to improve water use efficiency (wue), it is necessary to recognize factors restricting wue and then introduce appropriate approaches for mitigating the harmful effects of such factors (mccarthy et al., 2002). in this respect, balancing (*) corresponding author: ghasemnezhad@guilan.ac.ir citation: ashori m., ghasemnezhad m., biglouei m.h., 2019 influence of post-véraison water deficit on berries yield and quality of three table grape cultivars. adv. hort. sci., 33(1): 67-75 copyright: © 2019 ashori m., ghasemnezhad m., biglouei m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 april 2018 accepted for publication 17 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 67-75 68 between vegetative and reproductive phases in plants in favor of hindering excess vegetative growth is one of these approaches being used to improve not only wue but also fruit quality of grapevine, because excess growth of shoots may have adverse impact on fruit quality (chalmers et al., 1981). reduction in vegetative growth in favor of improving wue can be implemented by many approaches. one of such approaches is to constrict the amount of water required for plant growth through reducing the number of irrigation or decline in amount of water in each irrigation (chalmers et al., 1981; goodwin and jerie, 1992). for achieving this purpose, regulated deficit irrigation (rdi) was introduced (santesteban and mirandam royo, 2011). as more than 82% of iran territory is located in arid and semiarid zone with drought climate and extreme temperature changes in the world (amiri and eslamian, 2010), this approach may pave the way for planting crop in a vast majority of abandoned farmland deprived of adequate water distribution, although employing this approach may reduce crop production because of reduction in water use (chalmers et al., 1981). under drought condition, rdi abridges vegetative growth in favor of enhancing reproductive growth and this could improve yield (santesteban and mirandam royo, 2011). at critical stages of plant growth and development, change in water status certainly influences fruit quality by affecting vegetative growth and fruit development (van leeuwen et al., 2004; ezzhaouani et al., 2007). therefore, rdi improves cluster quality through reduction in berry drop (ojeda et al., 2002). under a mild drought stress, acevedo-opazo et al. (2010) proposed an appropriate threshold for managing rdi schedule to keep quality of grape fruits. the positive rdi effect on phenolic compounds, tss, and anthocyanins has previously been documented (ojeda et al., 2002; van leeuwen et al., 2004). the highest yield (48 kg a tree) with high fruit quality was observed in seedless grape cultivars experiencing rdi under drought condition (faci et al., 2014). according to the results obtained by zabihi and azarpajouh (2004) and dolatibaneh and norjo (2012), rdi probably decrease photosynthetic capability in leaves of grape; and as a result, it reduces (fruit) yield. in other research, it was shown that rdi increased berry quality and increase water use efficiency in grape trees pinillos et al. (2016). rabiei et al. (2003) pointed out that using rdi at the end of growth season significantly increased phenolic compounds in merlot cultivar. with respect to mentioned issues, the current experiment was designed to investigate effects of different rdi treatments on yield and fruit quality of the three grape cultivars (i.e. keshmeshi, sahebi and sharabi) during the onset of véraison to harvest under climate of khorramabad province, iran. 2. materials and methods vineyard site and experimental design this experiment was conducted during the 2015 and 2016 growing seasons at semiarid climate in lorestan province, iran (47°e, 51°n, 1400 m). ownrooted vitis vinifera l. cvs. keshmeshi, sahebi and sharabi were planted in 2007 by spacing of 3×2 m in n-s oriented row. vines were trained to spur-pruned bilateral cordon with 6, 4 and 4 nodes per vine in ‘keshmeshi’, ‘sahebi’ and ‘sharabi’, respectively according to flowering type. the vineyard was dripirrigated using pressure-compensated emitters (flow rate 4 lh-1). the vines were fertigated from june with a nutrient compound containing 150 kg ha-1 n, 25 kg ha-1 k, 6 l ha-1 s and 4 kg ha-1 fe chelate. three rdi strategies were compared with the full irrigation practice (control). specifically, the control received full irrigation over fruit growth and development (etc 100), whereas rdi treatments were: 80 % of etc, 60% of etc and 40% of etc. rdi treatments were applied at the onset of berries color change (véraison) to harvest time. daily metrological data were obtained from a database of khoramabad’s meteorology station located closely to the experimental vineyard and water requirement for each vine was calculated according to cropwat 8.0 software. furthermore, irrigation scheduling was based on soil water holding capacity, daily crop water requirement, effective depth of root, percentage of wetted soil surface according to defined treatments (100%, 80%, 60% and 40% etc) using the following relations. where in= net irrigation water content (mm), fc= field capacity (%), pwp= permanent wilting point (%), pb= soil bulk density (gr.cm-3), dr= effective depth of root (120 cm) i= irrigation interval (day), k= operation coefficient of rdi treatments, etc= plant ashori et al. post-veraison water deficit on table grape cultivars 69 water requirement. all vines were equally irrigated based on their water requirement before starting rdi treatment. in this study, the drip irrigation system was equipped with two droplets adjusted manually for 4 lh-1 per vine. in order to access required pressure, a floating pump was used and the amount of water allocated to each irrigating turn was measured by water counter with 0.1 l accuracy. directing water into each plot containing three grapevines was performed through stopcock water equipped in drip irrigation. for each irrigation treatment, three replications were established according to a randomized block design. each replication consisted of five lines of 60 vines each, and the experimental measurements were performed on 20 homogeneous vines from the 3 central lines chosen at the beginning of the study according to their trunk cross sectional area (tcsa). sampling and analysis the berries were harvesting from 10 vines per each treatment and three samples for each vine, based on minimum maturity index of sugar content and berry color change. the mean total yield, clusters weight, number of berry per cluster and berry weight was measured from 10 vines for each treatment and three samples for vines. soluble solid content (ssc) was measured by a digital refractometer (model eurromex rd). titratable acidity (ta) was measured with a digital titrator in presence of 0.1 naoh and ph was determined by a digital ph meter (model sclto tt). total phenolic was determined by using the folinciocalteau method (brand-williams et al., 1995) with slight modification in a uv-vis spectrophotometer (t80+, pg instruments ltd). briefly, 0.5 g of extract was mixed with 3 ml of methanol 85%, and then centrifuged for 10 min by 10000 per min. then, 300 µl of diluted extract was mixed with 1.5 ml of diluted folin-ciocalteau reagent (1:10 in distilled water). after vortexing, 1.2 ml of 7.5% sodium carbonate was added and allowed to stand for 90 min at room temperature. the absorption at 760 nm was measured and results were expressed as gallic acid equivalents (gae) per 100 g fresh weight. total flavonoids content were determined spectrophotometerically according to the zarrouk et al. (2012). catechin was used as a standard. the flavonoids content were expressed as mg catechin equivalents (re) per 100 g fw. total antioxidant capacity (tac) was measured using the 2, 2 diphenyl-1-pic-rylhydrazyl (dpph) radical scavenging method described by brand-williams et al. (1995) with slight modification. the amount of 75 μl of fruit extract was mixed with 2925 μl 0.1 mol l-1 dpph in methanol. after incubating at room temperature for 30 min in the dark, the absorbance of the mixture was measured at 517 nm using uv-vis spectrophotometer. for each sample, three separate determinations were recorded. antioxidant activity was expressed as the percentage decline in absorbance in comparison with the control, corresponding to the percentage of dpph scavenged (% dpphsc), which was calculated as: %dpphsc = [(acontrol-asample)/acontrol]× 100 data analysis the results obtained for each irrigation treatment were compared using anova and when significant differences were found, duncan’s test was used to identify differences in mean values. data from each year were analyzed separately; all analyses were performed with sas 17.0. 3. results and discussion fruit yield the results of anova showed that simple effect of rdi on total yield at first year was significant (p<0.01). compared to controls, 80% etc did not show a significant effect on yield at first year, whereas applying 40% and 60% etc reduced fruit yield by 33% and 48%, respectively. also, it was found that sahebi in comparison to sharabi gained higher fruit yield at first year (table 1). the results of this experiment showed that the interaction effect of cultivar × rdi had a significant effect on yield in second year of experiment (fig. 1). in ‘keshmeshi’ and ‘sharabi’, using 40% etc significantly resulted in a yield reduction, while 40% and 60% etc decreased fruit yield in sahebi. santesteban and mirandam royo (2011) and di vaio et al. (2001) showed that rdi brought about a reduction in grape yield, although they proposed rdi as a useful approach to enhance quality and quantity of fruits in the grapes grown especially in subtropical climate. on the contrary, dolatibane and norjo (2012) and zabihi and azarpajouh (2004) stated that rdi could reduce grape yield. the negative effect of rdi on fruit yield is associated with its harmful effect on photosynthesis activities and consequently supplying required assimilates for berry’s formation adv. hort. sci., 2019 33(1): 67-75 70 (conesa et al., 2016). also, a remarkable increase in transpiration rate of berries under severe drought stress can be accounted for losing berries weight and lowering grape yield. conesa et al. (2016) reported that rdi had not a significant effect on grape yield and rdi-experiencing plants had the same yield as controls. in this study, we also didn’t find any significant differences between 80% rdi and control during two consecutive growing seasons. berry weight and cluster weight the results of anova showed that berry weight was significantly affected by simple effects of cultivar and rdi (p<0.01). the interactional effect of cultivar × rdi had a significant effect on cluster weight in both years of experiment (p<0.01). in general, the weight of grape berry and cluster was significantly decreased as the percentage of rdi was gradually decreased (from 100% to 40% etc) in both experimental years. comparing to controls, although applying 40% and 60% etc reduced cluster weight of ‘keshmeshi’ at first year, all rdi treatments did not render a reduction in cluster weight of keshmeshi in second year of experiment. in ‘sahebi’, cluster weight was diminished by applying 40% and 60% etc at first year, whereas by just 40% etc in second year of experiment. in this regard, all rdi treatments reduced berry and cluster weight of sharabi while 80% etc did not significantly reduce cluster weight in sharabi only in second year (fig. 2). the results of this research revealed that effect of rdi on berry and cluster weight is strongly correlated to cultivars. according to coombe (1992), change in berry’s water status under drought is directly related with change in berry weight. rabiei et al. (2003) figured out that rdi may be responsible for a reduction in berry weight in merlot cultivar. in this regard, dolatibaneh and norjo (2012) stated that the highest berry weight was obtained at full irrigation; and, in spite of finding non-significant difference between 50% and 75% etc treatments, they concluded that berry weight is often depressed under rdi performance. as compared to controls, the cell size of fruit experienced different rdi regimes reduced due to reduction in vegetative growth, leaf area, and photosynfig. 1 effect of regulated deficit irrigation (rdi) on fruit yield of three grape cultivars in the second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. season treatment levels yield (kg vine-1) berry weight (g) ssc (%) ta (%) ph 2015 rdi control 23.70 a 1.11 a 17.55 a 0.68 a 3.51 a 80% etc 21.75 a 1.08 a 17.18 a 0.69 a 3.40 a 60% etc 15.90 b 0.89 ab 17.24 a 0.72 a 3.48 a 40% etc 12.24 d 0.72 b 17.12 a 0.66 a 3.48 a significance ** ** ns ns ns cultivar keshmeshi 19.47 ab 0.60 b 19.84 a 0.78 a 3.35 b sahebi 22.77 a 1.28 a 14.15 b 0.6 2b 3.59 a sharabi 15.20 b 0.98 a 17.83 a 0.67 b 3.46 ab significance ** ** ** ** ** 2016 rdi control 24.40 a 0.96 a 20.25 a 0.66 a 3.50 a 80% etc 23.56 ab 0.92 a 21.31 a 0.64 a 3.47 a 60% etc 21.82 b 0.78 b 20.33 a 0.68 a 3.45 a 40% etc 18.71 c 0.62 c 21.67 a 0.67 a 3.42 a significance ** ** ns ns ns cultivar keshmeshi 17.31 b 0.57 b 24.50 a 0.75 a 3.37 b sahebi 29.02 a 0.99 a 17.95 c 0.59 b 3.59 a sharabi 20.04 b 0.90 a 20.22 b 0.65 ab 3.41 b significance ** ** ** ** ** table 1 the effects of regulated deficit irrigation (rdi) on fruit yield and quality of grape cultivars ns= not significant, ** significant at p<0.01, * significant at p<0.05. ashori et al. post-veraison water deficit on table grape cultivars 71 thesis products, and this probably led to reduction in berry and cluster weight (ezzhaouani et al., 2007). in current research, the grape cultivars had different responses to rdi especially at 40% and 60% etc regimes. regarding these results, different responses to rdi treatments suggests the sensitivity of different cultivars to drought stresses. berry length and diameter the data presented in figure 3 showed a significant interaction effect of cultivar × rdi on traits of berry length and diameter. in both years of experiment, 40% etc reduced berry length and diameter, whereas 80% etc did not have a significant effect on both of them. accordingly, the cultivar response to rdi was found different (fig. 3). under rdi performance, the cell size was lower than that of at full irrigation. due to reduction in the vegetative growth and leaf area of rdi-subjected plants, the size of berry and photosynthetic products were reduced (deluc et al., 2009). zabihi and azarpajouh (2004) reported that water deficiency resulted in reduction in the size of berry, which is in agreement with our findings. total soluble solid the results of this research revealed that only simple effect of cultivar on tss was significant in both years of experiment, and the highest and lowest tss were found in ‘keshmeshi’ and ‘sahebi’, respectively (table 1). contrary to dolatibaneh and norjo (2012) who stated that rdi had a positive effect on tss of grape fruit, lanari et al. (2014) figured out a non-significant effect of rdi of tss in grape fruit. according to previous research, mild drought may stimulate aba production in favor of increasing some sugar production through different ways in order to cope with stressful condition. in this respect, some researchers pointed out that water deficiency could reduce photosynthesis activities and consequently constrict sugar production, but whenever water deficiency is exacerbated even fruits may not ripe properly and this probably affects tss status in fruits (dolatibaneh and norjo, 2012). titratabe acidity and ph the results of this experiment demonstrated that only cultivar treatment had a significant effect on ta at first year of experiment, and additionally sahebi and sharabi obtained the lower ta as compared to keshmeshi cultivar. contrary to the results obtained at first year of experiment (table 1), an interaction effect of cultivar × rdi had a significant effect on ta in second year of experiment. although, rdi did not have effect on ta in ‘keshmeshi’, rdi at 60% etc significantly increased ta in sahebi cultivar. in addition, applying 40% and 60% etc caused a reduction in ta of sharabi cultivar (fig. 4). in both years of experiment, ph was only affected by cultivar; and highest ph was observed in sahebi as compared to the two other cultivars. dolatibaneh and norjo (2012) pointed out that rdi could decline the rate of organic acid of grape fruit. during cluster ripping, coinciding with rising environmental temperature, drought stress is able to decrease acidity of berry and accordingly paves the way for minimizing the rate of ta in grape berries. lanari et al. (2014) reported that rdi did not have a significant effect on fig. 3 effect of regulated deficit irrigation (rdi) on rachis weight of three grape cultivars in two growing seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 2 effect of regulated deficit irrigation (rdi) on berry weight of three grape cultivars in the second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. 72 adv. hort. sci., 2019 33(1): 67-75 ta, whereas findings of zabihi and azarpajouh (2004) showed water availability increased organic acids in grape berries. total phenol the results of this experiment revealed that the interaction effect of cultivar × rdi had a significant impact on total phenol (fig. 5). comparing to controls, applying only 80% etc increased total phenol in ‘keshmeshi’ while applying all rdi treatments in sahebi enhanced total phenol in both years of experiment. in sahebi, the highest amount of phenol was found by employing 40% (at first year) and 80% (in second year) etc treatments. the results also revealed that using 40% and 60% etc (at first year) as well as 60% and 80% etc (in second year) had significantly affected total phenol in sharabi. rabiei et al. (2003) reported that applying rdi at the end of growth season significantly increased phenol content of merlot comparing to controls, which is in agreement with our findings. a prolonged drought stress was found to significantly decrease total phenol and other phenolic compounds such as frolic acid, coumaric acid, and caffeic acid (krol et al., 2014). by investigating effects of different rdi regimes on phenolic compounds in shirazi cultivar, ojeda et al. (2002) demonstrated that rdi, dependent on type of phenolic compounds, may change the amount of phenol in grape berries. in this regard, some factors including type of cultivar, climate, soil, media culture, maturely period, and yield are effective on the amount of phenols and other biocompounds in grapes (deluc et al., 2009; bindon et al., 2011; zarrouk et al., 2012). in our research, rdi treatments had different impacts on phenol; and the results of this research, dependent on type of cultivar, suggest that an appropriate rdi can increase total phenol in grape berries. flavonoids the results of this research revealed that flavonoid in berries was influenced by rdi treatments. in general, applying 60% and 80% etc significantly increased flavonoid in all the three cultivars. as compared to other cultivars, ‘keshmeshi’ was lowly affected by rdi, in a way that 60% etc at first year and 80% etc in second year of experiment significantly enhanced flavonoid content in berries. in contrast, applying 80% and 60% etc at first year and 40%, 60%, and 80% etc in second year significantly increased flavonoid content in sahebi. as compared to the second year of experiment, the rate of flavonoid in sharabi was significantly elevated by all rdi treatments at first year of experiment relative to controls (fig. 6). in grape berries, flavonoids are the abundant secondary metabolites influencing wine’s physical features (especially color and astringency) (brillante et al., 2017). the biosynthesis of such secondary metabolites is highly affected by environmental stresses (kuhn et al., 2013). the previous research has demonstrated that regulating vineyard irrigation is efficiently able to increase these metabolites (kennedy et al., 2002). similarly, findings of zarrouk et al. (2012) revealed that rdi significantly increased flavonoid compounds in grape’s aragonez cultivar in fig. 4 effect of regulated deficit irrigation (rdi) on titratable acidity percentage of three grape cultivars in second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 5 effect of regulated deficit irrigation (rdi) on total phenol of three grape cultivars during two seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. ashori et al. post-veraison water deficit on table grape cultivars 73 the two subsequent years, which are in consistent with ours. deluc et al. (2009) reported that effect of rdi on flavonoid content of chardonnay and cabernet sauvignon cultivars was different, in a way that its content in former cultivar increased while in latter one decreased. in current research, drought stress at 60% and 80% etc raised flavonoid content in the all grape cultivars and this manifests the positive effect of rdi on increasing grape quality. total antioxidant capacity the results of anova revealed that interactional effect of cultivar × rdi had a significant effect on total antioxidant capacity (tac) in both years of experiment (table 1). as compared to full irrigation, applying 60% and 80% etc at first year of experiment significantly increased tac in ‘keshmeshi’ and ‘sahebi’. in second year of experiment, all rdi treatments interestingly enhanced tac in sharabi relative to controls. moreover, tac was increased by using 60% and 80% etc in keshmeshi, as well as 60% and 80% etc in sahebi, and just 80% etc in sharabi (fig. 7). in a similar way, soukhtesaraee et al. (2017) figured out that drought stress significantly increased tac in chefta and yaghoti cultivars, but did not affect bidanehsefid. tangolar et al. (2015) showed that rdi increased tac at different growth stages of razaki cultivar. comparing to full irrigation, rdi depending upon some factors such as time of applying rdi and type of cultivar diminished tac in leaf and root of kiszmisz cultivar (krol et al., 2014). plant materials possessing antioxidant properties surely contain different phenolic compounds. the antioxidant properties of these materials, due to their redox ability and chemical structures are able to neutralize free radicals through forming a complex with metal ions (krol et al., 2014). in many previous research, the strong correlation between tac and phenolic content were reported (tangolar et al., 2015). in our research, a positive relationship was found between phenol and tac. anthocyanin the results of this research showed that the interaction effect of cultivar × rdi had a significant effect on anthocyanin content in both years of experiment (table 1). comparing to controls, all the three rdi treatments increased anthocyanin content in keshmeshi cultivar. in other words, applying 40%, 60%, and 80% etc at first year and 60% and 80% etc in second year increased anthocyanin contents in sahebi (fig. 8). anthocyanins are a class of phenolic compounds responsible for generating black and red colors in grape epidermis. these compounds possess powerful antioxidant property and also play a significant role in physiological process. according to previfig. 6 effect of regulated deficit irrigation (rdi) on flavonoid content of three grape cultivars on both seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 7 effect of regulated deficit irrigation (rdi) on antioxidant capacity of three grape cultivars on two seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. adv. hort. sci., 2019 33(1): 67-75 74 ous study, water shortage can strongly influence anthocyanin accumulation in grape cultivars (ozden et al., 2010; kyraleou et al., 2016). also, bindon et al. (2011) reported that an increase in time of irrigation could remarkably hinder anthocyanin biosynthesis. so far, the effect of drought stress on reduction in canopy’s density in favor of entrance more light radiation into tree canopy has been reported. through this, the light quantitatively required to anthocyanin biosynthesis is supplied (tangolar et al., 2015). previous research showed that rdi, through influencing physiological and hormonal responses, changed the pathway of anthocyanin biosynthesis (shellie, 2011; nelson et al., 2015). in present research, it was found that rdi significantly increased anthocyanin accumulation of iranian grape cultivars in both years of experiment, although the cultivar responses towards rdi were various. 4. conclusions overall, the results of this research revealed the positive effect of rdi (especially 80% etc) on yield and some fruit qualities in keshmeshi, sahebi, and sharabi cultivars. in this regard, rdi significantly increased not only yield but also some fruits’ qualitative features like anthocyanin, phenol, flavonoid, and total antioxidant capacity in comparison to controls. therefore, 80% etc can be advised to maintain and increase fruit qualities of keshmeshi, sahebi, and sharabi cultivars under regional conditions. references acevedo-opazo c., ortega-farias s., fuentes s., 2010 effects of grapevine (vitis vinifera l.) water status on water consumption, vegetative growth and grape quality: an irrigation scheduling application to achieve regulated deficit irrigation. agric. water manag., 97(7): 956-964. amiri m.j., eslamian s.s., 2010 investigation of climate change in iran. j. environ. sci. eng., 3(4): 208-216. bindon k., myburgh p., oberholster a., roux k., du toit c., 2011 response of grape and wine phenolic composition in vitis vinifera l. cv. merlot to variation in grapevine water status. afr. j. enol. vitiv., 32(1): 7188. brand-williams w., cuvelier m.e., berset c.l., 1995 use of a free radical method to evaluate antioxidant activity. lwt food sci. technol., 28(1): 25-30. brillante l., martinez-luscher j., yu r., plank c.m., sanchez l., bates t.l., kurtural s.k., 2017 assessing spatial variability of grape skin flavonoids at the vineyard scale based on plant water status mapping. j. agric. food chem., 65(26): 5255-5265. chalmers d.j., mitchell p.d., van heek l., 1981 control of peach tree growth and productivity by regulated water supply, tree density, and summer pruning (trickle irrigation). j. am. soc. hortic. sci., 106: 307-312. conesa m.r., falagán n., josé m., aguayo e., domingo r., pastor a.p., 2016 post-veraison deficit irrigation regimes enhance berry coloration and health-promoting bioactive compounds in ‘crimson seedless’ table grapes. agric. water manag., 163: 9-18. coombe b.g., 1992 research on development and ripening of the grape berry. am. j. enology vitic., 3(1): 101110. deluc l.g., quilici d.r., decendit a., grimplet j., wheatley m.d., schlauch k.a., cramer g.r., 2009 water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of cabernet sauvignon and chardonnay. bmc genomics, 10(1): 212. di vaio c., cirillo c., boselli m., masi e., 2001 dry matter accumulation and partitioning of cabernet sauvignon pot-grown vines under different water regimes. adv. hort. sci., 15(1-4): 25-30. dolatibaneh h., norjo a., 2012 effect of deficit irrigation on quantitative and quality traits of fruit and water productivity of three grapevine cultivars. seed plant prod. j., 27(4): 435-450. 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(in farsi). tangolar s.g., tangolar s., tarim g., kelebek h., topcu s., 2015 the effects of bud load and applied water amounts on the biochemical composition of the ‘narince’ grape variety. not. bot. hort. agrobot. cluj napoca, 43(2): 380-392. van leeuwen c., friant p., jaeck m.e., kuhn s., lavialle o., 2004 hierarchy of the role of climate, soil and cultivar in terroir effect can largely be explained by vine water status. joint int. conf. on viticultural zoning, pp. 433-439. zabihi h.r., azarpajouh e., 2004 grape response to different soil moisture regimes. j. soil water sci., 18(10): 34-39. zarrouk o., francisco r., pinto-marijuan m., brossa r., santos r.r., pinheiro c., chaves m.m., 2012 impact of irrigation regime on berry development and flavonoids composition in aragonez (syn. tempranillo) grapevine. agric. water manag., 114: 18-29. 51 adv. hort. sci., 2011 25(1): 51-63 received for publication 10 may 2010. accepted for publication 4 march 2011. review paper crop physiology of elephant foot yam [amorphophallus paeoniifolius (dennst. nicolson)] v. ravi, c.s. ravindran, g. suja, james george, m. nedunchezhiyan, g. byju, s.k. naskar central tuber crops research institute, sreekariyam, 659 017 thiruvananthapuram, india. key words: corm, dormancy, sprouting, elephant foot yam. abstract: amorphophallus paeoniifolius (dennst. nicolson), syn. a. campanulatus (roxb.) bl. exdence (also elephant foot yam) is largely cultivated in the philippines, java, indonesia, sumatra, malaysia, bangladesh, india and china. in india, it is cultivated in the states of andhra pradesh, west bengal, gujarat, kerala, tamil nadu, maharashtra, uttar pradesh and jarkhand. sree padma, gajendra, sree athira (a hybrid), bidhan kusum and nda-9 are some of the high yielding amorphophallus varieties released for cultivation. the corm production potential of this crop is 5080 t ha-1 and net economic return is about 2000 – 3000 us$ per ha. plant growth and corm yield is influenced by the size of planting material (corms/cormels/corm pieces), plant spacing, nutrient management and water availability. nevertheless, the production aspect of this crop is less understood as scanty research has been conducted in this crop. the available literature on growth and productivity of elephant foot yam is briefly described in this article. 1. introduction amorphophallus paeoniifolius (dennst.), syn. a. campanulatus (roxb.) bl. exdence (also elephant foot yam) is an herbaceous, perennial c3 crop. it is basically a crop of southeastern asian origin. it serves as a source of protein as well as starch. it has long been used as a local staple food in many countries such as the philippines, java, indonesia, sumatra, malaysia, bangladesh, india, china and southeastern asian countries (chandra, 1984; sugiyama and santosa, 2008). it is commercially cultivated due to its production potential and popularity as a vegetable in various indian cuisines. in india, it is cultivated in andhra pradesh, west bengal, gujarat, kerala, tamil nadu, maharashtra, uttar pradesh, and jarkhand states whereas in northern and eastern states, the wild, local cultivars grown are generally used for making vegetable pickles and medicine preparations for various aliments. the crop is also cultivated as an intercrop along with turmeric (fig. 1) and under coconut (fig. 2) or banana. in recent years, farmers in bihar and uttar pradesh have also begun cultivation. under improved cultural practices and high yielding varieties the production potential of this crop varies between 30 and 100 t ha1 and the net profit (economic return) is about 2000 – 3000 us$ per ha (aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). this crop also offers export potential in india since it is not commercially cultivated in other fig. 1 elephant foot yam as an intercrop with turmeric. arrows indicate elephant foot yam plants. fig. 2 elephant foot yam under coconut. 52 countries (misra and shivalingaswamy, 1999; misra, 2000; misra et al., 2001). in india, ‘sree padma’, ‘gajendra’, ‘sree athira’ (a hybrid), ‘bidhan kusum’ and ‘nda-9’ are some of the high yielding amorphophallus varieties released for cultivation (aicrp, 2006 a). the corms are usually eaten as a vegetable after boiling or baking and are rich in calcium, (50 mg g-1), phosphorus (34 mg g-1) and vitamin a (260 iu g-1). the leaves are used as a vegetable by local tribes in india because they contain a high concentration of vitamin a (rajalakshmi et al., 2001). elephant foot yam plants grow well in medium to light soils (coarse-textured sandy soils) with adequate amounts of organic matter because they prefer well-aerated soils. the crop can tolerate temporary flooding, but anaerobic water logging causes corm rot. in kerala, elephant foot yam is planted in february and harvested during november-december under rainfed conditions. in andhra pradesh, the crop is planted during september-october and harvested in june (winter season crop) or planted in june and harvested in january (rainy season crop) under irrigated conditions. in west bengal, the crop is planted in october and harvested in june under irrigated conditions. this review summarizes the available literature on growth and productivity of elephant foot yam. 2. shoot characteristics the new shoot (leaf) sprout emerges from the cut corm pieces or full corm used as planting material (plate 1 a and b). the time of emergence (sprouting) of new shoots depends on the dormancy status of the planting material. if the planting material has completed its dormancy before planting, then the new shoot sprout will emerge as soon as it is planted. leaf emergence is delayed when the apical buds of seed corms are damaged or cut pieces of corm are planted. leaves were found to emerge earlier when whole corms were planted than when cut corms were planted, irrespective of corm size (sen et al., 1996). when whole corms, bud portions or upper half sections were planted, buds sprouted 2-3 weeks after planting. however, buds started to sprout 47 weeks after planting when vertical 1/2, 1/4 and 1/8 corm sections and lower half corm sections were planted (sugiyama and santosa, 2008). once the sprout is initiated, further development of new shoots may be completed within 30 days (plate 2 a to f). leaves are basal, compound, pinnate, solitary and erect. leaves are medium to very large in size. the plant develops leaves by using preserved carbohydrates in seed corms (planting material) and then daughter corms (new corms) enlarge by using plate 1 -new sprout emerging from cut corm pieces (a) and full corm (b) of elephant foot yam before planting. emerging sprout from cut pieces of corm emerging sprout from the apical bud meristem zone 53 assimilates synthesized by the leaves. in general, amorphophallus corms have one apical bud, which exists inside the cavity in the head part of seed corms. three or four small cataphylls existing in the head part of corms cover the apical bud in which the first leaf primordium has already differentiated at planting. the cataphylls elongate concomitantly with leaf development. possibly, they protect a leaf from damage by soil impedance during development. furthermore, subepidermal cells of cataphylls may contain needle-like crystals of calcium oxalate which presumably offer protection to a young leaf from damage by pests. cataphyll size depends on corm size and plant age. the cataphylls wither after leaves become mature. leaves are composed of a petiole (pseudostem) and three rachises with many leaflets. the number of leaves which develop during the growing season is dependent on corm age. during a growing season up to 12 leaves may be produced successively. as such, more than two leaves may coexist at the same time. the number of leaves is plate 2 different stages (a-f) of leaf development from the sprout in elephant foot yam. canopy pseudostem newly developing corm mother corm c early stage of leaf emerging out from the cataphyll d leaf emerging out from the cataphyll e spreading canopy b cataphyll enclosing the leaf and the corn showing profuse root development a sprouting corm showing the newly forming corm f full plant. 54 also determined by the size of planting materials. plants originating from small corms (10 g) produce three to eight leaves, while large corms (500 g) usually produce one or two leaves during a growing season. under field conditions, weeds grow much before shoot development from planted corms because of corm dormancy and delay in sprouting. under weedy conditions, leaves are submerged under weeds (fig. 3) and the number of leaves, total leaf area, leaf thickness and fresh masses of corms decreases markedly (santosa et al., 2006 c). when preflowering and post flowering corms with similar fresh masses were planted both types of corms sprouted at about the same time; however, leaf sizes (length of petioles and rachis) were larger in preflowering corms than in postflowering corms (sugiyama and santosa, 2008). up to 150-250 leaflets may be produced per leaf and this may vary among accessions. the leaf area of any one of the three lobes of a. campanulatus leaves showed a highly significant correlation (r = 0.93 to 0.97) with total leaf area (patel and mehta, 1987). the number of stomata in the lower epifig. 3 a heavily weed-infested elephant foot yam field. plate 3 a and b stomata in elephant foot yam leaf. a) stomata on the abaxial leaf surface (10x10). b) single stoma with subsidiary cells (40x10). dermis increased from 10.22 per unit area at 50 days after planting (dap) to 17.78 per unit area at 150 dap (gopi et al., 2008). a stoma has two adjacent cells surrounded by four subsidiary cells (plate 3 a and b). the leaf area index increased with time and reached a maximum (6.1) at 120 dap at a planting density of 140 x 103 plants ha-1 (das et al., 1997). on the other hand, the lai reached 4.4 and 5.4 at a planting density of 100 x 103 and 120 x 103 plants ha-1 respectively. petioles (pseudostem) look like the stems of normal plants and are cylindrical in morphology. in general, large petioles indicate that the corm is also large. depending upon the variety, plant spacing or size of planting material used, the mean shoot length varied between 47.3 and 122.5 cm (mukhopadhyay and sen, 1986; ravindran and kabeerathumma, 1991; sen and das, 1991; goswami and sen, 1992; james george and nair 1993; geetha, 2001; suja et al., 2005, 2006; aicrp, 2004, 2005, 2006, 2007, 2008, 2009; saraswati et al., 2008). increases in n application from 50 to 150 kg ha-1 increased shoot length by 11%, (mukhopadhayay and sen, 1986) or did not increase shoot length and girth (geetha, 2001), while increases in k application from 50 to 150 kg ha-1 did not have any significant effect on shoot growth (mukhopadhayay and sen, 1986; geetha 2001). regardless of plant spacing, an increase in size of planting material increased plant (pseudostem) height; plant height was maximum (84.6 cm) when 1 kg cut corm piece was used as planting material. closer plant spacing (60 x 45 cm) increased plant height (53.8 cm) more than wider plant spacing (90 x 90 cm) (james and nair, 1993). plants produced from whole seed corms were taller than those produced from cut pieces of corm of the same size. this may be due to early sprouting and better root ramification (sen and das, 1991). canopy spread was found to vary between 70.2 and 143.8 cm (ravindran and kabeerathumma, 1991; sen and das, 1991; goswami and sen, 1992; james and nair, 1993; aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). regardless of plant spacing, increases in the 55 size of planting material increased canopy spread and canopy spread was maximum (132.7 cm) when 1 kg cut corm piece was used as planting material at wider plant spacing (90 x 90 cm) (sen and das, 1991). canopy spread was greater in plants raised by planting whole seed corms than in plants produced from cut pieces of corms of the same size. this was presumably due to early sprouting and better root ramification (sen and das, 1991). biomass production of shoots (leaf and pseudostem/petiole) increased up to 120 and 150 dap respectively and declined thereafter, whereas corm dry weight and total dry matter production (tdmp) showed a steady increase up to maturity. the corm drymatter production (cdmp) per ha increased with increases in planting material size or plant density and the highest cdmp (25.6 t ha-1 and 19.4 t ha-1 respectively) was observed at six months after planting (map) by using 250 g cut corm pieces as planting material or with high plant density (14 plants m-2) (das et al., 1997). crop growth rate (cgr) increased gradually up to 120-150 dap and sharply declined at maturity as crop growth ceased. however, the relative growth rate (rgr) continued to decrease with crop age and was the highest at the early growth stage (das et al., 1997). the leaf area increased with increases in planting material size or plant density and the highest leaf-area index (5.4) was observed between 4 and 5 map by using 250 g cut corm pieces as planting material or with high plant density (14 plants m-2) (das et al., 1997). similary cgr increased with increase in planting material size or plant density and highest cgr (25.3-32.2 g m-2day-1) was observed at 5 months by using 250 g cut corm piece as planting material. the cgr was 22. 4 g m-2 day-1 at a plant density of 14 plants m-2 (das et al., 1997). treating corm pieces from the bottom portion of corm with growth regulators thiourea, kno3 and ga3 effectively influenced the growth characters and ga3 gave the maximum corm yield (das et al., 1997). 3. plant growth regulators application of triazole compounds (systemic fungicides) triadimefon (tdm), paclobutrazole (pbz) and propiconazole (pcz) through soil drenching increased total root length (by 8.85-75.92%), dry weight of whole plant (by 71.44-84.91%), intercellular co2 concentration (by 25.12-27.91%), leaf thickness, number of spongy and palisade cells, number of chloroplasts per cell, net photosynthetic rate (pn) (by 15.7-28.92%) and water use efficiency (wue) (by 56.81-87.9%) as compared to untreated control plants. in contrast, total leaf area, transpiration rate (tr) and stomatal conductance decreased (gopi et al., 2005, 2008, 2009). 4. root characteristics roots grow out from the surface of newly developing daughter corms at the base of the pseudostem through the remnants of the cataphylls concomitantly with leaf emergence. these roots extend horizontally and are densely distributed at a shallow depth of the top 15-30 cm soil depth. the roots are cylindrical and 2 to 5 mm thick. roots grow more than 1 m in length under adequate soil moisture conditions or under adequate rain and are known as “rain roots”. under dry soil conditions, the root length decreases to less than 30 cm length. the transverse section (t.s.) of root shows about 25 layers of thin walled parenchymatous cortex cells surrounding a central stellar portion with eight protoxylem points (plate 4 a and b). plate 4 -a and b root anatomy of elephant foot yam (4x10). a) t.s. of root showing stele. b) t.s. of root showing cortex. 56 5. corm development and yield a new daughter corm is formed at the region between the petiole (pseudostem) and seed corm when a sprout grows out from the corm (fig. 4). then, roots appear from the surface of new corm and attain a maximum dry mass at 90 dap. the daughter corm begins to enlarge after a leaf has fully expanded (one to two months) and remarkable enlargement occurs later. the dry mass of seed corms (planting material) decreases gradually, finally decomposing within three months after the new shoot sprouts and develops. after the corm has been initiated, it continuously grows and bulks as long as there is adequate moisture in the soil. morphologically, the corm is a shortened stem with compressed nodes and internodes. there are many small lateral buds (about 1 mm in height) and one large lateral bud (5-15 mm in height) arranged in a definite pattern in concentric nodes of corms. the number of lateral buds per node ranges from 13.0 to 43.3. the number of lateral buds is larger in the middle region of corms than in the head and bottom regions. about 20% of visible lateral buds develop into cormels in the head and middle regions of corms, while about 8% of visible buds develop into cormels in the bottom region. therefore, the middle region of corms produces a larger number of cormels than other regions (sugiyama and santosa, 2008). corm growth rate (corm bulking rate) increased steadily between 1 and 5-6 map. maximum corm bulking rate (7.2-8.2 g plant-1 day-1 ) was observed during the fifth or sixth map (mukhopadhayay and sen, 1986; nair et al., 1991). corm bulking efficiency (final corm weight or size as compared to that planted) follows four rules of thumb: 1) at identical plant spacing, corm bulking efficiency decreases with increases in planting material size in both cut pieces and whole corm (table 1); 2) at a given constant planting material size, corm bulking efficiency increases with increases in plant spacing (table 2); 3) corm bulking efficiency is greater in the case of whole corm than cut pieces of corm used as planting material (table 3); and 4) under rainfed conditions, using constant size of planting material, corms harvested from a particular field or a plot show gradient sizes (table 4). nevertheless, the proportion of gradient sizes may narrow under the best management and soil conditions. thus, corms of desired size can be produced by using appropriate sized planting material and plant spacing. production of full corms of 1 kg size is suitable for home consumption because cut corms may perish rapidly. increasing the level of n from 100 to 200 kg ha-1 or k2o from 75 to 150 kg ha-1 increased the plant heightfig. 4 new corm formed at the base of the apical sprout before planting. cut pieces 100 100 200 250 250 300 500 500 500 750 750 full corm 250 500 750 1000 400-500 g (z) 1 kg (z) 5 kg (z) rajib et al., 2007 nedunchezhiyan, 2006, james and nair, 1993 rajib et al., 2007 james and nair, 1993 sen and das, 1991 ghosh et al., 2008 james and nair, 1990 sen and das, 1991 ghosh et al., 2008 james and nair, 1990 sen and das, 1991 sen and das, 1991 nedunchezhiyan, 2006 ravindran (pers. comm.) nedunchezhiyan (pers. comm.) size of corm planted (g) plant spacing size of corm harvested (g) corm bulking efficiency reference 50 x 50 cm 60 x 20 cm 50 x 50 cm 60 x 45 cm 50 x 50 cm 55 x 50 cm 60 x 45 cm 50 x 50 cm 55 x 50 cm 60 x 45 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 90 x 90 cm 1.2 x 1.2 m 1.5 x 1.5 m 513 500-700/1000 659 740 840 810 1063 950 1320 1017 1230 1360 1480 1800 2530 3-4 kg 5 kg 15 kg 5.1 5-7/10 3.3 3.0 3.4 2.7 2.1 1.5 2.6 1.4 1.6 5.44 3.0 2.4 2.5 6-10 5 3 table 1 at identical plant spacings, corm bulking efficiency decreases with increase in planting material (cut pieces or full corm) size (z) corm bulking efficiency in farmer’s field under the best management practices. new corm showing newly emerging roots from the surface sprout 57 100 100 100 200 200 200 250 250 250 300 300 300 300 300 500 500 500 500 500 500 500 500 750 750 750 rajib et al., 2007 james and nair, 1993 ghosh et al., 2008 ghosh et al., 2008 james and nair, 1993 james and nair, 1993 size of corm planted (g) plant spacing (cm) size of corm harvested (g) corm bulking efficiency reference 50 x 30 50 x 40 50 x 50 50 x 30 50 x 40 50 x 50 60 x 45 90 x 45 90 x 90 40 x 40 55 x 50 65 x 60 70 x 70 90 x 85 40 x 40 55 x 50 65 x 60 70 x 70 90 x 85 60 x 45 90 x 45 90 x 90 60 x 45 90 x 45 90 x 90 279 373 513 389 510 656 740 927 1143 810 1005 1265 1450 1585 1170 1320 1610 1800 1970 1063 967 1533 1017 1290 1880 2.8 3.7 5.1 1.9 2.6 3.3 3.0 3.7 4.6 2.7 3.4 4.2 4.8 5.3 2.3 2.6 3.2 3.6 3.9 2.1 1.9 3.1 1.4 1.7 2.5 table 2 at a given constant planting material size, corm bulking efficiency increases with increase in plant spacing 250 500 750 1000 size of corm planted (cut pieces) (g) size of corm harvested (g) multiplication ratio size of whole corm planted (g) 840 950 1230 1740 3.4 1.5 1.6 1.7 250 500 750 1000 1360 1480 1800 2530 5.4 3.0 2.4 2.5 size of corm harvested (g) corm bulking efficiency table 3 corm bulking efficiency is greater in the case of whole corm than the cut pieces of corm used as a planting material (sen and das, 1191) and corm bulking rate (sen et al., 1996). increases in n application from 50 to 150 kg ha-1 increased corm growth (corm bulking rate) by 10.6-27.6% during the six month growth period (mukhopadhayay and sen, 1986). the effect of n was more pronounced during the initial growth period than during the later growth period. the increase in corm bulking rate due to increase in n application from 50 to 150 kg ha-1 was highest (27.6%) during the four month growth period but declined to 15.3% and 10.6% during the fifth and sixth map respectively. the increase in n application from 50 to 150 kg ha-1 increased the mean corm weight per plant by 21.3%. the corm yield per ha increased by 20% with an increase in n application. the corm yield was 84.6 and 102.3 t ha-1 with n at 50 and 150 kg ha-1 application respectively. increases in k application did not significantly increase corm growth, mean corm weight per plant and corm yield per ha. however, n and k had significant interactive effects on corm growth (corm bulking rate), mean corm weight per plant and corm yield per ha and this appears to be mainly due to n (mukhopadhayay and sen, 1986). shoot height, basal shoot (pseudo-stem) girth, and dry matter accumulation in shoot increased and reached a peak at 120 dap. corm and total (shoot and corm) dry matter increased up to 150 days and declined thereafter. maximum shoot height (85.2 cm), shoot girth (16.4 cm), shoot dry matter (6.63 t ha-1) and corm yield (67.83 t ha-1) were obtained with the application of 150 kg ha-1 n and k in two splits (verma et al., 1995). treating planting material (corms) with 2% azotobacter solution at the time of planting and application of 9.0 kg ha-1 of culture mixed with 40 kg of soil at the root zone of the crop along with 150 kg n ha-1 resulted in high corm yield (64.9 and 62.2 t ha-1 respectively) (mukhopadhayay and sen, 1999). size of harvested corms proportion (%) table 4 under rainfed conditions, with use of constant size of planting material, corms harvested from a particular field or a plot shows gradient sizes 2-2.5 kg 1.5-2.0 kg 1.0-1.5 kg 900 g 700 g 600 g 500 g 400 g 300 g 200 g 100 g 50 g 1-2 5 5 5-6 6-7 7-8 15 12 10 10 10 10 58 corm yield varied between 30.9 and 85.4 t ha-1 depending upon the variety, cultural practices (particularly plant spacing) and manurial practices (mukhopadhayay and sen, 1986; nair et al., 1991; ravindran and kabeerathumma, 1991; goswami and sen, 1992; james and nair, 1993; kundu et al., 1998; geetha, 2001; suja et al., 2005, 2006, 2007, suja and sundaresan, 2008 a, b). corm yields between 39.6 and 98.9 t ha-1 were obtained due to application of 100-200 kg n and 100-150 kg k2o5 each per ha (nair et al., 1991; sen and das, 1991; kundu et al., 1998). application of farmyard manure at a rate of 30 t ha-1 increased the fresh mass or corms by 15 %, while application of n at 150 kg ha-1 increased yield by 6.5% (patel and mehta, 1984). kabeerathumma et al. (1987) reported that 100 kg ha-1 n, 38 kg ha-1 of p2o5 and 267 kg ha -1 of k2o were removed from the field every year when 33 t ha-1 of corms were produced. organic farming (fym at 35 t ha-1 + green manuring with cowpea to generate 2 0-2 5 t ha-1 of green matter + neem cake at 1 t ha-1and ash at 3 t ha-1) increased corm yield by 25.37% (62.67 t ha-1) as compared to traditional method (farmer’s practice, fym 25-30 t ha-1 + and ash at 3 t ha-1) (49.99 t ha-1) and by 19.21 % as compared to conventional method (fym 25 t ha-1 + npk @ 100: 50: 150 kg ha-1) of cultivation (52.57 t ha-1) (suja et al., 2005, 2006, 2007, suja and sundaresan, 2008 a, b, 2009). the corm yield was significantly influenced by the size of seed corm and higher yields were recorded from planting materials of 1 kg size (sen et al., 1984; asokan, 1984; sen and das, 1991). increasing the size of planting material from 2 50 g to 1 kg increased mean corm weight per plant from 0.75 to 1.74 kg whereas the corm yield per ha increased from 21.6 to 77.34 t (sen et al., 1984; asokan, 1984; sen and das, 1991; james and nair, 1993; das et al., 1995). comparatively more corm yield was obtained by planting whole seed corms: about 45% greater than the corm yield obtained from cut pieces of corms of the same size (table 5 and 6). this was presumably due to early sprouting and better root ramification (sen and das, 1991). nevertheless, a seed corm size of 400 500 g at 90 x 90 cm spacing would be ideal for economic cultivation of elephant foot yam (james and nair, 1993; yadav et al., 2008). for production of small size (< 1 kg) corms for home use, planting materials of 100-300 g may be used (das et al., 1995; mondal and sen 2004; rajib et al., 2007). to prevent decay after planting due to the presence of several soil borne pathogens, cut corm pieces are dipped in cow dung slurry mixed with mancozeb (0.2%) + monocrotophos (0.05%) for 10 min and surface dried under shade for 24 hr before planting. biofertilizers and other beneficial microorganisms may be added to the cow dung slurry for high productivity (nedunchezhiyan et al. 2006). it was found that planting depth affected plant growth and yield (santosa et al. 2004 a). deeper planting of seed corms led to deformation in daughter corms. at a depth of 30 cm, most corms were elongated or became pyriform. therefore it is desirable to have corms at a depth of 10 cm below the soil surface (sugiyama and santosa, 2008). the multiplication ratio in amorphophallus could be enhanced to 1:15, from the conventional 1:4, by adoptcut corm piece (g) 250 500 750 1000 whole corm (g) 250 500 750 1000 cd (0.05) 37.4 42.2 54.7 77.3 60.5 65.8 80.0 112.4 seed corm size (g) shoot length (cm) canopy spread (cm) mean corm weight (kg) corm yield (t ha-1) 62.6 72.3 81.0 84.6 69.1 75.4 88.8 96.8 0.8 85.8 89.0 114.8 132.7 88.6 99.7 117.9 134.9 0.8 0.84 0.95 1.23 1.74 1.36 1.48 1.8 2.53 0.06 250 500 750 14.1 18.9 23.2 seed corm size (g) shoot length (cm) canopy spread (cm) mean corm weight (kg) corm yield (t ha-1) 36.5 40.4 48.7 99.8 97.1 114.4 1.14 1.53 1.88 table 5 effect of seed corm size on shoot length, canopy spread, mean corm weight and corm yield source: sen and das, 1991. table 6 effect of seed corm size on shoot length, canopy spread, mean corm weight and corm yield source: james and nair, 1993. 59 ing the minisett technique developed in ctcri (james et al., 2004). minisetts produced corms in the range 600 g to 1.5 kg. treating setts of corm pieces from the bottom portion of corm with ga3 (200 ppm) resulted in maximum corm yield (das et al., 1997). various integrated nutrient management practices (combination of inorganic fertilizers, organic manures and biofertilizers) and weed management practices enhanced plant height, canopy spread, corm size, and corm yield per ha (aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). the mean starch content of amorphophallus corm varied between 9.2 and 23.8% and the increase in n or k application did not have a significant effect on starch content (mukhopadhayay and sen, 1986; geetha, 2001). organic practices favoured starch content of elephant foot yam corm (suja et al., 2005, 2006, 2007; suja and sundaresan, 2008 a, b). the starch content was found to range from 3.6 to 11.5% on a fresh mass basis in indonesian accessions (santosa et al., 2002), and from 7.0 to 14.3% in indian accessions (moorthy et al., 1994). little variation was noted in the average size of starch granules (9-13 µm) and amylase content (22-24%) among different accessions (moorthy, 2002). 6. corm dormancy amorphophallus corms exhibit dormancy for about three to five months after harvest. as a result, planting and harvesting are done at a particular time of the year. amorphophallus is propagated by corms as such or by cut corm pieces having a part of apical meristem. sprouting percentage was greater (98%) with top cut portion of corm than the cut corms from the lower half of the mother corm (dhua et al., 1988; nedunzhyan and mohankumar, 1997; mondal and sen, 2004; santosa et al., 2006 b). the bottom portion of the corm is not generally used as planting material due to its lower sprouting efficiency (dhua et al., 1988; nedunzhian and mohankumar, 1997; mohankumar and ravi, 2001). therefore, a greater portion (about 25%) of the harvested produce is again lost as source of planting materials. also, the apical bud from the corm can be excised and used as planting material. removal of the apical bud results in development of one or two adjacent buds within two weeks which also can be excised and used as planting material (fig. 5). ethrel or ethephon was reported to induce early sprouting in amorphophallus corm (dhua et al., 1988; bala and indira, 1992). treating cut pieces of corms from the lower half with chemicals significantly improved sprouting, subsequent growth and yield. among the different chemicals used, thiourea, potassium nitrate and ccc were effective in promoting sprouting. thiourea (200 ppm) and kno3 (1000 ppm) and kinetin (5 ppm) increased corm sprouting by 24.3-92.0, 17.8 and 13.4% respectively as compared to control (table 7) (dhua et al., 1988; kumar et al., 1998). however, mean corm weight was greater in plants from corms treated with thiourea (100 ppm), potassium nitrate (kno3) (500 ppm) and ccc (0.02 ml l-1) yielding 722, 821 and 806 g per plant respectively (dhua et al., 1988). however, corm yield per ha did not increase significantly in plants from corms treated with chemicals, as compared to plants from untreated corms. exposing the whole corms to smoke for 6 h per day for six weeks increased sprouting by 58.3% as compared to untreated corms presumably due to ethrel in smoke. similarly exposing the corms to high temperature (32-45oc) increased sprouting by 83.3% as compared to untreated corms (mohankumar and ravi, 2001; archana et al., 2009). pre-harvest, foliar application of potassium nitrate (2%) and thiourea (1%) had greater influence on breaking dormancy and inducing early sprouting (bhagavan et al., 2008). this may be due to an increase in the availability of sugars as a result of an increase in respiration at higher temperature. compared to smoke and heat treatments, soaking corms in different chemicals [kno3, thiourea, ammonium sulphate (nh 4so4)] for a short period (20-30 min) for 1-2 hr had no significant effect on inducing early sprouting (mohankumar and ravi, 2001). however, treating the apical portion of corm (after removing the apical bud) with thiourea and subsequently wetting the apical portion for a period of 10 days induced early sprouting with more sprouts (archana et al., 2009). darkness had an adverse fig. 5 emerging sprout from the lateral bud after the apical bud is scooped. emerging sprout from the lateral bud after the apical bud is scooped new daughter corm scar of the scooped apical bud 60 effect on sprouting (kumar et al., 1998). in a. konjac, abscisic acid (aba) and ferulic acid were extracted from the dormant corms and exogenous application of aba (10 mg l-1) and ferulic acid (400 mg l-1) inhibited sprouting and growth of the terminal buds of non-dormant corms, suggesting that aba and ferulic acid are inhibitors of sprouting of dormant corms (sun et al., 1996). corms are acrid before dormancy, but it decreases after dormancy (santosa et al., 2003). 7. ecological requirements elephant foot yam grows well under tropical, warm, humid conditions with maximum day-time temperature ranging between 25 and 35oc, minimum night-time temperature ranging between 20 and 25oc and annual rainfall ranging between 1000 and 3000 mm spread over a period of about six to eight months. it grows well in sandy loam or sandy clay loam soil with good drainage and ph of 6.0 to 7.0. it can also be grown in laterite soil (with about 40-50% gravel) but heavy clay soil is not suitable for this crop. soil with high organic matter favours good crop growth and corm yield. planting material (whole or cut pieces of corm) is planted shallow in pits of 60 x 60 x 45 cm size dug out in well-ploughed soil. the top soil dug out is then mixed with farm yard manure or compost (2.0-2.5 kg per pit) and the mixture is put back into the pit prior to placing the planting material over it. the planting material is placed vertically in the pits and is then covered with soil and compacted lightly. 8. response to shade elephant foot yam tolerates shade conditions. therefore, it can be intercropped between young trees. corm yield decreased by 66 % when light intensity was reduced to 2 5% of full sunlight (pushpakumari and sasidhar, 1992). on the contrary, santosa et al. (2006 a) reported that the fresh biomass of corms increased with a decrease in light intensity; 75 % shading produced the largest corms and 0% shading produced the smallest. under full sunlight necrosis and curling at either the edge or the tips of leaflets occurred causing 25 % loss of the crop. no damage was observed in the 25, 50 and 75 % shading. however, shading treatments significantly decreased the leaf number. the short life span of leaves might enhance the production of new leaves resulting in a larger number of leaves under full sunlight. shading treatments significantly affect the length of petioles and rachis. plants developed the shortest petioles under full sunlight but the longest under 75 % shading. 9. effect of water deficit stress little research work has been done on the response of amorphophallus to water deficit stress. soil moisture status does not influence sprouting but further development of new shoot depends on adequate soil moisture. elephant foot yam plants produce large corms and yield more when the water supply is adequate (aicrp, 2008). about 1000-1500 mm of rainfall per year is optimum for the crop. many plants enter dormancy earlier than usual when the rainy season is shorter than four months and supplementary irrigation is necessary for high productivity under the same conditions. plants produced a larger number of leaves under frequent watering (one-, threeand five-day intervals) than under sevenand 15-day intervals; the third leaves were produced in treatments up to seven-day intervals, but neither the second nor the third leaves were produced with 15-day intervals. furthermore, frequent watering produced large leaves and extended their life span compared to less frequent waterthiourea (100 ppm) thiourea (200 ppm) kno3 (500 ppm) kno3 (1000 ppm) ethrel (0.025 ml l-1) ethrel (0.125 ml l-1) kinetin (5 ppm) kinetin (10 ppm) ccc (0.02 ml l-1) ccc (0.1 ml l-1) control (soaked in water) control (unsoaked) top cut portion cd (0.05) 28.5 31.3 30.9 28.9 18.9 29.3 24.5 16.7 32.4 19.7 30.9 22.2 36.7 11.6 treatments sprouting (%) mean corm weight (g) total corm yield (t ha-1) 73.3 91.1 82.2 86.6 75.5 73.3 82.2 75.5 84.4 68.8 68.8 66.6 97.7 16.7 721.6 488.0 820.6 528.6 434.3 679.6 390.0 410.6 806.3 563.6 587.3 540.6 722.6 218.3 table 7 effect of chemicals on sprouting of corm, mean corm weight and corm yield (source: dhua et al., 1988) 61 ing (santosa et al., 2004 b). a decrease in the dry mass of seed corms was more evident with frequent watering, suggesting that reserved carbohydrates in seed corms are not easily metabolized under a limited water supply. the ratios of dry mass of daughter corms to that of seed corms are 6.1, 1.1, 0.6, 0.4 and 0.2 at one-, three-, five-, seven-, and 15-day intervals, respectively. the high ratios under frequent watering treatments could be ascribed to the fact that the soil water availability affects not only the utilization of dry matter in seed corms but also the production and translocation of photoassimilates into daughter corms (sugiyama and santosa, 2008). the roots dried earlier than usual when the soil water content decreased to less than 40 % of field capacity (santosa et al., 2004 b) and the crop tolerates water deficit stress conditions for about 30-60 days but prolonged stress may affect corm yield (santosa et al., 2004 b). in green-house conditions, plant growth was not affected when plants were watered at one-, threeor five-ay intervals. nevertheless, infrequent watering (watering at sevenor 15-day intervals) reduced corm yield and forced the corms to enter into dormancy. soil moisture conservation methods like mulching induced a higher percentage of early sprouting, greater canopy spread, plant height, greater mean corm weight and corm yield (mohankumar et al., 1973). in india, mulching the field with paddy straw resulted in maximum plant height (78.2 and 88.2 cm respectively), girth (14.1 and 14.4 cm respectively) and corm yield (47.44 and 56.74 t ha-1 respectively) as compared to control (aicrp, 2004, 2006 a, b). also cowpea live mulch produced greater yield (41.72 t ha-1) than control (aicrp, 2006 a, b). maximum corm yield was also obtained by black polythene mulching (82.48 t ha-1) and straw mulch (64.82 t ha-1) ranked second (aicrp, 2004). although the corm yield and net return in the straw mulch treatment was lower than polythene mulch, the cost:benefit ratio of straw mulch (1:3.18) was greater than polythene mulch and other treatments (aicrp, 2004). mulching with sesame leaves also resulted in better corm yield (41.8 t ha-1) than straw and black polythene mulch (aicrp, 2004, 2006 a, b). paddy straw mulch also resulted in greater corm yield (13.8 t ha-1) than control (aicrp, 2004, 2006 a, b). when considering mulching with straw, black polythene or cowpea, corm yield was significantly greater only in the first two cases (11.69-14.12 t ha-1) whereas live cowpea mulching significantly reduced corm yield (5.68 t ha-1) compared to control (7.98 t ha-1) (aicrp, 2004, 2006 a, b). maximum corm yield (44.3 t ha-1) was recorded with the application of 100% recommended dose of fertilizer (rdf) along with flood irrigation and the yield (43.5 t ha-1) was on par with the application of 100% rdf plus irrigation at 100% cpe (aicrp, 2009). corm yield was significantly reduced when irrigation was less than 100% cpe (aicrp, 2009). finally, the corm yield of elephant foot yam was greater (37.3 t ha-1) under micro-irrigation (drip-irrigation) at 60% cpe daily for the first 15 days and then on alternate days for the next 15 days, at 80% cpe between two and six months and then at 60% cpe between seven and eight months, than under surface irrigation (26.4t ha-1) (nedunchezhiyan et al., 2008). 10. seed dormancy successful seed production has been reported in amorphophallus (arakeri, 1950). a seed dormancy of five to six months has been reported in this crop (arakeri, 1956). exposing seeds to running water for six days resulted in greater sprouting (55.5%) than in control (2.7%). however, exposing seeds to water for more than six days led to a lower percentage of sprouting (rajendran and hrishi, 1976). 11. future thrust since the whole corm and cut corm pieces are used as planting material, a large portion of harvested produce is used for propagation. therefore, the development of plantlets through in vitro culture of apical/lateral buds (irawati et al., 1986; archana et al., 2009; unnikrishnan and mohan, 2009) should be further refined and exploited for planting material production. furthermore, more detailed investigation of the physiological aspects of growth and productivity of amorphophallus needs to be developed in the following areas: effect of photoperiod and temperature on leaf area development, crop growth rate, stomatal characteristics, photosynthetic rate, root development and rooting pattern, corm development and bulking rate, light interception, dry matter production and partitioning (harvest index), varietal variation in these aspects and physiological factors limiting corm yield. effect of exogenous application of growth regulators such as benzyl adenine and other such growth promoters on maximizing corm yield. since amorphophallus needs a long duration (8 months growing period) for maximum corm yield, studies on factors controlling corm bulking could reveal the physiological basis for developing rapid bulking, short duration varieties. studies to determine water, light and thermal degreeday requirements and the effect of water deficit stress, high temperature (>35oc) (heat stress), salinity and shade on growth and productivity. factors controlling corm dormancy, breaking of dormancy and sprouting and related gene expression. references aicrp, 2004 elephant foot yam. all india coordinated research project on tuber crops, annual report 2003-2004, pp. 54-62. aicrp, 2005 elephant foot yam. all india coordinated research project on tuber crops, annual report 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n., panda p.k., 1995 growth, corm development and uptake of n and k as influenced by doses and methods of n and k application in elephant foot yam (amorphophallus campanulatus). j. potassium res., 11: 68-74. yadav r.k., sanwal s.k., deka b.c., buragohain j., 2008 effect of seed size on tuber yield of elephant foot yam (amorphophallus paeniifolius) under meghalaya condition. nat. sem. amorphophallus: innovative technologies, abst., pp. 111. impaginato 183 1. introduction tomatoes are widely used, and those are a rich source of fiber, phenolics, vitamins a, c, and small amounts of vitamin e and lycopene. lycopene prevents the harmful effects of free radicals and different types of cancers as well as cardiovascular disease (pila et al., 2010; orabi et al., 2015). major problem of postharvest tomato is softening and ripening during storage, distribution, and marketing because of their susceptibility to damage (batu, 2004; agamy et al., 2013). fruit firmness and color are as effective factors of tomato quality, which are used as fruit quality indicator (batu, 2004; agamy et al., 2013). tomato is climacteric so its ripening continues after harvesting and it can become overripe quickly. hence, its quality decreases and its shelf life limits (batu, 2004). pila et al. (2010) reported that owing to lack of information on appropriate postharvest treatments, packaging, temperature, etc., the fruits not only lose their quality but also encounter a substantial postharvest loss. cold storage is one of the most efficient and most practical postharvest procedures that maintains quality of products from the harvest to consumption time (bourne, 2006), and extending the storage life of fresh horticultural products, tomatoes can be stored successfully for weeks (hatami et al., 2013), but the main problem is the postharvest handling, because the tropical and subtropical products are sensitive to chilling injury (ci) (soleimani aghdam et al., 2012). hatami et al. (2013) reported that improper temperature management is the primary cause of many postharvest diseases and disorders. elhadi and jeffrey (2012) reported that mature green tomatoes are the most sensitive to low temperatures among the commercial fruit, and there is a risk to develop chilling injury if they are held below 13°c or 12.5°c (rugkong, 2009) and 12°c (galvez et al., 2010; zhang et al., 2010). sevillano et al. (2009) reported that chilling injury reduced tomato fruit quality. commonly visible ci comprise several symptoms such as surface pitting (soleimani aghdam et al., 2012) and alteration of ripening process as indicated by delayed or even total failure offruit color development and softening (rugkong, 2009), adv. hort. sci., 2016 30(3): 183-192 doi: 10.13128/ahs-20281 effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage e. baninaiem (*), a.m. mirzaaliandastjerdi, s. rastegar, kh. abbaszade department of horticultural science, faculty of agriculture and natural resources, university of hormozgan, bandar abbas, iran. key words: ascorbate peroxidase, decay, lycopersicon esculentum, weight loss. abstract: nowadays, there is a considerable tendency to replace dangerous chemicals with natural compounds, compatible with plant, human, and nature. this study was aimed to assess the effect of salicylic acid on quality and storage life of tomato (lycopersicon esculentum cv. baraka). the salicylic acid application was including plant foliar application three weeks before harvest at concentration 4 mm, followed by the postharvest dipping fruits in salicylic acid at different concentrations (1, 2, 4 mm), then storing at 10°c for 40 days to investigate quantitative and qualitative characteristics. the chilling injury symptoms, electrolyte leakage, decay and a* (redness) value significantly decreased and activity ascorbate peroxidase increased. ascorbic acid content, total soluble solid, titratable acidity, firmness, and l* (lightness) retained by salicylic acid treatments. the salicylic acid application had no significant influence on weight loss and b*. application of salicylic acid in all concentrations, especially a combination of treatments preharvest to concentrations 4 mm as well as postharvest 4 mm, had the highest influence on qualitative and quantitative characteristics and increased the postharvest life of the tomato fruit. (*) corresponding author: b.elham.2013@gmail.com received for publication 21 april 2016 accepted for publication 17 november 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 183-192 184 increased susceptibility to alternaria rot and decay (ding et al., 2002), decrease mealy texture when ripened (alternaria and cladosporium rots are usually associated with chilling injury) (elhadi and jeffrey, 2012). salicylic acid (sa) is a phenolic compound and plant growth regulator (zavala et al., 2004) and defenses against biotic and abiotic environmental stresses. plants produce reactive oxygen species (ros) when exposed to biotic and abiotic environmental stresses conditions (agamy et al., 2013). ros includes superoxide, hydrogen peroxide and hydroxyl ions (dat et al., 2000). thus, ros cause to damage in cellular structures. there is a mixture of non-enzymatic antioxidants (carotenoids, ascorbate) and enzymatic antioxidants in plants, such as catalase (cat) and ascorbate peroxidase (apx) which inhibit harmful effects of these ross. the enzymatic action of apx reduces h2o2 using ascorbate as an electron donor (orabi et al., 2015). salicylic acid is an antioxidant defense system and regulates different physiological and biochemical processes in plants including: plant growth (khan et al., 2003), stomatal conductivity (hayat et al., 2010), photosynthesis (fariduddin et al., 2003), seed germination (babalar et al., 2007), disease resistance (janda et al., 2007), heavy metal stress, low temperature, high temperature and salinity (hayat et al., 2008). salicylic acid treatment could be used to enhance the chilling resistance of maize, cucumber and rice (kang and saltveit, 2002), pomegranate (sayyari et al., 2009) and tomato (ding et al., 2001, 2002). salicylic acid delays the ripening of banana and kiwifruit during storage (srivastava and dwivedi, 2000; zhang et al., 2003). babalar et al. (2007) reported that pre and postharvest sa treatments caused fruit quality maintenance in strawberry. fattahi et al. (2010) reported that losses in fruit quality are mostly due to its relatively high metabolic activity during storage. salicylic acid is known as a signal molecule in the induction of defense mechanisms in plants. due to the risk of inappropriate use of substance chemicals in postharvest technology, it is essential to study the application of safe postharvest treatments along with cold storage. since the time between tomato fruit harvest and consumption may take long weeks, and during this period many changes could happen that affect the postharvest behavior of fruits. therefore, the aim of this article was to appraise the effects of pre and postharvest sa application to maintain the qualitative characteristics of tomato fruits at cold storage and increase the postharvest life of the tomato fruit. 2. materials and methods plant material and salicylic acid treatment fruits of tomato (lycopersicon esculentum cv. baraka) produced in the greenhouse at university of hormozgan (iran) were used. baraka cultivar is a hybrid seed appropriates for the tropical region. fruits were harvested at mature green stage in april 2014 and transferred to laboratory where they were selected for health and size, weight, and color uniformity. fruits were divided randomly. then, they were washed and dried in the air. each treatment consisted of 60 fruits, and each treatment was composed of three replicates (20 fruit per replicate): three sa treatments were compared (4+1, 4+2, and 4+4 mm) consisting of a 4 mm sa plant foliar application three weeks before harvest followed by the postharvest fruit dipping for five minutes in sa solutions at different concentrations (1, 2, and 4 mm). fruits harvested from non-treated plants were used as control. tomatoes were stored at 10°c temperature and 8590% relative humidity (rh) for 10, 20, 30 and 40 days (hatami et al., 2013). samples were taken at every 10 days intervals during storage for quality evaluation. firmness and weight loss fruit firmness was measured using a penetrometer equipped with a 6 mm diameter flat probe exerting maximum force on fruit. units were expressed as kg cm-2 (shafiee et al., 2010). fruit weight loss was measured immediately after harvest and storage time. the results were calculated as percentage of weight loss at the start of the experiment and at different intervals during storage by this formula: %wl = [(w1-w2)/w1] × (100), where %wl= percentage weight loss, w1= initial fruit weight in (g), w2= final fruit weight in (g) (zhang et al., 2002). superficial color superficial color of tomato was measured using a minolta chronometer model cr 400 and average readings at three points against each other in the fruits were recorded. color indices inclusive (l*, a*, and b* values) were measured. superficial color of the fruit was expressed as l* (the ratio of white to black color), a* (the ratio of red to green color) and b* (the ratio of yellow to blue color) (shafiee et al., 2007). chilling injury index (scores) and electrolyte leakage chilling injury index of fruits was evaluated at 10°c after 10, 20, 30 and 40 days in cold storage. symptoms were manifested as surface pitting and baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 185 dehydration according to the method of sayyari et al. (2009). the severity of the symptoms was assessed with scores according to the following 3 stage scale: 0 (no symptom), 1 (1-25% of damaged area), 2 (2650% of damaged area) and 3 (>51% of the damaged area). the average extent of chilling-injury damage was expressed as a chilling-injury (ci) index, which was calculated using the following formula: ci = [(value of hedonic scale) × (number of fruit with the corresponding scale number)] / (4 × total number of fruit in the sample). the rate of electrolyte leakage (el) was measured according to the method of mirdehghan et al. (2007), using 6 discs (10 mm diameter) of peel tissue, cut with a cork borer. conductivity was measured after 4 h of incubation in 25 ml of 0.4 m mannitol under constant shaking. the conductivity of the solution (l1) was measured with a conductivity meter (ttracon wtw 325). after readings had been taken, the vials were autoclaved at 121°c for 15 min, and then cooled to 20°c. the conductivity of tissues (l2) was measured. ion leakage was calculated as the ratio of l1 to l2. fruit decay index (scores) decay incidence of each fruit was determined by scores. according to the amount of the decay on fruit surface scales from 1 to 5 were given to the each treatment where; 1= normal (no decay on fruit surface), 2= trace (up to 5% of fruit surface were decayed), 3= slight (5-20% of fruit surface were decayed), 4= moderate (20-50% of fruit surface were decayed), and 5= severe (>50% of fruit surface were decayed). results were expressed as fungal decay index (babalar et al., 2007). vitamin c (ascorbic acid) fruit vitamin c content was measured by using titrimetric method with the titration of filtrate against 2,6-dichlorophenol indophenol and the results of vitamin c content were expressed as mg/100 g (pila et al., 2010). titratable acidity and total soluble solids five ml of extracted fruit juice was diluted to 45 ml with distilled water. then, extract fruit juice was titrated with 0.1 n sodium hydroxide to a ph of 8.1. titratable acidity (ta) was determined as percentage of citric acid by this formula: ta (%)= [(v × n × meq)/y] × 100 where v= volume of sodium hydroxide used ml, n = sodium hydroxide normality, and meq = 0.064, y = volume of bulk fruit juice ml (saltveit, 2005). total soluble solid (tss) in the extracted fruits juice was measured with a portable refractometer (model dbr95), and the results were expressed as brix. ascorbate peroxidase ascorbate peroxidase (apx) was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm for 1 min in a uv-vis spectrophotometer (model unicuv2100). samples from pulp of 0.5 g fresh tissue homogenized and the homogenized samples were centrifuged at 14000 rpm for 15 min. the supernatant was used as crude enzyme extract for apx enzyme analyses. the 3 ml reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbic acid, 0.1 mm edta, 0.1 mm h2o2, and 0.1 ml enzyme. the reaction started with adding of 0.1 mm hydrogen peroxide. the enzyme activity was calculated using the extinction coefficient 2.8 mm-1 cm-1 for ascorbate (nakano and asada, 1981). statistical analysis statistical analysis was performed by sas software (sas institute inc., 1990) according to a split plot in time design on the basis of completely randomized design (crd), with 3 sa concentration and 3 replicates. data were analyzed by glm and differences among means of data were compared by with least significant difference (lsd) test at a significance level of 0.05. 3. results firmness and weight loss the results showed a rapid decrease in firmness in the control set compared to the fruits treated with sa during storage, and all treated fruits were firmer than the control set (p< 0.01). maximum fruit flesh firmness (3.2 kg cm-2) was recorded in pre and postharvest sa treatments 4+4 mm and control set had the softest fruits (0.6 kg cm-2) at the end of the experiment but, the difference was not statistically significant compared to the other sa concentrations (table 1). no significant changes were observed in weight loss during storage for any treatments, with the exception of weight loss in 30 days sa-treated fruit, which was lower than the control set (table 1). color assessment the results of this investigation showed that pre and postharvest sa treatments had an effect on fruit lightness (l*) and redness (a*) value in comparison adv. hort. sci., 2016 30(3): 183-192 186 with control set (p<0.01). no significant (b*) value were observed in treated fruits, except 20th days in 4+2 mm (34.49) and 40th days in 4+4 mm (39.76) satreated fruit, which were higher level than control set (31.34). during storage, the l* value decreased. the control set had the lowest l* value (22.60), and the highest value was recorded in pre and postharvest sa treatments 4+2-4+4 mm (28.21 and 28.87, respectively) at the end of the experiment, and their difference was not statistically significant compared to the other sa concentrations. our results indicate that sa treatments delayed the loss of l* value in tomato during storage. in general, a* value increased during storage as well as ripening. in the other hand, the color development rate of tomatoes increased with the increase in maturation. the most value of color index a* with a negative value (-6.13) was recorded in the green fruits of the control set. the negative values were observed in immature fruits and treated fruits showed negative/lower a* values than control fruits. the highest value of color index a* (19.87) was recorded in completely ripened tomatoes of the control set at 40th days. the index a* had a sharp increase control set at 10th days with a* value changing from negative (green color) to positive (red color) (table 1). storing mature-green fruits, treated with salicylic acid at 10°c increased the postharvest life up to 40 days. chilling injury and electrolyte leakage our results showed that ci increased during storage, but applying different concentrations of sa could significantly (p<0.01) affect chilling injury index in tomato fruit. salicylic acid treatments lowered the levels of chilling injury compared to that of the fruits of control set, and the highest chilling injury was observed in control set. no chilling injury symptoms were observed in tomato fruits with pre and postharvest treatments 4+4 mm (table 2). the results obtained from the present study showed that electrolyte leakage of control set (70.27%) was significantly higher than that of sa-treated fruits end of the storage period, and there was no significant difference between concentrations used for treatment (p<0.01) (table 2). fruit decay index decay increases during storage and the results of our evaluation showed that sa, at different concentrations, significantly affected fruits decay (p<0.01). fruits with sa treatments showed lower levels of decay as compared to that of the fruits of control set. highest decay was observed in our control set. in pre and postharvest treatments 4+4 mm, there were not any decay symptoms in tomato fruit after 40 days. we did not witness any significant differences between concentrations (4+1 and 4+2 mm) (table 2). means within each column with different superscript letters are significantly different (p = 0.05) for each sampling. table 1 effect of pre and postharvest treatments salicylic acid in firmness, weight loss, l*, a*, b* of tomato fruit cv. baraka and stored at 10°c for up to 40 days time storage (days) salicylic acid treatments firmness (kg cm-2) weight loss (%) color parameter l* a* b* 0 0 4.82±1.02 a 0.00±0.00 f 34.98±2.69 a -6.13±0.63 g 15.47±1.17 e 10 0 3.38±0.18 cdef 1.62±0.82 e 29.37±1.23 fg 3.83±0.35 d 15.76±1.51 e 4+1 4.11±0.07 abc 1.32±0.03 e 33.02±1.97 abc -5.73±0.17 g 13.42±0.59 e 4+2 4.18±0.03 abc 1.28±0.03 e 33.31±0.89 ab -6.03±0.82 g 11.75±0.42 e 4+4 4.29±0.021 ab 1.22±0.02 e 33.84±1.59 ab -6.86±0.36 g 16.27±2.87 e 20 0 2.88±0.78 f 2.38±0.54 cd 26.62±0.80 hi 5.86±1.58 cd 24.59±2.97 d 4+1 3.97±0.11 bcd 2.12±0.01 d 31.89±0.96 b-e -4.94±0.59 fg 24.34±6.31 d 4+2 4.08±0.08 abc 2.10±0.02 d 32.24±0.84 bcd -2.45±0.37 ef 34.49±0.08 ab 4+4 4.15±0.14 abc 2.09±0.01 d 32.46±1.00 bcd -5.73±0.45 g 24.72±7.10 d 30 0 1.70±0.67 g 3.11±0.14 b 24.41±0.86 ij 11.84±2.14 b 32.06±1.73 bc 4+1 3.64±0.07 b-f 2.69b±0.02 c 29.73±0.47 efg -1.99±0.71 ef 31.89±0.99 bc 4+2 3.75±0.07 b-e 2.66±0.03 c 30.16±0.95 d-g 2.94±0.11 d 28.14±4.10 cd 4+4 3.78±0.09 b-e 2.65±0.03 c 30.67±0.52 c-f -4.75±0.55 efg 27.53±9.01cd 40 0 0.60±0.10 h 3.92±0.62 a 22.60±1.07 j 19.87±1.85 a 31.34±3.77 bc 4+1 3.02±0.08 ef 3.67±0.02 a 27.95±0.96 gh 5.64±0.33 cd 36.85±2.79 ab 4+2 3.08±0.08 ef 3.64±0.04 a 28.21±0.16 fgh 5.54±0.38 cd 32.40±0.32 bc 4+4 3.20±0.04 def 3.63±0.02 a 28.87±0.71 fgh -1.70±0.70 e 39.76±0.70 a baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 187 vitamin c (ascorbic acid) tomato fruits vitamin c content was decreased during storage, and it was found to be maintained with pre and postharvest treatments of sa, and this result was statistically significant (p<0.01). tomato fruits treated with sa showed comparatively higher levels of ascorbic acid than the fruits of control set (9 mg/100 g), and the highest ascorbic acid content in pre and postharvest treatments 4+4 mm (45 mg/100 g) was observed, but there was no significant difference between two concentrations of our treatment (table 2). titratable acidity (ta) and total soluble solids (tss) tomato fruits ta content was maintained with pre and postharvest treatments of sa, and it significantly resulted in firmer fruits comparing to the controls (p<0.01). titratable acidity (ta) content in treated fruits was higher than the control set (0.86%), and our results showed that ta maintained with pre and postharvest treatments of sa and we observed the highest ta content in concentrations of 4+2-4+4 mm (1.02, 1.03%, respectively) at 40th days (table 3). also, tss increased during storage. highest and lowest tss were observed in control set and treated fruits, respectively, and this difference was significant (p<0.01). sa application in this experiment had a significant effect on soluble solids. therefore, soluble solids of control fruits (4.36 °brix) were more than of treated fruits (2.70 °brix in 4+4 mm) after 40th days storage (table 3). ascorbate peroxidase (apx) the results showed apx decreased in control fruits, it increased and then decreased again in treattable 2 effect of pre and postharvest treatments of salicylic acid in chilling injury, electrolyte leakage, decay, ascorbic acid of tomato fruit cv. baraka and stored at 10°c for up to 40 days means within each column with different superscript letters are significantly different (p = 0.05) for each sampling. time storage (days) salicylic acid treatments chilling injury electrolyte leakage (%) decay index ascorbic acid (mg/100 g) 0 0 0.00± 0.00 d 40.63±10.32 d 0.00±0.00 d 66±6.00 a 0 0.00±0.00 d 56.59±4.05 c 0.00±0.00 d 32±1.05 f 10 4+1 0.00±0.00 d 26.21±1.81 gh 0.00±0.00 d 65±1.73 a 4+2 0.00±0.00 d 24.39±0.74 h 0.00±0.00 d 66±3.00 a 4+4 0.00±0.00 d 23.98±2.00 d 0.00±0.00 d 68±3.46 a 20 0 0.00±0.00 d 61.80±1.30 bc 0.00±0.00 d 18±3.00 g 4+1 0.00±0.00 d 28.27±0.90 fgh 0.00±0.00 d 49±4.58 bcd 4+2 0.00±0.00 d 25.63±0.65 gh 0.00±0.00 d 52±4.58 bc 4+4 0.00±0.00 d 23.68±0.94 h 0.00±0.00 d 55±1.73 ab 30 0 0.60±0.20 b 65.11±5.47 ab 0.46±0.31 b 12±3.00 gh 4+1 0.20±0.00 c 33.25±2.71 defg 0.20±0.00 c 44±4.58 de 4+2 0.20±0.00 c 31.81±1.70 efgh 0.20±0.00 c 48±3.00 cd 4+4 0.00±0.00 d 30.59±0.79 efgh 0.00±0.00 d 50±1.73 bcd 40 0 1.06±0.23 a 70.27±0.92 a 1.07±0.23 a 9±3.00 h 4+1 0.33±0.12 c 37.53±1.21 de 0.40±0.20 b 39±3.00 e 4+2 0.27±0.12 c 36.65±2.95 de 0.33±0.23 bc 41±3.46 e 4+4 0.00±0.00 d 36.51±2.65 def 0.00±d 45±3.00 de means within each column with different superscript letters are significantly different (p=0.05) for each sampling. time storage (days) sa treatments ta (%) tss (°brix) apx (mg/g fw) 0 0 0.94±0.19 abcd 3.40±0.69 c 37.14±1.24 e 0 0.92±0.01 bcd 3.43±0.15 bc 20.90±19.03 f 10 4+1 1.03±0.01 ab 2.23±0.15 d 54.78±1.12 ab 4+2 1.04±0.01 ab 2.16±0.15 d 55.66±1.29 a 4+4 1.04±0.02 ab 2.46±0.46 d 56.12±2.49 a 20 0 0.90±0.01 bcd 4.00±0.10 ab 18.80±11.17 f 4+1 1.01±0.01 abc 2.66±0.12 d 52.31±2.06 abc 4+2 1.04±0.01 ab 2.60±0.20 d 53.83±4.36 ab 4+4 1.06±0.01 a 2.53±0.06 d 55.70±4.77 a 30 0 0.89±0.03 cd 4.26±0.21 a 7.38±2.89 g 4+1 1.01±0.01 abc 2.66±0.06 d 40.18±2.47de 4+2 1.02±0.01 abc 2.63±0.12 d 43.78±4.81 cde 4+4 1.03±0.01 ab 2.66±0.06 d 46.42±3.11bcd 40 0 0.86±0.03 d 4.36±0.40 a 6.95±1.38 g 4+1 1.00±0.01 abcd 2.63±0.12 d 38.06±1.09 de 4+2 1.02±0.02 abc 2.66±0.0 6d 38.61±1.31 de 4+4 1.03±0.02 ab 2.70±0.10 c 38.18±2.51 de table 3 effect of pre and postharvest treatments sa in ta, tss, apx of tomato fruit cv. baraka and stored at 10°c for up to 40 days adv. hort. sci., 2016 30(3): 183-192 188 ed fruits. the activity of apx in treated fruits was higher than the controls, and there was no significant difference between the three concentrations at the end of the experiment (p<0.01) (table 3). 4. discussion and conclusions the results of this study indicate that pre and postharvest treatment with sa produced the firmest fruits. softening of fruits is one of the most common physical parameters to assess the progress of ripening (srivastava and dwivedi, 2000; brummell, 2006) and softening is a major problem of tomato that limits the quality. key factors associating with fruit softening are the depolymerisation and degradation of cell wall components (brummell, 2006). srivastava and dwivedi (2000) reported polygalacturonase is primarily responsible for ripening associated pectin degradation and fruit softening. level of polygalacturonase activity has been positively correlated with fruit ripening and softening in banana and tomato fruits. application of salicylic acid is useful in inhibiting tissue softening in fruits by reducing cell wall hydrolases activities and maintaining cell membrane consistency (supapvanich, 2015). wei et al. (2011) reported that exogenous application of sa enhances defense mechanisms and production of antioxidants in fruits during storage that leads to a decrease in lipid peroxidation of the cell membrane and results in maintained cell membrane structure. this result was in agreement with the reports of babalar et al. (2007) and shafiee et al. (2010) that suggested pre and postharvest application of sa on strawberry could decrease the softening and keep them firm during storage. zhang et al. (2003) showed that sa effectively prevented kiwifruit softening during storage and rate of fruit ripening related to internal sa concentration as well as srivastava and dwivedi (2000) reported that salicylic acid treatment inhibited the process of banana fruit softening during ripening. srivastava and dwivedi (2000), zhang et al. (2003) and wang et al. (2006) reported that rapid softening of fruits during ripening was simultaneous with rapid decrease in endogenous sa of fruits. tomato fruit weight loss did not show changes in response to sa treatments (except in 30th days lower than the control set). the results of this study did not accord with the ones of babalar et al. (2007) and shafiee et al. (2010). the l* value decreased during storage. babalar et al. (2007) reported higher lightness in pre and postharvest sa treated strawberry fruits than control. shafiee et al. (2010) showed sa treatments were not effective on fruit lightness in comparison with the control set. fattahi et al. (2010) suggested that the decrease in l* value represented the formation of dark color in the pulp due to oxidative browning reactions or increasing in brown pigment concentrations. value a* increased during fruit ripening. the same results were obtained from pre and postharvest sa application on strawberry (babalar et al., 2007), but shafiee et al. (2010) reported that sa treatments were not effective on a* value in comparison with control. the a* value is a useful index of maturation and the degree of ripening in tomato (artes et al., 1999) and the external color is a key factor indicating the quality of tomato (supapvanich, 2015). changes in a* result increase the respiration rate during storage. the salicylic acid treatment causes a decrease in respiration and a delay in the appearance of the climacteric peak, which is concentration-dependent (srivastava and dwivedi, 2000). shafiee et al. (2010) reported that the effect of sa treatments might be due to the reduction of respiration, and it prevents from an increase in a* value, so it could have an advantage in delaying the senescence. the sa application did not affect b* value except 20th days in 4+2 mm and 40th days in 4+4 mm sa-treated fruit, which showed higher level than control set. there were not literature about the effect of pre and postharvest application of sa on b*changes. salicylic acid treatments lowered the levels of chilling injury compared to that of the fruits of control set, and the highest chilling injury was observed in control set. ding et al. (2001, 2002) reported that chilling injury was manifested in tomato fruit by some symptoms. severely injured fruit developed sunken areas (blemishes) an increased susceptibility to alternaria rot and decay. initially, ci affects the cell membrane with changes in the fatty acid of phospholipids. secondary damages are on the cell membrane that leads to disruption of the cell structure (soleimani aghdam et al. , 2012). asghari and soleimani aghdam (2010) suggested that treatment with sa prior to low-temperature storage induce heat shock proteins (hsps) biosynthesis and, at the same time, ci tolerance in tomatoes and peaches. accumulation of the heat shock proteins (hsps) in chilling-sensitive horticultural products with sa treatments would allow their storage at low temperatures without ci development. this membrane damage can be measured by the electrolyte leakage, which the results obtained from the present study showed that electrolyte leakage of control set was significantly baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 189 higher than that of sa-treated fruits. therefore, these results indicate that sa can maintain membrane consistency through enhancing the antioxidant potential of the plant. reduction of electrolyte leakage and prevention of oxidative damage to cells under stress conditions has been mentioned as primary mechanisms of stress tolerance. the same results were obtained from postharvest treatment with sa to prevent chilling injury (sayyari et al., 2009; soleimani aghdam et al., 2012) and electrolyte leakage (sayyari et al., 2009; soleimani aghdam et al., 2012; orabi et al., 2015). fruits with sa treatments showed lower levels of decay as compared to that of the fruits of control set and in pre and postharvest treatments 4+4 mm, there were not any decay symptoms in tomato fruit after 40th days. salicylates are major components of the signal transduction pathways of plants playing an important role in disease resistance (asghari and soleimani aghdam, 2010). different researches show that sa had no direct effect on the decrease of decay in pear fruits (fruits were sprayed with sa), but it might reduce fungus development (shafiee et al., 2010). babalar et al. (2007) reported sa in a concentration dependent manner from 1 to 2 mm effectively reduced fungal decay in selva strawberry fruit. salicylic acid applied to either plant’s vegetative stage, fruit development stages or postharvest stage could completely control decay and increased fruit shelf life. yao and tian (2005) showed preharvest and postharvest treatments of sweet cherry fruit with sa showed significantly lower disease percentages in storage at 25°c than the control. at 0°c, the inhibitory effects of preharvest sa treatments on postharvest disease were better than those of the postharvest treatment. shafiee et al. (2010) also obtained similar results for pre and postharvest sa treatments on strawberry fruit. tomato fruits vitamin c content was decreased during storage and fruits treated with sa showed comparatively higher levels of ascorbic acid than the fruits of control set. shahkoomahally and ramezanian (2014) reported that the utilization of ascorbic acid during later storage periods may be the reason for its decreased amounts. generally, when fruits become overripe, vitamin c content declines concurrently with the degradation of fruit tissues. the results obtained from this study indicate that the sa treatments were beneficial in delaying degradation of ascorbic acid content during storage. therefore, the sa treated fruits exhibited higher maintenance of ascorbic acid as compared to that of control set. sa prevents vitamin c destruction by increasing the antioxidant ability and resistant of plants and fruits (wang et al., 2006; shafiee et al., 2010). also, exogenous sa could be effective in reducing the rate of respiration and ethylene production (renhua et al., 2008). thus, the results of this study confirm previous reports of postharvest treatment with sa to preserve vitamin c content in tomato (pila et al., 2010), orange (huang et al., 2008), rambutan fruit (supapvanich, 2015) and pineapple fruit (lu et al., 2011). titratable acidity content in treated fruits was higher than the control set. titratable acid depends directly on the concentration of organic acids in the fruit as an important factor in maintaining the quality of fruits (kazemi et al., 2011). therefore, any treatment that slows the metabolism and aging of the product can slow down the changes during storage to reduce titratable acid (zokaee khosroshahi et al., 2007). a correlation between enhanced respiration and a decrease in ta has been suggested by shahkoomahally and ramezanian (2014) to be due to the use of organic acids as respiratory substrates in the respiratory cycle in fruits. organic acids have a higher ratio of oxygen to carbon compared to carbohydrates or fatty acids; therefore, those are easier to consume as an energy source in the process of respiration. salicylic acid reduces respiration and ethylene production, leading to the reduction in consumption of organic acids as respiratory substrates (serrano et al., 2003). salicylic acid application in this experiment had a significant effect on soluble solids. therefore, soluble solids of control fruits were higher than of treated fruits. bal and celik (2010) revealed that after harvest and during storage and ripening of the fruits was increased the tss content. asghari and soleimani aghdam (2010) and bal and celik (2010) reported that cell walls contain large amounts of polysaccharides, mainly pectins and cellulose, and are digested due to the activity of the cell wall degrading enzymes leading to a significant increase in tss content. salicylic acid effectively protects cell walls by decreasing the expression of degrading enzymes and as a consequence prevents from dramatic increase in tss content of the cells, and caused slow down of ripening. similar observation was reported with sa treated banana. salicylic acid treatments inhibited ethylene biosynthesis and delayed the senescence. this is because in the control fruits, due to the aging process (ripening), cell wall was digested and increased soluble solids. on the other hand, sa treatment reduces cellular metabolic activities, such as adv. hort. sci., 2016 30(3): 183-192 190 respiration and ethylene production, and thus maintains the membranes and cell walls, and prevents from an abnormal increase in the soluble solids (valero et al., 2006). an increase in tss content of fruits during storage due to the conversion of starch to be soluble sugars is one of the ripening indexes (fisk, 2006). this result was in agreement with babalar et al. (2007) who reported that the use of salicylic acid decrease tss of strawberry fruits and consequently, effectively delays fruit senescence process. treatment of kiwifruits maintained a lower tss content than the control fruits at the end of cold storage (soleimani aghdam et al., 2009). however, it is in disagreement with lu et al. (2011) results on pineapple fruit and shafiee et al. (2010), report on strawberry, who suggested that sa did not affect soluble solids content and titratable acidity. the activity of apx in treated fruits was higher than the controls. the study indicated the beneficial effects of sa by pre and postharvest treatments on tomato fruit quality. wang et al. (2006) and soleimani aghdam et al. (2012) reported that sa might mitigate postharvest ci in fruits and vegetables via different mechanisms. these mechanisms include: a) enhanced alternative oxidase (aox) gene expression as an efficient ros avoidance gene, b) increased ascorbate peroxidase (apx) and glutathione reductase (gr) activity, c) enhanced reduced-to-oxidized ascorbate (asa/dhasa) and reduced-to-oxidized glutathione ratios (gsh/gssg) and d) improved heat shock proteins (hsps) gene expression in peach fruits. orabi et al. (2015) suggested that there is an important link between plant antioxidant ability and the applied doses of the sa. the observed variation (increase) activity of apx is due to sa activates the resistance system and increases the cell antioxidant capacity. asadi et al. (2013) showed that exogenous application of salicylic acid lightened the toxic actions induced by stress and decreased lipid peroxidation rates with increasing antioxidant activity. there is not any report on the effects of pre and postharvest treatments with sa on fruits apx activity. as a whole, this study showed that pre and postharvest treatments of sa are an effective method of extending storability and postharvest life of tomato fruits at 10°c. the most effective treatment in reducing losses of fruit quality was found to be sa 4+4mm treatments during the storage period of tomato fruit. it was determined that under these conditions baraka tomato could be stored for 40 days without losing much of its quality. acknowledgements financial support for this work was provided by vice chancellor of faculty of agriculture and natural resources of hormozgan university. references agamy r.a., elsayed e.h., tarek h.t., 2013 acquired resistant motivated by salicylic acid applications on salt 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effect of exogenous putrescine on post-harvest life of strawberry (fragaria ananassa duch.) fruit, cultivar selva. sci. hortic., 114: 27-32. 253 1. introduction root-restricted cultivation is an effective technique for saving resources, to control root environment, to anticipate yield and to regulate the quality of vegetables. for all these reasons, its use has significantly increased during the last decades in vegetable nurseries (shi et al., 2008). root restriction (rr) may occur when container size and/ or rooting volume is physically constrained (tschaplinski and blake, 1985; ismail and noor 1996; saito et al., 2008; mugnai et al., 2009), especially in greenhouse-grown horticultural crops (thomas, 1993). a reduced container volume stimulates the formation of a denser root mass, with decreased root growth (ismail and noor, 1996). together with a strong limitation of the soil available to the root system for water and nutrients uptake, rr also reduces canopy growth (ismail and noor, 1996; shi et al., 2008) by affecting many plant physiological and biochemical processes. the mechanism behind the reduced shoot growth is not yet fully understood. several hypotheses have been proposed: water and nutrient stresses (hameed et al., 1987), decrease in root respiration (shi et al., 2007), reduction in photosynthesis (shi et al., 2008), and synthesis and translocation of plant hormones (liu and latimer, 1995). however, contradictory results are reported as to which of these factors plays a significant role in the response of aerial plant parts to restricted root growth, with strong differences between species. leaf photosynthesis strongly depends on environmental conditions such as radiation, co 2 concentration and temperature. in addition to these environmental conditions, photosynthesis is subjected to internal regulation associated with sink demand for assimilates (marcelis, 1991). in the presence of a physical restriction to root growth, a major metabolic sink for photosynthetically fixed carbon at seedling stage (thomas and strain, 1991) may result in feedback inhibition mechanisms (shi et al., 2008). this investigation was therefore planned to determine the morphological and physiological changes induced in response to rr conditions and to determine the time frame within which these changes occurred. in particular, this research aims to study the link between leaf gas exchange parameters and carbohydrate production in regulating growth in pepper (capsicum annuum l.) plants. 2. materials and methods plant material experiments were carried out at the department of plant biology, university of pisa (italy). seeds of pepper (capsicum annuum l.) cv. sienor were sown in seedling flats filled with vermiculite and placed in a germinating room at constant temperature (25°c) and light intensity (300 mol m-2 s-1 ppfd). after germination, seedlings with the first true leaves were selected for uniformity and single plants were transplanted into 7 ml (root restricted, rr) starch accumulation in the leaves of root-restricted pepper affects plant growth by a feedback-inhibition of the photosynthesis h. al-debei*, s. mugnai** * department of horticulture and crop science, university of jordan, amman 11942, jordan. ** dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: capsicum annuum l., carbohydrates, leaf gas exchange, root restriction. abstract: the mechanism behind the reduced growth occurring in plants subjected to root restriction is still not fully understood. therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root volume reduction and to determine the time frame within which these changes occurred. in particular, this research focused on the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in pepper (capsicum annuum l.). our results show that the reduced growth is mainly linked to a feedback inhibition of the photosynthesis, caused by a concurrent limited stomatal conductance (probably driven by both stomatal factors and hormonal substances) together with a strong accumulation of starch in the leaves. adv. hort. sci., 2011 25(4): 253-259 received for publication 30 september 2011 accepted for publication 26 october 2011 254 and 230 ml (control) speeding flats filled with vermiculite. flats were placed in a greenhouse and suspended 15 cm above the benches to facilitate air pruning of roots and to induce rr treatment throughout the experiment period. in each flat 24 seedlings were planted regardless of the original number of cells per flat to minimize the effect of mutual shading, to avoid light competition between plants and to allow for uniform plant density. in order to avoid any water or nutrient stress, a closed fertirrigation system controlled by a timer was established to supply water and nutrients at frequent and regular intervals. the nutrient solution was composed as follows: 10 mm no 3 -, 1 mm h 2 po 4 -, 8 mm k+, 4 mm ca2+, 1.5 mm mg2+, 1 mm so 4 2-, 0.04 mm fe2+ and microelements (ph 6.0, ec=1.2 ms/ cm). the nutrient solution was renewed every week. growth measurements five plants per treatment were sampled at weekly intervals. roots were carefully washed, and then plants were separated into leaves, stems and roots. leaf area was measured with an area meter (delta t-devices ltd., cambridge, uk), plant height was estimated using a ruler, and dry weight for each organ was obtained after oven drying (48 h at 70°c). leaf gas exchange measurements net co 2 assimilation (a), stomatal conductance (g) and transpiration (e) measurements were performed weekly (n=5) on the central sector of the youngest fully expanded leaf by using an open system (cms 400, heinz walz, effeltrich, germany) connected to an assimilation chamber and equipped with a high sensitivity irga (binos, leybold haeraeus, germany) under temperature (24°c) and growing light (400 μmol/m*s par) conditions provided by a mercury vapour lamp (osram hqi-ts 250 w/ndl). calculation of all the parameters was performed following von caemmerer and farquhar (1981) using a specific software (diagas 2.02, walz, effettrich, germany). water use efficiency (wue) was calculated as the ratio between a max and e max . for each crop species, e, a and g were also measured under different light intensities (0, 20, 50, 100, 200, 400, 600, 800 and 1000 μmol/m*s) as described above. a piece of black cloth was used to provide complete darkness, whereas different layers of wire mesh with very small holes were used to provide the required light intensity. chlorophyll content five leaf disks (10 mm diameter) were randomly taken from the uppermost fully expanded leaves at weekly intervals, and extracted in 2 ml of n,n-dimethylformamide for 24 h in the dark. absorbance was then determined for each sample using a spectrophotometer at 647 and 663 nm. chlorophyll a and b contents, and a/b ratio were calculated according to moran (1982). determination of total, osmotic and turgor potentials leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange (n=5). total water potential (ψw) was determined using a pressure chamber (pardossi et al., 1991). osmotic potential (ψs) of the leaf xylem sap was determined using an osmometer (precision system, usa) by determining the freezing point depression of the sample. leaf turgor potential (ψp) was calculated using the following equation (eq. 1): ψp = ψw ψs (eq. 1) measurement of sugar content leaf, stem, and root samples (approx. 50 mg each) were taken at weekly intervals (n=5) and directly freeze-dried in liquid nitrogen. samples were homogenized and extracted with 1 ml hot 80% ethanol, boiled for 5 min, centrifuged at 12000 rpm for 15 min and then the supernatant was collected. the pellet was extracted again as described above, and the supernatant was collected again. at the end, the pellet was evaporated to remove any excess of ethanol. particulates including starch were suspended in 1 ml of koh 20 mm, boiled and centrifuged at 8000 rpm for 15 min and the supernatant was collected. the extract from ethanol was used for sucrose, glucose and fructose determinations, and the extract from koh was used for starch determination. for sugar determination, two 200 μl aliquots from the ethanol extract were taken, one incubated for 30 min at 37°c with 100 μl solution containing invertase (1 mg invertase/ml na-acetate 50 mm at ph 4.6), and the other one with 100 μl solution containing na-acetate 50 mm at ph 4.6, they were then both brought to the final volume (1 ml) with a solution containing 100 mm tris-hcl, ph 7.6, 3 mm mgcl 2 , 2 mm atp, 0.6 mm nadp, 1 unit hexokinase and 1 unit glucose-6-p-dehydrogenase (incubated at 37°c for 30 min). absorbance at 340 nm was then measured by a spectrophotometer. the concentration of glucose in each solution was determined from glucose standard curves according to guglielminetti et al. (1995). the solution without invertase was used to calculate the amount of free glucose in the sample and the difference between the two gave the amount of sucrose (as glucose equivalent). for each of them, 10 μl of solution containing 15 μl of phosphoglucoisomerase in 150 μl of tris-hcl 300 mm at ph 7.6 were incubated at 37°c for 15 min, then absorbance at 340 nm was determined. the difference between the one without invertase and treated with phosphoglucoisomerase and the other without invertase at the first determination gave the amount of free fructose (as glucose equivalent). for starch determination, 100 μl of extract was incubated at 37°c for 1 h with 100 μl solution of na-acetate 100 mm ph 5.2/10 u α-amylase. this solution was then incubated with 100 μl of na-acetate 100 mm ph 4.6/10 u amyloglucosidase at 55°c for 1 h. finally, the solution was boiled and centrifuged to eliminate denaturated protein from α-amylase and amyloglucosidase. 100 μl of this solution was taken and brought to 300 μl with distilled water; starch analysis (as glucose equivalent) was then carried out as mentioned above for glucose. 255 statistical analysis data were analyzed by one-way anova, and means (n=5) were separated using duncan’s multiple range test (p≤0.05). statistical analysis was performed using graphpad prism 4.0 (graphpad software). 3. results and discussion root volume reduction greatly affected growth parameters, confirming several previous results concerning growth depression induced by rr in many horticultural crops (see for example kharkina et al., 1999; saito et al., 2008; shi et al., 2008), but scarcely in pepper (ismail and davies, 1998). total dry weight significantly decreased starting from day 30 after emergence with reducing container size (fig. 1a). in detail, rr pepper plants showed a 3.85-fold lower total dry weight compared to control at the end of the experiment. leaf area was also greatly affected by volume reduction: rr plants showed a 4.15-fold reduction (fig. 1b) at the end of the experiment. rr plants appeared to be smaller (reduced height values) (fig. 1c), denoting a slackened development compared to control plants, with a preferred allocation of dry matter in the root system than in the aerial system, as demonstrated by a slight increase in the root:shoot ratio (fig. 1d). rr generally caused an increase in root:shoot ratio (mugnai et al., 2000), with roots growing in smaller volume forming a highly branched mat. the increased root:shoot ratio reported by some researchers for many crop species subjected to rr might be attributed to an increased substrate temperature in smaller containers in conjunction with a possible temperature dependence of root elongation, as suggested by hurley et al. (1998). during the first month, no significant differences were noticed in leaf gas exchange parameters. stomatal conductance (g) significantly decreased in rr plants (fig. 2a) table 1 chlorophyll content (a, b and a/b ratio) measured at weekly intervals from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants day control plants root-restricted plants (rr) chl a (mg cm-2) chl b (mg cm-2) a/b chl a (mg cm-2) chl b (mg cm-2) a/b 23 12.469 4.485 2.780 11.453 4.170 2.746 30 13.387 4.756 2.814 13.107 4.642 2.823 37 14.671 5.639 2.601 13.845 5.259 2.632 44 16.274* 6.347* 2.564 14.058* 5.589* 2.515 51 17.784* 7.450* 2.387 11.493* 4.768* 2.410 * indicates significantly different values between the two treatments for the same parameter and date for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 1 growth parameters measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total dry weight (a), leaf area (b), plant height (c) and root:shoot ratio (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 2 leaf gas exchange parameters measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants: stomatal conductance (a), net co 2 assimilation (b) and transpiration (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 256 from day 36, leading to a significant reduction in both net co 2 assimilation (a, fig. 2b) and transpiration (e, fig. 2c) until the end of the experiment. the reduction in a was not related to a decrease in the chlorophyll content of rr plants (table 1), as significant differences between the two treatments were noticed for chlorophyll a, b, and a/b ratio only after 43 days from the beginning of the experiment. also, rr plants showed increased instantaneous water-use efficiency values (wue) (fig. 2d), as reported for several species under stress (blum, 2009). rr treatment also affected leaf gas exchange parameters’ response to light (fig. 3). all the parameters (g, a and e) strongly decreased their values, leading to less pronounced response curves. in details, photosynthetic parameters, such as dark respiration, light compensation point and maximum co 2 assimilation, started to significantly decrease after 37 days (table 2). leaf water status did not seem to be the cause of the stomatal closure in rr plants, as total water potential (fig. 4a) and turgor potential (fig. 4c) did not show any significant difference in either of the treatments, even if slight reductions in total water potential and osmotic potential were measured at the end of the experiment in rr plants. table 2 dark respiration (dr, µmol co 2 m-2 s-1), light compensation point (lcp, µmol m-2 s-1 par), maximum co 2 assimilation (amax, µmol co 2 m-2 s-1), water-use efficiency (wue, µmol co 2 mmol h 2 o-1) measured every two weeks from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants day control plants root-restricted plants (rr) dr lcp amax wue dr cp amax wue 23 -1.07 14.06 17.31 5.47 -0.9 12.83 15.11 5.51 37 -3.31* 41.56* 24.87* 5.59* -2.75* 71.50* 6.67* 6.34* 51 -2.93 42.17* 15.71* 5.55* -2.87 85.56* 6.46* 6.65* * indicates significantly different values between the two treatments for the same parameter and date for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 3 light saturation curves for the three leaf gas exchange parameters (g, a and e) measured every two weeks from day 23 to the end of the experiment in both control and root-restricted (rr) plants. 257 this result also confirmed that no symptom of water stress ever occurred during the experimental period, leading to a positive feedback about our experimental system. our results reveal that rr significantly reduces g, as previously noticed by other authors on different species (ismail and noor, 1996; kharkina et al., 1999), and that g is the primary cause of the reduction in a in rr plants, suggesting a stomatal factor limiting the photosynthetic rate under rr conditions (shi et al., 2008). however, the decline in g was not correlated to a concurrent decline in total water potential, as leaf tissues were able to maintain a high level of turgor during the whole experiment. therefore, other factors should be involved in the stomatal closure. it has been suggested that rr induces a reduction in g through a decrease in the supply of growth substances from roots to shoots and/or an imbalance in root and shoot hormones. for example, shi et al. (2008) reported that shoot growth suppression might be caused by the influence of aba originating from the restricted roots. ismail and davies (1998) found that the slight increase in xylem sap [aba] measured in pepper plants could not account for the reduction in leaf growth and g. they suggested that insufficient aba synthesis occurred to trigger the processes that cause reductions in leaf growth and g. sugar content determination led to interesting results. sucrose content significantly increased in rr plants starting from day 37 (fig. 5a). also, rr treatment led to a clear increase in glucose content (fig. 5b) and a concurrent decrease in fructose content (fig. 5c) together with a great accumulation of starch (fig. 5d). in particular, starch accumulation in the tissues began early in the developmental process (day 29). starch was mainly compartmentalised in the leaves (fig. 6a) of rr plants, whereas no significant differences were noticed both in stems (fig. 6b) and in roots (fig. 6c) between control and rr plants, except for day 33. the decline in a observed in rr conditions was often interpreted as a feedback inhibition by carbohydrate accumulation (pezeshki and santos, 1998). plant fig. 4 leaf water status determined at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total water potential (a), osmotic potential (b) and turgor (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 5 sugar content measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total sucrose (a), total glucose (b), total fructose (c) and total starch (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 258 growth is strongly affected by leaf photosynthetic activity, since photosynthates are essential either as the source of carbon used for the build-up of organic compounds or as the source of energy needed for biochemical reactions involved in growth and maintenance processes. growth rate may regulate photosynthesis either through effects on the supply of growth substances translocated into leaves or through effect on the translocation rate of photosynthates from leaves to the growing organs (carmi et al., 1983). the accumulation of photosynthates is influenced by the rate of their translocation to the sink organs (sonnewald and willmitzer, 1992), and sink demand for photosynthates has a marked influence on source leaf photosynthesis, which is greatly dependent on sink strength, considered as a product of sink size and sink activity (sonnewald and willmitzer, 1992). however, sink size is determined by different parameters. roots are recognized as a metabolic sink that influences the partitioning of photosynthetically fixed carbon (gifford and evans, 1981; robbins and pharr, 1988). sink limitation caused by rr can greatly reduce leaf photosynthetic rate in many crop species (hameed et al., 1987; ismail and noor, 1996; whiley et al., 1999; shi et al., 2008), and reduced translocation of assimilates from leaves (robbins and pharr, 1988; kharkina et al., 1999). rr often promotes an accumulation of non-structural carbohydrates in the stem and leaves in response to the lack of the active sinks (nishizawa and saito, 1998), meaning that the difference in the growth rate between rr and control treatments was not due to a decrease in assimilates’ supply to the organs whose growth was restricted (mandre et al., 1995). our results suggest that the role of the leaves as sink organs may increase when root growth is extremely limited by volume restriction and a relatively larger amount of carbohydrate may accumulate in the canopy. a new shoot to root equilibrium may be established for an increased function of leaves and stem, together with a concurrent diminished function of the roots. therefore, it can be concluded that as a result of reduced vegetative growth an excess of assimilates was produced which could not be used for growth, and thus accumulated in the form of starch, as also indicated by shi et al. (2008). accumulation of nonstructural carbohydrates in the leaves in response to rr could provide a feedback mechanism that reduces carbon metabolism (thomas and strain, 1991). starch accumulation may reduce net photosynthetic rate by avoiding intracellular co 2 transport (shi et al., 2008). however, contradictory results were obtained by rieger and marra (1994), suggesting that reduced co 2 assimilation cannot always be explained by a feedback inhibition of carbohydrates. the relatively low maximum assimilation (a max ) rates for container-grown plants compared to field-grown plants may be attributed to containers restricting the root sink, thus causing the photo assimilate supply to exceed the capacity of demand (i.e. end-product inhibition of photosynthesis) as indicated by whiley et al. (1999). in conclusion, our results show that growth reduction by rr is mainly linked to a photosynthetic limitation, caused by a reduced stomatal conductance (probably driven by both stomatal factors and hormonal substances) and a strong accumulation of starch in the leaf tissues, which led to a feedback inhibition of the photosynthetic process. acknowledgements the authors are particularly indebted to prof. franco tognoni for scientific support. references blum a., 2009 effective use of water (euw) and not water-use efficency (wue) is the target of crop yield improvement under drought stress. field crops research, 112(2-3): 119-123. fig. 6 starch content in the different plant organs measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:leaf (a), stem (b) and roots (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 259 carmi a., hesketh j.d., enos w.t., peters 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of horticultural science and landscape, faculty of agriculture, ferdowsi university of mashhad, mashhad, iran. key words: clove, hydraulic conductivity, peppermint, thyme, vase life. abstract: the effects of pulse and permanent treatments were studied on vase life, water content and hydraulic conductivity of gerbera cut flower cv. rosalin. this study was conducted as a factorial experiment based on completely randomized design with three replications. the first factor was pulse treatments, using nanosilver (ns) 5 and 10 mg/l, salicylic acid (sa) 50 and 100 mg/l and distilled water as control, and the second factor was permanent treatments applying distilled water, sucrose, peppermint, thyme and clove essential oils (eo). the results showed that ns 10 mg/l + peppermint eo 100 mg/l and ns 10 mg/l + thyme eo 100 mg/l treatments had the best effect on longevity and maintaining the water content and hydraulic conductance of rosalin cut flower, compare to other treatments. these solutions enhanced life of gerbera cut flowers to about 14 days. flower water content was high (about 90%) except in 4% sucrose permanent treatment flowers which decreased more rapidly during the vase life. the effective hydraulic conductivity was observed in ns 10 mg/l + peppermint eo 100 mg/l (0.16 cm/min) and ns 10 mg/l + thyme eo 100 mg/l (0.21 cm/min) solutions and had nearly stable trend even in day 8 after pulsing. 1. introduction gerbera (gerbera jamesonii bolus, asteraceae) commonly known as transvaal daisy, barberton daisy or african daisy, is one of the ten most popular and important commercial cut flowers grown in a wide range of climatic conditions. gerbera is a perennial, tropical, herbaceous plant with colorful and attractive flowers that are widely used as a decorative garden plant or cut flowers. cut gerbera flowers consist of a terminal composite floral head (inflorescence), called the capitulum, and a stem, which called scape and has no leaves (dole and wilkins, 2006). gerbera has the fourth place in the international cut flower market. the flowers are hardy and resist against transport conditions. however, the most important problem of the gerbera cut flowers is short vase life. the end of vase life of cut gerbera flowers is often due to bending of the scape, which precedes wilting of the ray florets (nair et al., 2003; van son, 2007; ansari et al., 2011; perik et al., 2012; kilic and cetin, 2014; aghajani and jafarpour, 2016). however, postharvest life of cut flowers could be affect(*) corresponding author: mahroo.motaghayer@gmail.com citation: motaghayer m.s., azizi m., teheranifar a., 2019 nanosilver, salicylic acid and essential oils effects on water relations of gerbera ‘rosalin’ cut flowers. adv. hort. sci., 33(2): 271-281 copyright: © 2019 motaghayer m.s., azizi m., teheranifar a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 august 2018 accepted for publication 6 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 271-281 272 ed by the application of various chemicals as preservatives (nair et al., 2003; prashanth et al., 2010). insufficient water uptake is one of the main reasons for water deficit and wilting during the vase life (knee, 2000; van ieperen et al., 2002). stem end blockage is a main factor in the imbalance between water uptake and water loss from cut flowers (he et al . , 2009). researches showed that bacteria (microbes) (van meeteren, 1978; he et al., 2006) or bacteria and decay products (liu et al., 2009) cause the blockage of cut gerbera flower. bacteria in vase water can block the vessels in the surface of cut stems (ferrante et al., 2007). bacterial inhibitors such as silver nanoparticles or salicylic acid could extend vase life of cut flowers (loubaud and van doorn, 2004; solgi et al., 2009; vahdati mashhadian et al., 2012). physiological substances such as lignin, mucilage or gum (van doorn and cruz, 2000; loubaud and van doorn, 2004, wang et al., 2014) and cavitation (van meeteren et al., 2006) decrease the vase life of some cut flowers. the air emboli or cavitation would be reduced if cut flowers were put into water after cutting immediately (van ieperen et al., 2002). cut flowers are sensitive to microbial contamination at the stem end and this determines a reduction of their vase life (van meeteren, 1978, van doorn and de witte 1994, balestra et al., 2005). nano technology is based on engineered particle of 1-100 nm (diameter). nanosilver (ns) is included in this technology and can have more chemical and biological activities in order to reduce size. in recent years, ns is being used as a new antiseptic for many industrial processes like medical industry, water purification and vegetable disinfection (rai et al., 2009). in addition, ns treatment has been proposed for improving the postharvest life of cut flowers (liu et al., 2009; solgi et al., 2009; ansari et al., 2011; danaee et al., 2013). the role of salicylic acid (sa), as an internal growth regulator and a natural phenolic compound, has been completely proved in multiple physiological processes like ethylene biosynthesis, stomatal conductance, respiration, senescence and the activation of defense systems against different pathogens. by activating antioxidant enzymes, sa delays the process of senescence in flowers. in addition, sa inhibits ethylene synthesis and action (raskin, 1992; hayat et al., 2010; an and mou, 2011; jamshidi et al., 2012). exogenous supply of carbohydrate can play an important role in lengthening the vase life and postharvest conditions of cut flowers. the gerbera cut flowers have short postharvest life. sucrose effect on enhancing the vase life of cut flowers is associated with water balance. the application of sucrose treatment and sugars accumulated in the flowers increase the sugar and osmotic concentration, improve water absorption and flower turgidity (reddy and singh, 1996; prashant et al., 2010; bhanusree et al., 2015). researches also showed that the combined use of ns 5 mg/l with 4% sucrose and 2.5 mg/l gibberellic acid increase postharvest life of gerbera (ansari et al., 2011). many chemicals have been used in cut flowers vase solutions for inhibiting microorganisms’ growth and extending the vase life by improving water uptake. these chemicals include silver nitrate, 8hydroxyquinoline sulfate and 8-hydroxyquinoline citrate, which are expensive and harmful for the environment and human health (nowak et al., 1990; ichimura et al., 1999; nair et al., 2003; motaghayer and esna-ashari, 2009; solgi et al., 2009; ansari et al., 2011). it is crucial to use natural, safe and inexpensive compounds for the large-scale application of preservatives improving cut flower vase life (kilic and cetin, 2014). essential oils (eo) are organic, natural, safe and eco-friendly substances that have strong anti-inflammatory, antibacterial, antifungal, antioxidant and anticarcinogenic effects. these properties are attributed to the high levels of phenolic compounds (solgi et al., 2009; bayat et al., 2011; raut and karuppayil, 2014). the application of different medicinal plants eos on increasing the vase life of cut flowers have been studied by many researches. the effect of peppermint (mentha pipperita l.) eo has increased freshness and quality of flower color and prevented the discoloration in alstroemeria (babarabie et al., 2016), flower’s quality and delay of leaf and flower senescence of tuberose cv. pearl (hoseini and korehpaz, 2015) and vase life of ‘utopia’ rose cut flowers (saghazadeh et al., 2014). thyme (thymus vulgaris l.) eo (solgi et al., 2009) and water extract of thyme (amini et al., 2014) was added to the preserving solution for extending the vase life of gerbera ‘dune’ cut flower and essence containing thymus vulgaris and cuminum cyminum increased solution uptake and quality of gerbera ‘sorbet’ cut flowers (dareini et al., 2014). clove (eugenia caryophyllata thunb.) eo increased lisianthus cut flower vase life (kazemi et al., 2014) and clove eo and water extract increased gerbera ‘ecco’ vase life (ziyaei movahed et al., 2010). there are two different ways for treating cut flowers; pulse and permanent treatment. pulsing is a motaghayer et al. nanosilver and essential oils effects on gerbera ‘rosalin’ cut flowers 273 short-term treatment that can be done by producers and it helps postharvest vase life and flowering after storage period. permanent treatment mostly is a long-term treatment, which can be done by consumers for enhancing cut flower vase life (abdelkader and rogers 1986; nowak et al., 1990; arora and singh, 2002). sucrose can maintain the cell’s turgor pressure and provide energy for cellular respiration, also is an important nutrient for microorganisms. therefore, it should not be used without anti-microbial agents in preservatives (nowak et al., 1990). the effect of ns (liu et al., 2009) and sa (jamshidi et al., 2012) treatments alone on extending cut flowers vase life was assessed in different researches. since the effects of different concentrations of various preservative solutions on the postharvest life of cut flowers are altering depend on plant species, the applied chemicals and interaction of their compounds in vase solution and the method of treatment, the determination of the effective preservatives as well as the method of application is very important. therefore the aim of this study was to screen the effects of ns and sa as pulse treatment and sucrose and thyme, clove and peppermint eos as permanent treatment on vase life and hydraulic conductivity of gerbera ‘rosalin’ cut flowers. 2. materials and methods plant growth conditions and treatments gerbera (g. jamesonii cv. ‘rosalin’) flowers were grown in standard hydroponic greenhouse conditions in ferdowsi university of mashhad, iran. the flowers were harvested during morning by pulling out the stems from the plants when 2-3 rows of stamens of the bisexual disc florets were mature. stems were pulled, not cut and the base of stem was removed before hydration (dole and wilkins, 2006). the stems were taken immediately to the laboratory and recut under water to 35 cm length. the cut flowers were immediately immersed individually into 500 ml vase solutions. in order to simulate the domestic use, the vase solutions were not changed and the stems were not recut during the experiment. the end of gerbera cut flower vase life was considered as the time in which more than one third of the outer petals of inflorescence start to be brown or wilted or curled or stem bending (≥90°) or breaking was occurred (dole and wilkins, 2006). this study was conducted as a factorial experiment based on completely randomized design with three replications and four stems in each replicate. the first factor was pulse treatments: distilled water (d), salicylic acid (sa) 50 and 100 mg/l (merck company), nanosilver (ns) 5 and 10 mg/l (nanocid company, iran). the second factor was permanent treatments: distilled water, sucrose 4% (merck company), peppermint eo 100 mg/l, thyme eo 100 mg/l, clove eo 300 mg/l (zardband company, iran) (table 1). pulse treatments were applied for 24 h. treated stems were then stood into vases containing permanent treatments. vase solutions were freshly prepared at the beginning of the experiment and not renewed during of the study. the eos constituents were determined by zardband company (iran) using gc-ms analysis table 1 pulse and permanent treatments used in the experiment pulse treatment/permanent treatment distilled water/distilled water distilled water/sucrose distilled water/peppermint eo distilled water/thyme eo distilled water/clove eo sa 50 mg.l-1/distilled water sa 50 mg.l-1/sucrose sa 50 mg.l-1/peppermint eo sa 50 mg.l-1/thyme eo sa 50 mg.l-1/ clove eo sa 100 mg.l-1/distilled water sa 100 mg.l-1/sucrose sa 100 mg.l-1/peppermint eo sa 100 mg.l-1/thyme eo sa 100 mg.l-1/ clove eo ns 5 mg.l-1/distilled water ns 5 mg.l-1/sucrose ns 5 mg.l-1/peppermint eo ns 5 mg.l-1/thyme eo ns 5 mg.l-1/ clove eo ns 10 mg.l-1/distilled water ns 10 mg.l-1/sucrose ns 10 mg.l-1/peppermint eo ns 10 mg.l-1/thyme eo ns 10 mg.l-1/ clove eo adv. hort. sci., 2019 33(2): 271-281 274 (table 2). gc-ms analysis revealed that the major constituents of the eos were: thymol (53.5%) in thyme eo; 1-menthol (41.22%) and menthone (24.01%) in peppermint eo and eugenol (62.4%) in clove eo. measuring hydraulic conductivity hydraulic conductivity was measured by a slight modification in the method of melcher et al. (2012) (fig. 1). a piece of 15 cm of flower stem end was cut with a sharp blade under distilled water. the upper part of the stem (part 1 in fig. 1) was inserted into a silicon tube (part 2 in fig. 1) (internal diameter 4 mm) filled with degas distilled water and the basal part of the stem (part 3 in fig. 1) was kept in the degas distilled water. using a three-way glass valve (part 4 in fig. 1), the silicon tube was connected from one side to the degassed distilled water tank (part 5 in fig. 1) and from the other side was attached into a ushaped pipe (part 6 in fig. 1) below the stems end. the whole set (stem, three-way glass valve, degas distilled water tank and u-shaped pipe) was fixed (van ieperen et al., 2002). the stem vase was placed on a digital scale (part 7 in fig. 1) connected to the computer and the stem and degas distilled water weight changes were recorded at time regular intervals (30 minutes). fifty cm head pressure of water (h; which made 5 kpa pressure) was applied, so that water had passed through the segments. the flow rate was then determined by measuring the volume of the passed water. three stem segments were used for each treatment (ichimura et al., 2005). measurements of stem hydraulic conductivity involves measuring the flux for a given driving force (q⁄δp; where δp is the pressure drop across the segment), normalized by the length of the stem segment and referenced either to the cross-sectional area of the stem (melcher et al., 2012). in equation 1, k (cm/min) is hydraulic conductivity, q (cm3/min) is the recorded flux (gravimetric or volumetric flow rate), l (cm) is the length of the measured segment, a (cm2) is the cross-sectional area of the stem segment and h (cm) is head pressure of water height. the data were collected at days 2, 4, 6 and 8 after pulse treatment. k = ql/ah) (1) to observe the microscopic effects of chemicals and eos on stem closure and hydraulic conductivity during the vase life, 2 cm of treated stem was used for histological study. the cut stem segments (3-5 cm in length) were stored in a solution of faa (formalin (40%): glacial acetic acid (50%): ethyl alcohol (70%): fig. 1 hydraulic conductivity system scheme; the upper part of the stem (1); silicon tube (2); the basal part of the stem (3); the three-way glass valve (4); the degas distilled water tank (5); the u-shaped pipe (6); the digital scale (7); h: water height. table 2 major chemical constituents of the eos peppermint eo thyme eo clove eo gcms analysis (%) gcms analysis (%) gcms analysis (%) limonene 2.25 terpinene gamma 7.20 alpha copaene 0.04 cineole 4.59 para-cymene 27.4 beta caryophyllene 3.79 menthone 24.01 thymol 53.50 alpha humulene 0.45 isomenthone 3.83 oxyde de caryophyllene 0.29 1-methyl acetate 4.38 eugenol 81.83 neomenthol 2.84 isoeugenol 0.13 1-menthol 41.22 acetate de eugenyle 12.50 pulegone 1.56 methyl eugenol 0.01 menthofuran 2.98 density (20°c) 0.9036 0.923 10.636 refractive index(20°c) 14.605 1.502 15.335 optical rotation (°) -23.68 -1.0 -0.35 batch 13/47/23 45109 68382 motaghayer et al. nanosilver and essential oils effects on gerbera ‘rosalin’ cut flowers 275 and permanent solutions had remarkable effect on flower longevity. the best treatments were ns 10 mg/l + thyme eo 100 mg/l (14.25 days) and ns 10 mg/l + peppermint eo 100 mg/l (14 days) which had significant difference with other treatments (table 4). it has been observed that sa 50 mg/l + 4% sucrose was less effective than control on flower postharvest life and sucrose had negative effect on gerbera flowers life (table 4). flower water content flower water contents for the pulse treatments with distilled water and 50 mg/l sa were a little higher than other treatments (table 5). in 4% sucrose per[13:5:200]) to preserve the tissue before sectioning. stem transverse sections, at 16-µm thickness, were made using a manual rotary microtome (leitz 1512, germany) after fixing in faa and permanent mounts were prepared in paraffin wax. cross sections were stained with safranin o/fast green stain method and embedded on microscope slides. digital images were made at 10x magnification with a digital camera (olympus dp71, japan) attached to a light microscope (olympus bh2, japan) and computer. flower water content flower water content (wc) was measured as mentioned in equation 2. flower fresh weights (fw) were assessed at the beginning of the experiment and flower dry weights (dw) were recorded after drying to constant weight in an oven for at least 48 h at 85°c. water content was calculated for three replicates (he et al., 2006; lu et al., 2012). wc = (fw-dw)/dw *100 (2) the experiment was conducted in the laboratory at 20-22°c, 40-50% rh, and 15 µmol/m2s light intensity (cool white florescent tubes) under a daily light period of 12 hours. the obtained data were analyzed using mstat-c program and mean comparison was done using lsd range test. 3. results flower vase life results of this study showed that in single effect of applied treatments, all ns pulse treatments markedly (p<0.01) extended vase life of gerbera ‘rosalin’ cut flowers. the 10 mg/l ns pulse treatment gave the longest vase life (12.20 days) as compared to the other treatments (table 3). sa 100 mg/l (9.08 days) pulse treatment significantly increased flower vase life compared to the control (8.13 days). however, there was no significant difference between ns 5 mg/l and sa 100 mg/l. the single effect of applied treatments indicated that peppermint and thyme eos (100 mg/l) application in preservative solutions as permanent treatment (fig. 2) could extend the vase life of gerbera cut flowers to 9.98 and 10.35 days respectively (table 3). however, there was no significant difference among these two treatments and control in extending vase life. flowers placed in sucrose 4% and clove eo 300 mg/l had the least (8.83 days) vase life (table 3). the results showed that the interaction of pulse table 3 the simple effect of pulse and permanent treatments on ‘rosalin’ gerbera cut flower vase life (day) the means showing similar letters in each column have no significant difference according to the lsd range test (p<0.01). treatments vase life (day) pulse treatment distilled water 8.13 d sa 50 mg.l-1 8.30 cd sa 100 mg.l-1 9.08 bc ns 5 mg.l-1 10.02 b ns 10 mg.l-1 12.20 a permanent treatment distilled water 10.02 a sucrose 8.55 b peppermint eo 9.98 a thyme eo 10.35 a clove eo 8.83 b fig. 2 gerbera ‘rosalin’ cut flowers treatment by ns (10 mg/l) and peppermint eo (100 mg/l). during pulse treatment, vase solution was covered by dark plastic coverage to prevent undesirable light reaction in ns. 276 adv. hort. sci., 2019 33(2): 271-281 manent treatment flowers, water content declined more rapidly during the vase life period and was significantly different from the others. generally, the interaction of pulse and permanent treatment showed that 4% sucrose had negative effect especially after 10 mg/l ns application as pulse treatment (table 4). hydraulic conductivity the hydraulic conductance of the stem end segments did not change over the first 2 days after pulsing and had very low rates. thereafter it changed over time and increased slightly on day 4. the rate of stem flower hydraulic conductivity sharply increased at day 6 and 8 after pulse treatment application. hydraulic conductivity of stems treated with 5 and 10 mg/l ns pulse markedly showed lower rate during the experiment compared to other treatments (table 6). in addition, permanent treatments had significant effect on hydraulic conductance throughout assessment. result showed that peppermint eo 100 mg/l had lower look rate in hydraulic conductance than other solutions (table 7). however, hydraulic conductance of the stem segment was nearly the same at the initial day of the experiment. the interaction of pulse and permanent treatment determined that ns 10 mg/l + peppermint eo 100 mg/l and ns 10 mg/l + thyme eo 100 mg/l had the lowest rate even in day 8 after pulsing (table 8). in the ns 10 mg/l + peppermint eo 100 mg/l flowers, the hydraulic conductance of the stem segments slightly increased thereafter. hydraulic contable 6 the effect of pulse treatments on trends of hydraulic conductivity of gerbera cut flower stem on day 2, 4, 6 and 8 of the experiment table 4 the effect of different treatments’ interactions on ‘rosalin’ gerbera cut flower vase life (day) and water content (%) table 5 the effect of pulse and permanent treatments on ‘rosalin’ gerbera cut flower water content (%) the means showing similar letters have no significant difference according to the lsd range test (p<0.01). treatment vase life (day) water content (%) distilled water/distilled water 9.58 efg 90.95 ab distilled water/sucrose 8.08 i 86.15 c distilled water/peppermint eo 7.83 ij 91.29 a distilled water/thyme eo 8 i 91.39 a distilled water/clove eo 7.17 jk 91.21 a sa 50 mg.l-1/distilled water 9.08 fgh 91.31 a sa 50 mg.l-1/sucrose 7 k 85.02 c sa 50 mg.l-1/peppermint eo 8.08 i 91.09 a sa 50 mg.l-1/thyme eo 8.92 gh 90.97 a sa 50 mg.l-1/ clove eo 8.42 hi 91.15 a sa 100 mg.l-1/distilled water 9.83 def 90.54 ab sa 100 mg.l-1/sucrose 8.58 hi 83.44 d sa 100 mg.l-1/peppermint eo 9.17 fgh 90.45 ab sa 100 mg.l-1/thyme eo 9.83 def 90.73 ab sa 100 mg.l-1/ clove eo 8 i 89.46 b ns 5 mg.l-1/distilled water 9.67 efg 90.55 ab ns 5 mg.l-1/sucrose 8.58 hi 82.68 d ns 5 mg.l-1/peppermint eo 10.83 c 90.81 ab ns 5 mg.l-1/thyme eo 10.75 c 90.56 ab ns 5 mg.l-1/ clove eo 10.25 cde 90.42 ab ns 10 mg.l-1/distilled water 11.92 b 90.52 ab ns 10 mg.l-1/sucrose 10.50 cd 81.1 e ns 10 mg.l-1/peppermint eo 14 a 91.36 a ns 10 mg.l-1/thyme eo 14.25 a 91.22 a ns 10 mg.l-1/ clove eo 10.33 cde 90.99 a the means showing similar letters in each column have no significant difference according to the lsd range test (p<0.01). treatment water content (%) pulse treatment distilled water 90.20 a sa 50 mg.l-1 89.91 a sa 100 mg.l-1 88.93 b ns 5 mg.l-1 89.00 b ns 10 mg.l-1 89.04 b permanent treatment distilled water 90.77 a sucrose 83.68 b peppermint eo 91.00 a thyme eo 90.97 a clove eo 90.65 a the means showing similar letters in each column have no significant difference according to the lsd range test (p<0.01). pulse treatment k (cm/min) day 2 day 4 day 6 day 8 distilled water 0.57 d 5.42 a 7.58 b 8.95 c sa 50 mg.l-1 1.04 b 5.35 a 12.96 a 9.17 c sa 100 mg.l-1 1.72 a 6.44 a 10.42 a 15.47 a ns 5 mg.l-1 0.84 c 2.50 b 4.45 c 13.08 b ns 10 mg.l-1 0.57 d 0.91 c 2.05 d 4.97 d motaghayer et al. nanosilver and essential oils effects on gerbera ‘rosalin’ cut flowers 277 ductivity of other treated flowers increased sharply after day 6 during the rest of the vase life (table 8). the survey of slope trend of each treatment during the experiment’s period, showed that the lowest slope was observed in ns 10 mg/l + peppermint eo 100 mg/l and ns 10 mg/l + thyme eo 100 mg/l respectively. while in other treatments, slope trend was enhanced so that the most slope trend was considered in 50 mg/l sa + 4% sucrose. in addition, histological study showed that in the ns 10 mg/l + peppermint and thyme 100 mg/l eos treated flowers stem remained healthy for longer period while the other stems became hollow after a few days (fig. 3 and 4). table 7 the effect of permanent treatments on trends of hydraulic conductivity of gerbera cut flower stem on day 2, 4, 6 and 8 of the experiment the means showing similar letters in each column have no significant difference according to the lsd range test (p<0.01). permanent treatment k (cm/min) day 2 day 4 day 6 day 8 distilled water 0.81 c 5.14 a 8.66 b 14.62 a sucrose 1.46 a 4.93 ab 13.50 a 12.76 a peppermint eo 0.84 c 2.74 c 3.61 d 6.25 c thyme eo 0.69 d 3.65 bc 6.00 c 8.77 b clove eo 0.94 b 3.52 bc 5.07 cd 9.25 b table 8 the trends of hydraulic conductivity of gerbera cut flower stem in all treatment on day 2, 4, 6 and 8 of the experiment the means showing similar letters in each column (day) have no significant difference according to the lsd range test (p < 0.01). treatment k (cm/min) slope trend day 2 day 4 day 6 day 8 distilled water/distilled water 0.42 ghi 5.60 bcd 9.41 cde 17.04 cd 53.664 distilled water/sucrose 0.64 fg 5.96 abcd 8.88 def 14.04 ef 43.117 distilled water/peppermint eo 0.21 hi 4.89 cde 4.62 ghi 0.00 j 5.16 distilled water/thyme eo 0.62 fg 4.52 de 6.41 fg 0.00 j 52.384 distilled water/clove eo 0.96 de 6.14 abcd 9.23 cde 13.69 f 41.263 sa 50 mg.l-1/distilled water 0.76 ef 7.91 a 11.28 cd 14.95 def 45.931 sa 50 mg.l-1/sucrose 1.14 d 7.58 ab 43.58 a 0.00 j 16.337 sa 50 mg.l-1/peppermint eo 1.51 bc 1.94 fgh 2.74 ij 0.00 j 12.728 sa 50 mg.l-1/thyme eo 0.57 fg 4.39 de 9.54 cd 14.21 ef 46.081 sa 50 mg.l-1/ clove eo 1.19 cd 5.67 bcd 12.34 c 16.71 cde 53.215 sa 100 mg.l-1/distilled water 1.53 b 7.30 ab 11.33 cd 14.69 def 43.499 sa 100 mg.l-1/sucrose 1.67 ab 6.87 abc 21.41 b 21.51 a 74.065 sa 100 mg.l-1/peppermint eo 1.83 ab 5.17 cd 6.60 efg 20.43 ab 57.224 sa 100 mg.l-1/thyme eo 1.67 ab 7.95 a 10.99 cd 20.72 ab 60.198 sa 100 mg.l-1/ clove eo 1.94 a 5.10 cd 5.31 gh 0.00 j 5.769 ns 5 mg.l-1/distilled water 1.04 de 4.22 de 8.54 def 18.74 bc 5.741 ns 5 mg.l-1/sucrose 1.91 a 3.18 ef 5.28 gh 22.59 a 64.126 ns 5 mg.l-1/peppermint eo 0.52 fgh 2.27 fg 4.69 ghi 10.40 g 32.056 ns 5 mg.l-1/thyme eo 0.45 ghi 1.97 fgh 4.26 ghi 7.48 hi 23.375 ns 5 mg.l-1/ clove eo 0.40 ghi 1.07 ghi 0.67 k 6.19 i 16.963 ns 10 mg.l-1/distilled water 0.40 ghi 1.51 ghi 3.93 hi 7.68 ghi 24.278 ns 10 mg.l-1/sucrose 1.91 a 1.86 fgh 4.79 ghi 5.65 i 14.158 ns 10 mg.l-1/peppermint eo 0.16 i 0.12 i 0.28 k 0.41 j 0.0904 ns 10 mg.l-1/thyme eo 0.21 i 0.57 hi 0.94 jk 1.42 j 0.4012 ns 10 mg.l-1/ clove eo 0.23 hi 0.67 hi 1.02 jk 9.65 gh 28.601 fig. 3 the gerbera cut flower healthy stem. adv. hort. sci., 2019 33(2): 271-281 278 3. discussion and conclusions flower vase life ns particles enter into cell, tissue and organs, so they can replace with silver salts (such as silver nitrate or silver thiosulfate) in preservative solutions. ns inhibits the respiration and electron transfer system and material transfer in microbial cell membrane (paull and lyons, 2008). various researches indicated that flowers treated with ns solution, had more vase life. silver ions, because of small size, have more contact with outer space and influence more on their environment. ns, in comparison with silver ions, showed antimicrobial property at inferior concentration (solgi et al., 2009; ansari et al., 2011) different studies determined antimicrobial effects of main components of thyme (nikolić et al., 2014 a), peppermint (kazem alvandi et al., 2011; nikolić et al., 2014 b) and clove eo (boukaew et al., 2017). in addition, amini et al. (2014) reported that thyme eo in pulsing with distilled water treatment showed the best results for extending cut gerbera flower vase life and preventing more weight loss. hydrophobicity is an important characteristic of thyme and peppermint eos. this enables them to separate the lipid components of the bacterial cell membrane and mitochondria, binding to membrane proteins and releasing lipopolysaccharides, which results in disturbing cell wall structures (solgi et al., 2009). researches indicated also that sa pulse treatment, followed by ns as permanent solution (danaee et al., 2013), sa utilization as permanent treatment (jamshidi et al., 2012) significantly promoted the vase life of gerbera cut flowers. however, in this study, sa had no special effect on flower longevity. although ziyaei movahed et al. (2010) reported that clove eo increased gerbera vase life. in this study, it had the least effect on postharvest life of gerbera cut flowers. despite of sucrose important role in extending the vase life of cut flowers, ansari et al. (2011) reported a negative effect on ‘rosalin’ gerbera cut flowers. the main reason could be severe bacterial growth in vase solution. flower water content cut flowers and foliage can have limited commercial value because they dehydrate during vase life because of water uptake decrease. water deficit could develop even when cut flowers are placed in water (nazari deljou, et al., 2012). gerbera cut flowers stem break of is mainly caused by water shortage in the flowers due to the increased difficulty of water flow from the water source to petals. it is also supposed to be a competition for available water between flower heads and stems. the increase in flow resistance leads to stem break as a result of microbial activity in the vase water (balestra et al., 2005). it could be concluded from the results of this study that in all treatments with antimicrobial agents such as ns, sa and thyme, peppermint and clove eos flower water contents were high and had no harmful decrease. hydraulic conductivity many experiments were performed to find out the cause of stem bend in gerbera cultivars. research showed that removal of the floral head prevented stem bending, indicating that bending is physically due to the gravitational pull on the floral head. stem bending in cut gerbera can be due to lack of mechanical support. bending might relate to lack of wall thickening, particularly in the xylem. at least two other factors might contribute to mechanical stem strength. the first factor is gerbera stems elongation during vase life. elongation zones usually have weakly developed xylem and sclerenchyma. since the stems are usually placed in water under an angle, stem elongation will increase the gravitational pull of the floral head result in earlier stem bending. the second factor is the presence of a cavity in the center of the gerbera stem. observations showed the cavity at the time of harvest, in several cultivars (perik et al., 2012). other factors affecting stem bending could be adverse water relations such as lack of turgor. after a few days of vase life, there are many bacteria in the vase solution. stem bending can be due to xylem blockage by bacteria, which results in low water uptake. as transpiration is not inhibited as fig. 4 the gerbera cut flower hollow stem. motaghayer et al. nanosilver and essential oils effects on gerbera ‘rosalin’ cut flowers 279 much as water uptake, net water loss occurs followed by the loss of turgor and stem bending (van meeteren, 1978). generally, in cut flowers fresh weight decreased before stem bent occurred, and this is accompanied by a decline in absorption of water by the flowers. stem break could be prevented by pretreatment of the stems with ns by adding to the vase water. van meeteren (1978) suggested that there are two different pathways for water uptake: a direct one through the xylem vessels at the cut surface and an indirect one through the cavity in the stem. only the direct water uptake is strongly inhibited by growth of bacteria in the vase water. stem bend occurs when the direct water uptake is inhibited by bacterial activity. van meeteren (1978) suggested that the minimum concentration of silver nitrate could avoid stem bending and inhibit bacterial growth in the water. there is an association between a high population density of bacteria in the water and scape bending (van doorn and de witte, 1994). the results of this experiment also showed that bacteria would block the main water pathway (xylem vessels) over the time and the stem would become hollow (fig. 3 and 4). so that active water uptake is effectively prevented. bacterial activity could be significantly inhibited by adding ns (as pulse treatment), peppermint (1menthol and menthone) and thyme (thymol) eos (as permanent treatment) to vase solution. based on the results of this study, new antimicrobial agents such as ns, thyme and peppermint eos had a positive effect on flower vase life and water content. it might be due to this fact that these are very effective antimicrobial agents, which inhibited the microbial growth and prevented bacterial plugging in conducting tissues. however, exogenous supply of sugars can increase water balance and osmotic concentration and plays an important role in lengthening the vase life of cut flowers, but in this research, sucrose had negative effect on flower water contents and gerbera ‘rosalin’ vase life period. this issue can be related to the negative effect of sucrose on microbial growth. although, there was no available data about the effect of eos on stem hydraulic conductivity changes in cut flowers, this research showed that eos could improve hydraulic conductance and keep it in a normal and stable condition for longer period. references abdel-kader h., rogers m.n., 1986 postharvest treatment of gerbera jamesonii. acta horticulturae, 181: 169-176. aghajani n., jafarpour m., 2016 effects of pre-and postharvest treatments of silicon and rice hull ash on vase life of 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gerbera cv. hongyan. j. agric. sci. technol., 16(2): 365-372. ziyaee movahed z., kafi m., khalighi a., azizi m., sharifi r., 2010 investigation of the possibility in replacing natural ingredients (essential oil and extracts of clove) instead of antibacterial chemicals ingredients in preservative solution of the gerbera cut flower. iranian j. hortic. sci., 41: 337-345 (in persian). impaginato 549 adv. hort. sci., 2018 32(4): 549-556 doi: 10.13128/ahs-23504 flower development and pollen vitality of moringa oleifera lam. grown in a humid temperate climatic condition s. radice 1, e. giordani 2 (*) 1 department of plant physiology, faycaum conicet, machado 914, university of morón, argentina. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: fertility, flower anatomy, microsporogenesis. abstract: moringa oleifera is a tropical tree cultivated in many countries. this species has acquired a great importance in human nutrition and it was recently indicated as a “novel food” by the european commission. recently, moringa plants have been introduced in humid temperate climatic areas, among which moreno (buenos aires province argentina). in such area, the cultivation is possible for the production of leaves, but plants need protection during winter time in order to overcome damages due to low temperatures and hence to produce capsules and seeds. the main objective of this research was to study flower morphology and anatomy of m. oleifera, as well as microsporogenesis and viability of pollen grains of plants cultivated in moreno in comparison with those produced in a humid sub-tropical climatic area of argentina (san miguel de tucumán). flowers grown in the temperate environment resulted similar for morphological parameters to those observed in the sub-tropical environment. nevertheless, pollen grain fertility depended directly on air temperature and it was negatively affected by the lower temperatures registered in the temperate site. according to the observed results, pollen viability increases with mean monthly temperatures above 16°c. 1. introduction moringa oleifera lam. (moringa) is a multipurpose small to mediumsized, evergreen or deciduous tree, native to northern india, pakistan and nepal. it has a spreading open crown with drooping, fragile branches, feathery foliage with tripinnate leaves, and a thick corky whitish bark (marcu, 2005). m. oleifera is utilised as animal fodder and employed in human nutrition due to its healthy properties (fuglie, 1999; palada and chang, 2003; ganatra et al., 2012; paula et al., 2017), as well as in the production of fuel (foidl et al., 2001), water sanitation (wilson, 1992; lekgau, 2009; padilla et al., 2012). moringa leaves are considered a “novel food” by the european commission, so confirming their valuable properties in terms of energy, nutrients, proteins and minerals, as reported by several authors (atawodi et al., 2010; tende et al., 2011; yameogo (*) corresponding author: edgardo.giordani@unifi.it citation: radice s., giordani e., 2018 flower development and pollen vitality of moringa oleifera lam. grown in a humid temperate climatic condition. adv. hort. sci., 32(4): 549-556 copyright: © 2018 radice s., giordani e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 june 2018 accepted for publication 28 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 549-556 550 et al., 2011; gopalakrishnan et al., 2016; vats and gupta, 2017). araujo et al. (2016) highlighted also the importance of m. oleifera in regions characterized by desertification and water deficit. moringa cultivation is expanding all over the world, including in climatic areas, which differ noticeably from those of its tropical origin. recently some experiments showed the feasibility of cultivating moringa in the humid temperate climatic conditions of buenos aires province (argentina) for leaf production. leaf extracts from trees grown in that conditions showed higher phenol content and antioxidant activity than those obtained from plants cultivated in typical tropical climates (arena and radice, 2016). nevertheless, flower differentiation, anthesis and fertility resulted negatively altered and the production of pods and seeds, both of them important source of nutrients, was very low. the main objective of this research was to study the effect of air temperature on flower morphology and anatomy of m. oleifera, as well as on the microsporogenesis and pollen grain viability, observed on trees cultivated at moreno (buenos aires province) in comparison with those grown in san miguel de tucumán (argentina), the first characterized by a humid temperate climate and the latter by a humid subtropical environment. 2. materials and methods plant material all plants were obtained from the same seed lot. homogeneous seedlings (n= 10) were grown in soil and in open air and cultivated in san miguel de tucumán (26° 49’59.00” s, 65° 13’00” w, elevation 456 m asl), while another similar set of seedlings (n=10) was planted in moreno (34° 39’ 0” s, 58° 47’ 0” w, elevation 14 m asl) in 25 l plastic pots under a glasshouse from april to september. successively pots were placed in open air. moreno has a humid temperate climate, with an average temperature of 23.4°c in january and 10.0°c in winter time (june); san miguel de tucumán, has humid subtropical climate (19.4°c the average annual temperature) with a hot and long summer and mild and dry winter. the precipitation pattern is monsoonal with an average of 997 mm (climate-data.org). a set of monthly air temperature parameters is reported in figure 1. flower morphology flowers (n = 100) on different phenological stages were observed on both groups of plants, and samples were collected monthly for further observations from september to december. button flower collected were used fresh and fixed in faa (formaldehyde, 100 ml; ethyl alcohol, 500 ml; acetic acid, 50 ml; distilled water, 350 ml). light microscopy button flowers (n= 10) were immediately frozen to -25°c and embedded in a medium consisting of polyethylene glycol and polyvinyl alcohol. successively they were cut frozen by microtome inside the cryostat. histologic slices were cut at 5 to 10 µm. a set of ten button flowers were dehydrated in an ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. sections were observed with a leica dm 2500 microscope. fluorescent microscopy flowers in anthesis phase (n= 50) fixed in faa were shaved with distilled water and softened with naoh (8n) as described by martin (1959). then, they were stained with aniline blue to study pollen tube growth. squash material was observed by a leica microscope (dm 2500) using fluorescence with excitation filter bp: 450-490. scanning electron microscopy (sem) button flowers fixed in faa (n= 10) were dehydrated in an ethanol series and critical point-dried with liquid co2 was employed. then it was sputtercoated with gold-palladium (40% gold and 60% palladium) for 3 minutes. samples were observed with philips xl30 sem. fig. 1 mean (a) and average minimum (b) air monthly temperatures (°c) in san miguel de tucumán and moreno locations. radice and giordani moringa in temperate climate 551 pollen viability pollen viability was performed with fluorescent microscopy according to radice and arena (2016) on fresh anthers taken from button flowers of two localities (moreno and san miguel de tucumán). pollen evaluation was expressed in percentage. to determine the statistical significance of the hypothesis the chi-squared test (χ2) was used. 3. results flower development flower development starts with the appearance of the flower on a clustered inflorescence (fig. 2). these first buds are green reddish and about 1mm long (fig. 2a). they grow up to about 10mm and turn to white greenish (fig. 2a). anthesis takes place sequentially among the flowers of the inflorescence (fig. 2b). at anthesis, flower shows a zygomorphy symmetry. the larger transversal petal is bent upwards, while the others are reflexed downwards together with the sepals (fig. 2c). anthers are yellow and no dehiscent; flowers have odour and nectar in this phase. as the flower develops, anthers change colour to dark yellow (fig. 2c) and finally to brown (fig. 2d). it was observed that 1-3 anthers were not developed in flowers collected from moreno field. pistil, which at the time of the anthesis is below the anthers, continues to grow until it protrudes several millimetres above the androecium (fig. 2c). finally, petals wither and fall, while the ovary enlarges and turns to reddish colour regardless its fertilisation (fig. 2e). flower structure moringa oleifera plants grown in moreno and san miguel de tucumán experimental fields developed flowers with average values of ≈5 petals, ≈6 sepals, ≈6 stamens, ≈5 staminoides and 22 ovules (data not showed). in the observed flowers, the unique largest petal (referred as “primordium petal”) stands right; the others are folded (fig. 2c). female part of the flower shows the complete pistil with the style and a hairy ovary (fig. 3a). ovary is tricarpelar and ovules are located in parietal placentation (fig. 3b). stigma is just a hole (fig. 3c). some nectarostomata surround the gynophore (fig. 4). glandular hairs, with the function of expelling the nectar, are present on the nectarostomata surface (fig. 4a). nectarostomata produces nectar in sub epidermal cells, that accumulate it and transfer it through the intercellular spaces (fig. 4b). frozen section of a button flower just before the anthesis phase allows to appreciate the state of the fig. 2 flower development of moringa oleifera lam. a, from button flower to anthesis. b, beginning of anthesis. c, anthesis; d, flower senescence; e, pod formation. bar = 1 cm. fig. 3 pistil of moringa oleifera lam. a, external view of the pistil; b, internal view of the ovary with ovules; c, detail of the stigma. bars = a-c, 1mm. adv. hort. sci., 2018 32(4): 549-556 552 structures and their normal coloration (fig. 5). in fact, it is possible to see anthers with pollen grains already formed wrapped in a yellow substance similar to sporopollenin (fig. 5b). pistil shows developing ovules attached to the carpelar wall (fig. 5a). finally, all internal organs are enveloped by sepals and petals (fig. 5a). flowers studied by sem showed there is no defined stigmata structure. pistil is coronate by a smooth cell structure as a continuation of the style (fig. 6a). the internal cavity of the ovary is covered with hairs (fig. 6c) and ovules adhere to their walls on the connection of two carpels. ovules appear to be campylotropous (fig. 6d). microsporogenesis button flowers from 1mm to 10 mm (fig. 2a) have been used to observe different steps of pollen grain formation. different stages of pollen grain differentiation were observed: microsporocytes mother cells, tetrads, microsporocytes and mature pollen (fig. 7). development was very fast and it accomplished in less than one week. moringa flowers have monothecal anthers. button flowers shorter than 5 mm contains inside the just formed pollen sac microspore mother cells (fig. 7a). these cells show a big and visible nucleus and a very dense cytoplasm indicating a high activity. fig. 4 nectary of moringa oleifera lam. (light micrographs). a, longitudinal section of the nectary; b, detail of the nectar cumulated in the intercellular spaces (arrows). bars = a, 100 µm; b, 10 µm. fig. 5 flower in pre anthesis phase of moringa oleifera lam. (frozen section). a, longitudinal section of flower with petals (pe), anthers (an) and ovary with ovules (arrows); b, mature pollen grain surrounded by sporopollenin. bars = a, 10 cm; b, 10 µm. fig. 6 sem micrographs of moringa oleifera lam pistil. a-b, style and stigma; b, detail of the stigma with pollen grain; c, ovarian cavity with ovules; d, detail of an ovule; e-f, pollen grains. bars = a, 50 µm; b, e, 20 µm; c, 500 µm; d, 100 µm; f, 10 µm. radice and giordani moringa in temperate climate 553 during this stage, the last inner layer of the anther wall corresponds to tapetal cells that are formed by large and binucleate cells (fig. 7a). as the buds lengthen, more advanced stages of microsporogenesis are observed. in fact, it was observed the tetrad (fig. 7b) and then the release of microsporocites (fig. 7c). at this point, tapetum degrades (fig. 7c). the young microspores show a central nucleus and a vacuolated cytoplasm. the last stage shows mature pollen grains and free orbicules (fig. 7d). when mature pollen grains are formed, tapetum disappears completely. mature pollen grains measure about 20 µm. it is possible to observe the exine of the grains well formed and the cytoplasm of the vegetative cell with a lot of amyloplasts (fig. 7d). pollen viability it was possible to differentiate green, red and orange yellowish pollen grains corresponding to viable, non-viable and sub viable pollen grains respectively. pollen viability among flowers at different dates (table 1) showed great variations on flowers collected in moreno; on the contrary, no differences were observed between flowers collected from san miguel de tucumán throughout the study period (table 2). moreno flowers showed a very low percentage of table 1 viability of pollen grains collected on different months and different locality values with different letters between the same column are significant different. tukey (p≤0.05). viable pollen grains respect to san miguel de tucumán flowers during september to november. although the number of pollen grains per anther in source month viable non viable sub-viable moreno september 10 c 79 a 11 a moreno october 28 c 53 a 19 a moreno november 48 b 32 b 20 a moreno december 74 a 3 c 23 a s.m.tucumán september 68 a 22 b 10 a s.m.tucumán october 63 a 25 b 12 a s.m.tucumán november 70 a 22 b 8 a s.m.tucumán december 69 a 28 b 13 a table 2 viability of pollen grains collected on september from anthers of the same flower values are expressed on percentage. values with different letters between the same column are significant different. tukey (p≤0.05). source anther viable non viable sub-viable moreno 1-1 10 a 84 a 6 a moreno 1-2 7 a 91 a 2 a moreno 1-3 10 a 82 a 8 a fig.. 7 microsporogenesis of moringa oleifera lam (light micrographs). microsporangium with conspicuous microspore mother cells (mmc) and tapethal cells (tc); b, tetrads (td); c, microporocytes free (m) and tapetal cells (tc) in metabolization phase; d, mature pollen grains (arrows) with amyloplast (a) in the cytoplasm and orbicules (circles). bars = a 10 µm; b-d, 20 µm. 554 adv. hort. sci., 2018 32(4): 549-556 the flowers was not evaluated, it was observed that flowers collected from san miguel de tucumán had more amount of pollen grain in each anther. some anthers of moreno flowers contained immature pollen and a viscous substance. additionally, according to the harvesting period of the flowers, some anthers developed only dead pollen grains. according to these results, a more detailed analysis of pollen viability was performed between pollen grains derived from different anthers of the same flower and between anthers from different flowers collected from moreno trial. viability of pollen grains of different anthers collected from the same flower was not statistically different (table 2), while viability of pollen from different flowers of different trees collected on the same date resulted significantly different between flowers (table 3). viable pollen grains were contained on great proportion in some flowers but scarce in others, with a random distribution among trees. pollination during anthesis, pistils both with or without germinated pollen were observed. when pollination occurs, pollen grains fall freely into the stigma cavity and then germinate (fig. 6b). in effect, pistils treated by martin technique showed that a mass of pollen grains is housed in the cavity and that many pollen tubes germinated (fig. 8a) and later on reached the ovary and fertilized the ovules (fig. 8b). pollination was rarely observed on flowers collected from moreno during spring time (september to november) but it was very frequent in summer time. on the contrary, it was observed that flowers collected on san miguel de tucumán were profusely pollinated in both periods and fruit production was continuous throughout the year. fruit production on moreno plants started in summer until may, while it resulted continuous in san miguel de tucumán. 4. discussion and conclusions flower morphology and anatomical structure observed in flowers collected from moreno field and san miguel de tucumán did not show significant difference with those described in l iterature (ramachandran et al., 1980). furthermore, flowers from the two localities studied in argentina have a normal external and internal development. frozen sections allowed to see the internal anatomy with its natural colorations and to show more clearly the presence of orbicules, that, in the traditional cuts, appear very confused because of the different colorations applied. on the other hand, flowers studied by sem allowed clarifying some concepts. in fact, it was clearly showed how pollen germination begins even in the absence of any connection to any structure of the stigma or style. bhattacharya and mandal (2004) found that some extra proteins and esterases contribute towards the stigmatic receptivity; furthermore, the occurrence of intraovarian trichomes, which is not widespread in the angiosperms, could facilitate the growth of the pollen tubes. this assessment made by dickison (1993) is based on that the trichomes functionally resemble obturators. pollen grain formation seems to be very variable depending on the geographical location and the time of year. in fact, muhl et al. (2011) showed that low temperature regime induces flowering but provokes low pollen viability and this statement would explain the results obtained in moreno spring flowers, thus confirming that pollen grain viability is affected by air temperature. in fact, the lowest values of pollen fig. 8 fertilization of moringa oleifera lam (fluorescent light micrographs). a, pollen tube growing toward the style (arrows); b, ovules (ov) and pollen tube crossing the micropile and entering the embryo sac (arrows). bars = a-b, 100 µm. table 3 viability of pollen grains from different flowers of the same plant source flower viable non viable sub-viable moreno 1 a 7 b 78 ab 15 a moreno 1 b 6 b 82 a 12 a moreno 1 c 18 a 67 b 15 a moreno 1 d 2 b 95 a 3 b moreno 1 e 15 a 72 b 13 a values are expressed on percentage. values with different letters between the same column are significant different. tukey (p≤0.05). 555 radice and giordani moringa in temperate climate grain viability were observed on september and october when mean temperatures were lower than 16°c. additionally, the higher percentage of pollen viability observed in san miguel de tucuman is consistent with the air temperatures registered in that location, which resulted about 4/5°c higher than those observed in moreno (fig. 1). the difference of the quality of pollen grains collected in spring and summer could be related to the amount of orbicules or ubisch bodies. studies on ubisch body formation in brachypodium support the evidence that they are formed in the tapetum and are involved in exine synthesis (sharma et al., 2014). actually, during spring time, anthers with mature pollen grains brings many orbicules. this fact suggests that when the exine was not well formed the quantity of dead pollen was important. on the other hand, pollen viability seems to have an influence on the efficiency of pollination. as directly observed, all plants were very visited by insects during blooming on both experimental locations, but pollination was rarely observed in moreno flowers, while it was very frequent in flowers collected in san miguel de tucumán. although tropical climates are those considered ideal for m. oleifera according to muhl et al. (2011), good results obtained with san miguel de tucumán flowers confirm that sub-tropical climates are also suitable for this species. taking into account reproductive functions, and namely the microsporogenesis, moreno environment seems to be just below the threshold of good temperature regime for m. oleifera during spring time despite the climatological predictions made by falasca and bernabé (2008). in conclusion, the reported results demonstrate the possibility of moringa cultivation unusual non tropical climates. flowers of the trees grown in moreno field were normal from the morphological point of view only in some periods of the year and the quality of their development was related to air temperatures. plants grown on san miguel de tucumán have an un-interrupted production of flowers despite not being in a tropical climate. on the other hand, moringa cultivated in marginal areas, as the locality of moreno can be considered, could offer important advantages. acknowledgements the authors thank ing. oscar dantur for the collection of plant material in san miguel de tucumán and mrs. isabel farías for her assistance with the histology. this research was supported by ifund university of florence project. references araújo m., santos c., costa m., moutinho-pereira j., correia c., dias m.c., 2016 plasticity of young moringa oleifera l. plants to face water deficit and uvb radiation challenges. j. photochem. photobiol., b. biol., 162: 278-285. arena m.e., radice s., 2016 seasonal variation in leaf growth and antioxidant content of moringa oleifera cultivated at buenos aires, argentina. int. j. agric. biol., 18: 719-725. atawodi s.e., atawodi j.c., idakwo g.a., pfundstein b., haubner r., wurtele g., owen r.w., 2010 evaluation of the polyphenol content and antioxidant properties of methanol extracts of the leaves, stem, and root barks of moringa oleifera lam. j. of medic. food, 13(3): 710-716. bhattacharya a., mandal s., 2004 pollination, pollen germination and stigma receptivity in moringa oleifera lam. grana, 43: 48-56. dickison wc., 1993 floral anatomy of the styracaceae, including observations on intra-ovarian trichomes. bot. j. linn. soc., 112: 223-255. falasca s., bernabé m.a., 2008 potenciales usos y delimitación del área de cultivo de moringa oleifera en argentina. revista virtual redesma, 2(1). foidl n., harinder p.s., becker k., 2001 potentiel du moringa oleifera pour les besoins agricoles et industriels, pp. 45-78. in: fuglie l.j. (ed.) l’arbre de la vie. les multiples usages du moringa. cta et cws, dakar, senegal. fuglie l.j., 1999 the miracle tree: moringa oleifera, natural nutrition for the tropics. church world service, dakar, senegal. ganatra t.j., joshi umang h., bhalodia payal n., desai tusharbindu r., 2012 a panoramic view on pharmacocnostic, pharmacological, nutritional, therapeutic and prophylactic value of moringa oleifera lam. int. res. j. pharm., 3(6): 1-7. gopalakrishnan l., doriya k., kumara d.s., 2016 moringa oleifera a review on nutritive importance and its medicinal application. food sci. human wellness, 5: 49-56. lekgau s., 2009 moringa oleifera a tree giving life to rural communities. national agricultural marketing council (namc), pretoria, south africa. marcu m.g., 2005 miracle tree. kos health publications, la canada, ca (usa). martin f.w., 1959 staining and observing pollen tubes in the style by means of fluorescence. stain technology, 34: 125-128. adv. hort. sci., 2018 32(4): 549-556 556 muhl q.e., du toit e.s., robbertse p.j., 2011 adaptability of moringa oleifera lam. (horseradish) tree seedlings to three temperature regimes. am. j. plant sci., 2: 776-780. padilla c., fraga n., suárez m., 2012 effect of the soaking time of moringa (moringa oleifera) seeds on the germination and growth indicators of the plant. cuban. j. agric. sci., 46 (4): 419-421. palada m.c., chang l.c., 2003 suggested cultural practices for moringa. international cooperator’ guide, asian vegetable research and development center, avrdc, pub., 3-545. paula p.c., sousa d.o.b., oliveira j.t.a, carvalho a.f.u, alves b.g.t, pereira m.l., farias d.f., viana m.p., santos f.a., marias t.c., vasconcelos i.m., 2017 a protein isolate from moringa oleifera leaves has hypoglycemic and antioxidant effects in alloxaninduced diabetic mice. molecules, 22: 271. radice s., arena m.e., 2016 characterization and evaluation of berberis microphylla g. forst pollen grains. adv. hort. sci., 30(1): 31-37. ramachandran c., peter k.v., gopalakrishnan p.k., 1980 drumstick (moringa oleifera): a multipurpose indian vegetable. econ. bot., 34: 276-283. sharma a., singh m., bhalla p., 2014 cytochemistry of pollen development in brachypodium distachyon. plant syst. evol., 300(7): 1639-1648. tende j.a., ezekiel i., dikko a.a.u., goji a.d.t., 2011 effect of ethanolic leaves extract of moringa oleifera on blood glucose levels of streptozotocin-induced diabetes and normoglycemic wistar rats. br. j. pharmacol. toxicol., 3: 1-4. vats s., gupta t., 2017 evaluation of bioactive compounds and antioxidant potential of hydroethanolic extract of moringa oleifera lam. from rajasthan, india. physiol. mol. biol. plants, 23(1): 239-248. wilson g., 1992 moringa oleifera (the kelor tree). agrovision publishing, mansfield, queensland, australia. yameogo c.w., bengaly m.d., savadogo a., nikiema p.a., traore s.a., 2011 determination of chemical composition and nutritional values of moringa oleifera leaves. pakistan j. nutr., 10(3): 264-268. 3 1. introduction world production of pineapple (ananas comosus l.) reached 19 million tonnes in 2008 with the industry dominated by brazil followed by thailand, the philippines and indonesia. in australia, pineapple, as an exotic species, is grown almost exclusively in queensland, producing 104,000 tonnes annually with an industry average yield of 37.9 t ha-1 in ~2700 ha (dhungel et al., 2009) contributing an annual farm gate value of au$50 million (fao, 2011). thanks to crassulacean acid metabolism (cam), pineapple is adapted to dry environments. however, pineapple is sensitive to water-logging and therefore requires a well-drained soil with good aeration when grown with irrigation. pineapple response to irrigation is generally high for yield and quality. industry interest in developing irrigation for pineapple has been triggered by recurring episodes of drought brought about by climate change, and a major shift in the historical rainfall pattern, and under such circumstances strategic and supplementary drip irrigation are imperative for a sustainable industry (camp et al., 1993). increased cost of water, reduced ground water reserves and vocal public pressure have forced growers to look for more effective alternatives to the traditional surface flood and furrow irrigation. in response, increasing adoption of localized micro irrigation such as sprinkler, drip irrigation (di) or subsurface drip irrigation (sdi) is taking place to improve water use efficiency (wue) and to minimize environmental impacts by reducing runoff and deep drainage (thompson et al., 2002). the irrigation efficiency of sprinkler systems is less than for drip systems due to evaporative losses and the tendency to promote foliar diseases in the former. although far more efficient, di and sdi can induce temporal hypoxic conditions in the rhizosphere due to their sustained wetting fronts, particularly in fine textured soil (machado et al., 2003) where oxygen may not be sufficiently available for root respiration. localized water-logging purges the soil pores of air and causes hypoxia, reducing root metabolic activity and function (goorahoo et al., 2002; bhattarai and midmore, 2009). alleviation of hypoxic rhizosphere conditions can be achieved through the use of aerated water for irrigation, increasing oxygen availability in the root zone (su and midmore 2006; dogan et al., 2008). oxygation, the term we use for aerated irrigation water with sdi, has been shown to benefit growth of a range of crops, particularly in heavier soils. this is because even under normal irrigations, roots can suffer due to a lack of soil oxygen. an increase in crop yields and wue has been aerated water irrigation (oxygation) benefits to pineapple yield, water use efficiency and crop health j. dhungel, s.p. bhattarai(1), d.j. midmore centre for plant and water science, faculty of science engineering and health, central queensland university, rockhampton, qld 4702, australia. key words: ananas comosus, cam photosynthesis, ‘d’ leaf, phytophthora, root respiration. abstract: pineapple roots need adequate oxygen to function, sustaining growth and yield. the crop is susceptible to soil saturation caused by natural rainfall or irrigation, or even with drip irrigation that creates sustained wetting fronts. drip and subsurface drip irrigation can develop sustained wetting fronts, particularly in low permeability soils, predisposing plant roots to a low oxygen environment. we evaluated the use of aerated irrigation water “oxygation”, employing mazzei air injectors which mix air with irrigation (12% air by volume of water) in-line, increasing oxygen concentration in the irrigation water stream. the effect of this treatment was evident in growth, development, and leaf gas exchange parameters. total fruit yield increased by 44 and 26% whereas industry yield increased by 11 and 6% due to oxygation compared to the control and no irrigation, respectively. high yield was associated with an increase in fruit size and not the number of fruits produced. phytophthora infestation in the oxygation (3% of plants) was significantly reduced compared to the control (4.9%), and without irrigation treatment (10.5%) suggesting that reasonable management of phytophthora, which is one of the major pathological problems for pineapple production in australia and elsewhere, can be addressed through aerated water irrigation. oxygation responses were mediated through root and soil processes involving greater root biomass, root respiration, increased microbial diversity and enhanced soil aeration status. adv. hort. sci., 2012 26(1): 3-16 (1) corresponding author: s.bhattarai@cqu.edu.au received for publication 8 april 2011 accepted for publication 18 december 2011 4 confirmed by bhattarai et al. (2004) with the use of aerated water irrigation in a number of annual crops with c 3 metabolism. other studies on other crops in a range of soil types with drip and sdi have also been implicated in creating a wetting front that can induce the possibility of hypoxia in the rhizosphere (goorahoo et al., 2002; midmore et al., 2006). drip irrigation which involves point source water application in the rhizosphere could lead to constantly poor aeration around the root zone and predispose pineapple plants to a number of physiological disorders and susceptibility to phytophthora root rot. oxygation, using aerated water that has potential to ameliorate the hypoxic/ anoxic conditions, may be of benefit not only to improve the wue, yield and quality but also to minimize disease caused by phytophthora, with symptoms of root and fruit rot in pineapple. previous research in annual crops has demonstrated the potential of oxygation to improve yield and wue (bhattarai and midmore, 2009). most of the effects of water-logging in pineapple are mediated through the roots, including predisposition of the root system to a number of diseases including root and fruit rot. generally field grown pineapples tend to develop roots in the upper soil layer, where they are confined and follow the wetting area. as the root activities are bound to the wetting fronts, the susceptibility to phytophthora is generally high. recent adoption of drip irrigation for pineapple in australia and elsewhere is encouraging. introduction of aerated drip systems can be pivotal in improving irrigation water use efficiency and minimising the infestation by phytophthora in pineapple. the main objective of this trial was to determine under what conditions there is a measurable positive effect of aeration and how that relates to impact upon incidence of phytophthora and on general crop growth and development. the study was designed to evaluate the effectiveness of oxygation in a perennial crop pineapple with the cam pathway for carbon fixation, evaluating the benefits on fruit yield, quality, and water use efficiency. we report the results of field research carried out in collaboration with valley syndicate pineapple farm in yeppoon, qld australia from 2007 to 2011 in order to evaluate the above mentioned effects of aerated water for drip irrigation. 2. materials and methods trial site and soil the field experiment was conducted at valley syndicate pineapple farm, yeppoon, central queensland, australia (23o9’31.12os, 150o42’51.36oe). the crop was grown over the period 2007-2011 from which two harvests were taken as main crop and first ratoon crop. total area of the experimental site was 2.15 ha on a calcareous sandy loam soil, with organic carbon 0.68-1.2%, total nitrogen 0.06-0.09%, potassium (colwell) 25-139 mg/kg, and phosphorus (colwell) content of 18-39 mg/kg. the crop seasons were relatively wet compared to long-term averages. the region is described as a semiarid tropical environment, with summer-dominant rainfall (fig. 1). fig. 1 total monthly rainfall (mm), monthly average maximum temperature (°c), and monthly minimum temperature (°c) over the experimental period at yeppoon. experimental design the experiment was a randomised block design, with two sdi irrigation treatments, with and without oxygation. air injection into the irrigation water for oxygation was supplied by a 1583™ air injector venturi (mazzei corp, usa) installed in-line immediately before the field plot, regulated to ingress 12% air by volume of water following bhattarai et al. (2006). the experiment was replicated seven times within 14 field plots (average dimensions 16 m x 70 m) where seven plots received aerated water by sdi and seven plots were irrigated without aerated water. an adjacent block, comprising three plots, received no irrigation (although all other inputs such as fertilizer, flowering regulation chemicals, fungicides and insecticides were identical) and was included as a third treatment (noirrigation) for comparison purposes. planting materials and crop management crowns of pineapple variety gc1 were planted on 24 october 2007, in a double row, raised bed (10 cm high) system to accommodate up to 53,333 plants/ha. inter row space was 50 cm and between plants was 25 cm, whereas centre to centre between the bed was maintained at 1.5 m. the oxygation treatment commenced 14 march 2008 (139 days after planting). ethylene (ethephon) was applied twice as a saturated solution in water and activated charcoal to enhance absorption to induce flowering, as pressurized spray late in the evening or at night for enhanced uptake through the stomata. plants were of an optimum size (~2 kg) for forcing in order to obtain even flowering. irrigation design and scheduling irrigation was blocked in four units. a mazzei air injector was installed near the block (3 m from the first plot), whereas the pump was installed near to reservoir about 1 km distance from the plot. the inlet pressure of 45 psi was achieved at the point of the mazzei air injector installation. a pressure differential across the air injector was maintained at 45 and 15 psi for the inlet and outlet, respectively, to maintain air injection at 12% by volume 5 of water (fig. 2). the drip tube (manufactured by plastro australia) consisted of pressure compensated emitters at 30 cm intervals emitting at the rate of 1.2 l/hr, buried at 150 mm from the soil surface, one drip tube per bed running between two 50-80 m length crop rows. fig. 2 mazzei air injector installed in a pressurised irrigation line close to the plot for air injection into irrigation water for oxygation (top), and the first year crop at fruit development stage (b) being marked for destructive sampling. irrigation and soil water monitoring soil water content was monitored at depths of 10, 20, 30 and 40 cm, logged at 15 min intervals but averaged daily, using a calibrated odyssey greenlight-redlighttm (glrl) sensor (capacitance probes manufactured by data flow system pvt ltd, nz). there was one monitoring site in each plot. soil moisture was also measured (at 10 cm intervals from 10-60 cm depths) during the diurnal measurement of photosynthesis over a period of five days in january 2010; a calibrated odyssey micro-gopher system, the probe of which consists of a capacitance sensor (soil moisture technology, australia) was used. scheduling of irrigation was based on the averaged readings from the glrl sensors at 20 cm depth; the amount applied was calculated to take soil moisture content when at c. <50% of the field capacity (fc 36 mm h 2 o per 100 mm soil depth) to refill the soil water reservoir by irrigation to reach field capacity. this required different durations to bring the soil to fc. water applied to individual blocks (oxygation and control) was measured with two calibrated water meters installed near the pumping station and the rainfall data were accessed from a nearby weather station (<1 km aerial distance). nutrients to the crop were supplied through an industry standard rate of basal application of macro and micro nutrients and top dressing by spreading in the non-irrigated plot and by fertigation on drip irrigated plots supplied at the equivalent dose for all treatments. the industry strategies for increasing pineapple yield and fruit size included side dressing of nitrogen (160 k/ha), phosphorus (60 kg/ ha) and potassium (193 kg/ha) over five split applications in a year. an additional dose of magnesium (12 kg/ha) was also applied once. soil oxygen monitoring the o 2 concentration in the soil was measured at 15 cm depth between two emitters and offset 5 cm from the drip tube using pst3 o 2 sensitive fibre-optic minisensors with fibox-3 oxygen meters (presens gmbh, germany) as described by klimant et al. (1995). sensors were installed in the soil for five days prior to data collection and soil oxygen monitoring took place for two days before oxygation, during oxygation and two days post oxygation event in the oxygation and control plots following the procedure of chen et al. (2010). soil oxygen monitoring was also performed in the ratoon crop over the period of four days before (-48 hr), during (0 h) and post (+48 hr) irrigation. destructive plant sampling for dry matter partitioning destructive plant sampling for dry matter accumulation and partitioning was carried out on 22 nov 2008 (392 dat) and 15 january 2010 (713 dat) to evaluate the treatment effects in the main and ratoon crops. plant samples collected from two whole 2 m linear lengths per bed were separated into leaf, stem, fruits and roots, oven-dried at 70°c, and the fresh and dry weights of each component were recorded. the ‘d’ leaf is always easy to pull from the plant and has leaf margins that are more-or-less parallel all the way to the leaf base (bartholomew, 2008). the ‘d’ leaf is defined as the youngest physiologically mature leaf on the plant and also is the tallest leaf on the plant. plant height and sla were measured on the ‘d’leaf. diurnal changes in gas exchange and plant parameters light interception by the canopy was measured using an accupar ceptometer (decagon, usa) and canopy temperature was recorded using an everest ag multimeter. leaf photosynthesis (a), transpiration (e) and stomatal conductance (sc) were measured at 6 hr intervals using an infrared gas analyser (irga) lca-4 (adc, uk) on two fully-expanded topmost sunlit leaves per plot on each occasion at early morning (dawn), midday, dusk and at night following adams et al. (2002). soil respiration was 6 measured in the soil 3-5 cm from the plant using irga principle with an egm-3 (pp systems (uk) following hanson et al. (2000) on 392 dat for the main crop and 713 dat for the ratoon crop. data collection was also carried out to measure the gas exchange responses of the crop before, during and after oxygation events in the aerated sdi, the control sdi and no irrigation treatments. pre-irrigation data were collected over a 24 hr period, on 14-15 january 2010 at 6 hr intervals to include dawn, midday, dusk/early evening and night to determine diurnal patterns of leaf gas exchange parameters during and post irrigation, and data on soil moisture and soil respiration were also collected using microgoopher and egm-3 soil respiration systems, respectively. harvesting and yield determination harvesting was performed at two scales, i.e. sample plot harvesting for total yield and industry harvest for marketable fruits yield. for sample plot harvests, all fruits were hand-picked at maturity from two rows of 2 m linear lengths (16 plants) selected from each of the seven replicated bordered plots, and from three plots in the non-irrigated area. the fruits were counted, weighted and processed for quality parameters for both main crop and ratoon crop. change in fruit colour, particularly to the eye, from green to yellowish was considered an index for maturity and harvesting. fruits without crown and peduncle were weighed. the industry harvest represents only marketable fruit yield harvested by the crew of pickers in a mobile harvester. commercially-harvested fruits were weighted in the load cells with wooden crates containing ~500 kg. commercial harvest was carried out for each plot separately at approximately weekly intervals. the main crop was harvested from january to april 2009 and it was left for ratoon which recommenced harvesting in 2010. the ratoon crop was harvested 28 june to 11 october 2010, and the whole crop was then uprooted for planting of a new crop, hence the crop spanned a period of 39 months from planting. fruit quality determination mature fruits harvested from sample areas were used for quality determination. fruit quality parameters measured included °brix, fruit size, volume, density, fruit height and width, flesh colour, skin colour, dry matter, translucency and flavour, following the standard analytical method described by bartolome et al. (1995). fruit quality was assessed on fruits from different harvests in the main and also in the ratoon crop. in total, 20-50 randomly selected mature fruits were assessed for quality in the main and ratoon crops. the flavor score was determined based on smell by a panel group following industry standard; 1= no flavor, 2= little flavor, 3= good flavor. water use efficiency crop water use efficiency was calculated to represent irrigation water use efficiency (iwue) and gross water use efficiency (gwue). iwue is calculated as fruit yield (t) per megaliter of irrigation input, whereas gwue is the fruit yield (t) per megaliter of crop water input, comprising both the inputs from irrigation and rainfall. instantaneous water use efficiency (wue i ) was calculated from the irga data which represent µmol co 2 assimilated for each mmol of h 2 o transpired during measurement of the photosynthesis process. soil physical and chemical parameters changes in soil physical and chemical properties were assessed by measuring soil compaction, bulk density and air filled porosity (following the method of usda, 2010) and soil organic carbon (kuhlbusch, 1995) and nitrogen (iso 13878 soil quality elemental analysis). soil compaction was determined with remek cp4011 soil cone penetrometers (ict international, australia) during the destructive plant sampling period to a depth of 35 cm from the soil surface. soil water samples were collected at 50 cm depth for sub-surface leaching and nutrient analysis particularly nitrate signature using wetting front detectors (csiro, australia). subsurface solution samples were also collected from ceramic solusamplers placed at depths of 20 cm and 50 cm to determine nutrient transfer through the soil profile. at the end of the crop season, soil cores (20 cm deep and 8.6 cm diameter) were collected to determine bulk density, root density, air filled porosity at field saturation, field capacity and in dry soil following the method by peverill et al. (2002). soil microbial and phytophthora determinations fluorescein diacetate hydrolysis activity (fda) analysis provides a surrogate measure of the soil microbial load. soil samples were collected during harvest at the depth 10 cm and 10 cm distance from emitter, and were used for fda analysis following the method described by adam and duncan (2001). the incidence of phytophthora infestation was assessed in the field crop. in each plot, a double row comprising ~500 plants was examined for infestation based on the visual symptoms following pegg (1977). plants with characteristic symptoms of phytophthora on fruit and crown rot were counted and percentage infestation calculated. data analysis data were analysed following the procedures for analysis of variance (anova) for randomised block design in genstat version 11 (vsn international, uk) as the no irrigation plots were contiguous to sdi plots. for most of the crop, only the main effects of soil and water parameters are presented, whereas for all other data collected during the diurnal events (leaf and soil gas exchange parameter) the effects of the treatment on the diurnal course were also analysed. hence some significant interactions between the treatments and diurnal effects have been analysed and presented. means were separated by the least significant difference (lsd) at p≤ 0.05. 3. results and discussion weather conditions rainfall was recorded as 917 mm, 1294 mm, 875 mm, and 1850 mm for 2007, 2008, 2009 and 2010, respective7 ly (fig. 1). 2010 had the highest rainfall, more than four times that of the driest years in the past 20 years. total rainfall input during the entire crop period was 4250 mm. irrigation input irrigation scheduling was based on measurement of the soil moisture deficit. the same delivery system through mazzei injector was utilized for air injection and water application (fig. 2). irrigation commenced when soil moisture reached the refill point. irrigation input during the crop period for the oxygation and control treatments was 252.4 and 240.5 mm per hectare respectively (fig. 3). rain contribution during the same period was 4250 mm per hectare (fig. 1). therefore, the proportion of irrigation to total crop water input was only 5.5%. all irrigation events were scheduled for supplementary and strategic applications to the crop. soil moisture in the profile soil moisture at 40 cm was consistently higher than at 20 cm, irrespective of the treatment (fig. 4). oxygation maintained somewhat less soil moisture compared to the control at both depths. on a number of occasions, the soil moisture content was well above the field capacity (36 mm for 100 mm soil depth) at greater depths, particularly in the control treatment. the results suggested that soil moisture increased with increasing depths (0-60 cm), irrespective of the treatment (fig. 5). the effect of irrigation treatments on soil moisture was evident, as soil moisture always remained lower with oxygation compared to the control for all the depths in spite of slightly higher irrigation input associated with oxygation, suggesting that water loss from the rhizosphere was greater through transpiration for plants with the oxygation treatment. soil oxygen dynamics soil oxygen concentration in the oxygated rhizosphere remained higher (7.5 vs 4.4 ppm) than the control. the highest oxygen concentrations (10.72 and 7.08 ppm) were noted during irrigation for the oxygation and control groups, respectively. the lowest concentrations for oxygation and control were 5.48 and 3.09 ppm, respectively fig. 4 change in soil moisture (mm water 100 mm-1 of soil depth) at two different depths (400 and 200 mm) over the period of two months (march-april 2009) in the oxygation and control treatments measured by red light green light soil moisture sensors. fig. 3 cumulative irrigation input (ml ha-1) over the crop period for oxygation and control treatments. fig. 5 change in soil moisture (mm water 100 mm-1 of soil depth) over a period of four days [before (2 days), during (upon completion of 2 hr irrigation) and after (2 days) irrigation] in the oxygation and control irrigation. 8 (fig. 6). a higher oxygen concentration in the rhizosphere during oxygation events compared to non-aerated water irrigation was also reported by chen et al. (2010) in cotton and wheat crops in both vertisol and ferrosol under similar climatic conditions. changes in soil physical properties soil compaction. there was less soil compaction in the oxygation treatment compared to the control, and it was higher in the non-irrigated treatment, particularly at depths below the position of emitters (fig. 5). soil resistance increased down the soil profile to 37.5 cm depth, and the treatment effect was consistent over this depth. the data suggest that the lower soil resistance down the profile was consistently maintained throughout the profile in the oxygation treatment (fig. 7) compared to the control and no-irrigation treatments, particularly in the positions below the emitter depths. this observation is in contrast to the report presented by bhattarai and midmore (2010 unpublished data) on cotton in vertisol where they showed a greater soil resistance in the wetting fronts of cotton under oxygation compared to the control. such a response was linked with rapid water uptake and transpiration by oxygated plants and, therefore, a drier soil in the wetting front region that contributed to greater soil resistance. soil bulk density and air filled porosity. a tendency for lower air filled porosity was recorded in the no-irrigation treatment compared to irrigated oxgation and control treatments. air-filled porosity at field capacity did not differ significantly between the treatments (table 1). however, the air field porosity at near saturation was significantly greater in the irrigated control and higher in oxygation compared to the no-irrigation treatment. while no significant difference between the treatments was noted for the soil bulk density, root density was significantly lower (reduced by one half) for the no-irrigation treatment compared to both oxygation and control treatment (table 1). non irrigated plants showed shallower root systems, and were easier to pull by hand. instead of putting down deep rooting, they tended to produce adventitious root in the soil surface. consistent with these results, schneider et al. table 1 soil physical and biological properties assessed at the end of the ratoon crop for oxygation, control and no-irrigation treatments treatments air filled porosity (%) bulk density (g/cm3) root density (kg/m3) fluorescence release (µg /g dwt soil/ hr) phytophthora (% plants)near saturation field capacity control 2.6 20.7 1.68 6.32 2.2 4.9 oxyation 2.3 20.6 1.61 7.50 2.2 3.0 no-irrigation 2.0 18.3 1.61 3.56 1.8 10.5 p value 0.021 0.102 0.243 0.047 0.890 <0.001 lsd (p≤0.05) 0.537 ns ns 1.811 ns 1.379 mean separated by lsd. ns= not significant. fig. 6 change in soil dissolved oxygen concentration (ppm) over the period of four days [before (2 days), during (upon completion of 2 hr irrigation cycle) and after irrigation (2 days)] in the oxygation and the control treatment measured at the wetting front. fig. 7 soil compaction measured in the soil at field capacity at the end of the ratoon crop as influenced by soil depth for oxygation (open circle), control (closed circle), and non-irrigated (closed diamond) treatments. 9 (1992) reported deep penetration of roots and greater root biomass near the drip line and major concentration of root mass at 30-40 cm depth in drip irrigated pineapple on a silty clay soil in hawaii usa. soil respiration in the ratoon crop, the rate of soil respiration did not differ significantly between the oxygation and control treatments three days after irrigation, however the rate was greater for the irrigated treatments compared to noirrigation treatment. the diurnal pattern of soil respiration showed a greater soil respiration rate during the day and during the night compared to the early morning and evening (table 2). in the fast growing pineapple crop, i.e. before the first crop harvest, soil respiration was significantly greater in the oxygation (2.2 g co 2 m-2 hr-1) compared to the control (1.4 g co 2 m-2 hr-1) treatment, an increase of 64%, at 394 days after planting (chen et al., 2010), and following 6 hr of irrigation. root respiration has been shown to be enhanced by aeration in a number of previous studies on oxygation. these findings are in accord with those of bhattarai et al. (2005) who showed that oxygation increases the amount of oxygen in the irrigation water and ultimately in the root zone, which drives greater root respiration, and therefore ameliorates the temporal hypoxia associated with wetting fronts. soil biological properties and phytophthora low fda values were noted in the no-irrigation treatment compared to irrigation treatments without significant differences. a marked effect of irrigation treatments was noted on the development of phytophthora symptoms in the field crop (table 2). the oxygation treatment showed significantly lower infection (3%) compared to the no oxygation sdi treatment (4.9%), whereas the highest phytophthora infestation (10.5%) was recorded in the noirrigation treatment. in spite of the lower water application rate and reasonably dry soil surface in the no-irrigation plot, development of phytophthora was more severe in this treatment. exposure of the roots to the soil surface provides poor anchorage to the plant. when the plant was loaded with fruit, the top-heavy weight of the plant resulted in crop lodging and damage to the roots. this may have predisposed the plants to phytophthora contamination particularly when the plot was wet due to rainfall. severe crop lodging was noted in the non-irrigated treatment in this trial site. a study by stirling (2004) also suggested that the pineapple crop in qld in a ferrosol treated with cane trash mulch and under minimum tillage, both of which contribute to maintaining greater soil aeration status, recorded a high fda level and a positive correlation with greater nematode suppression in the field compared to non-mulched traditional tillage treatments. soil chemical properties (nutrients) soil organic carbon, colwell k and colwell p contents were higher (the latter two not reaching significance at p<0.05) in the no-irrigation treatment compared to oxygation and control treatments when analysed at the end of the crop period. in contrast, soil ph (cacl 2 ) was significantly lower, and exchangable k was signifcantly higher in the no-irrigation treaments compared to irrigated control or oxygation treatments (table 3). no significant effects of irrigation treatments were detected for total n, electrical conductivity, and exchangeable calcium and magnesium concentration in the soil. table 3 macro nutrients, organic carbon, soil ph, conductivity (cond) and exchangeable calcium (exc. ca), exchangeable magnesium (exc. mg), exchangeable potassium (exc. k) in the soil sampled after the ratoon crop in oxygation, control and no-irrigation treatments treatments organic carbon (%) n total (%) colwell p (mg/kg) colwell k (mg/kg) cond (ds/m) ph (cacl 2 ) exc. ca (meq/100g) exc. mg (meq/100g) exc. k (meq/100g) control 0.900 0.06 19.00 61.50 0.04 3.45 0.22 0.06 0.04 oxygation 0.890 0.07 17.50 56.50 0.05 3.50 0.18 0.05 0.05 no-irrigation 1.210 0.08 45.00 107.00 0.05 3.30 0.31 0.08 0.12 p value 0.064 0.14 0.009 0.086 0.572 0.033 0.547 0.485 0.008 lsd ns ns ns ns ns 0.129 ns ns 0.031 mean separated by lsd. ns= not significant. table 2 soil respiration, soil temperature, and soil resistance for different irrigation treatments and diurnal sampling treatments soil respiration (g co 2 /m2) soil temperature (°c) soil resistance (kpa) (2.5-30 cm) control 0.82 26.8 1285.2 oxygation 0.82 26.5 1012.8 no-irrigation 0.37 27.1 2067.5 p value (aeration) 0.018 0.030 <0.001 lsd (p≤0.05) (aeration) 0.329 0.423 434.1 day (1300 hrs) 0.89 29.5 evening (1900 hrs) 0.51 26.2 night (2300 hrs) 0.99 26.1 morning (0500 hrs) 0.57 25.2 p value (diurnal) 0.008 <0.001 p value (a x d) 0.287 0.726 lsd (p≤0.05) (diurnal) 0.327 0.420 lsd (a x d) ns ns mean separated by lsd. ns= not significant. 10 plant growth and development dry matter partitioning during growth of the main crop the effect of oxygation was assessed on vegetative and reproductive biomass of the main crop during the early fruit growth stage prior to maturity (392 dat). dry weight in the root, leaf, fruits and total above-ground dry biomass increased significantly due to oxygation compared to the control. however, the stem dry weight was not affected by the treatments. the size of the immature fruits (measured as weight of individual fruit) was significantly larger with oxygation compared to the control harvested at the same time (table 4). the fruit dry weight was greater by 14% with oxygation compared to the control and, dry biomass was 13% greater in the oxygation compared to the control treatment (table 4). these results are consistent with yield increases in previous trials on other crops such as tomato, zucchini and cotton representing the c 3 pathway for co 2 assimilation (bhattarai et al., 2005). dry matter partitioning during the growth of the ratoon crop dry matter partitioning was carried out for the ratoon crop during the growth phase, at the same time as the diurnal measurements of gas exchange (713 dat). above-ground and total dry matter biomass increased with oxygation compared to the control, but without significant differences. however, the increase in leaf dry weight associated with oxygation, compared to the control, was significant (table 5). the leaf weight with oxygation at 22.9 t/ha was 15% greater than that for the control. this result in the ratoon crop was similar to that in the main pineapple crop, where leaf weight increased by 14%. the total biomass was 35.4 t/ha with oxygation, which was 7% more than in the irrigated control (31.2 t/ha). this result is also consistent with, but somewhat less than, the result in the main pineapple crop, where the biomass increase was 14%. the oxygation treatment improved root biomass in the main crop (79%) and much less so in the ratoon crop compared to the non-oxygated control. the result in the main crop was consistent with the hypothesis that oxygation improves oxygen availability in the rhizosphere, which positively influences the availability and uptake of water and nutrients favorable for increased root growth and enhanced soil microbial functions (goorahoo et al., 2002). the lower soil compaction in oxygation treatment plots could also have favoured root growth. crop physiological performance leaf chlorophyll leaf chlorophyll content was estimated using a spad meter. a standard calibration for spad was also made with acetone chlorophyll extraction method (arnon, 1954) and a close agreement was achieved between these two methods (fig. 8) as reflected by the coefficient of determination (r2=0.807). the chlorophyll content in the d leaf of the ratoon crop was recorded as higher with oxygation compared to the control and no-irrigation treatments. the increase in leaf chlorophyll content was to the order of 1117% with oxygation compared to that of the control and no-irrigation treatments (table 6). table 4 effect of oxygation on plant dry weight and its components at the sample harvest of the main crop (392 dat) from irrigated control and oxygation treatments (no irrigation treatments were not sample harvested in main crop) treatments stem (g/m2) root (g/m2 ) leaf (g/m2 ) agdb (z) (g/m2 ) fruit (g/fruit) total (g/m2 ) root/shoot control 585.9 582.3 1760.0 2345.9 833 2928.2 0.248 oxygation 671.8 1056.1 2232.0 2903.8 1002 3959.9 0.363  p value 0.633 0.004 0.025 0.001 0.016 0.004 0.071 lsd (p≤0.05) ns 343.5 555.8 323.7 124.7 367.5 0.109 (z) above-ground dry weight. mean separated by lsd. ns= not significant. table 5 effect of oxygation on dry plant weight and its components at harvest of the ratoon crop (713 dat) treatments stem (g/m2 ) root (g/m2 ) leaf (g/m2 ) crown (g/m2 ) fruit (g/m2 ) agdb (g/m2 ) total (g/m2 ) root/shoot control 549 528 1942 9.9 85 2587 3115 0.204 oxygation 598 533 2289 14.7 105 3007 3541 0.177 no-irrigation 623 409 2136 0.0 0.0 2760 3169 0.148 p value 0.76 0.32 0.08 0.62 0.53 0.20 0.22 0.34 lsd (p≤0.05) ns ns 150 ns ns ns ns ns mean separated by lsd. ns= not significant. 11 ‘d’ leaf characters a significant increase in plant height was recorded in oxygation and control treatments compared to no-irrigation plants. although in the first crop there was no effect of oxygation on the number of leaves per plant, nor on ‘d’ leaf area and the specific leaf area (sla) of the ‘d’leaf (nor on plant height, data not presented), a significant increase in ‘d’ leaf area due to sdi with or without oxygation compared to the non-irrigated control was evident in the ratoon crop (table 6). a larger ‘d’ leaf area has been linked with higher yield of pineapple fruits in a number of previous studies (e.g., fournier et al., 2007). canopy light interception light interception by the canopy increased significantly with oxygation compared to the control and no-irrigation treatments (table 6). light interception by the canopy was highest (92%) in the oxygation treatment and increased by 5% and 7% compared to the no-irrigation and control treatment respectively (table 6). leaf photosynthesis there was a distinct diurnal pattern for leaf gas exchange. no gas exchange activity was recorded during the daytime, while the carbon dioxide exchange rate ranged between 2.4-3.5 µmol m2 s-1 over the early morning, evening and night (table 7). a higher co 2 exchange rate was noted in the oxygation treatment compared to the control table 6 pineapple leaf characteristics of the ratoon crop as affected by irrigation treatments when harvested at the fruit developing stage treatments plant height (cm) leaf (no./plant) ‘d’leaf area (cm2) ‘d’leaf weight (g) sla(z) (cm2/g) chlorophyll (spad units) li (y) (%) control 98.0 9.6 263.23 5.81 46.8 44 87 oxygation 102.0 8.9 280.45 6.15 48.2 49 92 no-irrigation 93.3 7.9 215.31 5.03 45.4 42 83 p value 0.06 0.82 0.01 0.31 0.63 0.08 0.09 lsd (p≤0.05) 5.8 ns 33.11 ns ns 5.2 5.6 mean separated by lsd. ns= not significant. (z) sla = specific leaf area. (y) li = light interception by the canopy. table 7 diurnal variation for leaf co 2 exchange rate, stomatal conductance, transpiration rate, and leaf temperature for different treatments treatments leaf co 2 exchange rate (µmol/m2/s) stomatal conductance (mmol/m2/s) transpiration rate (mmol/m2/s) leaf temperature (°c) control 2.038 0.312 2.75 30.7 oxygation 2.198 0.376 2.80 30.6 no-irrigation 1.850 0.150 2.71 31.1 p value (aeration) 0.39 0.428 0.062 0.033 lsd (p≤0.05) ns ns 0.09 0.396 day (1300 hrs) -0.14 0.018 1.27 38.5 evening (1900 hrs) 3.53 0.196 3.21 27.7 night (2300 hrs) 2.39 0.353 3.69 27.6 morning (0500 hrs) 2.93 1.324 5.30 27.5 p value (diurnal) <0.001 <0.001 <0.001 <0.001 lsd (p≤0.05) (diurnal) 0.585 0.463 1.034 0.451 p value (a x d) 0.791 0.135 0.082 <0.001 lsd (p≤0.05) (a x d) ns ns ns 0.966 mean separated by lsd. ns= not significant. a x d = interactions between aeration and diurnal measurement for the given parameters. fig. 8 relationship between the spad unit and leaf chlorophyll concentration by acetone extraction method. (c hl or op hy ll μ /c m 2 ) spad unit 12 and no-irrigation. the diurnal pattern of leaf gas exchange in photosynthesis in pineapple is characteristic of the crassulacean acid metabolism (cam), in which carbon dioxide is temporarily fixed during the night and in conditions of very low light intensity as in other succulent plants (cushman, 2001). pineapple crops are able to cope with seasonal variations in weather such as rainfall, dry atmosphere and drought, all of which reduce productivity, due to their ability to assimilate co 2 via the cam pathway (san-josé et al., 2007). during the day stomata are closed and leaf surface transpiration is at its lowest (zhu et al., 1999). due to this unique cam physiology, pineapple exhibits high wue, several times higher than c 3 and c 4 plants (cushman, 2001). our leaf gas exchange data were collected as point source data, from small portions of the leaf and were instantaneous measurements, hence raising questions as to whether the observations made in a single leaf in instantaneous time frames can reflect the response of the whole plant over an integrated time scale. leaf transpiration, stomatal conductance and temperature leaf transpiration was low during the day and higher in early morning, evening and night (fig. 9), and was lower for control and no-irrigation treatments compared to oxygation (table 7). the stomatal conductance was somewhat higher for the oxygation compared to the control and noirrigation treatments; however, the difference in stomatal conductance between the treatments was not statistically significant (fig. 9). in contrast, the leaf temperature measured during gas exchange decreased significantly with irrigation compared to the no-irrigation treatment (table 7). higher leaf transpiration rate is associated with evaporative cooling of the leaf surface that reduces leaf temperature in relation to the ambient temperature of the leaf environment. a significant interaction between irrigation method and diurnal time scale was due to significantly higher predawn leaf temperature in no-irrigation treatment compared to aerated sdi and sdi control (table 7). this is due to low transpiration, and slow evaporative cooling of leaf in this treatment compared to other irrigated treatments in the experiment (table 7). fruit yield the harvest from sub-sample areas at maturity was comprised of all fruits irrespective of their size and marketability. the total pineapple fruit yield (consisting of main crop and ratoon) was significantly greater with oxygation (133.7 t/ha) compared to that of the irrigation control (106.4 t/ha), and least in no-irrigation treatment (90.4 t/ha). the total yield increase due to oxygation was 48% compared to no-irrigation, and 26% compared to the control (table 8). the harvest yield was greater in the main crop compared to the ratoon crop. the total harvest of the ratoon crop was only 51% that of the main crop, averaged over all three treatments. however, oxygation still maintained a higher yield compared to the control and no irrigation in the ratoon crop. table 8 pineapple fruit yield from the sample area (harvested yield) and industry marketable harvest (industry yield) for the main and ratoon crops treatments harvested yield (t/ha) (z) industry yield (t/ha) (y) main crop ratoon total main crop ratoon total control 68.20 38.17 106.37 50.92 18.25 69.17 oxygation 79.60 54.11 133.71 53.08 20.18 73.26 no-irrigation 71.30 19.07 90.37 49.50 16.42 65.92 p value 0.005 0.001 0.032 0.295 0.051 0.076 lsd (p≤0.05) 6.43 10.76 12.36 ns 3.17 7.39 mean separated by lsd. ns= not significant. (z) harvested yield is all fruits harvested from the sample area. (y) industry yield refers to only marketable yield harvested on whole plot basis by the industry picking process. fig. 9 leaf stomatal conductance (a), transpiration rate (b), photosynthetically active radiation (c), co 2 exchange rate (d), soil temperature (e), soil respiration (f), leaf temperature (g) and instantaneous water use efficiency (h) in oxygation, control and no-irrigation treatment over the time period of 24 hr (bar shows mean, line shows standard error of the means) in a ratoon pineapple crop. morning day evening night morning day evening night 13 total industry fruit yield was highest in the oxygation treatment (73.3 t/ha), followed by control (69.2 t/ha) and no-irrigation (65.9 t/ha). the marketable fruit yield in the industry harvest was greater by 11% due to oxygation compared to the no-irrigation treatment, and 6% compared to the control treatment (table 8). the industry yield as a proportion of the sampled yield was also greater in the main crop compared to the ratoon crop. the total industry harvest in the ratoon crop was only 36% that of the main crop, averaged over all three treatments. the sample plot yields were considerably higher than the commercially harvested yields due to the fact that commercial yield only considered fruit >1.5 kg; the sample plot yields included fruits which were smaller but mature. the benefits of oxygation for pineapple yields are in agreement with data from oxygation field trials on a vertisol, where lint yield of cotton increased consistently over a number of years and the benefit averaged >10% per annum (bhattarai and midmore, 2009). fruit quality the individual fruit weight increased significantly due to oxygation, particularly for the main crop, compared to the control and no-irrigation treatment. the fruits in the oxygation treatment were 230 g and 228 g larger than the control and no-irrigation treatments respectively in the main season crop. in the ratoon crop, the effect of treatments on mean fruit size was not as notable, and the mean fruit size in the no-irrigation treatment had improved considerably. greater annual rainfall (~1900 mm) that was more evenly distributed compared to the previous year (fig. 1) minimized crop water stress, and imparted a positive effect on the fruit size and quality in the no-irrigation treatment in the ratoon crop. other parameters of the fruit size such as fruit height and width were also significantly greater with oxygation compared to the control in the main crop. such increase in fruit size due to oxygation has also been reported for other crops such as tomato (bhattarai et al., 2006). the total soluble solid content measured as °brix and dry matter content remained quite consistent across seasons and treatments. the brix readings were much higher than the minimum brix standard set for golden circle (12° brix) and fresh market consumption. a number of other fruit quality parameters such as fruit translucency, flesh and skin color, flavor and fruit shape were also measured at harvest. fruit translucency at harvest in the main crop was lower with oxygation compared to that of the control treatment (table 9), whereas in the ratoon crop the effect of oxygation was significant in lowering the translucency. low translucency in pineapple at harvest is considered an indicator of better quality fruit. fruit quality measured by ranking of flesh colour was better under the oxygation: the score for flesh colour ranking was 11% higher (3.29) under oxygation than under the control (2.95). fruit quality measured by ranking of skin colour was also better in the oxygation treatment. the score for skin colour was 5% higher with oxygation than in the control. flavor quality of pineapple was also improved with oxygation. the flavor quality score for the sample from the oxygation treatment was 12% higher than the control sample. although a positive effect of oxygation was recorded on these quality parameters, the differences were not statistically significant in either crop, except for flavor in the main crop and translucency in the ratoon (table 10). water use efficiency season-long water use efficiency for the total harvested yield component, the irrigation water use efficiency (iwue), which includes only the irrigation component as the water input, increased by 20% due to oxygation (52.98 t/ml) compared to the control (44.23 t/ml). the gross water use efficiency (gwue), which includes both irrigation and rainfall inputs, increased by 39% due to oxygation (2.97 t/ml), and by table 9 fruit characteristics and quality parameters of pineapple as affected by irrigation treatments in the main crop treatments fruit weight (g/fruit) fruit height (cm) fruit width (cm) brix (º) density (g/cm3) dry matter (%) translucency (1-5) (z) flavor (1-3) (y) flesh colour (1-5) (x) skin colour (1-5) (w) control 832.0 12.71 10.32 16.41 0.89 17.51 1.28 2.45 2.95 2.93 oxygation 1061.8 13.77 10.85 16.55 0.91 17.76 1.10 2.75 3.29 3.07 no-irrigation 834.0 14.50 10.63 15.83 0.97 17.57 2.17 2.83 3.67 3.50 p value 0.045 0.014 0.084 0.719 0.185 0.547 0.11 0.005 0.197 0.564 lsd (p≤0.05) 209.5 0.748 ns ns ns ns ns 0.167 ns ns mean separated by lsd. ns= not significant. (z) translucency rating: 1 = 0% translucency, 2 = 25% translucency, 3 = 50% translucency, 4 = 75% translucency, 5 = 100% translucency. (y) flavor rating: 1 = no flavor, 2 = little flavor, 3 = good flavor. (x) ,flesh colour rating: 1 = 100% white, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. (w) skin colour rating: 1 = 100% green, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. 14 9% due to the control (2.37 t/ml) compared to the noirrigation treatment (2.20 t/ml) (table 11). for the marketable yield component (i.e. the industry harvest), iwue increased only marginally due to oxygation, while gwue increased by 6 % due to oxygation (1.63 t/ml), and by 5% due to the control (1.54 t/ml) compared to the no-irrigation treatment (1.55 t/ml) (table 11). table 11 water use efficiency (irrigation water use efficiency and total water use efficiency) of the harvested sample yield and industry yield in different irrigation treatments for the total yield averaged over the main and ratoon crop treatments wue harvested yield (tonnes/ml) wue industry yield (tonnes/ml) iwue (z) gwue (y) gwue gwue control 44.23 2.37 28.76 1.54 oxygation 52.98 2.97 29.02 1.63 no-irrigation na 2.13 na 1.55 mean 48.60 2.49 28.89 1.57 (z) iwue= irrigation water use efficiency presents tonnes of total harvested fruits per mega liter of applied irrigation. (y) gwue= gross water use efficiency presents tonnes of total harvested fruits per mega liter of applied irrigation + rain contribution in the crop for the entire crop duration. the wue has been presented for harvested yield (total harvest from the sample area), and industry yield harvested by the industry harvesting crew by the machine (represent total marketable fruits). these observations are consistent with, but much smaller than, the findings of bhattarai et al. (2005) where greater wue due to oxygation using sdi tomato was reported, and for cotton and vegetable soybean where season-long wue for fruit and biomass yield and instantaneous leaf transpiration rate were greater with oxygation (bhattarai and midmare, 2009). cost benefit analysis/decision support the additional cost for installing an oxygation unit in an already established sub-surface drip irrigation system involves the purchase of a mazzei air injector model mi 1583 (au$ 365), plus fittings and pressure gauges for an existing 3” irrigation pipe (au$ 135), totaling au$ 500/ ha. the installation cost per unit area can decrease with an increase in the size of the air injector. the estimated yield increment of 7.5 ton/ha/crop with oxygation over the average industry yield of 65.9 ton/ha without irrigation brings an additional return of au$ 3750/ha in the first crop at a sale value of au$ 500/ton of fruits for the investment of au$ 500 for oxygation. for a new sdi installation, however, the cost with oxygation for pineapple is au$ 6000. hence, the repayment period for the investment to oxygated sdi is two crop cycles (six years). sdi infrastructure lasts 15 years, covering five cycles of the crop (three years/crop cycle) with potential additional returns of $18,750/ha over the 15-year period. these comparative estimates have been based on a crop with no-irrigation, particularly in high rainfall years (4500 mm over three years). crop performance under drier years without irrigation is expected to be much less and under such circumstances sdi offers greater opportunity to deliver strategic irrigation. we conclude that oxygation can improve both yield and quality of ratoon pineapple for an industry scale of operation. 4. conclusions the total and marketable fruit yield increased with oxygation and the irrigation control compared to no-irrigation. aerated water increased the marketable fruit yield (73.25 t/ha) by 11% whereas control treatment increased yield (69.2 t/ha) by 6% compared to no-irrigation (65.9 t/ha). total yield (both marketable and unmarketable) was greater, significantly so, due to oxygation (133.7 t/ha), compared to the control (106.4 t/ha), and no-irrigation (90.4 t/ ha). yield gain with oxygation was attributed to the larger area and weight per leaf, greater plant height, higher specific leaf area, chlorophyll content and light interception by the canopy compared to the control and no-irrigation. greater co 2 exchange rates and instantaneous water use efficiency were recorded for the oxygation compared to table 10 fruit characteristics and quality parameters of pineapple as affected by irrigation treatments in the ratoon crop treatments fruit weight (g/fruit) fruit height (cm) fruit width (cm) brix (º) density (g/cm3) dry matter (%) translucency (1-5) (z) flavor (1-3) (y) flesh colour (1-5) (x) skin colour (1-5) (w) control 793.0 12.40 10.17 16.46 0.90 16.37 1.30 2.41 2.74 2.76 oxygation 961.0 13.43 10.62 13.80 0.92 16.61 0.92 2.29 2.74 3.22 no-irrigation 988.0 14.68 10.70 15.90 0.97 16.81 2.40 2.80 3.80 3.60 p value 0.061 0.007 0.138 0.09 0.003 0.674 0.003 0.117 0.077 0.473 lsd (p≤0.05) 203.0 1.16 ns ns 0.03 ns 0.638 ns ns ns mean separated by lsd. ns= not significant. (z) translucency rating: 1 = 0% translucency, 2 = 25% translucency, 3 = 50% translucency, 4 = 75% translucency, 5 = 100% translucency. (y) flavor rating: 1 = no flavor, 2 = little flavor, 3 = good flavor. (x) flesh colour rating: 1 = 100% white, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. (w) skin colour rating: 1 = 100% green, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. 15 the control and no-irrigation treatments. carbon dioxide exchange was not measurable during the day, only in the early morning, evening and night. aerated irrigation water also reduced phytophthora infestation in the field from 11% in the non-irrigated control to 3%, whereas 5% infestation was noted for control drip irrigation. hence, the use of aerated drip irrigation demonstrated multiple benefits for yield, quality and phytophthora disease management. the trial seasons were rather wetter than average years, and the in crop total rainfall was 4250 mm (42.5 ml), requiring only small amounts of irrigation (2.405 and 2.524 ml for control and oxygation respectively). supplementary and strategic irrigation contributed only 5.5% to total crop water input. nevertheless, this strategic use of irrigation and oxygation led to marked benefits for the pineapple crop yield and quality. 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samsampour 1, s. zarbakhsh 2 1 department of horticulture science, faculty of agriculture and natural resources, university of hormozgan, bandar abbas, iran. 2 department of horticulture science, plant breeding and biotechnology, faculty of agriculture, shiraz university, shiraz, iran. key words: anthocyanin, catalase, cut gladiolus flower, enzymatic antioxidant system, nitric oxide (no), sodium nitroprusside (snp) abstract: sodium nitroprusside (snp), as nitric oxide (no) donor, has been considered by postharvest researchers as one of the best option for slowing the processes controlling senescence in cut flowers. here, we investigate the role of no on postharvest physiology and vase life of the gladiolus grandiflorus cv. white prosperity. vase life markedly extended by snp at 150 μm from 3 day to 7.33 day and thus those inducer effects were doseand time-dependent. snp at 125 μm interdependent on vase life time period was observed to be the optimal dose for improving of relative fresh weight (rfw), peroxidase (pod), and total monomeric anthocyanin (tma) in cut flowers. supplementing vase solution with snp indicated significant increase in water uptake of cut flowers and consequently protected to decline in rfw due to alleviate water losses stress. snp was maintained the level of total soluble protein, lipid peroxidation, and pod, whereas it enhanced the level of catalase (cat) and tma in flower petals. summary of our results revealed that snp exogenous prolongs vase life via maintaining protein degrade, scavenging free radical in term of anthocyanin and enzymes antioxidant, decreasing polyphenol oxidase, inhibiting lipid peroxidation, and improving membrane stability in ‘white prosperity’ cut flowers. 1. introduction floriculture is an emerging and fast expanding globalized market and subsequently studies on postharvest handling of cut flowers occupy a fundamental position (gul and tahir, 2013). therefore, the postharvest longevity of flowers have a vital importance in evaluating the value of the each horticulture plant. this aspect can be particularly hold good with cut flowers and it is a necessity for extended handling and transportation periods. cut flowers are greatly perishable, and consequently they have short vase life and also are exposed to early senescence processing, which restricts efficient marketing of economically significant ornamental plants (*) corresponding author: kazemzadehhashem@yahoo.com citation: kazemzadeh-beneh h., samsampour d., zarbakhsh s., 2018 biochemical, physiological changes and antioxidant responses of cut gladiolus flower ‘white prosperity’ induced by nitric oxide. adv. hort. sci., 32(3): 421-431 copyright: © 2018 kazemzadeh-beneh h., samsampour d., zarbakhsh s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 june 2018 accepted for publication 16 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 421-431 422 (nasibi et al., 2014). however, postharvest senescence is a major restriction to the marketing of many species of cut flowers and so much appreciable efforts have been dedicated to developing postharvest treatments to extend the marketing period or increasing postharvest longevity (vajari and nalousi, 2013). sodium nitroprusside (snp), as donor nitric oxide (no, is one of the postharvest treatments which recently using from it for improving postharvest life of horticulture crops has exceptionally increased. postharvest application of snp has been shown to be effective in extending the postharvest life of a range of flowers, fruits and vegetables when applied as a short term fumigation treatment at low concentrations (wills et al., 2000). no is a short-lived bioactive molecule, which is considered to function as prooxidant as well as antioxidant in plants. no molecule is now documented as an important signaling molecule and reported to be involved in various key physiological processes such as plant defense mechanism, abiotic stress resistance, germination, stimulate antioxidant compounds, decrease lipid peroxidation, growth and development of plants etc. (zhao et al., 2004). furthermore, it was also revealed that plant response to such stress or like drought, high or low temperature, salinity, heavy metals and oxidative stress derived from reactive oxygen species (ros), is moderate by no (mandal and gupta, 2014 ). no is recognized as a biological messenger in plants and it has been proved that no is effective for increase the vase life of cut flowers because it can be may play role as anit-ethylene synthesized from wounded or non-wounded organ (abasi, 2014). liao et al. (2009) reported that no may act as an antagonist of ethylene in cut rose flowers senescence. optimum snp levels could postponement the climacteric phase of many tropical fruits and elongate the post-harvest shelf life of a wide range of horticultural crops by inhibiting ripening and senescence (singh et al., 2013). gladiolus is one of the four famous cut flowers in the world (bai et al., 2009). gladiolus cut flowers have extremely used to decorate graves and celebrate major life events in iran. likewise, the longevity of cut flowers is one of the main challenges of florists today. first data concerning about the effect of snp on differential activity of antioxidants and expression of sags (senescence associated genes) in relation to vase life of gladiolus cut flowers (gladiolus grandiflora cv. snow princess) has been reported by dwivedi et al. (2016). finding of their study suggested that the application of snp increases vase life by increasing the scavenging mechanism of reactive oxygen species (ros) in terms of antioxidants activity, membrane stability and down-regulation of ggcyp1 gene expression in gladiolus cut flowers. under condition in plants subjected to snp, not only the responses of various genotypes or cultivars to snp may be multiresponse, but also the responses rely on dose-and cultivar-dependent, physiological growth state, and environmental factors status. the same trend has been stated by naing et al. (2017) who found that snp promoted the vase life of the cut gerbera flowers via a delay in the time to stem bending; however, all three gerbera cultivars responded to snp and the effects were found to be doseand cultivar-dependent. in the previous study, it has been demonstrated that the snp dose that was best for one cultivar was not suitable for another; thus, variation in the optimal dose of snp among cultivars for the enhancement of their vase life could result from differences in their genetic background (naing et al., 2017). hence, whether snp participates in improving of cut flowers of white prosperity cultivar has not been yet reconnoitered. however, the purpose of the present study was to evaluate the effect induced by nitric oxide donor namely, snp, on the enzymatic antioxidant activity, biochemical and physiological processes of cut gladiolus (gladiolus grandiflorus cv. white prosperity) flowers in order to extend their vase life and postharvest shelf-life. 2. materials and methods plant material and snp treatments cut flowers used in the experiment were g. grandiflorus cv. white prosperity. cut gladiolus flowers were obtained from a commercial grower presented in mahallat city, as famous central commercial production of ornamental plant, in iran at normal harvest maturity and transferred immediately to laboratory of the postharvest physiology and technology research, faculty of agriculture and natural resources, hormozgan university at jun, 2017 and the experiments were established on the same day. flowers stems ends were recut under tap water to eliminate air emboli, to inhibit vascular blockage, and to trim to a uniform length of 70 cm. stock solutions of snp (enzo life sciences) were prepared following the manufacturer’s instructions. uniform cut flowers kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 423 were placed in holding solutions, that containing of snp, na2 [fe (cn) 5 no]. 2h2o (sigma-aldrich), as no donor (0, 25, 50, 75, 100, 125 and 150 μm) plus 3% sucrose as carbohydrate supplement. for control set, flowers were dipped in distilled water plus 3% sucrose. finally, the flowers stems were placed in 500 ml bottles with 250 ml of each mentioned solutions containing different concentrations of the snp solutions + 3% sucrose and they were maintained at a temperature of 23±3°c, 60±5% relative humidity and under a 12 h photoperiod using cool-white fluorescent lamps (24 μmol m-2 s-1 irradiance) during experimental period. there were three bottles (21 flowers) per treatment and the experiment was done seven treatments. to escape from photodegradation of snp (release of a nitrosyl ligand and a cyanide ion), the bottles were shielded with black nylons. snp treatment was applied as a continuous treatment and flower stems were kept in solutions till the end of vase life. vase life and water uptake the vase life was determined based on wilting of more than one-third of the petals of flower and vase life termination of each floret was considered as soon as the first symptom of wilting was observed. indeed, it was defined as the number of days in vase life required for one-third of the florets of each spike to lose its ornamental value (lost turgor and wilted). water uptake was measured by periodically weighting the vase of a control bottle without cut flowers and bottles containing flowers. finally, vase water uptake was determined using the formula (rezvanypour and osfoori, 2011): water uptake (ml day-1 g-1 fresh weight) = (st-1-st)/wt where st= solution weight (g) at = days 1, 4, 8 and st-1= solution weight (g) on the preceding day, and wt = fresh weight of the cut flower (g) on t days. number of opened, unopened florets and relative fresh weight on each spike, the number of opened and unopened florets was recorded from the beginning of the experiment to until 20 days after snp treatments. the fresh weight was measured every 4 days and relative fresh weight (rfw) of cut flowers was calculated by the following equation: rfw (%) = (wt/w t=1 ) × 100 where wt = weight of cut flowers (g) at t = days 1, 4, 8 and wt=1 = the initial fresh weight of the same cut flower (g) on day 1 (rezvanypour and osfoori, 2011). antioxidant enzyme assays antioxidant enzyme activities were determined in the third floret from the base of spike at three time points (days 1, 4, and 8). the 100 mg of floret tissue from controls and snp treatments were removed, were homogenized with mortar and pestle in 1 ml 50 mm epps buffer (ph 7.8) containing 0.2 mm edta and 2% pvp, and were ice-covered for the analysis of antioxidant activity. the homogenates were centrifuged at 4°c for 20 min at 12 000×g and the obtaining supernatants were used to evaluate of antioxidant enzyme activities. catalase (cat) activity was assayed as described by chance and mahly (1995) as follows: the assay reaction mixture of cat contained 50 mm phosphate buffer (ph 7.8), 15 mm h2o2, and crude enzyme. the decomposition of h2o2 was followed at 240 nm (e = 39.4 mm-1 cm-1). absorbance values were quantified using standard curve generated from known concentrations of h2o2. for the measurement of peroxidase (pod) activity, the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm guaiacol, 5 mm h2o2 and enzyme. the reaction was started by adding 300 μl of h2o2 (0.03%). the pod activity was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (e = 26.6 mm-1 cm-1) (chance and mahly, 1995). the polyphenol oxidase (ppo) activity was assayed in 2.8 ml of reaction mixture comprised 2.5 ml of 50 mm potassium phosphate buffer (ph 7.8), 0.3 ml substrate containing 0.2 ml pyrogallol and 0.1 ml crude enzyme (kar and mishra, 1976). the reaction mixture was mixed and the ppo activity was determined in absorbance at 420 nm (6.2 mm-1 cm-1). it’s to be remembered that the blank cuvette consisted of 3.0 ml potassium phosphate buffer (ph 7.8). the results of antioxidant enzymes activitie was expressed as units (u) per mg fw. anthocyanin content assay petals were cut from controls and snp treatments of at three time points (days 1, 4, and 8) and were frozen for the analysis of total monomeric anthocyanin (tma). tma content in petals extract was determined by the ph-differential method based on two buffer system described previously by giusti and wrolstad (2005). to measure the absorbance at ph 1.0 and 4.5, the samples were diluted 2 times with ph 1.0 potassium chloride buffer (0.025 m) and ph 4.5 sodium acetate buffer (0.4 m), respectively. therefore, the tma content analyses of prepared mixtures were performed following the methods of giusti and wrolstad (2005). adv. hort. sci., 2018 32(3): 421-431 424 lipid peroxidation the level of lipid peroxidation in petals tissue was measured by determination of malondialdehyde (mda), which is recognized to be breakdown products of lipid peroxidation, at the end of time points (days 8). the mda content was determined with the thiobarbituric acid (tba) reaction. temporarily, 0.2 g of sample tissue was homogenized in 5 ml 0.1% tca. the homogenate was centrifuged at 10000 g for 5 min. 4 ml of 20% tca containing 0.5% tba were added to 1 ml aliquot of the obtained supernatant. the mixture was heated at 95 ºc for 15 min and cooled immediately on ice. the absorbance was measured at 532 nm by a spectrophotometer. the value for the non-specific absorption at 600 nm was subtracted from the above value. the level of lipid peroxidation was expressed as mmol of mda formed using an extinction coefficient of 155 mmol-1 cm-1 (heath and packer, 1968). total soluble proteins total soluble proteins (tsp) content of petals at the three time point (days 1, 4, and 8) was determined according to the method of bradford (1976) using bovine serum albumin as standard. statistical analysis the experiment was carried out in completely randomized design (crd) with three replications. three flowers stems were used for each replication and thus, the experiment was done with seven treatments and three replication per treatment. the nonnormalize date of the total soluble protein, pod enzyme activity and rfw of cut flowers were normalized with kurtosis and skewness test; so, their transformed date used for analyzing. data were statistically analyzed using analysis of variance (anova) in sas software (version 9.4, sas institute inc., cary, nc, usa). correlations among the evaluated parameters were analyzed using pearson’s correlations (p<0.05 and p<0.01). mean comparisons to identify significant differences between treatments were performed using least significant difference (lsd) at the p<0.01 or 0.01 level of probability. 3. results vase life, rfw and water uptake application of snp markedly enhanced the time to vase life for white prosperity cultivar (p < 0.01). results showed that the bottle solution containing snp + sucrose, significantly increased the vase life of cut flowers compared to the control solution (distilled water), as maximum vase life with higher concentration of snp treatments was verified near the end of storage (table 1). however, it positive impacts on increasing vase life was dose-dependent: 150 and 125 μm were displayed to be the best concentration for vase life (7.33 and 5.66 days) of ‘white prosperity’, respectively, whereas the other concentrations lower than the 125 μm did not markedly influenced vase life as compared to control (p<0.01). generally, based on the results of vase life, ‘white prosperity’ exhibited a longer vase life (4.33 days) when exposed to 150 μm no as compared to control (3 days). the prolonged vase life in snp-treated cut flowers were approximately associated with increasing in floral opening of cut flower by 150 μm treatment (table 1). a direct significant relationship was detected between snp and floral opening (%); however, increasing in snp concentration resulted in increasing in floral opening percentage. statistically, the floral abscission and un-opened flower did not affected by snp treatments as compared to control (p<0.01). values followed by the same letter within a column indicate they are not significantly different (p< 0.01) by least significant difference (lsd). table 1 effect of different concentrations of sodium nitroprusside (as nitric oxide donor) on vase life, flower opening and floral abscission in cut gladiolus flowers (gladiolus grandiflorus cv. white prosperity) treatments vase life (days) full-opened flower (%) un-opened flower (%) floral abscission (%) sodium nitroprusside 0 µm 3.0 c 58.60 b 19.21 a 24.39 a sodium nitroprusside 25µm 3.0 c 60.33 ab 15.51 a 22.99 a sodium nitroprusside 50 µm 3.33 c 69.83 ab 14.83 a 18.02 a sodium nitroprusside 75 µm 4.0 bc 66.92 ab 15.87 a 17.19 a sodium nitroprusside 100 µm 3.33 c 72.38 ab 10.52 a 19.72 a sodium nitroprusside 125 µm 5.66 ab 74.81 ab 6.38 a 18.79 a sodium nitroprusside 150 µm 7.33 a 80.25 a 6.52 a 13.21 a kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 425 significantly increased water uptake (12.33±0.56 and 12.69±1.51 ml day-1 fw) compared to the control (9.16±0.81 ml day-1 fw) resulted in 34.60% and 38.53% increase in vase solution uptake on day 1, respectively; however, they were also conserved the same manner on day 4 or day 8. in contrast, the low concentration did not sufficiently play protective role to inhibit water losses on 4 days, which was also detected that the 25 and 50 μm accelerated water losses, even faster than controls, especially on 8 days. lipid peroxidation the data, belong to mda concentration of flowers petals representing the level of lipid peroxidation is revealed in figure 2a. measurement of mda demonstrated that vase solutions containing 125 and 150 μm snp significantly decreased mda production in comparison to control (p<0.01). overall, it was predictable that white prosperity without treatment (control) showed higher mda concentration than the snp treatments. results found inversely correlation between lipid peroxidation and higher snp concentration. at any specified level of snp concentration, the production of lipid peroxidation product was lesser in treatments at the end experiment (8 days) comparison to control. thus, the vase solutions having as shown in figure 1a, rfw (data normalized; 4.6 is equal to 100%) in cut gladiolus flowers of control gradually was declined during the vase life period, while the decline in rfw was no observed by snp treatments throughout the vase life. it is notable that the rfw of the snp treatments solutions except to 125 μm at three point time did not significantly difference with those in control solutions at initial point time (day 1). so, the presence of snp in vase solutions displayed a protective role to the inhibition of rfw decline in vase life period, even when the vase life of cut flowers ended. only snp with concentration 125 μm (5.60±0.08) in vase solution prolonged the rfw 22.55% higher than other concentrations on day 4 or day 8 in comparison to those in controls at initial time (fig. 1a). senescence is a process characterized by water loss and desiccation of plant tissues. during vase life period, water uptake gradually was declined in both some of the snp treatments (25, 50, and 75 μm) and control cut flowers (fig. 1b). generally, the water uptake with snp concentration 100, 125, 150 μm were higher than those under control condition, respectively (p<0.01). at the initial point time (day 1) of vase life, the white prosperity showed a rapid response to high snp concentrations for promoting water uptake; however, the water loss was not observed during its vase life period. the vase solutions containing snp at concentration 125, 150 μm fig. 1 the effects of different concentrations of sodium nitroprusside (as nitric oxide donor) on physiological changes of gladiolus grandiflorus cv. white prosperity cut flowers during vase life. data are means of three replications. vertical bars indicate standard deviation. fig. 2 effect of different concentrations of sodium nitroprusside (as nitric oxide donor) on malondialdahyde (mda), as an indicator of lipid peroxidation, total soluble protein in gladiolus grandiflorus cv. white prosperity cut flowers during vase life. vertical bars with the same letters did not show significantly different using lsd method at p<0.01 significant level. 426 adv. hort. sci., 2018 32(3): 421-431 snp at concentration 125, 150 μm significantly declined the lipid peroxidation of cell membrane (0.463±0.04 and 0.61±0.04 mmol g-1 fw) compared to the control (1.55±0.07 mmol g-1 fw) resulted in 70.13% and 60.65% decline in product induction of lipid peroxidation, mda, on days 8 prior to senescence appearance in cut flowers. total soluble proteins the chemical analysis for tsp of the flower petals exhibited that snp significantly increased the tsp during vase life period in comparison with control (p<0.01) (fig. 2b). the protein degradation of flower petals in control was higher than snp treatments; however, the total soluble protein gradually was declined in control across days. thus, not only snp lead to help to the inhibition of protein degradation in flowers petals on day 4 or day 8, but also it caused in delaying the senescence of gladiolus flowers. so, all of the snp treatments except to 150 μm displayed a protective or maintain role for protein degradation in cut flowers. overall, only increase in tsp was observed with 150 μm snp and also was recorded highest tsp for its on day 8; however, the protein degradation did not occurred by 150 μm snp treatment during vase life period. furthermore, the prolong vase life of white prosperity flowers can be strongly associated with increasing in tsp and inhibiting from its degradation in flower petals during vase life. enzymatic antioxidant and non-enzymatic antioxidant activities anova analysis with mean comparison showed that antioxidant enzymes and non-enzymatic antioxidant activities in flower petals differed significantly between control and treatments in white prosperity (p<0.01). as expected, the ppo activity (u/mg fw) was continually increased in control during vase life, which this tendency was also approximately found for 25 μm (fig. 3a). the low concentration from 25 to 75 μm did not sufficiently decrease the ppo activity in flower petals comparison to control (p<0.01). so, the decrease in ppo activity was observed by 100, 125, and 150 μm treatments on day 4 or day 8, respectively; however, increasing snp concentration in vase solution resulted in markedly decreasing ppo activity in comparison to controls at initial time of vase life (p<0.01). it can be predictable that the positive effect of snp on maintaining or decreasing ppo activity was high dose-dependent. it is now well recognized that high ppo activity accelerate to senescence and to induce browning in plant tissues. generally, the high concentrations of snp to white prosperity cut flowers, check the activity of ppo enzyme, lead to help in delaying the senescence of gladiolus flower via preventing the ppo activity compared to control (p<0.01). as shown in figure 3b, the pod activity (u/mg fw) significantly decreased in control flowers throughout vase life, while the cat activity (u/mg fw) in control flowers displayed a constant tendency at all of the 3 point time of vase life (fig. 3c) comparison to snp treatments(p<0.01). it is appears that all of the treatments except to 125 μm significantly played a protective role to conserve the decrease of pod activity during vase life (p<0.01). the highest pod activity obtained by 125 μm on 8 days, according to lsd test at p<0.01. however, the positive effect of snp on pod activity was doseand time-dependent: 125 μm was observed to be the optimal concentration for pod activity on day 4. thus, in white prosperity, low concentrations did not adequately increase pod activity, which was also found for concentrations higher than the optimal levels (fig. 3b). in concerning about cat activity, the positive relation was detected between cat activity and snp treatments; however, increasing in snp concentration resulted in increasing cat activity, especially on day 4 or day 8, compared to control (p<0.01) (fig. 3c). the results of the present study indicated that with more addition snp concentration by 100 to 150 μm into vase solution was lead to positively increase in cat activity at each of three time points during vase life. the results of lsd test (p<0.01) indicated that tma degradation was gradually happened in control flowers during vase life (fig. 3d). the snp treatments not only significantly prevented from the tma degradation but also they were greatly enhanced the tma production over 8 days, compared to control (p<0.01). at the during vase life, the low concentrations of snp demonstrated a protective role to inhibit tma degradation in flower petals in comparison to control (p<0.01). the furthest increase in the tma production was archived for 125 μm (0.273 ± 0.037 mg l-1), and 150 μm (0.193±0.015 mg l-1) with a significant difference compared to control, respectively. hence, improved tma in flower petals likely to pod activity was doseand time-dependent: 125 μm was observed to be the optimal concentration for tma content. thus, in white prosperity, low concentrations did not adequately increase tma content, which was also detected for concentrations higher than the optimal levels on day 4 or day 8. 427 kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” pearson correlation analysis reveals interactions between physiological, biochemical and antioxidant system related traits in order to arrange for an overview of the associations between physiological, biochemical traits, and antioxidant system activity, the pearson correlation test used for analyzing and thus was investigated all of the significant associations, as presented in table 2. from this analysis 23 positive and 11 negative significant correlations was achieved. among them, some correlations were expected, such as the positive and negative correlations observed between antioxidant system activity, for example, cat activity and tma content (r= 0.89, p<0.01), and ppo activity and tma content (r= -0.95, p<0.01) on days 8, respectively. with regard to physiological traits, the results of paired linear correlation indicated that rfw was positively correlated with cat (r= 0.85, p<0.05 on days 4), and pod activity (r= 0.86, p<0.05 on days 4 and r= 0.81, p<0.05 on days 8), and tma content (r= 0.79, p<0.05 on days 8), while was negatively correlated with ppo activity on day 4 (r= -0.80, p<0.05) and day 8 (r= -0.87, p<0.05) of the white prosperity vase life. also, the pearson correlation of water uptake with cat activity (r= 0.80, p<0.05 on days 1; r= 0.82, p<0.05 on days 4; r= 0.86, p<0.05 on days 8) and with tma content (r= 0.81, p<0.05 on days 8) was positive significant, whereas displayed a negative significant with ppo activity (r= -0.79, p<0.05 on days 4 and r= -0.85, p<0.05 on days 8) (table 2). however, suggesting that the snp treatment is a key inhibitor to water loss and an inducer to antioxidant system for delaying the senescence of gladiolus flowers during vase life, especially on 4 and days 8. total soluble protein had a positive correlation with tma content and a negative correlation with ppo activity. tma indicated a positive correlation with water uptake, rfw, total soluble protein, and cat activity and a negative correlation with ppo and pod activity. cat activity was positively correlated with physiological traits, tma and negatively correlated with ppo and pod activity. however, according to the results of pearson correlation, suggesting that snp might be an important protective or inducer involved in the physiological, biochemical process and antioxidant system in white prosperity vase life that can be alleviate to water loss, rfw, and to browning process, which lead to early senescence appearance. 4. discussion and conclusions the postharvest longevity of cut flower has a critical importance in determining the value of crop. recently, snp, a no donor known to be a signal molecule involved in biotic and abiotic stress tolerance, has been increasingly used to extend the vase life of fig. 3 evaluating the effects of different concentrations of sodium nitroprusside (as nitric oxide donor) on enzymatic and non-enzymatic antioxidant system changes during vase life period of gladiolus grandiflorus cut flowers. vertical bars indicate standard deviation. 428 adv. hort. sci., 2018 32(3): 421-431 cut flowers, such as rose, gladiolus, and carnation (naing et al., 2017). first data concerning about application exogenous snp to improve vase life of g. grandiflora cv. snow princess cut flower has been reported by dwivedi et al. (2016). it is generally accepted that different genotypes or cultivars might indicate different physiological or biochemical responses to exogenous snp, which is the effects induced by it may be rely on doseand cultivardependent. some published evidences supports no acting as a negative regulator during leaf senescence, but also there is opposite result in this regard; no enhances flower abscission and senescence in cut racemes of lupinus havardii wats (sankhla et al., 2003; guo and crawford, 2005). thus, the properly effects of snp on enhancing physiological and biochemical processes for one cultivar, may not be suitable for another, which is due to differences in their genetic background. this aspect has also been confirmed by naing et al. (2017), who found that snp dose that was best for one cultivar of gerbera cut flower was not suitable for another; thus, variation in the optimal dose of snp among cultivars for the enhancement of their vase life could result from differences in their genetic background. hence, whether snp participate in improving of cut flowers of white prosperity cultivar has not been yet reconnoitered. therefore, in the current study, we investigated the role of snp in the enhancement of physiological, biochemical responses, and antioxidant activity to extend vase life of gladiolus grandiflorus cv. white prosperity cut flower. cut flower senescence is linked to a sequence of highly regulated physiological and biochemical processes such as degradation of proteins, dna content, peroxidation lipids and membrane leakage, degradation of macromolecules, cellular decompartmentalization, floral abscission, color change, leaf yellowing, and weight loss (buchananwollaston et al., 2003; nasibi et al., 2014). in this study, results of our findings revealed that the physiological, biochemical, and antioxidant activity induced by snp in white prosperity cultivar were more different than those induced in snow princess cultivar, a previous study by dwivedi et al. (2016), which it may be due to differences in their genetic background. hence, in present study, snp was significantly promoted the vase life of ‘white prosperity’ cut flowers through help to delay the senescence appearance and desiccation on tissue or organ level; however, it effects were discovered to be doseand time-dependent. vase life positively associated with table 2 pearson correlation between physiological and biochemical characteristics of gladiolus grandiflorus cv. white prosperity cut flowers affected by sodium nitroprusside during vase life period wu= water uptake; rfw= relative fresh weight; tsp= total soluble protein; ppo= polyphenol oxidase activity; pod= peroxidase activity; cat= catalase activity; tma= total monomeric anthocyanin, the 1, 2, and 3 representing vase life time for each variable on day 1, day 4, and day 8. ns, *, ** non-significant, correlation is significant at the 0.05 and the 0.01level, respectively. rfw1 has no computed because at least one of the variables was constant. traits wu1 wu2 wu3 rfw2 rfw3 tsp1 tsp2 tsp3 ppo1 ppo2 ppo3 pod1 pod2 pod3 cat1 cat2 cat3 tma1 tma2 tma3 wu1 1 0.932 ** 0.919 ** 0.499 ns 0.595 ns -0.499 ns 0.261 ns 0.408 ns 0.288 ns -0.584 ns -0.713 ns -0.741 ns 0.152 ns 0.152 ns 0.803 * 0.746 ns 0.927 ** 0.421 ns 0.655 ns 0.732 ns wu2 1 0.849 * 0.698 ns 0.753 ns -0.383 ns 0.517 ns 0.616 ns 0.48 ns -0.579 ns -0.801* -0.666 ns 0.347 ns 0.347 ns 0.752 ns 0.829 * 0.981 ** 0.434 ns 0.752 ns 0.854 * wu3 1 0.627 ns 0.722 ns -0.388 ns 0.430 ns 0.553 ns 0.40 ns -0.791* -0.850* -0.787* 0.271 ns 0.271 ns 0.780 * 0.871 * 0.862 * 0.25 ns 0.697 ns 0.811 * rfw2 1 0.988 ** 0.208 ns 0.747 ns 0.687 ns 0.802 * -0.575 ns -0.809* -0.295 ns 0.866 * 0.866 * 0.296 ns 0.854 * 0.656 ns 0.233 ns 0.534 ns 0.745 ns rfw3 1 0.100 ns 0.713 ns 0.677 ns 0.774 * -0.663 ns -0.872* -0.406 0.817 * 0.817 * 0.352 ns 0.892 ** 0.724 ns 0.311 ns 0.565 ns 0.794 * tsp1 1 -0.009 ns -0.208 ns 0.071 ns 0.36 ns 0.342 ns 0.615 ns 0.585 ns 0.585 ns -0.514 ns -0.215 ns -0.436 ns -0.333 ns -0.319 ns -0.405 ns tsp2 1 0.971 ** 0.558 ns -0.64 ns -0.75 ns -0.432 ns 0.445 ns 0.445 ns 0.405 ns 0.685 ns 0.543 ns -0.062 ns 0.771 * 0.820 * tsp3 1 0.526 ns -0.693 ns -0799* -0.561 ns 0.299 ns 0.299 ns 0.591 ns 0.751 ns 0.642 ns -0.073 ns 0.851 * 0.889 ** ppo1 1 -0.272 ns -0.615 ns 0.056 ns 0.747 ns 0.747 ns 0.16 ns 0.775 * 0.365 ns 0.068 ns 0.154 ns 0.507 ns ppo2 1 0.897** 0.868* -0.21 ns -0.21 ns -0.486 ns -0.7 ns -0.682 ns -0.286 ns -0.734 ns -0.847* ppo3 1 0.739 ns -0.45 ns -0.45 ns -0.556 ns -0.910** -0.832* -0.36 ns -0.742 ns -0.956** pod1 1 0.152 ns 0.152 ns -0.688 ns -0.532 ns -0.786* -0.363 ns -0.817* -0.791* pod2 1 1.000** -0.14 ns 0.548 ns 0.269 ns 0.185 ns 0.086 ns 0.318 ns pod3 1 -0.14 ns 0.548 ns 0.269 ns 0.185 ns 0.086 ns 0.318 ns cat1 1 0.661 ns 0.749 ns -0.124 ns 0.779 * 0.682 ns cat2 1 0.786* 0.125 ns 0.646 ns 0.862 * cat3 1 0.482 ns 0.823 * 0.894 ** tma1 1 0.084 ns 0.3 ns tma2 1 0.887 ** tma3 1 kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 429 rfw, water uptake, tsp content, tma, enzyme antioxidant activity and lipid peroxidation. at the start of vase life, there was a noticeably increase and then a constant tendency in water uptake of white prosperity cut flowers during their vase life, which suggested that snp might have a protective role in cut flowers against water losses stress (fig. 1b). the rapid increase in initial water uptake was dose-and time-dependent, while increase in rfw was more dose-dependent (fig. 1a). the 125 μm concentration was observed to be the optimal concentration for increasing in rfw. the rfw increase obtained in white prosperity is in isagreement with results pronounced by dwivedi et al. (2016) in snow princess. the inhibition or improvement in rfw across days under no condition is probbly attributed to the excessive potential of water uptake, leading to a stability or promote in cell turgidity pressure, which restricts burning from reserved carbohydrates in respiration and limits fresh weight reduction. an association of improved water uptake and inhibited fresh weight reduction has been reported by vajari and nalousi (2013) in carnation and naing et al. (2017) in gerbera cut flower. overall, the ‘white prosperity’ in snp (150 μm) had prolonged vase life over control, which was strongly associated with increased water uptake and improved rfw. the damage to the plant cell’s biomembrane liable to senescence process, decrease in the ratio of unsaturated fatty acids, change mobility of the cell membrane, and generate free radicals are resulted in an increase in the concentration of malondialdahyde (mda), which is an indicator of lipid peroxidation and of injury to the plant cell membrane (chen, 2009). so, the higher membrane stability plays a key role in inhibiting leakage of electrolytes, sugars, pigment, solute leakage, and also lipid peroxidation as well as in delay senescence during gladiolus cut flowers postharvest (ezhilmathi et al., 2007; ghadakchiasl et al., 2017). our results showed that the change in membrane stability and lipid peroxidation occurrence resulting from the mda production were alleviated by snp concentrations (150, 125 μm) on day 7 after treatment and therefore protected and reduced white prosperity mda production in cell membrane (fig. 2a). these results are in agreement with those reported earlier by mansouri (2012), who suggested that snp prolonged the vase life of chrysanthemum flowers, which was accompanied by decreasing in the electrolyte leakage, levels of mda and lipid peroxidation. indeed, the role of no in prevention of lipid peroxidation is related to the ability of no to react with lipid alcoxyl (lo•) and lipid peroxyl (loo•) radicals and stop the chain of peroxidation in a direct fashion (beligni and lamatina, 1999). the role of snp in reducing membrane lipid peroxidation has previously been stated by liao et al. (2012) and dwivedi et al. (2016). many researchers have been shown that protein degradation and also shortage protein due to consumption it instead of soluble carbohydrate during senescence process for respiration in petals are the most important causes for shortening cut flowers vase life (rezvanypour and osfoori, 2011). in addition, snp significantly maintained the tsp degradation in cut flowers petals, while in absence snp increased the tsp degradation to a greater rate than snp treatments on day 4 and day 8 (fig. 2b). the tsp measured in vase solution supplemented by 150 μm was distinctly higher than those placed in controls on day 8. the proteins are the basic components of all cell activities, their reduction degrades enzymes and causes higher production of free radicals, as well as reducing protein synthesis (saed-moucheshi et al., 2014). therefore, it was clear that the proteins degradation during vase life significantly inhibited by snp supplements in the vase life of ‘white prosperity’. the increase or protect of tsp degradation by snp application in strawberry (ghadachiasl et al., 2017) and in peanuts (verma et al., 2010) has also been claimed. earlier studies have been confirmed that snp may either be directly scavenging ros and thus decreasing lipid peroxidation, or it may be modulating the activity of antioxidant system (beligni and lamatina, 1999; saed-moucheshi et al., 2014). various studies have demonstrated that the vase life of cut flowers is modulated by antioxidant enzymes and nonenzymatic antioxidant activities (vajari and nalousi, 2013). thus, supplemented vase solutions with snp stimulated a higher enzymatic or non-enzymatic antioxidant activity in flower petals during vase life period. the ppo catalyzes the browning reaction and results in the formation of quinine, which is subsequently polymerized to varying degree leading to production of brown pigments (dubravina et al., 2005). the ppo activity greatly was reduced by snp, while the pod and cat activity greatly promoted by snp during progress senescence (fig. 3). the results were in accordance with the findings of ghadakchiasl et al. (2017) and dwivedi et al. (2016). indeed, the no synthesized by snp in tissue plant acts signaling adv. hort. sci., 2018 32(3): 421-431 430 molecule to enhance the enzymatic antioxidant activity such as sod and cat and ultimately protects proteins degradation as well as lipid peroxidation against free radicals. so, pod and cat high activity induced by snp showed a negatively correlation with ppo activity and thus they blocked ppo activity during vase life (table 2). approximately, high and markedly negative between ppo and more traits examined in current study were also detected in pearson correlation analysis. furthermore, tma degradation in flower petals protected by snp, while tma degradation increasable induced in flower petals without presence snp during vase life period. however, positive effects induced by snp in both pod activity and tma were doseand time-dependent, therefore, the 125 μm was selected as an optimal concentration for tma and pod activity (fig. 3b, d). antioxidant compounds such as vitamin c, glutathione, and anthocyanin plays vital role, as non-enzymatic system, in protecting cell against destructive chemical compounds such as free radicals and reactive oxygen species (ros) that are constantly produced by the cell metabolism and their concentration increases under stress conditions (kazemzadeh et al., 2015). however, snp increased tam content at any time and level of snp concentration in comparison to controls. the high and significantly positive correlation between tma anthocyanin with cat activity and total soluble protein was obtained by pearson analysis (table 2). with progress senescence during vase life, tma probably scavenged free radicals due to oxidative stress, consequently, inhibited more deterioration of membrane and protein degradation in flower petals. in conclusion, vase life period in the g. grandiflorus cv. white prosperity cut flower is likely to be associated with many parameters, particularly fresh weight content, water uptake, enzymatic or nonenzymatic antioxidant activities, membrane stability and lipid peroxidation. furthermore, it was found that positive effects induced by snp on vase life distinctly were doseand time-dependent and were also genetic back ground cultivar-dependent in comparative responses between white prosperity with snow princess, which has previously been reported by dwivedi et al. 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viruses” was established, within the italian ministry of agriculture finalized project “arnadia”, with the purpose of producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest. in 2011, the methods proposed to perform phytosanitary testing were reassessed (dm 13 december 2011) and it was established that mother plants grown in italian nurseries (cultivars or rootstocks) have to be checked for virus infections 10 years after transplanting. the first deadline was set for 30 june 2012 to consider mother plants transplanted in 2001 or earlier. obviously, these mother plants were checked and produced according to older regulations (starting with dpr 24 december 1969) that define pathogens (absence of grapevine leafroll and fanleaf degeneration disease) and methods for their assay (biological indexing) differently compared to the most recent regulations. presently, plants are tested for the following viruses: grapevine leafroll associated virus -1 (glrav-1) and -3 (glrav-3), grapevine fanleaf virus (gflv), arabis mosaic virus (armv) and grapevine virus a (gva). the significant presence of these viruses was recently reported in tuscany after assays carried out in sanitary selection programs. from 1997 to 2004, health tests conducted on 172 uncertified plants selected from the tuscan costal area (elba island, lucca and maremma) showed a critical phytovirologic condition, with 97.1% infected plants (materazzi et al., 2006 a). in the period 20002004, health tests conducted on 318 uncertified grapevine plants selected in d.o.c. or d.o.c.g. areas of tuscany (chianti classico, montalcino and montepulciano) revealed that 58.8% vines were virus-infected (materazzi et al., 2006 b). these findings cannot be transferred to nurseries, considering their use of certified materials. in any case, the virologic status of uncertified grapevine in tuscany indicates the presence of grapevine viruses and related vectors, underlining the importance of periodic verifications in grapevine nurseries to guarantee the highest health standards of plant production and to help reduce the sustainability of old grapevine mother plants in relation to new mandatory diagnostic tests for virus control d. rizzo*, l. stefani*, m. paoli*, e. triolo**, a. panattoni**, a. luvisi**(1) * servizio fitosanitario regionale, servizi agro ambientale di vigilanza e controllo, regione toscana, via dei fiori, 8, 51010 pescia (pt), italy. ** dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: arabis mosaic virus, grapevine fanleaf virus, grapevine leafroll, grapevine virus a, phytosanitary test. abstract: in 2011, the methods to perform phytosanitary tests to check for viruses in grapevine nurseries were reassessed and new regulations were defined (dm 13 december 2011). the mandatory tests require serological assays for the diagnosis of five viruses in grapevine mother plants transplanted in tuscany in 2001 or before. the aim of the present paper is to report the impact of certification programs applied before 2001 in tuscany and the sustainability of older mother plants with relation to the new mandatory diagnostic tests. among the cultivars, virus infection was reported in 19.2% of pool samples, whereas 2.4% of rootstock pool samples showed a compromised health status. glrav-3 is the most frequently found virus (10.4% and 1.3% of cultivar and rootstock pools, respectively), and it is also included in the most frequent multiple infections. multiple infections represent about 25% of infected cultivar pools and almost 50% of infected rootstock pools. adv. hort. sci., 2012 26(3-4): 148-150 (1) corresponding author: aluvisi@agr.unipi.it. received for publication 6 november 2012 accepted for publication 26 february 2013 149 the spread of grapevine pathogens, as determined by the updated regulations. in this paper, we report the results obtained from serological (elisa) tests for the diagnosis of five viruses in grapevine mother plants (cultivars or rootstock) transplanted in 2001 or before, as dictated by dm 13 december 2011. the aim is to report the impact of certification programs applied before 2001 in tuscany and the sustainability of older mother plants as stated by the new mandatory diagnostic tests. 2. materials and methods plant sampling and elisa tests plant sampling and elisa tests were carried out following procedures defined by the working group arnadia grapevine viruses, included in dm 13 december 2011. in accordance with legislation, the following steps were undertaken. sampling was performed beginning in november 2011 in 33 nurseries, collecting sample pools composed by homogenous material from five plants. phloem tissue (2 g) was collected from pools and mechanically ground (tissue lyzer with 10 ml-grinding jar, qiagen, venlo, netherlands) with extraction buffer. elisa test was performed using commercial polyclonal antibodies as well as negative and positive controls (agritest, bari, italy). absorbance at od 405 nm was recorded by photometry (titertek multiskan, titertek instruments inc., huntsville, usa). readings were normalized as r value (od-treated explant/od-hc), identifying the r= 2 threshold which distinguishes the positive versus the negative response (monette, 1983). 3. results the elisa test (table 1) showed that 19.2% of cultivar pools were infected with at least one of the viruses. all five viruses were found in cultivar samples, but glrav-3 was considerably more frequent than the others, followed by gva. the combination of these two viruses also represent the most frequent multiple infection. multiple infections represent about 25% of infected pools and they are characterized by 13 different virus combinations. the least frequent virus was armv. with regard to rootstock pools, there was a low rate of infection (2.3%), even if glrav-3 was still the most frequent virus detected. this virus was also included in the most frequent multiple infections that, for rootstocks, represent almost 50% of infected pools. multiple infections were reported in ten different virus combinations. also in rootstocks, armv was the least frequent virus. 4. discussion and conclusions monitoring revealed glrav-3 as the most frequent virus in cultivar or rootstock mother plants, as reported in sanitary selection research previously carried out in tuscany (triolo and materazzi, 2004; materazzi et al., 2006 a) and other italian areas (digiaro et al., 2000; bica et al., 2002; martelli, 2002). similarly, the low frequency of armv is in agreement with other health checks performed in tuscany on uncertified plants (borgo et al., 2000; materazzi et al., 2006 a). viruses were frequently detected in multiple infections, in particular in rootstocks, with a wide range of combinations. table 1 rates of virus infection for cultivar or rootstock pools detected by elisa test mother plant no of checked pool no of infected pool % of virus infection cultivar 712 137 19.2 rootstock 1523 36 2.4 infected pool out of total (%) glrav-1 glrav-3 gflv armv gva cultivar 2.2 10.4 1.8 1.4 5.1 rootstock 0.9 1.3 0.9 0.6 0.7 pool infected by multiple viruses out of total (%) cultivar rootstock glrav-3/gva 2.11 glrav-1/glrav-3 0.33 glrav-1/glrav-3/gva 0.56 glrav-3/gva 0.13 glrav-1/glrav-3/gva/gflv 0.42 glrav-3/gflv 0.13 glrav-1/glrav-3 0.28 armv/gva 0.13 glrav-1/gflv 0.28 others 0.46 glrav-3/gva/armv 0.28 others 0.98 total multiple infections 4.91 total multiple infections 1.18 150 considering that these findings represent the first application of dm 13 december 2011, it is not possible to evaluate the health status of mother plants grown in tuscan nurseries. moreover, comparison to other italian areas is not relevant because no homogeneous data are available. in any case, these finding can be a starting point to evaluate the health trend of plants in tuscan nurseries. the current health status of mother plants may be due to re-infection events as all tested viruses are known to be vector-transmitted (golino et al., 2002; andret-link et al., 2005; zorloni et al., 2006; demangeat et al., 2010; tsai et al., 2010) and relative vectors have been found in tuscany. however, the significant improvement in diagnostic tests over the last 30 years do not seem to exclude that the primary source or basic material were originally infected. even if these categories are considered in dm 13 december 2011, there is no updated health information available that can reconstruct the propagation links. in this case, the application of traceability tools such as electronic identification (bandinelli et al., 2009; luvisi et al., 2012 a, b) could support retrieval of health information. moreover, the activity of local conservative breeders, such as the associazione toscana costitutori viticoli (tos.co.vit.) set up in tuscany in 2003 (triolo, 2011), can promote the use of certified plants selected according to the most recent regulations. considering that the rate of infected cultivars and rootstocks was found to be very low during verifications carried out during tuscan sanitary selections, these findings confirm that the use of certified plants helps reduce the spread of grapevine viruses. references andret-link p., fuchs m., 2005 transmission specificity of plant viruses by vectors. j. plant pathol., 87: 153-165. bandinelli r., triolo e., luvisi a., pagano m., gini b., rinaldelli e., 2009 employment of radiofrequency technology (rfid) in grapevine nursery traceability. adv. hort. sci., 23(2): 75-80. bica d., nicolosi e., costa a., colombo a., buonocore e., 2002 indagine sulla presenza dei principali virus e nematodi della vite in sicilia. informatore fitopatologico, 52(1): 64-67. borgo m., ferroni g., salvi g., scalabrelli g., 2000 clonal selection of “vermentino” grapevine in tuscany. acta horticulturae, 528: 731-738. demangeat g., komar v., van ghelder c., voisin r., lemaire o., esmenjaud d., fuchs m., 2010 transmission competency of single-female xiphinema index lines for grapevine fanleaf virus. phytopathology, 100(4): 384-389. digiaro m., simeone v., boscia d., savino v., 2000 stato sanitario delle varietà ad uva da tavola di recente introduzione in puglia. informatore fitopatologico, 50(7): 54-58. golino d.a., sim s.t., gill r., rowhani a., 2002 california mealybugs can spread grapevine leafroll disease. calif. agr., 56(6): 196-201. luvisi a., panattoni a., bandinelli r., rinaldelli e., pagano m., triolo e., 2012 b propagative material of grapevine: rfid technology for supporting traceability of “basic” and “certified” material along the wine production chain. adv. hort. sci., 26(1): 39-43. luvisi a., panattoni a., triolo e., 2012 a radio-frequency identification could help reduce the spread of plant pathogens. calif. agr., 66(3): 97-101. martelli g.p., 2002 le principali virosi della vite oggi. informatore fitopatologico, 52(4): 18-27. materazzi a., luvisi a., triolo e., 2006 b diffusione e peculiarità di agenti virali su ceppi di sangiovese selezionati in toscana. in: il sangiovese vitigno tipico e internazionale: identità e peculiarità, arsia, italy, pp. 339-343. materazzi a., triolo e., scalabrelli g., d’onofrio c., luvisi a., ferroni g., 2006 a clonal selection of cv. aleatico (vitis vinifera l.) along tuscan costal area. proc. international symposium on environment identities and mediterranean area, pp. 531-535. monette p.l., 1983 virus eradication through in vitro techniques. the international plant propagatoris society, 33: 90-100. triolo e., 2011 storia ed attualità di tos.co.vit. e del suo nucleo di premoltiplicazione. in: vivaismo viticolo: nuove performance di tracciabilità e sviluppi per il mercato vivaistico. debatte, italy, pp. 44-50. triolo e., materazzi a., 2004 malattie virali e simil-virali della vite in toscana: diffusione, pecularietà ed analogie in 5 vitigni. quaderno arsia, 1: 103-116. tsai c.w., rowhani a., golino d.a., daane k.m., almeida r.p.p., 2010 mealybug transmission of grapevine leafroll viruses: an analysis of virus–vector specificity. phytopathology, 100(8): 830-834. zorloni a., prati s., bianco p.a., belli g., 2006 transmission of grapevine virus a and grapevine leafrollassociated virus 3 by heliococcus bohemicus. j. plant pathol., 88(3): 325-328. impaginato 19 adv. hort. sci., 2018 32(1): 19-26 doi: 10.13128/ahs-21174 the impact of sinorhizobium meliloti and pseudomonas fluorescens on growth, seed yield and biochemical product of fenugreek under water deficit stress s. bolandnazar 1 (*), a. sharghi 2, h. naghdi badhi 3, a. mehrafarin 3, m.r. sarikhani 4 1 department of horticulture, faculty of agriculture, university of tabriz, tabriz, iran. 2 department of horticulture science, islamic azad university, science and research branch, teheran, iran. 3 medicinal plants research centre, institute of medicinal plants, acecr, karaj, iran. 4 department of soil science, faculty of agriculture, university of tabriz, tabriz, iran. key words: nicotinic acid, pgpr, seed, trigonelline, water use efficiency. abstract: bacteria that colonize plant roots and promote plant growth are referred to as plant growth-promoting rhizobacteria (pgpr). for a long-serving period, the pgprs have been applied as biofertilizers in crops culture. recent studies indicated the importance of pgpr for controlling the water deficit. the present study investigates the effects of two different pgprs on some morphophysiological characteristics in fenugreek under water deficit stress. the first factor was application of four pgpr levels including (1. sinorhizobium meliloti, 2. pseudomonas fluorescens, 3. combination of s. meliloti and p. fluorescens and 4. control without bacterial inoculation) and four levels of soil water content including 40%, 60%, 80% and 100% of field capacity (fc) was considered as second factor. the results showed that, leaf area, shoot fresh and dry weight, nitrogen, phosphorus and potassium content, and water use efficacy (wue) were significantly improved by pgpr inoculation and individual use of pgpr was more effective. decreasing of soil water content up to 0.40 fc and inoculation of two bacteria led to increase of secondary metabolites such as nicotinic acid and trigonelline. however seed yield was decreased in pgpr treated plants. 1. introduction insufficient water induces a stress in plants called water deficit stress (dodd and ryan, 2016). water deficit stress has major effects on plant growth and development, limiting crop production in the worldwide. (*) corresponding author: bolandnazar@tabrizu.ac.ir citation: bolandnazar s., sharghi a., naghdi badhi h., mehrafarin a., sarikhani m.r., 2018 the impact of sinorhizobium meliloti and pseudomonas fluorescens on growth, seed yield and biochemical product of fenugreek under water deficit stress. adv. hort. sci., 32(1): 19-26 copyright: © 2018 bolandnazar s., sharghi a., naghdi badhi h., mehrafarin a., sarikhani m.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 september 2017 accepted for publication 8 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 19-26 20 water deficit negatively affects the plant growth and reproduction and disrupts the whole-plant functions (bray, 2004; hummel et al., 2010). it causes cellular changes such as solutes concentration, cell volume alteration, disruption of water potential gradients, changes in membrane shape and disrupting its integrity, loss in turgor pressure, and protein denaturation (bray, 1997; bartels and sunkar, 2005). water deficit is a major threat to agricultural production and tolerance to drought conditions is one of the main targets for crop improvement (salekdeh et al., 2009). plant growth-promoting rhizobacteria (pgpr) are rhizosphere bacteria which constitute symbiotic relationships in large varieties of plants and are used as a biofertilizer (shaukat et al., 2006). pgpr have been reported to confer positive effects and induce plant resistances to environmental stresses and diseases caused by pathogens (kloepper et al., 2004; mayak et al., 2004 a, b; compant et al., 2005; he and yang, 2007; dimkpa et al., 2009; yang et al., 2009). a wide variety of mechanisms that can improve the plant growth, have been suggested to be impressed by pgpr. these involved mechanisms are as follows: nitrogen fixation (van loon, 2007), production of 1aminocyclopropane-1-carboxylate deaminase (acc) (govindasamy et al., 2008), production of volatile organic compounds (ryu et al., 2003), induction of systemic resistance (chandler et al., 2008), phytohormone production (vessey, 2003), siderophore production (el-tarabily and sivasithamparam, 2006), phosphate solubilization (ryu et al., 2003) and potassium releasing (sarikhani et al., 2016). fenugreek (trigonella foenum-graecum l.) is a member of the fabaceae family, cultivated worldwide as a semiarid crop, and traditionally used as a medicinal plant. fenugreek is grown as a spice and a vegetable crop and also have been used as a traditional therapy for the remedy of diabetes (miraldi et al., 2001; smith, 2003; fernández-aparicio et al., 2008). its effect as an antidiabetic and antiatherosclerotic have been documented (ajabnoor and tilmisany, 1988; sharma and raghuram, 1990). fenugreek’s leaves are a rich source of iron, calcium, β-carotene and other vitamins and its seeds contain tannic acid, diosgenin, trigocoumarin, alkaloids trigonelline, trigomethyl coumarin, gitogenin and vitamin a (warke et al., 2011). recently, published literatures indicated that pgpr ameliorate the plants tolerance to abiotic stresses through a variety of mechanisms (srivastava et al., 2008; sandhya et al., 2010). also beneficial effects of pgprs on medicinal plants have been reported (jaleel et al., 2007). shafighi et al. (2014) reported that inoculation of fenugreek with pgpr increases plant height, vegetative growth and seed yield in both well watered and water limited condition. rubin et al. (2017) emphasized that application of pgpr decreases abiotic stress especially drought stress in various plants. they summarized that pgpr inoculation not only improves root and shoot biomass in non-stress condition but also it can enhance aerial biomass and reproductive yield under drought stress condition, however root mass was not increased under stress. in enormous studies, the synergistic effect of nitrogen fixing bacteria especially rhizobia and phosphate solubilizing bacteria such as pseudomons has been reported but application of these pgpr in stressed condition needs more attention. therefore, in this study we focused to the inoculation effect of two native and endogenous pgpr (sinorhizobium meliloti tabriz and pseudomonas fluorescens tabriz) in fenugreek under drought stress condition. 2. materials and methods the seeds of fenugreek with good germination quality was provided from “jahad daneshgahi-iranian institute of medicinal plants”, karaj, iran. the present investigation, carried out in research greenhouse of faculty of agriculture at the university of tabriz during 2015-2016. two bacteria, including pseudomonas fluorescens tabriz and sinorhizobium meliloti tabriz were obtained from the laboratory of soil biology, university of tabriz (tabriz, iran). nutrient broth (nb) and yeast mannitol broth (ymb) were used to prepare a primary culture of pseudomonas and sinorhizobium respectively to inoculate seeds of plant. the experiments were conducted in a factorial design based on completely randomized block design with three replications. the first factor was application of pgpr in 4 levels including 1. s. meliloti, as nitrogen fixing bacterium 2. p. fluorescens, as phosphorous solubilizing bacterium 3. combination of s. meliloti and p. fluorescens 4. negative control without any bacteria and fertilizer treatment. the second factor was soil water content treatment based on field capacity (fc) in 4 levels (100, 80, 60 and 40% of fc). seed of fenugreek was sown in a plastic pot which had 5 kg soil and after establishment 5 plants remained in each pot. soil water content was maintained as aforementioned values by daily weighting of pots by digital scale and water loss by evapotranbolandnazar et al. the impact of s. meliloti and p. fluorescences on fenugreek 21 spiration was added to each pot. plants kept in a greenhouse under a 16 h photoperiod, 24±4/18±3°c day/night temperatures, and 40-60% relative humidity. at the end of the experiment leaf area was measured, by the leaf area meter (li 3100c area meter, li-cor, usa). dry weight of each part was determined after drying at 72˚c until constant weight. the fresh and dry weight plants were determined using a digital weighing scale. the composition of potassium and phosphorus was determined by nitric perchloric and nitric acid digestion methods (zasoski and burau, 1977; havlin and soltanpour, 1980). phosphorous was measured by a vanadate-molybdate method using a spectrophotometer (motic, cl-45240-00, china) and k was determined using a flame photometer (model 405g, iran). nitrogen was measured according to the kjeldahl method that involves changing the form of organic nitrogen to ammonium (nh4) by concentrated sulfuric acid and then measuring the amount of ammonium production (baker and thompson, 1997). also the seed yield was recorded at maturity. water use efficiency (wue) was calculated by the following formula: wue = dw/uw in this formula, dw and uw represent dry mass production and the amount of consumed water, respectively (karimi and roosta, 2014). analysis and quantization of trigonelline trigonelline in the seed sample was measured according to modified method of zheng and ashihara (2004). the samples were ground with 80% methanol and magnesium oxide (mgo) in a mortar and pestle. after incubation at 60°c for 30 min, the homogenates were centrifuged and the supernatant was collected. after complete evaporation of methanol, the methanol-soluble extracts were dissolved in distilled water. the samples were filtered using a disposable syringe filter unit and the aliquots were used for determination of trigonelline (tg) by hplc. the analyses of the samples were carried out using a knauer k2600a liquid chromatography (germany), equipped with a nucleosil c18 (150 mm × 4.6 mm i.d, 5 μm) column. a mixture of methanol: water (50:50 v/v) served as the mobile phase and ph of solution adjusted to 5.0 with 50 mm sodium acetate. the elution has been made in an isocratic mode at a flow rate of 1 ml min-1 and the detection made at 268 nm by uv detector from the above mentioned company (koshiro et al., 2006). one analysis requires 20 min. the retention time of this alkaloid was 4.4 min. before carrying out hplc analysis, we made calibration curve by using different concentrations (0.1, 0.2, 0.5, 0.7 and 1.0 mg ml-1) of trigonelline in phase media. then calibration curve made with trigonelline and the correlations were excellent for trigonelline. this process was performed according to united states pharmacopoeia (u.s. pharmacopeia, usa) by cold extraction method as directed for alcohol soluble material, except where water was used in place of alcohol. measuring nicotinic acid for the measurement of nicotinic acid, it was carried out according to modified martin et al. (1997). the 0.5 g of fenugreek seed powder was mixed with 0.5 g of magnesium oxide (mgo) and 30 ml of distilled water was added to it. the resulting mixture for 30 minutes at 100°c was placed in bath water bath. after cooling, the resulting mixture was filtered using filter paper (1) and was brought to a volume of 50 ml with distilled water. finally absorption at a wavelength of 263 nm of the samples was measured by a spectrophotometer. nicotinic acid concentrations were determined using the standard curve. statistical analysis all collected data were subjected to two-way analysis of variance (anova) through proc glm procedure, using a sas statistical package (sas institute, software version 9.4, cary, nc, usa). if interactions were significant, means were compared by duncan’s multiple range test to determine whether means of the dependent variable were significantly different at p<0.05. 3. results analysis of data variances indicated that the effect of pgpr and soil water content and their interaction on leaf area, shoot fresh and dry weight were significant (p≤0.01). means comparison showed that pgpr inoculation increased fenugreek leaf area, shoot dry and fresh weight especially s. meliloti (table 1). by increasing of water deficit stress, leaf area, shoot fresh and dry weight was decreased (table 2). in aspect of interaction between pgpr inoculation and water stress, the highest and lowest leaf area, shoot fresh and dry weight was observed in well watered (100% fc) and combination of s. meliloti and p. fluorescens treated plants and severe water stressed control plants respectively (fig. 1, 2 and 3). it seems that in normal condition s. meliloti adv. hort. sci., 2018 32(1): 19-26 22 improved aerial growth of fenugreek better than p. fluorescens, whereas combination use of two pgpr bacteria was successful in enhancement of shoot growth better than individual using (fig. 1). un-inoculated control plants produced significantly higher seed yield per pot than pgpr treated fenugreek (table 1). water limitation led to decrease in seed yield (table 2). the maximum seed weight was observed in control plants (fig. 4). the nitrogen, phosphorus and potassium concentration was significantly affected by pgprs and water deficit. as shown in the table of mean comparison (table 1) for the effects of pgpr treatments, the highest n, p and k were observed in plants treated with s. meliloti follows by p. fluorescens and treattable 1 results of mean comparison of different pgpr treatments sxp= treatment containing both s. meliloti and p. fluorescens. dissimilar letters indicating significant differences (duncan’s multiple range test p≤0.01). n, p and k were measured in dry shoot of plant. bacteria leaf area (cm2) shoot fresh weight (g) shoot dry weight (g) n (mg g-1) p (mg g-1) k (mg g-1) wue (g kg-1) seed yield (g pot-1) trigonelline (mg g-1) nicotinic acid (mg g-1) control 756 c 17.71 c 4.18 c 7.36 c 0.54 d 25.84 c 0.139 c 26.53 a 6.84 b 12.34 ab s. meliloti (s) 1212 a 32.14 a 6.00 a 14.27 a 0.71 a 31.73 a 0.226 a 14.23 b 6.97 b 12.69 b p. fluorescens (p) 959 ab 29.74 a 4.37 b 12.93 b 0.67 b 30.40 b 0.213 a 13.25 c 7.65 a 14.11 a sxp 937 b 21.14 b 5.05 ab 12.17 b 0.63 c 29.43 c 0.181 b 14.19 b 7.74 a 14.05 a table 2 results of mean comparison of different irrigation treatments dissimilar letters indicating significant differences (duncan’s multiple range test p≤0.01). n, p and k were measured in dry shoot of plant. soil water content (fc) leaf area (cm2) shoot fresh weight (g) shoot dry weight (g) n (mg g-1) p (mg g-1) k (mg g-1) wue (g kg-1) seed yield (g pot-1) trigonelline (mg g-1) nicotinic acid (mg g-1) 100% 1189 a 33.78 a 6.48 a 13.60 a 0.63 c 29.35 b 0.138 c 24.18 a 6.26 c 11.10 d 80% 1025 b 24.76 b 5.79 b 12.20 b 0.59 d 29.05 b 0.165 b 23.52 b 6.63 c 12.07 c 60% 913 c 18.17 c 3.97 c 12.19 b 0.65 b 30.25 a 0.169 b 12.83 c 7.55 b 13.84 b 40% 737 d 13.93 d 3.36 d 9.73 c 0.69 a 30.37 a 0.287 a 10.20 d 8.76 a 16.18 a fig. 1 interaction of pgpr and soil water content on leaf area of fenugreek. fig. 2 interaction of pgpr and soil water content on the shoot fresh weight fenugreek. fig. 3 interaction of pgpr and soil water content on the shoot dry weight of fenugreek. bolandnazar et al. the impact of s. meliloti and p. fluorescences on fenugreek 23 ments containing both s. meliloti and p. fluorescens. by decreasing of soil water content, n concentration was decreased significantly, but inverse, p and k concentration was increased significantly with an exception at 80% of fc treatment (table 2). the highest and the lowest n concentration was observed in plants treated with s. meliloti and dual application of pgpr bacteria at well watered (100% fc) and control plant at severe water stress treatment (40% fc) respectively (fig. 5) and the highest and the lowest p concentration was related to plants treated with s. meliloti and p. fluorescens in single form at well watered (100% fc) and control plant at severe water stress treatment (40% fc) respectively (fig. 6). according to the interaction effects between pgprs and drought stress the highest and lowest k concentration was observed in dual application of pgpr bacteria at well watered (100% fc) and control plant under sever water stress (40% fc) respectively (fig. 7). water use efficiency (wue) was significantly affected by pgpr and soil water content. mean comparison indicated that pgpr inoculate plants produced more shoot biomass per water unit than control ones (table 1). by increasing water deficit stress wue was increased significantly (table 2). in aspect of interaction between pgpr and water stress it was shown that both dual application of p. fluorescens and s. meliloti and separate application of bacteria under severe water deficit stress (40% fc) led to highest wue and well watered control plants produced the lowest wue (fig. 8). as it was shown in the table of mean comparison (table 1), individual and dual pgpr treatments improved trigonelline and nicotinic acid. by increasing water deficit stress trigonelline and nicotinic acid was significantly increased (table 2). the highest and lowest trigonelline and nicotinic acid was observed in p. fluorescens inoculated at 40% fc soil water content treatment and control in 100% fc soil water content treatment respectively (figs. 9 and 10). fig. 4 interaction of pgpr and soil water content on the seed yield of fenugreek. fig. 5 interaction of pgpr and soil water content on n concentration of fenugreek shoot. fig. 6 interaction of pgpr and soil water content on p concentration of fenugreek shoot. fig. 7 interaction of pgpr and soil water content on k concentration of fenugreek shoot. adv. hort. sci., 2018 32(1): 19-26 24 4. discussion and conclusions water deficit limits many crop production worldwide and negatively affects the plant growth and reproduction; recently published literatures indicated that pgpr ameliorate the plants tolerance to abiotic stresses through a variety of mechanisms (srivastava et al., 2008; sandhya et al., 2010). in keeping with our results, mishra et al. (2010) indicated that pgprs could ameliorate the negative effects of salinity stress conditions by positive effects on parameters such as increasing the germination in plants, and also increasing the yield, drought tolerance, and growth. it has also been reported that even in the presence of optimum levels of nitrogenous fertilizers, inoculating with pgpr containing acc-deaminase activity can improve the yield and growth of inoculated plants (shaharoona et al., 2006). according to the results of this investigation inoculation with pgpr containing acc-deaminase considerably decreased the damages caused by drought stress on the growth and yield. they reported that un-inoculated plants exposed to drought stress at vegetative growth stage had significantly decreased shoot growth by 41%, while in the inoculated plants the decreased shoot growth was only by 18 per cent. in present study control plants without any inoculation produced higher seed yield per pot than pgpr treated fenugreek (table 1). it should be noted that the experiment duration was 5 month and fenugreek has indeterminate flowering habit, so while plants flowering were continued it was harvested. it have been reported that pgpr could delay the flowering time (jaleel et al., 2007). water stress led to decrease in fenugreek seed yield. the reason may be due to this fact that in the absence of stress conditions, more photosynthesis material have been stored in the organs such as stems and leaves which by transferring to the seeds increased the grain weight. in contrary, under stress conditions, the water and mineral absorption by the plant is disrupted which decreases the plant growth and reduces the transmission of photosynthesis material in leaf and other organs to the grain (jaleel et al., 2007). similar to our findings, root bacterial inoculations significantly affected the plant nutrient element contents in apple compared to controls and increased the phosphorus content of treated plants (karlidag et al., 2007). ordookhani et al. (2010) reported that p. fluorescens improved potassium in tomato plant. water use efficiency was increased by pgpr in fenugreek in present investigation. similar to the present findings jaleel et al. (2007) demonstrated that the minimum wue was related to un-inoculated cases which was improved by inoculation with rhizobium and pgpr. in the present investigation leaf area, shoot weight, dry weight, nitrogen, phosphorus and potassium content, and wue increased significantly by treatment with both pgprs. pgpr fig. 8 interaction of pgpr and soil water content on the wue of fenugreek. fig. 9 interaction of pgpr and soil water content on the trigonelline of fenugreek seed. fig. 10 interaction of pgpr and soil water content on nicotinic acid of fenugreek seed. bolandnazar et al. the impact of s. meliloti and p. fluorescences on fenugreek 25 colonizes the plant’s root system and modulates its growth through increasing the availability of nutrients it also protects the plants from phytopathogens (lee et al., 2013). it has been reported that in induced drought stress condition, fixed nitrogen during photosynthesis, spend the production of secondary metabolites (aliabadi farahani et al., 2009). also beneficial effects of pgprs on medicinal plants have been reported (jaleel et al., 2007). it has been reported that the synthesis of secondary metabolites in medicinal plants is induced specific pathway by the impact of microorganisms (bouchereau et al., 1996). integrative use of pgprs and water deficit stress could be an enhance the eco-friendly strategy of pgprs and plants and could increasing the alkaloid yields in medicinal plants (lee et al., 2013). since the fenugreek is used as a medicinal plant this strategy could be applied for increasing its useful secondary metabolites. in conclusion, the results of the present investigation indicate that both s. meliloti and p. fluorescens could effectively increase vegetative growth, nitrogen, phosphorus and potassium content, secondary metabolites and wue in fenugreek regardless of water stress. also under water stress condition pgpr increased plant growth. however in present study seed yield because of delaying bolting time was decrease by application of pgpr. references ajabnoor m.a., tilmisany a.k., 1988 effect of trigonella foenum graceum on blood glucose levels in normal and alloxan-diabetic mice. j. ethnopharmacol., 22: 45-49. aliabadi-farahani h., valadabadi s.a., daneshian j., khalvati m.a., 2009 evaluation changing of essential oil of balm (melissa officinalis l.) under water deficit stress conditions. j. med. plants res., 3: 329-333. baker w.h., thompson t.l., 1997 determination of total nitrogen in plant samples by kjeldahl. in: plank c.o. 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seedlings. plant sci., 166: 807-813. impaginato 97 adv. hort. sci., 2019 33(1): 97-104 doi: 10.13128/ahs-22618 morphological traits and yield of ajowan affected by different irrigation intervals and growth regulators s. ghassemi 1 (*), s. zehtab-salmasi 1, k. ghassemi-golezani 1, s. alizadehsalteh 2 1 department of plant eco-physiology, faculty of agriculture, university of tabriz, tabriz, iran. 2 department of horticulture, faculty of agriculture, university of tabriz, tabriz, iran. key words: carum copticum, field, grain yield, growth regulator, stem diameter, water stress. abstract: two field experiments were carried out to evaluate the effect of salicylic acid and abscisic acid applications on some morphological traits and yield of ajowan (carum copticum l.) under different watering conditions. irrigation intervals (irrigation after 70, 100, 130 and 160 mm evaporation from class a pan) were located in main plots and exogenous applications of water (control), salicylic acid (0 and 1 mm) and abscisic acid (0 and 50 micro-molar) were allocated to sub plots. water stress negatively affected the morphological traits of the ajowan. stem diameter, branches per plant, leaves number per plant, biological yield and grain yield per plant of ajowan were considerably improved by application of salicylic acid and abscisic acid. salicylic acid treatment was more effective than abscisic acid treatment, in this study, salicylic acid treatment increased the stem diameter and leaves number per plant more than abscisic acid and caused a yield enhancement in ajowan plants. in general, foliar spray of salicylic acid and abscisic acid could alter morphological traits and yield of ajowan. 1. introduction medicinal plants have been used for centuries as remedies for diseases and human health, because they contain secondary metabolites of medicinal value. ajowan (carum copticum l.) is an annual herbaceous plant belonging to the umbelliferae family, which grows in the east of india, iran, and egypt, with white flowers and small, brownish seeds. its fruit has been widely consumed as a food flavoring agent and spice. during the past centuries in the iranian traditional medicine, several therapeutic effects including anti-vomiting, antiasthma and anti-spasm, is postulated for ajowan fruits (boskabady et al., 2005). water deficit, defined here as an unbalance between soil water availability and evaporative demand which can naturally occur in the field and causes a decrease in carbon assimilation, tissue expansion and actually (*) corresponding author: saeid.ghassemi67@gmail.com citation: ghassemi s., zehtab-salmasi s., ghassemigolezani k., alizadeh-salteh s., 2019 morphological traits and yield of ajowan affected by different irrigation intervals and growth regulators. adv. hort. sci., 33(1): 97-104 copyright: © 2019 ghassemi s., zehtab-salmasi s., ghassemi-golezani k., alizadeh-salteh s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 february 2018 accepted for publication 23 november 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 97-104 98 cell number. each of these macroscopic processes involves a large number of genes, enzymes, hormones and metabolites (skirycz and inze, 2010). abiotic stresses such as drought cause metabolic changes ranging from synthesis of limited quantities of specialized metabolites to large shifts in primary metabolite composition as well as many other physiological responses. during drought there is a need for osmoticum to accumulate inside the plant cell to retain water and maintain positive turgor pressure (verslues and juenger, 2011). it is generally accepted that, at whole plant level, adaptation to water depletion starts with a change in stomatal conductance, leading to reduced net co2 assimilation and impaired photosynthesis, which result in shoot growth termination. the result of nassiri et al. (2014) showed that seed yield, water use efficiency and harvest index of ajowan reduced in water stress treatment. also, fresh and dry weight of ocimum sp. significantly reduced in water stress conditions (khalid, 2006). adaptation to water stress can be highly controlled by plant growth regulators (popko et al., 2010). salicylic acid is a phenolic compound capable of enhancing plant growth and yield in some plants (arfan et al., 2007). salicylic acid can regulate various stress responses and development such as abiotic stress responses, flowering, senescence, thermogenesis and resistance to pathogens (vicente and plasencia, 2011). among abiotic stresses, this growth regulator has been reported to counter low temperature (tasgin et al., 2003), water stress (abbaspour and ehsanpour, 2016), salinity stress (el tayeb, 2005) and high temperature (he et al., 2005). salicylic acid plays diverse physiological roles in most of plants, which include nutrient uptake, stomatal movements, enzyme activities, thermogenesis, ethylene biosynthesis, photosynthesis, flower induction and plant growth (hayat and ahmad, 2007). applications of 1 or 2 mm sa significantly reduced chilling injury and fruit decay of apricot fruit as well as membrane electrolyte leakage and ascorbic acid content. fruits treated with sa resulted in high total polyphenolic content, antioxidant capacity and carotenoids content (ezzat et al., 2017). also, application of sa significantly enhanced activity of phenylalanine ammonialyase (pal) and content of hydrogen peroxide in apricot fruit. treated fruits showed significantly lower activity of catalase and ascorbate peroxidase but higher activity of superoxide dismutase and peroxidase than those in control fruits (wang et al., 2015). exogenous application of salicylic acid or its derivates affects diverse plant processes. since, this growth regulator is heavily involved in crosstalk with other plant hormones; its effect on some of these processes may be indirect (pieterse et al., 2009). abscisic acid plays critical roles in numerous biological processes, such as gene transcription, seed dormancy and stomatal closure (cutler et al., 2010). this growth regulator appears to be a major player in mediating the adaptation of the plant to water stress (pal et al., 2011). abscisic acid enhances drought tolerance in wheat and many of the other plant species (travaglia et al., 2010). that plays an important role in the regulation of abiotic stress resistance in plants and orchestrates complicated signalling pathways involved in the response to reduced water availability as well as in multiple developmental processes (kim et al., 2010). based on the aforementioned studies and other researchers, it is acceptable that exogenous application of salicylic acid and abscisic acid can improve abiotic stress tolerance in plants. thus, this study was designed to investigate the effect of salicylic acid and abscisic acid applications on some morphological traits of ajowan plants under water stress. 2. materials and methods at the research farm of the faculty of agriculture, university of tabriz, tabriz, iran, during the growing seasons of 2014 and 2015, two field experiments were carried out to evaluate the effect of salicylic acid and abscisic acid applications on some morphological traits and yield of carum copticum l. under different irrigation treatments. irrigation intervals [i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation from class a pan (a cylinder with a diameter of 120.7 cm that has a depth of 25 cm) as normal irrigation and mild, moderate and severe water deficit, respectively] were assigned to main plots and foliar applications of water (control), salicylic acid (0 and 1 milli-molar) and abscisic acid (0 and 50 micro-molar) were allocated to subplots. average maximum and minimum temperatures and rainfall during the experiment in 2014 and 2015 are shown in table 1. each plot had 6 rows of 4 m length, spaced 25 cm apart. seeds of this plant were treated with benomyl (benlat wp 50%; shanghai bosman industrial co. ltd, china) at a rate of 2 g kg-1 before sowing. then sown by hand on april 2014 and 2015 at a depth of about 1 cm in a sandy-loam soil. all plots were regularly irrigated after sowing until seedling establishment, and ghassemi et al. morphological traits of ajowan affected by watering and growth regulators 99 thereafter irrigations were carried out according to the treatments. during plant growth and development, weeds were controlled by hand as required. salicylic acid and abscisic acid were sprayed on plants at vegetative (once) and reproductive (once) stages. leaf number per plant was measured by hand at grain filling stage. also, at maturity stage, other traits such as plant height (by meter), stem diameter (by digital caliper) and branches per plant (count by hand) were determined. at maturity, 10 plants of the middle part of each plot were harvested and grain yield per plant were determined. then above ground biomass was oven-dried at 80°c for 48 hours and weighed and subsequently plant biomass was calculated. measurements for all morphological traits were normally distributed and confirmed through the kolmogorov-smirnov test. randomness was confirmed using the run test, and the descriptive statistics were calculated. mstatc software used to the data analyzed and the means of traits were compared using duncan multiple range tests at p≤0.05. 3. results and discussions combined analyses of variance showed significant effects of irrigation and growth regulators on stem diameter, branches per plant and leaf number per plant, biomass and grain yield per plant. plant height was affected by irrigation treatments and plant biomass was significantly influenced by year. the interaction of year × irrigation, year × growth regulator and year × irrigation × growth regulator for plant biomass were significant. grain yield per plant significantly affected by interaction of irrigation × growth regulator (table 2). plant height of ajowan decreased with increasing water stress. there were no significant differences between hormonal treated and control plants (fig. 1). stem diameter decreased with increasing irrigatable 1 averages of maximum and minimum temperatures and rainfall during the work in tarbiz, iran, 2014-2015 ns,*,**= no significant and significant at p≤0.05 and p≤0.01, respectively. month temperature (°c) rainfall (mm) 2014 2015 2014 2015 april 23.7 12.6 50.2 43.2 may 29.5 30.45 10.7 1.5 june 37.1 38.9 18 0.9 july 38.9 40.2 1.3 0 august 34.75 31.1 0 26.4 september 20.65 23.45 84.2 24.1 source df mean square plant height stem diameter branches per plant leaf number per plant plant biomass grain yield per plant year (y) 1 0.056 ns 0.001 ns 0.68 ns 74.01 ns 65.532 ** 10.178 ns repeat 4 213.63 0.001 7.94 233.44 24.849 5.580 irrigation (i) 3 1465.38 ** 0.169 ** 47.90 ** 5662.23 ** 709.482 ** 294.030 ** y×i 3 0.05 ns 0.001 ns 1.71 ns 24.82 ns 15.325 * 2.631 ns error 12 48.69 0.004 1.35 190.24 3.320 2.420 growth regulator (g) 2 0.72 ns 0.05 ** 6.12 * 1762.16 ** 80.355 ** 18.847 ** y×g 2 0.05 ns 0.001 ns 0.10 ns 73.39 ns 5.137 ** 0.296 ns i×g 6 72.50 ns 0.001 ns 2.40 ns 90.44 ns 16.430 ** 3.944 ** y×i×g 6 0.05 ns 0.001 ns 1.07 ns 24.93 ns 2.724 * 0.316 ns error 32 54.43 0.002 1.25 67.27 0.834 0.498 cv (%) 14.32 9.86 10.44 16.81 8.53 12.03 table 2 combined analysis of variance of morphological traits of ajowan under different irrigation and growth regulators in tarbiz, iran, 2014-2015 fig. 1 means of plant height of ajowan for different irrigation levels. different letters in each column indicate significant difference at p≤0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. adv. hort. sci., 2019 33(1): 97-104 100 tion intervals. hormonal application, especially treatment with salicylic acid significantly increased this trait (fig. 2a and b). with increasing water deficit branches per plant of ajowan decreased. however, difference between i2 and i3 was not significant. salicylic acid treatment increased this trait but, there was no difference between salicylic acid and abscisic acid (fig. 2c and d). similarly, leaf number per plant decreased as water deficit increased. salicylic acid treatment significantly increased this trait. however, there was no significant differences between abscisic acid treated and control plants (fig. 2e and 2f). in both years plant biomass of ajowan decreased with decreasing water supply. salicylic acid application enhanced this trait under i1 and i2 in 2014 and under i1 in 2015, but abscisic acid treatment only increased plant biomass under i1 in both years. salicylic acid was advantage that of abscisic acid on this trait (fig. 3). with increasing water deficit, grain yield of salicylic acid treated plants significantly decreased, compared with control plants. exogenous application of salicylic acid under i1 and i2 and also abscisic acid application under i2 treatment improved grain yield of ajowan plants. however, under i3 and i4 irrigation treatments there was no significant difference between hormonal treated and non-treated plants (fig. 4). reduction in plant height due to water stress (fig. 1) is related with the competition of plants for nutrients and water availability (ghassemi-golezani et al., 2010). water stress during vegetative stages has the greatest impact on biomass and plant height (ghassemi-golezani et al., 2008). it has been config. 2 means of stem diameter (a and b), branches per plant (c and d) and leaf number per plant (e and f) of ajowan for different irrigation and growth regulators. different letters in each column indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid; aba= abscisic acid. fig. 3 means of plant biomass of ajowan for year × irrigation × growth regulator. different letters in each column indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid aba= abscisic acid. ghassemi et al. morphological traits of ajowan affected by watering and growth regulators 101 firmed by many researchers that water stress lead to growth reduction, which was reflected in leaf area, plant height, dry mass, and other growth functions (fischer et al., 1980; kriedemann et al., 1981). the impact of water stress on plant growth can be explained as a method of adaptation to the conditions of water shortage to limit the rate of transpiration (lu and neumann, 1998), in order to maintain the water supply in the soil around plant roots to increases the chance of survival of the plant (passioura, 2002). the mechanism, by which plant height is reduced under water stress, is through the reduction of cell elongation, which leads to the reduction of cell size and therefore the reduction of plant height (schuppler et al., 1998). a reduction in leaf turgor and photosynthesis under water stress condition suppresses cell expansion and growth, leading to the diminution of stem diameter (fig. 2a) (anjum et al., 2011). the decrease in growth parameters under drought stress could be considered as an avoidance mechanism which minimizes water losses (rodriguez et al., 2005). stem diameter fluctuates daily because of transpirationinduced tension changes in the stem sap (irvine and grace, 1997; perämäki et al., 2001). salicylic acid application promotes cell division and enlargement (hayat et al., 2005). it has been reported that salicylic acid significantly enhances the average growth speed of tomato stems (stevens and senaratna, 2006), which is in line with the results of the present study. salicylic acid was reported to increase cytokinins in corn and these hormones increased the stem diameter (shakirova et al., 2003). foliar spray of abscisic acid is favouring vegetative growth of plants as shown for ajowan (fig. 2b) and soybean (travaglia et al., 2009). exogenous application of abscisic acid was able to increase plant adaptive response to various environmental conditions (abraham et al., 2008). number of branches and leaves per plant decreased under drought condition (fig. 2c and 2e). this might be related with the suppression of cell expansion and cell growth due to the low turgor pressure. reduced number of leaves by moisture stress is in line with the finding of stolf-moreira et al. (2010) who found that water stress affected number of leaves for soybean. this is because water stress leads to decreased rate of leaf initiation and reduction in leaf area of already formed leaves. this can be related with lower photosynthetic activity in the affected leaves. the overall effect is a decrease in the rate of new leaf initiation and increase in leaf shedding thereby resulting to reduction in number of green leaves per plant (yunusa et al., 2014). salicylic acid stimulatory effect on growth estimated characteristics could be related with the effect of this growth regulator on the endogenous phytohormones such as growth promoters i.e. cytokinins, gibberellins and auxins (waffaa et al., 1996; shehata et al., 2000). application of these growth regulators increased number of branches per plant (fig. 2d) and leaves (fig. 2f), which could lead to increment of number of flowers and subsequently seed yield. in agreement with these results fathy et al. (2003) on eggplant and gharib (2007) on basil and marjoram they mentioned that salicylic acid increased plant height, number of branches and leaves per plant and dry mass as well, respectively. abscisic acid by induction of genes that encode enzymes and other proteins involved in cellular dehydration tolerance, plays a critical role in regulating plant water status through guard cells and growth as well as (luan, 2002; zhu, 2002) which might be the main reason for increased branches per plant. treatment with abscisic acid in sesamum indicum l. increased number of branches to a large extent (abraham et al., 2008). plant biomass was reduced under water stress (fig. 3) due to leaf senescence and decline in the cell enlargement resulting from reduced turgor pressure (shao et al., 2008). severe water stress may result in arrest of photosynthesis and disturbance of metabolism (liang et al., 2006). water stress inhibits cell enlargement and that reduces various biochemical and physiological processes (shao et al., 2007). fig. 4 mean grain yield of ajowan for interaction of irrigation × growth regulator. different letters indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid, aba= abscisic acid. 102 adv. hort. sci., 2019 33(1): 97-104 drought stress leading to stomata closure and reduction in photosynthesis rate and leaf growth (ozturk, 1999), which ultimately decreases plant biomass. this reduction in plant biomass resulted in decreasing the plant height (fig. 1), stem diameter, branches per plant and leaf number per plant (fig. 2) and consequently grain yield per plant (fig. 4). salicylic acid improved the production of plant biomass (fig. 3) by increasing the stem diameter, branches per plant and leaf number per plant (fig. 2). salicylic acid influences a wide variety of plant processes, including stomatal regulation, chlorophyll content and photosynthesis (yildirim et al., 2008). also, by increasing ribulose 1,5-bisphosphate (rubp) content under drought condition, protecting the photosynthetic machinery from reactive oxygen species produced during drought stress (shehata et al., 2001) and maintaining lai and photosynthetic activity under stress (bayat and sepehri, 2012) increased plant biomass. abscisic acid plays a critical role in regulating plant water status through guard cells and growth as well as by induction of genes that encode enzymes and other proteins involved in cellular dehydration tolerance (zhu, 2002), which might be resulted in increasing dry mass under drought stress (fig. 3). reduction of grain yield under water stress (fig. 4) has been attributed to reduced plant height (fig. 1), stem diameter, branches per plant, leaf number per plant (fig. 2) and plant biomass (fig. 3). water stress severely limits growth and yield of plants by reducing chlorophyll content of leaves, photochemical efficiency of photosystem ii (ghassemi-golezani and lotfi, 2012), photosynthesis (munns et al., 2006) and ground green cover (ghassemi-golezani and ghassemi, 2013). water stress during vegetative stages largely reduces plant height and biomass, while during reproductive stages it has the greatest negative impact on grain yield (ghassemi-golezani et al., 2008). grain yield was improved by growth regulators (fig. 4). the increase in yield might be due to increased sink size mainly number and mass of grains. this could be related to increased photosynthetic efficiency by stabilization of chlorophyll and higher production and translocation of organic material from source to sink. salicylic acid stimulates physiological processes that were reflected on improving vegetative growth (fig. 2, 3) followed by active translocation of the photosynthesis products from source to sink. abscisic acid is also able to activate metabolism of carbohydrates temporally stored in the plant stem (yang et al., 2003; 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effects of moisture stress on the growth parameters of soybean genotypes. discourse j. agric. food sci., 2: 142-148. zhu j.k., 2002 salt and drought stress signal transduction in plants. ann. rev. plant biol., 53: 247-273. impaginato 105 adv. hort. sci., 2019 33(1): 105-111 doi: 10.13128/ahs-22596 propagation of rosa hybrida l. cv. dolce vita by stenting and stem cutting methods in response to different concentrations of iba m. pourghorban 1, s. khaghani 1 (*), p. azadi 2, 3 (*), a. mirzakhani 4, m. changizi 1 1 department of agriculture, arak branch, islamic azad university, arak, iran. 2 department of genetic engineering, agricultural biotechnology research institute of iran (abrii), agricultural research, education and extension organization (areeo), p.o. box 31535-1897 karaj, iran. 3 department of tissue culture, ornamental plants research center, horticultural science research institute, agricultural research, education and extension organization (areeo), mahallat, iran. 4 horticulture crops research department, markazi agricultural and natural resources research and education center, areeo, arak, iran. key words: indole-3-butyric acid (iba), propagation, rooting, rootstock, stenting. abstract: this study was conducted to investigate the effect of different concentrations of indole-3-butyric acid (iba) in propagation of rosa hybrida l. cultivar dolce vita by stenting (cutting and grafting) and stem cutting methods in greenhouse conditions. different concentrations of iba (0, 1500, 3000 and 4500 mg/l) were considered on rosa hybrida l. cv. dolce vita grafted onto rosa hybrida l. ‘natal briar’ rootstock. then, the stentings and stem cuttings were cultured in a cocopeat + perlite (in 1:1 ratio) medium under mist system. the research was conducted as a completely randomized design with three replications. the results suggested that all iba treatments significantly increase rooting percentage compared with the control plants, and the highest specifications of roots and shoots were observed in 1500 mg/l iba in both methods of propagation. the results approved the superiority of stem cutting in rooting. however, the higher content of chlorophyll a, b and total were obtained in stenting method. 1. introduction rose flower (rosa hybrida l.) is one of the world’s most popular flowers among ornamental plants (castilon et al., 2006), and cut rose flower industry is the most important aspect of rose culture industry in the world (bleeksma and van doorn, 2003) with a turnover of 735 million euros in 2015 (azadi et al., 2016). rose plants are propagated by seed and asexual methods such as stem cutting, grafting, budding, cutting-grafting (stenting), cutting-budding, root grafting and tissue culture (salehi and khosh(*) corresponding author: azadip22@gmail.com azadip@abrii.ac.ir shahab.khaghani@gmail.com citation: pourghorban m., khaghani s., azadi p., mirzakhani a., changizi m., 2019 propagation of rosa hybrida l. cv. dolce vita by stenting and stem cutting methods in response to different concentrations of iba. adv. hort. sci., 33(1): 105-111 copyright: © 2019 pourghorban m., khaghani s., azadi p., mirzakhani a., changizi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 february 2018 accepted for publication 30 november 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 105-111 106 khui, 1997 a, b; dole and wilkins, 2005; azadi et al., 2007, 2013). vegetative propagation is the sole method for maintaining desirable characters in a superior cultivar particularly when it is heterozygous and polyploid. the easiest and most common method of growing roses is the use of stem cuttings (anderson and woods, 1999). the success of rooting in stem cuttings depends on species and cultivar, growth season, the condition of cutting wood, type of cuttings like hardwood cuttings, semi hard wood cuttings, softwood and herbal cuttings, and many other factors (hartmann et al., 2002). however, stenting method is an efficient technique for quick propagation of plants. this method is an effective technique of rose propagation (van de pol and breukelaar, 1982), in which cutting and grafting is performed simultaneously and the scion is grafted onto a non-rooted rootstock. formation of the union and adventitious roots on the rootstock occurs simultaneously (nazari et al., 2009; karimi, 2011; babaie et al., 2014). cutting and grafting is also a valuable technique in propagation of several horticultural species including roses (koepke and dhingra, 2013), conifers, rhododendrons, and also a number of citrus fruits, apples, plums and pears (hartmann et al., 2002), therefore the use of rootstocks in propagation is a common practice. plant propagation by stenting has been practiced in several nurseries as a substitute for budding, since it has advantages such as better yield and quality, and resistance to crown gall disease (park and jeong, 2010). efficiency and flower productivity in grafted plants is higher than those in plants growing on their own roots and this is due to the use of rootstock (cabrera, 2002). because of different climatic and soil conditions in different areas of the world, different rootstocks are recommended to be the main subject of attention of compatibility between rootstock and scion (niu and rodriguez, 2008). ‘natal briar’ is a rootstock of unknown lineage, probably coming from south africa, and is becoming the desirable rootstock for hydroponic production systems in netherlands, usa and colombia (cabrera, 2002). ‘natal briar’ is the rootstock for grafted roses, which represents 60-70% of the world’s cultivation since it is easy to root and gives well stem length and head size (otiende et al., 2015). exogenous auxin is shown to have a necessary role in root formation in cuttings and has an effect on the speed and the percentage of rooting of cuttings. plants produce natural auxin in their fresh shoots and leaves, however, the synthetic auxin must be used for successful rooting to prevent cuttings death (stefanic et al., 2006; kasim and rayya, 2009), and has been reported in many species, e.g. hibiscus rosa sinensis (kumar and singh, 2012). for accelerating the formation of adventitious roots, auxin is widely used on the stem cuttings (galavi et al., 2013), and it increases the speed and percentage of rooting (kasim and rayya, 2009). photosynthetic pigments in plants comprise chlorophylls a and b and these pigments play an important role in light absorption during photosynthesis (lobato et al., 2009). carotenoids are a group of natural tetraterpenoid pigments distributed widely in plants, algae, fungi, and bacteria. many flowers, fruits, and roots owe their vivid orange, yellow, and red colors to carotenoids. carotenoids play necessary roles in photosynthesis and photoprotection (domonkos et al., 2013; niyogi and truong, 2013; hashimoto et al., 2016). this research was carried out to evaluate different concentrations of iba in propagation of rosa hybrida l. cultivar ‘dolce vita’ using stem cutting and stenting. 2. materials and methods greenhouse conditions this research was carried out in february 2017 in a greenhouse with plastic cover located in markazi province, arak, iran. the greenhouse was equipped with mist system (fig. 1 a) as well as hot water system for adjusting temperature and humidity. during the study cycle, the average temperature and relative humidity of day were maintained at 20±5°c and 85±5%, respectively. at night, the mist system was automatically switched off and temperature was maintained at 10-15°c and relative humidity of 65±5%. plant materials and propagation (stenting and stem cutting) plant materials were prepared from a rose commercial greenhouse (located in markazi province, arak, iran). the scions consisted of a single-node and one leaf (including 2 leaflets) were collected from rosa hybrida l. cv. dolce vita as soon as their mother plants entered to faded flower stage. the scions were grafted onto a 4.0 cm length internode taken from the semi-hard wood cuttings of the rootstock rosa hybrida l. ‘natal briar’. in this method, scions and rootstocks with a suitable flat cut could be grafted together with the maximum overlap of the cambium layer (fig. 1 b). the scions were selected based on thickness of the stem rootstock. scions then were pourghorban et al. propagation in rose 107 grafted using splice grafting method. scions and rootstocks with an appropriate smooth cut could be grafted together with the maximum overlap of the cambium layer. plastic tape was used for wrapping the graft union. the end bud in the rootstock was removed for better rooting and avoiding sucker production (fig. 1 c). stem cuttings with at least 3 nodes and of 5-8 mm diameter and 15-20 cm length were selected from the middle portion of the vigorously growing shoots of rosa hybrida l. cv. dolce vita as soon as their mother plants entered to faded flower stage. the end node of the stem cuttings were removed for better rooting. the distal end of rootstocks and stem cuttings were treated with different concentrations of iba (1500, 3000 and 4500 mg/l) by quick dip method, and control plants were treated with distilled water. the stentings and stem cuttings were rooted in a mixture of cocopeat and perlite medium (in 1:1 ratio) under mist system. to prevent fungal infection, the beds were disinfected with fungicides 0.2% ‘captan’ solution every two weeks. the grafted plants and stem cuttings were grown for 60 days under the mentioned greenhouse conditions. at the end of the study, the plants were removed from the culture medium and some of their morphological and biochemical traits including healing percentage, rooting, root number, longest root, fresh weight of roots, dry weight of roots, shoot percentage, leaf number, shoot number, longest shoot, content of chlorophyll (a, b and total) and carotenoid were measured. chlorophyll (a, b and total) and carotenoid contents measurement procedure to measure the chlorophyll content, 0.2 g of fresh leaves were ground to a fine pulp by adding 10 ml of 80% acetone. the supernatant was transferred to a 25 ml volumetric flask. the washings were collected in the volumetric flask and the volume was made up to 25ml with 80% acetone. the solution was centrifuged (5000 rpm) for five minutes. the absorbance of the solution was read at 663, 645 and 470 nm against the solvent (80% acetone) blank (lichtenthaler, 1987). chl a (mg/ml) tissue = [12.7(a663) 2.69 (a645)] v / 1000 w chl b (mg/ml) tissue = [22.9(a645) 4.68 (a663)] v / 1000 w total chl (mg/ml) tissue = [20.2(a645) + 8.02(a663)] v / 1000 w car (mg/ml) tissue = [(1000 a470 1.8 chl a 85.02 chl b)/198] v / 1000 w where chl= chlorophyll, chl a= chlorophyll a, chl b= chlorophyll b, car= carotenoid, a= absorbance at specific wavelengths, v= final volume of chlorophyll extract in 80 % acetone (25 ml), and w= fresh weight of tissue extracted (0.2 g). data collection and statistical analysis sixthy days after planting, the stentings and stem cuttings were taken out from the media and some traits such as rooting percentage, shoot number, root number, root length (cm), leaf number, shoot length (cm), contents of leaf chlorophyll (a, b and total) and carotenoid (mg/g), root fresh and dry weight (g), and healing (graft-take) percentage were recorded for each stenting and stem cutting. after calculating fresh weight of roots, they were wrapped in paper envelopes and dried in oven at 60°c for 24 hours to calculate their dry weight. content of leaf chlorophyll and carotenoid were determined using spectrophotometric method (saini et al., 2001). the experiment was conducted as a completely randomized design with three replications, and each replication consisted of 16 samles. the data were analyzed with sas software and means were compared through duncan test (p<0.05). 3. results the influence of treatments on morphological parameters and graft healing of stentings are shown fig. 1 greenhouse space and the stages of preparation of stentings. mist system in the greenhouse (a), flat cut in scions and rootstocks (b), connection scions and rootstocks by plastic tape, and remove end bud (c). adv. hort. sci., 2019 33(1): 105-111 108 in table 1. the presented data clearly revealed that different concentrations of iba significantly affected various traits of roots and shoots. maximum rooting percentage, root number, average root length, and fresh and dry weight of roots were recorded in 1500 mg/l of iba. the highest grafting success, number of leaves, number of shoots per stenting, and average shoot length were observed in 1500 mg/l of iba concentration (table 1). an example of rooted stentings is shown in figure 2 (a-d). the influence of treatments on morphological parameters in stem cuttings is also shown in table 2. maximum rooting percentage, root number, and fresh and dry weight of roots in stem cuttings were recorded in 3000 mg/l of iba. an example of rooted stem cuttings is shown in figure 3 (a-d). different concentrations of iba had significant effect on rooting percentage, root number, and fresh and dry weight of the roots. the highest rooting percentage, root number, fresh and dry weight of roots was obtained in stem cuttings treated with 3000 mg/l of iba. however, there was no significant difference between treatments (1500 and 3000 mg/l) in most of the traits of stem cuttings (table 2). results showed that contents of leaf chlorophyll (a, b and total) and carotenoid in stentings and stem cuttings were affected by different concentrations of iba (p<0.01). an example of rooted stentings is shown in figure 4 (a-d). healing percentage in stentings the presented data clearly revealed that maximum grafting success (66.66%) was in 1500 mg/l of iba, and the minimum healing percentage was observed in control and 4500 mg/l of iba (table 1). root indices and fresh and dry weight of roots the highest percentage of rooting were observed in stentings (81.25%) and stem cuttings (97.91%) treated with 1500 and 3000 mg/l of iba, respectively, and the minimum rooting percentages (35.41% and 83.33%) in both propagation methods were table 1 the effect of iba treatments on growth parameters and graft healing of rosa hybrida l. cv. dolce vita stentings fig. 3 rooting level at different concentrations iba in rosa hybrida l. cv. dolce vita stem cuttings. control plant (a), concentration of 1500 mg/l iba (b), concentration of 3000 mg/l iba (c) and concentration of 4500 mg/l iba (d). fig. 2 rooting level at different concentrations iba in rosa hybrida l. cv. dolce vita stentings. control plant (a), concentration of 1500 mg/l iba (b), concentration of 3000 mg/l iba (c) and concentration of 4500 mg/l iba (d). iba (mg/l) healing (%) rooting (%) root number longest root (cm) roots fresh weight (g) roots dry weight (g) shoot (%) leaf number shoot number longest shoot (cm) 0 37.50 b 35.41 b 1.96 b 0.63 c 0.25 c 0.02 c 37.5 b 1.25 b 0.44 b 2.22 b 1500 mg/l 66.66 a 81.25 a 9.60 a 3.00 a 0.95 a 0.12 a 66.66 a 2.50 a 0.70 a 5.32 a 3000 mg/l 43.75 b 50.00 b 4.54 b 1.93 b 0.60 b 0.07 b 43.75 b 1.96 ab 0.46 ab 3.51 b 4500 mg/l 37.50 b 41.66 b 3.73 b 1.71 b 0.51 b 0.06 b 37.50 b 1.59 b 0.37 b 2.80 b table 2 the effect of iba treatments on growth parameters of rosa hybrida l. cv. dolce vita stem cuttings iba (mg/l) rooting (%) root number longest root (cm) roots fresh weight (g) roots dry weight (g) shoot (%) leaf number shoot number longest shoot (cm) 0 83.33 b 13.95 d 5.06 b 1.23 d 0.12 c 93.75 a 4.8 a 1.69 a 7.87 b 1500 mg/l 95.83 a 22.94 b 8.21 a 1.76 b 0.16 b 95.83 a 4.83 a 1.61 ab 11.20 a 3000 mg/l 97.91 a 26.49 a 7.73 a 2.01 a 0.22 a 97.91 a 4.29 b 1.41 ab 10.46 a 4500 mg/l 85.41 b 20.72 c 5.80 b 1.55 b 0.15 bc 85.41 a 3.39 c 1.35 b 8.73 b pourghorban et al. propagation in rose 109 recorded in control plants (table 1, 2). based on this study, different concentrations of iba affected root number in stentings and stem cuttings. the maximum root number in stentings (9.60) and stem cuttings (26.49) were observed in 1500 mg/l and 3000 mg/l of iba, respectively (table 1 and 2). results showed the greatest average root length in stentings (3 cm) and stem cuttings (8.21 cm) on 1500 mg/l of iba, and the smallest rooting length in stentings (0.63 cm) and stem cuttings (5.06 cm) were observed in control plants (table 1 and 2). the greatest root fresh weights were found in 1500 and 3000 mg/l of iba respectively in stentings (0.95 g) and stem cuttings (2.01 g), and the lightest root fresh weights in both propagation methods were observed in control plants. the greatest dry weight of the root in stentings (0.12 g) and stem cuttings (0.22 g) were also observed at 1500 and 3000 mg/l of iba, respectively, and the lightest dry weight of the root in both propagation methods were recorded in the control plants (table 1 and 2). number of leaves, number and length of shoots in stentings, highest leaf number (2.50), shoot number (0.70) and shoot length (5.32 cm) were observed in 1500 mg/l of iba. the lowest leaf number (1.25) and shoot length (2.22 cm) were recorded in control plants, while the lowest shoot number (0.37) was observed in 4500 mg/l of iba (table 1). in stem cuttings, highest leaf number (4.83) and shoot length (11.20 cm) were observed in 1500 mg/l of iba, while highest shoot number (1.69) was observed in control plants (table 2). contents of leaf chlorophyll (a, b and total) and carotenoid in this study, contents of chlorophyll a, b and total and contents of carotenoid were measured in stentings and stem cuttings. in 0.2 g of fresh leaves, the highest content of chlorophyll a (1.03 and 0.69 mg/g), chlorophyll b (0.57 and 0.32 mg/g), total chlorophyll (1.62 and 1.03 mg/g) and carotenoid (0.77 and 0.49 mg/g) were observed in the 1500 mg/l of iba in stentings and stem cuttings, respectively (fig. 4 a-d). 4. discussion and conclusions physiologically, stenting is more complicated than cutting propagation since formation of the graft union must occur simultaneously with rooting and there are interactions between photosynthesis, root formation, and bud development (van de pol et al., 1986). karimi (2011) also reported that in stenting, the graft union must be formed before root initiation. therefore, after leaf formation on the scion, carbohydrates and natural hormones are produced and transmitted from the leaves to the rootstock for growing. another study reported that rootstock plays an important role in the entire process of root growth, and propagation through bench grafting will bring success. in propagation of chinese hibiscus through stenting showed that iba treatments significantly increased rooting percentage (izadi and zarei, 2014). the effect of different concentrations of auxin on rooting of stewartia pseudocamellia, reported that rooting percentages in cuttings treated with rooting hormones (71.9% to 93.6%) are higher than rooting percentages(53%) in the control plants (nair et al., 2008). for root formation on stem cuttings, natural or synthetic auxins are essential. auxin increases the formation of adventitious roots in many species through facilitating carbohydrates and nitrogen materials transfer to the cutting base and motivating primordial root. the high level of auxin may have a negative effect on root length. auxin leads to transfer of leaf carbohydrate and nitrogen to the roots and therefore causes an increase in the root dry weight (hartmann et al., 2002). al-salem and karam (2001) reported that auxin concentration had a significant effect on rooting regardless of its chemical structure or type. the maximum rooting percentage, number of root, length, and fresh and dry weight were obtained by basal cuttings treatment with 24 mm of iba. the maximum dry weight of roots may be fig. 4 comparison content of chlorophyll and carotenoid (mg/ml) in rosa hybrida l. cv. dolce vita stentings and stem cuttings treated with different concentrations of iba (mg/l). content of chlorophyll a (a), content of chlorophyll b (b), total chlorophyll content (c) and carotenoid content (d). 110 adv. hort. sci., 2019 33(1): 105-111 ascribed to increased roots length and number of roots (ingle and venugopal, 2009). auxins promote adventitious root formation and formed roots enhance the uptake of water and mineral nutrient and production of hormones (cytokinins) which are required for shoot growth and development (otiende et al., 2015). growth amount of shoots and roots are interdependent (tonutti and giulivo, 1990). the probable cause for increase in shoot length may be the better utilization of carbohydrates, nitrogen and other nutrients which has been assisted by growth regulators (chandramouli, 2001). izadi and zarei (2014) reported that the highest leaf number was observed in stentings with higher root number. buds and leaves are considered significant factors to improve root induction (hartmann et al., 2002). leaf chlorophyll content was affected by the interaction between cultivars and propagation methods and was significant in all grafted cultivars; as a result, leaf chlorophyll content and quality index were higher in grafted plants compared to those propagated by cuttings. this might be the effect of rootstock (nazari et al., 2009). in conclusion, the study demonstrated that the application of iba plays an important role in success of rosa hybrida l. cv. dolce vita propagation through stenting and stem cutting methods. iba had significant effect on rooting compared to control plants, and the highest rate of rooting percentage was observed in 1500 mg/l iba in both methods of propagation. superiority of stem cutting in the case of percentage of rooting was approved. however, the higher content of chlorophyll a, b, total and carotenoids were obtained in stenting which probably will affect the quality and flower size at reproductive stage. it is needed further study on the growth stages of the plant until to the flower harvesting in both cuttings and stenting method. acknowledgements authors are grateful to mr. feizian, dr. edrisi, mr. bahrami, and ms. heidari for their assistance during this experiment. the study was done in greenhouse of mr. feizian. references al-salem m.m., karam n.s., 2001 auxin, wounding, and propagation medium affect rooting response of stem cuttings of arbutus andrachne. hortscience, 36: 976978. anderson r.g., woods t.a., 1999 an economic evaluation of single stem cut rose production . 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a quick propagation by simultaneously cutting and grafting. sci. hort., 17: 187-196. van de pol p.a., joosten m.h.a.j., keizer h., 1986 stenting of roses, starch depletion and accumulation during the early development. acta horticulturae, 189: 51-59. impaginato 281 adv. hort. sci., 2017 31(4): 281-288 doi: 10.13128/ahs-20818 anatomical and morphological changes in scion of some olive grafting combinations under water deficit a. dadashpour 1, a. shekafandeh 1 (*), r. oladi 2 1 department of horticultural science, college of agriculture, shiraz university, p.o. box 65186-71441 shiraz, iran. 2 section of wood biology, faculty of natural resources, university of teheran, p.o. box 31585-4313 karaj, iran. key words: growth parameters, olive, olive grafting, rootstock, water deficit, xylem anatomy. abstract: effects of water stress deficit were studied on xylem anatomical features and some growth parameters among six olive grafting combinations; amygdalifolia/arbequina (am/ar), amygdalifolia/koroneiki (am/ko), amygdalifolia/zard (am/z), conservallia/koroneiki (co/ko), conservallia/zard (co/z) and conservallia/arbequina (co/ar) of about three-year-old olive trees (olea europaea l.) under greenhouse conditions. to realize this, a factorial experiment was conducted in a completely randomized design (crd). the results showed that rootstocks exhibited significant effects on scions xylem anatomical physiognomies, such as vessel lumen area (vla) and vessel diameter (vd) and additionally on some growth indices including main stem length (sl), lateral shoot number (lsn) and graft union-cross sectional area (gu-csa). xylem anatomical characteristics including vla, porosity, vessel frequency (vf) and vd of scions decreased when they were grafted onto arbequina and koroneiki rootstocks, but increased onto zard rootstock. all growth parameters showed a decrease under drought stress, while this reduction was more pronounced for zard rootstock than the other rootstocks. however, co/z showed the highest vf and the lowest vulnerability index (vi) and exhibited a better performance at the end of recovery. 1. introduction olive (olea europaea l.), as an evergreen tree, is often under severe drought stress conditions in summer. while withstanding a high level of drought situations (lo gullo and salleo, 1988), the resistance level depends on cultivar and genetic characteristics of the plant (bacelar et al., 2006; therios, 2009). the ability of acclimation to water deficit includes morphological, physiological, biochemical and anatomical mechanisms (bacelar et al., 2006; therios, 2009) as inhibition of cell expansion, leaf area limitation, dense cuticles and trichomes, shoot growth cessation and (*) corresponding author: shekafan@shirazu.ac.ir citation: dadashpour a., shekafandeh a., oladi r., 2017 anatomical and morphological changes in scion of some olive grafting combinations under water deficit. adv. hort. sci., 31(4): 281-288 copyright: © 2017 dadashpour a., shekafandeh a., oladi r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 june 2017 accepted for publication 5 october 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 281-288 282 foliar chemistry changes (bacelar et al., 2007; guerfel et al., 2009). rootstocks can enable the scion for normal growth under water deficit (turner, 1986). in fact, grafting of commercial cultivars on tolerant rootstocks is a specific technique of acclimation to environmental stresses as a rapid tool compared to breeding methods (flores et al., 2010). knowledge about anatomical characteristics of rootstock and scion is essential to understand their interactions under drought conditions. some researchers suggested that vigor reduction induced by dwarfing rootstock may contribute to change water transfer at the graft union (soumelidou et al., 1994; atkinson et al., 2003). other investigations have been focused on the role of tree water status, determining the vegetative structure of grafted tree (basile et al., 2003; solari et al., 2006). it has also been emphasized that sensitivity to drought is related to vessel dimension (lo gullo et al., 1995; trifilo et al., 2007). xylem vessels of plants subjected to drought are in danger of emboli and dysfunction. therefore, drought-adapted trees or shrubs usually have narrower vessels with concomitant in the higher number of vessels per mm2 compared to the drought-sensitive trees (carlquist, 2001). in a study by trifilo et al. (2007) it has been reported that dwarfing rootstocks effectively reduce grafted plant size, although they are not essentially responsible for higher tolerance to drought by scions improving tree resistance to water deficit. it is an ordinary trend to increase root, stem vessel diameter, and decrease vessel density with tree height in some fruit and forest trees (zach et al., 2010). many xylem traits such as xylem-to-phloem ratio, vessel density, and vessel size influence dramatically the rootstock growth ability (meland et al., 2007; trifilo et al., 2007), playing an important role in hydraulic conductance of root and stem (tombesi et al., 2010; zach et al., 2010). thus, number and diameter of vessels are two main characteristics determining hydraulic conductance (tyree and ewers, 1991). smaller and fewer vessels in the scion and graft union of cherry trees could be related to hydraulic resistance and a decreased growth of scion (olmstead et al., 2006). anatomical parameters such as vessel frequency, vessel lumen area and percentage of vessels on wood cross section are reliable to preselect tree vigor. it has been widely accepted that reduced plant growth via increased hydraulic resistance derives from graft union and xylem conduit structure (goncalves et al., 2007; tombesi et al., 2010). a comprehensive understanding of grafted trees vulnerability to drought derived from different scion/rootstock combinations will be useful in orchards situated in semi-arid regions experiencing drought stress. in fact, olive trees grow in mediterranean basin with low summer rainfall while irrigation is not a popular practice. using cloned rootstocks that potentially control scion vigor is a very necessary factor for establishing new olive orchard as an innovative cultivation method (baldoni and fontanazza, 1989; rugini et al., 1996). information on xylem anatomical responses of grafted olives under drought stress conditions are few. it has been assumed that different olive rootstocks may induce various anatomical and morphological modifications in the scions (therios, 2009). hence, the aim of the present study was to investigate xylem anatomical changes of scions’ stems and morphological alterations of some olive grafting combinations under water deficit conditions. 2. materials and methods plant materials and experiment location olive (olea europaea l.) current-season-growth shoots as scion (cvs. amygdalifolia and conservallia) were cleft grafted onto three rootstocks of two years old rooted cuttings (cvs. koroneiki, arbequina and zard) in winter 2015. these grafted plants were transplanted in 12 l plastic pots containing a substrate mixture of field soil, sieved sand, and humus in a 1:1:1 (v: v: v) proportions and was placed in a greenhouse 1100 m above sea level; with a latitude 35°56’ n, and longitude of 50°58’ e and temperature of 28±3°c during the day and 23±3°c at night. the physical and chemical properties of pots soil mixture are presented in table 1. treatments all grafted olive trees were irrigated at field soil characteristics value physical properties sand (%) 39 silt (%) 38 clay (%) 23 texture loam chemical properties organic carbon (%) 1.19 electrical conductivity (ds/m) 2.68 ph 7.78 n (%) 0.12 p (mg/kg) 28.6 k (mg/kg) 360 table 1 physical and chemical properties of the soil used in this study dadashpour et al. anatomical changes in scion of some grafted olives under water deficit 283 capacity (fc) until the start of experiment. moisture content of the substrate was calculated with time domain reflectometry (tdr) in two opposite sides of the containers in each pot at a depth of 20 cm and then an average was calculated. the experiment was carried out in july 2016. the graft combinations were amygdalifolia/koroneiki (am/ko), amygdalifolia/zard (am/z), amygdalifolia/arbequina (am/ar), conservallia/koroneiki (co/ko), conservallia/zard (co/z) and conservallia/arbequina (co/ar). then, they were divided into two groups; group one was irrigated at fc as control and the group two was subjected to water shortage by withholding irrigation for a period of 4 weeks (ws), (n=6). three grafted plants were used for anatomical measurements (n=3) and three were subjected to re-watering for determination of viability and recovery. after three weeks of rewatering (recovery period), the stem length was measured in different graft combinations. xylem anatomical measurements after 4 weeks of water deficit, stem pieces (2cm above the graft union) were collected early in the morning from experimental plants in both groups and immediately fixed in faa (formalin, acetic acid, ethanol, 1:1:1, v:v:v) in the late summer 2016. stem pieces were cross-sectioned using a sliding microtome (gls1, wsl, and switzerland). cross sections with the thickness of ~10 µm were stained with 0.1 % (w/v) safranin (staining in red lignified cell walls) and 1% (w/v) astra-blue (staining in blue-green cellulosic walls) and observed at different magnification under a light microscope (fluo3, bel engineering, italy) equipped with a digital camera (eurekam, bel engineering, italy) connected to a computer. the last annual rings in microscopic sections were investigated both under bright filed and fluorescence lights. the vessel lumen area (vla), vessel diameter (vd), vessel frequency (vf=number of vessels/mm2), vulnerability index (vi= vd/vf), and porosity (total vessel lumen area/ total analyzed area ×100) were measured using image j software (https://imagej.nih.gov /ij/). vi was calculated to assess vessel susceptibility to damage as a reliable indicator during the water deficit. vd was calculated using the mean value of the vessel lumen area [vla = (vd/2)2 π], to estimate idealized diameter. morphological parameters in order to evaluate the growth indices of grafted plants under the period of water stress deficit, main stem length growth (sl) was calculated based on a difference between the stem length above graft union at the beginning and at the end of the drought period. stem diameter (middle of the graft union) was measured with a hand caliper at the end of the drought period (4 weeks) in two opposite sides and an average was applied to calculate the graft union cross sectional area (gu-csa) in mm2. lateral shoot number (lsn) was recorded by counting the shoots in the main stem (above the graft union) at the beginning and at the end of the drought period. leaf number, above the graft union, was recorded at the beginning and at the end of the drought period and then a difference was calculated as ln (the leaves having more than 1 cm length were counted). leaf area (la, cm2) was measured using leaf area meter at the end of the drought period and an average of five full-expanded leaves was used for the analysis. data analysis a factorial experiment was conducted in a completely randomized design (crd) with 9 replications. treatments included 6 graft combinations and 2 levels of irrigation. normal distribution of data was investigated using shapiro-wilk test. data were analyzed by spss version 20.0 statistics software using multivariate analysis of variance (manova) and means were compared by duncan’s multiple range test at probability of 5%. pearson’s correlation coefficients were tested among the analyzed characteristics, using values from 36 graft combinations. 3. results xylem anatomical properties statistical analysis of the data showed significant effects of the rootstocks on the scion vla, sl, lsn, gu-csa and vd. the main effects of rootstocks showed that koroneiki induced the highest values of vla (514.5 μm2) and vd (25.4 μm) in the scion, which was significantly greater than those of induced by zard rootstock. however, there was no significant difference among three rootstocks in porosity, vf, and vi (table 2). the interactions between graft combinations and water stress on scion xylem anatomical characterisrootstock vessel lumen area (μm2) porosity (%) vessel frequency (n/mm2) vessel diameter (μm) vulnerability index arbequina 464.1 a 5.3 a 107.8 a 24.2 a 0.28 a koroneiki 514.5 a 6.9 a 133.4 a 25.4 a 0.27 a zard 371.7 b 4.5 a 119.4 a 21.2 b 0.23 a table 2 the effects of different rootstocks on xylem anatomical properties of scion zard rootstock (fig. 1a). arbequina and koroneiki rootstocks decreased porosity and vf of amygdalifolia and conservallia scions under water shortage conditions; however, zard rootstock increased porosity and vf of both scions (amygdalifolia and conservallia) (figs. 1b and 1c). water shortage increased vessel diameter in am/z and co/z combinations compared with the corresponding controls (fig. 1d). the highest vi was observed in co/ar and co/ko whilst the lowest gained in co/z under water stress conditions (fig. 1e). scion morphological characteristics regardless of the scion types and irrigation levels, rootstocks affected some growth characteristics of scion; koroneiki rootstock exhibited the highest value of scion stem length (sl), which was significantly greater than that of zard rootstock (table 3). this rootstock had the lowest graft union cross sectional area (gu-csa). arbequina presented the greatest gu-csa and lateral shoot number (lsn). koroneiki showed the highest leaf area (4.86 cm2) and leaf number (24.38), however, they were not significantly different from the other rootstocks (arbequina and zard) (table 3). in all graft combinations, water stress deficit significantly decreased the number of leaves and lateral shoot number; however, these reductions were not significant in co/ko combination (figs. 2a and 2c). drought stress also decreased leaf area in all graft combinations, although this reduction was only significant in co/ar combination (fig. 2d). water stress deficit declined scion stem length (sl) in all graft combinations such that there was no growth in am/z and co/z and stem length reduction was not significant in co/ko combination compared to the control (fig. 2b). drought stress significantly reduced gu-csa in am/ar and am/ko while had not significant effect on other graft combinations (fig. 2e). all grafted plants continued to grow as a result of re-watering on scions stem length growth. however, the resulting sl were significantly lower than the controls except in the case of co/z. am and co scions, exhibiting no sl growth under water stress deficit conditions onto zard rootstock, showed a sl elongation which were not significantly different from the adv. hort. sci., 2017 31(4): 281-288 284 tics showed that water shortage caused a decrease in the vessel lumen area (vla) of both scions (amygdalifolia and conservallia) grafted onto the arbequina and koroneiki rootstocks (fig. 1). on the contrary, it increased vla in both scions grafted onto fig. 1 interaction effects of graft combinations (am/ar, am/ko, am/z, co/ar, co/ko, co/z) and water stress deficit on vessel lumen area (a), porosity (b), vessel frequency (c), vessel diameter (d) and vulnerability index (e). am (amagdalifolia), ar (arbequina), ko (koroneiki), co (conservalia) and z ( zard). vertical bars indicate se. each value represents the mean ± se of 3 replicates. means with the same letters are not significantly different (p>0.05) using duncan multiple range test. rootstock leaf number leaf area (cm2) lateral shoot number stem lenght (cm) graft union cross sectional area (mm2) arbequina 18.33 a 4.10 a 1.00 a 3.95 ab 55.74 a koroneiki 24.38 a 4.86 a 0.71 ab 5.80 a 44.45 b zard 18.42 a 3.92 a 0.25 b 1.85 b 47.13 ab table 3 the effects of different rootstocks on xylem anatomical properties of scion dadashpour et al. anatomical changes in scion of some grafted olives under water deficit 285 (figs. 4c and 5a) or were different between these two sub-annual areas (fig. 5c). the fluorescence fig. 2 interaction of graft combinations (am/ar, am/ko, am/z, co/ar, co/ko, co/z) and water stress deficit on leaf number (a), stem length (b), lateral shoot number (c), leaf area (d) and gu-cross section area (e). am (amygdalifolia), ar (arbequina), ko (koroneiki), co (conservallia) and z (zard). vertical bars indicate se. each value represents the mean ± se of 3 replicates. means with the same letters are not significantly different (p>0.05) using duncan multiple range test. fig. 4 micrographs of am/ko (amygdalifolia/koroneiki) combination of olive stem (2 cm above graft union) under bright field (a, b, d) and blue fluorescence light (d). water-stressed samples (b and d) formed narrower rings with smaller vessels comparing with the control (a and c) (cr= current annual ring, ve= vessel, ph= phloem). fig. 3 stem length of graft combinations (am/ar, am/ko, am/z, co/ar, co/ko, co/z) under water stress deficit (ws) and recovery (re) and their controls (cws), (cre), respectively. am (amygdalifolia), ar (arbequina), ko (koroneiki), co (conservallia) and z (zard). vertical bars indicate se. each value represents the mean ± se of three replicates. means with the same letters are not significantly different (p>0.05) using duncan multiple range test. corresponding controls (fig. 3). fluorescence microscopy helped us to distinguish tree ring boundaries since the intensity of emitted light by these specimens diminished from early wood to late wood fig. 5 micrographs of co/ar (conservallia/arbequina) combination of olive stem (2 cm above graft union) under irrigation (a and b) and water deficit conditions (c and d). the right-hand photos (b and d) were taken from samples under bright field light while a and c are the corresponding samples, respectively, under uv and blue fluorescence light, cr= current annual ring. adv. hort. sci., 2017 31(4): 281-288 286 emission light in the late wood of control samples was different in intensity and/or color compared with those of under stress (fig. 5). it seemed that the late ed fibers in water stressed samples are chemically different from control samples. 4. discussion and conclusions xylem anatomical properties vessel number and vd are considered as the main factors determining the hydraulic conductance (tyree and ewers, 1991). in addition, it has been hypothesized that vd and vf may affect drought tolerance. thus, the highest vf value in zard might explain its better performance under drought stress in comparison with the two other rootstocks analyzed in this study (fig. 1c). these results are consistent with the anatomical analysis in cherry rootstocks (goncalves et al., 2007; meland et al., 2007; zoric et al., 2012), while dispute the results on apple tree (bauerle et al., 2011) due to genetic-dependent responses of different tree species. since all plants in this study were grown at the same conditions, xylem anatomical differences among the control rootstocks may have a genetic basis; as it has been supposed that low vigor rootstocks may hereditary produce smaller vessels (beakbane and thompson, 1947). correlation of an increase of vd and a decrease in vf with tree height demonstrated in many other investigations (trifilo et al., 2007; zach et al., 2010). cloned rootstocks are less vigorous, having changes in the anatomy of xylem vessel, which may clarify their effects on shoot behavior (atkinson et al., 2003). in the current research, zard induced a narrower vessel formation compared with arbequina and koroneiki, confirming the results of a previous study on olive tree (trifilo et al., 2007). interestingly, zard caused conservallia to produce the highest number of xylem vessels (vf) under water stress in comparison with the scions grafted onto other rootstocks (fig. 1c). zard rootstock also increased vf in am scion in lesser degree than co. vessel frequency (vf) is often increased by drought (sterck et al., 2008; fichot et al., 2011) through improving the hydraulic conductance (scoffoni et al., 2012). a decreased vf has also been reported under drought stress (corcuera et al., 2004), which is in line with the current results on the scions grafted onto arbequina and koroneiki rootstocks, suggesting drought may contradictorily influence olive different rootstocks. the drought stress increased the vi in am/ar, co/ar, co/ko and am/ko combinations, while decreased in two other combinations (am/z and co/z). it is generally accepted that cultivars with smaller and frequent vessels exhibit low vi (carlquist, 1977), a rationale for a better water transport by co/z. this finding is consistent with that of salvia (hargrave et al., 1994), and suggests wider vessels (greater vd) might be more sensitive to abnormal function performance compared with vessels having smaller diameter. additionally, it is supposed that scions may be more susceptible to show embolism onto invigorating rootstocks during water stress (hargrave et al., 1994). scion morphological properties leaf expansion is the most sensitive characteristic to water deficits, because turgor reduction is the earliest significant biophysical effect of water stress, and leaf expansion is a turgor-dependent activity (taiz and zaiger, 2003). limited leaf area resulted in a low photosynthesis and consequently decreased the shoot length (marron et al., 2002), the number of leaves and the number of lateral shoots. although sl reduction is expected under drought stress, differences among graft combinations can be originated from genetic elements of rootstocks (goncalves et al., 2007). vessel diameter reduction may cause a reduced growth in the scions grafted onto dwarf rootstocks and ultimately result in diminishing stomatal conductance and photosynthesis. the differences in scions growth can be caused by root hydraulic, which plays an important role in the control of olive plant growth (nardini et al., 2006). thus, it can be suggested that plant height and leaf area reduction may be correlated to a low cell development under water deficit in some species, implying the lowest values of sl and la by zard rootstock in this study. although zard rootstock stopped sl under water deficit conditions, however, it increased porosity and vf. it might be concluded that this rootstock employed assimilation for changing the pattern of xylem vessel as an alternative to elongating sl. drought reduces the leaf number per plant. in fact, leaf area extension depends to water status, temperature, and assimilation supplied for growth, which may be affected by drought in a plant. la reduction in the current study is in consistent with results in populus and ziziphus (suther and patel, 1992; thakur and sood, 2005). in relation to correlation between measured traits, vla and vf didn’t show any correlation (r= 0.19). on the other hand, porosity (%), is positively affected by both of these variables. usually, when dadashpour et al. anatomical changes in scion of some grafted olives under water deficit 287 vla is decreased in an angiosperm plant, a decrease in water flow capacity is compensated by increasing the number of produced vessels. hence, vla and vf are usually negatively correlated (oladi et al., 2014). however, in this research, the water deficiency resulted in a simultaneous decrease of both features (except for zard rootstock), suggesting a different strategy employed by olive tree. positive correlation between vd and gu-csa demonstrated that vigorous rootstocks with higher gu-csa had larger vessels, facilitating transfer of more water to aerial parts. a correlation value of 0.31 between ln and vd suggests that larger vessels might result in higher number of leaves in grafted trees. there is a relatively high correlation between ln and sl (r= 0.71), exhibiting a longer sl probably due to more leaf numbers on different rootstocks under irrigation and deficit conditions. recent studies have shown that differences among rootstocks in either root size or scion extension influence soil-water-plant relationship (clearwater et al . , 2007; cohen et al . , 2007; rodriguez-gamir et al., 2010). xylem vessel traits may determine hydraulic conductance extent and subsequently affect the current season vegetative growth. it seems vf is the main anatomical difference among the studied rootstocks. several investigations suggested smaller and fewer vessels in graft union can contribute to water flow resistance, resulting in growth reduction (olmstead et al., 2006; goncalves et al., 2007) as observed in the case of zard rootstock. in this research, water stress deficit decreased the growth indices of all grafting combinations and among them, zard rootstock inhibited any growth. considering anatomical analysis, zard rootstock induced higher porosity and vf in both scions (am and co) and exhibited the lowest vi under water stress conditions. in contrast, although koroneiki and arbequina showed some growth under water stress, however, induced high vi in both scions (am and co) and they did not attain the same performance compared to the control at the end of recovery. however, co/z combination showed the best performance compared to the control at 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33(3): 327-332 doi: 10.13128/ahs-23642 long-time storage pochota fendleri seeds with different packaging a.g. souza 1 (*), o.j. smiderle 2, c.a. pedrozo 2 1 department of biology, federal institute of roraima ‐ campus amajari‐ roraima, brazil. 2 brazilian agricultural research corporation ‐ embrapa rorarima, boa vista‐rorarima, brazil. key words: germination, pet, plastic, seed quality, vigour. abstract: the aim of this study was to evaluate the vigour and physiological quality of seeds of pochota fendleri in two weight classes, stored in different types of packaging over a period of 28 months. the experimental design was completely randomised, with treatments arranged in a 2 x 3 x 4 factorial scheme (2 seed sizes x 3 types of packaging x 4 storage periods), with four replications. the stored seeds were evaluated for germination percentage and germination speed index every six months up to 28 months. at 28 months, the percentage incidence of fungi on the seeds was also determined. the seeds of pochota fendleri remain viable and of high quality for a period of 28 months storage. a pet bottle kept in the refrigerator is recommended to store pochota fendleri seeds. 1. introduction a tree species belonging to the family malvaceae, pochota fendleri (jacq. ws alverson) is highly valued in various countries of central and south america; in brazil, the state of roraima is the only area where the species occurs naturally (smiderle et al., 2017). the high rates of deforestation in areas of natural occurrence, caused mainly by the high demand for wood, have classified the species as threatened with extinction (llamozas et al., 2003; fao, 2017). in addition to its use as wood in the production of manufactured boards, planks, panels, doors, windows and furniture, the plants of pochota fendleri are also used for the shade and shelter of livestock, for planting living fences, for the manufacture of handicrafts and in the recovery of degraded areas (briscoe, 1995; smiderle et al., 2017). planting brazilian native species when compared to exotic species, can contribute to the conservation of regional biodiversity, and may also present important technical and economic advantages due to the ease of acclimatising and perpetuating these species (smiderle and souza, 2016). however, the reduced number of seeds, the difficulty of their collection in areas of natural occurrence and the lack of basic information on native species make them difficult to cultivate. (*) corresponding author: aline.souza@ifrr.edu.br citation: souza a.g., smiderle o.j., pedrozo c.a., 2019 long‐time storage pochota fendleri seeds with different packaging. adv. hort. sci., 33(3): 327332 copyright: © 2019 souza a.g., smiderle o.j., pedrozo c.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 july 2018 accepted for publication 28 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 327-332 328 one of the most researched and controversial aspects concerns the influence of seed weight on physiological quality; in any one batch, seeds classified as large usually give better performance when compared to small seeds as they have a greater amount of nutrient reserves, which serve as input for the initial development of the plant (souza et al., 2017 a). therefore, based on morphological characteristics, germination tests and initial seedling vigour, it is possible to make better decisions about the quality of seed batches, resulting in a reduction in the production time of more-uniform seedlings, and the possibility of successful initial seedling establishment (pereira et al., 2011; dresch et al., 2013; oliveira et al., 2016; souza et al., 2016; souza et al., 2017 b). among the various stages of the seed-production process and seed technology, storage constitutes one of the critical phases and has a great influence on the viability and conservation of the seeds in the batch (nery et al., 2017). storage is important for maintaining seed quality over time, delaying the process of deterioration, so that the seeds maintain their longevity and vigour until they are sown (carvalho and nakawaga, 2000). knowing the storage capacity of seeds makes it possible to adopt the proper conditions for each species; however, due to the diversity of forest species, there is still a lack of information on the technology of such seeds in the literature, especially in relation to storage behaviour (balouchi et al., 2017) for the conservation of germinating power (smiderle et al., 2016; felix et al., 2017). the type of packaging used during storage is also important for maintaining both viability and vigour, since it is directly related to the physiological quality of the stored seeds (rodrigues et al., 2016). the physiological quality of seeds is often evaluated by the standard germination test, which is carried out under optimal environmental conditions to determine the maximum germinating potential and establish a limit for the performance of the batch after sowing (catão et al., 2016). conditions able to preserve seed quality for a certain period are essential; however, there is still little information available (smiderle et al., 2018) on seed technology when applied to native species such as pochota fendleri. the aim of this study was to increase information on the conservation of forest seeds by evaluating the vigour and physiological quality of the seeds of pochota fendleri in two weight classes stored over 28 months in different types of packaging. 2. materials and methods the research was carried out at the seed analysis laboratory and in the forest sector of embrapa roraima. the species used in the research was pochota fendleri (seem.) ws alverson & mc duarte, whose seeds were collected for quality analysis from trees at 10 years of age, in the experimental area of embrapa roraima located in the district of mucajaí, roraima (at 2°23’45.31” n and 60°58’44.34” w) during march and april of 2014. the fruit was harvested as soon as it opened. after the seeds were extracted, they were left to dry for 24 hours on a shaded canvas on the ground, and then packed in polyethylene bags and sent to the seed analysis laboratory of embrapa roraima located at 02°45’28n and 60°43’54” w, at an altitude of 90 m, in boa vista, roraima, for the experiments to be carried out. the collected seeds were selected and sorted as to weight (small seeds being those that passed through a 4 mm diameter sieve, having a mean individual weight of 0.027 g; and large seeds, those that were retained in a 4.5 mm diameter sieve, with a mean individual weight of 0.048 g). the seeds were then packed in individual paper bags, transparent plastic containers or pet bottles, and stored in a refrigerator at 10°c and a relative humidity of 60%. the temperature and relative humidity were monitored with a thermo hygrometer to obtain a monthly average. the packs of samples were divided into sufficient quantities for later evaluation. four samples of 10 previously weighed seeds of pochota fendleri were selected to determine the water content remaining in a drying oven at 105±3°c for 24 h (mapa, 2009). this determination was repeated at 16 and 28 months. the moisture content was calculated based on the fresh weight of the seeds (mapa, 2009). the experimental design was completely randomised, with treatments arranged in a 2 x 3 x 4 factorial scheme (2 seed classes x 3 types of packaging x 4 storage periods), with four replications. seed characteristics were determined every six months during the 28 months of storage, starting from the fourth month. the tests and methodologies to which the seeds were submitted and evaluated are described below. the germination test was carried out on four replications of 50 seeds, in plastic boxes (gerbox®) on souza et al. ‐ seeds storage of pochota fendleri 329 germination paper (germitest®) moistened with distilled water at 2.5 times the weight of the paper and kept in a germination chamber at 25±2°c under constant light. the germination test was evaluated by daily counts until the fourteenth day, considering the number of seeds that emitted a root greater than 2 mm (labouriau, 1983). from the data obtained with the germination test, the germination speed index (gs) was calculated as per the method recommended by maguire (1962). the seeds were also characterised for biometry, the small seeds showing mean values of 5.01 mm for length and 3.32 mm for diameter, and the large seeds, 5.49 mm and 4.20 mm. the values were obtained with the aid of a digital calliper. the means of the variables were submitted to the statistical analysis utilizing the software sisvar (ferreira, 2014), with variance analysis and the tukey test (p≤0.05%). regression analysis was performed for the time factor (months). the statistical analysis was carried out using the sisvar software. 3. results and discussion at the time of storage, the seeds of pochota fend‐ leri presented a mean water content of 10.5% for the seeds classified as small, and 8.1% for those classified as large. the results for seed water content after 4, 16 and 28 months storage in the different treatments can be seen in table 1. the water content of the seeds was determined by the storage conditions, and no statistical analysis was applied to the values. the relative humidity of the three storage containers varied during the 28 months, which contributed to maintaining the seed water content. the packages prevented or decreased the exchange of water vapour between the seeds and the storage environment. it is worth noting that before storage the seeds presented 98% germination for those classified as large and 85% for those classified as small. the results obtained in the present study demonstrated a strong relationship r2= 0.88, for germination percentage in both classes of seed packed in the different containers. for the study under analysis, the seeds classified as small showed inferior performance in relation to germination percentage when compared to the large seeds (fig. 1a and b), irrespective of container or storage period. table 1 mean water content (%) in seeds of pochota fendleri for different weight classes, packaging and storage in the column, means followed by different letters, lowercase letters between packages and uppercase between sizes, differ by tukey test at 5%. packaging months mean 4 16 28 large seeds paper bag 8.0 a b 7.8 a b 7.7 a a 7.8 plastic bag 8.1 a b 8.0 a b 7.8 a b 7.9 pet bottle 8.1 a b 8.0 a b 7.8 a b 7.7 mean 8.05 7.96 7.77 small seeds paper bag 10.0 a a 9.2 b a 8.0 b a 9.0 plastic bag 10.5 a a 10.0 a a 9.0 a a 9.7 pet bottle 10.5 a a 10.0 a a 9.5 a a 10.0 mean 10.25 9.90 8.79 fig. 1 mean values for germination (%) of large (a) and small (b) seeds of pochota fendleri for different packaging and storage periods (months). pet l= pet bottles, large; paper l= paper bags, large; plast l= transparent plastic containers, large; pet s= pet bottles, small; paper s= paper bags, small; plast s= transparent plastic containers, small. adv. hort. sci., 2019 33(3): 327-332 330 in table 2, the means of the squares and the levels of significance can be seen by the f test for the characteristics evaluated, at a level of 0.05% probability for all variables studied. at 28 months storage, the large seeds packed in the pet bottles presented on average a 68% yield in germination percentage when compared to those in the plastic packaging (fig. 1a). it was found however, that even after 28 months storage, the large seeds storage pet bottles (fig. 1a) maintained a germination percentage 83% below the minimum established for commercialisation, which is 85% (mapa, 1992). according to souza et al. (2017 b), for the same species the seeds of greater weight presumably have more reserves, a higher level of hormones and wellformed embryos, and are considered to have greater vigour. with the small seeds however, there was an expressive linear loss in germination percentage in the three containers during storage. the same results were seen for germination speed index in the small seeds (fig. 2b), demonstrating that this characteristic can be efficient in detecting differences in vigour among seed size classes in storage. evaluating germination capacity in seeds of enterolobium schomburgkii, horing et al. (2012) found that there was a linear decrease in germination percentage after 30 months of storage for seeds packed in waterproof plastic pots but stored under laboratory conditions. seeds of caesalpinia leiostachya (benth.) ducke (pau-ferro) maintained a higher germination percentage when stored for a period of eight months inside the fruit in a natural environment (biruel et al., 2007). the germination speed index obtained for large seeds stored for up to 10 months in pet bottles was 18.0 (fig. 2a); however, a greater reduction was found when the seeds were kept in paper or plastic bags (fig. 2a). fig. 2 mean values for the germination speed index of large (a) and small (b) seeds of pochota fendleri submitted to different packaging and months of storage. pet l= pet bottles, large; paper l= paper bags, large; plast l= transparent plastic containers, large; pet s= pet bottles, small; paper s= paper bags, small; plast s= transparent plastic containers, small. table 2 summary of the variance analysis (mean squares and significance by the f test), coefficients of variation, and general means obtained for germination (g) germination speed index (gsi) and water content (u%) of pochota fendleri for different packaging and storage periods (months) *significant difference in 0.05 levels; ns= not significant. source of variation df germination (%) germination speed index source of variation df water content (%) storage periods (sp) 4 6481.7708 ** 232.8101 ** storage periods (sp) 2 4918472 ** packing (p) 2 3954.1666 ** 183.7151 ** packing (p) 2 2040139 ** size (s) 1 8066.6666 ** 140.9604 ** size (s) 1 53388889 ** sp x p 8 288.8020 ** 3.3093 ns sp x p 4 0.270347 ns sp x s 4 272.3958 ns 3.8119 ns sp x s 2 2287639 ** p x s 2 1379.1666 ** 60.6049 ** p x s 1 1075972 ** sp x p x s 8 159.1145 ns 8.0687 ns sp x p x s 2 0.186597 ns error 120 1.237.500 40.788 error 54 0.183426 total 149 total 71 coefficient of variation (%) 16.08 17.31 coefficient of variation (%) 4.87 souza et al. ‐ seeds storage of pochota fendleri 331 quality assumes a prominent role in the cultivation of forest species. references balouchi h., baladi s., moradi a., movahhedi d.m., 2017 the influence of temperature and moisture con‐ tent on seed longevity of two genotypes of linum usitatissimum. seed sci. technology, 45: 130-138. bello e.p.b.c., 2005 influência de substratos, temperatu‐ ra, estresse hídrico e armazenamento na germinação de torresea acreana ducke [dissertação]. cuiabá: faculdade de agronomia e medicina veterinária. biruel r.p., aguiar i.b., paula r.c., 2007 germinação de sementes de pau‐ferro submetidas a diferentes con‐ dições de armazenamento, escarificação química, tem‐ peratura e luz. rev. brasil. sem., 29(3): 151-159. briscoe c., 1995 pespectivas personales para plantaci‐ nes. programa del silvicultura. catie. turrialba, 3051 costa rica. carvalho n.m., nakagawa j., 2000 sementes: ciência, tecnologia e produção. 4. ed. jaboticabal: funep, pp. 588. catão h.c.r.m., gomes l.a.a.a., guimarães, r.m., fonseca p.h.f., caixeta f., marodin j.c., 2016 physiological and isozyme alterations in lettuce seeds under different conditions and storage periods. j. seed scien., 38: 305-313. dresch d.m., scalon s. de p.q., masetto t.e., vieira m.d.o.c., 2013 germinação e vigor de sementes de gabiroba em função do tamanho do fruto e semente. pesq. agropec. trop., 43: 262-271. fao, 2017 faostat. statistics agricultural database. fao, rome, italy. felix c.f., araujo f.s., ferrari c.s., pacheco m.v., 2017 dessecação e armazenamento de sementes de adonidia merrillii (becc.) becc. agrária rev. brasil. ciênc. agrar., 12: 86-91. ferreira d.f., 2014 rewiew: sisvar: a guide for its boot‐ strap procedures in multiple comparisons. ‐ ciênc. agrotec., 38(2): 109-114. horing r.c., mojena pa., pelissari f., 2012 qualidade fisiológica de sementes e estudos morfológicos de plân‐ tulas de enterolobium shomburgkii (benth). congreso florestal, labouriau l.g., 1983 a germinação de sementes. série de biologia, monografia 24. programa regional de desenvolvimento científico e tecnológico. secretaria geral da organização dos estados americanos, washington, usa. llamozas s., duno de stefano r., meier w., riina r., stauffer f., aymard g., huber o., ortiz r., 2003 libro rojo de la flora venezolana. ‐ provita fundación polar. fundación instituto botánico de venezuela, “dr. tobías lasser”, caracas, venezuela, pp. 549. it is therefore interesting to classify seeds by size, since the procedure is quick, and can be carried out manually or with the use of specific sieves or even a densimetric table. the use of heavier seeds gives the consumer or producer a greater guarantee of the physiological quality of the material purchased and used in producing seedlings of pochota fendleri, since seed quality assumes a prominent role in the cultivation of forest species, and can be considered one of the principal bottlenecks, especially in the area of seedling production. in turn, seeds classified as small and packed in paper bags presented an index that was 35% smaller at 28 months than seeds stored in pet bottles. according to marcos filho (2005), a reduction in the germination speed index is the first symptom of a fall in seed performance, generally determined by disorganisation of the membrane system. bello (2005) found an increase in the mean germination time and a decrease in the germination speed index in seeds of torresea acreana (mirtaceae) over a storage period of 12 months, indicating a reduction in germination speed. the results obtained for the variables evaluated in the present study indicate that seeds should be stored in pet bottles in a refrigerated environment maintained at 10°c for different periods (months). there are currently no studies that might support the staggered production of pochota fendleri seedlings in the field or in the nursery. the practice of staggered sowing is not yet carried out by producers of pochota fendleri, but such a method, if based on scientific principles, could make a positive contribution to seedling production, making it possible to produce seedlings at different times throughout the year, as well as providing seedlings of differing quality standards, which would depend on the investment capacity of the producer. 4. conclusions the seeds of pochota fendleri remain viable with high physiological quality for up to 28 months storage in pet bottles. seeds of pochota fendleri, stored in a refrigerator at 8 to 10°c in pet bottles, retain their physiological quality. the vigour of large seeds of pochota fendleri is preserved for a longer time in pet bottles. the use of heavier seeds gives the consumer or producer a greater guarantee of the physiological quality of the material purchased and used in producing seedlings of pochota fendleri, since seed 332 adv. hort. sci., 2019 33(3): 327-332 maguire j.d., 1962 speed of germination‐aid in selection and evaluation for seedling emergence and vigor. crop scien., 2: 176-177. mapa, 1992 regras para análise de sementes. ministério da agricultura, pecuária e abastecimento, brasília, brazil. mapa, 2009 regras para análises de sementes. ministério da agricultura, pecuária e abastecimento, secretaria de defesa agropecuária, brasília, df, brazil, pp. 399. marcos filho j., 2005 fisiologia de sementes de plantas cultivadas. piracicaba: fealq, biblioteca de ciências agrárias luiz de queiroz, 12, pp.495. nery f.c., prudente d.o., alvarenga a.a., paiva r., nery m.c., 2017 storage of calophyllum brasiliense cambess. seeds. brazil. j. biol., 76: 1-6. oliveira d.l., smiderle o.j., paulino p.s., souza a.g., 2016 water absorption and method improvement concerning electrical conductivity testing of acacia mangium (fabaceae) seeds. rev. biol. trop., 64: 16511660. pereira s.r., giraldelli g.r., laura v.a., souza a.l.t., 2011 tamanho de frutos e de sementes e sua influên‐ cia na germinação de jatobá‐do‐cerrado (hymenaea stigonocarpa var. stigonocarpa mart. ex hayne, leguminosae caesalpinoideae). rev. brasil. sem., 33: 141-148. rodrigues d.b., radke a.k., rosa t.d., tunes c.d., gularte j., tunes l.m., 2016 adequação metodoló‐ gica da determinação do teor de água em sementes de cenoura. tecnol. & ciênc. agropec., 10: 40-43. smiderle o.j., souza a.g., 2016 production and quality of cinnamomum zeylanicum blume seedlings cultivated in nutrient solution. rev brasil. ciênc. agrar., 11: 104-110. smiderle o.j., souza a.g., pedrozo c.a., lima c.g.b., 2017 nutrient solution and substrates for ‘cedro doce’ (pochota fendleri) seedling production. rev. bras. eng. agríc. amb., 21: 227-231. smiderle o.j., souza a.g., pedrozo c.a., silva t.j., souza a.a., 2018 correlation between mass and vigor of pochota fendleri (malvaceae) seeds stored in different environments. ‐ rev. ciênc. agrar, 41: 93-99. smiderle o.j., souza a.g., souza a.a., 2016 morphological aspects of seeds, emergence and growth of plantlets of surinam cherry trees sown at dif‐ ferent depths. j. plant scien., 4: 119-125. souza a.g., smiderle o.j., bianchi v.j., 2017 a characterization biometric and morphophysiological of peach rootstock seeds using images of their seedling vigor. recent pat. food nutr. agric., 9: 010-018. souza a.g., smiderle o.j., muraro r.e., bianchi v.j., 2017 b morphophysiological quality of seedlings and grafted peach trees: effects of nutrient solution and substrates. recent pat. food nutr. agric., 9: 010-018. souza a.g., smiderle o.j., spinelli v.m., souza r.o., bianchi v.b., 2016 correlation of biometrical charac‐ teristics of fruit and seed with twinning and vigor of prunus persica rootstocks. j. seed scien., 38: 322-328. impaginato 133 adv. hort. sci., 2019 33(1): 133-138 doi: 10.13128/ahs-22530 impact of partial root-zone drying on growth, yield and quality of tomatoes produced in green house condition a. hakim 1 (*), z. qinyan 2, m. khatoon 3, s. gullo 4, 1 8624 festival drive, elk grove, ca, usa. 2 1613 holly lane, davis, ca, usa. 3 sacramento, california, usa. 4 oakwood university, cooper complex 7000 adventist blvd. nw, huntsville, al, usa. key words: conventional irrigation, fruit size, growth, tomato, yield. abstract: water resources are limited for irrigation worldwide especially for the arid and semi-arid regions; therefore, there is an urgent need to reassess an alternative technique for conventional irrigation. partial root-zone drying (prd) is considered a new water-saving irrigation technique which has been tested for some crop species. the prd technique simply requires wetting half of the rooting zone and leaving the other half dry, thereby utilizing reduced amount of irrigation water. the wetted and dry sides are interchanged in the subsequent irrigations. the focus of this article is to evaluate the effect of prd on growth, yield and quality of tomatoes as compared to conventional irrigation. to evaluate the effect of prd, a greenhouse experiment was conducted where two irrigation treatments were tested during a 160-day growing period: (1) control treatment where drip irrigation was applied to both sides of the plants; (2) prd treatment in which half of the irrigation water in drip irrigation was given alternately only to one side of the root system with each irrigation. prd treatment had 15% and 7% decreases in shoot fresh weight and leaf area of plant, respectively; however, prd had 20% higher fruit per cluster and 18% increase in fruit production in comparison to the control treatment. no significant difference was detected on fruit size between prd treated plants and control plants. but, fruits from prd treated plant exhibited better appearance, higher lycopene content, firmness, total soluble solid (tss), and tss/titratable acidity (ta) ratio than control ones. fruit from control treatment contained higher chlorophyll content than fruit from the prd treatment. postharvest storage results indicated that higher percentage of rot and chilling injury were observed in control fruits than prd treated fruits. the results of this study indicated that prd is a promising water saving irrigation technique which is able to produce higher yield and better quality tomatoes than conventional drip irrigation. 1. introduction worldwide, agriculture accounts for 70% of all water consumption, as compared to 20% for industry and 10% for domestic use. the world’s population is growing by roughly 80 million people each year. more than (*) corresponding author: hakim61@hotmail.com citation: hakim a., gullo s., khatoon m., qinyan z., 2019 impact of partial root-zone drying on growth, yield and quality of tomatoes produced in greenhouse condition. adv. hort. sci., 33(1): 133-138 copyright: © 2019 hakim a., gullo s., khatoon m., qinyan z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 january 2018 accepted for publication 5 october 2018 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 133-138 134 99% of the world’s food supply comes from the land. as the world population continues to grow geometrically, great pressure is being placed on irrigation water to provide an adequate food supply. therefore, there is a big challenge on how to increase food production with limited water resources. this is especially true for tomato (lycopersicon esculentum mill), which is the second most important vegetable crop with a total world production of 130 million tons in 2016 (euro-fresh, 2016). the challenge becomes even more severe in arid and semi-arid regions where water availability is decreasing and competition for water is increasing between agriculture and industry. therefore, water resources should be used with a higher efficiency as well as a higher productivity. partial root-zone drying irrigation (prd) is one of the new efficient and productive water-saving irrigation methods that can save irrigation water up to 50% in processing tomatoes (casa and rouphael, 2015). this technique has the potential to significantly reduce crop water use (el-sadek, 2014), reduce canopy vigor, but able to maintain crop yields and quality of crops (sun et al., 2014) as compared to conventional irrigation methods. although processing tomatoes accounts for the majority of tomato tonnage, while the comparatively higher prices of fresh market tomatoes make them higher ranked in terms of value. to evaluate the effect of prd on fresh market tomatoes, an experiment was conducted in greenhouse with the objective to compare the effect of conventional drip irrigation (cdi) to prd drip irrigation on the growth, yield and quality of the fresh market tomatoes. 2. materials and methods experimental conditions the experiment was conducted in a greenhouse (approximately 24°c in day time and 15°c at night time, relative humidity (rh) 65%, 15 hour’s photoperiod and ambient light condition) at the chateau fresno nursery, 13505 south fresno, california 93609, usa from april to september 2015. seeds of the fresh market tomato (lycopersicon esculentum mill cv. vibelco) were sown on march 1st, 2015. thirty days after seeding, uniform plants were transplanted into 12 wooden boxes (2.53 m length × 0.65 m width × 0.20 m height each). each box had 4 compartments (0.50 m length × 0.50 m width × 0.20 m height) with one experimental plant per compartment. to avoid lateral water movement, a small piece of plastic (0.50 m length × 0.025 m width × 0.04 m height) was placed centrally on the base of each compartment. plants were grown in a vermicast and coconut fiber mixture (70:30 v/v). plants were fertilized with 15-15-15 (npk) at 120 kg/ha, purchased from lowes, elk grove, california, usa. bees were used for pollination. irrigation treatments two weeks after transplanting two irrigation treatments were tested. treatments were: control (conventional drip irrigation, cdi) to both sides of the root system, and half of irrigation water in drip irrigation given alternately only to one side of the root system with each irrigation (prd) (fig. 1). irrigation treatment was given 0.10 m away from the main stem and on both sides of the row. irrigation covered a total area and soil volume of 0.24 m2 and 0.048 m3, respectively. but half of it at 10:00 h and the other half at 16:00 h by manual drip irrigation system. two irrigation lines were set up and operated separately for the prd treatment. two emitters per plant (one on each line) each emitting 4 l/h were placed 0.15 m away from the main stem of each plant. irrigation in cdi treatment covered a total area and soil volume of 0.018 m2 and 0.004 m3, but half of irrigated area and soil volume was wetted in prd treatment at each irrigation. there was some drainage in all treatments, but this was not measured. however, water losses by drainage were minimized by adjusting the amount of water as the crop developed. so, values of the irrigation use efficiency fig. 1 schematic diagram of partial root-zone drying (prd) irrigation in tomato plants. hakim et al. partial root-zone drying effects on tomatoes 135 presented here might have been under-estimated considering the water losses by drainage. growth, yield, yield component, fruit firmness, fruit water content and blossom-end rot measurements growth, yield, yield components and blossomend rot were measured from twelve (12) randomly selected plants/fruits from each treatment. the plant heights were measured with a tape from the base of the plant to the tip of the plant. plant growth and development data were taken on the sampled tagged plants monthly for three months. leaf area was measured using a destructive method. total leaf area (cm2) was measured by leaf area meter (model, delta-t, cambridge, uk). the numbers of clusters were counted per plant from the first to the last cluster during the growing period. the numbers of fruits were counted when the plants started fruiting. the fruit weights were determined after harvesting the tomato using a weighing balance. after 150 days, one plant per treatment per replication was destroyed and the total vegetative fresh weight was assessed and expressed as kg/plant. mature green tomato fruit firmness was measured using an instron universal testing machine with a 0.5 cm2 plunger, measurement was taken at the mid-section of the fruit. water content of fruit was expressed on a dry weight basis. fruit blossom-end rot incidence was recorded and calculated in percentage of fruit affected per plant. fruit quality at harvest or postharvest for postharvest quality evaluation, six (6) replicates of five (5) mature green (cascio, 2017) fruits from each treatment were randomly chosen approximately 130 days after transplanting and were stored in a dark refrigerated room at 3°c with 95% rh. after a storage period of 2 weeks, all fruits were moved to a ventilated room without supplemental light at 24°c with 65% rh and held for 7 days. the following attributes were checked for quality measurements: weight loss, chilling injury, decay, appearance/color change, total soluble solids (tss), ph, titratable acidity (ta), chlorophyll and lycopene content. fruit weight loss was determined prior to and after storage. it was calculated as the percentage of initial fresh weight. color development was observed visually using a subjective scale with mature green (mg) = 1, breaker (b) = 2, pink (p) = 3, light red (lr) = 5 and red (r) = 6 (usda, 2005). chilling injury (surface pitting) was rated visually by estimating the percentage of the injured fruits. decay (unidentified) was rated visually and calculated as a percentage of fruit affected. chlorophyll and lycopene content were determined from three randomly selected fruits from each treatment by grinding pericarp tissue (about 5 g) in 15 ml acetone. the extract was taken for centrifugation at 35,000 rpm for 10 minutes. before centrifugation, the tubes were covered with aluminum foil to prevent light-induced lycopene oxidation. after centrifugation, the supernatant was decanted and adjusted to 20 ml with acetone. absorbance of the extracts at 664 nm for chlorophyll and 503 nm for lycopene was measured with a spectrometer (model 160 a). total chlorophyll content in milligram per 100 gram of tissue was calculated according to the formula developed by holden (1976). lycopene content was calculated using the molecular extension coefficient of 3240 (davis, 1976) and expressed as micrograms per gram of fresh weight. total soluble solid (tss), ph and titratable acidity (ta) were measured on juice extracted from fruit. tss content was determined with a digital refractometer (atago, model 1, tokyo, japan). ta was determined by a metler auto titrator (model v 20) and ph was measured with an autocal ph meter (model phm 83). experimental design and data analysis a completely randomized design was used with the two treatments replicated six times with four plants per replication for each treatment. data were analyzed by a complete randomized model using the glm procedure of sas software version 8.2 (sas institute, cary, nc, usa). student’s t-tests were used to determine significant effects between two treatment means. 3. results and discussions mean plant height over the 3 months are illustrated in figure 2. the maximum height was obtained from tomato plant irrigated with cdi/full water regime. prd treated plants resulted in the lower height. similar results were reported by pal et al. (2016) who grown tomato plant under deficit irrigated conditions plus paclobutrazol application. prd treatment had 15% and 7% decreases in shoot fresh weight and leaf area of plant, respectively. however, prd had 20% higher fruit per cluster and 18% increase in fruit production in comparison to the cdi treatment (table 1). the difference between cdi and prd treated plant’s shoot fresh weight, leaf area, and fruit production were significantly different at adv. hort. sci., 2019 33(1): 133-138 136 0.001, 0.01 and 0.05% level respectively. the cdi treated plant appeared to have excess moisture in root zone, which causes root inactivity contributing to lower yield and delayed maturity of the crop as compared with the prd treated plant. mean cluster per plant, fruit per cluster, total fresh weight of fruit per plant and fruit weight (individual) were lower in cdi treated plants as compared to prd treated plants. the difference was statistically significant only in case of cluster per plant and total fresh weight per plant (table 1). the lower cluster number, less fruit per cluster and less weight of fruit per plant in cdi treated plant might be due to the excessive vegetative growth as a result of luxurious amount of water application. prd treated plant’s fruit exhibited significantly higher percentage of blossom-end rot as compared to the cdi treated ones. the blossom-end rot is a physiological disorder of tomato fruit caused by calcium deficiency or excessive soil moisture fluctuation which reduce uptake and movement of calcium into the plant (mathew and salvadore, 2007). the higher percentage of blossom-end rot in prd treated fruit might be due to the reduced movement of calcium to the prd treated plants. however, no calcium was analyzed either from leaf or from fruits in this study. fruit size, water content and fruit weight were influenced by prd treatment. there were some differences in fruit size, weight and water content in prd and cdi treated plant’s fruit; however, the difference were not statistically significant (table 1). reduced weight loss in the prd treated tomatoes during the storage is a positive quality attribute in tomato fruit especially for distant market (table 2). yadav and singh (2014) indicated that the weight loss of fruits in storage condition is mainly from water loss and from solid constituents. the lower water loss in prd treated fruit might be due to lesser incident of micro-cracks in the skin. however, no skin micro-cracks were examined in this study. in this study, when compared to fruits from cdi treated plants, fruits from prd treated plants exhibited significantly lower chlorophyll and higher lycopene content on the 7th day at 24°c followed by a 2-week storage period at 3°c (table 2). klunklin and savage (2017) also detected significantly higher lycopene content in prd treated tomato fruit. according to gindi et al. (2016), 63 percent of consumers’ purchase interest depends on color of fruits. table 2 demonstrated that fruits from prd treated plants exhibited less visible chilling injury (fruit surface pitting) and decay, but similar color change as fruits from cdi treated ones. in the regions of the world where irrigation water is expensive, such as arid and same-arid regions, prd treatment would be beneficial in terms of economic return, where water supply is crucial. soluble solids are an important tomato quality fig. 2 effect of cdi (conventional dripping irrigation) and prd (partial root-zone irrigation) on plant height over 3 months. physiological parameters treatment difference (prd-cdi) cdi group prd group fruit size, diameter (mm) 68.4 67.8 0.6 ns total fresh weight of fruit (kg/plant) 5.1 7.03 1.93 *** fruit water content (%) 95.2 94.8 0.4 ns fruit weight (g) 95.2 95.0 0.2 ns fruit firmness after harvest (kg/cm2) 9.1 9.0 0.1 ns cluster/plant 8.1 10.2 2.1 *** fruit/cluster 6.1 6.3 0.2 ns shoot fresh weight (kg) 10.6 9.1 1.5 *** leaf area (cm2) 451.54 418.26 33.28 * blossom end rot (%) 5.5 6.0 0.5 *** table 1 effect of prd on tomato fruit size, total fresh weight, water content, fruit firmness, cluster/plant, fruit/cluster, shoot fresh weight, leaf area, and blossom-end rot ns, *, **, *** non-significant or significant at t≤0.05, 0.01 or 0.001 respectively. hakim et al. partial root-zone drying effects on tomatoes 137 parameter. tomato flavor is generally determined by the content of soluble solids and acid (titratable acid). according to aoun et al. (2013), tomato flavor impact is co-related to total sugar and acid. in this study, total soluble solid (tss), tss/ta and ph increased, while ta decreased in prd treated fruits (table 2). in an earlier study, sun et al. (2014) also detected higher tss in tomato fruit produced under prd condition. the higher tss and lower ta in fruit from prd treated plants were probably due to the less retained water in fruit from prd treated plant than fruit from cdi treated plant. the higher ph in fruit from prd treated plant was compatible with the lower ta in fruits from prd treated plant than in fruits from cdi treated plant. 4. conclusion the result of this study demonstrated that “vibelco” tomato plant treated with prd slowed its vegetative growth, but produced higher yield and better quality fruit at the same time saved water by 50%. this study proved that prd is a noble water saving method which can prevent excess water use and increases economic returns due to the reduction of water use. acknowledgements the author wishes to thank dr. abu helal ibrahim for his experimental assistance. references aoun a.b., bechiheb b., benyahya l., ferchichi a., 2013 evaluation of fruit quality traits of traditional varieties of tomato (solanum lycopersicum) grown in tunisia. african j. food sci., 7(10): 350-354. casa r., rouphael y., 2015 effects of partial root-zone drying irrigation on yield, fruit quality, and water-use efficiency in processing tomato. j. hort. sci. biotech., 89(4): 389-396. cascio j., 2017 a harvest of green tomatoes. cooperative extension service, fnh-00024, university of alaska, fairbanks, usa. davis b.h., 1976 carotenoids, pp. 38-165. in: goodwin t.w. (ed.) chemistry and bio-chemistry of plant pigments. vol. 2. academic press, new york, usa, pp. 679. el-sadek a., 2014 water use optimization based on the concept of partial root zone drying. ain. shams. eng. j., 5(1): 55-62. euro fresh, 2016 tomatoes: around the world. the international distribution magazine for fresh produce & retail. euro-fresh, news, september 7. gindi a.a., abdullah a.m., ismail m.m., nawi n.m., 2016 factors influencing consumer’s retail formats choice for fresh fruits purchase in klang valley malaysia. int. j. agri. res. sustain. food sufficiency, 3(3): 52-61. holden m., 1976 chlorophyll, pp. 461-488. in: goodwin t.w. (ed.) chemistry and bio-chemistry of plant pigments. academic press, new york, usa, pp. 595. klunklin w., savage g., 2017 effect on quality characteristics of tomatoes grown under well-watered and drought stress conditions. foods., 6(8): 56. matthew d.t., salvadore j.l., 2007 blossom-end rot: a calcium deficiency. j. plant nutrition, 27(1): 123-139. table 2 effect of prd on weight loss, chilling injury, decay, chlorophyll content, lycopene content, color change, ph, titratable acidity (ta), total soluble solid (tss), and tss/ta in mature green tomatoes on the 7th day at 24°c following a 2-week storage period at 3°c physiological parameters treatment difference (prd-cdi) cdi group prd group weight loss (%) 2.15 1.96 0.19 *** chilling injury (%) 15.00 10.00 5.0 ns decay (%) 13.00 8.00 5.0 *** chlorophyll (mg/100 g fresh weight) 3.90 3.20 0.7 ** lycopene (µg/g fresh weight) 7.10 7.70 0.6 * color change 4.50 4.50 0.0 ns ph 4.40 4.55 0.15 ns ta (% citric acid) 0.49 0.46 0.03 * tss 4.30 4.50 0.2 ns tss/ta 8.77 9.80 1.01 *** ns, *, **, *** non-significant or significant at t≤0.05, 0.01 or 0.001 respectively. 138 adv. hort. sci., 2019 33(1): 133-138 pal s., zhao j., khan a., yadav n.s., batushansky a., barak s., rewald b., fait a., lazarovitch n., rachmilevitch s., 2016 paclobutrazol induces tolerance in tomato to deficit irrigation through diversified effects on plant morphology, physiology and metabolism. sci. rep., 6: 1-13. sun y., holm p.e., liu f., 2014 alternate partial rootzone drying irrigation improves fruit quality in tomatoes hort. sci., 41(4): 185-191. usda, 2005 tomatoes, shipping point and market inspection instructions. agricultural marketing service, specialty crops program, specialty crops inspection division, usda, pp. 78. yadav a.k., singh s.v., 2014 osmotic dehydration of fruits and vegetables: a review. j. food sci. technol., 51(9): 1654-1673. 43 1. introduction the world’s cultivated lands are increasingly affected by drought and salinity (barrett-lennard, 2000). halophytes growing in highly saline soils could thus serve as a resource for the identification and development of new crop systems for marginal saline soils (debez et al., 2011; ben hamed et al., 2013). the actual yield of halophytes remains largely unknown since their domestication is still limited, yet the economic potential of some of these plants has been positively assessed by various groups (aronson, 1989; ashour and thalooth, 1993; abdelly et al., 2006). numerous brassicaceae species are current and emerging biodiesel crops: in addition to the oil-rich seed, the ability of brassicaceae species to grow on marginal land with minimal inputs make them particularly attractive and potentially viable for this application. among halophyte species studied for their potential as oleaginous plants, the brassicaceae cakile edentula (o’leary et al., 1985), crambe abysinnica (mandal et al., 2002) or cakile maritima (ghars et al., 2005) have been reported to contain high amounts of oil. cakile maritima, the sea rocket, is an annual succulent halophyte frequently found from the black sea coasts to the mediterranean region, and from the atlantic coasts of north africa to the north of europe (clausing et al., 2000). tunisian accessions of c. maritima contain up to 40% seed-oil (ghars et al., 2005). plant growth, harvest index, silique number and seeds produced per fruit segment is maximal at 100 mm nacl (debez et al., 2008) but c. maritima can survive at up to 800 mm nacl (ellouzi et al., 2013) and successfully reproduces till 500 mm nacl salinity (debez et al., 2004). seed-oil content did not seem to be affected by salinity, although erucic acid level could increase (debez et al., 2006). these facts highlight the need to better understand the basis of adaptation to saline environments, as well as traits associated with oil production itself. in previous works we described some aspects of the response of c. maritima to salt stress (debez et al., 2004, 2006, 2008; ellouzi et al., 2011) and our data point out that c. maritima adopts a complex survival strategy at high salinity, however numerous data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. suspension culture cells offer an in vitro system that is widely used in plant biology as a convenient tool to investigate a wide range of phenomena. it consists in a model system as suspension culture provide a ready source of a homogenous cell type and avoids the complications of multicellular tissue types in planta (moscatiello et al., 2013). suspension culture cells were recently used in various studies and models concerning plant responses to salinity, such as proteomic studies (chen et al., 2012; liu et al., 2013), metabolomic studies (liu et al., 2013) or establishment of a cell suspension culture of the halophyte cakile maritima i. ben hamed*, **, b. biligui*, d. arbelet-bonnin*, c. abdelly**, k. ben hamed**, f. bouteau*(1) * université paris diderot, sorbonne paris cité, institut des energies de demain, umr 8632, paris, france. ** laboratoire des plantes extrêmophiles, centre de biotechnologie de borj cedria, university of carthage-tunis, bp 901, 2050 hammam lif, tunisia. keys words: biofuel, cakile maritima, cell suspension, halophytes, oil. abstract: cakile maritima is a member of the brassicaceae family also known as sea rocket. it is an annual succulent halophyte frequent in coastal dune vegetation in mediterranean regions and atlantic coasts from north africa to the north of europe. this halophyte presents a complex survival strategy at high salinity and its seeds contain up to 40% of an oil which could be suitable for biofuel production and other industrial applications. however, data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. cell suspension cultures offer an in vitro system convenient for cell biology studies and biotechnological methods are still not developed for this putative crop. the present paper reports initiation of c. maritima cell suspension cultures from callus obtained from aerial parts of seedlings. the establishment of a suspension culture which preserves its salt resistance provides an opportunity to gain insights into c. maritima biology. adv. hort. sci., 2014 28(1): 43-48 (1) corresponding author: francois.bouteau@univ-paris-dodierot.fr received for publication 17 march 2014 accepted for publication 4 june 2014 44 transcriptomic studies (matsuura et al., 2010; bae et al., 2012). it is also a convenient means to study transport system regulations and oxidative responses to various biotic and abiotic constraints (kadono et al., 2010; baz et al., 2012; yukihiro et al., 2012; tran et al., 2013), comprising salinity (cessna et al., 2007; wang et al., 2010; pons et al., 2011; queirós et al., 2011). here, we report the development of a cell suspension culture of c. maritime; growth performance was evaluated on control and nacl cultured cells to validate the biological system. these suspension culture cells could be a valuable tool to gain further insights into halophyte studies and their potential applications. 2. materials and methods establishment of callus of cakile maritima in this study we used cakile maritima seeds harvested in the raoued region in the north of tunisia. callus cultures were initiated from the aerial part of 14-day-old, light-grown seedlings. seeds were submerged in 70% ethanol for 1 min, then rinsed with sterile distilled water, submerged in chlorine bleach for 10 min and then rinsed three times (5 min each) with sterile distilled water. the seeds were placed in petri-dishes containing murashige and skoog medium including vitamins (ms) (murashige and skoog, 1962), supplemented with 30 g.l-1 sucrose, 8 g.l-1 agar. the ph was adjusted to 5.8 with koh. stem segments were finely cut and then placed on a solid callus-inducing medium (cim) containing 6.2 g.l-1 gamborg b5 (gamborg et al., 1968) supplemented with 20 g.l-1 glucose, 8 g.l-1 agar and with growth regulators 9.06 µm of 2.4 d and 0.46 µm of kinetin. the ph was adjusted to 5.7 with koh. after two to three weeks, callus appeared on the sides of the segments. when the size of callus became larger than 1 cm, they were divided and transferred to a new medium. establishment cell suspension cultures of cakile maritima approximately 5 g of callus were transferred to 125 ml flasks containing fresh gamborg b5 medium supplemented with 30 g.l-1 glucose, 0.2 µm 2,4-d and 0.45 µm kinetin. the ph was adjusted to 5.7 with koh. flasks were incubated on a rotary shaker at 120 rpm and maintained at 22°c in the dark. feeding of the cultures with fresh medium was done at 10-14 day intervals during which time the suspensions were allowed to settle under agitation (rotary shaker at 120 rpm). this procedure was repeated for about eight weeks. then, the suspension was subcultured every seven days by transferring 20 ml of the culture into 50 ml of fresh medium in 250 ml erlenmeyer flasks. arabidopsis thaliana cell suspension culture conditions arabidopsis thaliana l. cell suspensions were prepared from calluses of the cell line t87 generated from the ecotype columbia plant as previously described (tran et al., 2013). the suspension cells were obtained after about two months and five to six subcultures in 1 l round-bottom flasks containing 350 ml liquid gamborg b5 culture medium (ph 5.8). cell suspensions were sub-cultured weekly using a 1:10 dilution. growth evaluation growth of the culture was evaluated by measuring the fresh weight of cells and the density of cells using a nageotte cell. cell viability cell viability was assayed using the vital dye neutral red. cells (100 µl) were incubated for 5 min in 400 µl phosphate buffer ph 7 with neutral red to a final concentration of 0.001% (w/v). cells that did not accumulate neutral red were considered dead. at least 500 cells were counted for each replicate and the procedure was repeated at least three times for each treatment. external ph measurements measurements of extracellular ph were performed with ph-sensitive electrodes every 24 h for six days from 5 ml of cultured medium cells. the procedure was repeated on three independent subcultures. cell size cell images were recorded with a camera (kappa cf11dsp) on a light microscope (labophot-2 nikon) and sizes were measured using image analysis software kappaimagebase-2.2sp2-metreo (kappa optoelectronics gmbh, gleichen, germany). protoplast isolation protoplasts were isolated from suspension cultures six days after subculture. fifteen ml of suspension cells were used. after cell sedimentation, the supernatant was removed and replaced by 5 ml of gamborg b5 fresh medium containing 0.1 g cellulysin, 0.05 g macerase and 0.3 m sorbitol. the digestion was carried out under shaking at 120 rpm at 22°c for 30 min. after incubation, protoplasts were collected by centrifugation at 300 rpm for 3 min and re-suspended in 5 ml of gamborg b5 fresh medium supplemented with 0.6 m sorbitol or 0.3 m sorbitol. 3. results and discussion the different steps in the development of cakile maritima callus from stem segments are reported in figure 1. callus appeared on the sides of the discs after two to three weeks. although produced in light, callus became non-chlorophyllian and pale yellow in color (fig. 1c, d). thus cakile maritima loses its power of chlorophyll synthesis during its passage from plant stage to callus stage. to generate cell suspension, approximately 5 g of calluses were transferred in gamborg medium. these calluses progressively disintegrated to the smallest cell aggregates in the liquid medium. 45 large quantities of cells could be obtained after two months feeding (fig. 2a, b). at this stage suspension cells were subcultured every week in fresh gamborg medium and after an additional 1.5 months the culture became more homogeneous as the cell aggregates became smaller and the cell size increased (fig. 2c, d). the level of dead cells in the culture also progressively decreased reaching about 10% after four months of culture, in the same range as observed for tobacco or a. thaliana suspension cells (baz et al., 2012; yukihiro et al., 2012; tran et al., 2013). using four-month-old c. maritima culture, growth curves were established by measuring the fresh weight of cells (fig. 3a) and the cell density (fig. 3b). both curves showed a typical sigmoidal shape with a latency period of 24 h, then an exponential phase lasting about four days prior to a plateau phase (fig. 3a, b) supposed to be due to nutrient depletion. we monitored the medium ph during the culture procedure (fig. 3c). after a slight acidification at the beginning of the exponential phase, the ph became more alkaline, reaching 6.5 at the end of the culture (fig. 3c). this alkalization could be involved in the decrease in biomass production as ling et al. (2008) pointed out that ph of 6.7 lowered the growth of suspension culture of ficus deltoidea. however, the ph variations recorded during the culture should remain suitable for nitrate uptake as reported for ipomoea suspension cells (martin and rose, 1976). histological analysis on six-day-old suspension cells revealed distinct morphological features of the cultured cells (fig. 4). although the cell aggregate size diminished during culture establishment, a few large aggregates (>20 cells) remained. however the largest part of the culture consisted of compact small groups of less than 20 cells, 60% corresponding to groups of three to 10 cells, single cells representing about 10% of the population (fig. 4a). most of the cells were rounded in shape (fig. 4b, left and center); less than 2% were elongated (fig. 4b, right). interestingly, as observed during the establishment of the culture (fig. 2b, d), the size of the cells seemed to be dependent on the size of the groups, the isolated cells being the largest (fig. 4c). it is known that protoplasts can be used for transient expression, trafficking assay or ion homeostasis analysis, notably in studies on plant resistance to salinity (laohavisit et al., 2012; haro et al., 2013; morgan et al., 2013; mottaleb et al., 2013; son et al., 2013). thus, we evaluated the usefulness of the cell cultures for the isolation of protoplasts. protoplasts were isolated from six-day-old suspension cultures. based on the different cell sizes measured (fig. 4b, c) we observed protoplasts of different sizes. most of these protoplasts maintained in 0.6 m sorbitol apfig. 1 establishment of cakile maritima calluses. (a) three-weekold c. maritima seedling grown from sterilized seed. (b) slices of leaf and rod were finely cut for callus generation. (c) onemonth-old calluses derived from rod and leaf slices. (d) magnification of a callus. fig. 2 establishment of cakile maritima cell suspensions. (a) dense suspension cells grown in flask after two months feeding and (b) corresponding cell aggregates magnified 385x. (c) dense suspension cells grown in flask after 1.5 months of subculture after feeding and (d) corresponding cell aggregates magnified 385x. fig. 3 growth pattern of cakile maritima cell suspension determined by fresh weight (a) or cell density measurements (b). evolution of the medium ph during cell suspension growth (c). bars indicate mean ± sd of at least three experiments. 46 peared plasmolyzed and numerous protoplasts seemed to be shrunken (fig. 5a). we then used 0.3 m sorbitol and obtained rounded protoplasts with large vacuoled evidenced by neutral red staining (fig. 5b). finally, cakile maritima being a halophyte, we checked the salinity resistance of the suspension cells. suspension cells were subcultured in gamborg medium complemented with 100, 400 or 800 mm nacl. sigmoidal growth curves were obtained and no significant differences were observed between the control and the suspension cells growing in presence of 100 mm nacl when 400 mm and 800 mm strongly decreased the growth of the suspension cells (fig. 6a). these data are in accordance with what was described for seedlings, c. maritima even requiring the presence of a moderate salt concentration (50-100 mm nacl) to maintain a significant growth activity and plant development (debez et al., 2004, 2008). although strongly reduced at 400 mm or 800 mm nacl, the growth of the suspension cells was also in accordance with previous data indicating that c. maritima can survive up to 800 mm fig. 4 morphology of six-day-old cakile maritima suspension cells. (a) frequency of cell groups in the culture. (b) different morphologies of cells. (c) sizes of the cells according to the size of the cell groups. at least 250 cells were analyzed; bars indicate mean ± sd. fig. 5 protoplasts derived from six-day-old cakile maritima cell suspension maintained in 0.6 m sorbitol (a) or 0.3 m sorbitol (b). fig. 6 evaluation of cakile maritima cell suspension resistance to salinity. (a) growth pattern of cell suspensions grown in presence of 100, 400 or 800 mm nacl determined by fresh weight measurement (b). comparison of cell death extents of c. maritima and arabidopsis thaliana cell suspensions treated 6 h with nacl concentrations ranging from 100 to 800 mm. bars indicate mean ± sd of three experiments. 47 nacl (ellouzi et al., 2013) and successfully reproduces till 500 mm nacl salinity even if the biomass was reduced (debez et al., 2004). we further compared the extent of cell death induced 6 h after the addition of various nacl concentrations on c. maritima and a. thaliana suspension cells. cell death began to increase from 100 mm nacl to almost 100% at 600 mm for a. thaliana (fig. 6b). for c. maritima, the increase in cell death was significant only with 400 mm nacl and reached only 60% at 800 mm, remaining largely inferior when compared to a. thaliana (fig. 6b). it is worth noting that 40% of surviving cells probably go on dividing, which explains the growth of the culture, although reduced, at 800 mm nacl (fig. 6a). as a whole, these data demonstrate that c. maritima suspension cells preserve their ability to resist salinity. 4. conclusions the present study reports the development of c. maritima cell suspension cultures which maintained their salt resistance, offering greater understanding about adaptation to saline environments, as well as traits associated with biofuel production. acknowledgements ibh was supported by fellowships from tunisian higher education and scientific research ministry and université paris-sud. references abdelly c., barhoumi z., ghnaya t., debez a., hamed ben k., ksouri r., talbi o., zribi f., ouerghi z., smaoui a., huchzermeyer b., grignon c., 2006 potential utilization of halophytes for the rehabilitation and valorisation of salt-affected areas in tunisia, pp. 163-172. in: özturk m., y. waisel, m.a. khan, and g. görk (eds.) biosaline agriculture and salinity tolerance in plants. birkhäuser verlag, switzerland, pp. 205. aronson j.a., 1989 haloph: a database of salt tolerant plants of the world. in: whitehead e. 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bouteau f., kadono t., kawano t., 2012 peroxyacetyl nitrateinduced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line. plant signal. behav., 7: 113-120. impaginato 193 adv. hort. sci., 2018 32(2): 193-203 doi: 10.13128/ahs-21304 yield and yield components of coriander under different sowing dates and seed rates in tropical environment k.t. kassu, h.h. dawit, a.y. wubengeda, a.t. almaz, m.t. asrat ethiopian institute of agricultural research, ethiopia. key words: biomass yield, coriander, fruit yield, seed rate, sowing date. abstract: coriander makes use of favorable environmental conditions when it is sown at optimum time and rate. however, this information is very limited in the southeastern mid-highlands of ethiopia. field experiments were, therefore, conducted between 2011 and 2014 at three different research stations to determine optimum sowing dates and rates. the experiment had split plot design in randomized complete block with three replications, in which sowing dates and seed rates were the main and sub-plot treatments, respectively. the four sowing date treatments were june 20, july 10, july 30 and august 20 while the four seed rate treatments were 30, 40, 50 and 60 kg ha-1. coriander sowed in the third decade of july at arsi robe and from the first to the third decades of july at kulumsa and sagure gave the highest fruit and biomass yields. earlier sowing in the second decade of june, and delayed sowing in the second decade of august brought fruit yield reductions of 37-66 and 37% at arsi robe, 27-45 and 58-66% at kulumsa, and 24-40 and 26% at sagure, respectively. however, coriander did not respond to seed rates. owing to the enhanced yields of coriander, intermediate cultivation at a seed rate of 30 kg ha-1 was found optimum. 1. introduction coriander (coriandrum sativum l.), which belongs to the family of umbelliferae (apiaceae) is one of the most important annual spice and medicinal herb. it is grown in ethiopia and throughout the world for its seeds as well as leaves and has immense uses (diederichsen, 1996; hedburg and hedburg, 2003; parthasarathy et al., 2008; nowak and szemplinski, 2014). coriander originated from the mediterranean and western asian regions (burdock and carabin, 2009). along with central asia and near east countries, vavilov (1992) mentioned ethiopia in the lists of centers of origin for coriander. ivanova and stoletova (1990) also reported that india, northern africa, central asia and ethiopia are centers of formation and cradles for different types of coriander. there is a longstanding tradition of cultivation of coriander in ethiopia (diederichsen, 1996; geremew et al., 2014). (*) corresponding author: kasstad96@yahoo.com citation: kassu k.t., dawit h.h., wubengeda a.y., almaz a.t., asrat m.t., 2018 yield and yield components of coriander under different sowing dates and seed rates in tropical environment. adv. hort. sci., 32(2): 193-203 copyright: © 2018 kassu k.t., dawit h.h., wubengeda a.y., almaz a.t., asrat m.t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 193-203 194 the immense uses of coriander depend on the choice of fruits or green herbs, which are linked to their chemical compositions. the most important constituents are the essential and fatty oils (diederichsen, 1996). coriander has got significant importance as a spice in culinary, food, beverage, medicine, perfumery, pharmaceuticals and sanitary industries (jansen, 1981; diederichsen, 1996; delaquis et al., 2002; kubo et al., 2004). on the other hand, its green foliage is used in vegetables owing to its richness in vitamins and other minerals (singh et al., 2005). in ethiopia, coriander is widely used for domestic culinary. the seeds are used for flavoring the powder of hot red pepper locally called “berbere” and used for numerous meat and vegetarian dishes, leavened flat ethiopian bread locally called “injera”, cakes and bread. the leaves are added as an aromatic herb to tea and stew locally called “wot” (jansen, 1981; geremew et al., 2014). coriander is also a good melliferous plant since it produces a considerable quantity of nectar and thereby attracts many different insects for pollination. studies indicated that one hectare of coriander allows honeybees to collect about 500 kg of honey (diederichesen, 1996). the residues left after extraction of the essential oils are used as best ruminant feed since they still contain as nearly the same digestible fat and protein content as the whole fruits (diederichesen, 1996). the success of coriander production is influenced by genetic, weather and agronomic factors (nowak and szemplinski, 2014). the maximum fruit and essential oil yields are attained only when an appropriate combination of these factors are provided for the plant (rangappa et al., 1997; gil et al., 2002). coriander is among the tropical crops and generally sown in winter season if the objective is seed for production (sharangi and roychowdhury, 2014). as a temperature-sensitive crop, it generally requires a relatively cool, comparatively dry and frost-free weather during its early stage for good vegetative growth and relatively warm temperature during flowering and reproductive stage for high yields and good quality (peter, 2004; kalra, 2008; sharangi and roychowdhury, 2014). the ideal temperature for germination and growth of coriander is 20-25°c (singhania et al., 2006). coriander exploits the environment most favorably when it is sown at optimum time (kuri et al., 2015) since sowing date significantly affects the photoperiodic response of plants and determines yields and qualities (rasam et al., 2007). time of sowing controls the crop phenological development along with efficient conversion of biomass into economic yield (khichar and niwas, 2006). earliness in sowing leads to untimely flowering; however, it may also pose susceptibility to the damage of extreme cold and frost. on the other hand, delay in sowing hampers growth, yield and quality of the crop due to deficiency of soil moisture at latter stages (sharangi and roychowdhury, 2014; rashed and darwesh, 2015). determination of optimum seed rate is also a basic element for successful coriander production (rasam et al., 2007). many agronomic studies conducted in the world revealed that seed rate had a highly significant effect on the productivity and quality of coriander (diederichsen, 1996; kumar et al., 2007; ghobadi and ghobadi, 2010). both low and high seed rates resulted in reduced yield and oil concentrations. rapid life cycle of coriander allows it to fit into different growing seasons, making it possible to grow the crop under a wide range of conditions (lopez et al., 2007). cultivation of coriander in ethiopia; however, is limited to the mid to highlands (1500-2500 m a.s.l.), where sufficient soil moisture can be provided from rainfall. it can also be cultivated in the lowlands if the rainfall is sufficiently supplemented by irrigation (jansen, 1981; geremew et al., 2014). although coriander has got diverse uses, economic importance and one of the several plant species for which ethiopia is known as a center of origin and diversity (jansen, 1981; diederichsen, 1996), it is one of the most neglected or under-utilized aromatic and spice crop (beemnet and getinet, 2010). the wealth of coriander is not yet exploited in ethiopia. compared to other crops, there is no or very limited information available on the agronomic packages. this study was, therefore, carried out to determine optimum sowing date and seed rate for increased yield of coriander in the southeastern mid-highlands of ethiopia. 2. materials and methods description of the study sites the experiment was conducted at three locations, namely arsi robe, kulumsa and sagure in the southeastern mid-highlands of ethiopia. the sites are representatives of the region, where coriander cultivation can potentially be carried out, and optimum kassaye et al. response of coriander to sowing dates and seed rates 195 sowing dates and seed rate studied. it was conducted for two seasons in 2011 and 2012 at arsi robe, and 2011 and 2014 at kulumsa and sagure. due to infestation by unknown disease, the crop could not perform well in 2012 and 2013 at kulumsa and sagure, and harvesting could not be done. arsi robe, sagure and kulumsa are located from 8.4 to 8.6 n and 40.1 to 40.4 e, 8.01 to 8.15 n and 39.2 to 39.3 e and from 7.77 to 8.03 n and 38.94 to 39.31 e, respectively. the altitudes of the locations vary from 2200 m a.s.l. at kulumsa to about 2500 m a.s.l. at arsi robe and sagure. the dominant soil type of the three locations is characterized as vertisol (iuss working group wrb, 2014). climate long-term mean annual rainfall at arsi robe, kulumsa and sagure were 937, 812 and 653 mm, respectively. hence, arsi robe and sagure had the highest and lowest, respectively rainfall with intermediate values at kulumsa (fig. 1). 56, 55 and 67% of the annual rainfall concentrated in the months of july and august at arsi robe; june, july and august at sagure and july, august and september at kulumsa. the major crop production activities are conducted between june to november; therefore, the rainfall amount and distribution during these months have significant influence on the yield and yield attributes. the highest rainfall was recorded in august at all study sites (fig. 1). from august, the rainfall amount and distribution reduce sharply and reach the lowest in the month of december at all locations. november, december, january and february are dry months with the lowest records of rainfall amounts. long-term mean maximum temperature records of arsi robe, kulumsa and sagure were 22.3, 23.2 and 22.5°c, respectively. the corresponding values for mean minimum temperatures were 8.3, 10.5 and 8.8°c, respectively (fig. 2). february was the hottest month at arsi robe and sagure while the corresponding month at kulumsa was march. december was the coldest month at all locations with the lowest records of 4.9, 8.0 and 5.1°c at arsi robe, kulumsa and sagure, respectively. experimental set-up and procedure the experiment had split plot design in randomized complete block with three replications, in which the sowing dates and seed rates were the main and sub-plot treatments, respectively. the four sowing date treatments were june 20, july 10, july 30 and august 20 whereas the four seed rate treatments were 30, 40, 50 and 60 kg ha-1. the sowing dates were set to choose the optimum time by allowing coriander to make maximum benefit from the suitable environmental parameters, especially rainfall and temperature for its successful establishment, survival and performance. on the hand, the seed rates were set by making reference to the existing recommendation of 40 kg ha-1. the seedbed preparation started in early april and totally plowed four times prior to planting. all experimental plots at each location and season were planted with coriander (cv. keteba). the seedbeds were prepared in ridge and furrow, and seeds were drilled on raised beds by hand at 0.30m spacing between rows on the aforementioned sowing dates for all fig. 1 total mean monthly rainfall at arsi robe, kulumsa and sagure. fig. 2 mean annual maximum (a) and minimum (b) temperatures at arsi robe, kulumsa and sagure. adv. hort. sci., 2018 32(2): 193-203 196 sites in plot sizes of 1.8 m by 6 m. the spacing between plots and replications were 0.5 m and 1 m, respectively. the recommended phosphorus (20 kg p ha-1) and nitrogen (18 kg n ha-1) nutrients were uniformly applied to all plots close to the seed rows as basal dose at the time of sowing from di-ammonium phosphate (20-18 p-n). weeds were controlled by manual cleaning. data collection twenty-five randomly selected plant samples were manually cut at the ground level from the inner four rows by excluding the outer two to avoid any border effect, air-dried, the moisture content adjusted to constant level and used for measuring above ground total biomass. the harvest index was calculated by dividing the dry mass of seeds collected from the 25 plant samples and threshed manually by the dry mass of biomass, and multiplying the ratio by 100. for the measurement of fruit yield, the whole crop was harvested from a net plot area of 6 m2 (1.2 m by 5 m), subjected to air-drying and threshed manually. the fruits were detached from the biomass, cleaned and weighed. the seed moisture content was determined by placing samples from each plot in an oven at 105°c for 24 hours. the above ground total biomass and seed mass from each plot were then adjusted to 0 g kg-1 moisture content (dry weight) and expressed in kg ha-1 for statistical analysis purpose. plant height data were taken from each plot at physiological maturity from ten plant samples. data analysis all yield and yield components data were combined across sites and seasons and subjected to analysis of variance using the general linear model procedure (proc glm) of sas statistical package version 9.2 (sas institute, 2002). least significance difference (lsd) tests were employed to evaluate the means of the main and interaction effects of the treatments for each parameters measured (determined). mean separation for the interaction effects were conducted using minitab®18 statistical package (minitab inc.). when p<0.05, means values of treatments were declared as significantly different. 3. results the analysis of variance over two seasons indicated that sowing date and season as well as their interaction had very significant (p<0.001) effect on most of the traits measured at all locations (tables 1-3). sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns ns ns sowing date (sd) *** *** *** *** error (a) 45.64 11.12 19566.89 317296 year (y) *** ns *** *** seed rate (sr) *** ns ns ns sd x sr ns ns * ns y x sd *** *** *** *** y x sr ns ns ns ns y x sd x sr ns ns ns ns error (b) 32.67 13.5 16423.2 202979 cv 6.63 10.02 13.21 16.75 table 1 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at arsi robe in 2011 and 2012 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. table 2 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at kulumsa in 2011 and 2014 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns * ns sowing date (sd) *** *** *** *** error (a) 5.86 1.83 14609.46 870629 year (y) *** *** *** ns seed rate (sr) * ns ns ns sd x sr ns *** *** ns y x sd *** *** *** ns y x sr *** *** ns ns y x sd x sr * *** ns ns error (b) 7.09 8.84 46135.74 2447414 cv 3.02 10.06 13.2 25.05 table 3 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at sagure in 2011 and 2014 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns ns ns sowing date (sd) *** *** *** *** error (a) 11.04 4.24 295392.79 2299583 year (y) *** *** *** ns seed rate (sr) *** ns ns ns sd x sr *** *** *** *** y x sd *** *** *** *** y x sr ns *** ns *** y x sd x sr * *** ns * error (b) 19.36 11.21 69124.93 928593 cv 4.93 10.71 18.52 20.75 kassaye et al. response of coriander to sowing dates and seed rates 197 the effects of seed rate on fruit and biomass yields were not significant; however, its interaction with sowing date and season brought significant improvement on some of the variables measured. results further showed that the interaction effects among sowing date, seed rate and season were not significant on most of the yield and yield components. the amount of variance associated with the sowing date x season interaction was the most important for this study. effect of sowing date sowing dates and their interaction with season significantly (p<0.001) affected the fruit and biomass yields of coriander at all locations except for the biomass yield at kulumsa. the highest fruit yields of coriander were attained from coriander sowed on july 30 in 2012 and 2014 at arsi robe and kulumsa, respectively; and july 30 and july 10 in 2014 and 2011 at sagure, respectively (tables 4-6). the coriander sown on july 30 and june 20 in 2012 and 2011, respectively at ari robe and july 10 in 2014 at sagure gave the highest biomass yields. fruit yields of 2713, 2028 and 2006 kg ha-1 were obtained at kulumsa in 2014, sagure in 2014 and arsi robe in 2012, respectively from coriander sowed on july 30. july 10 sown coriander at sagure gave a fruit yield of 1928 kg ha-1, which was statistically equivalent to the july 30 sown coriander. similarly, biomass yields of 4732 and 4573 kg ha-1 were harvested at arsi robe in 2012 and 2011 from july 30 and june 20 sown coriander, respectively. the highest biomass yield at sagure, 7217 kg ha-1, was found from the coriander sowed on july 10 in 2011. the result further revealed that early (june 20) and late (august 20) sown coriander produced inferior fruit and biomass yields compared to the intermediate sowing dates. the effects of sowing date and its interaction with season on the harvest index were also very significant (p < 0.001) at all locations (table 1-3). the highest harvest indexes were obtained from the august 20 sown coriander at arsi robe and kulumsa in both years (tables 4-6). the values of harvest index from the august 20 sown coriander at arsi robe were 42.7 and 40.9% in 2012 and 2011, respectively. the corresponding values from the same sowing date at kulumsa were 42.1 and 41% in 2014 and 2011, table 4 influences of sowing date and season on yield and yield components of coriander at arsi robe in 2011 and 2012 sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2012 2011 2012 2011 2012 2011 2012 june 20 72 de 112 a 34 cd 28 e 239 f 1272 b 721 e 4573 a july 10 79 c 108 a 34.8 c 31.1 d 502 e 1147 c 1446 d 3702 b july 30 68 e 96 b 38.6 b 42.9 a 683 d 2028 a 1809 d 4732 a august 20 76 cd 80 c 40.9 ab 42.7 a 696 d 1193 bc 1736 d 2828 c table 6 influences of sowing date and season on yield and yield components of coriander at sagure in 2011 and 2014 table 5 influences of sowing date and season on yield and yield components of coriander at kulumsa in 2011 and 2014 sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2014 2011 2014 2011 2014 2011 2014 june 20 106 a 105 a 18.9 e 20.5 de 1366 c 1493 c 7841 ab 8013 ab july 10 100 b 101 b 29.2 c 27.5 c 1835 b 1991 b 6326 c 7206 bc july 30 88 c 105 a 22 d 35 b 1876 b 2713 a 8559 a 7859 ab august 20 48 d 50 d 41 a 42.1 a 793 d 934 d 1942 d 2220 d sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2014 2011 2014 2011 2014 2011 2014 june 20 96 c 108 a 33.4 b 26.5 e 1523 b 1162 c 4638 cd 4507 cd july 10 96 c 103 b 28.2 cde 29.1 cd 2006 a 1435 b 7217 a 4947 bc july 30 78 d 102 b 40.4 a 35 b 1557 b 1928 a 3846 de 5495 b august 20 64 e 66 e 30.2 c 27.4 de 888 d 845 d 3204 e 3320 e adv. hort. sci., 2018 32(2): 193-203 198 respectively. statistically equivalent harvest index (42.9%) was also provided at arsi robe from the coriander sowed on july 30 in 2012. the highest harvest index at sagure (40.4%) was obtained from the july 30 sown coriander in 2011. the influences of sowing date and its interaction on plant height was also very significant (p <0.001). generally, early and late sown coriander resulted in the tallest and shortest plant heights, respectively, which were consistent over years and locations. early sown coriander resulted in the tallest plant at arsi robe in 2012 (112 cm), kulumsa in 2011 (106 cm) and 2014 (105 cm), and sagure in 2014 (108 cm). the tallest plant heights at kulumsa in 2014 (105 cm) and arsi robe in 2012 (108 cm) were also attained from coriander sowed on july 30 and july 10, respectively. delay in sowing of coriander (august 20) brought the shortest plant height at kulumsa (48 and 50 cm in 2011 and 2014, respectively) and sagure (64 and 66 cm in 2011 and 2014, respectively). temporal variabilities significantly affected both yield and yield components of coriander at all locations (tables 1-3). most of the variables measured at arsi robe, kulumsa and sagure gained better advantage owing to sowing of coriander in 2012 than 2011, 2014 than 2011 and 2011 than 2014, respectively implied the influences of seasonal variabilities on yield and yield attributes. the effects of temporal variations on yield and yield attributes were more pronounced at arsi robe than kulumsa and sagure areas, which could be justified by the magnitudes of differences in the fruit yields recorded during the study period. the variances in the highest fruit yields between 2012 (2028 kg ha-1) and 2011 (696 kg ha-1) at arsi robe, 2014 (2713 kg ha-1) and 2011 (1876 kg ha-1) at kulumsa, and 2011 (2006 kg ha-1) and 2014 (1928 kg ha-1) at sagure were 1331, 837 and 78 kg ha1, respectively (tables 4-6). this implied that the magnitude of the variabilities associated with the fruit yields between the two years were so large at arsi robe compared to kulumsa and sagure. spatial variabilities were also accountable for large disparities in yield and yield components of coriander among the testing locations. fruit and biomass yields of coriander were superior at kulumsa compared to sagure and arsi robe. mean fruit yields of coriander combined over season at arsi robe, kulumsa and sagure were 970, 1625 and 1418 kg ha1, respectively. the corresponding values for biomass yields were 2693, 6246 and 4647 kg ha-1, respectively (tables 4-6). compared to arsi robe, kulumsa and sagure produced 68 and 46% more fruit yields and 132 and 73% more biomass yields of coriander, respectively. effect of seed rate seed rate did not bring significant effect on most of the yield and yield components of coriander measured at all locations (tables 1-3), which were consistent over years and locations. however, its interaction with sowing date and year had significant effect on some of the yield and yield attributes of coriander at all locations (tables 1-3). the fruit yields obtained from the july 30 sown coriander at a seed rate of 30 kg ha-1 at arsi robe and any one of the four seed rates at kulumsa were found to be statistically superior over all other possible sowing date x seed rate interactions. this implied that the lowest seed rate, 30 kg ha-1, could be sufficient for optimum yield of coriander in the study areas. sowing of coriander on july 30 at a seed rate of 30 kg ha-1 gave fruit yield of 1526 kg ha-1. similarly, coriander sowed on july 30 at seed rates of 30, 40, 50 and 60 kg ha-1 at kulumsa gave fruit yields of 2239, 2306, 2427 and 2204 kg ha-1, respectively, which were statistically equivalent to each other but significantly different from the other treatments (table 7). the seed rate x sowing date interactions at sagure were not significant for the fruit yields of coriander. seed rate interaction with year brought significant effect on the fruit and biomass yields of coriander at sagure only (tables 1-3). except for the biomass yield at sagure, the sowing date x seed rate x year interactable 7 influence of sowing date x seed rate interactions on the fruit yield of coriander at arsi robe, kulumsa and sagure from 2011 to 2014 sowing date seed rate (kg ha-1) arsi robe kulumsa sagure 30 40 50 60 30 40 50 60 30 40 50 60 june 20 907 de 746 f 695 f 674 f 1378 de 1293 e 1506 cde 1541 cd 1508 bcd 1272 def 1456 cde 1133 efg july 10 781 ef 909 de 822 def 786 ef 2176 b 2086 b 1702 c 1688 c 1846 ab 1708 abc 1677 abc 1650 abc july 30 1526 a 1356 b 1355 b 1184 c 2239 ab 2306 ab 2427 a 2204 ab 1544 bcd 1955 a 1683 abc 1787 abc august 20 936 d 958 d 958 d 926 de 969 f 834 f 850 f 802 f 783 h 942 fgh 917 gh 826 gh kassaye et al. response of coriander to sowing dates and seed rates 199 tions were also not significant at all locations for most of the attributes measured (tables 1-3). 4. discussion and conclusions the highest fruit and biomass yields from the july 30 sown coriander at arsi robe and kulumsa, and july 10 to 30 sown coriander at sagure attributed to the fulfillment of optimum soil moisture and thermal conditions from vegetative to reproductive stages (figs. 3-5). rainfall and its expected consequent soil moisture had significant impact on the performance of coriander at all locations. coriander seeded to the sowing dates within or after the highest precedent rainfall events provided the highest fruit and biomass yields. the third decade of july in 2011 and 2012 at arsi robe, and the first decade of july in 2011 and 2014 at sagure fell within the highest precedent rainfall amounts and linked with the highest fruit and biomass yields (figs. 3-5). the highest amount of precedent rainfall could lead to the retention of optimum amount of water in the soil, which soak the seeds, soften its cover and assist embryonic stem to be emerged very easily. compared to the other three sowing dates, the third sowing date (july 30) at arsi robe and the second sowing date (july 10) at sagure in both years were linked with the highest rainfall events of the previous 10 days, and the highest fruit and biomass yields. the highest rainfall events of the previous 10 days at kulumsa occurred in the second decade of august. though the performance of crop at its initial stages was greater, it faced acute moisture stress at its latter development stage and resulted in inferior fruit and biomass yields. compared to the remaining two sowing dates, the highest rainfall event at kulumsa was recorded in the third decade of july, which was associated with superior fruit and biomass yields of coriander. katar et al. (2016) reported increase in the fruit yield of coriander with increase in precedent rainfall. the variations in the responses of coriander to sowing time were also accounted for temperature. the superior yield and yield attributes of coriander were found to be linked to the sowing dates, when their maximum temperatures were the lowest. the maximum temperature records of the periods were the lowest in the third decade of july at arsi robe, kulumsa and sagure (figs. 3-5), which were associated with the highest fruit and biomass yields. the minimum temperatures at each location were not too low enough to affect the germination of seeds; rather it was modest for coriander. for successful germination, coriander requires low temperature since it favors germination by promoting the breakdown of reserve proteins in seeds to particular amino acids, which are necessary for growth of embryo (robinson, 1954; guha et al., 2014). temperature above optimum value hampers germination of seeds owing to the encouraged activities of several microorganisms such as bacteria and fungi. activation of microorganisms, in turn, adversely affects the embryo and endosperm of coriander seeds (naeem et al., 2002; ali et al., 2015). generally, winter crops like coriander are vulnerable to high temperature particularly during reproductive stages (kalra, 2008). earlier sowing (june 20) resulted in the fruit yield reductions of 37 and 66% in 2012 and 2011, respectively at arsi robe, 45 and 27% in 2014 and 2011, respectively at kulumsa, and 24 and 40% in 2011 and 2014, respectively at sagure. the corresponding declines in biomass yield were 60% in 2011 at arsi robe, and 36 and 18% in 2011 and 2014, respectively at sagure (tables 4-6). those reductions in yields could be because of the storage of excess soil moisture in the root zone during seeding and the adverse effect of intensive rainfall particularly in august on the leaves and flower of coriander. the relatively older leaves and flowers of coriander sown earlier (june 20 and july 10) at arsi robe and kulumsa, and (june 20) at sagure fell due to heavy rainfall in august (figs. 3-5). the premature shed of leaves and flowers reduced the performance of photosynthesis, fruit formation and ultimately the yield of coriander. the july 30 and august 20 sown coriander were not adversely affected due to the intensive rain fell in august as their leaves were still so young enough to recover soon, and the flowers were not yet blossomed. delayed sowing (august 20) of coriander also produced inferior fruit and biomass yields. with delay in sowing to august 20, fruit yield reductions of 37% in 2012 at arsi robe, 66 and 58% in 2014 and 2011, respectively at kulumsa, and 26% in 2011 at sagure were recorded. similarly, delayed sowing resulted in biomass yield reductions of 40% in 2012 at arsi robe, 77 and 72% in 2011 and 2014 at kulumsa, and 26% in 2011 at sagure (tables 4-6). the inferior yields of coriander attributed to the acute scarcities of rainfall in november at all locations (figs. 3-5). owing to delayed sowing and its consequential soil moisture stress, coriander had inadequate time to complete its vegetative growth since it entered to the reproducadv. hort. sci., 2018 32(2): 193-203 200 tive phase at quicker rates and generally the whole crop-growing period was shortened (carrubba et al., 2006; sharangi and roychowdhury, 2014). consequently, inferior development of shoots and reduced yield attributes occurred (tables 4-6), which were the coriander’s response to the acute shortage of rainfall and high temperatures that encountered at latter stages in the growing season (carrubba et al., 2006; nowak and szemplinski, 2014). shortage of rainfall and its resulting soil moisture stress during the growing season brought physiological disorders such as a reduction in transpiration and photosynthesis (sarker et al., 2005; carrubba et al., 2006). besides, reduced vegetative as well as reproductive growth of coriander and their consequent decrease in seed yield could be accounted for the above optimum temperatures. the optimum temperature might have been surpassed for the late sown coriander, and adversely influenced its physiological processes including photosynthesis and respiration (sharma et al., 2003). the influences of precipitation and air temperatures on yield and yield components of coriander were reported in the works of nowak and szemplinski (2014). bhadkariya et al. (2007) found 30.6 and 76.4% declines in the fruit yield of coriander owing to delay in sowing from march 30 to april 14 and april 29, respectively. moosavi et al. (2012) reported that with delay in sowing, coriander fruit and biomass yields reduced by 76.4 and 74.7%, respectively. moniruzzaman et al. (2015), carrubba et al. (2006) and zheljazkov et al. (2008) also reported that productivity declined as sowing postponed to latest dates. though earlier sowing resulted in the tallest plant heights, it could not increase the yields of coriander. the inferior yields attributed to logging of the crop, shed of leaves and flowers during the cropping season in response to heavy rain fell particularly in august. this result is in agreement with pan et al. (2003), carrubba et. al. (2006), bhadkariya et al. (2007), ghobadi and gobadi (2010), and moosavi et al. (2012) who reported that earlier sowing of coriander led to the highest plant height. delay in sowing resulted in stunted growth and finally led to reduced yields of coriander. the significant decrease in plant height because of delay in sowing could be correlated with higher temperatures that the plants experienced at their latter stages. this incidence shortened their growing period and assimilate-building process owing to earlier maturity. thus, plants could not maintain adequate opportunity for conducting photosynthesis; hence, their height and branch-bearing capacity could be decreased (moosavi et al., 2012). sharangi and roychowdhury (2014) found that with delay in sowing from october to december, plant height of coriander decreased significantly. moosavi fig. 3 ten-day values of rainfall, maximum and minimum daily air temperature records in arsi robe from june to october in 2011 and 2012 cropping seasons. fig. 4 ten-day values of rainfall, maximum and minimum daily air temperature records in kulumsa from june to october in 2011 and 2014 cropping seasons. fig. 5 ten-day values of rainfall, maximum and minimum daily air temperature records in sagure from june to october in 2011 and 2014 cropping seasons. kassaye et al. response of coriander to sowing dates and seed rates 201 (2012) also reported significant decrease in plant height with delay in sowing from march 30 to april 29. the major factors that attributed to the variations in yields of coriander among locations and seasons were related to climate particularly rainfall and soil. in their studies, rashed and darwesh (2015) found that microclimate significantly affected sowing dates implied the need for determination optimum sitespecific sowing date. rainfall had contrasting effect at arsi robe and kulumsa in 2011; it was excess at arsi robe, but optimum at kulumsa. compared to 2011, the rainfall amount and distribution in 2012 at arsi robe and 2014 at kulumsa were optimum; hence, fruit and biomass yields obtained could be superior. these results corroborate the findings of carrubba et al. (2006), who reported a highly significant dependence of coriander yields on precipitation during its growing period with linear correlation coefficient of 0.93. nowak and szemplinski (2014) also reported the significant influence of temporal variations on coriander yield and yield attributes. the rainfall amount and its influence need to be interpreted in relation to the soil types of the study areas. compared to arsi robe and sagure, where the soils are heavy vertisols, the soil type of kulumsa is relatively light vertisol. hence, the occurrence of water logging at kulumsa particularly during the months with heavy rainfalls was lower as compared to arsi robe and sagure. coriander gave the highest fruit and biomass yields in areas, where rainfall was modest and the soil was light vertisol. during the cropping season, rainfall was lower at kulumsa than sagure and arsi robe, and sagure was lower than arsi robe. for example, cumulative rainfall from the first decade of june to the end of the third decade of august at arsi robe, kulumsa and sagure were 508, 282 and 302 mm, respectively indicating rainfall at kulumsa was robust for the development of coriander. this further implied that rainfall amount had strong effect on fruit and biomass yields of coriander at the studied sites. as rainfall amount increased, fruit and biomass yields of coriander decreased; hence, fruit and biomass yield obtained from kulumsa was superior followed by sagure. among the tested seed rates, the lowest, 30 kg ha-1, has been found optimum because under lower seed rate, plants tended to compensate the reduced sowing density by producing new branches and result in optimum fruit and biomass yields (diederichsen, 1996). okut and yidirum (2005) reported that seed rate had no effect on harvest index of coriander while ghobadi and ghobadi (2010) observed no significant effect on 1000 fruit weights of coriander. the superior yield and yield attributes in response to the seed rate of 30 kg ha-1 signified the need for investigation under further reduced rates. diederichsen (1996) reported that based on the weight of 1000 fruits, 3 to 20 kg ha-1 fruits of coriander could be sufficient for optimum yield. the results of the current study indicated that sowing date significantly influenced the yield and yield attributes of coriander. however, the effects of seed rate and its interaction with sowing date were not significant on most of the variables measured. the optimum sowing dates that gave the highest fruit and biomass yields of coriander fall on third decade of july at arsi robe, and from first-third decades of july at kulumsa and sagure. earliness and delay in sowing resulted in inferior yields. though coriander did not respond to seed rate in this study, the lowest rate, 30 kg ha-1, was found sufficient to give optimum yield. however, further low seed rate studies need to be conducted. considering the demand of low input, fruit and biomass yields obtained throughout experimental periods, cultivation of coriander was satisfactory provided the crop was sown at appropriate sowing time. owing to its economic benefits, soil improvement, nutritional qualities and reproductive roles, wider cultivation of coriander in the southeastern mid-highlands of ethiopia and other areas with similar agro ecologies is encouraged. acknowledgements the funding and logistics provision of the ethiopian institute of agricultural research for the execution of this research activity is sincerely acknowledged. the authors greatly acknowledge all members of land and water resources research team of kulumsa agricultural research center for their field and laboratory assistances. we would also like to thank the anonymous reviewers, whose comments and suggestions significantly improved this manuscript. references ali h., ayub g., elahi e., shahab m., ahmed s., ahmed n., 2015 response of coriander (coriandrum sativum l.) to different nitrogen levels and sowing dates. asian j. 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vavilov n.i., and v.f. dorofeev (eds.) origin and geography of cultivated plants . cambridge university press, cambridge, uk, pp. 498. zheljazkova v.d., pickett k.m., caldwell c.d., pincock j.a., roberts j.c., mapplebeck l., 2008 cultivar and sowing date effects on seed yield and oil composition of coriander in atlantic canada. ind. crops prod., 28(1): 88-94. impaginato 81 adv. hort. sci., 2020 34(1s): 81­88 doi: 10.13128/ahsc­7669 effects of kaolin­based particle film on physiological, nutritional, nutraceuticals parameters and ceratitis capitata infes­ tations in peach fruit at harvest and after storage a. palma 1 (*), a.m. cicilloni 1, d. satta 2, l. de pau 2, s. d’aquino 1 1 institute of food production sciences, national research council, traversa la crucca 3, loc. baldinca, li punti, 07100 sassari, italy. 2 agris sardegna, agency for research in agriculture, loc. bonassai, 07100 sassari, italy. key words: decay, fruit fly, peach fruit, postharvest physiology, quality. abstract: the mediterranean fruit fly (ceratitis capitata wiedemann) is a worldwide pest of economic importance because attacks a large number of agricultural crops and for the extent of the damage it causes. among the alter­ native control strategies to the use of sprays with synthetic insecticides, a very important role can be played by powders obtained from rocks whose activity arise from the ability to form a film of white powder, which acts as a repellent and irritant to insects. this film can also interfere with plants’ physiology and affect quality of fruit. in this study the efficacy of a commercial kaolin­based formulation to control medfly infestations was compared to synthetic insecti­ cides commonly used against this pest (phosmet, alfa­cypermethrin, deltamethrin). the results showed a significant reduction of medfly attacks in fruits treated with insecticides (1.5% damaged fruit) or with kaolin (0.5% dam­ aged fruits) compared to the untreated sample (10% damaged fruits), while physiological and quality parameters did not show relevant differences between treatments and control fruit. overall results highlight how the use of kaolin represents a valid alternative to treatments with synthetic insecticides to control c. capitata attacks on peaches, while not affecting fruits’ quality. 1. introduction ceratitis capitata (diptera tephritidae), also known as the mediterranean fruit fly is one of the most harmful insect pests to several fruit crops of the mediterranean countries. it is a polyphagousic phy­ tophagous that is considered highly invasive for the wide of host species and the high tolerance to low temperatures, compared to other fruit flies (malacrida et al., 2007). the fruits can be attacked early or close to har­ vesting: in the first case, the fly may sting the fruit several times for the oviposition and the developing larvae cause rottenness, early ripeness (*) corresponding author: amedeo.palma@cnr.it citation: palma a., cicilloni a.m., satta d., de pau l., d’aquino s., 2020 ­ effects of kaolin‐based parti‐ cle film on physiological, nutritional, nutraceuti‐ cals parameters and ceratitis capitata infesta‐ tions in peach fruit at harvest and after storage. ­ adv. hort. sci., 34(1s): 81­88 copyright: © 2020 palma a., cicilloni a.m., satta d., de pau l., d’aquino s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 december 2019 accepted for publication 11 july 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 81­88 82 and fruit drop. in case of late stings, the eggs laid shortly before harvesting, hatch during storage. yet, even in case eggs would not hatch, wounds caused by stings could favor growth of decay causing fungi (d’aquino et al., 2011). given the extent of the dam­ age caused by this fly, early monitoring is required, when fruits are not yet ripe. in italy the control strat­ egy against c. capitata is mainly carried out with traps for adult specimens or with chemical sprays, whose use is increasingly discouraged after the european directive 2009/128/ec. moreover, the development of resistant strains to conventional pes­ ticides (sparks and nauen, 2015) has stimulated stud­ ies for alternative and biological methods based on low toxicity and environmentally friendly plant extracts and mineral products. one of these mineral products is the kaolin clay, a fine powder, mainly composed of kaolinite, which, sprayed onto trees as a water suspension, forms a white and thin particle film on leaves and fruit surface (glenn et al., 1999; mazor and erez, 2004). different mechanisms seem to be involved in contrasting medfly attacks on fruit. the particle film beside masking the colour of leaves, stems and fruits, making long­distance host recogni­ tion difficult (saour and makee, 2004), renders the host less attractive for the white color of the film, which is the least attractive colour for ovipositing females of c. capitata (katsoyannos, 1987). moreover, the hard and irritating surface of kaolin film exerts at some extent a repellent effect on sever­ al insects, included medfly (saour and makee, 2004; salerno et al., 2019). however, if the positive effect of kaolin to control several pests is well documented and consistent, its impact on physiological response of plants and fruit quality is contradictory, depending on several factors such as the commercial formulations of the powder, the climate conditions, the intensity and quality of solar radiation, and environmental temperature. several studies show that kaolin particle films do not reduce photosynthesis and plant growth but mitigate water stress and photorespiration caused by intense solar radiation (kerns and wright, 2000; glenn et al., 2002; jifon and syvertsen, 2003). kaolin treatments were also reported to reduce leaf temperature and increase water use efficiency in artichoke (basnizki and evenari, 1975) and to decrease the rate of co2 absorption (presumably due to a partial block of stomata opening) in sorghum and cotton (stanhill et al., 1976; moreshet et al., 1979). the aim of this study was to evaluate the effec­ tiveness of kaolin versus synthetic insecticides to control medfly infestation and quality in stone fruit at harvest and during a simulated marketing conditions (smc) of 7 days. 2. materials and methods plant material and treatment the experiment was carried out in a stone fruit orchard located in north­western sardinia (lat 39° 50’ n, long 09° 38’ e). seven years old peach trees [prunus persica (l.) batsch.] cv. o’henry, highly sus­ ceptible to medfly attacks, were chosen for the experiment. trees were trained to a palmetta sys­ tem, spaced 3 m along the rows and 4 m between the rows. to evaluate the efficacy of the different treatments, a randomized block design with 3 repli­ cates of 3 trees per treatment was used. each repli­ cate was separated by the next one by four untreat­ ed trees. the following treatments were compared: i) kaolin (surround® wp, geovita, turin, italy; dissolved in water at 30 g/l); ii) a sequence of synthetic insecti­ cides representing a local protocol to control medfly, applied in the following order: one treatment with phosmet (spada® 50 wg, gowan, ravenna, italy, at 1.5 g/l); two treatments with alpha­cypermethrin (fastac® 10 sc, basf, monza e brianza, italia, at 0.3 g/l) and two treatments with deltamethrin (decis® evo, bayer crop science, ravenna, italy, at 0.12 g/l); iii) untreated control. phosmet, an organophosphate insecticide, can penetrate through the plant surface with a limited transport in plant tissues (agrochemicals handbook, 1983), while alpha­cypermethrin and deltamethrin are synthetic insecticides belonging to the pyrethroid group, that kill insects for contact and are more sta­ ble than pyrethrins when exposed to air and sunlight (worthing and hance, 1991). all treatments, carried out by spraying the products on the plants to obtain a homogeneous coverage, started 42 d before har­ vest, when fruits were not susceptible to medfly attacks and repeated at week intervals until 7 d before harvest. this local protocol followed by grow­ ers relies on frequent treatments in order to main­ tain residue levels of insecticides sufficient to kill medfly adults on fruit surface. on the other hand, kaolin was also sprayed at week intervals to maintain a continuous and even film on fruit surface, whose homogeneity would be reduced and made discontin­ uous by fruit growth in case less treatments had palma et al. ‐ peach postharvest physiology 83 been done. evaluation of medfly damage and storage condition at harvest, the total number of fruits for each treatment showing visible damage by medfly, con­ firmed by the presence of larvae after dissecting the fruit, were counted and the percentages of damaged fruit on the total yield of each plant were calculated. the total number of fruit produced by each plant ranged between 278 and 321. one hundred and twenty sound fruits (divided in replicated of 40 fruits each) for each treatment, free of any visible defect, were selected for storage. the fruits were placed in plastic trays and stored in a ven­ tilated storage room kept at 20°c and 55­60% rh for 7 d. at the end of storage, fruits were inspected for damage by medfly (presence of larvae within the flesh after dissection) or for the presence of molds but with no evident sign of medfly’s attack. the per­ centage of fruit damaged by medfly and that of fruit with the presence of molds were calculated. physiological and chemical determinations respiratory activity and ethylene production rates were determined after 1, 2, 3, 5 and 7 d of storage at 20°c and 55­60% rh using 10 sound fruit for treat­ ment. fruit were individually placed in 1 l jars, whose lids were fitted with two silicon septa and closed for 1 h prior co2 determination. at sampling time, the headspace air was mixed for 1 min by an electrical fan fixed inside the jar. co2 concentrations were determined by a combined co2/o2 analyzer (combi check 9800­1, pbi­dansensor a/s, rinsted, denmarck). the analyzer was connected to each jar by two tubes, each one ending with a needle inserted in one of the two septa to form a closed system. respiration activity, as co2 release, was expressed as ml kg­1 h­1. to determine ethylene concentration a 1­ml sam­ ple headspace air from each jar was withdrawn with a gas­tight syringe from the same septa used for co2 determination. ethylene was assessed by a varian 3300 gc (australia ltd., victoria, australia) equipped with a flame ionization detector (fid), carbowax 20m 80/120 mesh carbograph 1 aw 30 column (alltech, italy, milan), and the column, injector and detector temperatures set at 60°c, 110°c and 180°c, respec­ tively. chemical analyses were performed in triplicate at harvest and after 7 d of storage at 20 °c from puree obtained by homogenizing the fruit with a domestic homogenizer. the results of all chemical analyses are the mean values of three replications. according to the type of analysis, detailed procedures of sample preparation are described below. titratable acidity (ta), total soluble solid (sst), total phenolic compounds, glucose, fructose, sucrose, antioxidant activity and organic acid were determined on supernatant obtained by centrifuga­ tion of the puree at 13,000 x g for 20 min and filtered through a 0.45 µm acetate cellulose filter. ta was measured using an automatic titrator (metrom 720 sm tritino, switzerland) by titrating aliquots (10 g) of samples to an endpoint of ph 8.2 with 0.1n naoh and expressing the result as g l­1 cit­ ric acid, while tss were measured by a digital refrac­ tometer (mod. pr­101, atago, tokyo, japan) and expressed as percentage. total phenolic content was determined according to the folin­ciocalteu colorimetric method (singleton and rossi, 1965) and expressed as mg 100­1 g­1 gallic acid equivalents. folin­ciocalteu phenol reagent was from fluka (buchs, switzerland). analyses of glucose, fructose and sucrose were performed according to palma et al. (2018). stock standard solutions of each carbohydrate were pre­ pared in ultrapure water and quantified according to the linear calibration curves of standard compounds. glucose, fructose, and sucrose were purchased from sigma­aldrich co. (milan, italy). antioxidant activity was assessed using the free radical dpph, according to bondet et al. (1997). the mixture containing 3ml of a methanol solution of 6×10­5 mol l­1 of dpph and 100 µl samples was allowed to react for 15 min in a cuvette. the decrease of absorbance at 515 nm of dpph solution added with the sample was measured and the results expressed as trolox equivalent antioxidant capacity (mmol l­1 teac). 2,2­diphenyl­1­picrydazyl (dpph), and trolox (6­hydroxy­2,5,7,8­tetramethylchroman­ 2­carboxylic acid), a water­soluble analog of vitamin e reagent were from fluka (buchs, switzerland). malic and citric acid measurement was performed according to palma et al. (2013). total carotenoids were extracted from puree using a mixture of hexane/acetone/ethanol (2:1:1). the homogenized sample (10 g) was weighed into 100 ml glass vials with 50 ml of extranet solution. samples were kept in constant agitation for 60 min. the solutions were left to separation into a distinct polar layer (35 ml) and non­polar layer (25 ml) con­ taining carotenoids. total carotenoids were deter­ mined by a spectrophotometric method using a uv­ vis spectrophotometer (cary 50, varian australia ltd., victoria, australia). total carotenoids content adv. hort. sci., 2020 34(1s): 81­88 84 fig. 2 ­ respiratory activity as carbon dioxide release in ‘o’henry’ peach as affected by pre­harvest treatments with kaolin or insecticides stored at 20°c. columns with different letters are significantly different at p≤0.05 according to duncan’s multiple range test. vertical bars represent standard deviation (n=10). was calculated by comparing the absorbance of the carotenoids hexane solution with a calibration curve obtained using different concentrations of standard �carotene at 451 nm (kopec et al., 2012). �carotene was from sigma­aldrich co. (milan, italy). firmness measurements were carried out by a testing machine (mod. do­fb 0.5 ts, zwick roell, ulm, germany) recording the highest resistance (f max) opposed to the penetration of an 8­mm­diame­ ter flat faced cylindrical plunger to a depth of 10 mm and moving at a speed of 3.3 mm s­1 and the defor­ mation of the fruit surface at the highest resistance opposed before penetration (l at f max). the two parameters, f max and l at f max, were expressed as newton (n) and mm respectively. ten fruits were used for each treatment. mass loss, expressed as percentage, was deter­ mined on 30 fruits for treatment, individually weighed at harvest and at the end of the storage period. statistical analysis statistical analysis was performed using statgraphics centurion software (herndon, va, usa), version xv professional statistical program. analysis of variance (anova) was carried out according to a single­factor design, after testing (skewness and kurtosis) data normality assumptions. appropriate data transformations were carried out when viola­ tions of normality assumptions were met. the num­ ber of replications differed depending on the type of analysis performed. mean comparisons were per­ formed using duncan’s multiple range test at p≤0.05. 3. results evaluation of medfly damage at harvest and after storage at harvest, kaolin and insecticides reduced the percentage of peaches with visible damages by med­ fly to 0.5 and 1.5%, respectively compared to 10% of untreated fruit (data not shown). at the end of storage, fruit with visible damage and the presence of larvae within the flesh, observed after cutting the fruit, were 0.5±1% and 3±1.6% in kaolin and insecticide treatments, respectively, while in control fruit the infested fruits were approximately 16±2.1% (fig. 1). decay incidence due to the pres­ ence of molds was 7.5±2 % in kaolin treated fruit, 13±3% in those treated with insecticides and 19±5.2% in untreated ones. consequently, the total loss was 8±3 % in kaolin treated fruit, 16±4.1 % in those treated with the insecticides and 35±7.3% in control ones (fig. 1). effect of treatments on physiological and chemical properties compared to harvest time, respiration rate almost doubled at the end of storage, but significant differ­ ences could not be detected among treatments (fig. 2). in contrast, overall ethylene production showed a decreasing trend during the first 2 d and then gradu­ ally increased with final values significantly higher than those recorded at harvest time (fig. 3). although significant differences were not detected among treatments, presumably due to the relatively high variability occurring among the fruit of the same treatment, fruit treated with insecticides showed constantly higher rates than the other two treat­ ments (fig. 3). fig. 1 ­ incidence of fruit damaged by ceratitis capitata in ‘o’henry’ peach after 7 d of storage. columns with diffe­ rent letters are significantly different at p≤0.05 according to duncan’s multiple range test. vertical bars represent standard deviation (n=4). palma et al. ‐ peach postharvest physiology 85 regarding the other compounds, negligible varia­ tions occurred over storage in citric acid (p=0.048), total carotenoids (p=0.084) and malic acid (p=0.253) levels, while total phenols and antioxidant activity increased with final values of about 129 mg 100­1 g­1 gallic acid equivalents (p=0.001) and 1.8 mmol l­1 teac (p=0.001), respectively. however, both total phenols (p=0.240) and antioxidant activity did not show any significant difference neither at harvest time (p=0.291, p=0.183, respectively) nor after 7 d of storage (p=0.571, p=0.982, respectively). fruit firmness was affected by storage time but not by treatments (table 2). in particular, f max and lat f max decreased during storage with final values about 90 % lower than harvest time. mass loss, which on average was around 3 and 6% in table 1 are reported data concerning the chem­ ical composition of peaches at harvest and after 7 d storage. ta showed an overall significant decline of about 9% (p= 0.024) after 7 d of storage, but differ­ ences among treatments were not significant neither at harvest time (p=0.720) nor at the end of storage (p=0.982). sst, differently than ta, showed an overall increase of about 6 % over storage (p=0.012). values of individual treatments were slightly but significant­ ly lower in kaolin treated fruit at harvest (p=0.042), while no significant difference could be detected among treatments after 7 d of storage (p=0.532). glucose, fructose and sucrose content did not show any difference among treatments neither at harvest time (p=0.952, p=0.914, p=0.241, respective­ ly) nor at the end of storage (p=0.412, p=0.119, p=0.264, respectively), but while overall fructose lev­ els increased with storage (p=0.018), sucrose decreased (p=0.041). fig. 3 ­ ethylene production rates in ‘o’henry’ peach as affected by pre­harvest treatments with kaolin or insecticides sto­ red at 20°c. columns with different letters are significan­ tly different at p≤0.05 according to duncan’s multiple range test. vertical bars represent the standard devia­ tion (n=10). table 1 ­ changes in chemical parameters in ‘o’henry’ peaches as affected by pre­harvest treatments with kaolin or insecticides at har­ vest or after 7 d storage at 20°c values within rows for each parameter not followed by the same letters are significantly different at p ≤0.05 according to duncan’s mul­ tiple range test. each mean is followed by the standard deviation (n=3). chemical parameter or compound treatments harvest 7 days at 20°c control kaolin insecticides control kaolin insecticides ta (%) 1.02 ± 0.05 a 1.02 ± 0.03 a 1.01 ± 0.01 a 0.92 ± 0.06 b 0.92 ± 0.05 b 0.92 ± 0.04 b sst (°brix) 14.7 ± 0.17 b 14.2 ± 0.25 c 14.7 ± 0.17 b 15.3 ± 0.30 a 15.1 ± 0.05 a 15.4 ± 0.30 a glucose (g 100 ml­1) 1.16 ± 0.10 a 1.16 ± 0.10 a 1.18 ± 0.04 a 1.20 ± 0.03 a 1.23 ± 0.01 a 1.24 ± 0.05 a fructose (g 100 ml­1) 1.61 ± 0.14 b 1.61 ± 0.14 b 1.57 ± 0.03 b 1.91 ± 0.01 a 1.95 ± 0.07 a 1.82 ± 0.07 a sucrose (g 100 ml­1) 9.51 ± 0.27 a 9.53 ± 0.56 a 9.03 ± 0.16 a 8.12 ± 0.07 b 8.12 ± 0.07 b 7.97 ± 0.17 b total phenols (g 100 ml­1) 121.7 ± 31.8 b 125.1 ± 49.5 ab 119.7 ± 31.0 b 128.8 ± 27.8 a 130.9 ± 29.4 a 129.5 ± 11.0 a tot carotenoids (g 100 ml­1) 7.60 ± 0.58 a 7.25 ± 0.50 a 6.81 ± 0.29 a 7.19 ± 0.44 a 7.36 ± 0.21 a 7.08 ± 0.76 a antioxidant (mmol l­1 teac) 1.76 ± 0.01 b 1.77 ± 0.01 b 1.77 ± 0.01 b 1.82 ± 0.01 a 1.86 ± 0.03 a 1.82 ± 0.01 a malic acid (g 100 ml­1) 1.23 ± 0.05 a 1.24 ± 0.05 a 1.24 ± 0.03 a 1.16 ± 0.13 a 1.14 ± 0.04 a 1.20 ± 0.05 a citric acid (g 100 ml­1) 0.23 ± 0.01 c 0.26 ± 0.02 ab 0.23 ± 0.03 bc 0.27 ± 0.03 ab 0.29 ± 0.01 a 0.27 ± 0.02 ab table 2 ­ evolution of firmness as f max (maximum force to penetration) and l at f max (deformation of the fruit surface at f max) and changes in weight loss (% reduc­ tion of the initial weight) in ‘o’henry’ peaches as affec­ ted by pre­harvest treatments with kaolin or insectici­ des at harvest or after 3 or 7 d of storage at 20°c treatment f max (n) l at f max (mm) weight loss (%) harvest 3 days control 46.97 a 4.79 a 4.23 a kaolin 53.83 a 5.49 a 4.04 a chemical 49.71 a 5.07 a 4.13 a 7 days 7 days control 4.60 b 0.47 b 6.36 b kaolin 5.88 b 0.60 b 5.97 b chemical 4.80 b 0.49 b 6.04 b values in column for each storage time not followed by the same letter are significantly different at p≤0.05 according to duncan’s multiple range test. 86 adv. hort. sci., 2020 34(1s): 81­88 after 3 and 6 d, respectively, was not affected by treatments (table 2). 4. discussion and conclusions different field as well as laboratory studies demonstrated the efficacy of kaolin treatments in reducing medfly punctures on fruit of different species (mazor and erez, 2004; d’aquino et al., 2011; lo verde et al., 2011) and its higher efficacy when compared to traditional (organophosphates, pyrethroids) or novel insecticides, despite the excel­ lent laboratory results of the last ones. for example, the high activity of spinosad (adan et al., 1996; vargas et al., 2002; mangan et al., 2006), was not confirmed in field experiments to control medfly in citrus fruit, while kaolin tested in the same experi­ ment, resulted very effective (braham et al., 2007). indeed, in contrast to traditional insecticides, whose efficiency may be affected by medfly developmental stage, population density, mode of action and environmental factors (ligth, temperature, rain) that can shorten their persistance (braham et al., 2007), kaolin efficiency seems stable over time, provided a uniform coverage of fruit surface and absence of abundant rains (mazor and erez, 2004; lo verde et al., 2011). d’aquino et al. (2011) found a significant lower percentage of damaged fruit at harvest in peaches and nectarines treated with kaolin com­ pared to those subjected to a conventional treat­ ment with organophosphates trichlorfon and fen­ thion, although the kaolin protective activity was higher in peaches rather than in nectarines owing to the lower adherence of the particles on nectarines surface and the difficulty to form a uniform film. our results showed a marked effect of both kaolin and insecticides in reducing the number of fruit with visible damages at harvest, but after one week of storage at 20°c, decay incidence caused by patho­ genic fungi was markedly higher in fruit treated with insecticides than in those treated with kaolin. the lower performance of fruit treated with insecticides compared to those treated with kaolin may depend on the short persistence of pyrethroids: due to their rapid degradation rate, the level of residues on fruit surface after one week might be not sufficient to completely prevent medfly oviposition. as a result, in fruit damaged just before harvest, decay incidence at the end of storage caused by the activity of develop­ ing larvae or pathogenic fungi penetrated through stings was higher in insecticides treated fruit than in kaolin ones. kaolin sprayed on leaves and fruit surface, depending on particles size and film uniformity may affect the transmission of photosynthetically active, ultraviolet and infrared radiations, resulting in changes in surface temperature, photosynthetic activity, selective production of pigments, chemical composition, susceptibility to decay and physiological disorders (jifon and syvertsen, 2003; lombardini et al., 2005; russo and díaz­pérez, 2005; cantore et al., 2009). our results showed no difference in respiration and ethylene production rates of kaolin treated fruit compared to control or insecticides treated ones. these results can be explained considering that the size and porosity of the particles deposited on fruit surface would not affect gas exchange and the fruit ripening process. kaolin did not affect firmness and mass loss. these results are in contrast with those reported by ergun (2012) with ‘galaxy’ apples, who attributed the reduction of mass loss to the small size of the kaolin particles which by partially blocking stomata and lenticels would have led to a reduction of gases and water vapor exchange with the environment. a reduced transpiration rate of kaolin was also detected in bean leaves (tworkoski et al., 2002), groundnut (khan and morey, 1980), and tomatoes (cantore et al., 2009). differently, either no or an inconsistent effect of kaolin film on transpiration and stomatal conductance were reported by others (kerns and wright, 2000; glenn et al., 2001; jifon and syvertsen, 2003; russo and díaz­pérez, 2005; glenn, 2012; lobos et al., 2015). genetic variability among species, differences in growth environment, possible kaolin­ induced physical skin modifications, kaolin formula­ tions, may be only some among the numerous fac­ tors leading to contrasting results (wand et al., 2006; conde et al., 2016). although no specific study at our knowledge has been set up to specifically evaluate the effect of kaolin treatments on fruit quality, generally results reported in the literature indicate a positive effect of kaolin on overall quality. these positive effects of kaolin, seems to rely on its ability to reduce organs’ surface temperature and to enhance solar radiation reflection, which, while reducing the risk of sunburns improves skin color development, lowers the photorespiration process and increases photosynthetic efficiency (glenn et al., 2002; wand et al., 2006; glenn, 2009). palma et al. ‐ peach postharvest physiology 87 in grapevine, kaolin treatments stimulated the phenylpropanoid, flavonoid­flavonol and antho­ cyanin­pathways thus increasing total phenolics and anthocyanins content in ripe berries, but had no effect on ph, ta and sst (conde et al., 2016). our results, in agreement with previous findings (glenn, 2012; lobos et al., 2015; conde et al., 2016), denoted no effect of kaolin on ta and sst, but also on juice antioxidant activity, glucose, fructose, sucrose and organic acids contents both at harvest time and during storage. despite kaolin treatment was not able to com­ pletely control medfly attacks, its performance was superior to synthetic insecticides in controlling direct damage by medfly due to the presence of larvae but also indirect damage even when larvae did not devel­ op, for the lower incidence of decay caused by wounds pathogens. therefore, kaolin seems to be a promising alternative to conventional insecticides to manage medfly infestation in peaches. one potential disadvantage of kaolin at commercial level is the fact that the white film persists on fruit surface after har­ vest and that to be removed fruit should be washed, an operation that normally is not done on peaches intended for fresh consumption. however, the pres­ ence of kaolin on fruit surface could be exploited positively commercially by reporting in the label that the white film covering the fruit is a proof that no synthetic insecticides were used in the growing process. on the other hand, in case fruit are destined to the processing industry rinsing the fruit would not be a problem. references adan a., del estal p., budia f., gonzalez m., vinuela e., 1996 ­ laboratory evaluation of the novel naturally derived compound spinosad against ceratitis capitata. ‐ pestic. sci., 48: 261­268. basnizki j., evenari m., 1975 ­ the influence of a reflec‐ tant on leaf temperature and development of the globe artichoke (cynara scolymus l.). ­ j. amer. soc. hort. sci., 100: 109­112. bondet v., brand­williams w., berset c., 1997 ­ kinetics and mechanism of antioxidant activity using the dpph free radical method. ­ lwt ­ food sci. technol., 30: 609­615. braham m., pasqualini e., ncira n., 2007 ­ efficacy of kaolin, spinosad 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inoculation may be mediated by depolarization-activated outward-rectifying k+ channels (shabala et al., 2010). in animals, the immunosuppressant drug mycophenolic acid (mpa) [(4e)-6-(4-hydroxy-6-methoxy-7-methyl-3oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid] depletes cellular guanine nucleoside (gn) by inhibition of inosine monophosphate dehydrogenase, modulating the activity of k atp channels (li et al., 2000). mpa is also an effective antiviral drug in plants such as grapevine (panattoni et al., 2007; skiada et al., 2009; luvisi et al., 2012 a; skiada et al., 2013; panattoni et al., 2014; guazzelli et al., 2015), but no reports about its effect on kconducting ion channels in plants are available. in terms of metabolic dependence of mpa action in plant cells, antiviral drug translocation across membranes was linked to the free energy available in a proton electrochemical potential difference (luvisi et al., 2012 b). nowadays, compounds that influence k atp channel activity are currently available for clinical use, and include diazoxide (do) (7-chloro-3-methyl-4h-1,2,4-benzothiadiazine 1,1-dioxide) (babenko et al., 1998; 2000) whose effectiveness was also confirmed in plants to investigate the inhibition of mitochondrial k atp channels (chiandussi et al., 2002). with regard to the potential effect due to gn depletion by mpa, cyclic derivatives of gn can be involved in plant k-conducting ion channels. in fact, inward-rectifier potassium ion channels have been cloned from hordeum vulgare, nicotiana tabacum and arabidopsis thaliana (leng et al., 1999). these channels open in the presence of cyclic nucleotides such as cyclic guanosine monophosphate (cgmp), and are therefore referred to as cyclic nucleotide gated channels (leng et al., 2002). the cgmp is thought to be present in the cytosol of plant cells, and to be involved in signal transduction pathways which regulate many aspects of cellular metabolism (assmann, 1995). the activity of antiviral drugs in grapevine cells was recently investigated using electrophysiological methods (panattoni et al., 2013 a). in the present paper we investigate the effects of mpa on depolarization induced by extracellular k+ in foliar samples of vitis vinifera cv. sangiovese. this grapevine cultivar is affected by various plant viruses (rizzo et al., 2012; rizzo et al., 2015) and the efeffects of extracellular k+ on grapevine membrane potential as influenced by the antiviral mycophenolic acid. an electrophysiological study a. luvisi1 (*), e. rinaldelli 2, a. panattoni 1 1 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: chemotherapy, virus host interaction, vitis vinifera. abstract: mycophenolic acid (mpa) is an effective antiviral drug in plants, and its action in modulating the activity of katp channels is already known in animals. in the present work an electrophysiological study was carried out to investigate mpa effects on plant k+ channels, through the measurement of trans-plasma membrane potential in samples of vitis vinifera cv. sangiovese treated with extracellular k+. tests confirmed that the administration of mpa (in preincubated samples or in those maintained under chemical treatment) can reduce the membrane depolarization induced by k+. however, mpa-induced alteration in membrane potential was sensitive to the katp channel opener diazoxide, as well to treatments with guanosine. this result confirms the effectiveness of mpa in influencing katp channel activity as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by mpa. adv. hort. sci., 2015 29(4): 207-212 (*) corresponding author: aluvisi@agr.unipi.it received for publication 28 september 2015 accepted for publication 5 november 2015 208 adv. hort. sci., 2015 29(4): 207-212 fects of antiviral drugs where investigated (panattoni et al., 2011; luvisi et al., 2011; panattoni et al., 2013 b). in addition, the effects of gnor dotreatments were evaluated in order to investigate the effect of gn depletion caused by mpa or its effects on k atp channels. 2. materials and methods plant material virus-free (with regard to viruses included in the european commission directive 2005/43/ec), eight-yearold v. vinifera cv. sangiovese were used for electrophysiological tests. the sanitary condition of each plant was confirmed by rt-pcr (nakaune and nakano, 2006; faggioli et al., 2013). symptomless plants were used. in june 2013, fully expanded leaves were excised and fresh freehand samples (3-5 mm diameter) were cut with an ethanol-cleaned razor blade for testing (rinaldelli et al., 2012; 2014). measurement of membrane potential (em) for chemical assays (table 1), preincubation for 1 h in basal solution (bs) or chemical-bs solutions (mpa-bs, gn-bs, do-bs at 0.5, 1.0, 2.0 mm) adjusted to ph 5.6 with tris (2-amino-2-hydroxymethyl-propane-1,3-diol) was followed by preparation of leaf segments according to luvisi et al. (2012 b). step 1 for em measurements was conducted perfusing aerated bs or chemical-bs through the chamber that fixes the sample at a flow rate of 10.0 x 10-3 l min-1. the measuring electrodes used were micropipettes (tip diameter < 1µm) obtained from single-barreled borosilicate capillaries (world precision instruments, sarasota, usa) as described in rinaldelli et al. (2012). insertion of the microelectrodes took place in the central zone of the mesophyll by way of a micromanipulator (world precision instruments, sarasota, usa). successful microelectrode impalement was determined by rapid attainment of a steady value without subsequent decay (ober and sharp, 2003), followed by a stabilized em for 5 min. em after stabilization was recorded to evaluate the effect of chemicals on membrane potential. membrane signal steadiness was calculated considering the number of successful cell impalements out of those attempted (%). step 2 began after em stabilization, using solutions enriched with kcl. two procedures were carried out in order to investigate the effects induced by extracellular k+ following mpa treatments. the first procedure was carried out on samples in which treatment involved only the preincubation step (li et al., 2000). thus, samples were preincubated in mpa-bs, followed by em measurement under bs (step 1) and k-bs (step 2). in this test, two concentrations of kcl (5, 10 mm) were evaluated. the effect of chemical treatment was calculated considering em after bs stabilization and maximum em achieved after k-bs administration, expressed as δ em (table 1). the second procedure was carried out following a conventional electrophysiological approach. in order to evaluate the interference of chemicals (mpa, gn or do) on effects induced by k+, tests were carried out using chemical-bs solution both in preincubation and in step 1, while in step 2 the solution was enriched with kcl (chemical-k-bs) at 10 mm (concentration chosen considering results of the first procedure). the interference of chemicals on membrane depolarization induced by k+ was calculated considering em after chemical-bs table 1 solutions used in membrane potential tests chemical assay solution abbreviation membrane potential (em) cacl 2 5.0x10-4 m, k 2 so 4 2.5x10-3 m, mes 5.0x10-3 m bs em bs bs with mpa mpa-bs em mpa bs with guanosine gn-bs em gn bs with diazoxide do-bs em do extracellular k+ assay solution abbreviation membrane potential (em) variation of membrane potential (%) bs with kcl k-bs em k em k em bs = δ em k mpa-bs with kcl mpa-k-bs em mpa-k em mpa-k em mpa = δ em mpa-k gn-bs with kcl gn-k-bs em gn-k em gn-k em gn = δ em gn-k do-bs with kcl do-k-bs em do-k em do-k em do = δ em do-k mpa-bs with guanosine mpa-gn-bs em mpa-gn mpa-bs with diazoxide mpa-do-bs em mpa-do mpa-gn-bs with kcl mpa-gn-k-bs em mpa-gn-k em mpa-gn-k em mpa-gn = δ em mpa-gn-k mpa-do-bs with kcl mpa-do-k-bs em mpa-do-k em mpa-do-k em mpa-do = δ em mpa-do-k 209 luvisi et al., effects of extracellular k+ on grapevine membrane potential stabilization and maximum em achieved after chemicalk-bs administration, expressed as δ em (table 1). preincubation and electrophysiological tests were carried out at 22±0.5°c under light (30 watt m-2). all tests were conducted on 15 healthy or infected samples. plots are representative of 15 equivalent experiments. measurements were performed under faraday cage to protect tests from external radio frequency interference. statistical analysis the effects of treatments on δ em were elaborated using sigma-plot software (version 11; systat software, san jose, ca). the software was used to perform oneor twoway analysis of variance (anova) in a random design and pairwise multiple comparisons on significant effects and interactions using the holm-sidak method. data expressed in percent were converted to arcsin values. p ≤ 0.05 was considered to be significant. 3. results and discussion em measurement in chemical preincubated samples foliar samples were preincubated in chemical-bs solutions and membrane potential was measured with microelectrodes. preincubation with mpa-bs solution did not change the resting membrane potential with regard to the chosen concentration (table 2). however, mpa seems to interfere with the signal steadiness at 1.0 and 2.0 mm, causing a reduction of successful impalement by 37.9 and 64.7 %, respectively. assays carried out with bs enriched by gn or do showed no effect on membrane potential and did not interfere with the steadiness of the signal. with regard to the effects induced by extracellular k+, em measurement was carried out perfusing aerated bs solution throughout the chamber with the fixed sample (step 1), followed by perfusion of k-bs solution (step 2), and recording the depolarization due to k+ treatment. in mpa-preincubated samples, the antiviral drug can cause a differential response to potassium effects, as shown in figure 1. the cell response confirms the nernstian changes in diffusion potential due to increased k+-concentration or activation of k+ channels. as reported in table 3, kcl caused different depolarization according to each concentration, but the preincubatable 2 effect of sample preincubation with chemical administered at different concentrations (0.5, 1.0, 2.0 mm) on membrane potential (em, mv) or membrane potential signal steadiness on samples of vitis vinifera cv. sangiovese, expressed as % of successful microelectrode impalement treatment em (mv) signal steadiness (%) bs -109.5±6.0 a (z) 53.6 mpa-bs (mm) 0.5 -110.6±7.2 a 51.7 1.0 -110.0±6.9 a 33.3 2.0 -107.6±5.7 a 18.8 gn-bs (mm) 0.5 -111.6±8.5 a 50.0 1.0 -112.0±9.5 a 48.4 2.0 -109.0±7.0 a 53.6 do-bs (mm) 0.5 -110.9±7.8 a 45.5 1.0 -113.5±8.5 a 48.4 2.0 -108.3±7.1 a 51.7 (z) values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p ≤0.05). bs= basal solution; mpa= mycophenolic acid; gn= guanosine; do= diazoxide. table 3 depolarization induced in samples of vitis vinifera cv. sangiovese by extracellular k+ at 5 or 10 mm following mpa-bs preincubation compared to untreated sample δ em (%) k-bs (mm) 5 10 bs 18.2±3.5a (z)a (y) 30.7±3.7 ab mpa-bs (mm) 0.5 17.9±4.1 aa 29.8±3.2 ab 1.0 12.0±3.2 ba 23.3±2.0 bb 2.0 11.1±2.3 ba 16.4±4.1 cb (z) values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p ≤0.05). (y) values in the same line followed by the same letter do not differ significantly according to duncan’s multiple range test (p ≤0.05). the effect was calculated considering em after bs stabilization and maximum em achieved after k-bs administration, expressed as δ em (%). bs = basal solution; mpa = mycophenolic acid. fig. 1 effects induced on samples of vitis vinifera cv. sangiovese by extracellular k+ (kcl 10 mm) subsequent to mpa 1.0 mm treatment compared to untreated sample. bs = basal solution; mpa = mycophenolic acid. plot is representative of 15 equivalent experiments. 210 adv. hort. sci., 2015 29(4): 207-212 tion with mpa was able to cause a reduction in depolarization. in particular, mpa at 1.0 or 2.0 mm significantly affected kcl at 10 mm, while mpa at 5 mm seemed not to alter the effect induced by extracellular k+. kcl at 10 mm was used for the subsequent test. em measurement in samples maintained under chemical treatment tests carried out while maintaining chemical administration throughout steps 1 (perfusion of chemical-bs solution) and 2 (perfusion of chemical-bs solution enriched by kcl) of em measurement confirmed the effectiveness of mpa in reducing the effect induced by k+ (table 4). mpa interference was concentration-dependent and at the lower concentration mpa did not interfere with the effect on membrane potential induced by k+. conversely, at higher dosages (2:1 mpa:kcl), mpa causes a δ em mpa-k of 2.5±0.7%, with a reduction of more than 90% of potassium effects. mpa effectiveness was maintained at equal concentration (1:1 mpa:kcl), showing δ em mpa-k at 9.5±1.1 %, with a reduction of almost 70 % of potassium effects. no effects on trans-plasma membrane depolarization due to k+ were registered by gn or do. simultaneous administration of gn with mpa (2:1) caused complete restoration of external k+ effect on membrane potential, as well as adding do (1:1) (data not shown). 4. conclusions membrane depolarization caused by mpa (rinaldelli et al., 2012) is a temporary effect. in fact, after 1 h of preincubation in mpa-bs solution, the trans-plasma membrane potential of the sample was not altered by the antiviral drug compared to the untreated control. similarly, gn or do did not change resting membrane potential. interference of signal steadiness caused by mpa at a higher concentration may be linked to cell toxicity induced by the antiviral drug (panattoni et al., 2007; luvisi et al., 2012 a). in fact, samples under stress conditions increase the difficulty of cell membrane measurements (vuletic et al., 1987; rawyler et al., 2002). with regard to effects induced by extracellular k+, preincubation tests showed how mpa at 1.0 mm or higher concentrations can interfere up to 10 mm of kcl. the mpa reduction of membrane depolarization caused by extracellular k+ was confirmed by following tests in which chemical administration was maintained throughout all steps of measurement. the reduction of potassium effects was higher compared to administering mpa only in the preincubation step. this result is probably linked to the variation in mpa intra-/extracellular gradient due to solution changes between preincubation and steps 1-2. our results indicate that the antiviral mpa inhibited the effects of extracellular k+ in plants, through specific channels. in fact, the mpa-induced alteration in membrane potential was sensitive to the k atp channel opener do, which hyperpolarized resting membrane potential in treated cells to a level similar to that achieved in control cells; results were also similar to those obtained in animal cells (li et al., 2000). moreover, gn was able to inhibit the mpa action against extracellular k+, suggesting that mpa could act also as an inward-rectifier potassium ion channel inhibitor through the depletion of gn. inhibition of the activity of k+ channels caused by mpa may be involved in programmed cell death (pcd) in table 4 two-way factorial analysis of variance of δ em caused by chemical-k-bs (mpa-k-bs, gn-k-bs, do-k-bs, kcl at 10 mm) on samples of vitis vinifera cv. sangiovese treaded chemical-bs (mpa-bs, gn-bs, do-bs). pairwise multiple comparison analysis with holm-sidak test was performed source of variation df ss p treatment (a) 2 0.836 <0.001 concentration (b) 3 0.228 <0.001 a x b 6 0.696 <0.001 residual 170 0.207 total 181 1.961 comparison for factor dm t p comparison for a do vs. mpa 0.149 23.461 <0.001 gn vs. mpa 0.139 21.880 <0.001 do vs. gn 0.010 1.5888 ns comparison for b within mpa 0.0 mm vs. 2.0 mm 0.284 22.276 <0.001 0.5 mm vs. 2.0 mm 0.250 19.586 <0.001 0.0 mm vs. 1.0 mm 0.231 18.160 <0.001 0.5 mm vs. 1.0 mm 0.197 15.470 <0.001 1.0 mm vs. 2.0 mm 0.052 4.116 <0.001 0.0 mm vs. 0.5 mm 0.034 2.690 ns comparison for b within gn 2.0 mm vs. 0.5 mm 0.00543 0.433 ns 2.0 mm vs. 0.0 mm 0.00467 0.366 ns 1.0 mm vs. 0.5 mm 0.00276 0.220 ns 2.0 mm vs. 1.0 mm 0.00267 0.209 ns 1.0 mm vs. 0.0 mm 0.00200 0.157 ns 0.0 mm vs. 0.5 mm 0.00076 0.061 ns comparison for b within do 2.0 mm vs. 0.5 mm 0.0473 3.771 ns 2.0 mm vs. 0.0 mm 0.0447 3.505 ns 2.0 mm vs. 1.0 mm 0.0406 3.189 ns 1.0 mm vs. 0.5 mm 0.0067 0.531 ns 1.0 mm vs. 0.0 mm 0.0040 0.316 ns 0.0 mm vs. 0.5 mm 0.0026 0.210 ns bs= basal solution; mpa = mycophenolic acid; gn = guanosine; do = diazoxide. ss= sum of square, df = degrees of freedom, dm = difference of means, t = t-value. ns= non significant at p> 0.05. 211 luvisi et al., effects of extracellular k+ on grapevine membrane potential response to viral infection. excessive k+ efflux and intracellular k+ depletion are the key early steps in apoptosis (one form of the pcd) in mammalian systems. a critical role for potassium homeostasis in the apoptotic process has also been endorsed for plants (huh et al., 2002; shabala et al., 2007; shabala, 2009) even if it has not yet been linked to the mechanism of action of mpa in plants. references assmann s.m., 1995 cyclic amp as a second messenger in higher plants. status and future prospects. plant 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cells. gen. physiol. biophys., 6: 203-207. impaginato 153 adv. hort. sci., 2019 33(2): 153-160 doi: 10.13128/ahs-23517 diversity of morpho-physicochemical traits in iranian sour cherry genotypes using multivariate analysis s. aliyoun nazari 1 (*), j. hajilou 1, m. zeinalabedini 2, a. imami 3 1 department of horticultural sciences, faculty of agriculture, university of tabriz, iran. 2 agriculture biotechnology research institute of iran (abrii), karaj, iran. 3 horticultural departments of seed and plant improvement institute (spii), karaj, iran. key words: fruit quality traits, genetic diversity, prunus cerasus l. abstract: in this study, morpho-physicochemical characterization of sour cherry genotypes from iran was investigated. thirty-four morphological and eight physicochemical traits were recorded. sour cherry genotypes had a high variability in traits related to fruit characters such as fruit weight, stone volume, total anthocyanin content and total soluble solid. as a result, sour cherry genotypes exhibit total phenolic content and antioxidant activity higher than “ciganymeggy” and “erdi botermo” cultivars. principal component analysis (pca) suggested that leaf dimensions, fruit weight, stone weight, and stone volume could be sufficient for identification of genotypes. hierarchical cluster analysis classified sour cherry genotypes and “ciganymeggy” and “erdi botermo” cultivars into two main clusters. the first cluster was characterized by a upright tree vigour, depressed fruit pistil end, reniform shape of fruit, high sweetness, dark red juice, flower high length and diameter, fruit and stone weight and length and diameter, total soluble solid, low total phenolic content, high total flavonoid content and high total anthocyanin content. 1. introduction sour cherry, prunus cerasus l., is known as tetraploid (2n =4x = 32), originated through natural hybridization of the large statured, cold sensitive sweet cherry (p. avium l., 2n = 2x = 16), and the low growing, cold tolerant ground cherry (p. fruticosa pall., 2n = 4x = 32) (olden and nybom, 1968). this species originated around the black and caspian seas and were cultivated in temperate and cold regions. sour cherry spread slowly from its origin to other regions due to human and animal migrations (pérez-sánchez et al., 2008). sour cherry fruit is mostly used for industrial preserves (jams, purees, juices and concentrates), while only a small portion is assigned to fresh consumption. sour cherry is also used as a sweet cherry rootstock. this rootstock is more resistant to soil wetness and cold (*) corresponding author: saliyoun66@gmail.com citation: aliyoun nazari s., hajilou j., zeinalabedini m., imami a., 2019 diversity of morpho-physicochemical traits in iranian sour cherry genotypes using multivariate analysis. adv. hort. sci., 33(2): 153-160 copyright: © 2019 aliyoun nazari s., hajilou j., zeinalabedini m., imami a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 june 2018 accepted for publication 12 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 153-160 154 climate than wild sweet cherry and mahaleb forms. in 2014, the total world production of sour cherry reached 1.1 million tons, being turkey, the russian federation, poland, ukraine, iran and serbia the most important producing countries (faostat, 2014). the main sour cherry producing areas in iran are the ardebil, azerbaijan, khorasan and alborz provinces. from the viewpoint of fruit quality, several studies for characterization of fruit traits have been accomplished recently. sour cherry is a valuable source of vitamins (a, b1, b2, c, e, k, and niacin), carotenoids like beta-carotene, minerals, fiber, various sugar like fructose, glucose, maltose, antioxidant agents such as caffeic acids, cyaniding-3-o-glucosylrutinoside and flavoids (mulabagal et al., 2009; ferretti et al., 2010). this products has positive effects on human health (ataie-jafari et al., 2008; saric et al., 2009; kuehl et al., 2010). analysis of flavonoids from p. cerasus identified kaempferol, quercetin, quercetin 3-o-glucoside, and isorhamnetin 3-o-rutinoside (piccolella et al., 2008). the main anthocyanins found in cherry are cyanidin-3-o-glucoside, cyanidin-3-o-rutinoside, cyanidin-3-o-glucosylrutinoside, cyanidin-3-osophoroside, pelargonidin-3-o-glucoside, peonidin-3o-rutinoside and cyanidin-3-o-arabinosylrutinoside (chaovanalikit and wrolstad, 2004). much of the genetic diversity is available in the wild types and natives from the center of origin. wild species are probable gene resources for the breeding objectives such as resistance to pests and diseases, more appropriate cultivars for table and industry, extending cherry season and developing new resistant and dwarfing rootstocks. therefore, it is necessary to characterize and preserve these species (demirsoy and demirsoy, 2004; aliyoun nazari et al., 2012). description of the morphological characteristics is the usual methodology accepted from a legal point of view for patenting and registration of varieties. several quantitative and qualitative evaluations showed a clear difference between sour cherry, with a more marked variability within the sour cherries group, probably due to the more intense domestication processes that have taken place. morphological characterization continues to be the first step for germplasm description and classification, and the statistical method of factor analysis is a useful tool for screening the accessions of a collection (badenes et al., 2000; hajilou and fakhimrezaei, 2011). several morphological characterization studies have been carried on the sour cherry (krahl et al., 1991; rodrigues et al . , 2008; rakonjac et al . , 2010; najafzadeh et al., 2014). as an origin of the subgenus cerasus, iran has a rich cherry germplasm. the area of this study is part of a large growing area in north west of iran. sour cherry has been cultivated in this area for many years. however, the conservation and characterization of local cultivars is important to avoid the loss of genetic variability and as a potential source of genetic variation for future sweet and sour cherry breeding programs. these genotypes show distinctive agronomic characters such as low susceptibility to fruit cracking, high levels of soluble solids and early fruit maturity. the objective of this study was to survey, identify and characterize sour cherry genotypes existing in the province of east azerbaijan shabestar (iran) for their later introduction into a germplasm bank. 2. materials and methods plant materials the plant material was located on the shabestar town in west side of the east azerbaijan province, in north-west of iran. a total of 15 sour cherry genotypes and two cultivars, “ciganymeggy” and “erdi botermo”, were used in this study. evaluation of morphological and physicochemical traits characterization of vegetal material and fruits was based on sour cherry descriptors developed by the international union for the protection of new varieties of plants upov (upov, 2006). thirty four morphological (16 qualitative and 18 quantitative) and eight physicochemical traits were recorded as described in table 1 and 2. in this study, a total of 17 sour cherry genotypes, including 15 local sour cherry genotypes and two cultivars, “ciganymeggy” and “erdi botermo”, with three replicates for each genotype were evaluated. the evaluation for morphological characters was based on 30 measurements of each trait. for the analysis of physicochemical traits, fruits were picked at the commercial maturity stage. all fruits were collected from a single plant, randomly from all cardinally oriented branches with different directions around the canopy. all samples were stored in a freezer at -20°c. the frozen fruit material (5 g) was homogenized with a polytron (2 min on ice) with 10 ml of extraction solution, consisting of 0.5 n hcl in methanol/milli-q water (80% v/v). the mixture nazari et al. iranian sour cherry characterization 155 was incubated overnight at 4°c and then centrifuged for 20 min at 4°c and 20000 g. supernatant was recovered and the volume measured. this hydroalcoholic extract was used for total phenolics, anthocyanins, flavonoids, and antioxidant capacity assays (cantin et al., 2009). the content of phenolic comno. trait 1 2 3 4 5 6 7 9 11 1 tree vigour very weak weak medium strong very strong 2 tree habit upright semi-upright spreading drooping 3 tree branching weak medium strong 4 tree bud distribution along entire branch only on middle distal part of branch only on distal part of branch 5 flower arrengment of petal free intermediate overlapping 6 flower shape of petal circular medium obovate broad obovate 7 flower arrangement solitary double in clusters irregular 8 starting bloom from april 9-11 day 11-13 day 13-15 day 15-17 day 17-19 day ›19 day 9 fruit ripening time from june 5-10 day 10-15 day 15-20 day 20-25 day 25-30 day › 30 day 10 fruit pistil end pointed flat depressed 11 stone shape narrow elliptic broad elliptic circular 12 fruit shape reniform oblate circular elliptic 13 fruit color of skin orange red light red medium red dark red brown red blackish 14 fruit color of flesh yellowish pink medium red dark red 15 fruit sweetness low medium high 16 color of juice colorless light yellow pink medium red dark red table 1 sixteen qualitative traits and their states and codes studied of sour cherry genotypes no. trait unit min max mean cv (%) 1 flower diametr mm 23.5 39.5 28.1 12.4 2 petal length mm 10.8 13.7 11.9 8.1 3 petal width mm 9.6 14.0 11.6 11.9 4 pestil length mm 11.4 13.6 12.4 5.1 5 number of stamens 31.9 37.0 34.5 4.1 6 fruit length mm 13.5 19.1 15.2 8.8 7 fruit diameter mm 13.1 22.6 17.6 10.9 8 fruit length/ diameter -mm 0.8 1.0 0.9 6.1 9 length of stalk mm 41.0 54.0 46.8 7.3 10 fruit weight gr 12.4 54.3 21.4 55.7 11 stone length mm 5.6 9.5 7.1 11.7 12 stone diameter mm 5.5 9.0 7.4 13.2 13 stone volume cm3 0.1 0.4 0.2 36.0 14 stone weight gr 2.2 3.5 3.0 13.5 15 leaf blade length mm 67.0 96.8 78.9 10.2 16 leaf blade width mm 36.8 53.4 43.4 9.8 17 leaf blade length/ blade width 1.7 2.0 1.8 4.0 18 petiole length mm 13.6 18.4 16.2 8.2 19 ph 2.0 3.6 3.3 13.9 20 total soluble solid % 12.1 23.3 16.8 20.9 21 vitamin c mg/100g fw 10.5 13.2 11.7 7.7 22 titratable acidity % 1.9 2.7 2.3 9.0 23 total phenolic content mg gae/100 g fw 228.0 289.0 241.8 6.0 24 total anthocyanin content mg cyanidin 3-glucoside /100 g fw 60.1 130.3 98.0 21.5 25 antioxidant activity μg te/ 100g fw 52.3 67.7 60.5 7.5 26 total flavonoids content mg qe/ 100 g fw 141.8 155.8 145.8 2.5 table 2 the range of 26 quantity variability in sour cherry genotypes traits, mean and coefficient of variations (cv %) pounds in methanol extracts was determined according to the folin-ciocalteu method (waterhouse, 2001). absorbance was measured at 725 nm using a spectrophotometer (uv-2100 spectrophotometer). total anthocyanin content (tac) was determined using the ph differential method (giusti and adv. hort. sci., 2019 33(2): 153-160 156 wrolstad, 2001). the absorbances of the extracts at 510 and 700 nm were measured against a blank. tac was calculated and expressed as mg cyanidin 3-glucoside equivalent/100 g of fw. total flavonoid content of each extract was determined following colorimetric method (chang et al., 2002). the antioxidant capacity was measured using the dpph method adapted from brand-williams et al. (1995). titratable acidity was established by titration with 0.1 n naoh and sugar content was measured as total soluble solids (tss) using digital refractometer (atago pr 100, japan). vitamin c content was estimated according to the titration with 2, 6-dichlorophenolindophenol method (aoac, 2000). statistical analysis statistical analysis were performed using spss 17.0 (spss inc., chicago, il). to obtain basic statistics for the entire plant material studied, maximum and minimum values, mean, and coefficients of variation (cv %) were calculated for each trait. relationships among the species were investigated by principal component analysis (pca). pca was performed using spss statistics software. scatter plot of the first two pcs and the cluster analysis were created by past statistics software (hammer et al., 2001). 3. results and discussion characteristics of cultivars several researchers have reported the morphological variation between some prunus subgenus cerasus genotypes such as for sweet cherry (p. avium), sour cherry (p. cerasus), mahaleb (p. mahaleb), marmareh (p. incana) and tomentosa cherry (p. tomentosa) (ganji-moghadam and khalighi 2007; khadivi-khub et al., 2008; perez-sanchez et al., 2008; zhang et al., 2008; rakonjac et al., 2010; aliyoun nazari et al., 2012). morphological characteristics of the studied genotypes are resumed in the table 1 and 2. results showed that high variation among studied genotypes was found for fruit weight (cv=55.7%) and stone volume traits (cv=36%). this result is compatible with zhang et al. (2008) report. they observed high morphological variation among populations, where the highest variations were in fruit weight, fruit width, and leaf width. tree habits of the studied genotypes are different. most of the genotypes have drooping, three have spreading and one has upright tree habit (“erdi botermo”). tpc values ranged between 228 and 289 mg gae/100 g fw of sour cherry genotypes, which is in good agreement with previously published results (dragovic-uzelac et al., 2007; khoo et al., 2011; alrgei et al., 2015). “erdi botermo” had the lowest tpc among the studied genotypes and n02 had the highest, that is consistent with the results of papp et al. (2010) that reported the “erdi botermo” have lowest tpc among all tested genotypes. behrangi et al. (2015) reported that tpc is versatile on the basis of fruit type, stage of growth, farm of landing, extraction method, component of tpc experiment and other factors. therefore tpc decreased by transforming fruit from first stages of growth to fully ripe form that is compatible with our results. as shown in table 2, total antioxidant capacity (ac) of sour cherries was between 52.3 and 67.7 μg te/100 g fw. the total ac of different sour cherry cultivars showed significant difference (blando et al., 2004; bonerz et al., 2007; khoo et al., 2011). the lowest tac was found in n02 genotype (60.1 mg cyanidin 3-glucoside/100 g fw), while “erdi botermo” had the highest tac (130.3 mg cyanidin 3-glucoside/ 100 g fw). these differences in tac showed that the plant growth region and the harvest period might have an impact on plant growth and metabolite concentration (premier, 2002). sour cherry is one of the richest source of flavonoid (marinova et al., 2005), that is consistent with our results. principals component analysis eighty percent of the variability observed was explained by seven components (table 3). for each trait, a factor loading of more than 0.51 was considered as being significant. pc1 represents mainly fruit pistil end, fruit color of skin, fruit length, fruit diameter, fruit weight, stone length, stone diameter, stone volume, leaf blade length, leaf blade width and total flavonoids content with significant positive effects, also tree habit, flower shape of petal, fruit ripening time with negative effects and account for 29.65% of the variance. the second principals component with 13.4% of total variance included traits of the tree branching, vitamin c, titratable acidity with negative impacts and the trait of flower diameter, petal length, petal width and stone weight with positive impacts. high absolute values of the correlations between variables related to the growth, fruit and leaf size, and pc1 or pc2 were also established by krahl et al. (1991) and rakonjac et al. (2010) in sour cherry, by lacis et al. (2010) and rakonjac et al. (2014) in sweet cherry, aliyoun nazari et al. (2012) in nazari et al. iranian sour cherry characterization 157 marmareh (p. incana) and by khadivi-khub et al. (2012) in prunus subgen. cerasus. pc3 was correlated with starting bloom, stone shape, pistil length, fruit length/diameter, ph and length of stalk. the remaining components explain less variability. grouping of cultivars hierarchical cluster analysis classified native sour cherry genotypes and “ciganymeggy” and “erdi botermo” cultivars in two main clusters (fig. 1). the first major cluster is divided into two subgroups; subgroups i consisted of erdi botermo cultivar and and subgroup ii contained ciganymeggy and some of the genotypes, indicating that these sour cherry genotype had high similarity to ciganymeggy cultivar. the second cluster included the native genotypes. the table 3 eigen values and cumulative variance for seven major factors obtained from principal component analysis (pca) and traits within each factor for sour cherry genotypes trait factors pc1 pc2 pc3 pc4 pc5 pc6 pc7 tree vigour -0.10 0.32 0.12 -0.62** 0.02 0.30 -0.52 tree habit -0.87** 0.05 0.19 0.14 0.14 -0.15 0.00 tree branching 0.14 -0.74** 0.21 0.21 0.02 -0.24 -0.08 tree bud distribution 0.32 0.02 0.15 -0.60** -0.04 -0.29 -0.39 flower arrangement of petal 0.32 -0.34 0.35 -0.02 -0.14 0.39 0.32 flower shape of petal -0.90** 0.10 0.19 -0.03 -0.19 -0.20 -0.04 flower arranement -0.36 -0.39 0.18 -0.22 0.13 0.64** -0.22 starting bloom from april 0.02 -0.15 0.61** -0.24 -0.48 0.14 0.27 fruit ripening time from june -0.87** 0.08 -0.10 0.15 0.20 0.02 -0.02 fruit pistil end 0.81** -0.13 -0.02 -0.29 -0.18 -0.13 -0.04 stone shape 0.18 0.34 0.62** 0.50 0.02 0.26 0.07 fruit shape -0.83** 0.22 0.26 0.25 0.07 -0.05 -0.06 fruit color of skin 0.80** -0.12 -0.27 0.10 0.34 0.11 0.09 fruit color of flesh 0.16 -0.50 0.14 0.26 -0.55** 0.26 -0.06 fruit sweetness 0.63** 0.02 -0.51 -0.08 0.28 0.15 0.16 color of juice 0.90** -0.10 -0.19 0.03 0.19 0.20 0.04 flower diameter -0.28 0.71** -0.15 0.17 0.05 0.15 0.39 petal length -0.10 0.77** -0.14 0.32 -0.32 -0.03 0.15 petal width -0.10 0.70** -0.12 0.14 -0.48 0.29 0.06 pestil length -0.18 0.08 0.70** 0.06 -0.36 0.02 -0.38 number of stamens -0.21 0.11 -0.46 0.46 0.40 -0.02 -0.19 fruit length 0.90** 0.33 0.09 0.03 0.04 -0.11 -0.07 fruit diameter 0.81** 0.23 -0.19 0.22 -0.13 -0.18 -0.15 fruit length/diameter -0.15 0.01 0.52** -0.38 0.33 0.24 0.28 length of stalk 0.01 -0.02 0.59** -0.13 0.40 0.07 0.11 fruit weight 0.86** 0.16 0.21 -0.14 0.28 -0.19 0.07 stone length 0.89** 0.11 0.25 0.14 -0.10 -0.03 0.09 stone diameter 0.60** -0.20 0.47 -0.02 -0.23 -0.34 0.05 stone volume 0.88** -0.05 0.31 0.01 -0.17 -0.16 0.09 stone weight 0.35 0.77** 0.26 -0.03 0.13 0.08 -0.05 leaf blade length 0.64** 0.50 0.11 -0.17 0.01 0.17 -0.28 leaf blade width 0.64** 0.41 0.03 -0.09 0.07 0.34 -0.49 leaf blade length/leaf blade widht 0.05 0.49 0.21 -0.23 -0.13 -0.39 0.43 petiol length 0.38 0.14 -0.39 -0.13 -0.12 0.18 0.54** total soluble solid 0.47 -0.17 0.31 0.45 0.45 -0.16 -0.13 ph 0.12 0.31 0.67** 0.59 0.09 0.25 0.01 vitam c -0.05 -0.56** -0.13 -0.09 -0.15 0.07 0.19 titratable acidity 0.21 -0.51** 0.47 0.04 0.39 0.21 0.31 total phenol content -0.10 0.14 0.40 0.12 0.27 -0.67** -0.07 total anthocyanin content 0.51 -0.20 -0.24 0.20 -0.59** -0.12 0.00 antioxidant activity 0.12 0.36 0.01 -0.42 0.34 -0.08 0.28 total flavonoids content 0.61** -0.32 -0.24 0.50 0.08 0.09 -0.25 eigen value 12.45 5.63 4.69 3.32 2.42 1.43 1.18 cumulative variance (%) 29.65 43.06 54.24 62.16 69.30 75.11 80.54 158 adv. hort. sci., 2019 33(2): 153-160 first cluster were characterized by a upright tree vigour, depressed fruit pistil end, reniform shape of fruit, high sweetness, dark red juice, flower high length and diameter, fruit and stone weight and length and diameter, total soluble solid, low total phenolic content, high total flavonoid content and high total anthocyanin content. perez-sanchez et al. (2008) suggested that dendrogram gained from morphological characteristics clearly showed the relationships among the cultivars of sweet, sour and duke cherries. in addition, khadivi-khub et al. (2012) reported that dendrogram obtained from morphological characteristics clearly separated some cerasus genotypes. the second cluster were characterized by small fruit and stone, drooping or spreading tree habit. scatter plot was prepared according to the pc1 and pc2 by past software (fig. 2). starting from the positive to the negative values of pc1, these genotypes indicated a gradual decrease in fruit pistil end, fruit color of skin, fruit length, fruit diameter, fruit weight, stone length, stone diameter, stone volume, leaf blade length, leaf blade width and total flavonoids and an increase in tree habit, flower shape of petal, fruit ripening time. starting from the negative towards the positive values of pc2, the genotypes indicated a gradual increase tree branching, vitamin c, titratable acidity and a decrease flower diameter, petal length, petal width and stone weight. 4. conclusions morphological characterization continues to be the first step for the description and classification of germplasm and statistical methods like principal components analysis (pca) are useful tools for screening the accessions of a collection (cantini et al., 1999; badenes et al., 2000). pca is used for data reduction that transforms the original variables into a limited number of uncorrelated new variables. this technique, producing a smaller set of composite variables, account for much of the variance among the set of original variables and allows visualization of the differences among the individuals, identification of possible groups and finding relationships among individuals and variables (martinez-calvo et al., 2008). high correlations were found between some traits and principal components, which could reduce the number of traits to be studied in sour cherry germplasm. for instance, measuring the traits of pc1 (such as fw, sl, sd. sv, fpe, and fcs) is suggested for future studied in sour cherry genotypes. dependent on the trait, a certain number of genotypes were observed that showed lower or higher values than the commercially grown cultivars involved in this study. especially in reference to the fruit weight, a high portion of genotypes were characterized by smaller fruits than that of “ciganymeggy” and “erdi botermo”. in addition, native genotypes showed higher values of total phenolic content and antioxidant activity traits than the commercial cultivars. we conclude that this is the first study of sour cherry native genotypes, which deals with the morphological and physicochemical variation basis of genetic diversity. although these accessions does not fig. 1 dendrogram of 17 sour cherry genotypes based on morphological traits by past software. fig. 2 factor scores for the first two principle components (pcs) for sour cherry genotypes. nazari et al. iranian sour cherry characterization 159 represent the whole sour cherry germplasm in iran, considerable genetic diversity observed in both morphological and physicochemical characteristics 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(ed.) current protocols in food analytical chemistry. john wiley & sons, new york, usa. zhang q., yan g., dai h., zhang x., li c., zhang z., 2008 characterization of tomentosa cherry (prunus tomentosa thunb.) genotypes using ssr markers and morphological traits. sci. hortic., 118: 39-47. impaginato 517 adv. hort. sci., 2018 32(4): 517-524 doi: 10.13128/ahs-20689 stability analysis of fruit yield of some olive cultivars in semi-arid environmental condition i. arji (*) crop and horticultural science research department, kermanshah agricultural and natural resources research and education center, areeo, kermanshah, iran. key words: adaptability, ammi, olea europaea l., olive, stability parameters, yield. abstract: this study was conducted to evaluate yield stability of 12 iranian and foreign olive cultivars in dalaho olive research station during 2006-2008. according to the variance analysis, significant variation (p<0.01) was observed between cultivars and years. classification based on duncan (p<0.05) showed that konservolia was superior variety and sevillano, koroneiki and zard were placed in the second group. cultivars were divided into 3 groups based on cluster analysis using ward method. the first principal component of the interaction between olive cultivars and the year’s show 69.25% of the variance and was statistically significant at 1% level based on ammi analysis. according to regression coefficient (bi) deviation from regression (s2 di), wricke’s ecovalence (wi), coefficient of determination (ri2) and shukla’s stability variance (δi 2) methods ‘mission’ and ‘zard’ had the higher stability. according to the ammi stability (asv) ranking, the following cultivars were the most stable, mission, amigdalolia and koroneiki, while the most unstable were ‘konservolia’, ‘sevillano’, ‘roghani’, ‘arbequina’ and ‘abou-satal’. ‘konservolia’ even showed the lowest stability but its stability in all parameters was significant different in terms of performance. generally ‘konservolia’, ‘sevillano’, ‘koroneiki’ and ‘zard’ were appropriate for fruit yield and will be introduced for breeding programs in semi-warm climate. 1. introduction olive (olea europaea l.) tree is an evergreen native to the mediterranean region. some olive wild genotypes are present in different region of iran like kermanshah province in the west of iran. there are more than 40 native olive genotypes in subtropical regions of kermanshah province like sarpool-e-zahab, gilan-e-gharb and paveh. marone and fiorino (2012) reported that olive (olea europaea l.) distributed across three continents from south africa to the central part of the africa and horn africa, from egypt and red sea to the mediterranean areas and asia from palestine, syria, mesopotamia and western and eastern areas of himalaya chain to the southwestern of china. this report (*) corresponding author: issaarji@gmail.com citation: arji i., 2018 stability analysis of fruit yield of some olive cultivars in semi-arid environmental condition. adv. hort. sci., 32(4): 517-524 copyright: © 2018 arji i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 may 2017 accepted for publication 19 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 517-524 518 revealed that there are some olive genotypes in three continents. in recent years, due to higher olive oil demand, the cultivation of olive has been expanded in various regions of iran. however, the cultivation of olive tree is limited because of harsh environmental conditions and water scarcity in most of the new olive plantation areas (arji and arzani, 2008). the limitation of water as well as long hot summers in the regions lead to poor fruit and oil quality (saadati et al., 2013; khaleghi et al., 2015). cheng et al. (2017) stated that low temperatures would be improved olive oil quality by increasing unsaturated fatty acid amounts in the fruit. temime et al. (2006) reported that more unsaturated fatty acid of chetoui olive variety was recorded in cooler regions than dry and warm regions. despite of good vegetative growth, some of the olive varieties do not show good performance as production in warm regions. this is due to lack of adapted and stable cultivars in such environmental conditions. check-adapted varieties and optimal stability are essential for the fruit yield. it is assumed that the stability of a genotype is very important over time in each region (finlay and wilkinson, 1963). homeostatic and agronomic are two genotypic stabilities. in homeostatic stability a certain genotype shows constant response under different conditions. but in agronomic stability, genotype yield is linked to productivity potential (hayward et al., 1993). generally, the stability is defined as the actual performance of a genotype under changing environmental conditions. reliable stability of production efficiency under environment changing is very important (kan et al., 2010). stability analysis methods are categorized in two parametric and non-parametric groups (sabaghnia et al., 2006). several methods such as regression coefficient (finlay and wilkinson, 1963), sum of squared deviations from regression (eberhart and russel, 1966), stability variance (shukla, 1972) and additive main effects and multiplicative interaction (ammi) (gauch and zobel, 1988) have been commonly used to parametric stability analysis. environmental sustainability of individual genotypes can also be estimated by regression analysis and cultivar will be stable when the deviation of regression was zero or at least (hayward et al., 1993). it is mentioned that regression analysis in bilinear models and analysis of variance in biadditive models have limitations in genotype and environment interaction. this restriction reduced by multiplicative components for interactions in generalized linear models (glm) such as additive effects and multiplicative interaction (ammi) (gauch, 1992). in this model the main additive effects was calculated by variance analysis and then genotypes and environment interaction, which is known as multiplicative interaction, are analyzed by principal components analysis (romagosa and fox, 1993). olive is one of the fruit trees with alternate bearing tendency in which it not bear regularly (lavee, 2007). this phenomenon is affected by different factors l ike genetic and physiological traits (goldschmidt, 2005). the degree of alternate bearing in olive is highly dependent on environmental conditions (lavee, 2007). fruit production in olive is more irregular by climate change where adverse environmental conditions are frequent (lodolini and neri, 2012). for this purposes stability of olive production is very important in new olive growing region like sarpool-e-zehab environmental conditions. ammi analysis was used to evaluate the stability of different crops (esmaeilzadeh-moghaddam et al., 2011), but there is lack of research in horticultural crops. weather conditions are variable during different years in new olive cultivation regions so that we need to find out more stable olive cultivars. in the present work, the year was considered as environmental variable. generally, the main goal of this study was the evaluation of yield stability of different olive cultivars in warm condition of kermanshah province. 2. materials and methods material, site characterization and experimental design this experiment was conducted in dalahv olive research station of sarpool-e-zahab (longitude: 45° 51´ e, latitude: 34° 30´ n, altitude: 570 m asl) to verify the yield stability of 12 iranian and foreign olive cultivars (table 1). two years old self-rooting plantlets were planted in the year 2000, with 6x6 m spacing table 1 name and codes of genotypes genotype name 1 amphisis 2 konservolia 3 zard 4 amigdalolia 5 koroneiki 6 roghani 7 manzanillo 8 abou-satal 9 mission 10 arbequina 11 sevillano 12 shenge arji olive fruit stability analysis 519 distance in a randomized complete block design with three replications. each experimental unit consisted of 5 trees so that 15 trees of each cultivar were evaluated. trees were pruned as vase shape and irrigated each three days with drip irrigation system. climate of sarpool-e-zahab is warm with relatively low humidity during summer as shown in figure 1. also soil and water analysis were reported (tables 2 and 3). data analysis methods fruit yield was measured during 5 years from 2004 to 2008. as fruit yield was low in the years 2004 and 2005, therefore 3 years (2006, 2007 and 2008) were analyzed to determine yield stability. spss, irristat and excel were used for statistical analysis and the mean comparison was done by duncan’s multiple range test at p<0.05. the model of ammi analysis is presented in equation (1). y ger = μ + α n + β e +σ n λ n α gn γ en + ρ ge + ε ger (1) where αn is the main effect of genotype; βe is the main effect of environment; n is the number of main components in ammi model; λn is a single value related to the n remained main components in the model; αgn is thespecific vector for the g genotype from n main component; γen is the specific vector for the e environment from n main components; ρ ge is the noise and ger is the error (clay et al., 1995). the following parameters were calculated to analyze yield stability, coefficient of variability (cvi) (francis and kannenberg, 1978), wricke’s (1962) ecovalance (wi2), shukla’s (1972) stability variance (σi 2), pinthus’s (1973) coefficients of determination (r2), and finlay and wilkinson (1963) regression coefficient (bi). alternate bearing index (abi) was calculated during three successive years from 2006 till 2008, using the following equation (2) (monselise and goldschmidt, 1982): (2) where n = number of years, and a1, a2... an = yields in the corresponding years. 3. results and discussion fruit yield analysis of variance the results of variance analysis for yield of olive (kg/tree) show that the genotype, environment (year) and interaction effects were significant (p<0.01) (table 4). specific response of the cultivars to ecological factors over a 3-year period were confirmed by the results of duncan multiple range-test, which proved that cultivar and year interaction effect was also significant (table 5). it is evident from data in table 5, for 3 study years, ‘konservolia’ had thehighest mean yield, 24.69 kg/tree, while ‘roghani’ had the lowest mean yield, 4.87 kg/tree. fruit yield variability was depending on the year but olive varieties show different responses (table 5). so, this indicates that the genotypes present different behavior in that environment. this may be due to differences in genetic basis of cultivars (rakonjac and živanovic, fig. 1 precipitation, mean temperature and relative humidity during five years of experiment. table 2 physical and chemical soil characteristics soil depth (cm) particle-size distribution (%) oc (%) ph tnv (%) ava. k (mg/kg) ava. p (mg/kg) total n (%)clay silt sand 0-30 34 52 14 2.25 7.7 41 520 6.2 0.18 31-60 40 37 23 0.78 7.7 45 275 2.6 0.06 table 3 irrigation water chemical characteristics ec (ds/m) tds (mg/l) ph meq/l s.s.p (%) s.a.r co 3 -2 co 3 h clso 4 -2 sum anions ca2++ mg2+ na+ sum cations 550 352 7.28 0 4.6 0.3 1.9 6.8 6.6 0.2 6.8 2.94 0.11 table 4 analysis of variance for olive fruit yield s.o.s df ss ms replication 2 2083 0.115 ns cultivar 11 3026.53 275.14 ** error 22 199.59 9072 year 4 1023.13 511.567 ** cultivar x year 44 1423.3 64.696 ** error 96 285115 5.94 cv% 19.18% adv. hort. sci., 2018 32(4): 517-524 520 2008). olive varieties with yield stability are important for sustainable production. stable cultivars have high yield with lower variation during the years. based on the results, ‘konservolia’, ‘zard’, ‘koroneiki’, ‘amigdalolia’, ‘arbequina’ and ‘sevillano’ have higher fruit yield with moderate yield fluctuation during the years. analysis of variance is only able to express the presence or absence of interaction and is not possible to interpret yield stability. for this reason, using univariate and multivariate nonparametric interpret better interaction of cultivars and years in the sustainability debate (gauch, 1992; falconer and mckay, 1996; arciniegas-alarcon et al., 2011; gauch, 2013). ammi analysis the anova for fruit yield using the ammi method is presented in table 6. there were significant differences among the genotypes, environments (years) and g × e interaction. in this experiment environments were the years based on citadin et al. (2013) method. combined analysis of variance (anova) for fruit yield of olive cultivars indicated that genotypes, year and genotype-by-year interactions (gei) were the most important source of fruit yield variation (table 6). the contribution of variation caused by the cultivar, year and gei were 52.56%, 17.77%, and 24.72%, respectively. this result showed that olive cultivars had different yield performance across years. the high share of interaction in the total sum of squares is very important to use stability analysis for fruit yield of olive varieties. similar results were reported in yellow passion fruit by oliveira et al. (2014) and peanuts (oliveira and godoy, 2006). maulión et al. (2014) stated that the significance of the environmental effect and gei were used as a starting point to study yield stability among peach accessions. ammi analysis indicated that two first ipca were significant (p<0.01). the ipca1 accounted for 69.25% of the ge interaction (table 6). however, based on these results most information can be graphically displayed using ipca1. biplot graph of the model (ipca1 vs. yield) is presented in figure 2. according to figure 2, ‘zard’ and ‘mission’ showed greater yield stability by values near the origin of the ipca1 axis. however, mean yield of ‘mission’ was lower than total mean yield. ‘konservolia’ with highest fruit yield and ‘roghani’ with the lowest fruit yield were unstable cultivars and the others were in the intermediate stability. one of the most important parameter in olive stability is alternate bearing. this index seems to be useful in determining the sustainability of production in fruit trees. based on biplot ammi1 analysis, ‘konservolia’ was more productive (fig. 2) in all years than the others and its alternate bearing index was low (table 5). so it is recommended to use this parameter in stability evaluation. in this experiment, variability due to the year was greater than variability table 5 fruit yield (kg tree-1), mean yield (kg tree-1) and alternate bearing index of olive cultivars during 20062008 cultivar 2006 2007 2008 mean alternate bearing index amphissis 7.03 hij 3.5 j 16.37 cdef 8.97 efg 0.16 konservolia 26.03 b 14.57 c-g 33.47 a 24.69 a 0.06 zard 17.37 cd 8.23 hij 16.67 cde 14.09 cd 0.01 amigdalolia 15.39 cdef 6.533 ij 10.43 fghi 10.78 def 0.1 koroneiki 23.8 b 13.33 d-h 16.7 cde 17.94 bc 0.08 roghani 7.1 hij 4.5 ij 3 j 4.87 g 0.21 manzanillo 24.53 b 7.86 hij 6.27 ij 12.89 de 0.31 abou-satl 7.78 hij 7.17 hij 9.1 ghij 8.02 fg 0.04 mission 14.53 c-g 8.07 hij 14.33 c-g 12.31 def 0.003 arbequina 7.36 hij 10.18 fghi 16.08 cdef 11.21 def 0.19 sevillano 25.13 b 10.8 e-i 20.48 bc 18.81 b 0.04 shenge 10.34 fghi 6.4 ij 7.13 hij 7.96 fg 0.09 mean 15.53 8.43 14.17 12.71 table 6 analysis of variance for fruit yield of 12 olive cultivars by ammi during 2006 -2008 s. o. v df ss ss% ms genotypes 11 3026.52 52.56 275.14 ** year 2 1023135 17.77 511.57 ** cultivar x year 22 1423305 24.72 64.7 ** ipc1 12 985614 69.25 82.13 ** noise 10 437688 30.75 43.77 ns error 96 285115 4.95 2.97 total 35 5758075 fig. 2 biplot ammi1 (means vs pc1) for the data on the yield of olive (ton ha-1) with 12 cultivars () and five years (δ). arji olive fruit stability analysis 521 caused by varietal effects based on scattered effect (fig. 2). ammi analysis method is highlighted to study g x e interaction which combines a univariate method for the additive effects of genotypes and years with a method for the multiplicative effects of the g x e interaction (zobel et al., 1988; citadin et al., 2013). gauch and zobel (1996) stated that this method can contribute to the identification of widely adapted genotypes with high yields, as to the agronomic zoning for regional cultivar recommendation. a genotype will be ideal with high yields and ipca1 values near zero. in general, according to the results of ammi analysis zard was the most stable cultivar with high yield and ipca1 values near zero. ‘konservolia’ and ‘sevillano’ had high yield but higher ipc1 values than zero, therefore we recommend them as superior cultivars for pickling purpose. ferreira et al. (2006) reported that an undesirable genotype has low stability as well as low yields. cluster analysis according to the obtained dendrogram from cluster analysis using ward method, genotypes were divided in three groups (fig. 3). this result is confirmed by biplot ammi1 (fig. 2). stability analysis results eberhart and russell’s (1966) stated that a stable cultivar is considered to be the one that has regression coefficient approximating 1.0 and standard error of regression as low as possible. according to this model a genotype with the higher mean fruit yield has general adaptability. in the present research, regression coefficients ranged from 0.02 to 2.11 for fruit yield (table 7). this variation in regression coefficients indicates that cultivars had different responses to year’s fluctuations. a genotype would be adapted to favorable conditions when regression coefficient is higher than one and other would be adapted to unfavorable conditions when regression coefficient is less than one. a genotype with regression coefficient equal to one would have an average adaptation to all environments. according to table 7, ‘amphissis’, ‘mission’ and ‘amigdalolia’ with regression coefficients near to one are most stable all the years. ‘koroneiki’, ‘zard’, ‘manzanillo’, ‘sevillano’ and ‘konservolia’ with regression coefficients higher than one were stable (table 7, fig. 2), while other cultivars like abequina, table 7 mean yields (kg/tree) and various stability measurements and their ranking orders of 12 olive cultivars evaluated during five years 2006-2008 cultivar fruit yield (kg/tree) rank bi rank s2 di rank wi rank δ i 2 rank cv i rank r i 2 rank asv rank amphissis 8.97 9 1.01 6 59.32 11 59.32 9 33.44 9 74.14 11 0.33 3 0.291 4 konservolia 24.69 1 2.11 12 54.93 10 89.87 11 51.77 11 38.57 6 0.7 7 1951 12 zard 14.09 4 1.34 9 0.66 2 3.94 2 0.21 1 36.08 5 0.99 10 0.546 6 amigdalolia 10.78 8 1.08 7 6.27 6 6.44 3 1.71 2 41.14 9 0.84 9 0.032 2 koroneiki 17.94 3 1.21 8 15.34 8 16.62 5 7.81 5 29.78 4 0.73 8 0.184 3 roghani 4.87 12 0.18 3 7.67 7 26.73 8 13.88 8 42.63 10 0.11 2 1345 10 manzanillo 12.89 5 1.57 10 134.4 12 143.68 12 84.05 12 78.49 12 0.34 4 0.5 5 abou-satal 8.02 10 0.16 2 1.22 3 21.27 6 10.61 6 12.33 1 0.37 5 1239 8 mission 12.31 6 0.96 5 0.65 1 0.69 1 1.74 3 29.86 3 0.98 11 0.027 1 arbequina 11.21 7 0.02 1 39.64 9 66.81 10 37.93 10 39.73 8 0.0004 1 1265 9 sevillano 18.81 2 1.92 11 2.13 4 26.21 7 13.57 7 38.88 7 0.98 11 1349 11 shenge 7.96 11 0.43 4 3.57 5 12.85 4 5.56 4 26.36 2 0.59 6 0.937 7 bi = finlay and wilkinson’s (1963) regression coefficient; sdi2 = eberhart and russell’s (1966) deviation from regression parameter; wi = wricke’s (1962) ecovalence; δi 2 = shukla’s (1972) stability variance; cv% = francis and kannenberg’s (1978) coefficient of variability; ri2= coefficient of determination; asv = ammi stability value fig. 3 dendrogram from cluster analysis based on ward method. 522 adv. hort. sci., 2018 32(4): 517-524 shenge, abou-satal and roghani with regression coefficients less than one were unstable (fig. 2). ‘konservolia’ (bi=2.11) was productive during 2006 and 2008 than the others. high yielding varieties were not found stable with regression coefficients (bi). similar results were found by maulión et al. (2014) in peach stability evaluation. as olives have alternate bearing, ‘konservolia’ had the highest fruit yield in non-bearing year (2007) in compare to the others (table 5). the most stable cultivars with the lowest s2 di values were mission and zard. the most unstable cultivarswith the highest s2 di values were manzanillo, amphissis and konservolia. according to the eberhart and russell’s (1966) model, regression coefficients (bi) approximating 1.0 coupled with s2 di of zero indicate an average stability. ‘mission’ and ‘zard’ with regression coefficients near to 1 and s2 di near to zero were most stable than the others. zard cultivar had higher mean yield so it has general adaptability all the years. concept of ecovalence was defined by wricke (1962), where the genotypes with low eco valence have smaller fluctuations across environments and therefore are stable. the most stable cultivars according to the ecovalence method of wricke (1962) were mission and zard. these cultivars were in the ranked 6 and 4 for mean yield, respectively. the most unstable cultivars according the eco valence method were manzanillo and konservolia with the mean yield rank of 5 and 1 respectively (table 7). this method would not be suitable to select high-yielding cultivars but it is useful to select cultivars with the same yield of the mean yield (table 5). for this reason, genotypes with a low wi value have smaller deviations from the mean across years and are thus more stable. shukla’s (1972) stability variance (δi 2) revealed that ‘zard’, ‘amigdalolia’ and ‘mission’ had the smallest variance across the years and were stable, while manzanillo and konservolia cultivars had the largest δi 2 and were unstable. the ‘konservolia’, ranked first for mean yield, showed insteadpoor stability based on shukla’s stability variance. the mean cv analysis was proposed by francis (1977) to study the physiological basis of yield stability. the stable cultivar is the one that provides a high yield performance and consistent low cv (crossa et al., 1990). according to this method, ‘abou-satal’, ‘shenge’, ‘mission’ and ‘koroneiki’ were the most stable; ‘zard’, ‘konservolia’, ‘sevillano’ and ‘arbequina’ were intermediate stable, while amigdalolia, roghani, amphissis and manzanillo were the most unstable cultivars (table 7). moghaddam and dehghanpour (2001) stated that the main problem with this method is that low-yielding cultivars are placed into the category of stable cultivars. in this experiment high yielding varieties were in intermediate parts of classification. a greater coefficient of determination (ri2) value is desired because higher ri2 values indicate favorable responses to environmental changes (sayar et al., 2013). in our study, zard, mission and sevillano cultivars had higher ri2 values for fruit yield and ‘amigdalolia’, ‘koroneiki’, ‘konservolia’ and ‘shenge’ with medium ri2 values have high and medium stability in yield, respectively while others with low ri2 values were unstable cultivars (table 7). according to the asv ranking, the following cultivars were the most stable, mission, amigdalolia and koroneiki, while the most unstable were ‘konservolia’, ‘sevillano’, ‘roghani’, ‘arbequina’ and ‘abou-satal’ . based on yield cluster analysis olive cultivars were classified into three categories. category 1 was cultivars having high yield and medium alternate bearing (‘konservolia’, ‘sevillano’ and ‘koroneiki’) (fig. 3). these cultivars are widely adapted around the world (barranco et al., 2000; therios, 2009). barranco et al. (2000) reported that ‘konservolia’ has a high productivity and alternate bearing but ‘sevillano’ is productive with constant production in mediterranean regions. also, therios (2009) stated that ‘sevillano’ is cultivated in warmer regions in spain and italy without any problems. our results revealed that ‘sevillano’ had relatively constant production during the experiment. koroneiki is one of the most important olive oil cultivar in the greece with high fruit yield and good oil quality (barranco et al., 2000). our results indicated that its productivity was relatively high and constant but oil content (data not presented) was low. category 2 was cultivars having medium yield and medium or high alternate bearing (‘zard’, ‘manzanillo’, ‘mission’, ‘arbequina’ and ‘amigdalolia’) (fig. 3). results showed that ‘arbequina’ had medium productivity with medium alternate bearing. our result was not confirmed by therios (2009) and barranco et al. (2000) findings, where ‘arbequina’ has a high productivity with constant yield and high oil content in the italy. therios (2009) stated that manzanillo is categorized as a good performance olive cultivar in the world. in our research, ‘manzanillo’ had medium productivity with high arji olive fruit stability analysis 523 alternate bearing. mission is a dual-purpose commercial olive cultivars in the world (therios, 2009). mission’s productivity was medium and alternate in our research. amigdalolia is an olive cultivar originated from greece with medium productivity and alternate bearing (barranco et al., 2000). our result represent that this cultivar show medium productivity and alternate bearing. category 3 was cultivars having low yield and low, medium or high alternate bearing (‘abou-satl’, ‘shengeh’, ‘roghani’ and ‘amfissis’) (fig. 3). we do not recommend these cultivars for planting in warm environmental condition. 4. conclusions in conclusion, one of major purpose of yield-trial 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pisa province, especially the area of lari, is one of the most important and famous districts for the sweet cherry production in tuscany (gargani et al., 2013). as with other fruit species, the introduction and diffusion of new varieties of sweet cherry in specialized crop systems has marginalized the local ones to the point that some have disappeared, while others are still present as single plants or in mixed orchards. in those situations, autochthonous and ancient varieties have formed plant populations, resulted from the selective pressure exerted by both natural environment and human cultural practices; importantly, these populations act as a natural reservoir of genetic variability and source of useful genes for the selection of new varieties, for the improvement of the existing ones, with the global objective to guarantee the levels of sustainability and stability of production systems (di matteo et al., 2016). concurrently, autochthonous and ancient varieties could represent source of new agro-economic systems, based on the use of crop residuals, or on an alternative use of the products. globally, during the last decades, worldwide biodiversity has been lost at an unprecedented rate in all the ecosystems, including agro-ecosystems. accordingly, a number of instruments and tools that contribute to a sustainable development while adv. hort. sci., 2017 31(2): 121-129 doi: 10.13128/ahs-21089 morpho-chemical and aroma investigations on autochthonous and highly-prized sweet cherry varieties grown in tuscany c. taiti, s. caparrotta, s. mancuso, e. masi (*) dipartimento di scienze delle produzioni alimentari e dell’ambiente dispaa, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: fruit aroma, morpho-chemical parameters, prunus avium l., ptr-tof-ms, volatile compounds. abstract: the morpho-chemical and aromatic characteristics of four sweet cherry cultivated varieties (prunus avium l.) grown in the area of lari (pisa, central italy) were evaluated with the aim to investigate their properties, mainly concerning volatile organic compounds (vocs). of these, three cultivars (‘di giardino’, ‘di nello’, and ‘marchiana’) represent ancient sweet cherries recovered through a private cultivation program (belonging to the group of the so-called ‘ciliegia di lari’); their evaluation was compared with the commercial cultivar ferrovia, highly-prized variety marketed in italy and abroad. morpho-chemical analyses highlighted statistical differences among the cultivars under study, mainly on total soluble solids (tss) and tritatable acidity (ta). aroma investigation was performed with ptr-tof-ms (proton transfer reaction time of flight mass spectrometer) approach, employed here for the first time in cherry fruits. about 50 vocs were detected; among them, those belonging to the chemical classes of aldehydes and alcohols were the most represented although with different intensities between samples. tentative identification of some key vocs for cherry fruit was also performed and preliminary conclusions on the characterization of ancient and wide spread italian cultivars were given. (*) corresponding author: elisa.masi@unifi.it received for publication 30 may 2017 accepted for publication 19 june 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2017 31(2): 121-129 122 addressing objectives and priorities related to biodiversity have been established at different levels, from global to national ones (fao, 2010); in italy, for example, since 2015, the law n. 194/2015 has been published with the aim to safeguard and enhance the protection of biological resources relevant to food and agriculture from the risks of extinction and genetic erosion. in this context, many studies have been performed to explore plant biodiversity, especially through the investigation of plant and fruit metabolomics. indeed, plants produce a wide range of metabolites, the main part being involved in secondary metabolic pathways; these are the result of different plants responses, through the course of evolution, to specific needs and stimuli. among such metabolites, volatile organic compounds (vocs) play a dominant role (dicke and loreto, 2010), being released by quite any kind of plant tissues (peñuela and llusia, 2001; dudareva et al., 2006) as green leaf volatiles, nitrogen-containing compounds and aromatic compounds. plants vocs can be emitted constitutively (holopainen and gershenzon, 2010; holopainen et al., 2010), or as a consequence of the interactions of plants with biotic and abiotic factors (spinelli et al., 2011). therefore, vocs can be considered as important metabolites for the characterization of biodiversity. accordingly, the study of vocs emitted by fruits, represents a strategic tool for discriminating varieties of the same species growing in the same environment. to this end, the aim of the present study was to investigate the properties, mainly concerning vocs, in fruits of a collection of autochthonous and ancient sweet cherry varieties, and to compare them with those of a commercial variety. 2. materials and methods plant material fruits of four sweet cherry cultivated varieties for fresh consumption (namely: ‘di giardino’, ‘di nello’, ‘marchiana’ and ‘ferrovia’) used in this study were collected at fully ripe grade from ten years old prunus avium l. trees grown in an experimental farm in lari (pisa province, italy), in late may-early june 2017. the genotypes were planted with a spacing of 6.0×4.0 m, trained as spindle-bushes, and managed according to standard cultural practices. yield per tree was comparable among cultivars (around 35-40 tons per hectare). the cherry orchards were located about 130 m above sea level (lat. 43° 33’ 08’’ n, long. 10° 35’ 21’’ e). three of these cultivars (‘di giardino’, ‘di nello’, ‘marchiana’) represent ancient native sweet cherry cultivars recovered through a private cultivation program (belonging to the group of the so-called ‘ciliegia di lari’); instead ‘ferrovia’, highly-prized variety marketed in italy and abroad, has been used as commercial cultivar in order to compare the morpho-chemical and the aromatic characteristics with the native ones. for each cultivar, 1 kg of homogeneous and healthy fruits were harvested randomly from multiple trees at a commercial ripening stage based on color change and fruit firmness. samples were transported to the laboratory in isothermal plastic bags within 2 h from harvesting and stored at 4°c until the analysis were performed (at least 24 h after the sampling). subsequently, the measurements were made in the following order, according to the degree of destruction: fruit skin color, morphological parameters, volatile compounds and chemical parameters. before the measurements, fruit samples were washed in deionized water. morpho-chemical parameters fifteen fruits from each sweet cherry cultivar were used to assess the morphological parameters and the skin color. the total weight (fruit and seed) was determined using a digital balance (sartorius te1502, usa) while their three linear dimensions (length, width and thickness) and the stalk length were measured using a electronic digital caliper (stainless hardened, sensitivity of 0.01 mm). the ripening stage of sweet cherries is characterized by fruit color changing from green to red (diaz-mula et al., 2009). the color characteristics were analyzed on the fruit surface (skin) using a minolta cr-200 chromatometer (minolta, ramsey, nj) and l (lightness), a (green to red) and b (blue to yellow) values were measured. subsequently, a and b values were used to calculate the color index (a/b) since this value shows a continuous increase during sweet cherry un grirororroriroro ripening (díaz-mula et al., 2009). furthermore, hue angle (tan-1 b/a) and the chroma index (√a2 + b2) were assessed as two main parameters used to describe visual color appearance (little, 1975; mclellan et al., 1995). results are reported as mean ± deviation standard. the chemical parameters were determined in triplicate thus each sample was represented by the pulp of three fruits. few drops of fruit juice were used to determine the total soluble solids (tss) with a refractometer (n1 atago co., japan) and expressed taiti et al. aroma investigations on sweet cherry varieties grown in tuscany 123 as °brix. subsequently, the fruit pulp was shredded and blended with 150 ml of deionized water. the obtained solution was filtered and used to measure ph with a digital ph meter (basic20, crison instruments) and titratable acidity (ta) by tritation with 0.1 n naoh up to ph 8.1 and expressed as percentage of malic acid. furthermore, tss/ta ratio has been assessed since this value is linked to the fruit flavor and is one of the main indicator of the fruit quality (alonso and alique, 2006). data are reported as mean ± deviation standard. ptr-tof-ms profiling the volatile profile of four different sweet cherry samples and the tentative identification of each detected compound was evaluated by ptr-tof-ms (model 8000, ionicon gmbh, innsbruck, austria) which guarantees high sensitivity with a very hightime resolution (taiti et al., 2017). a further description of ptr-tof-ms is given by lindinger et al. (1998). the analysis method and instrumental settings were carried out following the procedure previously used by taiti et al. (2016). briefly, each sample consisted of four freshly cut fruits (including the seed). for the analysis, each fruit was cut in 2 parts, inserted in a clear glass jar (3/4 l at 22°c, with a dynamic headspace flushing flow rate of 0.75 l per meter, lpm) equipped with two teflon inlet and outlet tubes on opposite side, connected respectively to a zero-air generator (peak scientific) and the ptr-tof-ms. before each analysis, the jar was cleaned for 1 minute with free vocs air and subsequently was incubated for 80 s. blank measurements were carried out between samples to monitor background air. the analyses were performed in independent triplicates and an averaged mass spectrum per sample was calculated after background and transmission correction (n=10). the mass spectral data (m/z = 20-210) of four sweet cherry cultivars was assessed after the removal from the dataset of masses m/z = 32 (o2 +) and m/z = 37 (water cluster ion), other interfering ions and their isotopologues. the instrument was operated at e/n value of 133 townsend (1 td = 10-17 cm2 v-1 s-1). the chamber ionization conditions were kept as follows: drift temperature 60°c, drift voltage 580 v and drift pressure 3.80 mbar. each sample measurement was performed with an acquisition rate of 1 spectrum/s for 80 s. the raw data were acquired by the tofdaq viewer® software (tofwerk ag, thun, switzerland) and the count losses due to the ion detector dead time were corrected off-line following the methodology based on poisson correction as previously reported by titzmann et al. (2010). moreover, to reach a good mass accuracy (up to 0.001 th), the instrumental calibration was based on m/z = 29.997 (no+), m/z = 59.049 (c3h7o+) and m/z = 137.137 (c10h17 +) and was performed off-line. finally, the vocs identification was based on models of fragmentation available in the literature (buhr et al., 2002; lee et al., 2006; aprea et al., 2007; maleknia et al., 2007; kim et al., 2009; jardine et al., 2010; tani, 2013) and compared with published vocs emitted from sweet cherry fruits (table 1). statistical analysis analysis of mean values and deviation standard, multivariate analysis of variance (anova, p≤0.05), and mean separation by tuckey’s test (p≤0.05) were performed using the statistical package graphpad prism 5.0 software, il, usa). 3. results and discussion as known, cherry fruit quality (expressed as concentration of nutritive and bioactive compounds) is mainly affected by genotype, environment and orchard management (predieri et al . , 2004; gonçalves et al., 2006). in this work all cherry cultivars were obtained from the same farm and all trees were grafted onto the same rootstock, therefore, the differences observed should be attributed almost exclusively to genetic characteristics. morpho-chemical parameters fruit color and size are the main parameters employed to visually evaluate the sweet cherries (romano et al., 2006). the morpho-chemical attributes of the four cherry cultivars are shown in table 2. among comparative cultivars the highest average fruit weight was measured in ‘ferrovia’ (7.24±0.82 g), while all other autochthonous accession showed lower values; furthermore, ‘di giardino’ (5.46±0.65 g) and ‘di nello’ (4.62±0.62 g) were also statistically different compared to ‘ferrovia’ and ‘marchiana’. as far as concerning other morphological parameters, cv. ferrovia confirmed to be the biggest (21.54±1.24, 24.33±0.84, 21.07±0.97 mm for length, width and thickness, respectively), while cv. marchiana was the smallest (15.59±0.99, 18.76±0.82, 15.12±0.73 mm for length, width and thickness respectively). fruits belonging to ‘di giardino’ and ‘di nello’ showed intermediate shape. interestingly, the stalk length was highest for ‘di nello’ which belongs to the category of varieties with ‘medium stalk length’, while the others (less than 39 mm) are cultiadv. hort. sci., 2017 31(2): 121-129 124 vars with ‘short stalk length’ (roselli and mariotti, 1999; fajt et al., 2005). color parameters, especially a/b, hue angle and chroma indices, were comparable to those reported by díaz-mula et al. (2009) for ripe cherry fruits. in this work, based on the hue angle values reported by crisosto et al. (2002), the analyzed varieties showed fruits with color tending to: full l ight red for ‘marchiana’ (25.29±1.22), between full light red and 50% bright red for ‘ferrovia’ (22.72±1.49), between 50% bright red and full dark red for ‘di nello’ and ‘di giardino’ (table 2; see also pictures inserted in figure 1, representing cherry samples for each cultivar under study). the chemical parameters of sweet cherries as total soluble solids (tss) and titratable acidity (ta), as well as indicators of the degree of ripening, are also important quality indexes for cherry cultivars evaluation (crisosto et al., 2003). the results of the chemical parameters are presented in table 2. significant differences were found in tss values especially with regard to cv. di nello (12.94±1.08 °brix) which showed values significantly lower than the other cultivars under study. moreover, the highest values were detected in ‘ferrovia’ (22.77±0.58 °brix) while ‘marchiana’ and ‘di giardino’ showed very similar values to each other (table 2). thus, excluding the cv. di nello, all cultivars analyzed were above the limit of 14-16°brix, considered acceptable for marketing cherries as suggested by crisosto et al. (2003). some differences (p≤0.05) were also found in ta among the sweet cherry cultivars (table 2). the highest average values were found in ‘ferrovia’ (0.90±0.05 as percentage of malic acid per fresh weight) followed by ‘di giardino’ and ‘marchiana’ (0.58±0.05 and 0.64±0.02, respectively), and finally by ‘di nello’ cultivars that showed the lowest values (0.37±0.05). accordingly, ‘ferrovia’ fruits showed the lowest ph value, and ‘di nello’ the highest one (p≤0.05). moreover, since the tss/ta ratio has being related to the consumer acceptance, giving that the sugar concentration increases while acidity remains relatively constant during the maturation or ripening process (spayd et al., 1986), such index can be used as well as a quality parameter in sweet cherry (alonso and alique, 2006). indeed, as it has been observed elsewhere for sweet cherry fruits (crisosto et al., 2003; garcia-montiel et al., 2010) the increase in tss/ta ratio during ripening process is due to the higher increase in tss than the increase in ta. table 1 list of vocs detected in the four sweet cherry samples. chemical classes and references, where available, are indicated m/z protonated chemical formula chemical class (tentative identification) references 27.022 c 2 h 3 + alkyl fragment 30.046 c 2 h 6 + alkene 31.018 ch 3 o+ aldehyde 31.054 c 2 h 7 + alkyl fragment 33.033 ch 5 o+ alcohol 39.022 c 3 h 3 + unknown fragment 41.038 c 3 h 5 + alcohol fragment/ester fragment 43.018 c 2 h 3 o+ ester fragment 43.054 c 3 h 7 + alcohol fragment 45.033 c 2 h 5 o+ aldehyde 1 47.049 c 2 h 7 o+ alcohol 2 51.038 ch 7 o 2 + alcohol 53.040 c 4 h 5 + alkyl fragment 55.055 c 4 h 7 + aldehyde fragment 57.033 c 3 h 5 o+ aldehyde 59.059 c 3 h 7 o+ aldehyde/ketone 3 61.028 c 2 h 5 o+ acid 4 65.038 c 5 h 5 + alkyl fragment 67.054 c 5 h 7 + alkyl fragment 69.033 c 4 h 5 o+ heterocyclic aromatic compound 5 69.069 c 5 h 9 + terpene 1 71.049 c 4 h 7 o+ aldehyde 71.085 c 5 h 11 + alcohol 73.065 c 4 h 9 o+ aldehyde/ketone 6 75.044 c 3 h 7 o 2 + ester 77.040 c 6 h 5 + alkyl fragment 79.049 c 6 h 7 + alkene 81.069 c 6 h 9 + terpene fragment 83.086 c 6 h 11 + alcohol fragment 85.065 c 5 h 9 o+ ald 87.044 c 4 h 7 o 2 + ketone 89.059 c 4 h 9 o 2 + ester 91.054 c 7 h 7 + unknown fragment 93.069 c 7 h 9 + alkyl 6 95.086 c 7 h 11 + unknown fragment 97.064 c 6 h 9 o+ aldehyde 1 99.080 c 6 h 11 o+ aldehyde 1 101.096 c 6 h 13 o+ aldehyde 2 103.075 c 5 h 11 o 2 + acid 4 107.049 c 7 h 7 o+ aldehyde 2 109.101 c 8 h 13 + unknown fragment 115.075 c 6 h 11 o+ acid 4 117.091 c 6 h 13 o 2 + acid 2, 7 119.101 c 9 h 11 + alkyl fragment 121.065 c 8 h 9 o+ aldehyde 6 127.111 c 8 h 15 o+ ketone 7 137.137 c 10 h 17 + terpene 1 refereces legend for data available on cherry fruit: (1) vavoura et al., 2015; (2) serradilla et al., 2012; (3) mattheis et al., 1992; (4) wen et al., 2014; (5) zhang et al., 2007; (6) bernalte et al., 1999; (7) sun et al., 2010. where legend is missing, the vocs identification was based on fragmentation models available in literature (buhr et al., 2002; lee et al., 2006; aprea et al., 2007; maleknia et al., 2007; kim et al., 2009; jardine et al., 2010; tani, 2013). taiti et al. aroma investigations on sweet cherry varieties grown in tuscany 125 specifically, this ratio was highest in ‘di nello’ (32.80±3.47) and lowest in ‘marchiana’ and ‘ferrovia’ (25.22±1.81 and 25.26±1.39, p≤0.05) (table 2). this data suggest that also cv. di nello, that had reported the lowest tss (12.94±1.08 °brix), given the low acidity (0.37±0.05) could be considered as qualitatively comparable to the other cultivars, from the point of view of the fruit maturity. ptr-tof-ms profiling beside fruit sweetness and skin color, aroma is perhaps the most appreciated fruit characteristics (romano et al., 2006). the typical aroma composition of each fruit is affected by their chemical composition (including phytonutrients) as: fatty acids, amino acids, carotenoids, phenols and terpenoids (sun et al., 2010). thus, the fruits aroma is often forfig. 1 vocs profile of the four sweet cherry cultivars under study. representative pictures of each cultivar are also shown. table 2 morpho-chemical proprieties of sweet cherry samples. data represent mean ± deviation standard ‘di giardino' ‘di nello' ‘marchiana' ‘ferrovia' morphological proprieties fruit weight (g) 5.46±0.65 b 4.62±0.62 c 6.68±0.64 a 7.24±0.82 a fruit length (mm) 22.47±0.94 a 20.33±0.92 b 15.59±0.99 c 21.54±1.24 a fruit width (mm) 19.24±0.75 bc 19.83±0.92 b 18.76±0.82 c 24.33±0.84 a fruit thickness (mm) 20.16±1.01 a 18.60±1.21 b 15.12±0.73 c 21.07±0.97 a stalk length (mm) 30.34±3.05 b 41.26±3.92 a 30.79±4.04 b 32.12±3.82 b skin color l 31.26±1.64 b 30.68±1.36 b 33.67±1.53 a 31.09±1.34 b a 13.66±2.85 b 13.36±2.71 b 19.72±2.06 a 13.50±2.99 b b 4.35±2.06 b 4.70±1.79 b 9.36±1.41 a 5.73±1.59 b a/b 2.94±0.51 a 2.69±0.45 ab 2.12±0.11 c 2.40±0.17 bc hue angle (deg) 18.74±2.72 c 20.10±4.22 bc 25.29±1.22 a 22.72 ±1.49 ab chroma 13.13±3.95 b 14.23±2.95 b 21.83±2.45 a 14.67±3.37 b chemical proprieties ph 3.68±0.10 b 4.03±0.10 a 3.47±0.01c 3.52±0.03 c tss (° brix) 16.34±2.57 b 12.94±1.08 c 16.10±0.65 bc 22.77±0.58 a ta 0.58±0.05 b 0.37±0.05 c 0.64±0.02 b 0.90±0.05 a tss/ta ratio 29.39±1.99 ab 32.80±3.47 a 25.22±1.81 b 25.26±1.39 b the letters after the values indicate the significant differences within the same row according to tuckey's test (p≤0.05). adv. hort. sci., 2017 31(2): 121-129 126 mulated by a complex mixture of vocs, whose composition is species-specific and sometimes is varietyspecific (el hadi et al., 2013). it follows that, it is important to identify at first the entire volatile imprint for all cherry types and subsequently investigate which compounds contribute to create the unique aroma/flavor of each sweet cherry, as it represents a fundamental quality parameter used by the consumer. by the ptr-tof-ms analysis 47 peak signals were identified (table 1); among them, a minimum of 40 peaks for ‘ferrovia’ to a maximum of 45 peaks for marchiana cultivar were detected (fig. 1). as a general overview, cultivars under study showed highest intensities for different vocs (fig. 1). in particular, among all samples, the biggest peaks detected, corresponding to vocs with highest intensity, were the following m/z: 27.022, 31.018, 33.033, 41.038, 43.018, 45.033, 57.033, 61.028, 75.044, 81.069, 83.086 (each present in percentages >1 on the total). in respect to each variety, cv. di giardino showed the main peak at m/z = 33.033 (2592 ppbv, on average over replicates), tentatively identified (ti) as methanol, l inked to mild alcoholic odour; cv. marchiana and di nello were rich in m/z = 45.033, ti as acetaldehyde (respectively 1459 ppbv and 816 ppbv, on average over replicates), linked to pungent/fruity odour (this volatile compound was well represented in all samples); cv. ferrovia had the biggest signal at m/z = 33.033, followed by m/z = 57.033 (568 ppbv, on average over replicates), ti as fragment of hexanal and/or hexyl acetate, linked to green leaf odour (fig. 1). in addition, ‘di giardino’ showed high signal intensities for terpene compounds compared to the other varieties used in study (fig. 1a). being monoterpenes (c10) and sesquiterpenes (c15) the compounds that most affect the aromatic profile in some fruits, their presence in ‘di giardino’ determines probably a characteristic aroma. the different terpenes composition in sweet cherry varieties has been also observed by vavoura et al. (2015) that showed, in five varieties, a different composition in types and amount of terpenes compounds. interestingly, peak detected at m/z = 121, ti as methylbenzaldehyde, referred as cherry-like scent (bernalte et al . , 1999), was observed only in ‘marchiana’ (fig. 1c). instead, ‘ferrovia’ showed an interesting peak at m/z = 75.044, ti as methylacetate linked to ether sweet fruity odour (fig. 1d). concerning the cultivar di nello, it is worth noting that intensities of vocs related to green leaf odour (m/z = 57, 81, 99 and 101) were inferior in respect to the other sample varieties (fig. 1b). as reported by sun et al. (2010), the aroma compounds in sweet cherry is determined by a great number of organic components, especially aldehydes, alcohols, esters, acids and terpenes. hence, each peak detected and tentatively identified was then clustered according to the chemical class (table 1). this allowed to drawing some further interesting consideration and to better appreciate the differences between aroma profiles of sweet cherry fruits belonging to different cultivars (fig. 2). aldehydes are the most abundant class of vocs (in percentage) in all cherry samples (73%, 66%, and 63% respectively in ‘marchiana’, ‘ferrovia’ and ‘di nello’, fig. 2b,c,d), excluding ‘di giardino’ where alcohols were the biggest class (47%, fig. 2a). among aldehydes, the peaks detected at m/z = 45, 57, 59, 99, 101 are linked to compounds known to be among the most important aroma compounds of sweet cherry fruit (mattheis et al., 1992; wen et al., 2014; vavoura et al., 2015). in contrast to the results of vavoura et al. (2015) but according to serradilla et al. (2012), 2-propanone (m/z = 59) was not the most abundant compound identified, although it has been detected in all cherry cultivars under study. on the contrary, the compounds known as ‘green leaf volatiles’ such as 2-hexenal (m/z = 99.080), hexanal (m/z = 101.096) and their main fragment (m/z = 57.033) were found. all of them, even in very small quantities as hexanal, give a strong odour due to their low perception threshold (matsui, 2006). besides aldehydes, alcohols revealed to be abundant in all cherry samples (fig. 2), and in fact they represent important compounds for the aroma in sweet cherry (sun et al., 2010). in this study, methanol (m/z = 33), ethanol (m/z = 47) and propanol (m/z = 61) were highlighted. such alcohol compounds are precursors of natural aroma and appear as a result of anaerobic respiration in fruits and are linked to the normal maturation process (taiti et al., 2015). in addition, both autochthonous and commercial cherries analyzed were rich in ethanol, which most probably derives from carbohydrates metabolism (glycolysis) (mattheis et al., 1992). these data also suggest that the intensity of ethanol emission is at least partially associated to tss content; in fact, the lowest intensity of ethanol was found in ‘di nello’ (4.05±1.40 ppbv), the cultivar with lowest tss (table 2), while the highest was found in ‘ferrovia’ (10.2±1.60 ppbv), the cultivar richest in tss (table 2); similarly, the other two varieties, ‘di taiti et al. aroma investigations on sweet cherry varieties grown in tuscany 127 giardino’ and ‘marchiana’, had similar ethanol content, at intermediate intensity in respect to ‘di nello’ and ‘ferrovia’, according to tss data (table 2). as for the determination of acids, a total of four acids were detected, acetic (m/z = 59), 3-methylbutanoic (m/z = 103), hexenoic (m/z = 115) and (e)-2hexanoic acid (m/z = 117), confirming results reported by sun et al. (2010). in our cherry samples, ‘ferrovia’ showed the highest percentage (6%) of this class of compounds (fig. 2d). finally, esters were found in all cherry cultivars (fig. 2) but in low concentrations (especially for ‘marchiana’) (fig. 2c). for example ethyl acetate (m/z 89.059), that is identified as impact volatile in sweet cherry fruits (zhang et al., 2007) and is associated to pineapple aroma, showed the highest emission level in ‘ferrovia’ (3.73±0.83 ppbv), followed by ‘di giardino’ (1.67±0.77 ppbv), ‘di nello’ (0.91±0.15 ppbv) and finally by ‘marchiana’ (0.16±0.08) with values below 1 ppbv. 4. conclusions in this study, fruit properties of a collection of autochthonous and ancient sweet cherry varieties (prunus avium l.) were investigated and compared to the properties of a commercial variety. morpho-chemical analyses showed statistical differences among the cultivars, the main ones found on fruit total soluble solids (tss) and acidity. concerning tss, two of the authoctonous cultivars investigated, as well as the commercial one ‘ferrovia’, resulted acceptable for the market (tss above the limit of 14-16 °brix, crisosto et al., 2003). fruits from the cultivar di nello, on the other hand, despite they turned out to be not so rich in sugars, showed the biggest tss/ta ratio, another important index related to fruit quality and acceptance (alonso and alique, 2006). concerning the analysis of aromatic compounds, the use of ptr-tof-ms (the first in our knowledge) allowed to distinguish each cultivar’s peculiarities. among autochthonous cultivars, ‘di nello’ was the weakest in emitting green leaf related odours; ‘di giardino’ showed to be rich in the chemical classes of terpenes, which most affect the aromatic profile in some fruits; methylbenzaldehyde (cherry scent) was detected only in ‘marchiana’. such results would suggest potentiality interest for all these autochthonous varieties, not only for commercial purposes, but also for breeding ones. in conclusion, this study on sweet cherry fruits provides preliminary information that can be useful tools for the enhancement and utilization of native cultivars. acknowledgements this study was supported by funds of the regione toscana ‘‘praf 2012–2015 misura 1.2 e)’’ program fig. 2 pie charts of the vocs emitted by the four sweet cherry cultivars under study, expressed as chemical classes according to table 1. adv. hort. sci., 2017 31(2): 121-129 128 (call ‘agrifood’, project volatosca). we gratefully acknowledge the ‘azienda agricola mastrociliegia’ (lari, pisa, italy) in the person of dr. leonardo puccioni for his technical support and as provider of cherry samples. references alonso j., alique r., 2006 sweet cherries, pp. 359-367. in: hui y.h. 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soil and containers for urban agriculture in developing areas a. pardini 1 (*), massolino f. 2, grassi c. 1 1 dipartimento di scienze delle produzioni alimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 ....missing.... key words: multipurpose species, prickly pear, strategic food species, urban agriculture. abstract: urbanization and poverty have brought to worse life conditions in towns of many developing countries, including difficult availability of food, especially fresh. urban agriculture and horticulture can contribute to the availability of fresh foods, officinal and medicinal plants, but the little availability of irrigation and surface to destine for cropping suggest the convenience of little water consuming species, with little needs of soil fertility and that can be eaten entirely. opuntia fiscus-indica (l.) mill. corresponds at all these requirements, and it is a very promising strategic species that can be eaten completely (green parts, fruits and even flowers), it has good nutritional values and also interesting medical properties. a trial has been done to compare the initial productivity of cladodes multiplied in pots, car tires and open field. our results suggest that the prickly pear can be cropped better in large exhausted tires than in small plots also saving money for the materials. 1. introduction urbanization and poverty cause food insufficiency in many tropical and subtropical countries and this problem is enhanced in towns and urban peripheries where land plots to crop are few and small and there is irregular availability of foods in local markets. a few examples: accra (ghana) is a growing town that loses about 2.600 hectares of farm land every year to buildings and consequently reduces agricultural land availability nearby and into the town. the peripheral agriculture in hanoi (vietnam) produces at least 150.000 tons of fruit and vegetables per year. in cuba urban and peripheral agriculture gives around 60 percent of horticultural produce consumption in large towns. still in cuba due to the constraints caused by trade embargo, a growing percentage of the agricultural production is provided by urban agriculture: in 2002 more than 14.000 ha of urban yards produced 3.100.000 tons of food, and 90% of the habana fresh produce comes from local urban farms. in 2003 more (*) corresponding author: andrea.pardini@unifi.it citation: pardini a., massolino m., grassi c., 2017 opuntia ficus-indica (l.) mill. growing in soil and containers for urban agriculture in developing areas. adv. hort. sci., 31(4): xxx-xxx copyright: © 2017 pardini a., massolino m., grassi c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 may 2017 accepted for publication 22 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): xxx-xxx 192 than 200.000 cubans worked in the expanding urban agriculture sector (cuban ministry of agriculture, 2013). in kinshasa (democratic republic of the congo), urban agriculture is producing an estimated 75.000 to 85.000 tons of vegetables per year that is 65% of the town supply. agriculture is being pushed far from many towns, with increasing costs of food transport, packing and conservation enhanced by the bad conditions of rural roads, and heavy losses in transit. in these areas, growing fruit and vegetables in and around cities increases the supply of fresh produce and improves the economic access to food for poor people (fao 2015) that spend 60-80% of their income on food. urban families may actually grow crops or raise small animals, and so produce some of their own food (cohen and garret, 2009). moreover many foods available in towns and peripheries of developing countries are introduced with international aid and consequently they are a constraint to food sovereignty. opuntia ficus-indica is a wild plant and a cultivated crop, whose value is largely underestimated. its green parts are commonly used in environmental protection against soil erosion and as forage (mulas and mulas, 2004), the biomass is used for biogas (rosato, 2014), industrial sectors use prickly pear fruits and leaves as raw materials for cosmetics, drinks and food additives (saenz et al., 2013). its flowers are useful to honey bees, but all its above ground parts are also good meals in form of fruits, fresh and cooked salads, soups, refreshing beverages, flour for breads and pastas. fruits and cladodes are very nutritional (saenz et al., 2013; rodriguez et al., 1996), and their use a human functional food dates back to pre-columbian times (ramirez et al., 2010). especially young cladodes (nopalitos in latin america) conserve water that is very useful in hot dry areas for both humans and livestock, around 9% crude protein, low fats (just around 1%), several minerals especially potassium (220 mg per 100 grams), calcium (16-33 mg), phosphorus (13-28 mg), and vitamins especially c vitamin (ascorbic acid 40 mg per 100 grams), with the best nutritive values being in the young cladodes (retamal et al., 1987). the seeds are commonly considered a waste of the food industry however the extraction of oil is under study for applications in food, pharmaceutical and cosmetic industries (de wit et al., 2017). calories content vary within 25 and 50 kcal per 100 grams dry which is comparable to the most common fruits, moreover it has important pharmaceutical effects including antioxidants and also anti-cancerogenic effects (livrea and tesoriere, 2006). for all this uses the prickly pear has to be considered a multipurpose species quite useful also in many agroforestry systems. opuntia ficus-indica is a species of the cactaceae with cam metabolism that favors water conservation, even if young cladodes have a c 3 metabolic pathway (mulas and mulas, 2004) that causes water recall from lower-older cladodes during the day reducing water availability for the whole plant on benefit of the growth of younger parts (wang et al., 1997). opuntia ficus-indica is a wild plant native of mexican deserts, with very little needs of soil fertility and capable of growth even without any management. few parasites treat this plant. all this makes this crop possible in most tropical-subtropical areas with enhanced hot and dry season, with poor soils and little management. it can be cropped also in permaculture and with this techniques it becomes an additional strategic plant useful as disasters relief crop beside short cycle cereals, beans and vegetables, after people displacement due to earthquakes (see in haiti capital town), hurricanes (as common in caribbean islands like cuba), or after war situations (like recently happened in somaliland, iraq, libya) when the whole national or regional agro-food system has to be restarted. one of the problems related to prickly pear food harvesting and processing is related to the spines or glochids (hichy hair) that can be removed by immersion in water and mechanical treatment or fire burning. however there are spineless varieties (var. inermis) commonly used for forage, and also varieties almost without glochids that can be handled safely and make easy harvesting and processing, even if the absence of defensive parts make the plants more sensible to animal predation. due to the good nutritional values, the little water and soil needs and the easy management, prickly pears can be advised as a strategic crop for urban and peripheral areas with difficult climate or poor soil, as a green fence and also cropped in pots and exhausted tires as common in many towns of tropical developing countries. the productivity of prickly pear has been studied for field production or in greenhouse but still oriented to later in field transplanting. researches have been done about how cladode size, their position at planting can influence initial growth and production (bakali et al., 2016) and has been suggested that pardini et al. opuntia ficus-indica growing in soil and containers for urban agriculture 193 horizontal planting is the best technique in arid regions, and that cladode size influence early dry matter production. the same authors proved that cladode orientation to the sun, and the planting depth had not significant effects on early production. however singh and vijai singh (2003) suggest that planting vertical is generally the best technique together with using cladodes 12 months old, and planting in spring. the effects of planting cladode parts instead of whole has been studied by stambouli-essasi et al. (2015), that suggest the possibility to use just parts in order to save planting material. concerning plant densities in open field, ruiz-espinosa et al. (2008), suggest plantations of 60.000 plants per hectare. concerning different clones, several authors have got diversified results in different environments, thus suggesting a relationship clone-environment (flores, 1992; floreshernandez et al., 2004; ruiz-espinosa et al., 2008). although there are few data available on opuntia cultivation in containers, these are not intended for food production. our research is oriented to the production of a strategic food plant and has compared the productivity of prickly pear cladodes grown in soil (as the case of green fencing) or in pots (the case of urban agriculture in balconies) and in exhausted tires (as common in backyards, small plots or balconies of urban peripheries of many developing countries). 2. materials and methods the trial was done on a spineless cultivar of opuntia ficus-indica (l.) mill. without glochids imported from cuba in the winter 2011, “milpa alta” that is the most cultivated in mexico (gallegosvasquez and mondragon-jacobo, 2011), it is an erected cultivar with elliptic cladodes, spineless and practically without glochids, with yellow fruits. the introduced parts have been multiplied for four years in order to have the necessary number of green parts, and then planted for the trial in central italy (florence hills, 250 m asl, exposed to south) on the first of may 2015 and then again on may 2016. the location has 750 mm average annual rainfall with 159 during the 90 summer days, 14.5c° average annual temperature, in the period 1971-2000 there was an average of 62 days with maximum temperatures above 30c°. the soils of the trial location are mainly clayey (45% clay). the initial plantation has been done using only young cladodes produced by mother plants the summer before (august 2014), planted after winter on 1st may 2015, thus 8 months old. a second plantation was done in 2016 after winter on 1st may, starting from cladodes produced by mother plants in the summer before (august 2015), thus also these 8 months old. all the cladodes used for first and second planting had similar size, 5-7 cm width and 20-25 cm length in order to have very similar conditions of all plants at the beginning. each cladode was planted in a pot or in a tire or straight in the soil. in extensive soil they were planted in three single lines at distances of 50 cm on the line, simulating a soil area available similar to what they have in plots or tires. pots were 20 cm large x 30 cm deep that is a size quite common in local canned food tins that are used as flower pots, exhausted tires were borrowed all the same size 70 cm diameter (45 inner) x 23 cm width that is a 4wd type very common in developing countries. one pot has been got from each tire. tires were put on cemented area to avoid rooting in external soil. the tires were brought back to the giver after the end of the trial for ecological elimination by existing regulations. pots and tires were filled with the same soil were cladodes were also planted straight. pots and tires were disposed in three lines alternated with the three lines of plants planted in the soil, and mixed in a randomized block design. treatments were not irrigated, in the attempt to reproduce conditions of absence or minimal management, common in strategic crops in harsh areas including home yards, balconies and peripheral areas where the water available serves mainly for human needs. thus we got three treatments: 1) extensive soil, 2) pots, 3) tires. each treatment was implemented with 8 initial cladodes (8 repetitions), for a total of 24 plants per year. measurements were done at the end of the growing season (30 september 2015 and 30 september 2016 for all treatments) on: total number of new cladodes originated from each planted (counting); fresh weight of new cladodes (harvesting all new cladodes and weighing fresh with scale), the fresh weight has been considered because this food is eaten fresh (like pineapple or papaya, as examples); dry weight (after fresh weighting, all new cladodes were split in two halves to favor dehydration and oven dried for 5 days at 80°c, then weighted again to calculate the percentage of dry matter). adv. hort. sci., 2017 31(4): xxx-xxx 194 fruits cannot be produced in the trial location during one growth season because it is too short. statistical analysis was done with lsd at p= 0.05. 3. results and discussion number of new cladodes the number of new cladodes (table 1) was the highest in extensive soil (6 in the average of the two years) and the least in pots (2.5 in the average). obviously roots explore a larger area of extensive soil than in pots or tires and consequently have higher water and nutrients availability, moreover soil temperature in pots and tires (that are black) rises more than in the soil and this in turn causes higher evaporation and reduces water availability. this somehow contrasts with the ability of a container to conserve water better than extensive soil, but this doesn’t happen in full summer when high temperatures cause strong evaporation. tires are larger than pots and probably have given intermediate yields because there is more soil than in pots, small plots have brought the plants to stressed conditions very rapidly whilst tires have provided better conditions for some longer time. the higher number of cladodes in the second year (5.0 in the average of treatments, in comparison to 3.7 of the first year) can be due to warmer temperatures that persisted up to the end of the summer 2016 whilst several cool and some rainy days happened in the year before. fresh weight of new cladodes the fresh weight (table 2) was higher in the extensive soil (778 grams in the average of two years) than in tires (508.5 grams) and in pots (only 172 grams). the higher yield in 2016 (554.7 grams in the average of all treatments) than in 2015 (417.7) is probably due to warmer summer temperatures of 2016 in comparison to 2015. dry weight of new cladodes and percentage of dry matter the percentage of dry matter (table 3) was higher in 2016 (14% in the average of all treatments) than in 2015 (12%) probably because the warmer temperatures of 2016 favored evaporation whilst the cooler days and some rains increased water content in 2015. the total dry matter yield was similarly higher in 2016 (75.4 grams in the average of all treatments) than in the year before (50.9 grams). the highest yield was got in 2016 in extensive soil (94.4 grams in the average of the two years, with a maximum of 114 grams in 2016) and the lowest was got in pots (24,3 grams in the average of the two years, with a minimum of 19.5 grams in 2015). 4. conclusions most households in towns, especially in developing countries, have not soil available and must rely on small and cheap containers for gardening, under this point of view, tires not only are cheaper then pots and they are easily available in many tropical towns, but they also provide better growing conditions for opuntia ficus-indica than commercial pots. of course extensive soil is a better condition for table 1 number of new cladodes in the three treatments in the two years of trial and average table 2 fresh weight (grams) of new cladodes in the three treatments in the two years of trial and average different letters show significantly different values at p=0.05. table 3 dry weight (dw, grams) and dry matter percentage (dm, %) in new cladodes in the three trial treatments in the two years of trial and average different letters show significantly different values at p=0.05. treatment number of new cladodes 2015 2016 average extensive soil 5 a 7 a 6.0 pot 2 c 3 c 2.5 tire 4 b 5 b 4.5 average 3.7 5.0 4.3 treatment fresh weight of new cladodes (grams) 2015 2016 average extensive soil 679 a 877 a 778.0 pot 150 c 194 c 172.0 tire 424 b 593 b 508.5 average not irrigated 417.7 554.7 482.2 treatment dry weight of new cladodes 2015 2016 average dw(g) dm(%) dw(g) dm(%) dw(g) dm(%) extensive soil 74.7 a 11 b 114.0 a 13 b 94.4 12.0 pot 19.5 c 13 a 29.1 c 15 a 24.3 14.0 tire 50.9 b 12 ab 83.0 b 14 ab 67.0 13.0 average not irrigated 48.4 12.0 75.4 14.0 61.9 13.0 pardini et al. opuntia ficus-indica growing in soil and containers for urban agriculture 195 growing prickly pears than into containers, at least referring to water needs and taking into account that in extensive soil problems can rise from weeds competition more than in pots or tires. the production in our trial in italy has not been much because of the short warm season (4 months, with only three having summer temperatures) and because we planted cladodes not yet rooted. we can reasonably consider that production would be much higher in the tropics and subtropics where temperatures are good all the year through, using, already rooted plants. the production could be increased much if some waste water can be used in the household for irrigation. the yield of new cladodes can integrate the diet and be a useful supply for a small family during the worst part of a dry season of 3-4 months, can be also a source of herbal medicine extremely useful to families. although the limiting climatic conditions of our trial, the results maintains their value of comparison within treatments also for tropical countries. a couple of issues to be investigated are whether food production in tires is economically more convenient than transforming them into handmade items such as sandals (it is a common practice), and check food quality for eventual absorption of toxic contaminants from exhausted tires into edible plants. finally, a next trial on the use of prickly pear as a strategic plant, could be to start growing cladodes in tires or pots and when grown use some of these to plant green fences. the use of prickly pear for green fences can also be advised in order to shift the common use of planting the poisonous euphorbia spp. (usually euphorbia trigona) that has been much diffused in many and large areas because its spines 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(cactaceae) growing under greenhouse hydroponics system. journal of food, agriculture and environment, 8(3-4): 660-665. retamal n., durán j.m., fernández j., 1987 seasonal variations of chemical composition in prickly pear (opuntia ficus-indica (l.) miller). journal of the science of food and agriculture, 38(4): 303-311. rodriguez s., orphee c., macias s., generoso s., gomez-garcia l., 1996 tuna: propriedades fisicoquimicas de dos variedades . la alimentacion latinoamericana, 210: 34-37. rosato m.a., 2014 il fico d’india: una biomassa trascurata. www.agronotizie.imagelinenetwork.com. web accessed 18 november 2016. ruiz, f., alvarado, j., murillo, b., garcía, j., pargas, r., duarte, j., beltrán, a., fenech, l., 2008 rendimiento y crecimiento de nopalitos de cultivares de nopal (opuntia ficus-indica) bajo diferentes densidades de plantación. yield and growth of green cladodes of prickly pear (opuntia ficus-indica) cultivars under different plant densities. -j. pacd: 22-35. adv. hort. sci., 2017 31(4): xxx-xxx 196 saenz c., berger h., rodriguez-felix a., galletti l., corrales garcia j., sepulveda e., varnero m.t., de cortiazar v.g., arias e., mondragon c., higuera i., rosell c., 2013 agro-industrial utilization of cactus pear. fao, rome, pp. 150. singh r.s., vijai singh, 2003 growth and development influenced by size, age, and planting methods of cladodes in cactus pear (opuntia ficus-indica (l.) mill.). j. pacd: 47-54. stambouli-essassi s., harrabi r., bouzid s., harzallah-skhiri f. 2015 evaluation of the efficiency of opuntia ficus-indica cladode cuttings for vegetative multiplication. not. bot. horti. agrobo., 43(2): 521527. wang n., zhang h., nobel p.s., 1997 phloem-xylem water flow in developing cladodes of opuntia ficus-indica during sink to source transition. j. exp. bot., 48: 675-682. impaginato 151 adv. hort. sci., 2017 31(3): 151-156 doi: 10.13128/ahs-21953 tuber yield and processing traits of potato advanced selections s. melito 1 (*), v. d’amelia 2, r. garramone 2, c. villano 2, d. carputo 2 1 dipartimento di agraria, università di sassari, via enrico de nicola, 07100 sassari, italy. 2 dipartimento di scienze agrarie, università di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: breeding, chipping ability, solanum tuberosum, tuber specific gravity. abstract: world potato production continuously requires new cultivars to satisfy farmers’ and consumers’ demand. tuber yield and quality are some of the main potato breeding targets. in this study, 27 advanced potato clones from 7 hybrid families were evaluated for yield, tuber specific gravity and chipping ability. variability in tuber yield was found between families as well as between clones. forty-eight percent of clones showed higher productivity compared to the best control (agria, 1.1 kg). families displayed significant differences also in terms of tubers specific gravity, with about 70% of clones with a score higher than 1.080, which was considered the minimum acceptable value for processing. chipping ability was evaluated at harvesting time and after cold storage with and without reconditioning. the majority of studied clones showed a good chipping ability score (<4.5) at harvest; five samples chipped well after cold storage with reconditioning, while good chippers were not identified after cold storage without reconditioning. the use of an arbitrary index calculated for each clone is proposed to assist the selection of materials with a good trait combination. 1. introduction potato, solanum tuberosum, is the most cultivated not cereal crop in the world, ranking fourth after rice, wheat and corn (fao statistics, 2012). it represents an important component of human diet, because tubers are able to supply several nutrients, such as essential amino acids, vitamins (as vitamin c) and minerals. it can be consumed either as fresh product or as processed ready-to-eat food (i.e. chips and french fries) (carputo et al., 2005). overall, the number of potato varieties cultivated for processing purposes is increasing and more than 50% of potato yield is driven to food industries. in order to follow the food market trends, new varieties with a specific combination of processing-related traits are continuously requested. a traditional potato breeding program begins with sexual hybridization of tetraploid varieties or élite clones, in order to generate biparental families. progenies are then evaluated for quality traits and the (*) corresponding author: smelito@uniss.it citation: melito s., d’amelia v., garramone r., villano c., carputo d., 2017 tuber yield and processing traits of potato advanced selections. adv. hort. sci., 31(3): 151-156. copyright: © 2017 melito s., d’amelia v., garramone r., villano c., carputo d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/ index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 january 2017 accepted for publication 5 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 151-156 152 most promising clones are then selected for further evaluations (mackay, 2005; rak and palta, 2015; melito et al., 2017). because of the autotetraploid nature of s. tuberosum and its heterozigosity level, the f1 presents trait segregation; as a consequence, vegetative propagation and field selection are needed for several years to identify superior clones (haynes et al., 2012). tuber traits considered by breeders in developing new varieties are different. among them, tuber yield and tuber quality characteristics, including both external (skin color, tuber size and shape, eye depth) and internal (dry matter content, chip ability) traits (carputo and frusciante, 2011) are deeply evaluated. external traits play an important role in potato fresh market, because they influence consumers’ choice. on the counterpart, internal traits mainly affect tuber processing. one of the principal goals of potato breeding programs is the selection of varieties with a good chipping ability following cold storage. indeed, following harvest, potato are normally stored at low temperatures (<8°c) to keep an interrupted supply used by the processing industries, to prevent sprouting, to reduce bacteria soft rot attack and to contain the loss of dry matter (malone et al., 2006; zhao et al., 2013). unfortunately, in response to cold storage most potato varieties convert tuber starch to reducing sugars (glucose and fructose). this reaction is known as “cold-induced sweetening” and is recognized as a serious problem for potato processing industry (dale and bradshaw, 2003). the accumulated reducing sugars undergo the non enzymatic maillard reaction with free amino acids when potatoes are fried in hot oil, resulting in dark unacceptable chips. furthermore, mottram et al. (2002) and stadler et al. (2002) reported high level of acrylamide because of the maillard reaction, which is potentially damaging for human health. equally important for the potato processing industry is the availability of varieties with high tuber specific gravity. tubers with specific gravity higher than 1.080 are generally considered suitable for processing. the main aim of this study was to test the performance of 27 potato advanced clones belonging to 7 families with different genetic background. tuber yield, specific gravity, chipping ability and earliness were determined to select the most promising clones. finally, an evaluation index useful for selection of genotypes with a good combination of traits was calculated. 2. materials and methods genetic material and experimental design the material used in this study derived from a conventional program of clonal selection started in 2009 in a single hill plot. from 2010 to 2013 selected clones were cultivated in larger unreplicated plots with spaced plants. clones with undesired characteristics (i.e. long stolons, deep tuber eyes and tuber defects) were discarded and the number of seed tubers was increased. three commercial varieties (spunta, adora and agria,) were used as control. the field trials were conducted at marigliano (district of naples) (lat. 40.927759°, long. 14.451370°). tuber production and quality evaluation control varieties and experimental clones were planted in a randomized complete block design with 3 replications. for each clone, 10 tubers were planted in a single row with spacing of 30 cm between plants and 70 cm between rows. plants were grown following the standard cultural practices of the geographic area. tubers were planted in march and harvested in july, when plants start senescing (roughly 120 days after plantation). total yield (ty) was evaluated at harvest. chips were produced by frying 10 longitudinally cut tuber slices from the center of each tuber and 2 tubers for each clone were used. the chips color was evaluated in 3 consequent times: at harvest, at three months of cold storage (7°c) and at 2 weeks of reconditioning (20 24°c) after cold storage. to optimize the chipping test, slices were washed in water before frying in soybean oil. tubers were considered completely fried when oil end to bubble. a colorimetric scale, from 1 (very light) to 10 (very dark) was used to determinate chipping ability. based on carputo et al. (2002), clones with a score ≤ 4.5 were considered suitable for chipping. the specific gravity of tubers (tsg) of each clone was estimated on 1 kg of tuber sample, evaluating the ratio weight in air/weight in water (woolfe, 1987). foliage earliness was evaluated at 90 days after planting. an earliness score (from 1= very late to 5= very early) was associated to each clone comparing foliage senescence to the control variety spunta (earliness score= 3). evaluation index to assist the selection of clones, an evaluation index (ei) was elaborated, associating an arbitrary score to each trait based on the value obtained: ty melito et al. tuber yield and processing traits of potato advanced selections 153 (kg/plant): 1= <0.5; 2= 0.51-1 ; 3= 1.1-1.5; 4= 1.51-2 ; 5= >2.1. tsg: 1= ≤1.080 (not suitable for processing); 2= 1.081-1.085; 3= 1.086-1.090; 4= >1.091. chipping color: 1= >4.5 at each test after cold storage; 2= <4.5 at least in one test after cold storage; 3= <4.5 at both tests after cold storage. average earliness score: 1= ≤1.0; 2= 1.1-2.0; 3= 2.13.0; 4=> 3.1. the ei was calculated summing the scores for each trait: the higher index values, the more desirable genotypes. the ei was calculated only for clones for which all the evaluation data were available. statistical analysis one-way anova was run using jmp 7 software (sas institute, cary, nc, usa). when a significant f was found (p<0.05), data were compared using tukey’s post hoc multiple comparison test. each trait was used to compare the mean values among clones and varieties and to varieties individually. 3. results and discussion the development of new potato varieties addressing production efficiency and sustainability requires a well-planned breeding program. the accumulation of multiple traits in a single variety is one of the main goal of potato breeding programs. among the available different strategies, those based on sexual hybridization between tetraploid varieties or clones, followed by selection, still represent a successfully and widely used option. in this research, 27 advanced clones were selected and evaluated for yield and processing traits. they belong to seven families obtained from crosses involving ten cultivated varieties (spunta, victoria, jenny, blondy, agria, bolesta, sandy, majestic, alcmaria, primura,) and one breeding clone (mc 329). as reported in table 1, clones under evaluation showed differences in terms of flesh color and tuber shape, whereas skin color, eye table 1 tuber characteristics of the potato clones under selection. for each clone, pedigree, tuber skin color (tsc), tuber flesh color (tfc), tuber shape (ts), eye and stolon characteristics are reported. families pedigree tsc tfc ts eyes stolons ‘spunta’ x ‘victoria’ s04-2-10 yellow yellow oblong superficial short s04-2-17 yellow yellow round superficial short s04-2-18 yellow yellow long superficial short s04-2-28 yellow yellow oblong superficial short s04-2-34 yellow yellow oblong superficial short s04-2-40 yellow yellow oblong superficial short s04-2-53 yellow yellow long superficial short s04-2-55 yellow yellow long superficial short s05-7-15 yellow light yellow oblong superficial short s05-7-4 yellow yellow long superficial short ‘jenny’ x ‘mc 329’ s04-7-2 yellow white round superficial short s04-7-6 yellow white round superficial short s04-7-27 yellow white round superficial short ‘blondy’ x ‘victoria’ s05-1-2 yellow yellow long superficial short s05-1-25 yellow white long superficial short ‘bolesta’ x ‘mc 329’ s05-2-10 yellow white round superficial short s05-2-11 yellow light yellow round superficial short s05-2-15 yellow yellow round hallowed short s05-2-18 yellow yellow round superficial short s05-2-23 yellow yellow round superficial short s05-2-3 yellow yellow round superficial short s05-2-4 yellow yellow oblong superficial short s05-8-5 yellow yellow round superficial short ‘agria’ x ‘sandy’ s04-5-32 yellow yellow round superficial short ‘majestic’ x ‘alcmaria’ s04-6-2 yellow white oblong superficial short ‘primura’ x ‘alcmaria’ s05-4-2 yellow yellow oblong superficial short solanum tuberosum adora light yellow yellow round superficial short agria yellow yellow oblong superficial short spunta light yellow yellow oblong superficial short adv. hort. sci., 2017 31(3): 151-156 154 depth and stolon length were uniform. this is the result of the previous selection pressure aimed at discarding clones with undesired characteristics (e.g. deep eyes). tuber yield, specific gravity, and chipping ability of material under evaluation are reported in table 2. data are summarized on a family basis. overall, significant differences in ty were found among families. the average ty was 1.2 kg/plant, ranging from 0.43 to 3.00 kg, detected in a clone from ‘spunta’ x ‘victoria’ and ‘majestic’ x ‘alcmaria’, respectively. by contrast, the comparison between the mean ty of clones and that of control varieties revealed not significant difference (table 3). however, analysis of ranges within each family revealed the presence of clones with good yield performances. in this research, 12 very promising clones for tuber yield were identified, belonging to ‘spunta’ x ‘victoria’ (5), ‘majestic’ x ‘alcmaria’ (1), ‘jenny’ x ‘mc 329’ (1), ‘blondy’ x ‘victoria’ (1), ‘bolestra’ x ‘mc 329’ (4) (not shown). this may outline the occurrence of allelic combinations providing satisfactory diversity, a prerequisite for heterosis in yield (mendoza and haynes, 1974). potato quality evaluation does not include only tuber yield, but also several qualitative and quantitative traits. among them, tuber dry matter and chipping ability are fundamental for processing. in particular, the chipping ability is a quality parameter highly important for food industries because it influences not only yield of the processed product, but also oil absorption rate in fried products (asmamaw et al., 2010). on the other hand, tuber specific gravity (tsg) is commonly accepted as measure of the dry matter content and it provides the suitability of potato varieties for processing (kabira and berga, 2003). based on this finding, fitzpatrick et al., (1964) identified three classes of tsg: low (less than 1.077), intermediate (between 1.077 and 1.086), and high (more than 1.086). furthermore, kabira and berga (2003) reported that tuber should have a specific gravity higher table 2 tuber yield (kg of tubers per plant) (ty), tuber specific gravity (tsg), chip category color and evaluation index (ei) of 27 potato advanced clones. chip category color was evaluated at harvest and after 90 days of cold storage at 7°c, with and without reconditioning at room temperature for two weeks (respectively + rec; rec) (see materials and methods). for each trait, the average family value (range) is reported material no. of clones ty * tsg * chip category colour * ei * direct cold storage rec cold storage + rec families ‘spunta’ x ‘victoria’ 10 1.09 (0.43-1.58) b 1.081 (1.073-1.088) bc 3.8 (2.0-7.0) 8.5 (7.0-10.0) b 6.1 (2.0-7.0) abc 10.1 (6.0-14.0) c ‘jenny’ x ‘mc 329’ 3 0.95 (0.48-1.51) b 1.095 (1.086-1.104) a 2.0 (2.0-2.0) 10.0 (10.0-10.0 a 4.3 (2.0-7.0) bc 13.0 (11.0-15.0) ab ‘blondy’ x ‘victoria’ 2 1.20 (1.13-1.28) b 1.087 (1.086-1.089) ab 2.5 (2.0-3.0) 8.5 (8.0-9.0) ab 6.0 (5.0-7.0) abc 11.5 (11.0-12.0) abc ‘bolesta’ x ‘mc 329’ 9 1.20 (0.98-1.45) b 1.086 (1.079-1.091) b 4.0 (7.0-2.0) 8.9 (7.0-10.0) ab 6.7 (3.0-8.0) a 10.6 (7.0-13.0) bc ‘agria’ x ‘sandy’ 1 0.76 b 1.080 bc 2 10.0 ab 8.0 ab 7.0 c ‘majestic’ x ‘alcmaria’ 1 3.00 a 1.079 bc 3 7.0 b 3.0 c 15.0 a ‘primura’ x ‘alcmaria’ 1 1.02 b 1.070 c 3 10.0 ab 8.0 ab 7.0 c varieties adora 1 0.80 b 1.071 c 2 9.0 ab 5.0 abc 8.0 c agria 1 1.18 b 1.075 bc 5 10.0 ab 5.0 abc 11.0 abc spunta 1 1.10 b 1.078 bc 4 8.0 ab 7.0 abc 10.0 abc f-ratio 13.52 8.92 ns 3.03 3.56 5.42 p-value <0.0001 <0.0001 0.0036 0.0009 <0.0001 table 3 means, ranges and comparisons between clones and control varieties (spunta, adora, agria) obtained evaluating tuber yield (ty), tuber specific gravity (tsg), chipping ability (direct, after cold storage, ± reconditioning, rec) and the evaluation index (ei) (z) number of clones with a significantly better score compared to the control. ns, *, ** indicates that means are not different or statistically different at p<0.05 and p<0.01, respectively (lsd 0.05). family ty (kg) (z) tsg (z) chip category color (z) ei (z) direct cold storage rec cold storage + rec selections 1.16 (0.43-3.00) 1.083 (1.070-1.104) 3.4 (2.0-7.0) 8.8 (7.0-10.0) 6.1 (2.0-8.0) 10.5 (6.0-15.0) cultivars 1.03 (0.80-1.18) 1.075 (1.071-1.078) 3.3 (2.0-4.0) 8.3 (8.0-9.0) 6.3 (5.0-7.0) 9.7 (8.0-11.0) comparisons selection vs cultivar ns 11 ** ns ns ns ns selection vs adora ns 13 ** ns ns ns ns selection vs agria ns 7 ** ns ns ns ns selection vs spunta ns 4 ** ns ns ns ns * means comparison using tukey’s test. levels not connected by the same letter are significantly different (p<0.05). ns indicates not statistically significant data. melito et al. tuber yield and processing traits of potato advanced selections 155 than 1.080 to ensure good processing ability. in this research, tsg significantly varied among families studied (table 2). on average, the specific gravity was 1.083, higher than the three control varieties used (1.075). it ranged from 1.070 (‘primura’ x ‘alcmaria’) to 1.104 (a clone from ‘jenny’ x ‘mc 329’). nineteen clones (70%) showed a specific gravity higher than 1.080; and 4 clones, belonging to ‘bolesta’ x ‘mc 329’ (2) and ‘jenny’ x ‘mc 329’ (2), revealed a tsg higher than 1.090 (table 2). the mean specific gravity of the studied potato clones was significantly higher than that of the controls, with 11 clones showing a higher specific gravity than the mean of the controls (table 3). the chipping ability of the selected clones was evaluated at three times (at harvest and 90 days of cold storage at 7°c, with and without reconditioning at room temperature), based on the requirement of the potato processing market. indeed, cold storage allows potato industries to process tubers when fresh product is not available, preventing sprouting and diseases (malone et al., 2006). meanwhile, cold storage induces degradation of starch, conversion of sucrose in glucose and fructose causing an accumulation of reducing sugars. this process is extremely disadvantageous for the potato processing industry because it induces browning of chips (dale and bradshaw, 2003). during frying, high temperatures on reducing sugars activate the maillard reaction on chips, which became dark colored and bitter, and so not marketable (kumar et al., 2004). variability in chipping ability was found among families, with significant differences after cold storage with or without reconditioning (table 2). by contrast, no significant contrast between the mean chipping value of clones and that of control varieties fried at harvest and after cold storage was found (table 3). however, the analysis of the chipping score ranges of clones indicated that at harvest and after cold storage with reconditioning, 11 and 5 clones respectively, presented a chipping score lower than 4.5 (not shown). therefore, they were all good chippers. by contrast, good chippers were not identified after chips were fried directly out of cold storage. these data were expected considering that during cold storage two principal phenomena occur. the first is called “reversion” and is caused by reducing sugar accumulation: potatoes that generally show a good chipping ability after harvest, give dark, not acceptable chips (oltmans and novy, 2002). the second event occurs when stored potatoes are subjected to a warm (room temperature) period. it is called “reconditioning” and it induces a decrease of reducing sugars in the tuber. this phenomenon happens because during the warming period about 80% of reducing sugars (glucose and fructose) are converted back to starch (oltmans and novy, 2002). consequently, tuber cold storage may produce lighter colored chips compared to the chips produced without a warming period. in order to support the selection of clones with interesting trait combination, an arbitrary evaluation index (ei) was estimated (melito et al., 2017) (table 2). the average family ei was 10.90, ranging from 7 in ‘spunta’ x ‘victoria’ (1) and ‘primura’ x ‘alcmaria’ (1) to 16 in ‘majestic’ x ‘alcmaria’ (1). the best control variety was agria, with an ei of 11. in general, high variability and significant differences in ei were observed among the seven families studied (table 2). analysis of ranges revealed that in almost all families clones with ei higher than that of control varieties were present. the development of a score to evaluate the performance of breeding materials can be used to summarize the analysis of multiple traits, providing a synthetic parameter to support and simplify the practical selection of potato: high ei could indicate interesting genotypes that can be further analyzed deeper 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application of calcium to decrease yellow sap contamination at different positions of garcinia mangostana l. y. tanari 1 (*), d. efendi 1, 2, r. poerwanto 1, 2, d. sopandie1, k. suketi 1 1 department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, 16144 bogor, west java, indonesia. 2 center for tropical horticulture studies, kampus ipb baranangsiang, jln. raya pajajaran, 16144 bogor, west java, indonesia. key words: ca pectate, sector, shaded, transpiration, well-exposed. abstract: the present research aimed at studying the effects of ca application, through soil fertilization, on yellow sap contamination based on the position of the fruits on the canopy of the tree. the tree was divided into 6 sectors based on the differences in light exposure i.e. sector 1, 2, and 3 for shaded fruit positions and sector 4, 5, and 6 for well-exposed (to light) fruit positions. the present study used a randomized complete block design (rcbd), consisting of 2 treatments i.e. 0 kg ca/tree and 4.8 kg ca/tree. the results revealed that ca treatment lead to an increase in ca-pectate content in pericarp. in addition, the exposed fruit position allegedly increase the absorption of ca-pectate to the fruit. thus, it is important to both apply ca on the soil and ensure that the fruit, in the canopy, gets enough light to decrease the occurrence of yellow sap contamination. the well-exposed position of the fruit, in the 4.8 kg ca/tree treatment during anthesis, had increased the ca-pectate content of the pericarp which, in turn, resulted in a decrease in yellow sap contamination in segment, aryl, and rind of the mangosteen fruit. 1. introduction yellow sap is a sap which is naturally produced in each organ of the mangosteen, excepts the root. indeed, it constitutes the main constraint in the indonesian mangosteen agribusiness industry due to the fact that it causes the fruit flesh (aryl) to have a bitter taste and a less attractive look (osman and milan, 2006). statistical data (2015) revealed that only 14.8% of the total indonesian mangosteen production is exported as a consequence of high percentage of yellow sap contamination. yellow sap is found in the yellow sap duct which is surrounded by typical epithelium cells (dorly et al., 2008). it will contaminate the surface of the fruit (or aryl) if the epithelial cells of the secretory duct break as a result of cadeficiency. the break of the epithelial cells is connected with the extreme changes in groundwater during the developmental process of the fruit (pechkeo et al., 2007), and the differences in growth rate (*) corresponding author: yulindatanari@yahoo.co.id citation: tanari y., efendi d., poerwanto r.. sopandie d., suketi k., 2018 application of calcium to decrease yellow sap contamination at different positions of gancinia mangostana l. adv. hort. sci., 32(2):169-175 copyright: © 2018 tanari y., efendi d., poerwanto r., sopandie d., suketi k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 169-175 170 between the seed and the aryl with the fruit pericarp during the growing phase of the fruit (poerwanto et al., 2010). according to previous research, the break of yellow sap duct is related to the concentration of ca. indeed, ca content of the pericarp of mangosteen contaminated fruit (by yellow sap) is lower compared to that of a normal fruit (poovaradom, 2009; kurniadinata et al., 2016). according to marshner (2012), ca structurally functions to strengthen the cell wall, plant tissues, and the stability of the membrane. mortazavi et al. (2016) reported that ca can minimize cell membrane injury. the increased effect of ca application can be explained by its role in cell membrane structure. meanwhile, seligmann et al. (2009) stated that ca in plant plays an important role regarding the strength of the mechanical tissue and the determination of the fruit quality. however, ca is an immobile nutrient that could not be translocated from plant tissues. thus, developing leaves and fruits fully depend on the transmission of ca in the transpiration stream of the xylem. according to qiang and ling (2005), transpiration is the main factor that promotes ca movements and a low transpiration rate will result in a low addition of ca2+ to aerial organs. mangosteen is a small or medium height tree with a straight, symmetrically branched to form a conical and very tight canopy which leads to both a low light intensity and temperature in the shaded internal part compared to the well light-exposed parts (sector). the described architecture is believed to influence the microclimate of various parts of the plant canopy, causing differences in transpiration rate, which in turn, is believed to cause differences in ca absorption rate to the fruit. crisosto et al. (1995) report the greater the light interception by an individual fruit and its surrounding leaves the better its quality. fruit that developed in the more shaded inner canopy positions have a greater incidence of internal breakdown than fruit from the high light, outer canopy positions. erez and flore (1986) reported that fruit exposed to light experienced a quality improvement by pigmentation of peach that is also assumed to relate to the sink strength of the fruit. research, on the relationship between both ca and the fruit position in the canopy and the occurrence of yellow sap, become capital in determining the sector with a high contamination potential. the present research was aimed to determinate the effects of ca on yellow sap contamination based on the position of the fruit in the tree canopy 2. materials and methods time and place the present research was conducted in tandolala village, poso district, central sulawesi, from october 2015 to april 2016. tandolala village is situated at an altitude of 508 mdpl with a ph (soil) of 4.8, and a rainfall of 192.8 mm/month. the observations on yellow sap contamination were carried out in the laboratory of natural science at the university of sintuwu maroso poso. analyses of total ca and pericarp capectate contents were performed in the laboratory of soil chemistry and fertility, bogor agricultural university. materials ten productive plants, at an average 40 years old (with a planting distance ranging from of 4x4 m to 6x6 m), an average height of 16 meters and a canopy diameter of 4 m, were used in the present study. dolomite [camg(co3)2], containing 30% ca, was used as ca source. the canopy of the tree was divided into 6 sectors as follows: sector 1 inner bottom, sector 2 outer bottom, sector 3 inner middle, sector 4 outer middle, sector 5 inner top, and sector 6 outer top. the tree was divided into sectors based on the modifications of setiawan et al. (2012) techniques (fig. 1). parameters, such as light intensity, temperature, and humidity, were measured on weeks 4, 8, and 12, on each sector by placing the measuring tool (thermohydrometer) in each sector. afterwards, the light intensity was recorded as displayed on the measuring fig. 1 fruit positions in the canopy of the tree. adapted from setiawan et al., 2012. s= sector. tanari et al. calcium application against yellow sap contamination in mangosteen 171 tool. light intensity, temperature and humidity averages for each sector are presented in table 1. experimental design a randomized complete block design (rcbd), consisting of 2 treatments i.e. 0 kg ca/tree and 4.8 kg ca/tree (equivalent to 16 kg dolomite/tree), was used as experimental design in the present study. fertilization with ca was carried out during anthesis by sowing in the path that was already made around the mangosteen tree (below the crown of the plant) and recovering it with soil. based on the light intensity measurement results in table 1, the observations on yellow sap contamination were carried out on both shaded and wellexposed positions. a fruit was considered shaded if the perceived light intensity is low. meanwhile, it was categorized as well-exposed if the average received light intensity is high. shaded fruit positions were identified in sectors 1, 2 and 3, with an average light intensity of 481 lux, while well exposed fruit positions were identified in sectors 4, 5 and 6 with an average light intensity of 1229 lux. the total sample from each sector was 12 fruits (72 fruits per tree). thus, a total of 720 mangosteen fruits were used as samples. the transpiration rates, of the leaves, were measured at both shaded and well-exposed positions. the results of the above measurements will be used to estimate the transpiration rates of the fruits in various positions. the transpiration rate was measured by means of a lux meter. measurements harvesting was carried out 16 weeks post-anthesis (wpa). the observations on the percentage of yellow sap (pys) were carried out in order to determine the percentage of contaminated fruit in the group of fruits that was observed. a fruit was considered contaminated although the yellow sap, that pollutes both the aryl and the rind just one spot (small patch). the percentages of contaminated aryl (pca), contaminated rind (pcr), and contaminated segment (pcs) was calculated by means of the following equations: pca = (total yellow sap contaminated aryl /total fruit sample) x 100 pcr = (total yellow sap contaminated rind/total fruit sample) x 100 pcs = (total yellow sap contaminated segment/total fruit segment sample) x 100 yellow sap contamination score shows the severity level of a fruit contaminated by yellow sap and ranges from 1 (very well) to 5 (very bad). observations on aryl fruit and yellow sap contaminated rind scores referred to kurniadinata et al. (2016 ) method as presented in table 2 and 3. ca pectate analyses referred to an analysis method developed by setyaningrum et al. (2011) which uses dry fruit sample. the samples were ground into partitable 1 light intensity, temperature, humidity, and transpiration rate at different fruit positions (exposed and shaded) score description 1 very good, clean white aryl, no yellow sap between aryl and rind, and fruit vessels as well 2 good, 1-2 yellow sap stains (small patch) on one end of the aryl, but does not make the fruit bitter 3 good enough, the presence of some yellow sap stains (patch) on one end of the aryl or between the segments and the littering aryl 4 bad, presence of yellow sap stains/blobs at the end of the segments, between the segments or the fruit vessels, making the fruit bitter 5 very bad, the presence of large yellow sap stains/blobs at the end of the segments, between the segments or at the fruit vessels, making the fruit bitter with a clear colored aryl table 2 yellow sap contamination score on aryl score 1= very good/without contamination, up to score 5= very bad /high contamination score. table 3 yellow sap contamination score on rind score 1= very good/without contamination, up to score 5= very bad /high contamination score. score description 1 very good, flawless rinds with no visible yellow sap. 2 good, flawless rinds with 1-5 yellow sap stains (small patch) which dry without affecting the color of the fruit 3 good enough, flawless rinds with 6-10 yellow sap drops which dry and do not affect the color of the fruit 4 bad, flawed rinds due to medium/large yellow sap clumps, there are 1-2 yellowing streams 5 very bad, flawed rinds with more than one large yellow sap clumps with lots of yellowing streams on the rind of the fruit and a dull fruit color fruit positions light intensity (lux) temperature (oc) humidity (%) transpiration rate (hpa/s) sector 1 414.44 26.54 70.98 shaded 0.03 sector 2 536.00 26.80 70.46 sector 3 492.76 26.08 71.54 sector 4 1581.51 27.04 69.77 well-exposed 0.06 sector 5 944.60 26.74 72.04 sector 6 1163.11 27.26 70.97 adv. hort. sci., 2018 32(2): 169-175 172 cles, added with ion free water and shacked for 2 hours. afterward, the solution was centrifuged for 15 minutes at a speed of 3000 rpm. the supernatant was then filtered to collect the pellet to which a 1 mol l-1 of nacl was added, shacked for 2 hours and centrifuged for 15 minutes. the extraction result was analyzed atomic absorption spectrophotometer (aas) in order to obtain data on ca pectate. statistical analysis pca, pcr, pcs, total ca contents, and ca-pectate were analyzed using sas 9.1.3 program, which was followed by duncan’s post-hoc comparison (at a significance level of 5%) test. meanwhile, data on fruit score, yellow sap contaminated aryl were analyzed by mean of a kruskal wallis test, which was followed by dunn test. 3. results the application of ca had an effect on the decrease in yellow sap contamination in mangosteen (table 4). the lowest percentage of yellow sap contaminated segment was observed in the 4.8 kg ca/tree (exposed fruit position), which significantly differed to those of other treatments. the percentage of yellow sap contaminated segment showed significant decline of 81% with application of 4.8 ca/tree on exposed position compared to treatment with 0 kg ca/tree on shaded position, 71% compared to treatment with 0 kg ca/tree on exposed position and 73% compared to 4.8 kg ca/tree on shaded position. the percentage of yellow sap contaminated aryl, in the 4.8 kg ca/tree treatment (exposed fruit position), showed an average percentage of 14.2%, which was not different to that of the 0 kg ca/tree treatment (23.9%). no differences were observed among treatments application of 4.8 kg ca/tree in terms of yellow sap contaminated rind percentage. the percentage of yellow sap contaminated rind was considerably high with treatment of 0 kg ca/tree, which was 79.9% on shaded position and 73.9% on exposed position. while the application of 4.8 kg ca/tree showed that yellow sap contamination was still considerably high at 61.3% on shaded position and 53.5% on shaded position. although the numbers were still high, there was a decline of 33% with application of 4.8 ca/tree compared to without ca application on shaded position. according to martias et al. (2012), percentage of yellow sap contaminated that was higher than 50% was considered very high. table 5 presents the yellow sap contamination scores of both aryl and rind. the best yellow sap contaminated aryl score was observed at the wellexposed fruit position with the application of 4.8 kg ca/tree, and significantly differed to other treatments. meanwhile, the yellow sap contaminated rind score in the 4.8 kg ca/tree treatment revealed similar results for both shaded and well-exposed fruit positions (table 5). accumulation of ca in fruit pericarp was a representation of adsorbed soil ca by plant. total ca content of the fruit pericarp did not significantly differ among treatments, but was significantly different to that of the ca-pectate contents. the ca-pectate contable 4 percentage yellow sap contamination in fruit segment, aryl and on rind (16 wpa) numbers followed by different letters within the same column showed significant differences in dmrt test (α= 5%). shaded (sector 1, 2 and 3) with a light intensity of 481 lux, exposed (sector 4, 5 and 6) with a light intensity of 1229 lux. table 5 score of yellow sap contamination in aryl and on rind (16 wpa) data were analyzed based on kruskal wallis test. numbers followed by different letters within the same column showed significant differences based on dunn test (1%). score 1: very good; score 2= good; score 3= good enough; score 4= bad; score 5= very bad. treatments fruits contaminated by yellow sap (%) pericarp ca content (ppm) segment aryl rind total pectate 0 kg ca/tree shaded 21.3 a 45.7 a 79.9 a 1088.9 552.02 b exposed 13.6 a 23.9 b 73.9 ab 2100.0 528.87 b 4.8 kg ca/tree shaded 15.0 a 34.9 a 61.3 bc 1500.0 576.40 b exposed 4.0 b 14.2 b 53.5 c 1566.7 754.60 a f-test * * ns * treatments yellow sap contaminated aryl and rind score in 1-5 aryl rind 0 kg ca/tree shaded 1.99 a 2.38 a exposed 1.67 c 2.17 b 4.8 kg ca/tree shaded 1.77 b 1.90 c exposed 1.23 d 1.74 c dunn-test * * tanari et al. calcium application against yellow sap contamination in mangosteen 173 tent was higher in the ca treated fruit (well-exposed position) (table 6). the percentage of yellow sap contaminated rind and aryl was reduced because of increased ca-pectate in fruit pericarp, proving that ca-pectate had a role in strengthening cell wall epithelium which compile the yellow sap duct making the cell stronger and kept yellow sap from leaking and contaminate the aryl and rind. leaves transpiration rate measurement, on week 4, 8, and 12, resulted in average transpiration rates of 0.03 hpa/s (shaded position) and 0.06 hpa/s (well exposed position) (table 1). this data supported the measurement result of light and temperature on table 1, that the higher the temperature and light intensity, the higher the transpiration rate. 4. discussion and conclusions ca is an immobile nutrient, for its absorption rate follows the transpiration pathway in the xylem. thus, a deficiency in ca often occurs in fruits that do not transpire as much as the leaves. in the present study, the transpiration rate of the fruits was estimated by observing parameters such as light intensity, temperature and humidity in each sector, and also the transpiration rates of leaves at both the exposed and shaded positions. data shown in table 4 showed that the percentage of yellow sap contamination on the rind was higher than aryl and segment. yellow sap contamination was caused by the same factor which was the lack of ca on epithelium cell of yellow duct sap, but triggered by different things. according to dorly et al. (2008), yellow sap contamination on the aryl was caused by turgor and mechanical pressure, that was the pressure of aryl and seed growth outward during fruit enlargement. while contamination on the rind was caused by turgor pressure of pericarp cell, or by insect, fungal, or bacterial attack. fruits that were exposed to a high light intensity (table 1) and treated with 4.8 kg ca/tree had highest ca-pectate content (table 6) which was a consequence of the high light intensity received. marschner (2012) stated that part of the pectin, in the leaves, is in the form of ca-pectate in a high light intensity condition. ca strengthens the main plant cell wall in a crosslinking with the pectin. formation of calcium pectate due to the binding of calcium with pectins has been found beneficial to increase the strength of cell wall and middle lamella (carpita and mccann, 2000). the high ca-pectate content is believed to be a result of light intensity not only on the leaves but also on the fruit that possibly increases photosynthesis rate and transpiration rate, as demonstrated by the results of the present research. such fruits (well-exposed, treated with ca, and showing highest ca-pectate), showed also lowest yellow sap contamination levels. a high ratio of ca-pectate over total ca content (48% of the total ca) (table 6) could decrease yellow sap contamination through the strengthening of the epithelial cell walls in the yellow sap duct. a research, conducted by setyaningrum et al. (2011), revealed that the capectate content represented 20% of the total ca, which tend to reduce yellow sap contamination in mangosteen. the improvement of fruits quality (low yellow sap contamination) is related to high light intensity of fruits exposition. physiologically, light has a direct influence by photosynthesis and indirect influence throught plant’s growth and development as the results of direct metabolic responses (fitter and hay, 1991). the improvement of fruits quality is caused by the distribution of photosynthate to the fruit exposed to light. the high temperature and light intensity in the exposed tree canopy resulted in higher transpiration rate at exposed positions compared to the shaded ones (table 1). similar results were observed by caleb et al. (2013) who demonstrated that temperature and humidity have significant effects on the transpiration rate. the high leaves transpiration rate at the exposed position is believed to be a consequence of higher ca translocation from the root to parts of the leaf (including part of the exposed fruit), for the ca is translocated along with water during the transpiration process. the high transpiration rate led to a high translocation of ca from the root to the fruit, since ca is translocated along with water during numbers followed by different letters within the same column showed significant differences in dmrt test (α= 5%). shaded (sector 1, 2 and 3) with a light intensity of 481 lux, exposed (sector 4, 5 and 6) with a light intensity of 1229 lux. table 6 calcium total and ca-pectate content in pericarp and percentage ratio of ca-pectate/total ca at 16 wpa treatment ca content in pericarp (%) percentage pectate/totaltotal pectate 0 kg ca/tree shaded 0.11 0.06 b 50.5 exposed 0.21 0.05 b 23.8 4.8 kg ca/tree shaded 0.15 0.06 b 40.0 exposed 0.17 0.08 a 47.1 f-test ns * ns adv. hort. sci., 2018 32(2): 169-175 174 the transpiration process. hansen (1980) stated that ca is transported in the fruit by means of water distribution through the xylem. gilliham et al. (2011) demonstrated that the mechanism of ca uptake is through the apoplast of the root along with the mass flow which follows the apoplast or the symplast pathway to the xylem. according to qiang and ling (2005) and white and broadley (2003), the transport through the apoplastic pathway mainly depends on the transpiration, while the symplast pathway is selective in controlling ca2+ to the xylem which depends on ca2+ requirement at the canopy. if there is a deficiency in ca supply of the plant, the plant may experience damages at the cell level. thus, the supplementation of ca is required on soil with low ca content. according to martias et al. (2012), yellow sap contamination could be directly prevented by the ca availability in the soil. amor and leo (2006) stated that the ca concentration of the plant significantly decrease due to a low supply. in addition, hocking et al. (2016) demonstrated that low supply and transport of ca would result in a ca deficiency, leading to damages of both membrane and cell walls of the fruit. the results revealed that ca treatment lead to an increase in ca-pectate content in pericarp. in addition, the exposed fruit position allegedly increase the absorption of ca-pectate to the fruit. thus, it is important to both apply ca on the soil and ensure that the fruit, in the canopy, gets enough light to decrease the occurrence of yellow sap contamination. the well-exposed position of the fruit, in the 4.8 kg ca/tree treatment during anthesis, had increased the ca-pectate content of the pericarp which, in turn, resulted in a decrease in yellow sap contamination in segment, aryl, and rind of the mangosteen fruit. acknowledgements the authors would like to thank the ministry of research, technology and higher education for funding and supporting the present research through doctoral dissertation program 2017. references amor f.m., leo f.m., 2006 differential effect of transpiration and ca supply on growth and caconcentration of tomato plants. sci. hort., 111: 17-23. caleb o.j., mahajan p.v., opara u.l., 2013 impact of temperature and relative humidity on the transpiration rate of pomegranate arils. journal of food, 11(3): 199-207. carpita n., mccann m., 2000 the cell wall, pp. 52-108. in: buchanan b.b., g. wilhelm, and r.l. jones, (eds.) biochemistry and molecular biology of plants. john wiley & son, chichester, uk, pp. 1280. central bureau of statistics, 2015 production of fruits in indonesia 2015. jakarta indonesia (in indonesian). crisosto c.h., mitchell f.g., johnson s., 1995 factors in fresh market stone fruit quality . postharvest news and information, 6(2): 17-21. dorly s., tjitrosemito s., poerwanto r., juliarni, 2008 secretory duct structure and phytochemistry coumpounds of yellow latex in mangosteen fruit. hayati j. of biosciences, 15: 99-104. erez a., flore j.a., 1986 the quantitative effect of solar radiation on ‘redhaven’ peach fruit skin color. hortscience, 21: 1424-1426. fitter ah., hay r.k.m., 1991 environmental physiology of plants. gadjah mada university press, yogyakarta, pp. 421. 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(in indonesian). white p.j., broadley m.r., 2003 calcium in plants. ann bot., 92 (4): 487-511. impaginato 23 adv. hort. sci., 2019 33(1): 23-31 doi: 10.13128/ahs-23015 biochemical characterization of artichoke (cynara cardunculus var. scolymus l.) spring genotypes from marche and abruzzo regions (central italy) a. galieni, 1 (*) f. stagnari 2, m. pisante 2, c. platani 1, n. ficcadenti 1 1 centro di ricerca orticoltura e florovivaismo, crea, via salaria, 1, 63077 monsampolo del tronto (ap), italy. 2 facoltà di bioscienze e tecnologie agroalimentari e ambientali, università degli studi di teramo, via r. balzarini, 1, 64100 teramo, italy. key words: antiradical activity, artichoke genotypes, capitula quality traits, total phenolic content. abstract: ten artichoke genotypes from marche and abruzzo regions [ascolano (as), castorano (cs), clone monsampolo, jesino (je), mazzaferrata (mz), montelupone a, montelupone b, urbisaglia1 (ub_1), urbisaglia2 and violetto tardivo di pesaro] were characterized for their quality traits and peculiar enduse attitudes, in comparison with the reference romanesco clone c3 (cl_c3). total polyphenols content (tpc), total flavonoid content (tfc) and antiradical activity were assessed in the receptacle and external bracts of both main and first order capitula. cl_c3 showed high tpc and tfc values in the receptacle of the main flower heads (7.4 mg gallic acid equivalents, gae, g-1 dry weight, dw, and 3.6 mg rutin equivalents, rue, g-1 dw, respectively), confirming its attitude for fresh consumption. je and ub_1 showed great and stable (among main and first order capitula) head quality, highlighting their potential for breeding programs to enhance the content of functional compounds. conversely, mz and as could be appreciable for processing or pharmaceutical applications, being characterized by great tpc (external bracts, first order capitula: 2.8 and 2.7 mg gae g-1 dw, respectively) and tfc (external bracts, first order capitula: 2.1 and 2.7 mg gae g-1 dw, respectively) values in the waste parts. high correlations between tpc and tfc with antiradical activity were also observed. our results suggest the possibility to promote the utilization in genetic breeding programs of the autochthonous artichoke populations, according to their peculiar characteristics, including also their biochemical composition. 1. introduction globe artichoke [cynara cardunculus l. var. scolymus (l.) fiori] belongs to the family of asteraceae (compositae) and it is an herbaceous perennial crop mainly cultivated in the mediterranean basin (about 65% of world production) followed by americas and china (sihem et al., 2015, lombardo et al., 2017). in italy it plays an important role in the agro-food (*) corresponding author: angelica.galieni@crea.gov.it citation: galieni a., stagnari f., pisante m., platani c., ficcadenti n., 2019 biochemical characterization of artichoke (cynara cardunculus var. scolymus l.) spring genotypes from marche and abruzzo regions (central italy). adv. hort. sci., 33(1): 23-31 copyright: © 2019 galieni a., stagnari f., pisante m., platani c., ficcadenti n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 april 2018 accepted for publication 12 december 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 23-31 24 chain with over 43.8 kha and approximately 366 kt of floral heads produced (fao, 2016). the italian gene pool of globe artichoke includes hundreds of varieties and ecotypes, grouped into four main types i.e. “catanesi”, “romaneschi”, “spinosi” and “violetti”. according to the harvesting period, they are classified as early or late distinct clonal varietal groups, with the former having a typical autumn-winter cycle in the southern regions, while late including spring genotypes mainly grown in central regions (ciancolini et al., 2013 a, b). the varietal constitution of artichoke is restricted to clonal selection carried out within local populations propagated by agamic way which represent a patrimony of agrobiodiversity and a biological, cultural and economic heritage (ficcadenti et al., 2013). however, several cases of homonymy and synonymy (different varieties are called with the same name in the first case, while the same variety comes call with different names in the second) as well as unsatisfactory, uniformity and identity of accessions occur (ficcadenti et al., 2013). traditional agricultural and food production must be safeguarded to avoid processes of globalization and homologation and, consequently, identification, collection, characterization and conservation of agrobiodiversity as well as development of genetic improvement strategies, particularly linked to nutritional and organoleptic traits, have been undertaken in these years (mauromicale and ierna, 2000; ficcadenti et al., 2010; ciancolini et al., 2012). recently, a renewed and growing interest for artichoke cultivation has been observed worldwide mainly due to its potential uses as functional food: its large immature inflorescences, called capitula or heads, represent a rich source of bioactive compounds including polyphenols with a strong antiradical activity (schütz et al., 2004) inulin, fibres and minerals (lattanzio et al., 2009; lombardo et al., 2010; pandino et al., 2011). furthermore, the utilization of by-products of artichoke processing (i.e. external bracts) involves animal feedstuff (megías et al., 2002) or extraction of functional molecules (larossa et al., 2002). it emerges that the types and amount of bioactive substances and their activity (i.e. polyphenols content and the related antioxidant activity) could be used to characterize and select specific genotypes. nowadays, only few studies have investigated on polyphenols content, discriminating among the different head parts of artichoke (see for example fratianni et al . , 2007; lombardo et al . , 2010; soumaya et al. 2013; sihem et al., 2015); besides, the simultaneous determinations of total polyphenols and flavonoids content with radical scavenging capability, have not been considered at all. consequently, in the present work we aimed at investigating such important biological properties in eleven spring accessions of artichoke collected in central italy (marche and abruzzo regions). the primary objectives were to obtain a preliminary: (i) genotype’s characterization of the selected central-italy artichoke accessions from a biochemical point of view; (ii) evaluation of the suitability of the selected artichoke genotypes for fresh consumption or by-products production. 2. materials and methods plant material, management practices and head sampling the study was carried out in 2014 at the experimental field of the research centre for vegetable and ornamental crops, council for agricultural research and economics (crea-of), located in monsampolo del tronto (ap) (latitude 42°52’59.1” n, longitude 13°48’01.9” e), in the coastal area of the marche region (central italy) a typical area for globe artichoke cultivation. ten artichoke accessions from marche and abruzzo regions, named as “clone monsampolo” (cl_msp), “ascolano” (as), “castorano” (cs), “jesino” (je), “mazzaferrata” (mz), “montelupone a” (ml_a), “montelupone b” (ml_b), “urbisaglia1” (ub_1), “urbisaglia2” (ub_2) and “violetto tardivo di pesaro” (vt_ps), were collected on the base of their peculiar sensory features and were compared with the reference genotype “romanesco clone c3” (cl_c3), characterized by high market standards of the flower heads (purple with green shades, round shape, regular size and thick consistency). the selected globe artichoke genotypes differ for their biological and morphological profiles, as briefly synthetized in table 1. plant material (shoots, named “carducci”) was transplanted in august 2011 in rows spaced 1.00 m apart with row spacing of 1.20 m; each plot (artichoke genotype) consisted of thirty plants. the fertilization program, typical of the area, consisted in: 150 kg ha-1 of n, 80 kg ha-1 of phosphorus pentoxide (p2o5) and 100 kg ha-1 of potassium oxide (k2o), respectively. the experimental field was kept weedfree by mechanical weed control and no pest control was needed. galieni et al. biochemical differences among artichoke genotypes 25 at the marketing stage, six capitula per artichoke genotype were harvested, without floral stem, in two subsequently times (10th april and 10th may, considered as early and mid-spring), allowing to compare both main (first sampling data) and first order (second sampling data) capitula. each flower head was separated into ‘external bracts (∼15 bracts)’ (waste part) and ‘receptacle’ (edible fraction), freeze-dried, homogenized and stored at -20°c until biochemical characterization. chemical analysis the extraction of polyphenols and flavonoids were carried out as described by gouveia and castilho (2012 a). the folin-ciocalteu reagent method was used to evaluate the total polyphenols content (tpc) of the external bracts and receptacle following the method of gouveia and castilho (2011). plant extracts were dissolved in methanol (10 mg ml-1); aliquots of 50 µl were added to 1.25 ml of folin-ciocalteu (dilution, 1:10) and 1.0 ml of a 7.5% na2co3 solution. solutions were maintained at room temperature for 30 min and the tpc was determined at 765 nm using a beckman du640b spectrophotometer (beckman coulter, brea, california, usa). gallic acid standard solutions were used to calibrate the method, so results were expressed as mg gallic acid equivalents (gae) per g-1 dry weight (dw). total flavonoids content (tfc) was calculated following the procedure described by gouveia and castilho (2012 a) and estimated as rutin equivalents (rue), i.e. expressed as mg rue g-1 dw. methanolic solutions (500 µl of sample solution) of the plant extracts (2.5 mg ml-1) were mixed with 1.5 ml of methanol, 2.8 ml of water, 100 µl of potassium acetate (1 m) and 100 µl of aluminium chloride (10% in methanol). the absorbance of reaction mixture was read after 30 min at room temperature and at 415 nm using a beckman du640b spectrophotometer. the radical scavenging activity of the extracts was determined using the stable radicals: (i) 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) teac/abts assay (abts) (re et al., 1999), modified as described by gouveia and castilho (2012 a); and (ii) 2,2-diphenyl-1-picrylhydrazyl dpph assay (gouveia and castilho, 2012 b). in each assay, trolox was employed as reference standard and results were expressed as µmol trolox equivalent (te) g-1 dw. reagents and solvents were purchased from sigma chemicals co. (st. louis, mo). all reagents were of analytical grade. statistical analysis in order to test (f-test) the effect of genotype on all the investigated variables, a one-way analysis of variance (anova) was performed. the experiment was conducted following a complete randomized design and each sampled capitula represented a single repetition. when significant differences were detected, the means were compared based on the standard error of the difference (sed) between means, with significance being assigned using the least significant difference (lsd) value at the 5% (p<0.05) level of significance. before the anova, the data were analyzed to test for normality and homoschedasticity assumptions, through graphical methods. to interpret and summarize the association between treatments (artichoke genotypes: cl_c3, cl_msp, as, cs, je, mz, ml_a, ml_b, ub_1, ub_2 and vt_ps) and variables (tpc, tfc, abts, dpph) the principal component analysis (pca) was applied. the pca was performed separately for main and first order capitula; each principal component (pc) was table 1 head characteristics of the eleven selected genotypes of globe artichoke genotype acronym colour of outer bracts colour of inner bracts bracts "clone c3" cl_c3 green with purple shades yellow spineless "clone monsampolo" cl_msp green yellowish-green spineless "ascolano" as purple with green shades yellow-greenish with purple shades spineless "castorano" cs purple with light green shades yellow-purple spineless (but mucronate) "jesino" je purple with green shades yellow purple spineless "mazzaferrata" mz purple with green shades yellow-greenish spineless "montelupone a" ml_a purple with green shades yellow-purple spineless "montelupone b" ml_b purple with green shades yellow-purple spineless (but mucronate) "urbisaglia 1" ub_1 purple yellow-purple spineless "urbisaglia 2" ub_2 purple yellow-purple spineless "violetto tardivo ps" vt_ps purple yellow-purple spine adv. hort. sci., 2019 33(1): 23-31 26 calculated as a linear combination of the standardized original variables by using the eigenvectors of the correlation matrix. the results were visually explored in a two-dimensional pca correlation biplot: standardized pc1 and pc2 scores were plotted as symbols, while the correlations between pcs and standardized variables (factor loadings) were plotted as vectors. statistical analyses were performed with the r software (r core team, 2017). 3. results and discussion the tpc and tfc in the receptacle and external bracts of main and first order capitula of the eleven artichoke genotypes, are reported in table 2. tpc ranged from 1.5 to 9.2 mg gae g-1 dw, while tfc ranged from 1.8 to 4.1 mg rue g-1 dw, matching with the literature data or, in some circumstances, resulting slightly higher (lombardo et al., 2010; pandino et al., 2011; gouveia and castilho, 2012 a; pandino et al., 2012 a; sihem et al., 2015; dabbou et al., 2017; marques et al., 2017; petropoulos et al., 2017). both traits were significantly (p<0.05) influenced by genotype: differences are related to both head part (receptacle or external bracts) and their location on plant architecture (main or first order capitula) (table 2). clear trends were observed: je gave the highest tpc content (5.1 mg gae g-1 dw, averaged over head parts and harvest time), followed by ub_1, ub_2 and vt_ps (5.0, 5.0 and 4.3 mg gae g-1 dw on average, respectively), while cl_msp resulted as one of the worst genotypes in terms of polyphenols concentratable 2 total polyphenols content [tpc, mg gallic acid equivalents (gae) g-1 dry weight (dw)] and total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and in the external bracts of different artichoke genotypes genotype§ main capitula first order capitula tpc (mg gae g-1 dw) tfc (mg rue g-1 dw) tpc (mg gae g-1 dw) tfc (mg rue g-1 dw) receptacle cl_c3 7.4 ± 0.11 3.6 ± 0.23 3.8 ± 0.57 2.5 ± 0.38 cl_msp 3.6 ± 0.28 1.9 ± 0.16 3.1 ± 0.09 1.9 ± 0.12 as 5.7 ± 0.42 2.4 ± 0.21 3.9 ± 0.55 2.2 ± 0.19 cs 6.3 ± 0.91 2.5 ± 0.30 2.6 ± 0.03 1.9 ± 0.08 je 8.2 ± 1.09 3.3 ± 0.44 5.4 ± 0.47 3.0 ± 0.34 mz 4.9 ± 0.73 2.8 ± 0.17 6.8 ± 1.35 3.5 ± 0.61 ml_a 5.8 ± 1.34 4.1 ± 1.01 2.1 ± 0.19 1.8 ± 0.19 ml_b 4.3 ± 0.22 2.3 ± 0.31 4.0 ± 0.64 3.1 ± 0.66 ub_1 8.0 ± 0.58 3.4 ± 0.36 4.7 ± 0.66 2.9 ± 0.69 ub_2 9.2 ± 1.55 3.9 ± 0.74 4.4 ± 0.15 2.6 ± 0.15 vt_ps 6.8 ± 0.45 3.5 ± 0.42 4.2 ± 0.45 3.2 ± 0.45 f-test ** * ** * sed 1.2 0.7 0.9 0.6 external bracts cl_c3 3.2 ± 0.12 2.6 ± 0.07 2.1 ± 0.06 2.3 ± 0.18 cl_msp 2.2 ± 0.15 2.0 ± 0.00 1.7 ± 0.11 2.0 ± 0.10 as 3.0 ± 0.19 2.4 ± 0.08 2.7 ± 0.42 2.7 ± 0.30 cs 2.9 ± 0.28 2.1 ± 0.17 2.1 ± 0.04 2.6 ± 0.20 je 3.9 ± 0.53 2.5 ± 0.18 3.0 ± 0.46 2.9 ± 0.56 mz 2.4 ± 0.14 2.5 ± 0.08 2.8 ± 0.16 2.1 ± 0.04 ml_a 2.1 ± 0.25 2.4 ± 0.25 1.5 ± 0.08 2.3 ± 0.02 ml_b 2.1 ± 0.16 2.0 ± 0.07 2.2 ± 0.28 2.3 ± 0.28 ub_1 4.8 ± 0.30 2.9 ± 0.17 2.4 ± 0.03 2.3 ± 0.22 ub_2 4.1 ± 0.61 2.7 ± 0.25 2.2 ± 0.19 2.3 ± 0.02 vt_ps 4.0 ± 0.11 3.2 ± 0.05 2.2 ± 0.05 3.0 ± 0.21 f-test ** ** ** ns sed 0.4 0.2 0.3 data refer to both main and first order capitula (two different harvest times, at early and mid-spring 2014). means ± standard errors of n=6 independent replicates are reported. * p<0.05; ** p<0.01; *** p<0.001; ns = not significant. sed, standard error of differences between means. § the list of the used acronomys is reported in table 1. galieni et al. biochemical differences among artichoke genotypes 27 tion in artichoke heads (on average 2.7 mg gae g-1 dw) together with ml_a and ml_b (on average 2.9 and 3.2 mg gae g-1 dw, respectively) (table 2). these results were quite confirmed by tfc data (table 2), indicating those genotypes’ suitable for fresh consumption rather than food processing. lower antioxidant compounds (i.e. polyphenols) is, indeed, considered a qualitative trait required by industry, thanks to the scarce propensity to enzymatic browning phenomena after cutting and storage operations (lattanzio et al., 1994; lombardo et al., 2010). the reference genotype (cl_c3) confirmed its high value for fresh consumption, registering higher tpc and tfc, only in the combination early-spring harvest (main capitula)/receptacle (table 2), mostly appreciated by consumers and with the highest commercial value. as previously observed (fratianni et al., 2007; lombardo et al., 2010; pandino et al., 2011; pandino et al., 2012 b; pandino et al., 2013 a; sihem et al., 2015), polyphenols were not uniformly distributed in the different floral head parts (fig. 1): regardless of the harvest time, higher tpc values were observed in the receptacle (5.2 mg gae g-1 dw, averaged over genotypes) while lower in the external bracts (2.7 mg gae g-1 dw, averaged over genotypes). besides, no differences emerged in terms of tfc (2.5 vs. 2.8 mg gae g-1 dw in external bracts and receptacle respectively, averaged over genotypes). the different amount of antioxidant compounds in the various head parts is of interest to identify genotypes rich in these molecules in the by-products (external bracts) and hence interesting for the industrial processes (i.e. animal feedstuff, fiber production, recovery of functional ingredients) (femenia et al., 1998; larossa et al., 2002; megías et al., 2002; lattanzio et al., 2009). nonetheless, we observed some differences in terms of relative tpc among artichoke accessions, with particular regards in terms of relative tfc (fig. 1): the genotypes cl_msp, as and cs were characterized by the highest relative tfc in the waste products (fig. 1), suggesting useful utilization for the by-products processes, with external bracts representing a potential innovative source for flavonoid extraction. lastly, tpc values lowered in both receptacle and external bracts shifting from main to first order capitula (fig. 2a); this trend was confirmed by all the accessions with the exception of mz (fig. 2a), which gave the highest tpc values in the first order flower heads (table 2), so maintaining a high content of functional compounds during the growing cycle. a similar behavior was recorded for some of the selected artichoke accessions in terms of tfc (see for example cl_msp, as and ml_b) (fig. 2b). this was probably attributable to the environmental conditions recorded during the harvest season. despite the solar radiation levels show the stronger effect on polyphenols accumulation in the artichoke’ receptacle (pandino et al., 2013 b), in our study the lower temperatures observed in april (-10% on average with respect to may considering the mean air temperatures recorded during the first 10 days of each month) could have affected tpc and tfc, as previously observed in other crops (klimov et al., 2008; hykkerud et al., 2018). fig. 1 relative proportion (as percentage) of total polyphenols content [tpc, gallic acid equivalents (gae) g-1 dry weight (dw)] and total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and external bracts averaged over both main and first order capitula of different globe artichoke genotypes (see table 1 for the list of acronyms). data represent means ± standard errors, n=12 independent replicates. fig. 2 variations normalized to main capitula values (dashed line) of (a) total polyphenols content [tpc, mg gallic acid equivalents (gae) g-1 dry weight (dw)] and (b) total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and external bracts of different globe artichoke genotypes (see table 1 for the list of acronyms). data represent means ± standard errors, n=6 independent replicates. 28 adv. hort. sci., 2019 33(1): 23-31 radical scavenging activity (abts and dpph assays) registered differences among thesis similar to those observed for tpc and tfc data (table 3). abts values ranged from 21.2 to 146.8 µmol te g-1 dw and dpph ranged from 12.8 to 204.3 µmol te g-1 dw, showing the same order of activity previously found in other antioxidant capacity assays on artichoke (gouveia and castilho, 2012 b; rouphael et al., 2017). the highest activity was concentrated in the receptacle (85.7 and 103.8 µmol te g-1 dw for abts and dpph, respectively vs. 41.9 and 38.3 µmol te g-1 dw for abts and dpph, respectively in the external bracts) regardless of genotype and harvesting time (table 3) (sihem et al., 2015). also for these biochemical traits, je, ub_1 and ub_2 ranged at the first positions while cl_msp as the genotype with the lowest abts and dpph values (table 3). moreover, we found significant (p<0.001) linear relationships between these variables, confirming previous results (alghazeer et al., 2012; lombardo et al., 2013). follows the higher measured pearson’s correlation coefficients: tpc vs. abts, r=0.84; tpc vs. dpph, r=0.91; tfc vs. abts, r=0.76; tfc vs. dpph, r=0.81. indeed, phenolic compounds are known to have the ability to block the chain reaction of reactive oxygen and nitrogen species through different pathways involving (i) direct reaction with free radicals, (ii) sequester metal ions able to spread the chain reaction, (iii) synergic action with other antioxidants (khasawneh et al., 2014). the relationships between tpc, tfc, abts and dpph, classified based on the analysed capitula parts table 3 radical scavenging activity (µmol trolox equivalents (te) g-1 dw) obtained from two different assays: trolox equivalent antioxidant capacity with 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (abts) and 2,2-diphenyl-1-picrylhydrazyl (dpph) in the receptacle and in the bracts of different artichoke genotypes data refer to both main and first order capitula (two different harvest times, at early and mid-spring 2014). means ± standard errors of n=6 independent replicates are reported. * p<0.05; ** p<0.01; *** p<0.001; ns = not significant. sed, standard error of differences between means. § the list of the used acronomys is reported in table 1. genotype§ main capitula first order capitula abts (µmol te g-1 dw) dpph (µmol te g-1 dw) abts (µmol te g-1 dw) dpph (µmol te g-1 dw) receptacle cl_c3 146.8 ± 6.91 165.5 ± 7.75 61.1 ± 14.29 61.3 ± 26.77 cl_msp 71.3 ± 8.85 47.3 ± 12.82 42.3 ± 7.39 31.7 ± 2.95 as 87.2 ± 14.10 109.9 ± 14.97 73.4 ± 7.23 78.2 ± 14.87 cs 97.4 ± 28.90 127.2 ± 31.56 34.4 ± 5.83 33.5 ± 7.20 je 119.5 ± 19.14 185.6 ± 34.58 76.0 ± 12.66 56.9 ± 11.52 mz 86.6 ± 19.63 108.4 ± 28.80 95.0 ± 12.93 178.2 ± 42.51 ml_a 104.2 ± 32.54 144.8 ± 57.25 31.4 ± 3.08 26.2 ± 9.39 ml_b 68.2 ± 8.77 80.0 ± 12.90 82.3 ± 19.76 47.2 ± 12.23 ub_1 140.3 ± 16.58 171.5 ± 19.28 67.5 ± 12.89 111.0 ± 26.09 ub_2 128.2 ± 27.06 204.3 ± 49.60 95.2 ± 5.22 91.7 ± 9.76 vt_ps 107.4 ± 7.40 138.2 ± 12.15 70.2 ± 16.40 84.3 ± 22.80 f-test ns * ** ** sed 42.4 16.7 28.5 external bracts cl_c3 57.3 ± 5.56 42.9 ± 4.49 37.6 ± 8.53 18.2 ± 2.77 cl_msp 27.6 ± 0.77 12.8 ± 3.78 21.2 ± 3.39 16.1 ± 2.57 as 45.5 ± 4.93 37.0 ± 4.82 40.8 ± 3.63 42.6 ± 10.25 cs 34.1 ± 8.87 38.1 ± 6.55 32.1 ± 2.04 24.2 ± 3.38 je 64.7 ± 12.85 80.5 ± 13.40 54.4 ± 10.96 53.7 ± 16.83 mz 27.5 ± 2.79 21.7 ± 5.90 48.1 ± 5.17 34.0 ± 8.95 ml_a 31.9 ± 6.81 36.6 ± 1.91 27.4 ± 3.67 17.9 ± 5.71 ml_b 27.5 ± 1.78 27.2 ± 5.04 31.5 ± 10.47 20.9 ± 7.52 ub_1 80.9 ± 8.72 93.9 ± 10.09 35.8 ± 4.19 38.4 ± 2.52 ub_2 58.8 ± 16.82 71.4 ±20.49 39.1 ± 6.21 19.1 ± 6.07 vt_ps 57.6 ± 2.45 59.4 ± 4.90 39.2 ± 6.46 36.7 ± 3.85 f-test ** ** ns ** sed 11.5 12.7 10.8 galieni et al. biochemical differences among artichoke genotypes 29 (i.e. receptacle tpc_rec, tfc_rec, abts_rec and dpph_rec and external bracts tpc_bra, tfc_bra, abts_bra and dpph_bra), and the eleven artichoke accessions, were summarized by pca. the results, on the basis of harvest time, are graphically displayed in two correlation bi-plots (figs. 3a and 3b, respectively); in table 4 are reported the factor loadings, the eigenvalues and the percentage of the explained variance. in early-spring harvesting time (i.e. referring to main capitula data), the first and second principal components explained 90.2% of the total data variability (80.7 and 9.5% for pc1 and pc2, respectively) (table 4). the variables were grouped into two distinct clusters, separated by pc2: in the upper right quadrant, we found all the chemical data related to the external bracts samples (tpc_bra, tfc_bra, abts_bra and dpph_bra) while in the bottom right section, those related to the receptacle ones (tpc_rec, tfc_rec, abts_rec and dpph_rec); all the variables reached high pc1 scores (scores from 0.952 to 0.788 for tpc_rec and tfc_rec, respectively) (table 4). regarding genotypes, cl_c3, ub_2, je, vt_ps and ub_1 clustered separately on the right along pc1 and were positively associated with all the investigated variables; conversely, cl_msp exhibited the highest negative pc1 score (-1.533) followed by ml_b, mz, cs, as and ml_a (fig. 3a). with respect to the mid-spring harvesting time (i.e. referring to first order capitula data), the eigenvalues for pc1 and pc2 were 5.28 and 1.71, respectively, thus capturing 87.4% of the total data variability (table 4). again, variables were clearly separated by pc2 while genotypes by pc1 (fig. 3b). in particular, all the variables reached high pc1 scores with the exception of tfc_bra, which was mainly correlated with pc2 (scores: 0.328 and 0.886 for pc1 and pc2, respectively; table 4). je and mz showed the higher pc1 scores (1.302 and 1.568, respectively) despite they performed very differently with respect to pc2 (scores: 1.668 and -1.679 for je and mz, respectively). as a consequence, je was related to higher tpc, abts and dpph values in the waste fractions (external bracts) while mz in the edible parts (receptacle). pca proved to be a useful tool to summarize the biochemical characteristics of the different investifig. 3 two dimensional principal component analysis (pca) correlation bi-plot [main capitula/first harvest time (a) and first order capitula/second harvest time (b)]: symbols show the standardized scores on pc1 (x-axis) and pc2 (yaxis) for the eleven artichoke genotypes (see table 1 for the list of acronyms); vectors coordinates represent the correlations between standardized variables [total polyphenols content in receptacle and external bracts (tpc_rec and tpc_bra, respectively), total flavonoids content in receptacle and external bracts (tfc_rec and tfc_bra, respectively), radical scavenging activity obtained from two different assays in the receptacle and external bracts (abts_rec and abts_bra, respectively; dpph_rec and dpph_bra, respectively)] and pcs. table 4 principal component analysis (pca): factor loadings, eigenvalues and percentage of the explained variance variables main capitula first order capitula pc1 pc2 pc1 pc2 tpc_core 0.952 -0.093 0.941 -0.288 tfc_core 0.788 -0.561 0.853 -0.242 dpph_core 0.927 -0.286 0.777 -0.477 abts_core 0.916 -0.163 0.831 -0.356 tpc_bratee 0.903 0.418 0.926 0.228 tfc_bratee 0.823 0.084 0.328 0.886 dpph_bratee 0.933 0.220 0.762 0.572 abts_bratee 0.930 0.315 0.912 0.226 eigenvalue 6.455 0.761 5.284 1.711 explained variance (%) 80.692 9.515 66.056 21.382 adv. hort. sci., 2019 33(1): 23-31 30 gated artichoke genotypes, and clear conclusions could be obtained, confirming the results in terms of single investigated biochemical parameters. in particular, the reference genotype cl_c3 and the accessions ub_1, ub_2, je and vt_ps confirmed higher tpc, tfc and, consequently, antiradical activity in the main capitula, highlighting their important attitude for fresh consumption. this greater head quality was maintained during all the growing season (i.e. as quality traits of the first order capitula) only for ub_1, je and vt_ps. other genotypes, such as as and, principally, mz, were clearly characterized by higher bioactive compounds in the first order flower heads and by smaller capitula. these accessions could represent a promising potential as germplasm for future breeding programs to select elite cultivars, characterized by: (i) higher and stable quality traits suitable for fresh consumption (i.e. je); (ii) high concentrations of biochemical compounds, especially in the waste products, to be used for processing or pharmaceutical applications although further investigations on smaller and waste flower heads are needed. 4. conclusions in conclusion, our results confirm that the capitula of globe artichoke could be considered a functional food thanks to its relevant content of bioactive compounds accumulated in both receptacle and external bracts. the properties of the external bracts could be usefully exploited for other end-use purposes, although they are still edible fractions (fratianni et al., 2007; pandino et al., 2011). a great and appreciable variation among genotypes in terms of chemical composition and nutritional value exists. such biodiversity of the accessions of abruzzo and marche regions should be exploited and utilized, taking into account the peculiarity of each genotype (in terms of both yield and quality) as well as the actual end-use which can be reached. acknowledgements we 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m., 2013 polyphenolic composition in different organs of tunisia populations of cynara cardunculus. l. and their antioxidant activity. j. food nutr. res., 1: 1-6. impaginato 363 adv. hort. sci., 2018 32(3): 363-369 doi: 10.13128/ahs-22320 assessment of vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers by application of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate s.a. mirjalili 1 (*), b. kavoosi 2, y. peyro 3 1 imam khomeini higher education center, agriculture research, education and extension organization, teheran, iran. 2 fars agricultural research and education center, agriculture research, education and extension organization, teheran, iran. 3 faculty of agriculture, science and research branch, islamic azad university, tehran, iran. key words: cumin, essential oil, hydroxyquinoline sulfate, rosa hybrida. abstract: natural preservatives such as herbal essential oils have potential ability for extending postharvest vase life of cut flowers. in this study, application effect of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate on vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers were investigated. a factorial experiment with three levels of each in different time after harvesting was conducted. results showed that usage of different level of cumin essential oil and hydroxyquinoline sulfate had significant effects on rose attributes at the level of 0.05. the results showed that the interaction effect of cumin essential oil and hydroxyquinoline sulfate in measuring time was significant (p<0.05) on all of parameters except for anthocyanin content in rose petals in a way that the highest amount for measured traits was obtained with treatment of 150 mg l-1 cumin essential oil and 400 mg l-1 8-hydroxyquinoline sulfate. 1. introduction roses have a critical role in the manufacturing of various medicinal and nutritional products. rosa, known as the symbol of affection and elegance in iran, is one of the leading cut flower in global floriculture trade including our country (butt, 2005; zamani et al., 2011). the genus rosa belongs to the family rosaceae and includes 200 species and more than 18,000 cultivars (ahmad et al., 2013). cut flower trading is the prime purpose of rose cultivation, but short vase life is the most crucial problem. commercially, post-harvest longevity of cut flowers is of importance. many studies have therefore focused on its quality both in pre and postharvest periods (mirjalili, 2015). the most common physiological and morphological responses after harvesting are wilting or bent neck caused by pathogens (*) corresponding author: abmirjalili@gmail.com citation: mirjalili s.a., kavoosi b., peyro y., 2018 assessment of vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers by application of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate. adv. hort. sci., 32(3): 363-369 copyright: © 2018 mirjalili s.a., kavoosi b., peyro y. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 363-369 364 especially bacteria, resulted in decreasing the vase life of cut rose flowers (leiv and hans, 2005; thwala et al., 2013). the development of such symptoms is resulted from vascular occlusion, mainly located in the basal stem end (lü et al., 2010; farahi et al., 2013). study on effects of natural plant products, including essential oils as preservatives hasted during last decades (elgimabi and ahmed, 2009). in nature, essential oils play an important role in the protection of the plants as antibacterial, antiviral, antifungal and insecticides (bakkali et al., 2008). cumin (cuminum cyminum l.) is an aromatic plant in the family apiaceae. cumin seeds are rich of essential oil especially cumin aldehyde, used as a stimulant as well as carminative and therapeutics (iacobellis et al., 2005; asghari marjanloo et al., 2009). there are reports on preservative effects of plant essential oils on other plants pathogens, such as tea essential oil on the botrytis in grape (jobling, 2000) and antifungal effect of persian thyme essential oil on strawberry (nabigol and morshedi, 2011). positive effects of plant essential oils have been reported on longevity of cut flowers’ vase life (deans and ritchie, 1987; dudai et al., 1999). thwala et al. (2013) used cumin essential oil for decreasing degradation and vessel boring in orchids resulted in delay of senescence. 8-hydroxyquinoline sulphate (8-hqs) as a very important germicide in preservatives is used in floral industry. hqs acts as an anti-microbial agent and increases water uptake (ali and hassan, 2014). the positive effect of 8-hqs and calcium chloride alone or in combination with 4% sucrose as chemical preservative solutions to improve postharvest quality of cut gerbera flowers has been shown (soad et al., 2011). it reported that hqs extended the vase life of rose cut flowers, whereas sucrose can promote the effect of hqs (ichimura et al., 1999). it documented that vase life and postharvest quality of different cut flowers were enhanced by 8-hqs treatment through improving water uptake, fresh weight and carbohydrate content (kim and lee, 2002; hassan et al., 2003; 2004; ali and hassan, 2014). despite the valuable reports on successful use of various phytochemicals for improving longevity of fresh cut flowers, screening for introducing and developing an exact, cheap and easy-to-use preservative is of importance for floriculture (wu et al., 2016). the objective of this study was to investigate the effect of different cumin essential oil concentrations and 8-hydroxyquinoline sulphate (8-hqs) on vase life and postharvest quality of cut rose flowers in different measuring time. 2. materials and methods cut rose (rosa hybrida cv. angelina) flowers were obtained from the commercial greenhouse around shiraz. cut rose flowers were harvested when florets were not opened but sepals were turned back and separated from petals during september 2014 and immediately transported to the laboratory. prior to insert in solutions, flowering stems of plants were cut under water to prevent air entrance into the xylem conduits that were opened by cutting. this factorial experiment was conducted in randomized complete blocks design with three replications. treatments were 8-hydroxyquinoline sulfate (8-hqs) at four level (0, 200, 400 and 600 mg·l-1) and cumin essential oil at three level (0, 100, 150 mg·l-1) in three measuring time (1st day, 8th day and 16th day) after treatment. after the duration of treatments, the flowers were placed in beakers containing 400 ml distilled water during the vase life evaluation period. the control flowers were kept in distilled water. replications included five flowers per treatment. vase life room conditions was 12 hours day length, 18±2°c, 60±5% rh and 12 μmol s-1 m-2 light intensity and measured traits were vase life determination (days), petal fresh weight/dry weight rate (%), flower diameter (mm), anthocyanin content (mg 100 g-1 f.w.), relative water content (rwc) (%), leakage of ions (%), catalase enzyme activity (cat) (ua·mg-1 pro), peroxidase enzyme activity (pod) (ua·mg-1 pro), membrane stability index (msi) (%). vase life determination in this study, vase life was considered as the time during which cut-flower can keep its marketability quality and before senescence symptoms including bending of petal margins and wilting are appeared (singh, 1994). cut-flower durability was evaluated from cut flower treatment till their ornamental value has disappeared. leakage of ions floret samples from each treatment were taken on first day and were repeated on day 7 for determining ions leakage by using the method of sairam et al. (1997). two florets samples (0.2 g) were taken and placed in 20 ml of double distilled water in two different 50 ml flasks. the first one was kept at 40°c for 30 min while the second one was kept at 100°c in boiling water bath for 15 min. the electric conductivity of the first (c1) and second (c2) samples were measured with a conductivity meter. the leakage of ions was expressed as the membrane stability index according mirjalili et al. vase life and postharvest quality of cv. angelina cut rose 365 to the following formula (ezhilmathi et al., 2007): membrane stability index (msi)=[1-(c1/c2)] × 100 (eq. 1) petal anthocyanin the amount of 200 mg petal samples was pulverized in 3 ml 99:1 (v/v) methanol and hydrochloric acid and obtained extracts were centrifuged at 12000 rpm for 20 min at 4°c. supernatants were kept in 4°c and under darkness condition for 24 h. after that, light absorption was estimated by spectrophotometer in 550 nm wavelength and using silence coefficient (ɛ =33000 mol2 cm-1) (krizek et al., 1993). petal membrane stability index for determining petal membrane stability, two samples of petals each including 200 mg of each replication were weighted and dipped in 10 ml double distilled water. one of them was placed in 40°c benmary for 30 min and second one at 100°c benmary for 15 min. after reaching to the room temperature, electrical conductivity of the solutions was measured with a ec meter and the stability percent of the membrane was determined according ezhilmathi et al. (2007), as equation 1. enzymes assays peroxidase (pod) enzyme was extracted from 200 mg homogenized samples in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 28 mm guaiacol, 5 mm hydrogen peroxide and crude extract was prepared and its absorbance at 470 nm was detected during 1 min, using spectrophotometer (bio-rad). enzyme activity was expressed as absorption delta of 470 nm per mg protein (chance and maehly, 1995). catalase (cat) enzyme was extracted from 200 mg samples homogenized in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. the supernatant was transferred to 15 ml tubes and referred to enzyme extract. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 10 mm hydrogen peroxide and crude extract was prepared and its absorbance at 240 nm was detected using a spectrophotometer (bio-rad). enzyme activity was described by measuring the conversion rate of hydrogen peroxide to water and oxygen molecules, as the decrease of absorbance per time per mg of protein (8). enzyme activity was expressed as absorption delta of 240 nm per mg protein. all steps of enzyme extraction were performed on ice. cumin essential oil and 8-hydroxyquinoline sulphate (8-hqs) were purchased from zardband pharmaceuticals medicinal plants production co., yasuj, iran and were used. statistical analysis all data were analyzed for significant differences using analysis of variance (anova) using the sas (statistical analysis system) statistical package (sas institute, cary, nc, usa). data were then subjected to mean separation by the least significant difference test (lsd) at p<0.05. 3. results according to results of variance analysis, interaction effects of cumin essential oil (ceo), 8-hydroxyquinoline sulfate (hqs) application and measuring times was significant (p<0.05) on measured traits of vase life, petal fresh/dry weight rate, flower diameter, relative water content (rwc), leakage of ions, catalase enzyme activity (cat), peroxidase enzyme activity (pod), membrane stability index (msi) except for anthocyanin content. interaction effect of hqs and measuring times was insignificant on anthocyanin content too, while main effects of each factor and interaction effects of ceo × hqs and hqs × t were significant (p<0.05) (table 1). s.o.v df mean squares vase life petal dry weight flower diameter anthocyanin content relative water content leakage of ions cat pod msi ceo 2 16.02 * 0.52 * 2.686 * 0.0020 * 17.33 * 37.31* 26.45 * 23.30 * 69.35 * hqs 3 45.99 * 0.30 * 1.542 * 0.0016 * 48.76 * 44.97* 51.20 * 54.21 * 47.07 * time 2 11.33 * 0.78 * 1.033 * 0.0011 * 93.32 * 52.47 ns 21.30 * 23.22 ns 99.33 * ceo×hqs 6 24.35 * 0.89 * 2.037 * 0.0034 * 58.25 * 41.25 * 35.15 * 35.81 * 49.99 * ceo×t 4 20.46 * 0.55 * 2.432 * 0.0037 * 48.39 * 39.56 * 28.14 * 38.92 * 58.23 * hqs×t 6 21.32 * 0.53 * 3.321 * 0.0061 ns 59.41 * 45.81 * 32.18 * 40.25 * 63.28 * ceo×hqs×t 12 25.41 * 0.24 * 1.421 * 0.0061 ns 39.99 * 21.34 * 45.23 * 39.48 * 48.49 * table 1 analysis of variance for measured traits in cut rose (rosa hybrida cv. angelina) flowers treated by cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate in different measuring times *,**, shows significant differences at 5%, 1%, respectively. ns= not significant. ceo = cumin essential oil. hqs= 8-hydroxyquinoline sulfate adv. hort. sci., 2018 32(3): 363-369 366 concerning the mean comparison, the maximum vase life was obtained by application of 100 mg·l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline sulfate. however, the minimum vase life was observed in control treatments (fig. 1). the greatest petal fresh/dry weight rate was evident in the treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and the least with control treatments (fig. 2). the results indicated that the highest flower diameter was found in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and the lowest diameter in control treatments (fig. 3). relative water content showed the maximum and minimum value in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and control treatment, respectively (fig. 4). the greatest amount of ions leakage in control treatments and the lowest amount in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate were found (fig. 5). according to results of mean comparison, the highest catalase enzyme activity was attained in 150 mg·l-1 cumin essential oil and 400 mg· l-1 8-hydroxyquinoline sulfate, while the lowest of that was reported in control treatments (fig. 6). the greatest peroxidase enzyme activity was observed in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8hydroxyquinoline sulfate and the least activity in control treatments (fig. 7). according to the obtained results, the highest membrane stability index was obtained in 150 mg·l-1 cumin essential oil and 400 mg l-1 8-hydroxyquinoline sulfate (fig. 8). fig. 1 changes of vase life under different levels of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs). fig. 2 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on petal dry weight (g) in different measuring times. fig. 3 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on flower diameter (mm) in different measuring times (days). fig. 4 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on relative water content (rwc) (%) in different measuring times (days). fig. 5 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on leakage of ions (%) in different measuring times (days). mirjalili et al. vase life and postharvest quality of cv. angelina cut rose 367 quality of cut rose (rosa hybrid cv. angelina) flowers in different measuring time. application of 100 mg·l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline sulfate increased vase life of cut rose flowers. this result was in accordance with results of hussein (1994) and knee (2000). the application of 8-hqs may prevent the accumulation of microorganisms in xylem vessels and suppressed the xylem occlusion due to its role as anti-microbial agent and hence, it might reduce stem plugging. essential oils like ceo play an important role in the protection of the plants as antibacterial, antiviral, antifungal, insecticides and also against herbivores by reducing their appétit for such plants (bakkali et al., 2008). petal fresh/dry weight rate was improved significantly by application of 100 mg·l-1 cumin essential oil and 600 mg.l-1 8hydroxyquinoline sulfate. these results are in line with results of ali and hassan (2014) on strelitzia cut flowers with application of 8-hydroxyquinoline sulfate and gibberlic acid treatments. the application of 8-hqs may reduce the plasmolysis of cells which occurred when the rate of cellular water loss is too rapid. the cut rose flowers reached to the highest diameter with application of 100 mg· l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline sulfate. these findings are in according to reports of kim and lee (2002). hqs not only prevents the vascular obstruction caused by the microorganisms, but also prevents the blockage stimulated by the plant itself. the highest relative water content in cut rose flowers was related to treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate. these results are similar to knee (2000) findings on cut carnation flowers. leakage of ions was occurred in control treatments in the highest amount. essential oil of cumin mainly conjugated to compounds that have known as phenolic compounds, are responsible for pathogen control in plants (plotto et al., 2003). these compounds prevent senescence and wilting by their antibacterial property and reducing the ph of the environment (elgimabi and ahmed, 2009). catalase and peroxidase enzymes activities increased significantly by treatments of 150 mg·l-1 cumin essential oil and 400 mg· l-1 8-hydroxy quinoline sulfate. these results are consistent with results of ranjbar et al., 2015. catalase is an important biological factor with major function in superoxide metabolism and plays an important role in releasing oxygen and hydrogen peroxide free radicals and prevents creation of hydroxyl radicals (spanou et al., 2012). peroxidase has different biologfig. 6 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on catalase enzyme activity (cat) (ua mg-1 pro) in different measuring times (days). fig. 7 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on peroxidase enzyme activity (pod) (ua mg-1 pro) in different measuring times (days). fig. 8 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on membrane stability index (msi) (5) in different measuring times (days). 4. discussion and conclusions the results showed that the application of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) had positive effect on vase life and postharvest 368 adv. hort. sci., 2018 32(3): 363-369 ical functions such as detoxification of hydrogen peroxide, lignin biosynthesis, hormonal signaling and response to stress (gao et al., 2010). maybe the treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate decreases oxidative stresses in cut rose flowers (hassan and ali, 2014). membrane stability index showed the highest percent in treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate. these findings are compatible to results of kazemi and ameri (2012). they showed the positive effect of herbal essential oils of thyme and lavender on the stability of the membrane and reduction of mda. the senescence of cut flowers with hormonal regulatory mechanism is involved in changing the physical and biochemical features of cellular membrane (buchanan wollaston, 1997). oxidative membrane injury allows the mixing of the normally separated enzyme (ppo) and oxidizable substrates (polyphenols), which lead to browning (hodges, 2003). according to palma et al. (2002), the herbal essential oils by preventing the activity of oxygen species reduce the lipid peroxidation in cell membrane and the concentration of mda. plant essential oils are bioactive in the vapor phase, and this makes them fumigants for postharvest rotting fungi control in fruits and grains (paster et al., 1995; hammer et al., 1999; feng and zheng, 2007). different studies showed postharvest disease control in different fruit species by using biological agents including essential oils (bishop and thompdon, 1997; feng and zheng, 2007; amiri et al., 2008). a limiting factor in cut flower marketing is postharvest senescence. there are many reports used different materials for extending rose cut flower vase life. we studied application of cumin essential oil and 8hydroxyquinoline sulfate. they had positive effects (p<0.05) on vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers. results showed they affect some growth and development parameters such as 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moghbeli et al., 2019). one of the farming practices is concurrent cultivation of two or more crops in the same field which is experienced in (*) corresponding author: yaegoob@yahoo.com citation: raei y., sayyadi ahmadabad m., ghassemi­ golezani k., ghassemi s., 2020 ­ pinto bean and black mustard responses to bio‐fertilizers under intercropping system. ‐ adv. hort. sci., 34(2): 175­182. copyright: © 2020 raei y., sayyadi ahmadabad m., ghassemi­golezani k., ghassemi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 175­182 176 many regions of the world (tüzel and öztekin, 2017). some reasons have been identified for farmers engaging in intercropping which are still valid today. first, it leads to increase in the utilization of environ­ mental factors. this has both space and time dimen­ sion. plants are different in rooting habitat and have different nutrient requirements. thus, the intercrop­ ping of plants can increase the utilization of nutri­ ents, water and light. also, intercropping can lead to reduction of adverse conditions in the agroecosystem (lithourgidis et al., 2011). intercropping may also lead to better soil management because of the fact that may crops overlap in terms of the time they are in the soil. other economic reasons such as depend­ ability of returns and increased returns from the same piece of land may make farmers adopt inter­ cropping (alabi and esobhawan, 2006). watikai et al. (1993) and willy (1990) confirmed that increasing the yield of biomass in intercropping is due to the more absorption of light. the highest performance is achieved when intercropping canopy is composed of two layers: (1) tall plants with narrow leaves and high photosynthetic capacity; (2) dwarf plants with lying leaves and low photosynthetic capacity. in general, the productivity in intercropping is more than sole cropping (raei et al., 2015). among nutrient elements, nitrogen is an impor­ tant nutrient and has vital functions in plant growth and development. nitrogen deficiency imposes most limits on crop production compared to other nutri­ ents. with large areas of the arable land in iran being located in arid and semiarid regions, most of them face low organic matter content as well as nitrogen deficiency and also, to achieve an economically sound production, nitrogen plays a significant role in these regions (joorabi et al., 2015). on the contrary, slow­release nitrogen fertilizers are effective and inexpensive alternative to soluble n (jiao et al., 2005). the yield of pea in intercropping of pea and wheat increased by application of slow release nitro­ gen fertilizer (abbady et al., 2016). in all around of the globe, for achieved high yield of plants, the chemical fertilizers are extensively being used. however, this type of fertilizers has devastating effects on the health of the soil animals. a better alternative of these chemicals might be to exploit the microbial capabilities to be served as bio­fertilizer (tomer et al., 2016). bio­fertilizers colonize at the rhi­ zosphere and improve nutrient accessibility of plants and increase the growth of plants. microorganisms residing in rhizosphere immensely facilitate trace ele­ ment’s uptake. they may act as biocontrol agent, by means of antagonistic activity against phytopatho­ genic microorganisms, interfering in the bacterial quorum sensing systems, etc. however, bio­fertilizers perform more than one mechanism for accomplish­ ing plant growth enhancement (kumar et al., 2014; dutta and patel, 2016). black mustard is an important oilseed crop. it is often grown as an intercrop or mixed crop either with pulses or cereals crops, but its productivity is very low due to improper combination (kumar et al., 2014). bean is also one of the most important food supplements for human, and its protein content is rich (arija et al., 2007). it is also tolerant to shadow and can be planted in intercropping system and grows well. it can increase the soil nitrogen by nitro­ gen fixation (kowal and kassam, 1978). intercropping of legumes with non­legumes increases yield per unit area, because they use different nitrogen sources and have low competition for nitrogen (haugard­ nielsan et al., 2001). the importance of this pulse crop is based on its good nutritive composition and its high market value, which mainly depends on the consumption quality of the product (nutritional and culinary quality of either the seed or the pod). thus, the present investigation was carried out to study pinto bean and black mustard responses to bio­fertil­ izers and chemical nitrogen fertilizer, intercropping system and interaction of intercropping system × nitrogen fertilizer. 2. materials and methods field conditions the experiment was conducted in 2016 at the research farm of the faculty of agriculture, university of tabriz, iran (latitude 38°05ʹ n, longitude 46°17’ e, altitude 1360 m above sea level with the mean annual rainfall of 285 mm). some physical and chemical properties of soil in experi­ mental area and averages of maximum and minimum temperatures and rainfall during the work in 2016 were shown in table 1. experimental design and treatments a factorial set of treatments was arranged with three replications. in this experiment, fertilizer treat­ ments were control (non­fertilizer), bio­fertilizers (azotobarvar 1 and barvar 2), bio­fertilizers + 50% the recommended chemical urea fertilizer (125 kg/ha) and bio­fertilizers + 100% chemical fertilizer (250 raei et al. ‐ pinto bean and black mustard intercropping and bio‐fertilizers 177 kg/ha). azotobarvar 1 contains the azoto bacter­ vinelandii (strain o4) and barvar 2contains the pan­ toea agglomerans (strain p5) and pseudomonas puti­ da (strain p13). the cropping patterns comprised pure stands of bean and black mustard, additive intercropping with a ratio of 50% black mustard + optimum density of pinto bean mono cultures and an additive intercropping with optimum density of two species in mono cultures. measurements yield and yield components. at maturity and when the moisture content of seeds decreased by about 18%, 10 plants were harvested from each plot and 100 grains weight of pinto bean and black mustard were recorded. also to determine of grain and bio­ logical yields, an area equal to 1 m2 was harvested from middle part of each plot considering marginal effect and dried in an oven at 75°c for 48 hours. subsequently, biological and grain yields per unit area were determined. harvest index was calculated by the following equation: harvest index = (grain yield/biological yield) × 100 evaluative indices of intercropping land equivalent ratio (ler), as an agronomic index, indicates the efficiency of intercropping for using the resources of the environment compared with mono cultures (mead and willey, 1980). the value of unity is the critical value. when the ler is greater than one, the intercropping improves the growth and yield of the cultivars. in contrast, when ler is lower than one the intercropping negatively affects the growth and yield of the plants grown in mixtures (caballero et al., 1995). the ler was calcu­ lated as: ler= ypb + ybp yp yb where yp and yb are the yields of pinto bean and black mustard, respectively, as sole crops and ypb and ybp are the yields of pinto bean and black mustard, respectively, as intercrops. relative value total (rvt) as an economic index proposed by schultz et al. (1982). this index is widely used now and has been used by many researchers. the rvt was calculated as: rvt = ap1 + bp2 ami where, p1 and p2 are the yields of two different crops in intercropping and m1 and m2 are the yields of those of these crops in monocultures (m1> m2). also, a and b are the market prices of crop 1 and 2 respec­ tively. if the rvt >1, the mixture crop has the advantage and if the rvt <1, pure stand will have an economic advantage. if rvt =1, then these two methods are not economically advantageous to each other. relative yield is the ratio of the species response in the mixture to the species response when grown in monoculture. relative yield total (ryt) is the total ry of the two associated species, as shown in below: ryt = rya + ryb rya = ya in mixture /ya in monoculture ryb = yb in mixture /yb in monoculture a ryt of 1 indicates that species a and b are mak­ ing demands on the same resources. if ryt is <1, this table 1 ­ some physical and chemical properties of experimental soil and averages of maximum and minimum temperatures and rain­ fall during the work in 2016 physical and chemical properties of experimental soil depth (cm) ec (ds/m) ph organic carbon (%) n (%) p (mg/kg) k (mg/kg) fe (mg/kg) ca (mg/g) sand (%) silt (%) clay (%) soil type 0­35 2.77 7.75 0.37 0.04 4.90 255 2.60 780 74 14 12 sandy loam months averages of maximum and minimum temperatures and rainfall temperature (°c) rainfall (mm) april 9.4 78.2 may 16.9 13.5 june 22 14.8 july 28 0 august 29.4 15 adv. hort. sci., 2020 34(2): 175­182 178 shows antagonism between species a and b. if the ryt is >1, the yield of the mixture is greater than that of the single and is preferred. the relative crowding coefficient (rcc) is a mea­ sure of the relative dominance of one species over the other in a mixture (de wit, 1960). the rcc was calculated as: rcc = (ypb/yp)/(ybp/yb) where yp and yb are the yields of pinto bean and black mustard, respectively, as sole crops and ypb and ybp are the yields of pinto bean and black mustard, respectively, as intercrops. if rcc= 1, the amount of crop in the mixture will be equal to monocropping. also, if rcc <1 indicates that the amount of the product in the mixture has decreased relative to solecrop and if rcc >1, the yield of the mixture is higher than that of pure stand of crops and the mixing is beneficial. statistical analysis analyses of variance for data based on theexperi­ mental design and comparison of means (duncan multiple range test) at p≤0.05 were carried out, using mstatc software. excel software 2013 was used to draw figures. 3. results analyses of variance showed significant effects of cropping pattern and fertilizers on 100 grains weight, biological and grain yields per unit area of pinto bean and also biological and grain yields per unit area of black mustard. 100 grains weight of black mustard was significantly affected by fertilizer treatments and interaction of cropping pattern × fer­ tilizers (table 2). the highest 100 grains weight, biological and grain yields per unit area of pinto bean and grain yield of black mustard were achieved in pure stands of bean and black mustard and also inbio­fertilizers + 100% chemical fertilizer (urea). maximum biological yield of black mustard was achieved inpure stands of black mustard culture, but there were no significant differ­ ences with additive intercropping with optimum den­ sity of two species in mono cultures treatment. also, maximum of this trait was achieved in bio­fertilizers + 100% chemical fertilizer (urea) but, there were no significant differences with bio­fertilizer + 50% chem­ ical fertilizer (table 3). significantly, maximum 100 grains weight of black mustard in different cropping patterns was observed in intercropping with a ratio of 50% black mustard + optimum density of pinto bean mono cultures and bio­fertilizers + 100% chemical fertilizer (urea). generally, in other cropping patterns there were no considerable differences between fertilizer treat­ ments (fig. 1). evaluation of intercropping efficiency of treat­ ments indicated that land equivalent ratio (ler) is >1 in all intercropping and fertilizer treatments and this showing the superiority of intercropping compared to single cropping. maximum of ler and relative yield total (ryt) were attended in optimum density of two species and bio­fertilizers + 100% chemical fertilizer (urea). maximum relative value total (rvt) is related to optimum density of two species with non­fertiliz­ er. maximum of relative crowding coefficient (rcc) was related to non­fertilizer treatment in 50% of optimum density of two species in pinto bean and optimum density of two species in black mustard (table 4). table 2 ­ analysis of variance of the agronomic traits in pinto bean and black mustard under different cropping patterns and fertilizer treatments ns, * and **: non­significant and significant at p≤0.05 and p≤0.01, respectively. source df mean square pinto bean (phaseolus vulgaris l.) black mustard (brassica nigra l.) 100 grains weight biological yield grain yield harvest index 100 grains weight biological yield grain yield harvest index replication 2 72.94 3680597 1131685 2.03 0.01 1786035 157796 2.60 cropping pattern 2 79.10 ** 40310058 ** 9924157 ** 0.70 ns 0.01 ns 122550980 ** 1961700 ** 5.77 ns fertilizer (f) 3 140.98 ** 37336605 ** 9970014 ** 17.55 ns 0.14 * 24340924 ** 832223 ** 2.44 ns c × f 6 4.78 ns 989636 ns 236278 ns 6.49 ns 0.17 ** 8957965 ns 11308 ns 6.06 ns error 22 2.75 559597 133108 10.29 0.04 5045637 21412 3.77 cv % ­ 5.23 16.85 16.27 6.40 4.46 17.12 8.53 14.59 raei et al. ‐ pinto bean and black mustard intercropping and bio‐fertilizersn 179 cantly increased the field performance of these plants (table 3, fig. 1), followed by bio­fertilizers + 50% chemical fertilizer. however, biological and grain yields for black mustard was affected as similar to bio­fertilizer + 50% chemical fertilizer (urea) with bio­ fertilizers + 100% chemical fertilizer (urea). therefore, bio­fertilizer application resulted in decreasing 50% of chemical fertilizing. chemical fer­ tilizer has various negative environmental effects such as soil, water and air pollution, which increase environmental production cost (moradi et al., 2011). bio­fertilizer as essential components of organic farming, play a vital role in maintaining long term fer­ tility and sustainability of soil. bio­fertilizers have the ability to access a major part of nutrients for growing plant along with growth promoting factors (cordovilla et al., 1999). significant reduction of grain and biological yields in intercropping (table 3) was attributed to interspe­ cific competition between two crops (bybee­finley and matthew, 2018). pilbeam et al. (1994) has noted that grain yield of maize in sole culture was greater than intercropping with bean. competition for nutri­ ent uptake and deficiency of nitrogen transport are responsible for the reduction of maize yield in inter­ cropping with legumes (tomar et al. , 1988). however, there were not significant differences between sole cropping and optimum density of two species in intercropping system. therefore, the pres­ ence of pinto bean plants hasn’t considerable inter­ specific competition on black mustard plants. always grain yield of plants did not reduce in intercropping. fig. 1 ­ mean 100 grain weight of black mustard for interaction of cropping pattern × fertilizers. different letters indicate significant difference at p ≤ 0.05 (duncan test). c1, c2, c3= pure stands of bean and black mustard,additive intercropping with a ratio of 50% black mustard + opti­ mum density of pinto bean mono cultures and additive intercropping with optimum density of two species in mono cultures, respectively. f1, f2, f3, f4= control (non­ fertilizer), bio­fertilizers (azotobarvar 1 and barvar 2), bio­fertilizer + 50% chemical fertilizer urea and bio­fertili­ zers + 100% chemical fertilizer (urea), respectively. table 3 ­ means of the agronomic traits in pinto bean and black mustard under different cropping patterns and fertilizer treatments different letters in each column indicate significant difference at p≤0.05. means are average values of three replicates ± standard errors. c1, c2, c3= pure stands of bean and black mustard, additive intercropping with a ratio of 50% black mustard + optimum density of pinto bean mono cul­ tures and additive intercropping with optimum density of two species in mono cultures, respectively. f1, f2, f3, f4= control (non­fertilizer), bio­fertilizers (azotobarvar 1 and barvar 2), bio­fertilizer + 50% chemical fertilizer (urea) and bio­fertilizers + 100% chemical fertilizer (urea), respectively. treatment pinto bean (phaseolus vulgaris l.) black mustard (brassica nigra l.) 100 grains weight (g) biological yield (kg/ha) grain yield (kg/ha) biological yield (kg/ha) grain yield (kg/ha) cropping pattern c1 34.08± 5.83 a 6505.70± 80.65 a 3269.90± 57.18 a 15181.70± 123.21 a 2066.60± 45.45 a c2 32.19± 5.67 b 3793.90± 61.59 b 1911.80± 43.72 b 9437.90± 97.14 b 1272.60± 35.67 c c3 29.00±5.38 b 3013.80± 54.89 c 1543.10± 39.28 c 14737.50±121.39 a 1802.10± 42.45 b fertilizer treatments f1 26.54±5.15 d 2117.40± 46.01 d 1048.90± 32.38 d 11022.00± 104.98 c 1372.80± 37.05 c f2 31.23± 5.58 c 3537.50± 59.47 c 1771.20± 42.08 c 12606.00± 112.27 1542.00±39.26 b f3 33.37± 5.77 b 5138.70± 71.68 b 2688.20± 51.84 b 14266.00± 119.44 a 1959.50± 44.26 a f4 35.87± 5.98 a 6777.60± 82.32 a 3458.10± 58.80 a 14581.00± 120.75 a 1980.70± 44.50 a 4. discussion and conclusions according to the results, bio­fertilizers + 100% chemical fertilizer (urea) was the best fertilizer treat­ ment in pinto bean and black mustard as, it signifi­ 180 adv. hort. sci., 2020 34(2): 175­182 as an illustration, long et al. (2001) showed that the grain yield of wheat increased 28 to 30% in intercrop­ ping with soybean compared to monoculture. the land equivalent ratio (ler) of the all inter­ cropping treatments was more than 1, which indicat­ ed an advantage of intercropping in comparison with monocultures of pinto bean and black mustard (table 4). this can be attributed to increasing plant densi­ ty/m2 and more use efficiency of environmental resources (nasrollahzadeh asl et al., 2009). bio­fertil­ izers improved ler at all plant population as were applied alone or along with chemical fertilizer. in intercropping system, root interaction could increase the root activity and microbial quantity in the rhizos­ phere (zhang, 2013). interspecific interaction between species in the rhizosphere can also affect nutrient availability and uptake in intercropping (haugard­nielsan, 2001). dua et al. (2005) found that intercropping potato and french bean in all inter­ cropping treatments enhanced yield compared to sole cropping and the amount of ler was more than one. specific competition usually includes competi­ tion for soil water, available nutrients, and solar radi­ ation (buxton and fales, 1993). competition can also have a significant impact on the growth rate of the presented species in intercropping. relative value total (rvt) of intercropping treat­ ments was higher than 1 which showed the econom­ ic advantage of intercropping compared to monocul­ tures. the highest rvt were observed in the non­fer­ tilizerwith optimum densities of two species. rvt was improved as plant density increased. on these biases rvt values of optimum densities for two species were higher than 50% optimum density at the same fertilizer treatments (table 4). it was attributed to more improvement intercropping yields compared to monocultures (javanmard et al., 2018). several indices such as ler, rvt, relative yield total (ryt), rel­ ative crowding coefficient (rcc) (table 4), competi­ tive ratio, aggressively, actual yield loss, monetary advantage, and intercropping advantage have been developed to describe competition and economic advantage in intercropping (ghosh, 2004; midya et al., 2005). rcc is ability of a species to use limited resource in intercropping with its ability to gain the same resource in intercropping system by using yield com­ paring and shows the competitive advantage of inter­ cropping components (snaydon, 1991). rcc of black mustard in most treatment was higher than rcc of pinto been. its maximum value was observed in treatment non­fertilizer and optimum density of two species about 2.803. the highest value of rcc of pinto been in treatment non­fertilizer and 50% of optimum density of two species. fertilizer application result in decreasing rcc of pinto bean and increasing table 4 ­ evaluation of intercropping efficiency of treatme fertilizer treatments land equivalent ratio (ler) relative value total (rvt) relative yield total (ryt) relative crowding coefficient (rcc) of pinto bean relative crowding coefficient (rcc) of black mustard inter­ cropping (50% of optimum density of two species) inter­ cropping (optimum density of two species) inter­ cropping (50% of optimum density of two species) inter­ cropping (optimum density of two species) inter­ cropping (50% of optimum density of two species) inter­ cropping (optimum density of two species) inter­ cropping (50% of optimum density of two species) inter­ cropping (optimum density of two species) inter­ cropping (50% of optimum density of two species) inter­ cropping (optimum density of two species) control (non­ fertilizer) 1.003 1.102 3.181 4.758 1.003 1.102 1.073 0.356 0.931 2.803 bio­fertilizers (azotobarvar 1 and barvar 2) 1.107 1.349 2.717 3.874 1.107 1.349 1.007 0.559 0.992 1.787 bio­fertilizer + 50% chemical fertilizer (urea) 1.253 1.381 2.661 3.229 1.253 1.381 0.879 0.515 1.136 1.940 bio­fertilizers + 100% chemical fertilizer (urea) 1.304 1.418 2.401 2.817 1.304 1.418 0.804 0.602 1.242 1.658 raei et al. ‐ pinto bean and black mustard intercropping and bio‐fertilizers 181 rcc of black mustard in 50% of optimum density of two species. also, with increasing black mustard den­ sity in intercropping, rcc of black mustard was high­ er than bean at all fertilizer treatments. generally, fertilizer application, change the superiority of bean toblack mustard (table 4). fertilizer treatments, particularly bio­fertilizers + 100% chemical fertilizer (urea) improved grain yields of pinto bean and black mustard via higher 100 grains weight and biological yield per unit area. resource use efficiency was increased in intercropping sys­ tems. intercropping diversify agroecosystem, and resulted in sustainable production and increase eco­ nomic income, in addition, can be effective the use of agricultural land considerably. finally, it was conclud­ ed that intercropping pattern was better than mono­ cultures of two species at different levels of fertilizers and also bio­fertilizers application could increase effi­ ciency of chemical fertilizer and it can reduce the environmental risk and increase field performance of pinto bean and black mustard. references abbady k.a., el­maaz e.i.m., ahmed h.m.r.m., zohry a.a., 2016 ­ carbon sequestration as a function of inter‐ cropping management practices and different nitroge‐ nous fertilizer types. ­ j. soil sci. agri. eng., 7: 565­586. alabi r.a., esobhawan a.o., 2006 ­ relative economic value of maize ‐ okra intercrops in rainforest zone, nigeria. ­ j. cent. eur. agr., 7: 433­438. arija i., centeno c., viveros a., brenes a., marzo f., illera j.c., silvan g., 2007 ­ nutritional evaluation of raw and extruded kidney bean (phaseolus vulgaris l. var. pinto) in chicken diets. ­ poult. sci., 85: 635­644. buxton c.l., fales s.l., 1993 ­ plant environment and quality, pp. 92­116. ­ in: fahey g.c. 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processes of many trees including plane trees (platanus orientalis l.). this experiment was conducted to study the relationship between leaf chlorosis disorder and the nutritional status and some important physiological characteristics of plane trees. the experiment was arranged in a randomized complete block design with six replications and four treatments including control, manure (m), manure + fertilizer (20-5-10) (mf), and manure + fertilizer + mycorrhizal fungi (mfa) (glomus intraradices + g. mosseae). the results showed that although all treatments significantly improved the nutrients content, soluble carbohydrates content, photosynthesis rate and chlorophyll content in the leaves, they mostly reached their peak in the mycorrhizal inoculated plants. nitrogen (n), phosphorus (p) and zinc (zn) were increased in the amf amended trees compared to the control plants. the photosynthesis rate was enhanced by all the mixtures at least by 60% compared to the control. the most chlorosis (17.5%) to the leaves recorded on the control plants, while leaf damage dropped to less than 2.9% at mycorrhizal treatment leading to the improved nutritional balance in the plane trees. the results proved the effectiveness of including mycorrhizal inoculation to the common fertilization practices to prevent leaf chlorosis in the plane trees. 1. introduction plane tree (platanus orientalis l.) is among the most common ornamental and street trees planted in the urban landscape in iran and some mediterranean countries (anselmi et al., 1994; khorsandi et al., 2016). they are known for their longevity and wide distribution in the temperate zones. however, the chlorosis as an important physiological disorder in the plane trees has affected a majority of them in iran in recent years (khorsandi et al., 2016). the problem is a common physiological disorder affecting many plants around the globe. it is especially a major problem in (*) corresponding author: h.ali@ag.iut.ac.ir citation: aalipour h., nikbakht a., etemadi n., 2019 relationship between chlorosis, photosynthesis and the nutrient content of plane trees in the presence of chemical and organic fertilizers. adv. hort. sci., 33(2): 171-177 copyright: © 2019 aalipour h., nikbakht a., etemadi n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 may 2018 accepted for publication 10 january 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 171-177 172 the calcareous soils and soils with high ph (wallace, 1982). the chemical properties of the soil and the adequate supply of nutrients are major factors affecting natural plant growth and extension (cekstere and osvalde, 2013), therefore important factor in nutrient uptake is the availability of the nutrients in the soil. most trees cultivated in the alkaline and calcareous soils are exposed to the incidence of chlorosis which is reported to be basically due to fe deficiency (mortvedt, 1986). several factors can contribute to leaf chlorosis including nutritional disorders and a disorder in the chlorophyll biosynthesis. indeed, the lack of some nutrient elements such as nitrogen (n), zinc (zn) and expecially iron (fe), lead to the chlorosis in plants (godde and dannehl, 1994). moreoever, following the lack of sufficient chlorophyll, the affected plant will not be able to operate photosynthesis process, resulting in stunted growth (miller et al., 1984). arbuscular mycorrhizal fungi (amf) are obligate biotrophs that colonize the roots of the most land plants and increase host nutrient acquisition (desiro et al., 2014) and it is claimed that virtually all trees acquire nutrients through symbiotic mycorrhizal fungi (brundrett, 2009). mycorrhizal inoculation is documented as a method to the improve nutrient uptake in many plants (lehmann et al., 2014; varga, 2015; young et al., 2015). amf are effective symbionts for plants, and their symbiotic relationship can increase plant growth (vafadar et al., 2014). moreover, there is a lack of information on symbiosis relationship between the plane tree and amf fungi. in the present study, we added amf to the common fertilization program of the plane trees in the urban landscape to study the following items. firstly study the effect of mycorrhizal association on the trees response and then, observe how nutrient content and different physiological processes are associated with the leaf chlorosis disorder. to the best of knowledge, this is the first report attempting to discover the correlation between different plane tree physiological processes and leaf chlorosis disorder under amf inoculation. 2. materials and methods experimental site and treatments the experiment was conducted during 2013-2014, on the campus of the isfahan university of technology in isfahan (32°39’ n, 51°40’ e; 1600 m), iran. the site is characterized as having an arid climate with cold winters, 122.8 mm average annual rainfall and 23.4°c average annual temperature. twenty-four uniform 15-year-old plane trees (p. orientalis l.) were selected. the experiment was a randomized complete block design (rcbd) with four treatments. treatments included control, manure (m), manure + fertilizer (water soluble 20-5-10 n-p-k compound fertilizer with 12.8% sulfur, 1.3% magnesium oxide, novatec solub, compo, germany) (mf), and manure + fertilizer + mycorrhizal fungi (mfa). six replications were prepared for each treatment. the plants were inoculated with two amf inoculations including glomus intraradices and g. mosseae (both of them have been transferred to new genera, so index fungorum considers them now as rhizophagus intraradices (n.c. schenck & g.s. sm.) and funneliformis mosseae (nicolson & gerd.) (schüßler and walker, 2010). the am fungi were provided by the institute of soil and water research, tehran, iran. inoculum was comprised of a mixture of spores (80 spores g-¹ for g. intraradices and 80 spores g-¹ for g. mosseae). the mixtures of filling materials were placed into 0.5 × 0.5 m holes, depending on the treatment in early spring. this technique provides a nutrients in a zone in and around each hole. with the first wetting, the nutrients are released from the fertilizer into the soil and the manure slowly lower the ph of the soil surrounding the hole. over a period of time, a zone of soil around each hole is modified to be lower in ph and rich in micronutrients in approximately the correct proportions. two identical holes were drilled around each tree about one meter away from the tree trunk and filled up with the corresponding mixture. during the process, we avoided drilling into large buttress roots. in m treatment, trees received 5 kg of manure per hole mixed with the soil of the drilled hole. in mf treatment, 100 g of fertilizer per hole was added to the manure. trees of mfa treatment received the amf inoculums by adding 250 g of mycorrhizal inoculums into each hole mixed with manure and fertilizer (500 grams of inoculum per each tree in total). the control group did not receive any treatment (two identical holes were drilled). the trees were irrigated once a week. some chemical and physical properties of the soil and cow manure are presented in table 1. measurements various morphological and physiological parameters were measured 5 months after treatment. the aalipour et al. platanus orientalis disorders and nutritional status 173 mineral contents of the plant leaves were determined in the second year of the experiment. plant samples were oven-dried at 65°c for 48 h and then were ground to determine their mineral composition. the determination of the total n in the leaf samples was based on the kjeldahl method (baker and thompson, 1992). the extraction of p, k, fe, and zn from the plant tissue material was performed by using 2 m hydrochloric acid (hcl) after dry ashing at 550°c for 5.5 h. the concentrations of fe and zn were determined by atomic absorption spectrophotometer (670 shimadzu, kyoto, japan) (aoac, 2006). p concentration was determined by vanado molybdate phosphoric acid method with a spectrophotometer (uv-160a uv-visible recording spectrophotometer, shimadzu, tokyo, japan) (cottenie, 1980). lci portable photosynthesis and transpiration rate analyzer (li 6400; licor, lincoln, ne, usa) was used to measure the net photosynthesis rate (a) between 09.30-11.30 h on 10 fully expanded current-season leaves situated at the midcanopy height. the soluble sugars were measured according to the phenol-sulfuric acid method (dubois et al., 1956). the extraction of the leaf chlorophyll pigments was carried out using 100% acetone according to lichtenthaler (1987). for evaluation of the leaf chlorosis extent, 100 leaves from each tree were selected randomly and both leaf surfaces were scanned by a scanner (canon i-sensys mf4010, canon inc., korea). the leaf chlorosis was determined by digital image processing using matlab software. a range of color was defined for the leaf chlorosis in the program and total leaf area was examined pixel by pixel by the software and the percentage of pixels which was defined as chlorotic areas were calculated by the software (rathod et al., 2013). data analysis data were assessed for normality and log-transformed used to make data conform to normality when necessary prior to analysis. non-homogeneity data were observed in leaf chlorosis, being the data transformed with the formula arcsin ö(leaf chlorosis/100) to obtain homogeneity. the experimental data were statistically analyzed by the analysis of variance (anova). the significance of the differences between treatments was estimated using the least significant difference (lsd) test at p≤0.05, and graphs were drawn using excel 2010. statistical correlation was calculated by pearson’s correlation coefficient (r). this test was used to measure the strength of a linear association between the leaf chlorosis and other variables including nutrients content, photosynthesis rate and chlorophyll and soluble sugar contents. the value r = 1 means a perfect positive correlation and the value r = -1 means a perfect negative correlation. the experimental data were statistically analyzed with statistical analysis systems (sas) software, version 9.1 and statistics, version 8.0. 3. results amf inoculation increased all nutrients content including p, n, fe and zn in the leaves of the treated trees (table 2). n, p and zn reached their peak value only when the fertilizer mix amended by mycorrhizal inoculums. p and zn increased by 424% and 425% factors texture ph ec (ds m-1) organic matter (%) n (%) p-available (mg kg-1) k-exchangeable (mg kg-1) fe (mg kg-1) zn (mg kg-1) soil clay 7.9 1.53 1.15 0.15 140 235 1400 21 manure 8.02 15.23 20.4 3.07 791 2030 12300 194 table 1 some chemical and physical properties of the soil and cow manure used in research treatment nutrient n (g kg-1) p (g kg-1) fe (mg kg-1) zn (mg kg-1) control 17.47 d 1.85 c 54.93 b 4.82 d manure 19.14 c 5.25 b 148.99 a 15.09 c manure + fertilizer 19.65 b 7.31 b 146.71 a 17.32 b manure + fertilizer + amf 20.39 a 9.71 a 170.14 a 25.31 a table 2 influence of arbuscular mycorrhizal (am) fungi and other treatments on nutrient uptake of plane tree (platanus orientalis l.) means in the same column followed by the same letters are not statistically different at p≤0.05 by the least significant difference test (lsd). adv. hort. sci., 2019 33(2): 171-177 174 respectively, compared to the control plants. all treatments (regardless of the composition of the mixture) successfully enhanced fe and n contents in the leaves compared to the control plants (table 2). all treatments significantly improved the soluble carbohydrates content in the leaves; however, it reached the peak in the mycorrhizal inoculated plants. the soluble carbohydrates content increased by 35.44% and 13.82% compared to the control of non-fertilized treatment and non-inoculated plants, respectively (fig. 1). all treatments increased the photosynthesis rate at least by 60% compared to the control, although no significant difference was observed between the treatments (fig. 2). the same trend was observed in the case of chlorophyll content (fig. 3), where it increased at least by 32% compared to the control. leaf chlorosis was influenced dramatically by the treatments (fig. 4). the most leaf chlorosis was recorded in control non-fertilized treatments. leaf chlorosis reduced to less than 2.9% on inoculated trees by amf, while 17.5% of leaves tissue were affected by chlorosis in the control plants. the amf inoculated plants showed an increase of 13.44% compared to the plants that received manure + fertilizer. a strong relationship was found between leaf chlorosis and fe, n and zn contents (fig. 5). a significant and linear relationship was also found between chlorosis and the chlorophyll content and net photosynthesis in the plane tree leaves (fig. 6). leaf chlorosis in the plane trees resulted in a dramatic and linear decline in the soluble carbohydrate in the leaves (fig. 6). 4. discussion and conclusions two explanations can be presented for increasing fe content under any treatments (except for the config. 1 influence of arbuscular mycorrhizal (am) fungi and other treatments on soluble carbohydrate content in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 2 influence of arbuscular mycorrhizal (am) fungi and other treatments on photosynthesis rates in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 3 influence of arbuscular mycorrhizal (am) fungi and other treatments on chlorophyll content in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 4 influence of arbuscular mycorrhizal (am) fungi and other treatments on chlorosis damage in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. aalipour et al. platanus orientalis disorders and nutritional status 175 trol) in this experiment (fig. 1). firstly, as the soil of the site was a calcareous and compacted soil, any treatment increasing the air flow in the soil could improve fe content in the plant (lucena, 2003). secondly, using the manure itself can increase fe content in the plants (mortvedt, 1986). it is documented that the manure provides micronutrients including fe and improves the structure of the soil (lucena, 2003). our results at least partly confirm the findings that combining fe fertilizers with the organic matter is more favorable in terms of fe uptake than fe sources applied alone (mortvedt, 1986). it has been reported that the application of feso4 (4-8 kg tree-¹) mixed with manure, cotton seed cake, or other organic substances in 8 to 10 holes in the soil around the crowns of apple trees (malus sylvestris mill.) resulted in marked correction of fe chlorosis (zheng-qing and cang-zhen, 1982). it is also well established that as a result of the decomposition of organic matters in the soil, compounds such as humic acid (ha) and fulvic acid (fa) are produced in the soil (nardi et al., 2002). these acids are well known as naturally-occurring chelating agents (mortvedt, 1986; nardi et al., 2002). there are many reports showing enhanced micronutrients uptake by the plants receiving ha, fa (nikbakht et al., 2008; 2014) or organic matter (atiyeh et al., 2002). it is shown that inoculation turfgrass (lolium prenne l.) with amf receiving ha not only improved plant growth but also showed more elevated nutrients content in the leaves than in non-inoculated (control) plants or plants receiving only ha (nikbakht et al., 2014). researchers believed that the role of amf in no-3 transport to the root surface is significant (subramanian and charest, 1999; javaid, 2009). they especially insist that the role of amf is of value and importance in nitrate uptake in mediterranean and (semi-) desert ecosystems which are characterized by calcareous soils. n, p and zn uptake reached their peak value when the fertilizer mixture amended by amf (table 2). these results confirm the well-documented effect of amf inoculation on nutrients uptake (brundrett, 2009; varga, 2015; young et al., 2015). a strong relationship between leaf chlorosis and fe, n and zn contents implies that the chlorosis is not only because of fe deficiency in the plant, but also other nutrients including n and zn play they own role (fig. 5). it indicates that leaf chlorosis in the plane trees was not simply due to fe deficiency. it is well documented that fe is an essential element for many vital processes in a plant including photosynthesis, respiration, n fig. 5 relationship between chlorosis with fe (a), zn (b) and n concentration (c) in plane tree (platanus orientalis l.). fig. 6 relationship between chlorosis with photosynthesis rate (a), chlorophyll content (b) and soluble carbohydrate (c) in plane tree (platanus orientalis l.). 176 adv. hort. sci., 2019 33(2): 171-177 fixation, chlorophyll and hormone synthesis; fe is also a constituent of heme proteins (cytochromes, catalase, and peroxidase) (briat and lobreaux, 1997). as a result, affected plants by fe deficiency suffer severe metabolic and structural disorders (javaid, 2009). there are also some reports indicating that the major cause of fe deficiency is the very low solubility of fe oxides in the soil (mortvedt, 1986). it shows the importance of the fact that the role and priority of each element in the plane tree chlorosis remain to be investigated further. fe deficiency depresses the synthesis of chlorophyll, which results in the decrease of photosynthetic products, which in turn affect plant growth (wang et al., 2008). as a result of carbohydrates synthesis reduction in chlorotic leaves, which slows the movement of k+ from the leaf to the phloem vessels, a decline in the production of biomass is reported (maldonado-torres et al., 2006). these explain why we found a relationship between chlorosis and chlorophyll content, net photosynthesis and soluble carbohydrate content of the leaves (fig. 6). moreover, increased photosynthetic capacity by amf is in agreement with the results of the previous study by birhane et al. (2012). it seems this process has improved nutrition, leading to higher photosynthetic rates (vafadar et al., 2014). to the best of our knowledge, no similar information has yet been provided for interaction effect of fertilizers and amf inoculation on plane trees and its relationship with the leaf chlorosis disorder. this study demonstrated that amf inoculation added to the common fertilizer program served successfully as a biological and environmental-friendly method to overcome chlorosis disorder of the plane trees. in addition, the findings of this study suggest that in calcareous soils drill hole nutrition should be considered as a standard method to prevent nutritional disorders in the urban landscape. the results also revealed that fe is not the only nutrient participating in the leaf chlorosis of plane trees. it suggests further investigations to study the weight and importance of each nutritional element in chlorosis disorder of the plane trees. in this study we mainly focused on the effect of improved media around the plane trees, rather than specific effect of amf. indeed, this study was one of the primary trail in a series of experiments we want done later. in the later, we specifically will studied the amf effect on the plane trees. our experiment creates a paradigm for future studies of relationship between plane trees and microorganisms. references anselmi n., cardin l., nicolott g., 1994 plane decline in european and mediterranean countries: associated pests and their interactions. eppo bull., 24: 159-171. aoac, 2006 official methods of analysis. association of official analytical chemists, 18th edition, gaithersburgs, md, usa. atiyeh r.m., lee s., edwards c.a., arancon n.q., metzger j.d., 2002 the influence of humic acids derived from earthworm-processed organic wastes on plant growth. bioresour. technol., 84: 7-14. baker w.h., thompson t.l., 1992 determination of total nitrogen in plant samples by kjeldahl, pp. 13-16. in: plank c.o. 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cang-zhen l., 1982 studies on the application of ferrous sulphate for controlling chlorosis of apple tree on calcareous soils. j. plant nutr., 5: 883896. impaginato 375 adv. hort. sci., 2019 33(3): 375­379 doi: 10.13128/ahs­24026 direct shoot regeneration of three petunia cultivars a.n. vakili 1, h. bagheri 1 (*), p. azadi 2 1 department of biotechnology, bu‐ali sina university, 6517838695, hamedan, iran. 2 department of genetic engineering, agricultural biotechnology research institute of iran (abrii), agricultural research, education and extension organization (areeo), karaj, iran. key words: bap, leaf disk, organogenesis, petunia hybrida, tdz. abstract: a tissue culture system for acquiring high­efficiency regeneration of petunia was optimized. leaf explants of alvan, large flower alvan (lf alvan) and mahalat cultivars of petunia hybrida were cultured separately on ms medi­ um including various concentrations of tdz and ba without auxin in order to assess direct shoot regeneration. alvan showed the highest frequency of shoot regeneration (100%) and the highest mean number of shoots per explant (25.33) on ms containing 2 mg/l tdz. for lf alvan cultivar the highest percent­ age of shoot organogenesis (100%) and the highest mean number of shoots per explant (18.20) were observed when ms medium containing 1 mg/l ba was used. with the mahalat cultivar the maximum rate of direct regeneration was obtained on ms supplemented with 0.5 and 1 mg/l ba (80%). the mean num­ ber of shoots per explant (9.63) was obtained when 2 mg/l tdz was used. regenerated shoots were successfully elongated (2 to 3 cm in length) and transferred into half­strength ms as the rooting medium supplemented with 0.1 mg/l naa. the shoots were successfully rooted, acclimatized and transferred to the greenhouse. 1. introduction petunia (petunia hybrida) is well known as an economically important ornamental plant and is grown worldwide for its beautiful and fragrant flowers. propagation techniques with modern approaches intend to give a hand to scientists to provide demands of ornamental industry (rout et al., 2006). an efficient plant regeneration system is necessary for the success­ ful genetic transformation (ntui et al., 2010). there are several reports for in vitro shoot regeneration of petunia hybrida species from several explants including leaf (preece, 2000; ntui et al., 2010; abu­qaoud et al., 2010; khan et al., 2011; abu­qaoud, 2012; burbulis et al., 2015), somatic cells (rao et al., 1973) cotyledon (dulien, 1991), embryo (dimasi­theriou et al., 1993), protoplast (auer et al., 1992; auer et al., 1999; abu­qaoud et al., 2010), petal (razdan, 2003), and microspore (li et al., 2013). various factors could affect organogenesis in p. hybrida such as light (reuveni and evenor, 2007), sugar and co2 (qu et al., 2007), ethylene (*) corresponding author: bagheri.hedayat@gmail.com citation: vakili a.n., bagheri h., azadi p., 2019 ­ direct shoot regeneration of three petunia cultivars. ­ adv. hort. sci., 33(3): 375­379 copyright: © 2019 vakili a.n., bagheri h., azadi p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 october 2018 accepted for publication 23 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 375­379 376 (dimasi­theriou et al., 1993), nitrogen and calcium (frett and dirr, 1996) and also hormonal combina­ tions (ying et al., 2005; xiao­feng et al., 2009; xian­ chun, 2010) petunia regeneration happens directly and indirectly by combinations of auxins and cytokinins in medium culture (michalczuk and michalczuk, 2000; ziv et al., 2005). adventitious bud formation from somatic cells of p. hybrida was induced by exogenous cytokinins such as ba (6­benzyladenine), zeatin, kinetin and tdz (thidiazuron) (rao et al., 1973; thirukkumaran et al., 2009). it is reported that tdz acted different from traditional cytokinins and was able to accomplish both the cytokinin and auxin requirements of differ­ ent plant species for regeneration (murthy et al., 1998; sanikhani et al., 2006). the highest frequency of direct shoot organogenesis of daady blue and white dreams cultivars of p. hybrida was obtained on ms medium supplemented with different concentra­ tion of tdz (abu­qaoud, 2012). also, tdz alone pro­ vided the highest percentage of shoot organogenesis and mean number of shoot per explant of p. hybrida cv. mitchell (thirukkumaran et al., 2009). it is also reported that exogenous cytokinin especially ba could control the commitment of petunia leaf explants to induce shoots in tissue culture (auer et al., 1992; abu­qaoud et al., 2010). therefore in this study, we investigated the effect of tdz and ba as well as genotype on direct shoot regeneration of three petunia cultivars. this efficient regeneration system is very useful in genetic transformation pro­ jects of p. hybrida. 2. materials and methods seed germination seeds of three local cultivars of petunia, alvan, large flower alvan (lf alvan) and mahalat, were sterilized with 70% ethanol for 30s, and sodium hypochlorite solution 1% for 10 minutes. they rinsed 3 times with sterilized water and cultured on ms medium. seeds were grown under 25 ± 2 °c with 16/8 hour photoperiod, under fluorescent illumina­ tions (40 μmol m­2s­1). organogenesis the newly formed leaves were cut 6­8 mm in length, and then cultivated on 5 modified msmedia: ms medium without hormones (ms1), ms + 0.5 mg/l ba (ms2) [sigma­aldrich, steinheim, germany], ms + 1 mg/l ba (ms3), ms + 1 mg/l tdz (ms4) [sigma­ aldrich, steinheim, germany] and ms + 2 mg/l tdz (ms5). abu­qaoud et al., (2010) got more regenera­ tion when they used 0.8 mg/l ba. therefore we selected 0, 0.5 and 1 mg/l ba to better estimate ba effect. also as thirukkumaran et al., (2009) reported more regeneration with 2 mg/l tdz, we selected 0, 1 and 2 mg/l tdz to investigate its effect. moreover, the ms was supplemented with 30 g/l sucrose and solidified with 7 g/l agar [duchefa, haarlem and the netherlands]. the optimum ph of all culture media was considered 5.8 which adjusted with 1n naoh before sterilization. then all media were sterilized using autoclave at 121°c for 20 min. explants were placed on regeneration medium with the adaxial side upward. the cultures were incubated at 25±2°c, with a light to dark period of 16/8 hours under cool­white fluorescent light at 40 μmol m­2 s­1. explants were sub­cultured every two weeks. they were investigat­ ed using binocular stereo microscope, regarding to the mean number of explants inducing shoots and the mean number of induced shoots and buds per explants after 4­5 weeks on regeneration medium. rooting and acclimatizing regenerated shoots were transferred into half­ strength ms supplemented with 30 g/l sucrose, 0.1 mg/l naa [duchefa, haarlem, and the netherlands] and solidified with 7 g/l agar. the rooted plantlets rinsed under tap water and planted on the plastic pots with combination of sterile peat moss and per­ lite mixture (2:1). they kept in greenhouse condi­ tions. statistical analysis the experiment was done based on completely randomized design with three replications and 10 leaf explants in each replication. data were normal­ ized through arcsin (√x) and (√x+0.5) transformation in spss. the normalized data were analyzed using sas statistical analysis package and were compared via duncan’s multiple range test at p ≤ 0.01 and p ≤ 0.05. 3. results effect of ba on organogenesis direct shoot formation was obtained in all three cultivars after 4­5 weeks. no regeneration occurred on ms1 medium which means hormones are neces­ sary to induce shooting (tables 1, 2). when 0.5 mg/l ba (ms2) was used no differences in frequency of regeneration was observed among cultivars. the low vakili et al. ‐ regeneration of three petunia cultivars 377 mean numbers of shoot per explant (5.05 and 6.21) were observed in ms2 for mahalat and alvan culti­ vars, respectively. when 1 mg/l ba (ms3) was used differences were observed in all three cultivars and lf alvan cultivar showed 100% shoot regeneration (table 2), with a mean number of 18.20 shoots per explants (fig. 1 a). effect of tdz on organogenesis significant differences were observed among three cultivars when tdz concentration was increased (tables 3, 4). the low shoot regeneration frequency was obtained on ms4 and ms5 media for mahalat cultivar (table 4). alvan cultivar showed the highest percentage of shoot regeneration (100%) and mean number of shoots per explant (25.33) on ms with 2 mg/l tdz (fig. 1 b) and the lowest one (6.61) was belong to mahalat cultivar on ms with 1 mg/l tdz. 4. discussion and conclusions we could show that auxin is not necessary for direct shoot regeneration of three cultivars of p. hybrida. it is already reported that the number of shoot per explants dramatically increased when explants exposed to the medium containing ba (auer et al., 1992). the highest shoot regeneration rate (45%) and the maximum average number of shoots per explant (7.5) from petunia leaf explants on ms with 2 mg/l ba + 0.5 mg/l naa has also been report­ ed (abu­qaoud et al., 2010). in the current study the highest shoot regeneration frequency in alvan culti­ var and the mean number of shoots per explant in both alvan and mahalat cultivars were observed when 2 mg/l tdz was used which is in conformity with thirukkumaran et al. (2009). the importance of tdz on regeneration and shoot induction frequency table 1 ­ effect of modified ms medium supplemented with different concentration of ba on shoot regeneration from leaf explants of p. hybrid the values represent the mean ± standard error of three replicates. different letters are showing considerable differences at p≤0.05. fig. 1 plant regeneration from leaf explants of different culti­ vars of petunia hybrida. (a) direct shoot regeneration of lf alvan on ms + 1 mg/l ba (bar: 2 mm); (b) direct shoot regeneration of alvan on ms + 2 mg/l tdz (bar: 4 mm); (c) root formation after 2 weeks on rooting media (bar: 5 mm); (d) a 4 weeks old plantlet after transfer to the pot (bar: 1 cm). ms media frequency of regeneration the mean number of shoots per explant cultivars cultivars ms1 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e ms2 83.33 ± 2.8 b 83.33 ± 1.8 b 80.00 ± 3.1 b 6.21 ± 0.12 d 13.31 ± 0.85 b 5.05 ± 1.00 d ms3 80.00 ± 3.7 b 100.00 ±0.00 a 80.00 ± 1.1 b 10.12 ± 0.41 bc 18.20 ± 0.85 a 7.76 ± 0.56 cd table 2 ­ effect of modified ms medium supplemented with different concentration of tdz on shoot regeneration from leaf explants of p. hybrid the values represent the mean ± standard error of three replicates. different letters are showing considerable differents at p≤0.05. ms media frequency of regeneration the mean number of shoots and buds per explant cultivars cultivars alvan lf alvan mahalat alvan lf alvan mahalat ms1 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 f 0.00 ± 0.00 f 0.00 ± 0.00 f ms2 83.33 ± 3.4 b 80.00 ± 1.0 b 66.66 ± 2.1 c 16.25 ± 1.00 b 12.00 ± 1.21 c 6.61 ± 0.08 e ms3 100.00 ± 0.00 a 83.33 ± 0.8 b 70.00 ± 1.7 c 25.33 ± 1.02 a 14.31 ± 0.96 bc 9.63.00 ± 0.11 d adv. hort. sci., 2019 33(3): 375­379 378 and the mean number of shoots per explant was also investigated in daddy blue and dreams white geno­ types (abu­qaoud, 2012). this study showed that a cytokinin source of tdz or ba may be enough for direct shoot regeneration of three mentioned culti­ vars of p. hybrida. application of tdz instead of both auxin and cytokinin requirements for organogenesis in the wide range of plant species has been support­ ed (murthy et al., 1998). probably tdz tends to make balance among endogenous growth regulators that is essential for inducing specific modes of regeneration. it was found that many factors such as genotype and exogenous growth regulators have the capability to influence on biochemical pathways controlling the endogenous cytokinin content (krikorian, 1995). in the present study a significant difference in regenera­ tion frequency was observed among studied cultivars probably due to the different level of endogenous hormones. for lf alvan cultivar, the maximum regen­ eration frequency (100%) and the highest number of shoots per explants (18.20) were obtained when ba concentration was increased from 0.5 to 1 mg/l while the other two cultivars showed less reaction. these findings confirm the report of jamshidnia and sayed tabatabaei (2013), and burbulis et al., (2015) on dif­ ferences in shoot regeneration frequency among three different genotypes of petunia. here we report an efficient direct shoot regeneration system in petunia hybrida using leaf explants of alvan cultivar. this cultivar can be considered as a suitable cultivar for transformation experiments. to conclude, the present study provided an effi­ cient direct shoot regeneration system without auxin in petunia using leaf explants that could be improve transformation studies. acknowledgements this work was supported by bu­ali sina university, hamedan, iran, and novin giti gene biotech. co. biotechnology incubator center of national institute of genetic engineering and biotechnology (nigeb), tehran, iran. references abu­qaoud h., 2012 ­ improving adventitious shoot regeneration from cultured leaf explants of petunia hybrida using thidiazuron. ­ afr. j. biotechnol., 11(51): 11230­11235. table 3 ­ analysis of variance of different concentrations of tdz on shoot regeneration of p. hybrida *, **, significant at 5% and 1% levels, respectively. table 4 ­ effect of tdz on shoot regeneration of p. hybrida using duncan’s multiple range test ms1= ms medium without hormones, ms4= ms + 1 mg/l tdz; ms5= ms + 2 mg/l tdz. means compared using duncan’s multiple range test. the values represent the mean ± standard error of three replicates. different letters are showing considerable differents at p≤ 0.05. source of variation df mean squares p­value frequency of regeneration mean number of shoots per explant frequency of regeneration mean number of shoots per explant tdz 2 3.1897 ** 11.4806 ** 0.000 0.000 cultivar 2 1.3250** 6.5896 ** 0.009 0.004 tdz × cultivar 4 0. 8015* 4.2010 ** 0.022 0.009 error 18 0. 215 0.9080 total 26 ms media frequency of regeneration the mean number of shoots and buds per explant cultivars cultivars alvan lf alvan mahalat alvan lf alvan mahalat ms1 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 f 0.00 ± 0.00 f 0.00 ± 0.00 f ms4 83.33 ± 3.4 b 80.00 ± 1.0 b 66.66 ± 2.1 c 16.25 ± 1.00 b 12.00 ± 1.21 c 6.61 ± 0.08 e ms5 100.00 ± 0.00 a 83.33 ± 0.8 b 70.00 ± 1.7 c 25.33 ± 1.02 a 14.31 ± 0.96 bc 9.63 ± 0.11 d vakili et al. ‐ regeneration of three petunia cultivars 379 ntui v.o., azadi p., supaporn h., mii m., 2010 ‐ plant regeneration from stem segment‐derived friable callus of “fonio” (digitaria exilis (l.) stapf.). ­ sci. hortic., 125: 494­499. preece j.e., 2000 ­ shoot organogenesis from petunia leaves, pp. 167­175. ­ in: trigiano r.n. 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(ed.) plant hormones: physiology, biochemistry and molecu‐ lar biology. kluwer academic publishers, pp. 833. li f., li c., li m., yu m., fang c., wang s., 2013 ­ in vitro culture of petunia hybrida microspores and agrobac‐ terium‐mediated transient expression of β‐glu‐ curonidase (gus) reporter gene. ­ int. j. agric. biol., 15: 1098­1104. michalczuk b., michalczuk l., 2000 ­ the effect of light quality on regeneration rate and plantlet development in transgenic petunia ‘revolution’ (surfinia type). ­ acta horticulturae, 530: 397­401. murthy b.n.s., murch s.j., saxena p.k., 1998 ­ thidiazuron: a potent regulator of plant morphogene‐ sis. ­ in vitro cell dev. biol. plant., 34: 267­275. impaginato 563 adv. hort. sci., 2018 32(4): 563-568 doi: 10.13128/ahs-22468 a preliminary investigation on developmental and biochemical responses of amsonia orientalis to ultraviolet-c irradiation a. acemi 1 (*), y. avcı duman 2, y. yüzügüllü karakuş 1, f. özen 1 1 department of biology, faculty of sciences and arts, kocaeli university, 41380 kocaeli, turkey. 2 department of chemistry, faculty of sciences and arts, kocaeli university, 41380 kocaeli, turkey. key words: antioxidant enzymes, european bluestar, plant development, tissue culture, uv-c irradiation. abstract: the present study aims to investigate the developmental and biochemical responses of ornamental amsonia orientalis irradiated with ultraviolet-c (uv-c). nodal explants of the species were exposed to uv-c irradiation on the first, 15th and the last days of the in vitro culture for 15 (3.47 kj m-2), 30 (6.94 kj m-2) and 60 min (13.87 kj m-2). in general, root lengths and numbers were negatively influenced by prolonged uv-c exposure. however, mean shoot numbers and lengths were slightly enhanced after 15 and 30 min of irradiation. high hydrogen peroxide (h2o2) and malondialdehyde (mda) levels were observed due to the uv-c exposure. activities of the antioxidant enzymes, pod (peroxidase) and cat (catalase) were found to be enhanced whereas sod (superoxide dismutase) was reduced. these results indicated that uv-c irradiation for shorter durations may be carefully used to improve in vitro shoot proliferation in a. orientalis. however, it should be noted that longer irradiation durations can trigger stress responses and lipid peroxidation-dependent cell membrane damage which will further result in the plant loss. 1. introduction ultraviolet (uv) irradiation is present in sunlight in three different wavelengths which are classified as uv-a (400-315 nm), uv-b (315-280 nm) and uv-c (280-100 nm). uv irradiation may affect growth and metabolic processes in plants due to its high quantum energy (kobashigawa et al., 2011). since wavelengths below 280 nm are absorbed by the ozone layer they do not reach to the surface of the earth (alexieva et al., 2001). however, reduction of stratospheric ozone and, a decrease in the ozone layer may lead to increase in uv-c irradiation reaching the biosphere. (*) corresponding author: arda.acemi@kocaeli.edu.tr citation: acemi a., avci duman y., yüzügüllü karakuş y., özen f., 2018 a preliminary investigation on developmental and biochemical responses of amsonia orientalis to ultraviolet-c irradiation. adv. hort. sci., 32(4): 563-568 copyright: © 2018 acemi a., avci duman y., yüzügüllü karakuş y., özen f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 january 2018 accepted for publication 30 may 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 563-568 564 beside its germicidal activity, application of uv-c irradiation at 254 nm wavelength is used for several purposes including regulation of fruit ripening in tomato (tiecher et al., 2013), fruit quality stabilization during storage of fresh-cut watermelon (artés-hernández et al., 2010) and retardation of fruit decay in strawberry (erkan et al., 2008). its role in growth regulation and modulation of flowering time in ornamental plants was declared since uv-c light application increased branching and the number of flowers in salvia splendens and viola tricolor (bridgen, 2016). also, the antioxidative function was found to be enhanced after uv-c application in spinacia oleracea (kobashigawa et al., 2011). however, the application of uv irradiation can be hazardous in higher doses. therefore, to better understand the developmental and metabolic responses of horticultural plants to uv-c irradiation, uv-dose-dependent studies in controlled environmental factors should be also conducted on other plant species. amsonia orientalis decne. [syn. rhazya orientalis (decne.) a. dc.] which is also known as european blue star or eastern rhazya is an ornamental plant with medicinal properties. it is cultivated more commonly by gardeners in the usa than those in europe since it is deer-resistant due to its slightly toxic latex content (acemi et al., 2016). however, the natural populations of the species were taken under conservation by the european council in the frame of bern convention since they are quite limited in nature (acemi et al., 2017). in the current study, the effects of dose-dependent uv-c irradiation applications on in vitro development and antioxidative enzymes of a. orientalis were investigated to shed light the usability of this treatment in horticultural plant propagation. 2. materials and methods plant material preparation and in vitro uv-c treatments nodal explants were excised from mature individuals of amsonia orientalis growing in the garden of kocaeli university. the shoots were multiplied by following the protocol described by acemi et al., (2013 a). nodal explants excised from in vitro-raised shoots were inoculated into the ms (murashige and skoog’s) medium (1962) supplemented with 30 g l-1 sucrose and 7 g l-1 of plant agar. the ph of the medium was set to 5.7, and the cultures were incubated for 30 days under the same conditions defined by acemi et al., (2013 a). the applied uv-c doses were determined from previous reports (lópez-rubira et al., 2005; artés-hernández et al., 2010; bridgen 2014; castronuovo et al., 2014; bridgen, 2016). the cultures were subjected to uv-c irradiation at the first, 15th and the last days of the culture period. the irradiation was applied from 30 cm distance for 15, 30 and 60 min per application day by using sylvania g15w uv-c lamp (λmax 253.7 nm). uv-c treatments were applied from the top of the culture vessels in a sterile biosafety cabinet, and lids of the culture vessels were kept opened during treatments to ensure penetration of the irradiation energy. the total accumulated irradiation levels were estimated as 3.47, 6.94 and 13.87 kj m-2 for 15, 30 and 60 min of uv-c irradiation, respectively (kobashigawa et al., 2011; severo et al., 2015). biochemical assays the optimized method of acemi et al. (2017) was followed to determine malondialdehyde (mda) as lipid peroxidation product, and h2o2 contents. the crude extract for antioxidant enzyme activities’ determination was prepared by homogenizing the tissue samples (shoots and roots together) in extraction buffer of 50 mm sodium phosphate (ph 7.0) containing 0.1 mm ethylenediaminetetraacetic acid (edta). after centrifugation of the homogenate at 14,000 g for 15 min at 4°c, the resulting supernatants were collected and used for catalase (cat), peroxidase (pod) and superoxide dismutase (sod) activity assays. cat activity was determined according to the method of aebi (1974). pod activity was assayed using the pyrogallol oxidation method (kar and mishra, 1976) while sod activity was determined by following the method of dhindsa et al. (1981). data collection and statistical analysis the morphometric evaluation was done using 30 explants in each repeat. all assays were repeated thrice and mean values were compared using duncan’s multiple range test at p<0.05 significance level. the enzyme assays and extract preparation were done on the same day to minimize the loss of enzymatic activities. 3. results plant growth and organ development at the end of the incubation period, the mean shoot length slightly increased in response to 15 and acemi et al. uv-c irradiation effects on amsonia orientalis 565 30 min uv-c exposure while this parameter was negatively affected by 60 min of exposure. however, increments in the mean shoot length were not statistically different than the control group (fig. 1a). the mean shoot numbers were influenced positively by 15 and 30 min uv-c exposure whereas only the result of 15 min exposure was found to be statistically different than the control group (fig. 1b). the mean root lengths were found to be decreased due to uv-c application even at the shortest duration. this reduction in root lengths was between 62.5% (15 min) and 95% (60 min) compared to the control (fig. 1a). in contrast to mean shoot numbers, the mean root numbers reduced gradually in response to the elevated uv-c exposure (fig. 1b). lipid peroxidation and h2o2 content application of uv-c irradiation significantly changed the lipid peroxidation level of the plant. fifteen and 30 min of uv-c exposures triggered mda accumulations without statistical differences between them. the highest accumulation level was observed from the cultures exposed to uv-c for 60 min (fig. 2a). h2o2 content increased following uv-c exposure maintaining similar levels within treatments (fig. 2b). antioxidant enzyme activities uv-c irradiation caused a decrease in sod activity without statistical differences between treatments (fig. 3a). pod activity significantly increased due to uv-c exposures. the highest pod activity was observed in cultures exposed to 15 min of uv-c followed by a significant decrease at 30 and 60 min of uv-c exposures, which were not significantly different (fig. 3b). cat activity exhibited a similar trend with pod activity. the shortest uv-c exposure caused the highest increase in the cat activity while longer exposures induced statistically same results (fig. 3c). fig. 1 effects of uv-c irradiation on amsonia orientalis growth parameters. a= length of shoot and root, b= number of shoot and root. the values are represented as means ± sd, different letters denote significant differences (p<0.05). fig. 2 oxidative effects of uv-c irradiation on amsonia orientalis. a= mda and b= h2o2 contents. the values are represented as means ± sd, different letters denote significant differences (p<0.05). adv. hort. sci., 2018 32(4): 563-568 566 4. discussion and conclusions uv irradiation can damage dna and affect plant growth through various regulatory and/or stressmediated processes. as one of these processes, cell cycle modulation sourced by dna damage may particularly slow down the progress from g1 to s phase (jiang et al., 2011). therefore, in our results the decrease in the mean shoot length after 60 min uv-c irradiations might be due to the uv-induced dna damage and impaired cell cycle progress. differently, mean shoot number was found to be increased in a. orientalis after 15 min of uv-c exposure, which was in accordance with the previous report that found an increment of axillary branching in viola tricolor (bridgen, 2016). our results also showed adverse effects of uv-c on the root growth of a. orientalis. this negative effect can be explained by oxidative degradation and/or inhibited-synthesis of indole acetic acid (iaa); a phytohormone responsible for root proliferation (berli et al., 2013). this possible inhibition of iaa might also be a result of photo-oxidation of the plant growth regulator (ciurli et al., 2017). the oxidative degradation of iaa starts with a peroxidase-involved decarboxylation process on the side chain or oxidation of the indole ring (normanly, 2010). in the enzyme assays, elevated peroxidase activity against uv irradiation supports this discussion which is still in need of further experiments to be clearly proven. mda content indicates reactive oxygen species (ros)-mediated cellular damage considering damage to membrane lipids of stress-exposed plants. the observed increase in h2o2 levels coincided with enhanced mda levels in a. orientalis. also, pod and cat activities were found to be increased to remove ros to limit mda production in a. orientalis . degradation of the enzyme proteins and nucleic acids can be started after ros-induced peroxidation of the cell membrane lipids. at the same time, h2o2 could activate mitogen-activated protein kinases (mapks) in plants, leading to an enhanced antioxidant defense system (nie et al., 2013). however, excess accumulation of h2o2 causes cellular damage (gong et al., 2001). the increase in both h2o2 and mda levels indicates cellular damage in a. orientalis. excessive ros in uv-c-exposed plants may be produced because of disruption in metabolic activities or increased activity of membrane-localized nadph-oxidase (kalbina and strid, 2006). the increment in the activities of cat and pod enzymes did not seem to limit ros production in a. orientalis since h2o2 accumulation was consistently found at high levels. as a defense mechanism against environmental stress factors, plants favor the production of antioxidant enzymes (berli et al., 2013). maintenance of the fig. 3 effects of uv-c irradiation on enzymatic antioxidant activities in amsonia orientalis. a= sod, b= pod and c= cat activities. the values are represented as means ± sd, different letters denote significant differences (p<0.05). acemi et al. uv-c irradiation effects on amsonia orientalis 567 antioxidant defense system to cope with ros plays a significant role in keeping the cell membranes stabilized. the antioxidant enzymes sod, cat, and pod are widely distributed in all higher plants and involved in decomposition of different forms of ros (foyer and noctor, 2000). excessive production of o2– triggers sod enzyme activity which converts superoxide radicals into either o2 or h2o2 while the excess accumulation of h2o2 is prevented by catalase and/or the ascorbate-glutathione cycle enzymes (ma et al., 2014). although the o2− content was not determined in our study, the inhibition of sod activity by uv irradiation might be due to increased o2− content. pod is involved in such processes like lignification and tolerance to environmental stresses in higher plants. during uv exposure, elevated pod and cat activities showed that activities of both enzymes can be triggered in a. orientalis. the bulk h2o2 removal activity of cat in the cell is followed by the scavenging action of pod on the h2o2 which is not taken by cat (willekens et al., 1997). in this report, this cooperation between both enzymes is also shown in a. orientalis. thanks to in vitro plant tissue culture technique, several conservation studies on a. orientalis were conducted and a high number of individuals were propagated thereby populations in turkey were conserved (acemi et al., 2013 a, b). the present study has revealed that although short-term application of uv-c irradiation can enhance shoot induction, it limits root growth and triggers oxidative stress at extended exposure durations. therefore, in further detailed studies on a. orientalis uv-c radiation should be applied for less than 30 min (6.94 kj m-2). however, it should be noted that short-term uv-c application can be considered as a promoter factor only in shoot multiplication phase in a. orientalis. the lower doses may be carefully applied to enhance branching in the horticultural industry. however, the possible developmental and biochemical responses given against uv-c irradiation would change species to species. acknowledgements this work 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chamnongpol s., davey m., schraudner m., langebartels c., van montagu m., inzé d., van camp w., 1997 catalase is a sink for h2o2 and is indispensable for stress defence in c3 plants. embo j., 16(16): 4806-4816. 121 1. introduction greenhouse soilless horticulture requires a high level of technology, considerable capital investment, and operators with adequate professional skills but it is highly productive, ergonomic, and uses water and space efficiently (savvas, 2003; resh, 2012). over the last few years research has aimed to achieve the most effective systems able to satisfy restrictions relating to the protection of ground water and soil. in fact, the conversion of soilless culture to a closed system is associated with environmental policies established to reduce environmental pollution (voogt et al., 2013). to avoid some of the risks (rapid spread of root pathogens, accumulation of na and mismanagement of electrical conductivity ec and ph), disinfection of recirculated nutrient solution (ns) and amendment of its chemical composition are carried out. however, discharge of part of the recirculated solution is still required since nacl, from the irrigation water, and organic compounds, from long cultivations, accumulate causing an inhibition of growth (voogt et al., 2013). the difficulties associated with managing the recirculated ns hinder the widespread adoption of the closed system, especially in areas where protected agriculture is characterized by a low level of technology. subirrigation can simplify the closed loop management of the ns, because unlike the drip-irrigation system, the elements that are not absorbed by the plant do not accumulate in the recirculated solution but rather in the upper part of the substrate, where roots are less present (venezia et al., 2001; santamaria et al., 2003; venezia et al., 2006; venezia and piro, 2007; venezia, 2010). spatial distribution of salts within the substrate makes it possible to minimize the effects of excessive salinity. only a few studies to date about soilless cultivations have assessed the possibility of reusing the substrate (urrestarazu et al., 2008; venezia et al., 2008). the use of inert inorganic substrates involves disposal problems that can be avoided by employing organic substrates. among them, peat has been widely used in the mediterranean area. in this context, it is necessary to identify ecologically-friendly alternatives, such as renewable organic substrates that can be reused for several crop cycles, which would provide great benefits to vegetable production. in the case of reusing the substrate, the presence of roots from the previous crop may result in adverse effects on the development of the crop, especially because in soilsoilless cultivation of cherry tomato with gutter subirrigation and reused substrate m. capodilupo, m. stipic, a. venezia(*) consiglio per la ricerca e l’analisi dell’economia agraria, centro di ricerca per l’orticoltura (creaort), via cavalleggeri 25, 84098 pontecagnano (sa), italy. key words: closed system, coconut fiber, horticulture, solanum lycopersicum l. abstract: soilless cultivation systems in horticultural production are modern technologies that involve the supply of water and minerals through the nutrient solution and plant growth on media. the soilless cultivation of vegetables ensures higher yields and better quality than with traditional soil crops. when managed in a closed system with irrigation water of good quality soilless cultivation can significantly reduce the environmental impact of nutrient solutions with respect to crops grown in open systems. compared with drip irrigation, gutter subirrigation simplifies the management of the closed system because disinfection of recirculated nutrient solution and correction of its chemical composition is not necessary. gutter subirrigation was developed in previous experiments for the cultivation of tomato on fresh substrate. the objective of this work was to evaluate the use of the same substrate after two consecutive crops of tomato and to study the effect of the presence, if any, of previous radical residues with the same cultivar and rootstock. the experiment was conducted in an unheated iron and polycarbonate greenhouse with forced ventilation. cherry tomato plants were transplanted into pots containing fresh or reused coconut fiber. the following parameters were considered: biomass, production, product quality (electrical conductivity, ph, titratable acidity, dry residue, dissolved solid content) and chemical analysis of recirculated and radical nutrient solution. the results obtained in this experiment reveal no significant differences in production and fruit quality between plants grown on fresh substrate and those grown on reused substrate (marketable yield was 5.4 kg m-2 vs 5.3 kg m-2, respectively). adv. hort. sci., 2015 29(2-3): 121-124 (*) corresponding author: accursio.venezia@entecra.it received for publication 26 september 2014 accepted for publication 11 may 2015 122 adv. hort. sci., 2015 29(2-3): 121-124 less cultivation farmers are specialized in and practice monoculture. the aim of this work was to evaluate the utilization of ecologically-friendly coconut fiber substrate for repeated crops of cherry tomato cultivated in closed loop gutter subirrigation. 2. materials and methods the experiment was conducted at crea vegetable crops research centre in pontecagnano, southern italy (40°38’ n, 14°52’ e, 28 m asl). cherry tomato plants (solanum lycopersicum l., cv shiren cois ’94 rootstock beaufort de ruiter) were grown in a closed-loop soilless system with gutter subirrigation in an iron-type greenhouse with polycarbonate covering under natural light conditions. air temperature thresholds for ventilation and cooling were 25 and 26°c, respectively. a fresh bulk coconut fiber substrate was compared with the same medium previously utilized for two cropping cycles of cherry tomato shiren/beaufort. the latest cultivation on the reused substrate was terminated 40 days before the experiment started. plots were composed of one row of 20 pots per trough for the fresh substrate and 17 for the reused one. the pots, which contained 10 l of substrate, were positioned on hydraulically independent gutter with 1% slope. for each gutter the ns was contained in an independent storage tank of 270 l. the ionic compositions of the ns supplied expressed in meq l-1 were: ns t1, used for irrigation before transplant in the fresh substrate: 0.3 na+, 0.4 nh4+, 2.5 k+, 4.9 mg++, 8.9 ca++, 0.4 cl-, 11.5 no 3 -, 2.2 h 2 po 4 -, 2.2 so 4 --. ec and ph values were 1.8 ds m-1 and 5.5, respectively. ns 0,3: 0.3 na+, 0.4 nh 4 +, 4.0 k+, 1.5 mg++, 4.4 ca++, 0.2 cl-, 6.8 no 3 -, 0.8 h 2 po 4 -, 1.8 so 4 --. ec and ph values were 1.2 ds m-1 and 5.7, respectively. ns 0,6: 0.3 na+, 0.1 nh 4 +, 8.0 k+, 3.0 mg++, 6.0 ca++, 0.3 cl-, 13.5 no 3 -, 1.5 h 2 po 4 -, 1.2 so 4 --. ec and ph values were 2 ds m-1 and 5.7, respectively. during the first month of growth, plants were irrigated with ns 0,3; ns 0,6 was used in the second month; subsequently ns 0,6 and ns 0,3 were used alternately in order to maintain a constant ec of 1.5 ds m-1 until the end of the cycle. plants were transplanted on 17 august 2013, trained to a single stem and grown up to the seventh truss at a density of 2.6 plants m-2. the experiment ended on 17 december 2013. a bumblebee colony was provided to aid pollination. consumption, ec and ph of the ns were recorded daily. ionic composition of the recirculated ns was determined fortnightly, with samples taken after refilling the tanks. electrical conductivity, ph and ionic composition of the aqueous extracts (1:1.5 v/v growth medium:deionized water) from three layers of the substrate (0-5, 5-17 and 1721 cm from pot bottom) were determined after harvesting. each layer was mixed well with water for 20 min and then filtered before measurements. electrical conductivity and ph values of all samples were determined by metrohm 856 conductivity module; ionic composition by ion chromatography using dionex ics-1500/ics-1600 rfic. the leaf chlorophyll concentration was determined weekly on the fifth true leaf of four plants per plot by using a hand-held spad-502 meter device that estimates chlorophyll in vivo by transmittance of red (650 nm) and infrared (940 nm) radiation through the leaf, and calculating a relative spad meter value that should “correspond to the amount of chlorophyll present in the same leaf” (minolta, 1989). dry above-ground biomass was determined on two plants per plot. plant organs were separated into fruits, stems, leaves, and roots and dried in a forced-air oven at 80°c for 72 h. fruit quality was assessed by measuring the ec, ph, titrable acidity, dissolved solid content and dry residue of blended fruit. to determine titrable acidity and dissolved solid content (°brix) the 905 titrando-metrohm and the refracto 30 px-mettler toledo were used, respectively. to determine the dry residue, the samples of blended tomato berries were dried on ceramic plates at 65°c for 72 h. 3. results and discussion the daily consumption of ns was highest at mid-cycle (1 l per plant per day) and there were no differences between the two treatments. with the recirculated ns, there were no significant effects of substrate reuse on ec. the average ec value was 1.3 ds m-1 in the first month after transplantation, it then increased to 1.7 ds m-1 in the second month and gradually decreased until the end of the cycle up to a value of 1.5 ds m-1 according to the management and composition of the ns supplied. in aqueous extracts from the three layers sampled at the end of the cycle, the ec of fresh substrate tended to increase from the base (0.7 ds m-1) towards the upper part of the vessel (2.5 ds m-1). in pots with reused substrate, ec values were higher for both the intermediate (2.6) and top (9.6 ds m-1) layer. this confirms that excess salts tend to accumulate upwards due to capillary rise. in aqueous extracts of the three layers, the concentration of all elements increased towards the upper part of the vessel and more intensely in the reused vessels. the initial average ph value of the recirculated ns was 5.7 and increased up to 6.3 for both treatments during the first 30 days after transplantation (dat). with reuse in the advanced stage of the cycle (80 dat), the ph of the recirculated solution decreased (5.7); the ph value was 6.2 for fresh and 5.7 for reused substrate at the end of the cycle. regarding aqueous extracts, ph values were lower in the upper layer: in pots with fresh substrate ph was 6.9 for the bottom and 5.9 for the top layer; in reused substrate, the average value was 6.5 for the bottom and 6.3 for the top. 123 capodilupo et al., tomato soilless cultivation with subirrigation for both treatments the initial average spad value was 39.1; at 75 dat for plants cultivated on fresh substrate it was on average 41.7, and 37.9 on reused substrate. at the end of the cycle, spad decreased to a value of 32.3 for both. reuse of substrate resulted in a slight decrease in production (3.4%): the average marketable yield was 5.4 kg m-2 on fresh substrate and 5.3 kg m-2 on reused substrate. the effect of reuse was minimal (3.7%) also on fruit weight with an average of 22.6 g on fresh and 21.7 g on reused substrate noted. with an average value of 106 g/m2 plants cultivated on reused coconut fiber showed a negligible increase of stem biomass (0.2%); also fruit biomass suffered a small change from 498 g/m2 to 519 g/m2 while leaf biomass was reduced by 12.2% with an average value of 266 g/m2 and 233 g/m2 on fresh and reused substrate, respectively. titratable acidity, ph and °brix decreased linearly with the order of the cluster: ph average value was reduced from 4.17 to 4.10; titrable acidity from 0.61 to 0.51% and °brix between 5.5 and 4.3. electical conductivity and dry residue had a more constant trend ranging between 4.54 4.21 ds m-1and 6.9-6.6%, respectively. urrestarazu et al. (2008) grew a tomato crop on almond shell residue reused for 530 days (after four crops) in an open, drip irrigated system without significant yield and fruit quality parameter differences compared to the fresh substrate. they did not encounter phytotoxicity problems because the run off from the open system eliminated root exudates and excess residual salts from previous tomato crops as observed in a tomato crop grown on a substrate added with a compost which was very rich in salts (stipic et al., 2012). in our work the system was closed with zero run off and for the subirrigation, salts and root exudates of the reused substrate accumulated in upper layers due to capillary rise, but still remained in the system. this spatial distribution and the presence of a suitable microflora allowed a normal yield compared to the fresh substrate despite the intense monoculture after two consecutive crops of tomato with the same cultivar and rootstock in pots containing 10 l of substrate with all previous radical residues. the experiment is still underway with a tomato crop growing on a coconut fiber substrate reused after four tomato crops to confirm these results and to characterize the microflora. 4. conclusions this work has demonstrated that it is possible to carry out a soilless monoculture of tomato with gutter subirrigation, a technique still not widespread for the cultivation of fruit vegetables. reuse of the coconut fiber did not induce phytotoxicity and there were no significant effects on the quantity and quality of the product obtained. subirrigation allows all the excess ions to accumulate in the upper part of the substrate; in this way there is no increase of the ec in the lower part of the vessel where roots are massively present nor is there alteration of the re-circulated nutrient solution composition. this confirms that, compared with traditional drip irrigation, gutter subirrigation with coconut fiber simplifies management of the re-circulated nutrient solution in soilless closed loop systems of a fast-growing species such as cherry tomatoes. the comparison between fresh and reused substrate showed that it is possible to reuse coconut fiber at least three times, thus reducing costs in terms of production and the environment. acknowledgements the work was conducted under the project mipaf ofralserpon01_01435. we are grateful to dr. ida chiancone, silvana comella, dr. carlo cesare, mario farina, andrea landi, dr. luigi santonicola and michele de maio for their collaboration. the work reported in this paper has been presented at the “postharvest2014 reducing postharvest losses to feed the world congress” held in barletta, italy, on 2223 may 2014. references minolta, 1989 chlorophyll meter spad-502. instruction manual. minolta co., ltd., radiometric instruments operations, osaka, japan. resh h.m., 2012 hydroponic food production: a definitive guidebook for the advanced home gardener and the commercial hydroponic grower. 7th ed. crc press, boca raton, fl, usa. santamaria p., campanile g., parente a., elia a., 2003 subirrigation vs drip-irrigation: effects on yield and quality of soilless grown cherry tomato. j. hort. sci. biotech., 78: 290-296. savvas d., 2003 hydroponics: a modern technology supporting the application of integrated crop management in greenhouse. j. food agr. environ., 1: 80-86. stipic m., di cesare c., chiancone i., farina m., landi a., piro f., venezia a., 2012 soilless cultivation of tomato using its composted crop residues in the substrate. acta italus hortus, 5: 109-113. urrestarazu m., mazuela p.c., martinez g.a., 2008 effect of substrate reutilization on yield and properties of melon and tomato crops. j. plant nutr., 31: 20312043. venezia a., 2010 pomodoro coltivato senza suolo: sistema aperto/chiuso e subirrigazione. ‘progetto di ricerca per potenziare la competitività di orticole in aree meridionali’ ed. consiglio per la ricerca e la sperimentazione in agricoltura, pp. 52. venezia a., piro f., 2007 coltivazione senza suolo di pomodoro per subirrigazione in canaletta. italus hortus, 14(2): 171. 124 adv. hort. sci., 2015 29(2-3): 121-124 venezia a., piro f., apicella a., di cesare c., stipic m., chiancone i., bacco a., conelli l., 2008 coltivare pomodoro fuori suolo con bassi costi e buone rese. l’informatore agrario, 6: 57-60. venezia a., piro f., tonini a., di cesare c., 2006 pomodoro senza suolo con subirrigazione in canaletta. colture protette, 8: 70-74. venezia a., tonini a., schiavi m., 2001 subirrigazione in vaso a confronto con nft per peperone e melanzana. italus hortus, 6: 39-43. voogt w., beerling e., van os e., blok c., van der maas b., 2013 sustainable nutrient management in soilless culture in dutch greenhouse horticulture. nutrihort: nutrient management, innovative techniques and nutrient legislation in intensive horticulture for an improved water quality. proceedings, september 16-18, ghent, belgium. impaginato 443 adv. hort. sci., 2018 32(3): 443-448 doi: 10.13128/ahs-23910 effects of ozonation on the phenolic fraction of olive oil mill wastewater (oomw): a study case a. ianni 1, e. marone 1 (*), c. martino 2, a. cichelli 3, g. martino 1 1 facoltà di bioscienze e tecnologie agro-alimentari ed ambientali, università di teramo, via r. balzarini, 1, 64100 teramo, italy. 2 dipartimento di medicina veterinaria, università di perugia, via s. costanza, 4, 06126 perugia, italy. 3 dipartimento di scienze mediche, orali e biotecnologie, università g. d’annunzio, via dei vestini, 31, 66100 chieti, italy. key words: antioxidant capacity, biotoxicity, hydroxytyrosol, seeds germination test, tyrosol. abstract: olive oil mill wastewater (oomw) is considered the most polluting fraction of olive processing residues, due to its high content in polyphenols. in this study it has been examined the possibility of using an ozone source as a strong oxidant agent, to lower the phenolic fraction of oomw, allowing its use in agriculture. the oomw, coming from a continuous 3-phase olive oil mill located in the province of arezzo (italy) was submitted to ozonation for 1, 3, and 8 hours, using an ozone generator. total polyphenols, antioxidant activity, and the amount of tyrosol and hydroxytyrosol were determined on the derived samples. to measure the biotoxicity of the treated oomw the germination test of radish seeds was used. the results of the chemical determinations highlighted the effect of dephenolyzation performed by ozonation of oomw, with a significant decrease of antioxidant activity. hydroxytyrosol was significantly lowered, depending on the duration of treatment, while tyrosol resulted less affected. the germination test showed that, with a 50% dilution of oomw, the biotoxicity decreases as the ozone treatment increases. 1. introduction the production of olive oil in the world since 2010 has exceeded 3 million of tons; in the last decades olive growing has expanded beyond its traditional cultivation areas, characterized by warm and arid climate (marone and fiorino, 2012), thanks to the development of continuous oil separation techniques during the 1960s and 1970s, determining a qualitative improvement and a faster processing of the product (kapellakis et al., 2008), the harvesting mechanization (fiorino et al., 2010), the improvement of cultivation techniques, as well as new training systems and canopy management (tous et al., 2010). as processing by-product, important amounts of pomace (solid residues) and olive oil mill wastewater (*) corresponding author: emarone@unite.it citation: ianni a., marone e., martino c., cichelli a., martino g., 2018 effects of ozonation on the phenolic fraction of olive oil mill wastewater (oomw): a study case adv. hort. sci., 32(3): 443-448 copyright: © 2018 ianni a., marone e., martino c., cicchelli a., martino g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 june 2018 accepted for publication 30 september 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(3): 443-448 444 (oomw) are obtained (table 1) (di giovacchino and preziuso, 2006), both considered environmental pollutants. in the past, the olive pomace obtained from pressure and continuous three-phase mills was used only to produce oil by means of chemical solvents (di giovacchino and preziuso, 2006), whereas with the evolution of agriculture practices, and the search for new renewable sources of energy, it was employed as raw material for biogas production and, more recently, it started to be considered a dietary component of dairy cattle in order to improve the characteristics of both milk (castellani et al., 2017) and derived products (castellani et al., 2018). the major problem related to the widespread use of pomace in agriculture depends on the high presence of phenolic compounds, with strong antimicrobial activity that determine a remarkable biotoxicity. phenols show antioxidant properties but also phytotoxic actions in soil and are credited to reduce microbial growth in both anaerobic and aerobic digester. this aspect was explained by girardi et al. (2014), who investigated different oxidative chemical treatments able to reduce both the phenolic content and the triglyceride fraction in various olive solid residues obtained through different extraction processes. in this way it was possible to obtain combined phenolfree compounds and reduced triglyceride, for a semi-finished product appropriated for agricultural purposes, using hydrogen peroxide (h2o2) alone or combined with fe2+ (the fenton system). the oomw is probably the most polluting and biotoxic by-products of the olive processing because of their remarkable antioxidant capacity deriving from the phenolic compounds (de marco et al., 2007), even if it represents a promising resource for agriculture, especially considering their contribution in terms of water intake, and the high quantities of nutritive minerals directly deriving from the fruit juices. an inco-med research project entitled ‘‘new technologies for olive mill waste water detoxification and product recovery’’ (newtech omw, contract ica3-ct-2002-10033) has been granted by the european commission to valorize the oomw. started in 2003 and involving four mediterranean countries, it deals with the treatment of oomw with the aim to develop low-cost, low technology, and environment-friendly treatments to recover high added-value products for different agricultural uses. among the explored solutions can be listed the addiction of fungal cells, soil minerals or pure oxidation catalysts (gianfreda et al., 2006). recently, kerasioti et al. (2017) presented a work focused on the investigation of the effects of livestock feed supplemented with omw on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheeps. therefore, in this case was presented a solution for the development of a low-cost intervention for pathological conditions associated with oxidative stress, while at the same reducing the potential risk of environmental pollution. among the oxidant agents commonly distributed in nature, the strongest is certainly represented by ozone (o3), a component of the atmosphere, generated by the absorption of uv radiation by oxygen. it is considered a strong oxidant which guarantees a broad spectrum of bactericidal effects both in the gaseous and aqueous states. high reactivity, penetrability, and spontaneous decomposition without leaving any harmful by-product, justify the numerous potential uses and make it preferable compared to other oxidizing agents, such as sodium hypochlorite and hydrogen peroxide (brodowska et al., 2017). in food industry, the ozone application is mostly related to decontamination of water and detoxification of product surface, such as the elimination of mycotoxins and pesticide residues from some agricultural products (jin-gab et al., 1999). because of its high oxidizing potential and the minimal environmental impact, this compound has been used for the first time in this work for the detoxification of oomw through the control of the phenolic fraction in order to induce a decrease of the antioxidant activity. the aim of this study was therefore the development of a simple, cheap and low environmental impact methodology, transferable on pressure decanter 3-phase decanter 2-phase and half decanter 2-phase olive pomace (kg t-1) 250-350 450-550 550-650 800-850 olive oil mill wastewater (l t-1) 400-500 600-800 150-300 table 1 quantity of olive pomace and olive oil mill wastewater (oomw) obtained in olive processing by different mechanical systems from di giovacchino and preziuso, 2006. ianni et al. olive oil mill wastewater ozonation 445 an industrial scale, for oxidative detoxification of oomw through ozonation. 2. materials and methods reagents and standards hydroxytyrosol, tyrosol, gallic acid, abts (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid), tptz (2,4,6-tripyridyl-s-triazine), trolox [(±)-6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid], folin-ciocalteu’s reagent, sodium carbonate, potassium persulfate, fecl3·6h2o, methanol and hplc-grade ethanol were purchased from sigma-aldrich (milan, italy), while ethyl acetate was obtained from carlo erba (milan, italy). all chemicals and reagent grade used in the present research were used without further purification steps. olive oil mill wastewater (oomw) collection the used oomw from season 2017 (from october to december) was obtained in march of the present year from a decanting tank of a continuous three-phase olive oil extraction system located in the province of arezzo (italy), where the main cultivars are, in order, moraiolo, leccino and frantoio. ozonation of oomw the oomw was treated with ozone for 1, 3 and 8 h. for each condition, 500 ml of crude oomw were placed in a glass beaker with a total capacity of 800 ml (base diameter: 10 cm; height: 13.5 cm), and ozone was insufflated at room temperature by using a ozosteril generator (p.m.g. depurazione, vercelli, italy) with a production capacity of 250 mg/h and an emission flow equal to 62±0.3 ml/min (power: 30 w max). samples were then aliquoted and stored at 20°c until use. seeds germination test to evaluate the effect of ozonation on the biotoxicity of oomw, a germination test was performed using radish seeds (raphanus sativus l.), according to the methodology used for assessing compost maturity (warman, 1999). comparison was made among the germination of seeds in three different substrates: distilled water (w), untreated oomw (c), and oomw treated with ozone for 1, 3, and 8 hours (without dilution and after a 50% dilution (v/ v) with distilled water). for each testing condition were prepared 10 petri dishes (diameter 9.0 cm, height 1.5 cm) with a double layer of filter paper (whatman® 41) soaked in 10 ml of each substrate, each dish containing 10 radish seeds appropriately arranged (fig. 1). the dishes were placed in a climatic chamber in the dark and at a temperature of 25±1°c. the surveys on germination were carried out after 1 day (t1), 3 days (t2) and 6 days (t3), counting the seeds for which it was possible to observe the appearance of the plumula. the obtained data were submitted to one way and multiway analysis of variance (anova). separation of means was performed by the fisher’s lsd test (p<0.05). computations were performed by statgraphics centurion xv v. 15.0.04. extraction of phenolic compounds from oomw the recovery of oomw phenolic compounds was performed according to the procedure reported by dammak et al. (2016) with slight modifications. ethyl acetate was added to oomw samples (1:1; v/v) and the resulting mixture was stirred for 30 min at room temperature. after centrifugation at 4000 rpm for 5 min, the organic phase was separated, filtered with paper filters (whatman® 41) and concentrated in a rotary vacuum evaporator (40°c). the residual was dissolved in methanol and filtered through a syringe filter (0.45 μm) before subsequent analysis. determination of total phenolic content the total polyphenol content in samples of treated oomw was estimated by the folin-ciocalteu colorimetric method, according to the procedure of singleton and rossi (1965). 10 µl of each sample were mixed with 90 µl of distilled water and 500 µl of freshly prepared 0.2 n folin-ciocalteu’s reagent (1:10 v/v with water). after 10 min, 400 µl of satured fig. 1 radish seeds germination at t3; on the left, petri dishes with untreated oomw; in the middle, oomw 8h; on the right, oomw 1h (diluted 1:1 v/v). adv. hort. sci., 2018 32(3): 443-448 446 sodium carbonate (75 g/l) were added. after incubation at 23°c (room temperature) for 1.5 h, the absorbance of the resulting blue coloured solution was measured at 765 nm with jenway 6305 uv/vis spectrophotometer. quantitative evaluations were performed by using a standard calibration curve of six points (r2 = 0.9944) ranging from 0 to 50 µg/ml of gallic acid in 80% methanol. the total phenolic content was expressed as gallic acid equivalents (gae), in milligrammes per milliliter of sample. evaluation of total antioxidant capacity the estimation of total antioxidant capacity (tac) in oomw extracts was carried by comparing two different approaches: the abts method and the ferric reducing antioxidant power (frap). with regard to the abts was used a modified methodology previously reported by ozgen et al. (2006). abts (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) 7 mm was incubated at room temperature for 12-14 h with an oxidant (2.45 mm potassium persulfate) to obtain a stable, dark blue-green radical solution. the solution was than diluted with 80% hplc-grade ethanol to an absorbance of 0.70±0.2 at 734 nm to form the test reagent. reaction mixtures containing 10 μl of sample and 1.990 µl of reagent were incubated in the dark at room temperature for 6 min, and the reduction of color deriving from the antioxidants action was measured at 734 nm with jenway 6305 uv/vis spectrophotometer. the absorbance was compared to that of the calibrated trolox standard (range 0-16 µm). additional dilution was needed if the absorbance values were over the linear range of the standard curve. results were expressed in terms of trolox equivalent antioxydant capacity (teac; mmol/ml). the frap assay was done according to thaipong et al. (2006). the fresh working solution was prepared by mixing in a 10:1:1 ratio 300 mm acetate buffer (ph 3.6), 10 mm tptz (2,4,6-tripyridyl-s-triazine) solution, and fecl3·6h2o solution. 10 µl of each oomw extract were allowed to react with 1.990 µl of the frap solution for 30 min in dark condition. the formation of colored product (ferrous tripyridyltriazine complex) was recorded at 593 nm. results are expressed in mm teac per ml of oomw extract. hplc analysis the evaluation of phenolic profile was performed by using a hplc method; attention was focused on the relative amounts of hydroxytyrosol and tyrosol before and after the oxidative treatments. the hplc instrument was a varian system coupled to a uv/vis detector set at 280 nm and equipped with a supelcosil lc-18 column (5 µm particle size, 25 cm x 4.6 mm; sigma aldrich, milan, italy) maintained at 40°c in an oven. the eluent flow was fixed at 1 ml/min and the used hplc grade solvents were water and methanol. the elution gradient starts with 80% of water and 20% of methanol; after 30 minutes the water starts to be excluded in favor of the methanol, until reaching the 100% of methanol after 50 minutes from the beginning of the analysis. this condition is then kept constant for further 20 minutes. at the end of the analysis (70 min) the ratio between the solvents was turned back to the initial 80:20 and kept for other 10 min (equilibration of the instrument). the biophenols of interest were identified by comparing the elution times of chromatograms obtained for oomw extracts with those of standards. 3. results chemical effects of oomw ozonation following the ozonation of oomw, a simple and efficient extraction of phenolic component from each sample was obtained by using ethyl acetate, allowing to analyze in an accurate way the total phenolic content, the antioxidant activity and relative quantities of two biophenols of particular interest: hydroxytyrosol and tyrosol. the first noteworthy finding concerns the fact that the oomw used in this study is characterized by a higher phenolic content than that reported in other studies (ochando-pulido et al., 2015; dammak et al., 2016). after treatment with ozone, a marked and timedependent reduction of the phenolic content was evidenced. as reported in table 2, the reduction of tpc is significant after 3 hours of ozonation (from 54.60 mg/ml to 35.34 mg/ml; p<0.01), and reaches the minimum value (16.19 mg/ml; p<0.01) after 8 hours of treatment. these results correlates with the tac which was evaluated in each sample through two different approaches; both the abts and the frap assay showed the lowest antioxidant potential after 8 hours of ozonation (p<0.01), that is precisely in the samples poorest in terms of phenolic compounds. the hplc analysis was performed with the aim of determining the influence of the ozone oxidation ianni et al. olive oil mill wastewater ozonation 447 ozonation, t3), the percentage of germination is statistically equal to that obtained by using distilled water as a substrate (fig. 2). the effect of the different ozonation times on the germination is highlighted in figure 2, that clearly shows as in oomw (c) germination starts only after the third day of incubation, while the seeds placed on ozonated substrates arrange in a sequence (oomw 1h, oomw 3h, and oomw 8h) which reflects the increases in the ozonation. the number of seeds that germinate continues to increase after the third day practically only in the oomw 8h test, and on the sixth day (t3) 87% of the incubated seeds germinated. treatments on the relative concentrations of three biophenols. in particular the attention was focused on hydroxytyrosol and tyrosol, two compounds commonly found in the by-products of the olive oil production industry, to which a high antioxidant activity is associated (de marco et al., 2007). the analysis showed only a partial reduction of hydroxytirosol and tyrosol content; as reported in table 2, hydroxytyrosol showed a minimum residual of 61% after 3 h of ozonation, while tyrosol did not fall below the 78% even after 8 hours of treatment. this datum is not in full agreement with the marked reduction of the tpc previously reported. unexpected is the data concerning ph (table 2), which does not seem to undergo significant changes. seed germination test table 3 shows the germination ability of the radish seeds, placed on different germination substrates: solutions of untreated oomw (c) and oomw derived from different ozonation times (1h, 3h, and 8h, respectively) diluted to 50%; as control, distilled water (w) was used. for each test, the number of seeds germinated after 1, 3 and 6 days (t1, t2, and t3, respectively), was counted. the trial was carried out also on the undiluted oomw, but in this case no germination was obtained: therefore these data have not been reported. from table 3, it is clear that the radish has a high germination ability and, after 1 day, all the seeds placed in distilled water (w) germinated. in the 50% diluted oomw (c) an inhibition is still measurable, and only after 6 days of staying on the substrate it was possible to observe the beginning of germination (on average 6.0%). it is also evident the “time effect” of ozonation, which allowed a germination percentage of 30% already on the first day for oomw treated for 8 hours (t1) and 17% for oomw treated for 3 hours (t1). in the case of the best result (8 hours of table 2 analytical parameters collected after oxidative treatments of oomw with ozone for 1, 3 and 8 hours values in the same row followed by different letters differ significantly (significance was set at p<0.05). * arbitrary unit. § % residual inside the round brackets. c 1h 3h 8h total phenolic compounds (gae mg/ml) 54.60±2.98 a 51.10±2.59 a 35.34±4.68 b 16.19±0.42 c total antioxidant capacity abts (teac µmol/ml) 6.26±0.41 a 5.16±0.63 a 4.52±0.49 b 1.75±0.35 c frap (teac µmol/ml) 3.72±0.12 a 3.03±0.06 b 2.13±0.02 c 1.62±0.14 d hydroxytyrosol*,§ 741 a 639 b (81.0) 471 c (61.4) 490 c (66.1) tyrosol*,§ 630 a 595 a (88.1) 511 b (78.0) 518 b (78.8) ph 4.52±0.03 a 4.49±0.02 a 4.51±0.04 a 4.48±0.03 a table 3 average number of seed germinated per petri dish (ten seeds) with different treatments (w, c, oomw 1h, 3h, 8h) after 1 (t1), 3 (t2), and 6 days (t3), respectively values in the same row followed by different letters differ significantly (significance was set at p<0.05). ozone treatment w c 1h 3h 8h t1 10.0±0.0 d 0.0±0.0 a 1.0±0.0 b 1.7±1.49 b 3.0±1.15 c t2 10.0±0.0 c 0.0±0.0 a 6.7±1.57 b 7.0±2.83 b 7.7±0.67 b t3 10.0±0.0 c 0.6±0.52 a 7.0±1.49 b 7.3±2.83 b 8.7±0.82 c fig. 2 interaction plot (treatment x time) from multiway anova. 448 adv. hort. sci., 2018 32(3): 443-448 4. discussion and conclusions ozonation has proven to be a valuable tool for lowering the phenolic fraction in the oomw. in the present study, using a laboratory instrument, after 8 hours of ozonation the phenolic fraction decreased to less than one third of the initial amount, with an almost equivalent lowering of the antioxidant activity, according to previous works; hydroxytyrosol showed to be reduced in a more marked way with respect to the tyrosol. the findings concerning the germination essay evidenced the detoxifying effect of ozonation; after 8 hours of ozonation the percentage of germinated seeds is statistically equal to that obtained using only distilled water as a substrate, while in the untreated solution the seeds germination is still inhibited after 6 days. these results are also confirmed by comparing the succession of the final germination percentages obtained using different ozonation times: 6% for untreated oomw, 70% after 1, 73% after 3, and 87% after 8 hours of ozonation, respectively. further investigations should be performed to deeply investigate the biochemical effects of ozonation on oomw, but the treatment is really promising for an effective dephenolyzation of the oomw, even for oil mill industrial plants allowing to transform this by-product, currently considered highly polluting, in a crop product suitable for energy production in bioreactors, as natural fertilizer, or foodstuff supplement. references brodowska a.j., novak a., smigielski k., 2017 ozone in the food industry: principles of ozone treatment, mechanisms of action and applications: an overview. crit. rev. food sci. nutr., 10: 1-26. castellani f., vitali a., bernardi n., marone e., grotta l., martino g., 2018 lipolytic volatile compounds in dairy products derived from cows fed with dried olive pomace. eur. food. res. technol., 244: 1-8. castellani f., vitali a., bernardi n., marone e., palazzo f., grotta l., martino g., 2017 dietary supplementation with dried olive pomace in dairy cows modifies the composition of fatty acids and the aromatic profile in milk and related cheese. j. dairy sci., 100: 8658-8669. dammak i., khoufi s., sayadi s., 2016 a performance comparison of olive oil mill wastewater enzymatic treatments. food and bioproducts processing, 100: 61-71. de marco e., savarese m., paduano a., sacchi r., 2007 characterization and fractionation of phenolic compounds extracted from olive oil mill wastewaters. food chemistry, 104: 858-867. di giovacchino l., preziuso s., 2006 utilization of olive mill by-products. proceedings second international seminar olivebioteq 2006. “recent advances in olive industry”. special seminars and invited lectures, mazzara del vallo (tp), italy, 5-10 november, pp. 379-389. fiorino p., marone e., ottanelli a., 2010 mechanical harvesting, productivity and superintensive planting systems in olive groves. adv. hort. sci., 24(1): 91-94. gianfreda l., iamarino g., scelza r., rao m.a., 2006 oxidative catalysts for the transformation of phenolic pollutants: a brief review . biocatalysis and biotransformation, 24(3): 177-187. girardi f., cichelli a., perri e., basti c., d’alessandro n., 2014 oxidative treatments of solid olive residues: effects on phenolic and fatty acid fractions. eur. j. lipid technol., 116: 352-359. jin-gab k., ahmed e.y., sandhya d., 1999 application of ozone for enhancing the microbiological safety and quality of foods: a review. j. food prot., 62(9): 1071-1087. kapellakis i.e., tsagarakis k.p., crowther j.c., 2008 olive oil history, production and by-product management. rev. environ. sci. biotechnol., 7(1): 1-26. kerasioti e., terzopoulou z., komini o., kafantaris i., makri s., stagos d., gerasopoulos k., anisimov n.y., tsatsakis a.m., kouretas d., 2017 tissue specific effects of feeds supplemented with grape pomace or olive oil mill wastewater on detoxification enzymes in sheep. toxicol. rep., 4: 364-372. marone e., fiorino p., 2012 oleiculture in progress. adv. hort. sci., 26(3-4): 163-175. ochando-pulido j.m., victor-ortega m.d., hodaifa g., martinez-ferez a., 2015 physicochemical analysis and adequation of olive oil mill wastewater after advanced oxidation process for reclamation by pressure-driven membrane technology. sci. total environ., 503-504: 113-121. ozgen m., reese r.n., tulio a.z., scheerens j.c., miller a.r., 2006 modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (abts) method to measure antioxidant capacity of selected small fruits and comparison to ferric reducing antioxidant power (frap) and 2,2‘-diphenyl-1-picrylhydrazyl (dpph) methods. j. agric. food chem., 54(4): 1151-1157. singleton v.l., rossi j.a., 1965 colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic., 16(3): 144-158. thaipong k., boonprakob u., crosby k., cisneroszevallos l., byrne d.h., 2006 comparison of abts, dpph, frap, and orac assays for estimating antioxidant activity from guava fruit extracts. j. food composition and analysis, 19: 669-675. tous j., romero a., hermoso j.f., 2010 new trends in olive orchard design for continuous mechanical harvesting. adv. hort. sci., 24(1): 43-52. warman p.r., 1999 evaluation of seed germination and growth tests for assessing compost maturity. compost science & utilization, 7(3): 33-37. impaginato 441 adv. hort. sci., 2019 33(3): 441-446 doi: 10.13128/ahs-23998 pollen viability and in vitro germination of six pistachio (pistacia vera l.) cultivars grown in northern jordan a. aldahadha (*), k. al sane, a. bataineh, a. abu alloush, z. hamouri national agricultural research center (narc), baq’a, jordan. key words: in vitro germination, jordan, pistachio, pollen storage, pollen viability. abstract: this study was conducted on six pistachio cultivars (lazaourdi, nab-el jamal, boundiki, batouri, marawhi and aschouri) to investigate the percentage of pollen viability and in vitro pollen germination under stored and non-stored (fresh) conditions. the results indicated that there was a significant interaction between pollen viability of pistachio cultivars and storage period. this study showed that the non-stored (fresh) pollen of cultivars batouri and lazaourdi had significantly the highest viability (87%) and in vitro pollen germination (69.7%), respectively; indicating that such cultivars could be used as best pollinators. on the other hand, cultivar nab-el jamal had the lowest viability (43.7%) and in vitro pollen germination (40.3%). it was found that pollen viability for all stored pollen cultivars were significantly reduced by 30% when compared with non-stored (fresh) pollen. however, stored pollen germination for one month was zero for all pistachio cultivars. in addition, the results of viability for all fresh pollen cultivars were poorly linearly correlated (r2=0.149) with the results of in vitro germination of fresh pollen. further research is required to examine both pollen viability and in vitro pollen germination under different short and long-term storage periods and methods. 1. introduction pistachio (pistacia vera l.) is a member of the family anacardiaceae (ak et al., 2016). pistachio trees are dioecious, meaning that the pistillate and staminate flowers are formed on different trees. pistachio trees are wind pollinated as flowers have no petals to attract insects (hosseini et al., 2015). one male tree is required for every eight female trees, but this ratio is usually not observed in orchards (bahramabadi et al., 2018). therefore, the amount of pollen produced in each cluster and germination rate of pollen must be high in male trees (ak et al., 2016). pistachio is mainly cultivated in warm-temperate to subtropical parts of the world for its commercially valuable and edible seeds (tilkat and onay, 2009). the center of origin of pistachio species in the near east includes the central asia and turkey. jordan ranks number 13 for pistachio production in the world and has a world share of 0.1 %. production quantity of pistachio in jordan increased from 10 tons in 1975 to 967 tons in 2016 (fao, 2016), (*) corresponding author: abdallah.aldahadha@narc.gov.jo citation: aldahadha a., al sane k., bataineh a., abu alloush a., hamouri z., 2019 pollen viability and in vitro germination of six pistachio (pistacia vera l.) cultivars grown in northern jordan. adv. hort. sci., 33(3): 441-446 copyright: © 2019 aldahadha a., al sane k., bataineh a., abu alloush a., hamouri z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 september 2018 accepted for publication 24 april 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 441-446 442 with an increase in harvested area up to 301 hectares in 2016. in vitro pollen germination is a very useful technique because it can unravel the physiological and biochemical conditions required for the successful pollen germination and pollen tube development (sanjay et al., 2016). in addition, in vitro pollen germination is one of the most convenient and reliable methods used to test the viability of fresh or stored pollen. however, pollen grains of pistacia vera l. have been considered to be difficult to germinate in vitro (golan-goldhirsh et al., 1991). on the other hand, pollen viability usually refers to the ability to distribute functional sperm cells to the embryo sac following compatible pollination (shivanna and ram, 1993). the quality of pollen is evaluated on the basis of viability and vigor. actually, pollen represents a critical stage in the life cycle of plants because viable pollen is essential for effective reproduction of sexual plants. there has been recurrent interest in developing reliable methods for short and long-term storage of pistachio pollen (ateyyeh, 2012). such methods would be useful in storing pollen to be used in pistachio breeding programs (vithanage and alexander, 1985) and supplemental pollination programs in pistachio production (crane and iwakiri, 1981) which requires collection of sufficient amounts of pollen and its storage for short (hours to weeks) or long (months to years) periods, while maintaining viability (shivanna and sawhney, 1997). in addition, storing pollen is very important for cross-pollination, crop breeding, physiology, biotechnology, plant biodiversity and its conservation (polito and luza, 1988). in this study, six pistachio cultivars were grown in maru agricultural research station, irbid, jordan since 1984 (all cultivars were introduced from syria). the percentages of pollen viability and in vitro pollen germination have never been tested in our orchard and these percentages may be varied among these tested cultivars. thus, the overall aim of this experiment was to investigate in vitro germination and pollen viability in six pistachio cultivars under non-stored (fresh) and stored pollen in refrigerator for one month. in addition, this experiment was performed to find a correlation between in vitro pollen germination and viability, to check efficiency of stored pollen and to determine which pistachio cultivar could be recommended as best pollinator. 2. materials and methods plant material and location this research was carried out on six pistachio cultivars: lazaourdi, nab-el jamal, boundiki, batouri, marawhi and aschouri from pistachio orchard at maru agricultural research station. this station is located in irbid governorate at 32° 33’ mn latitude, 35° 51’ e longitude and 589 m above mean sea level (al-ghzawi et al., 2018). maru has typical mediterranean climate conditions with hot and dry summer and an average annual precipitation of about 380 mm and represents an intermediate drought area. in vitro pollen germination and viability tests were carried out at the biotechnology laboratory of the national agricultural research center (narc), baqa’a, jordan. pollen collection pollen collection took place during the flowering period from 3rd to 6th of april, 2018. pollen was collected from six pistachio cultivars. upon anthesis, pistachio clusters of each cultivar were detached and shacked to a glass square. care was taken through this process to prevent contamination. afterwards, pollen of each cultivar was placed in a closed vial to be used later on. pollen storage for each pistachio cultivar, samples of pollen were placed in small glass vials and these samples of non-stored (fresh) pollen were immediately taken to laboratory for in vitro germination and viability tests. other samples were stored in refrigerator at 4°c for 1 month to compare in vitro pollen germination and viability with those of non-stored pollen. in vitro pollen germination test the medium used for in vitro germination testing as recently described by ateyyeh (2012) contained 1% agar, 15% sucrose and 100 ppm boric acid (h3bo3). pollen grains were placed on medium and incubated at 24°c for 24 hours. after this time, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of in vitro pollen germination. for each cultivar, 3 replicates (petri-dishes) were used. pollen is considered to be germinated if the developed pollen tube is exceed (23 times) of its diameter. pollen viability test pollen viability was estimated as described by ateyyeh (2012) by using 1% ttc (2, 3, 5-triphenyl aldahadha et al. ‐ pollen viability and in vitro germination of pistacia vera l 443 tetrazolium chloride) and 60% sucrose. ttc-sucrose solution was stored in brown glass bottle in a refrigerator. one drop of solution was placed onto microslide then a small amount of pollen was suspended in the drop and cover glass was placed onto the microslide, wrapped with aluminum foil and incubated in chamber room at 28°c for 60 minutes. after incubation, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of pollen viability. for each cultivar, 3 replicates (microslides) were used. pollen grains stained orange or bright red color were considered viable. experimental design and statistical analysis the experiment was performed in a factorial design with six pistachio cultivars and two storage treatments (non-stored and stored pollen for 1 month) to investigate pollen viability and in vitro pollen germination separately. there were three replicates for each cultivar and storage treatment. data were analyzed by factorial anova. when there were significant interactions, one-way anova was used and means were separated using least significant difference (lsd). 3. results and discussion in vitro pollen germination both proper pollination and pollen vigor are essential for pistachio productivity since the marketable portion is the seed. to obtain a good fruit set, pollination and fertilization are required. previous studies reported that the yield and quality of nuts were influenced by pollen performance in pistachio (acar and kakani, 2010). it has been indicated that the validity of the in vitro evaluation of pollen germination is a predictor of in vivo behavior (acar and kakani, 2010). the percentages of in vitro pollen germination for six pistachio cultivars are summarized in table 1. the results indicated that the germinability of pistachio pollen varies according to the cultivar under nonstored (fresh) condition (table 1). in particular, fresh pollen of cultivar lazaourdi had significantly the highest in vitro germination percentage (69.7%), followed by cultivars aschouri and boundiki, then cultivars batouri and marawhi. however, fresh pollen of cultivar nab-el jamal had significantly (p < 0.01) the lowest germination percentage (40.3%). in addition, acar et al. (2010) found that under in vitro conditions, pollen germination showed that atli, uygur and kaska male pistachio cvs were generally better than their f1 hybrids. contrary to the reports of polito and luza (1988) who found that pistachio pollen lost its germinability after several days, preliminary tests of vaknin and eisikowitch (2000) revealed that fresh pollen lost most of its germinability within several hours. results of vaknin and eisikowitch (2000) indicated that freshly collected pollen showed the highest germination rate (76.7%). in addition, günver-dalkılıç and dayıdoğru (2011) found that pollen grain germination ratio for pistachio was changed between 78.22% and 63.29% under room conditions at initial day (day 1) in pistachio. however, pollen grain germination ratios were found between 55.83% and 43.26% in pistachio at the 2nd day storage in refrigerator. after one month of pollen storage in refrigerator, in vitro pollen germination was zero for all pollen pistachio cultivars (table 1). this is supported precisely with findings of ateyyeh (2012), suggesting that the table 1 in vitro pollen germination and pollen viability percentage of six pistachio cultivars grown in maru agricultural research station under non-stored (fresh) and stored pollen for one month at 4°c data with the same letter in each column are not significantly different (least significant difference at p<0.05). cultivars in vitro pollen germination (%) pollen viability (%) non-stored pollen (fresh) stored pollen (1 month) non-stored pollen (fresh) stored pollen (1 month) lazaourdi 69.7 a 0 66.7 b 43.0 d nab-el jamal 40.3 d 0 43.7 d 34.3 e boundiki 60.0 b 0 55.0 c 35.7 e batouri 51.0 c 0 87.0 a 53.7 c marawhi 47.3 c 0 65.0 b 46.0 d aschouri 62.3 b 0 85.0 a 67.0 b mean 55 0 67 47 standard error 1.64 0 1.64 3.24 lsd (0.05) 5.142 0 7.547 7.547 adv. hort. sci., 2019 33(3): 441-446 444 stored pollen in refrigerator for one month is not effective method for in vitro pollen germination. furthermore, the pollen grain germination ratios for pistachio were dramatically decreased and reached to about zero starting from the 4th day of storage under room conditions and at 10th day of storage under refrigerated conditions (günver-dalkılıç and dayı-doğru, 2011). pistachio pollen could be stored in refrigerator just for two weeks, which is enough for artificial cross pollination purpose, if the difference in flowering period between males and females didn’t exceed two weeks. vaknin and eisikowitch (2000) found that germinability of pollen kept in the refrigerator for six days was reduced but it retained about 60%. pollen viability the results showed that the percentages of pollen viability are significantly different in regard to pistachio cultivar under non-stored (fresh) and stored conditions (table 1). specifically, non-stored pollen of cultivars batouri (87%) and cv. aschouri (85%) had significantly the highest pollen viability percentage, followed by cultivars (lazaourdi and marawhi), and then cultivar boundiki. nevertheless, cultivar nab-el jamal had significantly (p<0.01) the lowest pollen viability percentage (43.7%). on the other hand, stored pollen for one month significantly (p<0.01) reduced pollen viability percentage in all pistachio cultivars when compared with non-stored pollen. for example, the highest and lowest pollen viability were in cultivars aschouri (67%) and nab-el jamal (34.3%); respectively under stored conditions for one month (table 1). the mean percentage value for fresh pollen viability for all pistachio cultivars was 67%, while for those of stored pollen viability was 47%. thus, all pistachio cultivars lost approximately 30% of their pollen viability when pollen stored at 4°c in refrigerator. however, the mean value for fresh in vitro pollen germination for all pistachio cultivars was 55%. therefore, the percentage of pollen germination was 18% less than those for pollen viability under nonstored conditions. günver-dalkılıç and dayı-doğru (2011) found that the highest and lowest pollen grain viability ratios were obtained as 88.24% (in safranin test) and 70.18% (in ttc test), respectively, in pistachio types (4 male pistachios (pistacia vera l.) grafted on terebinth). ateyyeh (2012) found that fresh pollen viability of pistachio was 87.4% during 2006/2007 season. ateyyeh (2012) found that pollen viability of pistachio under refrigerated conditions for 4 weeks was 0% and 38.7% for seasons 2006/2007 and 2007/2008; respectively. in fact, many factors affect pollen viability and longevity such as genetic variation between species, abiotic environmental conditions, temperature, moisture content, oxygen pressure, nutritional and physiological conditions under which the plants are grown, and the methods of pollen collection and storage (barnabas and kovacs, 1997). it was suggested that the loss of pollen viability in the course of short-term storage is directly related to changes in the water content of the pollen grains, rather than to a deficiency of essential metabolites (barnabas and kovacs, 1997). correlation between tests there was a significant (p<0.05) regression of in vitro pollen germination on viability (fig. 1), meaning that there is a relationship (linear correlation) between in vitro germination and viability for the fresh or non-stored pollen but with poor fit (r2=0.149). particularly, in vitro pollen germination and viability percentages were similar for cultivars (lazaourdi, nab-el jamal and boundiki), however, for cultivars (batouri, marawhi and aschouri), in vitro pollen germination was less than viability percentages. this is why the overall correlation between pollen viability and in vitro germination is weak under non-stored pollen condition. on the other hand, there was no correlation between in vitro pollen germination and viability when pollen stored in refrigerator at 4°c as pollen germination was zero for all tested pistachio cultivars. pollen viability has been correlated with in vitro pollen germination in banksia and some other proteaceae plants (schori et al., 1992). a positive and highly significance correlation between different pollen viability stains and pollen germination test in momordica species fig. 1 correlation between viability and in vitro germination of fresh pollen in six pistachio cultivars grown in maru agricultural research station. significant regression at p<0.05. n=3. aldahadha et al. ‐ pollen viability and in vitro germination of pistacia vera l 445 (rathod et al., 2018), even though, very high percent pollen viability was reported but when the germination test was performed the pollen germination was not showed more than 70 %. it has been pointed out that in vitro pollen germination rates are considered the best indicator of pollen viability (shivanna et al., 1991). furthermore, a good correlation was revealed between in vitro pollen germination with fruit and seed setting in three ornamental tropical tree species (sanjay et al., 2016). 4. conclusions a significant interaction between pollen viability of pistachio cultivars and storage period was found in this experiment. pollen viability in all pistachio cultivars was reduced under storage conditions. pistachio cvs. batouri and lazaourdi might be used as best pollinators. based on our results, it is not recommended to store pollen in refrigerator for one month for germination purposes. further research is necessary to test pollen germination under different conditions and periods of storage. acknowledgements authors would like to warmly thank the director general of the national agricultural research center (narc), jordan for supporting and facilitating this study. thanks are also extended to the staff of maru agricultural research station /irbid. references acar i., ak b.e., sarpkaya k., 2010 effects of boron and gibberellic acid on in vitro pollen germination of pista‐ chio (pistacia vera l.). afr. j. biotechnol., 9: 51265130. acar i., kakani v.g., 2010 the effects of temperature on in vitro pollen germination and pollen tube growth of pistacia spp. sci. hort., 125(4): 569-572. ak b.e., acar i., sakar e., goursoz s., 2016 the impor‐ tance of pistacia species for pistachio production in turkey. acta horticulturae, 1139: 183-188. al-ghzawi a.a., bani khalaf y., al-ajlouni z.i., alquraan n.a., musallam i., bani hani n., 2018 the effect of supplemental irrigation on canopy tempera‐ ture depression, chlorophyll content, and water use efficiency in three wheat (triticum aestivum l. and t. durum desf.) varieties grown in dry regions of jordan. agriculture, 8(5): 1-23. ateyyeh a.f., 2012 effect of storage method on date palm and pistachio pollen viability. jor. j. agric. sci., 8: 573-582. bahramabadi e.z., jonoubi p., rezanejad f., 2018 some cytological and physiochemical features relating to non‐storability of pistachio (pistacia vera l.) pollen. grana, 57(6): 1-8. barnabas b., kovacs g., 1997 storage of pollen, pp. 293-314. in: shivanna k.r., and v.k. sawhney (eds.). pollen biotechnology for crop production and improvement. cambridge university press, uk, pp. 448. crane j.c., iwakiri b.t., 1981 morphology and reproduc‐ tion of pistachio. hort. rev., 3: 376-393. fao, 2016 faostat. agricultural statistics database. fao. food and agriculture organization of the united nations, rome, italy. golan-goldhirsh a., schmidhalter u., müller m., oertli j.j., 1991 germination of pistacia vera l. pollen in liquid medium. ‐ sex. plant reprod., 4(3): 182-187. günver-dalkiliç g., dayi-dogru ö., 2011 determination of pollen grain viability and germination levels for pistachio and terebinth in aydin/turkey ecolo‐ gy. pak. j. bot., 43(2): 841-848. hosseini n., bahramabadi e.z., rezanejad f., 2015 study of morphological and anatomical traits of male flower, developmental stages of anther and pollen grain of pistachio (pistacia vera l.). iran. j. biol., 28(1): 116-125. polito v.s., luza j.g., 1988 low temperature storage of pistachio pollen. euphytica, 39: 265-269. rathod v., behera t.k., munshi a.d., durgesh k., jat g.s., krishnan b.g., sharma n., 2018 pollen viability and in vitro pollen germination studies in momordica species and their intra and interspecific hybrids. ‐ inter. j. chem. studies, 6(6): 32-40. sanjay k.k., khanduri v.p., kar k., mohan sharma ch., riyal m.k., 2016 effect of growth regulators and time on in vitro pollen germination in three ornamental tropical tree species. j. agr. sci. tech., 18(5): 12471255. schori y., goren t., ben-jacov j., 1992 pollen germina‐ tion and storage in banksia and some other proteaceae plants. acta horticulturae, 316: 19-20. shivanna k.r., linskens h.f., cresti m., 1991 pollen viability and pollen vigor. theor. appl. genet., 81(1): 38-42. shivanna k.r., ram h.y.m., 1993 pollen biology: contribution to fundamental and applied aspects. current sci., 65(3): 226-233. shivanna k.r., sawhney v.k., 1997 pollen biology and pollen biotechnology: an introduction, pp. 1-12. in: shivanna k.r., and v.k. sawhney (eds.) pollen biotechnology for crop production and improvement. cambridge university press, uk, pp. 448. tilkat e., onay a., 2009 direct shoot organogenesis from in vitro derived mature leaf explants of pistachio. 446 adv. hort. sci., 2019 33(3): 441-446 in vitro cell. dev. biol. plant, 45: 92-98. vaknin y., eisikowitch d., 2000 effects of short term storage on germinability of pistachio pollen. plant breed., 119: 347-350. vithanage h.i.m.v., alexander d.mc.e., 1985 synchronous flowering and pollen storage techniques as aids to artificial hybridization in pistachio (pistacia spp.). ‐ j. hort. sci., 60: 107-113. impaginato 175 adv. hort. sci., 2017 31(3): 175-181 doi: 10.13128/ahs-21957 uniform and virus-free citrus rootstocks production via nucellus culture a. sepehrtaj, a.r. shahsavar (*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: citrus, nucellus culture, uniform rootstocks, virus-free. abstract: prevalence of various virus and virus-like diseases is among the main reasons for the decrease in quality and quantity of citrus crops. these diseases are mainly spread through the propagation method in citrus which is budding. using nucellus culture of bitter orange and mexican lime seeds, uniform and virus-free rootstocks could be produced so that the diseases prevalence could be prevented. in order to generate adventitious shoots from nucellus culture in each of the two rootstocks, direct organogenesis method is used. in all conducted experiments, murashige and skoog (ms) medium were used. two plant growth regulators of benzyl adenine (ba) in 0, 1, 1.5 and 2 mg l-1 concentration and gibberellins (ga) in 0, 1 and 2 mg l-1 concentration were used in the medium and the main effects of each plant growth regulator were studied separately and their interaction on shoot generation were also surveyed. considering the retrieved data, it was determined that the interaction of ba and ga have a higher impact on shooting, comparing to the cases where each of the regulators is used alone. in mexican lime rootstock, the best culture medium for generating shoots from nucellus culture is the culture medium containing 2 mg l-1 ba and 2 mg l-1 ga and in bitter orange rootstock, the highest shooting rate was attributed to the culture medium containing 2 mg l-1 ga and 1 mg l-1 ba. for the mexican lime and bitter orange shoots rooting, indole butyric acid (iba) was used. the concentrations of this plant growth regulator used in mexican lime were 0, 0.5, 1 and 1.5 mg l-1 and for bitter orange were 0, 1 and 1.5 mg l-1. the highest rooting rate for mexican lime was in culture medium containing 0.5 mg l-1 iba and for bitter orange, it was the culture medium containing 1 mg l-1 iba. the obtained plantlets were gradually adapted with the external environment. 1. introduction citrus is among the most important fruit tree groups in tropical and subtropical regions in the world (jajoo, 2010) and have a significant role in human diet as fruits which contain a high amount of vitamin c and other nutrients such as potassium. similar to other fruit trees, citrus is commercially propagated through grafting a scion on a proper stock and a grafted tree is generated which retains the traits of stock and scion (spreen, 2009). citrus propagation through this method is among the most important limiting factors in generating them, since it leads to the (*) corresponding author: shahsava@shirazu.ac.ir citation: sepehrtaj a., shahsavar a.r., 2017 uniform and virus-free citrus rootstocks production via nucellus culture. adv. hort. sci., 31(3): 175-181. copyright: © 2017 sepehrtaj a., shahsavar a.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 november 2016 accepted for publication 23 april 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 175-181 176 spread of various viral and virus-like diseases through generation of infected seedling (rangan et al., 1968). choosing stock and scion suitable for each environment and then ensuring plants free of any disease play a great role in optimal generation of this crop (shahsavar, 2005). hence, it is required to generate healthy and virus-free stock and scion so that a healthy plant combination is generated. generating healthy and virus-free scion is possible through shoot-tip-grafting (stg) (murashige et al., 1972; shahsavar and khosh-khui, 1994; shahsavar, 2005). however, virus-free rootstock production is not problematic since rootstocks are produced by seeds and seeds are not virus diseases vectors, even if the mother plant is infected (altaf et al., 2001; singh et al., 2006). another issue which is important in rootstocks is their uniformity. propagation from seeds could not lead to production of uniform rootstocks. although some of the rootstocks are produced by the nucellar embryos which are uniform and similar to the mother plant, there are plants produced by natural embryos which are not completely similar to the mother plant and this could lead to the lack of uniformity among the produced rootstocks. hence, in rootstock production, methods should ensure virus-free rootstocks, and uniformity, as well. the best method in reaching this objective is through planting nucellar embryos. fortunately there are nucellar embryogenesis and polyembryonic in most citrus species which produce true to type plants (wutscher, 1979). in 1958, rangan carried out the initiation of nucellar embryos in one of citrus genotypes for the first time and micro propagation in these genotypes provided the possibility for production of uniform plant populations (rangan swamy, 1958). nucellus culture is generally considered as an effective method for producing virus-free citrus and virus is not spread through nucellus cultured. also, the produced plants through nucellus culture and somatic embryogenesis have the potentials to produce plants with the traits of the mother plant (singh et al., 2006). in 2001, altaf reported that using nucellus tissue explants, they were able to produce virus-free plants showing minimum differences with the mother tissue. in the conducted research, the presence of cytokinin ba was necessary for shoot regeneration. however, the optimum concentration of ba is depending on explant genotype and other conditions. rooting of the generated shoots have been reported differently considering used genotype, culture medium and iba concentration (chaturvedi and mitra, 1974; barlass and skene, 1982; duran-vila et al., 1989; jakson and looney, 1999). the main objective of this research is to study mexican lime and bitter orange micropropagation using generation of adventitious shoots through nucellus culture of these seeds and reaching a uniform and virus-free rootstock and also determining the effect of plant growth regulators in stimulation and growth of shoots and rooting of these shoots. 2. materials and methods in this research two important citrus rootstocks including bitter orange (citrus aurantium l.) and mexican lime [citrus aurantifolia (christm.) swingle] were used. immature fruits (100-120 days after pollination) of these species were transferred from darab research station to the laboratory of horticultural sciences department at shiraz university. initially, the fruits were washed with water and a dish washing detergent (rika®) and rinsed. subsequently, they were put in 40% clorox® solution (regular commercial bleach which contains 5-6% sodium hypochlorite) for 15 minutes so that their surface was disinfected. the seeds of each fruit were separated and after washing the seed gel, they were transferred under laminar air flow cabinet. the seeds were left in clorox® solution for 10 minutes and rinsed with sterilized distilled water so that their surfaces were disinfected. subsequently, both seed shells were separated before planting. murashige and skoog (ms) medium (murashige and skoog, 1962) with various plant growth regulators concentrations were used in this research. in order to study the impact of ba and ga and their interaction on shoot generation from nucellus tissue of mexican lime and bitter orange, ba with concentrations of 0, 1, 1.5 and 2 mg l-1 and ga with concentrations of 0, 1 and 2 mg l-1 were used. therefore, there were 4 treatments and 8 replications per treatment and one explant per replication, totally 32 tubes per treatment. sucrose and agar concentration used in all culture media were 30 and 8 g l-1, respectively. the ph of the culture medium was set to be 5.75±0.05 after putting in autoclave and disinfected for 15 minutes at 120°c and pressure of 1.5 kg cm-2. culturing explants after separating the area of the seeds which contained zygotic embryo, remaining tissue of the seed seperhrtaj and shahsavar uniform and virus-free citrus rootstocks production via nucellus culture 177 which contained the nucellar embryo was cultured horizontally in 150×250 ml test tubes which had 20 ml of ms basal culture medium. one explant was sown per tube and exposed to 16-h daily of 20002500 lux illumination of fluorescent light at 27±1°c for 4 weeks. rooting of the generated adventitious shoots for rooting of the generated adventitious shoots, ms basal culture medium along with iba was used (0, 0.5, 1, 1.5 mg l-1 and 0, 1 and 1.5 mg l-1 of iba for mexican lime and bitter orange, respectively, in ten populations). the cultured shoots were kept in rooting culture medium for 20 days using day length of 16 h at 27±1°c. adaptation of rooted plantlets for adaptation, the rooted plantlets were transferred to pots containing a mixture of 50% soil and 50% sand and plastic bags were put over them and kept at 25±3°c and at light intensity of 2,000 lux. the pots were initially irrigated with one eigth of the ms salts concentration for 10 days and they were irrigated by tap water subsequently and they gradually adapted with the external environment by forming holes in the plastic bags. experiment design and data analysis at the end of regenerating shoots experiment, the number of shoots, the length of the shoots and the number of leaves were recorded for each explant. at the end of rooting experiment, the numbers of roots, the length of roots, roots fresh and dry weight were recorded. experiments were conducted in factorial arrangement in a completely randomized design with several replications. the number of replications and the number of explants in each replication are presented at the bottom of each table. data were analyzed by spss software and the means comparison was conducted by duncan’s multiple range test at probability level of 5%. 3. results generating adventitious shoot from nucellus culture of mexican lime explants of nucellar tissue began inflating after being placed on the culture medium in places of cuts and gradually adventitious seedlings and then shoots were emerged in the places of cuts. in this research, the highest number of adventitious shoots (fig. 1) were retrieved in 2 mg l-1 ba and 2 mg l-1 ga with the value of 3.33 (table 1) and there was no significant difference found on the generated adventitious shoots between the media which contained only ba or ga in various concentrations of these growth regulators. considering the derived results, media containing both growth regulators of ba and ga had more impact on shoot regeneration, comparing to the media containing one of the growth regulators only (table 1). the average shoot length in various treatments was different (table 1). the highest shoot length was derived in treatment of 2 mg l-1 of ba and 2 mg l-1 of ga with 6.33 cm which had significant difference with the control (0) treatment (table 1). the highest rate of leaf generation was 9 leaves in 2 mg l-1 of ba and 2 mg l-1 of ga treatment, which has a significant difference with the control treatment (table 1). fig. 1 proliferation of shoots of mexican lime nucellus culture. treatment number of shoots length of shoots number of leaves ga0 mg l-1 +ba0 mg l-1 0.0 c 0.0 e 0.0 d ga 0 mg l-1+ba1 mg l-1 2.28 ab 4.4 abc 8.0 ab ga 0 mg l-1 +ba1.5 mg l-1 1.8 abc 3.20 bcd 7.0 ab ga 0 mg l-1 +ba2 mg l-1 1.5 abc 1.75 dec 4.5 bc ga1 mg l-1 +ba0 mg l-1 2.75 ab 5.125 ab 8.2 ab ga 1 mg l-1 +ba1 mg l-1 0.0 c 0.0 e 0.0 d ga 1 mg l-1 +ba1.5 mg l-1 2.0 ab 4.167 abcd 5.7 abc ga 1 mg l-1 +ba2 mg l-1 0.0 c 0.0e 0.0 d ga2 mg l-1+ba0 mg l-1 1.0 bc 1.5 de 2.0 bc ga 2 mg l-1 +ba1 mg l-1 2.33 ab 3.167 bcd 5.1 abc ga 2 mg l-1+ba1.5 mg l-1 0.0 c 0.0 e 0.0 d ga 2 mg l-1+ba2 mg l-1 3.33 a 6.33 a 9.0 a table 1 the impact of ba and ga growth regulators and their interaction on the number of shoots, length of shoots and number of leaves derived from mexican lime nucellus culture ga= gibberellic acid; ba= benzyl adenine; the results are based on eight replications and one explant per replication. in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range tests. adv. hort. sci., 2017 31(3): 175-181 178 rooting of shoots derived from nucellus culture of mexican lime table 2 presents the rooting of separated mexican lime shoots in culture medium with all concentrations of iba. the highest amount of roots was 4.3 which was achieved in 0.5 mg l-1 of iba treatment, showing significant difference with the average number of roots in 1 and 1.5 mg l-1 and the control treatment. there was no significant difference found between the average root length in concentrations of 0.5, 1 and 1.5 mg l-1 and the average length of the control treatment. the highest fresh weight was 0.052 g in concentration of 0.5 mg l-1 of iba that had a significant difference with the control and other treatments. the highest root dry weight was 0.011 g in concentration 0.5 mg l-1 of iba that had no significant difference with the control and other treatments (fig. 2). generating shoot from nucellus culture of bitter orange the highest value of adventitious shoot regeneration was in the culture medium containing 1 mg l-1 ba and 2 mg l-1 ga which had a significant difference with the rest of treatments, including control treatment (table 3). in this study, no shoot was generated in the culture medium which contained ga only. also, in culture media which contained ba only, the shoot was generated in 1 mg l-1 growth regulator. considering the retrieved results, culture media containing both growth regulators of ba and ga impact the shoot generation better in bitter orange, similar to mexican lime (table 3). the highest generated shoot length average was in 1 mg l-1 of ba and 2 mg l-1 of ga treatment which had a significant different with the control treatment (table 3). the highest leaf generation was 5 leaves which was related to the 2 mg l-1 of ba and 1 mg l-1 of ga treatment that had a significant different with the control treatment (table 3). rooting of shoots derived from nucellus culture of bitter orange table 4 presents the rooting of separated bitter orange shoots in culture medium with concentrations of 0, 0.5, 1 and 1.5 mg l-1 of iba. the highest number of roots were 3 in 1 mg l-1 of iba treatment, such result did not have any significant difference indole butyric acid (iba) mg l-1 root dry weight (g) root fresh weight (g) root length (cm) number of roots 0 0.0072 a 0.024 b 5.01 a 1.66 b 0.5 0.011 a 0.052 a 4.9 a 4.3 a 1 0.0094 a 0.024 b 6.62 a 2.0 b 1.5 0.007 a 0.018 b 4.93 a 2.28 b table 2 the impact of iba growth regulator on rooting of shoots derived from mexican lime nucellus culture the results are based on eight replications and one explant per replication. in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range tests. fig. 2 rooting of the mexican lime adventitious shoots. table 3 the impact of ba and ga growth regulators and their interaction on the number of shoots, length of shoots and number of leaves derived from bitter orange nucellus culture treatment number of shoots length of shoots number of leaves ga0 mg l-1 + ba0 mg l-1 0 c 0 c 0 c ga 0 mg l-1 + ba1 mg l-1 1.33 b 2.6 ab 4 a ga 0 mg l-1 + ba1.5 mg l-1 0 c 0 c 0 c ga 0 mg l-1 + ba2 mg l-1 0 c 0 c 0 c ga1 mg l-1 + ba0 mg l-1 0 c 0 c 0 c ga 1 mg l-1 + ba1 mg l-1 0 c 0 c 0 c ga 1 mg l-1 + ba1.5 mg l-1 1 b 2 b 2 b ga 1 mg l-1 + ba2 mg l-1 1 b 4 a 5 a ga2 mg l-1 + ba0 mg l-1 0 c 0 c 0 c ga 2 mg l-1 + ba1 mg l-1 2.25 a 4 a 4 a ga 2 mg l-1 + ba1.5 mg l-1 1.5 b 3.5 a 4.5 a ga 2 mg l-1 + ba2 mg l-1 0 c 0 c 0 c ga= gibberellic acid; ba= benzyl adenine; the results are based on eight replications and one explant per replication. in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range tests. seperhrtaj and shahsavar uniform and virus-free citrus rootstocks production via nucellus culture 179 with the average number of roots observed in 1.5 mg l-1 treatments, while it had a significant difference with the control. the root length average in each treatment was 10.30 and 8.51 cm respectively for concentrations of 1 and 1.5 mg l-1 which showed a significant difference with the control treatment. the highest fresh weight of 0.05 g and the highest dry weight of 0.012 g were observed using concentration of 1 mg l-1 of iba, showing a significant difference with the control treatment. 4. discussion and conclusions generating shoot from nucellus culture of mexican lime and bitter orange some seedlings of some citrus cultivars were generated in vitro by culturing nucellus explants (rangan et al., 1968, 1969). however, this method has not been successful in all cases (button and kochba, 1977). the current study showed that nucellus in mexican lime and bitter orange species is not capable of generating shoot without plant growth regulators, and application of plant growth regulators could increase their potentials for generating shoot. usman et al. (2005) reported that the number of shoots induced in each explant depends on the citrus type. for instance, in kinnow mandarin, there is more shoots generated comparing to orange during direct organogenesis. in this experiment, it was indicated that mexican lime nucellus explants have higher potentials for generating shoot, comparing to bitter orange. considering the role of plant growth regulators for generating shoot from nucellus, two growth regulators of ba and ga were used in this research. results have suggested that ba is responsible for generating shoot from nucellus in both species, since in medium without the regulators, there was lower number of shoots in mexican lime, and there was no shoot in bitter orange. external application of ba is necessary in culture medium (raj-bhansal and arya, 1978). for direct organogenesis, rattanpal et al. (2011) put epicotyl and hypocotyl explants of citrus jambhiri lush. in culture medium containing ba and this hormone led to generation of shoot in these explants. ga leads to increase the length of the generated shoots (rattanpal et al., 2011). in studying ga impact, without the presence of ba in the culture medium in this research, it could be claimed that this hormone has been effective on shoot length in both species and the results from this research are in accordance with the results from saini et al. (2010). they reported that adding ga to the culture medium containing ba improves the number of elongated shoots (saini et al., 2010). results from this research suggested that using a combination of various levels of ba and ga has been more effective in both species, comparing to the application one of these hormone only. for instance, the highest number of shoots, shoot length and number of leaves in mexican lime was related to the treatment with 2 mgl-1 of ba and 2 mg l-1 of ga and in bitter orange, the highest number of shoots and shoot length was related to the use of 2 mg l-1 of ga and 1 mg l-1 of ba. there are various reports which show that the application of these two plant growth regulators have been effective on shoot generation, such as that of gill and gosal (2002) who generated a high rate of shoots in c. depressa by applying 1 mg l-1 of ba and 2 mg l-1 of ga. also, the highest percentage of shoot generation in explants of poncirus trifoliate was derived when 2 mg l-1 of ga and ba were used in culture medium (usman et al., 2005; tzatzani et al., 2009). rooting of shoots drived from nucellus culture of bitter orange and mexican lime the highest rate of rooting for bitter orange was obtained in the medium containing 1 mg l-1 of iba. in mexican lime the highest rate of rooting was obtained in the medium containing 0.5 mg l-1 of iba. by increasing the concentration of this hormone, there was a decrease in the number of generated roots. sandra and morehart in 1998 (in orange) and singh et al. in 2006 (in mexican lime and tangerine) reported that by the increase in iba concentration, the rooting rate decreases which are in accordance with the results from this experiment. iba has been highly successful in rooting in many citrus species, including citrus aurantifolia (raj-bhansal and arya, table 4 the impact of iba growth regulator on rooting of shoots derived from bitter orange nucellus culture indole butyric acid (iba) mg l-1 root dry weight roots fresh weight root length number of roots 0 0.0 b 0.0 b 0.0 b 0.0 b 1 0.012 a 0.05 a 10.30 a 3.0 a 1.5 0.011 a 0.039 a 8.51 a 2.16 a the results are based on eight replications and one explant per replication. in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range tests. adv. hort. sci., 2017 31(3): 175-181 180 1978). in addition to the above mentioned, the results about the impact of iba on rooting of nucellus shoots in mexican lime and bitter orange were in accordance with the findings of khalekuzzaman et al. (2008) on adhatoda vasica, wilson et al. (2010) on indoneesiella ecohides and purkayastha et al. (2008) on andrographis paniculata. a great number of lateral roots were generated in mexican lime in the concentration of 0.5 mg l-1 of iba, while there was no lateral root observed in other treatments. transfer and adaptation the plantlets adapted to the sterilized mixture of equal volume ratio of perlite and vermiculite in 2 months. since the humidity is high in culture tubes, the humidity for the plantlets is provided by plastic bag during adaptation period and over time the humidity is decreased by forming holes in the plastic bags. all adapted plants were transferred to the greenhouse with 100% success and stayed alive. these results were in accordance with the findings from dojam et al. (2001) in orange in and jajo (2010) in citrus limonia osbeck. interaction of ba and ga has a higher impact on shooting in mexican lime and bitter orange rootstocks. in mexican lime the best culture medium for generating shoots containing 2 mg l-1 ba and 2 mg l-1 ga and in bitter orange rootstock, the highest shooting rate was attributed to the culture medium containing 2 mg l-1 ga and 1 mg l-1 ba. the highest rooting rate for mexican lime was in culture medium containing 0.5 mg l-1 iba and for bitter orange; it was the culture medium containing 1 mg l-1 iba. references altaf n., murwat e.k., bhatti i.a., mohsin m., 2001 nucellar regeneration and polyembryony of citrus cultivars. pak. j. bot., 33(2): 211-215. barlass m., skene k.g.m., 1982 in vitro plantlet formation from citrus species and hybrids. sci. hort., 17: 333-341. button j., kochba j., 1977 tissue culture in citrus industry, pp. 70-92. in: reinert j., and y.p.s. bajaj (eds.) applied and fundamental aspects of plant cell tissue and organ culture. springer-verlag, berlin, germany, pp. 804. chaturvedi h.c., mitra g.c., 1974 clonal propagation of citrus from somatic callus cultures. hort sci., 9: 118120. dojam m., khosh-khui m., shekafandeh a., 2001 in vitro propagation of sweet orange (citrus sinensis l. osbeck) by direct organogenesis in epicotyl segments. j. hort. sci. and technol., 2: 85-94. duran-vila n., ortega v., navarro l., 1989 morphogenesis and tissue culture of three citrus species. plant cell, tiss. org. cult., 16: 123-133. gill m.i.s., gosal s.s., 2002 micropropagation of pectinifera (citrus depressa hayata. a potential citrus rootstock for sweet orange. indian j. citriculture, 1(1): 32-37. jajoo a., 2010 in vitro propagation of citrus limonia osbeck through nucellar embryo culture. j. biol. sci., 2(1): 6-8. jakson d.i., looney n.e., 1999 temperate and subtropical fruit production. cab international, wallingford, oxon, uk, pp. 321. khalekuzzaman m., rahmanm s., rashid m.h., hossainm s., 2008 high frequency in vitro propagation of adhatoda vasica nees through shoot tip and nodal explants culture. j. bio. sci., 16: 35-39. murashige t., bitters w.p., rangan e.m., nauer e.m., roistacher c.n., holliday p.b., 1972 a technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones. hort. sci., 7: 118-119. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue culture. physiol. plant., 15: 473-497. purkayastha j., sugla t., paul a., solleti s., sahoo l., 2008 rapid in vitro multiplication and plant regeneration from nodal explants of andrographis paniculata: a valuable medicinal plant. in vitro cell. dev. biol. plant., 44: 442-447. raj-bhansal i.r., arya h.c., 1978 shoot formation in stem and root callus of citrus aurantifolia (christm.) swingle̕, grown in cultures. curr. sci., 47: 775-776. rangan swamy n.s., 1958 culture of nucellar tissue of citrus in vitro. experienta, 14: 11-12. rangan t.s., murashige t., bitters w.p., 1968 in vitro initiation of nucellar embryos in monoembryo development by liquid culture in sunflower (helianthus annus l.). plant sci., 67: 73-82. rangan t.s., murashige t., bittersw p., 1969 in vitro studies of zygotic and nucellar embryogenesis in citrus, pp. 225-229. in: chapman h.d. (ed.) proceeding of the international citrus symposium. university of california at riverside, riverside, ca, usa. rattanpal h.s., kaur g., gupta m., 2011 in vitro plant regeneration in rough lemon (citrus jambhiri lush) by direct organogenesis. african j. bio., 8163: 1372413728. saini h.k., gill m.s., gill m.i.s., 2010 direct plant regeneration in rough lemon (citrus jambhiri. lush). indian j. biotech., 9: 419-423. sandra m.k., morehart a.l., 1998 tissue culture of osage orange. hort sci., 23(3): 613-615. shahsavar a.r., 2005 comparison of different citrus rootstocks for micro grafting. iran agr. res., 5: 109116. shahsavar a.r., khosh-khui m., 1994 the effects of sevseperhrtaj and shahsavar uniform and virus-free citrus rootstocks production via nucellus culture 181 eral variables on shoot-tip grafting of (clementine) mandarin on” troyer” citrange. iran agr. res., 13: 1-18. singh b., sharma s., rani g., zaidia a., hallan v., nagpal a., virk g.s., 2006 in vitro production of indian citrus ringspot virus-free plants of kinnow mandarin (citrus nobilis × c. deliciosa tenora) by ovule culture. j. plant biotech., 7: 259-265. spreen t.h., 2009 the world citrus industry, pp. 249-269. in: verheye w.h. (ed.) soil, plant growth and crop production. volume iii. encyclopedia of life support systems. unesco, uk, pp. 425. tzatzani t.t., theriou k., therios i., molassiotis a., 2009 the effect of ba, ga and iba on in vitro organogenesis of poncirus trifoliatafrom globular bodies. acta horticulturae, 825: 289-292. usman m., mahammad s., fatima b., 2005 in vitro multiple shoot induction from nodal explants of citrus cultivars. central european agri. j., 6: 435-442. wilson s., justinkoilpilla y., louis jesudass l., 2010 in vitro multiplication and field establishment of indoneesiella ecohides l. an important medicinal plant. int. j. biol. technol., 1: 64-68. wutscher h.k., 1979 citrus rootstocks. hort. rev., 1: 237-269. impaginato 233 adv. hort. sci., 2020 34(2): 233­240 doi: 10.13128/ahsc­7848 extraction of total protein from shoots of cereus morphological variants (cactaceae) for proteomic analysis v.n.a. fernandes 1, c.a. mangolin 2, a.f. neves 1, f.c. sousa nogueira 3, h. zeni neto 4, m.f.p.s. machado 2 (*) 1 postgraduate program in genetics and breeding, universidade estadual de maringá, maringá pr, brazil. 2 department of biotechnology, genetics and cell biology, universidade estadual de maringá, maringá pr, brazil. 3 department of chemistry of proteins, unidad proteomics, chemistry institute, universidade federal do rio de janeiro, rio de janeiro, rj, brazil. 4 department of agronomy, universidade estadual de maringá, maringá pr, brazil. key words: cactus, phenotypic variants, protein extraction, sds­page, succulent tissues. abstract: since there is a hypothesis that qualitative and/or quantitative differ­ ences of specific proteins may be associated with morphological variants in cacti of the cereus genus (phenotypes erect, tortuosus and monstruosus), in current study we tested three different methods for protein extraction from shoots of the phenotypic variants to obtain protein fractions for further pro­ teomic analysis. the tca/acetone method for protein extraction revealed a larger number of well­defined bands in sds­page system than the methods with phenol. the quantification of protein extracted by tca/acetone ranged between 0.488 (tortuosus) and 2.92 µg·ml­1 (monstruosus). although the use of phenol is the most appropriate procedure for protein extraction from recalci­ trant tissues, results have shown that extraction buffer containing two antioxi­ dant agents (edta and β­mercaptoethanol) and pmsf to prevent protein degradation was efficient to avoid proteolysis and lower protein yield, than using tca/acetone precipitation for protein extraction from shoots of cereus sp. the use of extraction buffer with appropriate combinations of antioxidant agents, phenol­complexing agents, and protease inhibitors may be an efficient alternative for proteins extraction from succulent and recalcitrant tissues (such as cactus plants) using a simple protein extraction method. 1. introduction since the 1960s the study on proteins from plant tissues is frequently restricted by extraction procedures from different tissues (loomis, 1969; 1974; kelley and adams, 1977). different procedures for protein extrac­ tion have been reported for different tissues and plant species to obtain (*) corresponding author: mfpsmachado@uem.br citation: fernandes v.n.a., mangolin c.a., neves a.f., sousa nogueira f.c., zeni neto h., machado m.f. p.s., 2020 ­ extraction of total protein from shoots of cereus morphological variants (cactaceae) for proteomic analysis. ‐ adv. hort. sci., 34(2): 233­240. copyright: © 2020 fernandes v.n.a., mangolin c.a., neves a.f., sousa nogueira f.c., zeni neto h., machado m.f.p.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 september 2019 accepted for publication 16 january 2020 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 233­240 234 protein fractions which are adequate for elec­ trophoresis and polypeptides characterization on gel. taxa and tissues have their own set of endoge­ nous tannins, phenols and phenoloxidases which interfere differently with protein stability. protein extraction from cactus tissues is particularly an arduous process due to the abundance of complex heteropolysaccharides, coupled to endogenous tan­ nins, phenols and phenoloxidases in the shoot tis­ sues of adult plants. heteropolysaccharides confer high viscosity to the extraction solution after shoot­ tissue homogenization and hinder the solubilization of proteins. protein extraction has been a primordial stage for the studies on proteomics. the detection of pro­ teins and protein variants found in cells of a given tissue under specific conditions (functional genom­ ic) (wilkins et al., 1996; westermeier and naven, 2002) requires adequate solubilization and stability of protein fractions. the first step should establish a procedure for protein extraction from shoots, or rather, for the proteome analysis in shoots of phe­ notypic variants of cactus from genus cereus. the cereus plants have been mainly used as forage for ruminants (silva et al., 2011) and fruits and pulp are used in the preparation of cookies, pies, and pastries (almeida et al., 2007). several studies have revealed the importance of the c. peruvianus species as fruit crop (mizrahi, 2014). while the c. jamacaru plants constitute a wild natural resource in the semiarid region of northeastern brazil, an industrial and eco­ nomic importance has been attributed to the c. peru‐ vianus species cultivated in the southern region of brazil (alvarez et al., 1995; barros and nozaki, 2002). medicinal importance is also attributed to c. peru‐ vianus since arabinogalactan extracted from the gum has been indicated for the treatment of gastric ulcers (tanaka et al., 2010). phenotypic variants of cacti from the genus cereus, tagged tortuosus and mon‐ struosus varieties, are frequently cultivated together with plants featuring typical columnar­erect shoots in home gardens and public parks and squares in brazil´s southern region. although the origin of the tortuosus and monstruosus varieties has not been reported in the literature, the qualitative and/or quantitative differences of specific proteins may be associated with the morphological variants. since the success of a proteomic experiment depend on the correct identification of proteins, three different methods described by he and wang (2008) for protein extraction from shoots of the three phenotypic variants of cereus were employed in the current study. the methods described by he and wang (2008) were used in protein extraction from aloe vera tissues, a succulent and recalcitrant plant similar to cacti. it is expected that protocols described by these authors may be suitable for pro­ tein extraction and quantification from shoots of cereus plants. 2. materials and methods samples of cereus plants with typically erect shoots and plants of the varieties tortuosus and mon‐ struosus (fig. 1), cultivated in south brazil (in maringá pr brazil, at 23°25’38” s; 51°56’15” w), were collect­ ed from home gardens and maintained in the experimental botanic garden of the state university of maringá (altitude 554.9 m; 23°25’s; 51°25ʹw). pieces of shoots (2 g) from erect, tortuosus and mon‐ struosus plants (four plants of each morphology) were collected and used as samples for protein extraction. the cuticle and the fractions of cells with chlorophyll were removed from the pieces of shoot to minimize the contamination of the samples by polysaccharides. fresh shoot sections (2 g) of each cereus plant with erect shoots (e1­e4) and plants of the varieties tortuosus (t1­t4) and monstruosus (m1­m4) were ground to a fine powder in liquid nitrogen and homogenized in 2 ml buffer 0.02 m tris­hcl ph 7.5, 0.25 m sucrose, 0.01m ethylene glycol tetraacetic acid (edta), 0.001 m phenylmethylsulfonyl fluoride (pmsf), 1% triton x­100 and 2% ß­mercaptoethanol, following method by he and wang (2008). proteins were extracted from two samples of each e1­e4, t1­ fig. 1 ­ different morphologies of cereus peruvianus plants showing stems: erect (a), tortuosus (b), monstruosus (c). fernandes et al. ‐ extraction of protein for proteomic analysis 235 t4, and m1­m4 plants (table 1). after homogenization, 1.0­ml aliquots from each cereus plants were transferred to 2 ml microtubes and prepared by three procedures: i) tca­acetone precipitation; ii) phenol extraction, and iii) improved phenol extraction method according to described by he and wang (2008). protein was quantified by the fluorometric method in a fluorometer qubit® 1.0 using qubit® protein assay kit from life technologies. polyacrylamide gel (12%) was prepared with 16.2 ml of 30% acrylamide and 0.8% bis­acrylamide dissolved in 4 ml of 1.5 m tris­hcl, ph 8.8, 107 µl 10% sds, 5.7 ml twice­distilled water, 320 µl 2% ammonium per­ sulfate and 16 µl temed. stack gel was prepared at a final concentration of 5%, ph 6.8: 3 ml of 10% acry­ lamide and 5% bis­acrylamide dissolved in 3 ml of 0.24 m tris­hcl ph 6.8, 30 µl twice­distilled water, 250 µl 2% ammonium persulfate and 3 µl temed. further, 25 mm tris/200 mm glycine, ph 8.3, and 0.1% sds were employed in the electrode chambers. samples were taken from the freezer and 2 μl loading dye [20% glycerol 10 mm, tris­hcl 1.5 m ph 8.8, 10% (w·v­1) bromophenol blue, 10% sds (w·v­1), β­mercaptoethanol 2% (v·v­1), and twice­distilled water q.s.p.] were added to 8 μl of sample and applied to the gel. electrophoresis was performed in tris­glycine buffer at 200 volts, for thirty minutes, for the stacking gel, followed by two hours and thirty minutes for the separation of the proteins. after elec­ trophoresis, the gel was fixed in a fixation solution (40% methanol; 70% acetic acid) and stained in silver 20% following protocol by laemmli (1970). after migration, gels were stained, photographed and the images were analyzed with gelanalyzer 19.1 softwa­ re (http://www.gelanalyzer.com/) to transform elec­ trophoretic bands into peaks, to calculate rf and esti­ mate the molecular weight of proteins. the protein quantification (µg·µl­1) by the fluoro­ metric method in duplicate of morphological variants of cereus with shoots erect (e1­e4), tortuosus (t1­ t4), and monstruosus (m1­m4) obtained by extrac­ tion with tca/acetone method described by he and wang (2008) were analyzed using the software r (r core team, 2019) with the packages: i) “nortest” (gross and ligges, 2015) to verify the normality of the errors by the lilliefors test (lilliefors, 1969), ii) “car” (fox and weisberg, 2019) to verify the indepen­ dence of errors by the durbin­watson test (durbin and watson, 1951), and iii) the “expdes” package (ferreira et al., 2018) to verify the homogeneity of variances by the o´neill and matthews test (o´neill and matthews, 2000). all these procedures were admitting the error at 1% of significance. 3. results and discussion the tca/acetone method for protein extraction from shoots of cereus plants revealed a larger num­ ber of well­defined bands in sds­page system than the methods with phenol and improved­phenol extraction. in the tca/acetone method, each homog­ enized tissue (plant tissues with shoots erect, tortuo‐ sus and monstruosus) in the extraction buffer was centrifuged at 15,000 rpm for 30 min at 4°c. the supernatant was then recovered and placed in a new tube where ¼ of the volume of acetone containing 50% trichloroacetic acid (tca) was added. the solu­ tion was incubated for 2 h, at ­20°c and centrifuged again at 15,000 rpm for 40 min, at 4°c. the super­ natant was then discarded and the pellet was table 1 ­ protein quantification (µg µl­1) by the fluorometric method in duplicate of morphological variants of cereus with shoots erect (e1­e4), monstruosus (m1­m4), and tortuosus (t1­t4) obtained by the extraction with tca/acetone method described by he and wang (2008) erect monstruosus tortuosus sample µg·µl­1 sam µg·µl­1 sample µg·µl­1 e1 0.824 m1 2.64 t1 1.090 0.658 2.68 0.990 e2 0.732 m2 1.71 t2 0.768 0.766 1.91 1.150 e3 0.964 m3 2.26 t3 1.740 0.874 2.40 1.520 e4 1.100 m4 2.54 t4 0.488 1.480 2.92 0.592 adv. hort. sci., 2020 34(2): 233­240 236 washed three times with acetone containing 0.2% 1,4­dithiothreitol (dtt). pellets were then dissolved in 50 µl lysis buffer (8 m urea, 4% np­40 and 1% dtt) and stocked at ­20°c. the quantification of protein extracted by tca/acetone method was made in duplicate (two samples of each shoot morphology: erect, tortuosus and monstruosus) and ranged between 0.488 (tortu‐ osus) and 2.92 µg·ml­1 (monstruosus) (table 1). the normality of the errors by the lilliefors test (lilliefors, 1969), the independence of errors by the durbin­watson test (durbin and watson, 1951) and the homogeneity of variances by the o´neill and matthews test (o´neill and matthews, 2000) showed that there were no restrictions by p­values regarding analysis of variance (anova) procedure (table 2). significant differences (at 1% level) in protein con­ centrations within and between cereus plants with the erect, tortuosus and monstruosus morphologies were detected by anova procedure (table 3). the non­significant experimental error indicates that the sampling error (different morphologies) may justify the differences in protein concentrations between cereus plant morphologies. non­significant experimental error assures the researcher more reli­ able results because the effect of uncertainty is smaller (patterson, 1946; cochran and cox, 1957; snedecor and cochran, 1980). a higher protein concentration in monstruosus plants than in erect and tortuosus plants has been detected by tukey´s test (tukey, 1953) employed to compare the averages of protein concentrations in erect, tortuosus and monstruosus plants (table 4). figure 2 also illustrates the highest protein concen­ tration in monstruosus plants and shows the ampli­ tude of concentration rates within each plant. differences in protein concentrations in cereus plants with the erect, tortuosus and monstruosus morphologies indicate that it is necessary to evaluate the protein concentration in more than one plant with the same morphology to specify the average protein concentration of each morphology. differences in protein concentrations among cereus plants with erect, tortuosus and monstruosus mor­ phologies detected in current study support the hypothesis that qualitative and/or quantitative differ­ ences of specific proteins may be associated with morphological variants in cacti of the cereus genus, and may be used to justify a proposal for further pro­ teomic analysis. the phenol extraction method described by he and wang (2008) revealed a number of protein frac­ table 2 ­ p­values of the normality of errors by the lilliefors test (lilliefors, 1969), independence of errors by the durbin­watson test (durbin and watson, 1951) and the homogeneity of variances by the o´neill and matthews test (o´neill and matthews, 2000) procedure normality independence variance lilliefors 0.8897 durbin­watson 0.0379 o´neill and matthews 0.4089 table 3 ­ mean squares (ms) of the comparison of protein con­ centrations from the three cereus morphologies (erect, tortuosus and monstruosus shoots) and their respective sampling and experimental mistakes ** p­value <0.01. ms morphologies 5.2477 ** sample error 0.2969 ** experimental error 0.0251 table 4 ­ mean protein concentrations (µg µl­1) from the three cereus morphologies (erect, tortuosus and monstruo‐ sus) revealed by the tukey´s test morphology mean protein concentraction (µg·µl­1) monstruosus 2.3825 a tortuosus 1.0423 b erect 0.9248 b fig. 2 ­ amplitude of the concentration values observed within monstruosus (m), tortuosus (t), and erect (e) plants of cereus. fernandes et al. ‐ extraction of protein for proteomic analysis 237 tions smaller than the number of protein fractions detected by the tca/acetone precipitation method in cereus plants, while protein fractions were absent with the improved phenol extraction method described by he and wang (2008)(fig. 3). the gelanalyzer software was useful to create calibration curves and to estimate the quantity of the protein from a band. figure 4 shows the calibration curve that make a correspondence between the quantity of the protein loaded on each lane and the areas of the peaks of each lane (the software name the area of the peak as raw volume and the conventional units are in pixels). the correlation coefficients of such cali­ bration curves were higher than 0.99 for proteins with molecular masses ranging from 24 until 180 kda (table 5). however, data obtained by ms (mass spectrometry analysis) indicated proteins with mole­ cular weights varying from 220 to 15 kda. in gel regions without clear band distinction in 1de sds­ page were detected considerable amounts of pro­ tein identified by ms. preliminary mass spectrometry analysis identified 753 proteins extracted by the tca/acetone precipitation method in the cereus plants (unpublished results). improved phenol extraction method was described as the most appropriate procedure for pro­ fig. 3 ­ protein extraction from shoots of cereus with erect (samples 1­2, 5­6, 9­10), tortuosus (samples 3, 7, 11) and monstruosus (samples 4, 8, 12) morphologies, with tca/acetone method (samples 1­4), with phenol extrac­ tion method (samples 5­8) and with the improved­phe­ nol extraction method by he and wang (2008) (samples 9­12), in the sds­page system 12%. fig. 4 ­ the main panels of gel analyser software showing the image of the entire electrophoresis gel (panel a); the bands from the selected lane from panel a are transformed in peaks (panel b) and their area are computed in panel c, as raw volume (conventio­ nal units). based on the molecular weights of the markers (in panel d), the correspondence between the molecular mass and the migration (as rf) of the protein is presented. 238 adv. hort. sci., 2020 34(2): 233­240 tein extraction from aloe vera tissues, a succulent plant, similar to cacti. phenol has also been consid­ ered as the most effective for the removal of unwanted interfering substances from the protein samples of tissues from other recalcitrant plants (he and wang, 2008; pavoković et al., 2012; riffel et al., 2012). it has been postulated that, in the case of par­ ticularly recalcitrant tissues, acetone and tca/ace­ tone precipitation do not sufficiently remove nucleic acids, carbohydrates and polyphenols, which cause co­precipitation and degradation of proteins. the phenol method, although more laborious and time­ consuming, resulted in higher protein yield and with the lowest contamination rate of samples than the tca/acetone precipitation method alone for protein extraction from recalcitrant tissues (he and wang, 2008; pavoković et al., 2012; wu et al., 2014). however, results from the current study are indicative that the use of an extraction solution con­ taining two antioxidant agents (edta and β­mercap­ toethanol) and pmsf to prevent protein degradation was efficient to avoid proteolysis and lower yield of proteins using tca/acetone precipitation for protein extraction from shoots of cereus sp. although the use of phenol is reported to be the most appropriate procedure for protein extraction from recalcitrant tis­ sues, a significant proportion on cellular proteins may be lost from the extracts during the extraction proce­ dure with phenol. the phenol extraction method involves more steps than the tca/acetone method while a ‘minimum­steps´ method is preferred with protein extraction procedures (isaacson et al., 2006). the tca/acetone method described by he and wang (2008) involved only one precipitation, two centrifu­ gations and three washes after homogenization of the tissues, whereas nine steps (including one overnight precipitation at ­20°c) were reported for phenol method. thus, the use of the tca/acetone method described by he and wang (2008) represent­ ed a minimum­steps method and, thus, a shorter time for the extraction of proteins from shoot tissues of cereus plants. a relatively simple extraction buffer containing one phenol­complexing agent (pvp) and two antiox­ idant agents (β­mercaptoethanol and edta) was also used for electrophoresis of the several isozymes in shoot tissues of the cactus cereus peru‐ vianus (mangolin and machado, 1997), while phenol and ammonium acetate were employed for the pro­ tein extraction from callus tissues of c. peruvianus (mangolin et al., 1999) for two­dimensional elec­ trophoresis of callus tissues grown in culture media containing different concentrations of auxin and cytokinin. only one phenol­complexing agent (pvp) and a cocktail of protease inhibitors (roche, usa) were used for protein extraction from tissues of the cactus mammillaria gracilis grown in vitro (rogic´ et al., 2015). the tca/acetone/β­mercaptoethanol method plus protease inhibitors (roche, usa) was also employed for protein extraction from several species of succulent plants and some cactus species (lophocereus marginatus, mammillaria magnimam‐ ma and opuntia fícus‐indica) instead of phenol pre­ cipitation method (lledías et al., 2017). the evidence from these studies actually demonstrates that, in the case of tissues with different levels of interfering sub­ stances, different phenol complexing agents and dif­ ferent antioxidant agents are required. the physio­ logical state of each tissue is the decisive factor of a greater or lesser complexity in protein extraction processes. as types and concentrations of polysac­ charides, polyphenols and other secondary metabo­ lites may be induced in response to environmental factors, different types and concentrations of phenol­ complexing agents and antioxidant agents may be needed for the same tissue in different environmen­ tal conditions. according to wendel and weeden (1989), extraction solutions for tissues with moderate table 5 ­ quantity of the protein loaded on each lane detected with tca/acetone method (samples 1­4) and with phenol extraction method (samples 5­8) in the sds­page system 12% from the cereus plants lane mw of each band (kda) lane 1 ­ ­ ­ ­ ­ ­ 45 ­ 24 lane 2 ­ ­ ­ 77 ­ ­ 45 ­ 24 lane 3 ­ 145 90 77 ­ ­ 45 39 ­ lane 4 180 145 90 77 ­ ­ 45 39 ­ lane 5 ­ ­ ­ ­ ­ ­ 45 ­ ­ lane 6 ­ 145 90 77 61 ­ 45 39 24 lane7 ­ ­ ­ ­ ­ ­ 45 ­ ­ lane 8 ­ ­ ­ ­ ­ 58 45 ­ ­ fernandes et al. ‐ extraction of protein for proteomic analysis 239 levels of interfering substances require at least two phenol­complexing and two antioxidant agents, while at least four phenol­complexing and three antioxi­ dant agents are needed for tissues with high levels of interfering substances. 4. conclusions the simplest method (tca/acetone precipitation), described by he and wang (2008), and the use of two antioxidant agents and a protease inhibitor showed a number of protein fractions greater than the number of protein fractions detected with the phenol method in shoots of cereus analyzed in cur­ rent study (plants with erect shoots and plants of the varieties tortuosus and monstruosus). the authors´ expectation is to use this simple method of extracting proteins for proteomic analysis of the phenotypic variants from the genus cereus. however, the use of phenol or other phenol­complexing and antioxidant agents to extract proteins from shoots of other cereus plants grown in different regions or in differ­ ent environmental conditions (different seasons of the year, e.g.) may be needed. consequently, the proposal to establish a ‘universal protocol’ for succu­ lent plants, such as cacti, seems unattainable. therefore, testing with extraction buffers and precip­ itation methods, which may be time­consuming and laborious, seems indispensable for further analysis of other cereus plants in a proteomic­based approach. acknowledgements the authors would like to thank the coordenação de aperfeiçoamento de pessoal de nível superior, brasília, df, brazil) for financial support (capes; finance code 001) and conselho nacional de desenvolvimento científico e tecnológico (cnpq), grant no. 306508/2013­3. references almeida c.a., figueiredo r.m.f., queiroz a.j.m., oliveira f.m.n., 2007 ­ características físicas e quími‐ cas da polpa de xiquexique. ­ rev. ciências agronômica, 38: 440­443. alvarez m., costa s.c., huber a., baron m., fontana j.d., 1995 ­ the cuticle of the 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portland, or, usa. westermeier r., naven t., 2002 ­ proteomics in practice: a laboratory manual of proteome analysis. ­ wiley­ vch, weinheim, germany. wilkins m.r., pasquali c., appel r.d., 1996 ­ from pro‐ teins to proteomes: large scale protein identification by two‐dimensional electrophoresis and amino acid analy‐ sis. ­ biotechnology, 14: 61­65. wu x., gong f., wang w., 2014 ­ protein extraction from plant tissues for 2‐de and its application in proteomic analysis. ­ proteomics, 14: 645­658. http://onlinelibrary.wiley.com/book/10.1002/3527600175 impaginato 21 adv. hort. sci., 2020 34(1s): 21­26 doi: 10.13128/ahsc­8492 modified atmosphere packaging to improve the shelf­life of goji berries during cold storage m. palumbo 1, 2, i. capotorto 1 (*), m. cefola 1, s. burbaci 1, b. pace 1 1 institute of sciences of food production, cnr, national research council of italy, c/o cs‐dat, via michele protano, 71121 foggia, italy. 2 department of science of agriculture, food and environment, university of foggia, via napoli, 25, 71122 foggia, italy. key words: lycium barbarum l., marketability, respiration rate, visual quality, wolfberries. abbreviation: aa= antioxidant activity; air= control in air; cie= commission internationale de l’eclairage; etoh= ethanol; fw= fresh weight; map= modified atmosphere packaging; meoh= methanol; pmap= passive modified atmosphere packaging; rr= respiration rate; tp= total phenols; tss= total soluble solids; vq= visual quality. abstract: this study was carried out to evaluate the effect of modified atmos­ phere packaging on the quality parameters and the shelf­life of fresh goji berries. fruits, placed in trays, were closed in passive modified atmosphere packaging (pmap) using polypropylene bags or kept in open polyethylene bags (air) as control. samples were analyzed just after harvest and during storage (5, 13 days) at 7°c for visual quality (vq), color parameters, weight loss, dry weight, total soluble solids (tss), antioxidant activity (aa) and total phenols (tp), while respiration rate (rr) was evaluated only after 5 days. changes in gas composition in pmap samples was measured daily. the use of pmap allowed to reduce the rr of about 26% compared to fresh sample, to preserve the berries weight loss during storage and their marketability until 13 day at 7°c, while air samples were not edible after 5 days due to mold growth on the berries surfaces. no changes of color parameters, dry weight, tss, aa and tp were observed during storage comparing treatments. in conclusion, the use of pmap was able to extend the shelf­life of goji berries for 13 days at 7°c, 8 days more than berries stored in air. 1. introduction the goji (lycium barbarum l.) berries, also known as wolfberries, are considered “superfruits” for their high nutritional value, richness in nutri­ ents, antioxidants and bioactive compounds of which the health promot­ ing properties are known (sidhu and zafar, 2012; jatoi et al., 2017; niro et al., 2017). the berries are mostly grown for dry fruit, but nowadays the (*) corresponding author: imperatrice.capotorto@ispa.cnr.it citation: palumbo m., capotorto i., cefola m., bur­ baci s., pace b., 2020 ­ modified atmosphere packaging to improve the shelf‐life of goji berries during cold storage. ­ adv. hort. sci., 34(1s): 21­ 26 copyright: © 2020 palumbo m., capotorto i., cefola m., burbaci s., pace b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2019 accepted for publication 26 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 21­26 22 goji market is significantly expanding, focus also on the fresh fruit. however, due to the tender peel and high­water content, fresh goji berries are easy to damage and rot, so their transport and storage are difficult (fan et al., 2019). for these reasons the use of postharvest handling to preserve the storability of this perishable fruit are required. very few studies are been conducted on the storage of fresh goji berries. jatoi et al. (2018) evaluated the postharvest quality of fresh goji berries stored at different tem­ perature, from ­2°c to 20°c, concluding that the opti­ mum storage temperature to preserve phytochemi­ cal and sensory attributes was 0°c. the application of the lecithin (jatoi et al., 2017) or edible coating based on lotus leaf extract (fan et al., 2019) were studied to improve the shelf­life of fresh goji berries. ban et al. (2015) reported that a combination of heat treat­ ment at 40°c for 30 min followed by chitosan coating protect goji berries from decay, extending their postharvest life up to 28 days of storage at 2°c. the use of additives or coatings, even though are natural, is often undesirable from consumers that are more attracted to fresh products without any additional ingredients. from this point of view, the use of modi­ fied atmosphere packaging (map) during storage, consisting in a reduction of o2 and/or an increase in co2 levels, can be a valid tool, in addition to the proper temperature, in order to improve the shelf­ life of goji berries. modified atmosphere act reducing respiration rate and weight loss, delaying ripening and softening, thus minimizing the incidence of some physiological disorders and decay (kader, 2002 a). kafkaletou et al. (2017) tested the effectiveness of a short­term treatments with different atmospheres enriched in co2 to prevent fungal decay in fresh goji berries, concluding that atmospheres with high co2, from 15 to 20%, were able to reduce fungal decay incidence in goji berries stored for 14 days at 1°c. to the best of our knowledge, no further studies on the application of modified atmosphere on goji berries are available, so the present study is aimed to evalu­ ate the application of map technology to extend the shelf­life of fresh goji berries. 2. materials and methods reagents extraction solvents (meoh, etoh), 2,2­diphenyl­1­ picrylhydrazyl (dpph), 6­hydroxy­2,5,7,8­tetram­ ethylchroman­2­carboxylic acid (trolox) and all stan­ dards used in the experiments were obtained from sigma­aldrich (st. louis, mo., usa). folin­ciocalteu’s phenol reagent was purchased from merck (germany). plant material and experimental set‐up fresh goji berries (lycium barbarum l.), about 2 kg, were provided from favella group located in the south of italy (corigliano calabro, italy), and trans­ ported in cold condition to the postharvest laboratory of cnr­ispa to be processed. after elimi­ nation of damaged fruits, 3 replicates of about 80 grams berries were used for the initial determination, while the remaining samples were placed in polyethylene terephthalate trays (model c250/50 carton pack®, italy), 80 grams per trays, and sealed in passive modified atmosphere packaging (pmap) using polypropylene bags (dimension 200 x 150 mm, 30 mm thickness), or in unsealed polyethylene bags (air) as control. for each packaging condition (pmap or air), 6 bags (3 replicates × 2 storage times) were stored at 7°c (±1) and analysed initially and after 5 and 13 days for visual quality, color parameters, weight loss, dry weight, total soluble solids, antioxi­ dant activity and total phenols, while respiration rate was evaluated initially and after 5 days, because of mould development in air samples after 13 days. in addition, changes in gas composition in pmap sam­ ples was monitored daily using a gas analyser (checkpoint, pbi dansensor, ringsted, denmark). respiration rate the respiration rate of goji berries was measured at 7°c using a closed system as reported by kader (2002 b). about 80 grams of berries for each replicate were put into 6 l sealed plastic jars (one jar for repli­ cate) where co2 was allowed to accumulate until the value of 0.1%. the time needed to reach this value was calculated, making co2 measurement at regular time intervals. for the co2 analysis, 1 ml gas sample was taken from the head space of the plastic jars through a rubber septum and injected into the gas chromatograph (p200 micro gc, agilent, santa clara, ca) equipped with dual columns and thermal con­ ductivity detector. co2 was analyzed with a retention time of 16 s and total run time of 120 s on a 10 m porous polymer (ppu) column at a constant tempera­ ture of 70°c. respiration rate was expressed as ml co2 kg­1 h­1. after the respiration rate evaluation, berries were used for the following analysis. visual quality and color analysis visual quality was evaluated by a group of ten palumbo et al. ‐ goji berries stored in passive modified atmosphere packaging 23 trained people, on a subjective 5 to 1 scale, with 5= excellent, no defects; 4= very good, minor defects; 3= fair, moderate defects; 2= poor, major defects; 1= inedible. a score of 3 was considered to be the limit of marketability, while a score of 2 represented the limit of edibility. color parameters (l*, a* and b*) were measured, for each replicate, on 3 random points on peel sur­ face of 5 goji berries using a colorimeter (cr­400, konica minolta, osaka, japan) in the reflectance mode and in the cie l* a* b* color scale. colorimeter was calibrated with a standard reference having val­ ues of l*, a* and b* corresponding to 97.55, 1.32 and 1.41, respectively. hue angle (h°=arctan b*/a*) and sat­ uration (chroma=ö a*2 + b*2) were then calculated from primary l*, a* and b* readings. weight loss, dry weight and total soluble solid con‐ tent goji weight loss was calculated at each storage time as percentage of variation from the initial fresh weight. to measure dry weight, goji berries were maintained in a forced­draft oven at 65°c until con­ stant weight was reached. total soluble solid con­ tent, expressed in °brix, was measured using a digital refractometer (model dbr35, xs instruments, carpi, italy) on a liquid extract obtained by whisking in a blender (1 min; 14,000 rev. min­1) 10 goji berries from each replicate and then filtering the juice. antioxidant activity and total phenols to determine both antioxidant activity and total phenol contents, the extraction procedure reported by cefola et al. (2012) was followed. in detail, 5 grams samples were homogenized (ultraturrax t­25, ika staufen germany) in a meoh:water (80:20) solu­ tion for 1 min, and then centrifuged at 5°c at 6440 ´ g for 5 min. the supernatant was therefore used for the assays. the antioxidant activity assay was per­ formed following the procedure described by brand­ williams et al. (1995) with minor modifications. briefly, the supernatant, proper diluted, was pipetted into 0.95 ml of dpph solution to start the reaction. the absorbance was read after about 30 min at 515 nm. trolox was used as a standard and the antioxi­ dant activity was expressed in milligrams of trolox per 100 g of fresh weight (fw) (mg trolox 100 g­1 fw). the total phenol content was determined according to the method of singleton and rossi (1965). each extract (100 μl), proper diluted, was mixed with 1.58 ml water, 100 μl of folin­ciocalteu reagent and 300 μl of sodium carbonate solution (200 g l­1). the absorbance was read after 2 h at 765 nm. total phe­ nol content was calculated on the basis of the cali­ bration curve of gallic acid and expressed as mil­ ligrams of gallic acid per 100 g of fresh weight (mg gallic acid 100 g­1 fw). statistical analysis in order to evaluate the effect of packaging condi­ tion (pmap or air) on quality parameters of goji berries, a one way anova was performed at each storage time (5 and 13 days), and mean values were separated applying least significant difference (lsd) multiple range test with significant difference when p≤0.05. 3. results at harvest, the respiration rate of goji berries was 23.6 (±3.5) ml co2 kg­1 h­1 at 7°c. after 5 days of stor­ age, respiration rate in air samples slightly increased, while the use of pmap allowed to reduce the rate of respiration of about 26% compared to fresh sample (table 1). in figure 1 changes in gas composition inside bags in pmap samples were reported. starting from air composition (21% o2 and 0.03% co2), oxygen was gradually consumed by the product, due to the respi­ ration process, with a consequent accumulation of co2. at the first sampling time, after 5 days at 7°c, o2 fig. 1 ­ changes in gas composition of goji berries stored in pmap for 13 days at 7°c (±1). values are means of three replicates for each storage time ± standard deviation. table 1 ­ respiration rate of goji berries at harvest and after 5 days at 7°c (±1) in pmap or air values are means of three replicates ± standard deviation. different letters indicate statistical differences for p≤0.05, accor­ ding to lsd test. respiration rate (ml co2 kg­1 h­1) at harvest fresh 23.6 ± 3.5 after 5 days at 7°c pmap 17.3 ± 0.2 b air 28.4 ± 1.9 a adv. hort. sci., 2020 34(1s): 21­26 24 fig. 2 ­ changes in visual quality of goji berries stored in pmap or air for 13 days at 7°c (±1). values are means of three replicates for each packaging condition at each storage time. within the same storage time, different letters indicate statistical differences, p≤0.05. visual quality score: 5=excellent, no defects; 4=very good, minor defects; 3=fair, moderate defects; 2=poor, major defects; 1=inedible. and co2 values were 10.0% (± 1.0) and 8.4% (± 0.6), respectively. then, the consumption of oxygen was slowdown, reaching the concentration of 3.3% (± 0.9) after 13 days at 7°c, while co2 was 10.4% (± 0.9) (fig. 1). the visual quality of the product at harvest was not optimal, in fact panelists gave an initial score of 4 (good) (fig. 2). at each storage time, significant dif­ ferences between treatments were observed. in par­ ticular, after 5 days at 7°c goji berries stored in air were not edible, mainly due to mold growth on the berries surfaces, while pmap samples were scored as more than acceptable, keeping their marketability until the end of the storage (fig. 2). regard the other physical (color, dry weight) and chemical (total soluble solid content, antioxidant activity, total phenols) parameters, the values mea­ sured at harvest are reported in table 2; however, no significant changes were observed during storage and comparing treatments (at the end of the storage dry weight, tss, aa and tp had mean values of 23.9%±0.6, 21.7±0.4°brix, 82.1±2.4 mg trolox 100 g­1 fw and 216.1±1.3 mg gallic acid 100 g­1 fw, respec­ tively), except for all the color parameters that slight­ ly decreased during storage (at the end of the stor­ age, l*, a*, b*, h° and chroma mean values were 46.8±0.7, 38.4±0.1, 37.3±0.1, 14.0±0.3, 4.8±1.3, respectively). 4. discussion and conclusions respiration rate represents one of the most important parameters that should be taken into account for the study of the postharvest perfor­ mance of fresh produce since it is inversely correlat­ ed with shelf­life and thus, it can be used as an indi­ cator of perishability. considering the initial respira­ tion rate of goji berries (23.6±3.5 ml co2 kg­1 h­1 at 7°c), these fruits have a high respiratory metabolism, according to the classification reported by kader (2002 c). in the proposed pmap, the o2 reduction and co2 accumulation, due to the high berries respi­ ration rate and the permeability properties of the packaging, slowed down the rate of respiration of goji berries. this positive effect of map was previous­ ly reported on different fruit by sandhya (2010). similar results on goji berries treated with different atmospheres of low oxygen and high co2 were the weight loss of goji berries stored in pmap was almost constant for all the storage period while fruits stored in air lost about 17.5% of the initial weight after 13 days at 7°c (fig. 3). fig. 3 ­ weight loss of goji berries stored in pmap or air for 13 days at 7°c (±1). values are means of three replicates for each packaging condition at each storage time. within the same storage time, different letters indicate statisti­ cal differences, p≤0.05. table 2 ­ color parameters, dry weight, total soluble solid con­ tent, antioxidant activity and total phenols evaluated in goji berries at harvest evaluated parameters data color parameters l* 49.1±0.6 a* 46.9±2.4 b* 48.2±2.6 h° 45.1±0.3 chroma 67.2±3.5 dry weight (%) 22.8±0.3 total soluble solid content (° brix) 21.4±0.5 antioxidant activity (mg trolox 100 g­1 fw) 85.8±1.5 total phenols (mg gallic acid 100 g­1 fw) 211.1±2.4 values are means of three replicates ± standard deviation. palumbo et al. ‐ goji berries stored in passive modified atmosphere packaging 25 weight loss, with the consequence depreciation of the product. on the other hand, the application of a modified atmosphere packaging resulted a valid tool to delay the loss of quality of goji berries, prolonging their shelf­life. results of the present study demon­ strated that the use of pmap allowed to reduce the respiration rate, preserved the berries weight loss and the heath properties, and control the mould development. as consequence berries goji stored in pmap showed a shelf­life of 13 days at 7°c, 8 days more than berries stored in air. acknowledgements the authors thanks the farm favella group for providing plant material. references ban z., wei w., yang x., feng j., guan j., li l., 2015 ­ combination of heat treatment and chitosan coating to improve postharvest quality of wolfberry (lycium bar­ barum). ­ int j food sci technol., 50: 1019­1025. brand­williams w., cuvelier m.e., berset c., 1995 ­ use of a free radical method to evaluate antioxidant activity. ­ lwt ­ food sci. technol., 28: 25­30. cefola m., d’antuono i., pace b., calabrese n., cari­ to a., linsalata v., cardinali a., 2012 ­ biochemical relationships and browning index for assessing the stor‐ age suitability of artichoke genotypes. ­ food res. int., 48: 397­403. donno d., mellano m.g., raimondo e., cerutti a.k., prgomet z., beccaro g.l., 2016 ­ influence of applied drying methods on phytochemical composition in fresh and dried goji fruits by hplc fingerprint. ­ europ. food res. technol., 242(11): 1961­1974. fan x.j., zhang b., yan h., feng j.t., ma z.q., zhang x., 2019 ­ effect of lotus leaf extract incorporated compos‐ ite coating on the postharvest quality of fresh goji (lycium barbarum l.) fruit. ­ postharvest biol. technol., 148: 132­140. jatoi m.a., fruk m., buhin j., vincekovic m., vukovic m., jemric t., 2018 ­ effects of different storage tem‐ perature on storage life, physic‐chemical and sensory attributes of goji berry (lycium barbarum l.) fruits. ­ erwerbs­obstbau., 60(2): 119­126. jatoi m.a., juric s., vidrih r., vincekovic m., vukovic m., jemric t., 2017 ­ the effects of postharvest appli‐ cation of lecithin to improve storage potential and quality of fresh goji (lycium barbarum l.) berries. ­ food chem., 230: 241­249. kader a.a., 2002 a ­ controlled atmosphere storage, pp. 22­25. ­ in: kenneth c.g., i.w. chien, and m. reported by kakfaletou et al. (2017). the positive effect of the application of pmap was reported also on the weight loss that was significant lower in pmap compared to air samples. this because the use of packaging represents a barrier to vapor diffusion which allows to maintain an adequate relative humidity within the package, so tissue dehydration is limited (zagory and kader, 1988). the presence of fungal decay was the main factor that influenced the loss of marketability of fresh goji berries during storage. the co2 accumulation (until 10%) inside the packages was able to inhibit the mold growth, as previously observed (kafkaletou et al., 2017). in particular these authors reported that an atmosphere with 15­20% of co2 applied as a short­ term treatment for 2 days at 1°c was able to reduce fungal decay incidence in goji berries stored for 14 days at 1°c. the red color of goji berries was not influenced by packaging condition (pmap or air), whereas a slight decrease in all the color parameters measured were observed during storage. similar results were report­ ed on goji berries by kafkaletou et al. (2017) and jatoi et al. (2017). regarding to dry weight, our data (22.8%±0.3) are in accordance with data reported by niro et al. (2017) that found a moisture of 77.4% on fresh goji berries, means 22.6% dry weight. also, the presented data of total soluble solids at harvest (21.4±0.5°brix) are similar to that reported by kafkaletou et al. (2017) (from 21 to 25°brix) and fan et al. (2019) (about 22°brix). goji berries is consid­ ered a “superfruit” for their antioxidant activity due to the high content of bioactive compounds and vita­ min c (sidhu and zafar, 2012; jatoi et al., 2017; niro et al., 2017). in the present research paper, the value of total phenols (211.1±2.4 mg gallic acid 100 g­1 fw) at harvest, is quite similar to that reported in litera­ ture on fresh goji berries by donno et al. (2016) (from 199.5 to 240.3 mg gallic acid 100 g­1 fw, depending on region of cultivation) and jatoi et al. (2017) (about 223 mg gallic acid 100 g­1 fw). as for antioxidant activity, our data at harvest were similar (85.8±1.5 mg trolox 100 g­1 fw) to data reported by jatoi et al. (2017) on the same fruit. both total phe­ nols and antioxidant activity remain unchanged dur­ ing storage in pmap and in air samples, as previous­ ly reported by jatoi et al. (2017) for total phenols. fresh goji berries are highly perishable fruits, with a high respiration rate, if stored in air. the loss of marketability is very fast, and it is mainly due to the development of molds on the peel surface. in addi­ tion, the high transpiration of the peel causes a rapid 26 adv. hort. sci., 2020 34(1s): 21­26 saltveit (eds.) the commercial storage of fruits, veg‐ etables, and florist and nursery stocks . usda, handbook no. 60. kader a.a., 2002 b ­ methods of gas mixing, sampling and analysis , pp. 145­148. ­ in: kader a.a. 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(ed.) postharvest technology of horticultural crops. division of agriculture and natural resources, university of california, california, usa. kafkaletou m., christopoulos m.v., tsantili e., 2017 ­ short‐term treatments with high co2 and low o2 con‐ centrations on quality of fresh goji berries (lycium bar­ barum l.) during cold storage. ­ j sci food agric., 97: 5194­5201. niro s., fratianni a., panfili g., falasca l., cinquan­ ta l., rizvi alam m.d., 2017 ­ nutritional evaluation of fresh and dried goji berries cultivated in italy. ­ ital. j. food sci., 29: 398­408. sandhya, 2010 ­ modified atmosphere packaging of fresh produce: current status and future needs. ­ lwt ­ food sci. technol., 43(3): 381­392. sidhu j.s., zafar t.a., 2012 ­ super fruits: pomegranate, wolfberry, aronia (chokeberry), acai, noni, and amla, pp. 653­ 679. ­ in: sinha n.k., j.s. sidhu, j. barta, j.s.b. wu, and m.p. cano (eds.) handbook of fruits and fruit processing. 2nd edition. wiley­blackwell, singleton v.l., rossi j.a., 1965 ­ colorimetry of total phenolics with phosphomolybdic‐phosphotungstic acid reagents. ­ am. j. enol. viticult., 16: 144­158. zagory d., kader a.a., 1988 ­ modified atmosphere pack‐ aging of fresh produce. ­ food technol., 42(9):70­77. https://www.worldcat.org/search?q=au%3asinha%2c+nirmal+k.%2c&qt=hot_author https://www.worldcat.org/search?q=au%3asidhu%2c+jiwan+s.%2c&qt=hot_author https://www.worldcat.org/search?q=au%3abarta%2c+jo%cc%81zsef%2c&qt=hot_author https://www.worldcat.org/search?q=au%3awu%2c+james+s.+b.%2c&qt=hot_author impaginato 471 adv. hort. sci., 2018 32(4): 471-477 doi: 10.13128/ahs-21989 essential oil production of murraya paniculata (l.) jack at different harvest times c.i.m. semarayani, s.a. aziz (*), m. melati department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, 16680 bogor, indonesia. key words: β-methylesculetin, caryophyllene, murralongin, solvent extraction. abstract: murraya paniculata (l.) jack has a fragrant flower, from which the fragrance is due to the essential oil. the study aimed to investigate the production of essential oil and its chemical compounds at different harvest times. the research was conducted at an organic experimental farm, bogor agricultural university, bogor, indonesia (6°30’-6°45’ s, 106°30’-106°45’ e) from october 2016 to february 2017 using randomized complete block design. the experiment consisted of one factor, namely the harvest times, comprised of harvest at 05.00-07.00 and 07.00-09.00 a.m. m. paniculata flowers were collected at three different flower ages, comprised of two days before anthesis, one day before anthesis and the day of anthesis (blooming). the different flower ages indicated by the flower size. ethanol extraction method was used to extract the essential oil of the flowers from different harvesting times and then chemical compounds were analyzed by gas chromatography-mass spectrometry. the result showed that flower number and weight were not affected by harvesting times. the flower collected on the day of anthesis had the highest flower number and weight. harvesting flowers at anthesis can be done at 05.00-09.00 a.m. the highest quantity and quality of essential oils were obtained by harvesting the flowers at anthesis. β-methylesculetin and murralongin were the primary compounds in m. paniculata flowers that harvested at 05.00-09.00 a.m. 1. introduction murraya paniculata (l.) jack well known as orange jessamine is an ornamental plant and belongs to family rutaceae (shah et al., 2014), it has white flowers with sweet fragrance (gilman, 1999). the plants are native to southeastern asia, i.e. cambodia, laos, myanmar, thailand, vietnam, indonesia, malaysia, and philippine (dosoky et al., 2016). m. paniculata has been used in traditional medicine because the plant has anti-amnesic, anti-inflammatory, anti-diabetic, anti-fungal, anti-bacterial, anti-helminthic, anti-cancer, and anti-oxidative properties (sharma and arora, 2015). beside as a source for perfumery, m. paniculata is also used as a source of flavors (el-sakhawy et al., 1998) because the flowers are highly aromatic and contain sufficient amount of essential oil (naseem et al., 2015). plants essential oils are aromatic components that composed (*) corresponding author: sandra.a.aziz@gmail.com citation: semarayani c.i.m., aziz s.a., melati m., 2018 essential oil production of murraya paniculata (l.) jack at different harvest times. adv. hort. sci., 32(4): 471-477 copyright: © 2018 semarayani c.i.m., aziz s.a., melati m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 november 2017 accepted for publication 18 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 471-477 472 of different chemical compounds (younis et al., 2011). different compositions of chemical compound of m. paniculata essential oils have been studied. rout et al. (2007) found that pentane extraction was the best method to obtain the highest amount of essential oil of m. paniculata flowers in india. this study also found that manool and (e)-nerolidol were the major component of essential oil (rout et al., 2007). the chemical compounds of m. paniculata flowers can also be extracted with liquid co 2 (rout et al., 2010). different compounds from m. exotica flowers were found, namely (e,e,e)-α-springene, (e)nerolidol, (e,e)-α-farnesene, methyl palmitate and germacrene b (raina et al., 2006). the variation of chemical composition in the essential oil from murraya flowers is affected by the place where the plants are planted (el-sakhawy et al., 1998) and harvesting time. concerning the latter, harvesting time may influence the quantity and quality of essential oils of m. paniculata flowers; this has been reported in four rosa cultivars (younis et al., 2009) and jasminum sambac flowers (younis et al., 2011). younis et al. (2011) reported that the best time to collect j. sambac flowers was in the morning before sunrise because highly volatile of jasmine oil. therefore, the purpose of this research was to investigate the effect of harvest time on the production and chemical compounds of m. paniculata essential oil. 2. materials and methods plant material the field experiment was conducted from october 2016 to february 2017. the experiment used 30 (62 month-old) plants that were planted in 1 m x 1 m on latosol soil, at the organic experimental farm, bogor agricultural university, bogor, indonesia (6°30’-6°45’ s, 106°30’-106°45’ e) at 250 m above sea level. a voucher specimen was deposit at the herbarium bogoriense, bogor, indonesia. type-a climate based on schmidt-ferguson with the average monthly rainfall, temperature and humidity of 305 mm, 26°c and 85%, respectively (mcga, 2017). before treatment, each plant was fertilized with 3.0 kg rice-hull ash, followed eliazar and aziz (2015). experimental design the experiment was arranged in randomized complete block design, with single factor (petersen, 1994) with harvest times as treatments, comprised of harvest at 05.00-07.00 and at 07.00-09.00 a.m. the flowers were collected at three different ages, comprised of two days before anthesis, one day before anthesis, and at the day of anthesis. the two ages of flower before anthesis were indicated by the size of flower buds which have been observed in the preliminary study. the anthesis of the flower bud with 1.00±0.06 cm length and 0.48±0.06 cm width will occur two days later; while flower bud with 1.16±0.06 cm length and 0.56±0.04 cm width will occur in the next day. the observations include developmental stages, number, fresh weight, the content of essential oils and chemical compounds of m. paniculata flowers. data were analyzed using t-student with α= 5% (petersen, 1994). essential oil extraction the analysis of essential oils was conducted at tropical biopharmaca research center, bogor agricultural university, bogor, indonesia. all harvested flowers (from different harvesting times) were extracted with solvent extraction method of ethanol. the flowers were immersed in ethanol until all samples were submerged, for two days. this mixture was filtered, then, the extract was separated from the solvent by rotary evaporator with a temperature of 40°c. the oil yield percentage was calculated from the weight of extract (g) divided by weight of fresh flower (g). gas chromatography-mass spectrometry analysis chemical compounds of essential oils were analyzed at health laboratory of dki jakarta province. the essential oils were injected into gas chromatography-mass spectrometry (gcms). an agilent technologies 7890 gas chromatography instrument with an auto sampler and 5975 mass selective detector and chemstation data system, equipped with a 30 m x 0.25 mm hp ultra 2 capillary column with 0.25 µm film thickness. helium was used as the carrier gas. the initial temperature was programmed at 80°c and then increased at the rate of 3°cmin-1 to 150°c held for 1 min and finally raised to 280°c at the rate of 20°c min-1 held for 26 min. the injector and interface temperatures were 250°c and 280°c, respectively. the ionization voltage was 70ev and a sample injection volume 5 µl. the individual peaks were identified by retention times, compared with those of compounds in health laboratory of dki jakarta province database. the percentage of each compound detected from samples was calculated according to the area of the chromatographic peaks. semarayani et al. essential oil production of murraya paniculata l. 473 3. results the results showed that the average of flower number (regardless the flower ages) harvested at 05.00-07.00 am was not significantly different to those harvesting at 07.00-09.00 a.m. (table 1). at both harvest times, it was found the following order of flower number based on flower stages: flowers at anthesis, flowers that would bloom two days later, and the least was flowers that would bloom in the next day. the differences in flower number between flower stages were not always significant. the difference of flower number between harvesting time was 8.96%. the average flower weight (regardless the flower stages) of m. paniculata was not significantly different between the times of harvesting (table 2). a similar trend as of flower number was also found in flower weight based on flower stages. the highest flower weight was blooming flowers (flowers at anthesis), and the lowest was flowers at the stage of one day before anthesis. the difference in flower weight between harvesting time was 16.59%. the extraction of m. paniculata flowers at different harvesting times with ethanol resulted in a yellowish-brown solution called concrete. these results were in line with that of paibon et al. (2011), that reported how the extraction of j. sambac flowers with ethanol produced a solution of yellowish brown to reddish. on the other hand, m. paniculata flowers that extracted with pentane produced a deep yellow waxy residue (rout et al., 2007). there was an indication that the essential oil percentage between harvest times was different. the percentage of essential oils from flowers harvested at 07.00-09.00 was higher than that at 05.00-07.00 a.m., regardless the flower stages, the difference was 0.38% (table 3). comparing among flower stages, it was found that anthesis flowers harvested at 05.0007.00 am had the highest percentage of essential oils. on the other hand, the highest percentage of essential oils at 07.00-09.00 am was obtained from flowers at the stage of one day before anthesis. from this calculation, the production of essential oils from flowers harvested at 07.00-09.00 was higher than that from 05.00-07.00 am. based on flower stages, blooming flowers (at anthesis stage) produced the highest amount of essential oils at both harvesting times, this related to the highest fresh flower weight. the analysis of chemical compounds showed 41 types that were contained in m. paniculata flowers at different harvesting times. the highest number of chemical compound types was found in flowers at anthesis when they were harvested at 07.00-09.00 am. this result was in line with younis et al. (2011), where j. sambac flowers harvested at anthesis had table 1 number of orange jessamine flowers at different harvesting times if p-value>α; α= 0.05, then means between the treatments were statistically equal; (k) = average of flower number at three flower age criteria; x= comparison between the flower 2 days before anthesis with 1 day before anthesis; y= comparison between the flower 2 days before anthesis with anthesis; z= comparison between the flower 1 day before anthesis with anthesis. harvesting time average of flower number/plant p-value percentage of flower number (%) at 05.00-07.00 am, flower ages 29.40 (k) 2 days before anthesis 24.67 x: 0.3349 ns 27.97 1 day before anthesis 14.07 y: 0.1742 ns 15.95 at anthesis 49.47 z: 0.0397 * 56.08 at 07.00-09.00 am, flower ages 31.11 (k) 2 days before anthesis 35.53 x: 0.0150 * 38.82 1 day before anthesis 12.87 y: 0.5056 ns 14.06 at anthesis 44.93 z: 0.0282 * 47.12 table 2 weight of orange jessamine flowers at different harvesting times if p-value>α; α= 0.05, then means between the treatments were statistically equal; k: average of flower number at three flower age criteria; x: comparison between the flower 2 days before anthesis with 1 day before anthesis; y: comparison between the flower 2 days before anthesis with anthesis; z: comparison between the flower 1 day before anthesis with anthesis. harvesting time average of flower weight (g/plant) p-value percentage of flower weight (%) at 05.00-07.00 am, flower ages 3.43 (k) 2 days before anthesis 2.05 x: 0.4572 ns 19.92 1 day before anthesis 1.34 y: 0.0884 ns 13.21 at anthesis 6.88 z: 0.0355 * 66.86 at 07.00-09.00 am, flower ages 4.72 (k) 2 days before anthesis 5.70 x: 0.0819 ns 40.35 1 day before anthesis 1.33 y: 0.4398 ns 9.38 at anthesis 7.13 z: 0.0029 ** 50.27 table 3 the percentage and production of essential oil of orange jessamine flowers at different harvesting times data were not analyzed statistically; (k ) = average of essential oils at three flower age criteria; * = production of essential oils based on the weight of the harvested flowers. treatment essential oil (%) production of essential oil (g/g fresh flower) * at 05.00-07.00 am, flower ages 3.13 (k) 4.83 2 days before anthesis 2.06 0.63 1 day before anthesis 3.40 0.69 at anthesis 3.94 4.07 at 07.00-09.00 am, flower ages 3.51 (k) 7.47 2 days before anthesis 3.49 3.00 1 day before anthesis 3.80 0.76 at anthesis 3.25 3.47 adv. hort. sci., 2018 32(4): 471-477 474 more chemical compounds than those in flower bud. the analysis on m. paniculata flowers revealed the presence of coumarins, esters, fatty acids, phenolics, triterpenes, sesquiterpenes and other compounds (table 4). coumarin was the most common compound found in all treatments. harvesting time at 05.00-07.00 a.m. gave the highest number of coumarins, esters, fatty acids, and sesquiterpenes. coumarin was the highest amount of bioactive compound found in m. paniculata essential oils, (table 4) and the dominant compounds in the coumarin group were β-methylesculetin and murralongin (table 5). the amount of β-methylesculetin from flowers harvested at 07.00-09.00 am was higher than those from 05.00-07.00 a.m. different flower stages had a different dominant compound. harvesting flowers at the stage of one day before anthesis gave the highest percentage of β-methylesculetin at both harvesting times, but harvesting at anthesis delivered the highest percentage of murralongin. the analysis of chemical compounds showed that terpenoid groups found in the essential oil of m. paniculata flowers were triterpenes and sesquiterpenes (table 4). the percentage of triterpenes was higher than sesquiterpenes, this finding was different from the previous study which showed that sesquiterpenes were the main compound in essential oils. the current study showed that sesquiterpenes derivatives found in the essential oils of m. paniculata flowers and found at both harvesting times were αzingiberene, α-bergamotene, and caryophyllene (table 6). flowers harvested at 05.00-07.00 am had a higher percentage of α-zingiberene, α-bergamotene, and caryophyllene compare to those harvested at 07.00-09.00 a.m. there was an indication that different flower ages have different compositions of chemical compounds, except caryophyllene that was found at the same flower age at both harvesting times. 4. discussion and conclusions the above results showed that there were no significant differences between the number and weight of flowers harvested at 05.00-07.00 and 07.00-09.00 am. the different harvesting times reflected the positable 4 chemical compounds of orange jessamine essential oils at different harvesting times data were not analyzed statistically; k= average of chemical compounds at three flower age criteria. treatment % peak area coumarin ester fatty acid phenolic triterpene sesquiterpene other compound at 05.00-07.00 am, flower ages 71.29 k 1.80 k 9.12 k 4.34 k 3.27 k 0.77 k 9.42 k 2 days before anthesis 69.68 3.16 12.39 5.52 1.55 0.47 7.24 1 day before anthesis 74.33 1.41 10.09 4.43 1.33 0.55 7.87 at anthesis 69.85 0.83 4.89 3.06 6.93 1.30 13.14 at 07.00-09.00 am, flower ages 68.07 k 1.60 k 10.40 k 5.09 k 1.93 k 0.72 k 12.20 k 2 days before anthesis 66.98 2.15 11.91 6.20 1.03 0.41 11.33 1 day before anthesis 70.52 1.21 5.87 5.13 1.21 0.71 15.34 at anthesis 66.72 1.44 13.41 3.94 3.55 1.04 9.92 table 5 coumarins compounds of orange jessamine essential oils at different harvesting times data were not analiyzed statistically; k: average of chemical compounds at three flower age criteria. treatment % peak area β-methylesculetin murralongin at 05.00-07.00 am, flower ages 60.65 (k) 7.36 (k) 2 days before anthesis 59.46 5.36 1 day before anthesis 61.59 7.76 at anthesis 60.89 8.96 at 07.00-09.00 am, flower ages 62.07 (k) 5.71 (k) 2 days before anthesis 61.00 5.12 1 day before anthesis 65.51 5.01 at anthesis 59.71 7.01 table 6 sesquiterpenes compounds of orange jessamine essential oils at different harvesting times data were not analyzed statistically; k: average of chemical compounds at three flower age criteria. zbn, zingiberene; bgn, bergamotene; cp, caryophyllene. treatment % peak area α-zbn α-bgn cp at 05.00-07.00 am, flower ages 0.75 (k) 0.55 (k) 0.27 (k) 2 days before anthesis 0.47 0.00 0.00 1 day before anthesis 0.00 0.55 0.00 at anthesis 1.03 0.00 0.27 at 07.00-09.00 am, flower ages 0.71 (k) 0.41 (k) 0.17 (k) 2 days before anthesis 0.00 0.41 0.00 1 day before anthesis 0.57 0.00 0.15 at anthesis 0.85 0.00 0.19 semarayani et al. essential oil production of murraya paniculata l. 475 tion of sunrise where higher light intensity was found at 07.00-09.00 am. the current study showed that the anthesis of m. paniculata flowers occurred before 05.00 am, therefore there was no increase in the number and weight of flower after that time. de souza et al. (2004) reported that the anthesis of metrodorea nigra st. hill. flowers, belonging to rutaceae family, occurs in the morning. time of harvesting is important because it is related to the amounts of essential oils produced. filho et al. (2006) reported that harvest at 08.00 am resulted in the highest yield of the essential oil from fresh leaves of basil (ocimum basilicum l.). the importance of harvesting time is also shown by dobreva and kovacheva (2010) where the essential oils content of rosa damascena mill. and r. alba l. drops dramatically when the flowers collected after noon. besides investigating the effect of harvesting time, this study also observed the essential oil production at different flower developmental stages of m. paniculata. the results showed that harvesting flowers at anthesis stage (blooming flower) yielded the highest percentage of essential oils. flowering plants released diverse blends of volatile to attract pollinator and seed disseminators. the floral scent is a signal, which pollinators can use to discriminate a particular flower. it may contain from one to 100 volatile substances, but most species emit between 20 and 60 different compounds, so there won’t be any identical floral scents (dudareva et al., 2006). therefore, the presence of essential oil at anthesis will ensure the reproductive success. azam et al. (2013) also reported that the highest amounts of volatile compounds were present in fully opened flowers of citrus reticulata blanco, c. unshiu marc., c. sinensis (l.) osbeck, c. limon (l.) burm., c. medica (l.), and c. changshanensis chen et. fu. in general, essential oils are a mixture of compounds belonging to different chemical entities such as terpenes, phenols, aliphatic compounds, benzenoid, and heterocyclic compounds (shakeel-urehman et al., 2018). chemical compounds found in m. paniculata flowers in india were monoterpenes, sesquiterpenes, benzenoids, diterpenes, and fatty acids (rout et al., 2007). different from those study, the current experiment showed that coumarins were the dominant compounds in m. paniculata flowers. coumarins are also present in leaves of m. paniculata in indonesia (kinoshita and firman, 1996) and taiwan (kinoshita et al., 1996). furthermore, from the current study, it was found the presence of two coumarins derivatives in essential oils of m. paniculata, β-methylesculetin and murralongin. βmethylesculetin compounds can function as antioxidants (kontogiorgis and hadjipavlou-litina, 2005) and anti-inflammatory (kontogiorgis and hadjipavloulitina, 2005; zuoqi et al., 2008). murralongin is thought to be a chemical compound identifier of m. paniculata essential oils. harvesting at anthesis produced the highest percentage of murralongin. the previous study reported that murralongin was found in essential oils from leaves and flowers of m. paniculata (gill et al., 2014), and leaves of m. omphalocarpa in taiwan (chen et al., 2003). terpenoids compounds that were identified in this study were triterpenes and sesquiterpenes. in general, terpenoids are the dominant compounds in essential oils (sangwan et al., 2001). the current study showed that the percentage of triterpenes compounds is higher than sesquiterpenes (table 4). this was not in line with butu et al. (2014) who reported that the basic compound in the essential oil was sesquiterpenes. terpenoids have many volatile compounds that have high enough vapor pressures at normal atmospheric conditions to allow significant release into the air (dudareva et al., 2004). therefore, despite the same plant, may have different types of compounds. sesquiterpenes compounds that could be identified in this current study were α-zingiberene, α-bergamotene, and caryophyllene. there were similarity and difference between this finding and the previous study. the similarity was reported by raina et al. (2006) where those three compounds were also found in m. exotica essential oils from flowers. raina et al. (2006) found that caryophyllene had the highest percentage, on the contrary, the current study showed that caryophyllene had the lowest percentage. the different finding indicates that the chemical composition and yield of essential oils are affected by many factors, such as provenance, weather, soil conditions, time of harvest, and the extraction method (boira and blanquer, 1998). caryophyllene is one of the compounds in perfume ingredient (salvadorcarreno and chisvert, 2005), but it also used as a mixture of spices, citrus scents, soaps, detergents, lotions as well as in various food products (sabulal et al., 2006). flamini et al. (2007) and darjazi (2012) reported that caryophyllene is also present in flower of c. limon and c. nobilis lour var. deliciosa swingle. it can be concluded that the harvest of m. paniculata flowers can be done at 05.00-09.00 am to obtain the highest quantity and the best quality of essential oil. flowers must be harvested at anthesis stage to reach the highest production of essential oil with β476 adv. hort. sci., 2018 32(4): 471-477 methylesculetin and murralongin, as the main compounds of m. paniculata flowers. references azam m., song m., fan f., zhang b., xu y., xu c., chen k., 2013 comparative analysis of flower volatiles from nine citrus at three blooming stages. int. j. mol. sci., 14: 22346-22367. boira h., blanquer a., 1998 environmental factors affecting chemical variability of essential oils in thymus piperella l. biochem. syst. ecol., 26: 811-822. butu m., butnariu m., rodino s., butu a., 2014 study of zingiberene from lycopersicon esculentum fruit by mass spectometry. dig. j. nanomater biostruct., 9(3): 935-941. chen k.s., wu c.c., chang f.r., chia y.c., chiang m.y., wu y.c., 2003 bioactive coumarins from the leaves of murraya omphalocarpa. planta med., 69: 654-657. darjazi b.b., 2012 a comparison of volatile component of flower, leaf and peel of citrus reticulata blanco (citrus nobilis lour var. deliciosa swingle). j. med. plants res., 6: 2365-2372. de souza l.a., moscheta i.s., mourao k.s.m., da rosa s.m., 2004 morphology and anatomy of the flower and anthesis of metrodorea nigra st. hill. 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46: 854-860. impaginato 183 adv. hort. sci., 2021 35(2): 183­194 doi: 10.36253/ahsc­7493 stem and leaf anatomical and physio­ logical characteristics of ‘colín v­33’ avocado seedlings n.v. useche­carrillo 1, a.f. barrientos­priego 1 (*), c.a. núñez­colín 2, e. campos­rojas 1, j. ayala­arreola 1 1 departamento de fitotecnía, universidad autónoma chapingo, km 38.5 carr. méxico‐texcoco, texcoco, c.p. 56230 méxico. 2 universidad de guanajuato, mutualismo 303, col. la suiza, celaya, guanajuato, c.p. 38060 méxico. key words: gas exchange, persea americana mill., stem anatomy, stomatal conductance, transpiration, xylem vessels. abstract: the anatomical and physiological structure of the ‘colín v­33’ avoca­ do stem and leaf is described from samples from plants obtained from seed in order to identify genotypes and early selection parameters in a rootstock improvement program for avocado. eighty­nine plants of 12 months of age were used, where a total of 25 anatomical variables of the stem, leaf, and phys­ iological of leaf were evaluated. a cluster analysis was conducted that generat­ ed a hierarchical dendrogram that suggested six groups of plants. furthermore, from the 25 variables, eight were selected as discriminant when performing a canonical discriminant analysis, the variables that most discriminated for the first canonical component were: stem diameter and density of xylem vessels, for the second: thickness of the stem epidermis, temperature of the stem leaf and stomata length, while for the third: thickness of the cambium, transpira­ tion rate, and stomatal conductance. the genotypes showed a great variation between the groups, the characteristics of these indicated that the genotypes of group 4 showed some that could be related to small or dwarf plants (smaller stem diameter, high density of xylem vessels, a higher rate of transpiration and stomatal conductance). in contrast to the genotypes of group 3 which present­ ed opposite characteristics in the previous variables, being able to associate with vigorous plants. the anatomical traits of the stem showed to be highly related to the behavior of the avocado plants. associating genotypes with phys­ iological and anatomical variables in leaf and stem can have great value for the selection of rootstocks at an early stage of development. 1. introduction fruit trees used for the establishment of plantations are formed by a variety/rootstock combination, where the variety provides the productive part and the rootstock provide the root system of the tree (dolgun et al., 2009). the rootstocks provide a simplified management of the orchard, (*) corresponding author: abarrien@correo.chapingo.mx citation: useche­carrillo n.v., barrientos­priego a.f., núñez­colín c.a., campos­rojas e., ayala­arreola j., 2021 ­ stem and leaf anato‐ mical and physiological characteristics of ‘colín v‐ 33’ avocado seedlings. ­ adv. hort. sci., 35(2): 183­194. copyright: © 2021 useche­carrillo n.v., barrientos­priego a.f., núñez­colín c.a., campos­rojas e., ayala­ arreola j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 november 2019 accepted for publication 6 may 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-7493 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 183­194 184 increase the productivity, ensure the survival of the trees, control the vigor conferring dwarfism and allowing the use of high planting densities and the water balance of the plant (solari et al., 2006), influ­ ence the qualitative and nutritional attributes of the fruits (remorini et al., 2008), also influence the scion development, as well as its adaptation to different types of soil, water stress conditions and other cli­ matic conditions (giorgi et al., 2005), as well as salt stress tolerance (massai et al., 2004). selection of the rootstock and the cultivar or vari­ eties to be exploited is an economically important decision, since the degree of productivity and quality of production will depend on the variety/rootstock combinations (pinochet, 2010), these combinations are in function of the degree of affinity in terms of vascular connections between the graft and the root­ stock, because vascular regeneration is a complex process that includes the differentiation of xylem and phloem (aloni et al., 2010). avocado production in mexico is based on the use of seed­derived rootstocks which generate large trees that, over time, complicates the agronomic management. for this reason, one of the priorities in avocado research is to find dwarf cultivars that are highly productive, with good fruit quality and resis­ tant to pests and diseases in order to establish high­ density plantations and reduce production costs (sánchez­colín et al., 1992). the use of rootstocks in intensive avocado cultivation is of great importance and rootstocks for this crop have been reported to influence the size of the tree and its productivity (bergh and whitsell, 1962). potentially dwarfing root­ stocks such as ‘wilg’ and ‘colin v­33’ have been eval­ uated against ‘duke 7’, for their ability to limit the growth of ‘hass’ (roe et al., 1995). ‘colín v­33’ avocado is a mexican selection that has low vigor and an expanded growth habit (roe et al., 1995), considered as a possible dwarf rootstock since it has been used as an interstock, with reported reduction of 43% of the tree height on ‘fuerte’ (barrientos priego et al. , 1987) and ‘hass’ (barrientos­villaseñor et al., 1999). on the other hand, given the genetic variability for the height of the seedlings of this cultivar (rubí arriaga, 1988), it makes it a valuable material for the improvement of dwarf avocado rootstocks. in this regard, barrientos­ priego et al. (1992) have found that the cultivar seedlings produce some dwarfing individuals when used as rootstocks. efforts have been made mainly with ‘colín v33’ for the selection of dwarf rootstocks that meet the needs of the new production systems, where stoma­ ta density has been proposed as a possible pre­selec­ tion index (barrientos­pérez and sánchez­colin, 1982; barrientos­priego and sánchez­colín, 1987). other important features to be investigated are the ones that contribute to the hydric status of the plant and its productivity, in which a series of physio­ logical, anatomical and morphological characteristics of the stem can be measured. at the physiological level, gas exchange can be studied (vilagrosa et al., 2010) and to determine the affinity between vari­ ety/rootstock combinations, the anatomical structure of xylem can be studied (sory et al., 2010; leal­ fernández et al., 2013). the anatomical characteristics of the dimensions of vessel elements and the proportions of xylem and phloem, in stems, are important to be able to define the amount of water that can be transported through them, since as the tissues become larger and the presence of smaller diameter of the vessels (higher pressure for water movement), the amount of water transported will be greater, therefore, there will be a better adaptation of the plants to low humidity con­ ditions (vasconcellos and castle, 1994; reyes­ santamaría et al., 2002). reyes­santamaría et al. (2002) found vessel ele­ ments with smaller diameters, for the avocado geno­ types that have less vulnerability to drought. the stomatal density (ed) and the thickness of the epi­ dermis are characteristics that may be related to drought tolerance (baas, 1982), as they are the most exposed anatomical characteristics of the plant, which represent the last link in the transpiration tor­ rent towards the atmosphere (faust, 1989). the use of avocado seedling rootstocks of a local type called “criollo” is very common but rarely stud­ ied from their anatomical and physiological charac­ teristics on stem and leaf, variables that can be useful as a preliminary study to understand their possible role when grafted. the objective of this research was to describe the anatomical and physiological struc­ ture of the stem and leaf of plants derived from avo­ cado seed of ‘colín v­33’, to select individuals at the seedling level with distinctive characteristics. 2. materials and methods the research was carried out in a greenhouse of the experimental field of the chapingo autonomous useche‐carrillo et al. ‐ anatomical and physiological characteristics of avocado 185 university, in chapingo, state of mexico, located at 19° 29’25.7’’ and 98° 52’24.5’’ with an altitude of 2240 meters above sea level. plant material as plant material, 89 avocado seedling plants of ‘colín v­33’ were used, that were donated by the germplasm bank of the salvador sánchez colín foundation­cictamex, s.c. located in coatepec harinas, state of mexico. the seeds were established in a black bag of caliber 600 of 26 cm x 35 cm with perforations in the first lower third; using soil, perlite and compost as a substrate (3:1:1; v:v:v), watered twice a week, and located in a glass greenhouse with oscillating temperatures between 35 ± 4°c. leaf gas exchange variables to each of the plants the eleventh leaf, fully expanded and healthy, counted from the base towards the apex of the plant was selected as recom­ mended by barrientos­priego et al. (2003). once marked, the variables co2 assimilation rate (a), tran­ spiration rate (e), leaf temperature, internal co2 con­ centration and, stomatal conductance was evaluated. a punctual measurement was made between 11:00 and 13:30 hours a day with an infrared gas analyzer (model ci­340, cid bio­science). this was done dur­ ing three days of the month of september of the year 2017 and the measurement was taken in the same plant order every day to avoid more variation in the data taken for each plant. the average of the three values obtained was used in the analysis. the water use efficiency index (wue) was calculated based on the variables co2 assimilation (a) and tran­ spiration rate (t), using the formula wue = a/t. leaf anatomical variables after the measurements of gas exchange vari­ ables, on the same leaf previously marked, an impression of the underside of the middle part of the lamina using sil icone for dental impressions (exactoden) was taken. by applying transparent nail varnish on the (negative) impression, the positive impression was obtained, which was placed on a slide and fixed with a coverslip. in the positive impressions of each sample, the image area was calculated with an object microme­ ter, stomatal density (sd) per mm2 and epidermal cell density (ecd) per mm2 were determined. these vari­ ables were evaluated in five fields [400x, ayala­ arreola et al. (2010)] in a motic b3 professional series microscope, with the adaptation of a moticam 480 camera with a 16 mm adapter. in addition, 10 stomata were measured in each sample. stomata counts, epidermal cells and stomata length measure­ ment were performed with the help of imagej 1.52a image analyzer. with all this information the stomatal index (ei) was calculated, which is equal to: ei = [sde/ (sd + ecd)] x 100. stem anatomical variables transverse stem samples were obtained from plants of approximately 1 cm each, which were fixed in 96 % ethanol:100 % glacial acetic acid (2:1; v:v) and processed in an automatic tissue exchanger (tissuematon fisher) with 2­ethoxyethanol (cello­ solve) and xylene, then transfer to paraffin (55°c) staying 72 hours inside a stove. the paraffin pyramid was made according to sass (1968) and in a rotary microtome (american optical, model 820), trans­ verse cuts were made with a thickness of 10 µm. the cut sections were stained for 30 min at room temper­ ature in a mixture of equal volumes of 0.1% aqueous solutions of safranin and fast green, then washed in distilled water for 5 minutes and washed in 2 changes of absolute alcohol for 2­3 min (bryan, 1955). the stained sections then mounted on slides with coverslips by means of haupt adhesive and 10 % formalin (sass, 1968). fifteen fields were observed in each preparation per replication (three repetitions). for which the 4x, 10x and 40x lenses were used as appropriate, in a motic b3 professional series microscope, with the adaptation of a moticam 480 camera with a 16 mm adapter and digital images were obtained. the area of the images was calculated with a slide micrometer and then the cell layers of the tissues and the dimen­ sions of xylem vessel elements were measured with the help of the imagej 1.52a image analyzer. the tissues evaluated were epidermis, parenchy­ ma, phloem fibers, phloem, cambium, xylem, and pith. the thickness of each layer and the total diame­ ter of the stem was measured. for the xylem dimen­ sions number of vessel elements per area (vessel density), vessel element area, major axis of vessel element, minor axis of vessel element, cell wall thick­ ness of two contiguous vessel elements, roundness index (ri) of vessel and feret diameter were obtained. statistical analysis a cluster analysis was performed using ward’s minimum variance agglomeration method to gener­ ate a hierarchical dendrogram (núñez­colín and adv. hort. sci., 2021 35(2): 183­194 186 escobedo­lópez, 2011). the resulted dendrogram was divided according to hotelling’s pseudo­statisti­ cal t2 (johnson, 1998). a canonical discriminant analy­ sis (cda) with the mahalanobis distance was per­ formed to determine the most discriminating vari­ ables with the greatest importance that describe the groups (núñez­colín and escobedo­lópez, 2014). all statistical analysis were performed with the sas v.9.2 statistical package (sas institute, 2009). 3. results and discussion in order to explore the homogeneity within each variable evaluated and based on the coefficients of variation (cv), it was determined that the physiologi­ cal variables of leaf: co2 assimilation rate, stomatal conductance and water use efficiency index (wue), were the characteristics with the greatest variation (table 1), presenting cv of 60.84%, 57.65%, and 62.5%, respectively. these characteristics being less stable within the plants studied (more heteroge­ neous). on the contrary, all the anatomical variables of stem and leaf showed lower coefficients of varia­ tion that were below 28%, so they could be consid­ ered more homogeneous but still show contrasting features (fig. 1 and 2). cluster analysis based on hotelling’s t2 pseudo­statistic (fig. 3), the grouping by ward’s method showed the identifica­ tion of six groups of plants (fig. 4). group 1 com­ posed of 21 plants, group 2 by 9, group 3 with 13, group 4 with 13, group 5 with 16 and group 6 with 17 plants (table 2). in the obtained dendrogram a partition of six groups was used approximately at a cut­off distance r2 semi­partial close to 0.050 (fig. 3). the greater dis­ tance corresponds to group 4 confirming that are dif­ ferent from the rest, that was also reinforced with table 1 ­ mean values of some leaf and stem anatomic variables and leaf gas exchange variables of ‘colín v­33’ avocado seedling plants. the highest values are presented in bold for the coefficient of variability wue= water use efficiency. cv= coefficient of variability. variables maximum mean minimum cv (%) epidermis (µm) 1.72 1.12 0.74 18.43 parenchyma (µm) 36.00 19.79 12.48 21.66 phloem fibers (µm) 22.96 15.99 9.19 20.36 phloem (µm) 66.46 39.72 21.46 22.56 cambium (µm) 10.69 7.14 4.24 21.01 xylem (µm) 115.22 76.05 28.40 25.42 pith (µm) 98.49 60.02 19.41 27.20 stem ø (µm) 197.78 152.08 107.85 14.36 vessel density by area (vessels/µm2) 6.13 3.11 1.73 25.20 vessel area (µm2) 2688.49 1868.91 1096.34 16.99 mayor axis of vessel (µm) 40.59 29.40 20.27 14.68 minor axil of vessel (µm) 22.80 17.63 12.05 15.04 wall thickness of two cells (µm) 3.57 2.80 2.19 11.06 roundness index 0.59 0.35 0.18 24.03 feret ø 87.80 49.53 30.63 18.48 leaf temperature (°c) 28.53 26.52 23.23 4.47 co2 assimilation rate (umol·m­2·s­1) 3.97 1.28 ­0.06 60.84 transpiration rate (mmol·m­2·s­1) 2.00 0.89 0.32 45.69 stomatal conductance (mmol·m­2·s­1) 105.18 33.04 9.33 57.65 internal co2 concentration (ppm) 352.87 232.58 67.33 20.53 wue 4.69 1.56 ­0.19 62.50 stomatal density (stomata/mm2) 317.65 190.66 105.88 19.80 epidermis cell density (cells/mm2) 1267.65 818.88 582.35 17.56 stomatal index (%) 25.16 19.03 11.78 13.89 stoma length (µm) 19.64 15.99 13.11 8.60 useche‐carrillo et al. ‐ anatomical and physiological characteristics of avocado 187 fig. 1 ­ transverse section of xylem tissue of main stem from plant numbered as 110 (a) and 88 (b) of ‘colin v­33’ avocado seedlings. arrows show vessel elements. plants were developed. generally, plants with a greater number of relatively small vessel elements are associated with drought­resistant genotypes (vasconcellos and castle, 1994; reyes­santamaría et al., 2002; núñez­colín et al., 2006). the cc 2 represented 28.90% of the total varia­ tion and was associated with: epidermis thickness, leaf temperature, and stoma length (table 3 and 4). the anatomical stem variable (epidermis thickness) present in cc 2 is associated with drought resistance (baas, 1982) and it is possible to relate it as acquired adaptation, to tolerate prevailing environmental con­ ditions of the greenhouse (38°c). on the other hand, the size of stomata is one of the anatomical variables of the leaf that could be sensitive to the change in environmental conditions (hetherington and fig. 2 ­ epidermis replicates of leaf lamina from plant numbered as 162 (a) and 96 (b) of ‘colin v­33’ avocado seedlings. arrows show stomata. fig. 3 ­ hotelling's pseudo­statistical t2 and the recommended number of groups as a proof of dendrogram division using physiological and anatomical of leaf and stem variables of 89 ‘colin v­33’ seedling plants. 188 adv. hort. sci., 2021 35(2): 183­194 woodward, 2003). consequently, these characteris­ tics can be considered as representative of the adap­ tation to the environment of the plant, as a constant feature of taxonomic value, that the relationship between these two anatomical variables (epidermis thickness and stoma length) have with the variable air temperature. the cc 3 was associated with the variables thick­ ness of the cambium, transpiration rate, and stom­ atal conductance. stomatal conductance refers to the control exerted by stomata on the rate of transpira­ tion, representing the ability of a water molecule to diffuse through the leaf per unit of time. a minor stomatal conductance may be the expanation for lower co2 assimilation and at the same time a lower transpiration rate (sholefield et al., 1980; barrientos­ villaseñor et al., 1999). stomata size is considered a key physiological variable on stomatal conductance (holland and richardson, 2009), which is why the stomatic conduc­ tance use can be considered as a rapid test in geno­ type selection, as an indirect measure of stomatic density, since this can also be a way to perform a genotype separation towards dwarf types as pro­ posed by barrientos­pérez and sánchez­colín (1987). the f significance test of the mahalanobis dis­ tance (table 5) indicated that there are highly signifi­ cant differences between the groups of plants at a level of p<0.0001. to have a better and clearer visualization (distrib­ ution) of the groups of plants, the graphic represen­ tation was made in the first factorial plane with the first two canonical components (fig. 5). the graphic representation of the groups in the first factorial plane showed that the group 4 is the only one that is isolated from the rest and was very different from the other five groups, with most of its table 2 ­ groups formed according to the cluster analysis with 89 plants derived from ‘colín v­33’ avocado seedlings, derived from anatomical and physiological characteristics of stem and leaf group seedings clustered group 1 16, 103, 106, 113, 118, 121, 126, 129, 131, 133, 138, 139, 142, 143, 145, 146, 147, 148, 150, 152, 156 group 2 1, 73, 110, 112, 137, 141, 144, 157, 159 group 3 5, 13, 14, 21, 22, 23, 57, 72, 74, 80, 86, 96, 123 group 4 8, 18, 65, 76, 81, 85, 88, 93, 105, 127, 155, 160, 162 group 5 3, 7, 10, 17, 19, 28, 32, 33, 41, 52, 56, 77, 78, 79, 82, 111 group 6 62, 87, 92, 99, 101, 104, 107, 114, 115, 117, 119, 120, 122, 124, 128, 130, 158 fig. 4 ­ cluster by the ward method into six groups formed by the anatomical and physiological characteristics of the stem and leaf of ‘colín v­33’ avocado seedlings. the black dotted line represents the dendrogram cut accor­ ding to hotelling’s pseudo­statistic t2. useche‐carrillo et al. ‐ anatomical and physiological characteristics of avocado 189 plants in the lower left quadrant (fig. 5). groups 1, 2 and 6 are probably clustered because they are plants with anatomical and/or physiological similarities according to the variables evaluated in this study (fig. 5). these groups are those that have dis­ persion in both planes, so they are considered the most heterogeneous groups within the plants studied. groups 3 and 5 were slightly more separated from the rest of the groups, but between them, there is some closeness (less dispersion), which could be inferred as similar groups according to the behavior of the variables evaluated (fig. 5). wue= water use efficiency index. table 3 ­ values of the canonical discriminant analysis of stem and leaf anatomical variables and physiological leaf variables of 89 ‘colín v­33’ avocado seedlings table 4 ­ total canonical coefficients of the canonical discriminant analysis (cda) of anatomical characteristics of the stem and leaf, as well of physiological characteristics of the leaf of ‘colín v­33’ avocado seedlings. the highest values are presented in bold let­ ters in three canonical components canonical component proper value variance (%) accumulated variance (%) canonical correlation approximated calculated f p>f 1 47.504 39.91 39.91 0.909 4.93 <0.0001 2 34.405 28.90 68.81 0.880 4.02 <0.0001 3 18.683 15.69 84.50 0.807 3.09 <0.0001 4 13.703 11.51 96.01 0.760 2.45 <0.0001 5 0.4746 3.99 100 0.567 1.42 0.1419 studied variable can 1 can 2 can 3 can 4 can 5 epidermis (µm) 0.166274 0.677758 0.107810 ­0.136872 0.076201 parenchyma (µm) 0.250557 0.442521 ­0.254777 ­0.069388 0.232325 phloem fibers (µm) 0.431579 0.577674 0.439686 ­0.030758 0.020814 phloem (µm) 0.432130 ­0.365082 0.103285 0.028883 0.067597 cambium (µm) 0.136514 ­0.145255 0.545261 0.168825 ­0.111704 xylem (µm) 0.408444 ­0.551603 ­0.038947 0.244313 ­0.159389 pith (µm) 0.061974 0.521382 0.194067 0.087974 ­0.233069 stem ø (µm) 0.601144 ­0.122358 0.233650 0.369948 ­0.235378 vessel density by area (vessels/µm2) ­0.642113 0.285704 ­0.040889 ­0.295962 0.234301 vessel area (µm2) 0.198248 0.296278 0.035828 0.557918 0.230066 mayor axis of vessel (µm) 0.540765 ­0.053647 ­0.094319 0.682230 0.145237 minor axil of vessel (µm) 0.464509 0.339487 0.238300 0.596176 0.043128 wall thickness of two cells (µm) 0.262160 0.553793 0.219939 0.168747 ­0.051240 roundness index 0.521700 ­0.516643 ­0.056714 0.381362 ­0.115487 feret ø ­0.415188 0.247956 ­0.183468 ­0.143826 0.263973 leaf temperature (°c) ­0.055325 0.671497 0.197631 ­0.080977 0.002190 co2 assimilation rate (µmol·m­2·s­1) ­0.346322 0.209754 ­0.090718 0.653934 ­0.383860 transpiration rate (mmol·m­2·s­1) ­0.538189 0.110232 0.546988 0.242327 0.023917 stomatal conductance (mmol·m­2·s­1) ­0.530059 ­0.016900 0.502987 0.237708 0.022604 internal co2 concentration (ppm) ­0.035307 ­0.155754 0.408420 ­0.518668 0.491616 wue 0.010946 0.092531 ­0.434341 0.556483 ­0.488497 stomatal density (stomata/mm2) 0.059033 ­0.043825 0.165589 0.198190 0.023354 epidermis cell density (cells/mm2) ­0.032255 0.110568 0.025203 ­0.166117 0.273310 stomatal index (%) 0.098199 ­0.132837 0.100880 0.400103 ­0.233939 stoma length (µm) 0.087852 0.636119 ­0.014346 ­0.140672 ­0.173689 adv. hort. sci., 2021 35(2): 183­194 190 in the pattern of dispersion of the plants in a three­dimensional graph formed by the three canoni­ cal components (cc), it was observed that group 4 is completely separated from the rest and is located towards the most negative values of cc 1 and cc 3 (fig. 6), it was also observed that groups 3, 5 and 6 were not so separated from the rest of groups (less dispersed), such as 4, and if they could be easily dif­ ferentiated in the graph located towards positive val­ ues of cc 1 and cc 2; on the other hand, the most dispersed groups were 1 and 2, mixing with each other, indicating that they probably have similar characteristics. group 1 is formed by plants with the greatest epi­ dermis thickness and the greatest stoma length but the lowest stomatal conductance of all other groups (fig. 6). the thickness of the epidermis is a character­ istic that may be related to drought tolerance accord­ ing to baas (1982), as it is the most exposed anatomi­ cal characteristic of the plant along with the stomatic density, representing the last link of the transpiration stream into the atmosphere (faust, 1989). the plants of group 2 were characterized by hav­ ing the cambium with less thickness, high average values of stem diameter and stomata length. group 3 was characterized by having plants with greater stem diameter, a lower density of vessel ele­ ments and shorter stomata length, low average val­ ues in transpiration and stomatal conductance. this group of plants is likely to be more vulnerable to physiological problems such as drought as reported by reyes­santamaría et al. (2002). the plants in group 4 had the lowest values for stem diameter, but the highest density of xylem ves­ sel elements, also the highest rate of transpiration and the highest stomatal conductance, this being the group that separated completely from the rest (fig. 4). the results could indicate that the plants belong­ table 5 ­ mahalanobis distance and its significance, from anatomical characteristics of stem and leaf, as well of physiological character­ istics of leaf from 89 ‘colín v­33’ avocado seedlings group 1 2 3 4 5 6 1 ­ 2 18.20 ­ 3 24.83 *** 36.28 *** ­ 4 37.84 *** 39.65 *** 55.11 *** ­ 5 22.39 *** 34.87 *** 16.52 *** 29.83 *** ­ 6 12.92 *** 22.02 *** 16.19 *** 36.78 *** 23.52 *** ­ *** significant at p<0.0001 of probability. fig. 5 ­ dispersion of the six groups of ‘colín v­33’ avocado seed­ lings in the first factorial plane of the first two canonical components of the canonical discriminant analysis, deri­ ved from anatomical and physiological characteristics of stem and leaf. fig. 6 ­ dispersion of 89 ‘colín v­33’ avocado seedlings in the first three canonical functions based on physiological and anatomical variables of leaf and stem. useche‐carrillo et al. ‐ anatomical and physiological characteristics of avocado 191 ing to this group probably use more water and can be more adapted to condition of more availability, because by decreasing the stomatal opening and closing it contributes to reducing the loss of water due to evapotranspiration (núñez­colín et al., 2006). previous results (sholefield et al., 1980; barrientos­ villaseñor et al., 1999) indicated that there is a posi­ tive correlation between the variables co2 assimila­ tion, stomatal conductance and transpiration rate, thus an increase in stomatal conductance promotes the increase in the transpiration rate and the photo­ synthetic rate (damián­nava et al., 2009). group 5 was represented by plants with the low­ est thickness of the epidermis, the lowest leaf tem­ perature, low stomatal conductance values, and tran­ spiration. group 6 (fig. 5) was characterized by hav­ ing the greater cambium thickness, high average val­ ues in the variables stem diameter, epidermis thick­ ness, leaf temperature, stomata length, but low den­ sities values of vessel elements, transpiration, and stomatal conductance. groups 1 and 6 were closest to each other, according to the mahalanobis distances (table 4), while the most distant groups were 3 and 4 (fig. 5). for any type of plant, the conducting tissue of the stem, the size of the xylem vessel elements, the per­ centage of the xylem and phloem, and the relation­ ship between xylem and phloem, are anatomical fea­ tures that define the transport capacity of water in plants. it has been observed in some tree species, that as the percentage of vascular tissues increases and the diameter of the vessels are smaller, the amount of water transported is higher, and this may be an indicator of greater adaptation from plants to low humidity conditions in the soil (vasconcellos and castle, 1994; reyes­santamaría et al., 2002). for the case of avocado rootstocks, it has been found that stems of ‘duke 7’ (1248.7 µm2) had narrowed xylem element cells diameters than ‘toro caynon’ (1536.1 µm2), were ‘duke 7’ showed higher daily sap flow (2.8 kg day­1) compared to ‘toro caynon’ (2.0 kg day­ 1) on ungrafted plants and in the case of grafted plants it also increased the sap flow when ‘duke 7’ was used (fassio et al., 2009). several authors compared normal (vigorous) trees with dwarf trees of citrus, olive, mango, and copaifera langsdorfi found that dwarf trees are char­ acterized by narrow (small) vessel elements and a higher density of xylem vessel elements (saeed et al., 2010; el said et al., 2013; rashedy et al., 2014; longui et al., 2014, respectively) which has also been found in avocado (reyes­santamaría et al., 2002). the results found showed that the plants of group 4 are the ones that had the highest densities of vessels and with this, probably less vessel element diameter, increasing transport and conferring a low probability of suffering cavitation and embolism (núñez­colín et al., 2006). on the other hand, goncalves et al. (2007) and tombesi et al. (2010) indicated that in cherry and peach dwarf rootstocks, respectively, the diameter of xylem vessel elements is smaller. it has been found in peach that large diameters of xylem vessel elements are found in the more invigorating rootstocks (bruckner and dejong, 2014). for all the above, it is inferred that genotypes belonging to group 4 are small size plants or with dwarfing characteristics, this statement is reinforced by studies that showed that intermediate and small size plants frequently have a smaller stem diameter, such as the case of the group 4 plants that had the lowest average stem diameter values (fig. 7). these results show a similar trend to the study of lópez jiménez and barrientos priego (1987) in trees of ‘colín v­33’ where the trunks in dwarf trees had a circumference and average diame­ ter smaller than the tall trees. fig. 7 ­ discriminant variables of plants derived from ‘colín v­33’ avocado seedlings in the six groups generated according to canonical discriminant analysis (adc) and ward clu­ ster, derived from anatomical and physiological characte­ ristics of stem and leaf. adv. hort. sci., 2021 35(2): 183­194 192 the classification results show that 97.8 % of origi­ nally grouped individuals were correctly categorized (table 6), which indicated that the six groups based on this are consistent, showing the stability of belonging to each group. where only two individuals were atypical of their group. these results give cer­ tainty to the analysis performed and the congruence of the groups obtained. 4. conclusions the variables stem diameter, the density of vessel elements, thickness of the epidermis, leaf tempera­ ture, stomata length, thickness of the cambium layer, transpiration rate and stomatal conductance, dis­ criminated the groups of ‘colín v­33’ avocado seedling plants correctly. the main correlations obtained were a positive correlation between the density of xylem vessel ele­ ments with the rate of transpiration and with stom­ atal conductance. these correlations indicated a lin­ ear relationship of these variables and this last can be used as an index of preselection to discriminate the vessel element density in seedlings. a higher density of elements of xylem vessel ele­ ments and smaller stem diameter appear to be indi­ cators of dwarf plants in avocado, so these character­ istics could allow the selection of genotypes with behavior of this type of growth. group 4 genotypes presented anatomical and physiological characteristics probably associated with dwarf types unlike group 3, which were plants with typical anatomical and physiological characteristics of vigorous plants. to demonstrate the use of the different contrast­ ing groups it is required further studies with grafted plants to determine their potential according to their rootstock characteristics. references aloni b., cohen r., karni l., aktas h., edelstein m., 2010 ­ hormonal signaling in rootstock‐scion interac‐ tions. ­ sci. hort., 127(2): 119­126. ayala­arreola j., barrientos­priego a.f., colinas­ león m.t., sahagún­castellanos j., reyes­ale­ mán j.c., 2010 ­ relaciones injerto‐interinjerto y carac‐ terísticas anatómicas y fisiológicas de la hoja de cuatro genotipos de aguacate. ­ rev. chapingo ser. hort., 16(2): 147­154. baas p., 1982 ­ systematic phylogenetic and ecological wood anatomy: history and perspectives, pp. 23­58. ­ in: baas p. 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symplast tol‐ erance to intense drought conditions and xylem vulner‐ ability to cavitation coordinated? an integrated analy‐ sis of photosynthetic, hydraulic and leaf level processes in two mediterranean drought‐resistant species. ­ environ. exp. bot., 69: 233­242. impaginato 183 adv. hort. sci., 2017 31(3): 183-189 doi: 10.13128/ahs-20575 field evaluation of three biopesticides for control of the raspberry cane midge, resseliella theobaldi (barnes) in bulgaria m. mohamedova department of entomology, agricultural university plovdiv, 12 mendeleev str., 4000 plovdiv, bulgaria. key words: azadirachtin, bacillus subtilis, biocontrol, efficacy, resseliella theobaldi, spinosad. abstract: the raspberry cane midge, resseliella theobaldi is a key pest on red raspberry, rubus idaeus. the larvae of the insect severely attack the raspberry canes, resulting in premature death of the plant canes. in the last decade, organic production of raspberry fruits has significantly increased in bulgaria. at the same time there are few products of botanical or microbiological origin that might be used for control of this pest. in present study the effect of neemazal® t/c (azadirachtin a), sineis 480 sc® (spinosad), and bacillus subtilis on r. theobaldi was evaluated. the experiments were conducted in two raspberry fields at different altitude. in the field at lower altitude (196 m), the raspberry cane midge has developed four generation per year, while in the field at higher altitude (960 m) three generations of the pest have been completed. lowest number of larvae in raspberry canes was observed after application of neemazal® t/c, and b. subtilis in both raspberry fields. both products demonstrated highest efficacy at 7th day after treatment, when the number of larvae per splits was 67.1-82.5% for neemazal® t/c, and 75.1-81.2% for b. subtilis lower compared with the control at the two experimental sites. 1. introduction among the small fruit crops grown in bulgaria, the red raspberry (rubus idaeus l.) is the most valuable. during the last five years, the total area of raspberry plantation has increased by 43% and the yield has reached 3620 kg-1 ha. at the same time approximately 54% of raspberry production is organic. in 2015 and 2016, about 75% of the total raspberry yield was exported mainly to western european countries, but also to several markets in asia (agrostatistics, 2015). the most serious pest of raspberry is the raspberry cane midge, resseliella theobaldi (barnes) (diptera: cecidomyiidae), which causes premature death of the plant canes. in bulgaria, the insect was first reported by stoyanov in 1960. the author examined the life cycle of r. theobaldi (*) corresponding author: m.mohamedova@au-plovdiv.bg citation: mohamedova m., 2017 field evalutation of three biopesticides for control of the raspberry cane midge, resseliella theobaldi (barnes) in bulgaria. adv. hort. sci., 31(3): 183-189. copyright: © 2017 mohamedova m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 april 2017 accepted for publication 15 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 183-189 184 under different conditions and reported the development of three-five overlapping generations a year. the larvae of the insect attack the plant primocanes, both of those fruiting in june-july as well as those fruiting in august-september (stoyanov, 1963). the larvae feed under the bark of canes and clearly define dark brown spots appearing on the green surface of the canes. pitcher (1952) stated that damage to raspberry plants caused by r. theobaldi was usually associated with fungal pathogens such as botrytis cinerea, fusarium avenaceum and didymella applanta. the fungi cause necrosis of vascular cylinder through the larval feeding sites (williamson, 1987). the complex of damage involved the raspberry cane midge and mycoses is known as “midge blight” (pitcher and webb, 1952). as a result of usually present of midge blight, there is no established relationship between population level of r. theobaldi and degree of plant damage caused by the insect (williamson and hargreaves, 1979). according to shternshis et al. (2002), the effect of the control treatments against the pest should be assessed by estimating the severity of midge blight including fungal lesions. to date, the biological control of raspberry cane midge is still poorly investigated. there are few reports concerning alternatives to chemical control for r. theobaldi. sex-pheromone-based strategies are promising techniques to control of many economically important pests. pheromone traps for raspberry cane midge were used for the first time in the uk in 2005 (milenković et al., 2006). cross and hall (2006) and hall et al. (2009) identified 2-acetoxy-5-undecanone as a major component of r. theobaldi female sex pheromone. over the past ten years, the sex pheromones have been tested for monitoring the male emergence (cross et al., 2008, tanasković and milenković, 2010, sipos et al., 2012). therefore, little information concerning the application of biopesticides for control of r. theobaldi in raspberry organic production is currently available. for instance, use of products based on entomopathogenic bacteria, bacillus thuringiensis (bt) and streptomyces avermitilis against raspberry midge blight have been reported (shternshis et al., 2002). further, there are no publications on the biological control of this raspberry pathogen complex. the objective of this study was to evaluate the possibility to control raspberry cane midge using two commercial biopesticides and one noncommercial bacterial strain under field conditions. in particular, i attempted to assess the role of used products in reducing the number of r. theobaldi larvae in raspberry canes. 2. materials and methods biopesticides and bacterium cultivation commercial formulations neemazal® t/c (azadirachtin a, trifolio-m, germany), and sineis 480 sc® (spinosad, dowagrosciences, bulgaria), and a bacterium bacillus subtilis were used against midge in raspberry fields. the strain of b. subtilis was grown in the dark for 48 h at 24°c on tryptic soy broth agar (tsba). for inoculum production a loop of the bacteria was transferred into 100 ml of tsb and allowed to multiply for 48 h on a rotary shaker (160 rpm) at the same temperature. bacterial suspensions were centrifuged at 4000 rpm for 20 min, and the bacterial pellet was resuspended in sterile¼ strength ringer’s solution (merck). the bacterial suspension was adjusted to a final concentration of 108 cfu ml-1 by dilution with ringer’s solution. the bacterial strain identification was determined by fame analysis, following by biolog analysis. experimental design the trials were conducted in 2016 in two commercial raspberry plantations in the regions of bogdanovo (196 m) and samokov (960 m). the first location (bogdanovo) has a flat topography, with small hills. the soil type is leptosols (bulgarian soil taxonomy), and the landscape is dominated by agricultural land use. according to the climatic data (national institute of meteorology and hydrology, bas), the average air temperature from april through october 2016 was 22.4°c. the average rainfall for the investigation period was 284 mm. the geographical coordinates of the raspberry field in bogdanovo are 42°36’ n, 26°00’ e. the second location (samokov) is a valley between two mountains rila and verila. the soil type is fluvisols, and the landscape is dominated by arable land. the average air temperature from april through october 2016 was 15.4°c. for the same period, the average rainfall was 531 mm. the geographical coordinates of the raspberry field in samokov are 42°21’ n, 23°34’ e. the cultivar heritage (usa) was grown in both three years old fields. plants were located in spacing of 50 cm within rows and 2.0 m between rows. experimental plots were 10 m2, each plot containing approximately 100-120 raspberry canes. the size of mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 185 the buffer zone between the plots was 4 m. the treatments were arranged in a completely randomized block design with four replications. treatment and application methods the pheronorm® standard large delta traps (andermatt biocontrol ag, switzerland) were used to determine the population dynamic of the cane midge. the traps containing 10 µl cane midge sex pheromone lure per trap were mounted on bamboo sticks at height of 60 cm the 10th of april. three traps were used in bogdanovo (11 ha), and two in samokov (7,5 ha). two applications were made in samokov, one against the first generation (on the 17th may), and one against the second generation (on the 14th july). three applications were made in bogdanovo on 9th may, 8th july and 13th august against the first, second and third generations of r. theobaldi, respectively. the timing of each treatment was chosen according to the number of males caught by the traps. the treatments were made during the period of midge oviposition and larvae hatching. the test suspensions were: neemazal® t/c (0.2%), sineis 480 sc® (0,025%), and b. subtilis (20 ml). the suspensions were applied at a volume application rate of 0.1 l m-2, using a hand held sprayer. the plants in the control were treated with equal quantity (0.1 l m-2) of water. data collection and analysis the observations were made on the 3rd, 6th, and 12th day after treatments. twenty canes per replicate, 20 canes were examined under stereomicroscope and the number of larvae in the natural splits was counted. obtained data were subjected to one-way analysis of variance (anova) and the treatment means were compared with the control plants, according to the duncan’s test (p<0.05). 3. results in 2016, the raspberry midge cane flight pattern demonstrated four generations in bogadonovo (196 m) and three generations in samokov (960 m) (fig. 1). in both commercial fields, the flight of midges started in the second half of april, a week later in samokov (25.04) compared with bogdanovo (18.04). at lower altitude, the first, the second, and the third generations of the pest showed three pronounced peaks of its flight dynamic. in the higher altitude, there were two peaks of midge cane flight the first one between 19th and 25th of may, and the second one between 18th and 21th of july, for the first and second generations respectively (fig. 1). in general, the population density of the males was higher in bogdanovo than at samokov. the highest number of males was recorded during the intensive flight of the first generation of the midge in both bogdanovo (824) and samokov (683) sites (fig. 1). later in the season, the number of the males attracted by the traps in bogdanovo was 623, 698 and 193 for the second, third and fourth generation, respectively. in samokov, the number of the males was 528 and 267 for the second and third generation, respectively. the flight of the fourth generation of r. theobaldi continued until 12th of october in bogdanovo, while the flight of the third generation of the midge in samokov was completed on 27th of september (fig. 1). the results obtained from this observation allowed finding the most appropriate date for treatments. the evaluated bioinsecticides demonstrated biological activity against the raspberry midge cane applied during the period of pest oviposition and larvae hatching. except for the treatment against the second generation of r. theobaldi in samokov, in both raspberry fields, bogdanovo and samokov, the highest efficiency was found for the insecticide neemazal® t/c (tables 1 and 2). this biopesticide also demonstrate the rapid initial effect against the penetration of larvae into raspberry canes. the number of midge larvae per split in raspberry canes treated with neemazal® t/c was significantly lower than the control at observations at 3rd, 7th and 12th day after applying the insecticide in bogdanovo (table 1, p<0.05). there was no significant difference between the efficacy demonstrated by neemazal® t/c and b. subtilis. after the treatment against the first generation the number of midge larvae per split varied fig. 1 trap catches of males of resseliella theobaldi in the fields of bogdanovo and samokov during the spring, summer and autumn of 2016. adv. hort. sci., 2017 31(3): 183-189 186 between 1.08 and 1.54 for neemazal® t/c, and between 1.55 and 2.07 for b. subtil is . similar results were observed after the second and third treatments. the insecticide sineis 480 sc® demonstrated the lowest efficacy compared with neemazal® t/c and b. subtilis (table 1, p<0.05). at the observation at 3rd day after treatments against the first and third generation of raspberry midge in bogdanovo, the number of larvae in plots treated with sineis 480 sc® was not significantly different from the number of larvae in control plots. in the second generation there was a statistical difference between the variant with the bioinsecticide and the control variant. after the treatments at 7th and 12th day, the insecticide showed better effect, and the number of larvae into the canes was significantly lower than the control but still higher compared with neemazal® t/c and b. subtilis (table 1, p<0.05). in the experiments conducted in samokov sineis 480 sc® demonstrated higher efficacy against raspberry cane midge and the number of larvae per split was statistically different than the control at all three observations (table 2, p<0.05). in this field, b. subtilis was more effective than neemazal® t/c and the number of larvae per split varied between 2.86 and 2.91 after first treatment. after the same treatment, the number of larvae into the canes treated with neemazal® t/c varied between 3.15 and 2.94. after the treatment against the second generation of r. theobaldi, b. subtilis showed rapid initial effect than neemazal® t/c. there was significant difference in number of larvae at 3rd day 2.63 and 4.86 for b. subtilis and neemazal® t/c, respectively (table 2, p<0.05). table 1 efficacy of three biopesticides using against raspberry midge cane in bogdanovo means within each column followed by the same letter are not significantly different, duncan’s test (p<0.05). treatments products/active ingredients number of midge larvae/split (day after treatment) 3rd (±sd) 7th (±sd) 12th (±sd) first generation neemazal® t/c (azadirachtin a) 1.54 (±0.18) a 1.12 (±0.07) a 1.08 (±0.37) a sineis 480 sc® (spinosad) 4.25 (±0.45) b 2.93 (±1.04) b 3.41 (±1.35) b b. subtilis 2.07 (±0.67) a 1.35 (±0.11) a 1.55 (±0.64) a control 5.62 (±1.02) b 6.28 (±1.22) c 6.78 (±0.56) c second generation neemazal® t/c (azadirachtin a) 2.48 (±1.11) a 1.74 (±0.64) a 1.62 (±0.19) a sineis 480 sc® (spinosad) 5.77 (±1.32) b 3.82 (±1.04) b 3.87 (±0.94) b b. subtilis 3.19 (±0.44) a 2.17 (±0.05) a 2.28 (±0.14) a control 8.44 (±1.12) c 8.92 (±1.65) c 9.41 (±0.27) c third generation neemazal® t/c (azadirachtin a) 1.24 (±0.72) a 0.92 (±0.08) a 0.98 (±0.34) a sineis 480 sc® (spinosad) 3.48 (±1.13) b 2.14 (±0.22) a 2.21 (±0.75) a b. subtilis 1.37 (±0.18) a 1.22 (±0.31) a 1.43 (±0.86) a control 4.05 (±0.93) b 4.89 (±1.16) b 5.12 (±0.99) b table 2 efficacy of three biopesticides using against raspberry midge cane in samokov means within each column followed by the same letter are not significantly different, duncan’s test (p<0.05). treatments products/active ingredients number of midge larvae/split (day after treatment) 3rd (±sd) 7th (±sd) 12th (±sd) first generation neemazal® t/c (azadirachtin a) 3.15 (±1.11) a 2.13 (±0.82) a 2.94 (±0.54) a sineis 480 sc® (spinosad) 6.05 (±1.32) b 4.74 (±1.08) b 5.38 (±0.67) b b. subtilis 2.92 (±0.97) a 2.39 (±0.17) a 2.86 (±0.91) a control 10.21 (±1.24) c 12.17 (±1.98) c 14.71 (±1.15) c second generation neemazal® t/c (azadirachtin a) 4.86 (±0.46) b 3.77 (±0.21) b 3.58 (±1.06) a sineis 480 sc® (spinosad) 5.14 (±1.11) b 3.98 (±0.87) b 3.43 (±0.35) a b. subtilis 2.63 (±1.07) a 2.05 (±0.57) a 2.73 (±0.97) a control 8.73 (±0.48) c 10.84 (±1.54) c 13.22 (±1.18) b mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 187 4. discussion and conclusions r. theobaldi was described by theobald in 1920 (barnes, 1926). since then, almost a century later, it has become a pest of economic importance of raspberry crop throughout europe. the midge cane is widely distributed in bulgaria, greece, rumania, italy, france, ireland, uk, sweden, czech republic, slovakia, hungary and poland. in these countries the insect has been introduced mainly with infested planting materials and somewhere with infested soil. when establishing a new raspberry plantation, it is critical to choose the cultivar that is well adapted to local soil and climatic conditions and it is less susceptible to infestation by the raspberry cane midge, as well. normally, infested raspberry plants have demonstrated the symptoms of dark brown, clearly defined spots in the canes 3 to 5 weeks after laying the eggs (stoyanov, 1963). for this reason, it is important to determine the most appropriate timing for control of r. theobaldi. the treatments have to be done before the larval feeding sites become visible. organic production of raspberry in many countries, including bulgaria, is a challenge because of lack of products for plant protection, which have nonchemical origin. moreover, in bulgaria there are no officially registered biopesticides or even chemical insecticides, that can be specifically used for control of the midge cane. meanwhile, neemazal® t/c has been registered for control of tomato borer, tuta absoluta, meyr. considering this need, the present study met its objective the results showing the possibility of biological control of the r. theobaldi. the tested biologically based preparations demonstrated their efficacy against the larvae of midge cane. the pest had four generations in the plantation at lower altitude (bogdanovo) and three generation in the plantation at higher altitude (samokov). further, the data from the pheromone traps showed that the first generation had the highest population density in both raspberry plantations. the forth and the third generations in both sites showed the lowest population density. according to this information, the time and the number of treatments were determined. among the tested biopesticides, neemazal® t/c and b. subtilis demonstrated the highest efficacy, causing up to 82.5% (the former) and 81.2% (the later) reduction of number of midge larvae in the splits compared to the control. sineis 480 sc® demonstrated slower initial effect than neemazal® t/c and b. subtilis, but comparatively high efficacy, causing up to 63.26% reduction in number of larvae in the splits compared with the control. in fact, the present evaluation is the second attempt to apply only environmentally safe products for control of r. theobaldi. the first one was made by shternshis et al. (2002). the authors tested the preparations based on bt (bacticidae®), and s. avermitilis (phytoverm®) for control of the raspberry midge blight and reported significant reduction of disease complex severity compared with the control variants. s. avermitilis is the base of spinosad, the active ingredient of sineis 480 sc®. spinosad as an active ingredient of the product audienz®, was tested by barrofio et al. (2011) against the raspberry midge cane. the result from this experiment is ambivalent, showing comparatively high efficacy against the midge larvae, but not significantly differ compared to the control. in this study spinosad showed to be less effective to the raspberry cane midge compared with both, azadirachtin a and b. subtilis. the result is interesting, because the spinosad penetrates translaminarly in plant tissues and this suggests higher efficacy compared with the other tested products. deleva and harizanova (2014) stated the rapid initial effect of spinosad against the larvae of tomato borer, t. absoluta. the authors reported 73.33% larval mortality in tomato leaves at 3rd day after treatment. neem-based products have been evaluated for their efficacy against the different pest on berry plants. kim (2014) reported insufficient activity of azadirachtin against the rednecked cane borer, agrilus ruficollis f. on blackberries in usa. contrary, aguilera et al. (2009) commented the high efficacy of neem against the raspberry weevil, aegorhinus superciliosus g. in chile. the authors reported significant embryogenesis inhibition after applying the neem. the lowest number of larvae in raspberry canes observed in this evaluation is probably due to affect of azidarachtin on both larvae and adult of raspberry cane midge. the results obtained after application of, b. subtilis indicate that the bacterium might be considered as an effective agent for control of r. theobaldi. b. subtilis was originally isolated from the soil and has been tested as a biocontrol agent of root-knot nematode, meloidogyne arenaria on tomato (mohamedova and samaliev, 2011). the bacterium is able to colonize successfully the rhizosphere of the plants and affect different pathogens in this zone. this suggests that b. subtilis could influence the pupae of the raspberry cane midge in the soil. i have not observed any phytotoxicity or negative adv. hort. sci., 2017 31(3): 183-189 188 influence of the three biopesticides on raspberry plants and beneficial insects. in several raspberry canes collected from the plantation in samokov was observed parasitized 4th instar midge larvae of the third generation (fig. 2). the midge larvae probably were infested by the parasitic larvae of aprostectus genus. therefore the results of the present evaluation show the possibility of the tested biopesticides to control of the raspberry cane midge. these products are a good alternative to chemical insecticide and might be successfully integrated in the control strategies of r. theobaldi in both, conventional and organic raspberry production. further research should focus on screening the pesticides and bioagents, which could be able to control the disease complex “midge blight”, causing very often the dead of raspberry plants. acknowledgements the present study was partially supported by association of raspberry growers in bulgaria. the author would like to thank to s. velikova for her kind help and the growers who allowed access to their plantations. i am grateful to prof. svetoslav bobev for his comments concerning the “midge blight” symptoms and his advice and experience during the process of writhing of this manuscript. references agrostatistics, 2015 fruit production. ministry of agriculture and food, sofia, bull. no., 289. aguilera p.a., zampezzi v.m., araneda d.x., klein k.c., rebolledo r.r., 2009 azadirachtin effectivity in embryogenesis inhibition of aegorhinus superciliosus (guérin) (coleoptera: curculionidae). idesia, 27(1): 47-55. barnes h.f., 1926 the gall midges of blackberries and raspberries. j. pomol. hort. sci., 5: 137-140. baroffio c.a., mittaz c., bedard f., 2011 flight monitoring and efficacy trials against ressliella theobaldi in switzerland. iobc/wprs bulletin, 70: 45-49. cross j.v., baroffio c., grassi a., hall d.r., łabanowska b., milencović s., nilsson t., shternshis m., tornéus c., trandem n., vétek g., 2008 monitoring raspberry 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on plant protection. zlatibor, 27 november 2 december 2006. book of abstracts, pp. 117-118 (in serbian). mohamedova m., samaliev h., 2011 effect of the rhizobacterium, bacillus subtilis on meloidogyne arenaria at different temperature. agric. sci. technol., 3(4): fig. 2 parasitized and nonparasitized 4th instar larvae of resseliella theobaldi in splits of raspberry canes collected from samokov raspberry plantation (september, 2016) mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 189 378-383. pitcher r.s., 1952 observations on the raspberry cane midge (thomassiniana theobaldi barnes). i. biology. j. hortic. sci., 27: 71-94. pitcher r.s., webb p.c., 1952 observations on the raspberry cane midge thomassiniana theobaldi barne. ii. “midge blight”, a fungal invasion of the raspberry cane following injury by t. theobaldi. j. hortic. sci., 27: 95100. shternshis m.v., beljaev a.a., shpatova t.v., bokova j.v., duzhak a.b., 2002 field testing of bacticidae®, phytoverm® and chitinase for control of the raspberry midge blight in siberia. biocontrol, 47: 697-706. sipos k., madár s., markó, m., pénzes b., 2012 the possibility of automation of sex pheromone trapping: tested on resseliella theobaldi (barnes) (diptera, cecidomyiidae). afr. j. agric. res, 7(5): 1410-1413. stoyanov d., 1960 protection against the pests and diseases of raspberry in due time. ovostarstvo, 7: 24-29 (in bulgarian). stoyanov d., 1963 investigations on the raspberry cane midge thomassiniana thebaldi barnes in bulgaria. izvestija na instituta za zastita na rastenijata, gara kostinbrod, 4: 41-46 (in bulgarian). tanasković s., milencović s., 2010 monitoring of flight phenology of raspberry cane midge resseliella theobaldi barnes (diptera: cecidomyiidae) by the pheromone traps in western serbia. acta entomol. serbica, 15(1): 81-90. williamson b., 1987 effect of fenitrothion and benomyl sprays on raspberry cane midge (resseliella theobaldi) and midge blight, with particular reference to leptospheria coniothyrium in the disease complex. j. hortic. sci., 62: 171-175. williamson b., hargreaves a.j., 1979 a technique for scoring midge blight of red raspberry a disease complex caused by resseliella theobaldi and associated fungi. ann. appl. biol., 91(3): 297-301. impaginato 311 adv. hort. sci., 2018 32(3): 311-318 doi: 10.13128/ahs-22302 postharvest performance of cut rose cv. lovely red as affected by osmoprotectant and antitraspirant compounds e. di stasio, y. rouphael (*), g. raimondi, c. el-nakhel, s. de pascale dipartimento di agraria, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: β-aminobutyric acid, l-proline, rosa spp., stomatal conductance, transpirational flux, water balance. abstract: in cut flowers, the post-harvest turgor is a critical aspect in a system in which, in the absence of the root system, transpiration water losses must be compensated. two experiments were conducted in order to elucidate the effect of osmoprotectants (l-proline) as well as of molecules with antitranspirant behavior (ß-aminobutyric acid baba or pinolene) on the water relations and vase life of rose cut stems. applications of l-proline enhanced water fluxes, water conductivity, relative water content and stomatal conductance of rose cut stems in comparison to untreated plants, thus increasing the vase-life of rose cut flowers. baba treatment reduced the stomatal conductance in rose as well as the daily water consumption, on the other hand senescence phenomena occurred earlier. the water used by pinolene treated stems was lower compared to the control and this was associated with a medium increase of the vase life. overall, enhanced osmoregulation prolonged the vase life of cut flowers since the improved water status allowed the cut stem to partially continue its metabolic functions. on the other hand, the control of transpirational flux was functional in maintaining cellular turgor in pinolene treated cut stems, whereas with baba, senescence phenomena occurred probably due to the activation of biochemical pathway of senescence involving abscisic acid. taking all together, osmoregulation or direct control of transpirational fluxes may provide a promising avenue for improving the post-harvest longevity of cut roses. 1. introduction post-harvest efficiency is a crucial point of the cut flowers commercial value since it is related to growth and storage conditions interacting with the plant genetic background; those aspects will overall contribute to maximize the stems qualitative performance after cutting (fanourakis et al., 2013). cut flowers are subjected to a wide range of post-harvest losses as developmental senescence, leaf and petal abscission, leaf discoloration, premature wilting and disease from moulds and fungal pathogens (scariot et al., 2014). however, among all, water balance is yet a major factor influencing the longevity of cut flowers, in a system in (*) corresponding author: youssef.rouphael@unina.it citation: di stasio e., rouphael y., raimondi g., elnakhel c., de pascale s., 2018 postharvest performance of cut rose cv. lovely red as affected by osmoprotectant and antitraspirant compounds. adv. hort. sci., 32(3): 311-318 copyright: © 2018 di stasio e., rouphael y., raimondi c., elnakhel c., de pascale s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 311-318 312 which water losses must be compensated by water uptake and transport in the absence of the root system (singh and moore, 1992; lu et al., 2010). in particular, the post-harvest life of cut flowers is strongly dependent on their ability to maintain tissues hydration overtime, and water deficit or wilting mainly occur if the amount of transpiration exceeds the volume of water uptake (halevy and mayak, 1981; van doorn, 2012). one of the first plant responses to abiotic stress (as for cutting) is the stomatal closure, and this mechanism provides protection against tissues dehydration by reducing transpiration from the leaf surface (hare et al., 1998). however, in cut stems stomata are not completely closed after cutting, leading to a residual stomatal transpiration that together with cuticular transpiration, determine additional water losses from the stem (van doorn, 2012). on the other hand, a phenomenon that can severely undermine the cut stems post-harvest performance, is the lowering of water uptake that is mainly due to occlusions located in the basal stem end (he et al., 2006). in rose, one of the main causes of the cut flower wilting is the vascular occlusion determined by bacteria, air emboli and physiological responses to cutting (fanourakis et al., 2013). in rose cut flowers, the regulation of water balance has been in the past decades a key aspect in the improvement of the stems vase life (alaey et al., 2011; reid and jiang, 2012). among several mechanisms suggested that may improve water uptake in response to a stress, hydraulic conductivity variations and accumulation of compatible solutes are the most documented (chen and murata, 2002; ehlert et al., 2009). compatible solutes or osmolytes are organic compounds which help in raising the osmotic pressure and thereby maintaining both the turgor pressure and the driving gradient for water uptake (serraj and sinclair, 2002). common osmolytes found in plants mainly include proline, trehalose, fructan, mannitol and glycinebetaine and these compounds also help in maintaining the structural integrity of enzymes, membranes and other cellular components during the stress regime (zhao et al., 2007; chen and jiang, 2010). compatible solutes may be constitutively overproduced in transgenic plants (zhang et al., 2004) or directly applied on plants to improve stress tolerance under both open-field and protected cultivation (okuma et al., 2004; barbieri et al., 2011; cirillo et al., 2016). in addition to physiological mechanisms that can be exploited to improve water uptake, the reduction of transpirational flux has traditionally been one of the main objectives for controlling the cellular turgor after harvesting of fresh-cut vegetables and ornamentals (prakash and ramachandran, 2000). the reduction of transpiration can improve the water balance of cut flowers and extend their vase life, whereby artificial closure of the stomata might be an efficient strategy to reduce water losses (lu et al., 2010; van doorn, 2012). one method of limiting water loss involves the use of antitranspirants, which reduce plant transpiration forming a vapour-impermeable film on the leaf surface and, among these, the natural terpene polymer di-1-p-menthene (pinolene) is widely applied on different crops (francini et al., 2011; abdel-fattah, 2013). these polymers, also called “film forming antitranspirants”, sprayed on crops in a form of water emulsion, are generally employed to reduce weathering and extend pesticide efficacy, improving distribution and adherence of agrochemicals and decrease water loss and wilting of young transplants (gale and hagan, 1996; percival and boyle, 2009). research conducted on cut roses revealed that treatments with a film-forming antitranspirant are able to reduce the degree of fresh weight loss and water loss during transpiration, delay the process of flower opening and slow down the rate of stomatal conductance reduction (song et al., 2011). alternatively to traditional antitranspirants, the use of compounds which may induce a series of stress adaptation mechanisms, such as stomatal closure, could also be considered. beta-aminobutyric acid (baba) is a non-proteinogenic amino acid known for its ability to increase plant resistance to biotic (jakab et al., 2001; baider and cohen, 2003) and abiotic stresses (jakab et al., 2005). applications of baba may improve the plant tolerance to stress by activation of defense mechanisms mediated by abscissic acid (aba) and salicilic acid (sa) (zimmerli et al., 2000; jakab et al., 2005; baccelli and mauchmani, 2016). taking this background into consideration, it is clear that an efficient control of water balance is crucial to improve cut flowers vase-life and this can be achieved by using molecules that activate water transport in stem or inhibit transpiration. therefore, the aim of this study was to assess the influence of exogenous applications of osmolytes such as proline or anti-transpiring solutions like βaminobutyric acid and pinolene on the water balance, vase-life and also to shed light on the potential di stasio et al. postharvest performance of cut rose cv. lovely red 313 physiological mechanism(s) involved in cut stems of rose. 2. materials and methods plant material and growth conditions two experiments were carried out in order to assess the effects of exogenous applications of lproline (experiment 1) or antitranspirants [specifically: an active compound film forming (pinolene) and a stomatal closure inducing active compound βaminobutyric acid ; experiment 2] on water control in cut stems of rose plants (rosa spp. l.) cv. lovely red. cut flowers of rose were harvested from two years plants grown in closed soilless system in a heated greenhouse located in naples, south italy (40°51’55.5”n 14°20’30.1”e). rose plants were grown in plastic channels containing pumice and lapillus. the basic nutrient solution was supplied through a drip-irrigation system at a flow rate of 2 l h-1. irrigation frequency was regularly adjusted during the growing cycle based on the crop water requirements. at marketable harvest, cut stems were immediately transferred to the laboratory under room conditions, re-cut at the base (2-3 cm) and placed in graduated glass cylinders with 300 ml of deionized water and sodium hypochlorite (50 mg l-1). application of compatible solutes and antitranspirants compounds in the first experiment, two days before harvest, rose plants were treated with 10 mm l-1 of l-proline (sigma-aldrich, saint louis, missouri, usa) in 200 ml of deionized water per plant, applied in the growth substrate. control plants were treated with deionized water only. the treatment was performed at the end of the last daily irrigation and repeated after 24 h. in the second experiment, two days before harvest and at the end of the last daily irrigation, a substrate treatment was performed on rose plants, with of 0.5 mm l-1 of ß-aminobutyric acid (baba sigmaaldrich, saint louis, missouri, usa) in 200 ml of deionized water per plant, whereas control consisted of plants treated with deionized water. on a second plot of plants, the pinolene treatment was performed in post-harvest once transferred to the laboratory. stems were sprayed with a solution of 50 g l-1 of pinolene (96% poly-1-p-menthene, nu-film, intrachem bio, italy) in deionized water. control stems were sprayed with deionized water only. storage and physiological measurements part of the stems, weighted and sized based on length and diameters, were placed on ten precision balances (ek-410i, a&d instruments ltd, abingdon, uk) connected via usb to a computer for automatic monitoring of weight through a specific software (rscom®, corby, u.k.). these cylinders were sealed with parafilm to avoid water losses through evaporation. rs-com® software was set to record three daily weights in order to determine stems water consumptions over storage. cut stems were stored for 12 days under room conditions measuring daily mean temperature and relative humidity using a thermohygrometer (do 9847k, delta ohm srl, padova, italy). at storage days 2, 4 and 6, water flux measurements were recorded by using a scholander pressure chamber (3005f01 plant water status console, soil mosture equipment corp., santa barbara, california, usa). twenty centimeter stem segments (5 cm below the calix after measuring stems diameter) were immersed into a cylinder containing distilled water, placed in the pressure chamber, while the other extremity, out of the chamber, was connected to falcon tubes to collect and weight the water efflux. the system was then subjected to increasing pressure (p= 0.05, 0.1, 0.2, 0.3 mpa) and maintained at each pressure value for 5 minutes up to a constant outflow from the stem. water flux (jv) was expressed as jv= kg h2o m-2 s-1 m-1. water conductivity (lp) of stems was then expressed by the slope of the regression function of jv vs. p. volumes of collected efflux per unit of time (jv) were normalized to the cutting section surface. stomatal conductance (gs) was determined at storage days 2, 4 and 6 using a diffusion porometer (delta p-4, delta-t devices, cambridge, u.k.) in three daily measurements (h 9:00 am; h 1:00 pm; h 7:00 pm). osmotic potential (yπ) was measured using a dewpoint psychrometer (wp4, decagon devices, washington) on frozen/thawed leaf samples. relative water content (rwc) value was calculated as: rwc= (leaf fresh weight leaf dry weight)/(leaf saturated weight leaf dry weight) (morgan, 1984). leaf area was estimated by scanning cut stems leaves and using the image j® software (abramoff et al., 2004) for image processing. the cut stems vase life was assessed visually using a “quality score” from 0 to 4. statistical analysis all data were statistically analyzed by anova using the spss software package (spss 10 for windows, 2001). the rwc data were transformed in adv. hort. sci., 2018 32(3): 311-318 314 arc-sin before anova analysis. 3. results experiment 1. effect of l-proline application on postharvest performance of cut rose exogenous applications of l-proline enhanced significantly water fluxes of rose cut stems in comparison to the untreated control, for all the 3 days of measurements (fig. 1a). during storage, water flux decreased from day 2 to day 6 in treated stems (fig. 1a). moreover, stems water conductivity (lp) was 3.05 [(kg h2o m-2 s-1 m-1) mpa-1] in control and 3.55 [(kg h2o m-2 s-1 m-1) mpa-1] in l-proline treatment (table 1). significant increase in stomatal conductance (gs) as well as in rwc were observed in l-proline treatment compared to the control (table 1). similarly to the physiological measurements, water consumptions normalized per leaf area were higher in treated stems (fig. 1b). as a result of cellular osmotic adjustment due to l-proline application, leaf osmotic potential was lower for treated stems in comparison to untreated control (table 1). the improved water status of l-proline treated stems influenced positively cut stems longevity extending their vase life by 2 days compared to the untreated control. experiment 2. effect of antitraspirants application on postharvest performance of cut rose in our current study, stomatal conductance was reduced by baba treatment respect to the control (table 2). consequently, daily water consumption, normalized per leaf area, was lower for baba treated stems (fig. 2b). the application of 0.5 mm of baba significantly reduced water fluxes during storage (fig. 2a) as well as the water conductivity (lp) of rose cut stems (2.6 [(kg h2o m-2 s-1 m-1) mpa-1] in control vs. 1.54 [(kg h2o m-2 s-1 m-1) mpa-1] in baba treatment). rwc was significantly higher in the control and it decreased during storage (table 2). furthermore, the water potential decreased over time and it was lower for rose stems treated with baba (table 2). the vase life, however, was not influenced by the treatment since in baba treated stems the improvement of the water balance was accompanied by premature yellowing of the leaves. as a consequence of the mechanical stomatal closure, the water use of pinolene treated stems was always lower compared to control (fig. 3). this was associated with a significant increase of the vase life by 1.5 days (fig. 4). 4. discussion and conclusions it is well established that osmotic adjustment contributes to maintain water uptake and cellular turgor (maggio et al., 2002; heuer, 2003). among all the osmolytes involved in this process, it has been sugfig. 1 effect of exogenous l-proline on water flux (a) and water consumptions per leaf area (b) of rose cut stems during storage. vertical bars indicate ± se of means. table 1 effect of exogenous l-proline on stomatal conductance (gs), relative water content (rwc), osmotic potential (ψπ) and water conductivity (lp) of rose cut stems treatment gs (cm s-1) rwc (%) ψπ (mpa) lp (kg h 2 o m-2s-1 m-1) control 0.38 b 84 b -0.17 b 3.05 b proline 0.42 a 89 a -0.34 a 3.55 a significance * * * * ns,*, not significant or significant at p≤0.05 respectively. within each column, different letters indicate significant differences. table 2 effect of exogenous β-aminobutyric acid (baba) on stomatal conductance (gs), relative water content (rwc), osmotic potential (ψπ) and water conductivity (lp) of rose cut stems ns,*, not significant or significant at p≤0.05 respectively. within each column, different letters indicate significant differences. treatment gs (cm s-1) rwc (%) ψπ (mpa) lp (kg h 2 o m-2 s-1 m-1) control 0.41 a 79 a -0,17 2.60 baba 0.25 b 74 b -0,27 1.54 significance * * * * di stasio et al. postharvest performance of cut rose cv. lovely red 315 gested that proline, exogenously applied via foliar spraying or through the irrigation water, could localize into the cytoplasm to reduce the cellular osmotic potential and to restore cellular hydration (gadallah, 1999; barbieri et al., 2011). in our experiment, lproline treatment on rose plants has shown to substantially improve the hydration state of the cut stems, with an observed decrease of the leaf osmotic potential and increased stomatal conductance and leaf rwc. consequently, the improved hydration state of tissues and stomatal conductance enhanced the water consumption in plants treated with lproline. the decline in stem water conductivity, is one of the main reasons for impaired water balance, as well as water stress is the most common reason for reduced cut roses vase life (halevy, 1976; joyce and jones, 1992). the increase in water fluxes and water conductivity for the l-proline treated flowers was an evidence of improved water status of the plant tissues that probably was the main factor involved in the extended vase life of l-proline treated stems. it has been demonstrated that, in cut flowers, osmolytes are fundamental compounds in maintaining water balance, a key factor to extend their longevity (ichimura et al., 1997) as well as accumulation of these solutes, such as proline, is one of the main mechanisms to alleviate the detrimental effects of dehydration (morgan, 1984; anjum et al., 2011). in fact, the role of osmolytes includes mainly protection against the deleterious effects of the low water activity, preserving appropriate cellular volume (csonka and hanson, 1991). however, even though it is not yet clear if an extension of the cut flowers vase life may be more related to the ability of stem to maintain sustained water uptake rates or to control water losses, the control of the stomatal conductance is a fundamental determinant of the tissue water balance (fuchs and livingston, 1996; woodward et al., 2002). in nature, it is well known that plants control water losses by regulating transpiration in response to environmental factors (chaerle et al., 2005). in some respect, cut flowers respond to the same stimuli and the difference between the rate of water uptake and the tranfig. 2 effect of exogenous β-aminobutyric acid (baba) on water flux (a) and water consumptions per leaf area (b) of rose cut stems during storage. vertical bars indicate ± se of means. fig. 3 effect of pinolene on water consumptions per leaf area of rose cut stems during storage. vertical bars indicate ± se of means. fig. 4 effect of pinolene on the vase life of rose cut stems expressed as decay of 50% of the stems quality. different letters indicate significant differences at p≤0.05, vertical bars indicate ± se of means. 316 adv. hort. sci., 2018 32(3): 311-318 spiration rate is one of the parameters that will define their hydration state. as documented in different cases, baba acts through potentiation of aba-dependent signaling pathways (ton and mauch-mani, 2004) and for this reason, we supposed that applications of ß-aminobutyric acid (baba) may increase the tolerance to water shortage through the induction of functions associated to the synthesis of aba, such as stomatal closure (jakab et al., 2001; desikan et al., 2004). applied on rose plants, baba treatment has induced a decrease in stomatal conductance, with the consequent reduction of the stems water consumptions. along with this decrease of the transpirational flux, water fluxes and rwc decreased over time and they were generally lower in baba treated stems. in addition, lp was lower in baba-treated stems. the decreased rwc and water potential, together with a reduction of the cut stems hydration state, may be associated to senescence phenomena that occurred with the premature yellowing of the leaves, which is also mediated by aba (hunter et al., 2004; ferrante et al., 2006). accordingly, mayak and halevy (1972) reported that exogenous application of aba to rose cut flowers accelerate senescence phenomena. another strategy to control the plants transpiration and sustain a favorable plant water status is the utilization of antitranspirants compounds (del amor and rubio, 2009). our results confirmed that filmforming antitranpirants are effective in reducing water losses providing a thin coating on the leaves surfaces leading to an improved tissues water status in cut roses (moftah and al-humaid, 2005; song et al., 2011; mikiciuk et al., 2015). consistently, this mechanical effect on the transpirational flux regulation was observed on water consumptions normalized per leaf area rose cut stems, that were significantly reduced by pinolene application during the vase life. the reduced water use was correlated to an extended vase life compared to the water-treated control. in conclusion our results demonstrated that both osmoregulation and direct transpirational control were effective strategies in maintaining an enhanced hydration state of rose cut stems, leading to a prolongation of the stems vase life. treatment with 10 mm l-proline has allowed the maintenance of higher stomatal aperture and improved cut stems rwc and lp during storage. the positive effects on cut stems were measured as decrease of osmotic potential and increased stomatal aperture consequent to osmoregulation. these physiological conditions are crucial for prolonging the vase life of cut flowers because, despite the absence of the root system, allow the stem to partially continue its metabolic functions. on the other hand, the reduction of transpiration that is considered a functional target for controlling cellular turgor after harvest thus prolonging cut flowers. since aba is involved in the induction of physiological mechanisms that facilitate adaptation to abiotic stress, it has been hypothesized that the administration of baba, a mediator of aba functions, may confer a stress protection that could result in enhanced turgor and vase life of cut stems. in fact, our results also demonstrated that applications of 0.5 mm baba on rose has reduced water consumption by inducing stomatal closure. however, this was associated with a more rapid decay of the cut stems quality probably for earlier oncoming of senescence 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alimentari, ambientali e forestali, dagri, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: coffea arabica, coffea canephora, ptr-tof-ms, volatile organic compounds. abstract: the quality of coffee is linked to the aroma created by the chemical reactions that occur during the roasting process. while it is generally thought that roasted coffee is a stable product with a relatively long shelf-life, little information is available on the evolution (kinetic) of the volatile organic compounds (vocs) in the days immediately following the process. the aim of this study is to determine the evolution of vocs released by coffee beans, on samples of coffea arabica (three different origins) and coffea canephora (1 single origin), by using a proton transfer reaction time-of-flight mass spectrometer (ptr-tof-ms) 24 hours after roasting, and for the next 9 days. results confirmed the differences already highlighted in previous studies between the vocs spectra of the two species. there were also significant differences in the intensity of emissions for the different origins of coffea arabica, with the highest vocs amount over time always detected in the honduras arabica samples. the involved detected protonated ions were grouped into three classes: compounds (ppbv) present with decreasing quantity; weakly increasing; almost constant trend; or always increasing. a complex dynamic emerged for the different protonated ions over time, which not only affects the mass spectra of the different species but also influences the configuration of the mass spectra of the different geographical zones of production. 1. introduction two species contribute to the production of roasted beans, coffea arabica (commercially known as arabica) and coffea canephora var. (*) corresponding author: emarone@unite.it citation: colzi i., marone e., magnelli s., mancuso s., azzarello e., taiti c., 2019 kinetics of volatile organic compounds (vocs) released by roasted coffee during the first ten days after processing. adv. hort. sci., 33(1): 145-150 copyright: © 2019 colzi i., marone e., magnelli s., mancuso s., azzarello e., taiti c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 november 2018 accepted for publication 15 march 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 145-150 146 robusta (commercially known as robusta). these two species are characterized by different economic value, with arabica having the higher costs, due to both the different aromatic properties and the higher production costs. the two species are distinguishable by aromatic profiles (gloess et al., 2014; yener et al., 2014; colzi et al., 2017), and our previous work (colzi et al., 2017) demonstrated that the proton transfer reaction time of flight mass spectrometry (ptrtof-ms) can be successfully used to distinguish between the aromatic profiles of these two species in each step of the processing chain (green beans, roasted beans, ground coffee, brews). little known are the changes in volatile organic compounds (vocs) detected through headspace analyses, that could be used to recognize the genetic origin of the coffee stock and, within the same species, the geographical provenience as macro zone of production. the quality of coffee is linked to the particular aroma created by the chemical reactions that occur during the roasting process (maillard reaction). in addition to the roasting conditions, the quality of the product also depends on the genetic origin of the raw material. roasted coffee is generally considered a stable product with a relatively long shelf-life. nevertheless, little is known about the possible changes in the days immediately following the roasting process, which could alter the complex kinetics of vocs determining the coffee aroma and therefore its value. indeed, evidence indicates that chemical changes can still occur following the roasting process (nicoli et al., 2009), affecting the aroma of the brews. confirming this, coffee made from freshly ground beans was found to have a stronger aroma, less bitterness and was preferred by expert panelist when compared with beans stored for 1 or 2 weeks (ross et al., 2006). the present study has two goals: 1) to define the vocs emission in coffee beans of different species and origins in the first days after roasting; 2) to ascertain if differences in the vocs kinetics depend on the physical parameters of the roasted bean, such as porosity and the surface; 3) to explore the variation of the vocs spectra emitted over time as a way to improve the possibility of distinguishing the different species and, within them, the origins of the different stocks. 2. materials and methods coffee samples coffee was sampled from medium-dark roasted beans of 3 certified commercial stocks of c. arabica having different origin (honduras, india, and nicaragua) and from one of c. canephora var. robusta (congo). all just roasted coffee samples were provided by caffè magnelli s.r.l. (florence, italy) and stored in laminate packaging in the dark at room temperature (21-23°c) before the analysis. mass spectrometry analyses were thus performed on 120 samples, subdivided in 90 c. arabica and 30 c. canephora. vocs analysis voc measurements on roasted beans were performed by using a commercial ptr-tof 8000 model, from ionicon analytik gmbh (innsbruck, austria). the analyses were carried out starting approximately 25 hours after roasting, and proceeded for the next 9 days, with 24-hour intervals. three 5 g samples were taken each day from the different coffee stocks for the analyses. the analyses were performed following the methodology reported by colzi et al. (2017). each sample was put into a 0.75 l glass container plugged with a cover in which two teflon tubes are inserted, respectively connected to a zero-air generator (peak scientific, glasgow, scotland) and to the ptr-tof-ms. before each analysis, the jar was cleaned for 60 seconds with free vocs air and after was incubated for 60 s. the measurement was carried out for 60 seconds using a dynamic headspace flushing flow rate of 0.75 l per minute (lpm). since, the “temperature” is critical for the release of vocs, the analisys was performed in a climatized room (22°c, 90% rh). the raw data were acquired by the tofdaq viewer® software (tofwerk ag, thun, switzerland) and subsequently vocs were converted in ppbv using the formula described by lindinger et al. (1998) and constant values for the reaction rate coefficient (kr=2.109 cm3 s-1) to calculate concentration for each voc (cappellin et al., 2011). all detected compounds were tentatively identified by comparing the measured protonated ions with those reported in the extensive literature regarding the composition of vocs released from coffee and in other previous ptr-ms studies (colzi et al., 2017; flament and bessière-thomas, 2002; gloess et al., 2014; lindinger et al., 2008; lópez et al., 2016; maeztu et al., 2001; mondello et al., 2005; romano et al., 2014; yener et al., 2014; yeretzian et al., 2002). statistical data analysis one-way analyses of variance (anova) was percolzi et al. vocs kinetic in roasted coffee beans 147 formed to compare the total vocs content in 4 coffee stocks over 10 days; the separation of means was calculated by the fisher’s least significant difference (lsd) test. computations were performed by statgraphics centurion xv v. 15.0.04. on 90 arabica and 30 robusta samples, a multivariate partial least square-discriminant analysis (plsda) was applied, as supervised classification method, on the spectral data (n = 129) obtained from a total of 120 coffee samples taken from 4 different stocks (arabica honduras, arabica india, arabica nicaragua, and robusta congo), to determine whether it is possible to distinguish the genetic and geographical origin of the samples based on the voc profiles. for a more detailed description of the method, see (colzi et al., 2017; taiti et al., 2017). data were preprocessed by a logarithmic transformation (log+1). the statistical analysis was performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab® r2015b (mathworks inc., natick, ma, usa). to monitor voc trend in real-time in each coffee batch over the 10 days after roasting, a batch statistical process control (bspc) was performed after logarithmic transformation (log+1) on spectral (ptr-tof-ms) data of the 120 samples. bspc is the analysis of process data as a function of both correlation among the measured variables and correlation in time (also known as the “batch trajectory”). in this analysis the “measured variables” were 54 protonated ion signals selected on a total of 129 as tentatively identified in previous studies (maeztu et al., 2001; flament and bessière-thomas, 2002; yeretzian et al., 2002; mondello et al., 2005; lindinger et al., 2008; gloess et al., 2014; romano et al., 2014; yener et al., 2014; lópez et al., 2016; colzi et al., 2017), while the “time” was represented by the 10 days of surveys. the vocs data were subdivided into 4 “batches” (experiments) (arabica honduras, arabica india, arabica nicaragua, and robusta congo) that were subsequently subdivided into “steps” (10 steps = 10 days). a summary principal component analysis (pca) was then applied, after mean centering, summarizing the change in measured variables over the batch progress based on the “mean” as a reference statistic, sufficiently sensitive to the trajectory profile, to capture the trends in the trajectories of the variables. as unsupervised classification method a hierarchical clustering (ward method, euclidean distance) was performed on the data expressed as percentage of the highest value of each protonated ions, related to the 90 arabica samples, which represent all the samples unequivocally related to the same species. computations were performed by xlstat 2014.5.03 software. 3. results and discussion figure 1 shows the amount of the vocs emitted from the 4 stocks during the 10 days of testing, and the related trend curves (2nd grade polynomials: for arabica honduras y = -61.937x2 + 656.19x + 4604; for arabica india y = -95.22x2 + 1126x + 455.4; for arabica nicaragua y = -108.42x2 + 1250.8x + 1382.7; for robusta congo y = -7.1514x2 + 111.47x + 1705.4). the compared samples of the 4 batches were strongly differentiated both for amounts of emitted vocs and for trends (table 1). the robusta (congo) recorded the lowest emissions, that did not statistically change during the considered time period. by contrast, the comparison between the arabica samples highlighted key differences between the 3 batches. coffee samples from honduras were very rich in vocs. the nicaraguan samples had a less intense emission, and quantitatively increased only until the 5th day. although the amount of voc emitted resulted lower compared with the nicaraguan samples, the arabica samples of indian origin had a similar voc emission trend with a peak on the 5th day after roasting (fig. 1) (table 1). the data represented in figure 1 were explored through two different methods of multivariate analysis: a pls-da (fig. 2), and a pca applied to the results of a batch process (fig. 3). these approaches allow to highlight the two different aspects related to the facfig. 1 total vocs emission (ppbv) in the different coffee stocks over the time (dotted line) and related trend curves (whole line). adv. hort. sci., 2019 33(1): 145-150 148 tors influencing the process: 1) species and origins (pls-da applied to 120 coffee samples based on 129 detected signals), and 2) the processes that develop over time expressed as coefficients of 54 selected variables with pc1 during the 10 days of surveys after roasting (pca applied to a bspc). the score plot from pls-da shown in figure 2 confirmed that voc analysis using the ptr-tof-ms technique can indeed be used to distinguish robusta from arabica samples. furthermore we found that the evaluation of the whole spectrum of voc emission during 10 days of surveys can also be used to determine within the same species, albeit with some overlapping, the different origins (fig. 2). in the figure 3, where the dynamics of the single 54 tentatively identified protonated ions related to vocs found in the arabica samples of the three different origins are reported, a downward shift of the vocs pool on the 4th day is observed (fig. 3, red circle) while, in general, a progressive slight decrease and compression of the vocs complex can be seen in the time, up to the 10th day. fig. 2 score plot of pls-da model and summary statistics for the 4 coffee stocks samples based on 129 protonated m/z. table 1 trend of total vocs emission (ppbv) in 4 coffee batches over 10 days time total vocs (ppbv) average * arabica honduras arabica india arabica nicaragua robusta congo day 1 4654.6 ± 340.3 1928.8 ± 353.9 3055.9 ± 125.0 1634.7 ± 249.7 2818.5 ± 1261.3 a day 2 6588.9 ± 516.63 2321.3 ± 392.1 3023.0 ± 465.0 1653.1 ± 267.0 3396.5 ± 2022.5 ab day 3 5200.3 ± 92.8 2315.7 ± 5.19 3609.9 ± 250.3 2797.1 ± 123.0 3480.8 ± 1150.8 ab day 4 6694.6 ± 2105.3 2461.9 ± 756.2 4398.1 ± 931.3 1916.7 ± 491.7 3867.8 ± 2223.7 abc day 5 7087.2 ± 2509.9 4844.2 ± 314.3 5677.0 ± 912.4 2267.03 ±197.3 4968.8 ± 2162.8 c day 6 5680.3 ± 1146.0 3856.5 ± 449.9 5048.2 ± 906.3 1408.3 ± 148.6 3998.4 ± 1826.5 abc day 7 6706.1 ± 576.0 3915.7 ± 1614.9 4792.3 ± 747.9 2166.0 ± 684.7 4395.0 ± 1907.6 bc day 8 4600.6 ± 522.9 3517.5 ± 481.2 4867.8 ± 632.7 2218.1 ± 355.4 3801.0 ± 1173.7 abc day 9 5704.1 ± 1286.2 2393.8 ± 681.6 3127.7 ± 890.6 2315.0 ± 586.2 3385.2 ± 1630.0 ab day 10 5368.0 ± 862.5 2271.3 ± 646.7 3281.8 ± 934.5 2055.4 ± 459.3 3244.1 ± 1510.8 ab average * 5828.5 ± 1345.7 d 2982.7 ± 1102.6 b 4088.2 ± 1126.8 c 2043.1 ± 513.0 a 3735.6 ± 1764.0 average ± s.d. *different lower case letters within a row or a column indicate difference by the lsd test at the 95.0% confidence level (p=0.05). fig. 3 score plot from a pca applied to a bspc, showing the dynamic over the 10 days of surveys evidenced by the vocs complex (54 protonated ion signals tentatively identified) related to 120 coffee samples. outlier samples were highlighted by a red circle. colzi et al. vocs kinetic in roasted coffee beans 149 in the dendrogram obtained by the hierarchical classification of the three arabica stocks (90 samples) based on 54 protonated ions, two main clusters (a and b) were identified and, within the cluster b, a further division into two subgroups of compounds (b1 and b2) emerged (fig. 4). each group was characterized by a particular trend in the 10 days of the test. cluster a combined compounds characterized by a decreasing trend over time. on the other hand, cluster b grouped compounds with increasing trend (b1), or compounds at first increasing and then decreasing their amount over the time, in slightly different way (b2). figure 5 shows the trends, for the three arabica stocks, of three protonated ions chosen as representative of the groups emerged by the dendrogram reported in figure 4. in particular, mass m/z = 45.030, representative of the group a, was always decreasing, while mass m/z = 99.040 (b1) showed an upward trend and mass m/z = 82.060 (b2) initially increased and and then decreased to finally stabilize on intermediate values. 4. conclusions roasted coffee beans are considered resistant to spoiling phenomena, and as such are marketed, or considered for subsequent treatments. nevertheless, little is known about the possible changes in the days immediately following the roasting process, which can determine complex variations in the kinetics of voc emission within the different species and of different origins. the data set collected in this work highlights that samples of arabica and those coming from honduras production area have the highest level of voc emissions and the greater dynamism in the kinetics of change. these changes in the voc kinetics in the 10 days following the roasting process found within the arabica samples also improved the separation of the samples based on their geographical origin. the arabica species samples hierarchical classification applied to the 54 selected protonated ions related to vocs and/or fragment of vocs allowed to pool them together, independently of the chemical family they belong to, in relation to their kinetics over time. three groups of vocs were highlighted from the dendrogram (fig. 4). the first, which clustered together those that decrease over time. on the other hand, the second and third group respectively combined vocs that either had an upward trend but then stabilized over time, or those that constantly increased. the analysis of the vocs highlighted dynamics that changed over time. this was associated with the simultaneous presence of voc whose amount fig. 4 dendrogram from cluster analysis of 90 arabica sample based on 54 selected protonated m/z. fig. 5 trend of m/z = 45.030, m/z = 99.040, and m/z = 82.060, representative of group a, subgroup b1, and subgroup b2, respectively. average values for each of the 4 stocks from normalized data. 150 adv. hort. sci., 2019 33(1): 145-150 in the spectrum decreased while others tended to increase. the combined resulting variability determined changes of coffee aromas in the 10 days immediately following the roasting. acknowledgements the authors would like to thank caffè magnelli s.r.l. for providing 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a., cappellin l., märk t.d., del pulgar j.s., gasperi f., navarini l., biasioli f., 2014 ptr-tof-ms characterisation of roasted coffees (c. arabica) from different geographic origins. int. j. mass spectrom., 49(9): 929-935. yeretzian c., jordan a., badoud r., lindinger w., 2002 from the green bean to the cup of coffee: investigating coffee roasting by on-line monitoring of volatiles. eur. food res. technol., 214(2): 92-104. impaginato 77 adv. hort. sci., 2019 33(1): 77-85 doi: 10.13128/ahs-23298 apple seed stocks affected scion tree vigor and performance based on maternal self(in)compatibility h. hajnajari temperate and cold fruits research institute (karaj), horticulture sciences research institute, ag. research education and extension organization (areeo), karaj, iran. key words: breeding, dwarfness, genetic purity, inductive effect, malus × domestica borkh., seed rootstock. abstract: in a breeding program to increase uniformity of apple saplings size, shape and vigor the genetic improvement of seed rootstocks was considered as a key point. the half-sib seeds of two selected native crab parents, self-compatible “morabbaei” and self-incompatible “azayesh”, both dwarf, were considered as the main sources of variances into the six grafted scions. the inductive effects of the two seed masses were assessed on vegetative traits of 6 and 7-year-old scion trees. according to the results, shoot length, shoot thickness and internode length as main components of tree vigor showed significant differences at 1% level in all the six scions. the shoot length mean of the grafted scions on “azayesh” seed progeny was higher than “morabbaei” seed stock. the grafted scions on self-compatible “morabbaei” seed stock gave place to the shoots with higher thickness than the same grafted on “azayesh” seed source. combinations of “red delicious”-“morabbaei” and “braeburn”-“azayesh” demonstrated max and min values of internodes length during two consecutive years. both rootstock and scion trunk diameters on self-incompatible “azayesh” seeds resulted higher than seed progeny of self-compatible “morabbaei”. shoot number was not affected by rootstock type, whereas the effect of rootstock × scion, scion and year resulted significant. 1. introduction malus genus is characterized by great phenotypic and genetic diversity. high heterozygosity is reinforced by dominant self-incompatibility besides inbreeding depression (korban and skirvin, 1984). among 6000 documented apple cultivars, a large part of commercial production still relies on few cultivars including “golden delicious”, “granny smith”, “fuji”, “gala” and delicious group (o’rourke, 2003). rootstocks can influence vigor, habit and cropping of the scion cultivar as well as tolerance to unfavorable climatic or edaphic conditions (webster, 2005). rootstock performance is highly correlated with the genetic potential of rootstock to provide anchorage, adaptaion to pedo-climatic conditions and resist to abiotic stress efficiently (fazio, 2014). trees with limited height are requested due to facilitate orchard management. smaller trees are also (*) corresponding author: hassanhajnajari@yahoo.com citation: hajnajari h., 2019 apple seed stocks affected scion tree vigor and performance based on maternal self(in)compatibility. adv. hort. sci., 33(1): 77-85 copyright: © 2019 hajnajari h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 may 2018 accepted for publication 12 december 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 77-85 78 easier to target with sprays, reducing undesirable spray drift and increasing efficiency of usage (jobir, 2016). good performance, dwarfness, precociousness, scion compatibility, free from suckers and burrknots are some of the attributes of an ideal rootstock (wertheim and webster, 2005). clonal rootstocks are being developed rapidly while seed rootstocks have gradually decreased. still today, many nurseries in canada, belarus and finland use apple seed rootstocks. “antonovka” was selected as a seed source due to the freeze resistance and its cytoplasmic heritability (żurawicz and lewandowski, 2014), the system for which some traits are transmitted to the next generation by maternal parent (lorenzetti and ceccarelli, 1980). seed rootstocks improvement is applied for dwarfing effect, and tolerance to dry soil conditions (brown and maloney, 2005). in iran seed rootstocks are used because of the alkaline and dry soils and also due to bad topography and exaggerated fragmentations. in absence of a specialized sector for pure seed production, the mixture of recycled seeds of processing industry characterized by high genetic diversity are used in saplings production (hajnajari and tarrahi, 2009; hajnajari, 2010). increasing genetic purity of seed rootstocks will enhance scion tree size and uniformity by taking advantage of their neglected aspects (hajnajari and mizani, 2015). similarly, almond trees are raised on wild peach and bitter almond rootstock (sharma et al. , 2004). “mazzard” seedlings followed by “mahaleb” are the most widely used rootstocks for cherry tree production (demirsoy et al., 2017). each open pollinated seed has its specific hormonal status due to the different genetic provenience different from the rest which will cause a gradient of tree growth habits and vigor within the same scion cultivar. while tree vigor is provoked by hybrid heterosis (lorenzetti et al., 2011), the mandatory inbreeding ruling the self-compatible cultivars as maternal seed source may cause vigor decline of scion trees. it was suggested that the inbred f1 seed stocks could weaken the vigor of scion. vigorous trees with higher canopy size need increased planting distance (kosina, 2010). selection of proper parents as seed source was the unique pass to way out from the emblematic situation (hajnajari, 2018). cultivar evaluation program led to selection of few native dwarf crabs “morabbaei”, “zinati” and “azayesh” as seed source parents (hajnajari, 2010). contemporaneous field screening program for determination of self-compatibility level of the 92 cultivars followed by fluorescent microscope studies showed complete self-compatibility of “morabbaei”, “zinati” and “azayesh” as selfincompatible (forughikia et al., 2014; hajnajari and moradi, 2014). using such maternal parents that profit high level of self-compatibility accompanied with dwarfness can minimize seed genetic variability leading to production of the f1 seed progeny marked by high genetic purity. both traits of dwarfism and low vigor within the seedlings can be used for production of uniform saplings. the same parents were used for production of clonal rootstocks resistant to the crown rot disease (hajnajari et al., 2012). the effects of two seed masses, as f1 progeny of “azayesh” (self-incompatible) and “morabbaei” (selfcompatible) on growth characteristics of six apple scion cultivars were investigated. practically, the two open pollinated seed masses derived from two different maternal parents are assumed as two family’s genetically, diverse within the lots and among them, however two various sister masses belonging to different families. nevertheless, both parents as crab apples carry jointly dwarfness trait and are native to iranian habitat. here is shown that the seed rootstocks bred by selfcompatible parent incur more morphological uniformity and control of tree vigor in scion trees compared with the rival sister seeds produced by heterozygote parent “azayesh” which afford more vigorous and less homogenous tree architecture due to the need of self-incompatible parent for foreign pollen. 2. materials and methods the mature apple trees, 6 and 7-year-old (“golden delicious”, “red delicious”, “granny smith”, “braeburn”, “gala” and native “golab-e kohanz”) pruned in spindle form, grafted on two selected seed masses were studied to understand the effects of seed sources on vegetative characteristics of the scion cultivars. genetically, the two seed lots were identified as f1 half-sib progenies derived from two different maternal parents including the self-compatible cultivar “morabbaei” and the other self-incompatible “azayesh”, revealed as homozygote and heterozygote, correspondingly. the two seed lots were obtained from the maternal parents exposed equally to open pollination, so forming two genetically diverse seed masses as two families considered as the main treatments. the investigated traits included annual diametric growth of scion hajnajari seed rootstocks improvement in apple 79 trunk, graft point and rootstock. for each tree, 4 annual shoots were selected and labelled, and internodes number, internodes length, shoot length, shoot thickness and lenticels number were assessed. also, shoot numbers per tree and crotch angle were studied. the entire comparative inter and intra varietal measurements were achieved to distinguish the influences of the two seed rootstocks as the main treatments. interaction of scion per rootstock was recorded to study also the role of scion tree vigor against dwarfing effect of the seed source. six trees, 3 × 3 m, were assigned for each combination of “scion cv-seed progeny” within six replications, large enough to explain seed source inductive effects. growth traits of grafted scions were measured in two successive vegetative seasons (2014 and 2015). the experimental orchard was established in meshkin-abad station under temperate and cold fruit research center located in karaj, alborz province (iran). the experiment was set as according to a completely randomized blocks design (rcbd) with six replications for statistical analysis. anova was performed by combining the analysis of the collected data regarding all growth traits in exam, both seed rootstocks and cultivars. all statistical analyses were carried out using the general linear model (glm) procedure of the sas version 9.0. the duncan’ multiple range test (p≤0.01) was used to evaluate differences between treatments. 3. results shoot length and thickness shoot length and shoot thickness were affected significantly by rootstock, scion and year interactions (table 1). all variables are measured at annual shoots. shoot length in 2014, “red delicious”-”azayesh” combinations showed the longest shot lenght, while “golden delicious” on “morabbaei” had the lowest value, in 2015. comparing the treatments, no significant differences were observed for effects of the two seed progenies “azayesh” and “morabbaei” on the single cultivars, at the end of the first growing season. in the second year, significant differences were observed by different sources of seed rootstocks for grafted cultivars (table 2). in 2014, it was found that shoot length value for all scions of “golab-e kohanz”, “gala”, “granny smith” and “braeburn” combined with “morabbaei” seed rootstocks showed lower values than “azayesh” f1 progeny; albeit “braeburn” demonstrated significant difference, “golden delicious” and “red delicious” showed no significant differences on the 2 seed sources for shoot length. these results indicate that the maternal self-incompatible “azayesh” gave place to more vigorous seed progeny. whereas in 2015, the entire scions had lowered values of shoot length showing significant difference affected by seed source (table 2). considering the prevailing traits of dwarfness associated with homozygosis carried by “morabbaei” it becomes clear how these are transmitted to the relative f1 seed progeny whom could control the vigor of the combined scion trees. though both parents may produce seeds which reinforced vigor control of scion trees but the “morabbaei” seed progeny showed the capability both to induce dwarfism and uniformity in size and shape into the scion cultivars (table 2). parallel researches of artificial self-pollination followed by florescent microscopy studies put in evidence how the pollen tubes of “morabbaei” could penetrate to its own ovary confirming the total selfcompatibil ity of selected maternal parent (forughikia, et al., 2014). future investigations are source df mean square shoot length shoot thickness internode number internode length rootstock diameter graft diameter trunk diameter shoot number branch angle lenticels number year (y) 1 44376.9 ** 3.92 ** 7.65 ** 1.14 ** 1912.11 ** 4460.61 ** 0.22 ** 0.25 * 67.79 ns 0.007 ns block (year) 10 4982.37 ** 0.04 ** 0.17 ** 0.07 ** 223.66 ns 108.48 ns 0.01 ns 0.03 ns 420.71 ** 0.07 * rootstock (r) 1 39402.37 ** 0.67 ** 0.04 ns 0.56 ** 1005.01 * 911.83 * 0.03 ns 0.03 ns 3679.76 ** 1.29 ** scion (s) 5 10396.58 ** 0.063 ** 0.24 ** 0.12 ** 28.27 ns 111.47 ns 0.02 ns 0.14 * 833.27 ** 0.24 ** r × s 5 6614.24 ** 0.067 ** 0.06 ns 0.23 ** 409.78 * 143.15 ns 0.02 ns 0.17 ** 1749.75 ** 0.76 ** y × r 1 17580.61 ** 0.003 ns 0.05 ns 0.56 ** 56.99 ns 31.91 ns 0.03 ns 0.006 ns 667.52 * 0.10 ns y × s 5 7960.64 ** 0.06 ** 0.12 ** 0.08 ** 82.02 ns 79.06 ns 0.02 ns 0.10 ns 440.02 * 0.20 ** y × r× s 5 11061.31 ** 0.07 ** 0.12 ** 0.06 ** 52.05 ns 46.51 ns 0.01 ns 0.01 ns 668.91 ** 0.37 ** cv (%) 25.62 12.16 16.52 10.74 24.007 23.11 7.63 22.32 16.80 14.83 table 1 analysis of variance regarding the effects of year, rootstock and scion on vegetative traits **= significant differences at 1% level, *= significant differences at 5% level, ns= not significant. adv. hort. sci., 2019 33(1): 77-85 80 required to elucidate the mechanisms and biochemical pathways by which a self-compatible cultivar can accept insider pollen leading to fertilization, within a self-incompatible species. shoot thickness combination of “red delicious”-”morabbaei”, in the first year, showed the highest shoot thickness, while both combinations of “golden delicious” and “red delicious” on “azayesh”, in the second year, showed the lowest rates. it could be inferred that low vigor of “morabbaei” f1 progeny inhibited shoot length growth rising inversely shoot thickness. in 2014, converse growth rhythm was registered in “golden delicious” and “braeburn” on heterozygote seeds of “azayesh” inducing significant differences, while “golden delicious”, “red delicious” and “gala” were affected significantly by “azayesh” f1 progeny for the measured variables in 2015. moreover, in 2014 “morabbaei” seeds induced significant differences between “golden delicious” and “granny smith”. similar differences were observed among “golden delicious”, “red delicious” and “gala”, in 2015 (table 2). such differences in successive years within the combinations are attributed to the normal annual growth. internode number year and scion main effects and year × scion and year × rootstock × scion interaction significantly affected internode number (table 1). the highest number of internode was found in 2014 in combination of “golden delicious”-“morabbaei” confirming the role of homozygosity on lowering vegetative growth, changes in metabolic pathways and relative hormonal modifications at intra-cellular level. however, the lowest expression of the genes for internode number was found in “gala”-“azayesh” combination, in 2015. as a reason, the latter low vigor of “gala” occurred in “on” year could influence such result. in 2014, there were no significant differences among the cultivars for internode number on “azayesh” seeds, while in the second year significant differences were observed caused by annual growth. contrarily, “morabbaei” seed stocks induced significant differences in “gala”, “golden delicious” and “granny smith”, in 2014, which confirms relatively lower vigor of “gala”, whilst in 2015 significant differences was observed among the grafted cultivars (table 2). in 2014, there were no significant differences between rootstocks for each scion combination, but in 2015, “golden delicious” and “red table 2 the interaction effects of year × scion × rootstock on some vegetative traits of the apple scion cultivars year seed stock scion shoot length mm shoot thickness mm internodes number internodes length mm rootstock diameter mm graft diameter mm trunk diameter mm shoot number crotch angle lenticels number 2014 azayesh 'golden delicious' 179.24 b-g 5.70 ef 21.06 ab 14.66 b-e 45 a 43 a 30.2 a 7.75 a 61.67 cd 16.25 ij 'red delicious' 208.22 a 6.43 de 19.26 a-d 17.20 bc 43.6 a 47.2 a 38.5 a 10.2 a 68.33 bcd 24.23 cde 'granny smith' 177.39 b-h 6.87 bcd 20.17 abc 17.19 bc 57.5 a 58.25 a 52.25 a 17.66 a 73.75 a-d 33.12 a 'braeburn' 175.25 b-h 5.10 ghi 18.08 b-e 15.54 b-e 49.4 a 56 a 40.5 a 11.16 a 83.33 a-d 14.53 j 'golab-e kohanz' 168.75 b-h 6.80 cd 19.40 a-d 14.66 cde 47.33 a 51.66 a 50.83 a 8.5 a 87.50 ab 10.22 k 'gala' 178.45 b-g 6.86 bcd 18.16 b-e 17.83 b 62.83 a 60.83 a 47.66 a 10.5 a 85.00 abc 18.16 e-i morabbaei 'golden delicious' 193.63 abc 6.18 de 22.86 a 16.83 bcd 47 a 43.66 a 35.83 a 14.5 a 82.17 a-d 21.21 c-f 'red delicious' 195.06 ab 8.00 a 19.46 a-d 20.33 a 56 a 41 a 51 a 17.16 a 95.00 a 21.94 c-g 'granny smith' 174.61 b-h 6.36 de 20.20 abc 13.73 b-e 40.25 a 49.6 a 41.8 a 13 a 90.00 ab 21.40 c-h 'braeburn' 159.70 e-i 7.63 ab 19.58 a-d 15.75 b-e 43.4 a 49.6 a 43.4 a 9.33 a 60.00 d 23.80 bcd 'golab-e kohanz' 165.74 c-h 7.41 abc 19.56 a-d 15.30 b-e 44.6 a 54.5 a 43.16 a 6.33 a 82.50 a-d 29.27 ab 'gala' 171.44 b-h 7.86 ab 15.57 d-g 15.03 b-e 41.2 a 44.5 a 36 a 8.00 a 73.33 a-d 15.50 hij 2015 azayesh 'golden delicious' 176.64 b-h 3.94 k 13.39 fgh 13.24 ef 69.33 a 70.33 a 58.66 a 6.33 a 68.33 bcd 14.18 ij 'red delicious' 149.59 hi 4.03 k 9.52 hi 11.36 fg 51 a 62 a 52.33 a 10.16 a 70.00 bcd 22.65 c-g 'granny smith' 164.23 d-h 4.47 h-k 14.17 efg 14.89 b-e 63.2 a 70.2 a 58.8 a 10.66 a 69.00 bcd 19.34 c-h 'braeburn' 181.71 a-f 4.31 jk 11.32 ghi 9.97 g 62.16 a 69.16 a 61 a 9.4 a 59.00 d 16.30 hij 'golab-e kohanz' 187.42 a-e 4.30 ijk 14.11 efg 10.23 g 60.33 a 68.66 a 58 a 7.5 a 76.67 a-d 18.22 d-i 'gala' 185.61 a-e 4.63 g-j 8.25i 10.36 g 68.33 a 68.5 a 57.5 a 9.16 a 83.33 a-d 17.28 ghi morabbaei 'golden delicious' 143.23i 6.10 de 9.11i 14.67 cde 53 a 58.33 a 45.83 a 10.00 a 90.00 ab 21.08 c-g 'red delicious' 191.06 a-d 4.33 h-k 16.10 c-f 21.24 a 60 a 62 a 53 a 17.16 a 95.00 a 22.56 bc 'granny smith' 116.40 j 4.63 g-j 14.70 efg 13.79 ef 44.6 a 57.5 a 44.66 a 7.33 a 85.00 abc 17.26 hij 'braeburn' 151.71 ghi 4.68 g-j 11.31 ghi 16.79 bc 51.66 a 64 a 50 a 6.66 a 76.67 a-d 23.19 bc 'golab-e kohanz' 156.16 f-i 4.97 gh 14.43 efg 15.61 b-e 58.25 a 65.75 a 53 a 7.83 a 73.33 a-d 19.94 c-h 'gala' 150.88 ghi 5.24 fg 11.82 f-i 11.50 fg 44 a 57.66 a 44 a 9.33 a 83.33 a-d 17.50 f-i in each column, means with similar letters are not significantly different at the 1% probably level using duncan multiple range test. hajnajari seed rootstocks improvement in apple 81 delicious” showed significant differences on “morabbaei” and “azayesh” seed sources (table 2). internode length internode length was affected by year, scion cultivar and rootstock main effects and their interactions (table 1). the longest internode was found in “red delicious” on “morabbaei” rootstocks in both years and the lowest value was registered in combination of “braeburn”-“azayesh”, in 2015 (table 2). in the first year, among all the scions combined with “azayesh” rootstocks, there was significant difference between “golab-e kohanz” and “gala” and among the grafted cultivars on “morabbaei” sole “red delicious” showed significant difference. “golab-e kohanz” is the most vigorous iranian cultivar early and “gala” with moderate tree vigor. tree vigor, denoted as potential of vegetative growth, is measured based on height and spread of adult trees self-rooted, or relative to reference cultivars on the same rootstock in apple descriptors (watkins and smith, 1997). “golden delicious” and “red delicious” were used as reference cultivars to assess rootstocks efficiency for tree vigor, the trait that encompasses other components as shoot length, shoot thickness, internode number, internode length, shoot number, and crotch angel by which the tree overall size, shape and architecture will be reflected. fazio (2014), assumed that rootstock performance is highly correlated with the genetic potential to impart positive architectural properties to the scion like vigor control. considering the ruling genetic purity carried by “morabbaei” seed rootstocks, it imparted uniformity in the size of internode length in all the scion cultivars. in the second year, “golden delicious” and “granny smith” showed significant differences against other scions grafted on “azayash”, while “red delicious” and “gala” had significant difference relative to the other scions on “morabbaei” (table 2). such differences are attributed to the different genetic vigor of scion materials. rootstock, graft point and scion diameter according to the table 1, the effect of year was significant for rootstock, graft union and scion trunk diameters. overall, rootstock diameter, graft and trunk were higher in the second year. rootstock and graft point were affected equally by rootstock factor. interaction of rootstock × scion was significant for rootstock (table 1). interaction effect of scion × rootstock on rootstock demonstrated that combination of “granny smith” on “azayesh” and “morabbaei” rootstocks endured modifications on the trait, for example 60.66 mm to 42.66 mm, respectively. there was only significant difference between “red delicious” (47.30 mm) and “gala” (65.58 mm) for rootstock limiting factor to “azayesh” rootstock. similar comparison within seed lots of “morabbaei” revealed significant difference in combination with “granny smith” (42.66 mm) and “gala” (60.22 mm). surprisingly, both seed rootstocks influenced “gala” increasing rootstock to the highest values of 65.58 mm and 60.22 mm (table 3). moreover, significant difference was observed between the combinations of “granny smith”-“azayesh” and “granny smith”“morabbaei” for the effect of rootstock type on each scion for rootstock, while no difference was observed between seed rootstocks for other combinations (table 3). this result is assumed positive for uniform apple tree saplings production on the improved seed rootstock. shoot number shoot number was not affected by rootstock type, while the effects of year, scion and rootstock × scion were significant (table 1). in 2014, more lateral branches were grown than 2015. among scionsstocks combinations, “morabbaei” seed stock showed both max. and min. shoot number, on “red delicious” (17.16) and on “golab-e kohanz” (7.08), respectively (table 3). no significant difference was found among the six scion cultivars for shoot number on “azayesh”, whilst “morabbaei” rootstock induced significant differences of shoot number on “golden delicious” and “red delicious”. crotch angle in this study crotch angle was affected by year, rootstock scion rootstock diameter (mm) shoot number 'azayesh' 'golden delicious' 55.43 abc 7.14 c 'red delicious' 47.30 bc 10.18 bc 'granny smith' 60.66 ab 13.00 bc 'braeburn' 56.36 abc 10.36 bc 'golab-e kohanz' 53.83 abc 8.00 bc 'gala' 65.58 a 9.83 bc 'morabbaei' 'golden delicious' 50.27 abc 12.25 ab 'red delicious' 58.00 abc 17.16 a 'granny smith' 42.66 c 10.16 bc 'braeburn' 46.50 bc 8.00 bc 'golab-e kohanz' 50.67 abc 7.08 c 'gala' 60.22 ab 9.25 bc table 3 the interaction effect of scion × rootstock on rootstock diameter and shoot number in each column, means with similar letters are not significantly different at the 1% probably level using duncan multiple range test. 82 adv. hort. sci., 2019 33(1): 77-85 rootstock and scion interaction (table 1). the combination of “red delicious”-“morabbaei” showed the widest value of crotch angle probably as a result of heavy cropping, while light bearing trees in the combination of “braeburn”-“azayesh” had the lowest crotch angle rates, in both years. in general, the scion cultivars grafted on “morabbaei” had wider crotch angle than on “azayash” progeny (table 2). except partial genetic role of the scions regarding growth habit, this effect might be result of higher tree vigor control by self-compatible parent “morabbaei” rootstocks and subsequent decrement of woody parts in favor of flower bud initiation and fruit set (wertheim and webster, 2005). clearly, bearing shoots under higher fruit weight were bent downward increasing crotch angle. lenticels number lenticels number was affected by year × rootstock × scion interaction (table 1). the max and min values of lenticels number were found in “granny smith” and “golab-e kohanz” both on half-sib seed stocks of “azayesh”, in the first year. it can be deduced that the increased leaf lenticels number of the “golab-e kohanz” as the most vigorous scion is a normal physiological trend appeared trough interaction of the grafting with as much vigorous seed stock “azayesh” in the year. more vegetative growth induced by vigorous seeds demands higher respiration rate for which a higher lenticels number equilibrate the defined biological status. in both years, significant difference was observed among the fruit scions on “azayesh” and “morabbaei”. this confirms significant effect of rootstock seed masses for lenticels number. in 2014, comparisons of rootstocks for each cultivar showed that “golden delicious”, “granny smith”, “braeburn” and “golab-e kohanz” were affected by seed stocks for lenticels number. also in 2015, “golden delicious” and “braeburn” were affected for this trait (table 2). the comparison between two seed sources put in evidence of high dominant uniformity of lenticels number trait for scions grafted on the seed stocks and originated from self-compatible “morabbaei”. 4. discussion and conclusions according to the results, shoot length, shoot thickness and internode length as 3 main components of tree vigor showed significant difference in all the six cultivars grafted on two seed masses. dominant homozygosity carried out by self-compatible “morabbaei” and relative progeny overcame heterosis effects which induced by “azayesh” seeds and tree vigor interaction. our results regarding control of tree vigor “morabbaei” seed stocks demonstrated that it could reduce shoot length; infact this trait decreased in all the cultivars except for “golden delicious”, in 2014. this cultivar showed the same opposite rhythm for all growth traits like crotch angle, internode thickness, internode number, roostock, and scion diameter on “morabbaei” seed stock. also, other four scion cvs. decreased shoot length value and were affected by dwarfing seeds in the range of 13 to 3 mm for “red delicious” and “golab-e kohanz”, 7 and 3 mm for “gala” and “granny smith”, respectively. these results confirm the reciprocal influences of two genomes, scion and seed rootstock within the same genus, each one specialized differently for which were selected in the selection procedure. there is no clear reason for reaction of “golden delicious” for measured trait when compared with five scion cultivars grafted on the same seed pure line progeny. this may not be related to the distant geographical origin of two genomes, iran-usa. with studying scion tree vigor was suggested that j-te-e as an acceptable rootstock for “rubin” among 14 rootstocks (kosina, 2010). the scion cultivars on “morabbaei” seeds showed higher shoot thickness than the same scions on “azayesh” seed progeny. increased shoot thickness as the diametric growth of annual shoots on “morabbaei” seeds indicated accumulation of carbohydrates for productive functions. however the final canopy size is determined genetically by scion-rootstock interaction. higher dwarfing efficiency raised in “morabbaei” than “azayesh” progeny is in agreement with tworkoski and miller (2007 a), who indicated that malling rootstocks have different size-controlling effects on different apple scions. the lowest interned number (8.25) was found in “gala”-“azayesh” combination, in 2014, while the highest (22.86) was found in combination of “golden delicious”-“azayesh”, in 2015. the biennial comparisons evidenced that the higher mean of internode number was induced by “morabbaei” in both years, 22.86 and 9.11, related to “azayesh” equal to 21.06 and 8.25. these finding confirmed stronger dwarfing effects of the self-compatible parent. dwarfing mechanism acted through an increase of internode length and contrarily shortening internode length. highest value of interned length was found in “red hajnajari seed rootstocks improvement in apple 83 delicious”-“morabbaei” combination for 2014 and 2015 and the lowest in “braeburn” on “azayesh” in 2015 (table 2). others reported that wild peach rootstocks influenced shoot growth and leaf area for the scions only during the first year (sharma et al., 2004). in general, in the first year, the internode number and internode length were higher than the second year. it can be attributed also to the difference between orchard management during two years. interesting results were obtained in 2015 comparing effects of two seed stocks on major part of the scions. dwarfing property of “morabbaei” seed stocks overcame in the next season which caused shoot length shortening in appropriate growth rhythm and shoot thickness increasing and internode number and decreasing rootstock and scion diameter. a part of less expected behavior of “red delicious” on “morabbeai” stock which shoot length and internode length was both increased, but other traits like shoot thickness, internode numbe , and scion diameter followed normal dwarfing trend. all the other growth traits assessed in the rest of four scion cultivars were highly affected by dwarfing effect of “morabbeai” seed progeny, without exception (table 2). weibel et al. (2003) indicated that differences in peach shoot length were related primarily to the internode length rather than the node number, whereas seleznyova et al. (2003) attributed the differences in apple branch size to the reduction in neoformed internode length and node number. mean of internode length depended on node number. it means that shorter internodes are caused by lower node number (costes and garcia-villanueva, 2007). measured diametric values of graft union and scions on self-incompatible “azayesh” seed stocks showed higher than those on self-compatible “morabbaei” f1 seed progeny. scion-rootstock discontinuity may lead to burrknot formation and high diametric difference between rootstock and trunk that may be a site where xylem may be affected by modified cell diameters or cell disorganization between different scions and rootstocks (atkinson et al., 2003; tworkoski and fazio, 2011). such morphological differences may in turn affect hydraulic conductivity, root hormones production, or nutrient transport and explain the differences in growth. tworkoski and fazio (2015) reported that the scion cultivars “gala”, “sm.9”, and “sm.27” reduced rootstock of “g.41” and “g.11” compared with “fuji” scion. in the second year, the most of fruit scions on “morabbaei” seed had smaller diameter than “azayesh” without significant difference, except for “red delicious” (table 2). this implies that rootstocks may affect scion. so, shoot number was not affected by rootstock type, whereas the effect of rootstock×scion, scion and year resulted significant. “red delicious” and vigorous “golab-e kohanz” on “morabbaei” seed rootstock had the highest and lowest brn, respectively (table 3). “morabbaei” f1 seeds induced more new shoots than “azayesh” seed stocks. generally, the mandatory cut back of leader in spindle pruning method activates the latent buds, forming new lateral shoots due to removal of apical dominance. this practice however was performed equally on the leader of all combinations. thus, it may be concluded that while, on one side, homozygosis affects canopy size and decreases shoot length, on the other side, the reserved carbohydrates might be used in activation of latent buds in absence of apical dominance. obviously, winter pruning obliges crossing out of adventitious shoots for flower initiation purposes. across all scions, the dwarfing m.9, and seedling rootstock induced the lowest and the highest tree height and scion diameter, respectively. significant interactions indicated that effects of sizecontrolling rootstock on components of shoot growth vary with apple tree growth habit (tworkoski and miller, 2007 a). in this study all the six scions carried the spread habit, for which crotch angle was investigated. meantime, it might be attributed to the hormonal factor due to the genetic factor derived from seed stocks, scions and relative interaction. seedling rootstock might affect growth habits by increasing the auxin/cytokinin ratio. changing in hormonal ratio might be a factor regulating the development of growth habit in apple scions, and rootstock might modify the hormone concentrations in shoot tips (tworkoski and miller, 2007 b). as prerequisite, crotch angle is mentioned as a stable trait and used in cultivar differentiation and cultivar/progeny evaluation (watkins and smith, 1997), and less affected by external factors which is agreed with our results of crotch angle among five scions on two diffrent stocks, except for “red delicious”. in general, the grafted scions on “morabbaei” seeds showed wider crotch angle than “azayash” progeny, probably for higher fruit weight as a result of higher set. the significant differences of crotch angle between “golden delicious” and “granny smith” on “azayesh” seed rootstocks, in the first year, can be attributed to the stability of this trait, whereas lack of differences in the second year would be justified by pruning effect. it is however very important that the single cultivars on “azayesh” didn’t show significant differences in adv. hort. sci., 2019 33(1): 77-85 84 terms of crotch angle with the same cultivar on “morabbaei”, except for “red delicious” (table 2). however, endogenous growth regulators mainly auxins and cytokinins play a major role in the control of lateral bud development (sachs and thimann, 1967). there are numerous reports of growth-regulating chemicals affecting branch crotch angle (verner, 1938; williams and billingsley, 1970; elfving and forshey, 1977). warner (1991) reported that rootstock affects primary scaffold branch crotch angle of apple trees, which is consistent with our results. also lenticels number of annual shoots unit, defined as stable trait like crotch angle, was affected by rootstocks and year, and interaction of rootstock × scion × year. comparisons of rootstocks for each cultivar, in 2014, showed that “golden delicious”, “granny smith”, “braeburn” and “golab-e kohanz” were affected by seed stocks for lenticels number, and in 2015, “golden delicious” and “braeburn” were affected for this trait (table 2). however, the use of lenticels number may be successfully applied for cultivar/hybrid evaluations when more cultivars are grafted on similar rootstocks. the self-compatible parents are able to supply seeds characterization with high genetic purity. furthermore, the improved seed rootstocks seem to be the preferred solution to combat genetic variability, suitable for heavy and alkaline soils and inappropriate slopes. conventional breeding remains the main method to release new clonal or seed rootstock (wertheim and webster, 2005). seed rootstock improvement program was started to impede use of a mixture of seeds with unknown origin and with high genetic variability and evolved into adequate genetic purity, which is the basic element for standard apple tree production. references atkinson c.j., else m.a., taylor l., dover c.j., 2003 root and stem hydraulic conductivity as determinants of growth potential in grafted trees of apple (malus pumila mill.). j. exp. bot., 54: 1221-1229. brown s.k., maloney k.e., 2005 malus × domestica apple, pp. 475-511. in: litz r.e. 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95: 649-651 żurawicz e., lewandowski m., 2014 controlled freezing as a low-temperature tolerance test for apple rootstocks. acta horticulturae, 1058: 451-456. impaginato 139 adv. hort. sci., 2019 33(1): 139-143 doi: 10.13128/ahs-22813 effects of packing materials and ethanol concentrations on removal of astringency of ‘rojo brillante’ at room temperature z. hejazi 1 (*), m.i. sadat 1, b.k. karimi 2, m. fawad 3, a. muneer 3 1 department of horticulture, faculty of agriculture, nangarhar university, afghanistan. 2 directorate of agriculture, irrigation and livestock (dail), jalalabad, afghanistan. 3 perennial horticulture development and research center (phdc), jalalabad, afghanistan. key words: calcium carbide, de-astringency, diospyros kaki, ethanol dipping. abstract: most of the persimmons grown in afghanistan are astringent, hence their fruits require de-astringency treatments to become edible at harvest time. the co2 with or without ethanol treatment provides an optimal method for the rapid removal of the persimmon astringency. however, this method is sophisticated and currently not feasible for most of the farmers. the present study is therefore carried out to assess a simple and suitable technique for deastringency of the persimmons at farmer level. the fruits of ‘rojo brillante’, a pollination variant astringent (pva) spanish cultivar, were quickly dipped in 0, 10, 20, and 40% ethanol. the treated fruits were packed and sealed in the paper cartons, polyethylene bags or left open at the room temperature. the firmness, total soluble solids and astringency level of fruits were measured after every three days for nine days. the astringency was removed when fruits treated with 20% or 40% ethanol and packed in the polyethylene bags for nine days. the total soluble solids content of the fruits treated with ethanol was lower in any of the used concentrations than the untreated fruits. the commercial firmness of the fruits (1.5-2.5 kg) obtained by packing the fruits in the polyethylene bags or treated with 20% ethanol concentration. 1. introduction persimmon (diospyros kaki thunb.) is one of the fruits cultivated in the east of afghanistan. the subtropical climate of this region provides a favorable environment for the persimmon production. on the other hand, the naturally grown wild population of diospyros lotus can be seen in the mountainous area, lies at an altitude between 1500 and 1900 m asl (samadi et al., 2009). in the frame of perennial horticulture development project (phdp), an (*) corresponding author: zia.hejazi@yahoo.com citation: hejazi z., sadat m.i., karimi b.k., fawad m., muneer a., 2019 effects of packing materials and ethanol concentrations on removal of astringency of ‘rojo brillante’ at room temperature. adv. hort. sci., 33(1): 139-143 copyright: © 2019 hejazi z., sadat m.i., karimi b.k., fawad m., muneer a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 april 2018 accepted for publication 5 october 2018 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 139-143 140 ex situ germplasm of 24 persimmon accessions was established in farme-jaded, jalalabad (masini and giordani, 2016). currently, this center preserved 22 different varieties, of which ten were donated by university of florence, italy (afghanhorticulture.org; samadi et al., 2009). these cultivars contained both astringent and non-astringent types of the persimmons. because of the good adaptation with the climate and affinity with a more common rootstock of diospyros lotus, the astringent type is widely spread in the country. non-astringent persimmons are normally edible at harvest, whereas fruit of the astringent cultivars as ‘rojo brillante’ (pollination variant astringent pva) can be consumed after astringency is removed. natural loss of the astringency of the latter type is achieved by leaving the fruits on the tree until they are overripe, with soft flesh (taira, 1995). however, it is not desirable for the fruit growers, because it takes a long time, attracts birds eating the fruits and increases protection expenses; furthermore, the handling and marketing of soft fruits is very time consuming and expensive. due to these limitations, the ripening of the astringent persimmons by the artificial method is required. in afghanistan, the persimmon producers used calcium carbide (cac2) to hasten ripening process of the fruits (samadi et al., 2009). this agent is also commonly used in ripening of mangoes and bananas in several other developing countries (asif, 2012). although cac2 is widely available, easy to use and cheap, it is associated with health hazards. recent findings have proved that calcium carbide is carcinogenic (per et al., 2007; asif, 2012). moreover, fruits ripened with cac2 have negative quality attributes. they are often soft and less tasty, and have a shorter storage life. therefore, secure and simple methods that can be used by farmers to remove astringency of persimmons are needed. today, the world top persimmon producers make use of either co2 or ethanol (etoh) for the removal of the persimmon astringency (kato, 1984, 1990; eaks, 1967). the use of co2 is safe and induces rapid ripening, but maintaining a strict control condition of its application wouldn’t be easy for the persimmon growers, particularly in remote countryside orchards. de-astringency of the ‘rojo brillante’ persimmons by co2 with or without ethanol is already established (salvador et al., 2007; novillo et al., 2015), but the removal of astringency using alcohol without co2 has not been explored. the aim of the present study was to assess the use of etoh without co2 in different packaging material. 2. materials and methods the fruits of ‘rojo brillante’ from two clones of 5year-old trees grown in jalalabad perennial horticulture development and research center, formerly named phdp, were harvested at the yelloworange color on october 2017. after sorting, fruits (n=180) were mixed for uniformity of size and used for the experiments. the fruits were quickly immersed in 0 (distilled water), 10, 20, and 40% aqueous ethanol solutions and then air dried. sixty fruits were packed and sealed in the paper catons (2 kg net weight), another 60 fruits were sealed in transparent polyethylene bags (0.018 mm thickness), and the remaining fruits were left open at the ambient temperature. the fruits within each group were further sub-divided into five per pack. subsequently, all the packaged fruits left at the room temperature. room temperature measured at 15 min interval using a data logger with thermo-hygro sensor (rc4hc, elitech, jiangsu jingchuang electronics co., ltd, china). the data were recorded after every three days for the fruit firmness, total soluble solids (tss) and astringency levels. fruit firmness was determined for each fruit at two pared equatorial positions after peeled, using a hand-held penetrometer equipped with an 8 mm tip. to measure the astringency and tss, fruits were cut into two halves. one half was used to estimate astringency and the remaining halve was used to express the juice for the brix with a digital atago refractometer. the astringency was estimated by tannin print method (eaks, 1967), where the freshly cut surface of the fruit soaked in 5% ferric chloride and then the black color scored from 0 (non-astringent) to 5 (very astringent). the data were subjected to repeated measures anova of general linear model of spss (16.0) statistical software and mean separation was performed using tukey’s (hsd) method at p≤ 0.05. 3. results and discussion the room naturally kept almost a consistent temperature that averaged 20.9°c during the experiments. this degree is recommended if faster deastringency of the persimmons is aimed with co2 or co2-ethanol treatment (besada et al., 2010; novillo hejazi et al. astringency removal at room temperature in ‘rojo brillante’ 141 et al., 2015). the results of the anova showed that firmness of the ‘rojo brillante’ fruits were significantly affected by packing and ethanol concentrations; however, the interaction between packing and ethanol was found insignificant (fig. 1). the fruits packed and sealed nine days in the polyethylene bags were softer (2.5 kg) than of paper cartons (3.0 kg) and unpacked persimmons (2.9 kg). likewise, fruits treated with 20% ethanol was significantly softer (2.5 kg) than the fruits treated with distilled water (3.1 kg). kato (1990) stated that high-quality persimmon fruit has a firmness of 1.5 to 2.5 kg; fruit firmer than 2.5 kg is too hard and fruit with a firmness <1.5 kg are too soft to tolerate the physical handling. hardness of the persimmon fruits is a concern both for the consumers and fruit shelf life, but people in our region are not yet familiar with eating of crispy persimmons. the content of soluble solids was significantly affected by the ethanol concentrations, while the effects of packing and the interaction were not considerable (fig. 2). the tss of the fruits immersed in 20% ethanol solution were significantly lower (17.9) than of distilled water (19.5). more in a similar pattern, ethylene treatment decreased total soluble solids of the astringent type of ‘hiratanenashi,’ but increased in fruits of ‘fuyu,’ which is a non-astringent persimmon (takata, 1975). except for the ethanol concentrations, the astringency of the ‘rojo brillante’ fruits was significantly influenced by packing and the interaction between packing and ethanol concentration at p≤0.002. despite that after six days of the treatment, recurrence of the astringency (edagi et al., 2009; sestari et al., 2009) was evident in some treatments, fruits treated with 20% or 40% ethanol concentrations and placed nine days in the polyethylene bags remained slightly to non-astringent (fig. 3 and 4). these findings are in accordance with kato (1987) who attained de-astringency of the persimmons by ethanol vapor method. in our study, packing alone was not effective to remove the astringency, because the fruits treated with distilled water and placed nine days in the paper cartons or polyethylene bags were similar as those exposed to the room ambient. although the anaerobic condition of the package or chamber promoted de-astringency of the persimmons (eaks, 1967; novillo et al., 2015; monteiro et al., 2017), no effect of the polyethylene bags or paper cartons fig. 1 changes in the firmness of ‘rojo brillante’ fruit treated with 0, 10, 20, and 40% ethanol and then packed and sealed in paper cartons, polyethylene bags or left open during three days at the room ambient. bars indicate se (n= 5). fig. 2 changes in total soluble solids of ‘rojo brillante’ fruit treated with 0, 10, 20, and 40% ethanol and then packed and sealed in paper cartons, polyethylene bags or left open during three days at the room ambient. bars indicate se (n= 5). adv. hort. sci., 2019 33(1): 139-143 142 would be associated with materials permeability. monteiro et al. (2017) reported that among three different polyethylene packages, polyethylenepolyamide succeeded to remove ‘giombo’ astringency faster, whereas low-density polyethylene and polypropylene didn’t work because of their greater permeability to gases. moreover, the result of deastringency of the closed chamber with no-addition was alike with the chamber added ethyl alcohol (eaks, 1967). astringency will be removed when soluble tannins are becoming insoluble because of the polymerization with acetaldehyde in the fruit flesh during the treatments (taira, 1995). since acetaldehyde is known to be generated in situ, it can be triggered by exogenous ethanol application (novillo et al., 2015). kato (1984) revealed that during de-astringency of the persimmons, acetaldehyde increased with the increase of ethanol concentration. he further explained that alcohol treatment caused a rise in ethylene evolution and induced carbon dioxide. thus, the effectiveness of the polyethylene bags to astringency removal in higher doses (20%, 40%) of the ethanol solutions of this study might be referred to ethylene and through co2 production, which probably preserved better by the polyethylene bags than of paper cartons. 4. conclusions this study suggests that it is feasible to remove astringency of the ‘rojo brillante’ persimmons by packing them nine days in the polyethylene bags after immersed in 20% ethanol solution. however, to assure complete sweetness, time might be extended for two or three more days, depending on fruit maturity, temperature, and package permeability. future researchers may test polyethylene bags in different thickness along with some modifications to improve this protocol for a more rapid loss of the persimmon astringency. acknowledgements we would like to appreciate the effort of the european commission (ec), in the particular university of florence for granting the elite cultivars of persimmons to the national fruit collection of jalalabad, afghanistan. references asif m., 2012 physico-chemical properties and toxic effect of fruit-ripening agent calcium carbide. ann. trop. med. public. health, 5: 150-156. besada c., arnal l., salvador a., martĺnez-jávega j.m., 2010 optimization of the duration of deastrinfig. 3 changes in astringency level of ‘rojo brillante’ fruit treated with 0, 10, 20, and 40% ethanol and then packed and sealed in paper cartons, polyethylene bags or left open during three days at the room ambient. bars indicate se (n= 5). fig. 4 tannin prints of the freshly cut surface of ‘rojo brillante’ fruits treated with 20% ethanol solution, then left open at the room ambient (a), or packed and sealed in paper cartons (b), or polyethylene bags for nine days (c). hejazi et al. astringency removal at room temperature in ‘rojo brillante’ 143 gency treatment depending on persimmon maturity. acta horticulturae, 858: 69-74. eaks i.l., 1967 ripening and astringency removal in persimmon fruits. proc. amer. soc. hort. sci., 91: 868875. edagi f.k., sestari i., terra f.a.m., chiou d.g., kluge r.a., antoniolli l.r., 2009 effect of ripening stage on astringency removal of ‘rama forte’ persimmon. acta horticulturae, 833: 269-274. kato k., 1984 astringency removal and ripening as related to ethanol concentration during the de-astringency by ethanol in persimmon. j. jpn. soc. hort. sci., 53: 278-289. kato k., 1987 large-scale trials for the short term deastringency in persimmon fruits by ethanol. j. jpn. soc. hort. sci., 56: 92-100. kato k., 1990 astringency removal and ripening in persimmons treated with ethanol and ethylene. hortsci., 25(2): 205-207. masini g., giordani e., 2016 from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan. adv. hort. sci., 30(4): 197-205 monteiro m.f., aguila j.s.d., pessoa c.d.o., kluge r.a., 2017 vacuum packaging is efficient to remove astringency and to maintain the firmness of ‘giombo’ persimmon. rev. bras. frutic., 39: e-358. novillo p., gil r., besada c., salvador a., 2015 astringency removal of ‘rojo brillante’ persimmon by combining co2 and ethanol application. acta horticulturae, 1079: 599-604. per h., kurtoğlu s., yağmur f., gümüş h., kumandaş s., poyrazoğlu m.h., 2007 calcium carbide poisoning via food in childhood. j. emerg. med., 32(2): 179180. salvador a., arnal l., besada c., larrea v., quiles a., pérez-munuera i., 2007 physiological and structural changes during ripening and deastringency treatment of persimmon fruit cv. ‘rojo brillante’. postharvest biol. technol., 46: 181-188. samadi g.r., miakhel i.m., abdiani s.a., 2009 status of persimmon cultivation in afghanistan. acta horticulturae, 833: 31-34. sestari i., edagi f.k., terra f.a.m., chiou d.g., kluge r.a., antoniolli l.r., 2009 influence of ripening stage and astringency removal on quality of cold stored ‘rome forte’ persimmon. acta horticulturae, 833: 263-268. taira s., 1995 astringency in persimmon, pp. 97-110. in: linskens h.f., and j.f. jackson (eds.) fruit analysis. springer, hannover, germany, pp. 160. takata m., 1975 effects of ethylene on the respiration and ripening of fruits. j. jpn. soc. hort. sci., 44(1):8288. impaginato 273 adv. hort. sci., 2018 32(2): 273-280 doi: 10.13128/ahs-20654 direct and indirect in vitro plant regeneration of two commercial cultivars of perennial ryegrass s. esmaeili, h. salehi (*), m. khosh-khui department of horticultural science, school of agriculture, shiraz university, shiraz, iran. key words: abscisic acid (aba), ʻgrasslandʼ, lolium perenne l., maltose, meristem tip, ʻnumanʼ. abstract: experiments were conducted on direct and indirect regeneration from the meristem tip and mature caryopsis explants of lolium perenne l. ʻnumanʼ and ʻgrasslandʼ. de-husked caryopses were cultured both intact and longitudinally sliced on ms media supplemented with 2,4-d alone, and in combinations with ba. the highest percentage of callus induction obtained from intact-sliced caryopses were 71 and 87% for ʻgrasslandʼ on ms basal medium supplemented with 6 mg l-1 2,4-d+ 0.02 mg l-1 ba, and 5 mg l-1 2,4-d. while, for ʻnumanʼ, the highest callus induction was achieved by the same explants as 55% and 72% with 5 mg l-1 2,4-d + 0.02 mg l-1 ba, and 4 mg l-1 2,4-d + 0.02 mg l-1 ba, respectively. the best regeneration medium for ʻgrasslandʼ was ms medium supplemented with 10 g l-1 maltose and 2 mg l-1 aba. in a separate experiment, meristem tip cultures were incubated on two type combination of plants growth regulators along with control treatment. the best regeneration rate was obtained in both cultivars on ms medium supplemented with 0.1 mg l-1 2,4-d + 0.5 mg l-1 kin. plantlets with well-developed roots were transferred to greenhouse condition. four weeks later, all acclimatized plants were survived. 1. introduction turfgrasses control the soil erosion, carpet lawns, cover athletic fields, and beautify the environment. perennial ryegrass (lolium perenne l.) is one of the most important turfgrass species for sports fields, golf course fairways, as well as urban landscapes in the areas with temperate climate. that is the fast establishing component of lawn seed mixtures comprising slow growing species such as kentucky bluegrass (poa pratensis l.), and is used for winter overseeding on warm-season turfgrasses as well. however, the poor ability to survive in drought regions have limited its distribution. therefore, improving drought tolerance is an important goal in perennial ryegrass breeding programs via classic or modern (genetic transformation) techniques. callus production with good quality and efficient plant regeneration of various cultivars of perennial ryegrass is a requisite to the grass transformation techniques. the formation of embryo(*) corresponding author: hsalehi@shirazu.ac.ir citation: esmaeili s., salehi h., khosh-khui m., 2018 direct and indirect in vitro plant regeneration of two commercial cultivars of perennial ryegrass. adv. hort. sci., 32(2): 273-280 copyright: © 2018 esmaeili s., salehi h., khosh-khui m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 october 2017 accepted for publication 20 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 273-280 274 genic calli and plant regeneration were affected by several factors including genotype, explant tissue, culture medium and its supplements (bhaskaran and smith, 1990). many studies have been done on improving tissue culture of perennial ryegrass. in perennial ryegrass, various explants including immature inflorescence, mature seed, leaf base, meristem tip and axillary bud have been used for callus induction, and direct and indirect plant regeneration (dale, 1977; dale and dalton, 1983; torello and symington, 1984; creemers-molenaar et al., 1989; altpeter et al., 2000; bradley et al., 2001; can et al., 2004; salehi and khosh-khui, 2005; newell and gray, 2005; altpeter, 2006). among these explants, mature seeds are most commonly used for callus induction and related subsequent studies. perennial ryegrass is an outcrossing, wind-pollinated and highly self-incompatible species with abundant genetic variation in each cultivar (mohr et al., 1998; smith et al., 2001; wang et al., 2003; bolaric et al., 2005). replacing the seed explants with other vegetative explants would omit this variation. in vitro meristem tip culture is an efficient method for obtaining virus free and identical to mother plant materials. in addition, meristem tip culture is an appropriate method for genetic transformation. in this method, the exposed meristem could directly be used for transformation; then after, will multiplied in vitro, or can form multiple shoot clumps that can be bombarded with the gene(s) of interest, and then each meristem will regenerated the mature plants. therefore, development of an optimized and short time tissue culture protocol plays a critical role in successfully transformation of the grass species. to the best of our knowledge, there is no report on meristem tip culture of ʻgrasslandʼ and ʻnumanʼ cultivars of perennial ryegrass. in addition to, it has been shown stimulating effect of aba on somatic embryogenesis of some grass species calli such as wheat (qureshi et al., 1989), maize (close and ludeman, 1987), kentucky bluegrass (van ark et al., 1991) and bermudagrass (li and qu, 2002). the objectives of the present investigation were comparison of the callus induction on intact and longitudinally sliced caryopses, and evaluation of the effects of aba and maltose combinations on plant regeneration of two lolium sp. cultivars to optimize their tissue culture condition. furthermore, a separate experiment was conducted to find a direct regeneration and fast tissue culture method for these cultivars. 2. materials and methods seed surface sterilization seeds of the two commercial cultivars of perennial ryegrass, ʻgrasslandʼ and ʻnumanʼ, were purchased from iran bazr seed company (karaj, iran). seeds were socked in 50% h2so4 for 15-20 min to remove the husks (salehi and khosh-khui, 2003). the de-husked seeds were surface sterilized with 70% ethanol for 1 min and immediately were treated with a house-hold bleach (5.25% available chlorine) for 20 min, then rinsed 6 times with sterile distilled water. callus induction the surface sterilized seeds were used both intact and longitudinally sliced. for callus induction, ms basal medium (murashige and skoog, 1962) plus 3% sucrose supplemented with 4, 5, 6, 7, 8, 9 and 10 mg l-1 2,4-d alone and with 0.02 mg l-1 ba. intact and longitudinally sliced seeds were cultured on 80 mm petri dishes containing callus induction media. after 4 weeks, the calli were separated, transferred to the same fresh media and maintained for 3 weeks. then, the calli were transferred to optimum ms media, supplemented with 4 mg l-1 2,4-d + 0.02 mg l-1 ba and 5 mg l-1 for ʻnumanʼ and ʻgrasslandʼ, respectively. the number of seeds that induced callus was recorded according to seed viability. the callus induction rate was calculated as the number of caryopses which induced callus over the total number of explants plated × 100. all cultures were kept at 24±1°c in dark. plant regeneration to determine the best regeneration media for ʻnumanʼ and ʻgrassland ,̓ the embryogenic calli were transferred to ms media containing 3% sucrose and 0, 10, 15 and 20 g l-1 maltose with 0, 1.7, 2 and 2.3 mg l-1 aba. filter-sterilized aba was added after autoclaving when the media temperature dropped to 50°c. plant regeneration was scored 7-10 days after transferring the calli to the regeneration medium. the criterion used to determine regeneration was the formation of a distinguishable shoot at least 1 cm in length. plant regeneration rate was defined as the percentage of callus that had regenerated shoots. the regeneration cultures were maintained at 24±1°c with a 16 h light/8 h dark cycle under a light intensity of 20 µmol m-2 s-1. meristem tip culture in a second experiment, surface sterilized intact caryopses were incubated on 80 mm diameter petri dishes containing ms media, 3% sucrose, solidified esmaeli et al. in vitro plant regeneration of perennial ryegrass cultivars 275 with 0.8% plant agar and were kept at 24±1°c in dark. after emerging radicles, the petri dishes were transferred to 16h/8h light/dark photoperiodic condition. meristem tips of 7-10 days old seedlings were excised by a stereo microscope (c121506 zeiss stemi 1000 binocular stereo zoom 0.7-3.5x microscope 10x eyepieces, germany) and cultured on three different ms media including hormone-free ms, ms supplemented with 0.01 mg l-1 2,4-d + 0.2 mg l-1 kinetin, ms supplemented with 0.1 mg l-1 2,4-d + 0.5 mg l-1 kinetin. all media were supplemented with 3% sucrose and ph was adjusted to 5.8 prior to autoclaving. the length of the meristem tips cultured ranged from 0.1-1 mm and consisted of the meristem dome plus one or several leaf primordia. after 2 weeks, the number of direct regenerated plantlets was recorded in each treatment based on green and healthy plantlets produced and then the percentage of direct regeneration of meristem tips was calculated. one hundred meristem tips were cultured in each petri dish. all media were placed in dark for 3 days at 24±1°c and then were transferred to 16h/8h light/dark photoperiodic condition. light intensity was provided by cool-white fluorescent lamps at a photon flux density of 20 µmol m-2 s-1. statistical analysis complete randomized design with factorial arrangements was used for all experiments. for callus induction, each treatment had four replications, each replicate consisted of 100 seed. experiments on direct and indirect regeneration were done with three replications. all data were analyzed with sas 9.1 software and means were compared using lsd test at 5% level. 3. results and discussion effects of cultivars, explants and hormonal treatments and their interaction on callus induction plant growth regulators (pgrs) are specific molecules used in plants and supplemented at relatively low concentrations to work as signaling compounds for plant growth and development (sauer et al., 2013). the most extensively used and studied class of pgrs in plant tissue cultures are auxin and cytokinin (ikeuchi et al., 2013). among auxin sources, 2,4-d is known as the most effective pgr to induce callus formation with stimulating cell elongation and enlargement in many plant species especially turfgrasses. in addition, low levels of cytokinins such as ba and bap enhanced callus regeneration ability in several grass species (zhong et al., 1991; van der valk et al., 1995; chaudhury and qu, 2000; bradley et al., 2001). calli were observed in 7 to 15 days after placing the explants on callus induction media. their morphological appearance was hyperhydrated and white. after 4 weeks, three types of calli were observed including: i) white and hyperhydrated; ii) yellowish and friable; iii) compact, nodular, yellowish to opaque (fig. 1 b-d). this variability is probably due to different tissues origin. results showed that callus induction was notably increased by 28% in sliced caryopses (61.37) compared to intact caryopses (48.04). in addition, there was a significant difference fig. 1 steps of caryopses culture of two perennial ryegrass cultivars. a) the de-husked caryopses and surface sterilization of caryopses, right ‘grassland’, left ‘numan’. b) calli derived from longitudinally sliced caryopsis tissue, c) embryogenic callus type ii. d) embryogenic callus type iii. e) green shoots produced on embryogenic callus. f) shoot proliferation on the regeneration medium after two weeks. g) plants acclimatized in soil mixture after one week. h) healthy and green plants maintained in greenhouse after one month. adv. hort. sci., 2018 32(2): 273-280 276 between two cultivars. the callus induction raised by 24% in ʻgrasslandʼ (60.57) compared to ʻnumanʼ (48.83) (data not shown). in ʻgrasslandʼ, the highest good quality callus induction was obtained at 5 mg l-1 2,4-d in sliced caryopses, while ʻnumanʼ had the highest callus induction at 4 mg l-1 2,4-d with 0.02 mg l-1 ba treatment in sliced caryopses (table 1). in ʻgrasslandʼ, higher concentrations of 2,4-d declined callus induction with explants sliced and entire caryopses. however, more calli showed white color with increasing 2,4-d concentration in mentioned media. the effectiveness of 2,4-d in inducing the formation of callus is attributed to its main characteristic which can stimulate cell division of plant tissues and strongly suppress their organogenesis. in a similar study, liu et al. (2006) used 2,4-d and bap for callus induction in perennial ryegrass. they found that the highest callus induction rate with the best callus quality was obtained on ms medium containing 5 mg l-1 2,4-d and 0.05 mg l-1 bap. preliminary studies on the effectiveness of bap on somatic embryogenesis in callus culture medium in several grass species were reported (zhong et al., 1991; chaudhury and qu, 2000; bradley et al., 2001). according to previous reports, the optimal bap concentration in the induction medium should be 0.020.05 mg l-1 for perennial ryegrass. in this experiment, no significant difference was found on media supplemented with 0.0 or 0.02 mg l-1 ba. however, this amount of ba had positive effect on morphology and cellular structure of calli and their capacity to regenerate plantlets. as previously stated, faster and more callus induction of longitudinally sliced caryopses in comparison with intact caryopses were detected. increase in contact surface of tissue could be an important reason for callus induction. these findings are similar to other studies which have been conducted by altpeter et al. (2000), altpeter and xu (2000), bai and qu (2001) and bradley et al. (2001) on lolium sp. and festuca sp. bai and qu (2001) improved both callus production and regeneration rate of tall fescue, festuca arundinacea schreb. with slicing seeds in callus culture medium. also, similar inclusion on chopping the mature seeds of lolium sp. and festuca sp. to suppress germination and stimulation of callus production were reported (altpeter et al., 2000; altpeter and xu, 2000). although, salehi and khoshkhui (2005) obtained contrary results by using horizontally sliced caryopses of lolium sp. cultured on callus induction medium. these results might be related to genotypic differences and callus origin. these results might be used as a general application for other grass species. interaction effect of aba and maltose on plant regeneration the highest regeneration rates were recorded for both cultivars on ms medium supplemented with 30 g l-1 sucrose, 10 g l-1 maltose and 2 mg l-1 aba. as shown in figure 2, ʻgrasslandʼ has higher regeneration rate (~13%) than ʻnumanʼ. incidentally, maltose had not notably effect on plant regeneration percentage in both cultivars lonely but aba concentrations showed significant effects on plant regeneration rate (fig. 2). this result can be referred to the role of aba in maturation of embryos (finkelstein et al., 1985). the most important source of energy for plant growth in the culture medium is carbon. several table 1 interaction of different concentrations of 2,4-d and ba on callus induction of intact and sliced caryopses in two cultivars of lolium perenne l. 2,4-d (mg l-1) ba (mg l-1) ‘grassland’ ‘numan’ intact seeds sliced seeds intact seeds sliced seeds 4 0 49± 3.9 de 69± 6.0 bc 49±2.4 de 54±3.5d 5 0 61±2.7 c 84±3.2 a 46±1.9 de 61±3.8 c 0.02 59±5.2 c 73±2.0 ab 55±1.8 d 41±2.9 e 6 0 51±3.4 de 77±3.8 a 30±2.9 g 62±3.6 c 0.02 62±5.4 c 71±2.3 b 53±6.7 d 54±4.7 d 7 0 71±0.6 ab 60±4.8 c 41±3.2 e 58±1.7 cd 0.02 55±3.5 d 75±5.5 ab 53±2.7 d 65±4.9 bc 8 0 53±1.1 d 73±6.3 ab 54±1.2 d 67±3.5 b 0.02 46±5.0 e 69±3.4 bc 45±6.6 e 53±1.7 d 9 0 46±5.0 e 53±6.4 d 35±1.6 f 38±1.9 f 0.02 38±6.6 f 57±2.5 c 29±2.4 g 53±1.9 de 10 0 43±7.9 e 54±4.1 de 38±2.8 f 41±3.2 e 0.02 54±7.4 d 56±4.1 d 34±4.9 f 54±2.5 d values represent mean ± se. means with the same letter are not significantly different at p ≤ 0.05. fig. 2 regeneration rates of two cultivars of lolium perenne l. in combination concentrations of aba (0, 1.7, 2 and 2.3 mg l-1), and maltose (0, 10, 15 and 20 g l-1 mal) under same concentration of sucrose (30 g-1 suc). esmaeli et al. in vitro plant regeneration of perennial ryegrass cultivars 277 studies have reported the effects of carbohydrates such as sucrose and maltose on different plant species like wild cherry, rubber tree, oilseed rape and sugarcane (reidiboym-talleux et al., 1998; blanc et al., 1999; slesak and przywara 2003; gill et al., 2004). maltose is the best carbon source for callus induction and regeneration medium. furthermore, maltose, as an osmoticum, plays a major role in the optimization of callus cellular environment and protect calli by reducing ethylene (darachai et al., 2004; zaidi et al., 2006). the regeneration rate was affected by using maltose, to almost five-fold increase at 20 g l-1 maltose compared to 30 g l-1 sucrose in both cultivars studied (fig. 2). in addition, aba increased the regeneration rate ~25 and 16 fold at 2 mg l-1 in comparison with 0 mg l-1 in ʻgrasslandʼ and ʻnumanʼ, respectively. the combination of maltose and sucrose and aba at high concentrations had no promoting effect on plant regeneration in both cultivars. in ʻgrasslandʼ, significant declines was observed at 2.3 mg l-1 aba + 20 g l-1 maltose (71.3%) and 1.7 mg l-1 aba + 10 g l-1 maltose (100%). according to the results, both cultivars showed different responses at high concentrations of maltose and aba combination. generally, the regeneration rate dramatically reduced by 100% at 2.3 mg l-1 aba + 20 g l-1 maltose in ʻnumanʼ compared to ʻgrasslandʼ (fig. 2). a probable reason for reducing the regeneration percentage is related to sucrose in regeneration media that stimulates the ethylene production in plant tissues which can cause browning of calli. on the contrary, maltose might protect the calli from browning. however, beneficial effect of maltose was observed on embryogenesis and regeneration of cereals such as rice, wheat and perennial ryegrass, but the mechanisms of maltose role in tissue and cell culture media is not yet completely known. there were significant differences between two cultivars according to plant regeneration rates. our results are in accordance with previous studies on kentucky bluegrass (van ark et al., 1991), zoysiagrass (dhandapani et al., 2008), and bermudagrass (li and qu, 2002). according to the results of van ark et al. (1991), by adding aba to regeneration medium, the percentage of calli with somatic embryos or embryolike structures increased (up to 29.6%) as compared to the control (16.4%). in addition, dhandapani et al. (2008) found that aba significantly increased embryogenic callus formation from stem nodes, but not from young inflorescences. aba not only promotes the transition of somatic embryos from the proliferation to the maturation phase (langhansova et al., 2004), but it also enhances embryo quality by increasing desiccation tolerance and preventing precocious germination (li et al., 1997; robichaud et al., 2004; vahdati et al., 2006; rai et al., 2008). ultimately, based on mentioned authors results, the role of aba on increasing somatic embryogenesis and regeneration were affected by several factors including origin of explant, physiological status and environmental condition. direct regeneration in meristem tip culture an efficient and rapid method was developed for direct regeneration of two cultivars of perennial ryegrass. the results of meristem tip culture provided good information regarding changings in pgrs and their effects on plantlets growth and development. there was significant difference between two cultivars in plantlet regeneration rate. in ʻgrasslandʼ, the overall regeneration rate of meristem tips was about 63.5%, while this parameter was approximately 55.7% in ʻnumanʼ. meristem elongation and development as a single plantlet on ms medium without pgrs was observed (fig. 3c). more plantlets were obtained from ʻnumanʼ meristem tips in comparison to ʻgrasslandʼ on hormone-free ms medium (fig. 3d). the same results were previously reported on curculigo latifolia (babaei et al., 2014). in the present study, overall regeneration percentage of both cultivars on ms media was obtained approximately 41%, while in fig. 3 steps of meristem tip culture. a) ten days old seedling. b) isolated meristem tips. c) meristem tip cultured on ms medium. d) meristem tips cultured on ms medium supplemented with 0.01 mg l-1 2,4-d + 0.2 mg l-1 kin. e) meristem tips on ms medium supplemented with 0.1 mg l-1 2,4-d +0.5 mg l-1 kin. adv. hort. sci., 2018 32(2): 273-280 278 l-1 ba was selected. in sliced caryopses more callus induction was observed in both cultivars. the best regeneration medium for them was also recommended. generally, low concentrations of maltose and aba was more effective on regeneration rate of two cultivars. in addition, an efficient and rapid procedure for direct in vitro regeneration of two perennial ryegrass cultivars has been established. despites, ms hormone-free medium was an acceptable medium for regeneration of meristem tips but the highest regeneration rate of meristem tips was observed with 0.1 mg l-1 2, 4-d + 0.5 mg l-1 kin in both cultivars. there was a positive relation between length of meristem tips and their survival percentage. it is now possible to test the effectiveness of this technique to produce virus-free plants. moreover, the results can definitely improve the transformation efficiency of these cultivars. references altpeter f., 2006 perennial ryegrass (lolium perenne l.). agrobacterium protocols. volume 2. methods mol. biol., 344: 55-64. altpeter f., xu j., 2000 rapid production of transgenic turfgrass (festuca rubra l.) plants. j. plant physiol., 157: 441-448. altpeter f., xu j., ahmed s., 2000 generation of large numbers of independently transformed fertile perennial ryegrass (lolium perenne l.) plants of forage-and turftype cultivars. mol. breed., 6: 519-528. babaei n., psyquay abdullah n.a., saleh g., lee abdullah t., 2014 an efficient in vitro plantlet regeneration from shoot tip cultures of curculigo latifolia, a medicinal plant. the sci. world j., 2014: 1-9. bai y., qu r., 2001 factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue. plant breed., 120: 239-242. bhaskaran s., smith r., 1990 regeneration in cereal tissue culture: a review. crop sci., 30: 1328-1336. dale (1977) investigation, overall regeneration percentage of nine species of grasses was reported about 11% on hormone free ms medium. by adding plant growth regulators to the media, more plantlets and tillers grew in both cultivars. as shown in table 2, an increased regeneration percentage of 35% and 37% by adding 0.01 mg l-1 2,4-d + 0.2 mg l-1 kin and 53% and 48% by increasing kin from 0.2 to 0.5 mg l-1 was observed in ʻgrasslandʼ and ʻnumanʼ, respectively. these results show that an exogenous supply of growth regulators is required or can be beneficial for the regeneration of whole plants from stem apices as stated in several previous studies (dale, 1977). in his first study on meristem tip culture of lolium species, dale (1977) stated that the best regeneration rate was obtained on ms medium compared to the other media. the higher regeneration rate was obtained for l. multiflorum (92%) on the medium containing 0.01 mg l-1 2,4-d + 0.2 mg l-1 kin and for l. perenne (54%) on medium containing 0.5 mg l-1 2,4d + 0.02 mg l-1 kin. according to our results, the size of the meristem tips cultured usually affects their response in culture. in ʻnumanʼ large meristem tips 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dev. biol. plant, 39: 277-282. zaidi m.a., narayana m., sardana r., taga i., postel s., johns r., altosaar i., 2006 optimizing tissue culture media for efficient transformation of different indica rice genotypes. agron. res., 4: 563-575. zhong h., srinivasan c., sticklen m.b., 1991 plant regeneration via somatic embryogenesis in creeping bentgrass (agrostis palustris huds.). plant cell rep., 10: 453-456. impaginato 265 adv. hort. sci., 2018 32(2): 265-272 doi: 10.13128/ahs-21988 beneficial effects of foliar application of organic chelate fertilizers on french bean production under field conditions in a calcareous soil m.k. souri (*), m. aslani department of horticultural science, tarbiat modares university, tehran, iran. key words: aminochelate, amino acid, calcareous soil, nutrient uptake, pod yield, quality. abstract: aminochelate are organic-based chelate fertilizers with higher efficiency for agricultural applications. in the present study, foliar application of three organic-based chelate fertilizers, a macro-micro mixture and soil applied npk were evaluated on french bean growth characteristics under open field in a calcareous soil. the results showed that plant growth, pod yield (79%) and pod quality were improved by application of chelate fertilizers. growth parameters as plant height, number of leaves and lateral shoots, shoot dry weight, pod number and pod length were significantly increased by foliar application of the chelate fertilizers. the concentrations of nitrogen, potassium and iron in pods and above all in leaves were increased by foliar application of chelate fertilizers compared to control and soil applied npk. pod ph and tss were not influenced by treatments; however, foliar application of the chelate fertilizers resulted in higher titratable acidity (40%), vitamin c (112%) and protein (35%) content of pods. the results indicate that organic-based chelate fertilizers can be effective safer alternatives for simple chemical salts in calcareous soils. 1. introduction calcareous or lime soils are the dominant type of soil in many parts of the world, and are characterized by high ph as well as high levels of carbonates and bicarbonates. nutrients and particularly iron uptake in such soil conditions is restricted. in many cases lime-induced chlorosis refers to iron deficiency (souri, 2015). the leaf chlorosis is mainly due to high ph of soil solution and sap solution induced by high concentrations of carbonates and bicarbonates, which results in precipitation of nutrients in soil and cell apoplast (mengel, 1994; nikolic and römheld, 2002). plant cultivation in calcareous soils requires especial management techniques and strategies particularly regarding micronutrients supplementation (souri, 2015). various fertilizers, as well as different forms of nutrient elements do not have the same uptake efficiency (jeppsen, 1991; fernández and (*) corresponding author: mk.souri@modares.ac.ir citation: souri m.k., aslani m., 2018 beneficial effects of foliar application of organic chelate fertilizers on french bean production under field conditions in a calcareous soil. adv. hort. sci., 32(2): 265272 copyright: © 2018 souri m.k., aslani m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 november 2017 accepted for publication 20 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 265-272 266 ebert, 2005). since decades chelate fertilizers have been used due to their better suitability and efficiency to meet plant’s need of nutrient elements particularly under calcareous soil conditions. simple nutrient salts are in inorganic form, which for utilization by plant roots must be restructured to ionic form in soil solution. this makes them very vulnerable to various inactivation processes, resulting in their low efficiency rate (mengel, 1994; nikolic and römheld, 2002; souri, 2016). on the other hand, high price of commercial synthetic chelate fertilizers such as edta or eddha restricts their application by many farmers. in addition, there is also great doubt on their impacts on plant, environment and ecosystem health issues (souri, 2015). aminochelate fertilizers are claimed as suitable alternatives for simple salt fertilizers. for nutritional purpose the safety of fertilizer products is very important. organic chelates such as aminochelate fertilizers are formulated mainly for foliar application, even if in various studies their soil application also resulted in higher growth compared to soil applied npk (souri and yarahmadi, 2016) or a more complete fertilizer (salwa, 2011; garcia et al., 2011). the method of fertilizer application also plays an important role in uptake efficiency by plant roots (fernández and ebert, 2005). there are generally two methods of soil and foliar application for most fertilizers. most of micronutrients and nitrogen fertilizers including amino acids can simply be applied to leaves with acceptable uptake efficiency (jeppsen, 1991; marschner, 2011). nevertheless, foliar application cannot fully replace soil application of fertilizers in agriculture (souri, 2015; dehnavard et al., 2017). aminochelates are composed of amino acids and a single or several nutrient elements (metals) and represent a more suitable form of fertilizers for sustainable production than routine fertilizers (souri, 2015). in cultivation systems, application of nitrogen and micronutrients needs a precise and intelligent management, in which various organic chelates can play important role. however, general chemical properties of various aminochelate fertilizers have not been yet studied in soil or within the plant tissues. in recent studies, it was shown that application of amino acid chelates of nutrients in nutrient solution or on plant foliage, significantly increases plant growth and biomass production (zeid, 2009; ghasemi et al., 2014; souri et al., 2017), leaf number, leaf area, leaf spad index (garcia et al., 2011; souri and yarahmadi, 2016), fruit yield (naseri et al., 2013; pourebrahimi, et al., 2013; fahimi et al., 2016), fruit quality (machado et al., 2008; souri et al., 2017) and composition of plants (zhou et al., 2007; garcia et al., 2011; ghasemi et al., 2014). moreover, application of organic-based fertilizers can also result in higher soil microbial activity and fertility, whereas chemical forms of fertilizers with higher salt effects generally reduce soil microbial activity and soil fertility (salwa, 2011; souri, 2015). it was shown that soil application of amino acids has suppressive effect on some soil pathogens such as meloidogyne incognita (saeed et al., 2005). green bean is a relatively sensitive plant to lime soil, showing dwarf growth and low yield due to restriction of flower development and fruit set. in the present study effects of foliar application of three organicbased chelate fertilizers were investigated on french bean growth characteristics and pod quality factors under calcareous soil conditions. 2. materials and methods experimental site this study was performed at faculty of agriculture, tarbiat modares uni., tehran-iran during 2013. the field in which the experiment was conducted had a calcareous soil. the soil sample was collected from three parts of the field (0-30 cm depth), mixed together and analyzed for some physicochemical characteristics that are presented in table 1. application of treatments the experiment was arranged in complete randomized blocks using six treatments and four replications. treatments were: 1) control (without any fertilizer application), 2) soil application of npk, 3) foliar application of biomin (amino acid based fertilizer; arbico-co, texas, usa), 4) foliar application of humifolin (humic acid based fertilizer; tradecrop co., spain), 5) foliar application of delfanplus (only amino acid; tradecrop co., spain) and 6) foliar application of a mixture of macro-micro solution. each replicate physico-chemical caracteristics data texture loam-clay ph 7.8 ec ds/m 2.6 total c (%) 0.77 total n (%) 0.09 caco3 (%) 5.5 extractable p (mg kg-1) 15.2 exchangeable k (mg kg-1) 250 table 1 some properties of the soil used in the experiment souri and aslani foliar application of organic chelate fertilizers on green bean 267 was a plot of 0.7×1m consisting of 12 plants. green bean seeds (phaseolus vulgaris l) were directly sown in the soil, and after germination they were thinned to 12 plants per plot. throughout the growing period all plots and plants were treated the same regarding irrigation, and pest-disease control. in npk treatment, a final amount of 6 g per plant from a 20:10:20 formulation was used in three split applications that were incorporated into the soil just near the root systems using 200 ml water. the first application was before sowing and the rest was applied in one week interval after second week of emergence. foliar application of organic-based and mix fertilizers were done five times during growth period, in a constant concentration of 0.2%. plants were foliar sprayed at 6-7 o’clock in the morning using a portable sprayer, by which the upper and the lower surface of leaves were treated. the first spray was done at four leaf stage, and the remaining foliar sprays were applied at one-week intervals. all the organic-based commercial fertilizers used in present study were in liquid form, consisting of one or several nutrient elements. composition of fertilizers the composition of various fertilizers is presented as follows: biomin: a liquid fertilizer containing 2% n, 2.5% zn, 1.5% mn, 1% fe, 0.4% mg, 0.4% cu, and 0.02% mo; humifolin, a liquid fertilizer containing 42% organic compounds including 37% fulvic and humic acid, 5% various vitamins, 0.5% phosphorus, 0.28% fe, 0.041% zn, 0.0035% mn, 0.0023% cu, 0.0012% mg, and 0.0012% b; delfanplus, a liquid fertilizer consisting of 24% free amino acids, 9% total nitrogen, 5% n-protein and 43% organic carbon. macro-micro mixture consisted of 5% n, 2.5% zn, 2.5% fe, 2% mn, 0.5% mg, and 0.5% cu. plant sampling and measurements french bean plants were grown for ten weeks, and various growth traits were measured during growth period, as well as at harvest time. plant pods were harvested several times during their growth period. cumulated yield of plants during 2-3 harvests (in each replicate) was recorded and the average was presented as plant final yield of fresh pods. accordingly, total number of pods per plant was recorded. the average number of leaves and lateral shoots of plants were counted per replicate (plot) and calculated per plant. chlorophyll index of leaves was recorded using a portable spad meter (model 502 plus, illinois, usa) with 10 reading per plant and at least 120 readings for each replicate, by which the average was recorded in the results. plant stem diameter was measured by caliper (model mitutoyo japan). average shoot dry weight per plant was calculated after drying four randomly chosen plants from each replicate at 65°c for 24 hours. total soluble solid (tss) as brix index was determined with a refractometer using a drop of pod extracted juice. for determination of pod ph and titratable acidity, 10 g of fresh pods (from each replicate) was cut, crushed and centrifuged at 9000 rpm. ten ml of supernatant was titrated using 0.1 m naoh until a final ph of 8.2, and the pod acidity based on citric acid in 100 g fresh pods was calculated using the following formula: ta= 100 ×m×n×v/s×n where ta is the amount of pod acidity (mg/100 g fw), m the molecular weight of dominant pod acid (citric acid= 64 g) n: valence of dominant acid, n: normality of naoh, s: weight of pod sample (g) and v the volume of consumed naoh. for measurement of pod vitamin c, 10 g of fresh pods from final harvest was gently washed, cut in small pieces and then crushed in a mortar in presence of 10 ml of 2% metaphosphoric acid. the mixture was immediately centrifugated at 9000 rpm (eppendorf centrifuge 5810r, hamburg, germany) for 5 minutes at 4°c. the supernatant was used for titration by 2,6 dichloro indophenols, and the amount of vitamin c for 100 g fresh pod was calculated in relation to records of a standard curve of l-ascorbic acid concentration of 0, 25, 50, 100 and 200 mg l-1. determination of pod protein content was done using coomassie brilliant blue g250 dye according to bradford’s method (1976). the nutrient concentration of n, k and fe were determined in plant leaves and green pods using kjeldahl, flame photometer and atomic absorption spectrophotometer methods. statistical analysis data were analyzed using spss 16 and differences among treatments were determined at 5% level by duncan’s test. graphs were prepared using excel microsoft. 3. results plant vegetative growth vegetative growth characteristics of plants are presented in table 2. application of various fertilizers had significantly improved plant growth. all the most important growth factors were improved especially adv. hort. sci., 2018 32(2): 265-272 268 by foliar application of organic-based chelate fertilizers (table 2). in particular, plant height was increased by application of three organic chelates, while there was no difference among control plants and those plants treated with soil applied npk or by foliar application of macro-micro mixture. stem diameter was significantly higher in plants that received foliar application of organic chelates (table 2). number of leaves was maximum in plants treated with biomin and humifolin; however they had no significant difference with delfanplus treated plants. the least number of leaves was in control plants. number of lateral shoots was increased by foliar application of biomin and humifoline; however, there was no significant difference among all organic fertilizers. there was no significant improvement of lateral shoots by soil applied npk or foliar application of macro-micro mixture (table 2). plants treated with delfanplus produced longest internodes that showed no difference with biomin or humifolin treated plants (table 2). application of all fertilizer treatments, except foliar application of macro-micro mixture, resulted in significantly higher spad values compared to control plants (table 2). determination of shoot dry weight revealed that plant growth and biomass production was significantly improved by foliar application of the organic fertilizers, as well as by soil applied npk treatment. on the other hand, the maximum shoot dry weight was recorded for biomin treatment, which showed significant difference with control and all other fertilization treatments (table 2). plant yield and nutrient status application of all fertilizers increased the pod yield compared to control plants (table 3). plants produced significantly higher yield when they received foliar application of organic chelate fertilizers (table 3). number of seeds per pod and number of pods per plant had a similar trend, in which foliar application of three organic chelate fertilizers recorded higher values. regarding pod length, plants treated with foliar application of three organic chelates had the longest pods compared to all other treatments. all fertilizer treatments increased the pod dry weight. plants treated with biomin produced significantly higher pod dry weight (table 3), although application of all the organic chelates significantly increased pod dry weight compared to soil applied npk or foliar application of macro-micro mixture. nutrient profile of plant leaves and pods were significantly increased by foliar application of organic fertilizers, only for n and k, and also by npk soil table 2 effects of various fertilization treatments on vegetative growth traits of french bean plants in a calcareous soil under field conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. treatments plant height (cm) stem diameter (cm) number of leaves number of lateral shoots length of internode (cm) spad index shoot dry weight (g) control 26 b 0.62 b 14 c 4 b 5.6 b 36 b 7.2 d npk 31 ab 0.79 b 19 b 5 b 5.5 b 40 a 11.3 c biomin 39 a 1:05 24 a 8:00 6.7 ab 41 a 22.1 a humifolin 39 a 0.96 a 24 a 7:00 6.1 ab 41 a 15.6 b delfanplus 38 a 0.98 a 22 ab 6 ab 7:02 42 a 17.5 b macro-micro mixture 29ab 0.67 b 17 bc 4 b 5.1 b 38 ab 8.5 d table 3 effects of various fertilization treatments on yield and fruit characteristics of french bean plants grown in a calcareous soil under field conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. treatments plant pod yield (g) number of seeds/pod number of pods per plant pod length (cm) pod dry weight (g) pod ph pod tss control 31.4 c 2.9 c 17 c 5.5 b 8.3 d 6.20 a 1.7 a npk 38.3 b 3.8 b 28 b 6.5 b 10.8 c 6.30 a 1.8 a biomin 59.1 a 5.1 a 49 a 9.1 a 21.1 a 6.21 a 2.4 a humifolin 53.5 a 4.7 a 43 a 9.3 a 16.2 b 6.22 a 2.1 a delfan plus 56.6 a 4.8 a 45 a 9.6 a 16.6 b 6.23 a 2.0 a macro-micro mixture 36.7 b 3.9 b 27 b 6.5 ab 11.4 c 6.24 a 2.1 a souri and aslani foliar application of organic chelate fertilizers on green bean 269 application (table 4). foliar application of biomin and delfanplus resulted in significantly higher k and fe concentration in leaves, and k concentration of pods, compared to humifolin. the lowest nutrient concentrations were recorded in control and macro-micro mixture treatments. determination of nutrients in green pods (table 4) showed that the significant highest amount of n was in plants sprayed with biomin aminochelate, and then by delfanplus, humifolin and soil applied npk, respectively. potassium concentration was significantly higher in foliar spray of biomin and delfanplus, while the lowest concentration was in pods treated with macro-micro mixture and in control plants. iron concentration of green pods was significantly increased by organic fertilizers. pod quality there was no significant difference in pod ph and pod total soluble solids (tss) among treatments (table 3). however, determination of titratable acidity (fig. 1) revealed that plants treated with humifolin resulted in significantly higher pod acidity (but not different from biomin and delfanplus), while the least titratable acidity was measured in soil applied npk, control and macro-micro treatments, that showed no difference with biomin and delfanplus treatments (fig. 1). foliar application of three organic fertilizers resulted in significantly higher vitamin c content of pods compared to control plants (fig. 2). pod protein content was higher in plants treated with humifolin and biomin, and lower in control and plants treated by foliar application of macro-micro mixture. pod protein content was intermediate for soil applied npk and foliar application of delfanplus (fig. 3). 4. discussion and conclusions in the present study plant growth was improved by foliar application of three organic chelate fertilizers. many parameters of vegetative growth as well as plant yield, nutrient content and fruit quality were improved by application of these three commercial fig. 1 effects of various fertilization treatments on titratable acidity of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. treatments leaf pod n (%) k (%) fe (mg kg-1 dw) n (%) k (%) fe (mg kg-1 dw) control 2.1 c 1.7 c 55.3 c 3.7 c 2.8 c 77.2 b npk 2.7 b 2.6 a 64.1 c 5.2 b 4.6 b 85.1 b biomin 3.5 a 2.8 a 126.3 a 6.5 a 5.3 a 108.3 a humifolin 2.9 b 2.3 b 97.7 b 5.6 b 4.7 b 96.6 a delfanplus 3.2 ab 2.6 a 118.5 a 5.7 b 5.1 a 99.0 a macro-micro mixture 2.3 c 1.8 c 66.1 c 3.7 c 2.7 c 82.4 b table 4 effects of various fertilization treatments on some nutrients in leaves and pods of french bean grown in field with calcareous soil conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. fig. 2 effects of various fertilization treatments on vitamin c content of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. fig. 3 effects of various fertilization treatments on protein content of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. adv. hort. sci., 2018 32(2): 265-272 270 and nutrient uptake has been shown (david et al., 1994; el-ghamry et al., 2009; salwa, 2011; canellas et al., 2015). the biostimulant effects of humic substances are characterized by both structural and physiological changes in roots and shoots related to nutrient uptake, assimilation and distribution (nutrient use efficiency traits). in addition, they can induce shifts in plant primary and secondary metabolism related to abiotic stress tolerance which collectively modulate plant growth as well as promoting fitness (canellas et al., 2015). in the present study, improved growth and plant performance of french bean might be also due to the higher nitrogen and micronutrients content of plant leaves. the n, k and fe concentrations in plant leaves were significantly improved by foliar applications of organic chelate fertilizers. metal ions such as fe, zn, mn and cu are essential for healthy plant growth, being required for various metabolism reactions (marschner, 2011). they have direct and distinct effects on plant performance, as well as on yield and quality parameters. however, uptake of micronutrients such as iron by roots from soil could be limited due to low chemical stability and precipitation of these elements particularly in calcareous soil (fernández and ebert, 2005; souri, 2015). in calcareous soils, with high ph and carbonate-bicarbonates levels, plants are prone to iron and other micronutrient deficiencies. lime-induced chlorosis is one of the most important nutritional disorders, affecting many plant yield and quality traits. aminochelate fertilizers represent an excellent n source for plant, in both foliar and soil applications. in present study, n concentration of leaves and pods were significantly improved by organic fertilizers. higher nitrogen content of plant was also reported for radishes (liu et al., 2008) and marigold (souri and yarahmadi, 2016), when plants were treated by foliar application of aminochelate fertilizers. improvement in nutrient elements profile of tomato was observed when amino acids were applied in nutrient solution, which finally improved plant growth and nutrient concentrations, particularly n status of plants (garcia et al., 2011). nitrogen has an important role in growth, yield and quality of crops and must be usually applied to meet the plant needs. french bean can fix atmospheric n2, so it may need less n fertilization; however, in some parts of iran farmers use also high rates of urea to enhance plant growth. on the other hand, application of high amount of n fertilizers could lead to significant reduction in yield and quality, as well as organic fertilizers. increasing in plant growth and various yield traits were also reported in other studies (machado et al., 2008; garcia et al., 2011; naseri et al., 2013; salwa, 2011; ghasemi et al., 2014; sadak et al., 2015; souri and yarahmadi, 2016). in a recent study it was shown that foliar application and to lesser extent soil application of a commercial aminochelate fertilizer increased the growth, yield and quality of tomato, cucumber and bean plants (souri et al., 2017). foliar application of a mixture of amino acid on bean plants significantly improved the tolerance to seawater salinity stress (sadak et al., 2015). similarly, application of amino acids significantly improved growth parameters of shoots and fresh weight as well as pod yield of soybean plants under pathogen infection (saeed et al., 2005). vegetative growth of plant height and dry weight of potato plants were increased by foliar application of amino acids (el-zohiri and asfour, 2009). three foliar application at 6-7 leaves stage and two more sprays in two weeks intervals using fe and zn aminochelates with different concentrations up to 0.3%, resulted in 15-35% higher potato tuber yield per hectare (pourebrahimi et al., 2013). stimulatory effects of amino acids on plant growth characteristics have been well documented, particularly under adverse climatic conditions such as salt and drought stresses (rai, 2002; zhou et al., 2007; garcia et al., 2011; salwa, 2011; sadak et al., 2015). amino acids are key important player in plant metabolism. they are the intermediate compounds in nitrogen assimilation, and represent the main form by which nitrogen is translocated within the plant through phloem (marschner, 2011). various amino acids and peptides are precursor of physiologically important phytohormones (cobbett and goldsbrough, 2002; marschner, 2011) or they are involved in detoxification of different toxins within the plant cells (cobbett and goldsbrough, 2002; souri, 2015). in addition to the amino and carboxyl groups, amino acids have a side chain or r group that is attached to the α-carbon. each amino acid has unique characteristics arising from the size, shape, solubility and ionization properties of its r group. nevertheless, the side chain of amino acids exerts a deep effect on their biological activity as well as on the structure and activity of proteins. by far, glycine is the main and widespread used amino acid in manufacturing aminochelate fertilizers, despite frequently a mixture of amino acids may be included (souri, 2015). similarly, the stimulating effect of foliar or soil application of humic acid on increased plant growth souri and aslani foliar application of organic chelate fertilizers on green bean 271 various pollutions. leaf greenness, as the best health indicator of plants, depends on chlorophyll biosynthesis and concentration, which in part is affected by n and micronutrients. aminochelates generally contain all these effective nutrient elements. application of aminochelates, as a source of n fertilizer, can also result in lower nitrate accumulation in plant tissues. high nitrate content of plant tissues is a negative factor, particularly in leafy vegetables that are fresh consumed. application of reduced form of nitrogen (such as ammonium or amino acids) instead of oxidized form (nitrate) can lead to less nitrate accumulation in plant tissues (marschner, 2011; souri et al., 2017). on the other hand, plant might have different response to various fertilizers of a given nutrient (souri, 2015). however, there are always distinct clear responses of vegetative and reproductive growth, as well as quality parameters to nitrogen fertilizers (marschner, 2011; souri, 2016). the higher efficiency of aminochelate fertilizers can be due to the chelating effects of amino acids or organic acids on nutrient elements. the chelating effects of amino acids on nutrients has been commercially used for improving nutritional status of animals and human for more than 6 decades, and for plants in recent years (souri, 2016). as it is well known, amino acids are “zwitterions” in biological systems, and have distinct different behavior in acidic and basic solutions, to maintain the ph of the system. the structure of an amino acid allows it to act both as an acid and a base, depending on solution ph. this behavior is quite important in plant nutrition, as ph is one of the main factors responsible for nutrient use efficiency, and frequently high ph (in calcareous soils) and low ph (in acidic soils) restrict nutrients solubility and bioavailability (souri, 2015). nevertheless, in present study organic chelate fertilizers had various composition of one or several components of amino acid or nutrients. conducting scientific research with such amino or organic chelates due to their mix nature of various components is quite difficult (souri, 2016). in conclusion, foliar application of organic chelate fertilizers resulted in higher plant growth under calcareous soil conditions. aminochelates are composite fertilizers of amino acids and various nutrient elements particularly iron and zinc and separating the effects of each component is relatively difficult. the effect of organic chelate fertilizers particularly aminochelates on many physiological and molecular responses of plants has not been well studied. application of organic chelate fertilizers can avoid all negative effects of routine chemical salt fertilizers such as urea or ammonium nitrate, including leaching, 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technology, al-balqa applied university, al-salt 19117, jordan. key words: 41b, p1103, r110, ros, salinity. abstract: grapevine rootstocks that can absorb brackish water and maintain satisfactory growth of the grapevine scion might be a feasible management practice in areas suffering scarce water resources. the objective of this study was to evaluate the expression of antioxidant genes in ‘superior seedless’ leaves grafted on r110 (vitis berlandieri x v. rupestris), 41b (v. berlandieri x v. vinifera) and p1103 (v. berlandieri x v. rupestris) in response to diluted brackish water irrigation at three levels: 1.5, 3.0 and 5.0 ds m-1 in addition to the 0.8 ds m-1 control. results revealed that after salinity exposure for two weeks, the transcript levels of apx, mn-sod and mdar increased in ‘superior seedless’ leaves grafted on the different rootstocks. however, their expression levels in response to salinity were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat gene showed obvious enhanced level in plants grafted on p1103 in response to salt exposure. meanwhile, the expression of cat gene in ‘superior seedless’ scion grafted on 41b or r110 showed almost unchanged level in control and stressed conditions. down-regulation of cuzn-sod was recorded in leaves of ‘superior seedless’ grafted on p1103. slight up-regulation of this gene in response to saline condition was recorded when scion was grafted on 41b or r110. the expression of gpx was enhanced in scion grafted on p1103 and 41b. on the other hand, scion grafted on r110 showed decreased expression of gpx in response to salt treatment. grapevine rootstocks that have v. rupestris and v. berlandieri in their parentage are good candidates for salinity tolerance. (*) corresponding author: iqrunf@bau.edu.jo citation: qrunfleh i.m., abu-romman s., ammari t.g., 2017 ‘superior seedless’ grapevine grafted on three rootstocks grown on calcareous soil under diluted brackish water irrigation. ii. expression of antioxidant genes adv. hort. sci., 31(4): 275-280 copyright: © 2017 qrunfleh i.m., abu-romman s., ammari t.g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 may 2017 accepted for publication 29 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 275-280 276 1. introduction grapes (vitis sp.) are considered as one of the world’s major commercially grown fruit crops. in jordan, grapes are ranked in second place after olives regarding the total area planted. the total area planted with grapes is 3806 ha (fao, 2014). more than half of that area is under irrigation and jordan is now ranked as the world’s second water-poorest country. irrigation with low quality water during the whole growing season of the crops, even the tolerant ones, does not always produce high yield. mixing low quality water; such as dam brackish water, with conventional quality irrigation water in ratios to keep the salinity of the irrigation water below the threshold of the target crop might be an acceptable management practice and was used by many researchers (abdel gawad and ghaibeh, 2001). alternating conventional quality water with brackish water is another management practice. its application would be easier because it does not need containers for mixing two different sources of irrigation water. the conventional quality irrigation water can be used during the sensitive stages of plant growth and the brackish water during the non-sensitive or less sensitive stages. considerable yields were obtained using saline irrigation water (4-12 ds m-1) in crops that had been previously defined as moderately sensitive to salt stress (bustan et al., 2004). furthermore, in some crops (e.g., tomato) the reduction in the fresh yield was compensated by an increase in fruit dry weight and other quality parameters (mizrahi et al., 1988). bustan et al. (2005) reported that the combination of fresh (1.2 ds m-1) and brackish (7 ds m-1) irrigation water increased the yield level of melon to that of fresh water plants whereas it brought about the improvement of fruit quality typical to brackish water plants, thus providing an attractive approach to optimize late-summer melon production. plants subjected to saline environment use different mechanisms to overcome such abiotic stress. to avoid a disorder of ion homeostasis under saline conditions, plant cells have to maintain a low na+ concentration and keep a high h+ concentration in the cytosol where enzymes for metabolism are located (zörb et al., 2005). usually, plants use different ways to maintain a low cytosolic sodium concentration, including restricting na+ influx, maintaining active na+ efflux, and compartmentalizing na+ into the vacuole, etc. (rubio et al., 1995). these mechanisms involve a number of na+/h+ antiporter proteins that are localized in plant plasma and vacuolar membranes (vasekina et al., 2005). they catalyze the exchange of na+ for h+ across membranes and the energy needed is generated by the h+-adenosine triphosphatase and h+-pyrophosphatase (niu et al., 1995; shi and zhu, 2002). plants exposed to salt stress showed enhanced formation of reactive oxygen species (ros) such as superoxide anion, hydrogen peroxide and singlet oxygen (mittler, 2002). the harmful effects of these molecules are referred to as oxidative stress (halliwell, 2006). plants have evolved an enzymatic antioxidant system to reduce the ros levels. this enzymatic system includes superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase (apel and hirt, 2004). the development of salt-tolerant crops is a practical solution to sustain agricultural productivity. because of the complexity of the trait, traditional crop-breeding programs aimed at improving tolerance to salinity have limited success. therefore, understanding the cellular and molecular bases of salinity-tolerance mechanisms is essential for marker-assisted selection and genetic engineering of salt tolerance in economic crops. understanding the responses of plants to the major environmental stressor salinity is an important topic for the biotechnological application of functional mechanisms of stress adaptation. plant engineering strategies for cellular and metabolic reprogramming to increase the efficiency of plant adaptive processes may either focus on (1) conferring stress tolerance by directly reprogramming ion transport processes and primary metabolism or (2) by modulating signaling and regulatory pathways of the adaptive mechanisms. the second approach seems to be more perspective because it is likely that signaling and regulatory factors orchestrate as key signaling components the transcriptional and translational control of group (1) adaptive mechanisms (diédhiou et al., 2008; popova et al., 2008). most knowledge on molecular mechanisms involved in plant salt responses and adaptation has been derived from analyses of the glycophytic models arabidopsis thaliana and rice. such knowledge is lacking in vitis species, therefore detecting expression changes of antioxidant defense genes is the objective of this study. since the understanding of a plants response to a stress requires an evaluation of stress induced changes in gene expression, the expression of major defense genes (superoxide disqrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. ii. expression of antioxidant genes 277 mutase, ascorbate peroxidase, catalase, glutathione peroxidase, monodehydroascorbate reductase) in ‘superior seedless’ grafted on different rootstocks has been examined by rt-pcr. 2. materials and methods plant material three grape rootstocks were evaluated in this study: r110 (vitis berlandieri x v. rupestris), 41b (v. berlandieri x v. vinifera) and p1103 (v. berlandieri x v. rupestris). the rootstocks were purchased from les pépiniéristes du comtat, sarrians, france. after being imported, ‘superior seedless’ bud cultivar was grafted on the rootstocks in a local nursery; al-bushra nurseries, may, 2013. grafted plant materials were planted in polyethylene bags filled with peatmoss. the one year old grafted grapevine rootstocks were grown for several months to allow for the formation of a well developed root system before applying treatments. fertilizers and fungicides were applied as necessary. soil and water the soil was brought from the southern jordan valley. soil was crushed and sieved through 1 cm sieve and plastic pots (the working volume of the pots was 44 l) were filled with 50 kg each in order to roughly have a bulk density of 1.14 g cm-3. the bulk density is within the typical range of bulk densities of agricultural soils. if bulk density was higher, infiltration would be very slower and hydraulic conductivity would be slower as well. low hydraulic conductivity would exacerbate the osmotic effect and create anoxic conditions. the pots were placed in a controlled greenhouse. grafted grapevines were transplanted in february and the growth was unified based on the number of buds and root length. the root system was cut back to 15 cm in length and the vegetative system was cut back to eight buds. the water was brought from al-karameh dam located in the jordan valley and stored in a galvanized tank. three levels of irrigation water salinity; in terms of electrical conductivity (ec), were applied: 1.5, 3.0 and 5.0 ds m-1 in addition to the 0.8 ds m-1 control. these three levels of diluted brackish water were used to find out the salt level that would result in a tolerable “adverse” effect to design alternate irrigation that would contribute to water saving. these concentrations were selected based upon the threshold ec of grapes (i.e. ec<2 ds m-1). the treatments were prepared by mixing the dam water with tap water. a portable conductivity meter (model cond 3210, wtw, germany) was used to measure the ec and to obtain the determined salinity levels. the twelve treatments were arranged in a randomized complete block design with three replicates. the grafted grapevines started to break the dormancy period during spring. composite fertilizer (20:20:20), urea and ammonium sulphate were also applied to the grapevines and growth was again unified before applying the assigned treatments. irrigation with the assigned treatments started in may. all pots received the same amount of water whenever irrigation was applied. each pot received a total amount of irrigation water equal to 446 mm. irrigation was scheduled according to evaporation readings from free water surface (in mm) taken every 48 hours and corrected using proper grapevine crop coefficient of 0.30 (according to food and agriculture organization). dna and rna extraction leaves were sampled starting from february until november, 2014 (every two weeks) and frozen in liquid nitrogen for analyzing dna and rna using kits (intron, korea). total rna was extracted from frozen leaf samples with the iqeasy™ plus plant rna extraction mini kit (intron biotechnology, korea) according to user’s manual. rna concentration and purity were estimated based on absorbance at 260 and 280 nm. two microgram of rna was used to reverse transcribe the first strand cdna using power cdna synthesis system (intron biotechnology, korea) according to the manufacturer’s protocols with oligo (dt)15 as a primer in a reaction volume of 20 µl. the first strand cdnas generated for all the samples were used for semi-quantitative rt-pcr to monitor the transcript levels. the gene-specific primers for rt-pcr were designed based on the basis of the sequences published in genbank using the software primer-blast (http://www.ncbi.nlm.nih.gov/ tools/primer-blast/index.cgi?link_loc=blasthome). the ef-1α gene was selected as a reference gene. the primer sequences are listed in table (1). the pcr reaction was performed using intron i-maxtm ii system (intron, korea). the same thermal profile was used for all pcr reactions; pcr was initiated with enzyme activation at 95°c for 2 min followed by 32 cycles of 40s at 95°c, 40s at 56 °c and 1 min at 72°c. different amplification cycles were tried and the product of the 32 cycles was selected to be presented. all rt-pcr products were loaded in ethidium bromide-stained 1.5 % (w/v) agarose gel. adv. hort. sci., 2017 31(4): 275-280 278 3. results and discussion as an abiotic stress, salinity induces oxidative damage in plant cells through the increased generation of reactive oxygen species (ros) in different cell compartments (mittler, 2002). the activation of antioxidant defense system reduces the level of ros and minimizes the impact of oxidative stress and its associated damage. plant cells possess antioxidant enzymes which have the ability to detoxify toxic ros (apel and hirt, 2004). rootstock choice should be taken with careful consideration since the scion is dependent on the rootstock (creasy and creasy, 2009). novel rootstocks are frequently used to confer resistance to environmental adversities in horticultural crops (albacete et al., 2015). a promising approach to improve salt tolerance of horticultural species is the use of grafting on salt-tolerant rootstocks (colla et al., 2010; giuffrida et al., 2014; simpson et al., 2015; zrig et al., 2016). grapevine rootstock selection is a key factor and could be considered an important strategy to mitigate salinity stress. grape rootstocks do influence the scion cultivar in many growth and physiological aspects (gu, 2003). however, some contradictions can be found in the literature in terms of the salt tolerance of grapevine rootstocks implying that various factors are involved, which eventually determine grapevine response to salt stress. for example, southey and jooste (1991) found that american hybrids performed poorly in response to salinity when used as rootstocks for the cultivar ‘colombard’. in addition, cavagnaro et al. (2006) concluded that argentinean cultivars performed better than european cultivars in an in vitro salinity evaluation study. regarding differences in ranking rootstocks, dardeniz et al. (2006) indicated that 41b was the most salt resistant rootstock, followed by 140ru and p1103, and the least resistant was 5 bb. on the other hand, walker et al. (2002) showed that the highest salt resistance was obtained when p1003 was used as a rootstock. to determine whether nacl-induced stress and rootstock type were able to regulate the expression levels of genes responsible for antioxidant defense response, a semi-quantitative rt-pcr assay was performed for the analysis of six major antioxidant enzyme genes (apx, cat, cuzn-sod, mn-sod, gpx, and mdar) (fig. 1). table 1 primer pairs used in gene expression analysis gene genebank id primer pairs (5'→3') amplicon size (bp) ascorbate peroxidase eu280159 f: gacaatgaagcacccagaggag 542 (apx) r: aatgggcttcagcatagtcagc cuzn-superoxide dismutase af056622 f: ctgctccatctcgtgtctttct 452 (cuzn-sod) r: atccacaattgttgcttcagcc mn-superoxide dismutase np_001268135 f: agaaaatcgctagggttagggc 538 (mn-sod) r: tacccagcaatggaaccaagtt catalase af236127 f: aggcccagttcttcttgaggat 860 (cat) r: aggcaagcatctcattctcagc glutathione peroxidase xm_002272900 f: atgtcgaagcaaatacagcagg 472 (gpx) r: tgagaggggaagttgttgggta monodehydroascorbate reductase np_001267971 f: tcatgtttgtgttggaagcgga 868 (mdar) r: ggacagatcaaaggcacgagag elongation factor 1α xp_002277159 f: attgtggtcattggccatgttg 566 (ef-1α) r: ccttcgaaaccagagatgggaa fig. 1 semi-quantitative rt-pcr expression analysis of major antioxidant enzyme genes in ‘superior seedless’ grafted on different rootstocks and exposed to salinity for two weeks. the ef-1α gene was used as the internal control for normalization of loading. qrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. ii. expression of antioxidant genes 279 plants utilize antioxidant enzymes, such as: superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), ascorbate peroxidase (apx) and monodehydroascorbate reductase (mdar), as defense mechanisms against salinity and do have the ability to detoxify toxic ros (apel and hirt, 2004). their expression has been studied in many crops as previously mentioned in the introduction part. however, such knowledge is lacking in vitis species, specifically in vitis berlandieri and rupestris . meanwhile, their expression is extensively studied in both vinifera rootstocks and cultivars (carvalho et al., 2015). after salinity exposure for two weeks, the transcript levels of apx , mn-sod and mdar were increased in leaves of ‘superior seedless’ grafted on the different rootstocks. however, their expression levels in response to salinity were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat gene showed obvious enhanced level in plants grafted on p1103 in response to salt exposure. however, the expression of cat gene in ‘superior seedless’ scion grafted on 41b or r110 showed almost unchanged level in control and stressed conditions. down-regulation of cuzn-sod was recorded in leaves of ‘superior seedless’ grafted on p1103, while, slight up-regulation of this gene in response to saline condition was recorded when scion was grafted on 41b or r110. in response to salinity stress, the expression of gpx was enhanced in scion grafted on p1103 and 41b. on the other hand, scion grafted on r110 showed decreased expression of gpx in response to salt treatment. the expression of these genes was evaluated every two weeks, and only the results after the first two weeks of the stress treatment were presented since no differences in expression were noticed at the other time points between different treatments. this indicates early differential responses to salt stress at the level of antioxidant defense genes. such responses were previously reported in other plant species (ellouzi et al., 2014; ranjit et al., 2016). 4. conclusions rootstock choice is critical in determining grapevine performance and productivity under saline conditions. expression levels of apx, mn-sod and mdar were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat and gpx genes showed obvious enhanced level in plants grafted only on p1103 in response to salt exposure. cuzn-sod was down-regulated in leaves of ‘superior seedless’ grafted on p1103 and up-regulated when scion was grafted on 41b or r110. rootstocks with enhanced expression levels of antioxidant defense genes, such as superoxide dismutase (sod) and catalase (cat) can possibly eliminate or reduce detrimental effects of ros. thus, grapevine rootstocks that possess this defense line are more preferable to be utilized for soils subjected to salinity or grapevines irrigated with diluted brackish water. moreover, additional studies are needed to investigate the salt-stress responses of enzymatic and non-enzymatic antioxidant components in grapevine grafted on contrasting rootstocks. acknowledgements this research was funded by the scientific research support fund under the number agr/2/04/2011. we wish especially to acknowledge the help of ayah awad for her technical assistance. references abdel gawad g., ghaibeh a., 2001 use of low quality water for 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(cv. colombard) on a relatively saline soil. s. afr. j. enol. vitic., 12(1): 32-41. vasekina a., yershov p., reshetova o., tikhonova t., lunin v., trofimova m., babakov a., 2005 vacuolar na+/h+ antiporter from barley: identification and response to salt stress. biochemistry, 70(1): 100107. walker r., blackmore d., clingeleffer p., correll r., 2002 rootstock effects on salt tolerance of irrigated field grown grapevines (vitis vinifera l. cv. sultana). 1. yield and vigor inter-relationships. australian j. grape and wine res., 8: 3-14. zörb c., noll a., karl s., leib k., yan f., schubert s., 2005 molecular characterization of na+/h+ antiporters (zmnhx) of maize (zea mays l.) and their expression under salt stress. j. plant physiol., 162(1): 55-66. zrig a., mohamed h.b., tounekti t., khemira h., serrano m., valero d., vadel a.m., 2016 effect of rootstock on salinity tolerance of sweet almond (cv. mazzetto). south african journal of botany, 102: 50-59. impaginato 105 adv. hort. sci., 2018 32(1): 105-111 doi: 10.13128/ahs-22807 volatile compounds from different fruit parts of two cultivars of cydonia oblonga c. taiti 1, e. giordani 1, e. palm 1, w.a. petrucci 1, g. bennati 2, g. gestri 2, e. marone 3 (*), e. azzarello 1, s. mancuso 1 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 associazione oasi apistica le buche, via regina margherita, 26, 59016 poggio a caiano (po), italy. 3 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, via r. bazarini, 1, 64100 teramo, italy. key words: fruit tissues, proton transfer reaction time-of-flight mass spectrometer, physicochemical fruit parameters, quince, vocs. abstract: quince is characterized as a fragrant fruit which, unlike other pomes (apple, pear), is not used for fresh consumption due to its astringency and compactness, but only in its processed form (jams, jelly, distillery products, and nutraceutical compounds). as a consequence, there is little knowledge currently available concerning the characteristics of the fruit, and in particular its aromatic and chemotaxonomic patterns. in this work, carpometric, chemometric and spectrophotometric measurements were performed on quince fruits. vocs emitted by different tissues or parts of the fruit were studied to describe its aromatic profile. the study was carried out on the fruits of an old, well-known cultivar (‘gigante di wranja’, commonly called ‘wranja’) and a new tuscan accession. intact, halved and solely pulp (cubed) samples were evaluated for each individual fruit. data obtained from voc analysis through proton transfer reaction time-of-flight mass spectrometer (ptr-tof-ms) were evaluated by multivariate statistical analysis. the spectra obtained from the intact fruit samples showed a higher amount of masses corresponding to terpenes or terpenoid compounds, which fundamentally characterize the aroma of this type of fruit; these substances were found to be much less present in the vocs emitted by the pulp, where high values of masses linked to the maturation processes were instead found. 1. introduction plants are not only a dietary source for both human beings and animals but also serve as medicinal, nutritional, and ornamental purposes (*) corresponding author: emarone@unite.it citation: taiti c., giordani e., palm e., petrucci w.a., bennati g., gestri g., marone e., azzarello e., mancuso s., 2018 volatile compounds from different fruit parts of two cultivars of cydonia oblonga. adv. hort. sci., 32(1): 105-111 copyright: © 2018 taiti c., giordani e., palm e., petrucci w.a., bennati g., gestri g., marone e., azzarello e., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 december 2017 accepted for publication 6 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 105-111 106 (ashraf et al., 2016). the potential of plants as medicine is supported by abundant scientific evidence (lattanzio et al., 2009) and researchers are currently focused on isolating new aromas and active phytochemicals produced by different plant organs. quince is the fruit of an ancient tree, cydonia oblonga miller of the rosaceae family, and has a very wide origin area that ranges from anatolia to the caucasus. it was known to the greeks and romans, and it seems that the name of the genus cydonia comes from the ancient name of the town of chania on the island of crete (roversi, 1991). it is a climacteric, large-sized (up to 500 g) fruit, namely pome (false fruit), and generally oblong (pyriform types) or rounded (appleshared types) (bellini et al., 2007). the harvest is still carried out by hand, although the desire to mechanize this operation with systems similar to those used for the mechanical harvesting of the apple tree is increasing (fiorino et al., 2010). when ripening, the color of the fruit epidermis turns from greenish-yellow to bright yellow when fully ripe. the pulp is very firm, and not directly edible due to the presence of tannins and pectic substances (bellini et al., 2007). ripe fruits are commonly used as a source of pectin, and to produce jam or jelly, liquors, and distilled products with aromatic notes typical of quince (silva et al., 2004). the fruits can also be beneficial to human health thanks to their high phytochemical content (ashraf et al., 2016). many studies have reported cydonia oblonga as an excellent natural source of phenolic and flavonoid compounds which are considered potent antioxidants (silva et al., 2004; oliveira et al., 2007). quince fruit is considered an important dietary source of health-promoting compounds due to its antioxidant, antimicrobial and antiulcerative properties (magalhães et al., 2009). although the aroma is one its most important fruit-derived parameters, only a few studies have focused on the volatile emission by cydonia (tateo and bononi, 2010), and in particular, by different tissues of the fruit. indeed, the intense and pleasant aroma released by cydonia may represent an important starting point in the genetic improvement of this species. recently, proton transfer reaction-time of flightmass spectrometry (ptr-tof-ms) was proposed as an innovative analytical technology for volatile organic compound (voc) detection and quantification on fruit matrices due to its capacity to rapidly provide a comprehensive mass spectrum with high-time resolution and without sample treatment (mayr et al., 2003; taiti et al., 2017 a). the aim of the current study was to analyze the aroma profile of different fruit parts of two cydonia oblonga genotypes, both grown in italy, to increase the chemotaxonomic knowledge, fruit management and quince product trade. 2. materials and methods plant materials quince fruits belonging to an old, well-known cultivar (‘gigante di wranja’, commonly called ‘wranja’) and a new tuscan accession were harvested in tuscany (italy) in the last week of september 2017. for each cultivar, 12 homogeneous and healthy fruits were selected, gently brushed to eliminate the hair cover and washed in deionized water. fruits were stored in a climatic chamber (14±1°c, 85-90% relative humidity) for three days prior to laboratory analysis. for all the fruits, color, weight, maximum diameter and height were determined. six fruits were used for the spectrophotometric tests by ptr-tof-ms and the remaining six for the physicochemical measurements. for chemical analyses (lugol starch test and refractometric grade), fruits were divided in half so that one half of each fruit was used for the starch test and the other to evaluate refractometric grade. color and physicochemical fruit parameters peel color. the identification of the different peel color of quince fruits was assessed using a minolta cr-200 chroma-meter (minolta, ramsey, nj, usa) according to the hunter scale, as previously described by taiti et al. (2017 b). fruits dimensions and firmness. for each cultivar, the weight (g), maximum diameter (cm), and height (cm) of twelve samples were measured. the firmness of each fruit (expressed as kgf) was measured as the force needed to reduce fruit diameter by 2 mm using a penetrometer (model 53205d, turoni, italy). total soluble solids (tss). to evaluate tss concentration, six quince halved fruits for each genotype were squeezed to collect a few drops of juice; then tss levels were measured with an n1 atago refractometer (atago co., japan) and expressed as °brix. starch iodine test. starch-iodine test (lugol solution) was performed by visual evaluation on halved fruits and by scoring samples on a ‘golden delicious’ standardized 1-9 scale (smith et al., 1979). volatile compounds detection vocs analyses were carried out with a high-resotaiti et al. vocs from different fruit parts of two quince cultivars 107 lution ptr-tof-ms (ionicon analytik gmbh, austria) using h3o+ as ion donor. for each genotype, six fruits were used; the same fruit was first analyzed as intact whole, then divided into two parts, analyzing the halves together; finally, two cubes were got from each one of the halved fruit respectively, without peel (5 × 5 × 5 cm, about 10 g each one); all the measurements were performed in triplicates on the same sample. all whole and halved samples were weighed, and the vocs data were based on 100 g samples for whole and halved samples to allow for comparisons between the obtained results. the drift tube of ptr-tof-ms instrument was set to 80°c and operated with a drift pressure of 2.30 mbar and a voltage of 550 v. these settings lead to an e/n ratio (e, electric field strength in the drift tube; n, buffer gas number density in the drift tube) equal to 135 townsends (1 td = 1017 vcm2). mass/charge ratio of peaks detected at m/z 21 (signal for h3o+) and m/z 37 [signal for water clusters (h2o)h3o+] were monitored during all measurements to check the instrument’s stability and cluster ion formation. to reach a good mass accuracy (up to 0.001 th), internal calibration was based on three points and was performed off-line. acquisition and post-processing data were performed as reported by marone et al. (2017). statistical analyses one-way analyses of variance (anova) were performed (1) to compare the physicochemical parameters of two quince genotypes, and (2) to compare the voc emission profiles whole, halved, and cubed fruit samples. separation of means was performed by the fisher’s lsd test (p= 0.05). computations were performed by statgraphics centurion xv v. 15.0.04. a principal component analysis (pca) was applied to the whole spectral data of 36 quince samples, previously submitted to a logarithmic transformation and mean centering as pre-processing. computations were performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab_ r2015b (mathworks inc., natick, ma, usa). a correspondent analysis (ca) was applied to the spectral data of the 36 quince samples to build up a simultaneous ordination of quince samples and protonated m/z, thus facilitating the evaluation of their reciprocal relationships. the analysis preserves the χ2-distances between the data matrix rows and columns. data were weighted using the symmetric option (module hierclus, syn-tax 2000 program package). 3. results and discussion physicochemical analyses the results of the physicochemical analyses on the different cultivars are reported in table 1, and are largely in agreement with a previous study on different cultivars (leonel et al., 2016). among the physicochemical parameters analyzed, some significant differences were observed between the two cydonia accessions, with regard to firmness, °brix values and starch contents. in particular, the °brix content was lower for ‘wranja’ (12.2±0.9) compared to tuscan accession (17.2±0.9); vice versa the starch content was higher for ‘wranja’. the starch and sugar contents measured for the tuscan accession indicate that these fruits may have been further along in the ripening process than the ‘wranja’ fruits. vocs headspace analysis samples (whole, halved and cubed) belonging to two different cydonia genotypes were analyzed by ptr-tof-ms detecting a total of 67 peak signals (fig. 1). these signals represent different groups of volatile compounds including hydrocarbons, esters, alcohols, terpenoids, aldehydes, ketones and lactones. these results show that, even if no different volatile compounds were identified, different emission intensities were found between the two genotypes (fig. 1). on the other hand, among fruit tissues, both differences in number and emission intensity of each detected compound were instead observed. figure 1 shows that for both cultivars, the intensity and number of compounds emitted were higher in table 1 one-way analysis of variance (anova) related to the quince fruits physicochemical parameters (average ± sd) different lowercase letters within a column indicate differences by the lsd test at the 95% confidence level (p= 0.05). cydonia accessions weight (g) ø (mm) high (mm) firmness (kgf) °brix starch overcolor background color l* a* b* l* a* b* wranja 268.9 82.8 77.8 6.1 12.2 4.25 77.7 -12.5 62.6 72.6 -15.9 58.4 ±54.5 ±7.0 ±4.5 ±0.9 a ±0.9 a ±0.5 a ±2.4 ±2.4 ±3.3 ±4.1 ±2.3 ±3.1 tuscan 320 86.5 85.4 7.6 17.2 2.75 76.9 -13.1 57.6 76.6 -12.8 60 ±92.6 ±9.2 ±9.7 ±0.4 b ±0.9 b ±0.5 b ±4.7 ±2.3 ±5.6 ±2.6 ±2.2 ±2.9 adv. hort. sci., 2018 32(1): 105-111 108 the whole fruit followed by halved fruit, and lowest in cubed fruit. these results are in agreement with a previous study of imayoshi et al. (1995) on nashi pear, where differences in the total number of vocs among pulp, peel, and whole fruit were found. indeed, as reported by paillard (1981) for many fruits species, voc production changes among different fruit tissues, being highest in the skin and nearby tissues. it is noteworthy that fruit’s aroma depends on the combination of vocs produced, and on the concentration and odor threshold of each in the blend. a further investigation of the spectral data was performed by a multivariate ordination. the pca (fig. 2) approach applied to the whole dataset (ppbv) of 67 detected protonated masses (data not shown) give a general view of the quince sample ordination, based on the analysis of defined fruits parts (intact, halved, and cubed fruits) related to the two chosen accessions (tuscan accession and ‘wranja’). the first three components accounted for 96.4% of the total variability (66.1%, 17.7%, 12.7%, respectively). a strong effect related to the type of the sample used rather than the cultivar is evident. the whole fruit samples of both cultivars, joined in the lower right quadrant of the diagram, strongly differ from those obtained from the head space of the pulp only, both correlated with the negative part of the x-axis (pc1), but clustered based on the cultivar, and respectively related to the positive part (tuscan accession) and to the negative part (wranja) of the y-axis (pc2). the halved fruit group is placed in the upper right quadrant, with the two cultivars partially overlapped, indifig. 1 typical ptr-ms mass spectrum (average, n=6) obtained by different fruit parts of two cultivars of cydonia. (a) tuscan accession, (b) ‘wranja’; (1) whole fruit, (2) halve fruit, (3) cube fruit. fig. 2 pca scores of the quince samples based on the full spectra distribution of the ‘tuscan accession’ and ‘wranja’. (a1) = ‘tuscan accession’ whole fruits; (a2) = ‘tuscan accession’ halved fruits; (a3) = ‘tuscan accession’ cube fruits; (b1) = ‘wranja’ whole fruits; (b2) = ‘wranja’ halved fruits, (b3) = ‘wranja’ cube fruits. taiti et al. vocs from different fruit parts of two quince cultivars 109 cating a greater similarity of masses with the intact whole fruit, even if positively correlated with pc2. to better evaluate the effect of specific chemical family compounds, the 20 masses with statistically significant (p= 0.05) discriminating power among the different parts of the fruit were selected by anova within the whole spectrum (table 2), upon which a correspondence analysis was subsequently applied. the join plot from ca (fig. 3) simultaneously displays quince different sample and protonated m/z ordinations based on the results of the variable selection. the intact fruits of both cultivars are characterized by a group of masses in which terpenes and terpenoids are well represented. in particular, the highest presence of monoterpene (m/z 135, m/z 137), terpenoid (m/z 153) and sesquiterpene (m/z 205) compounds distinguished the whole fruit from the other two types of cut samples. according to taiti et al. (2017 b), the higher emission intensity and number of vocs detected in whole fruits was associated to the presence of the peel. it is well known that a great diversity of vocs linked to the pericarp tissue of the peel are emitted, such as esters, alcohols, aldehydes and terpenes (chervin et al., 2000; rodriguez et al., 2013). the vocs linked to cutting, such as the green leaf volatiles (glvs) (i.e., hexenal, hexanal), are highest in halved and cubed fruits, compared to intact fruits where the intensities of these signals were very low (table 2), and confirmed by the fact that these compounds were well represented in pulp tissue. moreover, halved fruits occupy an intermediate position, while cubed fruit samples are shifted to the right in the diagram (fig. 3), with a differentiation along the axis of the y (axis 2) determined by the masses m/z = 45.033, m/z = 47.049 and m/z 33.033 (only for the tuscan accession). as reported by taiti et al. (2015) the methanol emission in fruits increase table 2 protonated selected masses discriminating whole, halved and cubed fruit samples, identified via ptr-tof-ms: mass/charge (m/z) ratios, chemical formula, tentative identifications, minimum and maximum values detected (ppbv) and voc, as previously reported in the literature (cydonia*; ptr-ms#) no. protonated m/z chemical formula tentative identification min detected value (ppbv) max detected value (ppbv) references 1 33.033 ch 5 o+ methanol 11.7 2724.1 khoubnasabjafari and jouyban (2011)* 2 45.033 c 2 h 5 o+ acetaldehyde 35 16416.9 umano et al. (1986)* 3 47.049 c 2 h 7 o+ ethanol 1.5 199.8 umano et al. (1986) 4 55.054 c 4 h 7 + c4 aldehydes fragment 65.3 433.1 taiti et al. (2017 b)# 5 57.033 c 3 h 5 o+ alkyl fragment (hexanal/hexyl acetate) 8.5 25.4 taiti et al. (2017 b)# 6 57.069 c 4 h 9 + alkyl fragment (hexanol/valeric acid) 18.1 75.2 taiti et al. (2017 b)# 8 75.044 c 4 h 11 o+ 2-butanol/2-methyl-1-propanol 0.5 19.7 umano et al. (1986)* 9 81.069 c 6 h 9 + terpenes and c6 fragments 49.8 203.5 maleknia et al. (2007) # 10 83.085 c 6 h 11 + c6 fragments /hexenol fragment 8.8 50.7 maleknia et al. (2007)# 11 85.064 c 5 h 9 o+ 2-methyl-2-butenal/1-penten-3-one 4.6 26.2 khoubnasabjafari and jouyban (2011)* 12 99.080 c 6 h 11 o+ (e)-2-hexenal 1.6 8.9 umano et al. (1986)* 13 101.096 c 6 h 13 o+ hexanal/(z)-3-hexen-1-ol 1.7 44.8 tateo and bonomi (2010)* 14 109.101 c 8 h 13 + terpene fragments 0.4 23.6 maleknia et al. (2007) # 15 117.091 c 6 h 13 o 2 + isobutyl acetate/ethyl butyrate/butyl acetate 1.5 176.7 tateo and bonomi (2010)* 16 135.116 c 10 h 15 + p-cymene 0.4 11.9 tateo and bonomi (2010)* 17 137.132 c 10 h 17 + monoterpenes (e.g. limonene, g-terpinene) 0 5.1 tateo and bonomi (2010)* 18 145.122 c 8 h 17 o 2 + ethyl hexanoate 0.5 153.9 tateo and bonomi (2010)* 19 153.127 c 10 h 17 o+ oxygenated terpenes (e.g. geranial) 0 0.4 tsuneya et al. (1983)* 20 205.195 c 15 h 25 + sesquiterpenes (e.g. bergamotene) 1.1 49.8 tateo and bonomi (2010)* fig. 3 join plot from correspondence analysis. (a1) = ‘tuscan accession’ whole fruits; (a2) = ‘tuscan accession’ halved fruits; (a3) = ‘tuscan accession’ cube fruits; (b1) = ‘wranja’ whole fruits; (b2) = ‘wranja’ halved fruits, (b3) = ‘wranja’ cube fruits. numbers correspond to the protonated m/z. adv. hort. sci., 2018 32(1): 105-111 110 steadily throughout the ripening process. on the other hand, ethanol is the product of anaerobic metabolism and, together with acetaldehyde, accumulates in pome fruit under imposed hypoxia and poor gas exchange in the pulp tissue (pinto et al. 2001). pome fruit contains ethanol and acetaldehyde as part of their aroma (ritenour et al. 1997) and as reported by rapparini and predieri (2003) these compounds increase together with the ripening process and their emission increased at a faster rate with the onset of fruit senescence. 4. conclusions the typical quince fruit aroma depends on a mix of volatile compounds which originate by different parts of the pome. by the analyses of different fruit tissues, differences in vocs number and in their emission intensity were observed, while little difference was observed between the two varieties analyzed. moreover, it has been shown that the intensity and number of compounds emitted was highest in the whole fruit, followed by halved fruit and cubed fruit. through multivariate analysis, some masses have been highlighted that determine the differentiation of the spectral composition between the vocs emitted by the pulp (halved and cubed fruit samples) and those obtained from intact fruit that could be specifically responsible for the particular aroma of quince fruit. the four masses referring to terpenes and terpenoids are prevalent in the peel, and are moderately related to the cultivar. further work should be done to increase the knowledge concerning the chemotaxonomic profiles of the different cultivars, and to understand how through this tool, innovations can be developed and moved to quince genetically improved and to the food industry. references ashraf m.u., muhammad g., hussain m.a., bukhari s.n., 2016 cydonia oblonga m., a medicinal plant rich in phytonutrients for pharmaceuticals. frontiers in pharmacology, 7: 163. bellini e., giordani e., giannelli g., picardi e., 2007 quince. cydonia oblonga mill., pp. 439-457. in: bellini e. (ed). the fruit woody species. descriptor list. arsia regione toscana, press service, sesto fiorentino, firenze, italy, pp. 1070. chervin c., speirs j., loveys b., patterson b.d., 2000 influence of low oxygen storage on aroma compounds of whole pears and crushed pear flesh. postharvest biol. technol., 19(3):279-285. fiorino p., marone e., ottanelli a., 2010 mechanical harvesting, productivity and superintensive planting systems in olive groves adv. hort. sci., 24(1): 91-94. imayoshi y., yukawa c., iwabuchi h., baser k.h.c., 1995 volatile constituents of chinese pear ‘yali’. proc. 13th int. congress of flavours, fragrances and essential oils, turkey, 2: 15-19. khoubnasabjafari m., jouyban a., 2011 a review of phytochemistry and bioactivity of quince (cydonia oblonga mill.). j. med. plants res., 5(16): 3577-3594. lattanzio v., kroon p.a., linsalata v., cardinali a., 2009 globe artichoke: a functional food and source of nutraceutical ingredients. j. func. foods, 1: 131-144. leonel m., leonel s., tecchio m.a., mischan m.m., moura m.f., xavier d., 2016 characteristics of quince fruits cultivars (cydonia oblonga mill.) grown in brazil. australian j. crop sci., 10(5): 711. magalhães a.s., silva b.m., pereira j.a., andrade p.b., valentao p., carvalho m., 2009 protective effect of quince (cydonia oblonga miller) fruit against oxidative hemolysis of human erythrocytes. food and chemical toxicology, 47(6): 1372-1377. maleknia s d., bell t.l., adams m.a., 2007 ptr-ms analysis of reference and plant-emitted volatile organic compounds. inter. j. mass spectrometry, 262(3): 203210. marone e., masi e., taiti c., pandolfi c., bazihizina n., azzarello e., fiorino p., mancuso s., 2017 sensory, spectrometric (ptr-tof-ms) and chemometric analyses to distinguish extra virgin from virgin olive oils. j. food sci. technol., 54(6): 1368-1376. mayr d., mark t., lindinger w., brevard h., yeretzian c., 2003 breath-by-breath analysis of banana aroma by proton transfer reaction mass spectrometry. inter. j. mass spectrometry, 223: 743-756. oliveira a.p., pereira j.a., anddrade p.b., valentao p., seabra r.m., silva b.m., 2007 phenolic profile of cydonia oblonga miller leaves. j. agric. food chem., 55: 7926-7930. paillard n.m.m., 1981 factors influencing flavour formation in fruits, pp. 479-507. in: schreier p. (ed.) flavour ’81. 3rd weurman symposium proceedings of the international conference, munich, april 28-30, walter de gruyter, berlin, new york. pinto k., lentheric i., vendrell m., larrigaudiere c., 2001 role of fermentative and antioxidant metabolism in the induction of core browning in controlledatmosphere stored pears. j. sci. food agr., 81: 364370. rapparini f., predieri s., 2003 pear fruit volatiles. hort. reviews, 28: 237-324. ritenour m.a., mangrich m.e., beaulieu j.c., rab a., saltveit m.e., 1997 ethanol effects on the ripening of climacteric fruit. postharvest bioi. technol., 12: 35-42. taiti et al. vocs from different fruit parts of two quince cultivars 111 rodriguez a., alquezar b., pena l., 2013 fruit aromas in mature fleshy fruits as signals of readiness for predation and seed dispersal. new phytologist, 197(1): 36-48. roversi a., 1991 cotogno, pp. 164-172. in: avanzato d., g. barbera, g. bargioni, e. bellini, a. bergamini, f. calabrese, a. de michele, r. di lorenzo, e. faedi, c. fideghelli, g. fontanazza, a. godini, r. guerriero, o. insero, f. monastra, g. paesano, r. paglietta, a. roversi, g. tamponi, and a. tombesi frutticoltura speciale. reda, roma, italy, pp. 783. silva b.m., andrade p.b., valentao p., ferreres f., seabra r.m., ferreira m.a., 2004 quince (cydonia oblonga miller) fruit (pulp, peel, and seed) and jam: antioxidant activity. j. agric. food chem., 52: 44054712. smith r.b., lougheed e.c., franklin e.w., mc millan i., 1979 the starch iodine test for determining stage of maturation in apples. can. j. plant sci., 59: 725-735. taiti c., colzi i., azzarello e., mancuso s., 2017 a discovering a volatile organic compound fingerprinting of pouteria lucuma fruits. fruits, 72(3): 131-138. taiti c., costa c., menesatti p., caparrotta s., bazihizina n., azzarello e., petrucci a.w., masi e., giordani e., 2015 use of volatile organic compounds and physicochemical parameters for monitoring the post-harvest ripening of imported tropical fruits. european food res. technol., 241(1): 91-102. taiti c., marone e., lanza m., azzarello e., masi e., pandolfi c., giordani e., mancuso s., 2017 b nashi or williams pear fruits? use of volatile organic compounds, physicochemical parameters, and sensory evaluation to understand the consumer’s preference. european food res. technol., 243(11): 1917-1931. tateo f., bononi m., 2010 headspace-spme analysis of volatiles from quince whole fruits. j. essential oil res., 22(5): 416-418. tsuneya t., ishihara m., shiota h., shiga m., 1983 volatile components of quince fruit (cydonia oblonga mill.). agric. biol. chem., 47(11): 2495-2502. umano k., shoji a., hagi y., shibamoto t., 1986 volatile constituents of peel of quince fruit, cydonia oblonga miller. j. agric. food chem., 34(4), 593-596. impaginato 35 adv. hort. sci., 2020 34(1): 35­42 doi: 10.13128/ahsc­7651 different substrates for seedling production of euterpe oleracea mart. o.j. smiderle 1, a.g. souza 2 (*), r.d. menegatti 3, t.j. silva 4 1 brazilian agricultural research corporation embrapa roraima, boa vista, brazil. 2 federal university of paraíba/ufpb, campus ii areia‐pb, brazil. 3 federal university of pelotas, capão do leão, brazil. 4 federal university of roraima, ufrr, brazil. key words: alternative substrates, morphological characteristics, seedling pro­ duction. abstract: the aim of this study was to evaluate the effect of substrates involv­ ing different combinations of soil, cattle manure, burnt rice husks, sand and commercial substrate on growth in the açai palm (euterpe oleracea mart.) under nursery conditions in the state of roraima. the experiment was conduct­ ed in the fruit­farming sector of embrapa roraima, located in the district of boa vista. the experimental design was completely randomised, with nine treatments and four replications of five plants per replication. the treatments were t1: sand, t2: commercial substrate (organoamazon©), t3: 25% t1 + 75% manure, t4: 50% t1 + 50% manure, t5: 75% t1 + 25% manure, t6: 25% t1 + 75% burnt rice husks, t7: 50% t1 + 50% burnt rice husks, t8: 75% t1 + 25% burnt rice husks and t9: 25% soil + 25% sand + 25% manure + 25% burnt rice husks. the morphological characteristics plant height (h), stem diameter (sd) and number of leaves (nf) were evaluated every 30 days from transplanting to the end of the experiment (210 days). the root dry weight (rdw), shoot dry weight (sdw), total dry weight (tdw) and the dickson quality index (dqi) were obtained at 210 days. the substrates composed of 50% sand + 50% manure and 25% soil + 25% sand + 25% manure + 25% burnt rice husks are indicated for the production of seedlings of euterpe oleracea mart., as they provide superior growth in plant height and stem diameter, and improve total dry­weight pro­ duction in the plants. 1. introduction the species euterpe oleracea mart., known throughout the world as açaí, is a palm tree, native to the brazilian amazon, belonging to the arecaceae family (oliveira et al., 2019). it has recently been the target of several studies, as it is a functional food rich in proteins, fibre, lipids, vita­ mins and minerals, and has antioxidant properties, making it an indis­ pensable part of the most varied diets and recommended to those who practise physical exercise and enthusiasts of healthy living (kang et al., 2010; oliveira et al., 2019). according to honorio et al. (2017), the açai palm is considered a species with multiple uses, however, its economic (*) corresponding author: souzaufpel@gmail.com citation: smiderle o.j., souza a.g., menegatti r.d., silva t.j., 2020 ­ different substrates for seed‐ ling production of euterpe oleracea mart. ­ adv. hort. sci., 34(1): 35­42. copyright: © 2020 smiderle o.j., souza a.g., menegatti r.d., silva t.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 december 2019 accepted for publication 10 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 35­42 36 potential is mainly related to marketing the fruit and stems, the origin of the palm heart. since brazil is the largest producer, consumer and exporter of these products, large­scale cultivation is attractive to both industry and rural producers, who can obtain a varied income from the products and by­products through­ out the year (monteiro et al., 2019). considering the health benefits as well as the high added value of the products of this species, studies that contribute with information concerning cultiva­ tion are extremely relevant, especially as the açai palm occurs naturally in such areas as low­lying plains and flooded woodlands that have unique cli­ mate conditions which can compromise the initial planting stage of the crop if quality seedlings are not used (neves et al., 2019). both the planting and maintenance of homogeneous plantations of the açai palm therefore require the acquisition of vigorous seedlings to guarantee a return on investment and success in establishing the plantation; for this, it is necessary to improve the techniques for producing açai seedlings of a high commercial standard (silva et al., 2017). the production of quality seedlings depends on various factors such as the composition of the sub­ strate, which has the role of supporting the plants and providing suitable chemical and physicals condi­ tions for the initial development of the roots and shoots (bilderback et al., 2005). normally, the large­ scale production of seedlings makes use of commer­ cial substrates; however, besides the high cost of acquiring the substrate, the great majority contain only small concentrations of nutrients and require the use of fertilisers, a factor that increases the costs of the activity (olle et al., 2012). as an alternative, several authors propose the use of substrates consisting of agricultural by­products that can be used mixed with commercial substrate or other products, for example, rice husks, charcoal, coffee chaff and animal manure, as well as by­prod­ ucts from agroindustry, which vary according to the region (rinaldi et al., 2017). according to olle et al. (2012), the choice of sub­ strate, in addition to considering the cost of acquisi­ tion and availability for seedling production, should be based on technical and scientific results that show the material is able to promote high rates of initial plant growth and survival after planting; this will reduce the costs of establishing the crop, making it possible to expand and/or set up new plantations. there are several materials that can be used as sub­ strates for the production of seedlings of forest species, either alone or in combination, such as sand, soil, expanded clay, vermiculite, sawdust, rice husk, pine bark, bark fiber among others (olle et al., 2012). according to silva et al. (2018) when compared to exclusive use, the combination of different materials may result in satisfactory results, especially regarding the maximization of seedling growth, a fact possibly related to the combination of factors that favorfavor­ able conditions for availability, absorption, transloca­ tion, and nutrient use by plants. based on the above, the aim of this study was to evaluate the effect of substrates involving different combinations of soil, cattle manure, burnt rice husks, sand and commercial substrate on growth in the açai palm (euterpe oleracea mart.) under nursery condi­ tions in the state of roraima. 2. materials and methods the experiment was conducted in the fruit­ farming sector of embrapa roraima (at 2°23’45.31” n and 60°58’44.34” w), located in the district of boa vista in the state of roraima. mature fruit of the açai were harvested in the town of anori, in amazonas, and taken to the seed laboratory of embrapa roraima for the experiment. the fruit was first pulped with the help of a pulp processor for mechanical extraction of the seeds, which were then washed in running water until the residue was completely eliminated, and kept at room temperature. the propagating material was later sown in a bed containing washed sand as a substrate for germination and initial development of the seedlings. substrate moisture was maintained by manual irrigationwith distilled water, four times a day. the process of seedling emergence began around 30 days after sowing. once the seedlings had reached a height of approximately 2.0 cm, they were trans­ planted into polyethylene bags (17 x 22 cm) contain­ ing different combinations of commercial substrate, soil, cattle manure, burnt rice husks and sand accord­ ing to the predetermined treatments. the plants were then housed in a nursery under 50% shading, and irrigated by sprinkler for five minutes, three times a day. the experimental design was completely ran­ domised (crd), with nine treatments, four replica­ tions and five plants per replication, for a total of 180 plants. the treatments were t1: sand, t2: commer­ cial substrate (organoamazon©), t3: 25% t1 + 75% smiderle et al. ‐ substrates effects on euterpe oleracea 37 manure, t4: 50% t1 + 50% manure, t5: 75% t1 + 25% manure, t6: 25% t1 + 75% burnt rice husks, t7: 50% t1 + 50% burnt rice husks, t8: 75% t1 + 25% burnt rice husks and t9: 25% soil + 25% sand + 25% manure + 25% burnt rice husks. according to silva et al. (2009), a composite sample of substrate for each treatments was collected, air­dried and sieved through a 2.0 mm mesh, for chemical characteriza­ tion, whose results are presented in table 1. the morphological characteristics of plant height (h), stem diameter (sd) and number of leaves (nl) were evaluated every 30 days from transplanting to the end of the experiment. plant height was mea­ sured with a ruler graduated in centimetres (cm), and considered the height of the plant from the surface of the soil to the apex of the plant. the stem diame­ ter was measured with the aid of a digital calliper in millimetres (mm), 1 cm above the surface of the sub­ strate. at the end of the experiment (210 days after transplanting), the following plant characteristics were evaluated: shoot dry weight (sdw), root system dry weight (rdw) and total dry weight (tdw). for this, the plants were removed from the polyethylene bags, the roots were separated from the substrate by washing under running water and the shoots then separated from the root system. to obtain the dry weight, the two materials were placed in a forced air circulation oven at ±70°c to constant weight. the dickson quality index (dqi) was then determined (dickson et al., 1960). the data were submitted to analysis of variance (anova) and the mean values of the treatments compared by tukey’s test at 5% prob­ ability with the aid of the sisvar software. 3. results and discussion the analysis of variance revealed significant differ­ ences between the plants submitted to the different combinations of substrates for all the morphological characteristics under evaluation (table 2), demon­ strating that the different combinations can have a direct influence on the growth characteristics of plants of the açai palm (euterpe oleracea mart.) under nursery conditions. it can be seen from the result of the mean­value comparison test that the plants grown in t4, compris­ ing 50% sand + 50% cattle manure, obtained on aver­ age greater or statistically equal values for all the morphological characteristics under evaluation. a similar result, except for the variable sd that was slightly lower, was obtained for the plants submitted to t9, a substrate consisting of a mixture of 25% soil + 25% sand + 25% manure + 25% burnt rice husks. among the components in the treatments (t4 and t9) that resulted in better results for all the morpho­ logical characteristics under evaluation, cattle manure and sand should be highlighted. these results reinforce those obtained in an experiment by menezes and oliveira (2009) with the organic pro­ duction of açai seedlings; the authors determined that the greatest growth in seedling height was obtained in the substrate containing cattle manure. honorio et al. (2017), carrying out the test for ger­ mination and emergence in seeds of the açaí in alter­ native substrates, suggested a combination of cattle manure + sand (1:1) as the most efficient in promot­ ing the variables of physiological seed quality. araujo table 1 ­ chemical composition of the different combinations of soil, cattle manure, rice husks, and commercial substrate using açaí seedlings (euterpe oleracea mart.) growing seedlings, under nursery conditions * t1= sand, t2= commercial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks. treatments h potential om dag/kg k mg/dm³ p mg/dm³ ca cmol/dm³ mg cmol/dm³ al cmol/dm³ h+al cmol/dm³ zn mg/dm³ fe mg/dm³ mn mg/dm³ cu mg/dm³ b mg/dm³ s mg/dm³ t1 * 6.7 2.6 108.0 145.0 10.4 0.5 0.0 1.0 26.7 40.4 139.0 1.1 0.6 18.8 t2 5.7 10.0 312.0 263.9 13.8 7.4 0.0 1.9 26.9 62.3 160.2 0.6 0.7 49.1 t3 5.8 6.2 112.0 314.9 10.2 5.0 0.0 1.7 24.4 13.5 90.9 0.6 0.8 50.7 t4 6.5 4.0 92.0 218.2 10.0 2.9 0.0 1.3 23.5 20.3 107.0 0.8 0.8 34.9 t5 6.2 3.8 92.0 151.2 9.9 1.6 0.0 1.2 24.3 27.9 111.2 1.0 0.7 25.9 t6 6.1 4.9 106.0 71.7 12.2 1.4 0.0 1.9 16.5 13.5 88.6 0.3 0.5 17.2 t7 6.6 4.3 120.0 93.0 11.0 0.9 0.0 1.1 20.8 18.0 127.3 0.6 0.6 17.7 t8 6.7 3.7 122.0 132.9 11.0 0.7 0.0 1.1 23.3 33.5 132.3 0.8 0.4 19.4 t9 6.4 4.1 120.0 170.2 9.9 1.8 0.0 1.2 23.1 15.1 100.5 0.7 0.6 28.1 adv. hort. sci., 2020 34(1): 35­42 38 et al. (2019) describes how cattle manure is a basic substrate for obtaining vigorous seedlings, since its main function is to improve water retention, in addi­ tion to supplying macro and micronutrients and increasing their availability to the plants, resulting in improvements in seedling performance. according to olle et al. (2012), another basic com­ ponent to be used in small proportions when com­ bining substrates for plants is sand, as it provides the ideal conditions for aerating the substrate and gives good drainage. furthermore, promoting the use of this material is based on its low cost and easy avail­ ability in some regions. the superior results obtained for rdw, sdw, tdw and dqi in the plants submitted to t3, com­ posed of 25% sand + 75% cattle manure, should also be pointed out. according to menegatti et al. (2019), the dqi is a good indicator of plant quality, since robustness and the balance of biomass distribution in the plants are considered in its calculation and it includes several parameters which are regarded as important; the higher its value, the better the quality standard of the seedling. however, since the performance of the plants in t3, despite an expressive dqi, was poor for the other characteristics under analysis (h and sd), and consid­ ering their importance in evaluating seedling quality, this shows the need for combining characteristics to obtain a better evaluation. conversely, plants submitted to treatment t1, comprising 100% sand, displayed inferior values for all the morphological characteristics under evalua­ tion. as mentioned above, sand is a conditioner to be used in small proportions in the composition of a substrate, as it is practically an inert material and not associated with any nutrients that can be made avail­ able to the plant. in addition, sand shows high drainage potential, which may reduce water avail­ ability to the plant, making it impossible to maintain all the physiological processes essential to growth. furthermore, according to smiderle et al. (2015), high­density materials such as sand, when used alone or in large proportions within a mixture, are unsuit­ able due to their excessive weight, which makes it difficult to handle the plants in their containers and to trade or transport them to their final planting site. similar to the results obtained for the plants sub­ mitted to the substrate of 100% sand, the t7 sub­ strate, comprising a mixture of 50% sand + 50% burnt rice husks also resulted in poor plant performance for the morphological characteristics under evalua­ tion. according to smiderle et al. (2015), caution is needed when using combinations of burnt rice husks and sand in the composition of plant substrates, since both materials allow high water drainage and may result in water deficiency in the plants, which would compromise the processes of photosynthesis and respiration and consequently the maintenance of cellular elongation, resulting in smaller plant growth (souza et al., 2020). table 2 ­ mean values for plant height, stem diameter, number of leaves, root dry weight, shoot dry weight, total dry weight and dickson quality index, in açaí seedlings (euterpe oleracea mart.) grown in different combinations of substrates under nursery * mean values followed by the same lowercase letter in a column do not differ at 5% probability by tukey’s test. t1= sand, t2= commer­ cial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks. substrate plant height (cm) stem diameter (mm) number of leaves root dry weight (g plant­1) shoot dry weight (g plant­1) total dry weight (g plant­1) dickson quality index t1 8.50 e * 3.77 f 3 e 2.14 c 2.39 c 4.53 c 1.34 c t2 16.47 bc 6.80 c 5 b 6.67 bc 2.09 c 8.76 bc 1.56 c t3 17.43 b 6.18 d 4 c 25.99 a 20.90 a 46.89 a 12.94 a t4 24.19 a 8.21 a 5 a 28.62 a 13.76 ab 42.38 a 12.37 a t5 15.35 c 5.77 d 4 c 10.24 b 9.39 bc 19.63 b 5.49 b t6 11.47 d 5.00 e 4 d 5.05 bc 4.46 c 9.50 bc 2.99 c t7 9.71 de 3.88 f 3 e 3.50 c 2.82 c 6.32 bc 1.91 c t8 11.02 d 3.93 f 3 e 4.37 bc 3.17 c 7.54 bc 2.14 c t9 24.16 a 7.57 b 5 a 29.00 a 21.26 a 50.26 a 12.81 a mean 15.37 5.68 4.00 12.84 8.91 21.76 5.95 cv % 22.01 16.06 14.76 24.16 29.91 31.38 27.10 smiderle et al. ‐ substrates effects on euterpe oleracea 39 capacity for the formation and growth of new roots. according to nascimento et al. (2019), the substrate that provides the seedlings with a balance between stem diameter growth and height, also provides greater robustness and more resistance to the adverse conditions found in the field, resulting in a higher survival rate and consequently reducing the costs of replanting. as a result of the water deficit and the reduction in photosynthetic rates, the plants accumulate a smaller amount of biomass, thereby showing a reduction in total dry mass ­ the behaviour shown by the plants in t1 and t7 (4.53 and 6.32 g plant­1 respectively). the plants grown in these two sub­ strates had a total dry mass of less than around 6.5 times the dry mass found in the plants from the treatments considered superior, t9 and t4 (50.26 and 42.38 g plant­1 respectively). the total dry weight is the sum of sdw and rdw; the higher this value, the better the quality of the produced seedlings (adamipour et al. , 2019). according to chiomento et al. (2019), the total dry weight indicates the hardiness of a seedling, with the highest values representing more lignified and hardier seedlings, which may take hold faster under field conditions soon after planting. it should be emphasized that the satisfactory mor­ phological characteristics showed by plants grown in the commercial substrate (organoamazon©) (t2) can be related to the chemical composition of the sub­ strate, which showed higher levels than the other treatments for the macro and micronutrients, includ­ ing organic matter (om), which when decomposed tends to release nutrients, especially nitrogen, and other mineral elements such as phosphorus, magne­ sium, calcium, sulphur (aalipour et al., 2019). however, it is also assumed that this substrate did not result in plants with superior morphological char­ acteristics to the other plants due to the ph of the substrate, which was less than 6. according to aalipour et al. (2019), soils with a ph below 6 tend to reduce the availability of some nutri­ ents, compromising the absorption and supply of the required amount of each element to enhance some of the physiological processes that culminate in growth. the growth in plant height and stem diameter of the plants cultivated in different combinations of substrates throughout the period of the experiment can be seen in figure 1 and 2, respectively. all the curves, irrespective of the morphological characteris­ tic under evaluation, show a linear trend, but the plants in t4 and t9 showed faster growth when com­ pared to the plants of the other treatments being tested, achieving greater values for these variables by the end of the experiment (table 3). chiomento et al. (2019), among the morphologi­ cal characteristics under evaluation, the stem diame­ ter is the most indicated for evaluating the survival capacity of the seedling in the field, due to a greater fig. 1 ­ effect of different combinations of substrates on plant height in açai seedlings (euterpe oleracea mart.) under nursery conditions. t1= sand, t2= commercial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks (n=4). fig. 2 ­ effect of different combinations of substrates on stem diameter in açai seedlings (euterpe oleracea mart.) under nursery conditions. t1= sand, t2= commercial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks (n=4). 40 adv. hort. sci., 2020 34(1): 35­42 according to silva et al. (2017), the presence of manure may have improved both the structural char­ acteristics of the soil and the water retention, and may also have acted as a nutrient reservoir, including larger quantities of nitrogen, nutrient which may be indirectly discriminated for variable o.m. (table 1). for figueiredo et al. (2019), n is an essential element for components of the photosynthetic system, such as the chlorophylls, proteins and enzymes that allow satisfactory rates of carbon assimilation to be main­ tained (taiz et al., 2017; figueiredo et al., 2019), and thereby guarantee the production of photoassimi­ lates that drive plant growth. in turn, cattle manure mixed with soil has been widely used as a substrate for the production of seedlings of various species, as it provides ideal nutri­ tional conditions for plant development, demonstrat­ ing that the presence of organic material in the sub­ strate may be decisive in the growth of seedlings in the nursery and better initial start­up in the field (steffen et al., 2010; silva et al., 2017). considering further the relationship between the chemical characteristics of the substrate in each treatment and growth over time, it can be seen that treatments t1 and t7, which had lower values for these two morphological characteristics (h and sd), had low levels of organic matter and mg. this result can be understood in view of the mate­ rials used in the substrate of each treatment. t1, con­ sisting entirely of sand, can be described as a sub­ strate devoid of mineral nutrients; this may have compromised the production of several enzymes, proteins and other compounds which are indispens­ able to the metabolism of the plant. whereas t7, a substrate composed of a mixture of sand and burnt rice husks (1:1), induced higher val­ ues for the variables h and sd, these are not statisti­ cally superior to obtained for t1 (table 3 and 4), sug­ gesting that burnt rice husks, being an organic mate­ rial, may have provided the plants with a certain level of nutrients. however, according to smiderle et al. (2015), sand and burnt rice husks degrades relatively slowly, which together with the high drainage it exhibits, table 3 ­ mean values for plant height (cm), stem diameter (mm), number of leaves and nivel significative for treatments obtained in açaí seedlings (euterpe olera‐ cea mart.) grown in different combinations of substra­ tes under nursery conditions * means followed by the same small letter in the column do not differ from one another by the tukey test (p≤0.05%). t1= sand, t2= commercial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks. treatments plant heigh (average) stem diameter (average) number of leaves (average) t1 8.50 e * 3.77 f 3.24 e t2 16.47 bc 6.80 c 4.56 b t3 17.43 b 6.18 d 4.03 c t4 24.19 a 8.21 a 5.01 a t5 15.35 c 5.77 d 4.30 bc t6 11.47 d 5.00 e 3.71 d t7 9.71 de 3.88 f 3.13 e t8 11.03 d 3.93 f 3.23 e t9 24.16 a 7.57 a 4.97 a dms 1.78 0.48 0.31 error 0.40 0.11 0.07 cv % 22.01 16.06 14.76 table 4 ­ linear equations, r2 values and significance for the plant heigh, stem diameter and number of leaves of açai seedlings (euterpe oleracea mart.) grown in different combinations of substrates under nursery conditions ** significative for p<0.01; * significative for p<0.05. t1= sand, t2= commercial substrate (organoamazon©), t3= 25% t1 + 75% manure, t4= 50% t1 + 50% manure, t5= 75% t1 + 25% manure, t6= 25% t1 + 75% burnt rice husks, t7= 50% t1 + 50% burnt rice husks, t8= 75% t1 + 25% burnt rice husks, t9= 25% soil + 25% sand + 25% manure + 25% burnt rice husks. treatment plant heigh stem diameter number of leaves equation r2 (sign) equation r2 (sign) equation r2 (sign) t1 0.0286x + 5.0629 0.96 ** 0.0157x + 1.88 0.97 ** 0.0137x + 1.60 0.98 ** t2 0.1055x + 3.805 0.90 ** 0.0257x + 3.7129 0.95 ** 0.0139x + 2.8857 0.81 ** t3 0.1173x + 1.9929 0.90 ** 0.0311x + 2.4486 0.92 ** 0.0161x + 2.10 0.93 ** t4 0.177x + 2.95 0.98 ** 0.0403x + 3.3686 0.98 ** 0.0098x +3.828 0.47 * t5 0.0869x + 4.921 0.96 ** 0.0266x + 2.5514 0.98 ** 0.0130x + 2.728 0.51 * t6 0.0545x + 4.927 0.96 ** 0.0195x + 2.7043 0.98 ** 0.0121x + 2.257 0.83 ** t7 0.0358x + 5.4 0.89 ** 0.0191x + 1.5857 0.98 ** 0.0133x + 1.5286 0.99 ** t8 0.0411x + 6.1 0.89 ** 0.0205x + 1.4543 0.99 ** 0.0121x + 1.7714 0.98 ** t9 0.1799x + 2.571 0.95 ** 0.0452x + .1329 0.98 ** 0.0100x + 2.771 0.51 * smiderle et al. ‐ substrates effects on euterpe oleracea 41 may lead to leaching of the available nutrient con­ tent, explaining the smaller growth. it should be noted that the low levels of mg in treatments t1 and t7 may also have compromised plant growth in these substrates, since mg is the cen­ tral atom in the chlorophyll molecule. substrates with less available mg therefore tend to a reduced synthe­ sis of this molecule. the reduction in leaf chlorophyll decreases the capacity for light absorption, promoting the fixation of a smaller amount of carbon, which reduces the production of carbohydrates and conse­ quently reduces the energy for maintaining the rate of plant growth (cartelat et al., 2005). the number of remaining leaves per plant over the experimental period for each substrate under test can be seen in figure 3. the treatments showed different behaviour according to the materials used in the substrate. treatments t9 and t4 displayed a quadratic trend when adjusting the regression equa­ tions, showing that throughout the growing period the number of leaves per plant tend to decrease due to the fall of older leaves. these results suggest that plants submitted to these treatments (t4, consisting of 50% sand + 50% cattle manure, and t9, a substrate consisting of 25% soil + 25% sand + 25% manure + 25% burnt rice husks) throughout the development period, employ a greater amount of energy for growth in h and rd, instead of increasing the number of leaves. a reduction in the number of leaves during plant growth can be beneficial, since after transplanting the seedlings in the field, the smaller number of leaves per plant will result in lower rates of transpira­ tion enabling the plant to direct the energy produced during the process of photosynthesis towards root growth and development, initially helping the plants to take hold under climate conditions which differ from those of the greenhouse. 4. conclusions the use of different materials in the substrate composition influenced the growth of euterpe oler‐ aceae mart., seedlings, highlighting for treatment 50% sand + 50% manure and the most diverse com­ position (25% soil + 25% sand + 25% manure + 25% burnt rice husks). the substrates which consisted of the combina­ tions 50% sand + 50% manure and 25% soil + 25% sand + 25% manure + 25% burnt rice husks are indi­ cated for the production of seedlings of euterpe oler‐ acea mart., as they provide superior growth in plant heightand stem diameter, and improve total dry­ weight production in the plants. these treatments suggest the plants employ a greater amount of energy for growth in h and sd, instead of increasing the number of leaves, and this can be a strategy will result in lower rates of transpi­ ration enabling the plant to direct the energy pro­ duced during the process of photosynthesis towards growth faster and ensuring high field survival rates. these results could have of great interest to pro­ ducers of seedling euterpe oleracea mart., as they show an increase in the quality of the seedlings pro­ duced, which is an advantage when planting, as bet­ ter­quality seedlings tend to take hold faster and dis­ play better growth in the field, besides helping to reduce production costs. acknowledgements we thank cnpq (national council for scientific and technological development) for the productivity and research scholarship to the corresponding first author. references aalipour h., nikbakht a., etemadi n., 2019 ­ 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the seedlings of euterpe oleracea mart. seeds of different ecotypes in function on the substrate. ­ j. agric. sci., 10: 325­332. smiderle o.j., silva v.s., chagas e.a., souza a.g., ribeiro i.g., chagas p.c., souza o.m., 2015 ­açai seedling production: effect of substrates and seeds size on germination and growth of seedlings. ­ j. adv. agric., 4: 316­323. souza a.g., smiderle o.j., montenegro r.a., morya­ ma t.k., dias t.j., 2020 ­ controlled‐release fertiliser and substrates on seedling growth and quality in agonandra brasiliensis in roraima. ­ j. agric. studies, 8: 233­242. steffen g.p.k., antoniolli z.i., steffen r.b., machado r.g., 2010 ‐ casca de arroz e esterco bovino como subs‐ tratos para a multiplicação de minhocas e produção de mudas de tomate e alface. ­ acta zool mex., 2: 333­ 343. taiz l., zeiger e., moller i.m., murphy a., 2017 ­ plant physiology and development. sixth edition. ­ sinuaer, oxford university press, uk, pp. 856. http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x#! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! http://https://www.sciencedirect.com/science/article/pii/s037842900400139x" /l "! impaginato 27 adv. hort. sci., 2020 34(1s): 27­33 doi: 10.13128/ahsc­7855 short­term low temperature treat­ ments of harvested wine grapes (cv. vermentino) affect the volatile organic compound profile of the berries m. modesti (*), r. shmulevitz, s. brizzolara, p. tonutti life sciences institute, scuola superiore sant’anna, piazza martiri della libertà, 33, 56124 pisa, italy. key words: aroma, post­harvest, temperature conditioning, terpenoids, vitis vinifera. abstract: in the recent years, due to the climate change and the effects of greenhouse gases average temperatures are increasing. grapes cultivated in mediterranean areas are exposed to high temperatures especially during the late growing season and at harvest. this may induce undesirable biochemical processes (e.g. aroma losses and oxidative reactions) with negative effects on the berry composition and specific quality traits of the resulting wine. in the present study the effects of short­term low temperature treatments on har­ vested grapes before vinification have been evaluated. bunches of wine grapes cv. vermentino have been hand­harvested and then refrigerated at 4°c and 10°c for 24 and 48 hours, while 22°c has been applied as control temperature. grapes were analysed in terms of technological parameters (weight loss, total soluble solids, titratable acidity, ph and total polyphenols) and volatile organic compound profile by hs­spme gc­ms. low­temperature post­harvest treat­ ments affect total polyphenols content of the berries and appear to reduce the heat­related aroma loss, increase the content of four volatile terpenoids and decrease the accumulation of ethyl acetate. 1. introduction several challenges characterize the wine industry and have a marked impact on the production chain, final quality of the wines and consumer acceptance. one major problem, in particular in warm­temperate cli­ mates, is represented by the increase of the average temperatures, main­ ly due to the accumulation of greenhouse gases, that often leads to differ­ ent pheno/physiological processes in grape berry and strongly affects berry development. high temperatures induce anticipated and unbal­ anced ripening and, at harvest, undesirable biochemical changes such as aroma losses and oxidative processes (ribéreau­gayon et al., 2006). this negatively affects grape composition and wine quality. hence, it is crucial to find and develop effective strategies for mitigating these negative effects. one option is represented by the application of postharvest cool­ (*) corresponding author: margherita.modesti@santannapisa.it citation: modesti m., shmulevitz r., brizzolara s., tonutti p., 2020 ­ short‐term low temperature treatments of harvested wine grapes (cv. vermentino) affect the volatile organic compound profile of the berries. ­ adv. hort. sci., 34(1s): 27­ 33. copyright: © 2020 modesti m., shmulevitz r., brizzolara s., tonutti p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 january 2020 accepted for publication 4 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 27­33 28 ing treatments of the bunches. postharvest protocols based on controlling (low­ ering) temperature are used for the management and storage of fresh horticultural crops with the main goal of prolonging commercial life and freshness (tonutti, 2013). the effects of low temperature on harvested fruits are diverse and depend on a number of factors including the fruit type, pre­harvest fac­ tors, ripening stage, the applied temperature and the duration of the treatment (kader, 1999). both prima­ ry and secondary metabolisms are affected (brizzolara et al., 2020), with changes in the composi­ tion and quality parameters, including those related to polyphenols and volatile organic compounds (vocs) (valenzuela et al., 2017, brizzolara et al., 2018). this is also the case of table grapes that, when stored at 0°c to prolong commercial life, show changes in the voc profiles and related­metabolic pathways, resulting in altered overall flavours (maoz et al., 2019). with other goals, post­harvest treatments can also be applied on specific crops undergoing process­ ing. this is the case of wine grapes on which tech­ niques such as controlled dehydration, high carbon dioxide, ozone, ethylene and pre­cooling treatments have been applied or studied to modulate the com­ position of the harvested berries and the style of the resulting wines (mencarelli and tonutti, 2013; becatti et al., 2014; mencarelli and bellincontro, 2018). maintaining harvested wine grapes at low tempera­ ture is a practice that is already applied in certain production areas and for specific enological purpos­ es. low temperature treatments prior to vinification appear to have a positive effect on the aromatic pro­ file of the wines, especially when white­skinned berries are processed. this empirical approach has, so far, very little scientific evidence and, differently from table grapes, just few studies report the effects of such treatments on technological parameters and secondary metabolism (including aroma compounds) of wine grapes. marais (2003) showed that keeping grapes (cv. pinotage) overnight at 10°c and then maintaining the same temperature during skin con­ tact with the must prior to fermentation resulted in the production of the most typical and highest quali­ ty pinotage wines, compared to the same treatments carried out at 15°c. this effect appears to be related to changes in ester metabolism occurring in the berries. mencarelli and bellincontro (2018) reported that following a 10°c treatment applied on wine grapes during post­harvest partial dehydration (a practice used to produce special wines, such as the “passiti”) an up regulation of genes involved in the phenylpropanoid pathway occurs together with a slight increase of stilbenes and a decrease of polyphenol oxidase activity. the present study aimed at evaluating the effect of a short­term low tempera­ ture conditioning on harvested wine grapes cv. vermentino in terms of technological parameters and vocs profile. 2. materials and methods grapes samples and cooling treatments bunches of white­skinned wine grapes (vitis vinifera l.) cv. vermentino were hand harvested in 2018 in correspondence of an average total soluble solid (tss) value of 21°brix. the grapes were collect­ ed from a commercial vineyard (lodolina) located in the hills of candia (massa province, tuscany, italy. 44°02’197.6” n, 10°11’265.9” e). the vines are trained at simple guyot, and all agronomic practices follow the disciplinary of production for the appellation of controlled origin (doc) candia dei colli apuani. after harvest, grapes were immediately transported to the laboratory and selected based on absence of evident defects or diseases. grapes were randomly distributed into six lots (of 5 kg each) and subjected to post­harvest low temperature treat­ ments as follow: two lots were cooled at 4°c (±0.5) for 24 (4°c 24 h) and 48 (4°c 48 h) h; two other lots were cooled at 10°c (±0.5) for 24 (10°c 24 h) and 48 (48°c 48 h) h. the last two lots were used as a control and kept at 22°c (±0.5) for 24 (22°c 24 h) and 48 (22°c 48 h) h. immediately after harvest (t0) and at the end of each treatment,30 berries per biological replicate (three biological replicates per lot) were col­ lected and immediately analyzed for technological parameters. for vocs analysis 30 berries per biologi­ cal replicate (five biological replicates for each treat­ ment) were homogenized and a nacl buffer solution (1 m) has been added (1:1) by using an ultraturrax (mod. t25, ika),immediately frozen in liquid nitrogen and stored at ­80°c. technological parameters the weight loss (wl) of 5 bunches from each lot was measured by using a technical balance. these 5 bunches were tagged and weighed at t0 and at the end of each treatment. for each of the three biologi­ cal replicates a total of 30 berries were manually pressed and the obtained must was centrifugated modesti et al. ‐ low‐temperature conditioning of harvested wine grapes 29 (8,000 rpm, 5 min, 22°c), filtered with syringe filters (0.22 µm pore size, 33 mm diameter, sigma­aldrich, italy) and used for the following analyses: ph, using a ph meter (ph­metro glp21; crison instruments); tss employing an optical refractometer; titratable acidity (ta), titrating 7.5 ml of filtered must with 0.1 n sodi­ um hydroxide (naoh), expressed in g/l of tartaric acid equivalent. for each of the three biological repli­ cates, 30 berries were powdered with liquid nitrogen and total polyphenols were then extracted from 250 mg of berries powder with 1.25 ml of 80 per cent methanol and then centrifugated at 4°c, 10,000 rpm for 15 min. the total polyphenols content (tpc) was then measured using the folin­ciocalteau method (singleton and rossi, 1965), expressed as mg of gallic acid equivalents (gae) x 100 g−1 fresh weight. hs‐spme gc‐ms analysis the pre­homogenized (as described above) sam­ ples were thawed and 10 g were weighed in a 20 ml glass crimpvial for headspace analysis (cat. no. su860049, sigma­aldrich, italy) sealed with silicone septa for spme (cat. no. 27362, sigma­aldrich, italy). the grape samples were incubated under agitation for 30 minutes at 40°c. vocs were sampled at the same temperature for 30 min using an spme fiber (50/30 μm, dvb/car/pdms, 1 cm long; supelco, bellefonte, pa, usa). the fiber was desorbed into the injector of the gc set at 250°c for 5 min (splitless mode). a clarus 680 gas chromatograph equipped with a split/splitless injector (perkinelmer®, waltham, massachusetts) was used for the analysis. volatiles were separated on a fused­silica capillary column (db­wax, 60 m, 0.32 mm id, 0.25 μm film thickness; restek, bellefonte, pa). helium was used as carrier gas with a flow rate of 1 ml min­1. the gc­ ms settings employed were the same adopted by genova and montanaro (2012). for the identification of the compounds, a mass spectrometer (clarus 500 mass spectrometer, perkinelmer®, waltham, massachusetts) coupled to the gc was used. each chromatogram was deconvoluted using amdis soft­ ware (national institute of standards, gaithersburg, md, usa). each peak was identified by comparing the experimental spectra with those of the national institute for standards and technology (nist98, version 2.0, usa) data bank including only com­ pounds with 75 per cent of identity or more. the peaks were quantified using turbomass software (turbomass®, version 5.4.2 perkinelmer inc., usa, 2008), by integration of the peak’s areas. the area of each peak was normalized on the sum of the areas of all peaks detected in the same chromatogram to eliminate variations in fiber adsorption. the efficien­ cy of the fiber was monitored by running on daily bases a quality check (qc) sample, calculating the percent of variance in the total area of the qc chro­ matograms. for each sampling time and treatment five biological replicates were analyzed. statistical analysis each set of replicates was tested to detect out­ liers performing principal component analysis (pca) employing metaboanalyst online tool (chong et al., 2019). one­way anova was performed on technological parameter and gc­ms data following a post hoc tukey’s honestly significant difference (hsd) test (with p= ≤ 0.05) for multiple comparation using graphpad prism version 7 (graphpad software, la jolla california usa).vocs revealing statistically sig­ nificant differences between treatments were then analyzed by means of partial least square discrimi­ nant analysis (pls­da) using metaboanalyst online too (chong et al., 2019). 3. results considering technological parameters, as expect­ ed all samples lost weight after 24 and 48 h, follow­ ing both cooling treatments (4 and 10°c) and control conditions (22°c) (table 1). the wl percentage was higher in the control, which showed the highest value after 48 h. the lowest wl value was recorded for grapes cooled at 4°c for 24 h. the wl of grapes cooled at 10°c for 24 h was not significantly different from samples kept at 4 and 22°c for the same time. both cooled samples at 48h showed significantly lower wl values than the respective control. compared to t0 samples the ph values were slightly lower in all samples except for grapes kept at 22°c for 48 h, while ta values significantly decreased only in berries kept for 24h at 4°c and in the 48 h control sample (table 1). with the exception of this latter sample, compared to t0 a general reduction of tss values was observed in comparison with t0 sample. tpc was significantly lower in comparison to t0 in control berries kept at 22°c for 24 and 48 h (table 1). low temperature treatments induced variable effects on this parameter with increases in 10°c 24 h and decreases in 10°c 48 h samples. the grapes vocs profile was acquired by hs­spme gc­ms. a total of 35 vocs has been detected. among adv. hort. sci., 2020 34(1s): 27­33 30 ment is reported in figure 2. the model explains 55.3 per cent of the variability with still an overlapping of the treatments (fig. 2a). fig. 2b reports the vip scores for the employed features. the highest score is attributed to isoledene, which is markedly accumu­ lating in berries kept at 10°c. furthermore, cooled grapes showed again a higher content of terpenoids comparing with the t0. as far as ethyl acetate is con­ cerned, this compound shows the highest level in the control grapes and the lowest in t0 samples. 4. discussion and conclusions in detached fruits, wl progresses with time and is dependent on the vapour pressure deficit, the evapo­ them, 14 terpenes, 7 esters, 4 alcohols, 3 aldehydes, 2 alkanes, 2 benzene derivates, 1 ether, 1 alkane and 1 phenol were identified. one­way anova test was run on the whole vocs dataset: a total of 11 com­ pounds resulted significantly different (p≤0.05) between treatments (data not shown). among the 11 statistically significant vocs, 5 compounds of inter­ est, known for their impact on grapes and wine aroma, were present and so used for a pls­da analy­ sis. these compounds were three sesquiterpenes (cadinene, cubebene and isoledene) and the monoterpene dihydro­citronellol, which are generally associated with floral and spicy notes, and ethyl acetate, which is considered an off­flavor and associ­ ated with the anaerobic metabolism. the pls­da was carried out separately for the two sampling times. cadinene, cubebene and isoledene, dihydro­citronel­ lol and ethyl acetate levels were used as predictor variables, while the different treatments and t0 were used as response variable. the effect of the 24 h treatment is reported in figure 1. after 24 h of treat­ ment, the model explained 51.6 per cent of the vari­ ability present in the dataset and in this projection the different treatments and t0 samples partially overlaps (fig. 1a). figure 1b reports the vip scores for the employed features. the highest score is attributed to cubebene, which seems to be strongly accumulated in berries held at 10°c. noticeably, the level of all the terpenoids considered is higher in the cooled grapes, regardless the temperature, with the only exception of the monoterpene dihydro­citronel­ lol which showed the lowest level in 4°c sample. interestingly, the most marked variation for the three sesquiterpenes (cubebene, cadinene and isoledene) is observed when comparing cooled with t0 samples. on the other hand, control grapes kept at 22°c are characterized by an accumulation of ethyl acetate. a slight accumulation of this compound is found also in grapes held at 10°c (fig. 1b).the effect of 48 h treat­ table 1 ­ technological parameters in vermentino wine grapes at harvest (t0) and after post­harvest treatments at 4°c for 24 h (4°c 24 h), 10°c for 24 h (10°c 24 h), 4°c for 48 h (4°c 48 h) and 10°c for 48 h (10°c 48 h). 22°c is the temperature of the control sam­ ples different letters indicate statistically significant differences at p≤0.05 according to the results of the tukey’s hsd test. values are the mean of three biological replicates +/­ sd. technological parameters t0 4°c 24 h 10°c 24 h 22°c 24 h 4°c 48 h 10°c 48 h 22°c 48 h weight loss (%) ­ 1.4±1.5 c 2.3±0.6 bc 4.5±1.9 b 2.8±1.5 bc 4.5±1.5 b 9.3±2.1 a ph 3.46±0.02 b 3.40±0.0 c 3.33±0.0 d 3.34±0.0 d 3.39±0.0 c 3.36±0.01 d 3.50±0.0 a titratable acidity (g/l­1) 4.6±0.2 ab 4.2±0.0 c 5±0.1 a 4.5±0.0 b 5±0.1 a 4.7±0.0 a 3.7±0.0 c total soluble solid (° brix) 21±0.0 a 19±0.0 b 16.5±0.4 d 19±0.0 b 17.6±0.3 c 18±0.0 c 20.4±0.2 a total polyphenols content (gae/100 gr fw) 613.6±60.4 b 550±47.3 bc 722.7±84.5 a 560.4±9 c 555.6±83 bc 492.1±77.2 c 363.3±46.7 d fig. 1 ­ a) partial least squares discriminant analysis (pls­da) performed on vocs detected in vermentino grapes fol­ lowing cooling treatment at 4 and 10°c for 24 h and con­ trol treatment at 22°c. cadinene, cubebene, dihydro­ citronellol, isoledene and ethyl acetate levels were used as predictor variables while the different treatments and t0 were used as response variables. each color repre­ sents different treatment with five replicates. 95% confi­ dent intervals are presented in ellipses. b) the variable importance in projection scores of pls­da (vip scores). the coloured boxes on the right indicate the relative con­ centrations of the corresponding metabolite in each group under study. modesti et al. ‐ low‐temperature conditioning of harvested wine grapes 31 eral genes involved in the phenylpropanoid pathway and to the accumulation of stilbenes and flavonoids. maintaining harvested berries at 4­10°c can be con­ sidered as mild stress: it is well known that posthar­ vest cold stress induces changes in fruit secondary metabolic pathways and compounds, including phenylpropanoids (dixon and paiva, 1995; ruiz­ garcía and gómez­plaza, 2013; mencarelli and bellincontro, 2018). concerning the volatile compounds, our results indicate that, as general effect, low temperature con­ ditioning of vermentino grapes has an impact on the volatile terpenoid content of the berries. it is well known that the presence of terpenoids significantly affects the aroma of grapes and wines (d’onofrio et al., 2017), and this is particular important for wines vinificated from neutral variety such as vermentino. terpenoids are classified based on the number of carbons present in the chemical structure: monoter­ penes (10 carbons), sesquiterpenes (15 carbons), diterpenes (20 carbons), triterpenes (30 carbons), and carotenes (40 carbons) (yu and utsumi, 2009; li et al., 2019). among the different classes, a signifi­ cant influence on the aroma of grapes and wine has been attributed to the monoterpene class which, in wine, is generally associated with pleasant floral notes (d’onofrio, 2011). along with monoterpenes, sesquiterpenes are another important subclass. to date, there has been limited research on sesquiter­ penes since they are considered less volatile and aroma­active than monoterpenes (may and wüst, 2012; black et al., 2015). however, sesquiterpenes have been recently correlated with significant organoleptic characteristics of grapes (d’onofrio et al., 2017). indeed, their concentrations in berries can be crucial for the final wine quality (luo et al., 2019) since sesquiterpenes are more stable than monoter­ penes and once extracted from the berry they can be retained in the finished wine (dunlevy et al., 2009). it has been suggested that they provide balsamic, woody and spicy notes (slaghenaufi and ugliano, 2018). based on our preliminary results, it can be hypothesized that low temperature post­harvest treatment is effective in improving specific aromatic traits of vermentino berries and, possibly, wines. the observed increase of terpenoids could be the result of changes in specific metabolic steps of this chemi­ cal class. a key reaction is the conversion of farnesyl pyrophosphate (fpp) to sesquiterpenes, catalyzed by the different members of the terpene synthase (tps) family (tholl, 2006; muhlemann et al., 2014). it is well known that tps activity and so terpenoids rative driving force for water movement and affected by both temperature and relative humidity (cirilli et al., 2012). harvested fruits, including grape berries, already react at low wl values with metabolic changes eventually affecting grape composition (costantini et al., 2006; rizzini et al., 2009; tonutti and bonghi, 2013). in the present trial, control grapes that showed the highest wl values most likely under­ went specific water stress­related reactions more pronounced than those occurring in low temperature samples. previous studies (bellincontro et al., 2009) have demonstrated that a temperature between 5 and 10°c helps to reduce weight loss and to maintain the cellular structure of the berries, with a general reduction of metabolic events. in both control sam­ ples (kept for 24 or 48 h at 22°c), high values of tss well correlate with the loss of weight and the conse­ quent concentration of solutes. the variability pre­ sent among samples for this parameter but also for ph and ta might be the consequence of the hetero­ geneity of the samples, collected in a commercial vineyard. the effects of cooling grapes before vinification on these technological parameters appear to be more clearly defined after 48 h of treatment. this appears also true concerning tpc that increased in cooled samples after 48 h. the effect of postharvest low temperature on tpc has been reported for table grapes by maoz et al. (2019) who showed that stor­ age at 0°c for 6 weeks, led to an upregulation of sev­ fig. 2 ­ a) partial least squares discriminant analysis (pls­da) performed on vocs detected in vermentino grapes fol­ lowing cooling treatment at 4 and 10 °c for 48 h and con­ trol treatment at 22°c. cadinene, cubebene, dihydro­cit­ ronellol, isoledene and ethyl acetate levels were used as predictor variables while the different treatments and t0 were used as response variables. each color represents different treatment with five replicates. 95% confident intervals are presented in ellipses. b) the variable impor­ tance in projection scores of pls­da (vip scores). the coloured boxes on the right indicate the relative concen­ trations of the corresponding metabolite in each group under study. 32 adv. hort. sci., 2020 34(1s): 27­33 biosynthesis strongly depends on both endogenous and environmental factors (robinson et al., 2014). specific studies performed on grape berries in the field showed that high temperatures could reduce terpenoids biosynthesis, and also induce the degra­ dation of thermolabile compounds (d’onofrio, 2011). concerning specifically post­harvest, tps have been studied in toon buds by zhao et al. (2019). they found a strong increase of transcripts related to ter­ penoid biosynthesis under low temperature condi­ tion, which resulted with sesquiterpenoid accumula­ tion. additional studies are so needed to understand if the observed increase of terpenoids in vermentino berries is due to a low­temperature induced biosyn­ thesis or just a maintenance of the pre­accumulated compounds. the use of low temperature in post­harvest and food production is widespread. this preliminary study shows that cooling treatment immediately after harvest have significant metabolic effect on wine grapes. as general effect, it seems that cooling treatments are effective in improving the terpenoids­ related aroma pool of the neutral variety vermentino. this effect appears to be strongly dependent on the applied temperature as well as on the treatment duration: the specific effects of cooling and the interplay between these two parameters (temperature x treatment duration) need to be fur­ ther 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terpenoid biosynthesis during cold storage. ­ sci. hortic., 257: 108747. impaginato 557 adv. hort. sci., 2018 32(4): 557-561 doi: 10.13128/ahs-22026 in vitro responses of gerbera (gerbera jamesonii) cultivars multiplied under different photoperiods j.c. cardoso (*) department of biotechnology, plant and animal production, centro de ciências agrárias, universidade federal de são carlos (ufscar), rodovia anhanguera, km 174, cp 153, cep 13600-970, araras, sp, brazil. key words: cost reduction, in vitro development, light, micropropagation, shoot multiplication. abstract: light affects several aspects of micropropagation. five cultivars of gerbera were subjected to photoperiodic regimes of 10, 12 and 16-h of light for the shoot multiplication. the reduction of photoperiod from 16-h to 12-h resulted in increases in 41.8% to 97.2% of shoot multiplication in all gerbera cultivars. 1. introduction gerbera is one of the most important species for cut flower market in the world, together with lilies, tulips and roses (bhatia et al., 2011; university of kentucky, 2015), owing to several breeding programs that have resulted in a rich diversity of colors (cardoso and teixeira da silva, 2013). although gerbera can be propagated in vivo using seeds or rhizome division (son et al., 2011), micropropagation using apical shoots or through organogenesis is the only way that can provide realistic large scale clonal propagation with genetic fidelity and disease free plantlets (kanwar and kumar, 2008; bhatia et al., 2009) to meet the current demand of highly technological floriculture market. multiplication stage in micropropagation is one of the most crucial steps to establish in vitro propagation. in this regard, most of the earlier studies on gerbera micropropagation were focused on establishment of different types and concentrations of plant growth regulators. several factors affected gerbera in vitro shoot multiplication, as culture medium, plant growth regulators (especially citokinins as benziladenine), growth conditions and the propagation method. however, genotype is the most important factor influencing in vitro development, including the rate of multiplication of gerbera (son et al., 2011; cardoso and teixeira da silva, 2013). the basal culture medium ms (*) corresponding author: jeancardosoctv@gmail.com citation: cardoso j.c., 2018 in vitro responses of gerbera (gerbera jamesonii) genotypes multiplied under different photoperiods. adv. hort. sci., 32(4): 557-561 copyright: © 2018 cardoso j.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 november 2017 accepted for publication 21 may 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 557-561 558 (murashige and skoog, 1962) added 3% sucrose and benziladenine (0.5 to 3.0 mg l-1) is the most used for gerbera shoot multiplication (kanwar and kumar, 2008; cardoso and teixeira da silva, 2013). although the chemical factors have been well studied in relation to the micropropagation for several species, including gerbera, the physical factors such as temperature and light (wavelength, ppfd and photoperiod) conditions are among the less discussed ones, despite their great biological and economic influence on the different development stages of micropropagation. the electrical energy used in laboratories, especially in growth rooms, is the second most important cost factor of in vitro plantlet production, after labor cost (cardoso and teixeira da silva, 2012). therefore, an optimum light source is significant for this cost factor (cardoso et al., 2013). in this study, we tested different photoperiodic regimes at multiplication stage of micropropagation of gerbera cultivars to determine its effect on plant development and energy cost reduction in growth room conditions. 2. materials and methods five cultivars of gerbera were used in this study: ‘basic’ and ‘kiserian’ with both rose and pink inflorescences, and ‘orange dino’, ‘orca’ and ‘onedin’ with orange, white and yellow inflorescences, respectively. the plant material used as donor was cultivated under greenhouse conditions and prepared as described by cardoso and teixeira da silva (2012). briefly, the plants in reproductive phase were completely defoliated and the rhizomes were maintained in cold chamber at 4°c for seven days, followed by transplanting these rhizomes to plastic pots containing organic substrate consisting of coconut fiber. after 14 days of transplantation, the first shoots were observed, and 21-28 days old shoots (5-10 cm in length and 2-3 leaves) were excised from the mother plants and were taken to the laboratory for surface disinfestations. the shoots were reduced to 1.0-1.5 cm and washed for 5 min with tap water, followed by immersion in alcohol 75% (v/v), then in sodium hypochlorite with 1-1.25% of active chlorine for 20 min. finally, the explants were washed three times in autoclaved distilled water. shoot-tips of 2-3 mm length were used as explants to start gerbera micropropagation. the plants were multiplied to obtain the required quantity for the experiment. the experiment was carried out during the multiplication stage of micropropagation. the culture medium used was murashige and skoog (1962) supplemented with 30 g l-1 of sucrose, 0.1 g l-1 of myoinositol, 0.5 mg l-1 of benzyladenine (ba) and 6.0 g l-1 of agar-agar (algagel, type 900, barueri, brazil). the flasks containing the culture medium and the individual shoots were maintained in growth room at 25±1°c and cultivated under cool white fluorescent lamps (philips®, barueri, brazil) as light source with photon flux density of 20-25 µmol m–2 s–1. the plants were subjected to three different photoperiods as follows: 10-h light/14-h dark, 12-h light/12-h dark and 16-h light/8-h dark. the last photoperiod condition was taken as control, as this photoperiod condition is regularly used for many species, including gerbera, for in vitro micropropagation (cardoso and teixeira da silva, 2013). in total, five borosilicate flasks (10 cm height x 5.5 cm diameter) containing four shoots, and covered with transparent polypropylene caps (repetitions), for each treatment were used. the experimental design was 5 × 3 factorial with five cultivars and three photoperiods. the experiments were conducted for three months with time of transplanting of shoots each time for 30 days, resulting in three cultivation periods. in each pricking time, the multiplication factor was determined by counting the number of shoots obtained for the next subcultivation divided by the number of plantlets used at the start of the experiment. five homogenized flasks with four plantlets in each turn were separated for the next evaluation, maintaining the plantlets in the same photoperiodic regime used in the previous experiment. the data were subjected to the analysis of variance (anova) and the shapiro-wilk test; if necessary, the data were normalized before comparison of means. the means were compared by the scott-knot test at 5% of probability. 3. results, discussion and conclusions around 20% of the explants were successfully established in vitro, regenerated into plantlets, and transferred to the multiplication medium. shoot apices with first leaf developed and without signals cardoso photoperiods in gerbera micropropagation 559 of bacterial or fungal contamination in culture medium were transferred to the light condition and used to obtain the first shoots for the experiment. around 60% of the explants were contaminated and around 20% formed callus with no regeneration into plantlets. the shoots so obtained were successfully in vitro multiplicated and used for the experiments of multiplication. these results were similar to those observed in other works at the establishment stage (cardoso and teixeira da silva, 2012, 2013). genotype is one of the most important factors that affect the micropropagation, including the rate of multiplication and the responses to the factors affecting in vitro development of explants being micropropagated. this was also observed in our work with a positive interaction between the genotypes and photoperiods (fig. 1). under 16-h photoperiod, the cultivars ‘onedin’, ‘orange dino’ and ‘orca’ showed a higher multiplication rate (5.5, 5.0 and 4.8 shoots/explant, respectively) than the rose inflorescences cultivars basic and kiserian, which showed a lower multiplication rate of 3.6 and 2.4 shoots/ explant, respectively. interestingly, when the plantlets were subjected to the photoperiod of 12-h (4-h less than control photoperiod), only the cultivar kiserian showed the multiplication rate less than 7.0 shoots/explant. thus, it can be concluded that the response to the photoperiods has a direct correlation with the genotype used for micropropagation (fig. 1). in fact, some cultivars of gerbera with rose and red inflorescences showed recalcitrance characteristics during in vitro multiplication, limiting their propagation and requiring protocol modification to accelerate the production of shoots and the efficiency of multiplication stage, compared with other cultivars. as example, a historical mean of multiplication along three years of in vitro cultivation showed that gerbera cv. lamborghini (red inflorescences) was 3.8 shoots/explant compared with 6.8 and 4.6 shoots/explant for ‘suzzane’ (orange) and ‘dino’ (yellow) (cardoso, personal observations in commercial lab). the influence of genotype toward shoot multiplication was also observed by hartl et al. (1993). they classified gerbera cultivars according to the rate of multiplication into three multiple-shoot induction cultivars: high (8 shoots/inoculum), moderate (6-7 shoots/inoculum) and low (4-5 shoots/inoculum). we also observed all these classes in our cultivars. however, the different photoperiodic conditions had different effects on the position of the cultivars in different classes of multiplication as proposed by hartl et al. (1993). in our work the classification of cultivars using the scott-knot test with the best photoperiod for multiplication (12-h photoperiod) showed at least two classes of multiplication: (a) easily multiplied with number of shoots/explant >7.0; and (b) recalcitrant for multiple shoot induction, with the number of shoots/explant <4.0. an intermediary classes can also be considered as (ab), where the multiple shoot induction rates was 4 to 7 shoots/explant, but this was observed only in 10-h and 16-h photoperiod conditions. the photoperiod had significant effects on gerbera in vitro multiplication (fig. 1 and 2). normally, the multiplication at 25±2°c associated with 16-h photoperiod is most used for gerbera (cardoso and teixeira da silva, 2013), but in our actual work, we observed that for all five cultivars tested, the use of 12-h photoperiodic regime resulted in significant increase in multiple shoot induction when compared with 16-h standard photoperiod conditions (fig. 1). the maximum positive effects (97.2%) were obtained in one of the most recalcitrant gerbera cultivar basic, followed by ‘orca’ (68.8%), ‘kiserian’ (62.5%), ‘orange dino’ (54.0%) and ‘onedin’ (41.8%). similar results were observed in micropropagation of ornamental pineapple annanas comosus var. erecfig. 1 in vitro multiplication of gerbera (gerbera jamesonii) cultivars under different photoperiodic regimes. the means were obtained from three multiplication times (experiments) considering five replicates (flasks) with four plants. different small and capital letters show the differences among genotypes and photoperiod regimes, respectively, by the scott-knot test at 5% probability. the coeficient of variation is 13.52. the data were considered normal by shapiro-wilk normality test and the f was significative for all factors, including the interaction genotype x photoperiod. adv. hort. sci., 2018 32(4): 557-561 560 tifoilius, in which the reduction of photoperiod for 12-h resulted in better multiplication rate (6.6 shoots/explants) than 16-h photoperiod (4.2 shoots/explants) (santos et al., 2015). the influence of photoperiod on in vitro development was also noted for other species and found affecting other in vitro plantlet characteristics of development, such as micro-tuberization in potato cultivars (seabrook et al., 1993). in amaryllis (hippeastrum johnsonii), the authors observed that 16-h photoperiodic regime resulted in better in vitro root length and number, bulblet diameter, and leaf length, compared with 14h and 12-h of photoperiod (zakizadeh et al., 2013). several amendments to reduce the cost have been proposed for gerbera micropropagation, such as chemical sterilization in place of autoclaving during all stages of micropropagation (cardoso and teixeira da silva, 2012) and the use of greenhouse rooting and elongation, called pag culture, in place of laboratory growth conditions (cardoso et al., 2013). the reduction in photoperiodic regime resulted in low energy consumption and so the low cost for the multiplication stage of gerbera. this might be useful for the production of low cost plantlets from micropropagation of this important cut flower. the major conclusion can be drawn from this study is that a reduction in photoperiod by 4-h of light per day along with increase in multiplication rate may increase the efficiency of the propagation system. chen (2016) concluded that the electrical energy for control environmental conditions on growth room, associated with the low efficiency of multiplication, is the most important factor that leads to increase cost of micropropagated plantlets of phalaenopsis. this author showed that increase in multiplication rate from 1.5 to 2.5 could lead to a 50% cost reduction in micropropagated phalaenopsis. photoperiod has a direct effect on the development of greenhouse cultivated gerbera genotypes, affecting plant width, height, shoot dry weight and number of flowers in a condition of 16-20-h of photoperiod (gangnon and dansereau, 1990). however, the interaction of photoperiod and the cultivation and development of gerbera could be more complex involving possible role of other environmental factors (pettersen and gislerød, 2003), and only few of them have been understood. our results showed for the first time that the in vitro development of gerbera plantlets can be affected by photoperiod regimes (fig. 1 and 2). increases in axillary shoot production was observed in reduced photoperiods (12 and 10-h) compared with 16-h. in addition, the use of white fluorescent lamps in the in vitro cultivation of gerbera resulted in a higher multiplication rate compared to different colors of light-emitting diodes (leds) lamps (gök et al., 2016). our actual experiments showed that reducing the photoperiod to 12-h produced slightly increased shoot height with no visual effect on the size of leaves (fig. 2). on the other hand, a further reduction of photoperiod to 10-h reduced the size of leaves (diameter and length) and resulted in etiolated development, showing negative effects on quality of gerbera shoots produced for rooting stage (fig. 2). as example, the fresh weight of gerbera shoots in 10-h photoperiod is 20.0% (‘onedin’), 29.7% (‘orca’) and 11.6% (‘orange dino’) less than compared with shoots produced in 12 or 16-h photoperiodic regimes (data not showed). only gerbera cv. kiserian showed practically the same fresh weight in 10-h and 12-h photoperiod. plantlets obtained from all treatments were successfully in vitro rooted (95-100% of rooting) in ½ms with 30 g l-1 sucrose and 0.5 mg l-1 of indol-butyric acid and were acclimatized in greenhouse conditions using polypropylene trays with coconut powder as organic substrate. no morphological alterations were observed in acclimatized plantlets obtained, independent to the cultivar or photoperiod used, and the plantlets maintained the main characteristics of the donor plants until the flowering stage. fig. 2 morphological characteristics of in vitro cultivation gerbera (gerbera jamesonii) cv. onedin under different photoperiodic regimes: 10-h and 12-h. shoots from 10-h photoperiod presented smaller leaves (sl) and smaller and etiolated shoots (ses), compared to 12 and 16-h photoperiod. bar = 1.0 cm cardoso photoperiods in gerbera micropropagation 561 acknowledgements special thanks to uniplant co. for the infrastructural support to this research. jcc thanks the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the research grants (process number 304174/2015-7). references bhatia r., singh k.p., jhang t., sharma t.r., 2009 assessment of clonal fidelity of micropropagated gerbera plants by issr markers. sci. hortic., 119: 208211. bhatia r., singh k.p., sharma t.r., jhang t., 2011 evaluation of the genetic fidelity of in vitro propagated gerbera (gerbera jamesonii bolus) using dna-based markers. plant cell tiss. organ cult., 104: 131-135. cardoso j.c., rossi m.l., 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2015 field-grown cut flowers. cooperative extension service of university of kentucky, university of kentucky, usa. zakizadeh s., kaviani b., onsinejad r., 2013 role of photoperiod on some growth characters of amaryllis (hippeastrum johnsonii), a bulbous plant. eur. j. exp. biol., 3(1):289-291. impaginato 257 adv. hort. sci., 2017 31(4): 257-265 doi: 10.13128/ahs-20832 solar radiation levels modify the growth traits and bromatological composition of cichorium intybus f. schwerz 1 (*), j. sgarbossa 2, t. olivoto 3, e.f. elli 4, a.c.m. aguiar 2, b.o. caron 2, d. schmidt 2 1 luiz de queiroz college of agriculture, university of são paulo (esalqusp), department of plant science, piracicaba, sp, brazil. 2 federal university of santa maria, frederico westphalen campus, departament of agronomic and environmental sciences, frederico westphalen, rs, brazil. 3 federal university of santa maria, department of plant science, santa maria, rs, brazil. 4 luiz de queiroz college of agriculture, university of são paulo (esalqusp), department of biosystems engineering, piracicaba, sp, brazil. key words: chicory, growth rate, radiation use efficiency, shading screens. abstract: shading greenhouse may be an effective method to achieve a suitable environment for crop growth and to enhance crop yield and quality in places or seasons where there is high light intensity. therefore, solar radiation levels may modify the biomass accumulation and bromatological composition. different solar radiation levels (100%, 70% and 50% of available solar radiation) were simulated in order to determine crop responses to these factors in chicory (cichorium intybus l. var. foliosum). a hydroponic experiment was conducted in an experimental greenhouse in the city of frederico westphalen, rio grande do sul, brazil. plants grown in lower solar radiation levels are more efficient in converting solar radiation into dry matter, had a higher lipid content, increased chlorophyll indices a, b and total, as well as reduced leaf thickness, acid detergent fiber, cellulose, and lignin content, presenting more attractive bromatological features for commercial production. in this study it was demonstrated that the use of shading screens is an effective method to attenuate the solar radiation, this is especially relevant in places or seasons where there is high light intensity, which contribute to achieve better characteristics of the chicory produced. 1. introduction growth rate, dry matter production and radiation use efficiency are considered important variables when analyzing yield-limiting factors and their interactions in different production environments. dry matter accumulation under non-limiting conditions is directly related to the amount (*) corresponding author: felipe_schwerz@hotmail.com citation: schwerz f., sgarbossa j., olivoto t., elli e.f., aguiar a.c.m., caron b.o., schmidt d., 2017 solar radiation leves modify the growth traits and bromatological composition of cichorium intybus. adv. hort. sci., 31(4): 257265 copyright: © 2017 schwerz f., sgarbossa j., olivoto t., elli e.f., aguiar a.c.m., caron b.o., schmidt d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2016 accepted for publication 29 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 257-265 258 of intercepted photosynthetically active radiation (caron et al., 2012). therefore, variation in total biomass due to limiting biotic and abiotic factors may be attributed to changes in the solar radiation levels. changes in the solar radiation level of a given environment may affect the photosynthetic apparatus of plants. lower solar radiation level reduces the photosynthetic rate and hence plant growth. when radiation levels is above the light saturation point, the reaction center of the psii is inactivated and frequently damaged, constituing a phenomenon called photoinhibition (taiz et al., 2017). due it, studies have identified a reduction of quantum efficiency, photosynthetic rate, and possible damage to the photosynthetic apparatus (zhang et al., 2008; murchie et al., 2009) beyond changes on the yield, bromatological and nutritional aspects, when plants are exposed both to suboptimal or super optimal radiation levels (dario et al., 2015; bianculli et al., 2016). achieving an appropriate environment in greenhouses in subtropical regions has become one of the main challenges for chicory producers, due to a large amount of solar radiation transmitted into the greenhouse and then converted into sensible and latent heat. thus, greenhouse cooling methods and their impact present a considerable problem that requires a solution (abdel-ghany and al-helal, 2010). a strategy used in greenhouses in order to provide an appropriate environment for plant growth and to increase crop yield is the shading screens (sethi and sharma, 2007; ganguly and ghosh, 2011). shading is one of the most inexpensive ways to reduce heat accumulation and to modify the greenhouse environment (sethi and sharma, 2007; holcman and sentelhas, 2012), besides promoting higher rates of diffuse radiation. solar radiation use efficiency (ɛb) of crops is determined by the slope of the linear regression between produced dry matter and photosynthetic active radiation (par) intercepted by the leaves (monteith, 1965, 1972, 1977; van heerden et al., 2010). reduced solar radiation availability within the greenhouse environment may influence various traits of chicory, especially the leaf area index, the thickness of leaves and chlorophyll content, because cultivation in shaded environments often results in thinner leaves (wherley et al., 2005; liu et al., 2016). these leaves contain a greater total chlorophyll content per unit of fresh weight when compared to leaves grown under full sunlight. chlorophyll content of each leaf, per unit area, may be smaller in environments with reduced incident solar radiation (wild and wolf, 1980; taiz et al., 2017). research showing the change of yield, and bromatological and nutritional traits as a function of incident solar radiation level, both as suboptimal and optimal levels were shown by bianculli et al. (2016), dario et al. (2015), and kataria and guruprasad (2015). to the authors’ knowledge, this study is the first to investigate the impact of different solar radiation levels on plant growth, radiation use efficiency and biomass partitioning in chicory. information that reveals the impact of solar radiation levels on chicory as well as the bromatological composition are relevant in order to improve some management in a greenhouse environment. in addressing this lack of information, the following hypotheses were investigated: (i) solar radiation use efficiency is reduced in the plants grown under high solar radiation level; and (ii) shading screens provide an appropriate environment for plant growth, resulting in plants with better bromatological composition. these hypotheses justify the following aims: (i) to determine the influence of different solar radiation level on plant traits; and (ii) to evaluate the bromatological compositon of chicory plants under different solar radiation levels. 2. materials and methods study area the study was conducted in an experimental greenhouse in the city of frederico westphalen, rio grande do sul, brazil. a hydroponic experiment was performed between the months of march 2016 to june 2016. the geographical location of the experiment was 27°23’ s, 53°25’ w, 490 m asl. according to the köppen climate classification, the climate is cfa, i.e., humid subtropical with mean annual temperatures of 19.1°c, and varying maximum and minimum temperatures of 38 and 0°c, respectively (alvares et al., 2013). management of hydroponic system chicory (chicory intybus l. var. foliosum) seeds were inserted into phenolic foam board on 25 march 2016. seedlings were transplanted into a system called “seedling tray” on 3 april 2016, when they reached two to three true leaves; seedling tray had 40 mm-hydroponic channels (3-cm deep, spaced by 7 cm between channels and 10 cm between plants in the channels), with 3% declivity. seedlings remained schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 259 in this system until they reach a developmental level of five true leaves. after the seedling tray stage, the final transplant to the growth tray was performed on 11 april 2016. each final growth tray was formed by 11 hydroponic channels (6-m long, 0.10-m wide and 0.05-m deep), subjected to a 4% declivity. the spacing was 0.20 m between plants in the channels and 0.20 m between channels. thus, three hydroponic benches had 33 channels, with 242 plants per bench, 726 plants throughout the experiment composed the system. a 1-hp pump coupled to a fiberglass tank with a capacity of 1000 liters powered the hydroponic system. the nutrient solution used was prepared with 400 mg l -1 of a commercial hidrogood® fert (hidrogood modern horticulture, brazil); these values were considered to be a full dose. nutrient solution was pumped inside the hydroponic channels and was collected at the end of each channel by a system of closed system gutters. the nutrient solution used was prepared with 400 mg l-1 of a commercial hidrogood® fert (hidrogood modern horticulture, brazil); these values were considered to be a full dose. irrigation was performed in an on-and-off system in periods of 15 minutes throughout the day (6 am 7 pm.); and 15 minutes each two hours during the night (7 pm 6 am). potential of hydrogen (ph) and electric conductivity of the nutritive solution were assessed daily with a digital ph meter (ph009ia model), and with a conductivity meter (az8301 model), respectively. the ph of the nutrient solution was kept at 6.0 (±0.5) using sulfuric acid (10% concentration of h2so4) or very low sodium hydroxide (2% of naoh). nutrients were replaced when the electrical conductivity of the nutritive solution reached 50% of its initial concentration. solar radiation level and experimental design the experimental design was a randomized complete block, arranged in a factorial arrangement (solar radiation level x evaluation periods) with four replications. different solar radiation level (srl) were simulated using black polyethylene meshes, fixed 1.0 m above the hydroponic benches. the following treatments were applied: 100% of available srl (without mesh over the plants), 70% of available srl (30% transmissivity mesh), and 50% of the available srl (50% transmissivity mesh). each simulated radiation level treatment composed a different hydroponic bench and the meteorological conditions in all treatment were the same, differing only the solar radiation levels in each hydroponic bench. for the growth traits analysis, the chicory plants were collected from the central hydroponic channels of each treatment, beginning seven days after the transfer to the final growth tray. the evaluations were performed weekly until the average fresh mass of experimental plants hit 250 g (harvest point). destructive evaluation consisted of two whole plants in each replication, totaling eight plants per treatment in each period. in laboratory the sectioning plants was performed, including the preparation of leaf discs in order to determine the leaf area and dry matter partitioning. the total dry matter (tdm) of the plants was determined from the sum components (root, stem and leaves). each component was gathered and placed into pre-identified individual paper sacks. the sacks were then kept in a forced circulation oven at 60°c until a consistent mass was obtained. the samples were later weighed on a precision balance in order to obtain the dry mass of each component, which together resulted in the tdm. growth rates and bromatological analysis in each evaluation period, the following variables were determined using the average values of dry matter (dm) and leaf area index (lai): specific leaf area (sla), leaf area ratio (lar), leaf weight ratio (lwr), absolute growth rate (agr), relative growth rate (rgr) and net assimilation rate (nar). for more details about the used metrics of evaluation and the determination of these variables, see thornley (1976) and gardner et al. (1985). the chlorophyll index a, b, total and a/b ratio were determined with a cfl 1030 chlorophyll meter. we selected, in the last evaluation period, fully expanded leaves from the upper third of 10 plants in each replication. all collected dry matter samples were properly prepared and subjected to bromatological analysis; the following traits were determined: ash (ash, % of dm), lipids (lip, % of dm), crude protein (cp, % of dm), neutral detergent fiber (ndf, % of dm), acid detergent fiber (adf, % of dm), lignin (ln, % of dm), hemicellulose (hc, % of dm), cellulose (cel, % of dm) and soluble carbohydrates in neutral detergent (scnd, % of dm). for the ash content determination, the method aoac 923.03 (1995) was used, which consider a temperature of 550°c. lipid content was quantified according to the method proposed by bligh and dyer (1959). the values of ndf, adf and ln were determined, followed by the calculations for estimating hc, cel and csnd as proposed by senger et al. (2008), and the crude protein (n x 6.25) was determined by micro-kjeldahl method (method 960.52) of aoac (1995). adv. hort. sci., 2017 31(4): 257-265 260 radiation use efficiency production of dry matter was based on the model proposed by monteith (1977), where dry matter production was calculated from intercepted photosynthetically active radiation (ipar) multiplied by the use efficiency (εb). the εb was calculated by the ratio between the average production of accumulated tdm and the ipar involved in the production of biomass according to the following expression: dm= b*ipar where dm = dry matter, in g; = conversion efficiency of in biomass produced, in g mj–1 and = intercepted photosynthetically active radiation, in mj m–2. estimation of accumulated photosynthetically active radiation was determined according to the expression proposed by varlet-grancher et al. (1989): *ipar=0.95*(inpar)*(1-e (-k*lai)) where ipar = intercepted photosynthetically active radiation, in mj m-2; inpar = incident photosynthetically active radiation, in mj m-2; k = light extinction coefficient. the k value calculated in the current study for the chicory plants was 0.13. lai = leaf area index. the light extinction coefficient (k) was calculated using the following equation: k= in(rn/rt) lai where k= light extinction coefficient, rn= solar radiation measured under the plant canopy (mj m-2); rt= radiation above the plant canopy (mj m-2); lai= leaf area index. leaf area index was calculated by the following expression: lai= la/ua where lai = leaf area index; la = leaf area, in cm2; ua = useful area per plant, in cm2. the fraction of photosynthetically active radiation was considered 47% of the incident solar global radiation found in rio grande do sul (assis and mendez, 1989). the estimation of accumulated photosynthetically active radiation was based on monteith (1977) and varlet-grancher et al. (1989). the par transmissivity of greenhouse cover was calculated based on an assessment using a quantum sensor, 50 cm height from the ground level, measuring 40 random points inside and outside of the greenhouse weekly during the trial period. to calculate transmissivity, the following equation was used: t = (100*pari)/paro where t= transmissivity, in %; pari= photosynthetically active radiation inside of the greenhouse; paro= photosynthetically active radiation outside of the greenhouse. with the transmissivity data of greenhouse and polyethylene meshes, pari was estimated for each treatment, according to the following expressions: par100%=pari par70%=pari*0.7 par50%=pari*0.5 where photosynthetically active radiation inside of the greenhouse; environment with 70% of photosynthetically active radiation available and environment with 50% of photosynthetically active radiation available. the values of incident global solar radiation during the study were obtained with the automatic climatological station of the national institute of meteorology, located at 300 m from the study site (27°39’ s and 53°43’ w). chicory growth and nutritional composition variables were statistically analyzed with the software sas 9.0 (sas institute 2002). data were initially examined for homogeneity of variance and then subjected to analysis of variance. tukey test (p>0.05) was used to compare the difference between the treatments. 3. results radiation use efficiency in chicory the radiation use efficiency and photosynthetically active radiation accumulated values in the different solar radiation level during the conduct of the study are shown in figure 1. it was observed that the fig. 1 radiation use efficiency (εb) in g mj-1 and photosynthetically active radiation levels (par) accumulated over cichorium intybus cycle. adm = accumulated dry matter, pariac = photosynthetically active radiation intercepted accumulated. schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 261 plants growing under reduced radiation availability (50% and 70%) presented respectively 51.6% and 40.1% higher radiation use efficiency than those growing under 100% of solar radiation level. plants grown in low levels of photosynthetic active radiation were more efficient in converting the radiant energy into accumulated dry matter. growth rates and leaf traits the absolute growth rate values were greater in the higher level of solar radiation from 42 dat up to the crop harvest (fig. 2a). the relative growth rate showed changes depending on the age of the plants, but for the solar radiation levels was observed difference only at 14 and 21 dat (fig. 2b). in relation to the net assimilation rate was not observed changes in the values in function of the solar radiation level, however, the largest net assimilation rate values were observed at 21 dat (fig. 2c). the attenuation of 50% and 70% of solar radiation reduced the leaf area index of the plants by 51.8% and 19.8% on average when compared with the plants growing under 100% of solar radiation level from 42 dat up to the crop harvest (fig. 3a). for the specific leaf area, significant differences can be observed from transplanting up to 21 dat, where the plants under lower radiation levels showed the higher specific leaf area (fig. 3b). in relation to the leaf area ratio, it was identified higher value only at 7 dat for the plants under 100% of solar radiation level (fig. 3c). additionally, for the leaf weight ratio was not verified difference in the different shading levels (fig. 3d). plants grown in lower radiation levels showed higher chlorophyll index a, b, and total, which were 19.4, 36.4 and 25.3% higher than those observed for the 100% of solar radiation level, respectively. fig. 2 absolute growth rate (agr), relative growth rate (rgr) and net assimilation rate (nar) in cichorium intybus grown under different solar radiation levels. lowercase letters denote differences within each day after transplanting by tukey test (p<0.05). bars represent average values ± se (n=8). fig. 3 leaf area index (lai), specific leaf area (sla), leaf area ratio (lar) and leaf weight ratio (rlw) in cichorium intybus grown under different global solar radiation levels. lowercase letters denote differences within each day after transplanting by tukey test (p<0.05). bars represent average values ± se (n=8). adv. hort. sci., 2017 31(4): 257-265 262 however, plants without light restriction showed higher chlorophyll-a/b ratio (fig. 4). biomass partitioning and bromatological composition the pattern of dry matter accumulation in the leaves, roots and stem of the chicory plants was similar for all solar radiation levels; however, there was observed an increase in the proportion of leaves during the crop cycle, with a decrease in the percentage of roots and stem of the plants. this pattern of partitioning was observed for all solar radiation levels (fig. 5). reduced levels of solar radiation increased the lipids and ash contents and decreased the neutral detergent fiber, acid detergent fiber, lignin, hemicellulose and cellulose values that characterized less rigid leaves. the crude protein content and soluble carbohydrates in neutral detergent were not affected by shading levels (fig. 6). in overall, the bromatological traits of chicory plants were affected by the solar radiation levels. 4. discussion and conclusions solar radiation levels modify the radiation use efficiency and growth rates the results showed that growing conditions have a striking effect on the radiation use efficiency in relation to its capacity to convert solar radiation into dry biomass. the efficiency of plants to convert solar energy into biomass was higher in low solar radiation fig. 4 chlorophyll-a, chlorophyll-b, and total chlorophyll index and chlorophyll-a/b ratio in cichorium intybus grown under different solar radiation levels. lowercase letters denote differences between solar global radiation levels by tukey test (p<0.05). bars represent average values ± se (n=8). fig. 5 biomass partitioning (%) in each days after transplanting (dat) among leaves, roots and stem in cichorium intybus grown under different solar radiation levels. bars represent average values ± se (n=8). fig. 6 ash percentage (ash), lipids (lip), crude protein (cp), neutral detergent fiber (ndf), acid detergent fiber (adf), lignin (ln), hemicellulose (hm), cellulose (cel), soluble carbohydrates in neutral detergent (csnd) in cichorium intybus leaves growing in different solar radiation levels. lowercase letters denote differences between solar global radiation levels by tukey test (p<0.05). bars represent average values ± se (n=8). schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 263 level. the higher efficiency of chicory plants was not enough to compensate the limitations of solar radiation. this is justified by the higher leaf area index observed under high levels of radiation. the highest leaf area index observed for plants growing under 100% of solar radiation may be related to the higher rate of photosynthesis, since there were no limitations on the availability of solar radiation. according to taiz et al. (2017), sun leaves increase co2 assimilation having more availability of rubisco, and can dissipate excess of light energy due to a large pool of components in the xanthophyll cycle. in this sense, it is important to emphasize that plants with higher conversion efficiency are not always the ones that result in higher yield. this affirmation was confirmed in this study, where the chicory plants grown under reduced solar radiation levels were more efficient into convert solar radiation into biomass, however, showed lower leaf area index. this response can be explained due the morphological changes occurred in function of the shading conditions. the shading effect provided by the solar radiation level of 50% significantly increased the specific leaf area of the plants mainly up to 14 days after transplantation. the decrease in leaf thickness of plants grown at higher levels of shading maybe linked to the fact that plant prefers to spend photoassimilated for the expansion of the leaf area (cooper and qualls, 1967; carvalho et al., 2006; gondim et al., 2008; lenhard et al., 2013), thus ensuring itself a greater possibility of intercepting solar radiation. in chicory, the attenuation of solar radiation above the plants with the use of shading screens resulted in an alteration in leaf thickness which was responsible for changes in the chlorophyll content and bromatological composition (sims and pearcy, 1992; terashima et al., 2001; oguchi et al., 2003). this plant response is correlated, where a reduction in the leaf thickness can imply in changes in the chlorophyll content and bromatological composition depending on the intensity of leaf thickness alteration in function of the level of shading. the morphological changes were considered positive, as the leaves of these plants were more tender. this characteristic may be considered in the market commercialization due to a better acceptability of the consumer’s for products with greater bromatological features. reduced solar radiation levels improve morphological and bromatological traits of chicory the current study confirmed the difference in the bromatological quality of chicory plants grown in different solar radiation level. in overall, a higher lipids content and lesser fiber, lignin, hemicellulose and cellulose components were observed under reduced solar radiation levels. this result reveals an important bromatological change, which confers more quality to the chicory produced. the reduction of lipid content in chicory plants under high solar radiation levels may be associated with the occurrence of light saturation and photoinhibition. according to taiz et al. (2017), the light saturation in the leaves of most species occurs between 500 and 1000 µmol m−2 s−1, whereas the full solar radiation can provide around 2000 µmol m−2 s−1. excessive light causes damage to chloroplasts, one of the locations of fatty acids synthesis (halliwell, 1987; mittler, 2002; gill and tuteja, 2010). the higher content of lipids in chicory plants produced under reduced solar radiation level is an important finding that can help people health, once lipids deliver energy and essential fatty acids, being essential for fat-soluble vitamin absorption, and may contain natural antioxidants (lindley, 1998). considering the consumer’s point of view, the higher content of lipids and reduction of fiber, lighin, hemicellulose and cellulose components are favorable, since the leaves will present better characteristics for consumption. such effects produced by the attenuation of solar radiation can be explained due to the fact that shading reduces the availability of assimilates used for the development of secondary cell walls (kephart and buxton, 1993). additionally, this fact is relevant because the lignin and cellulose components are important substances against abiotic stress, including uv-b radiation (rozema et al., 1997). in phalaenopsis orchids, e.g., an increase in the lignin content in the leaves and roots of the plants where observed when the light intensity was increased, being this result associated with the induction of pal, cad and pod activities (copur and tozluoglu, 2008). regarding the morphological and physiological changes, the highest content of chlorophyll-a, b and total observed for the plants grown under 50% of solar radiation level is related to the need to increase the utilization of the available radiation for the plants. the current study confirmed the ability of plants to compensate the low level of radiation by increasing the number of photosynthetic pigments, which resulted in a higher radiation use efficiency by plants (fig. 1). higher chlorophyll content in plants grown under adv. hort. sci., 2017 31(4): 257-265 264 low solar radiation level is consistent with the findings of minotta and pinzauti (1996), cardillo and bernal (2006), and hazrati et al. (2016). a lower chlorophyll a/b ratio in the lowest light intensity treatment is due to chlorophyll-b being degraded more slowly in shaded environments (lee et al., 2000). information generated in the current study is relevant, as it provides valuable information to vegetable’s producers, assisting in the strategy to minimize the effect of the high solar radiation inside of the greenhouses. we proved the attenuation of solar radiation is an effective method to improve morphological and bromatological aspects of chicory. by using this simple method, both researchers and farmers might have a competitive advantage in planning their cropping systems, mainly in sites or seasons where the high light intensity is a limiting factor for chicory cropping. chicory plants grown in lower solar radiation levels are more efficient in converting solar radiation into dry matter, have a higher lipid content, chlorophyll index a, b and total, reduced leaf thickness, acid detergent fiber, cellulose, and lignin content, which confirm the hypothesis tested. in this study it was demonstrated that the use of shading screens is an effective method to attenuate the solar radiation; this is especially relevant in places or seasons where there is high light intensity, which contributes to achieve better characteristics of the cichory produced. acknowledgements the authors are grateful to the national council for scientific and technological development (cnpqbrazil), for the productivity scholarship of the coauthor braulio otomar caron and for their financial support for the authors felipe schwerz and tiago olivoto. references abdel-ghany a.m., al-helal i.m., 2010 characterization of solar radiation transmission through plastic shading nets. j. sol. energy mater. sol. cells., 94: 1371-1378. alvares c.a., stape j.l., sentelhas p.c., moraes gonçalves j.l., sparovek g., 2013 köppen’s climate classification map for brazil. meteorol. z., 22: 711728. aoac, 1995 official methods of analysis. 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properties. j. agric. food chem., 56: 670-675. impaginato 123 adv. hort. sci., 2020 34(2): 123­132 doi: 10.13128/ahsc­7835 growth, yield and fruit quality of tomato under different integrated management options against tuta absoluta meyrick a. ndereyimana 1, 2 (*), s. nyalala 1, p. murerwa 1, s. gaidashova 2 1 department of crops, horticulture and soils, egerton university, po box 536, 20155 egerton, kenya. 2 department of agriculture research and technology transfer, rwanda agriculture and animal resources development board, po box 5016, kigali, rwanda. key words: azadirachtin, beauveria bassiana, integrated pest management, metarhizium anisopliae, phytolacca dodecandra, solanum lycoper‐ sicum l., steinernema, tephrosia vogelii. abstract: this study evaluated the effect of entomopathogens and plant extracts, used against tuta absoluta, on growth, yield, and fruit quality of tomato. two field trials were carried out in a randomised compled block design, replicated thrice. the treatments were steinernema sp. rw14­m­c2a­3, steinernema sp. rw14­m­c2a­3, metatech®wp (metarhizium anisopliae, strain fcm ar 23b3), beauvitech®wp (beauveria bassiana, strain j25) as ento­ mopathogens, tephrosia vogelii and phytolacca dodecandra as plant extracts, and azadirachtin 0.03% ec. imidacloprid and water also were included as posi­ tive and negative controls, respectively. the best growth and yield parameters were recorded with the entomopathogens and azadirachtin, which were insignificantly different in most cases. the increase in yied of healthy fruit per plant (average of two trials) compared to the negative control (water spray) was 11.4, 10.8,10.1, 9.6, 3.96, 2.2, 11.7 and 2.4 folds for steinernema sp. rw14­ m­c2a­3, steinernema sp. rw14­m­c2a­3, metatech®wp, beauvitech wp, t. vogelii, p. dodecandra, azadirachtin, and imidacloprid, respectively. there was no significant difference in number of leaves per plant and fruit quality parame­ ters. the entomopathogens and azadirachtin, which exhibited a capacity to enhance tomato growth and reduced yield losses due to t. absoluta, are rec­ ommended to be included in integrated pest management programme on tomato. 1. introduction the increasing world population requires food security, which can be partly achieved by reducing the portion of food lost every year as a result of pests (kumar and omkar, 2018). however, yield losses inflicted by crop pests have been observed to increase constantly despite different strate­ gies being implemented globally (dhaliwal et al., 2010). tomato (solanum lycopersicum l.) is one of the most popular vegeta­ (*) corresponding author: assinapol@gmail.com citation: ndereyimana a., nyalala s., murerwa p., gaidashova s., 2020 ­ growth, yield and fruit quality of tomato under different integrated management options against tuta absoluta meyrick. ­ adv. hort. sci., 34(2): 123­132 copyright: © 2020 ndereyimana a., nyalala s., murerwa p., gaidashova s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2020 accepted for publication 18 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 123­132 124 bles in the world and its fruits are a rich source of nutrients and health­promoting compounds (luna­ guevara et al., 2014; asensio et al., 2019). one aver­ age­sized tomato fruit offers 40% and 20% of the rec­ ommended daily amount of vitamins c and a, respec­ tively. it also provides a significant amount of dietary fibres and minerals like calcium and potassium (tigist et al., 2013). furthermore, the antioxidant activity of ascorbic acid, carotenoids, and phenols protects humans against cancers and cardiovascular diseases (tigist et al., 2013; luna­guevara et al., 2014). therefore, any technology used on tomato crop has to be investigated not only for its effect on growth and yield but also on fruit quality parameters. several pests have been reported to attack toma­ to throughout its production cycle (kumar and omkar, 2018). the tomato leaf miner, tuta absoluta meyrick (lepidoptera: gelechiidae), was recognised among the major pests since 1964 in argentina from where it invaded the rest of south america (desneux et al., 2010). following spain invasion during the year 2006, the pest spread to many other european coun­ tries, the middle east, more than 40 african coun­ tries, and almost all southern west and central asian countries neighbouring china, the world’s largest tomato producer (biondi et al., 2018; mansour et al., 2018). in only one decade, t. absoluta spread drasti­ cally and the world tomato production area under its invasion increased from 3% to 60% (biondi et al., 2018). in rwanda, t. absoluta was first recorded in bugesera district in 2015 (fao, 2015), after which it quickly spread in all tomato production areas of the country. the damage inflicted by t. absoluta affects negatively its growth and development and can lead to total crop failure (desneux et al., 2010; biondi et al., 2018). this calls for concerted efforts from diffe­ rent stakeholders in developing effective manage­ ment strategies against this devastating pest. synthetic pesticides have been observed to be less effective against t. absoluta (roditakis et al., 2013) and are associated with various challenges and harmful effects (brahman et al., 2012; kumar and omkar, 2018). the concept of integrated pest man­ agement (ipm) was developed to address the draw­ backs of solely relying on chemical control. in this perspective, alternatives to synthetic insecticides with reduced negative effects have been the object of research in several parts of the world (biondi et al., 2018). a lot has been done on natural enemies, which are used in biological control of t. absoluta in some parts of the world (desneux et al., 2010; el­ ghany et al., 2016; giorgini et al., 2019). different biopesticides based on entomopathogens and botan­ ical insecticides have also been evaluated and shown to be effective against this pest. however, these studies have been limited to specific biocontrol strains/species and also have been carried out mainly in the pest’s area of origin (jallow et al., 2019). besides, many other studies have been limited to lab­ oratory conditions (youssef, 2015; el­ghany et al., 2016; giorgini et al., 2019). there is also a scarce information on the effects on different t. absoluta management options on growth, yield and fruit quali­ ty of tomato. entomopathogenic nematodes (epns), ento­ mopathogenic fungi (epfs) and plant extracts (pes) are among the claimed options for effective manage­ ment of t. absoluta (mansour et al. , 2018). laboratory studies in rwanda recommended some epns, epfs, and pes which can be advanced to field evaluation stage (ndereyimana et al., 2019 a, b, c). to this aim, the current study investigated the growth, yield and fruit quality of tomato as affected by entomopathogens and plant extracts against t. absoluta. 2. materials and methods study site this study was carried out in bugesera district of rwanda, in a farmer’s field located at 02° 32’ 355” south latitude, 30° 26’ 963” east longitude and an elevation of 1338 m above sea level. the average annual rainfall and temperature are 854 mm and 21.4°c, respectively (kabirigi et al., 2017). experimental design, trial establishment, and treat‐ ments application the study evaluated nine treatments in a ran­ domised complete block design with three replica­ tions. the individual experimental plots were 3 m long and 2 m wide, with 1.5 m wide paths between them. thirty days old, healthy and uniform tomato cv. roma seedlings were transplanted into the plots applied with 20 t of organic manure per hectare and mulched with dry grass. transplanting for trials one and two was carried out on 3rd april 2019 and 28th june 2019, respectively. the treatments included: two local epn isolates (steinernema sp. rw14­m­c2a­3 and steinernema sp. rw14­m­c2b­1), two commercial formulations of epfs [metatech® wp: metarhizium anisopliae (metsch.) sorok, strain fcm ar 23b3, 5 x 109 cfus/g, ndereyimana et al. ‐ different management options against tuta absoluta in tomato 125 and beauvitech® wp: beauveria bassiana (bals.) vuill., strain j25, 1 x 1010 cfus/g], two local plant extracts (tephrosia vogelii and phytolacca dodecan‐ dra), azadirachtin 0.03% ec (nimbecidine), imidaclo­ prid (confidor sl 200) and water. the two last treat­ ments were included as positive and negative con­ trols, respectively. the two epn isolates used were obtained from biological control laboratory ­ epn production facility at rwanda agriculture and animal resources development board (rab) (yan et al., 2016). mass production of the epns was done through in‐vivo method using galleria mellonella lar­ vae (kaya and stock, 1997). for field applications, these epns were formulated into sponges and were used at a concentration of 5 x 109 ijs/ha (gözel and kasap, 2015). the epf formulations were obtained from dudutech division, flamingo horticulture (k) ltd, naivasha, kenya and were used at a concentration of 250 g/ha. the two local plant extracts were prepared from leaves of local plants (t. vogelii and p. dodecan‐ dra). the fine powder was obtained (using an electric grinder) from the leaves dried in a shaded area, mixed with boiled water and kept for 12 hours. the concentration used for field application was 15% weight/volume (w/v) and filtration was done using a muslin cloth. azadirachtin 0.03% ec (nimbecidine) and imidacloprid (confidor sl 200) were used at the rates of 5 ml and 1 ml, respectively, per litre of water. all these treatments were applied weekly using a knapsack sprayer and the application volume was 1000 l/ha (brusselman et al., 2012). cultural operations apart from the difference in applied treatments, all other cultural operations were uniformly done in all the experimental plots. fungicide application was done every week by alternating copper oxychloride 50% wp with fungicides containing mancozeb 80% or mancozeb (640 g/kg) + metalaxyl (80 g/kg). each tomato plant was fertilised with 10 g of npk 17­17­17 as basal fertiliser, supplemented with 4 g of urea 46% on 30th day after transplanting as per rab rec­ ommendation. other cultural practices like watering, weeding, and pruning were carried out conventional­ ly. data collection and analysis data were collected on growth, yield, and fruit quality parameters. plant growth parameters: plant height, stem diameter and number of leaves per plant, were recorded every two weeks. plant height (cm) was measured from the ground to the tip of each of five randomly selected plants using a metre tape. stem diameter (mm) was measured from the collar using a digital vernier caliper. the number of leaves arising from the main stem was counted. for yield parameters, the numbers of flower trusses per plant and flowers per truss were recorded 40 days after transplanting, while the number of fruits per truss was recorded 60 days after transplanting. the number and yields of healthy and bored fruits were recorded during the harvesting period, which started 72 and 70 days after transplanting in trials one and two, respectively. all the above parameters were taken from five plants selected randomly in the mid­ dle of each plot. fruit quality parameters, namely fruit firmness (kg f/cm2), total soluble solids (tss) (°brix), beta­ carotene (mg/100 g of fruit), lycopene (mg/100 g of fruit), and ascorbic acid (mg/100 g of fruit), were recorded. to determine fruit firmness, tomatoes were harvested at the pink stage and stored at room temperature until the uniform red ripe stage. then, five fruits were randomly selected from each treat­ ment lot and fruit firmness measured in the equatori­ al zone of each tomato using a penetrometer (ritenour et al., 2002). total soluble solids were determined on the same fruits used for the determi­ nation of fruit firmness using a refractometer (rhw refractometer, optoelectronic technology company limited, uk) (majidi et al., 2011). beta­carotene was obtained following the method described by delia et al. (2004). lycopene was extracted using acetone and analysed in a spectrophotometer at 503 nm. lycopene content was then calculated using the for­ mula given by ranganna (1997) as follows: lycopene content = 3.1206 x a x v x d x 100 (w x 100) where a = absorption, v = volume made up, d = dilution, w = weight of sample. ascorbic acid was determined by titration with 2,6­dichlorophenolin­ dophenol dye (aoac, 1990). the distribution of the collected data was assessed and the appropriate transformation was undertaken, where necessary, before subjecting them to analysis of variance. in both trials, the num­ bers of healthy and bored fruits per plant were square­root transformed, while the yield of healthy and bored fruits per plant were log­transformed. the number of fruits per truss was log­transformed in trial one, and arcsine­transformed in trial two; while the number of flowers per truss was arcsine­ transformed in trial two. all other parameters were adv. hort. sci., 2020 34(2): 123­132 126 fig. 2 ­ stem diameter of tomato cv. roma under different treat­ ments against tuta absoluta in trials one (a) and two (b). t1: steinernema sp. rw14­m­c2a­3, t2: steinernema sp. rw14­m­c2b­1, t3: metatech®wp (metarhizium aniso‐ pliae , strain fcm ar 23b3), t4: beauvitech® wp (beauveria bassiana, strain j25), t5: tephrosia vogelii, t6: phytolacca dodecandra, t7: azadirachtin 0.03% ec, t8: imidacloprid, t9: water; dat: days after transplan­ ting; different letters above the bars indicate significant difference according to tukey’s test (p≤0.05). analysed without transformation. to determine the effect of the treatments on tomato fruits yield and quality, analysis of variance was carried out; and the means for significantly different treatments (at p≤0.05) were separated using tukey’s honestly sig­ nificant difference test. the data analysis was car­ ried out using the statistical analysis system pack­ age, sas software version 9.2 (sas institute, 2010). 3. results tomato growth parameters plant height was significantly (p≤0.05) influenced by the studied treatments from 30 days after trans­ planting (dap) (fig. 1). in both trials, the plant height was not significantly different at 15 dat; with an average of 14.9 and 15.3 cm for trials one and two, respectively. plant height increased with time but became almost constant at 45 dat. in trial one, there was no significant difference among the ento­ mopathogens (epns and epfs) and azadirachtin on all days of observation. tephrosia vogelii was not signifi­ cantly different from all the above at 30, 45, and 60 dat, except steinernema sp. rw14­m­c2a­3. lower plant height was recorded with p. dodecandra and the controls, which were insignificantly different. in trial two, plant height did not significantly differ among the treatments, except p. dodecandra and the controls which had lower plant height than others. stem diameter did not significantly differ among the treatments at 15 and 30 days after transplanting (dat) in trial one and at 15 dat in trial two (fig. 2). in addition, only the stem diameter in the negative con­ trol was significantly lower as compared to the other treatments at 45 dat in trial one. phytolacca dode‐ candra and imidacloprid were similar to the negative control, with significantly lower stem diameter (p≤0.05) compared to the other treatments at 60 dat. for trial two, p. dodecandra and negative con­ trol had significantly lower stem diameter as com­ pared to the other treatments at 30 dat; but at 60 dat it was only the negative control which had signif­ icantly lower stem diameter as compared to azadirachtin and all entomopathogens except beauvitech® wp. the number of leaves per plant was not signifi­ cantly affected by the evaluated treatments in both fig. 1 ­ plant height of tomato cv. roma under different treat­ ments against tuta absoluta in trials one (a) and two (b). t1: steinernema sp. rw14­m­c2a­3, t2: steinernema sp. rw14­m­c2b­1, t3: metatech®wp (metarhizium aniso‐ pliae, strain fcm ar 23b3), t4: beauvitech® wp (beauveria bassiana, strain j25), t5: tephrosia vogelii, t6: phytolacca dodecandra, t7: azadirachtin 0.03% ec, t8: imidacloprid, t9: water; dat: days after transplan­ ting; different letters above the bars indicate significant difference according to tukey’s test (p≤0.05). ndereyimana et al. ‐ different management options against tuta absoluta in tomato 127 trials. however, the general trend observed in both trials was that slightly higher (but not significantly dif­ ferent) number could be obtained in plots treated with metatech® wp (m. anisopliae, strain fcm ar 23b3) and azadirachtin in trial one; and with steinernema sp rw14­m­c2a­3 and beauvitech® wp (b. bassiana, strain j25) in trial two (fig. 3). the aver­ age numbers of leaves per plant recorded at 60 dat in trial one were 13.0, 12.8,14.0, 12.5, 12.8, 12.6, 13.3, 11.9, and 12.6; while in trial two they were 11.8, 11.6, 11.7, 11.9, 11.1, 11.1, 11.5, 11.2, and 11.3, in plots treated with steinernema sp. rw14­m­c2a­3, steinernema sp. rw14­m­c2b­1, metatech®wp (m. anisopliae, strain fcm ar 23b3), beauvitech® wp (b. bassiana, strain j25), t. vogelii, p. dodecandra, azadirachtin, imidacloprid, and water, respectively. effect of entomopathogens and plant extracts on tomato yield the evaluated treatments significantly (p<0.001) influenced tomato yield parameters in both trials (table 1). generally, plots treated with the ento­ mopathogens or azadirachtin had higher perfor­ mance as compared to those with plant extracts or controls. a similar number of flower trusses per plant was recorded by steinernema sp. rw14­m­c2a­3, steinernema sp. rw14­m­c2a­3, metatech®wp, fig. 3 ­ number of leaves per plant for tomato cv. roma under different treatment against tuta absoluta in trials one (a) and two (b). t1: steinernema sp. rw14­m­c2a­3, t2: steinernema sp. rw14­m­c2b­1, t3: metatech®wp (metarhizium anisopliae, strain fcm ar 23b3), t4: beauvitech® wp (beauveria bassiana, strain j25), t5: tephrosia vogelii, t6: phytolacca dodecandra, t7: azadi­ rachtin 0.03% ec, t8: imidacloprid, t9: water; dat: days after transplanting; similar letters above the bars indica­ te non­significant difference according to tukey’s test (p≤0.05). table 1 ­ yield parameters (mean ± sd) of tomato under different entomopathogens and plant extracts treatments t1: steinernema sp. rw14­m­c2a­3, t2: steinernema sp. rw14­m­c2b­1, t3: metatech®wp (metarhizium anisopliae, strain fcm ar 23b3), t4: beauvitech® wp (beauveria bassiana, strain j25), t5: tephrosia vogelii, t6: phytolacca dodecandra, t7: azadirachtin 0.03% ec, t8: imidacloprid, t9: water. means followed by the same letter (s) are not significantly different (tukey’s test, p≤0.05) treatments number of flower trusses/plant number of flowers/truss number of fruits/truss number of healthy fruits/plant number of bored fruits/plant yield of healthy fruits (g/plant) yield of bored fruits (g/plant) trial one t1 11.9 ± 0.2 a 9.7 ± 0.4 a 4.1 ± 0.3 a 5.9 ± 0.2 a 5.8 ± 0.2 a 406.3 ± 10.9 a 333.3 ± 33.4 a t2 11.6 ± 0.4 abc 8.5 ± 0.1 bc 4.0 ± 0.1 a 5.6 ± 0.2 a 5.1 ± 0.4 a 381.0 ± 22.3 a 286.8 ± 8.8 a t3 11.7 ± 0.2 ab 7.7 ± 0.2 cd 4.1 ± 0.1 a 5.5 ± 0.3 a 6.1 ± 0.4 a 374.4 ± 23.5 a 328.8 ± 34.9 a t4 11.4 ± 0.1 abc 7.3 ± 0.3 de 3.8 ± 0.5 a 5.4 ± 0.2 a 5.6 ± 0.4 a 335.0 ± 34.5 a 313.9 ± 17.3 a t5 10.9 ± 0.2 bc 7.4 ± 0.1 de 3.0 ± 0.3 b 2.5 ± 0.3 b 3.2 ± 0.2 b 151.0 ± 12.3 b 161.7 ± 9.7 b t6 10.8 ± 0.3 c 6.4 ± 0.2 ef 2.9 ± 0.2 b 1.5 ± 0.1 c 2.6 ± 0.2 cb 81.5 ± 12.5 b 126.2 ± 10.1 b t7 12.7 ± 0.1 a 9.4 ± 0.6 ab 4.3 ± 0.2 a 6.5 ± 0.2 a 4.9 ± 0.6 a 402.9 ± 12.7 a 275.7 ± 29.5 a t8 10.8 ± 0.3 c 6.5 ± 0.3 ef 3.0 ± 0.4 b 1.7 ± 0.2 c 2.3 ± 0.5 cb 86.6 ± 9.0 b 109.9 ± 26.2 bc t9 10.8 ± 0.5 c 5.6 ± 0.7 f 2.5 ± 0.3 b 0.7 ± 0.4 d 1.9 ± 0.5 c 32.5 ± 8.2 c 83.7 ± 22.1 2 b cv 2.5 4.89 6.53 4.39 5.76 4.2 2.23 p <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 trial two t1 9.0 ± 0.2 a 7.7 ± 0.3 a 3.8 ± 0.3 a 5.5 ± 0.2 a 4.6 ± 0.7 a 367.8 ± 5.2 a 249.5 ± 22.1 a t2 9.0 ± 0.2 a 7.4 ± 0.2 a 3.7 ± 0.2 a 5.4 ± 0.2 a 4.7 ± 1.0 a 350.9 ± 12.1 a 255.9 ± 38.1 a t3 8.8 ± 0.2 ab 5.6 ± 0.2 cb 3.8 ± 0.4 a 4.9 ± 0.4 a 5.6 ± 0.8 a 309.7 ± 26.3 a 302.4 ± 45.9 a t4 8.8 ± 0.5 ab 5.5 ± 0.2 cb 3.6 ± 0.2 a 4.9 ± 0.4 a 5.3 ± 1.0 a 319.4 ± 33.5 a 273.6 ± 64.8 a t5 8.8 ± 0.2 ab 5.9 ± 0.2 b 2.8 ± 0.1 b 2.0 ± 0.4 b 4.4 ± 0.4 a 113.1 ± 13.4 b 220.9 ± 18.2 a t6 8.1 ± 0.2 b 5.2 ± 0.3 cb 2.4 ± 0.4 b 1.4 ± 0.2 b 2.1 ± 0.1 b 67.0 ± 8.5 c 114.8 ± 17.0 b t7 9.3 ± 0.2 a 8.0 ± 0.3 a 4.0 ± 0.2 a 6.0 ± 0.4 a 4.7 ± 0.1 a 392.5 ± 38.1 a 266.6 ± 19.6 a t8 8.7 ± 0.1 ab 5.5 ± 0.3 cb 2.7 ± 0.2 b 1.6 ± 0.3 b 1.9 ± 0.5 b 74.7 ± 8.1 c 96.7 ± 22.5 b t9 8.1 ± 0.2 b 5.0 ± 0.2 c 2.2 ± 0.3 b 0.8 ± 0.2 c 1.7 ± 0.2 b 35.7 ± 6.7 d 80.0 ± 10.10 b cv 3.02 2.31 4.17 5.11 7.94 2.3 3.05 p 0.0004 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 128 adv. hort. sci., 2020 34(2): 123­132 beauvitech wp, and azadirachtin. these values were significantly (p<0.001) higher than t. vogelii, p. dode‐ candra, imidacloprid, and water spray in trial one. in trial two, the effect of t. vogelii and imidacloprid was similar to all the treatments but the plot treated with epns and azadirachtin recorded a significantly higher number of flower trusses per plant than the negative control. the number of flowers per truss was signifi­ cantly higher with steinernema sp. rw14­m­c2a­3 and azadirachtin in trial one, and with all ento­ mopathogenic nematodes and azadirachtin in trial two. higher numbers of fruits per truss, healthy and bored fruits per plant were recorded with all ento­ mopathogens and azadirachtin, in both trials. a simi­ lar trend was observed in the yield of healthy and bored fruits per plant. effect of entomopathogens and plant extracts on tomato fruit quality tomato fruit quality parameters were not signifi­ cantly influenced by the applied treatments against t. absoluta. the results obtained were so close to each other that it is not easy to find any trend amongst the treatments (fig. 4). the overall average values obtained were 3.2 and 3.3 kg f/cm2 for fruit firmness, 4.2 and 4.4obrix for tss, 8.3 and 8.1 mg/100 g of fruit for beta­carotene, 5.4 and 5.5 mg/100 g of fruit for lycopene, 14.36 and 14.6 mg/100 g of fruit for ascorbic acid in trials one and two, respectively. 4. discussion and conclusions scarce studies have been conducted on the effects of entomopathogens and plant extracts on growth, yield and fruit quality of tomato. the signifi­ cant differences observed in plant height and stem diameter could be due to the differences in the effi­ cacy of studied treatments against t. absoluta. the damages inflicted by t. absoluta larvae my have affected the physiological and biochemical reactions of tomato plants, so that plant growth was conse­ quently affected (desneux et al., 2010). beauveria bassiana which was reported to exhibit endophytic activity by colonising vascular tissues would be expected to impair the normal plant growth. however, different researchers reported that b. bassiana does not impede tomato growth (klieber and reineke, 2016; allegrucci et al., 2017). on the other hand, since t. vogelii is a rich source of nitro­ gen, fixed through biological nitrogen fixation (stevenson et al., 2012), more growth would be expected in this treatment compared to the others because nitrogen is more involved in plant growth and biomass production (larbat et al., 2016). this was, however, not observed in this study and could be explained by the fact that the amount sprayed as an insecticide was too little to have a direct signifi­ cant effect on plant growth. finally, the insignificant difference in the number of leaves per plant despite the treatments could be because this parameter is associated with the genetic makeup of the plant (kaushik et al., 2011) and not with cultural practices including pest management. the significant difference in flower­related parameters could also be due to the difference in the efficacy of the studied treatments. by attacking the floral parts, t. absoluta larvae might have dam­ aged some of them before they differentiate into flowers and caused others to drop; which could be the explanation for the flower abortion observed in this study. these results are in agreement with cherif et al. (2013) who reported that t. absoluta larvae can damage tomato flower parts and cause flower drop. the observed significant difference in yield parameters may also have arisen from the indirect effect of t. absoluta larvae through their feeding fig. 4 ­ fruit quality parameters of tomato cv. roma under diffe­ rent treatment against tuta absoluta in trials one (a) and two (b). t1: steinernema sp. rw14­m­c2a­3, t2: steinernema sp. rw14­m­c2b­1, t3: metatech®wp (metarhizium anisopliae, strain fcm ar 23b3), t4: beauvitech® wp (beauveria bassiana, strain j25), t5: tephrosia vogelii, t6: phytolacca dodecandra, t7: azadi­ rachtin 0.03% ec, t8: imidacloprid, t9: water; similar let­ ters above the bars indicate non­significant difference according to tukey’s test (p≤0.05). ndereyimana et al. ‐ different management options against tuta absoluta in tomato 129 activity in leaf mesophyll (biondi et al., 2018), which might have slowed down the process of assimilates synthesis and partitioning for their utilisation by dif­ ferent plant organs, including flower parts and fruits. in agreement with the above observation, desneux et al. (2010) and el­ghany et al. (2016) also reported that a tomato attack by t. absoluta dis­ turbs its normal growth, development and the sub­ sequent yield. thus, higher numbers of flower truss­ es per plant, flowers per truss and fruits per truss recorded with entomopathogens and azadirachtin suggest that these treatments can reduce tomato yield loss as compared to the plant extracts and the controls (imidacloprid and water spray). in their study, rab and haq (2012) found that the number of flowers per truss varied from 17.1 to 30.8 while the number of fruits per cluster was 4.1­6.4 for tomato cv. roma. however, in the present study, a range of 5.6­9.7 flowers per truss and 2.2­4.3 fruits per truss was obtained. this indicates the ability of t. absoluta to negatively affect the flower and fruit­ bearing capacity of tomato plant. this is one of the reasons for high yield losses frequently observed with t. absoluta infestations (cherif et al., 2013; biondi et al., 2018). the higher numbers and yield of healthy fruits that were obtained with epns, epfs, and azadirachtin support our earlier findings in laborato­ ry experiments (ndereyimana et al., 2019 a, b, c). in line with the findings of this study, braham et al. (2012), gözel and kasap (2015), youssef (2015), and el­ghany et al. (2016) reported that epns, epfs, and azadirachtin result in better control of t. absoluta. the performance of plant extracts and imidacloprid (positive control) remained low as it was in our pre­ vious laboratory studies (ndereyimana et al., 2019 a, b). negative control also recorded very low yield, which was consistent with desneux et al. (2011) and biondi et al. (2018) who emphasized that if there are no serious pest management strategies that are meticulously implemented, the yield loss might reach 100%. higher number and yield of bored fruits obtained from plots treated with entomopathogens and plant extracts, as compared to plant extracts and controls, might have resulted from the reduced number of aborted and damaged flowers by t. absoluta in the plots where these treatments were applied. although these fruits survived from early abortion and the dropping of progenitor flowers, they were more exposed to t. absoluta because they were many, and thus a group of them was later bored by the pest that spoiled their quality. compared to the negative control, the yield of healthy fruits obtained with steinernema sp. rw14­m­c2a­3, steinernema sp. rw14­m­c2a­3, metatech®wp, beauvitech®wp, and azadirachtin increased 12.5, 11.7, 11.5, 10.3 and 12.4 folds, respectively. while compared to the posi­ tive control, it was 4.8, 4.5, 4.2, 4.1 and 5.0 folds, respectively. this confirms that, despite the invasive nature of t. absoluta, different management options can reduce significantly its negative impact on the crop. however, dependence on synthetic insecti­ cides should be discouraged as evidenced by the results of this study, which are consistent with sev­ eral other researchers (desneux et al., 2010; roditakis et al., 2013; biondi et al., 2018). the commercial value of bored fruits is lost because they are not preferred by customers as external appearance and absence of defects are among the factors determining consumer preference (asensio et al., 2019). in addition to the larvae that enter inside the fruits, also some pathogens like fungi often get inside through the created holes and cause fruit decaying before or after harvest (desneux et al., 2010). the findings of this study are supported by previous researchers who worked on other pests and reported that crop pests are among the main factors reducing the yield and quality of field horticultural produce by direct feeding or by favouring several dis­ eases (kumar and omkar, 2018). thus, implementa­ tion of ipm is worth to ensure better yield and quality of tomato crop. since the damage inflicted by tuta absoluta on the leaves of tomato plants negatively affects its physiological processes (desneux et al., 2010; biondi et al., 2018) and fruit total soluble solids are translo­ cated from the photosynthetic activities in the leaves (beckles, 2012), significant difference in fruit quality parameters was expected among treatments with different t. absoluta infestation levels. similarly, the entomopathogens and azadirachtin that exhibited better t. absoluta control would have also resulted in higher quality fruits as compared to the plant extracts and the controls´ treatments. the observed non­significant difference in tomato fruit quality parameters: firmness, total soluble solids, beta­carotene, lycopene, and ascorbic acid among treatments against t. absoluta, therefore may be attributed to other factors such as variety, crop nutrition, climatic conditions, fruit ripening stage, and storage period (marsic et al., 2011; rab and adv. hort. sci., 2020 34(2): 123­132 130 haq, 2012; tigist et al., 2013; asensio et al., 2019). fruit firmness results obtained in this study fall in the range of the values obtained by rab and haq (2012). fruit firmness is an important quality parame­ ter that determines fruit shelf­life and resistance to mechanical damage (tigist et al., 2013). in line with the current study, parmar et al. (2018) also obtained a tss value of 4.8 obrix for tomato cv. roma under organic management system. also, tss values obtained by rab and haq (2012) ranged from 4.08 to 6.10 obrix under different rates of calcium chloride and borax. the values of beta­carotene and lycopene recorded in this study are close to what was obtained by parmar et al. (2018) (8.34 mg/100 g and 5.38 mg/100 g of fruit, respectively) for the same variety (roma) produced organically. the ascorbic acid results obtained in this study agree with the earlier findings of tigist et al. (2013) who obtained the val­ ues of 13.2 and 14.8 mg/100 g after four and eight days of room temperature storage, respectively, for tomato cv. roma fruits harvested at the green mature stage. according to tigist et al. (2013), these quality parameters develop into fruit during the pre­harvest period and they do not get improved after harvest­ ing. however, they can be maintained by proper post­harvest handling and storage. since pre­har­ vest activities are responsible for the development of quality parameters in tomato fruits, any technolo­ gy used to improve its production should also be assessed for its effect on fruit quality. as a conclusion, the studied entomopathogens and plant extracts significantly affected tomato growth and yield but not the fruit quality parame­ ters. better yield performance can be obtained with the entomopathogenic nematode isolates (steinernema sp. rw14­m­c2a­3 and steinernema sp. rw14­m­c2a­3), commercial formulations of entomopathogenic fungi (metatech®wp: metarhizium anisopliae, strain fcm ar 23b3 and beauvitech®wp: beauveria bassiana, strain j25) and azadirachtin 0.03% ec, which were not significantly different. these biorational control agents are rec­ ommended to be included in the ipm of tuta abso‐ luta. the results of this study will guide producers to select the best control options that can result in higher comparative growth and yield without com­ promising fruit quality. further studies should be conducted to confirm the effects of the studied entomopathogens and plant extracts under varied agro­climatic conditions. acknowledgements this work was sponsored by united states agency for international development, as part of the feed the future initiative, under the cgiar fund, award num­ ber bfs­g­11­00002, and the predecessor fund the food security and crisis mitigation ii grant, award number eem­g­00­04­00013. the authors are grateful to egerton university and rwanda agriculture and animal resources development board for providing opportunities to conduct this study. much apprecia­ tion is due to p. rukundo, b. kagiraneza, b. uzayisenga, and j. kajuga for their technical support. authors are also thankful to d. bazagwira, g. ingabire and 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putrescine, spermidine, spermine. abstract: the most critical problems of temperate fruit trees are poor fruit set and low yield. to solve the problem, some major chemical compounds such as polyamines and sics (self-incompatibility control substance, mn+b) can be used. popular polyamines including putrescine (0.1 and 0.25 mm), both spermine and spermidine (0.05 and 0.25 mm), and sics (1 and 2 mg l-1) were used alone or with cotton coverage bags to cover branches in order to investigate self-incompatibility in malus domestica. results showed that spermidine (0.25 mm) led to higher yields in comparison with that of the control. sics (2 mg l-1), also, demonstrated the highest yield compared with that of the control. at june fruit set, treatment with spermidine (0.25 mm) led to the highest percentage of fruit set and also the highest index of self-incompatibility and percentage of final fruit set among treatments. 1. introduction alternate bearing and self-incompatibility are the main issues in apple. alternate bearing is vigorous in some apple cultivars like ‘golden delicious’. most apple cultivars are self-incompatible and self-unfruitful; therefore, they need another cultivar for pollination and fertilization. gametophytic self-incompatibility (gsi) occurs when the s allele of the pollen grain matches either of the s alleles of the stigma. in such a case, the pollen tube begins to develop but stops before reaching the micropyle (asatryan and tel-zur, 2013). gametophytic self-incompatibility is controlled by glycoprotein with rnase (s-rnase) activity expressed in the pistil. s-rnase (qing-qing et al., 2009; duca et al., 2010; uchida et al., 2012) encoded by the s-locus gene is named sfb (s-haplotype-specific f-box gene) in the rosaceae. to date, in rosaceae family, si has mainly been studied in japanese pear (pyrus pyrifolia) and almond (prunus dulcis) (qing-qing et al., 2009; uchida et al., 2012). however, some requirements are necessary for suitable cross pollination: having (1) compatible pollen grains with enough quantity and high quality (in fact, pollination with (*) corresponding author: davarynej@um.ac.ir citation: sayyad-amin p., davarynejad g.h., abedy b., 2018 the effect of polyamines and sics on the compatibility, fertility and yield indices of apple cv. golden delicious. adv. hort. sci., 32(2): 213219 copyright: © 2018 sayyad-amin p., davarynejad g.h., abedy b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 november 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 213-219 214 semi-compatible pollen resulted in lower fruit-set than that with fully compatible pollen) (sapir et al., 2008), (2) an overlap period of pollination between pollinizer and pollinator (pollinizer trees must be cultivated along with the pollinator trees) (3) proper time of flowering and blooming, and finally, (4) having attractive flowers in order to reduce the number of bees visiting weeds on the orchard floor (the pollen grain of fruit trees is sticky and heavy causing not to be carried by the wind) (bekey and burgett, 1981). hand pollination is a type of cross pollination that can be effective on producing crop in adverse weather conditions. when the king flower opens, it is necessary to place bee colonies in order to do cross pollination by bees and then remove them at petal fall in fruit orchards: however, this method is also timeconsuming and needs a big number of workers (bekey and burgett, 1981; maib et al., 1996). despite the presence of hives, pollen transfer limitation and subsequent seed set reductionwas observed in orchards (quinet et al., 2016). the other suitable technique applied to do pollination involves chemical methods; in fact, some chemical compounds can be used in order to achieve enough fruit set and high yield. plant bio regulators (pbrs) had significant effects on increasing pollen germination and pollen tube length in almond pollen: the action of these pbrs significantly increased the percentage of fruit set at both the bud pink and petal fall phenological stages (maita and sotomayor, 2015). as an alternative, the use of polyamines (putrescine, spermine, spermidine), that are natural compounds involved in plant growth and development process, has been suggested reduce flowers and fruits drop; polyamines competes with ethylene synthesis with whose they share the same precursor called s-adenosyle methionine (crisosto et al., 1988; khezri et al., 2010). polyamines as plant bio regulator revealed to increase pollen tube growth and fruit set by stimulating pollen germination (crisosto et al., 1988; liu et al., 2006) and were effective on pollen tube elongation (aloisi et al., 2015). also, self-incompatibility control substance (sics), which is a mixture of manganese and boron (son et al., 2009), could be useful for increasing fruit set. polyamines and sics were effective on increasing yield and fruit set in crops such as pear (crisosto et al., 1988; son et al., 2009), apple and apricot (asadi et al., 2013), olive (costa et al., 1986), sweet cherry (grant sheard, 2008), sweet orange (saleem et al., 2008), pistachio (khezri et al., 2010), mango (malik et al., 2005), and date palm (tavakoli and rahemi, 2014). with regard to the effect of these compounds on pollen tube growth, spermidine (spd) and spermine (spm) influence the promotion of the pollen tube elongation at polyamines concentrations up to 50 mm in rosaceae family, whereas higher concentration of spd and spm resulted inhibitory for pollen tube elongation in rosaceae family and correlate with male sterility in actinidia deliciosa (aloisi et al., 2015). they are compounds useful in enhancing ovule longevity (crisosto et al., 1988; liu et al., 2006), without having any deleterious and toxic effects on human life (azh et al., 2014). in the initial stage of fruit development, an active cell division takes place, which possibly needs sufficient polyamines. at the later stage of fruit development, polyamine synthesis is reduced. as biosynthesis of polyamines takes place before pollen tube emergence, low level of free polyamines in cytoplasmic male sterile plants influences cell division and its enlargement, leading to abnormal development and low viability of pollens (liu et al., 2006). polyamines increase pollen tube growth and fruit set by stimulating pollen germination. as well, they play a role in carbohydrates and nitrogen regeneration, followed by increasing chlorophyll content and leaf area (baninasab and rahemi, 2008). use of polyamines (put, spm, spd) at bloom improves ovule longevity in fruit crops such as apricot and pear. higher endogenous polyamine contents have been correlated with improved ovule viability in apricot and sour cherry (grant sheard, 2008). boron increased pollination activity (nyomora et al., 1997). in boron deficiency, phenolic compounds aggregates on stigma. the accumulation of these compounds due to the activation of dehydrogenase enzyme led to the pollen grain not to be germinated. boron increases pollen grain viability by increasing the flavenoids content of pollen grain (marschener, 1995). the percentage of fruit set as a vital factor is as important as other quantitative and qualitative traits to achieve an acceptable yield in apple (malus domestica). it seems the contemporary application of polyamine and sics together can enhance their effects. although there are numerous researches on the individual application of polyamine and sics, there was not found any literature comparing the effect of polyamines and sics on yield indices, especially along with cotton coverage bags on the index of fertility (if) and the index of self-incompatibility (isi). the aim of this study was to increase yield indices in apple cultivar ‘golden delicious’, accompanying the decrease of fruit drop using polyamines and sics. sayyad-amin et al. polyamines and sics effect on compatibility, fertility and yield indices of apple cv. golden delicious 215 2. materials and methods the study was carried out on thirteen year-old apple trees cv. golden delicious in an orchard located in mashhad (latitude of 36°20’and altitude 59° 34‘). it is an area with arid and semiarid climate and annual average precipitation of 255 mm. the foliar application was done with 5 l sprayer on four selected branches between bud swollen and flower opening pheological stages at early morning. the compounds included polyamines [putrescine (put) 0.1 or 0.25 mm; spermine (spm) 0.05 or 0.25 mm; spermidine (spd) 0.05 or 0.25 mm] and sics [1 mg/l (3.5 mg boric acid, 6.8 mg manganase sulphate) and 2 mg/l (6.8 mg boric acid, 13.6 mg manganese sulphate)] (son et al., 2009) with (+) or without (-) cotton coverage bags (ccb), and control (untreated) plants. cotton coverage bags were used in order to prevent cross and open pollination. in this study, the following traits were measured: yield, percentage of fruit set [initial (2 weeks after petal fall); june (fruit drop in june); final (at harvest)] and fruit drop. index of fertility (if) was measured based on the percentage of initial fruit set ratio in each treatment compared to control. index of self-incompatibility (isi) was evaluated based on the percentage of final fruit set ratio in each treatment in comparison with control; in this regard, the ratios of 0.2, between 0.2-1, and higher than 1 represent incompatibility, semi-compatibility and full self-compatibility, respectively (zeinanlo et al., 2001; azimi et al., 2008; seifi 2008; taslimpour and aslmoshtaghi, 2013). the randomized complete block design with four replicates was applied in this study. at final, the data were analyzed by sas software ver. 9.1 (sas institute, 2004) and the means were compared using lsd test at 0.05. fruit diameter and length were measured with non destructive method during the fruit growth period until the harvest time. this method was better than the destructive technique. non destructive method are less time consuming and no need to laboratory space, and without harvesting. according to this method, fruit length and diameter were measured every 2 weeks without harvest, whereas in destructive method, these parameters are measured in fruits harvested every 2 weeks and their length and diameter were measured. therefore, non destructive method was used in the present study (arzani et al., 1999; dehghani et al., 2012). 3. results the effects of polyamines and sics on yield and fruit set without cotton coverage bags. data showed that spd treatment (0.25 mm) led to a higher yield in comparison to the control (p<0.05). sics (2 mg l-1) demonstrated the highest yield among all of the treatments (p<0.05). it was also found that put (0.1 mm) and spm (0.05 mm) showed increase in yield compared with the control, but it was statistically non-significant at 0.05 level. sics (1 mg l-1) increased initial fruit set when compared to control. the highest june and fruit set related to spd (0.25 mm) among all of the treatments (p<0.05). with cotton coverage bags. there was significant increase in percentage of initial fruit set by application of sics (2 mg l-1), and spm (0.25 mm) (table 1) in comparison to the control. the results also demonstrated the higher percentage of initial fruit set under treatment with spd (0.05 mm) than the control, but it was statistically non-significant at 0.05 level. percentage of final fruit set showed statistically significant difference (p <0.05) in most of the treatments in comparison with the control, except for put (0.1 mm), sics (2 mg l-1), spm (0.25 mm), spm (0.05 mm) and spd (0.25 mm). the effects of polyamines and sics on if and isi the index of fertility increased in treatments with spm (0.25 mm + ccb) and sics (2 mg l-1 + ccb). all of the treatments could led to semi or full fertility and compatibility, due to the percentage of fruit set ratio in each treatment compared to the control was larger than 0.2. the effects of polyamines and sics on fruit drop in general, final fruit drop decreased significantly in treatments with put (0.1 mm) and spd (0.05, 0.25 mm). without coverage. sics (1, 2 mgl) decreased percentage of initial fruit drop in comparison with the control of 12 and 10%, respectively (p <0.05). final fruit drop decreased significantly in treatments with put (0.1 mm) and spd (0.05, 0.25 mm). with cotton coverage. spm (0.25 mm) demonstrated the significantly (p<0.05) decrease of 14% in percentage of initial fruit drop in comparison with the control. adv. hort. sci., 2018 32(2): 213-219 216 the effects of polyamines and sics on fruit growth habit based on figure 1, fruit growth totally increased in all of the treatments; in fact, fruit length and diameter increased gradually from july 9 to harvest (fig. 1 b and d) whereas spm (0.25 mm + ccb) showed a different trend. 4. discussion and conclusions the effects of polyamines and sics on yield and fruit set in the present study, application of polyamines [put (0.1, 0.25 mm), put (0.25 mm + ccb), spd (0.05, 0.25 mm) and spd (0.05 mm + ccb)] between periods of swollen buds and the start of flowering phenological stages could increase fruit set at final fruit set stage. several researchers studied about the effects of polyamines on yield indices. polyamines (put, spm, spd) enhanced ovule longevity at bloom in fruit crops such as apricot, pear and sour cherry (grant sheard, 2008). polyamine synthesis had also positive influence on development and viability of pollen and it occurred before pollen tube emergence. therefore, lower content of free polyamines caused male sterility in flowers (liu et al., 2006). exogenous application of different polyamines at full bloom had influence on increasing fruit set and total yield in apples, olive, litchi and mango. increase in fruit set and yield by polyamines was due to raising pollination, fertilization and fruit retention (costa et al., 1986). saleem et al. (2008) stated that polyamines significantly increased initial fruit set and yield and maximum fruit set was observed in spd, spm and put, respectively. put had positive effect on increasing ovule longevity, epp (effective pollination period), n and b and might raise the pollen tube growth rate in the styles of pears (crisosto et al., 1988), but their role on raising ovule longevity might be due to the improved nutritional status of the flower (grant sheard, 2008). application of put raised fruit set and yield of ‘comice’ pear at the start of flowering (crisosto et al., 1988). fruit set, crop density and yield efficiency under low fruit set conditions were improved by put application at flower opening stage in pear (crisosto et al., 1988). according to our results, the effects of polyamines on fruit set and yield were in agreement with crisosto et al. (1988) on pear, malik et al. (2005) on mango, grant sheard (2008) on sweet cherry, saleem et al. (2008), khezri et al. (2010) and asadi et al. (2013) on pistachio. in present study, sics (2 mg l-1) had also a positive influence on fruit set and yield. in boron deficiency, phenolic compounds aggregate on stigma. accumulation of these compounds due to activation of dehydrogenase enzyme did not lead to germination of pollen grain. boron increased pollen grain viability by increasing the flavenoids content of pollen grain (marschener, 1995). boron increased pollination activity (nyomora et al., 1997). foliar application table 1 the effect of polyamines (putrescine, spermine and spermidine) and sics on yield, fruit set and first drop means with the same letters were not significantly different according to lsd (0.05). spm= spermine, spd= spermidine, put= putrescine, if=index of fertility, isi=index of incompatibility. treatments yield (g) initial fruit set (%) june fruit set (%) final fruit set (%) if isi first drop (%) final drop (%) control 1230 cde 55.893 d-g 40.395 b-e 18.02 f 1 b-e 1 bcd 30.7 cde 68.25 a-e without cotton coverage bag put (0.1 mm) 1416.7 c 50.179 e-h 39.046 c-f 28.636 bc 0.8625 def 1.4872 b 34.091 cd 42.424 g put (0.25 mm) 1101 def 15.152 j 37.231 c-f 27.778 bc 0.5565 f 0.3715 e 31.548 cde 55.357 efg spd (0.05 mm) 728.5 ghi 34.953 hi 32.749 c-g 22.281 de 0.6352 ef 1.2683 bcd 25.417 d-g 51.25 fg spd (0.25 mm) 1990.8 b 41.692 ghi 61.722 a 34.722 a 0.7621 def 2.4166 a 18.75 gh 20.313 h spm (0.05 mm) 1274.5 cd 74.605 abc 43.367 bcd 29.592 bc 1.3588 ab 1.0409 bcd 24.107 efg 76.259 ab spm (0.25 mm) 1130 def 69.86 bcd 23.077 g 19.268 f 1.2552 abc 1.529 b 64.245 a 68.593 a-e sics (1 mg l-1) 452 ij 75.63 abc 52.241 ab 20.26 f 1.3693 ab 1.2772 bcd 19.063 fg 56.818 d-g sics (2 mg l-1) 2349 a 59.127 c-e 40.857 b-e 29.241 b 1.0773 bcd 1.4317 bc 9.43 h 58.772 c-f with cotton coverage bag put (0.1 mm) 556 ij 42.361 f-i 28.03 fg 18.374 f 0.8869 c-f 1.4954 b 35.714 c 64.249 b-e put (0.25 mm) 1116 def 33.145 i 27.083 fg 22.917 de 0.7276 def 1.2771 bcd 48.889 b 72.222 a-d spd (0.05 mm) 696 hi 60.348 cde 30.925 efg 20.049 c 1.0839 bcd 1.4104 bc 50.379 b 63.258 b-f spd (0.25 mm) 984 efg 56.25 d-g 43.75 bc 18.5 f 1.015 b-e 0.7663 de 16.193 gh 77.399 ab spm (0.05 mm) 355 j 51.667 e-h 43.089 bcd 20.365 f 0.9567 cde 0.8757 cde 56.034 ab 74.353 abc spm (0.25 mm) 599 ij 84.858 ab 31.579 efg 17.526 f 1.5371 a 1.2116 bcd 22.5 efg 55 efg sics (1 mg l-1) 900.7 fgh 57.143 defg 32.712 c-g 25.173 cd 1.054 bcd 1.3247 bcd 28.333 c-f 56.667 d-g sics (2 mg l-1) 720 ghi 87.326 a 44.355 bc 19.903 f 1.5973 a 1.1082 bcd 49.958 b 83.953 a sayyad-amin et al. polyamines and sics effect on compatibility, fertility and yield indices of apple cv. golden delicious 217 of boron increased yield (mashayekhi and atashi, 2008) and fruit set in comparison with control and fruit abscission was lower than control (khoshghalb et al., 2011). maz ardalan and savaghebi firoozabadi (1997) reported that foliar application of mn increased fruit yield. high levels of boron in floral organs such as the stigma and style, may aid pollen germination and make faster pollen tube growth down the style and into the ovary. application of sics a day before full bloom at 1 or 2 mg l-1 on three pear cultivars increased fruit set especially at 1 mg l-1 sics. furthermore, sics at 2 mg l-1 ascended the number of seeds (son et al., 2009). in addition, regarding fruit yield, foliar application of mn alone showed significant increase in fruit yield of sweet oranges due to its effect on increasing the number of fruit/tree as well as fruit average weight (hasani et al., 2012). our results for sics were in accordance with maz ardalan and savaghebi firoozabadi (1997), khoshghalb et al. (2011) on pear, mashayekhi and atashi (2008) on strawberry, son et al. (2009) on pear, hasani et al. (2012) on pomegranate. the effects of polyamines and sics on if and isi in our experiment, all of the treatments could led to semi or full fertility and compatibilty. so, this result was in line with duca et al. (2010) on pear. if the isi is lower than 0.2 means (self or cross) incompatibility, if it is between 0.2 to 1 interpret as semi (self or cross) compatibility, and if it is higher than 1, reveals full (self or cross) compatibility (zeinanlo et al., 2001; azimi et al., 2008.; seifi, 2008). duca et al. (2010) reported that, in compatible pollinated styles, the levels of put and spm were similar and higher than spd, whereas in self-incompatible pollinated styles, put was the highest. in the compatible pollinated styles, these three polyamines showed higher content when compared to selfincompatible pollinated styles (duca et al., 2010). pollen germination and pollen tube development are important for fertilization (kuruki et al., 2017). extremely low pollen germination rates may cause fruit setting failure because of ovule degradation before the pollen tube reaches the ovary (kuruki et al., 2017). full compatibility is superior to semi-compatibility for ensuring high fruit set, even when environmental conditions are favorable for growth and pollination (sapir et al., 2008). among the natural polyamines, spm showed strongest effects on tube growth (aloisi et al., 2015). the effects of polyamines and sics on fruit drop put treatments significantly reduced secondary fig. 1 effects of foliar application of polyamines and sics on fruit length without ccb (a), with ccb (b) and fruit diameter without ccb (c) and with ccb (d) of apple (cv. ‘golden delicious’). put= putrescine, spm= spermidine, spr= spermine, ccb= cotton coverage bags. adv. hort. sci., 2018 32(2): 213-219 218 fruit drop on date palm, apple, pear, mango, sweet orange and avocado (asadi et al., 2013). a reduction in secondary fruit drop was observed in date palm, apple, pear, mango, sweet orange and avocado (tavakoli and rahemi, 2014). due to preventing enzymatic conversion of 1aminocyclopropane-l-carboxylic acid (acc) to ethylene by polyamines, the ethylene production reduced and is followed by fruit drop (tavakoli and rahemi, 2014). decreased fruit drop one week before full bloom in the “on” year and increased yield per shoot two weeks before full bloom in the “off” year were observed by application of spm (0.1 and 1 mm), but spd (1 mm) just lowered fruit abscission one week before full bloom in the “on” year (khezri et al., 2010). in our study, spd (0.25 mm) was the best treatment on fruit set (june and final), yield, isi and fruit drop. asadi et al. (2013) reported that the most effective treatment on raising fruit set were spm and spd in apricot, respectively (asadi et al., 2013). the content of spd was the highest at four development stages, followed by put and spm, respectively (valero, 2010). tavakoli and rahemi (2014) stated that treatment with spd 1 mm led to the highest fruit yield. the effects of polyamines and sics on fruit growth habit regarding the effects of polyamines on fruit length and diameter, fruit growth totally increased because of increasing fruit length and diameter in all of the treatments in our study. in the case of fruit growth, malik et al. (2005) demonstrated that the amount of polyamines increased during initial fruit growth period of apple, pear, apricot and strawberry followed by gradually decrease near maturity. polyamine content of pericarp declined from fruit set to maturity. spd and spm were higher than put during initial fruit growth compared to later during fruit development (malik et al., 2005). put application had a positive effect on fruit size and weight, which might due to its role in cell division leading to improved weight and diameter of fruit (saleem et al., 2008). mn application in sics increased fruit diameter and length but only the 0.6% rate of manganese was significant on fruit diameter (hasani et al., 2012) and influenced fruit growth habit. control trees produced significantly lower fruit yield, so naturally the fruit size was greater compared to fruit from polyamines-treated trees (saleem et al., 2008). spermidine in concentration of 0.25 mm, with/without cotton coverage bags, was the best 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(hsri), agricultural research, education and extension organization (areeo), mahallat, iran. key words: amylases, chitosan, forcing, flowering, magnetite, magnetic supplement of fertilizer, sugars. abstract: lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is requested as potted of cut flowers. it is so important to consider its quality along with its production rate in terms of yield (quantity). however, it always needs to intend the product costs aside from quantity and quality. fertilizer utilization is so important and this may be improved by compounds that promote it and also have synergetic effects themselves. we examined both carboxymethylated chitosan (cmc) and magnetic nano-carbocymethylated chitosan (mncc) to produce lilium bulb and advised them especially magnetic chitosan. in this study, these compounds (magnetic and non-magnetic chitosan) at concentrations of zero, 2.5, 5, 10 and 15 mg/l were examined during lily forcing in three cultivars, including cherbourg, navona, brunello, which are from asiatic and oriental lilies. the results showed that highest concentrations (10 and 15 mg/l) among between examined concentrations regardless of compounds types and cultivars did not make toxicity and had significant effects on plants biology and physiology [contents of carbohydrates and enzymes affecting these carbohydrates (amylases)]. however, for observing morphological changes may be need to use higher concentrations of these compounds. note that this needs to examine the non-toxicity of higher concentrations in future studies. 1. introduction flowers have always had a valuable place among different classes of society and various ceremonies and rituals. lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is mostly used as cut flowers throughout the year. furthermore, it is used as potted plants in gardens and green landscapes (shafiee-masouleh et al., 2014). it is necessary to consider the nutrition of lilies like other plants in the greenhouse production. and also, optimization and improvement of photosynthetic efficiency of plants can be effective in increasing photosynthetic storages and thus increasing its quanti(*) corresponding author: shafiee.masouleh@areeo.ac.ir citation: hatamzadeh a., shafiei-masouleh s.-s., 2019 the use of organic nano-supplements of fertilizer for lily forcing period. adv. hort. sci., 33(2): 215-226 copyright: © 2019 hatamzadeh a., shafiei-masouleh s.-s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 septmeber 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 215-226 216 tative and qualitative yields in terms of flower number, plant size and vase life. different groups of lilies (asiatic, oriental and l. longiflorum) have different nutritional requirements. for example, asiatic lilies has been reported that have the best growth when are fertilized by lower levels during forcing (treder, 2003). chitosan particles in nano-scale can be important in the delivery of drugs in medicine, because these compounds promote the absorption of active molecules or compounds through the cell membrane and allow organs to have bioavailability to molecules. chitosans can be used as encapsulated nanoparticles or used directly and therefore, the surface of these particles with chelating structure of the modified chitosan can play an important role in sustainable agriculture (kashyap et al., 2015). magnetic chitosan nanoparticles with the effect of chelating elements in the experiment conducted by shafiee-masouleh et al. (2014) increased the biomass of lilium and the storage organs (bulbs) during bulb production. the effect of magnetic fluids on living systems has been studied both in medicine and in the world of plants, and it has been shown that magnetic fluids can be effective in the production of calluses and metabolic activities. the effects of magnetism on plants by earth’s magnetic field, pulsed and inductive constant magnetic fields, an electromagnetic field, and effects of magnetic nanoparticles have been studied by researchers and theirs roles have been confirmed to enhance photosynthesis and growth and development of plants (pavel et al., 1999; răcuciu and creangă, 2007 b; răcuciu et al., 2009; shafieemasouleh et al., 2014). it has also been shown that superparamagnetic nanoparticles, which are as permanent magnets effected by external magnetic fields or ambient temperature of their environment, affect membrane systems and membrane ion exchange (pavel and creangă, 2005; faeghi and seyedpour, 2013). many researchers investigated the effects of chitin and the chitosan polymers and oligomers as spraying, soil application or seed treatment and fertilizer treatments, they reported that photosynthesis and plant growth were enhanced (el-tantawy, 2009; dzung et al., 2011; farouk and amany, 2012). the allocation of carbon in plants is important that is affected by various factors such as the level of photosynthetic compounds, the number and size of competing sink (flowers, seeds, fruits, bulbs and tubers, etc.) and their location in plants and the potential for initial storage in the leaves and re-translocation in plants. understanding the components that affect the initiation and development of organs and the balance between source and sink organs are essential to regulate the allocation of assimilates (du toit, 2001). the chl content of the leaf affects photosynthesis, and chl a/b is the best index to understand the photosynthetic capacity and direct information on the activity of the enzymes involved in the photosystem ii in the chloroplast membrane (răcuciu and creangă, 2007 b; răcuciu et al., 2009). iron deficiency reduces the amount of photosynthetic pigments. in addition, the electron transfer in the photosystems i and ii undergoes a change. also, the activity of 1, 5-d-phosphate-carboxylase and the photosynthetic function of plant decrease. therefore, it is necessary to provide iron for the plant, because the biochemical properties of iron and its effect on the metabolic pathway of the plant are important. uptaking iron in alkaline soils is difficult; this is due to the formation of ferric hydroxide in the presence of oxygen, because plant cannot uptake it (thoiron and briat, 1999). the main component in reducing no3 absorption is iron deficiency. ferrous enzymes (nitrite and nitrate reductases) affect the no3 absorption. in addition, growth of plants decreases due to decreased no3 absorption and the synthesis of metabolites (proteins, nucleic acids, chlorophylls, etc.) (borlotti et al., 2012). the use of chelating agents is useful to remove iron uptake problems by plants (abadía et al., 2011). magnetic fluids at suitable concentrations have a positive effect on the photosynthetic capacity of plant. iron ions in the structure of magnetic fluids can be an important source of iron for the development of plants (răcuciu and creangă, 2007 b). iron is not in the chl structure, but it is one of the essential elements to synthesize chl (răcuciu et al., 2009). in general, magnetic fluids are nanoparticles dispersed in water or in a hydrocarbon fluid such as citrate. biocompatibility of magnetite (fe3o4) has been confirmed (răcuciu et al., 2009). however, treating plants with electromagnetism or any other magnetic field with these particles is destructive and changes the genotype and phenotype of plants and causes chromosomal deviations (pavel et al., 1999; pavel and creangă, 2005; răcuciu and creangă, 2007 a, b; răcuciu et al., 2009). chitosan is one of deacetylated derivatives of chitin. as a natural polymer is abundant and can be degraded by biological agents and it can be used in agriculture. this molecule is environmentally friendly and non-toxic that is used in the formulation of slow release fertilizers (wu and liu, 2008). in vitro use of chitosan in a suitable concentration in vitis vinifera l. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 217 stimulated the photosynthesis and increased plant growth with increasing root and shoot biomasses. it also protected the plant against botrytis cinererea fungus and cytological changes (barka et al., 2004). we utilized the effects of chitosan, as nanoparticle with magnetic properties on plant growth and development in lily bulb production (shafiee-masouleh et al., 2014) and cucumber (the data were not published yet). modern agriculture should look for factors that while having positive effects as well as its use to be easy, affordable and reasonable. it is not possible to use a large magnetic field in farms. also, use of macromolecule of chitosan as foliar application will cause stomata obstruction and reduce gas exchange and photosynthesis. therefore, the achievement of a soluble compound or nano-structured composite with the synergic effects of two compounds (magnetite and chitosan) will be very valuable at the same time. to achieve these purposes, this research studies the following: i) increasing yield and post-harvest quality of cut lily by increasing the efficiency of nutrient uptake and photosynthetic performance of the plant; ii) institutionalization of chitosan use at the nanoparticle scale during forcing of lily flowers; iii) investigating the effect of magnetic nanoparticles on yield and quality of lily flowers; and iv) physiological and morphological understanding of the effect of chitosan nanoparticles on the yield and quality cut flowers of lily. 2. materials and methods plant material and cultivation conditions in this study, bulbs of three cultivars of lilium, including brunello (asiatic, 16-18 cm in circumferences), navona (asiatic, 16-18 cm in circumferences) and cherbourg (oriental, 18-20 cm in circumferences) were used to study effects of two organic supplements of fertilizer (osfs). after melting of vernalizing substrate of bulbs, the bulbs were disinfected with 0.1% benomyl fungicide for 15 min and dried in the air for one day. thirty uniform bulbs in size were selected per cultivars to force. the culture substrate contained cocopeat and perlite (1:1, v/ v) that was completely homogeneous. the bulbs were planted in one-kilogram plastic bags with some pores into 10 cm depth and irrigated with tap water until emergence. after emergence every 23 days depending on the weather and substrate conditions were fertigated. greenhouse experiments in the research greenhouse of the faculty of agricultural sciences, university of guilan, rasht, iran in latitude of 37° 12’ 3.94’ n and longitude of 49 38’ 55.78’ e were performed. preparation of fertilizer supplements carboxymethylation of chitosan . the carboxymethylated chitosan (cmc) was prepared from chitosan [poly (d-glucosamine), (c 6 h 11 no 4 ) n , deacetylated chitin] with low molecular weight 100,000300,000 (acros, acros organics, geel, belgium) to increase solubility of chitosan by carboxymethylation. the procedure of preparing of cmc was presented by shafiee-masouleh et al. (2014). an ir (ir470, shimadzu, kyoto, japan) (fig. 1) was used to analyze the produced h-form cmc (not salt containing na ion). preparation of nano-particles magnetite according to shafiee-masouleh et al. (2014), nano-particles of fe3o4 were synthesized. fecl3 (merck, germany) and fecl2.4h2o (merck, germany) were used to prepare fe3o4. the whole procedure of preparing fe3o4 can be observed at the mentioned paper. preparation of magnetic nano-carboxymethylated chitosan the magnetic nano-carboxymethylated chitosan (mncc) was prepared with encapsulation of fe3o4 by carboxymethylated chitosan (cmc) according to fig. 1 ir spectrum of carboxymethylated chitosan. this spectrum shows that the carboxylmethyl groups (-ch3cooh; 1741 cm-1; stretching vibration feature of c=o groups) resulted from monochlroacetic acid salt are linked to o atom in -oh group, and -nh2 exists on carboxymethylated chitosan. adv. hort. sci., 2019 33(2): 215-226 218 shafiee-masouleh et al. (2014). this was carried out by carbodimiide (merck, germany) and saline phosphate buffer in sonication conditions. the details of laboratory procedures can be studied in the mentioned paper. analyses of the size and morphology of nano-particles were performed with sem (fig. 2), and ir and xrd were used to identify the coating and structure of mncc (figs. 3 and 4). statistical design a split plot factorial experiment was conducted in a completely randomized design with 15 main plots and 9 (1 + 8) sub plots. the main factors included three lilium cultivars and sub factors were two types of compounds as organic supplements of fertilizer (osfs); including carboxymethylated chitosan (cmc) and magnetic nano-carboxymethylated chitosan (mncc) both of them at concentrations of 0, 2.5, 5, 10 and 15 mg/l. one group of zero concentration was considered as control treatment for both of compounds. the experiments were carried out by 5 replications and with 135 pots in the research greenhouse of the faculty of agricultural sciences, university of guilan, iran. at the end of forcing period, the growth and morphological characteristics were measured for each plant (in puffy bud stage: when first flower bud showed the color and little opening in top of bud) and were harvested at a height of 45 cm from the final flower bud and transferred to the laboratory. after the stems were cut diagonally under water, they were placed into a 1000 ml bottles containing 500 ml distilled water and transferred to the growth chamber to study the postharvest life and biochemical properties. variance analysis of data was measured using sas software (version 9.1, 2003). mean comparisons were performed with tukey’s test at probability levels of 5 or 1% according to variance analysis. 3. results and discussion magnetic nanoparticles (pavel et al., 1999; pavel and creangă, 2005; răcuciu and creangă, 2007 a, b; răcuciu et al., 2009) in maize and barley, constant magnetic field (dhawi et al., 2009) in a palm tree, pulsed magnetic field (radhakrishnan and kumari, 2012) in soybeen, chitosan (górnik et al., 2008) in grape and chitosan oligosaccharides (dzung et al., 2011) in coffee seedlings have been investigated. all of researchers reported an increase in mineral uptakfig. 2 the image of magnetic nano-carboxymethylated chitosan particles by sem microscopy. size and morphology of fe3o4 coated by carboxmethylated chitosan. fig. 3 ir spectrum of magnetic nano-carboxymethylated chitosan. transferring the absorbance band of -nh2 group from 1589 cm-1 to 1602 cm-1 and increasing absorbance intensity in wavelength number 1741 cm-1 show ester bond into some parts of carboxy groups in carboxymethyl chitosan on the surface of fe3o4 nanoparticles. fig. 4 xrd pattern of magnetic nano-carboxymethylated chitosan. the widths of large peaks report crystal properties of magnetic nanoparticles. the average of particles sizes based on debye scherrer equation was calculated 10 nm. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 219 unlike variance analysis, significant differences between three factors were observed on the vegetative length, total length and chl intensity. differences between treatments on vegetative development showed that morphological characteristics are less affected by the experimented osfs and their concentrations or these compounds may have decreasing effects on these characteristics. in cherbourg cultivar, the highest length of vegetative stem was observed in the absence of osfs or at low concentration (5 mg/l) of cmc, which showed a significant difference with application of 2.5 mg/l cmc in navona. of course, these differences were not significant coming by the effects of magnetism or chitosan, separately. based on the results of 3-way anova test (table 1), effects of osfs on vegetative development of three lily cultivars due to the uniform variances of data around the mean and type i error did not show a significant differences for 3-way interactions between cultivars, compounds and their concentrations. only a significant 2-way interaction (between cultivar and concentrations of osfs) was observed on the chl intensity by anova test. and also, it was observed significant differences between cultivars (table 1). n=5. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. table 1 effects of osfs on vegetative development of three lily cultivars cultivars (a) treatments vegetative length (cm) total length (cm) w chl intensity (spad index)osfs (b) cons of osfs (mg/l) (c) cherbourg cmc 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 54.48±5.14 ab 81.36±5.40 ab 48.52±2.43 defg 5 57.70±2.14 a 84.60±3.52 a 42.46±2.74 ef 10 52.30±3.31 ab 79.80±3.98 ab 42.16±2.22 ef 15 54.10±1.49 ab 81.40±3.34 ab 46.70±2.35 efg mncc 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 55.30±2.91 ab 82.00±3.29 ab 46.50±3.21 efg 5 53.00±3.41 ab 81.40±4.41 ab 46.32±1.99 efg 10 53.12±1.36 ab 80.60±1.93 ab 39.98±2.83 g 15 50.84±2.73 ab 82.20±2.65 ab 46.02±1.99 efg navona cmc 0 48.30±2.23 ab 81.20±3.01 ab 59.52±0.31 abcd 2.5 47.60±2.01 b 78.00±1.92 ab 60.16±1.54 abcd 5 50.80±2.00 ab 81.80±1.57 ab 61.60±0.58 abc 10 50.30±1.51 ab 81.90±2.65 ab 61.98±1.15 ab 15 46.20±1.83 ab 76.00±2.07 ab 61.40±1.57 abc mncc 0 48.30±2.23 ab 81.20±3.02 ab 59.52±0.31 abcd 2.5 39.60±0.80 ab 69.90±0.48 ab 57.10±3.82 abcde 5 49.30±2.22 ab 80.00±1.25 ab 60.16±1.13 abcd 10 45.60±1.83 ab 75.30±2.33 ab 62.72±1.83 a 15 50.10±1.86 ab 80.00±3.58 ab 57.86±0.97 abcde brunello cmc 0 50.90±2.24 ab 70.70±2.29 ab 53.38±3.54 abcdefg 2.5 49.10±4.14 ab 68.50±5.34 ab 51.10±2.14 abcdefg 5 50.90±3.12 ab 69.20±4.53 ab 50.12±1.74 abcdefg 10 52.60±4.01 ab 72.30±5.27 ab 53.76±1.81 abcdef 15 49.50±1.90 ab 66.80±2.81 b 49.34±0.50 bcdefg mncc 0 50.90±2.24 ab 70.70±2.89 ab 53.38±3.54 abcdef 2.5 54.50±2.23 ab 77.30±1.72 ab 49.70±0.61 bcdefg 5 49.90±2.14 ab 69.80±2.18 ab 51.84±1.78 abcdefg 10 50.90±3.60 ab 69.60±3.14 ab 51.60±1.68 abcdefg 15 52.80±3.18 ab 71.80±3.99 ab 49.22±2.08 cdefg anova (3-way) a ** ** ** b ns ns ns c ns ns ns a×b ns ns ns a×c ns ns * b×c ns ns ns a×b×c ns y ns y ns y cv (%) 10.46 8.68 8.58 220 adv. hort. sci., 2019 33(2): 215-226 pared to other treatments. total length showed similar reaction with vegetative length to treatments but the least value was observed in brunello treated by 15 mg/l cmc. however, chl intensity under effects of treatments showed the most value in navona treated by 10 mg/l mncc and the least were observed in cherbourg treated by 10 mg/l mncc (table 1). it seems that morphological characteristics of cultivars treated by two types of osfs in different concentrations are under dominant effect of cultivar; on the other hand, morphological characteristics of different lily cultivars show the various reactions to osfs and their concentrations. generally, from the above observations on vegetative development, it can be interpreted that firstly, size of the plant is affected by genotype, and secondly, magnetism may increase the synthesis of chl and chl density, but it does not affect the size of the shoots and this needs to be investigated more with higher concentrations. nguyen van et al. (2013) stated that chitosan nanoparticles can easily penetrate into plant cells and increase the biological activity of cells. they pointed to an important effect of chitosan nanoparticles on the biophysical properties of the coffee plant such as increasing chl content and plant growth and development. răcuciu and creangă (2007 b) used low-density (less than 100 μl/l) ferrofluids without electromagnetic treatment or any other magnetic field for corn seedlings. they observed that due to their super-paramagnetic effects (a special effect of magnetic nanoparticles) on the structure of photosynthetic enzymes, growth was enhanced. răcuciu and creangă (2007 a, b) and răcuciu et al. (2009) used two fluids of magnetic nanoparticles of fe3o4 coated with or without tetraethylammonium hydroxide in petri dishes for germination of corn seeds without the use of a magnetic field (50 μl per liter). they observed that plant height increased, but when the concentration increased, toxicity was reported. they referred that magnetic iron is a source for plant growth. in addition, magnetism has a good effect on photosynthesis. el-sayed (2014) reported the increasing effect of magnetic water treatment on plant growth of vicia faba, chl a and b, and carotenoids, and contents of gibberellic acid and kinetin it is known that kinetin (cytokinins) is effective in preservation and prevention of the chlorophylls destruction (taiz and zeiger, 2002). farouk and amany (2012) reported an increase in chl a and total chl by spraying the chitosan (250 mg/ l) on chickpea compared to low concentrations (50 and 125 mg/l), and their interpretation about increasing the content of photosynthetic pigments was the increase of cytokinin level and stimulation of the synthesis of chlorophyll, and they interpreted this as the role of amino groups in chitosan on the mentioned processes. chlorophyll density below 40 (spad index) indicates a disruption to photosynthetic process (netto et al., 2005). however, health of the photosynthetic apparatus in all treatments, especially high concentrations of two osfs (magnetic and non-magnetic) can be seen. răcuciu et al. (2009) reported that when concentration of fe3o4 magnetic nanofluid was increased up to 300 μl/l, content of chl a decreased by about 20 mg/l of fresh weight. in addition, răcuciu and creangă (2007 b) stated that the magnetic nanofluid of fe3o4 coated with tetramethylammonium hydroxide (50 μl per liter) produced more pigments (chl a and b and carotenoid), and higher concentrations of fluids destroyed the photosynthetic process. limpanavech et al. (2008) reported that chitosan affected the expression of chloroplast genes in dendrobium orchid and altered the chloroplast size. dzung et al. (2011) sprayed chitosan at appropriate concentrations on coffee seedlings and reported an increase in content of chlorophyll and also in uptake of mineral elements (nitrogen, phosphorus, potassium, calcium and magnesium), and enhancement of photosynthesis as well as the synthesis of chlorophyll of leaves. as can be seen in table 2, 3-way interactions between cultivars, osfs and their concentrations in anova test due to type 1 error could not affect reproductive development of lily plants. however, mean comparisons showed significant effects at hsd0.01 on these characteristics. in cherbourg, the most inflorescence lengths were observed in the plants treated with 15 mg/l mncc, but in navona without significant different with cherbourg, the most lengths of inflorescence was observed in control and 10 mg/l cmc. the shortest length of inflorescence was observed in brunello. however, this cultivar with the least concentration of magnetic osf had the most length and generally, it seems that on this characteristic, the effect of genotype was dominant toward osfs and their concentrations. how to make a reaction of a plant as flower bud number affected by 3-way interactions of factors shows dominant effects of genotype toward osfs and their concentrations. we can statistically analyze each cultivar separately to observe 2-way interactions and/or single effects of osfs on the responses of lily plants. cherbourg and navona could not show response to osfs and their concentrations for bud number. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 221 however, brunello reaction to osfs and their concentrations was obvious. it can be said that this cultivar could respond to the osfs compared with two other cultivars, because produced the most bud number with 2.5 mg/l of magnetic compound. in the case of bud length, although 3-way interaction showed significance, it can be seen for this morphological characteristic that the effect of genotype was dominant again, and cherbourg produced larger buds compared with two others. furthermore, this response was observed about flowering date, and cherbourg flowered later (table 2). from the observations of the effects of three factors, including cultivar, osfs and concentrations of them, it can be interpreted that this range of the tested concentrations could not affect vegetative and reproductive morphology, and it seems that more concentrations of osfs must be investigated in future research. it is known that one of the factors that contribute to growth of the reproductive part (flower bud length) is potassium. amino groups in the carboxymethylated chitosan and its derivatives can table 2 effects of osfs on reproductive development of three lily cultivars cultivars (a) treatments inflorescence length (cm) bud no. bud length (mm) flowering date (days)osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 28.08±0.56 abc 6.80±0.58 abc 116.63±0.24 a 94.60±0.24 a 2.5 26.88±0.84 abcdef 6.80±0.37 abc 116.31±0.77 a 93.00±0.77 a 5 26.90±1.57 abcdef 6.20±0.20 b 123.27±0.49 a 93.20±0.49 a 10 27.50±1.47 abcd 7.00±0.32 abc 116.14±0.55 a 92.5±0.55 a 15 27.30±1.87 abcde 6.40±0.40 abc 118.64±0.37 a 93.80±0.37 a mncc 0 28.08±0.56 abc 6.80±0.58 abc 116.64±0.24 a 94.60±0.24 a 2.5 26.70±1.09 abcdef 6.40±0.51 abc 122.06±0.51 a 93.60±0.51 a 5 28.40±1.41 ab 6.80±0.37 abc 120.39±0.58 a 93.80±0.58 a 10 27.48±0.88 abcd 6.80±0.37 abc 110.43±0.68 a 93.40±0.68 a 15 31.36±2.06 a 7.00±0.58 abc 119.86±0.81 a 94.40±0.81 a navona cmc 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.40±0.53 ab 9.50±0.29 ab 80.02±0.73 b 60.20±0.73 b 5 31.00±2.26 ab 9.20±0.49 a 79.80±0.49 b 59.80±0.49 b 10 31.60±1.35 a 9.00±0.63 a 81.16±0.63 b 59.00±0.63 b 15 29.80±1.11 ab 9.40±0.24 a 78.72±0.49 b 58.80±0.49 b mncc 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.30±0.89 ab 9.20±0.97 a 76.04±0.58 b 59.20±0.58 b 5 30.70±1.07 ab 9.00±0.32 a 77.69±0.60 b 59.60±0.60 b 10 29.70±1.07 ab 10.00±0.71 a 78.95±1.02 b 59.20±1.02 b 15 29.90±2.34 ab 8.40±0.68 a 80.34±0.60 b 59.60±0.60 b brunello cmc 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 19.40±1.37 defg 5.80±0.97 b 86.39±1.47 b 61.60±1.47 b 5 18.30±1.52 g 5.80±0.66 b 79.46±1.71 b 62.20±1.71 b 10 19.70±1.38 defg 5.80±0.97 b 80.04±1.56 b 62.80±1.56 b 15 17.30±1.28 g 5.60±1.20 c 84.75±1.38 b 61.00±1.38 b mncc 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 22.80±1.23 bcdefg 6.40±0.75 abc 84.40±1.59 b 62.20±1.59 b 5 19.90±0.89 cdef 5.40±0.75 c 78.47±1.17 b 60.60±1.17 b 10 18.70±0.85 fg 4.60±0.24 c 76.75±0.20 b 61.20±0.20 b 15 19.00±0.96 g 5.50±0.96 b 80.59±1.72 b 61.60±1.72 b anova (3-way) a ** ** ** b ns ns ns c ns ns ns a×b ns ns ns a×c ns * ** b×c ns ns ns a×b×c ns y ns y ns y cv (%) 17.61 6.69 1.63 n=5. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. adv. hort. sci., 2019 33(2): 215-226 222 serve as a place for the absorption of some metal cations. this compound is used to remove heavy metals and water purification and this shows its chelating role (chang and chen, 2005; chang et al., 2006). chelating sites of chitosan may be effective in absorbing essential metals ions such as manganese, iron (fe+2 and fe+3), potassium, magnesium, etc. for plants. the abilities of polymer or oligomer chitosan to stimulate plant growth under in vitro conditions; vitis vinifera l. (barka et al. , 2004); orchid dendrobium phalaenopsis (nge et al., 2006); and the growth of protocorm like bodies in the dendrobium orchid (pornpienpakdee et al., 2010) have already been reported. pornpienpakdee et al. (2010) used chitosan macromolecules, which had been deacetylated up to 70%, at a concentration of 10 mg/l under in vitro culture conditions for dendrobium orchid and produced plants with more length. in addition, limpanavech et al. (2008) when used the polymer or oligomer chitosan (with deacetylated degrees of 70, 80 or 90%) at concentrations of 1-100 mg/l, they observed that polymers at concentrations of 1-10 mg/l and oligomers at concentrations of 50-100 mg/l resulted in more inflorescences production. gornik et al. (2008) applied a type of commercial compound of chitosan, biochikol 02.pc containing 2% chitosan, at concentration of 0.5% on grape cuttings and reported that root system expanded and it increased the number of new branches and their lengths. radhakrishnan and kumari (2012) reported an increase in leaf number and growth of soybean, and an increase in pod length and grain weight under the influence of pulsed magnetic field. el sayed (2014) reported that magnetic water treatment increased the sink yield in bean (seed and number of seeds per plant). he suggested this effect may be due to increased photosynthetic function of the plant under influence of magnetic treatment. ohta et al. (1999) reported an increase in the photosynthetic reservoir (flower number) in lisianthus with plant seed treatment by chitosan acid solution. limpanavech et al. (2008) also demonstrated that mechanism of chitosan effect on increasing the number of plant photosynthesis reservoirs (number of flowers) in the dendrobium orchid may be caused by effect on the development of photosynthetic apparatus and increasing the size of chloroplasts after chitosan spraying. kananont et al. ( 2010) reported that different types of chitosan as polyand oligosaccharides with varying degrees of deacetylation at a concentration of 10 mg/l are effective for in vitro culture medium of seed germination of dendrobium orchids and growth of the protocorm like bodies. they stated that chitosan amino groups may be effective factor on the growth of the protocorm like bodies. pornpinpakdee et al. (2010) described role of concentration of different types of chitosan on growth rate of orchid dendrobium. the results of 3-way anova (table 3) show significant differences in content of soluble carbohydrates of terminal bud of inflorescence in three cultivars of lily treated with osfs and their concentrations. the highest content of glucose was significantly observed in brunello treated with 15 mg/l osfs regardless of their magnetic or non-magnetic properties. navona was in second rank in response to treatments compared to brunello. however, cherbourg under effects of osfs and their levels showed the least glucose content regardless of being magnetism or not compared with two other cultivars. fructose synthesis in the terminal bud had different reaction to the type of osfs based on genotypes. generally, the most concentrations of osfs regardless of their structures had the most effect on fructose content (table 3). sucrose approximately had similar response to glucose toward treatments, but about this carbohydrate after ‘brunello’, ‘cherbourg’ had second score for the most content not ‘navona’. starch and other sugars and proteins (enzymes) are photosynthetic products that are transported to storage organs (flowers, seeds or bulbs). iron acts as a cofactor for many enzymes, forming part of cytochromes and involves in biochemical reactions, including respiration, photosynthetic material transfer, nitrate synthesis, nitrogen fixation, and dna synthesis (he et al., 2011). el sayed (2014) reported that photosynthetic function of bean plant increases with magnetic water treatment. he reported that contents of glucose and sucrose as well as polysaccharides in the leaves, stems and the whole plant of bean were higher toward irrigation without magnetic field. in addition, positive effect of chitosan on plant growth may be due to its effect on increasing the phosphorus content. phosphorus is an essential element in the biosynthesis and carbohydrate transfer for cell division and the formation of dna and rna (farouk and amany, 2012). according to table 4, variance analysis of osfs effects (type and concentration) on the α-amylases activities of three cultivars of lily showed the significance of 3-way interactions. osfs effects on α-amylase in ‘navona’ compared with two other cultivars showed the highest activity of this enzyme in the highest tested concentration of non-magnetic osf. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 223 while, the highest activity recorded for this enzyme in ‘brunello’ was observed in magnetic osf (mncc) unlike ‘navona’. cultivar cherbourg unlike two other cultivars showed the highest activity of α-amylase regardless of being magnetic of osf with the high concentrations of two osfs, in tissue of the last flower bud of inflorescence. therefore, responses types of cultivars toward being magnetic or not of osfs were different. on the other hand, the amount of activity of this enzyme in ‘navona’ was higher regardless of the type of osf and their concentrations. according to 3-way anova, β-amylase activity in tissue of the terminal flower bud of inflorescence was significantly affected by three factors (table 4). the activity of this enzyme unlike α-amylase was more in ‘brunello’, and like α-amylase, the activity of this enzyme was more with the highest concentration of magnetic osf. the β-amylase activity in ‘cherbourg’ was like α-amylase in the same cultivar. however, in ‘navona’, this enzyme showed the highest activity affected by the both of magnetic and table 3 effects of osfs on soluble carbohydrates into the terminal bud tissue of inflorescence in three lily cultivars n=3. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. cultivar (a) treatments glucose fructose sucrose osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 ghi 2.5 1.95±0.24 fg 2.21±0.18 ghij 5.70±0.28 def 5 3.54±0.18 de 3.34±0.17 efg 8.21±0.35 ab 10 4.50±0.23 bcd 3.33±0.20 efgh 8.19±0.28 abc 15 3.50±0.24 de 4.61±0.21 bcd 5.70±0.23 def mncc 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 gh 2.5 0.89±0.13 g 3.33±0.16 efghi 5.71±0.24 def 5 1.98±0.23 fg 3.27±0.18 efghi 5.67±0.15 def 10 3.50±0.11 de 4.60±0.28 bcd 8.23±0.28 ab 15 2.01±0.29 g 5.51±0.23 ab 8.22±0.44 ab navona cmc 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 2.66±0.24 ef 4.03±0.24 cde 3.03±0.20 hi 5 3.49±0.22 de 4.03±0.20 cde 3.07±0.19 hi 10 5.03±0.31 abc 4.83±0.26 bc 4.53±0.25 fgh 15 5.02±0.28 abc 6.05±0.31 a 5.81±0.17 de mncc 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 3.51±0.23 de 1.54±0.13 jk 1.59±0.15 i 5 5.02±0.32 abc 2.60±0.11 fghij 3.10±0.16 ghi 10 5.84±0.23 ab 2.75±0.19 fghi 3.06±0.19 hi 15 5.88±0.23 ab 4.02±0.17 cde 4.49±0.23 fgh brunello cmc 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 fgh 2.5 2.72±0.18 ef 0.83±0.14 k 6.80±0.34 bcd 5 4.03±0.25 cde 2.12±0.14 ij 06.80±0.31 bcd 10 4.81±0.23 abcd 3.65±0.19 cdef 9.81±0.41 a 15 6.03±0.30 a 3.57±0.19 def 9.84±0.46 a mncc 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 efg 2.5 4.02±0.23 cde 0.88±0.18 k 4.81±0.24 efg 5 4.81±0.25 abcd 2.13±0.18 hij 6.46±0.19 cde 10 4.84±0.21 abcd 3.58±0.20 def 9.82±0.40 a 15 6.02±0.26 a 4.70±0.25 bcd 9.79±0.36 a anova (3-way) a ** ** ** b ns ** ** c ** ** ** a×b ** ** ** a×c ** ** ** b×c ns ns ** a×b×c ** ** ** cv (%) 10.88 10.28 7.96 adv. hort. sci., 2019 33(2): 215-226 224 non-magnetic osfs with different manner toward αamylase in the same cultivar. the earth’s magnetic field affects orientation of the ferromagnetic particles and modulations of reactions. it has been reported that the magnetic field has an effect on the biochemical processes and stimulation of the activity of proteins and enzymes on increase of the seed vigor (dhawi et al., 2009). tham et al. (2001) reported an increase in shoot growth of rice seedling in the hydroponic medium using acid solution of chitosan. magnetic fields have been reported to increase sugar content in sugar beet roots (beta vulgaris) and the content of gluten in wheat (triticum aestivum) (dhawi et al., 2009). radhakrishnan and kumari (2012) in their experiment on soybeans indicated a positive effect of a pulsed magnetic field on the increased activity of the αand β-amylases. in our experiment, mncc with its synergetic effects of magnetism, chitosan, and iron, was remarkably increased the photosynthetic structures, table 4 effects of osfs on amylases (n=3) into the terminal bud tissue of inflorescence and vase life (n=5) of three lily cultivars z the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 and n=5 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. cultivars (a) treatments α-amylase β-amylase vase life (days)wz osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 4.42±0.21 gh 7.97±0.30 g 8.20±0.97 ab 5 6.61±0.33 ef 12.31±0.90 de 7.00±0.71 ab 10 6.61±0.20 ef 13.49±0.68 d 7.20±0.37 ab 15 9.00±0.32 cd 12.33±0.99 de 6.00±0.63 ab mncc 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 6.56±0.21 ef 6.01±0.43 g 6.60±0.51 ab 5 7.14±0.17 def 8.04±0.30 g 6.67±0.67 ab 10 8.99±0.49 cd 12.04±0.95 def 8.00±0.00 ab 15 11.07±0.44 ab 8.07±0.33 g 6.40±0.51 ab navona cmc 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 8.09±0.40 cde 9.11±0.45 ef 7.20±0.49 ab 5 8.02±0.41 cde 12.15±0.63 de 7.80±0.20 ab 10 9.63±0.34 bc 15.21±0.65 cd 8.60±0.51 a 15 12.04±0.53 a 15.11±0.73 cd 8.20±0.49 ab mncc 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 3.07±0.20 hi 12.10±0.50 def 6.80±0.37 ab 5 5.57±0.22 fg 14.92±0.68 cd 8.80±0.58 a 10 5.59±0.27 fg 18.09±0.79 abc 7.80±0.37 ab 15 8.03±0.34 cde 18.13±0.74 abc 9.25±0.48 a brunello cmc 0 2.04±0.14 i 6.52±0.37 g 5.80±0.58 ab 2.5 2.18±0.12 i 8.16±0.44 fg 4.80±0.73 b 5 4.27±0.24 gh 13.38±0.53 d 6.20±0.37 ab 10 6.82±0.32 ef 15.27±0.75 cd 7.00±0.77 ab 15 6.81±0.35 ef 19.30±0.69 ab 7.67±0.67 ab mncc 0 2.04±0.14 i 6.52±0.37 g 5.80±2.58 ab 2.5 2.09±0.07 i 12.99±0.61 de 6.20±0.20 ab 5 4.18±0.22 gh 15.43±0.81 bcd 7.60±0.51 ab 10 6.80±0.24 ef 15.11±0.70 cd 5.80±1.02 ab 15 9.29±0.50 bc 19.41±0.79 a 5.80±1.02 ab anova (3-way) a ** ** * b ** * ns c ** ** ns a×b ** ** ns a×c ** ** ns b×c ** ** ns a×b×c ** ** nsy cv (%) 7.84 7.99 19.18 hatamzadeh and shafiei-masouleh organic fertilizer for lilium 225 the transfer of photosynthetic products and the storage of carbohydrates; so that takeda et al. (1983) stated that the increase of substrate promotes the activity of amylase enzymes, especially α-amylase. according to table 4, the longer vase life was observed in ‘navona’ in the high concentrations of osfs (magnetic and non-magnetic) based on 3-way interactions compared with two other cultivars. hajnorouzi et al. (2011) reported that using a combination of earth’s magnetic field and weak pulsed electromagnetic field on corn seedlings can increase the growth rate and decrease the iron content of the plant and maintain the membrane’s health and decrease oxidative burst. since the magnetic field is the natural property of the earth, plants and other living creatures are permanently responding to the magnetic field during their lives. earth acts as a magnet with its northern and southern poles, and natural effects of the magnetic field can change the growth and yield of plants on the ground. in particular, the electromagnetic spectrum of solar radiation stimulates plant growth through the process of photosynthesis. the possible mechanism is the changing of the electrostatic balance of the plant system at the membrane surface of the cell, which is the primary site for any plant growth restriction or promotion (radhakrishnan and kumari, 2012). therefore, the role of superparamagnetic nanoparticles can be interpreted in our experiment based on the influence of the earth’s magnetic field. it can be stated, instead of the iron salt (which is often difficult to uptake by plants), a chelating agent in the nutrition solution can be used both as the iron source for the plant and also is suitable for the absorption of other elements. between two tested compounds, mncc is a more suitable compound than cmc because it has a positive synergic effect in addition to providing iron in the plant, which is effect of both chitosan and magnetism. in addition, the characteristics of particle size in this type of supplement, i.e. nanosize, affect the biophysical characteristics and biological activities of the plant. 4. conclusions our experiment showed that utilizing the magnetic composite of chitosan (mncc) or modified and chelating macromolecule of chitosan (cmc) in horticulture can significantly affect the growth and development physiology of plants. we introduced this compound, especially magnetic compound, as fertilizer supplement in production period of lily bulb (shafiee-masouleh et al., 2014); now in this experiment for three cultivars forcing period of lily shows that high concentration of both osfs can be used as a supplement in nutrition solution. the highest concentrations (10 and 15 mg/ l) regardless of osf types and the cultivars response caused significant physiological effects on the content of carbohydrates and also the enzymes that are influence on carbohydrates (amylases). it seems that observing the remarkable morphological changes needs to use the higher concentrations of both osfs without toxic effects. however, this subject (the use of higher concentrations) must be examined in horticultural plants. the examined cultivars (cherbourg, navona and brunello) showed almost different responses to osfs, but generally magnetic osf can be advised and for security utilization needs to be examined with higher concentrations at future research. references abadía 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particular taste (lópez et al., 2009). the raw plant samples originating from m. officinalis l. species are also used in the traditional medicine for the treatment of headache, flatulence, colic, nausea, indigestion, anaemia, nervousness, vertigo, malaise, asthma, bronchitis, syncope, amenorrhea, cardiac failure, insomnia, epilepsy, depression, psychosis, hysteria, ulcers and wounds (who, 2004; karasová and lehotay, 2005; dastmalchi et al., 2008). therefore, its aqueous and alcoholic extracts are traditionally used for their spasmolytic, nervous sedative, antiviral and antioxidant activities (lópez, et al., 2009; atanassova et al., 2011; de carvalho et al., 2011; lin et al., 2012). all of these properties of lemon balm have been related to the high levels of phenolic acids found in this species, mainly hydroxycinnamic acid derivatives such as rosmarinic acid (fecka and turek, 2007). some studies already reported other phenolic compounds in lemon balm. heitz et al. (2000), isolated luteolin 3-o-glucuronide as the major flavonoid presented in m. officinalis from france. in 2002, patora and klimek isolated and determined the structure of six major flavonoids (apigenin and luteolin derivatives) in lemon balm from poland based on spectral data. lemon balm has a complex chemical composiadv. hort. sci., 2017 31(2): 141-147 doi: 10.13128/ahs-21091 short note hplc/dad, gc/ms and gc/gc/tof analysis of lemon balm (melissa officinalis l.) sample as standardized raw material for food and nutraceutical uses f. ieri 1 (*), l. cecchi 1, 2, p. vignolini 1, m.f. belcaro 3, a. romani 1, 3 1 dipartimento di statistica, informatica, applicazioni “g. parenti”, disia, università degli studi di firenze, viale morgagni, 59, 50134 firenze, italy. 2 dipartimento di neuroscienze, psicologia, area del farmaco e salute del bambino (neurofarba), università degli studi di firenze, viale pieraccini, 6, 50139 firenze, italy. 3 laboratorio qumap, qualità delle merci e affidalibilità di prodotto, polo universitario città di prato, piazza ciardi, 25, 59100 prato, italy. key words: aromatic compounds, comprehensive two-dimensional (2d) gas chromatography, hs-spme-gc×gc-tof fingerprint analysis, hydroxycinnamic acids, lemon balm, phenols. abstract: melissa officinalis l., commonly known as lemon balm, is a perennial herb belonging to lamiaceae family. traditionally administered in infusion form, it has therapeutic properties, such as sedative, carminative and antispasmodic, but also it is used for treatment of headache, rheumatism, indigestion and hypersensitivities. lemon balm has a complex chemical composition. the aim of this work was the comprehensive characterization of secondary metabolites of a dried lemon balm (melissa officinalis l.) sample, through hplc/dad, gc/ms and gc/gc/tof analysis, as raw material for the standardized phyto-complexes production useful for food and nutraceutical application. this sample contained rosmarinic acid (caffeic acid dimer) as the main compound of phenolic fraction (32.4 mg g-1). citronellal was the most abundant compound in the volatile fraction, followed by α-citral and β-caryophyllene. the total citral amount, in terms of sum of αand β-citral, was 149.4 mgcitral kg-1. comprehensive two-dimensional gc fingerprint analysis of lemon balm produced rationalized peak patterns for up to 200 volatile compounds. (*) corresponding author: francesca.ieri@unifi.it received for publication 30 may 2017 accepted for publication 6 july 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 141-147 142 tion. it contains hydroxycinnamic acids (up to 6% of rosmarinic acid, p-coumaric and caffeic acids) and up to 0.37% of an essential oil composed of monoterpenes (more than 40%) and sesquiterpenes (more than 35%). among the most significant terpenoids there are citral, citronellal, geraniol, nerol, linalool, farnesyl acetate, humulene, caryophyllene and eremophilene (escop, 2003; who, 2004). the essential oil is considered to be a therapeutic principle usually responsible for most of the biological activities, such as spasmolytic, antimicrobial, antitumour andantioxidant ones, but also plant polyphenols, especially rosmarinic acid, are involved as well (sadraei et al., 2003; de sousa et al., 2004). phenolic acids are secondary metabolites, which create a large group of naturally occurring compounds, showing a broad spectrum of biological activities. the phenolic acids are important bioactive constituents of m. officinalis l.; among them rosmarinic, caffeic, chlorogenic and ferulic acids are especially interesting. a literature screening showed that rosmarinic acid exhibits anti-inflammatory, antibacterial, and antiviral activities, it reduces atopic dermatitis and prevents alzheimer’s disease (huang et al., 2009; fujimoto and masuda, 2012). ferulic acid has strong antioxidant, antimicrobial, anti-inflammatory, anti-thrombotic, and anti-cancer activities (peng et al., 2012). chlorogenic acid shows anti-inflammatory, anti-bacterial, and antiobesity properties (sun et al., 2013), whereas caffeic acid has anti-inflammatory, antioxidative and immunomodulatory effects (anwar et al., 2012). chemical quality evaluation of a herbal medicine should consist of two aspects (jin et al., 2008). the first is identification and quantitation of one or more constituents that occurred in high quantities. the other is development of chemical fingerprint, which has been introduced and accepted by the who and other authorities as a strategy for quality assessment of herbal medicines (chang et al., 2008; li et al., 2010). among the separation methods, e.g. hplc, hptlc, gc and ce, which have been recognized as a rapid and reliable means for the identification and quantitation of the herbal medicine constituents, hplc is the most popular method and is widely used in the fingerprint analysis (kong et al., 2009; peng et al., 2011). up to now, coherent data is missing about the evaluation of quality consistency of the medicinal raw plant samples of m. officinalis l. obtained from different manufactures. the aim of this work is the comprehensive characterization of secondary metabolites of a dried lemon balm (melissa officinalis l.) sample, through hplc/dad, gc/ms and the innovative gcxgc/tof analysis, as raw material for the standardized phytocomplexes production useful for food and nutraceutical application. therefore, in this study a combinative method is developed based on the reference hplc, gc and gcxgc fingerprint and quantitation of selected phenolic and volatile compounds to assess the quality consistency of lemon balm. 2. materials and methods plant material the dried vegetal tissues of melissa officinalis l. were collected by officinali agribioenergia factory (medicina, bologna, italy). the dried sample were finely chopped with a grinder (mulinex ar 11, groupe seb, france) with a particle size of about 1.0 mm. determination of volatile composition and polyphenolic compounds gc-ms analysis. in the first step of gc-ms analysis, hs-spme-gc-ms was selected as the most suitable technique to recover and analyze the highest number of volatile organic compounds (vocs) in melissa officinalis l. samples. consequently, a dried foliar sample was ground and a homogenous powder was obtained. fifty mg of the powdered sample, together with 2 g of nacl and 5 ml of deionized water were placed into a 20-ml screw cap vial fitted with ptfe/silicone septa. after 5 min of equilibration at 60°c, vocs were absorbed exposing a 2-cm trivalent spme fiber (dvb/car/pdms by supelco) for 10 min into the vial headspace under orbital shaking (500 rpm) and then immediately desorbed at 280°c in a gas chromatograph injection port operating in split less mode. the chromatographic analysis was performed in a gc system coupled to quadrupole mass spectrometry using an agilent 7890a gc equipped with a 5975c msd. the separation of analytes was achieved by an agilent db innowax column (length 50 m, id 0.20 μm, df 0.40 μm). chromatographic conditions were: initial temperature 40°c, then 10°c min-1 up to 260°c, hold for 6.6 min. compounds were tentatively identified by comparing calculated kovats retention index and mass spectra of each peak with those reported in mass spectral databases, namely the standard nist08/wiley98 libraries. each sample was analyzed in triplicate. in the second step of gc-ms analysis, solid-liquid extraction followed by liquid injection was selected to ieri et al. hplc/dad, gc/ms and gc/gc/tof analysis of lemon balm for food and nutraceutical uses 143 quantify the main vocs identified by hs-spme-gc-ms, namely citronellal, α-citral (geranial), β-citral (neral) and β-caryophyllene. to this aim, 0.5 g of powdered sample was extracted with 3 ml of heptane; the extraction was performed for 15 min in an ultrasound bath and for 24 hours in a shaker at 1000 rpm and 24°c. the mixture was than centrifuged at 6.000 rpm for 30 min and the supernatant was recovered and used for the gc-ms analysis, which was performed by the same chromatographic system used for the hs-spme-gc-ms analysis. each sample was analyzed in triplicate. two five-level calibration curves were built using β-caryophyllene standard (range 0.625-40 ppm, r2 0.9961) and citral standard (range 1-100 ppm, r2 0.9995). citronellal, α-citral and β-citral was expressed as mgcitral kg-1, and β-caryophyllene was expressed as mgcaryoph kg-1. gcxgc-ms analysis. vocs were absorbed exposing a 2-cm trivalent spme fiber as described in gc-ms analysis. an agilent 7890a gc equipped with a 5975c msd was used and comprehensive gc×gc analyses were carried out on an agilent gc 7890b, with an agilent flow modulator system, coupled to an tof-ds markes detector. the analytes separation was achieved with a hp-5ms ui column (0.18x0.18mm, 20 min) coupled with a innowax column 0.23x0.32 mm, 5 min. a tentative compounds identification was performed by comparing mass spectra of each peak with those reported in mass spectral databases. extraction and hplc/dad analysis of phenolic compounds. an aqueous extract was prepared adding 50 ml to 2.5 g of dried material. the solution was heated at 75°c and kept at that temperature for 60 min, then, the mixture was centrifuged and analyzed by hplc/dad. standards and solvents. authentic standards of kaempferol 3-o-glucoside and rosmarinic acid were purchased from extrasynthèse s.a. (lyon, france), βcaryophyllene and citral were purchased from sigmaaldrich. all solvents used were of hplc grade purity. hplc/dad analysis. analyses of flavonols and phenolic acids were carried out using an hp 1200 liquid chromatograph equipped with a dad detector (agilent technologies, palo alto, ca, usa). compounds were separated using a 250x4.6 mm i.d., 5 mm luna c18 column (phenomenex, usa). uv/vis spectra were recorded in the 190-600 nm range and the chromatograms were acquired at 250, 280, 330 and 350 nm. the samples were analyzed by gradient elution at a flow rate of 0.8 ml/min. the mobile phase was a multi-step linear solvent gradient system, starting from 95% h2o (adjusted to ph 3.2 by hcooh) up to 100% ch3cn in 55 min. identification and quantification of individual phenolic compounds. the identity of phenols was ascertained using data from hplc-dad, by comparison with bibliographic data and combination of retention times and uv/vis spectra with those of authentic standards. the quantification of individual phenolic compounds was performed directly by hplc-dad using a five-point regression curve (r≥0.998) in the range of 0-30 mg on the basis of authentic standards. in particular, flavonols were determined at 350 nm using kaempferol 3-o-glucoside as a reference compound, while the phenolic acid derivatives were determined at 330 nm using rosmarinic acid as reference compound. each sample was analyzed in triplicate. 3. results and discussion gc-ms is widely recognized as the most suitable analytical technique for the analysis of vocs; in particular, the hs-spme-gc-ms allows the identification of the most representative compounds. we selected the dvb/car/pdms fiber since it proved to be the most universal assembly for sufficient isolation of compounds with different physicchemical properties (cui et al., 2009). figure 1 shows the total ion chromatogram (tic) fig. 1 total ion chromatogram (tic) by hs-spme-gc-ms analysis of melissa officinalis l. foliar sample. adv. hort. sci., 2017 31(2): 141-147 144 obtained by hs-spme-gc-ms analysis of the dried powdered foliar sample of melissa officinalis l. up to 50 volatile organic compounds were identified and their abundance was reported as percentage on the total area of the identified peaks (table 1). terpenes were the most representative class of compounds, in terms of both number of molecules and relative abundance (15 monoterpenes, 71.91%; 8 sesquiterpenes, 19.01%). among them, the most abundant compounds were citronellal (27.54%), α-citral (25.00%), β-caryophyllene (9.24%) and β-citral (7.61%). the other main classes of identified vocs were 11 aldehydes (total 3.64%), 6 alcohols (3.60%), 4 ketones (0.96%) and 3 esters (0.76%). starting from these data, we selected the most representative vocs from the hs-spme-gc-ms analysis and quantified them in terms of mg kg-1 on dried foliar sample basis. to this aim, sample was extracted and analyzed by liquid injection gc-ms, as described in the experimental section. table 2 summarizes the obtained results. citronellal was the most abundant compound in the heptanoic extract, followed by α-citral and βcaryophyllene. the total citral amount, in terms of sum of αand β-citral, was 149.4 mgcitral kg-1, that was more than citronellal amount. hs-spme and gc×gc-ms fingerprint analysis are ideal tools to analyze complex volatile matrices, and provide a sensitive method for the direct comparison and chemical visualization of plant volatile components. gc×gc-ms is currently adopted as separation technique not only because of its high separation power and sensitivity but also for its ability to produce more widely distributed and rationalized peak patterns (cordero et al., 2008) for chemically correlated group of analytes. hs-spme gc×gc-tof-ms analysis of the complex volatile fraction of lemon balm was submitted to rt (min) tentative identification abundance (% on the total) 23.2 citronellal 27.54 29.61 α-citrale (geranial) 25.00 26.73 β-caryophyllene 9.24 28.42 β-citrale (neral) 7.61 29.44 germacrene d 3.00 24.55 linalol 2.82 35.7 caryophyllene oxide 2.70 15.81 (e)-2-hexenal 2.65 21.96 1-octen-3-ol 2.47 29.88 geranyl acetate 2.42 25.22 methyl citronellate 2.34 30.37 δ-cadinene 1.44 16.63 e-ocimene 1.35 30.55 γ-cadinene 0.91 23.87 copaene 0.88 31.75 geraniol 0.79 25.68 isopulegol isomer b 0.64 20.24 (z)-3-hexen-1-ol 0.57 16.77 3-octanone 0.52 34.34 β-ionone 0.48 25.58 isopulegol isomer a 0.46 6.52 ethyl acetate 0.46 20.39 3-octanol 0.44 19.18 6-methyl-5-hepten-2-one 0.38 22.77 α-cubebene 0.36 19.7 2,6-dimethyl-5-heptenal 0.36 20.84 nonanal 0.33 11.61 hexanal 0.25 16.08 z-ocimene 0.21 18.5 (e)-3-hexen-1-yl acetate 0.20 13.95 β-myrcene 0.17 37.78 nerol 0.15 17.86 methyl hex-2-enoate 0.10 17.81 octanal 0.10 10.44 2,5-diethyltetrahydro-furan 0.09 7.11 2-methyl-butanal 0.07 7.21 3-methyl-butanal 0.06 15.21 d-limonene 0.05 9.85 1-penten-3-one 0.05 13.62 1-penten-3-ol 0.05 8.73 pentanal 0.05 4.40 dimethyl sulfide 0.04 15.02 3-methyl-1-butanol 0.04 18.34 (z)-2-penten-1-ol 0.03 13.11 (e)-2-pentenal 0.03 13.37 5-methyl-hexanal 0.03 14.71 heptanal 0.03 16.41 (z)-4-heptenal 0.02 5.22 2-methyl-propanal 0.02 8.6 2-pentanone 0.01 table 1 relative abundance of the vocs identified by hsspme-gc-ms analysis data are expressed as area % on the total area of all the identified peaks. table 2 content of the main vocs by liquid injection gc-ms analysis data are the mean of three determinations (standard deviation <3%) expressed in mg kg-1 dry weight. voc amount citronellal 119.2 (mgcitral kg-1) α-citral (geranial) 109.7 (mgcitral kg-1) β-citral (neral) 39.7 (mgcitral kg-1) β-caryophyllene 74.3 (mgcaryoph kg-1) ieri et al. hplc/dad, gc/ms and gc/gc/tof analysis of lemon balm for food and nutraceutical uses 145 advanced fingerprinting analysis of 2d chromatographic data. in figure 2a is reported a “contour plot” from gc 2d-ms/tof analysis where the “blobs” correspond to a single volatile compound detected. 417 blobs were detected and, after subtracting base line blobs corresponding to fiber blending or background interferences, 203 blobs/compounds were identified. an advanced, effective and reliable non-targeted analysis approach known as comprehensive template matching fingerprinting (cordero et al., 2012) was adopted (fig. 2b). this method considers, as comparative feature, each individual 2d peak together with its time coordinates, detector response and ms fragmentation pattern, and includes them in a sample template that is created by the analyst and can be used to compare plots from different samples directly and comprehensively. in this case a template was created for a comprehensive comparative analysis of 2d chromatographic data and to correctly interpret visual differences in further analysis. the most intense blobs corresponded to citronellal, α-citral and β-citral as evidenced in the gc-ms analysis. also the sesquiterpene βcaryophyllen was an intense blob. up to 24 blobs/compounds belonging to the class of sesquiterpenes were distributed in a defined part of the contour plot (fig. 2b). gc×gc-ms analysis produced distributed and rationalized peak patterns for sesquiterpenes, monoterpenes and oxigenated monoterpenes (fig. 2b). the analysis can be see also in the 3d view, showing the complex volatile fraction of lemon balm leaves (fig. 3). data of the retention time and λ max in the visible region of specific standard (rosmarinic acid) obtained by hplc-dad analysis allowed the identification of phenolic compounds of the cultivated lemon balm sample. all these derivatives were calculated based on rosmarinic acid calibration curve (35.23 mg g-1). fig. 2 contour plot from gc 2d-ms/tof analysis of lemon balm. a: graphic view of the blobs/compound detected; b: sample template of the main compounds detected. adv. hort. sci., 2017 31(1): 141-147 146 data are the mean of three determinations (standard deviation <3%) expressed in mg g-1 dry weight. the lemon balm sample studied presented the rosmarinic acid (caffeic acid dimer) as the main compound. few studies report the existence of rosmarinic acid as being the most abundant phenol in this species (caniova and brandsteterova, 2001; ziaková et al., 2003; fecka and turek, 2007; lee, 2010). the rosmarinic acid content was slightly high (32.4 mg g-1) and in accordance with those reported by fecka and turek (2007) that presented values of rosmarinic acid in m. officinalis ranging from 32.6 to 5.1 mg g-1 of infusion. it was also identified a flavone, the only flavonoid found in this sample (1.7 mg g-1). this peak presented a uv spectra with λ max at 350 nm, and was tentatively identified as a luteolin derivative, described as the major flavonoid in m. officinalis by heitz et al. 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research, education and extension organization (areeo), karaji, iran. 2 horticulture department agriculture faculty, university of hormozgan, bandar abbas, iran. key words: flower, kernel, multiple regression, nut, path analysis, yield. abstract: the present research aimed to study the flower and fruit properties of 60 almond’s cultivars and genotypes. all fruit and kernel traits had high heritability (ranged from 20.73 to 92.13%). double kernel and pistil length showed the most and few genotypic variation, respectively. the most yield belonged to filippo ceo, k8-24, fragiulo and k9-24 (6.9, 6.84, 6.78 and 7.6 kg/tree respectively). multivariate analysis was the applied technique to determine the relationship among important traits. kernel weigh caused yield directly and kernel width indirectly. to estimate kernel weight, kernel variables had less r square (0.61) than nut variables (0.94). 1. introduction the efficiency of plants breeding program depends on selection of desirable parents and progenies. almond cultivars differ in the growth and branching pattern, as well as bearing habit (duval and grasselly, 1994). self-compatibility (socias i company and felipe, 1988; ortega and dicenta, 2003), late blooming (vargas and romero, 2001), flower density, production rate (dicenta et al., 1993 a; gradziel and kester, 1998), fruit maturity period (kester and asay, 1975; dicenta et al., 1993 b) and kernel properties (spiegel-roy and kochba, 1974, 1981; kester et al., 1991) are the main almond breeding objectives, to be informed by the relationship among these traits, facilitates the breeding process, and selection of the desirable almond genotypes (dicenta et al., 1993 a). multivariate analysis is the common technique to evaluate almond genotypes according to quantitative and qualitative characteristics (de giorgio and polignano, 2001; de giorgio et al., 2007). lansari et al. (1994) used this method in order to evaluate morphological variation of almonds varieties. their results indicated that nut and kernel traits, compare to (*) corresponding author: shamili@ut.ac.ir citation: imani a., shamili m., 2018 phenology and pomology of almond’s cultivars and genotypes using multivariate analysis. adv. hort. sci., 32(1): 27-32 copyright: © 2018 imani a., shamili m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 august 2017 accepted for publication 16 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 27-32 28 vegetative characteristics, has important role in genotype distinction. de giorgio and polignano (2001) mentioned fruit characteristics are the most effective variables to differentiate almond genotypes. de giorgio et al. (2007) divided 88 almond varieties, into seven separate groups based on morphological traits. besides, it has been reported that almond nut length and thickness, and kernel length have significant correlations with kernel length, moreover kernel width have most positive effect on kernel weight (spiegel-roy and kochba, 1981; kester et al., 1977). the direct and indirect interaction of the traits which affect the yield can be used as a breeding tool, in this work we analyzed 60 local and foreign almond genotypes and cultivars to understand the casual relation among some important traits. 2. materials and methods plant materials this research carried out as randomized complete blocks design, during the years 2013 -2014, in horticultural sciences research institute, karaj, iran (51°35’e, 48°n, 1297 m above sea level, average annual max/min air temperature of 31.3/22.5°c, mean rh of 60%, the average annual precipitation of 148 mm, shallow soil ph=7.5). the average of minimum daily temperature (°c) of january to april (years 2013-2014) is given at figure 1 (based on iranian meteorological organization data). we evaluated 60 almond genotypes (table 1) using almond descriptors (gülcan, 1985) (table 2). all genotypes were 7-year-old and except tuono, supernova, filippo ceo and fragiulo, the others were self-incompatible. data analysis descriptive statistics (minimum, maximum and mean) was used to describe and summarize the data by spss 22 and sas9. variance components, variance coefficients and heritability were calculated by excel 2013. multivariate regression analysis was performed to assess the relationship among variables by adjusted r square (adjrsq) procedure. briefly, the subsets of fig. 1 average of minimum daily temperature (°c) from january to april (years 2013-2014). cultivar and genotypes origin foreign local nonpareil boty k1-25 k14-24 k9-2 a-200 mission roby d124 k13-40 k9-32 k9-20 perlice karmel k8-24 k9-24 k6-5 k1-16 padre sh-21 shekofeh k8-b k16-23 12-3 tuono sh-6 mamaie k11-9 sahand 8-3 filippo ceo ne plus ultra sefid k4-13 k9-7 d101 marcona sh-17 k8-32 k3-8 k5-6 supernova k1-5 k6-4 k5-17 rabie fragiulo sh-13 k2-22 k5-27 d-99 sh-12 sh-15 k3-12 k3-19 d-8 a-230 saba talkh asli z-3 table 1 almond cultivars and genotypes evaluated in the project table 2 quantitative traits using in 60 almond cultivars and genotypes trait measuring unit measuring method flower characteristics flower size cm caliper petal length cm caliper petal width cm caliper stamen number number counting pistil length cm caliper pistil diameter cm caliper nut characteristics nut length mm caliper nut width mm caliper nut thickness mm caliper nut weight g digital balance kernel characteristics kernel length mm caliper kernel thickness mm caliper kernel width mm caliper kernel weight g digital balance kernel percentage % calculating double kernels % counting yield kg/tree digital balance imani and shamili phenology and pomology of almond’s cultivars 29 the independent variables, which have the best estimation of some important dependent variables (like flower size, double kernel percentage) were selected. then path analysis was used to describe the direct relation among variables focusing on causality. 3. results table 3 represents the statistical descriptive and variance component. based on the results, the most genotypic variance (126.37 and 99.08 respectively) belonged to the double kernel and the kernel percentage, while pistil length and thickness had the least (0.01). the most heritability belonged to kernel percentage and double kernel (92.13% and 89.74% respectively). the rest of the variables had heritability over 20.73%. various researches reported different almond kernel weigh heritability such as 64% (kester et al., 1977), 45% (spiegel-roy and kochba, 1981) and 78% (dicenta et al., 1993 b); so to have high kernel weight (5.1 grams) progenies, the parents with high kernel weight should be chosen. in the present study kernel weigh heritability was 70.15%. filippo ceo (9.6 kg/tree), k8-24 (6.84 kg/tree), fragiulo (6.78 kg/tree) and k9-24 (6.7 kg/tree) had the most yield and talkh asli (2.33 kg/tree), d101 (2. 44 kg/tree), 12-3 (2.45 kg/tree), 8-3 (2.8 kg kg/tree) and sefid (2.82 kg/tree) had the lowest. in some environmental conditions (e.g. low temperature before flowering) almond progenies have been produced double kernel nuts even if the parents did not appearance the trait (egea and burgos, 1995; sánchez-pérez et al., 2007). it has been found that complexity and dominant inheritance of the mentioned trait is affected by genotype and phenotype, which have been reported by several researchers (dicenta et al., 1993 a, b; arteaga and socias i company, 2001; sánchez-pérez et al., 2007). in our study, the double kernel varied from 0 to 57%, suggests it have been influenced by the genotypes differences. food industries seeks for almond progenies with the medium thickness and smooth surface nuts (sánchez-pérez et al., 2007). we found nut thickness between 9.08-20 mm. kester et al. (1977) reported nut thickness heritability about 0.71, whereas we estimated it about 0.41. in the present investigation, stepwise regression was used to identify the yield causal system which separates independent variables into direct and indirect ones (table 4 and fig. 2). according the data, nut weight had the most standardized beta (0. 944). as well as the nut related traits (length, width, thickness table 3 descriptive statistic, heritability, phenotypic and genotypic coefficients of almond traits traits max mean min variance components heritability coefficient of variance phenotypic environmental genotypic genotypic phenotypic flower size 5 3.88 2.9 0.39 0.11 0.28 71.04 13.57 16.10 petal length 2.5 1.65 1 0.13 0.05 0.08 59.08 16.81 21.87 petal width 1.8 1.35 0.98 0.08 0.04 0.04 52.38 15.54 21.47 stamen number 37 26.83 15 25.57 4.00 21.57 84.35 17.31 18.84 pistil length 1.72 1.46 1.1 0.03 0.02 0.01 45.65 8.10 11.98 pistil thickness 0.52 0.26 0.1 0.04 0.03 0.01 23.08 38.21 79.54 nut length 47.17 33.35 26.59 24.05 4.67 19.38 80.60 13.20 14.70 nut width 28.82 20.08 13.28 15.66 4.00 11.66 74.45 17.00 19.70 nut thickness 20 14.07 9.08 9.64 5.67 3.98 41.23 14.17 22.06 nut weight 4.76 2.72 0.97 5.47 4.33 1.13 20.73 38.99 85.65 kernel length 30.89 24.29 18 8.50 1.00 7.50 88.23 11.27 12.00 kernel width 16.78 12.10 9.19 4.94 1.63 3.31 66.97 15.03 18.37 kernel thickness 9.12 6.98 4.61 1.38 0.63 0.74 54.02 12.35 16.81 kernel weight 1.67 1.06 0.56 0.13 0.04 0.09 70.15 28.82 34.41 kernel percentage 68.88 42.02 23.52 137.17 10.80 126.37 92.13 26.75 27.87 double kernel 57 9.95 1 110.41 11.33 99.08 89.74 100.00 105.56 yield 7.4 5.21 2.62 2.69 0.95 1.74 64.72 25.31 31.46 adv. hort. sci., 2018 32(1): 27-32 30 and weight); the kernel variables (length, width and percentage) significantly influenced the yield (table 4). the most direct effect related to kernel weight, nut weight, nut thickness, nut width and nut length, respectively (fig. 2). it is recommended that these traits can be assumed as the selection criteria to improve almond commercial yield. also, direct and indirect variables which effect kernel weight (table 5), double kernel and flower size have been evaluated via multivariate linear regression. nut characteristics (nut weight and width) had more regression coefficients. kernel weight estimation entering three independent variables (for example, kernel length, width and thickness) had more r2 than two variables (like kernel length and width). double kernel estimation using nut traits did not have acceptable r2, neither kernel traits. although the flower size was influenced by the number of stamens, petal length, and width significantly, but the r2 was very low (0.353). 4. discussion and conclusions yield improvement is the most important objective of the almond breeding programs. the commercial yield of almond fruit trees (kg kernel per tree) results the interaction of self (in)compatibility, flower table 4 yield causality analysis and stepwise correlation coefficients yield nut weight kernel weight kernel thickness kernel width nut width nut weight 0.488** 1 kernel weight 0.655** 0.780* 1 kernel thickness 0.565** 0.630** 0.697** 1 kernel width 0.612** 0.625** 0.752** 0.778** 1 nut width 0.466** 0.568** 0.587** 0.594** 0.832** 1 kernel length 0.266* 0.608** 0.524** 0.281* 0.516** 0.381** nut length 0.266* 0.580** 0.489** 0.203 kernel percentage -0.570** -0.176 flower size 0 pistil length -0.088 petal width -0.053 stamen number -0.153 pistil length -0.110 nut thickness -0.022 double kernel 0.140 fig. 2 path analysis for traits that affect almonds yield (beta coefficients have given at parenthesis). table 5 kernel weight estimation based on different independent variables dependent variable independent variables entering to equation r2 kernel weight nut weight nut width kernel thickness kernel percentage 0.9461 kernel weight nut length nut weight nut width double kernel 0.9455 kernel weight nut weight nut width kernel percentage 0.9454 kernel weight kernel length kernel width kernel thickness 0.6193 kernel weight kernel length kernel thickness 0.5888 kernel weight kernel width kernel thickness 0.5827 kernel weight kernel length kernel width 0.5759 *, **, denote statistical significance at 5 and 1% levels, respectively. imani and shamili phenology and pomology of almond’s cultivars 31 buds density, fruit abscission, kernel dry weight and kernel size (garcia et al., 1996). although some small kernel cultivar (such as ‘felisia’) are ideal for chocolate bars and almond drops (socias i company and felipe, 1999), large kernel almond are valued almost. the trait, as well as its correlated trait, varies each year. high correlation between almond in shell weight and kernel weight (-0.82) (sánchez-pérez et al., 2007), in shell kernel ratio and in shell weight (-0.72) and in shell kernel ratio and kernel weight (0.7) (dicenta and garcía, 1993) have been reported previously. based on our result nut length significantly correlated with nut width (0.59) and nut weight (0.71). moreover kernel weight showed high correlation with kernel length (0.64) and nut weight (0.69). the kernel weight influenced the yield directly and nut weight indirectly. while nut width, weight and diameter influenced nut length. based on the results k8-24 had relatively high nut length (33.5 mm), nut width (28.82 mm), kernel length (25.01 mm) and kernel width (7.48 mm). fragiulo, k8-24 and filippo ceo had relatively high kernel weight (1.486, 1.378 and 1.373 gr respectively). k8-24 and fragiulo had relatively high nut and kernel weight. according to our findings, kernel weight can be used as a selection criterion for almond breeding programs. regression models for kernel weight estimation revealed that nut characteristics like length, width and weight had more r2 (0.94) than the kernel characteristic such as weight, width and percentage (r2= 0.61). acknowledgements the research was funded by the iranian agricultural research, education and extension organization (areeo. project number: 8204-1. 2013/5/2). references arteaga n., socias i company r., 2001 heritability of fruit and kernel traits in almond. acta horticulturae, 591: 269-274. de giorgio d., leo l., zacheo g., lamascese n., 2007 evaluation of 52 almond (prunus amygdalus batsch.) cultivars from the apulia region in southern italy. j. hort. sci. biotech., 82(4): 541-554. de giorgio d., polignano g.b., 2001 evaluating the biodiversity of almond cultivars from a germplasm collection field in southern italy. sust. glob. farm, 56. dicenta f., garcía j.e., 1993 inheritance of kernel flavour in almond. heredity, 70(3): 308-312. dicenta f., garcía j.e., carbonell e., 1993 a heritability of flowering, productivity and maturity in almond. j. horti. sci., 68: 113-120. dicenta f., garcía j.e., carbonell e., 1993 b heritability of fruit characters in almond. j. hort. sci., 68: 121-126. duval h., grasselly c., 1994 behaviour of some selffertile almond selections in the south-east of france. acta horticulturae, 373: 69-74. egea j., burgos l., 1995 double kernelled fruits in almond (prunus dulcis mill.) as related to pre-blossom temperatures. ann. appl. biol., 126: 163-168 garcía j.e., dicenta f., berenguer t., egea j., 1996 programa de mejora del almendro del cebas-csic (murcia). fruticultura profesional, 81: 64-70. gradziel t.m., kester d.e., 1998 breeding for self-fertility in california almond cultivars. acta horticulturae, 470: 109-117. gülcan r., 1985 descriptor list for almond (prunus amygdalus) (revised). fao, ibpgr secretariat, rome, italy. kester d.e., asay r., 1975 almonds, pp. 387-419. in: janick j., and j.n. moore (eds.). advances in fruit breeding. purdue univ. press, west lafayette, ind., usa, pp. 623. kester d.e., gradziel t.m., grasselly c., 1991 almonds (prunus). acta horticulturae, 209: 701-758. kester d.e., hansche p.e., beres w., asay r.n., 1977 variance components and heritability of nut and kernel traits in almond. j. amer. soc. hort. sci., 102: 264266. lansari a., iezzoni a.f., kester d.e., 1994 morphological variation within collections of moroccan almond clones and mediterranean and north american cultivars. euphytica, 78: 27-41. ortega e., dicenta f., 2003 inheritance of self-compatibility in almond: breeding strategies to assure selfcompatibility in the progeny. theor. appl. genet., 106: 904-911. sánchez-pérez r., ortega e., duval h., martínezgómez p., dicenta f., 2007 inheritance and relationships of important agronomic traits in almond. euphytica, 155: 381-391. socias i company r., felipe a.j., 1988 self-compatibility in almond: transmission and recent advances in breeding. acta horticulturae, 224: 307-317. socias i company r., felipe a.j., 1999 ‘blanquerna’, ‘cambra’ y ‘felisia’. tres nuevos cultivares autógamos de almendro. inf. técn. econ. agrar., 95(2): 111-117. adv. hort. sci., 2018 32(1): 27-32 32 spiegel-roy p., kochba j., 1974 the inheritance of bitter and double kernel characters in the almond. z. pflanzenzücht., 71: 319-329. spiegel-roy p., kochba j., 1981 inheritance of nut and kernel traits in almond. euphytica, 30: 161-174 vargas f.j., romero m.a., 2001 blooming time in almond progenies. ciheam, options méditerranéennes, 56: 29-34. impaginato 319 adv. hort. sci., 2021 35(3): 319­327 doi: 10.36253/ahsc­10346 prospects for improvement of plectranthus edulis (vatke) agnew: a high potential food security crop t. feyissa institute of biotechnology, addis ababa university, addis ababa, ethiopia. key words: genetic diversity, micropropagation, neglected crops, nutritional composition, tuber. abstract: plectranthus edulis (vatke) agnew is a tuber crop originated and culti­ vated in ethiopia for food and medicinal uses. it has higher quality nutrient composition than other tuber crops. the cultivation of p. edulis is under threat because of introduction of exotic species, land use change, habitat destruction, population pressure, selection of only few local varieties by farmers and clima­ te change. although p. edulis is a high potential food security crop, there is no any improved variety as there has been very little research on the crop. this lack of research attention including absence of improved varieties and lack of conservation strategy may lead to significant genetic erosion of this crop. previous studies of p. edulis include documentation of indigenous knowledge, analysis of nutritional composition and the use of its starch in drug formulation, traditional cultivation practices, micropropagation, in vitro regeneration, and morphological and molecular genetic diversity. however, more extensive research in all aspects is required for its improvement, use and conservation. therefore, it is imperative to review the research results of this crop for the research community. this article reviewed research findings of this crop and presents it in a comprehensive way so that the readers will get ample informa­ tion and can refer original research findings for the details. 1. introduction plectranthus edulis (vatke) agnew (synonym. coleus edulis) is an ancient indigenous tuber crop that occurs both as cultivated and wild spe­ cies in ethiopia. although it is known by different local names depending on the place where it is cultivated, oromo dinich is the most popular local name. the cultivation of p. edulis is restricted to ethiopia. it is mainly cul­ tivated in the south and south western parts of the country at altitudes ranging between 1880 and 2200 m a.s.l. as food, and sometimes medicine (demissie, 1988; taye et al., 2007; megersa, 2010). p. edulis is a dicotyle­ donous plant that grows up to a height of 1.5 m (taye, 2008). p. edulis is a high energy food crop and the tubers contain substantial amounts of micro­ and macro­nutrients. when compared to irish potato (*) corresponding author: tileye.feyissa@aau.edu.et citation: feyissa t., 2021 ­ prospects for improvement of plectranthus edulis (vatke) agnew: a high potential food security crop. ­ adv. hort. sci., 35(3): 319­327. copyright: © 2021 feyissa t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. author contributions: all authors contributed equally to the work. competing interests: the authors declare no competing interests. received for publication 17 january 2021 accepted for publication 16 july 2021 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-10346 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 319­327 320 (solanum tuberosum), p. edulis has higher food energy and contains twice the amount of fat and cal­ cium (ehnri, 1997). it contains a similar amount of protein to that of irish potato and almost twice that of sweet potato (ipomoea batatas) after cooking (ehnri, 1997). the cultivation of p. edulis is under threat because of a decline in diversified local food production systems as a result of introduction of exotic species, land use change, habitat destruction, population pressure, selection of only a few local varieties by far­ mers, farming system intensification and climate change (smolders, 2006; fao, 2010). the decline in cultivation of the crop may result in erosion of the genetic base preventing the use of distinctive useful traits that are used for crop adaptation and improve­ ment (padulosi et al., 2002; ibc, 2005). there is enormous untapped potential in ethiopia to exploit the rich and diverse plant genetic resour­ ces of underutilized root and tuber crops including p. edulis. in spite of this potential, very little research has been done to improve the productivity of p. edu‐ lis (ibc, 2007). currently, however, p. edulis is reali­ zed as a high potential food security crop that is attracting research attention. the poor storability of tubers is found to be the major production constraint of p. edulis. it is propa­ gated by vegetative methods so that it is susceptible to diseases that are transmitted from one generation to the next through infected planting material (taye, 2008). this problem can be solved by integration of new technologies including biotechnological approa­ ches. documentation of information on indigenous knowledge, uses, and distribution of this crop is essential for its conservation, further utilization, and improvement (mathenge, 1995). it is also important to initiate research on seed production of p. edulis. so far, there is no any improved variety of p. edulis. the objective of this article is to review some aspects of this crop, with particular regard to biotechnologi­ cal approaches and present it in a comprehensive way so that the readers will get summarized informa­ tion to design research or policy on conservation and improvement of the crop. 2. production practices farmers select seed tubers from the previous har­ vest or they buy from the market. most farmers plow the land more than three times before planting and mix farmyard manure with the soil (mekbib and weibull, 2012). farmers do not use chemical fertili­ zers and other commercial inputs for production of p. edulis. the seed tubers are planted from march to april as there is brief rain during this time. farmers commonly slice a single tuber into pieces and plant the pieces because they claim planting tuber pieces increases tuber yield (taye et al., 2007; mekbib and weibull, 2012). research findings also showed that planting tuber pieces results in higher yields than whole tubers (taye, 2008). in southern ethiopia, almost 50% of the farmers plant the tubers with their sprouts whereas the others plant after removing the sprouts (taye, 2008). most p. edulis growing farmers remove the shoot apex with one or two pairs of leaves from the main stem and branches to increase stem number. research results show that this practice significantly increases the number of stems and enhances faster stolen formation. most farmers commonly build up the soil around the stem to enhance plant growth and increase tuber yield. the absence of positive cor­ relation between frequency of removing the shoot apex and amount of the soil built up around the stem was reported, although both activities serve the same purpose (taye, 2008). weeding is carried out at least three times at dif­ ferent growing stages of p. edulis. following harvest of p. edulis, farmers plant other crops, and the yields of crops that follow p. edulis in the rotation are high. the reason for increase in yield could be due to the slowly released nutrients into the soil and improved soil structures as the result of the organic manure applied in previous cropping seasons (mekbib and weibull, 2012). depending on the type of variety, p. edulis is harvested from six to eight months after planting. although the cultivation is mainly perfor­ med by male farmers, female farmers also play important roles in selecting tubers for different end­ use criteria related to the household food require­ ments (mekbib and weibull, 2012). farmers used to maintain several local varieties of p. edulis for different purposes. however, the num­ ber of local varieties preserved by farmers has been declining. so far, the total number of local varieties all over the country is not known. however, there are at least six local varieties of p. edulis that grow in spe­ cific areas as study results on three kebeles (the smallest administrative units in ethiopia), namely delebo­atewaro, delebo­wogene and kokete­mare­ chare of sodo zuria district, south ethiopia have feyissa ‐ plectranthus edulis for food security 321 shown (mekbib and weibull, 2012). growth habits, taste, tuber skin color, days to maturity, drought tolerance, storage period and marketability are the main characteristics of the plant used by farmers to identify local varieties (mekbib and weibull, 2012). distribution and seed exchange of local varieties by farmers play key roles in conservation and utilization of local varieties (de boef, 2008). younger farmers mostly purchase the planting material from markets whereas older farmers (older than fifty years) use their own saved seed tubers from the previous har­ vest. older farmers have much richer indigenous knowledge than younger farmers about the cultural value of the crop, indicating a limitation in the docu­ mentation of knowledge on the management. these knowledge gaps may lead to cultivation of only few local varieties (negri, 2003; mekbib and weibull, 2012). this calls for more research on documentation of local knowledge and transferring this knowledge to the young farmers. one of the major challenges of p. edulis produc­ tion is seed tuber storage. the tuber is stored in the field where it is produced until used for the required purpose. this storage method results in significant tuber yield loss due to excessive heat especially during dry seasons. the land where the tubers are stored remains idle until all the tubers are removed. therefore, as farmers do not want their land to be idle, most of them often obtain seed tubers from other sources during planting season (mekbib and weibull, 2012). farmers cover p. edulis fields with mulching materials to protect the tubers from direct sunlight until the next planting season. after the pre­ paration of the land for planting, tubers are transfer­ red to the pits for a day to facilitate the germination and performance of seedlings. most seed tubers are obtained from farmers who have enough land to pro­ duce p. edulis as they produce more than a hou­ sehold requirement and save the surplus for sale (negri, 2003). although different cultural practices have been designed to overcome the production con­ straints of p. edulis, very little research has been done to tackle the problems. another important con­ straint is shortage of land for planting (mekbib and weibull, 2012). this can be solved by amending land usage policy. some efforts have been made to evaluate perfor­ mance of p. edulis with regard to yield and yield rela­ ted characters and reports show different perfor­ mance of the crop at different environments (taye et al., 2013). tuber fresh weights of 4556­4933 g m­2, or 45­49 mg ha­1 were obtained at experimental sites, which is higher than yields produced by farmers (740­1480 g m­2). this higher experimental yields compared to farmers’ yields is because farmers har­ vest the tubers early late emergence, slow develop­ ment of the canopy, full coverage of the ground in a short period of time and decline in soil cover during senescence caused poor radiation interception. radiation interception of a crop can be enhanced by increasing ground cover by the canopy. this could be achieved by planting large seed tuber pieces (wiersema and cabello, 1986; lommen and struik, 1994), by planting more tuber pieces per area (spitters, 1990) or by using traditional practices that enhance the canopy development (taye et al., 2012). 3. nutritional content p. edulis contains substantial amounts of nutrients. moisture content of the tuber dry matter ranges from 14.1% to 17.5%, whereas the protein content ranges from 0.70% to 1.76% (hellemans et al., 2017). these values are higher than cassava tuber, which contains 0.3% protein and 0.1% to 0.3% starch (alvani, 2011; waterschoot et al., 2015). another study reported 11.2% moisture, 0.14% ash, 0.21% lipid, 0.43% protein, and 99.22% starch con­ tent on a dry weight basis (assefa, 2015) (table 1). table 1 ­ proximate composition of plectranthus edulis tubers on dry weight basis in g per 100 g (z) ethiopian health and nutrition research institute sample type moisture content (%) crude protein total ash crude fiber crude fat carboydrate energy (calories) references raw 81.9 1.5 1.1 0.70 0.20 15.3 69 ehnri (z), 1997 boiled 73.8 1.0 1.3 1.00 0.20 23.7 101 ehnri, 1997 starch 11.2 ­ 0.14 ­ 0.21 99.2 ­ assefa, 2015 starch 15.5 1.09 3.0 0.24 ­ ­ alvani et al., 2011 adv. hort. sci., 2021 35(3): 319­327 322 samples collected from different regions of ethiopia showed variation in fiber content based on their geographic location. starch extracted from tuber samples collected from wolaita zone (chenqoua, inuka and lofua areas) showed signifi­ cantly lower fiber content than tuber samples collec­ ted from arjo white, chencha and jarmet. moreover, the samples collected from arjo white, chencha and jarmet contain higher fiber content than cassava or potato. whether this variation is due to the environ­ ment or genetic variation needs further investigation. p. edulis contains amylase ranging from 14.2% to 23.9% (waterschoot et al., 2015). tuber samples collected from abay chomen district of oromia region, ethiopia, showed variation in mineral content may be due to genotype or envi­ ronmental variation. p. edulis is found to be rich in potassium followed by magnesium, calcium, zinc, manganese, and copper. mineral content of p. edulis is presented in table 2. the trace metals (chromium and nickel) and the heavy metals (lead and cadmium) were not detected, indicating p. edulis is safe with regard to the toxic heavy metal health concerns (lema, 2016). the phosphorus content of most p. edulis samples is twice as much as in irish potato. high phosphorus content is linked to resistant starch, paste viscosity and gel strength (lu et al., 2012). higher phosphorus content is responsible for the gra­ nular structure, and especially for the crystalline region to be less rigid and therefore enabling absorp­ tion of more water (lin et al., 2013). high phospho­ rus content was also found to impart high viscosity to the starch and increase gel strength. this indicates p. edulis starch can be used in food processing that requires high gel strength and can provide resistant starch in functional food preparation (hellemans et al., 2017). however, further research should be con­ ducted to confirm this finding. p. edulis is found to be richer in most of the mineral elements than irish potato. starch physicochemical properties scanning electron microscopy (sem) of the starch granules showed elliptical and some oval shapes. the starch showed a normal monomodal granule size distribution. x­ray diffraction pattern of the starch showed typical b­type with a distinctive peak (assefa, 2015). on the contrary, other authors reported distinct bimodal distribution pattern of starch granu­ lar characteristics, which indicates the presence of small (b­type) and large (a­type) granules (hellemans et al., 2017). the shape of b­type granules is spheri­ cal whereas that of a­type is elliptical when visuali­ zed by the cryo­sem. for starch extracted from p. edulis tuber, such bimodal distribution is not com­ mon. starch extracted from irish potato and cassava shows normal distribution pattern (waterschoot et al., 2015). therefore, p. edulis can be used in food industry and as an alternative for potato starch, which is less suitable to be used in pasteurized foods. typical water absorption (wa) pattern, swelling power and relative humidity of p. edulis is reported to be higher than irish potato starch but its solubility at all studied temperatures is lower than irish potato (assefa, 2015). the physicochemical analysis of the starch showed 1.08 g/g wa of p. edulis (hellemans et al., 2017). this means that the starch granules absorb on average of 1.08 times their own weight after it is completely hydrated. only a small difference was observed in wa among p. edulis starch samples of dif­ ferent accessions. the high water binding ability of the starch could reduce the stickiness of dough, increase moistness, improve handling and soften the texture of baked products, which makes p. edulis starches more suitable for use in baked goods (taggart, 2004). differential scanning calorimeter (dsc) thermo­ grams of p. edulis starch showed higher onset tempe­ rature (to) of 69.2°c, peak temperature (tp) of 74.3°c and ending temperature (te) of 83.3°c than the starch of irish potato (assefa, 2015). similarly, the pasting properties showed p. edulis starch has table 2 ­ mineral content of plectranthus edulis in mg per 100 g w ppm/dm­ parts per million/dry matter. sample k ca fe mg zn p references raw ­ 29.0 9.30 ­ ­ 90.0 ehnri, 1997 boiled ­ 19.0 1.10 ­ ­ 62.0 ehnri, 1997 starch w 513 397 20.8 175 4.5 1803 alvani et al., 2011 starch 3.54 0.62 ­ 0.49 0.24 ­ lu et al., 2012 feyissa ‐ plectranthus edulis for food security 323 (bryan, 1983). these problems might be solved by using the benefits of modern plant biotechnology such as plant tissue culture that can supply a large number of disease­free planting materials to growers who continually acquire it. micropropagation and in vitro shoot regeneration increased use of rapid multiplication techniques enables production of large amounts of pathogen­ free planting material. most rapid multiplication techniques in root and tuber crops involve the use of aerial portions of the plant so that contact with soil and tuber is broken and most non­systemic patho­ gens can be eliminated (bryan, 1983). meristem cul­ ture is the most popular tissue culture technique for production of disease free planting materials. it can be used alone or in combination with thermotherapy to eliminate a number of viruses and bacteria (kartha and gamborg, 1975). meristem culture technique eli­ minates systemic pathogens found in the mother stock plant. the first micropropagation protocol of p. edulis was developed from meristem culture (tsegaw and feyissa, 2014) followed by in vitro shoot regenera­ tion from leaf derived callus (aschale and feyissa, 2019). shoots were initiated from meristem explants using murashige and skoog (ms) (murashige and skoog, 1962) medium. the ms medium was supple­ mented with 1.0 mg/l gibberellin (ga3) and 0.1 mg/l a­naphthalene acetic acid (naa) in combination with different concentrations of benzylaminopurine (bap) (tsegaw and feyissa, 2014). according to the authors, ms medium containing the aforementioned hormones was proved to be the best in terms of shoot initiation from meristem for mother plants col­ lected from two locations, holeta and wolaita. the shoots were transferred to multiplication medium and the highest mean shoot number per explant (7.2) was obtained on medium containing 1.0 mg/l kinetin in combination with 0.1 mg/l naa for shoots derived from mother plants collected from holeta while, 6.2 shoots per explant was obtained on medium containing 3.0 mg/l kinetin in combination with 0.05 mg/l naa for shoots derived from mother plants collected from wolaita. the effect of solid and liquid media on in vitro axillary shoot proliferation was investigated by culturing shoots on ms medium containing different concentrations of bap in combi­ nation with thidiazuron (tdz) (yimam, 2013). the highest mean shoot number per explant, 5.85 and 6.07, were obtained on solid and liquid media, higher pasting temperature than irish potato (hellemans et al., 2017). a rapid increase in viscosity occurred at tp range of 70.7 to 74.0°c compared to irish potato starch (52.2°c). the disintegrating abilities of starch of p. edulis and irish potato were compared in paracetamol tablet formulations prepared by wet granulation method (assefa, 2015). the granules were prepared with the starches at different disintegrant concentra­ tions and characterized for particle size distribution and flow properties. the prepared tablets showed excellent flow property as manifested by the weight uniformity. after studying the crushing strength, fria­ bility, disintegration time, and dissolution rate of the tablets using standard methods, the results showed that p. edulis starch favorably competed with potato starch as a disintegrant in the paracetamol tablet for­ mulations. in other recent study, carboxymethylated p. edulis starch was evaluated as a suspending agent in metro­ nidazole benzoate suspensions by comparing with sodium carboxymethyl cellulose (nacmc) (brhane, 2020). the author reported viscosities of the formu­ lations prepared with carboxymethylated p. edulis starch were significantly lower than that of nacmc. the flowability of the suspensions was in the order of carboxymethylated p. edulis starch (cmps) greater than nacmc. at 1% concentration, carboxymethyla­ ted p. edulis starch resulted in significantly higher sedimentation volume than nacmc. potassium dihy­ drogen phosphate (kh2po4), which is employed as a flocculating agent, significantly increased the sedi­ mentation volume of the suspensions prepared with carboxymethylated p. edulis starch and nacmc. the redispersibilities of cmps were found to be better than nacmc. the author pointed out that all suspen­ sions resulted in a release of greater than 85% of drug within 1 h. all suspension formulations were found to be stable (brhane, 2020). the author con­ cluded carboxymethylated p. edulis starch can be used as alternative suspending agent. 4. prospects of biotechnology for improvement of p. edulis in addition to the shortage of seed tubers and the poor storability of the tubers, systemic diseases, viru­ ses, viroids and mycoplasma as well as several patho­ genic bacteria are the most devastating root and tuber crops including p. edulis, in terms of yield loss 324 adv. hort. sci., 2021 35(3): 319­327 liquid medium containing 0.1 mg/l iaa. the highest mean root number per shoot of 10.15 on ms medium supplemented with 2.0 mg/l iba was also reported (aschale and feyissa, 2019). all in vitro regenerated plants of holeta origin and 96% of wolaita origin survived after one month of acclimatization in a greenhouse. similar study repor­ ted 100% and 82.85% survival of plantlets derived from solid and liquid media, respectively, after accli­ matization in greenhouse (yimam, 2013). all in vitro rooted and 78 % ex vitro rooted microshoots survi­ ved in the greenhouse (aschale and feyissa, 2019). the development of these protocols for micropropa­ gation and in vitro regeneration of shoots from callus is the prerequisite for further advanced biotechno­ logy research such as genetic transformation and genome editing of this crop. 5. genetic diversity of p. edulis for improvement of any crop, the existence of adequate genetic diversity is imperative. this could be achieved if and only if there is appropriate conser­ vation strategy. selection and cultivation of only few local varieties of p. edulis and its current replacement by other tuber crops has been causing serious gene­ tic erosion of the local gene pool of this crop. genetic diversity analyses using morphological traits there are several studies on morphological gene­ tic diversity of p. edulis. genetic diversity of 36 acces­ sions of p. edulis using 16 morphological traits was assessed and highly significant variation among the accessions for all the analyzed traits except length of tuber was reported (garedew et al., 2013). in another study, 20 p. edulis accessions that were assessed using 29 morphological traits showed 75.7% variance among the accessions (mekbib, 2007). this study showed that there are accessions collected from different regions of the country but showed genetic similarity, and some of those collected from the same geographic area showed different genetic background. this indicates geographic diversity does not necessarily represent genetic diversity. similarly, genetic diversity analysis of 20 accessions using 13 traits revealed significant variation of all traits except flower length and leaf width (soresa, 2017). recent extensive genetic diversity study of 174 accessions at three environments based on 12 qualitative and 16 quantitative traits also showed a highly significant respectively, containing 1.5 mg/l bap in combination with 0.5 mg/l tdz. developing in vitro regeneration protocol through callus phase or somatic embryogenesis is a prerequi­ site to genetically improve a crop through biotechno­ logical approaches including genetic engineering, genome editing and creating somaclonal variants. recently, in vitro regeneration protocol of p. edulis have been developed by using leaf explants (aschale and feyissa, 2019). the highest percentage of callus induction (100%) was obtained on ms medium sup­ plemented with 1.5 mg/l naa in combination with 1.0 mg/l bap and 2.0 mg/l naa in combination with 0.5 mg/l bap. the highest shoot regeneration per­ centage (46.6%) from callus was obtained on ms medium containing 0.1 mg/l bap in combination with 0.5 mg/l tdz. the highest mean shoot number per callus (1.66) was obtained on medium containing 1.5 mg/l bap in combination with 1.0 mg/l tdz. the highest mean shoot length (0.63 cm) was obtained on medium supplemented with 0.5 mg/l bap in com­ bination with 0.1 mg/l tdz. the highest mean shoot number per explant (58.68) was obtained on medium supplemented with 0.5 mg/l bap in combination with 0.4 mg/l ga3. rooting and acclimatization shoots cultured on half salt strength ms medium rooted better than those cultured on full salt strength (tsegaw and feyissa, 2014). shoots derived from explants collected from holeta resulted in the highest percentage of rooting (100%) and 3.12 mean root number per shoot whereas shoots whose explants were collected from wolaita resulted in 76.7% rooting and 2.2 mean root number per explant. microshoots were also directly planted in a greenhouse for rooting and acclimatization by bypas­ sing the in vitro rooting stage and the overall best result in rooting was obtained in ex vitro rooting con­ dition. it was promising in cost reduction as it bypas­ ses the in vitro rooting stage. reducing the cost of in vitro production is a key for increasing the applica­ tion of the method. rooting of microshoots in vitro is expensive and can even double the price of the plan­ tlets (zimmerman, 1988; de klerk, 2002). in another study, shoots were cultured on half strength ms medium containing different concentrations of indole acetic acid (iaa) and indole butryic acid (iba) for roo­ ting (yimam, 2013). the highest mean root number per shoot (10.55) was obtained on solid medium and 13.11 mean root number per shoot was obtained in feyissa ‐ plectranthus edulis for food security 325 variation among the populations and non­significant environment­population interaction for most of the quantitative traits (gadissa et al., 2020). the first six principal axes of principal components analysis accounted for 77% of the total variation. the popula­ tions were grouped into four clusters but there was no strong groupings based on geographical locations from where the genotypes were collected. this indi­ cates the historical or contemporary gene flow, parti­ cularly tuber exchange among the different geo­ graphical regions. although there are reports of rela­ tively high genetic diversity, still there is a need for more collections and evaluation over multiple loca­ tions and seasons for well­refined genetic diversity estimation. however, as the analyzed traits are affec­ ted by the environment, the morphological genetic diversity results should be complemented by molecu­ lar genetic diversity studies. genetic diversity analyses at molecular level assessment of molecular genetic diversity of p. edulis was conducted by using inter simple sequence repeat (issr) markers for the first time in 2015 (shiferaw, 2015). the shannon information index ranged from 0.25 to 0.39 with overall index of 0.48. nei’s gene diversity ranged from 0.18 to 0.30 with overall mean diversity of 0.33, and 74.23% within population variation. analysis of molecular variance (amova) showed within population diversity range of 41.30% to 65.22%. another study using the same marker exhibited overall percent polymorphism of 95%, 0.62 shannon information index and 0.40 nei’s gene diversity (gebrehiwet et al., 2019). more exten­ sive study of genetic diversity assessment was con­ ducted by using expressed sequence tag simple sequence repeats (est­ssrs) derived from p. barba‐ tus (gadissa et al., 2018). the author identified 128 alleles in 12 populations that consisted of 287 indivi­ dual plants. the results showed gene diversity index range of 0.31 to 0.39 with overall mean of 0.35. among the 12 populations, wenbera, awi and wolaita populations showed the highest genetic diversity, and hence these populations can be consi­ dered for in situ conservation and identification of genotypes with traits of interest that can be used in breeding programs. amova showed low population differentiation with only 3% of the total variation accounting for variation among populations. struc­ ture and cluster analyses did not group the popula­ tions into distinct clusters, which may be attributed to historical and contemporary gene flow and also the reproductive biology of the crop. 6. conclusions p. edulis is a high potential food security crop con­ taining substantial amounts of nutrients. however, there is no any improved variety of the crop, and cur­ rently its cultivation is declining. shortage of tuber seed is the major problem of this crop. recently, the farmers are turning to produce other cash crops that generate more income. with advances in plant research, it is important to integrate both conventio­ nal and modern technologies to improve this crop. unless urgent attention is given to such neglected and underutilized crops, these crops are extremely subjected to genetic erosion as the farmers shy away from producing them. using biotechnological resear­ ch tools such as plant tissue culture, genetic enginee­ ring, genome editing and plant molecular breeding might be a future hope for improvement of this crop, although the international funding opportunities for such orphan crops are highly limited. in vitro propa­ gation and in vitro regeneration protocols from diffe­ rent explants have already been developed as reviewed in this article and these protocols can be used for improvement of p. edulis. in addition, infor­ mation from the genetic diversity results reported so far could be used as a baseline for more research in the area and improve this crop using genomic tools including marker assisted breeding. acknowledgements the author acknowledges addis ababa university for providing the necessary resources during writing of this review. the author is also grateful to graduate students who have been working with him and other researchers who generated interesting information on plectranthus edulis that served as resources to write this review. 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resources university of khuzestan, khuzestan, iran. keywords: antioxidant enzyme, ethylene, plant growth regulator, post-harvest, sugar. abstract: the present study investigates the roles of nitroprusside (snp), salicylic acid (sa), methyl jasmonate (mj), and their interaction with 8-hydroxyquinoline sulfate (8-hqs) in regulating the peroxidase activity (pox), water uptake, the relative water content (rwc), the contents of malondialdehyde (mda), soluble sugar, proline, and protein content in the petals and the stem bending of gerbera jamesonii ‘sun spot’ cut flowers. cut flowers were treated with various concentrations (50, 100, and 200 µm) of hold-solutions containing 8-hqs, sa, mj, and snp. hold solutions were used alone or in combination with 100 µm 8-hqs for 24 h. distilled water was used as control and sucrose (4%, w/v) was added to all solutions. the findings showed that 50 µm sa+ 100 µm 8hqs markedly improved the rwc, the contents of proline, anthocyanin, carotenoid, protein, and soluble sugar, and activities of pox in the petals and markedly reduced water loss and the contents of mda in the petals, compared with other treatments, especially the control. meanwhile, the combination of plant growth regulators (pgr) with 8-hqs markedly improved positive indexes than use alone pgr. this phenomenon seemed to be due to more absorption of pgr. among different concentrations of pgr, 50 μm is the most effective treatment for the improvement of the vase life of gerbera jamesonii cut flowers. the results also demonstrated that sa+8-hqs improves the vase life of gerbera cut flowers by enhancing the membrane stability and water retaining capacity as well as increasing proline, antioxidant activity, and pigment contents. 1. introduction vase life and quality are key factors that contribute to the aesthetic and benefits of cut flowers (mansouri, 2012). the short vase life of most cut flowers is mainly due to the water loss, which is an important physiological process that affects the main quality characteristics of cut flowers, such as appearance (salehi salmi et al., 2018). gerbera jamesonii is a commercially popular cut flower that ranks 10 in the globe auctions. this plant is a member of the family asteraceae that (*) corresponding author: mrsalehisalmi@gmail.com citation: hemati e., salehi salmi m.r., daneshvar m.h., heidari m., 2019 the roles of sodium nitroprusside, salicylic acid, and methyl jasmonate as hold solutions on vase life of gerbera jamesonii ‘sun spot’. adv. hort. sci., 33(2): 187-195 copyright: © 2019 hemati e., salehi salmi m.r., daneshvar m.h., heidari m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 november 2018 accepted for publication 15 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 187-195 188 originates from africa and madagascar and extends to china (parthasarathy and nagaraju, 1999; hind, 2007). sensitivity to microbial contamination at the stem base is a major postharvest problem of this plant (balestra et al., 2005). microorganisms cause stem end blockage in cut flower (he et al., 2006; liu et al., 2009) and also secretion of toxic compounds, and thereby accelerated wilting (williamson et al., 2002). vase life of gerbera has been studied extensively with different treatments (nair et al., 2003; solgi et al., 2009; shabanian et al., 2018). witte et al. (2014) reported that treating cultivars of gerbera stems by sucrose in combination with an antimicrobial compound (hqc, chlorine) resulted in less bending than the same concentration of the antimicrobial compounds alone. perik et al. (2014) noted that other factors might also be involved in bending and showed that a mixture of chemicals delayed the time to bending in six tested cultivars of gerbera. sodium nitroprusside (snp), the inorganic nitrous compound (nitroferricyanide) with the formula na2[fe(cn)5no].2h2o, is an important signaling molecule. this molecule has diverse physiological functions for plants such as inducing tolerance to adverse environmental factors (shi et al., 2016; kumar rai et al., 2018). application of snp in vase solution resulted in extending the vase life of gerbera cut flowers (shabanian et al., 2018); however, there is very limited information regarding the positive effects of exogenously applied snp in extending the vase life. ortho-hydroxybenzoic acid or salicylic acid is an endogenous plant growth regulator. exogenous application of salicylic acid (sa) can affect the antioxidant capacity of plant cells and prolong vase life of cut flowers, such as rose (alaey et al., 2011) and anthurium (promyou et al., 2012). however, little work has been reported on the role of sa on cut flower vase-life improvement and physio-chemical attributes related to senescence. methyl jasmonate (mj) has been found as a naturally occurring substance in higher plants. in this context, the application of mj induced antioxidant system activity can suppress fungal infection and enhance stress resistance (kanani and nazarideljou, 2017). to our knowledge, mj effects on specific physiological and biochemical processes in gerbera cut flower have not been studied yet. as a derivative of 8-hydroxyquinolines, 8-hydroxyquinoline sulfate is widely used as antibacterial since the beginning of the 1950s. this compound is active against gram-negative bacteria of the enterobacteriaceae family, fungi of the candida genus, and mycoplasma (chupakhina et al., 2012). in previous studies, 8-hydroxyquinoline sulfate (8-hqs) remarkably increased vase life of rose cut flowers (ichimura et al., 1999). in the present study, the role of three plant growth regulators (i.e., snp, mj, and sa) in regulating the activities of the antioxidant enzyme, relative water content (rwc), water uptake, the contents of malondialdehyde (mda), protein, pigments and soluble sugar in the petals, and time of stem bending of gerbera cut flowers were investigated. the objective of the present study is to provide a theoretical basis for the application and optimization dosage of plant growth regulators in combination with an antimicrobial compound, i.e., 8-hqs, in improving the vase life of gerbera cut flowers during the vase-holding period. 2. materials and methods plant material and treatment ‘sun spot’ cut-gerberas (gerbera jamesonii), harvested at normal harvest maturity, were obtained from a commercial grower (dezfol, khuzestan, iran). the length of the stem varied between 65 and 70 cm. the harvested flowers were packed into parchment paper and transported to the laboratory within 1-2 h. then, stems were re-cut to a uniform length of 55, under distilled water to avoid air embolism. each sixflower sample was placed randomly in 250 ml of various concentrations (50, 100, and 200 µm) of holdsolutions containing 8-hqs, sa, mj, and snp. hold solutions were used alone or in combination with 100 µm 8-hqs for 24 h. distilled water was used as control and sucrose (4%, w/v) was added to all solutions. to maintain the proper concentrations of holdsolutions, the mouths of the vases were covered with plastic wrap (around the stem) to minimize evaporation and to prevent contamination. then, flowers were individually sited in glass bottles of 25 cm height, approximately 150 ml of distilled water in each bottle, under laboratory condition. the laboratory was maintained at 22°c, 60±5% relative humidity, and 16 mmol m-2 s-1 photons irradiance using cool fluorescent lamps for a 12 h photoperiod (07:0019:00 h). measurements time of stem bending was determined as described by perik et al. (2012). relative fresh weight (rfw) was calculated by the hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 189 following formula: rfw (%) = [(fw t=10 -fw t=0 )/ fw t=0 ] ×100 where fwt=10 is the fresh weight of flower (g) at 10th day and fwt=0 is the fresh weight of the same flower (g) at first day (he et al., 2006). water uptake (ml) was calculated by subtracting in the weight of the remaining water at the end of the experiment from the initial weight. soluble carbohydrate content in petals was measured by the anthrone colorimetric method according to the method of xue (1985). total anthocyanin content of petal was measured by the ph differential method of yang et al. (2009). total carotenoid content of gerbera petal tissue was estimated using the method of wellburn (1994). flavonoid petal tissue was measured according to the method of markham (1982). protein content in petal was estimated by the method of bradford (1976) using bovine serum albumin as the protein standard. malondialdehyde (mda) content in petals was measured by the thiobarbituric acid reaction following the procedure of hodges et al. (1999). peroxidase (pox) activity was evaluated by oxidation of guaiacol, as a substrate, according to chance and maehly (1955). statistical analysis data were analyzed using analysis of variance (anova) in sas software. means were compared by one-way anova and duncan’s multiple range test at the 5% level of significance. 3. results and discussion vase life as shown in figure 1, hold-solutions affected senescence of gerbera jamesonii in a dose-dependent manner. compared with control, different concentrations of sa, with or without 8-hqs, all prolonged the senescence of gerbera as shown by the tighter stem and more showy flowers. compared with other concentrations of sa, 50 µm sa+ 100 µm 8-hqs markedly prolonged the longevity of the cut flowers. as shown in figure 1, different concentrations of snp, with or without 8-hqs, all prolonged the length of vase life of gerbera cut flower, compared with control. compared with control, 50 µm, 100 µm, and 200 µm snp noticeably increased the length of vase life of gerbera jamesonii cut flower by 65%, 78%, and 43%, respectively. however, there was no significant difference in the stem bending of flowers among different concentrations of snp. among various concentrations of 8-hqs, stem bending of cut flowers treated by 100 μm 8-hqs was significantly lower than those treated by other concentrations. compared with control, 50, 100, and 200 μm 8-hqs increased the vase life by 69%, 100%, and 78%, respectively. different concentrations of mj all significantly increased vase life, compared with the control (fig. 1). however, mj treatments were less effective on vase life compared with 8-hqs, sa, and snp treatments. shabanian et al. (2018) showed that snp extended the vase life of gerbera cut flowers as compared with their respective control treated with water alone. in agreement with this finding, treatments with snp extended the vase life of other cut flowers, e.g., carnation (zeng et al., 2011), chrysanthemums (mansouri, 2012), gladiolus (dwivedi et al., 2016), and rose (liao et al., 2013). the mechanism of sa action, as a hold solution, in vase life of cut flowers has not been clarified; however, published data suggest some association with ethylene production. zhang et al. (2003) showed that application of sa resulted in suppression acc synthase and acc oxidase activities and biosynthesis of ethylene in kiwifruit. in gladiolus, the maximum vase-life was obtained once flowers treated with a solution containing 100-ppm 5-sulfosalicylic acid + 4% sucrose (ezhilmathi et al., 2007). 8-hqs is a subclass of quinolones with a wide variety of biological effects. the 8-hydroxyquinoline derivatives emerged as a hold-solutions being widely explored for several biological functions such as antifungal effects (oliveri and vecchio, 2016) and antimicrobial (abouelhassan et al., 2017). according to van doorn (1997), the bending of gerbera cut flowers was caused by low turgescence of the flower scape when facing water uptake problems. in addition, he notified that bactefig. 1 effect of the different preservative solutions (µm) on time of stem bending in gerbera cut flowers. columns followed by different letters are significantly different at p=0.05. adv. hort. sci., 2019 33(2): 187-195 190 ria in the vase water were the most common cause of xylem blockage affecting water uptake. accordingly, antimicrobial compounds such as 8hydroxyquinoline citrate (elhindi, 2012) and essential oils (salehi salmi et al., 2018) were applied to improve vase-life of cut flowers. the effect of the application of mj on vase life of cut flowers varies widely among species and cultivars. these reports indicated that the ethylene production rate might change with the kind of genes, which were stimulated by mj (salimi et al., 2016). the results of the present study indicated that the decline in the vase life was significantly less in cut flowers in mj-treated, compared with other treatments. water loss and water uptake comparing the results of the different hold-solutions revealed statistically significant differences such that the maximum and minimum amounts of water loss, in the 10th day, occurred on cut gerbera hold in 100 µm 8-hqs+ 200 µm mj and 100 µm 8-hqs+ 100 µm snp, respectively (fig. 2). from figure 2 the maximum water loss occurred also on cut gerbera hold in mj 200 µm alone (together with 100 µm 8-hqs+ 200 µm mj). also, data showed that cut flowers treated with 50 and 100 µm 8-hqs; 200 µm sa; 50 and 100 µm snp; 50, 100, and 200 sa+ 100 µm 8-hqs; 50 µm mj+ 100 µm 8-hqs; 50, 100, and 200 µm snp + 100 µm 8-hqs hold solutions lost lower water the control (fig. 2). the lowest amount of water uptake appeared on 10th day in the cut flowers treated with 100 µm 8hqs+ 200 µm mj (fig. 3). however, there was no significant difference between this treatment and 50 µm mj, 200 µm mj, and 200 µm snp control treatments. in other treatments, water uptake was increased over 10 days of postharvest life in comparison with control. however, water uptake amounts showed significant differences among hold-solutions such that the maximum amount of it was observed in a cut flower treated with 100 µm 8-hqs+ 100 µm snp (fig. 3). cut flower senescence is closely associated with water uptake stem and rwc of petals, whereas, these characteristics are closely related with the contents of osmoregulation substances such as soluble sugars and soluble proteins (hou et al., 2018). soluble carbohydrates of petal changes of sugars content of gerbera petals are shown in figure 4. maintenance of elevated total soluble carbohydrates content exhibited by the flowers under hold-solutions treatments can be correlated with the delay in senescence and the increase in vase life of gerbera flowers. the results indicate that treatment with hold-solutions, except the high concentration of mj, with or without 8-hqs, caused a significant decrease in reducing sugars compared with the control. reducing carbohydrate starvation or its symptoms led to unwanted color changes and eventually increased susceptibility to microorganisms. postharvest treatments can reduce carbohydrate starvation during the vase life phase. postharvest treatments like sugar feeding often havefig. 2 relative water loss of cut flower of gerbera in various hold solutions with different concentrations (µm). the indicator was determined on 10st day. columns followed by different letters are significantly different at p=0.05. fig. 3 effects of different concentrations (µm) of various hold solutions on the water uptake. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 4 total soluble carbohydrates in petals of hold-solutionstreated and untreated gerbera cut flowers stored at 22°c for 10 days. hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 191 est amount of carotenoid content among all treatments at the end of the experiment. figure 7 depicts the contents (expressed as mg/g fresh weight) of flavonoid obtained from petals of gerbera cut flowers untreated and treated with hold solution at different concentrations. by comparing untreated-control samples, hold solutions differences could be clearly established (fig. 7). in particular, the concentrations of petal flavonoid were significantly (p<0.05) higher in treated cut flowers with snp, sa or mj; except 50 µm sa, 200 µm mj, 50 µm snp treatments; than control. as can be seen, cut flowers treated with 100 and 200 µm snp+ 100 µm 8-hqs exhibit higher concentrations of flavonoid than the other treatments. despite the results of this study, most investigations on vase life of cut flowers do not present data on the changes in pigmentation and those that use subjective color grades for evaluation. browning and discoloration are important factors in determining display quality of cut flowers and in many cases are the major reason for the termination of vase life (elhindi, 2012; khalaj et al., 2017; salehi salmi et al., 2018). petal coloration is caused by the accumulation of pigment, including carotenoids, flavonoids, and betacyanins, within epidermal cells. anthocyanins are synthesized via the phenylpropanoid and flavonoid pathways (tanaka et al., 2008). carbon metabolite levels, directly and indirectly, affect almost every metabolic process in a plant life. anthocyanin and carotenoid biosynthesis occur concomitantly with sugar accumulation in plant tissue (hara et al., 2003; zhang et al., 2015). similarly, in our study, some vase solutions promoted soluble carbohydrates contents in the petals of gerbera cut flowers (fig. 4), accompanying higher anthocyanin contents and presenting better ornamental quality of petal color (figs. 5, 6, and 7). it is reported that application of mj enhanced a positive effect on vase life in general. it seems that 8-hqs, by preventing vascular blockage, caused sugars directly to reach flowers in the transpiration stream via xylem. increased sugar caused by exogenous sucrose is well known from earlier studies (ichimura et al., 1999; promyou et al., 2012). han et al. (2018) have illustrated that snp treatment inhibited significantly the degradation of sucrose of peach fruit at the end of storage. they suggested sugars were significantly affected by snp treatment probably due to the activities of sucrose metabolism enzymes. yu et al. (2016) found that mj treatment could increase the encoding level and enzyme activity of sucrose phosphate synthase, which resulted in the enhancement of sucrose content. petal pigments the highest concentration of anthocyanin in gerbera florets was in the sa+ 8-hqs treatments, followed by the 8-hqs and sa treatments. the mj, with or without 8-hqs, treatments did not result in a significant increase in anthocyanin concentration compared with that of the control (fig. 5). carotenoid content of petals were increased under treatment with all concentrations of sa with or without 8-hqs, all concentrations of snp with or without 8-hqs, high concentrations of 8-hqs, and all concentrations of mj with 8-hqs (fig. 6) while the control had the lowfig. 5 effects of different concentrations (µm) of various hold solutions on anthocyanin content. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 6 effects of different concentrations (µm) of various hold solutions on amount of carotenoid. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 7 effects of different concentrations (µm) of various hold solutions on flavonoid. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. 192 adv. hort. sci., 2019 33(2): 187-195 accumulation of flavonoids in daucus carota (sircar et al., 2012). pox activity the pox activity in the petal of gerbera flowers that were treated with different concentrations of all hold-solutions, except 100 µm mj+ 100 µm 8-hqs, slightly increased during vase life (fig. 8). the highest activity of pox was observed in cut flowers hold in 50 µm 8-hqs, 100 µm sa, and 200 µm 8-snp solutions. the enzymatic antioxidant system can work against the accumulation of reactive oxygen species (ros). regulation of the antioxidant status and ros production by snp in plant cells subjected to either biotic or abiotic stressors is well established (vidal et al., 2018). previously, it has been shown that snp provides protection in broccoli florets against rapid yellowing after harvest (shi et al., 2016). this study was carried out to provide evidence for the ability of snp to regulate flower senescence through regulation of the antioxidant status of gerbera petal cells. salicylic acid can also act as a protector against several stressful impacts, scavenge free oxygen radicals, and counteract oxidative damage by regulating cellular redox balance and accelerating the transformation of superoxide anion and enhancing the activities of antioxidant enzymes (zhang et al., 2003). sa treatment reduced chilling injury in anthurium via improving the activities of sod, cat, and pox (promyou et al., 2012). kumar rai et al. (2018) reported that sa and snp enhanced tolerance to heat stress in lablab purpureus, by elevating antioxidant enzyme activity of pox, sod, and cat and thus alleviating heatinduced oxidative damage. the present study, for the first time, indicated that sa and snp treatment delayed the senescence of gerbera flowers via improving the activity of antioxidant enzymes of pox. mj was reported to stimulate pox activity in banana plants and reduce the level of o2 and h2 o2 (sun et al., 2013). in this regard, fan et al. (2016) reported inducing resistance responses in eggplant fruit by increasing the expression of pox genes. protein there was a significant difference among the type of hold-solution treatments on protein in the 10th day, although the protein content showed no significant differences among concentrations of a holdsolution. protein content for sa treatments, with or without 8-hqs, increased compared with the control; however, it was not significantly different from other treatments to control (fig. 9). the increase observed in the protein content, through treatment with sa, was likely the result of less protein degradation (alaey et al., 2011) or an increase of protein synthesis (ezhilmathi et al., 2007). under flower senescence, the stimulation of protein synthesis leads to protein accumulation that may involve in the enhanced activity of enzymes as a defense mechanism (promyou et al., 2012). to support the accumulation of proteins due to sa treatment, it was reported that sa results in a pronounced increase in total protein content and the formation of new proteins in roses (alaey et al., 2011). it seems that mj by increasing antioxidant defense enzymes leads to maintaining carbohydrate at high levels, as antioxidant inhibits the oxidation of cell biomolecules like proteins and carbohydrates (kanani and nazarideljou, 2017). in the present study, different concentrations of snp and sa, with or without 8-hqs, could markedly increase the contents of soluble sugars and soluble proteins, which increased the rwc of petals and water uptake. these increases were helpful in increasing the water retaining capacity and also played an important role in increasing vase life of gerbera cut flowers. in addition, schouten et al. (2018) suggested that snp enhances flow through xylem vessels by increasing the ionic strength of the vase water. fig. 8 effects of different concentrations (µm) of various hold solutions on activity of pox. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 9 petal proteins of gerbera cut flower in various hold solutions with different concentrations (µm). the indicator was determined on day 10. columns followed by different letters are significantly different at p=0.05. hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 193 mda in the present experiment, a noticeable decrease in the mda in control treated cut flowers compared with the other vase-solutions (fig. 10). accumulation of elevated amounts mda in control treated cut flower was recorded. this accumulation indicates the presence of oxidative stress in gerbera petals. zhang et al. (2015) reported over-reduction of the electron transport chain in mitochondria as the main source of o2 production under specific stress conditions. production of h2o2 can occur during lipid catabolism as a side-product of fatty acid oxidation. ross are also involved in the detoxifying reactions catalyzed by cytochromes in both the cytoplasm and the endoplasmic reticulum (kumar rai et al., 2018). obviously, the observed high activity of pox in saand snptreated cut flowers are as a protective mechanism against senescence. it has been revealed that mj mitigates the ros effects in maize seedlings subjected to oxidative stress (ahmadi et al., 2018). 4. conclusions treatment with sa+8-hqs extends the vase life of gerbera cut flowers at relatively low sa concentrations and leads to generating the maximum costeffectiveness. in conclusion, our results demonstrated that 8-hqs improves the vase life through increasing water uptake and consequently increases total soluble carbohydrates. also, 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supply improves petal coloration and anthocyanin biosynthesis in paeonia suffruticosa ‘luoyang hong’ cut flowers. postharvest biol. technol., 101: 73-81. zhang y., chen k., zhang s., ferguson i., 2003 the role of salicylic acid in postharvest ripening of kiwifruit. postharvest biol. technol., 28: 67-74. impaginato 343 adv. hort. sci., 2018 32(3): 343-351 doi: 10.13128/ahs-22380 water spectral pattern as a marker for studying apple sensory texture m. vanoli 1 (*), f. lovati 1, m. grassi 1, m. buccheri 1, a. zanella 2, t.m.p. cattaneo 1, a. rizzolo 1 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (creait), via venezian, 26, 20133 milano, italy. 2 centro di sperimentazione laimburg, laimburg 6, 39040 ora (bz), italy. key words: aquaphotomics, crispness, juiciness, malus x domestica borkh, mealiness, nir. abstract: aquaphotomics is a scientific discipline which investigates the waterlight interactions in biological systems by using nir spectroscopy and multivariate analysis to relate water absorption patterns to bio-functionalities. this work aimed at evaluating the feasibility of aquaphotomics to study apple fruit sensory texture. ‘braeburn’, ‘gala’ and ‘kanzi®’ apples were analyzed by a micronir spectrometer and for mechanical, structural and texture sensory characteristics. cluster analysis on sensory texture attributes showed four different profiles for each cultivar having different water spectral patterns (wasp). on average, the wasp of mealy apples showed the highest absorbance values at 1364, 1372 and 1382 nm and the lowest in the 1438-1492 nm range suggesting a preponderance of water structures with weak-hydrogen bonds; the opposite was found in crispy and juicy apples indicating the presence of more organized water structures with medium-strong hydrogen bonds. this wasp difference could be due to a different softening rate: apples clustered as firm/crispy/juicy had the highest firmness and the lowest intercellular spaces, while mealy apples had low firmness and high intercellular spaces indicating a more advanced softening. the chemical changes due to the pectin hydrolyzation could affect the water structures. the aquaphotomics approach could be a useful tool for studying the sensory texture of fruits as water structures actually change in apples with different textural characteristics whatever the cultivars. 1. introduction the preferences of apple consumers are generally based on a combination of texture and flavor (harker et al., 2003). textural properties can be considered as the main factors responsible for fruit freshness, having crunchiness and juiciness a positive effect and mealiness a negative effect on consumer choice (péneau et al., 2006). texture consists of a number of different properties perceived by human senses and depends on mechanical and structural characteristics of the fruit pulp. texture changes with (*) corresponding author: maristella.vanoli@crea.gov.it citation: vanoli m., lovati f., grassi m., buccheri m., zanella a., cattaneo t.m.p., rizzolo a., 2018 water spectral pattern as a marker for studying apple sensory texture. adv. hort. sci., 32(3): 343-351 copyright: © 2018 vanoli m., lovati f., grassi m., buccheri m., zanella a., cattaneo t.m.p., rizzolo a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 december 2017 accepted for publication 17 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 343-351 344 fruit softening due to degradation and modifications of the cell wall and middle lamella structures (gwanpua et al., 2014). during softening, pectins undergo extensive structural modifications so that cell wall polymers are less bound together and become highly hydrated (goulao and oliveira, 2008). the hydrolysis of pectin requires water as substrate and occurs in the intercellular water (de smedt et al., 2002). all these changes influence the organization of water molecules (free water, dimers, trimers, solution shells) in the fruit tissue depending on the strength of hydrogen bonding, and produce changes in the water absorbance pattern. thus, when an aqueous system, such as apple fruit, is measured by near infrared (nir) spectroscopy, the light absorbance reflects the water vibrations and the contribution (concentration and structure) of the other molecules interacting with water. tsenkova (2009) proposed the aquaphotomics approach to relate water absorption patterns to bio-functionalities. in a series of experiments, it was found that vis-nir spectra acquired in living systems under various perturbations (temperature, ion concentrations, oxidative stress, illumination, disease, damage, etc) are characterized by twelve common water bands repeatedly occurring in different combinations in the spectral regression or classification models predicting the investigated perturbations. these water absorption bands (6-20 nm width, each) called water matrix coordinates (wamacs) corresponded to 1344 (c1), 1364 (c2), 1372 (c3), 1382 (c4), 1398 (c5), 1410 (c6), 1438 (c7), 1444 (c8), 1464 (c9), 1474 (c10), 1492 (c11) and 1518 (c12) nm (tsenkova, 2009). the variation of wamacs defines the water spectral pattern (wasp) which can be visualized by star-charts called ‘aquagrams’ (tsenkova, 2010). changes in water absorbance pattern have been used as biomarkers to monitor water quality (kovacs et al., 2016), to reveal the presence of bioactive compounds from propolis in smart packaging materials (barzaghi et al., 2017), to detect honey adulteration with high fructose corn syrup (bázár et al., 2016), to distinguish the effects of various coatings on the quality of different types of cheese and of winter melon (cattaneo et al., 2016). aquaphotomics has also been shown to be a useful tool for discriminating fresh and stored apples and for distinguishing apples stored under different atmospheres. differences between fresh and stored apples involved c5, c7, c9 and c12 water matrix coordinates, while differences between apples stored in normal and in controlled atmosphere activate the c8 and c10 wamacs (barzaghi et al . , 2014). significant relationships were found between vis/nir wavelengths and some sensory attributes of apples, involving water absorption bands. absorption measured at 720, 1440, and 2338 nm was positively correlated and at 1940 nm negatively related to mealiness, while the opposite was found for crunchiness and chewiness. the absorbance in the 630-700 nm range and at 1940 nm was positively correlated with juiciness, while negative correlations were found at 940 and 1450 nm (mehinagic et al., 2004). the aim of this work was to evaluate the feasibility of aquaphotomics to study the texture sensory profiles of apple fruit belonging to three cultivars having different texture characteristics. 2. materials and methods fruit ‘braeburn’ apples (808 fruits) picked in 2014, ‘gala’ apples (270 fruits) picked in 2015 and ‘kanzi®’ apples picked in 2014 (540 fruits) and in 2015 (270 fruits) at the experimental orchard of laimburg (bz) were stored for 4 (‘gala’) or 6 months (‘braeburn’ and ‘kanzi®’) under controlled atmosphere (‘gala’: 1% o2, 1.5% co2; ‘braeburn’: 1.8% o2, 1.3% co2; ‘kanzi®’: 1% o2, 1.5% co2) and analyzed after 1 and 7 days of shelf life at 20°c (‘kanzi®’2014) or after 1, 7 and 14 days at 20°c (‘braeburn’, ‘gala’, and ‘kanzi®’2015). all fruits of the experiment were individually measured by a micronir spectrometer and individually analyzed for texture sensory properties (firmness, crispness, mealiness, juiciness), mechanical characteristics and relative internal space volume (risv). sensory analysis sensory analysis was carried out separately per cultivar according to eccher zerbini et al. (1999) and rizzolo et al. (2010). each sensory session corresponded to a time of analysis, that was 9 sampling times for ‘braeburn’, ‘gala’ and ‘kanzi®’2015 apples and 6 sampling times for ‘kanzi®’2014 apples. as for ‘braeburn’, ‘gala’ and ‘kanzi®’2015 apples, in each session 9 fruits were presented to each panelist, while for ‘kanzi®’2014 apples 3 fruits per panelist were used. when 9 fruits were presented to each panelist, two sensory sessions were scheduled, the first with 5 fruits and the second with 4 fruits, at 10:30 and at 12:00 am, respectively. a total of 810 (‘braeburn’), 540 (‘kanzi®’ 2014) and 270 (‘gala’, ‘kanzi®’ 2015) apples were tested. the sensory tests vanoli et al. water spectral pattern and sensory texture 345 were performed by a panel of ten short-term-trained judges from research centre for engineering and agro-food processing of the council for agricultural research and economics (crea-it, milano), who had participated in prior studies on sensory evaluation of apples, in a sensory lab equipped with six computerized individual booths under white artificial lighting at room temperature (20°c). the fizz-network 2.47b (biosystemes, couternon, france) software was used for test implementation and results collection. fruits (one peeled slice/apple) were presented to the judges at most 1 h after the cut to avoid the browning process, coded with three-digit random numbers and placed on a white flat-bottomed dish in randomized order for each assessor. at the beginning of each session, a peeled slice of a fruit not included in the experimental plan was tasted to eliminate the first tasting effect. drinking water was provided as a palate cleaner between samples. as the quality of consumer perception is influenced by extrinsic attributes (e.g., price, dimension, size, origin, ripening stage), none of these attributes were mentioned to the judges (taiti et al., 2017). each sample was evaluated for the intensity of four sensory attributes related to fruit structure: firm, crispy, mealy and juicy. evaluation was based on a form with a continuous open linear scale, consisting of 40 characters, each panelist rated the intensity of each attribute on the open linear scale anchored to 0 (no presence) and 120 (maximum intensity). definitions of the sensory attributes are as follows: firm, the resistance to mastication perceived at the first and successive bites; crispy, the textural property perceived at the first bite when the fruit yields suddenly with a characteristic sound; mealy, the textural property consisting of the presence of lumps formed during mastication; juicy, the textural property giving the sensation of progressive increase in the free fluids in the oral cavity during mastication (eccher zerbini et al., 1999). prior to statistical analyses, the rating scores of each attribute were standardized by panelist in order to remove the variability due to panelists using different parts of the scale according to bianchi et al. (2009). mechanical properties and relative internal space volume individual fruit were analyzed for flesh firmness, compression and intercellular spaces (risv). flesh firmness and compression were measured on two opposite sides of each fruit (the blush side and the opposite one) in the equatorial region and data were averaged per fruit. firmness was measured with an 11 mm diameter cylindrical plunger mounted on a ta-xt plus texture analyzer (stable micro systems, godalming, uk) at the cross-head speed of 3.33 mm/s to a depth of 8 mm. mechanical properties were also nondestructively assessed by a uniaxial compression test. in this test, each apple was compressed between two steel parallel plates to a fixed deformation of 1 mm at a speed of 25 mm/min on an instron universal testing machine and the modulus of deformability (ed) was computed according to eccher zerbini (1981): ed = f 1-µ2 (d l /2)3/2 d1/2 where f is the force at 1 mm of compression (n), dl is the total deformation (mm), d is the fruit diameter (mm); µ is the poisson’s ratio. as poisson’s ratio was not measured in this work, it was fixed at a value equal to 0.3 (ahmadi et al., 2016). relative internal space volume (risv) was computed according to risv=100×[1−(d f −d j )] where d f is the density of the fruit (i.e. ratio fruit mass to fruit volume under water), and d j is the density of the fruit juice (baumann and henze, 1983). nir analysis nir spectra were acquired on each intact fruit in reflectance mode using a diode array spectrometer (micronir 1700 viavi, diessechem instrument, italy) over the 900 to 1670 nm range (50 scans, 128 reading points) on two opposite sides of each fruit (the blush side and the opposite one) in the equatorial region and data were averaged per fruit. the spectra were pretreated using multiplicative scatter correction (msc) to remove scatter effects, followed by savitsky-golay second derivative (15 points, secondorder polynomial) after converting the spectra from reflectance to absorbance (the unscrambler software, ver. 10.0.1, camo process as, norway). aquagrams were built using the 12 characteristic water absorption wavelengths (1344, 1364, 1372, 1382, 1398, 1410, 1438, 1444, 1464, 1474, 1492 and 1518 nm) within each cluster sensory profile of each cultivar (see sensory analysis paragraph). the values of the aquagrams (aq) were obtained according to: aqλ = aλ µλ σλ where aλ is absorbance, µλ is the mean value of all adv. hort. sci., 2018 32(3): 343-351 346 spectra and σλ is the standard deviation of all spectra at wavelength λ (cattaneo et al., 2016). aquagrams were calculated using ms excel 2010 (microsoft, usa). the aquagram displays normalized absorbance values from different sample groups at several water bands on the axes originating from the center of the graph. absorbance values at the wamacs were placed on the respective radial axes. statistical analysis risv, mechanical and texture sensory data were submitted to analysis of variance (anova) considering apple cultivar as factor and means were compared by bonferroni’s test at p≤0.05. data of texture sensory attributes were also analyzed by using an agglomerative hierarchical clustering of observations. ward’s clustering method and squared euclidean distance were applied to create four data sets having distinctive sensory texture profiles. to form the clusters, the procedure begins with each observation in a separate group and then combines the two observations which are closer together to form a new group. after re-computing the distance between groups, the two groups then closest together are combined. this process is repeated until only the n fixed groups remained. as cultivars had different texture characteristics, cluster analysis on sensory texture attributes was carried out within each cultivar and year. data of firmness, compression and risv were submitted to anova considering sensory cluster as factor and means were compared by bonferroni’s test at p≤0.05. all the statistical analyses were performed using statgraphics version 7 (manugistic inc., rockville, md, usa) software package. 3. results mechanical and sensory analyses showed that the three cultivars had different texture characteristics (tables 1 and 2). ‘braeburn’ apples had very low firmness, intermediate risv and the highest ed, along with the lowest scores for firm, juicy and crispy and the highest scores for mealy. ‘gala’ apples had the highest risv, the lowest ed and firmness, showing intermediate scores for juicy, crispy and mealy . ‘kanzi®’ fruit had the highest firmness, the lowest risv, intermediate ed and were perceived as the most firm, juicy and crispy and the least mealy; kanzi®’2015 showed lower firmness and ed, higher risv and higher scores for mealy than ‘kanzi®’2014. from an exploratory analysis carried out by dividing the standard score of each texture sensory attribute into five arbitrary classes according to: very low (<20), low (21-39), medium (40-59), high (60-80) and very high (>80) intensity of the attribute, and by pairing the classes of all attributes for every fruit, it was found that the minimum number of combinations of intensity classes between attributes was four. so clustering analysis was applied with the aim of creating the four data sets having distinctive texture sensory profiles, according to the descriptions and centroid values reported in table 3. profile w1 corresponded to a very firm, juicy and crispy texture for ‘kanzi®’ apples, and to a very firm/juicy texture with a medium crispness for ‘braeburn’ and ‘gala’. profile w2 of ‘kanzi®’ apples was very similar to profile w1 of ‘braeburn’ and ‘gala’ apples, while that of ‘gala’ and ‘braeburn’ was characterized by a medium juicy and crispy texture, with low scores for mealiness in ‘braeburn’ fruit. profile w3 of ‘kanzi®’ apples showed a soft (2014) or a medium texture (2015) without mealiness, while that of ‘gala’ and ‘braeburn’ was quite firm with low (‘gala’) or medium scores (‘braeburn’) for mealiness. profile w4 was characterized by a soft, dry and mealy texture with high scores for mealiness in ‘braeburn’ and ‘kanzi®’2014. considering the cluster distribution in relation to storage time and shelf life period, profile w1 was found in 65% of fruit at harvest and at storcultivar firmness (n) ed (n/mm2) risv (%) 'braeburn' 56.2 bc 10.5 a 17.6 b 'gala' 55.2 c 6.5 d 19.6 a 'kanzi®'2014 61.5 a 9.8 b 15.0 d 'kanzi®'2015 57.6 b 7.5 c 15.9 c table 1 mechanical properties and intercellular spaces (risv) of 'braeburn', 'gala' and ' kanzi®' apples means in the same column followed by different letters are statistically different at p≤0.05 (bonferroni's test). table 2 mean scores of texture sensory attributes of 'braeburn', 'gala' and 'kanzi®' apples means in the same column followed by different letters are statistically different at p≤0.05 (bonferroni's test). cultivar firm juicy mealy crispy 'braeburn' 47.0 b 40.9 c 46.2 a 33.3 c 'gala' 55.0 a 50.0 b 29.0 b 40.0 b 'kanzi®'2014 57.2 a 62.0 a 18.1 d 53.4 a 'kanzi®'2015 59.5 a 59.4 a 24.5 c 51.5 a vanoli et al. water spectral pattern and sensory texture 347 age removal in ‘braeburn’ and in ‘gala’, and in 84% of ‘kanzi®’ apples; profile w2 of the three cultivars and profile w3 of ‘kanzi®’2014 were equally distributed among the storage times and the days of shelf life; profile w3 was found in 75% of ‘kanzi®’2015, ‘braeburn’ and ‘gala’ apples kept at 20°c; profile w4 was characteristic of fruit held for 14 days at 20°c and was found in 75% of ‘kanzi®’2015, 80% of ‘gala’ and 90% of ‘braeburn’ apples. each cluster showed different mechanical properties and intercellular spaces (fig. 1). firmness significantly decreased from w1 to w2, w3 and w4 profiles in ‘braeburn’ and in ‘kanzi®’ 2014 and 2015 apples, while in ‘gala’ had the same values in w1 and w2 profiles and decreased in w3 and w4, where showed the lowest values. risv significantly increased from w1 to w2, w3 and w4 profiles in braeburn and in ‘kanzi®’ 2015, while in ‘gala’ had the same values in w1 and w2 and increased in w4; in ‘kanzi®’ 2014, risv showed the lowest values in w1 and the highest in w3 and w4. ed distinguished only profile w1 from the other ones, except for ‘kanzi®’2014 (fig. 1). nir spectra of the three cultivars (fig. 2) show high variability in the 1344-1518 nm range corresponding to the first water overtone considered in the aquaphotomics approach. aquagrams showed a different water organization according to the different texture sensory profiles and to the cultivars (fig. 3). ‘braeburn’ apples table 3 texture sensory profiles of the four clusters within each cultivar cluster number and texture sensory profile firm juicy mealy crispy % obs 'braeburn' w1 very firm/juicy, medium crispy, not mealy 66.0 59.8 21.2 52.3 26.7 w2 medium firm/juicy, quite crispy/mealy 52.4 40.7 35.4 37.3 23.9 w3 quite firm/juicy/crispy, mealy 38.5 36.6 55.5 24.1 29.0 w4 very soft, not juicy, not crispy, very mealy 28.0 22.4 78.2 16.9 20.4 'gala' w1 very firm/juicy, medium crispy, not mealy 75.5 67.9 19.2 58.6 31.9 w2 very firm, medium juicy/crispy, not mealy 65.1 46.4 24.5 45.0 20.0 w3 medium firm/juicy, quite crispy/mealy 43.4 44.4 32.5 30.6 30.4 w4 very soft, not juicy, not crispy, mealy 26.7 31.6 45.7 17.1 17.8 'kanzi®'2014 w1 very firm/juicy/crispy, not mealy 75.5 85.3 7.0 82.7 32.3 w2 very firm/juicy, medium crispy, not mealy 61.6 64.5 17.0 54.7 34.8 w3 low firm/juicy/crispy, not mealy 34.1 35.9 16.8 24.1 25.1 w4 very soft, not juicy, not crispy, very mealy 35.5 37.8 73.8 20.8 7.8 'kanzi®'2015 w1 very firm/juicy/crispy, not mealy 80.2 78.5 15.2 74.2 23.3 w2 very firm/juicy, medium crispy, not mealy 67.2 61.7 20.0 55.6 36.7 w3 medium firm/juicy/crispy, not mealy 51.9 48.9 25.7 41.7 20.0 w4 very soft, not juicy, not crispy, mealy 28.8 43.4 42.3 27.5 20.0 for each cluster are reported: the description of the texture sensory profile, the values of centroids for each descriptor and the percentage of observations (% obs) grouped in the cluster. fig. 1 mechanical properties and intercellular spaces of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples according to the texture sensory profiles described in table 3. 348 adv. hort. sci., 2018 32(3): 343-351 showed the greatest changes in the water absorbance pattern (wasp) according to the different texture profiles, while ‘kanzi®’2014 the lowest ones. mealy apples belonging to ‘braeburn’ w3 and w4, ‘gala’ w4 and ‘kanzi®’2015 w4 profiles were associated to very high values in the 1364-1382 nm range and to very low values in the 1410-1518 nm range, while mealy apples belonging to ‘kanzi®’2014 w4 profile showed a balanced distribution of water structures with highest absorbance at 1344 nm and fig. 2 raw nir spectra of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples. fig. 3 aquagrams of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples according to the texture sensory profiles described in table 3. 349 vanoli et al. water spectral pattern and sensory texture lowest absorbance values at 1518 nm. in low mealy apples (‘braeburn’ w2, ‘gala’ w3) the absorbance decreased at 1344-1382 nm while increased at 14101492 nm comparing to mealy apples of the same cultivars. in contrast, the wasp of firm, juicy and crispy sensory profiles (‘kanzi®’2014 and 2015 w1 and w2, ‘gala’ w1) were characterized by a regular distribution of the different water structures with lower absorbance values at 1344-1382 nm and higher absorbance values at 1410-1492 nm comparing to the other textural profiles, while the wasp of ‘braeburn’ w1 showed the lowest values at 1344 and at 1364 nm and the highest in 1398-1438 nm range. 4. discussion and conclusions ‘braeburn’, ‘gala’ and ‘kanzi®’ apples showed different texture characteristics and were clustered according to the texture sensory attributes in four different profiles, ranging from a very firm/crispy/ juicy texture to a mealy or a very mealy one. each texture profile was characterized by specific mechanical and structural properties and showed different water spectral patterns. the differences in the textural profiles could be due to different softening rates occurring in apples. it is well known that fruit softening involves degradation and modifications of the cell wall and middle lamella structures, loss of turgor pressure, starch degradation and modification in the symplast/apoplast relations affecting the textural characteristics of the pulp: when cell-to-cell adhesion is weaker than the individual cell walls, cell separation occurs and the intact cells are responsible for the mealy texture, while when the individual cell walls are weaker than cell-to-cell adhesion, cell wall breakage occurs and the cellular content is released producing a juicy texture (goulao and oliveira, 2008; harker et al., 2002; vanoli et al., 2009). in this work it was found that apples clustered as very firm and firm had the highest firmness and the lowest risv, whereas apples clustered as mealy showed low firmness and high risv indicating a more advanced softening as reported by ting et al. (2013), vanoli et al. (2011) and rizzolo et al. (2016). moreover, mealy texture mainly belongs to apples held at 20°c for 7-14 days, that are fruits in which the softening process has already occurred, while the firm texture was typical of apples just picked or at storage removal, when softening was only at the beginning and apples had a rigid cellular structure with intact and adherent cell walls. the chemical changes occurring with softening also affected the water structures with a similar pattern for the three cultivars and for the two seasons in ‘kanzi®’ apples. the wasp of mealy apples were characterized by a preponderance of water structure with weak-hydrogen bonds (1364-1382 nm), whereas the wasp of crispy and juicy apples indicated the presence of more organized water structures (dimers, trimers) with medium-strong hydrogen bonds (1410-1492 nm). peirs et al. (2005) found that when cell walls deteriorate, water molecules may relocate into the intercellular spaces, and this phenomenon changes the refractive indices at the cell walls. møller et al. (2013) reported that apples treated with 1-mcp clearly differed in water state and dynamics compared to untreated fruit, as water in the cytoplasm and extra-cellular compartments and water in the vacuole were less restricted in treated apples, suggesting that a high firmness is associated with a low amount of vacuole water. the involvement of the first overtone of the oh vibration and pectin metabolism was observed by boeriu et al. (1998) who found that the absorptions in the 14401445 nm range varied with the percentage of the degree of pectin esterification in green beans. similarly, sirisomboon et al. (2007) found a correlation between the absorption in the 1418-1464 nm range and the oxalate soluble pectin fractions in the ais of intact pears and between the absorptions at 1368 and at 1452 nm and the total pectin content in pear juice. barzaghi et al. (2014) observed that in stored apples the organization of water molecules involved more hydrogen-bonded water than in fresh fruit. this is contrary to our results; this difference could be due to the fact that in this work intact fruits were measured while barzaghi et al. (2014) measured apple slices. in conclusion, the aquaphotomics approach could be a useful tool and the water spectral pattern could be a marker for studying the texture sensory profiles in apple fruit as water structures actually change along with texture characteristics whatever the cultivar. furthermore, being aquaphotomics based on nir spectroscopy it could be possible to discriminate apples with different texture sensory properties in a nondestructive way. however, further studies are needed to better understand the relationships between the water spectral pattern and pectin metabolism and the different water structure organizations and the sensory profiles. 350 adv. hort. sci., 2018 32(3): 343-351 acknowledgements the present work was carried out in the framework of the project ‘monitoring key environmental parameters in the alpine environment involving science, technology and application’ (monalisa), funded by the autonomous province of bolzano (italy). references ahmadi a., 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spectroscopy nondestructively reveals structural changes in ‘pink lady®’ apples during storage. procedia food science, 1: 81-89. impaginato 541 adv. hort. sci., 2018 32(4): 541-548 doi: 10.13128/ahs-22211 yield, quality, antioxidants and mineral nutrients of physalis angulata l. and physalis pubescens l. fruits as affected by genotype under organic management n.a. golubkina 1 (*), h.g. kekina 2, m.r. engalichev 1, m.s. antoshkina 1, s.m. nadezhkin 1, g. caruso 3 1 agrochemical research center, federal scientific center of vegetable production, 143080 moscow region, odintsovo district, vniissok, selectsionnaya 14, russia. 2 medical academy of postgraduate education, 123995 moscow, russia. 3 dipartimento di scienze agrarie, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: antioxidants, mineral nutrients, physalis angulata l., physalis pubescens l., production, sugars, taste. abstract: introduction and selection of unconventional plants with high concentration of biologically active compounds is one of the worthy ways for producing functional food, which is beneficial to human health. research was carried out in northern europe (russia) with the purpose to assess yield, quality and biologically active compounds concentration in physalis angulata and physalis pubescens fruits. p. angulata cultivars konditer and konditer 2 gave the highest yield (11.3 and 11.0 t·ha-1 respectively), due to the highest mean fruit weight (80 and 70 g respectively); p. pubescens variety zolotaya rossip had the worst outcome due to the very small berries (3 g), in spite of their highest number per plant (165). ‘zolotaya rossip’ fruits overall attained higher values of quality indicators compared to all p. angulata cultivars. positive correlations were recorded between dry matter and polyphenols as well as between total sugars and polyphenols. physalis fruits showed to be a good source of antioxidants, k, mg and p for human beings. taste index turned out highly reliable in evaluating fruit quality and it was dependant on dry matter, total sugars, polyphenols and ca. 1. introduction the availability of new functional food has been becoming more and more important for human health regulation and protection against the major diseases of the millennium. physalis fruit is mostly an exotic product, imported from tropical and subtropical regions, particularly from central america, where this genus is characterized by the highest biodiversity (medina-medrano et al., 2015). the popularity of this genus plants has been increasing in several world (*) corresponding author: segolubkina45@gmail.com citation: golubkina n.a., kekina h.g., engalichev m.r., antoshkina m.s., nadezhkin s.m., caruso g., 2018 yield, quality, antioxidants and minearl nutrients of physalis angulata l. and physalis pubescens l. fruits as affected by genotype under organic management. adv. hort. sci., 32(4): 541-548 copyright: © 2018 golubkina n.a., kekina h.g., engalichev m.r., antoshkina m.s., nadezhkin s.m., caruso g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2017 accepted for publication 12 july 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 541-548 542 countries, due not only to its high nutritional value and exclusive taste (puente et al., 2011), but also to its effectiveness as a medical plant. indeed, all plant parts are useful in modern medicine for their properties: antiasthma, antihepatitis, antimalaria and antidermatitis (chang et al., 2008), antiallergenic, anticarcinogenic, anti-inflammatory, antihyperglycemic, antimicrobial, antiseptic, antiviral, cardio-protective, diuretic, expectorant, febrifuge (bastos et al., 2006; sharma et al., 2015). in this respect, a negative correlation has been shown between the level of physalis consumption and the risk of cardiovascular, pulmonary and gastrointestinal diseases (leenders et al., 2014; hjartaker et al., 2015). moreover, the high antioxidant activity of water and alcoholic extracts of physalis is reportedly beneficial to patients suffering from alzheimer’s disease as well as from memory and concentration deficit (susanti et al., 2015). among the tens of physalis species, p. angulata and p. pubescens are the most suitable for production in the northern europe, due to their frost resistance and tolerance to fungal and bacterial diseases (mamedov et al., 2017). notably, the genetic selection carried out in russia allowed to create interesting varieties, suitable for cultivation both in central russia (kondratieva and engalichev, 2013; mamedov et al., 2017) and in siberia (makarov, 2002), mainly belonging to p. angulata. however, a few results concerning the variability of interspecies quality characteristics and even no cultivar comparisons of biologically active compounds accumulation in physalis fruits have been published so far. taking into account the scarcity of literature reports, the present research was aimed at evaluating the varietal differences in yield, quality and antioxidant content of p. angulata and p. pubescens fruits grown in northern europe. 2. materials and methods growth conditions six p. angulata l. cultivars (violet, lakomka, konditer, konditer 2, lezhky, korolek) and one p. pubescens l. cultivar (zolotaya rossip) were compared in a research carried out, under the organic farming management, in the experimental fields of the federal scientific center of vegetable production (moscow region, 55° 39’ 23’’ n, 37° 12’ 43’’ e) in 2015 and 2016. the trial was conducted on a clay-loam soil, with рн 6.8, 2.1% organic matter, 108 mg·kg-1 n, 450 mg·kg-1 p2o5, 357 mg·kg-1 k2o, exchangeable bases sum as much as 95.2%. the air temperature values, recorded at plant level, were: 14.2°c and 13.5°c in may, 16.9°c and 17.1°c in june, 21.2°c and 20.4°c in july, 19.3°c and 18.0°c in august, 12.1°c and 12.2°c in september, in 2015 and 2016 respectively. a randomized complete blocks design, with three replicates, was used for treatments distribution in the field; each treatment had a 19.25 m2 (5.5 × 3.5 m) surface area, including 50 plants. physalis seeds were sown in peat boxes on 14 april and the seedlings were transplanted in the field on 23 may, spaced 55 cm along the rows, the latter being 70 cm apart. as for p. angulata, the six cultivars chosen for the trial are the most spread in russia, whereas within p. pubescens only the cultivar zolotaya rossip tested in our research is cultivated in this country. the organic farming practice complied with ec regulation 834/2007. physalis crops were preceded by pea and each year the fertilization supplied the crops with 56 kg ha-1 of n, 16 of p2o5 and 98 of k2o. half of the fertilizers dose was given just before transplanting and the remaining 50% on dressing at two week intervals. drip irrigation was practiced for watering the crops when needed. plant protection from fungal diseases and insects was achieved by adopting trichoderma suspensions, copper, sulphur, azadirachtin. harvests were carried out from mid-august to the end of september. general analytical methods ripe, undamaged and regularly shaped fruits were classified as “marketable”. at each harvest, the weight and number of marketable fruits in each plot was recorded and the mean weight was assessed on random samples of 50 fruits per plot. cumulative plant biomass was calculated as the sum of the above-ground plant biomass at the end of the experiment plus the total fruit production from the beginning of the harvest period. dry weight was assessed after dehydration of the fresh samples in an oven at 70°c until they reached constant weight. in each plot, a sample of twenty-five fruits was collected and transferred to the laboratory for analysis. samples preparation prior to analyses, physalis fruits were extracted from papery husk and homogenized with a stainless steel blender for 1 min. the resulting homogenates golubkina et al. physalis spp. under organic management 543 were immediately subjected to the analysis. total soluble solids (tss) and sugars determination of total soluble solids was carried out by a refractometer (irf-22, russia). the results were reported as °brix at 20°c. monoand disaccharides were determined using cyanide method (kidin, 2008). titratable acidity (ta) ta was measured using 20 ml of the water physalis extract (1:1), titrated to ph 8.1 using 0.1 n naoh (gost, 1996). the following formula was used for calculation: ta= v 1 x 0.1x0.064x100:20 where v-1 is the volume of naoh used; 0.1 is the naoh normality; 0.064 is the weight of a citric acid milliequivalent in g; 20 is the volume of the physalis extract used. mineral nutrients k, mg, mn, ca, na and p contents in dried homogenized fruit samples were assessed using icp-ms on quadruple mass-spectrometer nexon 300d (golubkina et al., 2017). nitrate content in fresh fruits of physalis species was determined using ionselective electrode on ionomer expert-001 (russia), as previously described (golubkina et al., 2017). antioxidants total polyphenols were assessed using folinciocalteu colorimetric method (sagdic et al., 2011). the ascorbic acid content was determined by visual titration of fruit extracts in 6% trichloracetic acid with tillmans reagent (aoac, 2012). taste the assessment of physalis taste was performed using two methods: chemical and organoleptic. taste index was calculated starting from the brix degree and acidity values, using the equation proposed by navez et al. (1999) and nielsen (2003) for tomato fruits: ti = (brix/20 ta) + ta where ti is the taste index and ta is the titratable acidity calculated referring to citric acid. though organoleptic perception of taste depends on the cultural background of judges and cannot be considered universally objective, physalis fruit taste was evaluated additionally by 10 experts, via sensory analysis using 5 balls scales (krueger and casey, 2000). statistical analysis data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 21. data expressed as percentage were subjected to angular transformation before processing. notably, the factor “year of research” had no significant effects on the variables examined, both in terms of main effects and of interactions with the other experimental factor. therefore, we have reported the results obtained from the data statistical processing as means of the two years of research. 3. results and discussion plant growth and yield as it can be seen in table 1, a correspondence between plant height and dry matter was recorded in p. pubescens variety zolotaya rossip, which had the smallest plants and the lowest dry matter, whereas within p. angulata the cultivar korolek showed the table 1 biometrical, growth and yield parameters of p. angulata and p. pubescens cultivars within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar plant height (cm) plant dry weight (g·m-2) planting to harvest beginning (days) fruits per plant (no.) yield (t·ha-1) mean fruit weight (g) violet (p. angulata) 90 d 296.6 c 105 bc 58 cd 9.0 b 60 c korolek (p. angulata) 130 a 320.3 c 108 b 64 b 9.3 b 56 cd lakomka (p. angulata) 105 c 200.7 d 90 d 47 f 7.3 c 60 c konditer (p. angulata) 125 ab 402.5 a 115 a 62 bc 11.3 a 70 b konditer 2 (p. angulata) 120 b 368.3 b 102 c 53 e 11.0 a 80 a lezhky (p. angulata) 110 c 223.6 d 118 a 55 de 7.7 c 54 d zolotaya rossip (p. pubescens) 80 e 133.3 e 85 e 165 a 1.3 d 3 e adv. hort. sci., 2018 32(4): 541-548 544 tallest plants but ‘konditer’ and ‘konditer 2’ produced the highest dry matter amounts. the latter cultivar was characterized by the longest crop cycle, similarly to lezhky, and p. pubescens zolotaya rossip resulted in the earliest fruit ripening; among p. angulatum varieties, only ‘lakomka’ showed early ripeness comparable to ‘zolotaya rossip’ one. correspondently to plant dry matter production, p. angulata cultivars konditer and konditer 2 also gave the highest yield (11.3 and 11.0 t·ha-1 respectively), due to the highest mean berry weight (80 and 70 g respectively), whereas p. pubescens variety zolotaya rossip had the worst outcome, in spite of the huge number of fruits per plant which were, however, very small (3 g). among p. angulatum varieties, lakomka and lezhky showed the lowest values (7.3 and 7.7 t·ha-1 respectively), due to the lowest prolificity and the smallest fruits respectively. quality indicators and mineral nutrient content as reported in table 2, the fruits of p. pubescens cultivar zolotaya rossip overall attained higher values of quality indicators than the six p. angulata varieties examined; compared to the average of the latter six cultivars, p. pubescens fruits showed 1.6 and 1.95 times higher content of total sugars and monosaccharides respectively as well as 1.27 times higher titratable acidity. among p. angulata cultivars, in ‘violet’ fruits the highest levels of dry residue, soluble solids and total sugars were recorded and in ‘lezhky’ the lowest. otherwise, ‘lakomka’ showed the highest monosaccharide content (32.2% out of total sugars) and cultivar violet the lowest (18.8%); the variation coefficient relevant to monosaccharide content in p. angulata species attained 18.3%. as for quality indicators, the physalis cultivars tested showed high variability in the content of dry matter (15.0%) and sugars (28.2%) as well as in juice acidity (16.7%), whereas the juice ph variability was low (2.1%). in our research, dry matter and sugar content in p. pubescens are similar to those detected in physalis berries grown in tropical and subtropical countries (yildiz et al., 2015). notably, soluble sugars highly affect flavour quality of tomato fruits (doras et al., 2001) and, according to olivares-tenorio et al. (2016) reports, the main carbohydrates of physalis fruits are sucrose and glucose, whereas fructose content is neglectable. the analysis of mineral nutrient content performed in our research showed the close element concentrations of p. pubescens and p. angulata fruits grown in the same environmental conditions (table 3). as for varietal differences in mineral nutrient table 2 quality indicators of physalis angulata and p. pubescens cultivars fruits table 3 mineral nutrient concentrations in physalis angulata and p. pubescens cultivars fruits (mg kg-1 d.w.) within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar dry matter (%) soluble solids (°brix) total sugars (%) reducing sugars (%) titratable acidity (%) рн violet 10.5 b 8.1 b 7.8 b 1.8 d 0.79 c 4.82 ab korolek 9.3 d 5.9 c 5.6 d 1.8 d 0.70 d 4.65 bc lakomka 8.7 e 6.1 c 6.0 c 2.9 b 0.45 f 4.94 a konditer 10.0 bc 6.0 c 5.9 cd 1.6 d 0.88 b 4.62 bc konditer 2 9.7 cd 6.1 d 5.9 c 2.2 c 0.79 c 4.51 c lezhky 8.5 e 4.9 d 4.7 e 1.7 d 0.63 e 4.70 ac zolotaya rossip 15.5 a 9.7 a 9.6 a 3.9 a 0.90 a 4.72 ac mean 9.5 6.2 6.0 2.0 0.71 4.71 within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar ca k mg na p no 3 violet 415 b 2465 ab 1449 c 51 b 3483 c 2390 a korolek 722 a 2013 d 1740 ab 48 b 3825 b 2108 b lakomka 763 a 1284 f 1845 a 53 b 4345 a 2365 a konditer 720 a 2300 bc 1809 a 34 b 4277 a 1990 bc konditer 2 739 a 1723 e 1614 b 6 c 3503 c 1876 c lezhky 797 a 2152 cd 1286 d 61 b 3509 c 2287 a zolotaya rossip 414 b 2561 a 1708 ab 293 a 3572 c 1181 d golubkina et al. physalis spp. under organic management 545 accumulation, in p. angulata fruits the variability was high in na concentration (33%) and low in mg, p and nitrates (11.6, 8.5 and 6.9%). recently, the element composition of vegetable edible parts has been drawing attention as one of the most important factors affecting human being ingestion of mineral nutrients. unfortunately, the few literature reports available do not give the opportunity to perform correct interspecific or varietal comparisons, due to research carried out under different environmental conditions and species (el-sheikha et al., 2010; eken et al., 2014). referring to recommended dietary allowance values (institute of medicine, 2001), the consumption of 300 g fresh physalis fruits per day results in the ingestion of 24.8% potassium, 14.4% phosphorous and 12.3% magnesium needed by human organism. the latter benefits from these mineral nutrients in terms of optimization of carbohydrates, protein and lipid metabolism, bone integrity and brain activity, protection against cancer as well as cardiovascular diseases, obesity and diabetes. in this respect, p. angulata and p. pubescens fruits as well as the p. peruviana ones (zhang et al., 2013) may be considered as good sources of several elements. antioxidants polyphenols. in the present research, p. pubescens and p. angulata cultivars growing in the same geochemical environment (central russia) resulted in fruit polyphenols accumulation ranging between 18.7 and 25.1 mg gae/g d.w. (fig. 1). notably, the top concentration detected in the cultivar violet was 34.2% higher than the lowest level recorded in ‘korolek’, both belonging to p. angulata species; p. pubescens cultivar zolotaya rossip ranked third (20.5 mg gae/g d.w.). compared to our findings, in previous research (medina-medrano et al., 2015) higher phenolic values were detected in the fruits of five physalis wild species grown in mexico (32 to 86 mg age/g d.w.), with the lowest concentrations recorded in p. angulata berries. moreover, p. peruviana fruits produced in colombia accumulated 400 to 600 mg gae/g f.w. of polyphenols (narvaez-cuenca et al., 2014), but unfortunately this species is not suitable for cultivation in northern europe (kondratieva and engalichev, 2013). indeed, among natural secondary plant metabolites, polyphenols are considered to be the strongest antioxidants, which are able to inhibit carcinogenesis at initial and development stages, thus suggesting the great importance of their accumulation in agricultural plants (yang et al., 2001). the correlations between polyphenols and total sugars concentrations and between polyphenols and dry matter content were positive and highly significant (r= 0.99 and r= 0.91, respectively, at p≤0.001). the first one is supposed to reflect the existance of phenolic glycosides, identified as the main phenolics in wild physalis species (medina-medrano et al., 2015). the second correlation explains the higher dry matter and phenolics contents detected in physalis fruits produced in southern countries compared to those obtained in central russia. the high statistical significance of the correlations between the above parameters gives the opportunity to highlight the quality performances of cultivar violet, which showed the highest concentrations of polyphenols as well as the highest carbohydrates and dry matter content among p. angulata varieties and p. pubescens cultivar zolotaya rossip, with the highest content of dry matter and carbohydrates. these parameters may be used in physalis breeding for increasing fruit nutritional quality. ascorbic acid. in our research, vitamin c concentration in p. angulata cultivars ranged from 0.96 mg/g d.w. (cultivar violet) to 1.33 mg/g d.w. (cultivar korolek); however, the lowest value was recorded in the berries of p. pubescens cultivar zolotaya rossip (fig. 2). these results show that the synthesis of this antioxidant in russia is much lower than that reported for p. peruviana and p. pubescens in tropical and subtropical areas: i.e. 10 to 30 vs 20 to 50 mg/100 g f.w. (el sheikha et al., 2008, 2010; olivares-tenorio et al., 2016). indeed, the higher light intensity occurring at lower latitudes enhances ascorbic acid accumulation (bartoli et al., 2006), whereas in previous investifig. 1 polyphenol concentration as a function of physalis cultivar. means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. 546 adv. hort. sci., 2018 32(4): 541-548 gations carried out in colombia, kenia and southern africa (fischer et al., 2000), no relationship of this antioxidant with the altitude was found. moreover, the low vitamin c variability obtained in the present intestigation prove the predominance of environmental effects on the genetic one in affecting this antioxidant accumulation in physalis fruits (fig. 1). notably, the outer husk of physalis fruits is known to prevent ascorbic acid oxidation (valdenegro et al., 2012) and, despite the relatively low vitamin c content, 100 g of fresh physalis fruits produced in the northern hemisphere can supply human organism with 14 to 21% of the required vitamin c consumption (70 mg per day). taste among p. angulata and pubescens cultivars examined, the highest taste index (ti) and nutritional values were recorded in ‘zolotaya rossip’ (p. pubescens) and ‘violet’ (p. angulata). as far as taste assessment is concerned, it is a critical point in nonconventional plants produce and, indeed, this determination has never been performed on physalis fruits up to date. a general approach based on tomato berry testing suggests several significant factors affecting taste: dry matter, soluble solids, juice electrical conductivity, carbohydrates and organic acids contents (adams and ho, 1989; clement et al., 2008). in our research, we have assessed that the taste index (ti) used for tomato fruits can be successfully used for physalis berries. indeed, the organoleptic analysis and ti approach in evaluating physalis fruit quality show a good convergence of the results and suggest significant prospects of ti utilization in determining new varieties quality (fig. 3). in this respect, several correlations arose between the taste index and the following quality parameters of physalis fruits: total sugar content (r=+0.98; p<0.001), dry matter (r = +0.92; p<0.001), polyphenol content (r= +0.96; p<0.001); calcium (r= -0.91; p<0.001). notably, the two latter correlations had never been reported previously, neither in physalis nor in tomato. 4. conclusions the present research, carried out in central russia, allowed to assess interspecies and varietal differences in yield and quality characteristics of p. angulata and p. pubescens fruits grown under organic management. in this respect, this investigation provided with interesting clues, mainly concerning the nutritional and antioxidant properties of the cultivars tested and their growing prospects by organic farming procedures in northern europe. the variability of biologically active compounds, macroelement content and taste index, as well as their significant correlations may serve as the basis for enhancing the high potential of the physalis varieties examined for functional food production. acknowledgements we would like to thank dr. m. mamedov for his effective help with the research team arrangement. fig. 2 ascorbic acid concentration as a function of physalis cultivar. means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. fig. 3 correlation between taste index (ti) and organoleptic evaluation of physalis taste. golubkina et al. physalis spp. under organic management 547 references adams p., ho l.c., 1989 effects of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. j. hort. sci., 64(6): 725-732. aoac, 2012 the official methods of analysis of aoac 22 vitamin c. association official analytical chemists gaithersburg, md, usa. bartoli c.g., yu j., gomez f., fernandez l., mcintosh l., foyer c.h., 2006 inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in arabidopsis thaliana leaves. j. exp. bot., 57(8): 1621-1631. bastos g.n., santos a.r., ferreira v.m., costa a.m., bispo c.i., silveira a.j., do nascimento j.l., 2006 antinociceptive effect of the aqueous extract obtained from roots of physalis angulata l. on mice. j. ethnopharmacol., 103: 241-245. chang j.c., lin c.c., wu s.j., lin d.l., wang w.w., miaw c.l., ng lt., 2008 antioxidative and hepatoprotective effects of physalis peruviana extract against acetaminophen-induced liver injury in rats. pharm. biol., 46: 724-731. clement a., dorais m., vernon m., 2008 multivariate approach to the measurement of tomato maturity and gustatory attributes and their rapid assessment by visnir spectroscopy. j. agr. food chem., 56: 1538-1544. doras m., papadopoulos a.p., gosselin a., 2001 greenhouse tomato fruit quality. hort. rev., 26: 239319. eken a.e., unlu-enderlik b., baldemir a., ilgen s., soykut b., erdern o., kayseri c., 2014 antioxidant capacity of physalis peruviana l. fruit sold in markets. j. clin. anal. methods., 104328/gcam 2709. el sheikha a.f., piombo g., goli t., montet d., 2010 main composition of physalis (physalis pubescens l.) fruit juice from egypt. fruits, 65: 255-265. el sheikha a.f., zaki m., bakr a., el-habashi m., montet d., 2008 physico-chemical properties and biochemical composition of physalis (physalis pubescens) fruits. food global science books, 2(2): 124-130. fischer g., ebert g., ludders p., 2000 provitamin a, carotenoids, organic acids and ascorbic acid content of cape gooseberry (physalis peruviana l.) ecotypes grown at two tropical altitudes. acta horticulturae, 531: 263268. golubkina n.a., kosheleva o.v., krivenkov l.v., dobrutskaya h.g., nadezhkin s., caruso g., 2017 intersexual differences in plant growth, yield, mineral composition and antioxidants of spinach (spinacia oleracea l.) as affected by selenium form. sci. hortic., 225: 350-358. gost р., 1996 fruit and vegetable juices. method for determination of titratable acidity. european standard ен 12147. 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0.5; 2.5; and 5.0 mm as nasio3. after seven days of exposure to treatments, leaf area, leaf number, shoot length, and fresh and dry weight of roots, stem and leaves were determined. the application of 0.5 mm si promoted an increase in growth parameters of plants used in this work, mainly in leaf area, leaf number, and leaf and stem dry weight. however, the application of higher concentrations of si (2.5 mm) promoted reduction in the growth parameters, mainly in leaf area. it was also possible to observe a genotypic variation with respect to si, smij319-7 and smif212-3 genotypes being the most responsive to si. therefore, the concentration of 0.5 mm si is considered optimal for potentiating the growth of potato plants, and smij319-7 and smif212-3 genotypes are the most responsive to si. 1. introduction potato (solanum tuberosum l.) is one of the most produced food crops in the world, after rice, wheat and maize (faostat, 2014), and because of its popularity it is known as “the king of vegetables” (roy et al., 2017). the current potato production in brazil exceeds three million tons per year, with a planted area of more than 130 thousand hectares (faostat, 2014). many variables affect the performance of potato plants. among the ones manageable by man, nutritional management is one of the most important (westermann and davis, 1992). in southern brazil, potato is cultivated predominantly in acid soils, poor in calcium, magnesium and with high aluminum (al) and manganese contents. accordingly, there is evidence that silicon (si) interacts with aqueous al, thereby reducing its bioavailability (and thus toxicity) and at the same time increasing the (*) corresponding author: athos_odin@hotmail.com citation: dorneles a.o.s., pereira a.s., possebom g., sasso v.m., rossato l.v., tabaldi l.a., 2018 growth of potato genotypes under different silicon concentrations. adv. hort. sci., 32(2): 289295 copyright: © 2018 dorneles a.o.s., pereira a.s., possebom g., sasso v.m., rossato l.v., tabaldi l.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(2): 289-295 290 bioavailability of the essential element phosphorus (dietzel, 2002; farooq and dietz, 2015). after oxygen, si is the second most abundant element in lithosphere and soil, representing about 28% of earth’s crust (gunes et al., 2007). silicon is considered an essential element only for some species belonging to poaceae and cyperaceae families (liang et al., 2005), but the beneficial effects of this element in growth, development, yield and resistance to disease have been observed in a wide variety of plant species (ma, 2004). yet it is not possible to demonstrate its essentiality to all higher plants due to the fact that there is no direct evidence that it participates of a molecule or of a constituent or metabolite essential for plants (epstein, 1999). the beneficial effects of si to plants are most evident under conditions of stress (ma and yamaji, 2008). published data suggest that si increases tolerance to drought (gong et al., 2005), toxic metals (gu et al., 2011), uv-b radiation (li et al., 2004), salt stress (liang et al., 2003) and resistance of plants to pests and pathogens (gao et al., 2011). silicon also alleviates mineral stress, such as manganese (mn) and aluminum (al) toxicity, and p deficiency (iwasaki et al., 2002). a considerable amount of si is found in various food products, like grains/cereals and their products (e.g. breakfast cereals, bread, beer), fruit and certain vegetables (bananas, currants, beans, lentils), and all natural waters (bissé et al., 2005). according to nielsen (2009), a high consumption of si may be beneficial, facilitating the absorption or the use of some minerals, including magnesium and copper, which are essential for growth and bone maintenance, cardiovascular health and wound healing. there is no current information available on beneficial effects of si on the growth of potato genotypes. taking into account that about 40% of the world’s arable soils have acid ph and toxic concentrations of al (ramanujan, 2014), including the soils in rio grande do sul, and that potatoes are grown in a large area in this state, it is important to analyze the effect of si on potato plant growth. the objective of this study was to analyze the effect of different si concentrations on the growth parameters of potato genotypes, seeking to select potato genotypes more responsive to si and the optimal si concentration. 2. materials and methods the tests were carried out at the laboratory of plant biotechnology and in greenhouses belonging to the department of biology at the federal university of santa maria. four potato (solanum tuberosum l.) genotypes were used: smij319-7, dakota rose, smif212-3 and sminia793101. microtubers of four potato genotypes obtained from the genetics program and improvement of potato, ufsm, santa maria, rs, were propagated in plastic cups of 300 ml, using sand as substrate, and irrigated daily with nutrient solution. after about three weeks, 40 uniform plants of each genotype (shoot length of 5 cm) were chosen, the roots were washed in running water to remove the substrate and plants were transferred to hydroponic system consisting of a vessel with capacity of one liter. the plants were exposed to complete nutrient solution for three days for acclimatization. the nutrient solution was as follows (in µm): 6090.5 n; 974.3 mg; 4986.76 cl; 2679.2 k; 2436.2 ca; 359.9 s; 243.592 p; 0.47 cu; 2.00 mn; zn 1.99; ni 0.17; b 24.97; 0.52 mo; 47.99 fe (feso4/na edta). after this acclimatization period, the plants were cultured for seven days in a new nutrient solution (ph 4.5±0.1) exposed to four different concentrations of silicon (si): 0; 0.5; 2.5; and 5.0 mm, as nasio3. this ph was used to ensure greater availability of si, and to prevent its interaction with cations in the solution. the treatments were arranged in a randomized design, with 10 replicates per treatment and one plant for repetition. the nutrient solution with si treatments was replaced every 48 hours and ph was adjusted daily. seven days after the start of exposure to treatments, the plants were collected to determine leaf area (with leaf area integrator am 300, adc bioscientific ltd.), leaf number, shoot lenght (with scale graduated in millimeters) and dry weight of leaves, stem and roots. for the statistical analysis, the data was checked for normal distribution of errors by the andersondarling test and homogeneity of variances by the bartlett test for all experiment variables. when these assumptions were met, it was proceeded to variance analysis and scott-knott test for treatments at 5% error probability, using sisvar application (ferreira, 2008). 3. results and discussion for smij319-7 and sminia793101 genotypes, the concentration of 0.5 mm silicon (si) promoted an dorneles et al. silicon in hydroponic potato cultivation 291 increase in leaf area compared with control (without si) (fig. 1a). this result is consistent with studies of other species showing the si ability, when applied at low levels (barcelo et al., 1993), to stimulate growth and development of plants, particularly under abiotic and biotic stress (epstein, 1999; ma, 2004; liang et al., 2007; dorneles et al., 2017). the increase in leaf area promoted by si can result in greater interception of solar radiation, and consequently greater accumulation of biomass in these plants. for smif212-3 and dakota rose genotypes, there were different responses in leaf area among treatments (fig. 1a), indicating that there is a genotypic variation in relation to si effects on potato plants. the concentrations of 2.5 and 5.0 mm si promoted a reduction in leaf area in dakota rose genotype compared with control, suggesting that in this genotype, this parameter is more sensitive to high si concentrations. besides, the concentration of 2.5 mm of si also promoted leaf area reduction for smij319-7 and sminia793101 genotypes. silicon is able to interact with essential ions to plants, such as mg, zn and fe (liang et al., 2005; da cunha et al., 2008; naeem et al., 2014), thus the excess of si may lead to the immobilization of these elements in the apoplast of these plants. for the smij319-7, smif212-3 and sminia793101 genotypes, 0.5 mm si promoted an increase in the leaf number compared to the control (fig. 1b), while for the dakota rose genotype there was no significant difference among treatments. the literature reports that si plays a favorable role in growth by improving the acquisition of mineral nutrients (lee et al., 2010), possibly due to a kinetic relationship with some nutrient absorption, thereby causing an increase in biomass production. thus, due to the ph level used in this work, the effect of si should take place in the plant, which may lead to a better distribution of nutrients throughout the plant. the presence of si in the growth medium significantly influenced on shoot length of potato genotypes (fig. 1c), where there was significant difference between si concentrations and control (without si). for the dakota rose genotype, in all si concentrations, it was observed decreased shoot length compared to control (fig. 1c). besides, for the sminia793101 genotype, there was a reduction in plant height at 2.5 mm si. on the other hand, si concentration of 0.5 mm promoted increase in plant height for smij319-7 and smif212-3 genotypes, thus this concentration can be optimal for the growth of potato plants. these data suggest that si has significant and beneficial effect on this parameter for all genotypes, except for dakota rose, which did not obtain a positive response to si application. fig. 1 leaf area (a), leaf number (b) and shoot length (c) of potato genotypes exposed to different silicon concentrations in growth medium. different lowercase letters indicate significant differences between treatments in the same genotype. different capital letters indicate significant differences between genotypes in the same treatment. adv. hort. sci., 2018 32(2): 289-295 292 silicon at 0.5 and 5.0 mm caused a significant increase in yield of leaf dry weight only for smij319-7 and smif212-3 genotypes compared to the control (without si) (fig. 2a). in smij319-7 plants, increased biomass accumulation induced by si coincided with increase in leaf area, which may have promoted a greater area of interception of sunlight. for smif2123 genotypes, the increase of leaf dry weight may be explained by si accumulation in the leaves, because this genotype did not present increase in leaf area. the increase in biomass of potato plants observed in our study might be due to a higher rate of photosynthesis, chlorophyll content and increased activity of enzyme ribulose 1,5-bisphosphate carboxylase caused by si (lee et al., 2010). on the other hand, 2.5 mm si promoted a reduction on leaf dry weight for sminia793101 and dakota rose genotypes. this reduction in leaf dry weight accumulation caused by the application of 2.5 mm si in the genotypes sminia793101 and dakota rose may be due to this concentration not being sufficient for the activation of possible si transporters, which may be more active in higher concentrations. some non-accumulating si species like cucumbita moschata duch. and solanum tuberosum present differences in the expression of si transporters with increased application of this element (mitani et al., 2011 a, b; vulavala et al., 2016). for the smij319-7 genotype, si concentrations (0.5 and 5.0 mm) promoted an increase in the production of stem dry weight compared to control (fig. 2b). the concentration of 0.5 mm si also promoted an increase in stem dry weight production for sminia793101 genotype. silicon deposited in tissues can improve light trapping features for maintaining more upright the leaf blade (epstein, 1999), resulting in increase of plant biomass. this deposition of si in tissues may have contributed to the increase of biomass due to possible formation of a barrier to transpiracional flow that could lead to higher efficiency use of water (lux et al., 2002; shi et al., 2005). on other hand, for the smif212-3 and dakota rose genotypes, there was a negative effect of si on the production of stem dry weight. for the root dry weight, there was significant difference between the si concentrations in all genotypes, where the presence of 0.5 mm si promoted an increase in root dry weight compared to control (fig. 2c). on the other hand, higher concentrations of si (2.5 and 5.0 mm) generally promoted a reduction in root dry weight compared to control. this indicates that lower si concentration (0.5 mm) is beneficial for this parameter, but higher concentrations may be detrimental to root dry weight. in addition, this accumulation of root dry weight is in agreement with low stem dry weight for the dakota rose genotype, indifig. 2 leaf dry weight (a), stem dry weight (b) and root dry weight (c) of potato genotypes exposed to different silicon concentrations in growth medium. different lowercase letters indicate significant differences between treatments in the same genotype. different capital letters indicate significant differences between genotypes in the same treatment. dorneles et al. silicon in hydroponic potato cultivation 293 cating a si effect on the mobilization of resources and their accumulation in roots. the behavior pattern of the genotypes in relation to si was similar. however, the dakota rose genotype has a higher root dry weight accumulation when compared to the other genotypes. nevertheless, it is possible to observe that the effect of si concentrations on root dry weight accumulation for the dakota rose and sminia793101 genotypes follow a similar behavior pattern. the same may be observed when comparing the behavior pattern of the smij319-7 and smif212-3 genotypes, which are also similar. these data show that genotypes smij319-7 and smif212-3 are more responsive to si compared to genotypes dakota rose and sminia793101. these behavior patterns may be observed on all parameters of this work, and may be explained by differences on expression of putative si-transporter, which may differ among genotypes and si concentrations (mitani et al., 2011 a, b; vulavala et al., 2016). at high concentrations (2.5 and 5.0 mm), silicon led to reduction of some growth parameters evaluated in the genotypes used in this study. this response may be due to si effects on immobilizing and complex cations in plants apoplast (shi et al., 2005; moussa, 2006; farooq and dietz, 2015). some of these cations may be nutrients such as zinc, magnesium and manganese, thereby the reduced absorption by the plant may lead to nutritional stress in the long term. in addition, the silicon deposited in leaves may reduce the transpiration avoiding water loss by plants (hodson et al., 2005; farooq and dietz, 2015). however, the excess of silicon on leaf surface may possibly reduce transpiration so that it reduces photosynthetic processes that depend on gas exchange and constant water flow. most research currently focus on si potential in abiotic stresses mitigation (dorneles et al., 2016; pavlovic et al., 2016; ashfaque et al., 2017). however, studies demonstrating si effects at high concentrations are scarce and show that high concentrations of this element may reduce the productive and qualitative potential of plants (marodin et al., 2016; de souza ferraz et al., 2017). in addition, some plant species are specialized in absorbing, transporting and accumulating si in their tissues due to the presence of specific transporters (farooq et al., 2015; sanglard et al., 2016). such carriers may also be present in other species (mitani et al., 2011 b). vulavala et al. (2016) demonstrated that the genotype ‘winston’ of solanum tuberosum has mechanisms of transport responsive to si. it is possible that the differences in the responses of the genotypes of this study are due to differences in the expression of these transporters. this reinforces the importance of the present study, which provides data for future recommendations for si application in potato plants. 4. conclusions the concentration of 0.5 mm of silicon may be considered optimal, since it induced an increase in growth parameters in potato plants, especially in leaf area, leaf number and biomass of leaves and stems. furthermore, the most responsive genotypes to si were smij319-7 and smif212-3, possibly due to molecular characteristics such as presence of si transporters. however, high si concentrations may lead to unfavorable responses in some potato genotypes. references ashfaque f., inam a., iqbal s., sahay s., 2017 response of silicon on metal accumulation, photosynthetic inhibition and 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h., zhang f., 2005 effect of si on the distribution of cd in rice seedlings. plant and soil, 272(1): 53-60. vulavala v.k., elbaum r., yermiyahu u., fogelman e., kumar a., ginzberg i., 2016 silicon fertilization of potato: expression of putative transporters and tuber skin quality. planta, 243(1), 217-229. westermann d.t., davis j.r., 1992 potato nutritional management changes and challenges into the next century. american potato journal, 69:753-767. impaginato 33 adv. hort. sci., 2019 33(1): 33-38 doi: 10.13128/ahs-22953 combination of alginate based edible coating-betel essential oil in extending the shelf life of rose apple cv. dalhari (syzygium samarangense) c.k. setiawan (*), n.a. utama, b.a. pradana agrotechnology department, faculty of agriculture, universitas muhammadiyah yogyakarta, indonesia. key words: alginate, betel essential oil, edible coating, rose apple cv. dalhari. abstract: rose apple cv. dalhari is a local fruit grown in berbah district, special region of yogyakarta. this fruit perishable, easily loses its water content and is attacked by microbes. this research aimed to determine the best combination between alginate and betel essential oil to inhibit the growth of microbes and maintain quality of rose apple cv. dalhari. alginate and betel essential oil treatments were used at three concentration levels, alone and in combinations, respectively of 2%, 2.5%, 3% and 0%, 0.1%, 0.2%. the results showed that combination treatment of 2.5% alginate and 0.1% betel essential oil was the most effective to maintain the fruit quality. the sole addition of betel essential oil was not able to inhibit microbial growth. furthermore, combination of alginate based edible coating and betel essential oil was able to maintain the quality of rose apple cv. dalhari up to nine days. 1. introduction rose apple cv. dalhari is locally cultivated at berbah district, yogyakarta. rose apple cv. dalhari is one of the most famous fruits due to its large size, slightly fresh sour taste, and high water content. while, rose apple has thin skin makes low ability to maintain its water content and also is easily growth by microbes when it is kept at room temperature (pertiwi et al., 2012). edible coating is an alternative technology to overcome the problem and its widely known as the ability to maintain the atmosphere condition around the fruit, such as control gas exchange and control water loss, maintain the fruit’s texture, and also reduce the risk of microbial attack (campos et al., 2011). edible coating formulation can be derived from polysaccharides, lipids, proteins as long as the material are tasteless, odourless, and transparent. polysaccharides include alginate that is widely used as primary ingredient for edible coating due to its ability in holding high oxygen and in resulting good quality of edible coating (quirós-sauceda et al., 2014). alginate is obtained from marine brown algae (phaeophyceae) extraction. recently, the use of edible coating in fruit preservation is combined with another (*) corresponding author: chandra_fp@.umy.ac.id citation: setiawan c.k., utama n.a., pradana b.a., 2019 combination of alginate based edible coating-betel essential oil in extending the shelf life of rose apple cv. dalhari (syzygium samarangense). adv. hort. sci., 33(1): 33-38 copyright: © 2019 setiawan c.k., utama n.a., pradana b.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 january 2018 accepted for publication 18 september 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 33-38 34 active ingredient, such as antibrowning agent, nutrients, and antimicrobial agent to decrease microbial growth (campos et al., 2011; guerreiro et al., 2015; atarés and chiralt, 2016; hamedi et al., 2017). some essential oils have shown their effectiveness as food preservatives due to their antimicrobial activity and antioxidant content. betel essential oil contains chavicol, eugenol, acetyl eugenol as the main compounds to prevent pathogen growth in different edible commodities (prakash et al., 2010; basak and guha, 2015). however there are still limited information available regarding the use of edible coating to maintain the freshness of rose apple cv. dalhari. the only report from previous authors refers to effect of storage temperature of rose apple cv. dalhari (widoyo, 2013) , and none mentioned the use of edible coatings. this study aimed to determine the effect of betel essential oil combined with alginate-based edible coatings to prevent pathogen growth, and extend the shelf life of rose apple cv. dalhari. 2. materials and methods rose apple fruit cv. dalhari was harvested in berbah district, special region of yogyakarta, indonesia in the middle of april, 60 days after flowering. harvested fruit has characteristic red colour skin and average weight 125 g/fruit from 243 fruits. the fruit was immediately brought to postharvest laboratory at the universitas muhammadiyah yogyakarta. the fruits were selected based on defect and size, then stored in a refrigerator (14°c) until treatment. food grade sodium alginate (al) (iis, indonesia) was used for coating treatment, glycerol as a plasticizer, and betel essential oil (be). preparation and application of edible coating edible coating was prepared with diluted alginate into aquadest solution and heated in 85°c. solution was added with 1.5% glycerol and betel essential oil in different concentration. alginate-betel essential oil solution were formulated into nine treatments: al 2% (w/v) (a1s0); al 2% (w/v)-be 0.1% (v/v) (a1s1); al 2% (w/v)-be 0.2% (v/v) (a1s2); al 2.5% (w/v) (a2s0); al 2.5% (w/v)-be 0.1% (v/v) (a2s1); al 2.5% (w/v)-be 0.2% (v/v) (a2s2); al 3% (w/v) (a3s0); al 3% (w/v)-be 0.1% (v/v) (a3s1); al 3% (w/v)-be 0.2% (v/v) (a3s2). each treatment was performed in several steps as follow: the fruit was washed using sodium hypochlorite 200 µl l-1 and dried at room temperature followed by dipping in edible coating solution for 2 min. the excess of edible coating solution was dripped off for 30 s before being dipped in cacl 2% solution for 1 min. three treated fruits were packaged with polypropylene trays with perforated cover and stored at 14°c until analysis. analysis was performed on days 0, 3, 6, 9, 12, and 15. fruit quality assessment twenty seven groups of rose apple (9 treatments x 3 replicates) were weighed during the storage time. the weight loss (wl) of rose apple was determined using the equation: wl (%)= [(w0-wt)/w0] x 100 where w0 is the initial weight and wt is the sample weight at time t. fruit firmness was measured using a fruit hardness tester fht200 (extech instruments, usa). each fruit was tested in three different sides with a 6 mm diameter probe on equatorial position and recorded in n/mm2. total acidity (ta) determination was performed with methods according to iso 750-1998. five grams of rose apple slices were homogenised and diluted to 100 ml with distilled water. the mixture was filtered and was added 3-5 drops of phenolphthalein (1% in 95% ethanol) to 10 ml solution. the solution was then stirred and titrated with 0.1 n naoh. ta was determined as a percent of citric acid. total soluble solid (tss) was determined using a handrefractometer (atago, japan). the tools determines the juice tss content after being homogenised the flesh and it was expressed as a brix (%). reducing sugar (rs) were determined according to nelsonsomogyi technique (nelson, 1944) and the result showed in percentage. the analysis was performed in 540 nm abs using spectrophotometer uvmini-1240 (shimadzu, japan). antibacteria and antiyeast activity bacteria and yeast were isolated from decayed rose apple using paper discs and plate count. suspension was obtained from diluting 1 gr samples on destilled water in seven-fold dilution series. a petri dish was prepared with poured 10 ml nutrient agar medium and allowed to solidify. the 0.1% inoculum suspension was poured and flattened on the medium. sterilized filter paper disc dipped with 0.1% betel essential oil for 15 min and then placed on the surface of a medium. the petri incubated at room temperature for 48 h. after incubation time, measurement the inhibition zones were done with a setiawan et al. shelf life of rose apple cv. dalhari 35 ruler. in the other hand, 0.1% of betel essential oil was poured in medium before inoculated with 0.1 ml inoculum. after 48 h incubation at room temperature, the visible microbe was counted and the result was presented in log colony forming unit (cfu). after treatment was done, all samples were analyzed with microbial count for bacterial and yeasts based on mola et al. (2017). one gram of homogenized sample was diluted using 9 ml sterilized water. sample then diluted in seven-fold dilution series before plating. a 0.1 ml solution was poured on plate count agar (pca) surface and potato dextrose agar (pda) for enumerating total bacteria and molds. microbiological analysis was performed in three replication and the results were expressed as cfu (colony forming units) per grams fresh weight. sensory analysis the sensory was analysed by 15 semi-trained panellists on the based of 5-point hedonic scales (1=very dislike; 2=dislike; 3=neither like or dislike; 4=like; 5= like very much) for the parameters of texture and appearance according to guerreiro et al. (2015) with some modification. the panellists were recruited from staff and students of the department. they were trained in the initial test to be familiar with the fruit. all samples were analyzed on 0, 3, 6, 9, 12, 15 days after treatment. statistical analysis the experiment was carried out in randomised experimental design. the experiment data were analysed using sas statistical software package 9.4 for windows and duncan multiple-range test (p<0.05) was performed for mean comparisons. 3. results weight loss is one of the most important freshness indicators in fruit. as shown in figure 1, the weight loss increases in all fruit treatment during 15 days storage. however, a combination of alginate 2.5% and betel essential oil 0.1% can significantly (p<0.05) prevent the fruit weight loss, while the treatment of alginate 2% has shown the highest weight loss. weight loss of rose apple has no relation to its firmness. it can be shown in figure 2. although it shows that the value of firmness is not constant, overall the trend tends to show the similar pattern during 15 days of storage. alginate can maintain the fruit firmness’ decline even there is no significant effect between all treatment (p>0.05). a similar trend was also shown in pineapples (azarakhsh et al., 2012), and apple (rojas-graü et al., 2007 a) (omsoliu et al., 2008). the ta, tss and rs content show a regular decreasing during 15 days of storage time. the ta decreases after three days storage time in all treatment. however, as can be seen in figure 3a, the fruit treated with 3% alginate shows slower ta reduction. the value of tss slowly decreases on all treatments during 15 days storage (fig. 3b) despite no significant effect (p>0.05) is observed. rose apple fruit being non-climacteric fruit, tends to maintain similar tss content during storage. concerning rs content, there is no significant effect (p>0.05) in all treatments. rs value decreased after three days storage as showed in figure 3c. the effect of the edible coating containing a different concentration of betel essential oil on microbial growth is shown in figure 4a and 4b. as fig. 1 effect of alginate based edible coating-betel essential oil on weight loss of rose-apple cv. dalhari. fig. 2 effect of alginate based edible coating-betel essential oil on fruit firmness of rose-apple cv. dalhari. adv. hort. sci., 2019 33(1): 33-38 36 shown in the figure, edible coating can inhibit both bacteria and yeast in rose apple cv. dalhari. the increase in the population of both bacteria and yeast is observed during 15 days of storage time. the addition of betel essential oil can inhibit the yeast growth until nine days, while bacteria can be shown in no essential oil addition treatment after six days storage (fig. 4a). the sensory quality evaluation is not significantly different (p>0.05) during 15 days of storage time based on both treatments. the result indicates that different changes happen in the treatment during storage time although panellists did not apprehend those differences. the result also showed that rose apple is not suitable for consumption at 9 d after storage since most panellists give a score under minimum acceptable value in overall score (fig. 5). 4. discussion and conclusions reducing effect from essential oil to weight loss can be described with conjunction of betel essential oil with polysaccharides based edible coating. this relation make the edible coating more stable and permeable. similar result was described in the experiment using lemongrass (azarakhsh et al., 2014), cinnamon and palmarosa (raybaudi-massilia et al., 2008), oregano and vanillin (rojas-graü et al., 2007 a). the use of alginate as edible coating is expected can decrease the weight loss. in the present study, the results show that increasing fig. 3 effect of alginate based edible coating-betel essential oil on ta (a), tss (b) and rs (c) of rose-apple cv. dalhari. fig. 4 effect of alginate based edible coating-betel essential oil on bacteria (a) and yeast (b) population of rose-apple. fig. 5 effect of alginate based edible coating-betel essential oil on panelist preferences of rose-apple cv. dalhari. setiawan et al. shelf life of rose apple cv. dalhari 37 alginate concentration determine an increase in weight loss. in a previous study, this phenomenon has been reported and weight loss was explained as coming from edible coating itself (guerreiro et al., 2015). addition of betel essential oil does not affect the firmness (fig. 3). in a previous study, the use of lemongrass essential oil on alginate based edible coating decreases the fruit firmness due to its lower ph that triggers pectic acid hydrolysis (rojas-graü et al., 2007 a). fresh cut pineapple which are treated with alginate-lemongrass essential oil edible coating shows a decrease in firmness value during storage (azarakhsh et al., 2014). in ta observation, the result show that high concentration of alginate can decrease oxygen permeability which led reduction on the respiratory process (campos et al., 2011). ta changes are related to organic acid production and an acidity reduction maybe represent as a metabolic changes result in fruit or caused by the use of organic acid as respiratory substance (selcuk and erkan, 2015). tss content is a critical attribute which has contribution to quality and consumer acceptability. our result demonstrate that alginate based edible coating shows ability to maintain the tss content of rose apple. this findings are in line with the previous study in sweet cherry (díaz-mula et al., 2012), guava (nair et al., 2018), cantaloupe (zhang et al., 2015), blueberry (mannozzi et al., 2017) and ber fruit (ramana rao et al., 2016). rs value is an essential indicator for determining respiratory process in the fruit. betel essential oil addition does not affect the rs value. however, many studies showed alginate can maintain rs value which is reported in carambola (gol et al., 2015), cherry (díaz-mula et al., 2012) fresh fruit has high nutrient and high water content which allow microbial to multiply (rojasgraü et al., 2007 a). betel essential oil can inhibit the microbiological growth in food preservation (prakash et al., 2010). in a previous study, the result showed that there is no fungi that can grow in media which have been added with betel essential oil more than 0.6% (v/v) (basak and guha, 2015). however, our previous study in preferences (unpublished data) on alginate-betel essential oil edible coating showed that most panellists accepted to consume rose apple with the addition of essential oil less than 0.2%. addition betel essential oil shows proper appreciation up to 9 d in appearance value. this result is related to low microbial spoilage compared to no addition of essential oil. similarly, changes are shown in rose apple cv. dalhari texture preference. the panellist show acceptable score up to 9 d. this overall result is consistent with in previous study where it is no differences between fruit with low concentration essential oil addition with control in sensory test (raybaudi-massil ia et al. , 2008; azarakhsh et al., 2014; bustos et al., 2016; guerreiro et al., 2016). in conclusion, edible coating treatment with alginate 2.5% and betel essential oil 0.1 % maintained the quality of rose apple cv. dalhari by reducing weight loss, fruit firmness, microbial growth and appearance. in a study of microbial growth inhibition using paper disc test, betel essential oil has shown to inhibit both yeast and bacteria. however, in this experiment, a combination of alginate-betel essential oil has not shown the different effect to microbial growth on rose apple cv. dalhari. the main limitation of this study is the lack of information in the fruit. future research needs to be done for more findings and better understanding. acknowledgements the authors thank to the universitas muhammadiyah yogyakarta for financially supporting this research. the research grant was 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(in indonesian). zhang y., ma q., critzer f., davidson p.m., zhong q., 2015 effect of alginate coatings with cinnamon bark oil and soybean oil on quality and microbiological safety of cantaloupe. int. j. food microbiol., 215: 25-30. impaginato 241 adv. hort. sci., 2017 31(4): 241-247 doi: 10.13128/ahs-22376 genetic variability and correlation studies in grapes (vitis vinifera l.) in leh district of jammu and kashmir t. dolkar, m.k. sharma, a. kumar (*), m.s. mir, s. hussain division of fruit science, sher-e-kashmir university of agricultural science and technology, kashmir, shalimar campus, 190 025, srinagar, jammu and kashmir, india. key words: genotypic coefficient, heritability, phenotypic coefficient, yield. abstract: the present investigations on genetic variability and correlation in wild grape accessions were carried out in the most favourable regions of leh district of jammu and kashmir. fifty wild grape accessions from five different villages of the district were marked and evaluated for important morphological traits i.e. yield and quality parameters. data on various vegetative, fruit physical and fruit chemical characters were taken. significant variations were observed for all the characters studied except number of seeds per berry. high genotypic coefficient of variance (gcv) and phenotypic coefficient of variance (pcv) were recorded for yield (21.73 and 22.81), yield efficiency (41.19 and 52.73), bunch weight (21.07 and 21.23), number of berries per bunch (22.31 and 25.28), berry length (21.69 and 22.07), berry weight (21.13 and 21.28) and total sugars (22.69 and 22.70). out of twenty studied characters, thirteen characters registered high heritability estimates, while five recorded moderate heritability estimates and number of seeds per berry and acidity showed low heritability estimates. however, cane length (59.40), leaf area (31.43), bunch weight (43.16), number of berries per bunch (27.18) and tss/acid ratio (25.81) exhibited high genetic advance as percentage of mean indicating additive gene effect. fruit or berry yield was positively and significantly correlated with bunch length (0.652), bunch breadth (0.584), bunch weight (0.946), number of berries per bunch (0.672), berry breadth (0.363) and number of seeds per berry (0.612). this study revealed that the characters such as yield, bunch length, bunch weight, number of bunch per vine, berry weight, number of berries per bunch, total soluble solids and total sugars are the most important traits for selecting best grape accessions. 1. introduction grape (vitis vinifera l.) is an economically important and widely cultivated fruit crop in the world and is the first fruit crop to be cultivated by man to produce table fruits, dry fruits, juice and wine (frederique et al., 2010). it is a fairly good source of minerals like calcium, phosphorus, iron and vitamins like b1 and b2. the area under grape cultivation in india is 118.700 hectares with production of 2585.300 kg (nhb, 2015). in jammu and kashmir, grapes are grown over an area of 315 ha with annual production of 1299 mt (department of horticulture, 2016). the grapes are (*) corresponding author: khokherak@rediffmail.com citation: dolkar t., sharma m.k., kumar a., mir m.s., hussain s., 2017 genetic variability and correlation studies in grapes (vitis vinifera l.) in leh district of jammu and kashmir. adv. hort. sci., 31(4): 241-247 copyright: © 2017 dolkar t., sharma m.k., kumar a., mir m.s., hussain s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2016 accepted for publication 29 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 241-247 242 quite heterozygous and seedling off-springs exhibit wide genetic variability not only in fruit quality but also in vegetative vigour. because of these variations, seeds are not used for propagation of vines meant for commercial purpose. more than 9,600 grape cultivars exist around the world (galet, 2000) and as per international variety catalogue almost 16,000 prime names appear in the genus vitis (maul and eibach, 2003). to preserve the current genetic pool and to use it judiciously, it is necessary to evaluate the extent of this diversity by identification and distinction of grape accessions, as well as the determination of genetic relationship between local cultivars and wild relatives (negrul, 1973). wild grapevines (vitis vinifera l.) are heavily threatened in their natural habitats and high priority is given to the collection and preservation of this germplasm (forneck et al., 2003). indeed, the preservation of wild populations is considered essential for the maintenance of genetic variability and the resistance to genetic erosion (cunha et al., 2009). grape is grown under a variety of soil and climatic conditions in three distinct agro-climatic zones namely mild humid tropical region, sub-tropical region and dry temperate region. under dry temperate region of jammu and kashmir the maximum area is in lower belt of leh district (angchok et al., 2009). during the early period, when ladakh was the transit point on the central asian trade route, the traders, nomads and invaders from yarkand, baltistan, punjab, kashmir, china and tibet used to pass through this region (jolden, 2012) and the grape vines got disseminated or introduced in the lower belt of leh district. the cultivation of grapes in leh offers a greatest advantage by being ready for harvesting during the month of august-september which is an off season for the rest of the country and can therefore fetch higher remuneration. keeping in view the economic importance of grapes and to boost its cultivation in the leh district, the present investigation was carried out to generate the vital information on the existing germplasm of grape vine in the leh district and selection of elite clones for further multiplication and distribution among the farmers. 2. materials and methods experimental material and area thorough surveys of different areas of ladakh region was carried out during 2014 and out of a large population of wild grape only fifty accessions were selected from five different villages [warseedo (4), achinathang (21), yokmathang (7), hanuthang (5) and dha (13)] of leh district. the vines were marked on the basis of health, vigour, bearing habit and desirable berry characters. all the marked vines were of seedling origin ranging an age of between 20 to 60 years. the weather and geographical features of the studied location are represented in figure 1. observations recorded observations were recorded on vegetative, foliage, fruit physical and chemical characters. data was recorded on the various characters such as cane length (m), cane diameter (cm), internodal length (cm), leaf area (cm2), yield (kg/vine). yield efficiency was calculated as per westwood (1993). where, trunk cross sectional area (tcsa) = girth2/4 π. with respect to bunch and berry physical characters i.e. bunch length (cm), bunch breadth (cm), berry length (cm) and berry breadth (cm) were recorded with the help of vernier caliper. for these characters randomly ten bunches and ten berries were taken from the vines. randomly selected samples were subjected to bunch weight (g) and berry weight (g) with the help of digital weighing balance. all bunches from the vine were counted and number of bunches per vine were observed while for number of berries per bunch randomly ten bunches were taken and counted. number of seeds per berry was also counted. berry chemical characters i.e. tss (°b), acidity (%), total sugars (%), tss/acid ratio, and juice content (%) were observed as per the standard procedure as given in aoac (1998). fig. 1 weather and geographical features of the area surveyed. dolkar et al. genetic variability and correlation studies in grape in leh district, india 243 statistically analysis data collected on various parameters were statistically analyzed as per the procedure given by snedecor and cochran (1994). the genotypic coefficients of variation and heritability (in broad sense) were calculated (singh and chaudhary, 1979) while genetic advances were estimated as per the procedure of johnson et al. (1955). 3. results and discussion wide range of variation was observed in all the studied characters among different accessions. the extent of variability was measured in terms of range, mean, standard deviation, coefficient of variation, pcv, gcv, heritability, genetic advance and genetic gain (table 1). cane length and cane diameter varied from 115.82 cm (gl-49) to 228.43 cm (gl-27) and 1.21 cm (gl-2) to 2.70 cm (gl-13) with a mean value of 156.27 cm and 1.67 cm, respectively. the standard deviation and coefficient of variation for cane length was 29.34 and 18.78 per cent whereas for cane diameter was 0.29 and 16.87 per cent. maximum and minimum values for intermodal length were recorded in accessions gl-1 (29.93 cm) and gl-23 (10.50 cm) with a mean value of 23.76 cm. accession gl-31 (124.67 cm2) recorded minimum leaf area while gl14 (184.00 cm2) recorded maximum leaf area. average mean value for leaf area recorded was 148.62 cm2 and coefficient of variation was 10.89 per cent. kadu et al. (2007) also reported variation in leaf area as well as in vine vigour among fifteen grape cultivars. maximum yield was observed in accession gl15 (23.16 kg/vine) whereas accession gl-39 (11.02 kg/vine) scored minimum yield with a average mean value of 13.33 kg/vine. thakur et al. (2008) and joshi et al. (2015) also reported similar results with respect to yield. yield efficiency varied from 0.02 kg/cm2 (gl9, gl-42 and gl-43) to 0.19 kg/cm2 (gl-33) however mean value for yield efficiency was 0.08 kg/cm2 (table 1 and fig. 2). standard deviation and coefficient of variation for yield and yield efficiency was 2.94 and 22.07 per cent and 0.04 and 42.85 per cent, respectively. the high coefficient of variation obtained in case of yield and yield efficiency may be due to variation in the age of vine and other yield attributes such as height of the vine and vine girth. with respect to bunch and berry characters all the accessions had wide variation. maximum bunch length (23.50 cm) and bunch breadth (13.07 cm) was registered in gl-1 accession whereas minimum bunch length was recorded in gl-9 (11.58 cm) and minimum bunch breadth in gl-32 (7.03 cm) (table 1). average mean, standard deviation and coefficient of variation for bunch length was 18.30 cm, 2.84 and 15.52 per cent, respectively and for bunch breadth these values were 9.68 cm, 1.70 and 17.56 per cent, respectively. accession gl-37 recorded minimum (76.70 g) bunch weight whereas maximum bunch weight was recorded in gl-15 (155.50 g). average mean value and coefficient of variation for bunch table 1 genetic variability components for major characters in various grape accessions selected in leh district characters range mean sd cov coefficient of variance (%) heritability (%) genetic advance genetic gain (%)gcv pcv cane length (cm) 115.82-228.43 156.27 29.34 18.78 18.69 18.94 97.4 59.4 38.01 cane diameter (cm) 1.21-2.70 1.67 0.29 16.87 17.02 17.47 94.9 0.57 34.13 internodal length (cm) 10.50-29.93 23.76 4.76 20 19.92 20.24 96.9 9.59 40.36 leaf area (cm2) 124.67-184.00 148.62 16.2 10.89 10.73 11.22 91.5 31.43 21.15 yield (kg/vine) 11.02-23.16 13.33 2.94 22.07 21.73 22.81 90.8 5.68 42.61 yield efficiency (kg/cm2) 0.02-0.19 0.08 0.04 42.85 41.19 52.73 61 0.05 62.5 bunch length (cm) 11.58-23.50 18.3 2.84 15.52 15.16 15.95 90.3 5.43 29.67 bunch breadth (cm) 7.03-13.07 9.68 1.7 17.56 16.93 18.77 81.3 3.05 31.51 bunch weight (g) 76.70-155.50 100.19 21.16 21.11 21.07 21.23 98.5 43.16 43.08 no. of bunches/vine 113.00-152.00 134.38 9.89 7.35 6.94 8.14 72.8 16.39 12.2 no. of berries/bunch 38.00-110.00 67.02 15.64 23.33 22.31 25.28 77.9 27.18 40.56 berry length (cm) 0.81-2.06 1.12 0.24 21.42 21.69 22.07 96.5 0.49 43.75 berry breadth (cm) 0.80-1.64 1.05 0.17 15.23 15.96 16.31 95.7 0.34 32.38 berry weight (g) 0.92-1.92 1.37 0.29 20.59 21.13 21.28 98.6 0.59 43.07 number of seeds/ berry 1-feb 1.21 0.3 21.7 19.83 32.74 36.7 0.3 24.79 tss (ob) 13.70-23.10 20.15 2.03 10.01 9.95 10.25 94.2 4.01 19.9 acidity (%) 0.15-0.27 0.21 0.03 9.52 11 16.25 45.9 0.03 14.29 total sugars (%) 5.81-15.62 10.93 2.48 22.7 22.69 22.7 99.9 5.11 46.75 tss/acid ratio 62.48-137.33 97.35 17.75 17.97 16.53 21.23 60.6 25.81 26.51 juice content (%) 60.00-93.33 77.7 8.7 11.18 11.09 11.4 94.7 17.27 22.23 adv. hort. sci., 2017 31(4): 241-247 244 weight was 100.19 g and 21.11 per cent. havinal et al. (2008) and mukhtar et al. (2011) also reported similar results for bunch weight while studying twelve grape cultivars, however kamiloglu and polat (2009) observed much higher bunch weight which might be due to the age and cultural practices adopted. number of bunches per vine and number of berries per bunch ranged between 113 (gl-10) and 152 (gl-1) and 38 (gl-18) and 110 (gl-1) with a mean value of 134.38 and 67.02, respectively. coefficient of variation for number of bunches per vine and number of berries per bunch was 7.35 and 23.33 per cent, respectively. the difference in the yield per vine in different grape cultivars might be due to differences in weight of the bunch, number of bunches, weight of the berries and age of the vines besides their successful adoption to the varying agroclimatic conditions under which they are cultivated (havinal et al., 2008). gl-11 recorded maximum berry length (2.06 cm) whereas gl-32 and gl-33 recorded minimum berry length (0.81 cm) with a mean value of 1.12 cm and coefficient of variation was 21.42 per cent. gl-29 registered maximum berry breadth (1.64 cm) however minimum was registered in gl-32 (0.80 cm) with average mean value and coefficient of variation of 1.05 cm and 15.23 per cent. berry weight varied from 0.92 g (gl-4 and gl-33) to 1.92 g (gl-45) (table 1 and fig. 2). average mean value and coefficient of variation for berry weight was 1.37 g and 20.59 per cent. similar values for berry characters were also reported by al-shawish (2010). number of seeds per berry ranged between 1 and 2 with mean value of 1.21. out of fifty studied accessions, forty accessions were with one seed and only ten accessions had two seeds. coefficient of variation for number of seeds per berry was 21.70 per cent. total soluble solids ranged between 13.70ob (gl41) to 23.10ob (gl-43) whereas acidity varied from 0.15 (gl-40) to 0.27 per cent (gl-18). average mean value for total soluble solids and acidity was 20.15ob and 0.21 per cent whereas coefficient of variation was 10.01 per cent and 9.52 per cent (table 1). thakur et al. (2008) and jiang et al. (2012) also reported similar range for tss in the commercial cultivars of grape. maximum total sugar was recorded in gl-30 and gl-31 (15.62%) and minimum was recorded in gl-42 (5.81%) with a average mean and coefficient of variation of 10.93 per cent and 22.70 per cent. gl-41 (62.48) recorded minimum tss/acid ratio whereas gl-13 (137.33) recorded maximum tss/acid ratio. juice content ranged between 60 per cent (gl32) to 93.33 per cent (gl-1, gl-12 and gl-29). mean values and coefficient of variation for tss/acid ratio was 97.35 per cent and 17.97 per cent whereas these values for juice content was 77.70 per cent and 11.18 per cent. sharma and bist (1993) and ghosh et al. (2005) also reported similar results for tss, total sugars and juice content while evaluating eight local accessions of dry temperate areas of himachal pradesh. a perusal of the data revealed that the magnitude of the pcv was higher than gcv for all the characters. the estimates of pcv and gcv were high for yield (21.73 and 22.81), yield efficiency (41.19 and 52.73), bunch weight (21.07 and 21.23), number of berries per bunch (22.31 and 25.28), berry length (21.69 and 22.07), berry weight (21.13 and 21.28) and total sugar (22.69 and 22.70) indicated the presence of adequate genetic variation among the genotypes and suitability of these attributes for further improvement by selection. bist and sharma (1995) and gupta et al. (2015) also reported high phenotypic and genotypic coefficient of variance for bunch weight, berry weight and yield characters. pcv was high and gcv was moderate for number of seeds per fig. 2 yield, yield efficiency and berry weight in various grape accessions selected in leh district. dolkar et al. genetic variability and correlation studies in grape in leh district, india 245 berry. gcv in general, were lower than the pcv (table 1) which indicated close association between genotype and phenotype. high value of pcv and gcv indicated the existence of substantial variability, ensuring ample scope for their improvement through selection. these results further confirmed with the findings of earlier researchers for kumar et al. (2002) and wei et al. (2003). low gcv and pcv were recorded for leaf area (10.73 and 11.22), number of bunch per vine (6.94 and 8.14) and total soluble solids (9.95 and 10.25). the estimates of pcv and gcv for leaf area, number of bunch per vine and total soluble solids were low in magnitude yet these were close to each other indicating little effect of environment in the inheritance of these traits. heritability is a parameter of huge importance to the breeders as its magnitude indicates the reliability with which a genotype can be recognized through its phenotype expression (table 1). most of the characters studied had high heritability estimates, however they are moderate for yield efficiency (61.0%), number of bunches per vine (72.8%), number of berries per bunch (77.9%), and tss/acid ratio (60.6%) and low for number of seeds per berry (36.7%) and acidity (45.9%). the heritability of highest magnitude was noticed for total sugar (99.9%) closely followed by berry weight (98.6%) and bunch weight (98.5%). high estimates of heritability in broad sense indicate that substantial improvement can be made using standard selection procedures. high heritability estimates for yield, bunch weight, berry weight, tss were observed by kumar et al. (2002), wei et al. 2003 and gupta et al. (2015) and are in consonance with the present study. heritability estimates alone are not an ideal parameter for predicting the effect of selecting the desired individual. heritability estimates along with genetic advance are more useful than heritability value alone in predicting the selection of best individuals. in the present investigations cane length (97.4 and 59.40), leaf area (91.5 and 31.43) and bunch weight (98.5 and 43.16) exhibited high genetic advance as a percentage of mean along with high heritability. these results indicated the influence of additive gene action and hence these characters are likely to respond to selection. high heritability and low genetic advance were observed for yield (90.8 and 5.68), berry length (96.5 and 0.49), berry breadth (95.7 and 0.34), berry weight (98.6 and 0.59) and tss (94.2 and 4.01) which may be attributed to the non additive gene effects and these traits can be improved through hybridization and use for hybrid vigour. number of seeds per berry (36.7 and 0.30) and acidity (45.9 and 0.03) showed low heritability associated with genetic advance indicating the role of non additive gene for these traits suggesting thereby that their improvement could be achieved through heterosis breeding. a highly significant positive correlation was recorded between bunch length and bunch breadth (0.953), bunch weight (0.727) and number of berries per bunch (0.421) resulting in getting high yields (table 2). cane length was highly significant and positive correlated with yield (0.356), bunch length (0.356) and bunch weight (0.447). yield registered table 2 correlation coefficients (genotypic) in different characters in various grape accessions selected in leh district characters cane lenght (cm) cane diamter (cm) internodal lenght (cm) leaf area (cm2) yield (kg/ vine) yield efficieciency (kg/cm2) bunch lenght (cm) bunch breadth (cm) bunch weight (g) no. of bunches/ vine no. of berries/ bunch berry lenght (cm) berry breadth (cm) berry weight (g) no. of seeds/ berry tss (°b) acidity (%) total sugar (%) tss/ acid ratio juice content (%) cane lenght (cm) 1.000 0.272 0.122 0.244 0.356** -0.520** 0.356** 0.254 0.447** -0.174 0.156 0.202 0.287* 0.274* 0.224 -0.01 0.268 -0.224 -0.225 0.409** cane diameter (cm) 1.000 0.001 0.389** 0.353* -0.336* 0.135 0.052 0.359** 0.163 0.318* 0.262 0.266 0.023 0.428** -0.067 -0.144 -0.226 0.099 0.465** internodal lenght (cm) 1.000 0.309* 0.333* -0.052 0.328* 0.353* 0.315* 0.092 0.177 0.219 0.227 0.13 0.336* 0.027 0.056 0.154 -0.04 0.12 leaf area (cm2) 1.000 0.315* -0.106 0.286* 0.197 0.326* 0.071 0.172 0.222 0.198 0.137 0.26 -0.025 0.04 -0.288* 0.008 0.326* yield (kg/vine) 1.000 0.049 0.652** 0.584** 0.946** 0.289* 0.672** 0.337* 0.363** 0.173 0.612** -0.024 0.052 -0.148 -0.058 0.535** yield efficiency (kg/cm2) 1.000 -0.025 -0.011 -0.094 0.377** 0.202 -0.201 -0.278* -0.308* -0.233 -0.07 -0.101 0.115 0.039 -0.284* bunch lenght (cm) 1.000 0.953** 0.727** -0.196 0.421** 0.144 0.219 0.254 0.342* 0.159 -0.012 -0.255 0.096 0.435** bunch breadth (cm) 1.000 0.639** -0.206 0.294* 0.196 0.299* 0.309* 0.404** 0.163 0.151 -0.248 -0.035 0.408** bunch weight (g) 1.000 -0.018 0.647** 0.383** 0.430** 0.263 0.648** 0.021 0.013 -0.293* -0.004 0.583** number of bunches/vine 1.000 0.209 0.173 0.035 -0.26 0.091 -0.302* 0.048 0.234 -0.23 -0.066 number of berries/bunch 1.000 0.261 0.301* -0.603** 0.530** -0.129 0.031 -0.129 -0.11 0.282* berry lenght (cm) 1.000 0.945** 0.064 0.389** 0.008 0.144 -0.24 -0.109 0.396** berry breadth (cm) 1.000 0.08 0.465** 0.031 0.147 -0.242 -0.107 0.456** berry weight (g) 1.000 0.089 0.162 -0.055 -0.156 0.141 0.264 number of seeds/berry 1.000 0.13 0.274* -0.214 -0.166 0.566** tss (°b) 1.000 -0.102 0.323* 0.665** -0.086 acidity (%) 1.000 -0.097 -0.808** 0.266 total sugar (%) 1.000 0.255 -0.305* tss/acid ratio 1.000 -0.22 juice content (%) 1.000 adv. hort. sci., 2017 31(4): 241-247 246 highly significant and positive correlation with bunch length (0.652), bunch breadth (0.584), bunch weight (0.946), number of berries per bunch (0.672), berry breadth (0.363) and number of seeds per berry (0.612) while showed simple positive correlation with number of bunches per vine (0.289) and berry length (0.337) depicting that all these characters are yield contributing characters. kliewer and dokoozlia (2000) and gupta et al. (2015) also reported positive and significant correlation between yield and bunch length, bunch breadth, bunch weight. bunch weight showed positive and highly significant correlation with number of berries per bunch (0.647), berry length (0.383), berry breadth (0.430) and number of seeds per berry (0.648). a highly significant but negative correlation was observed between number of berries per bunch and berry weight (-0.603). berry length and berry breadth showed highly significant and positive correlation with number of seeds per berry (0.389 and 0.465) and juice content (0.396 and 0.456). acidity was highly but negatively correlated with tss/acid ratio (-0.808). these results are in conformity with those reported by kumar et al. (2002) and gupta et al. (2015) who advocated that the importance should be given to bunch length, bunch breadth, number of bunches per vine, number of berries per vine, berry weight during selection process because these characters contribute towards the yield. 4. conclusions most of the traits under study showed significant variations from low to high magnitude to heritability and genetic advance. these can facilitate selecting and utilizing the most preferred traits of interest and also hint the potential of grape for further improvement. some traits with high phenotypic and genotypic coefficient of variation those are detrimental which make possible grape improvement. a significant positive correlation of economic traits like bunch length, bunch weight, number of berries per bunch, berry breadth, number of seeds per berry with yield was recorded suggesting that selection for these characters would lead to crop improvement. references al-shawish f.a., 2010 evaluation of some yemeni grape cultivars according 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pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut gerbera flowers s.-s. shafiee-masouleh department of genetics and breeding, ornamental plants research center (oprc), horticultural sciences research institute (hsri), agricultural research education and extension (areeo), mahalatt, iran. key words: antioxidant, flavonoid, ga4+7, gerbera jamesonii. abbreviations: nano-silver= ns; deionized water= di; calcium sulfate= cs; gibberellin4+7 = ga; anthocyanin leakage= al; total soluble solids= tss. abstract: the aim of this study was to evaluate interactions between ns coupled with cs and ga on flavonoid, cell membrane behavior and extending the vase life of cut gerbera. pulse treatments of flowers were conducted in ns at concentrations of 0 (di), 3 or 9 mg/l for 24 h. then, flowers were treated with preservative solutions containing calcium sulfate (0, 10 or 20 mm) and ga4+7 (0 or 20 mg/l), plus 1.5% sucrose in all preservative solutions. pulse treatments with 3 or 9 mg ns/l and holding in solution containing 20 mm cs compared to the control treatment (holding in the solution of sucrose following pulse treatment in di) significantly extended vase life by 8 days. according to the antioxidant role of flavonoids, and lower amounts of flavonoid in the flowers that pretreated with ns, therefore, it may be said that ns prevented from microbial attack. 1. introduction gerberas (gerbera jamesonii) are well-known flowers for the variety of their colors, and are popular in the world flower trade (liu et al., 2009 a; solgi et al., 2009). however, often the growers and florists suffer further loss from short vase life of gerberas. for example, the mean of vase life in some cultivars of gerbera (‘bayadere’ and ‘sunway’) are reported only between 6 to 10 days when water tap is used (shabanian et al., 2018). gerberas are ethylene insensitive, but bacterial plugging of the xylem is a main cause of early and rapid senescence in their cut flowers (liu et al., 2009 a). the decrease of water uptake and consequently the increase of the ratio between transpiration and water uptake (i.e., high value of (*) corresponding author: shafiee.masouleh@areeo.ac.ir citation: shafiee-masouleh s.-s., 2018 effects of nanosilver pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut gerbera flowers. adv. hort. sci., 32(2): 185-191 copyright: © 2018 shafiee-masouleh s.-s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 185-191 186 water balance) will be caused as a result of xylem obstruction, and will end the cut flower vase life. nano-silver (ns) pulse and continuous treatments for cut flowers are newly used and are known as novel agents of anti-microbial (liu et al., 2009 a; solgi et al., 2009; lü et al., 2010). as a novel antiseptic, ns is used in the medical industry, silver embedded fabrics, water purification and vegetable disinfection. due to their high surface area to volume ratio, among other unique chemical and physical properties, ns formulations provide full contact with microorganisms and are highly effective as germicides. ns particles can connect the cell membranes and penetrate into bacteria. then, ns can disrupt the respiration and cell division and cause the cell death. ns releases silver ions (ag+) within bacterial cells, silver ions have bactericidal activity (liu et al., 2009 a; solgi et al., 2009; nair et al., 2010; sharon et al., 2010; lü et al., 2010; liu et al., 2012). naghsh (2010) described the inhibited meiosis in aspergillus niger due to ns activity. alavi and dehpour (2010) reported that the nano-silver solution is effective on greenhouse cucumber downy mildew disease. liu et al. (2009 a) and lü et al. (2010) observed that ns pulse treatments extended vase life of the cut gerbera and the rose flowers. also, solgi et al. (2009) reported that ns continuous treatments inhibited the growth of bacteria in the solution and xylem vessels and increased vase life of cut gerbera flowers. calcium increase postharvest longevity of fresh cut flowers (gerasopoulos and chebli, 1999; de capdeville et al . , 2005; sosa nan, 2007). this increased postharvest longevity may be due to a delay of physiological events related to senescence, such as a decrease in water uptake, increased water transpiration loss, decreased fresh weight, stem bending (sosa nan, 2007). since the level of soluble carbohydrates will be maintained by the treatment of gibberellin (ga) (ranwala and miller, 2000; whitman et al., 2001; hatamzadeh et al., 2010), therefore, ga can have a positive effect on the water balance. furthermore, sucrose in the preservative solutions maintains water balance, in addition to act as a food source (solgi et al., 2009). the objective of this research was to evaluate the interactions of calcium sulfate and gibberellin continuous treatments by ns pulse treatments on flavonoid as an antioxidant component, and vase life of cut gerberas. flavonoids have antioxidant effects and can be effective on the vase life. antioxidant molecules can be efficient systems to protect cells against pathogen and water deficit-induced oxidative stress, and this prevents the senescence and cell death (shabanian et al., 2018). 2. materials and methods plant material cut gerbera (gerbera jamesonii cv. pink elegance) flowers that were grown in standard hydroponic greenhouse conditions were purchased from a flower and plant growing company (pakdasht, tehran, iran). flowers were harvested by pulling the stems off in the plants when 2-3 rows of stamens of the bisexual disc florets were mature (gerasopoulos and chebli, 1999; solgi et al., 2009) in the morning. stem bottom of harvested flowers was put in the flower capsule containing deionized water (di). flowers were packed and transported within 8 h to the laboratory. in the laboratory, stems were re-cut to a length of 45 cm into the di to remove air emboli (liu et al., 2009 a; solgi et al., 2009). flowers were re-cut 2-3 times, when it was necessary. the flowers were placed in a controlled environment room at 20±2°c with 60±10% r.h. and 12 µmol/m.s. light intensity (cool white fluorescent lamps; 12 h/day). experimental design and treatments solutions of pulse treatment were prepared in two concentrations of ns (3 or 9 mg/ l), and di was used as a control treatment. flowers were treated for 24 h with the two concentrations of ns (nanonasb-pars company, iran) or di. each pulse treatment contained 18 flowers. following pulse treatment, the flowers were individually kept into 1000 ml glass vases containing 500 ml of fresh solutions (as continuous treatment) that were prepared at second day of the experiment and were not renewed. in the continuous treatments, three concentrations (0, 10 or 20 mm) of calcium sulfate, cs, (caso42h2o, merck company), and two concentrations (0 or 20 mg/ l) of ga4+7 (serva company, usa) were used. in the all continuous treatments, sucrose 1.5% was used. there were three replications and three samples per treatment in a completely randomized design as factorial experiment. each sample was one flower per bottle. data were analyzed using three-way analysis of variance (proc glm), and the means shafiee-masuleh antioxidant molecule and vase life of cut gerbera flowers 187 were compared by tukey’s test (hsd) at p≤0.05 using sas (9.1) statistical software. correlation coefficients between vase life and cell conditions and flavonoid were conducted by spss (version 11.5). regression analysis (path analysis) was taken to determine the major factors that affect vase life (dependent variable). the independent explanatory variables were anthocyanin leakage, tissue ph, tss and flavonoid. the software used for path analysis was spss/pc+ “stepwise” (version 11.5). vase life vase life was recorded from harvest time by the time the flowers showed symptoms of petal wilting or curling, stem bending (≥90°) or breaking, therefore, the flowers were visited daily. total flavonoid assay in termination of the vase life, to extract total flavonoid, 20 ml of acidic methanol (1% hcl) was added to 0.2 g fresh weight of petals, and the mixture was stirred for 48 h in the dark. the extract was used to measure total flavonoid content immediately (chang et al., 2002). total flavonoid content was measured by aluminum chloride colorimetric assay (chang et al., 2002; kumar et al., 2008). an amount of 200 µl of plant extract was added to 600 µl of methanol, 40 µl alcl3 (10%), 40 µl of potassium acetate (1 m) and was made to 2000 µl by distilled water. the solution was vigorously mixed and after keeping at room temperature in the dark for 30 min, the absorbance was measured against reagent blank at 510 nm with a spectrophotometer (t80+ uv/vis spectrometer, pg instruments ltd). the calibration curve of standard solutions of catechin (5-40 µg/ ml of 1% hcl in methanol) was drew (y= 0.0003x + 0.1654, r2= 0.9989). total flavonoid content of flower was expressed as mg catechin equivalent per 100 g of fresh weight. anthocyanin leakage to evaluate the effects of treatments on the cell membrane structure, anthocyanin leakage was measured to observe the stability of plasma membrane. at tenth day of vase life, 0.5 g of petals was sliced to pieces of 1×1 cm, these pieces were washed in di water two times and within a period of 2 h. then 10 ml of di water was added to samples. after 12 h in 25°c, the absorbance was recorded with a spectrophotometer (t80+ uv/vis spectrometer, pg instruments ltd) at 525 nm (poovaiah, 1979). tissue ph measurement tissue ph was measured according to the method of hill (1999) to consider the treatment influences on the conditions of cell reactions. in termination of vase life, 2 g of petals was crushed in liquid n2, and then was placed at -80°c for 48 h. the frozen tissues were removed from -80°c, thawed at 20°c, then were frozen in liquid n2 again and placed at -80°c for a further 36 h. after thawing at 20°c again, 25 ml distilled water was added to tissues in the test tube, then were frozen at -20°c for 24 h. the ph of filtered fluid was recorded after thawing with a ph meter. total soluble solid total soluble solid (tss) of petal juice was measured with a refractometer (ceti, belgium) as °brix (roein et al., 2009). 3. results and discussion interactions between ns and cs significantly extended the vase life (fig. 1). ns pulse treatments and then holding in preservative solutions (10 or 20 mm cs) extended the vase life compared to di pulsing and preservative solution containing sucrose (control treatment) (fig. 1). however, no significant (p<0.05) difference was found among various concentrations of ns and cs to extend the vase life. also, interactions between cs and ga4+7 had significant effect on the extension of the vase life (p<0.05) (fig. 2). the longest vase life was found in the pulse treatment with 3 mg ns/l and preservative solution containing 20 mm cs. it was 7.5 days (i.e., 62.5%) added to vase life compared to the control (fig. 1). gerbera is insensitive to ethylene (liu et al., 2009 a); therefig. 1 interactions between nano-silver (0, 3 or 9 mg/l) and calcium sulfate concentrations (= 0 mm; = 10 mm; ▲= 20 mm) on the vase life (days) in cut gerbera flowers. the means with same letters have no significant differences (msanova = 6.27; type 1 error, hsd0.05), n=3. adv. hort. sci., 2018 32(2): 185-191 188 fore, ns effects for extending the vase life of gerbera are not related to anti-ethylene effects of ns. therefore, it must be explained that the basic role of ns is to prevent from bacterial plugging of the xylem (liu et al., 2009 a, b; solgi et al., 2009; chaloupka et al., 2010; lü et al., 2010); then increasing water uptake and calcium. according to gerasopoulos and chebli (1999), post-calcium uptake prevents appearing the symptoms of the end of vase life in gerbera [wilting, petal curling, stem bending (in this cultivar was not observed) and breaking (in this cultivar was observed partially)]. the longest vase life was obtained in preservative solutions containing 10 mm cs without ga4+7 [approximately 19 days, i.e., 32.3% more than treatment with 20 mg/l ga, without cs, which had significant (p<0.05) differences with solutions containing 20 mg l-1 ga4+7 without cs] (fig. 2). gibberellin can enhance hydrolization of starch to glucose, and during enzymatic process sucrose will be produced. more production of sucrose causes strength of cell walls. having more sugar in tissues preserves them of early disruption and increases their longevity (halevy and mayak, 1981). also, whitman et al. (2001) determined that ga4+7 sprayed to lil ium longiflorum had a positive effect to decrease foliar chlorosis and increased vase life contrary to our results; moreover, it decreased the effect of cs. there were significant interactions (p<0.05) between ga, cs and ns on total flavonoid at the end of vase life (table 1). the highest flavonoid content table 1 the effect of nano-silver pulsing and continuous treatments by calcium sulfate and gibberellin on the biochemistry factors of cut gerbera flowers fig. 2 interactions between calcium sulfate (0, 10 or 20 mm) and ga4+7 concentrations (= 0 mg ga4+7/l; = 20 mg ga4+7/l) on the vase life (days) in cut gerbera flowers. the means with same letters have no significant differences (msanova = 1.41; type 1 error, hsd0.05), n=3. treatments flavonoid (mg equivalent catechin per 100 g f.w.) anthocyanin leakage (absorbance in 525 nm) tissue ph tss (° brix)pulse treatment continuous treatment ns (mg/ l) csz (mm) ga (mg/ l) 0 0 0 3933.3 bcd 0.165 a 4.98 abcd 11.2 a 20 2350.0 d 0.160 abc 5.15 a 10.8 ab 10 0 4516.7 abcd 0.161 abc 4.94 bcd 11.1 a 20 3877.8 bcd 0.161 abc 4.98 abcd 11.3 a 20 0 8183.3 ab 0.160 abc 4.91 bcd 11.2 a 20 8933.3 a 0.157 bc 4.94 bcd 9.9 ab 3 0 0 5711.1 abcd 0.159 abc 4.98 abcd 9.4 ab 20 2988.9 cd 0.163 ab 5.01 abc 10.0 ab 10 0 5655.6 abcd 0.155 c 4.88 bcd 9.0 ab 20 3433.3 bcd 0.157 bc 4.83 cd 8.9 ab 20 0 7544.4 abc 0.156 bc 4.86 cd 8.8 ab 20 5377.8 abcd 0.155 c 4.83 cd 7.9 b 9 0 0 2933.3 cd 0.156 bc 4.96 abcd 9.8 ab 20 3100.0 cd 0.157 bc 5.05 ab 10.4 ab 10 0 2766.7 cd 0.155 c 4.85 cd 8.3 ab 20 5322.2 abcd 0.156 bc 4.89 bcd 8.5 ab 20 0 6488.9 abcd 0.156 bc 4.82 d 9.5 ab 20 3711.1 bcd 0.157 bc 4.86 cd 8.9 ab anova (mean of square) ns 815234.8 ns 0.0001 ** 0.04 ** 18.97 ** cs 50446502.1 ** 0.0001 ** 0.12 ** 4.47 * ga 12438400.2 * 0.00000002 ns 0.02 * 0.74 ns ns x cs 6853137.9 y 0.00002 * 0.0008 y 1.56 y ns x ga 696546.46 y 0.00002 * 0.01 y 0.47 y cs x ga 2485082.3 y 0.000003 y 0.01 y 1.52 y ns x cs x ga 5213477.41 y 0.00001 y 0.0009 y 0.1 y the means with different letters are significant (hsd0.05). n= 3.** = significant at p≤0.01; * = significant at p≤0.05; ns = not significant. y= type i error. shafiee-masuleh antioxidant molecule and vase life of cut gerbera flowers 189 was measured in the petals of flowers that were treated with di and kept in the solution containing 20 mm cs and 20 mg/l ga. whereas treated flowers with di and kept in the solution containing 20 mg/l ga showed the lowest total flavonoid. the antioxidant role of flavonoids was revealed for the flowers that were not pulsed by ns, because, the microbial attack might be a signal to synthesize the flavonoids (khatiwora et al., 2010). when calcium (meyer et al., 1973) and gibberellin (ranwala and miller, 2000; hatamzadeh et al., 2010) were made available, they activated the reducing of nitrate to produce phenylalanin, and to form simple carbohydrates, respectively. therefore, the pathway of flavonoid synthesis was completed and total flavonoid was increased. phenylalanie transforms into 4-coumaroyl-coa in the phenylpropanoid pathway, and finally enters the flavonoid synthesis pathway (falcone ferreyra et al., 2012). the most significant interactions (p<0.05) were observed between ga, cs and ns on the anthocyanin leakage. in the flowers which were not treated with ns and/or cs, anthocyanin leakage was highest (table 1). the stability of cell membrane will have been decreased by factors as senescence, microbial (attacking by micro-organism) or no microbial (deficit of calcium) diseases. the measurement of anthocyanin leakage at half of vase life could be a gauge to evaluate the stability of cell membrane. the accumulation of calcium in middle lamella of cell wall increases the stability of cell membrane and decreases anthocyanin leakage (nikbakht et al., 2008). ns prevents microbial attack and decreases senescence and keeps stability (liu et al., 2009 a; solgi et al., 2009; lü et al., 2010). the most tissue ph was recorded at pre-treatment with di and keeping in solution containing 20 mg ga/l, and the least tissue ph was at pulsing with 9 mg ns/l and keeping in calcium sulfate solution (20 mm). generally, significant differences were observed between treatments (p<0.05) (table 1). schmitzer et al. (2010) explained that increasing the cell sap ph causes the developing flowers from the bud to senescence stage. the most tss was measured in the flowers of control treatment, and the least tss was recorded in the flowers which were pulsed with 3 mg ns/l and kept in solution containing 20 mm cs and 20 mg ga/l. significant differences (p<0.05) were observed between two mentioned treatments. however, no one has the significant differences with other treatments. gebremedhin et al. (2013) interpreted that tss will be increased by more water uptaking to provide the required substrate for respiration. the analysis of correlation coefficients (table 2) shows the negative significant correlation (p≤0.01) between vase life and anthocyanin leakage and tss. furthermore, there is positive significant correlation between anthocyanin leakage and tss (table 2). for the last parameter, the coefficient of multiple determinations (r2) was 0.479 in linear model for the vase life (table 3). this coefficient gives the proportion of the total variation in the dependent variable (vase parameters vase life al tissue ph tss flavonoid vase life 1 al -0.656 ** 1 tissue ph -0.353 0.412 1 tss -0.692 ** 0.757 ** 0.375 1 flavonoid -0.338 0.253 -0.373 0.206 1 vase life linear model variable b se b standard β t significance constant 32.265 4.04 7.987 0 tss -1.586 0.414 -0.692 -3.832 0.001 multiple r 0.692 r2 0.479 adjusted r2 0.446 standard error 1.82 anova sum of squares df mean squares f significance regression 48.834 1 48.834 14.688 0.001 residual 53.197 16 3.325 total 102.031 17 ** significant in p<0.01 (two-tailed correlations), n=18. table 3 vase life of gerbera flowers regressed (stepwise regression) against anthocyanin leakage, tissue ph, tss and flavonoid table 2 correlation coefficients between vase life (days), anthocyanin leakage (al), tissue ph, total soluble solids (tss) and flavonoid (mg equivalent catechin per 100 g f.w.), n=3 adv. hort. sci., 2018 32(2): 185-191 190 life) explained by the predictors included in the model. thus, from among four independent variables, total soluble solids explained 47.9% of the observed total variation in the vase life, and other independent variable (anthocyanin leakage, tissue ph and flavonoid) had a lesser role in the vase life. furthermore, the test statistic in linear model showed that coefficient of tss negatively and significantly (p≤0.01) influenced vase life (table 3, fig 3). therefore, the factors that cause the increased tss and anthocyanin leakage lead to decrease vase life. this is also confirmed by other researchers (gebremedhin et al., 2013). according to the results, we recommend pulse treatment with 3 mg ns/l and then, continuous treatment with 20 mm cs for increasing the vase life of cut gerbera by 8 days. references alavi s.v., dehpour a.a., 2010 evaluation of the nanosilver colloidal solution in comparison with the registered fungicide to control greenhouse cucumber downy mildew disease in the north of iran. acta horticulturae, 877: 1643-1646. chaloupka k., malam y., seifalian a.m., 2010 nanosilver as a new generation of nanoproduct in biomedical applications. trends biotech., 28: 580-588. chang c.c., yang m.h., wen h.m., chern j.c., 2002 estimation of total flavonoid content in propolis by two complementary colorimetric methods. j. food drug anal., 10: 178-182. de capdeville g., maffia l.a., finger f.l., batista u.g., 2005 pre-harvest calcium sulfate applications affect vase life 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chemical composition of organically grown leek n.a. golubkina 1 (*), t.m. seredin 1, m.s. antoshkina 1, h.v. baranova 1, v. stoleru 2, g.c. teliban 2, g. caruso 3 1 agrochemical research center, federal scientific center of vegetable production, 143072 moscow region, odintsovo district, vniissok, selectsionnaya 14, russia. 2 department of horticulture technology, university of agriculture sciences and veterinary medicine, 3m sadoveanu, 700490 iasi, romania. 3 dipartimento di agraria, università degli studi di napoli federico ii, 80055 portici, napoli, italy. key words: allium porrum l., ascorbic acid, greenhouse, mineral elements, polyphenols, sugars. abstract: the research was carried out in order to assess the effects of nine cultivars in factorial combination with open field or greenhouse growing on yield, quality indicators, antioxidants and elemental composition of leek in moscow region. greenhouse management resulted in higher yield compared to open field cultivation, due to higher mean pseudo-stem weight, and cultivar giraffe gave the highest production. pseudo-stem dry matter was better affected by greenhouse cultivation, whereas the content of monosaccharides, total sugars, nitrates, ascorbic acid and polyphenols was enhanced by open field growing. the cultivars vesta and summer breeze showed the highest dry matter and total sugar content, whereas goliath had the highest antioxidant, selenium and potassium concentration. among the mineral elements, k and mg in pseudostems were better affected by greenhouse conditions, whereas ca attained a higher concentration under open field growing. the antioxidant system of allium porrum was characterized by significant positive correlations between se, polyphenols, ascorbic acid and potassium. 1. introduction leek (allium porrum l.) is a major crop among allium species and it is mainly grown in indonesia, turkey and, within europe, in france and belgium for producing edible pseudo-stems. the latter have high nutritional value, also due to the high content of potassium and iron (koca and tasci, 2016), and high biological activities connected with the remarkable concentration of antioxidants comparable with that of allium cepa (*) corresponding author: segolubkina45@gmail.com citation: golubkina n.a., seredin t.m., antoshkina m.s., baranova h.v., stoleru v., teliban g.c., caruso g., 2019 effects of crop system and genotype on yield, quality, antioxidants and chemical composition of organically grown leek. adv. hort. sci., 33(2): 263-270 copyright: © 2019 golubkina n.a., seredin t.m., antoshkina m.s., baranova h.v., stoleru v., teliban g.c., caruso g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 25 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 263-270 264 (sekara et al., 2017), such as polyphenols (ben arfa et al., 2015), glucosinolates, s-alkenyl-l-cysteine sulfoxides and pectic polysaccharides (ozgur et al., 2011). accordingly, leek shows antimicrobial, cardio-protective, hypo-cholesteremic, hypoglycemic, antirheumatic, hypotensive, antianemia, and anticancer action, improves liver, gastro-intestinal and brain efficiency, decreases blood pressure, inhibits platelets aggregation and prevents neural tube defects as well as prostate diseases (radovanović et al., 2015). protected cultivation may be appropriate to organic horticulture which is more susceptible to the environmental unbalances due to the milder farming practices and is usually more profitable than the conventional management (caruso et al., 2012; conti et al., 2015). within the crop system, cultivar assessment in terms of content of antioxidants as well as macroand micro-elements in leek pseudo-stems raises the interest of establishing the relations between the mentioned substances and, accordingly, identifying the most interesting genotypes, also based on their yield. due to the fragmented investigations relevant to varietal differences in biologically active compounds (bernaert et al., 2012) and elemental composition (koca and tasci, 2016), we carried out research aiming to evaluate the effect of both crop system and cultivar on yield, quality, antioxidant content and elemental composition of a. porrum grown either in greenhouse or in open field. 2. materials and methods plant material and growth conditions research was carried out on leek (a. porrum l.) grown in greenhouse at the experimental fields of federal scientific center of vegetable production, in odintsovo (moscow, russia, 55°40’ n, 37°12’ e) in 2015 and 2016 on a clay-loam soil, with рн 6.8, 2.1% organic matter, 108 mg kg-1 n, 450 mg kg-1 p2o5, 357 mg kg-1 k2o, exchangeable bases sum as much as 95.2%. mean temperature values from may to october were: 13.0, 16.1, 19.8, 18.6, 12.3, 6.4°c in open field; 20.4, 21.4, 23.7, 20.0, 14.5, 8.3 in greenhouse. the experimental protocol was based on the factorial combination between two crop systems (open field, greenhouse) and nine cultivars (goliath, summer breeze, premier, casimir, kalambus, camus, vesta, giraffe, bandit), using a split-plot design with three replicates. the sowing was performed on 5 december in 8 x 8 cm trays and the plantlets were transplanted in the field on 14 may, spaced 15 cm along the rows, the latter being 40 cm apart. leek crops were preceded by organically grown vegetables in the previous four years, such as carrot, bean, rape and pea. prior to planting, plough at 30 cm depth, hoeing at 15 cm and fertilization with 180 kg ha-1 n, 80 p2o5 and 120 k2o were practiced; during the growing period, 40 kg ha-1 n were supplied in three times at two-week intervals, starting at bulbification stage, and just in the last n application 7 kg ha-1 of p2o5 and of k2o were also provided. drip irrigation was activated at 80% soil available water. the organic farming practice complied with ec regulation 834/2007 and 889/2008. plant protection was achieved by applying copper oxychloride against rust and azadirachtin against aphids. harvests of ripe plants were performed from 5 to 10 october in greenhouse and from 12 to 19 october in open field, when the pseudo-stems had reached their maximum growth, and the leaf blades were trimmed at 15 cm length for obtaining the marketable product. in each plot, determinations were made of the marketable product weight (pseudostems with 15 cm long leaf blades) and the mean pseudo-stem (with 15 cm long leaf blades) weight on twenty-plant samples. further plant samples were collected, gently washed with water to remove surface contaminants and dried with filter paper. pseudo-stems and leaves were separated, cut with plastic knife, dried to constant weight and homogenized; the resulting powders were subjected to laboratory analysis. dry matter the dry matter content in leaves and pseudostems of a. porrum was assessed after dehydration of the fresh samples in an oven at 70°c, until they reached constant weight. sugars monosaccharides were determined using ferricyanide colorimetric method, based on the reaction of monosaccharides with potassium ferricyanide (swamy, 2008). total sugars were determined after acidic hydrolysis of water extracts with 20% hydrochloric acid (swamy, 2008). fructose was used as an external standard. polyphenols the concentrations of the total polyphenols in each sample of leaves and pseudo-stems were determined in 70% ethanol extract (1 hour at 80oc) using the folin-ciocalteu colorimetric method, according to golubkina et al. crop system and genotype effects in allium porrum 265 golubkina et al. (2018 b) by unico 2804 uv (usa) spectrophotometer. the polyphenol content was expressed as milligrams of gallic acid equivalents per 100 grams of dry weight (mg gae 100 g-1 d.w.). ascorbic acid the ascorbic acid content in leek leaves and pseudo-stems was assessed by visual titration of fresh plant extracts in 6% trichloracetic acid with tillmans reagent (caruso et al., 2009; aoac, 2012). antioxidant activity the antioxidant activity of leek leaves and pseudo-stems was assessed using redox titration method (maximova et al., 2001; golubkina et al., 2018 b), via titration of 0.01 n kmno 4 solution with ethanolic extracts of leaves and pseudo-stems. the values were expressed in mg gae 100 g-1 d.w. nitrates the nitrate content was assessed in fresh pseudostems using ion selective electrode on ionomer expert-001 (econix, russia). elemental composition the content of al, as, b, ca, cd, co, cr, cu, fe, i, k, li, mg, mn, na, ni, p, pb, se, si, sr, v and zn in leek pseudo-stems was assessed using icp-ms on quadruple mass-spectrometer nexion 300d (perkin elmer inc., shelton, ct 06484, usa) in the biotic medicine center in moscow (golubkina et al., 2017). statistical analysis data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 21. the data expressed as a percentage were subjected to angular transformation before processing. as the year of research had no significant effect on the yield, quality, antioxidant and elemental composition variables examined, both as main factor or in interaction with the experimental factors “crop system” and “cultivar”, the results are reported as average values of the two years of investigation. 3. results and discussion growth, yield and quality indicators of pseudo-stems the crop system showed significant effects on leek plant biomass, pseudo-stem yield and mean weight, as these variables attained higher values in greenhouse compared to open field (table 1); as reported in the previous section, the plant commercial ripeness was anticipated by 8 days on average in the protected environment. these trends are consistent with those recorded in previous research (conti et al., 2015). differences between the varieties were recorded with regard to: biomass, which was highest in cultivar summer breeze and lowest in premier; mean pseudo-stem weight and, accordingly, yield which ranged from 22.0 to 36.1 mg ha-1, with the cultivar giraffe showing the best performance, premier and kalambus the worst. the greenhouse growing resulted in higher concentration of dry matter, ash and nitrates, but lower monosaccharides and total sugars in pseudo-stems, compared to those detected in open field (table 1), similarly to previous reports (conti et al., 2015). notably, the values of dry matter recorded in our table 1 growth and yield indicators, and content of dry matter, sugars and nitrates in a. porrum pseudo-stems * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. treatment plant biomass (kg m-2 d.w.) marketable pseudo-stems yield (mg ha-1) mean weight (g) dry matter (%) monosaccharides (g 100 g-1 d.w.) total sugars (g 100 g-1 d.w.) ash (%) nitrates (mg kg-1 f.w.) crop system open field 11.0 25.3 162.8 16.7 3.81 12.0 4.8 44.7 greenhouse 15.8 30.8 185.7 19.7 3.41 10.7 5.2 66.3 * * * * * * * * cultivar goliath 8.7 ef 27.6 de 177.0 de 12.1 e 5.11 a 7.5 e 8.5 b 48.0 d premier 8.3 f 22.0 g 132.6 g 14.6 d 4.62 ab 10.6 c 12.4 a 45.9 d bandit 12.0 d 31.0 bc 192.8 bc 14.9 d 3.73 c 8.9 d 4.7 c 65.3 a kalambus 9.9 e 22.3 g 134.6 g 17.1 c 4.13 bc 10.5 c 2.8 e 43.1 d cazimir 11.6 d 24.3 f 150.1 f 18.4 c 2.98 d 11.0 bc 4.1 cd 57.5 bc giraffe 18.6 b 36.1 a 225.4 a 19.8 b 3.58 c 11.2 bc 3.1 e 54.7 c camus 16.0 c 29.9 cd 188.0 cd 20.6 b 2.62 d 12.3 b 3.2 e 64.9 a vesta 15.4 c 26.3 ef 163.8 ef 22.6 a 2.74 d 14.6 a 2.9 e 60.1 ab summer breeze 20.3 a 33.1 b 204.5 b 23.7 a 2.97 d 15.5 a 3.4 de 60.5 ab adv. hort. sci., 2019 33(2): 263-270 266 research fell within the 12.1 to 23.7 % range (table 1) which is much wider than that relevant to a. porrum grown in czech republic (9-11%) (lundegardh et al., 2008). moreover, the cultivars with high dry matter content (summer breeze and vesta) have a long shelf-life and are even suitable as dry spice source, whereas the varieties showing low dry matter (goliath, premier and bandit) better fit the salad industry target. significant varietal differences in ash content were recorded (table 2), with the ratio between leaf and pseudo-stem related to this variable decreasing as follows: summer breeze > cazimir > vesta > giraffe > bandit > kalambus > camus > premier > goliath. the higher nitrate accumulation in pseudo-stems grown in greenhouse is connected with the lower light intensity occurring in the protected environment compared to open field conditions, which limits the nitrate reductase activity; however, it was much lower (105 mg kg-1 f.w.) than that relevant to the topaccumulator species (caruso et al., 2011) and referred to previous reports (santamaria, 2006). antioxidants ascorbic acid and polyphenols highly affect plant antioxidant activity (proteggente et al., 2002); in our research, the open field conditions resulted in higher content of both antioxidant compounds in leek pseudo-stems compared to greenhouse (table 2). the crop system did not affect the selenium concentration either in pseudo-stems or leaves of a. porrum. the high average content of ascorbic acid recorded in our research presumably makes the product safe and healthy, as ascorbic acid participates in producing essential nitrogen oxide for human organism, thus preventing nitrosamine formation from nitrate accumulating in plants (santamaria, 2006). moreover, wide varietal differences were found in ascorbic acid concentration, unlike the polyphenol content which was characterized by higher stability in pseudo-stems and even more in leaves (table 2). among the cultivars examined, goliath showed the highest content of ascorbic acid, polyphenols and selenium in pseudo-stems (table 2). notably, these cultivars from domestic selection are characterized by lower levels of polyphenols compared to literature references, which may be connected with the different crop cycle and harvest time (biesiada et al., 2007). bernaert et al. (2012) also reported a higher polyphenol concentration in pseudo-stems of thirty leek cultivars grown in belgium (7.3 to 11.3 mg ga g-1 d.w.) compared to our values (3.3 to 6.3), but a lower content of ascorbic acid ranging between 90 to 350 mg 100 g-1 d.w. in the latter research no correlation was recorded between ascorbic acid and polyphenol concentration in leek pseudo-stems, whereas a significant positive relationship has been found in our investigation (r = 0.94 at p<0.01). the lack of correlation relevant to the thirty leek cultivars grown in belgium (bernaert et al., 2012) presumably depends on varietal heterons not significant; * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. table 2 concentrations of ascorbic acid, polyphenols and selenium in leek treatment ascorbic acid in pseudo-stems (mg 100 g-1 f.w.) polyphenols (mg ga 100 g-1 d.w.) selenium (µg kg-1 d.w.) pseudo-stems leaves pseudo-stems leaves crop system open field 57.6 466.5 887.1 71.9 61.2 greenhouse 47.2 376.8 699.5 76.2 64.4 * * * ns ns cultivar goliath 169.3 a 626.1 a 827.8 a 106.4a 14.1 e premier 72 b 497.6 b 751.8 b 79.1 b 64.8 c bandit 50.6 c 455.6 bc 739.8 b 74.1 bc 47.2 d kalambus 32.1 de 368.6 de 858.5 а 70.6 bc 74.8 b cazimir 37 d 334.1 e 843.1 а 59.7 e 76.9 ab giraffe 31.3 ef 386.1 d 760.8 аb 72.9 bc 48.4 d camus 27.1 fg 401.6 cd 786.3 ab 63.1 de 81.4 ab vesta 24.3 g 345.3 de 855.5 a 68.1 cd 85.4 a summer breeze 28.0 fg 379.7 de 716.3 b 72.1 bc 72.3 bc golubkina et al. crop system and genotype effects in allium porrum 267 tion between selenium and polyphenol content was found in wheat (lachman et al., 2011) and a negative correlation between quercetin and selenium was recorded in a. cepa (golubkina et al., 2016). however, moderate doses of selenium are deemed to enhance the content of antioxidants such as polyphenols, flavonoids and carotenoids (malagoli et al., 2015). in our research, the nine leek genotypes examined showed significant correlations between the components of the antioxidant system, i.e. selenium, ascorbic acid and polyphenols: se and ascorbic acid (r= 0.93 at p≤0.01); se and polyphenols (r= 0.92 at p≤0.01); ascorbic acid and polyphenols (r = 0.94 at p≤0.01). the latter correlations relevant to leek pseudo-stems may be significantly useful in leek selection based on high antioxidant content. elemental composition the beneficial effect of many mineral elements to human health has arisen a remarkable interest to the chemical composition of vegetable crops, such as leek (koca and tasci, 2016). investigations of element content in a. porrum plants have disclosed this species ability to accumulate high concentrations of minerals, but so far assessments of the leek whole profile relevant to mineral elements and to the varietal features connected to their accumulation have been lacking. the analysis of twenty-five element content in leek pseudo-stems (tables 3-5) has allowed to assess the varietal differences in elemental profile. the concentration of calcium was higher in pseudo-stems grown in greenhouse, whereas potassium and magnesium attained higher levels in open field. sodium, geneity, consequent to genotype selection based on morphological types (light-green summer type, darkgreen winter type and intermediate autumn type). interestingly, in our research the concentration of polyphenols in leek leaves has always been higher than the pseudo-stem one, raising the issue of possible crop waste valorization as a source of these antioxidants. among the components of plant antioxidant system, selenium also plays a significant role. indeed, though it is not an essential element for plants, selenium is able to provide a powerful antioxidant defense to plants against drought, salinity, frost, flooding, uv light and herbivore (malagoli et al., 2015). notably, allium species belong to the secondary selenium accumulators, which show a remarkable tolerance to high concentration and consequent accumulation of this element due to se ability to substitute sulfur in natural compounds, as previously reported in leek (koca and tasci, 2016). in our research, a. porrum grown in moscow region showed a se accumulation range from 60 to 107 µg kg1 d.w., which is much lower than the values recorded in turkey (koca and tasci, 2016). this suggests the significant effect of selenium status in the environment on plant ability to concentrate this microelement. the adverse correlation between selenium content in leaves and pseudo-stems (r= 0.95 at p≤0.01), similar to that recorded for polyphenols, entails a rather stable level of selenium accumulation in plant. reports relevant to selenium in plant secondary metabolites, as well as to polyphenols particularly in absence of selenium uptake are rather scarce and often controversial. in this respect, a positive correlatable 3 macroelement concentration in a. porrum pseudo-stems (g kg-1 d.w.) ns not significant; * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. treatment calcium potassium magnesium sodium phosphorus crop system open field 4.2 22.5 1.0 0.27 2.83 greenhouse 4.8 18.9 0.8 0.28 2.73 * * * ns ns cultivar goliath 3.7 bc 58.7 a 0.8 c 0.32 bc 3.12 b premier 3.1 cd 5.4 e 0.8 c 0.36 b 2.74 bc bandit 10.4 a 26.3 b 2.1 a 0.75 a 2.53 cd kalambus 4.0 b 15.2 c 0.6 d 0.16 e 2.43 cd cazimir 4.3 b 9.0 d 0.6 d 0.15 e 2.08 d giraffe 2.6 d 17.9 c 0.7 cd 0.12 e 2.49 cd camus 4.4 b 19.6 c 1.1 b 0.16 e 4.07 a vesta 3.9 bc 16.9 c 0.7 cd 0.18 de 2.77 bc summer breeze 4.4 b 17.7 c 0.8 c 0.26 cd 2.80 bc 268 adv. hort. sci., 2019 33(2): 263-270 phosphorus and all the microelements and heavy metals analyzed were not significantly affected by the crop system (tables 3-5). moreover, the potassium concentration was positively correlated with the ash content (r= 0.78 at p≤0.01). from the comparison between the nine leek cultivars it arose that the three cultivars premier, goliath and cazimir had contrasting features from each other. indeed, goliath was characterized by the highest content of k, fe, b, zn and se, and the lowest of cd. premier accumulated preferably со, i, al, as, cd, ni, pb and sr, but poorly cu and zn. cazimir showed the highest concentration of na and zn, but the lowest of k, i, se, cr and ni. among the minerals examined, the highest correlation coefficients were recorded between al and as, pb, v, со and li. indeed, the physiological role of al in plants has not been completely understood so far, though this element is supposed to both activate at low doses some enzymes and control membrane permeability (ahn and matsumoto, 2006). lithium also showed wide varietal differences, consistently with previous reports (kabata-pendias and pendias, 2010). the correlations recorded in our research are in agreement with those found in five species grown both in ecological unpolluted and in oil-polluted areas of nigeria (essiett et al., 2010). as for selenium, though the leek varietal differences are rather low compared to other elements, the significant correlation recorded between se and k is a remarkable characteristic of this allium species and it has been very scarcely investigated so far. in spinach, the fertilization with sodium selenate increased the potassium content in the female plants but not in the male ones (golubkina et al., 2017), whereas in other research (põldma et al., 2011) garlic biofortification led to selenium antagonistic activity towards k. taking into account that potassium participates in plant protection against all forms of biotic and abiotic stress along with selenium and other antioxidant compounds (wang et al., 2013), the close relationship between the two minerals in leek suggests intensive interactions between all components of the defense system. indeed, potassium was predominant in leek elemental composition, showing significant correlations with both ash (r= 0.78 at p≤0.01) and polyphenols (r= 0.96 at p≤0.01). the known ability of potassium to decrease the activity of polyphenol oxidase in plants and enhance polyphenol accumulation (mudau et al., 2007) may be a good explanation of the positive correlation between polyphenols and potassium in leek plants. the active table 4 microelements concentration in a. porrum pseudo-stems (mg kg-1 d.w.) table 5 heavy metal concentration in a. porrum pseudo-stems (mg kg-1 d.w.) within each row, means followed by different letters are significantly different according to duncan test at p≤0.05. within each row, means followed by different letters are significantly different according to duncan test at p≤0.05. element goliath cazimir premier vesta kalambus summer breeze bandit giraffe camus b 20.5 a 14.8 bc 16.3 b 9.5 de 8.5 e 9.4 de 9.4 de 12.3 cd 10.7 d co 0.08 b 0.05 d 0.10 b 0.03 d 0.29 a 0.04 d 0.04 d 0.04 d 0.10 b cu 4.7 df 4.4 ef 3.4 g 5.7 bc 6.4 ab 5.0 ce 6.8 a 4.0 fg 5.3 cd fe 206 a 110 c 168 b 97 bd 74 e 80 de 93 ce 100 cd 215 a i 0.06 bc 0.04 c 0.31 a 0.04 c 0.06 bc 0.06 bc 0.04 c 0.08 b 0.08 b li 0.11 b 0.04 c 0.15 a 0.03 c 0.01 c 0.03 c 0.02 c 0.03 c 0.11 b mn 12.2 c 12.0 c 22.6 a 9.8 c 6.3 d 9.5 c 10.6 c 19.2 b 21.7 ab si 14.1c 10.5 d 27.3 a 9.2 e 12.8 cd 13.1 cd 11.0 de 18.8 b 15.4 c sn 0.15 c 0.22 b 0.02 d 0.16 c 0.48 a 0.18 bc 0.51 a 0.22 b 0.23 b zn 23.5 ab 26.7 a 11.8 f 18.1 de 16.0 e 19.2 ce 21.5 bc 22.2 bc 21.2 bd element goliath cazimir premier vesta kalambus summer breeze bandit giraffe camus al 78.0 c 29.5 d 126.0 a 21.0 df 7.7 g 24.1 de 12.2 fg 19.3 ef 88.4 b as 0.03 b 0.02 bc 0.05 a 0.02 bc 0.01 c 0.02 bc 0.01 c 0.02 bc 0.06 a cd 0.08 d 0.10 bc 0.18 a 0.11 b 0.08 d 0.06 d 0.11 b 0.17 a 0.11 b cr 0.13 c 0.08 g 0.48 a 0.10 df 0.10 eg 0.12 cd 0.09 fg 0.15 b 0.11 ce ni 1.05 a 0.46 c 0.96 ab 0.98 ab 0.56 c 0.57 c 0.83 b 0.60 c 1.08 a pb 0.34 b 0.28 bc 0.83 a 0.10 e 0.10 e 0.13 de 0.20 cd 0.12 e 0.83 a sr 28.3 ab 25.3 c 30.4 a 24.6 c 28.2 ab 17.3 d 28.8 ab 26.4 bc 28.5 ab v 0.21 b 0.07 cd 0.28 a 0.07 cd 0.04 d 0.09 c 0.07 cd 0.07 cd 0.30 a golubkina et al. crop system and genotype effects in allium porrum 269 participation of potassium in the antioxidant defense system of this allium species is also characterized by positive correlation of the element with the ascorbic acid content (r= 0.95 at p≤0.01). in this respect, the results of the present work reveal the close relationship between the main components of the leek antioxidant system, including polyphenols, ascorbic acid, selenium and potassium. in our research, the lowest negative correlation coefficients were recorded between selenium, chromium and iodine (table 6). se is known as an antagonist of cr and its protective role towards cr has been previously reported (qing et al., 2015). the interaction between se and i is more complex; both the elements are not essential for plants, but at low concentrations they may improve plant growth, development and protection from biotic and abiotic stresses (pilon-smits, 2015). separate plant fortification with se and i showed the possibility of mutual stimulation by the two elements (golubkina et al., 2018 a). the selective accumulation of selenium in the spinach male plants and of iodine in the female ones suggests the participation of phytohormones in the interactions between selenium and iodine (golubkina et al., 2017). with regard to heavy metals, highly significant correlations were found between v and al, as, со, pb and fe (table 6). 4. conclusions from research carried out in moscow region with the aim to assess the effects of open field or greenhouse conditions on yield and quality performances of nine leek (a. porrum) cultivars under organic farming, useful remarks have been drawn. the genotypes examined had a uniform behavior with both the crop systems, showing higher yield and dry matter when grown in the protected environment, but better quality and antioxidant performances in 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lime (citrus aurantifolia), and sour orange (citrus aurantium) fruit peel (500, 1000 and 2000 µl l-1 concentrations), cinnamon (cinnamomum cassia) bark and summer savory (satureja hortensis) aerial parts (400, 500 and 600 µl l-1 concentrations) were used on penicillium digitatum mycelium. none of the eos extracted from tested citrus in this study could inhibit mycelial growth completely even at concentration of 2000 µl l-1. the best results were obtained with cinnamon and summer savory eos at concentration of 500 and 600 µl l-1. so, based on the results, cinnamon and summer savory eos can be ideal candidates to replace the synthetic fungicides to control postharvest green mold of citrus fruit. gc-ms analysis showed that the most abundant of all constituents in eo extracts were carvacrol and γ-terpinene in summer savory and (e)-cinnamaldehyde in cinnamon. 1. introduction citrus spp. are the most important produced fruits in the world (sharma and saxena, 2004), due to their good taste, useful nutrients, and widespread availability (liu et al., 2012). nevertheless, the high water content and nutrient composition make them also very susceptible to decay by pathogens after harvest (tripathi and dubey, 2004). one of the most common diseases that infects citrus fruit is green mold caused by penicillium digitatum (zheng et al., 2005). the yield losses and the worsening of the quality caused by the fungus are economically important. this pathogen infects the fruit through wounds on the peel inflicted during harvest, transportation, handling or commercialization. penicillium digitatum is one of the most important pathogen in citrus industry, because one generation of green mold complete during 7-10 days in rotten fruit at 20-25°c, and the large amounts of spores are disseminated easily by air currents (palou, 2014). currently, the use of synthetic fungicides is the primary and most sim(*) corresponding author: ramezanian@shirazu.ac.ir citation: khorram f., ramezanian a., saharkhiz m.j., 2018 in vitro activity of some essential oils against penicillium digitatum. adv. hort. sci., 32(4): 487-493 copyright: © 2018 khorram f., ramezanian a., saharkhiz m.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 january 2018 accepted for publication 21 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 487-493 488 ple method for the control of postharvest diseases of citrus fruit (palou et al., 2008). however, fungicides consumption is strongly becoming restricted because of residual toxicity, carcinogenicity, long degradation period and increasing human health concerns (tripathi and dubey, 2004; palou et al., 2008). recently, researchers have been interested in development of alternative methods to manage postharvest decay. the essential oils (eos) are one of non-chemical and useful control options for the management of fungal postharvest diseases (sassi et al., 2008). essential oils are complex compounds that are natural and environmentally friendly, having antioxidant, antimicrobial and medicinal properties (bakkali et al., 2008). so, they can be ideal candidates to replace synthetic antimicrobials for maintenance of harvested horticultural crops (tripathi and dubey, 2004). many studies reported the beneficial effects of eo treatments for the control of postharvest decay caused by p. digitatum, such as thymus vulgaris at concentration of 1000 ppm (fatemi et al., 2012), mentha spicata and lippia scaberrima at concentrations of 1000 and 3000 μl l-1, respectively (du plooy et al., 2009), bubonium imbricatum at concentration of 1000 ppm (alilou et al., 2008), citrus spp. at concentration of 10% (badawy et al . , 2011), and cinnamomum zeylanicum at concentration of 0.5% (kouassi et al., 2012), thereby enhancing shelf life of fruits and vegetables. the purpose of this study was to investigate the in vitro activity of eos obtained from sweet orange (citrus sinensis), lemon (citrus limon), lime (citrus aurantifolia), and sour orange (citrus aurantium) fruit peel, cinnamon (cinnamomum cassia) bark and summer savory (satureja hortensis) aerial parts for the control of green mold caused by p. digitatum as a preliminary study to find a suitable and effective eo as alternative to synthetic fungicides to control green mold in citrus postharvest management. 2. materials and methods extraction of essential oils plant materials used in this study are shown in table 1. the air-dried plants material (300 gr) were cut into pieces, grounded into powder by blender, then the eos extracted through hydro-distillation for 3-4 hours using a clevenger apparatus (miquel et al., 1976). then the eos were dehydrated with anhydrous sodium sulfate and stored in dark bottles at -20°c before using for antifungal study. isolation of fungus the fungus used throughout this study was p. digitatum, the cause of citrus green mold. for isolation of fungus colony, p. digitatum spores were isolated from a decayed orange and cultured on potato dextrose agar (pda) by the single spore procedure at 25°c. the isolates were maintained on pda until needed. in vitro antifungal assay the antifungal assay was performed on pda plates amended with three concentrations (500, 1000 and 2000 µl l-1) of sweet orange, lemon, lime and sour orange eos and three concentrations (400, 500 and 600 µl l-1) of cinnamon and summer savory eos. tween 80 (merck-kgaa, germany) as an emulsifier was mixed with 80 ml of sterilized and molten pda media, cooled to about 45°c, and then enriched with eos. there were four 80 mm plates/replicates per treatment. after one day, the mycelia of p. digitatum from 4-days-old cultures were put in the center of amended pda petri plates with a cork borer. all of the plates were sealed with parafilm. inoculated plates were kept at 25°c for 8 days. colony diameter was determined daily by measuring the average radial growth (obagwu and korsten, 2003). in order to evaluate its effect on fungal growth, tween 80 (emulsifier) was also considered as a treatment in the experiment. table 1 plant materials used for eos extraction name family used part origin sweet orange (citrus sinensis cv. thomson navel) rutaceae fruit rind tissue (flavedo and albedo) fars-iran lemon (citrus limon cv. lisbon) rutaceae fruit rind tissue (flavedo and albedo) fars-iran lime (citrus aurantifolia cv. mexican lime) rutaceae fruit rind tissue (flavedo and albedo) fars-iran sour orange (citrus aurantium cv. amara) rutaceae fruit rind tissue (flavedo and albedo) fars-iran cinnamon (cinnamomum cassia) lauraceae tree bark china summer savory (satureja hortensis) lamiaceae aerial parts fars-iran khorram et al. essential oils against penicillium digitatum 489 inhibition percentage (ip) of fungal growth was calculated as the radial growth of treated fungus (t) relative to the growth in control (c) treatment (plates without eo and tween 80) according to the following formula: essential oils analysis at the end of the study, the main components of the most effective eos on p. digitatum were analyzed by gas chromatography (gc) and gas chromatography-mass spectrometry (gc-ms). the gc analysis was carried out by the use of agilent gc (7890-a, perkinelmer, usa) and a flame ionization detector. it was done on fused silica capillary hp-5 column. the temperatures of injector and detector were held at 250°c and 280°c, respectively. nitrogen was selected as carrier gas; oven temperature was 60-210°c at a rate of 4°c/min, which was then increased to 240°c at a rate of 20°c/min, and finally, kept for 8.5 min. the gc-ms analysis was performed using an agilent gc series 7890-a (perkinelmer, usa) with a fused silica capillary hp-5ms column and 5975-c mass spectrometer (unico, usa). carrier gas was helium. ion source and interface temperatures were set at 230°c and 280°c, respectively. mass range was programmed from 45 to 550 amu. oven temperature was 60-210°c at a rate of 4°c/min. n-alkanes was used as a standard to determine the retention indices for all constituents. the constituents were recognized by comparing their retention indices with literature reports, and their mass spectra comparison with the wiley, adams and mass finder 2.1 library data (adams, 1997). statistical analysis the experiment was distributed according to a split plot in time design. the analysis of variance (anova) was performed. mean comparisons were conducted by lsd (least significant difference) at p≤ 0.01. data were analyzed by sas software (v. 9.1). 3. results and discussion inhibitory effects of different treatments on penicillium digitatum growth the in vitro activity of the tested eos on colony diameter of p. digitatum during 8 days of incubation is summarized in table 2. our results indicated that colony radial growth of p. digitatum was inhibited completely (100%) under in vitro condition by both cinnamon and summer savory eos at 500 and 600 µl l-1 concentrations during 8 days of incubation. also, the mycelial growth table 2 inhibition percentage (%) of plant essential oils on in vitro radial growth of penicillium digitatum * for each column, similar letters (lower case, superscript) are not significantly different according to lsd (p≤0.01) test. ^ means followed by similar letters (subscript), are not significantly different according to lsd (p≤0.01) test. treatment eo concentration (µl l-1) time (day) 1 2 3 4 5 6 7 8 control 0.00 g * s ^ 0.00 g s 0.00 h s 0.00 g s 0.00 e s 0.00 g s 0.00 e s 0.00 e s tween 80 0.80 g q-s 0.43 g rs 0.38 h rs 1.29 g p-s 1.24 e p-s 5.09 fg n-s 7.08 e l-s 8.13 d e k-s sweet orange 500 22.42 f w-i 5.81 fg l-s 10.34 gh i-s 12.73 fg d-q 6.59 e l-s 9.37 fg j-s 6.55 e l-s 0.00 e s sweet orange 1000 23.81 f w-g 5.75 fg m-s 18.02 e-g b-k 23.07 ef w-h 12.56 e d-r 16.26 e-g c-n 11.55 e g-s 0.00 e s sweet orange 2000 75.80 b b 54.74 b e-i 48.24 c i-o 50.72 c g-k 32.09 cd r-b 38.42 cd l-s 41.76 c j-s 36.56 bc o-v lemon 500 32.81 ef r-a 17.41 d-g c-m 20.79 e-g z-j 23.18 ef w-h 13.03 e d-q 12.79 fg d-q 12.19 e f-s 0.00 e s lemon 1000 46.91 c-e l-p 24.46 c-f v-e 38.18 cd m-s 41.15 cd j-s 17.42 c-e c-m 22.27 d-f x-i 20.14 de b-k 12.50 c-e e-r lemon 2000 61.38 b-d d-h 23.54 c-f w-h 45.84 c i-q 50.87 c g-k 34.39 c q-x 44.26 c i-r 34.66 cd p-w 25.31 b-e t-c lime 500 36.98 ef m-u 20.71 d-f a-i 21.95 e-g y-i 17.98 f c-m 15.74 de c-n 16.98 e-g c-n 17.25 de c-n 0.00 e s lime 1000 54.54 b-e e-i 30.49 c-e s-b 44.03 c i-r 48.19 c i-o 30.72 cd s-b 33.27 c-e r-y 33.22 cd r-y 30.85 b-d s-b lime 2000 70.43 bc b-d 42.48 bc i-s 49.22 c h-m 50.62 c g-l 34.11 c q-y 41.72 c j-s 40.39 c k-s 32.50 b-d r-a sour orange 500 48.92 c-e i-n 36.82 b-d n-u 14.64 fg c-o 12.61 fg d-r 11.36 e h-s 16.16 e-g c-n 17.89 de c-m 0.00 e s sour orange 1000 50.94 c-e g-k 24.00 c-f w-f 30.77 de s-b 33.67 de q-y 13.36 e c-p 15.97 e-g c-n 14.85 de c-o 9.37 de j-s sour orange 2000 61.44 b-d d-h 33.01 c-e r-z 44.12 c i-r 53.39 c f-j 33.12 c r-y 37.28 cd m-t 41.08 c j-s 40.94 b k-s cinnamon 400 40.52 d-f k-s 12.63 e-g d-r 24.77 ef u-d 22.22 ef x-i 3.34 e o-s 6.48 fg l-s 9.40 e j-s 3.44 e o-s cinnamon 500 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a cinnamon 600 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a savory 400 100.00 a a 89.61 a a 74.01 b bc 70.91 b b-d 66.01 b b-e 65.25 b b-f 61.83 b c-g 51.25 b g-k savory 500 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.0 0 a a 100.00 a a 100.00 a a 100.00 a a savory 600 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a adv. hort. sci., 2018 32(4): 487-493 490 was decreased by cinnamon and summer savory eos at concentrations lower than 500 µl l-1, but it was not suppressed completely. the main activity of the eos in the postharvest fruit are derived from their ability to inhibit pathogen growth (periago et al., 2004). cinnamon eo has the potential to be employed as a natural antifungal agent for fruit disinfectation, as cinnamaldehyde is its main constituent (xing et al., 2010). furthermore, it has been reported that summer savory contains some substances with antibacterial properties (deans and svoboda, 1989). in this research, cinnamon and summer savory eos have the strongest effect on p. digitatum growth (table 2). it has been reported that eucalyptus and cinnamon (cinnamomum zeylanicum, blume) oil vapour (500 ppm) reduced decay almost by 50% in tomatoes after 10 days of storage (tzortzakis, 2007). moreover, win et al. (2007) presented that eos from cinnamon at the concentration of 5.0 g l-1 completely inhibited conidial germination and mycelial growth of all fungi on banana (colletotrichum musae, fusarium spp. and lasiodiplodia theobromae). in addition, lopez-reyes et al. (2010) showed that summer savory, oregano and thyme eos at 10% showed significant inhibitory effect (similar to chemical control) against p. expansum and botrytis cinerea on four cultivars of apples. the mechanism by which eos suppress the microbial growth is not fully understood, but a number of possible explanations have been postulated. essential oils are lipophilic and this property enables them to preferentially move from an aqueous phase into fungi membrane. this action leads to membrane expansion, increasing in membrane fluidity and permeability, membrane proteins disorder, respiration rate control, change of ion transportation in fungi and induced cellular contents leakage (burt, 2004; oonmetta-aree et al., 2006; khan et al., 2010; fadli et al., 2012). in the present study, the lowest inhibition was observed in control plates that contained only pda (0%); however, this was not significantly different from plates containing pda and the tween 80 without the eos during 8 days. so, results indicated that the tween 80 used as an emulsifier had no effect on the mycelial growth (table 2). we observed an increase of antifungal effects of the tested citrus fruits peel eos such as sweet orange, lemon, lime and sour orange as the eos concentration increased, but the fungi growth was not inhibited completely even at concentration of 2000 µl l-1. so, as the results showed, none of the tested concentrations of citrus fruits eos in this study could inhibit radial growth completely (table 2). essential oils are present in great quantities in the flavedo of citrus fruit (caccioni et al., 1998). the citrus fruits eo consists a mixture of components such as terpenes, hydrocarbons, ketones, aldehydes, alcohols, acids, and esters. the amount of them depends on the citrus cultivar, the extraction and separation techniques (fisher and phillips, 2008). the positive effect of the volatile components of citrus fruit essential oils on p. digitatum and italicum growth has been reported (caccioni et al., 1998). the spore germination and mycelium growth of p. italicum and digitatum were stimulated by the essential oil of citrus reticulata blanco at concentration of more than 2.5 μl ml-1 (wang et al., 2012). moreover, badawy et al. (2011) reported that citrus aurantifolia eos had the antifungal effects against p. digitatum pathogens at concentration of 10% (v/v). however, in our study the application of citrus spp. could not provide acceptable control of green mold disease. analysis of the summer savory and cinnamon eos the analysis of the volatile profiles in summer savory and cinnamon eos are listed in table 3 and 4, table 3 chemical composition of the summer savory essential oil * retention index number component ri* (%) 1 αthujene 924 1.15 2 α-pinene 932 0.64 3 camphene 946 0.06 4 hepten-1-ol 958 0.05 5 sabinene 969 0.01 6 β-pinene 974 0.21 7 3myrcene 988 1.15 8 phellandrene 1002 0.23 9 α-terpinene 1014 3.75 10 p-cymene 1020 2.19 11 sylvestrene 1025 0.37 12 e-βocimene 1044 0.07 13 γ-terpinene 1054 31.98 14 terpinolene 1086 0.05 15 trans-α sabinene hydrate 1098 0.07 16 isoborneol 1155 0.06 17 terpinene-4-ol 1174 0.2 18 α-terpineol 1186 0.1 19 carvacrol methyl ether 1241 0.09 20 thymol 1289 0.8 21 carvacrol 1298 55.66 22 thymol acetate 1349 0.03 23 carvacrol acetate 1370 0.07 24 caryophyllene 1417 0.36 25 aromadendrene 1439 0.08 26 α–humulene 1454 0.01 27 bicyclogermacrene 1500 0.18 28 bisabolene 1505 0.21 29 unknown 0.01 30 spathulenol 1577 0.04 khorram et al. essential oils against penicillium digitatum 491 respectively. a total of 30 different components of summer savory, and 37 components of cinnamon were identified and isolated by gc and gc-ms from the eos. the principal components of the summer savory eo were carvacrol (55.66%), γ-terpinene (31.98%), α-terpinene (3.75%), p-cymene (2.19%), 3myrcene (1.15%), and α-thujene (1.15%). the major components of the cinnamon eo were (e)-cinnamaldehyde (70.04%), α-copaene (10.82%), δcadinene (5.35%), α-muurolene (4.23%), and γmuurolene (1.25%). other constituents which were less than 1% have been shown in table 3 and 4. as shown in table 2, both summer savory and cinnamon eos were equally effective in inhibiting the growth of p. digitatum. this is in accord with the reported in vitro inhibitory effect of carvacrol against pathogens (periago et al., 2004). in fact, the main component of summer savory eo is a phenol (sacchetti et al., 2005), and its most important mechanism of antimicrobial activity is connected with the phenolic ring in its chemical structure (ultee et al., 2002). furthermore, it has been reported that the toxicity rate of the phenol ring is due to the site (s) and number of hydroxyl groups (cowan, 1999). concerning cinnamon eos, its major volatile compound is cinnamaldehyde. moreover, it has been reported that cinnamon eo had potent anti-bacterial and anti-fungal activities due to cinnamaldehyde (ooi et al., 2006), because it acts as membrane irritants (nabavi et al., 2015). 4. conclusions in this study, the in vitro activity of plants eos against p. digitatum were tested at different concentrations during 8 days of incubation at 25°c. as showed by the results, the stronger inhibitions were obtained by cinnamon and summer savory eos at concentration of 500 and 600 µl l-1. none of the citrus eos could inhibit fungus radial growth completely compared with cinnamon and savory eos. gc-ms analysis showed that the most abundant of all constituents in eo extracts were carvacrol and γ-terpinene in summer savory and (e)-cinnamaldehyde in cinnamon. acknowledgements we thank shiraz university research council for financial supports, fars agricultural and natural resources research and education center, shiraz, iran for technical assistance. references adams r.p., 1997 identification of essential oil components by gas chromatography/mass spectroscopy. j. am. soc. mass spect., 6(8): 671-672. alilou h., akssirar m., hassani l.m.i., chebli b., el hakmoui a., mellouki f., rouhi r., boira h., blázquez m.a., 2008 chemical composition and antifungal activity of bubonium imbricatum volatile oil. phytopathol. mediterr., 47(1): 3-10. badawy f.i., sallam m.n., ibrahim a., asran, m., 2011 efficacy of some 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lu y., 2010 antifungal activities of cinnamon oil against rhizopus nigricans, aspergillus flavus and penicillium expansum in vitro and in vivo fruit test. int. j. food sci. technol., 45(9): 1837-1842. zheng x.d., zhang h.y., sun p., 2005 biological control of postharvest green mold decay of oranges by rhodotorula glutinis. eur. food res. technol., 220(34): 353-357. impaginato 113 adv. hort. sci., 2018 32(1): 113-135 doi: 10.13128/ahs-21330 bio-techniques for improvement of qualitative and quantitative traits in walnut (juglans regia) u.n. shah 1, j.i. mir 2, n. ahmed 3, a. zaid 4, s. jan 3, k.m. fazili 1, s.h. wani 5 (*) 1 department of biotechnology, university of kashmir, srinagar, jammu and kashmir, india. 2 central institute of temperate horticulture (icar), srinagar, jammu and kashmir, india. 3 sher-i-kashmir university of agricultural science and technology, jammu and kashmir, india. 4 plant physiology and biochemistry laboratory, department of botany, faculty of life sciences, aligarh muslim university, 202002 aligarh, india. 5 mrcfc, khudwani, sher-i-kashmir university of agricultural science and technology, jammu and kashmir, india. key words: antioxidants, molecular markers, nutraceuticals, polyphenols, phytochemicals, walnut. abstract: walnut, juglans regia (l.) is an economically significant plant for its immense nutritive and economic value. the breeding character of walnut has lent it a wide diversity in genetic characteristics. the principal vegetative and common traditional agronomic traits together with biochemical characterization i.e., karyotyping and isoenzyme expression have been the early research methods. however, these techniques are time-consuming and susceptible to the environmental variations. literature is meager in the distribution, applied applications in general and the use of agriculture biotechnology in particular in case of walnut plants. the bio-techniques like molecular markers are adequate in number and there is little or no diversity in the method employed for research on walnuts. despite basic research method, the organization of information, its retrieval and presentation structures, form elaboration experienced immense advancement via molecular markers such as rflp, issr, rapd aflp, ssr and snp. this appraisal in its first part provides detailed information regarding the present scenario of data on biogeographical distribution, health benefits of walnut worldwide and current applications in the agroforestry management, biochemical evaluations and applied uses of a walnut tree which is relevant for both basic and applied research. the review in its second part sheds light on the application of sophisticated agricultural biotechnology techniques such as use of molecular markers to evaluate, realize the full potential of walnut for increasing its quality, quantity and for its sustainable production which cannot be obtained through usual breeding techniques to meet the demands of a projected world population. (*) corresponding author: shabirhussainwani@gmail.com citation: shah u.n., mir j.i., ahmed n., zaid a., jan s., fazili k.m., wani s.h., 2018 bio-techniques for improvement of qualitative and quantitative traits in walnut (juglans regia). adv. hort. sci., 32(1): 113-135 copyright: © 2018 shah u.n., mir j.i., ahmed n., zaid a., jan s., fazili k.m., wani s.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 september 2017 accepted for publication 27 october 2017 ahs advances in horticultural science review paper adv. hort. sci., 2018 32(1): 113-135 114 1. origin and history of walnut (juglans regia l.) walnut juglans regia (l.) is commonly recognized as “jupiter’s royal acorn”. english walnut is commonly known as “persian walnut” as it originated from ancient persia and was adorned as for royalty. juglans regia (l.) species is extensively cultivated in the temperate zones of the world for its best quality nuts and procurement of commercial timber (mcgranahan and leslie, 1991, 2009). lateral fruit bearing walnut exhibit more genetic variation particularly among familial varieties. in ancient times, the recognition of walnut was spread vigorously through caravans via sea trade. greek and romans have lately acquired trend towards walnut cultivation to avert the speckled walnut growing along the coastal areas. 2. botanical description of walnut plant morphology juglans regia (l.) is a deciduous perennial tree belonging to the family juglandaceae and grown chiefly for its edible seeds. walnut tree is predominantly an extensive canopy-dweller decurrent tree with trunk size ranging from 1.5-2 meters in diameter, 25-35 m in height and its survival rate is about 200 years. walnut bears smooth bark with olive brownish color adapted to moderate light conditions. leaves are 30-40 cm long, compound, alternately arranged and odd-pinnate. male flowers are produced in drooping catkins and the female flowers, in clusters of two to five, are terminal, which ripens in the autumn into a fruit with a semi-fleshy husk and a brown nut. the kernel of the nut is protected by a corrugated woody shell. flowering, pollination and fruiting juglans are monoecious species, having male and female reproductive organs on separate flowers on the same tree. juglans regia is self-fertile, heterogamous and it can be protandrous or protogynous depending on cultivar. the characteristic inflorescence is catkin bearing about 2-3 pistillate flowers born terminally or laterally. walnut is self-fertile however occasionally it requires different cultivar due to dichogamy. majority of walnuts are protandrous and anemophilous. nuts are borne individually or in groups enumerating 2-3 on shoot tips. walnut is properly shielded by a fleshy covering which cracks at maturity. walnut shape is variable from ovoid to round attaining a maximum diameter of 2 meters enclosing two kernels divided by extremely delicate covering expanding from inner cover of the shell. taxonomical classification walnut is a member of juglandaceae family and includes approximately 60 species, among which 21 species are classified under genus juglans (table 1). different varieties of walnut and their commercial uses are given in table 2. all walnut species are edible and english walnut is easily crackable and enormous in size. different sections and species of walnut genus juglans is classified into four types: juglans sect. cardiocaryon, juglans sect. juglans, juglans sect. rhysocaryon, and juglans sect. trachycaryon. juglans sect. cardiocaryon. this variety originated from the northeast asia. this walnut variety bears enormous leaves containing 15 leaflets. the wood is commercially soft, and nuts are thick shelled borne in a raceme inflorescence, such as, for example, j. mandshurica and j. ailantifolia. juglans sect. juglans. originated from the southeast europe to central asia. this variety bears large leaves with 9 broad hairless leaflets having entire margins. the wood is commercially hard, for example, j. regia (l.) and j. sigillata. juglans sect. rhysocaryon. (black walnuts) originated from the north america and the south america. this variety bears enormous leaves with 20 table 1 taxonomic classification of walnut kingdom plantae phylum tracheophyta class magnoliopsida order fagales family juglandaceae subfamily juglandoideae tribe juglandeae subtribe juglandinae genus juglans species ● j. regia linnaeus ● juglans ailanthifolia carr. ● juglans ailantifolia carriere ● juglans bixbyi rehd. ● juglans boliviana (c. dc.) dode ● juglans californica s. wats. ● juglans cinerea linnaeus ● juglans hindsii (jepson) jepson ex r. e. sm. shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 115 table 2 different varieties of walnut and their commercial uses no. species description commercial uses 1 english/persian walnut (juglans regia) native to the region of california, oregon, himalayas, europe and china. most ubiquitous cultivated in foothills having high water table. characterized by a relatively thin, gnarled shell enclosing a smooth, large, ivory-colored nut and rich flavor. as an effective nutraceutical with a high nutritive value. procurement of high quality timber in the furniture industry for high-end flooring, guitars, furniture, veneers, knobs and handles as well as gunstocks 2 eastern black walnut (juglans nigra) native to eastern north america. characterized by a thick, hard shell with sharp, jagged edges and a darker color. black nuts are known for their pungent aroma and robust flavor. used in timber industry as well as nut production. the hardblack walnut shell is also used commercially in abrasive cleaning, a filtering agent in scrubbers in smoke stacks, cleaning jet engines, cosmetics, and oil well drilling and water filtration 3 white/ butternut (juglans cinerea) native to the eastern united states and southeast canada. characterized by slow-growing species, and rarely lives longer than 75 years. bark is light grey in colour and the whole leaf is downy-pubescent, and a somewhat brighter, yellower green than many other tree leaves. leaves are alternate and compound and have odd number of leaflets with a terminal leaflet used in lumber industry to make furniture, and is a favorite of woodcarvers. it is used to dye fabrics. used as a medicine owing to its cathartic properties 4 japanese walnut/ heartnut (j. ailantifolia) native to japan and sakhalin. characterized by light grey bark, pinnate and brighter yellower green leaves, nuts produced in bunches of 4-10 and are of spherical shape the edible nuts have an oily texture. the husks are also used to make a yellowish dye 5 manchurian/ chinese walnut (j.mandshurica) native to the eastern asiatic region (china, russia and korea). characterized by odd, alternate, pinnate, long and broad leaves. shell is thick and the kernels are edible but small and difficult to extract. also contain less quantities of allelopathic compounds like juglone the timber is in use but is less valuable as compared to english or black walnut. cultivated as an ornamental in colder temperate regions 6 iron walnut (juglans sigillata) native to the yunnan, guizhou, sichuan and xizang in china. characterized by oval-shaped nuts with bumps and ridges. cultivated for its edible nuts used as ornamental plant in gardens and parks 7 california walnut (juglans californica) endemic to california and generally found in southern california coast ranges, transverse ranges, and peninsular ranges, and the central valley. characterized by a large shrub or a small tree. it has a small hard nut in a shallowly grooved, thick shell that is difficult to remove. it has a better flavour than juglans nigra raw as well as cooked seeds are used in pies, cakes, biscuits, confections etc. an attractive wood, but the frequent branching pattern of the trunk limits the use of this wood commercially. also has a medicinal value 8 brazilian/ argentine walnut (j. australis) native to argentina and bolivia. characterized by dense, hard and strong wood. its more frost resistant than j. regia nutritive value. the immature fruits are pickled whole for human consumption. the mature nuts are also eaten. the concentrated extract of the husk is also used as a vermifuge 9 northern california walnut (juglans hindsii) endemic to northern california. characterized by smooth, brown, thick shell, that contains a small edible nutmeat commercially important as a rootstock for orchard stock of juglans regia. also used as an ornamental tree in wildlife gardens, and for habitat gardens, natural landscaping projects. its wood commonly called claro walnut is used in the lumber industry 10 west indian walnut (west indian walnut) not native to jamaica but found in cuba, the dominican republic, haiti, and puerto rico. characterized by a drupe 2-3 cms long with a black husk and a seed, which is an edible walnut meat, inside. it is listed as an endangered species in the endangered species act of the united states used in timber industry. the attractive wood is similar to that of the black walnut 11 cedro negro (juglans olanchana) native to costa rica, guatemala, el salvador, honduras, mexico and nicaragua. commonly called cedro negro. characterized by long branches bearing twigs tipped with 40-50 cm long, glabrous, pinnately compound leaves, darker on the top than on the bottom. the base of the trunk sometimes has buttresses used in the timber industry for light construction, cabinetmaking, parquet floors, luxurious furniture, turnery, musical instruments, and veneer. the husk is used to dye leather 12 colombian walnut (juglans neotropica) native to colombia, ecuador, and peru. characterized by slowgrowing tree up to 40 mts height with grooved, red-brown bark and an oval-shaped canopy. leaves are compound with a serrated border nuts are used as food. used in timber industry because the hard, durable wood is highly prized in cabinetry, flooring, veneers, utensils, and other forms of decoration adv. hort. sci., 2018 32(2): 113-135 116 leaflets, serrate margins. the wood is commercially very hard, such as, j. californica, j. hindsii, j. australis, j. nigra, and j. olanchana. juglans sect. trachycaryon. originated from the eastern north america. this variety bears wide leaves containing 20 leaflets having serrate margins. the wood is commercially soft and the fruits are borne in clusters of two to three. the nuts have a thick and rough shell such as j. cinerea (l.). 3. world walnut production walnut grows abundantly in temperate areas of the world and cultivated commercially in 48 countries on an area of 1.6 million acres. the worldwide production of walnuts has reached maximum particularly in countries of asia. global walnut production for the year 2015-16 rose 155,000 tons from the previous year to 2.1 million tons, with china and the united states accounting for over 75 percent of total production (usda, 2016). china is the world’s chief producer of walnut and accounts for 51.49% of world’s walnut production followed by the usa (26.86%), european union (6.02%), ukraine, chile, turkey and india contribute 5.25%, 3.09%, 3.09%, 1.96%, respectively to the world’s walnut production (usda, 2016). global walnut production for the year 2016-17 is nearly unchanged from the october 2016 record forecast of 2.1 million tons in-shell basis, with china and the united states accounting for nearly 80 percent of total production (usda, 2016). figure 1 gives the schematic representation of year wise production of walnuts across the world. 4. status of walnut in india walnuts are commonly called “akhrot” in india and they are grown in the northwestern himalayan belt, expanding up to sikkim and darjeeling. but the commercial farming of walnut is limited to the states of arunachal pradesh, himachal pradesh, uttarakhand and jammu and kashmir. the area and production of walnut in india for the year 2014-15 were recorded to be 125,000 ha and 206,000 tons respectively (government of india, 2015). india has exported 3,291.71 mt of walnuts worth of rupees. 117.92 crores during the year 2015-16 (apeda, 2016). figure 2 gives the schematic representation year wise area versus production of walnut in india. the major walnut importing countries from india are vietnam social republic (15%) followed by egypt arab republic (11%), netherland (11%), united kingdom (9%), spain (8%), united states (7%), germany (6%), france (4%), thailand (3%), australia (3%) and others (23%) (ministry of agriculture, 2015) (fig. 3). jammu and kashmir is the main center for commercial walnut production in india and contributes pretty nearly 98% of the country’s output and the fig. 1 schematic representation of walnut year wise production across the world. fig. 2 shows the schematic representation of walnut production during three consecutive years in india. fig. 3 showing principal countries which imports walnuts from india. shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 117 state has been declared as the “agri. export zone for walnuts” (isher et al., 2016). most walnuts that are produced for export by india are principally produced in this state. according to the united nations development programme special unit for southsouth cooperation (undp su/ssc), 63,000 hectares of jammu and kashmir state is under walnut cultivation. these plantings produce around 60,000 tones of walnuts worth an estimated 25 million rupees. 5. walnut as phytochemical walnut is popularly known for its abundant phytochemicals for instance phenolics, fatty acids, melatonin and serotonin (christopoulos and tsantili, 2015). in addition to kernels, green shells, walnut husk, seeds, bark and leaves are utilized in the cosmetic and pharmaceutical sector (negi et al., 2011; fernández-agulló et al., 2013; vinson and cai, 2012). table 3 gives a description of walnut and its phytoconstituents in diverse walnut varieties. the level of phenolics in walnut depends on diverse ecological factors, genotype, type of cultivar, geographical site, climatic parameters and developmental stages (solar et al., 2006; amaral et al., 2008). toivonen and hodges (2011) illustrated tissue and age specific variation in phenolic acids. walnut polyphenols are potential candidates which work against in vitro plasma and low-density lipoprotein ldl oxidation. regueiro et al. (2014) reported several individual phenolic compounds in walnut kernels and characterized approximately thirty-seven compounds. colaric et al. (2005) demonstrated a greater amount no. walnut name part used phytochemical extracted reference 1 persian walnut kernels phenolic acids (gallic acid, ellagic acid, syringic acid, chlorogenic acid, p-coumaric acid) and flavanols (catechin, epicatechin, gallocatechin, procyanidin b2, epigallocatechin, epicatechin gallate) figueroa et al., 2016 2 persian walnut kernels ellagic acid fukuda et al., 2004; christopoulos and tsantili, 2012 3 heartnut juglans ailanthifolia var. cordiformis) and persian walnut (juglans regia l.) kernels ellagic acid li et al., 2006 4 persian walnut leaves phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, ellagic and trans-cinnamic) flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone nour et al., 2012 5 persian walnut bark gallic acid, chlorogenic acid, catechine hydrate;resorcinol; caffeic acid, rutin trihydrate, syringic acid, vanillicacid,p-coumaric acid,quercitine dihydrate, naphto-resorcinol, trans-cinnamic acid etc noumi et al., 2012 6 persian walnut fruit phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salicylic, ellagic and trans-cinnamic acid) flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone. cosmulescu et al., 2014 7 persian walnut walnut husks chlorogenic acid, caffeic acid, ferulic acid, sinapic acid, gallic acid, ellagic acid, protocatechuic acid, syringic acid, vanillic acid, catechin, epicatechin, myricetin, and juglone. stampar et al. 2006 8 persian walnut kernels and pellicle chlorogenic, caffeic, p-coumaric, ferulic, sinapic, ellagic, and syringic acid. syringaldehyde and juglone colaric et al., 2005 9 persian walnut leaves and husks chlorogenic acid, ferulic acid, gallic acid and trans-cinnamic acid chrzanowski et al., 2011 10 persian walnut leaves juglone thakur, 2011; babula et al., 2006 11 persian walnut axillary shoots (microshoots) phenolic acids (gallaicacid, chlorogenic acid, p-coumaric acid, syringic acid, vanillin) and flavanoids (quercetin, naphthoquinone) and juglone cheniany et al., 2013 12 persian walnut fruit gallic acid, chlorogenic acid, sinapic acid, protocatechuic acid, catechin and juglone jakopič et al., 2009 table 3 description of walnut and its phytoconstituents in diverse walnut varieties adv. hort. sci., 2018 32(1): 113-135 118 of phenolics in walnut skin than in kernels. oliveira et al. (2008) confirmed protective properties of walnut husk exhibiting higher anti-microbial activity. walnut kernels are known to be an immense resource of ellagitannins having higher anti-oxidant property (shimoda et al., 2009). amaral et al. (2008) confirmed that presence of 96% of tannins as major causal agent for the medicinal potential of walnuts along with phenolics and flavanoids. phenolic compounds are known to confer health benefits. juglans regia l. leaves are good sources of flavonoids. many types of research are mainly focused on the extraction/isolation and the antioxidant effect of flavonoids. walnut flavonoids have not been extensively analyzed, however, english walnut leaf was subjected to vigorous chromatographic evaluation (pereira et al., 2007). amaral et al. (2008) and pereira et al. (2007) analysed quercetin 3-galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside and two other partially identified quercetin 3-pentoside and kaempferol 3-pentoside derivatives in walnut leaves along with myricetin as exclusive flavonoid in walnut husk (lugasi et al., 2003; stampar et al., 2006). myricetin exhibit exceptionally high anti-oxidant property along with the potential of imparting beneficial effects on bone heath thereby preventing bone resorption and elevating osteoblastic potential and bone formation (hsu et al., 2007). rutin (quercetin-3-rutinoside) is another significant flavonoid in walnut leaves having elevated levels of anti-oxidant potential that prevent dna oxidation (nour et al., 2012). the previous studies on flavonoids present in juglans regia (l.) leaves provided a conjectural source for an additional study on the organic decoction from j. regia leaves. the promising results from the medicinal point of view were procured from flavonoids. estimation and characterization of phenolic compounds in walnut leaves can be utilized as an important resource. carvalho et al. (2010) reported phenolics and associated anti-oxidant characteristics of methanolic and petroleum ether extracts procured from walnut green shells and leaves. seeds methanolic extracts exhibited highest phenolic content approximately 116 mg gallic acid equivalent (gae)/g of extract with ec50 of 0.143 mg/ml using dpph (2,2-diphenyl-1-picrylhydrazyl) scavenging activity followed by leaf and green husk. solvent polarity was directly correlated with the distinct disparity in the activity of samples as observed by the solubility of phenolics compounds in respective solvents. hence, methanol solvent is preferred for phenolics extraction (do et al., 2014) while petroleum ether is nonpolar normally employed for extraction of lesser polar constituents for instance plant pigments, sterols and fatty acids (carvalho et al., 2010). carvalho et al. (2010) reported maximum phenolics fraction (116.22±3.76 mg of gae/g of seed extract followed by leaf 94.39±5.63 mg of gae/g of extract) and green husk (50.18±2.69 mg of gae/g of extract). previous research reports have conformed methanolic extracts as most efficient in radical scavenging activity. al-okbi et al. (2014) reported phenolics content of 47.42 g gae/100 g in ethanolic extract of walnut seed. jalili and sadeghzade (2012) illustrated the average value of 22.16±0.66 (mg/g) of phenolic content in aqueous extracts in diverse walnut cultivars. ghasemi et al. (2011) illustrated the phenolic content of 15.15 to 108.11 mg gallic acid equivalents g-1 of extract and flavonoid content ranging from 3.59 to 22.91 mg quercetin equivalents (qe)g-1 of extract. rahimipanah et al. (2010) reported total flavonoids and phenolics in walnut green husks via aluminum nitrate and folin-ciocalteu colorimetric protocol and their content were 1.44±0.2 mg quercetin and 34.28±1.35mg gallic acid equivalent per gram of dry sample correspondingly. kale et al. (2010) demonstrated flavonoid fraction of walnut bark extracts with maximum quercetin between 3.50 to 32.81 mg/g and phenolics content varied from 20.32 to 44.87 mg/g in the extract. ethyl acetate extract of walnut showing maximum phenolics and flavonoid sustaining antioxidant potential walnut extracts and recommend their utilization for advanced research to identify secondary metabolites. 6. phytopharmacology of walnut all the parts of walnut i.e., bark, leaves, flowers are exclusively used widely in ayurveda, unani, homeopathy and allopathic medicine system (shah et al., 2014). forino et al., 2016 reported conventional uses of walnut in health maintenance. diverse activities of walnut utilized are due to enormous phytoconstituents such as saponins, glycosides, alkaloids, and steroids (muthaiyah et al., 2011). all parts of walnuts particularly leave and husk is extensively used in the pharmaceutical and cosmetic industries (ribeiro et al., 2015). table 4 gives the description of walnut as phytochemical and its activity against diverse pathogens. shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 119 antimicrobial activity antimicrobial potential of walnut especially bark (farooqui et al., 2015) leaves (pereira et al., 2007) and fruits (pereira et al., 2008) have been extensively utilized against various infections. studies have also demonstrated the antimicrobial activity of walnut products, particularly of bark, leaves, fruits and green husk of juglan regia (l.) was subjected to hot and cold extraction and resulting solution exhibited high antibacterial activity against pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, staphylococcus epidermidis, micrococcus luteus, salmonella typhimurium, enterococcus faecalis, bacillus thuringiensis, protomonas extroquens, and proteus sp., as evaluated through agar streak and disc diffusion assays (biyik, 2010; deshpande et al., 2011). antifungal activity aqueous extracts of leaves and bark display antifungal activity against an extensive spectrum of fungi using disc diffusion, agar dilution, agar streak dilution and reddish assays. pereira et al. (2008) demonstrated the enormous anti-fungal potential of walnut extracts refluxed with petroleum ether (b.p. 40-60°c) against candida albicans and cryptococcus neoformans. noumi et al. (2010) demonstrated antifungal properties of juglans regia (l.) alongside oral candida strains. antiviral activity zhai et al. (2007) confirmed inhibition of tobacco mosaic virus (tmv) by 95% ethanol and ethyl acetate leaves extract of j. regia. at a minimum concentration of 1.5 µg/ml of methanolic extract of j. regia, there was distinct inhibition sindhis (mouhajir et al., 2001). antioxidant activity several research reports demonstrated antioxidant activity of ethyl acetate, butanol, ether and aqueous methanol extract of walnut kernels, husks and leaves as evaluated by dpph radical scavenging assay and lipid oxidation inhibition by β-carotene linoleate (oliveira et al., 2008; pereira et al., 2008; carvalho et al., 2010; rahimipanah et al., 2010; table 4 description of walnut as phytochemical and its activity against diverse pathogens walnut part used target cell/ pathogen activity reference juglans regia bark staphylococcus aureus, e. coli antibacterial farooqui et al., 2015 juglans regia bark candida albicans, candida glabrata, and candida parapsilosis strains antifungal noumi et al., 2010 juglans regia kernels mcf-7 (estrogen receptor positive breast adenocarcinoma), kb (oral and mouth), hepg-2 (liver), caco2 (colon), and wrl-68 (liver) anti-proliferative negi et al., 2011 juglans regia leaves tobacco mosaic virus antiviral zhai et al., 2007 juglans regia leaves sindbis virus herpessimplex (hsv), sindbis (sinv), and poliovirus antiviral mouhajir et al., 2001 juglans regia leaves 769-p renal and caco-2 colon cancer cells anti-proliferative carvalho et al., 2010 juglans regia leaves a375 human melanoma cell anti-proliferative shah et al., 2015 juglans regia kernels colo-205 anti-proliferative anjum et al., 2016 juglans regia walnut oil herpes simplex (hsv), parainfluenza (pi-3) escherichia coli, pseudomonas aeruginosa, proteus mirabilis, klebsiella pneumoniae, acinetobacter baumannii, staphylococcus aureus, and enterococcus faecalis, fungi: candida albicans and candida parapsilosis antiviral, antibacterial, antifungal orhan et al., 2011 juglans regia leaves bacillus cereus, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, fungi: candida albicans, cryptococcus neoformans antibacterial and antifungal pereira et al., 2007 juglans regia husks bacillus cereus, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, klebsiella pneumonia, fungi: candida albicans, cryptococcus neoformans antibacterial and antifungal oliveira et al., 2008 adv. hort. sci., 2018 32(1): 113-135 120 qamar and sultana, 2011). consumption of walnuts and walnut skin diet by c57bl/6 mice showed decline oxidative stress as observed by a change in the enzymatic and non-enzymatic system (bulló et al., 2010). furthermore, research report demonstrated ameliorating effect of walnut consumption on ldl oxidizing potential owing to their elevated levels of omega-6 pufas. antidiabetic activity in mediterranean and asiatic countries, air-dried leaves of juglans regia have been employed to treat diabetic symptoms (hosseini et al., 2014), and the potential of walnut leaves to ameliorate hyperglycemia in humans has been evaluated by both in vitro and in vivo. pitschmann et al. (2014) reported inhibition of protein-tyrosine phosphatase 1b and no effect on peroxisome proliferator-activated receptors-gamma (pparγ) in myocytes fed with methanolic extracts from walnut leaves. this study confirmed walnut leaf extracts drastically decline levels of serum fasting hba1c, blood glucose and insulin (hosseini et al., 2014). hence the study concluded hypoglycemic potential of walnut leaves wielding lowering effect on sugar levels in the liver as well as kidney. the mechanisms of action underlying the antihyperglycaemic effect of the walnut leaf extracts were investigated by in vitro studies. inhibition of significant enzymes involved in sugar metabolisms like α-amylase (rahimzadeh et al., 2014), ptp1b (ahmad et al., 2012) and associated processes like glycation and oxidation reactions have been reported with walnut leaves (pitschmann et al., 2014). antihelmintic activity a wide range of anti-worm activities was also reported from bark extracts of walnut. kale et al. (2011) reported antihelminthic activity against eiciniafeotida using reference albendazole. methanolic, ethanolic and benzene extracts of juglans regia (l.) exhibited inhibitory activity against annelids like pheretimaposthuma using standard drug piperazine citrate (upadhyay et al., 2010). anti-inflammatory activity anti-inflammatory potential of walnut is comparatively inadequate. papoutsi et al. (2008) demonstrated anti-inflammatory activity because of ellagic acid. hepatoprotective activity ameliorative effect of walnut polyphenols from kernel pellicle against a decline in glutamyl oxaloacetic transaminase (got) and glutamyl pyruvic transaminase (gpt) was reported in mice model after a single oral administration (200 g/kg) (shimoda et al., 2008). these results confirmed higher values of a hepatoprotective effect than curcumin. 7. walnut and cancer due to the increasing inclination towards plant based drugs, more vigorous initiatives have been taken to action to develop more advanced plant based drug formulations. among the diverse phytochemicals procured from plants, polyphenols are known to be most active candidates against cancer (kim et al., 2016). singh and sukhla (2015) demonstrated polyphenols as potent cancer inhibiting phytoconstituents as revealed by inhibitory effects of walnut on cancer promotion and progression. polyphenols inhibit cancer progression by hampering nuclear factorkappa b (nf-kb) activation. nf-kb is a nuclear transcription factor exhibiting significant role in gene regulation implicated in inflammation and carcinogenesis. upregulation of nf-kb is implicated in cell-cycle progression. polyphenol suppresses nfkb and activator protein-1 (ap-1), its inhibition is considered as a potential step in chemoprevention. polyphenols further induce inhibition in mitogen activated protein kinases (mapk), protein kinases (pk), of the growth-factor receptor (gfr) leading to cell cycle arrest, the onset of apoptosis and decline in angiogenesis (bonfili et al., 2008). mertens-talcott et al. (2005) illustrated on the synergistic, additive, or antagonistic interface of polyphenolic compounds to counteract cancer growth. shah et al. (2015) reported numerous chemical constituents possessing anticancer activity due to omega-3 fatty acids (gerber, 2012), vitamin e (mainly the c-tocopherol form) (albanes et al., 2014), phytosterols (ramprasath and awad, 2015), ellagic acid (eskandari et al., 2016), gallic acid (ho et al., 2013) and flavonoids namely quercetin (firdous et al., 2014), carotenoids (virtamo et al., 2014), and melatonin (travis et al., 2014). hardman (2014) reported 80% inhibition of breast cancer in mice following walnut consumption. due to the presence of omega-3 fatty acids along with phytosterols in walnut breast cancer was declined remarkably. phytosterols bind with estrogen receptors and subsequently decline breast cancer. consumption of walnut diet declined a breast tumour by about 60% in transgenic mouse. hardman (2014) confirmed presence of numerous complex constituents in walnut exhibiting additive or synergistic effect on cancer suppression. further presence of shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 121 γ-tocopherol in walnut exhibit anti-cancer activities against prostate, colon, renal and lungs as demonstrated by anti-proliferative and anti-angiogenic mechanisms (rafieian-kopaie and nasri, 2015). walnut inhibits inflammation by inhibiting endothelin (ma et al., 2010). research on prostate cancer established higher levels of endothelin in men indicating the need for further research to decipher relation between walnut and prostate cancer. reiter et al. (2013) investigated prostate cancer and a determined decline in tumor and lncap (lymph node carcinoma of the prostate) xenograft growth. negi et al. (2011) determined anti-proliferative activity of chloroform and ethyl acetate fractions of walnut in human cancer cell lines such as mcf-7 (michigan cancer foundation-7) (estrogen receptor positive breast adenocarcinoma), kb (oral and mouth), hepg2 (liver), caco2 (colon), and wrl-68 (liver) cancer cell lines. all the cell lines exhibited inhibition against kidney and colon cancer however for 769-p renal and caco-2 colon cancer cells, walnut leaf revealed the elevated rate of anti-proliferative competence than walnut seeds and husk. the observations procured confirmed lack of relationship among total phenols and anti-proliferative activities signifying some class of compounds implicated in cancer inhibition (carvalho et al., 2010). this study was further validated by tsoukas et al. (2015) who demonstrated a converse association between walnut intake and colon cancer incidence. paur et al. (2010) reported dose dependent inhibition in lipopolysaccharide(lps)induced nf-κb activity in the monocytic cell line (u937-κb) by blending clove, oregano, thyme, walnut and coffee. this study confirmed the comparative efficacy of the combination of different extracts than individual plant constituent. hence, walnut can be utilized as a natural source of anti-oxidants as well as chemo preventive candidate. thakur (2011) demonstrated inhibition of carcinogenesis in rats by azoxymethane and can be utilized as chemo preventive agent against neoplasia. during recent years, juglone is established as effective cytotoxic agent and induce cell apoptosis via mitochondria dependent pathway via human lung cancer (a549) cells (cenas et al., 2006), human leukemia (hl-60) cells (xu et al., 2012 a), and human cervical carcinoma (hela) cells (zhang et al., 2012). ji et al. (2008) reported inhibition of growth and induction of apoptosis of sarcoma 180 cells in vivo. juglone, being an inhibitor of peptidyl-prolyl cis-trans isomerase nima-interacting 1 (pin1) is an impending remedial target for anticancer research. role of pin1 in relation to tamoxifen-resistant breast cancer has been an important determinant factor in drug resistance. these observations demonstrate pin1 augment e2f-4 and egr-1-driven expression of lc-3, an autophagosome marker is over expressed in cancer cells than non-cancerous. namgoong et al. (2010) further established juglone as potent inhibitor having a distinct declining effect on the tpa and induced overexpression of e2f-4 and egr-1 transcription factors controlling lc-3 gene expression. 8. walnut as a nutraceutical the term “nutraceutical” is used to illustrate food or food nutrient supplement that suggests a medical or health benefit by providing simple nutrition (wong et al., 2015). functional foods are known to be a remedy for chronic diseases (luciano, 2014). walnut has been used as functional food. they are important components of the mediterranean diet (ley et al., 2014). nutritional profile of walnut is tabulated in table 5. not only the kernel of walnut but also green shells and bark have also been used in the cosmetic and pharmaceutical industries (ribeiro et al., 2015; adeel et al., 2017). walnuts are chiefly consumed for their organoleptic and nutritional aspects since they are resources of important anti-oxidants and preserve food attributes (martinez and maestri, 2016). walnut seeds are now established as major nutraceutical owing to their effects on coronary heart disease (nasri et al., 2015; schwingshackl et al., 2017). walnuts possess numerous nutritional aspects including a maximum fraction of polyphenolics due to which they are incorporated into recommended dietary supply (sanchez-gonzalez et al., 2017). christopoulos and tsantili (2015) reported numerous phytochemicals in walnut for example phenolics, fatty acids, melatonin and serotonin. in addition to phytochemicals, various hydrolyzable and condensed tannins have been reported in walnut (figueroa et al., 2016). high oil and protein content of kernel of juglans regia (l.) (juglandaceae) validates this fruit requisite for human nutrition. fao has categorized walnut as a deliberate candidate for human nutrition and phytomedicine (gandev, 2007). most of the energy in walnuts generates from fats which account for 65% which makes them energy-rich and highcalorie food (tapsell et al., 2009). martinez and maestri (2008) reported triacylglycerols, in which monounsaturated fatty acids (mufas) mainly oleic acid (18:1 n-9) and polyunsaturated fas (pufas; adv. hort. sci., 2018 32(1): 113-135 122 linoleic (18:2 n-6) and α-linolenic acids (18:3 n-3) in walnut oil are present in high amounts in all genotypes. walnuts are a chief resource for omega-6 fatty acid called linoleic acid. they also contain a relatively high percentage of omega-3 fat called alpha-linolenic acid (ala) contributing up to 8-14% of the total fat content. deckelbaum and torrejon (2012) reported walnut as the sole source of ala which declines inflammation and augments blood profile. muradoglu et al. (2010) and martinez et al. (2010) reported average protein estimation of about 18.1% comprising of 70% of total seed protein along with lesser amounts of globulins (18%), albumins (7%) and prolamins (5%). walnut proteins possess all essential amino acids requisite for humans. amino acids exist in proper ratio in walnuts e.g., lysine/arginine ratio in walnut proteins is comparatively lower than in daily vegetable proteins. the lower lysine/arginine ratio declines atherosclerosis development (martinez et al., 2010). in addition to macronutrients, walnuts are richest sources of vitamins and minerals, potassium (k), phosphorus (p), magnesium (mg) and iron (fe) (cosmulescu et al., 2009). calcium (ca), sodium (na), zinc (zn) and copper (cu) are present in moderate fractions (siahnouri et al., 2013). macro elements are necessary for proper development and growth of organisms (jeszka-skowron et al., 2016). most of macroelements assist in enzymatic reaction involved in the general metabolism of the organism. trace elements promote heart health, maintains bone, nerve and immune system functions (abdel-aziz et al., 2016). vitamins are indispensable organic substances and in addition to their role in important metabolic functions, they are also required for proper functioning of hormone regulation. anjum et al. (2016) confirmed presence of ellagic acid, gallic acid, tocopherol (vitamin e), ellagitannins (tannins) and phytochemicals with potential antioxidant activity, for instance, melatonin. walnut also comprises of elevated levels of αtocopherol, a vitamin e, which prevents lipid oxidation process and decline risk of cancer and coronary heart disease (jiang, 2014). health effects of walnut (table 6) cardiovascular benefits. nijike et al. (2015) reported daily consumption of walnut lowers risk of heart diseases. higher anti-oxidant and elevated levels of the fat fraction in walnut are important for heart health. brain health. brain function is immensely advanced by eating nuts. walnuts are also associated with alleviation of depression and age-linked diseases (grosso and estruch, 2016). arab and ang (2015) reported daily consumption of walnuts enhance memory retention. walnuts help reduce problems in metabolic syndrome. metabolic syndrome is complex disorder encompassing increased triglycerides, high blood nutrients total amount /100 gm walnut calories 654 water 4% protein 15.2 g carbs 13.7 g sugar 2.6 g sucrose 2.4 g glucose 0.1 g fructose 0.1 g fiber 6.7 g fats 65.2 g saturated 6.13 g 16:00 4404 mg 18:00 1659 mg monounsaturated fatty acids 8.933 g 18:01 8799 mg 20:01 134 mg polyunsaturated fatty acids 47.174 g omega-3 9.08 g omega-6 38.09 g trans fat 0 vitamins vitamin a 1 µg vitamin c 1.3 mg vitamin d 0 µg vitamin e 0.7 mg vitamin k 2.7 µg vitamin b1 (thiamine) 0.34 mg vitamin b2 (riboflavin) 0.15 mg vitamin b3 (niacin) 1.13 mg vitamin b5 (panthothenic acid) 0.57 mg vitamin b6 (pyridoxine) 0.54 mg vitamin b12 0 µg folate 98 µg choline 39.2 mg minerals calcium 98 mg iron 2.91 mg magnesium 158 mg phosphorus 346 mg potassium 441 mg sodium 2 mg zinc 3.09 mg copper 1.59 mg manganese 3.41 mg selenium 4.9 µg cholesterol 0 mg table 5 nutritional profile of walnut shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 123 pressure, insufficient high-density lipoproteins, cholesterol, and obesity. saneei et al. (2013) reported daily consumption of one ounce of walnuts for 2-3 months can lower risk of metsassociated disorders. benefits in treatment of type 2 diabetes. type 2 diabetes is a growing problem in developed and industrialized countries. a better understanding of dietary changes is needed to improve this disorder. the significant diet regime for type 2 diabetes patients for lowering cardiovascular issues has been reported by consumption of walnuts on daily basis (farr et al., 2017). anti-cancerous effects. walnuts contain a wide variety of antioxidant and anti-inflammatory bioactive components that may have anti-cancerous properties (byerley et al., 2017). presence of polyphenolic compounds, phytosterols, gamma-tocopherol, omega-3 fatty acids and ellagic acid decline threat of chronic oxidative stress and ameliorate inflammatory properties to counteract intimidation of cancer advancement (perugu and vemula, 2014). choi et al. (2016) and al-mahmood et al. (2016) reported a lower incidence of colon, breast and prostate cancer associated with walnut consumption. other health benefits. walnut nutrients exhibit significant function in the maintenance of bone structure (kajarabille et al., 2013). it has been recently confirmed that walnut consumption decline blood levels of n-terminal telopeptidentx of type 1 collagen. more collagen composition signifies higher bone stability as well as lower mineral loss from bones (griel et al., 2007). katz et al. (2012) confirmed the potential significance of walnut consumption in body weight management. 9. application of molecular markers in horticultural crops horticultural crops are important for dietary purpose and source of revenue for farmers in the emerging countries (behera and france, 2016). genetic table 6 health effects of walnut cardiovascular aspect walnut benefit blood quality lowering ldl, the “bad” cholesterol; decreased total cholesterol; increased gamma-tocopherol; increased omega-3 fatty acids in red blood cells (alpha-linolenic acid) vasomotor tone decreased aortic endothelin; improved endothelial cell function risk of excessive clotting decreased maximum platelet aggregation rate; decreased platelet activation risk of excessive inflammation decreased c reactive protein (crp); decreased tumor necrosis factor alpha (tnf-a) blood vessels improves the function of blood vessels by cutting the risk of plaque buildup in the arteries brain tonic relieves depression and age-related decline in brain function decline in metabolic syndrome reduction of various mets-related problems treatment of type 2 diabetes regulates blood sugar and insulin metabolism development of body structure decrease blood levels of n-telopeptides of type 1 collagen (ntx); weight maintenance and prevention of obesity anti-cancer activities anti-inflammatory properties help lower risk of chronic inflammation adv. hort. sci., 2018 32(1): 113-135 124 improvement in horticultural crops is not yet as noteworthy as it has been achieved in case of cereals and grasses. the advancement and accessibility of genomic resources in these crops can be utilized resourcefully to conserve and investigate existing genetic diversity and to understand the association between genotype-phenotype and to accelerate breeding (chaturvedi and sahijram, 2015). current progress in mechanization and high-throughput sequencing can be utilized to decipher ambiguous and intricate genomes. gene pyramiding and polygenic resistance evaluation in diverse genotypes with extensive resistance have been made possible through the advent of molecular markers (ansari, 2015). walnut is valued for its wood and nut, phytochemicals but so far as its transcriptomics and genomic data is concerned, it is very limited (hu et al., 2016). from the past few decades, a significant progress has been achieved by the use of molecular markers in plant biotechnology and unraveling their genomic intricacies for detecting and exploiting dna polymorphism in the plants. there are different markers, i.e. markers based on the morphology of plants, on biochemical functions and dna-based molecular markers (parveen et al., 2016). these molecular markers are classified as hybridization-based or non-pcrbased markers. by using, these dna based molecular markers, one can get complete information about a particular plant from the level of the nucleotide to a segment on dna and frequencies of alleles, population structure and distribution of genetic diversity. molecular markers explicitly reveal the polymorphism at dna level (deoxyribonucleic acid). they are routinely used in various crop improvement programs, ecological, physiological, genetic studies of plants and to progress the effectiveness and accuracy of traditional plant breeding through marker assisted selection (allwright and taylor, 2016). important information for genetic diversity can be evaluated for different horticultural crops by using rapd markers, for example, eggplant and watermelon (trivedi et al., 2016), moringa oleifera (l.) (kumar et al., 2017). aflp markers have effectively been utilized for evaluating genetic diversity in horticultural crops like celosia argentea (olawuyi et al., 2016), rosa platyacantha (yang et al., 2016) and olive (mnasri et al., 2017). owing to their hyper variability and competence in distinguishing of polymorphisms, ssr have become ideal markers for assessment of genetic diversity in horticultural crops l ike potato (ghebreslassie et al., 2016), grapes (rao, 2017), high density genetic map construction (xue et al., 2008), population and conservation genetic studies, clonal identification (liu et al., 2016 a, b), controlled crosses certification, species and hybrid identification, paternity determination (de la rosa et al., 2013), marker assisted selection (ashraf and foolad, 2013) and genotyping (billot et al., 2013). issr is extensively utilized in population genetics of horticultural crops i.e., elaeis guineensis (chagas et al. , 2015) and rhododendron triflorum (xu et al., 2017) and determining the genetic diversity of horticultural crops such as bael (aegle marmelos corr.) (mujeeb et al., 2017). this section of the chapter provides a detailed account of the exploitation of different molecular markers used in case of walnut study. use of some like random amplified polymorphic dna (rapd), simple sequence repeat (ssr), inter-simple sequence repeat (issr), single nucleotide polymorphism (snp) are briefly discussed in improving, increasing the quality and quantity of walnut is summarized. single nucleotide polymorphism (snps) have been widely used in plant germplasm management and breeding of fruit tree crops (van nocker and gardiner, 2014). these benefits have resulted in snps gradually becoming the markers of choice for exact genotype identification and diversity analysis in horticultural crops such as cacao (theobroma cacao) (fang et al., 2014 a), grapevine (vitis vinifera) (cabezas et al., 2011), pummelo (citrus maxima) (wu et al., 2014), strawberry (fragaria spp.) (longhi et al., 2014) and tea (camellia sinensis) (fang et al., 2014 b). 10. molecular characterization in walnut molecular characterization of walnut germplasm is mandatory requirement for establishing breeding purposes and establishment of proprietary rights. conventional approaches for germplasm characterization are evaluated based on comparative morphological analysis. due to the ecological effects on phenotypic expression together with juvenile phase of walnut, there are ample hindrances in proper classification of walnut. to surmount these restrictions, molecular markers are employed for discrimination and identification of walnut accessions. these dna based markers are not influenced by environment and can be identified in all development stages of plant tissues. characterization of genetic variability in walnut was done using isozymes (malvolti et al., 1994), to identify interspecific hybrids (arulsekar et shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 125 al., 1985), in species and cultivars differentiation (vyas et al., 2003) and to evaluate mating aspects (rink et al., 1994). rflp markers has been employed for evaluating parentage and ascertaining phylogenetic associations and discriminate among species and cultivars in the genus juglans (aly et al., 1992; fjellstrom et al., 1994; fjellstrom and parfitt, 1995). description of various molecular markers used in molecular characterization of various walnut genotypes is given in table 7. randomly amplified polymorphic dna (rapd) rapd is immensely significant in deciphering closely associated cultivars in walnut and results obtained are harmonized with preexisting data procured from rflps owing to more vigorous polymorphism exhibited by rapd (nicese et al., 1998). furthermore, rapd markers were also employed to assess polymorphism intensity at interspecific level among persian walnut (j. regia) and in the northern california black walnut [j. hindsii (jeps.)] (woeste et al., 1996 a) and to recognize a marker associated with hypersensitivity to the cherry leaf-roll virus (woeste et al., 1996 b). nicese et al. (1998) evaluated eighteen rapd primers among nineteen walnut genotypes that comprised of strongly associated cultivars and parents in breeding programs. following cluster analysis of closely associated genotypes, genotypes can be segregated into two classes having association with their closely related alleles. rapd markers can investigate polymorphism to decipher variation among walnut genotypes particularly strongly associated genotypes and genetic similarity subsisting on rapds revealing can detect enough polymorphism to differentiate among walnut genotypes, even among closely identified (hayward et al., 2015). rapd markers are highly useful for evaluating genetic variability in genus juglans through dna fingerprinting to differentiate between valuable genotypes for selection (pop et al., 2010; ahmed et al., 2012). rapd was employed to evaluate association among eight walnut genotypes thriving in turkey and discrimination among indigenous and exotic genotypes together with their early bearing progenies (erturk and dalkilic, 2011). xu et al. (2012 b) illustrattable 7 description of various molecular markers used in molecular characterisation of various walnut genotypes walnut species/variety molecular marker used traits associated references juglans regia rflp inheritance and linkage fjellstrom et al., 1994 juglans regia rapd and ssr phenotypic pop et al., 2013 juglans regia ssr morphological mahmoodi et al., 2013 juglans regia ssr morphological karimi et al., 2014 butternut (juglans cinerea l.), japanese walnut (juglans ailantifolia) and buartnut (juglans cinerea × juglans ailantifolia) ssr nut phenotype chen et al., 2014 juglans regia rapd butternut canker disease resistance zhao et al., 2014 juglans nigra, juglans regia, and hybrid (juglans x intermedia (carr) rapd association between native/foreign genotypes with their early-bearing natural hybrids erturk and dalkilic, 2011 juglans regia ssr identification of interspecific hybrid pollegioni et al., 2014 juglans regia ssr morphological ebrahimi et al., 2011 juglans regia issr morphological and biochemical traits malvolti et al., 2010 juglans regia issr distinction between native and international cultivated genotypes christopoulos et al., 2010 juglans regia slaf disease resistance to anthracnose zhu et al., 2015. juglans regia aflp geographical proximity bayazit et al., 2007 juglans regia snp fingerprinting of 30 walnut genotypes ciarmiello et al., 2013 juglans regia snp genome sequencing liao et al., 2014 adv. hort. sci., 2018 32(1): 113-135 126 ed rapd and aflp for deciphering genetic diversity of walnuts in western sichuan plateau and qinba mountainous regions. the walnut genotypes were procured from 8 different regions and 32 rapd primers and 28 aflp primers in combination were recognized with polymorphic bands in entire walnut. in this study, high allelic number and elevated genetic diversity identified with rapd and aflp signify western china has significant genetic diversity resource and profuse genetic variance among walnuts. zhao et al. (2014) employed rapd as chief molecular implement for characterizing genome of butternut (juglans cinerea), japanese walnut (juglans ailantifolia), black walnut (juglans nigra), persian walnut (juglans regia), manchurian walnut (juglans mandshurica), and an interspecific hybrid (j. ailantifolia cinerea) for species-specific markers. the study revealed 38 amplicons were exclusive to japanese walnut and buartnut hybrids lacking in butternut. rapd markers were relatively insufficient to differentiate intraspecific variability inside japanese walnut, butternut, or their hybrids. rapd can also decipher hybrid lineage as in the case for discriminating whether japanese walnut or manchurian walnut was the progenitor of hybrid line or not. the results demonstrated that some of the hybrids can be distinguished through morphological and genetic resemblances through rapd as it demonstrates pedigree data. the resulting rapd markers provide their utility in the conservation of butternut, an endangered north american species. simple sequence repeat (ssr) ssr is enumerated among most precise molecular marker owing to their high polymorphism, co-dominant transmission, higher reproducibility, exhibiting higher resolution and more convenient pcr detection in deciphering genetic relationship (miah et al.. 2013). wang et al. (2015), ali et al. (2016), ebrahimi et al. (2016), and pang et al. (2017) illustrated research reports where in genetic characterization of walnut was evaluated using ssr. han et al. (2016) described genetic diversity and population structure study of j. regia germplasm using ten primers developed from expressed sequence tags (est-ssr) and sequence polymorphisms among the phenylalanine ammonia lyase (pal) gene. the result showed high level of population differentiation. in addition, ssr markers informative in juglans regia may also be polymorphic in other juglans (l.) species (aldrich et al., 2003) and have utility for breeding hybrid rootstocks. ssr are more consistent and trustworthy system for molecular characterization. ssr has wide numerous relevance in case of walnut comprising of cultivar characterization, cultivar identification, pedigree substantiation for cultivar and in deciphering evaluation of superior rootstock for breeding and paternity analysis (dangl et al., 2005). ruiz-garcia et al. (2011) employed 32 ssr primer pairs to characterize 57 walnut cultivars originated from spain and usa. in this research reports, 32 primer pairs flanking simple sequence repeats, were established in juglans nigra to screen elite variety with high rate of polymorphism. further selection of 19 selected microsatellite markers provided differentiation of evaluated cultivars exhibiting 97 alleles and on an average 5 alleles per locus establishing ssr as primary choice for characterization of walnut germplasm. the data generated from ssr characterization further confirmed molecular parameters of spanish walnut to be different from californian genotypes. ahmed et al. (2012) illustrated genetic association of 82 walnut genotypes using 13 ssr and 20 rapd primers exhibit higher level of genetic diversity in these walnut cultivars thriving rigorously in climate of jammu and kashmir. these results demonstrate applications of ssr markers in walnut breeding and conservation. pollegioni et al. (2014) illustrated genetic diversity and spatial genetic structure of 39 autochthonous persian walnut populations analyzed transversely along asian range via 14 neutral microsatellite markers. shah et al. (2016) reported genetic characterization of 96 walnut genotypes growing in north western himalaya as investigated by 19 ssr markers and the study displayed high polymorphism rate of 89.6%. chen et al. (2014) demonstrated potential of ssr markers in the cultivar characterization together with intellectual property rights. topcu et al. (2015) developed genomic libraries augmented with ca, ga, aac, and aag repeats via genomic dna from j. regia cv. maraş-18 to develop ssr markers for walnut. this study confirmed ga-enriched library as preferred alternative in terms of allelic number, polymorphism, productivity, and information content. inter simple sequence repeat (issr) markers issr-pcr is a most convenient approach that surmounts most of these restrictions encountered by other molecular markers (karimi et al., 2014). issr is most popular among plant improvement. potter et al. (2002) has demonstrated the application of intersimple sequence repeat (issr) markers in genetic characterization of english or persian walnut (juglans regia l.). in this study eight issr primers presented exclusive fingerprint for 48 cultivars investigated. the dendrogram developed from data presented classes shah et al. bio-techniques for improvement of qualitative and quantitative traits in walnut. a review 127 pertaining to known pedigrees which does not provide this data anticipating the fact that there is restriction in context to utility of issr in determining genetic association among species. christopoulos et al. (2010) reported immense application of issr markers in genetic characterization for deciphering genetic diversity among greek natives of walnut (juglans regia l.). in this study, similarity coefficient values signified presence of a higher extent of genetic variability. majority of international cultivars were classified together whilst majority of greek native populations cannot be classified in separate class. international cultivars exhibit lesser diversity than greek native population genotypes. the pairwise regional phipt values signified that most geographically isolated regions are the most genetically differentiated. the intense variability in greek germplasm along with their desired traits anticipated that native germplasm can be utilized for breeding purposes and preservation of walnut germplasm. li et al. (2011) employed issr markers to evaluate the genetic variation and genetic structure to provide a theoretical basis and technical support for appropriate conservation and application of existing genetic resources of walnut. analysis of anova (analysis of variance) also showed genetic variance among populations was larger than within a population. mantel test and upgma (unweighted pair group method with arithmetic mean) dendrogram based nei’s genetic distance displayed that genetic distance between populations had more significant correlation with geographic distance. ji et al. (2014) developed issr primers to assess the degree and design of genetic diversity among eight populations of north china mountain walnut (ncmw). this diversity was further confirmed through anova analysis. structure of ncmw and upgma cluster analysis demonstrated restricted gene flow, habitat devastation and geographical isolation might be determining factors for population structure. upgma cluster further signify that eight populations of walnut can be classified into three discreet groups as per similarity coefficient and geographic origin but exhibited significant association with morphological traits especially nuts. hu et al. (2016) in their classical experiments use transcriptomic information from rna-seq to understand development of polymorphic simple sequence repeats (ssrs, microsatellites) for understanding the population genetics of walnut. they studied more than 47.7 million clean reads, 99,869 unigenes having length of 747 bp. they further identified 39,708 (42.32%) genes, 63 new transcriptome-derived microsatellite markers. the identification and characterization of microsatellite markers in their study could help to explore the diversity, genetic structure, population genetics for improved walnut future breeding practices, besides providing a useful genetic resource information for studying and understanding the genomic and transcriptome aspect of walnut. doğan et al. (2014) evaluated genetic association of 59 walnuts (juglans regia l.) genotypes, international and turkish by using three different types of molecular markers i.e. rapd, issr and ssr primers. these results exhibited that ssr markers offer elevated rank of polymorphism as compared to rapd and issr. single nucleotide polymorphism (snp) markers snp is the most abundant type of dna variation in most species. the introduction of next generation sequencing (ngs), together with high throughput genotyping technology, makes it relatively easy to identify and use snps (elshire et al., 2011). the new generation of molecular markers based on single nucleotide polymorphisms (snp) represent a promising and effective tool for fast and accurate species identification. ciarmiello et al. (2013) developed a simple amplification refractory mutation technique, based on snp markers of rdna and cox2 intron i sequences, to fingerprint 30 walnut genotypes. rdna sequences revealed the presence of 402 variations and cox2 intron i sequences showed 769 variable positions. the findings revealed that the cox2 intron i region, either alone or in conjunction with rdna, could be used effectively in identifying these walnut genotypes. liao et al. (2014) performed genome sequencing of walnut and then all the sequence reads were mapped against genome assembly of walnut. in total, 49,202 nucleotide variations were detected including 48,165 single nucleotide polymorphisms (snp5) and 1037 insertions/deletions (indels). 11. conclusions the nutritional factors of walnut are improved due to the presence of numerous micro elements. the notable nutritive feature of walnuts relates to a rich range of polyphenols. the breeding character of walnut has bestowed it an inclusive diversity in genetic features. these results have been attained after an extended progression of evolution and by going through intricate environmental abnormalities. however, these techniques are susceptible to the environmental variations. the markers are adequate adv. hort. sci., 2018 32(1): 113-135 128 in number and there is little or no diversity in the method employed for research on walnuts. despite basic research method, the organization of information, its retrieval and presentation structures, form elaboration experienced immense advancement via molecular markers such as rflp, issr, rapd aflp, ssr and snp. this assessment offers data regarding health benefits of walnut at the global level and existing applications in the horticulture. the present work comprehensively describes the utilization of molecular markers in walnut plants which could help to improve/enhance the traits linked to its optimum/yield growth and sustainable production of innumerable essential metabolites having the ability to find applications in horticulture and allied science (fig. 4). acknowledgements the authors express their gratitude to central institute of temperate horticulture (indian council of agricultural research, (icar), aligarh muslim university and university grants commission new delhi, india for financial support of this work. references abdel-aziz s.m., aeron a., garg n., 2016 fortified foods and medicinal plants as immuno modulators, pp. 143-162. in: garg n., s.m. abdel-aziz, and a. aeron (eds.) microbes in food and health. springer international publishing, cham (zg) szitzerland, pp. 362. adeel s., rafi s., salman m., 2017 potential resurgence of natural dyes in applied fields, pp. 1-26. in: ulislam s. 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fragment markers and identification of a quantitative trait locus for anthracnose resistance in walnut (juglans regia l.). bmc gen., 16: 1. impaginato 433 adv. hort. sci., 2018 32(3): 433-441 doi: 10.13128/ahs-22932 effect of wounding intensity on physiological and quality changes of strawberry fruit m.t. solomon, f. piazzolla, m.l.v. de chiara, m.l. amodio (*), g. colelli dipartimento di scienze agrarie, degli alimenti e dell’ambiente, università degli studi di foggia, via napoli, 25, 71122 foggia, italy. key words: ascorbic and dehydroascorbic acid, ‘candonga’, cutting degree, fresh-cut, respiration rate, total phenolic content. abstract: wounding makes fresh-cut product more perishable than whole fruit. the effect of wounding intensity on respiration rate and nutritional quality of fresh-cut ‘candonga’ strawberries was investigated. fruit were submitted to six levels of cutting intensity whole fruit (who), 4, 16, 64, and 128 pieces and chopped (cho) samples. respiration rate, and the main nutritional parameters were evaluated at the processing day and after 2 days of storage at 5°c. results showed that wounding intensity significantly influenced respiration rate, ascorbic and dehydroascorbic acids, total phenolic content, and antioxidant capacity. respiration rate increased with wounding intensity up to the level of 64 pieces (10.01 µg kg-1 s-1) compared to who (5.5 µg kg-1 s-1) and then decreased in the cho samples (2.81 µg kg-1 s-1). at day 2, the stress caused by the high intensity of cutting (64 pieces and cho) induced a higher degradation of ascorbic acid, phenolic compounds, and antioxidant capacity. stress-related changes decrease when the wounding damage was so high that it completely compromises the functionality of the cells (from 64 pieces up). these results should be considered for processing and packaging optimization of minimally processed strawberries-based products. 1. introduction the continuous physiological activity of living plant tissues induces severe compositional and structural variations, also associated to ripening and senescence of fresh produce during postharvest life. tissue responses cannot be blocked but it is possible to delay them within certain limits in order to prolong fruit shelf life (el-ramady et al., 2015). the physiological stresses due to physical damage and wounding occurring during minimal processing, make fresh-cut products more perishable than whole fruit (nicola and fontana, 2014). immediate response of plant cells start from a wound signal formed in adjacent and distant tissues, which gives rise to a wide range of different physiological and biochemical reactions. common are respiration rate and ethylene production increase, variation in product quality, synthesis and/or loss of phytochemicals with consequent decrease of nutritional content, stimulation of enzymatic activity (*) corresponding author: marialuisa.amodio@unifg.it citation: solomon m.t., piazzolla f., de chiara m.l.v., amodio m.l., colelli g., 2018 effect of wounding intensity on physiological and quality changes of strawberry fruit. adv. hort. sci., 32(3): 433-441 copyright: © 2018 solom m.t., piazzolla f., de chiara m.l.v., amodio m.l., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 march 2018 accepted for publication 28 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 433-441 434 and bacterial spoilage (brecht, 1995; surjadinata and cisneros-zevallos, 2003). the complex interrelationship among the different effects of wounding on physiological processes of fresh-cut products, are comprehensively described by saltveit (1997). wound responses could vary depending on different factors: species and cultivar of the product, maturity stage, temperature of processing and storage, cutting-type and sharpness of the blades, but also on process temperature (el-ramady et al., 2015), o2 and co2 levels, and water vapor pressure (brecht, 1995). the basis for the wound-induced changes include altered genes expression and changes in enzyme activities involved in an effort to heal the damaged tissues providing defense mechanisms of the plant aimed to prevent further and more serious damages (chung et al., 2006). during storage time the increase in respiration rate is usually responsible for the aging of the products due to consumption of reserve energy during redox process. as a consequence, the higher the respiration rate, the shorter is the storage life and therefore the faster is the quality deterioration. respiration is a function of the climacteric or non-climacteric behavior of the product and of the physiological age of climacteric fruit (gunes and lee, 1997). as already reported wounding induces an increase in enzyme activity, in particular higher activities of phenylalanine ammonia-lyase (pal), peroxidase (pod) and polyphenol oxidase (ppo) are observed. (saltveit et al., 2005 b). the main effect of the postcutting interaction of substrates with enzymes, such as ascorbate oxidase, ppo, and pod, is the degradation of phytonutrients. phenols oxidation and the resulting browning may induce a reduction in nutrient content resulting often in degradation of color, texture and flavor of fresh-cut products (saltveit, 1997; francis et al., 2012). it is possible to reduce wound-induced browning with the application of antioxidant or calcium-based active compounds and treatments that interfere with the synthesis or oxidation mechanisms of the phenolic compound precursors (brecht, 1995; saltveit, 1997; saltveit et al., 2005 a). however, according to francis et al. (2012), the induced synthesis of phenolic compounds after cutting caused an increase in nutritional value for lettuce, celery, carrot, parsnips, and sweet potato, while in the same study a decrease of phenols was observed in cut zucchini, radish, potato, and red cabbage, pointing out the influence of product on the wound-induced response type. amodio et al. (2014) described the consecutive reaction mechanism that regulates the phenolic content in fresh-cut produce during storage and how their variation is related to cut intensity. they observed, for example, that there was an increase in k1 values (the rate constant for the de novo synthesis of the phenols), when fresh-cut lemons were cut as half-slices rather than slices. this result was also in agreement with the increase in pal activity resulting from the higher level of wounding on vegetable tissues. at the same time, the cut intensity did not affect the rate of phenolic oxidation. the authors stated that one of the most important factor affecting the phenolic content and synthesis is the biological variability; in fact, each product shows a particular combination of factors that can contribute to the amount and composition of wound-induced phenolics (francis et al., 2012). fernando reyes et al. (2007) stated that the final concentration of phenolic compound in cut products is strongly affected also by the type of tissue and the initial level of reduced ascorbic acid. strawberry-based product attracted in the recent years the food industry due to the high amount of bioactive compounds (vitamin c, anthocyanins and flavonols). for this reason strawberries are one of the richest fruits in term of antioxidant capacity (cordenunsi et al., 2002; pertuzatti and barcia, 2015). understanding the stress-induced changes is important in order to develop reliable approaches to control the stress responses, and improve the quality of minimally processed fresh products, particularly when the fruit is subjected to a pronounced mechanical damage. this makes very interesting the investigation on the effect of wounding on soft and fresh fruit like strawberries. therefore, the main objective of the present study was to determine the effect of wounding intensity, on physiological and quality changes of fresh strawberry fruit, with a particular focus on the respiration rate and nutritional compounds. 2. materials and methods sample preparation ‘candonga’ strawberries (fragaria x ananassa duch.) were purchased from local stores in foggia (south italy) and stored at 5°c overnight. in the next morning, fruit with uniform color and size, free of physical defects and decay, were divided into six groups (treatments), each one corresponding to one different level of wounding intensity: whole fruit (no cutting), cutting into 4, 16, 64, 128, pieces and chopped defined as who, p4, p16, p64, p128 and solomon et al. effect of wounding intensity on quality of strawberry fruit 435 cho, respectively. about 12-15 fruit were cut (300 g) for each replicate and 150 g of product were used for initial determinations, whereas the remaining samples were stored at 5°c under a continuous flow of humidified air for 2 days. three replicates were used for each cutting intensity treatment and for each quality parameter determination. vitamin c, total phenolic and anthocyanin content, antioxidant capacity, soluble solid content, ph value, titratable acidity and sugar/acid ratio were evaluated at the processing day (day 0) and after storage (day 2). separate samples were used for respiration rate measurement. respiration rate respiration rate of fresh-cut strawberries was measured in static conditions as described in kader (2002). respiration rate (expressed as µg kg-1 s-1 of co2) was determined at 120 min after cutting. separate samples (about 300 g each) of strawberries were placed in 5 l sealed glass jars with a plastic septum for sampling gas; jars were closed after an equilibration time of about 1 hours. from each jar, a gas sample (0.5 ml) was collected after the required time to accumulate co2 in the headspace up to a concentration of 0.1-0.2%, and injected into a gas chromatograph (shimadzu, model 17 a, kyoto, japan), equipped with a thermal conductivity detector (200°c). separation of co2 was achieved on a carboxen 1006 plot (30 m × 0.53 mm, supelco, bellefonte, pa, usa), with a column flow of 7 ml min−1, and an oven temperature of 180°c. compositional attributes vitamin c content was measured in 5 grams of fresh homogenized strawberry tissue as l-ascorbic acid (aa) and l-dehydroascorbic acid (dha) contents expressed as g of aa, dha or total vitamin c (aa + dha) per 1 kg of fresh weight (g kg-1) following the procedure by zapata and dufour (1992) with slight modifications. total phenolic content (tpc) was analyzed using the folin-ciocalteau method of singleton and rossi (1965), with some modifications where five grams of fresh tissue were homogenized in an ultraturrax (ika t18 basic, wilmington, nc, usa) with 10 ml of extraction buffer containing 200 ml of distilled water, 800 ml of methanol and 2 mm (84 mg l-1) of sodium fluoride (naf). the absorbance was read at 725 nm compared with a blank (prepared in the same way, replacing the sample with 100 µl of distilled water) using a spectrophotometer (uv-1700 shimadzu, jiangsu, china). tpc was calculated based on the calibration curve of gallic acid and results were expressed as g gallic acid equivalents per 1 kg fresh weight (g kg-1). total anthocyanin content (tac) was determined following the protocol described by cordenunsi et al. (2002), with small modifications using hydrochloric acid/methanol mixture as extraction medium. 700 µl of extract plus 300 µl of 1% hcl-meoh solution were put in cuvettes and absorbance was read immediately in a spectrophotometer at 510 nm. results were expressed g of pelargonidin-3-glucoside (pg-3-glu) equivalents per 1 kg of fresh weight (g kg -1). the antioxidant capacity (ac) assay was conducted on the same extract made for tpc, following the method of brand-williams et al. (1995), with slight modifications. fifty µl of extract were mixed with 950 µl of dpph (2, 2-diphenylpicrylhydrazyl) solution and absorbance was read at 515 nm after 24 h. trolox (6-hydroxy-2, 5, 7, 8-tetramethlychromane-2carbxylic acid) was used as a standard and results were expressed in g of trolox equivalents per 1 kg of fresh weight (g kg-1). total soluble solids (tss) were obtained by measuring the refractive index of fresh strawberry juice using a digital refractometer (atago rx-7000cx; atago co. ltd., japan) at 25°c and expressed as percentage. one gram of sample was used to determine the ph and titratable acidity (ta), with an automatic titrator (t50 m terminal, mettler toledo, switzerland) against a volume of 0.1 n naoh until reaches the final ph of 8.2. ta was expressed as g of citric acid equivalent per 1 kg of product (g kg-1). tss/ta ratio was also calculated. statistical analysis data were subjected to a 2-way anova (for treatment and sampling time); treatment means were separated by tukey’s test at p<0.05 using stat graphics centurion xvi.i (stat point technologies, inc., warrenton, va usa) software. 3. results wounding intensity had a significant effect on all parameters except ph; the same for storage time with exception of total phenolic content, while the interaction between the two factors showed a significant effect on respiration rate, ascorbic and dehydroascorbic acids, total vitamin c, total phenolic content and antioxidant capacity but no significant effect adv. hort. sci., 2018 32(3): 433-441 436 on tac, tss, ph, ta and tss/ta ratio. following, these effects are described in detail. the effect of wounding intensity on the respiration rate of ‘candonga’ strawberry fruits after 120 min post-cutting at 5°c is shown in figure 1. wounding induced a significant raise in respiration of strawberries tissues that resulted to be increasing with cutting intensity up to a certain point from 5.5 (who) to 10.01 µg kg-1 s-1 of co2 (p64). further increase of wounding beyond p128 did not stimulate respiration rate: a significant decrease up to a minimal level of 2.81 µg kg-1 s-1 was observed for chopped samples. the effect of wounding intensity on aa, dha and total vitamin c content (aa+dha) of fresh-cut ‘candonga’ strawberries is shown in figure 2. at day 0, aa and total vitamin c were not significantly affected by wounding intensity, with almost similar values referred to ascorbic acid among treatments ranging from 0.37 (p64) to 0.44 g kg-1 (p4). on the other hand, an increase in dha content could be observed already at day 0 with a significant difference among treatments ranging between 0.067 g kg-1 in the who and 0.22 g kg-1 in the treatment p128. the mean values of total vitamin c content at day 0 ranged from 0.51 (who) to 0.61 (p128) g kg-1. p128 and cho samples showed in fact significantly lower values of ascorbic acid (0.27 and 0.17 g kg-1, respectively) than other treatments, with 64-piece sample showing an intermediate behavior (0.41 g kg-1). moreover a significant increase of dha with the increase of wounding intensity was observed for all samples starting from p16. the lowest and highest dha level at day 2 were found with the who and cho samples (0.12 and 0.55 g kg-1, respectively). the highest level of wounding intensity (cho) induced about a 5-fold increase in dha content. the effect of wounding intensity on tpc, tac, and ac of fresh-cut ‘candonga’ strawberries is shown in table 1. the different cutting stress significantly affected the tpc of fresh-cut strawberries. at day 0, p128 had significantly higher (p<0.05) total phenolic content compared to other treatments with the exception of p64, with p16 showing the lowest amount (2.27 g kg-1 of gallic acid equivalent) and, together with p4 (2.37 g kg-1), resulting in significantly lower content of phenolic compounds than whole fruit. there was no significant difference in tac both at day 0 and 2 although the mean tac value of sample p4 showed slight increase during storage time. the mean value of tac ranged from 0.19 (p4) to 0.22 (cho) g kg-1 of pg-3-glu (table 1). wounding intensity also significantly affected antioxidant capacity of fresh ‘candonga’ strawberries. at day 0, samples cut into 16 (6.35 g kg-1 of trolox equivalent) and 64 (6.59 g kg-1) pieces had significantly higher ac than the cho (5.41 g kg-1) and who (5.20 g kg-1) samples. a similar trend was also observed after 2 days from cutting when p16 sample showed significantly higher ac value when compared to the cho samples, although this difference was not significant if compared to the rest of the treatments. a particular behavior was observed for cho strawberries (table 1): no significant effect of storage time was observed on the tpc, tac, and ac values of this sample indifig. 2 effect of wounding intensity on ascorbic acid (aa, black bars, g kg-1), dehydroascorbic acid (dha, grey bars, g kg-1) and total vitamin c content (sum of aa and dha, g kg-1) of fresh-cut ‘candonga’ strawberries at day 0 and day 2 of storage at 5°c. who, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped. mean value (n=3) is reported. different lowercase and uppercase letters, indicate significant differences among treatments and storage times, respectively, according to tukey’s test (𝑃≤0.05). different italics letters indicate significant differences among treatments for total vitamin c content for each storage time. fig. 1 effect of wounding intensity on respiration rate at 5°c of ‘candonga’ strawberry fruit. who, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped. error bar represent st. dev of mean values (n=3). different lowercase letters indicate significant difference among treatment according to tukey’s test (𝑃≤0.05). solomon et al. effect of wounding intensity on quality of strawberry fruit 437 respiration rate gives an immediate overview of the metabolism of a commodity (fig. 1), where higher respiration is an indicator of accelerated metabolism which is usually inversely related to shelf-life. strawberry is among the commodities with highest respiration rate (saltveit, 2002), that can be further increased as a cut consequence. according to surjadinata and cisneros-zevallos (2003), an increase in respiration may occur due to simultaneous enzyme synthesis and decrease. the newly synthesized enzymes could in fact be degradated by an inactivation system. it is possible that, after a certain cutting degree, the very high tissue damage compromised the cell functionality. the transition from respiring to non-respiring tissues after wounding is probably related to the damage of the membrane system or mitochondria and consequent disruption of oxidative phosphorylation. changes in ammonium dihydrogen phosphate (adp) and ammonium transferase phosphate (atp) concentrations in wounded tissue indicate that oxidative phosphorylation failed to keep place with atp utilization in injured tissues (lafta and fugate, 2011). according to these authors, a 41% reduction in atp concentration and a simultaneous increase in adp (31%) were observed between day 1 and day 4 after incremental injury of sugar beet root. costa et al. (2011), reported that respiration rates of fresh-cut strawberries was higher than whole fruit and that low storage temperature significantly influenced this parameter. this is possibly due to the fact that cutting increases the surface exposed to the air, and as a consequence, oxygen is able to diffuse into the internal cells more rapidly. moreover, injured cells show an increased metabolic activity (nilsson and hedenqvist, 2011; saltveit, 1997). thus, respiration is stimulated by physical damages given to the cating that these compounds were quite preserved during cold storage, and did not show after two days of storage great variations if compared with other samples. in addition, this sample showed also the lowest respiration and no significant difference in total vitamin c if compared to the who samples. these characteristics may be therefore exploited to maintain the quality of fresh-blended products. tss, ph value, ta and tss/ta ratio of fresh-cut strawberry subjected to different wounding intensity and stored for two days at 5°c were significantly affected by wounding intensity (with exception of ph) and storage time, but not by their interaction (data not shown). the mean values of tss ranged from 7.6 (who at day 0) to 8.6% for who, p16 and cho samples at day 2. almost similar ph values (3.9 to 4.0) were determined for all treatments and sampling times. ta ranging from 0.07 to 0.08 g kg-1 of citric acid showed almost no differences among treatments and sampling time except for p64 which had a slightly lowest value after two days. similarly, almost no differences in the tss/ta ratio were recorded. the highest and lowest values were 9.4 (p4 at day 0) and 12.1 (p16 at day 2). 4. discussion and conclusions the effect of wounding intensity on respiration rate and compositional values of fresh ‘candonga’ strawberry fruit was clearly determined. in general, high degree of wounding intensity (p128 and cho) caused a significant decrease in respiration rate. in addition, the chopped sample did not show any significant difference in tpc, tac and ac during storage time. days at 5 °c wounding intensity who p4 p16 p64 p128 cho total phenolic content 0 2.88±0.07 ab 2.37±0.16 bc 2.27±0.04 bc 2.95±0.02 aab 3.23±0.08 aa 2.94±0.05 ab 2 2.62±0.12 bb 3.19±0.18 aa 3.25±0.17 aa 2.52±0.15 bb 2.87±0.10 bab 2.58±0.07 ab total anthocyanin content 0 0.19±0.004 ns 0.19±0.007 ans 0.20±0.004 ns 0.21±0.018 ns 0.20±0.019 ns 0.20±0.010 ns 2 0.21±0.015 ns 0.21±0.002 bns 0.21±0.014 ns 0.22±0.002 ns 0.22±0.021 ns 0.22±0.012 ns antioxidant capacity 0 5.89±0.03 abc 6.02±0.05 aabc 6.35±0.12 aa 6.59±0.09 aa 6.27±0.12 aab 5.41±0.06 ac 2 5.20±0.22 bab 5.98±0.44 bab 6.16±0.02 ba 5.44±0.03 bab 5.67±0.09 aab 4.62±0.10 ab table 1 effect of wounding intensity on total phenolic ontent (tpc, in g kg-1 of gallic acid equivalent), total anthocyanin content (tac, in g kg-1 of pelargonidin-3-glucoside), and antioxidant capacity (ac, in g kg-1 of trolox equivalent) of fresh-cut ‘candonga’ strawberries at day 0 and day 2 of storage at 5°c iwho, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped; mean values (n=3) ± standard deviations are reported. different lowercase and uppercase letters, indicate significant differences among treatments and storage times, respectively, according to tukey’s test (𝑃≤0.05). 438 adv. hort. sci., 2018 32(3): 433-441 fruits: the more the severity of damage, the more the degree of respiration rate increase (kader, 1987; zhu et al., 2001). the main consequence of an increase in co2 and ethylene production as a response to cutting process (saltveit, 1997) could be a reduction of the fresh-cut product shelf life. moreover, moisture in the cut surface may impede gas diffusion, and this, together with increased respiration, could possibly lead to anaerobiosis, causing further deterioration of the tissues (saltveit, 1997; surjadinata and cisneroszevallos, 2003). however, wound-induced respiration depends on the type of tissue, temperature, controlled atmospheres and degree of cutting (zhu et al. 2001). as described by surjadinata and cisneroszevallos (2003) respiration rate of carrot tissues after wounding showed a typical increase (resulting in a maximum peak) and then a decrease reaching steady-state respiration values similar to that of whole carrot. in some plant tissues, such as potato, this behavior may be related to the oxidation of fatty acids and carbon dioxide, being these reactions responsible for increased respiration after wounding (gunes and lee, 1997). according to surjadinata and cisneros-zevallos (2003), wounding stimulates enzymatic activity of phosphofructokinase and cytochrome oxidase from the respiration pathway, which catalyse the phosphorylation of fructose-6phosphate to fructose-1,6-bisphosphate (a key regulatory step in the glycolytic pathway) and the electrons transfer to oxygen, respectively. a higher enzymatic activity could be due to activation of already present enzyme or to de novo synthesis, as suggested by surjadinata and cisneros-zevallos (2003). their higher activity results in an increase of respiration rate in wounded tissues (lafta and fugate, 2011). on the processing day total vitamin c content did not vary among the treatments, most probably because ascorbic acid was oxidized to dehydroascorbic. the same vitamin c content trend showed in figure 2 was observed by costa et al. (2011), and may be ascribed to the fact that tissue had only slightly responded to the stress. after 2 days from cutting a slight increase in aa was observed for samples p16 and p64 while a noticeable reduction was found with the increase of cutting intensity. as a results of enzymatic and non enzymatic oxidation of ascorbic acid, prolonged storage period, mechanical damages or thermal treatment, dha amount is known to increase. the oxidized form thus represents the majority of vitamin c (davey et al., 2000; lee and kader, 2000). in the present study different behaviors were observed: as for p128 and cho samples the decrease in aa amount due to oxidation was accompanied by an increase in dha amount ending in a rise of total vitamin c content of the products. since this increase was proportional with the decrease in aa concentration, it can be supposed that no further oxidation of dha into 2,3-diketogulonic acid occurred. regarding p16 and p64 samples at day 2, higher aa amounts were detected, suggesting the occurrence of new synthesis or the presence of different sources of ascorbic acid. cordenunsi et al. (2005), reported that ascorbic acid synthesis in strawberries occurs during the storage period, and that temperature may affect it. as for sweet pepper fruit, it was observed that wounding stress activated both biosynthesis and metabolism reduction of ascorbic acid, leaving unaffected the level of aa in the product (imahori et al., 1997). moreover, wolucka and van montagu (2003) proposed a new vitamin c biosynthesis pathway in which l-gulose and l-gulono-1,4-lactone act as direct precursors of ascorbic acid in plant tissue during storage. as for the present work it was possible to suppose that new synthesized ascorbic acid replaced the amount which was oxidized. as a result, ascorbic acid content did not decrease over time while a huge increment in dha occurred for p64 sample. some authors observed in different strawberry cultivars that dha evolution with storage time, when associated with ascorbic acid retention, can be considered an evidence of a redox system (aa/dha) triggered during cold storage, reported to be a cultivar-specific more than fruit-specific process (cordenunsi et al., 2005). moreover, to date, many of the products deriving from dha degradation are still unclear, although 2,3diketogulonic acid, threose and oxalic acid, glyoxal, methyl glyoxal and diacetyl have been identified or hypothesized as byproduct of dehydroascorbic acid decomposition (fayle et al., 2000). ascorbate, its product of oxidation (dha) and consequently dha metabolism play significant roles in the apoplast (lin and varner, 1991). for this reason dha results to be a key-factor in ascorbate catabolism, and it could be oxidized to oxalate or hydrolyzed to 2,3-diketogulonate and downstream carboxypentonates. the prevalence of one of the two reactions (oxidation or hydrolysis) is dependent on the status of the reactive oxygen species (parsons et al., 2011). in general, dha/aa ratio tends to rise during storage time although the oxidized form is unstable and is easily decomposed, this leading to a decrease in its biological activity. the changes in the form of ascorbic acid result to be important from a technological and a 439 solomon et al. effect of wounding intensity on quality of strawberry fruit nutritional point of view (lee and kader, 2000; cordenunsi et al., 2005). a possible explanation for what is showed in table 1 may be found in the prevalence of phenolics de novo synthesis with respect to their oxidation as response to cutting stress. as described by kang and saltveit (2002), the cutting related to processing of fresh-cut fruit and vegetables may induce an increase in their antioxidant capacity, enhancing synthesis and accumulation of phenols. in fact, wounding stimulates the activity of phenylalanine ammonia-lyase, which is responsible for the catalyzation of the first step of phenylpropanoid metabolism due to which tartaric acid is converted into chlorogenic acid. an increase in its activity leads to accumulation of phenols, enzymatic oxidation and tissue discoloration (saltveit, 2000; adams and brown, 2007). in contrast, however, wounding also induces antioxidant degradation, resulting in oxidation of active compounds such as ascorbic acid and phenolic compounds. their final content is the balance result between production and oxidation rates, being these rates affected by storage temperature or cutting intensity as modeled by amodio et al. (2014). however, in the case of a more serious damage, as for cho samples, tpc degradation was supposed to be faster than their production, ending in the lowest value of tpc. at day 2 however, tpc increased more clearly in p4 and p16 samples, which showed significantly higher values than other treatments, including the who. this is supported by literature, torres-contreras et al. (2014) report that phenolic content in white potato tubers that were subjected to different wounding stresses showed an accumulation of 100% and 65% tpc for slices and pieces, respectively, whereas shredded potatoes stored at 10°c for 96 hours showed 40% lower phenolic content if compared to the starting product. similarly, an accumulation of wounding-induced tpc (approximately 60%) and an increase in the activity of pal enzyme were found in purple-flesh sliced potato tissues stored at 15°c for 48 hours (reyes and cisneros-zevallos, 2003). according to surjadinata and cisneros-zevallos (2003), phenolic antioxidant accumulation is dependent on the level of wounding intensity. the phenolic content increased with wounding intensity by 97, 76, and 252% when cut as slices, pieces and shreds, respectively compared to non-wounded carrots (0.45-0.52 g kg-1). moreover, the same behavior was observed by hu et al. (2014) on fresh-cut lotus root. after 7 days of storage pal activity of fresh-cut lotus root slices was 68% higher compared to control. at the same time 130% increase in phenolic content was detected. antioxidant activity also increased due to total phenol accumulation; however, wounding resulted in a significantly higher browning by increasing the ppo activity of the slices. phenols represent the main structural and defense-related functions in plant cells via the phenylpropanoid pathway. wounding of fruit and vegetables tissues obviously causes rupture of the cell membrane and this induce several physiological responses causing the combination of phenolics with the oxidative enzymes and/or the synthesis of different classes of phenolics to repair the wounding damage. in strawberries, tac was reported to be in the range between 0.15 to 0.80 g kg-1 (padmanabhan et al., 2016) and cold storage may induce their biosynthesis and accumulation (holcroft and kader, 1999). accordingly, about 60% accumulation of tac in sliced potatoes was described by reyes and cisneros-zevallos (2003), and a similar increase was reported for anthocyanin content in pomegranates stored at low temperatures (arendse et al., 2014). the high amount of vitamin c, phenolics including anthocyanins and therefore antioxidant activity in strawberries, even after severe cutting stress, is very important for beneficial effects on consumer health (giampieri et al., 2012). the relatively higher values of ac determined for cho sample could probably be due to the higher values of total vitamin c and phenolics. antioxidant capacity of fruit is, in fact, strictly related to the presence of vitamin c and phenolic compounds, being these effective systems to scavenge oxygen radical, ac level is influenced by the occurrence of different active phytochemical compounds (giampieri et al., 2012). as reported by tulipani et al. (2008), vitamin c in strawberries is the greatest contributor (>30%) to the total antioxidant capacity followed by anthocyanins (contributing for 25-40%). the remaining part was composed mainly of ellagic acid derivatives and flavonols (padmanabhan et al., 2016). antioxidant activity is therefore an expression of total vitamin c and total phenolic content of the product, including anthocyanin and ellagitannins groups (giampieri et al., 2012). as for ph, tss and ta, no clear trend and very slight differences were observed, as also reported by li et al. 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isoascorbic acid simultaneous determinations by reverse phase ion interaction hplc. j. food sci., 57: 506-511. zhu m., chu c.l., wang s.l., lencki r.w., 2001 influence of oxygen, carbon dioxide, and degree of cutting on the respiration rate of rutabaga. j. food sci., 66: 30-37. impaginato 151 adv. hort. sci., 2021 35(2): 151­164 doi: 10.36253/ahsc­9624 single and combined effects of bacillus spp. and brown seaweed (sargassum vulgare) extracts as bio­stimulants of eggplant (solanum melongena l.) growth r. aydi­ben­abdallah 1 (*), n. ammar 1, f. ayed 1, 2, h. jabnoun­ khiareddine1, m. daami­remadi 1 1 lr21agr03‐production and protection for a sustainable horticulture, university of sousse, regional research centre on horticulture and organic agriculture, bo 57, 4042 chott‐mariem, tunisia. 2 technical centre of organic agriculture, 4042 chott‐mariem, sousse, tunisia. key words: bacillus spp., bio­stimulant, compost tea, eggplant growth, extracts, sargassum vulgare. abstract: bacillus subtilis sv41, b. amyloliquefaciens subsp. plantarum sv65 and sargassum vulgare extracts were evaluated for their plant growth­promot­ ing potential on eggplant (solanum melongena l.) plants. bio­treatments applied singly and/or in combination were further compared to a compost tea and to a commercial bio­fertilizer (acadian™). results clearly showed that the combined treatments based on the two bacillus spp. strains and the aqueous algal extract and the last one mixed with b. amyloliquefaciens subsp. plan‐ tarum sv65 induced the highest enhancements in the plant height and the maximum root length which were estimated at 32.4­33.9%, 23.9­25.5% and 23.4­25% and at 36.8­41%, 32.9­37.4% and 36.3­40.5% compared to water, compost tea and acadian™ based treatments, respectively. furthermore, the combined treatment based on the aqueous algal extract and b. amyloliquefa‐ ciens subsp. plantarum sv65 had significantly improved eggplant growth where the recorded increments in the stem diameter, the aerial part fresh weight, and the root fresh weight varied from 17.5 to 24.6%, 38.4 to 46.1%, and 32.3 to 50% as compared to the three controls, respectively. as for single treatments test­ ed, the aqueous extract had induced a significant improvement in the major growth parameters measured. developed bio­stimulant was found to be more effective than compost tea and commercial bio­fertilizer based treatments. 1. introduction the eggplant (solanum melongena l.) contributes to the diversification of market gardening products and constitutes a new product requested by foreign markets. in tunisia, the exported quantities over the past five (*) corresponding author: raniaaydi@yahoo.fr citation: aydi­ben­abdallah r., nawaim ammar, fakher ayed, hayfa jabnoun­khiareddine, mejda daami­remadi., 2021 ­ single and com‐ bined effects of bacillus spp. and brown seaweed (sargassum vulgare) extracts as bio‐stimulants of eggplant (solanum melongena l.) growth. ­ adv. hort. sci., 35(2): 151­164 copyright: © 2021 aydi­ben­abdallah r., nawaim ammar, fakher ayed, hayfa jabnoun­khiareddine, mejda daami­remadi. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 august 2020 accepted for publication 28 may 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9624 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 151­164 152 years were estimated at 187 tons. the export rate remains low compared to 56 thousand tons recorded in 2013/2014 agricultural campaign concentrated in tomato, watermelon, potato, and salad crops. to meet the requirements of consumers and increase the competitiveness of our exports at the interna­ tional markets level, significant efforts have been made in terms of improving quality and productivity of this crop (gil, 2020). the increasing demand for eggplants has gone along with the rapid population growth (maghfoer et al., 2014). eggplants contain low calories and high nutrient potential (sowinska and krygier, 2013). according to gandhi and sundari (2012), eggplant is widely used for medicinal features to reduce blood cholesterol and to regulate hypertension. thus, due to these benefits, the demand of eggplant and its production is expected to increase (sowinska and krygier, 2013). long term use of inorganic fertilizer has altered soil fertility leading to decreased efficiency of nutri­ ent absorption and productivity and adverse effects on environment and human health (jagatheeswari, 2013; waseem et al., 2013). therefore, research efforts are concentrated on alternative nutrients to improve soil physical, chemical, and biological traits through the application of chimerical organic fertiliz­ ers (maghfoer et al., 2014) and/or various organic soil amendments such as compost (d’hose et al., 2012), plant extracts (bijarniya, 2011), algae (eyras et al., 2008), and microbial inoculants (arora et al., 2020). application of chemical fertilizers with inocu­ lants has been also explored (carvajal­muñoz and carmona­garcia, 2012). application of microbial inoc­ ulants has gained an increased interest in the last three decades (babalola and glick, 2012). microbial inoculants are the formulations of bene­ ficial living microorganisms that, when added to the soil, they can improve the availability of nutrients to host plant either directly or indirectly, thereby lead­ ing to improved plant growth (gaind, 2011). various microorganisms are explored for the production of microbial inoculants such as azotobacter , azospirillum, bradyrhizobium, mycorrhizae, phospho­ rus solubilizing bacteria, and rhizobium. these bio­ inoculants can colonize the soil and perform various biophysical and biochemical soil activities that facili­ tate the availability and the uptake of nutrients to plants (alori et al., 2017). microbial inoculants could be grouped into nitrogen fixers i.e. rhizobium and bradyrhizobium , phosphate solubilizers i.e. pseudomonas, bacillus, aspergillus etc., cellulose degraders such as cytophaga, and phosphate mobi­ lizers such as mycorrhizae. recent demands of organic farming enhanced the application of organic treatments such as seaweed extracts in agriculture. seaweeds are aquatic plants belonging to the plant kingdom of thallophyta (arioli et al., 2015). at least 59 species of seaweeds can stimulate germination, growth, and yields of some horticultural plants (sunarpi et al., 2010). seaweed application in the agricultural field has numerous benefits such as stimulation of seed germination, promoting plant growth, improvement of water and nutrient uptake, enhancement of frost and saline resistance, biocontrol and resistance towards phy­ topathogenic agents, and remediation of pollutants of contaminated soil (nabti et al., 2016). fresh and dry seaweed or its derived products i.e extracts, com­ posts, and soil conditioners, have been long used in agriculture to enhance plant growth and productivity (eyras et al., 2008). seaweeds applied, singly or in combination with other macroalgae and/or bacteria, enhance crop productivity. sridhar and rengasamy (2010) successfully applied a brown marine alga s. wightii combined with a green seaweed ulva lactuca to enhance peanut growth. additionally, a mixture of two bacteria azotobacter chrocoocum and bacillus megaterium var. phosphaticum combined with sea­ weed extracts increased growth of bitter orange plants (ismail et al., 2011). in view of previous studies, aqueous and methanolic extracts from a brown macroalgae (s. vul‐ gare) were assessed singly and in combination with two endophytic bacteria i.e b. subtlis sv14 and b. amyloliquefaciens subsp. plantarum sv65 for egg­ plant growth. both bacillus spp. used in this study showed growth and health bio­stimulating effects on tomato plants through their capacity to produce indole­3­acetic acid, organic acids siderophore and their ability to solubilize phosphate and to biocontrol fusarium wilt disease in tomato (aydi ben abdallah et al., 2017, 2018). furthermore, ammar et al. (2017) demonstrated s. vulgare aqueous and methanolic extracts’ ability to efficiently control fusarium dry rot disease in potato. phenolic acids and flavonoids are the major components in the methanolic extract of s. vulgare using hplc­dad analysis (ammar et al., 2017). the main objective of this study was to evaluate the ability of two bacillus spp. strains applied singly and/or combined with s. vulgare aqueous or aydi‐ben‐abdallah et al. ‐ bacillus spp. and sargassum vulgare extracts as biostimulants of eggplant growth 153 methanolic extracts on eggplant growth and produc­ tivity. 2. materials and methods bacillus spp. culture b. subtilis sv41 (accession number kr818071) and b. amylolequifaciens subsp. plantarum sv65 (accession number kr818073) isolated from two wild solanaceous species datura metel and solanum nigrum, respectively, were used in this study. their isolation protocol, characterization and identification analysis were mentionned in aydi ben abdallah et al., (2017) and (2018) studies. they were previously selected based on their growth bio­stimulating effect and ability to control tomato fusarium wilt disease when tested in pot experiment or under field condi­ tions (aydi ben abdallah et al., 2017; 2019). the plant growth­promoting traits of both bacillus strains are detailed in table 1. stock cultures of both bacterial strains, were con­ served at ­20°c in nutrient broth (nb) medium amended with 40% glycerol. bacillus spp. colonies of a 1­day­old culture on nutrient agar (na) medium were transferred to luria­bertani broth (lb) and incu­ bated at 28 ± 2°c for 48 h and under continuous shaking at 150 rpm. the bacterial strains were tested at the exponential stage of growth (data unpub­ lished). the concentration of bacillus spp. was adjust­ ed at 108 cells/ml using spectrophotometre at do 600 nm. preparation of aqueous and methanolic extracts from sargassum vulgare brown seaweed was sampled during february 2014 from monastir, tunisia (n 35°46’47.754”; e 10°47’9.312”). the alga sampling and processing are detailed in a previous study (ammar et al., 2017). grounded samples were packed and stored at 4°c until use. for aqueous extraction, 1 kg of powder sample of s. vulgare was soaked in 20 l of sterile distilled water (sdw) and boiled at 100 ± 2°c for 1 h. after cooling, extracts were filtered twice through whatman n°1 sterile filter paper and further sterilized by filtration through sterile microfilter (0.22 μm pore size). the collected aqueous extracts, prepared at the concen­ tration 50 g/l, were stored at 4°c until further use within a week to avoid any chemical alteration (ammar et al., 2017). for methanolic extraction, samples of the brown seaweed (1 kg each) were subjected to a series of maceration in methanol (3 l) for three days under ambient room conditions. after filtration, the solvent was evaporated using a rotary evaporator under reduced pressure (at 60°c). one gram of the methanolic dry residue was separately dissolved into 10 ml of methanol. methanolic extracts used at the concentration 1 g/l were stored at 4°c until further use (ammar et al., 2017). eggplant seedling preparation the cultivar bonica, the most used by agricultures in the tunisian centre­east regions, was used in this study. eggplant cv. bonica seeds were disinfected by immersion into 0.2% sodium hypochlorite for 3 min. they were washed several times with sdw. disinfected seeds were subsequently treated with bacterial suspensions (~108 cells/ml) and/or aqueous and methanolic s. vulgare extracts using 20 μl per seed for 1 h. the same volume of sdw was used for treatment of control seeds. eggplant treated and untreated seeds were sown in alveolar plastic trays (7×7 cm) filled with sterilized peat™ (floragard vertriebsgmbh für gartenbau, oldenburg). seeds were further treated at trays with 5 ml of bacterial suspensions (~108 cells/ml) and/or aqueous and methanolic extracts from the brown seaweed. control seeds were treated with the same volume of sdw. during all the growing period, trays were watered regularly to avoid drought stress and seedlings were kept under greenhouse conditions (20­30°c with a 16 h light and 8 h dark cycle, and 60­ table 1 ­ plant growth­promoting (pgp) traits of bacillus subtilis sv41 and bacillus amyloliquefaciens subsp. plantarum sv65 used in the current investigation (aydi ben abdallah et al., 2017, 2019) z iaa = indole­3­acetic acid; +: production of iaa. y + = presence of phosphatase activity; ­: absence of phosphatase activity. x = organic acids; +: production of oxalic and malic acids. w + = presence of siderophore activity. pgp traits bacterial strain b. subtilis sv41 b. amyloliquefa‐ ciens subsp. plantarum sv65 iaa production z + + phosphatase activity y ­ + organic acids x + + siderophore production w + + adv. hort. sci., 2021 35(2): 151­164 154 70% relative humidity) until reaching the two­true leaf growth stage. screening of the effects of bacillus spp. and sargassum vulgare extracts on eggplant growth effect of single bio‐treatments each bacillus spp. strain (b. subtilis sv41 or b. amyloliquefaciens subsp. plantarum sv65) was singly inoculated to eggplant seedlings by dipping roots for 30 min in a bacterial suspension (108 cells/ml) pre­ pared as described above (aydi ben abdallah et al., 2017). control seedlings were dipped in sdw only and lb medium. treated and control seedlings were transplanted into individual pots (12.5 cm × 14.5 cm) containing sterilized peat. treated seedlings were re­ treated as substrate drenching with 50 ml of each bacterial cell suspension or with 50 ml of s. vulgare aqueous and methanolic extracts prepared as described above. four weeks after transplanting, eggplant seedlings were re­treated with 50 ml of each bacterial suspension and/or tested aqueous and methanolic extracts. seven replicates of one seedling each were used for each individual treatment and the whole experi­ ment was conducted twice. treated and control seedlings were grown for 60 days under greenhouse conditions as described above (botta et al., 2013). after 60 days of growth, the plant height, the stem diameter, the aerial part fresh and dry weights, the maximum root length, the root fresh and dry weights, the flower number, the fruit number, and the fruit fresh and dry weights were noted. effect of combined bio‐treatments for combined bio­treatments, equal volumes of cell suspensions of each bacterial strain from 2 d­old lb cultures were mixed and adjusted to 108 cells/ml with sdw. equal volumes of each aqueous and/or methanolic extract from the brown seaweed were mixed with an equal volume of bacterial suspension of b. subtilis sv41and b. amyloliquefaciens subsp. plantarum sv65 adjusted at 108 cells/ml or their com­ bination. seven combined bio­treatments were test­ ed and detailed in table 2. eggplant cv. bonica seedlings were treated by dip­ ping roots for 30 min in each combined bio­treat­ ment prepared as described above. control seedlings were dipped in sdw only and lb medium. treated and control seedlings were potted in commercialized sterile peat. treated seedlings were re­treated as substrate drenching with 50 ml of each tested com­ bined bio­treatment. four weeks after transplanting, eggplant seedlings were re­treated with 50 ml of each combined bio­treatment as described above. seven replicates of one seedling each were used for each individual treatment and the whole experi­ ment was conducted twice. after 60 days of growth under the same greenhouse conditions, the same growth parameters detailed above were measured. comparative efficacy of tested bio‐treatments (bacillus spp. and sargassum vulgare extracts) and organic amendments bacillus spp. strains and s. vulgare extracts even applied singly or in combination were compared to a compost tea and to a commercial bio­fertilizer for their growth­promoting potential on eggplant seedlings. comparative efficacy of tested bio‐treatments and a compost tea the compost used in this study contained 70% of bovine manure, 25% of sheep manure and 5% of olive­mill solid waste. the characterization of com­ post and the preparation procedure of compost tea (1:5 w/v) were described in a previous study (ayed et al., 2018). the physico­chemical and microbial char­ acterization of compost are listed in table 3. the compost used in this study had significantly improved the plant height, the leaf number, the aerial part dry table 2 ­ the seven combined bio­treatments tested bio­treatment code of bio­treatment bacillus subtilis sv41 + bacillus amyloliquefaciens subsp. plantarum sv65 b1 + b2 sargassum vulgare aqueous extract + b. subtilis sv41 e aq + b1 s. vulgare aqueous extract + b. amyloliquefaciens subsp. plantarum sv65 e aq + b2 s. vulgare aqueous extract + b. subtilis sv41 + b. amyloliquefaciens subsp. plantarum sv65 e aq + b1 + b2 s. vulgare methanolic extract + b. subtilis sv41 e meth + b1 s. vulgare methanolic extract + b. amyloliquefaciens subsp. plantarum sv65 e meth + b2 s. vulgare methanolic extract + b. subtilis sv41 + b. amyloliquefaciens subsp. plantarum sv65 e meth + b1 + b2 aydi‐ben‐abdallah et al. ‐ bacillus spp. and sargassum vulgare extracts as biostimulants of eggplant growth 155 weight of tomato plants compared to the untreated control plant (ayed et al. 2018). eggplant seedlings were treated by dipping roots for 30 min in compost tea (ct). control seedlings were dipped in sdw only. treated and control seedlings were transplanted into individual pots (12.5 × 14.5 cm) containing sterilized peat. treated seedlings were re­treated as substrate drenching with 50 ml of compost tea. four weeks after trans­ planting, eggplant seedlings were re­treated with 50 ml of compost tea. after 60 days of growth under greenhouse condi­ tions, the growth parameters were measured as described above. comparative efficacy of tested bio‐treatments and a commercial bio‐fertilizer the commercial bio­fertilizer used in this study was the acadian™ seaweed extract powder used at 2 g/l. the procedure of seedling treatment, the green­ house conditions and the noted growth parameters were the same as described above. statistical analysis a one­way analysis of variance (anova) was used for data analysis. the software used is the statistical package for the social sciences (spss) for windows version 16.0. each experiment was conducted twice yielding similar results. no significant interactions between treatment and experiment were noted. therefore, one representative trial of each experi­ ment is reported. experiments were undertaken according to a completely randomized design. means were compared using multiple range duncan test at p≤0.05. 3. results growth‐promoting potential of tested single bio‐ treatments b. subtilis sv41 and b. amyloliquefaciens subsp. plantarum sv65 based treatments and the aqueous and methanolic s. vulgare extracts were screened singly for their plant growth­promoting (pgp) ability on eggplant plants. as shown in figs. 1, 2 and 3, the plant growth parameters (plant height, stem diame­ ter, fresh and dry weight of the aerial part, maximum root length, root fresh weight, flower and fruit num­ ber, and fruit fresh weight), noted 60 days post­treat­ ment, varied significantly (at p≤0.05) depending on tested bacterial and/or algal extracts. plants treated separately with the whole bacterial cells of both bacillus strains and the aqueous extracts from s. vulgare were significantly 19 to 29.2% taller than the untreated control plants (fig. 1a). the treat­ ments based on b. amyloliquefaciens subsp. plan‐ tarum sv65 cells, the aqueous and the methanolic extracts from the brown seaweed led to a significant increase by 14.3 to 20.9% in the stem diameter as compared to water control (fig. 1b). treatments with the methanolic and the aqueous algal extracts had stimulated by 33.8 and 43.4% the aerial part fresh over the untreated control (fig. 1c). only the aque­ ous extract had significantly enhanced the aerial part dry weight by 32.3% compared to control (fig. 1d). it should be highlighted that eggplant aerial part devel­ opment was similar for lb medium­ treated plants and water control ones (figs. 1a, 1b, 1c, 1d). as for their effects on the root development, all tested bio­treatments induced a significant (at p ≤ 0.05) increment in the maximum root length and the root fresh weight when compared to control (figs. 2a, 2b). the maximum root length was significantly increased by 16.6 to 27.7% with both bacillus spp. strains and s. vulgare aqueous extract when applied separately as compared to control (fig. 2a). the root z, y bacteria and fungal counts from compost during the matura­ tion phase of composting after 72 h of incubation at 35°c onto pca and pda, respectively. table 3 ­ the physico­chemical and microbial characterization physico­chemical characterization total organic carbon (%) 25 organic matter (%) 43 water retention (%) 33 total porosity (%) 50 bulk density (g/cm3) 0.55 dry matter (%) 70 electrical conductivity ( ms/cm) 3.6 potential of hydrogen (ph) 7.3 ambient temperature (°c) ̴ 34 nutrient content (% of dry matter) nitrogen (n) 1.82 phosphorus (p) 0.07 potassium (k) 1.2 calcium (ca) 3.39 sodium (na) 0.59 microbial characterization bacterial count z (105 cfu/g of compost) 3.92 fungal count y (104 cfu/g of compost) 6.6 156 adv. hort. sci., 2021 35(2): 151­164 fresh weight was enhanced by 29.9 and 38.2% over control following treatments with b. amyloliquefa‐ ciens subsp. plantarum sv65 (b2) and s. vulgare methanolic extract, respectively (fig. 2b). it should be highlighted that eggplant root development parame­ ters were comparable on plants treated with lb medium as well as water control plants (figs. 2a, 2b, 2c). data illustrated in figure 3a indicated a significant increase by 65.5 to 78.7% over the untreated control in the flower number following the individual applica­ tion of all tested bio­treatments where the highest increment, of about 78.7% over control, was achieved using the algal aqueous extract. the fruit number was 30% higher than control in plants treat­ ed with the aqueous extract (fig. 3b). eggplant plants treated separately with s. vulgare extracts and the whole cell suspensions of b. subtilis sv41 showed 25.7­28.7% higher fruit fresh weight relative to con­ trol (fig. 3c). it should be highlighted that plants treated with lb medium behaved similar than water control plants for eggplant flower and fruit produc­ tion (figs. 3a, 3b, 3c, 3d). growth‐promoting potential of tested combined bio‐ treatments seven combinations of the tested bio­treatments were evaluated for their effect on eggplant growth. analysis of variance revealed a significant (at p ≤ 0.05) variation in the plant height, the stem diame­ ter, the fresh and dry weights of the aerial part, the maximum root length, the root fresh weight, the flower number, and the fruit fresh weight, depending on tested treatments. as shown in figure 1a, a signifi­ cant increment in plant height, by 23.7 to 33.9% compared to control, was noted on eggplant plants treated with s. vulgare aqueous extract combined with each of bacillus strains (eaq+b1 and eaq+b2), fig. 1 ­ comparative efficacy of single and combined bio­treatments with sargassum vulgare extracts and selected bacillus spp. strains on the aerial part development of eggplant plants compared to the untreated control and to two organic amendments. water= plants treated with water; lb= plants treated with luria­bertani broth medium; ct= plants treated with a compost tea; biof= plants treated with a commercial bio­fertilizer (acadian™); b1= single treatment with b. subtilis sv41; b2= single treatment with b. amyloliquefaciens subsp. plantarum sv65; b1+b2= combined treatment with b. subtilis sv41 and b. amyloliquefaciens subsp. plantarum sv65; e aq: single treatment with aqueous s. vulgare extract; e aq+b1= combined treatment with s. vulgare aqueous extract and b. subtilis sv41; e aq+b2= combined treatment with aqueous s. vulgare extract and b. amyloliquefaciens subsp. plan‐ tarum sv65; e aq+b1+b2= combined treatment with aqueous s. vulgare extract and both bacillus spp. strains. e meth= single treatment with methanolic s. vulgare extract. e meth+b1= combined treatment with methanolic s. vulgare extract and b. subtilis sv41; e meth+b2: combined treatment with methanolic s. vulgare extract and b. amyloliquefaciens subsp. plantarum sv65; e meth+b1+b2: combined treatment with methanolic s. vulgare extract and both bacillus spp. strains. bars sharing the same let­ ters are not significantly different according to multiple range duncan test at 5%. (a) comparative efficacy of tested bio­treat­ ments on eggplant height; (b) comparative efficacy of tested bio­treatments on eggplant stem diameter; (c) comparative efficacy of tested bio­treatments on eggplant aerial part fresh weight; (d) comparative efficacy of tested bio­treatments on eggplant aerial part dry weight. aydi‐ben‐abdallah et al. ‐ bacillus spp. and sargassum vulgare extracts as biostimulants of eggplant growth 157 fig. 2 ­ comparative efficacy of single and combined bio­treatments with sargassum vulgare extracts and selected bacillus spp. strains on the root development of eggplant plants compared to the untreated control and to two organic amendments. water= plants treated with water; lb= plants treated with luria­bertani broth medium; ct= plants treated with a compost tea; biof= plants treated with a commercial bio­fertilizer (acadian™); b1= single treatment with b. subtilis sv41; b2= single treatment with b. amyloliquefaciens subsp. plantarum sv65; b1+b2= combined treatment with b. subtilis sv41 and b. amyloliquefaciens subsp. plantarum sv65; e aq= single treatment with aqueous s. vulgare extract; e aq+b1= combined treatment with s. vulgare aqueous extract and b. subtilis sv41; e aq+b2= combined treatment with aqueous s. vulgare extract and b. amyloliquefaciens subsp. plan‐ tarum sv65; e aq+b1+b2= combined treatment with aqueous s. vulgare extract and both bacillus spp. strains. e meth= single treatment with methanolic s. vulgare extract. e meth+b1= combined treatment with methanolic s. vulgare extract and b. subtilis sv41; e meth+b2= combined treatment with methanolic s. vulgare extract and b. amyloliquefaciens subsp. plantarum sv65; e meth+b1+b2= combined treatment with methanolic s. vulgare extract and both bacillus spp. strains. bars sharing the same let­ ters are not significantly different according to multiple range duncan test at 5%. (a) comparative efficacy of tested bio­treat­ ments on eggplant maximum root length; (b) comparative efficacy of tested bio­treatments on eggplant root fresh weight; (c) comparative efficacy of tested bio­treatments on eggplant root dry weight. fig. 3 ­ comparative efficacy of single and combined bio­treatments with sargassum vulgare extracts and selected bacillus spp. strains on the eggplant flowers and fruit production compared to the untreated control and to two organic amendments. water= plants treated with water; lb= plants treated with luria­bertani broth medium; ct= plants treated with a compost tea; biof= plants treated with a commercial bio­fertilizer (acadian™); b1= single treatment with b. subtilis sv41; b2= single treatment with b. amyloliquefaciens subsp. plantarum sv65; b1+b2= combined treatment with b. subtilis sv41 and b. amyloliquefaciens subsp. plantarum sv65; e aq= single treatment with aqueous s. vulgare extract; e aq+b1= combined treatment with s. vulgare aqueous extract and b. subtilis sv41; e aq+b2= combined treatment with aqueous s. vulgare extract and b. amyloliquefaciens subsp. plan‐ tarum sv65; e aq+b1+b2= combined treatment with aqueous s. vulgare extract and both bacillus spp. strains. e meth= single treatment with methanolic s. vulgare extract. e meth+b1= combined treatment with methanolic s. vulgare extract and b. subtilis sv41; e meth+b2= combined treatment with methanolic s. vulgare extract and b. amyloliquefaciens subsp. plantarum sv65; e meth+b1+b2= combined treatment with methanolic s. vulgare extract and both bacillus spp. strains. bars sharing the same let­ ters are not significantly different according to multiple range duncan test at 5%. (a) comparative efficacy of tested bio­treat­ ments on eggplant flower number; (b) comparative efficacy of tested bio­treatments on eggplant fruit number; (c) comparative efficacy of tested bio­treatments on eggplant fruit fresh weight; d: comparative efficacy of tested bio­treatments on eggplant fruit dry weight. adv. hort. sci., 2021 35(2): 151­164 158 the aqueous and the methanolic algal extracts com­ bined each one with both bacterial strains (eaq+b1+b2 and emeth+b1+b2) and the methanolic extract mixed with b. subtilis sv41 (emeth+b1). the highest increase of this parameter, of about 32.4­ 33.9% over control, was recorded following treat­ ments with s. vulagre aqueous extract combined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) and the algal aqueous extract mixed with the two bacillus strains (eaq+b1+b2). a significant enhancement of the stem diameter of the treated plants, estimated at 13.6 to 24.7% over control, was also noted following the seven test­ ed combined bio­treatments. the highest increment (24.7%) was induced by the aqueous extract com­ bined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) and at a lesser extent this extract when mixed with both bacillus strains (eaq+b1+b2) (20.9%) (fig. 1b). the aerial part fresh weight was also significantly increased by 29.6 to 46.1% over control following treatments with the two bacterial strains (b1+b2), the aqueous extract combined with each bacterial strain separately (namely eaq+b1 and eaq+b2) or in combination (eaq+b1+b2) and the methanolic extract mixed with b. subtilis sv41 (emeth+b1) (fig. 1c). the highest increment (by 46.1% relative to the control) was induced by the aqueous extract com­ bined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2). eggplant plants treated with the algal aqueous extract combined with b. amyloliquefaciens subsp. plantarum sv65 cells (eaq+b2) or mixed with both bacillus strains (eaq+b1+b2) showed a signifi­ cant enhancement in their aerial part dry weight by 25.4­26.5% compared to control (fig. 1d). as shown in figure 2a, the maximum root length increase over control ranged between and 18.4 to 41% following all tested combined bio­treatments and the highest improvement, of about 36.8­41% over control, was induced by s. vulgare aqueous extract mixed with both bacillus strains (eaq+b1+b2) or with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2). all tested bio­treatments combined with the algal aqueous extract induced a significant enhancement in the root fresh weight of about 30.6­ 50% as compared to control (fig. 2b). furthermore, the combined treatment based on s. vulagre methanolic extract and b. subtilis sv41 (emeth+b1) had also significantly improved this parameter by 41.4% over control. the highest increment in the root fresh weight, of about 50% relative to control, was noted on plants treated with the combined treat­ ment composed of the aqueous extract and b. amy‐ loliquefaciens subsp. plantarum sv65 (eaq+b2). when screened for their effects on the flower number, the seven combined bio­treatments led to 54.5­73.7% increment in this parameter compared to control (fig. 3a). also, the fruit fresh weight was sig­ nificantly increased by 24.2% over control on egg­ plant plants treated with s. vulgare methanolic extract mixed with b. amyloliquefaciens subsp. plan‐ tarum sv65 (emeth+b2) (fig. 3c). comparative efficacy of tested bio‐treatments with a compost tea and a commercial bio‐fertilizer eleven tested bio­treatments, applied singly or in combination, were evaluated for their growth­pro­ moting potential on eggplant seedlings as compared to a compost tea and to a commercial bio­fertilizer (acadian™). aerial part development analyses of variance of all growth parameters measured (plant height, stem diameter, aerial part fresh and dry weights) showed a significant variation (at p ≤ 0.05) between tested bio­treatments as com­ pared to compost tea­ and acadian™ based treat­ ments. data showed a significant enhancement by 18.3 to 25.5% over compost tea based treatment in plant height of eggplant plants treated with s. vulgare aqueous extract applied either singly or in combina­ tion with both bacillus spp. strains and/or singly with each tested bacterial strain (fig. 1a). furthermore, plants treated with the methanolic extract combined with b. subtilis sv41 and b. amyloliquefaciens subsp. plantarum sv65 (emeth+b1+b2) were 19.3% taller than those treated with the compost tea. similarly, compared with the tested commercial bio­fertilizer, the recorded increment varied from 17.7 to 25% depending on treatments. the highest increase of plant height, of about 23.4­25.5% and 23.3­25% over the commercial bio­fertilizer (i.e. acadian™ treat­ ment), were induced by the aqueous extract mixed with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) or with both bacterial strains (eaq+b1+b2), respectively. all treatments with s. vulgare aqueous extract, applied either singly or in combination with each bacillus strains separately or both strains combined, showed significant improvement in eggplant stem diameter by 9.9­17.5% and 11.2­18.7% over compost aydi‐ben‐abdallah et al. ‐ bacillus spp. and sargassum vulgare extracts as biostimulants of eggplant growth 159 tea and the commercial bio­fertilizer treatments, respectively (fig. 1b). b. amyloliquefaciens subsp. plantarum sv65 applied singly (b2) induced a signifi­ cant increment in this parameter by 12.2% versus compost tea­based treatment. s. vulgare methanolic extract mixed with b. subtilis sv41 (emeth+b1) led to a significant improvement in the stem diameter by 9.2% when compared to acadian™ based treatment (fig. 1b). as compared to both tested organic amend­ ments, eggplant plants treated with s. vulgare aque­ ous extract combined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) showed an incre­ ment by 17.5­18.7% in this growth parameter. as shown in fig. 1c, the aerial part fresh weight was significantly improved by 24.4 to 38.5% over the compost tea treatment in plants treated separately with the aqueous (eaq) and the methanolic (emeth) s. vulgare extracts, the aqueous one combined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) or mixed with both bacillus strains (eaq+b1+b2), and the methanolic extract associated with b. subtilis sv41 cells (emeth+b1). compared to the commercial bio­fertilizer, tested bio­treatments had also significantly improved this parameter by 24.9 to 38.9%. the highest increments in the fresh weight of the aerial part, of about 38.5 and 38.9% compared to compost tea­ and acadian™ based treatments, were induced by the aqueous extract combined with b. amyloliquefaciens subsp. plan‐ tarum sv65 (eaq+b2), respectively. the aerial part dry weight was enhanced by 26.7­ 33.4% and 31.2­37.5% over the compost tea and the acadian™ controls, using s. vulgare aqueous extracts either singly or in combination with b. amyloliquefa‐ ciens subsp. plantarum sv65 and both selected bac­ terial strains (fig. 1d). plants treated singly with the aqueous extract showed the highest increment of this parameter of about 33.4 and 37.5% compared to those amended with compost tea and acadian™, respectively. root development the maximum root length and the root fresh weight varied significantly (at p ≤ 0.05) depending on tested bio­treatments. plants treated with seven bio­ treatments (the aqueous and methanolic extracts combined with each bacterial strain and/or with both strains and the aqueous extract applied singly) showed significant improvement in the maximum root length of about 15.9 to 37.4% compared to ones treated with the compost tea (fig. 2a). as compared to acadian™ treatment, nine bio­treatments (same as previously, combined bacillus spp. strains and b. amyloliquefaciens subsp. plantarum sv65 applied singly) induced a significant enhancement by 17.7 to 40.5% in this parameter. the highest increments of the maximum root length, of about 37.4 and 40.5% over the compost tea and the commercial bio­fertiliz­ er controls, were induced by s. vulgare aqueous extract combined with b. amyloliquefaciens subsp. plantarum sv65 (eaq+b2) or with both bacillus strains (eaq+b1+b2), respectively, (fig. 2a). the root fresh weight was significantly improved by 32.3% compared to compost tea using the aqueous extract mixed with b. amyloliquefaciens subsp. plantarum sv65 and by 36.1 and 25% versus acadian™ treat­ ment using the last bio­treatment and the methano­ lic extract combined with b. subtilis sv41, respective­ ly (fig. 2b). the highest increments on this parame­ ter, by 32.3 and 36.1% compared to compost tea­ and acadian™ based treatments, were induced by the aqueous extract from s. vulgare mixed with b. amy‐ loliquefaciens subsp. plantarum sv65, respectively (fig. 2b). fruit production anova analyses performed for the flower num­ ber, fruit number and fruit fresh weight showed a sig­ nificant variation (at p ≤ 0.05) between the eleven bio­treatments tested and compost tea and acadian™ based treatments. as compared to compost tea control, the seven tested bio­treatments had significantly improved the flower number by 40.6 to 59.6%. the highest increase (59.6%) was noted on plants treated singly with the aqueous s. vulgare extract. compared to the commercial bio­fertilizer (acadian™), only the treat­ ment with the algal aqueous extract had significantly enhanced this parameter by 38.3% (fig. 3a). this aqueous extract when applied singly had also induced a significant improvement of the fruit num­ ber by 40% compared to compost tea and acadian™ based treatments (fig. 3b). as shown in fig. 3c, the average fruit fresh weight was significantly enhanced by 15.8 to 20.8% over the compost tea control using separately b. subtilis sv41, the aqueous and the methanlic s. vulgare extracts and the last one com­ bined with b. amyloliquefaciens subsp. plantarum sv65. these bio­treatments had significantly improved this production parameter by 35.8 to adv. hort. sci., 2021 35(2): 151­164 160 39.6% relative to the commercial bio­fertilizer. 4. discussion and conclusions the use of eco­friendly resources has been a major focus of attention in the past three decades. although reports on the benefits of using microbial inoculants for the promotion of plant growth and health in agricultural soil have been inconsistent, there is a promising trend for microbial inoculants to meet the sustainable agricultural production needs (alori et al., 2017). the use of seaweeds as bio­fertil­ izers in horticulture and agriculture has increased in the recent years basmal et al. (2019). this study was aimed to evaluate the efficacy of combining bacillus spp. strains and s. vulagre extracts (aqueous and methanolic extracts) in order to select the best combination for the bio­stimulation of eggplant growth. furthermore, bio­treatments (bacteria and algae extracts) tested singly and/or in combination were compared against two organic amendments i.e compost tea and acadian™ (a com­ mercial bio­fertilizer) to select the most effective bio­ stimulant among the tested treatments. bio­treatments (bacteria and/or algae extract) could be applied either singly as seed priming prior sowing, seedlings root dipping prior transplanting, soil drenching and foliar spraying or combination of two or more methods of application (papenfus et al., 2013). in this study, bio­treatments either used singly or in combination were applied as seed priming, then seedlings root dipping and finally as substrate drenching. the recommended method, timing and the rate of applications were greatly different accord­ ing to plant variety and growth stages (lola­luz et al., 2013). according to matysiak et al. (2011) study, the stimulatory potential is more efficient at the early stage of plant growth. in this study, all bio­treat­ ments were applied early at pre­sowing, the first application occurred at the two­true leaf stage and the second one four weeks post­planting. as single application, the aqueous extract from s. vulgare used at 50 g/l showed higher growth­pro­ moting potential based on major growth parameters of eggplant than b. subtilis sv41, b. amyloliquefa‐ ciens subsp. plantarum sv65 and the methanolic extract compared to the untreated control, and to compost and acadian™ based treatments. as demon­ strated by michalak and chojnacka (2015), water extraction was found the most effective for better release of micro­ and macro­elements from seaweed biomass even used as fertilizer and bio­stimulant. the application of seaweed extracts exhibit stimulat­ ing activities of plant growth, yield and fruit quality in a variety of horticultural crops (battacharyya et al., 2015; kocira et al., 2018; mahmoud et al., 2019). indeed, the use of water extract from algae as plant growth bio­stimulant was described in several crops such as wheat, tomato, arabidopsis, spinach, and vigna sinensis and this under normal and stressed environments (nabti et al., 2010; craigie, 2011; kavipriya et al., 2011). in this study, the boiling aque­ ous extract from s. vulgare at 100°c for 1 h did not affect its growth­promoting potential and the con­ tents of polyphenol and flavonoids (ammar et al. 2017). water extracts prepared by autoclaving or heating previously washed marine alga in distilled water are found to have beneficial growth stimulat­ ing effects in cereal and flowering plants (nabti et al., 2010; craigie, 2011). the aqueous s. vulgare extract applied singly had significantly improved the majority of growth para­ meters as compared to the untreated control and to the two tested organic amendments. some seaweeds have been successfully used as soil conditioners and fertilizers in agriculture (duarte et al., 2018). commercially, extracts from brown algae such as acadian are good sources of fertilizer (hurtado et al., 2008). fertilizers derived from seaweeds such as fucus, laminaria, ascophyllum, sargassum etc. are known to be biodegradable, non­polluting and non­ hazardous to human and environment (dhargalkar and pereira, 2005). mathur et al. (2015) study demonstrated the benefical effects of seaweed liquid fertilizer from sargassum wightii, ulva lactuca and enteromorpha intestinalis on stimulation of seed ger­ mination and growth, and enhancement of biochemi­ cal traits of glycine max plants. seaweeds extracts were found to be more active than chemical fertiliz­ ers in enhancing seed germination and growth para­ meters (godlewska et al., 2016). vasantharaja et al. (2019) found that foliar spraying of cowpea plants with the brown seaweed extract at 3% significantly improves the shoot length, the number of leaves per plant, yield, the total phenolic and flavonoid contents and the antioxidant activity as compared to control plants. foliar spray of liquid fertilizer based on s wightii extract has successfully enhanced the chloro­ phyll content, the internodes and the shoot length of tomato and chilli pepper plants compared to seed soaking (murugan et al., 2020). the mechanisms of aydi‐ben‐abdallah et al. ‐ bacillus spp. and sargassum vulgare extracts as biostimulants of eggplant growth 161 stimulation of plant growth by the marine algal extracts may be due to the diverse compounds observed in their extracts. indeed, macronutrients, organic substances such as amino acids and plant growth regulators substances are presents in the sea­ weed liquid fertilizer of sargassum species (zodape et al., 2008; nabti et al., 2016; murugan et al., 2020). furthermore, seaweed based treatments are able to increase the level of nutrient in soil such as nitrogen, phosphorus and potassium and other compounds as polysaccharides wich are necessary for plant growth that are highly diverse and constitute the major com­ pounds of algae cell walls (heltan et al., 2015; mirparsa et al., 2016; nabti et al., 2016). to improve the plant growth­promoting ability of both selected bacillus spp. used in the current study, they were combined either single or in combination with the aqueous and/or the methanolic s. vulgare extracts. microbial inoculants, applied singly or in combination, are able to improve nutrient availability and uptake, and to strengthen plant health (alori et al., 2017). as compared to untreated control and to the two tested organic amendments (compost tea and acadian™), eggplants treated with combined formu­ lations of b. amyloliquefaciens subsp. plantarum sv65 and aqueous s. vulgare extract showed the highest enhancements in plant height, stem diame­ ter, aerial part fresh weight, maximum root length, and root fresh weight. furthermore, the combination of b. subtilis sv41, b. amyloliquefaciens subsp. plan‐ tarum sv65 and the aqueous extract had significantly increased the plant height, the stem diameter and the maximum root length as compared to water, compost tea and acadian™ based treatments. when applied on seeds, plant surfaces or soil, microbial inoculants are shown able to enhance root exuda­ tion, increase the availability and supply essential nutrients to host plants, and thereby promoting their growth (trabelsi and mhamdi, 2013). the phytohor­ mones synthetized by microbial inoculants can result in development of the root system, expansion and elongation of the root hairs and lateral roots, leading to improved uptake of water and nutrients (halpern et al. 2015). fixation of atmospheric nitrogen, solubi­ lization of minerals such as phosphorus by microbial inoculants are also involved in plant growth promo­ tion (babalola, 2010). indirectly, microbial inoculants also affect the status of plants by eliciting the induced systemic resistance (isr) or the systemic acquired resistance (sar) thus improving their health. these acts prevent soil­borne pathogens from inhibiting plant growth (yang et al., 2009). the ability to trigger a salicylic acid (sa)­independent pathway controlling systemic resistance is a common trait of isr­inducing bio­control bacteria. both bacillus spp. used in this study, have been demonstrated as promising bio­stimulants when challenged to tomato plants and their ability to produce the indole­3­acetic acid, organic acids and/or siderophores, to solubilize phosphate, and to control fusarium wilt disease was evidenced (aydi ben abdallah et al., 2017, 2018). plant growth­promoting rhizobacteria (pgpr) applied singly and/or in combination reduced appli­ cation rates of chemical fertilizers. as demonstrated by adesemoye et al. (2009), a mixture of pgpr strains b. amyloliquefaciens in937a and bacillus pumilus t4, and the arbuscular mycorrhizae (am) glomus intraradices added to 75% fertilizer success­ fully enhance growth, yield, and nutrient (nitrogen and phosphorus) uptake of tomato plants compared to the 100% fertilizer control. in the same way, three bio­stimulants consisting of a mix of rhizospheric microorganisms i.e. pseudomonas sp. 19fv1t, p. fluo‐ rescens c7 and am fungi, tested in conditions of reduced fertilization, induced an increment in the yield, the fruit quality and the nutritional value of tomato fruits (bona et al., 2018). el­yazeid et al. (2007) demonstrated that the double inoculation with paenibacillus polymyxa and bacillus megaterium associated with a foliar spray of boron led to an enhancement of growth­promoting hormone levels including gibberellic acid, 3­indole acetic acid and cytokinines associated with a decrease in the abscisic acid inhibitor. double inoculation especially with the mycorrhizal fungus g. intraradices and boron spray improved sex ratio and early production of fruits accompanied with high yield of squash. several investigations support different aspects of potential macro algal applications in agriculture. currently, seaweed extracts are the new type of prod­ ucts used in plant cultivation (elsharkawy et al., 2019). it should be highlighted that the improvement of growth parameters in eggplant plants treated with combined bacillus spp. and aqueous extract from s. vulgare, recorded in the current study, is higher than that induced following the single application of aque­ ous extract. hence, the combinations of bio­treat­ ments enhance either the efficacy of bacteria and algal aqueous extract more than when applied singly. however, the combinations of the methanolic extract with tested bacillus spp. strains did not induce signifi­ adv. hort. sci., 2021 35(2): 151­164 162 cant increments in the major growth parameters. the synergism occurring between both tested bacterial strains and the aqueous s. vulgare extract was con­ firmed based on various growth parameters. crocker (2018) investigation clearly demonstrated the in vitro ability of seaweed extract to enhance pgpr growth which may explain the synergism noted. also, basmal et al. (2019) found that the biological fertilizer formu­ lation based on sargassum sp. extract enhance the growth rate of beneficial pseudomonas fluorescens. through the in planta experiments, combined pgpr inocula and seaweed extract enhanced significantly the root growth parameters of treated soybean plants compared to the untreated ones (crocker, 2018). the addition of bio­fertilizer containing multi­strains of bacillus acting as phosphorus­fixing agents and azotobacter, azospirllum and rhizobium as nitrogen­ fixing inoculants combined with a foliar spay with mixed seaweed extracts from ulva lactuca, ulva facia‐ ta and peterocladia caplicia at 10 ml/l led to incre­ ment of growth characters and to enhancement of the total yield of pea plants (elsharkawy et al., 2019). as conclusions, the use of plant­growth promoting bacteria especially bacillus strains and the brown seaweed extracts (aqueous and methanolic extracts) as bio­stimulants on eggplant plants was emphasized as compared to untreated ones. the combined treat­ ment based on bacillus spp. strains and the aqueous s. vulgare extract was found to be the most efficient bio­stimulant as compared to a compost tea and a commercial bio­fertilizer tested i.e. acadian™. the beneficial roles of the above combined bio­treat­ ments on growth parameters were higher than their single applications. the influence of the combined bio­stimulant developed based on the two tested bacillus spp. strains and the brown seaweed aqueous extract on the soil microbial community need to be explored in the future to find out ways to more effec­ tively apply this combined bio­treatment and to elu­ cidate its effects on soil microbiome including phy­ topathogenic and beneficial microorganisms. acknowledgements this work was funded by the ministry of higher education and scientific research of tunisia through the funding allocated to the research unit ur13agr09­integrated horticultural production in the tunisian centre­east, regional research centre on horticulture and organic agriculture of chott­ mariem, tunisia. references adesemoye a.o., torbert h.a., kloepper j.w., 2009 ­ plant growth‐promoting rhizobacteria allow reduced application rates of chemical fertilizers. ‐ microb. ecol., 58: 921­929. alori e.t., dare m.o., babalola o.o., 2017 ­ microbial inoculants for soil quality and plant health, pp. 281­ 328. ­ in: lichtfouse e. 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apple trees in mountainous and plain regions of algeria a. abed 1, 2 (*), m. bonhomme 3, a. lacointe 3, g. bourgeois 4, d. baalicherif 1 1 national school of agronomic sciences, algiers, algeria. 2 laboratory water‐ rock ‐plant, khemis‐miliana university, algeria. 3 clermont auvergne university, inra, piaf, f‐63000 clermont‐ferrand, france. 4 agriculture and agri‐food canada, saint‐jean‐sur‐richelieu research and development centre, saint‐jean‐sur‐richelieu (qc), canada. key words: budburst, flowering, golden delicious, modelling, temperature. abstract: global warming is a strongly felt reality in recent years in algeria. the fruit trees crop is particularly exposed to the impact of this warming, especially apple trees. a comparative study has been realized between a chronological daily temperature series from 1980 to 2016, and phenological data series (budburst and flowering) from 2000 to 2016, regarding the apple tree variety of golden delicious in two zones of northern algeria, sidi lakhdar (town of ain defla, in an altitude of 211 m) and benchicao (town of médéa, in an altitude of 1133 m). some contrasting tendencies according to sites and periods have been demonstrated: very significant warming at sidi lakhdar site in autumn and spring, in particular in october and april, disturbing thus the entrance of the buds in the endodormancy and ecodormancy. the result is a late action of the cold until february, which proved to be insufficient. however, no average warming has been demonstrated at the benchicao site, where the temperatures between november and january were cold enough to satisfy the need of cold units and raise the endodormancy. it seems that the failure to fulfill the need of cold units at sidi lakhdar site has strongly affected the goodness of fit of the classic phenological models, confirming indirectly the existence of more complex physiological processes (not taken in consideration by models), which manifest themselves in limited zones such as sidi lakhdar site. 1. introduction according to the experts of the intergovernmental group of the climate evolution (igce), from now on to the end of the 21st century, the average temperature will be raising from 2 to 6°c in europe following the regions, the climatic models and the socio-economic scenario. the summer droughts will be more intense as well (giannakopoulos et al., 2005; (*) corresponding author: abedlila24@yahoo.fr citation: abed a., bonhomme m., lacointe a., bourgeois g., baali-cherif d., 2019 climate chan‐ ge effect on the bud break and flowering dates of the apple trees in mountainous and plain regions of algeria. adv. hort. sci., 33(3): 417-431 copyright: © 2019 abed a., bonhomme m., lacointe a., bourgeois g., baali-cherif d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 january 2019 accepted for publication 3 september 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 417-431 418 gleizer et al., 2007). according to legave (2009), a worrying acceleration of the global warming has appeared during the 1990 decade and much more during 2000 decade. in occidental europe and the mediterranean basin, on a recent period of 30 years (1973-2002), we can estimate the average increase of the annual temperature at about 1°c since the end of 1980’s. regional differences are noticed though, with a warming relatively marked in the region of meknes in morocco (2.3°c) (balaghi, 2017), definitely higher than the one marked in the south-west of france (+1.3°c in nimes)(legave, 2009). the global warming will affect, and has already notably done, a wide range of physical/biological systems and human activity sectors, among which agriculture (including livestock) and its principal function of producing and nutrition (seguin, 2010). for algeria, the 21st century shall be characterized by temperatures increase, in the order of 1.0 to 1.5°c at the horizon of 2020 (fourth report of the igce in bourchef, 2013) and precipitations decrease in the order of 15 to 20%. extreme climatic phenomena are already affecting the region, like the rain and thunderstorms of november 2001 in algiers and october 2008 in ghardaia, and the cold waves in january 2005 and february 2001 in all algeria. all these events can be qualified as historic at least regionally. some simulations realized for two types of agricultural years in terms of pluviometry (normal and dry), show at the horizon of 2020 a decrease in the yield of winter cereals from 6% to 14% according to the geographical regions and the year type in algeria (tabet-aoul, 2000; tabet-aoul and bessaoud, 2009). phenology is the study of the occurrence of periodic events in animal and plant life in relation with the climate variations. those are characters that interpret the organisms’ adaptation to the climatic variation (chuine, 2005). the task of plant-phenology is to observe and record the periodically recurring growth stages. leaf unfolding, flowering of plants in spring, fruit ripening, colour changing and leaf fall in autumn are all examples of phenological events (koch et al., 2006). it has been designated as a key point to evaluate the global warming impact on the agricultural cultivations (moriondo and bindi, 2008). many studies have pointed an agreement which many species advanced the spring phenology events (budburst and blooming dates) particularly (doi and katano, 2008; gordo and sanz, 2010; malagi, 2014). since distinct phenological stages were defined decades ago (baggiolini, 1952; lichou et al., 1990; meier et al., 1994), a comparison of available definitions of phenological stages in cherry used independently throughout europe showed overlaps and shortcomings; hence, harmonisation was reached in this respect in the cost cherry fa 1104 working group 2 (cherry phenology and climate change) based largely on the acceptance of the bbch scale and agreed standard cultivars for phenology monitoring. cultivars were selected on the basis of early, medium and late flowering and most widely grown throughout europe. this contribution presents the agreed phenology stages in both visual and wording evidence. similarly, this contribution presents the agreed cultivars to be monitored in future for phenology and climate change effects for harmonization (wenden et al., 2017). in this context, the fruit arboriculture seems relatively vulnerable from the fact of some of its characteristics, rather biological (eg: the fruit trees sustainability and their need to many years of growth before fruiting) than economic. compared to other productions (annual cultivations), the fruit arboriculture is particularly exposed to unfavorable climatic impacts from the fact of multiannual consequences (alternation of production after ceasing) and accumulative (repeated impacts on the tree architecture). on the socio-economic level, strong links have been woven during the time between the product and its production place (eg: provence almonds, roussillon apricots…etc). this characteristic developed in france, for commercial valorization reasons, in a regulatory form of origin and quality naming (eg: agen prunes, lorraine plums…etc.). from this fact, the substitution of varieties and much more species for long-term climatic adaptation reasons seems relatively difficult to be implemented, probably risking to encounter regulatory and human obstacles (legave, 2009). these characteristics constitute an obstacle to the fast changes, not only to the variety range but also to the cultivation systems to cope with rising temperatures or other constraints from climate change. this climate vulnerability has already been expressed in the 2000s by strong production irregularities. unprecedented accumulations of unfavorable climatic conditions (frost, high temperatures, excessive rainfall) have been observed during key phases of the annual cycle of trees, from flowering to fruiting. thus, in southern france, very significant production losses were provoked, especially in 2007 for cherry trees following stormy episodes in may and june, which strongly penalized the french production, and in 2008 for apricot following episodes of excessive abed et al. ‐ climate change effect in apple flowering 419 heat as blooming approached. sensitive varieties have had abortion rates that strongly penalize the national production. phenological notes on flower buds of fruit trees, collected under contrasting temperature (time and place) conditions in europe, showed a significant advance of the different phenological stages, especially the flowering dates, for all the places. modeling work on spring phenology strongly suggests that warming has two opposite effects: (1) in autumn and early winter, a slowdown in the satisfaction of cold unit needs, delaying endodormancy; (2) at the end of winter and in spring, an acceleration of the satisfaction of the heat needs during the ecodormancy phase. the more pronounced intensity of this latter effect, consistent with the more pronounced increases in temperature at the end of winter-early spring than in autumn, largely explains the advances in flowering (legave et al., 2009). the analysis of flowering dates over long periods in western europe for the golden delicious apple variety reveals more significant progress in the north of the continent (10 days) than in the oceanic west (6-7 days) and a shortening of flowering time in continental regions (legave et al., 2012). these regional differences across western europe led to a decrease in spatial variability, that is to say, smaller differences between the flowering dates in the contrasting regions (decrease of 8-10 days for complete flowering between the mediterranean and continental regions). modeling studies, based in particular on the correlations between the average temperature of the period of ecodormancy and the observed flowering dates, confirm the notion that flowering advances and shortenings are mainly due to a faster satisfaction of the demand for heat units (legave et al., 2015). however, delayed endodormancy has also been noted in the oceanic and mediterranean regions, which may explain the shorter advances in these areas despite similar or greater warming and ultimately lead to delayed flowering. the joint statistical analysis of flowering date series for the golden delicious variety and temperature dynamics reveals a geographical diversity of responses to warming from autumn to spring. temperate climates in europe are characterized by flowering progress, while soft climates are characterized by flowering progress or stationary flowering dates (eg. morocco and brazil), (legave et al., 2015). at the same time, legave et al. (2015) and el yaacoubi et al. (2016) have shown in mild winter conditions, a longer flowering time associated with the high average temperature of the endodormancy period. in the same context, a comparison of dormancy dynamics of vegetative and floral buds of apple and almond trees was recently conducted between southern france, southern brazil, and northern morocco. differences in dormancy intensity and kinetics have been identified in relation to regional differences in the satisfaction of cold needs and different levels of requirements of the genotypes studied. the observed diversity of dormancy patterns suggests that genotypes adapted to mild climates (eg, almond trees, apple trees with low cold needs) are characterized by the ability of vegetative buds to remain in a state of low dormancy and ability of flower blanks to grow rapidly, guaranteeing the absence of phenological anomalies subsequent to foliage and flowering (el yaacoubi et al., 2015). the apple tree is currently an important fruit species in algeria. production is the most important fruit production, but it does not sufficiently cover the demand. the central region (medea blida ain defla) totals about 7400 ha or about a quarter of the total area. apple cultivation has grown considerably, from 30,000 ha in 2003 to 41,000 ha in 2013, with a production reaching 400,000 tons (f.a.o 2013, mentioned by meradi, 2015). due to the levels of yield and quality obtained, the golden delicious variety is one of the three varieties that dominate the algerian market, particularly in the region of medea (golden delicious 70%, starkrimson 20% and granny smith 5%) (hadj sahraoui, 2014). the apple “hanna”, of its real name “anna”, is a new variety of apple trees introduced in algeria. it is planted in less cold areas, in the center of algeria on the perimeter of high chellif in ain defla, in the west on the sebaou valley of telemcen and in the east to khenchela and m’sila. they are among the varieties less demanding in cold and generally give apples of lesser quality, hardly storable (hamdani et al., 2016). however, apart from regionalized studies aimed at predicting climate change through time series of temperature and rainfall and estimating its impact on crops through the increase of yields in all regions of algeria including constantine region in the east of the country (kherief nacereddine and alatou, 2004; tabet, 2008; zekri et al., 2009) and oran region in the west (benabadji and bouazza, 2000; labani et al., 2006), no study on phenological development as a key element to characterize the impact of climate change has been undertaken. we therefore wanted to begin to fill this gap with this study aiming at first, adv. hort. sci., 2019 33(3): 417-431 420 the characterization of climate change via temperature series and the search for a possible impact on the phenology of the apple tree and, in a second step, the determination of the critical periods with regard to the accumulation of cold units and units of heat, by the implementation of the classical phenological models. for this, we analyzed the time series of phenological data of the golden delicious variety in two contrasting zones from the climatic point of view: a zone of plain with a rather warm climate, sidi lakhdar (town of ain defla) and a cooler zone in altitude, benchicao (town of medea). 2. materials and methods sites and climatic data the temperature data recorded for each site are shown in table 1. the two zones selected in this study are: sidi lakhdar (town of ain defla, latitude: 36° 15 ‘50’’ n, longitude: 2° 09’ 39’’ e, altitude 211 m, located in the center of algeria 145 km south-west of algiers) known for its semi-arid climate with a mild winter and a very hot summer, and benchicao (town of médea, latitude: 36° 11 ‘59’’ n, longitude: 2° 50’ 55’’ e, altitude 1133 m, located 80 km south-west of algiers) in a mountainous area with a warm temperate climate. daily maximum and minimum temperature data obtained over a period of 37 years (1980 to 2016) were collected at weather stations near selected sites belonging to the national office of meteorology. average temperatures were calculated using maximum and minimum temperatures. missing daily data were estimated using two methods: a linear interpolation for some values over 1 to 3 days (means to fill the missing values of tmax and tmin were carried out): correlations with another site for the longest periods namely; between the station of sidi lakhdar and the station of chlef (latitude 36° 10’ n, longitude 1° 20’ e, altitude 116 m) for the month of may of the year 2005, and between the station of benchicao (médéa) and the station of bordj bou arreridj (latitude 36° 04 ‘23’’ n, longitude 4° 45’ 39’’ e, altitude 930 m) for the months of january, february, march and april of the year 1980 and the month of may of the year 2001. similarly, a correlation was made between the médéa site and the sétif meteorological station (latitude 36° 11 ‘28’’ n, longitude 5° 24’ 49’’ e, altitude 1038 m) for the months of september, october and november of the year 1981, and february and december of the year 1990. phenological data data collected from 2000 to 2012 were provided by specialized state agencies. these are average dates that represent all the orchards visited. those from 2013 to 2016 were collected directly from the same orchards, which were among the most apple orchards planted at both sites. phenological monitoring 3 to 4 times a week was carried out on adult trees, the number of which sufficiently covered the total area of a given orchard (50%), respecting the two orientations (north-south and west-east). these orchards have not undergone any chemical treatment to break endodormancy or accelerate flowering. phenological stages were described according to the bbch scale (meier et al., 1997, 2001). the phenological stages of bud break (bud burst, baggiolini stage c and stage 51 of the bbch scale) and early flowering (10% open flowers, baggiolini f1 stage and 61 bbch scale) were observed from 2000 to 2016 on the two apple orchards maintained according to conventional horticultural practices. both stages were reported affected when 60% of the trees in the orchard had reached the given stage. table 1 phenological and temperature data collected in climate-contrasting sites for ‘golden delicious’ apple trees site benchicao (md) sidi lakhdar (sd) region (town) medea ain defla latitude/longitude 36°11' 59'' n / 2°50' 55'' e 36°15' 50'' n / 2°09' 39'' e altitude (m) 1133 211 climatic area sub-humid semi-arid period recorded of temperature 1980-2016 1980-2016 bud burst stage and observation period a bbch 51 / 2000-2016 bbch 51 /2000-2016 flowering stage and observation period a bbch 61 / 2000-2016 bbch 61 /2000-2016 a: bbch 51, 61; stages in phenological code bbch (meier, 1997), are respectively swelling buds of inflorescences and 10% of flowers open. abed et al. ‐ climate change effect in apple flowering 421 a, b and c. the parameter a determines the width of the window on which the function is not zero. the larger the value, the larger the temperature range over which the cold units are wide. parameter b determines the sharpness of the response curve and its asymmetry. the more b differs from zero, the sharper the image (and more asymmetric). parameter c determines the value of the average response when b is close to zero and represents a limit to the temperature range over which cold units accumulate, when b is significantly different from zero. the wang model was first defined by wang and engel (1998). it is characterized by an optimum and is not symmetrical. this concerns the family of the beta function. it is composed of three parameters, namely tmin, topt and tmax (minimum, optimal and maximum temperatures). the sigmoid model was introduced by hänninen (1990). it consists of two parameters, d and e. the d parameter defines the sharpness of the response. values far from zero induce a sharper response curve. the parameter e is the average response temperature. smooth utah/ wang and smooth utah/ sigmoid the smooth utah model was introduced by bonhomme et al. (2010) and is a smoothed version of the utah function proposed by richardson et al. (1974). this function assumes that cooling can occur only over a range of temperatures and has four parameters: tm1, topt, tn2 and min. negative cooling values can be accumulated on hot days, increasing the amount of cold to reach. tm1: this parameter defines the sharpness of the decrease of the cold effect on the endodormancy of the buds. the lower tm1, the slower is the decrease. topt: this parameter corresponds to the optimum average daily temperature, for which a cooling unit is accumulated each day. tn2: this parameter defines the intermediate response, i.e. the temperature (above topt) that has half of topt’s effectiveness for inducing endodormancy. min: this parameter defines how much the impact of high temperatures can be negative. when min = 0, high temperatures do not have a negative impact on endodormancy release. when min = 1, the negative impact of a day that is too hot is equivalent to the positive effect of a day in topt. each model is characterized by efficiency (eff), an estimated time (t0) and a quadratic error (rmse: root mean square error). modeling and data analyses to better explain the phenological behavior of the golden delicious variety in the two sites studied and to highlight the effect of the temperatures on the latter in terms of satisfaction in cold units and heat units, statistical analyses were carried out under r (r development core team 2008), concerning regression curves between the different temperature components (minimum, average and maximum) and the year as well as the two phenological stages (bud burst and flowering). similarly, parametric name correlation tests of spearman were performed between two variables namely annual and monthly temperature (minimum, average and maximum) and year on the one hand and phenological stages on the other hand. calculation and establishment of cold unit accumulation curves were performed using the utah model (richardson et al., 1974). utah model the utah model was designed by richardson et al. (1974). this model combines the cold units for temperatures between 0 and 16°c and associates a negative value with temperatures higher than 16°c. this model is built to use fixed degree-days (independent of cold units) to predict bud break. the utah model (richardson et al., 1974) transforms the hourly temperature into a cold unit from -1 to 1. the cumulative number of utah cold units at time t is expressed as follows: ucutot = with (u= 0 for t≤1.4°c, u= 0.5 for 1.4°c <t≤2.4°c, u= 1 for 2.4°c <t≤9.1°c, u= 0.5 for 9.1°c <t≤ 12.4°c, u= 1 for 12.4°c <t≤15.9°c, u = -0.5 for 15.9°c <t≤18.0°c, u = -1 for t≥18.0°c) (ricard, 2014). phenological modeling platform pmp5.5 the phenological models were adjusted using the phenology modeling platform (pmp5.5) proposed by chuine et al. (2013). pmp5.5 is an environmental-use interface aimed solely at managing the construction of a phenological model, fitting a phenological model to the data and simulating using a phenological model. the best results of the bud burst and flowering date prediction in the two studied sites are obtained by two-phase models (knowing that phase 1 corresponds to the accumulation of cold units and phase 2 corresponds to the accumulation of heat units ) quoted below. chuine/wang and chuine/sigmoid the chuine model has been described in chuine (2000) and is composed of three parameters, namely tu t 1  422 adv. hort. sci., 2019 33(3): 417-431 3. results general climatology. annual tendencies the annual average maximum, medium, and minimum temperatures for both sites are shown in figure 1. the linear regression of mean annual temperatures over the 36 available years revealed a significant warming (p = 0.004) at the sidi lakhdar (sd) site, with a significant increase in the mean annual minimum temperatures (p = 0.001), and less for the average annual maximum temperatures (p = 0.04). there is rather a cooling tendency at mountain site of benchicao, although not significant (p = 0.09), concerning the annual average and where the maximum temperatures experienced some significant regression (p = 0.001). the hottest years were 1990, 2010 and 2007 at the site of sidi lakhdar and 2016, 1997 and 2000 at benchicao site. tendencies for the autumn‐spring period a seasonal analysis from the 36 years available shows marked differences between the two sites. the monthly average temperature tendencies for the months of october to may (minimum, mean and maximum daily temperatures) measured during the period 1980 to 2016 for the two sites (sidi lakhdar and benchicao) are summarized in table 2 and figure table 2 temperature data collected characteristics in the two studied sites in algeria during 36 years * p<0.05, ** p<0.01. fig. 1 average annual temperatures (minimum, average, maximum) for the two sites, sidi lakhdar (sd) and benchicao (md) from 1980 to 2016. *, ** indicate the significance level of the correlation for p <0.05 and for p <0.01, respectively. sidi lakhdar: (sd) tmin: y = 0.0403x 66.906 (r² = 0.33**), taverage : y = 0.0305x 41.183 (r² = 0.21**), tmax: y = 0.0234x 21.077 (r² = 0.0864). médéa:(md) tmin: y= 0.0122x 12.479 (r² = 0.03), taverage: y = -0.019x + 54.187 (r² = 0.090), tmax: y = -0.0503x + 121.02 (r² = 0.31**). month daily temperature mean daily temperature during 36 years in °c correlation coefficient of trends and level of significance sidi lakhdar benchicao sidi lakhdar (sd) benchicao (md) r p r p october average 21.3 17.4 0.40 0.01* 0.07 0.50 maximum 27.0 21.6 0.38 0.018* 0.14 0.40 minimum 15.6 13.3 0.44 0.006** 0.36 0.028* november average 15.7 11.4 -0.004 0.1 0.40 0.007** maximum 20.3 14.7 0.03 0.842 0.20 0.008** minimum 10.8 8.1 0.11 0.48 0.17 0.30 december average 12.0 8.0 0.17 0.28 0.16 0.16 maximum 16.4 10.9 0.10 0.556 0. 17 0.31 minimum 7.6 5.2 0.19 0.26 0.009 0.966 january average 10.9 7.3 0.35 0.03* 0.20 0.08 maximum 15.3 10.2 0.24 0.143 -0.20 0.23 minimum 6.4 4.6 0.32 0.051 0.10 0.54 february average 11.9 8.1 0.11 0.48 -0.32 0.005** maximum 16.8 11.2 0.047 0.783 -0.47 0.003** minimum 6.8 5.0 0.16 0.34 0.22 0.18 march average 14.2 10.5 0.01 0.51 -0.23 0.03* maximum 20.0 14.3 0.008 0.961 -0.47 0.030* minimum 8.5 6.8 0.25 0.13 0.025 0.88 april average 16.6 13.3 0.42 0.008** -0.07 0.51 maximum 23.0 17.5 0.36 0.027* 0.012 0.565 minimum 10.4 9.03 0.49 0.002** 0.18 0.28 may average 21.0 17.9 0.32 0.04* -0.1 0.38 maximum 27.9 22.7 0.21 0.19 0.027 0.403 minimum 14.3 13.0 0.40 0.012* 0.089 0.600 abed et al. ‐ climate change effect in apple flowering 423 2. table 2 generates spearman parametric name correlation values between maximum, average and minimum monthly temperatures and year, and where p is the level of significance and r is the correlation coefficient. we focused on the period from october to may, which is the period that most affects the physiological processes associated with the spring phenology of flower buds of fruit trees in our region. average october temperatures are high and increase significantly at sidi lakhdar site. the month of april is also warming significantly on this site (table 2, fig. 2). no significant tendency is recorded for the other months. correlations on monthly temperature tendencies also clearly showed significant summer warming in july at sidi lakhdar site (data not shown). at mountain site of benchicao, on the other hand, as already indicated above, during the 36 years, no significant warming is recorded in average temperature. on the contrary, average temperatures decreased in november and february (p = 0.007 ** and p = 0.005 *, respectively). regarding average minimum temperatures, the lowest values were recorded during the month of january for both sites with 6.4 and 4.6°c. the highest minimum temperature value of 12.1°c and the lowest value of 8.2°c were recorded by order in 2006 and 1991 at the site of sidi lakhdar for this month of january. at mountain site benchicao, the highest value of 9.7°c is reported in 2000 compared to a lower value of 6.6°c in 1980. the site of sidi lakhdar experienced extreme maximum temperatures during the months of october and april which explains the significance of the increase in average temperatures during these two months (fig. 2a). significant regressions of maximum temperatures were recorded at the benchicao site during the months of november, february and march. phenological development comparisons of the phenological tendencies of the apple tree (in terms of bud burst and flowering) in the two contrasting environments were made from the 16 years available. for budding dates of flower buds at sidi lakhdar site, some variation between years was revealed with marked tardiness during the years 2002, 2007, 2012, 2013 and 2016, when there was a bud break between the end of march and the first days of april and an early fruit maturity in 2000, 2003 and 2006, but the overall tendency for all years is not significant (p = 0.07). the tendency towards the advancement of flowering dates (fig. 3) is also not significant (p = 0.24), the earfig. 2 evolution of average monthly temperatures, on both sites, for months where the trend is significant at sidi lakhdar (october and april) and at benchicao (november and february). *, ** indicate the significance level of the correlation for p <0.05 and for p <0.01, respectively. sidi lakhdar: (sd) average april: y = 0.068x 119.18 (r² = 0.2252**), average october: y = 0.0742x 126.9 (r² = 0.1909**). médéa:(md) average february: y = 0,0799x + 167,72 (r² = 0, 1833), average november: y = 0.0483x + 107.88 (r² = 0.1376). fig. 3 trends in bud burst and flowering dates at the sidi lakhdar (sd) and benchicao (md) sites. *, ** indicate the significance level of the correlation for p<0.05 and for p<0.01, respectively. sidi lakhdar: (sd) bud burst/years: y = 0,1397x + 80, 86 (r² = 0, 0104), flowering/years: y = -0.4412x + 105.03 (r² = 0.0872). médéa: (md) bud burst/years: y = 0.7623x + 83.257 (r² = 0.2496), flowering/years: y = 0.8971x + 100.4 (**r² = 0.3263). adv. hort. sci., 2019 33(3): 417-431 424 liest years being 2003, 2009, 2010 and 2011, and the later years 2001, 2004 and 2005 at benchicao site, a significant tendency (p = 0.010) at the late flowering dates of the apple tree is to be reported (fig. 3b). on the other hand, differences in historical trends were shown in the bud break dates, oscillating between advancement during the years from 2000 to 2003, and a delay in the years 2004 to 2008. accumulation of chilling units in winter the utah cold unit (cu) accumulation curves reveal significant interannual differences at the sidi lakhdar site and show that for the years 2001, 2007, 2010 and 2016, this accumulation was insufficient because at below 600 cu, which is well below the estimated needs of the golden delicious variety (900 cu) (fig. 4). on the other hand, at mountain site of benchicao, the needs are always quickly satisfied. in order to analyze the influence of the different months in terms of cold units, we calculated the correlations between the dates of bud burst or flowering and the monthly temperatures (minimum, average and maximum) (table 3). very schematically, the tendencies can be summed up as follows: at the benchicao site, the month of january and the whole period from november to january and february have a strong influence on meeting the needs in cold units because the correlation is positive (the warmer it is, the more the budding/flowering is late), whereas it is not the case in november and december (october remains quite neutral with a negative correlation). the impact of january is preponderant because if we look at the influence of the period from november to january, we find a negative effect of high temperatures on the precocity (r² = 0.40) while the months of november and december have an inverse effect. at the site of sidi lakhdar, the balance sheet is fig. 4 cumulative cold unit according to the utah model at the sidi lakhdar site. a) years when cumulative cold is less than 500 hrs; b) years when cumulative cold exceeds 800 hrs. table 3 spearman's correlation between bud burst and flowering for ‘golden delicious’ apple tree and mean temperature from october to april * p<0.05, **p<0.01. variable sidi lakhdar (sd) benchicao (md) mean temperature minimum temperature maximum temperature mean temperature minimum temperature maximum temperature bud burst flowering bud burst flowering bud burst flowering bud burst flowering bud burst flowering bud burst flowering october -0.031 0.11 -0.21 0.13 -0.20 0.033 0.015 0.019 -0.106 0.135 0.081 0.14 november -0.41 * -0.12 -0.55 * -0.21 -0.38 -0.036 -0.54 * -0.225 -0.57* -0.232 -0.47* 0.18 december -0.18 -0.43 * -0.11 -0.232 -0.2 -0.50 * -0.40 * 0.054 -0.52* -0.11 -0.322 0.18 january 0.31 * -0.002 0.006 -0.24 0.46 * 0.14 0.46 * 0.3 0.232 0.15 0.54* 0.41 february 0.10 0.01 -0.09 0. 12 0.01 0.13 0.063 0.073 0.021 -0.015 0.13 0.15 march -0.43 * -0.34 * -0.27 -0.147 -0.36 -0.36 * -0.60 * -0.65 * -0.60* -0.64* -0.55* -0.56* april -0.45 * -0.30 -0.41 * -0.145 -0.48 * -0.33 * 0.08 -0.072 -0.101 -0.30 0.12 0.041 november-january 0.30 * -0.26 0.14 -0.45 * 0.30* -0.30* 0.43 * 0.40 * 0.28 0.33 0.52* 0.53* november-february 0.06 -0.052 0.21 -0.14 0.18 -0.14 0.48 * 0.44* 0.233 0.212 0.30 0.21 november-march 0.041 -0.29 0.01 -0.20 0.132 -0.30 0.184 0.27 0.062 0.104 0.33 0.30 march-april -0.56 * -0.45 * 0.08 -0.025 -0.54 * -0.330 -0.26 -0.44* -0.304 -0.54* -0.133 -0.20 abed et al. ‐ climate change effect in apple flowering 425 globally the same. the correlations between mean and maximum temperatures in january on the one hand, and bud break dates on the other, are significantly positive, indicating the importance of this month’s temperatures for the satisfaction of cold unit requirements, the month of october remains little determinant. a hot january is a delay in meeting cold needs and bud break. a negative tendency of the high temperatures of the period from november to january on the precocity (r2 = 0.30) was recorded whereas the other months go rather in the direction of a gain of precocity. significant and negative relationships were observed between maximum temperatures in december and flowering, with a correlation coefficient of -0.50. accumulation of forcing units in spring march-april period (-0.44) show the link between the early flowering period and the average temperature it is rather march that plays the main role for both sites. at the benchicao site, only march temperatures show a significant negative correlation with bud break and flowering dates. negative correlations obtained between average temperatures of the period, with a very high prevalence of march temperatures. at the site of sidi lakhdar, march and april strongly influence the precocity via maximum temperatures and average temperatures. phenology modeling for both sites, the best sequential models selected are given in table 4. they were chosen on the basis of efficiency (eff) and rmse (rmse), but also taking into account the physiological relevance of the temperature response curves for the cold unit stacking phase and the heat unit stacking phase. rmse may appear acceptable (~ 5 days) on a flowering date but the efficiencies are less good. the correlation between the observed values and the predicted values confirms this diagnosis (fig. 5). the efficiency is very slightly improved (reaching a difference of 0.03 to 0.09) by eliminating the years when the cumulative cold units are not satisfactory at sidi lakhdar site. table 4 modeling bud burst and flowering for ‘golden delicious’ apple tree by chuine/wang and smooth utah/sigmoid in the two studied sites with all years and without years where chilling does not satisfied site two sites with all years two sites without years where chilling dose not satisfied, 2001, 2007, 2010, 2016 phenological stage bud burst flowering bud burst flowering model chuine / wang smooth utah / sigmoid chuine / wang smooth utah / sigmoid t0 starting date -107.8 -75.3 110.6 61.4 rmse 5.39 4.86 5.93 4.16 eff 0.49 0.47 0.50 0.56 settings chuine wang smooth sigmoid chuine wang smooth sigmoid a 0.50 topt 20.69 tm1 -37.54 d -40 a 0.68 topt 17.09 tm1 9.72 d -2.72 topt 18.45 b 12.70 tmin 3.30 topt 23.52 e 6.40 b -28.90 tmin 2.40 tn 2 27.52 e -21.80 min -0.86 c 27.04 tmax 43.14 tn 2 24.40 c -15.36 tmax 26.36 min -0.99 fig. 5 comparison between the observed and the predicted flowering date of apple in two sites. a) for all years by the smooth utah/sigmoid model. y = 0.5286x + 49.199; r² = 0.4796; b) without the years where the cumulative cold units are not satisfied by the smooth utah/sigmoid model. y = 0.5041x + 51.647; r² = 0.5555. adv. hort. sci., 2019 33(3): 417-431 426 by examining separately the two sites, and in particular that of sidi lakhdar, which shows years when the accumulation of cold units is not satisfied (fig. 6a), we see that the withdrawal of these years greatly improves the results of modeling (fig. 6b) and in particular for flowering. the relevance of the response curve obtained for the cold unit function is also greatly improved (table 5). at benchicao site (table 6), the efficiency of the two-phase model is medium and figure 7 shows that it is difficult to predict the early years. in fact, the two-phase model is not much better than a onephase model on bud break and not better for flowering. the results of the one-phase model (only the heat-accumulation model: degree-day growth, parabolic, richarsdon, wang, sigmoid, threshold and smooth utah) yielded non-significant results. 4. discussion and conclusions the sensitivity of phenophases to temperature changes is a good indicator of the long-term biological impacts of climate change and terrestrial ecosystems (richardson et al., 2013). several studies have shown that the phenophases most sensitive to temperature variations are those occurring in spring or summer and that there is a relatively linear relationship between the occurrence of these phenophases site sidi lakhdar with all years sidi lakhdar without years where chilling dose not satisfied, 2001, 2007, 2010, 2016 phenological stage bud burst flowering bud burst flowering model chuine/ sigmoid chuine/ sigmoid chuine/ sigmoid chuine/ sigmoid t0 -102.3 61 -119 -59.7 rmse 3.79 5.02 3.30 2.10 eff 0.72 0.44 0.80 0.90 settings chuine sigmoid chuine sigmoid chuine sigmoid chuine sigmoid a 1.61 d -32.51 a 2.95 d -11.93 a 0.96 d -2087 a 3.21 d -40 b 5.67 e -21.66 b -25.47 e 0.50 b -29.7 e -25.47 b 12.47 e 14.93 c 19.42 c 8.60 c -6.72 c 16.93 table 5 modeling bud burst and flowering for ‘golden delicious’ apple tree in sidi lakhdar by chuine/sigmoid model with all years and without years where chilling does not satisfied table 6 modeling bud burst and flowering for ‘golden delicious’ apple tree in benchicao by smooth utah/wang and chuine / sigmoid models with all years fig. 6 comparison between the observed and the predicted bud burst date of the apple tree at the sidi lakhdar site. a) for all years by the chuine/sigmoid model. y = 0.8316x + 13.544; r² = 0.7207; b) without the years when the cumulative cold units are not satisfied by the chuine/sigmoid model. y= 0.8165x + 15.548; r² = 0.8066. site benchicao with all the years phenological stage bud burst flowering model smooth utah / wang chuine / sigmoid t0 -104.9 -78.7 rmse 5.43 4.91 eff 0.48 0.46 settings smooth utah wang chuine sigmoid tm1 -34.85 topt 22.31 a 0.45 d -39.99 topt 21.15 tmin 3.60 b -10.58 e 6.39 tn2 29.42 tmax 49.9 c 1.03 min -0.81 abed et al. ‐ climate change effect in apple flowering 427 and temperature (gordo and sanz, 2010; morin et al., 2010; beaubien and hamann, 2011). our study is a first attempt to evaluate the impact of global warming on the bud break and flowering of the apple tree in northern algeria. the study of the chronological series of temperatures (minimum, average and maximum) from 1980 to 2016 (36 years) in the two important apple production sites, namely: sidi lakhdar and benchicao, showed a very significant warming in october and april at the site of sidi lakhdar (sd) likely to strongly disrupt the entry into endodormancy and ecodormancy. strangely, the benchicao (md) site does not show global warming and even a cooling trend in november and february, which could lead to an endodormancy and a start of satisfaction in cold units and then an acceleration of the recovery of growth leading to greater precocity. establishing the time of endodormancy emergence is important (guerriero et al., 2002), but is hampered by the complexity of the process, including the fact that, under natural conditions, cold and warm temperatures alternate, causing “inversions” in the process of endodormancy emergence (overcash and campbell, 1955; couvillon and erez, 1985; erez and couvillon, 1987). some results suggest that partial dissatisfaction with cold can be compensated by heat unit supplementation for bud break (dantec, 2014). to lift the endodormancy, the bud must accumulate enough cold (campoy et al., 2011). when the plant has reached its cold needs, the endodormancy is lifted and the buds can resume their growth as soon as the conditions become favorable, i.e. under certain (rather high) temperature conditions, soil moisture and nutrients, and for photosensitive species, certain photoperiod conditions (körner and basler, 2010; polgar and primack, 2011). these favorable conditions must last for a certain period of time for the bursting of the buds to appear. for our case, the cold needs were always met to lift the endodormancy of the apple tree at the benchicao site, and this as from the end of december or the first half of january (fig. 4). conversely, at the sidi lakhdar site, values below the threshold for satisfying cold needs estimated at 900cu for the golden delicious apple tree were obtained for the years 2001, 2007, 2010 and 2016. when it takes place, the satisfaction of cold needs is later, around midfebruary (fig. 4) and does not really start until the end of november. everything leads to a dominating importance of the month of january in the course of the endodormancy lifting process and it will be interesting to look at what the future climate scenarios give for this particular period for the choice of future apple varieties. the months of november and december play a precocious role at the benchicao site (negative correlation with the date of bud burst or flowering). this can only be understood if the organogenesis in the buds continues during these two months and therefore there is no endodormancy at this time. the observed increase in average november and october minimum temperatures (table 2) is consistent with this. at the site of benchicao, the month of march is crucial for the precocity, it is the temperatures of this month which allow the growth after the satisfaction of the needs of cold towards the end of december and the beginning of january all the more so as the temperatures of the february remain rather low (and tend towards a cooling). the tendency to tardiness with the site of benchicao is thus coherent with the cooling in the month of february. the october warming at sidi lakhdar site may explain later entry into endodormancy. gentle temperatures (12°c) in february can then accelerate bud burst and flowering, at least for years when cold needs are met. otherwise endodormancy will be greatly delayed or disrupted. according to the study of legave et al. (2015), carried out in three geographically contrasting countries of the mediterranean region, in morocco (meknes), france (nimes) and italy (forlì) over the last 40 years in order to understand the impact of climate change, especially the increase in temperature, on the golden delicious apple tree, the forcing period is shorter in meknes. legave et al. (2012) also found a marked trend towards shorter simulated duration of forcing period and late fig. 7 comparison between the observed and the predicted flowering date of the apple tree at the benchicao site by the chuine/sigmoid model. y = 0.4501x + 57.365; r² = 0.4632. adv. hort. sci., 2019 33(3): 417-431 428 endodormancy period. the physiological functioning of the golden delicious apple tree during the dormant and growing season may explain, in part, the regional differences observed in the flowering dates (heide, 1993). in the same context, kauffman and blanke (2018) have reported after a study conducted on three cherry cultivars at different levels of cold needs (minimum, medium and high) that, in optimum chill, the optimum forcing was ca. 8.000 gdh (>12  °c), irrespective of variety, allowing up scaling of the results to possibly other varieties. overall, the results have shown that diminishing chilling as a result of climate change can be compensated for, in part up to 50%, by a larger amount of forcing to obtain natural flowering in the orchard. these results may explain the good progress of flowering on the site of sidi lakhdar, although the cold needs were not often satisfied. el yaacoubi et al. (2014) also reported that spring temperatures appear to be essential for complete flowering in mild climates. in the latter case, early full flowering dates occurred when the average temperature during the forcing period rapidly exceeded 15°c provided adequate satisfaction of the cold requirements. phenological models predicting the occurrence of different phenophases as a function of environmental conditions (mainly temperature and photoperiod), predict that the global increase in temperature during the winter will slow or even jeopardize the endodormancy emergence due to lack of cold (chuine et al., 2016). the onephase and two-phase models for all years do not give good results at the sidi lakhdar site. this is explained by the negative influence of years when cold unit needs have not been met. if these years are removed, the two-phase chuine/sigmoid model for bud burst and flowering gives good results. this may mean that in these cases of partial non-fulfillment of cold unit requirements, the physiological processes involved in bud break-up and flowering are different or that “something” in addition occurs. at benchicao site, the efficiency of the two-phase models is average, since the requirements in cold units are often met; only the forcing period can have an effect on the precocity. this study aimed to show the effects of the anticipated increase in temperature on two phenological phases of the apple tree (golden delicious) in two algerian sites with contrasting climates. we highlighted contrasting trends by site and by period. warming at sidi lakhdar site in autumn and spring, however, the statistical data of temperatures did not raise any average warming at the benchicao site. rather surprising and never described before, there has been a tendency to cool down some months at the benchicao site. critical periods for cold units were identified, concerning the period between november and january at benchicao site, but january temperatures were more important in l ifting endodormancy. at the site of sidi lakhdar, buds enter late into endodormancy and the result is a late action of cold that extends until february without always being sufficient. forced side, it is the temperatures of the month of march that have a discriminating effect on bud burst and flowering at the site of benchicao combined with those of april at the site of sidi lakhdar. on this site, despite the warming in april, we do not gain in precocity probably because of a satisfaction of cold needs “to the limits” as describe legave et al. (2012) for the nimes region. we have also highlighted, particularly at the site of sidi lakhdar that more complex physiological processes must be at work especially the years of low cumulative cold units. it is not excluded that other factors, not included in this work, could be involved in the budburst process such as photoperiod or precipitation (vitasse et al., 2009; grab and craparo, 2011; el yaacoubi et al., 2014). except for the twophase chuine/sigmoid budburst and flowering model, which gave better results at sidi lakhdar site after the elimination of the years when the cold unit requirements were not met, all the models give rather weak efficiencies indirectly confirming the non-taking into account of a complexity of factors associated with physiological functioning for sites like sidi lakhdar’s. the study of the impact of global warming on the apple tree requires a precise determination of the accumulations in cold units necessary for the emergence of endodormancy and budding in various environments. this involves highlighting these two phases by forcing techniques at the laboratory level and anatomical studies of meristematic bud tissues to see their ability to bud. references baggiolini m., 1952 les stades repères dans le dévelop‐ pement annuel de la vigne et leur utilisation pratique. revue romande d’agriculture et d’arboriculture, 8(1): 4-6. balaghi r., 2017 le changement climatique dans la région de fés‐meknés. etat de l’art, vulnérabilité, impact sur les terres et les principales cultures. résumé à l’attention des décideurs. i.n.r.a., rabat, morrocco, abed et al. ‐ climate change effect in apple flowering 429 pp. 1-14. beaubien e., hamann a., 2011 spring flowering response to climate change between 1936 and 2006 in alberta, canada. bioscience, 61: 514-524. benabadji n., bouazza m., 2000 quelques modifica‐ tions climatiques intervenues dans le sud‐ouest del’oranie (algérie occidentale). rev. energ. ren., 3: 117-125. bonhomme m., rageau r., lacointe a., 2010 optimization of endodormancy release models using series of endodormancy release data collected in france. acta horticulturae, 872: 51-60. bourchef m., 2013 axes stratégiques en matière de changements climatiques sur le nord de l’algérie. ‐ rapport sur l’élaboration de la stratégie nationale de gizc pour l’algérie, pp. 69. campoy j.a., ruiz d., egea j., 2011 dormancy in tem‐ perate fruit trees in a global warming context : a review. sci. hortic., 130: 357-372. chuine i., 2000 a unified model for the budburst of trees. j. theor. biol., 207: 337-347. chuine i., 2005 un reseau d’observations phenologiques pour la gestion du changement climatique, pp. 49-58. in: landmann g., and s. landeau (eds.) de l’observa‐ tion des écosystèmes forestiers à l’information sur la forêt. editions quae, paris, france, pp. 93. chuine i., bonhomme m., legave j.m., de cortazaratauri i.g., charrier g., lacointe a., ameglio t., 2016 can phenological models predict tree phenology accurately in the future? the unrevealed hurdle of end dormancy break. ‐ global change biology, 22: 34443460. chuine i., de cortazar-atauri i.g., kramer k., hänninen h., 2013 plant development models, pp. 275-293. in: schwarz m.d. 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laboratoire de biomathématiques, biophysique, biochimie et scientométrie, faculté des sciences de la nature et de la vie, université de bejaia, 06000 bejaia, algeria. 2 université de tizi ouzou, faculté des sciences biologiques et des sciences agronomiques, département d’agronomie, tizi ouzou (15000), algeria. 3 université chadli bendjedid d’el‐tarf, faculté des sciences de la nature et de la vie, département d’agronomie, bp 73 (36000), el‐tarf, algeria. 4 centre de recherche en technologies agro‐alimentaires, route de tergua‐ouzemour, 06000 bejaia, algeria. key words: antioxidant activity, eggplant, extraction condition, optimization, phenolic compound. abstract: the optimum conditions for extraction of total phenolic contents (tpc) and maintaining the highest antioxidant activity from eggplant were determined. extraction experiments were carried out by investigating the effects of the solvent nature (acetone, ethanol, methanol, or water), solvent concentration (30­90%), extraction temperature (30­100°c), extraction time (30­120 min), solid to solvent ratio (1/25­1/100 g/ml), and number of extrac­ tions (1, 2 and 3) on the recovery of phenolic compounds and antioxidant activ­ ity of the extracts. the tpc was assessed to determine the polyphenolic compo­ nent while free radical scavenging activity (frsa) and ferric­reducing power (frp) were used to evaluate the antioxidant activity of eggplant extracts. all extraction parameters had significant effects (p<0.05) on the tpc extraction and the antioxidant activities. the best conditions were obtained using three extraction steps with aqueous acetone 70% (v/v) at 25°c for 60 min and with 1 g/50 ml solid to solvent ratio. the optimum extraction conditions exhibit the tpc concentrations of 794.94 mg gae/100 g and antioxidant activities of 737.86 mg te/g (frsa) and 28.00 mg te/g (frp). the free radical scavenging and ferric­ reducing potentials were found to be positively significantly correlated with phenolic content under the influence of all extraction parameters. 1. introduction eggplant (solanum melongena l.) commonly known as brinjal, is an economically important common vegetable grown and consumed (*) corresponding author: dlynda2002@yahoo.fr citation: arkoub­djermoune l., benmeziane f., mada­ ni k., boulekbache­makhlouf l., 2019 ­ optimization of phenolic compounds recovery and in vitro antioxidant activity of algerian egg‐ plant (solanum melongena l.). ­ adv. hort. sci., 33(4): 567­580. copyright: © 2019 arkoub­djermoune l., benmeziane f., madani k., boulekbache­makhlouf l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 june 2019 accepted for publication 30 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 567­580 568 throughout the world. it is a good source of phenolic compounds (jung et al., 2011; salerno et al., 2014; arkoub­djermoune et al., 2016; dranca and oroian, 2017; sharma et al., 2019), vitamins and minerals, especially iron, compared to other commonly con­ sumed vegetables, and it is nutritionally comparable to tomato (kalloo, 1993). it is ranked amongst the top ten vegetables in terms of oxygen radical absorbance capacity due to the fruit phenolic con­ stituents (cao et al., 1996) which are powerful antioxidants (jung et al., 2011; piao et al., 2014; scorsatto et al., 2017; sharma et al., 2019). studies have shown that eggplant extracts sup­ press the development of blood vessels required for tumor growth and metastasis (matsubara et al., 2005), and inhibit inflammation that can lead to ath­ erosclerosis (han et al., 2003). extracts from eggplant fruit skin have been demonstrated to possess high capacity in scavenging of superoxide free radicals and inhibition of hydroxyl radical generation by chelating ferrous iron (kaneyuki et al., 1999; noda et al., 2000; boulekbache­makhlouf et al., 2013). superoxide radi­ cals generated in vivo are usually converted into hydrogen peroxide, and like other free radicals, can damage lipids, proteins, and dna (halliwell et al., 1995). from the 120 vegetable species evaluated for antioxidant activity using four different assays (2,20­ azinobis­[3­ethylbenzthiazoline­6­sulphonic acid), 2, 2­diphenyl­1­picrylhydrazyl radical, inhibition of lipid peroxidation, and superoxide scavenging), eggplant ranked among the top 10 species for superoxide scav­ enging (sos) activity (hanson et al., 2006). nasunin, an anthocyanin isolated from the skin of purple egg­ plant fruit, is one phenolic compound implicated in both inhibition of hydroxyl radical generation and sos activity (kaneyuki et al., 1999; noda et al., 2000). extraction is the first step in isolation of phenolic compounds from plant materials. considering the compositional diversity of the natural sources of polyphenols, as well as the structure and physico­ chemical properties of these compounds, specific processes must be designed and optimized for each phenolic source (santos­buelga and williamson, 2003; pinelo et al., 2005). the extraction protocol must enable complete extraction of phenolics, as well as minimization of oxidation, degradation, and polymerization of desired products (zuo et al., 2002). many factors, such as type of solvent (water, methanol, ethanol, ethyl acetate, acetone, and hexa­ ne), ph, temperature, time, solid/solvent ratio, and extraction number, can affect the efficiency of the extraction process (zuo et al., 2002; mo et al., 2011). there are several efficient extraction methods for determination of phenolic compounds in solid sam­ ples. these include supercritical fluid extraction (sfe), accelerated solvent extraction (ase), solid­phase microextraction (spme), ultrasound­assisted extrac­ tion (uae) (dranca and oroian, 2017; ferarsa et al., 2018; nipornram et al., 2018), and microwave­assist­ ed extraction (mae) (mahugo santana et al., 2009; dahmoune et al., 2013; koyu et al., 2018). solid­liquid extraction (sle), which was used in this investigation, has been the method of choice of numerous researchers for extraction of phenolics from many sources (liyana­pathirana and shahidi, 2005; durling et al., 2007; bachir bey et al., 2013; benmeziane et al., 2014; mokrani and madani, 2016; caldas et al., 2018; mohd hazli et al., 2019). effect of extraction process can be generally evaluated based on a one factor one time approach, also known as single experiment, in which only one factor is variable at one time while all others are kept constant. the availability of phenolic compounds in eggplant as an antioxidant source is documented. however, no optimal protocols have been established so far for phenolic extraction from these vegetable. to the best of our knowledge, no data was reported about the effect of extracting para­ meters on the survey of phenolics from eggplant. hence, the objective of this study was to optimize the extraction of total phenolics compounds (tpc) maintaining the highest antioxidant capacity (dpph free radical­scavenging activity; frsa and ferric reducing power; frp) from eggplant (solanum melon‐ gena l.) using single factor experiments approach under conditions compatible with food use. the objective in extracting phytochemicals from their plant sources is to liberate these compounds from the vacuolar structures where they are found, either through rupturing plant tissue or through a process of diffusion. the factors that contribute to the effi­ ciency of extraction are, in particular, solvent type (ethanol, methanol, acetone and water), solvent con­ centration (30%, 50%, 70% and 90%, v/v), tempera­ ture (25, 50, 75 and 100°c), time (30, 60, 90 and 120 min), solid to solvent ratio 1/25, 1/50, 1/75 and 1/100 g/ml), and number of extraction (1, 2 and 3). the stability of antioxidant activities (frsa and frp) in extracts was also investigated. 2. materials and methods chemicals folin­ciocalteu reagent was provided from arkoub‐djermoune et al. ‐ optimization of phenolic compounds recovery of algerian eggplant 569 biochem, chemopharma (montreal, quebec). sodium carbonate (na2co3), acetone, ethanol and methanol were obtained from prolabo (made in ce). potassium ferricyanide (c6n6fek3), ferric chloride (fecl3­6h2o), trichloroacetic acid, gallic acid and trolox from biochem­chemopharma (uk). 1,1­ diphenyl­2­picrylhydrazyl (dpph) was obtained from sigma­aldrich (steinheim, germany). plant material the fresh eggplant (solanum melongena l.) used in this study was purchased from local market of bejaia city (algeria) at february 2010. after transferring at the laboratory, the sample was washed with distilled water and wiped then some tests were done on fruits in order to determine their physico­chemical charac­ teristics. eggplant fruits presented a ph of 4.15, water content of 92.8 g/100 g fw, titratable acidity of 1.24 g/100 g dw, total sugar content of 20.55 g/100 g dw, total soluble solids content (brix) of 31.48 g/100 g dw and ash content of 0.55 g/100 g dw (arkoub­ djermoune et al., 2016). the sample was frozen at ­ 20°c before analysis and a quantity of eggplant were mixed and used for each extraction step. plant material extraction approximately 0.1 g of crushed eggplant was weighed in a glass vial and extracted with 10 ml of the extracting solvent. the mixture was shaken at a constant rate using a water bath shaker, centrifuged for 20 min at 4000 g (nüve nf 200, ankara, turkey), and filtered through a watman filter paper. the extraction process was carried out in duplicate. the obtained filtrates extracts were subsequently used for the determination of total phenolic compounds (tpc) and antioxidant activities: dpph­free radical scavenging activity (frsa) and ferric reducing power (frp) measurements. experimental design in the present study, single factor experiments was used to determine the optimum conditions for extracting phenolic compounds from eggplant. a total of six parameters namely extraction solvent (ethanol, methanol, acetone and water), solvent con­ centration (30­90%; v/v), extraction temperature (30­ 100°c), extraction time (30­120 min) solid to solvent ratio (1/25­1/100 g/ml), and number of extractions (1, 2 and 3) were studied in which one parameter was varied at a while the other parameters were fixed. the optimal extracting conditions were select­ ed on the basis of the tpc, frsa and frp measure­ ments. solvent nature and concentration by setting extraction time (30 min) and tempera­ ture (25°c) and sample/solvent ratio (0.1 g/10 ml), samples were extracted with acetone (30%, 50%, 70%, and 90%; v/v), ethanol (30%, 50%, 70%, and 90%; v/v), methanol (30%, 50%, 70%, and 90%; v/v) and water. extraction temperature using the best solvent type and solvent concen­ tration, eggplant samples were extracted at tempera­ tures ranging from 25°c to 100°c (25, 50, 75 and 100°c). the best extraction conditions studied were selected according to the value of three responses (tpc, frsa and frp). extraction time eggplant samples were extracted using the best solvent concentration and extraction temperature determined previously. the extracts were prepared by varying the extraction time from 30 min to 120 min (30, 60, 90 and 120 min). the best extraction conditions studied were selected according to the value of three responses (tpc, frp and frsa). solid to solvent ratio eggplant samples were extracted using the best solvent concentration. the extraction procedure was repeated by varying the sample/solvent ratio 1/25, 1/ 50, 1/75, and 1/100 g/ml, while fixing the extrac­ tion time and temperature at 60 min and 25°c as determined previously. tpc and antioxidant activity values for consecutive extractions were added for determination of this parameter. number of extractions the final step of this experiment was to deter­ mine the effect of the extractions number. in order to determine this effect, the extraction setting with the optimal conditions selected previously: solvent (acetone 70%; v/v), temperature (25°c), time (60 min) and the solid to solvent ratio (1/50 g/ml), the extraction was repeated three times on the solid residue after centrifugation of the mixture at 4000 rpm during 20 min. total phenolic compound determination (tpc) the amount of tpc in eggplant extract was deter­ mined using the folin­ciocalteu reagent and gallic acid as standard as described by velioglu et al. (1998). in brief, 200 µl of each extract were intro­ duced into test tubes then added with 1.5 ml of folin­ciocalteu reagent (previously diluted ten times). after 5 min, 1.5 ml of sodium carbonate (60 g/l) were added. the tubes were mixed and allowed adv. hort. sci., 2019 33(4): 567­580 570 to stand in darkness at room temperature for 30 min. absorption at 760 nm against a blank was measured using a shimadzu uv­vis spectrophotometer (kyoto, japan). the concentration of phenolic compounds extracts is determined by reference to the calibration curve obtained under the same conditions using the gallic acid as the standard, expressed as milligram gallic acid equivalent per one hundred gram of the fresh weight (mg gae/100 g fw). all measurements were carried out in triplicate. antioxidant activity free radical scavenging activity of dpph. (frsa). in the 1,1­diphenyl­2­picrylhydrazyl (dpph.) assay, antioxidants were capable to reduce the stable radi­ cal dpph. to the yellow colored diphenylpicrylhy­ drazine (dpph­h). the test is based on the reduction of an alcoholic solution of dpph. in the presence of a hydrogen donating antioxidant due to the formation of the non­radical form dpph­h (gülçin, 2007). the dpph. radical­scavenging activity of eggplant extracts was estimated as described by milardović et al. (2006). briefly, 100 µl of samples extract were mixed with 3 ml of dpph in methanol (6.10­5m). the mixtures were left for 30 min at room temperature and its absorbance then measured at 517 nm against a blank. all measurements were carried out in tripli­ cate. the percentage scavenging was calculated using the following equation: dpph­frsa (%) = [(acontr ­ aextr)/acontr] × 100 where acontr is the absorbance of the control (without extract) after 30 min and aextr is the absorbance of extract. the inhibition percentage were expressed as milligram trolox equivalent per gram of the fresh weight (mg te/g fw). ferric reducing power (frp) the ferric reducing power of eggplant extract was performed by using the potassium ferricyanide­ferric chloride method. substances, with reduction poten­ tial, react with potassium ferricyanide (fe3+) to form potassium ferrocyanide (fe2+), which subsequently reacts with ferric chloride to form ferric ferrous com­ plex who has an absorption maximum at 700 nm (jayanthi and lalitha 2011). the reducing power of the extracts was evaluated according to the protocol of oyaizu (1986). one millitre of different concentra­ tions of the samples was mixed with phosphate buffer (1 ml, 0.2 m, ph = 6.6) and potassium ferri­ cyanide [k3fe(cn)6] (1 ml, 1 g/100 ml). the mixture was incubated at 50°c for 20 min. trichloroacetic acid (tca) (1 ml, 10 g/100 ml) was added to the solution which was then centrifuged for 10 min at 3000 g. the supernatant was gathered and mixed with distilled water (1.5 ml) and fecl3 (150 µl, 0.1 g/100 ml), and the absorbance was measured at 700 nm, increased absorbance of the reaction mixture indicate an increase in reducing power. results are expressed as milligram trolox equivalent per gram of fresh weight (mg te/g fw). the values are present­ ed as the means of triplicate analyses. statistical analysis results were analyzed using statistica software (version 5.5.fr; statsoft, inc, tulsa, usa). all values are expressed as mean ± standard deviation (sd) of duplicate extractions and triplicate assays. one­way analysis of variance (anova) with the lsd (least significant difference) test was used to determine significant differences (p<0.05) among the means. 3. results and discussion solvent nature and concentration the selection of extraction solvent is critical for the complex food samples as it will determine the amount and type of phenolic compounds being extracted. aqueous alcohols particularly acetone, ethanol and methanol are the most commonly employed in phenolic extraction from botanical materials (chan et al., 2009). in this study, diverse solvents, in mixtures with water, were used to extract antioxidant phenolic compounds from eggplant. all solvents used in the present work at different concentrations (30%, 50%, 70%, and 90%) under the same extraction conditions (30 min at 25°c) were capable of extracting phenolic compounds (table 1). pure organic solvents were not used because obtained extracts were cloudy. therefore, dilution with water to 90% was necessary. solvent type had a significant influence (p<0.05) on tpc and antioxidant activities (frsa and frp). the aqueous acetone (70%; v/v) extracts showed the highest yield of tpc (1032.16 mg gae/100 g). similar results were reported in our previous study (boulekbache­makhlouf et al., 2013) that 70% ace­ tone was better for phenolic extraction from egg­ plant byproduct (peel) than 70% ethanol, and 70% methanol. kallithraka et al. (1995) found that 70% acetone was the best solvent for the extraction of grape seed phenolics especially proanthcynidins. similar to the present study, acetone:water mix­ arkoub‐djermoune et al. ‐ optimization of phenolic compounds recovery of algerian eggplant 571 in agreement with polarity of the solvent used for the extraction and solubility of phenolics in them since the polarity of acetone, ethanol and methanol is 0.355, 0.654 and 0.762, respectively (tan et al., 2013). therefore, there is no single solvent able to extract all of the classes of phenolic compounds from a sample, simultaneously. acetone is a more efficient solvent for extracting phenolic compounds with a high molecular weight such as condensed tannins. it is strongly believed that the higher molecular weight of the solvent, the lower the polarity which enable other substances of about the same molecular weight to be easily extracted. this can be associated to ‘‘like dissolves like” or ‘‘polarity versus polarity” principle as both acetone and tannins are of high molecular weight. acetone has the lowest polarity but contains the highest total phenolic compounds value (alasalvar et al., 2006; uma et al., 2010). this would explain why acetone was found to be more efficient for extracting phenolics from eggplant. in addition, the lowest value of tpc was obtained with 70% ethanol, 90% ethanol and 50% methanol. this fact is due to a polarity of the solvent used for the extraction and solubility of phenolic compounds in them because these solvents due to their polarity are more effec­ tive for extracting polyphenols linked to polar fibrous matrices (tabart et al., 2007) and their antioxidant activity depends not only on the concentration of polyphenols but also on their chemical structure (the number and position of hydroxyl groups) (sroka and cisowski, 2003). in addition, the effectiveness of phe­ nolic compounds as antioxidants does not only tures have been reported to be one of the most effective solvents for extracting phenolics from dif­ ferent natural sources. meneses et al. (2013) and mokrani and madani (2016) demonstrated that 60% acetone was the best solvent for extracting antioxi­ dant phenolic compounds from brewer’s spent grains and peach fruit, respectively. acetone:water mixture is capable to break polyphenol­protein complexes. this fact would explain the high efficiency of this sol­ vent to extract phenolic compounds. downey and hanlin (2016) demonstrated that mixtures of acetone ranging from 50% to 70% are more effective in extracting condensed tannins from grape skin. in the other hand, it has been also demonstrated that acetone is more effective than other organic sol­ vents for extracting phenolics from different raw materials such as berries and apples (kähkönen et al., 2001), star fruits (shui and leong, 2006), onions (curcic et al., 2012), barley seeds (liu and yao, 2007), beach peas (chavan and amarowicz, 2013), pistachio byproducts (mokhtarpour et al., 2014), fenugreek (mashkor, 2014) and soybean (lien et al., 2015). the result also showed that there were no signifi­ cant difference between aqueous ethanol 50%, methanol (30%, 70%, and 90%), and water extracts. however, the use of water as single solvent provides a cloudy extracts with a high content of impurities (chirinos et al., 2007). generally, acetone is the best solvent for extract­ ing proanthocyanidins and tannins; ethanol efficient­ ly extracts flavonoids and their glycosides, catechols and tannins; whereas phenolic acids and catechin were better extracted with methanol. these facts are table 1 ­ effect of the solvent type and concentration on the extraction efficiency for tpc and antioxidant activities (frsa and frp) of eggplant values are presented as means ± sd of six measurements. values with different letters are significantly different (p<0.05). n= 2. solvent concentration tpc (mg gae/100 g fm) frsa (mg te/g fm) frp (mg te/g fm) acetone 30% 809.06 ± 29.65 c 410.23 ± 19.78 e 18.61 ± 0.59 d 50% 785.06 ± 31.22 c 200.11 ± 10.01 h 18.78 ± 1.44 d 70% 1032.16 ± 40.04 a 648.59 ± 14.57 a 26.06 ± 0.42 a 90% 775.18 ± 26.45 c 365.85 ± 28.37 f 21.33 ± 1.09 c ethanol 30% 871.19 ± 32.90 b 360.30 ± 12.01 f 15.44 ± 1.58 ef 50% 641.04 ± 25.53 de 458.18 ± 34.53 d 13.78 ± 1.44 f 70% 539.38 ± 37.65 f 298.12 ± 29.32 g 17.23 ± 0.29 de 90% 512.18 ± 21.32 f 537.80 ± 25.99 bc 24.78 ± 1.13 ab methanol 30% 615.62 ± 38.20 e 288.55 ± 18.88 g 16.67 ± 0.44 e 50% 532.32 ± 47.23 f 305.20 ± 25.32 g 15.50 ± 1.36 ef 70% 669.28 ± 13.62 de 518.87 ± 19.36 c 22.33 ± 0.50 c 90% 708.81 ± 33.89 d 638.89 ± 19.85 a 14.00 ± 1.26 f water ­ 660.81 ± 23.58 de 574.80 ± 17.96 b 23.39 ± 0.48 bc 572 adv. hort. sci., 2019 33(4): 567­580 depend on their composition but also influenced by the degree of polymerization, concentration and interaction of their various chemical structures with colorimetric analysis substances (moure et al., 2001). however, the acetone:water mixtures are more use­ ful for extracting polyphenol from protein matrices, since they appear to degrade the polyphenol protein complexes (tabart et al., 2007). according to grujic et al. (2012), mixtures of organic solvent and water have been revealed to be more efficient in extracting phenolic compounds than mono­component sol­ vents. inevitably, total phenolic content is also influ­ enced by the solubility of phenolic compounds in the solvent used, as their diverse chemical structures might alter their solubility (chaalal et al., 2012). the antioxidant activity of the eggplant phenolic extracts was determined by two methods, namely the 1,1­diphenyl­2­ picrylhydrazyl free radical scav­ enging activity (frsa) and the ferric reducing power (frp) assays, which have been widely used for the assessment of antioxidant capacity of various plant extracts and natural products. determination of scavenging stable dpph. free radical is a very quick way to evaluate the antioxidant activity of the extracts in a very short time. with this method, it was possible to assess the antiradical abili­ ty of an antioxidant by measuring the reduce in the absorbance of dpph. at 517 nm. as a result of a color change from violet to yellow, the absorbance dimin­ ishes when the dpph. radical is scavenged by an antioxidant through hydrogen donation to form a stable dpph­h molecule. in the radical form this mol­ ecule had an absorbance at 515 nm which disap­ peared by receiving an electron or hydrogen from an antioxidant to become a stable diamagnetic mole­ cule. aqueous acetone 70% was also observed to be the solvent presenting the highest antioxidant activi­ ty (table 1). the percentage of the dpph. radical­ scavenging activity (frsa) of acetone was 648.59 mg te/g fm, more three times than acetone 50% (200.11 mg te/g fm), which represents the lowest value. however, there were no significant difference between 90% acetone and 30% ethanol; 70% ethanol, methanol (30%, 50%) and water extracts with methanol (70%) and ethanol (90%) extract. our results are in accordance of those reported by mokrani and madani (2016), chaalal et al. (2012) and gonzález­montelongo et al. (2010) who shown that aqueous acetone were more efficient for extracting peach, prickly pear seeds and banana peel phenolics, respectively and therefore, produced extracts with higher antioxidant activity. this can be explained by the solubility of phenolic compounds which depends widely on the nature of the solvent (polarity) used, their degree of polymerization, as well as their inter­ action with other food components and the forma­ tion of insoluble complexes (naczk and shahidi, 2004). since acetone was the solvent presenting the highest yield of tpc and antioxidant activity simulta­ neously, it is believed that phenolics contributing effi­ ciently to the total antioxidant activity of eggplant extract are phenolic compounds with higher molecu­ lar weight and lower polarity, according to ‘‘like dis­ solves like” principle. ferric reducing power (frp) of eggplant extracts was measured by the direct reduction of fe3+(cn–)6 to fe2+(cn–)6 and was determined by measuring absorbance of the resulting perl’s prussian blue com­ plex formed after the addition of ferric ions (fe3+). in this method, the yellow color of the test solution changes to various shades of green and blue depend­ ing on the content of reductants (antioxidants) in the sample. these reducing agents reduce the fe3+/ferri­ cyanide complex to the ferrous form. thus, fe2+ can be monitored by measuring the formation of perl’s prussian blue at 700 nm (gülçin et al., 2006; gülçin, 2011). the same thing as frsa, 70% acetone extract was observed to have significantly the highest frp (26.06 mg te/g fm), two times approxymatively more than 50% ethanol (13.78 mg te/g fm), which represents the lowest value. some extract have showed no significant differences at p<0.05 (table 1). a similar result has been reported by mokrani and madani (2016) and chaalal et al. (2012) in peach and prickly pear seeds extracts, respectively. the differ­ ences observed in the antioxidant activity (frsa and frp) of eggplant extracts could be due the variation of the quantity and quality of phenolic compounds present in the different extracts. thus, by compro­ mising between the yield of tpc and antioxidant activities (fras and frp), 70% acetone was chosen as the best solvent to optimize the following extraction conditions. extraction temperature as depicted in figure 1, extraction temperature demonstrated a significant effect (p<0.05) on pheno­ lic content and antioxidant activities of eggplant extracts. the efficiency of tpc extraction and the antioxidant activities were influenced by the temper­ ature. the tpc dropped when the temperature arkoub‐djermoune et al. ‐ optimization of phenolic compounds recovery of algerian eggplant 573 increased from 25°c to 100°c. with the increase of temperature from 25 to 100°c, tpc, frsa and frp decrease from 1032.16 to 886.72 mg gae/100 g fm (fig. 1a), from 648.59 to 203.05 mg te/g fm (fig. 1b) and from 26.06 to 21.17 mg te/g fm (fig. 1c), respectively. this decrease may be attributing to a degradation of phenolic compound by increasing the temperature which has a great effect on the antioxi­ dant activity, in particular the antiradical activity. in this study, the optimal temperature for antioxi­ dant extraction from eggplant was 25°c. similar to the result reported by naczk and shahidi (2004) and mokrani and madani (2016) who found that heating cannot increase the phenolic extraction indefinitely and at temperature above 50°c, the stability of these compounds decreases with dramatic effects with the antioxidant activity. it should be noted that increas­ ing temperature beyond a certain value can lead to decomposition of some phenolic compounds. rostagno et al. (2007) reported a decomposition of isoflavones in soybean during heat treatments. malonyl isoflavones also degrade when extraction is performed between 75 and 100°c. extraction between 100­125°c affects acetyl isoflavones and higher temperatures sharply reduced the glucosides concentrations. it is not surprising to find out that the antioxidant activities results showed a similar trend to the total phenolic concentration. this could be due to the fact that each assay measures different kind of phenolics, and each phenolic compound shows different antioxidant properties, which depends on the chemical structure and substitution position (pokorny, 2003). according to liyana­pathirana and shahidi (2005) and hismath et al. (2011), heating mobilizes certain antioxidants while promoting concurrent decomposi­ tion of antioxidants, which are already mobilized at lower temperatures. when an acetone:water solvent is used for extraction, the evaporation of acetone will change the acetone­water ratio because acetone, with a boiling point of 56.2°c, becomes volatile (al­ farsi and lee, 2008). moreover, santos­buelga and williamson (2003) have reported that some pheno­ lics are thermosensitive, particularly certain flavonoids, such as anthocyanin and flavan­3­ol deriv­ atives which are the most prevalent polyphenols in eggplant. furthermore, cacace and mazza (2003) reported that temperature affected the extraction of anthocyanins and increasing the temperature over 30­35°c resulted in the decomposition of antho­ cyanins. this could be explained by a higher vulnera­ bility of anthocyanins to high temperature. nevertheless, several other studies reported that heat enhanced tpc recovery ju and howard (2003), pinelo et al. (2005), al­farsi and lee (2008) and benmeziane et al. (2014). this was probably due to the increased phenolic solubility, faster diffusion rate, better mass transfer, extraction yield, reduced solvent viscosity and surface tension (richter et al., 1996). according to wissam et al. (2012) an increase fig. 1 ­ impact of temperature on the extraction efficiency for total phenolic compounds (a), dpph free radical scanen­ ging activity (b) and ferric reducing power (c) of eggplant (n= 2). values with different letters are significantly diffe­ rent (p<0.05). adv. hort. sci., 2019 33(4): 567­580 574 in temperature increases the efficiency of the extrac­ tion since heat render the cell permeable, increase solubility and diffusion coefficients of the compounds to be extracted and decreases the viscosity of the solvent, thus facilitating its passage through the solid substrate mass but the use of temperatures higher than 50°c decreases the total polyphenols yield which is probably due to their degradation. the improvement of the antioxidant extraction with tem­ perature was probably due to the increasing diffusivi­ ty of the solvent in the solid matrix and the solubility of the phenolic compounds in the solvent, which favour the extraction (herrero et al., 2005; juntachote et al., 2006). in fact, the polarity of water (dielectric constant) is decreased when the tempera­ ture is increased, due to the breakdown of hydrogen bonds when water is subjected to high temperatures, changing the water properties. under these condi­ tions, the water becomes less polar and acts like an organic solvent such as methanol or ethanol, increas­ ing the solubility of the organic materials in it (ballesteros et al., 2017). the effect of extraction temperature is very variable from plant material to another; it depends especially on their composition on phytochemicals and to their resistance to heat. based on the results obtained with the effect of extraction temperature, we deduced that the pheno­ lic compounds present in eggplant extracts were thermally unstable. it is well known that the use of water in infusion is very common and easy process used for preparing tea, which extracts the polyphenols from tea with hot water. which can’t be applied in our case to extract phenolics from eggplant because they are very sensitive to heat treatment. while, the use of pure water as single solvent for extraction presents some problems and provides an extract with a high content of impurities (organic acids, sugars, soluble proteins) particularly at high temperatures, that could interfere in the phenolic identification and quantification (chirinos et al., 2007). when mem­ brane filtration was used, the presence of protein and polysaccharide reduced the filterability. moreover, the cumulative cost of the concentration operation increases since water is more difficult to remove than acetone and other organic solvents (bachir bey et al., 2013). furthermore, water dis­ solves many nutrients l ike sugar and protein. therefore, aqueous extracts are more susceptible to microorganism invasion during storage. several stud­ ies have reported that acetone:water mixtures are good solvent systems for extraction of polar antioxi­ dants and are more useful for phenolic extraction from protein matrices since phenolic­protein com­ plexes dissolve more easily (kallithraka et al., 1995; sun et al., 2002). since the highest tpc value was extracted at the temperature of 25°c with an extraction yield of 1032.16 mg gae/100 g fm, this temperature was cho­ sen as the best temperature for extracting phenolic compounds from eggplant and the extracts provide high phenolic content and maintaining a highest antiox­ idant activity with health benefits and could be great interest for application in pharmaceutical products. extraction time extraction time is essential in economizing energy and cost of the extraction process. the extraction time only slightly influenced the tpc and antioxidant activities of eggplant (fig. 2). the tpc, frsa and frp increased when extraction time was increased from 30 to 60 min. after 60 min, further increase of the fig. 2 ­ impact of the extraction time on the extraction efficiency for total phenolic compounds (a), dpph free radical sca­ nenging activity (b) and ferric reducing power (c) of egg­ plant (n= 2). values with different letters are significantly different (p<0.05). arkoub‐djermoune et al. ‐ optimization of phenolic compounds recovery of algerian eggplant 575 processing time significantly decreased (p<0.05) the rate of phenolics and antioxidant activity. a time of 60 min was selected as the optimal time for extrac­ tion. this duration allowed extraction of 1111.23 mg gae/100 g fm of phenolics, which corresponded to frsa and frp of 692.47 and 35.11 mg te/g fm, respectively. the optimal time for phenol extraction changes depending upon the material used for extraction but it is an important factor that influence tpc extraction and, hence, antioxidant activity. moreover, the type of phenolic compounds extracted and the tempera­ ture of extraction were also important factors. a sim­ ilar trend was shown by liyana­pathirana and shahidi (2005) on whole grains, bran of both soft and hard wheat and al­farsi and lee (2008) on date seeds. this observation was well explained by fick’s second law of diffusion, which predicts a final equilibrium between the solute concentrations in the solid matrix (plant matrix) and in the bulk solution (solvent) after a certain time (silva et al., 2007). in addition, liyana­pathirana and shahidi (2005) showed that the total antioxidant activity increased with an increase in extraction time from 15 to 60 min. beyond 70 min, the total antioxidant activity decreased sharply and reached a minimum at 105 min, probably due to decomposition of the active com­ pounds during the prolonged extraction time. in this investigation, 1 h for tpc extraction was optimal, and prolonged extraction up to 2 h decrease the tpc or the antioxidant activity at 25°c. our results revealed that an excessive time beyond 60 min is not useful to extract more phenolic compounds from eggplant. taking into account these facts, an extraction time of 60 min was selected as the best extraction time for extracting phenolic compounds and antioxidant activities of eggplant. solid to solvent ratio the choice of the solid to solvent ratio was investi­ gated because it influences phenolic recovery and the antioxidant activity. the total phenolic content and antioxidant activity of eggplant extracted by 70% ace­ tone at 25°c for 60 min using four solid/solvent ratios: 1/100, 1/75, 1/50 and 1/25 g/ml are shown in figure 3. sample/solvent ratio had a significant effect (p<0.05) on tpc and the antioxidant activities (frsa and frp). the tpc, frsa and frp radicals scavenging capaci­ ty increased from 1111.23 to 1258.07 mg gae/100 g, from 692.47 to 855.50 mg te/g and from 35.11 to 39.33 mg te/g, respectively, with the increase of solid/solvent ratio from 1/25 to 1/50 g/ml. the tpc recovery using a ratio of 1/25 was poor. extraction of antioxidants reached a maximum with a ratio of 1/50, which produced phenolic concentrations of 1258.07 mg gae/100 g (fig. 3a). the antioxidant activity changes with the same patherns with pheno­ lic concentrations with a changing ratio from 1/25 to 1/50 g/ml, but with ratio of 1/75 and 1/100 g/ml, the frsa and the frp decreased (fig. 3 b, c). ćujić et al. (2016) have noted that the higher solid to solvent ratio generate a decrease in the consumption of plant material and decrease in the cost of extrac­ tion. additionally, bucić­kojić et al. (2007) reported a significant difference of the polyphenols recovery yield from grape seeds depending on liquid­to­solid ratio, with the highest polyphenols concentration obtained using a ratio of 40:1. furthermore, prasad et al. (2012) employed a factorial design approach to identify the significant factors contributing to high extraction yield, antioxidant capacity and phenolic content in the extracts from mangifera pajang peri­ fig. 3 ­ impact of the solid to solvent ratio on the extraction effi­ ciency for total phenolic compounds (a), dpph free radi­ cal scanenging activity (b) and ferric reducing power (c) of eggplant (n= 2). values with different letters are signi­ ficantly different (p<0.05). adv. hort. sci., 2019 33(4): 567­580 576 carp. liquid­to­solid ratio was reported as highly sig­ nificant contributor. since the solid to solvent ratio of 1/50 g/ml was selected as the best ratio for phenolics extraction and antioxidant activities of eggplant. number of extractions a series of successive extractions were performed under operating conditions favoring the best extrac­ tion: 70% acetone as solvent, a temperature of 25°c, extraction time of 60 minutes and solid/solvent ratio 1/50 g/ml. three sequential extractions appear suffi­ cient; the first extract contain approximatively 60% of total extractable polyphenols (1258.07 mg gae/100 g) and exhibits an antioxidant activity of 855.61 mg te/g (frsa) and 39.33 mg te/g (frp) (fig. 4). this rate relatively was completed by the second (27%) with a tenor of 558.44 mg gae/100 g and gives an antioxidant activity of 415.30 mg te/g (frsa) and 18.44 mg te/g (frp) and third (13%) extraction with a concentration of 264.98 mg gae/100 g which exhibits an antioxidant activity of 245.95 mg te/g (frsa) and 9.33 mg te/g (frp). a similar result to that found in the present study was obtained by benmeziane et al. (2014) on the table grape (vitis vinifera l.) with percentages of 60%, 22%, 13% in the three fractions respectively. according to bonnaillie et al. (2012), depletion of the raw material and the concentration of the extraction medium are conduct­ ed mainly in the first three stages, the last stage, rather dilutes the volumes retained in the solid. consequently, the number of extractions can be lim­ ited to three successive contacts with fresh solvent, these authors for their part, found in a series of three successive extractions of dandruff peanut following results: 60%, 35% and 5% respectively. the maximum amount of extractable total phe­ nols in the extract prepared with the mixture of the three successive extractions was assessed to 794.94 mg gae/100 g fw and maintaining a highest antioxi­ dant activity of 737.86 mg te/g (frsa) and 28.00 mg te/g (frp) which can be probably attributed to the dilution factor in the three mixture extracts. pearson correlation analysis in order to more appreciate the relationships between antioxidant capacities and phenolic content of eggplant extracts, correlations between assays under different extracting conditions were analyzed. under the parameter of solvent type (table 2), no significant correlations were found between the tpc and the antioxidant activities (frsa and frp). this can be explained by a synergism of eggplant pheno­ lics present in the extract which may contribute to the overall observed antioxidant capacity. under the influence of extraction temperature (table 2), tpc was correlated positively with frsa assay (r= 0.82) at p<0.05 and frp assay (r= 0.66) at p<0.001. concerning the influence of extraction time condi­ tion (table 2), the tpc was observed to be correlated positively with frsa (r= 0.95) and frp (r= 0.83) at p<0.001. from this correlation, we can believe that eggplant phenolic compounds extracted at different times display antioxidant capacities. about the parameter solid to solvent ratio, tpc were observed to be positive significantly (p<0.001) correlated with frsa assay with pearson correlation coefficients of 0.90 and 0.90, respectively (table 2). previous studies showed that antioxidant activity of fig. 4 ­ impact of the number of extraction on the extraction efficiency for total phenolic compounds (a), dpph free radical scanenging activity (b) and ferric reducing power (c) of eggplant (n= 2). values with different letters are significantly different (p<0.05). arkoub‐djermoune et al. ‐ optimization of phenolic compounds recovery of algerian eggplant 577 phenolic compounds depends widely on their struc­ ture. therefore, the solid to solvent ratio affect posi­ tively the antioxidant capacity of eggplant phenolics. under the influence of number of extraction, cor­ relations between tpc and antioxidant assays (frsa and frp) were positively high (0.99, p<0.001). we can suggest that the hydrogen electron donating abil­ ities of eggplant extracts were directly proportional to the concentration of total phenolics. this relation­ ship suggested that the phenolic compounds of egg­ plant extracts might be the major contributors to the analyzed antioxidant activities. 4. conclusions in the present study, single factor experiments approach was used to determine the optimization of the extraction process of eggplant phenolics, investi­ gating some variables which such as effect of solvent extraction (solvent type and solvent/water mixture concentration), extraction temperature, extraction time, solid to solvent ratio and number of extrac­ tions. to the best of our knowledge, no data was reported about the effect of extraction parameters on the recovery of phenolic compounds from egg­ plant. the results of the present investigation demonstrated that all the extraction parameters exhibited significant effects (p<0.05) on the extrac­ tion efficiency of tpc and the antioxidant activity (frsa and frp) of eggplant extracts. the optimal extraction conditions, selected by compromising between the rate of total phenolic compounds (tpc) and their antioxidant activities (frsa and frp), were extraction with 70% aqueous acetone at 25°c for 60 min using a 1g/50 ml solid to solvent ratio and three successive extractions seem necessary for the deple­ tion of plant material. therefore the maximum extractions of polyphenols present in eggplant were the optimum conditions for tpc recovery and main­ taining the highest antioxidant activity for eggplant vegetable. these conditions allowed recovery of 794.94 mg gae/100 g fm and produced dpph free radical scavenging activity (frsa), ferric reducing power (frp) of 737.86 mg te/g fm and 28.00 mg te/g fm, respectively. a significant pearson correla­ tion coefficients were found between tpc and frsa and frp of eggplant extracts under the influence of extraction parameters. the results obtained in this study indicate that eggplant can be considered as a natural source of phenolics compounds known for their good antioxidant capacity. since antioxidant compounds provide health benefits, eggplant extracts could be of great interest for application in pharmaceutical products. this study will provide bases for future investigations on the optimization of the extraction of phenolic compounds from eggplant using other models such as response surface methodology. however, it is interesting to test other extraction methods such as microwave and ultra­ sound extractions, ultrafiltration, supercritical fluid and subcritical water extractions on extracting phe­ nolic compounds from eggplant. these alternative new technologies use less solvent and energy and may increase the safety and the quality of products. acknowledgements the authors gratefully acknowledge all partici­ pants enrolled in the study and express their appreci­ ation to algerian ministry of higher education and scientific research for the financial support. references al­farsi m.a., lee c.y., 2008 ­ optimization of phenolics and dietary fibre extraction from date seeds. ­ food chem., 108(3): 977­985. alasalvar c., magdalena k., ryszard a., fereidoon s., 2006 ­ antioxidant and antiradical activities in 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gene expression under watershortage stress d. jawdat 1 (*), a.w. allaf 2, n. taher 1, a. al-zier 2, n. morsel 1, z. ajii 2, b. alsafadi 1 1 department of molecular biology and biotechnology, atomic energy commission, po box 6091, damascus, syria. 2 department of chemistry, atomic energy commission, po box 6091, damascus, syria. key words: cotton, fiber quality, flowering, ftir, gene expression, tga, xrd. abstract: two accredited cotton (gossypium hirsutum l.) cultivars (aleppo 118 and deir al-zour 22) have been investigated to assess physiological, morphological, and molecular responses under water shortage conditions. both cultivars have shown early flowering. however, higher percentage of treated ‘aleppo 118’ plants kept flowering towards the end of the growing season compared to treated ‘deir al-zour 22’ plants. both cultivars kept consistent micronaire and cohesion under normal and water shortage conditions. the cultivar aleppo 118 displayed more consistent fiber quality between control and treated plants, while ‘deir al-zour 22’ showed variation in fiber length and strength between control and treated plants. results have demonstrated an increase in fold expression of dreb1a, tps and hspcb genes in the flowering stage of treated plants compared to the controls. results also showed a continuous activation of dreb1a gene in the two critical growing stages of a cotton life cycle, flowering and boll development in treated plants of ‘deir al-zour 22’. this work illustrates the different responses of two cotton cultivars under water shortage stress and its impact on flowering and fiber traits. 1. introduction the supply of groundwater in agriculture is reduced due to intersecting environmental, industrial and domestic sectors. groundwater-dependent agro-economies are mostly affected by drought stress, water deficits and aquifer depletion, specifically in the arid and semi-arid regions. drought is considered a major constraint to crop productivity and yield stability around the world. it is therefore, a major challenge to farmers in drought subjected regions. water saving and stable crop production are the main challenges that urge researchers to develop new cultivars and irrigation strategies for a sustainable use of water in agriculture. developing new cultivars that tolerate water stress has been assisted (*) corresponding author: ascientific2@aec.org.sy citation: jawdat d., allaf a.w., taher n., al-zier a., morsel n., ajii z., al-safadi b., 2018 cotton flowering behavior, fiber traits and gene expression under water-shortage stress. adv. hort. sci., 32(1): 79-92 copyright: © 2018 jawdat d., allaf a.w., taher n., al-zier a., morsel n., ajii z., al-safadi b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 may 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 79-92 80 and supported by wide range of strategies and procedures that enable the selection of candidate parents and an efficient screening of targeted progenies. genomics, marker-assisted selection, the manipulation of qtls using genetic engineering and transcriptomics can contribute in the release of improved, drought-tolerant cultivars (tuberosa and salvi, 2006). the metabolomics-assisted breeding is another candidate approach for the development of crops with increased tolerance to abiotic stresses (fernie and schauer, 2009). improving drought tolerance in crops requires understanding the biochemical responses under water deficit (reddy et al., 2004) and the characterization of drought patterns in the growing regions along with the physio-morphological traits of these crops under such environments (fukai and cooper, 1995; khan et al., 2010). plants have evolved their drought-adaptive strategies (meyre et al., 2001), between drought escape (early flowering) and drought tolerance. comprehending the flowering pattern of each crop under stressed and non-stressed environments is a key to which drought-adaptive strategy the crop plant follows. early flowering and maturity can help in saving groundwater for irrigation and can help in avoiding early rainfall that usually affects the harvest of certain crops such as cotton (jawdat et al., 2012). therefore, a vigilant assessment of crop responses to alteration in irrigation regimes is needed. at any rate, amendments and management of irrigation and water saving methods has a great potential in saving water sources in the arid and semi-arid regions (oweis et al., 2011; ünlü et al., 2011). cotton is a major cash oilseed and fiber crop with a world production estimated to be 25.01 million tonnes in 2013-2014 as announced at the official website of the international cotton advisory committee (icac) (https://www.icac.org). the widely famous, allotetraploid gossypium hirsutum l. has a distinct growth manner and can be useful in monitoring growth and developmental changes under diverse conditions (jawdat et al., 2012). cotton is grown in around 70 countries where the irrigated system dominates in arid regions; and is used to supplement rainfall in the humid regions to ensure yield stability. seed cotton production in syria (mostly semi-arid where cotton is irrigated) was around one million tonnes in 2000. a decrease in production from around 670 k tonnes in 2009 to around 472 k tonnes in 2010 and then up to about 671 k tonnes in 2011 in the same cultivation area (~200 k hectares), was attributed to the drought wave that hit the country in 2010. a rapid decline in production was recorded between 2011 and 2014, from around 670 to around 162 k tonnes respectively. this was concurrent with the unfortunate events taking place in syria. the principal objective of this paper is to assess physiological, morphological, molecular, and biochemical responses of two accredited cotton cultivars in syria under deficit water regime, in benefits of future breeding programs. 2. materials and methods plant materials plant material consisted of cotton seeds of two g. hirsutum l. accredited local cultivars: aleppo 118 [aleppo 40 (aleppo 1 x acala sj1) x american cultivar bw 76-31] and deir al-zour 22 (a selected line from deltapine 41). drought stress treatment seeds were sown in rows, where the distance between rows and plants were kept at 75 and 25 cm respectively. experimental plots were designed according to a randomized complete block design (rcbd) with each treatment is replicated once in each block. the experiment site was located in doubaya in damascus country side (980 m above sea level, 33° 29´ 36´ n and 36° 04´ 57´ e). the experiment consists of two irrigation treatments (control with full irrigation and treated with 50% of control irrigation), two cultivars (aleppo 118 and deir al zour 22) and three replicates per treatment. the soil (total n: 0.08%, available p: 12 ppm, k: 1.30 meq/100 g, and organic matter: 1.19%) was prepared by tractor ploughing deep for 45 cm, followed by two surface ploughings for 15 cm deep. fertilization was conducted following the cotton research administration (cra) guidelines and recommendations. nitrogen fertilizer as urea 46% [co(nh2)2] was applied at a rate of 415 kg/ha in four unequally split applications, 20% before sowing, 40% 30 days after sowing, 20% at floral bud emergence, and 20% at bolls initiation phase. phosphorus fertilizer [ca(h2po4)2] was applied at a rate of 130 kg/ha before sowing. potassium fertilizer was applied at the rate of 170 kg/ha in the form of potassium sulphate (k2so4). drip irrigation was applied at 250 m3/ha, at first, for both control and treated blocks. consequent irrigations were applied during the growing season of cultivated plants once a week at 250 m3/ha (full irrigation) in control blocks, and once in every 10 days at 125 m3/ha (deficit irrigajawdat et al. cotton behaviour under water stress 81 tion; receiving water at 50% of control treatment) in the treated blocks. except for one week where temperatures reached around 45 degrees in the afternoon, we had to irrigate both control and treatment blocks with emergency full irrigation. the distance between emitters was 25 cm and the emitter discharge was 8 l/h. thinning of plants was conducted around 4 weeks of germination. physiological data chlorophyll a and b content. leaves samples (100 mg/sample) were each immersed in 4 ml of 95% acetone and incubated at 6-8°c for 24 hrs. chlorophyll a and b concentration was determined spectrophotometrically by measuring the absorbance (optical density-od) at 662 and 644 nm respectively. the concentrations of chlorophyll a and chlorophyll b (μg. g-1 fw) in leaf tissues were calculated using the following equations (cha-um et al., 2006): chlorophyll a= 9.784*d 662 0.99*d 644 chlorophyll b= 21.426*d 644 4.65*d 662 where di is an optical density at the wavelength i. osmotic potential op) and osmotic adjustment (oa). osmometer readings in mosm/kg, were taken using the freezing point osmometer, the microosmoettetm (precision systems inc., usa). frozen 100 mg of leaf samples in liquid nitrogen were ground (a pool of three leaves per treatment). two milliliters of autoclaved d.d. water were added to each sample and samples were vortexed briefly and centrifuged for one minute at 13000 rpm. fifty microliters of the supernatant were used for each reading. the osmotic potential ψs was calculated using the following formula: op (mpa)= {r * t * osmometer reading}/1000 where r is the gas constant (0.008314), and t is the laboratory temperature in kelvin (298). the osmotic adjustment (oa) was calculated as the difference between the osmotic potential of non-stressed plants and the water stressed plants. morphological data twenty plants of each replicate were randomly tagged and flowering behavior data for days after planting (dap) were recorded such as: first floral bud emergence, plant height at first flower, and nodes above white flower (nawf). the percentage of plants bearing flowers and plants bearing floral buds were recorded towards end of season. end of season data also included: plant height, root depth (randomly selected plants from each replicate were carefully pulled out of ground), and number of secondary roots. physical and chemical fiber properties fiber quality testing. thirty bolls of each replicate were picked and sent to the fiber quality lab at the cotton research administration (cra) for testing: micronaire, fiber length, fiber strength, fiber cohesion, and ginning percentage. cra fiber quality lab conducts its testing according to the international testing standards with a temperature of 22±2°c and relative humidity of 64%±4%. the lab uses the following instruments: micronaire 775, digital fibrograph, pressley tester and stelometer for fiber testing. fourier transform infrared (ftir) acquisition spectra of cotton fiber ftir was conducted to study the chemical variance, if any, between fibers from control and treated plants of the two studied cultivars. the infrared spectra were recorded on nicolet 6700 in the range 4000 to 400 cm-1 with a resolution of 4 cm-1 at 32 scans as direct measurements of the cotton fibers (three replicates per treatment) samples using universal attenuated total reflectance technique ftir-atr with krs-5 crystals. the instrument is equipped with dtgs detector and kbr beam splitter. the operating system used was the omnic software (version 7.3, thermo nicolet, usa). the infrared spectra of fiber samples for control and treated plants of aleppo 118 and deir al zour 22 cultivars were recorded. the recorded spectra were repeated three times for each investigated sample. measurement of crystallinity by conventional x-ray powder diffractometry (xrd) xrd was applied to determine the crystallinity of fiber samples from control and treated plants of ‘aleppo 118’ and ‘deir al-zour 22’ samples. x-ray powder diffraction patterns were obtained on a stoe transmission stadi-p diffractometer with monochromatic cu kα1 radiation (λ = 1.5406 å) selected using an incident-beam curved-crystal germanium ge (111) monochromator, with the stoe transmission geometry (horizontal set-up) and a linear position-sensitive detector (psd). the determination of crystallinity of the samples was performed with the program winxpow (stoe and cie, 1999) using single-sample method. this method requires defining the portions of the diagram due to air scatter and inelastic scattering, amorphous scattering and bragg or crystal scattering. if ic(2q) and ia(2q) are known for the whole diagram, the crystallinity index is calculated from the relation: xc=(∑ic(2θ)⁄(lp∙f∙t)⁄(∑[(ic (2θ)⁄t+ia (2θ)]⁄(lp∙f) eq. 1 adv. hort. sci., 2018 32(1): 79-92 82 the summing is over bragg angle (2q). the lorentz-polarization factor lp, the average form factor f and the temperature factor t are all 2q-dependent functions: lp(2θ)= 1 + cos2 (2θ) ⁄ (sin2 (2θ) cosθ eq. 2 f(2θ)= ∑f (n, 2θ) eq. 3 t(2θ)= exp(-2b sin22θ) ⁄ λ2 ) eq. 4 the summing in eq. 3 is over all atoms in the formula unit. the sample’s overall formula and average temperature factor (b) have to be known in order to be able to apply this method. it’s noteworthy to mention that sample absorption is always neglected; therefore, this method works well for transmission data of organic samples within ± 3% error of the true crystallinity. the formula of cellulose (c6h12o5) and b = 4.0 å2 was used for our cotton fiber samples. thermogravimetry analysis (tga) and differential scanning calorimetry (dsc) the dynamic weight loss of fiber samples as a function of increasing temperature tests were conducted using a mettler instrument (tg50). the tests were carried out in a nitrogen atmosphere, purged (30 ml/min) using sample weights of 10-15 mg at a heating rate of 10oc/min. the resolution of the balance is given, as 1 microgram for weights less than 100 milligrams, and the temperature precision of the instrument is ± 2oc. dsc was used to determine the glass transition temperatures of the prepared samples. a mettler instrument (dsc20) was utilized to record the dsc spectra. all samples were tested in aluminum pans at a heating rate of 10oc/min over a temperature range from room temperature to 400oc. the precision of the used instrument is ± 0.2oc. scanning electron microscopy (sem) scanning electron microscopy (vegaii xmu, tescan, czech republic) with an accelerating voltage of 30 kv, was operated to capture sem images of cotton fibers coming from control and treated plants of both cultivars. gene expression analysis rna isolation and cdna synthesis. total rna was isolated from leaves of cotton plants using the modified hot borate method (jawdat and karajoli, 2012). quality of total rna was tested by agarose-formaldehyde gel electrophoresis using standard protocols. the iscripttm select cdna synthesis kit (biorad, usa) was used to obtain first strand cdna starting from 1 µg total rna following the manufacturer’s protocol. the cdna was diluted 4x and stored at -20°c. relative quantification of gene expression the cdna samples (control and treated) were used as a template to quantify target genes (dreb1a, tps and hspcb) expression level. realtime pcr was performed in two replicates in a 25 µl of a reaction mixture composed of 12.5 µl iq sybr super mix (biorad, usa), 1 µl of cdna, 1 µl of each of the forward and reverse primer (10 µm), and 9.5 µl of d.d. water. the quantification of mrna levels was normalized with the level of mrna for ghef1α5 (artico et al., 2010). the relative expression (fold expression) was calculated using the -∆∆ct method as follows: 2-∆∆ct = 2[(ct treated target gene – ct treated reference gene ) (ct untreated target gene ct untreated reference gene )] specific target and reference gene primers are listed in table 1. 3. results physiological, morphological, molecular and chemical data were recorded to identify main changes in two cash cultivars of cotton. the physiological and molecular analysis were carried out on leaf material from control and treated plants of ‘aleppo 118’ and ‘deir al-zour 22’, harvested 45 dap table 1 target and reference genes accession numbers and primers sequences gene genbank accession no. forward and reverse primers (5’-3’) dehydration responsive element binding gene (dreb1a) ay321150.3 f-agctatagcactgagagggaag r-gcttcttcgtccaagtaaaacc trehalose-6-phosphate-synthase gene (tps) ay628139.1 f-ttcactacatgctgcccatgtg r-ggctgtggaggaaaaaaccaag heat-shock protein calmodulin binding gene (hspcb) ay819767.1 f-ctccttgaatgtatttactgcc r-gtgcgtcctctagtgtcttt elongation factor 1-alphagene (ef1α5) dq174254 f-tccccatctctggttttgag r-cttgggctcattgatctgggt jawdat et al. cotton behaviour under water stress 83 in the course of floral bud initiation stage. chlorophyll a and b content and osmotic potential the chlorophyll a content showed a non-significant decreasing trend in treated plants compared to control plants in both cultivars. however, the chlorophyll b content showed a small increment in the treated plants. the chlorophyll a/b ratio showed a reduction in treated plants of both cultivars. a larger reduction (18%) was observed in treated plants of ‘deir al-zour 22’ compared to the small reduction (4%) in treated plants of ‘aleppo 118’ (fig. 1 inset graph). a non-significant drop in the osmotic potential of leaves in treated plants was observed in both cultivars and the osmotic adjustment was higher (0.016 mpa) in ‘aleppo 118’ compared to ‘deir alzour 22’ (0.006 mpa) (fig. 1). plant morphology early floral bud emergence was observed in both treated cultivars compared to control plants (fig. 2 inset graph i). the control plants of aleppo 118 cultivars flowered earlier than control plants of ‘deir alzour 22’. nawf counts, another indicator of flowering earliness, was also recorded in the third week of first flower emergence (fig. 2 inset graph ii). recorded nawf counts showed a significant decrease in treated plants of both cultivars. a final record of plants with flowers, and plants with floral bud percentages were also taken five months after planting towards the end of season (fig. 2). end of season flowering counts have shown ‘aleppo 118’ tendency to keep initiating floral buds (~ 21%) in treated plants compared to control plants (5.6%), and produce fully opened flowers (~ 31%) in treated compared to control plants (~ 2%). however, a similar but less potent flowering counts pattern was observed in deir al-zour 22 cultivar. end of season measurements have also included plant stem height, taproot depth and number of secondary roots (table 2). treated plants in both cultivars showed a slight increase in plant stem height and minor decrease in root depth. however, non-significant increase in number of secondary roots was observed in treated plants of both cultivars. physical and chemical fiber properties fiber quality testing. the two tested cultivars have presented slightly different behavior in terms of fiber properties. seed cotton yield showed no significant difference between treated and control plants in each cultivar. the cultivar deir al-zour 22 showed a significant increase in both fiber length and strength in the treated plants compared to control ones. fiber fig. 1 the inset graph is chlorophyll a/b ratio in leaves of control (c) and treated (t) ‘aleppo 118’ and ‘deir-al zour 22’ plants. main graph represents osmotic potential and osmotic adjustment in leaves of control and treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. plants were under drip irrigation system, where the control plants were given full irrigation each week, and treated plants have been given deficit irrigation each 10 days. data represent means and standard error of three replications. fig. 2 the inset graph-i shows floral buds emergence in days after planting (dap) in control and treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. the inset graph-ii represents nawf counts in control and treated plants of both cultivars. data were subjected to duncan’s test with a confidence level of 95% using statistica program. columns sharing a letter are not significantly different. data represent means and standard error of three replications. the original graph is the percentages of plants with flowers and plants with floral buds of both control and treated aleppo 118 and deir al-zour 22 cultivars, five months after planting. data were subjected to duncan’s test with a confidence level of 95% using statistica program. line markers sharing a letter are not significantly different (capital letters for plants with floral buds and small letters for plants with flowers. adv. hort. sci., 2018 32(1): 79-92 84 elongation showed no significant difference between control and treated plants in each cultivar. furthermore, no significant difference was recorded in the cultivar aleppo 118. two fiber properties, fiber cohesion and micronaire, exhibited no significant difference between cultivars and between treatments. a significant increase in fiber maturity was observed in the treated plants of aleppo 118 cultivar, whereas, a non-significant increase was recorded in treated plants of ‘deir al-zour 22’ (fig. 3). ftir-atr spectra acquisition of the cotton fiber. the ftir-atr spectra of fiber samples from control and treated plants of aleppo 118 and deir al-zour 22 cultivars were recorded. the spectra showed a typical characteristic reflectance bands for common cotton fiber substances. the spectra of fiber samples from control and treated ‘aleppo 118’ plants were aligned for comparison and showed minimum variation (fig. 4 a). the spectra of fiber from ‘aleppo 118’ control plants showed 15 distinctive bands centered at 3310, 2890, 1740, 1605, 1429, 1372, 1315, 1203, 1160, 1103, 1055, 1020, 675, 554 and 431 cm-1. the vibration at 1429 cm-1 is designated and assigned as a “crystalline” absorption band of the fiber (abidi et al. , 2014). the band at 1372 cm-1 vibration is assigned to c-h bending, and it may be most suitable for indicating cellulose crystallinity associated with the band at 2890 cm-1. the spectra of control and treated samples were highly similar except for the band at 1740 cm-1 which was absent in the treated sample. similar spectra range was observed in deir al-zour 22 cultivar. however, few noticeable new bands with distinctive structures were observed in the treated sample at 2890, 1740, and 1605 cm-1. the band centered at 2890 cm-1 contains two prominent peaks vibrations at 2910 and 2847 cm-1, which are assigned to ch2 asymmetrical and symmetrical stretching, cultivar plant height (cm) root depth (cm) no. secondary roots control treatment control treatment control treatment aleppo 118 99.3±3.5 ab 105.9±5.5 a 19.8±2.2 17.7±2.1 10.4±0.8 ab 13.1±0.8 a deir al-zour 22 80.3±3.8 c 87.6±5.1 bc 18.3±2.8 17.0±0.7 9.9±0.9 b 11.4±1.1 ab table 2 end of season measurements of plant stem height, taproot depth and number of secondary roots in control and treated plants of aleppo 118 and deir al-zour 22 cultivars data were subjected to duncan’s test with a confidence level of 95% using statistica program. numbers in rows and columns sharing a letter in each block (plant height and no. secondary roots) are not significantly different. data represent means and standard error of three replications. fig. 3 fiber properties of control and treated aleppo 118 and deir alzour 22 cultivars. fiber length (a), fiber strength (b), fiber cohesion (c), fiber elongation (d), micronaire (e), and maturity index (f). data were subjected to duncan’s test with a confidence level of 95% using statistica program. columns sharing a letter are not significantly different. data represent means and standard error of three replications. jawdat et al. cotton behaviour under water stress 85 respectively. as for the second band at 1740 cm-1, which is assigned to a carboxylic ester group (c=o stretching), a clearer structure is noticed in the treated sample in comparison with the control. another distinctive banding pattern is observed at 1605 cm-1 (adsorbed water), which is neither seen in the treated aleppo 118 cultivar and nor observed in deir alzour 22 cultivar control sample. this band contains two prominent peaks vibrations at 1577 and 1540 cm-1 (fig. 4 b). measurement of crystallinity by conventional x-ray powder diffractometry. fiber analysis using the xrd procedure showed a typical cellulose pattern in both control and treated plants for each cultivar (fig. 5). the amount of crystalline cellulose in studied samples was between 66-75% (table 3). as seen in table 3, there was a minor difference between the ci values of the treated and control ‘deir al-zour 22’ samples. however, there is no variation between control and treated samples of aleppo 118 cultivar. thermogravimetry analysis (tga) and differential scanning calorimetry (dsc). typical dynamic tga thermograms of the studied samples have been recorded and are shown in figure 6 a. the tga thermograms show a slight decrease in the weight and one significant step at high temperature. differential scanning calorimetry (dsc) was used to locate a possible glass transition temperature (tg) of the samples. the glass transition temperature could not be observed in all dsc thermograms. the endothermic peak at around 100oc could be due to evaporation of adsorbed water (fig. 6 b). scanning electron microscopy (sem). sem images reveal the longitudinal morphology of fibers coming from control and treated plants of aleppo 118 and deir al-zour 22 cultivars under three magnifications table 3 fiber crystalline index in control and treated aleppo 118 and deir al-zour 22 cultivars fig. 4 ftir-atr spectra of the cotton fibers obtained from (a) ‘aleppo 118’ control and treated plants and (b) ‘deir alzour 22’ control and treated at room temperature in the range 400-4000 cm-1. the recorded spectra were repeated three times and showed a typical and characteristic absorption bands for common cotton fibers substances. fig. 5 x-ray powder diffraction patterns of fibers in control and treated aleppo 118 and deir al-zour 22 cultivars. cultivar crystallinity index (%) control treatment aleppo 118 66.5±3.0 66.8±3.0 deir al-zour 22 73.7±3.0 68.5±3.0 adv. hort. sci., 2018 32(1): 79-92 86 (1.00 kx, 3.00 kx and 20.00 kx) (fig. 7). images of the two cultivars show the ribbon fiber shape rolled in a helicoid manner, a typical cotton morphological feature. the spiral and twisting fiber nature is present in both cultivars under both conditions. expression analysis of dreb1a, tps and hspcb drought-related genes fold expression of three drought-related genes in cotton (dreb1a, tps and hspcb) was analyzed, assisted by ghef1α5 gene expression for normalization (fig. 8). leaf samples of control and treated plants of both cultivars were harvested at two points (stages), s1 and s2. where s1 represents leaf material harvested at 40 dap (during floral bud to flower transition stage) and s2 represents leaf material harvested at 60 dap (bolls opening and maturation). gene expression analysis showed an activation of the studied genes in treated plants compared to the controls of both cultivars during s1. the s2 stage, experienced a drop in genes activity in both treated cultivars. analysis showed a major significant drop in gene activity (hspcb and tps) in treated ‘deir al-zour 22’ plants (s2) compared to samples of the earlier stage (s1). exceptionally, dreb 1a gene showed a slight decrease in gene expression keeping its high activity in treated plants ~4 fold higher than the config. 6 (a) tga thermograms of cotton fibers in presence of nitrogen. (b) dsc thermograms of cotton fibers in presence of nitrogen. fig. 7 sem images of cotton fibers. a, b and c. fibers of control ‘aleppo 118’ under 1.00, 3.00 and 20.00 kx magnifications. d, e and f. fibers of treated ‘aleppo 118’ under 1.00, 3.00 and 20.00 kx magnifications. g, h and i. fibers of control ‘deir al zour 22’ under 1.00, 3.00 and 20.00 kx magnifications. j, k and l. fibers of treated ‘deir al zour 22’ under 1.00, 3.00 and 20.00 kx magnifications. jawdat et al. cotton behaviour under water stress 87 trols. on the other hand, aleppo 118 cultivar showed an almost steady expression pattern of the three genes in the two stages (high expression in s1 and low in s2). this continuous high expression activity of dreb 1a needs to be further investigated in ‘deir alzour 22’ to study its relation to water deficit tolerance. 4. discussion and conclusions this study overlooks the behavior of two accredited cotton cultivars subjected to two irrigation regimes along their life cycle. it is anticipated that water scarcity in the mediterranean region will increase under climatic change and this can be addressed by evaluating the impacts of climate change on water resources and their management, the adaptive capacity and the policy responses (iglesias et al., 2011). improving water resources management is challenged by the physiology and biology of the crop, irrigation practices, environment, farmers’ perspectives, and government funding. increasing crop water productivity, i.e. producing more food, income, better livelihoods and ecosystem services with less water (molden et al., 2010), has been a major interest to agronomists, farmers, environmentalists and economists. our research is mainly interested in understanding the responses of two credited syrian cotton cultivars grown under water-shortage stress conditions, motivated by the concept of reaching a better quality and quantity of cotton fiber coupled with saving ground water resources in a cotton growing country. recently, drip irrigation systems have been widely employed, due to their improvement of water use efficiency (patanè et al., 2011). hence, our field experiments were irrigated using the drip irrigation system to reduce water runoff losses. two credited cotton local cultivars, aleppo 118 and deir al-zour 22, have been investigated in our study. the two cultivars are assigned by the ministry of agriculture to be grown in their pertinent environments in the syrian land. ‘deir al-zour 22’ is the featured cultivar for cultivation in deir al-zour province, along the euphrates, where high temperature is the dominant feature of the area especially during the growing season. ‘aleppo 118’, an early maturity cultivar, is cultivated in the arid and semi-arid northernwest regions of syria, mostly in aleppo province. control (full irrigation) and treated (deficit irrigation) plants of both cultivars were tested using some physiological, morphological, molecular, and biochemical parameters. the restricted water regime applied on our treated plants showed mild, non-significant effects on chlorophyll a and b content, which indicates a weak impact of the water-shortage stress regime on the photosynthesis process. however, the cultivar aleppo 118 showed a slight reduction in chlorophyll a/b ratio in treated plants compared to a larger reduction in treated plants of ‘deir al-zour 22’, which indicate the presence of higher chlorophyll b content in treated ‘deir al-zour 22’ plants. this in turn may suggest a slower degradation process of chl b coming from a decrease in the enzymatic activities that are known to render the conversion of chl b to chl a (folly and engel, 1999). it is suggested that drought stress has to be prolonged and severe before the chloroplast start to break down (jiang et al., 2010). another non-significant drop was also observed in the osmotic potential values of treated plants in both cultivars. however, the cultivar aleppo 118 showed a greater osmotic adjustment value which may indicate its stronger turgor maintenance mechanism. osmotic adjustment has been also associated with maintenance of membranes and protein structure, protection against oxidative damage (dacosta and huang, 2006). it has been reported that the greater the stress duration (increasing the number of stress cycles), the larger the osmotic adjustfig. 8 fold expression of dreb1a, tps and hspcb genes in leaves of treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. leaf samples were collected in two stages, s1 (during floral bud to flower transition stage) and s2 (bolls opening and maturation). data were subjected to duncan’s test with a confidence level of 95% using statistica program. significant fold expression change of tps and hspcb genes was observed between s1 and s2 treated ‘deir al-zour 22’. data represent means and standard deviation of three replications. adv. hort. sci., 2018 32(1): 79-92 88 ment in cotton leaves (oosterhuis and wullschleger, 1987). the morphology and phenology of plants can be significant indicators of drought tolerance. changes in plant growth rate and/or in flowering time are two common strategies that plants use to cope with drought (schmalenbach et al., 2014). the allotetraploid g. hirsutum l. species has a distinct growth manner in which the plant keeps a systematic morphological architecture (khan, 2003) that can be useful in monitoring growth and developmental changes under diverse environmental conditions (jawdat et al., 2012). in general, physiological processes are induced in plants under stress conditions to reduce the cellular damage and to alter developmental timing to complete their life cycle fittingly (yaish et al., 2011). stress-mediated flowering has been reported in several plant species; stresses like ultraviolet c, poor nutrition, low temperature and drought (wada and takeno, 2010). our results noted earliness in treated plants of both varieties and a tendency of ‘aleppo 118’ to bear higher percentage of flowers and floral buds toward the end of season. ‘aleppo 118’ has been reported to show excessive vegetative growth under excess water and nitrogen compared to other local cultivars (cra, personal communication). this may suggest that water status (excess and shortage) may hold back or trigger floral cues, and this needs to be investigated fully taking into consideration our result of the higher osmotic adjustment in ‘aleppo 118’ compared to ‘deir al-zour 22’. the small increase in stem height in treated plants of both cultivars can be explained that plants accumulate reserves in shoots and stems to cope with water stress (chavez et al., 2002). the water deficit regime, applied in our study through drip irrigation, encouraged the increase in number of secondary roots in treated plants of both cultivars on the expenses of a decrease in tap root length. fiber quality, which is a result of interactions between genetics and environment, controls fiber prices of cotton textile products (wang et al., 2014). the quest for cotton growers is the balance between fiber quality and quantity. achieving such a balance in cotton cultivation regions with growing water shortages is a main concern to the agricultural and industrial sectors. an escalating number of publications aim at understanding the mechanism of cotton fiber development and adaptation to abiotic stresses, such as drought, for the improvement of cotton fiber yields and quality (zhu et al., 2011; deeba et al., 2012; padmalatha et al., 2012; bowman et al., 2013; riaz et al., 2013; wang et al., 2013; zhang et al., 2013; sekmen et al., 2014; xie et al., 2015). cotton growth and yield are severely affected by excessive water-shortage stress, especially at critical growth stages such as the flowering phase (dağdelen et al., 2009). soil water was found to be correlated with fiber strength, elongation (johnson et al., 2002) and fiber maturity (davidonis et al., 2004). our results showed that two fiber properties, cohesion and micronaire, had no significant differences between control and treated plants of the two cultivars. it is with fiber cohesion, a property that causes materials to cling together, yarn spinning from staple fiber is possible (wakelyn, 2007). a previous study showed fiber cohesion stability of ‘deir al-zour 22’ between seasons and locations; whereas, ‘aleppo 118’ showed cohesion stability between seasons in one location (jawdat et al., 2012). as for fiber micronaire, it reflects fiber fineness and is a measure of internal fiber thickness and deposits of cellulose. it has a desired value range in the international market between 3.5 and 4.9 (jawdat et al., 2012). fiber micronaire, showed stability in both ‘aleppo 118’ and ‘deir al-zour 22’ between seasons and locations (jawdat et al., 2012). this indicates that environmental factors including water deficit have weak impact on the two fiber properties, cohesion and micronaire. deficit irrigation showed no significant effect on fiber length and strength in aleppo 118 cultivar. however, it had significant impact on the two fiber properties, causing increment in both strength and length of fiber in treated plants of deir al-zour 22 cultivar. fiber strength is an indicator of fiber resistance to stretching (wang et al., 2014) and is determined by environmental conditions and cultivar traits (zhou et al., 2011). it has been found that fiber strength is correlated with daily mean temperature during fiber development (hanson and ewing, 1956; ma et al., 2006; wang et al., 2009). this has been also observed on a previous study where a difference of 4-5°c showed a significant effect on fiber strength in both ‘aleppo 118’ and ‘deir al-zour 22’ (jawdat et al., 2012). however, this temperature difference did not affect fiber length in both cultivars (jawdat et al., 2012). this shows the impact of each of water deficit and temperature on fiber strength of ‘deir al-zour 22’ compared to the impact of only temperature on fiber strength of ‘aleppo 118’. fiber maturity can be defined as the relative wall thickness and wall development (wakelyn, 2007). in our work, we observed an increase in fiber maturity factor values in treated jawdat et al. cotton behaviour under water stress 89 plants compared to the controls, which suggests that water deficit did not affect the maturity of cotton fiber in a negative way and hence their quality as related to dye-ability and ease of processing. the ftir-atr spectra of the cotton fibers obtained from control and treated aleppo 118 and deir al-zour 22 cultivars showed similar spectra range except for few noticeable bands. this can be due to a reduction in surface water adsorption which leads to stronger fibers. the minimized accessibility of water molecules to the internal hydroxyl groups can be mostly due to the formation of cellulose macromolecules that induce a re-organization of cellulose and also increase the ordered cellulose (or crystalline) portion to produce a stronger fiber (liu, 2013) in deir al-zour 22 treated cultivar. results of xrd showed minor ci variation between fibers from control and treated deir al-zour 22. both tga and dsc showed no variation between fibers of treated and control plants in both cultivars. our work has also investigated gene fold change in expression of the transcription factor gene (dreb1a), the molecular chaperone gene (hspcb) (sotirios et al., 2006), and the trehalose biosynthesis gene (tps) (kosmas et al., 2006). the two genes hspcb and tps have been found in few studies to be expressed differently between drought-tolerant and drought-sensitive cotton genotypes. while the hspcb showed differential expression during the drought period in leaves of drought tolerant genotypes and not in the sensitive ones (nepomuceno et al., 2002; voloudakis et al., 2002), the tps gene was induced during the water stress period in both tolerant and sensitive cultivars (nepomuceno et al., 2002). interestingly, our study showed a significant reduction in hspcb and tps genes activity in leaves during bolls opening and maturation in both cultivars. whereas, dreb 1a gene kept a high active mode in leaves of treated ‘deir al-zour 22’ plants compared to ‘aleppo 118’ treated plants during that stage. dehydration-responsive element binding proteins (drebs) are transcription factors known to activate the expression of abiotic stress-responsive genes in divergent species via specific binding to the dehydration-responsive element/c-repeat (dre/crt) cis-acting element in promoters of target genes (mizoi et al., 2012). in arabidopsis, the overexpression of dreb1a revealed both freezing and dehydration tolerance in transgenic plants (liu et al., 1998). a better drought and salt tolerance was observed in transgenic wheat plants with dreb1 from glycine max (shiqing et al., 2005). the overexpression of oryza sativa dreb1 in rice transgenic plants has also showed improved tolerance to drought, salt and low temperature stresses (dubouzet et al., 2003; ito et al., 2006). a group of researchers found that dreb 1a was induced at a high level in pistils, three days after drought treatment which suggests its possible role as an early drought response regulator in the arabidopsis flower (su et al., 2013). our study adds to such findings and points to dreb 1a constant upregulation in leaves of ‘deir al-zour 22’ under drought stress, during flowering and boll maturation. our study has presented the responses of two accredited cotton cultivars, aleppo 118 and deir alzour 22 which have shown different behavior under water shortage. both cultivars have shown early flowering. however, a larger percentage of treated ‘aleppo 118’ plants kept flowering towards end of season compared to treated ‘deir al-zour 22’ plants. this may indicate a potential survival mechanism and fruiting cycle extension in aleppo 118 cultivar under water deficit regime, compared to the cultivar deir al-zour 22. aleppo 118 cultivar displayed more consistent fiber quality between control and treated plants, while ‘deir al-zour 22’ showed variation in fiber length and strength between control and treated plants. both cultivars kept consistent micronaire and cohesion under normal and water deficit conditions. two critical stages in cotton life cycle, flowering and boll development, were screened for hspcb, tps and dreb 1a gene activity using leaf material. results have demonstrated an increase in fold expression of these genes in the flowering stage of treated plants compared to the controls. a large regression in genes activity was noticed during boll development except for dreb1a gene in treated ‘deir al-zour 22’ plants. dreb1a gene showed continuous activation in the two stages and can be considered a candidate gene to support water deficit stress tolerance mechanism in this cultivar. the question of which cultivar is more droughttolerant than the other is to be deeply investigated in both 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by data-mining and expression pattern analysis. agri. sci. china, 10(4): 499-508. impaginato 61 adv. hort. sci., 2018 32(1): 61-69 doi: 10.13128/ahs-21860 extending vase life of cut rose (rosa hybrida l.) cv. bacara by essential oils m.r. salehi salmi (*), m. falehi hoseini, m. heidari, m.h. daneshvar department of horticulture science, khuzestan ramin agriculture and natural resources university, khuzestan, iran. key words: antioxidant, bacteria, bent-neck, hold solutions. abstract: recent studies showed that some essential oils functions as antibacterial compounds. in this study results showed treatment with essential oils promoted vase life of cut roses via decreasing bacteria number inside the stem. we investigated components in the hydrodistilled essential oils of bunium persicum bioss, mentha spicata l., thymus vulgaris l. and satureja hortensis l., as hold solutions, and their effects on relative fresh weight, water uptake, vase life, electrolyte leakage, anthocyanin content, soluble sugar content and number of bacteria at stem end of cut flowers of rose. gc-ms analysis of the extracted essential oil of b. persicum, m. spicata, th. vulgaris and s. hortensis l. led to the identification of 14, 20, 13 and 14 major compounds, respectively. in cut rose, the treatment containing the essential oils extended flower opening period longer than the control. the 200 µl l-1 essential oil of m. spicata treatment almost doubled the vase life of cut roses. hence these essential oils might be powerful, environmentally friendly substitutes for the chemical compounds currently added to vase waters to control bacterial content. 1. introduction rose (rosa hybrida l.) cut flowers play an important role in the florist trade (cairns et al., 2000). the cut flowers can have limited marketable value because they dehydrate during vase life as a result of decreased water uptake and the vase life is often very short. the cut flowers wilt and the floral axis becomes bent just under the flower head (bent neck). the appearance of such symptoms is considered to be caused by various factors such as bacteria, physiological responses of stems to cutting and air emboli. the development of this occlusion is correlated with the growth of bacteria at the cut surface and inside the stem (van doorn et al., 1989, 1990) and accumulation of bacteria in vase water shortens the vase life of cut rose flowers (de witte and van doorn, 1988). the addition of chemicals, including some trace elements such as silver nitrate, aluminum sulphate and 8-hydroxyquinoline sulphate into holding solutions has been tried with various accomplishments in efforts to prolong the vase life of cut roses (van doorn et al., 1990). the use of essential oils has recently become a common practice (*) corresponding author: mrsalehisalmi@gmail.com citation: salehi salmi m.r., falehi hoseini m., heidari m., daneshvar m.h., 2018 extending vase life of cut rose (rosa hybrida l.) cv. bacara by essential oils. adv. hort. sci., 32(1): 61-69 copyright: © 2018 salehi salmi m.r., falehi hoseini m., heidari m., daneshvar m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 october 2017 accepted for publication 11 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 61-69 62 because of strong antimicrobial activities against several pathogens. some essential oils of plants have been reported to be effective in extending the vase life of gerbera cut flowers (solgi et al., 2009). these natural organic substances have high levels of phenolic compounds such as carvacrol (sharififar et al., 2007). essential oils indicate a wide range of protective properties against disease states, oxidative stress, and microbial infection (botelho et al., 2007; yahyazadeh et al., 2008). and have reportedly been used for controlling plant diseases, particularly on fruits (yahyazadeh et al., 2008; ramezanian et al., 2016). however, there is little available information (solgi et al., 2009) on the use of essential oils for the control of microbial contaminations and extending the vase life of cut flowers. appropriate use of chemical fungicides during the postharvest could help minimize fungal infections; however, the use of chemical compounds is being discouraged for economic aims and because of growing concern about environment safety issues (wagacha and muthomi, 2008). in this study, an attempt has been made to investigate the protective role of carvacrol during vase life in ‘bacara’ cut-roses. this information will help to elucidate the best concentrations of these essential oils to be used in holding solutions to obtain remarkable beneficial results. 2. materials and methods extraction, analysis and antioxidant activity of essential oils the essential oils from fresh leaves and flowers of bunium persicum bioss, mentha spicata l., thymus vulgaris l. and satureja hortensis l. were extracted following hydro-distillation method. the leaves and flowers (100 g) were hydrodistilled in a clevenger apparatus with 400 ml of water. the extraction was carried out for 3.5 h. the extracted oils were collected from the graduated receiver. for the deletion of water traces from the oil, the extracts were dried over anhydrous na2so4 and were stored in sealed ampoule bottle in a refrigerator at 4°c until for analysis. gas chromatographic analysis was performed with agilent 6890a with helium as a carrier gas with a linear velocity of 30 cm/s on hp-5 column (30 m × 0.25 mm i.d, 0.25 µm film thickness). the oven was programmed to rise 50°c (3 min) isotherm, and then to 265°c at a rate of 5°c/min. injector and detector temperatures were 300°c and 265°c, respectively. the gc mass analysis was carried out on agilent 5975 equipped with a hp-5 column with the same characteristics as the one used in gc. unknown essential oil was recognized by comparing its gc retention time to that of known compounds and by comparison of its mass spectra either with identified compounds or available spectra in the literature. the antioxidant activity of essential oils was determined based on the dpph (1,1-diphenyl-2picrylhydrazyl) free radical scavenging capacity by the method described by brand-williams et al. (1995). the amount of 180 µl dpph reagent and 20 µl samples were mixed and shocked. decline of absorbance of tested fusions was monitored for 30 min at 517 nm. for each essential oil, the tests were repeated three times. plant material and general processing rose flowers (rosa hybrida l.) ‘bacara’ grown under standard commercial conditions in a glass covered greenhouse were used as plant material. flowers were harvested at commercial stage to 0.45 m in length. flower stems were placed in tap water after harvest and transported by non-refrigerated car to the laboratory within 2 h. the flowers were placed randomly in 3 l glass vases containing 1 l of essential oils solutions of 100, 200 and 400 µl.l-1 concentrations for 24 h. distilled water was used as control. sucrose (4%, w/v) was added to all solutions and the concentrations of essential oils were prepared in tween 20 (0.1%). then, flowers were individually placed in glass bottles of 25 cm height. each bottle contained approximately 200 ml of distilled water. bottles were standing in a controlled room under the following conditions: 12 h photoperiod (06.00-18.00) at photosynthetically activated radiation of 12 µmol m-2 s-1 provided by fluorescent lamps, 25±1°c and relative humidity of 60-70%. in total, nine floin three replications were placed in bottles. measurements relative fresh weight was calculated by the formula: rfw (% initial fresh weight)= (fwt/fwt=0)×100 where rfw was relative fresh weight, fwt was the fresh weight of flower (g) at t (in day) = 0, 1, 2, 3, etc., and fwt=0 was the fresh weight of the same flower (g) at t (in day) = 0 (he et al., 2006). total vase solution uptake. weight of vases containing vase solution without the cut stems were measured during the experiment period and flower opening was measured daily until the flowers salehi salmi et al. effect of essential oils on vase life of cut rose 63 attained their largest size in the vase and began loss their size. cut flower longevity was documented as days vase life from the stage flowers were placed into glass bottles till the end of vase life defined as the time that flowers showed indications of petal wilting or curling, fall of one or more petals (van der sman et al., 1996). electrolyte leakage was measured using an electrical conductivity meter. leaves were excised and washed with deionized water. after drying with tissue paper, 1 g fresh weight of leaves were cut into small pieces (about 1 cm2) and then immersed in 20 ml deionized water and incubated at 25°c. after 24 h, electrical conductivity (ec1) and after 48 h (ec2) of the bathing solution were recorded for the samples (lutts et al., 1996). determination of anthocyanins. samples of petal (100 mg) were powdered in a pre-chilled pestle and extracted into methanol: hcl (99:1). samples were incubated overnight at 4°c in darkness. the content of anthocyanins determined spectrophotometrically at 535 nm using an extinction coefficient (ε) of 29,600. the final concentration of anthocyanin was calculated based on weight of sample used and total volume of the extract. for determination of soluble sugars, fresh petals (0.30 g) were put into test tubes with 10 ml distilled water and sealed. the tubes were incubated in a water bath at 90°c for 30 min, then the tubes were removed and the volume set at 25 ml. the amount of 0.5 ml of supernatant was mixed with 1.5 ml distilled water, 5 ml concentrated sulfuric acid and 0.5 ml anthrone. the mixed solution was read at 620 nm (frohlich and kutscherah, 1995). ascorbate peroxidase activity. the fresh leaves (1 g) were mixed in 100 mm potassium phosphate buffer (ph 7.8) containing 1% (w:v) pvp (polyvinylpyrrolidone), 0.1 mm edta (ethylenediaminetetraacetic acid), and 0.5% (v:v) triton x-100 at 4°c, except that in the case of apx activity leaves were homogenized in 100 mm sodium phosphate buffer (ph 7.0) containing 1 mm edta and 5 mm ascorbate. the homogenate was filtered through 4 layers of cheesecloth and centrifuged at 18000 g for 20 min at 4°c. peroxidase activity was assayed as per the method of kar and mishra (1976). determination of bacterial numbers. the lowermost 2 cm (about 0.5 g in weight) of the stems were cut. the samples were washed 3 times with sterile deionized water. they were ground and then dilution was made with a 0.9% normal salt solution. liquid extract (0.1 ml) was spread on plate count agar (pca) plates. before counting of bacteria, pca plates were incubated at 38°c for 2 days (balestra et al., 2005). statistical analysis completely randomized experiment designs were used. statistical significance between mean values was assessed using analysis of variance (anova) and a conventional tukey’s test at p≤0.05 using sas (9.1) statistical software. for data of maximum flower opening, standard deviation (sd) was calculated and data are expressed in mean±sd of three replicates. 3. results and discussion gc-ms analysis of the extracted essential oil of b. persicum, m. spicata, th. vulgaris and s. hortensis l. led to the identification of 14, 20, 13 and 14 major compounds, respectively (table 1). the results revealed that 5 major components of essential oil from b. persicum contained γ-terpinene, βcaryophyllene, cuminyl acetate, p-cymene and βpinene. investigation is limited about the chemical composition of the b. persicum essential oil. it has been reported that γ-terpinene and β-caryophyllene are main components (shahsavari et al., 2008). five major components of mentha spicata l. essential oil were carvone, 1,8-cineole, borneol, limonene and pulegone. mentha essential oil was characterized by the dominant presence of carvone (56.9%) in agreement with results from other authors, who reported the presence of 76.65% in samples collected from india (chauhan et al., 2009) and 64.4% in samples collected in montenegro (scherer et al., 2013). carvacrol (67.3%), thymol (12.7%), α-pinene (4.25%), γ-terpinene (3.53%) and eucalyptol (3.32%) were most abundant in essential oil of th. vulgaris. similar results have been obtained by ben el hadj ali et al., (2014) with th. numidicus. the principle compounds identified in essential oil of satureja hortensis l. were carvacrol (66.4%), p-cymene (18.1%), linalool (4.5%), γ-terpinene (3.95%) and borneol (1.8%). a comparison of our study with a previous report (ghasemipirbalouti et al., 2014) suggests that the few differences in the volatile composition of the plant material could be attributed to a series of factors such as the genotype, plant developmental stage, environmental conditions and the methods of extraction. all assayed essential oils were able to reduce adv. hort. sci., 2018 32(1): 61-69 64 dpph, reaching 50% of reduction with ic50 values ranging 54.19±0.87 mg/ml for m. spicata, to 258.16 ± 1.53 mg/ml for s. hortensis (table 2). the variance analysis performed on the dpph scavenging activity of the essential oils showed significant differences among species (p≤0.05) and that the essential oil of m. spicata was the most potent of all the oils. therefore, some compounds such as compound carvone, 1,8-cineole and borneol were responsible for the dpph scavenging effects of m. spicata (mahdavikia and saharkhiz, 2015). the essential oil of th. vulgaris possessed slightly higher dpph scavenging effects than that of b. persicum and s. hortensis. this meant that the common compound thymol and high value of carvacrol played a leading role (ghasemi-pirbalouti and dadfar, 2013). bacterial numbers essential oils treatment, particularly 200, 400 µl l-1 m. spicata and 200, 400 µl.l-1 th. vulgaris, had negative effect on bacterial numbers. preventing bacterial division in vase water can reduce the occurrence of stem bending (solgi et al., 2009), suggesting that bacteria are a main cause. a positive correlation between the number of bacteria and water uptake of the flower stem have been reported (van doorn et al., 1989). vascular occlusions in cut rose flower usually develop when the number of bacteria in vase water reach 7-11 log10 cfu/ml (van doorn et al., 1990). some antimicrobial compounds have been used in tests with cut flowers. these compounds included silver nitrate (nair et al., 2003), nano-silver (nazemi rafi and ramezanian, 2013), 8-hydroxtable 1 major natural volatile components in the hydrodistilled essential oils of b. persicum, m. spicata, th. vulgaris and s. hortensis no. component retention indices percentage (%) b. persicum m. spicata th. vulgaris s. hortensis 1 α-thujene 924 0.05 0.03 0.18 0.3 2 α-pinene 932 1.75 1.09 4.25 0.5 3 camphene 946 0.56 4 sabinene 971 0.8 0.74 5 β-pinene 977 3.68 1.59 0.44 0.1 6 myrcene 989 0.71 0.41 0.86 0.6 7 3-octanol 922 0.21 0.1 8 eucalyptol 1018 3.32 9 p-cymene 1021 6.91 0.83 18.1 10 limonene 1036 3.28 5.69 0.44 11 1,8-cineole 1039 0.1 13.53 0.2 12 γ-terpinene 1059 27.61 0.58 3.53 3.95 13 linalool 1086 1.1 0.71 4.5 14 menthone 1154 0.26 15 borneol 1159 8.15 1.8 16 α-terpineol 1169 0.98 3.25 0.54 1.02 17 pulegone 1231 0.2 3.28 18 carvone 1237 56.94 19 thymol 1287 3.4 0.67 12.79 0.3 20 carvacrol 1293 0.35 0.47 67.36 66.46 21 β-caryophyllene 1415 25.1 0.93 0.85 22 cuminyl acetate 1434 16.68 23 caryophyllene oxide 1580 0.47 0.16 1.23 1.2 mean separation by tukey test, p≤0.05. table 2 antioxidant activity of essential oil extracts of b. persicum, m. spicata, th. vulgaris and s. hortensis dpph b. persicum m. spicata th. vulgaris s. hortensis ic 50 (mg/ml) 183.5 ± 2.15 b 54.19 ± 0.87 d 153.52 ± 2.66 c 258.16 ± 1.53 a salehi salmi et al. effect of essential oils on vase life of cut rose 65 yquinoline citrate (solgi et al., 2009), chlorine bleach, dichloroisocyanuric acid (jones and hill, 1993) and essential oils such as carvacrol (nazemi rafi and ramezanian, 2013) and thymol (solgi et al., 2009). the inhibitory effect of some essential oils on bacterial growth has been attributed to alcohols, esters, aldehydes, phenols, carvacrol, thymol, and eugenol (bassole and juliani, 2012). burt (2004) reported that essential oil components including thymol and carvacrol partition in the lipids of the cell membrane, rendering the membrane permeable and leading to leakage of cell contents, thus exerting their antibacterial action. relative fresh weight and total water uptake as expected, relative fresh weight continuously increased early days of experiment (table 3). nevertheless, such increases were significantly prolonged by using essential oils in hold solution. the relative fresh weight in 200 µl.l-1 m. spicata essential oils treated cut flowers was initially about 105.2% (day 1), and increased to 130.1% over 6 days of holding, but in untreated (control) cut flowers, relative fresh weight was initially about 111.3% (day 1), and decreased over 4 days of holding to 105%. similarly, as with different concentrations of m. spicata essential oil, a positive effect of other essential oils on relative fresh weight was confirmed by their impact on duration of increase and percentage of fresh weight. total water uptake of cut flowers under various treatments from day 1 to 8 in control as well as in all treated samples (table 3). the maximum uptake of vase solution was found 1.14-fold increase in the treated flower with 200 µl.l-1 th. vulgaris as compared to untreated (control) cut flowers. all treated flowers always took up water more than the control. water status is a factor directly correlated with the vase life of rose cut flower. this factor is determined by the balance between water uptake and loss due to transpiration (fanourakis et al., 2016). water uptake depends on variation of cultivars (fanourakis et al., 2016), the viscosity of the vase solution, vascular conductivity and the osmosis gradient between vase solution and stem solute (alaey et al., 2011). the improved water uptake in this study may be due to possible antibacterial activity of essential oils by inhibiting vascular blockage and/or increasing osmosis gradient. the water lose was affected by treatments and essential oils increased water retaining capacity compared to control treatment (without essential oil). it may be due to positive regulatory role of essential oils on stomatal closure which regulates the rates of transpiration and decreases the water loss of leaves and petals (solgi et al., 2009). flower opening generally, the opening of cut flowers kept in essential oils vase solutions was more than those flowers kept in distilled water (control). also maximum opening of control flowers occurred earlier. time and diameters of maximum opening flower were different between treatments. however, maxitable 3 vase life, total solution uptake, electrolyte leakage, petal anthocyanin content, soluble sugars of petal and bacterial numbers of cut rose (rosa hybrida l.) cv. bacara treated with b. persicum, m. spicata, th. vulgaris and s. hortensis mean separation for each parameter within rows by tukey test, p≤0.05. characteristic vase life (days) total solution uptake (g) electrolyte leakage (%) petal anthocyanins content (mg g-1 fw) soluble sugars of petal (mg g-1 fw) ascorbate peroxidase activity (units min-1 mg-1 protein) bacterial numbers (log10 cfu/ml) relative fw in 10th day (%) control 6.70 e 98.350 f 60.6 a 5.24 d 1.41 f 22.6 h 8.2 a 79.41 g b. persicum 100 µl.l-1 8.70 bc 103.25 e 51.4 b 6.52 bc 1.70 f 25.1 g 8.1 a 94.72 cd b. persicum 200 µl.l-1 9.80 b 106.44 b 48.5 bc 7.37 ab 2.29 e 24.6 gh 7.8 ab 100.80 ab b. persicum 400 µl.l-1 7.90 cd 100.23 e 45.7 c 6.07 c 2.14 e 22.9 h 7.3 d 84.22 f m. spicata 100 µl.l-1 11.2 a 110.31 a 19.8 i 7.95 a 4.52 b 54.9 a 6.1 f 104.50 a m. spicata 200 µl.l-1 10.4 ab 108.65 ab 28.3 g 7.68 a 4.05 bc 46.7 b 5.7 g 95.00 c m. spicata 400 µl.l-1 8.70 bc 106.71 b 33.1 f 6.03 c 3.77 c 43.3 bc 5.5 gh 91.48 de th. vulgaris 100 µl.l-1 8.30 c 105.94 c 22.4 h 7.19 b 4.35 b 38.6 d 7.5 cd 91.71 de th. vulgaris 200 µl.l-1 10.6 ab 112.67 a 17.4 j 7.70 a 5.18 a 40.8 cd 5.5 gh 104.50 a th. vulgaris 400 µl.l-1 9.20 b 107.36 b 32.0 f 7.36 ab 3.95 c 41.0 c 5.3 h 98.54 bc s. hortensis 100 µl.l-1 7.80 d 98.670 f 41.5 d 5.83 c 2.35 e 33.7 ef 7.8 bc 83.54 fg s. hortensis 200 µl.l-1 8.60 bc 103.68 de 38.5 e 6.44 bc 3.28 d 32.1 f 7.2 d 87.83 ef s. hortensis 400 µl.l-1 9.10 bc 104.86 d 37.5 e 6.87 b 3.46 cd 35.9 e 6.8 e 97.87 bc adv. hort. sci., 2018 32(1): 61-69 66 mum diameter was observed with flowers kept in 100 µl.l-1 m. spicata essential oil vase solution (fig. 1). mechanisms of flower opening vary for different flowers and are sensitive to various environmental conditions such as temperature, light, carbohydrate supply (kumar et al., 2008) and water relations. during the opening, numerous proceedings take place in a well-defined sequence, representing all parts of plant development, such as division, differentiation and elongation of cell (kumar et al., 2008) or gene expression (hoeberichts et al., 2005). in an experiment, essential oils at some concentrations had a noticeable effect on promoting flower opening of cut rose flowers. however, the treatments with a relatively higher concentration of essential oils showed a less effect on flower opening. according to mahdavikia and saharkhiz (2015) the function of essential oils as an effective antioxidant mainly depends on its concentration. it has been demonstrated that a relatively high dose of essential oils may be associated with injury of membranes and nucleic acids and cell death (ghasemi-pirbalouti and dadfar, 2013). the results suggest that essential oils at effective concentrations maintained water uptake and inhibited vascular occlusions development. thus, essential oils may improve water relations and supplementation of carbon sources, leading to cell expansion and flower opening. petal anthocyanin content petal anthocyanin content in cut flowers was determined. cut flowers treated with 100 µl.l-1 m. spicata essential oil in hold solution were observed to accumulate the highest rates of anthocyanin in the petals. other treatments led to higher amounts of anthocyanins compared to untreated (control) cut flowers. generally, all cut flowers treated with essential oils showed a similar tendency to maintain anthocyanin petal, however, essential oils of th. vulgaris and m. spicata were more efficient than b. persicum and s. hortensis. one of plant defense system is the presence of endogenous antioxidative compounds such as anthocyanins (apel and hirt, 2004). singlet oxygen produced can be detoxified by anthocyanins and affect the vase life. accumulation of these pigments in cell vacuoles, their hue and intensity depend on external conditions. anthocyanin biosynthesis is an essential part of flower development (dela et al., 2003). in the primary developmental phases of the flower, the anther produces a signal or ga, which promotes petal pigmentation (kumar et al., 2008). so, in this study, flower opening and development could be effect on anthocyanins accumulation. additionally, earlier studies have indicated that sugars such as sucrose are required as substrates for anthocyanin biosynthesis (nagira and ozeki, 2004). here, we showed that essential oils increased petal soluble sugar content. it seems that there is an interaction between the soluble sugars concentration and anthocyanins. ascorbate peroxidase activity with cut roses, the highest ascorbate peroxidase activity in petals occurred in those treated with m. spicata essential oils at the concentration of 100 µl l-1. as shown in table 3, the activity of ascorbate peroxidase in control flowers was less than that of the flowers treated with essential oils, however, there were no significant difference between control flowers and flowers treated with different concentrations of b. persicum essential oils. ascorbate peroxidase enzyme is considered to play significant roles in cellular defense against stresses (sevillano et al., 2009). during the later stages of vase life, petals contain higher levels of reactive oxygen species leading to oxidative damage (hossain et al., 2006). generally, treatment with essential oils increased ascorbate peroxidase activity compared the control. higher levels of the enzymatic activity in treated flowers were likely to counteract the oxidative stress and to scavenge the active oxygen species alleviate to the oxidative stress induced in cut roses and thus delayed flower senescence. these results are in agreement with the findings of hasan et al., (2014) who mentioned that cut roses were of an efficient antioxidant defense system. electrolyte leakage the ability to increase membrane stability is a characteristic of drought tolerance strategy (turner, 1986). with control flowers, a significant increase in fig. 1 time of maximum flower opening. values are given as mean±sd of three replicates. salehi salmi et al. effect of essential oils on vase life of cut rose 67 electrolyte leakage was detected; however, treatment with any essential oils level significantly inhibited electrolyte leakage increase relative to the control (table 3). the treatment with 200 µl l-1 th. vulgaris essential oils resulted in the lowest electrolyte leakage followed by 100 µl l-1 m. spicata essential oils. wilting in cut flower is often accompanied by membrane damage resulting in the leakage of solutes (ye et al., 2000). with studied cut flower, such as day lily (panavas et al., 1998), the most changes in the rate of electrolyte leakage were observed during the late stage of vase life. maintenance of membrane stability in response to essential oils application was most likely due to induced reduction of lipid peroxidation. this is supported by a lower level of electrolyte leakage in essential oil treatments. soluble sugars another factor controlling vase life of cut rose flower is sugar content, as the carbon supply is cut (halevy and mayak, 1979). the essential oils had a strong influence on the soluble sugars concentration in the petal of cut flowers and all treatments led to significantly more soluble sugars. soluble sugars increased approximately 3.6 fold from 1.41 mg g-1 fw for the control to 5.18 mg g-1 fw for pulsing flowers in solutions containing essential oil of th. vulgaris (200 µl l-1) and to 4.52 mg g-1 fw using 100 µl l-1 m. spicata essential oil. with cut flowers treated with preservative solution, minimum soluble sugar contents were observed in flowers kept in solutions containing 100 µl l-1 b. persicum essential oils. it seems that essential oils can change the capacity for sugar uptake in petals and stimulate active sucrose uptake. translocation of soluble sugars is considered as a key factor affecting the vase life (khayat and zieslin, 1989). the addition of chemicals including some componds such as silver nitrate, aluminum sulphate and 8-hydroxyquinoline sulphate to holding solutions has been tried with varied success in efforts to control vascular blockage and prolong the vase life of cut roses (van doorn et al., 1990). however, there is no available evidence on the use of essential oils for control of microbial contaminations and holding soluble sugars petal of cut flowers such as rose. vase life cut flower wilting is a widely reported problem during vase life of rose, particularly when xylem vessels are blocked by microorganisms (damunupola and joyce, 2008). in this study, longevity of treated cut flowers was significantly improved compared with the untreated (control) cut flower. also, significant differences were found in longevity conferred by the essential oils of species and concentrations. however, b. persicum, m. spicata and th. vulgaris decreased vase life at concentrations more than 200 µl l-1, 100 µl l-1 and 200 µl l-1, respectively. the treatment with m. spicata 200 µl l-1 essential oil almost doubled the vase life of cut roses. proper doses of some essential oils at proper dose could delay senescence in selected fruits (ramezanian et al., 2016) and cut flowers (solgi et al., 2009). for example, solgi et al. (2009) concluded that the vase life of gerbera flowers was extended by the 50 or 100 mg l-1 carvacrol from 8.3 to 16 days. in the present study, similar results was obtained with the vase life of cut rose. 4. conclusions the naturally occurring compounds in essential oils, such as thymol, carvone, carvacrol and menthol showed different levels of antibacterial activity. the major qualitative trait of cut roses is their vase life, which mainly depends on the water uptake of the stems after harvest and is hampered by the presence of bacteria. also, at relatively high concentration of the essential oils tested, early flower opening and senescence were related to an effect other than bacteria. this effect was correlated with low relative fresh weight and water uptake. it cannot be excluded at present that this early senescence might be due to a toxic effect. more information regarding the action of essential oils during vase life is required for better understanding of the mechanism of 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glycol, sucrose, wild species. abstract: for the first time components of the nutrient medium were identified for the successful germination of pollen in such ornamental species of the linum genus as l. grandiflorum, l. hirsutum, l. pubescens and l. thracicum. it was shown that the media with peg­2000 in concentrations of 20­30% in combination with boric acid and calcium chloride in the concentrations of 200 mg/l ensure germination of linum pollen up to 40­50%. the addition of sucrose and peg with higher molecular weight adversely affects the germination of pollen. this will allow anyone to evaluate the quality of flax pollen quickly and efficiently and more successfully implement many genetic and breeding programs. 1. introduction the linum genus has between 100 and 230 species with the main rep­ resentative of linum usitatissimum l., which is one of the oldest cultivated plants, whose products have long been used for a variety of human needs. not less diverse is the use of wild flaxes (jhala et al., 2008; lyakh and soroka, 2008). moreover, some annual and perennial wild relatives of the linum genus, such as l. grandiflorum, l. pubescens, l. hir‐ sutum, possessing fairly large flowers of various colors, are highly ornamental plants and are successfully applied in landscaping, flower bed arrangement, and gardening (lyakh, 2013). a number of wild linum species is a producer of valuable substances for the pharmaceutical industry (mohammed et al., 2009). ushijima et al. (2012) reported that many wild flax species exhibit distinct floral polymorphism, which allows them to be used for elucidation the mechanisms of such a phenomenon as heterostyly. wild flax relatives are also actively involved in phylogenetic analysis of the linum genus (muravenko et al., 2009; sveinsson et al., 2014). (*) corresponding author: lyakh@iname.com citation: lyakh v.a., soroka a.i., 2021 ­ artificial medium for in vitro pollen germination of some ornamen‐ tal linum species. ­ adv. hort. sci., 35(3): 329­332. copyright: © 2021 lyakh v.a., soroka a.i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 may 2021 accepted for publication 21 august 2021 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-10854 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 329­332 330 a broad use of wild flax species and active breed­ ing work with them provides for knowledge of the quality of pollen, produced by the plant. the ability of pollen to grow and germinate on an artificial medi­ um allows estimating its quality fast and effectively (jayaprakash, 2018). in flax, separate attempts were made to germi­ nate pollen in vitro by pandey and kumar (2013). however, for the pollen of both cultivated flax and its wild relatives, the medium, which ensures the emer­ gence of properly­shaped pollen tubes during pollen germination, has not yet been developed. in this respect the purpose of this work was to develop a nutrient medium suitable for the germination of pollen from a number of ornamental flax species. 2. materials and methods wild species l. grandiflorum desf., l. hirsutum l., l. pubescens banks and solander and l. thracicum degen were used in our studies as pollen sources. experiments were carried out during 2017­2018. the medium containing boric acid and calcium chloride in the concentrations of 200 mg/l was used as a basic one. a medium, consisting of boric acid, calcium chloride and sucrose as osmotic agent is commonly used for pollen germination of different species. we, however, excluded sucrose as according to our preliminary experiments with linum species it inhibited completely pollen germination. polyethylene glycol (peg) of various molecular weights was supplied to the basic media (boric acid and calcium chloride) as osmotic agent. in some cases we also used sucrose as an addition to peg. the following additions to the basic medium have been made: (a) peg 2000, 20%; (b) peg 2000, 30%; (c) peg 2000, 30% + sucrose, 5%; (d) peg 2000, 30% + sucrose, 15%; (f) peg 6000, 30% + sucrose, 5%; (g) peg 20000, 5% + sucrose, 15%. pollen was collected from 20­40 flowers and ger­ minated for 3­4 hours in a drop of an artificial medi­ um placed on a slide at the temperature of 25±1 °c in the dark. the pollen was then viewed under a light leica microscope (germany) with a 20x objective. pollen grains were counted as germinated if the pollen tube length was more than a pollen grain diameter. in each 3­5 replication of each treatment several fields of view were analyzed to count from 300 to 400 pollen grains. pollen grains near the mar­ gin of the medium were not recorded. after that a mean value of pollen germination percentage and a standard error of the mean were calculated (lyakh and soroka, 2008). the results of the experiments were analyzed statistically applying a t­test, according to wasserman (2005). 3. results and discussion figure 1a shows that a medium containing peg­ 2000 as an osmotic in the concentration of 20% ensured a sufficiently good germination of the pollen for the species under study. the percentage of germinated pollen grains ranged from 22.9±2.44 in l. thracicum to 51.0±2.84 in l. grandiflorum. the elevation in concentration of peg­2000 from 20% to 30% did not reduce this indicator in all species, except l. hirsutum, where an increment in the pollen germination was observed. addition sucrose to the fig. 1 ­ influence of peg­2000 concentration and sucrose addi­ tion (a) and peg molecular weight (b) on pollen germina­ tion of some ornamental linum species: −−♦−− l. grandi‐ florum, −−■−− l. hirsutum, −−▲−− l. pubescens, −−x−− l. thracicum. lyakh and soroka ‐ in vitro pollen germination of linum species 331 nutrient medium with peg­2000 adversely affected pollen germination as a whole. elevating the sucrose concentration up to 15% in a medium with peg 30% reduced the number of germinated grains in all the species. figure 1b data grant an idea on the influence of peg with different molecular weight on pollen germination. as revealed, the percentage of pollen germination was significantly larger in the case of an osmotic with a lower molecular weight. it is characteristic that the number of germinated pollen grains on a medium supplied with peg­6000, compared to peg­2000, for l. grandiflorum and l. thracicum decreased by a factor of 6.6 and 20.5, whereas for l. pubescens and l. hirsutum ­ the reduction amounted to 34.0 and 24.1% only. figure 2 demonstrates pollen germination pattern on a media with peg of different molecular weight and sucrose, showing the proportion of pollen grains with normal and burst pollen tubes. it can be seen that the percentage of pollen grains with burst tubes both in l. grandiflorum and l. hirsutum on a medium containing a high molecular weight polyethylene glycol (peg­20000) at the concentration of 5%, against the background of 15% sucrose, was quite large. it is characteristic that for some species it even exceeded the number of pollen grains with normal tubes. when the medium included peg­20000 at the concentration of 30%, pollen of all the studied species failed to germinate. as can be seen, in our experiment sucrose negatively affected the germination of pollen when it was added to the media with peg. with sucrose concentration increasing, the number of germinated pollen grains decreased notably. at the same time there are successful examples in the literature of the joint use of sucrose and peg. thus, for sunflower a nutrient medium was developed suitable for pollen germination which simultaneously included 15% of sucrose and 30% of peg (keshava murthy et al., 1994). such medium was successfully used to evaluate pollen response of various sunflower genotypes to the action of low temperature while selecting pollen for cold resistance (lyakh and totsky, 2014). analyzing the pollen germination of ornamental flax species on the media with peg of different molecular weights, it is clearly noticeable that with an increase in the peg molecular weight the degree of reduction of the studied indicator was different for different species. it can be assumed that such a difference in pollen response is due to the different osmotic potential of the pollen grains considering the natural habitat and presence a number of xeromorphic traits in l. hirsutum and l. pubescens as apposed to l. grandiflorum and l. thracicum (tutin et al., 1968). pandey and kumar (2013) have investigated in fig. 2 ­ pollen germination of l. grandiflorum (1) and l. hirsutum (2) on the media with peg of different molecular weight and with sucrose: (a) peg­2000, 30% + sucrose, 5% (b) peg­6000, 30% + sucrose, 5%; (c) peg­20000, 5% + sucrose, 15%. http://www.thesaurus.com/browse/notably adv. hort. sci., 2021 35(3): 329­332 332 vitro pollen germination in l. usitatissimum on a medium containing as an osmotic only sucrose. however, despite the high ability of the pollen of this species to germinate under the given conditions, pollen grains emitted mostly pollen tubes with mal­ formed morphology. in our experiment pollen of ornamental flax species germinated well and devel­ oped normal pollen tubes on the media containing, in addition to the basic components, peg­2000 as an osmotic agent. 4. conclusions wild species of the genus linum are widely repre­ sented on the ornamental plant market. moreover, their relatives can be used as a source material for fiber and oil flax breeding. for genetic and breeding programs pollen quality assessment is an important, and often necessary, procedure for their successful implementation. in vitro germination of pollen on artificial nutrient media is the simplest, but at the same time reliable way to determine the pollen via­ bility. our studies have shown that pollen of some wild species of the genus linum germinates well on the media containing, in addition to boric acid and calcium chloride, an osmotic agent in the form of polyethylene glycol­2000. replacing this osmotic with polyethylene glycol of a higher molecular weight or adding sucrose significantly impairs pollen germina­ tion rates. the patterns revealed allow to propose the composition of an artificial nutrient medium for germinating flax pollen, which will ensure its better germination than is known from the available scien­ tific literature. references jayaprakash p., 2018 ­ pollen germination in vitro, pp. 81­96 ­ in: mokwala p.w. (ed.) pollination in plants. ­ intechopen, london, uk. jhala a.j., hall l.m., hall j.c., 2008 ­ potential hybridiza‐ tion of flax with weedy and wild relatives: an avenue for movement of engineered genes? ­ crop sci., 48: 825­840. keshava murthy m.n., nanja reddy y.a., virupak­ shappa k., 1994 ­ development of suitable germina‐ tion medium for trinucleate pollen grains: an illustra‐ tion with sunflower. ­ j. oilseeds res., 11(20): 304­307. lyakh v., 2013 ­ genetics of flower color in linum grandif‐ lorum desf. ­ indian j. genetics plant breeding, 73: 335­ 337. lyakh v.a., soroka a.i., 2008 ­ botanical and cytogenetic peculiarities and biotechnological techniques for work with linum l. genus species. ­ zaporozhye national university, zaporozhye, ukraine. lyakh v.a., totsky i.v., 2014 ­ selective elimination of gametes during pollen storage at low temperature as a way to improve the genetic structure of sporophytic population for cold tolerance. ­ helia, 37(61): 227­235. mohammed m.m.d., christensen l.p., ibrahim n.a., awad n.e., zeid i.f., pedersen e.b., 2009 ­ new acyla‐ ted flavone and cyanogenic glycosides from linum grandiflorum. ­ natural products research, 23: 489­ 497. muravenko o.v., yurkevich o.y., bolsheva n.l., samatadze t.e., nosova i.v., zelenina d.a., vol­ kov a.a., popov k.v., zelenin a.v., 2009 ­ comparison of genomes of eight species of sections linum and adenolinum from the genus linum based on chromosome banding, molecular markers and rapd analysis. ­ genetica, 135(2): 245­255. pandey s., kumar g., 2013 ­ hazardous effect of gamma‐ rays on in vitro pollen germination and pollen tube growth in linum usitatissimum l. ­ chromosome botany, 8: 31­34. sveinsson s., mcdill j., wong g.k.s., li j., li x., deyho­ los m.k., cronk q.c.b., 2014 ­ phylogenetic pinpoin‐ ting of a paleopolyploidy event within the flax genus (linum) using transcriptomics. ­ annals of botany, 113: 753­761. tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a., 1968 ­ flora europea. volume 2 rosaceae to umbelliferae. ­ university press, cambridge, uk, pp. 489. ushijima k., nakano r., bando m., shigezane y., ikeda k., namba y., kume s., kitabata t., mori h., kubo y., 2012 ­ isolation of the floral morph‐related genes in heterostylous flax (linum grandiflorum): the genetic polymorphism and the transcriptional and post‐tran‐ scriptional regulations of the s locus ­ the plant journal, 69: 317­331. wasserman l., 2005 ­ all of statistics: a concise course in statistical inference. ­ springer natura, new york, usa, pp. 442. impaginato 165 adv. hort. sci., 2017 31(3): 165-174 doi: 10.13128/ahs-21956 nuclear 2c dna and genome size analysis in somatic embryo regenerated gladiolus plants using flow cytometry a. mujib(*), m. ali, d. tonk, n. zafar cellular differentiation and molecular genetics section, department of botany, hamdard university, 110062 new delhi, india. key words: callus, embryo maturation and germination, genetic stability, pgr. abstract: gladiolus is a valuable asexually propagated plant of horticultural importance. here, in the present communication, in vitro embryogenesis protocol and 2c dna content of embryo regenerated plant are described. callus was first induced from corms on 2, 4-d and naa + bap amended ms medium and the frequency was maximum (75.12%) in 0.5 mg/l naa + 0.5 mg/l bap added medium. the callus differentiated into embryos on 2, 4-d at variable numbers (3.13-5.32/callus mass); the addition of 1.0 mg/l bap and 0.25 mg/l naa was found very efficient in proliferating embryos (7.99/callus mass). direct somatic embryos were also formed on corm surfaces on 2, 4-d (0.5-1.0 mg/l) amended medium at varying numbers. the embryos did not progress to maturity in same induction medium, so other pgr treatments containing ga3 and aba were added. the amendment of ga3 was more responsive compared to aba and 0.51.0 mg/l of ga3 was identified as best effective treatment. the embryos showed a maximum of 62.15% maturity in 0.5 mg/l ga3 added medium after 8th of culture. on bap containing medium the mature embryos converted into plantlets and highest germination (42.65%) was noticed on 0.5 mg/l bap added medium. the 2c dna content of regenerated plant was measured by flow cytometry and was noted to be 1.34 pg. the somatic embryo derived plantlets are true-to-type, stable and grew normally in outdoor conditions, genome size is identical to corm derived gladiolus plants. this is the first flow cytometric dna analysis description in somatic embryo regenerated gladiolus plant. 1. introduction ornamental is a major group of plants, comprises of dicot, monocot, gymnosperm, fern and other lower groups, grown in indoor-, outdoor decoration and for aesthetic values. these plants are mostly annual and perennials. a large number of ornamentals are used in cut-flower industry, some common examples are carnation, rose, dahlia, lilies, amaryllis etc. (sarkar, 2010). gladiolus, often called as sword lily, is a perennial bulbous flowering plant, belongs to the family iridaceae. it comprises of about 260 species, of which 250 are native to african continent and some (*) corresponding author: amujib3@yahoo.co.in citation: mujib a., ali m., tonk d., zafar n., 2017 nuclear 2c dna and genome size analysis in somatic embryo regenerated gladiolus plants using flow cytometry. adv. hort. sci., 31(3): 165174. copyright: © 2017 mujib a., ali m., tonk d., zafar n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 december 2016 accepted for publication 8 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 165-174 166 are to eurasia (ohri and khoshoo, 1986). beside its ornamental value, a few species are described to be wild, distributed in south scandinavians hill to the coast of mediterranean sea. the gladiolus imbricatus, one of the wilds, is known to be a rich source of vitamin c and minerals; the flowers are edible and its taste is very similar to lettuce (elena et al., 2012). the different species of gladioli are also used as food plants by lepidoptera larvae especially the large yellow underwing. gladiolus is primarily cultivated worldwide as cut flower because of its beautiful spike with moderate to long vase-life. the gladiolus flower spikes are long, one sided bearing variable flowers of attractive colours. the hybrid gladioli, in particular, demonstrate a wide range of colours that make the plant a success in cut flower market. the plant grow well in winter months, when temperature hovering around 10-25°c. the major gladiolus producing countries are holland, poland, bulgaria, romania, israel, australia, denmark and us; in us, the annual sale of gladiolus is estimated to be around 370 million corms (narain, 2004). in india, too, around 130.000 ha are under ornamental cultivation as the growth of floral market is high, upward and crossed 1000 crore mark, as per 2006 data (singh, 2009, 2011). the gladiolus is predominantly distributed in asia, south and tropical africa and in europe (goldblatt and manning, 1998). most species are diploid showing 2n = 30 chromosome number; a large number of species are tetraploid hybrids, exhibiting chromosome count of 60 (krahulcová, 2003; elena et al., 2012). the various species of gladiolus are propagated vegetatively by corms and cormlets separation, which is a very slow process while seeds are only used for raising new hybrid varieties (memon, 2012). beside conventional propagation, the gladiolus is often multiplied by tissue culture in which leaf, nodal segment, apical shoot tip, inflorescence and corm pieces are cultured in various nutrient media (xu et al., 2009). yelda and bengi (2007) obtained profuse callus from leaf on 5.0 mg/l naa added ms in dark condition. the ms amended with 1.0 mg/l bap + 0.5 mg/l kin + 0.5 mg/l naa was found very effective in producing callus from cormlet (pragya et al., 2012). in gladiolus, the plantlets are regenerated directly from cultured explants or plants arise de novo from callus following organogenesis and embryogenesis pathway (stefaniak, 1994). remotti (1995) obtained primary and secondary embryos in gladiolus x grandifloras cv. peter pears from cell suspension, derived from cormlet embryogenic callus on 0.25 µm bap or zeatin added ms medium. the incidences of somatic embryogenesis have also been reported in a number of other studies (iantcheva et al., 1999; wu et al., 2015) in which genetic transformation method was utilized to make the plant resistant to gray mold, dry rot, root rot and other plant diseases (massey, 1928). particle bombardment mediated genetic transformation was earlier attempted in improving traits such as shortening dormancy (kamo and joung, 2007; kamo et al., 2009). agrobacterium mediated genetic transformation was also tried but was noted to be less efficient as fast regeneration protocols from transformed tissues are not fully optimized, which is a primary requirement for successful transformation studies (wu et al., 2015). moreover, gladiolus is a member of monocot, the tissue of monocotyledonous plant is noted to be less responsive/receptive in up taking foreign dna during transformation mechanism (smith and hood, 1995), although there are some current exceptions (zhao et al., 2000; zhang et al., 2006). there are several factors, controlling the success of plant regeneration the explant, medium and plant growth regulators (pgrs). the somatic embryogenesis way of plant regeneration has several advantages such as lower risk of somaclonal variation and higher rates of plant regeneration (vergne et al., 2010; zhao et al., 2013) and thus has been successfully practised in a variety of research programmes of biotechnological interest. although in vitro embryogenesis has been reported in some specific gladiolus species, the plantlet recovery from embryos is still not too high. this is primarily because of non-synchronous embryo induction, and poor quality of embryos, preventing early maturation and subsequent germination in culture. embryo (developed on explants directly) derived plantlets are true-to-type; genetic fidelity has often been maintained in somaclones (jayanthi and mandal, 2001). however, plants obtained from callus-mediated embryos demonstrate genetic and phenotypic variability and this instability increases with older culture of callus (endemann et al., 2001). the accumulation of higher levels of stress and the presence of pgrs are considered the signalling elements in inducing embryos and subsequent genetic variability in tissue culture raised plants (clarindo et al., 2008; delporte et al., 2013). thus, there is an urge to check somaclones’ fidelity in regenerated populations. a large number of conventional and modern techniques have been used recently to identify alteration in cultivating tissues and in vitro raised plants (das et al., 2013). flow mujib et al. nuclear 2c dna and genome size analysis in somatic embryo regenerated gladiolus by flow cytometry 167 cytometry, an important technique has frequently been used for measuring nuclear dna content in plant cells and tissues (zhang et al., 2005). the technique offers simple, fast and precise way of determining 2c dna in plant cells. it has several other fundamental and applied uses including the initiative of genome sequencing of known and unknown plant materials (galbraith, 2009; rewers et al., 2012). in the present study, flow cytometric investigation was conducted to measure and compare the 2c dna level of somatic embryo regenerated plantlets with naturally grown gladiolus, which served as tissue source for in vitro study. the fast and large-scale plant regeneration was also described by studying the role of pgrs in developing callus and embryos and in the event of maturation and germination of embryo in gladiolus. 2. materials and methods plant material and cultural conditions the gladiolus corms (gladiolus hybridus) from horticulture society of india, calcutta, were used as experimental material. first, the healthy gladiolus corms were selected and washed with cetrimide (a liquid detergent) for 10-15 min, followed by washing with running tap water for 30 min. this was followed by a treatment with 70% ethanol for 5 min, rinsed with distilled water for 3-4 times. finally, the corms were surface sterilized with 0.05% hgcl2 for 3 min, and washed 3-4 times with sterilized distilled water to remove the traces of mercuric chloride. the gladiolus corms were sliced into explants (8-10 mm), and were cultured in ms (murashige and skoog, 1962) amended with different concentrations of 2, 4dichlorophenoxyacetic acid (2, 4-d), indole acetic acid (iaa) and 1-naphthaleneacetic acid (naa). the medium ph was adjusted to 5.7 before sterilization at 121°c. the cultures were kept at 25±2°c in a culture room under 16 h photoperiod provided by cool white fluorescent light of intensity of 40 wm-2 s-1. induction of callus and embryogenic tissue corm pieces were cultured on ms, amended with 3% sucrose, 100 mg/l inositol and 2, 4-d, iaa and naa at different concentrations (0.25, 0.5, 1.0 and 2.0 mg/l) for callus induction. bap (0.5 and 1.0 mg/l) was also used in combination with naa (0.5, 1.0 mg/l) in separate experiments. within 7-10 days, brown yellowish callus was formed from the cut end of corms, which later engulfed the whole surface. the same level of pgrs was also used for callus maintenance. the induced callus transformed into embryogenic callus, which showed small, globular or ‘nearly globular’ or elongated, cylindrical structures on callus surface. these embryo structures were visible with naked eye. the effectiveness of pgrs and their levels were optimized by scoring callus induction percentage. the ineffective pgr treatments were discontinued. somatic embryo differentiation and proliferation embryogenic callus (about 50 mg) was cultured on ms amended with 2, 4-d (0.5, 1.0 and 2.0 mg/l), naa (0.5, 1.0 and 2.0 mg/l) and bap (0.5, 1.0 and 2.0 mg/l) separately for embryo differentiation and proliferation. in other experiments, naa (0.25, 0.5 mg/l) in combination with bap (1.0, 2.0 mg/l) were also used. the embryo numbers increased with time and these cultures were maintained by regular sub-culturing at interval of 3-4 weeks. the callus showing embryogenesis (the percentage) and embryo numbers (number/callus mass) were recorded at periodic intervals. maturation of embryos the developed embryos on callus were placed on a medium, containing all the essential components of ms, vitamins, inositol but without any pgrs for maturation of embryos. in other experiments, the ms was additionally added with abscisic acid (aba) at 0.25, 0.5, 1.0 mg/l and gibberellic acid (ga3) at 0.25, 0.5, 1.0 mg/l. in both ga3 and aba added medium, the somatic embryos transformed into green and elongated structures (maturing embryos), which germinated into plantlets later. the percentage of embryos showing maturation was recorded. embryo germination and plant recovery apparently green somatic embryos were separated and cultured on medium, added with various concentrations of bap (0.5, 1.0, 2.0 mg/l) either alone or with naa (0.25, 0.5 mg/l). in another set of experiments, the embryos were cultured on maturation medium for germination in which best maturation had noticed. a separate control medium (without pgr) was also prepared to make the comparison. the number of somatic embryos germinated and developed into plantlets (root and epicotyl development) were recorded. as the germinated plants had very little roots, plants were incubated in root promoting pgrs like indole-3-butyric acid (iba). roots were formed at variable numbers soon after iba treatment. embryo regenerated plantlets were transferred to 250 ml conical flask containing ½ ms + 3% sucrose but without pgr and kept for a week for acclimatization. adv. hort. sci., 2017 31(3): 165-174 168 transplantation of plants finally, the plantlets were removed from the conical flasks, were transferred to small coffee cups filled with sterilized soil rite and sand (1:1), and wrapped with pored polythene bags for maintaining humidity. plants were later transplanted to plastic pots (15 cm), kept for 3 weeks in incubation at room temperature of 25±2°c with 16 h photoperiod, provided by cool white fluorescent illumination. finally, plants were transferred to plastic pots (15 cm) filled with soilrite and sand (1:1) in outdoor condition. 2c dna and flow cytometry corm derived gladiolus plants, grown naturally in garden and somatic embryo regenerated plants were used for 2c dna determination study. leaf samples from both sources were processed according to the dolezel et al. (2007) method. about 1.0 cm2 of gladiolus young leaf and pongamia pinnata, 2c= 2.51 pg dna (in which 2c dna analysis study was optimized in the laboratories of our university) were harvested the day of analysis and chopped in 0.5 ml otto i buffer (0.3% citric acid monohydrate, 0.05% np-40), 50 µm propidium iodide and 100 µm rnase (sigmaaldrich, usa) following choudhury et al. (2014) method. the homogenate was filtered through a 100-µm nylon filter, and was analyzed by a (cfm) bd facs calibur (bd biosciences, san jose, ca, usa) flow cytometer. the 2c nuclear dna level of this experimental material was determined as follows: statistical analysis the data on the effects of pgrs on callus induction, embryogenesis and embryo numbers were analysed and expressed as mean ± standard error. each set of experiments consisted of three replicates and each experiment was repeated twice. the flow cytometry experiment was performed by randomly selecting regenerated plants. the presented mean values were separated using duncan’s multiple range tests at p≤0.05. 3. results induction of callus and callus biomass growth corms were used as explant on various auxin amended ms medium; the explant started to swell and callus was formed (fig. 1 a) form the cut ends later. three different auxins and their concentrations were tested of which 1.0 mg/l 2, 4-d showed highest callus induction percentage (72.66%), followed by the treatment with 0.5 mg/l 2, 4-d, in which 67.85% of cultured explants callused (table 1). other two fig. 1 a) corm callus induced in 2,4-d added ms medium; b) numerous somatic embryos developed on callus; c) directly induced somatic embryos on corm surface and d) somatic embryo regenerated plantlets (bars: a-c: 2 mm; d: 0.5 cm). table 1 callus induction frequency in gladiolus. corm explants was cultured on ms medium, added with 2, 4-d, iaa, and naa alone and bap and naa in below indicated combinations 2,4-d (mg/l) iaa (mg/l) naa (mg/l) callus induction (%) 0.25 0.0 0.0 48.25±4.44 f 0.5 0.0 0.0 67.85±4.65 c 1.0 0.0 0.0 72.66±5.76 b 2.0 0.0 0.0 58.64±3.87 e 0.0 0.25 0.0 0.0 g 0.0 0.5 0.0 0.0 g 0.0 1.0 0.0 0.0 g 0.0 2.0 0.0 0.0 g 0.0 0.0 0.25 0.0 g 0.0 0.0 0.5 0.0 g 0.0 0.0 1.0 0.0 g 0.0 0.0 2.0 0.0 g bap (mg/l) 0.5 0.5 75.12±5.55 a 1.0 0.5 63.16±3.89 d 0.5 1.0 64.66±4.85 d values are expressed as means ± standard errors of three replicates of two experiments; within each column means followed by the same letter are not significantly different at p≤0.05 according to duncan’s multiple range test (dmrt). mujib et al. nuclear 2c dna and genome size analysis in somatic embryo regenerated gladiolus by flow cytometry 169 auxins i.e. naa and iaa were observed to be nonresponsive when used alone. with bap (0.5 mg/l), naa was very active in producing callus, maximum efficient concentration being 0.5 mg/l naa, followed by 1.0 mg/l naa. corm induced callus was cultured on a variety of 2,4-d added concentrations (0.25, 0.5, 1.0 and 2.0 mg/l) for fast growth of callus and the moderate level was observed to be very efficient in producing good callus biomass growth. at the end of 12th weeks culture, 1.12 g of callus biomass was observed in 1.0 mg/l 2,4-d added medium (fig. 2), next effective concentration was 0.5 mg/l 2,4-d in which callus biomass was 0.91 g, little more of biomass (0.84 g) recorded at 2.0 mg/l 2,4-d. direct and indirect somatic embryogenesis in order to study plant regeneration, hard compact callus was placed on a variety of pgr amended medium and within a few weeks of incubation, callus started to differentiate and produced somatic embryos at variable numbers (fig. 1 b). three different concentrations were tested, of which 1.0 mg/l 2, 4-d was observed to be very efficient in which 79.13 percentage embryogenesis with an average of 5.34 embryos/callus mass was noted. equally effective 2,4-d concentration was 0.5 mg/l that showed good embryo numbers and embryogenesis percentage (table 2). unlike 2,4-d, naa another important auxin was noted to be inactive in inducing embryos. bap, a member of cytokinin, was however, noted to be active in inducing embryo, although low in number with lower rate, most efficient concentration was 0.5 mg/l bap in which 15.45 embryogenesis percentage and 2.93 embryo numbers were noted. the addition of naa in bap amended medium improved embryo numbers and embryogenesis percentage. the best treatment identified was 0.5 mg/l naa and 2.0 mg/l bap, which showed 69.18 embryogenesis percentage with 12.13 embryo numbers; next best treatment was 0.25 mg/l naa and 1.0 mg/l bap in which a mean of 7.99 embryo number/callus mass was noticed. the numbers of embryos however, increased with time. beside callus induced embryos, direct embryo formation on corm surface (fig. 1 c) was also noticed at lower frequency. the embryos were roundish, oval, elliptical in structure, often had white cotyledonary apex with yellowish radicle primordia at other ends. somatic embryo maturation and germination, and the role of various pgrs embryos were cultured on medium amended with ga3 and aba at different concentrations for embryo maturation. the application of ga3 was more efficient in comparison with aba treatment (table 3). in 0.5 mg/l ga3 amended medium, 54.55% embryo maturation was noticed after 4 weeks, which increased to 62.15% after 8 of incubation. at the same level of aba (0.5 mg/l), the embryo maturation percentage was low (18.75%) and with culture time the maturation percentage marginally improved i.e. 20.15% was noted after 8th weeks. higher concentration of ga3 and aba reduced embryo maturation frequency quite sharply. well matured somatic embryos, grew well in maturation medium but the germination frequency was low. the best maturation conditions i.e. the medium amended with 0.5 mg/l ga3 and 0.5 mg/l aba fig. 2 callus biomass growth on ms medium, added with various concentrations of 2, 4-d. values are means ± standard deviation of three replicates. within each bar, means followed by the letter, are not significantly different at p≤0.05 according to dmrt. 2,4-d (mg/l) naa (mg/l) bap (mg/l) embryogenesis (%) embryo numbers/ callus mass 0 0.5 0 0.0 g 0.0 h 0 1 0 0.0 g 0.0 h 0 2 0 0.0 g 0.0 h 0 0 0.5 15.45±2.12 e 2.93±0.79 f 0 0 1 14.61±2.42 e 2.38±0.16 f 0 0 2 8.66±1.73 f 1.96±0.10 g 0 0.25 1 80.15±6.66 a 7.99±1.21 b 0 0.5 1 70.12±5.22 c 6.46±1.76 c 0 0.5 2 69.18±3.44 c 12.13±2.28 a 0.5 0 0 75.42±6.43 b 5.32±0.87 d 1 0 0 79.13±5.38 a 5.34±2.10 d 2 0 0 39.33±3.58 d 3.13±1.06 e table 2 embryogenesis percentage and somatic embryo numbers in gladiolus callus, ms was amended with below indicated pgr concentrations and combinations. data were scored after 4 weeks of culture values are expressed as means ± standard errors of three replicates of two experiments; within each column means followed by the same letter are not significantly different at p≤0.05 according to duncan’s multiple range test (dmrt). adv. hort. sci., 2017 31(3): 165-174 170 showed very poor level of embryo germination (table 4) i.e. 8.50 and 4.84% respectively. so other pgr combinations were tested for better and early plantlets recovery from somatic embryos. three different concentrations of bap were added in ms and the germination ability was observed (table 4). in 0.5 mg/l bap amended medium the maximum somatic embryo germination percentage (42.65%) was noticed, followed by a treatment with 1.0 mg/l bap (31.85%), while higher level (2.0 mg/l) declined somatic embryo germination (18.16%) rate. the optimized bap (0.5 mg/l) level with naa was also similarly tested to improve embryo germination and a moderate germination percentage (33.25-33.25%) was noted in those pgr treatments. the somatic embryo regenerated plantlets (fig. 1 d) grew well in culture, but did not have strong root systems always, so a medium amended with root promoting pgrs were added. although in almost all treatments, roots were formed at variable numbers, iba (0.5, 1.0, 2.0 mg/l) was observed to be very effective in inducing roots (data not shown). the plants were finally transferred to outdoor conditions. genome size analysis of gladiolus for the analysis of genome size, the 2c dna content of somatic embryo regenerated plantlets was measured and was compared with naturally grown gladiolus using flow cytometric method. nuclear homogenate of juvenile leaves from naturally grown and somatic embryo regenerated plants were used for flow cytometry and the obtained results are presented in figure 3. the somatic embryo regenerated plant of gladiolus showed 2c dna content of 1.34 pg while corm grown plant obtained naturally had 2c dna content of 1.29 pg. the 2c nuclear dna content values of both the two sources are nearly the same that suggests no major alteration in genome size in embryo regenerated plants when compared with corm derived gladiolus. the genetic stability is thus maintained in the somatic embryo regenerated population. table 3 influence of ga3 and aba on somatic embryo maturation in gladiolus. data were scored after 4th and 8th weeks of culture values are expressed as means ± standard errors of three replicates of two experiments; within each column means followed by the same letter are not significantly different at p≤0.05 according to duncan’s multiple range test (dmrt). ms+ pgr (mg/l) maturation after 4 weeks (%) maturation after 8 weeks (%) control 4.21 f 6.30 f ga 3 (mg/l) 0.25 30.65±2.11 c 38.60±3.33 c 0.5 54.55±3.88 a 62.15±4.87 a 1 42.62±2.66 b 48.62±4.88 b aba (mg/l) 0.25 12.15±1.86 e 16.65±2.11 e 0.5 18.75±2.01 d 20.15±1.89 d 1 13.12±1.12 e 15.34±1.78 e table 4 somatic embryo germination in various pgrs added medium in gladiolus. data were scored after 6 weeks of culture values are expressed as means ± standard errors of three replicates of two experiments; within each column means followed by the same letter are not significantly different at p≤0.05 according to duncan’s multiple range test (dmrt). ms+ pgr (mg/l) germination (%) control 0.0 g ga 3 (mg/l) 0.5 8.50±0.89 e 1 6.66±1.11 f aba (mg/l) 0.5 4.84±1.02 g 1 4.68±1.82 g bap (mg/l) 0.5 42.65±2.67 a 1 31.85±1.89 c 2 18.16±1.22 d bap nap 0.5 0.25 33.25±2.23 b 0.2 0.25 31.66±2.24 c fig. 3 histogram of relative 2c dna content of corm grown (field) and somatic embryo regenerated gladiolus with standard pongamia pinnata plant. mujib et al. nuclear 2c dna and genome size analysis in somatic embryo regenerated gladiolus by flow cytometry 171 4. discussion and conclusions in the present study, plant regeneration through somatic embryogenesis and 2c dna content of regenerated plant were investigated in gladiolus. callus was first induced from corm on 2, 4-d added ms in which low to moderate doses were observed to be very effective. the amendment of auxins especially 2, 4-d was found very active in inducing callus in a number of investigated plants including gladiolus (kamo and joung, 2007; su et al., 2009; feher, 2015). on 2, 4-d added medium, embryos were differentiated on callus, and thus the origin of embryos are indirect in gladiolus. this callus mediated embryo induction incidence has been noted in a number of investigated ornamentals of horticultural importance (jiang and zhang, 2007; gow et al., 2009).the process of in vitro embryogenesis is often controlled by a variety of pgrs and its analogues. the impact of 2, 4-d in inducing callus and somatic embryos are reported widely in literature (gaj, 2004; feher, 2015). at lower level, 2, 4-d activates yucca (yuc) gene, which helps in synthesizing iaa and its transport in developing embryos in cultivated tissues (su et al., 2009; bai et al., 2013). the application of 2, 4-d also induces stress in culture, which provokes vegetative cells to acquire embryogenic state (gliwicka et al., 2013), although the exact molecular mechanism influencing this transition is still not understood clearly. although the embryo formation was more frequent on 2, 4-d added medium, the addition of naa and bap at various levels was noted to be equally responsive in inducing callus and in promoting embryo numbers in gladiolus, this is in agreement with stefaniak (1994) findings where naa was identified to be more effective in inducing embryos compared to other auxins tested. the same level of naa with bap was earlier reported to be very effective in a number of bulbous/ tuberous ornamental monocots (mujib et al., 2006; 2008). similar use of naa with bap was noted to be very beneficial for shoot formation in gladiolus, both during direct organogenesis and callus mediated regeneration (ascough et al., 2009; memon, 2012). wu et al. (2015) obtained globular somatic embryos on embryogenic callus on 3.0 mg/l tdz and 0.2 mg/l ba amended ms without any requirement of naa. yelda and bengi (2007) earlier reported somatic embryo induction on 0.1 mg/l bap added ms medium, very similar to bap induced embryo induction noted in our observations. thus, for acquiring somatic embryo, the cytokinin signalling is also equally important, often sufficient as against common perception of auxins, which induce embryos in culture (mujib et al., 2016). cytokinin induced somatic embryo formation has been observed in a number of plants and was reported (iantcheva et al., 1999). although the molecular role of cytokinin in triggering in vitro embryogenesis is not fully elucidated, cytokinins induced wuschel (wus) gene expression and transcription factor synthesis at early embryogenesis and shoot apical meristem development time were reported in several studied organisms (su et al., 2009; gordon et al., 2009; wang and chong, 2016). the clear demarcation and establishment of shoot and root apical meristem are important steps in embryogenesis/somatic embryogenesis (scheres, 2007). although the role of auxin and auxin-cytokinin interaction in determining shoot apical meristem is known, cytokinin mediated root apical meristem development needs further investigation. su et al. (2012) however, indicated that cytokinin induces the synthesis of arabidopsis response regulator, arr7 and arr15, the two essential transcription factors, for root apical meristem development during somatic embryogenesis. induced embryos did not grow fast therefore other pgr treatments were added to improve embryo quality, which facilitates in vitro plant regeneration. in the present study, various concentrations of ga3 and aba were used, both influenced embryo maturation but the role of ga3 was more profound compared to aba. the observation is very similar to vieitez findings (1995) in which poor role of aba on embryo maturation was noted. aba induced improved embryo maturation was, however, reported in other groups of plants including model arabidopsis (maruyama et al., 2007; bai et al., 2013). in contrast, the incorporation of ga3 facilitated embryo maturation by growing embryo size and by synthesizing more chlorophyll, which help in building protein, lipid, triglycerides and other energy reserves necessary during seed germination (santos-mendoza et al., 2008). the combination of ga3 and aba has also been reported to activate leafy cotyledon, lec and fus3 gene expression, help making energy reserves essential for seed/embryo germination (braybook and harada, 2008). embryo maturation and germination steps are both very important in somatic embryo based plant propagation. mature somatic embryos were transferred to a medium primarily amended with bap or with naa for plantlet formation. within a few days, leaflets were coming out from coleoptiles and later roots were developed. the use of bap alone or with naa was earlier noted adv. hort. sci., 2017 31(3): 165-174 172 to be very effective for conversion of plantlets from somatic embryos in other groups of plants (mujib et al., 2013). the regenerated shoots were transferred to iba added medium for better induction of roots as the embryo derived shoots had with less developed roots. iba promptly induced roots in gladiolus, like many other investigated materials (mujib et al., 2008). in gladiolus, it appears therefore that various pgrs control in vitro embryogenesis and plant regeneration and their requirement varies considerably. the embryo forming ability from callus in gladiolus was high and reproducible, which could efficiently be exploited in developing transgenics. the plants obtained from callus through organogenesis and embryogenesis demonstrate genetic variation and these changes are identified by traditional and modern techniques (das et al., 2013). here, in g. hybridus, plants were developed from callus via somatic embryogenesis pathway; the callus and the embryos are influenced by pgrs and stresses, both affect cell cycle/dna synthesis. thus, a study of regenerated plant status was indeed necessary. we analysed 2c dna content in gladiolus plants, derived from corm and somatic embryo by flow cytometry. the level of 2c dna of somatic embryo regenerated and corm obtained plants are the same in gladiolus. the amount of nuclear dna was unchanged and the genetic fidelity was maintained. due to its precision, the flow cytometric technique has widely been used in genetic stability studies (2c dna analysis) in a number of investigated materials including in vitro regenerated plants (loureiro et al., 2007; sliwinska and thiem, 2007). the in vitro cultural condition, however, cause genetic irregularities in cultivating tissues (giorgetti et al., 2011); using flow cytometry this alteration can easily be identified (dolezel et al., 2007). despite of the large number of applications and its ease, the 2c-dna estimation has not been conducted widely, rather restricted to only about 2% of the flowering angiosperms (galbraith, 2009). thus, any attempt with obtained 2c value, even from in vitro raised culture, will significantly enrich c-value database in angiosperm. similarly, the development of embryogenesis protocols discussed here can also be used in preparing synthetic seeds 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biochar on melon plants (cucumis melo l.) under drought stress conditions s. bagheri 1, m.r. hassandokht 2 (*), a. mirsoleimani 3, a. mousavi 4 1 department of horticultural sciences, faculty of agriculture, tehran science and research branch university, tehran, iran. 2 department of horticultural sciences, faculty of agriculture, university of tehran, karaj, iran. 3 department of plant production, faculty of agriculture and natural resources of darab, shiraz university, shiraz, iran. 4 national institute of genetic engineering and biotechnology, tehran, iran. key words: macro­elements, melon, micro­elements, morphological traits, proline. abstract: in order to investigate the effect of palm leaf biochar on some charac­ teristics of cucumis melo l. under drought stress, a split plot experiment was conducted in a completely randomized block design with three replications for two consecutive years. the main plot was irrigation level (60, 85, and 100% water requirement) and subplot was biochar in four levels (0, 0.18, 0.24, and 0.36 kg/m2). results showed that treatment of 0.24 kg/m2 biochar and 100% water requirement increased the characteristics of water use efficiency as 88%, shoot fresh weight as 77%, shoot dry weight as 32%, root fresh weight as 100%, root dry weight as 84%, root length as 54%, and average fruit weight 84% com­ pared to treatment without biochar and 60% water requirement. the highest level of leaf n, mn and k, shoot length, leaf area, leaf number, fruit diameter and fruit flesh thickness in the treatment of 0.36 kg/m2 biochar and 100% water requirement were higher 58%, 48%, 65%, 18%, 50%, 95%, 43% and 55%, than to of treatment without biochar and 60% water requirement respectively and had no significant difference with the treatment of 0.24 kg/m2 biochar and 85% water requirement. the highest rates of fe, zn and cu were related to 0.36 kg/m2 biochar and 60% water requirement as 60, 44 and 66% respectively com­ pared to treatment without biochar and 100% water requirement. the biochar­ free treatment with 60% water requirement accounted for the highest amount of proline due to high stress, and the proline content reduced with increasing biochar and decreasing stress in treatments. generally, the treatments of 0.24 and 0.36 kg/m2 of biochar increased most of the characteristics, however no significant difference was observed between these treatments. moreover, in 85% water requirement the drought stress conditions could compensate with the application of biochar. thus, using 0.24 kg/m2 of biochar and 85% of water requirement, recommended for the best result. 1. introduction melon (cucumis melo l.) is from cucurbitaceae family that requires (*) corresponding author: mrhassan@ut.ac.ir citation: bagheri s., hassandokht m.r., mirsoleimani a., mousavi a., 2019 ­ effect of palm leaf bio‐ char on melon plants (cucumis melo l.) under drought stress conditions ­ adv. hort. sci., 33(4): 593­603. copyright: © 2019 bagheri s., hassandokt m.r., mirsoleimani a., mousavi a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 june 2019 accepted for publication 24 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 593­603 594 warm weather and high light to grow (sangeetha et al., 2006). drought is one of the most important envi­ ronmental stresses that adversely impact plant growth and crop production. more than 45% of worldʼs agricultural lands are permanently exposed to drought and 38% of the world’s population resides in those places (ashraf and foolad, 2007). therefore, the majority of efforts will be focused on producing more crops in water shortage conditions in the future (sinaki et al., 2007). in arid and semi­arid conditions, which consist of the major part of iran, the lack of sufficient and proper vegetation causes reduction of the return of plant remnants and organic matter to the soil (tate, 2000). most of the soils in the arid and semi­arid areas in iran contain less than 1% organic matter (asghari, 2011). the organic matter shortage reduces the stability of the soil structure and its flak­ ing, eventually creating a hard and dense soil (hemmat et al., 2010). the use of organic fertilizers such as animal manure is a way of increasing the organic matter content in agricultural soils, however the application of this material cannot meet the needs of these soils (mesa and spokas, 2011). therefore, in order to improving the soil, the use of organic resources such as agricultural waste, com­ post, urban waste, and sewage sludge is necessary, so that while increasing agricultural products, sus­ tainable development can be achieved in agriculture (yin chan and xu , 2009; nazmi et al., 2012). in recent years, biochar has been used as a soil reformer, an organic carbon source, and somehow a method for carbon sequestration in agricultural soils. biochar is a char produced from plant biomass and agricultural waste like wheat straw, corn, rice, which is produced during the thermochemical process of pyrolysis; this process is referred to the slow burning of organic matter under low or lack of oxygen condi­ tion (glaser and birk, 2012). it has been reported in several studies that biochar is a useful reformer to improve the soil physical and chemical characteristics and is effective in preserving soil organic matter, increasing fertilizer efficiency, and enhancing crop production, especially in the soils of subtropical and tropical areas that have long been cultivated (van zwieten et al., 2010). biochar enhances the water holding capacity of the soil (basso et al., 2013) and change the particle size distribution and porosity of the soil due to its high specific surface area (ssa) (sun et al., 2014) and also is a direct source for k, ca, p, zn, and cu (chan et al., 2008). in addition, biochar increase the soil nutrient availability due to increas­ ing the cation­exchange capacity (cec), changing the soil ph. using a biochar produced from rice plant residues increased the plant fresh and dry weight, root fresh and dry weight, stem length, and leaf num­ ber in lettuce and cabbage plants (carter et al., 2013). addition of biochar increased the soil ph, ec, organic carbon, cec and n, p, k, na, ca, and mg con­ centration of the soil and also the p, n, and k con­ tents of the lettuce plants in this soil (nigussie et al., 2012). depending on the variety and farming condi­ tions during the year, each palm produces about 15­ 25 dry leaves, each weighting 1.5 to 2.5 kg. the gen­ eralization of this amount of plant residues to several million palms in iran leads to a great deal requiring the management of productivity and optimal use. these wastes can be converted into biochar and then used in soil. in recent years, many areas of iran have been faced with water shortages and droughts, thus increasing soil water holding capacity by adding organic matter and biochar to soil can increase the potential of land use in these areas. therefore, the present study was accomplished aiming to exploit the palm leaf biochar in order to increase soil organic matter and diminish the adverse effects of drought stress and investigate its effect on some characteris­ tics of melon plants. 2. materials and methods this experiment was carried out in 2016 and 2017 in an agricultural farm in zarrindasht region of fars province, iran, with a longitude of 54°, 20ʹ and a lati­ tude of 28°, 20ʹ with an altitude of 1021 m from the sea level. in this experiment, the samsouri varamin early variety melon was used. the remains of palm leaves from zarrindasht orchards were collected, air dried, and crushed and then packed in aluminum sheets to limit the oxygenation and packs were placed in the oven for four hours at 560 °c to pro­ duce biochar (hall et al., 2008). table 1 shows some chemical properties of biochar used in the experi­ ment. this experiment was conducted in the split table 1 ­ some chemical characteristics of biochar used in the experiment ph (1:7) ec (ds m­1) (1:7) mn (ppm) cu (ppm) zn (ppm) fe (ppm) k (%) p (%) n (%) 9 7.5 0.74 0.09 0.83 983.2 32.4 2 1.39 bagheri et al. ‐ effect of palm leaf biochar on melon plants 595 plot form in completely randomized block design with three replications. the main plot was irrigation level in three levels (60, 85, and 100% water require­ ment) and biochar as subplot in four levels (0, 0.18, 0.24, and 0.36 kg/m2). i1b1 = without biochar with 60% water requirement; i1b2 = 0.18 kg m2 biochar with 60% water require­ ment; i1b3 = 0.24 kg m2 biochar with 60% water require­ ment; i1b4 = 0.36 kg m2 biochar with 60% water require­ ment; i2b1 = without biochar with 85% water requirement; i2b2 = 0.18 kg m2 biochar with 85% water require­ ment; i2b3 = 0.24 kg m2 biochar with 85% water require­ ment; i2b4 = 0.36 kg m2 biochar with 85% water require­ ment; i3b1 = without biochar with 100% water require­ ment; i3b2 = 0.18 kg m2 biochar with 100% water require­ ment; i3b3 = 0.24 kg m2 biochar with 100% water require­ ment; i3b4 = 0.36 kg m2 biochar with 100% water require­ ment. in the year before planting, the farm was fallow plowed well and leveled. before planting, a soil sam­ ple prepared and some its chemical properties were evaluated (table 2). biochar was mixed with soil at 10 cm depth and then seeds were planted at an appro­ priate depth on the rows at distance of 2.5 m and 0.5 m on the row. the drip irrigation was applied, so that, a dripper was placed beside each plant in order to measure the amount of water consumed by the plant. in the 4 to 5 leaf stage, 50 kg/ha of nitrogen, 40 kg/ha of phosphorus and 40 kg/ha of potassium were added to the soil from sources of urea, potassium sulfate and triple super phosphate respectively. to estimate of the plant water requirement, the meteorological data including minimum and maxi­ mum temperature, minimum and maximum humidi­ ty, solar radiation, and wind speed were taken from the zarrindasht meteorological office (table 3). then, the amount of evapotranspiration of melon plant was measured and the daily water requirement of the plant was obtained using the appropriate formulas for two years. for estimation of potential evapo­ transpiration parameters (etos) and water requirements by the proposed method of fao using meteorological data and field surveys related to agronomic calendar and different stages of plant growth. it is then calculated by introducing the vegetation coefficient (kc) according to plant type, stage and duration of growth and its effect on (eto), evapotranspiration (etc). finally, by reducing the effective rainfall, the net requirement of irrigation water (in), which is the soil moisture deficiency, is estimated to be offset by irrigation. etc = eto*kc, where: etc = actual evapotranspiration of the plant (mm/day) eto = reference evapotranspiration (mm/day) kc = plant coefficient. before the melon fruit ripens, parameters of stem length, plant length, leaf area, and number of leaves per plant were measured. at harvest time, the para­ meters of total yield, average fruit weight, shoot fresh weight, shoot dry weight, root fresh weight and root dry weight determined by scale. fruit length, fruit diameter and root length determined by a ruler and fruit skin thickness, fruit flesh thickness, deter­ mined by a caliper after slicing the fruits. since it was not possible to separate the leaf at all stages to mea­ table 2 ­ some chemical characteristics of the farm soil ph (1:7) ec (ds m­1) (1:7) n (%) p (%) k (%) 7.8 0.54 0.05 0.122 0.014 year month mean minimum temperature (°c) mean maximum temperature (°c) precipitation (mm) potential evapotranspiration (mm) 2016 mar. 6.4 29.8 30 187.6 apr. 12.2 42.8 0.2 314.9 may. 17.6 44.2 0 397.8 2017 mar. 9.8 37.2 25.9 185.2 apr. 15.2 39.8 3.2 300 may. 17.8 44.8 0 387.4 table 3 ­ some meteorological characteristics during the two years of experiment adv. hort. sci., 2019 33(4): 593­603 596 sure the leaf area, first several leaves were separated and their length, width, and length by width were calculated. then, the area of the leaves was mea­ sured using the graph paper (mm) and the surface area relation was obtained using the excel software. the following relation, which has the highest regres­ sion coefficient (r2), was used to calculate the leaf area: y= 1.03 x + 44 where: y (cm2) = leaf area x (cm2) = length (cm) * width (cm) leaf proline content were determined by bates method (bates et al., 1973). leaf samples washed with distillated water, dried at 65°c for 48 h in an oven and ground. total n in the leaves was deter­ mined by micro­kjeldahl method (bremner, 1996). the grounded leaf samples were ashed at 550°c and digested with 2 n hydrochloric acid. p concentration in the extracts was determined by the yellow color method and k using flame photometer (helmke and sparks, 1996). concentrations of fe, zn, mn and cu were determined by an atomic absorption spec­ trophotometer (pg 990, pg instrument ltd. uk) as well. the water use efficiency was calculated as a cor­ relation between plant yield and plant water use dur­ ing the treatment period (liu et al., 2015). wue = y/v where: wue, y, and v were water consumption effi­ ciency in kg/m3, plant yield in kg per plant, and total water consumption in m3, respectively. statistical analysis was performed using the sas software (statistical analysis system) (v9) (sas institute inc. cary, nc, usa). differences among the mean values were detected by least significant differences (lsd) test at %5 level. 3. results the results revealed that the effects of drought stress and biochar and also the interaction of them were significant on water use efficiency and all physi­ ological characteristics (table 4). i3b3 and i3b4 treat­ ments increased 88% and 76% in water use efficiency respectively compared to i1b1 treatment, however there was no significant difference compared to treatments of i3b2, i2b3 and i2b4 (table 5). source of changes degree of freedom mean square water use efficiency shoot fresh weight shoot dry weight root fresh weight root dry weight shoot length root length leaf area leaf number fruit diameter fruit flesh diameter average fruit weight total yield r (replication) 2 125.59 42833.5 * 3253.9 ** 53.3 ** 1.0 ** 112.5 ** 35.5 ** 115.1 1626.1 ** 10.2 0.07 60257.037 ** 1506425.93 ** stress (a) 2 103.08 ** 228545.6 ** 6954.6 ** 158.6 ** 2.9 ** 184.2 ** 72.8 ** 4135.6 ** 3603.2 ** 351.9 ** 2.5 ** 597026.20 ** 14925655.00 ** r (year) 2 5.69 9183.5 15.3 1.02 0.1 1.2 0.2 76.5 2.2 4.1 0.09 29931.55 748288.89 biochar (b) 3 37.19 ** 229634.7 ** 3071.4 ** 81.0 ** 1.1 ** 117.8 ** 20.0 ** 6129.9 ** 3079.3 ** 82.0 ** 0.6 ** 225506.06 ** 5637651.49 ** a*b 6 7.44 ** 59484.6 ** 358.1 ** 10.0 * 0.3 ** 60.9 ** 4.2 * 1087.7 ** 618.2 ** 25.5 ** 0.15 * 38834.35 ** 970858.94 ** a*r ( year ) 8 4.9 11845.3 367.0 4.0 0.1 28.5 2.3 608.7 47.4 6.3 0.2 18493.98 462349.70 error 36 1.43 13001.4 69.7 4.19 0.0825 13.47 1.777 244.9 117.4 4.78 0.0561 5284.20 132105.19 table 4 ­ results of analysis of variance (anova) of biochar on some properties of melon plants under drought stress table 5 ­ effects of biochar and drought stress on some properties of melon plants under drought stress treatment water use efficiency shoot dry weight (g) root dry weight (g) shoot length (cm) root length (cm) leaf area (cm2) leaf number per plant fruit diameter (cm) fruit flesh thickness (cm) average fruit weight (g) i1b1 7.19 c 188.3 e 1.71 d 75 d 10.2 e 167.7 d 76.5 e 36 e 2.15 e 565.2 d i1b2 7.75 c 193.3 de 2.52 bc 77 cd 12.7 d 186.9 cd 91.5 de 43.3 d 2.76 cd 612.7 cd i1b3 7.92 c 206 cd 2.34 bc 76.8 cd 13.4 cd 188.3 cd 103.4 bcd 43.3 d 2.64 d 624.6 cd i1b4 7.88 c 205.2 cd 2.06 cd 77 cd 14.1 bcd 190.2 cd 106.5 bcd 43.4 d 2.84 bcd 637.5 cd i2b1 7.68 c 200.8 cde 2.5 bc 77 cd 14.7 bcd 193.1 cd 98.5 cd 46.4 cd 2.81 bcd 603.7 cd i2b2 8.73 bc 216 bc 2.68 ab 81.2 bcd 14.2 bcd 198.8 c 104.2 bcd 46.1 cd 2.83 bcd 687.9 cd i2b3 11.77 ab 238.7 a 3.14 a 85.5 ab 15.8 abc 212.2 bc 119.7 ab 48.2 bc 3.23 ab 929.3 ab i2b4 12.01 ab 209.8 bc 2.44 bc 79.6 bcd 15.2 abc 207.5 bc 110 bcd 46.5 cd 3.19 abc 934.7 ab i3b1 9.02 bc 213.3 bc 2.77 ab 78.1 cd 14.5 bcd 202.4 c 103 bcd 47 c 3.14 abc 723.1 c i3b2 12.01 ab 222.7 b 2.68 ab 80.8 bcd 15.9 abc 203.1 c 106 bcd 46.5 cd 3.16 abc 922.7 b i3b3 13.55 a 250 a 3.15 a 83.4 abc 17.5 a 233.9 ab 117.4 abc 50.7 ab 3.3 a 1057 a i3b4 12.69 a 249.2 a 2.71 ab 88.5 a 16.1 ab 252.7 a 132.5 a 51.7 a 3.35 a 994.7 ab bagheri et al. ‐ effect of palm leaf biochar on melon plants 597 well as the interaction of them on leaf proline con­ tent and all chemical characteristics except for the interaction of p were significant (table 6). the treat­ ment of i3b4 increased the n (fig. 4), k (fig. 5), and mn (table 7) as 58, 65, and 48%, respectively, com­ pared to the treatment of i1b1. regarding n ele­ interaction of the treatments indicated that the treatment of i3b3 increased shoot fresh weight (fig. 1), shoot dry weight (table 5), root fresh weight (fig. 2), root dry weight and root length (table 5) by 77, 32, 100, 84, and 71% compared to the treatment of i1b1, respectively. the highest shoot length, leaf area, leaf number per plant, fruit diameter, and fruit flesh thickness (table 5) were associated with i3b4 treatment, which increased these characteristics 18, 50, 95, 43, and 55% compared to i1b1 treatment respectively and there was no significant difference compared to the i3b3 treatment. regarding the shoot length, leaf number per plant, and fruit flesh thickness (table 5), there was no significant differ­ ence between i3b4 treatment with the treatment of i2b3. the treatment of i3b3 increased the average fruit weight (table 5) and total yield (fig. 3) by 84% compared to the treatment of i1b1, however there was no significant difference compared to the treat­ ments of i3b4 and i2b3 with increase rates of 73, 63, and 62%, respectively. the biochar­free treatment with 60% water requirement also accounted for the lowest rates in all characteristics. the results indicat­ ed that the effects of drought stress and biochar as fig. 1 ­ effects of the interaction of biochar and drought stress on shoot fresh weight of melon. fig. 3 ­ effects of the interaction of biochar and drought stress on yield of melon. fig. 2 ­ effects of the interaction of biochar and drought stress on root fresh weight of melon. table 6 ­ results of analysis of variance (anova) of chemical characteristics of melon leaf source of variation degree of freedom mean square n p k fe zn cu mn proline r (replication) 2 1.0 ** 0.004 * 0.2 * 3505.7 ** 144.7 * 4 68.2 * 1.5 * drought stress (a) 2 3.1 ** 0.06 ** 2.1 ** 14716.9 ** 1321.4 ** 24.9 ** 970.7 ** 166.2 ** r (year) 2 2.3 0.02 1.2 20.0 39.9 0.2 5.8 0.04 biochar (b) 3 1.2 ** 0.01 ** 1.0 ** 34202.0 ** 836.3 ** 63 ** 363.5 ** 13.7 ** a*b 6 0.3 ** 0.008 ns 0.2 ** 9413.6 ** 184.6 * 11.8 ** 200.9 ** 2.2 ** a*r ( year ) 8 0.08 0.001 0.07 1356.1 46 0.66 53.2 2.5 error 36 0.0734 0.00123 0.0446 623.32 76.479 3.135 15.31 0.528 * and ** indicate significant difference in 1 and 5% level respectively; ns= not significant. 598 adv. hort. sci., 2019 33(4): 593­603 ment, the treatments of 0.36, 0.24 and 0.19 kg/m2 and 100% water requirement was not significantly different in comparison to treatments of i2b3 and i2b4 (fig. 4). in terms of k, there was no significant difference between the treatments of i3b3 and i3b4 (fig. 5). in addition, the mn content in the treatments of i3b3 and i3b4 was not significantly different in compared to i2b3 and i2b4 treatments and the low­ est rate was also related to the biochar­free treat­ ment with 60% water requirement (table 7). the treatment of 0.36 kg/m2 increased the p level by 20% compared to the treatment without biochar and accounted for the highest rate, although it was not significant compared to the treatment of 0.24 kg/m2 (15%) (figs. 6 and 7). mean comparison of drought stress treatments suggested that 100% and 85% water requirement increased the leaf p content 36% and 10% compared to the treatment of 60% water requirement respectively and were significantly dif­ ferent compared to each other (figs. 6 and 7). the interaction of drought stress and biochar treatments showed that the treatment of 0.36 kg/m2and 60% water requirement increased leaf fe (fig. 8), zn, and cu (table 7) by 60, 44, and 66%, respectively, com­ pared to treatment without biochar with 60% water requirement, with the lowest rate being associated fig. 4 ­ effects of the interaction of biochar and drought stress on melon leaf n content. treatment zn (ppm) cu (ppm) mn (ppm) i1b1 44.07 bcd 9.51 d 44 d i1b2 46.87 bcd 10.18 d 47.17 d i1b3 54.97 ab 14.38 ab 51.51 bcd i1b4 63. 7 a 15.75 a 51.98 bcd i2b1 38.83 cde 9.98 d 49.68 cd i2b2 35.2 de 10.55 d 50.9 bcd i2b3 51.3 abc 11.92 bcd 56.88 abc i2b4 45.5 bcd 13.98 ab 59.1 ab i3b1 25.27 e 8.41 d 50.9 bcd i3b2 42.37 bcd 11.35 bcd 52.73 bcd i3b3 40.03 bcd 11.01 bcd 59.37 ab i3b4 43.53 bcd 10.85 cd 65.34 a fig. 5 ­ effects of the interaction of biochar and drought stress on melon leaf k content. table 7 ­ mean comparison interaction of biochar and drought stress on some characteristics of melon leaf fig. 6 ­ effects of drought stress on melon leaf p. in each column, mean values with the same letters do not have a significant difference in 1% probability level of the duncan’s test. fig. 7 ­ effects of biochar on melon leaf p. bagheri et al. ‐ effect of palm leaf biochar on melon plants 599 with the biochar­free treatment with 100% water requirement. for fe, the treatments of i1b2, i1b3 and i1b3 had not significantly difference to each other and with compared to i2b3 and i2b4 (fig. 8). the leaf zn content of the treatments of i1b3 and i1b4 and i2b3 was not significantly different (table 7). in the case of cu, the treatments of i1b3 and i1b4 were not significantly different from i2b4 (table 7). the lowest level of proline was related to i3b3, and it was not significantly different from the treatment of i3b4. moreover, the highest rate of proline was relat­ ed to the biochar­free treatment and 60% drought stress and was significantly different from other treatments (fig. 9). 4. discussion and conclusions reduction in water resources affects the physio­ logical processes of the plant and hence reducing the growth and yield. in this experiment, the water shortage of root media was compensated with the application of biochar and hence increasing in water holding capacity, and the water use efficiency of plant improved without decreasing growth and by increasing the nutrient supply and hence total yield. the treatments of i3b3 and i3b4 increased water use efficiency by 88 and 76% compared to the treatment of i1b1 respectively, but there was no significant dif­ ference compared to the treatments of i3b2, i2b3 and i2b4. in this experiment, no significant difference was observed between 85% and 100% water require­ ment, particularly in 0.24 and 0.36 kg/m2, indicating the fact that biochar application in 85% water requirement significantly reduced plant water use and hence, a significant effect on the water use effi­ ciency of the plant. akhtar et al. (2014) reported that the use of biochar obtained from rice bran and flaxseed increased water use efficiency in all irriga­ tion treatments compared to the biochar­free condi­ tions. uzoma et al. (2011) indicated that the applica­ tion of 10, 15, and 20 tons cow manure biochar per hectare significantly increased the water use efficien­ cy of corn plants in a sandy soil. i3b3 treatment increased the shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and root length by 77, 32, 100, 84, and 71%, respectively compared to the treatment of i1b1. the treatment of i3b3 had no significant difference in comparison to the treat­ ments of i3b4 and i2b3. roots grew well in biochar beds, which can be due to the improvement of the physical and chemical conditions of the soil and therefore, reducce the soil resistance to the root growth (chan et al., 2008). biochar can improve water permeability of the soil and facilitate root infiltration and increase root weight and length. the highest shoot length, leaf area, and leaf num­ ber per plant were related to i3b4 in compared to i1b1 treatment, however treatment of i3b4 was not significantly different from the treatment of i3b3. in this experiment, decrease in irrigation and increase in plant stress caused decrease in the shoot length, leaf area, and leaf number per plant, and with increased irrigation and biochar application, and hence decreasing stress, these values increased. in the stress conditions, the plant size reduces due to reduced transpiration, hence reducing its leaf cells fig. 8 ­ effects of the interaction of biochar and drought stress on melon leaf fe. fig. 9 ­ effects of the interaction of biochar and drought stress on leaf proline content of melon plants. adv. hort. sci., 2019 33(4): 593­603 600 and leaf size. with increasing water use efficiency and thus decreasing stress, the biochar leads to an increase in the leaf area and leaf number per plant (olympios, 1992). the shoot length increases because of the effect of biochar in increasing avail­ able p that causes increase in root growth and absorption of nutrients (hossain et al., 2010). moreover, sang and gio (2012) showed that the biochar with increasing chlorophyll content, leads to the improvement of photosynthesis, carbohydrate synthesis and biomass production; the result of which include the increase in the leaf area, leaf num­ ber per plant, hence increase in weight and length of the root and shoot of the plant. the treatment of i3b4 increased fruit diameter and fruit flesh diameter by the rate of 43 and 55 in compared to the treat­ ment i1b1, although there was no significant differ­ ence between the treatments of i3b3. in this experi­ ment, fruit diameter, fruit flesh diameter and hence the yield increased with the application of biochar. this is due to the nutritional elements available in the palm leaves (direct) and also the improvement of soil physical, chemical and biological characteristics (indirect) by biochar (major et al., 2010). biochar sig­ nificantly leads to increase in the organic carbon and soil fertility (kumar et al., 2013), increased growth and crop yield (spokas et al., 2010), and increased plant dry matter (van zwietn et al., 2007). the treat­ ment of i3b3 increased the average fruit weight and plant yield by 84% compared to the i1b1 treatment, however it was not significantly different compared to the treatment of i3b4, i2b4, and i2b3. other researchers (zhang et al., 2010) also attributed the increase in corn growth and yield in biochar treat­ ments to increased availability of the nutritional ele­ ments and improved physical properties of the soil, such as decreasing the apparent density. furthermore, biochar improves soil chemical proper­ ties including functional groups and cec (kharea et al., 2013), in addition to increased plant access to nutrients and improved plant growth (lehmann and joseph, 2009). uzoma et al. (2011) indicated that biochar application increased the growth and yield of corn compared to control, and had a significant effect on shoot length and number of leaves in different stages of corn growth in sandy soil. in this experi­ ment, addition of 0.19, 0.24, and 0.36 kg/m2, espe­ cially the treatments of 0.24 and 0.36 kg/m2and with­ out stress, increased other vegetative characteristics. moreover, the application of biochar with 85% water requirement did not significantly change these char­ acteristics, but increased stress (60% water require­ ment) decreased plant vegetative properties. n is considered as a mobile element, so n level reduces in conditions of water shortage. accordingly, it can be conclusively claimed that the addition of biochar to the soil, by increasing water retention, decreases the nitrate leaching from the soil and increases the avail­ ability of n in the soil, and this effect is stable for at least five months (clough et al., 2013). generally, there are varying reports on the effect of drought stress on nutrient content in plant species. the decreased rate of n in water shortage conditions (muni ram and singh, 1995; alam, 1999) and its strengthening under drought stress have been reported (abdel rahman et al., 1971). in this experi­ ment, the treatments of i1b4, i1b3 and i1b2 increased n content of the plant as 34, 32 and 10%, the treatments of i2b4, i2b3 and i2b2 increased n content of the plant as 52, 54, and 28%, and finally the treatments of i3b4, i3b3 and i3b2 increased n content of the plant by 58, 50 and 41% compared to treatment i1b1. it is concluded that the plant n con­ tent increases with application and increasing the biochar level and decreasing drought stress. results showed that biochar utilization increased leaf p content under stress and non­stress condi­ tions. the effects of organic matter on increasing p availability in the soils depend on their phosphorus content. due to the low amount of absorbable phos­ phorus in palm leaf biochar, this increase can be attributed to acids released from organic matter. these acids reduce the p stabilization in the soil and transform it into an absorbable form. the absorption of nutrients and available water by plant roots are closely related to each other. water relations affect all physiological processes related to the solubility and availability of nutrients (alam, 1999). in this experiment, application of 0.36, 0.24, and 0.19 kg/m2 increased 20, 15, and 5% of plant p, respectively, in comparison to the control (without biochar). moreover, the treatments of 100% and 85% of water requirement increased the plant’s p rate as respec­ tively 36% and 10% in comparison to the treatment of 60% of water requirement. it can be concluded that the leaf p content increased with increasing biochar level and decreasing drought stress. biochar application increased k under stress and non­stress conditions. increasing the soluble k due to the appli­ cation of biochar depends on their composition, especially their k content, the rate of k release, and the effect of organic molecules on the release of k from soil minerals (jalali, 2011; najafi­ghiri, 2015). at the presence of higher water rate, univalent ions bagheri et al. ‐ effect of palm leaf biochar on melon plants 601 such as k in the soil solution increase relatively more than bivalent ions such as ca and mg, however as the soil becomes dry gradually, clay colloids absorb k (univalent ions) more strongly to their surface and prevent the separation of these ions (kafi et al., 2009). in addition, since the overall growth of the plant, including the absorption activity of roots reduces due to stress, they will not be able to absorb k from the surface of clay colloids and, hence, the rate of absorption of these elements decreases (radin and eidenbock, 1984). in the present experi­ ment, treatments of i1b4, i1b3 and i1b2 increased the k rate as 13, 18, and 9%, also, treatments of i2b4, i2b3 and i2b2 increased the k rate as 39, 27, and 23%, and finally treatments of i3b4, i3b3 and i3b2 increased the k content by 65, 47, and 30%, respec­ tively. this results leads to the conclusion that the addition of biochar reduces stress and, as a result, increases the k content of the plant. biochar applica­ tion increased fe, zn, and cu under stress and mn under non­stress conditions. the researchers have suggested that drought stress stops the activity of older roots and only the tip of the roots absorb nutri­ ents, hence the bivalent cations such as iron are absorbed more than the univalent ones and adsorp­ tion of the anions is limited (martins et al., 2003). in the case of zn and cu elements, maybe in conditions of drought stress, continuous wetting and drying in the soil leads to the release of these elements from the clay layers and their concentration increases in the soil, hence increasing the adsorption phenome­ non (logan et al., 1997). mn and fe have an inverse relationship with each other in terms of absorption by the plant, that is, increasing the mn absorption decreases the fe absorption (martins et al., 2003). changes in the availability of micro elements in the soil are affected by the characteristics of organic mat­ ter and soil. the nutrients of organic matters are released through its decomposition. although various mechanisms are responsible for increase or decrease of retaining nutrients in the soil (sposito, 1984), stud­ ies have shown that adding biochar to the soil is effective on the capability of use of ions due to affecting ion exchangable capacity and microbial activity (atkinson et al., 2010). in an experiment, drought stress increased soil zn and cu and reduced mn (alizadeh et al., 2008). drought stress increased zn, fe, and cu content in the sage plant (sodaeizadeh and mansouri, 2014). in an experiment, biochar application increased fe and mn elements in ama­ ranth plant (habibi et al., 2017). in this experiment, 60 and 85% water requirement increased the fe con­ tent (76 and 107%), zn (74 and 13%), and cu (13 and 18%) compared to the 100% water requirement (without stress) and the treatments of 0.24 kg/m2 in 60 and 85% water requirement and 0.36 kg/m2in 60 and 85% stress, increased the fe rate as 33%, 48%, 60%, and 32%, the zn as 24, 16, 44, and 3%, and cu as 51, 25, 65, and 47% respectively, compared to the treatment of i1b1. moreover, in the present experi­ ment, the mn level increased with the use of biochar and decrease in the drought stress, so that treat­ ments of i1b4, i1b3 and i1b2 increased the mn by 18, 17, and 7%, the treatments of i2b4, i2b3 and i2b2 increased the mn by 34, 29, and 15%, and eventually, the treatments of i3b4, i3b3 and i3b2 increased the mn by 38, 44, and 19%, respectively. the use of biochar reduced proline content under stress and non­stress conditions. this finding sug­ gests that biochar decrease the water evaporation and keeping moisture in the root media, because of its large pores on its surface or improving the soil tex­ ture, and can improve root growth and hence reduce stress. under drought stress conditions, the water potential of the leaf decreases substantially, which, solutions such as proline accumulation in the leaf in order to adapt to the osmotic conditions. proline decreases in leaves under stress due to decreased synthesis and increased oxidation. it was observed that drought stress caused reduction in leaf water capacity of grape and, thus, increased proline and proline rate was reduced through the use of biochar in cultural media (rasouli and golmohammadi, 2009). in this experiment, the proline content decreased with increasing biochar treatments from 0.19 to 0.36 kg/m2 and the increase of water require­ ment from 60 to 100%, with the lowest amount of proline being related to 0.24 and 0.36 kg/m2and 100% water requirement. the results of this study revealed that adding palm leaf biochar to the soil especially in drought stress conditions 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production of td waste of approx. 2-3 tons/month is a major problem for the company. plant growth media containing peat (p) + 0, 5, 10, 25 or 50% td were prepared and tested on geranium plant growth and development. the use of td increased ec and ph of the final medium. plants of the cvs “ml diego” and “ml sailing ‘12” grown in p+5% or in p+10% td had similar height, number of leaves, number of flowers, photosynthetic activity and transpiration rates to the p alone (control) indicating that solid agro-industrial waste of tobacco could be used to partially substitute peat in growing medium for floricultural crop production. 1. introduction pelargonium x hortorum or ‘‘zonal geranium’’ is an ornamental species that originate from south africa, perfectly adapted to the mediterranean region. it is propagated by cuttings and it is a hybrid between p. inquisaus (l.) l’herit and p. zonale (l.) l’herit (dole and wilkins, 2005). zonal geraniums attract an increased commercial interest as they are extensively used in landscape designs and terrace gardens (berninger, 1993). flowering is strongly dependent on growth stage (i.e. juvenility), temperature (i.e. cold requirement) and sunlight (i.e. intensity and duration), but zonal geraniums are not described as longor short-day plants (fonteno, 1992; dole and wilkins, 2005). agricultural waste such as cotton gin trash, olive mill and green waste have been used in combination with peat for cultivation of ornamental plants (papafotiou et al., 2004; 2005; grigatti et al., 2007). the residue from tobacco processing (i.e. the tobacco dust; td) is buried to landfields, but in high w/w concentrations can be toxic due to its high tannin and alkaloid content (briski et al., 2003). compared to other waste material, td contains higher n and k and has ph values ranging between 5.0 and 6.0 (aderidan et al., 2003). the application of td waste in soil cultivated with lettuce increased yield compared to control plants (okur et al., (*) corresponding author: tassosdarras@yahoo.co.uk citation: tzavara s., darras a.i., assimakopoulou a., 2019 tobacco dust waste as an alternative medium to grow geranium (pelargonium x hortorum) plants. adv. hort. sci., 33(2): 295-298 copyright: © 2019 tzavara s., darras a.i., assimakopoulou a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 september 2018 accepted for publication 20 march 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 295-298 296 2008). it was suggested that incorporation of td waste as an alternative organic amendment might improve soil chemical and biological parameters, as well as crop yield in soils containing low organic matter content. apparently, no previous research has been conducted on utilization of tobacco byproducts as alternatives to grow ornamental plants. in the present study, we tested growing mediums of peat + increasing concentrations of td on growth and flowering response of zonal geraniums. 2. materials and methods plant material, media preparation and experimental lay-outs zonal geranium (pelargonium x hortorum) rooted cuttings (3-5 leaves; up to 12 cm height) of cvs ‘ml diego’ (red inflorescences) and ‘ml sailing’12’ (white inflorescences) were provided by selecta-one ltd (kavala, greece). single rooted cuttings were transplanted in plastic 2.5 l pots filled with growing mediums of peat + td. td was provided by karelias s.a. (kalamata, greece) and samples were analyzed in a continuous flow analyzer (cfa αα3; seal-analytical ltd., germany) (table 1). peat (hawita, germany) was mixed with 0, 5, 10, 25 and 50% (w/w) td (table 2). six-replicate pots per treatment with geranium plants were placed on the ground of a non-heated greenhouse at the premises of the university of peloponnese (lat. 37° 2’ 20’’ n, long. 22° 6’ 51’’ e) in a completely randomized design. two individual experiments were carried out (one for each cultivar) from november 2017 to february 2018. medium properties, plant assessments and statistical analysis medium ph and ec (ms/cm) were measured using a ph/mv meter (delta ohm hd 2105.2, padova, italy) and a conductivity handheld meter (eutech instruments, ecoscan con 5, singapore), respectively. plant height (cm), number of leaves and number of inflorescences were recorded weekly over the entire cultivation period of nine weeks. chlorophyll fluorescence, net co2 assimilation (as; μmol m-2 sec) and transpiration (e; mmol m-2 sec) were recorded using a handheld fluorimeter (os-30p, opti-sciences, inc. u.s.a.) and a lcpro+ portable photosynthesis system (adc bioscientific ltd. great amwell, herts, uk), respectively, on the 5th, 6th, 7th and the 8th week from transplanting (i.e. week-1). data means were separated using duncan’s multiple range test at p = 0.05. statistical analysis was performed in spss v. 21. 3. results tobacco dust contained nicotine, tss and small amounts of nitrates and ammonia (table 1). ec and ph values ranged between 8.92 and 9.46, and between 5.3 and 5.6, respectively. after mixing peat with 0 50% td, ph and ec of the final growing medium increased linearly to reach the values of 5.42 and 6.18 ms/cm, respectively (table 2). table 1 means and range values of tobacco dust content tobacco dust samples were analyzed in a continuous flow analyzer tobacco dust content means range nicotine (mg/l) 158.18 157.84 -160.14 tss (mg/l) 583.12 581.36 -584.04 nitrates (mg/l) 32.06 30.15-33.02 ammonia (mg/l) 6.38 6.25 6.52 ec (ms/cm) 9.17 8.92-9.46 ph 5.5 5.3-5.6 table 2 ph and ec (ms/cm) values of growing media after mixing peat with tobacco dust at 0 (control), 5, 10, 25 and 50% (w/w) growing media ph ec (ms/cm) mixing peat 4.00 0.06 mixing peat + 5% tobacco dust 4.51 1.30 mixing peat + 10% tobacco dust 4.85 3.22 mixing peat + 25% tobacco dust 5.09 4.84 mixing peat + 50% tobacco dust 5.42 6.18 peat replacement with 5 or 10% td in the growing medium positively affected growth and flowering of cvs. “ml diego” and “ml sailing’12” geraniums (table 3). plants of cv. “ml diego” grown in p+5% or p+10% td maintained similar or higher height, number of leaves, number of inflorescences, net co2 assimilation and transpiration rates to the control plants (i.e. p+0% td) (table 3). however, plants grown in p+25 or p+50% td suffered reductions in growth and flowering compared to the controls. plants grown in p+25 or p+50% td showed reduced number of inflorescences and transpiration rates, compared to the plants grown in p+5 or p+10% td (table 3). stunting growth and reduced flowering of plants cultivated in p+25 or p+50% td were justified tzavara et al. tobacco dust an alternative medium to grow geranium 297 by decreases in chlorophyll fluorescence (fv/fm) ratios (table 3). the fv/fm ratios ranged between 0.571 and 0.893 for plants grown in p+25% td and between 0.538 and 0.835 for plants grown in p+50% td indicating damage in plants’ photosystem (ps ii) that induced stress responses. plants of cv. “ml sailing’12” grown in p+5, p+10 or p+25% td, showed similar or higher number of leaves, number of inflorescences, net co2 assimilation and transpiration rates to the control plants (table 3). however, plants grown in p+50% td showed reduced height, number of leaves, number of inflorescences, indicating damage in plants’ photosystem (ps ii) recorded as low fv/fm ratio ranging between 0.538 and 0.773 (table 3). plants in p+50% td failed to reach minimum growth requirements and standard commercial size. 4. discussion and conclusions td successfully replaced part of peat in growth medium for zonal geranium plant production. plants of cvs. “ml diego” and “ml sailing 12” responded well when grown in p+5, p+10 and in few cases in p+25% td and, therefore, could potentially replace part of peat in the growth medium. the concept of peat replacement with agricultural waste material for the cultivation of ornamental plants has been examined the past 20 years. papafotiou et al. (2004) showed that olive-mill waste composts (owc) could partially replace peat for the production of euphorbia pulcherrima (poinsettia), although, at concentration of >12.5% delayed growth compared to plants cultivated in peat/perlite medium. tropical potted plants such as syngonium podophyllum, ficus benjamina and codiaeum variegatum could be successfully grown in 75% owc without showing symptoms of toxicity or other negative effects on growth and development (papafotiou et al., 2005). cotton gin trash compost (cgtc) and rice hulls (rh) were tested as peat replacements for the production of nerium oleander, pelargonium zonale, dedranthema grandiflora and lantana camara (papafotiou et al., 2001). replacing peat with 60% of gctc resulted in plant height decrease of all species, except those of p. zonale and increase in number of flowers to all species, except those of d. grandiflora. the use of green waste and sewage sludge compost (wssc) at 80% 20% (v:v) as a 25%-replacement of white peat, had positive effects on growth and flowering of begonia semperflorens, mimulus hybridus, tagetes patula x erecta and salvia splendens (grigatti et al., 2007). all species grown in 25% wssc showed greater height, number of flowers and plant dry weight compared to plants grown in 100% white peat. td is a potent agricultural byproduct that could be used in concentrations of <25% without affecting growth and quality of ornamentals. replacing peat with byproducts of the agricultural sector, merits an eco-biological prospect of environmental-friendly ornamental production. further research is needed to test td as peat replacement for cultivation of other ornamental species. data are means ± se of 9-week recordings and letters indicate the statistical differences according to duncan’s multiple range test at p = 0.05. not measured. plants failed to reach minimum growth requirements. table 3 effect of growing medium of peat amended with 0, 5, 10, 25 and 50% td on number of leaves, plant height, number of inflorescences, chlorophyll fluorescence, net co2 assimilation and transpiration of p. x hortorum plants of cvs "ml diego" and "ml sailing'12" treatments plant height (cm) range (cm) number of leaves range number of inflorescences range net co 2 assimilation (μmol m-2.sec) range (μmol m-2.sec) transpiration (mmol m-2.sec) range (mmol m-2.sec) chlorophyll fluorescence (f v /f m ) range (f v /f m ) ml diego' 0 8.83±0.25 ab 4-16 11.82±0.41 c 4-24 0.85±0.07 a 0-3 2.18±0.26 a 0.34-6.11 1.00±0.087 a 0.61-1.92 0.805±0.001 a 0.778-0.834 5 8.89±0.27 ab 2-18 13.50±0.66 b 4-33 0.86±0.09 a 0-6 3.01±0.34 a 0.24-7.14 1.36±0.170 a 0.71-2.57 0.801±0.002 a 0.685-0.828 10 9.12±0.24 a 5-17 15.12±0.68 a 2-36 0.93±0.10 a 0-5 2.40±0.29 a 0.22-5.32 1.28±0.184 a 0.42-2.34 0.802±0.002 a 0.753-0.878 25 8.39±0.23 b 4-15 12.11±0.55 bc 4-28 0.60±0.08 b 0-3 2.08±0.20 a 0.28-4.00 0.65±0.060 b 0.33-1.06 0.786±0.004 b 0.571-0.893 50 7.01±0.19 c 4-18 5.79±0.32 d 2-20 0.31±0.05 c 0-2 2.23±0.68 a 0.14-4.90 0.37±0.082 c 0.13-0.61 0.731±0.006 c 0.538-0.835 ml sailing'12' 0 9.70±0.33 a 5-16 14.11±0.62 bc 5-24 0.83±0.10 ab 0-3 2.07±0.32 b 0.34-4.07 0.89±0.096 a 0.29-1.40 0.801±0.001 a 0.778-0.819 5 9.53±0.38 a 2-18 15.79±1.00 ab 4-33 0.76±0.11 ab 0-3 3.40±0.52 a 0.24-7.14 1.04±0.161 a 0.30-2.72 0.793±0.003 a 0.685-0.818 10 9.07±0.34 a 5-17 16.68±1.01 a 4-36 0.98±0.16 a 0-5 2.40±0.62 ab 0.22-5.32 1.17±0.232 a 0.32-2.27 0.799±0.001 a 0.777-0.824 25 7.64±0.30 b 4-14 12.94±0.83 c 4-28 0.53±0.11 b 0-3 1.77±0.40 b 0.28-3.69 0.48±0.057 b 0.26-0.71 0.779±0.005 a 0.668-0.893 50 6.25±0.14 c 4-9 5.01±0.16 d 3-7 0.20±0.06 c 0-1 0.678±0.008 b 0.538-0.773 adv. hort. sci., 2019 33(2): 295-298 298 acknowledgements we sincerely thank karelias s.a. for providing tobacco dust used in the present study references adediran j.a., baets n.d., mnkeni p.n., kiekens l., muyima n.y.o., thys a., 2003 organic waste materials for soil fertility improvement in the border region of the eastern cape, south africa. j. biol. agric. hortic., 20: 283-300. berninger l.m., 1993 status of the industry, pp. 1-2. in: white j.w. (ed.) geraniums iv: the grower’s manual. fourth edition. ball publishing, batavia, illinois, usa, pp. 412. briski f., horgas n., vukovic m., gomzi z., 2003 aerobic composting of tobacco industry solid waste simulation of the process. j. clean tech. environ. policy, 5: 295-307. dole j.m., wilkins h.f., 2005 floriculture. principles and species. second edition. pearson, prentice hall, new jersey, usa, pp. 1048. fonteno w.c., 1992 geraniums, pp. 451-475. in: larson r.a. (ed.) introduction to floriculture. second edition. academic press, california, usa, pp. 636. grigatti m., giorgioni m.e., ciavatta c., 2007 compost-based growing media: influence on growth and nutrient use of bedding plants. biores. technol., 98(18): 3526-3534. okur n., kayikçioğlu h.h., okur b., delibacak s., 2008 organic amendment based on tobacco waste compost and farmyard manure: influence on soil biological properties and butter-head lettuce yield. turk. j. agric. for., 32(2): 91-99. papafotiou m., chronopoulos j., kargas g., voreakou m., leodaritis n., lagogiani o., gazi s., 2001 cotton gin trash compost and rice hulls as growing medium components for ornamentals. j. hortic. sci. biotech., 76(4): 431-435. papafotiou m., kargas g., lytra i., 2005 olive-mill waste compost as a growth medium component for foliage potted plants. hortscience, 40(6): 1746-1750. papafotiou m., phsyhalou m., kargas g., chatzipavlidis i., chronopoulos j., 2004 olive-mill wastes compost as growing medium component for the production of poinsettia. scientia hortic., 102(2): 167175. impaginato 511 adv. hort. sci., 2018 32(4): 511-516 doi: 10.13128/ahs-20537 effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars z. bakhtshahi-dizgahi 1, m. alizadeh 1 (*), e. seifi 1, d. javadi 2, a. hosseinpour 1 1 gorgan university of agricultural sciences and natural resources, faculty of plant production, department of horticulture, gorgan, iran. 2 guilan research center of agriculture and natural resources, rasht, iran. key words: chemical treatment, cold stratification, corylus avellana l., filbert, germination. abstract: propagation of european hazelnut by seed is influenced by some seed treatments. in this investigation, effect of stratification period and some chemicals on seeds of four hazelnut cultivars were studied. ga3 and four months of stratification, each individually resulted in the highest germination percentage at 82.73% and 83.75%, respectively. there were significant differences between cultivars and treatments in terms of germination percentage and rate. the highest germination percentage and rate were observed in the local cultivar gerd under ga3 treatment at 100 mg/l and also after four months of stratification. 1. introduction propagation by seeds is a conventional method to produce new plants. this is one of the most recognized efficient methods which is widely applied for different plant species. although sexual reproduction do not result in true to type plants, in breeding programs it is inevitable to apply it to grow hybrid seedlings. for example, in hazelnut, interspecific hybridization is necessary to transfer superior characters from wild species to the commercial european hazelnut (corylus avellana l.) (erdogan and mehlenbacher, 2000). seed germination is a main step in plant life cycle, and is influenced by various biotic and abiotic factors (yuan and wysocka-diller, 2006). it is essential to investigate different aspects in sexual propagation for all plant species. however, there are some common difficulties in using such approach to propagate many plants such as hazelnut, including seed dormancy and inconsistent seed germination which make some problems and retard improvement programs and sometimes end in hybrids loss. therefore, studying beneficial treatments to remove dormancy and subsequent uniform seed germination is considered of great importance (*) corresponding author: mahdializadeh@gau.ac.ir citation: bakhtshahi-dizgahi z., alizadeh m., seifi e., javadi d., hosseinpour a., 2018 effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars. adv. hort. sci., 32(4): 511-516 copyright: © 2018 bakhtshahi-dizgahi z., alizadeh m., seifi e., javadi d., hosseinpour a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 april 2017 accepted for publication 22 july 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 511-516 512 (wang and berjak, 2000; copeland and mcdonald, 2001). generally, germination process is controlled through a balance between inducing and inhibiting factors. provided that concentration of inducers is higher than inhibitors, seed dormancy will predominate. some stimulants such as temperature and light are necessary to lower the effect of inhibitors in seed. in such case, an inducing factor such as gibberellic acid (ga3) could have cumulative influence so that germination process will commence (bradbeer, 1988). in many temperate zone species, dormancy prevents the seeds from germinating (derkx, 2000). a dormant seed would not germinate even in favorable environmental conditions. several approaches have been suggested in literature to overcome this phenomenon, including cold stratification (bewley and black, 1994), and seed treatment by some chemicals such as gibberellic acid (ga3), polyamines and thiourea (frankland, 1961; çetinbaş and koyuncu, 2006; mello et al., 2009). cold stratification plays a major role as a stimulant to break seed dormancy. also its effect is accelerated in combination with chemicals or physical removing of seed coat (bewley and black, 1994). this technique is usually performed at temperatures between 0 and 10°c; which vary depending on different species. however, the best reported temperature for this kind of seed treatment is 5°c (bewley and black, 1994). aygun et al. (2009) suggested that hazelnut seeds need two to six months of pre-germination cold stratification. dormancy in hazelnut seeds is diminished through cytological, hormonal and biochemical changes during cold stratification period. for example, mobilization of phytic acid and phosphate was observed during this treatment (vasilios et al., 2005). ga3 treatments could remove various seed physiological dormancies and induce germination of dormant seeds (frankland, 1961). aygun et al. (2009) showed that in hazelnut seeds treated by ga3 at concentrations from 0 to 200 mg/l, the highest seed germination percentage was obtained by 100 mg/l ga3. the main polyamines existing in plant cells are putrescine, spermine and spermidine (davies, 2004). based on some evidences, polyamines have a role in seed dormancy process. sinska and lewandowska (1991) found that putrescine, spermine and spermidine decreased in apple seeds during cold stratification. in fact, putrescine and spermidine had inducing effect and spermine had inhibiting effect on apple seed germination. although thiourea is not applied commonly in seed germination experiments, it is able to enhance germination of some kinds of seeds (gul and weber, 1998; çetinbaş and koyuncu, 2006). stidham et al. (1980) showed that thiourea had an inducing effect on germination of 18 shrub species. according to cetinbaş and koyuncu (2006), this property of thiourea is attributed to its cytokinin-related effect in removing inhibitors. this investigation aimed to study the effects of some treatments including some chemicals and cold stratification on percentage and rate of seed germination in four hazelnut cultivars grown in iran. 2. materials and methods seeds of four hazelnut cultivars including a local cultivar gerd and three introduced cultivars barcelona, ronde (= ronde du piemont) and segorbe were collected from astara hazelnut research station, in astara, guilan province, iran. defected seeds were discarded and proper seeds were separated to study. treatments treatments applied in this study included ga3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm), thiourea (1000 and 2000 mg/l) and stratification for two and four months. the control treatment was distilled water. germination test the seeds were soaked for 24 hours in ga3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm) and thiourea (1000 and 2000 mg/l), and also some seeds were soaked in distilled water. in order to prevent seeds from rotting, the treated seeds were surface sterilized with sodium hypochlorite (10% v/v for 5 min.) and then rinsed by sterilized water. afterwards, the seeds were cultured in plastic pots containing pre-autoclaved sand as the medium (diameter of 2 mm). germination test was conducted in a factorial completely randomized design with three treatments including “no stratification”, “two months of stratification” and “four months of stratification”. emerging radical was considered as the index of germination. in chemical treatments, the number of germinated seeds up to 40 days from the culture date, and in stratification treatments, the number of germinated seeds up to 40 days from outing from refrigerator were recorded. stratification treatments were just performed by keeping them in refrigerator (5°c) for two to four months. cultured seeds were inspected regularly and in case of moisture decrease of media, bakhtshahi-dizgahi z. seed germination in four halzenut cultivars 513 autoclaved water was sprayed on them. the measured characteristics were percentage and rate of seed germination. the following formula was used to calculate germination percentage. germination percentage = (number of germinated seeds / total number of seeds) × 100 germination rate is defined as the time to reach 50 percent of germination, which was calculated by the formula below: germination rate = (1/time of reaching 50 percent of germination) for each cultivar, 210 seeds were used. the data were analyzed by analysis of variance (anova) and the software germin-g was applied to measure the target parameters (soltani et al., 2004). 3. results and discussion according to the analysis of variance, there was a significant difference among cultivars and chemical treatments with regard to germination percentage (p < 0.01) (fig. 1). even so, no significant difference was observed among cultivars and chemical treatments regarding germination rate (fig. 2). in addition, effect of stratification on germination rate and percentage was significantly different between the cultivars (p<0.01) (fig. 3 and 4). for germination percentage, the difference between mutual effects of “cultivar × chemical treatment” (p<0.05) (fig. 1), “cultivar × stratification” (p<0.05) (fig. 4) and “chemical treatment × stratification” (p<0.01) (fig. 5) was significant. for germination rate, the mutual effect of “cultivar × chemical treatment” showed a significant difference (p<0.05) (fig. 2). of the four hazelnut cultivars, ‘gerd’ had the highest germination percentage (82.73%) at 100 mg/l ga3, which showed 50.76% increase compared to the control (31.97%). although applying both ga3 concentrations was resulted in higher germination percentages, but no difference was observed between these two levels (fig. 1). between stratification and cultivar, the highest germination percentage was exhibited after four fig. 1 effect of chemical treatments and cultivars on seed germination percentage of hazelnut cultivars. fig. 2 effect of chemical treatments and cultivars on seed germination rate of hazelnut cultivars. fig. 3 effect of stratification treatments on seed germination rate of hazelnut cultivars. fig. 4 effect of stratification treatments and cultivar on seed germination percentage of hazelnut cultivars. fig. 5 effect of stratification and chemical treatments on seed germination percentage of hazelnut cultivars. adv. hort. sci., 2018 32(4): 511-516 514 months of stratification in the local cultivar gerd. no difference existed in four months of stratification between two local cultivars and barcelona (fig. 6). figure 5 reveals that between mutual effect of chemicals and stratification treatments, the highest germination percentage occurred through four months of stratification and in the control treatment. nevertheless, in treatments of two months of stratification and no stratification, both levels of ga3 resulted in the highest germination percentage. in all cultivars, the highest germination rate was recorded in ‘gerd’ (fig. 6). four months of stratification had the greatest influence on germination rate (fig. 3). of chemical treatments, the fastest germination was obtained by using the first level of ga3 (100 mg/l) in the cultivar gerd (fig. 2). various studies have demonstrated a direct relationship between cold stratification length and increasing in germination percentage. as can be clearly seen in figures 4 and 5, when stratification period increased, as a result, germination percentage of hazelnut seeds increased in all treatments and all cultivars. similar results were observed by aygun et al. (2009) which suggested that 120 days of cold stratification ended in higher germination percentage in hazelnut seeds in comparison with control. furthermore, bradbeer (1988) reported that three months of cold stratification led to a rise in hazelnut seed germination percentage. in other studies, three months of stratification without any warming improved germination percentage in seeds of jasminum fruticans (pipinis et al., 2009). another example for such impact has been reported by chin et al. (1992) in kiwifruit. in addition, there are many other studies that support the influence of applying some chemicals and plant growth regulators on rising germination percentage of seeds, which could be used singly or in combination with chilling. as a matter of fact, these chemicals are considered as substitutes for chilling requirement of seeds, and also can decrease length of chilling period. overall, gibberellins and cytokinins are able to promote seed germination in plants (davies, 2004; miransari and smith, 2014). in contrast, abscisic acid (aba) plays an inhibiting role in seed germination (miransari and smith, 2014). among types of gibberellins, ga3, ga4 and ga7 have the most impact on germination enhancement. however, cytokinins and auxins have so lower effect compared to gibberellins and cytokinins in terms of stimulating germination. in fact, the effect of all these growth regulators depends upon other factors such as light, temperature and oxygen; and also there are some mutual effects between them. gibberellins and cytokinins are capable of neutralizing the inhibiting effect of abscisic acid. since all these growth regulators naturally exist in seed in different ratios, so their observed effects on seed germination could be interpreted by the state of hormone balance (kucera et al., 2005). gibberellins are able to enhance seeds of different species to germinate through different ways. that is, external use of gibberellins could induce germination in seeds in which lack of germination is due to seed coat (e.g. legumes), or seed dormancy is because of seed embryo (e.g. apple, birch, hazelnut) (davies, 2004; miransari and smith, 2014). besides, in seeds that their germination depends on exposure to light (e.g. arabidopsis, lettuce), ga3 could promote seed germination even in the dark (cao et al., 2005). based on the results obtained in this study, the highest germination percentage and rate were gained through ga3 treatment at 100 mg/l (fig. 1, 2), which corresponded with results reported by aygun et al. (2009) that showed higher germination percentage after treatment by 100 mg/l gibberellin. in addition, there are some reports on gibberellin application on hazelnut seeds in order to enhance germination results (bradbeer and pinfield, 1967; jarvis and wilson, 1977; pinfield and stobart, 2006). in addition to gibberellins, other substances such as thiourea can also overcome seed dormancy. several investigations have demonstrated the effect of thiourea on dormancy breaking and increasing germination percentage of seeds in different plant species (gul and weber, 1998; çetinbaş and koyuncu, 2006). also ojha et al. (2010) showed that after using four treatments including gibberellin, potassium nitrate, ascorbic acid and thiourea on seeds of abrus precatorius, despite increasing germination percentage in all treatments, the highest and fig. 6 seed germination rates in four hazelnut cultivars. bakhtshahi-dizgahi z. seed germination in four halzenut cultivars 515 lowest germination percentage were obtained by gibberellin and thiourea treatments, respectively. polyamines are also a group of growth regulators and could improve seed germination in some species (szczotka and lewandowska, 1989; sinska and lewandowska, 1991). furthermore, in this research, putrescine had a significant difference compared to the control, in terms of germination percentage and germination rate of hazelnut seeds (fig. 2). 4. conclusions the results of this study revealed that the hormone ga3 and four months of cold stratification resulted in the highest germination percentage in hazelnut seeds at 82.73% and 83.75%, respectively. the cultivars and chemical treatments had significant effect on seed germination which were the highest in the local cultivar gerd under ga3 treatment at 100 mg/l and also after four months of cold stratification. acknowledgements the authors would like to express their gratitude to gorgan university of agricultural sciences and natural resources for financial and technical support. references aygun a., erdogan v., bozkurt e., 2009 effect of some pretreatments on seed germination of turkish hazel (corylus colurna l.). acta horticulturae, 845: 203-206. bewley j.d., black m., 1994 seeds: physiology of development and germination. 2nd ed. plenum press, new york/london. bradbeer j.w., 1988 seed dormancy and germination. chapman and hall, new york. bradbeer j.w., pinfield n.j., 1967 studies in seed dormancy iii. the effects of gibberellin on dormant seeds of corylus avellana l. new phytol., 66(4): 515-523. cao d.n., hussain a., cheng h., peng j.r., 2005 loss of function of four della genes leads to lightand gibberellin-independent seed germination in arabidopsis. planta, 223(1): 105-113. cetinbaş m., koyuncu f., 2006 improving germination of prunus avium l. seeds by gibberellic acid, potassium nitrate and thiourea. hortic. sci., 33: 119-123. chin k.l., blanche c.a., bachireddy v.r., 1992 gibberellic acid and cold stratification treatments affect kiwi seed germination and root elongation. hortic. sci., 27(6): 689. copeland l.o., mcdonald m.b., 2001 principles of seed science and technology. kluwer academic publishers, norwell, massachusetts, usa, pp. 488. davies p.j., 2004 plant hormones: biosynthesis, signal transduction, action! kluwer academic publishers, dordrecht, the netherlands, pp.750. derkx m.p.m., 2000 pre-treatment at controlled seed moisture content as an effective means to break dormancy in tree seeds, pp. 69-78. in: viemont j.d., and j. crabbe (eds.) dormancy in plants. cab international, wallingford, oxforshire, uk, pp. 400. erdogan v., mehlenbacher s.a., 2000 interspecific hybridization in hazelnut (corylus). j. amer. soc. hort. sci., 125(4): 489-497. frankland b., 1961 effect of gibberellic acid, kinetin and other substances on seed dormancy. nature, 192: 678-679. gul b., weber d.j., 1998 effect of dormancy relieving compounds on the seed germination of non-dormant allenrolfea occidentalis under salinity stress. ann. bot., 82: 555-560. jarvis b.c., wilson d., 1977 gibberellin effects within hazel (corylus avellana l.) seeds during the breaking of dormancy. i. a direct effect of gibberellin on the embryonic axis. new phytol., 78(2): 397-401. kucera b., cohn m.a., leubner-metzger g., 2005 plant hormone interactions during seed dormancy release and germination. seed sci. res., 15: 281-307. mello a.m., streck n.a., blankenship e.e., paparozzi e.t., 2009 gibberellic acid promotes seed germination in penstemon digitalis cv. husker red. hortic. sci., 44(3): 870-873. miransari m., smith d.l., 2014 plant hormones and seed germination. environ. exp. bot., 99: 110-121 ojha s., mondal p., konar j., 2010 effect of promoters and pretreatment with chemicals on seed germination of abrus precatorius l. plant archives, 10(2): 641-645. pinfield n.j., stobart a.k., 1969 gibberellin-stimulated nucleic acid metabolism in the cotyledons and embryonic axes of corylus avellana (l.) seeds. new phytol., 68(4): 993-999. pipinis e., milios e., aslanidou m., mavrokordopoulou o., smiris p., 2009 the effect of stratification on seed germination of jasminum fruticans l. 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effects of moist chilling on the seeds of black spruce (picea mariana [mill.] b.s.p.). ann. bot., 86: 29-36. yuan k., wysocka-diller j., 2006 phytohormone signalling pathways interact with sugars during seed germination and seedling development. j. exp. bot., 57(12): 3359-3367. impaginato 205 adv. hort. sci., 2017 31(3): 205-214 doi: 10.13128/ahs-21959 effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber m. golabadi 1, p. golkar 2 (*), a.-r. eghtedari 3 1 department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, 81595158 isfahan, iran. 2 research institute for biotechnology and bioengineering, isfahan university of technology, 8415683111 isfahan, iran. 3 department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, 81595158 isfahan, iran. key words: cucumber, flowering, male, sex expression, silver ions. abstract: male flower expression is considered an important aim in greenhouse cucumber breeding for creating paternal lines as a base for hybrid progeny. the study was carried out to evaluate the effects of different treatments on sex expression and fruit yield of cucumber in two different season (autumn-winter and spring-summer), in particular this research focuses on the influence of 1) usage of two chemical agents: silver thiosulphate [ag(s2o3)2 -3] and silver nitrate (agno3) at different concentration, respectively 200 and 500 ppm and 100, 200 and 300 ppm 2) plant development stages at the moment of the treatment (5, 10, and 15-leaf growth stages) and 3) application of single or double sprayings. analysis of variance showed that season, chemical applications and number of spaying had significant effect on the induction of a higher number of male flowers. a positive significant effect of season suggested that longer days and higher temperature promote the formation of male flowers in cucumber. this study showed that male flower production was induced by all concentrations of silver ions, especially high concentrations. important traits related to change of sex expression such as the number of days to male flowering and the number of male flowers are more affected by different interactions of studied factors in contrast to vegetative and yield related traits. also, the quadruple interaction effects indicated that silver ion could change sex expression at higher temperatures and longer days (second season in summer) with high concentration when applied in 15 leaf stage for agno3 and 5 and 15 leaf growth stage for ag(s2o3)2 -3 with double spraying. consequently, female lines can be induced to male flowering with silver ions, thus increasing the feasibility of large scale seed production of gynoecious × gynoecious cucumber hybrid. 1. introduction commercial cucumber (cucumis sativus l., 2n= 2x= 14) is a member of cucurbitaceae that is indigenous to india (renner et al., 2007). it is one of (*) corresponding author: golkar@cc.iut.ac.ir citation: golabadi m., golkar p., eghtedari a.-r., 2017 effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber. adv. hort. sci., 31(3): 205-214. copyright: © 2017 golabadi m., golkar p., eghtedari a.-r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 november 2016 accepted for publication 19 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 205-214 206 the most economically important cucurbit vegetable plants (tatlioglu, 1993, robinson and deckerwalters, 1997) and among the most widely-grown vegetable crop in the world, after tomato, onion and cabbage (plader et al., 2007). cucumber is used in different types (harvesting, slicing and fresh eating) that are used as fresh or processed vegetable (shetty and wehner, 2002). the cucumber is a thermophilic and frostsusceptible crop, usually cultivated in fields during the spring-summer period. its high demand has also made it an important crop to be widely grown in glasshouses or plastic houses (sarkar and sirohi, 2011). sex expression is an important factor that has a positive effect on yield and that constitutes a major component of cucumber improvement programs (yamasaki et al., 2003). in gynoecious cultivars, all flowers are female, so, male flowers are required for new line production via female flowers crossing. the sex expression of cucumis sativus l. is determined by genetics as well as environment (e.g. photoperiod, temperature) (yamasaki et al., 2003; bano and khokhar, 2009). change from vegetative growth to reproductive stages is a complex process regulated by many factors, and could be influenced by the application of plant growth regulators (ainsworth, 1999; sure et al., 2013). growth regulators have tremendous effects on sex modification and flowering in cucurbits that leading to either suppression of male flowers or an increase in the number of female flowers (al-masoum and al-masri, 1999). some researchers have already reported the effects of plant growth regulators on modification of sex expression in cucumber flowers (vadigeri et al., 2001; rafee kher et al., 2002; bano and khokhar, 2009). line production is important in breeding programs, because many cultivars in cucumber are hybrids (golabadi et al., 2015). in gynoecious cucumbers, male flower induction is necessary for production of f1 hybrid seeds (yamasaki et al., 2003; wang et al., 2011). therefore, change of sex expression in gynoecious cucumber is necessary. in general, auxin, ethylene and cytokinins promote female sex expression in various monoecious and dioecious systems (mohan ram and sett, 1982). exogenous application of plant growth regulators could alter the sex ratio if applied at the twoor fourleaf stage, which is the critical stage at which the suppression or proportion of either sex is possible (hossain et al., 2006). the effect of ethylene can be antagonized by specific ethylene inhibitors (hirayama and alonso, 2000). silver ions (ag+) restrain the physiological effect of the ethylene by blocking the ethylene receptors (hirayama and alonso, 2000). it is known that silver ions (ag+) applied as silver nitrate (agno3) or as silver thiosulfate [ag(s2o3)2 -3] replace copper ions (cu+) which is a part of the ethylene receptor preventing the receptor from responding to ethylene (abeles et al., 1992). another inhibitor of the ethylene action, agno3, suppresses the development of female flowers and induces the male ones (kumar et al., 2009; stankovic and prodanovic, 2002). male flower formation increased proportionally with the concentration of agno3 applied (stankovic and prodanovic, 2002). according to previous reports, agno3 induced more male flowers than ga3 on gynoecious breeding lines of cucumber (kalloo and franken, 1978) and summer squash (yongan et al., 2002). the positive effects of agno3 on male flower production in cucumber have reported by karakaya and padem (2011). thappa et al. (2011) used different plant growth regulators (ethephon, naphthalene acetic acid and maleic hydrazide) at the two, four and sixleaf and full-bloom stages in cucumber to induce male flowers. law et al. (2002) reported that silver thiosulfate, an ethylene inhibitor, enhanced stamen development in female white campion (silene latifolia). however, little effort seems to have been directed toward the study of the effects of silver ions on male flower production sprayed at different growth stages and if the numbers of spray applications could improve the effectiveness of the induction. on the other hand, it seems that there are few studies that correlate the effect of environmental conditions, especially season, to the number of spraying and leaf-stage growth on male flower induction of plant treated with silver ions. although silver is a toxicity ion, however the amount of its toxicity depends on species, silver concentration, times of application and plant growth stage spraying and exposure media. for exmple fuente et al. (2014) applied different concentrations of agno3 (0, 30, 60, 90, 200 mg l-1) at intervals of 8 days throughout the crop cycle (90 days) in watermelon. their results showed that silver accumulate in root more than other parts of plants and antioxidant in fruits increased in plants exposed to 30 mg l-1 agno3, but lycopene content decreased. therefore the main objectives of this study was to investigate the effects of chemical application [agno3 and ag(s2o3)2 -3], different leaf growth stages and number of spraying on flower and fruit characteristics of greenhouse cucumber in two different golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 207 seasons to identify the best condition and the optimal level of chemical application to boost the formation of male flowers. our findings will help to develop practical recommendations on these issues for improve male flowers production in cucumber breeding programs. furthermore, this work aimed to evaluate the stability of sex expression of a gynoecious cultivar adrian in different seasons. the present investigation was done as a first step to a hybridization program of cucumber. 2. materials and methods planting material and field evaluation the seeds of cv. adrian as a gynoecious genotype were sown at the mid autumn of 2014 and mid spring of 2015 at research greenhouse of agriculture department at islamic azad university isfahan branch, isfahan, iran (51° 36´ longitude and 32°63 latitude). the soil used was loam with ph 7.7 and electrical conductivity (ec) of 4.1 (ds/m). seeds were planted to soil directly and covered with peat moss and coco peat. plants were disposed in two rows, where the spaces between cucumber seedling was 50 cm and within the rows were 90 cm respectively, and 180 cm was left between every couple rows. the greenhouse air temperature at the growing period was maximum 29°c/18°c (day/night) in first season (autumn-winter) and 34°c/20°c (day/night) in second season (spring-summer) with a relative humidity of about 55% and 60%, respectively. nutrient levels in the irrigation solution water were n:216 (mg l-1), p:58 (mg l-1), k:286 (mg l-1), ca:185 (mg l-1), mg:185 (mg l1), s:43 (mg l-1), fe:5.59 (mg l-1), mn:1.97 (mg l-1), b:0.7 (mg l-1), zn:0.2 (mg l-1), cu:0.07 (mg l-1) and mo:0.05 (mg l-1). different fertilizers were used based on soil analysis that included: potassium nitrate, ammonium nitrate, magnesium nitrate, iron and other mineral elements such as sulphate dissolved in the irrigation water. dichlorvos, trigard, abamectin and organic neem oil were applied for insect control. the same fertilizers and pest management were used for all plants in the same time. drip irrigation was applied when needed. the source of water was urban water with electrical conductivity (ec) of 0.4 (ds/m). the ph of the irrigation water was adjusted to 6.5 by adding nitric acid. silver application different chemical treatments including agno3 [h1=100 ppm (0.59 mm), h2=200 ppm (1.18 mm) and h3=300 ppm (1.77 mm)], and ag(s2o3)2 -3 [h4=200 ppm (0.6 mm) and h5=500 ppm (1.5 mm)] and control (h6) were applied to induce changes in sex expression. agno3 was purchased from merck chemical company and ag(s2o3)2 -3 was synthesized according to law et al. (2002). the solutions were applied as a spray on the whole plant. water was sprayed on the control plants. spraying was accomplished at 5, 10, and 15-leaf growth stages (five leaves stage: ls1, ten leaves stage: ls2, and fifteen leaves stage: ls3). chemical treatments were employed in single or double spraying at one week interval. agno3 and ag(s2o3)2 -3 applications were conducted early in the morning (before sunrise) to avoid plant sunburn. each treatment was applied with adequate amounts of the solutions to assure that all the leaves were completely wetted in each spray event. the total time of experiment in every season was about 4 months after sowing and measurement of traits were done in this period. studied traits the traits of interest were measured on eight plants per replication and included: days to male flower expression (days to first male flower appearance after sowing) (dmf); the node number that the first male flower was appear (nnmf); the mean of internode length (the mean of five internode from node number 15 to 20) (il); the number of male flower (total male flower that opened in whole plant) (nmf), male flowering period (days from observation the first male flowering until the last male flowering observation (mfp); male flower diameter (was recorded at the widest points) (mfd); single fruit weight (from dividing total fruit weight to total fruit number in every harvesting) (sfh); fruit number per harvesting (fnh); fruit weight per harvesting (fwh) (the numbers and the weights of all the fruits harvested in each plot in each harvesting). statistical analysis the experiment was conducted as a combined analysis of variance with three different factors (chemical treatments, leaf growth stage, and number of spraying) under two different seasons. the data collected were subjected to analysis of variance (anova) based on a completely randomized block design with three replications using the general linear model (glm). statistical analysis system program (sas ver. 9) was used for data analysis. the differences between applications were decided on the basis of the least significant difference (lsd) test adv. hort. sci., 2017 31(3): 205-214 208 (p<0.05) according to their importance at the 0.05 confidence level. 3. results and discussion the result of analysis of variance based on a factorial experiment is presented in table 1. according to table 1, the season (s) had significant effect on all of the studied traits, except for single fruit weight. this result is logical, since phenotypic expression of sex is strongly modified by environmental and hormonal factors (mohan ram and sett, 1982). long days, high temperature, and silver ion promote formation of male flowers, whereas short days and low temperature promote the formation of female flowers (perltreves, 1999). clearly, chemical treatments (ch) had significant effects on all the traits studied, except for fruit number per harvesting (table 1). number of spraying (single or double) also had significant effects on the days to male flowering, the node number of first male flower, the mean of internode length, the number of male flower and male flowering period (table 1). hallidri (2004) reported that increased number of spraying events had significant effects on male flower production. he suggested that doses of 400 and 500 ppm of agno3 produced the highest number of male flowers. the leaf growth stage (lgs) of spraying showed significant differences on days to male flowering, the node number of first male flower, male flowering period, male flower diameter and fruit weight per harvesting (table 1). the interaction effect of environment × silver application had a significant effect on all traits with the exception of male flower period and fruit number per harvesting (table 1). interaction of season × leaf stage was significant for days to male flowering, the node number of first male flower, the number of male flower and male flower diameter. interaction of number of spray events × chemical application had a significant effect on days to male flowering, the node number of first male flower, the number of male flower and single fruit weight (table 1). interaction of chemical application × leaf stage of spraying showed significant differences on days to male flowering and the node number of first male flower (table 1). the interaction of environment × chemical application × leaf stage of spraying showed significant difference on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering. study of these interactions df= degree of freedom; s= season; ch= chemical treatments [silver nitrate (agno3) and silver thiosulphate ag(s2o3)2 -3); sp= number of spraying (single or double); lgs= leaf growth stage (5, 10 and 15). dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). ns= non significant. *, ** significant at p<0.05 and p<0.01, respectively. table 1 analysis of variance for single and combined effects of different studied traits in greenhouse cucumber source of variation mean squares of studied traits df dmf nnmf il nmf mfp mfd sfw fnp fnh season (s) 1 ** ** ** * ** ** ns ** ** replication (season) 4 ns ns ** ns ns ns ** ns ns chemical treatments (ch) 5 ** ** ** ** ** ** ** ns ** number of spraying (s0) 1 ** * ** ** ** ns ns ns leaf growth stage (lgs) 2 ** 9** ns ns ** ** ns ns ns** sp × ch 5 ** ** * ** ns ** ** ns ns s×sp 1 ns ns ns * ns * ns ns ** s×lgs 2 ** ** ns * ns ** ns ns ns ch×sp 5 * * ns ** ns ns * ns ns ch ×lgs 10 * ** ns ns ns ns ns ns ns sp×lgs 2 ns ns ns ns * ns ns ns ns s×ch×sp 5 ns ns ns ns ns ns ns ns ns s×ch×lgs 10 ** ** ns ** * ns ns ns ns ch×sp×lgs 10 ns ns ns ns ns ns * ns ns s×sp×lgs 2 ns ns ns ns * ns ns ns ns s×chsp×lgs 10 ns ns ns ns ns ns ns ns ns residual 140 ns ns ns ns ns ns ns ns ns golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 209 showed that important traits related to change of sex expression such as days to male flowering, node number of first male flower, and number of male flower more affected by different interactions in contrast to vegetative and yield related traits. therefore selection of best treatments should be done based on other treatments. for example numbers of male flower are affected by silver application, leaf stage, number of spraying and all their interactions. there was no significant difference for other triple interactions and environment × chemical application × leaf stage of spraying × number of spray events interaction in all of the studied traits. effects of season on the studied traits the comparison between two seasons showed that the first season had superior mean on days to male flowering, the node number of first male flower, male flower diameter and single fruit weight. it could be concluded that in the first season male flowers expressed later than second season and therefore dmf and nnmf were increased (table 2). on the other hand, in second season the number of male flower was increased (from 36 to 100 male flowers) and also the time of male flowering was earlier that is related to high temperature and long days. this result showed that environmental conditions, especially low temperature, could retard male flower formation and confirmed also that cucumber is a thermophilic plant. accordingly, stankovic and prodanovic (2002) reported that sowing season affected the number of male flowers. the interaction effect of season × chemical application × leaf stage was significant for number of male flower, days to male flowering and period of male flowering that all of them are related to sex expression, even though this interaction was not significant for vegetative related traits (table 2). effects of silver application on the studied traits in this study, different doses of chemical treatments [agno3 and ag(s2o3)2 -3] were used. the mean comparisons showed that chemical application increased the mean values of days to male flowering (table 3). there was a narrow variation in days to male flowering between different chemical treatments. the least day to male flowering was at a high concentrations of agno3 and ag(s2o3)2 -3 (table 3). this result showed that high concentrations of different chemicals agent caused earlier male flowering. dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 2 the mean comparison of different studied traits in cucumber in different seasons season traits dmf nnmf il nmf mfp mfd sfw fnh fwh s1 23.08 a 20.17 a 7.13 b 36.24 b 12.20 b 5.46 a 73.17 a 1.93 b 138.80 b s2 19.89 b 11.96 b 7.63 a 99.78 a 13.35 a 4 74.95 a 3.60 a 272.96 a table 3 the effects of various chemical treatments application on studied traits in cucumber means followed by the same letter in each row were not significantly different at 0.05 level using lsd test. traits control chemical treatments agno3 ag(s2o3)2 -3 100 ppm 200 ppm 300 ppm 200 ppm 500 ppm days to male flowering 0.23 c 26.41 a 26.08 a 25.04 b 26.02 a 25.17 b node number of male flower 0.13 b 20.18 a 19.11 a 19.23 a 19.08 a 18.68 a the mean of internode length 6.25 b 7.38 a 7.8 a 7.64 a 7.28 a 7.65 a the number of male flower 0.18 d 20.89 cd 68.07 b 129.76 a 44.79 bc 144.4 a male flowering period 0.074 e 10.16 d 16.13 bc 17.366 ab 14.71 c 18.21 a male flower diameter 3.29 d 5.36 c 5.45 bc 5.65 ab 5.36 c 5.71 a single fruit weight 84.7 a 73.9 b 70.95 b 72.15 b 70.68 b 71.89 b fruit number per harvesting 2.69 a 2.71 a 2.65 a 2.88 a 2.84 a 2.84 a fruit weight per harvesting 219.74 a 205.88 a 190.87 a 212.9 a 199.72 a 206.22 a adv. hort. sci., 2017 31(3): 205-214 210 the highest value for male flower diameter observed at 500 (ppm) of ag(s2o3)2 -3 and 300 (ppm) of agno3 (table 3). therefore these concentrations of ag (s2o3)2 -3 and agno3 could affect both vegetative and reproductive growth of flower. however there was no significant difference between all treatments for internode length. hallidri (2004) reported that the greatest number of staminate nodes was produced on plants sprayed two or three times with 400 to 500 (ppm) of agno3. rafee kher et al. (2002) reported that 200 (ppm) of ga increased the length of internode in cucumber. according our results the highest value for the number of male flower was denoted at 300 (ppm) of agno3 and 500 (ppm) of ag(s2o3)2 -3 about 130 and 144 male flower, respectively. law et al. (2002) reported that application of ag2s2o3 produced stamens in female flowers of white campion (silene latifolia) with longer filaments and larger anther locules. yongan et al. (2002) reported that agno3 had more significant effects than ga3 on male flower production in summer squash. stankovic and prodanovic (2002) reported that increasing concentrations of agno3 from 0.01% to 0.04% enhanced the number of male flowers in gynoecious lines, which is similar with the results obtained in the present study. jadav et al. (2010) reported that ethrel (200 ppm) had the greatest effect on male flower production among the hormones they investigated (ga3, ethrel, naphthalene acetic acid, and absisic acid). jutamanee et al. (1994) reported that the effects of different doses of ga3 and agno3 depended on the genotype and photoperiod. similar to their results, agno3 in the present study induced the formation of male flowers in all concentration. hallidri (2004) reported the concentration of 100 (ppm) of agno3 was ineffective on male flower induction. kalloo and franklen (1978) reported that different doses of agno3 (50, 200, and 500 mg l-1) led to non-significant effects on male flower production, fruit weight and fruit number. they showed similar trends in all the treatments. however in this study all concentrations of agno3 (100, 200, 500 ppm) produced male flower with significant differences. one explanation which might account for disparities observed between experiments was the difference in average quanta of solar radiation received by the plants during two different environmental conditions in two seasons. this result confirms the inducing effects of different chemical compounds used on male flower production. the highest value for single fruit weight was observed at control. mean comparison showed that chemical application has reduced the mean of single fruit weight in comparison to control, because production of male flower in every treated plant will reduce female flower number in contrast to control. there were no significant differences between all treatment for fruit number per harvesting and fruit weight per harvesting (table 3). so, this result could demonstrated that chemical application has only effects on flower number and its sex expression. effects of number of sprays on the traits studied number of sprays (single or double) showed significant effects on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering (table 4). according to table 4, double spraying treatment gave rise to higher mean values for days to male flowering, the node number of first male flower, the number of male flower and period of male flowering. on the other hand, double spray treatment was found to affect only sex expression while it had no effect on the traits related to morphology and fruit yield. effects of leaf stage on the studied traits the comparison of the mean values for the studied traits at different stages of leaf growth [five leaf dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 4 the effects of single and double spraying on studied traits in cucumber number of spraying traits dmf nnmf il nmf mfp mfd sfw fnh fwh single spraying (sp) 20.85 b 15.49 b 7.31 a 49.15 b 11.61 b 5.11 a 74.04 a 2.76 a 205.96 a double spraying (dp) 22.13 a 16.64 a 7.44 a 86.86 a 13.94 a 5.16 a 74.08 a 2.77 a 205.82 a golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 211 stage: ls1, ten leaf stage: ls2, and fifteen leaf stage: ls3] is presented at table 5. the highest mean for dmf and nnmf were observed at ls3 stage (table 5).this logical result is resulted from the most distance (days) between planting time to fifteen leaf stage. on the other hand, the highest dmf at 15 leaf growth stage belonged to 200 ppm ag(s2o3)2 -3 treatment (fig. 1). el-ghamriny et al. (1988) confirmed that sex differentiation in cucumber takes place at the 2-true leaf stage and that this was the best time for studying the effects of growth regulators on sex expression. there was no-significant difference between three leaf growth stages for number of male flower (table 5). therefore use of silver ions at different three growth stages had similar effects on modification of flower sex type in cucumber. therefore, if it is necessary to separate monoecious or androecious from gynodioecious plant types in breeding programs (for example in line production), breeder could apply these chemical agents at 15 leaf stage, when sex form of plant has been expressed. raymond (2004) proposed that 1000 ppm of ga3 at the 2-leaf stage had the best effect on male flower production with 3 hormone applications at 2-week intervals in cucumber. also, the highest values for period of male flowering and male flower diameter were obtained at the five leaf stage; that is logical, since the period between male flower observation and end of male flowering in ls1 was higher than ls2 and ls3. as already mentioned, male flower diameter was greater at the 5-leaf stage, which might be caused by the higher production of pollen and the increased capacity for crossing in the breeding programs. effect of different factor interactions on number of male flower as the number of male flower trait is the most important trait related to male flower induction in cucumber, the different significant interaction effects in this trait was assayed (table 2). the highest number of male flower was recorded at double spraying in 500 ppm of ags2o3 and double spraying at 300 ppm of agno3, respectively of 187.36 and 179 flowers (fig. 2 a). in the two chemical treatments at higher concentration, double spraying caused increasing of male flower number, although differences between one and two spraying in low concentrations of chemical agents was not significant. the highest (119.2) and the least (24) number of male flower was observed at second season and the first season at fifteen leaf stage, respectively (fig. 2 b). in all stages, high number of male flower was observed in second season and pointed out significant differences with first season. also, the most number of male flowers was obtained in high concentration of chemical treatments at the second season, however in all chemical treatments the number of male flowers was more than at second season (fig. 2 c). again, the second season showed the highest number of male flower with double spraying in contrast to first season, although double spraying in two seasons was more than single spraying (fig. 2 d). the triple interaction effects of season, leaf growth stage and chemical treatment on male flower number showed that male flower number in all three leaf growth stages and all chemical treatments under second season were more than first season, specially high concentration dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 5 the mean comparison for some of the studied traits in different leaf growth stages in cucumber developmental stages traits dmf nnmf il nmf mfp mfd sfw fnh fwh five leaf stage (ls1) 19.44 c 8.73 c 7.55 a 68.94 a 14.21 a 5.40 a 74.63 a 2.88 a 216.06 a ten leaf stage (ls2) 21.40 b 17.10 b 7.40 a 63.47 a 12.40 b 5.05 b 73.00 a 2.69 a 195.85 a fifteen leaf stage (ls3) 23.62 a 22.38 a 7.18 a 71.60 a 11.71 b 4.95 b 74.55 a 2.73 a 205.76 a fig. 1 the interaction effect of chemical treatment × leaf growth stage on the days to male flowering in cucumber adv. hort. sci., 2017 31(3): 205-214 212 of chemical treatments in fifteen leaf growth stage under second season (table 6). these results demonstrated that the second season had progressive effect on induction and development of male flower and environmental condition could be having a significant effect on this phenomenon. as mentioned before, in second season the temperature of greenhouse was higher than first season and also days were longer. these two reasons changed sex expression in second season more than first season. 4. conclusions phenotypic expression of sex determining loci in cucumber is strongly modified by environmental, chemical and hormonal factors. the results of the experiment revealed that agno3 and ag(s2o3)2 -3 have similar and positive effects on the male expression of cucumber. this study showed that male flower induction was induced by all concentrations of silver ions. these two chemical agents could induce a high number of male flowers in 300 and 500 ppm concentration [agno3 and ag(s2o3)2 -3], respectively. chemical treatments with silver ions as agno3 and ag(s2o3)2 -3 application can, therefore, be recommended for enhancing male flower production in cucumber, if further study will confirm the accumulations of silver in the fruit is negligible for the human health. as the time of agno3 spraying in this experiment was only three times, so the toxicity of this ion was low in a short time of period. on the other hand, as this chemical agent only was applied in experimental greenhouse, therefore the fruits of treated chemical treatment seasons season 1 season 2 ls1 ls2 ls3 ls1 ls2 ls3 control 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n agno3 (100 ppm) 6.83 mn 8.00 mn 9.17 mn 42.42 i-n 31.75 j-n 27.17 k-n agno3 (200 ppm) 43.00 i-n 20.33 lmn 14.67 mn 64.33 h-m 123.33 d-h 142.75 d-g agno3 (300 ppm) 122.33 d-h 91.00 e-j 19.33 lmn 145.33 def 147.42 cde 253.17 a ag(s2o3)2 -3 (200 ppm) 16.331 mn 26.00 k-n 19.00 lmn 84.58 f-k 38.00 i-n 84.67 f-k ag(s2o3)2 -3 (500 ppm) 77.33 h-l 97.17 e-i 81.83 g-k 224.83 ab 178.75 bcd 206.5 abc ls1= five leaf growth stage; ls2= ten leaf growth stage; ls3= fifteen leaf growth stage. means followed by the same letter were not significantly different at 0.05 level using lsd test. table 6 the effects of interaction between chemical treatment, seasons and leaf growth stage on male flower number in cucumber fig. 2 the effect of different interactions (a, b, c and d) on the number of male flower in cucumber. golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 213 plants were disposed from toxic effects of ag ion. also, the morphologic appearance of the cucumbers showed no abnormality after ion application. overall, this result indicates that silver ion could change sex expression in higher temperatures and longer days with high concentration and when applied in 15 leaf stage for agno3 and in 5 and 15 leaf stage for ag(s2o3)2 -3, although this ion is able to modify sex form in lower concentration and earlier growth stage in cucumber. although double spraying led to late male flowering, however this treatment could increase number of male flower significantly. therefore, the double spray treatment is proposed for male flower production in greenhouse conditions. this can be beneficial for breeding programs that need high numbers of male flowers and flowering periods, especially for parents that have different periods of growth stage. the interaction effect showed that the highest number of male flowers was induced by applying ag(s2o3)2 -3 (500 ppm) in 5 and 15 leaf stage in high temperature about maximum 35°c at day and minimum 20˚c at night. another finding of this study was that the number of male flowers showed significant increase with increasing doses of agno3 from 100 ppm to 300 ppm and ag(s2o3)2 -3 from 200 ppm to 500 ppm. therefore, high dosages of agno3 and ag(s2o3)2 -3 should be selected for inducing male flowers in cucumber. the highest dosage of chemical agents led to decrease in days to male flowering and the mean of internode length that is related to the effect of silver ion on sex form earlier than low dosage of this ion. leaf stage of the spray event was found to have a toxic effect on fruit. over all, this result indicates that high concentration of silver ion could change sex expression at higher temperatures and longer days in 15 leaf stage for agno3 and in 5 and 15 leaf stage for ag(s2o3)2 -3. because the highest level of number of male flower and male flower period and the lowest level of days to male flowering were obtained in these treatments. finally, male flowering started about 3 weeks after treatment and lasted for a period of up to 3 weeks thereafter. plants treated with silver ions did not elongate more than normal plants and grew normally; 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[schefflera arboricola (hayata) merr.] via direct shoot regeneration o. rezaei baghbidi, a. jowkar (*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: in vitro propagation, naa, schefflera arboricola, shoot induction, tdz, variegated leaves. abstract: schefflera [schefflera arboricola (hayata) merr.] is one of the most popular ornamental house plants conventionally propagated by seeds. rapid multiplication of elite clones is an important driving force for the pot plant’s market. in this regard, in vitro clonal propagation of three schefflera cultivars, ‘luseane’, ‘charlotte’ and ‘gold capella’, was examined. sterilization was done by 70% ethanol for 2 min and 1% sodium hypochlorite solution for 15 min. shoot proliferation of the nodal segments was dependent on cytokinin supply. the greatest number of shoots was obtained when nodal explants were cultured on the ms medium with 0.5 mg l-1 tdz for ‘luseane’, or 8 mg l-1 ba for ‘charlotte’ and ‘gold capella’. subculture of nodal segments harvested from the in vitro derived axillary shoots on the multiplication medium enabled continuous production of healthy shoots with similar frequency. plantlets of ‘luseane’ and ‘gold capella’ demonstrated 100% rooting using 2 mgl-1 naa, while ‘charlotte’ showed 93.75% root induction by 1 mgl-1 naa. plantlets were acclimatized successfully using peat moss and sand mixture (‘luseane’), loam soil, sand and leaf compost (‘charlotte’) or peat moss and perlite mixture (‘gold capella’). 1. introduction dwarf schefflera [schefflera arboricola (hayata) merr.] is an evergreen ornamental plant of the araliaceae family, native to china and taiwan (ohashi, 1993). it is mostly used indoors as a foliage pot plant because of the attractiveness of the umbrella-like palmately compound leaves and variegated cultivars (gilman and watson, 1994; chen et al., 2002). schefflera’s ability to clean the air and its tolerance to harsh interior environments has further increased its worldwide popularity (yang et al., 2009; dela cruz et al., 2014). to satisfy grower’s calls for potted plants of schefflera, methods for rapid propagation of selected cultivars are crucial. multiplication of this house plant is mainly by seeds which results with the segregation of the progeny traits and limited to its native plantation in the tropics (griffith, 1998; chen at al., 2002) and only to cultivars of (*) corresponding author: ajowkar@gmail.com citation: rezaei baghbidi o., jowkar a., 2018 micropropagation of dwarf schefflera [schefflera arboricola (hayata) merr.] via direct shoot regeneration. adv. hort. sci., 32(2): 205-212 copyright: © 2018 rezaei baghbidi o., jowkar a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 november 2017 accepted for publication 24 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 205-212 206 schefflera which have non-variegated green leaves (marcotrigiano, 1997). other propagation means are leaf-bud cuttings (hansen, 1986), air layering (gilman and watson, 1994) and stem cuttings (hansen, 1986). these practices are hindered by difficulties such as low number of propagules per plant, increased time of production and the risk of disease spread from several pathogens such as pseudomonas cichorii, xanthomonas campestris, phytophthora parasitica, pythium splendens and alternaria panax (chase and poole, 1986). micropropagation is a reasonably more efficient way for schefflera production as a greater number of plants can be produced faster compared to the traditional cuttings. moreover, in vitro culture of tropical ornamental plants has been recommended as a tool to eradicate the diseases that are frequently widespread in the mother plants (hartmann and kester, 2011). tissue culture has furthermore resulted in enhanced features compared to common propagations. it was revealed that micropropagated foliage pot plants such as syngonium, spathiphyllum, dieffenbachia (conover, 1992) and philodendron (chen et al., 2012) had a packed and denser plant forms in comparison to the conventional stem cuttings. consequently, small tissue culture-grown scheffleras may be readily suitable for the limited space available in the terrariums or bonsai pots. despite the fact that micropropagation is extensively exploited by the floriculture industry, their formulas are not released to the general public. to our knowledge, there is no tissue culture protocol available in the literature for s. arboricola, though a few number of articles have been published so far for other araliaceae species with horticultural importance such as cussonia paniculata (tetyana and van staden, 2001), fatsia japonica (choi et al., 2005), eleutherococcus senticosus (amin et al., 2003; you et al., 2005), panax quinquefolius (uchendu et al., 2011) hedera helix (sivanesan et al., 2011) polyscias balfouriana (ilyas et al., 2013) and polyscias fruticosa (sakr et al., 2014). the current research developed, for the first time, a micropropagation procedure for three cultivars of shefflera with green, white and yellow variegated leaves. in order to reach a high propagation rate and get well plantlets, the effects of various sterilization treatments, plant growth regulators’ type and concentration were studied on the survival rate, shoot proliferation and rooting of scheffleras. subsequent acclimatization of the micropropagated plantlets was also investigated using different potting media. 2. materials and methods plant material and culture medium three marketable cultivars with green, white and yellow variegated leaves (i.e. ‘luseane’, ‘charlotte’ and ‘gold capella’, respectively) were used for micropropapagation. the explants were cultured on ms (murashige and skoog, 1962) medium supplemented with 3% (w/v) of sucrose, and 0.65% (w/v) of agar. the ph of the medium was set to 5.7 before autoclave sterilization (i.e., 20 min, at 121°c). the plant growth regulators were added to the medium later by filter sterilization. the sterile nodal segments were established in culture medium containing 2 mg l-1 benzyl adenine (ba). in order to proliferate the shoots, the emerging buds were removed after 30 days and subcultured to media containing different cytokinin treatments. sterilization of three schefflera cultivars the stock plants were sprayed with ridomil fungicide (0.3%) one week before explant excision. stem cuttings were soaked in 0.1% detergent solution for 10 min, then washed under running tap water for 30 min. the explants were moved to laminar flow cabinet and 1.5-cm long nodal segments were excised from the stem. for surface sterilization of the explants, 70% etoh was tested for 0, 30, 60, 90, 120, 150 and 180 seconds. explants were then disinfected using 0, 0.5, 0.75, 1 or 1.25% (v/v) sodium hypochlorite containing 0.01% tween-20 for 5, 10 or 15 min on a shaker. after four rinses with sterile water and excision of the damaged edges, the explants were cultured on pda medium (300 g potato extract, 30 g l-1 of dextrose and 8 g l-1 of agar) for the evaluation of the contamination. five replicates were examined for each treatment with three explants in each flask. contaminations and survival rates were recorded after 1 month and those having noticeable infection signs were instantly discarded. effect of cytokinin type and concentration on shoot proliferation inducing new shoots was carried out using ba and kinetin (kin) at 0, 1, 2, 4 and 8 mg l-1 concentrations while thidiazuron (tdz) was tested at 0, 0.125, 0.25, 0.5 and 1 mg l−1 concentrations. five replicate flasks were used for each treatment with three explants in each flask. the number and length of the proliferated shoots, and number of leaves produced per explant were recorded after 30 days. the regenerated shoots were later subcultured to the best cytokinin-containing ms medium. rezaei baghbidi and jowkar micropropagation of dwarf schefflera 207 effect of auxin type and concentration on root induction shoots from the finest proliferation treatment were put in the glass flasks of ms medium containing 0, 0.1, 0.5, 1, 2 or 4 mg l−1 of indole-3-butyric acid (iba) or naphthaleneacedic acid (naa). after 15 days the shoots were transferred to half-strength ms medium devoid of auxins. five replicate flasks were tested in each treatment with three shoots in each flask. the number and length of the induced roots and rooting percentage of the shoots were recorded 15 days later. effect of potting media on plant acclimatization rooted shoots were carefully taken out from the glass flasks and washed under distilled water to get rid of attaching agar from the roots. the plantlets were then transferred to 10-cm plastic pots filled with different potting media: peat moss, peat moss and perlite (1:1), peat moss and sand (1:1), loam soil, sand and leaf compost (1:1:1). ten replications were tested for each of the treatments. potted plants were grown in a greenhouse with temperatures ranging from 24 to 28°c, relative humidity of between 70 and 90%, and light intensity of 35 µmol m-2 s-1 under a 12 h photoperiod. after 45 days the survival rate and plant height were analyzed. culture conditions, experimental design and data analysis the in vitro researches were carried out with an environment temperature of 25±2°c and a 16/8 h light/dark photoperiod delivered by cool fluorescent lamps at 35 µmol m-2 s-1. all the tests were arranged in a completely randomized design. analysis of the variances was done by sas software (sas institute inc., 2002) and means were compared by lsd test at 5% probability level. 3. results sterilization of three schefflera cultivars for the determination of the most effective surface sterilization, explants were soaked in 70% etoh solution for 0-180 sec. as the soaking time was increased to 180 sec, surface sterilization was increased gradually and eventually reached 100% in all three cultivars, however viability was rapidly decreased after 120 sec (table 1). thus, 120 sec, providing the highest disinfection (72.8%, 79.6% and 72.8% for ‘luseane’, ‘charlotte’ and ‘gold capella’, respectively) and viability (72.8%, 74.6% and 72.8% for luseane’, ‘charlotte’ and ‘gold capella’, respectively) altogether, was selected. the results were not significant between the cultivars. the explants of ‘luseane’ were effectively disinfected when 1.25% sodium hypochlorite was applied for 15 min, however this application resulted with a significant decrease of the viability to 13% (table 2). the most effective combination of disinfection (93.2%) and viability (86.4%) was obtained using 1% sodium hypochlorite for 15 min. ‘charlotte’ demonstrated a similar pattern of sterilization. the utmost control of contamination (93.2%) and viability (86.4%) was observed using 1% sodium hypochlorite for 15 min or 1.25% sodium hypochlorite for 10 min. similarly, also for ‘gold capella’, the best percentages of sterilization (93.2%) and viability (86.4%) were obtained by using 1% sodium hypochlorite for 15 min. effects of cytokinin type and concentration on shoot proliferation effects of benzyl adenine and kinetin treatments on each schefflera cultivars. the increase of ba concentration to 8 mg l-1 significantly increased the numtable 1 effects of ethanol treatments on surface sterilization and viability of three schefflera cultivars data (±se) are the mean values of five replicates with three explants in each. for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). 70% ethanol (sec) ‘luseane’ ‘charlotte’ ‘gold capella’ bacterial sterilization (%) viability (%) bacterial sterilization (%) viability (%) bacterial sterilization (%) viability (%) 0 0±0 e 0±0 e 0±0 d 0±0 d 0±0 e 0±0 d 30 19.8±8.08 d 19.8±0 d 13.2±8.08 d 13.2±0 d 13.2±8.08 de 13.2±0 cd 60 33±0 d 33±0 cd 46.2±8.08 c 46.2±0 b 26.4±6.60 d 26.4±0 c 90 52.8±8.08 c 52.8±0 b 53.2±8.25 c 53.2±0 b 59.6±6.65 c 59.6±0 a 120 72.8±6.80 b 72.8± 0 a 79.6±8.33 b 74.6±6.6 a 72.8±6.80 bc 72.8±0 a 150 93.2±6.80 a 39.6±6.60 c 100±0 a 33±0 c 86.4±8.33 ab 39.6±6.6 b 180 100±0 a 19.8±8.08 d 100±0 a 13.2±8.08 d 100±0 a 19.8±8.08 c adv. hort. sci., 2018 32(2): 205-212 208 ber of shoots per explant of ‘luseane’ to 3.75 (table 3). this concentration of ba also produced 12.5 leaves per explant. however, the largest shoot length of 0.88 cm was obtained using 4 mg l-1 ba. on the contrary, increasing kin concentration to 4 mg l−1 and above resulted with the decrease in the shoots number. kin also produced significantly lesser leaves at all the concentrations tested, and did not significantly increase the shoot length. treatment of ‘charlotte’ with cytokinins revealed a similar pattern as ‘luseane’. indeed, the greatest shoot proliferation of 3.75 shoots per explant was obtained using 8 mg l−1 ba, which also produced 15.3 leaves per explant and maximum shoot length of 0.90 cm. also kin increased the shoots number at the same concentration, though it was significantly lower than what was obtained with ba. the maximum shoot length and number of leaves observed with 8 mg l-1 kin were 0.73 cm and 5.67, respectively, and both were significantly lower than what was observed with ba. proliferation of the ‘gold capella’ with 8 mg l-1 ba resulted with maximum of 1.75 shoots per explant, the largest shoot length of 0.83 cm and 7.58 leaves per explant, which were all significantly higher than kin treatments (fig. 1). adding 2 or 4 mg l-1 kin to the medium slightly increased the shoot length compared to the control. table 2 effects of sodium hypochlorite treatments on sterilization and viability of three schefflera cultivars data (±se) are the mean values of five replicates with three explants in each. for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). cultivar minutes sodium hypochlorite (%) sterilization (%) viability (%) 0 0.5 0.75 1 1.25 0 0.5 0.75 1 1.25 ‘luseane’ 5 13.2±8.08 g 24.75±6.39 fg 33±0 e-g 41.25±6.39 d-f 49.5±7.38 de 13.2±0 f 24.75±0 ef 33±0 d-f 41.25±0 c-e 49.5±0 cd 10 13.2±8.08 g 33±0 e-g 52.8±8.08 de 74.5±6.58 bc 86.4±8.33 ab 13.2±0 f 33±0 d-f 52.8±0 cd 74.5±0 ab 79.6±6.80 ab 15 13.2±8.08 g 49.5±7.38 de 59.6±12.51 cd 93.2±6.80 ab 100±0 a 13.2±0 f 49.5±0 cd 59.6±0 cd 86.4±6.80 a 13.2±8.08 f ‘charlotte’ 5 19.8±8.08 f 33±10.44 ef 33±0 ef 41.25±6.39 de 49.5±7.38 de 19.8±0 ef 33±0 d-f 33±0 d-f 41.25±0 c-e 49.5±0 cd 10 19.8±8.08 f 41.25±6.39 de 52.8±8.08 de 74.5±6.58 bc 93.2±6.80 ab 19.8±0 ef 41.25±0 c-e 52.8±0 b-d 74.5±0 ab 86.4±6.80 a 15 19.8±8.08 f 49.5±7.38 de 59.6±12.51 cd 93.2±6.80 ab 100±0 a 19.8±0 ef 49.5±0 cd 59.6±0 bc 86.4±6.80 a 13.2±8.08 f ‘gold capella’ 5 13.2±8.08 g 19.8±8.08 fg 26.4±6.60 e-g 39.6±6.60 d-f 52.8±8.08 cd 13.2 0 f 19.8±0 ef 26.4±0 d-f 39.6±0 c-e 52.8±0 bc 10 13.2±8.08 g 26.4±6.60 e-g 52.8±8.08 cd 66±0 bc 86.4±8.33 ab 13.2±0 f 26.4±0 d-f 52.8±0 bc 66±0 ab 86.4±6.80 a 15 13.2±8.08 g 46.2±8.08 c-e 59.6±12.51 cd 93.2±6.8 0ab 100±0 a 13.2±0 f 46.2±0 b-d 59.6±0 bc 86.4±6.80 a 13.2±8.08 f cultivar control ba (mgl-1) kin (mgl-1) 1 2 4 8 1 2 4 8 ‘luseane’ no. of shoots 0.83±0.1 f 1.25±0.05 de 1.5±0.07 c 2.75±0.08 b 3.75±0.25 a 1.42±0.03 cd 1.42±0.05 cd 1.17±0.06 e 0.92±0.08 f shoot length (cm) 0.24±0.03 f 0.50±0.02 c 0.39±0.01 d 0.88±0.01 a 0.73±0.02 b 0.27±0.03 ef 0.27±0.02 ef 0.31±0.005 e 0.31±0.01 e no. of leaves 0.66±0.36 f 8±0.64 c 8.5±0.4 c 10.42±0.81 b 12.5±2.06 a 1.42±0.32 e 2±0.49 e 2.75±0.52 d 1.67±0.27 e ‘charlotte’ no. of shoots 0.45±0.21 g 1.67±0.14 f 1.92±0.36 e 2.5±0.48 c 3.75±0.55 a 2.25±0.28 d 1.67±0.41 f 1.67±0.14 f 3±0.36 b shoot length (cm) 0.36±0.11 f 0.36±0.12 f 0.45±0.16 de 0.72±0.11 b 0.90±0.15 a 0.47±0.05 d 0.40±0.08 ef 0.38±0.05 f 0.73±0.13 b no. of leaves 1.17±0.4 g 10.5±0.74 c 10.83±0.1 c 11.83±0.74 b 15.3±1.6 a 2.25±0.31 f 3.67±1.03 e 3.33±0.36 e 5.67±0.62 d ‘gold capella’ no. of shoots 0.42±0 g 0.75±0.04 de 0.83±0.1 cd 1.08±0.08 b 1.75±0.08 a 0.67±0.08 ef 0.75±0.08 de 0.92±0.08 c 0.58±0.08 f shoot length (cm) 0.19±0.05 e 0.59±0.04 b 0.47±0.04 c 0.76±0.06 a 0.83±0.03 a 0.25±0.02 e 0.39±0.02 cd 0.33±0.01 d 0.23±0.01 e no. of leaves 0.58±0.08 e 4.41±0.64 c 5.66±0.56 b 5.83±0.52 b 7.58±0.75 a 0.75±0.21 e 0.83±0.22 e 2±0.38 d 1.66±0.14 d table 3 effects of benzyl adenine and kinetin on shoot proliferation of each schefflera cultivars data (±se) are the mean values of five replicates with three explants in each. for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). fig. 1 effect of different cytokinin treatments on shoot proliferation of schefflera cvs. luseane (a), charlotte (b) and gold capella (c). shoots were produced using ms medium supplemented with 0.5 mg l-1 tdz for ‘luseane’ and 8 mg l-1 ba for the others. rezaei baghbidi and jowkar micropropagation of dwarf schefflera 209 comparison of thidiazuron treatments on shoot proliferation of schefflera cultivars. the highest shoot proliferation (6.25 shoots per explant) was observed in ‘luseane’, using 0.5 mg l-1 tdz (table 4) (fig. 1). the maximum shoot proliferation of ‘charlotte’ and ‘gold capella’ significantly lower than ‘luseane’, were 3.33 and 1.33 shoots per explant, using 0.5 and 1 mg l-1 tdz, respectively. the largest shoot length of 2.33 cm was observed in the ‘charlotte’ when 0.5 mg l-1 tdz was added to the medium. the other cultivars’ shoot length were significantly lower. furthermore, ‘charlotte’ produced the maximum leaves per explant (i.e. 13.5 leaves) using 0.5 mg l-1 tdz, while other cultivars yielded significantly fewer leaves per explant (8.75 for ‘luseane’, 2.42 for ‘gold capella’) at 0.25 mg l-1 tdz and 1 mg l-1 tdz, respectively. effect of auxin type and concentration on root induction rooting of ‘luseane’ was 100% when 2 or 4 mg l−1 iba as well as 0.5, 2 or 4 mg l−1 naa treatments were applied (table 5). the maximum number of roots per explant (i.e. 18.67 roots) was observed using 2 mg l-1 naa (fig. 2). moreover, the largest root length of 2.74 cm was produced by this treatment. however, 0.5 or 2 mg l-1 naa appeared to have no significant differences with the former treatment. also, the highest shoot length of 2.96 cm was recorded using 1 mg l-1 naa, albeit no significant differences were found with 0.5, 2 and 4 mg l-1 naa treatments. the rooting of ‘charlotte’ raised to the maximum 93.75% using 2 mg l-1 iba or 1 mg l-1 naa. other contdz (mgl-1) luseane' charlotte' ‘gold capella’ no. of shoots shoot length (cm) no. of leaves no. of shoots shoot length (cm) no. of leaves no. of shoots shoot length (cm) no. of leaves 0 0.83±0.1 gh 0.24±0.03 i 0.66±0 36 j 0.45±0.21 i 0.36±0.11 h 1.17±0.4 i 0.42±0 i 0.19±0.05 i 0.58±0.08 j 0.125 1.65±0.02 e 0.71±0.01 ef 5.40±0.48 f 1.75±0.08 e 1.28±0.19 c 5.75±0.67 f 0.67±0h i 1.02±0.01 d 1.92±0.16 h 0.25 5.58±0.08 b 1.05±0.05 d 8.75±0.76 c 1.83±0.1 e 1.34±0.13 c 7.33±0.79 d 0.75±0.08 h 0.58±0.04 g 1.75±0.21 h 0.5 6.25±0.08 a 0.66±0.04 fg 6.25±0.82 e 3.33±0.47 c 2.33±0.25 a 13.5±2.58 a 1.08±0.08f g 0.36±0.02 h 1.75±0.16 h 1 5.75±0.25 b 0.80±0.02 e 5.75±0.61 f 2.33±0.24 d 1.76±0.15 b 9.5±1.31 b 1.33±0.02 f 0.41±0.02 h 2.42±0.08 g table 4 effects of thidiazuron on shoot proliferation of schefflera cultivars data (±se) are the mean values of five replicates with three explants in each. for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). cultivar control iba (mgl-1) naa (mgl-1) 0.25 0.5 1 2 4 0.25 0.5 1 2 4 ‘luseane’ rooting (%) 8.25±6.39 d 33±0 c 66.25±10.59 b 74.5±6.58 b 100±0 a 100±0 a 33±10.44 c 100±0 a 91.75±6.39 a 100±0 a 100±0 a no. of roots 0.25±0.11 h 2.58±0.26 g 7.43±0.24 f 11.24±0.35 d 13.83±0.56 c 10.38±0.37 de 2.75±0.12 g 9.75±0.54 e 15.08±0.34 b 18.67±0.33 a 13.83±0.44 c root length (cm) 0.31±0.24 c 0.58±0.14 c 1.78±0.16 b 1.78±0.2 1.94±0.1 b 1.49±0.08 b 0.66±0.2 c 2.46±0.17 a 2.67±0.23 a 2.74±0.08 a 1.95±0.11 b shoot length (cm) 1.85±0.1 c 1.5±0.05 d 1.95±0.08 c 2.03±0.14 bc 1.94±0.06 c 1.89±0.06 c 2.27±0.08 b 2.87±0.12 a 2.96±0.19 a 2.77±0.12 a 2.82±0.14 a ‘charlotte’ rooting (%) 58.25±12.44 c 62.5±7.76 bc 83.5±7.38 ab 87.5±5.59 a 93.75±4.84 a 85.5±6.61 a 62.5±7.76 bc 91.75±6.39 a 93.75±4.84 a 91.75±6.39 a 83.5±7.38 ab no. of roots 3.75±0.58 e 5.02±0.33 d 9.49±0.36 c 12.25±0.45 b 12.28±0.33 b 10.18±0.56 c 6.33±0.86 d 15.06±0.46 a 16.46±0.69 a 13.33±1.21 b 9±0.32 c root length (cm) 1.13±0.12 c 1.04±0.14 c 1.88±0.04 b 1.86±0.1 b 1.88±0.24 b 1.17±0.06 c 1.32±0.19 c 2.49±0.06 a 2.47±0.15 a 2.49±0.33 a 1.49±0.09 b shoot length (cm) 2.41±0.26 b 1.53±0.07 c 1.99±0.07 b 2.28±0.11 b 2.19±0.13 b 1.52±0.1 c 2.28±0.1 b 2.92±0.1 a 3.34±0.16 a 3.21±0.18 a 2.26±0.14 b ‘gold capella’ rooting (%) 0±0 41.75±12.44 b 85.5±6.61 a 100±0 a 100±0 a 100±0 a 50±16.70 b 100±0 a 100±0 a 100±0 a 100±0 a no. of roots 0±0 0.72±0.14 d 6.99±0.3 c 6.86±0.25 c 9.39±0.46 b 7.14±0.35 c 1±0 d 8.69±0.62 b 8.88±0.26 b 11.83±0.76 a 9.13±0.32 b root length (cm) 0±0 0.75±0.06 f 1.63±0.03 e 1.91±0.1 d 2.09±0.07 cd 1.92±0.02 c 0.90±0.08 f 2.15±0.04 c 2.53±0.14 b 2.78±0.09 a 2.55±0.03 b shoot length (cm) 1.29±0.01 g 1.3±0.03 g 1.66±0.13 d-f ** 1.45±0.07 fg 1.92±0.14 cd 1.52±0.06 e-g 1.77±0.11 de 2.47±0.18 b 2.17±0.09 bc 2.83±0.19 a 2.26±0.08 b table 5 effects of auxins on rooting and young shoot growth of three schefflera cultivars data (±se) are the mean values of five replicates with three explants in each. for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). fig. 2 root induction of schefflera cvs. luseane (a), charlotte (b) and gold capella (c) after using 2, 1 and 2 mg l-1 of naa, respectively. note: the roots were submerged in distilled water for better visibility. adv. hort. sci., 2018 32(2): 205-212 210 centrations of iba and naa did not show significant discrepancies. supplementing the medium with 1 mg l-1 naa produced the greatest number of roots per explant with an average of 16.46 roots (fig. 2). on the other hand, 0.5 mg l-1 naa resulted with 15.06 roots which had no significant differences with the former treatment. furthermore, using 0.5 and 2 mg l-1 naa produced the largest root length of 2.49 cm, though 1 mg l-1 naa produced similar root length of 2.47 cm with no significant differences. the maximum shoot length of 3.34 cm was measured using 1 mg l-1 naa in the medium, although 0.5, and 2 mg l-1 naa treatments did not yield significant differences with the former. one hundred percent rooting was observed with ‘gold capella’ shoots when iba was applied at 1, 2 or 4 mg l-1, and naa at 0.5, 1, 2 or 4 mg l-1. the greatest number of roots per plantlet (i.e. 16.46 roots) was obtained using 2 mg l-1 naa treatment (fig. 2). moreover, the highest shoot and root length (2.78 and 2.83 cm respectively), were measured by addition of 2 mg l-1 naa, which showed significant differences compared to the other auxin treatments. effect of potting media on plant acclimatization schefflera’s survival rate was not significantly affected by different potting media. the highest survival rate of 100% was obtained for ‘luseane’ and ‘charlotte’ using peat moss and sand (1:1) and loam soil, sand and leaf compost (1:1:1), respectively (table 6). ‘gold capella’ showed a survival rate of 90% using peat moss or peat moss and perlite medium (1:1). 4. discussion and conclusions infection of tissue cultures of ornamental pot plants is a common phenomenon and an important barrier for their mass production. contaminations in aglaonema (chen and yeh, 2007), anthurium (kunisaki, 1980), dieffenbachia (brunner et al., 1995), spathyphyllum and syngonium (kneifel and leonhardt, 1992), zantesdeschia (kritzinger et al., 1998), as well as philodendron (fisse et al., 1987, chen et al., 2012) are commonly reported. thus, the current research was designed by setting up a disease-free shoot stock culture. following surface sterilization with 70% etoh for 120 sec and disinfection with 1% sodium hypochlorite for 15 min, more than 93% of the cultures were devoid of visible contaminations and they demonstrated 86.4% of viability for all tested cultivars. these results are consistent with the findings on fatsia japonica decne. (choi et al., 2005), polyscias balfouriana (ilyas et al., 2013) and polyscias fruticosa (sakr et al., 2014) from the araliaceae family. the green schefflera cultivar ‘luseane’, showed a significantly greater number of shoot induction (6.25 shoots per explant) compared to the whiteand yellow-variegated cultivars (tables 3, 4). this phenomenon could be ascribed to the fact that variegated plants have a decreased propagation rate (marcotrigiano, 1997). vitrification problem and further necrosis of the new shoots was observed in ‘charlotte’ and ‘gold capella’ when applying maximum concentration of 8 mg l-1 ba (fig. 3). thus, a comparable 0.5 mg l-1 tdz was suggested for in vitro propagation of these cultivars instead of the former treatment. sivanesan et al. (2011) reported increased number of shoots in hedera helix ‘mini’ by using a combination of 0.5 mg l−1 tdz and 0.1 mg l−1 naa. the mode of action of tdz may be through alteration in energy levels, nutrient uptake, nutrient assimilation, or cell membranes of plants (murthy et al., 1998). however, tetyana and van staden (2001) demonstrated that 2.5 mg l-1 ba supplement to the media in fig. 3 vitrification and necrosis of schefflera plantlets using 8 mg l-1 of ba in ‘charlotte’ (a) and ‘gold capella’ (b). mixture survival (%) shoots length (cm) ‘luseane’ ‘charlotte’ ‘gold capella’ ‘luseane’ ‘charlotte’ ‘gold capella’ peat moss 90±10 ab 90±10 ab * 90±10 ab 3.86±0.07 b 3.66±0.25 b 3.48±0.18 bc peat moss and perlite 90±10 ab 90±10 ab * 90±10 ab 4.81±0.22 a 4.49±0.2 a 4.92±0.17 a peat moss and sand 100±0 a 90±10 ab * 80±13.33 ab 3.60±0.1 bc 3.23±0.18 cd 2.94±0.23 d loam soil, sand and leaf compost 90±10 ab 100±0 a * 70±15.28 b 3.65±0.08 bc 3.72±0.24 bc 3.79±0.17 b table 6 effect of potting media on acclimatization of three schefflera cultivars for each cultivar, different lowercase letters indicate significant differences among treatments (lsd multiple range test, p≤0.05). rezaei baghbidi and jowkar micropropagation of dwarf schefflera 211 cussonia paniculata of the araliaceae family, resulted with the highest shoot induction of 3.5 shoots per explant. on the other hand, the outcomings of you et al. (2005) in eleutherococcus senticosus, the other member of the araliaceae family, were different than our findings. it was revealed that 2 mg l−1 ba proliferated shoots more effectively than tdz, kin and 2ip. various cytokinin types and concentrations affect shoot proliferation of plant species, though all belong to the same family. therefore, for each plant species, the shoot induction has to be newly investigated. it was revealed that after the tdz treatment, shoots’ length of all cultivars were comparatively longer than those of the kin and ba treatments. the white variegated schefflera ‘charlotte’ showed the greatest length (2.33 cm) using 0.5 mg l-1 tdz. this effect could be related to further increase of auxin production by tdz treatment (murthy et al., 1998). it is assumed that tdz has the ability to affect the amount of internal plant hormones (murch and saxena, 2001). zaytseva et al. (2016) reported a similar high activity for low concentrations of tdz in rhododendron. although the highest number of leaves was recorded by using 8 mg l-1 ba in all of the cultivars, other treatments are suggested due to the aforementioned vitrification problem. for ‘charlotte’, 0.5 mg l-1 tdz treatment and for the two other cultivars 4 mg l-1 ba produced maximum leaves without necrosis. generally, cytokinins enhance the photosynthesis and help translocate the nutrients to the leaves (taiz and zeiger, 2006). a sought-after plant tissue culture protocol essentially depends on sufficient rooting along with a successful acclimatization of the young plants. the results of the current study show that in all of the cultivars naa is more effective in rooting than iba. there are inconsistent reports on the rooting efficacy of different auxins. the research of you et al. (2005) indicated that 0.5 mg l−1 naa is more effective than iba for rooting of e. senticosus shoots, while a concentration of 0.75 mg l−1 iba or 1 mg l−1 naa have proven useful for c. paniculata (tetyana and van staden, 2001). auxin treatment of ‘charlotte’, ‘luseane’ and ‘gold capella’ increased the rooting percentage of the shoots, compared to the control by 35%, 92% and 100%, respectively. all shoots of ‘charlotte’ and ‘luseane’ were well acclimatized with a survival rate of 100% using media of loam soil, sand and leaf compost (1:1:1) and a mixture of peat moss and sand (1:1), respectively, while ‘gold capella’ demonstrated a none-significant lower survival frequency of 90% in the peat moss or peat moss and sand media (1:1). to our knowledge, the current research is the first report on micropropagation of different cultivars of schefflera. results demonstrated that green cultivar ‘luseane’ had a greater shoot proliferation than the other two cultivars with average shoot number of 6.25 shoots per explant using 0.5 mg l−1 tdz. the multiplicated shoots could lengthen on pgr-free ms medium and later showed effective rooting on naacontained media. although the yellow variegated cultivar ‘gold capella’ revealed a smaller proliferation rate of shoots compared to the control, its rooting was 100% improved. subsequent relocation of the ‘luseane’ and ‘charlotte’ plantlets to the acclimatization greenhouse resulted with 100% survival rate of the rooted shoots. generally, peat moss and perlite media (1:1) had a more positive effect on the young acclimatized plants of schefflera. the protocol developed in this research can be used for the aseptic production of schefflera. references amin m.n., rahman m.m., manik m.s., 2003 in vitro clonal propagation of paederia foetida l. a medicinal plant of bangladesh. plant tiss. cult., 13: 117-123. brunner i., echegaray a., rubluo a., 1995 isolation and characterization of bacterial contaminants from dieffenbachia amoena bull, anthurium andreanum linden and spathiphyllum sp. shoot cultured in vitro. sci. hortic., 62: 103-111. chase a.r., poole r.t., 1986 effects of fertilizer rate on severity of alternaria leaf spot of three plants in the araliaceae. plant dis., 70(12): 1144-1145. chen f.c., wang c.y., fang j.y., 2012 micropropagation of self-heading philodendron via direct shoot regeneration. sci. hortic., 141: 23-29. chen j., henny r.j., mcconnell d.b., 2002 development of new foliage plant cultivars, pp. 466472. in: janick j., and a. whipkey (eds.) trends in new crops and new uses. ahsh press, alexandria, usa. chen w.l., yeh d.m., 2007 elimination of in vitro contamination, shoot multiplication, and ex vitro rooting of aglaonema. hortscience, 42: 629-632. choi k.m., hwang s.j., ahn j.c., lee h.y., kim j.h., hwang b. 2005 in vitro propagation from axillary bud explants of fatsia japonica deene. et planeh. k. j. med. crop sci., 13: 300-303. conover c.a., 1992 foliage plants, pp. 571-598 in: larson r.a. 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volatile organic pollutant removal efficiency. hortscience, 44: 1377-1381. you x.l., choi, y.e., yi j.s., 2005 micropropagation of eleutherococcus senticosus through axillary bud culture. forest sci. tech., 1: 38-44. zaytseva y.g., poluboyarova t.v., novikova t.i., 2016 effects of thidiazuron on in vitro morphogenic response of rhododendron sichotense pojark. and rhododendron catawbiense cv. grandiflorum leaf explants. in vitro cell dev. biol. plant, 52: 56-63. impaginato 353 adv. hort. sci., 2018 32(3): 353-362 doi: 10.13128/ahs-21927 influence of soil and soilless agricultural growing system on postharvest quality of three ready-to-use multi-leaf lettuce cultivars b. pace 1, 2, i. capotorto 1, 2, m. gonnella1, f. baruzzi 1, m. cefola 1, 2 (*) 1 istituto di scienze delle produzioni alimentari, consiglio nazionale delle ricerche (cnr), via g. amendola, 122/o, 70126 bari, italy. 2 istituto di scienze delle produzioni alimentari, consiglio nazionale delle ricerche (cnr), urt c/o cs-dat, via m. protano, 71121 foggia, italy. key words: ammonium, lactuca sativa l., nitrate, microbial quality, postharvest storage, soilless cultivation. abstract: in this study the influence of soil and soilless growing systems (substrate 3:1 v/v perlite:peat) on quality and microbial traits of three multi-leaf lettuce cultivars (two green, ‘eztoril’ and ‘ezabel’, and one red, ‘ezra’) was evaluated at harvest and after 7 and 13 days of storage at 8°c. at harvest, ‘ezra’ showed a respiration activity and a total phenol content respectively 2-fold and 25% significantly higher than the green cultivars. soil lettuces resulted more stressed than those grown in soilless, as indicated by their initial content in antioxidants. as for nitrate content, soilless grown lettuces at harvest showed an average concentration higher than soil-grown ones, although values are generally lower than limits imposed by the eu regulation (no. 1258/2011). during storage, soilless lettuces showed no ammonium accumulation, differently from those cultivated in soil. in addition, lettuce cultivars grown in soilless condition showed unchanged content in the antioxidant activity and total phenols, and lower microbial counts than soil lettuces. results of the present study showed that soilless growing system can positively affect qualitative and microbiological parameter of lettuces studied, and it can be considered a good soilless growing technique in order to obtain high quality multi-leaf lettuces for readyto-use industry. 1. introduction the consumer’s demand of ready-to-use fruits and vegetables, and in particular that for minimally processed leafy vegetables, is continuously growing. although iceberg lettuce is still the main lettuce used in the ready-to-use industry, consumers are requesting other types of lettuce with attractive colours and shapes combining the best quality characteristics from all varieties (rijk zwaan, 2009). the new baby-sized leaves, (*) corresponding author: maria.cefola@ispa.cnr.it citation: pace b., capotorto i., gonnella m., baruzzi f., cefola m., 2018 influence of soil and soilless agricultural growing system on postharvest quality of three ready-to-use multi-leaf lettuce cultivars. adv. hort. sci., 32(3): 353-362 copyright: © 2018 pace b., capotorto i., gonnella m., baruzzi f., cefola m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 353-362 354 babyand multi-leaf have been developed recently as high quality lettuce varieties for the ready-to-use market. some benefits of baby-sized lettuce, when compared with whole-head lettuce, include: i) greater efficiency with higher percentage of usable product; ii) easier and faster processing; iii) more attractive colour and shapes, and iv) minimal oxidation due to smaller stem diameter (martínez-sánchez et al., 2012). moreover, for both multiand baby-leaf lettuces, no physical wounding was undertaken, except that of the harvesting, avoiding the physical damage that occurs during preparation of fresh-cut lettuce that causes an increase in respiration activity, biochemical changes and microbial spoilage, which may result in degradation of colour, texture and flavour of the ready-to-use produce (cantwell, 2004). likewise, cultivar selection is of great importance in the ready-to-use industry since quality characteristics (such as leaf colour, shape, freshness, texture and browning potential) can change largely depending on the genotype (nicola et al., 2009). the quality and shelf-life of ready-to-use leaves depend on genotypic traits of raw material and on several aspects from preharvest to postharvest processing (clarkson et al., 2003; cantwell, 2004). some physical and chemical indicators can be used for objective assessment of visual quality (barrett et al., 2010; salinas-hernández et al., 2015). among these, ammonium (nh4 +), produced during storage as a consequence of senescence in various vegetables (cefola et al., 2010; pace et al., 2014), might be used as predictors of shelf-life (cefola et al., 2017). in general, preharvest factors should be aimed to optimize their impact on postharvest quality (crisosto and mitchel, 2002). from this point of view, soilless system is becoming of high interest since it can improve both, preharvest and postharvest quality of vegetables (rodríguez-hidalgo et al., 2010). in particular, soilless agricultural growing system allows to set optimal conditions and nutrient concentration for plant growth (silberbush and ben-asher, 2001; selma et al., 2012) with the following advantages: higher yields (lopez-medina et al., 2004; recamales et al., 2007), better quality vegetables (recamales et al., 2007; cefola et al., 2011) and higher earliness (recamales et al., 2007; valenzano et al., 2008), compared to soil cultivation. the success of lettuce production depends to a great extent on the maintenance of a continuous growth rate by the optimal management of nutrients (luna et al., 2013). in addition, especially for leafy vegetables, the use of soilless system can avoid soil contaminants and improve the sanitary quality respect to traditional soil cultivation, leading benefits on raw materials for postharvest industry (selma et al., 2012). starting from these findings, the aim of this work was to evaluate the influence of two growing system (soil and soilless) on postharvest quality of three multi-leaf lettuce genotypes, including two green and one red, stored under refrigeration for 13 days. 2. materials and methods reagents extraction solvents (meoh, etoh), 2,2-diphenyl-1picrylhydrazyl (dpph), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and all standards used in the experiments were obtained from sigma-aldrich (st. louis, mo., usa). folin-ciocalteu’s phenol reagent was purchased from merck (germany). plant material and growing system three types of dutch multi-leaf lettuces (lactuca sativa l.), two green (cv. eztoril and ezabel) and one red (cv. ezra) (enza zaden, enkhuizen, netherland) were cultivated in an unheated plastic tunnel under soil (s) or soilless (sl) agricultural growing system in the same greenhouse at the experimental farm “la noria” of the institute of sciences of food production (cnr-ispa) located in the south of italy (mola di bari). a split-plot design with three replications was applied, randomizing the growing systems (gs) in the main plots and cultivars in the subplots. main plots were of 3.6 m2 (0.9 m wide and 4 m long). the sl system consisted of three single benches (4 m long x 0.3 m wide x 0.1 m high, with a slope of 2%) each plot containing a 3:1 (v:v) perlite:peat mixture as substrate. the nutrient solution was supplied to the sl system without recirculation and had the elemental composition given in table 1, where the soil characteristics are reported too. the irrigation water had the following composition (expressed in mmol l-1): 0.3 n-no3, 0.23 k, 0 p, 1.73 mg, 1.82 ca, 7.39 cl, 4.05 na. nutrient solution and water were supplied to sl and s units based on a timer controlled schedule, using minimum substrate water content values, monitored by tensiometers. furthermore, as additional reference control taking into account two different threshold levels of -5 and -25 kpa to start the irrigation supply in sl and s, respectively. for sl system a nutrient supply level criterion was additionally pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars 355 applied. at transplant soil plots were fertilized with ammonium nitrate and monopotassium phosphate giving the equivalent of 50, 80, 50 kg ha-1 of n, p2o5 and k2o, respectively, and after a month a 30 kg ha-1 integration of n from ammonium nitrate was applied. seedlings were produced in greenhouse in polystyrene trays on peat and were transplanted 25 days after sowing on february 22. harvest was performed 55 days after transplanting (on april 18) for the sl and after 73 days (on may 6) for the s system. greenhouse ventilation temperature was 20°c. in figure 1 the climatic parameters measured in the greenhouse are reported. daily air temperature was on average 21°c, and minimum and maximum air temperature ranged from 0.2 to 17.5 and from 17.5 to 46.0°c, respectively (fig. 1a). air relative humidity was on average 50.5%; daily minimum and maximum relative humidity ranged from 6 to 56% and from 45 to 85% (fig. 1b). the average photosynthetically active radiation was 282 µmol m-2 s-1; its mean and maximum values changed from 80 to 420 and from 400 to 2,080 µmol m-2 s-1, respectively (fig. 1c). after harvest, lettuces were immediately transported under refrigerated condition in polystyrene boxes to the cnr ispapostharvest laboratory. processing and storage after harvest, for each multi-leaf lettuce cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’), and for each agricultural growing system (s or sl), leafs were selected in order to avoid damaged samples, and no washing or pre-treatment were applied. for each cultivar and gs about 600 g of leaves were used for quality evaluation at harvest, whereas about 1.2 kg were used for the quality evaluations during storage. thus, leaves were put in open polyethylene bags (about 200 g each bag), and stored at 8°c in dark conditions. for each cultivar, 12 bags (3 replicates ´ 2 gs, s or sl, ´ 2 storage periods, 7 and 13 days) were prepared. fig. 1 climatic parameters (a= temperature; b= air relative humidity; c= photosynthetically active radiation) measured in the greenhouse during the experiment, from seedling transplantation to harvest days. the blue and red perpendicular lines indicate the harvest day for lettuce cultivated in soilless and soil condition, respectively. mineral composition soil composition soilless nutrient solution sand 24.30% silt 31.90% clay 43.80% ph 7.6 6.5 ec (ds m-1) 2.5 2.3 cl7.39 mg2+ 1.73 na+ 4.05 k+ 5.12 ca2+ 4.74 nh 4 + 0.5 no 3 9.43 p-h2po41.61 s-so420.81 cec (cmol kg-1 dw) 31.8 organic matter (g kg-1 dw) 14 total n (g kg-1 dw) 0.95 available p (g kg-1 dw) 110 available k (g kg-1 dw) 244 caco 3 (g kg-1 dw) 0.11 table 1 soil and soilless nutrient solution composition. soil classified as clay soil (usda textural soil classification, 1987). values of nutrient solution are expressed in mmol l-1. micronutrients were supplied according to johnson et al. (1957) adv. hort. sci., 2018 32(3): 353-362 356 respiration activity and the analysis of nitrate content were performed at harvest. in addition, antioxidant activity, total phenols, ammonium content and microbiological analysis were evaluated at harvest and after 7 and 13 days of storage. respiration activity the respiration activity of each cultivar was measured using a closed system as reported by kader (2002). about 100 g of leaves for each replicate were put into 6 l sealed plastic jars (one jar for replicate) where co2 was allowed to accumulate until the value of 0.1%. the time needed to reach this value was calculated, making co2 measurement at regular time intervals. for the co2 analysis, 1 ml gas sample was taken from the head space of the plastic jars through a rubber septum and injected into the gas chromatograph (p200 micro gc, agilent, santa clara, ca) equipped with dual columns and thermal conductivity detector. co2 was analyzed with a retention time of 16 s and total run time of 120 s on a 10 m porous polymer (ppu) column at a constant temperature of 70°c. respiration activity was expressed as ml co2·kg-1·h-1. antioxidant activity, total phenol, ammonium and nitrate content to determine both antioxidant activity and total phenols content, the extraction procedure reported by cefola et al. (2012), was followed. in detail, 5 g samples were homogenized (ultraturrax t-25, ika staufen, germany) in a meoh: water (80:20) solution for 1 min, and then centrifuged at 5°c at 6440 x g for 5 min. the supernatant was therefore used for the assays. the antioxidant activity assay was performed following the procedure described by brand-williams et al. (1995) with minor modifications. briefly, the supernatant, proper diluted, was pipetted into 0.95 ml of dpph solution to start the reaction. the absorbance was read after about 30 min at 515 nm. trolox was used as a standard and the antioxidant activity was expressed in g of trolox equivalents per kg of fresh weight (g teac kg-1 fw). the total phenol content was determined according to the method of singleton and rossi (1965). each extract (100 μl), proper diluted, was mixed with 1.58 ml water, 100 μl of folin-ciocalteu reagent and 300 μl of sodium carbonate solution (200 g l-1). the absorbance was read after 2 h at 765 nm. total phenol content was calculated on the basis of the calibration curve of gallic acid and expressed as g of gallic acid per kg of fresh weight (g ga kg-1 fw). for ammonium content the method reported by weatherburn (1967) was used. in detail, 5 grams of chopped sample were homogenized (ultraturrax t25, ika staufen, germany) with 20 ml distilled water for 2 min, centrifuged at 6440 x g for 5 min, and 0.5 ml extract was used for the analysis. color development, caused by the reaction with a phenol nitroprusside reagent and alkaline hypochlorite solution, was determined after an incubation of 20 min at 37°c, by reading the absorbance at 635 nm (uv1800, shimadzu, kyoto, japan). ammonium content was expressed as µmole nh4 + per kg of fresh weight (µmole nh4 + kg-1 fw). as for nitrate content, samples (about 100 g for replicates) were dried in the oven (65°c until constant weight) and were ground to fine powder. the powder (0.5 g for each replicate) were extracted on orbital shaker for 20 minutes with 50 ml of a solution containing 3.5 mmol l-1 of sodium carbonate and 1 mmol l-1 sodium bicarbonate. analysis were carried out using a ion exchange chromatography (dionex dx 200, dionex corp, sunyvale, ca, usa) with a conductivity detector, using an ionpac ag14 precolumn and an ionpac as4a separation column (dionex corporation). results were expressed in mg of nitrate per kg of fresh weight (mg no3 kg-1 fw). microbiological analysis samples (30 g for replicates) were homogenized for 1 min in 0.1% sterile buffered peptone water (difco laboratories, detroit, mi, usa) (1:5 dilution) using a stomacher (seward, london, uk). total aerobic mesophilic bacteria count was evaluated using plate count agar (difco) incubated at 30°c for 48 h. yeasts and moulds were counted on sabouraud dextrose agar (difco) supplemented with chloramphenicol and chlortetracycline (both 0.05 g l-1) and incubated at 25°c for 5-7 days. total counts of enterobacteriaceae were obtained by pour-plating dilutions (1 ml) in violet red bile glucose agar (difco) and plates were incubated at 37°c for 24 h. microbiological counts were expressed as log cfu g-1 of fresh weight (log cfu g-1 fw). statistical analysis in order to study the effect of gs (s or sl), cultivars, cv (‘ezra’, ‘eztoril’ and ‘ezabel’) and their interaction (gs x cv) on quality parameters at harvest, and the effect of gs, cv, storage (0-7-13 days) and their interaction (gs x cv x storage) on quality parameters, two multifactor anova were performed (statistica software). when significant effect of factors were detected, the student newman keuls (snk) test was applied to separate means. for a visual pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars 357 analysis of the data, principal component analysis (pca) (princomp procedure, sas software, cary, nc, usa; biplot by xlstat, addinsoft, paris, france) was performed on mean centered and standardized (unit variance scaled) data prior to analysis. the data matrix submitted to pca was made up of 18 observations 3 cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’) x 2 growing system (s and sl) x 3 storage times (0-7-13 days) and 6 quality parameters (antioxidant activity, total phenols, ammonium, mesophilic bacteria, yeasts and moulds, enterobacteriaceae). 3. results effect of growing systems and cultivars on lettuces quality traits at harvest yield response was influenced by genotypes more than gs (s or sl), since a lower fresh weight was produced by the red lettuce compared to the other two cultivars (2.7 vs 3.6 kg m-2) and only in cv. eztoril there was a higher yield in s compared to sl system (4.3 vs 3.3 kg m-2). the effect of gs, multi-leaf lettuce cv (‘ezra’, ‘eztoril’ and ‘ezabel’) and their interaction on the quality parameters measured at harvest was investigated (table 2). ammonium content, antioxidant activity, total phenols and nitrate content were significantly affected by gs and cv, while respiration activity was affected only by cv. the interaction gs x cv was statistically significant only for nitrate content (table 2). the respiration activity of cv. ezra was twofold higher than the green cultivars (‘eztoril’ and ‘ezabel’). regarding ammonium content, the values found for lettuces cultivated in sl were statistically higher respect to s and, between cultivars, red multileaf lettuce had mean values statistically higher than green cultivars (table 2). growing system affects significantly the antioxidant activity and total phenols: plants cultivated in s showed significantly higher mean content than sl samples (table 2). regarding cv, there were no differences between green multileaf lettuces in antioxidant activity and total phenols, while the red cultivar ezra had lower values of antioxidants and higher values of total phenols respect to the green cultivars (table 2). as for nitrate content was almost double in sl lettuces than s grown ones (838 vs 432 mg no3 kg-1 fw). the red multi-leaf lettuce (cv. ezra) had mean values of nitrate statistically higher than cv. eztoril but not different from cv. ezabel (table 2). effect of growing systems and cultivars on lettuces’ quality traits and microbial parameters during cold storage the results of multifactor anova on antioxidant activity, total phenols and ammonium content as affected by gs, cv, storage time (0, 7 and 13 days) and their interaction were reported in table 3. growing system affected antioxidant activity and total phenols, cv affected total phenols and ammonium, while storage time affected antioxidant activity respiration activity (ml co 2 kg-1 h-1) ammonium content (µmole nh 4 + kg-1fw) antioxidant activity (g teac kg-1 fw) total phenols (g ga kg-1 fw) nitrate content (mg no 3 kg-1 fw) growing system (gs) soilless 47.44 66.7 2.33 1.32 838.12 soil 45.73 49.6 4.82 1.84 431.96 cultivar ezra 72.67 a 72.80 a 3.07 b 1.89 a 765.71 a eztoril 35.82 b 48.10 b 3.82 a 1.41 b 524.32 b ezabel 31.26 b 53.60 b 3.84 a 1.44 b 615.09 ab gs ns ** *** ** *** cultivar *** ** ** * * gs x cultivar ns ns ns ns * table 2 effect of growing system (soil and soilless) and cultivar (ezra, eztoril and ezabel) on quality parameters measured at harvest when interaction among factors was not significant, the results of the mean separation test (snk test) are reported. different letters indicate statistical difference within cultivars, respectively, for p≤0.05. ns, not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 358 adv. hort. sci., 2018 32(3): 353-362 and ammonium (table 3). considering the interaction among factors, antioxidant activity was affected by gs x storage and cv x storage; total phenols were influenced by gs x cv and by gs x cv x storage, and ammonium was affected only by gs x storage (table 3). in figure 2, changes in antioxidant activity (a), total phenols (b) and ammonium content (c) during storage of the three multi-leaf lettuce cultivars, cultivated in s and sl conditions, are reported. at harvest, lettuces cultivated in s showed values of antioxidant activity significantly higher than sl lettuce. however, during storage, antioxidant activity of lettuces cultivated in s decreased rapidly, reaching approximately the same values of samples cultivated in sl after 7 day of storage at 8°c; after it remained unchanged for cv. ezabel (about 2 g teac kg-1 fw) and slightly increased for cv. eztoril (about 3 g teac kg-1 fw). the cultivar ezra cultivated in s showed a content in antioxidant activity almost constant during storage, with a 30% reduction at the end of storage (fig. 2a). whereas, lettuce cultivated in sl showed unchanged values of antioxidant activity during time, with a slight reduction at the end of storage for cv. ezabel (about 1.3 g teac kg-1 fw) (fig. 2a). regarding the content of total phenols (fig. 2b), green multileaf lettuces cultivated in s showed a slight decrease during the first week of storage, after then values rise again until the end of storage, reaching values of about 1.6 and 1.7 g ga kg-1 fw for cv. eztoril and ezabel, respectively (fig. 2b). a specular trend for green multi-leaf lettuce cultivated in sl was observed (fig. 2b). the cv. ezra cultivated in sl showed the same behavior of green lettuces; whereas ‘ezra’ cultivated in s showed an initial total phenol content of about 2.3 g ga kg-1 fw, which increased during the first week of storage, reducing to initial values until the end of the storage (fig. 2b). as regard data of ammonium content (fig. 2c) lettuces cultivated in sl showed unchanged values during postharvest storage, starting from the initial mean values of about 79.9±7.5, 57.0±9.3 and 63.2±9.6 mmole nh4 + kg-1 fw in cv. ezra, eztoril and ezabel, respectively. whereas, lettuces cultivated in s showed an increase in ammonium content during storage, which doubled for all cultivars, starting from initial mean values of 65.5±15.7, 39.2±3.5 and 44.0±7.0 mmole nh4 + kg-1 in cv. ezra, eztoril and ezabel, respectively. fig. 2 changes in antioxidant activity (a), total phenols (b) and ammonium content (c) of three multi-leaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’), cultivated in soil or soilless condition, during storage at 8°c. mean ± sd. table 3 multifactor anova of antioxidant activity total phenols and ammonium content as affected by growing system (soil or soilless), cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’) and storage time (0, 7 and 13 days) antioxidant activity (g teac kg-1 fw) total phenols (g ga kg-1 fw) ammonium content (µmole nh 4 + kg-1fw) growing system (gs) *** ** ns cultivar (cv) ns ** ** storage time *** ns * gs x cv ns ** ns gs x storage * ns * cv x storage *** ns ns gs x cv x storage ns * ns ns= not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 359 pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars as concerns microbial populations evaluated during the trial, results of multifactor anova statistical analysis of mesophilic bacteria, yeasts and moulds and enterobacteriaceae as affected by growing system (gs), cultivars (cv) and storage time are reported in table 4. all factors and their interactions affected significantly the microbial populations evaluated, with the exception of gs x cv interaction for yeasts and moulds. as concerns total mesophilic bacteria, lettuces cultivated in sl showed an increase of about 1.5 log unit during storage, starting from initial mean values of 3.7±0.05 log cfu g-1 (cv. eztoril), 4.9±0.22 log cfu g-1 (cv. ezabel) and 6.1±0.29 log cfu g-1 (cv. ezra), whereas lettuces cultivated in s growing condition showed an higher significant increase in mesophilic population during storage, of about 3 log unit, starting from a mean initial count of about 4.67±0.68 log cfu g-1 (fig. 3a). yeast and mould loads from s and sl were not found to be significantly different. however, during 13 days of cold storage their amount increased significanly for all cultivars, resulting statistically higher in multi-leaf lettuces cultivated in s respect to the sl ones (fig. 3b). also in the case of enterobacteriaceae, lettuces cultivated in s showed a significant increase of about 4 log unit at the end of the storage, starting from initial values of 3.4±0.9, 2.4±0.26 and 4.10±0.10 log cfu g-1 for cv. ezra, eztoril and ezabel, respectively. in lettuces cultivated in sl conditions, the increase of about one magnitude order was found for this microbial population (fig. 3c). principal component analysis principal component analysis revealed that almost 84% of the total variability of data was explained by the first two principal components. pc1 resulted mainly and positively correlated to ammonium and the counts of the three microorganisms groups. each of them contributed to pc1 for 20-25% of the total variability (fig. 4). on the other hand, antioxidant activity and total phenols contributed to pc2 for more than 45% each. among observations, all s growing system samples after 13d storage showed a stronger and positive correlations with pc1, ammonium and the microbiological counts. at a proximate position collocated samples of the red cultivar ezra collected from sl and stored for 7 and 13 days. on the contrary, all cultivars grown on the s system, at fig. 3 changes in total mesophilic bacteria (a), yeasts and moulds (b) and enterobacteriaceae (c) of three multileaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’), cultivated in soil or soilless condition, during storage at 8°c. mean ± sd. mesophilic bacteria yeasts and moulds (log cfu g-1 fw) enterobacteriaceae growing system (gs) ** * * cultivar (cv) *** * *** storage time *** *** *** gs x cv *** ns *** gs x storage *** *** *** cv x storage *** ** *** gs x cv x storage *** * *** table 4 multifactor anova of mesophilic bacteria, yeasts and moulds and enterobacteriaceae as affected by growing system (soil or soilless), cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’) and storage time (0, 7 and 13 days) ns= not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 360 adv. hort. sci., 2018 32(3): 353-362 harvest, were negatively correlated to pc1, on the opposite site of ammonium and microbial parameters. it seems that no observations were strongly correlated to antioxidant activity and total phenols, with the exception of soil-grown ezra leaves sampled at harvest and at 7 days storage, followed by the other two cultivars coming from s at harvest and soilgrown ezra at 13 days storage (fig. 4). among observations negatively correlated to pc2, one result noteworthy, the sl-grown ‘ezabel’ sampled at 13 days after storage, since it showed a sharp decrease in the antioxidant activity at the end of the storage, as described in figure 2a. 4. discussion and conclusions the three multi-leaves lettuces studied in this research showed different qualities at harvest. in particular, the green cultivars resulted very similar and suitable for postharvest processing, on the basis of respiration activity and ammonium content. at contrary, the red cultivar was considered more perishable than the green ones, due to the high respiration activity (kader, 2002) even though it showed an higher content in polyphenols than the green cultivars, as previously reported by other authors (martínez-sánchez et al., 2012; selma et al., 2012). as regards antioxidant activity and total phenols, the higher contents measured in s cultivated lettuces could be a plant response to applied stress treatments (oh et al., 2009). compared to the sl growing system, s irrigation management implies necessarily different water conditions, keeping s plants in not optimal and constant water availability all-day. as a consequence, s plants may have occasionally experienced a water stress combined to heat stress during the highest temperature hours under greenhouse, in the few days preceding harvest (fig. 1). even a timelimited stress can activate the antioxidant synthesis in the plant metabolism (oh et al., 2009). on the other hand, the more constant availability of water and nutrients in sl grown plants allowed a higher nitrogen uptake, partially accumulated as nitrate in the vacuoles at higher rate than in s cultivated lettuces. however, the nitrate content measured at harvest was generally low in both gs compared to the limits imposed by the eu regulation no. 1258/2011. in compliance with the current regulation, nitrate accumulation in lettuce grown in the spring-summer period under greenhouse should not exceed 4,000 mg kg-1 fw. this limit is in agreement with the potentially high nitrate accumulating capacity of lettuce. however, at our latitude, the optimal light conditions found by plants during the spring months allow an efficient and fast assimilation of the up-taken nitrate. during cold storage, the sl growing system resulted able to preserve the quality of lettuces since no increase in ammonium content (senescence indicator) was registered whereas multi-leaf lettuces cultivated in s that resulted more senescence-prone. ammonium accumulates in leafy vegetables during storage, as consequence of protein catabolism. thus, ammonium was used as indicator of quality and shelf-life of green vegetable. (chandra et al., 2006; pace et al., 2014; cefola et al., 2015; cefola and pace, 2015; cefola et al., 2017). data from ammonium confirms that sl could be considered a suitable growing system to preserve postharvest quality of the cultivars analysed, although genotyping characteristics of each cultivar need also to be taken into account (selma et al., 2012). during storage nitrate measurements were not carried out, since in preliminary trials performed on the same lettuce genotype (cv. ezra) no nitrate changes after storage at 8°c for 10 days were detected. in particular nitrate remained unchanged at 1550 and 1800 mg kg-1 fw, in soilless and soil lettuce, respectively (data not shown). this was supported by several contributes in literature, referring about no modification of nitrate concentration in lettuce and other species during storage at temperature in a range from 1 to 10°c (siomos et al., 2002; chung et al., 2004; konstantopoulou et al., 2010). the sl cultivation showed a positive effect fig. 4 pca biplot (pc1 vs pc2) describing the spatial distribution of quality and microbiological parameters of three multi-leaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’) grown in soil (s) and soilless (sl) system during storage; a= ezra; b= eztoril; c= ezabel. aa= antioxidant activity; phenols= total phenols; 0d – 7d – 13d: 0-7-13 days of storage. 361 pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars also on microbiological quality of the green cultivars during storage. similarly, results were reported by other authors on table grape (cefola et al., 2011) and on soilless growing systems (scuderi et al., 2011; selma et al., 2012). in conclusion, the three multi-leaves lettuces studied in this research showed different qualities at harvest. in particular, green cultivars resulted very similar and suitable to postharvest processing, whereas, the red one was considered more perishable. at harvest, lettuces grown in soil showed the higher content in antioxidant activity and total phenols and the lower in nitrate than soilless samples. however, the nitrate content measured at harvest was generally low in both growing systems compared to the limits imposed by the eu regulation no. 1258/2011. regarding the postharvest storage, ready-to-use lettuces cultivated in soilless showed microbiological and qualitative performance better than those grown in soil. in particular, soilless growing system improved the storability of lettuces and allowed to the production of clean raw material, particularly suited for ready to use industry. it is interesting to note, as soilless system resulted able to limit ammonium accumulation (senescence indicator), also in red cultivar, which for genotipic traits resulted more senescence prone than green lettuces. acknowledgements this research was financed by miur research projects: “high-convenience fruits and vegetables: new technologies for quality and new products,” pon01_01435. references barrett d.m., beaulieu j.c., shewfelt r., 2010 color, flavor, texture, and nutritional quality of fresh cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing. cr. rev. food sci., 50: 369-389. brand-williams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. lwt food sci. technol., 28: 25-30. cantwell m., 2004 evaluating the quality of fresh-cut products. in: cantwell m. 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adv. hort. sci., 2019 33(1): 57-65 doi: 10.13128/ahs-23328 effects of pre-harvest applications of different source of calcium on the cell wall fractions and stem bending disorder of gerbera (gerbera jamesonii l.) cultivar flowers m. aghdam 1, m. hassanpour asil 2 (*), m. ghasemnezhad 2, s.a.a. mousavi mirkalaei 3 1 university campus 2, university of guilan, rasht, iran. 2 faculty of agricultural science, university of guilan, rasht, iran. 3 chalous branch, islamic azad university, chalous, iran. key words: catalase, cellulose, lignin, membrane stability index, superoxide dismutase. abstract: gerbera flower belongs to the composite family and is one of the top five cut flowers in the world in terms of production and consumption, which has a great economic value in the international flowering industry. this study was designed to evaluate whether calcium pre-harvest application, provided through 0, 0.5, 1 and 1.5% of calcium chloride (cacl2) and calcium nitrate (cano3), could extend the day of stem bending of gerbera cut flower. in the present study, we used two gerbera cultivars ‘intense’ and ‘rosaline’ as resistant and sensitive to stem bending, respectively. for evaluation of associated traits with stem bending, the produced flowers were kept in a vase solution containing 200 mg/l hydroxyquinoline with temperature conditions of 20°c. the results showed that day of stem bending of flowers extended 9.62 and 10.37 days by application of 1% cacl2 for ‘rosaline’ and ‘intense’ respectively. all treatments were effective in the increasing relative water content of flower due to increase water uptake. the results also revealed that the calcium pretreatment delayed flowers senescence and increased antioxidant enzyme activity. application of calcium resulted in an increase in membrane stability index in the cut flowers of both cultivars, providing evidence for delay of senescence in calcium-treated cut flowers. also, results showed that calcium application significantly increased lignin, cellulose and hemicellulose content of both cultivars. the maximum and the minimum lignin and cellulose content were observed in resistant and sensitive cultivars, respectively. in general, pre-harvest application of calcium (especially 1% cacl2) with increasing of antioxidant enzyme activity and stem lignification led to decreasing of stem bending disorder in both cultivars. 1. introduction gerbera is one of the most important ornamental plants in the world (*) corresponding author: m.hassanpour150@gmail.com citation: aghdam m., hassanpour asil m., ghasemnezhad m., mousavi mirkalaei s.a.a., 2019 effects of pre-harvest applications of different source of calcium on the cell wall fractions and stem bending disorder of gerbera (gerbera jamesonii l.) cultivar flowers adv. hort. sci., 33(1): 57-65 copyright: © 2019 aghdam m., hassanpour asil m., ghasemnezhad m., mousavi mirkalaei s.a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2018 accepted for publication 4 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 57-65 58 used widely as cut flower or potted flower (çelikel and reid, 2002; perik et al., 2014). gerbera, followed by rose, chrysanthemum, tulip, and lily, occupies fifth position in top ten cut flower in world in terms of its production and consumption (nair et al., 2006; perik et al., 2014). the longevity or postharvest life of gerbera flowers depends on the extent of stem bending and the lack of stem strength (naing et al., 2017; nazarideljou and azizi, 2015). in this regard, although flowers of some gerbera cultivars are apparently not wilted or faded, their stems have drooped due to their inappropriate stem strength; and based on flower quality and grading, flowers with stem bending more than 30° are not worth maintaining (çelikel and reid, 2002; perik et al., 2014). the lack of mechanical protection and cell wall lignification (especially secondary cell wall) as well as lowering stem strength are of more likely reasons for emerging stem bending in gerbera (perik et al., 2012). in addition to its remarkable effect on stem hardening and growing gerbera uprightly, lignin plays an utmost role in promoting water flow inside xylem vessels (vanholme et al., 2010). also, there is a negative relationship between the rate of stem lignification and the extent of stem bending (perik et al., 2012). accordingly, increasing stem lignification directly influences stem strength and thereby reducing stem bending of gerbera cut flowers (nazarideljou and azizi, 2015). in past years, many attempts have been made to increase postharvest life and quality of cut flowers through different techniques. in cut flowers industry, inappropriate condition and inadequate nutrition often lead to a reduction in the quality and quantity of produced gerbera flowers; and regarding these issues can economically improve gerbera flowers production (khangoli, 2001). calcium is an essential element affecting on plant growth and development; and its accumulation in plants facilities pectin polymers’ linkage so as to improve mechanical strength of stem in line with reducing stem bending and extending flowers vase life (gerasopoulus and chebli, 1999; helper, 2005; li et al., 2012). the role of intra and intercellular calcium on cell metabolism change is attributed to its effect on the membrane structure and function and cell walls (ferguson and drobak, 1988). the results of previous studies showed that calcium, due to its role in the synthesis of pectin located in cell wall of xylem vessels, facilitates water translocation in stem and inhibits stem bending (van ieperen and van gelder, 2006). also, applying calcium as pre or postharvest treatments caused to increase the vase life of many cut flowers (gerasopoulos and chebli, 1999; hepler, 2005; li et al., 2012; geshnizjany et al., 2014). in spite of existing such documents, the effects of calcium on reducing stem bending of gerbera cut flowers are in needs of more investigations. therefore, in present research, the effect of different calcium concentrations in forms of chloride and calcium nitrate on stem bending and other related parameters in two gerbera cultivars were scrutinized. 2. materials and methods this research was conducted using a factorial experiment in completely randomized design (crd) with four replicates and fourteen plots in each replicate and five pots in each plot (totally 280 pots) in local commercial greenhouse (persian gulf, tehran, iran). based on the sensitivity to stem bending, two cultivars of gerbera, namely ‘intense’ as a tolerant cultivar and ‘rosaline’ as a sensitive cultivar to stem bending (nazarideljou and azizi, 2015), were used in this experiment. gerbera seedlings were cultivated in pots with 18 cm diameter filled with coco peat and perlite (1:1) maintained in greenhouse with 2025/15-18°c (day/night), 70±5% relative humidity and 14 h with a light intensity of 40 µmol-2 m-2 s-1 (naing et al., 2017). before receiving the treatments, the seedlings were feeded based on a nutritional formula used by most commercial greenhouses in iran (table 1). elements content (mmol/l) macro-elements nitrogen 10 phosphorous 1.8 potassium 5.5 magnesium 2 sulphate 3 micro-elements iron 40 magnesium 5 zinc 5 copper 1 molybdenum 1 boron 30 table 1 nutrient elements used in gerbera plant fertilization during excremental period aghdam et al. stem bending disorder of gerbera 59 after planting seedlings in pots and undergoing their perfect establishment, experimental seedlings received calcium chloride and calcium nitrate treatments at three levels (0.5%, 1%, and 1.5%) in form of foliage spray (mehran et al., 2007) while controls received distilled water. the flowers were harvested when two to three rows of ray floret got color but the stamens still did not open. after preparing of flowers stem in the length of 40 cm, they were placed in the erlenmeyer containing 500 cc distilled water and hydroxiqinolin sulphate (hqs) in concentration 200 mg/l. then, they were transferred into the ambient temperature and a light with intensity of 28 µmol-2 m-2 s-1 and 60% relative humidity (gerasopoulos and chebli, 1999). the treatments were measured on 9th of experiment. stem bending was determined by assessing the accompanying change in the position of the flower head as described previously (perik et al., 2012). ten excised petals per each flower were weighed (fresh weight, fw) and placed in distilled water in the dark for 6 hours to allow them to reach full turgidity and, hence, to determine their turgid weight (tw). these samples were then dried at 70°c for 24 h and their dry weight (dw) was recorded. finally, relative water content was calculated using the fw-dw/tw-dw (abdolmaleki et al., 2015). membrane stability index (msi) was recorded using an electrical conductivity meter based on ezhilmathi et al. (2007). petal samples were rinsed and immersed in 10 ml of distilled water. the samples were incubated at room temperature for 60 min with shaking (150 rpm). the electrical conductivity of the solution (ec1) was read after shaking using a conductivity meter. samples were incubated in 95°c water bath for 20 min the second reading (ec2) was taken after the solutions had cooled to room temperature. msi was calculated as [1-(ec1/ec2)] ×100. petal samples (0.2 g) was homogenized with 2 ml of 50mm phosphate buffer (ph=7) containing 1 mm edta, 1mm phenylmethylsulfonyl fluoride (pmsf) and 2% polyvinylpyrrolidone (pvp) (w/v) in ice waterbath and then centrifuged at 12000 g for 20 min at 4°c. the supernatant was collected and used for enzyme assay with a uv-visible spectrophotometer (cary 100, varian, usa). the activities of enzymes were determined according to standard methods previously reported for cat (ec1.11.1.6) (aebi, 1984) and sod (ec 1.15.1.1) (giannopolitis and ries, 1977). enzyme activities were expressed as enzyme units.mg-1 protein. protein content was determined according to the method of bradford (1976). the cell wall materials of the proximal end of flower stem were fractioned following the method of li et al. (2012). briefly, the stems were ground into fine powder in liquid nitrogen and extracted with 95% alcohol, then washed twice with boiling alcohol and methyl alcohol: chloroform (1:1, v/v), respectively. finally, the cell wall residues were dried over night at 50°c and used for analysis. hemi-cellulose content was determined by the phenol colorimetric assay (dubois et al., 1956) and the cellulose content was measured by the anthrone colorimetric assay (updegraff, 1969). lignin content was determined following the method of müse et al. (1997). all measurements were reported as µg/mg dw. stem calcium concentration was determined by atomic absorption spectrophotometer (abdolmaleki et al., 2015). the data were analyzed using sas software and the comparing means was carried out using duncan multiple range test at 5% probability and the drawing figures were carried out using excel 2013 software. 3. results and discussion stem bending the results of variance analysis showed that the main effects of treatment and cultivar (p<0.01) and their interaction effects (p<0.05) were significant on stem bending (table 2). all calcium treatments, except 0.5% calcium nitrate in ‘intense’, significantly increased the days until onset of stem bending. also, calcium chloride had better effect on stem bending in ‘rosaline’. stem bending is the most important factor affectdependent variable cultivar (c) treatment (t) c × t stem bending ** ** * relative water content ns ** ns membrane stability index ** ** * catalase activity ** ** ** superoxid dusmutase activity * ** * lignin content ** ** * cellulose content ** ** ** hemicellulose content ns ** * calcium content ** ** ns table 2 analysis of variance (anova) for cultivar, calcium treatment and their interactions effects on evaluated dependent variables in gerbera cut flower ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non-significance at p<0.05. adv. hort. sci., 2019 33(1): 57-65 60 ing the quality of cut flowers, flower vase life, and flower loss during harvesting in gerbera. genetic, nutrition, storage temperature, and the lack of water balance in xylem vessels are of the most important factors facilitate stem bending (van meeteren, 1978; perik et al., 2012; 2014). reduction in rigidity of the stems carrying flower is associated with the reduction in the formation of lignin compounds. in this regard, it has been stated that the higher-vase life cultivars of chrysanthemum contain more lignin compared to lower-vase life ones (lv et al., 2011). it has been described that tolerance to stem bending is contributed to the presence of a sufficient amount of sclerenchyma and lignin in the stems of flowers (perik et al., 2012). so, each factor being able to increase the stem rigidity also reduce stem bending. calcium ions maintain cell wall rigidity as they bind to pectin molecules, thereby increasing cell wall stiffness. the positive effects of calcium chloride on the reduction of stem bending in gerbera were proved by gerasopoulos and chebli (1999). also, perik et al. (2014) reported that calcium treatments decreased stem bending in gerbera cultivars likely due to enhancing stem rigidity. it seems that calcium inhibits vascular blockage and escalates stem strength in line with reduction in stem bending and consequently increasing flower vase life (fig. 1). relative water content the results showed that just treatment had a significant effect on relative water content (rwc). the effects of cultivar and its interaction with treatment were not statistically significant (table 2). compared to controls, all applied calcium treatments effectively fig. 2 the main effects of calcium treatments on petals relative water content of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 1 the effects of calcium treatments on day of stem bending of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. increased rwc and among all applied treatments, 1% calcium chloride had the better effect on rwc while 1.5% calcium nitrate gained the lowest effects on rwc (fig. 2). the results of this research are in agreements with those obtained by cortes et al. (2011) who reported that calcium treatments increased the relative fresh weight of cut flowers. in gerbera, applying calcium treatments extended flowers vase life by enhancing water uptake by stem and maintaining flower water content (geshnizjany et al., 2014). also, it has been stated that the flower fresh weight and solution absorption decreased whereas flower wilting increased, these mainly occur due to water loss by different organs of flowers (borochov and woodson, 1989; urban et al., 2002). in other words, when the stem vessels are blocked, flower spent the absorbed water through transpiration and subsequently reduced rwc (urban et al., 2002; van meeteren, 1978). in results, upholding a proper water uptake by cut flowers is of crucial issues in order for extending the vase life of flowers (urban et al., 2002). whenever the rate of transpiration exceeds more than that of water uptake, water deficiency occurs and leads to flower wilting and reduction in flower weight (van meeteren, 1978). in current research, the significant effect of calcium treatments on increasing the petal rwc of cut flowers was observed, it can be resulted from a reduction in stem blockage owing to increasing lignin biosynthesis. membrane stability index the results of variance analysis revealed that the main effects of cultivar and treatment as well as their interaction cultivar × treatment on msi was significant (table 2). also the mean comparison of the aghdam et al. stem bending disorder of gerbera 61 interaction effect revealed that calcium treatments used in both cultivars significantly increased msi. it was found a significant different among calcium treatments of both forms on msi. the highest rate of msi was observed in ‘intense’ cultivar by 1% calcium chloride which increased as much as 13% to controls, but the highest rate of msi was found in ‘rosaline’ cultivar by applying 1% calcium nitrate which increased as much as 15% to controls (fig. 3). in general, the senescence in plants is an oxidative and controlled process including biological, physiological, hormonal, and structural changes destroying macro molecules such as protein, nucleic acids, and lipids (iqbal et al., 2017). the lack of balance between generating reactive oxygen species (ros) and cleaning ros by plant defensive systems paves the way for inducing oxidative stresses which inflect damage on different parts of cells and destroy them. the destruction of cell membrane is one of the processes which intensifies in the presence of different ros, resulted in increasing cell electrolyte leakage (abdolmaleki et al., 2015). in our research, pretreatment of different sources of calcium increased msi in petals of gerbera cut flowers. calcium is supposed to alleviate the adversary effect of ethylene on initiating cell senescence through inhibiting cell membrane destruction and consequently to extend the vase life of cut flowers (de capdeville et al., 2005). also, the structural and functional roles of calcium on maintaining cell membrane and cell walls have been proved (ferguson and drobak, 1988). in other words, according to the results of our experiment, calcium treatments were found to enhance antioxidant enzyme activities resulted in decreasing oxidative stress and preserving cell membrane structure. our results are in agreement with those of abdolmaleki et al. (2015) who reported that applying calcium preharvest treatment retarded damage to cell membrane and hence increased vase life of rose cut flowers. it appears that calcium treatments directly increase cell wall stability and indirectly influence enzyme processes in order to increase the content of msi in gerbera cut flowers. cat and sod activity the results showed that the interaction effects of cultivar × treatment was significant in terms of catalase (cat) and superoxide dismutase (sod) (table 2). compared to controls, all applied treatments significantly increased cat in both cultivars and the effects of the treatments in both cultivars were the same (fig. 4a). also, calcium nitrate and calcium chloride significantly improved sod activities on both cultivars. the highest sod activity was observed in ‘intense’ cultivar by using 1.5% calcium nitrate, whereas in ‘rosaline’ cultivar it was found by applying 1.5% calcium chloride (fig. 4b). the process of senescence in cut flowers is usually accompanied with a modulation in the antioxidant enzymes’ activities (borochov and woodson, 1989). fig. 3 the effects of calcium treatments on petals membrane stability index of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 4 the effects of calcium treatments on catalase (cat) (a) and superoxide dismutase (sod) (b) activities of gerbera petals. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. 62 adv. hort. sci., 2019 33(1): 57-65 the results of different studies showed that increasing antioxidant enzyme activity extended flower longevity because these antioxidants retarded the process of senescence (ezhilmathi et al., 2007). it has been demonstrated that calcium treatments, through changing in antioxidants activities, delayed flower senescence and maintained chlorophyll and protein contents in gladiola flower during storage (sairam et al., 2011). in this respect, zhao et al. (2006) showed that calcium chloride enhanced the activities of cat and sod in the cut flowers of rosa hybrida and dendrobium phalaenopsis. in current research, both forms of calcium increased cat and sod activities in gerbera flowers (fig. 4). it is obvious that improving antioxidant activities prevents cell membrane from breakdown and maintains membrane integrity. calcium may serve as a secondary messenger in a pathway to stimulate the synthesis of antioxidant enzymes (jiang and zhang, 2003). with respect to what mentioned above, calcium may indirectly stimulate the antioxidant activities and consequently reduce the oxidative stresses through lessening the peroxidation activity and maintaining the membrane integrity, or it may directly keep the membrane integrity and cell wall and delay the process of senescence in cut flowers. lignin, cellulose, and hemicellulose the results showed that the interaction effect of cultivar × treatment was significant on lignin (p<0.05), cellulose (p<0.01), and hemicellulose (p<0.05) (table 2). comparing to ‘rosaline’, more lignin and cellulose were observed in ‘intense’, but the rate of hemicellulose in both cultivars was not significantly different (fig. 5). application of both forms of calcium at all their concentrations significantly improved the rates of lignin and hemicellulose in both cultivars (fig. 5a and 5b). the highest rate of lignin was found in ‘intense’ by 0.5% calcium nitrate while it was obtained in ‘rosaline’ by 1.5% calcium chloride (fig. 5a). on the other way, all calcium treatments increased cellulose in ‘intense’ in comparison to control, but just 1% and 1.5% calcium chloride and 0.5% of calcium nitrate increased cellulose in ‘rosaline’ as comparing to controls (fig. 5b). one of probable factors on providing stem bending in cut flowers is contributed to the lack of sufficient lignin formation in cell wall, especially in the secondary wall besides its effect on stem rigidity, causes to take up water continuously by flowers (vanholme et al., 2010). peroxidase (pod) and phenylalanine ammonia lyase (pal) are of key enzymes involving in the pathway of lignin biosynthesis; their roles in stem strength and delaying stem bending in gerbera flowers have been proved (nazarideljou and azizi, 2015). it has been reported that the cut flowers prepared from the long-lasting cultivars contain more pod and pal compared to short -lasting cultivars (lv et al., 2011; nazarideljou and azizi, 2015). in the present research, ‘intense’ showed a higher tolerance to stem bending than ‘rosaline’ because of containing more lignin and cellulose. the results of this research also revealed that calcium treatments significantly increased lignin, cellulose, and hemicellulose contents in gerbera. perik fig. 5 the effects of calcium treatments on lignin (a), cellulose (b), and hemicellulose (c) of gerbera petals. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. aghdam et al. stem bending disorder of gerbera 63 et al. (2014), by investigating the effects of different treatments on stem rigidity, figured out that using calcium chloride lessened stem bending in gerbera flowers through increasing stem strength resulted from lignin formation. in this regard, li et al. (2012) reported that applying calcium chloride at pre-harvest stage significantly enhanced the rate of pectin, lignin, cellulose, and hemicellulose in the cut flowers of herbaceous peony. also, it has been stated that using some materials such as ethylene and salicylic acid, through affecting on the some enzymes like pal involving in lignin biosynthesis, had a direct influence on stem bending of flower (ferrante et al., 2007). also, naing et al. (2017) reported that applying sodium nitroprusside augmented the gene expression of lignin biosynthesis; and likewise pal extended flower vase life and diminished stem bending of gerbera flowers. calcium content the interaction effect of cultivar × treatment was not significant in terms of calcium, but the main effects of treatment and cultivar were significant (table 2). the rate of calcium in ‘intense’ was observed higher than of that in ‘rosaline’ (fig. 6a). in other words, all calcium treatments significantly increased the amount of calcium in comparison to controls. there was a significant difference among all treatments and 0.5% calcium nitrate and calcium chloride had the lowest effect on calcium content of the stem cut flower (fig. 6b). calcium is an essential element effecting on the plants’ growth and development processes in a way that its accumulation in them facilitates creating the connections among pectin polymers; and accordingly it escalates the mechanical strength of stem and lignin production. eventually, they lead to reducing stem bending and increasing flower longevity (gerasopoulos and chebli, 1999; li et al., 2012). the internal and inter cellular roles of calcium are contributed to its part in plants leading to the changes on cell metabolism as well as its effect on the structure and function of cell wall and membrane (ferguson and drobak, 1988). the results showed that application of calcium, due to its role in synthesis of the pectin located in cell wall of xylem vessels, improved water translocation in stem and prevented cut flowers from stem bending (van ieperen and van gelder, 2006). in reality, a reduction in the resistance towards water conductivity in plants is one of calcium duties for extending vase life of cut flowers (cortes et al., 2011). calcium improves the flower longevity and this may delay many issues such as physiological phenomena related to senescence, reduction in water uptake, and losing more water from flower through transpiration, and thereby reducing flower fresh weight and stem bending (gerasopoulos and chebli, 1999; de capdeville et al., 2005; sosanan, 2007). in current research, applying calcium increased calcium concentration in stem of cut flowers. in a similar way, using different forms of calcium at growth stage of herbaceous peony enhanced the internal calcium, lignin formation, and consequently the rigidity of the stems carrying flowers (li et al., 2012). nikbakht et al. (2008) reported that calcium accumulation in the stem of gerbera delayed stem bending. therefore, it seems that an increase in the content of calcium inside stems is associated with the reduction of stem bending and extending flower longevity. 4. conclusions in general, the results showed that applying calcium in different forms of nitrate and chloride reduced the stem bending in gerbera cultivars. both forms of calcium had a positive effect on gerbera cultivars, but mostly calcium chloride had better effect on stem fig. 6 the main effects of cultivar (a) and calcium (b) treatments on calcium content of two gerbera flower stem. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. adv. hort. sci., 2019 33(1): 57-65 64 bending in comparison to calcium nitrate; and its 1% concentration compared to other its concentrations gained the best results. the positive effect of calcium 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(in japan). impaginato 319 adv. hort. sci., 2017 31(4): 319-327 doi: 10.13128/ahs-20545 phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds e. abedi gheshlaghi 1 (*), v. rabiei 2, m. ghasemi 3, j. fattahi 3, f. razavi 2 1 horticulture crops research department, guilan agricultural and natural resources research and education center, areeo, rasht, iran. 2 department of horticulture, faculty of agriculture, university of zanjan, iran. 3 horticulture research institute, citrus and subtropical research center, agricultural research, education and extension organization (areeo), ramsar, iran. key words: actinidia deliciosa, biochemistry, chilling, enzyme. abstract: bud dormancy is an adaptability process in woody plants that enables them to survive in unfavorable conditions. in the present study, the phenols, antioxidant capacity, and activity of three enzymes were evaluated during endodormancy phases in two hayward and tomuri cultivars and two female and male golden genotypes of kiwifruit buds. the buds were collected from ten-year-old own-rooted vines from the end of october 2015 until the end of january 2016 in the north of iran. the results revealed that phenols, antioxidant capacity (rsa), phenylalanine ammonia-lyase (pal), and polyphenol oxidase (ppo) activities of buds significantly increased at the beginning of endodormancy and subsequently decreased at the end of the endodormancy. the pod activity increased in hayward and tomuri from the onset of endodormancy and continued for two weeks after the endodormancy release. the total phenol had a positive and significant correlation with rsa and pal enzyme activity. furthermore, higher antioxidant capacity and phenols in both male and female golden genotypes were attributed to the higher pal enzyme activity in both genotypes. this study proposes that the rsa%, pal activity, and phenol concentration could be employed as a biomarker to indicate bud dormancy phases in kiwifruit. 1. introduction the first axillary buds are initiated on the developing shoots of kiwifruit shortly after bud break in the first growing season (walton et al., 1997). similar to temperate fruits, the axillary buds of kiwifruit can be induced into endodormancy by short days and low temperatures at the end of the summer or the beginning of autumn (mcpherson et al., 1995). bud endodormancy is an adaptability process in woody plants that (*) corresponding author: eabedig@yahoo.com citation: abedi gheshlaghi e., rabiei v., ghasemi m., fattahi j., razavi f., 2017 phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds. adv. hort. sci., 31(4): 319327 copyright: © 2017 abedi gheshlaghi e., rabiei v., ghasemi m., fattahi j., razavi f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2017 accepted for publication 19 october 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 319-327 320 enables trees to survive in unfavorable conditions such as drought and extremely hot and cold weather (arora et al., 2003); in addition, more importantly, it encourages the reproductive processes such as the formation of flowers and fruit set to be accomplished in favorable condition and guarantees the reproductive growth and survival of the plants (campoy et al., 2011). endodormancy (winter dormancy) is a true dormancy in which the bud growth is prevented by an inhibitory system within the bud (horvath et al., 2003). overcoming endodormancy and maximum bud break and flowering in favorable environmental conditions are achieved by accumulating the minimum amount of chilling hours (mcpherson et al., 1997). after that endodormancy is released, the growth of buds is prevented directly by external environmental factors. this type of dormancy mainly occurs in late winter and is named ecodormancy (horvath et al., 2003). generally, horticultural specialists determine the time of bud break by chill ing and heat units. however, this method is based on ambient temperature, varies according to the environmental conditions, and cannot express the internal situation of buds (dennis, 2003). during bud endodormancy, there is no visible growth, but physiological changes in respiration, growth regulators, carbohydrate metabolism, the amount of water, and other compounds occur, influencing bud endodormancy control (ben mohamed et al., 2010). a series of the changes occurring in the biochemistry of the buds appear to indicate the shift from the endodormancy stage to the ecodormancy stage (pakish et al., 2009; szecskó et al., 2002). richardson et al. (2010) stated that high and stable sucrose concentrations are likely to be a good indicator of the true dormancy of the buds in kiwifruit (actinidia deliciosa). investigating the relationship between biochemical compounds and the beginning of the endodormancy in nine varieties of apricot revealed seasonal changes in phenolic compounds and peroxidase (laslo and vicas, 2012). these changes are caused by the accumulation of chill ing units during the endodormancy, that is necessary for the development of some phenological stages. pakish et al. (2009) studied peroxidase and oxidase activities in the varieties of pistachio buds during the winter and observed seasonal variation in the november-march interval. there is a significant difference in terms of the phenols of buds in different dormancy stages within cultivars of the same species. the total phenol of the flowering buds in peach (szalay et al., 2005) and apricot (laslo and vicas, 2012) increased at the beginning of endodormancy, showed a gradual increase in dormancy period, and disappeared during flowering. large variations in the content of polyphenols can be observed in certain varieties of pistachio (pistacia vera l.) in the november-march interval. the phenol of all cultivars was reduced in the swollen buds (pakish et al., 2009). factors controlling the onset, maintenance, and termination of endodormancy are varied and have not been studied much (luedeling et al., 2009). moreover, the starting point of endodormancy, the end of endodormancy, and the start of ecodormancy are not clearly defined in plants; therefore, the study of changes in biochemical compounds in this field could be useful (pakish et al., 2009; ben mohamed et al., 2010). a number of studies have been done on the changes in carbohydrates (richardson et al., 2007, 2010), nitrogen, and amino acid (walton et al., 1991) of kiwifruit in the endodormancy period; however, the activities of enzymatic and non-enzymatic antioxidants such as phenol and its metabolism have not been studied yet. thus, the aim of this study was to inspect the changes in antioxidant capacity, total phenol, and activity of three enzymes during endodormancy and the early ecodormancy in four cultivars and genotypes of kiwifruit. 2. materials and methods plant material and sampling bud samples of hayward and tomuri kiwifruit cultivars (actinidia deliciosa) and two male and female golden genotypes (a. chinensis; mass selection of golden kiwifruit cultivar seedlings) were collected from canes of ten-year-old own-rooted vines from the end of october 2015 until the end of january 2016 with a 7-day interval at 11 steps. from the first to the last sampling, the vines received 0, 222, 309, 443, 570, 740, 864, 1003, 1125, 1272, and 1450 chilling units, respectively (richardson et al., 1974). kiwifruit vines were located at the national citrus and subtropical research institute of iran (latitude 75.36° north and longitude 33.51° east) and were trained on a t-bar training system with a planting distance of 4×6 m. the samples were immediately frozen in liquid nitrogen and kept for subsequent analyses at -80°c. at every sampling date, 30 buds were collected from 1-year-old canes at nodes 6 to 20 starting from the basal end of canes. ten buds abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 321 were selected randomly in three replications for further analyses of biochemical compounds (richardson et al., 2010). the activities of phenylalanine ammonia-lyase (pal), peroxides (pod), polyphenol oxidase (ppo), antioxidant capacity (rsa), and total phenol content were determined in buds during endodormancy. estimation of endodormancy period chilling requirements and bud endodormancy period of kiwifruit cultivars and genotypes were estimated via single-node cuttings test. simultaneously, fifteen cuttings in three replications of each cultivar and genotype were collected and the buds were taken for biochemical measurements. cuttings from each treatment were transferred into a forcing chamber at 25°c, with 16 h of light (wall et al., 2008). the lowest mean time budburst (mtb) for half of the buds were considered as an endodormancy release (tisne-agostini et al., 1992). extraction and determination of total phenol and antioxidant capacity the total phenol of each extract was determined according to the folin-ciocalteu procedure reported by meyers et al. (2003). moreover, the spectrophotometric method introduced by wettasinghe and shahidi (2000) was employed for the chemical determination of antioxidant. extraction and assay of pal one hundred mg of kiwifruit buds powdered by liquid nitrogen were mixed with 2 ml of 1.0 mm borate buffer containing 1.0% of polyvinyl pyrrolidone. after homogenization by a homogenizer (model ika-t8, germany), the samples were centrifuged for 15 min at 4°c and 13,000 rpm. the supernatants were slowly transferred into the tubes by pipette. extracts for the ensuing measurements were maintained at -80°c. pal (ec 4.3.1.24) activity was determined according to the study carried out by yu et al. (2012). extraction and assay of ppo and pod activities the two hundred-mg samples of fresh buds, which were collected, were ground in liquid nitrogen and homogenated with 2 ml of potassium phosphate buffer (50 mm, ph= 7.0) containing 1% polyvinyl pyrrolidone (pvp) (w/v) and 0.05% edta at 4°c. the homogenate was centrifuged at 14000 rpm for 15 min at 4°c. the supernatant was used as a crude enzyme solution for assay and was maintained at -80°c for the following measurements. the activity of ppo enzyme (ec 1.14.18.1) was quantified by the method described by in et al. (2007). the activity of pod enzyme (e.c 1.11.1.7) was measured by the method employed by srivastava et al. (1983). the activities of these enzymes were calculated, using the beer-lambert law on the basis of a single enzyme unit (μmol) per mg of fresh weight according to the following formula: u/g fwmin = absorption changes per minute × reaction mixtur/ supernatant volume × extinction coefficient statistical analyses this study was conducted as a two-factor factorial in a completely randomized design. the first factor is 11 sampling dates, and the second factor is four cultivars having three replications in the period of 20152016. the anovas and standard errors of the mean (se) were generated, using sas 9. all significant means were separated, using the duncan (p≤0.01). the correlation between the total phenol and antioxidant capacity, pal, ppo, and pod activities was calculated via the software spss 22. 3. results and discussion the beginning date for the chilling accumulation was considered to be when a stable chilling accumulation occurred and the temperatures causing a negative effect were infrequent (richardson et al., 1974; guerriero et al., 1990). this date corresponded with 27th october 2015. the first samples were conducted on this date. mean time budburst was more evident on this date than on the date of the endodormancy release; thus, the kiwifruit axillary buds may inter endodormancy sooner than the end of the summer or the beginning of autumn (mcpherson et al., 1995). the results revealed that the maximum depth of endodormancy in this study was in late november. the duration of bud endodormancy was different in the cultivars and genotypes. the end of endodormancy was 21st december 2015 for female golden genotype (740-unit chilling), 28th december 2015 for male golden genotype (864-unit chilling), and 4th january 2016 for hayward and tomuri cultivars (1003-unit chilling) as shown in figure 1. the antioxidant capacity indicated a significant difference between the buds of genotypes and cultivars sampled on different dates (p≤0.01). the highest value in the antioxidant capacity was observed in female golden genotype on 28th december 2015 (fig. 2); however, there were no significant differences in the antioxidant capacity of all buds samples adv. hort. sci., 2017 31(4): 319-327 322 collected in december (i.e. 07th, 14th, 21st and 28th). the lowest value in the antioxidant capacity was observed in tomuri cultivar on 11th january after endodormancy release (fig. 2). the antioxidant capacity of buds changed considerably at the beginning and during the maintenance and release of endodormancy (p≤0.01) (fig. 2). the increase in antioxidant capacity from the end of october to the early november was simultaneous in four cultivars and genotypes; however, the peak period of the antioxidant capacity of buds was different between these cultivars and genotypes (fig. 2). the antioxidant capacity showed a significant reduction (p≤0.01) in the male and female genotypes at the end of december, being simultaneous with the end of bud endodormancy of both genotypes. the reduction in the antioxidant capacity in hayward and tomuri cultivars occurred about two weeks later, coinciding with the completion of their chilling requirements and the end of endodormancy in the buds (fig. 2). the activity of pal enzyme has been shown in figure 3 and demonstrated a significant difference in buds of all kiwifruit cultivars and genotypes (p≤ 0.01). the activity of this enzyme in the male and female golden genotypes was more than that of the hayward and tomuri cultivars (fig. 3). female golden genotype had the highest pal activity in middecember. the lowest activity of this enzyme was in tomuri cultivar on 30th november 2015. pal enzyme activity in all four kiwifruit cultivars and genotypes began an upward trend at the end of november (p≤0.01) (fig. 3). it remained relatively stable during endodormancy in cultivars and genotypes, but was associated with a fluctuation in male and female golden genotypes during ecodormancy. however, the enzyme activity of all cultivar showed a significant decrease (p≤0.01) at the end of endodormancy compared to the stable periods of endodormancy. plants release hydrogen peroxide (h2o2) in response to the environmental stress. low temperature stress has also been shown to induce h2o2 accumulation in cells (okane et al., 1996). hydrogen peroxide, as the second messenger of the increase in the activity of pal enzyme, can activate pal enzyme activity, as a key enzyme in the phenylpropanoids pathway, ultimately leading to higher total phenol and accumulation of flavonoids (wang et al., 2015). oxidative stress caused by chill ing during the endodormancy of kiwifruit buds increased the activity of this enzyme and the production of antioxidant compounds such as phenols, resulting in a higher antioxidant capacity in the buds during the endodormancy. it has been reported that cold acclimation of fig. 1 effect of sampling date of cuttings on average of 50% bud break and endodormancy end period in four kiwifruit cultivars and genotypes during dormant season in 2015-2016. the arrows show the endodormancy end date for each cultivar and genotype. fig. 2 effects of kiwifruit cultivars and different sampling dates on antioxidant capacity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 3 effects of kiwifruit cultivars and different sampling dates on phenylalanine ammonia-lyase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds moreover, ± the standard error of mean is shown on the vertical bar. abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 323 plants leads to a remarkable increase in pal activity, depending upon the range of low temperature to which the plants are subjected (stefanowska et al., 2002). the amount of total phenol have been shown in figure 4 and varied between cultivars and genotypes of kiwifruit, and there were significant differences in their buds (p≤0.01). male and female golden genotypes had higher total phenol than hayward and tomuri cultivars (fig. 4). total phenol content showed substantial changes at the beginning, during the maintenance, and at the end of the endodormancy of kiwifruit buds (p≤0.01) (fig. 4). the amount of total phenol showed an increasing trend from the late october. in the late november-early december period, coinciding with the onset of true endodormancy, the amount of total phenol reached its maximum (fig. 4) and its value remained at a high level in the buds of cultivars and genotypes during this period. by reaching the end of endodormancy, the amount of phenol had decreased significantly at the end of december and at the beginning of january in the buds of male and female golden genotypes and hayward and tomuri cultivars, respectively (p≤0.01). phenolic compounds are a valuable piece of evidence used in determining the differences between diverse varieties of myrtus communis and pistacia lentiscus and have a key role in detecting the genetic differences in biochemical methods (tattini et al., 2006). thus, it appears that the significant differences in terms of the total phenol content between hayward and tomuri cultivars and male and female golden genotypes (p≤0.01) (fig. 4) are the result of their genetic differences. endodormancy is developed gradually after the cessation of growth; additionally, the severity of the endodormancy deepens in autumn and then gradually disappears by removing the physiological barriers of growth through the chilling process (dennis, 2003). total phenol concentration and antioxidant capacity enhance along with the development of endodormancy and reach their maximum value in the deepest stage of endodormancy (fig. 2, 4). the changes in these two variables are similar in the establishment, maintenance, and release of endodormancy. a high total antioxidant activity in male and female golden genotypes may be attributed to the high amount of phenol. phenolic compounds are synthesized in plant cells in favorable environmental conditions, but environmental stresses change their levels in cells (kliebenstein, 2004). mid-autumn cold and the start of endodormancy period causes an increase in oxidative stress in plants. this stress results from reactive oxygen species that affect the growth of plants (scalabrelli et al., 1991; mittler et al., 2004). plants possess a protective system composed by the enzymatic antioxidant system such as peroxidase and catalase (anderson et al., 1995) and the non-enzymatic systems (agarwal and pandey, 2004). phenols are non-enzymatic antioxidants and their antioxidant activities are mainly due to their redox properties which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quencher (huda-faujan et al., 2009). phenols play an important role not only during cold resistance, but also during breaking the endodormancy of peach (siller-cepeda et al., 1992) and apricot (viti and bartolini, 1998) buds as an antioxidant. total phenol changes in kiwifruit buds are in agreement with the results of the studies on peach flower buds (szalay et al., 2005), apricot vegetative buds (laslo and vicas, 2012), and pistachio flower buds (pakish et al., 2009). correlation analyses showed a positive significant correlation between total phenol, antioxidant capacity and pal activity in kiwifruit cultivars and genotypes (table 1). reduction in antioxidant capacity (fig. 2) and total phenol (fig. 4) after receiving chilling and a stable period in winter could be considered as a biomarker of endodormancy release in the cultivars which were studied. in cultivars and genotypes of kiwifruit buds, ppo activity changed substantially (p≤0.01) as shown in figure 5. this enzyme had the highest activity in the tomuri variety on 7th january 2016 and the lowest activity in male and female golden genotypes at the fig. 4 effects of kiwifruit cultivars and different sampling dates on phenol content of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. adv. hort. sci., 2017 31(4): 319-327 324 first sampling date (fig. 5). at the end of october 2015 and contemporaneous with the development of bud endodormancy, the activity of ppo enzyme, in all cultivars except hayward, increased and reached its peak in mid-december and then decreased (p≤0.01; fig. 5). ppo activity in the hayward variety peaked a week earlier than the other cultivars. in tomuri cultivar, ppo activity was stable for three weeks in the dormancy period and reduced at ecodormancy. ppo is a copper-containing enzyme which catalyzes the oxidation of phenolic compounds to quinone or quinine-like compounds in the presence of molecular oxygen. a high ppo enzyme activity after endodormancy is probably due to the removal of some growth-inhibiting phenols (wang et al., 1991), and the phenolic substances such as inhibitors or stimulants change enzyme activity (thirugnanasambantham et al., 2013). the increased activity of the ppo enzyme at the early stage of endodormancy period and its declined activity at the end of the endodormancy of grape buds (scalabrelli et al., 1991), plums (szecskó et al., 2002), and pistachio flower buds (pakish et al., 2009) were reported, corresponding with the results of this experiment. it appears that the effect of antioxidant compounds is to inhibit free radicals and reactive oxygen species in cultivars and genotypes during stress. pod activity was notably different in buds (p≤0.01). this enzyme had the highest activity in the tomuri cultivar after the endodormancy release (fig. 6). the lowest activity was observed at all cultivars and genotypes at the first sampling date (the 27th october). the pod activity pattern was not constant during the endodormancy of buds in the cultivars and genotypes which were studied (fig. 6). pod activity increased in golden genotypes later than that in the hayward and tomuri cultivars. the activity of this enzyme increased significantly in early december and mid-december 2015 in male and female golden genotype buds, respectively (p≤0.01). however, pod activity decreased significantly in both genotypes at the bud endodormancy release (fig. 6). the increased activity of pod at the end of october, in ** correlation is significant at the 0.01 level. table 1 correlation coefficient of kiwifruit cultivars and genotypes between phenol, antioxidant capacity (rsa %), phenylalanine ammonia-lyase (pal), peroxidase (pod), and polyphenoleoxidase (ppo) fig. 5 effects of kiwifruit cultivars and different sampling dates on polyphenol oxidase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 6 effects of kiwifruit cultivars and different sampling dates on peroxidase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten bud. moreover, ± the standard error of mean is shown on the vertical bar. cultivars and genotypes phenol pal rsa pod ppo hayward phenol 1 0.25 ns 0.95 ** -0.11 ns -0.34 ns pal 0.25 ns 1 0.17 ns 0.24 ns -0.18 ns rsa 0.95 ** 0.17 ns 1 -0.22 ns -0.27 ns pod -0.11 ns 0.24 * -0.22 ns 1 0.44 ns ppo -0.34 ns -0.18 ns -0.27 ns 0.44 ns 1 tomuri phenol 1 0.38 ns 0.94 ** 0.38 ns 0.28 ns pal 0.38 ns 1 0.42 ns 0.20 ns -0.22 ns rsa 0.94 ** 0.42 ns 1 0.27 ns 0.28 ns pod 0.38 ns 0.20 ns 0.27 ns 1 0.28 ns ppo 0.28 ns -0.22 ns 0.28 ns 0.28 ns 1 female golden phenol 1 0.76 ** 0.93 ** 0.20 ns 0.16 ns pal 0.76 ** 1 0.67 * -0.19 ns -0.16 ns rsa 0.93 ** 0.67* 1 0.19 ns 0.14 ns pod 0.20 ns -0.19 ns 0.19 ns 1 0.88 ** ppo 0.16 ns -0.16 ns 0.14 ns 0.88 ** 1 male golden phenol 1 0.75 * 0.83 ** 0.36 ns 0.26 ns pal 0.75 * 1 0.74 * 0.40 ns 0.37 ns rsa 0.83 ** 0.74 * 1 0.61 ns 0.49 ns pod 0.36 ns 0.40 ns 0.61 ns 1 0.95 ** ppo 0.26 ns 0.37 ns 0.49 ns 0.95 ** 1 abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 325 requires further investigation. this study indicated a significant and stable increase in the pal and rsa activities. furthermore, we found that phenol concentration could be associated with the transition to, and maintenance of, bud in true endodormancy. due to the positive and significant correlation between total phenol, antioxidant capacity, and pal activity, we concluded that antioxidant capacity, in both male and female golden genotypes, is attributed to the high phenol which was a result of high pal enzyme activity. acknowledgements the paper is resulted of ebrahim abedi 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nano-silicon dioxide. j. food. eng., 113: 408-414. impaginato 399 adv. hort. sci., 2018 32(3): 399-406 doi: 10.13128/ahs-22299 ready-to-eat raspberries: qualitative and nutraceutical characteristics during shelf-life g. cortellino 1, p. de vecchi 1, r. lo scalzo 1, v. ughini 2, g. granelli 3, m. buccheri 1 (*) 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (crea), centro di ingegneria e trasformazioni agroalimentari, via venezian, 26, 20133 milano, italy. 2 dipartimento di scienze delle produzioni vegetali sostenibili, facoltà di scienze agrarie alimentari ed ambientali, università cattolica sacro cuore, via emilia parmense, 84, 29100 piacenza, italy. 3 dipartimento di scienze agrarie e ambientali, produzione, territorio, agroenergie, università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: anthocyanins, firmness, phenols, rubus, scavenging activity. abstract: raspberry (rubus idaeus l.) fruits are characterised by a high content of nutraceuticals, such as vitamin c, polyphenols and anthocyanins, which are considered antioxidant compounds. the ready-to-eat raspberry product could increase the market opportunities and the consumption of this high-valueadded fruit. the aim of this research was to evaluate the evolution of qualitative and nutraceutical characteristics during the shelf-life of ready-to-eat raspberries. samples from three raspberry cultivars (‘glen magna’, ‘tulameen’ and ‘heritage’) were sanitized and then packed in polypropylene bowls. the analyses were carried out at harvest (raw material) and after 3, 6 and 8 days of storage at 3°c. the study indicated the loss of fruit firmness as the most problematic aspect, followed by a less important change in hue values from light red to dark red. the modifications of chemical-physical parameters (soluble solids content, ph and titratable acidity) during shelf-life did not compromise the product quality. processing and cold storage affected only slightly the nutraceutical profile (scavenging activity, phenols and anthocyanin content), except for ascorbic acid, therefore, the ready-to-eat raspberries could be considered a good source of compounds with potential health benefits. some handling difficulties were highlighted during processing due to the high fragility of fruit which caused a high percentage of waste. 1. introduction increasing epidemiological data suggested the correlation between the consumption of fresh fruits and vegetables with the prevention, delay or onset of chronic degenerative diseases, including cancer (kaur and kapoor, 2001). fruit and vegetables are, in fact, rich of the so-called (*) corresponding author: marina.buccheri@crea.gov.it citation: cortellino g., de vecchi p., lo scalzo r., ughini v., granelli g., buccheri m., 2018 ready-to-eat raspberries: qualitative and nutraceutical characteristics during shelf-life. adv. hort. sci., 32(3): 399-406 copyright: © 2018 cortellino g., de vecchi p., lo scalzo r., ughini v., granelli g., buccheri m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 december 2017 accepted for publication 19 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 399-406 400 nutraceuticals, which are compounds with a significant biological action, especially as antioxidants (szeto et al., 2002; fu et al., 2011). phenolics compounds, as an example, have a significant antioxidant activity (szajdek and borowska, 2008). they can donate electrons to the reactive oxygen species (ros), converting them into innocuous molecules (haminiuk et al., 2012) and they can also exert beneficial modulatory action in cells (williams et al., 2004). ascorbic acid (aa) is an essential vitamin that is found in fruits and vegetables and it has several positive functions: in plant and animal systems, aa interacts enzymatically and non-enzymatically with the reactive oxygen species and it is able to terminate the radical chain reactions by converting the ros in non-toxic products (davey et al., 2000). red raspberry fruit (rubus idaeus l.) are highly appreciated by consumers for their aromatic taste, especially when they are fully ripe. they are an important dietary source of bioactive compounds (kähkönen et al., 1999) such as anthocyanins, hydrosoluble pigments belonging to the class of polyphenols (benvenuti et al., 2004; pantelidis et al., 2007; szajdek and borowska, 2008) and vitamin c (mazur et al., 2014 a, b). unfortunately the postharvest life of this small fruit is short, because of its high respiration rate, loss of firmness and freshness, susceptibility to fruit rot and darkening (krüger et al., 2011); consequently it loses very early its market viability. ready-to-eat fruits and vegetables are one of the most important novel products introduced into the italian agro-food system over the last 30 years and they represent a dynamic and innovative sector. in the last few years, the consumption of these products has increased, because the doubt and mistrust that characterised them in the 90’s have now disappeared. ready-to-eat products are very convenient because they do not need any preparatory operation (washing, cutting) before consumption. there are obviously some disadvantages in using this kind of products: they are usually costly and the processing operation can shorten the postharvest life of the fruit/vegetable. however, since fruit are poorly eaten by kids and teens (oecd, 2012) the diffusion of ready-to eat products is often encouraged in order to increase fruit consumption among young people. raspberries are generally eaten fresh and a ready-toeat product could increase the consumption of this bioactively rich fruit. the aim of this research was to evaluate the qualitative and nutraceutical characteristics during the shelf-life of ready-to-eat raspberries. 2. materials and methods raw material investigations were carried out on raspberry fruits of three different cultivars picked at commercial maturity: ‘heritage’ and ‘glen magna’, grown at the fruit experimental centre of milan university (italy) and ‘tulameen’, grown at the experimental comparative field for berry fruits of the catholic university of piacenza (italy). on arrival at the laboratory the fruits were stored at 3±1°c till the processing time, always within 24 h. processing raspberries were manually selected in order to remove damaged fruits. fruits were dipped in a disinfectant solution (0.022% of active chlorine) for 10 min and successively rinsed in tap water for 1 minute. raspberries, after being softly dried with adsorbent paper, were packed and hermetically sealed in polypropylene (transpiration rates (tr): o2tr: 0.117 mm3 m-2 d-1 pa-1; co2tr: 1.847 mm3 m-2 d-1 pa-1) bowls (100 g per bowl), previously sanitized by uv radiation, and hermetically sealed with a film (o2tr: 0.476 mm3 m-2 d-1 pa-1; co2tr: 2.17 mm3 m-2 d-1 pa-1) using a packaging machine mod. tsm 95 (minipack-torre, dalmine, bergamo, italy). a rack, made of the same material was inserted inside the bowl in order to drain any dripping liquid. the packed fruits were then stored at 3±1°c for 8 days. chemical and physical analyses chemical and physical analyses were carried out on six samples (100 g each) of raspberry fruits per treatment at harvest (raw material) and after 3, 6 and 8 days of storage at 3°c. fruit of each bowl were weighed and the amount of weight loss was measured by the percentage alteration between the initial (packaging) and final weight of the fruit of each bowl. tititratable acidity [g citric acid/100 g fresh weight (fw)] and ph were assessed according to the official methods of analysis (aoac, 1985) and the soluble solids content (ssc, %) was measured by a multiscale automatic refractometer (rfm91 model, bs, uk). colour parameters l*, a* and b* were measured by using the spectrophotometer cm-2600-d minolta and hue was calculated as arctangent (b*/a*). texture was measured on 15 fruit per treatment and sampling date, using an instron universal testing machine (model 1140, instron, high wycombe, uk), as maximum force (g) used to compress by the fruit (crosshead speed 10 cm/min). cortellino et al. nutritional quality of ready-to-eat raspberries 401 analysis of the nutritional compounds nutritional compounds were analyzed on 3 samples (100 g each) per treatment and sampling date. the fruit extracts (2 per sample) were prepared as follows: 2 g of homogenized flesh were extracted with 18 ml of solution made of etoh 95% and hcl 0.02n (1:1 v/v), vortexed for 30 sec, centrifuged (15 min, 4°c, 10000 g) and filtered through a cheesecloth. since the most important nutraceuticals in rubus fruits belong to the phenols, this class of compounds was analysed by two different assays. i) total phenols content (tpc) was measured by folin-ciocalteu method, as described by singleton and rossi (1965). an aliquot of 150 µl of fruit extract, 5 ml of distilled water and 1 ml folin-ciocalteu reagent were incubated for 8 min, followed by the addition of 2 ml of 20% na2co3. after 2h in the dark, the absorbance was measured at 730 nm against blank with a uv-uvidec 320 spectrophotometer (jasco, japan). results were expressed as mg of gallic acid equivalent (gae) per 100 g of fresh weight (fw). ii) total monomeric anthocyanin pigment was estimated by the ph differential method described by giusti and wrolstad (2001) using a uv-uvidec 320 spectrophotometer (jasco, japan). extracts were diluted 1:10 with two buffers at ph 1.0 and 4.5; readings of each sample were made at 510 nm and 700 nm and the absorbance (a) of the diluted sample was calculated using the formula: a= (a 510 a 700 ) ph 1.0 (a 510 a 700 ) ph 4.5 the anthocyanin concentration was calculated using the cyanidin 3-glucoside (c3g) molar extinction coefficient 26,900 and it was expressed as mg of c3g equivalents in 100 g of fw. ascorbic acid (aa) was determined according to the method described by davey et al. (2003) with some modifications. briefly, 300 ml of the extract were added to 600 ml of 6% meta-phosphoric acid solution, diluted 1:20 in ortophosphoric acid solution (0.02m) and analyzed by an hplc system (jasco, japan). aa was separated on an inertsil ods-3 (gl science) column kept at 37°c and connected to a variable wavelength detector (uv1575, jasco) set at 254 nm, using as mobile phase 0.02m orthophosphoric acid at the flow rate of 0.7 ml/min. ascorbic acid concentration was expressed as mg in 100 g of fw. samples were also subjected to one of the most used free radical scavenging assays, which is relatively easy and cheap in execution and is strongly correlated with the sample’s nutraceutical content: the 2,2-diphenyl-1-pycrilidrazyl (dpph.) assay (brandwilliams et al., 1995). the amount of 500 µl of a solution 0.5 mm dpph (dissolved in etoh) 2.0 ml etoh and 100 µl of sample was mixed in a 1-cm path cuvette and the absorbance at 517 nm against blank (2.5 ml etoh and 100 µl of sample) was recorded at time 0 s and 180 s. the percentage of dpph decrease was computed as: dpph %=[(a t0 – a t180 )/a t0 ] × 100 and the results were expressed as mg trolox equivalents in 100 g of fw. statistical analysis statistical analyses were carried out with the statgraphics software v.5.1 package (manugistics, rockwell md). data were submitted to multifactor anova evaluating the main effects of the factors “cultivar” and “shelf-life”. differences among the treatments were determined by tukey multiple range test or by least significant distance (lsd) test (p≤0.05). data were also examined using multivariate analysis by principal component analysis (pca). 3. results at the end of the shelf-life of the ready-to-eat raspberries, a fairly limited weight loss (0.1-0.3%), was noticed in all samples, mostly due to a slight juice dripping (data not shown). samples from different cultivars were characterized by different soluble solids content (table 1): the cultivar tulameen showed higher ssc values, followed by ‘heritage’ and ‘glen magna’. during shelflife, this parameter decreased slightly in the cultivars tulameen and glen magna while it did not change in the ‘heritage’ fruit. total acidity decreased during shelf-l ife in ‘heritage’ and ‘glen magna’ and slightly in ‘tulameen’ (table 1). no important changes in ph values were shown by ‘heritage’ and ‘tulameen’, while a significant decrease of ph was found in ‘glen magna’ fruit during the storage at 3°c (table 1). the compression test showed similar firmness for all the cultivars (table 1). after only 3 days of shelflife at 3°c, firmness decreased markedly in ‘heritage’ (-32%) but only slightly in ‘tulameen’ and ‘glen magna’ (table 1). at the day 6 a significant decrease in firmness was observed in all the cultivar and, at end of the shelf-life (8 days) all the fruits had a firmness loss between -43% and -48%. adv. hort. sci., 2018 32(3): 399-406 402 visual appearance is a key issue for commercial shelf-life as consumer acceptance is often based on fruit colour which is used to make conclusions on the freshness of the product. the colour of ‘heritage’ and ‘tulameen’ fruits was characterized, at harvest, by lower l*, a* and b* values (table 2) than those reported in literature (çekic and özgen, 2010; krüger et al., 2011). fruit of ‘glen magna’ showed higher lightness (l*) and lower a* and hue values than the other two cultivars (table 2). during shelf-life, the trend of l*, a* and b* values was not well defined, while hue decreased significantly in ‘glen magna’ and ‘heritage’ indicating a darker red colour of the fruit. ‘tulameen’ sample was, instead, more stable from the chromatic point of view. at harvest the amounts of total anthocyanin of ‘heritage’ (granelli et al., 2010), ‘tulameen’ (kruger et al., 2011) and ‘glen magna’ (mazur et al., 2014 b) were within the range of the values reported in literature (fig. 1a) and the cv. heritage showed the highest content. the cold storage induced a slight but significant loss of anthocyanin in all samples: after only 3 days (-20%) for ‘tulameen’ and after 8 days for ‘heritage’ (-12%) and ‘glen magna’ (-13%). the average total polyphenols content (fig. 1b) was higher in ‘glen magna’, followed by ‘tulameen’ and ‘heritage’. tpc resulted quite stable over the shelf-life and no significant decrease was recorded. in our study the ‘tulameen’ fruits showed the highest ascorbic acid content (fig. 1c) at the harvest. aa content decreased significantly after only 3 days in in ‘tulameen’ fruit which showed a loss of 26%. the final losses were about 26% for ‘heritage’ and ‘tulameen’ and even higher (-42%) for ‘glen magna’. the cultivar heritage showed a significantly lower scavenging activity value (fig. 1d) than the other cultivar at harvest and this difference was confirmed over the shelf-life. during storage the free radical scavenging activity had an up and down trend, and the end of the shelf-life it resulted slightly, even though significantly, decreased only in the ‘tulameen’ samples. principal components analysis (pca) can provide an overview of the shelf-life behaviour of ready-toeat raspberries from different cultivars (fig. 2). from the pca analysis of the raspberry samples three functions were extracted, explaining the 91.2% of total variance. considering the first two principal components pc1 (54.3% of the total variance) grouped the table 1 maturity indices of ready-to-eat fruit from different raspberry cultivars, at harvest (raw) and after 3-6-8 days shelf-life at 1°c different letters indicate significant differences among the days of shelf-life of the same cultivar or among the main factors (tukey test). cultivar day of shelf-life ph ssc (%) acidity (g citric acid/100 g fw) firmness (g) heritage raw 2.66 a 10.78 a 2.13 b 14.38 b 3 2.62 a 10.68 a 2.03 a 9.82 a 6 2.61 a 10.47 a 2.01 a 8.75 a 8 2.67 a 10.49 a 1.97 a 7.46 a tulameen raw 2.77 a 11.52c 1.55 b 14.50 b 3 2.71 a 11.13 b 1.55 b 12.29 b 6 2.75 a 10.83 ab 1.52 b 9.30 a 8 2.76 a 10.74 a 1.38 a 8.23 a glen magna raw 2.82 a 9.54 b 1.72 c 15.05 b 3 2.82 a 9.31 ab 1.56 b 11.89 ab 6 2.90 ab 9.18 ab 1.49 ab 10.13 a 8 2.97 b 9.01 a 1.42 a 8.63 a main factors cultivar heritage 2.64 a 10.60 b 2.03 b 10.13 a tulameen 2.75 b 11.05 c 1.50 a 11.07 a glen magna 2.87 c 9.26 a 1.54 a 11.42 a day of shelf-life raw 2.74 a 10.61 b 1.80 c 14.64 c 3 2.71 a 10.37 ab 1.71 b 10.97 b 6 2.75 a 10.10 a 1.67 b 9.74 ab 8 2.80 a 10.13 a 1.58 a 8.10 a table 2 color parameters of ready-to-eat fruit from different raspberry cultivars, at harvest (raw) and after 3-6-8 days shelf-life at 1°c different letters indicate significant differences among the days of shelf-life of the same cultivar or among the main factors (tukey test). day of shelflife a* b* l* hue (h°) heritage raw 21.75 ab 9.75 a 27.44 ab 24.03 b 3 22.84 ab 9.10 a 27.60 ab 21.58 a 6 21.65 a 8.23 a 26.20 a 20.73 a 8 24.38 b 9.67 a 29.77 b 21.54 a tulameen raw 18.90 a 8.36 a 29.51 a 23.64 a 3 20.53 a 8.99 a 29.83 a 23.58 a 6 19.23 a 8.20 a 29.33 a 23.07 a 8 20.48 a 8.90 a 30.31 a 23.44 a glen magna raw 18.17 a 7.25 a 30.83 b 21.89 b 3 19.76 a 7.12 a 28.18 a 19.45 a 6 19.13 a 6.83 a 28.96 a 19.53 a 8 19.34 a 6.43 a 31.30 b 18.32 a main factors cultivar heritage 22.65 b 9.18 b 27.75 a 21.96 b tulameen 19.78 a 8.61 b 29.74 b 23.43 c glen magna 19.09 a 6.90 a 29.81 b 19.79 a day of shelf-life raw 19.60 a 8,45 a 29,25 ab 23,18 b 3 21.04 ab 8.39 a 28.53 a 21.53 a 6 20.00 ab 7.75 a 28.16 a 21.11 a 8 21.40 b 8.33 a 30.45 b 21.09 a cortellino et al. nutritional quality of ready-to-eat raspberries 403 colour parameters a* and b*, anthocyanins, acidity and ssc, opposite to scavenging activity, polyphenols content, l* and ph. pc2 (23.7%) was positively related to ascorbic acid, hue and ssc. in the biplot pc1 versus pc2 the three cultivars are very well distinguished. heritage samples showed positive pc1 scores, linked to their high values of anthocyanins, acidity, a* and b* values. on the other side of the plot, ‘glen magna’ samples were characterized by low values on pc1 , due to their high dpph scavenging activity and polyphenols content, l* parameter and ph values. all the ‘tulameen’ samples had positive scores on pc2, linked to high ascorbic acid, hue and ssc. all the raw samples showed positive pc2 scores, being characterized by higher ascorbic acid content and more brilliant red colour (higher hue value). these values decreased markedly during shelf-life and, after only 3 days, all the samples showed lower pc2 scores. 4. discussion and conclusions the weight loss can be an important problem during the postharvest of raspberry fruit (haffner et al., 2002) however, in this experiment it always remained far below the limit of marketability (6%) (haffner et al., 2002) and all fruit showed a good hydration state at the end of the shelf-life. as it is visually shown in the pca analysis, the raspberry cultivars were characterized by different quality parameters: in particular ‘heritage’ by a higher anthocyanin content, acidity, a* and b* value, while ‘glen magna’ by a higher phenolic content and scavenging activity and ‘tulameen’ by a higher ascorbic acid content. at harvest ssc of raw fruit of ‘heritage’ and ‘tulameen’ was lower than those reported by the literature (çekic and özgen, 2010; granelli et al., 2010; krüger et al., 2011;) while the values of ‘glen magna’ were similar to those described by mazur et al. (2014 a). the decrease in soluble solids content showed by all the cultivars during storage, as well as the reduction in acidity, was probably due to the normal respiration activity of the fruit (giuggioli et al., 2015). in any case, ssc was still at a good level at the end of shelf-life, showing a fairly good potential for these raspberry cultivars as a ready-to-eat product. it is well established, in fact, that ssc value is generally correlated with desirable flavor quality (kader, 1997). the decrease of total acidity and the drop of the ph over time were also observed in other fresh-cut fig. 1 total content (mean±standard error) of anthocyanins (a), phenols (b), ascorbic acid (c) and total scavenging activity (d) measured in ready-to-eat samples from three raspberry cultivars, at harvest (raw) and after 3-6-8 days of shel-life at 3°c. the least significant distance (lsd) for p≤0.05 is indicated on every single graph. 404 adv. hort. sci., 2018 32(3): 399-406 products (wright and kader, 1997). tissue softening is a very serious problem and it often represents a limiting factor for many ready-toeat products. since consumers tend to want firm fruit, it is essential to evaluate fruit firmness in order to better assess the global quality of the product during shelf-life. firmness of fresh-cut fruits has been widely reviewed but no data about this kind of readyto-eat whole product are available. besides, raspberry fruit is very fragile and the firmness is difficult to measure. the decrease in the firmness showed after only 3 days, even though the fruit tissues weren’t injured by cutting, could be due to the necessary washing and sanitizing step. this operation consisted of a first dip in chlorinated water, to reduce the microbial loads on the fruit surface, and a second rapid dip in tap water, to eliminate residual chlorine and keep the sensorial properties of the untreated fruit. moreover, the gentle drying of wet surfaces by adsorbent paper, a crucial step to remove the excess water, could cause mechanical damage to such delicate raspberries structures even though it is carried out carefully. the stress caused from the washing step might also have affected the fruit color. by the way, the darkening of the fruit, which could affect the consumer choice, is reported by other authors (haffner et al., 2002; giovanelli et al., 2014) for fresh or packed raspberries stored in normal atmosphere at low temperature. in addition, haffner et al. (2002) showed that storage in controlled atmosphere kept the berries more attractive than when stored in normal atmospheres. raspberry red colour is usually related to their anthocyanin composition (garcía-viguera et al., 1998). the higher initial anthocyanin content (fig. 1a) and the higher value of the red parameter a* (table 2) observed in ‘heritage’, if compared to the other two cultivar, would confirm this positive correlation. the slight decrease of anthocyanin showed by the packed raspberry fruit during storage is in accordance with the results of nunes et al. (2005) on strawberry stored at 1°c for 8 days. the author asserted that pigment degradation may be caused by the increased polyphenols oxydase activity as a result of physiological stress due to water loss during storage. conversely, other authors reported an increase of the total anthocyanin content both in raspberry packed and stored at 4°c for 7 days (giovanelli et al., 2014) and in fresh fruit stored at 20°c for 1 day (krüger et al., 2011). the total phenolics content (tpc) (fig. 1b) of raw fig. 2 pca biplot of quality and nutritional parameters during shelf-life of ready-to-eat raspberry fruit from the cultvars heritage (h), tulameen (t) and glen magna (g) after 0(raw)-3-6-8 days of shelf-life at 3°c (ssc=soluble solids content; l*, a*, b*, hue=colour parameters; dpph=scavenging activity). 405 cortellino et al. nutritional quality of ready-to-eat raspberries ‘heritage’ reflected the findings of cekic and özgen (2010) whereas de ancos et al. (2000) and granelli et al. (2010) reported lower tpc values for this cultivar. tpc observed in the cv. tulameen (cekic and özgen, 2010; kruger et al., 2011) and ‘glen magna’ (mazur et al., 2014 a) were in accordance with those reported in the literature. the stability of phenolics showed by the analyzed cultivar during shelf-life was also reported by giovanelli et al. (2014) for fresh fruit and by de ancos et al. (2000) for another type of processed raspberries product such as long-term frozen stored. ascorbic acid is one of the most important parameters used to control plant-derived food quality. reduced temperature after harvest is considered to be important for its content in fruit (krüger et al., 2011). the decrease trend of the ascorbic acid of the analyzed cultivars during storage fully reflects the findings of de ancos et al. (1999) and krüger et al. (2011). our results are, instead, not in accordance with kalt et al. (1999) and haffner et al. (2002) who found that aa level of raspberries, not washed and kept in normal atmosphere, was not reduced by several days of cold storage. a reasonable explanation for this discrepancy may be found in the washing step involved in the ready-to-eat processing: the hypochlorite contained in the sanitizing solution used could have oxidized the ascorbic acid, as reported by bielski (1982). it is well known that the scavenging activity of raspberry fruit is due to their high content of anthocyanins, phenolics and ascorbic acid (krüger et al., 2011). a significant correlation (r2 = 0.857) was observed between the dpph values and those of total phenol whereas the scavenging activity were not significantly correlated with total anthocyanins nor ascorbic acid. our results, although lower than those found by benvenuti et al. (2004), confirmed that the radical scavenging is related mainly to total phenol content. the results of this study indicated the loss of firmness as the more problematic aspect of the ready-toeat raspberries, followed by a less important change of the fruit colour from light red to dark red. the variations of chemical-physical parameters such as soluble solids content, ph and total titratable acidity, did not compromise the product quality. the processing (washing and packing) and the cold storage affected only slightly the nutraceutical profile, except for ascorbic acid, therefore the ready-to-eat raspberries could be considered a good source of compounds with potential health benefits. some handling difficulties were highlighted during the processing steps of the samples (washing, drying and packing) due to the high fragility of fruit which caused a high percentage of waste. the hollow structure of raspberries made the drying step particularly difficult, however this is necessary as 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club, khoram-abad branch, islamic azad university, khoram-abad, iran. key words: alkali stress, pepper, salicylic acid, salt stress. abstract: salinization and alkalization of soils are agricultural problems in arid and semiarid regions of the world such as iran. in this experiment the effects of salicylic acid (sa) on resistance of pepper plants under salt stress (ss) and alkali stress (as) were evaluated. treatments include 0 and 150 mm of ss, 0, 50 and 100 mm of as and 0, 0.75 and 1.5 mm sa. results showed that ss and as imposed negative effects on pepper plant growth and productivity. reduction in growth and yield in ss was higher than as and maximum reduction occurred in high mixed stresses. sa application improved growth parameters and increased yield, relative water content (rwc) and chlorophyll of plants subjected to ss and as and provided significant protection against stress compared to non-sa-treated plants. for most traits, 0.75 mm of sa was more effective than 1.5 mm concentration. sa ameliorated the injury caused by ss and as by increasing chlorophyll and rwc and inhibiting proline accumulation and leaf electrolyte leakage (el). in general, results indicate that salinity and alkalinity have negative effects on growth and yield of pepper plants and these negative effects can be ameliorated by application of sa. 1. introduction salinity and alkalinity of soil seriously affect about 932 million hectares of land globally, reducing productivity in about 100 million hectares in asia (rao et al., 2008). in iran 12.5% of the agricultural lands in arid and semi-arid areas are alkaline. while salt stress (ss) in a soil generally involves drought stress and ion-induced injury (munns, 2002), alkali stress (as) exerts the same salt stress influences with the added effects of highph stress (shi and yin, 1993). the high-ph caused by as directly affects the mineral absorption and interferes with the re-establishment of ionic balance. it can strongly affect the absorption of inorganic anions such as (*) corresponding author: f.ghanbari92@basu.ac.ir citation: amirinejad a.-a., sayyari m., ghanbari f., kordi s., 2017 salycilic acid improves salinityalkalinity tolerance in pepper (capsicum annuum l.). adv. hort. sci., 31(3): 157-163. copyright: © 2017 amirinejad a.-a., sayyari m., ghanbari f., kordi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 septmeber 2016 accepted for publication 6 april 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 157-163 158 cl–, no3 – and h2po4 –, and can disrupt the selective absorption of k+-na+, and ph homeostasis in the plant tissues (yang et al., 2007). to resist as, plant not only have to regulate the intercellular ph to maintain ionic balance, but also have to spend material and energy to regulate ph in their root environment. thus osmo-regulation and ion balance play key influence in plants ss and as resistance (yang et al., 2007). there has been considerable study of ss on plants growth and development, however, relatively little attention has been given to as despite its importance. like to other environmental adverse conditions, as can lead to emerge reactive oxygen species (ros), as a result of closing stomata and reducing co2 into cells and consequently blocking photosynthesis activities (yan et al., 2011). as caused metabolic disturbance, lipid peroxidation and chlorophyll breakdown and proline accumulation in plant tissue (gao et al., 2012). various techniques have been tested to improve saline-alkali soils, including chemistry, physics, biology and engineering improvements to increase soil fertility and crop yield (li-ping et al., 2015). in recent studies a number of plant growth regulators (pgrs) have been under trial to alleviate the environmental stresses in plants. salicylic acid (sa) is a phenolic compound which is considered as a pgr and plays an important role in defensive mechanisms against biotic and abiotic stresses in plants. flowering induction, plant growth and development, synthesis of ethylene, opening and closure of stomata and respiration are some of the important roles of sa in plants (raskin, 1992). sa protects plants from damages caused by oxidative stresses through increasing antioxidants enzymes activities (el-tayeb, 2005; idrees et al., 2011). sa has received much attention due to its function in plants’ responses to environmental stresses. literature exists about some beneficial effects of sa on plants under drought (jafari et al., 2015), low temperature, high temperature (wang and li, 2006; sayyari, 2012), salinity (shakirova et al., 2003; el-tayeb, 2005; stevens et al., 2006; idrees et al., 2011), heavy metal (metwally et al., 2003) and biotic stresses (makandar et al., 2012). however, no information exists on the effects of the mentioned compounds on salt-alkali stress defense mechanisms, until now. thus the purpose of this experiment was to examine the possibility that application of sa would protect pepper plants from damaging effects of salt-alkali stresses. specific objectives of this research were: (1) to compare ss and as on pepper plant growth and development (2), to determine some physiological responses of pepper plant to ss and as and (3) evaluate the protective effect of a pre-treatment with sa. 2. materials and methods plant material and growing conditions seeds of capsicum annuum l. cv. plenty were obtained from pakan bazr co. (isfahan, iran) and cultured in bed to obtain seedlings for experiment. when the seedlings had 2-4 true leaves, the seedling with similar size were selected and transferred into plastic pots (20 cm height, and 23 cm diameter) which were filled with about 8 kg of 1:1:2 mixture of fine sand, leaf mound and garden soil. the pots were then transferred to the greenhouse with av erage temperature of 25.5/19.5°c (day/night) and natural light. salt stress, alkali stress and salicylic acid treatments when the seedlings have been established in pots (4-6 true leaves), they were sprayed with 0 (as control), 0.75 and 1.5 mm sa until both sides of the leaves were completely wet. three days later, plants were subjected to ss and as treatments until end of each experiment. two neutral salts (nacl and na2so4) and two alkaline salts (nahco3 and na2co3) were selected for salinity and alkalinity stresses imposition. the two neutral salts were mixed in a 9:1 molar ratio (nacl:na2so4), and applied in 0 mm and 150 mm to the ss group, and two alkaline salts were also mixed in a 9:1 molar ratio (nahco3:na2co3) in 0 mm, 50 mm and 100 mm, and applied to the as group. ss and as treatment were conducted with daily watering of plants by mentioned treatment. control plants (0 mm ss and 0 mm as) were watered with distilled water (yang et al., 2007; rao et al., 2008). chlorophyll determination chlorophyll was assessed by taking fresh leaf samples (0.1 g) of plants in each replicate from young and fully-developed leaves. the samples were homogenized with 5 ml of ac etone (80% v/v) using pestle and mortar and centrifuged at 3000 rpm. the absorbance was measured with a uv/visible spectrophotometer at 663 and 645 nm and chlo rophyll were calculated (mg/g fw) using the equations pro posed by strain and svec (1966) given below: total chlorophyll = 20.2×(a645)+8.02(a663). amirinejad et al. salicylic acid improves salinity-alkalinity tolerance in pepper (capsicum annuum l.) 159 electrolyte leakage electrolyte leakage was determined according to lutts et al. (1995) method. ten leaf discs of randomly chosen plant were taken from the youngest fullyexpanded leaf. the leaf discs were then placed in test tubes containing 10 ml of distilled water. these samples were incubated at 25°c on a shaker for 24 h. electrical conductivity (ec) of bathing solution (ec1) was read after incubation. the same samples were then placed into a water bath (100°c) for 20 min and the second reading (ec2) was determined after cooling of the solution to room temperature. the el was calculated as ec1/ec2 and expressed as a percentage. proline content determination the proline was determined according to the method described by bates et al. (1973). the amount of 0.5 g of samples were homogenized in 10 ml of 3% (w/v) sulfosalicylic acid. after centrifuging at 10000 rpm, 2 ml of the supernatant was mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid in a test tube. the mixture was placed in a water bath (100°c) for 1 h. the reaction mixture was extracted with toluene and the chromophore-containing toluene was aspirated and cooled to 25°c. the absorbance was measured at 520 nm with a uv/visible spectrophotometer. proline concentration was expressed as μg/g fw. measurement of relative water content relative water content was measured according to costes et al. (2006). leaves sample in each treatment was weighed (fw) and then immediately floated on distilled water for 5 h in the dark. turgid weight (tw) of leaf disks was obtained after drying excess surface water with paper towels. dry weight (dw) of disks was measured after drying at 75°c for 48 h. the rwc was calculated using the following formula: relative water content = (fw-dw)/(tw-dw)×100 statistical analysis data were analyzed for significant differences using a factorial analysis of variance with ss and as levels and sa concentrations as main factors with three replications and 5 seedlings per each. statistical analysis was performed using sas program and the means compared using the duncan’s multiple range test at p=0.05. 3. results and discussion leaf area and total yield the leaf area and pepper yield displayed a significant reduction in response to the increasing levels of ss and as treatments. among all stress treatments tested, 100 mm as+ss exhibited the strongest reduction of leaf area and yield followed by 50 mm as+ss. foliar sprayed sa plants exhibited significant response to improve yield as compared to unsprayed ones in all stress levels (fig. 1 and 2). the general effect of soil salinity in plants is to decrease the growth resulting in smaller leaves, shorter stature, and sometimes fewer leaves. the primary effect of salinity, especially at low to moderate levels, is due to its osmotic stress (munns, 2002). the as exerts the same stress factors as ss but with the added effect of high-ph stress. the effect of ss and as in reducing the growth and yield of various plants was reported in (yang et al., 2007; chen et al., 2011) studies that are in agreement with the findings of this study. however, in the present study, adverse effects of as on yield and leaf area were lower than that of ss. this results implies not only that ss and as are different stresses, but also that resistance of pepper plant to as is stronger that to ss. the treatment of sa caused a significant increasing in yield and leaf area in comparison to control plants at all stresses levels (fig. 1 and 2). the positive effect of sa on plants under salinity stress have been reported (shakirova et al., 2003; stevens et al., 2006; idrees et al., 2011) which their upshots are in agreefig. 1 effect of salicylic acid (sa) on leaf area of pepper plant under salt stress (ss) and alkali stress (as). fig. 2 effect of salicylic acid (sa) on total yield of pepper plant under salt stress (ss) and alkali stress (as). adv. hort. sci., 2017 31(3): 157-163 160 ment with our findings. application of sa improved wheat plants under water stress by activities of cell division in apical meristem (shakirova et al., 2003). exogenous sa treatment also improved chlorophyll and photosynthesis rate and created stability of plant cell membrane, reduction of el in barely plants and finally tolerance plants to water stress (el-tayeb, 2005). the ability of sa to increase yield and growth parameters, ameliorating the adverse effects of stress, may have important implications in improving the plant growth and overcoming the growth barrier arising from ss and as conditions. chlorophyll content some of the environmental stresses symptoms in the plants are reduction of chlorophyll content and this reduction depends upon the plant species (colom and vazzana, 2001). in this experiment, the reduction of chlorophyll was occurred due to ss and as and 100 mm ss+as treatment resulted in having the lowest rate of total chlorophyll (fig. 3). in this respect, there are same reports over reducing chlorophyll due to exposing plants to ss and as (chen et al., 2011; gao et al., 2012). environmental stresses leads to increase the ros production in cells. these free radicals cause peroxidation and consequently destructing the photosynthesis pigments (schütz and fangmeier, 2001). because of these events, the growth of plant will be affected and declined. treated plants with sa as foliar spray increased chlorophyll content in all concentrations, but 0.75 mm sa treated plants showed significant increases in chlorophyll compared with control and 1.5 mm sa (fig. 3). these results are in agreement with those of sayyari (2012) who found that sa foliar and soil applications increased chlorophyll content in cucumber plants following chilling stress. salicylic acid by eliminating of ros may improve chlorophyll content in pepper plants under stressful conditions. chen et al. (1993) showed that in response to environmental stresses sa accumulates to high concentrations, and prevent antioxidant enzyme such as cat activity, thereby leading to an enhancement in hydrogen peroxide (h2o2) content, which could then induct the development of systemic acquired resistance (sar), induce activity of rosdetoxifying enzymes and antioxidant metabolites. also, idrees et al. (2011) reported that sa-induced salinity tolerance in periwinkle plants might be associated with an increase in the antioxidant activity. therefore, impact of sa on plants chlorophyll under stresses condition may be related to its effect on the antioxidative enzyme activities and h2o2 metabolism (idrees et al., 2011). proline content proline is sensitive physiological index of plants responding to some stresses. salinity and alkalinity treatments had significant effects on proline contents in pepper plant leaves. proline accumulation in leaves was markedly increased in salt and alkali stressed plants in comparison with that of the control plants. the results showed that mixed ss-as can cause heavy accumulation of proline content compared with ss and as single stresses (fig. 4). in general, the accumulation of proline, relates closely with osmotic stress intensity. osmo-regulation is a physiological phenomenon during which osmosis potential of stressed tissues are reduced due to the accumulation of some material such as elements, sugar, amino acids (proline) and organic acids. thus, turgor pressure of the cells is kept well (irigoyen et al., 1992). proline by osmosis control, maintaining enzymes activity and removal of hydroxyl radicals, increases the tolerance of the plants against stresses (kuznetsov and shevyakova 1999). the results showed that proline accumulation increased not only with increasing ss, but also when as imposed (fig. 4). this suggested that the proline accumulation, as the major osmolyte, correlates closely with the intensity of the osmotic stress induced by ss and as. fig. 3 effect of salicylic acid (sa) on chlorophyll of pepper plant under salt stress (ss) and alkali stress (as). fig. 4 effect of salicylic acid (sa) on proline content of pepper plant under salt stress (ss) and alkali stress (as). amirinejad et al. salicylic acid improves salinity-alkalinity tolerance in pepper (capsicum annuum l.) 161 results showed that sa application increased proline content in pepper plants under ss and as. a highest amount of proline (44.67 μm/gfw) was achieved in 1.5 mm sa treatment and 100 mm as+ss, and lowest amounts (9.54 μm/gfw) were observed in 0.75 mm sa and 0 mm as (fig. 4). these results are in agreement with those of el-tayeb (2005) who showed that sa treatment increases the proline content in the leaves of barely plant subjected to salinity stress. in the present study, sa induced an accumulation of proline in the leaves under ss and as, and when sa was applied, a stress tolerance occurred in the pepper plant. thus, osmo-regulation can be considered to be one of the important phenomenon involved in sa induced protective mechanism in pepper leaves in response to salt and alkali stresses. relative water content one of the indices showed the water status of plants is rwc. measuring of relative water content is an important physiological parameter in evaluation of plant response to environmental stresses (nautiyal et al., 2002). results indicated that by increasing ss and as, rwc in pepper leaves decreased slightly. reduction under ss was greater than those under as and greatest rwc reduction was achieved in highest mixed stress level (100 mm as+ss) (fig. 5). these results are in agreement with yang et al. (2007) who found that salt-alkali stresses decreases the rwc in the leaves of kochia sieversiana. plants can reduce rwc as a quick and economical approach to osmotic adjustment in response to osmotic stress (lissner et al., 1999). reduction of rwc of the plants due to stress is related to the reduction of soil humidity; in these conditions, plants close the stomata to avoid more water waste. the reason of stomata closure is aba that is made in the root in stress conditions and is accumulated in stomata cells (chaves et al., 2002). therefore, maintaining a high rwc might be a key characteristic of plants that allow it to absorption osmolytes with minimum energy consumption. results showed that sa treatment increased leaf rwc in pepper plants under ss and as. a highest amount of rwc (89%) was achieved in 1.5 mm sa treatment and 0 mm as, and lowest (54%) were observed in 0 mm sa and 100 mm as+ss (fig. 5). parida and das (2005) reported that the rwc, water potential and osmotic potential of plants under stress become more negative with an increase in salinity. this study showed that sa treatments induced an increase in rwc of plants as compared to the un-treated sa plant. increases in rwc of plants treated with sa were also reported for other crops grown under stress including tomato (stevens et al., 2006) and barely (el-tayeb, 2005). increasing of rwc may be related to the role of sa in accumulation of compatible osmolytes in plants subjected to stress, as, this effect was observed in the results of proline. electrolyte leakage in order to assess membrane permeability, electrolyte leakage (el) was determined. its relative conductivity can be used to evaluate the damage on structure and function of cell membranes under stresses. results showed that el significantly higher under ss than as. on the other hand, both stresses increased the el but the extent of the increment under ss was much greater than under as and maximum of el was achieved in highest mixed stress level (100 mm as+ss) (fig. 6). the results of the present study are in agreement with gao et al. (2012) who determined that el of oat (avena sativa l.) was intensively increased by as. gao et al. (2012) showed that the el of alfalfa seeds gradually increased with increasing salinity and alkalinity, this was attributed to the damage seeds cell membranes resulting from mixed salt-alkali stress. these results suggested cell membrane structure of pepper leaves under ss and as received damage after treatment with salts. application of sa significantly decreased leaf el in salt and salt × alkali stressed plants. plants treated by sa foliar spray at 0.75 and 1.5 mm had shown significantly less el than control plants (fig. 6). these results indicated that, sa reversed the adverse effects of stress and caused a significant decrease in el. the results of the present study are in agreement with stevens et al. (2006) who showed that sa facilitated the maintenance of membrane functions in tomato under salinity stress. this effect could be attributed to the stimulation of antioxidant responses and elevated calcium absorption that protects thefig. 5 effect of salicylic acid (sa) on relative water content of pepper plant under salt stress (ss) and alkali stress (as). adv. hort. sci., 2017 31(2): 157-163 162 plant from the oxi dative damage (el-tayeb, 2005). also, jafari et al. (2015) showed that exogenous application of sa in cucumber (cucumis sativus l.) subjected to osmotic stress lead to a decrease in el and induced drought tolerance. these results suggested that pepper leaves cell membrane structure in during salt and alkali stresses received less damage after application of sa. 4. conclusions in summary, our study clearly showed that saline and alkaline stresses as two types of abiotic stresses, have negative effects on pepper plant growth and productivity. on the other hand, the negative effect of mixed salt-alkali stress is more severe than that of only salt or alkali. the results of this research demonstrated that treating plants with 0 mm as (control) is resulted in increasing plant growth parameters and rwc in comparison to other treatments. the harmful effect of saline stress on the pepper plants was significantly greater than that of alkaline; this harmful effect might have resulted from the higher concentration of salinity or higher sensitivity of pepper to ss compared to as. sa pre-treatment of pepper seedlings via foliar spray was effective in saline and alkaline resistance. in most of evaluated treatments employed in this research, they did not show significant differences on sa concentrations. the best protection was obtained in plants treated with 0.75 mm sa, however 1.5 mm sa is more effective in 100 mm as+ss in some trait for example proline content. this sa effect was associated with alter of physiological parameters such as increase of photosynthetic pigments and proline accumulation and decrease el of plants by addition of sa. further, the results indicate that sa can be considered as a potential growth regulator for improving plant growth and yield under ss and as, and it may be recommended in arid and semiarid regions. references bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress studies. plant and soil, 39(1): 205-207. chaves m.m., pereira j.s., maroco j., rodrigues 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salt and alkali conditions. plant and soil, 294(12): 263-276. impaginato 93 adv. hort. sci., 2018 32(1): 93-103 doi: 10.13128/ahs-21874 physio-morphological variations of pummelo genotype (citrus grandis l. osbeck) m.m. hossain*, r.f. disha, m.a. rahim department of horticulture, bangladesh agricultural university, mymensingh, bangladesh. key words: citrus grandis l. osbeck, correlation coefficient, heritability, path analysis. abstract: the study was conducted to evaluate the physio-morphological variations of 21 pummelo genotype. the experiment was carried out at the existing plantation of bangladesh agricultural university germplasm centre, mymensingh, during september 2014 to june 2015. results showed that different genotype exhibited differently in their physio-morphological features. genotype thai jambura exhibited highest leaf and petiole wing length (16.77 cm and 11.63 cm, respectively), while maximum number of anthers (44.33) were recorded in genotype green skin. the heaviest and lightest fruits were recorded in genotype hybrid (1283.33 g) and accession-52 (300 g). while the maximum weight of non-edible portion (463.33 g), pulp to peel ratio (3.97), thickness of pulp (11.50 cm), amount of juice (366.67 ml), total soluble solids (tss) (18.67%), number of seeds (114) and weight of seeds (58 g) were found in genotype hybrid. correlation coefficient study indicated that leaf length, breadth, petiole wing length, fruit weight, weight of non-edible portion, seed weight, seed number/fruit had positive and highly significant association with leaf breadth, petiole wing breadth, weight of non-edible portion, pulp thickness, total weight of seeds/fruit and number of fruits/plant, respectively. in respect of path analysis, leaf breadth, petiole wing length, fruit weight, average weight of seed, %tss, seed number/fruit had positive direct effect on fruits/plant indicating its importance as a selection criteria. 1. introduction pummelo (citrus grandis l. osbeck) belonging to the family rutaceae, is one of the most distinctive and easily recognized species of the genus citrus (verdi, 1988). it is one of the most important fruit in bangladesh because of its taste, aroma and nutrient value. citrus carry some vitamins like vitamins c, b, a and some minerals like calcium, iron, citric acid etc. the pummelo is an exotic large citrus fruit that is an ancestor of the common grapefruit. in bangladesh it is locally known as jambura, batabilabu, (*) corresponding author: mokter.agr@bau.edu.bd citation: hossain m.m., disha r.f., rahim m.a., 2018 physio-morphological variations of pummelo genotype (citrus grandis l. osbeck). adv. hort. sci., 32(1): 93-103 copyright: © 2018 hossain m.m., disha r.f., rahim m.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 93-103 94 badami, jamir. the pummelo is significantly larger than the grapefruit. its flesh is sweet and it has a thick skin and rind. the fruit of the pummelo has a light green colored rind but this gradually becomes mostly yellow when it has fully ripened. the inside of the fruit has a pink color when it is ripe. the pummelo tree thrives well in tropical or near tropical climates. like other citrus fruits, the pummelo usually ripen in winter. it is an important commercial citrus fruit, grown and available almost everywhere in bangladesh. the availability of this fruits helping people to overcome the malnutrition problem. there is a special pummelo fruit based diet to treat asthma. pummelo is a dietary fruit; its caloric value is 25-58 kcal/100 g (morton, 1987). in vietnam, the aromatic flowers are used in making perfume. the wood is used for tool handles and firewood while leaves, flowers, fruits, and seeds are sometimes used as herbal medicine to treat cough, fever and gastric disorders (verheij and coronel, 1992). pummelo grows well everywhere in bangladesh and is comparatively more tolerate to insects and diseases than other citrus fruits. but the number of trees with good quality fruit is very negligible in comparison to other citrus growing countries of the world. in the year 2015, bangladesh produces 31,036 metric tons pummelo from 4159.10 hectares of land (bbs, 2015). although it is one of the most important citrus fruit in bangladesh, production as well as yield of pummelo fruits is very low due to lack of high yielding and good quality variety. it is reported that a single pummel tree can yield 70-100 fruits/year which is equivalent to 20 tons/ha/year (verheij and coronel, 1992). therefore, selection of high yielding genotype is necessarily important to increase the yield of pummelo in bangladesh. characterization of pummelo using morphological traits will help in the selection of genetically potential genotype for cultivation and also for their exploitation in plant breeding program. selection of superior genotype from the collected accessions will help in increasing production of pummelo in this country. for improving the production as well as yield of pummelo fruits in this country, bangladesh agriculture university germplasm center (bau-gpc) has already been collected some pummelo genotype from different corner of bangladesh and also from thailand, vietnam, and malaysia. this experiment has been undertaken to study the physio-morphological characteristics of those pummel accessions to evaluate their relative performance. in plant breeding program, knowledge of the interrelationship among and between yield contributing characters is necessary. correlation and the path coefficient analysis will provide a true picture of genetic associating among different traits (bhatt, 1973). path coefficient analysis specifics the cause and effect and measures their relative importance. therefore, correlations in combination with the path co-efficient analysis quantify the direct and indirect contribution of one character upon another (dewey and lu, 1959). this experiment was therefore, undertaken to study the physio-morphological characteristics of pummelo genotype collected from different parts of the world and to access the interrelationship between yield and yield contributing characters and to select better quality genotype for cultivation in bangladesh. 2. materials and methods the experiment was conducted at bangladesh agricultural university germplasm center, department of horticulture, bangladesh agricultural university, mymensingh during september 2014 to june 2015. the location of bau-gpc was medium high land, well drained and slightly acidic soil with ph range from 5.5 to 6.8. the study was conducted in an established orchard where 21 indigenous and exotic pummelo genotype were collected and established (table 1). the average age of plants were 3-4 years. treatment pummelo genotype sources of origin t 1 bau-1 bangladesh t 2 bau-2 ,, t 3 bau-4 ,, t 4 hybrid ,, t 5 jambura (seeded) ,, t 6 mohini ,, t 7 green skin vietnam t 8 malaysian malaysia t 9 thai jambura thailand t 10 accession-51 bangladesh t 11 accession-52 ,, t 12 accession-57 bangladesh t 13 accession-58 ,, t 14 accession-59 ,, t 15 accession-62 ,, t 16 accession-63 ,, t 17 accession-76 ,, t 18 accession-87 ,, t 19 accession-93 ,, t 20 accession-101 ,, t 21 accession-103 ,, table 1 collected pummelo genotype with their source of origin hossain et al. physio-morphological variations of pummelo genotype 95 these 21 genotype were considered as experimental treatments. the experiment was carried out in a randomized complete block design with three replications. data on different physio-morphological characters of leaves, flowers, fruits and seeds were recorded from sample plants of each genotype following citrus ipgri descriptor (ipgri, 1999). leaf length, breadth, petiole wing length, petiole wing breadth were measured by using measuring scale. individual fruit weight was recorded by a digital balance and expressed in grams (g). for determination of pulp to peel ratio, fruits were peeled off and weight was taken for separated pulp and peel and expressed in g. thickness of fruit pulp and peel was measured by using measuring tape after cross-sectioning of the fruits and expressed in centimeter (cm). the pulp vesicles were removed and blended in a blenderin addition with 150 ml of water. then juice was filtered by a sieve. except juice the rest of waste materials was considered as non-edible portion and recorded the weight. the amount of juice was measured by measuring cylinder and expressed in milliliter (ml). total soluble solids (tss) was determined by abbe hand refractometer. a drop of juice squeezed from the pulp vesicles of fruit then placed on the prism of the refractometer and percent total soluble solids was observed from reading. temperature correction was made using the method describe by ranganna (1978) .total number of fruits of each plant was counted during 15 days interval. thereafter, the average number of fruits per plant was recorded. the total number of seeds per fruit was counted. the total number of seeds, which was obtained from fruits, was weighted in a balance and expressed in g. after counting and weighing the total seeds average weight of seed was determined by the following formula and expressed in g. average weight of seed (g) = weight of seeds (g) total number of seeds estimation of simple correlation coefficient association of different characters under the study was analyzed by working out simple correlation coefficient for all the possible pairs of character combination. simple correlation coefficient (r) among the important characters of pummelo genotype was estimated with the formula stated by singh and chaudhary (1985). estimation of path coefficient path coefficient analysis was done according to the procedure stated by dewey and lu (1959) using simple correlation values. in path analysis, correlation coefficient is partitioned into direct and indirect effects of independent variable on the dependent variable. in order to estimate direct and indirect effects of the correlated characters, i.e.x1, x2 and x3, yield y, a set of simultaneous equations (three equations in the examples) is required to be formulated as shown below: ryx 1 =pyx 1 + pyx 2 rx 1 x 2 + pyx 3 rx 1 x 3 ,ryx 2 = pyx 1 rx 1 x 2 + pyx 2 + pyx 3 ,ryx 3 =pyx 1 rx 1 x 3 + pyx 2 rx 2 x 3 + pyx 3 where, ‘r’ denotes simple correlation coefficient and ‘p’ denotes path coefficient. total correlation, say between x1 and y is thus partitioned as followed: pyx1 = indirect effect of x1, pyx2rx1x3 = indirect effect of x1 via x2 only, pyx3rx1x3 = indirect effect of x1 via x3 only. after calculating the direct and indirect effect of the characters, residual effect (r) was calculated by using the formula stated by singh and chaudhary (1985). component of variance the genotypic and phenotypic variances were calculated according to johnson et al. (1955). estimation of genotypic and phenotypic coefficient of variation were calculated according to burton (1952). estimation of heritability heritability in broad sense can be calculated by using following formula: h2 b (%) = genotipic variance x 100 penotypic variance statistical analysis data on different physio-morphological parameters were statistically analyzed to find out the significance of difference among genotype means. the analyses of variances for most of the characters under consideration were performed by f variance test. the significance of the difference between treatments means was evaluated by least significance difference test describe by gomez and gomez (1984). 3. results morphological features of leaves as regard to leaf length it was observed that leaf length varied significantly among the genotype. the longest value was observed in genotype thai jambura (16.77 cm) and minimum value in genotype adv. hort. sci., 2018 32(1): 93-103 96 bau-2 (7.67 cm) (fig. 1). it was revealed that the leaf breadth of the plant also varied significantly and ranged from 5.00 to 11.63 cm (table 2). the genotype thai jambura showed the highest leaf breadth (11.63 cm) and the lowest leaf breadth (5 cm) was observed in accession-62 (table 2). it was revealed that the petiole wing length of the leaf varied significantly and ranged from 1.23 to 3.60 cm (table 2). the genotype thai jambura showed the highest petiole wing length (3.60 cm) and the lowest petiole wing length (1.23 cm) was observed in accession-62 (table 2). petiole wing breadth of pummelo genotype varied significantly. the genotype thai jambura showed the highest petiole wing breadth (4.17 cm) and the lowest petiole wing breadth (0.90 cm) was observed in accession-63 (table 2). morphological features of flowers in case of number of petals per flower it was observed that petals varied significantly genotype to genotype. the maximum number of petals was found in genotype hybrid (6) and minimum value (4) in genotype bau-2, bau-4, mohini, accession-59, 63, 93 and 103 (table 2). similarly, calyx number was also found significantly varied among the genotype. the height number of calyx was observed in geno** = significant at 1% level of probability. table 2 leaf breadth, petiole wing length, petiole wing breadth, number of petals, calyx, anthers per flowers of 21 pummelo genotype genotype leaf breadth (cm) petiole wing length (cm) petiole wing breadth (cm) no. of petals/flower no. of calyx/flower no. of anthers/flower bau-1 5.70 2.00 1.60 5.00 5.00 42.00 bau-2 5.83 2.00 1.50 4.00 4.00 35.67 bau-4 5.93 2.83 2.87 4.00 4.00 39.00 hybrid 5.93 1.67 0.93 6.00 6.00 32.33 jambura (seeded) 6.23 3.33 2.33 5.00 5.00 32.33 mohini 6.17 2.17 1.67 4.00 4.00 38.00 green skin 5.93 2.50 1.73 5.00 5.00 44.33 malaysian 5.63 1.73 1.17 5.00 5.00 43.00 thai jambura 11.63 3.6 4.17 5.00 5.00 41.33 accession-51 5.90 1.33 1.17 4.00 4.00 34.33 accession-52 5.17 2.70 2.23 5.00 5.00 42.67 accession-57 5.80 2.40 1.40 5.00 5.00 32.33 accession-58 8.17 1.67 1.50 5.00 5.00 41.00 accession-59 5.80 2.40 1.40 4.00 4.00 43.67 accession-62 5.00 1.23 1.10 5.00 5.00 31.33 accession-63 5.63 1.40 0.90 4.00 4.00 33.33 accession-76 5.17 2.40 2.50 5.00 5.00 22.67 accession-87 6.27 2.73 3.33 5.00 5.00 43.67 accession-93 7.13 2.10 2.00 4.00 4.00 35.33 accession-101 7.07 2.00 1.47 5.00 5.00 37.67 accession-103 5.43 1.83 1.33 4.00 4.00 29.00 lsd 0.01 0.69 0.62 0.76 0.78 0.78 6.07 level of significance ** ** ** ** ** ** fig. 1 leaf length of 21 pummelo genotype. vertical bar indicates lsd at 1% level of probability. type hybrid (6) and lowest number (4) in genotype bau-2, bau-4, mohini, accession-51, 59, 63, 93 and 103 (table 2). however, number of anthers/flower significantly varied among 21 pummelo genotype and ranged from 22.67 to 44.33. the maximum value was recorded in genotype green skin (44.33) and the minimum value in accession-76 (22.67) (table 2). physio-morphological features of fruits individual fruit weight varied significantly among the genotype and it ranged from 300 to 1283.33 g. fruit with the heaviest weight (1283.33 g) was observed in genotype hybrid and the lowest (300 g) hossain et al. physio-morphological variations of pummelo genotype 97 fruit was recorded in accession-52 (table 3). in respect of non-edible portion weight, statistically significant variations were noticed among the genotype. the maximum weight of non-edible portion (463.33 g) was obtained from the genotype hybrid. on the other hand, the minimum weight of non-edible portion (100 g) was recorded from the genotype accession-52 (table 3). pulp to peel ratio of fruits varied significantly among the 21 pummelo genotype. the amount of pulp to peel ratio was highest in genotype hybrid and lowest in accession-87 (fig. 2). among the genotype thickness of fruit pulp differed significantly and ranged from 6.50 to 11.50 cm (table 3). the pulp of the fruits of genotype bau-1 and bau-2 was the thickest (11.50 cm) followed by the hybrid genotype (11.37 cm) and thai jambura (11.00 cm) and the thinnest (6.33 cm) pulp was observed in accession-51 (table 3). the thickness of peel also differed significantly among the genotype. the peel of the genotype jambura (seeded) and accession-93 was thickest (2.17 cm) and the thinnest (1.00 cm) peel was observed in genotype accession76 and accession-103 (table 3). in terms of juice content of fruits it was noticed that pummelo genotype varied significantly. the highest amount of juice (366.67 ml) was measured in genotype bau-1 and hybrid followed by bau-2 (350 ml) and thai jambura (336.67 ml) while the lowest (90 ml) found in accession-52 (table 3). total soluble solids (tss) contents of fruits was significantly different in different pummelo genotype. the highest tss was recorded in genotype hybrid (18.67% brix) followed by accession-62 (18.23% brix), accession-63 and accession-87 (17.33% brix) and the lowest tss was observed in genotype mohini (13.5% brix) (fig. 3). table 3 fruit weight, weight of non-edible portion, thickness of pulp, peel, average weight of seed/ fruit, amount of juice/fruit and number of fruits/plant of 21 pummelo genotype ** = significant at 1% level of probability. genotype fruit weight (g) non-edible portion weight (g/fruit) pulp thickness (cm) peel thickness (cm) juice amount (ml/fruit) number of fruits/plant seed weight average (g) bau-1 1160.00 450.00 11.50 2.03 366.67 20.16 0.50 bau-2 1250.00 426.67 11.50 1.83 350.00 17.38 0.48 bau-4 1060.00 266.67 10.17 1.83 143.33 33.33 0.47 hybrid 1283.33 463.33 11.37 1.80 366.67 29.56 0.51 jambura (seeded) 696.67 290.00 8.97 2.17 233.33 47.16 0.24 mohini 543.33 206.67 7.33 1.57 101.67 35.00 0.08 green skin 866.67 243.33 10.50 1.30 223.33 10.00 0.33 malaysian 1020.00 290.00 10.83 1.67 206.67 25.86 0.43 thai jambura 1013.33 346.67 11.00 1.50 336.67 31.16 0.29 accession-51 516. 67 233.33 6.33 1.83 130.00 14.16 0.55 accession-52 300.00 100.00 6.50 1.33 90.00 14.53 0.58 accession-57 906.67 380.00 9.50 2.00 200.00 15.60 0.39 accession-58 490.00 236.67 7.33 1.33 140.00 11.36 0.06 accession-59 696.67 276.67 8.67 1.50 206.67 13.53 0.65 accession-62 613.33 306.67 8.17 2.00 123.33 9.20 0.42 accession-63 760.00 323.33 9.33 1.83 233.33 10.58 0.47 accession-76 640.00 136.67 9.73 1.00 193.33 12.24 0.45 accession-87 720.00 276.67 8.50 1.17 170.00 12.73 0.47 accession-93 876.67 310.00 9.50 2.17 216.67 24.00 0.58 accession-101 776.67 333.33 9.21 1.93 150.00 27.53 0.42 accession-103 816.67 236.67 9.90 1.00 156.67 23.33 0.39 lsd 0.01 80.19 64.57 1.77 0.48 63.96 1.81 0.11 level of significance ** ** ** ** ** ** ** fig. 2 pulp to peel ratio of 21 pummelo genotype. vertical bar indicates lsd at 1% level of probability. adv. hort. sci., 2018 32(1): 93-103 98 the number of fruits/plant varied significantly and ranged from 9.20 to 47.16 with the mean value of 20.88. among the genotype jambura (seeded) bears the maximum number of fruits (47.16) followed by mohini (35.00) and bau-4 (33.33) whereas accession-62 (9.20) bears the minimum number of fruits (table 3). in respect of number of seeds/fruit it was observed that different genotype differed significantly. the maximum number of seeds per fruit was recorded in the genotype hybrid (114) followed by seeded jambura (110), bau-4 (108.67) whereas the minimum number of seeds was found in accession58 (10) (fig. 4). like number of seeds, seed weight/fruit was also differed significantly from genotype to other genotype. the maximum weight of seeds/fruit was found in the genotype hybrid (58 g) followed by bau-4 (51.67 g), accession-93 (50.00 g) whereas the minimum weight of seeds was found in the accession-58 (1.00 g) (fig. 5). average weight of seed differed significantly ranging from 0.06 to 0.65 g with the mean value of 0.42 g. the heaviest average weight of seed (0.65 g) was found from the accession-59 whereas accession-58 gave the lowest average seed weight (0.06 g) (table 3). correlation coefficient estimation of simple correlation coefficient was made among some important fruit producing characters of 21 pummelo genotype. the values of ‘r’ and the characters correlated are presented in table 4. it was observed that leaf length had highly significant positive correlation with leaf breadth (r= 0.853**) and significant positive association with petiole wing length (0.519*) and petiole wing breadth (0.544*). it had also significant negative correlation with number of fruits/plant (-0.464*) (table 4). leaf breadth had highly significant positive correlation with wing breadth (0.584**) and significant positive correlation with wing length (0.450*). on the other hand, this character had negative correlation with %tss, number of seeds, weight of seed and average weight of seed (table 4). correlation coefficient revealed that petiole wing length had highly positive significant correlation with petiole wing breadth (0.846**) and number of fruits/plant (0.773**). on the other hand, this character had negative correlation with weight of non-edible portion, pulp to peel ratio, average weight of seed and %tss (table 4). it was observed that wing breadth had positive correlation with numfig. 3 total soluble solids (tss) of pummelo genotype. vertical bar indicates lsd value at 1% level of probability. fig. 4 number of seeds per fruit (a) and weight of seeds per fruit (b) of pummelo genotype. vertical bars indicates lsd at 1% level of probability. fig. 5 weight of seeds per fruit of pummelo genotype. vertical bar indicates lsd at 1% level of probability. hossain et al. physio-morphological variations of pummelo genotype 99 ber of anthers, thickness of pulp, total soluble solids, number of seeds/fruit and number of fruits/plant. on the other hand, this character had negative correlation with weight of fruit, weight of non-edible portion, pulp to peel ratio, average weight of seed and weight of seeds/fruit (table 4). it was observed that number of anther had simply positive correlation with weight of fruit and weight of non-edible portion. this character had highly negative significant correlation with number of fruits/plant (-0.686**) and had negative significant correlation with number of seeds/fruit (-0.455*) (table 4). correlation coefficient revealed that weight of fruit had highly positive significant correlation with weight of non-edible portion (0.818**) and thickness of pulp (0.930**). on the other hand, this character had negative significant correlation with %tss (-0.487*) (table 4). it was observed that weight of non-edible portion had highly positive significant correlation with thickness of pulp (0.671**) and number of fruits/plant (0.655**). on the other hand, this character had only negative correlation with %tss (table 4). correlation coefficient revealed that pulp to peel ratio had simply positive correlation with thickness of pulp, %tss, number of seeds and weight of seed/fruit. this character had negative correlation with average weight of seed and number of fruits/plant (table 4). it was observed that thickness of pulp had significant positive correlation with number of seeds/fruit (0.440*) and had significant negative correlation with %tss (-0.452*) (table 4). correlation coefficient revealed that average weight of seed had highly positive significant correlation with weight of seeds/fruit (0.588**) and number of fruits/plant (0.605**). this character had simply positive association with number of seeds/fruit (table 4). it was observed that tss had highly positive significant correlation with number of fruits/plant (0.823**) and had simply negative association with number of seeds/fruit (table 4). correlation coefficient revealed that number of seeds/fruit had highly positive significant with total weight of seed (0.822**) and had highly negative significant with number of fruits/plant (table 4). path coefficient analysis a path coefficient is simply a standardized partial regression coefficient and as such measures of the influence of one variable upon another permits separation of correlation coefficient into components of traits leaf breadth (cm) petiole wing length (cm) petiole wing breadth (cm) number of anthers/ flower weight of fruit (g) weight of non-edible portion (g/fruit) pulp to peel ratio thickness of pulp (cm) average weight of seed (g/fruit) tss (% brix) number of seeds/ fruit weight of seeds (g/fruit) no. of fruits/ plant leaf length (cm) 0.853** 0.519* 0.544* 0.176 0.011 -0.043 0.236 0.071 -0.393 -0.093 0.18 -0.052 -0.464* leaf breadth (cm) 0.450* 0.584** 0.292 0.121 0.169 0.301 0.132 -0.39 -0.151 -0.097 -0.18 0.192 petiole wing length (cm) 0.846** 0.259 0.045 -0.15 -0.102 0.151 -0.095 -0.161 0.231 0.069 0.773** petiole wing breadth (cm) 0.227 -0.002 -0.208 -0.128 0.082 -0.099 0.014 0.051 -0.018 0.121 number of anthers/flower 0.021 0.024 -0.334 -0.035 -0.01 -0.242 -0.455* -0.414 -0.686** weight of fruit (g) 0.818** 0.195 0.930** 0.143 -0.487* 0.413 0.387 0.208 weight of nonedible portion (g/fruit) 0.195 0.671** 0.1 -0.298 0.26 0.308 0.655** pulp to peel ratio 0.235 -0.059 0.079 0.162 0.234 -0.106 thickness of pulp (cm) 0.094 -0.452* 0.440* 0.351 0.056 average wt. of seed (g/fruit) 0.306 0.152 0.588** 0.605** tss (% brix) -0.114 0.139 0.823** number of seeds/fruit 0.822** -0.754** weight of seeds (g/fruit) -0.822** table 4 correlation coefficient between fruits per plant and fruit producing characters in pummelo genotype * = indicates 5% level of significance (using mean values). ** = indicate 1% level of significance (using mean values). adv. hort. sci., 2018 32(1): 93-103 100 direct and indirect effects. the simple correlation values were used to compute the path coefficient analysis. direct and indirect effects of different characters on fruits per plant have been presented in table 5. 4. discussion and conclusions the physio-morphological features of 21 pummelo genotype were found significantly different among each other. the variations were observed in leaves, flowers, fruits and seeds of all genotype. a wide variation of morphological characters of trees, leaves, fruits and seeds were identified among the pummelo clones (paudyal and haq, 2008). hossain (1983) noticed varied leaf length, breadth, wing length and breadth of citrus. however, minor differences were observed between genotypic (4.043 cm) and phenotypic (4.542 cm) variance as well as genotypic (20.10%) and phenotypic (21.30%) coefficient of variation indicating minimum environmental effect upon the expression of this character. heritability in broad sense (h2b) was also calculated and it was moderately high as 89.01% (table 6). in case of leaf breadth, table 5 path coefficient between fruits per plant and fruit producing characters in pummelo genotype residual effetct: 0.1579, bold and underlined direct effect. traits leaf length (cm) leaf breadth (cm) petiole wing length (cm) petiole wing breadth (cm) number of anthers/ flower weight of fruit (g) weight of non-edible portion (g/fruit) pulp to peel ratio thickness of pulp (cm) average weight of seeds (g/fruit) tss (% brix) number of seeds/ fruit weight of seeds (g/fruit) number of fruits/ plant leaf length (cm) -0.660 0.312 0.613 -0.940 -0.044 0.032 0.069 -0.091 -0.135 -0.366 -0.062 0.647 0.161 -0.464* leaf breadth (cm) -0.270 0.650 0.531 -0.101 -0.073 0.350 -0.272 -0.116 -0.253 -0.363 -0.100 -0.349 0.558 0.192 petiole wing length (cm) -0.380 0.640 0.180 -0.146 -0.065 0.130 0.242 0.039 -0.289 -0.088 -0.107 0.831 -0.214 0.773** petiole wing breadth (cm) -0.145 0.213 0.999 -0.173 -0.057 -0.006 0.335 0.049 -0.157 -0.092 0.009 0.184 0.056 0.121 number of anthers/flower -0.468 0.107 0.306 -0.392 -0.252 0.061 -0.038 0.128 0.066 -0.009 -0.160 -0.163 0.128 -0.686** weight of fruit (g) -0.029 0.441 0.053 0.004 -0.005 0.289 -0.132 -0.075 -0.177 0.133 -0.322 0.149 -0.120 0.208 weight of non-edible portion (g/fruit) 0.114 0.616 -0.177 0.359 -0.006 0.237 -0.161 -0.075 -0.128 0.093 -0.197 0.935 -0.955 0.655** pulp to peel ratio -0.628 0.109 -0.121 0.229 0.084 0.564 -0.314 -0.385 -0.449 -0.055 0.052 0.583 -0.726 -0.106 thickness of pulp (cm) -0.188 0.481 0.178 -0.141 0.009 0.269 -0.108 -0.090 -0.191 0.087 -0.299 0.157 -0.108 0.056 average weight of seeds (g/fruit) 0.105 -0.142 -0.112 0.171 0.003 0.414 -0.161 0.023 -0.179 0.930 0.193 -0.457 -0.182 0.605** tss (% brix) 0.247 -0.551 -0.190 -0.024 0.061 -0.141 0.480 -0.030 0.864 0.285 0.663 -0.410 -0.431 0.823** number of seeds/fruit -0.479 -0.354 0.473 -0.088 0.114 0.119 -0.418 -0.062 -0.842 0.752 -0.075 0.361 -0.255 -0.754** weight of seeds (g/fruit) 0.138 -0.656 0.082 0.031 0.104 0.112 -0.496 -0.090 -0.671 0.546 0.092 0.296 -0.310 -0.822** table 6 genotypic variance, phenotypic variance, genotypic coefficient of variation, phenotypic coefficient of variation and per cent heritability of fruit producing characters of 21 pummelo genotype traits genotypic variance phenotypic variance genotypic cv phenotypic cv heritability (%) leaf length (cm) 4.043 4.542 20.10 21.30 89.01 leaf breadth (cm) 2.004 2.103 22.61 23.16 95.29 petiole wing length (cm) 0.362 0.441 27.46 30.30 82.09 petiole wing breadth (cm) 0.651 0.771 44.25 48.16 84.44 number of anthers/flower 30.870 38.432 15.06 16.80 80.32 weight of fruit (g) 66033.637 67396.887 31.73 32.06 97.98 weight of non-edible portion (g/fruit) 8092.547 8976.507 30.80 32.44 90.15 pulp to peel ratio 0.350 0.406 23.28 25.08 86.20 thickness of pulp (cm) 2.218 2.884 15.97 18.21 76.91 average weight of seed (g/fruit) 0.021 0.024 35.01 37.40 87.67 tss (% brix) 1.436 1.890 7.47 8.56 75.98 number of seeds/fruit 754.160 804.840 37.21 38.44 93.70 weight of seeds (g/fruit) 195.793 209.793 43.17 44.69 93.33 number of fruits/plant 103.963 104.635 48.84 49.00 99.36 hossain et al. physio-morphological variations of pummelo genotype 101 petiole wing length and petiole wing breadth little differences were observed between genotypic (2.004 cm, 0.362 cm, 0.651 cm) and phenotypic variances (2.103 cm, 0.441 cm, 0.771 cm) as well as genotypic (22.61%, 27.46%, 44.25%) and phenotypic coefficient of variation (23.16%, 30.30%, 48.16%) indicating minimum environmental effect upon the expression of these characters. heritability in broad sense (h2b) was also calculated and it was high as 95.29%, 82.09%, 84.44% (table 6). flower characters of different pummelo genotype were also varied significantly in this experiment. hoque (2015) reported that petals number of pummelo genotype ranged from 4 to 4.5. considerable differences were observed between genotypic (30.870) and phenotypic (38.432) variance as well as genotypic (15.06%) and phenotypic (16.80%) coefficient of variation indicating considerable environmental effect upon the expression of this character. h2b was also calculated and it was very high as 80.32% (table 6). regarding fruit weight, it was found that fruit weight varied significantly among the pummelo genotype. rahman et al. (2003) reported that fruit weight of local pummelo accessions varied from 718.33 g to 2160 g. while hays (1966) noticed that the pummelo fruit is larger than the other important commercial citrus species, somewhat weighing more than 1 kg. purseglove (1968) also observed that the fruit of pummelo is large to very large. the weight of pummelo fruit were ranged from 250 to 1218.75 g (hossain, 1983). in bangladesh, weight of fruit varied from 396 to 1418 g. this variation might be due to genetical, physiological, nutritional or environmental influences. the genotypic and phenotypic variances of weight of fruits were 66033.637 g and 67396.887 g, respectively. the genotypic coefficient of variation (31.73%) was lower than phenotypic coefficient of variation (32.06%), which indicated more influence of environment on the performance of particular trait. h2b was also calculated and it was 97.98% which was high. the genotypic and phenotypic variances of weight of non-edible portion were 8092.547 g and 8976.507 g, respectively. the genotypic coefficient of variation (30.80%) was lower than phenotypic coefficient of variation (32.44%), which indicated more influence of environment on the performance of particular trait. heritability in broad sense (h2b) was also calculated and it was 90.15%, which was moderately high. the genotypic and phenotypic variances of ratio of pulp and peel were 0.350 and 0.406, respectively. the genotypic coefficient of variation (23.28%) was lower than the phenotypic coefficient of variation (25.08%), which indicated more influence of environment on the performance of particular trait. h2b was also calculated and it was 86.20%, which was very high. the genotypic and phenotypic variances of thickness of pulp were 2.218 cm and 2.884 cm, respectively. the genotypic coefficient of variation (15.97%) was lower than the phenotypic coefficient of variation (18.21%), which indicated more influence of environment on the performance of particular trait. h2b was calculated and it was 76.91%, which was very high. tss content significantly varied among the genotype. but little differences were observed between genotypic (1.436%) and phenotypic (1.890%) variances as well as genotypic (7.47%) and phenotypic (8.56%) coefficient of variation indicating low environmental influence on this trait. h2b was calculated and it was 75.98% which was high. the number of fruits/plant varied significantly among the genotype. verheij and coronel (1992) reported that a single tree can yield 70-100 fruits/year. the genotypic and phenotypic variances of thickness of number of fruits/plant were 103.963 and 104.635, respectively. the genotypic coefficient of variation (48.84%) was lower than the phenotypic coefficient of variation (49.00%), which indicated more influence of environment on the performance of particular trait. h2b was also calculated and it was 99.36% which was very high. maximum number of seeds/fruit was observed in hybrid (114) among the studied genotype and minimum seed number was found in accession-58. hossain (1983) reported that the number of seeds in pummelo fruits varied from 8 to 94/fruit and also he found both seedless and seeded genotype. the genotypic and phenotypic variances of number of seeds/fruit were 754.160 and 804.840, respectively. the genotypic coefficient of variation (37.21%) was lower than the phenotypic coefficient of variation (38.44%), which indicated more influence of environment on the performance of particular trait. h2b was also calculated and it was 93.70% which was very high. the genotypic and phenotypic variances of total weight of seeds were 195.793 g and 209.793 g, respectively. the genotypic coefficient of variation (43.17%) was lower than the phenotypic coefficient of variation (44.69%), which indicated more influence of environment on the performance of particular trait. h2b was also calculated and it was 93.33%, which was very high. correlation coefficient revealed that leaf breadth, petiole wing length and breadth will be increased with the increase of leaf length. on the other hand, this trait had negatively associated with weight of non-ediadv. hort. sci., 2018 32(1): 93-103 102 ble portion, average weight of seeds/fruit, %tss and weight of seeds/fruit. this indicate that petiole wing length and breadth will be increased with the increase of leaf breadth. on the other hand, this trait had negatively associated with tss, number of seeds/fruit and total weight of seeds/fruit. correlation coefficient revealed that petiole wing breadth and number of fruits/plant will be increased with the increase of petiole wing length. correlation coefficient revealed that weight of non-edible portion and thickness of pulp will be increased with the increase of fruit weight. this indicate that thickness of pulp and number of fruits/plant will be increased with the increase of weight of non-edible portion. on the other hand, this trait had negatively associated with %tss. it was observed that correlation coefficient revealed that thickness of pulp indicates that number of seeds/fruit will be increased with the increase of thickness of pulp. correlation coefficient revealed that average weight of seed/fruit indicates total weight of seeds and number of fruits/plant will be increased with the increase of average weight of seed. correlation coefficient revealed that %tss indicates number of fruits/plant will be increased with the increase of %tss. correlation coefficient revealed that number of seeds/fruit indicates weight of seeds/fruit will be increased with the increase of number of seeds/fruit. leaf length showed low direct negative effect (-0.66) on fruits/plant whereas it also contributed indirect positive effect via leaf breadth, petiole wing length, fruit weight, weight of non-edible portion, number of seeds/fruit and weight of seeds/fruit. leaf breadth contributed low direct positive effect (0.65) on fruits/plant. this trait had also indirect positive effect on wing length, fruit weight, weight of seeds/fruit and number of fruits/plant. petiole wing length showed low direct positive effect (0.18) on fruits/plant whereas it also contributed indirect positive effect on fruits/plant via leaf breadth, fruit weight, weight of non-edible portion, pulp to peel ratio and number of seeds/fruit. petiole wing breadth contributed low direct negative effect (-0.173) on fruits/plant. this trait had also indirect positive effect on number of fruits/plant via leaf breadth, petiole wing length, weight of non-edible portion, pulp to peel ratio, %tss, number of seeds/fruit and weight of seeds per fruit. number of anthers/flower showed low direct negative effect (-0.252) on fruits/plant. this trait had also indirect positive effect on leaf length, wing length, weight of fruit, pulp to peel ratio, thickness of pulp and weight of seeds/fruit. fruit weight showed low positive effect (0.289) on fruits/plant whereas it also contributed indirect positive effect on number of fruits/plant via leaf length, leaf breadth, wing length, average weight of seed and number of seeds/fruit. weight of non-edible portion contributed direct negative effect (-0.161) on fruits/plant. this trait had also indirect positive effect on number of fruits/plant via leaf length, leaf breadth, petiole wing breadth, weight of fruit, average weight of seed and number of seeds/fruit. pulp to peel ratio showed direct negative effect (0.385) on fruits/plant whereas it also contributed indirect positive effect via leaf breadth, petiole wing breadth, number of anthers/flower, weight of fruit, %tss and number of seeds/fruit. thickness of pulp contributed direct negative effect (-0.191) on fruits/plant. this trait had also indirect positive effect on number of fruits/plant via leaf breadth, petiole wing length, weight of fruit, number of anthers, average weight of seeds and number of seeds/fruit. average weight of seeds/fruit showed direct positive effect (0.93) on fruits/plant whereas it also contributed indirect positive effect on fruits/plant via leaf length, petiole wing breadth, number of anthers/flower, fruit weight, pulp to peel ratio and tss. tss showed direct positive effect (0.663) on fruits/plant. this trait had also indirect positive effect on number of fruits/plant via leaf length, number of anthers/flower, weight of non-edible portion, thickness of pulp and mean weight of single seed. number of seeds/fruit contributed direct positive effect (0.361) on fruits/plant whereas it also contributed indirect effect via petiole wing length, number of anthers/flower, weight of fruit and average weight of seeds/fruit. weight of seeds/fruit showed direct negative effect (-0.31) on fruits/plant. this trait had also indirect positive effect via leaf length, petiole wing length, petiole wing breadth, number of anthers/flower, weight of fruit, average weight of seeds/fruit, tss (%) and number of seeds/fruit. it was noticed that a wide variations existed among the pummelo genotype and these variability could be useful for varietal improvement of pummelo in bangladesh. it appeared from the study that grouping of the genotype differed based on morphological traits. in respect on different traits genotype hybrid was found potential followed by bau-1. references bbs, 2015 monthly statistical bulletin. bangladesh hossain et al. physio-morphological variations of pummelo genotype 103 bureau of statistics, statistics division, ministry of planning, government of people’s republic of bangladesh, dhaka, bangladesh, pp. 144. bhatt g.m., 1973 significance of path coefficient analysis in determining the nature of character association. euphytica, 22(2): 338-343. burton g.w., 1952 quantitative inheritance in grasses. proceedings of 6th international grassland congress, 1: 277-283. dewey d.r., lu k.h., 1959 a correlation and path-coefficient analysis of component of crested wheatgrass seed production. agron. j., 51: 515-518. gomez k.a., gomez a.a., 1984 statistical procedure for agricultural research. 2nded., john willey and sons, new york, usa, pp. 28-192. hayes w.b., 1966 fruit growing in india. 3rd ed., allahabad agricultural institution, allahabad, india, pp. 209. hoque m.a., 2015 floral biology of indigenous pummelo genotypes. bangladesh j. agril. res., 40(2): 177-188. hossain m.m., 1983 morphological studies of different citrus plants. m. sc. (ag.) thesis, department of horticulture, bau, mymensingh, bangladesh, pp. 112. ipgri, 1999 descriptors for citrus. international plant genetic resources institute, rome, italy, isbn 92-9043425-2. johnson h.w., robinson h.f., comstock r.e., 1955 estimates of genetic and environmental variability in soybeans. agron. j., 47: 314-418. morton j.f., 1987 pummelo, pp. 147-151. in: dowling c.f. (ed.) fruits of warm climates. florida flair books, miami, fl, usa, pp. 505. paudyal k.p., haq n., 2008 variation of pomelo (citrus grandis l. osbeck) in nepal and participatory selection of strains for further improvement. agroforst. syst., 72(3): 195-204. purseglove j.w., 1968 tropical crops dicotyledon. lonmans green and company limited, london, uk, pp. 502. rahman m.m., rabbani m.g., khan a.s.m.m.r., ara n., rahman m.o., 2003 study on physio-morphological characteristics of different local pummelo accessions. pakistan j. biol. sci., 6: 1430-1434. ranganna s., 1978 manual of analysis of fruit and vegetable products . tata mcgraw-hill publishing company limited, new delhi, india, pp. 634. singh r.k., chaudhary b.d., 1985 biometrical methods of quantitative genetic analysis. haryana j. hort. sci., 12(2): 151-156. verdi a., 1988 application of recent taxonomical approaches and new techniques to citrus breeding. proceeding of the 6th international citrus congress, tel aviv, israel, march 6-11, vol. 2, pp. 303-315. verheij e.w.m., coronel r.e., 1992 plant resources of south-east asia no. 2. edible fruits and nuts. prosea foundation, bogor, indonesia. impaginato 433 adv. hort. sci., 2019 33(3): 433-439 doi: 10.13128/ahs-23476 efficient and easy micropropagation of morus nigra and the influence of natural light on its acclimatization w.n. duarte ¹ (*), c.a. zanello ¹, j.c. cardoso ² ¹ master’s graduate program in plant production and associated bioprocesses, centro de ciências agrárias, ufscar, araras, brazil. ² department of biotechnology, plant and animal production, centro de ciências agrárias, ufscar, araras, brazil. key words: benzyladenine, black mulberry, greenhouse, growth room, plant growth regulators. abstract: micropropagation, which employs various plant growth regulators (pgrs) in the culture medium for the induction of multiple shoots as well as adventitious roots, is a widely-used technique for the propagation of the genus morus. the main objective of the present study was to evaluate the effect of pgr-free culture medium on the quantitative and qualitative characteristics of the micropropagation of morus nigra under the growth room and greenhouse environmental conditions. although a higher rate of multiplication (4.7–5.2 shoots/explant) was obtained from the treatments using benzyladenine (ba) as the pgr, the pgr-free culture medium also exhibited comparable multiplication rate (4.1 shoots/explant) with a higher quality of shoots and without any symptoms of hyperhydricity. furthermore, the use of pgr-free culture of m. nigra for in vitro propagation combined the steps of shoot multiplication and rooting phase using the same culture medium, further simplifying the process. in this study, the micro-shoots were also assessed for their in‐vitro rooting and acclimatization potential. the incubation of an in‐vitro culture of nodal explants in a controlled growth room for 28 days exhibited optimum response with about 90% rooting and 100% plantlet survival during the acclimatization phase. these results were better than those incubated under greenhouse conditions. 1. introduction morus nigra is an exotic plant of unknown origin, with wild populations occurring in the aegean region of turkey (browicz, 2000). its fruits are a rich source of phenolic compounds such as flavonoids (arfan et al., 2012). its propagation via seeds is limited due to a high degree of heterozygosity and a long period of sexual immaturity to reach fruit production (vijayan, 2014). mulberry (morus sp.) is commercially propagated via the rooting of stem cuttings. however, in vitro clonal production of plantlets via micropropagation could also be achieved using ms medium supple(*) corresponding author: willnduarte@gmail.com citation: duarte w.n., zanello c.a., cardoso j.c., 2019 efficient and easy micropropagation of morus nigra and the influence of natural light on its acclimatization. adv. hort. sci., 33(3): 433-439 copyright: © 2019 duarte w.n., zanello c.a., cardoso j.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 september 2018 accepted for publication 24 april 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 433-439 434 mented with benzyladenine (ba) at concentrations of 0.5 to 2.5 mg l-1 for multiple shoot induction and indole butyric acid (iba) or naphthalene acetic acid (naa) at concentrations of 0.25 to 2.0 mg l-1 for root induction, in different species of this genus (chitra and padmaja, 2005; ahmad et al., 2007; ji et al., 2008; chattopadhyay et al., 2011). some protocols have also used gibberellic acid (ga3) for the elongation of shoots (gogoi et al., 2017). although plant growth regulators (pgrs) such as ba, iba, and naa promote and regulate the in vitro development and improve the efficiency of micropropagation, the addition of pgrs to the culture medium may also result in undesirable effects, varying from the loss of quality of generated plantlets and phenotypic variations, to certain undesirable genetic mutations called somaclonal variations (bairu et al., 2011). the control of environmental conditions in a growth chamber or culture room for in vitro propagation is another requirement of commercial micropropagation, which increases the costs of micropropagated plantlets (chen, 2016). cardoso et al. (2013) suggested that the use of photoautotrophic micropropagation following pre-acclimatization in a greenhouse using natural light could reduce the costs of this technique, along with enhanced in vitro plantlet quality. this study aimed to develop a simple protocol for the micropropagation of black mulberry (m. nigra) without the use of plant growth regulators (pgr-free micropropagation). the study also evaluated the use of in vitro pre-acclimatization under greenhouse conditions for the acclimatization of generated m. nigra explants. 2. materials and methods establishment of in vitro cultures of morus nigra apical and axillary buds procured from mature (4year-old) field-grown plants of m. nigra cv. ‘portuguesa’ were used for the establishment of in vitro cultures. young shoots of 1 to 2 cm length were immersed in 70% (v/v) ethyl alcohol for 30 sec, followed by surface steril ization in 40% sodium hypochlorite solution (containing 2.0-2.5% of active chlorine) for 15 min. after asepsis, the explants were thoroughly washed with sterile distilled water and trimmed further to a length of 3-5 mm using a scalpel blade, under a laminar airflow hood. the explants were then inoculated in flasks containing 30 ml ms medium (murashige and skoog, 1962) with half strength of macronutrients (½ ms) and supplemented with 30 g l-1 sucrose, 0.1 mg l-1 myo-inositol, 1.0 mg l-1 ba, and 0.1 mg l-1 naa. the ph of the culture medium was adjusted to 5.8±0.05, after which 6.4 g l-1 agar-agar was added and the medium was autoclaved at 121°c and pressure of 1 kgf/cm² for 20 min. the culture flasks were incubated in a growth room with a light intensity of 2,500 lux (cold fluorescent lamps of 20 watts), 16-h photoperiod, and temperature of 25±2°c. after in vitro establishment, the explants were maintained every 30 days by sub-culturing the nodal segments in ½ms culture medium without the addition of plant growth regulators, until the numbers of segments obtained were sufficient to execute the experiments. effect of different pgrs on in vitro shoot multiplica‐ tion the basal culture medium described for the in vitro establishment (½ms medium supplemented with 30 g l-1 sucrose, 0.1 mg l-1 myo-inositol) was used throughout the study, with variations in the types and concentrations of the most commonly used pgrs for the genus, viz., pgr-free; ba at 0.25, 0.50, and 1.0 mg l-1; thidiazuron (tdz) at 0.10 mg l-1; and ga3 at 3.0 mg l-1 (vijayan, 2014). for the experiment, the nodal explants (1.0±0.2 cm) with one axillary bud were obtained from in vitro cultures previously grown on pgr-free ½ms medium for 30 days. the growth conditions were identical to those described previously. the experiments were conducted using a completely randomized design (crd), with five replicates (flasks) containing four nodal explants in each. after 30 days of incubation of the culture, growth parameters such as shoot length, number, and width of leaves, and multiplication rate (number of new nodal segments obtained from each inoculated explant) were evaluated for each treatment. all the measurements were carried out using a digital caliper. in vitro rooting and acclimatization the nodal explants (1.0±0.2 cm) containing one axillary bud were inoculated in flasks containing ½ms basal medium without pgrs. eight treatments in a 2×4 factorial design, with two environmental conditions, i.e., culture room and greenhouse, and four different durations of in vitro culture (7, 14, 21, and 28 days) were used. the culture room conditions were identical to those described in the establishment phase, while the greenhouse was covered with duarte et al. ‐ efficient and easy micropropagation of morus nigra 435 diffusion agricultural plastic sheets of 150 µm thickness, with a light intensity of 65000±3000 lux, and average temperature ranging from 28.1 to 29.5°c during the period of in vitro rooting and acclimatization phase. each treatment comprised of four flasks containing five nodal segments in each. the in vitro assessments were conducted after each period of growth (7, 14, 21, or 28 days). the number of nodal segments with new sprouts and roots was the parameter used for the evaluation. the field-based assessments were also conducted after 30 days of plant growth under greenhouse conditions on a commercial substrate consisting of peat (pindstrup®, denmark). the evaluation parameters included the height of plantlets, and the number and width of leaves. all the measurements were performed using a digital caliper. the obtained data were analyzed using analysis of variance (anova) and the means were subjected to tukey’s comparison post-test at 5% probability. the software assistat version 7.7 beta was used for the statistical analysis (silva and azevedo, 2006). 3. results and discussion successful establishment of morus nigra the development was initiated through the growth of shoots with fresh leaves after 14 days of inoculation, without any contamination or oxidation of explants. the first subculture was carried out after 30 days of inoculation and the shoots were multiplied until sufficient quantity of the explants was obtained for the ensuing micropropagation experiments. pgr‐free culture medium resulted in single‐step micropropagation and higher quality of plantlets the nodal segments cultured on pgr-free ½ms medium showed the best average height of plants (6.4 cm), with a good number (4.4) and width of leaves (1.8 cm). the addition of 0.25 mg l-1 and 1.0 mg l-1 ba increased the multiplication rate from 4.1 (pgr-free) to 4.7 and 5.24, respectively (table 1). however, the addition of ba, even at the lowest concentration used in culture media (0.25 mg l-1), negatively affected the quantitative (height of plants and number and width of leaves) (table 1) as well as qualitative parameters of regenerated shoots, showing callus formation at the basal cut ends and yellowish leaves (fig. 1 a-c), compared with pgr-free medium (fig. 1 f). these symptoms were more intense (yellowish-brown leaves) in case of tdz, even at a low concentration of 0.1 mg l-1 (fig. 1 d). the addition of ga3 resulted in a reduction in the number of leaves and axillary buds, leading to a lower multiplication rate (table 1; fig. 1 e) than the pgr-free medium. in most of the protocols related to in vitro micropropagation of morus sp., the addition of cytokinins table 1 effects of culture medium with different pgrs on the in vitro development and multiplication of nodal segments of morus nigra * multiplication rate referring to the mean number of nodal segments obtained by each segment inoculated in vitro. average values followed by the same letter in the column do not differ from each other by tukey's test at 5% probability. treatment plantlet leaves height (cm) multiplication rate* number width (cm) pgr-free 6.4 a 4.1 bc 4.5 a 1.8 a ba 0.25 3.5 b 4.7 ab 3.7 b 1.2 b ba 0.50 3.5 b 5.0 a 3.7 b 1.1 b ba 1.00 3.3 b 5.2 a 3.6 b 1.1 b ga3 3.6 b 2.2 d 2.4 c 1.2 b tdz 2.3 c 3.6 c 3.4 b 1.1 b f value (pgrs) 18.24 22.66 7.86 16.59 coefficient of variation (%) 38.3 26.4 30.8 26.6 fig. 1 shoots of morus nigra in different types and concentrations of plant growth regulators (pgrs): (a) 0.25 mg l-1 ba; (b) 0.50 mg l-1 ba; (c) 1.0 mg l-1 ba; (d) 0.10 mg l-1 tdz; (e) 3.0 mg l-1 ga3. (f) pgrs-free ms½ medium. bar = 1 cm. adv. hort. sci., 2019 33(3): 433-439 436 to the culture medium had shown positive effects on the multiple shoot induction (chattopadhyay et al., 2011; lalitha natarajan et al., 2013). until now, no report was available on the use of pgr-free culture medium for the in vitro multiplication of morus sp. yadav et al. (1990) observed a high shoot multiplication rate from shoot apex (4.2) and nodal explants (11.3) in m. nigra using ms medium supplemented with 1.0 mg l-1 ba. they also recorded a high shoot multiplication rate (6.3) with nodal explants using kinetin (kin). anis et al. (2003) recorded in vitro multiplication rates ranging from 2.4 to 5.2 using 2.0 mg l-1 ba and 0.2 mg l-1 naa in m. alba. on the other hand, although akram and aftab (2012) did not observe multiple shoot induction in m. macroura using either ba or kin singly in ms medium, the supplementation of ba with iba in the culture medium resulted in a high multiplication rate of 4.7 shoots/ explant. in the current study, the excision of shoot apical meristem (sam) of m. nigra helped in breaking the dormancy of axillary buds in nodal explants cultured in vitro and increased the level of cytokinin. excision of the sam also caused a reduction in the endogenous levels of auxins and enhanced the expression of genes encoding isopentenyl transferase (ipt), a key precursor enzyme for the biosynthesis of endogenous cytokinin. this increased the level of endogenous cytokinin in the axillary buds, resulting in stimulation of cell division and shoot development (tanaka et al., 2006). the highly resistant nature of ba to the degradation activity of cytokinin oxidases explained the largely improved in vitro shoot productivity when this pgr was added to the culture medium (ashikari et al., 2005). the use of pgr-free medium resulted in the production of shoots and roots without any supplementation. however, yadav et al. (1990) observed that iba was more effective in the promotion of rooting in m. nigra as compared to iaa and naa, and increased the number of roots from 1.2 (auxin-free) to 6.1 (0.25 mg l-1). these results are strikingly similar to our findings, as we also obtained only 1-2 roots per stem nodal segment without using auxins (data not shown). nevertheless, although a lower number of roots were produced, even a single root was enough for the acclimatization of morus nigra plantlets. the plants obtained from the culture medium containing ba exhibited some morphological alterations compared with those developed in the pgrfree medium. the abnormalities included difficulty in in vitro manipulation during subculture due to the breakage of brittle tissues, reduced internodal length, and yellowish leaf appearance. these symptoms of hyperhydricity were observed irrespective of the ba concentration (fig. 1a-c). the repeated subcultures on ba containing medium led to somaclonal variations in the micropropagated plants (nasser and mahmood, 2014). nanism, hyperhydricity, and chlorosis were some of the abnormalities reported in the in vitro‐raised plants with continuous use of high concentrations of ba (israeli et al., 1991; kumar et al., 1999). in the current study, no morphological abnormality was observed in the obtained plantlets, even after 24 months with mensal subcultures, when pgr-free culture medium and nodal explants of 1.0±0.2 cm were used. greenhouse pre‐acclimatization did not show optimal development of plantlets a notable difference was observed in the shoot development from in vitro nodal segments of mulberry after seven days of inoculation and 21-28 days of growth in growth room and under greenhouse conditions, with about 100% of nodal explants producing new shoots in the growth room compared to only 80% under greenhouse conditions (fig. 2 a). however, the highest impact of environmental conditions was observed on the rooting, where only 40% of the nodal segments produced roots in the greenhouse compared to 90% in growth room conditions (fig. 2 b). although up to 80% shoot regeneration from the nodal explants was recorded after 14 days of culture in greenhouse conditions, the rate later reduced to 70% and 40% after 21 and 28 days, respectively. this could be attributed to the death of plantlets after cultivation for 14 days in greenhouse conditions, possibly due to excessive ambient light intensity (above 68,000 lux), which caused maximum daytime temperatures reaching between 35.1 to 36.7°c. this temperature was around 10°c higher in comparison to the standard growth room conditions, i.e., 25±2°c temperature at a light intensity of 2,500 lux. high temperatures may lead to the alteration of cellular processes, such as protein expression (koini et al., 2009; stavang et al., 2009) and the hormonal regulation of development by, for example, cytokinin (macková et al., 2013). the acquisition of thermotolerance (acclimatization) requires the expression of heat shock proteins (hsps) in order to avoid oxidative stress and involves the synthesis of abscisic acid (aba) (penfield, 2008). aba is a well-known phytohormone, which inhibits the growth of plants under duarte et al. ‐ efficient and easy micropropagation of morus nigra 437 environmental stress or pathogen attack (sharp and lenoble, 2002). therefore, it could result in the reduced shoot and root formation in the nodal segments of m. nigra under the extreme light and temperature in greenhouse conditions. future experiments should use covers to reduce the light intensity and extreme temperatures in the flasks, similar to those used for the gerbera pre-acclimatization tests (cardoso et al., 2013). the in vitro culture of micropropagated plantlets during elongation and rooting phase under greenhouse conditions is called as pre-acclimatization. this has been successfully executed in gerbera (gerbera jamesonii) and was reported to promote the growth and acclimatization rate of gerbera plantlets and reduce the costs of micropropagation (cardoso et al., 2013). the major differences between the previous and present studies lie within the partial control of environmental factors, which in the previous study were maintained as 25±5°c, ppfd 100 μmol m-2 s-1 and a 12-h photoperiod, i.e., natural growing conditions for gerbera. in contrast, in our study with m. nigra, the incidence of high light intensity (900-1800 μmol m-2 s-1), a temperature exceeding 30°c, and longer photoperiod (12:30 h) proved detrimental to in vitro as well as field-based development of plantlets. growth room environment for 28‐days of in vitro cul‐ tivation provided optimum conditions for in vitro root‐ ing and acclimatization as shown in table 2, the previously observed effect of in vitro conditions on the development of plantlets was also reflected during the acclimatization stage under greenhouse conditions. the nodal explants maintained in vitro under greenhouse conditions also resulted in reduced development of plantlets in the acclimatization stage. the duration of in vitro culture incubation also affected the development of acclimatized plantlets of m. nigra. although a 7-day-incubation period resulted in 96% survival, plantlets were better developed with a 28-day-incubation period before acclimatization (table 2). the plants maintained for different durations under in vitro greenhouse conditions showed improvement only in leaf width (0.57 cm at 7-d versus 1.79 cm at 28-d), with no gain in terms of shoot length and number of leaves (table 2; fig. 2c). on the other hand, plants grown under culture room conditions showed better ex vitro acclimatization of m. nigra plantlets, as the shoots obtained after 28 days of in vitro incubation were longer (9.2 cm) and had greater leaf width (3.2 cm) compared to those incubated in vitro in the greenhouse (3.8 cm and 1.8 cm, respectively). fig. 2 number of nodal segments with new shoots (a) and roots (b) along the period of 28-days of in vitro cultivation. plants after 30 days from ex vitro acclimatization (c) and previous in vitro cultivated under growth room (left) and greenhouse (right). 438 adv. hort. sci., 2019 33(3): 433-439 the culture room conditions of programmed and controlled temperature with low light intensity were conducive enough for the production of plantlets with healthy shoots and roots, and consequent successful acclimatization (100%) of morus nigra. 4. conclusions a single-step protocol using pgr-free culture medium for the in‐vitro shoot induction, multiplication, and rooting was optimized for efficient largescale production of m. nigra plantlets. the micropropagated shoots of m. nigra were maintained in pgr-free culture medium for two years without any symptoms of morphological abnormalities or somaclonal variations. the in vitro rooting under culture room conditions after 28 days presented better response during the acclimatization phase, with the production of plantlets of superior quality. since preacclimatization in the greenhouse did not result in enhanced adaptation of plantlets, further extensive experiments with more variations in the controlled conditions of temperature and light intensity should be undertaken in order to optimize a viable and efficient method for the cost-effective micropropagation of morus sp. acknowledgements wnd and caz thanks to capes for master’s scholarship and jcc thanks to cnpq for the process number 304174/2015-7. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001 references ahmad p., sharna s., srivastava p.s., 2007 in vitro selection of nahco3 tolerant cultivars of morus alba (local and sujanpur) in response to morphological and biochemical parameters. hort. sci. 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mathur and ahuia, 1991; mathur, 1992). also, plant regeneration from adventitious shoots, seedlings, suspension cultures of v. edulis (enciso-rodriguez, 1997; castillo-españa et al., 2000), shoot regeneration from leaf segments of v. officinalis (abdi and khosh-khui, 2007), shoot organogenesis and somatic embryogenesis from leaf explants of v. jatamansi (rong et al., 2014) and from shoot buds of v. jatamansi (kaur et al., 1999) and shoot (*) corresponding author: abdi@pgu.ac.ir citation: abdi gh., khush-khui m., shekafandeh a., 2019 embryogenesis in valerian (valeriana officinalis l.) using leaf segments. adv. hort. sci., 33(2): 179-185 copyright: © 2019 abdi gh., khush-khui m., shekafandeh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 november 2018 accepted for publication 28 january 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 179-185 180 organogenesis and somatic embryogenesis from leaf explants of valeriana jatamansi jones have been described (chen et al., 2014). somatic embryogenesis is an efficient and high volume propagation system for the large number of plants within a short period. successful genetic transformation attempts have mostly employed embryogenic callus or cell cultures as the target tissue in several medicinal plants (leena and jaindra, 2003). however, a major limitation of this callus system is the repeated subculture to select embryogenic callus portions among highly proliferating non-embryogenic tissue. this process is not highly producible and furthermore increases the chance of somaclonal variation. as these limitations have become unavoidable, strategies to improve plant regeneration must necessarily include manipulation of the medium to embark upon new morphogenetic pathways (pedroso and pais, 1995). direct somatic embryogenesis offers several advantages in medicinal plant improvement, as cost effective and largescale clonal propagation is possible using bioreactors, ultimately leading to automation of somatic seed production and development of artificial seeds. besides, such a system could provide a new source for use in genetic transformations. the plant derived from direct somatic embryogenesis usually is unicellular in origin and hence genetically uniform. the leaf segments of valerian are an excellent source for the induction of indirect embryogenesis and the factors affecting this process had been studied (castilloespaña et al., 2000). direct somatic embryogenesis and factors controlling it have been studied in many plant species (pedroso and pais, 1995; chen et al., 1999; desai et al., 2004; kuo et al., 2005; quirozfigueroa et al., 2006; thengane et al., 2006; jayanthi et al., 2011). based on our knowledge, there is no report about direct somatic embryogenesis on valeriana officinalis. the aim of this study was to establish a method for asexual multiplication of valeriana officinalis through direct somatic embryogenesis. 2. materials and methods plant material and culture methods fresh leaves of valerian were collected from 4month old greenhouse-grown plants (fig. 1a). they were washed with tap water and a few drops of rica (a commercial detergent). they were surface sterilized by 70% ethyle alcohol for 1 min. and rinsed twice with sterile distilled water. the leaves were immersed in a solution of 1.5% sodium hypochlorite for 10 min and rinsed four times with sterile distilled water. the leaves were cut into 7-8 mm2 segments and transferred to 150 ml glass jars with 25 ml of half-strength ms medium (murashige and skoog, 1962). the ph of media was adjusted to 5.8 by 0.1 n hc1 before autoclaving for 15 min at 121°c and 1.5 kg·cm-2 pressure. cultures were placed initially under the dark condition for 2 weeks and thereafter they were maintained at 25±3°c under 16 h photoperiod provided by cool white fluorescent lamps (45 µmol·m-2·s-1) with relative humidity of 75-85%. embryo induction and germination for embryo induction, after preliminary experiments, ms media were supplemented with 0.5 mg·l-1 2,4-d, 0.5 mg·l-1 naphthalene acetic acid (naa), 100 mg·l-1 glutamic acid (glu) or different concentrations of sucrose (3, 4, and 5%) (table 1). for embryo germination, two separate experiments were conducted. in the first experiment, the explants that formed embryo were divided to 4 segments (each fragment was about 0.2±0.03 g) and transferred to ms medium containing naa (0.5, 1, 2 and 5 mg·l-1), gibberellic acid (ga3) (0.5, 1 and 2 mg·l-1), or ms added to different combinations of naa (0.1 and 0.2 mg·l-1) and fig. 1 effects of different treatments on in vitro culture of valerian, (a) 4-month old greenhouse-grown plant of valerian, (b) initiation of small embryo-like structures in the 10 to 13 day after culture in induction media, (c) and (d) embryo formation in top and cut end of valerian leaf explant after 4 weeks. (e) callus initiated in m2 medium with great potential for regeneration. (f) germination of embryo mass in medium supplemented with 2 mg l-1 kin and 0.1 mg l-1 naa. abdi et al. valeriana officinalis l. embryogenesis 181 kinetin (1.5 and 2 mg·l-1). in the second experiment, embryos were transferred individually to ms medium supplemented with naa (0.1 and 0.2 mg·l-1) and kinetin (1.5 and 2 mg·l-1) to stimulate the germination and roots and shoots development. acclimatization the 4 cm plantlets were transferred to small pots containing 1/3 vermiculite, 1/3 perlite and 1/3 sand (v/v). the pots were placed in transparency box and maintained under 25±5°c temperatures and 70% relative humidity for 4 weeks and then transferred to greenhouse. data analysis the experiment was conducted as a completely randomized design in a factorial arrangement with 4 replications and each replicate with 12 explants. the means were compared with duncan’s new multiple range test (dnmrt) at 5% probability level. to determine the efficiency of embryo induction medium, responsive leaves that formed embryo was recorded after 4 weeks. for embryo germination experiments the percent of germinated embryo was recorded after 3 weeks and the number of plantlets was recorded 6 weeks after culture. plantlets were recognized when they developed roots and shoots. 3. results embryo induction the leaf segments showed swelling and initiation of small embryo-like structures in the 10-13 days after culture (fig. 1b). in the following weeks, embryogenic clumps were visible at the cut end and surface of the explants (fig. 1c and d). embryoid formation in cut edges of leaf explants was higher than other parts of leaves. well-developed embryos were observed all over the cultured explants within four weeks of culture. different medium showed different embryogenesis response. when glu and sucrose (4%) were added to medium, embryogenesis response increased. maximum embryogenic response of leaf explants (57.21±2.7%) was observed on m8 medium supplemented with 2, 4-d, 4% sucrose, and 100 mg·l-1 glutamic acid. naa addition instead of 2, 4-d decreased percentage of explants response in m9 (43.31±2.33%) medium. increasing sucrose concentration more than 4% reduced embryogenic response on m 12 (25.28±1.24%), and m13 (13.39±2.28%) (table 1). the explants on m2 and m3 media did not exhibit any embryogenic response. however, explants on these media initially showed slight swelling and subsequently resulted to callus production. m2 showed higher percentage of callus proliferation compared to m3 (data not shown). callus initiated in m2 medium had a great potential for regeneration (fig. 1e). the explants on m1 medium did not exhibit any response. embryo germination for embryo germination experiment, two kinds of explants were used. in the first experiment, the explants that formed embryo were divided to 4 segments (each fragment was about 0.2±0.03 g) and used as mass embryo. in the second experiment the embryos were transferred individually to ms medium supplemented with various supplements. somatic embryo germination response varied greatly in various hormone supplements (table 2). ms without growth regulators showed low response to embryo germination. in this treatment, germination percentage per number of embryoid pieces and also number of plantlet per pieces was not considerable (table 2). ms+naa (0.5, 1, and 2 mg·l-1) led to rooting of somatic embryos (fig. 2a). the inclusion of ga33 (0.5, 1, and 2 mg·l-1) in the germination medium increased the germination percentage and consequently plantlets with both well-developed shoots and roots. the combination of kin-naa enhanced somatic embryo germination percentage more than other treatments. among kin-naa treatments, 2 mg·l-1 kin and 0.1 mg· l-1 naa showed high frequency of embryos and also plantlet formation (table 3). the somatic embryos were easily isolated with a pair of forceps, each developing into a single plant. the highest recovery table 1 different media used for induction of direct somatic embryogenesis in valeriana officinalis l. pgr additives (mg·l-1) m 1 m 2 m 3 m 4 m 5 m 6 m 7 m 8 m 9 m 10 m 11 m 12 m 13 naa 0.5 0.5 0.5 0.5 0.5 0.5 2,4-d 0.5 0.5 0.5 0.5 0.5 0.5 glutamic acid 100 100 100 100 100 100 sucrose (%) 3 3 3 3 3 4 4 4 4 5 5 5 5 response (%) 12.4±0.3 11.21±1.1 5.28±0.1 3.4±0.03 57.21±2.7443.31±2.33 9.8±0.15 8.8±0.1 25.28±1.2413.39±2.28 adv. hort. sci., 2019 33(2): 179-185 182 of somatic embryos (78+3.21%) was possible on ms medium supplemented with naa (0.1 mg·l-1) and kin (2 mg·l-1), this condition showed considerable germination percentage and plantlet formation while the fewest somatic embryos germination and developing into a single plant (41+1.73%) was observed in ms medium supplemented with a low concentration of naa (0.1 mg·l-1) and kin (1.5 mg·l-1) (table 4). 4. discussion and conclusions using different plant tissue culture technique and plantlet regeneration have been reported in different species of valeriana genus, such as v. officinalis (abdi and khosh-khui, 2007; abdi et al., 2008; reza et al., 2009), valeriana wallichii (mathur et al., 1988), valeriana edulis ssp. procera (enciso-rodriguez, 1997), v. jatamansi (kaur et al., 1999; das et al., 2013), valeriana glechomifolia (salles et al., 2002; bello de carvalho et al., 2004), valeriana glechomifolia (bello de carvalho et al., 2004). however, direct somatic embryogenesis have never been reported from any explant of the species. in this study, the effects of different plant growth regulators (pgrs) and different supplement on the induction of direct somatic embryogenesis were investigated. we established an efficient embryo induction, somatic embryogenesis and plant regeneration system from leaf explants using various supplement and different types and concentrations of pgrs. the induction and development of in vitro somatic embryos comprise complex processes including cell division, differentiation, growth, and pattern formation (capron et al., 2009)., the composition of the basal culture medium, the type and levels of plant growth regulators (pgr), the level of carbon sources and the balance of organic and inorganic nitrogen sources are key factors for in vitro embryogenesis system. primary nitrogen sources, including glu, is very important in plant tissue culture in order to stimulate the cellular growth fig. 2 effects of different treatments on in vitro culture of valerian (a) rhizogenesis response of embryo in medium containing naa. (b) individual embryos in medium containing 2 mg l-1 kin and 0.1 mg l-1 naa. (c) well germinated embryos in medium containing 2 mg l-1 kin and 0.1 mg l-1 naa. (d) plantlets with developed shoots and roots obtained from embryo germination. table 2 effects of various supplements on germination of valeriana officinalis l. embryos media response ms + without growth regulators germination and plantlet production was low ms + naa rhizogenesis ms + kin and naa plantlets with both well-developed shoots and roots ms +ga 3 plantlets with both well-developed shoots and roots with vigorous growth of plantlets table 3 effects of various supplements on frequency and plantlet/embryo mass in valeriana officinalis l. plant growth regulator explants response (%) plantlet per each embryo mass pgr free 13.71 g 1.27+0.1 e ga3 (mg·l-1) 0.5 23.24 f 2.71+0.01 d 1.0 38.00 d 3.00+0.10 c 2.0 31.70 e 2.71+0.11 d kin+naa (mg·l-1) 1.5+0.1 43.37 d 3.21+0.12 c 1.5+0.2 37.75 d 4.00+0.02 b 2.0+0.1 61.32 a 5.21+0.12 a 2.0+0.2 51.37 b 4.27+0.13 b pgr (mg l-1) explant response (%) plantlet per each embryo mass kin + naa 1.5 + 0.1 49+1.2 c 23 bc 1.5 + 0.2 41+1.73 d 19 c 2.0 + 0.1 78+3.21 a 37 a 2.0 + 0.2 58+2.41 b 25.6 b table 4 effects of growth regulators on percentage of embryo germination and number of plantlets abdi et al. valeriana officinalis l. embryogenesis 183 embryos induction (luo et al., 1996; nakagawa et al., 2001). the positive effect of sucrose may related to the vital function of the sucrose such as controlling the several developmental processes in the cells (gibson, 2000; smeekens, 2000), nutritional function (serve as a carbon source during somatic embryogenesis) and osmotic regulator or cell osmolarity (biahoua and bonneau, 1999 ). the positive role of sucrose in the present study may be interpreted as both nutritional and osmotic regulatory functions of this carbohydrate. the conversion of somatic embryos to plantlets is a multi-step process. in many embryogenetic systems the transfer of somatic embryos into pgr free culture medium enhances the development of the somatic embryos and their conversion to plantlets. one of the determinative factors for the low rates of somatic embryo conversion to plantlets is associated with the residual effects of 2, 4-d. prolonged expositions to this pgr normally reduces the conversion and increases the number of abnormal somatic embryos (cruz et al., 1990). when somatic embryos of v. officinalis were culture in medium supplemented with kin-naa and ga3, the number of plantlets increased. the positive role of cytokinins may be related to reversion of negative effects caused by 2, 4-d to the cultures (parrot et al., 1988). the role of the ga3 in promoting the germination of somatic embryos is well documented in other embryogenic systems (deng and cornu, 1992). our results are in agreement with the findings of castilloespaña et al. (2000) who used combination of kinnaa for embryo germination in valeriana edulis ssp. procera. in conclusion, the induction of direct somatic embryogenesis in valerian using leaf segments as described in this study could be useful in rapid propagation of the elite plant, which has best characters for medicinal purposes. furthermore, direct embryogenesis can be beneficial for gene transformation via particle bombardment or agrobacterium in short time, avoiding somaclonal variation. in addition, the identified protocol does not seem induce a proliferation of callus before the differentiation of somatic embryos. regenerated plants with well-developed root and shoot systems were successfully (72%) transferred to greenhouse. acknowledgements the authors are thankful to the prof. tafazoli for useful comments. and the connection between cells and tissues (young et al., 1999). the productions of direct somatic embryos on leaf explants of v. officinalis are possible by the addition of the glu to the culture medium and increasing the sucrose level to 4%. absence of any callus formation indicated that the process of embryo development was direct. glutamate occupied a central role in amino acid metabolism in plant. it can form by action of the glutamate synthase utilizing glutamine (bohinski, 1991). glutamine as a nitrogen source for purins and pirimidins biosynthesis, was significantly stimulated the direct embryogenesis in valeriana officinalis in this study. this finding is in parallel with the results of chowdhry et al. (1993), gex et al. (2006) and shahsavari (2011). this could explain the enhanced rate of induction and development of somatic embryos in the present investigation. additionally, during the metabolism and protein synthesis the nitrogen originated from amino acids is quickly assimilated into carbonic skeletons (lea, 1993). this stimulation suggested that organic nitrogen was a growth-limiting factor in valerian cultures and the inclusion of glutamine decreased the culture lag phase, which indicated that glutamine was much more readily assailable than inorganic nitrogen. the somatic embryos in this study were formed more in the cut edges of leaf explants. increasing in embryogenic competence of wounded tissue probably was related to the endogenous growth regulators changes in leaf tissue (ivanova et al., 1994). this study also demonstrated that 2, 4-d is more efficient than naa to induce somatic embryos formation in valerian. shoot organogenesis and somatic embryogenesis were also reported in cucumber in the presence of 2,4-d and naa (kuijpers et al., 1996) and the same results were reported in cassava leaf explants by sofiari et al. (1997). plant tissue studies on v. edulis revealed that 2,4-d induced somatic embryos while naa induced shoots (castillo-españa et al., 2000). increasing the level of the sucrose from 3% to 4% showed a positive effect in embryo induction, while the induction response decreased at the presence of 5% sucrose in the culture medium. similar results have been also reported for the other plant species (luo et al., 1996; biahoua and bonneau, 1999; nakagawa et al., 2001) sucrose as common carbohydrate in the phloem sap of many plants have affects in plant tissue culture and the formation of somatic embryos in culture medium (luo et al., 1996; nakagawa et al., 2001) for this reason adding high concentration of sucrose can enhance the somatic 184 adv. hort. sci., 2019 33(2): 179-185 references abdi g., khosh-khui m., 2007 shoot regeneration via direct organogenesis from leaf segments of valerian (valeriana officinalis l.). int. j. agric. res., 2: 877-882. abdi g., salehi h., khosh-khui m., 2008 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burchi crea, centro di ricerca orticoltura e florovivaismo,via dei fiori, 8, 51017 pescia (pt), italy. key words: germplasm, monkey puzzle, phenology, phenotyping, climate, pistoia’s nurseries. abstract: araucaria araucana is a south american endemic conifer of conservation concern. after its introduction to europe, this species has been often planted for ornamental purposes in parks and gardens, where its unusual appearance was admired. after the mid-1970s this tree received increasing attention from the nursery growers of pistoia (tuscany) and became of considerable economic importance. however, being one of the iconic threatened trees listed in cites appendix i, the international trade of these species is rigorously regulated. this study was aimed at developing a first morphological descriptor list for further phenotyping of in loco produced plant material. a first step of the research focused on the description and a better understanding of a. araucana phenological phases inferred from mediterranean climate conditions. the second phase regarded the analysis of observed or measured morphological characteristics of the tree, branches, scales, inflorescences, fruits and seeds observed on a subset of 4 selected putative populations over a 3-year period of vegetative growth. the results allowed to select 39 most discriminant descriptors, which are presented together with their range of variability and classes. the achieved descriptor list represents a suitable tool for the selection of genotypes and for the breeding of a. araucana. 1. introduction araucaria araucana (molina) k. koch, commonly known as ‘monkey puzzle tree’ or ‘pehuèn’, is an endangered conifer species native to southcentral chile and south-western argentina, where it has a relatively limited distribution, split between the main area spanning both sides of the andes and two other disjunct small subpopulations in the coastal cordillera of chile (donoso, 1993, 2006; donoso et al., 2008; drake et al., 2009). the present distribution is a remnant of a more extensive former distribution, which has been severely diminished by logging, human-set (*) corresponding author: maurizio.antonetti@crea.gov.it citation: antonetti m., nin s., burchi g., 2019 first insight into araucaria araucana (molina) k. koch under its southernmost european growing condition: a proposed descriptor list for morphological characterization. adv. hort. sci., 33(2): 283-294 copyright: © 2019 antonetti m., nin s., burchi g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 december 2018 accepted for publication 11 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 283-294 284 fires and land clearance since european colonization in the mid-19th century (veblen, 1982; burns, 1993; rechene, 2000). in particular, the intense human seed collecting and animal grazing have led to a lack of natural reproduction by seed, and when any regeneration occurs it is principally asexual with tree sprouting from roots (schilling and donoso, 1976; gallo et al., 2004). since 1976, this species has been protected in chile under the status of a chilean national monument and, since 1997, it is also protected internationally under the convention on international trade in endangered species of wild fauna and flores (cites) (farjon and page, 1999; herrmann, 2006). araucaria araucana was first introduced in england by the scottish naturalist archibald menzies in 1795. its unusually straight cylindrical bole and whorled branches as well as its 10-15 cm thick tortoise-shell-like bark made it internationally popular as an ornamental plant. the following decades saw a rapid spread of this impressively large and long-lived conifer throughout all the european continent. it was introduced in italy from paris in 1822 and the first a. araucana tree was planted in the garden of the marquis pucci’s favourite property in florence, tuscany. this italian region represents the southernmost limit of its distribution area. thanks to its perfect adaptability to the tuscan soil and climate conditions, this majestic slow-growing tree was included in the important ornamental horticulture district of pistoia (nw from florence) starting from the ii postworld war period. thereafter, there was a notable and rapid increase of a. araucaria commercial propagation in the pistoia district in line with the steadilygrowing demand. this tree became of considerable economic importance and the period between the 1970s till the early 2003 saw the maximum expansion of commercialization rate and tree planting in private properties and public gardens. due to its constantly declining distribution, together with its slow growth and its limited dispersal ability, in 2003, according to the regulation (ce) n. 1497, the listing of this species was transferred from appendix ii to appendix i of cites (http://www.cites.org/eng/app/appendices. html; valid from october 4th, 2017), which strictly regulates the trade in its timber and seeds, and listed in the 2008 iucn red list of threatened species (http://www.iucnredlist.org; march 2017) as an endangered species currently on risk of extinction. as a consequence, plant nurseries had to adopt a mandatory stock register of alive and dead specimen, where both entries and exits (including origin, quantity, causes of death, etc.) had to be specified. a progressive and ongoing reduction of monkey puzzle tree propagation in the pistoia district has resulted from increased regulation, complex management, high risk of penalties, increased costs for staff training, and risks from plant diseases associated with climate change. ultimately, the number of nurseries holding and propagating araucaria plants conspicuously declined during the last fifteen years. the identification of a new tuscan variety could offer the opportunity to disengage from the procedural constraints imposed by the cites convention. hence, the production of a descriptor list for the characterization of tuscan selected germplasm is a necessary first step towards the definition of genetic diversity based on morphological variation and for varietal identification in araucaria. the descriptor list might represent the first attempt at achieving an unified documentation system thereby enabling through a standardized format an easier exchange of information between researchers and collection curators. although there is a high demand for new descriptor lists to be developed for many forest conifer species, up to our knowledge there is only an upov descriptor l ist available for picea abies l. (https:// www.upov.int/test_guidelines/en/fulltext_tgdocs.jsp?q= picea; copyright ©2011, upov). despite the conservation interest in this species, little is known of its phenotypic and genetic variation. the genetic diversity of monkey puzzle between andean and coastal chilean populations has been investigated in previous studies by delmastro and donoso (1980) and rafii and dodd (1998). more recently, advanced biotechnologies, such as rapds, isozymes, microsatellite and rflp analysis, were used to characterize genetic heterogeneity within and among some south american populations (bekessy et al., 2002; ruiz et al., 2007; marchelli et al., 2010; martín et al., 2012). however, no reports were found in the world literature on morphological traits. this study was part of the caraviv project ‘characterization of araucaria araucana germplasm selected by the nursery industries of the pistoia’s district for commercial development’, supported by the ministero delle politiche agricole alimentari e forestali (mipaaf-oiga), aimed at contributing to a better understanding of araucaria growing and to enhance the commercial exploitation of local genetic resources. this paper provides a brief account of araucaria araucana phenological phases under italian climatic conditions and is focused on the development of a first descriptor lists in order to antonetti et al. auracaria araucana. first insight 285 characterize in loco produced plant material and make information available to other growers in a systematic and unambiguous form. 2. materials and methods plant material the plant material used in this study was the a. araucana germplasm available in the pistoia’ nursery district, covering an area of approx. 965 sq km, ranging from 50 m to 550 m above sea level, located in northern tuscany. phenology throughout three-year growing cycles (20152017), the phenological phases (onset of flowering, full bloom, fertilization, fruit ripening and seed production) of a. araucana trees belonging to 8 putative populations were observed every two weeks from march to june (during the flower maturation), and monthly in the rest of the year. taking into account the peculiar structure of the reproductive buds of this species, we decided to consider as onset of flowering the inflorescence appearance and as full bloom the inflorescence maturity, i.e. the pollination phase. four out of the selected populations belong to pistoia’s hinterland in a plain area (43°53’ n; 10°55’ e; 60 m a.s.l.), while the remaining four are located in high hills (44°0’ n; 10°52’ e; 550 m a.s.l.). the number of plants for each population ranged from 10 to more than 200, varying in age, gender and sexual maturity. morphological descriptor list twenty/twenty-five-year-old specimens belonging to a subset of 4 putative populations were randomly defined in order to perform a morphological description. the considered populations were derived from seeds of different origin (unknown, dutch fair, spanish fair, local selected progeny) and grown in three private nurseries, located very close to each other in a plain area under the same organic regime. local selected progeny refers to seeds collected from a couple of old trees located in villa lodolo (s. marcello pistoiese, 44°03’ n; 10°47’ e; 623 m a.s.l.). these trees were introduced from argentina in 1920 and represent the main genetic source of a. araucana local germplasm. climatic data, i.e. atmospheric pressure (ap), medium (avg t), maximum (max t) and minimum (min t) air temperatures, relative air humidity (rh), wind run (wr), global horizontal irradiation (ghi), rainfall (rain), evaporation (ev), for the same area were collected monthly from january 2015 to december 2017 (table 1). minimum, maximum, average, standard deviation and coefficient of variability (%) values of each morphometric character were calculated for the whole set of plants by using one-way analysis of variance (anova). this in turn led to the definition of classes for all the measured traits by subdividing the total range into intervals 2 times the standard deviation, i.e. whether above or below the average value of each parameter as described in bassi (2003). statistical analysis was performed using spss 20 software (chicago, il, usa). table 1 seasonal averages (2015-2017) of main meteorological data collected in pistoia’s nursery areaa ap= atmospheric pressure; avg t= medium air temperature; max t= maximum air temperature; min t= minimum air temperature; rh= relative air humidity; wr= wind run; ghi= global horizontal irradiation; rain= rainfall; ev= evaporation. a processed from ce.spe.vi database, pistoia (http://www.cespevi.it/meteo.htm; paolo marzialetti © 1996/2018 ce.spe.vi. pistoia). year season ap (mbar) avg t (°c) max t (°c) min t (°c) rh (%) wr (km) tsi (kwh/m²) rain (mm) ev (mm) 2015 winter 1009.33 8.33 14.73 3 68.33 86.7 1.53 2.7 1.6 spring 1011 18.7 26.67 11.03 62.33 73.37 3.93 1.73 4.9 summer 1009 24.77 33.13 16.87 57 69.6 4.17 1.23 6.5 fall 1016.33 11.5 18.07 6.97 78.33 33.73 1.17 3.67 1.07 2016 winter 1007.33 9.1 14.9 4.47 74 77.6 1.37 5.9 1.4 spring 1007 16.83 25.2 9.83 65 77.9 3.7 2 5 summer 1010.67 24.2 33.03 16.37 57.67 66.9 4.2 1.57 6.5 fall 1014.67 10.43 18.3 5.1 78 24 1.3 3.57 1.03 2017 winter 1012.33 8.47 15.77 2.43 67.33 66.4 1.67 3.53 1.77 spring 1009.67 19.5 27.7 11.53 60 76.03 4.07 1.4 6.3 summer 1009.33 23.67 32.47 15.43 57 65.6 4.03 1.7 6.43 fall 1011.33 9.93 18 4.3 76 28.3 1.27 5.13 1.1 adv. hort. sci., 2019 33(2): 283-294 286 3. results phenology the results of our observations throughout the growing cycles of male and female a. araucana trees in the pistoia district are shown in figure 1. the main collected phenological data (flowering onset, flower maturation, fertilization, fruit ripening and seed production) were compared with those found in previous published surveys on native andean populations (table 2). morphological descriptor list starting from field observations over a three-year growing cycle, a total number of 39 descriptors were developed for further germplasm phenotyping, divided into 6 sections: 1) tree, 2) branches, 3) leaves, 4) male inflorescences, 5) female strobiles, 6) seeds and productivity. these descriptors apply to twenty/twenty-five-year-old trees (i.e. approx. the age of first fruit bearing) grown in nurseries as ornamental plants. the descriptor list has been enriched by images and drawings for a better understanding fig. 1 araucaria araucana male (♂) and female (♀) phenological stages under tuscan climate conditions. table 2 differences between the maturation stages of female and male inflorescences and seed production in araucaria araucana populations in their origin area and in italy i = first year; ii second year; iii = third year. a tortorelli, 1956; montaldo, 1974; donoso and cabello, 1978; rodriguez et al., 1983; marticorena, 1995. phenological phases chile/argentinea italy ♀ ♂ ♀ ♂ flowering onset nov. i aug. i / sep. i mar. i jul. /aug. i maturation dec. i dec. i may i may ii fertilization jan. ii apr. ii seed production dec. ii / feb. iii sep. / oct. ii antonetti et al. auracaria araucana. first insight 287 of the less familiar and discernible traits. descriptor list examples a 288 adv. hort. sci., 2019 33(2): 283-294 4. discussion and conclusions very few studies have examined botanical aspects in a. araucaria following individual plants over their lifetimes, and all are rather dated and referred to plants grown under south america climatic conditions (montaldo, 1974; donoso and cabello, 1978; hoffman, 1982; rodríguez et al., 1983). no data concerning the phenology and growing of this species under both european and italian environmental conditions have been reported elsewhere, except for a very old contribution to the understanding of cytology and sexual reproduction in a. araucana plants grown in northern france (favre-duchartre, 1960). søndergaard (2003) reported about new introductions of monkey puzzle to scandinavia and the west coast of norway, while kubus et al. (2014) evaluated hardiness of a. araucana trees grown in open ground in poland. however only data on annual shoot growth, tree height and degree of frost damages were given. on the other hand, phenological observations are some of the most sensitive data in identifying how plant species respond to regional climate conditions and to climatic changes. like all gymnosperm, monkey puzzle has extremely simple flowers without any ornamental value. the male and female reproductive structures are carried by ‘cones’ and are as a rule separated, in a araucaria draws have been taken and modified from the website http://www.eryprihananto.com (© ery prihananto, indonesia). b mean of 25 seeds. antonetti et al. auracaria araucana. first insight 289 fact monkey puzzle is usually dioecious (martinez, 1957; bekessey et al., 2002). nevertheless, although being basically female, 4 out of 15 flowering trees were found to produce both male and female cones during recording in southwestern of norway; moreover, on completely isolated male and female trees, a couple of cones of the other sex were observed in some years in the northernmost area (søndergaard, 2003). the author suggested a possible correlation between stress (by isolation and climatically exposed situations) and monoecious behavior in a. araucana. in our census, only one monoecious tree was found among all considered specimen (approx. 700 plants) of the pistoia province. the ratio of females to males in the examined populations grown in plain area was 1:4 (on a total of approx. 25% differentiated trees) being biased towards the male sex, while a rather balanced sex ratio was observed in older (approx. forty-five-year-old) totally differentiated trees grown in high hills (fig. 2). this finding is almost in accordance with sex occurrence reported by søndergaard (1975), who determined half female and half male trees in a population with 76% flowering trees in the west coast of norway. no other comparison with literature was possible for sex ratio, which has never been reported in elsewhere published data. since the relationship between tree growth and climate appeared to be sex-dependant, in that male trees were more sensitive to land precipitation and female fig. 3 araucaria araucana mature catkins during pollination. fig. 2 totally differentiated araucaria araucana female (a) and male (b) trees grown in pistoia’s high hills, showing strobiles and catkins formed in consecutive years. trees appeared more sensitive to air surface temperature during the prior period of growth (hadad and roig juñent, 2016), it is probably realistic to assume that gender imbalance in favor of male or female at the beginning of sexual differentiation might vary according to different climatic environments, such as those found in norway and italy. on the contrary, day length did not seem to have a strong influence on the growth and development of the monkey puzzle (søndergaard, 2003); as a matter of fact, day length dependant trees would never survive scandinavian latitudes, since growth would begin too early and cease too late causing extensive frost damage and eventually killing the plant. under the tuscan environmental conditions, male catkins start out erect in july-august, stop growing during the winter season and then become elongated in shape, pendant and reddish-brown at maturity in may of the following year. formed by many small scale-shaped leaflets, called microsporophyllus, they gave rise to a large quantity of pollen. in barely adult trees male caktins are found mostly in groups of 1-3 cones (fig. 3), while older adult trees have groups of 2 up to 7-8 cones. first differences in the apex of potentially vegetative or female flower buds become visible at the end of march (fig. 1d). in flower buds the apex develops into a round dome of 3-4 cm, with more elongated and less tight scales provided with long yelloworange appendixes. female inflorescences, having fertile scales which contain the ovules, called macrosporophylls, are distinguishable in april (fig. 1e), grouped in light green strobiles at the extremity of the new sprouts. greatest frequency of full bloom adv. hort. sci., 2019 33(2): 283-294 290 and pollination was observed in may (fig. 1c and 1f). some differences were found in flower appearance among growing areas. generally, flower buds developed two up to three weeks later when trees were grown at higher altitudes and experienced rigid winters. after fruit set, that occurs in april of year ii (fig. 1g), the sessile and generally solitary and immature female strobiles are erect, globular, with a symmetrical shape, and green colored. they take usually about 4-5 months to develop into ripe globular dark brown mature cones (fig. 1i) and remain closed until the complete maturation of the seeds. scales usually fall off at maturity in august-september of the same year, although an ongoing trend towards the postponement of fruit drop towards november-late december was observed as a consequence of the gradual rise in mean temperatures (fig. 4). as already assessed in previous studies (søndergaard, 2003; sanguinetti, 2014), cones occurrence was found to vary in relation with sun exposure; more in details, cones were most abundant in the south part of the crown. almost all trees showed a marked alternate bearing; especially in males, yearly fluctuation in cone number seemed too large to be imputable only to climatic variations among years, particularly evident for rainfall (fig. 5). alternate bearing was found to be much more pronounced in barely sexually differentiated trees compared to plants older than fifty years, suggesting that in young plants alternate bearing represents a strategic mechanism to save nutrient reserves for significant vegetative growth. approx. 200-250 seeds, reddish to brown and oblong to obconical in shape, were released from each strobile; these range of seeds is fully included in those found out from the literature (150-300) (montaldo, 1974; donoso and cabello, 1978; salazar et al., 2000). negative effects of heavy rainfall on pollination and seed production have been reported (sanguinetti et al., 2002). fructification began when plants were twentytwenty-five-year-old, partially in agreement with reproductive organs appearance reported by salazar et al. (2000) in chilean a. araucana plants, while trees in neuquén-argentina have been reported to become sexually mature after thirty years of age, once the trunk has reached a diameter larger than 20 cm (muñoz ibáñez, 1984). conversely, according to søndergaard (2003), flowering was not initiated before the trees were forty/fifty-year-old in northern europe and this discrepancy might be related to the altitude and latitude difference. the ontogenetic stages presented here (fig. 6) were in accordance with the a. araucaria biological fig. 4 minimum, maximum and average temperature values and trends in pistoia (years 1951-2017) (http://www.cespevi.it/meteo.htm; paolo marzialetti © 1996/2018). fig. 5 annual rainfall values and trends in pistoia (years 19512017) (http://www.cespevi.it/meteo.htm; paolo marzialetti © 1996/2018) fig. 6 schematic representation of the ontogenetic cycle of araucaria araucana. solid lines represent diploid phases (female and male gametogenesis) and embryogenesis after fertilization, while dashed lines represent haploid phases (ovules and pollen). (from favre-duchartre, 1960, modified). antonetti et al. auracaria araucana. first insight 291 cycle highlighted by favre-duchartre (1960) in northern france; moreover, differences in springtime temperatures between france and norway corresponded fairly well to the differences in time (about one month) for release of pollen and seedfall at the two sites, as reported by søndergaard (2003). on the contrary, as shown in table 2, a. araucana phenology was found to differ strongly from that observed in its natural distribution in the andes mountains. obviously, differences between months were principally due to the reversal of the hemispheres and consequentially of the seasons: when it is spring in europe, it is autumn in the andes and vice versa. however, changes seemed probably to be associated also to other factors, such as altitude, climate changes, growing conditions, etc. the effect of climate on the phenology, and in particular on the timing of reproduction, is well known for plants and extensively documented (beebee, 1995; stenseth and mysterud, 2002). these studies have demonstrated that onset of reproduction in spring may have advanced by a week or two due to recent changes in climate over much of europe, but to a much lesser extend in south america (walther et al., 2002; stenseth et al., 2002). contrary to expectations, it was noticed that in males the appearance of the catkins takes place approximately in the same months (august-september in chile and july-august in italy), which, however, correspond to the end of summer in italy and the end of winter in chile. moreover, in chile the male catkins reach full maturity within 3-4 months, whereas in our environmental conditions full bloom occurred within 9-10 months. this could be explained by the fact that in italy the further development of male inflorescence is stopped during the winter, while in chile, where the season turns towards spring, caktins keep growing without interruption. on the other hand, maturation seems to be related to the photoperiod coinciding in both hemispheres with long days in late spring (beginning of december in chile and end of may in italy). similar considerations can be drawn for females as well. the time between female flower appearance (march in italy and november in chile) and maturation (end of may in italy and beginning of december in chile, as for males) is about 3 months in our country and 1 month in chile. in both cases, the entry of the pollen tube into the ovary takes place within a month from the pollination. on the other hand, in our experimental conditions the true fertilization has been reported to occur after approx. 11 months (april of year ii) from pollination (favre-duchartre, 1960), whereas timing of fecundation has never been mentioned in the literature under andean environments. there are contradictory records about the time of seed maturation in chile, as it is unclear if fruits release the seeds after 11-13 months (donoso and cabello, 1978) or 16-18 months (tortorelli, 1956; montaldo, 1974) from pollination. data herein obtained showed that gravity seed dispersal, which usually takes place at the end of the summer (approx. 12-13 months after pollination), moved towards autumn (14-15 months after pollination) in 2016, and then towards early winter (16-18 months after pollination) in 2017. descriptor lists include the basic description of the traits, and the different classes of their expression (characterization) or how to measure the range of their variation (evaluation). most of the descriptors for characterization and evaluation are species-specific, but should be preferentially evaluated under homogeneous growing conditions in order to obtain comparable data and to avoid potential environmental influence on the phenotype. in our study, in order to find the most appropriate descriptors able for distinguishing effectively between individual phenotypes without loss of discriminating power, repeated observations were made on all plant material available in the pistoia’s nursery district. it immediately became clear that there were no evident differences among the expression of growing characters of young potted seedlings (fig. 7), despite the very high number of individuals within the various collections, whereas a high degree of betweenand within-population variability was noted among older trees (fig. 8). a first analysis of the available specimen clearly evidenced that plant habitus as well as various other vegetative traits consistently varied with plant age, fig. 7 araucaria araucana seedlings in pistoia’s nurseries (a: two-year-old seedlings; b: six-year-old seedlings). adv. hort. sci., 2019 33(2): 283-294 292 being this species a long-lived and massive tree up to 50 m tall and 2 m in diameter and attaining maximum ages of at least 1300 years (montaldo, 1974). moreover, different climatic patterns and growing conditions, such as plant density and weed control, might have influenced plant growth and habitus as well. the variability observed here is consistent with the outbreeding dioecious reproductive habit of this species and suggests that these populations should continue to be viable and able to respond to moderate levels of environmental change (hadad and roig juñent, 2016). however, recent phytopathological analysis in tuscany have revealed an increase in mortality, especially of female individuals, without a specific pathogen responsible (rizzo, 2017, personal communication). the most widely accepted theory is that weaker plants, due to climate-related stress, are more vulnerable to damage caused by aspecific pathogens. well as plants grown in nearby locations. with respect to the 39 descriptors detailed in the descriptor list, some very peculiar characters, i.e. insertion angle of the scales on the trunk (n. 8), uniformity of the apparent diameter on the primary and secondary branch (n. 19 and 20), catkin bending (n. 26), were individuated beside some of the most common traits, such as shape and density of canopy (n. 3 and 4), maximum length and width of the leaves (n. 22 and 23). undoubtedly, one of the tree’s most distinguishing feature is its scales, which are stiff, dark green and glossy with a spiny tip and completely cover each branch, closely overlapping each other. but we found surprisingly interesting the size together with the insertion angle of the scales on primary and secondary branches, resulting in evident dissimilar apparent diameters of branches. on the contrary, female inflorescence didn’t show any distinctive feature among populations, therefore it was not taken into account as a descriptor. in fact, while the true flower represents the main distinctive character in magnoliophyta, the pinophyta flowers, that are extremely simple, are not particularly relevant for the description of the species. in our study, morphological traits of a. araucana were observed, measured and documented for the first time under tuscan growing condition. in particular, the research was developed using trees from putative populations growing in the pistoia’s nursery district. the comparison of the observed phenotypic characteristics showed a wide range of variability among and within the considered populations. the resulting data allowed to classify accessions, and to build a catalogue of specific descriptors with embedded biological information that is an essential step towards germplasm phenotyping (in particular new variety description), management or for direct use in agriculture. limitations linked to the potential environmental influence on the phenotype are presented as well. apart from this preliminary study, nothing is known of the patterns of morphological variation within this species. the development of this descriptor list will assist in the systematic and objective recording and exchange of information, which in turn will increase utilization of genetic resources along with a better screening and use of a. araucana biodiversity for breeding programs. in order to analyze relationships between individuals or groups of specimens within locally grown populations, the morphometric characterization of a. araucana trees is in progress based on the defined descriptor list and suitable statistical multivariate approaches. genetic fig. 8 habitus variability in araucaria araucana trees grown in pistoia’s hinterland in a plain area. a correct germplasm characterization should consider adult plants, being necessary the presence of flowers and fruits. however, as the aim of the present study is the phenotyping of young cultivated plants for sale, only trees sufficiently young for trade but at the beginning of sexual differentiation were suitable for characterization. this is why only twenty/twenty-five-year-old specimen belonging to a subset of 4 putative populations grown under similar environmental and agronomic conditions were considered. plants over 25 year-old were discarded as antonetti et al. auracaria araucana. first insight 293 analysis of the same populations are being performed and will be processed in order to validate the discriminating efficiency of the presented descriptor list. acknowledgements authors acknowledge to the vivai bartolini, azienda macchia tommaso and the kind collaboration of andrea tozzi (villa lodolo) for providing plant material and hystorical information. this research was supported by the ministero delle politiche agricole alimentari e forestali (mipaaf-oiga), project caraviv “characterization of araucaria araucana germplasm selected by the nursery industries of the pistoia’s district for commercial development”. references bassi d., 2003 growth habits in stone-fruit trees. cnr, roma, italy. beebee t.j.c., 1995 amphibian breeding and climate. nature, 374: 219-220. bekessy s.a., allnutt t.r., premoli a.c., lara a., ennos r.a., burgman m.a., cortes m., newton a.c., 2002 genetic variation in the monkey puzzle tree 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parisi 3, l. spinelli 4, a. torricelli 4, 5, a. rizzolo 2, m. vanoli 2 (*) 1 agricultural engineering research institute (aenri), agricultural research center (arc), nadi el‐seid st, 12311 dokki‐giza, egypt. 2 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (crea‐ it), via g. venezian, 26, 20133 milano, italy. 3 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca cerealicoltura e colture industriali (crea‐ci), via di corticella, 133, 40128 bologna, italy. 4 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn‐cnr), piazza leonardo da vinci, 32, 20133 milano, italy. 5 politecnico di milano, dipartimento di fisica, piazza leonardo da vinci, 32, 20133 milano, italy. key words: absorption coefficient, bruise, internal brown spot, solanum tubero‐ sum cv. el beida, trs. abstract: aiming at investigating the feasibility of time­resolved reflectance spectroscopy (trs) for the non­destructive detection of internal brown spot (ibs) and other defects in ‘el beida’ potatoes, 90 tubers were measured in 8 points by trs for the absorption coefficient at 730 nm (µa730) and then trans­ versally cut open for recording presence and position of internal defects and ibs severity. the µa730 was lower in healthy tissue than in defected ones and increased with increasing ibs severity with no difference between healthy and slightly ibs tissues. tubers having at least one out of the eight µa730 measures ≥0.04262 cm­1 were considered “defected”. therefore, trs tubers classification performance were: defected, 73.5%; healthy, 45.5%; slightly ibs, 57.1%; moder­ ate ibs, 60%; and severe ibs, 100% of the cases. misclassification could be due to the high variability in flesh color of ‘el beida’ potatoes, as some healthy tubers showed l*, b* and c* color parameters very similar to that of defected ones, especially when ibs severity was slight or moderate, resulting in µa730 values not significantly different between healthy and ibs tissues. the feasibili­ ty of trs in detecting internal disorders in potatoes must be investigated in other susceptible cultivar to see if flesh color can represent a real problem in the detection of defects linked to browning development. 1. introduction detecting internal defects (internal brown spot, hollow heart, heat (*) corresponding author: maristella.vanoli@crea.gov.it citation: ibrahim a., grassi m., lovati f., parisi b., spi­ nelli l., torricelli a., rizzolo a., vanoli m., 2020 ­ non‐destructive detection of potato tubers internal defects: critical insight on the use of time‐resolved spectroscopy. ­ adv. hort. sci., 34(1s): 43­51 copyright: © 2020 ibrahim a., grassi m., lovati f., parisi b., spinelli l., torricelli a., rizzolo a., vanoli m. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 december 2019 accepted for publication 30 june 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 43­51 44 necrosis, black heart) in potatoes (solanum tubero‐ sum l.) is an important challenge for food engineer­ ing as potato is one of the main consumed products in the world: potato occupies the fifth position in terms of production after sugarcane, maize, wheat and rice (fao, 2019).so, it is crucial to ensure tuber quality along the potato supply chain. the presence of internal defects is not visible until tubers are cut or peeled and determines economic losses in potato industry as growers are not able to separate healthy from defected potatoes, causing waste during processing, and negatively influences consumer confidence. usually only a representative sample is cut and, if internal defects are present, the whole lot is removed without verifying the real inci­ dence of affected tubers, so increasing food waste. this problem can be tackled by using non­destructive techniques which potentially allow to segregate raw potato tubers according to the actual presence of internal defects, before the product reaches the fresh market or is processed. many noninvasive techniques (spectroscopic tech­ niques, computer vision systems, ultrasound meth­ ods) have been investigated to assess internal defects in potato tubers with various performance results depending on the type of defect, as reviewed by rady and guyer (2015). spectroscopic techniques can detect potato defects as changes in absorbance have been found comparing sound and damaged tissues. however, the similarity of absorption characteristics between skin and damaged tissue represents a limiting factor for the segregation of defected tubers. rady and guyer (2015) reported that the classification rate of defect­ ed tubers by using spectroscopic techniques ranged from 50 to 98%. recently, a transmission spectrum system in the visible/near infrared region was able to classify blackheart potatoes with an overall classifica­ tion rate of 96.5% by using six selected wavelengths (711, 817, 741, 839, 678, and 698 nm) (zhou et al., 2015). very good performances were reached by using imaging techniques. infrared hyperspectral imaging was able to detect hollow heart in ‘agria’ potatoes achieving an accuracy of 89.1% of correct classification (dacal­nieto et al., 2011). visible­near infrared and short wave infrared hyperspectral imaging coupled with pls­da (partial least square discriminant analysis) were successfully used to detect black spot in raw tubers of three potato culti­ vars achieving an overall correct classification rate of 95.5% and 98.6%, respectively (lópez­maestresalas et al., 2016). internal brown spot (ibs) is a physiological disor­ der of potato tubers which has an important eco­ nomic impact in italy. ibs incidence up to 50% has been observed under inductive environmental con­ ditions and in susceptible cultivars (parisi et al., 2014; pentangelo et al., 2017). ibs is characterized by the presence of punctiform and/or enlarged rust­ colored necrosis in the parenchymal tissues of the tubers. irregular­shaped spots already appear in the vascular ring during the tuber bulking growth stage; ibs symptoms increase from the end of tuber filling to the complete tuber maturity (raimo et al., 2018). ibs can affect different areas of the tuber depending on variety. in some cultivar, such as ‘luminella’, necrosis areas are localized in the apical position, while in other varieties, such as ‘ricciona di napoli’, the symptoms can affect large portions of the parenchymal tissues, compromising potato tuber appearance and taste and altering the processing features (specific gravity and frying quality) of the tubers (pentangelo et al., 2017; raimo et al., 2018). positive correlations have been found between ibs incidence and severity with tuber size and skin roughness (parisi et al., 2014; raimo et al., 2018). environmental conditions, soil properties and irriga­ tion rate also strongly affect ibs development, mak­ ing difficult the prevention, the prediction and the cure of this disorder (parisi et al., 2014; pentangelo et al., 2015, 2017; raimo et al., 2018). in addition, potatoes affected by ibs do not show external symp­ toms and at present it is not possible to segregate healthy from ibs tubers during mechanical grading and packaging (parisi et al., 2014; raimo et al., 2018). recently, vanoli et al. (2012) investigated the possibility of using time­resolved reflectance spec­ troscopy (trs) to non­destructively detect ibs in ‘luminella’ potato tubers, a well­known susceptible genotype. trs is a non­destructive optical technique which, in combination with proper models of photon migra­ tion, explores the fruit tissue at a depth of 1­2 cm with no or limited influence from the skin, allowing the measurement of the absorption (µa) and reduced scattering coefficients (µs) (cubeddu et al., 2001; torricelli et al., 2008). the absorption properties are related to the chemical composition (water, pig­ ments), whereas the scattering properties are related to the structure (intercellular spaces, cell size and shape, starch granules). trs has been mainly applied in postharvest studies for estimating the internal fruit ibrahim et al. ‐ non‐destructive detection of potato internal defects 45 attributes related to maturity in apples, peaches, nectarines, plums, mangoes and pears, for discrimi­ nating fruit with different texture and sensory char­ acteristics and for the detection of internal defects in fruits and vegetables (rizzolo and vanoli, 2016). the development of internal disorders induces changes in the optical properties, as absorption increases with browning development (vanoli et al., 2014) and scattering properties vary when a defect affects the fruit structure, as for mealiness, woolli­ ness or watercore (vanoli et al., 2010; lurie et al., 2011; vangdal et al., 2012; rizzolo and vanoli, 2016). comparing healthy and browned fruit, the latter show higher µa values in the 670­940 nm range, as found in apples (internal browning), pears (brown heart), peaches (browning), plums (browning) and potatoes (ibs). the highest differences between the absorption spectra of browned and healthy tissues were found in the 670­780 nm range, and hence, these wavelengths were selected to distinguish healthy product from defected ones. high positive correlations were found among µa measured at 670 nm (µa670) and at 780 nm (µa780) and browning scores in plums (vangdal et al., 2012), nectarines (lurie et al., 2011) and in apples (vanoli et al., 2014). it was possible to use µa750 to distinguish healthy ‘granny smith’ apples from those affected by internal browning with the former being characterized by µa750<0.030cm­1; similarly, healthy ‘braeburn’ apples had µa740<0.030 cm­1 while in ‘conference’ pears, fruit with µa720≤0.034 cm­1 were not affected by brown hearth (eccher zerbini et al., 2002; rizzolo and vanoli, 2016). in potatoes, µa690 was used to segre­ gate healthy from ibs tubers : tubers having µa690 values equal or higher than 0.039 cm−1 were consid­ ered as ibs and were correctly classified in the 81% of the cases. healthy tubers showed µa690 values of 0.031±0.0032 cm­1 (mean±standard deviation) and were all correctly classified by trs (vanoli et al., 2012). these promising results were limited by the fact that the most part of the tubers had a small size and a round shape making quite easy the detection of ibs measuring each tuber by trs in correspon­ dence of four equidistant points around the equator. the aim of this work was to detect ibs in ‘el beida’, an oval shaped variety characterized by large size tubers with white flesh, in order to find the most suitable trs set­up which allow to probe the whole bulk of each potato revealing also the presence of other defects such as necrosis, black spot and bruis­ es. 2. materials and methods potatoes potato tubers cv. el beida were supplied by a local grower in bologna province (italy) who found ibs in some potato samples. all potatoes were washed and dried with a paper towel. defective samples showing bruises, rots, holes and greening were removed, and 120 tubers were selected for the experiment: 90 tubers were used for defect detection and 30 tubers for flesh color measurements. the tubers for defect determination were labeled, and morphological para­ meters including weight and diameters (x=longest axis, y= longest axis normal to x; z= longest axis nor­ mal to y) were measured. the geometric mean diam­ eter (gmd) and the sphericity of each tuber were cal­ culated according to mohsenin (1986) as following: gmd = (xyz)1/3 sphericity = gmd x­1 then, each tuber was measured by trs and cut open for detecting ibs. time‐resolved reflectance spectroscopy (trs) each tuber was measured by trs for the absorp­ tion coefficient at 730 nm (µa730), being this wave­ length suitable for detecting ibs in potato (vanoli et al., 2012). considering both biological (large size of ‘el beida’ tubers, ibs randomly distributed within the flesh), and the instrumenal characteristics (geometry of the trs fibers), trs measurements were per­ formed in two regions of the tuber, at 15 mm dis­ tance from the sample center (on the right side­ ring1 and on the left side­ring2), rotating the tuber 90° each time (0°, 90°, 180°, 270°) for a total of 8 measurement points. a portable compact setup working at discrete wavelengths developed at politecnico di milano (martinenghi et al., 2016) was used. the light source is a supercontinuum fiber laser (sc450­6w, fianium, uk) providing white­light picosecond pulses, with the duration of a few tens of picoseconds. a custom­ made filter wheel loaded with 14 band­pass interfer­ ence filters (nt­65 series, edmund optics, new jersey, usa) is used for spectral selection in the range 540­940 nm. light is delivered to and collected from the sample by 1 mm fiber placed at 1.5 cm dis­ tance from the illumination point. a second filter wheel identical to the first one is used for cutting off the fluorescence signal originated from the sample when it is illuminated in the visible spectral region. adv. hort. sci., 2020 34(1s): 43­51 46 the browned areas. flesh color thirty tubers were transversally cut open and color was measured on two opposite sides of the flesh with a spectrophotometer (cm­2600d, minolta co., japan), using the primary illuminant d65 and 2° observer in the l*, a*, b* color space. from a* and b* values, hue (h°) and chroma (c*) were computed according to: h° = arctangent (b*/a*) × 360/(2×3.14) and c*=(a*2 + b*2)−2. 3. results and discussion on average, ‘el beida’ tubers had gmd = 67.2±0.6 mm (mean±se) and sphericity=0.77±0.05 showing that potatoes studied in this experiment had medi­ um­large size and cylindrical shape (table 1), and were different from ‘luminella’ potatoes used in the previous experiment (vanoli et al., 2012) which had gmd= 51.8±0.5 mm and sphericity= 0.91±0.01. ibs was found in 26.7% of the tubers and the other defects were observed in 48.9% of the tubers, while 24.4% of tubers were healthy. tubers affected by other defects showed small brown or grey spots under the skin and only in one case there was some internal necrosis area. the light then is detected with a photomultiplier (hpm­100­50, becker&hickl, germany) and the pho­ ton time­of­flight distribution is measured by a time­ correlated single­photon counting board (spc­130, becker&hickl, germany). the instrumental response function has a full width at half maximum of about 260 ps and the typical acquisition time is 1 s per wavelength. a model for photon diffusion in turbid media was used to analyze trs data to assess the bulk optical properties of the samples (martelliet al., 2009) to obtain the estimates of μa and μs at each wavelength. ibs assessment after trs measurements, each tuber was trans­ versally cut open in correspondence of the central part of the tuber, and at 15 mm from the center on the right and on the left side where the trs fibers were positioned. then, each equatorial section of each tuber was photographed, and the presence and position of internal defects and the ibs severity in correspondence of each trs measurement point were recorded. tuber without any visual ibs were considered healthy (h) while those affected by ibs at least in one section out the eight trs measured sec­ tions were considered ibs (ibs). ibs was also scored according to its severity as slight, moderate and severe considering the size of the tissue affected by ibs and the color intensity of table 1 ­ morphological characteristics of potato tubers cv. el beida table 2 ­ absorption coefficient measured by trs at 730 nm (µa730, cm­1) in healthy and in defected tubers and in relation to ibs severity weight (g) diameter x (mm) diameter y (mm) diameter z (mm) gmd (mm) sphericity mean 196.4 87.8 64.8 53.5 67.2 0.77 min 96.7 65.4 52.1 44.2 54.0 0.67 max 382.7 116.0 80.6 64.7 83.0 0.88 sd 55.5 11.0 6.3 4.9 6.1 0.04 se 5.8 1.2 0.7 0.5 0.6 0.005 tissue type ibs severity healthy other defects ibs slight moderate severe mean 0.0375 0.0413 0.0420 0.0386 0.0404 0.0468 min 0.0204 0.0228 0.0309 0.0309 0.0338 0.0338 max 0.0542 0.0563 0.0651 0.0454 0.0517 0.0651 sd 0.0042 0.0053 0.0066 0.0033 0.0041 0.0082 se 0.0002 0.0005 0.0007 0.0007 0.0007 0.0016 ibrahim et al. ‐ non‐destructive detection of potato internal defects 47 tubers were correctly classified in 57.1% of the case, moderate ibs in 60% of the cases, while 100% of severe ibs affected tubers were identified. probably when ibs was slight and moderate, the single brown spots within the potato flesh and/or brown area with slightly brown color make the ibs detection by trs difficult. figure 2 shows some examples of trs measure­ ments at 730 nm in correspondence of the 8 points (4 points for ring 1 and 4 points for ring 2) around the tuber in comparison with the actual localization of the defect within the flesh. figures 2a and 2b show two ibs affected tubers correctly classified by trs: when ibs spot are present, then the µa730 val­ ues were above the threshold values of 0.04262 cm­1 (see points 1, 3, 5 and 7 ring 1 and points 4 and 8 ring 2 for panel a; points 3, 5 and 7 ring 1 for panel b), while in healthy regions, the µa730 values were below the threshold values (see points 2 and 6 ring 2 for panel a; points 1 ring 1 and points 2, 4, 6 and 8 ring 2 for panel b). figure 2c shows that trs was able to reveal the presence of a bruise spot under the skin as only in point 4 ring 2 the value of µa730 was above the threshold of 0.04262 cm­1. however, in panel d of figure 2 is reported an example of an ibs affected tuber not correctly classified by trs, as ibs spots are present in correspondence of the points 1 and 5 ring 1 and point 8 ring 2 but all the µa730 values are below the threshold of 0.04262 cm­1. it seems that the detection of defects in ‘el beida’ potatoes did not depend on tuber size. considering the distribution of potato tubers within 4 gmd class­ es (50­60 mm, 60­70 mm, 70­80 mm, 80­90 mm), 86.5% of the tubers belongs to 60­70 mm and 70­80 considering the severity of ibs among ibs tubers, 29% showed slight severity, 42% moderate severity and 29% severe symptoms. in a previous work on ibs detection in potatoes by trs (vanoli et al., 2012), healthy and ibs affected tubers were measured in the 540­900 nm range and the highest relative percentage differences between the absorption coefficient values of these tissues were found in the 580­690 nm range, thus µa690 was chosen for ibs detection. however, also in correspon­ dence of µa730 the relative percentage difference between healthy and ibs tissue was high. so, we choose to measure potato for the absorption coeffi­ cient at 730 nm. we select 730 nm also because in the current trs set­up a higher power was available compared to 690 nm. the µa730 was significantly lower in healthy tissue than in defected ones (p ≤ 0.05), even if no difference was found between ibs affected tubers and those affected by other defects (table 2).in ibs tubers, µa730 significantly increased with increasing ibs severity; however, no significant difference was found between healthy and slightly ibs tissues (table 2). similarly, µa740 and µa750 measured in apples affected by internal browning, increased with the development of browning with healthy fruit showing the lowest µa740 and µa750 values and those affected by brown flesh the highest ones (vanoli et al., 2010, 2011). in addition, µa740 and µa750 values increased with increasing browning severity, even if this increase was significant only in fruit with moderate and severe browning while no difference was found between healthy flesh and that affected by slightly browning (vanoli et al., 2010, 2011). in order to fix the threshold value of µa730 above which a potato tuber can be classified as affected by ibs or by other defects, the mean and the 95% confidence intervals of µa730 value of defected tis­ sues were computed (µa730= 0.04368 ± 0.00105 cm­1). hence, tubers having at least one out of the eight µa730 measures equal or higher than 0.04262 cm­1 were classified as affected by ibs or by other defects. figure1 shows the µa730 values for the eight mea­ surement points for each of the 90 tubers under examination. only 73.5% of the tubers affected by defects was correctly classified by trs measurements: 70.8% of tuber affected by ibs and 75.0% of tubers with other defects. considering ibs potatoes, slightly affected fig. 1 ­ values of the absorption coefficient measured by trs at 730 nm in each tuber. the horizontal dashed line corre­ sponds to the threshold value of µa730 for defect detec­ tion. 48 adv. hort. sci., 2020 34(1s): 43­51 mm classes, where tubers correctly classified as defected by trs are 70.0% and 75.0%, respectively (fig. 3). on the other hand, there were some problems in the identification of healthy tubers, as only 45.5% of healthy tubers was correctly classified by trs. in this case it can be hypothesized a kind of relationship with tuber size, as the percentage of tuber correctly classified as healthy increased with tuber size, being correctly classified 0% of tuber for 50­60 mm class, 45.5% for 60­70 mm size and 71.1% for 70­80 mm size (fig. 3). healthy tubers considered by trs as affected by defect were characterized by µa730 val­ ues ≥0.04262 cm­1 in at least 1 point out 8 measured ones by trs (fig. 4). this misclassification could be due to differences in the flesh color of potato tubers. in fact, changes in the absorption coefficients measured by trs in the 540­780 nm range reveal variations in the flesh color due to the presence of pigments (carotenoids, antho­ cyanins, chlorophylls) or to browning development. fig. 2 ­ comparison between localization of defects in potato tubers (left) and the corresponding trs measurements at 730 nm (right). ibs tubers correctly classified by trs (panels a and b); tuber with bruise correctly classified by trs (panel c); ibs tuber not identi­ fied by trs (panel d). the dashed lines correspond to the threshold value of µa730 for defect detection. fig. 3 ­ healthy and defected tubers distribution according to 4 gmd classes correctly (i) or not correctly (ni) identified by trs. ibrahim et al. ‐ non‐destructive detection of potato internal defects 49 good correlations were obtained between trs absorption spectra and total carotenoids content (r2 cv=0.83 ­0.93) and flesh color parameters (r2 cv=0.78­0.96) in different mango cultivars (vanoli et al., 2016). in fruit affected by browning, high negative corre­ lations were found between µa measured at 720 nm (‘conference’ pears), 740 nm (‘braeburn’ apples ), 750 nm (‘granny smith’ apples) and l* and h°, while positive correlations were observed for a*, b* and l* (eccher zerbini et al., 2002; vanoli et al., 2010, 2011). flesh color was significantly different between browned and healthy tissues, showing the former higher a*, b*, c* and lower l* and h° values than the healthy ones (eccher zerbini et al., 2002; vanoli et al., 2010, 2011). l* and h° significantly decreased and a* significantly increased with increasing browning severity, even if no clear distinction was found between healthy and slightly browned tissues. in this experiment, 30 tubers not used for ibs detection were cut open and flesh color was mea­ sured. six tubers showed ibs: in this case flesh color was measured on browned areas. ‘el beida’ potatoes were characterized by a white flesh color (table 3). as expected, tissue affected by ibs had lower values of l* and h° and higher values of a*, b* and c* than healthy tubers (table 3), as previously observed in pears affected by brown heart (eccher zerbini et al., 2002) and in apples with internal browning (vanoli et al., 2010, 2011). considering healthy potatoes, a high variability in flesh color is observed, with l*, b* and c* values close to those of browned tissues. this sce­ nario could explain why healthy tubers with darker flesh color have been classified by trs as affected by defects. in the previous work on ‘luminella’ potatoes (vanoli et al., 2012), in which all healthy tubers were correctly classified, flesh color was not measured, so it can only be hypothesized that flesh color of this cultivar showed less variation than in ‘el beida’ and with values of healthy tissue not so close to slightly affected tubers, making easier the correct tubers classification. fig. 4 ­ values of the absorption coefficients measured at 730 nm in healthy tubers. the dashed lines correspond to the threshold value of µa730 for defect detection. table 3 ­ color of healthy and ibs potato flesh healthy flesh ibs flesh l* a* b* c* h° l* a* b* c* h° mean 71.78 ­2.21 15.80 15.95 97.93 62.96 0.21 19.23 19.27 89.74 min 66.93 ­2.85 14.38 14.49 96.90 56.30 ­1.39 17.12 17.19 85.02 max 74.80 ­1.80 19.01 19.21 98.77 68.93 1.82 21.63 21.67 94.57 sd 1.91 0.24 1.04 1.05 0.63 4.28 1.19 1.99 1.98 3.53 se 0.39 0.05 0.21 0.13 0.13 0.49 0.49 0.81 0.81 1.44 adv. hort. sci., 2020 34(1s): 43­51 50 4. conclusions time resolved reflectance spectroscopy has shown to be a feasible tool for detecting internal defects in potato tubers of medium­large size. however, there are some problems to be solved. first at all, ‘el beida’ potatoes showed high variability in flesh color, with some healthy tubers having color very similar to those affected by internal defects and so healthy tubers were misclassified by trs measure­ ments. on the other side, when ibs severity was slight or moderate, it was difficult to find significant differences between the absorption coefficients mea­ sured at 730 nm in healthy and ibs tissues, as flesh color was not so different. so, the feasibility of trs in detecting internal disorders in potatoes must be investigated in other susceptible cultivar in order to see if flesh color can represent a real problem in the detection of internal defects linked to browning development. in this study, eight measurement points were used to explore each tuber in a non­destructive way. this trs set­up allowed to better explore the whole bulk of each potato: in fact, ibs detection did not depend on the tuber size. however, when ibs devel­ oped through some small and brown spots, the detection by trs was very difficult. on the other hand, it’s not possible to increase the regions explored by trs, as when the fibers are positioned too close the tuber ends, the trs signal is not reliable due to boundaries effect. at boundaries, photon can escape from the tissue and, if it not properly mod­ elled, this might introduce overestimation of the absorption coefficient. the trs set­up used in this study is based on the contact between the tubers and the optical fibers; the positions and the distance between the fibers determine the volume explored by trs; perhaps, a non­contact system could allow to better localize the defect, if properly coupled to advanced modelling of the boundaries effect. it is worth noting that these effects can influence classical (not time resolved) nir spectroscopy system since absorbance estimate are influenced not only by absorption and scattering properties of the tissue but also by the geometrical properties (size and shape). acknowledgements we thank società agricola aia s.s. (castenaso, bologna) for supplying the potatoes. we are grateful to science and the technology development fund (stdf), ministry of state for scientific research, egypt for financial support to ayman ibrahim. this research was carried out within the activity of the agrofiliere project funded by italian ministry of agriculture (d.m. 36503/7305/2018). references cubeddu r., d’andrea c., pifferi a., taroni p., torri­ celli a., valentini g., dover c., johnson d., ruizaltisent m., valero c., 2001 ­ nondestructive quantification of chemical and physical properties of fruits by time‐resolved reflectance spectroscopy in the wavelength range 650‐1000 nm. ­ appl. opt., 40: 538­ 543. dacal­nieto a., formella a., carrión p., vazquez­ fernandez e., fernandez­delgado m., 2011 ­ non‐ destructive detection of hollow heart in potatoes using hyperspectral imaging. ­ proc. 14th computer analysis of images and patterns, 6855: 180­187. eccher zerbini p., grassi m., cubeddu r., pifferi a., torricelli a., 2002 ­ nondestructive detection of brown heart in pears by time‐resolved reflectance spec‐ troscopy. ­ postharvest biol. technol., 25: 87­97. fao, 2019 ­ faostat. ­ food and agriculture organization of the united nations, rome, italy. lópez­maestresalas a., keresztes j.c., goodarzi m., arazuri s., jarén c., saeys w., 2016 ­ non‐destruc‐ tive detection of blackspot in potatoes by vis‐nir and swir hyperspectral imaging. ­ food control, 70: 229­ 241. lurie s., vanoli m., dagar a., weksler a., lovati f., eccher zerbini p., spinelli l., torricelli a., feng j., rizzolo a., 2011 ­ chilling injury in stored nectarines and its detection by time‐resolved reflectance spec‐ troscopy. ­ postharvest biol. technol., 59: 211­218. martelli f., del bianco s., ismaelli a., zaccanti g., 2009 ­ light propagation through biological tissue and other diffusive media: theory, solution, and software. ­ spie press, washington, dc, usa, pp. 298. martinenghi e., di sieno l., contini d., sanzano m., pifferi a., dalla mora a., 2016 ­ time‐resolved sin‐ gle‐photon detection module based on silicon photo‐ multiplier: a novel building block for time‐correlated measurement systems. ­ rev. sci. instrum., 87, 073101. mohsenin n.n., 1986 ­ physical properties of plant and animal materials. ­ gordon and breach science publishers, new york, usa. parisi b., mandolino g., raimo f., pentangelo a., 2014 ­ la maculatura ferruginea, insidia nascosta per la patata. ­ l’informatore agrario, 70(42): 41­44. pentangelo a., parisi b., raimo f., mandolino g, pane c., 2017 ­ maculatura ferruginea, nuovi studi per ibrahim et al. ‐ non‐destructive detection of potato internal defects 51 la prevenzione. ­ l’informatore agrario, 73(41): 43­47. pentangelo a., raimo f., cavallaro f., mandolino g., parisi b., 2015 ­ maculatura ferruginea su patata: tecniche di contenimento. ­ l’informatore agrario, 71(42): 43­46. rady a.m., guyer d.e., 2015 ­ rapid and/or nondestruc‐ tive quality evaluation methods for potatoes: a review. ­ comput. electron. agric., 117: 31­48. raimo f., pentangelo a., pane c., parisi b., man­ dolino g., 2018 ­ relationships between internal brown spot and skin roughness in potato tubers under field conditions. ­ potato res., 61: 327­339. rizzolo a., vanoli m., 2016 ­ time‐resolved technique for measuring optical properties and quality of food, pp. 187­224. ­ in: lu r. 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anderson et al., 2003). this perennial, herbaceous, warm season, c4 grass is commonly known as ‘chaiar’ or (*) corresponding author: hsalehi@shirazu.ac.ir citation: akbari m., salehi h., 2018 biochemical and physiological evaluations of common bermudagrass [cynodon dactylon (l.) pers.] iranian accessions under cold stress adv. hort. sci., 32(2): 157-168 copyright: © 2018 akbari m., salehi h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 157-168 158 ‘margh’ in iran. common bermudagrass’s high density, recuperative ability, high tolerance to drought, heat, salinity, wear, flood and most of soils cause the species to be extensively used in tropical and subtropical regions of iran. this species is a major turfgrass for livestock herbage, golf courses, sport fields, public parks and soil conservation. despite its good characters, c. dactylon has a considerable tendency to be damaged or killed by frost, especially in transition zone (munshaw et al., 2006). an important process in the winter perpetuity of common bermudagrass is acclimatization, which is an adaptation process to overcome the environmental stresses (levitt, 1980). the most favorable temperature for root and shoot growth of cool-season turfgrass species varied from 10 to 18°c and 18 to 24°c, respectively. warm season turfgrasses have c4 photosynthetic pathway and are best adapted to warm climatic region of the world and grows well at temperatures between 24 to 29°c and 27 to 35°c for root and shoot growth, respectively (beard, 1973). during cold stress, plants exhibit different mechanisms to develop their cold hardiness and increase their freezing tolerance (zhu et al., 2004; knight and knight, 2012). some of these processes includes changes in the concentration of amino acids, sugars, proteins, compatible solutes, certain hormones, and changes in the degree of fatty acid saturation level and antioxidant capacity that affect the freezing tolerance (karpinski et al., 2002; munshaw et al., 2006; zhang and ervin, 2008). genetic resources and wild plant species that genetically related to cultivated variety have gross value in plant breeding programs (hajjar and hodgkin, 2007). today, many investigations have focused on the naturally occurring genetic differences in stress tolerance of many plants such as lolium perenne l., brachypodium distachyon l. and festuca arundinacea schreb. (luo et al., 2011; hu et al., 2012; salehi et al., 2013). for many years, improvement of warm season turfgrass quality and cold tolerance are the main goals in breeding programs. natural populations of bermudagrass should have considerable genetic variation for tolerance to environmental stresses. since bermudagrass is cosmopolite plant, its considerable genetic variation is predictable. there is great diversity among wild populations and cultivars of common bermudagrass for tolerance to freezing (anderson et al., 2003) and other environmental stresses. in spite of that, there is little data about the cold tolerance of common bermudagrass, and many researches are being conducted to improve cold tolerance of this species (zhang et al., 2011; shi et al., 2015). the aims of the present research were to evaluate iranian common bermudagrass accessions to find accessions with good freezing tolerance and examine physiological changes during cold stress. 2. materials and methods forty-nine accessions of natural common bermudagrass were collected from 18 provinces of iran with different climatic conditions ranging from shiraz city with subtropical condition and tabriz city with temperate climatic condition (fig. 1), and one foreign cultivar ‘blackjack’ was used as control. all accessions were collected originally from grasslands, roadside, seaside, and around orchards and agriculture fields, and transferred to the school of agriculture, shiraz university, under natural greenhouse condition (52°32’ e and 29°36’ n, elevation fig. 1 map showing the sampling locations of common bermudagrass accessions from different regions of iran. 1: boroujerd, 2: malayear, 3: ghidar, 4: zanjan, 5: tabriz, 6: sarein, 7: talesh, 8: anzali, 9: nour, 10: sari, 11: gorgan, 12: minoudasht, 13: chenaran, 14: mashhad, 15: maiami, 16: damghan, 17: semnan, 18: tehran, 19: arak, 20: malayear intersection, 21: nahavand, 22: firouzan, 23: kamiaran, 24: dehgolan, 25: sanandaj abidar, 26: kermanshah taghbostan, 27: mahidasht, 28: islamabad gharb, 29: homail, 30: ilam saymareh bridge, 31: holailan, 32: poldokhtar, 33: mamoulan, 34: khoram abad, 35: foreign cultivar, 36: doroud nahalestan, 37: doroud daneshjo park, 38: doroud siahvel, 39: doroud babahour, 40: azna, 41: aligoudarz, 42: daran, 43: naein, 44: ardakan 1, 45: ardakan 2, 46: yazd, 47: taft, 48: abarkouh, 49: safashahr, 50: shiraz. akbari and salehi evaluation of common bermudagrass iranian accession under cold stress 159 1810 m a.s.l.). each accession was transplanted into 14 cm diameter pots filled with uniform mixture of 1:1:1 (v:v:v) of sand, loamy soil and decomposed manure. turfgrasses were kept in natural greenhouse condition and were clipped to a height of 5 cm every 2 weeks. low and freezing temperature treatments were conducted at 24/17, 7.5/0, -7.5/-12 and -15/-15°c day/night cycles and a 10 h light (300 µmol m-2 s-1) for 7 days, using a controlled-environment chamber. after each temperature regime, physiological traits including: superoxide dismutase (sod) (beauchamp and fridovich, 1971), catalase (cat) (dhindsa et al., 1981), ascorbate peroxidase (apx) and peroxidase (pod) (chance and maehly, 1995) activities, proline (bates et al., 1973), protein (bradford, 1976), electrolyte leakage (saadalla et al., 1990) and chlorophyll content (saini et al., 2001) were measured. turfgrass color and visual quality were rated visually after each treatment (beard, 1973). to extract antioxidant enzymes, fresh leaf or stolon samples (0.5 g) were collected and ground to a fine powder in a mortar by adding liquid nitrogen and then homogenized with an ice cold enzyme extraction buffer containing 0.5% polyvinylpyrrolidone (pvp), 3 mm edta, and 0.1 m potassium phosphate buffer (ph=7.5). the extracted samples were centrifuged for 10 min at 13500 rpm and 2-4°c and stored on ice until used. the resulting supernatant was used for enzyme analysis. sod activity was determined according to the procedure used by beauchamp and fridovich (1971), cat activity was determined as described by dhindsa et al. (1981), ascorbate peroxidase and peroxidase activities were determined according to the method described by chance and maehly (1995). proline was determined according to the method described by bates et al. (1973). using spectrophotometer (uv-120-20, japan) at 520 nm wavelength, appropriate proline standards were included in calculation of its content in samples. the protein content was quantified using bradford method with bovine serum albumin (bsa) as standard. electrical leakage measured, as described by saadalla et al. (1990), using an electrical conductivity meter (metrohm 644, swiss) and calculated with the following formula: electrolyte leakage = ec1/ec2 × 100 chlorophyll content was measured according to the method of saini et al. (2001) using the following formula: mg chl/g f.w. = [(20.2(od 645 nm) + (8.02(od 663 nm)] x v f.w. x 1000 where: od is optical density, v is the final solution volume in ml, and f.w. is tissue fresh weight in mg. turfgrass color and visual quality were measured after each treatment on a 1 to 9 scale where 1 was very poor quality turf, 6 was minimally acceptable turf, and 9 was exceptional turf quality (beard, 1973). this study was conducted in a factorial experiment based on completely randomize design (crd) with three replications. factors were fifty accessions and four different concentrations of low and freezing temperatures (24/17, 7.5/0, and -7.5/-12 day/night cycles). in the case of treatment with -15/-15°c day/night cycles, only the seven most cold tolerant accessions were evaluated. mean comparisons were performed using the least significant difference (lsd) at p= 0.05 probability level. physiological traits were evaluated for accession clustering to determine cold sensitive or cold tolerant using ward’s method of hierarchical cluster analysis in spss software. 3. results the ward cluster analysis based on physiological traits before low temperature treatments (at 24/17°c day/night cycles) grouped the 49 accessions and the foreign cultivar into two major groups (fig. 2). the first group contained 18 accessions with low antioxidant enzymes activity, proline, protein and chlorophyll content including: abidar sanandaj, boroujerd, holailan, malayear, ghidar, nour, saymareh bridge, anzali, islamabad gharb, tehran, tagh bostan, kermanshah, homail, maiami, minodasht, mashhad, poldokhtar, safashahr and shiraz. the second group contained other accessions with more antioxidant enzymes activity, proline, protein and chlorophyll content. two major groups were formed based on physiological characters after cold stress (at 7.5/0°c day/night cycles) (fig. 3). the first group contained 36 accessions with low proline, protein, chlorophyll content and low antioxidant enzymes activity. other accessions were in second group and had more proline, protein, chlorophyll content and more antioxidant enzymes activity included fourteen accessions: arak, doroud daneshjo park, azna, taft, safashahr, ardakan 2, mahidasht, mamoulan, naein, yazd, chenaran, semnan, homail and daran. the dendrogram from physiological characters after cold stress (at -7.5/-12°c day/night cycles) grouped the 50 accessions in two main clusters (fig. 4). the dendrogram from physiological characters at -15/-15°c day/night cycles grouped the 7 most cold-tolerant genotypes into two main groups (fig. 5). the first group contained 3 accessions with lower proline, proadv. hort. sci., 2018 32(2): 157-168 160 tein, chlorophyll content and antioxidant enzymes activity than other elite accessions were: aligoudarz, gorgan and safashahr. other accessions were in second group and had more antioxidant enzymes activity, proline, protein, chlorophyll, color and visual quality, and the least el included: foreign cultivar, malayear, naein and taft. pod, cat, sod, apx, proline, protein, el, chlorophyll, color and visual quality were influenced by cold and freeze temperatures. with drop in temperature from 24 to 7.5°c, pod, cat, sod, and apx activities, and proline and protein content increased in all accessions; and from 7.5°c to -7.5°c significantly decreased the same parameters. the highest antioxidant enzymes activity, chlorophyll content, color and visual quality at -7.5°c were observed in taft, foreign fig. 2 dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar before cold stress (at 24/17°c day/night cycles). fig. 3 dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar aftrer cold stress at 7.5/0°c day/night cycles. akbari and salehi evaluation of common bermudagrass iranian accession under cold stress 161 cultivar, naein, malayear, aligoudarz, safashahr and gorgan accessions. peroxidase the results of peroxidase assay at 24°c showed that its maximum and minimum activity were belonged to gorgan and arak accessions, respectively (table 1). the highest peroxidase activity at 7.5°c and -7.5°c was observed in malayear accession. the maximum peroxidase activity at -15°c was observed in naein accession and the second rank was belonged to common bermudagrass accession collected from taft. catalase the results of catalase assay at 24°c showed that fig. 4 dendrogram of the physiological relationships between 50 accessions of common bermudagrass and control cultivar after cold stress at -7.5/-12°c day/night cycles. fig. 5 dendrogram of the physiological relationships between 7 cold-tolerant accessions of common bermudagrass after cold stress at -15/-15 °c day/night cycles. 162 adv. hort. sci., 2018 32(2): 157-168 its maximum activity was belonged to ghidar and nour accessions, and its minimum activity was belonged to abarkouh accession (table 1). the highest catalase activity at 7.5°c and -7.5°c was observed in doroud daneshjo park and taft accessions, respectively (tables 2 and 3). the maximum catalase activity at -15°c was observed in taft accession and the second rank was belonged to common bermudagrass accession collected from malayear (table 4). superoxide dismutase the results of sod assay showed that there was difference between sod activities of different accessions in all temperature regimes. the maximum and minimum superoxide dismutase activity at 24°c was belonged to doroud nahalestan and mamoulan turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. table 1 amount of pod, cat, sod, apx, chl, proline, el, protein, color and visual quality of different iranian accessions and foreign common bermudagrass before cold stress (at 24/17°c day/night cycles) location of accession pod (u mg−1 f.w.) cat (u mg−1 f.w.) sod (u mg−1 f.w.) apx u mg−1 f.w.) chl proline (µmol g−1 f.w.) el (%) protein (mg g−1 f.w.) color vq doroud nahalestan 13.26 36.11 120.50 81.25 4.06 13.20 11.90 15.20 7.2 7.1 abarkouh 13.17 34.12 119.10 79.12 3.68 13.10 13.40 14.30 6.3 6.5 abidar sanandaj 12.45 35.62 118.60 59.85 3.95 13.20 11.80 14.60 7.1 7.2 aligoudarz 13.54 36.11 119.50 79.56 4.02 13.40 11.30 14.80 7.0 7.1 anzali 13.36 34.25 116.40 69.55 3.78 12.80 12.60 14.90 6.2 7.3 arak 12.11 35.25 119.70 75.25 3.92 13.10 12.70 14.90 7.0 7.1 ardakan 1 12.26 36.11 116.80 72.12 3.97 12.70 12.30 14.30 7.1 7.2 ardakan 2 13.21 35.17 117.50 81.21 4.02 13.10 12.50 14.80 7.0 7.0 azna 13.12 36.24 118.60 80.12 3.96 13.50 11.30 14.90 7.6 8.1 boroujerd 12.25 35.24 118.50 59.21 4.36 12.10 10.50 14.30 8.5 9.0 chenaran 12.18 35.14 118.90 78.56 4.35 13.40 11.10 14.30 8.4 8.6 damghan 12.17 36.24 117.50 77.56 3.81 12.50 13.70 13.80 6.0 6.2 daran 13.18 35.59 117.30 74.21 4.09 12.90 11.80 13.50 7.0 7.2 dehgolan 13.11 34.35 117.90 74.32 3.89 13.10 11.60 14.50 7.6 7.2 doroud babahour 13.14 36.12 116.50 79.65 3.94 13.40 11.20 15.20 7.8 8.0 doroud daneshjo park 13.21 35.59 117.40 84.21 4.41 13.50 10.80 15.30 8.2 8.5 doroud siahvel 13.54 36.15 114.50 85.17 4.51 13.30 10.40 15.10 8.5 9.0 firouzan 12.45 35.14 118.50 77.89 3.89 13.20 11.30 14.30 7.0 7.0 foreign cultivar 13.55 36.25 119.50 79.81 4.47 13.10 11.20 15.10 8.7 8.5 ghidar 13.31 36.28 115.80 67.18 3.85 12.70 12.30 13.80 7.7 7.5 gorgan 13.65 35.17 116.50 77.21 4.38 13.10 10.60 13.90 8.2 8.3 holailan 12.58 35.18 115.50 58.52 4.12 12.40 12.50 14.50 7.2 7.4 homail 13.26 35.26 117.80 68.74 3.96 12.90 12.40 14.70 7.6 7.4 islamabad gharb 13.14 34.16 118.60 69.52 3.76 13.10 13.40 15.30 6.1 6.2 kamiaran 13.89 34.23 117.60 75.12 4.02 13.40 11.50 14.50 7.2 7.3 khorram abad 13.23 35.35 115.40 74.25 4.39 12.50 11.90 14.70 8.1 8.4 mahidasht 13.45 36.21 118.30 72.32 4.32 13.50 10.60 15.10 8.6 9.0 maiami 12.54 35.25 118.20 68.25 3.98 12.90 11.60 13.90 7.4 7.5 malayear 13.12 36.12 119.40 63.14 4.18 13.20 11.10 15.20 7.3 8.0 malayear intersection 13.24 36.25 118.60 81.32 4.32 13.40 11.20 14.50 8.0 8.2 mamoulan 13.14 35.21 113.70 81.23 3.92 12.30 12.40 14.60 7.4 7.2 mashhad 13.11 36.15 119.20 69.65 4.32 13.10 10.80 14.40 8.1 8.2 minodasht 13.14 36.15 117.70 69.54 4.06 13.20 10.90 13.80 7.2 8.4 naein 13.85 35.16 115.30 75.16 4.06 13.20 11.20 14.20 7.3 7.1 nahavand 13.55 36.12 116.50 75.14 4.07 13.10 11.70 15.10 7.6 7.4 nour 12.51 36.28 115.30 65.23 4.41 11.90 13.10 14.80 8.0 7.6 poldokhtar 12.57 35.18 119.40 69.85 4.37 12.10 11.70 14.80 8.0 8.2 safashahr 13.35 35.12 118.70 71.21 4.05 13.10 12.20 15.10 7.4 7.5 sarein 13.15 35.15 119.20 81.25 4.81 13.20 10.90 15.30 9.3 8.4 sari 13.12 35.21 117.50 78.54 3.96 12.50 11.20 14.60 7.8 8.2 saymareh bridge 13.21 35.62 117.50 64.21 4.09 12.60 13.10 14.60 7.0 6.7 semnan 13.32 35.27 116.50 76.5 3.35 12.40 14.70 13.80 5.3 4.2 shiraz 12.56 35.52 119.10 70.25 3.89 12.80 12.50 14.80 7.1 7.2 tabriz 12.74 34.18 120.10 75.65 4.38 12.90 11.10 14.60 8.2 7.6 taft 13.54 36.18 118.60 82.35 4.49 13.20 11.20 14.90 8.3 8.1 tagh bostan kermanshah 12.46 35.23 117.60 68.65 3.81 12.80 13.10 14.10 6.2 5.7 talesh 12.18 36.14 117.50 74.32 4.85 13.50 10.80 14.70 9.0 8.1 tehran 13.21 34.56 118.80 69.95 3.89 12.50 13.80 14.10 6.5 6.2 yazd 12.96 35.11 116.40 81.24 3.95 12.80 12.60 14.50 7.2 7.5 zanjan 12.25 35.62 117.20 81.25 3.96 13.40 11.40 15.10 7.5 8.0 lsd (5%) 0.458 0.264 1.860 0.650 0.508 0.320 1.520 0.458 1.452 1.286 163 akbari and salehi evaluation of common bermudagrass iranian accession under cold stress accessions, respectively. the highest superoxide dismutase activity at 7.5°c and -7.5°c was observed in doroud daneshjo park accession (tables 2 and 3). the maximum superoxide dismutase activity at -15°c was observed in taft accession and the second rank was belonged to common bermudagrass accession collected from naein (table 4). ascorbate peroxidase the results of ascorbate peroxidase assay showed that there was difference between ascorbate peroxidase activity of different accessions in all temperature regimes. the maximum and minimum ascorbate peroxidase activity at 24°c was belonged to doroud siahvel and holailan accessions, respectively. the turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. table 2 amount of pod, cat, sod, apx, chl, proline, el, protein, color and visual quality of different iranian accessions and foreign common bermudagrass after cold stress (at 7.5/0°c day/night cycles) location of accession pod (u mg−1 f.w.) cat (u mg−1 f.w.) sod (u mg−1 f.w.) apx (u mg−1 f.w.) chl (mg g−1 f.w.) proline (µmol g−1 f.w.) el (%) protein (mg g−1 f.w.) color vq doroud nahalestan 20.90 40.86 143.0 125.00 2.12 19.6 59.6 19.1 4.8 4.9 abarkouh 21.73 42.29 136.0 108.57 2.28 21.9 57.8 21.8 5.7 5.6 abidar sanandaj 25.64 40.86 132.5 59.29 2.17 20.8 59.3 19.0 5.0 5.1 aligoudarz 22.18 42.75 138.5 148.57 3.11 26.4 52.5 21.2 7.0 7.1 anzali 26.39 41.02 147.5 91.43 2.28 21.6 58.2 18.7 5.5 5.6 arak 23.38 41.17 128.5 249.29 2.31 22.2 56.4 18.4 5.7 5.9 ardakan 1 22.86 41.12 132.5 105.15 2.27 19.8 57.5 18.5 5.1 5.2 ardakan 2 22.78 40.96 136.0 185.71 2.31 21.7 56.8 19.2 5.3 5.3 azna 23.38 42.50 144.0 224.29 2.41 25.3 56.2 20.3 6.0 6.2 boroujerd 15.19 41.48 140.0 91.43 2.24 21.3 57.4 19.8 5.2 5.3 chenaran 24.36 43.16 136.0 156.43 2.31 21.9 57.5 18.9 5.8 5.9 damghan 18.20 41.48 135.5 137.14 2.21 22.3 58.1 19.2 5.1 5.2 daran 22.78 43.46 139.7 427.14 2.41 27.3 55.1 18.6 6.0 6.2 dehgolan 23.53 44.69 137.0 42.14 2.34 21.9 58.2 18.6 5.7 5.9 doroud babahour 15.94 40.71 132.0 81.43 2.29 21.9 58.2 19.7 5.7 5.6 doroud daneshjo park 17.22 44.94 151.0 245.00 2.41 21.6 54.9 20.2 5.9 6.1 doroud siahvel 17.82 41.12 137.8 127.86 2.31 21.5 57.5 19.8 5.7 5.8 firouzan 24.36 43.52 132.5 135.69 2.36 22.4 56.8 19.1 5.7 5.8 foreign cultivar 22.11 42.24 137.5 122.86 3.28 27.9 51.1 21.5 7.5 7.5 ghidar 20.08 42.44 138.5 132.71 2.26 21.5 58.5 18.7 5.4 5.5 gorgan 24.14 41.99 129.5 135.00 2.35 27.3 56.5 18.8 6.4 6.5 holailan 26.92 42.35 145.5 75.24 2.31 21.9 56.4 18.7 5.8 5.9 homail 19.40 42.95 142.0 170.00 2.19 19.7 57.2 18.5 5.0 5.2 islamabad gharb 21.82 41.85 141.4 120.24 2.15 19.5 57.5 18.8 5.0 5.1 kamiaran 26.62 43.21 139.0 120.00 2.31 21.7 57.4 19.4 5.6 5.5 khorram abad 24.59 42.55 130.0 85.71 2.35 21.8 57.5 18.7 5.7 5.8 mahidasht 23.31 44.08 135.5 189.29 2.37 24.9 54.5 18.9 6.1 6.2 maiami 22.41 41.37 136.0 132.72 2.35 23.2 55.5 18.5 6.0 6.1 malayear 34.14 41.78 138.0 134.29 3.21 27.2 51.5 21.5 7.4 7.4 malayear intersection 19.55 41.83 146.0 57.14 2.33 22.4 56.5 19.3 5.8 5.9 mamoulan 22.78 43.98 139.5 191.43 2.39 24.3 55.5 19.1 6.0 6.1 mashhad 22.56 42.95 136.0 62.86 2.28 21.5 57.2 19.1 5.7 5.8 minodasht 20.38 42.09 147.0 140.00 2.25 21.5 57.9 19.1 5.2 5.4 naein 23.08 42.29 139.0 191.43 3.26 27.5 51.2 18.7 7.5 7.6 nahavand 24.74 41.22 135.5 110.71 2.26 21.8 57.2 20.8 5.5 5.4 nour 24.66 42.90 133.5 89.14 2.21 20.7 57.1 18.6 5.0 5.3 poldokhtar 22.33 40.81 139.2 121.43 2.37 22.5 54.5 18.6 6.0 6.2 safashahr 21.95 41.53 132.5 212.86 2.38 27.1 53.5 21.3 6.4 6.3 sarein 23.91 43.31 144.0 140.71 2.31 22.6 56.1 20.5 5.5 5.8 sari 21.73 43.06 129.5 71.43 2.32 21.8 57.2 18.5 5.8 5.7 saymareh bridge 25.86 40.96 129.5 75.00 2.34 19.8 57.8 19.4 5.7 5.1 semnan 23.98 42.18 128.6 161.43 1.86 17.8 59.5 19.5 4.1 4.2 shiraz 23.08 41.42 137.3 115.29 2.15 19.8 57.8 20.1 5.0 5.2 tabriz 25.41 41.22 136.5 142.14 2.15 19.8 59.4 18.4 4.9 4.9 taft 25.26 42.80 142.4 232.14 3.36 27.2 50.5 22.1 7.7 7.7 tagh bostan kermanshah 21.23 41.46 134.5 81.74 2.14 26.4 59.8 17.1 5.0 4.9 talesh 21.34 41.36 135.5 102.20 1.15 18.9 62.4 14.9 3.3 3.4 tehran 19.85 41.27 143.5 140.71 2.24 21.4 57.3 18.7 5.2 5.2 yazd 25.94 42.65 126.0 184.29 2.11 21.7 58.5 17.4 4.9 4.8 zanjan 23.76 42.80 145.5 101.43 2.34 26.1 56.1 19.1 6.0 5.9 lsd (5%) 1.014 1.382 7.674 0.458 0.512 0.7279 4.086 1.825 0.988 1.027 164 adv. hort. sci., 2018 32(2): 157-168 highest ascorbate peroxidase activity at 7.5°c and 7.5°c was observed in daran accession (tables 2 and 3). the maximum ascorbate peroxidase at -15°c was observed in taft accession and the second rank was belonged to common bermudagrass accession collected from naein (table 4). total protein the results presented in tables 2, 3 and 4 revealed that there was difference between total protein of different accessions in all temperature regimes. the maximum total protein at 24°c was belonged to doroud daneshjo park, sarein and table 3 amount of pod, cat, sod, apx, chl, proline, el, protein, color and visual quality of different iranian accessions and foreign common bermudagrass after cold stress (at -7.5/-12°c day/night cycles) location of accession pod (u mg−1 f.w.) cat (u mg−1 f.w.) sod (u mg−1 f.w.) apx (u mg−1 f.w.) chl (mg g−1 f.w.) proline (µmol g−1 f.w.) el (%) protein (mg g−1 f.w.) color vq doroud nahalestan 20.90 40.86 143.0 125.00 2.12 19.6 59.6 19.1 4.8 4.9 abarkouh 21.73 42.29 136.0 108.57 2.28 21.9 57.8 21.8 5.7 5.6 abidar sanandaj 25.64 40.86 132.5 59.29 2.17 20.8 59.3 19.0 5.0 5.1 aligoudarz 22.18 42.75 138.5 148.57 3.11 26.4 52.5 21.2 7.0 7.1 anzali 26.39 41.02 147.5 91.43 2.28 21.6 58.2 18.7 5.5 5.6 arak 23.38 41.17 128.5 249.29 2.31 22.2 56.4 18.4 5.7 5.9 ardakan 1 22.86 41.12 132.5 105.15 2.27 19.8 57.5 18.5 5.1 5.2 ardakan 2 22.78 40.96 136.0 185.71 2.31 21.7 56.8 19.2 5.3 5.3 azna 23.38 42.50 144.0 224.29 2.41 25.3 56.2 20.3 6.0 6.2 boroujerd 15.19 41.48 140.0 91.43 2.24 21.3 57.4 19.8 5.2 5.3 chenaran 24.36 43.16 136.0 156.43 2.31 21.9 57.5 18.9 5.8 5.9 damghan 18.20 41.48 135.5 137.14 2.21 22.3 58.1 19.2 5.1 5.2 daran 22.78 43.46 139.7 427.14 2.41 27.3 55.1 18.6 6.0 6.2 dehgolan 23.53 44.69 137.0 42.14 2.34 21.9 58.2 18.6 5.7 5.9 doroud babahour 15.94 40.71 132.0 81.43 2.29 21.9 58.2 19.7 5.7 5.6 doroud daneshjo park 17.22 44.94 151.0 245.00 2.41 21.6 54.9 20.2 5.9 6.1 doroud siahvel 17.82 41.12 137.8 127.86 2.31 21.5 57.5 19.8 5.7 5.8 firouzan 24.36 43.52 132.5 135.69 2.36 22.4 56.8 19.1 5.7 5.8 foreign cultivar 22.11 42.24 137.5 122.86 3.28 27.9 51.1 21.5 7.5 7.5 ghidar 20.08 42.44 138.5 132.71 2.26 21.5 58.5 18.7 5.4 5.5 gorgan 24.14 41.99 129.5 135.00 2.35 27.3 56.5 18.8 6.4 6.5 holailan 26.92 42.35 145.5 75.24 2.31 21.9 56.4 18.7 5.8 5.9 homail 19.40 42.95 142.0 170.00 2.19 19.7 57.2 18.5 5.0 5.2 islamabad gharb 21.82 41.85 141.4 120.24 2.15 19.5 57.5 18.8 5.0 5.1 kamiaran 26.62 43.21 139.0 120.00 2.31 21.7 57.4 19.4 5.6 5.5 khorram abad 24.59 42.55 130.0 85.71 2.35 21.8 57.5 18.7 5.7 5.8 mahidasht 23.31 44.08 135.5 189.29 2.37 24.9 54.5 18.9 6.1 6.2 maiami 22.41 41.37 136.0 132.72 2.35 23.2 55.5 18.5 6.0 6.1 malayear 34.14 41.78 138.0 134.29 3.21 27.2 51.5 21.5 7.4 7.4 malayear intersection 19.55 41.83 146.0 57.14 2.33 22.4 56.5 19.3 5.8 5.9 mamoulan 22.78 43.98 139.5 191.43 2.39 24.3 55.5 19.1 6.0 6.1 mashhad 22.56 42.95 136.0 62.86 2.28 21.5 57.2 19.1 5.7 5.8 minodasht 20.38 42.09 147.0 140.00 2.25 21.5 57.9 19.1 5.2 5.4 naein 23.08 42.29 139.0 191.43 3.26 27.5 51.2 18.7 7.5 7.6 nahavand 24.74 41.22 135.5 110.71 2.26 21.8 57.2 20.8 5.5 5.4 nour 24.66 42.90 133.5 89.14 2.21 20.7 57.1 18.6 5.0 5.3 poldokhtar 22.33 40.81 139.2 121.43 2.37 22.5 54.5 18.6 6.0 6.2 safashahr 21.95 41.53 132.5 212.86 2.38 27.1 53.5 21.3 6.4 6.3 sarein 23.91 43.31 144.0 140.71 2.31 22.6 56.1 20.5 5.5 5.8 sari 21.73 43.06 129.5 71.43 2.32 21.8 57.2 18.5 5.8 5.7 saymareh bridge 25.86 40.96 129.5 75.00 2.34 19.8 57.8 19.4 5.7 5.1 semnan 23.98 42.18 128.6 161.43 1.86 17.8 59.5 19.5 4.1 4.2 shiraz 23.08 41.42 137.3 115.29 2.15 19.8 57.8 20.1 5.0 5.2 tabriz 25.41 41.22 136.5 142.14 2.15 19.8 59.4 18.4 4.9 4.9 taft 25.26 42.80 142.4 232.14 3.36 27.2 50.5 22.1 7.7 7.7 tagh bostan kermanshah 21.23 41.46 134.5 81.74 2.14 26.4 59.8 17.1 5.0 4.9 talesh 21.34 41.36 135.5 102.20 1.15 18.9 62.4 14.9 3.3 3.4 tehran 19.85 41.27 143.5 140.71 2.24 21.4 57.3 18.7 5.2 5.2 yazd 25.94 42.65 126.0 184.29 2.11 21.7 58.5 17.4 4.9 4.8 zanjan 23.76 42.80 145.5 101.43 2.34 26.1 56.1 19.1 6.0 5.9 lsd (5%) 1.014 1.382 7.674 0.458 0.512 0.7279 4.086 1.825 0.988 1.027 turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. 165 akbari and salehi evaluation of common bermudagrass iranian accession under cold stress islamabad gharb accessions, and minimum total protein was belonged to daran accession. the highest total protein at 7.5°c and -7.5°c was observed in taft accession (tables 2 and 3). the maximum total protein at -15°c was also observed in taft accession and the second rank was belonged to common bermudagrass accession collected from malayear (table 4). proline our results revealed that there was difference between proline content of different accessions in all temperature regimes. the maximum proline content at 24°c was belonged to doroud daneshjo park, azna, mahidasht and talesh accessions, and the minimum proline content was belonged to nour accession. the highest proline content at 7.5°c and -7.5°c was observed in foreign cultivar and taft accession, respectively (tables 2 and 3). the maximum proline content at -15°c was observed in naein accession and the second rank belonged to common bermudagrass accession collected from taft (table 4). electrolyte leakage as shown in tables 1, 2, 3 and 4, drop in temperature severely increased el in all accessions. the highest el at 7.5°c and -7.5°c was observed in talesh and abidar accessions, respectively, and the least was seen in taft accession (table 2 and 3). the minimum el at -15°c was observed in taft accession and the second rank was belonged to common bermudagrass accession collected from naein (table 4). chlorophyll content, color and visual quality the highest chlorophyll content, color and visual quality were observed at 24°c and fall in temperature under 24°c severely decreased these characters in all accessions (tables 1, 2, 3 and 4). the highest chlorophyll content, color and visual quality at -7.5°c were observed in taft, foreign cultivar, naein, malayear, aligoudarz, safashahr and gorgan (table 3). the maximum chlorophyll content, color and visual quality at -15°c were observed in taft, naein and malayear accessions. 4. discussion and conclusions the 50 c. dactylon accessions were clustered into two major groups by ward’s method on the basis of physiological characters at all temperature regimes. accessions with high pod, cat, sod, apx, proline, protein, chlorophyll, color and visual quality, fall in same group. no complete relationships were found between the clustering of the common bermudagrass accessions in dendrograms based on physiological characters and their geographical affiliations. these patterns of physiological variations within common bermudagrass accessions might be due to different genetic background because of various ploidy levels, cross pollination, genetic overlap, germplasm exchange and gene flow. seven accessions collected from taft, naein, malayear, foreign cultivar, aligoudarz, safashahr and gorgan were the most cold-tolerant genotypes. because, the highest antioxidant enzymes activity, chlorophyll content, color and visual quality at -7.5°c were observed in these accessions. iran has a variable climate and we collected these species from different climatic regions of the country that shows its adaptation to wide ranges of climates. the results obtained from our physiological analysis demonstrated that the level of variation was great among iranian c. dactylon accessions. the best color and visual quality in all common bermudagrass accessions were observed before cold stress. reducing the temperature below 24°c severely decreased chlorophyll content, visual quality and color in all accessions. the results reporttable 4 amount of pod, cat, sod,apx, chl, proline, el, protein, color and visual quality of the most cold-tolerant accessions of bermudagrass from iran after cold stress (at -15/-15°c day/night cycles) in each column, means with the same letter are not significantly different according to least significant difference (lsd) test at p= 0.05. turfgrass visual quality and color based on a scale of 1-9, 1= brown/dead turf, 6= minimal acceptable turf, 9= ideal green, healthy turf. location of accession pod (u mg−1 f.w.) cat (u mg−1 f.w.) sod (u mg−1 f.w.) apx (u mg−1 f.w.) chl (mg g−1 f.w.) proline (µmol g−1 f.w.) el (%) protein (mg g−1 f.w.) color vq taft 10.12 a 31.35 a 96.00 a 71.45 c 1.85 a 16.350 a 78.1 d 14.20 a 6.20 a 6.30 a naein 10.81 a 26.25 bc 93.86 a 85.39 a 1.74 a 16.41 a 82.3 c 13.70 a 6.10 a 6.00 ab malayear 9.55 a 27.28 b 87.08 ab 74.65 c 1.36 e 14.21 ab 87.5 ab 13.90 a 5.36 b 5.30 ab foreign cultivar 11.16 a 24.24 cd 76.18 bc 81.26 b 1.24 bc 13.82 ab 85.6 bc 12.80 a 5.10 b 5.10 bc aligoudarz 10.13 a 23.36 d 71.22 c 67.25 d 0.94 cd 11.87 b 89.4 ab 11.85 a 4.30 c 4.30 c gorgan 8.21 a 23.57 d 74.39 c 52.39 f 0.89 cd 11.68 b 91.2 a 12.10 a 4.20 c 4.50 cd safashahr 9.17 a 22.11 d 69.00 c 57.48 e 0.75 d 11.89 b 90.8 a 12.80 a 4.10 c 4.10 d adv. hort. sci., 2018 32(2): 157-168 166 ed by esmaili and salehi (2012) correspond to the results we obtained. the best temperature for growth and development of tropical grasses is ranging from 27 to 35°c (beard, 1973). in warm-season turfgrasses, if temperature drops to 10-12.8°c their growth decrease considerably and enters dormancy at close to 0°c (christians, 2004). mccarty (2001) suggested that a sudden air temperature decline to -5°c or a less rapid fall to below -12°c can cause damage to tropical turfgrasses. photosynthetic apparatus directly affected by cold stress. in our study, the highest pod, cat, sod and apx activity were observed at 7.5°c. as temperature diminished from 7.5 to -15°c, antioxidant enzymes activity decreased. this is in agreement with manuchehri et al . (2014) for cynodon dactylon (california origin). the study made by zhang et al. (2006) on common bermudagrass ‘riviera’ and ‘princess-77’ showed an increase in the sod activity during the first seven days of cold acclimation and then after a decline was observed. coldacclimatized plants can tolerate freezing stress better than non-acclimated ones due to rapid development of metabolic defenses against freezing stress (zhang et al., 2006). overall, cold stress produces large amounts of ros which causes oxidative injury to plants through vast destruction of proteins, carbohydrates, lipids, cellular membranes, dna and major decline of atp reserve, and finally cell death (dionne et al., 2001; gill and tuteja, 2010). plants protect their cells from ros damage by raising the activity of antioxidant enzymes like apx, sod, cat and pod (apel and hirt, 2004). results of many studies indicate a positive correlation between freezing tolerance in bermudagrasses and antioxidant enzymes activity (zhang et al., 2006, manuchehri et al., 2014). over-production of ros during chilling periods increase oxidative stress and can enhance the activity of antioxidant enzymes and stimulate synthesis of antioxidant metabolites (karpinski et al., 2002). our results regarding an increase in pod, sod, cat and apx activity found in this study is assumed to defense mechanism against oxidative damage caused by cold stress. the central role in the antioxidant defense system is perform by sod via catalyzing o2 ·into h2o2 and o2, while other antioxidant enzymes are also essential for breakdown of h2o2 through various pathways (mittler, 2002; apel and hirt, 2004). cat can produce o2 from h2o2. apx play a key role in ros detoxification by conversion of h2o2 in to h2o. the balance between ros production and elimination is vital mean for the protection of plant cells, and apx plays a major role in maintaining this balance (asada, 1992; lin et al., 2004). karpinski et al. (2002) showed that tolerant plants have higher antioxidant enzyme activity that has dilatory effect on photooxidative injury during cold stress periods. it is postulated that under cold stress condition, cold tolerant plants increase the activity of their antioxidant enzymes that support active photosynthesis and development of carbohydrate reservation and other compounds such as protein and proline with protective functions. our findings revealed that higher antioxidant enzymes activity can be attributed to better cold tolerance in common bermudagrass. decreasing the temperature increased el, proline and protein content. this negative correlation between temperature and el can be due to cell membrane damage caused by cold temperatures. the el method is commonly used to quantify the degree of cell membrane damage induced by cold temperatures and to assay the cold stress tolerance of turfgrasses (shashikumar and nus, 1993; anderson and taliaferro, 2002). our results indicated that leaf el increased during cold acclimatization. cold acclimatization may induce ross production and oxidative stress, and may cause slight damage to cell membrane and later will cause an increase in el. the production of free radicals under cold stress conditions may initiate the signaling pathways of plant metabolic defense responses, which may reduce cell membrane disruption and el (zhang and ervin, 2008). one of the most important differences between cold tolerant and cold sensitive accessions may be greater development of defense responses to scavenging ross and lowering the el in cold tolerant accessions. cold acclimation increase proline content in all accessions, but we observed much higher proline content in cold tolerant accessions. this results for proline content in our research, is in accordance with the results presented by munshaw et al. (2004), where they reported a significant increase of stolon proline concentration of the common bermudagrass ‘princess-77’ during cold acclimation period. other supporting results include those of munshaw et al. (2006) and zhang et al. (2006), where they found that higher proline content in bermudagrass cultivars during the winter can be related to greater freezing tolerance. many studies have pointed to the cryoprotectiveness of proline and their function as osmolytes or compatible solutes (koster and leopold, 1988; santarius, 1992; karpinski et al., 2002). proline is a cryoprotectant for chloroplast membranes of spinach (spinacia oleracea l.) and plays a crucial role in plant protection against freezing stress (santarius, 1992). proline contributes akbari and salehi evaluation of common bermudagrass iranian accession under cold stress 167 to the acclimatization of plants to cold stress by increasing osmotic potential (reducing the water potential) and decreasing the freezing point of cells. protein content of all accessions was high during cold acclimation. higher protein content was observed in cold-tolerant accessions compared to cold-sensitive ones. one of the studies that confirm our results also, is a study by zhang et al. (2006), with their study on c. dactylon ‘riviera’ as a cold tolerant cultivar with higher protein content, and ‘princess-77’ as a cold sensitive cultivar with lower protein content following acclimatization. during cold acclimation plants increase their capacity for synthesis of novel proteins (cloutier, 1983). this increase in protein content can be attributed to their determinant role in freezing tolerance of common bermudagrass. our results showed rapid physiological alterations in common bermudagrass accessions in response to cold stress. our results also revealed that cold-tolerant common bermudagrass accessions showed higher proline, protein, antioxidant enzymes, color, visual quality and chlorophyll content, and cold-sensitive accessions showed more severe cell membrane damage (el) under cold stress conditions. we identified drastic natural variations in tolerance between common bermudagrass accessions of iran in response to freezing stress. comparative study between 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arabidopsis homeodomain transcription factor gene, hos9, mediates cold tolerance through a cbf-independent pathway. proc. nation. acad. sci., 101: 98739878. impaginato 451 adv. hort. sci., 2018 32(4): 451-458 doi: 10.13128/ahs-22456 yield, fruit quality and physiological responses of melon cv. khatooni under deficit irrigation t. barzegar (*), n. heidaryan, h. lotfi, z. ghahremani department of horticulture science, faculty of agriculture, university of zanjan, zanjan, iran. key words: antioxidant enzyme, irrigation, melon, proline, water use efficiency. abstract: to evaluate the effect of water deficit stress on growth, yield, fruit quality and physiological traits of melon cv. khatooni, field experiments were conducted in split plot randomized complete block design with three replications. in 2014, irrigation treatments consisted of two deficit irrigation regimes, 33% and 66% of etc (crop evapotranspiration), and 100% etc as the control (di33, di66 and i100). in 2015, irrigation treatments applied were: 40, 70 and 100% etc (di40, di70 and i100). the results showed that plant height and leaf area decreased from treatment i100 to di40 and di33. the highest average fruit weigh and yield were obtained from irrigation 100% etc for both years. the water use efficiency (wue) significantly increased in response to increase water deficit stress. deficit irrigation treatments significantly decreased leaf relative water content, vitamin c and fruit firmness, whereas antioxidant enzymes activity, proline and total soluble solid contents increased. these results suggest that the crop is sensitive to water deficits, that moderate water stress (di70 and di66) reduced yield by about 28.5-38.2% and severe water stress (di40 and di33) had a much more marked effect, reducing yield by 48.1-61.4%. 1. introduction melon (cucumis melo l.) is an important horticultural crop in iran, generally cultivated in arid and semi-arid regions. iran is the third largest melon-producing country in the world with more than 1476801 tonnes (fao, 2014) of production. melon plants are highly productive under adequate irrigation conditions; however water for irrigation is not always available at the time and amount needed by the crop, so water scarcity is a major constraint to horticultural production in arid and semiarid regions (sharma et al., 2014). deficit irrigation regime, a practice that supplies water below evapotranspiration (et) demands, can optimize water productivity when full irrigation is not possible (fereres and soriano, 2007). when water supply is limited, plant growth and yield is reduced and plant structure is modified by decreasing in leaf size (kirnak et al., 2002; chaves et al., 2003). the effect of deficit irrigation on fruit yield and quality has been reported by numerous researchers with different results. in melon, deficit (*) corresponding author: tbarzegar@znu.ac.ir citation: barzegar t., heidaryan n., lofti h., ghahremani z., 2018 yield, fruit quality and physiological responses of melon cv. khatooni under deficit irrigation. adv. hort. sci., 32(4): 451-458 copyright: © 2018 barzegar t., heidaryan n., lofti h., ghahremani z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 december 2017 accepted for publication 31 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 451-458 452 irrigation reduced marketable fruit number and yield, average fruit weight, fruit diameter and did not affect rind thickness and seed cavity, but increased total soluble solids content (sharma et al., 2014). although deficit irrigation reduce crop yield, may be able to save a significant amount of irrigation water (sharma et al., 2014). fabeiro et al. (2002) stated that deficit irrigation during blooming stage affected mainly fruit yield, at setting stage both quantity and quality, and the deficit imposed at ripening stage affected sugar content. rouphael et al. (2008) indicated that water deficit significantly reduced yield, biomass production and leaf water status of mini-watermelon, but increase the water use efficiency. the soluble solids concentration (ssc) is probably the most important quality parameter that is commonly evaluated by consumers (cabello et al., 2009). water deficit studies in melon have been reported to increase (sharma et al., 2014), decrease (long et al., 2006), or had no effect (hartz, 1997) on soluble solid content. vitamin c content, as a secondary metabolite of plants, did not change with deficit irrigation in watermelons (75% etc) (leskovar et al., 2004) and melons (50% etc) (sharma et al., 2014). oxidative stress is one of the major causes of cellular damage in plants during stress (miller et al., 2010). however, plants can avoid the drought damage by promoting antioxidant enzymes activity, such as superoxide dismutase (sod), peroxidases (pod), and catalase (cat), to scavenge for free radicals and, or accumulate osmotic regulators such as soluble sugar, and proline may play a role in protection of cellular machinery against photo-oxidation by reactive oxygen species (ros) that increase the drought resistance of plants under water stress (foyer and noctor, 2005; veljovic-jovanovic et al., 2006). although the effects of water stress have been studied on growth and yield of different crops during the last years, recent information on the response of iranian melon yield and quality to deficit irrigation remains limited, particularly about the results of restricted water distributions in arid and sub-arid environments. the main goal of this study was to evaluate the effect of controlled deficit irrigation on the physiological parameters and yield of the khatooni melon cultivar. 2. materials and methods experimental site two field experiments were conducted during the growing season of 2014 and 2015 from june to september at research farm of agriculture faculty, university of zanjan (iran), to study the effect of water deficit on fruit yield and quality, antioxidant enzymes activities, water use efficiency (wue), proline and vitamin c content. the soil texture was silty loam with 7.8 ph. some soil characteristics and irrigation water chemical properties were showed in table 1 and 2. the daily climate data during the growing seasons (2014 and 2015) was shown in table 3. table 1 soil physical and chemical properties at the experiment site ph ec (ds m-1) n (%) ca (g kg-1) na (g kg-1) k (g kg-1) om (%) soil texture sand (%) silt (%) clay (%) 7.40 1.49 0.07 0.12 0.13 0.2 0.94 silt loam 25 38 37 table 2 irrigation water chemical properties at the experiment site om= organic matter. bicarbonate (mg l-1) carbonate (mg l-1) cl (mg l-1) mg (mg l-1) ca (mg l-1) k (mg l-1) na (mg l-1) ec (ds m-1) ph 195.2 0.0 582.2 103.7 258.45 0.0 50 2.35 6.5 table 3 climatic parameters during the growing seasons climatic parameters june july august sept 2014 2015 2014 2015 2014 2015 2014 2015 minimum air temperature (°c) 7.60 12.90 10.70 18.53 13.10 16.14 6.80 12.58 maximum air temperature (°c) 35.80 31.90 39.50 34.46 39.10 35.50 35.40 30.28 rainfall (mm) 7.30 0.33 17.30 1.13 0.10 0.00 4.00 2.93 relative humidity (%) 41.50 44.00 43.40 42.00 37.00 39.00 41.40 52.00 barzegar et al. deficit irrigation effects on melon quality 453 plant materials and irrigation treatments the experiment was done on a completely randomized block design whit three irrigation levels and three replications. ‘khatooni’, yellow-green netted skin color and chimeric stripes, an iranian melon from the inodorous group widely cultivated in iran, was selected for study. the seeds were sown on 1th july 2014 and 23th may 2015 at recommended spacing of 50 cm in row with 200 cm between rows. the irrigation system consisted of one drip line every crop row. fertilizers was delivered as a pre-plant base comprising 80 kg n/ha, 50 kg p/ha and 80 kg k/ha. at a very early stage, plants were pruned (removing the apex of the main stem), and trained to have two lateral branches. three irrigation levels were calculated, based on actual evapotranspiration (etc). in 2014, irrigation treatments were control or irrigation at 100% etc (i100), deficit irrigation at 66% etc (di66) and at 33% etc (di33) of control. according to 2014 results, when water deficit stress treatments strongly reduced fruit yield, in 2015 deficit irrigation treatments were changed, and the irrigation treatments were: 100% etc (i100), 70% etc (di70) and 40% etc (di40). before starting the differential irrigation at five-leaf stage, all treatments were supplied with similar amount of water to maximize stands and uniform crop establishment. all other necessary operations such as pests and weeds control were performed according to recommended package of practices during the crop growth. measurements. plant growth and leaf area after 30 days of irrigation treatments, the average of leaf area was recorded whit leaf area measurement (delta-t device ltd, england). after fruit harvest, vine length of each plant was measured. for estimate leaf dry weight, at first fresh weight of leaf was measured; then they were dried in a hot-air oven for 2 days at 72°c, after which the dry weights (%) of leaf was recorded. yield and productivity components the fruits were harvested when color changed from green to yellow and after the appearance of the netted pattern. each melon fruit was weighed to determine mean fruit weight (fw). the fruit number per plant and fruit yield per plant was measured to determine of total yield, expressed in t ha-1. fruit yield was calculated by the mean fruit weight (kg), fruit number per plant and the density (20,000 plants/ha). fruit quality immediately after harvest, flesh ratio (fr), fruit firmness (ff), total soluble solid (tss) and vitamin c (vc) were determined. the flesh ratios were calculated using the formulae: fr (%)=[(a+b)2-(a’+b’)2/(a+b)2]×100 where a is the fruit length, a’ is the seed cavity length, b is the fruit diameter and b’ is the seed cavity diameter. from the liquid extract obtained by liquefying the mesocarp of each fruit, tss content was determined by a handheld refractometer and expressed as °brix. fruit firmness was measured on the mesocarp tissue at three random locations per fruit using a digital penetrometer (mc cormic-ft 327) and recorded as kg cm-1. proline content proline content in leaf tissue was determined according to the method of bates et al. (1973). mature leaves of plant were sampled 30 days after the onset of the deficit irrigation treatments. proline was extracted from a sample of 0.5 g fresh leaves material samples in 3% (w/v) solution sulphosalycylic acid and estimated using the ninhydrin reagent. after reading the absorbance of fraction at a wave length of 520 nm, proline concentration was determined using a calibration curve and expressed as mg g-1 fw. catalase and peroxidase enzymes activity samples were taken from the fully expanded leaf and transferred to the laboratory in the ice. leaf sample (0.5 g) was frozen in liquid nitrogen and ground using a porcelain mortar and pestle. catalase (cat) activity was measured by following the decomposition of h2o2 at 240 nm with a uv spectrophotometer (havir and mchale, 1987). samples without h2o2 were used as blank. the activity of cat was calculated by the differences obtained at od240 values at 30 second interval for 2 min after the initial biochemical reaction. peroxidase (pod) activity was measured using modified method of the tuna et al. (2008) with guaiacol at 470 nm. a change of 0.01 units per minute in absorbance was considered to be equal to one unit pod activity, which was expressed as unit g-1 fw min-1. leaf relative water content the relative water content (rwc) in leaves was determined whit sampling fully expanded young leaves at noon according to yamasaki and dillenburg, (1999). leaf relative water content was calculated adv. hort. sci., 2018 32(4): 451-458 454 using the following formula: rwc -(%) =[(fw-dw)/(sw-dw)]×100 where fw stands for fresh weight, dw for dry weight, and sw for saturated weight. water use efficiency water use efficiency (wue) was calculated for all treatments based on total crop yield and amount of water applied during growth period. wue was estimated as the ratio of fruit yield (y, kg ha-1) and irrigation water applied (w, m-3) (stanhill, 1986). wue=y/w. statistical analysis all data were analyzed statistically using a oneway anova. because of differences in the treatments, the data for each year were submitted to anova separately. for data analysis, a completely randomized block design was used (3 irrigation levels × 3 replications × 10 observations per experimental unit). data were analyzed using the sas statistical program (sas institute inc., cary, nc, usa), and means were compared by duncan’s multiple range tests at the 5% and 1% probability levels. 3. results and discussion plant growth leaf area, vine length and leaf dry weight (ldw) data of the treatments were presented in table 4. leaf area significantly decreased in the water deficit stress treatments in both years, reduction 20.38% (di33) and 30.4% (di40) in 2014 and 2015, respectively. in 2014, deficit irrigation stress had no effect on ldw. on the contrary, in 2015, ldw was affected significantly by the irrigation treatments, decreasing 22.05% in i100 treatment. also, water deficit stress significantly reduced vine length in 2014, but no significant effect was observed by water deficit stress in 2015. these findings are similar the results obtained by pew and gardner (1983) and ribas et al. (2001) who found that vegetative growth was higher under full irrigation instead of limited irrigation. growth is an irreversible increase in volume, size, or weight, which includes the phases of cell division, cell elongation, and differentiation. a decrease in plant growth may be due to the limitation of cell division, cell enlargement caused by loss of turgor and inhibition of various growth metabolisms (farooq et al., 2012), and also decrease in photosynthesis (huang et al., 2011). yield, productivity components and water use efficiency fruit yield was affected significantly by the irrigation treatments in both years (table 4). the highest value of fruit yield (40.37 and 43.43 t h-1) was obtained in the irrigation 100% etc in 2014 and 2015, respectively. fruit number and fruit weight significantly reduced under deficit irrigation (table 4). the mean fruit number per plant was lower in 2014 (1.8, i100) compared to 2015 (2.7, i100). in contrast, fruit mean weight was higher in 2014 (2.18 kg) against 2015 (1.60 kg) that was obtained under irrigation 100% etc. the lowest fruit number and fruit weight (1.25 kg) was observed respectively, with irrigation 33% etc in 2014 and irrigation 40% etc in 2015. this result agrees with the findings of ribas et al. (2001), cabello et al. (2009) and sharma et al. (2014), who reported that limited irrigation reduced fruit yield of melon. table 4 effect of deficit irrigation on average leaf area (la), vine length (vl), fruit weight (fw), number of fruits per vine (fn), yield (y), and water use efficiency (wue) in 2014 and 2015 seasons i33, i40, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) la (cm2) ldw (%) vl (cm) fn fw (kg) y (t ha-1) wue (kg m-3) 2014 100 151.53 a 16.24 a 185.6 a 1.8 a 2.18 a 40.37 a 14.14 ab 66 130.11 b 16.45 a 133.3 ab 1.3 ab 1.91 ab 24.94 b 15.11 ab 33 120.64 b 17.23 a 116.33 b 1.1 b 1.36 b 15.55 c 17.65 a 2015 100 183.74 a 16.4 b 148.33 a 2.7 a 1.60 a 43.43 a 14.24 b 70 151.72 b 19.59 a 138.33 a 2.2 ab 1.42 ab 31.03 b 14.53 b 40 127.88 b 21.04 a 115.5 a 1.8 b 1.25 b 22.50 c 18.45 a barzegar et al. deficit irrigation effects on melon quality 455 the reduction in fruit yield under deficit irrigation treatments compare to i100 treatment can be explained by the decrease in both mean fruit weight and numbers of fruits per vine (table 4). cabello et al. (2009) and sharma et al. (2014) also reported the reduction in fruit number and fruit weight under deficit irrigation. previous studies indicated that fruit weight in melon is more sensitive to water stress than fruit number (long et al., 2006; dogan et al., 2008). figure 1 presents the correlation between irrigation and fruit yield, fruit weight and fruit number per vine. correlation between irrigation and fruit yield (r2= 0.93) was stronger than the correlation with fruit weight (r2= 0.58) and fruit number per vine (r2= 0.51) which indicates that the reduction in fruit yield with deficit irrigation was attributed to the significant decrease in average fruit weight and fruit number per vine (fig. 1 b and c). wue is the relation between yield and the quantity of irrigation water (zeng et al., 2009). in both years, wue was lowest for irrigation 100% etc. overall; deficit irrigation resulted in 19.88% and 22.81% wue increased in di33 and di40, respectively (table 4). wue had negative correlation (r2 = 0.64) with irrigation water amount (fig. 2). higher wue has also been achieved in watermelon (leskovar et al., 2004), muskmelon (kirnak et al., 2005; zeng et al., 2009), mission and da vinci melon cultivars (sharma et al., 2014) in response to deficit irrigation. fruit quality fruit quality as indicated with fruit firmness, flesh ratio, total soluble solid (tss) and vitamin c was presented in table 5. in both years, fruit firmness decreased as the irrigation was restricted. the lowest fruit firmness was 1.49 kg cm-1 under irrigation 33% etc, although there was no significant difference fig. 1 relationship between irrigation by fruit yield (a), fruit weight (b) and fruit number per plant (c) in 2014 and 2015. values are the mean of 3 replications/10 observations each irrigation level, in two years. fig. 2 relationship between irrigation by water use efficiency (wue) in 2014 and 2015. values are the mean of 3 replications/10 observations each irrigation level, in two years. table 5 effect of deficit irrigation on fruit firmness (ff), flesh ratio (fr), total soluble solid (tss) and vitamin c (vc) in 2014 and 2015 seasons i33, i440, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) ff (kg cm-1) fr (%) tss (°brix) vc (mg 100 ml-1) 2014 100 2.38 a 49.55 a 10.06 b 10.002 a 66 1.79 ab 49.53 a 11 ab 8.082 b 33 1.49 b 48.29 a 12.06 a 6.98 c 2015 100 3.15 a 54.07 a 9.03 b 10.68 a 70 3.00 a 49.04 ab 10.7 ab 9.21 b 40 2.1 b 45.77 b 11.76 a 7.88 c 456 adv. hort. sci., 2018 32(4): 451-458 between i100 whit di66 and di70 in 2014 and 2015, respectively. these results was agreement with cabello et al. (2009) in melon, who reported that increasing irrigation water improved flesh firmness, but obtained a reduction in flesh firmness when irrigation water increased in following year. also, sharma et al. (2014) did not obtain a significant difference of irrigation treatments on fruit firmness with approximately positive effect of optimal irrigation. the flesh ratio was unaffected by the irrigation rates in 2014 season. however, flesh ratio varied significantly in 2015. the largest flesh ratio (54.07%) was obtained under irrigation 100% etc in 2015. these results are in agreement with the results of dogan et al. (2008) in melon. the results indicated that optimal irrigation water could increase flesh thickness while water stress has a negative effect on it. it is not in accordance with ribas et al. (2003) who reported that the flesh and skin ratios are not usually affected by the irrigation levels. tss is a very important index of quality in melon fruits (zeng et al., 2009). in both years, larger amounts of irrigation water resulted in lower tss. the highest tss was recorded with 12.06 and 11.76 °brix in irrigation 33 and 40% etc, respectively. the similar results were also observed by some other researchers (lester et al., 1994; fabeiro et al., 2002). dogan et al. (2008) showed that fruit sugar content affected positively by water stress. furthermore, other studies have shown that in muskmelon, tss decreased with the decrease in irrigation water levels (long et al., 2006; zeng et al., 2009; li et al., 2012). gonzalez et al. (2009) found no significant differences for watermelon fruit soluble solids between well-watered and regulated deficit irrigation treatments, although it was 9.5% higher, for the regulated deficit irrigation treatment. deficit irrigation markedly (p<0.05) reduced vitamin c content. the highest value of vitamin c was found in treatments i100 in both years (table 5), which high decrease value (30.21%) was recorded in irrigation 33% etc. the results indicated that vitamin c content was highly sensitive to deficit irrigation. our results are agreement with li et al. (2012) and wang et al. (2017) who showed that severe water deficit stress reduced significantly the fruit vitamin c content, but these results differ from the findings of cui et al. (2008) who stated that water deficit during the fruit growth and maturation stages increased significantly vitamin c content. proline accumulation the exposure to water deficit stress significantly (p<0.05) increased proline content (table 6). the maximum value of proline content was 1.97 and 1.8 mg g-1 fw under irrigation 33 and 40% etc, respectively. accumulation of proline plays an important role in plants to adaptive on environmental stresses, particularly low water stress (kavas et al., 2013). the proline that accumulated in the leaves under waterlimited environment is a cellular regulator that helping to sustain the activity of the cell and tissue in water deficit condition by preventing injuries in the internal apparatus of cell (ahmed et al., 2009). catalase and peroxidase enzymes activity significant differences among treatments were observed for cat enzyme activity (table 6). cat activity was the highest (7.47 and 6.97 µmol h2o2 g-1 fw min-1) with di33 and di40 treatments. similar to cat, the pod activity in both seasons increased in response to an increase in water deficit stress (table 6), which high pod activity was found by irrigation 33% etc in 2014. in present study, the antioxidant enzyme activates increased with the decrease of irrigation water table 6 effect of deficit irrigation on proline, catalase enzyme activity (cat), peroxidase enzyme activity (pod) and relative water content (rwc) i33, i440, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) proline (mg g−1fw) cat (µmol h 2 o 2 g-1 fw min-1) pod (unit g-1 fw min-1) rwc (%) 2014 100 0.77 b 4.52 b 0.422 b 78.63 a 66 1.5 a 5.62 b 0.5 b 67.45 ab 33 1.97 a 7.47 a 0.789 a 55.19 b 2015 100 0.97 c 4.4 b 0.356 b 73.13 a 70 1.302 b 5.19 b 0.486 a 64.26 ab 40 1.808 a 6.97 a 0.511 a 58.74 b 457 barzegar et al. deficit irrigation effects on melon quality applied. as found by kavas et al. (2013) in melon and huseynova (2012) in wheat, the antioxidant activity of cat significantly increased by drought stress. antioxidative enzymes like pod and cat play a major role in conferring drought tolerance and cta and pod activity of drought tolerance genotypes were higher than sensitive genotypes under drought stress (hameed et al., 2013). relative water content as applied irrigation water decreased, the relative water content of leaf decreased (table 4). the results showed that different irrigation treatments had similar effects on rwc in both seasons. the highest value of rwc was recorded in irrigation 100% etc. the decrease in rwc being respectively, 29.8 and 19.67% for di33 and di40 compared to i100. rwc decreased linearly in response to an increase in water deficit stress in melon (kavas et al., 2013), watermelon (kirnak and dogan, 2009) and mini-watermelon (rouphael et al., 2008). the results indicated that the rwc was improved by the increasing irrigation water. kirnak and dogan (2009) stated the higher leaf relative water content values are generally indication of enough soil water in root zone. 4. conclusions water deficit has been shown to adversely affect leaf area, yield, and leaf water status of melon, but led to increase the wue and tss. since the water scarcity is a key factor for plant production under arid and semi-arid regions, thus achieving great values of wue is more reasonable than maximum yield. wue in di40 and di33 was greater than full irrigation treatment. irrigation water increased yields not only by increasing the mean weight of the fruits, but also by increasing fruit number per vine. in both years, the physiological parameters showed significant differences. results indicated that the change of cat and pod activity and proline accumulation cooperated with water deficit; indeed cat and pod activities and proline content (60.1% and 46% in di33 and di40, respectively) increased with enhancement of drought intensity, and in stressed plants were significantly higher than full irrigated plants. the results suggested that antioxidant enzyme activities (cat and pod) as well as proline accumulation may play an important role in protecting ‘khatooni’ melon plants against drought stress. references ahmed c.b., rouina b.b., sensoy s., boukhris m., abdallah f.b., 2009 changes in gas exchange, proline accumulation and antioxidative enzyme activities in three olive cultivars under contrasting water availability regimes. environ. exp. bot., 67: 345-352. bates l., waldren r.p., teare i.d., 1973 rapid determination of free proline for water stress studies. plant soil., 39: 205-207. cabello m.j., castellanos m.t., romojaro f., martinez-madrid c., ribas f., 2009 yield and quality of melon grown under different irrigation and nitrogen rates. agric. water manag., 96: 866-874. chaves m.m., maroco j.p., pereira j.s., 2003 understanding plant responses to drought from genes to whole plant. fun. plant biol., 30: 239-264. cui n.b., du t.s., kang s.z., li f.s., zhang j.h., wang m.x., li z.j., 2008 regulated deficit irrigation improved fruit quality and water use efficiency of pearjujube trees. agric. water manag., 95: 489-497. dogan e., kirnak h., berekatoglu k., bilgel l., surucu a., 2008 water stress imposed on muskmelon (cucumis melo l.) with subsurface and surface drip irrigation systems under semiarid climatic conditions. irrigation sci., 26: 131-138. fabeiro c., martin d.e., santa olalla f., de juan j.a., 2002 production of muskmelon (cucumis melo l.) under controlled deficit irrigation in a semi-arid climate. agric. water manag., 54: 93-105. fao, 2014 faostat. faostat3.fao.org/home/index.html. farooq m., hussain m., wahid a., siddique k.h.m., 2012 drought stress in plants: an overview, pp. 1-33. in: aroca r. 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angustifolia. rev. bras. fisiol. veg., 11: 69-75. zeng c.z., bie z.l., yuan b.z., 2009 determination of optimum irrigation water amount for drip-irrigated muskmelon (cucumis melo l.) in plastic greenhouse. agric. water manag., 96: 595-602. impaginato 389 adv. hort. sci., 2018 32(3): 389-398 doi: 10.13128/ahs-23229 postharvest control of aspergillus niger in mangos by means of essential oil s. javadpour 1, a. golestani 2, s. rastegar 2 (*), m.m. dastjer 2 1 food and cosmetic health research center, hormozgan university of medical sciences, bandar abbas, iran. 2 department of horticultural science, college of agriculture, university of hormozgan, hormozgan, iran. key words: aspergillus niger, essential oil, mango, postharvest. abstract: the use of essential oil as an alternative mean to synthetic fungicides has been considered in the past years for management of the postharvest decay of fruits in order to ensure more safe and long storage life of these perishable commodities. aspergillus niger is one of the most dangerous fungal pathogen which can cause postharvest diseases in fresh mangos. the aim of this study was to assess the effectiveness of essential oil from four aromatic plants (thymus vulgaris, salvia mirzayanii, artemisa persica, and rosmarinus officinalis) in comparison to fungicide ‘mancozeb’ against a. niger under in vitro and in vivo conditions. after inoculation of mango fruits with an isolate of a. niger followed by curative treatments with essential oil, the main physical and chemical attributes of mangoes were determined under postharvest condition. the in vitro results showed that colonies of a. niger were totally inhibited by application of essential oil of t. vulgaris (at all the tested concentrations) and a. persica (1500 μl/l). while, s. mirzayanii showed the lowest effect at 1000 μl/l if compared with the other essential oils. the results of the in vivo experiments showed that treatments with t. vulgaris and s. mirzayanii essential oil had significant (p<0.05) effects in preventing fruit decay at 1000 μl/l after 10 days of storage, while, r. officinalis essential oil significantly (p<0.05) reduced deterioration of mango fruits at 500 μl/l, followed by a. persica. rosemary also showed the highest fruit firmness in comparison with other treatments. also, the essential oils maintained higher chlorophyll content. the results of this work showed that application of essential oil on mangos assurance both a significant preservation on their quality attributes by controlling, at the same time, decaying caused by a. niger during the postharvest phase. 1. introduction postharvest diseases are among the major causes of losses of mangos (mangifera indica l.) fresh produce throughout the supply chain. the incidence of the postharvest diseases can also affect the quality of mangos limiting their shelf life up to 3-4 days. in literature is reported that about 17-37% of fresh mangos is wasted after harvesting and marketing (madan (*) corresponding author: rastegarhort@gmail.com citation: javadpour s., golestani a., rastegar s., dastjer m.m., 2018 postharvest control of aspergillus niger in mangos by means of essential oil. adv. hort. sci., 32(3): 389-398 copyright: © 2018 javadpour s., golestani a., rastegar s., dastjer m.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 may 2018 accepted for publication 12 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 389-398 390 and ullosa, 1993). sharma et al. (1994) have reported that about 17.7% of this fresh produce is lost during the storage and marketing. mango decay caused by the plant pathogenic fungus aspergillus niger is one of the most dangerous postharvest diseases, leading to the losses of fruit quality during storage (duamkhanmanee, 2008). it well known from published reports as more negative effects associated to use of chemical fungicides for controlling postharvest diseases have been reported on the human’s health and environment (wightwick et al., 2010). furthermore, consumers believe that fruits not treated (or minimally-treated) with fungicides are safer for fresh consumption (du plooy et al., 2009). in the past 20 years there has been a great interest in using essential oils (eos) to control postharvest diseases, such increasing shelf life of stored fruits (tripathi and dubey, 2004). several studies have also reported on the antifungal activity of thymus vulgaris against different strains of colletotrichum gloeosporioides, rhizopus stolonifer, penicil l ium digitatum (abdolahi et al . , 2010; sellamuthu et al. , 2013), aspergillus flavus , aspergillus niger, aspergillus fumigatus, and alternaria alternata (kumar et al., 2008). different species of salvia are yet used as antimicrobial agents (fiore et al., 2006; kelen and tepe, 2008), however salvia mirzayanii is an endemic plant which grows only in some parts of iran, and thus there is no an exhaustive information regarding its effect on a. niger (rechinger, 1986). centeno et al. (2010) have reported that extracts from rosmarinus officinalis and t. vulgaris could have a significant effect on the control of fungal decaying. previous studies have also confirmed the interesting antimicrobial activity of r. officinalis eo against spoilage and pathogenic foodrelated fungi (abdolahi et al., 2010). de sousa et al. (2013), for instance, detected the strong effect of the origanum vulgare and r. officinalis eos in controlling a. flavus. on the other hand, fewer papers report issues regarding to postharvest control of a. niger on mango fruits using eos as an alternative mean to synthetic fungicides. this study was focused to evaluate the effectiveness of eos derived from four aromatic plants (t. vulgaris, s. mirzayanii, artemisa persica and r. officinalis) to in vitro and in vivo suppress the growth of one pathogenic strain of a. niger by preserving the mango fruit quality attributes under postharvest condition. 2. materials and methods plant material and extraction of essential oil s. mirzayanii and a. persica samples were collected from lar region of fars province, iran (lat. 27°41’ 3’’ n and long. 54°2’ 10’’e). a t. vulgaris sample was collected from geno region of hormozgan province, iran (lat. 25° 38’ 37.9’’ n and long. 57° 46’ 28’’ e) and r. officinalis samples from kerman province, iran (lat. 30° 17’ 2.1’’ n and long. 57° 5’ 0.1’’ e). samples were harvested in vegetative stage (before flowering). the leaves of samples were cut into small pieces and shade-dried at room temperature. the material was then ground to fine powder. the 80 g of plant material were subjected to extraction of eos by hydro-distillation method for 6 h using a clevenger’s apparatus (moghaddam et al., 2011). the eos were separately collected, dehydrated using sodium sulphate (na2so4), and finally stored in a dark bottle at 4°c until tested. in vitro experiments fungi were isolated from mango and their identity was confirmed the aspergillus niger (ptcc 5010) was supplied by iranian research organization for science and technology (irost). culture of the pathogen organism was maintained on potato dextrose agar (pda) medium. stock cultures were grown at 25°c for 7 days to allow for sufficient sporulation. antifungal effects of eos were carried out by the solution method (sm) according to pitarokili et al. (1999). inhibitory effects of eo extracted from s. mirzayanii, a. persica, r. officinalis and t. vulgaris were determined by in vitro antifungal assays. to measure the direct fungal inhibition of each eo on mycelial growth of a. niger, three different concentrations of them (1000, 1200 and 1500 μl/l) were added to potato dextrose agar (pda; provided by scharlau) media before solidification into petri dishes (8 cm diameter) at 45-50°c. fungal disks with 5 mm diameter were placed on the middle of petri dishes and incubated at 25°c for 10 days. three replicates per each treatment (4 eo × 3 concentrations) including control plate without eo were prepared. inhibition percentage was determined at the end of incubation time by the index: ip = (dc-dt)/dc ×100 ip = inhibition percentage, dc = mycelium diameter in the control plate, and dt = in the eos-treated plate. javadpour et al. postharvest control of aspergillus niger in mangos 391 in vivo experiments mangos cv. halily were harvested from minab (lat. 27°07’51’’ n and long. 57°05’13’’ e) at the maturity stage of development. fruit surface was before disinfected with 2% sodium hypochlorite for 3 min, and then artificial inoculations were done by puncturing fruit surface (4 mm deep and 2 mm wide for each inoculation point) with a sterile needle on two sides of each fruit with 40 μl of a conidial suspension containing 106 ufc/ml of a. niger that it has been sprayed above each wound. after one-day of incubation at 25°c to allow conidia germination into fruit tissue, fruits were treated with 500 and 1000 μl/l eo of each plant in comparison to 0.5 and 1.0 mg/l mancozed. after the treatment (curative), all fruit trials, including the control (fruits were inoculated with conidial suspension without the treatment with essential oils), were placed into boxes and kept at 25°c for 1, 2, and 3 weeks. decay percentage of fruit was calculated as the number of decayed fruit/ total number of fruit at each replication* 100 (elanany et al., 2009). physical-chemical analysis firmness of each fruit was measured at two points of the equatorial region by using a texture analyzer with a 5 mm probe (lurton, taiwan) with units expressed in kg/cm2. surface color was measured on each fruit at two opposite sides using a chromameter (cr 400, minolta) which provided cie l*, a*, and b* values. l* is color lightness (0= black and 100= white). the a* scale shows in the maximum the red (+a*) and in the minimum the green color (a*) while the b* ranged from yellow (+b*) to blue (b*). the content of ascorbic acid (aa) expressed as mg/100 g fruit weight was determined as described by molla et al. (2011). aliquots of 10 ml of each sample was homogenized in 100 ml of extraction buffer containing 3% metaphosphoric acid. aliquots of 10 ml of homogenate was titrated against standard dye 2,6-diclorophenol indophenols to a faint pink color. the method proposed by lichtenthaler (1987) was used to determine the total chlorophyll and carotenoids content of fruit. the ‘total soluble solids’ (tss) content was determined at 20°c using a digital refractometer, and expressed as °brix. the ph of fruit juice was measured using a jenway 3320 ph meter calibrated by ph 4 and 7 buffer solutions. the ‘titratable acidity’ (ta) was determined by titration of 5 ml extract with 0.1 mol l−1 sodium hydroxide at ph 8.1 and expressed as percent citric acid (molla et al., 2011). weight loss, fruit firmness, surface color change, content of aa, total chlorophyll, and carotenoids, and tss, ta and ph were determined after 1, 2, and 3 weeks of storage at 25°c. these characteristics was done with 3 replicates (3 large fruits for 1 replicate). statistical analysis the experiment was conducted in a randomized factorial designed whit essential oils treatment and storage time as the two factors. data were submitted to one-way analysis of variance (anova) using sas version 16.0 and means were separated by the duncan test at p<0.05 (n=3). 3. results and discussion aspergillus niger mycelia inhibition in vitro experiments showed that mycelia growth of a. niger was significantly suppressed (p<0.05) when treated with the different concentrations of each eo (fig. 1). the fungal growth was totally inhibited (ip= 100%) by all the concentrations of t. vulgaris eo and with 1500 μl/l a. persica eo. on the other hand, s. mirzayanii eo showed lower effect (ip= 72%) than other eos when tested at 1000 μl/l concentration. our findings are in agreement to the ones described by kohiyama et al. (2015) who reported that t. vulgaris eo was able to control the growth of a. flavus. similar observations on the prevention of fig. 1 inhibitory effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 1000, 1200 and 1500 μl/l on mycelia growth of aspergillus niger cultures incubated at 25°c for 10 days. bars indicate the sd of the mean. different letters indicate significant differences in mean values (p<0.05). adv. hort. sci., 2018 32(3): 389-398 392 different pathogenic fungi by using eos have been reported in the previous studies, such as those conducted by tripathi and dubey (2004) and pawar and thaker (2006). boubaker et al. (2016) reported the antifungal activity of four thymus species eos against penicillium digitatum, penicillium italicum and geotrichum citriaurantii. pawar and thaker (2006) showed that cinnamomum zeylanicum, cinnamomum zeylanicum, cinnamomum cassia, cymbopogon citratus and syzygium aromaticum were the best plant sources for eos extraction showing a noticeable inhibitory effect against a. niger. it was also reported that the mycelial growth of a. niger was inhibited by application of 2.5 and 3.0 μg/ml of citrus sinensis oil (sweet orange) in potato dextrose broth and agar water medium, respectively (sharma and tripathi, 2008). modifications on fungal structures induced by the eos afore-quoted might be due to interactions of their components (carvacrol, thymol, eugenol, vanillin and etc.) with cell wall synthesis, which could affect fungal growth and its morphology (rasooli et al., 2006; rao et al., 2010). some researchers have stated that some phenolic compounds present in the eos could affect the plasma membrane and the cellular organelles, such as mitochondria of the fungi by decreasing the lipid and saturated fatty acid levels and increasing the unsaturated fatty acids, resulting in the leakage of ca2+, mg2+ and k+ (sharma and tripathi, 2008). in addition, the existence of the hydroxyl groups and the aromatic nucleus could be a other important factor for the eos antimicrobial activity (numpaque et al., 2011). fruit decay suppression as shown in figure 2, the decay percentage of fruits increased with the storage time, variable from 1 to 3 weeks of incubation. the percentage of decay significantly decreased (p<0.05) with increasing of the concentration of t. vulgaris and a. persica eos after 3 weeks of storage. after one week, no significant differences were observed between control and treated fruits (data not shown). after two weeks, significant differences were found among treatments with r. officinalis eo at 500 μl/l and t. vulgaris at 1000 μl/l. at the end of experiment, after two weeks, the maximum level of decay was related to the control fruits (70%), and the minimum one was attributed to r. officinalis (500 μl/l) and a. persica (1000 μl/l) eos, reaching 12% and 13.3% decay, respectively. these data agree with those obtained by ramezanian et al. (2016) who showed the possibility of using z. multiflora and t. vulgaris eos to control postharvest citrus alternaria decay (black rot). in addition, elshafie et al. (2015) reported that o. vulgare eo can control the brown rot of peach. jhalegar et al. (2015) addressed their study on the influence of lemon grass, eucalyptus, clove and neem eos against p. digitatum and p. italicum in ‘kinnow’ mandarin. these authors showed that the decay rot during storage was less in the treated fruits than in the control ones. duamkhanmanee (2008) reported that 4000 ppm lemon grass eo could control anthracnose by c. gloeosporioides decay of mangos. phenolic compounds such as carvacrol and thymol (rao et al., 2010) contained in eos have a lipophilic molecular structure, therefore it interfer with membrane-catalyzed enzymes and cell wall, causing the cell death of microbes (shirzad et al., 2011). many researchers believe that the type and the amount of phenolic compounds present in the oil can determine the antifungal activity of the eos (tripathi and dukey, 2004). weight loss the weight loss of fruit was increased strongly during the early weeks, but this increase was gradual throughout the storage period (fig. 3). after two weeks of storage, the highest and lowest weight loss was observed in the control samples and those treated with 1000 μl/l s. mirzayanii (12.7% and 9.8% respectively). during the storage, the main mango weight loss (13.2%) was found in the control fruits, while the lowest one (10.8%) was observed in r. officinalis treated fruits at 500 μl/l concentrations. the mechanism of eos for reducing physiological fig. 2 suppressive effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos decay after 10 and 15 days of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). javadpour et al. postharvest control of aspergillus niger in mangos 393 cell wall components. breakdown and the enzymatic degradation of insoluble protopectins into more simple soluble pectin can be associated with softening (willats et al., 2001). ramezanian et al. (2016) found that the eos reduced the activity of polygalacturonase and galactosidase, which are softening enzymes in the cell wall components, and maintained orange fruit firmness through the storage. the results obtained in the present study agree with those of maqbool and alderson (2010), who showed that by the application of lemongrass oil (0.05%) and cinnamon oil (0.4%), the firmness of banana and papaya fruits was maintained during storage. however, tzortzakis (2007) reported that eucalyptus and cinnamon eos had no effect on the tomato and strawberry firmness. surface color change the results related to the changes in the fruit color (in terms of l*, a* and b*) of the treated mango showed that the lightness of the fruits peel was decreased throughout the storage time (table 1). the fruits treated with 1000 μl/l r. officinalis eo retained higher l* over other treatments and control samples, after three weeks of storage. the highest and lowest l* was found in r. officinalis and a. persica at 1000 μl/l concentration, respectively. the results showed that a* was significantly decreased during the storage. however, the fruits treated with eos maintained a higher a* than did the control ones. at the end of the storage, the lowest a* (7.46) and the highest a*(11.73) were found in the control and r. officinalis treated fruits at 1000 μl/l concentraloss in weight might be related to the reduction of ethylene production and the respiration rate. also, eos cover the peel of fruit, creating the water barrier between the fruit and the environment, thereby reducing water exchange (morillon et al., 2002). this agrees with previous studies showing the efficacy of eos in reducing the weight loss of cherries and grapes (serrano et al., 2005). similarly, du plooy et al. (2009) reported that the use of mentha spicata and lippia scaberrima eos reduced weight loss in ‘valencia’ oranges. fruit firmness a continuous decline in mangos firmness was observed throughout storage (fig. 4). however, the fruits treated with eos showed higher firmness than the control ones. in each stage of storage, no significant difference was identified in the firmness of fruits, at different concentrations of eos. after 3 weeks of storage, the firmness of control fruits was around 3.06 kg/cm2, while the treated fruits were significantly firmer (p<0.05). in this stage, fruits treated with mancozeb showed no significant difference, as compared with those treated with 500 μl/l t. vulgaris eo. however, r. officinalis in both concentrations showed the highest firmness, as compared with other treatments. firmness, as one of the fruits properties, is a complex sensory attribute that also includes crispiness and juiciness; it is important in determining the acceptability of horticultural crops. it has been accepted that the loss of fruit firmness throughout the storage is mainly due to the depolymerization of fig. 3 effect of the thymus vulgaris , salvia mirzayanii , artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos weight loss after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). fig. 4 effect of the thymus vulgaris , salvia mirzayanii , artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos firmness after 1, 2, and 3 weeks of incubation compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). 394 adv. hort. sci., 2018 32(3): 389-398 tion, respectively. the results also showed that b* was increased during the storage time, but the trend in fruits treated by eos was slower than that in the control. at the end of storage, s. mirzayanii and r. officinalis, at 500 μl/l concentrations, showed the minimum b* value (40.7 and 40.6), respectively. however, control and mancozeb samples showed the maximum b* value (56.6 and 55.9), respectively. the results obtained in the present study showed that eos treatment could have better litheness with lower a* and b*, as compared to mancozed and control groups. ramezanian et al. (2016) showed the effect of the zataria multiflora and t. vulgaris eos on the black rot of ‘washington navel’ orange fruit. they found that the best color was related to zataria at 300 μl/l and thyme eos at 400 μl/l concentrations. in agreement with our findings, marjanlo et al. (2009) showed the effect of cumin eo on the postharvest quality of strawberries, finding that the essential oil treated fruits maintained a higher l* during storage in comparison with the controls. aa content ascorbic acid (vitamin c) content was gradually decreased during storage; however, its strength was lower in the treated samples (table 2). different concentrations of the eos significantly maintained ascorbic acid content, as compared to the control. overall, the most (14 mg/100 g/1) and the least (9 mg/100 table 1 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos color change ± sd after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). testing index treatment (concentration) storage time week 1 week 2 week 3 l* control 63.3±0.43 a 47.5±0.56 n 46.2±0.56 n mancozeb 0.5 mg/l 64.9±0.5 a 59.8±0.48 c-g 54.1±0.59 j-m mancozeb 1 mg/l 55.3±.23 f-l 55.9±0.75 f-l 56.7±0.76 f-l thymus vulgaris 500 μl/l 62.1±0.33 a-d 54.8±0.46 i-m 51.4±0.36 mn thymus vulgaris 1000 μl/l 60.6±0.54 b-f 57.8±0.49 e-j 51.8±0.66 mn salvia mirzayanii 500 μl/l 54.6±0.32 i-m 51.9±0.58 l-n 56.1±0.54 f-k salvia mirzayanii 1000 μl/l 59.9±0.33 c-g 58.4±0.44 d-g 49.7±0.38 mn artemisa persica 500 μl/l 59.7±0.43 c-g 56.9±0.58 f-l 48.7±0.65 n artemisa persica 1000 μl/l 57.9±0.23 e-j 58.8±0.75 d-g 41.4±0.65 o rosmarinus officinalis 500 μl/l 59.8±0.19 c-g 60.7±0.66 a-d 54.1±0.39 j-m rosmarinus officinalis 1000 μl/l 62.2±0.25 a-d 65.3±0.76 a 59.5±0.53 c-g a* control 16.4±0.87 12.6±0.87 g-i 7.4±0.87 l mancozeb 0.5 mg/l 17.2±0.87 15.8±0.88 a-e 8.1±0.98 l mancozeb 1 mg/l 16.8±0.99 16.1±0.98 a-d 8.6±0.99 kl thymus vulgaris 500 μl/l 16.4±0.67 16.1±0.76 b-f 8.6±0.76 kl thymus vulgaris 1000 μl/l 17.2±0.89 15.3±0.87 a-c 11.2±0.85 ij salvia mirzayanii 500 μl/l 17.1±0.78 16.7±0.88 c-f 11.1±0.76 ij salvia mirzayanii 1000 μl/l 17.6±0.77 15±0.68 e-g 8.4±0.87 kl artemisa persica 500 μl/l 17.1±0.77 14.2±0.87 f-g 10.8±0.97 ij artemisa persica 1000 μl/l 17.4±0.69 13.5±0.88 f-h 11.6±0.97 h-j rosmarinus officinalis 500 μl/l 16.8±0.90 15.8±0.98 a-e 11.7±0.98 ij rosmarinus officinalis 1000 μl/l 14.6±1.02 16.3±0.96 a-d 10.1±1.03 jk b* cntrol 25.5±0.78 ij 34.7±1.02 e 56.6±2.2 a mancozeb 0.5 mg/l 20.7±1.03 l-n 30±1.03 fg 55.9±3.2 a mancozeb 1 mg/l 21.5±0.85 l-n 32.9±1.07 ef 46.7±2.9 bc thymus vulgaris 500 μl/l 20.8±0.87 mn 25.1±0.84 h-j 46.7±3.8 bc thymus vulgaris 1000 μl/l 20.6±0.78 mn 30.6±1.06 fg 49.4±3.5 b salvia mirzayanii 500 μl/l 17.0±0.78 ab 30.7±0.94 fg 40.6±2.6 d salvia mirzayanii 1000 μl/l 23.8±1.03 i-l 27.3±0.99 gh 48.3±3.5 bc artemisa persica 500 μl/l 19.4±0.86 n 26.6±0.95 hi 47.8±3.6 bc artemisa persica 1000 μl/l 20.8±0.98 l-n 30.8±1.04 fg 47.6±3.2 bc rosmarinus officinalis 500 μl/l 20.7±1.03 l-n 24.4±0.90 i-k 40.7±2.8 d rosmarinus officinalis 1000 μl/l 22.8±0.99 j-m 23.4±1.05 i-l 45.5±3.5 c 395 javadpour et al. postharvest control of aspergillus niger in mangos g/1) amount of ascorbic acid content was detected in the fruits treated with s. mirzayanii at the concentration of 500 μl/l and control after three weeks of storage, respectively. in general, fruits treated with mancozeb showed lower ascorbic acid content in comparison with those treated with eos throughout the storage. in agreement with our findings, geransayeh et al. (2012) showed that the vitamin c content of grapes was decreased significantly during the storage; however, a higher vitamin c amount was observed in the samples treated with t. vulgaris eo. our results were nevertheless in contrast with those of marjanlo (2009) who did not detect any significant difference in the amount of ascorbic acid in strawberry fruits treated by the essential oils. carotenoids and total chlorophyll content analysis of the variance of carotenoids content revealed a significant difference (p< 0.05) between treatments (table 2). the concentration of carotenoids content was low at the initial time of storage and then significantly increased during storage. at the end of the process, control fruits showed the highest content of carotenoids (1.94 mg 100 g-1). r. officinalis and t. vulgaris, tested at 500 μl/l showing 1.09 and 1.15 (mg 100 g-1) as carotenoids content, respectively, were the lowest one, as compared with other treatments. the total chlorophyll content was gradually decreased to a lower concentration in all table 2 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on the ascorbic acid, carotenoids and total chlorophyll content ± sd in mangos after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). active metabolite treatment (concentration) storage time (week) week 1 week 2 week 3 ascorbic acid control 12.3±3 cd 11±2 cd 9.5±4 d mancozeb 0.5 mg/l 13.3±4 c 12.6±3 cd 11±3 cd mancozeb 1 mg/l 13.5±4 c 12.3±3 cd 11±2 cd thymus vulgaris 500 μl/l 14.8±7 bc 13.6±4 c 12±4 cd thymus vulgaris 1000 μl/l 14.5±4 bc 13.7±4 c 12±5 cd salvia mirzayanii 500 μl/l 17.4±4 a 16.4±4 b 14±5 bc salvia mirzayanii 1000 μl/l 13.3±6 c 12.9±3 cd 12±4 cd artemisa persica 500 μl/l 15.9±7 bc 14.8±2 bc 12±5 cd artemisa persica 1000 μl/l 13.4±4 c 12.6±5 cd 11±5 cd rosmarinus officinalis 500μl/l 17.5±4 a 17±5 b 13±3 c rosmarinus officinalis 1000 μl/l 15.8±3 bc 14.5±4 bc 11±c 2 d carotenoids control 0.85±0.03kn 1.64±0.06 b 1.94±0.03 a mancozeb 0.5 mg/l 0.69±0.04 m-o 1.57±0.03 b 1.43±0.03 b-e mancozeb 1 mg/l 0.74±0.03 m-o 1.52±0.04 bc 1.48±0.08 b-d thymus vulgaris 500 μl/l 0.74±0.07 m-o 1.12±0.05 g-j 1.15±0.03 f-i thymus vulgaris 1000 μl/l 0.55±0.03 o 1.28±0.08 c-g 1.91±0.06 a salvia mirzayanii 500 μl/l 0.69±0.08 m-o 1.26±0.05 d-h 1.39±0.07 b-f salvia mirzayanii 1000 μl/l 0.7±0.06 m-o 1.1±0.06 g-k 1.53±0.03 bc artemisa persica 500 μl/l 0.56±0.05 o 0.85±0.04 l-n 1.31±0.04 c-g artemisa persica 1000 μl/l 0.6±0.06 o 0.87±0.08 j-m 1.22±0.03 e-h rosmarinus officinalis 500 μl/l 0.52±0.04 o 0.93±0.05 i-m 1.09±0.04 g-l rosmarinus officinalis 1000 μl/l 0.5±0.07 o 1.01±0.03 h-l 1.16±0.05 f-i total chlorophyll control 2.72±0.2 f 1.19±0.02 gh 0.03±0.002 i mancozeb 0.5 mg/l 3.98±0.4 e 1.31±0.02 g 0.53±0.03 h mancozeb 1 mg/l 3.44±0.2 f 1.72±0.02 g 0.31±0.02 h thymus vulgaris 500 μl/l 4.86±0.5 de 2.5±0. 6 f 0.11±0.01 h thymus vulgaris 1000 μl/l 4.33±0.2 de 2.66±0. 5 f 0.11±0.02 h salvia mirzayanii 500 μl/l 5.43±0.3 d 3.55±0.2 f 0.16±0.01 h salvia mirzayanii 1000 μl/l 7.62±0.2 bc 3.47±0.2 f 0.15±0.02 h artemisa persica 500 μl/l 9.61±0.2 a 2.58±0.7 f 0.16±0.03 h artemisa persica 1000 μl/l 8.38±0.3 b 2.35±0.5 f 0.22±0.02 h rosmarinus officinalis 500 μl/l 8.69±0.2 ab 3.27±0.8 f 0.16±0.04 h rosmarinus officinalis 1000 μl/l 8.19±0.4 b 3.3±0.6 f 0.32±0.5 h 396 adv. hort. sci., 2018 32(3): 389-398 treatments throughout the storage (table 2); however, the highest reduction was observed in the control fruits. at the end of storage, the minimum chlorophyll content (0.03) was recorded in the control, while the highest was found in mancozeb with 0.5 mg/l concentration. tss, ta and ph a gradual increase in tss percentages was determined in all treatments (table 3). generally, the fruits treated with eos had lower tss percentages than the control fruits throughout the storage. however, the treated fruits did not show any significant difference in tss, as compared with the controls. these results were in agreement with those in other studies (marjanlo et al., 2009). nevertheless, they are in discordance with rabiei et al. (2011), who reported that thyme eos treatment had a significant effect on the ph of apples. as shown in table 3, ta values were also gradually decreased during storage. at the end of storage, the maximum ta values (0.27%) were observed in a. persica (1000 μl/l), while the minimum (0.10%) was in the control fruits. among the eos treatments, r. officinalis (1000 μl/l) resulted in the lowest acidity (0.17%), this was followed by t. vulgaris (0.16%) with 1000 μl/l concentration during storage; however, no significant difference was observed between the treatments. these results are in agreement with those reported by maqbool and alderson (2010), who showed that the table 3 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on total soluble solids (tss), ph, and titratable acidity (ta) ± sd in mangos after 1, 2, and 3 weeks of incubation compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). quality parameter treatment storage time (week) week 1 week 2 week 3 tss control 8.9±3 c 9.6±3 b 9.9±2 a mancozeb 0.5 mg/l 8.06±3 cd 9.23±5 b 9.5±3 b mancozeb 1 mg/l 8.33±4 c 9.13±5 bc 9.5±2 b thymus vulgaris 500 μl/l 8.96±5 c 9.63±4 b 9.7±2 ab thymus vulgaris 1000 μl/l 8.83±4 c 9.76±4 ab 9.8±4 ab salvia mirzayanii 500 μl/l 8.7±4 c 9.26±2 b 9.3±3 b salvia mirzayanii 1000 μl/l 8.33±3 c 9.26±5 b 9.6±2 b artemisa persica 500 μl/l 8.66±3 c 9.3±3 b 9.6±3 b artemisa persica 1000 μl/l 8.1±5 cd 9.53±5 b 9.7±4 b rosmarinus officinalis 500 μl/l 7.5±3 d 9±4 bc 9.2±2 b rosmarinus officinalis 1000 μl/l 8.66±4 c 9.53±5 b 9.5±3 b ph control 1.15±0.03 cd 2.89±0.2 c 4.75±1.4 a mancozeb 0.5 mg/l 1.06±0.02 cd 2.38±0.4 c 4.3± 2 ab mancozeb 1 mg/l 1.09±0.02 cd 2.59±0.3 c 4.3± 2 ab thymus vulgaris 500 μl/l 1.06±0.02 cd 2.51±0.2 c 4.18± 2. 2 ab thymus vulgaris 1000 μl/l 1.26±0.02 cd 2.54±0.3 c 4.31±1.8 ab salvia mirzayanii 500 μl/l 0.99±0.02 d 2.74±0.2 c 4.24±1.9 ab salvia mirzayanii 1000 μl/l 1.12±0.02 cd 2.26±0.2 c 4.42±2 ab artemisa persica 500 μl/l 1.03±0.02 cd 2.45±0.3 c 4.48±3 ab artemisa persica 1000 μl/l 1.08±0.02 cd 2.17±0.2 c 4.47±1.4 ab rosmarinus officinalis 500 μl/l 1.02±0.02 cd 1.76±0.1 cd 4.36±1.2 ab rosmarinus officinalis 1000 μl/l 1.07±0.02 cd 2.52±0. 5 c 4.56±2.1 ab ta control 0.208±0.03 c 0.196±0.01 c 0.10±0.009 d mancozeb 0.5 mg/l 0.254±0.02 bc 0.255±0.01 bc 0.23±0.02 bc mancozeb 1 mg/l 0.27±0.02 bc 0.234±0.02 bc 0.23±0.02 bc thymus vulgaris 500 μl/l 0.328±0.02 b 0.26±0.03 bc 0.23±0.02 bc thymus vulgaris 1000 μl/l 0.308±0.01 b 0.262±0.02 bc 0.16±0.01 cd salvia mirzayanii 500 μl/l 0.384±0.02 b 0.267±0.03 bc 0.25±0.02 bc salvia mirzayanii 1000 μl/l 0.352±0.03 b 0.299±0.03 bc 0.24±0.02 bc artemisa persica 500 μl/l 0.288±0.02 bc 0.277±0.02 bc 0.23±0.02 bc artemisa persica 1000 μl/l 0.405±0.02 b 0.307±0.01 b 0.27±0.02 bc rosmarinus officinalis 500 μl/l 0.471±0.02 a 0.302±0.02b 0.26±0.02 bc rosmarinus officinalis 1000 μl/l 0.328±0.01 b 0.261±0.01 bc 0.17±0.01 cd javadpour et al. postharvest control of aspergillus niger in mangos 397 maximum reduction of the ta values was observed in the control fruits of bananas and papayas. data related to the changes in the ph of fruits during storage revealed a significant increase in all treatments (table 3). in each time, a higher ph value was found in the control groups. these results were in line with those reported by du plooy et al. (2009) and tzortzakis (2007), showing no significant differences between the ph of control and treated fruits 4. conclusions the present study proves that the t. vulgaris, a. persica, r. officinalis and s. mirzayanii eos could be employed under postharvest condition to control a pathogenic isolate of a. niger on mango fruits. the effectiveness of these eos was more than mancozeb. so that, the eos here tested could be used as a natural fungicide to control an isolate of a. niger during postharvest mangos. however, further studies are needed to fully understand the antimicrobial mechanisms incited by these eos on a wide range of a. niger isolates, and evaluate their commercial implementation in order to increase the storage lifetime and quality of this marketable commodity. acknowledgements this study as research project was financially supported by hormozgan university of medical sciences, bandar abbas, iran. authors are thankful for providing necessary facilities for carrying out this work. references abdolahi a., hassani a., ghosta y., bernousi i., meshkatalsadat m.h., 2010 study of the potential use of essential oils for decay control and quality preservation of tabarzeh table grape. j. plant prot. res., 50: 45-52. boubaker h., karim h., el hamdaoui a., msanda f., leach d., bombarda i., vanloot p., abbad a., boudyach e.h., aoumar a.a.b., 2016 chemical characterization and antifungal activities of four thymus species essential oils against postharvest fungal pathogens of citrus. ind. crops prod. 86: 95-101. centeno s., calvo m.a., adelantado c., figueroa s., 2010 antifungal activity of extracts of rosmarinus officinalis and thymus vulgaris against aspergillus flavus and a. ochraceus. pak. j. biol. sci., 13(9): 452455. de sousa l.l., de andrade s.c.a., athayde a.j.a.a., de oliveira c.e.v., de sales c.v., madruga m.s., de souza e.l., 2013 efficacy of origanum vulgare l. and rosmarinus officinalis l. essential oil in combination to control postharvest pathogenic aspergill i and autochthonous mycoflora in vitis labrusca l. 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shirzad h., hassani a., ghosta y., abdollahi a., finidokht r., meshkatalsadat m.h., 2011 assessment of the antifungal activity of natural compounds to reduce postharvest gray mould (botrytis cinerea) of kiwifruits (actinidia deliciosa) during storage. j. plant protect. res., 51: 1-6. tripathi p., dubey n.k., 2004 exploitation of natural products as an alternative strategy to control post-harvest fungal rotting of fruit and vegetables . postharvest biol. technol., 32(2): 235-245. tzortzakis n.g., 2007 maintaining postharvest quality of fresh produce with volatile compounds. innov. food sci. & emerg. technol., 8: 111-116. wightwick a., walters r., allinson g., reichman s., menzies n., 2010 environmental risks of fungicides used in horticultural production systems, pp. 273-304. in: carisse o. (ed.) fungicides. intech, rijeka, croatia, pp. 538. willats w.g.t., mccartney l., mackie w., knox j.p., 2001 pectin: cell biology and prospects for functional analysis. plant molecular biology, 47: 9-27. impaginato 157 adv. hort. sci., 2020 34(2): 157­166 doi: 10.13128/ahsc­7775 different environments and doses of controlled­release fertilizer in peach rootstocks production r.d. menegatti (*), a.g. souza, v.j. bianchi department of botany, institute of biology, federal university of pelotas, rs, brazil. key words: fertilization, plant nutrition, prunus persica. abstract: the objective of this study was to evaluate the effects of different environments and doses of controlled­release fertilizer (crf) on the initial growth of peach rootstocks [prunus persica l. (batsch)] cv. capdeboscq. the experimental design was completely randomized, in a 2 x 4 factorial design, four replications and five plants per plot. the treatments were the combination of two cultivation environments (on open­air benches and greenhouse bench­ es) and four doses of crf (0, 2, 4 and 8 g l­1 of substrate), in the 19­06­10 npk formulation. ninety days after their transplanting, the variables plant height, stem diameter, number of leaves per plant, shoot dry matter, root dry matter, total dry matter, plant height and stem diameter ratio were evaluated in addi­ tion to the dickson quality index. all morphological variables evaluated pre­ sented a quadratic positive response to the increase of the applied fertilizer until the dosage of maximum technical efficiency (around 6.2 g l­1). the mainte­ nance of the plants in greenhouse benches and the incorporation of 4 g l­1 crf to the substrate ensures greater efficiency in the input use, reducing the amount of time necessary for peach trees cv. capdeboscq to achieve their graft­ ing point and to be used as rootstocks. 1. introduction southern brazil is the greatest national peach producer and this region is recognized as one of the main production centers of stone fruit trees in the country, especially peach trees. however, the traditional production system of these species is mostly performed in the field (bianchi et al., 2014; ibge, 2018), strongly influenced by climatic conditions. the use of protected environments is an alternative for the production of stone fruit trees, which can ensure the survival of the plants during the most critical phase of the tree production chain. especially in rootstock production by seeds, the seedlings emergence stage and initial growth require environment control, as well as optimal nutrition and irrigation, and protection against pests and diseases in order to assure the fast growth and development of the plants (souza et al., 2017). the production of fruit plants in protected environments, such as (*) corresponding author: renata.d.menegatti@gmail.com citation: menegatti r.d., souza a.g., bianchi v.j., 2020 ­ different environments and doses of controlled‐ release fertilizer in peach rootstocks production. ‐ adv. hort. sci., 34(2): 157­166. copyright: © 2020 menegatti r.d., souza a.g., bianchi v.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 january 2020 accepted for publication 1 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 157­166 158 greenhouses, allows the maintenance of optimum conditions for cultivation throughout the year, antici­ pating and extending the grafting period, reducing production costs and increasing the plants quality standard for sale (oliveira et al., 2017). in brazil, the traditional production system of stone fruit trees requires 360­540 days to grow a plant that is suitable for trading (mayer et al., 2015). in this system, the necessary time for rootstocks to reach the grafting point is of approximately 240 days (fischer et al., 2016). in part, this long period is due to the slow growth of plants in the field during the winter and early spring. in order to reduce the time between seed germi­ nation and the production of rootstocks suitable for grafting, the cultivation in a protected environment, as well as the use of appropriate substrates and fer­ tilizers in the proper doses for the crop are alterna­ tives to optimize the initial phase of the plants growth (bianchi et al., 2014; jamal et al., 2017; menegatti et al., 2019 b). the environmental conditions of the plant pro­ duction system directly influence the plants physio­ logical processes and can directly affect plants growth (souza et al., 2017). protected environments can promote greater uniformity for the plants growth in comparison with plants produced in the field or in an unprotected environment (reis et al., 2010; fischer et al., 2016; oliveira et al., 2017). different environments can also affect the germination of seeds, and the growth and quality of the seedlings produced. the interaction between environmental conditions with the application of fertilizers may con­ tribute to optimize the space for plant production in nursery and to reduce the plant production systems impact on the environment. in addition to the use of commercial substrates, the increase of the nutrient supply is recommended because the substrate alone does not provide enough nutrients for the complete development of the plants (dutra et al., 2016). among the many types of fertilizers available, the controlled­release fertilizer (crf) is the most efficient. crf promotes the slow release of nutrients and the absorption of the ideal amount throughout the plants’ growth period, allowing them to achieve maximum strength (zamunér filho et al., 2012; menegatti et al., 2017 a). the concomitant use of fertilizers with the sub­ strate favors the formation of more vigorous plants in shorter time, which reduces the period in which they stay in the nursery and, consequently, the pro­ duction costs (muniz et al., 2013; menegatti et al., 2017 b). however, few are the researches that report the use of fertilization as an additional factor to the production of stone fruit trees (zhang et al., 2014; jamal et al., 2017; menegatti and bianchi, 2019). even scarcer are the studies that consider plant prop­ agation in a protected environment, such as a green­ house in comparison with open environments (picolotto et al., 2007; reis et al., 2010). the scarce information about the use of con­ trolled­release fertilizers in the production of peach rootstocks in protected environments encouraged the accomplishment of this study, whose objective was to verify the effect of different environments and doses of crf on the initial growth of peach trees cv. capdeboscq for rootstock purposes. 2. materials and methods ripe peach fruits of cv. capdeboscq were harvest­ ed in january 2017 from clonal mother plants kept in the germplasm collection of peach rootstocks at the federal university of pelotas (ufpel), brazil. the experiment was conducted between october (2017) and january (2018), at the department of botany­ ufpel, capão do leão, rs, brazil, at 21° 48’ south lati­ tude, 41° 20’ west longitude and an altitude of 11 m. after the harvest of the fruits, the post­harvest management of the pits was carried out according to picolotto et al. (2007). then the seeds were stratified, as described by souza et al. (2017). after the stratifi­ cation period (35 days at 7°c), the seeds were sown, 1.0 cm deep, in 72­cell polystyrene trays (114 cm3 per cell) containing a mixture of orchard soil + vermi­ culite + medium sand + commercial substrate plantmax® (1:1:1:1) as substrate, and kept in a green­ house. when the seedlings, hereinafter referred as “plants”, reached the transplant point (15 cm between collar and apex), they were transplanted into 1­liter plastic bags containing washed sand, which was used as substrate (table 1) and whose crf (osmocote®) doses had the n­p­k formulation of 19­ 06­10 (4­6 months), which were previously incorpo­ rated into the sand. the experimental design was completely random­ ized, in a 2 x 4 factorial design, with two environ­ ments (on open air benches and on benches inside the greenhouse) and four doses of osmocote® (0, 2, 4 and 8 g l­1 substrate), with four replications and five plants per replication. “protected environment” refers to the arco menegatti et al. ‐ peach rootstocks production 159 pampeana metallic structure greenhouse model, cov­ ered with a 150­millimeter thick, low­density poly­ ethylene plastic film, arranged at the north­south direction and with the following dimensions: 10.0 m x 21.0 m and with the maximum height of 5.0 m. the benches used in the two environments were 1­meter high metallic structures, positioned at ground level. the environmental open air conditions and the ones in the greenhouse during the period of the experi­ ment are described in table 2. ninety days after transplantation, when 75% of the plants of one of the treatments reached the grafting point (at least 5 mm of stem diameter and 10 cm above the soil), the plants were evaluated for the variables stem diameter (sd), plant height (h) and number of leaves (nl). based on these data, it was possible to calculate the plant height and stem diam­ eter (h/sd) ratio. the height of the rootstocks was measured using a graduated ruler, and the stem diameter was measured with a digital caliper. the plants were dried in a forced air circulation oven at 70°c for 72 hours to obtain the shoot dry matter (sdm), root dry matter (rdm) and total dry matter (tdm) per plant. the dickson quality index (dqi) was obtained by the formula: dqi = tdm/ [(h/sd) + (sdm/rdm)], according to gomes and paiva (2011). the stem diameter increase (δsd) was obtained through the data collected every 15 days until the end of the experiment (90 days after transplanta­ tion). possible differences between treatments were verified by analysis of variance (anova). the vari­ ables that exhibited significant differences were sub­ mitted to regression analysis in order to verify the plants growth response in proportion to the crf increasing doses in both growing environments. the data analysis was performed in the statistical pack­ age sisvar (ferreira, 2011). 3. results and discussion at the end of the experiment (90 days after trans­ plantation), the survival rate of the peach rootstock plants was of 100% for all treatments. all variables exhibited interaction (p <0.05) between the environ­ ment factors and the crf (osmocote®) doses (table 3), indicating that the study of factor interaction is important to define the best condition to stimulate plant growth and development. table 1 ­ average chemical composition of the sand substrates used in the production of peach tree rootstocks *sand; **om= organic matter; v= base saturation; sb= sum of bases; cec= cation exchange capacity (cec). substrates om** % v % h+al mg dm­3 sb mg dm­3 cec mg dm­3 p mg dm­3 k μg dm­3 ca μg dm­3 mg μg dm­3 zn μg dm­3 fe μg dm­3 mn μg dm­3 sand* 0.00 67.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.00 0.00 table 2 ­ environmental conditions: temperature (t°c), relative humidity (rh%) and global radiation (w m­²) in the two cultivation environments (in open­air benches and greenhouse benches) during the cultivation period of peach trees cv. capdeboscq environment temperature (°c) rh (%) global radiation (w m­²) greenhouse 23.42 66.81 348.50 open air 21.15 78.62 477.17 table 3 ­ summary of the variance analysis for contrasts between the environmental factors of cultivation and crf (osmocote®) doses for the variables stem diameter (sd), plant height (h), number of leaves per plant (nl), shoot dry matter (sdm), root dry matter (rdm), total dry matter (tdm), plant height and stem diameter (h/sd) ratio, and the dickson quality index (dqi) of peach trees cv. capdeboscq 90 days after transplantation * significant at the probability level (p<0.01) and ** significant at the probability level (p<0.05) by the f test. source of variation df mean square sd (mm) h (cm) nl sdm (g) rdm (g) tdm (g) h/sd dqi environment (e) 1 128.2 ** 1897.8 ** 56.2 ** 603.8 ** 96.9 ** 216.2 ** 474.4 ** 42.0 ** dose of crf (d) 3 137.7 ** 549.8 ** 109.8 ** 233.8 ** 37.9 ** 84.1 ** 103.0 ** 60.7 ** e x d 3 8.0 ** 162.9 ** 11.6 ** 70.1 ** 12.6 ** 26.9 ** 31.4 ** 3.9 * mean 4.3 60.8 55.0 5.9 9.6 15.5 13.4 1.7 cv (%) 4.0 4.3 11.9 10.1 25.5 17.0 6.4 9.7 mean greenhouse 4.6 80.9 64.0 8.48 13.8 22.3 16.7 1.8 mean open air 3.9 40.7 46.0 3.3 5.3 8.6 10.0 1.5 adv. hort. sci., 2020 34(2): 157­166 160 all morphological variables exhibited a quadratic behavior in the adjustment of the regression equa­ tions (figs. 1, 2, 3 and 4), proving that the highest dose test results decreases the variables values, that is, increasing the fertilizer dosage allows the increase of the plants growth up to the maximum technical efficiency dose (mted). the mted for plant height in the protected envi­ ronment was of 6.43 g l­1, corresponding to the height of 113.8 cm, which was three times higher than the control treatment (substrate without the addition of osmocote®) in the same environment, 90 days after transplantation (fig. 1). the mted for plant height in the external environment was of 5.09 g l­1, whose plants reached the height of 52.1 cm, in contrast to the 23.9 cm high of the treatment with­ out the addition of crf (fig. 1). similar height growth was also observed with the application of osmocote® in the studies performed by silva et al. (2011) in the production of rangpur lime rootstock [citrus limonia l. (osbeck)] and by dutra et al. (2016) in the growth of canafistula [peltophorum dubium (spreng.) taub.]. however, the environments and doses tested in their studies were different of this one. the positive effects of crf application on plant growth in different species reinforce the necessity of specific studies to enable the definition of the mted for each species and cultivar, which may provide superior growth and efficiency in the use and exploitation of fertilizers by the plants. the estimated mted for the sd variable of the plants cultivated in greenhouse was of 7.29 g l­1, cor­ responding to a diameter of 5.51 mm (fig. 2). according to the current legislation of the secretary of agriculture and food of rio grande do sul (ordinance 302/98), the grafting must be performed when the rootstocks reach a sd over 5 mm and a height of 10 cm from the ground. thus, the mted estimated for plant grown in greenhouse allows the rootstocks to reach the minimum diameter for graft­ ing at 90 days after transplantation. on the other hand, in the external environment, the plants have not reach the minimum stem diame­ ter required for grafting during the experiment, even at the highest crf dose (fig. 2). in peach rootstock production, it is extremely important to define the environment and the fertiliz­ er mted to obtain the ideal sd for grafting in a short­ er period. the minimum diameter of 5 mm is used to make sure that the rootstocks phloem and xylem have greater competence to perform rapid vascular connection between the scion/rootstock (santarosa et al., 2016), which will allow the effective transloca­ tion of the nutrients absorbed by the roots (root­ stock) to the aerial part (scion cultivar). this condi­ tion will result in a higher percentage of graft­take and growth of the grafted plants, and it will reduce the period to obtain commercial plants. souza et al. (2013) stress the influence of the rootstock diameter to reduce the time to develop grafted plants of “ponkan” tangerine. the plants rapid growth was obtained with the use of rootstocks with larger diameter in the appropriate cultivation conditions in the greenhouse. the best plant growth in both height and stem diameter was obtained in plants grown in a green­ house with the mted. this suggests that the green­ house environment provided better conditions of temperature, humidity and luminosity for the plant growth. associated with the mted, these conditions fig. 1 ­ plant height (cm) of the peach plants cv. capdeboscq, in relation to the dosage of the controlled­release fertilizer (crf) and two cultivation environments, 90 days after transplanting. fig. 2 ­ stem diameter (mm) of peach plants cv. capdeboscq, in relation to the dosage of the controlled­release fertilizer (crf) and two cultivation environments, 90 days after transplanting. menegatti et al. ‐ peach rootstocks production 161 allowed the plants to reach the ideal point for rapid grafting, which is a desired aspect in the production system of peach tree rootstocks. paricá seedlings [schizolobium amazonicum huber ex ducke] grown in a protected environment also exhibited superior performance for plant height and stem diameter (frigotto et al., 2015). these authors concluded that greenhouse cultivation significantly increases the growth variable values in comparison with external environment cultivation. the highest mean number of leaves per plant (97) was obtained with the estimated mted (4.69 g l­1) for the plants grown in the greenhouse (fig. 3a). as for the variable number of leaves, it was found that the plants of cv. capdeboscq cultivated in an open sky achieved dmet higher than plants kept in a greenhouse (fig. 3), a fact that may be related to phytosanitary problems, such as, for example, small leaf spots and necroses detected in the leaves of this treatment, during the conduction of the plant experi­ ment. these leaf damage possibly induced damage to the leaf photosynthetic apparatus, however, this damage may have been efficiently reversed through the emission and growth of new leaves. this hypothesis can be supported by the fact that, at this moment, the plant enhances the production of photoassimilates and destines most of it, the maintenance and maximization of the aerial part, making this organ the drain of greater energy fig. 3 ­ number of leaves per plant (3a), shoot dry mass (3b), root dry mass (3c) and total dry mass (3d) of peach plants cv. capdeboscq, in relation to the dosage of crf and two cultivation environments. demand, both to stimulate the leaf growth maximi­ zing the capture of light, as well as to boost the thic­ kening of the stem diameter ensuring the robustness of the rootstock. it should also be noted, according to the results obtained in this work, that plants grown in the open suggest that they have prioritized the increase in the diameter of the stem at the expense of growth in height, as shown in table 4, a strategy that can increase the robustness of the plants and decrease the exposure of the aerial part, guaranteeing their survival for a longer period, as well as the maintenan­ ce of the physiological processes in this cultivation environment, which expose the plants to sudden environmental variations. the cultivation environment has a strong influen­ ce on environmental conditions, such as temperature and global radiation, parameters that are indirectly related to the efficiency of plants in terms of light absorption capacity, and later conversion to energy, as well as in the absorption and use of nutrients. a gradual production increase of shoot dry mat­ ter, root dry matter and total dry matter up to the crf mted was registered (fig. 3b, 3c and 3d), regardless of the cultivation environment. however, the plants grown in greenhouse presented higher val­ ues for total dry matter. these results corroborate the effects of osmocote® in the growth of rangpur lime rootstocks, as reported by scivittaro et al. 162 adv. hort. sci., 2020 34(2): 157­166 (2004). they found that as controlled­released fertil­ izer doses increased, the dry matter production of the rangpur lime rootstocks increased as well. the leaf area has not been quantified in this study. however, there are previous studies that sup­ port the increase of the number of leaves per plant is directly proportional to the growth of the leaf area (menegatti et al., 2017 a). the greatest leaf area of plants grown in greenhouses provides greater effi­ ciency in solar energy uptake for photosynthesis and photoassimilate production, which is directly related to the nutrient supply, including nitrogen (n), present in the crf formulation used in this study. the use of crf in the mted ensures the availabili­ ty and efficient utilization of n by the plants because the leaching level of n is reduced in comparison with conventional fertilizers (zamunér filho et al., 2012; muniz et al., 2013). n is an essential element to the components of the photosynthetic system, such as chlorophylls, carboxylase activity/oxygenase of ribu­ lose 1.5­bisphosphate and carboxylase of phospho­ enolpyruvate (bassi et al., 2018), thus maintaining satisfactory rates of carbon assimilation (taiz and zeiger, 2017) and consequently guaranteeing the production of photoassimilates that support plant growth. the crf effects on the production of rangpur lime plants for use as rootstocks were registered by serrano et al. (2006) and silva et al. (2011). they con­ cluded that the fertilizer mted increases the vari­ ables shoot dry matter, root dry matter and total dry matter, stressing the importance of fertilization for the maintenance of the photosynthetic process in order to increase the total dry matter and conse­ quently the plants growth. the relation between plant height and stem diam­ eter (fig. 4a) presents a balanced growth of the plants raised in greenhouses. the h/sd ratio is one of the parameters most used in the plant’s quality evaluation. moreover, it fig. 4 ­ ratio between height and stem diameter and dickson quality index of peach plants cv. capdeboscq, in relation to the dosage of controlled­release fertilizer and two cultivation environments. table 4 ­ mean values of the differences between greenhouse and open air for the variables stem diameter (sd), plant height (h), num­ ber of leaves per plant (nl), shoot dry matter (sdm), root dry matter (rdm), total dry matter (tdm), plant height and stem diameter (h/sd) ratio, and dickson quality index (dqi) of peach plants cv. capdeboscq 90 days after transplantation dose sd (mm) h (cm) nl sdm (g) rdm (g) tdm (g) h/sd dqi 0 0.47 10.6 4.6 0.7 1.2 1.9 1.9 0.1 2 0.35 33.4 19.8 4.1 5.8 9.9 6.3 0.2 4 0.84 51.6 38.9 7.0 11.9 19.0 8.4 0.5 8 1.11 64.9 6.4 8.7 14.9 23.7 9.7 0.5 menegatti et al. ‐ peach rootstocks production 163 reflects the accumulation of reserves and ensures greater resistance and adequate potential of the rootstock (souza et al., 2013). the index considers two parameters in a single indicator, and it can be used as a guide to determine the quality of the plants. the balance of the plants growth confirmed by the h/sd ratio may have been favored by the use of crf. the encapsulated fertilizers, such as crf, allow the slow release of nutrients through a porous struc­ ture (serrano et al., 2006), becoming available to the plants root system over time and according to their nutritional need, avoiding the leach and loss observed in conventional fertilizers. in addition to the h/sd ratio, the dqi is a good indicator of plant quality. for its calculation, it con­ siders the plants robustness and biomass distribution balance. therefore, the higher the value, the better the quality standard of the plants will be (gomes and paiva, 2011). the ideal value considered for the dqi is approxi­ mately of 2.00 (gomes and paiva, 2011). in our study, the highest dqi was close to 2.2 for the estimated mted of 6.18 g l­1 in plants grown in a greenhouse. however, for the plants kept in the external environ­ ment, the highest dqi value was of 1.83 with an mted of 5.72 g l­1, which is lower than the ideal value (fig. 4b). previous research from dutra et al. (2016) and zamunér filho et al. (2012), in addition to the results obtained in the present study, agree with the results for all morphological variables evaluated. a quadratic positive response was obtained proportionally to the increase of the crf doses up to the mted (of approx­ imately 6.2 g l­1). it proves that the plant will not have higher responses if a dose higher than the mted is applied. the δsd, evaluated every 15 days after the begin­ ning the experiment, is presented in figure 5. for the plants cultivated in a greenhouse, the use of the doses of 4 and 8 g l­1 of osmocote®, incorporated into the substrate, were proven efficient for the pro­ duction of rootstocks suitable for grafting after 90 days because they presented a final sd mean of 5.1 mm and 5.5 mm, respectively. considering the aforementioned results and the relevance of the sd variable in the production of peach rootstocks, we suggest the incorporation of at least 4 g l­1 of osmocote® into the commercial sub­ strate and the cultivation of the plants in a green­ house. those conditions can increase the efficiency of the fertilizer use to obtain plants that can be graft­ ed after 90 days. the effects of different environments on the pro­ duction of peach rootstocks cv. okinawa were reported by reis et al. (2010). they reached the ideal point for grafting after 179 days in a protected envi­ ronment. schmitz et al. (2014), evaluating the pro­ duction of peach rootstocks cvs. capdeboscq and okinawa, in three production systems, reached the grafting point after 154 days. fischer et al. (2016), researching the influence of the stratification period on wet cold in the emer­ gence and production of several peach rootstocks in the field, obtained materials suitable for grafting after 240 days. these results indicate that the use of crf and a protected environment are promising in the shortening of the productive cycle, as the grafting fig. 5 ­ increase of stem diameter, over time, of peach plants cv. capdeboscq, in relation to the dose of the crf and two cultivation envi­ ronments (a) open air and (b) greenhouse. adv. hort. sci., 2020 34(2): 157­166 164 rates and accelerates the respiratory metabolism, reducing the growth rate not only for sd, but for all morphological variables (afonso et al., 2017; bassi et al., 2018). another factor that may have contributed to reduce the plants growth in external environment was the crf formulation. the granules contain a homogeneous combination of nutrients and are covered by an organic resin that regulates the nutrients release proportionally to the substrates temperature and humidity (melo júnior et al., 2014). in addition to the high tempera­ tures, the heavy rainfall can contribute to accelerate the release of crf nutrients, resulting in leaching losses. according to the data provided by the agroclimatology station of pelotas (eapel, 2017), between october 2016 and january 2017, the cumu­ lative rainfall reached 549 mm, with a monthly aver­ age of approximately 137 mm, concentrated in three to four days of each month. the intense and concen­ trated precipitation in a short period of time may have caused greater leaching of the nutrients present in the soil solution that had the plants in external environment, reducing the efficiency of the fertilizer use and resulting in lower values for the evaluated variables in comparison with the plants grown in a greenhouse, which did not suffer the influence of the precipitation variable. considering the results obtained in this study, it was suggested that the control of the environment for plant cultivation provides greater efficiency in the use of productive resources (nutrients, water, tem­ perature, light and others). in addition to these fac­ tors, the use of crf incorporated into the substrate contributes to cause precocity in the production of peach rootstocks (reduction of the period to reach the grafting point) with a high­quality standard. 4. conclusions considering the results obtained in this study, the cultivation of plants in a greenhouse is proposed, since it provides the best conditions for the use of crf by the plants, and the concomitant use of the minimum dose of 4.0 g l­1 because it reduces the period to reach the grafting point to 90 days. acknowledgements this study was partially financed by the coordination for the improvement of higher point can be reached after 90 days. this outcome is probably due to the ready availability of nutrients and the maintenance of the plants under favorable environmental conditions, although these results must be validated for other cultivars of [prunus persica l. batsch] rootstocks with potential use in brazil, such as cvs. flordaguard, okinawa and the tsukuba series (menegatti et al., 2019 a). the greatest δsd occurred between 30 and 60 days after transplantation (fig. 5), regardless of the dose employed, which may be due to the slow releasing of the nutrients, a main characteristic of the fertilizer used, which resulted in the greater amount of nutrients available to the plants during the experi­ ment (huett and gogel, 2000). this result also sug­ gests that it is necessary to replace the mineral ele­ ments around 60 days after the first application in order to maintain the plants optimal growth rate. therefore, studies to elucidate the best crf replace­ ment period are necessary in order to ensure contin­ ued growth through the plants different develop­ ment stages. the crf used in the production of peach root­ stocks proved to be a promising alternative in com­ parison with conventional fertilizers. the crf slowly and continuously releases nutrients to the plant, avoiding leach losses and volatilization. furthermore, it ensures a better use of nutrients and reduces the environmental and economic impact (especially by the nitrogen economy, which is an expensive and easily leachable element that has a great potential to pollute the environment). the negative aspect of using crf is the higher cost in comparison with conventional fertilizers. however, the application of the mted, with the purpose of maximizing the input use in the production of root­ stocks, has been proving to be an economically viable alternative if we consider the price increase of basic inputs. other characteristics to be considered are the conventional fertilizers high susceptibility of leaching due to the frequency of irrigation in the nursery and the need for parcelled applications, which are driven by higher production costs (melo júnior et al., 2014). the lowest δsd 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c. taiti 1 (*), e. marone 2 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 bioscienze e tecnologie agro-alimentari e ambientali, università di teramo, via r. balzarini, 1, 64100 teramo, italy. key words: flavors and off-flavors, panel test, partial least square-discriminant analysis (pls-da), ptr-tof-ms, volatile organic compounds (vocs). abstract: international olive oil council (iooc) states chemical and organoleptic parameters to classify the commercial grade of olive oil. finding tools or analytical procedures able to support the organoleptic evaluation would be helpful to streamline and facilitate the commercial classification. the aim of the present study was to evaluate a new tool and validate a procedure that allows a fast and non-invasive volatile compounds detection system, able to assign each sample to its right trade category. moreover, we tried to test the capability of ptr-tof-ms in grading olive oils according to their fruity intensity levels. a total of 273 olive oil samples collected from argentina (21), chile (10), italy (191), morocco (17), tunisia (4) and eu (30) were analyzed and classified through: (1) panel test and (2) ptr-tof-ms analysis. on the whole ptr-tof-ms data evoo and not evoo as resulted by panel test were clustered by pca in two main groups and correctly classified by pls-da model, confirming the high confidence level (95%) in utilizing analytical spectral data for helping panel test and able to easy monitoring the quality formation in the oils, by a fast and cheap control from harvest until the store. the eight protonated masses detected as vip by the model may be linked to negative olfactory notes. finally, pca applied on the volatile profile of 122 classified evoo highlighted a shift of the samples distribution following the trend of the fruity intensity as assessed by the panelists. in conclusion, this trial confirmed the availability of a new, precise and simple analytical tool as the ptr-tof-ms, which coupled with an appropriate multivariate data analysis, allows to classify evoo according to their trade category and fruity intensity. 1. introduction the virgin olive oil is the only vegetable oil consumed without any refinement and characterized by a peculiar synthesis between taste and aroma. the importance of extra virgin olive oil (evoo) is due to its high content of oleic acid and phenolic compounds, which act as natural (*) corresponding author: cosimo.taiti@unifi.it citation: taiti c., marone e., 2017 evoo or not evoo? a new precise and simple analytical tool to discriminate virgin olive oils. adv. hort. sci., 31(4): 329-337 copyright: © 2017 taiti c., marone e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2017 accepted for publication 28 november 2017 ahs advances in horticultural science invited paper adv. hort. sci., 2017 31(4): 329-337 330 antioxidants (bendini et al., 2007). its composition makes it not only a food and dressing, but also a product able to protect the human organism from some dysfunctions and pathologies (marone and fiorino, 2012). as reported by aued-pimentel et al. (2013), evoo has unique characteristics compared to other vegetable oils, such as exceptional sensory and nutritional attributes, therefore worldwide the olive oils are the most valuable ones with a price (normally 3-5 times) higher than other edible oils (zou et al., 2009). as a consequence, in the last years, some adulterations of evoo with olive oils of lower quality, or with oils of different botanical origin (catharino et al., 2005; vlachos et al., 2006) have been found. as defined by the international olive oil council (iooc), olive oil is split in trade categories of different quality and commercial value. because the high commercial value, and the relatively low availability against a high consumption, some traders and bottlers are prompted to sell as evoo inferior olive oils that does not reflect the parameters established by the iooc. according with the iooc rules, the trade class attribution depends not only on chemically defined parameters (i.e. free acidity and peroxides index) but also on a sensory evaluation (se) that assesses off-flavors and fruity presence and intensity. therefore the evoo is the only traditional food that must be tested through a panel test. taste and aroma are determined by the presence and the amount of peculiar volatile organic compounds (vocs), giving to the product unique appealing proprieties. on average the olive oil contains more than 100 volatile compounds belonging to different chemical categories (guadarrama et al., 2000). vocs emission by the olive fruit and/or olive oil is mostly related to oxidative reactions (i.e. due to injuries during the fruits crushing and malaxation processes). vocs develop according to distinctive biosynthetic pathways and, among these, the ‘‘lipoxygenase (lox) cascade’’ determines the enzymatic splitting of polyunsaturated fatty acids (linoleic and linolenic) with the ‘‘controlled’’ production of aldehydes, ketones, alcohols, carboxylic acids, esters and other vocs (angerosa et al., 2004; kalua et al., 2007). the importance of the se for the olive oils, is due both to its ability to identify the positive attributes and also to evaluate the defects (peri and rastelli, 1994). indeed, the volatile compounds can be used: (a) to discriminate evoo and virgin olive oil (voo); and (b) as quality parameters, being the vocs responsible especially for the green notes and fruity of high-quality evoo oils (gomez-rico et al., 2006). while the chemical parameters are easily evaluated through chemical analyses, the flavor and off-flavors are assessed with more subjectivity through the sensory analyses. the se by the panel test is based on strict and laborious rules, and needs trained peoples; therefore, as currently planned, the panel test is time consuming and very expensive. thus, while the chemical analyses guarantees objectivity, repeatability and speediness, the sensory analysis does not allow this result. indeed, as reported by marone et al. (2017) the se presents some disadvantages such as: (1) subjectivity of the analysis which could influence the overall evaluation; (2) the need of a large number of trained panelist (8-12) to allow the statistical validation of the results; (3) a limited number of samples evaluable by each panelist a day. moreover, the results are difficult to generalize, because a lack of a common standard shared in the world, neither easy exploitable in any situations, nor to apply at any step of chain of olive oil making before sale (i.e. processing, storage). consequently, there is no doubt that the detection of each type of olive oil manipulation needs to be addressed to ensure a correct trade classification, quality and consumer price. currently, the most common used analytical techniques to detect vocs emitted by olive oil are both chromatographic and spectrophotometric methods, as the dynamic headspace gas chromatograph (dhsgc) (procida et al., 2016), electronic nose and electronic tongue (aparicio et al., 2000; cosio et al., 2007) and the proton transfer reaction-time of flight-mass spectrometer (ptr-tof-ms) (aprea et al., 2006; marone et al., 2017). the ptr-tof-ms shows a high resolution coupled to a rapid screening power of samples, it is easy to handle and does not need any sample manipulation (blake et al., 2009; taiti et al., 2017). moreover, this tool is applicable to any step of the olive oil production, from the processing to the market, including the product storage (marone et al., 2017). furthermore, as the ptr-tof-ms could work at temperature near those of the tasting, it should give as output a bulk of vocs at least similar to those perceived by panelists or consumers. a first attempt to directly link spectral data from ptr-tof-ms as protonated masses to the olfactory sensations perceived by the panelists, to distinguish evoo from not evoo, and consequently correctly classify the virgin olive oils in their trade category, was recently carried out by marone et al. (2017). in this cited work, although employing a low number of samples, it was possible to build up, in a taiti and marone evoo or not evoo? 331 statistically meaningful way, a color codified card highlighting some specific vocs that seem to characterize the off flavors as perceived by the panelist. starting from this result, the aim of the current work was to develop and to test a fast analytical method that combines efficiency, accuracy and reliability for a rapid screening and quantification of volatile compounds in olive oil samples. this analysis method should be helpful to: (1) detect the main defective odors and distinguish the olive oils trade categories; (2) understand if there is an accurate and precise correlation between the judgment provided by the panel test and the analysis of the volatile component by ptr-tof-ms; (3) evaluate different quality and types of evoo using the positive attributes (i.e. fruity and green notes). 2. materials and methods oil sampling analyses were carried out during 3 years of surveys (from 2015 to 2017) on the whole spectra of 273 olive oil samples, produced from 2012 to 2017 (table 1). the olive oils came from three different continent (africa, south america, and europe); most samples were obtained from producers or supermarkets, both blend or monocultivar stocks; in this last case, the most of the olive oil samples were obtained at the olive mill. to enhance and enlarge the samples set variability, evoo from supermarkets labeled as origin were acquired together with “aged” samples (certainly processed two or more years before to be analyzed). for each sample, two filled dark bottles of 250 ml were collected and quickly sent to the storage refrigerated room (17°c) until the organoleptic and vocs analyses were carried out. finally, for some samples, the vocs and se analyses were repeated during the three years of analysis. panel test after the spectrometric determinations all samples were submitted to a panel test. all panels were organized according to the official e.u. olive oil sensory analysis regulation (n. 2568/91 and its successive modifications) (table 1). each taster on the panel shall enter the intensity of the negative and positive attributes on the 10-cm scale in the profile sheet. the oil is graded by the panel leader in line with the median of the defects and the median for the fruity attribute. according to the reference ranges, an olive oil is graded as extra virgin if the median of the defects is 0 and the median of the fruity attribute is above 0. in the present work, all the samples that did not result evoo were classified as not evoo, without any further distinction. volatile compounds detection measurements were performed with a chemical ionization mass spectrometer (ptr-ms) equipped with a time-of-flight (tof) analyzer (ptr-tof 8000 model, ionicon analytik, innsbruck, austria) in its standard mode and using h3o+ as ions for the chemical ionization. ptr-tof-ms has some advantages compared to the other traditional electron ionization such as: reduced fragmentation which eases compound identification and guarantees high sensitivity with a very high time resolution and no need of sample treatments (taiti et al., 2017). previously blake et al. (2009) provided a complete and detailed description of the ptr-ms technology. all the instrumental parameters used during the measurement were set as follow: a constant drift voltage of 600 v and a pressure of 2.20 ± 0.02 mbar were maintained in the reaction chamber and the instrument operated at a standard e/n value (electric field strength/gas number density) of 138 td (1 td = 10-17 cm2 v-1 s-1). each sample was prepared on the basis of the following protocol: 15 g of oil (t 25°c) were introduced in apposite glass jar (750 ml), afterwards were fluxed with clean air (zero air generator, peak scientific) for 120 seconds and subsequently were hermetically sealed and incubated for 60 seconds at 25°c inside an incubator. each jar was provided with inlet and outlet teflon pipes, which connect the glass jar to the ptr-tof-ms system and to the zero-air generator, respectively. two replicates of each sample were analyzed and the order of samples was randomized. besides, at the beginning of the experiment and always after three oils sample an identical empty jar was analyzed for background subtraction. headspace concentrations of each oil sample were subsequently averaged over the two replicates and used for further statistical analysis. the range of mass spectra was recorded in the range of 20-210 m/z at 1 spectrum per 1 second, and the mass calibration was based on m/z = 21.022 (h3o+), m/z = 59.049 (c3h7o+) and m/z = 137.132 (c10h17 +); the calibration was made before starting each files and, subsequently, all files were recalibrated off-line. data were recorded with the tofdaq software (tofwerk ag, switzerland) and all spectra were acquired and analyzed using a procedure previously reported by taiti et al., 2017. data were expressed in ppbv following a procedure adv. hort. sci., 2017 31(4): 329-337 332 described by lindinger and jordan (1998). then, the data obtained were filtered by eliminating peaks that were lower than a threshold of 0.50 ppbv and eliminating all signals relative to ions hard to quantify precisely. after filtration, data have been sent to the statistical analysis (fig. 1). provenience zone cultivar/blend number of samples get from: producer (1), supermarkets (2) processing campaign (a), or acquisition (b) year ptr-tof-ms analysis year evoo (0)/not evoo (1) argentina arbosana 4 1 2017 a 2017 1 (4) argentina blend 4 2 2012 b 2016 1 (4) argentina blend 4 1 2013 a 2016 1 (4) argentina blend 4 1 2014 a 2016 1 (4) argentina coratina 2 1 2015 a 2016 1 (2) argentina coratina 1 1 2017 a 2017 0 (1) argentina koroneiki 2 1 2017 a 2017 0 (2) chile arbequina 2 1 2012 a 2017 1 (2) chile arbosana 4 1 2012 a 2017 1 (4) chile arbosana 1 1 2013 a 2017 1 (1) chile koroneiki 1 1 2012 a 2017 1 (1) chile koroneiki 2 1 2013 a 2017 1 (2) italy arbequina 4 1 2012/13 a 2016 1 (4) italy arbequina 3 1 2013/14 a 2016 1 (3) italy arbequina 7 1 2015/16 a 2016 0 (7) italy arbequina 5 1 2016/17 a 2017 0 (5) italy arbosana 3 1 2015/16 a 2016 0 (3) italy arbosana 2 1 2016/17 a 2017 1 (2) italy blend 2 1 2012/13 a 2017 1 (2) italy blend 11 1 2015/16 a 2015 0(11) italy blend 12 1 2015/16 a 2016 0(12) italy blend 14 1 2016/17 a 2017 0(10) 1(4) italy blend 17 2 2017 b 2017 0(13) 1(4) italy carolea 5 1 2013/14 a 2015 1(5) italy frantoio 8 1 2013/14 a 2015 1(8) italy frantoio 2 1 2015/16 a 2015 0(2) italy gentile di chieti 9 1 2015/16 a 2015 0(9) italy gentile di chieti 4 1 2015/16 a 2016 0(4) italy intosso 7 1 2015/16 a 2015 0(7) italy intosso 4 1 2015/16 a 2016 0(4) italy itrana 7 1 2015/16 a 2015 0(7) italy itrana 4 1 2015/16 a 2016 0(4) italy koroneiki 3 1 2015/16 a 2016 0(3) italy koroneiki 4 1 2016/17 a 2017 0(4) italy leccino 5 1 2013/14 a 2015 1(5) italy maurino sel. vittoria 3 1 2015/16 a 2016 0(3) italy maurino sel. vittoria 4 1 2016/17 a 2017 0(4) italy oliana 5 1 2016/17 a 2017 1(5) italy olivastra seggianese 18 1 2015/16 a 2016 0(3) 1(15) italy peranzana 5 1 2015/16 a 2015 0(5) italy peranzana 4 1 2015/16 a 2016 0(4) italy sikitita 5 1 2015/16 a 2016 0(5) italy sikitita 5 1 2016/16 a 2017 1(5) morocco arbequina 2 1 2012/13 a 2017 1(2) morocco picholine maroccaine 3 1 2014/15 a 2016 1(3) morocco picholine maroccaine 3 1 2014/15 a 2017 1(3) morocco picholine maroccaine 3 1 2015/16 a 2016 1(3) morocco picholine maroccaine 6 1 2015/16 a 2017 1(6) tunisia koroneiki 4 1 2012/13 a 2017 1(4) u.e. blend 5 2 2016 b 2016 1(5) u.e. blend 25 2 2017 b 2017 1(25) table 1 description of 273 olive oil samples in relation to provenience zone, cultivar, acquisition from producer or supermarkets, processing campaign or getting year, ptr-tof-ms analysis year, and panel test judgement (evoo/not evoo) taiti and marone evoo or not evoo? 333 multivariate data analysis a principal component analysis (pca, unsupervised method) was applied to the spectral data of 273 olive oil samples, submitted to a logarithmic transformation and mean centering as pre-processing. computations were performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab® r2015b (mathworks inc., natick, ma, usa). a multivariate partial least squares-discriminant analysis (pls-da, supervised method) was applied on the spectra of the 273 olive oil samples, to develop a model for differentiating evoo from not evoo. as pre-processing data, they were submitted to a logarithmic transformation and auto-scaling. the training set (85% of the samples) allowed to select the optimal number of latent variables (lvs) throughout the calibration and cross validation phases. the training and validation subsets were obtained by the euclidean distances based on the algorithm of kennard and stone (1968). the test set (prediction) consisted of 15% of the samples previously removed from the dataset. as cross validation procedure, venetian blind with 10 splits and 1 sample per split was chosen. the performances of the model were evaluated by the number of correct assignments and the root-mean-squared error of cross-validation (rmsecv), and prediction (rmsep). the optimal number of lvs resulted associated to the minimum error and misclassification rate of the calibration dataset. the reliability of the model was tested by confusion matrices. the threshold to assign a sample to a class was chosen based on the bayes theorem, minimizing the number of false positives and false negatives. variable importance in projection (vip) scores (p=0.01) were also calculated. a random permutation of the class labels (permutation test) was also performed (500 iterations), so to generate nonsense datasets for comparison with the true model, to evaluate the probability that the model is significantly different from one casually built up under the same conditions. pls-da analysis was performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab® r2015b (mathworks inc., natick, ma, usa). a pca was then applied to the spectral (ptr-tof-ms) data of the 122 samples resulting evoo based on the panel test, previously submitted to a logarithmic transformation and auto-scaling. 3. results and discussion evoo or not evoo vocs emission by olive oil is characterized by the presence of different compounds belonging mainly to alcohols, esters, aldehydes, ketones, terpenes and hydrocarbons. c6 molecules are the main volatile compounds derived from polyunsaturated fatty acids fig. 1 schematic representation of oil samples analyses and classification using the ptr-tof-ms. this technique allows rapid and nondestructive vocs detection throughout the entire food-to-fork chain (e.g. oils) without any sample pretreatment. all data acquired by ptr-tof-ms were used to obtain analytical information regarding the quality of product and for trade categories, varieties and geographical origin classification applying different multivariate analyses. adv. hort. sci., 2017 31(4): 329-337 334 through the lipoxygenase pathway (cecchi and alfei, 2013), generally characterized by low molecular weight. these compounds easily come in contact with the olfactory cells and help to create flavor and sometimes off-flavor. according to marone et al. (2017), there is the possibility to directly relate the volatile profile obtained by ptr-tof-ms to distinguish evoo from not evoo, and, as a consequence, to correctly classify the virgin olive oils in their trade category. to confirm the preliminary results obtained by marone et al. (2017), and validate the new procedure and methodology, we used a huge number of samples that were collected and analyzed in different years. in the present work, to define their trade category, 273 olive oil samples were submitted to the se, that classified 151 samples as not evoo, and 122 as evoo. by analyzing each oil sample (table 1), 63 volatile compounds were detected within a mass range of m/z = 20-210 (data not shown). pca applied to the whole dataset (ppbv) allowed to get a first general overview of the data distribution. two main groups of evoo and not evoo were clearly highlighted (fig. 2) in the bidimensional space of the first two components, despite the great variability present in the original data set, due to the great diversification in the olive oil samples. this variability is also evidenced by the need to consider the first 7 components to justify 90.17% of the total variance (respectively: 60.26%, 12.56%, 5.17%, 3.81%, 3.39%, 2.78, and 2.20%). the data ordination clearly highlights that the vocs spectra provided by not evoo samples were well distinguishable from those of the evoo, with a few partially overlapping zones in the upper right and bottom left quadrants. this behaviour indicates a different spectral distribution between flavors and off-flavors, confirming the same result obtained by the se. subsequently, a partial least squares discriminant analysis (pls-da) approach was applied to determine the trade category of the olive oil samples. a seven-component pls-da model, evaluated by its performances indicators (table 2), resulted robust to discriminate the not evoo from the evoo samples in the model/validation data set, and in the independent test set. the optimal number of latent variables (lvs), associated to the minimum error rate and the minimum number of not assigned samples, resulted in 7 (table 2). the permutation test table 2 pls-da statistics for each y-block (class 1 = not evoo; class 0 = evoo) related to 273 olive oil samples. sensitivity (se); specificity (sp); class error, rmsec, rmsecv, and rmsep for calibration (cal), cross validation (cv), and prediction (pred), respectively. confusion matrices for calibration, cross validation, and prediction statistics lvs se (cal) sp (cal) se (cv) sp (cv) class. error (cal) class. error (cv) class. error (pred) rmsec rmse cv rmse p not evoo 7 0.972 0.968 0.954 0.960 0.029 0.043 0.042 0.206 0.261 0.226 evoo 0.968 0.972 0.960 0.954 confusion matrices classes matthew's correlation coefficient1-not evoo 0-evoo calibration results p re d ic te d a s 1-not evoo 105 4 0.940 0-evoo 3 121 cross validation results 1-not evoo 103 5 0.914 0-evoo 5 120 prediction results 1-not evoo 16 2 0.903 0-evoo 0 22 fig. 2 pca ordination of 273 olive oil samples. green = not evoo, red = evoo. taiti and marone evoo or not evoo? 335 pls-da model also allowed to evidence the significant (>1.5) vip scores, indicating the role of the selected protonated masses to differentiate the two classes (fig. 3). vip scores reported in figure 3 confirm the results of our preliminary work (marone et al., 2017). in particular, the masses m/z = 47.050 (tentatively identified (ti) as: ethanol), m/z = 61.030 (ti: acetic acid), m/z = 75.040 (ti: propanoic acid) and m/z = 89.060 (ti: butanoic acid) resulted as factors able to distinguish evoo from not evoo. indeed, ethanol and acetic acid are generally considered as compounds deriving from microbial alterations due to a long time of olive storage before processing (morales et al., 2000) and therefore represent a known defect. likewise propanoic acid and butanoic acid are both considered defective compounds, that can be linked to fermentation processes in olive fruits as a long time of storage (angerosa et al., 1996) or related to the sugar fermentation (morales et al., 2013). overall, for all the samples evaluated, it is interesting to note that the procedure applied in this study to discriminate evoo from not evoo is not affected by factors such as: year under analysis, harvesting year, variety and geographical origin. in fact the model resulted significant at 95% confidence level only considering as classification (variability) factor the evoo/not evoo distinction. classification of different evoo fruity intensity evoo are currently also labeled according to the fruity intensity perceptions (robust, medium, delicate), based on the iooc regulation (coi/t.20/doc. no 15/rev. 8, 2015). thus, we tried to evaluate the different evoo fruity intensity using the dataset provided only by the vocs profile of the 122 evoo samples (according to the panel test) (table 1) through a indicated that the model is significant at 95% confidence level. in fact, the probability of model insignificance vs. permuted samples resulted 0.0 based on the wilcoxon and sign test, both in self-prediction and cross-validated, and 0.005 by the rand t-test. the model successfully classified 96.9% of samples into their trade category based on the panel test results in fitting, 95.5% in cross validation (internal validation) and 95% in prediction (external validation). that is, as reported in the confusion matrices (table 2), in the calibration, on a total of 233 samples, 226 were correctly classified, while 3 resulted false positive (predicted as evoo from the panel test, but classified as not evoo by the spectrometer) and 4 false negative (predicted as not evoo from the panel test, but classified as evoo by the spectrometer). in the cross validation, 223 samples were correctly classified, while 5 resulted false positive, and 5 false negative. in the prediction results, on 40 samples, 38 were correctly assigned to their right class, while only 2 resulted false positive. the occurring of false positive (3 samples in prediction, judged as evoo by the panel test, and classified as not evoo by the spectrometer), can be related to the fact that all compounds (including off-flavors) are only perceived by the human olfactory when they exceed their specific threshold values (morales et al., 2013). thus, we can assume that, below this threshold values, the presence of a given compound linked to a defect is not perceived by the human olfactory, but is inexorably detected by the spectrometer. on the other hand, only a few borderline olive oils judged as not evoo by the panelists but classified as evoo by the tool (false negative) were detected. a scores plot of the first two components of the pls-da model for all oil samples is shown in figure 3. the fig. 3 score plot (lv1, lv2) of the pls-da model. green = not evoo, red = evoo; vip scores > 1.5. adv. hort. sci., 2017 31(4): 329-337 336 pca analysis. the first two components explained about 63% of the total variability, and the derived scatterplot (fig. 4) showed three groups of samples that are rather well separated. it is interesting to note that linking the fruity score assigned by the panel test to each sample, the three groups distributed in the chart according to their fruity intensity (fig. 4), even if the samples grouped by the tool at the bottom of the figure (fig. 4) show vocs profiles relatively close, while the fruity intensity scores attributed to the same samples by the panelists ranged from 4 to 7. according to this chemometric approach, the subjectivity of the panel test becomes evident at intermediate values of fruity intensity. pca analysis also underlines two outliers group. in the first one, labeled with “m”, the three samples belonging to cv. maurino sel. vittoria harvested in year 2016 are found; this can be linked to peculiar flavor notes characterizing this tuscan clone, that showed the highest amount of terpene compounds compared to all other samples (data not shown). the second one, represented by a few samples separated from the central bulk and shifting to the right part, labeled with “s” (fig. 4), is formed by samples with particular flavor notes (data not shown). these samples, belonging to the cv. sikitita, as reported by garcía-gonzalez et al. (2010), are in fact characterized by typical aromas. associating the fruity score assigned by the panel test to each sample, it is remarkable as the entire aromatic profile detected by the ptr-tof-ms seems to be linked to changes in the amount of masses within the spectra rather than to the presence of specific compounds in the human olfactory perception of the fruity intensity. 4. conclusions the chemometric classification model proposed in this trial and based on the vocs fingerprint acquired by the ptr-tof-ms allows to distinguishing olive oil samples of different trade category. in particular, it was demonstrated that: (1) the entire volatile profile can be useful to classify oils belonging to different commercial categories (as the panel test), (2) the different qualities and types of evoo can be split by using the fruity intensity. the accuracy of classification proposed is very high and it is more efficient than that obtained by other authors using different tools. indeed, this tool does not require any sample pre-treatment and allows identifying compounds with low molecular weight (i.e methanol, ethanol, etc.) compared to other ones. given our results and the emerging need of the olive oil sector that requires the developmental analytical tools to support or integrate the panel test, this work opens the way for the use of ptr-tof-ms coupled with an appropriate multivariate analysis, as a quick and cheap tool with high confidence level and fig. 4 pca ordination of 122 extra virgin olive oil samples. the objects key color indicates fruity intensity scores as evaluated by the panel test increasing from blue (fruity = 1) to yellow (fruity = 8.5). black circled samples indicate: maurino sel. vittoria (m) and sikitita (s). taiti and marone evoo or not evoo? 337 comparable to the panel test, for the olive oil quality identification. references angerosa f., lanza b., marsilio v., 1996 biogenesis of ‘‘fusty’’ defect in virgin olive oils. grasas aceites, 47: 142-150. 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completely randomized design, in a 4x5 factorial scheme, with four pepper cultivars [brs moema biquinho yellow (biq. yellow), airetama biquinho red (biq. red), boyra habanero red (boyra hab. red), brs seriema tupã bode red (tupã bode red)] and five light spectral qualities, being blue leds (b-leds); red leds (rleds); blue+red leds (br-leds); white leds (w-leds) and fluorescent lamp (fl) carried out germination and vigor analysis, with four replicates of 50 seeds. for this, the seeds were conditioned inside gerbox® boxes and kept in a growth room. the biq. yellow and boyra hab. red peppers showed the highest potential of germination and vigor, respectively, indicating high physiological quality. in general, the light spectral qualities provide differentiated responses in the initial development of the pepper cultivars, being the reduction of the percentage of dead seeds favored by the spectrum br-leds and w-leds. the root fresh mass is increased by all lights, except r-leds. the fresh mass of the aerial part presents positive results in the fl lamps. shoot length is favored by the r-leds. 1. introduction capsicum peppers are closely related to the brazilian richness culture and are a valuable part of the biodiversity heritage, being cultivated an immense variety, sizes, colors, flavors and pungences (neitzke et al., 2008). the brazilian production is around 11,071 tons (conab, 2015), being the state of são paulo considered the largest producer. among the factors that regulate plant production, the light plays an important role because it is an important regulator of growth and development of the plant, as it regulates morphological characteristics and acts as an energy source in the primary metabolism and in the photosynthetic process (simlat et al., 2016). the qualitative or quantitative characteristics of growth and morphogenesis are influenced by the quality of the supplied light, affecting the plants development, mainly, by photomorphogenic alterations (heo et al., 2002; rezende et al., 2008). (*) corresponding author: daani_fontana@hotmail.com citation: fontana d.c., becker c.e., pinheiro m.v.m., pretto m.m., dos santos j., caron b.o., schmidt d., 2019 impact of light quality on the physiological characteristics of capsicum chinense seeds. adv. hort. sci., 33(2): 235-243 copyright: © 2019 fontana d.c., becker c.e., pinheiro m.v.m., pretto m.m., dos santos j., caron b.o., schmidt d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 march 2018 accepted for publication 22 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 235-243 236 in recent years, light-emitting diodes (led) have been widely used as alternative light source potential for plants (simlat et al., 2016). among the advantages of leds systems are visible light emission and low heat production for long periods, with a specific wavelength, color and lighting flexibility, reduction of electrical consumption and toxic substances, as well as improved lifetime (carvalho, 2007; yeh and chung, 2009). this technology becomes promising for the growth of plants in a controlled environment, such as in tissue culture and also in the supplementation of growth chambers and greenhouses (yeh and chung, 2009). different wavelengths of light can trigger a variety of responses in plants (simlat et al., 2016). for example, red, blue, green and white leds lights were tested in different species, mainly forest ones, demonstrating the promotion of seed germination and subsequent development (gonçalves et al., 2006; victório and lage, 2009). red light may promote seed germination and root development (daud et al., 2013), shoot elongation (kim et al., 2004; araújo et al., 2009), fresh mass increment (sorgato et al., 2016), and an increase in shoot length (cybularzurban et al., 2007), among others. combinations of blue and red leds may promote biomass increase (gu et al., 2012; maluta et al., 2013; da silva et al., 2016) and increase of the root system (gu et al., 2012). the blue wavelengths tend to improve stomatal conductance (hogewoning et al., 2010), affect phototropism (johkan et al., 2010), and increase the rate of photosynthetic pigments production. however, plants exhibit a wide range of morphological and phytochemical plasticity in response to each type of wavelength of light (macedo et al., 2011). many studies report that leds can modify seed germination and plant growth and development (gonçalves et al., 2006; victório and lage, 2009; daud et al., 2013; da silva et al., 2016). although there are reports on the development of seedlings of the genus capsicum spp. in light qualities (da silva et al., 2016), the effects of the light spectral qualities on the germination and vigor of capsicum chinense have not yet been analyzed. thus, the objective of this work was to verify the impact of the light spectral qualities on the physiological quality of capsicum chinense seeds. 2. materials and methods plant material and conduction of the experiment the work was conducted at the plant tissue culture laboratory of the universidade federal de santa maria, campus frederico westphalen rs (federal university of santa maria, campus frederico westphalen rs), in november 2016. the experiment was conducted in a completely randomized design, in a 4x5 factorial scheme, with four pepper cultivars (capsicum chinense) and five light spectral qualities, totaling 20 treatments, with four replicates of 50 seeds each tratament, totalizing 4000 seeds tested. four cultivars of pepper were used [brs moema biquinho yellow (biq. yellow), airetama biquinho red (biq. red), boyra habanero red (boyra hab. red) and brs seriema tupã bode red (tupã bode red)], and five light spectral qualities [tec-lamp® blue leds (450 nm) b-leds; red leds (660 nm) r-leds; blue (450 nm) + red (660 nm) brleds in the ratio of 40% and 60%, respectively; white leds w-leds; and special daylight type fluorescent fl (osram®, brazil)]. the seeds were placed inside gerbox® boxes with lids (11x11x3 cm) containing two sheets of germitest® paper (in box dimensions), moistened with 0.2% kno3 solution (dissolved in distilled water), in proportion to 2.5 times the dry paper weight, as described in the regras analis de sementes (rules for seed analysis) (mapa, 2009). the gerbox boxes were maintained in a growth room under temperature of 25±2°c and a luminous intensity of 36 µmol m-2 s-1 for 14 days. analyzed variables for the germination test, counting was performed by seven and 14 days after the test installation. at the first count (fc), the normal seedlings were counted and the values expressed as a percentage (%), at 14 days the following variables were analyzed: percentage of germination (pg), percentage of abnormal seedlings (pas), percentage of hard seed (phs) and percentage of dead seeds (pds) (mapa, 2009). according to mapa (2009), dead seeds are the seeds that do not germinate at the end of the test, are neither hard nor dormant, and are usually softened, attacked by microorganisms and show no signs of germination. already, the hard seeds are those that remain without absorbing water for a longer period than normal and are therefore at the end of the test with the appearance of seeds newly placed on the substrate. for the root length (rl) and shoot length (sl) variables, 10 seedlings of each replicate were measured for all light qualities, being measured with a digital caliper. for the fresh mass of the aerial part (fmap) and root fresh mass (rfm), the same seedlings were fontana et al. light quality impact on capsicum chinense seeds 237 used for the sl and rl measurement, with the values referring to the 10 seedlings. afterwards, the seedlings were conditioned in paper bags and kept in a forced air oven at 60°c, until constant weight was reached, to determine the dry mass of shoot (dms) and dry mass of the root (dmr). the germination speed index (gsi) was calculated by the sum of the number of germinated seeds per day, divided by the number of days between sowing and germination, following the maguire’s methodology (1962). gsi= (g1 / n1) + (g2 / n2) + ⋯ + (gn / nn) (1) where gsi = g 1 , g 2 , ..., gn = number of seedlings computed in the first, second, third and last count; n 1 , n 2 , ..., nn = number of days of sowing to the first, second, third and last count. the obtained data were submitted to analysis of variance, and the interaction between pepper cultivars and light spectral qualities was evaluated, and when they were significant, the averages were compared by the tukey’s test, at 5% of error probability, using the statistical program assistat 7.7 beta. 3. results the analysis of variance showed significant interaction for the pepper cultivar factors x light spectral qualities only for the fresh mass of the aerial part (fmap) and shoot length (sl) variables. the variables of the first count (fc), percentage of germination (pg), normal seeds (ns), abnormal seeds (as), hard seeds (phs), germination speed index (gsi), root fresh mass (rfm) and root length (rl) were significant only for the pepper cultivars factor. root length (rl), root fresh mass and percentage of dead seeds (pds) variables were significant for the light spectral qualities factor. on the other hand, the dry mass of the aerial part (dmap) and dry mass of the root (dmr) variables were not significant (data not shown) by the f test, at 5% of error probability. the pepper cultivars differed for the percentage of normal seeds (ns) and abnormal seeds (as). the highest percentages of ns were observed for biq. yellow pepper with approximately 88%, being the same as biq. red pepper (85%), and differing from the others (p<0.05) (fig. 1a). for the variable as, the tupã bode red cultivar presented the highest values, with 12.30% of abnormality, being higher than the others, and the lowest percentages were verified for biq. yellow pepper, with 4.40% (fig. 1b). regarding the percentage of hard seeds, the boyra hab. red cultivar had the highest values, with an average of 11.8%, differing significantly from the others (fig. 1c). the biq. yellow pepper cultivar showed the highest percentages of germination in the evaluation of the first count (fc), with 94.8%, differing significantly from the boyra hab. red cultivar (fig. 1d). fig. 1 percentage of normal seeds (ns-a), abnormal seeds (asb), hard seeds (hs-c) and germinated (gs-d) of four pepper cultivars, being biq. yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities. *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). adv. hort. sci., 2019 33(2): 235-243 238 for the root length variable, the boyra hab. pepper presented the highest values with approximately 51 mm in length, being statistically different from the other cultivars. the lowest averages were observed in biq. yellow and biq. red peppers with 36 and 35 mm, respectively (fig. 2a). the boyra hab. red pepper again stood out for the root fresh mass variable, with 0.072 gram for 10 seedling differing significantly from the other ones evaluated (fig. 2b). for the germination speed index (gsi), the biq. yellow pepper stood out, presenting 23.70, being significantly similar to the biq. red and tupã bode red peppers, differing only from boyra hab. red pepper (fig. 2c). for the first counting of normal seeds the biq. yellow pepper showed the highest values with 84.1% of normal seedlings, differing from the other ones (fig. 2d). the light spectral qualities of br-leds and w-leds provided higher root length (rl), with 43.37 and 42.97 mm respectively, differing statistically from the fl (fig. 3a). for the root fresh mass variable, it was observed that the red spectrum promoted mass fig. 2 root length (a), root fresh mass (g for 10 seedlings) (b), germination speed index (c) and first counting of normal seeds (d) of four pepper cultivars, being biq. yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities. *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). fig. 3 root length (a), root fresh mass (g for 10 seedlings) (b) and percentage of dead seeds (c) of four pepper cultivars submitted to different light spectral qualities, being b-leds, r-leds, br-leds, w-leds and fl lamps. *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). fontana et al. light quality impact on capsicum chinense seeds 239 (table 1). r-leds spectral quality provided the highest averages for the shoot length variable, being higher to the other spectra. in this spectrum, the boyra hab. red and tupã bode red were superior to the other peppers. for biq. yellow pepper the r-leds, fl lamp and br-leds spectra conditioned the larger shoot length, statistically differing from the b-leds and w-leds. for biq. red pepper, the r-leds spectrum was statistically superior to the others (table 2). the boyra hab. red pepper presented superior performance to the others, presenting the highest average in shoot lenght (sl), being favored by the leds spectra and in disadvantage by the fl, differing significantly. shoot length of tupã bode red cultivar was favored by the r-leds light spectra qualities, differing significantly from the other spectra (table 2). 4. discussion and conclusions the biq. yellow pepper cultivar was superior to gsi, fc, ns, pg, presenting the lowest percentage of abnormal (as) and hard (hs) seeds (fig. 1). however, the boyra hab. red pepper cultivar has been highlighted for the rl (figs. 2a, 3a), sl (table 2), fmap (table 1), rfm (figs 2b, 3b) variables. the results sugreduction, with 0.013 gram for 10 seedling, differing statistically from the other light spectra, which presented higher mass (fig. 3b). correspondingly, it was observed that the red spectrum conditioned the highest percentage of dead seeds, with 6%, differing statistically from the br-leds and w-leds. the beleds and w-leds spectra conditioned low percentage of seed mortality (fig. 3c). for the biq. yellow pepper cultivar the light spectral qualities of w-leds, fl and br-leds showed the highest values of fresh mass of the aerial part (fmap), differing significantly from r-leds (table 1). for biq. red and boyra hab. red peppers, the fl light provided greater accumulation of fresh mass, presenting 0.194 and 0.283 gram for 10 seedlings, respectively, being statistically higher to the others (table 1). as for the tupã bode red pepper, the wleds light conditioned the largest fresh mass of the aerial part, differing significantly from the b-leds and r-leds (table 1). it was observed that the br-leds and fl lights provided a greater increment of fresh mass for boyra hab. red pepper, with 0.236 and 0.283 g for 10 seedling, respectively, differing significantly from the r-leds spectrum. the tested peppers presented similar responses in relation to the w-leds light spectrum, not statistically different from each other table 1 fresh mass of the aerial part (g for 10 seedlings) of four pepper cultivars, being biquinho yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities, b-leds, r-leds, br-leds, w-leds and fl lamps cultivars fresh mass of the areal parts b-leds r-leds br-leds w-leds fl biq. yellow 0.144 bab 0.114 bb 0.182 ba 0.191 aa 0.192 ba biq. red 0.1233 bc 0.139 abbc 0.179 bab 0.169 aabc 0.194 ba boyra hab. red 0.196 abc 0.176 ac 0.236 aab 0.204 abc 0.283 aa tupã bode red 0.099 bc 0.138 abbc 0.160 bab 0.200 aa 0.183 bab cv 15% * different lowercase letters in the column or uppercase letters in the row represent significant statistical difference (tukey’s test p<0.05). table 2 shoot lenght of four pepper cultivars, being biquinho yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities, being b-leds, r-leds, br-leds, w-leds and fl lamps * different lowercase letters in the column or uppercase letters in the row represent significant statistical difference (tukey’s test p<0.05). cultivars shoot lenght (mm) b-leds r-leds br-leds w-leds fl biq. yellow 28.66 abb 34.28 ba 30.62 abab 29.26 bcb 34.15 aa biq. red 26.56 bc 37.65 ba 30.34 bbc 30.65 abbc 33.33 ab boyra hab. red 30.74 ab 43.14 aa 34.59 ab 33.92 ab 25.52 bc tupã bode red 29.075 abc 42.10 aa 33.89 abb 26.18 cc 26.53 bc cv 6.71% 240 adv. hort. sci., 2019 33(2): 235-243 gest that biq. yellow pepper has a higher germinative potential, while boyra hab. red pepper has greater vigor. the characteristics of germination and vigor are individual for each cultivar and variables between them. for most of them, the speed, uniformity and germination rate depends on external and internal factors to the seed (plue et al., 2010; demotesmainard et al., 2016), such as temperature, humidity, light, viability of the embryo and genetic factors, characterizing in this way, the differences verified between the pepper cultivars. in general, the use of high vigor seeds results in a good performance of the crops in the field through better establishment of seedlings and survival of seedlings. in this way, germination and vigor tests are important in order to choose the best pepper to be used. specific light spectra can act positively stimulating the germination process in some species (gonçalves et al., 2006), or can be indifferent to others. in general, species whose seeds present sensitivity to the light quality, the positive photoblastics, are considered pioneers in nature, since they require light stimulus to initiate their germination process (rebouças and santos, 2008). for the capsicum chinense pepper plant, as observed in this study, only the percentage of dead seeds was influenced by the luminous spectra tested where the r-leds spectrum caused a high number of dead seeds whilst the br-leds and wleds reduced the percentage of mortality. the other germination variables evaluated did not present responses to the spectra. red light has been reported to stimulate seed germination and root development (bewley and black, 1994; abdullateef and osman, 2011; daud et al., 2013). in addition to the quality, the luminous intensity in which the seeds and plants are submitted can also promote differentiated responses in the plant, as verified for capsicum chinense habanero, which presented increase in growth with the light intensity of 28 μmol m-2 s-1 (barrales-lópez et al., 2015). the luminous spectra used in this experiment were larger, with 36 µmol m-2 s-1, which may have masked the plant response. it is known that light in excess can result in reduction of the net photosynthetic rate, causing oxidative damage to the foliar tissues; only under appropriate light plants can be fully self-regulated to obtain the best status for absorption and transformation of light energy (yao et al., 2017). it is known that excess light can also promote photovoltaic changes in plants, leading to the production of reactive oxygen species (ros), which may have promoted mortality in seeds submitted to the red spectrum. after germination, it was possible to observe changes in the morphological characteristics due to the different light spectral qualities in which they were submitted. fluorescent light (fl), for example, provided a higher increase of fmap in the evaluated peppers. this spectral quality is the most used for in vitro growth of plant species. positive results in the increase of fresh mass of the aerial part were already found for curcuma longa in fluorescent light, followed by red light (~ 625-440nm) and yellow light (~565590nm) (de souza ferrari et al., 2016). naturally plants develop themselves under varied lights composed of a mixture of quality and quantity, which promotes the activation of several photoreceptors, among them phytochromes (rockwell et al., 2006). the highest values of shoot length were verified in r-leds spectral quality for capsicum chinense peppers, corroborating with other studies, which found an increase in root formation in cultures such as jatropha curcas and protea cynaroides (daud et al., 2013; wu and lin, 2013), and stevia rebaudiana (simlat et al., 2016). kim et al. (2004), observed stretching of the aerial part of chrysanthemums cultivated in vitro, under light in the red band. when the plants were submitted to the r-leds spectrum, some authors verified elongation in cattleya loddigesii (araújo et al., 2009), increase of fresh mass in dendrobium phalaenopsis (sorgato et al., 2016) and increase of shoot length for cattleya (cybularz urban et al., 2007), corroborating with the results found in this work. red light is effective for photosynthesis as the red emission spectrum fits perfectly with the photon energy required to reach the first excited status of a and b chlorophyll (singh et al., 2015). lights in blue and red spectra too are strongly absorbed by phytochrome through specific photoreceptors (mathews, 2010). these photoreceptors activate enzymes associated with the synthesis of auxins, growth hormone, and greater photosynthetic efficiency (sun et al., 1998), promoting an increase in growth, justifying the results found. in this way, plants that grow under these conditions have a good initial development, such as a well-formed root system, which allows for faster acclimation and better survival rates in the field (chandra et al., 2010; gruszecki et al., 2010). positive results for root fresh mass and root length were observed in br-leds spectrum. some studies indicate that combinations of leds with blue (30%) and red (70%) spectrum promoted an increase fontana et al. light quality impact on capsicum chinense seeds 241 in biomass of fragaria x ananassa and saccharum officinarum (nhut et al., 2003; maluta et al., 2013). results for anthurium andraeanum favored gains in fresh and dry matter in the combination of rb-leds, followed by white light (gu et al., 2012). when rbleds have been used in vitro propagation, a large part of the works observed a mass gain in both the root and the aerial part (gu et al., 2012; maluta et al., 2013; da silva et al., 2016). for chili culture (capsicum annuum l. cv. rubi gigante), shoot length and collar diameter were favored by treatment with br-leds light compared to white fluorescent light (fl) (da silva et al., 2016). for this species, the authors also point out that the different qualities of the light spectrum have little effect on the growth and development of seedlings. the photosynthetic pigments absorb light in the red and blue range, the red quality increas the photosynthetic rate, while blue quality improves the chloroplast development, chlorophyll biosynthesis and stomatal opening, thus, increasing the content of photosynthetic pigments (johkan et al., 2010; hogewoning et al., 2010; daud et al., 2013). these luminous spectra influence the primary and secondary metabolism in plant development, however, plants exhibit a wide range of morphological plasticity, and phytochemical in response to a given type and wavelength of light (macedo et al., 2011). in this way, some cultures are favored by the supply of these wavelengths, increasing their growth. the tested peppers showed good results for the root length and root fresh mass variables when submitted to the w-leds spectrum. positive results with the use of w-leds were obtained by wilken et al. (2014), in which they found more vigorous growth of musa spp. compared to the use of fluorescent lamps. for sugarcane plants, w-leds lamps promoted a higher number of shoots, in addition to a higher content of chlorophylls and carotenoids (ferreira et al., 2017). the emitted photons in the combination of brleds, suppose that more photoreceptors of the pepper seedlings received stimuli, which may have triggered some morphogenetic mechanism in more photoreceptor cells than when exposed to only one spectrum. similar assumptions were made by shimokawa et al. (2014) and chen et al. (2017). in this way, the positive results found for rfm and rl and the low seed mortality (sm), in the br-leds combination were explained. light is a signal that is received by photoreceptors, which regulate plant differentiation and growth (li et al., 2013). the quality of the emitted light by leds has promoted significant improvements in morphogenesis and differentiation in different species grown in vitro (gupta and jatothu, 2013). however, the effects and mechanisms associated with light quality may be peculiar to each species or cultivar (li et al., 2013; da silva et al., 2016). some explanations may be generalized, however, this specificity seems to be related to the different responses that were found for the vigor of the pepper cultivars. the biq. yellow and boyra hab. red present high potential of germination and vigor, respectively, indicating high physiological quality. in general, the light spectral qualities provide differentiated responses in the initial development of the peppers, being the reduction of the percentage of dead seeds favored by the spectrum br-leds and w-leds. the root fresh mass is increased by the all lights, except r-leds. the fresh mass of the aerial part presents positive results in the fl lamps. shoot length is favored by the r-leds. acknowledgements the authors would like to thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarship. references abdullateef r.a., osman m.b., 2011 effects of visible light wavelengths on seed germinability in stevia rebaudiana bertoni. int. j. biol., 3(4): 83. araújo a.g., pasqual m., rodrigues f.a., rodrigues j.d., castro e.m., santos a.m., 2009 crescimento in vitro de cattleya loddigesii lindl. em diferentes espectros luminosos associado a ácido giberélico. rev. ceres, 56(5): 542-546. barrales-lópez a., robledo-paz a., trejo c., espitiarangel e. rodríguez-de la o.j.l., 2015 improved in vitro rooting and acclimatization of capsicum chinense jacq. plantlets. 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cultivation. renew. sust. energ. rev., 13(8): 2175-2180. impaginato 13 adv. hort. sci., 2020 34(1s): 13­20 doi: 10.13128/ahsc­7444 postharvest aptitude of begonia semperflorens and viola cornuta edible flowers s. demasi, n.m. falla, m. caser, v. scariot (*) department of agricultural, forest and food sciences, university of torino, largo paolo braccini, 2, 10095 grugliasco (to), italy. key words: anthocyanins, antioxidant activity, fresh cut flowers, polyphenols, postproduction, pot plants, shelf­life. abstract: the edible flowers are sold as pot plants or fresh cut produce and are attracting interest recently thanks not only to their organoleptic characteristics but also to their content in bioactive molecules. however, there is little infor­ mation about the variations that these characteristics undergo during posthar­ vest. in this study, the productivity and longevity of begonia x semperflorens‐ cultorum hort. and viola cornuta l. pot plants were evaluated in an interior environment simulating the house conditions. besides, the effect of cold stor­ age (4°c) was evaluated on the aesthetic quality and the bioactive compounds content (total polyphenols, total anthocyanins, antioxidant activity through frap assay) of b. semperflorens and v. cornuta fresh cut flowers, using two dif­ ferent packaging, modelling a plastic box or a flowpack. the results suggest that v. cornuta could be a better choice for retailers because of its longer shelf life and better maintenance of its content in bioactive compounds, especially in the flowpack packaging. conversely, b. semperflorens could be more suitable as pot plant, showing more adaptability and flower production in a domestic envi­ ronment. 1. introduction numerous flowers have been used in culinary arts since ancient times both in europe, including rosa l. spp., calendula officinalis l., viola spp., and taraxacum officinale f.h. wigg (mlcek and rop, 2011; grzeszczuk et al., 2016; fernandes et al., 2017; scariot et al., 2018), and in asia and south­america, such as begonia spp. (laferrière, 1992; basurto­peña et al., 2003; zheng et al., 2018). nowadays, edible flowers are horticultural niche products, sold as pot plant or as fresh cut flowers, with increasing appeal for the food industry due to their organoleptic and healthy proper­ ties (kaisoon et al., 2012; grzeszczuk et al., 2016; lu et al., 2016). edible flowers improve the sensorial qualities of food by adding colour, fragrance, flavour and visual appeal to culinary preparations (kelley et al., 2001a; mlcek and rop, 2011; koike et al., 2015). in the third millennium, several studies revealed the chemical compo­ sition of many wild and cultivated flowers, highlighting the presence of (*) corresponding author: valentina.scariot@unito.it citation: demasi s., falla n.m., caser m., scariot v., 2020 ­ postharvest aptitude of begonia semper‐ florens and viola cornuta edible flowers ­ adv. hort. sci., 34(1s): 13­20 copyright: © 2020 demasi s., falla n.m., caser m., scariot v. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 november 2019 accepted for publication 4 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 13­20 14 important bioactive compounds, such as carotenoids and phenolics (lu et al., 2016). these phytochemicals with antioxidant activity are very important for plants, since they inhibit their natural senescence process, mainly caused by the presence of reactive oxygen species (ros) (mlcek and rop, 2011). during metabolism ros and other free radicals are produced in human body too, normally inactivated by an endogenous antioxidant system (mlcek and rop, 2011; loizzo et al., 2016). however, under stress con­ ditions, in high load situations, because of lifestyle or pathological situations, these free radicals can accu­ mulate, generating oxidative stress (loizzo et al., 2016) by reacting and damaging all types of biomolecules such as lipids, proteins, carbohydrates, and dna (kaisoon et al., 2012). if damaged dna is left unrepaired, it may become cancerous (mlcek and rop, 2011; kaisoon et al., 2012; li et al., 2014). thus, a diet rich in antioxidants, which can scavenge free radicals, can reduce the oxidative stress and may be a strategy to prevent some chronic conditions (kaisoon et al., 2012; loizzo et al., 2016; lu et al., 2016). epidemiological data showed that dietary pat­ terns were significantly associated with the preven­ tion of these chronic diseases, especially when rich in antioxidants (kaisoon et al. , 2012), including carotenoids and phenolics (koike et al., 2015; grzeszczuk et al., 2016). as a result of the increased knowledge of the edi­ ble flowers’ properties, the consumers’ demand of this kind of product is increasing worldwide (fernandes et al., 2017; pires et al., 2019; falla et al., 2020), thanks to the increased attention to the quali­ ty of foodstuffs and to the content of individual com­ pounds (rop et al., 2012; lu et al., 2016). however, edible flowers are highly perishable and have a short shelf life (petal abscission and discol­ oration, flower wilt, dehydration, and tissue brown­ ing start to appear 2­5 days after harvest), which lim­ its their marketability (koike et al., 2015; fernandes et al., 2018). fresh cut edible flowers are typically packaged in small, rigid, plastic boxes, in order to protect them from desiccation and to preserve their frail structure (kelley et al., 2003; kou et al., 2012). it is noteworthy that consumers eat with their eyes well before they taste with their mouths, thus it is important to maintain the visual appeal of a flower on market; quality is essential: consumers want more varieties of top quality plants with a longer shelf life (kelley et al., 2001 b). nevertheless, edible flower’s postproduction technology still receives less atten­ tion than that of other horticultural products, such as vegetables and fruits, because edible flowers’ pro­ duction is still low and it is a niche market (fernandes et al., 2018). temperature is usually the most important envi­ ronmental factor limiting shelf life of horticultural products (kelley et al., 2003): both respiration and transpiration processes are considered as the major causes of postharvest losses and poor quality in pro­ duce. thereby, controlling temperature of storage is very important since these factors directly influence the two metabolic processes mentioned above, extending the product’s shelf life (flores­lópez et al., 2016). a flower’s short shelf life may cause not only a rapid decrease in visual quality, but also a rapid loss of its nutraceutical compounds, however very few articles reported the effects of storage on quality of edible flowers, and even less investigated these effects on their nutraceutical compounds (landi et al., 2015). therefore, it would be interesting to deep­ en the knowledge on whether the loss of nutraceuti­ cal compounds in edible flowers during storage occurs more or less quickly than the loss of visual quality. this information could promote the con­ sumption of edible flowers at visual quality levels less than perfect, with minor flaws (kelley et al., 2001 b). thus, the aim of this work was to evaluate two common edible flowers’ species (begonia x semper‐ florens‐cultorum hort., commonly referred to as begonia semperflorens, and viola cornuta l.) as 1) pot plants, by evaluating the productivity and longevity in an interior environment simulating the domestic conditions; and 2) fresh cut flowers by eval­ uating the shelf life and the content in biologically­ active compounds (total polyphenols, anthocyanins) and antioxidant activity, when stored at 4°c, testing two types of packaging (a plastic box closed with its own lid or closed with a plastic film in a flowpack). 2. materials and methods pot plant postproduction the potted flowering plants of begonia semperflo‐ rens (10 plants) and viola cornuta (36 plants) were obtained from the nursery fratelli gramaglia (collegno, italy; 45°05’22.4’’ n, 7°34’26.4’’ e, 302 m a.s.l.). plants were kept at room temperature (about 20°c), in a peat­based substrate, hand­watered when needed, throughout the harvest period: 30 days for begonias and 11 days for violas. every two days demasi et al. ‐ edible flowers post‐harvest quality 15 opened flowers were harvested and weighed to eval­ uate the flowering longevity and productivity. for each plant, the number of flowers produced was counted and the weight of flowers suitable to be con­ sumed (opened flowers in good visual conditions) was calculated: namely each plant’s productivity. fresh cut flower postharvest fresh flowers harvested when fully open and in good visual conditions were put into plastic packages (ondipack 250 cc, 123x114x50 mm, polypropylene, plemet, france), 5 g of flowers for each box (fig. 1). two packaging methods were assessed, for both species: ­ plastic box closed with its own plastic lid (abbr. pp; 8.96 g); ­ plastic box without its lid, inserted into a bi­orient­ ed polypropylene plastic film (abbr. bopp; 6.04 g). the plastic films were folded and closed on three sides through heat­sealing (fr400, ferplast, cuneo, italy), modelling a flowpack. flowers were stored at 4°c, in refrigerators with a glass door [fiocchetti fridge, luzzara (re), italy], sim­ ulating markets’ shelves conditions, with four repeti­ tions per packaging type. every two days, each package was weighed, in order to obtain data about the flowers’ weight varia­ tion. the visual appeal of flowers was scored on a 9­ point scale based on visual observation of the degree of decay (aquino­bolaños et al., 2013; landi et al., 2018), where 9 was assigned to flowers without imperfections, 5 was the limit of marketability of the product (the limit of acceptability for the consumer), while 1 was the value of a decomposing flower, at the end of its life cycle. ultrasound extraction at the beginning of the trial, so that the day of harvest (t0) could be represented, about 5 g of flow­ ers of both b. semperflorens and v. cornuta were col­ lected from pot plants; the same was done after stor­ age, when the flowers reached grade 5 of the visual scale. one of the four packages stored per method was taken and the 5 g of flowers contained in it were stored at ­80°c until analysis. flower samples were grinded with liquid nitrogen, then 0.5 g of grinded plant material were put into a glass tube, to which 25 ml of a 50% aqueous meoh (methanol) solution were added. three repetitions were carried out for each sample. the tubes were put into the ultrasound extractor (23 khz, reussarl, drap, france) for 15 min­ utes at room temperature. the obtained phyto extract was filtered with paper filters (whatman filter papers no. 1, whatman, maidstone, uk) and the obtained solution was stored at ­20°c for further analysis. total polyphenols the total phenolic content was determined fol­ lowing the folin­ciocalteu method (singleton et al., 1999). the analysis was performed as follows: 750 µl of diluted 1:10 folin reagent were mixed with 150 µl of phytoextract and 600 µl of na2co3 (7.5%) in each plastic tube. samples were left in the dark at room temperature for 30 minutes. absorbance was mea­ sured at 765 nm by means of a spectrophotometer (agilent technologies, cary 60 uv­vis, santa clara, ca, united states), and the results were expressed in milligrams of gallic acid equivalents per 100 g of fresh weight (mg gae/100 g fw). total anthocyanins the total anthocyanin content in the extracts was determined through the ph­differential method as indicated by lee et al. (2005) and giusti and wrolstad (2005). the analysis was performed as follows: 1 ml of phytoextract was put into a 10 ml flask, and then made up to volume with an aqueous buffer solution at ph 1 (kcl and hcl). the same was made in a sec­ ond flask with an aqueous buffer solution at ph 4.5 (c2h3nao2and c2h4o2). samples were put in the dark at room temperature for 20 minutes. absorbance of both flasks was measured at 515 nm and 700 nm by means of a spectrophotometer (agilent technologies, cary 60 uv­vis, santa clara, ca, united states), and the results were expressed in milligrams of cyanidin­3­o­glucoside per 100 grams of fresh weight (mg c3g/100 g fw). fig. 1 ­ flowers of v. cornuta (a) and b. semperflorens (b) freshly harvested and put in the plastic package. adv. hort. sci., 2020 34(1s): 13­20 16 fig. 2 ­ flower production of b. semperflorens (red) and v. cor‐ nuta (purple). data are shown in a cumulative curve. antioxidant activity ‐ frap assay the method used to evaluate the antioxidant activity is the frap (ferric ion reducing antioxidant power) assay as indicated by benzie and strain (1996). the antioxidant activity was determined mixing 30 µl of phytoextract with 90 µl of deionised water and 900 µl of frap reagent. the samples were then placed at 37°c for 30 minutes. absorbance was mea­ sured at 595 nm by means of a spectrophotometer (agilent technologies, cary 60 uv­vis, santa clara, ca, united states). results were expressed as mil­ limoles of ferrous iron equivalents per kilogram of fresh weight (mmol fe2+/kg fw). statistical analysis all data were subjected to the statistical analysis for the homogeneity of variance (levene test). weight variations were compared using a one­ way anova test. mean comparisons between data obtained from the two different packages during postharvest were performed using an independent samples t­test, by means of the spss 25 software (version 25.0; spss inc., chicago. illinois). 3. results pot plant productivity begonia semperflorens and v. cornuta pot plants showed differences in the number of flowers pro­ duced over time. on average, from the day of arrival at the laboratory, plants of b. semperflorens pro­ duced flowers for 30 days (with an initial production of about 38 flowers per plant, and a final production of 1­2 flowers per plant) (fig. 2), with an average total productivity of 11 flowers per plant per day. pot plants of v. cornuta bloomed for 11 days (starting with an average of 21 flowers per plant up to 3­4 flowers per plant) (fig. 2), with an average total pro­ ductivity of 9 flowers per plant per day. cut flower shelf life as shown in figure 3, the shelf life of b. semperflo‐ rens and v. cornuta assessed at 4°c is quite different: the first species reached the limit of marketability (grade 5) after 9 days (both in pp and in bopp) (fig. 3, red lines), while the viola flowers remained accept­ able for the consumer up to two weeks (both for pp and for bopp) (fig. 3, purple lines). during storage up to grade 5, the flowers did not show significant weight variations in both packaging type (table 1). bioactive compounds the total polyphenol and anthocyanin content, and the antioxidant activity (frap) of b. semperflo‐ rens and v. cornuta’s flowers are reported in table 2. values are referred to flowers freshly picked, corre­ sponding to grade 9 (t0), and after storage at 4°c, when they reached grade 5 of the visual scale (i.e. limit of marketability), that corresponded to 9 days for begonias and 16 days for violas (fig. 3). comparisons aimed to highlight flower differences between the two species and along time within the same packaging and between the two types of pack­ aging (table 2). variations are visualized in figure 4. concerning the pot plants, v. cornuta flowers at t0 showed higher values of both polyphenols (p<0.001) and antioxidant activity (p<0.001) than b. semperflo‐ rens, while this latter showed a higher content in anthocyanins (p<0.05) than v. cornuta. fig. 3 ­ trend of visual quality during storage for b. semperflo‐ rens (red) and v. cornuta (purple). data are shown as mean values. the intersection of the curves with the dot­ ted horizontal line corresponds to the marketability limit (grade 5). pp= plastic box + lid; bopp= plastic box without its lid, inserted into a bi­oriented polypropylene plastic film. demasi et al. ‐ edible flowers post‐harvest quality 17 regarding the bioactive compound’s evaluation during the post­harvest, begonia flowers kept in pp encountered a decrease of all parameters (polyphe­ nols: ­45.78%; anthocyanins: ­85.33%; antioxidant activity: ­52.35%) while in bopp only the antho­ cyanins decreased (­99.44%). viola flowers kept in pp encountered an increase in anthocyanins (+202.5%) and a decrease of antioxidant activity (­34.21%) while total polyphenols were constant (table 2). in bopp all the parameters decreased (phenolic content ­ 76.51%, antioxidant activity ­88.05%, anthocyanins ­ 32.52%). table 1 ­ flower weight variation during cold storage (4°c) up to the grade of marketability table 2 ­ total polyphenols, total anthocyanins, and antioxidant activity (frap) at grade 9 (day of harvest) and grade 5 (i.e. limit of marketability, corresponding to 9 days for begonias and 14 days for violas) of visual quality scale of b. semperflorens and v. cornuta flowers stored at 4°c in two different packaging pp= plastic box + lid; bopp= plastic box without its lid, inserted into a bi­oriented polypropylene plastic film). data are shown as mean values. comparisons between data were performed using a one­way anova analysis. pp= plastic box with its own lid; bopp= flowpack. data are shown as mean values. * p≤0.05. ** p≤0.01. *** p≤0.001. mean comparisons between data were performed using an independent samples t­test. flower species day weight (g) pp weight (g) bopp begonia semperflorens 0 5.25 5.04 2 5.25 5.04 4 5.25 5.03 7 5.24 5.18 9 5.23 5.01 p ns ns viola cornuta 0 5.18 5.43 2 5.18 5.43 4 5.14 5.44 7 5.16 5.47 9 5.12 5.41 11 5.10 5.40 14 5.17 5.38 16 5.17 5.37 p ns ns flower species packaging total polyphenols (mg gae/100 g fw) total anthocyanins (mg c3g/100 g fw) antioxidant activity frap (mmol fe²⁺/kg fw) grade 9 grade 5 p grade 9 grade 5 p grade 9 grade 5 p begonia semperflorens pp 246.71 133.75 ** 378.67 55.57 *** 95.23 45.38 ** bopp 246.71 264.77 ns 378.67 2.12 *** 95.23 83.49 ns p ‐ *** ­ *** ­ *** viola cornuta pp 767.26 877.01 ns 27.76 83.99 ** 391.89 257.84 * bopp 767.26 180.26 *** 27.76 18.74 * 391.89 46.83 * p ‐ * ­ ** ­ * fig. 4 ­ percentage variation of the content of a) total anthocya­ nins, b) total polyphenols, c) antioxidant activity (frap) in b. semperflorens (red) and v. cornuta (purple), up to grade 5 of visual quality scale, depending on the type of packaging (stored at 4°c). the statistical analyses were made separately on b. semperflorens and v. cornuta values. mean comparisons between data were perfor­ med using an independent samples t­test. * p≤0.05. ** p≤0.01. ***p≤0.001. axis 0 uses the t0 value as a refe­ rence. pp= plastic box + lid; bopp= plastic box without its lid, inserted into a bi­oriented polypropylene plastic film. 18 adv. hort. sci., 2020 34(1s): 13­20 4. discussion and conclusions this study aimed to evaluate the aptitude of two common edible flower species, i.e. b. semperflorens and v. cornuta, to be sold as pot plants or fresh cut flowers. the growing conditions adopted in this study (i.e. 18­20°c and low lighting) were useful to simulate the maintenance of pot plants in a domestic environ­ ment, so to give information on productivity to the final consumer. these conditions are unlikely to be fully appropriate, especially for violas. indeed, v. cor‐ nuta plants are more productive at temperatures from 4 to 10°c (ball, 1991; nau, 1998). cooper and watson (1952) noticed that flowers sizes are also big­ ger when plants grow at night­time temperatures of 10°c. a frequent harvest could concur to cause a gen­ eral reduction in productivity and in flowers size too. in this study, where flowers were picked every 2­3 days, a change in flowers weight was observed. begonia flowers weighted 0.5­0.6 g at the beginning of the experiment and 0.3­0.4 g at the end, while viola flower varied from 0.2 g to 0.1 g. begonias flow­ ers, moreover, showed petals discoloration during the last days of harvest. few data are available in literature about b. sem‐ perflorens and v. cornuta so that comparisons with results of other studies are difficult. some informa­ tion could be found in other congener species. low temperature (4°c) and natural lighting during storage mimed the retailer conditions. data about b. semper‐ florens agreed with those assessed by friedman et al. (2007) in flowers of begonia elatior and b. semperflo‐ rens that were stored in plastic trays for about ten days at 2­5°c. viola cornuta shelf life was in accor­ dance with the data found by kelley et al. (2003) in viola wittrockiana, that was considered marketable after two weeks of storage at 5°c. regarding the phy­ tochemical content, results were partially discordant with those found by benvenuti et al. (2016) in v. wit‐ trockiana that showed a higher antioxidant activity but also a higher anthocyanins content than b. sem‐ perflorens. results of works that evaluated the content of edible flower phytochemicals during cold storage are sometimes conflicting. aquino­bolaños et al. (2013) observed a reduction in the nutraceutical values of squash (cucurbita pepo l.) edible flowers, conversely friedman et al. (2007) found no differences in antho­ cyanins content in b. semperflorens flowers. landi et al. (2018) analysed b. semperflorens flowers too, finding a general constancy in the nutraceutical val­ ues during storage. data obtained in this study showed that the phytochemical content of b. sem‐ perflorens decreased during storage in the plastic box closed with its own lid, while in the flow pack the total phenolic content, and the antioxidant activity, remained constant. the best way to store flowers of b. semperflorens could be therefore the flowpack, preferably a perforated one to prevent condensation of vapours on their inner surface (mlcek and rop, 2011). conversely, v. cornuta flowers seemed to bet­ ter preserve its characteristics in the plastic box closed with its own lid, showing a certain constancy in polyphenols and antioxidant activity, and a signifi­ cantly increased level of anthocyanins, while in the flow­pack showed a significant reduction in all three parameters. in conclusion, our data confirm that b. semperflo‐ rens and v. cornuta are suitable for edible flower pro­ duction both in term of shelf life and phytochemical characteristics. begonia semperflorens seems prefer­ ably marketable as pot plants, thanks to its better adaptability to grow in the domestic environment and longer flowering. conversely, v. cornuta flowers resulted more suitable as fresh cut produce, showing a longer shelf life and preserving better the phyto­ chemical characteristics during storage at 4°c. new technological approaches (ethylene inhibitors, modified atmosphere packaging, edible film coatings, high hydrostatic pressure, irradiation, etc.) could further improve the distribution and mar­ keting efficiency of edible flowers, contributing to their success in the market (fernandes et al., 2018). acknowledgements the research was funded by the program interreg alcotra francia italia v­a, project n. 1139 “antea ­ attività innovative per lo sviluppo della filiera tran­ sfrontaliera del fiore edule” references aquino­bolaños e.n., urrutia­hernández t.a., lópez del castillo­lozano m., chavézservia j.l., verdalet­guzmán i., 2013 ­ physicochemical param‐ eters and antioxidant compounds in edible squash (cucurbita pepo) flower stored under controlled atmo‐ spheres. ­ j. food qual., 36: 302­308. ball v., 1991 ­ plugs ‐ the way of the 1990s, pp. 137­153. ­ in: bail v. 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parida and das, 2005). photosynthesis is one of the physiological processes that is affected by salinity stress (munns et al., 2006; chaves et al., 2009). salinity stress may reduce the photosynthesis rate by decreasing in stomatal factors such as stomatal conductance (bethke and drew, 1992; parida et al., 2004), internal co2 partial pressure (bethke and drew, 1992; iyenger and reddy, 1996) and non-stomatal factors such as inhibition and degradation of photosynthetic pigments (lee et al., 2004; chaves et al., 2009), photosynthetic electron transport reactions, quenching ability of excessive energy through chlorophyll fluorescence (lee et al., 2004), efficiency of rubisco for carbon fixation (liu et al., 2011), and photophosphorylation (stoeva and kaymakanova, 2008). adverse effects of salinity on plant growth may also result from impairment of photosynthetic apparatus (ashraf, 2004). hydrogen peroxide and nitric oxide are bioactive molecule involved in the signaling process within plants (leshem, 2000; uchida et al., 2002; azevedo neto et al., 2005; hung et al., 2005; li et al., 2011). researches have shown that hydrogen peroxide and nitroxide at low concentrations, play an important role as signaling molecules (gechev and hille, 2005; quan et al., 2008). studies have shown that hydrogen peroxide and nitric oxide are involved in acclamatory signaling triggering tolerance against salt stress (hayat et al., 2013; semida, 2016). azevedo-neto et al. (2005) reported that the pretreatment with h2o2 in nutrient solution induces acclimation to salinity stress in maize. semida (2016) observed that exogenous h2o2 application enhanced salt stress tolerance in onion plants by improving the photosynthetic efficiency and plant water status as evaluated by relative water content and membrane stability index. the use of no increased the resistance of pinus eldarica to salinity and improved its growth characteristics (zamani et al., 2014). uchida et al. (2002) reported that h2o2 and no are the important signaling molecules in rice for resistance to salinity stress. tomato is one of the most important vegetable crop in the world. in iran, the tomato also holds the number one position among vegetables, with almost 6.4 million metric tons of production (fao, 2014). the cultivated tomato has been classified as moderately sensitive to salinity. salinity affects tomato plant growth at various stages including seed germination, root and shoot development and fruit production (cuartero and fernandez-munoz, 1999). this research was undertaken to assess changes in plant growth, water relations, cell membrane stability and photosynthesis parameters in salt-treated tomato plants and to examine neutralizing effects of no and h2o2 to exposure to salt. 2. materials and methods plant material tomato seeds, cv. falat were surface-sterilized in 2.5% sodium hypochlorite for 10 min, followed by four washes with distilled water. seeds were sown in the plastic tray filled with a silica sand in the greenhouse under controlled conditions (photoperiod of 16/8 h day/night, 60-65% humidity and 25-30°c temperature). seeds were irrigated with tap water daily. seedlings with 2 true leaves were transplanted to 25×25 cm pots (one plant per pot) maintained under similar conditions as the tray containing developing seedlings and fertilized alternate days with halfstrength hoagland solution (hoagland and arnon, 1950) until solution drainage occurred at the bottom of the pot at each fertigation. treatment and experimental design seven days after transplanting, uniform seedlings of tomato cultivars were sprayed to run off with distilled water, 10 mm h2o2 or 0.1 mm snp in 0.025% tween 20 (as a surfactant) at 6:30 am and then the sprays were repeated at 7 and 14 days later. the concentrations of h2o2 and snp and the number and timing of sprays were based on results from a preliminary experiment (data not shown). after the last spraying, irrigation was done with half strength hoagland solution supplemented with 0, 25, 50 and 75 mm of nacl solution. the experimental procedures were completely randomized in 3 × 5 factorial design, with three foliar spray (sodium nitroprusside [snp], h2o2 and distilled water) and four salt concentrations (0, 25, 50, and 100 mm nacl in nutrient solution), performed in triplicate. the number of plants were six in each replicate. plants were sampled at 90 day after seeding. three samples were analyzed for each replication (9 samples in each treatment). the fully-expanded leaves were used for the determination of all experimental parameters. determination of plant growth traits ninety-day-old tomato plants were carefully removed from each pot and the leaves, stems and roots of plants were weighed to record their fresh hajivar and zare‐bavani ‐ alleviation of salinity stress in tomato plants 411 weights and then placed in an oven at 70°c till the constant weight to record their dry weights. determination of relative water content (rwc) rwc was estimated using 2-cm-diameter fullyexpanded leaf discs, excluding midrib according to the method of hayat et al. (2013). the discs were weighted for fresh mass (fm) and immediately floated on double-distilled water in petri dishes for 24 h, in the dark, to saturate them with water. water adhering to discs was blotted and the turgid mass (tm) was measured. the dry mass (dm) of discs was recorded after dehydrating them at 70°c until the constant weight. the rwc was then calculated using the formula: rwc = [(fm − dm)/(tm − dm)] × 100. determination of proline content free proline content was determined according to the method of bates et al. (1973). samples (0.5 g) were homogenized in 5 ml 3% sulfosalicylic acid and extracts were centrifuged at 8000 x g for 15 min. the amount of 1 ml filtrate was mixed with equal volumes of acetic acid and ninhydrin reagent (1.25 g ninhydrin, 30 ml of glacial acetic acid, 20 ml 6 m h3po4) and incubated for 1 h at 100°c. the reaction was stopped by placing the test tubes in ice cold water. the samples were vigorously mixed with 3 ml toluene. after 50 min, the light absorption of the toluene phase was estimated at 520 nm on a uv-vis spectrophotometer. the proline concentration was determined using a standard curve. free lproline content was expressed as μg/g dry weight. determination of total soluble sugar content total soluble sugar content was determined by phenol-sulfuric acid according to the method of dubois et al. (1956). dry leaves sample (0.1 g) were extracted with 5 ml of 80% ethanol, by boiling the samples in glass tubes in a 95°c-water bath for 10 min. after extraction, the tubes were centrifuged at 500 x g for 5 min, and the supernatants of the extractions were used for sugar analysis. one hundred ml of sample was added to 900 ml of distilled water then the mixture was vortexed. one ml of 5% phenol and 5 ml of h2so4 were added to 1 ml of sample and the mixture was stirred. after cooling under room temperature for 15 min, the absorbance of the sample was recorded at 490 nm. determination of membrane stability index (msi) the msi was determined according to methods of sairam and srivastava (2002). leaf disc (0.2 g) were thoroughly washed in double distilled water and thereafter placed in a test tube containing 10 ml of double distilled water in two sets. one set was heated at 40°c in a water bath for 30 min and the electrical conductivity (ec1) of the solution was recorded using an electrical conductivity meter. another set was boiled at 100°c for 10 min and their electrical conductivity was recorded as above (ec2). the msi was calculated as: msi= [1-(ec1/ec2)] ×100 determination of leaf photosynthetic pigments chlorophyll a, b and total chlorophyll were extracted and determined (in mg/ g fw) following the procedure is given by lichtenthaler and buschmann (2001). fresh leaf samples (0.2 g) were homogenized in 50 ml acetone (80%) and then centrifuged at 10,000 × g for 10 min. the absorbance of the acetone extract was measured at 663, 645 and 470 nm using a uv-visible spectrometer (shimadzu, kyoto, japan). determination of leaf photosynthetic attributes photosynthetic attributes (stomatal conductance [gs], internal co2 concentration [ci], transpiration rate [e], and net photosynthetic rate [pn]) in intact leaves were measured by a infrared gas analyzer (ci340, photosynthesis system, cid bio-science, usa) between 10:00 and 12:00 h under a clear sky. photosynthetic pigments and attributes were measured on three samples of leaves in each pot and three pot in each replication. statistical analysis the experimental design was a completely randomized factorial, four salinity levels (0, 25, 50 and 100 mm nacl) and two levels of h2o2 and snp (10 and 0.1 mm respectively). all measurements were carried out in three replicates and data were subjected to one-way analysis of variance using sas program (sas 9.1; sas institute inc., cary, nc). significant differences between means were determined by tukey’s tests. p values less than 0.05 were considered statistically significant. 3. results growth parameters salinity markedly decreased fresh weight and dry weight of root, leaf and shoot (fig. 1, a-h). however, the h2o2 and snp spraying were able to reduce the adv. hort. sci., 2019 33(3): 413-416 412 adverse effects of salt stress. moreover, the fresh weight and dry weight of root, leaf and shoot from h2o2 and snp-sprayed plants were higher than the ones stressed plants (fig. 1). relative water content (rwc) when salinity was absent, rwc was not significantly altered by h2o2 and snp pretreated plants (fig. 2, i). under salinity condition, plants sprayed with snp or h2o2 displayed higher rwc when compared to water sprayed ones. plant pretreated with h2o2 was not significantly affected by salinity of 25 mm nacl. salinity did not promote any significant alteration in snp pretreated plants in 25 and 50 mm nacl stress. under 100 mm nacl stress conditions, the rwc was reduced in all evaluations (fig. 2, i). proline content the proline-specific increase in plants exposed to nacl (fig. 2, j). pretreatment to either h2o2 or snp resulted in an increase in proline levels of plants under salinity stress. interestingly, among unstressed plants, treatment of h2o2 and snp also increased the proline levels (fig. 2, j). total soluble sugar content (tssc) salinity stress significantly increased the tssc (fig. 2, k). pretreatment of h2o2 or snp significantly decreased the tssc compared to water sprayed plants. the highest amount of tssc was observed with 100 mm nacl without h2o2 or snp, while the lowest amount of tssc was observed with snp and h2o2 application without salinity (fig. 2, k). membrane stability index (msi) under non saline conditions, the msi were not affected by h2o2 and snp spraying (fig. 2, l). although salinity had decreased the leaf msi, it did not promote any significant alteration in h2o2 and snp sprayed plants in 25 mm nacl stress compared to non saline conditions. the msi was significantly decreased by 50 and 100 mm nacl stress, however, plants treated with h2o2 and snp were less affected by salinity stress compared to water sprayed plants. the snp-sprayed plants showed values of msi higher than the stressed plants sprayed with water and h2o2 (fig. 2, l). leaf photosynthetic pigments there were significant decreases in the chlorophyll a, b and total chlorophyll contents in saltstressed plants. plants treated with h2o2 and snp fig. 1 effect of salt treatment and application of exogenous h2o2 and snp on growth parameter of tomato plants. leaf fresh weight (a), stem fresh weight (b), root fresh weight (c), leaf dry weight (d), stem dry weight (e), root dry-weight (f), total fresh weight (g), and total dry weight (h). data shown are the mean (±se) of three independent experiments. significant differences among treatments were determined by tukey's test (p<0.05). fig. 2 effect of salt treatment and application of exogenous h2o2 and snp on relative water content (i), proline (j), total soluble sugar (k) and membrane stability index (l). three plants were analyzed for each treatment. data shown are the mean (±se) of three independent experiments. significant differences among treatments were determined by tukey's test (p<0.05). hajivar and zare‐bavani ‐ alleviation of salinity stress in tomato plants 413 4. discussion and conclusions salt stress is a major abiotic stress that imposes osmotic and toxicity stress to plants and consequently induces a reduction in plant photosynthesis and growth (acosta-motos et al., 2017). in this study, our results confirmed that salinity at the tested concentrations inhibited the growth of tomato plants (fig. 1, a-h). h2o2 and no are bioactive molecules that are known as important signals not only in plant disease resistance, but also in the process of growth, development, and responses against abiotic stress (mazid et al., 2011; niu and liao, 2016). salinity stress is known detrimental effect on the overall growth and productivity of plants (ashraf, 2004) and may inhibit plant growth due to reduction of water uptake by plants (kaya et al., 2003). several studies have shown the beneficial effects of h2o2 and snp pretreatment on salt tolerance in plants (uchida et al., 2002; azevedo-neto et al., 2005; wahid et al., 2007). potikha et al. (1999) suggested that h2o2 increases cell division and is involved in the differentiation of the cell wall. our results are in agreement with those previously reported for maze (azevedo-neto et al., 2005; gondim et al., 2013), rice (sathiyaraj et al., 2014), cotton, cowpea and sorghum (freitas et al., 2011). terasaki et al. (2001) noted that snp possibly enhances exoand endo-β-d-glucanase activities in cell walls, where the glycosidic linkage between glucose units within cell walls is broken by these enzymes (zhang et al., 2003), and growth enhance by increasing internal turgor pressure and water content. similarly to our results, growth stimulation by exogenous no was demonstrated in tomato (wu et al., 2010; hayat et al., 2013). in this study h2o2 or no resulted in higher increase of relative water content, proline and membrane stability index in leaf of tomato plant and decrease of total soluble sugar content (fig. 2, i-l) which could promote plant growth under salt stress (duan et al., 2007) and non saline conditions (zhang et al., 2005), indicating that h2o2 and no are involved in the intrinsic mechanism of growth under different conditions. the higher relative water content in h2o2 and snp-sprayed stressed plants (fig. 2, i) appears to be the result of h2o2 and no-induced increased levels of compatible solutes under salt-induced osmotic stress (tan et al., 2008; hayat et al., 2013), which resulted in better growth of stressed plants. proline accumulation is an essential indicator for plant response to salinity stress (sathiyaraj et al., 2014). the h2o2 and snp-pretreated plants showed a signifispray had higher values compare to the watersprayed plants (fig. 3, m-o). the chlorophyll content (a, and total chlorophyll) in the water-sprayed plants and plants receiving h2o2 and snp were similar in 0 and 25 mm nacl treatment (fig. 3, m and o). leaf photosynthetic attributes the h2o2 and snp-sprayed plants showed higher net photosynthesis (pn), stomatal conductance (gs) and intercellular co2 concentration (ci) than the water-sprayed plants under non saline conditions (fig. 1a-1c). although the pn, e, gs and ci were strongly decreased by salinity stress, plants sprayed with h2o2 and snp were less affected than the watersprayed ones. the pn in the plants receiving h2o2 and snp were higher at 25 mm nacl treatment compared with the water-sprayed ones. plants receiving 100 mm nacl had lower pn, gs, ci and e than other treatment. fig. 3 effect of salt treatment and application of exogenous h2o2 and snp on photosynthesis pigment and attributes of tomato plants. chlorophyll a (m), chlorophyll b (n), total chlorophyll (o), photosynthetic rate (p), stomatal conductance (q), transpiration (r) and intercellular co2 concentration (s). data shown are the mean (±se) of three independent experiments. significant differences among treatments were determined by tukey's test (p<0.05). 414 adv. hort. sci., 2019 33(3): 413-416 cantly higher amount of proline than the saltstressed ones (fig. 2, j). stress-induced proline accumulation in plants help in osmotic adjustment (sathiyaraj et al., 2014). in addition to the role as a compatible osmolyte, proline can also increase membrane stability, confer enzyme protection and help in non-enzymatic free radical detoxifications (khan et al., 2002; sathiyaraj et al., 2014). thus, the increase of proline may trigger tolerance to salt stress in tomato plants. the salt stressed plant showed an increase in total soluble sugar content, but h2o2 and snp sprayed plants showed a significantly decreased of tssc compared with the water-sprayed plants (fig. 2, k). the reduction in tssc by h2o2 and no application in this experiment (fig. 2, k) may be attributed to the crucial role of h2o2 and no in mitigating the negative effect of salinity stress (semida, 2016). similarly, tssc reduction by exogenous h2o2 was demonstrated in onion (semida, 2016). h2o2 and snp sprayed plants showed a significantly increased of membrane stability index compared with the water-sprayed ones (fig. 2, l). the salt stressed plant showed an decreas in msi, and the decrease in msi reflects the extent of lipid peroxidation caused by active oxygen species. the rate of lipid peroxidation has been widely used as an indicator of oxidative damage (sathiyaraj et al., 2014). result showed exogenous h2o2 and snp treatment are able to prevent lipid peroxidation and thus protect the cells from the damage of salinity stress. h2o2 and no are known to enhance chlorophyll content in plant (gondim et al., 2013; hayat et al., 2013). in this experiment, the chlorophyll content was negatively affected by salinity (fig. 3, mo). singh and dubey (1995) showed that the loss of chlorophyll content could be related to photoinhibition or oxidative damages that acts as a cellular marker of salinity stress. therefore, the pretreatment with h2o2 and snp was effective to reduce the detrimental effects of salinity in chlorophyll content (fig. 1a). salinity stress is also known to reduce photosynthesis, due to an increase in reactive oxygen species formation, water status alteration, and a decrease in chlorophyll content and co2 diffusion through stomatal guard cells (chaves and oliveira, 2004; munns and tester, 2008). silva et al. (2011) reported that reduction in photosynthesis by stomatal closure occurs during early exposure to salinity stress, while biochemical limitations concern due to long-term nacl exposure. thus, the reduction of photosynthesis in plants was caused by reduction in stomatal conductance, decreasing the intercellular co2 concentration for rubisco activity (shahbaz et al., 2010). some studies reported the maintenance of gas exchange correlate with salt tolerance in plants (james et al., 2006; munns and tester, 2008). in this experiment, results showed all gas exchange parameters were less affected by salinity in plants previously treated with h2o2 and snp (fig. 3, p-s). therefore, our data indicate that h2o2 and no-pretreatment increased stomatal conductance, which enabled high net photosynthetic rate and improved growth parameters. in addition, the higher leaf msi induced by the h2o2 and snp pretreatment in nacl stressed plants is an evidence that plants were able to control oxidative damages caused by ros in the photosynthetic apparatus and maintain leaf gas exchange (fig. 2, l). similarly, it is observed that that the h2o2 and snp-pretreatment caused increases in net photosynthetic rate, transpiration rate, stomatal conductance and intercellular co2 concentration in plants subjected to salinity when compared to non-treated seedlings (wahid et al., 2007; gondim et al., 2013; hayat et al., 2013). as a whole, exogenous h2o2 and no are able to improve plant growth of tomato and 0.1 mm snp produces the most effective improvement. exogenous h2o2 and no greatly alleviated the oxidative stress induced by salt stress in tomato plant. therefore, exogenous h2o2 and no treatment on tomato seedling may be an option to improve photosynthesis and growth under saline conditions. foliar application of h2o2 and snp provides an easy, low cost, and effective strategy to overcome environmental stress problems. exogenous h2o2 and snp application is a convenient and effective approach 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horticulture, college of agriculture, isfahan university of technology, isfahan, iran. 2 department of plant, food, and environmental sciences, faculty of agriculture, dalhousie university, 50 pictou road, po box 550, truro, b2n 5e3, nova scotia, canada. key words: endemic accession, nutrient absorption, n metabolism, photosynthe­ sis. abstract: the use of an endemic plant as rootstock has many merits but its application for cucurbits production has not been extensively investigated. the present study determined the growth responses of grafted cucumber using two endemic rootstocks from cucurbita pepo l. fertilized with different ratios of nitrate (no3 ­): ammonium (nh4 +) fertilizer. a greenhouse study was carried out using cucumber (c. sativus ‘dominos’) grafted on two accessions of cucurbita pepo l. collected from babol and isfahan with the control being ungrafted c. sativus ‘dominos’. different ratios of no3 ­/nh4 + as follows: 100:0 (no3 − alone), 25/75, 50/50, 75/25 and 0/100 (nh4 + alone) were applied. it was found that dif­ ferent rootstock has the same physiology but different growth attributes. the growth of the ungrafted cucumber was lower than the grafted ones, and babol showed better or equal growth compared to the isfahan rootstock. the no3 ­ /nh4 + effect on growth of the cucumber shoot and root fresh and dry weights, root and shoot lengths, nodes, and number of leaves were increased in the 75/25 ratio compared to the other treatments. grafting on the isfahan and babol showed the same effect of n metabolism i.e., grafting increased nitrate reductase activity and no3 ­ concentration in the 75/25 and the 100/0. protein content and amino acids content of leaves increased in the grafted cucumber treated with 50/50 no3 ­/nh4 +. the same response of photosynthesis parame­ ters was observed in the different rootstocks. in conclusion, the result suggest­ ed that the grafted ‘dominos’ on babol endemic rootstock at 50/50 no3 ­/nh4 + ratio gave the high growth. 1. introduction grafting is a horticultural technique performed by joining two plants i.e., a scion and a rootstock. grafting of plants can minimize the detrimen­ tal effects of biotic and abiotic stress if the proper rootstock is used (lee (*) corresponding author: mhaghighi@iut.ac.ir citation: haghighi m., zamani o., abbey l., 2021 ­ growth response nitrogen metabolism of grafted cucumber fertilized with different ratios of nitra‐ te: ammonium fertilizer. ­ adv. hort. sci., 35(2): 165­174 copyright: © 2021 haghighi m., zamani o., abbey l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2020 accepted for publication 24 dcember 2020 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8343 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 165­174 166 and oda, 2003; rouphael et al., 2008; keshavarzi and shekafandeh, 2019). many studies have shown that grafting promotes root and shoot growth, increase plant resistance against diseases, increase plant tol­ erance to temperature extremities and soil salinity, increase nutrient and water absorption, and plant productivity (colla et al., 2010; lee et al., 2010). nutrient uptake like nitrogen (n) and phosphorus (p) absorptions improved by grafting of cucumber to fig leaf gourd (cucurbita ficifolia l.) (pogonyi et al., 2005). cucumber ‘adrian’ was grafted onto three root­ stocks of lagenaria siceraria, cucurbita maxima × c. moschata, and zucchini. results showed that grafting improved total yield,leaves number, total soluble solids and titratable acidity. the effect of grafting is highly dependent on the choice of rootstock (goreta et al., 2014 ; dadashpour et al., 2017) to improve growth and fruit production. the benefits of grafting on vegetative growth have been reported for cucurbit­type crops (goreta et al., 2014) but using the endemic cucurbits as a rootstock for this family has not been extensively investigated. ammonium, nitrate and urea are the three forms of inorganic nitrogen that increase plant growth (niu et al., 2011). most plants prefer nitrate to ammoni­ um or a combination of them. the optimal no3 ­/nh4 + ratio depends on many factors such as plant species, plant growth and maturity stages, and environment condition (marschner, 2012). yet, little is known about how the no3 ­/nh4 + ratio may affect grafted plant and their nitrogen metabolism. higher no3 ­/nh4 + ratio produce higher biomass than other ratios in mesquite (prosopis velutina) (hahne and schuch, 2006). conversely, higher no3 ­ improves growth and flowering in phalaenopsis orchid (phalaenopsis) (wang et al., 2008). in some plants, n form has no significant effects on growth. for instance, the dry weight of shoots and roots, and root/shoot ratio were not affected by no3 ­/nh4 + ratio in sophora secundiflora (niu et al., 2011). however, using high no3 ­ in plant nutrition is a risk for the envi­ ronment and human health. replacing no3 ­ in plant nutrition with an appropriate amount of nh4+ may alleviate these concerns. plants usually grow on no3 ­ and nh4 + as nitrogen sources, which eventually influ­ ence the synthesizing of amino acids and proteins (xing et al., 2015). the optimum level of n and the best ratio of no3 ­ /nh4 + are required by each species according to their respective growth and productivity (fernandez­nava et al., 2010). on the other hand, the process of up taking no3 ­ or nh4 + has a substantial impact on the uptake of other cations and anions and rhizosphere­ ph. when roots take up no3 ­ with a negative charge and nh4 + with a positive charge, they release a specif­ ic charged molecule to keep a balanced ph inside the plant cells. nh4 + reduces rhizosphere ph while no3 ­ increases ph (marschner, 2012). high levels of nh4 + can also inhibit the uptake of cations such as calcium and magnesium (siddiqi et al., 2002). ammonium application reduces the rate of iron deficiency but increases phosphate and sulfate uptake due to changing substrate ph. in contrast, nitrate reduces the absorption of those essential anions. thus, most of the time, supplying the proper no3 ­/nh4 + ratio results in the highest growth rates and plant yield by balancing nutrient absorption (marschner, 2012). cucumber has a shallow root system and could not uptake nutrient well (causin et al., 2004). information on the influence of endemic accession as a rootstock on cucumber nutrient absorption and growth is limited. to better understand the effect of no3 ­/nh4 + ratio for grafted cucumber, the present study was designed. the present study determined plant growth response of grafted cucumber using two endemic rootstocks from cucurbita pepo l. fertil­ ized with different ratios of no3 ­/nh4 + under green­ house conditions. 2. materials and methods production of rafte seedlings and experimenta design the greenhouse experiment was carried out at the isfahan university of technology, isfahan, iran. cucurbita pepo l. accession as the rootstocks were collected from the babol (babol), isfahan region (isfahan), and c. sativus ‘dominos’ is a common culti­ var used as scion. the ungrafted dominos used as control. total nitrogen in the nutrient solution was equal and comprised no3 ­/nh4 + ratio 100:0, 25:75, 50:50, 75:25, or 0:100. rootstock and scion seeds were sown in cocopeat and perlite (50/50 v/v). scion seeds had been cultured 10 days before the root­ stock seeds in cocopeat: perlite ratio of 1:1. scion plants and rootstocks were cut beneath and above the first true leaves, respectively. the hole insertion grafting method as described by lee (1994) was adopted. firstly, true leaves and meristem tissue were removed at the growing tip of the rootstock haghighi et al. ‐ nitrogen metabolism of grafted cucumber 167 before making a slit across the growing point from the bottom of one cotyledon to the other side of the hypocotyl. the newly grafted cucumber plant was kept in a grafting chamber with approximately 65% relative humidity and exposed at 16 h fluorescent light at 25­30°c for two weeks. the grafted plants were moved to the greenhouse and maintained at 30­35°c and 60­65% relative humidity for one week to gradually adapt to the greenhouse conditions (kashi et al., 2008). fertilization with a half­modified johnson nutrient solution was applied to the grafted plants including (mm): mgso4 (2), kh2po4 (1), h3bo3 (50), mncl2 (10), cacl2 (1), mnso4 (10), cuso4 (1.5), znso4 (0.8), na2moo4 (0.4), co(no3)2 (0.1), kno3 (10), fecl3 (0.1), edta (0.3) and h3bo3 (50.5) mm (jones, 2005). ten (10) days after adaptation of the grafted plants, they were treated with the no3 ­/nh4 + ratios on a daily basis. the control treatment was irrigated with a complete johnson nutrient solution. the plants were kept in the nutrient solution at varying no3 ­/nh4 + ratios with 5 min airing in every 15 min in 2liter container. the ec and ph of the nutrient solu­ tion were kept at 2.0±0.2 ds m­1 and 6.0±0.3, respec­ tively, by adding hno3 and h3po4 into the nutrient solution. plants were maintained for six more weeks before final harvest. data collection greenness. chlorophyll index was measured with portable spad (spad­502 plus, minolta, japan). growth trait assay. all the leaf and nod number were counted. the shoots of seedlings were separat­ ed from the roots and after recording the fresh weight, they were oven dried at 70°c to constant weight. root volume was measured by change in water volume in a graduated container (haghighi et al., 2012). shoot and root length measured by ruler and the stem thickness was measured using a pair of caliper. photosynthesis traits assay. gas exchange para­ meters including photosynthesis rate, transpiration, stomata conductivity, and intercellular co2 of stoma­ ta were measured by a portable photosynthesis meter (li­cor li­3000, usa) on a sunny day. photosynthetically active radiation (par) intensity was 1000 μmol m­2 ꞏs­1 and co2 concentration was 350 μmolꞏmol. the same leaves of each plant was used for chlorophyll measurement using chlorophyll meter (spad­502, minolta corp., nj, usa). mesophyll conductance (mmol co2 m­2 s­1) was measured according by using the formula: photosynthetic rate/sub­stomatal co2 concentration (ahmadi and siosemardeh, 2005). phenolic content. the folin­ciocalteu method was used for measuring the total phenolic content of the root exudate. the absorbance was measured at 725 nm with a spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan) (motamedi et al., 2019). prolin. plant leaves were homogenized in 3% sul­ fosalicylic acid at 4°c. then the solution incubated and centrifuged at 5000 rpm for 20 min. the super­ natant was mixed with 2.5% ninhydrin, 60% phos­ phoric acid (v/v) and 1 ml of glacial acetic acid (100%). the absorbance was measured at 518 nm by spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan) (bates et al., 1973). total amino acid. total amino acids measured by high­performance liquid chromatography (hplc). samples were hydrolyzed with 6 m hcl and 10 mg phenol (for protection of tyrosine) at 110 °c for 24 h. hplc system was equipped with md­1510 diode­ array detector and set to 263 nm (λmax). the sam­ ples were injected with a 20 μl loop using a 7125 valve (rheodyne, cotati, ca) onto a purospher rp­18 column and operated at 25°c with a flow rate of 1.0 ml/min using 50 mm acetate buffer (ph 4.2) as elu­ ent a and acetonitrile as eluent b (el­abagy et al., 2014). the level of amino acids present in 100 g of leaves. protein. na­phosphate buffer (ph 7.2) was used to homogenized 1 g fresh leaf samples then centrifuged at 4°c. the absorbance of the supernatants and dye measured using a spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan) at 595 nm. bovine serum albumin (bsa) used as protein standard (bradford, 1976). nitrate reductase enzyme. the activity of nitrate reductase enzyme was measured according to the method proposed by (cazetta and villela, 2004). the amount of 400 mg of leaf samples was placed in a phosphate solution (100 mg, ph=7.5) containing 4% propanol and potassium nitrate, and stored in dark­ ness for an hour at a temperature of 30°c. then, 1 ml of the solution containing sulfanilic acid was dis­ solved in 2 ml of chloride acid and 1 ml of naph­ thylethylene diamide solution (0.02%) and after 20 min, the absorbance was measured at 540 nm wave­ length. sodium nitrite (nano3) was used to prepare the standard solution and the enzyme activity was calcu­ lated based on µmol nitrite/gr fw h. adv. hort. sci., 2021 35(2): 165­174 168 nitrate concentration. the leaf nitrate content was determined following the procedures described by atanasova (2008). nutrient concentrations. the amount of calcium was measured by an atomic absorption device (model: perkin elmer, aa200) (sharifi et al., 2016). zk concentration was determined by atomic absorption after digestion with hcl (murillo­amador et al., 2007). nitrogen concentration of leaves measured by kjeldahl method. phosphorus was estimated by the vanadomolybdo phosphoric acid colorimetric method at 460 nm (estan et al., 2005). ca concentrations. twelve fruits for each treat­ ment when reached market ripe harvested at the end of the experiment for measurements. four leaf samples, consisting of young, fully expanded leaves were collected, washed thoroughly with tap water, gave a final rinsing with deionized water, dried at 65°c to constant weight. the extraction of ca from the plant tissue material was performed using 1 n hcl after dry ashing at 550°c for five h. the amount of calcium was measured by an atomic absorption device (model: perkin elmer, aa200) (sharifi et al., 2014). k, mg, and p concentrations. the concentrations of k, mg, and p were measured (shield torch system, agilent 7500a). meanwhile, phosphorus was estimat­ ed by the vanadomolybdo phosphoric acid colorimet­ ric method at 460 nm (sharifi et al., 2014). p was col­ orimetrically determined using a spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan). statistical analysis the factorial experiment was arranged in a com­ pletely randomized design with 10 replications. data were analyzed with statestix 8 (tallahassee fl, usa) and treatment means were separated using the least significant difference (lsd) test at the 5% level of sig­ nificance when the analysis of variance indicated sig­ nificant difference between treatments at p≤0.05. 3. results the anova in table 1 showed that all growth and development characteristics were affected by no3 ­ /nh4 + ratio and rootstock. the interaction of no3 ­/nh4 + ratio and rootstock significantly affected all measured variables except rootstock stem thickness and root volume. except for leaf greenness, all physiological parameters were affected by no3 ­/nh4 + ratio and rootstock and their interaction (table 2). table 2 ­ analysis of variance effect of no3:nh4 ratio on physiological characteristics of cucumber ns, *, ** not significant or significant at 5% or 1%. table 1 ­ analysis of variance effect of no3:nh4 ratio a rootstock on growth characteristics of cucumber ns, *, ** not significant, significant at 5% or 1%, respectively. source df shoot length rootstock stem thickness number scion leaves number scion nodes shoot fresh weight root fresh weight root volume root length shoot dry weight no3:nh4 ratio (n) 4 59.92 ** 3.03 ** 7.23 ** 12.88 ** 24.69 ** 1.08 ** 2.44 ** 100.49 ** 0.06 ** rootstock (r) 2 290.70 ** 6.51 ** 36.87 ** 24.01 ** 14.03 * 1.16 ** 2.94 ** 92.88 ** 0.26 ** nxr 8 37.40 ** 1.67 ns 6.19 ** 4.03 * 24.73 ** 0.64 ** 2.04 ns 44.01 ** 0.09 ** error 30 26.78 0.40 3.44 1.60 3.40 0.05 0.11 5.07 0.01 cv 20.31 14.22 28.98 20.89 31.31 38.97 39.76 18.63 24.10 source df greeness photo­ synthesis tran­ spiration inter­ cellular co2 concentra­ tion of stomata stomata conductivity meso­ philic conductivity nitrate reductase activity proline root phsenol protein nitrate amino acid no3:nh4 ratio (n) 4 14.34 ns 9.59 ns 3.01 ns 19146.7 ns 0.0041 ns 0.0051 ns 0.006 ns 2.33 ns 1.61 ns 3307 ns 0.0000013 ns 35460 ns rootstock ( r) 2 3.19 ns 11.17 ns 1.97 ns 34138.4 ns 0.0043 ns 0.0033 ns 0.013 ns 1.77 ns 1.56 ns 29073 ns 0.000075 ns 251790 ns nxr 8 23.42 ** 19.16 ** 4.11 ** 13750.9 ** 0.005 ** 0.005 ** 0.003 ** 2.39 ** 2.05 ** 59005 ** 0.000015 ** 236095 ** error 22 0.92 9.17 9.17 9.17 9.17 9.19 9.17 9.18 9.17 30518 0.000043 72919 cv 38 12.52 4.69 7.41 3.23 0.16 0.15 0.02 0.65 0.08 17.18 11.08 24.17 haghighi et al. ‐ nitrogen metabolism of grafted cucumber 169 the main effect of nutrient absorbtion was not affected by no3 ­/nh4 + ratio and rootstock, but the interaction showed significant changes (table 3). the main result of the n source indicated that the thickest rootstock stem was at 75/25 no3 ­/nh4 + ratio and the most significant root volume was influenced by treatment 50/50 no3 ­/nh4 + ratio. the ungrafted plants had the least root volume and babol has the most root volume (table 4). shoot length increased when the ratio of no3 ­ /nh4 + increased, which led to higher no3 ­ in shoot length compared to the root length (fig. 1a and b). the shoot length was highest in the 0/100 and then in the 75/25 of no3 ­/nh4 + ratio in all rootstock. it seemed the root had the best growth in the 50/50 no3 ­/nh4 + ratio, and when the no3 ­/nh4 + ratio was increased by more than 75% of total­n, the root length decreased. shoot and root fresh and dry weights were not fig. 1 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) shoot length and b) root length. improved with only no3 ­ or nh4 + (fig. 2a­d). the babol rootstock caused the best shoot and root growth in the 50/50 no3 ­/nh4 + ratio. ungrafted plant showed the lower growth in all the treatment n ratios. it seemed that the different no3 ­/nh4 + ratio did not affect the growth in ungrafted plants. isfahan rootstock increased both shoot and root growth in the 75/25 no3 ­/nh4 + ratio. the highest root and shoot growth were seen in the babol and the 50/50 no3 ­/nh4 + ratio compared to all the other treat­ ments. the numbers nodes and leaves increased by increasing the no3 ­ portion of the nutrient solution for the grafted plants, but there were no significant changes in the ungrafted plants in the different no3 ­ /nh4 + ratios (fig. 3a and b). the lowest number of table 4 ­ effects of no3:nh4 ratio and rootstocks on root volume and cucumber rootstock stem ns, *, ** not significant or significant at 5% or 1%. table 3 ­ analysis of variance effect of no3:nh4 ratio on nutrient concentration of cucumber ns, *, ** not significant, significant at 5% or 1%, respectively. source df n concentration p concentration k concentration ca concentration mg concentration no3:nh4 ratio (n) 4 230. 9 ns 244.56 ns 43.12 ns 78.12 ns 6.12 ns rootstock (r) 2 23.45 ns 150.01 ns 35.93 ns 602.45 ns 13.08 ns n×r 8 26.54 ** 111.77 ** 4.65 ** 81.12 ** 4.20 * error 22 5.27 67.38 3.23 76.32 1.35 cv 38 25.62 13.04 15.50 23.97 44.38 treatments rootstock stem thickness (cm) root volume (mm3) no3:nh4 ratio 100/0 4.51 b 0.35 c 25/75 3.62 c 0.47 bc 50/50 4.50 b 1.85 a 75/25 5.46 a 0.80 b 0/100 4.40 b 0.78 b rootstock un­grafted 3.71 b 0.49 b babol 4.66 a 1.40 a isfahan 5.13 a 0.67 b 170 adv. hort. sci., 2021 35(2): 165­174 leaves and nodes was seen in the ungrafted plants. these findings were in line with the increase in shoot length, which was higher in treatments with increased no3 ­/nh4 + ratio for grafted plants. photosynthesis was more significant in both graft­ ed cucumbers in all the no3 ­/nh4 + ratio compared with the ungrafted plant, and it seemed that it is not related to the changes of the greenness index (fig. 4a). the greenness index did not change between treatments significantly, except for the high increase in babol from the50/50 no3 ­/nh4 + ratio. furthermore, the photosynthesis rate increased in the 50/50 no3 ­/nh4 + ratio in the ungrafted cucumber (fig. 4b). transpiration in all the rootstock was increased by the 25/75 no3 ­/nh4 + and decreased with increasing no3 ­/nh4 + ratio in the nutrient solution. it seemed fig. 2 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) shoot fresh weight, b) shoot dry wei­ ght, c) root fresh weight and d) root dry weight. fig. 3 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) number of leaves and b) number of nodes. fig. 4 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) greenness (spad), b) photosynthe­ sis, c) transpiration d) stomata conductivity, and e) inter­ cellular co2 concentration of stomata. haghighi et al. ‐ nitrogen metabolism of grafted cucumber 171 that by increasing transpiration in all plants stomata conductivity was increased by the25/75 no3 ­/nh4 + treatment (fig. 4c). stomata conductivity was highly raised in the isfahan rootstock and the ungrafted plants (fig. 4d). the stomata conductivity was enhanced in the ungrafted cucumber, especially at 25/75 no3 ­/nh4 +. conversely, the co2 concentration in the stomata reduced in the 25/75 no3 ­/nh4 + and increased with no3 ­ increment in the nutrient solution (fig. 4e). phenol exudate of the root was highest in 25/75 no3 ­/nh4 + and decreased by increasing the no3 ­ por­ tion (fig. 5a). it was highest in the ungrafted com­ pared to the grafted cucumber plants. the highest proline content was seen in the 100/0 and the 25/75 no3 ­/nh4 + and decreased by increasing the no3 ­ con­ centration (fig. 5b). nitrate reductase activity was highest in the 25/75 no3 ­/nh4 + and was reduced by increasing the no3 ­ portion. the lowest no3 ­ concentration was recorded by the 0/100 and the 25/75 no3 ­/nh4 +. nitrate con­ centration was higher in the 0/100, and 100/0 no3/nh4 and was the same in between grafting plants in 25/75 and 50/50 no3/nh4 (fig. 6b). the amino acid was higher in grafted cucumber, especial­ ly in 5/75 and 50/50 no3/nh4. protein content was higher in 50/50 no3/nh4 in all rootstock and un­ grafted (fig. 6a). it seemed that the most nutrient absorption was recorded by the babol and the isfahan rootstock, especially in the 100/0, 75/25 followed by the 50/50 no3 ­/nh4+ for n and k (fig. 7). conversely, the high­ est ca absorption was recorded by the 50/50 no3 ­ /nh4 + and to lesser extent by the 75/25 no3 ­/nh4 +. fig. 5 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) proline and b) root phenol. fig. 6 ­ the interaction effects of different rootstocks and no3:nh4 ratio on a) protein, b) nitrate and c) amino acid d) nitrate reductase (nr) activity. adv. hort. sci., 2021 35(2): 165­174 172 the mg and p absorption were less absorbed, but highest in the babol rootstock. 4. discussions and conclusions the effect of no3 ­/nh4 + and grafting on the cucumber growth a wide range of morphological and physiological characteristics are influenced by scions, rootstocks and their interactions (he et al., 2009). in this study, it was observed that growth in terms of shoot and root fresh and dry weights, root and shoot lengths, numbers of node and leaves were increased by the 75/25 treatment. however, some parameters were not significantly affected. all the growth parameters were improved by the 75/25 treatment in isfahan rootstock. on the other hand, the best result in plant growth improvement was seen in the 50/50 treat­ ment for babol. these results revealed that different rootstock act differently in different no3 ­/nh4 + ratio to promote growth. in all the growth parameters, the ungrafted cucumber was lower than the grafted ones and babol showed a better or similar increase than isfahan rootstock. when using no3 ­ or nh4 + alone, plant growth was not significantly different except for the numbers of node and leaves and root length in the grafted cucumber following application of nh4 + alone. it noted that the use of both sources of n indi­ vidually was not economical. less plant growth was seen in the ungrafted cucumber by using no3 ­ or nh4 + alone compared to the grafted cucumber. different rootstocks have different root size and different absorbance abilities which can affect vege­ tative growth rates. rootstocks improve photosyn­ thetic ability and increase yield in grafted plants (massai et al., 2004). rootstocks improve growth in plant by improving nutrient uptake, hormonal status and root growth (lee et al., 2010). grafting, especial­ ly babol rootstocks, contributed to better vegetative growth of ‘dominos’ due to higher root distribution, perhaps resulting in more nutrient uptake. it should be considered that increasing number of nodes is a sign for more yield because the flower initiate in nodes so the more node means more flowers and fruits as obtained with grafted cucumber. the effect of no3 ­/nh4 + and grafting on the metabo‐ lism of cucumber results revealed that grafting on the isfahan and babol showed the same effect on n metabolism, i.e., nr activity decreased and no3 ­ concentration increased in plants treated with the 75/25 and the 100/0 no3 ­/nh4 +.protein and amino acids contents of leaves was increased at the 50/50 treatment in graft­ ed cucumber and protein showed the same trend like amino acid. it can be concluded that the healthiest cucumber plant was obtained from the 50/50 treat­ ment in the grafted cucumber although the most nitrate metabolism was achieved by treatment 75/25 no3 ­/nh4 +. despite the effect of rootstock on growth, it was seen that the rootstock has no difference in the metabolism n in the cucumber plants. the other reason for the promotion of plant growth by chang­ ing the no3 ­/nh4 + ratio could be that after nr reduced no3 ­ to nitrite, it was changed to ammoni­ um, and amino acids were produced, which can later combine to produce proteins (haghighi et al., 2012). on the other hand, nitrate through producing active forms of cytokinins, as an osmolyte in vacuoles, stim­ ulates leaf function and growth, causing cell exten­ sion and improved growth (wang et al., 2008). increasing root length and cytokinin production helped the plant to absorb more water and nutrients to improve vegetative growth (haghighi et al., 2016 a). the effect of no3 ­/nh4 + and grafting on the stress photosynthesis traits of cucumber photosynthesis was not affected by n ratio but increased by grafting. stomata conductance, internal co2 of stomata and transpiration increased with increasing no3 ­/nh4 + ratio and was reduced by graft­ ed cucumber. it seemed changes in photosynthesis traits were more related to stomata status, which can be associated with the rootstock. photosynthesis was improved with grafting due to an efficient root system of the rootstock with regards to nutrient uptake compared to the ungrafted plants fig. 7 ­ the effects of different rootstocks on nutrients (n, p, k, ca and mg) absorption in different no3:nh4 ratio. haghighi et al. ‐ nitrogen metabolism of grafted cucumber 173 (lee et al., 2010). furthermore, more vigorous root­ stocks could absorb more water and nutrients. consequently, photosynthesis improved when these rootstocks were used compared to non­grafted plants as reported previously by haghighi et al. (2016 b). in all no3 ­/nh4 + ratios, grafting reduced stomata conductivity, which resulted in lower transpiration and improved water use efficiency so plants could deal with challenged conditions more efficiently (duan et al., 2001). in conclusion, the performance of the different cucumber plant accessions used as rootstocks i.e. nutrient absorption and growth parameters varied. the two accessions used in this study i.e. isfahan and babol responded similarly to n metabolism and pho­ tosynthesis traits. therefore, it seems that different rootstock has the same physiology but different growth pattern due to their pre­existing genetic dif­ ferences. more noticeably, we found that, shoot and root fresh and dry weights, root and shoot lengths, nodes, and number of leaves were increased by the 75/25 ratio of no3 ­/nh4 +. grafting on the isfahan and babol increased nitrate reductase activity and no3 ­ concentration, protein content, amino acids content of leaves and photosynthesis parameters. our find­ ings suggested that the grafting ‘dominos’ on babol endemic rootstock using 50/50 no3 ­/nh4 + ratio achieved better vegetative growth, which may result in better yield. references ahmadi a., siosemardeh a., 2005 ­ investigation on the physiological basis of grain yield and drought resis‐ tance in wheat: leaf photosynthetic rate, stomatal con‐ ductance, and non‐stomatal limitations. ­ int. j. agric. biol., 7: 807­811. atanasova e., 2008 ­ effect of nitrogen sources on the nitrogenous forms and accumulation of amino acid in head cabbage. ­ plant soil environ., 54(2): 66. bates l.s., waldren r.p., teare i.d., 1973 ­ rapid deter‐ mination of free proline for water‐stress studies. ­ plant soil, 39: 205­207. bradford m.m., 1976 ­ a rapid and sensitive method for the quantitation of microgram quantities of protein uti‐ lizing the principle of protein‐dye binding. ­ anal. biochem., 72: 248­254. causin h.f., tremmel d.c., rufty t.w., 2004 ­ growths nitrogen uptake, and metabolism in two semiarid shrubs grown at ambient and elevated atmospheric co2 concentrations effects of nitrogen supply and source. ­ am. j. bot., 91(4): 565­572. cazetta j.o., villela l.c.v., 2004 ­ nitrate reductase activity in leaves and stems of tanner grass (brachiaria radicans napper). ­ sci. agric., 61(6): 640­648. colla g., rouphael y., cardarelli m., salerno a., rea e., 2010 ­ the effectiveness of grafting to improve alka‐ linity tolerance in watermelon. ‐ environ. exp. bot., 68: 283­291. dadashpour a., shekafandeh a., oladi r., 2017 ­ anatomical and morphological changes in scions of some olive grafting combinations under water deficit. ­ hortic. sci., 31(4): 281­288. duan b., ran f., zhang x., zhang y., korpelainen h., li ch., 2001 ­ long‐term acclimation of mesophyll con‐ ductance, carbon isotope discrimination and growth in two contrasting picea asperata populations exposed to drought and enhanced uv‐b radiation of tree years. ­ agric. for. meteorol., 151: 116­126. el­abagy h.m., el­tohamy w.a., abdol­mawgoud a.m.r., abouhussein sh., 2014 ­ effect of different amino acid sources and application rates on yield and quality of onion in the newly reclaimed lands. ­ middle east j. agric. res., 3(1): 81­88. estan m.t., martinez­rodriguez m.m., perez­alfo­ cea f., flowers t.j., boalrin m.c., 2005 ­ grafting raises the salt tolerance of tomato through limiting the transports of sodium and chloride to the shoot. ­ j. exp. bot., 56: 703­712. fernandez­nava y., maranon e., soons j., castril­ lon l., 2010 ­ denitrification of fight nitrate on centra‐ tion waste water in alternative carbon sources. ­ j. hazard. mater., 173: 682­688. goreta ban s., žanić k., dumičić g., raspudić e., vuletin selak g., ban d., 2014 ­ growth and yield of grafted cucumber in soil infested with root‐knot nema‐ todes. ­ chilean j. agric. res., 74(1): 29­34. haghighi m., heidarian s., teixeira j., silva da a., 2012 ­ the effect of titanium amendment in‐n‐with‐ holding nutrient solution on physiological and photo‐ synthesis attributes and micro‐nutrienut uptake of tomato. ­ biol. trace elem. res., 150: 381­390. haghighi m., mohammadnia s., pessarakli m., 2016 a ­ effects of mycorrhiza colonization on growth, root exudates, antioxidant activity and photosynthesis trait of cucumber grow in johnson modified nutrient solu‐ tion. ­ j. plant nutr., 39: 2079­2091. haghighi m., sheibanirad a., pessarakli m., 2016 b ­ cucurbits grafting: methods, physiology, and responses to stresses, pp. 255­272. ­ in: pessarakli m. 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tion of spinach. ­ expl. agric., 51(1): 151­160. 229 adv. hort. sci., 2018 32(2): 229-238 doi: 10.13128/ahs-20845 colchicine-induced autotetraploidy and altered plant cytogenetic and morphophysiological traits in catharanthus roseus (l.) g. don h.r. hosseini 1, m. chehrazi 2 (*), d. nabati ahmadi 3, m. mahmoodi sorestani 2 1 department of horticultural science, agriculture faculty, shiraz university, shiraz, iran. 2 department of horticultural science, agriculture faculty, shahid chamran university of ahvaz, ahvaz, iran. 3 department of agronomy and plant breeding, agriculture faculty, shahid chamran university of ahvaz, ahvaz, iran. key words: chromosome number, flow cytometry, polyploidy. abstract: artificially induced polyploidy is often used to alter plant growth pattern and genetic makeup of certain plant species. this experiment was conducted to induce autotetraploidy in catharanthus roseus (‘alba’) which contains diploid chromosomes. application of four levels (0, 100, 200 and 400 mg/l) of colchicine concentrations were utilized at the two true leaf stages of c. roseus. it has been observed that 200 mg/l colchicine treatment had the most striking effect on producing polyploid plants. this concentration was able to boost yield performance and survival of tetraploids to 35% and 79% respectively. increasing of ploidy level was confirmed by flow cytometry and chromosome number. but, plant survival significantly decreased with increased of colchicine concentration. chromosome number, length and diameter of stomata and chloroplast number in stomata of guard cells increased with increased ploidy level, whereas the numbers of stomata decreased from 390 to 177 mm2 in tetraploid plants. the overall consequence of colchicines treatment appeared to be a beneficial approach. it elucidated that the chlorophyll content, diameter of the lateral branches, leaf length and width, petal length and width, duration length of flowering, durability of flowering, root diameter, fresh and dry weight of roots, seed length and seed diameter significantly increased in tetraploid as compared to diploid plants. 1. introduction morpho-physiological characters of ornamental and flower plants can be promoted via duplicating their chromosome numbers. plants with polyploidy chromosomes are usually capable of producing larger organs than diploid chromosomes. it has been proved that the chromosome (*) corresponding author: chehrazi_m@yahoo.com citation: hosseini h.r., chehrazi m., nabati ahmadi d., mahmoodi sorestani m., 2018 colchicine-induced autotetraploidy and altered plant cytogenetic and morpho-physiological traits in catharanthus roseus (l.) g. don. adv. hort. sci., 32(2): 229-238 copyright: © 2018 hosseini h.r., chehrazi m., nabati ahmadi d., mahmoodi sorestani m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 june 2017 accepted for publication 7 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 229-238 230 numbers in certain plant species can be naturally doubled due to autoploid and alloploid mechanisms that caused to generate plant with polyploidy chromosome (dhawan and lavania, 1996). polyploidy not only occur naturally, it also can be induced artificially using mutation agents. colchicine is one of these agents (blakeslee and avery, 1937). application of mutation agents on plant is not only able to multiple the chromosomes, but also to induce variations due to changes in chromosome numbers, used to manipulate morphological and biochemical production of plants (yasuda et al., 2008). a research report indicated that the results have shown colchicine and laecv treatments to induce different types of mitotic abnormalities including: c-metaphase, vagrant chromosomes, sticky chromosomes, anaphase bridges and increased frequency of micronuclei, along with a reduction in mitotic index in allium cepa root apical meristem cells (kundu and ray, 2016). induction of polyploidy tends to expand nucleus and cell size of organs which causes to have positive impact on enlargement of leaves, branches and flowers of plant. so, a suitable procedure capable of altering chromosomes numbers can become an important approach for improvement of traits such as plant size, flowers size and duration of flowering in horticultural plants (shao et al., 2003). in overall, polyploidy induction is a beneficial trend for those plant tissues that contain effective compounds in such way that these tissues become enlarge, thus able to sustain more chemical substances than diploid plants (adaniya and shirai, 2001). catharanthus roseus (l.) g. don (apocynaceae) is an ornamental plant that grows up to 30-100 cm in height. it was previously known as vinca rosea (l.) and commonly known as madagascar periwinkle. although, it is originated from madagascar but, widely distributed throughout the world due to survival ability in various habitats and recognized as an ornamental plant (van bergen and snoeijer, 1996). it is a perennial plant with diploid (2n=2x=16) chromosomes (verma et al., 2011). the apocynaceae family contains 114 genera and 4650 species. catharanthus roseus is a tropical plant and very sensitive to cold climate. if it grew under a favorite condition, it would produce flowers and its growth development remain over long period of times (jaleel et al., 2007). it has been reported that polyploidy induction using colchicine and sodium azid on clustered bean plants (cyamopsis tetregonoloba) caused to promote germination, flowering time, plant height, leaf number, cluster numbers of pods, pod length, pod and dry weight (velu et al., 2008). similar study of colchicine treatment on lace plant seedling indicated that morphological growth and cytogenetic components significantly changes. these alterations were included: increase in leaf area index, length, thickness and dark leaves, stomata size increases, reduction in the number of stomata and leaf epidermis, increases of chloroplast number in stomata guard cells, increase in the diameter of the flower, pollen diameter, petal length, size of capsules and seeds, and doubling of the chromosomes (ye et al., 2009; niu et al., 2016). it has also been observed that application of 0.1% colchicine concentration on jatropha curcas l. plants had the most striking effect on producing polyploidy with yielding improvement of 15% in tetraploids but showed that there was no significant difference between tetraploid and diploid plants in considering plant height. on the other hand, increasing the level of chromosomes in the plant, the stomata numbers and pollen grains became abundant, while stomata density decreased and the leaves became thicker. in general, the tetraploid plants had larger leaves, flowers and seeds as compared to diploid plants (niu et al., 2016). when the apical meristems and seedlings of hyssop (agastache foeniculum l.) plants received 17.500 μm colchicines and 50 μm trifluralin, a maximum growth of 16% tetraploid induction was obtained. size of stomata, chloroplast number, morphological traits, leaf length and width, distance between the nodes, leaf area, plant height, fresh and dry weight, and spikes length significantly increased in polyploidy plants (talebi et al., 2017). an investigation on tissue of dendrobium plant in vitro showed that, when culture medium received 0.075 % colchicines concentration for 14 hours, significant alterations occurred with polyploidy plant. it has revealed that tetraploid plants contained wider and dark leaves, reduce leaf angle and increase the diameter of the roots and stems as compared to diploid plants (sarathum et al., 2010). the aim of the present investigation is to evaluate the effect of colchicine treatment on c. roseus ‘alba’ and to study the impact of cytogenetically and morph-physiological alterations in both polyploidy and diploid plants. 2. materials and methods plant material and autotetraploidy induction this experiment was conducted as a completed randomized design. c. roseus ‘alba’ seeds were purchased from seed-pakan company and olchicines hosseini et al. plant cytogenetic and morpho-physiological traits alteration in catharanthus roseus 231 was obtained from sigma company. the seeds were planted into culture trays containing cocopeat medium (with ec 0.2 ds/m) and kept in greenhouse with 26±2°c day temperature and 19±2°c night temperature, relative humidity of 70±5% in both dark as well as light (16/8 h photoperiod) conditions, with irrigation round 2 days. the seeds were germinated after 14 days. after seeds germination, 300 apical meristems of the seedlings were treated with different concentrations of colchicine (0, 100, 200 and 400 mg/l with ph=6) at the two true leaf stages using micro spray. tween 20 (500 mm/l) was also added to colchicine solution in order to increase the surface contact of colchicine with plant’s leaves. colchicine treatments were repeated during seven consecutive days. the treated seedling then kept in greenhouse condition as mentioned above. when the plants reached the sixth-leaf growth stages, the treated plants were transferred into individual pot (22 diameter × 28 cm length) containing sand: clay: rotten manure (1:1:1) and they remained in the pots till the end of the experiment. all the plants within the pots received similar completed hoagland nutrient solution at flowering stage. the irrigation was applied with intervals of 3 days and same day and night temperatures and photoperiod as mentioned above, except relative humidity of 40±5% in greenhouse condition. flow cytometry analysis flow cytometry apparatus (model pa, partec, germany d-48161) was used to detect ploidy levels in plant tissues according to ju et al. (2005). the ploidy level of plants was determined at full blooming stage, exactly 15 weeks after transplanting. chemical agents such as nuclear extraction buffer solution and 4, 6 diamid-2-phenyl indol (dapi), under common name of cystain uv persices, were provided from partec companies. parsley (petroselinum crispum mill.) which has nuclear weight of 4.46 pg was used as a standard plant for calibration of the apparatus. nuclei suspensions were obtained when approximately 100 mg of fully developed fresh leaf tissue from different parts of the plant was chopped by a sharp razor blade in a specific buffer on ice, according to gao et al. (1996). nuclear suspensions were filtered through a 50 μm nylon filter and rnase a (sigma aldrich co.) at a concentration of 2 μg/ml was added to each sample (gao et al., 1996). prior to running the experiment all the prepared samples were kept in ice till analyzes via flow cytometry initiated. the internal software of the fcm (bd fac station data processing system) was used to analyze histograms for determination of peak position and the relative ploidy index of the samples for each individual plant (gao et al., 1996). the ploidy level analysis (dna content) was performed using ratio (peak 1= unknown plants and peak 2= index plant) (valente et al., 1998). determination of the ploidy levels of each of the samples were performed in three replicates. measurement of stomata three matured and developed leaves were cut off from different parts of each tetraploid and diploid plant. nail varnish technique was used with some modifications to isolate samplings from surface epidermises in order to observe stomata (smith et al., 1989). the epidermis were mounted on glass slides and a light microscopy “olympus u-da” with a digital camera “dpi 12” was used to photograph and measure stomata density. light microscope was used to assess stomata (numbers/mm2) and size of stomata (length and diameter) (fig. 1) with magnification of 100x and 400x respectively (smith et al., 1989). since feature of stomata density on the leaf was not uniform at the surrounding nervure, photographs from seven sections of stomata where obtained by rotating each individual sample under the microscope. then the mean was calculated for each measurement. observation and measure of chloroplast in stomata guard cells forceps was used to isolate epidermis from leaf surface in order to observe and count chloroplast in stomata guard cells in the detached leaf. the isolated epidermis was then placed in logol solution (1%) for 5 min. after coloring, each individual sample mounted on glass slides and light microscopy “olympus u-da” fig. 1 measurement of stomata (length and diameter) with light microscope magnification 400x. adv. hort. sci., 2018 32(2): 229-238 232 with a digital camera “dpi 12” at the 400x magnification was used to count chloroplasts and simultaneously take photograph for the following counting of the number of chloroplasts in the stomata guard cells. comparison of morphological traits of diploid and tetraploid plants after identification of tetraploid plants, morphological and physiological characteristics, as well as growth behavior of both tetraploid and diploid plants were recorded in order to characterize the differences. number of lateral branches of each sample was counted accurately. a rule (with accuracy 1 mm) and a digital caliper (with accuracy 0.1 mm) were used to measure branch and root diameters. length of leaf and petal were measured by using the caliper digital (with accuracy 0.1 mm). as phenological characteristics, it was measured flowering period, which was considered from initial time of flowering to seeds formation, flowers durability on plants was evaluated according to the number of days. number of seeds per follicle was counted accurately. fresh root and dry weight was measured using a digital scale (accurately 0.01 g). oven (48 hours at 70°c) dry weight of root was measured. seed dimensions including length and diameter were measured using a dial binocular microscope magnification of 40x. evaluation of chlorophyll content fresh samples of apical leaves were collected from both tetraploid and diploid plants and washed thoroughly with distilled water. approximately, 0.1 g of leaves was weighed and placed in a mortar then 2 ml of 80% acetone added to the samples and gently crushed the leaves till the mixture was formed in a uniform state. the samples were centrifuged at 6000 rpm for 15 min. a spectrophotometer device with wavelength of 663 and 645 nm was set to read the absorption of chlorophyll. chlorophyll a and b then were calculated using the following formulas (arnon, 1949). 1) chlorophyll a=12.25(a663)-2.55(a645) × v/w 2) chlorophyll b=20.31(a645)-4.91(a663) ×v/w where v = volume of extract (ml) and w = weight of tissue (mg) observation of chromosome numbers study of cytogenetic event was implemented based on counting the set of chromosomes in individual plant cells of diploid and tetraploid plants. the cells of root tip tissue from germinated seeds were used to observe chromosomes numbers. the seeds of each individual plants (diploid and tetraploid) were sterilized separately with sodium hypocolorid solution (5%) for 5 min (24°c) and rinsed completely with distilled water for 10 min. the seeds were then placed on filter paper in petri dish to germinate at 25±1°c temperature inside the growth chamber. after germination, when the root length reached 5 mm, the roots were separated and washed with distilled water and placed in the pretreatment solution of 8-hydroxyquinoline citrate at temperature of 4°c for two hours. ethanol and acetic acid in ratio of 1:1 (v:v) were used to stabilize the samples at temperature of 4°c for 20 hours. the samples were washed once with distilled water for 30 min, then with 40% ethanol for 15 min, and kept in 50% ethanol for 15 minutes. after this time the samples were removed and kept in 70% ethanol for 15 min. ethanol and xylene were used to detect transparency of the samples (chehrazi et al., 2012). hydrolysis of the samples hydrolysis of the samples occurred when samples were immersed in 70% ethanol and hydrochloric acid (vol. 2:1) solution for 15 minutes. the samples were then washed with distilled water for 15 minutes and used acetocarmine solution to stain the samples for 5 hours at 25±2°c. approximately, 2 mm of tip apex of root was removed at the end of the root tip and placed on glass slides. a light microscopy “olympus u-da” which capable of magnifying of 400x with a digital camera “dpi 12” was used to get photograph and observe number of chromosome (chehrazi et al., 2012). statistical analysis spss software 16.0 was used to perform statistical analysis of the data. the t-test also was applied for the mean comparison at level of 1% of probability. 3. results and discussion effect of colchicine on the rates of survival and tetraploid plants application of 200 and 400 mg/l colchicine solutions on true two-leaf growth stages of c. roseus ‘alba’ diploid plants tended to induce tetraploidy. the concentration of 200 mg/l had the highest survival (79%), whereas the highest percentages of tetraploid plants were induced at the 400 mg/l concentration colchicines. the information from results of flow cytometry analysis, cytogenetic and morphological evaluations indicated that concentration of hosseini et al. plant cytogenetic and morpho-physiological traits alteration in catharanthus roseus 233 400 mg/l was not only able to promote autotetraploidy, but caused to generate the highest mortality among treated plants (table 1). this observation showed the potential of c. roseus ‘alba’ in responding to colchicines treatments reacted differently, so the certain of plants can’t preserve their chromosome sets in balance within the cells, in particular at the high concentration rate of colchicines. flow cytometry analysis elucidated that the majority of those plants tetraploid with high dose of colchicines developed defective chromosomes; mixed ploidy with abnormal structure and the plants die before reaching the maturity. an investigation implemented to induce polyploidy in challistephus chinensis nees. (hanzelka and kobza, 2001) and agastache foeniculum l. (talebi et al., 2017) was indicated that the increase of colchicine concentration on the targeted plants decrease the rate of plant survival. another experiment reported that there is a positive correlation between various concentrations of colchicine applications and mortality in seedlings of chamomile (tanacetum parthenium) (saharkhiz, 2007). it has been documented that when high dose of colchicines used as a mutation agent for plant, toxic contamination, phytotoxicity and abnormality became main cause of death in the plants (han et al., 1999). identification of tetraploid plants using flow cytometry results on analysis of ploidy levels by flow cytometry are shown in figures 2 and 3. the ploidy level was detected by placing plant tissue from tetraploid and diploid plants into flow cytometry, and then the dna content was recorded (sari et al., 1999). the amount of dna content was calculated according to formula: which is described by (bharathan et al., 1994). the rate of dna contents was equal to 0.350.45 and 0.7-0.9 for diploid and tetraploid plants respectively. these findings were similar to the results which were reported by talebi et al. (2017), niu et al. (2016). stomata size and density and chloroplast number of stomata guard cells the results of comparison of stomata at the different locations on the dorsal surface of fully developed leaves have shown that there were significant (1% probability level) differences between diploid and tetraploid plants in relation to density and size of stomata. the results also indicated that density of stomata in diploid and tetraploid plants were 390 and 177 numbers in mm2 (table 2 and fig. 4 b and f). while, the length of stomata and diameter were 17 and 25 µm in diploid and 22.5 and 35.5 µm in tetraploid plants respectively (table 2 and fig. 4 a and e). numbers of chloroplasts of stomata per guard cells were equal to 10 and 20 in diploid and tetraploid plants respectively (table 2 and fig. 4 c fig. 2 flow cytometry analysis of c. roseus ‘alba’ cell nuclei in diploid (peak 1) and index plant of parsley in diploid status (peak 2). colchicine concentration (mg/l) plant survival (%) tetraploid plants (%) 0 98±2 a 0 c 100 96±4 b 0 c 200 79±3 c 35±2 b 400 55±3 d 44±3 a table 1 percentage of plant survival and tetraploid plants of c. roseus ‘alba’ seedlings treated with colchicine fig. 3 flow cytometry analysis of c. roseus ‘alba’ cell nuclei in tetraploid (peak 1) and index plant of parsley in diploid status (peak 2). ± standard error (se). table 2 mean of stomata size, density and chloroplast numbers in diploid and tetraploid plants of c. roseus ‘alba’ ± standard error (se). genotype stomata length (µm) stomata diameter (µm) stomata density (n/mm2) chloroplasts number diploid 22.5±0.6 b 17±0.3 b 390±2.4 a 10±0.2 b tetraploid 35.5±0.4 a 25±0.3 a 177±2.3 b 20±0.3 a adv. hort. sci., 2018 32(2): 229-238 234 fig. 4 stomata size (a), stomata density (b), chloroplast numbers (c), chromosome numbers (d) in diploid and stomata size (e), stomata density (f), chloroplast numbers (g) and chromosome numbers in tetraploid (h) of c. roseus ‘alba’ plants. hosseini et al. plant cytogenetic and morpho-physiological traits alteration in catharanthus roseus 235 and g). early research reports on the size of stomata in the guard cells indicated that these cells are more dependent on genetic than environmental factors as compared to other cells in plant (yasuda et al., 2008). another study on jatropha curcas l. indicated that with increase ploidy, the stomata and pollen grains became larger, but stomata density decreased (niu et al., 2016). a study on the increase polyploidy level of japanese persimmon (diospyrus kaki l.) has shown that with increasing in ploidy level, plant’s stomata guard cells have become enlarge but with less density (tamura et al., 1996). in a report by roy et al. (2001) it has been shown that the length and diameter of the stomata were set to be standard parameters for identification of tetraploid plants. similar work has been done to identify autotetraploid using colchicines treatments in humulus lupulus plant (roy et al., 2001). several other studies showed that application of colchicines treatments tend to increase of ploidy level in plant, the length and diameter of stomata and chloroplast numbers increased (gu et al., 2005; talebi et al., 2017). observation and chromosomes counting in order to determine the ploidy level in the treated plants, the cells of root tip were isolated from both diploid and tetraploid plants after the series of preparation, stained and then light microscope used to count chromosomes sets. chromosome numbers of root tip cells of diploid and tetraploid were 16 and 32 respectively (fig. 3). application of colchicine tends to prevent the activity of the subunits join of microtubule (tubulin protein) and/or keep them apart that impedes the formation of the spindle fibers during the cell division and stops chromosomes movement in metaphase stage (kundu and ray, 2016). thus, cell division occurs without cell wall formation which leads to double the number of chromosomes in plant cells. colchicine treatment could induce different types of mitotic abnormalities including c-metaphase, vagrant chromosomes, sticky chromosomes, anaphase bridges and increased frequency of micronuclei (gupta, 2002; kundu and ray, 2016). phenotypic variation between diploid and tetraploid plants the t-test at the 1% probability level showed significant differences between diploid and tetraploid plants. as chromosome numbers duplicated, stem number, stem diameter, leaf area, leaf number, flower diameter, diameter of flower ovary, total chlorophyll content, fresh and dry weight of roots, duration of flowering length, durability of flowering, root diameter, length and diameter of seeds were significantly increased in the tetraploid as compared to diploid plants while. on the other hand, increases ploidy level from diploid to tetraploid decreased length of lateral branches and root and number of seed in follicle (table 3). in the present experiment, promotion of ploidy level caused to reduce lateral branch length, but tended to increase diameter and number of branches (table 3). in fact, the formation of polyploidy in plant is simply due to reduction of the frequency of cell division during initiation of growth and development which caused to lowering growth rate in tetraploid when compared to diploid plants. in an experimental study that was conducted to induce polyploidy in cumin plant it has been reported that autotetraploid plants have lower growth at the early growth stages and shorter time to flowering stage than diploid plants (dijkestra and speckmann, 1980). while, at the same time, the diameter of lateral branches and its numbers significantly enhanced in tetraploid plants (table 3). rubuluza et al. (2007) in their research proved that colchinice treatments are capable of producing similar out puts on seedling of colophospermum mopane l. plants. characteristics diploid tetraploid length of lateral branches (cm) 24.5±0.5 a* 13.3±0.6 b* diameter of lateral branches (mm) 3.2±0.2 b 5.3±0.2 a lateral branch number 3.3±0.2 b 8.4±0.3 a leaf length (mm) 60±1 b 97±3 a leaf width (mm) 20±0.04 b 47±0.2 a petal length (mm) 22±0.25 b 27±0.4 a petal width (mm) 15±0.05 b 25.3±0.03 a a chlorophyll (mg/g) 0.6±0.015 b 0.9±0.01 a b chlorophyll (mg/g) 0.21±0.01 b 0.37±0.01 a duration length of flowering (day) 148±2 b 181±2 a durability of flowering (day) 3.6±0.24 b 7±0.15 a root length (cm) 30.8±1.8 a 17.5±1.7 b root diameter (mm) 3.5±0.2 b 6±0.3 a fresh weight of roots (g) 8.5±0.2 b 14.2±0.3 a dry weight of root( g) 1.4±0.03 b 2.3±0.05 a number of seeds in follicle 17.5±0.4 a 7.7±0.4 b seed length (mm) 2±0.04 b 3±0.06 a seed diameter (mm) 1.1±0.02 b 1.5±0.04 a table 3 comparison of morphological and physiological traits in diploid and tetraploid plants of c. roseus ‘alba’ ± standard error (se). * in each row, means with similar and dissimilar letters are no significant and significant respectively according to t-test. adv. hort. sci., 2018 32(2): 229-238 236 the length and width of leaves were larger in tetraploid than diploid plants (table 3 and fig. 5). so, a plant with such characteristic is being able to sustain more chemical substances in vegetative organs. artificially induction of polyploidy in many plant species caused to increase cell size and consequently enhance flower size, inflorescence, leaves, vegetative and generative organs (watrous and wimber, 1988; omidbaigi, 2009; niu et al., 2016; talebi et al., 2017). other researchers reported the beneficial effect of artificially inducing polyploidy in the melaleuca alternifolia (zhang, 2000), tanacetum parthenium l. (saharkhiz, 2007), jatropha curcas (niu et al., 2016) and agastache foeniculum l. plants (talebi et al., 2017) which caused to enlarge size of leaves and branches. the amount of chlorophyll a and b were significantly increased in the leaves of tetraploid (table 3). the accumulation of high chlorophyll contents in the leaves of tetraploid plants may be relevant to increase number of chloroplasts in the stomata guard cells (fig. 4 c and g). as shown above, the number of chloroplasts in stomata guard cells of tetraploid plants was two folds greater than those in diploid plants. in a similar experiment conducted on acacia (acacia mearnsii), mathura et al. (2006) have shown that chlorophyll content was significantly higher in tetraploid than diploid plants. polyploidy tended to have positive effect on size of reproductive organs. the growth of length and width of petals developed significantly higher in tetraploid than diploid plants (table 3 and fig. 5). a considerable growth increases was occurred in petal and flower size of tetraploid when carnation and jatropha curcas plants received leaf foliar application of colchicines (roy chowdhury and tah, 2011; niu et al., 2016). in the present study, colchicine treatments caused to make the length of flower duration and durability of flowers longer in tetraploid than diploid plants (table 3). generally, in tetraploid plants, the beginning of flowering is delayed and the flowering period is longer than the diploid plants (blakeslee and avery, 1937; lavania and srivastava., 1991). the roots length in tetraploid (17.5 cm) is less than diploid plants (30.8 cm) (table 3). this reduction may be due to reduction of cell division in the longitudinal direction, but it has been reported that with reduction of root length, root diameter increased in these plants (sarathum et al., 2010). it has been found that when ploidy level was increased in salvia plant, root diameter significantly increased as compared to control plants (gao et al., 1996). increase in fresh and dry weight of root in tetraploid can be due to production of lateral roots and enhance of root diameter (table 3). a series of experimental studies which were carried on hyoscyamus niger l. and agastache foeniculum l. plants, elucidated that doubling level of chromosome from diploid to tetraploid caused to increase fresh and dry weight of root and shoot (lavania and srivastava, 1991; talebi et al., 2017). in the present experiment, colchicines treated plants were able to produce seeds with a bigger size than untreated plants. lengths of seeds in diploid and tetraploid plants were 2 and 3 mm, respectively. and seed diameter measurement indicated that tetraploid had thicker seed in diameter than diploid, but the number of seeds per follicle has decreased from 17.7 to 7.7 numbers in tetraploid plants (table 3). the implication behind the reduction of the seed number per follicle may be attributed to increasing the seed size in the plant. it has been reported that artificially induced polyploidy plant usually caused to produce seeds with bigger size as compared to diploid plants, whereas the seed numbers simply reduced. niu et al. 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propagation and polyploidy induction in melaleuca alternifolia. j. southwest agric. univ., 22: 507-509. impaginato 345 adv. hort. sci., 2019 33(3): 345-358 doi: 10.13128/ahs-23714 effect of vermicompost on morphological and physiological performances of pot marigold (calendula officinalis l.) under salinity conditions n. adamipour 1 (*), m. khosh-khui 1, h. salehi 1, h. rho 2 1 department of horticulture science, school of agriculture, university of shiraz, iran. 2 plant stress laboratory texas, a&m agrilife research at amarillo, 6500 amarillo blvd., west amarillo, tx 79106‐1796, usa. key words: antioxidative enzymes, chlorophyll content, fertilizer, nutrient elements. abstract: a greenhouse study was conducted in order to evaluate the interactions of vermicompost and salinity effects on morphology and physiology of pot marigold. the experiment was conducted with vermicompost treatments at five levels (0%, 5%, 10%, 15% and 20%) and salinity treatments at five levels (0, 50, 100, 150 and 200 mm nacl) in a completely randomized factorial design arrangement with four replications. results showed that increasing levels of salinity led to decline in leaf area, fresh and dry weights of flower, shoot, and root, n, p, k, fe, mg and zn concentrations, chlorophyll and carotenoid contents, while proline content increased in the plants. apx, sod, pod and cta enzyme activities significantly increased with increasing salinity from 0 to 150 mm nacl, then declined in 200 mm treatment in the plants. application of vermicompost increased the morpho-physiological indices and mineral nutrient uptake in the plants and could increase the plant yield by alleviating the harmful effects of salinity. 1. introduction calendula officinalis, known as “pot marigold”, is a plant in calendula genus of asteraceae family. it is perhaps native and widely naturalized further northern to southern europe and elsewhere in warm temperate regions of the world, and it may possibly be planted widely in gardens and landscapes (gharineh et al., 2013). among ornamental bedding plants, pot marigold is known to grow well under saline conditions. in fact, some pot marigold cultivars that are used as cut flowers or as bedding plants in landscaping can be grown by maintaining the quality of plants under saline conditions with an ecw of <8 ds m-1 (koksal et al., 2016). plants are exposed to ever-changing and often unfavorable environmental conditions, which cause both biotic and abiotic stresses such as extreme temperatures, flood, drought, and salinity. overexploitation of (*) corresponding author: nader.adamipour@shirazu.ac.ir citation: adamipour n., khosh-khui m., salehi h., rho h., 2019 effect of vermicompost on morphologi‐ cal and physiological performances of pot mari‐ gold (calendula officinalis l.) under salinity condi‐ tions. adv. hort. sci., 33(3): 345-358 copyright: © 2019 adamipour n., khosh-khui m., salehi h., rho h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 july 2018 accepted for publication 23 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 345-358 346 available water resources as well as environmental factors such as low precipitations, high temperatures and contamination from parental rocks are leading to increases in soil salinization (aroca et al., 2013). soil salinization is one of the most important agricultural and eco-environmental problems nowadays, which is increasing steadily in many parts of the world. saline soils have been estimated to occupy more than 7% of the earth’s land surface and it is expected to be increased by up to 50% by the middle of the twentyfirst century (ruiz-lozano et al., 2012). salinity stress is one of the major abiotic threats to plant life and agriculture worldwide and significantly reduces crop yield in the affected areas. excessive salt above what plants need limits plant growth and productivity and can lead to plant death. about 20% of all irrigated land is affected by soil salinity, decreasing crop yields. plants are affected by salinity stress in two main ways: osmotic stress and ionic toxicity. these stresses affect all major plant processes, including photosynthesis, cellular metabolism, and plant nutrition (aslamsup et al., 2011). amelioration of salt-affected soils can be accomplished through many effective methods, such as water leaching, chemical remediation, and phytoremediation (qadir et al., 2007). the amelioration of salt-affected soils using chemical agents, including gypsum, calcite, calcium chloride and organic matter, is a successful approach that has been implemented worldwide (sharma and minhas, 2005; tejada et al., 2006). according to a study, the application of organic matter conditioners has become a common practice in salt-affected areas in the last several decades and constitutes an important method of soil regeneration and fertility enhancement (melero et al., 2007). organic matter is very important for maintaining structural stability in soils as well as improving the physical, chemical and biological properties of soils. salt-affected soils generally exhibit poor structural stability due to low organic matter. the addition of organic materials (e.g. green, farmyard and poultry manures, compost, food processing wastes, etc.) has been suggested for improving structural stability of soils by many researchers (tejada et al., 2006). barzegar et al. (1997) found that addition of plant residues improved the water-stable aggregate in soils because of increased organic matter content and decreased soil salinity. the application of organic matter for soil remediation is considered essential for sustainable land use and crop productivity. given the importance of pot marigold in green space, and their being placed in saline soils in most cultivable regions, so far, enough research has not been conducted to understand morpho-physiological properties of pot marigold under salinity stress conditions and determine the status of vermicompost in reducing the devastating effects of salinity stress in pot marigold. for this purpose, an experiment was conducted to determine the effects of vermicompost on some morphological and physiological characteristics of pot marigold under salinity stress conditions. 2. materials and methods to investigate the effects of vermicompost on some physiological characteristics of pot marigold (calendula officinalis l. cv. candyman orange) under salinity stress, a greenhouse experiment was conducted in a completely randomized factorial design including vermicompost at five levels (0%, 5%, 10%, 15% and 20%) and salinity stress at five levels (including 0, 50, 100, 150 and 200 mm nacl) with four replicates. this study was conducted at the research greenhouse of the department of horticultural science, college of agriculture, shiraz university, shiraz, iran (52°32´e and 29°36´n). after disinfection of pots (20 and 30 cm in diameter and length, respectively), 5%, 10%, 15% and 20% (v/v) vermicompost from kian pars shiraz company, were dried in the shade and were mixed with four kilograms of a native soil. the main physical and chemical characteristics of the vermicompost and soil mixture are shown in table 1. then, four marigold seeds were planted in each pot. the pots containing seeds were kept in a greenhouse with 27/18°c (day/night) temperature, 16 h light conditions, and 35% relative humidity. after germination of seeds one plant was selected in each pot and the three other plants were removed. when the plants reached the four-leaf stage, the plants were treated with four levels of salinity stress. table 1 some physico-chemical properties of vermicompost and soil sample vermicompost soil organic matter % 33 0.41 ph 8.1 7.05 ec (d/sm) 1.7 0.6 p (%) 1.8 pwp (%) 5.1 k (%) 1.2 fc (%) 14 c/n 13 total n (%) 1.5 soil texture sandy-loam adamipour et al. ‐ effect of vermicompost on performances of pot marigold 347 salinity stress treatment was applied by adding net quantities of sodium chloride (nacl) to the irrigation water so that pots were irrigated with 50, 100, 150, and 200 mm nacl containing water based on field capacity of the soil, and the amount of the decreased water obtaining by the salinity treatment resulted in the average electrical conductivity (ec) of 3.62, 6.27, 9.36 and 12.71 ds/m in each level of the treatment, respectively. the control treatment was applied using distilled water. after 35 days of the treatments, the plants were harvested in order to measure morphological and biochemical traits. growth parameters growth parameters including, flower diameter (mm), leaf area (cm2) and fresh and dry weights of flower, shoot, and root (g) were measured. for dry weight determination, the shoots, roots, and flowers were dried in an oven at 70°c for 48 h and weighed. proline content the leaf proline content was determined using the method of bates et al. (1973). proline was extracted from leaf samples of 100 mg weight fresh with 2 ml of 40% methanol. 1 ml of the extract was mixed with 1 ml of a mixture of glacial acetic acid and orthophosphoric acid (6 m) (3:2, v/v) and 25 mg ninhydrin. after 1 h of incubation at 100°c, the tubes were cooled, and 5 ml toluene was added. the absorbance of the upper phase was spectrophotometrically determined at 528 nm. the proline concentration was determined using a standard curve. chlorophyll and carotenoid contents chlorophyll and carotenoid contents were estimated by the method of hiscox and israelstam (1979). fresh leaf and petal material (1 mg) and 10 ml dmso were taken in vials and kept in an oven at 65°c for 4 h. absorbance was read at wavelengths of 665 and 649 for leaves, and 480 nm for petals using a spectrophotometer (beckman du 640 b, fullerton, usa). the following equations were used to calculate each compound. chl a= (12.47×a665)-(3.62×a649) chl b= (25.06×a649)-(6.5×a665) total chlorophyll (mg g-1 f.w.) = chla + chlb carotenoids (mg g-1 f.w.) = where a stands for the absorbance reading of a sample at 665, 649 and 480 nm of wavelength. antioxidant analysis fresh leaf samples were homogenized in extraction buffer (0.1 m phosphate buffer ph 6.8) with a mortar and pestle on ice. the homogenate was then centrifuged at 12,000 g for 15 min at 4°c and the supernatant was used as the crude extract for the superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase. the superoxide dismutase (sod), guaiacol peroxidase (pod), ascorbate peroxidase (apx) and catalase (cat) enzymes were estimated using the methods previously described by beauchamp and fridovich (1971), chance and maehly (1955), nakano and asada (1981), and dhindsa et al. (1981), respectively. plant nutrient element analysis leaf material was ground to pass 0.5 mm sieve in a cyclone laboratory mill, weighed into ceramic crucibles, ashed overnight at 550°c in a muffle furnace, and the ash was suspended in 2m hcl for determination of mineral nutrients. then, total nitrogen (n), phosphorus (p) and potassium (k) were determined in the leaf using the kjeldahal, colorimetrically and ammonium acetate methods respectively., zinc (zn), magnesium (mg) and iron (fe) were determined by atomic absorption spectroscopy (baumard et al., 1998). statistical analyses the data were analyzed using one-way analysis of variance at p < 0.05 significance with sas version 9.4 software (sas institute inc., cary, nc). fisher’s lsd test was conducted to determine the statistical differences among different treatments. 3. results and discussion leaf area and fresh and dry weight of shoots the growth (leaf area and fresh and dry weight of shoots) was significantly affected by the treatments (table 2). the treatment of plants with nacl significantly reduced the growth parameters and the decrease was proportional to the concentration of nacl. the highest concentration (200 mm nacl) was the most deleterious and decreased the leaf area by 53.32%, fresh weight by 72.76%, and dry weight by 48.74% as compared to those of the control plants. however, all the above parameters were significantly enhanced by the vermicompost treatments. the highest and the lowest leaf area and fresh and dry weight of shoots were obtained in the 20% vermicompost and the control treatment, respectively (table 2). leaf area and fresh and dry weight of shoots increased (12.03%, 39.52% and 38.96%, respectively) at the 20% vermicompost treatment compared to the control plants. researches showed 1000 480 1 29 53 78 220      a chla chlb. . adv. hort. sci., 2019 33(3): 345-358 348 that soil salinity reduces the growth of plant shoots. this water potential reduction in the soil or osmotic effect is due to the presence of salt in the soil which limits the root water absorption (nguyen et al., 2015). in the salt stress, the plant’s hormonal system which synthesizes and transmits a number of hormones such as cytokinins is impaired and their transport from roots to the upper parts of the plant is limited. the termination of the hormone transfer from roots to branches and the consequent reduced water absorption capacity can lead to a decline in plant growth. reduction of leaf area can be explained by decreased cell growth or a reduced cell division rate due to decreased cellular turgor. decrease in leaf area also reduces the rate of photosynthesis, resulting in decreased fresh and dry weight of plants (zarei et al., 2016). in addition, alshammary et al. (2004) showed that decrease in growth was due to reduction of flexibility, development of cells, and reduction of auxins. vermicompost, due to its high microbial activity resulting from the presence of fungi, bacteria, yeasts, actinomycetes, and algae, can produce different growth regulators such as auxins, gibberellins, and cytokinins all of which may have positive effects on plant growth and development (xu et al., 2016; ullah et al., 2018). thus, the cause of rises in height and leaf area of the plants treated with vermicompost is probably from the stimulation of production of growth regulators including auxins and gibberellins. furthermore, vermicompost contains humic substances that increase the availability of plant n, p, k, and in particular zn for the synthesis of tryptophan, a precursor to auxins that are used for rooting and plant growth (sharifianpour et al., 2015; scaglia et al., 2016). fresh and dry weight of roots the fresh and dry weight of roots of the pot marigold plants was lower with the salinity stress treatments as compared to that of the plants under non-saline conditions (table 3). fresh and dry weight of roots decreased (51.91% and 77.98%, respectively) table 2 effect of salinity and vermicompost and their interaction on leaf area and shoot fresh and dry weight * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 leaf area 0 36.10±1.33 gh 36.10±0.62 gh 35.12±0.62 i 27.15±0.62 m 15.91±0.62 q 30.05±8.10 e 5 37.05±0.62 f 36.71±0.62 fg 35.01±0.62 gh 27.85±0.62 m 16.61±0.62 q 30.03±8.08 d 10 38.39±0.62 de 38.05±0.62 e 37.16±0.62 f 29.19±0.62 l 17.95±0.62 p 32.14±8.08 c 15 39.28±0.62 bc 38.94±0.62 cd 38.05±0.62 e 30.08±0.62 k 18.84±0.62 o 33.03±8.08 b 20 40.40±1.18 a 40.06±1.18 ab 39.17±1.18 cd 31.20±1.18 j 19.96±1.18 n 34.16±8.08 a mean salinity 38.24±1.73 a 37.95±1.65 a 37.06±1.65 b 29.09±1.65 c 17.85±1.65 d shoot fresh weight (g) 0 59.92±1.18 h 57.79±1.18 i 54.37±1.18 k 27.19±1.18 n 9.79±1.18 p 44.05±1.92 e 5 60.62±0.62 h 58.49±0.62 i 55.07±0.62 kj 39.11±0.62 m 10.49±0.62 p 44.75±1.92 d 10 70.35±0.82 e 68.22±0.82 f 64.22±0.82 f 48.84±0.82 l 20.22±0.82 o 54.49±1.92 c 15 76.27±0.91 ab 74.14±0.91 c 74.14±0.91 c 54.76±0.91 kj 26.14±0.91 n 60.41±1.92 b 20 77.32±0.75 a 75.19±0.75 bc 75.19±0.75 bc 55.8±0.751 j 16.22±0.75 n 61.46±1.92 a mean salinity 68.89±7.67 a 66.76±7.67 b 63.34±7.67 c 47.38±7.67 d 18.76±7.67 e shoot dry weight (g) 0 13.18±1.18 gh 12.84±1.18 hi 11.38±1.1 8 jk 8.92±1.18 mn 5.8±1.18 1p 10.42±3.01 e 5 13.88±0.62 fg 13.54±0.62 fgh 12.08±0.62 ij 9.62±0.62 lm 6.5±0.621 p 11.12±2.87 d 10 15.22±0.62 cd 14.88±0.62 de 13.42±0.62 gh 10.96±0.62 k 7.85±0.62 o 12.46±2.87 c 15 16.11±0.62 b 15.77±0.62 bc 14.31±0.62 ef 11.85±0.62 j 8.74±0.62 n 13.35±2.87 b 20 17.23±0.62 a 16.89±0.62 a 15.43±0.62 bcd 12.97±0.62 h 9.62±0.62 lm 14.48±2.87 a mean salinity 15.12±1.65 a 14.78±1.65 a 13.32±1.65 b 10.86±1.65 c 7.75±1.6 5 d adamipour et al. ‐ effect of vermicompost on performances of pot marigold 349 vated the amount of biomass; the total fresh and dry weightwere 31.37% and 79.04% higher than those of the control plants, respectively. the vermicompost treatments also improved the amount of biomass in the plants that were subjected to the salinity stress treatments (table 3). edwards and burrows (1988) reported that increase in fresh and dry weight of roots depends on increase in the activity of hormonal substances such as auxins, cytokinins, and gibberellins as well as vitamin b12. flower diameter and fresh and dry weight of flower flower diameter and fresh and dry weight of flower of the pot marigold plants were found to significantly decrease as the salt concentration was raised. these parameters decreased (33.58%, 67.94% and 27.48%, respectively) at the salinity level with 200 mm nacl compared to the control treatment (table 3 and 4). several reports have shown that salinity decreases the flower diameter and fresh and dry weight of flower in (zinnia elegans) (carter and grieve, 2010), madagascar periwinkle (cathasanthus roseus) (jaleel et al. , 2008) and garden mum (chrysanthemum× morifolium) (lee and van iersel, 2008). the vermicompost applications significantly at salinity level of 200 mm nacl compared to the control treatment. ionic toxicity, nutritional imbalance, and osmotic osmolality adjustment are the negative effects of salinity stress on plant metabolism. roots are the organ responsible for absorbing water and minerals, and salinity stress affects plant shoots more than the root system; however, the root system is the first organ that is exposed to salinity stress (demiral and türkan, 2005). one of the most effective indices in salinity tolerance is the maintenance of cellular turgor and osmotic regulation due to salt absorption and organic matter production. plants, for production of organic materials such as glycine betaine, sorbitol, mannitol, and proline, spend a large amount of energy to regulate osmotic resistance in response to salinity stress. as a consequence, plants under salinity stress conditions reduce root efficacy in supplying nutrients and water to other organs. this reduces growth of shoots and dry matter production, and eventually reduces the transfer of nutrients from roots to shoots, and thus leads to a reduction in dry weight of roots and stems of plants (claussen, 2005; butt et al., 2016; sattar et al., 2016). the vermicompost treatments of the plants grown under stress-free conditions significantly eletable 3 effect of salinity and vermicompost and their interaction on root fresh and dry weight and flower diameter * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 root fresh weight (g) 0 13.33±0.54 bcd 13.25±0.54 bcd 13.18±0.54 cde 9.28±0.54 fg 6.26±0.54 i 11.06±2.96 d 5 13.33±1.32 bcd 13.46±1.32 bcd 12.08±1.32 ij 9.49±1.32 fg 6.30±1.32 i 11.24±3.18 d 10 14.46±0.73 b 14.38±0.73 bc 14.31±0.73 bc 10.41±0.73 f 6.47±0.73 i 12.00±3.34 c 15 16.01±1.24 a 15.93±1.24 a 15.86±1.24 a 11.96±1.24 e 7.85±1.24 h 13.52±3.48 b 20 17.02±0.66 a 16.94±0.66 a 16.87±0.66 a 12.97±0.66 de 8.86±0.66 gh 14.53±3.36 a mean salinity 14.87±1.69 a 14.79±1.69 a 14.72±1.69 a 10.82±1.69 b 7.15±1.36 c root dry weight (g) 0 6.50±0.54 d-g 6.38±0.54 efg 6.07±0.54 fg 2.17±0.54 jk 0.83±0.54 l 4.39±2.51 d 5 6.71±1.32 e-g 6.59±1.32 e-g 6.28±1.32 fg 2.38±1.32 ijk 0.89±1.32 l 4.57±2.73 d 10 7.63±0.73 cd 7.51±0.73 de 7.20±0.73 def 3.30±0.73 ij 1.30±0.73 ik 5.38±2.76 c 15 9.18±1.24 ab 9.06±1.24 ab 8.75±1.24 bc 4.85±1.24 h 3.43±1.24 i 6.85±3.02 b 20 10.19±0.66 a 10.07±0.66 a 9.76±0.66 ab 5.86±0.66 gh 7.85±0.66 h 7.86±2.87 a mean salinity 8.04±1.69 a 7.92±1.69 a 7.61±1.69 a 3.71±1.69 b 1.77±1.28 c flower diameter (mm) 0 65.50±1.91 cde 61.50±1.91 def 59.50±1.91 fg 52.50±1.91 i 41.50±1.91 k 55.50±8.20 c 5 62.50±2.08 cde 61.50±2.08 def 59.50±2.08 fg 52.50±2.08 i 41.50±2.08 k 55.50±8.23 c 10 63.25±2.98 bcd 62.25±2.98 c-f 60.25±2.98 ef 53.25±2.98 i 42.25±2.98 k 56.25±8.45 c 15 65.00±3.55 abc 64.00±3.55 bcd 62.00±3.55 def 55.00±3.55 hi 44.00±3.55 kj 58.00±8.62 b 20 67.00±1.41 a 66.00±1.41 ab 64.00±1.41 bcd 57.00±1.41 gh 46.75±1.41 j 60.15±7.85 a mean salinity 65.05±2.85 a 63.05±2.85 a 61.05±2.85 b 54.05±2.85 c 43.20±3.03 d 350 adv. hort. sci., 2019 33(3): 345-358 increased these parameters compared to the control under both absence and presence of the salinity stress treatments. the 20% vermicompost treatment under salinity stress conditions gave the higher values for these parameters than those with the other treatments (table 3 and 4). the extent of the increase in the values mentioned above was by 8.37%, 56.53% and 6.19 in the 20% vermicompost treatment as compared with those of the control, respectively. hidalgo et al. (2006) stated that using vermicompost fertilizer increased flower diameter and fresh and dry weight of flower in marigold which is consistent with the findings of this study. proline content as shown in table 4, proline content of the pot marigold plants was markedly increased by the salinity stress treatments. the maximum and the minimum proline content were observed in the 200 mm nacl and the control treatments, respectively. proline content increased 447.87% at 200 mm nacl compared to the control treatment (table 4). proline content increase is one of the protective mechanisms of this plant against salinity stress. it has been reported when plants are exposed to salinity stress, the breakdown of proteins and thus the increase of amino acids and amides accelerates, one of which is proline (iqbal et al., 2015). protein degradation, decrease in proline oxidase enzyme activity, and exacerbation of p5cs gene expression are the most important factors affecting proline concentration under stress conditions. increase in p5cs gene expression is one of the most important factors affecting proline concentration under stress conditions (kubala et al., 2015). the proline content was clearly affected by the vermicompost treatments. as compared to the control, the increase was 1.28% at 20% vermicompost. interaction between levels of salinity and vermicompost resulted in the highest and the lowest proline content in the plants with 200 mm nacl and 20% vermicompost treatment and with 0 mm nacl and 0% vermicompost treatment, respectively (table 4). in this study, the application of vermicompost increased proline content and reduced the damaging effects of salinity stress. vermicompost increases the amount of n available in plants due to the presence of n in the proline structure, which leads to increased proline synthesis under salinity conditions and increased plant resistance to salinity stress (rafiee et al., 2017). table 4 effect of salinity and vermicompost and their interaction on flower fresh and dry weight and proline content * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 flower fresh weight (g) 0 4.35±0.57 ef 4.28±0.57 ef 3.88±0.57 fg 2.89±0.57 ij 1.04±0.57 m 3.29±1.37 d 5 4.71±0.65 de 4.64±0.65 de 4.24±0.65 ef 3.25±0.65 ih 1.56±0.65 lm 3.68±1.33 c 10 5.07±0.65 d 5.00±0.65 d 4.60±0.65 de 2.37±0.65 jk 1.92±0.65 kl 3.79±1.51 c 15 6.25±0.47 bc 6.18±0.47 bc 5.78±0.47 c 3.55±0.47 gh 2.03±0.47 kl 4.76±1.77 b 20 6.93±0.82 a 6.86±0.82 a 6.46±0.82 ab 3.27±0.82 ih 2.22±0.82 kl 5.15±2.16 a mean salinity 5.46±1.14 a 5.39±1.14 a 4.99±1.14 b 3.07±0.74 c 1.75±0.65 d flower dry weight (g) 0 1.26±0.07 e-h 1.25±0.07 e-h 1.20±0.07 hi 1.11±0.07 jkl 0.84±0.07 p 1.13±0.07 c 5 1.27±0.06 e-g 1.26±0.06 e-g 1.21±0.06 gh 1.12±0.06 jk 1.05±0.06 lm 1.16±0.07 b 10 1.30±0.05 cde 1. 30±0.05 cde 1.23f±0.05 gh 1.14±0.05 ji 0.88±0.05 op 1.18±0.07 ab 15 1.30±0.01 cde 1.30±0.01 cde 1.28±0.01 e-g 1.07±0.01 klm 1.04±0.01 m 1.20±0.08 a 20 1.40±0.05 a 1.38±0.05 ab 1.36±0.05 bcd 0.96±0.05 n 0.92±0.05 no 1.20±0.09 a mean salinity 1.31±0.22 a 1.29±0.13 a 1.25±0.16 b 1.08±0.10 c 0.95±0.16 d proline content (µmol g‐1 f.w.) 0 4.19±0.12 k 4.31h±0.12 ij 4.76±0.12 f 10.32±0.12 d 23.18±0.12 b 9.35±7.47 d 5 4.20±0.11 k 4.32±0.11 hi 4.77±0.11 f 10.33±0.11 d 23.19±0.11 b 9.36±7.47 d 10 4.22±0.10 k 4.35±0.10 h 4.80±0.10 f 10.35±0.10 d 23.22±0.10 b 9.39±7.47 c 15 4.28±0.08 j 2.40±0.08 g 4.85±0.08 e 10.41±0.08 c 23.27±0.08 a 9.44±7.47 b 20 4.30±0.07 ij 4.42±0.07 g 4.87±0.07 e 10.43±0.07 c 23.30±0.07 a 9.47±7.47 a mean salinity 4.24±0.10 e 4.36±0.10 d 4.81±0.10 c 10.37±0.10 b 23.23±0.10 a adamipour et al. ‐ effect of vermicompost on performances of pot marigold 351 chlorophyll content the plants treated with nacl exhibited a significant decrease in chlorophyll content and the greatest damage was caused by 200 mm nacl treatment. in comparison with the control, chlorophyll content decreased by 28.12% with 200 mm nacl treatment (table 5). whereas, the vermicompost treatments reversed the adverse effects of nacl and caused a significant increase in chlorophyll content in the salttreated plants (table 5). the highest value of chlorophyll content in leaves (18.71%) was recorded in the vermicompost-treated plants over the control plants. usually, decreasing chlorophyll when plants face stress conditions may be due to an alternation in n metabolisms in relation to the production of compositional compounds such as proline, which are used in osmosis regulation, because an increase in proline production causes glutamate to less involve in the chlorophyll biosynthesis pathways (jaleel et al., 2008; håkanson and eklund, 2010). in addition, increased oxidative stress that is caused by reactive oxygen species damage to the chloroplast structure reduces the concentration of chlorophyll. reduction of chlorophyll content has been reported in plants under salinity stress conditions due to the activity of chlorophyllase enzyme. furthermore, some growth regulating agents such as abscisic acids and ethylene stimulate the activity of this enzyme (ali et al., 2004; zhao et al., 2007). the application of vermicompost significantly increased chlorophyll content in the leaves in the present study. vermicompost increases the synthesis of chlorophyll under salinity stress conditions by providing nutritious elements such as fe, zn, mg, and n directly and indirectly (nadi et al., 2011; narkhede et al., 2011). total carotenoid content the content of carotenoid decreased in the plants that received 50, 100, 150 and 200 mm nacl concentrations. the content of carotenoid was the lowest (37.16% over control) for the plants that received the highest level of the salinity treatment (200 mm nacl) (table 5). the vermicompost treatments not only improved the production of total carotenoid under saline-free conditions, but also successfully ameliorated the adverse effects caused by the salinity stress treatments on the plants (table 5). the content of carotenoid increased in the vermicompost-treated plants grown with the 20% level by 1.30% compared to that in the control plants. carotenoids act as * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. table 5 effect of salinity and vermicompost and their interaction on chlorophyll content, total carotenoid content and superoxide dismutase vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 chlorophyll content (mg g‐1 f.w.) 0 2.09±0.11 k 2.06±0.11 l 1.97±0.11 m 1.77±0.11 r 1.46±0.11 v 1.87±0.26 e 5 2.17±0.11 g 2.14±0.11 i 2.05±0.11 l 1.85±0.11 p 1.54±0.11 u 1.95±0.26 d 10 2.24±0.11 e 2.21±0.11 f 2.12±0.11 j 1.92±0.11 o 1.60±0.11 t 2.02±0.26 c 15 2.27±0.11 d 2.24±0.11 e 2.15±0.11 h 1.95±0.11 n 1.64±0.11 s 2.05±0.26 b 20 2.44±0.11 a 2.41±0.11 b 2.32±0.11 c 2.12±0.11 j 1.81±0.11 q 2.22±0.26 a mean salinity 2.24±0.15 a 2.21±0.15 b 2.13±0.15 c 1.92±0.15 d 1.61±0.15 e total carotenoid content (μg∙g‐1 f.w.) 0 12.48±0.92 k 12.46±0.92 l 12.43±0.92 o 12.35±0.92 r 7.82±0.92 w 11.51±2.06 e 5 12.50±0.92 i 12.49±0.92 j 12.45±0.92 m 12.37±0.92 q 7.84±0.92 v 11.53±2.06 d 10 12.52±0.92 f 12.51±0.92 h 12.48±0.92 k 12.40±0.9 2p 7.87±0.92 u 11.55±2.06 c 15 12.57±0.92 d 12.55±0.92 e 12.52±0.92 g 12.44±0.92 n 7.91±0.92 t 11.60±2.06 b 20 12.63±0.92 a 12.61±0.92 b 12.58±0.92 c 12.50±0.92 i 7.97±0.92 s 11.66±2.06 a mean salinity 12.54±0.82a 12.52±0.82 b 12.49±0.82 c 12.41±0.82 d 7.88±0.82 e superoxide dismutase (ug‐1 f.w.) 0 146.00±1.92 m 152.00l±1.92 m 156.00±1.92 kl 440.00±1.92 d 104.00±1.92 q 199.60±1.25 c 5 155.50±0.80 kl 161.50±0.80 ijk 165.50±0.80 ghi 449.50±0.80 c 113.50±0.80 p 209.10±1.24 b 10 15.50±0.80 jkl 163.50±0.80 hij 167.50±0.80 f-i 451.50±0.80 bc 115.50±0.80 o 211.10±1.24 b 15 163.50±0.80 hij 169.50±0.80 e-h 173.50±0.80 ef 457.50±0.80 ab 121.50±0.80 n 217.10±1.24 a 20 165.50±0.80 ghi 171.50±0.80 efg 175.50±0.80 e 459.50±0.80 a 123.50±0.80 n 219.10±1.24 a mean salinity 157.60±1.22 d 163.60±1.22 c 167.60±1.22 b 451.60±1.22 a 115.60±1.22 e adv. hort. sci., 2019 33(3): 345-358 352 helper pigments in chloroplasts, but their most important role is antioxidant properties. because of the oxidative stress caused by salinity stress in plant tissues, carotenoid activity in both antioxidant enzymatic and non-enzymatic systems decrease (pant et al., 2009). in similar studies in summer savory (satureja hortensis l.) (najafi and khavari-nejad, 2010), wheat (triticum vulgare l.) (reddy and vora, 2005), and marjoram (origanum majorana) (baatour et al., 2010), reduction of carotenoids under salinity conditions has been reported. in a study consistent with the findings of the present study, ayyobi et al. (2014) stated that vermicompost increased carotenoid content in peppermint (mentha piperita l.) leaves. enzyme activities the activities of apx, pod, cat and sod in pot marigold plants were significantly affected by the salinity stress and vermicompost treatments. with the increasing extent of salinity stress, the activities of apx, pod, cat and sod increased markedly and then significantly decreased (table 5 and 6). apx, pod, cat and sod enzyme activities increased (12.08%, 13.67%, 36.08% and 186.54%, respectively) in the plants at salinity level of 150 mm nacl compared to those in the control plants. usually one of the biochemical changes that occur in plants under stress conditions is the accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and radical hydroxyl, all of which are highly toxic and reactive, and disrupt the normal metabolism of the cells. these radicals create secondary oxidative stress, through peroxidation of lipids, resulting in membrane degradation, protein degradation, deactivation of enzymes, elimination of pigments, and disruption of dna, leading to serious damage to the structure of cells and eventually to the whole plant. one strategy of plants to counteract this stress is the accumulation of antioxidant enzymes (kang et al., 2014). similar results have been reported in pot marigold (calendula officinalis l.) (hemmati et al., 2018) and chickpea (cicer arietinum l.) (sadak et al., 2017). the vermicompost treatments significantly enhanced the activities of apx, pod, cat and sod under salinity stress conditions, and the increases in the activities of apx, pod, cat and sod were 2.90%, 21.08%, 2.41% and 9.76% in the plants under the 20% vermicompost treatment compared with those in the control plants (table 5 and 6). similar results table 6 effect of salinity and vermicompost and their interaction on catalase, peroxidase and ascorbate peroxidase * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. salinity (mm) sanility mean vermicompost 0 50 100 150 200 catalase (ug‐1 f.w.) 0 31.65±1.58 r 31.85±1.58 p 32.16±1.58 m 43.18±1.58 e 20.78±1.58 w 31.92±7.4 e 5 31.80±1.58 q 32.00±1.58 o 32.31±1.58 k 43.33±1.58 d 20.93±1.58 v 32.07±7.4 d 10 31.85±1.58 p 32.05±1.58 n 32.36±1.58 j 43.38±1.5 8 c 20.98±1.58 u 32.13±7.4 c 15 32.05±1.58 n 32.26±1.58 l 32.56±1.58 h 43.58±1.58 b 21.19±1.58 t 32.33±7.4 b 20 32.41±1.58 i 32.61±1.58 g 32.92±1.58 f 43.94±1.58 a 21.54±1.58 s 32.69±7.4 a mean salinity 31.95±1.43 d 32.16±1.43 c 32.46±1.43 b 43.48±1.43 a 21.08±1.43 e peroxidase (ug‐1 f.w.) 0 68.40±0.93 o 69.07±0.93 no 70.80±0.93 mn 78.62±0.93 gh 41.48±0.93 t 65.68±12.99 e 5 70.75±3.98 nm 71.42±3.98 lm 73.15±3.98 kl 80.97±3.98 ef 43.83±3.98 s 68.03±13.43 d 10 75.03±3.98 jk 75.71±3.98 ij 77.44±3.98 hi 85.26±3.98 c 48.12±3.98 r 72.31±13.43 c 15 77.59±3.98 hi 78.27±3.98 gh 80.00±3.98 fg 87.82±3.98 b 50.67±3.98 q 74.87±13.43 b 20 82.25±3.98 ef 82.93±3.98 de 84.66±3.98 cd 92.48±3.98 a 55.33±3.98 p 79.53±13.43 a mean salinity 74.80±5.96 c 75.48±5.96 c 77.21±5.96 b 85.03±5.96 a 47.89±5.96 d ascorbate peroxidase (ug‐1 f.w.) 0 869.60±1.51 s 875.40±1.51 q 880.40±1.51 n 976.10±1.51 e 459.60±1.51 x 812.20±1.85 e 5 873.20±1.51 r 878.90±1.51 p 883.90±1.51 m 979.60±1.51 d 463.20±1.51 w 815.80±1.85 d 10 879.60±1.51 o 885.40±1.51 l 890.40±1.51 k 986.10±1.51 c 469.60±1.51 v 822.20±1.85 c 15 885.40±1.51 l 891.10±1.51 j 896.10±1.51 h 991.80±1.51 b 475.40±1.51 u 827.90±1.85 b 20 893.20±1.51 i 898.90±1.51 g 903.90±1.51 f 999.60±1.51 a 483.20±1.51 t 835.80±1.85 a mean salinity 880.20±1.6 d 885.90±1.6 c 890.90±1.6 b 986.60±1.6 a 470.20±1.6 e adamipour et al. ‐ effect of vermicompost on performances of pot marigold 353 have been reported in cowpea(vigna unguiculata), rice (oryza sativa l.), and tall fescue (festuca arundi‐ nacea schreb.) (cavalcanti et al., 2004; garcía et al., 2014; adamipour et al., 2016). nutrients in leaf tissue n, p, k, fe, mg and zn concentrations significantly declined by the increasing salinity stress level from 0 to 200 mm nacl (table 7 and 8). their concentrations table 7 effect of salinity and vermicompost and their interaction on nitrogen, phosphorus and potassium table 8 effect of salinity and vermicompost and their interaction on zinc, magnesium and iron * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity mean vermicompost 0 50 100 150 200 nitrogen (%) 0 2.01±0.04 ghi 1.99±0.01 hi 1.90±0.01 j 1.01±0.01 op 0.97±0.01 q 1.58±0.49 e 5 2.03±0.02 g 2.02±0.009 gh 1.93±0.009 j 1.04±0.009 o 1.00±0.009 pq 1.60±0.49 d 10 2.15±0.04 e 2.10±0.01 f 2.01±0.01 ghi 1.12±0.01 m 1.08±0.01 n 1.69±0.49 c 15 2.40±0.08 c 2.38±0.02 c 2.29±0.02 d 1.40±0.02 k 1.36±0.02 l 1.97±0.49 b 20 3.01±0.02 a 2.99±0.01 a 2.90±0.01 b 2.01±0.01 gh 1.97±0.01 i 2.58±0.49 a mean salinity 2.32±0.38 a 2.30±0.38 b 2.21±0.38 c 1.32±0.38 d 1.28±0.38 e phosphorus (%) 0 0.15±0.005 ijk 0.14±0.005 lm 0.12±0.005 no 0.11±0.005 p 0.08±0.005 r 0.12±0.02 e 5 0.15±0.006 hi 0.15±0.006 jkl 0.13±0.006 mn 0.11±0.006 op 0.09±0.006 qr 0.13±0.02 d 10 0.16±0.009 gh 0.15±0.009 ij 0.14±0.009 klm 0.12±0.009 no 0.10±0.009 q 0.13±0.02 c 15 0.19±0.006 e 0.18±0.006 f 0.16±0.006 g 0.15±0.006 ij 0.12±0.006 no 0.16±0.02 b 20 0.24±0.005 a 0.23±0.005 b 0.22±0.005 c 0.20±0.005 d 0.18±0.005 f 0.22±0.02 a mean salinity 0.18±0.03 a 0.17±0.03 b 0.15±0.03 c 0.14±0.03 d 0.11±0.03 e potassium (%) 0 3.93±0.14 fgh 3.89±0.14 ghi 3.84±0.14 hi 2.93±0.14 mn 2.11±0.14 p 3.34±0.74 d 5 4.11±0.18 d-h 4.07±0.18 d-h 4.02±0.18 e-h 3.11±0.18 lm 2.29±0.18 op 3.52±0.75 c 10 4.22±0.12 de 4.18±0.12 def 4.13±0.12 d-g 3.22±0.12 kl 2.40±0.12 o 3.63±0.74 c 15 4.63±0.22 b 4.59±0.22 bc 4.54±0.22 bc 3.63±0.22 ij 2.81±0.22 n 4.04±0.76 b 20 5.32±0.31 a 5.28±0.31 a 5.23±0.31 a 4.32±0.31 cd 3.50±0.31 jk 4.73±0.78 a mean salinity 4.44±0.54 a 4.40±0.54 a 4.35±0.54 a 3.44±0.54 b 2.62±0.54 c * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity mean vermicompost 0 50 100 150 200 zn (mg/kg) 0 91.47±0.47 efg 91.42±0.47 fg 91.34±0.47 fg 90.35±0.47 i 88.88±0.47 k 90.69±1. 10e 5 91.78±0.51 def 91.73±0.51 def 91.65±0.51 def 90.66±0.51 hi 89.19±0.51 jk 91.00±1. 10d 10 92.14±0.32 d 92.09±0.32 d 92.01±0.32 de 91.02±0.32 gh 89.55±0.32 j 91.36±1. 06c 15 93.25±0.86 c 93.20±0.86 c 93.12±0.86 c 92.13±0.86 d 90.66±0.86 hi 92.47±1. 28b 20 95.44±0.51 a 95.39±0.51 a 95.31±0.51 a 94.32±0.51 b 92.85±0.51 c 94.66±1. 11a mean salinity 92.82±1. 56 a 92.77±1. 56 a 92.69±1. 56 a 91.70±1. 56 b 90.23±1. 56 c mg (mg/kg) 0 1.69±0.09 efg 1.64±0.09 e-h 1.60±0.09 e-h 1.46±0.09 ghi 1.23±0.09 i 1.52±0.18 d 5 1.72±0.06 efg 1.69±0.06 efg 1.65±0.06 e-h 1.51±0.06 f-i 1.28±0.06 i 1.57±0.17 cd 10 1.82±0.12 cde 1.79±0.12 c-f 1.75±0.12 d-g 1.61±0.12 e-h 1.38±0.12 i 1.67±0.20 c 15 2.10±0.20 c 2.07±0.20 c 2.03±0.20 cd 1.89±0.20 cde 1.66±0.20 e-h 1.95±0.24 b 20 3.38±0.58 a 3.35±0.58 a 3.31±0.58 a 3.17±0.58 ab 2.94±0.58 b 3.23±0.54 a mean salinity 2.14±0.70 a 2.11±0.70 a 2.07±0.70 a 1.93±0.70 b 1.70±0.70 c fe (mg/kg) 0 122.05±1.68 ij 121.92±1.68 ij 120.69±1.68 j 115.50±1.68 k 100.27±1.68 m 116.09±8.60 e 5 124.19±1.96 hi 124.06±1.96 i 122.83±1.96 ij 117.64±1.96 k 102.41±1.96 m 118.23±8.65 d 10 128.42±2.33 g 128.29±2.33 g 127.06±2.33 gh 121.87±2.33 ij 106.64±2.33 l 122.45±8.72 c 15 154.19±2.69 d 154.06±2.69 d 152.83±2.69 d 147.64±2.69 e 132.41±2.69 f 148.23±8.80 b 20 196.53±4.15 a 196.40±4.15 a 195.17±4.15 a 189.98±4.15 b 174.75±4.15 c 190.56±9.24 a mean salinity 145.07±29.02 a 144.94a±29.02b 143.71±29.02 b 138.52±29.02 c 123.29±29.02 d adv. hort. sci., 2019 33(3): 345-358 354 decreased (44.82%, 36.06%, 40.99%, 15.01%, 20.56% and 2.79%, respectively) in the plants at 200 mm nacl compared to those in the control plants. n, p, k, fe, mg and zn concentrations in the leaf tissues of pot marigold increased significantly with increasing vermicompost composition in the media compared to those in the control plants. the highest and the lowest concentrations of the elements were observed in the 20% and the 0% vermicompost treatments, respectively (table 7 and 8). furthermore, n, p, k, fe, mg and zn concentrations in leaves increased 46.20%, 80.32%, 4.61%, 64.14%, 112.50%, and 4.37% in the plants with the 20% vermicompost treatment compared to those in plants with the control treatment, respectively. interaction between the levels of salinity and vermicompost resulted in the highest and the lowest nutrient concentrations in the plants with the 0 mm nacl and 20% vermicompost and with the200 mm nacl and 0% vermicompost treatments, respectively (table 7 and 8). plant growth is reduced in salt-affected soil because of the excess uptake of potentially toxic ions. soil salinity is characterized by high amounts of na, mg, ca, cl, hco3 and b ions in soil which have negative effects on plant growth. eventually, high salt concentrations in soil reduce the absorption of nutrients of plants which negatively affects the fertility of the soil (zhao and ren, 2007). in this study, the increase of vermicompost levels increased the mineral nutrition. vermicompost contains humic substances that have multiple effects in the soil. it may improve soil properties such as micronutrient transport and availability (aşık et al., 2009). chen and aviad (1990) summarized the effects of humic substances on plant growth and mineral nutrition, pointing out the positive effects on the uptake of macro(such as n, p, and k) and micro-elements. vermicompost may enhance the uptake of nutrients and reduce the uptake of some toxic elements of plants under salinity stress conditions. chamani et al. (2008) reported that addition of vermicompost to soils resulted in increased mineral contents in the substrate and higher concentrations p, ca, mg, cu, mn and zn in shoot tissues of red clover and cucumber. correlation analysis the results of the pearson correlation analysis between vermicompost abundance and the measured morphological traits and elements showed a positive and significant correlation. therefore, a positive relation between the increases of the amount of vermicompost and improvement in the morphological traits and measured elements can be inferred. according to the results, shoot fresh weight compared to shoot dry weight (r=0.89**) and flower diameter (r=0.57**) showed the highest and the lowest correlations among the morphological traits, respectively (table 9). in the measured elements under the vermicompost treatments, the highest correlation was observed between shoot fresh weight and k content (r=0.81**) while the lowest correlation was seen in mg content (r=0.67**) (table 9). this improvement can be attributed to the presence of macro-micro elements, the release of elements in the soil and soil amendment of physical and biological conditions which have been previously reported in other plants table 9 correlation coefficients for evaluated traits on pot marigold in vermicompost treatment traits shoot fw shoot dw leaf area root fw root dw flower diameter fruit fw fruit dw zn fe mg ca k p n shoot fw 1 shoot dw 0.891** 1 leaf area 0.886** 0.992** 1 root fw 0.798** 0.799** 0.777** 1 root dw 0.798** 0.799** 0.777** 1.00** 1 flower diameter 0.574** 0.603** 0.586** 0.519* 0.519* 1 fruit fw 0.811** 0.795** 0.801** 0.618** 0.618** 0.334 ns 1 fruit dw 0.618** 0.728** 0.695** 0.734** 0.734** 0.536* 0.417 ns 1 zn 0.789** 0.883** 0.875** 0.864** 0.864** 0.623** 0.778** 0.722** 1 fe 0.809** 0.836** 0.822** 0.825** 0.825** 0.624** 0.818** 0.686** 0.951** 1 mg 0.679** 0.797** 0.781** 0.764** 0.764** 0.538* 0.733** 0.749** 0.927** 0.907** 1 ca 0.789** 0.883** 0.875** 0.864** 0.864** 0.623** 0.778** 0.722** 1.00** 0.951** 0.927** 1 k 0.818** 0.851** 0.842** 0.799** 0.799** 0.561** 0.778** 0.765** 0.911** 0.929** 0.884** 0.911** 1 p 0.815** 0.847** 0.835** 0.815** 0.815** 0.554* 0.831** 0.698** 0.937** 0.981** 0.921** 0.937** 0.938** 1 n 0.805** 0.834** 0.814** 0.831** 0.831** 0.604** 0.810** 0.704** 0.956** 0.989** 0.933** 0.956** 0.916** 0.981** 1 fw = fresh weight; dw= dry weight. ns= not significant, * and **: significant at 5% and 1% respectively. adamipour et al. ‐ effect of vermicompost on performances of pot marigold 355 (bachman and metzger, 2008). in this correlation analysis, peroxidase activity (r=0.79**) and proline content (r=0.44**) had the highest and the lowest correlations with shoot fresh weight. further, insignificant correlations were observed in carotenoid content and catalase enzyme activity (table 10). it seemed the positive correlations in activities of antioxidant enzymes, chlorophyll and proline content in the plant was due to increased concentrations of the elements which could be provided by vermicompost. because in the structure of the mentioned traits, there are a variety of macroand microelements that vermicompost provides directly and indirectly to plants (hidalgo et al., 2006). the results of a simple correlation analysis between the morphological traits and the studied elements under salt stress conditions showed that shoot fresh weight had a positive and significant correlation with all the morphological indices and concentration of elements. in this analysis, the highest and the lowest correlations were seen in leaf area (r=0.99**) and fresh flower weight (r=0.91**), respectively (table 11). also, the highest correlation of shoot fresh weight was observed in fe content (r=0.98**) and the lowest correlation was found in n content (r=0.88**) (table 11). there are many reports of positive correlations between plant yield and concentration of elements under salinity stress conditions. increase in soil ph, reduction in the amount of absorbent elements in the soil, increase in toxic elements of soil such as cl and na, and osmotic stress in the soil that prevents water absorption are the most critical factors for this phenomenon in plants (edwards et al., 2010). analysis of correlations of shoot fresh weight with chlorophyll and proline contable 11 correlation coefficients for evaluated traits on pot marigold in salinity stress conditions traits shoot fw shoot dw leaf area root fw root dw flower diameter fruit fw fruit dw zn fe mg ca k p n shoot fw 1 shoot dw 0.939** 1 leaf area 0.995** 0,956** 1 root fw 0.966** 0.886** 0.961** 1 root dw 0.923** 0.868** 0.921** 0.986** 1 flower diameter 0.986** 0.977** 0.991* 0.952** 0.919** 1 fruit fw 0.918** 0.830** 0.906** 0.844** 0.798** 0.865** 1 fruit dw 0.942** 0.947** 0.948** 0.901** 0.855** 0.972** 0.779** 1 zn 0.938** 0.947** 0.956** 0.850** 0.802** 0.941** 0.923** 0.892** 1 fe 0.985** 0.940** 0.987** 0.914** 0.851** 0.977** 0.909** 0.948** 0.963** 1 mg 0.895** 0.910** 0.803** 0.762** 0.897** 0.919** 0.856** 0.969** 0.912** 0.857** 1 ca 0.938** 0.947** 0.956** 0.850* 0.802** 0.941** 0.923** 0.892** 1.00** 0.963** 0.969** 1 k 0.981** 0.961** 0.991* 0.954** 0.928** 0.982** 0.915** 0.927** 0.966** 0.967** 0.932** 0.966** 1 p 0.936** 0.877** 0.911** 0.933** 0.926** 0.919** 0.843** 0.858** 0.804** 0.879** 0.756** 0.804** 0.894** 1 n 0.885** 0.870** 0.892** 0.941** 0.976** 0.886** 0.809** 0.796** 0.831** 0.811** 0.781** 0.923** 0.817** 0.891** 1 fw = fresh weight; dw= dry weight. ns= not significant, * and **: significant at 5% and 1% respectively. table 10 correlation coefficients for evaluated traits on pot marigold under salinity stress conditions (above diameter) and vermicompost treatment (below diameter) ns= not significant, * and **: significant at 5% and 1% respectively. traits shoot fresh weight chlorophyll proline carotenoid ascorbate perodidase peroxidase catalase superoxide dismutase shoot fresh weith 1 0.913 ** -0.904 ** -0.874 ** 0.872 ** 0.990 ** 0.905 ** 0.861** chlorophyll 0.664 ** 1 -0.992 ** 0.857 ** -0.856 ** 0.888 ** 0.888 ** 0.626** proline 0.444 ** 0.176 ns 1 0.854 ** 0.680 ** -0.816 ** 0.714 ** 0.571** carotenoid 0.059 ns 0.488 * 0.454 * 1 0.801 ** 0.651 ** -0.544 * -0.346 ns ascorbate peroxidase 0.490* 0.064 ns 0.038 ns -0.435 ns 1 0.760 ** 0.407 ns -0.085 ns peroxidase 0.794 ** 0.480 * 0.540 * 0.093 ns 0.746 ** 1 0.458* -0.020 ns catalase 0.131 ns 0.444 * -0.341 ns 0.252 ns 0.430 ns 0.317 ns 1 -0.006 ns superoxide dismutase 0.514 * 0.793 ** -0.210 ns 0.131 ns -0.002 ns 0.241 ns 0.355 ns 1 adv. hort. sci., 2019 33(3): 345-358 356 tent, and antioxidant enzyme activity showed significant positive and negative correlations. from the viewpoint of biochemistry, most of significant correlations were found in content of chlorophyll (r=0.91**), proline (r= -0.90**), carotenoid (r= -0.87**), in activity of ascorbate peroxidase (r=0.87**), peroxidase (r=0.99**), catalase (r=0.90**), and superoxide dismutase (r=0.86**) compared to the shoot fresh weight (table 10). there were also few negative correlations in which proline and carotenoid content can be attributed to their function in dealing with osmotic stress in the plants due to increased salinity in the soil. in similar studies, positive correlation of antioxidant enzyme activity under salinity stress conditions in german chamomile (matricaria recutita l.), sunflowers (helianthus annuus l.) and basil (ocimuum basilicum l.) have been reported (baghalian et al., 2008; heidari et al., 2011; heidari, 2012). 4. conclusions salinization of soil is a serious land degradation problem and is increasing steadily in many parts of the world, particularly in arid and semiarid areas. soil salinity affects the establishment, growth, and development of plants leading to huge losses in productivity. vermicompost is one of the major organic fertilizer which can improve growth and salinity tolerance with containing plenty nutrition elements, hormones, and organic materials. in this study, increase in salinity stress significantly led to decline in morphological and physiological indices of pot marigold. application of vermicompost under salinity conditions increased morpho-physiological indices in this plant. vermicompost increased the activity of the antioxidant system, the content of proline and chlorophyll in the plant by increasing the nutrients in the soil environment that could increase the plant yield and alleviating the harmful effects of salinity. references adamipour n., heiderianpour m., zarei m., 2016 application of vermicompost for reducing the destructi‐ ve effects of salinity stress on tall fescue turfgrass (festuca arundinacea schreb. 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1427-1434. zhao g., ma b., ren c., 2007 growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. crop sci., 47: 123-131. impaginato 379 adv. hort. sci., 2018 32(3): 379-387 doi: 10.13128/ahs-23129 yield, quality and antioxidants of peeled tomato as affected by genotype and industrial processing in southern italy f. de sio 1, m. rapacciuolo 1, a. de giorgi 1, a. trifirò 1, b. giuliano 2, l. vitobello 1, a. cuciniello 3, g. caruso 3 (*) 1 stazione sperimentale per l’industria delle conserve alimentari (ssica), via faustino tanara, 31, 43100 parma, italy. 2 associazione nazionale industriali delle conserve alimentari vegetali (anicav), viale della costituzione isola f/3, 80143 napoli, italy. 3 dipartimento di agraria, università di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: long-type fruit hybrids, lycopene, polyphenols, pre-processing and post-processing production, sensorial features, solanum lycopersicum l. abstract: research was carried out on processing tomato in san severo (tavoliere delle puglie, foggia, italy) in order to compare four long-type fruit hybrids oriented to peeled produce (abbundo, umex, superpeel, taylor), using a randomized complete block design with three replicates. the hybrid superpeel reached the highest marketable yield due to the highest fruit number and mean weight; along the peeling chain, umex and taylor showed the highest processing efficiency. titratable acidity and sodium were highest in taylor fruits, whereas the highest fiber content was detected in abbundo fruits. compared to pre-processing fruits, peeled tomatoes showed increased values of total and soluble solids as well as reducing sugars, but decreased sugar ratio and colour. the highest concentrations of antioxidants in processed fruits were recorded in umex for lycopene and in superpeel for β-carotene. compared to pre-processing fruits, in peeled tomatoes lycopene and β-carotene concentrations remained stable and polyphenols increased referring to fresh weight. the hybrids examined did not show univocal trends in terms of sensorial features. 1. introduction tomato is the most cultivated vegetable species worldwide with 5,023,810 ha (fao, 2014); italy is a major european producer of processing tomato with a surface area as much as 79,761 ha devoted to this crop (istat, 2017 dati.istat.it), of which 32% are located in emilia-romagna and 25% in apulia. the italian industry of tomato and its derivatives (peeled, diced and sauce) attained an export gross marketable yield as (*) corresponding author: gcaruso@unina.it citation: de sio f., rapacciuolo m., de giorgi a., trifirò a., giuliano b., vitobello l., cuciniello a., caruso g., 2018 yield quality and antioxidants of peeled tomato as affected by genotype and industrial processing in southern italy. adv. hort. sci., 32(3): 379-387 copyright: © 2018 de sio f., rapacciulo m., de giorgi a., trifirò a., giuliano b., vitobello l., cuciniello a., caruso g. a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 april 2018 accepted for publication 7 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 379-387 380 much as about 1.5 billion of euros in 2017 (istat, 2017). these products are traditionally addressed to united kingdom, usa, france, australia, japan, germany, belgium, but also to growing demanding markets, i.e. eastern europe, south america (brazil and argentina), south africa, south corea, united arab emirates. the interest of processing factories in southern italy to valorize peeled-oriented tomato type matches the seed company goal to provide farmers with hybrids showing high yield, improved taste and flavour features, and better industrial requirements compared to the current genotypes. with these prospects, the new hybrids are evaluated with regard to the main traits, such as: plant architecture and parasite resistance; morphology, number, weight, ripeness uniformity, technological and quality performances of fruits. in order to carry out tomato genotype selection, some authors suggested to perform a comprehensive evaluation using synthetic agronomic and quality indexes (carli et al., 2011), upon assessing an appreciable number of related variables such as dry matter, soluble solids, sugars, acidity and antioxidants. notably, high dry matter and soluble solids are desirable characteristics for the canned tomatoes industry since they improve the quality of the processed product (de pascale et al., 2001). indeed, soluble solid content and titratable acidity are the main components responsible for tomato flavor (kader, 1986; flores et al., 2008) and they are most likely to match the consumer perception of the internal quality (baldwin et al., 1998; arazuri et al., 2007). in this respect, the balanced ratio between sugars and organic acids is important to sweetness, sourness and overall flavor intensity in tomatoes (de bruyn et al., 1971; stevens et al., 1977). in fact, high acids and low sugars will produce a tart tomato while high sugars and low acids will result in a tasteless tomato (kader, 1986). due to their antioxidant attributes in addition to sensorial appealing (raiola et al., 2016), tomatobased products are associated with a low risk of both cancer and incidence of coronary heart disease (pernice et al., 2010; ilahy et al., 2011). the present research was carried out since new hybrids for peeled tomato with improved features are needed for the cultivation in southern italy and, in this respect, they were compared with a widespread cultivar in southern italy, in terms of yield, technological, quality and sensorial characteristics. 2. materials and methods field conditions research was carried out on processing tomato at de maio farm, located in san severo (tavoliere delle puglie, foggia, southern italy) in 2017 on silty-sandy soil. the experimental protocol was based on the comparison between 4 long-type hybrids oriented to peeled produce: abbundo (hm clause italia spa., italy); umex (syngenta italia spa., italy); superpeel (united genetics italia spa., italy); taylor (nunhems italy srl, italy) as a control. a randomized complete block design was used for the treatment distribution in the field, with three replicates, and the elementary plot had a 67 m2 surface area. the transplant was performed on 28 april, arranging a double-row layout, with 35 cm between the plants spacing along the rows, 40 cm between the two rows in each couple and 150 cm between the outer rows of two adjacent couples, thus achieving a density of 3 plants per m2. the ordinary farming technique related to processing tomato in tavoliere delle puglie was followed and harvests were practiced between 3 and 7 august. yield, quality and technological determinations when the 90% fruits were ripe, the following agronomic determinations were made in each plot: weight of marketable fruits (red + colour turning point) and waste berries (green + rotten); mean fruit weight on a random 100 fruit sample; middle length and width on a random 20 fruit sample; percentage of fruit coverage exerted by plant canopy, referred to the ranges of 0-25%, 25-50%, 50-75%, 75-100%. quality analyses of pre-processing fruits sampled in each field plot, as well as technological and quality determinations of processed fruit samples of each hybrid were performed at the laboratories of stazione sperimentale per l’industria delle conserve alimentari in angri (salerno). as for technological determinations, the processing yield was assessed, representing the ratio between the canned tomato fruit amount, after selection and technological process, and the marketable yield obtained in the field. the fruit processing was performed using the pilot plant available at ssica. in this respect, tomato peeled production was carried out on a semi-industrial scale, with juice addition, packaged in painted tinplates of 1 kg; notably, the juice obtained by the same cultivar was added de sio et al. peeled tomato for processing industry 381 after partial concentration at 7.5°brix. in order to assess the processing yield, weights were recorded at each different step, the product to be peeled underwent selections and weighing of each fruit fraction such as yellow and necrotized, rotten, broken, undersized; moreover, skins were weighed after peeling. as concerns the product submitted to the juice chain, yellow, necrotized and rotten fruits, as well as skins and refining seeds were weighed. next, the drained fruit percentage was assessed, calculated as a mean of five cans; all the determinations were performed in triplicate and averaged. the fruit quality features assessed on both fresh and peeled fruits, referred to fresh weight, were: total solids (ts), soluble solids (ss), reducing sugars (glucose and fructose) and sucrose, colour (a/b ratio), antioxidants (lycopene, b-carotene, total polyphenols). in addition, the components included in the current european nutritional label, according to eu regulation 1169/2011, were determined: proteins, titratable acidity, lipids, fatty acids, fiber, salt and sodium. the analytical procedures were performed according to caruso et al. (2012) for total and soluble solids, sugars, proteins, fiber, ash and sodium; mipaf (1973) for titratable acidity and fats; golubkina et al. (2015) for fatty acids; conti et al. (2015) for colour; de sio et al. (2001) for carotenoids; golubkina et al. (2017) for polyphenols. briefly, total solids were assessed in oven at 70°c under vacuum until steady weight, whereas soluble solids by means of a digital refractometer. sugars were determined by hplc, using the 600e waters chromatographic system and a column sugar-pak waters at 85°c, edta-ca in water solution as eluent (50 mg l-1). proteins were assessed with the kjeldahl method, by a foss tecator digestor with a kjeltec 2300 distiller. fibre was determined after the samples were weighed, dried (105°c), gelatinized in the presence of heat-resistant α-amylase and enzymatically digested by proteases and amydoglucosydase, to remove proteins and starch, whereas soluble fibre was precipitated by ethanol; the residue was filtered, washed with ethanol and acetone, dried, weighed and split into two fractions to determine proteins and ash, and fibre content as the difference to the residue weight. sodium was assessed by atomic adsorption spectrophotometry, after sulpho-nitric mineralization, with a model 1100 perkin-elmer spectrophotometer. fatty acids were detected by gas chromatography via appropriate methyl ethers chromatography on capillary glass column, using an agilent 6890 gas cromatograph, equipped with a flame ionization detector; the peaks of fatty acids methyl ethers were identified by comparison to the retention times of reference standards. colour was assessed by a hunter associate laboratories d25-a model colourmeter, using a suitable measurement cell with the standard bcr n. 1266 reference whose values are l= 25.7, a= 23.7, b= 14.8; chromatic parameter values are expressed in the hunter scale as a/b. carotenoids were assessed through hplc, using a waters alliance chromatograph equipped with photodiode array detector mod. 996, performing the determinations at 450 nm on a reversed phase column ymc-pack c30 (250 x 4.6 mm i.d.) filled with 5 µm average particle size. polyphenols were determined in water extract through a spectrophotometer (unico 2804 uv, usa); the concentration was calculated according to the absorbance at 730 nm, using 0.02% gallic acid as an external standard. sensorial determinations sensorial or organoleptic determinations were performed on processed tomato samples of each hybrid, which were coded and anonymously analyzed by a panel test team composed of ten specialists in tomato derivatives and five fellows at the first panel experience. each expert evaluated the samples under neutral light (4000 k) and his opinion was reported in a form including 11 sensorial variables. among the latter, five were considered of primary importance and the remaining as their detailing. with regard to primary variables, the score ranged from zero (extremely unpleasant) to ten (extremely pleasant) and, in particular: colour score zero was matched to brick red and ten to bright red; firmness score zero was associated to chewiness resistance absence and ten to extremely tough product. as for secondary variables, the form delivered to the panel test team was elaborated in order to minimize the fluctuations caused by the first-experience fellows. moreover, the following perceptions and the related scores in brackets were taking into account: absence (2), mild presence (4), medium presence (6), strong presence (8); the scores were used to calculate the average value per each sample. statistical processing the data relevant to agronomic, technological, quality and sensorial determinations were statistically processed by analysis of variance, with the ensuing duncan’s multiple range test for mean separation at 0.05 probability level. the percentage values were subjected to angular transformation before processing. adv. hort. sci., 2018 32(3): 379-387 382 3. results and discussion the hybrids did not significantly differ in terms of crop duration, presumably due to the high temperatures and lack of rainfall during the crop cycles (fig. 1), which led to fruit ripeness and harvest anticipation. from yield and biometrical data reported in table 1, it arises that hybrid superpeel attained the highest marketable yield (175.2 t·ha-1), as much as 95.2% of the total yield, due to the very low waste production. the productive result derived from the combination of the fruit number per plant (90.7) and mean weight (77.4 g), with the berries showing higher values of diameter and length compared to abbundo and umex respectively, and higher thickness than abbundo and taylor. the hybrids abbundo and umex gave 32% lower marketable yield than superpeel due to lower values of the fruit number, size and weight as well as a higher waste production. the hybrid taylor ranked in the middle between the highest yielding superpeel and the least productive abbundo and umex. indeed, it provided with a 19.6% lower yield than superpeel but 15.4% higher production than the other two genotypes; this result was the consequence of the lowest fruit number (71) per plant but the highest berry dimensions and mean weight (79.3 g) as well as also the lowest fraction of waste produce. no statistically significant differences were recorded between the hybrids in terms of fruit covering by vegetation, which exceeded 75%. consistently with our findings, in previous investigation (portugal et al., 2015) hybrid productive performances ranged between 110 and 160 t·ha-1, whereas in other research (caruso et al., 2016; peixoto et al., 2017) tomato genotypes showed a wide range of yields under the 70 t·ha-1 threshold. with regard to processing efficiency (table 2), umex showed the highest value (87.0%) though not statistically different from the control. similarly, along the peeling chain umex and taylor showed the best performances (81.5% as an average), whereas fig. 1 ten-day means of temperatures and rainfall in san severo (foggia, italy) in 2017. ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. table 1 yield and biometrical parameters of long-type processing tomato hybrids hybrid total fruits marketable fruits waste fruits weight (%/total) weight (t·ha-1) number per plant weight (t·ha-1) weight (%/total) number per plant mean weight (g) diameter (cm) length (cm) length/ width flesh thickness (mm) abbundo 128.7 c 93.0 b 119.3 c 92.7 82.7 b 57.8 b 4.1 c 7.8 ab 1.89 7.0 b 7.3 a umex 126.0 c 91.0 b 118.9 c 94.3 78.5 b 60.4 b 4.3 bc 7.7 b 1.79 7.5 ab 5.7 b superpeel 184.1 a 97.7 a 175.2 a 95.2 90.7 a 77.4 a 4.6 ab 8.1 ab 1.76 7.8 a 4.8 c control 146.8 b 79.0 c 140.8 b 95.9 71.0 c 79.3 a 4.7 a 8.4 a 1.77 7.0 b 4.1 d ns ns ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. table 2 processing yield of four hybrids for peeled tomato hybrid processing yield (%) waste fruits along peeled chain (%) waste fruits along juice chain (%) total peeled juice yellow and necrotized rotten broken undersize skins yellow and necrotized rotten skins and seeds abbundo 81.5 b 72.4 b 90.1 13.3 a 0.6 a 6.3 a 1.3 6.3 c 5.0 a 3.0 b 2.0 c umex 87.0 a 82.0 a 92.2 4.0 d 0.0 b 5.8 b 0.8 7.4 b 2.8 c 2.2 c 2.9 a superpeel 82.7 ab 75.1 b 90.5 9.3 b 0.7 a 6.1 ab 0.6 8.2 a 4.3 b 2.9 b 2.3 b control 86.3 ab 80.9 a 91.8 7.9 c 0.2 b 3.8 c 0.7 6.5 c 2.9 c 3.4 a 1.9 c ns ns de sio et al. peeled tomato for processing industry 383 no statistically significant differences were recorded between the hybrids referring to: total solids (7% on average), soluble solids (6.1%), reducing sugars (3.8%), sugars ratio (54.3%), proteins (1.6%), lipids (0.3 %), energetic value (24.5 kcal·100 g1 or 104,5 kj·100 g-1), colour (1.82), glucose (1.5%), fructose (1.6%), sucrose (0.04%), fat acids expressed as saturated (0.07%), monounsaturated (0.06%) and polyunsaturated (0.14%). compared to pre-processing fruits, peeled tomatoes showed increased values of total and soluble solids as well as reducing sugars, but decreased levels of sugar ratio and colour; moreover, ph of fresh fruits was not significantly affected by the hybrid, ranging between 4.4 to 4.5. in previous research (raiola et al., 2018) the comparison between the values recorded before and after processing was genotype-dependent, except for titratable acidity and sugar ratio which always decreased and increased respectively, upon processing. high total solids content in fruits is an industrial target, as it reduces the processing costs; this parameter in tomato varies around the 5-6% average and it is affected by some factors such as cultivar, soil type and climate conditions during the growing and no significant differences arose as concerns the juice yield (91.2% as an average). in terms of waste along the peeling chain, abbundo attained the highest occurrence of yellow and necrotized fruits, umex the lowest and taylor the lowest broken berry percentage. the undersized and rotten fruits were very few and superpeel had the highest skin fraction. as for waste along the juice chain, abbundo showed the highest percentage of yellow and necrotized fruits, which was 79% higher than the lowest one corresponding to umex; the latter hybrid also had the lowest value of rotten berries and the highest of skin and seeds. the results relevant to the quality indicators of processed tomato (tables 3 and 4) show that significant effects of the hybrid were recorded on the following parameters: titratable acidity and sodium attained the highest value in taylor fruits (0.37% and 7.4 mg·100 g-1 respectively) and lowest in abbundo; the fiber content recorded in abbundo fruits was 17% higher than taylor one; ash and salt concentrations showed the highest levels in umex (0.54% and 20.4 mg·100 g-1 respectively) though the latter variable was not significantly different from taylor. table 3 quality features (referred to fresh weight) of peeled tomato fruits obtained from four hybrids ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid total solids (g·100 g-1 ) soluble solids (°brix ) reducing sugars (g·100 g-1) titratable acidity (g anhydrous citric acid 100 g-1) sugar ratio (%) proteins (g·100 g-1) fats (g·100 g-1) fiber (g·100 g-1) energetic value (kcal·100 g-1) colour (a/b) abbundo 7.0 6.1 4.0 0.27 c 56.7 1.56 0.27 1.32 a 24 1.82 umex 7.0 6.1 3.8 0.29 c 53.5 1.52 0.31 1.23 b 25 1.81 superpeel 7.0 6.1 3.7 0.33 b 53.5 1.59 0.26 1.19 bc 24 1.80 control 7.1 6.2 3.8 0.37 a 53.6 1.64 0.32 1.13 c 25 1.85 ns ns ns ns ns ns ns ns average 7.0 6.1 3.8 54.3 1.82 peeled/fresh (%) + 24.7 + 21.8 + 18.2 5.2 30.6 table 4 sugars, fatty acids and mineral components (refereed to fresh weight) in peeled tomato fruits produced by four hybrids ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid sugars (g·100 g-1) fatty acids (g·100 g-1) ash (g·100 g-1) sodium (g·100 g-1) salt (g·100 g-1)glucose fructose sucrose saturated monounsaturated polyunsaturated abbundo 1.48 1.57 0.04 0.06 0.06 0.13 0.47 c 5.6 c 10.3 b umex 1.54 1.58 0.04 0.07 0.07 0.15 0.54 a 6.4 b 20.4 a superpeel 1.46 1.58 0.04 0.06 0.05 0.13 0.51 ab 5.9 c 9.8 b control 1.49 1.60 0.04 0.08 0.07 0.15 0.50 bc 7.4 a 19.5 a ns ns ns ns ns ns 384 adv. hort. sci., 2018 32(3): 379-387 harvesting season (siddiqui et al., 2015). other authors (majkowska-godomska et al., 2008) recorded a total solids content of tomato fruits ranging from 3.83 to 7.00%. campos et al. (2006) and kader et al. (1987) reported that values of soluble solids below 4.5% are considered low for industrial tomatoes; in this respect, turhan and seniz (2009) found this quality indicator ranging between 5.0 to 5.5 % in processing tomato fruits and in other studies (cramer et al., 2001; de pascale et al., 2001) soluble solids values of tomato fruits ranged from 4 to 6%. sugar content is positively and highly correlated with total soluble solids in tomato fruit and, notably, fructose has a large impact on the sweetness perception (tieman et al., 2012). in our research the sum of glucose and fructose accounted for the 80% of the total sugars, whereas it attained about 65% in previous investigations (stevens et al., 1977; jones and scott, 1984; malundo et al., 1995). other authors found the total sugar content of ripe tomato ranging from 1.7 to 4.7% (petro-turza, 1987; turhan and seniz, 2009) or from 0.54 to 3.44% (melkamu et al., 2008) of fresh weight. in previous research, titratable acidity (ta) in tomato fruits ranged from 0.22 to 0.40% (turhan and seniz, 2009) or even from 0.25 to 0.70 (george et al., 2004). according to beckles (2012), values of total soluble solids and titratable acidity as much as 5.0 and 0.4% respectively are considered desirable to produce a good-tasting tomato. titratable acidity is mainly affected by citric and malic acids which reportedly attain about 15% of the dry content in ripe fresh tomatoes (petro-turza, 1987). kamis et al. (2004) states that taste and flavour of tomato fruits are positively correlated to sugars and organic acids content. moreover, in addition to flavour the organic acids influence ph, the latter being an important factor in canned tomato products to control the growth of thermophilic microorganisms (yousef and juvik, 2001). the influence of ph on the thermal conditions applied along the tomato processing chain is mainly associated to producing safe products and, in this respect, values below 4.5 prevent microorganism proliferation in the final product (campos et al., 2006; garcia and barrett, 2006). notably, some authors did not detect varietal dependent ph differences in tomato berries (kerkhofs et al., 2005), whereas in other research ph showed differences among cultivars (benal et al., 2005; frusciante et al., 2007) even in the wide range of 3.78 to 5.25 (turhan and seniz, 2009). mineral element concentration in tomato fruits may reach 8% of dry matter and influences tomato fruit taste through the effect on ph and titratable acidity (siddiqui et al., 2015). with regard to antioxidants (table 5), lycopene attained a 16% higher concentration in umex compared to abbundo; superpeel showed a 43% higher β-carotene content than abbundo; polyphenols concentration was the lowest in the control fruits, but did not significantly differs between the three hybrids examined. compared to fresh fruits, in peeled tomatoes lycopene and β-carotene concentrations remained stable and polyphenols increased referring to fresh weight, whereas in relation to total solids lycopene had a 19.1% decrease whereas polyphenols just a slight reduction (5.2%). in previous research (binoy et al., 2004) significant differences were found in lycopene and phenolic contents between the different genotypes, with lycopene showing 1 to 4 fold and 1 to 2 fold variation on fresh and dry weight basis respectively. moreover, unlike our findings where lycopene remained stable and polyphenols increased upon industrial processing table 5 antioxidants concentration in peeled tomato fruits obtained from four hybrids within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid lycopene β-carotene mg·kg-1 total polyphenols mg·kg-1 mg·100 g-1 ts mg eq. gallic acid 100 g-1 mg eq gallic acid g-1 total solids abbundo 127.0 c 181.9 c 2.1 c 36.7 a 5.3 a umex 146.6 a 208.3 a 2.3 bc 36.0 a 5.1 a superpeel 136.2 b 195.3 b 3.0 a 35.7 a 5.1 a control 131.0 bc 184.7 bc 2.4 b 31.7 b 4.5 b average 135.2 192.6 2.5 35.0 5.0 peeled/fresh (%) 0.9 -19.1 1.0 17.6 -5.2 385 de sio et al. peeled tomato for processing industry and referred to fresh weight, dewanto et al. (2002) reported the increase of lycopene concentration with no changes in polyphenols content in processed tomato fruits compared to raw berries. however, in other research (pavlović et al., 2017) the antioxidants content in tomato fruits decreased upon processing thermal treatment, though the significance and amplitude of the differences are genotype dependent, and it is also affected by temperature settings (jabbari et al., 2018). the scores resulted from the panel test performed by an expert team in tomato organoleptic evaluation and their subsequent statistical processing allowed to obtain the sensorial profiles which convey the immediate and clear quantitative and descriptive perception of the analyzed products. the graphic representation of qda (quantitative descriptive analysis) obtained by processing the evaluation forms filled in by the experts is shown in figure 2 a. taking into account the high number of data and in order to make it easier to interpret the profiles, the sensorial variable data considered negative for the relevant hybrids were extrapolated. in particular, the data related to strange taste and flavour and to acidity were clustered (fig. 2 b): the profiles and the statistical processing performed show that there are no significant differences between the hybrids. the sensorial profiles of the positive variables are shown in figure 2 c. moreover, from data statistical processing reported in table 6, it can be observed that the peeled product obtained from the hybrids abbundo and umex is statistically different in terms of colour, aspect and flavor; in addition, abbundo is statistically different from taylor for the colour and from superpeel for the sensation of fresh flavor. 4. conclusions from research carried out on the comparison between long-type hybrids oriented to peeled produce in tavoliere delle puglie (foggia, southern italy), superpeel showed the best yield performances (175.2 t·ha-1) with about 21% higher production than the average value attained by abbundo, umex and taylor, as a consequence of the combined effects of fruit number and mean weight. however, the highest processing efficiency was recorded for the hybrid umex and taylor, whereas no hybrid displayed an overall superiority in terms of quality features and sensorial profile. acknowledgements the authors wish to thank: the seed companies h.m. clause italia, syngenta italia spa and united genetics italia for the financial contribution intended for carrying out the research; the plant nursery “aniello cerrato” in sarno (salerno) for producing the fig. 2 organoleptic evaluations of peeled tomato fruits: comparison of the sensorial profiles of the four hybrids (a), sensorial profiles of the undesired features, named negative (b); sensorial profiles of the desired features, named positive (c). variable cultivar δ s aspect abbundo umex -1.43 0.007 colour abbundo umex -1.30 0.013 colour abbundo control -1.10 0.035 flavour abbundo umex 1.00 0.050 flavour 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tecnologie biologiche ed ambientali, università del salento, via provinciale monteroni, 73100 lecce, italia. key words: bois noir, flavescence dorée, grapevine yellows, phytoplasma. abstract: quantitative pcr protocols for phytoplasma detection were used to monitor grapevine yellows (gy) in 373 vineyards located in nine tuscan districts. among more than 70,000 plants visually monitored, 1.867 plants were sampled and “flavescence dorée” phytoplasmas (fd) were detected in 122 plants and mainly identified as trains belonging to 16srv-c subgroup. the “bois noir” (bn) phytoplasma was found in 734 samples, with prevalence of tufb type-b strains. the 2013–2015 monitoring program was strongly influenced by the first survey (2012) in which fd was found consistently in the north west (15 samples), whereas only a few cases were observed in the east territory (2 samples). both areas were thoroughly monitored in the following years: few foci were found in the east (2 in 2014, 1 in 2015), while several infected areas were found in the north west (6, 10 and 22 foci in 2013, 2014 and 2015, respectively). definitely, the novel fd foci detected in the survey (17, 6, 12 and 23 in each year of survey) and the widespread of bn, suggest a dangerous distribution of gy in tuscany. 1. introduction “flavescence dorée” (fd), the most harmful grapevine yellows (gy) in europe, is present in the northern part of italy and in some winegrowing areas it occurs simultaneously with “bois noir” (bn) (bianco et al., 2002; baric and dalla via, 2007). both diseaases are associated with the presence of phytoplasmas enclosed in 16srv group, subgroups c and d (martini et al., 1999) and ‘candidatus phytoplasma solani’ (quaglino et al., 2013), respectively. disease control programs for fd are very expen(*) corresponding author: andrea.luvisi@unisalento.it citation: rizzo d., materazzi a., stefani l., panattoni a., pierro r., marchi g., cinelli t., de bellis l., luvisi a., 2018 the monitoring program of phytoplasmas in tuscany (italy): results of a four year survey. adv. hort. sci., 32(2): 281-288 copyright: © 2018 rizzo d., materazzi a., stefani l., panattoni a., pierro r., marchi g., cinelli t., de bellis l., luvisi a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 january 2018 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 281-288 282 sive in italy because, due to mandatory uprooting of infected plants, growers must be refunded for yield losses and replanting (belli et al., 2010), while bn control depend on management of wild plants indeed, grapevines become dead-end hosts for this phytoplasma, therefore the spatial spread of bn most likely does not rely on transmission of the phytoplasma from vine to vine but on other plant species frequently observed in vineyards (maixner, 1994; marchi et al., 2015). other phytoplasmas have also been found sporadically in grapevines in italy, such as strains belonging to ribosomal subgroups 16sri-b (alma et al., 1996) and 16sri-c (landi et al., 2013) of ‘ca. p. asteris’ (aster yellows, ay). as reported by belli et al. (2010), control measures against gy are being implemented in italy, targeted mainly to fd because the economic importance of bn infections has emerged only recently, following the extensive use of molecular diagnostic assays. moreover, the present knowledge of the epidemiology and control of bn (mori et al., 2015; chuche et al., 2016) and fd (rashidi et al., 2014; casati et al., 2017) is not completely defined. however, compulsory control measures involve uprooting and destruction of any vine with gy type symptoms in the area, even before confirmation of fd infection by laboratory tests. in tuscany, fd was not known since 2003 (bertaccini et al., 2003) when it was detected in the north-western areas of the region. thereafter, compulsory control measures against fd were enforced. but successful eradication of fd relies on accurate diagnosis, which should be considered when the monitoring is planned. a strategic role is played by effective sampling of plants to reduce the risk of false negatives, and disease recognition is easier in grapevines affected by single infections; conversely, discrimination between diseases is more difficult in abandoned vineyards or when plants are affected by mixed infections such as mixed virus infections, frequently observed in cv. sangiovese (rizzo et al., 2012, 2015). the major risks of fd spread may derive from vineyards that are poorly protected against vectors. thus, badly managed vineyards may represent a good target for fd detection, however such vineyards are frequently characterized by symptomatic or poorly cultivated plants, which result in increasing difficulties in recognizing fd. in this paper the identification of novel foci of fd in tuscany during four years of monitoring is reported together with the estimation of the monitoring activity effectiveness. 2. materials and methods districts sampled in 2012-2015, 373 vineyards were selected in nine tuscan districts in the most important grape for wine production areas. where available, small vineyards (<1 ha) were included in the monitoring, as well as poorly managed vineyards. gy symptomatic samples from 200 plants in each vineyard (20 plants in 10 rows, randomly selected) were collected. thus, more than 70,000 plants were included in this symptoms survey. districts were grouped in five areas: northwest (massa-carrara, lucca, and pistoia), north-east (prato, firenze), west (livorno, pisa), east (arezzo, siena), and south (grosseto) (fig. 1). sampling procedures the overall number of vineyards and samples included in the gy monitoring was reported in table 1. in view of the pre-2012 surveys (bertaccini et al., 2013) the north west areas were considered the most susceptible to fd. in the following years, the number of samples and their distribution was determined based on the results of the previous year. pathogen findings in north west in 2012 (fig. 2 a), and first sporadic evidence of fd in eastern areas (fig. 2 b) led to increased monitoring activities in these areas, while monitoring in north east and east was drastically increased in 2013 (table 1). indeed, in fig. 1 tuscan districts were grouped in five territories: northwest (massa-carrara, lucca, pistoia), north-east (prato, firenze), west (livorno, pisa), east (arezzo, siena), south (grosseto). rizzo et al. monitoring grapevine phytoplasmas in tuscany 283 2013 north east sampling was increased by +574.1% in order to locate and eradicate any further infection site within a previously fd-free territory. sampling in west was also increased (+125.0%) on the supposition that the disease would spread towards the southern territories. in 2014, due to the results of the previous year, sample collection was globally similar (+2.3%). in 2015, sampling was concentrated in north west (+43.1%), where the presence of fd was alarming after three years of limited but constant findings (fig. 2 a). conversely, sampling in western territory was reduced (-88.8%) (fig. 3 a). sangiovese was the predominant cultivar sampled (more than 80% of samples). sampling was never redone in vineyards in which fd was found. leaf samples were collected from symptomatic grapevine plants during september and october of each year. each sample, consisted of 10-15 leaves showing typical sectorial reddening of the laminae processed independently (fig. 4). detection methods total nucleic acid was extracted from grapevine leaf veins tissues using a variant of the ctab method (angelini et al., 2001) and a mm400 steel bead mixer table 1 tuscan vineyards (vy) and samples included in grapevine yellows monitoring vy position 2012 2013 2014 2015 no. vy no. sample no. vy no. sample no. vy no. sample no. vy no. sample northwest 40 202 37 186 43 216 62 309 north east 5 26 21 106 20 98 6 32 weast 5 27 36 182 29 145 17 83 east 6 32 14 72 23 116 3 13 south 0 1 4 18 0 2 0 1 total 58 288 113 564 115 577 88 438 fig. 2 number of samples positive to “flavescence dorée” (fd) out of total samples tested in 2012-2015 in (a) north west and (b) east areas of tuscany; (c) comparison of incidence of fd in lucca district in north western tuscany in 2012 and 2015. adv. hort. sci., 2018 32(2): 281-288 284 mill (retsch, bergamo, italy). tna was re-suspended in te solution (10 mm tris; 0.1 mm edta; ph 8.0) and aliquots stored at -18°c until further use. quantitative pcr protocols targeting the 16s ribosomal rna (16sr rrna) gene were used to determine the presence of the phytoplasmas belonging to ribosomal groups 16sri, 16srxii-a and 16srv (angelini et al., 2007). leaves collected by v. vinifera plants, previously found infected by ‘ca. p. solani’ (subgroup 16srxii-a), flavescence dorée phytoplasmas (subgroups 16srv-c or -d) and ‘ca. p. asteris’ (subgroups 16sri-b or -c) were used as infected controls (ics). the infected controls were characterized following angelini et al. (2007) or berger et al. (2009) and conserved by phytosanitary service of the tuscany region. a set of ribosomal primer pairs for universal detection of phytoplasma associated to fd was used in nested-pcr: the direct was performed with p1/p7 (smart et al., 1996) followed by the nested pcr with 16r758f/m23sr primers (gibb et al., 1995; padovan et al., 1995). the nested amplicons obtained were digested with taqi (new england biolabs, usa), according to the manufacturer’s instructions, and digestion fragments were separated through electrophoresis on 3% agarose gel in tris-borate-edta (tbe) buffer. pcr conditions and protocols were as described by angelini et al., 2007. taqman allelic discrimination assay were performed following the protocol as described by berger et al 2009, using tufb type-specific probes carrying different fluorescent dyes. the concentrations of the reagents in the pcr mix, as well as the cycling conditions, were as originally described. reactions were performed in a cfx96 real-time thermocycler (biorad, usa). data were analyzed by measuring the threshold cycles (ct). once fd was detected in a vineyard, that vineyard was not further included in the monitoring and pps started the plant uprooting program. 3. results gy monitoring activity in 2012 among collected samples, 14.9% and 7.4% were positive for fd in north west and eastern samples respectively (fig. 2 a, b). a total of 17 novel fd foci were detected, 15 in the north west (mainly in massa-carrara and lucca districts) and 2 in both of the districts in the east (table 2). no fd was detected in samples from the north east, west or south. in 2012, the highest level of bn presence was found in the eastern samples (62.9%), while the lowest level of infection was observed in the north west (22.8%) (fig. 5 a-e). at the regional level, fd was found in 11.1% of the samples, while bn was found in 31.3% (fig. 3 b, c). ay was found at 0.3%. gy monitoring activity in 2013 in 2013, sampling was increased in the north east, east and west (+95.8%, globally) (fig. 3 a). north eastern vineyards were thoroughly investigated (+307.7% of sampling) because they are situated between the historically infected territory (north west) and the newly infected one (east) (fig. 2 a, b). no fd-infected sample was found in north east territory, while fd was found only in north west (5.4%). fig. 3 distribution of (a) sampling among tuscan provinces in 2012-2015. samples infected by (b) “bois noir” and (c) “flavescence dorée”. rizzo et al. monitoring grapevine phytoplasmas in tuscany 285 globally, 6 novel fd foci were identified, 5 of them in the lucca district (table 2). in 2013, infection rate of bn was generally high, reaching more than 80% of collected samples in north east and east territories, while the rate was lower in west (43.1%) (fig. 5 a-e). at regional level, fd was set at 1.8% and while bn was found in 73.4% of samples (fig. 3 b, c). gy monitoring activity in 2014 sample distribution was also similar to 2013, but more sampling was carried out in west (+61.1%) (fig. 3 a). fd was still found in north west (13.4%) and few samples was infected in east (1.4%) (fig. 2 a, b), confirming widespread infection sites in north west and sporadic (but difficult to eradicate) fd presence in eastern tuscany. in fact, 12 new foci were detected, 10 in lucca district and 2 in siena district (table 2). with regard to bn, disease rates were lower in 2014 compared to the previous year. more than 50% of samples were positive only in north east or south, while very low infection rate was observed in north west (15.2%) and west (10.3%) (fig. 5 a-e). at regional level, bn infection rate was quite low (28.8%), while fd apparently (5.4%) increased (fig. 3 b, c). fig. 4 symptoms of gy on cv. sangiovese. gy monitoring activity in 2015 in 2015, the largest number of fd-infected samples was detected since this survey was started, with 15.5% of positive samples in north west and 1.2% in east territory (fig. 2 a, b). unfortunately, infected samples were found in many different vineyards, thus 23 novel foci were detected, most of them in lucca (15), but a consistent number (6) in pistoia, the eastern district of north west territory (table 2). further decrease in bn detection was observed in north east, east and west, where about 25% of samples were bn-positive, while a lower level was recorded elsewhere (fig. 5 a-e). at regional level, a further increase in fd-positive samples was observed (11.1%), while bn infection was very low (14.6%) (fig. 3 b, c). additional observations on fd monitoring a comparison between fd findings in 2012 and 2015 in lucca district of north west territory (where fd findings were numerous) were reported (fig. 2 c) and pathogen spread seems to be directed towards the south eastern territories. moreover, the fd eradication was not achieved in the north west and east territories, despite application of intense monitoring adv. hort. sci., 2018 32(2): 281-288 286 programs. with regard to gy characterization, fd-c was most frequently found (fig. 6 a). few samples of fd-d were found in north west (lucca district). among bn, tufb type-b strains were significantly more frequent in all territories except south, were only tufb type-a was found (fig. 6 b). 4. conclusions even though a few fd infected plants were detected in the four years of monitoring, novel foci fig. 6 distribution of (a) “flavescence dorée” and (b) “bois noir” subgroups and tufb types respectively in tuscany. territory/districts novel flavescence dorée foci 2012 2013 2014 2015 north west 15 6 10 22 massa-carrara 8 0 0 1 lucca 6 5 10 15 pistoia 1 1 0 6 north east 0 0 0 0 west 0 0 0 0 east 2 0 2 1 siena 1 0 2 1 arezzo 1 0 0 0 south 0 0 0 0 total 17 6 12 23 table 2 findings of novel flavescence dorée foci fig. 5 samples positive to “bois noir” in tuscan areas in 20122015. rizzo et al. monitoring grapevine phytoplasmas in tuscany 287 continued to appear (17, 6, 12 and 23 in each year of survey), suggesting a more severe health condition of tuscan vineyards and a dangerous distribution of fd in tuscany. indeed, gy constitutes a serious concern for tuscan viticulture, considering the repeated finding of novel fd foci in the northern part of tuscany, the first detection of fd in the eastern territories and the frequent presence of bn in all districts. comparison between the 2012 and 2015 data in lucca district indicate how the disease is spreading in the north west of tuscany despite monitoring. in lucca district, novel foci were observed each year, whereas the consistent findings of novel fd foci in the eastern district of north west in 2015 was also worrisome. fortunately, no fd infected samples were found in north east, besides novel fd findings in the east. with regard to gy characterization, the prevalence of fd-c confirms the results of surveys in northeastern territories of italy (veneto) (borgo et al., 2001) and north-western territories of italy (piedmont) (marzachì et al., 2001). in tuscany, tufb type-b was predominant over tufb type-a in most districts. this evidence is in accordance with data reported by pierro et al. (2018), where the presence of the only tufb type-b was identified in a case vineyard in the chianti classico area (tuscany). this strain has, as main host plant of the phytoplasma and of the vector, convolvulus arvensis which high abundance was also reported in tuscan vineyards (marchi et al., 2015). true positive rate of gy was overestimated in 2012, 2014 and 2015, probably due to simultaneous foliar symptoms caused by virus and fungal disease. viruses, which are frequently found in tuscany (rizzo et al., 2012, 2015), as well as damage due to leafhopper, may mistake sampler. nevertheless, the percentage of infection was comparable to those obtained in northern italy (marzachì et al., 2001; marzachì and pacifico, 2006). eradication of fd from tuscany seems a difficult task even in recently colonized territories, probably due to the jeopardized distribution of the pathogen. that may lead, in the subsequent years, to the discovery of many further foci characterized by only a few plants. 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effects of frozen storage of olive fruit on extracted olive oils m. asheri 1 (*), m.m. sharifani 1, g. kiani 2 1 faculty of plant production, gorgan university of agricultural sciences and natural resources, basij square, 49189-43464 gorgan, iran. 2 department of agronomy and plant breeding, sari agricultural sciences and natural resources university, po box 578, sari, iran. key words: fatty acids, freezing, olea europaea. abstract: this study was achieved to examine the effects of freezing olive fruits of the arbequina, koroneiki and mission cultivars (the most common olive oil producing cultivars in iran) on the standard indices used for assessing virgin olive oil quality. oil was obtained from olive fruits stored at -4˚c for 1 week and 3 weeks, and compared with oil obtained immediately after harvest (control). the quality indices of oils obtained from frozen fruit showed no significant degradation in quality compared with the control samples. in fact the peroxide value of the frozen fruits decreased compared to the control, which is considered to have a positive effect on oil quality. in addition, compositions of the main fatty acids are not altered by freezing which demonstrate frozen storage as a viable option. oil derived from frozen olive fruit is not of inferior quality to non-frozen fruit in the production of olive oil. 1. introduction conservation of foods prior to processing by means of cold and frozen storage has been a relatively recent technique coming to prominence over the last 50 years (poerio et al., 2008). olive oil consumption is increasing throughout the world, even in countries that traditionally have not used olive oil. this trend has been promoted due mainly to the nutritional value of the mediterranean diet (patumi et al., 2002). olive fruits (olea europaea l.) undergo some mechanical procedures (milling, malaxation and centrifugation) to extract extra virgin olive oil. the quality of virgin olive oil is intimately related to the characteristics and composition of the olive fruit at the time of its processing (inarejos-garcía et al., 2010). highest quality extra virgin oils require optimum harvest stage, reduction in the time between harvest and milling, high quality oil extraction procedures and optimum storage conditions. the storage time between harvest and processing is one of the most important postharvest factors in oil quality. this becomes a significant problem when the volume of olive fruit exceeds the capacity of the mill plants. olive fruits are often stacked (*) corresponding author: maisa.asheri@gmail.com citation: asheri m., sharifani m.m., kiani g., 2017 an examination into the effects of frozen storage of olive fruit on extracted olive oil. adv. hort. sci., 31(3): 191-198 copyright: © 2017 asheri m., sharifani m.m., kiani g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 march 2017 accepted for publication 16 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 191-198 192 into large heaps at ambient temperature for several weeks prior to milling, which exceeds the storage limits (~48 hr) for the highest quality oil (garcia et al., 1996 a; ranali et al., 2000; angerosa, 2002). during this period of time fermentation may also occur, and pressure and heat within the piles provide a medium for fungi and bacteria growth (olias and garcia, 1997). anaerobic and aerobic processes take place inside and outside the piles of olive fruits, respectively, which causes deterioration of the fruit. this deterioration increases the acidity and reduces stability of the recovered oils (garcia et al., 1996 a). increase in volatile acids (acetic and butyric acid) during decomposition results in an unpleasant musty smell (olias and garcia, 1997). pigment content also decreases during this period, and additional refining needs to be done to clear all these unfavorable characteristics, increasing production cost and lowering market value (gutierrez-rosales et al., 1992). it is important to increase storage duration before milling the olive fruits to permit higher yields of better quality oil (petruccioli and parlati, 1987). studies have shown that cold storage can increase storage time without significantly affecting oil quality. clovodeo et al. (2007) stored ‘coratina’ olives used for oil production for 30 days at different temperatures and under different atmospheric conditions. they found that storage at 5°c, both under a flow of humidified air and a flow of 3% o2 + 5% co2, produced oils that maintained their initial chemical qualities until the end of the experimentation. however, the olives stored at room temperature deteriorated after 15 days of storage and the extracted oil had significantly reduced quality. poerio et al. (2008) frozed olives at -18˚c for 24 hours and extracted the oil with and without thawing the fruits. results were significantly different regarding peroxide value (pv), free fatty acids and polyphenols compared to control samples. oils from frozen olives had lower free fatty acids and pvs and freezing reduced the oxidative stability of the oil. the iranian government supports and subsidizes the expansion of olive cultivation to see about a sixfold increase in olive cultivated lands, i.e. from approximately 103,000 in 2014 to 600,000 ha by 2025, starting at 4,800 ha in 1993 at the launch of the expansion of olive cultivation plan (asheri et al., 2016). this has resulted in the spread of olive cultivation to areas where olive is not traditionally cultivated. therefore, many of the olive groves produce small amounts of fruit and have no olive processing and oil extraction facilities, and the fruit must be transported to an extraction facility. the ability to reduce deterioration during the storage time between harvest and processing is important to maintain quality. hence, it is worthwhile to discover ways to preserve olive fruits during this critical period. the aim of this study is therefore, to find methods that allow olive fruits to be stored for longer periods of time, without compromising the quality of extracted oils. this study investigated the effect of freezing olive fruits at moderate temperatures (-4°c) on quality indices of extracted oil, to understand if it was possible to extract high quality oil from fruits frozen for extended periods of time. the study is aimed to examine how susceptible the oil quality of different cultivars is to freezing, and to establish if the fruit of a particular cultivar responds better to freezing. the study considered 3 freezing treatments for 3 cultivars: fresh samples (control), freezing for 1 week and 3 weeks. 2. materials and methods the olive cultivars mission, arbequina, and koroneiki were selected for study as they are commonly used for the production of olive oil in iran. fruits were hand-picked from fadak grove located near qom, iran (34° 30´ n, 51° 00´ e). the grove is located about 15 km south of qom, a city at 150 km south of tehran, in the central hyper-arid to arid parts of iran. the fruits selected were at similar stages of ripening according to their ripening index which were 3.93, 3.84 and 4.23 for ‘arbequina’, ‘koroneki’ and ‘mission’, respectively. the fruits were immediately transferred to the university lab, washed and de-leafed. olives used to produce control samples were processed immediately, and the remaining olives stored in a freezer at -4°c. oil extraction for oil extraction, only sound and undamaged fruits were used. olives in the control samples were crushed using a hammer mill to form a paste. the paste was malaxed at 30˚c, then centrifuged at 4000 rpm for 10 minutes. olives used for treatment 1 were kept in freezer at -4˚c for one week and treatment 2 for 3 weeks at the same temperature. frozen samples were crushed and malaxed until they reached 25˚c (due to not thawing the samples), and then centrifuged at 4000 rpm for 10 minutes. extracted oil was collected using asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 193 a pipette and stored in dark glass jars in a refrigerator until analysis. determining oil content fifteen olive fruits from each replicate were placed in a petri dish and then placed in an oven at 105°c for 48 h. the dried olives were used to determine oil content in three replicates. olives were ground to a paste by mortar and pestle. the amount of 10 g of paste was placed in a soxhlet cartridge and oil extracted with 150 ml hexane at 70°c. after 6 h of extraction, hexane was collected and oil content was calculated on dry mass basis (agar et al., 1998). peroxide value the peroxide value of the oils was measured using the methods of garcia et al. (1996 b). a 5 g sample of the extracted olive oil was placed in a 250 ml erlenmeyer flask. the sample was shaken and then dissolved in 25 ml solution of acetic acid and chloroform (2:1, v/v). one milliliter of saturated potassium iodide (ki) solution was added. the mixture was placed in darkness for 5 min and then 75 ml of distilled water was added to stop the reaction. half of one milliliter of freshly prepared starch indicator solution (0.5%) was added to each sample. finally, the mixture was titrated with 0.01 n sodium thiosulfate until the blue indicator color disappeared. the peroxide value was expressed as milliequivalents of active oxygen per kilogram of oil (meq o2 kg-1) ( agar et al., 1998). coefficients of specific extinction values (k232 and k270) coefficients of specific extinction at 232 and 270 nm were measured by the methods reported in regulation eec/2568/91 of the european union commission (eec, 1991). the amount of 1 g of oil sample was diluted in 100 ml isooctane. the sample was then homogenized using a vortex and the solution was transferred to a 10 mm cuvette. absorbance at 232 and 270 nm was measured in a spectrophotometer using pure isooctane as a blank. fatty acid composition fatty acid composition in the oil was determined by the aocs official method (1997). methyl esters were prepared by vigorous shaking of a solution of oil dissolved in hexane (0.5 g in 7 ml) with 2 ml of 2n methalonic potash, and analyzed by gas chromatography. chromatographic analysis was performed on a trace gc (gas chromatograph), equipped with a flame ionization detector and split/splitless injector (trace gc, thermofinnigan, italy), using a silica capillary column, bpx-70 (30 m × 0.25 mm i.d. × 0.25 µm film thickness). the injector temperature was set at 250˚c and samples were injected manually (1 µl) with a split ratio of 1:80. the oven temperature was maintained at 175˚c for two min, and increased gradually to 230˚c at 3 ˚c/min and maintained for 10 min. nitrogen was used as carrier gas at a flow rate of 0.8 ml/min. the detector temperature was maintained at 270˚c. fatty acids were identified by comparing retention times with those of standard compounds. oxidizability (cox value) was calculated based on the fatty acid content of three unsaturated fatty acids [oleic acid (c18:1), linoleic acid (c18:2), and linolenic acid (c18:3)] using following relation (fatemi and hammond, 1980). oxidizability = [1 × (c18:1%) + 10.3 × (c18:2%) + 21.6 × (c18:3%)] determining oil chlorophyll and carotenoid content pigment contents were assayed using the spectrophotometric method of minguez-masquera et al. (1991). one gram of oil was dissolved in 10 ml of isooctane and the resulting solution transferred to a cuvette. absorbance at 470 and 670 nm (for carotenoid and chlorophyll, respectively) was measured in a spectrophotometer (unico/2800 uv/vis) using pure isooctane as a blank. the results are expressed as milligram of carotenoid or chlorophyll per kilogram of oil. statistical analysis experimental layout was factorial with three levels of freezing treatments and three olive cultivars. data were analyzed by spss software. comparison of means is performed using duncan’s multiple range test at a 95% confidence level and tables for analysis of variance (anova) are provided. 3. results and discussion oil content in fruit from the koroneiki and arbequina cultivars, there is no statistically significant difference in oil content between the control and the frozen samples (table 1). however, the mission cultivar fruit frozen for 1 and 3 weeks showed a significant decrease in mean oil content compared with the control under duncan’s multiple range test at 5%. since the independent variables (i.e. cultivar and freezing) act independently, freezing had no significant overall effect on the content of extracted olive oil (table 2). however in interpreting the results, it needs to be adv. hort. sci., 2017 31(3): 191-198 194 considered that oil extraction of frozen treatments was performed at a different temperature (25°c) in respect to the control (30°c). peroxide value the peroxide value (pv) is a measure of primary oxidation. table 1 shows the measured mean pv (meq o2 /kg) of the oils obtained from olives stored for the different time periods. our data showed that freezing reduces the peroxide value. the reduction was significant when olive fruits were frozen for three weeks, but not significant when frozen for only 1 week. cultivar’s effect in reduction of pv was significant at a 95% statistical level, while freezing had a very significant effect at a 99% level (table 2). no oil had peroxide values above the limit for extra virgin olive oil (20 meq o2/kg). poerio et al. (2008) and gomez and escoda (2010) also found reduced peroxide values in oil from olive paste and fruits that had been frozen. the lower peroxide value of oil from frozen fruit could be due to reduced enzymatic activity during crushing-malaxation due to the initial low temperature. specific extinction coefficients at 232 nm and 270 nm the k232 and k270 values for all treatments were below the maximum permitted values for extra virgin olive oil [2.50 and 0.20 respectively, according to regulation eec (1991)]. across all cultivars there was a net decrease in the k232 values due to freezing, but the difference was statistically insignificant. no change was detected in the k270 values between treatments (table 2). between cultivars however, the difference in the k232 and k270 values was significant with the arbequina cultivar showing the highest values, ‘koroneiki’ showing the lowest k232 and intermediate k270, and ‘mission’ showing intermediate k232 and the lowest k270 (table 1). these differences were consistent with the variations between olive cultivars shown by asheri et al. (2016). gomez and escoda (2010) also showed freezing to have no effect on the k270 and k232 values independent of cultivar. pigment content (chlorophyll and carotenoid) the slight decrease in the pigment content observed between the frozen treatments and the control was not statistically significant (tables 1, 2). also different cultivars did not demonstrate different levels of chlorophyll content. carotenoid content, on the other hand, was significantly different among the cultivars at 99% statistical level. chlorophylls and carotenoids play crucial roles in health and also in the oxidative activity of processed food stuff, due to their antioxidant nature in the dark and pro-oxidant activitable 1 means of chemical characteristics of olive oils derived from fresh and frozen olive fruits and their comparison with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample; t1= 1 week freezing sample; t2= 3 week freezing sample; pv= peroxide value. means within a column with the same lowercase letters are not significantly different. table 2 results of anova for chemical characteristics of olive oils derived from fresh and frozen olive fruits pv= peroxide value. ns= not significant; * significant at 5% level; ** significant at 1% level. samples oil content (% of dry matter) pv (meq o2 kg-1) k232 nm k270 nm chlorophyll (mg kg-1) carotenoid (mg kg-1) m c 47.21 a 7.33 a 1.13 bc 0.08 c 2.22 a 1.84 a m t1 43.08 b 6.63 ab 1.06 c 0.08 c 2.14 a 1.86 a m t2 42.73 b 5.47 bcd 1.05 c 0.08 c 2.06 a 1.79 ab k c 43.34 b 6.97 a 0.82 d 0.13 ab 2.20 a 1.53 bc k t1 41.80 b 6.53 ab 0.80 d 0.12 b 2.03 a 1.50 bc k t2 41.65 b 5.2 cd 0.76 d 0.12 b 2.02 a 1.43 c a c 43.57 b 6.53 ab 1.33 a 0.15 a 2.12 a 1.52 bc a t1 45.00 ab 5.63 bc 1.24 ab 0.14 ab 2.01 a 1.48 c a t2 42.82 b 4.47 d 1.22 ab 0.14 ab 1.98 a 1.48 c source mean square oil content pv k232 k270 chlorophyll carotenoid treatments 8.83 ns 2.63 ** 0.129 ** 0.002 ** 0.023 ns 0.091 * cultivar (a) 10.46 ns 2.11 * 0.50 ** 0.009 ** 0.024 ns 0.352 ** freezing (b) 12.14 ns 8.35 ** 0.16 ns 0.00004 ns 0.066 ns 0.008 ns ab interaction (cv *fr) 6.37 ns 0.04 ns 0.001 ns 0.000004 ns 0.002 ns 0.002 ns error 3.56 0.39 0.004 0.00009 0.058 0.026 asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 195 ty in the light (fakourelis et al., 1987). the higher the amount of these pigments, the higher the resistance to oil oxidation. our results indicated that freezing did not change olive oil resistance to oxidation based on pigment contents. kiritsakis et al. (1998) found that storage at lower temperatures reduced the pigment content of the extracted oils and morello et al. (2003) found slight decreases in chlorophyll and carotenoid concentration of oils obtained from frosted olives. these authors suggested that this could be due to the involvement of chlorophyllase and lypoxygenase. amongst cultivars, the mission cultivar shows significantly higher carotenoid content than ‘koroneiki’ and ‘arbequina’, as observed by asheri et al. (2016). fatty acid composition this paper reports 12 fatty acids detected in gc analysis including myristic acid (c14:0), palmitic acid (c 16:0), palmitoleic acid (c 16:1), heptadecanoic acid (c 17:0), heptadecenoic acid (c 17:1), stearic acid (c 18:0), oleic acid (c 18:1), linoleic acid (c 18:2), linolenic acid (c 18:3), arachidonic acid (c 20:4), arachidic acid (c 20:0), and erucic acid (c 22:1) (table 3). analysis of variance (anova) shows that freezing olive fruits did not have significant effects on the fatty acid composition of the oils, while cultivars had very significant influence on all the fatty acids except for myristic acid. in addition, freezing and cultivar variables acted independently on fatty acids level, except for myristic and palmitic acids in which the interaction of variables were very significant (at 99%) and significant (at 95%), respectively (tables 4, 6). comparison of the means demonstrate that ‘koroneiki’ and ‘mission’ had significantly higher levels of oleic acid (74.85 and 72.96% in fresh state, respectively) than ‘arbequina’ (60.76%). oleic acid is the most prominent fatty acid in olive oil and is a monounsaturated fatty acid (mufa) with demonstrated qualities in the stability of the oil. oleic acid levels did not change significantly during the storage except for the 1 week freezing of ‘arbequina’ (table 5). the other predominant fatty acids are palmitic acid [a saturated fatty acid (sfa)] and linoleic acid [a polyunsaturated fatty acid (pufa)]. ‘arbequina’ had the highest amounts of palmitic and linoleic fatty acids. this indicates lower quality of its oil, especially when it is considered together with its lower oleic samples myristic acid palmitoleic acid heptadecanoic acid heptadecenoic acid stearic acid linolenic acid arachidic acid arachidonic acid erucic acid m c 0.010 d 0.730 bc 0.0255 b 0.044 b 2.55 b 1.05 a 0.290 cd 0.409 a 0.068 c m t1 0.014 d 0.701 bc 0.024 b 0.042 b 2.44 b 1.00 ab 0.281 d 0.387 ab 0.065 c m t2 0.051 a 0.682 c 0.022 b 0.038 b 2.38 b 0.95 b 0.271 d 0.382 abc 0.063 c k c 0.030 b 0.798 bc 0.031 b 0.050 b 3.26 a 0.76 c 0.465 a 0.379 abc 0.137 a k t1 0.018 bcd 0.828 b 0.035 b 0.051 b 3.20 a 0.80 c 0.459 a 0.356 c 0.132 a k t2 0.011 d 0.817 b 0.032 b 0.050 b 3.16 a 0.77 c 0.448 a 0.359 bc 0.136 a a c 0.028 b 2.512 a 0.070 a 0.169 a 1.43 c 0.68 d 0.324 b 0.159 d 0.114 b a t1 0.027 bc 2.515 a 0.070 a 0.170 a 1.45 c 0.68 d 0.319 bc 0.151 d 0.103 b a t2 0.016 cd 2.579 a 0.072 a 0.169 a 1.44 c 0.69 d 0.319 bc 0.167 d 0.107 b table 3 fatty acid (myristic, palmitoleic, heptadecanoic, heptadecenoic, stearic, linolenic, arachidic, arachidonic and erucic acids) content (%) of olive oils derived from fresh and frozen olive fruits and comparison of the means with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample. t1= 1 week freezing sample; t2= 3 week freezing sample. means within a column with the same lowercase letters are not significantly different. source mean square oil content pv k232 k270 chlorophyll carotenoid treatments 8.83 ns 2.63 ** 0.129 ** 0.002 ** 0.023 ns 0.091 * cultivar (a) 10.46 ns 2.11 * 0.50 ** 0.009 ** 0.024 ns 0.352 ** freezing (b) 12.14 ns 8.35 ** 0.16 ns 0.00004 ns 0.066 ns 0.008 ns ab interaction (cv *fr) 6.37 ns 0.04 ns 0.001 ns 0.000004 ns 0.002 ns 0.002 ns error 3.56 0.39 0.004 0.00009 0.058 0.026 table 4 results of anova for fatty acids content (reported in table 3) of olive oils derived from fresh and frozen olive fruits pv= peroxide value. ns= not significant; * significant at 5% level; ** significant at 1% level. adv. hort. sci., 2017 31(3): 191-198 196 acid content. palmitic and linoleic acids amounts in the mission cultivar and linoleic acid in ‘koroneiki’ show significant changes after three weeks of frozen storage (table 5). among the fatty acids of lower content, only linolenic and myristic acids demonstrate significant changes in their mean values (table 3). linolenic and myristic acid contents of the mission cultivar show significant increases after three weeks of frozen storage. myristic acid levels of ‘koroneiki’ and ‘arbequina’ decreased due to freezing. total sfas, mufas and pufas did not demonstrate any statistically significant change under frozen storage, and all the change is due to cultivar (table 6). cultivar had very significant effect on the ratios of mufa/pufa and oleic/linoleic acids, and the effect of freezing on these ratios was significant at 95% confidence level. also, the analysis indicated that cultivar and freezing have significant interaction during the storage of olive fruit on these ratios. cox value, however was not influenced significantly by freezing (table 6). mean total sfa levels for ‘koroneiki’ and ‘arbequina’ did not change, while ‘mission’ showed a significant reduction in total sfa after 3 weeks of freezing. this change could be due to the reduction of palmitic acid. means comparison of total mufa content did not detect any significant changes. ‘koroneiki’ shows a significant increase in the total pufa content after 3 weeks of frozen storage, which leads to a reduction of mean mufa/pufa and oleic/linoleic acids ratios. similar to mufa/pufa and oleic/linoleic acids ratios, cox values did not change significantly for ‘mission’ and ‘arbequina’, while ‘koroneiki’ experienced a significant decrease for 3 weeks freezing treatment. the higher the mufa/pufa and oleic/linoleic acids ratios and the lower the cox values, the higher the oxidative stability of the oil. the implication of the reduction in mufa/pufa and oleic/linoleic acids ratios and increase in cox values after 3 weeks freezing for ‘koroneiki’ is that longer periods of freezing storage could reduce oxidative stability of its oil. however, it is important to note that despite the changes after 3 weeks of storage, ‘koroneiki’ oil still possessed signiftable 5 fatty acid (palmitic acid, oleic acid, linoleic acid, ∑sfa, ∑pufa, mufa/pufa, oleic/linoleic and cox value) compositions and comparison of the means with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample. t1= 1 week freezing sample; t2= 3 week freezing sample. sfa= saturated fatty acids; mufa= monounsaturated fatty acids; pufa= polyunsaturated fatty acid; cox value: calculated oxidizability. means within a column with the same lowercase letters are not significantly different. table 6 results of anova for fatty acids (reported in table 5) composition of olive oils derived from fresh and frozen olive fruits sfa= saturated fatty acids; mufa= monounsaturated fatty acids; pufa= polyunsaturated fatty acid. cox value: calculated oxidizability; ns= not significant; * significant at 5% level; ** significant at 1% level. samples palmitic acid oleic acid linoleic acid ssfa smufa spufa mufa/pufa oleic/linoleic acid cox value m c 11.11 c 72.96 c 10.47 c 14.25 c 73.80 c 11.93 b 6.19 c 6.97 c 2.03 b m t1 10.73 c 73.49 bc 10.53 c 13.76 74.30 c 11.91 b 6.24 c 6.98 c 2.03 b m t2 10.23 d 73.60 bc 11.00 b 13.24 d 74.39 bc 12.34 b 6.03 c 6.69 c 2.07 b k c 12.48 b 74.85 a 6.51 e 16.51 b 75.83 a 7.65 d 9.92 a 11.52 a 1.58 d k t1 12.48 b 74.74 a 6.62 e 16.43 b 75.75 a 7.78 cd 9.74 a 11.29 a 1.60 cd k t2 12.47 b 74.15 ab 7.09 d 16.37 b 75.15 ab 8.22 c 9.14 b 10.46 b 1.64 c a c 16.63 a 60.76 e 15.96 a 18.73 a 63.56 d 16.80 a 3.78 d 3.81 d 2.40 a a t1 16.54 a 61.76 d 15.94 a 18.65 a 64.55 d 16.77 a 3.85 d 3.87 d 2.41 a a t2 16.96 a 61.53 de 15.68 a 19.05 a 64.39 d 16.54 a 3.89 d 3.92 d 2.38 a source mean square palmitic acid oleic acid linoleic acid ssfa smufa spufa mufa/pufa oleic/linoleic acid cox value treatments 14.46** 80.35 ** 31.51 ** 9.76 ** 58.30 ** 29.31 ** 12.70 ** 19.90 ** 0.23 ** cultivar (a) 57.37 ** 320.35 ** 126.63 ** 38.45 ** 232.15 ** 116.90 ** 50.43 ** 78.93 ** 0.93 ** freezing (b) 0.06 ns 0.338 ns 0.13 ns 0.13 ns 0.33 ns 0.10 ns 0.14 * 0.30 * 0.001 ns ab interaction (cv*fr) 0.21 * 0.365 ns 0.14 ns 0.24 * 0.36 ns 0.12 ns 0.11 * 0.19 * 0.001 ns error 0.03 0.142 0.04 0.05 0.12 0.05 0.03 0.04 0.0004 asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 197 icantly higher mufa/pufa and oleic/linoleic acid ratios and lower cox values than the other cultivars, and therefore indicated the highest resistance to oxidative stability among these three cultivars. 4. conclusions the oils obtained from olive fruits stored at -4˚c are demonstrated to maintain the same characteristics of the control. among the biochemical indices, the decrease in pv was the only factor that varied significantly from the control samples. this decrease is received positively, as less oxidation occurs. pigment content remained stable during freezing period which is considered to be good to maintain quality oil. no differences in characteristics evaluated in this study were observed between cultivars, suggesting that freezing the olive fruits of all three cultivars in this research did not have any negative effect on the studied characteristics of the extracted oil. based on this study, freezing could be a suitable method of preserving olive fruits in the waiting period between harvesting and processing, which could assist in the maintenance of characteristics evaluated in this study during olive oil production. however, it may be worthwhile extending this experiment with different cultivars and to test characteristics such as sensory quality evaluation and oxidative stability. nonetheless, our results suggest that olive fruit may be stored frozen before processing into oil. so the products could be harvested at the optimal stage of ripening and preserved frozen while shipping them to the mill plant stations. this preservation system could be extended to the pre-processing waiting period at the mill plant, so that the processing of olives could be optimized. acknowledgements this research was funded by gorgan university of agricultural sciences and natural resources. we thank ahmad bolandnazar, the owner and manager of fadak olive grove, for his permission to access the grove. we would like to express our sincere gratitude to justin parker for proofreading the manuscript and also to naser mostafavi for his assistance in improvement of the earlier drafts of the manuscript. references agar i.t., hess-pierce b., sourour m.m., kadar a.a., 1998 quality of fruit and oil of black-ripe olives is influenced by cultivar and storage period. j. agr. food chem., 46: 3415-3421. angerosa f., 2002 influence of volatile compounds on virgin olive oil quality evaluated by analytical approaches and sensor panels. eur. j. lipid sci. tech., 104: 639-660. aocs, 1997 preparation of methyl esters of long-chain fatty acids from sampling and analysis of commercial fats and oils. aocs press, champaign, il, usa, pp. 266. asheri m., sharifani m.m., yamchi a., kiani g., ahmadi golsefidi m., hosseini-mazinani m., 2016 influence of olive cultivar on oil attributes in the arid region of qom, iran. agric. conspec. sci., 80(3): 173179. clodoveo m.l., delcuratoro d., gomes t., celelli g., 2007 effect of different temperatures and storage atmospheres on coratina olive oil quality. food chem., 102: 571-576. eec, 1991 characteristics of olive oil and olive-residue oil and the relevant methods of analysis.commission regulation (eec) no. 2568. offic. j. eur. commun., l248: 1-83. fakourelis n., lee e.c., min d.b., 1987 effects of chlorophylls and ß-carotene on the oxidation stability of olive oil. j. food sci., 52: 234-235. fatemi s.h., hammond e.g., 1980 analysis of oleate, linoleate and linolenate hydroperoxides in oxidized ester mixtures. lipids, 15: 379-385. garcia j.m., gutierrez f., barrera m.j., albi m.a., 1996 a storage of mill olives on an industrial scale. j. agr. food chem., 44: 590-593. garcia j.m., seller s., perez-camino m.c., 1996 b influence of fruit ripening on olive oil quality. j. agr. food chem., 44: 3516-3520. gomez m.a., escoda c., 2010 european patent ep1978078a1. gutierrez-rosales f., perdiguero s., gutierrez r., olias j.m., 1992 evaluation of the bitter taste in virgin olive oil. j. am. oil chem. soc., 69: 394-395. inarejos-garcia a.m., gomez-rico a., desamparados salvador m., fregapane g., 2010 effect of preprocessing olive storage conditions on virgin olive oil quality and composition. j. agr. food chem., 58(8): 48584865. kiritsakis a., nanos g.d., polymenopulos z., thomai t., sfakiotakis e.m., 1998 effect of fruit storage conditions on olive oil quality. j. am. oil chem. soc., 75: 721-724. minguez-mosquera m.i., rejano-navarro l., gandul-rojas b., sanchez-gomez a.h., garrido-feradv. hort. sci., 2017 31(1): 191-198 nandez j., 1991 color-pigment correlation in virgin olive oil. j. am. oil chem. soc., 68: 332-336. morello j.r., moltilva m.j., ramo t., romero m.p., 2003 effect of freeze injuries in olive fruit on virgin olive oil composition. food chem., 81: 547-553. olias j.m., garcia j.m., 1997 postharvest physiology and storage of tropical and subtropical fruits. cab international, wallingford, uk, pp. 229-243. patumi m., d’andria r., marsilio v., fontanazza g., morelli g., lanza b., 2002 olive and olive oil quality after intensive monocone olive growing (olea europea l., cv. kalamata) in different irrigation regimes. food chem., 77: 27-34. petruccioli g., parlati m.v., 1987 studies on olives storage before oil extraction. iii. effect of storage conditions of olives on the chemical and organoleptic characteristics of oils. third subproject: conservation and processing of foods a research report (1982-1986). national council of milano, milano, italy, abstract 20, pp. 83-84. poerio a., bendini a., cerretani l., bonoli-carbognin m., lercker g., 2008 effect of olive fruit freezing on oxidative stability of virgin olive oil. eur. j. lipid sci. tech., 110: 368-372. ranalli a., modesti g., patumi m., fontanazza g., 2000 the compositional quality and sensory properties of virgin olive oil from a new cultivar i-77. food chem., 69: 37-46. impaginato 569 adv. hort. sci., 2018 32(4): 569-573 doi: 10.13128/ahs-22942 field evaluation of new kabuli chickpeas lines for the production of canned seeds p. casini dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: canned products, canned seeds, chickpea, cicer arietinum l., seed grade, tuscany. abstract: there is an increased demand for canned chickpeas worldwide, which has also resulted in increased chickpea cultivation in italy. the availability of italian chickpea varieties, suitable for industrial transformation, is limited. the objective of the present study was to evaluate the field production of new kabuli chickpea accessions and their suitability for industrial transformation. thirteen accessions, provided by the international centre for agricultural research of turkey, together with ‘blanco sinaloa’, a commercial mexican variety, were cultivated in cesa, tuscany, in 2016 and 2017. the average seed yield was 2.8 and 3.4 t ha-1, respectively, in 2016 and 2017. increased yield in 2017 was attributable to a more balanced rainfall pattern, compared to that in 2016 which induced increased vegetative growth. based on production stability over the two-year trial, the varieties flip08-69c, flip-160c and flip05-157c turned out particularly suitable for cultivation. the climatic conditions in 2017 favored an increased presence of a grade b seed classification [220-250 seeds per 100 g (caliber 8)] which is preferred by industry. most varieties showed potential regarding seed dimension classification, however, of the varieties with a good production, flip05-157c was also characterized by seed gradation stability. 1. introduction in recent years, there has been a change in the style of food consumption by italian people. this includes the growing interest in legumes as an alternative protein source to meat. the lower fat content of legumes appears to be a more appreciated characteristic. here, we are referring to “food safety”, where consumers are prepared to incur higher costs when purchasing foods for health safety and towards guaranteeing environmentally friendly production techniques (dixon and sumner, 2003; fratianni et al., 2014). the industrial uses of legumes vary, ranging from animal feed (flour (*) corresponding author: paolo.casini@unifi.it citation: casini p., 2018 field evaluation of new kabuli chickpeas lines for the production of canned seeds. adv. hort. sci., 32(4): 569-573 copyright: © 2018 casini p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 march 2018 accepted for publication 4 july 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 569-573 570 and supplements) to the processing of the seeds for human consumption, especially in the form of preserved (canned) products. according to 2016 statistics, the italian market for canned vegetables is valued at approximately 400 million euros, representing an increase of +1.2% compared to the previous year (limonta, 2016). the same data showed that, in the face of production decreases in sweet corn, green beans and peas, there is an increase in beans and chickpeas. more specifically, the categories “beans” and “other legumes” (chickpeas and lentils) constitute more than 60% of the market. as regards chickpeas, with a 62% share, an increase of 10.2% was recorded. as an inevitable consequence, there was an increase in the area dedicated to chickpea cultivation in italy (palumbo, 2017), from 5.000 to almost 16.000 ha, from 2008 to 2017, respectively. this notable increase is attributable, not only to the aforementioned food trends, but also to agronomic benefits such as reducing the use of both fertilizers and pesticides. in turn, this serves to improve the structure and fertility of the soil and, above all, a more sustainable return to crop rotation with cereals (palumbo, 2017). these benefits were already sought after, as early as the beginning of 2000 (watson et al., 2017). concomitant with italian scenario for chickpea cultivation, there has been a steady increase in the world production of chickpeas from 63.4 to 77.5 million tons, from 2009 to 2015, respectively (muehlbauer and sarker, 2017). this positive trend in the chickpea market has naturally resulted in the involvement of the italian canning industry, always attentive to the needs of the market. an increasing number of industries, through their own incentive, are producing the raw material directly in italy just to meet the needs of “food safety”. in this way, through cultivation contracts, they are certain to direct cultivation towards the production of the raw material, adhering, as closely as possible to both product requirements and processing techniques for the production of canned chickpeas. the italian consumer prefers kabuli chickpeas. the characteristics of “kabuli” include a light color, preferably with a caliber of 8 (220-250 seeds per 100 g), a typical shape with rough surface, and with a thin integument that still adheres to the seeds once cooked (palumbo, 2017). the availability of italian chickpea varieties, suitable for industrial transformation, is rather limited. with regard to central italy, more specifically tuscany, experimental tests conducted in the ‘80s and ‘90s (casini, 1987, 1989), showed that either autumn or end of winter sowing subjected the cultivation to high risks of anthracnose [ascochyta rabiei (pass.) labr. trot.]. more recent research (radicetti et al., 2012), has highlighted that “pascià” and “principe” are the two most widespread italian varieties, suitable for industrial transformation with yields varying between 2.5 and 2.7 t ha-1. however, industry is continually looking for new varieties either for use in genetic improvement programs or for use towards better production, tolerance to diseases and technological qualities. the aim of the present study was to evaluate the field production of new accessions of kabuli chickpeas and their suitability for industrial transformation based on the preferred seed dimensions classification characteristics. 2. materials and methods the field experiments were carried out in tuscany, central italy, in 2016 and 2017 at the “centro per i l collaudo ed il trasferimento dell’innovazione di cesa (arezzo)”, 43° 18’ n; 11° 47’ e; 242 m a.s.l. the cultivation environment was characterized of a neutral, loamy-sandy soil. the principle physical and chemical characteristics of the soil were as follows: sand 36%, loam 38%, and clay 26% respectively. the soil ph was 7.0. total n was 0.11% and p (olsen) 13 ppm. exchangeable ca, mg and k were 4123, 595 and 141 ppm, respectively. thirteen accessions, provided by the international centre for agricultural research (icarda) of ankara (turkey), were used, in addition to “blanco sinaloa”, a commercial variety from mexico. based on previous experiments carried out in central italy (casini, 1989), the autumn-winter sowing period was not taken into consideration due to serious damage caused by anthracnose blight. as a result, the sowing dates were march 14, 2016 and february 22, 2017, respectively. plots were arranged according to a complete rcb design, with three replicates. the size of the plots were 2.0 x 4.0 m (four rows wide with 0.5 m row spacing). the sampling area was comprised of the two central rows of 3.0 m long. a seed quantity of 380 per plot was used. in order to obtain the correct planting density of 25 plants m-2, seedlings were thinned soon after complete emergence. plots were hand-weeded twice (45 and 65 days after emergence casini new kabuli chickpeas for canned seeds production 571 [dae]) during the growth cycle. the agricultural interventions performed during the two-year experimental period are reported in table 1. plant height, number of stems, height of the first pod and number of pods per plant were determined at maturation stage, using a total of 10 plants per sample plot. yield calculation and the number of seeds amounting to 100 g were performed using seed samples at standard humidity of 12%. grading of seeds was carried out according to the standard tables of conserve italia (2015) for the italian market as follows: grade a [<220 seeds per 100 g (caliber 6)]; grade b [220-250 seeds per 100 g (caliber 8)] and non standard or off-type (>250 seeds per 100 g). both grades a and b are considered suitable for processing, but b is the preferred grade. data collected in the experiments were processed utilizing a mixed-model analysis of variance (anova), where accession was considered as a fixed effect factor, and year as random effect factor. statistical differences were tested at p≤0.05, p≤0.01 or p≤0.001. the tukey’s hsd test was used to stress significant differences between means and homogenous groups. 3. results, discussion and conclusions the climatic trends over the two-year experimental period were very different, especially with regard to rainfall. in the first year, the crop benefited from 562 mm of rainfall, of which 142 mm were evenly distributed during the period may-mid-june. in the second year, total rainfall over the cultivation cycle was 290 mm. in 2017 the maximum temperatures were particularly high with an average of 24.8°c (31.0°c in the summer). principle biometric characteristics of the lines (table 2) were significantly different at either p≤0.01 or p≤0.001, with the exception of the number of empty pods per plant. as regards the main biometric characteristics, the higher rainfall of 2016, contributed to a greater vegetative growth of the plants, compared to that in 2017. the height of the plants 2016 2017 previous crop wheat sunflower plowing september 6, 2015 rippering and rolling october 22, 2016 harrowing september 28, 2015 november 2, 2016 grubbering january 10 harrowing march 14 february 22 pre-sowing fertilization march 14 february 22 n 52 and p 2 o 5 114 kg ha-1 n 52 and p 2 o 5 114 kg ha-1 sowing march 15 february 23 emergence april 11 march 14 harvesting august 22 august 19 table 1 agronomic technique, date of sowing, date of emergence and harvesting of the two field trials source of variation plant height (cm) stems per plant (n) height of first pod (cm) filled pods (n) empty pods (n) seeds per pod (n) lines (l) flip05-69c 58.3 a 5.1 bcd 34.3 a 43.5 bc 3.5 a 1.2 ab flip05-156c 55.1 ab 5.3 a-d 28.2 bcd 45.6 bc 4.9 a 1.1 ab flip05-157c 53.9 ab 6.7 ab 29.2 bc 39.1 cd 3.4 a 1.1 ab flip07-230c 58.7 a 6.9 a 30.7 ab 45.1 bc 5.0 a 1.1 ab flip07-318c 52.7 ab 5.5 a-d 30.0 abc 42.0 cd 3.0 a 1.3 a flip08-69c 55.7 ab 6.6 ab 28.9 bcd 48.5 bc 3.9 a 1.2 ab flip08-160c 52.6 ab 6.2 abc 29.3 bc 47.2 bc 4.4 a 1.1 ab flip08-170c 56.8 a 5.1 bcd 27.5 bcd 58.5 a 4.5 a 1.2 ab flip08-200c 55.4 ab 4.7 cd 25.8 cde 43.7 bc 4.9 a 1.2 ab w6-12861 59.0 a 6.0 a-d 31.3 ab 63.3 a 4.9 a 1.1 ab w6-9484 54.9 ab 5.1 bcd 24.3 de 37.2 cd 4.3 a 1.0 b w6-30 54.0 ab 5.7 a-d 25.4 cde 46.2 bc 4.1 a 1.0 b w6-25 52.4 ab 4.5 d 28.0 bcd 57.2 ab 4.6 a 1.2 ab blanco sinaloa 49.1 b 5.1 bcd 21.4 e 29.4 d 2.6 a 1.2 ab f ** *** *** *** ns ** year (y) 2016 42.1 6.7 31.8 65.8 5.8 1.2 2017 42.9 4.6 24.5 28.0 2.4 1.2 f ** ** *** *** *** ns l x y *** ** *** *** *** ns table 2 principle biometric characteristics of the lines ns= not significant; ** significant at p≤0.01; ***significant at p≤0.001. means followed by the same letter(s) are not different for p≤0.05 according to tukey test. adv. hort. sci., 2018 32(4): 569-573 572 was almost 20 cm higher (average of 64.7 cm) with a corresponding increased height of first stage pods (31.8 cm from the ground), more favorable for mechanical harvesting. increased vegetative growth was also expressed in the greater ramification number of 6.7 stems per plant in 2016 as compared to 4.6 in 2017, respectively. the height of the plants was both positively and significantly correlated to the number of full pods per plant (r2 = 0.799**), which in the first year was more than double than that observed for the second year: 65.8 vs. 28.0 cm, respectively. the average seed yield was 2.8 and 3.4 t ha-1, respectively, in 2016 and 2017 (fig. 1). the yield increases in 2017 were observed for all varieties. varieties showing a yield increase that exceeded 25% included flip 69c, w6 12861, w6 9484 and w6 25, respectively. these increases were attained despite a lower production of full pods per plant in 2017. this yield increase was likely attributable to the more favorable climatic conditions of 2017, in comparison to those of 2016, which induced increased vegetative growth. table 3 shows an increase in the number of seeds per each 100 g weight for almost all varieties, with the exception of w6 25, in which the number seeds (195-196 seeds per 100 g) is maintained. table 3 also provides the commercial classification values for varieties and their suitability for processing into precooked products. in this regard, all the lines were found to be suitable for both years with a grade classification of either a or b, with the prevalence of the more desired latter classification in 2017. the results of this experiment highlighted a good production capacity of the majority of the lines tested. some varieties, such as flip08-69c, flip-160c and flip05-157c, are particularly suitable, as based on production stability over the two-year trial. moreover, among these lines, flip05-157c was also characterized by seed gradation stability, suitable for industrial transformation. conditions of balanced rainfall, evident in 2017, favored an 8 caliber (b) seed yield, which is preferred by industry. in conclusion, with some exceptions, all the lines tested are considered suitable for use in selection and/or for breeding programs, aimed at obtaining varieties dedicated to the production of pre-cooked seeds for the italian market. references casini p., 1987 confronto varietale di cece (cicer arietinum l.) in semina primaverile. agricoltura ricerca, 9 (80): 33-42. casini p., 1989 comportamento di alcune varietà di cece (cicer arietinum l.) in semina autunnale. agricoltura ricerca, 11(95): 27-42. conserve italia, 2015 allegato capitolati di acquisto prodotti di classe 1, pp. 16-18. dixon r.a., sumner l.w., 2003 legume natural products: understanding and manipulating complex pathways for human and animal health. plant physiol., fig. 1 seed production of tested lines in 2016 and 2017 lines 2016 2017 seeds per 100 g (n) grade seeds per 100 g (n) grade flip05-69c 241 ab b 232 ab b flip05-156c 236 ab b 243 ab b flip05-157c 233 ab b 238 ab b flip07-230c 229 bc b 224 bcd b flip07-318c 219 cd a 231 bc b flip08-69c 202 e a 240 ab b flip08-160c 200 ed a 245 a b flip08-170c 215 cd a 238 ab b flip08-200c 219 cd a 246 a b w6-12861 216 cd a 231 bc b w6-9484 198 ed a 229 bc b w6-30 195 d a 196 ed a w6-25 225 bcd b 231 bc b blanco sinaloa 198 ed a 229 bc b table 3 number of seeds per 100 g and the corresponding grade for canned products means followed by the same letter(s) are not different for p≤0.05 according to tukey test. casini new kabuli chickpeas for canned seeds production 573 131: 878-885. fratianni f., cardinale f., cozzolino a., granese t., albanese d., di matteo m., zaccardelli m., coppola r., nazzaro f., 2014 polyphenol composition and antioxidant activity of different grass pea (lathyrus sativus), lentils (lens culinaris), and chickpea (cicer arietinum) ecotypes of the campania region (southern italy). j. functional foods, 7: 551-557. limonta m., 2016 la crescita dell’alimentare tra opportunità e rischi. iri. tuttofood, milano, april 19, italy. muehlbauer f.j., sarker a., 2017 economic importance of chickpea: production, value, and world trade, pp. 5-12 in: varshney r.k., m. thudi, and f. muehlbauer (eds.) the chickpea genome. springer international publishing ag, berlin, germany, pp. 142. palumbo m.g., 2017 il cece da industria si coltiva così. ortive da industria. terra e vita, 25: 48-52. radicetti e., mancinelli r., campiglia e., 2012 combined effect of genotype and inter-row tillage on yield and weed control of chickpea (cicer arietinum l.) in a rainfed mediterranean environment. field crops research, 127: 161-169. watson a., reckling m., preissel s., bachinger j., bergkvist g., kuhlman t., lindström k., nemecek t., topp c.f.e., vanhatalo a., zander p., murphybokern d., stoddard f.l., 2017 grain legume production and use in european agricultural systems. advances in agronomy, 144: 235-303. impaginato 299 adv. hort. sci., 2018 32(3): 299-309 doi: 10.13128/ahs-22260 postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage f. azadshahraki 1 (*), b. jamshidi 1, s. mohebbi 2 1 agricultural engineering research institute, agricultural research education and extension organization (areeo), karaj 31585-845, iran. 2 department of horticultural sciences, college of agriculture and natural resources, university of tehran, karaj 31587-77871, iran. key words: enzyme activity, lycopene, melatonin, proline, tomato fruit. abstract: in this study, tomato fruit was treated with 50, 100 or 200 µm melatonin and then stored at 5°c for 28 days to investigate the effect of melatonin treatment on chilling injury, nutritional quality and changes in the antioxidant system. tomato fruit developed chilling injury, manifested as surface pitting and irregular red color development during storage. these chilling injury symptoms, ion leakage and malondialdehyde content were significantly reduced, and proline and carotenoids contents were significantly increased by melatonin treatment. meanwhile, melatonin substantially reduced o2 production rate and h2o2 content, which result from significantly higher activities of superoxide dismutase, catalase, and peroxidase than control during the storage. these results suggest that melatonin treatment can effectively enhance chilling tolerance and reduce chilling injury. the reduction in chilling injury by melatonin may be associated with enhanced enzymatic and non-enzymatic antioxidants, in favor of membrane integrity and thus low cellular and tissue damage. 1. introduction cold storage is one of the most effective postharvest technologies to preserve the quality of fresh produces from the time of harvest until final preparation for human consumption in food chain (bourne, 2006). however, cold storage imposes great risk on postharvest commodities sensitive to chilling injury (ci). tomato (lycopersicon esculentum), as one of the most important tropical crops, is typically cold sensitive (hong and gross, 2006). the most common visual injury symptoms of ci depicted for tomato fruit include irregular ripening and red color development as well as surface pitting on the fruit. furthermore, as the chilled tissues are weakened, they become prone to decay and microbial spoilage. this phenomenon limits postharvest life and leads to significant degradation of (*) corresponding author: farzad_shahrakiazad@yahoo.com citation: azadshahraki f., jamshidi b., mohebbi s., 2018 postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage. adv. hort. sci., 32(3): 299-309 copyright: © 2018 azadshahraki f., jamshidi b., mohebbi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 december 2017 accepted for publication 31 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 299-309 300 produce quality (wang, 1993). melatonin, first discovered in tomato in 1995, accumulates in the fruits as they mature (dubbels et al., 1995; hattori et al., 1995). melatonin content, as an endogenous signaling molecule, increases in response to abiotic and biotic stress, such as drought, salinity, chilling, and pathogens to protect against damage caused by them (zhang et al., 2014; arnao and hernández-ruiz, 2015; liu et al . , 2016). accumulation of higher levels of melatonin in horticultural crops is beneficial not only for human health, but also for prolonging storability (tan et al., 2012). melatonin, a naturally occurring indoleamine, acts as endogenous elicitor and signaling molecule for plants growth and development, decreasing of biotic and abiotic stress, as well as a potent hydroxyl radical scavenger and antioxidant (zhang et al., 2014; zhang and zhang, 2014; manchester et al., 2015; zhang et al., 2015). melatonin contribution has been evidenced in a semilunar rhythm in macroalgae guarding this plant against high temperature stress (tal et al., 2011). melatonin treatment decreased apoptosis chilling-induced in carrot suspension cells. moreover, melatonin treatment alleviated chilling-induced shrinkage and disruption of carrot cell plasma membranes (lei et al., 2004). it has been reported that melatonin treatment reduced chilling injury in peach fruits by enhancement of chilling tolerance and provoking of defense response during cold storage (cao et al., 2016). soleimani aghdam and rezapour fard (2017) reported that melatonin treatment at 100 µm decreased strawberry fungal decay resulting from higher superoxide dismutase (sod) activity, associated with lower catalase (cat) and ascorbate peroxidase (apx) activities as well as higher phenylalanine ammonia lyase (pal) enzyme activity leading to higher total phenols and anthocyanins accumulation along with higher dpph scavenging capacity. likewise, marssonina apple blotch caused by fungus diplocarpon mali decreased by melatonin treatment at 0.1 mm which is caused by higher h2o2 accumulation leading to enhancing pathogenesis related (pr) proteins accumulation such as peroxidase, chitinase and b-1,3-glucanase, and triggering phenylpropanoid pathway by enhancing phenylalanine ammonia lyase (pal) enzyme activity (yin et al., 2013). it has been reported that the attenuating of postharvest physiological deterioration in cassava roots by melatonin treatment, obtained by lower h2o2 accumulation as a result of increasing antioxidant enzymes; sod, cat and gr activities causing higher membrane integrity indicated by lower malondialdehyde (mda) accumulation (ma et al., 2016). gao et al. (2016) reported that lower o2 and h2o2 accumulation in melatonin treated peach fruits resulted from higher antioxidant enzymes sod, cat, apx activities, concurrent with lower lipoxygenase (lox) enzyme activity leading to higher membrane integrity indicated by lower mda accumulation. in addition to antifungal and antioxidant activities, melatonin is useful in increasing postharvest sensory and nutritional quality of fresh produces (meng et al., 2015; cao et al., 2016; gao et al., 2016; liu et al., 2016; ma et al., 2016). it has been reported that preveraison melatonin-treated grape berries showed higher endogenous melatonin accumulation, which not only enhances berry size and weight, indicated by higher sugars accumulation and higher endogenous hormones ga/aba ratio, but also enhances synchronicity of berry ripening (meng et al., 2015). it has been observed that preharvest melatonin-treated tomato fruits showed higher fruits weight caused by higher sugars accumulation, as well as higher organic acids accumulation results in tomato fruits with favorable flavor. moreover, higher lycopene and ascorbic acid contents were observed in preharvest melatonin-treated tomato fruits (liu et al., 2016). since there is a lack of knowledge about the influence of melatonin on chilling tolerance of tomato fruit during low temperature storage, the present work was initiated to determine the efficacy of postharvest melatonin treatment on chilling demonstrations and enhanced fruits visual and nutritional qualities through augmenting antioxidant capacity of tomato fruits frequently encountered under cold storage. 2. materials and methods fruit and treatment tomato fruit (lycopersicon esculentum mill. cv banemi) were harvested at the mature green stage (i.e., liquefying locular tissue, seeds not cut with a knife) (saltveit, 1991) from a local producer in mohammad shahr, karaj (iran) and then immediately transported to karaj agricultural engineering and engineering research institute laboratory. the green stage of maturity with homogeneous size and randomly allotted into three groups (100 fruits per grop) for treatment in triplicate by dipping of fruits at 0 (control), 50, 100 and 200 µm melatonin solution for azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage 301 5 min at 20°c. the selected concentrations were based on published effects of these compounds on peach, strawberries and cherry tomatoes (sun et al., 2015; cao et al., 2016; soleimani aghdam and rezapour fard, 2017). following immersion, the fruits were dried for 1 h at room temperature. the tomato fruits were then put in plastic baskets, covered with a perforated plastic bag to retard weight loss and stored at 5°c with 80-85% relative humidity for 4 weeks. the seven-day intervals during storage at 5°c followed by shelf life at 20°c for 1 and 3 days, the development of chilling injury and ripening characteristics as well as enhanced fruits nutritional quality through augmenting antioxidant capacity were measured, respectively (ding et al., 2002). measurements of chilling injury and ripening characteristics chilling injury (ci) of fruits was evaluated at 20°c for 1 day after the 7-, 1421 or 28-day cold-storage periods. symptoms of tomato fruit chilling injury were manifested as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (wang, 1993). the severity of the symptoms was assessed visually according to the following four-stage scale: 0= no pitting; 1= pitting covering <25% of the fruit surface; 2= pitting covering <50%, but >25% of surface; 3= pitting covering <75%, but >50% of surface and 4= pitting covering >75% of surface. the average extent of chilling-injury damage was expressed as a chillinginjury (ci) index, which was calculated using the following formula: ci index (%) = {[(ci level) × (number of fruit at the ci level)]/(total number of fruits) × 4} × 100. for determining the effect of different treatments on ripening, fruits following 28 days storage, were incubated in diffused light at 20°c for 3 days to full red color development (ding et al . , 2002). measurement of full red color development in terms of carotenoids accumulation was conducted. lycopene and β-carotene were determined by the method described by nagata and yamashita (1992). the amount of 0.1 g of fruit tissue was mixed with 20 ml of hexane:acetone solution (3:2). an aliquot was taken from the supernatant and measured at 453, 505, 645, and 663 nm in a spectrophotometer. the content of lycopene and β-carotene was estimated using the following equations: lycopene = -0.0458 a 663 + 0.204 a 645 + 0.372 a 505 0.0806 a 453 β-carotene = -0.216 a 663 1.220 a 645 + 0.304 a 505 + 0.452 a 453 the results were expressed in milligrams per 100 g fresh weight (mg 100 g-1 fw). measurements of ion leakage and malondialdehyde content ion leakage was measured at 20̊°c for 3 days after the 7-, 14-, 21or 28day cold-storage period according to the method of zhao et al. (2009). 3 mm thick of mesocarp tissues were excised from equator part of 5 fruits. disks were put into aqueous 0.1 m mannitol and shaken at 100 cycles/min for 2 h. the conductivity of the solution (l1) was measured with a conductivity meter. solutions were boiled for 10 min and then cooled to 20°c. the conductivity of tissues (l2) was measured. ion leakage was calculated as the ratio of l1 to l2. malondialdehyde (mda) content was measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period using the thiobarbituric acid method described by zhao et al. (2009) with modification. absorbance at 532 nm was recorded and corrected for nonspecific absorbance at 600 nm. the amount of mda expressed as μmol mda per gram of pulp. measurement of proline content proline content was measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period using the acid ninhydrin method described by shan et al. (2007). proline in tissues was extracted with 30 ml l-1 sulfosalicylic acid at 100°c for 10 min with shaking. the extract was mixed with an equal volume of glacial acetic acid and acid ninhydrin reagent and boiled for 30 min. after cooling, the reaction mix was partitioned against toluene and the absorbance of the organic phase was recorded at 520 nm. the resulting values were compared with a standard curve constructed using known amounts of proline and expressed as μg proline g−1 fresh weight (fw). measurements of o2 production rate and h2o2 content the o2 production rate and h2o2 content were measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period. o2 production was measured using the method of elstner (1976) with modification. 4 g of fruit tissue was homogenized with 5 ml of 50 mm phosphate buffer (ph 7.8) and then centrifuged at 8000×g for 20 min at 4°c. the supernatant was used for the determination of o2 production and expressed as nmol g-1 fw min-1. for h2o2 measurement, 2 g of fruit tissue was homogenized with 5 ml of cold acetone and then centrifuged for 15 min at 8000×g at 4°c, the supernatant was collected immediately for h2o2 analysis according to the method of patterson et al. (1984). adv. hort. sci., 2018 32(3): 299-309 302 h2o2 content was expressed as nmol g-1 fw. enzyme extraction and analysis enzyme activities were measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period. 5 g of fruits tissue were homogenized with 50 mmol/l phosphate buffer (ph 7) containing 0.2 mmol/l edta and 2% pvp. the homogenate was centrifuged at 12,000×g for 20 min at 4°c and the supernatant was used. sod (ec 1.15.1.1) activity was determined according to giannopolitis and ries (1977) with modification. one unit of sod activity was defined as enzyme that caused 50% inhibition of nitro blue tetrazolium reduction by recording the absorbance at 560 nm. according to zhang et al. (2013) with modification, 1 unit of cat (ec 1.11.1.6) activity was defined as 0.01 decrease in absorbance at 240 nm per min. pod (ec 1.11.1.7) activity was determined according to maehly and chance (1954) with modification. one unit of pod was defined as 0.01 increase of absorbance at 470 nm as a result of guaiacol oxidation. statistical analysis experiments were performed using a completely randomized design. all statistical analyses were performed with sas 9.2 software package. data were analyzed by one-way analysis of variance (anova). mean comparisons were performed using hsd in tukey’s test for comparing treatment group at level of 1% (p<0.01) on three biological replicates. 3. results chilling injury and ripening characteristics the chilling injury (ci) symptoms were expressed on control group as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (wang, 1993), only after 7-day of cold storage and following shelf life at 20°c for 1 and 3 days, respectively (fig. 1). no significant difference in ci was observed between the control and 50 μm melatonin-treated fruit. whereas, melatonin-treated groups with 100 or 200 μm underwent normal ripening at 20°c and only few visual chilling-injury symptoms were observed after 14 days storage at 5°c (fig. 1). fruits treated with 100 or 200 μm melatonin maintained the same quality as before chilling-temperature storage except for developing a slight yellow pigmentation, and the effect of the used formulas increased with increasing their concentrations (fig. 2a). the results indicate that a 14-day storage was the maximum that could be tolerated by untreated mature green fruit. in this experiment, treatments with higher concentrations (100 or 200 μm) of melatonin were more effective in protecting against chilling injury than lower concentration (50 μm). for examining the effect of melatonin treatment on color development of fruit after cold storage, mature green tomatoes were transferred to 20°c for 3 days for ripening. treatment with 100 or 200 μm melatonin, prior to 5°c storage, was effective at alleviating chilling injury; this treatment category resulted in normally fruit ripening and uniform red color development caused by significantly (p<0.01) more lycopene and β-carotene accumulations, and the effect of the used formulas on fruit ripening and eventually visual quality increased with increasing their concentrations (fig. 2). however, control and melatonin-treated group with 50 µm failed to develop the normal red color, with lower lycopene and βcarotene values, demonstrated by large green patches or blotchy yellow areas. interaction effects and time of storage had no meaningful influence on these traits. ion leakage, malondialdehyde content ion leakage and mda, as a consequence of membrane damage, are credible parameters for ci development and degree for postharvest tomato fruit fig. 1 chilling injury (ci) index (%) of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 1 day of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage 303 (zhao et al., 2009). in this experiment, significantly the highest ion leakage was detected in control group (p<0.01) (fig. 3a). however, no significant differences were statistically found in the ion leakage incidence between melatonin-treated groups with 100 or 200 μm. as shown in figure 2b, mda content showed a similar pattern of change during storage. mda content was significantly (p<0.01) lower in melatonin-treated groups with 100 or 200 μm compared with control at the same time of cold storage, and the highest level observed about 14 to 21 days (fig. 3b). proline content there was a peak of proline content appearing in 14-day in all groups, which suggested that low temperature induced the proline synthesis mechanism in fruits (zhao et al., 2009). however, proline accumulation was about 2 times high in the melatonin-treated groups with 100 and 200 µm compared to control from 14-day to the end of storage period, and the effect of the used formulas on proline content increased with increasing their concentrations (fig. 4). no significant difference in proline content was observed between the control and 50 μm melatonintreated fruit. fig. 2 fruit ripening uniformity (%), lycopene (a) and β-carotene (b) contents of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. fig. 3 ion leakage (%) (a) and mda (b) content of tomato fruits treated with 100 and 200 µm melatonin during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 304 adv. hort. sci., 2018 32(3): 299-309 o2 production and h2o2 content in figure 5, the measured levels of o2 and h2o2 were shown as an influence of low temperature to ros generation in fruits exposure to chilling stress. contents of o2 and h2o2 remained relatively unchanged in control and melatonin-treated groups within the first 14 days of cold storage. thereafter, both o2 and h2o2 contents increased rapidly, treatment with 100 and 200 µm melatonin significantly (p<0.01) restrained the enhancement of o2 and h2o2 contents, and again the effect of the used formulas on o2 production rate and h2o2 content decreased with increasing their concentrations (fig. 4). no significant difference in o2 and h2o2 contents was observed between the control and 50 μm melatonintreated fruit. sod, cat, pod activities as depicted in figure 6a, the sod activity in both control and melatonin-treated groups steadily increased during storage, nonetheless significantly the highest sod activity was observed in melatonin treated groups with 200 and 100 µm throughout the storage, respectively (p<0.01). the changes of cat and pod activities in tomato fruit showed a similar pattern during the cold storage. the activities of both enzymes in control and melatonin-treated groups oscillatory increased with storage time. melatonin treatment significantly promoted the increases in activities of cat and pod, the activities of both enzymes were significantly higher (p<0.01) in these groups than those in control group during the whole storage (fig. 6). 4. discussion and conclusions little information is available on melatonin treatment of horticultural commodities, even though there are many reports suggesting that melatonin is an endogenous signaling molecule for the activation of certain plant defense responses and the onset of the tolerance has often been correlated with the accumulation of defense-related enzymes and compounds (zhang et al., 2014; arnao and hernándezfig. 4 prolin content of tomato fruits treated with 100 and 200 µm melatonin during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. fig. 5 o2 production (a) and h2o2 (b) content of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 305 azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage ruiz, 2015; liu et al., 2016). exogenous melatonin application has been shown to result in an improved chilling tolerance and reduced incidence of chilling injury in peach and strawberry fruits (cao et al., 2016; soleimani aghdam and rezapour fard, 2017). in this experiment, we found that melatonin treatment could effectively not only reduce development of surface pitting on the fruit and irregular ripening and full red color development (large green patches or blotchy yellow areas), the typical chilling injury symptoms in tomato fruit, but also enhance fruits nutritional quality. this indicates that postharvest treatment with melatonin increased chilling tolerance in tomato fruit. since melatonin treatment is easy to set up, inexpensive and safe, even if higher amounts are accumulated in the plant (tan et al., 2012), it could be a functional method to decrease chilling injury, maintain quality and prolong shelf life of tomato fruit. carotenoids, highly characteristic phytochemicals, known to be potent ros scavengers and antioxidants, act as a cell proliferation inhibitor and hindering of cancer cell growing (tijskens and evelo, 1994; levi et al., 2001; giovannucci et al., 2002; stahl and sies, 2005). during maturation/ripening, the green pigment chlorophyll degrades and carotenoids are synthesized. carotenoids, particularly lycopene and β-carotene, represent the primary components of ripe fruit pigmentation in tomato pericarp and are responsible for the characteristic color of ripe tomatoes, conferring deep red and orange colors, respectively. these carotenoids largely influence flavor and nutritional qualities as well as commercial value and enhances consumer acceptance of fresh tomato fruit (tijskens and evelo, 1994). in this study, higher accumulation of lycopene and β-carotene in melatonin treated groups with 100 and 200 µm not only contributed to alleviate chilling injury to fruit, but also lead to normally fruit ripening with uniform red color development (panels b and c of fig. 2). it has been reported that in tomatoes, the contents of lycopene and β-carotene increase from the green to the fully ripe stage (fraser et al., 1994). melatonin may affect directly or indirectly other carotenoid genes and/or enzymes in tomato fruit. this could be the case for example of lycopene cyclases, which is responsible for the formation of β-carotene from lycopene, which its accumulation is a ripening-related event in tomato (giovannoni, 2001). the higher levels of these compounds in melatonin-treated red ripe fruits may be associated with a general acceleration in ripening and with some of the associated transcriptional events, leading to the color change of tomato fruit (guo, 2015; sun et al., 2015). therefore, the improved capability of full red color development in chilling-faced melatonin-treated group is one of the most important outcome of this study for the quality of tomato. proline, an important amino acid, has been considered as a cellular osmotic regulator, protein stabilizer, free-radical scavenger, and lipid peroxidation inhibitor in plant (sun et al., 2015). the elevated level fig. 6 superoxide dismutase (a), catalase (b) and peroxidase activities (c) of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 306 adv. hort. sci., 2018 32(3): 299-309 of proline found to be associated with improved cold tolerance in chilling-sensitive plants (zhao et al., 2009; shang et al., 2011; zhang et al., 2013; cao et al., 2016). our findings are in agreement with the above reports (zhang et al., 2010), because a significantly (p<0.01) higher content of proline was observed in melatonin-treated tomato fruits with 100 or 200 µm during the whole storage period along with the reduced ci incidence (fig. 4). cao et al. (2016) reported that higher transcripts of ppp5cs and ppoa were observed in melatonin-treated peach fruits which provokes proline accumulation. zhao et al. (2009) claimed that proline levels in a tissue may be an effective indicator for ci analysis in postharvest tomato fruits. the reduction of cell energy and/or induction of alterations in membrane integrity are occurred in chilling-sensitive horticultural commodities at low temperatures. reducing scavenging potency through such factors as chilling-related inactivation of antioxidants and/or obstructed antioxidant turnover may result in the enhanced ros generation. chilling temperatures destroy the balance between ros formation and defense mechanisms which cause oxidatively chilling injury and consequent cellular damage (hodges et al., 2004). it is figured that antioxidant enzymes, sod, cat, and pod are the primary enzymatic scavenging mechanism of ros that contribute to attenuate chilling injury to fruit (sala and lafuente, 2000; mondal et al., 2004; ding et al., 2007; imahori et al., 2008). thus, this balance between the generation and scavenging of ros is crucial to cell survival during cold storage and is thought to be a major mechanism of resistance to chilling stress. it has been reported that in harvested commodities enhanced enzymatic antioxidant activities result in the improved chilling tolerance. a higher antioxidant enzyme activity was indicated in the chilling-tolerant mandarins compared with the chilling sensitive cultivars (sala, 1998). in many other studies, enhancement of antioxidant enzyme activity through a number of postharvest treatments (e.g. heat shock, low temperature conditioning and superatmospheric oxygen treatment) provoked chilling tolerance and alleviated chilling injury to fruit (wang, 1995; sala and lafuente, 2000; zheng et al., 2008). neutralizing of the o2 by sod is the initial step of cell defense against free radicals (bowler et al., 1992). cat is one of the enzymes that protect cells against ros because it catalyzes the decomposition of h2o2 to form o2 and h2o2 (imahori et al., 2008). pod catalyzes h2o2 dependent oxidation of substrate (fu and huang, 2001). in the present work, the higher increases in activities of sod, cat, and pod concurrent with reduced o2 and h2o2 content in melatonintreated groups than those in control group were indicated (fig. 6). while, the levels of h2o2 and o2 significantly increased during the development of irreversible chilling injury symptoms surface pitting and irregular ripening and full color development in control group. treatment with melatonin significantly alleviated these chilling-induced damages and increased the activities of sod, cat, and pod under cold stress. the increased sod activity could enhance the ability of the fruit to dismutate superoxide radicals, while the increases in cat and pod activities would contribute to the stronger omitting of hydrogen peroxide (lukatkin, 2002), which may give an explanation for the lower levels of o2 and h2o2 observed in melatonin-treated groups. these results suggest that effect of melatonin in reducing the incidence of chilling injury was correlated to enhanced enzymatic scavenging mechanism of ros. in melatonin-treated tomato fruits the continues functions of sod, cat, and pod may be associated with higher stress resistance and eventually extended shelf life. membrane lipid peroxidation may be one of the first events in the manifestation of ci, in which phase mda as a final product of polyunsaturated fatty acid oxidation was produced and damaged to cell membrane, resulted in ion leakage (lukatkin, 2002; imahori et al., 2008). as depicted in panels a and b of figure 3, the increase in ion leakage and mda from 14-day of storage period indicates that cold storage caused a distinct deterioration of membrane integrity and activation of lipid peroxidation in the non-treated control group, which could be attributed to the decreases in sod and cat activities as well as in antioxidant compounds including lycopene, βcarotene and proline. these reductions induced by chilling stress favor accumulation of o2 and h2o2, which can result in lipid peroxidation. ion leakage and mda content may be a reflection of ci development and fruit cold tolerance (zhao et al., 2009). furthermore, posmyk et al. (2005) reported that ion leakage intensity and mda content in a tissue can be a reliable indicators of the structural integrity of the membranes of plants exposed to low temperature. given to these results, prevention of mda accumulation and subsequent ion leakage by melatonin treatment could be related to a low degree of lipid peroxidation, which could result from the maintenance of high enzymatic and non-enzymatic antioxidants. it has been reported that melatonin efficiently con307 azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage tributes to membrane integrity maintenance, and in turn, alleviates symptoms and severity of ci (cao et al., 2016; soleimani aghdam and rezapour fard, 2017). treatment with melatonin attenuated chilling induced shrinkage and disruption of carrot cell plasma (lei et al., 2004). as a whole, the results of this study show that melatonin treatment can effectively enhance chilling tolerance and reduce chilling injury of 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wang c.y., 2008 transcript levels of antioxidative genes and oxygen radical scavenging enzyme activities in chilled zucchini squash in response to superatmospheric oxygen. postharvest biol technol., 47: 151-158. impaginato 525 adv. hort. sci., 2018 32(4): 525-534 doi: 10.13128/ahs-22424 diversity of plant growth promoting rhizobacteria of rhus tripartitus in arid soil of algeria (ahaggar) and their physiological properties under abiotic stresses a. benaissa 1, 3 (*), r. djebbar (1), a. abderrahmani (2) 1 laboratory of plant physiology, department of biology and physiology of organisms, faculty of biological sciences, university of sciences and technologies of houari boumediene el-alia, bp 16011 bab ezzouar, algiers, algeria. 2 laboratory of microbiology, department of cellular and molecular biology, faculty of biological sciences, university of sciences and technologies of houari boumediene el-alia, bp 16011 bab ezzouar, algiers, algeria. 3 laboratory of sciences and environment research, universitary center of amine elokkal elhadj moussa eg. akhamoukh, 11039 sersouf, tamanrasset, algeria. key words: abiotic stresses, ahaggar, plant growth promoting rhizobacteria, rhus tripartitus. abstract: plant growth promoting rhizobacteria (pgpr) associated with ucria (rhus tripartitus) represents a good alternative for including this crop in revegetation programs in arid area. in this study, 137 bacterial strains were isolated in tryptic soy agar medium (tsa) from six samples of ucria’s rhizospheric soil (ahaggar, algeria), based on colony characteristics, gram © reaction, oxidase and catalase tests. to evaluate their pgp activities and their physiological characteristics under stress environment, ten tests were made. sixty strains of 16 genera were selected for their pgpr abilities, which represent 43.79% to the total of rhizobacteria isolated. the maximum bacterial population were bacillus (35%). 71.66% of isolates were able to solubilize the phosphate, 31.66% were able to produce indole acid acetic (iaa), 58.33% were siderophore producers, 28,33% were able to produce cyanhydric acid (hcn) and 70% were able to grow without any source of nitrogen. indeed, pgpr strains have shown tolerance and/or resistance to several experimental environmental conditions. as a conclusion, the pgpr strains of ucria’s rhizosphere were shown a good potential for biofertilization and biocontrol of crops, and their tolerance to abiotic stresses is an interesting step to support their utilities. 1. introduction rhus tripartitus (called ucria, african sumac and tahounek in tamahaq) is an important medicinal plant belonging to the anacardiaceae fam(*) corresponding author: benaissa.asmaa@yahoo.fr citation: benaissa a., djebbar r., abderrhmani a., 2018 diversity of plant growth promoting rhizobacteria of rhus tripartitus in arid soil of algeria (ahaggar) and their physiological properties under abiotics stresses. adv. hort. sci., 32(4): 525-534 copyright: © 2018 benaissa a., djebbar r., abderrhmani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 december 2018 accepted for publication 27 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 525-534 526 ily. this species often grows in areas of marginal agricultural capacity. it encountered in algeria, in arid areas especially in the mountains of the ahaggar where the indigenous people (touaregs) use it to treat gastric disorders (chermat and gharzouli, 2015). this plant is also found in the north-eastern part of saudi arabia and in tunisia where it spreads in the center to the far southern part of the country. it is used in the arabian traditional medicine for centuries to treat cardiovascular and gastrointestinal disorders and inflammatory conditions (chetoui et al., 2013; shahat et al., 2016). the study of rhizosphere bacteria from medicinal plants is very important, as they are well known to have impact on plant growth and also produce industrially important metabolites and improve quality of medicinal products (bafana and lohiya, 2013). considerable numbers of studies were focused on the beneficial effects of bacterial species that colonize the rhizosphere of many plant species and proved their beneficial effects on plant growth, yield, and productivity as well as their role in the reduction of their susceptibility to diseases caused by phytopathogenic bacteria, fungi, viruses and nematodes and even against abiotic stresses. these bacteria have been called «plant growth promoting rhizobacteria» (kloepper et al., 2004; orhan et al., 2006; miransari, 2014; nadeem et al., 2014; gupta et al., 2015). these pgpr’s can enhance the plant growth by direct mechanisms such as the fixation of atmospheric nitrogen, the solubilization of minerals like phosphorus and iron, the production of siderophores and enzymes, the synthesis of phytohormones like the auxin, indole-3-acetic acid (iaa), cytokinins and gibberellins, their role in lowering of ethylene levels and the induction of systemic resistance. indirect mechanisms are used by pgpr to benefit the plant growth by the induction of the disease resistance by producing antibiotics or hydrogen cyanide, competition for nutrients, extracellular enzymes production and others (glick, 1995; vessey, 2003; adesemoye et al., 2009; saharan and nehra, 2011; saha et al., 2016). to the best of our knowledge, there are no studies conducted on the rhizospheric bacteria associated with rhus tripartitus, a medicinal plant that grows in ahaggar (algeria). we hypothesize, that this plant harbors a diverse group of rhizospheric bacteria that can help ucria to cope with harsh environmental conditions. so, the main objectives of this study were to characterize the isolated rhizobacteria associated with the rhizosphere of rhus tripartitus, their propreties as plant growth promoting bacteria and their capacities to tolerate abiotics stresses. 2. materials and methods sample collection, isolation and characterization of rhizobacteria six soil samples are taken from the rhizosphere of the wild ucria shrubs from ilaman region in tamanrasset, an arid area, which is located in the national culturel parc of ahaggar in the south of algeria (22° 49’ 59’’ north, 5° 19’ 59’’ east) during march, 2017. each soil sample was collected at a depth of 15 cm, around the root and placed in a sterile container. the samples collection was transported to the laboratory in an ice box set at 4°c. tenfold serial dilution of the samples was made by mixing the soil with sterile water, and plating on a tryptic soy agar medium (tsa). the plates were inverted and incubated, at 30°c for five days. the maximum of bacterial colonies present on plates were purified and characterized. the gram reaction, oxidase reaction and catalase test were performed as per standard procedure. in vitro screening and identification of plant growth promoting rhizobacteria the collected rhizobacterial isolates associated with rhus tripartitus were tested for their capacities to produce plant growth promoting effects. the pgpr were identified by using api galeries e20, ne20, chb, staph and nh (api, biomerieux sa, lyon, france). all the strains were preserved in nutrient broth added with 20% glycerol at -80°c. nitrogen fixation. the fixation of molecular nitrogen is tested on a free nitrogen medium. the bacterial isolates are inoculated on the plates and incubated at 25°c for 24-48 h. the growth on this medium after being transferred ten times in the same medium reflects the ability of bacteria to fix nitrogen (haahtela et al., 1983). production of hcn. the strains ability to produce hydro cyanic acid (hcn) is carried out according to the method of lorck (1948) on solid bennett agar amended with 4.4 g/l glycine is inoculated with a loop of the bacterial culture. 90 mm whatman paper are dipped in sodium picrate solution (0.5% picric acid and 2% sodium carbonate) for one minute and then placed underneath the petri plates lids. the plated were sealed with parafilm and incubated at 30°c for four days. the appearance of an orange to benaissa et al. pgpr of rhus tripartitus 527 red color indicates the production of hcn. solubilization of phosphates. qualitative phosphate solubilization activity was tested on nbrip (national botanical research institutes phosphate) medium by applying a spot of 20 μl of bacterial suspension on the surface of the agar and incubated at 30°c for 15 days (nautiyal, 1999). a clear halo zone around the colony is an indication of phosphate solubilization. the calculation of the solubilization index (s.i.) is carried out according to the formula developed by kumar and narula (1999): s.i. = diameter of the halo around the spot/diameter of the spot production of siderophores. the production of siderophores on solid medium is carried out qualitatively on chrome azurol s (cas) medium as described by schwyn and neilands (1987). the cas plates were prepared and divided into sectors and inoculated with bacterial culture spots (10 μl of 106 cfu/ml) and incubated at 25±2°c for 48-72 h. the development of an orange yellow halo around the colony was considered positive for the siderophore production. the change in color is due to the transfer of ferric ions from the cas to the siderophores. the calculation of the ratio (halo diameter/diameter of the bacterial colony) makes it possible to compare production differences between bacterial strains. production of indole acetic acid (iaa). the production of iaa was determined according to the method of holt et al. (1994). the principle is to inoculate the selected strains on the nutrient broth containing 0.1 g/l of l-tryptophan. the change of the solution color from yellow to pink or red when we add the reagent of salkowski (50 ml, 35% perchloric acid; 1 ml 0.5 fecl3) is an indication of positive result. screening of pgpr isolates for stress tolerance influence of salinity. to evaluate the strains ability of these strains to grow in salinity levels, different concentrations of nacl (1%, 5% and 8%). the salt were added to tsa medium in the liquid stage (infusion at 40°c) and deposited on the magnetic stirrer to dilute it. influence of ph and temperature. in order to test the ability of the bacteria to grow in alkaline and/or acidic environment, two media were prepared with the addition to 250 ml of tsa of 3 g of solid naoh 6.4 m and 1 ml of kcl 12 n to obtain a ph of 8.8 and 6 respectively. for studying the effect of incubation temperature on growth of the isolates, the bacterial cultures were grown on tsa medium and incubated at a variables temperatures (10, 20, 30, 40 and 50°c). tolerance of heavy metal. the sensitivity of the selected strains to copper (cu), zinc (zn), bromine (br), cyanide (cn), fluorine (f) and silicon (si) were tested. for this test, six tsa media of 250 ml were prepared with respectively three grams of solid kbr, caf2, sio2 and one gram of k3[fe(cn)6], znso4.7h2o and cuso4.5h2o. any growth on this media reflects the ability of strains to tolerate the heavy metal toxicity. antibiotics resistance. the selected strains were tested for their susceptibility to antibiotics on mullerhinton medium. it consists of bringing the germ into contact with disks of blotting paper impregnated with a given antibiotic at concentrations determined by the standardization of the antibiogram according to the clinical and laboratory standards institute (clsi), to determine the sensibility of this germ to antibiotics which allows classifying it in the category: r (resistant), s (sensible) or i (intermediate). ten different antibiotics were used: fosfomycin (fos) 50 µg, rifampin (ra) 5 µg, nalidixicid (na) 30 µg, spiramycin (sp)100 µg, 30 µg, novobiocin (nv) 30 µg, teicoplycin (tei) 30 µg, kanamycin (k) 30 µg and erythromycin (e) 15 µg (all from sigma chemical co., st. louis, mo.). 3. results in this study, we focused on the diversity of bacterial community of ucria’s rhizosphere and the evaluation of their plant growth promoting abilities, also under abiotic stresses. isolation and characterization of rhizobacteria one hundred and thirty seven (137) culturable bacteria were isolated from the rhizosphere of six healthy ucria plants using tsa medium. the rhizobacteria isolates showed a diversity of phenotypic and cultural characteristics of their colonies. infact, 54.19% of the isolated rhizobacteria were gram negative. 100% of strains showed positive test to catalase and variability to oxidase reaction (67.88% negative). screening and identification of pgpr biochimical characterization of pgpr isolates. the isolates were grouped into 16 genera of bacillus, adv. hort. sci., 2018 32(4): 525-534 528 pseudomonas, ewingella, staphylococcus, alcaligenes, micrococcus, kocuria, chryseomonas, chryseobacterium, cedecea, shigella, yersinia, providencia, acinetobacter, haemophilus and aeromonas based on cultural, morphological and biochemical characteristics. the maximum and minimum populations were bacillus (35%), and haemophilus and aeromonas with a percentage of 1.66% each (table 1). table 1 functional diversity of pgpr strains isolated from ucria’s rhizosphere sample no division/strains functions 1 gamma proteobacteria cedecea lapagei solubilization of phosphate, production of hcn and siderophores chryseomonas luteola solubilization of phosphate, production of siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production siderophores and nitrogen fixation firmicutes bacillus megaterium solubilization of phosphate, production of siderophores and nitrogen fixation bacillus megaterium solubilization of phosphate, production of aia and siderophores and nitrogen fixation bacillus circulans solubilization of phosphate, production of aia and siderophores, 2 gamma proteobacteria ewingella americana solubilization of phosphate, production of aia and siderophores chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation cedecea lapagei solubilization of phosphate, production of siderophores and nitrogen fixation cedecea lapagei solubilization of phosphate, production of aia and siderophores and nitrogen fixation firmicutes bacillus licheniformis solubilization of phosphate, production of aia and siderophores bacillus subtilis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of aia and siderophores and nitrogen fixation. bacillus licheniformis production of aia and siderophores actinobacteria kocuria varians production of aia 3 gamma proteobacteria chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation providencia rattgeri production of aia ewingella americana production of siderophores firmicutes bacillus subtilis production of hcn and fixation of azote bacillus non reactiv production of siderophores and fixation of azote bacillus licheniformis production of hcn and fixation of azote 4 gamma proteobacteria yersinia pestis solubilizationof phosphate, production of siderophores and nitrogen fixation haemophilus aphrophilus solubilization of phosphate, production of aia, and nitrogen fixation aeromonas salmonicida solubilization of phosphate, production of hcn and siderophores pseudomonas aeruginosa solubilization of phosphate, production of hcn and nitrogen fixation cedecea lapagei solubilization of phosphate, production of siderophores and nitrogen fixation acinetobacter colcoaticus solubilization of phosphate and production of aia, acinetobacter baumanii solubilization of phosphate, production of aia and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of hcn and siderophores. acinetobacter baumanii solubilization of phosphate and nitrogen fixation firmicutes staphylococcus lentus solubilization of phosphate and production of aia. staphylococcus lentus solubilization of phosphate, production of siderophores and nitrogen fixation actinobacteria micrococcus ssp solubilization of phosphate, production of siderophores and nitrogen fixation béta proteobacteria alcaligenes faecalis production of aia and nitrogen fixation 5 gamma proteobacteria chryseomonas luteola production of siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of hcn and aia. to be continued benaissa et al. pgpr of rhus tripartitus 529 plant growth promoting traits.the isolated rhizobacteria were screened for various pgp features responsible for plant growth promotion. the pgpr isolates represent 43.79% of the total of the rhizospheric bacteria. they belong to four divisions (gamma-proteobacteria, firmicutes, actino-bacteria and béta-proteobacteria). the strains mostly belonging to the gamma-proteobacteria, most of them affiliated to the enterobacteriaceae. according to the figure 1, species of gamma proteobacteria division were predominant with 45% among the isolats of pgpr of ucria behind firmicutes with 38.33%. infact, the species cedecea lapagei, chryseomonas luteola and ewingella americana were the most representative pgpr of gammaproteobacteria division. the species chryseomonas luteola found in five samples of six rhizospheric soils (table 1). different combinations of pgp effects have been found, 18.33% strains have been able to produce up to 4 pgp effects against 40% were able to produce 3 pgp effects, 25% showed 2 traits of pgp and 16.66% were able to produce only one pgp effect. in fact, the genera bacillus, ewingella, alcaligenes, the rhizobacteria were identified through six samples and five plant growth promoting treatments: solubilization of phosphate, production of cyanhydric acid, production of indol-acid-acetic, fixation of nitrogen and production of siderophores. fig. 1 classification of rhizospheric bacteria of ucria on the pgp traits. the main bacteria divisions of plant growth promoting rhizobacteria from the rhizosphere of ucria. sample no division/strains functions 5 firmicutes bacillus circulans solubilization of phosphate, production of hcn and siderophores and nitrogen fixation bacillus circulans solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus subtilis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of hcn and siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate and nitrogen fixation bacillus circulans production of hcn bacteroidetes chryseobacterium meningosepticum nitrogen fixation actinobacteria kocuria varians nitrogen fixation micrococcus ssp solubilization of phosphate, production of hcn and siderophores and nitrogen fixation 6 gamma proteobacteria ewingella americana production of aia and nitrogen fixation shigella spp solubilization of phosphate and nitrogen fixation pseudomonas aeruginosa solubilization of phosphate, production of siderophores and nitrogen fixation ewingella americana solubilization of phosphate, production of siderophores and hcn and nitrogen fixation ewingella americana solubilization of phosphate and nitrogen fixation providencia rattgeri production of aia firmicutes bacillus subtilis solubilization of phosphate and nitrogen fixation bacillus licheniformis solubilizationof phosphate, production of hcn and siderophores and nitrogen fixation bacillus subtilis solubilization of phosphate, production of hcn and nitrogen fixation bacillus non reactiv nitrogen fixation actinobacteria micrococcus ssp production of hcn and siderophores and nitrogen fixation béta proteobacteria alcaligenes faecalis solubilization of phosphatase, production of hcn and siderophores alcaligenes faecalis production of hcn table 1 functional diversity of pgpr strains isolated from ucria’s rhizosphere continued chryseomonas and cedecea have showed positive screening for all the pgp traits, according to the table 2. screening and assessment of phosphate solublizers and iaa producers. among the total isolates screened for phosphate solublization, 71.66% were able to solubilize inorganic phosphate (fig. 2) and were identified as potential phosphate solubilizing 530 adv. hort. sci., 2018 32(4): 525-534 bacteria that showed a clear halo zone around the colonies on nbrip’s agar plates amended with bromophenol blue. 20 isolates out of total rhizospheric flora had the capacity to produce were iaa in the presence of l-tryptophane. molecular nitrogen fixation. the ability of the isolates to grow on n-free medium indicated positive results for nitrogen fixation. in fact 70% of the isolates were able to grow even being transferred ten times in this medium which indicated their capacity to fix molecular nitrogen (fig. 2). siderophore and hcn production. siderophore production was registered at 58.33% of the strains based on the appearance of a halo zone of yellow orange color around the colony inoculated on casagar plates. seventeen isolates were positive for hcn production (fig. 2). physiological properties of pgpr isolates under abiotic stresses. the isolated bacteria were tested for their ability to tolerate abnormal growth conditions after incubation in a wide range of salt, ph and temperature stress condition, heavy metal toxicity and antibiotics. influence of salinity. according to the figure 3, the most percentages of strains which can grow in the 1% and 5% salinity are respectively 96.66% and 81.66%. in addition, only 16.66% of strains can tolerate % of salinity. influence of ph and temperature. the rhizobacterial isolates seems to tolerate the alkalinity better than the acidity with 66.66% and 46.66% respectively. however, these isolates could grow up to higher temperature of 50°c with 85%, which means that the temperature hadn’t a remarkable effect on bacterial growth (fig. 3). tolerance to heavy metal. depending on the metal tested and the species, the tolerance to heavy metals is different. in fact, we recorded a very good tolerance to silicon and bromine with respectively 80% and 78.33%. 55% of our strains have well tolerated fluoride followed by 30% for cyanide. the lowest percentages recorded were for copper and zinc with 13.33% and 3.33%, respectively. these results showed that heavy metals affected the bacterial growth (fig. 4). table 2 plant growth promotion activities of rhizobacteria genera isolated from ucria’s rhizosphere genera n 2 fe2 hcn aia po 2 bacillus + + + + + pseudomonas + + + + ewingella + + + + + staphylococcus + + + + alcaligenes + + + + + micrococcus + + + + kocuria + + chryseomonas + + + + + chryseobacterium + cedecea + + + + + shigella + + yersinia + + + providencia + acinetobacter + + + haemophilus + + + aeromonas + + + n2 = nitrogen fixation, fe2= siderophore production, hcn production, aia and po= phosphate solubilisation. fig. 2 siderophores production, phosphate solubilization, fixation of nitrogen, hcn and aia productions of the rhizobacteria isolates of ucria (ahaggar). fig. 3 estimation of tolerance of pgpr strains to alkalinity, acidity, salinity and temperature. fig. 4 estimation of tolerance of pgpr strains to heavy metals toxicity. br= bromine, zn= zinc, si= silicon, f= fluorine, cn= cyanide, cu= copper. 531 benaissa et al. pgpr of rhus tripartitus antibiotics resistance. the collection of pgpr isolates was tested for susceptibility to 10 antimicrobials. high frequency of resistance was observed for métronidazole (mt) followed by amoxyclav (amc) with respectively 93.33% and 91.66% of strains. the minimum resistance was recorded for fosfomycin (fos) with 16.66% (fig. 5). 4. discussion and conclusions the diversity of native bacterial species in the southern algerian soils remains largely unknown, especially for the rhizosphere of rhus tripartitus. most of the rhizobacteria isolated from ucria’s rhizospheric soils have been dominated by gram-negative (54.19%) and all were catalase positif. these results are in-line with studies realized on rhizosphere of brassica campestris (poonguzhali et al., 2006) and eragrostis tef zucc. trotter (woyessa and assefa, 2011). plant growth promoting rhizobacteria (pgpr) represent a diverse range of soil bacteria that stimulate the growth of their host when grown in association. such rhizosphere microbes benefit by utilization of metabolites secreted by plant roots as a nutrient for their growth and promote plant growth through more than one mechanism, including production of phytohormones and biocontrol of plant pathogens (rana et al., 2011). this present study showed a high diversity among the isolates of these species under different combination of pgp effects. the most often genera identified was bacillus (35%) with a high diversity of species, such as bacillus megaterium, b. licheniformis, b. subtilis, b. circulans and b. non-reactiv. pgpr of the genera bacillus have been reported in many studies (trivedi and pandey 2008; zou et al., 2010; liang et al., 2011; woyessa and assefa, 2011; nadeem et al., 2012; mishra et al., 2014; susilowati et al., 2015). the second pgpr group found throughout this study was belonging to species chryseomonas luteola (11.6%) and ewingella americana (8.3%). the latter was able to increase the growth of pipper and spinach (hou and oluranti, 2013). the third and last pgpr group (45.1%) belonged to the genera cedecea, providencia, yersinia, heomophilus, aeromonas, acinetobacter, micrococcus, alcaligenes, chryseobacterium, shigella, staphylococcus and pseudomonas. earliers studies showed that pseudomonas pgpr is a producer of hcn (castric, 1975), improving the availability of necessary nutrient (islam et al., 2014) and an agent of biocontrol (weller and thomashow, 1993). the genera acinetobacter promotes production of wheat, pea, chickpea, maize and barley through nitrogen fixation, siderophore production and mineral solubilization (gulati et al., 2009; sachdev et al., 2010). in constrat, cedecea have never shown in the litterature as pgpr potential and molecular analysis is necessary to confirm the identification. the industrialization of chemical fertilizers such phosphate and nitrogen has increased in the agricultural sector. these minerals are considered important limiting factors for many crops (ahmad et al., 2008). however, the aim of this study is also to evaluate the ability of rhizobacteria to promote the biodisponibility of n and p, in order to reduce of industrial fertilizers. in fact, most of ucria’s rhizobacteria could fix atmospheric nitrogen (70%) and 71.66% were able to solubilize inorganic phosphate in nbrip’s medium. similary, many studies shown that phosphorus-solubilizing microorganisms are ubiquitous in soils (chandra et al., 2007; tsavkelova et al., 2007; banerjee et al., 2010). pgpr can produce auxin-like compounds that increase the development of root system thus improving nutrient uptake by plants (voisard et al., 1989); however, out of total isolated rhizobacteria, 31.66% exhibited as producers of iaa in medium supplemented with l-tryptophan. the number of pgpr producers of hcn represent 28.33% of total isolates strains. these pgp traits have the capacity to enhance indirectly plant growth and protect them from phytopathogens (lugtenberg and dekker, 1999; shahat et al., 2016). rhizobacteria producing siderophores are of great importance for the plant because they make bioavaible iron. the siderophores decreases the metal bond and formation of free radicals in the roots zone, which prevented the degradation of iaa (yang et al., 2009). among the isolates of pgpr obtained in this study, 58.33% fig. 5 estimation of resistance of pgpr strains to antibiotics. adv. hort. sci., 2018 32(4): 525-534 532 are producers of siderophores. the production of siderophores in the rhizosphere increases the bacterial competition as well the root colonization (abrol et al., 1988). the rhizosphere is characterized by large environmental fluctuations, which may promote high diversity in the rhizosphere microbial community by maintaining high niche diversity. thus, microbial community diversity may be important especially in extreme condition, like high temperature, salinity or ph changes. under stress conditions, bacterial rhizosphere may promote the plant growth (cheikh and jones, 1994). in the present research, these pgpr isolates were traited under several environmental conditions. they have showed a good potential of tolerance for the previuosly conditions, which is a value, added to their beneficial effect. therefore, the aim of this work is not only to deal with the diversity of pgpr but also the selection of resistant strains at the most extreme conditions. the ucria’s rhizobacteria, were isolated from an arid area of ahaggar (algeria) and salinity is a natural feature of ecosystems in arid and semi-arid regions (curl and truelove, 1986). the 81.66% of pgpr studied exhibited as tolerant to the presence of 5% nacl and 85% could growth until 50°c. the variation on temperature is an important factor that can affect the hormonal balance of the plant (lovley, 1995). then, certain beneficial microorganisms can influence plants response to abiotic stresses like drought and high temperature (grover et al. , 2011). therefore, the necessity of discovering species able to grow under salt stress conditions and in a high temperature, are important to include them in revegetation system in arid area. the ph is one the obvious influencing factors of microbial activity and populations in soil (woyessa and assefa, 2011). most of these isolates (66.66%) can growth over alkaline ph and more than 46% of growth recorded in acidity condition, it suggests that there is a good potential to inoculate them over a range of wide ph. the growth of rhizobacteria at acidic ph values could be explained by their adaptation at arid soil. effectively, low rainfall can probably cause an increase in acidity of soil. on the other hand, an acidic environment of roots due to co2 and organic acid can be included in soil acidity (gururani et al., 2012). moreover, the pgpr isolates of this work, demonstrated a good tolerance in-vitro for heavy metals toxicity, with an average of 43.33% for chemicals forms of six metals. the accumulation in soil of heavy metals can perturbate the growth and the diversity of bacterial communities. many studies are shown the potential of application of pgpr in resistance and uptake of heavy metal by certain plants (lovley, 1995; yang et al., 2009; gururani et al., 2012). the ability to colonize roots and antibiotic resistance are other parameters needed to detect effective pgpr strains (siddiqui, 2005). effectively, the study of the sensitivity of rhizobacteria to antibiotics adds pgp potential. indeed, our strains have showed some resistance to the majority of antibiotics tested which can be involve high microbial competition in the rhizosphere. this resistance increased the chances of survival and colonization of the rhizospheric soil. the present study reflects the preliminary work done on the rhizosphere of rhus tripartitus in arid soils of algeria. indeed, the selection of pgpr strains, which can effectively grow under abiotics stresses conditions, can be used as promising biofertilizers and biocontroling of plants and 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it is traditionally used by the tuaregs (local inhabitants) of ahaggar (algeria). this saharomediterranean shrub is widespread from north africa to egypt (sahki and sahki, 2004). ferchichi (1999) has described the shrub as a very droughtresistant species characterized by abundant foliage throughout the year despite the soil’s moisture status, and it can be planted successfully on poor and marginal lands. on the geomorphological level of the algerian (*) corresponding author: benaissa.asmaa@yahoo.fr citation: benaissa a., djebbar r., boucelha l., 2019 eco-physiological and biochemical characterization of rhus tripartita (ucria) grande growing in algerian sahara under arid climate. adv. hort. sci., 33(1): 13-21 copyright: © 2019 benaissa a., djebbar r., boucelha l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 february 2018 accepted for publication 7 september 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 13-21 14 saharan environment, this plant belongs to the grouping of the ahaggar mountainous massifs where it grows on shallow soils. plant growth is strongly influenced by many biotic and abiotic factors. ecologists and physiologists have long been interested in the effects of environmental factors that lead to plant adaptation and distribution. therefore, phyto-ecophysiological studies are important to understand the species response in extreme environments and especially their valuation in these areas. by staying in extreme environments, the sahara is one of the driest and hottest deserts in the world (dutil, 1971). the desert fact is explained by extreme values of climatic parameters which constitute the essential factors of vegetation’s distribution and the grounds evolution (daoud and halitim, 1994). moreover, aridity is a natural selection force that influences plant adaptive strategies to water stress (sayed, 1998). hence different adaptive strategies of plants to aridity have been developed through considerable survival mechanisms. these strategies are divided into three general categories: drought escape, drought avoidance and dehydration tolerance (levitt, 1972; turner, 1979). drought escape is employed by plants under conditions where water limitation late in the growing season is likely, and it ensures that the plants can complete their life-cycle quickly during the brief period of favorable conditions. drought avoidance is a strategy for avoiding lower water status during drought by maintaining relatively high tissue water content due to reduced evaporatory water loss and an efficient water uptake (levitt, 1972). the third category, drought tolerance, is the ability of plants to withstand water deficit and maintain metabolism at low tissue water content (valliyodan and nguyen, 2008). in this strategy, osmotic adjustment, osmoprotection, antioxidation and scavenging defense system have been the most important physiochemical and biochemical bases responsible for drought tolerance. cell tissue and water conservation, antioxidant defense, cell membrane stability, compatible solutes and plant growth regulators mainly contributes in above said physiochemical and biochemical mechanisms. the three strategies are not mutually exclusive, and the same plant may use more than one strategy in order to adapt to periods of drought stress (nilsen and orcutt, 1996). therefore, to adapt with these natural constraints, plants have had to produce primary metabolites such as proteins and soluble sugars. this includes photosynthetic mechanisms, osmoregulation and antioxidant enzymes (liu et al., 2011; guo and gan, 2012). furthermore, water stress has a positive effect on the production and accumulation of secondary plant metabolites (ncib et al., 2018). their biosynthesis is often stimulated in response to biotic or abiotic stress (naczk and shahidi, 2004). the aim of this work is to study the adaptive physiological response of rhus tripartita to its naturally arid ecosystem (ahaggar, algeria). for this purpose, we will content to discuss our results in relation to water deficit and oxidative stress. 2. materials and methods presentation of study area localisation. the native plants of rhus tripartita (fig. 1) are located in the ilamane region (100 km away from tamanrasset city) which is situated in the ahaggar national and cultural park (22°49’59” north, 5°19’59” east). this park with an area of 4.500.000 ha, was created by the decree n. 87-231 of 03 november 1987. it is located in the mountainous center part of the volcanic ahaggar in the extreme algerian south (fig. 1). fig. 1 location of rhus tripartita growing in the rocky mountains of ilamane in the ahaggar national and cultural park. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 15 features edapho-climatic. before looking more specifically at plant physiology that interests us, it is necessary to present the main characteristics of the ahaggar. the bioclimate is arid, with very varied winter and summer temperatures and very low rainfall. the rains occur from may to september and usually increase in august. the annual rainfall varies between 180 mm and 250 mm. the thermal regime varies between the two very short winter and longer summer seasons. dubief (1959) noted the maximum average on the ground, in july, at 52°c towards 2700 m of altitude (assekrem) and estimates at 60°c this value at 1376 m (tamanrasset). these high temperatures condition the biology of plants in this environment. on the edaphic plane, soil encountered in this area is very little evolved because of a low rainfall. however, this park is marked by a geomorphological distinction (mountainous mass, wadis, hammadas, gueltates, plains and rocky ravines); this has resulted in a high heterogeneity in the soil composition (sandy, sandy-loamy, sandy-clayey, shallow and rocky). therefore, in the ilamane region, edaphic variability is a result of shallow skeletal soils that characterize this area. determination of physiological parameters in order to study the rhus tripartita physiology and biochemistry characteristics in relation to its arid environment, different measurements of primary and secondary metabolites are performed. all analyzes were performed on three replicates of six shrubs naturally growing in this area. the sampling was conducted during december 2017 (winter season). therefore, we consider that 6 individuals are largely sufficient given the physiognomic homogeneity of the population. relative water content (rwc) the rwc measurement is an old method (slatyer, 1967) which is currently widely used to estimate the plant water status and in particular water deficit. a 12 mm leaf disc taken from the second leaf by means of a punch is weighed directly (weight of the fresh vegetable material, wfvm). it is then placed in the refrigerator in a test tube containing distilled water for 24 hours and weighed to obtain the turgor weight (tw). the fragments are finally placed in an oven at 80°c for 48 hours and then weighed to obtain the weight of dry vegetable matter (wdvm). the rwc is calculated according to the following equation: rwc (%) = (wfvm wdvm) x 100/(tw wdvm) metabolites compositions for the analysis of sugars, proteins and proline, 100 mg of fresh material from each repeat was used. total hydro-soluble protein. soluble proteins are assayed according to bradford method (bradford, 1976). this method is a colorimetric assay based on the color change of coomassie blue after binding to aromatic amino acids (tryptophan, tyrosine and phenylalanine) and the hydrophobic residues of amino acids present in proteins. proline free foliar. the technique used for proline determination is that of troll and lindsley (1955) modified by magné and larher (1992). it is based on the ability of proline to react in acidic and hot conditions with ninhydrin (revealing aromatic amino acids including proline) to give a pink compound soluble in organic solvents such as toluene. ethanol soluble sugars. the soluble sugars assay method is based on the technique of mccready et al. (1950). in the presence of 91% sulfuric acid (h2so4) and hot conditions, the oses produce furfural derivatives that react with the anthrone to give a bluegreen compound. photosynthetic pigments. the assay was performed by colorimetric method; the pigments being naturally colored. to do this, read the od of the samples using the spectrophotometer previously calibrated with 80% acetone (corresponding to od= 0). the od readings were carried out at wavelengths 647 and 663 nm for chlorophylls and 470 nm for carotenoids. the concentrations were determined according to lichtenthaler (1987). antioxidant system total anti-oxidant capacity (cat). the total antioxidant capacity of rhus tripartita leaf was estimated using the method described by prieto et al. (1999). the amount of 0.1 ml of methanolic extract (2 g of dry matter in 10 ml of methanol) was mixed with 1 ml of molybdate reagent (0.6 m sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the absorbance was measured at 695 nm after incubation in boiling water bath about 90 min. the total antioxidant capacity was expressed as the milligramme gallic acid equivalent per gram of dry matter (mg gae/g dm). lipid peroxidation. lipid peroxidation is estimated according to the method of cakmak and horst (1991) which consists in grinding 100 mg of fresh plant material in 1 ml of 1% trichloroacetic acid (tca) foladv. hort. sci., 2019 33(1): 13-21 16 lowed by centrifugation at 12000 rpm/20 min. then, 1 ml of thiobarbituric acid (tba) at 0.5% (prepared in 20% tca) is added to 500 μl of extract and incubated with marie-bath at 95°c/30 min. after cooling, the optical densities (od) reading is carried out at 532 and 600 nm. the mda measurement content is calculated using its molar extinction coefficient (ƹ= 155 mmol/l cm). electrolyte leakage. electrolyte leakage (el) was estimated by measuring the electrolytic conductivity according to modified method of pike et al. (1998). this technique consists in placing ten foliar disks 12 mm in diameter in 20 ml of distilled water. a first measurement of the conductivity (ec) is carried out after stirring for 3 hours. then, a second measurement of the conductivity (et) is conducted, after marie-bath heating (95°c) for one hour. electrolyte leakage (%) is determined by the ratio (ec/et). total flavonoid content. total flavonoid content was estimated using the method reported by dewanto et al. (2002). it consists to mix 250 μl of vegetable methanolic extract with 25 μl of 5% nano2, added with 150 μl of alcl3 (2%). after 5 min, 0.5 ml of 1m naoh is added to the solution. after 10 min of incubation, the absorbance was measured at 510 nm. statistical analysis the data were subjected to statistical analysis using the microsoft excel 2010 program. all values of biochemical compounds and secondary metabolites are the mean ± se (standard error) of three replicates of a single sample. unidirectional analysis of variance (anova) was used and differences between individual means were considered significant at p<0.05. 3. results and discussion aridity depends on several climatic factors (temperature, wind, radiations) and above all on evaporation, an essential factor to calculate the water deficit. therefore, a rocky environment such as that of our study site, expresses accentuated drought effects because the soil scarcity. moreover, relatively rapid soil drought after rains causes water stress even during the rainy season (pimienta-barrios et al., 2001). to this end, we will discuss our results in relation to the influence of hydric and oxidative stress as the main parameters of aridity. therefore, plant eco-physiological study depends on several criteria, depending on environmental changes and their adaptation to them. indeed, plant survival in arid environments depends on different coping mechanisms. in this work, we have analyzed the variation of physiological parameters of rhus tripartita in relation to the arid climate of ahaggar. these parameters are often measured to study the different plants responses to abiotic stress. relative water content the low water availability can cause tissue dehydration, for this purpose, plant can control its hydric potential to cope with these high temperatures of arid zones. therefore, the relative water content of the plant gave a high value ranging from 54.79% to 81.55% (fig. 2). the highest content is recorded in shrub n. 5 with a significant difference compared to the same species shrubs. poole and miller (1975) reported that species of the same genus: rhus ovata, rhus laurina and rhus interguifolia; showed high water potential in water-deficit conditions. metabolite content in extreme temperatures, the plant can produce metabolites to protect itself from harmful and damaging effects. the total protein assay of r. tripartita showed levels that ranged from 20.03 mg/g in shrub n.3 to 33.76 mg/g in shrub n. 1 (fig. 3), which represent fairly high levels compared to periploca angustifolia labill, a shrub growing in tunisian arid areas with a rate of 5.204 mg/g dm (dghim et al., 2015). however, this amount is twice as high as that of the same species collected in libya (10.1 mg/g dm) (le houérou, 1991) and four times higher than that found in other species of the same genus such as rhus lancea (7.79 mg/g ms) (aganga and mosase, fig. 2 relative water content (rwc%) of rhus tripartita leaves according to climatic aridity conditions. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 17 2001) but it is one and a half times higher in other forage species: medicago sativa (19.4 mg/g ms) (le houérou, 1991). on the other hand, proline is an amino acid whose rate increases proportionally more rapidly than other amino acids in plants under water stress. it has been suggested as a study parameter for the selection of drought-resistant plants (bates et al., 1973). the proline level recorded in r.tripartita showed a significant difference (p<0.05) between the six shrubs of the same species with an interval of 212.45 µg/g 851.28 µg/g (fig. 3). plants tolerant to stress, have relative stability or low accumulation of proline compared to sensitive plants (lemzeri, 2006). according to dix and pearce (1981), the proline accumulation is not an adaptation reaction to stress, but rather a sign of metabolic disturbance. alternatively, proline may confer a protective effect on the plant by induction of stress-protecting proteins (vinocur and altman, 2005). however, osmotic stress can produce harmful effects in the plant’s cell compartments. a wide range of metabolites can intervene to avoid these effects including a variety of sugars and alcoholic sugars such as mannitol and trehalose (vinocur and altman, 2005). the study of sugars compound in rhus tripartita leaves was relatively high (938.93 µg/g) (fig. 3). several studies have investigated the soluble sugars accumulation in several stressful species (garg et al., 2002; penna, 2003; silva and arrabac, 2004). ahaggar is a region known for its continuous sunshine all year round. in the presence of strong radiation, several plants use protective mechanisms to reduce the absorption of solar radiation (harrison et al., 2010). the study of rhus tripartita photosynthetic pigments leaves showed results of 28.1 µg/g, 31.24 µg/g and 53.56 µg/g for chlorophyll a, b and total chlorophyll respectively (table 1). similar to those recorded in rhus typhina under water stress conditions (liping, 2007), these results indicate that dry conditions do not have significant effects on the degradation of photosynthetic pigments, which explains the dark green colour of the leaves. nevertheless, photosynthesis of plants in arid zones that believe under permanent conditions of water deficiency; are subject to photoinhibition risk (voronin et al., 2003). furthermore, the amount of photosynthetic pigments in the leaves of these plants has been shown to be relatively low (valladares and sanchez-gomez, 2006) which contradicts the findings of this study. the water stress is considered one of the environmental factors limiting photosynthesis, therefore plant growth (ozturk et al., 2010). fig. 3 soluble sugar content (μg/g), total proteins content (mg/g) and proline content (μg/g) in rhus tripartita leaves according to climatic aridity conditions. shrub chlorophyll a chlorophyll b total chlorophyll carotenoids shrub n° 1 17.367 ± 0.284 24.800 ± 0.527 41.300 ± 0.584 8.480 ± 0,006 shrub n° 2 23.600 ± 0.156 29.967 ± 0.295 53.560 ± 0.383 8.022 ± 0.025 shrub n° 3 24.133 ± 0.157 18.880 ± 0.271 43.067 ± 0.415 5.797 ± 0.017 shrub n° 4 24.967 ± 0.151 31.243 ± 0.564 56.473 ± 0.378 11.237 ± 0.079 shrub n° 5 24.033 ± 0.390 20.783 ± 0.533 44.577 ± 0.443 6.619 ± 0.089 shrub n° 6 28.100 ± 0.340 21.203 ± 0.363 49.503 ± 0.257 6.841 ± 0.039 table 1 content of photosynthetic pigments (µg/g) in the fresh leaves of rhus tripartitus (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) 18 adv. hort. sci., 2019 33(1): 13-21 antioxidant system in this study, the oxidative stress effect is evaluated through the quantification of mda and the electrolyte leakage since secondary aldehyde products from lipid peroxidation are generally considered to result from oxidative stress (del rio et al., 2005). the results presented in figure n. 10 showed that the mda content in the shrub is significantly higher (7.007 nmol/g) than other shrubs [5.165-5.673 nmol/g]. the electrolyte leakage was studied using conductivity measurement. it varies between 12.39 and 14.293 in the six shrubs of rhus tripartita as shown in figure n. 4. similary, in rhus typhina, high levels of mda have been recorded in water stress conditions (liping, 2007). therefore, mda is a degradation product of lipid peroxidation reactions that are formed during the attack of polyunsaturated lipids by reactive oxygen species (ros). however, this is the most widely used assay to characterize oxidative damage in plants (shulaev and oliver, 2006). moreover, high temperatures can produce metabolic disturbances based on reactive oxygen species and antioxidant systems. however, the present work showed a variability in total anti-oxidant capacity from one shrub to another and is within a range of 19 mg/g 100 mg/g (fig. 4). therefore, it is known that tac is mainly due to phenolic compound (tlili et al., 2014). on the other hand, the flavonoids constitute the main group of polyphenols, ubiquitous in plants, they are formed from aromatic amino acids (hernandez et al., 2009). they act as antioxidant molecules that ensure the binding of ros produced during stress and thus neutralize their effects before the manifestation of oxidative damage at the cellular level (lovdal et al., 2010). the determination of total flavonoids contents in rhus tripartita leaves gave result of 35.89 mg/g (fig. 4). this level is relatively similar to the same species under water stress conditions (39.2 µg/mg) (ncib et al., 2018) but higher than in other species of the same family such as rhus punjabensis (30.50 µg/mg) (tabassum et al., 2017). several studies have demonstrated the richness of rhus tripartita in flavonoids (mahjoub et al., 2007, 2010; tlili et al., 2014). therefore, flavonoid is considered as a phytochemical adaptation to the biotic and abiotic environment (dixon and paiva, 1995). in the same perspective, the carotenoids are soluble antioxidant lipids that play a very important role in abiotic stress tolerance (sieferman-harms, 1987; gill and tuteja, 2010). the carotenoids content found in our shrub leaves is relatively low (11.63 µg/g) (table 1) compared to another shrub of tunisian arid zones: periploca angustifolia labill (dghim et al., 2015). it is known that carotenoids have a protective role against photooxidation (ladygin et al., 2008) which may explain the adaptive power of this shrub. the relationship between aridity and the results found is certainly evident. it can be explained by the fig. 4 malondialdehyde content (nmol/g), total antioxidant capacity (mg/g), electrolyte leakage and flavonoid content (mg/g), of rhus tripartita leaves in relation to climatic aridity conditions. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 19 fact that the plant increases in both primary and secondary metabolite production under drought conditions. it should be noted that a difference in the metabolites composition has been recorded among different individuals of the same species. that said, not all shrubs had the same orientation towards the sunshine and some were grouped with the species myrtis nivellei and periploca laevigata. these two parameters can explain the difference between the results of the six individuals. 4. conclusions in summary, we presented a simple preliminary work on the physiological information of rhus tripartita in arid environments. therefore, our results have shown that this shrub presents an interesting adaptation to its climatic and edaphic environment. the approaches developed in this study certainly contribute to the understanding of shrub ecophysiology and can be improved plant productivity in arid regions. in conclusion, and according to classification of jenks and hasegawa (2005) of plants from desert and semi-arid zones, rhus tripartita can be considered as a xeromorphic plant, a species adapted to the desert that is not limited by water availability. references aganga a.a., mosase k.w., 2001 tannin content, nutritive value and dry matter digestibility of lonchocarpus capassa, zizyphus mucronata, sclerocarya birrea, kirkia acuminata and rhus lancea seeds. anim. feed sci. tech., 91: 107-113. bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress studies. plant soil, 39(1): 205-207. bradford m.m., 1976 a rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. anal. biochem., 72: 248-254. cakmak i., horst w.j., 1991 effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (glycine max). physiol. plant., 83(3): 463-468. daoud y., halitim a., 1994 irrigation et salinisation au sahara algérien. institut national de la recherché agronomique d’algérie (inraa), sécheresse, 5(3): 151160. del rio d., stewart a.j., pellegrini n., 2005 a review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. nutr. metab. cardiovasc. dis., 15(4): 316-328. dewanto v., wu x., adom k.k., liu r.h., 2002 thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. j. agricul. food chem., 50(10): 3010-3014. dghim f., bouaziz m., mezghani i., boukhris m., neffati m., 2015 laticifers identification and natural rubber characterization from the latex of periploca angustifolia labill. 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(ed.) plant genetics/genomics. volume 2. genetics and genomics of soybean. springer, new york, usa, pp. 406. vinocur b., altman a., 2005 recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. curr. opin. biotechnol., 16(2): 123132. voronin p.y., ivanova l.a., ronzhina d.a., ivanov l.a., anenkhonov o.a., black c.c., p’yankov v.i., 2003 structural and functional changes in the leaves of plants from steppe communities as affected by aridization of the eurasian climate. russian j. plant physiol., 50(5): 604-611. impaginato 143 adv. hort. sci., 2018 32(1): 143-147 doi: 10.13128/ahs-22715 comparative analysis of volatile compounds (potential aromatic ability) in the fruit of 15 olive italian cultivars c. taiti 1, m. redwan 1, e. marone 2 (*), g. atzori 1, e. azzarello 1, s. mancuso 1 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, via r. balzarini, 1, 64100 teramo, italy. key words: olive fruits, ptr-tof-ms, volatile organic compounds (vocs). abstract: virgin olive oils (voos) are characterized by peculiar flavors appreciated by the consumers all over the world. their organoleptic characteristics depend on the aromatic properties of the fruits of the different cultivars, which will originate the final products. vocs spectra of fifteen certified italian olive cultivars of the university of florence germplasm collection, chosen as their different geographical origin, diffusion, and product purpose, were acquired using a proton transfer reaction time-of-flight mass spectrometer (ptr-tofms). the vocs analyses highlighted a great variability among the fifteen cultivars, mostly due to compounds (c6 and c5) deriving from polyunsaturated fatty acids through the lox pathway. the early identification in the olive fruit of these compounds which are considered among the major contributors to the positive voos attributes, would be useful to produce high quality olive oils, and get useful information to individuate the best parents for the genetic improvement. 1. introduction virgin olive oil is worldwide considered a “commodity”, and until the second world war, a strategic food (fiorino et al., 2010). the complex flavor of virgin olive oil is mainly produced by volatile organic compounds (vocs) whose formation is related to olive fruit cell destruction (morales et al., 1996; angerosa, 2004). the total amount and types of vocs emitted change during the processing steps of olive fruit in the olive mill (morales et al., 1997). in particular, vocs emission is linked to the destruction of the cell structure of olive fruits which activates a specific chain of enzymatic reaction (lox cascade) (angerosa et al., 2004). the c6 and c5 are the compounds which most affect the aroma of olive oil gen(*) corresponding author: emarone@unite.it citation: taiti c., redwan m., marone e., atzori g., azzarello e., mancuso s., 2018 comparative analysis of volatile compounds (potential aromatic ability) in the fruit of 15 olive italian cultivars. adv. hort. sci., 32(1): 143-147 copyright: © 2018 taiti c., redwan m., marone e., atzori g., azzarello e., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 february 2018 accepted for publication 6 march 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(1): 143-147 144 erating the positive attributes such as fruity and herbaceous notes (marone et al., 2017). these compounds are usually responsible for typical aromas and flavors and play a dominant role in determining the peculiar aroma and quality of olive oils (zunin et al., 2005). the synthesis of c5 and c6 compounds is linked to the level of lipoxygenase (lox) activity to be catabolized through its pathway during the mill process (garçia-vico et al., 2017). different c6 straight chains are produced by the action of hydroperoxide lyase on polyunsaturated c18 fatty acids (linoleic c18:2 and linolenic c18:3). while, c5 seem to be synthesized through another branch of the lox pathway starting from 13-hydroperoxides derived from linolenic acid (garçia-vico et al., 2017). currently, the most innovative analytical technique used to detect vocs emitted by fruits, which provides a high resolution coupled to a rapid screening power of samples and easy to handle without sample manipulation, is the proton transfer reaction-time of flight-mass spectrometer (ptrtof-ms) (mayr et al., 2002; masi et al., 2015; taiti et al., 2017 a). currently, a little information is available and at the best of our knowledge one study was carried out to understand the vocs profile emission from olive fruits related to two cultivars (masi et al., 2015). the aim of this study was to develop a quick, accurate and relatively simple evaluation method based on the ptr-ms technique, to define the aromatic potentiality of each olive cultivar starting from olive fruits. this method could potentially help to address the choice of the cultivars in new plantations and to identify parents for future crosses, aimed to improve some organoleptic characteristics of the oils. 2. materials and methods plant material fruits of 15 certified italian olive cultivars, clonally propagated, different in their geographical origin, distribution and fruit’s use were obtained from plants of olive germplasm collection deriving from the italian and world olive gene bank (montepaldi experimental farm (43° 40’ 39’’ north latitude, 11° 08’ 46” east longitude, 210 m asl), university of florence) (table 1). the rainfed, bush trained, fourteen years old olive plants are ordinarily cultivated, grown on a slight slope of a sedimentarious gipsyarenaceous soil; the annual rainfall average is 867 mm, the average annual temperature results 13.3°c. fruit sampling for each cultivar, 1 kg of sound and healthy fruits was collected on october 20th from different parts of the trees of each cultivar. the day after, 100 washed fruits from each cultivar were weighted (w) and the color index (ci) was determined (uceda and hermoso, 1998). subsequently, for each cultivar nine whole and cut samples of fruits (~10 g) were submitted to the vocs analysis. statistical analyses one way analysis of variance (anova) was percultivar origin product use fruit average weight (g) color index (0-7) ascolana tenera marche table 4.80 ± 0.51 2.2 bianchera friuli venezia giulia olive oil 2.68 ± 0.12 1.5 carolea calabria olive oil/table 3.91 ± 1.17 1.2 coratina apulia olive oil 2.05 ± 0.60 1.5 fasolona basilicata olive oil/table 3.80 ± 0.58 3.1 frantoio tuscany olive oil 1.96 ± 0.36 1.5 itrana lazio olive oil/table 2.19 ± 0.94 1.4 leccino tuscany olive oil 2.02 ± 0.19 2.2 maiatica di ferrandina basilicata olive oil/table 2.75 ± 0.93 1.2 moraiolo tuscany olive oil 1.42 ± 0.28 2.1 nocellara del belice sicily olive oil/table 3.84 ± 0.76 1.8 palmarola apulia olive oil/table 2.15 ± 0.72 3.1 sant’agostino apulia table 4.36 ± 0.81 0.7 santa caterina tuscany table 5.80 ± 0.48 0.4 san francesco tuscany table 2.68 ± 0.22 1.5 table 1 list of the characteristics of the genetic material taiti et al. comparative vocs analysis of 15 italian olive cultivars 145 formed to compare the considered groups of chemical compounds: c5, c6, other vocs, and total vocs. separation of means was performed by the fisher’s lsd test (p = 0.01). computations were performed by statgraphics centurion xv v. 15.0.04. a factor analysis (fa) was applied to the spectral data of 273 olive oil samples, considering as factors three grouping of compounds: total vocs, c5, and c6, respectively. computations were performed by xlstat 2014.5.03. a principal component analysis (pca, unsupervised method) was applied to the whole spectral data of 273 olive oil samples, submitted to a logarithmic transformation and mean centering as pre-processing. computations were performed by plstoolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab_ r2015b (mathworks inc., natick, ma, usa). volatile compounds detection through a ptr-tof-8000 (ionicon analytik, gmbh, innsbruck, austria) measurements were performed in a similar way to the one reported by taiti et al. (2017 b). each analyzed sample consisted of ~10 g of olive fruits intact or cut into 4 parts. subsequently the sample was inserted into a 3/4 l glass container plugged with a cover in which two teflon tubes are inserted, connected respectively to a zero-air generator (peak scientific) and to the ptrtof-ms. the drift tube had the following ionization conditions: pressure of 2.20 mbar, voltage 600v and temperature 110°c. mass spectrometric data were collected over a mass range of m/z 21 to 210 and the acquisition time for each samples was 0.1 ns, for 120 seconds. the instrument internal calibration was based on: m/z = 29.997 (no+); m/z = 59.049 (c3h7o+) and m/z = 137.132 (c10h17 +) and was performed offline. the raw data were acquired with the tofdaq software (tofwerk ag, switzerland) as cps (count per second), and subsequently were converted in ppbv following the formula described by lindinger and jordan (1998) on the basis of the primary ion signal. 3. results and discussion the whole intact olive fruits of the fifteen cultivars showed no detectable quantities of vocs emission; in fact, the lipoxygenase pathway is activated when the fruit is damaged by the cut, producing a large quantity of volatile compounds, among which different c5 and c6 compounds, which are the main volatiles responsible for the positive aroma of olive oil (angerosa et al., 2004). meanwhile a great variability exists among the total vocs emitted by the cut fruits of fifteen different cultivars (table 2), ranging from 1061.8 ppbv in ‘ascolana tenera’ to 8767.5 ppbv for ‘carolea’. therefore, the total emission from ‘carolea’ was significantly higher compared to ‘ascolana tenera’. the cultivars can be arbitrarily divided in three main groups according to their total vocs emission: (a) low vocs emission, including together with the cv. ascolana tenera, fasolona and cultivar c5 compounds (ppbv) c6 compounds (ppbv) total vocs (ppbv) vocs emission (arbitrary ranking) ascolana tenera 8.01±0.61 a 180.73 ± 47.17 a 1061.77 ± 120.13 a low bianchera 35.29±2.15 g 1143.77 ± 148.60 f 5101.34 ± 305.77 g high carolea 53.90±7.83 i 2864.58 ± 543.60 i 8767.54 ± 1024.09 g high coratina 12.02±1.15 d 261.36 ± 34.62 abc 3177.13 ± 281.13 f medium fasolona 6.64±0.44 ab 229.19 ± 35.91 ab 1845.41 ± 124.20 de low frantoio 11.86±1.43 d 525.42 ± 96.22 de 2076.17 ± 149.65 abc medium itrana 43.63±1.75 h 2497.20 ± 222.50 h 7877.17 ± 491.75 g high leccino 9.07± 0.51 bc 264.10 ± 58.53 abc 3380.28 ± 148.72 f medium maiatica di ferrandina 24.49±3.21 f 1581.16 ± 262.15 g 5339.86 ± 472.56 ef high moraiolo 17.41±1.52 e 533.93 ± 67.89 de 2768.21 ± 150.04 bc medium nocellara del belice 16.77±1.15 e 544.07 ± 104.33 e 2986.40 ± 128.63 d medium palmarola 9.01±0.57 b 404.83 ± 64.22 cde 3142.02 ± 218.24 de medium sant’agostino 7.65±1.66 ab 148.79 ± 27.75 a 2309.40 ± 141.08 de medium santa caterina 8.73±1.61 b 402.58 ± 55.28 cde 2121.11 ± 148.07 ab medium san francesco 11.54±0.76 cd 368.17 ± 43.71 bcd 1934.07 ± 65.61 c low table 2 analysis of variance (anova) for the c5, c6, and total vocs related to the fifteen cultivars different upper case letters within a column indicate the difference by the lsd test at the 99% confidence leve (p= 0.01). adv. hort. sci., 2018 32(1): 143-147 146 san francesco; (b) medium vocs emission, including ‘leccino’, ‘coratina’, ‘palmarora’, ‘nocellara del belice’, ‘moraiolo’, ‘sant’agostino’, ‘santa caterina’, ‘frantoio’ and (c) high vocs emission, including ‘carolea’, ‘itrana’, ‘maiatica di ferrandina’, ‘bianchera’. also c6 and c5 straight chain showed an ample variability; in particular, c6 compounds ranged from 148.79 ppbv for the cv. sant’agostino to 2864.58 ppbv for the cv. carolea, with a fluctuation among the different cultivar of about 1:20. as the high amount of developed c6 compounds it is noteworthy to highlight the cv. carolea, coratina, and itrana. on the other hand, the c5 compounds showed a minimum value of 6.64 ppbv (cv. fasolona), and a maximum of 53.90 ppbv (cv. carolea). a biplot from factor analysis (fig. 1) simultaneously represents the relationship between total vocs, c6, and c5 compounds, highlighting the relative distances among the fifteen olive cultivars. the first axis explains the 97.25% of the total variability in the spectral data. the samples are clustered in two main groups (fig. 1), characterized by higher vocs emission (right quadrants), and lower vocs emission (left quadrants). although the different total amount, and despite the quantitatively different responses of the different cultivars, c5 and c6 compounds resulted directly related to the total vocs emission (0.952 ppbv, and 0.964 ppbv, respectively), as a consequence of a more general phenomenon, the lipoxygenase (lox) pathway, common to all the examined cultivars. therefore, by evaluating the total vocs amount, it is possible to analytically extrapolate the contribution of c5 and c6 in the total volatile compounds emission. moreover, by the factor analysis plot it can be noted that some cultivars are characterized by higher c6 compounds emission (e.g. ‘itrana’ and ‘maiatica di ferrandina’), while other cultivars showed higher c5 emission (e.g. ‘bianchera’ and ‘carolea’). subsequently a pca (fig. 2) was performed on the whole vocs data set to (1) give a general overview of the cultivars ordination and (2) to detect if the cultivars can be grouped basing on their geographical origin and/or final product use. anova results confirm (table 2) that a high variability appears in the vocs emission from the different cultivars, widely distributed without any particular tendency to clustering, excluding any possible link of the different cultivars due to their geographical origin or to the final product use. the first two components justified 58.32% for pc1 and 9.43% for pc2 of the total variability, respectively, indicating that the greatest amount of the total variance is explained by the pc1. the most important pc1 loadings resulted in eight m/z as reported in figure 2. in particular, m/z = 81.069 [tentatively identified (t.i.) as hexenal fragment] and m/z = 99.080 (t.i. as 2,3-hexenal) directly deriving from linolenic acid and responsible for the freshly cut grass odors, are previously reported as positive attributes in oils by marone et al. (2017) and taiti and marone (2017). fig. 1 biplot from factor analysis. relationships among the fifteen cultivars and the three groups of chemical compounds (c5, c6, total vocs). fig. 2 score plot from pca related to the vocs dataset (ppbv) for the fifteen cultivars; in the table the main pc1 loadings are reported. taiti et al. comparative vocs analysis of 15 italian olive cultivars 147 the reported data confirmed the possibility to predict and quickly evaluate during all stages of the fruit ripening the presence of vocs characterizing the positive flavors of the olive fruits obtained from defined cultivars or new breeding. 4. conclusions the data show a great variability in both amount and type of vocs emitted by olive fruits, simultaneously collected in the same environment from fifteen different italian cultivars. for example the total emission by ‘carolea’ was significantly higher compared to ‘ascolana tenera’. on the other hand, c5 and c6 compounds which most affect the aroma adding positive attributes to olive oil, were respectively 95% and 96% correlated to the total vocs. among the cultivars it is possible to note a different relationship between c5 and c6: the cv. bianchera showed the highest c5/total vocs ratio, while the cv. itrana, and carolea the highest c6/total vocs ratio. the presence of these two groups of compounds (c5 and c6) confirms that the cut of the fruit triggers the same phenomena that verifies at the moment of the olive processing, that is the activation of the lox cascade, which gives rise to vocs considered to be positive attributes for the olive oil aroma. further studies would be necessary to deepen and to develop the information that can be obtained from the use of the fruits to: (1) understand the behavior of the derived oils, and (2) to widen the genetic platform from which information can be acquired and (3) to start verifying the responses of fruits deriving from predetermined crosses. in conclusion, the possibility to early identify these compounds at the fruit level can allow to individuate the best conditions to produce high quality olive oils, and get useful information to individuate the best parents for the olive genetic improvement. acknowledgements the authors would like to thank the montepaldi experimental farm of university of florence for providing the samples used for these trials. references angerosa f., servili m., selvaggini r., taticchi a., esposto s., montedoro g.f., 2004 review: volatile compounds in virgin olive oil: occurrence and their relationship with the quality. j. chromatogr. a, 1054(1-2): 17-31. fiorino p., marone e., ottanelli a., 2010 mechanical harvesting, productivity and superintensive planting systems in olive groves. adv. hort. sci., 24(1): 91-94. garçia-vico l., belaj a., sanchez-ortiz a., martinezrivas j.m., perez a.g., sanz c., 2017 volatile compound profiling by hs-spme/gc-ms-fid of a core olive cultivar collection as a tool for aroma improvement of virgin olive oil. 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morales m.t., rios j.j., aparicio r., 1997 changes in the volatile composition of virgin olive oil during oxidation: flavors and off-flavors. j. agric. food chem., 45(7): 2666-2673. taiti c., colzi i., azzarello e., mancuso s., 2017 a discovering a volatile organic compound fingerprinting of pouteria lucuma fruits. fruits, 72(3): 131-138. taiti c., marone e., 2017 evoo or not evoo? a new precise and simple analytical tool to discriminate virgin olive oils adv. hort. sci, 31(4): 329-337. taiti c., marone e., lanza m., azzarello e., masi e., pandolfi c., giordani e., mancuso s., 2017 b nashi or williams pear fruits? use of volatile organic compounds, physicochemical parameters, and sensory evaluation to understand the consumer’s preference european food res. techn., 243(11): 1917-1931. uceda m., hermoso m., 1998 la calidad del aceite de oliva, pp. 699-728. in: barranco d., r. fernándezescobar, and l. rallo (eds.) el cultivo del olivo. mundi-prensa, madrid, spain, pp. zunin p., boggia r., salvadeo p., evangelisti f., 2005 geographical traceability of west liguria extravirgin olive oils by the analysis of volatile terpenoid hydrocarbons. j. chromatography a, 1089(1-2): 243-249. impaginato 161 adv. hort. sci., 2019 33(2): 161-170 doi: 10.13128/ahs-23363 the responses of enzymatic and nonenzymatic antioxidant systems of scion on different rootstocks under water stress deficit m. keshavarzi , a. shekafandeh (*) horticultural science, college of agriculture, university of shiraz, iran. key words: chlorophyll, ficus carica, graft, growth. abstract: selecting of the specific type of rootstock is an appropriate and shortterm method for increasing drought tolerance by improving the antioxidant systems in plants. in this research, the responses of the antioxidant systems of fig scion ‘sabz’ were investigated on various rootstocks at different irrigation levels. graft combinations were ‘sabz’ on ‘sabz’ (sa/sa), ‘siah’ (sa/si) and ‘torch’ (sa/t) rootstocks, plus ‘sabz’, ‘siah’ and ‘torch’ cultivars with no grafting, for a total of six groups. the plants were irrigated with 4 levels of 25, 50, 75 and 100% of water requirement (wr) for a duration of 12 weeks. the experiment was performed in a randomized complete design with 5 replications per treatment. the results showed that the ‘torch’ rootstock induced the greatest amount of anthocyanin, glutathione and ascorbic acid in ‘sabz’ (sa/t) at 25% wr. superoxide dismutase and catalase activities of ‘sabz’ grafted on ‘siah’ were more evidence compared to ‘sabz’ grafted on ‘torch’ rootstock at 25% of wr. the cv. sabz grafted on both ‘siah’ and ‘torch’ rootstocks indicated higher chlorophyll content, chlorophyll stability index and shoot growth than cv. sabz with no grafting. as a result, both rootstocks (t and si) with the activation of enzymatic and non-enzymatic antioxidant systems caused the scion protects its integrity and be able to tolerate more water stress. 1. introduction water scarcity is one of the most important environmental stresses, with its greatest impact on agriculture worldwide, especially in arid and semiarid regions (knapp et al., 2001; alizadeh et al., 2011). in recent years, the issue of climate change, combined with global warming, has been a major contributor to the increased water scarcity and plant losses in many parts of the world (kramer and boyer, 1995). since most of a plant’s processes are directly or indirectly affected by water, it is clear that most plants are affected by moderate to long-lasting drought throughout their life cycles (bhattacharjee and saha, 2014). the effects of drought stress on plants depend on genotype, rate and severity of the stress, age and stage of plants growth and development (rostami and (*) corresponding author: shefakan@shirazu.ac.ir citation: keshavarzi m., shekafandeh a., 2019 the responses of enzymatic and non-enzymatic antioxidant systems of scion on different rootstocks under water stress deficit. adv. hort. sci., 33(2): 161-170 copyright: © 2019 keshavarzi m., shekafandeh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 june 2018 accepted for publication 14 december 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 161-170 162 rahemi, 2013). plant tolerance to water stress depends on their morphological, physiological and biochemical mechanisms that determine the responses of the plant under stress conditions (penella et al., 2014). the fig (ficus carica l.) belongs to the moraceae family, a perennial fruit tree, which generally has a high tolerance to water deficit (faghih and sabet sarvestani, 2001). this tree is managed in two ways, irrigated or rain-fed, and is cultivated in many parts of the world with diverse climatic conditions such as iran, turkey and other mediterranean countries. the total area in the world cultivated with figs is reported to be about 388368 hectares with a production of 918813 tons per year (el-shazly et al., 2014). iran, with an annual production of more than 76414 tons of fig, ranks fourth in the world (fao, 2012). most fig trees in iran are cultivated in estahban, a semi-arid region in the southeast of fars province (shirbani et al., 2013). drought stress stimulates the accumulation of active oxygen species in plants. these reactive oxygen species are active forms of the oxygen molecule that are produced by the excitation of an oxygen molecule, or the transfer of one, two or three electrons to oxygen molecules, which ultimately lead to the formation of superoxide (o2 -), hydrogen peroxide (h2o2) and hydroxyl radical (oh) (gill and tuteja, 2003) these reactive oxygen species may be the first step in the degradation of oxidative processes, such as lipid peroxidation, chlorophyll breakdown, protein oxidation, and nucleic acid damage (anjum et al., 2011; gu et al., 2013). plants have specific defense mechanisms that provide protection on the molecular level. these mechanisms comprise non-enzymatic antioxidants, such as ascorbic acid (asa), glutathione (gsh) and anthocyanin and enzymatic antioxidants include catalase (cat) and superoxide dismutase (sod) (guerfel et al., 2009; liu et al., 2012 a). superoxide dismutase is a major scavenger that counters superoxide damage. catalase catalyzes the decomposition of the excess of hydrogen peroxide to water and oxygen and protects cells from oxidative damage. these two enzymes play an important role in the degradation of reactive oxygen species (mittler, 2002). with regard to the climate change models that predict an increase in drought and excessive groundwater depletion in the future, water scarcity appears to be the main limiting factor in agriculture (alizadeh, 2005). in this regard, the use of drought tolerant rootstocks can play an important role in managing water absorption, water use efficiency, survival potential, growth capacity and the success of grafting in dry conditions. different rootstocks show different levels of tolerance in response to drought, and their ability to have high tolerance depend on improving the vegetative growth of the scion, the horizontal and vertical expansion of the roots, the ability to absorb water and minerals and antioxidant system activities for scavenging of active oxygen species (ballesta et al., 2010; corso and bonghi, 2014). how the rootstock genotypes may be effective in response to water deficit stress has been investigated in many studies on fruit trees, such as apples and grapes (alizadeh et al., 2011; liu et al., 2012 b; corso and bonghi, 2014). most of these researches focus on the effects of rootstock on vegetative growth, fruit yield and quality, nutrition and hormones, and the scion’s water content in response to drought. however, there are limited investigations on how rootstock affects the antioxidant systems of the scion in response to water deficit. the aim of this study was to investigate the effect of different fig rootstocks on the enzymatic and non-enzymatic antioxidant systems of the ‘sabz’ scion in response to different levels of irrigation. 2. materials and methods in early november, offshoots semi-hard woodcuttings of different cultivars (‘sabz’, ‘siah’ and ‘torsh’) were disinfected with a commercial bleach (clorax) solution containing 5% naocl and 2% benomyl for 20 min. then, they were wrapped in a wet cotton cloth and placed at 4°c for 15 days to ensure that their chilling requirement was satisfied. after that, the bottoms of the cuttings treated with iba solution (1000 mg/l) and cultured in cartonplast boxes containing perlite. they were put in a greenhouse with natural sunlight, relative humidity of 64% and an average temperature of 38/16±2°c day and night respectively. after 2 weeks, rooted cuttings were transferred to 1 l plastic pots containing a mixture of soil: peat: sand (1:1:1, v/v/v). after 8 weeks, when the length of the new shoots reached 20 cm, cultivar sabz as scion were grafted (cleft grafting) onto the three rootstocks (‘sabz’, ‘siah’ and ‘torsh’). the place of grafting was firmly enclosed with cellophane. to maintain moisture, grafted plants were covered with plastic bags to ensure grafting success. after 15 days, the plastic bags were removed and the cellophanes were keshavarzi and shekafandeh scion antioxidants responses on different rootstocks 163 opened. subsequently, all grafted and non-grafted plants (controls) were transferred to 10 l pots without drainage that contained the aforementioned ratio of the soil mixture. the field capacity of the soil used for potting was determined according to the protocol described by richards (1949). the pots were irrigated daily at field capacity by the help of a balance. after complete deployment, the experiment was carried out by selecting 40 plants of each grafted combination (‘sabz’/ ‘sabz’, ‘sabz’/ ‘siah’, ‘sabz’/ ‘torsh’) and 40 plants from cultivars with no grafting (‘sabz’, ‘siah’ and ‘torsh’) in a randomized complete design with 4 levels of irrigation 100%, 75%, 50% and 25% of water requirement (wr). three months after the start of treatments, various indices were measured as follows. anthocyanin extraction solution containing methanol, water and concentrated chloric acid (hcl) was prepared by a ratio of 80: 20: 1. leaf samples were kept in the extract solution at 4°c in the dark for 48 h. then, the extract passed through a whatman filter paper (no. 1) and was read at 530 and 657 nm. anthocyanin content was measured using the microplate reader spectrophotometer according to the method described by alexieva et al. (2001). the amount of anthocyanin was calculated as μg g-1 fresh weight. glutathione the method used by moron et al. (1979) was followed to measure glutathione concentration. an amount of 200 mg of fresh leaves was homogenized with 2 ml of cooled trichloroacetic acid (tca) (5%), and they were centrifuged for 30 min at 15,000 rpm at 4°c. from supernatant extract, 75 μl was transferred to a vial containing 300 μl sodium phosphate buffer (0.2 m, ph 8) and 750 μl dtnb (5, 5-di-tiobis2-nitrobenzoic acid) 0.6 mm. the extract was read at 412 nm by a spectrophotometer microplate reader. glutathione concentration was calculated using standard curve. ascorbic acid to determine the concentration of ascorbic acid, 1 g of fresh leaf tissue was thoroughly crushed in 5 ml of tca (10% cold), and then centrifuged at 3500 rpm for 20 min. the supernatant was isolated and diluted to reach 10 ml. one ml of aqueous extract was mixed with 0.2 ml of dtc reagent (2, 4-di-nitrohydrazine, thiourea, copper sulfate) and was incubated at 37°c for 3 h. then, 1.5 ml of sulfuric acid (65%) was added and mixed thoroughly. the extract was allowed to stand at room temperature for 20 min. the absorbance was read at 520 nm using a spectrophotometer. the ascorbic acid was calculated in μg g-1 f.w. (elavarthi and martin, 2010). antioxidant enzymes to prepare leaf extract containing enzymatic antioxidant, 200 mg of fresh leaf tissue was ground with liquid nitrogen in a mortar. then, 1.2 ml of k2po4 buffer 0.2 m (ph 7.8) containing 0.1 mm edta (ethylenediaminetetraacetic acid, c10h16n2o8) was added to the samples and homogenized. the samples were centrifuged at 15,000 g for 20 min at 4°c. the supernatants were separated and then the same operation was repeated on residual. the resulting extract was used for determining the activity of antioxidant enzymes (elavarthi and martin, 2010). catalase activity catalase activity was determined by the method of aebi (1984) as described by elavarthi and martin (2010) by measuring the amount of hydrogen peroxide (h2o2) degradation via reducing the absorbance at 240 nm with a spectrophotometer microplate reader. to this purpose, 3 ml of the reaction mixture was made up of 2 ml of leaf extract, diluted with 50 mm k2so4 buffer at ph=7 and h2o2 10 mm, which reached 3 ml by distilled water, the reaction started and the absorbance of the samples were recorded for 1 min. superoxide dismutase superoxide dismutase activity (sod) was evaluated using a modified nitro-blue tetrazolium (nbt) method (elavarthi and martin, 2010). accordingly, 2 ml of the reaction mixture including 50 mm phosphate buffer (ph= 7.8), 2 mm edta, 9.9 mm methionine, 55 μm nbt (nitro blue tetrazolium chloride, c10h330cl2n10o6 ), 0.025% triton x-100 to was added to 40 μl of diluted sample (×2) and then, added 20 μl of riboflavin (1 mm). the reaction began by exposing the samples under a fluorescent tube (15 watts) for 10 min. the blank received the same chemical mixture but without leaf samples throughout the steps. the absorbance of the specimens was read at 560 nm in a microplate reader spectrophotometer and one unit of enzyme activity was taken as the quantity of enzyme, which reduced the absorbance reading of the sample to 50% in comparison to control. finally, sod was calculated as u g-1 fresh weight. chlorophyll stability index chlorophyll stability index (csi) was measured adv. hort. sci., 2019 33(2): 161-170 164 according to the method used by murty and majumber (1962). the fresh leaf samples were emerged in 20 ml of distilled water and placed in a warm water bath of 56±1°c for 30 min, and the chlorophyll contents of the samples were determined. the chlorophyll stability index was obtained from the following formula. csi (%)= 1-(chlorophyll content without heating/chlorophyll content after heating) × 100. chlorophyll content total chlorophyll, chlorophyll a and chlorophyll b content of leaves were measured using the dimethyl sulfoxide (dmso) method introduced by hiscox and israelstam (1979). leaf samples (100 mg fresh leaf) were submerged in 7 ml of dimethyl sulfoxide solution and put in the dark for 17 h. after, they were incubated in an oven at 60°c. by adding 3 ml of dimethyl sulfoxide to the samples, the volume was adjusted to 10 ml. then, 200 μl of the samples extract were transferred to a plate and read by microplate reader spectrophotometry at 633 and 645 nm wavelengths. the following formulas were used in order to determine the total chlorophyll concentration as mg g-1 fresh weight (fw). ch a (mg g-1 fw)= 12.7(a663) 2.69(a645) x volume made w.t of the sample x 10 ch b (mg g-1 fw) = 22.9(a645) 4.68(a663) x volume made w.t of the sample x 10 tch (mg g-1 fw)= 20.2(a645) + 8.02 (a663) x volume made w.t of the sample x 10 in these formulas, cha represents chlorophyll a, chb = chlorophyll b, tch = total chlorophyll; a = absorbance in the wavelength (nm). cell membrane injury the cell membrane damage index, measured by electrolyte leakage, is an indicator of estimating the tolerance of cellular protoplasts and the ability of the membrane to maintain integrity under conditions of water scarcity (bajji et al., 2002). this index was measured using the method followed by kocheva and georgiev (2003). a punching machine was used in order to prepare leaf samples measuring 1 cm in diameter. the leaf sample discs of 1 cm in diameter were washed three times with distilled water to remove surface contamination. then they were transferred to vials containing 20 ml of deionized water and kept at 10°c for 24 h. after measuring their electrical conductivity, they were placed in an autoclave of 120°c for 15 min, after cooling at 25°c, the electrical conductivity was re-measured. cell membrane damage (cmi) was obtained from the following formula. cmi ( %) = [1( t1 / t2)] / [1(c1 / c2)]×100 t and c are the stressed and control samples, respectively, while 1 and 2 are the primary and secondary ec measurements. the experiment had a factorial layout, based on 4 irrigation levels and 6 combinations of grafted and non-grafted plants in a randomized complete design with 5 replications and 2 plants per replicate. data analysis was performed using sas software version 9.3 and duncan’s multiple range test at 5% of probability was used for the means comparison. 3. results analysis of variance showed that the interaction between cultivars and different levels of irrigation were significant on all measured traits except anthocyanin (table 1). anthocyanin the results showed that increasing drought stress from 100% to 25% of wr the anthocyanin level increased in all grafted and non-grafted plants (fig. table 1 the variance analysis of some traits of fig cultivars under water stress condition source of variation df mean square sod cat ant gsh asc cultivar (a) 5 0.0259 ** 10.9 ** 0.008 ** 14.8 ** 68.3 ** water stress (b) 3 0.4569 ** 5.5 ** 0.014 ** 3.8 ** 42.2 ** a × b 15 0.0293 ** 2.3 ** 0.0001 ns 3.3 ** 15.8 ** error 96 0.0028 0.044 0.0001 0.55 1.29 c.v. 3.63 19.33 14.07 22.41 10.64 sod= superoxide dismutase; cat= catalase; gsh= glutathione; asc= ascorbic acid; ant= anthocyanin. **significant at 1% probability, ns; non-significant. keshavarzi and shekafandeh scion antioxidants responses on different rootstocks 165 1a). a comparison of cultivars with no grafting (‘sabz’, ‘siah’, and ‘torsh’) showed that ‘torsh’ and ‘siah’ had anthocyanin levels higher than the amount found in the ‘sabz’. however, ‘torsh’ rootstock increased the amount of the anthocyanins in the scion (‘sabz’) compared to the non-grafted ‘sabz’ cultivar (fig. 1b). glutathione the amount of glutathione in the leaves of nongrafted ‘torsh’ increased with increasing the stress intensity from 100% to 25% of wr, while the ‘siah’ cultivar reduced the glutathione significantly. the decrease in glutathione concentration in the leaf of the ‘sabz’ non-grafted and the ‘sabz’/‘sabz’ combination was not significant with increasing drought stress (fig. 2a). furthermore, both ‘siah’ and ‘torsh’ rootstocks significantly increased the glutathione level of the ‘sabz’ scion at 75% of wr. however, there was no a significant difference in glutathione rate between 75%, 50% and 25% of wr in sa/to combination. ascorbic acid the results showed with increasing stress from 100% to 25% of wr, no increase in ascorbic acid content was observed in non-grafted ‘siah’ and ‘sabz’ cultivars. the non-grafted ‘torsh’ showed an increase of 47.63% in the ascorbic acid content in the severe water stress compared with control. the ‘sabz’ and ‘siah’ rootstocks reduced the ascorbic acid content of the ‘sabz’ scion at 50% and 25% of wr, compared to the non-grafted ‘sabz’. nonetheless, ‘torsh’ rootstock also reduced the ascorbic acid content of the ‘sabz’ scion when 100% of wr was supplied, but this rootstock caused a significant increase in the ascorbic acid content (15.55%) of the ‘sabz’ scion in the 25% of wr (fig. 2b). superoxide dismutase superoxide dismutase activity increased with increasing drought stress in all non-grafted and grafted cultivars (fig. 3a). comparison of cultivars with no grafting showed that ‘siah’ at 50% of wr had the highest enzyme activity compared with ‘torsh’ and ‘sabz’ cultivars. at 25% of wr, the sod activity in the ‘sabz’ rootstock was 1.78 times higher than the ‘siah’ rootstock, and in the ‘siah’ rootstock it was 1.63 times higher than in the ‘torsh’ rootstock. each of the three rootstocks reduced the activity of the enzyme compared to the non-grafted ‘sabz’ cultivar when supplied with 50% and 25% of wr, although the differences in these values were not statistically significant (fig. 3a). catalase activity the activity of catalase enzyme increased significantly in all grafted and non-grafted cultivars in fig. 1 main effects of irrigation levels (a) and grafted and nongrafted cultivars (b) on leaf anthocyanin rate. sa= sabz; si= siah; to= torsh. values are means ± se (n=5). fig. 2 interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50, 25% wr) on: glutathione (a) and ascorbic acid (b). sa= sabz; si= siah; to= torsh. values are means ± se (n=5). 166 adv. hort. sci., 2019 33(2): 161-170 response to intense drought stress (fig. 3b). comparison of non-grafted cultivars showed that catalase enzyme activity in ‘torsh’ at 75%, 50% and 25% of wr was significantly higher than the two other cultivars (‘sabz’ and ‘siah’), which did not differ significantly. however, only ‘siah’ rootstock increased the activity of catalase enzymes (by 31.13%) in the ‘sabz’ scion when 25% of wr was applied compared to the non-grafted ‘sabz’ cultivar. analysis of variance showed that the interaction between cultivars and different levels of irrigation were significant on all measured traits (table 2). total leaf chlorophyll the results showed that in non-grafted cultivars, ‘torsh’ at 100%, 75% and 50% of wr showed higher chlorophyll contents than the other two cultivars. in grafted rootstocks, ‘siah’ and ‘torsh’ significantly increased the chlorophyll content in the leaves of the scion ‘sabz’ in 100% and 75% of wr. ‘siah’ rootstock also increased the chlorophyll content of the ‘sabz’ scion by 42.52% in 50% of wr compared with nongrated ‘sabz’ (fig. 4a). chlorophyll stability index the results showed that the chlorophyll stability index decreased as the drought stress intensified table 2 the variance analysis of total chl, csi, cmi, shoot fw and shoot dw of fig cultivars under water stress condition fig. 3 interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50, 25% water requirement: wr) on superoxide dismutase (sod) activity (a) and catalase activity (b). sa= sabz; si= siah; to= torsh. values are means ± se (n=5). source of variation df mean square total chl csi cmi shoot fw shoot dw cultivar (a) 5 685.6 ** 5449.9 ** 469.7 ** 13355.4 ** 1895.5 ** water stress (b) 3 1963.6 ** 8539.5 ** 11707.8 ** 22405.1 ** 2749.35 ** a × b 15 108.2 ** 134.7 ** 180.4 ** 691.71 ** 83.24 ** error 96 12.45 41.75 26.47 119.36 19.38 c.v. 23.83 12.45 5.59 13.03 12.77 chl= chlorophyll; csi= chlorophyll stability index; cmi= cell membrane injure; fw= fresh weight; dw= dry weight. **significant at 1% probability fig. 4 interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% wr) on leaf chlorophyll content (a) and chlorophyll stability index (b). sa= sabz; si= siah; to= torsh. values are means ± se (n=5). keshavarzi and shekafandeh scion antioxidants responses on different rootstocks 167 (fig. 4b). among the non-grafted cultivars, ‘torsh’ showed more stability in the leaf chlorophyll content than the other two cultivars at different levels of wr. ‘sabz’ scion on the ‘siah’ and ‘torsh’ rootstocks showed the higher chlorophyll stability than nongrafted ‘sabz’ and ‘sabz’/‘sabz’ graft combination at all levels of wr (fig. 4b). cell membrane injury the results showed that high levels of water stress increased leaf cell membrane damage in all grafted and non-grafted plants (fig. 5). the differences in the damage to the cell membranes of the ‘siah’, ‘torsh’ and ‘sabz’ were not significant at 100% of wr, but the 75% and 50% of wr induced significantly greater damages to the leaf cell membrane of ‘siah’ than the other two cultivars. at 25% of wr, the non-grafted ‘sabz’ cultivar showed lower levels of membrane damage in its leaf cells, compared to ‘siah’ and ‘torsh’. the results also showed that the effect of the ‘sabz’, ‘torsh’, and ‘siah’ rootstocks on the ‘sabz’ scion was not significant at all levels of water requirement, compared to the non-grafted ‘sabz’ cultivar (fig. 5). shoot fresh and dry weight the results showed that with increasing water stress deficit the shoot growth decreased in all nongrafted and grafted combinations of plants. however, ‘siah’ and ‘torsh’ rootstocks are associated with higher scion shoot fresh and dry weight compared to non-grafted (sa) and self-grafted (sa/sa) sabz cultivar (fig 6. a and b). 4. discussion and conclusions the increase in anthocyanin content in stressed leaves in this study confirms the protective role of anthocyanin against sunlight and active oxygen species in stressed plants (gholami et al., 2012 a). stimulation of anthocyanin production has been proven by osmotic pressure and it is believed (in most cases) that tissues that contain more anthocyanins are more tolerant to drought (chalker-scott, 1999). anthocyanins are able to protect cells against environmental damage through protection of cell membranes, organelles and nucleic acids (neil et al., 2002). in this study, ‘torsh’ rootstock increased the leaf anthocyanin in the ‘sabz’ scion. accordingly, the type of rootstock can increase the production of anthocyanins and thus counteract the reactive species of oxygen, increasing the ability of the plant to tolerate conditions of water stress. in the green tissues of plants, ascorbic acid is a major antioxidant soluble in water. leaf ascorbic acid changes seasonally, but as the leaf ages it remains at a constant level. however, exposure to stress can significantly change this situation and increase it (sircelj and batic, 2007). ascorbic acid, like carotenoids, plays an important role in protecting the photosynthetic system against the harmful effects of reactive fig. 5 interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% of wr) on membrane injury index. sa= sabz; si= siah; to= torsh. values are means ± se (n=5). fig. 6 interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% wr) on shoot fresh weight (a) and shoot dry weight (b). sa= sabz; si= siah; to= torsh. adv. hort. sci., 2019 33(2): 161-170 168 oxygen species (ros). in this research, the amount of glutathione and ascorbic acid increased in ‘torsh’ rootstock, and this effect was transmitted to the ‘sabz’ scion, which may be due to the rapid reaction of this rootstock to severe drought stress and the need for glutathione and ascorbic acid, which can confront ros more efficiently. even though the changes in glutathione and ascorbic acid were not significant at different levels of irrigation in the nongrafted ‘sabz’ cultivar, by grafting the ‘sabz’ scion on the ‘siah’ rootstock, the glutathione level in the ‘sabz’ scion increased significantly in 75% of water requirement and did not show any changes in ascorbic acid. in this regard, tausz et al., (2004) also reported that drought stress leads to a decrease in glutathione concentration, and the oxidation and reduction of compounds ultimately leads to the system’s degradation. an increase in the amount of glutathione in conditions of water shortage may be necessary to adjust the level of ascorbic acid in the plant (gholami et al., 2012 b). it seems that increased levels of glutathione in non-grafted ‘torsh’ cultivar and ‘sabz’ grafted on ‘torsh’ rootstock have been effective in regulating ascorbic acid levels at mild and severe stress levels. sircelj et al. (2005) also reported an increase in the level of glutathione and ascorbic acid at moderate levels, indicating compatibility with oxidative stress in apple trees, and stated that the decrease in glutathione levels at severe stress levels indicates severe oxidative stress. many researchers have shown that drought stress leads to oxidative stress in the plant (mittler, 2002; gill and tuteja, 2010; gholami et al., 2012 b; shirbani et al., 2013). excessive forms of stress can damage the plant by producing reactive oxygen species. the antioxidant defense system and the breakdown of active oxygen species are known to be under dry conditions (zarafshar et al., 2014). under mild and moderate water deficit conditions, a number of compatible plant species increase the activity of enzymatic antioxidants such as superoxide dismutase and catalase, although severe drought stress may cause damage to cells by stronger stimulations or impairments via reactive oxygen species that suppress enzymatic antioxidant activity (guerfel et al., 2008). superoxide dismutase and catalase are active enzymes for the elimination and degradation of harmful oxygen species in plants and maintain oxidative equilibrium during oxidative stress (gill and tuteja, 2010). superoxide dismutase enzymes have been reported to play an important role in the antioxidant metabolism of plants under environmental stress conditions, such as water deficit, via regulating their gene expression or activities (xu et al., 2010). high sod activity in ‘siah’ and ‘sabz’ nongrafted cultivars were achieved in 50% and 25% of wr respectively, and the activity of catalase in the non-grafted ‘torsh’ cultivar and in the ‘sabz’/‘siah’ graft combination (at 25% of wr) were also comparatively high. these results depend on the plant’s better protection against oxidative damage caused by water stress. it seems that the scion of the ‘sabz’ cultivar on the ‘siah’ rootstock led to greater improvements in the antioxidant system. chlorophyll content has a positive correlation with the rate of photosynthesis. therefore, the decrease in chlorophyll content under drought stress condition is a common symptom of oxidative stress, which may be due to photo-oxidation of pigments and chlorophyll degradation (anjum et al., 2011; shirbani et al., 2013). the reduction of chlorophyll content was observed in all cultivars under stress in this study. according to guerfel et al. (2009) the reduction of chlorophyll content can be attributed to its susceptibility to environmental stresses, especially drought. chlorophyll levels tend to decrease or otherwise remain unchanged during the period of drought stress in many species. this, however, depends on the duration and severity of the drought (anjum et al., 2011). in this study, chlorophyll contents at 75% and 50% of wr decreased less sharply in ‘sabz’/siah graft combination. this indicates that the light dissipation and antioxidant systems may prevent the degradation of chlorophyll molecules (niu et al., 2008). sircelj et al. (2005) also reported no reduction in the chlorophyll content of apple leaves ‘elastar’ under water stress is due to a strong antioxidant system and an efficient light dissipation system. anjum et al. (2011) attributed the decrease in the content of chlorophyll to the chloroplast membrane damage, swelling, lamella distortion and the occurrence of small droplets of lipids under drought stress conditions. the chlorophyll stability index is an indicator of measuring the membrane’s integrity and the pigments stability under stress conditions (ananthi et al., 2013). surendar et al. (2013) reported that a decrease in chlorophyll content under stress was due to the destruction of the chloroplast membrane with increasing phosphatase activity, which is located on the membrane. in this study, intense water stress (25% of wr) reduced the chlorophyll stability in all grafted and non-grafted rootstocks compared to keshavarzi and shekafandeh scion antioxidants responses on different rootstocks 169 100% of wr. this could be due to the degradation of chlorophyll when proteolytic enzymes, such as the chlorophyllase enzymes, are produced. the values of the high chlorophyll stability index in the non-grafted ‘torsh’ cultivar and the ‘sabz’/‘siah’ or ‘sabz’/’torsh’ graft combinations indicate a higher chlorophyll content in the leaves, which leads to an increase in the rate of photosynthesis and the production of more dry weight which helps the plant with stand dehydration (babu et al., 2008; ananthi et al., 2013; surendar et al., 2013). under conditions of water scarcity, the cell membrane of the leaves are subject to changes such as increased permeability and reduced selectivity, which can be observed through an increase in electrolyte leakage (zarafshar et al., 2014). in this study, the overall increase for leakage under stressful conditions was observed in all grafted and non-grafted rootstocks as compared to fully irrigated plants. the non-grafted ‘siah’ cultivar experienced a greater damage to the cell membrane of its leaf, compared to the non-grafted ‘sabz’ and ‘torsh’ cultivars. this indicates that the rootstock’s ability to maintain the integrity of its cell membrane in severe stress conditions is considered an important factor in determining tolerance to drought (bolat et al., 2014). undesirable performance of the cell’s metabolism during periods of drought stress leads to the stimulation of reactive oxygen species or the disruption of systems that prevent or reduce the activity of reactive oxygen species, which would damage the cell membrane and increase electrolyte leakage (guerfel et al., 2008; karimi et al., 2013). therefore, the fact that damage to the cell membranes of the non-grafted ‘sabz’ cultivar occurred less than in the non-grafted ‘siah’ rootstock may indicate a more tolerance to drought in the ‘sabz’ cultivar. however, shahidi-rad et al. 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g.g., 2014 morphological, physiological and biological responses to soil water deficit in seedling of three populations of wild pear tree (pyrus biosseriana). biotech. agro. soc. environ., 18: 1370-1400. impaginato 215 adv. hort. sci., 2017 31(3): 215-221 doi: 10.13128/ahs-20460 commercial advantages on basil architecture by ultraviolet-b irradiation a. ciurli 1, t. huarancca reyes1 (*), l. guglielminetti 1, 2 1 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via mariscoglio, 34, 56124 pisa, italy. 2 centro interdipartimentale di ricerca “nutraceutica e alimentazione per la salute”, università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: fresh market advantages, ocimum basilicum, plant architecture, uv-b. abstract: sweet basil (ocimum basilicum l.) is one of the most important herbs widely used for its medicinal properties and as food ingredient. the marketing of this product highlights the problem that these plants have long and slender stems, which are easy to break off and thus making difficult their market distribution. in this work, two cultivars of basil (genovese and profumo) at the adequate development stage for sale were used. we evaluated the effect of supplemental ultraviolet (uv)-b irradiation (15 w m-2; 3 h day-1) on plant growth and market quality. both cultivars of basil plants under uv-b irradiation resulted in increased leaf size and biomass, and decreased shoot length in comparison to that of under control growth conditions. these results indicate that the application of uv-b irradiation beneficially influenced plant architecture in basil improving their greenhouse production for fresh market. 1. introduction many species in the genus ocimum (labiatae) are ranked among the most important herbs for their medicinal properties, that are associated to high content of secondary metabolites including essential oils and caffeic acid derivatives (gülҫin et al., 2007). the most significant species of the genus is sweet basil (ocimum basilicum l.), which originated from tropical areas, such as india, africa and southern asia. sweet basil is an annual herbaceous species that is usually cultivated as an aromatic plant. the minimum temperature for the growth of sweet basil has been determined to be 10.9°c (vågen et al., 2003; chang, 2004). it is typically used in italian and asian cuisines because of the pronounced scent of its leaves, which depends on genotype (chang et al., 2009), nitrogen nutrition (sifola and barbieri, 2006) and harvesting system (may et al., 2008). sweet basil is not only cultivated for the use of aroma additives in food but also for other house-hold purposes, pharmaceuticals, cosmetics and folk medicine. numerous different chemo-types exist in both wild and cultivated (*) corresponding author: thais.huarancca@agr.unipi.it citation: ciurli a., huarancca reyes t., guglielminetti l., 2017 commercial advantages on basil architecture by ultraviolet-b irradiation. adv. hort. sci., 31(3): 215-221 copyright: © 2017 ciurli a., huarancca reyes t., guglielminetti l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 march 2017 accepted for publication 22 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 215-221 216 basil. for instance, sweet basil contains high levels of phenylpropanoids, e.g. eugenol and methyleugenol, and terpenoids e.g. l inalool and 1,8-cineole (lachowicz et al., 1997). in recent years, the consumption of fresh basil has been expanded in supermarkets where young seedlings are directly sold in pots obtained from greenhouses. these plants are intended for family use, which after to be transplanted, are placed on balconies, vegetable gardens or gardens. the marketing of fresh basil highlights the problem that these plants have long and slender stems, which are easy to break off and thus making difficult their market distributions. therefore, the objective of this study was to facilitate the sale of fresh basil in pots resulting not only in plants more compact and resistant to movements along the supply chain, but also healthy products in the market because of the avoidance of chemicals to control the plant height (körner and van straten, 2008; nagashima et al., 2011). the interest on the effects of ultraviolet (uv) irradiation on plants has considerably increased in the last ten years due to the continuous depletion of the ozone layer (ballaré et al., 2011). the stratospheric ozone layer completely absorbs solar uv-c (200-280 nm) which is extremely active and biologically lethal. uv-b (280-320 nm) is the most susceptible to ozone layer depletion because it is efficiently absorbed although small proportion is transmitted to the earth surface. uv-a (320-400 nm) is hardly absorbed by ozone and thus passes almost unaltered through the stratospheric layer reaching the earth surface (houghton et al., 2001; solomon et al., 2007). despite the small proportion of uv-b in the natural daylight and higher energy than uv-a, uv-b has substantive effects on plant growth and metabolism (kolb et al., 2001). moreover, plants are differently sensitive to uv-b levels which strongly depend on the latitude, hours of direct sunlight and variation in the thickness of the ozone layer (ballaré et al., 2011). for instance, plant species grown in mediterranean and tropical environments and/or at high altitudes have developed defensive mechanisms to protect themselves against uv radiation (zheljazkov et al., 2008). many studies found that uv-b irradiation significantly affects secondary compounds such as the biosynthetic pathway of phenylpropanoids, which are antioxidant agents (korkina, 2007) that also act as protection against uv (johnson et al., 1999; ioannidis et al., 2002). previous studies reported that uv-b irradiation also increases essential oils and total phenolic compounds content in plants (kumari et al., 2009; kumari and agrawal, 2011). moreover, it has been demonstrated that the uv-b irradiation stimulates the production of volatiles organic compounds in fresh herbs which usually are depleted when plants grown under glass or plastic greenhouses conditions in the absence of natural levels of uv-b (johnson et al., 1999; ioannidis et al., 2002). in the case of plant morphology, several studies have been demonstrated the role of uv-b in controlling the growth of various plants such as lycopersicon esculentum mill. and salvia splendens l. (garner and björkman, 1996; giannini et al., 1996; del corso and lercari, 1997). additional techniques to control plant growth have been proposed: temperature manipulation, induction of mild water or mechanical stress, and the use of chemical compounds, however all these methods present many drawbacks (moe and mortensen, 1992; garner and björkman, 1996; barreiro et al. 2006; sun et al., 2008). therefore, we focused on the role of uv-b as a growth regulator of potted basil intended for fresh consumption. 2. materials and methods plant material and growth conditions two cultivars of basil were used in this study, genovese and profumo. basil seeds were sowed into peat-based growing medium in 8 cm diameter pots (10 seeds each pot). pots were covered with nonwoven fabric and incubated in a growth chamber at 23±1°c, relative humidity 60-65%, 12 h light (150 μmol m-2 s-1). after one week of incubation, the coverage was removed and half of the pots were kept at the same conditions while the other half were under supplementary uv-b irradiation (fluorescent tubes with uv-b 10%, 15 w m-2, exo terra). uv-b was applied for 3 h starting 1 h before darkness, and lamps were placed at 0.5 m above plants. basil plants were regularly irrigated. control and uv-b treated plants were sampled 11 and 22 days after the onset of uv treatment. morphological analysis leaf length and width, shoot length, fresh and dry weight of shoot and leaves were determined upon treatment completion. all measurements were the mean of three independent experiments. analysis of pigments pigments were extracted and analysed from full expanded leaves as previously described (pompeiano et al., 2013). all the analyses were conducted in tripciurli et al. commercial advantages on basil architecture by uv-b 217 licate. statistical analysis the statistical analyses of biometric and physiologic traits were subjected to an analysis of variance (anova). differences between treatments were assessed using the f-test, and the least significant difference (lsd) was calculated at p≤0.05. all computations were performed with r 2.14.2 r development core team 2012. 3. results in this study, we analyzed the plant architecture and photosynthetic pigments content of two basil cultivars, profumo and genovese, at time zero (t0) and after treatment. two different time points of treatment were analyzed: 11 and 22 days after uv-b treatment (uv) and their respective control (c) conditions. morphological analysis was determined on leaves and shoots of both basil cultivars (fig. 1, 2). length and width of cotiledonary leaves did not show differences between cultivars at t0, and also between treated and control plants during all the experimental time (fig. 1). the first true leaves, which appeared at the first time point of the experiment, showed a significant increased size from 11 to 22 days at control conditions in both cultivars (fig. 1). it was also observed that uv treated plants significantly increase, about two times, the leaf expansion in both cultivars in comparison with their respective control (fig. 1). interestingly, only 22 days uv treated seedlings showed the second leaves pair without morphological differences between the cultivars (fig. 1). in the case of shoot length evaluation, no differences were observed between cultivars at t0, although ‘genovese’ showed longer shoot than ‘profumo’ at 11 and 22 days control conditions (fig. 2). moreover, the uv irradiated plants showed a considerably shorter shoots than that of control plants at both 11 and 22 days after treatment, and in all cases ‘genovese’ still longer than ‘profumo’ (fig. 2). the effect of uv irradiation on basil biomass of both cultivars was also analyzed (fig. 3). at t0, no differences were exhibited in fresh weight (fw) and dry weight (dw) leaves (only cotyledon) between cultivars, while fw and dw shoots were almost double in ‘genovese’ in comparison with ‘profumo’ (fig. 3 a-b). fig. 1 effect of uv-b radiation on the morphology of basil leaves. two cultivars of basil were use: (a, c) cv. profumo and (b, d) cv. genovese. (a, b) length and (c, d) width of basil leaves were measured at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. cotyledonary (white bars), first (grey bars) and second leaves (black bars) were measured separately. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). fig. 2 effect of uv-b radiation on the length of basil shoots. two cultivars of basil were use: (a) cv. profumo and (b) cv. genovese. shoot length of basil cultivars was measured at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). adv. hort. sci., 2017 31(3): 215-221 218 at 11 days after treatment, no differences were reported in fw and dw of shoot or leaves (cotyledon plus first pair) between cultivars (fig. 3 c-d). both cultivars treated with uv irradiation showed significant increase of fw and dw leaves in comparison with that of the control conditions, while no differences were reported in shoots (fig. 3 c-d). at 22 days after treatment the biomass in both cultivars was increased in comparison with that of 11 d, while similar pattern was maintained when it was compared the biomass before and after uv irradiation (fig. 3 ef). the photosynthetic pigments analyzed in both cultivars include chlorophyll a, chlorophyll b and carotenoids (fig. 4). all pigments were detected at t0 without differences among cultivars. then, the amount was dramatically reduced after 11 days under control conditions as well as uv irradiation, showing no statistical differences even between cultivars (fig. 4). at 22 days after treatment, the level of chlorophyll b was maintained similar to that of 11 days and no differences were registered between control and uv conditions (fig. 4). chlorophyll a and carotenoids levels at 22 days under control conditions were increased in respect to that of 11 days, and no differences were detected when compared with that of after uv treatment (fig. 4). 4. discussion and conclusions results of the present study pointed towards a positive impact of supplementary uv-b irradiation on basil architecture, resulting on the reduction of stem elongation, increase of leaf length and width, increase of biomass, induction of second leaf sproutfig. 3 effect of uv-b radiation on fresh and dry biomass of basil. two cultivars of basil were use: (a, c, e) cv. profumo and (b, d, f) cv. genovese. measurements were monitored at (a, b) time zero (7 days old basil seedlings) and after treatment. two different time points of treatment were evaluated: (c, d) 11 days and (e, f) 22 days after uv-b treatment (uv) or control (c) conditions. s: shoot, l: leaf, fw: fresh weight, dw: dry weight. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). fig. 4 effect of uv-b radiation on photosynthetic pigments in basil leaves. two cultivars of basil were use: (a) cv. profumo and (b) cv. genovese. measurements were monitored at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. chlorophyll a (white bars), chlorophyll b (grey bars) and carotenoids (black bars) content are expressed as mg g fw-1. each value is the mean ± sd of three independent experiments ciurli et al. commercial advantages on basil architecture by uv-b 219 ing and with a similar trend of photosynthetic pigments content in comparison to that of plants under control conditions. these uv-b effects on plants are in accordance with previous studies (giannini et al., 1996; del corso and lercari, 1997). the reduction of stem elongation found in our research was also reported previously in other species such as wheat (yuan et al., 1998). this phenomenon could be related to the impact of uv-b radiation on phytohormones metabolism, such as photo-oxidation of indole-3-acetic acid which plays an important role in stem elongation and lateral shoots (ros and tevini, 1995; mark and tevini, 1996). in addition, effects of uv-b radiation on leaf thickening, leaf elongation and biomass accumulation are highly dependent on uv-b dose and source, experimental parameters and species of study (kakani et al., 2003). therefore, in this study, we used growth chamber instead of open fields and greenhouses in order to control all the environmental conditions, making this condition replicable in any situation and avoiding the influence of environmental and seasonal variations. our results showed also an increase of leaf thickening, leaf elongation and biomass after uv-b irradiation, which could be related to a morphological architecture strategy to protect plants to deleterious effects of uv-b radiation (maffei and scannerini, 2000; jansen, 2002; santos et al., 2004; chang et al., 2009). moreover, the generation of second leaves in basil plants was earlier promoted after uv-b treatment which may also involve phytohormones metabolism changes; this effect was also previously reported (barnes et al., 1988). thus, structural changes on leaves in response to the applied uv-b were observed, although no changes on the content of chloroplast pigments was registered between uv treated and control plants. photosynthetic pigments are useful indicators of uv-b tolerance or sensitivity (kataria et al., 2014). lower pigments content was obtained at 11 days of treatment in comparison with t0, which could be due to the stress arising from the removal of the non-woven fabric that protected plants from excessive light. however, this phenomenon was recovered in a tendency to the start point t0, suggesting the acclimatization of plants, as observed similarly in previous report (teramura and sullivan, 1994; radyukina et al., 2012). therefore, our data suggest that genovese and profumo basil cultivars are tolerant to the supplementary uv-b irradiation (15 w m-2; 3 h day-1) at chlorophyll level, but producing other strategy to prevent uv-b penetration to the mesophyll cell at plant architecture level. although the impacts of uv-b radiation on plant growth and development have been widely studied (strid et al., 1994; ballaré et al., 1995; giannini et al., 1996; del corso and lercari, 1997; santos et al., 2004; zu et al., 2004; körner and van straten, 2008), the detailed mechanism of how uv-b radiation affects plant morphogenesis is still unclear. however, previous evidences found that uv-induced morphological changes are associated with the induction of the phenylpropanoids pathway resulting in accumulation of flavonoids (jansen et al., 1998). moreover, it has been demonstrated that flavonoids regulate auxin transport affecting plant architecture (jansen, 2002; robson et al., 2015). in conclusion, the results of this work have practical implications for greenhouse production of pot basil. in fact, supplementary uv-b radiation reduced plant height but increased biomass and leaf number, width and length with no significant effect on photosynthetic pigments. therefore, the application of uv lamps could replace chemical growth retardants and/or other techniques adopted for growth control in the production of potted basil for fresh market. acknowledgements we thank prof. alberto pardossi (university of pisa, italy) for helpful discussions. this research was supported by a grant from the social cooperative parvus flos. references ballaré c.l., barnes p.w., flint s.d., 1995 inhibition of hypocotyl elongation by ultraviolet-b radiation in deetiolating tomato seedlings. i. the photoreceptor. physiol. plant., 93(4): 584-592. ballaré c.l., caldwell m.m., flint s.d., robinson s.a., bornman j.f., 2011 effects of solar ultraviolet radiation on terrestrial ecosystems. patterns, mechanisms, and interactions with climate change . photochem. photobiol. sci., 10(2): 226-241. barnes p.w., jordan p.w., gold w.g., flint s.d., caldwell m.m., 1988 competition, morphology and canopy structure in wheat (titicum aestivum l.) and wild oat (avena fatua l.) exposed to enhanced ultraviolet-b radiation. funct. ecol., 2(3): 319-330. barreiro a.p., zucareli v., ono e.o., rodrigues j.d., 2006 growth analysis of basil plants submitted to plant growth regulators. bragantia, 65(4): 563-567. chang x., 2004 effects of light and temperature on adv. hort. sci., 2017 31(3): 215-221 220 volatile oil compounds and growth in basil (ocimum basilicum l.). ph.d. thesis, university of nottingham. chang x., alderson p.g., wright c.j., 2009 enhanced uv-b radiation alters basil (ocimum basilicum l.) growth and stimulates the synthesis of volatile oils. j. hort. sci. biotech., 1(2): 27-31. del corso g., lercari b., 1997 use of uv radiation for control of height and conditioning of tomato transplants (lycopersicon esculentum mill.). sci. hortic., 71 (1-2): 27-34. garner l.c., björkman t., 1996 mechanical conditioning for controlling excessive elongation in tomato transplants: sensitivity to dose, frequency, and timing of brushing. j. amer. soc. hort. sci., 121(5): 894-900. giannini a., pardossi a., lercari b., 1996 the use of uv radiation to control the architecture of salvia splendens plants. i. effects on plant growth, water relations and gas exchange. photochem. photobiol., 64(1): 123130. gülçin i̇., elmastaş m., aboul-enein h.y., 2007 determination of antioxidant and radical scavenging activity of basil (ocimum basilicum l. family lamiaceae) assayed by different methodologies. phytother. res., 21(4): 354-361. houghton j.t., ding y., griggs d.j., noguer m., van der linden p.j., dai x., maskell k., johnson c.a., 2001 climate change 2001. report of the intergovernmental panel on climate change (ipcc). cambridge university press, cambridge, uk. ioannidis d., bonner l., johnson c.b., 2002 uv-b is required for normal development of oil glands in ocimum basilicum l. 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sci., 2018 32(4): 495-501 doi: 10.13128/ahs-21934 onion crop response to different irrigation and n-fertilizer levels in dry mediterranean region i. mubarak (*), a. hamdan department of agriculture, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: bulb shape index, bulb yield, deficit irrigation, water productivity. abstract: due to the water scarcity in dry mediterranean condition, determination of water and nitrogen (n) fertilizer needs is a major challenge for crop production and environment protection. pot experiments under open field conditions were conducted for two consecutive years (2016 and 2017) to assess the effects of various levels of n-fertilizer and irrigation on onion crop, following a 4×3 factorial experiment arranged in a randomized block design with four nfertilizer rates (0, 40, 80, and 120 kg n ha-1), and three irrigation levels (100, 80, and 60% of the seasonal water use), with three replications. results indicated that the initial soil n-content (about 60 kg n ha-1) was sufficient to meet crop nitrogen requirements. however, results indicated that onion crop was sensitive to water stress, so that the highest total bulb yield (by, 19.1 t ha-1), dry matter in bulbs (dm, 2.97 t ha-1), and water productivity (wp, 1.9 kg m-3) were found under full irrigation compared to the deficit conditions. by, dm, and wp were predicted to be increased linearly with increasing levels of irrigation. the developed equations could be used for predicting onion crop yields under similar agro-pedo-climatic context, and as a tool for rational management of limited irrigation water. 1. introduction onion (allium cepa l.) is one of the most important horticultural crops worldwide. many studies have been carried out regarding the water and nitrogen fertilizer requirements of onion crop and the effects of deficit irrigation on yield and yield components (abdissa et al., 2011; igbadun et al., 2012; patel and rajput, 2013; tsegaye et al., 2016). a nitrogen fertilizer level of less than 100 kg ha-1 was found to be sufficient for onion crop production as in abdissa et al. (2011) and tsegaye et al. (2016). russo (2008) reported that nitrogen fertilizer had no significant effect on onion yield. moreover, the onion crop was found to be more moderately responsive to water deficit during the total growing season; and it is better to partition the water stress throughout the growing season (regulated deficit irrigation, rdi) rather than creating a stress during the (*) corresponding author: ascientific4@aec.org.sy citation: mubarak i., hamdan a., 2018 onion crop response to different irrigation and n-fertilizer levels in dry mediterranean region. adv. hort. sci., 32(4): 495-501 copyright: © 2018 mubarak i., hamdan a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2017 accepted for publication 28 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 495-501 496 critical stages of crop growth (kirda, 2000; kadayifci et al., 2005; patel and rajput, 2013). the deficit irrigation given at different levels (up to 40%) was found to be economically recommended. this wide range in allowable deficit levels could be due to the various agro-pedo-climatic context of their studied regions. in other word, onion grown in different soil and crop management factors responded differently to the application of both deficit irrigation and n-fertilizer. therefore, there is a continuous need to select both optimum n-fertilizer rate and irrigation level for onion crop in ever changing agro-pedo-climatic conditions. in dry areas of the mediterranean region, scarcity of water is the most limiting factor for onion crop production, due to the lack of rainfall over the production period between april and august (ragab and prudhomme, 2002; turner, 2004). the onion crop is grown in arid and semi-arid area in that region, with many cultivars. in the eastern mediterranean, as in syria, the ovalto elongated-shape onion, referred to as ‘selmouni red’, is the famous variety used for its long-day storage capacity. growers have targeted the larger bulb size and the higher marketable yield. they imagined the greater yields would require increased n fertilizer and water amount. high nitrogen fertilizer and irrigation water amounts may allow nitrate and other components to be most likely deeply percolated. so, research findings are an urgent need to determine water and n-fertilizer requirements for the onion crop in the production areas. in this context, the study reported herein has been designed to predict onion crop response to various levels of regulated deficit irrigation and n-fertilizer rates in dry mediterranean areas. results may contribute to introduce practical alternatives that would sustain onion productivity while using less water and fertilizer in the context of water scarcity and environmental protection. 2. materials and methods pot experiments under open field conditions were carried out at the deir al-hajar agricultural experiment station, damascus, syria (33°20′ n, 36°26′ e, altitude 600 m), for two consecutive growing seasons 2016 and 2017. table 1 shows some climatic data for the studied site during the course of these experiments. no rainfall was recorded during both growing seasons. the chemical and physical soil properties are: ph 8.0; ece 0.58 ds m-1; organic matter 1.19%; available p 6 ppm; total n 0.06%; no3 51.8 ppm; nh4 + 39.2 ppm. particle-size distribution analysis showed that the soil contains on average 27.8% sand, 42.7% silt and 29.5% clay, and therefore classified as a clay loam. average volumetric soil water contents at field capacity (fc) and permanent wilting point (pwp) are 0.36 and 0.18 m3 m-3, respectively. each pot was of diameter and depth of 25×30 cm, and contained 8 kg of natural soil from the field. three small bulb sets (also called as bulbils or bulblets) of onion (allium cepa l. ‘selmouni red’) were grown in each pot. after germination, plants were thinned to two bulb sets per pot, making a plant density of about 400000 plants ha-1. the pots were set outdoors under natural climatic conditions. the experiment was started on the planting day (march 31st and april 2nd for the 2016 and 2017 seasons, respectively) with the soil water content (swc) of all pots at field capacity (measured by pot’s weight). the experiment was laid out following a 4×3 factorial experiment arranged in a randomized complete block design (rcb design) with four n-fertilizer rates (n0, n40, n80, and n120), and three irrigation levels (fi, di80, and di60), with three replications, making a total of 36 pots. the n-fertilizer rates composed of n0, n40, n80, and n120 with 0, 40, 80, and 120 kg n ha-1 added to the soil, respectively. the three distinct irrigation treatments were: fi treatment (full irrigation) in which plants received 100% of accumulated crop evapotranspiration (100% of etc) and the root zone was replenished to field capacity; di80 and di60 treatments (regulated deficit irrigation) were irrigated at the same frequency as fi treatment but with season variable apr. may jun. jul. aug. 2016 tmin (°c) 11.6 14.9 18.7 19.9 21.0 tmax (°c) 29.2 30.5 36.6 38.1 37.8 taverage (°c) 21.2 23.6 30.6 28.9 29.5 rh (%) 67.0 58.0 69.0 64.0 65.0 precipitation (mm) 0.0 0.0 0.0 0.0 0.0 2017 tmin (°c) 9.7 14.4 17.3 20.6 20.0 tmax (°c) 26.2 31.6 35.8 40.6 38.5 taverage (°c) 19.2 24.9 28.4 31.1 28.9 rh (%) 63.1 57.9 56.3 55.6 59.3 precipitation (mm) 0.0 0.0 0.0 0.0 0.0 tmin= minimum temperature, tmax= maximum temperature, taverage= average temperature, rh= relative air humidity. table 1 some climatic data of the experimental site during both growing seasons mubarak and hamdan irrigation and n-fertilizer levels for onion crop 497 water amounts equal to 80 and 60% of the accumulated etc, respectively. in other words, the three watering treatments received at each irrigation event 1.0, 0.8 and 0.6 times the amount of soil water depleted under fi conditions, respectively. irrigation was applied 3 times per week. the pots were weighed before and after each irrigation event. the water amounts were regulated by weight (eq. 1). the depleted water amount (crop evapotranspiration, etc) (mm) between two successive irrigations was calculated as: etc = w1 w2 [1] pw x s where w1 was the weight of the pot (kg) after irrigation; w2 was the weight of the pot (kg) just before the next irrigation; ρw is the water density (g cm-3); and s is the soil surface area in the pot (m2). the daily crop evapotranspiration (mm day-1) was estimated by dividing the crop evapotranspiration estimated from eq. (1) by the number of days between two successive irrigation events. the seasonal crop evapotranspiration, i.e., the total crop water use during the growing season, was the summation of the daily etc. full doses of phosphorous and potassium were applied as basal dose at the time of planting. nitrogen was divided into two equal doses (according to the studied n-fertilizer rate) and applied with the irrigation water during early vegetative growth stage. irrigation was stopped at the end of july when over 50% dropping of leaf-tops was observed as signs of maturity. the onions were lifted to field cure about two weeks after (up to mid-august). after the leaves were completely dried, they were cut leaving about 2.0 cm above the bulb. the length, diameter, and weight of both matured onion bulbs from each pot were measured. the total bulb yield (by, t ha-1) was estimated. the dry matter in bulbs (dm, t ha-1) was also estimated by drying bulbs at 50°c to a constant weight. water productivity (wp, kg m-3) was computed by dividing the total bulb yield by the seasonal evapotranspiration. bulb shape index (sh i) was also calculated as the relationship between bulb length and diameter. no multi-centred bulbs were observed at harvest even under deficit irrigation. with two factors (n-fertilizer rate and irrigation level), the two-way analysis of variance (anova) was conducted using the dsaastat add-in version 2011 (onofri, 2007). a combined analysis of data over both years was performed to verify if n-fertilizer rate and irrigation level may have a significant and stable effect over year. mean comparison was made only for data after combined analysis using duncan’s multiple range test (dmrt) at the 1% level of significance. trend analysis (regression analysis) was also used to examine the relationship between measured variables and the quantitative factors showing a significant effect. the trend analysis was done based on the method of orthogonal polynominals as described by gomez and gomez (1984). 3. results and discussion the effects of years, n-fertilizer rates, and irrigation levels on the measured variables of onion crop (by, dm, wp, and sh i) were summarized in table 2. since no significant interaction year × treatments was observed, data are shown as the averages of the two years (table 3 and figs. 1-3). total bulb yield the combined analysis of data over years indicated that only the main effect of irrigation was significant, and highly influenced by (p<0.01) (table 2 and 3). no significant change in by was recorded with the addition of n fertilizer (table 3), indicating that the initial soil nitrogen content (about 60 kg n ha-1) was sufficient to meet the crop nitrogen requirements, and no extra n-application was needed. this is in agreement with the results of abdissa et al. (2011) who found that a rate of 69 kg n ha-1 was sufficient * = significant at 5% level, ** = significant at 1% level, ns = nonsignificant at 5% level. b = the degree of freedom of reps. within year is not adequate for valid test of significance (gomez and gomez, 1984). df = degree of freedom, by = total bulb yield, dm = dry matter in bulbs, wp = crop water productivity, and sh i= shape index. table 2 analysis of variance of the combined data of measured variables as affected by years, n-fertilizer rates, and irrigation levels (f-test values) source of variation df by dm wp sh i year (y) 1 b b b b reps. within year 4 irrigation level (i) 2 213.4 ** 75.44 ** 41.35 * 12.30 ns n-fertilizer rate (n) 3 <1 <1 <1 1.90 ns y x n 3 2.20 ns 1.24 ns 1.65 ns 1.30 ns y x i 2 <1 <1 1.03 ns <1 n x i 6 <1 1.82 ns <1 <1 y x n x i 6 2.89 ns 1.47 ns 2.58 ns 2.61 ns residual (pooled error) 44 total 71 cv (%) 18.6 22.8 18.9 14.9 adv. hort. sci., 2018 32(4): 495-501 498 for onion crop production. tsegaye et al. (2016) reported that a n level of 100 kg ha-1 was economically recommended for onion in southern ethiopia. according to russo (2008), nitrogen fertilizer did not affect onion yield. the effect of irrigation level resulted in the highest by (19.1 t ha-1) in fi treatment, while under di80 conditions, total bulb yield decreased by 36.4%, reaching 63.0% of decrease (7.1 t ha-1) under di60 conditions (table 3). similar results were also obtained by kumar et al. (2007) and bekele and tilahun (2007). trend analysis indicated that the relationship between total bulb yield and irrigation level was linear within the range of irrigation levels tested (r2=0.991 with p<0.01) (fig. 1). a significant linear decrease in the total bulb yield was predicted with increasing water deficit. in other word, by increased with increasing irrigation level (fig. 1). this is in agreement with other published findings (kadayifci et al., 2005; kumar et al., 2007; bekele and tilahun, 2007; nagaz et al., 2012). for instance, nagaz et al. (2012) observed that applying 60% of crop evapotranspiration (etc) caused significant decreases in fresh yield, dry matter, bulbs per hectare and bulb weight, compared to full irrigation (100% etc). this result confirmed the sensitivity of the onion crop to water deficit during the total growing season. on the contrary, other studies showed that onion crop is responsive to deficit conditions as compared with full irrigation. for example, tsegaye et al. (2016) and igbadun et al. (2012) found that deficit irrigation given at 75% of etc was economically recommended. also, nagaz et al. (2012) found no significant differences between regulated deficit irrigation at 80% etc and full irrigation (100% etc). these differences in onion crop response to deficit irrigation levels could be due to the various agro-pedo-climatic conditions. dry matter yield in onion bulbs (dm) also dm was found to be highly affected only by the main effects of irrigation levels (table 2 and 3). the mean values of dm were 2.97, 2.16, and 1.27 t ha-1 under fi, di80 and di60, respectively. that is to say, significant decreases of 27.2 and 57.1% in dm could be attained when onion crop was under di80, and di60, respectively, compared with fi (p<0.01) (table 3). trend analysis designated that the response of dm to various irrigation levels followed a linear relationship (r2=0.999 with p<0.01). dry matter yield increased with increasing levels of irrigation as can be seen in figure 2. thus, dry matter yield could be maximized whatever the n-fertilizer rate used in this study, when full irrigation was applied. several studied reported similar results, showing that dry matter yield was maximized under full irrigation rather than under deficit conditions (e.g., nagaz et al., 2012). concerning fertilizer rates, as mentioned above, anova did not detecte any significant change in dm yield with the addition of nitrogen fertilizer (table 3). this is in agreement with the findings of russo fig. 1 response of onion total bulb yield (by) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of by averaged over both years and all levels of n-fertilizer, at the specific irrigation level. in each column and for each studied factor, means followed by different letters are significantly different according to the dmr test. table 3 effect of n-fertilization rate and irrigation level on total bulb yield (by), dry matter in bulbs (dm), crop water productivity (wp), and shape index (sh i) in onion ‘selmouni red’ (data are the averages of the two years of experimentation) studied factor by (t ha-1) dm (t ha-1) wp (kg m-3) sh i (cm cm-1) n-fertilization rate n0 (0 kg n ha-1) 13.47 a 2.26 a 1.59 a 1.78 a n40 (40 kg n ha-1) 12.26 a 2.14 a 1.47 a 1.86 a n80 (80 kg n ha-1) 12.73 a 2.06 a 1.52 a 1.86 a n120 (120 kg n ha-1) 12.61 a 2.08 a 1.47 a 1.65 a irrigation level full irrigation (100% etc) 19.08 a 2.97 a 1.90 a 1.72 a deficit irrigation at 80%of etc 12.14 b 2.16 b 1.50 b 1.67 a deficit irrigation at 60%of etc 7.08 c 1.27 c 1.14 c 1.98 a mubarak and hamdan irrigation and n-fertilizer levels for onion crop 499 (2008). this result newly indicated that the initial soil nitrogen content was sufficient to meet the n-fertilizer needs of onion crop. irrigation water applied and water productivity with no rainfall received during both growing seasons (table 1), large water amounts were applied to meet the high crop water demand. the irrigation water applied to fi, di80, and di60 were, 1014, 822, and 634 in the 1st season, and 1039, 849, and 566 mm in the 2nd one, respectively. seasonal crop evapotranspiration (etc), as calculated using eq. (1), during the 2016 season was 993, 803, and 615 mm, and during the 2017 season was 1013, 823, and 630 mm, for fi, di80, and di60, respectively. as it can be seen, the seasonal etc values were very close to the irrigation water amounts even in di80 and di60 treatments. therefore, wp can be considered also a good estimate of irrigation water use efficiency (iwue). the combined analysis over years detected that wp was highly significantly influenced by the irrigation levels. it was unchanged with the addition of n fertilizer (table 2 and 3). the mean value of wp under fi (1.90 kg m-3) was significantly higher than both deficit irrigation levels, i.e., di80 (1.50 kg m-3) and di60 (1.14 kg m-3) (table 3). trend analysis indicated that the relationship between wp and irrigation level was linear with values of r2 of 0.998 at the 1% level (fig. 3). results indicated that wp was not ameliorated under deficit irrigation. this could be due to high onion crop sensitiveness to the water deficit during the total growing season. thus, the water savings under deficit irrigation could not balance the huge decrease in the total bulb yield under the dry conditions. this result is in agreement with similar results obtained by kadayifci et al. (2005), who found that high water use efficiencies were observed with increasing levels of irrigation. contrariwise, this result was in disagreement with the findings of other researchers (fereres and soriano, 2007; kumar et al., 2007; bekele and tilahun, 2007; patel and rajput, 2013; tsegaye et al., 2016). for example, patel and rajput (2013) reported that with 40% deficit irrigation throughout the growing season, water productivity can be significantly improved with 272-mm water saving which may be used to irrigate additional cropped area (half a hectare). kumar et al. (2007) found that irrigation water use efficiency and water productivity both were highest under 80% of etc and then declined with the increase in irrigation with microsprinkler irrigation system. igbadun et al. (2012) showed that higher water productivity in terms of water supplied could be obtained by irrigating onion crop at 50 and 75% of etc. these different results could be due to fig. 2 response of onion dry matter yield in bulbs (dm) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of dm averaged over both years and all levels of n-fertilizer, at the specific irrigation level. fig. 3 response of onion crop water productivity (wp) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of wp averaged over both years and all levels of n-fertilizer, at the specific irrigation level. 500 adv. hort. sci., 2018 32(4): 495-501 the differences in tested onion cultivars, soil type, and climatic conditions. they indicated that onion crop produced in different agricultural managements responded differently to water stress. bulb shape index no significant effects of both tested factors were observed on this variable (table 2 and 3). as above mentioned, the ovalto elongated-shape onion, referred to as ‘selmouni red’, was tested. its shape index generally varies from 1.5 to 3.0, according to several environmental conditions including planting dates, plant density, planting depth, and soil water availability. the lower the shape index, the better the bulb shape for marketing purposes (appearance and ease of packaging). the mean values of sh i, found in this study, were 1.72, 1.67, and 1.98 under fi, di80, and di60, respectively (table 3). the average sh i under severe water stress (di60) was 14.7% larger than that under non-water stress conditions (fi). in other word, sharp deficit irrigation tended to increase the bulb length, although this increase not significant, compared to its diameter. thus, the recommended agricultural management to produce better shape index of onion bulbs, is to irrigate onion plants using irrigation level of 100 or 80% of etc. 4. conclusions the following conclusions can be drawn from the results obtained in onion subjected to different nfertlizer rates and irrigation levels in the studied agro-pedo-climatic context: no additional n application over the initial soil n content (about 60 kg n ha-1) was found to be required for the tested onion cultivar. the highest total bulb yield, dry matter, and water productivity were recorded under full irrigation compared to the deficit conditions, indicating that onion crop was sensitive to water stress. total bulb yield, dry matter, and water productivity were predicted to be increased linearly with the increment in irrigation water amount. the developed equations could be useful for predicting onion crop yields under similar agro-pedo-climatic context and for rational management of limited irrigation water. with huge crop water requirements, further studies should focus on how to improve the regulated deficit irrigation practices in order to address water shortage and sustainable crop production in dry areas of the mediterranean region. acknowledgements the authors would like to thank the aec of syria for financial support. references abdissa y., tekalign t., pant l.m., 2011 growth, bulb yield and quality of onion (allium cepa l.) as influenced by nitrogen and phosphorus fertilization on vertisol. i. growth attribute biomass production and bulb yield. afr. j. agric. res., 6(14): 3252-3258. bekele s., tilahun k., 2007 regulated deficit irrigation scheduling of onion in a semiarid region of ethiopia. agric. water manag., 89: 148-152. fereres e., soriano m.a., 2007 deficit irrigation for reducing agricultural water use. j. exp. bot., 58: 147159. gomez k.a., gomez a.a., 1984 statistical procedures for agricultural research. second edition. wiley, new york, ny, usa, pp. 680. igbadun h.e., ramalan a.a., oiganji e., 2012 effects of regulated irrigation deficit and mulch on yield, water use and crop water productivity of onion in samaru, nigeria. agric. water manag., 109: 162-169. kadayifci a., tuylu g.i., ucar y., cakmak b., 2005 crop water use of onion (allium cepa l.) in turkey. agric. water manag., 72(1): 59-68. kirda c., 2000 deficit irrigation scheduling based on plant growth stages showing water stress tolerance. deficit irrigation practices, fao water reports, rome, italy, 22: 3-10. kumar s., imtiyaz m., kumar a., singh r., 2007 response of onion (allium cepa l.) to different levels of irrigation water. agric. water manag., 89: 161-166. nagaz k., masmoudi m.m., ben mechlia n., 2012 yield response of drip-irrigated onion under full and deficit irrigation with saline water in arid regions of tunisia. isrn agronomy, 2012: 1-8. onofri a., 2007 routine statistical analyses of field experiments by using an excel extension. proceedings 6th national conference italian biometric society “la statistica nelle scienze dellavita e dell’ambiente”, pisa, italy, june 20-22, pp. 93-96. patel n., rajput t.b.s., 2013 effect of deficit irrigation on crop growth, yield and quality of onion in subsurface drip irrigation. inter. j. plant prod., 7(3): 417-436. ragab r., prudhomme c., 2002 climate change and water resources management in arid and semi arid regions: prospective and challenges for the 21st century. biosyst. eng., 81: 3-34. russo v.m., 2008 plant density and nitrogen fertilizer rate on yield and nutrient content of onion developed from greenhouse-grown transplants. hortscience, 43(6): 1759-1764. 501 mubarak and hamdan irrigation and n-fertilizer levels for onion crop tsegaye b., bizuayehu t., woldemichae a., mohammed a., 2016 yield and yield components of onion (allium cepa l.) as affected by irrigation scheduling and nitrogen fertilization at hawassa area districts in southern ethiopia. j. agric. sci. food technol., 2(2): 15-20. turner n.c., 2004 sustainable production of crops and pastures under drought in a mediterranean environment. ann. appl. biol., 144: 139-147. impaginato 239 adv. hort. sci., 2022 36(3): 239­245 doi: 10.36253/ahsc­12192 contrasting aspects of the physical and physiological dormancy by seeds from four peach rootstocks r.d. menegatti 1, m.a. lima 1, a.g. souza 2 (*), o.j. smiderle 3, v.j. bianchi 1 1 federal university of pelotas, capão do leão, brazil. 2 centro university of maringá, maringá, brazil. 3 brazilian agricultural research corporation embrapa roraima, boa vista, brazil. key words: pre­germination treatment, prunus persica, seed propagation. abstract: considering that cultivars of peach rootstocks selected to be propa­ gated by seeds show variation in the degree of physiological dormancy and peculiarities regarding the limitation imposed by the endocarp, it is essential to define the cold stratification period and the ideal temperature to be used dur­ ing the pre­germination treatment. isolated for each cultivar, knowledge of these variables which will provide viability in the production of peach root­ stocks via seeds, in the presence of the endocarp, at a larger scale. two germi­ nation tests were carried out, in a completely randomized experimental design and 4 x 3 factorial scheme, with four replications, each one consisting of 25 endocarps. the first experiment was performed with four cultivars (aldrighi, capdebosq, okinawa roxo and tsukuba 1) and three stratification tempera­ tures (1°c, 4°c and 7°c) for a period of 90 days. for the second experiment, the same cultivars were used, two temperatures (7°c for ‘aldrighi’ and ‘capdebosq’ and 4°c for ‘okinawa roxo’ and ‘tsukuba’) and three stratification periods (40, 80 and 120 days), followed by sowing in substrate under greenhouse conditions for a period of 45 days, with subsequent endocarp breaking and re­sowing. the pre­germination treatment at 7°c for 90 days is sufficient to obtain high germi­ nation percentage of ‘aldrighi’ and ‘capdeboscq’ seeds in the presence of the endocarp. under the stratification conditions tested, the seeds of ‘okinawa roxo’ and ‘tsukuba 1’ require rupture of the endocarp to reactivate the germi­ native embryonic process. 1. introduction the production of peach rootstocks [prunus persica (l.) batsch] is tra­ ditionally done by seed, being the simplest and the most inexpensive method when compared to clonal propagation (martins et al., 2014; fischer et al., 2016). despite the fact that stone fruit production is very important in brazil, the country still presents relatively low orchard aver­ age productivity, mainly in rio grande do sul state (fischer et al., 2016). factors associated with this low productivity include pest incidence, dis­ (*) corresponding author: alineufla@hotmail.com citation: menegatti r.d., lima m.a., souza a.g., smi­ derle o.j., bianchi v.j., 2022 ­ contrasting aspects of the physical and physiological dor‐ mancy by seeds from four peach rootstocks. ­ adv. hort. sci., 36(3): 239­245. copyright: © 2022 menegatti r.d., lima m.a., souza a.g., smiderle o.j., bianchi v.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/ index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the origi­ nal author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 october 2021 accepted for publication 29 june 2022 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-12192 http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 239­245 240 eases, climate and seedling quality, mainly in the form of rootstock production; in general, they are obtained from seeds discarded by the canning indus­ try and, therefore, with no genetic identity (souza et al., 2018). currently, at federal university of pelotas and embrapa temperate climate, a wide range of root­ stock cultivars for peach seedling production are available to growers (souza et al., 2018; menegatti et al., 2019 a; souza et al., 2019 a; menegatti et al., 2020). however, peach seeds of different genotypes exhibit considerable divergence in germination rate as a result of the variability in the degree of physical and physiological dormancy of the seeds, constituting a limitation for commercial seedling production of the specie (souza et al., 2017; menegatti et al., 2019 b). souza et al. (2017) and souza et al. (2019 b) sug­ gest that the combination of physical and physiologi­ cal dormancy presented by seeds of temperate cli­ mate species, such as peach, may be one of the main factors responsible for the variation in the germina­ tion percentage of rootstocks seeds. this variation requires the exposure of seeds to different combina­ tions of time and low temperatures to overcome dor­ mancy and, consequently, to the maximizing and homogeneity of germination. according to souza et al. (2016) the overcoming of this dormancy can be achieved through techniques that promote the rup­ ture of the endocarp, popularly known as the stone. endocarp removal, suggested as a method for obtain­ ing high germination percentages in peach seeds (souza et al., 2017), is an operation that results in an additional cost for the nurseryman, mainly due to the high cost of the procedure, which must be performed manually and individually in order not to compromise the physical integrity of seed structures (wagner júnior et al., 2013). considering that cultivars of peach rootstocks selected to be propagated by seeds show variations in the degree of physiological dormancy and peculiar­ ities regarding the limitation imposed by the endo­ carp, it is essential to define the cold stratification period and the ideal temperature to be used during the pre­germination treatment, isolated for each cul­ tivar (cv.), which will provide viability in the produc­ tion of peach rootstocks via seeds, in the presence of the endocarp, at a large scale. therefore, this study aimed to evaluate contrasting aspects of physical and physiological dormancy shown by seeds of different peach rootstock cultivars. 2. materials and methods the present study was carried out with plant material obtained from ripe fruits harvested from clonal matrix plants of peach cvs. aldrighi, capdeboscq, okinawa roxo and tsukuba 1, from federal university of pelotas peach rootstock germplasm collection. two different experiments were performed, both referring to germination tests. in the first one, the endocarps were packed in tulle bags and placed in plastic boxes (40 x 27 x 10 cm) and covered with fine vermiculite, previously and periodi­ cally moistened with distilled water and fungicide solution (benlate 500 ­ 2 g l­1). then the plastic boxes were placed in biochemical oxygen demand (bod), in different temperatures, where they remained for 90 days in cold weather for stratification, with four cvs. (aldrighi, capdebosq, okinawa roxo and tsukuba 1) and three stratification temperatures (1°c, 4°c and 7°c), with four repetitions, each one consisting of 25 endocarps. at the end of the germination test (at 90 days), the percentage of seed germination was determined, with germinated seeds considered as those that pre­ sented radicle protrusion of at least two millimeters (mapa, 2009). with these data, it was possible to determine the ideal temperature to be used during the pre­germinative treatment of the seeds of each peach rootstock cv. in the presence of the endocarp. from these results, the second experiment was performed in which the endocarps were conditioned and kept under similar conditions to the previous experiment but were subjected to different stratifica­ tion periods using the temperatures predetermined in experiment i, specifically 7°c for cvs. aldrighi and capdebosq and 4°c for cvs. okinawa roxo and tsukuba 1, and three stratification periods (40, 80 and 120 days), with four repetitions, each one con­ sisting of 25 endocarps. at 40, 80 and 120 days, the germination percentage was determined according to mapa (2009), and the endocarps in which the seeds did not germinate during the cold stratification process were sown, at 1.0 cm depth, in polystyrene trays of 72 cells (114 cm3 per cell) containing as sub­ strate composed of 25% orchard soil + 25% vermi­ culite + 25% medium sand + 25% commercial. the trays were kept under controlled greenhouse condi­ tions, with irrigation as needed, for a period of 45 days. after this period, the percentage of seedlings menegatti et al. ‐ contrasts of physical and physiological domancy of peach rootstock seeds 241 emerged under greenhouse conditions was deter­ mined (mapa, 2009), and the endocarps in which the seeds did not germinate were removed from the trays and broken to extract the seeds using a manual lathe. the seeds were then re­sown in the polystyrene trays, under the same conditions men­ tioned above, and kept under greenhouse conditions to evaluate seedling emergence. the percentage of seedling emergence was daily monitored, and the experiment was finished at 15 days after the direct re­seeding during which, in three consecutive evaluations, no new seedlings emerged in any of the treatments. all analyses was performed with the program sigmaplot version 10.0 (systat, 2006). 3. results and discussion the seeds of cvs. aldrighi and capdeboscq pre­ sented superior results regarding to germination per­ centage in the presence of endocarp when stratified at 7°c for 90 days (table 1), suggesting that such con­ ditions are adequate to overcoming seed dormancy and appropriate for inducing germination. a temper­ ature of 7°c employed in cold stratification, for a period of 90 days the cvs. aldrighi and capdeboscq provided average values of germination percentage of 82 and 80% respectively, results considered satis­ factory according to mapa (2009). souza et al. (2017) evaluated the stratification of the seeds of these same cultivars, under identical conditions, but for a period of 60 days, registered lower values, which were: 56.5 and 21%, respectively, indicating that the period of 60 days is not sufficient to overcome the dormancy process and, consequently, to obtain satis­ factory germination results. these results also suggest that the 30­day increase, from the 60 days suggested by souza et al. (2017), to 90 days proposed in the present study, for the pre­germinative treatment of seeds from these cultivars. in the presence of endocarp, might have promoted significant changes in the hormonal bal­ ance that controls the process of physiological dor­ mancy, thereby making the seed metabolism more active and the embryo able to resume development. additionally, it was observed that when the stratifi­ cation temperature was reduced to 4°c and 1°c, the germination percentages decreased significantly for ‘capdeboscq’ and ‘aldrighi’, being about 54% and 15%, respectively (table 1). the seeds of cvs. okinawa roxo and tsukuba 1 showed low average germination percentages at the end of the stratification period, regardless of the temperature tested. the best results were obtained in the treatment in which the endocarps remained in stratification at 4°c for 90 days, which were: 10% and 11%, respectively (table 1). these results are superi­ or to those obtained by souza et al. (2017), who reported zero germination rate for these two cvs., when the seeds in the presence of the endocarp were stratified at 7°c, for a period of 60 days. although these results suggest that the prolonga­ tion of seed stratification period in the presence of the endocarp and the reduction in temperature from 7°c to 4°c, allow expressive increases in the germina­ tion percentage of seeds of these cultivars, these val­ ues are still considered unsatisfactory according to mapa (2009). based on the best results obtained in the first experiment, seeds from the four rootstocks were subjected to stratification temperatures that were considered most appropriate, with reductions and extensions of the stratification period. thus, ‘aldrighi’ and ‘capdeboscq’ endocarps were placed to germinate at 7°c and ‘tsukuba 1’ and ‘okinawa roxo’ were stratified at 4°c. in figure 1 a and b, the effects of the prolongation of the cold stratification period (in days) on the seed germination percentage in the presence of the endo­ carp are shown under the ideal temperatures of 7°c for ‘aldrighi’ and ‘capdeboscq’, and 4°c for ‘okinawa roxo’ and ‘tsukuba 1’, that were previously described as ideal for pre­germinative treatment. the prolongation of the cold stratification period induced an increase in the germination percentage, with the maximum obtained at 120 days independent of cv. evaluated, however, the most significant increase in germination rate was registered for ‘aldrighi’ (fig. 1). these results corroborate with the research done by wagner júnior et al. (2013), in which they observed superiority in the effect of cold stratification on seeds table 1 ­ mean germination values (%) of four peach rootstocks, in accordance of the temperature used in the pre­ger­ mination cold stratification treatment for 90 days temperature (°c) germination (%) aldrighi capdeboscq okinawa tsukuba 1 1°c 15 12.5 2 4 4°c 54 52.5 10 11 7°c 82 80 2 0 adv. hort. sci., 2022 36(3): 239­245 242 of some prunus cvs. according to the number of hours of accumulated cold increases. it is important to highlight that at 120 days of stratification at 7°c, ‘aldrighi’ and ‘capdeboscq’ pre­ sented an average germination percentage of 82% (fig. 1a), similar to that obtained before 90 days of cold stratification at this same temperature (table 1). these results indicate, with greater reliability, the use of pre­germinative stratification treatment at 7°c for 90 days to overcome seed dormancy of these culti­ vars, in the presence of the endocarp, and may have greater impact on the large­scale production of root­ stocks. of ‘capdeboscq’ as a rootstock in the production of peach seedlings in southern brazil (souza et al., 2018) for a long time. however, the use of this genotype is not currently recommended due to attributes such as the high susceptibility to different species of the genus meloidogyne (almeida et al., 2015, souza et al., 2019 c), a parasitic nematode, and the adherence of the fruit pulp in the endocarp. removal of the pulp necessitates the cleaning of the endocarps, facilitat­ ing the potential development of pathogens that may compromise the phytosanitary quality of the posthar­ vest seeds. these characteristics are also exhibited by ‘aldrighi’, discouraging the use of these particular cultivars, and promoting of the use of cutlivars with opposite characteristics. the increase in germination percentage with an extension of the stratification period was also observed in ‘okinawa roxo’ and ‘tsukuba 1’ seeds kept at 4°c (fig. 1b). however, at 120 days of stratifi­ cation, satisfactory germination rates were not reached (mapa, 2009), indicating that the increase in the number of cold hours beyond this period is not the main factor determining the low germination rate of these cultivars. thus, it is suggested that physical limitations imposed by the presence of the endocarp may con­ tribute decisively to the low seed germination rate of these cvs., as already evidenced by wagner júnior et al. (2013) and fischer et al. (2016), which suggests that the endocarp is a highly lignified structure, that confers high resistance to water absorption. this fac­ tor could compromise seed germination due to the difficulty of the embryo to overcome such a barrier during initial stages of the germination process when imbibition of water is critical. for these same four cultivars, souza et al. (2017) obtained germination rate of 100% when seeds were stratified without endocarp at 7°c for 25 days. on the other hand, when the seeds were stratified at 7°c for 60 days, without endocarp removal, there were 0 and 0,7% of germination rates for ‘tsukuba 1’ and ‘okinawa roxo’, respectively, suggesting that in addi­ tion to the presence of the endocarp, the early matu­ ration of the fruits of these cvs., in relation to the other genotypes, directly influence into the physio­ logical quality of the seeds, especially in relation to the embryo maturation. according to pérez­jiménez et al. (2012) when an embryo is not fully developed, it requires maintenance under specific conditions for a period of time to induce full development in order to ensure the establishment of a new seedling. fig. 1 ­ mean values of seed germination (%) in the presence of the endocarp, of the peach rootstocks cultivars, in biochemical oxygen demand (bod), in accordance of the period used in the pre­germination treatment. besides ensuring germination efficiency, the use of this pre­germination treatment to overcome seed dormancy of these cutlivars excludes mechanical pro­ cedures such as endocarps rupture, a method com­ monly indicated for many of the seeds of the prunus genus of commercial interest in which the endocarp acts as a physical limitation to the initiation of the germination process (souza et al., 2019 c). the superior seed germination rate and the lower degree of physical dormancy has promoted the use menegatti et al. ‐ contrasts of physical and physiological domancy of peach rootstock seeds 243 the differences in germination percentage of seeds previously treated with cold stratification by different temperatures and periods, followed by sow­ ing in the presence of the endocarp in trays contain­ ing commercial substrate, and kept for 45 days under greenhouse conditions are shown in figures 2 and 3. seeds of ‘aldrighi’ stratified previously at 7°c during the periods of 40 and 80 days (fig. 2), and the seeds of ‘capdeboscq’ stratified for 40 days (fig. 2b), fol­ lowed by sowing in the greenhouse did not demon­ strate a continuity in seed germination. for these treatments, a higher percentage of germination was expected during maintenance in the greenhouse given that during the period they were kept in bod the values were considered low. different behaviour was exhibited by seeds submitted to stratification treatment at 7°c for 120 days, for these two cultivars (fig. 2a and b), since they had already shown high germination percentage in bod, not showing signifi­ cant increases in the percentage of germination in the greenhouse. on the other hand, the seeds of ‘okinawa roxo’ and ‘tsukuba 1’ did not show high germination per­ centage either under bod conditions or when sub­ mitted to different stratification periods at 4°c fol­ lowed by a period under greenhouse conditions (fig. 3). this suggests once more the necessity of ruptur­ ing of the endocarps for the continuity of the germi­ native process by the embryo. souza et al. (2019 c) and menegatti et al. (2019 a) point out that in some peach rootstock cvs. the physical barrier imposed by the endocarp may be more determinant in germina­ tion than the low temperature and the cold stratifica­ tion period. the results show that physiological seed dormancy is always present and it needs to be over­ come with cold stratification so that the germination occurs, but besides physiological dormancy, the pres­ ence of the endocarp may also limit or even prevent fig. 2 ­ mean values of increase in percentage (%) of germinated seeds after pre­germination stratification treatment in accordance with the period, (a) 'aldrighi' and (b) 'capdeboscq', at 7°c, followed by sowing of the seeds in the presence of the endocarp in trays containing com­ mercial substrate and kept for 45 days in greenhouse (gr). dotted lines on darker bars indicate the limit of the average germination percentage obtained during stratifi­ cation in biochemical oxygen demand (bod), and the additional germination obtained after 45 days in greenhouse. fig. 3 ­ mean values of percentage increase (%) of germinated seeds after pre­germination stratification treatment in accordance of the period, (a) 'okinawa roxo' and (b) 'tsukuba 1', at 4°c, followed by seed sowing in the pre­ sence of the endocarp, in trays containing commercial substrate and kept for 45 days in greenhouse (gr). dotted lines on darker bars indicate the limit of the ave­ rage germination percentage obtained during stratifica­ tion in biochemical oxygen demand (bod), and the addi­ tional germination obtained after 45 days in greenhouse. 244 adv. hort. sci., 2022 36(3): 239­245 seed germination of some cultivars resulting in non­ uniform seedlings, as demonstrated by souza et al. (2019 c), souza et al. (2017), and the present study. among non­germinated seeds of ‘aldrighi’ and ‘capdeboscq’ after pre­germination treatment, fol­ lowed by sowing and maintenance in trays for 45 days in the greenhouse, when the endocarp was rup­ tured, a high percentage of nonviable seeds was found (mostly rotten), this percentage was highest with the cold stratification treatment for 40 days (table 2). moreover, the results showed that the increase of the cold stratification period induced a reduction in the number of dead seeds of these same cultivars. these results show that the presence of the endocarp, involving the seeds of these cultivars., doesn’t totally restrict the entry of water and cold to overcome dormancy, but probably the accumulation of 40 and 80 days of cold was not enough to promote effective changes in hormonal balance. this response was not observed in the great majority of ‘okinawa roxo’ and ‘tsukuba 1’ seeds (table 2), which when extracted from the endocarp were intact, with no turgidity, i.e., no evidence of water entering, which allowed the re­seeding and maintenance in the greenhouse again, for a period of 15 days. after this period, it was possible to observe a significant increase in the germination rate directly proportional with the extension of the stratification period employed, reaching a maximum value of 60 and 58%, respectively, after the stratification period of 120 days. the results obtained in this study reinforce the results published by souza et al. (2017), which com­ pared the seed germination rate of ‘tsukuba 1’ after cold stratification in the presence and absence of the endocarp and found that the germination rate was zero in the presence of the endocarp. in contrast, the authors obtained approximately 95% germination when the physical barrier (endocarp) was eliminated. similar response was also obtained by souza et al. (2017), having found that seeds without endocarp of ‘okinawa roxo’ and ‘tsukuba 1’ achieved germina­ tion percentages greater than 90%. in ‘okinawa roxo’ and ‘tsukuba 1’, the presence of the endocarp, besides preventing the entry and absorption of water by the embryo, evidenced by the absence of turgidity in the seeds present inside the endocarp even after stratification for 120 days, may also have prevented the leaching of the inhibitor substances present in the seeds. this factor would make the germination process in the pres­ ence of the endocarp more demanding regarding the stratification period. this behavior occurs espe­ cially in cultivars with greater resistance to rupture of the endocarp, as is the case with these two previ­ ously mentioned cultivars (souza et al., 2017) and ‘okinawa roxo’ according to work by fischer et al. (2016). 4. conclusions pre­germinative treatment at 7°c for 90 days is efficient to obtain high germination percentages of seeds of ‘aldrighi’ and ‘capdeboscq’ in the presence of the endocarp. total removal of the endocarp from the seeds of cultivars ‘okinawa roxo’ and ‘tsukuba 1’ enables a more efficient overcoming of physical and physiological dormancy and allows for a greater opti­ mization germination rate these cultivars that could benefit large­scale peach rootstock production. pre­germination treatment at 4°c for 80 days is not efficient to obtain high germination percentages of seeds of ‘okinawa roxo’ and ‘tsukuba 1’ in the table 2 ­ mean values of the percentage of non­germinated seeds after pre­germination stratification treatment, 'aldrighi' and 'capdeboscq', at 7°c (b) 'okinawa roxo' and 'tsukuba 1' at 4°c, for different periods, and kept for 45 days in greenhouse, per­ centage of dead seeds after this period, percentage of seeds germinated after the whole process followed by endocarp rup­ ture and re­sowing and percentage of lost seeds %s= percentage of seeds; gr= greenhouse; bod= biochemical oxygen demand. days in bod at 7°c + 45 days in gr days in bod at 4°c + 45 days in gr aldrighi capdeboscq okinawa roxo tsukuba 1 40 80 120 40 80 120 40 80 120 40 80 120 % s non­germinated 100 46 17 82 16 13 100 96 75 96 89 79 % s dead 94 12 11 78 12 8 0 2 1 0 3 2 % s germinated after rupture­endocarp 0 17 2 0 2 3 32 44 60 20 40 58 % s lost 6 17 4 4 2 2 68 5 14 76 46 19 menegatti et al. ‐ contrasts of physical and physiological domancy of peach rootstock seeds 245 presence of the endocarp. acknowledgements the authors would like to thank cnpq and capes for funding this study. references almeida c.b., souza a.g., argenta j.c., fachinello j.c., bianchi v.j., 2015 ­ the effect of rootstocks on the vigor, yield, and fruit quality of maciel peach trees. ­ rev. ciênc. agrár., 58: 301­307. fischer d.l.o., picolotto l., rocha m.s., souza a.g., bianchi v.j., 2016 ­ influência do período de estratifi‐ cação em frio úmido sobre a emergência e produção de porta‐enxertos de pessegueiro a campo. ­ rev. cong. urcamp., 1: 11­18. mapa, 2009 ­ regras para análise de sementes. ­ mapa, ministério da agricultura, pecuária e abastecimento, secretaria de defesa agropecuária, brasília, mapa­acs, pp. 395. martins a.s., bianchi v.j., zanandrea i., spinelli v.m., fachinello j.c., 2014 ­ efeito da estratificação de sementes na emergência e desenvolvimento inicial de plântulas de porta‐enxertos de pessegueiro. ­ científica, 42: 366­375. menegatti r.d., souza a.g., bianchi v.j., 2019 a ­ estimating genetic divergence between peach roots‐ tock cultivars using multivariate techniques based on characteristics associated with seeds. ­ genet. mol. res., 18: 1. menegatti r.d., souza a.g., bianchi v.j., 2019 b ­ growth and nutrient accumulation in three peach rootstocks until the grafting stage. ­ comun. sci., 10: 467­476. menegatti r.d., souza a.g., bianchi v.j., 2020 ­ different environments and doses of controlled‐release fertilizer in peach rootstocks production. ­ adv. hort. sci., 34(2): 157­166. pérez jiménez m., cos­terrer j., carrillo­navarro a., russell d., hamill s., eccleston k., bruce topp b., 2012 ­ use of embryo rescue in peach breeding in spain and australia. ­ aust. stonef. grower., 2: 12. souza a.g., brida a.l., garcia f.r.m., bianchi v.j., 2019 b ­ prospect for nematodes associated to diferent prunus rootstocks in brazil. ­ j. agric. sci., 11­253­262. souza a.g., smiderle o.j., bianchi v.j., 2018 ­ characterization biometric and morphophysiological of peach rootstock seeds using images of their seedling vigor. ­ recent pat. food nutr. agric., 10: 33­39. souza a.g., smiderle o.j., fischer l., bianchi v.j., 2019 c ­ post‐harvest management of prunus persica stones and the effects on seed and seedling quality. ­ agric. conspec. sci., 84: 1­6. souza a.g., smiderle o.j., menegatti r.d., lima m.a.c., neves t.r., bianchi v.j., 2019 a ­ patents for the physiological quality in seeds of peach rootstock classified by weight and stored for different periods. ­ recent pat. food nutr. agric., 10: 52­59. souza a.g., smiderle o.j., spinelli v.m., souza r.o., bianchi v.j., 2016 ­ correlation of biometrical charac‐ teristics of fruit and seed with twinning and vigor of prunus persica rootstocks. ­ j. seed sci., 38: 322­328. souza a.g., spinelli v.m., souza r.o., smiderle o.j., bianchi v.j., 2017 ­ optimization of germination and initial quality of seedlings of prunus persica tree roots‐ tocks. ­ j. seed sci., 39: 166­173. wagner júnior a., bruckner c.h., silva j.o.c., santos c.e.m., pimentel l.d., mazaro s.m., 2013 ­ necessidade de frio para estratificação das sementes e observações fenológicas em cultivares de pessegueiro. ­ rev. ceres., 60: 234­241. impaginato 459 adv. hort. sci., 2018 32(4): 459-470 doi: 10.13128/ahs-22479 combining ability and gene action of some tomato genotypes under low light condition s. emami 1, s.h. nemati 1 (*), m. azizi 1, m. mobli 2 1 department of horticultural science and landscape, faculty of agriculture, ferdowsi university of mashhad, mashhad, iran. 2 department of horticultural science, faculty of agriculture, isfahan university of technology, isfahan, iran. key words: combining ability, diallel, heritability, light intensity, reciprocal effects, solanum lycopersicum. abstract: limitations in access to electricity in rural areas and substantial cost of supplemental lightning necessitate breeding as response to low light conditions. seven inbred lines of tomato (solanum lycopersicum l.) and their f1 hybrids, including reciprocals, developed through a 7×7 full diallel cross were evaluated under two different levels of light. mean square for light (l) effect was significant for total yield, average fruit weight and days to first flower. variation attributable to genotypes and genotype × light (g×l) interaction had significant effect on all studied traits except days to ripening for which g×l interaction was not significant. diallel analysis across two environments indicated that general (gca), specific (sca) and reciprocal combining ability (rec) were significant for all characters implying importance of additive and nonadditive gene action along with cytoplasmic effects on genetic expression of yield, yield components and earliness. ratio of sca variance to sca variance and estimates of narrow sense heritability (h2 n.s) demonstrated higher weight of additive effects in inheritance of yield, fruit number and days to ripening, while indicating predominance of non-additive effects for fruit weight and early flowering. interactions gca×l and sca×l were significant for almost all studied features. a particular genotype could not be recommended for all traits, but variation among genotypes in response to ambient light was promising for feasibility of plant breeding for non-optimal light intensity and duration. 1. introduction tomato (solanum lycopersicum l.), a member of solanaceae family, is a wide cultivated vegetable used for fresh and processing market. this day neutral plant is grown in various regions of the world with different climates (mizoguchi et al., 2007; gerszberg et al., 2015). iran is among the top 10 countries producing tomato (faostat, 2014), but it performs very poorly in terms of tomato seed production and a large portion of the seeds, particularly hybrid cultivars, is imported to the country. the major obstacle to seed production in iran is the absence of breeding programs (*) corresponding author: nemati@um.ac.ir citation: emami s., nemati s.h., azizi m., mobli m., 2018 combining ability and gene action of some tomato genotypes under low light condition. adv. hort. sci., 32(4): 459-470 copyright: © 2018 emami s., nemati s.h., azizi m., mobli m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 january 2018 accepted for publication 16 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 459-470 460 for most of the vegetables including tomato; therefore, more attention to vegetables breeding in order to produce high quality seeds is required. hybrid breeding technique is one of the most important methods used for crop improvement. the information needed to develop proper f1 hybrid cultivars via hybrid breeding could be achieved through different methods including diallel analysis, a method to analyze crosses made among (n) lines in all possible combinations (griffing, 1956 a, b). the analysis is mainly adopted when dealing with limited number of parental lines and determines genetic parameters such as heterosis, general combining ability (gca), specific combining ability (sca), heritability, and nature of gene action. heterosis demonstrates the superiority of f1 progenies compared to the average of their parents. general combining ability (gca) shows the average performance of a parental line while specific combining ability (sca) refers to the best combination of crosses. general combining ability (gca) and specific combining ability (sca) are the indicators of additive and non-additive gene effects, respectively. these parameters help plant breeders in the selection of suitable parental lines and appropriate breeding method (sprague and tatum, 1942). it should be noted that despite of the advantages of hybrid cultivars over open pollinated ones, the high price of hybrid seeds developed via hybridization programs makes the use of them more economical for intensive and indoor cultivation (zengin et al., 2015). one of the major problems affecting plants growth in greenhouses is the decreased level of light received by plants due to significant loss of solar radiation caused by reflection and absorption by greenhouse covering material (baeza and lópez, 2012) and high plant density, typically executed in greenhouse cultivations (laurent et al., 2017). since light is one of the most crucial factors influencing growth rate, production quality and quantity of plant, supplying plants with adequate light intensity with suitable quality is of importance in greenhouses particularly during autumn and winter seasons (hangarter, 1997). in developing countries such as iran, most of the greenhouses do not benefit from high technology and suffer from lack of accessibility to electricity, hence; supplemental l ightning is not applied. moreover, substantial price of energy resources and tendency toward lower energy consumption (oz and atilgan, 2015) necessitate hybrid breeding for low light conditions. to our knowledge breeding for low light condition has not been conducted in tomato, therefore, this study aimed to investigate if there existed any differences among various tomato genotypes in response to two different light conditions while considering the introduction of suitable parental lines and hybrids for each condition as well as determination of stable genotypes over two environments. genetic parameters including combining ability, gene action and heritability were estimated to help breeders in choosing the best approach for the improvement of tomatoes regarding increased yield and earliness. 2. materials and methods plant material seven inbred lines of tomato consisted of ‘perimoga’, ‘la1793’, ‘ac06’, ‘ct6’, ‘mc3’, ‘c20’ and ‘kingstone’ (table 1) were cultivated in a research greenhouse of ferdowsi university of mashhad, mashhad, iran under optimum conditions. the lines were crossed in all possible two-way combinations (full diallel cross system) to develop f1 hybrids with their reciprocals. experimental design the experiment was performed in research greenhouse of ferdowsi university of mashhad with computerized temperature control system. a year round was divided into two growing seasons: the first growtable 1 description of parental inbred lines crossed in 7×7 full diallel cross system plant material abbreviation origin growth habit leaf type fruti shape perimoga p1 russia determinate vulgare oval la1793 p2 usa indeterminate vulgare round ac06 p3 iran indeterminate vulgare round ct6 p4 russia semi-indeterminate grandifolium oval mc3 p5 russia indeterminate vulgare round c20 p6 russia indeterminate vulgare oval kingstone p7 italy semi-indeterminate vulgare oval emami et al. tomato genotypes under low light condition 461 ing season was from march to august including warm seasons with high light intensity and long photoperiod (more sunny hours a day) and the second season covering cold seasons with low light intensity and short photoperiod (less sunny hours per day) started in september and ended up in january. to investigate the performance of tomato plants under two different light conditions, 21 f1 progenies together with reciprocals (42 f1 hybrid progenies) developed via a 7×7 full diallel cross system were cultivated during each of the two aforementioned growing seasons. daily average of light intensity of experimental greenhouse during each month is represented in table 2. the experiment was conducted in a completely randomized design with three replications per genotype in two different time span mentioned above (split plot). measurement of characters the observations concerning the following characteristics were recorded as described below. total yield per plant (kg) it was calculated by summing up the weight of fruits obtained from all pickings during 8 weeks from each plant. average fruit weight (g) the average fruit weight was an index of fruit size. all fruits collected from each harvest were weighted and the total weight of the fruits was divided into the number of the weighted fruits. number of fruits per plant harvested fruits of each plant in a period of 8 weeks were counted. days to first flower the number of days from seeding until the formation of first flower on the plants was recorded. days to ripening the number of days from flower anthesis to fruit ripening stage was determined through the date tagging of five flowers per plant at the time of anthesis. fruit ripening was considered the time when genetically red fruits turned pink and yellow ones turned yellow (garg et al., 2008). statistical analysis obtained data in each environment were analyzed using excel software (microsoft office 2010) using griffing’s (1956, a, b) method 3, model 1 (fixed effects) formula. combined analysis of data over two seasons was performed based on modified method 3, model 1 for several environments proposed by singh (1973) as described below: where yijk = observation of trait value of parents i and j in year k; µ = population mean; g i / g j = gca effect of parent i / j; sji / sji = sca effect of the hybrid developed from parent i × parent j / parent i × parent j; rij = rec effect of the hybrid produced by parent i × parent j; lk=effect of environment k; (gl)ik / (gl)jk = interaction between gca effect of parent i / j with environment k; (sl)ijk = interaction between sca effect of cross ij with environment k; and eijk = error of observation ijk. broad-sense heritability (h2 b.s) and narrow-sense heritability (h2 b.s) over environments were estimated using following formula (sharifi et al., 2010): where l indicated light condition; σ2g, σ2s and σ2r stand for variance components of gca, sca and rec, respectively; σ2gl, σ2sl and σ2rl represent variance components of gca×l, sca×l and rec×l, respectively. where f1 and yij are the mean performances of hybrids and parents, respectively. the average of light intensity per day was calculated based on 24 hours a day including night hours (n= 24, one measurements every hour). table 2 average of daily light intensity for each months (foot candle intensity/24h) average light intensity first growing season with high light intensity and duration second growing season with low light intensity and duration mar. apr. may june july aug. sept. oct. nov. dec. jan. feb. 680.7 694 750.5 863.3 893 760 650.7 547.3 550 527 535.5 655.7 adv. hort. sci., 2018 32(4): 459-470 462 3. results anova analysis the variance analysis of genotypes for each season separately showed that genotypes were highly significant (table 3); therefore, the compound analysis of variance over two environments was conducted (table 4). light condition was considered as main plot; therefore, error a refers to whole plot error. some genotypes, namely 42 f1 hybrids grown under each light treatment along with the interaction between genotypes and environments, were the sub plot of the experiment and error b represent whole plot error. compound anova analysis for two seasons represented in table 4 showed that light (l) effect was highly significant for total yield per plant, average fruit weight and days number to first flower but not significant for fruit number per plant and days number for fruit ripening. genotypes and the interaction of genotypes with light (g×l) were highly significant for all studied traits except for days to ripening in which genotypes did not show any interaction with environment. estimation of genetic parameters compound analysis of variance for general combining ability (gca), specific combining ability (sca) and reciprocal combining ability (rec) over two seasons for yield, yield components and earliness varied (table 4). the results indicated that variances due to table 3 mean squares of anova analysis over two light conditions for yield, yield components and earliness of f1 hybrids with their reciprocals developed via a 7×7 full diallel cross ** significant at p<0.01 level. sources of variation degree of freedom total yield per plant (kg) average of fruit weight (g) fruit number per plant days to first flower days to ripening light (l) 1 16.69 ** 14654.49 ** 913.52 359.53 ** 5.43 error a 4 0.74 261.6 140.91 0.61 8.8 genotypes (g) 41 2.05 ** 1944.73 ** 1593.50 ** 34.74 ** 23.38 ** g×l 41 0.17 ** 146.54 ** 96.24 ** 2.50 ** 3.99 error b 164 0.07 34.01 18.33 0.97 2.81 table 4 compound analysis of variance for combining ability over two environments for yield, yield components and earliness of f1 hybrids with their reciprocals developed via a 7×7 full diallel cross and estimates of heritability in broad and narrow senses as well as heterosis * significant at p<0.05 level, ** significant at p<0.01 level. sources of variation degree of freedom total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening gca 6 1.28 ** 3343.91 ** 2063.32 ** 54.66 ** 23.20 ** sca 14 1.35 ** 430.82 ** 644.60 ** 9.55 ** 8.68 ** rec 21 0.07 ** 23.00 ** 17.79 ** 0.62 * 2.80 ** gca×l 6 0.14 ** 120.47 ** 70.33 ** 2.86 ** 1.95 sca×l 14 0.06 ** 53.49 ** 42.07 ** 0.94 ** 1.68 * rec×l 21 0.03 25.29 * 14.49 ** 0.19 0.92 error 164 0.02 11.34 6.11 0.32 0.94 variance components σ2g 0.06 166.63 102.86 2.72 1.11 σ2s 0.33 104.87 159.62 2.31 1.94 σ2r 0.01 2.92 2.92 0.07 0.47 σ2g/ σ2s 0.19 1.59 0.64 1.18 0.57 σ2gl 0.01 10.91 6.42 0.25 0.1 σ2sl 0.02 21.08 17.98 0.31 0.37 σ2rl 0 6.98 4.19 -0.07 -0.01 h2 broad-sense (%) 94.77 94.94 95.7 94.38 90.38 h2 narrow-sense (%) 26.02 72.22 53.89 66.25 48.34 heterosis (%) 37.75 -22.95 55.04 3.05 -6.38 emami et al. tomato genotypes under low light condition 463 gca, sca and rec were highly significant for all studied characters. the significance of both gca and sca variances implies that all studied characters are controlled by either of additive or non-additive gene action. the effect of interaction gca×l and sca×l on all evaluated features were significant but fruit ripening period, which showed non-significant variance of gca×l. the interaction of rec×l showed remarkable effect only on average fruit weight and fruits number per plant. for total yield, fruits number and days to ripening, the gca (σ2g) was less than variance component of sca (σ2s) in a way that σ2g/σ2s was less than unity, demonstrating the predominance of nonadditive gene action in controlling mentioned traits (baker, 1978). the ratio of σ2g/σ2s for fruit weight and days to first flower was higher than unity, illustrating the more important role of additive gene action for inheritance of these attributes. estimates of broad-sense heritability percentage (h2 %) over environments was high for all studied traits ranged from 90 to 95% (table 4). narrow-sense heritability percentage (h2 %) was low for total yield, fruits number and days to ripening while relatively high for fruit weight and days to flowering. total heterosis percentage was high for yield and fruit number while negative for fruit weight. unlike yield and yield components, in earliness characters negative values show superiority but heterosis for early flowering was positive. negative but low heterosis was recorded for early maturity. estimation of mean values mean values of f1 hybrids for plant yield showed that hybrid ‘ct6×mc3’ (p4×p5) had the highest rate under both light conditions, while lowest amount was for hybrid ‘c20×mc3’ (p6×p5) (table 5). ‘perimoga×kingstone’ (p1×p7) cross and its reciprocal produced heaviest fruits over two environments. lightest fruits produced under normal light were for ‘mc3×la1793’ (p5×p2), but under low light, the fruits of hybrid ‘mc3×ac06’ (p5×p3) had the least weight. the highest number of fruits per plant was produced by hybrid ‘la1793×c20’ (p2×p6) and the lowest was for ‘kingstone×perimoga’ (p7×p1). hybrid ‘perimoga×ct6’ (p1×p4) commenced flowering earlier than other progenies. latest flowering under adequate light intensity was for ‘mc3×c20’ (p5×p6), while under low light, it was for hybrid ‘la1793×ac06’ (p2×p3). overall, according to pooled data over two seasons, hybrid ‘c20×mc3’ (p6×p5) took more days to flower compared with other progenies. longest fruit ripening duration was for hybrid ‘mc3×la1793’ (p5×p2) and shortest period for ripening was observed in the cross of ‘aco6×kingstone’ (p3×p7). estimation of gca, sca and rec effect for yield and yield components, positive values of gca, sca and rec indicate the superiority of genotypes while for earliness characters negative values are desired (table 6). most of the parental lines were not stable during two growing seasons and superior parents for each character differed with environmental changes. parental line of ‘ct6’ (p4) was the best combiner for achieving higher yield in both seasons, and the lowest gca in low light and high light condition was for ‘kingstone’ (p7) and ‘la1793’ (p2), respectively. in total ‘kingstone’ (p7) was the weakest genitor for yield across two environments. for fruit weight, although ‘perimoga’ (p1) had the highest gca in warm season, parental line ‘kingstone’ (p7) acted as the best combiner in both seasons. the best donors for increased fruits number during sunny and cloudy seasons were ‘la1793’ (p2) and ‘mc3’ (p5), respectively and ‘la1793’ (p2) had the best gca over both seasons. for days to first flower, parental genotype ‘perimoga’ (p1) possessed the highest negative gca and ‘mc3’ (p5) had the highest positive and significant gca under each ambient light and over both of them. the best combiner concerning days to ripening in both tested environments was ‘la1793’ (p2) and the weakest performance was for ‘kingstone’ (p7). in yield and related trait, a positive value of sca is repetitive of a successful cross between parental lines of that hybrid, while a negative value demonstrates that parental lines did not make up a good couple. for earliness, negative values of sca are indicative of prosperity .estimation of sca for each evironment and over two environments indicated that the best combination for total yield was ‘la1793×c20’ (p2×p6) and the worst was for ‘mc3×c20’ (p5×p6) (table 7). for fruit weight, hybrid ‘ac06×kingstone’ (p3×p7) had the highest sca during sunny seasons, and the hybrid developed from ‘perimoga×kingstone’ (p1×p7) had the highest magnitude during cloudy seasons and over two seasons. the highest and lowest sca estimations for fruits number were similar to total yield. the cross between ‘mc3×c20’ (p4×p6) was the most successful cross for decreased days to flowering during sunny months. ‘la1793×mc3’ (p2×p5) not only had the highest negative sca during cold months, but also possessed the best sca in total. for this trait, 464 adv. hort. sci., 2018 32(4): 459-470 table 5 means values and standard deviation for yield components and earliness of f1 hybrids developed via 7×7 diallel cross over two light conditions (part a) p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. values in parenthesis represent standard errors. ** significant at p<0.01. genotypes ♀ × ♂ total yield per plant (kg) average of fruit weight (g) fruits number per plant l1 l2 pooled l1 l2 pooled l1 l2 pooled p1×p2 2.72(±0.28) 1.48(±0.44) 2.10(±0.36) 70.13(±8.70) 45.90(±9.34) 58.02(±8.83) 38.93(±3.02) 31.90(±3.92) 35.42(±3.07) p1×p3 3.54(±0.36) 2.87(±0.48) 3.20(±0.42) 85.63(±10.48) 55.17(±8.89) 70.40(±8.53) 41.33(±1.64) 52.27(±7.01) 46.80(±2.69) p1×p4 3.81(±0.14) 3.50(±0.06) 3.65(±0.10) 88.53(±2.05) 56.47(±4.97) 72.50(±1.63) 43.00(±2.55) 62.20(±5.20) 52.60(±1.96) p1×p5 3.61(±1.02) 3.18(±0.20) 3.40(±0.60) 84.73(±7.22) 57.33(±3.14) 71.03(±2.79) 42.10(±8.98) 55.70(±6.58) 48.90(±7.65) p1×p6 2.24(±0.19) 2.23(±0.36) 2.24(±0.26) 40.60(±4.06) 43.80(±4.88) 42.20(±2.08) 55.27(±2.15) 51.87(±13.90) 53.57(±5.97) p1×p7 2.47(±0.36) 2.13(±0.33) 2.30(±0.34) 114.20(±14.97) 96.80(±10.83) 105.50(±8.28) 21.80(±3.97) 21.87(±2.20) 21.83(±2.05) p2×p1 2.29(±0.18) 1.58(±0.22) 1.94(±0.20) 64.33(±16.06) 42.17(±2.35) 53.25(±7.55) 36.60(±5.97) 37.50(±6.41) 37.05(±0.52) p2×p3 2.69(±0.17) 1.60(±0.04) 2.14(±0.08) 58.53(±7.25) 33.77(±2.03) 46.15(±2.64) 46.20(±3.10) 47.27(±2.75) 46.73(±1.05) p2×p4 3.27(±0.31) 2.77(±0.23) 3.02(±0.27) 46.13(±2.06) 39.83(±4.11) 42.98(±2.72) 70.70(±5.05) 70.07(±9.36) 70.38(±7.17) p2×p5 2.81(±0.25) 2.55(±0.20) 2.68(±0.19) 36.90(±3.33) 35.03(±2.84) 35.97(±1.21) 76.10(±4.58) 72.67(±3.25) 74.38(±3.01) p2×p6 4.20(±0.33) 2.77(±0.31) 3.49(±0.32) 44.63(±1.65) 32.63(±3.39) 38.63(±2.32) 94.03(±5.54) 84.80(±3.99) 89.42(±4.13) p2×p7 3.24(±0.33) 2.74(±0.33) 2.99(±0.33) 51.80(±4.54) 56.80(±4.54) 54.30(±4.54) 62.57(±1.72) 48.17(±2.29) 55.37(±1.94) p3×p1 3.33(±0.31) 2.42(±0.31) 2.87(±0.31) 76.60(±8.52) 54.03(±11.95) 65.32(±10.03) 43.50(±3.21) 45.27(±4.35) 44.38(±2.98) p3×p2 3.14(±0.30) 1.98(±0.25) 2.56(±0.19) 61.00(±2.11) 37.07(±3.62) 49.03(±1.37) 51.33(±3.18) 53.17(±2.90) 52.25(±2.83) p3×p4 3.34(±0.24) 2.62(±0.19) 2.98(±0.21) 68.20(±8.89) 54.23(±7.73) 61.22(±8.29) 49.17(±3.73) 48.57(±3.27) 48.87(±3.48) p3×p5 2.97(±0.25) 2.44(±0.30) 2.70(±0.27) 40.07(±1.75) 33.47(±4.46) 36.77(±3.07) 73.97(±3.10) 72.83(±1.05) 73.40(±1.15) p3×p6 2.81(±0.24) 2.48(±0.12) 2.65(±0.18) 61.50(±1.71) 37.90(±3.73) 49.70(±2.66) 45.63(±3.85) 65.67(±6.37) 55.65(±4.15) p3×p7 3.03(±0.18) 2.50(±0.20) 2.77(±0.19) 99.03(±5.56) 78.47(±2.90) 88.75(±1.66) 30.63(±0.59) 31.93(±3.61) 31.28(±1.89) p4×p1 4.27(±0.34) 3.69(±0.15) 3.98(±0.16) 83.03(±12.08) 64.67(±4.61) 73.85(±3.83) 51.77(±3.88) 57.20(±3.72) 54.48(±0.33) p4×p2 2.86(±0.2) 2.66(±0.20) 2.76(±0.09) 47.97(±4.71) 38.27(±3.84) 43.12(±1.66) 59.60(±1.73) 69.50(±1.85) 64.55(±0.15) p4×p3 3.18(±0.36) 3.01(±0.22) 3.09(±0.29) 66.53(±1.46) 54.90(±6.05) 60.72(±3.02) 47.80(±6.17) 54.83(±1.93) 51.32(±2.63) p4×p5 4.57(±0.26) 3.80(±0.34) 4.19(±0.27) 63.20(±4.30) 47.50(±3.76) 55.35(±1.30) 72.40(±3.41) 80.00(±5.73) 76.20(±3.21) p4×p6 3.32(±0.32) 3.14(±0.25) 3.23(±0.24) 63.83(±3.40) 50.20(±7.27) 57.02(±3.95) 52.27(±7.31) 62.77(±4.40) 57.52(±2.45) p4×p7 3.03(±0.31) 2.75(±0.17) 2.89(±0.21) 87.20(±11.27) 62.80(±3.24) 75.00(±5.56) 34.90(±2.80) 43.77(±0.51) 39.33(±1.57) p5×p1 3.37(±0.31) 2.97(±0.35) 3.17(±0.58) 76.83(±8.81) 47.27(±5.53) 62.05(±1.91) 44.03(±3.81) 62.77(±2.74) 53.40(±3.14) p5×p2 2.52(±0.40) 2.35(±0.27) 2.44(±0.58) 30.27(±4.99) 29.53(±2.17) 29.90(±1.41) 83.33(±3.32) 79.47(±7.33) 81.40(±5.24) p5×p3 3.29(±0.21) 2.10(±0.22) 2.69(±1.53) 44.13(±4.23) 26.87(±2.87) 35.50(±0.79) 75.00(±9.37) 77.90(±3.30) 76.45(±3.36) p5×p4 4.00(±0.20) 3.60(±0.15) 3.80(±2.08) 56.17(±4.99) 45.23(±1.63) 50.70(±2.92) 71.53(±8.22) 79.47(±0.49) 75.50(±4.29) p5×p6 1.87(±0.23) 1.51(±0.18) 1.69(±0.58) 42.17(±4.32) 37.67(±2.97) 39.92(±1.66) 44.33(±1.12) 40.10(±6.67) 42.22(±3.82) p5×p7 2.34(±0.25) 1.97(±0.35) 2.16(±0.58) 54.40(±3.10) 32.77(±3.08) 43.58(±3.09) 42.87(±2.34) 59.70(±5.28) 51.28(±3.73) p6×p1 2.57(±0.29) 1.97(±0.34) 2.27(±0.58) 55.63(±3.27) 36.90(±3.24) 46.27(±3.25) 46.10(±3.57) 53.10(±5.48) 49.60(±4.45) p6×p2 3.79(±0.39) 3.06(±0.25) 3.43(±1.53) 44.53(±2.34) 37.33(±4.52) 40.93(±3.41) 84.90(±4.27) 82.13(±4.31) 83.52(±1.90) p6×p3 2.99(±0.15) 2.59(±0.18) 2.79(±1.53) 55.77(±2.89) 43.10(±4.45) 49.43(±0.78) 53.70(±5.11) 60.27(±2.29) 56.98(±1.75) p6×p4 2.96(±0.18) 2.69(±0.24) 2.82(±1.53) 51.57(±0.46) 40.37(±6.80) 45.97(±3.35) 57.23(±3.45) 67.10(±5.23) 62.17(±0.94) p6×p5 1.77(±0.16) 1.24(±0.22) 1.51(±0.58) 50.93(±2.78) 40.23(±9.32) 45.58(±5.95) 34.67(±1.35) 31.03(±1.76) 32.85(±0.88) p6×p7 3.27(±0.35) 3.17(±0.25) 3.22(±0.58) 69.43(±3.52) 54.47(±3.54) 61.95(±3.53) 46.97(±2.77) 58.43(±8.39) 52.70(±3.27) p7×p1 2.18(±0.16) 1.83(±0.20) 2.00(±0.58) 116.40(±13.25) 99.53(±8.03) 107.90(±9.74) 18.97(±3.54) 18.30(±1.01) 18.63(±2.14) p7×p2 2.94(±0.12) 2.47(±0.17) 2.70(±1.00) 48.90(±0.35) 45.97(±3.68) 47.43(±1.95) 59.97(±2.03) 53.73(±2.06) 56.85(±1.55) p7×p3 3.25(±0.33) 2.62(±0.18) 2.93(±0.58) 108.60(±11.61) 72.67(±1.29) 90.62(±6.45) 29.93(±1.27) 35.90(±2.20) 32.92(±1.70) p7×p4 2.86(±0.09) 2.60(±0.16) 2.73(±0.58) 70.83(±2.00) 73.50(±2.56) 72.17(±1.24) 40.40(±2.40) 35.27(±0.99) 37.83(±1.63) p7×p5 2.67(±0.45) 2.18(±0.21) 2.42(±0.58) 60.07(±12.53) 39.57(±1.89) 49.82(±7.03) 44.73(±3.43) 54.97(±3.85) 49.85(±0.39) p7×p6 2.82(±0.27) 2.77(±0.27) 2.80(±1.15) 65.97(±8.25) 54.10(±5.91) 60.03(±1.24) 42.77(±1.37) 51.57(±7.46) 47.17(±3.05) f 11.42 ** 16.70 ** 16.47 ** 25.61 ** 28.12 ** 45.39 ** 49.16 ** 33.15 ** 76.95 ** lsd 0.05 0.50 0.41 0.40 11.51 8.69 7.52 6.80 8.11 5.23 to be continued 465 emami et al. tomato genotypes under low light condition table 5 means values and standard deviation for yield components and earliness of f1 hybrids developed via 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. values in parenthesis represent standard errors. ** significant at p<0.01. genotypes ♀ × ♂ days to first flower days to ripening l1 l2 pooled l1 l2 pooled p1×p2 56.00(±1.00) 54.33(±0.58) 55.17(±0.76) 14.00(±1.00) 15.67(±0.58) 14.83(±0.29) p1×p3 56.67(±0.58) 54.00(±1.00) 55.33(±0.58) 16.67(±2.08) 18.67(±1.53) 17.67(±1.61) p1×p4 54.00(±1.00) 50.67(±0.58) 52.33(±0.58) 18.33(±1.15) 18.33(±1.15) 18.33(±1.15) p1×p5 62.67(±0.58) 60.33(±0.58) 61.50(±0.50) 19.00(±2.65) 20.67(±1.15) 19.83(±1.76) p1×p6 58.00(±1.00) 54.67(±0.58) 56.33(±0.58) 18.00(±3.00) 18.00(±3.00) 18.00(±3.00) p1×p7 57.33(±0.58) 53.33(±1.53) 55.33(±0.76) 19.33(±2.08) 18.67(±1.53) 19.00(±1.00) p2×p1 56.67(±0.58) 54.67(±0.58) 55.67(±0.58) 19.00(±1.00) 16.67(±1.53) 17.83(±1.15) p2×p3 60.33(±1.15) 60.67(±1.53) 60.50(±0.87) 18.33(±1.53) 17.33(±1.53) 17.83(±1.44) p2×p4 60.67(±0.58) 58.67(±0.58) 59.67(±0.58) 16.33(±1.15) 15.67(±1.15) 16.00(±1.00) p2×p5 60.67(±0.58) 58.67(±0.58) 59.67(±0.58) 15.67(±2.08) 15.67(±2.08) 15.67(±2.08) p2×p6 61.67(±1.53) 60.33(±0.58) 61.00(±1.00) 17.00(±2.00) 17.00(±1.00) 17.00(±0.50) p2×p7 58.33(±0.58) 58.00(±1.00) 58.17(±0.29) 16.33(±1.15) 17.00(±2.00) 16.67(±1.53) p3×p1 57.33(±0.58) 54.67(±0.58) 56.00(±0.00) 18.33(±2.08) 18.33(±2.08) 18.33(±2.08) p3×p2 60.00(±0.00) 59.67(±0.58) 59.83(±0.29) 20.33(±2.52) 19.33(±2.31) 19.83(±2.36) p3×p4 56.00(±1.00) 55.67(±2.31) 55.83(±1.61) 16.67(±2.08) 16.67(±2.08) 16.67(±2.08) p3×p5 60.00(±1.00) 56.33(±1.53) 58.17(±1.04) 20.00(±1.73) 17.33(±2.08) 18.67(±1.53) p3×p6 60.67(±0.58) 59.00(±1.00) 59.83(±0.76) 17.00(±1.73) 17.00(±1.73) 17.00(±1.73) p3×p7 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 22.67(±2.08) 22.67(±2.08) 22.67(±2.08) p4×p1 54.67(±1.15) 51.33(±1.15) 53.00(±0.00) 17.00(±2.00) 17.00(±1.15) 17.00(±2.00) p4×p2 60.00(±1.00) 58.00(±1.00) 59.00(±1.00) 15.00(±1.00) 14.00(±1.00) 14.50(±0.87) p4×p3 58.00(±1.73) 57.00(±1.00) 57.50(±0.50) 14.67(±0.58) 15.33(±1.00) 15.00(±0.00) p4×p5 61.67(±1.15) 59.67(±1.15) 60.67(±1.15) 18.00(±2.00) 18.00(±1.15) 18.00(±2.00) p4×p6 56.33(±0.58) 54.33(±0.58) 55.33(±0.58) 20.00(±2.65) 21.00(±0.58) 20.50(±1.80) p4×p7 57.67(±0.58) 55.67(±0.58) 56.67(±0.58) 18.67(±1.53) 18.67(±0.58) 18.67(±1.53) p5×p1 62.00(±1.00) 59.33(±0.58) 60.67(±0.76) 20.33(±1.53) 19.33(±0.58) 19.83(±1.04) p5×p2 61.67(±0.58) 58.33(±0.58) 60.00(±0.00) 13.00(±1.00) 12.67(±0.58) 12.83(±1.04) p5×p3 61.00(±0.00) 57.33(±1.53) 59.17(±0.76) 19.67(±2.52) 16.00(±1.53) 17.83(±1.04) p5×p4 60.67(±2.08) 58.67(±2.08) 59.67(±2.08) 19.00(±3.00) 20.33(±2.08) 19.67(±2.08) p5×p6 63.67(±1.53) 58.67(±0.58) 61.17(±0.58) 21.00(±1.00) 20.33(±0.58) 20.67(±1.15) p5×p7 61.00(±0.00) 58.33(±0.58) 59.67(±0.29) 21.33(±1.53) 15.00(±0.58) 18.17(±0.29) p6×p1 58.33(±1.53) 55.33(±0.58) 56.83(±0.76) 17.67(±2.52) 17.67(±0.58) 17.67(±2.52) p6×p2 62.33(±1.53) 59.67(±1.53) 61.00(±1.32) 15.00(±2.00) 15.33(±1.53) 15.17(±1.76) p6×p3 59.67(±0.58) 59.33(±1.53) 59.50(±0.87) 16.00(±1.73) 19.67(±1.53) 17.83(±1.04) p6×p4 56.67(±0.58) 54.33(±1.53) 55.50(±0.87) 20.00(±2.65) 18.33(±1.53) 19.17(±2.02) p6×p5 63.33(±0.58) 60.33(±0.58) 61.83(±0.58) 18.67(±1.53) 18.67(±0.58) 18.67(±1.53) p6×p7 59.00(±1.00) 55.33(±0.58) 57.17(±0.29) 20.67(±1.53) 21.33(±0.58) 21.00(±1.50) p7×p1 58.00(±1.00) 52.67(±0.58) 55.33(±0.76) 20.67(±2.08) 20.67(±0.58) 20.67(±1.76) p7×p2 60.00(±0.00) 59.00(±1.00) 59.50(±0.50) 20.00(±1.00) 18.00(±1.00) 19.00(±0.00) p7×p3 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 20.67(±0.58) 20.67(±0.58) 20.67(±0.58) p7×p4 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 17.33(±1.15) 18.67(±0.58) 18.00(±0.00) p7×p5 62.00(±0.00) 59.33(±0.58) 60.67(±0.29) 20.00(±1.00) 17.67(±0.58) 18.83(±1.61) p7×p6 58.67(±1.15) 53.33(±1.15) 56.00(±1.00) 19.00(±1.00) 19.00(±1.15) 19.00(±1.00) f 18.69 ** 19.82 ** 28.14 ** 4.20 ** 19.82 ** 4.82 ** lsd 0.05 1.52 1.66 1.28 2.98 2.59 2.53 continued 466 adv. hort. sci., 2018 32(4): 459-470 table 6 general combining ability (gca) effects of parental lines for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. * significant at p<0.05 level, ** significant at p<0.01 level. a difference between gca of two parental lines at p<0.05 level. parental lines total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p1 -0.02 -0.06 -0.04 18.32 ** 10.97 ** 14.64 ** -13.46 **-11.38 **-12.42 ** -1.93 ** -2.70 ** -2.31 ** -0.06 0.42 0.18 p2 -0.02 -0.24 ** -0.13 ** -16.82 ** -11.61 ** -14.22 ** 14.63 ** 6.67 ** 10.65 ** 0.74 ** 1.77 ** 1.25 ** -1.90 ** -2.11 ** -2.00 ** p3 0.09 -0.12 ** -0.02 5.22 ** -0.87 2.17 ** -2.98 ** -1.78 * -2.38 ** -0.46 ** 0.40 * -0.03 0.2 0.36 0.28 p4 0.48 ** 0.64 ** 0.56 ** 1.98 3.76 ** 2.87 ** 3.28 ** 6.70 ** 4.99 ** -1.63 ** -1.20 ** -1.41 ** -0.80 * -0.34 -0.57 ** p5 -0.08 -0.06 -0.07 ** -13.35 ** -11.79 ** -12.57 ** 8.71 ** 10.29 ** 9.50 ** 2.94 ** 2.30 ** 2.62 ** 0.67 * -0.38 0.15 p6 -0.20 ** -0.08 -0.14 ** -12.68 ** -8.17 ** -10.42 ** 3.99 ** 4.51 ** 4.25 ** 0.74 ** 0.24 0.49 ** 0.1 0.79 ** 0.45 * p7 -0.25 ** -0.07 -0.16 ** 17.34 ** 17.71 ** 17.52 ** -14.15 **-15.01 **-14.58 ** -0.40 * -0.83 ** -0.61 ** 1.77 ** 1.26 ** 1.51 ** lsd 0.05 gi gj a 0.16 0.13 0.09 3.56 2.68 2.07 2.1 2.51 1.52 0.47 0.51 0.35 0.92 0.8 0.6 table 7 specific combining ability (sca) effects of f1 hybrids for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. *significant at p < 0.05 level. ** significant at p<0.01 level. a difference between two sca of two hybrids, with a common parent. b difference between two sca of two hybrids, with non-common parent. genotypes ♀ × ♂ total yield per plant (kg) average o fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p1×p2 -0.51 ** -0.70 ** -0.60 ** 1.29 -4.52 * -1.62 -14.90 **-15.90 ** -15.40** -1.72 ** -1.43 ** -1.58 ** 0.21 -0.1 0.06 p1×p3 0.31 ** 0.29 ** 0.30 ** -6.87 ** -4.69 ** -5.78 ** 7.36 ** 6.62 ** 6.99** 0.14 -0.23 -0.04 -0.89 -0.23 -0.56 p1×p4 0.53 ** 0.48 ** 0.50 ** 1.03 -3.36 -1.16 6.07 ** 9.07 ** 7.57** -1.36 ** -1.97 ** -1.66 ** 0.28 -0.37 -0.04 p1×p5 0.55 ** 0.66 ** 0.60 ** 11.36 ** 3.93 * 7.64 ** -3.68 ** 5.01 ** 0.67 2.08 ** 3.37 ** 2.72 ** 0.81 2.00 ** 1.41 ** p1×p6 -0.42 ** -0.29 ** -0.36 ** -21.97 **-11.65 **-16.81 ** 8.66 ** 4.04 * 6.35** 0.11 0.6 0.36 -0.46 -1.33 * -0.89 * p1×p7 -0.45 ** -0.43 ** -0.44 ** 15.16 ** 20.30** 17.73 ** -3.51 * -8.84 ** -6.17** 0.74 * -0.33 0.21 0.04 0.03 0.04 p2×p3 -0.21 * -0.38 ** -0.30 ** 6.92 ** -1.3 2.81 * -14.38 ** -9.97 ** -12.18** 0.64 * 1.13 ** 0.89 ** 2.78 ** 2.13 ** 2.46 ** p2×p4 -0.46 ** -0.22 * -0.34 ** -2.56 -2.3 -2.43 -4.25 ** 1.11 -1.57 1.98 ** 0.90 ** 1.44 ** 0.11 -0.67 -0.28 p2×p5 -0.29 ** 0.21 * -0.04 -0.69 6.48 ** 2.90 * 4.88 ** 3.80 * 4.34** -1.76 ** -2.43 ** -2.09 ** -2.69 ** -1.30 * -1.99 ** p2×p6 1.16 ** 0.70 ** 0.93 ** 9.64 ** 5.56 ** 7.60 ** 19.35 ** 16.98 ** 18.17** 1.28 ** 1.13 ** 1.21 ** -0.46 -0.47 -0.46 p2×p7 0.31 ** 0.38 ** 0.35 ** -14.61 ** -3.91 * -9.26 ** 9.29 ** 3.99 * 6.64** -0.42 0.70 * 0.14 0.04 0.4 0.22 p3×p4 -0.37 ** -0.24 ** -0.30 ** -4.28 2.48 -0.9 -3.31 * -8.53 ** -5.92** -0.16 0.27 0.06 -1.99 ** -1.97 ** -1.98 ** p3×p5 0.07 -0.09 -0.01 -14.22 ** -6.37 ** -10.29 ** 17.26 ** 11.55 ** 14.41** -1.22 ** -2.73 ** -1.98 ** 0.71 -1.27 * -0.28 p3×p6 -0.04 0.20 * 0.08 1.65 0.34 1 -2.84 * 4.93 ** 1.05 0.64 * 1.67 ** 1.16 ** -2.06 ** -0.77 -1.41 ** p3×p7 0.25 * 0.22 * 0.23 ** 16.80 ** 9.54 ** 13.17 ** -4.09 ** -4.60 ** -4.34** -0.06 -0.1 -0.08 1.44 * 2.10 ** 1.77 ** p4×p5 0.83 ** 0.58 ** 0.71 ** 6.60 ** 5.20 ** 5.90 ** 8.48 ** 7.43 ** 7.96** 0.61 1.20 ** 0.91 ** 0.38 1.93 ** 1.16 ** p4×p6 -0.2 -0.18 * -0.19 ** 3.95 0.49 2.22 -4.01 ** -1.59 -2.80** -1.86 ** -1.57 ** -1.71 ** 2.44 ** 1.27 * 1.86 ** p4×p7 -0.34 ** -0.43 ** -0.38 ** -4.75 * -2.51 -3.63 ** -2.98 * -7.48 ** -5.23** 0.78 * 1.17 ** 0.97 ** -1.22 * -0.2 -0.71 p5×p6 -0.95 ** -1.02 ** -0.98 ** 8.13 ** 9.71 ** 8.92 ** -24.69 **-34.54 **-29.62 ** 0.58 0.1 0.34 0.81 1.13 0.97* p5×p7 -0.21 -0.33 ** -0.27 ** -11.20 **-18.95 **-15.07 ** -2.26 6.75 ** 2.24 * -0.29 0.5 0.11 -0.02 -2.50 ** -1.26 ** p6×p7 0.45 ** 0.59 ** 0.52 ** -1.4 -4.45 * -2.93 * 3.53 * 10.19** 6.86 ** -0.76 -1.93 ** -1.34 ** -0.29 0.17 -0.06 lsd 0.05 s ij − s ik a 0.29 0.23 0.17 6.44 4.87 3.75 3.81 4.54 2.75 0.85 0.93 0.63 1.67 1.45 1.08 s ij − s kl b 0.25 0.2 0.14 5.58 4.21 3.25 3.3 3.93 2.38 0.74 0.81 0.55 1.44 1.26 0.93 emami et al. tomato genotypes under low light condition 467 ‘perimoga×mc3’ (p1×p5) was the weakest combination across both environments. the most negative value of sca for ripening period under low light belonged to ‘la1793×mc3’ (p2×p5) and under high light was for ‘kingstone×mc3’ (p7×p5). pooled value of sca in this character showed that generally ‘la1793×mc3’ (p2×p5) and ‘ac06×la1793’ (p3×p2) had the highest and the lowest negative values, respectively. the results of rec indicated that the best reciprocal combinations over two environments for total yield, average of fruit weight, fruits number, days to flowering and days to ripening was for ‘ac06×la1793’ (p3×p2), ‘kingstone×mc3’ (p7×p5), ‘mc3×la1793’ (p5×p2), ‘kingstone×c20’ (p7×p6) and ‘mc3×la1793’ (p5×p2), respectively (table 8). the lowest pooled rec in foregoing characters was for ‘kingstone×c20’ (p7×p6), ‘c20×ct6’ (p6×p4), ‘c20×mc3’ (p6×p5), ‘mc3×ac06’ (p4×p3) and ‘la1793×perimoga’ (p2×p1), respectively 4. discussion and conclusions the results indicated that total yield, average fruit weight and flowering time were influenced by the amount of received light, while fruit number and fruit ripening period were not affected. genotype effect was highly significant for all studied traits implying the feasibility of breeding. despite of simultaneous influence of light and genotype on yield, fruit weight and days to flower, a comparison between magnitude of environment and genotype effects revealed that the genotype variation played more important in the expression of studied traits. the significance of interaction genotype × light condition (g×l) for almost all characters except days to ripening revealed that there is a genotype variation in response to light intensity as regards yield, yield components and early flowering. previous studies reported genotype variation regarding reaction to environmental light in different species (stratton, table 8 reciprocal effect (rec) for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. * significant at p<0.05 level, ** significant at p<0.01 level. a difference between two rca of two hybrids. genotypes ♀ × ♂ total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p2×p1 -0.22 0.05 -0.08 -2.9 -1.87 -2.38 -1.17 2.8 0.82 0.33 0.17 0.25 2.50 ** 0.5 1.50 ** p3×p1 -0.11 -0.23 * -0.17 * -4.52 -0.57 -2.54 1.08 -3.5 -1.21 0.33 0.33 0.33 0.83 -0.17 0.33 p4×p1 0.23 0.1 0.17 * -2.75 4.1 0.67 4.38 * -2.5 0.94 0.33 0.33 0.33 -0.67 -0.67 -0.67 p5×p1 -0.12 -0.11 -0.11 -3.95 -5.03 * -4.49 ** 0.97 3.53 2.25 -0.33 -0.5 -0.42 0.67 -0.67 0 p6×p1 0.16 -0.13 0.02 7.52 * -3.45 2.03 -4.58 ** 0.62 -1.98 0.17 0.33 0.25 -0.17 -0.17 -0.17 p7×p1 -0.15 -0.15 -0.15 * 1.1 1.37 1.23 -1.42 -1.78 -1.6 0.33 -0.33 0 0.67 1 0.83 p3×p2 0.22 0.19 0.21 ** 1.23 1.65 1.44 2.57 2.95 2.76 * -0.17 -0.5 -0.33 1 1 1.00 * p4×p2 -0.21 -0.06 -0.13 0.92 -0.78 0.07 -5.55 ** -0.28 -2.92 * -0.33 -0.33 -0.33 -0.67 -0.83 -0.75 p5×p2 -0.14 -0.1 -0.12 -3.32 -2.75 -3.03 3.62 * 3.4 3.51 ** 0.5 -0.17 0.17 -1.33 -1.50 * -1.42 ** p6×p2 -0.21 0.14 -0.03 -0.05 2.35 1.15 -4.57 ** -1.33 -2.95 * 0.33 -0.33 0 -1 -0.83 -0.92 p7×p2 -0.15 -0.14 -0.15 * -1.45 -5.42 * -3.43 * -1.3 2.78 0.74 0.83 * 0.5 0.67 * 1.83 * 0.5 1.17 * p4×p3 -0.08 -0.19 0.06 -0.83 0.33 -0.25 -0.68 3.13 1.23 1.00 * 0.67 0.83 ** -1 -0.67 -0.83 p5×p3 0.16 -0.17 -0.01 2.03 -3.3 -0.63 0.52 2.53 1.52 0.5 0.5 0.5 -0.17 -0.67 -0.42 p6×p3 0.09 0.06 0.07 -2.87 2.6 -0.13 4.03 * -2.7 0.67 -0.5 0.17 -0.17 -0.5 1.33 * 0.42 p7×p3 0.11 0.06 0.08 4.77 -2.9 0.93 -0.35 1.98 0.82 0 0 0 -1 -1 -1.00 * p5×p4 -0.29 * -0.1 -0.19 ** -3.52 -1.13 -2.33 -0.43 -0.27 -0.35 -0.5 -0.5 -0.5 0.5 1.17 0.83 p6×p4 -0.18 -0.22 * -0.20 ** -6.13 * -4.92 * -5.53 ** 2.48 2.17 2.32 0.17 0 0.08 0 -1.33 * -0.67 p7×p4 -0.09 -0.08 -0.08 -8.18 ** 5.35 * -1.42 2.75 -4.25 * -0.75 0.33 0.33 0.33 -0.67 0 -0.33 p6×p5 -0.05 -0.13 -0.09 4.38 1.28 2.83 -4.83 ** -4.53 * -4.68 ** -0.17 0.83 * 0.33 -1.17 -0.83 -1.00 * p7×p5 0.17 0.1 0.13 2.83 3.4 3.12 0.93 -2.37 -0.72 0.5 0.5 0.5 -0.67 1.33 * 0.33 p7×p6 -0.23 -0.2 -0.21 ** -1.73 -0.18 -0.96 -2.1 -3.43 -2.77* -0.17 -1.00 * -0.58 * -0.83 -1.17 -1.00 * lsd 0.05 r ij − r ik a 0.31 0.25 0.18 7.04 5.32 4.1 4.16 4.96 3.01 0.93 1.02 0.69 1.82 1.59 1.18 adv. hort. sci., 2018 32(4): 459-470 468 d.a., 1998; martínez-ferri et al., 2001) promising for plant improvement regarding maintenance of high yield and earliness under lower level of light intensity. the remarkable effect of interaction g×l on studied traits except ripening period demonstrated that genotypes were not stable across two environments and should be evaluated in a range of environments. importance of genotype selection across different environments for tomato improvement concerning yield and earliness attributing traits was reported by chadha and kumar (2001), and biswas et al. (2011). according to mean performances of hybrids, superior genotypes for various characteristics differed and none of them could be considered as the best for all of the attributes. in this regard, in order to commercialize f1 hybrids, breeding programs should be conducted to collect suitable features in one plant (breseghello and coelho, 2013). either of general combing ability (gca) or specific combining ability (sca) was highly significant for all of evaluated features illustrating both additive and non-additive gene action were involved in controlling yield, yield components and earliness. our findings supports additive-dominance model reported by chishti et al. (2008) and biswas et al. (2011) for production and earliness traits. significant contribution of rec to total sum squire is indicative of inter-allelic interactions in the expression of studied traits. similarly, rec effect on fruit weight and number was reported by hannan et al., (2007 b). higher magnitude of sca variance (σ2s) in comparison with gca variance (σ2g) for total yield, fruit number and days to ripening indicated that these traits are mainly under the control of dominant effects. similar findings were reported by solieman (2009) and el-gabry et al. (2014) for fruit yield and number. the predominance of non-additive gene action over additive effects for days to ripening was in agreement with hannan et al. (2007a) but inconsistent with garg et al. (2008) who found additive gene action to be more effective on days to ripening over two environments. the ratio of σ2g/σ2s over two light conditions was greater than unity for fruit weight and days to flowering indicative of more weight of additive effects in inheritance of these features. garg et al. (2008), rai and asati (2011) and nadeem et al. (2013) also contributed the expression of fruit weight and early flowering to both additive and dominance gene actions with preponderance of additive effects. biswas et al. (2011) who examined tomato genotypes across two environments, different in terms of temperature and light intensity, reported more important role of additive gene action in control of fruit weight. the interaction gca×l was significant for all characters except for days to ripening indicating the sensibility of additive effects to light condition. the significant interaction sca×l was indicative of instability of dominance effects under different environmental light. rec×l varied for fruit weight and number demonstrating the susceptibility of cytoplasmic effects to environment in some traits and necessity of reciprocal crosses for choosing the most suitable genotypes for target environment. estimates of broad-sense heritability percentage (h2 b.s%) across two different light conditions was high, demonstrating the low effect of environment and high response of studied traits to breeding programs. relatively high narrow sense heritability percentage (h2 n.s%) for fruit weight and early flowering indicated that these traits are largely controlled by additive effects; while, low h2 n.s% of yield, fruit number and early maturity demonstrated higher weight of non-additive effects in inheritance of these traits. these findings agree with earlier work of biswas et al. (2011) and dutta et al. (2013) who reported low narrow sense heritability for yield and fruit number over two environments. importance of both additive and non-additive gene action with predominance of additive effects in expression of fruit weight and days to flowering revealed that selection breeding programs could be an effective strategy for genetic improvement of tomato for these characters, while exploitation of hybrid vigor should not be neglected (grilli et al., 2003). yield, fruit number and early ripening were controlled by additive-dominance effects with higher weight of dominance effects implying hybrid breeding could be adopted for improvement of these characters (gul et al., 2010). abd el-maksoud et al. (2013) proposed recurrent selection program for improvement of traits controlled by both additive and nonadditive effects. for such traits, hybridization in segregating generations followed by selection for outperforming genotypes has been recommended (dutta et al., 2013; bhattarai et al., 2016). limitation in access to clean energy resources in rural areas and global interest toward lower energy consumption necessitate breeding for low energy input. in the current study, genetic variation among tomato genotypes under different light conditions was observed and some genotypes showed more stability than others. none of parental lines or f1 emami et al. tomato genotypes under low light condition 469 hybrids exhibited high performance for all studied features, therefore, a particular genotype cannot be recommended. however, genetic knowledge obtained from this research could be used in planning tomato breeding programs. more important role of additive gene action in inheritance of fruit 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horticulture, faculty of agriculture, university of zanjan, zanjan, iran. 2 department of horticulture, faculty of agriculture, university of tabriz, tabriz, iran. 3 department of horticultural science, imam khomeini international university, qazvin, iran. key words: antioxidant enzymes, postharvest, prunus persica l., total phenols. abstract: peach trees were treated with salicylic acid at 0 (control) and 1.5 mm at 15 days before harvest to study the impacts of salicylic acid on nutritional quality of peach fruits at harvest and during storage at 1°c for 28 days. total phenols, flavonoids, and ascorbic acid contents were significantly higher in salicylic acid treated peach fruits after cold storage, leading to fruits with higher dpph• and frap radicals scavenging capacity. in addition, peach fruits treated with salicylic acid exhibited higher antioxidant enzymes catalase (cat), ascorbate peroxidase (apx) and superoxide dismutase (sod) activity after storage at 1°c for 2-4 week, leading to fruits with higher firmness and lower weight loss. thus, salicylic acid treatment of peach trees could increase nutritional quality of peach fruits consumption, due to its effect on increasing antioxidant molecules, with additional effect on delaying the fruit postharvest senescence by increasing the ros scavenging enzymes activity. 1. introduction peach (prunus persica l.) is highly perishable climacteric stone fruit and is a rich source of ascorbic acid, carotenoids, and phenolics that are good sources of antioxidants (tomas-barberan et al., 2001). however, the rapid softening of fruit during storage at ambient temperature results in a short shelf-life of the commodity and reduced commercial fruit quality and consumer acceptance (nunes, 2008). due to its economic impact and also human health, great efforts have been done by researchers for delaying postharvest senescence of peach fruits during cold storage leading to fruits with higher sensory and nutritional quality by applying postharvest treatment such as modified and controlled atmosphere storage, heat treatment, glycine betaine, nitric oxide, brassinolide; 1-methylcyclopropene, methyl jasmonate, oxalic acid and salicylic acid (cao et al., (*) corresponding author: razavi.farhang@znu.ac.ir citation: razavi f., hajilou j., aghdam m.s., 2018 salicylic acid treatment of peach trees maintains nutritional quality of fruits during cold storage. adv. hort. sci., 32(1): 33-40 copyright: © 2018 razavi f., hajilou j., aghdam m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 september 2017 accepted for publication 8 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 33-40 34 2010; liu et al., 2015; kang et al., 2016; gao et al., 2016; razavi and hajilou, 2016; shan et al., 2016; yu et al., 2016). fruit ripening with oxidative fact is associated with reactive oxygen species (ros) such as superoxide radical (o2 -), hydrogen peroxide (h2o2) and hydroxyl radicals (oh-) accumulation leading to membrane deterioration, lipid peroxidation and dna mutation and ultimately economical quality and quantity losses of fruits results from deterioration of their cellular metabolism (halliwell and gutteridge, 1989). for overcome to oxidative stress during ripening which is crucial for delaying fruits deterioration and maintaining fruits sensory and nutritional quality, fruits cells employed an antioxidant system, superoxide dismutase (sod), catalase (cat), and ascorbate peroxidase (apx) as enzymatic and ascorbate and glutathione, tocopherols, phenolics, flavonoids, alkaloids and carotenoids as non-enzymatic antioxidants (apel and hirt, 2004). sod vocalizes the first line of ros scavenging and catalyzes the dismutation of o2 _ to h2o2. then, h2o2 is eliminated by the actions of apx and cat. apx dismutes h2o2 by conversion of aa to dha (foyer and noctor, 2005). due to extra ros production and or incapable antioxidant system activity, fruits may encounter oxidative damage during ripening leading to quality losses. thus, delaying fruits ripening and maintaining fruits quality, due to its economic impact and also human health, can be results from effective antioxidant system activity in fruits during ripening, which is achievable by using of environmentally friendly technologies such as salicylic acid (sa) as safe signaling molecule (hodges et al., 2004; asghari and aghdam, 2010; kumar et al., 2014), which have potential in delaying ripening, enhancing quality and attenuating biotic and abiotic stress of fruits (asghari and aghdam, 2010). according to capability of sa in using as preharvest treatment, gimenez et al. (2014) reported that sweet cherry fruits treated at preharvest with sa at 0.5 mm and asa at 1 mm exhibited higher total phenolics and total anthocyanins, as well as higher hydrophilic and lipophilic antioxidant activity at commercial harvest (gimenez et al., 2014). also, gimenez et al. (2017) reported that sweet cherry fruits treated at preharvest with sa at 0.5 mm and asa at 1 mm exhibited higher total phenolics and total anthocyanins, as well as higher hydrophilic antioxidant activity during storage at 2°c for 28 days. also, cherry fruits treated at preharvest with sa and asa exhibited higher antioxidant enzymes catalase (cat), ascorbate peroxidase (apx) and superoxide dismutase (sod) during storage at 2°c for 28 days. valverde et al. (2015) reported that the sweet cherry fruits treated with preharvest 1 mm methyl salicylate exhibited higher total phenolics and anthocyanins content at harvest and during storage at 2°c for 28 days, leading to fruit with higher hydrophilic taa (h-aa). also, sweet cherry fruits treated with preharvest 1 mm methyl salicylate exhibited higher antioxidant enzymes cat, apx and sod activities during storage at 2°c for 28 days. thus, salicylates treatment of sweet cherry trees enhances health boosting attributes of cherry fruits consumption, by increasing antioxidant molecules, with supernumerary impacts on delaying the sweet cherry fruits postharvest senescence by enhancing ros scavenging enzymes activities (gimenez et al., 2017). it has been suggested that the preharvest salicylates treatments would have commercial fondness with low earnings costs and with considerable profits in fruit nutritional quality (gimenez et al., 2017). moreover, postharvest treatment of peach fruit with sa at 2 mm enhanced higher levels of pod, cat and sod activities during cold storage as compared with control peaches, which were accompanied by lower polyphenol oxidase (ppo) activity. in addition, sa treated fruits exhibited higher firmness and radical scavenging activity (tareen et al., 2012). then, the aim of this research was to evaluate for the first time the impacts of preharvest sa treatment of peach trees on bioactive molecules and the antioxidant enzymes sod, cat, and apx activities at harvest and during cold storage. 2. materials and methods fruits and treatments the experiment was carried out on 5-year-old peach [prunus persica (l.) batsch ‘anjiry maleki’] trees grafted on gf 677 rootstock, in a commercial orchard located in the north-west iran. the trees were spaced at 6×5 m, receiving identical cultural practices and trained to an open vase system. twelve trees were selected for uniform size and fruit load and sprayed with sa at concentrations of 1.5 mm on whole tree and control trees receiving only water. a surfactant (tween-20) was added to each solution as a wetting agent for maximum sa absorption, and sprays were applied at 15-day before commercial harvest. fruit from control and sa treated trees were razavi et al. salicylic acid and nutritional quality of peach fruit 35 harvested at commercial maturity and immediately transported to the laboratory. the fruits were selected for uniform size, color and absence of mechanical damage, and then one group was analyzed 24 h after harvest and another groups stored at 1±0.5°c and 90% rh for 28 d. at 7-day intervals, 5 fruits from each of three replications were selected, and left for a further 24 h at 20°c (shelf-life), and subjected to physicochemical analysis. flesh firmness and weight loss at each sampling date, flesh firmness (n) was measured on the opposite sides of the fruit after peel removal using an effegi penetrometer (model ft 011) equipped with an 8 mm diameter probe. to determine the weight loss, five fruits for each replicates were weighed at harvest and at 7 day intervals during cold storage. results were expressed as percentage of weight loss relative to the initial fruit weight. antioxidant enzymes activity assays crude extract for apx enzymes was performed by homogenizing 1 g of frozen fruits tissue with 5 ml of phosphate buffer 100 mm, ph= 7.8 containing 1% (w/v) pvp, 1 mm edta and 5 mm ascorbic acid. the homogenate was centrifuged at 18,000 g for 10 min at 4°c and the supernatant used for enzyme assay. apx activity was determined by the method of nakano and asada (1987) with some modification. the reaction mixture consisted of 3 ml of 50 mm potassium phosphate, ph 7.0, 0.2 mm ascorbic acid, 0.2 mm edta and 0.5 ml of crude extract, and the reaction was allowed to start by adding 0.5 ml of 0.5 mm h2o2. the decrease in absorbance at 290 nm was recorded spectrophotometrically for 3 min and apx activity expressed as u mg protein-1. crude extract for cat and sod enzymes was performed by homogenizing 1 g of frozen fruits tissue with 3 ml of phosphate buffer 50 mm, ph= 7.8 containing 2% (w/v) pvp, 1 mm edta. the homogenate was centrifuged at 14,000 g for 20 min at 4°c and the resulting supernatant was used for enzyme assay. cat activity was quantified following the method described by zhang et al. (2013). the reaction mixture consisted of 50 mm phosphate buffer (ph 7), 15 mm h2o2 and 0.1 ml of crude extract in a final volume of 3 ml. decreases in absorbance at 240 nm at intervals of 30 s were recorded spectrophotometrically. cat activity expressed as u mg protein-1. sod activity was assayed according to the method described by zhang et al. (2013). one unit of sod activity was defined as the amount of enzyme that causes a 50% inhibition of nitro blue tetrazolium reduction under assay conditions and the results were expressed as u mg protein-1. total protein content in the enzyme extract was assayed according to the method described by bradford (1976). total phenolics, flavonoids and ascorbic acid contents the amount of total phenolics in extracts was determined according to the folin-ciocalteu reagent method (singleton and rossi, 1965). gallic acid was used as a reference standard, and the total phenolic contents of extract were expressed as mg gallic acid equivalent 100 g-1 fresh weight (fw). total flavonoids content was determined in accordance with a protocol described by kaijv et al. (2006). a calibration curve was obtained using quercetin as a standard, and the results were expressed as µmol quercetin equivalent (qe) 100 g-1 fw. ascorbic acid content in the fruits was measured by 2,6-dichlorophenol indophenol dye method (aoac, 1984). for each sample, 10 g fresh fruits pulp was homogenized with 3% metaphosphoric acid solution and the mixture was made up to 100 ml. an aliquot of 10 ml was then titrated against the dye (2,6-dichlorophenol indophenol) till the pink color appeared. ascorbic acid content was estimated from a calibration curve of lascorbic acid and results expressed as mg ascorbic acid equivalents 100 g-1 fw. total antioxidant activity the frap assay was carried out using tptz (2,4,6tripyridyl-striazine) solution according to the procedure described by benzie and strain (1999). frap reagent was prepared freshly by mixing 2.5 ml of solutions tptz (10 mm, dissolved in 40 mm hcl) and fecl3 (20 mm) in 25 ml of acetate buffer (300 mm concentration and 3.6 ph). a 50 μl of the diluted sample was added to 1.5 ml of frap reagent. the absorbance of the mixture was measured at 593 nm after 4 min. incubation at 37°c using a uv-visible spectrophotometer (t-60, pg instrument uk). a calibration curve was built using a standard solution of feso4 and the frap values of extract were expressed as mmol fe(ii)/g fresh weight. the method of dehghan and khoshkam (2012) was used for measuring the dpph radical scavenging ability of peach extracts. the amount of 50 μl of peach extract was allowed to react with 1.95 ml of dpph radical solution (0.1 mm in methanol) for 30 min. the decrease in absorbance from the resulting solution (as) was monitored at 517 nm in a uv-visible spectrophotometer (t-60, pg instrument uk). absorbance of the blank solution of dpph (2 ml) was adv. hort. sci., 2018 32(1): 33-40 36 used as an experimental control (ac). the radical scavenging activity (rsa %) of the peach fruits extracts was calculated according to the following formula: rsa % = 100 (ac-as) ac statistical analysis the experiment was performed using a factorial design with sa treatment and storage time as the two factors. differences among means of data were analyzed by duncan’s test at p≤0.05 (n=3). all statistical analyses were performed with spss version 20.0. 3. results and discussion as shown in figure 1, fruits weight loss increased and fruits firmness decreased during cold storage in control and treated fruits, but fruits weight loss was significantly lower (p<0.05) and fruits firmness were significantly higher (p<0.05) after storage at 1°c for 2-4 weeks in peach fruits coming from sa treated trees than in controls (fig. 2). cell wall degradation by cell wall hydrolases such as polygalactosidase (pg), pectin methyl esterase (pme), β-galactosidase (β-gal) and xylanase along with cell membrane deterioration led to fruits softening that are associated with climacteric rise in ethylene production (srivastava and dwivedi, 2000). zhang et al. (2003) reported that kiwifruit treated with acetyl salicylic acid exhibited lower ethylene biosynthesis during fruit ripening, results from lower acc oxidase (aco) and acc synthase (acs) activity. kiwifruits during postharvest softening of at 20°c exhibited lower endogenous sa content, which was concurrent with higher lox activity and higher ethylene production. lox by generation of o2 have a regulatory role in ethylene biosynthesis via a contribution in acc conversion to ethylene (xu et al., 2000). kiwifruit treated with acetyl salicylic acid exhibited higher endogenous sa accumulation associated with lower lox activity, o2 accumulation, and acs and aco activities and eventually delayed ethylene biosynthesis. zhang et al. (2003) suggested that the higher fruits endogenous sa accumulation leads to lower ethylene biosynthesis and higher firmness. also, srivastava and dwivedi (2000) reported that sa treatment delayed banana fruits ripening, results from lower ethylene biosynthesis due to lower acs and aco enzymes activity. they also reported that sa treatment maintains fruits firmness, results from lower pg, xylanase and cellulase activity. maintaining firmness in peach fruits treated with sa may be result of directly inhibition of cell wall degradation enzymes activity, indirectly decreasing ethylene production, and also higher firmness in peach fruit treated with sa could be attributed to endogenous sa accumulation which lead to lower lox activity and ros accumulation. loss of weight in stored peach is mainly due to evaporation of water from the fruits and becomes apparent as shriveling. the lower weight loss in peach fruits treated with sa could be attributed to stabilization of cell membrane as well as cell wall integrity and the permeability of tissues. maintaining ascorbic acid content in fruits during postharvest ripening is crucial for human health, due to antioxidant function of ascorbic acid and also for human disability for ascorbic acid synthesis (davey et al., 2000; hassanpour et al., 2011). as shown in figure 3, ascorbic acid content decreased in control and treated fruits during storage at 1°c for 28 days, but ascorbic acid content was significantly higher during cold storage in sa treated peach fruits than in controls (p<0.05) (fig. 3). huang et al. (2008) reported fig. 2 firmness of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). fig. 1 weight loss of peach fruits treated with preharvest sa at 1.5 mm stored at 1 ± 0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). razavi et al. salicylic acid and nutritional quality of peach fruit 37 that navel orange fruit treated with sa exhibited higher ascorbic acid content, results from increasing cytosolic ca+2, enhancing gr enzyme activity, which in turn could increase gr/apx system activity leading to higher ascorbate/dehydroascorbate (aa/dha) and glutathione/glutathione disulfide (gsh/gssg) ratios. also, rao et al. (2011) reported that the sweet pepper treated with sa and cacl2 exhibited higher ascorbic acid content, which results from lower ascorbic acid oxidase (aao) enzyme activity. higher aa content in peach fruits treated with sa may be attributed to higher gr/apx system activity due to increase of cytosolic ca+2 concentrations and or lower aao enzyme activity. enhancing phenols accumulation in fruits during postharvest ripening is crucial not only due to their contribution in nutritional quality attributes of fruits such as color, astringency, bitterness and flavor, but also phenols are superior antioxidants and display ros scavenging activity (hassanpour et al., 2011). due to phenols ros scavenging capacity and their function in decreasing low-density lipoproteins (ldl), consumption of fruits with higher phenols would be associated with lowered risk of heart disease (vinson et al., 2001). as shown in figure 4 and 5, total phenols and flavonoids contents were significantly higher after storage at 1°c for 2-4 week in sa treated peach fruits than in controls (p<0.05). it has been reported that sweet cherry fruits treated at preharvest with sa at 0.5 mm and asa at 1 mm exhibited higher total phenolics and total anthocyanins, as well as higher total antioxidant activity at commercial harvest (gimenez et al., 2014) and during storage at 2°c for 28 days (gimenez et al., 2017). also, valverde et al. (2015) reported that the total phenolics and anthocyanins content were significantly higher in methyl salicylate treated sweet cherry fruits at harvest and during storage at 2°c for 28 days, leading to fruits with higher hydrophilic taa (haa). due to higher pal enzyme activity in cornelian cherry fruits treated with sa and cacl2, which was associated with higher total phenols, flavonoids and anthocyanins accumulation (aghdam et al., 2013; dokhanieh et al., 2013), it can be postulated that the higher total phenols and flavonoids contents in sa treated peach fruits may attributed to higher pal activity. wang et al. (2015) reported that the apricot fruits treated with sa exhibited higher total phenols and flavonoids accumulation and higher hydrophilic antioxidant capacity. higher hydrophilic antioxidant capacity in apricot fruits treated with sa was associated with higher pal enzyme activity. also, apricot fruits treated with sa exhibited higher sod enzyme activity and lower cat and apx enzymes activity, which leads to lower o2 and higher h2o2 accumulation. h2o2 as second messenger can activate pal enzyme activity, as a key enzyme in phenylpropanoids pathway, and ultimately higher total phefig. 4 total flavonoids content of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). fig. 5 total phenolics content of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). fig. 3 ascorbic acid content of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). adv. hort. sci., 2018 32(1): 33-40 38 nols and flavonoids accumulation (wang et al., 2015). since peach fruits treated with sa exhibited higher cat and apx enzymes activity, higher total phenols and flavonoids contents cannot be attributed to higher h2o2 accumulation. as shown in figure 6 and 7, dpph and frap scavenging capacity of the peach fruits treated with sa were significantly enhanced during storage at 1°c for 28 days (p<0.05), showing that sa treatment stimulated the scavenging capacity of the peach fruits on dpph and frap radicals, which may be results from higher total phenols and flavonoids accumulation (razavi and hajilou, 2016). dokhanieh et al. (2013) and aghdam et al. (2013) reported that the cornelian cherry fruits treated with sa and cacl2 exhibited higher total phenols, flavonoids, and anthocyanins accumulation results from higher pal enzyme activity as key enzyme in phenylpropanoid pathway which is responsible for antioxidant molecules biosynthesis. we proposed that sa treatment may stimulate the accumulation of phenol, and flavonoid in the peach fruits by activating phenylpropanoid pathway. higher dpph and frap scavenging capacity of the peach fruits treated with sa may be results from higher total phenols and flavonoids accumulation due to higher pal enzyme activity concurrent with higher ascorbic acid accumulation due to higher cytosolic ca2+ and or lower aao enzyme activity. as shown in figure 8, 9 and 10, antioxidant enzymes cat, sod and apx activity increased during storage at 1°c for 28 days in control and treated fruits, and sod and apx activities were significantly higher during all the storage period at 1°c for 28 days in sa treated peach fruits than in controls (p<0.05), while concerning cat, higher activity was observed only after 3-4 weeks of storage. valverde et al. (2015) reported that sweet cherry fruits treated at preharvest with methyl salicylate exhibited higher antioxidant enzymes cat, apx and sod during storage at 2°c for 28 days. gimenez et al. (2017) reported that sweet cherry fruits treated at preharvest with sa at 0.5 mm and asa at 1 mm exhibited higher antioxidant enzymes cat, apx and sod during storage at 2°c for 28 days. thus, salicylates treatment of peach trees enhances health boosting attributes of peach fruits consumption, by increasing antioxidant fig. 8 cat activity of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). fig. 9 sod activity of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). fig. 6 dpph scavenging capacity of peach fruits treated with preharvest sa at 1.5 mm stored at 1 ± 0.5°c for up to 28 days. data shown are mean±standard deviation of three replicate (n = 3). fig. 7 frap scavenging capacity of peach fruits treated with preharvest sa at 1.5 mm stored at 1±0.5°c for up to 28 days. data shown are mean ± standard deviation of three replicate (n = 3). razavi et al. salicylic acid and nutritional quality of peach fruit 39 molecules, with supernumerary impacts on delaying the peach fruits postharvest senescence by enhancing ros scavenging enzymes activities. higher antioxidant enzymes activity, together with higher antioxidants molecules accumulation, in peach fruits during storage, as a results of preharvest sa treatment, could contribute to ros scavenging during the postharvest ripening, which in turn, leads to delaying peach fruits postharvest ripening and senescence. sa enhance antioxidant systems activity by avoiding and/or scavenging ros, which led to decrease oxidative stress during peach fruits ripening and ultimately maintain postharvest quality by prevention of adverse effects of ros on fruits quality. 4. conclusions sa, as safe signaling molecule, could enhance nutritional quality and improve health promoting attributes of peach fruits consumption. in addition, 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507-514. yu l., liu h., shao x., yu f., wei y., ni z., xu f., wang h., 2016 effects of hot air and methyl jasmonate treatment on the metabolism of soluble sugars in peach fruit during cold storage. postharvest biol. technol., 113: 8-16. zhang y., chen k., zhang s., ferguson i., 2003 the role of salicylic acid in postharvest ripening of kiwifruit. postharvest biol. technol., 28: 67-74. zhang z., huber d.j., rao j., 2013 antioxidant systems of ripening avocado (persea americana mill.) fruit following treatment at the preclimacteric stage with aqueous 1-methylcyclopropene. postharvest biol. technol., 76: 58-64. impaginato 131 1. introduction the ability of plants to tolerate salinity stress is facilitated by a series of biochemical pathways which maintain or absorb water, protect plants chloroplast function and sustain an ionic balance. some of these pathways include the synthesis of active osmotic metabolites (zhifang and loescher, 2003). some proteins and enzymes destroy free radicals (mittova et al., 2003). carica papaya l. is the only species belonging to the carica genus. it can be found in tropical region of america. the cultivation of this crop is common in southern mexico, central america and south america, as well as most countries in the tropics. papaya is a fast-growing tree (it produces fruit in the third year after planting), extremely sensitive to cold, and planted only in the tropics. papaya fruit is rich in carotenoids, vitamins b, c, lycopene and mineral fibers. the skin, flesh and seeds of this product contain a number of phenolic compounds. salinity stress in soil or water, especially in hot, arid regions, could limit plant growth and reduce its yield (koca et al., 2007). plants growing in areas with extreme salinity are divided into halophytes and glycophytes. most glycophyte plants do not have the ability to tolerate salinity stress (sairam and tyagi, 2004). during salinity stress, all the main processes, including photosynthesis, lipid metabolism and energy, are affected (sairam and tyagi, 2004). the first response is to reduce the development rate of leaf area and then complete cease. however, growth process resumes as soon as the problem is fixed (parida and das, 2005). the plant either tolerates the stress, or avoids it. the former generally occurs at the cell level while the latter occurs at the plant level. during salinity stress, a plant can undergo dormancy (avoidance) or make certain cellular adjustments in order to resist drought stress (yokoi et al., 2002). the adv. hort. sci., 2017 31(2): 131-139 doi: 10.13128/ahs-21090 effects of salinity stress on certain morphological traits and antioxidant enzymes of two carica papaya cultivars in hydroponic culture a. refahi, a.r. shahsavar (*) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: antioxidant enzymes, carica papaya, hydroponic, morphological factors, salinity stress. abstract: carica papaya l. is the only species belonging to the carica genus. salinity stress in soil or water, especially in hot, arid regions, could limit plant growth and reduce its yield. this research studied six-month old seedlings of two cultivars of papaya (‘sinta’ and ‘solo’), in solid, disease-free form for two weeks inside a half-dose of hoagland solution. results obtained from the effects of salinity stress indicated that the longest root and shoot were observed in the control treatment in ‘sinta’. moreover, there was no significant difference between the two cultivars in terms of root length, shoot length, fresh weight of roots and fresh weight of shoots in different salinity levels. the highest dry weights of roots and shoots were found in the ‘sinta’ control treatment, while the lowest was observed in ‘solo’ 6 ds/m treatment. there was no significant difference between the two cultivars in terms of dry weights of roots and shoots. finally, the interaction of salinity levels showed that increasing salinity in both cultivars led to higher peroxidase, catalase, superoxide dismutase and ascorbic peroxides activity. by increasing the salinity level, the total protein and proline greatly increased in both cultivars, where the maximum value was found in the ‘sinta’ 6 ds/m salinity treatment, and this was significantly different from other treatments. a comparison of the different salinity levels showed that there was a significant difference between the 6 ds/m salinity treatment and other treatments. (*) corresponding author: shahsava@shirazu.ac.ir received for publication 16 january 2017 accepted for publication 21 june 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 131-139 132 aim of this study is to survey the effect of salinity stress on certain morphological traits and also on antioxidant enzymes of two carica cultivars in hydroponic culture condition. 2. materials and methods six-month old seedlings of two cultivars of papaya (‘sinta’ and ‘solo’), solid and disease-free, were placed for two weeks in a glass containing 700 ml of a half-dose hoagland solution, and were then transferred to the hydroponic system. afterwards, 500 ml of the half-dose hoagland solution per seedling was added to the hydroponic system. after two weeks, the salinity treatments were initiated at ph=6 as salinity stress continued for 8 weeks. then, the leaf samples were placed in aluminium foil and frozen at 20°c for further measurements of protein and antioxidant enzymes. plant height was measured with a ruler. at the end of the experiment, the fresh weight of plant organs was measured and then rinsed through distilled water and finally kept in the oven at 70°c until the dry weight was stabilized. then, shoots and roots dry weights were measured. a factorial experiment was conducted based on completely randomized design with 5 replications in the greenhouse of the horticultural science department, university of shiraz. the factors included salinity treatments: 0 (control), 2, 4, 6, 8 and 10 ds/m nacl which were added to half dose hoagland solution and 2 cultivars of papaya (‘sinta’ and ‘solo’). data analysis was done using sas (version 9.2; sas institute, cary, nc, usa), mean comparisons were carried out using lsd test at 5% of probability. extraction for measuring the amount of protein and antioxidant enzymes for extraction, 0.5 g of root or leaf sample was first ground in liquid nitrogen and then 2 ml of extraction buffer was added and homogenized in a porcelain mortar. then this mixture tube was centrifuged at 13,000 rpm for 15 min at 4°c. the upper phase was isolated for the purpose of reading the protein content and enzyme activity. for the preparation of the extraction buffer (50 ml), 0.607 g of tris(hydroxylmethyl)aminomethane and 0.05 g of polyvinylpyrrolidone (pvp) were dissolved into 45 ml of distilled water (ph 8.0). total protein the bradford assay (1976) was used to determine the protein concentration. to measure the protein concentration, 20 ml extract was diluted in 80 µl of extraction buffer; 5 ml of fresh reagent coomassie was added, stirred for 2 min, and finally, after 5 min, the optical density was read at a wavelength of 595 nm. besides, the extraction buffer was used as control. the concentration of protein in the sample was obtained according to the absorption, using the standard curve. bovine serum albumin was used as the standard and total soluble protein concentrations were expressed in mg g -1 fresh weight. proline the method of bates et al. (1973) was employed to measure the concentration of proline. according to this method, 0.5 g of leaves from each sample was placed in 10 ml of an aqueous solution of sulfosalicylic acid (3%) and the mixture was completely homogenized in a porcelain mortar. then, the homogenized mixture was filtered through paper no. 2. in the next stage, 2 ml of solution was mixed with 2 ml of a reagent, creatininedimenhydrinate and 2 ml of acetic acid was added to each tube. then, the samples were placed in bain-marie bath for 1 h at a temperature of 100°c and were immediately transferred into an ice bath for a few minutes. afterwards, 4 ml of toluene was added to each tube and the samples were stirred through vortex for 15 to 20 s until they were completely homogeneous. the supernatant phase was used to determine proline concentration based on the proline standard curve in the spectrometer at a wavelength of 520 nm. proline concentration was calculated using l-proline for the standard curve. guaiacol peroxidase (pod) activity to measure the quantitative concentrations of this enzyme, the method of chance and mahly (1955) was used with minor modifications. measurements were done according to the oxidation of guaiacol by that enzyme. in this method, 33 mol of extract was dissolved into 1 ml of a peroxidase solution containing 13 mm guaiacol, 5 mm hydrogen peroxide (h2o2) and 50 mm potassium phosphate buffer (ph=7), and the absorbance values were read for one minute at 10-s intervals and at a wavelength of 470 nm. to prepare 1000 ml of potassium phosphate buffer, 39 ml potassium phosphate and 50 mm monohydrate were mixed with 61 ml potassium dihydrogen and 50 mm phosphate. ascorbic peroxidase (apx) activity to measure the quantitative concentrations of this enzyme, the method of nakano and asada refahi and shahsavar salinity stress on morphological traits and antioxidant enzymes of two carica papaya cultivars 133 (1981) was used. according to this method, 50 ml of the extract was mixed with 1 ml of ascorbic peroxidase containing 50 mm potassium phosphate buffer (ph=7), 0.1 mm edta, 0.5 mm ascorbic acid (asa), and 0.15 mm peroxide hydrogen (h2o2). then, the absorption at a wavelength of 290 nm was read within one minute through a spectrophotometer. an enzymatic unit of ascorbic peroxidase is equivalent to a dissolution of 1 mm ascorbic acid in a minute. catalase (cat) activity to measure the quantitative concentrations of this enzyme, the method of nakano and asada (1981) was used. according to this method, 50 ml of the extract was mixed with 1 ml of catalase containing 50 mm potassium phosphate buffer (ph=7) and 15 mm hydrogen peroxide (h2o2). then, the absorption at a wavelength of 240 nm was read within one minute through a spectrophotometer. an enzymatic unit of catalase is equivalent to a dissolution of 1 mm hydrogen peroxide (h2o2) in a minute. superoxide dismutase (sod) activity to measure the quantitative concentrations of this enzyme, the method of beauchamp and fridovich (1971) was used. the measurement was based on the ability of sod enzyme to stop the photochemical reduction of nbt by superoxide radicals in the presence of riboflavin in light. according to this method, 50 ml of the extract was mixed with 1 ml of superoxide dismutase containing 50 mm potassium phosphate buffer (ph 7.8), 75 mm of nbt, 13 mm of l-methionine, 0.1 mm of edat and 2 mm riboflavin. it should be noted that the solution was stored separately in a dark container and after the addition of soluble extract and measurement solution, superoxide dismutase was added to qt. the mixture reacted when placed in the light chamber for 15 min. the solution was then placed in a spectrophotometer and the absorbance was measured and read at a wavelength of 560 nm. 3. results the interaction between salinity levels and cultivars on root length showed that the highest root length was found in the control treatment in sinta, while the minimum value was found in the 6 ds/m treatment of both ‘sinta’ and ‘solo’. seedlings of both cultivars in the 8 and 10 ds/m treatments exhibited no salinity resistance and were dried, therefore, these two treatments were excluded from the results; results also showed no significant differences between the two cultivars, in terms of root length, at various salinity levels (fig. 1). as shown in figure 2, the highest shoot length was observed in the ‘sinta’ control treatment, which was significantly different from other treatments. moreover, there was no significant difference between the two cultivars in terms of salinity levels. the control group of both cultivars generally had a significant difference with other treatments, while there existed no significant difference between concentrations of 2, 4 and 6 ds/m. evaluation of salinity effect on roots fresh weight between the two cultivars showed that the highest and lowest values were observed in the control treatment and the sinta 6 ds/m treatment, respectively. there was no significant differences between the two cultivars in terms of root fresh weight. the comparison of the various salinity levels, showed a significant difference between the control and other salinity treatments, while there was no significant difference fig. 1 interaction of cultivar and salinity levels on roots length. the means followed by the same letters were not significantly different at p≤0.05. fig. 2 interaction of cultivar and salinity levels on shoots length. the means followed by the same letters were not significantly different at p≤0.05. adv. hort. sci., 2017 31(2): 131-139 134 between treatments 2, 4 and 6 (ds/m) (fig. 3). in this study maximum root dry weight was observed in the ‘sinta’ control (fig. 4) while the minimum was observed in the ‘solo’ 6 ds/m treatment. the comparison between the two cultivars showed a significant difference between them in terms of roots dry weight. the mean salinity concentrations indicated that the control had a significant difference with other concentrations, while there was no significant difference between concentrations of 2 and 4 ds/m. evaluation of treatments applied in this study on shoots fresh weight indicated that control treatment in both ‘sinta’ and ‘solo’ had the highest values, while treatments 4 and 6 ds/m had the lowest values. a comparison of the two cultivars showed no significant differences in term of shoot fresh weight. in the comparison of salinity concentrations, no statistically significant difference was found between the control and 2 ds/m (fig. 5). according to figure 6, the findings of this study regarding the shoots dry weight showed that the highest dry weight was found in the control ‘sinta’, where there was no significant difference between this treatment and the 2 and 4 ds/m salinity treatments. the lowest shoot dry weight was found in the ‘solo’ 6 ds/m salinity treatment. a comparison of the two cultivars represented a significant difference in the shoots dry weight. moreover, the comparison of various salinity levels showed no significant difference between the control and 2 ds/m salinity treatments for both cultivars. the interaction effect of cultivar and salinity levels in this study showed that an increase in the salinity levels enhanced peroxidase activity in both cultivars. the highest enzyme activity was observed in ‘sinta’ 6 ds/m salinity treatment while the lowest was in ‘solo’ control treatment. a comparison of values between the cultivars showed a significant difference in terms of peroxidase activity. moreover, the comparison of various salinity levels showed a significant difference between the 6 ds/m salinity treatment and other treatments for both cultivars (fig. 7). according to figure 8, the increased salinity levels in both cultivars enhanced catalase activity. the maximum level of this enzyme was found in the ‘sinta’ 6 fig. 3 interaction of cultivar and salinity levels on root fresh weight. the means followed by the same letters were not significantly different at p≤0.05. fig. 4 interaction of cultivar and salinity levels on root dry weight. the means followed by the same letters were not significantly different at p≤0.05. fig. 5 interaction of cultivar and salinity levels on shoot fresh weight. the means followed by the same letters were not significantly different at p≤0.05. fig. 6 interaction of cultivar and salinity levels on shoot dry weight. the means followed by the same letters were not significantly different at p≤0.05. refahi and shahsavar salinity stress on morphological traits and antioxidant enzymes of two carica papaya cultivars 135 ds/m salinity treatment, which showed a significant difference with other treatments, while the minimum values were observed in the control treatment of both cultivars. a comparison of the two cultivars showed no significant differences. in the showed results, there was a significant difference between the different levels of salinity in the 6 ds/m treatment and the control 2 ds/m, while there was no significant difference when compared to the 4 ds/m treatment. in this study, the interaction effect of salinity stress on superoxide dismutase activity showed an increase at higher salinity levels in both cultivars. the maximum activity of this enzyme was found in the solo 6 ds/m salinity treatment which was not significantly different when compared with other treatments and the ‘sinta’ 4 and 6 ds/m salinity treatments. the comparisons between the cultivars were significantly different. the comparison of different salinity levels showed no significant difference between the 4 and 6 ds/m treatments (fig. 9). results reported in figure 10 showed that as salinity levels increased, the ascorbic peroxidase activity also increased. an evaluation of the interaction of salinity levels on the enzyme activity showed that the highest value was found in sinta 6 ds/m treatment while there was no significant difference with the same treatment in ‘solo’. the comparison between the cultivars showed no significant difference. moreover, a comparison of various salinity levels showed a significant difference between 6 ds/m salinity treatment and other treatments for both cultivars. from figure 11, the interaction effect of salinity levels on activity of total protein in this study showed that as salinity levels increased there was higher protein content in both cultivars. the maximum value was observed in ‘sinta’ of 6 ds/m salinity treatment, which was significantly different from other treatments, while the lowest was in the control for the two cultivars which were not significantly different. no significant difference was found when the cultifig. 7 interaction of cultivar and salinity levels on peroxidase activity. the means followed by the same letters were not significantly different at p≤0.05. fig. 8 interaction of cultivar and salinity levels on catalase activity. the means followed by the same letters were not significantly different at p≤0.05. fig. 9 interaction of cultivar and salinity levels on superoxide dismutase activity. the means followed by the same letters were not significantly different at p≤0.05. fig. 10 interaction of cultivar and salinity levels on ascorbic peroxidase activity. the means followed by the same letters were not significantly different at p≤0.05. adv. hort. sci., 2017 31(1): 131-139 136 vars were compared. as for the various salinity levels, the results showed that the 6 ds/m salinity treatment was significantly different from other treatments. evaluation of interaction effect between salinity levels on proline showed an increase in salinity level in both cultivars, followed by higher proline. in fact, the maximum amount of proline was observed in the sinta 6 ds/m salinity treatment which was significantly different from other treatments. there was no significant difference when both cultivars were compared. moreover, the comparison of various salinity levels showed a significant difference between the 6 ds/m salinity treatment and other treatments for both cultivars (fig. 12). 4. discussion and conclusions as mentioned in the results, the vegetative indicators decreased upon initiation of salinity treatments. literature indicates that plants, especially glycophytes, are highly sensitive during their early vegetative growth, for instance chickpea (khan et al., 2016), pepper (penella et al., 2016), grapevine (ikball et al., 2014), tomato (manai et al., 2014), rice (horie et al., 2012), cantaloupe (botia et al., 2005). increased salt leads to na+ sediment into root growth area, thus reducing the ability to select k+ versus na+ for root cells, which ultimately reduces their growth rate (zhong and lauchli, 1994). when exposed to saline conditions, plants show reduced uptake and low tissue retention of k+. (chakraborty et al., 2012; gharsallah et al., 2016). accordingly, k+ is considered as a key regulatory elements in plant metabolic process by promoting na+ exclusion and osmotic adjustment (chakraborty et al., 2016; gharsallah et al., 2016). reduced shoot growth due to salinity usually appears as shoots with low growth and reduced leaf area (lauchli and epstein, 1990). saline conditions reduce root growth and reduce water movement through the root with a decrease in hydraulic conductivity (acosta-motos et al., 2017). root hydraulic conductance is expressed in terms of root dry weight. root dry weight values which determine the root length and surface area, may vary greatly, thus affecting the water absorption (jonathan et al., 2006; zobel et al., 2007; acosta-motos et al., 2017). the results of our study are in accordance with these results. decreasing in fresh weight or dry weight has been observed in all plant tissues subjected to salt stress especially in the aerial part (acosta-motos et al., 2017). the stem growth is also reduced by salinity conditions. one of the important reason for decreasing root and shoot growth under saline condition could be the decreasing of nitrogen uptake in response to external nacl salinity due to antagonism between na + and nh4 + or between cland no3 (parihar et al., 2015; salachna and piechocki, 2016). another reason responsible for the reduction of vegetative index in the shoots, similar to what was mentioned for roots, is ion imbalance and increased ratio of na to ca (neves-piestun and bernstein, 2005). in the current experiments, the concentration of proline, which has an important role in eliminating free radicals and enzymes, increased at higher salinity levels. accumulation of proline under salinity conditions has been indicated to correlate with salt tolerance (mansour and ali, 2017). in fact, to eliminate osmotic stress created by high salinity, plants need to synthesize compatible organic solutes such as proline in the cytosol (gharsallah et al., 2016). besides its role as an osmolyte, proline contributes to scavenging ros, stabilizing sub cellular structures and functioning as a signal (szabados and fig. 11 interaction of cultivar and salinity levels on protein content. the means followed by the same letters were not significantly different at p≤0.05. fig. 12 interaction of cultivar and salinity levels on proline. the means followed by the same letters were not significantly different at p≤0.05. refahi and shahsavar salinity stress on morphological traits and antioxidant enzymes of two carica papaya cultivars 137 savoure, 2010). generally, plants under salinity conditions need to sustain their water potential (below potential of ground water) because they can continue absorbing water from the soil in order to maintain their turgor (tester and davenport, 2003). to maintain osmotic potential as well as ionic balance, plant cells during stress tend to accumulate any substance compatible with metabolism which does not interfere with other biochemical processes and are active in osmotic terms (zhifang and loescher, 2003). these substances cover a wide range of compounds such as carbohydrates, proteins and amino acids, including proline. this amino acid accumulates at a higher concentration than other amino acids in plant cells (abraham et al., 2003). it is possible that proline as a signaling molecule or regulator can activate the response as an adjustment process (maggio et al., 2002). hence, given the foregoing facts, there is a positive relationship between proline and curtailed damage of salt sensitivity, where the current study was consistent with previous works. in relation to salt damage to the overall growth of plant, the concentration of enzymes eliminating free radicals is very important. in their resistance against the damaging effects of reactive oxygen species, plants possess anti-free radical enzymes such as catalase, peroxidase, superoxide dismutase and other enzymes which eliminate reactive oxygen species and free radicals (mittova et al., 2003). in our study, the concentration of eliminating free radicals enzymes such as peroxidase, catalase, superoxide dismutase and ascorbic peroxidase increased at higher salinity levels. the increases in theses enzymes activities are an adaptive trait to overcome salt damage by reducing toxic levels of h2o2 and provide protection against oxidative stress (chawla et al., 2013; gharsallah et al., 2016). catalase, ascorbic peroxidase and glutation peroxidase have been reported as antioxidant enzymes in different plant tissues (chawla et al., 2013). during salinity stress, the balance between production and consumption of reactive oxygen species (ros) is disrupted, leading to the formation of condensation oxide (spychalla and desborough, 1990). ros has the potential to damage cellular structures, perchloric acids, fats and proteins (valko et al., 2006). in some salt-tolerant plants, increased in catalase activity have been recored after increasing nacl, such as those described in myrtle, suggesting increased photorespiratory activity (acosta-motos et al., 2015, 2017). catalase is often related to an enhanced tolerance to salt stress (gao et al., 2008; gharsallah et al., 2016). similarly, ascorbate peroxidase activity under salinity stress increases (mittova et al., 2004; gharsallah et al., 2016). these responses to salinity were the results of differentially increased activities of ascorbate peroxidase and catalase over that of superoxide dismutase (mittova et al., 2004; gharsallah et al., 2016). the results of our study were in accordance with these results. the results of this study indicated also that as salinity levels increased, there was higher protein content in both cultivars. this result was in agreement with that of abdel-haleem (2007) who reported as increase in protein band which might be involved in mungbean tolerance. plants growing in saline environments show distinct changes in the pattern of synthesis and accumulation of proteins. salinity causes either decreased or increase in the level of soluble proteins or completely disappears in some proteins when compared to the control treatment (win and zaw, 2017). salinity is an important stress in arid and semiarid region that reduces crops productivity, including that of the cultivars of carica papaya l. here investigated (‘sinta’ and ‘sola’). shoots and roots growth were decreased under salinity conditions. there was no significant difference between the two cultivars in terms of root length, shoot length, fresh weight of roots and shoots in different salinity levels. interestingly, on the other hand, in both cultivars increasing salinity led to higher peroxidase, catalase, superoxide dismutase and ascorbic peroxidase activity to provide protection against oxidative stress. also the increases in those enzymes activities are an adaptative trait to overcome salt damage by reducing toxic levels of h2o2. furthermore, in this experiment the concentration of proline, which has an important role in eliminating free radicals, increased at higher salinity levels in both cultivars; 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change in response to changes in nutrient concentrations. plant and soil., 297: 243-254. impaginato 319 adv. hort. sci., 2018 32(3): 319-324 doi: 10.13128/ahs-21978 shelf life of iceberg lettuce affected by hydro cooling and temperature of storage c.f.m. frança 1, m.n.s. santos 2, w.s. ribeiro 3, p.r. cecon 4, f.l. finger 3 (*) 1 universidade federal do oeste da bahia, campus multidisciplinar da barra, 41100-000, barra, ba, brazil. 2 universidade federal de viçosa, departamento de biologia vegetal, viçosa, mg, 36570-900, brazil. 3 universidade federal de viçosa, departamento de fitotecnia, viçosa, mg, 36570-900, brazil. 4 universidade federal de viçosa, departamento de estatística, viçosa, mg, 36570-900, brazil. key words: carbohydrates, chlorophyll, cooling curve, lactuca sativa l., relative water content, weight loss. abstract: pre cooling is applied to remove the field heat of harvested horticultural produces. the goal of this work was to determine the cooling curve and the effects of hydro cooling on quality and shelf life of iceberg lettuce ‘lucy brown’ stored at 5°c and 22°c. through shelf life, it was determined the changes on accumulated fresh weight loss, leaf relative water content and total chlorophyll, total soluble sugars, reducing and non-reducing sugars, and starch. the field heat from iceberg lettuce heads was removed within the first 10 min when submerged into cooled water at 4°c. hydro cooled lettuce heads accumulated water over the leaf surfaces resulting in higher rate of fresh weight loss during storage when compared to control. lettuce stored at 5°c kept higher relative water content in the leaves throughout the shelf life. hydro cooling treatment delayed the wilting of the external leaves in three and two days when stored at 5 and 22°c, respectively. hydro cooling did not influence the decrease on total soluble sugars, reducing sugars, non-reducing sugars and starch throughout shelf life, but affected the leaf chlorophyll content. independent of the temperature in which the ‘lucy brown’ iceberg lettuce will be stored, hydro cooling is recommended to prolong quality and shelf life. 1. introduction senescence is a natural process common to all fresh vegetables, which is intensified after harvest, by handling and storage conditions. in addition, the rate of deterioration is quickly intensified if a vegetable or a fruit is stored under stressed conditions. storing fresh horticultural products under extremes of high temperature or under low relative humidity, results in intense water loss, triggers senescence and finally the death of the tissues. (*) corresponding author: ffinger@ufv.br citation: frança c.f.m., santos m.n.s., ribeiro w.s., cecon p.r., finger f.l., 2018 shelf life of iceberg lettuce affected by hydro cooling and temperature of storage. adv. hort. sci., 32(3): 319-324 copyright: © 2018 frança c.f.m., santos m.n.s., ribeiro w.s., cecon p.r., finger f.l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 november 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 319-324 320 the major important factor affecting the postharvest shelf life for most of fresh horticultural products is the temperature during storage or display. to preserve the quality and prolong the perishables commercial quality, it is necessary to rapidly remove the field heat to an optimum temperature for subsequent storage (brosnan and sun, 2001). independent of the pre cooling method to be used, the premises as an useful postharvest practice, is based on the quick reduction of product temperature. in addition, it is recommendable to reduce the temperature as soon as possible after harvest, which will increase the beneficial effects of the rapid cooling. among the many benefits of pre cooling, in keeping the quality of a produce, is the lower respiration rate, the reduction of water loss by the product, and less contamination by pathogenic microorganisms (brosnan and sun, 2001). the rapid loss of quality and limited shelf life of leafy vegetables, like lettuce, parsley and jute leaves is mainly due to their fast postharvest dehydration (tulio jr. et al., 2002; finger et al., 2008; aguero et al., 2011). in these products wilting of leaves occur even faster when the storage is done under high storage temperatures or without any refrigeration and inadequate packaging. there is a variety of pre cooling techniques available including cooling rooms, hydro cooling systems, air forced cooling, ice packaging, vacuum and cryogenic cooling (brosnan and sun, 2001). hydro cooling is relatively inexpensive and very effective method recommended to remove the field heat of several leafy vegetables including kale, green onions and spinach (sargent et al., 2007). álvares et al. (2007) determined that hydro cooled parsley leaves had less water loss, resulting in longer shelf life without the appearance of wilting symptoms compared to control. hydro cooling also proved to be a faster method for cooling peach pulp to 1°c compared to forced air and conventional cooling room methods (brackmann et al., 2009). the expansion of large cities in developing are pushing the vegetable farms farther away from the markets, making harder for them to deliver products with good quality to urban population. this situation demands the incorporation of appropriated postharvest handling, but most of the small farmers have no capital to purchase refrigeration systems. iceberg lettuce is the most popular leaf vegetable used in burgers, sandwiches and salads by fast food stores in most of the countries, including brazil. thus, there is the need to evaluate the influence of pre cooling on the shelf life of this lettuce. therefore, the objective of this work was to determine the cooling curve and the effects of hydro cooling on quality and shelf life of iceberg lettuce heads stored under cold and room temperature conditions. 2. materials and methods heads of iceberg lettuce ‘lucy brown’ were harvested from the field at federal university of viçosa (642 m asl, 20°45’ lat. s and 42°51’ long. w) in the morning between 7 and 7:30 hours. the heads of lettuce were taken to the laboratory quickly and the heads with external leaves with brown spots, wilted, or dirty leaves were discarded. the lettuce heads weighting between 300 to 400 g were subjected to the following treatments: 1) hydro cooling followed by storage at 5°c; 2) control without hydro cooling and storage at 5°c; 3) hydro cooling followed by storage at 22°c; 4) control without hydro cooling and storage at 22°c. hydro cooling was performed by submerging the heads in a mixture of tap water and crushed ice at proportion of 3:1 (v:v) kept at 4oc. temperature of lettuce heads was determined at every five minutes with the help of a digital infrared thermometer. the temperature of the heads before initiating the hydro cooling treatment was between 20 to 22°c. at every 5 minutes, two heads were quickly removed from the cold water to determine the changes in the temperature, repeating the procedure up to fifty minutes. at the end of hydro cooling, the heads were removed from the cold water and allow draining for 5 min in the air before storage in the plastic boxes. hydro cooled and control lettuces were kept in plastic boxes at 5 and 22°c for the whole experiment. the boxes (18 cm height, 25 cm wide × 48 cm length) were covered with perforated (12 holes 1.1 cm in diameter) low density polyethylene plastic sheets to protect from excessive dehydration. the relative humidity inside the boxes was always between 85 and 90%. loss of fresh weight of heads, leaves relative water content, chlorophyll, total soluble sugar, reducing sugar, non-reducing sugar and starch leaf contents were determined at every 12 h up to the first 48 h and then at every 24 h until the end of the lettuce shelf life. the end of shelf life was determined when the heads were wilted, yellowed or with signs of deteriofrança et al.storability of lettuce iceberg 321 ration, being unfit for commercialization. the wilting, yellowing or deterioration of 50% or more heads was used as the discard parameter. the accumulated loss of fresh weight was obtained in relation to initial fresh mass of heads and during storage period. the leaf relative water content (rwc) was determined as described previously by álvares et al. (2007) with modifications. fifteen leaf discs with 1.1 cm in diameter were removed from the external, middle and internal position in the lettuce head, which were kept between two layers of wet sponge sheets until to obtain the leaf turgid fresh weight and then, they were oven dried at 70°c to obtain the total dry mass. the fresh weight of the disc, the turgid weight and the dry weight were used to estimate the rwc according to the formula established by barr and weatherley (1962). total chlorophyll content was determined in a combine sample of leaf discs removed from the external, middle and internal position in the head, following the method described by inskeep and bloom (1985) using 5,5-dimethylformamide as extractor. the absorbance of the filtrate was determined in a spectrophotometer at 647 and 664.5 nm and the results expressed in µg cm-2. samples of five grams of leaves from the external, middle and internal position of the lettuce head were homogenized in 80% hot ethanol and centrifuged at 2000 rpm for 15 min. the pellet was then re-extracted twice with 80% ethanol, and the total soluble sugars were determined by the phenol-sulfuric acid reaction (dubois et al., 1956). from the same extract was determined the reducing sugars content by the somogyi-nelson method (nelson, 1944). for total soluble sugars analysis sucrose was used as standard and glucose for the reducing sugar analysis. afterwards, the pellet from the ethanolic extraction was dried at 65°c and then the starch was hydrolyzed in 52% perchloric acid for 30 min with shaking (mccready et al., 1950). the procedure was repeated three more times. the quantification of starch was performed by the phenol-sulfuric acid reaction using sucrose as standard using the correction factor of 0.9. the total of non-reducing sugars was obtained by the difference between the total soluble sugars minus the content of reducing sugars. the experiment was conducted in a split-plot scheme, with the treatments in the plots and shelf life in the subplots in a randomized block design with four treatments and four replicates per treatment. each replicate was composed by one lettuce head. individual analysis of variance was performed to evaluate the effect of the hydro cooling and temperature of storage by using the saeg/ufv software, and the mean separation was done by scott knott test at 5% probability. the regression analysis was based on the regression coefficient using the t-test at 5% or 10% probability to establish the significance for the chosen regression model. 3. results and discussion initial temperature of the lettuce head showed a sharp drop within the first 10 min of hydro cooling time. the model that better explained the changes in temperature was exponential, with an estimated final temperature of 4.8°c after 10 min of hydro cooling (fig. 1). longer periods of cooling time did not remove additional field heat from the lettuce head. based on the lettuce temperature record data, the total amount of heat removed from the lettuce, by the cold water under this experiment conditions, corresponded to 71% from the initial temperature (fig. 1). the 87.5% or seven eighths cooling times recognized as the ideal theoretical reduction for the field heat presented by brosnan and sun (2001) was not achieved in this experiment, even after keeping the heads submerged in the cold water mixture for 50 min (fig. 1). using the same cooling technique of this experiment, álvares et al. (2007) reported the removal of only 43% of the initial temperature in bunched parsley leaves. the reason why the hydro cooling of lettuce was much more efficient in removing the field heat than parsley remains to be the subject of further studies. furthermore, the cooling time varied according to varieties of lettuce, as found by frança et al. (2015) working with butter lettuce where the ideal hydro cooling time was 5 min instead fig. 1 influence of hydro cooling time period treatment on the temperature of iceberg lettuce 'lucy brown'. adv. hort. sci., 2018 32(3): 319-324 322 of 10 min found for iceberg lettuce in this work (fig. 1). the difference of hydro cooling time between the two cultivars of lettuce may relay on the thickness and compactness of the whole head. the leaves from iceberg lettuce are thicker and more compact head than butter lettuce, which can restrict the access of cold water into the more internal leaves of the head. in others leaf vegetables, including peppermint, coriander and basil, the ideal time of hydro cooling was 20, 10 and 16 min, respectively (oliveira et al., 2015; barbosa et al., 2016; teixeira et al., 2016). the end of shelf was established when symptoms of wilting and discoloration appeared in the external leaves (data not shown). shelf life of hydro cooled lettuce and stored at 5°c was increased by 75% compared to not cooled lettuce, comprising a total of 168 h for the hydro cooled and 96 h for not cooled heads. for the lettuce that was hydro cooled and then stored at 22°c, the gain of shelf life was 50% or 72 h for hydro cooled and 48 h for not cooled lettuce. álvares et al. (2007) also found beneficial effects to the shelf life of hydro cooled bunched parsley leaves followed by cold storage. the result of this study shows the importance of keeping the cold chain for fresh vegetables, but also shows the contribution of hydro cooling on extending the shelf life even without further cold storage. because of the hydro cooling positive effects, the external leaves of the lettuce had a 72 h delay in showing wilt symptoms if stored at 5°c and 24 h delay for the lettuce stored at 22°c (data not shown). the increased shelf life of hydro cooled lettuce was determined by the higher leaf relative water content of hydro cooled lettuce (table 1). the smaller effect of hydro cooling in the lettuce kept at 22°c compared to 5°c may be due to the greater gradient of water vapor between the leaf surface and the atmosphere of storage at 22°c. but, in a similar experiment with butter lettuce, the beneficial effect of hydro cooling on shelf life was greater for the lettuce stored at 22°c compared to the shelf life of hydro cooled heads followed by storage at 5°c (frança et al., 2015). regardless the treatment, the rate of fresh weight loss was constant, resulting in linear accumulation up to the end of the lettuce shelf life (fig. 2). the lowest rate of weight loss was determined in the lettuce stored at 5°c without hydro cooling (0.109% h-1) and the highest for the heads hydro cooled and stored at 22°c (0.26% h-1), as previously observed in a similar experiment with butter lettuce by frança et al. (2015). the higher weight loss rate of hydro cooled lettuce was due to the water accumulated at surface and in between the leaves after being removed from the cooled water. regardless if the lettuce was hydro cooled or not, the lower rates of weight loss found for heads stored at 5°c was determined by the smaller gradient of water vapor compared to the storage room at 22°c (wills et al., 2010). like in this experiment, when coriander leaves were stored at 5°c also had lower rates of weight loss compared to leaves stored at 20°c, regardless if the leaves were submitted to hydro cooling treatment (oliveira et al., 2015). during the whole period of storage, the leaves of hydro cooled lettuce had higher relative water content when stored at 5°c (table 1). this higher content of water found in the hydro cooled lettuce leaves was clearly observed in the appearance of lettuces, which were more turgid than those not hydro cooled, which resulted in longer shelf life due to fresher appearance. similar results were found for hydro cooled butter lettuce heads, peppermint and coriander leaves, which also had higher relative fig. 2 accumulated fresh weight loss of iceberg lettuce 'lucy brown' submitted to the following treatments: t1) hydro cooling for 10 min + storage at 5°c; t2) control without hydro cooling + storage at 5°c; t3) hydro cooling for 10 min + storage at 22°c; t4) control without hydro cooling + storage at 22°c. te= time. table 1 influence of the hydro cooling and temperature of storage on the leaf relative water content during storage period of iceberg lettuce heads 'lucy brown' means followed by the same letter do not differ by the scott knott test at 5% probability. treatments rwc (%) hydrocooled + storage at 5°c 96.4 a storage at 5°c 92.8 b hydrocooled + storage at 22°c 94.0 b storage at 22°c 92.4 b cv % 3.2 frança et al.storability of lettuce iceberg 323 water content during their shelf life (frança et al., 2015; oliveira et al., 2015; barbosa et al., 2016). in conclusion, the hydro cooling treatment and storage 5°c resulted in higher level of water compared to not cooled heads, indicating the importance of field heat removal and followed by continuous cold chain. although hydro cooling reduced significantly the leaf chlorophyll content throughout storage for the lettuce stored at 5°c compared to the remaining treatments (table 2), and the coefficient of variation was high (35.7%). this reflects the positions internal, middle and external which the leaf samples were taken. because the relative water content present in the leaves of the hydro cooled lettuce stored at 5°c was higher, a much more favorable water status existed during storage. and at the same time, for the other treatments, the lower relative water content reflects a bigger dehydration rate of the cells, which resulted in higher chlorophyll concentration in the leaves (table 1). but, the same effect on chlorophyll content induced by hydro cooling was not present on parsley and ora-pro-nobis leaves (álvares et al., 2007; barbosa et al., 2015). these differences may be related to the lower trend of parsley and ora-pro-nobis leaves in loosing water from the cell to the environment during storage. hydro cooling had no effect on leaf carbohydrate changes during storage. however, there was significant decrease in the total soluble, reducing, nonreducing sugars and starch contents in the first 12 h of storage, either at 5 or 22°c (table 3). in the first few hours after harvest, a much greater amount of carbohydrate is required to maintain high respiratory demand, coinciding with the highest physiological activity (wills et al., 2010). in the study, during the first 48 h of storage there was a drop of 37.2, 24.5, 52.1 and 23.7% in the total soluble sugars, reducing, non reducing sugars and starch, respectively (table 3). in a similar work, frança et al. (2015) found similar decreases on non-reducing sugars and starch content of butter lettuce on the first 12 hours, but not on reducing sugars. the reduction of all carbohydrates found in this work, reflects the high demand of glucose and fructose to keep the respiratory activity even at low temperature of 5°c. since, leafy vegetables do not store large amounts of carbohydrates; their storage potential for longer shelf life is much smaller than tubers and fruits, which have large amount of stored carbohydrates. thus, further work with the use of controlled and modified atmosphere should be applied to increase iceberg lettuce shelf life. 4. conclusions regardless the temperature of storage, application of hydro cooling treatment removed most of the field heat with beneficial effects on quality, prolonging the shelf life of ‘lucy brown’ iceberg lettuce, by keeping higher water status in the cells and reducing discolorations in the leaves. hydro cooling had no influence on carbohydrate metabolism of the leaves throughout storage either 5 or 22°c. acknowledgements to cnpq, fapemig and capes for their financial support. references aguero m.v., ponce a.g., moreira m.r., roura s.i., 2011 lettuce quality loss under conditions that favor the wilting phenomenon. postharvest biol. technol., table 2 influence of the hydro cooling and temperature of storage on the total chlorophyll content during storage period of iceberg lettuce ‘lucy brown’ means followed by the same letter do not differ by the scott knott test at 5% probability. treatments total chlorophyll (µg cm-2) hydrocooled + storage at 5°c 6.00 b storage at 5°c 7.74 a hydrocooled + storage at 22°c 7.69 a storage at 22°c 7.81 a cv (%) 35.7 table 3 general mean values of total soluble sugars (tss), reducing sugars (rs), non-reducing sugars (nrs) and starch contents in 'lucy brown' icerberg lettuce stored at 5 or 22°c during the first 48 h of shelf life storage means followed by the same letter do not differ by the scott knott test at 5% probability. time (h) tss* rs * nrs * starch ** 0 2.72 a 1.73 a 1.25 a 3.36 a 12 1.84 b 1.12 b 0.71 b 2.65 b 24 1.82 b 1.21 b 0.60 b 2.55 b 36 1.62 b 1.06 b 0.55 b 2.32 b 48 1.71 b 1.11 b 0.59 b 2.57 b cv (%) 24.79 25.71 57.92 19.33 324 adv. hort. sci., 2018 32(3): 319-324 59: 124-131. álvares v.s., finger f.l., santos r.c.a., negreiros j.r.s., casali v.w.d., 2007 effect of pre-cooling on the postharvest of parsley leaves. j. food, agric. environ., 5(2): 31-34. barbosa c.k.r., finger f.l, casali v.w.d., 2015 handling and postharvest shelf life of ora-pro-nobis leaves. acta scientiarum agronomy, 37(3): 307-311. barbosa c.k.r., fonseca m.c.m., silva t.p., finger f.l., casali v.w.d., cecon p.r., 2016 effect of hydrocooling, packing, and cold storage on the postharvest quality of peppermint (mentha piperita l.). rev. bras. plantas med.: 18(1): 248-255. barr h.d., weatherley p. e., 1962 a re-examination of the relative turgidity technique for estimating water deficit in 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j., silveira v., owens h.h., 1950 determination of starch and amylose in vegetables. anal. chem., 22: 1156-1158. nelson n.a., 1944 photometric adaptation of somogyi method for determination of glucose. -j. biol. chem., 135: 136-175. oliveira l.s., silva t.p., ferreira a.p.s., pereira a.m., finger f.l., 2015 efeito do hidroresfriamento na conservação pós-colheita de coentro. hort. bras., 33(4): 448-452. sargent s.a., ritenour m.a., brecht j.k., bartz j.a., 2007 handling, cooling and sanitation techniques for maintaining postharvest quality. vegetable production handbook, document hs719, uf/ifas: 97-109. teixeira d.a., gomes j.a.o., bonfim f.g.g., pardo p.i., mayobre m.t., 2016 técnicas de conservação póscolheita para o manjericão. rev. bras. plantas med., 18( 1): 168-171. tulio jr. a.z., ose k., chachin k., ueda y., 2002 effects of storage temperatures on the postharvest quality of jute leaves (coschorus olitorius l.). postharvest biol. technol., 26: 329-338. wills r., graham d., mcglasson b., joyce d., 2010 postharvest: an introduction to the physiology and handling of fruit, vegetables and ornamentals. 4th ed. cabi, new york, pp. 280. impaginato 87 adv. hort. sci., 2019 33(1): 87-95 doi: 10.13128/ahs-23794 comparison of salinity effects on grafted and non-grafted eggplants in terms of ion accumulation, mda content and antioxidative enzyme activities m. talhouni 1 (*), k. sönmez 2, s. kiran 3, r. beyaz 4, m. yildiz 5, ş. kuşvuran 6, ş.ş. ellialtıoğlu 7 1 national agricultural research center (narc), horticulture directorate, amman, jordan. 2 eskişehir osmangazi university, faculty of agriculture, department of horticulture, eskişehir, turkey. 3 soil, fertilizer and water resources central research institute, ankara, turkey. 4 ahi evran university, faculty of agriculture, department soil science and plant nutrition, kırşehir, turkey. 5 ankara university, faculty of agriculture, department of agronomy, ankara, turkey. 6 cankiri karatekin university, kizilirmak vocational high school, cankiri, turkey. 7 ankara university, faculty of agriculture, department of horticulture, ankara, turkey. key words: apx, cat, eggplant, lipid peroxidation, nacl, na+, cl-, k+, ca++, scion/rootstock combination, sod. abstract: grafting onto resistant/tolerant rootstocks is known to alleviate the negative effects of abiotic stress factors like salinity by enhancing their enzymatic antioxidant defense system and having more efficient nutrient uptake. this study was carried out under greenhouse conditions, different rootstock/scion eggplant combinations were grown under two salinity treatments 1.8-2 ds/m (control) and 6-7 ds/m (stress) with seven eggplant genotypes as rootstocks (commercial and turkish genotypes). two genotypes were used as the scion. leaf mda and ions (na+, cl-, k+ and ca++) content, antioxidant enzymes activity were evaluated as indicators for plant tolerance level. it was found that the rootstock-grafted plants were more efficient in preventing na+ ions to be transferred to the plants upper parts and had higher sod, cat, and apx activity levels compared to the selfand non-grafted plants which resulted in better tolerance and growth in these plants. 1. introduction eggplant (solanum melongena l.) is an important vegetable crop (*) corresponding author: manar.alhouni@gmail.com citation: talhouni m., sönmez k., kiran s., beyaz r., yildiz m., kuşvuran ş., ellialtioğlu ş.ş., 2019 comparison of salinity effects on grafted and non-grafted effplants in terms of ion accumulation, mda content and antioxidative enzyme activities. adv. hort. sci., 33(1): 87-95 copyright: © 2019 talhouni m., sönmez k., kıran s., beyaz r., yıldız m., kuşvuran ş., ellialtıoğlu ş.ş. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 april 2018 accepted for publication 12 december 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 87-95 88 worldwide, its production reaches about 48.5 million ton while in turkey eggplant production reaches 800900 thousand tons (tuik, 2015). one of the most stress factors that affect eggplant production is salinity. salinity is a major environmental factor limiting plant growth and productivity, especially in the arid and semiarid regions (parida and das, 2005). conflicting literature exists on eggplant tolerance to soil salinity and this difference could be related to the varieties or cultivars used and to the different environmental conditions in those studies (ünlükara et al., 2010). overcoming salt stress problems would have a positive impact on agriculture production. attempts have been made to improve salt tolerance of crops by traditional breeding programs but with limited success due to the complexity of the trait (flowers, 2004). even the use of genetic transformation of plants to raise their tolerance, despite its success in some cases (rus et al., 2001); the complexity of the trait and lack of public acceptance, limiting its wide spread and use (munns, 2002). one way of avoiding or reducing losses in production caused by salinity would be to use the tolerant rootstocks. in relation to salt tolerance, many studies have been conducted to determine the response of grafted plants to salinity. according to these studies, the improvement of salt tolerance by grafting is related to the capability of rootstocks to reduce toxicity of na+ and/or clthrough exclusion and/or reduction of absorption of clby the roots, and the replacement or substitution of total k+ by total na+ in the foliage (estañ et al., 2005; martinez-rodriguez et al., 2008). it is supposed that useful rootstocks should be able to reduce the uptake and transport of saline ions to the shoot, which will slow or prevent the accumulation of the toxic salt ions in the leaves (usanmaz and abak, 2018). salt stress causes a range of adverse effects in plants, mainly ionic disorders, osmotic stress and nutritional imbalance. a common feature of these effects is the overproduction of reactive oxygen species (ros) such as singlet oxygen (1o2), superoxide anion (o2−), hydrogen peroxide (h2o2), and hydroxyl radical (oh) which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and dna which ultimately results in oxidative stress (ashraf and foolad, 2007). salt stress causes stomatal closure, which reduces the co2/o2 ratio inside leaf tissues and inhibits co2 fixation (hernández et al., 2000). plants antioxidant enzymes such (superoxide dismutase, sod; catalase, cat; ascorbate peroxidase, apx; glutathione reductase, gr; monodehydroascorbate reductase, mdhar; dehydroascorbate reductase, dhar; glutathione peroxidase, gpx; guaicol peroxidase, gopx and glutathione-stransferase, gst) work in concert to control the cascades of uncontrolled oxidation and protect plant cells from oxidative damage by scavenging of ros (scandalios, 1997; dixit et al., 2001; shalata et al., 2001). superoxide dismutase (sod) reacts with the superoxide radical at almost diffusion-limited rates to produce h2o2 (scandalios, 1993). h2o2 is scavenged by peroxidases, especially ascorbate peroxidase (apx), and catalase (cat). in the present study, we exposed non-grafted, self-grafted and rootstock-grafted eggplants to conditions of salt stresses to investigate whether grafted plants could improve tolerance to salinity by alleviating the expression of antioxidant enzymes. using local genotypes in breeding programs is of vital importance to find new rootstocks with the ability to alleviate the effects of salinity and reduce its effect on plant growth and productivity. in the present study, two eggplant genotypes were grafted onto seven rootstocks to compare the ability of the different rootstock genotypes in increasing eggplant tolerance as it is related to the ability of the rootstock to 1) control the transport of na+ and cl-, 2) to maintain better k+ and ca++ uptake, 3) to increase the enzymatic defense mechanism scavenging the ros induced by oxidative stress resulting in less leaf malodialdehyde (mda) content. another goal was to evaluate the potential of the turkish genotypes burdur and mardin as rootstocks under salinity conditions in comparison to the commercial genotypes. 2. materials and methods the field part of the experiment was carried out between august-november 2014, in a 300 m2 plastic house belongs to the private sector (genta general agricultural products marketing co.) in antalyaturkey while laboratory works and analysis were carried out in ankara university faculty of agriculture, departments of horticulture and agronomy laboratories. plant material two eggplant (solanum melongena l.) genotypes, naomi f1 cv., a commercial cultivar, and artvin, a salt sensitive breeding line, were used as scions. and for rootstocks five commercial genotypes were used, talhouni et al. salinity effects on grafted and non-grated effplants 89 agr703 (solanum aethiopicum), köksal f1, yula f1 and vista (s. incanum x s. melongena hybrids), and hawk (s. torvum), these genotypes are the most common used rootstocks in grafting eggplants due to their tolerance. in addition two local salt-tolerant genotypes burdur and mardin (s. melongena l.) were used as rootstocks, their tolerance to salinity were confirmed in previous studies screening for turkish tolerant genotypes (yaşar, 2003). grafting and salt treatment eggplant seeds were sown in germination trays filled with 2:1 peat:perlite. after sowing trays were kept under controlled conditions of temperature (25°c) and humidity (80%). when the seedlings reached 2-4 true leaves stage, grafting was carried out. the tube-grafting was used in this study because it is the most widely used grafting method in solanaceae family (rivard et al., 2009). then grafted seedlings were kept under controlled conditions of humidity (90%) for four days, then seedlings were transferred to the greenhouse under shading before planting for acclimatization. ten days after grafting seedlings were placed in the plastic house and were ready for transplantation in 8 l plastic pots filled with 3:1 perlite:vermiculite. in one of our earlier studies we examined these genotypes tolerance under salinity conditions at seedling stage in a hydroponic experiment (talhouni, 2016), in this study we wanted to assess these same genotypes at flowering and fruit set stage. when plants reached the flowering and fruit set stage salinity treatment began. 6-7 dsm-1 water ec was used as the stress treatment by solving nacl into the nutrition solution; according to the volume of barrels used in fertigation about 8 kg of iodine-free sodium chloride were required , while for the control the ec level was kept at 1.8-2 dsm-1 (no nacl was added). leaf ion concentrations after 60 days from salinity stress application, samples were taken from the control and the salinity treated plants for the different analysis. for the leafna+, cl-, k+ and ca++ concentration measurements, leaves were dried at 65°c for 48 hours, grounded, dissolved in 1% (v/v) hcl. for the analysis of na+, k+ and ca++ contents, atomic absorption spectrophotometer (varian spectra aa 220 fs) was used (kuşvuran, 2012). while for cl-, titration procedure was followed as described by taleisnik et al. (1997) using buchler cotlove chloridometer. enzyme extractions and assays fresh leaf samples were submersed for 5 min in liquid nitrogen. the frozen leaves were kept at -80°c for further analyses. enzymes were extracted from 0.5 g leaf tissue using a mortar and pestle with 5 ml extraction buffer containing 50 mm potassium phosphate buffer, ph 7.6 and 0.1 mm na-edta. the homogenate was centrifuged at 15,000 g for 15 min and the supernatant fraction was used to assay for the various enzymes. all steps in the preparation of enzyme extracts were performed at 4°c. apx activity was determined by measuring the consumption of ascorbate by following absorbance at 290 nm. one unit of apx activity was defined as the amount of enzyme required to consume 1 μmole ascorbate min-1 (cakmak and marschner, 1992). sod was assayed according to cakmak and marschner (1992), by monitoring the superoxide radical-induced nitro blue tetrazolium (nbt) reduction at 560 nm. one unit of sod activity was defined as the amount of enzyme which causes 50% inhibition of the photochemical reduction of nbt. catalase (cat) activity was measured as the decline in absorbance at 240 nm due to the decomposition decline of extinction of h2o2. the reaction was started by adding h2o2. lipid peroxide content lipid peroxidation was measured as the amount of malondialdehyde (mda) determined by the thiobarbituric acid (tba) reaction. frozen samples were homogenized in a pre-chilled mortar with two volumes of ice-cold 0.1% (w/v) tricloroacetic acid (tca) and centrifuged for 15 min at 15000 x g. assay mixture containing 1 ml aliquot of the supernatant and 2 ml of 0.5% (w/v) thiobarbituric acid in 20% (w/v) tricloroacetic acid (tca) was heated to 95°c for 30 min and then rapidly cooled in an ice-bath. after centrifugation (10000 x g for 10 min at 4°c), the supernatant absorbance (532 nm) was read and values corresponding to non-specific absorption (600 nm) were subtracted. the mda content was calculated according to the molar extinction coefficient of mda (155 mm-1 cm-1). statistical analysis randomized complete block design with three replicates was used. each replicate included 108 pots (18 rootstock/scion combination * 2 salinity level * 3 plants of each combination) with one plant/pot. data were subjected to duncan’s multiple range tests adv. hort. sci., 2019 33(1): 87-95 90 using the sas program (p≤0.01)(version 6.12, sas institute inc., cary, usa). 3. results na+ concentrations in general, the concentrations of na+ in the leaves increased significantly due to increased nacl concentration (table 1) with significant differences between grafting combinations, and a significant ‘salinity x scion/rootstock combination’ interaction at p≤0.01. after 60 days of salinity stress application, the combinations that showed the least concentrations of na+ in their leaves were; (rootstock/scion) köksal/artvin, vista/naomi, köksal/naomi, agr703/naomi and agr703/artvin (9.75, 9.65, 9.92 10.02 and 10.46 µg/mg fw respectively) with increase rates of 3511, 1035, 4623, 1721, 3506% respectively (fig. 1) which indicated that these rootstock genotypes could limit na+ to the leaves more successfully. no significant effects of grafting per se were noticed, no differences were observed between non-grafted and self-grafted combinations. clconcentration as in leaf na+ concentration, leaf clconcentration also increased significantly under salinity treatment in all combinations, with a significant differences and a significant ‘salinity x scion/rootstock combination’ interaction at p≤0.01 (fig. 1). the highest clconcentration was observed in artvin, naomi, (rootstock/scion) artvin/artvin, naomi/naomi, mardin/artvin, mardin/naomi (11.83, 10.81, 9.54, 9.06, 8.83 8.12 µg/mg fw) combinations, while the lowest was observed köksal/artvin, agr703/artvin, agr703/naomi and burdur/artvin (5.06, 5.06, 5.27, 5.49 µg/mg fw respectively) and there were no significant differences between nonand self-grafted combinations (table 1). k+ concentrations the amounts of k ion measured in leaf samples taken from plants treated with ec 6-7 ds / m nacl gave lower values in some combinations than control plants (table 1), the highest decrease in leaf k+ content was observed in non-grafted artvin (-9.82%).followed by (rootstock/scion) artvin/artvin with decrease rate of (-6.25%) (fig. 1). the highest values were obtained from (rootstock/scion); köksal/artvin, köksal/naomi, mardin/naomi, agr703/artvin, yula/naomi (4.86, 4.64, 4.36, 4.30, 4.27 µg/mg fw, respectively) (table 1). among these combinations, köksal/artvin, agr703/artvin and yula/naomi had the highest k+ ions increase rate (47.27%, 22.16%, 20.96%) (fig. 1). combinations that gave the lowest k+ ion amount measurements were (rootstock/scion) hawk/artvin, naomi, burdur/naomi, naomi/naomi, table 1 leaf ions concentration (µg/mg fw); in the different grafting combinations under control and salinity treatments grafting combination na+ k+ ca++ clcontrol salinity control salinity control salinity control salinity köksal/artvin 0.27±0.00 a 9.75±0.25 a 3.30±0.15 ab 4.86±0.33 b 0.50±0.03 f 0.48±0.03 e 0.06±0.02 a 5.06±0.64 a agr703/artvin 0.29±0.03 a 10.46±0.53 a-c 3.52±0.34 a-c 4.30±0.26 ab 0.49±0.02 f 0.41±0.04 de 0.06±0.02 a 5.06±0.90 a vista/artvin 0.51±0.03 ab 12.14±0.86 b-e 3.89±0.11 a-e 3.93±0.53 a 0.46±0.02 b-f 0.41±0.03 de 0.12±0.04 a-d 6.30±1.10 a-c yula/artvin 0.24±0.02 a 11.08±0.58 a-e 3.74±0.18 a-e 3.89±0.12 a 0.43±0.01 a-c 0.35±0.03 b-d 0.19±0.02 c-f 6.63±0.75 a-d burdur/artvin 0.49±0.01 b 10.91±0.53 a-d 3.57±0.12 a-c 3.97±0.24 a 0.49±0.02 ef 0.39±0.05 c-e 0.09±0.03 ab 5.49±0.13 ab mardin/artvin 0.61±0.09 b-d 12.36±0.29 c-e 3.70±0.23 a-d 4.22±0.09 ab 0.44±0.01 a-d 0.35±0.06 b-d 0.14±0.03 a-e 8.83±1.08 c-f hawk/artvin 0.26±0.05 a 10.89±0.18 a-d 4.05±0.28 c-e 3.84±0.31 a 0.46±0.02 c-f 0.41±0.03 de 0.11±0.02 a-c 6.45±1.50 a-d artvin/artvin 0.83±0.08 e-g 12.36±0.70 c-e 4.32±0.05 de 4.05±0.15 ab 0.41±0.01 a 0.35±0.02 b-d 0.19±0.02 c-f 9.54±1.21 e-g artvin 0.88±0.04 fg 12.77±0.46 de 4.38±0.28 e 3.95±0.13 a 0.40±0.01 a 0.29±0.02 ab 0.22±0.02 ef 11.83±0.62 g köksal/naomi 0.21±0.02 a 9.92±0.37 ab 4.11±0.22 c-e 4.64±0.23 ab 0.49±0.02 d-f 0.31±0.01 a-c 0.09±0.04 ab 6.06±0.38 ab agr703/naomi 0.55±0.12 ab 10.02±1.26 ab 3.87±0.06 a-e 3.96±0.28 a 0.48±0.01 d-f 0.36±0.02 b-d 0.10±0.04 a-c 5.27±0.55 a vista/naomi 0.85±0.05 fg 9.65±0.71 a 3.67±0.15 a-d 4.15±0.08 ab 0.44±0.02 a-e 0.36±0.02 b-d 0.15±0.03 b-f 6.13±0.19 ab yula/naomi 0.73±0.04 d-f 10.93±0.97 a-d 3.53±0.10 a-c 4.27±0.29 ab 0.42±0.01 a-c 0.25±0.02 a 0.18±0.05 b-f 7.51±0.54 a-e burdur/naomi 0.49±0.05 b 11.14±1.16 a-e 3.50±0.36 a-c 3.88±0.23 a 0.48±0.01 d-f 0.40±0.01 c-e 0.13±0.01 a-d 6.37±1.01 a-c mardin/naomi 0.68±0.04 c-e 12.07±0.13 b-e 3.95±0.32 b-e 4.36±0.24 ab 0.43±0.01 a-c 0.34±0.03 a-d 0.14±0.03 a-e 8.12±0.63 b-e hawk/naomi 0.80±0.10 e-g 11.14±1.20 a-e 3.80±0.11 a-e 4.09±0.27 ab 0.41±0.01 ab 0.35±0.01 b-d 0.20±0.02 d-f 7.29±0.24 a-e naomi/naomi 0.94±0.04 g 12.34±0.84 c-e 3.33±0.20 ab 3.96±0.36 a 0.40±0.01 a 0.34±0.01 a-d 0.22±0.02 ef 9.06±1.06 d-f naomi 0.52±0.06 ab 13.30±0.73 e 3.27±0.29 a 3.85±0.30 a 0.40±0.02 a 0.28±0.03 ab 0.23±0.03 f 10.81±1.41 fg cv (%) 43.07 9.78 8.81 6.83 8.01 15.22 38.38 27.17 treatment ** ** ** ** combination ** ** ** ** combination x treatment ** ** ** ** talhouni et al. salinity effects on grafted and non-grated effplants 91 agr703/naomi, artvin (3.84, 3.85, 3.88, 3.96, 3.96, 3.96 85 µg/mg fw). no significant differences were obtained for self-grafted combinations. ca++ concentrations ec 6-7 dsm-1 nacl treatment led to a decrease in leaf ca++ concentration in all combinations (fig. 1) with significant differences between treatments and combination (p≤0.01) and with a significant ‘salinity x scion/rootstock combination’ interaction. the highest leaf ca++ concentrations were obtained in (rootstock/scion) agr703/artvin, köksal/artvin, burdur/naomi, hawk/artvin and vista/artvin (0.48, 0.42, 0.41, 0.41 µg/mg fw, respectively). no significant effects were observed in self-grafted combinations. antioxidant enzyme activities salt treatments increased superoxide dismutase (sod) activities in all of the plants (table 2). however, fig. 1 leaf ions concentration (µg/mg fw); in the different grafting combinations under control (blue) and salinity treatments (red). table 2 sod, cat, and apx enzymes activities (µmol/g fw) in the different grafting combinations under control and salinity treatments eggplant sod cat apx mda control salinity control salinity control salinity control salinity köksal/artvin 217.03±8.00 a 635.46±27.84 gh 139.90±13.74 ab 593.91±28.44 h 2042.77±12.67 h 5387.96±560.98 e 5.12±0.29 a-d 10.44±0.54 a agr703/artvin 207.06±6.28 a 645.64±38.29 h 129.82±10.00 ab 576.91±27.73 gh 2020.91±97.38 h 4820.66±545.40 de 5.02±0.13 ad 10.23±1.24 a vista/artvin 215.92±11.75 a 464.88±44.39 c-f 113.03±11.99 a 484.17±25.96 c-f 1758.36±28.48 e-h 3829.29±568.57 a-d 4.86±0.27 ab 12.83±0.26 ab yula/artvin 193.54±6.29 a 414.49±26.02 b-d 103.86±6.46 a 409.13±1.88 bc 1765.71±141.18 e-h 3307.96±550.14 a-c 5.12±0.26 a-d 10.14±0.61 a burdur/artvin 208.97±10.32 a 508.88±55.72 d-f 131.44±4.26 ab 548.67±19.02 e-h 1882.51±52.64 f-h 3839.81±57.28 a-d 5.19±0.10 a-d 11.16±0.44 a mardin/artvin 195.91±10.91 a 470.99±16.48 c-f 138.76±14.25 ab 434.69±6.55 b-d 1395.16±235.41 b-d 3105.27±93.76 ab 5.49±0.25 a-e 11.50±0.21 a hawk/artvin 203.44±10.29 a 436.05±50.00 b-e 118.68±15.42 a 464.12±25.38 c-e 1926.37±199.83 gh 4303.30±32.77 b-e 4.80±0.31 a 11.95±0.98 ab artvin/artvin 186.29±10.04 a 341.33±58.60 ab 123.87±6.38 ab 350.16±52.43 ab 1134.59±102.54 ab 2731.28±37.49 a 6.32±0.13 fg 12.54±0.15 ab artvin 189.95±12.89 a 295.15±16.97 a 113.49±7.29 a 374.84±27.07 b 903.43±37.94 a 2614.61±37.06 a 6.34±0.33 g 14.81±1.23 b köksal/naomi 190.92±25.52 a 553.92±32.30 f-h 116.33±14.37 a 553.17±30.84 f-h 1968.59±112.34 g-h 4427.68±48.03 c-e 4.95±0.18 a-c 10.93±1.20 a agr703/naomi 213.81±4.16 a 491.39±39.05 d-f 165.69±17.37 b 503.21±14.17 d-g 1924.53±144.55 gh 4326.69±614.68 b-e 4.87±0.23 ab 10.54±1.04 a vista/naomi 194.09±17.87 a 457.84±29.92 c-f 124.91±15.83 ab 405.95±29.79 bc 1547.82±154.87 c-f 3072.32±48.96 ab 5.59±0.25 b-f 11.18±0.16 a yula/naomi 200.41±10.91 a 427.66±11.33 b-e 99.34±6.30 a 401.18±19.92 bc 1499.31±168.98 c-e 3663.61±635.13 a-d 5.26±0.28 a-d 11.06±0.93 a burdur/naomi 196.48±16.49 a 534.86±35.10 e-g 117.08±28.01 a 503.48±27.99 d-g 1813.06±32.67 e-h 3789.02±90.92 a-d 5.09±0.39 a-d 10.36±0.85 a mardin/naomi 191.44±20.27 a 378.05±10.08 a-c 104.70±15.90 a 367.67±14.99 ab 1608.48±112.47 d-g 3448.81±529.02 a-c 5.71±0.19 d-g 10.86±0.74 a hawk/naomi 193.35±9.53 a 421.65±43.89 b-d 140.49±24.51 ab 360.07±7.01 ab 1695.49±80.45 d-h 4471.40±613.60 c-e 4.80±0.12 a 10.89±0.92 a naomi/naomi 201.11±19.08 a 272.74±22.75 a 125.68±6.97 ab 366.67±43.39 ab 1205.04±58.49 a-c 2693.56±51.33 a 6.05±0.16 e-g 14.66±2.05 b naomi 199.68±10.99 a 271.57±36.99 a 123.98±8.26 ab 289.02±39.88 a 1033.83±54.71 ab 2782.04±593.00 a 5.66±0.18 c-g 14.91±1.65 b cv (%) 4.65 24.53 12.87 19.94 21.9 21.7 9.37 13.58 treatment (t) ** ** ** ** combination (c) ** ** ** ** c x t ** ** ** ** 92 adv. hort. sci., 2019 33(1): 87-95 in the rootstock-grafted plants, sod activity increased faster and with higher rates than in the nonand selfgrafted plants. köksal/artvin and agr703/artvin had the highest sod activity level (645.64 and 635.46 umol/min/mg fw respectively). followed by köksal/naomi, burdur/naomi, and burdur/artvin combinations (553.92, 534.8, and 508.88 umol/min/mg fw respectively). the highest increase rate in sod activity was obtained for agr703/artvin combination (211.8%) while naomi/naomi and naomi combinations had the lowest increase with rates of 35.62 and 36% respectively (fig. 2). naomi and self-grafted plants had the lowest values of the enzyme activity with no significant differences between nonand self-grafted combinations. catalase (cat) activity increased under salt treatment in all combinations compared to the control plants (table 2), (rootstock/scion) köksal/artvin and agr703/artvin had the highest cat activity (593.91 and 576.91 μmol/min/mg fw respectively). followed by köksal/naomi, burdur/artvin, agr703/naomi and burdur/naomi. on the other hand artvin and naomi (non-grafted) and the self-grafted plants had the lowest cat activity. burdur/naomi had the highest increase rate in cat activity (330%) while non-grafted naomi had the lowest rate (133%) (fig. 2). no significant differences were observed between nonand self-grafted combinations. under nacl-salinity conditions, ascorbate peroxidase (apx) activity was increased in all plants (fig. 2). however, agr703/artvin and köksal/artvin had the highest apx activity levels indicating their better tolerance level (4820.66 and 5387.96 μmol/min/mg fw respectively) as shown in table 2, while the lowest apx enzyme activity was found in non-grafted plants naomi and artvin indicating their poor tolerance. no significant differences were obtained between nonand self-grafted combinations. malondialdehyde (mda) salinity resulted in a significant increase on the mda content compared to the controls due to oxidative stress induced peroxidation (fig. 2). with regard to the mda, significant differences were found among grafting combinations and a significant ‘salinity x rootstock/scion combination’ interaction at p≤0.01 (table 2). according to the results naomi, naomi/naomi and artvin were found to be more sensitive with mda content increase rate of 163.4, 142.3, 133.6% respectively (fig. 2). no significant differences were observed between nonand self-grafted combinations. 4. discussion and conclusions as a result of high salinity level, na+ and clions can be accumulated in toxic levels in plant tissues depends on the plant species. even though these two ions are suitable for osmotic adjustment, excess concentrations will be toxic enough to prevent plant growth. in the study, combinations vista/artvin, köksal/artvin, vista/naomi, and köksal/naomi had less na+ accumulation in their tissues which indicates that these combinations were able to keep na+ ions away from their leaves. concerning leaf cl -, vista/artvin combination had the least concentration. grafted plants tend to hold na+ and clions in their root tissues preventing them from being translocated to the shoots and leaves in high concenfig. 2 sod, cat and apx enzymes activities (µmol/g fw) in the different grafting combinations under control (blue) and salinity treatments (red). talhouni et al. salinity effects on grafted and non-grated effplants 93 trations (levitt, 1980; estañ et al., 2005). most vegetables like, cucumbers, melons, tomatoes and eggplant are injured by excess na+ ions (tester and davanport, 2003). in giuffrida et al. (2009), increased nacl level led to na+ concentration increase in tomato leaves and fruits. kuşvuran et al. (2007), under salinity conditions na+ and clwere accumulated in higher rates in the salinity-sensitive melon plants compared to the salinity-tolerant ones. k+ decrease rate was different between the different combinations. akinci and lösel (2012), different eggplant genotypes showed different tolerant level to salinity. pala cv. showed better tolerance to salinity compared to kemer and aydın siyahı cultivars with better k+/na+ ratio. yaşar et al. (2006), in tissue culture study on eggplant, there was an increase in na+ and cltissues concentrations with decrease in k+ and ca++ due to salinity. however there were significant differences between different genotypes, the salinitytolerant mk and bb showed higher k+ and ca++ concentrations compared to the salinity sensitive gr and ah genotypes. consequently mk and bb had higher k+ and ca++ uptake decreased under stress treatment (savvas and lenz, 2000). in a similar study on pepper, the same results were obtained (aktaş et al., 2002). combinations with the highest leaf ca++ concentrations under salinity were vista/naomi, agr703/naomi and burdur/artvin. and maybe for this reason burdur genotype can be considered a potential rootstock for increasing eggplant tolerance against salinity. the decrease in ca++ uptake due to nacl salinity was observed by many authors, and in contrary to k+, the decrease was not due to the competition between na+ and k+ at the absorption site on the root surface, it was always found because of the decline in the transpiration rate under stress conditions (maggio et al., 2007). in gao et al. (2005), under stress conditions of low temperatures (5°c) grafted eggplants maintained higher leaf ca++ concentrations compared to the non-grafted plants which gave the grafted plants higher tolerance under such stress conditions. plant adaptation to salinity may depend on different mechanisms, including the capacity to maintain high levels of antioxidants and/or through the induction of antioxidant enzymes (sod, cat, gr, and apx, etc.) (sevengör, 2010). in the present study, rootstock-grafted plants had higher activity of antioxidant enzymes under salinity conditions, which was translated to lower mda content in their leaves which means these combinations, were less affected by the ros-induced lipid peroxidation and they were more tolerant to salinity than the nonand self-grafted plants. mda content always found higher in salinity-sensitive plants compared to salinity-tolerant ones (yaşar, 2003; kuşvuran et al., 2015) and a significant relation between mda content and antioxidant enzymes activity is first proven by shalata and tal (1998). meloni et al. (2001) in cotton, yaşar (2003) in eggplant, doğan (2004) in tomato, and sevengör (2010) in pumpkin, all found that mda content was low in plants with high antioxidant enzymes activity under salinity stress conditions. in this study, antioxidant enzymes activity showed a higher increase in rootstock-grafted plants compared to the nonand self-grafted plants, this increase was significantly different between grafting combinations. in another study where cucumber was grafted onto salinity-tolerant rootstock, h2o2 level was found to be low, whereas sod, cat, pod enzymes activity level were found higher. öztekin and tuzel (2011), cat activity level differed according to the rootstock genotypes, but always was higher in the grafted plants compared to the non-grafted plants. all results indicated that grafting per se had no significant role in alleviating negative effects of salinity as there were no significant differences between nonand self-grafted combinations in all parameters measured in this study. in general, local genotypes (landraces) are adapted to prevailing environmental conditions like salinity. in this work, the local turkish genotype burdur showed a good potential to compete commercial genotypes. on the other hand, mardin was way behind and did not show enough potential in this study. acknowledgements this research was supported by the coordination unit of the scientific research projects of the university of ankara (project no: 15h0447001). references akinci ş., lösel d.m., 2012 plant water-stress response mechanisms, pp. 15-42. in: rahman i.m.m. 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yıl university, institute of natural and applied sciences, van, turkey, pp. 138. yaşar f., ellialtioğlu s., kuşvuran s., 2006 ion and lipid peroxide content in sensitive and tolerant eggplant callus cultured under salt stress. europ. j. hort. sci., 71(4): 169-172. impaginato 365 adv. hort. sci., 2019 33(3): 365-373 doi: 10.13128/ahs-24266 impact of aerobic rice-leafy vegetables intercropping systems on weed management s. habimana 1 (*), k.n. kalyana murthy 2, y.a. nanja reddy 2, m. mudalagiriyappa 2, r. vasantha kumari 2, d.c. hanumanthappa 2 1 university of rwanda, cavm, p.o. box 210, musanze, rwanda. 2 university of agricultural sciences, gkvk, 560065 bengaluru, india. key words: amaranth, coriander, fenugreek, growth, return, spinach, yield. abstract: field experiments were carried out in summer 2017 and 2018 at zonal agricultural research station (zars), university of agricultural sciences, gkvk, bengaluru, india on red sandy loam soil. the main objective was to evaluate the impact of rice grown in aerobic conditions intercropped with leafy vegetables on weed management, wherein the sole rice and intercrops of four leafy vegetables: palak (spinacia oleracea l.), coriander (coriandrum sativum l.), amaranth (amaranthus spp l.), methi (trigonella foenum‐graecum l.) were designed in randomized complete block design (rcbd) of 9 treatments replicated four times. the results revealed that the greater the crop biomass, the higher the weed suppression achieved. sole rice was densely populated by weeds and also had higher weed biomass compared to the intercrops. however, intercrops suppressed efficiently the weeds, increased growth and rice equivalent yield over the sole rice crop. the intercropping systems with leafy palak (spinach) were the most suppressive of weeds. the rice + leafy vegetable palak recorded significantly lower weed density (138.4 no. m-2), dry weight at harvest (148.04 g m-2), higher rice grain, rice straw and palak leaf yield (7651; 9687 and 25508 kg ha-1, respectively) and higher net monetary return (₹ 156269 ha-1) over the sole rice. 1. introduction the rice is the world most important staple food for more than a half of the earth’s population. it contains high amount of carbohydrates, proteins and calories. more than 90 per cent of rice is cultivated and consumed in asia, where more than 60 per cent of the world’s population lives. according to fao, the world rice consumption demand increases as the population increases too. by 2025, the amount of 800 m t of rice grain will be needed for consumption, which is a bit higher than the current rice grain production of 718 m t. the rice production is facing several challenges such as population explosion, urbanization, industrialization, shrinkage of cultivable land, water resources, etc., which make difficult to meet the rice food production demand. increasing the rice productivity (*) corresponding author: shabimana@gmail.com citation: habimana s., kalyana murthy k.n., mudalagiriyappa m., nanja reddy y.a., vasantha kumari r., hanumanthappa d.c., 2019 impact of aerobic rice‐leafy vegetables intercrop‐ ping systems on weed management. adv. hort. sci., 33(3): 365-373 copyright: © 2019 habimana s., kalyana murthy k.n., mudalagiriyappa m., nanja reddy y.a., vasantha kumari r., hanumanthappa d.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2018 accepted for publication 8 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 365-373 366 per unit area is the need of the hour to bridge the gap between production and consumption. the production of rice in aerobic condition by using the same production method used for other rain fed cereals like maize, wheat, etc., can be one of the resorts to mitigate the challenges. the rice grown in aerobic conditions known as aerobic rice provides several benefits of saving the inputs and human labour resources and reduces the greenhouse gases among the others. however, this rice production system faces a lot of problems including the weed infestation since the weed competition for resources starts from day one. weeds can reduce the production of rice by 10-100% (rao et al., 2014; yaduraju et al., 2015). controlling weeds satisfactorily increases the cost of cultivation of the crop as well as deplete resource base (buriro et al., 2003). reduced weed biomass in intercropping systems has been reported by several workers in various field crops such as soybean, maize, sorghum, sunflower, green gram, red gram, groundnut, etc., but they did not explore yet the leafy vegetables as intercrops. there must be continuing attention paid to study the weed dynamics and crop-weed interference in intercropping systems with smother leafy vegetables. more information is needed concerning crop diversification on weed dynamics and weed flora and differential resources consumption by crops and associated weeds. most of the farmers of the developing world are small, marginal and are unable to bear the high costs associated in carrying out weed management operations. chemical weed control creates many problems such as development of herbicide resistant weeds, shifting weed flora and environmental pollution. the crop diversity can improve crop growth (kirkegaard and hunt, 2010), thus increasing crop competitiveness and tolerance to weeds (anderson, 2011). cropping systems composed of a diversity of crops with different life cycles are a great option to manage weeds and critical component of integrated weed management (colbach et al., 2014). to ensure safeguard against environmental pollution and to reduce chances of shifting of weed flora and the development of herbicide-resistant weeds, an intercropping system which allows minimum weed infestation and yield losses, appear to have great importance. although intercropping is practiced to maximize land use, it has also a significant effect in suppressing weed growth. but intercropping system alone is not sufficient to ensure adequate weed management practices, because of diverse canopy coverage occurred by intercrop. labour is becoming a scarce and costly input in agriculture. this has resulted in increased technical grade herbicide consumption. hence, the present thrust in weed studies is to minimize the herbicide use and to formulate integrated management practices by combining non-chemical methods, which are efficient, economically viable and eco-friendly sound. therefore, based on this background, the field experiments were planned. 2. materials and methods field experiments were carried out in summer seasons of 2017 and 2018 at zonal agricultural research station (zars), university of agricultural sciences, gkvk, bengaluru, india on red sandy loam soil to evaluate the performance of leafy vegetables smothering efficiency in weed management. experimental design and treatment details the experiment had rcbd design which was composed of 9 treatments replicated 4 times. the treatments included the four of rice intercropped with 4 leafy vegetables namely rice + amaranth, rice + coriander, rice + palak (spinach) and rice + methi (fenugreek) and the 5 treatments of sole crops including sole rice crop and 4 sole vegetable crops. the experimental site had ph (6.93), ec (0.36 dsm-1), medium in organic carbon (0.58 %), available nitrogen (362 kg ha-1), p2o5 (43 kg ha-1) and k2o (289 kg ha-1). land preparation and layout during january and february 2017, december 2017 and january 2018 the land was tilled with tractor-drawn cultivator followed by passing rotovator to bring the soil to the fine tilth. the plots were arranged as per the experimental design with 50 cm width bunds around each plot. the gross plot size was 5 m x 3.5 m (17.5 m2) accommodating 20 rows at a spacing of 25 cm, each row consisted 20 hills of rice plants with an intra-row spacing of 25 cm. one side row and one adjacent row on each side were left as border rows and the remaining 4 m x 2.5 m (10.00 m2) was retained as the net plot. the spacing between plots and replications were 0.5 m and 1 m, respectively. fertilizers application the recommended dose of farmyard manure (fym) at 10 t ha-1 was applied 15 days prior to sowing and fertilizer dose of 100: 50: 50 kg n, p2o5, k2o ha-1 habimana et al. ‐ weed management by intercropping system 367 was applied through urea, single superphosphate (ssp) and muriate of potash (mop), respectively. the 50 per cent of n was applied as basal dose and remaining 50 per cent was applied in two splits i.e., at tillering and panicle initiation stage of the rice crop. whereas, ssp and mop fertilizers were applied as basal dose at the time of sowing. for all intercrops, only recommended dose fertilizer of base crop was used. seeds and sowing shallow furrows spaced at 25 cm apart were created using marker during summer 2017 and 2018. the aerobic rice variety used was mas 946-1. two rice seeds per spot were dibbled by maintaining the inter and intra row spacing of 25 cm with a seed rate of 7 kg ha-1 on 8th march and 13th january for 2017 and 2018, respectively. the leafy vegetable seeds were sowed by the broadcasting method. amaranth variety used for sowing was arka suguna, seed rate used for broadcasting was 2.5 kg ha-1. coriander variety used was dwd-3, seed rate 10 kg ha-1 for broadcasting and duration of the crop was 30 days for vegetable and 90 days for grain. it provides green leaf productivity of 15 t ha-1. palak variety used was pusa jyoti with the yield over 200 to 400 quintals per hectare, seed rate used for broadcasting was 13 kg ha-1. methi variety used was co-1 variety; seed rate used for broadcasting was 12 kg ha-1. it provides green leaf productivity of 20 t ha-1. the seeds were covered with soil and gently compacted. irrigation was provided immediately after sowing to encourage uniform germination. irrigation irrigation was scheduled at 3-4 days interval through drip based on the rainfall, soil and crop appearance during the crop periods. drip irrigation system was set out which included the pump, filter units, mainline and sub-main lines for each replication and laterals for each plot. the water source was a bore well. water was pumped through 7.5 hp motor it was conveyed to the main field using 90 mm pvc pipes after filtering through sand and screen filter. from the mainline water was taken to the field through sub-main of 63 mm diameter pvc pipes. from the sub-main, 12 mm laterals were fixed at a spacing of 50 cm. the emitters in the inline laterals were fixed at 40 cm. the discharge rate of emitters was 3 lph. irrigation was withheld 10 days before the crop attained maturity. weed management weeds were controlled by manual cleaning. weed density and weed dry weight were recorded category wise with respect to grassy, broad leaved weeds and sedge weeds at 30, 60, 90 das and at harvest. weed density was recorded in 0.5 m × 0.5 m quadrate randomly at one spot in each plot. weeds were uprooted, washed with tap water, sun-dried, oven-dried at 65°c for 48 h. after attaining the constant weight, the samples were weighed and expressed in grams per m2. the weed smothering efficiency (wse) was worked out by following the below formula: wse (per cent) = (w1-w2) x 100 w1 where, w1: weed dry weight in sole rice crop stand plots; w2: weed dry weight in intercropped leafy vegetable plots uptake of nutrients nitrogen, phosphorus and potassium by weeds and different parts of paddy and leafy vegetables were calculated by multiplying the nutrient content and dry matter of weeds or yield of plant part using the following formula and expressed in kg ha-1. nutrient uptake by weed = nutrient content (per cent) x weed dw (kg ha-1) 100 nut. uptake by rice plant = nutr. cont. (per cent) x biological yield (kg ha-1) 100 rice and vegetable intercrops harvest the rice crop was harvested on 28thjuly, 2017 and on 14th june 2018 as the ear heads changed into brown color coupled with yellow colored straw in more than 90 per cent of plant population of each plot. all borderlines in every treatment plot were harvested as bulk by leaving net plot area. later, the net plot area was harvested treatment-wise separately by cutting at 2 inches above the ground, sun-dried for 3 days and threshed. the harvested produce was threshed manually. the grains were winnowed; sun-dried to bring the moisture up to 10-12 per cent and recorded the grain weight treatment-wise. the rice grain and straw dry weight from the net plot was recorded and expressed as kg ha-1. the threshed straw was left in the same field and same plot for sun drying for 10 days. the weight of straw was also recorded treatment-wise and computed for hectare basis. regarding the leafy vegetables, before harvesting between 30-40 days after sowing, 5 plant samples were taken for dry weight; thereafter the whole plot leaves were harvested, weighed and sold for consumption purpose. index of biological efficiencies of intercropping sys‐ tems different system productivity parameters of interadv. hort. sci., 2019 33(3): 365-373 368 cropping systems were worked out. the below are the formulas used. the land equivalent ratio (ler) is used to decide which crop is suitable among the intercropping components. it denotes relative to land area under sole crop required to produce the same yield as obtained under a mixed or an intercropping system at the same level of management. it is the ratio of land required by the pure crop to produce the same yield as intercrop. the ler was worked out by using the following formula given by willey (1979). ler = la + lb = (ya/sa + yb/sb) where, ler = yield of intercrop over yield of pure crop; la and lb = ler’s for the crops a and b; ya, yb is the yield of a and b crop grown as an intercrop; sa, sb is the yield of a and b crop grown as a sole crop. when ler >1 intercropping is advantageous, the reverse means the 2 crops are mutually antagonistic. rice equivalent yield was also worked out. normally, crop equivalent yield refers to the yields of different intercrops/crops are converted into the equivalent yield of any one crop based on the price of the produce. efforts have also been made to convert the yields of different crops into an equivalent yield of the main crop such as rice (verma and modgal, 1983). the rice equivalent yield of an intercropping system was calculated by taking into account the grain yield of component crops and the prevailing market price of both rice and intercrops as follows: the production efficiency was also calculated based on the rice equivalent yield and the duration of the cropping system and expressed as kg day-1 pe (kg day-1) = rice equivalent yield duration of cropping system data analysis to compare the performance of sole rice treatments with the rest of intercrops, an rcbd with 5 treatments was used. weed density was expressed on a square metre basis and was square root transformed before the analysis of variance (anova) as described by cochran and cox (1957). weed biomass was expressed in g m-2 and weed density was expressed in numbers m-2 before anova. rice grain, straw and leafy vegetable yield were expressed in ha1 before the anova. means were separated using least significant difference (lsd) at p<0.05. the productivity of intercropping was assessed by calculating the rey, ler, pe and net monetary return from component crop yields 3. results effect of the rice‐leafy vegetable intercropping sys‐ tem on weed density total weed density in aerobic rice was significantly influenced by different leafy vegetables intercropping systems at 30, 60, 90 days after sowing (das) and at harvest (table 1). total weed density at 30 das was significantly lower in rice intercropped with palak and sole palak (20.00 and 25.33; 25.08 and 30.88 no. m-2 during 2017 and 2018, respectively) as weed management practices 30 das 60 das 90 das at harvest 2017 2018 2017 2018 2017 2018 2017 2018 t1: rice+amaranth 5.34(29.33) 5.89(34.25) 7.42 (54.67) 7.73(59.23) 8.57(73.33) 8.93(79.25) 8.51(72.00) 8.76(76.30) t2: rice+coriander 6.75(45.33) 7.16(50.70) 8.81(78.67) 9.14(82.98) 9.95(98.67) 10.24(104.28) 9.89(97.33) 10.04(100.28) t3: rice+palak 4.32(20.00) 5.06(25.08) 5.71 (34.67) 6.52(42.05) 7.33(53.33) 7.73(59.33) 7.24(52.00) 7.56(56.70) t4: rice+methi 6.23(38.67) 6.67(43.98) 8.19(66.67) 8.51(72.00) 9.11(82.67) 9.32(86.45) 8.97(80.00) 9.28(85.70) t5: sole rice 7.77(61.33) 8.19(66.55) 9.74(96.00) 10.24(104) 10.35(106.67) 10.59(111.68) 10.28(105.33) 10.55(110.80) t6: sole amaranth 6.04(36.00) 6.55(42.43) t7: sole coriander 7.05(49.33) 7.38(53.90) t8: sole palak 5.08(25.33) 5.60(30.88) t9: sole methi 6.56(42.67) 7.00(48.50) s.em.± 0.46 0.027 0.56 0.026 0.19 0.007 0.16 0.014 cd (p=0.05) 1.36 0.08 1.76 0.08 0.61 0.021 0.51 0.043 table 1 total weeds density (no./m2) in rice as influenced by different intercropping systems during 2017 and 2018 values in parentheses are original values; data analysed using transformation -√x + 0.5. das= days after sowing. habimana et al. ‐ weed management by intercropping system 369 was followed by intercropping of rice and leafy vegetable amaranths (0.47, 140.15, 228.05 and 225.15 g m-2, respectively). effect of the rice‐leafy vegetable intercropping sys‐ tem on weed smothering efficiency weed smothering efficiency is a measure of the effect of intercropping on the suppression of weeds in comparison to sole crop stand. the data pertaining to the weed smothering efficiency are presented in table 2. in the present experiment, the higher weed smothering efficiency was found in intercropping of compared to the sole rice crop (61.33 and 66.55 no. m-2 during 2017 and 2018, respectively). among different intercropping practices, intercropping of rice and leafy vegetable palak recorded significantly lower density of total weeds at 60 das (34.67 and 42.05 no. m-2 during 2017 and 2018, respectively). it was followed by intercropping of rice and leafy vegetable amaranths: at 60 das (54.67 and 59.23 no. m-2, respectively). however, the sole rice registered a bit higher number of total weeds (96.00 and 104 no. m-2, respectively). the similar trend was noticed at the further crop growth stage of aerobic rice at 90 days after sowing and at harvest. effect of the rice‐leafy vegetable intercropping sys‐ tem on weed dry weight the weed dry weight is the useful parameter to assess the extent of weed competition with the crop plants. the total weeds were differed significantly at different growth stages of aerobic rice due to different intercropping practices. the 2 years pooled data are here under depicted in figure 1. total weed dry weight in aerobic rice was significantly lower in rice-palak leafy vegetables intercropping systems at 30 das, 60, 90 and at harvest (0.25, 115.34, 151.20 and 148.04 g m-2, respectively) as compared to the sole rice crop (1.56, 204.07, 302.09 and 279.08 g m-2, respectively). however, the latter table 2 rice grain, straw and leaf vegetable yield, rice equivalent yield (rey), land equivalent ratio (ler), production efficiency (pe), net monetary income and weed smothering efficiency (wse) in rice as influenced by different intercropping systems, during 2017 and 2018 t1: rice+amaranth, t2: rice+coriander, t3: rice+palak, t4: rice+methi, t5: sole rice, t6: sole amaranth, t7: sole coriander, t8: sole crop palak, t9: sole crop methi ; na=not analysed; (indian rupees: ₹ converted into us$ at the rate of 69.35₹ against 1 us$ on 29th march 2019). fig. 1 total weed dry weight at different growth stages as influenced by different intercropping systems, pooled data 2017 and 2018. t1: rice+amaranth, t2: rice+coriander, t3: rice+palak, t4: rice+methi, t5: sole rice. practices grain yield (kg ha-1) straw yield (kg ha-1) vegetable yield (kg ha-1) rey (kg day-1) ler pe (kg day-1) net income (₹ &us$ha-1) wse (%) 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 t1 6730 5755 8382 8167 14248 13811 11479 10358 1.88 1.82 76.53 69.05 76010₹ 1096 us$ 74749₹ 1078 us$ 20.07 18.60 t2 6018 5443 8030 7841 12057 11226 10037 9185 1.82 1.79 66.91 61.24 59357₹ 856 us$ 56543₹ 815 us$ 8.15 6.88 t3 7951 7351 9794 9580 25810 25207 16554 15753 2.12 2.15 110.36 105.02 158025₹ 2278 us$ 154515₹ 2228 us$ 48.09 45.86 t4 6444 5644 8196 8010 14133 12056 11155 9663 1.87 1.88 74.37 64.42 72095₹ 1039 us$ 70098₹ 1011 us$ 12.54 11.64 t5 5978 5403 7382 7174 t6 19010 18407 t7 15086 14482 t8 32705 32105 t9 18743 15893 s.em.± 92.32 183.89 260.38 259.15 1555 1427 684 713 0.09 0.06 4.08 4.19 na na na na cd @5% 296.99 572.91 811.20 807.36 4604 4191 2252 2313 0.28 0.21 12.7 13.06 na na na na 370 adv. hort. sci., 2019 33(3): 365-373 rice and leafy vegetable palak (48.09 and 45.86%, 2017 and 2018, respectively) followed by intercropping of rice and leafy amaranthus (20.07 and 18.60%, 2017 and 2018, respectively). effect of the rice‐leafy vegetable intercropping sys‐ tem on nutrient uptake by crops and weeds the weeds withdraw the nutrients that would have normally available to the crop. as the nutrient uptake is increased by weeds on account of higher weed population, the harmful effect could be expected on the crop. when the weed growth is effectively managed through integrated weed management, a decline in nutrient uptake by weeds is a natural consequence. uptake of major soil nutrients by weeds and crops indicated that the rate of increase in the uptake was proportional to the dry matter production. the 2 years pooled data on uptake of nitrogen, phosphorus and potassium by rice, leafy vegetables at harvest and weeds at 60 das as influenced by different intercropping practices are given in figures 2 and 3. the total nitrogen (153.78 kg ha-1), phosphorous (45.27 kg ha-1) and potassium (152.22 kg ha-1) uptake by rice crop at harvest were significantly higher in intercropping of rice with leafy vegetable palak as compared to sole rice (109.90, 29.49 and 109.25 kg npk ha-1, respectively). similar trend was seen with uptake by leafy vegetable crop (fig. 3) wherein leafy vegetable palak intercropped with rice significantly recorded higher amount of the total nitrogen (101.09 kg ha-1), phosphorous (14.18 kg ha-1) and potassium (38.97 kg ha-1) followed by leafy vegetable amaranthus intercropped with rice (77.08, 8.98 and 28.81 kg npk ha-1, respectively). at 60 days of crop growth (fig. 2), the nitrogen, phosphorous and potassium uptake by weeds were significantly influenced by different intercropping practices. significantly lower nitrogen (31.88 kg ha-1), phosphorus (19.90 kg ha-1) and potassium (28.92 kg ha-1) uptake were recorded with the intercropping of rice+leafy vegetable palak. it was followed by intercropping of rice with leafy vegetable amaranth: nitrogen (46.72 kg ha-1), phosphorus (27.39 kg ha-1) and potassium (42.68 kg ha-1) uptake as compared to sole rice (94.93, 46.48 and 91.83 kg npk ha-1, respectively). effect of the rice‐leafy vegetable intercropping sys‐ tem on rice and vegetable yield and efficiencies the rice grain yield, straw yield, leafy vegetable yield, rice equivalent yield (rey), land equivalent ratio (ler), production efficiency (pe) and net monetary income differed significantly due to different intercropping practices. the data are accessible in table 2. significantly higher grain yield, straw yield, vegetable leaf yield (7951, 9794, 25810 kg ha-1 and 7351, 9580, 25207 kg ha-1 for 2017 and 2018, respectively) were recorded in intercropping of rice with leafy vegfig. 2 nutrient uptake by rice crop at harvest and weed at 60 das as influenced by different intercropping systems. t1: rice+amaranth, t2: rice+coriander, t3: rice+palak, t4: rice+methi, t5: sole rice. fig. 3 nutrient uptake by leafy vegetable crops at harvest as influenced by different intercropping systems, pooled data 2017 and 2018. t1: rice+amaranth, t2: rice+coriander, t3: rice+palak, t4: rice+methi, t5: sole rice, t6: sole amaranth, t7: sole coriander, t8: sole crop palak, t9: sole crop methi. habimana et al. ‐ weed management by intercropping system 371 etable palak as compared to grain and straw yield of sole rice (5978, 7382 and 5403, 7174 kg ha-1 for 2017 and 2018, respectively). it was followed by intercropping of rice with leafy vegetable amaranth (6730, 8382, 14248 kg ha-1 and 5755, 8167, 13811kg ha-1 for 2017 and 2018, respectively). rice equivalent yield is the best tool to determine the overall productivity potential of an intercropping system. the data presented in table 2 reflected visible variation in rey among the intercropping systems showing the highest rey (16554 and 15753 kg ha-1 in 2017 and 2018, respectively) for intercropping of rice with leafy vegetable palak followed by intercropping of rice with leafy vegetable amaranthus (11479 and 10358 kg ha-1) which was on par with intercropping of rice with leafy vegetable methi (fenugreek) (11155 and 9663 kg ha-1). the data on land equivalent ratio of different intercropping systems indicated that ler values were greater than one in all the intercropping practices and the range of yield advantage over sole cropping of rice was between 79 and 115 per cent with the highest in case of intercropping of rice with leafy vegetable palak (115 per cent) followed by intercropping of rice with leafy vegetable methi (88 per cent) compared to monocropping of rice. significantly higher production efficiency was recorded in intercropping of rice with leafy vegetable palak (110.36 and 105.02 kg day-1 in 2017 and 2018, respectively) and was closely followed by intercropping of rice with leafy vegetable amaranthus (76.53 and 69.05 kg day-1) in both years. net monetary income (indian rupees: ₹ converted into us$ at the rate of 69.35₹ against 1 us$ on 29th march 2019) was higher in intercropping of rice with leafy vegetable palak (₹ 158025 equivalent to us$ 2278) ha-1; ₹154515 equivalent to us$ 2228) ha-1 for 2017 and 2018, respectively) followed by intercropping of rice with leafy vegetable amaranthus (₹ 76010 equivalent to us$1096); ₹ 74749 equivalent to us$ 1078) ha-1 for 2017 and 2018, respectively). 4. discussion and conclusions the weed population and total dry weight of weeds differed significantly due to different intercropping systems (table 1, fig. 1). the decline in weed density and lower weed dry matter accumulation in rice + palak intercropping systems may be attributed to shading effect and competition stress generated by the canopy of leafy vegetable in a unit area having smothering effect on associated weeds, thus preventing the weeds to attain the full growth (banik and ravi, 2013). the intercropping system suppressed the weed growth due to their spreading canopy coverage. the increased populations per unit area and crop competition in intercropping were also the possible reason for effective weed control (jha and dinesh, 1982; ibni et al. 2005; abdul et al. 2009; mian et al. 2011). higher weed smothering efficiency (table 2) in rice + palak intercropping systems resulted from less space available for the growth of weeds due to quick coverage of ground and more shading effect which led to the lower total weed population and its dry weight. similar findings were also reported by musthafa and potty (2001); vyas and kushwah (2008) and mian et al. (2011). the higher rice grain yield could be attributed to better yield attributing parameters namely higher no. of productive tillers hill-1, higher panicle length, higher panicle weight hill-1, higher total no. grain panicle1, higher 1000 grain weight and higher harvest index as compared to sole rice. the above increment in yield was attributed to increased growth attributes such as higher total dry matter production and distribution in various parts of the plant and higher leaf area as well. in addition to this, the higher canopy coverage by palak has resulted in a reduction in total weed population which turned the equilibrium in favor of crop for the use of the available resources. similarly, in the intercropping system, significantly higher fresh leafy vegetable yield (25508 kg ha-1) was recorded in intercropping of rice with leafy vegetable palak followed by intercropping of rice with leafy amaranthus (14029 kg ha-1, respectively). however, the sole leafy vegetable palak registered higher yield (32405 kg ha-1) followed by sole leafy amaranthus (18708 kg ha-1). these findings are in the similar trend with ibni et al. (2005); ahmed et al. (2006); mian et al. (2010). the higher nutrients uptake by rice crop and leafy vegetables in intercropping of rice with leafy vegetable palak might be attributed to minimum cropweed competition as a result of higher weed smothering efficiency, gave the better control of weeds from initial stages which led to lower weed population and their dry weight, this helped the crop to grow in weed-free environment and absorb more nutrients from the soil. hence, resulted in better growth and development of leafy vegetable and rice crops leading to better nutrient uptake. a similar report was also reported by abdul et al. (2009) and adv. hort. sci., 2019 33(3): 365-373 372 mian et al. (2010). the lower nutrient uptake by weeds in intercropping of rice with leafy vegetable palak was mainly due to better control of weeds as a result of lower weed competition leading to lower weed dry matter production as also noticed by ibni et al. (2005); abdul et al. (2009) and mian et al. (2010). the percentage increase over sole cropping of rice as a result of different intercropping systems, however, varied from 46.74 to 76.83 % clearly indicating substantial yield advantage of intercropping. the variation in rey under different cropping systems was ascribed to their variable utilization of soil and agro-resources. higher yield benefit in terms of rey of intercropping over monocropping of rice has also been revealed by abdul et al. (2009), mian et al. (2011), nagwa et al. (2014), rayhan et al. (2014), and gurpreet singh et al. (2018). the data on ler of different intercropping systems indicated that ler values were greater than one in all the intercropping treatments and the range of yield advantage over sole cropping of rice was between 79 and 115 per cent with the highest in case of intercropping of rice with leafy vegetable palak (115 per cent) followed by intercropping of rice with leafy vegetable amaranthus (85 per cent) compared to monocropping of rice was attributed to better utilization of natural resources (land, co2 and light). higher ler in intercropping compared to monocropping of rice was also reported by abdul et al. (2009), mian et al. (2011), nagwa et al. (2014), udhaya and kuzhanthaivel (2015) (table 2). significantly higher pe was recorded in intercropping of rice with leafy vegetable palak (107.69 kg day1) and was closely followed by intercropping of rice with leafy vegetable amaranthus (72.79 kg day-1). the result indicated that the intercrops remained in the field for a shorter time (30 das) and yields were also high leading to higher production per day. the similar tendency was noted by ibni et al. (2005); nazrul and shaheb (2011) and rayhan et al. (2014) (table 2). the higher net income increases in intercropping of rice with leafy vegetable palak was mainly due to the higher rice grain, rice straw yield, rice equivalent and higher leafy vegetable yield which in turn increased gross and net returns. these results are in agreement with the findings of ibni et al. (2005); abdul et al. (2009); mian et al. 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evaluation of an alternative mean for controlling postharvest rhizopus rot of strawberries u. de corato 1 (*), r. salimbeni 2, a. de pretis 2 1 agenzia nazionale per le nuove tecnologie, l’energia e lo sviluppo economico sostenibile (enea), divisione bioenergia, bioraffineria e chimica verde, ufficio territoriale di bari, viale japigia, 188, 70126 bari, italy. 2 bio-planta srl, ss. 96, 70100 bitonto (ba), italy. key words: crude extract, fatty acid, laminaria digitata (huds.) lamouroux, phenolic compound, peroxidase activity. abstract: crude extract of the brown seaweed laminaria digitata was tested as an alternative mean to control postharvest rhizopus rot of strawberries. the antifungal activity of four extracts (one un-fractionated and three soluble by hexane, ethanol, and water) was in vitro measured against one pathogenic isolate of rhizopus stolonifer at a concentration range from 10 g l-1 to 30 g l-1. the antifungal activity of the un-fractionated extract was in vivo measured into a climatic room at the same concentration range in comparison to fungicide fenhexamid in preventive and curative treatments. the peroxidase activity in l. digitata-treated inoculated strawberries was performed. a significant inhibition of mycelia growth applying 30 g l-1 of un-fractionated extract (until 80% after 5 days) and suppression of sporangia germination (until 95% after 24 hours) were found by a dose-dependent manner of the treatment. only the extracts fractionated by hexane and ethanol were likewise suppressive at the same concentrations against mycelia (until 71% and 66% respectively) and sporangia (until 82% and 69% respectively) such involving a direct toxicity induced by lipids and phenolic compounds to r. stolonifer suppression. fruit decay inhibition of the r. stolonifer/strawberries pathosystem increased from 10 g l-1 to 30 g l-1 until 75% after 4 days only in preventive treatment. an increased peroxidase activity (4.84 δod420 g-1 min-1) seen in fruit tissue after one-day from the application of 30 g -1 raw extract suggests that in vivo suppression could also be related to induced systemic resistance phenomena. 1. introduction strawberry fruits are a perishable commodity due to industrial processing chain that occurs immediately after harvesting which causes mechanical injury, desiccation, physiological disorders, deterioration of quality and nutrient composition, decaying, abiotic stress, mycotoxin contamination, and reduction of their market value (de cicco et al., 2008). the known fungi that are considered responsible of strawberry fruit (*) corresponding author: ugo.decorato@enea.it citation: de corato u., salimbeni r., de pretis a., 2018 evaluation of an alternative mean for controlling postharvest rhizopus rot of strawberries. adv. hort. sci., 32(3): 325-334 copyright: © 2018 de corato e., salimbeni r., de pretis a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 october 2017 accepted for publication 16 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 325-334 326 postharvest decay are botrytis cinerea pers. ex fr. and colletotrichum spp., the causal agents of gray mold and anthracnose respectively; penicillium spp., the agent of green and bleu mold; rhizopus spp., mucor spp., and alternaria alternata f. sp. fragariae, the main agents of rots (husaini and neri, 2016). among the most important postharvest decay agents that occur in strawberries, the soft rot caused by the zygomycete fungus rhizopus stolonifer (ehrenb.: fr.) vuill. induces economically severe losses both in the field and overall during long-distance transport, especially if storage temperature was more than 45°c and when strawberries are damaged during handling (maas, 1998). control of postharvest decay on strawberry (fragaria × ananassa duch.) fruit can be usually achieved by physical, chemical and biological methods (ippolito et al., 1997; droby et al., 2009). requirements from consumers for fruit free by chemical residues from synthetic fungicides have recently stimulated the researchers (mari et al., 2016) to find safer alternatives for environmental and human health based on the wide range of natural antifungal compounds, as phenolic substances given from wild edible herbs (gatto et al., 2011, 2013), and terpenic compounds provided from aromatic plants (de corato et al., 2010). in this context, seaweed extract can stimulate growth in strawberries by protecting them from pathogens and physiological hazards under storage condition (tuhy et al., 2013). crude extract from brown seaweed contains a wide range of antifungal substances, mainly lipids (triglycerides), phenolic compounds (phlorotannins) and water-soluble polysaccharides (laminarans, fucoidans, and alginates), which were investigated for their antifungal properties in preventing postharvest fruit losses caused by plant pathogenic fungi (washington et al., 1999). recently, the use of crude extracts from brown and red seaweeds obtained by a supercritical carbon dioxide technique in preventing the severe fruit postharvest losses caused by several plant pathogenic fungi, has been investigated (de corato et al., 2017). in addition to that, an excellent paper provides an overview of the most recent findings on the potential use of extracts from macroalgae for strawberry management, concerning both their biostimulant effects and antifungal properties against postharvest pathogens (righini et al., 2018). from this review, it is clear that very few papers about the practical use of algal extract as an effective alternative strategy to syntetic fungicides, such as fenhexamid, for controlling postharvest strawberries rot by r. stolonifer are reported in literature, because this fungus is really most invasive and it shows a faster development of aerial mycelia in comparison to other fungi afore-mentioned, especially if storage temperature varies from 6°c to 10°c. the purpose of this work was to assess a potential use of crude extract from the brown seaweed laminaria digitata (huds.) lamouroux for effectively controlling strawberry fruit rot caused by r. stolonifer under postharvest condition. the first objective was to investigate the potential suppressive, under in vitro and in vivo conditions, of crude extracts of l. digitata in preventing postharvest losses of strawberries caused by infections of r. stolonifer which are of greater importance in the italian markets. the second objective was to clarify the most probable hypothesis about the mechanisms explaining the suppressive effect observed. to reach these two objectives, the direct antifungal activity of four extracts (one un-fractionated and three fractionated by hexane, ethanol, and water) was in vitro, measured by a microbiological method within an increasing concentration range from 10 g l-1 to 30 g l-1. the antifungal activity of the raw extract was in vivo, measured at the same concentration range by a phytopathological method into a climatic room in comparison to fenhexamid during preventive and curative treatments. finally, the peroxidase (pod) activity in l. digitata-treated inoculated strawberries was assessed by an enzymatic method to investigate an indirect antifungal activity of the raw extract by induced systemic resistance mechanisms. 2. materials and methods experimental trials an amount of about 80 kg of fresh algal biomass, collected from healthy and matured l. digitata cultures performed into a photo-bioreactor of 900 l capacity, was purchased from a marine biorefinery located near the coastal areas of gibraltar and morocco. algal biomass was immediately refrigerated after harvesting thoroughly washed with seawater, and washed with tap water to remove all extraneous particles and epiphyte organisms. fresh biomass was dried into an industrial drier located at enea trisaia research centre (policoro, matera, italy) chopped, finely pulverized, heat-treated for 24 hours with sodium hydroxide (1:10 w/w) for de corato et al. control strategies of fruit postharvest diseases 327 triglycerides saponification, weighed, and stored at 4°c until extraction of biologically active substances. the extraction was carried out by using a mixture of un-polar and polar solvents in appropriate proportions (hexane:ethanol:water, 1:2:2 v/v) into a clevenger apparatus. extraction was repeated three times at room temperature, and total processing time was about 4-5 hours (selvi et al., 2014). stocks of raw crude extract were collected, suspended in methanol, collected into bottles, and stored at 4°c in the dark until tested. crude seaweed extract yield was calculated on three independent replicated samples of 50 g each after drying at 55±5°c for 2 hours, and expressed as a percentage on the basis of fresh biomass weight. stocks of crude seaweed extract were dried, weighed, and stored at -20°c in the dark till further uses. aliquots of raw extract were fractionated into three fractions using three solvents with a different affinity towards fatty acids, phenolic compounds and water-soluble polysaccharides (khanzada et al., 2007). samples of 50 g of dry extract were suspended in n-hexane, or distilled water, or pure ethanol (1:5 w/v) in separating funnel for 20 days at room temperature to separate lipids, polysaccharides and phenolic substances, respectively from the crude extract. suspensions were then filtered using whatman filter paper and concentrated under reduced pressure at 35°c using a rotary evaporator (strike 202, steroglass, perugia, italy) till the extract become as a syrup. three different fractions were finally separated from this residue. each fraction was individually collected from the respective funnel, and each stock was air-dried, weighed, and stored at -20°c in the dark till in vitro and in vivo assayed by using a certified pathogenic strain of r. stolonifer (provided by the collection of microorganisms and cell cultures institute, dsmz, braunschweig, germany) causing severe rots on strawberries under postharvest condition. stocks of total extract, or fraction of it, suspended in sterile 0.1 m k-phosphate buffer were in vitro tested at the concentration range of 10 g l-1, 20 g l-1 and 30 g l-1 for determining a minimum bioactive concentration by poison food technique (shahi et al., 1999). mycelia inhibition was quantitatively assessed measuring radial growth in petri plates (100 mm diameter) containing potato dextrose agar (pda, sigmaaldrich, milan, italy) adding 18 ml pda per plate. in treated plates, aliquots of 2 ml of sterile stock suspension containing extract (both raw and fractionated) were added to pda at 42±3°c before solidification. in untreated plates (control), 2 ml of sterile buffer was added to pda in place of the extract. three mycelia plugs measuring 5 mm diameter each were cut out by the margin of 3-day-old fungal cultures actively growing, and then aseptically placed on the upper pda surface. treated and control plates were incubated in the dark at 22±1°c for 5 days. mycelia growth inhibition was measured at three time set points (after 1 d, 3 d and 5 d of incubation) with respect to the control plates by the index mgi% = [(dco-dse)/dco] × 100 where, dco is the average of colony diameter (mm) in the control plates, and dse is the average of colony diameter in the plates amended with seaweed extract at the three afore-mentioned concentrations. all experiments were carried out with three replications of 10 plates for each. sporangia germination was evaluated on micro-cultures by a microassay on glass slides that allowed the quantitative analysis of sporangia suppression using an optical microscopy technique (gatto et al., 2011; 2013). assays on 96microwell (100 μl volume) plates purchased from aes laboratory (milan, italy) were performed. each micro-well was set up with three replicates each containing 10 μl of potato dextrose broth (pdb) provided from sigma-aldrich, 2 μl of sporangial suspension containing 108 cfu ml-1, and 88 μl of extract (both raw and fractionated) suspended in sterile 0.1 m k-phosphate buffer to be tested at the concentrations of 10 g l-1, 20 g l-1 and 30 g l-1. one micro-well row used as a control was filled with 10 μl pdb, 2 μl sporangial suspension, and 88 μl buffer. each plate was incubated at 22±1°c for 24 hours. after incubation, aliquots of 5 μl sporangia cultures taken from each micro-well were sampled and mounted on the upper surface of glass slides sterilized with denatured ethanol. number of the total, un-germinated, and collapsed sporangia were counted by a burker’s hemocytometer using a photomicroscope (40 × magnification) (bx60, olympus, milan, italy). sporangia germination suppression was measured at four time set points (after 2 h, 10 h, 18 h and 24 h of incubation) by the index sg% = su/st × 100 where, su is the average of the sum of un-germinated + collapsed sporangia in un-supplemented cultures (control) or amended with the extracts at the three afore-mentioned concentrations, and st is the average of number of total sporangia in the same sample. all measurements were performed with three replications of 5 glass slides taken from each micro-well. adv. hort. sci., 2018 32(3): 325-334 328 preventive and curative treatments were in vivo evaluated on strawberries (cv. camarosa) harvested from growers located in basilicata (policoro, matera, italy) under tunnel condition. healthy fruit untreated with synthetic fungicides and selected for uniform size, same ripening stage, and absence of visible defects and injuries, were washed under running tap water, surface-disinfected by dipping for 1 min in 2% sodium hypochlorite solution, rinsed with tap water and allowed to dry. each fruit was injured in the equatorial zone in two opposite points at the fixed dimensions (wide= 2 mm, deep= 2 mm), treated with the crude seaweed extract, and inoculated with a sporangial suspension of r. stolonifer. trials treated with the stocks containing 10 g l-1, 20 g l-1 and 30 g l-1 of raw extract suspended in sterile 0.1 m k-phosphate buffer were set up. two controls replacing the extracts, the first one with buffer alone, and the second one with one commercial fungicide containing as active substances 50% fenhexamid (1.2 g l-1) were both included for preventive and curative treatments. aliquots of 30 μl of the stock suspension of extract were dispensed over each wound of the treated fruit allowing the droplet to be absorbed into the fruit. aliquots of 30 μl of sterile buffer alone, or fungicide, were dispensed over the wound of the control fruit. each wound was inoculated with 10 μl of a sporangial suspension containing 106 cfu ml-1. in preventive treatments, the pathogen was inoculated over the injured area 2 days later from the application of the extract, or the fungicide, or the buffer alone, for enhancing plant defences before inoculation. instead, in those curative, the extract, or the fungicide, or the buffer alone, were applied over the wound 8 hours later from inoculation of the pathogen allowing sporangia germination before treatment. fruit were placed in trays, packaged in plastic bags and maintained into a climatic room for 4 days at the temperature of 20±2°c and relatively humidity (rh) of 96±2% in the dark. trials were arranged in a completely randomized experimental design including six replicates per treatment, whenever thirty strawberry fruit with two wounds per fruit were considered per each replication. disease incidence was assessed by counting the number of the infected wounds on each fruit. the disease incidence data were converted into the strawberries soft rot suppression data by the index di% = [(nco−nse)/nco] × 100 where, nco is the average of number of infected wounds in the control plots treated with buffer alone, and nse is the average of number of infected wounds in the plots treated with extract at the three afore-mentioned concentrations, or with the fungicide (arras et al., 1999). trials were also arranged to assess the pod activity including thirty strawberry fruit treated with 30 g l1 un-fractionated extract in preventive treatment, and sampled after one-day and five-days of incubation at temperature of 20±2°c and rh of 96±2% in the dark. inoculated and healthy fruit (not injured) treated with only sterile 0.1 m k-phosphate buffer were both included as positive and negative control, respectively. small pieces of tissue (diameter= 2-3 mm, deep= 3-4 mm) randomly collected from six points of each fruit were powdered with liquid nitrogen. samples of one-gram of tissue were extracted with 2 ml of 0.1 m sodium-phosphate buffer (ph 7.0) at 4°c and used for assessing pod activity. all experiments were performed with three replications of 10 fruit for each. pod activity was assayed according to hammerschmidt et al. (1982). the reaction mixture consisted of one-gram of tissue, 1.5 ml of 0.05 m pyrogallol, 0.5 ml of enzyme (1.5 u ml-1) and 0.5 ml of 1% h2o2 that it was incubated at 28°c. absorbance measured at a wavelength of 420 nm were recorded every 30 s for 3 minutes and the boiled enzyme preparation served as blank. pod activity was expressed as change in absorbance of the reaction mixture on the fresh weight basis (δ od420 g-1 min-1). statistical analyses the suppressive effects of each l. digitata crude extract to mycelia growth and sporangia germination of r. stolonifer (using both raw extract and soluble extracts by hexane, ethanol and water), and versus strawberries soft rot and peroxidase activity (using only raw extract), were assessed by using variance analysis (anova). in particular, two-way anova was applied to test the interactions either among three extract concentrations (10 g l-1, 20 g l-1 and 30 g l-1) and three incubation times (1 d, 3 d and 5 d) on mycelia growth inhibition index (mgi%), either between three extract concentrations (10 g l-1, 20 g l-1 and 30 g l-1) and two treatment types (preventive and curative) on strawberries soft rot suppression index (di%). for performing two-way anova we have used the average data of mgi% and di% assessed at the different extract concentrations, incubation times, and treatment types at p-value ≤0.05. value percentage of the two indexed parameters was before transformed into arcsine for satisfying the assumption of normality, and then compared by applying the de corato et al. control strategies of fruit postharvest diseases 329 duncan multiple test range (dmrt) whenever twoway anova revealed a significant difference among the means at a p≤0.05 level. however, these data has been shown as un-transformed values. one-way anova was instead used to test difference among four extract concentrations, including control (0 g l-1), at the same incubation time (2 h, 10 h, 18 h and 24 h) on sporangia germination suppression index (sg%). sg% values were before transformed into arcsine and then compared by dmrt whenever one-way anova revealed a significant difference among the means at p≤0.05. one-way anova was also used to test difference among the three experimental trials at the same sampling time to assess peroxidase (pod) activity at the first and fifth day, and dmrt was used to compare data whenever one-way anova revealed a significant difference among the means at p≤0.05. regards to better clarify the effect of seaweed extract at the three considered concentrations including control on the un-germinated and collapsed sporangia vs. incubation time, the sg rates were submitted to regression analysis in order to obtain curves that were compared for slope and elevation for each set points. all statistics were managed by the 12.0 spss programme (statistics base™, chicago, illinois, usa). 3. results the seaweed extract yield obtained at the end of the extraction process was about 1.22±0.1 g on 100 g of fresh biomass. r. stolonifer mycelia growth was affected by extract concentration and incubation time showing a significant interaction between these two factors after supplementation to pda with l. digitata crude extract (table 1). the mycelia growth inhibition percentage by increasing doses of raw extract resulted of 10%, 31% and 48% after one-day of incubation; it increased until 22%, 45% and 58% after three-days; finally it reached 40%, 61% and 80% at the end of incubation time (after five-days) applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract at each sampling time (fig. 1a). the mycelia growth inhibition percentage after supplementation to pda with increasing doses of extract purified with hexane resulted of 4%, 15% and 22% after one-day of incubation; it increased until 16%, 31% and 53% after threedays, reaching 20%, 45% and 71% at the end of incubation time applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract at each sampling time (fig. 1b). the mycelia growth inhibition percentage after amendment with increasing doses of extract soluble in ethanol resulted of 10%, 14% and 16% after one-day of incubation; it increased until 17%, 30% and 43% after three-days, it reached 19%, 43% and 66% after five-days applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract at each sampling time (fig. 1c). no inhibition effect after amendment with extract purified by water was seen (data not shown). the un-germinated and collapsed sporangia percentage of r. stolonifer after supplementation to pdb with increasing doses of l. digitata un-fractionated extract resulted of 16%, 24% and 36% after 2 h of incubation, while it increased until 50%, 77% and 95% at the end of incubation time (after 24 h) applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract at each sampling time (fig. 2a). the un-germinated and collapsed sporangia percentage following to supplementation with increasing doses of extract soluble in hexane resulted of 9%, 15% and 25% after 2 h of incubation, while it reached 40%, 65% and 82% after 24 h of incubation applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract (fig. 2b). the un-germinated and collapsed sporangia percentage after adding of increasing doses of extract soluble in ethanol resulted of 5%, 13% and 14% after 2 h of incubation, while it reached 42%, 55% and 69% at the end of incubation applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 extract (fig. 2c). no reduction of sporangia germination was found with respect to control after adding of extract soluble in water (data not shown). strawberry fruit soft rot suppression was affected by extract concentration and treatment type with a significant interaction between these two factors after adding of l. digitata raw extract over strawbertable 1 synthetic values to different two-way anova analysis to rhizopus stolonifer mycelia growth inhibition and strawberries rhizopus soft rot suppression at p-value ≤ 0.05 a extract concentration (10 g l-1, 20 g l-1 and 30 g l-1) and incubation time (1 d, 3 d and 5 d) are the two factors considered on mycelia growth inhibition. b extract concentration (10 g l-1, 20 g l-1 and 30 g l-1) and treatment type (preventive and curative) are the two factors considered on strawberry fruit rot suppression. c degree of freedom. effect df c f p-value on mycelia growth inhibition a: 1) extract concentration 2 44 0.02 2) incubation time 2 26.2 0.03 extract concentration × incubation time 4 2.8 0.02 on strawberries soft rot suppression b: 3) extract concentration 2 31.6 <0.01 4) treatment type 1 6.6 0.01 extract concentration × treatment type 2 9.4 <0.01 330 adv. hort. sci., 2018 32(3): 325-334 ries wound (table 1). the fruit decay inhibition percentage of the r. stolonifer/strawberries system after preventive treatment with l. digitata was of 22%, 49% and 75%; but it remained of 2%, 11% and 21% in curative treatment applying respectively 10 g l-1, 20 g l-1 and 30 g l-1 raw extract (figs. 3, 4). the chemical treatment with fenhexamid suppressed the strawberries rot until 100% in those preventive and 70% in those curative (figs. 3, 4). fig. 1 suppressive effect of laminaria digitata raw extract (a), and soluble extracts by hexane (b) and ethanol (c), on rhizopus stolonifer mycelia growth inhibition index (mgi%). the suppressive activity of extracts applied at three different concentrations was tested after incubation at 22±1°c for 5 days into petri plates on pda medium. each value, ranging from 0% (no inhibition) to 100% (total inhibition), represents the pooled mean of three replicates with 10 plates for each. mgi% values were collected after 1 d, 3 d and 5 d and analysed by two-way anova. asterisk indicates p-value ≤0.05 according to table 1. histogram points with different letters are significantly different according to duncan multiple test range (dmrt) at a probability p≤0.05 level. fig. 2 suppressive effect of laminaria digitata raw extract (a), and soluble extracts by hexane (b) and ethanol (c), on rhizopus stolonifer sporangia germination suppression index (sg%). the suppressive activity of extracts applied at four different concentrations (including control) was tested after incubation at 22±1°c for 24 hours into micro-well plates filled with pdb medium. each value, ranging from 0% (no suppression) to 100% (total suppression), is the pooled mean±sd (bars) of three replicates of 5 glass slides taken from each micro-well. sg% values were collected after 2 h, 10 h, 18 h and 24 h and submitted to regression analysis. for each incubation time, sg% values analysed by one-way anova with different letters are significantly different according to dmrt (p≤0.05). 331 de corato et al. control strategies of fruit postharvest diseases finally, as regards to measurement of peroxidase activity in strawberries (table 2), a significant increment of absorbance in plots treated with l. digitata raw extract was seen yet after one-day of incubation (6.84 δod420 g-1 min-1) if compared to those of the untreated-inoculated strawberries (5.01 δod420 g-1 min-1). moreover, the untreated-healthy fruit shows a significant lower pod (1.12 δod420 g-1 min-1) with respect to untreated-inoculated strawberry fruit at the same sampling time. 4. discussion and conclusions in vitro experiments performed with the l. digitata hexane-soluble extract fraction suggests that inhibition on mycelia growth and sporangial germination of r. stolonifer could be due to direct toxicity of the fatty acids found in extracts of l. digitata purified with chloroform (løvstad holdt and kraan, 2011). by supporting this hypothesis, in ours finding the hexane-soluble extract fraction exerted a similar antifungal effect in comparison to those seen with the total extract. a very similar effect was also found testing the ethanol-soluble extract, suggesting that suppression could be due to direct toxicity exerted by the phenolic substances found in ethanolic extracts of l. digitata (løvstad holdt and kraan, 2011). in fact, the ethanol-soluble extract fraction incited a similar antifungal effect in comparison to those seen either with the hexane-soluble fraction either with the un-fractionated extract. instead, no suppressive effect on mycelia and sporangia was seen applying the watersoluble extract fraction, suggesting that the watersoluble polysaccharides (laminarans, fucoidans and fig. 3 strawberries rhizopus soft rot suppression index (di%) after 4 days of incubation at 20±2°c and 96±2% rh in the dark following to preventive and curative applications of 10 g l-1, 20 g l-1 and 30 g l-1 laminaria digitata raw extract, with respect to trial treated with fenhexamid. values ranging from 0% (no suppression) to 100% (total suppression) are the pooled mean of six replicates per treatment, each one carried out with thirty strawberries with two wounds per fruit. di% values were analysed by two-way anova. asterisk indicates p-value ≤0.05 according to table 1. histograms with different letters are significantly different according to dmrt (p≤0.05). fig. 4 rhizopus soft rot symptom on strawberries cv. camarosa after 4 days of incubation at 20±2°c and 96±2% rh in the dark after a preventive application of 20 g l-1 and 30 g l-1 laminaria digitata raw extract with respect to the control trial treated with buffer alone and fenhexamid (1.2 g l-1). table 2 peroxidase (pod) activity in strawberries inoculated with r. stolonifer and preventively treated with 30 g l-1 laminaria digitata un-fractionated extract. pod assessment was carried out after one-day and five-days of incubation at 20±2°c and 98% rh in the dark values are the pooled mean±sd of three replicates of 10 fruit for each analyzed by one-way anova. in each column, values followed by different letters are significantly different according to duncan multiple test range (dmrt) at a probability p≤0.05 level. change in absorbance on the fresh weight basis (δod420 g-1 min-1). experimental trial one-day five-days l. digitata-treated inoculated strawberries 6.84±0.3 a 53.15± 1.5 a untreated-inoculated strawberries 5.01±0.2 b 20.38±0.9 b untreated-healthy strawberries (not injured) 1.12±0.06 c 5.27±0.2 c 332 adv. hort. sci., 2018 32(3): 325-334 alginates) present in aqueous extracts of l. digitata (løvstad holdt and kraan, 2011) could not be involved in mycelia and sporangia suppression under in vitro condition. moreover, mycelia inhibition was closely related to extracts concentration added into growing media, showing an increasing of antifungal activity as the dose of extract increased. a dosedependent manner of the treatments with l. digitata extracts was therefore found at least in the concentration range considered here. finally, our findings showed that mycelia inhibition increased from the first to the last day of incubation in a time-dependent manner, showing that the extracts (both un-fractionated and fractionated by hexane and ethanol) were really efficacy in suppressing mycelia growth during all incubation time. in vivo experiments performed with crude extract employed at 30 g l-1 dose showed a stronger efficacy of l. digitata in suppressing decay on infected strawberries by r. stolonifer in preventive treatments with respect to those curative. findings coming from in vivo experiments highlighted an interesting and very competitive antifungal efficacy of l. digitata raw extracts against r. stolonifer on strawberries when compared to action of fenhexamid in preventive treatments. our findings show that strawberries soft rot suppression was closely related either to extract dose or treatment type, since a remarkable increasing of suppressivity was seen as the concentration of extract applied over the wounds increased, as well as passing from the curative treatment into those preventive. therefore, an evident dose-effect of the preventive treatment was observed at least in the concentration range here considered. finally, a significant pod activity in l. digitata-treated inoculated strawberries was early found with respect to untreated-inoculated fruit. moreover, healthy strawberries treated with only sterile buffer showed a significant pod decreasing with respect to untreated-inoculated fruit. increments of pod activity early found after one-day of incubation after treatment with 30 g l-1 crude extract, and confirmed after five-days of incubation, could be related to activation of induced systemic resistance mechanisms into the r. stolonifer/strawberries system. in fact, the artificial inoculation with the pathogen without extract application induced a lower absorbance change with respect to l. digitatatreated inoculated strawberries, while not injured fruit showed a lower absorbance change in comparison to untreated-inoculated strawberries. our findings show that a direct antifungal activity exerted by crude extract of l. digitata could be attributed to its content of fatty acids, accordingly with de corato et al. (2017), which have investigated on the antifungal properties of five crude seaweed extracts obtained by a supercritical carbon dioxide technique, including those derived from l. digitata, on three fruit/pathogen pathosystems in preventing postharvest losses caused by b. cinerea, monilinia laxa (aderh. & ruhland) honey and penicillium digitatum (pers.) sacc. on strawberries, peaches and lemons, respectively. in our findings, phenolic compounds could be considered as good candidates able to suppress mycelia growth and sporangia germination of the pathogen together to fatty acids accordingly with the in vitro tests. this finding is nevertheless divergent if compared to the observations of de corato et al. (2017), whenever ethanolic extracts of l. digitata were weakly suppressive against b. cinerea and m. laxa when applied at 30 g l-1 dose with respect to extracts purified by hexane. the discordance between these two experimental evidences probably is due to the two different extractive techniques used, being employed a supercritical carbon dioxide technique to prevalently extract lipids rather than phenolic substances and water-soluble polysaccharides, while extraction by mixtures of polar and un-polar solvents in appropriate rates are generally less selective and more suitable to extract lipids, phenolic substances, and polysaccharides without preferences among them (selvi et al., 2014). several damaging mechanisms induced by exposure to vapour of essential oils derived from various aromatic plants, as well as after longer treatments with fatty acids and various phenolic substances were reported in literature, such as a partition of lipid layer of the cell membrane due to their hydrophobic nature, and affection of permeability of the cell membrane that cause leakage of cell components (rasooli et al., 2006; soylu et al., 2006; laird and phillips, 2011; da cruz et al., 2013; shao et al., 2013). in our findings, in vitro inhibition were not found by using aqueous extract revealing that the water-soluble polysaccharides fraction does not exert direct toxicity against mycelia development and sporangia germination. therefore, is reasonable affirm that laminarans, fucoidans and alginates present in aqueous extracts of l. digitata (løvstad holdt and kraan, 2011) could be involved in pod increasing by working most as resistance inducers (or elicitors) rather than as toxic chemicals. peroxidases usually employ hydrogen peroxide as a substrate causing defence reactions which earlier occur in the fruit tissue after infection. hydrogen peroxide has an antimicrobial properties due to its de corato et al. control strategies of fruit postharvest diseases 333 strong oxidizing power and its capacity to generate other oxidizing species (hydroxyl radicals, singlet oxygen species and hydrogen peroxides), on the whole well known as ros (‘reactive oxygen species’), which are toxic to living cells. inactivation of membrane respiratory chain enzymes and damage to dna are the probable mechanisms of action for hydrogen peroxide and related ros (imlay and linn, 1988; tatsuzawa et al., 1998). the laminarin, a storage polysaccharide (β-1,3-glucan) isolated for the first time from cell walls of l. digitata, elicits host defence responses in grapevine against b. cinerea (aziz et al., 2003), and the use of various chemical resistance inducers, including laminarin, for controlling postharvest gray mold and rhizopus rot in strawberry fruit, was studied in the past years (de miccolis et al., 2009; santini et al., 2009). we can conclude that the antifungal activity showed by crude extract from l. digitata could be mainly attributed to content of fatty acids and phenolic compounds extracted from this profitable algal biomass source by appropriate mixtures of polar and un-polar solvents; but, also an increased peroxidase activity probably elicited by the water-soluble polysaccharides content, as laminarin, could be related to activation of an induced systemic resistance mechanism able to suppress postharvest rhizopus soft rot of strawberries under in vivo condition. references arras g., nicolussi p., ligios c., 1999 non-toxicity of some antifungal yeasts (pichia guill iermondii , rhodotorula glutinis and candida oleophila) in laboratory animals. ann. microbiol. enzimol., 49: 125-131. aziz a., poinssot b., daire x., adrian m., bazier a., lambert b., joubert j.m., pugin a., 2003 laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and plasmopara viticola. molecular plant microbe interac., 16: 1118-1128. da cruz c.l., pinto v.f., patriarca a., 2013 application of plant derived compounds to control fungal 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an efficient transient vector for expressing heterologous proteins in nicotiana tabacum and n. benthamiana m. modarresi 1, m. jalali-javaran 1 (*), m. shams-bakhsh 2, s. zeinali 3, m. mirzaee 1 1 department of plant breeding and biotechnology, faculty of agriculture, tarbiat modares university, tehran, i.r. iran. 2 department of plant pathology, faculty of agriculture, tarbiat modares university, tehran, i.r. iran. 3 department of molecular medicine, pasteur institute of iran, tehran, i.r. iran. key words: green fluorescent protein, recombinant protein, tobacco plant, transient expression, viral vector. abstract: nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. it has some benefits such as fast and large quantity production of recombinant proteins with low cost. in this research, the green fluorescent protein (gfp) was transiently expressed in two tobacco species via turnip mosaic virus (tumv) derived vector, a virus which can infect a wide range of plant species. florescence microscopy results indicated that tumv could infect tobacco plants and accumulate gfp protein in plant leaves. in addition, rt-pcr, dot-blot and elisa assays demonstrated the recombinant gene transcription, translation and stability. this is the first report of using tumv-based viral vectors for producing recombinant proteins in tobacco. optimized tumv-based viral vectors could be used for producing recombinant proteins in tobacco. 1. introduction various expression systems, such as bacteria, yeast, plants, insects and mammalian cell cultures can produce recombinant proteins. the benefits of expressing recombinant proteins in plants include economic, agricultural scale, safe and authentic production (sijmons et al., 1990; ma et al., 2003; mardanova et al., 2015;). molecular farming (also known as molecular pharming or biopharming) uses genetically engineered plants for the production of biopharmaceutical products, vaccine subunits, industrial enzymes therapy peptides and other compounds of interest (boothe et al., 1997; wang and ma, 2011; yarbakht et al., 2015). recombinant proteins in plants may be gained by stable genetic transformation (nuclear or plastid) or through transient expression. transient expression is usually used for fast and flexible expression of genes of (*) corresponding author: m_jalali@modares@ac.ir citation: modarresi m., jalali-javaran m., shamsbakhsh m., zeinali s., mirzaee m., 2019 tumv as an efficient transient vector for expressing heterologous proteins in nicotiana tabacum and n. benthamiana. adv. hort. sci., 33(2): 257262 copyright: © 2019 modarresi m., jalali-javaran m., shamsbakhsh m., zeinali s., mirzaee m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 march 2018 accepted for publication 7 august 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 257-262 258 interest (goi), evaluation of expression system performance and components such as promoter and enhancers (chiera et al., 2008). in plants, a number of virus-based vectors are utilized for the transient expression of foreign genes, such as tobacco yellow dwarf virus (tydv) for transient expression of chalcone synthase in petunia hybrida (atkinson et al., 1998), tobacco mosaic virus (tmv) for transient expression of gfp (jellyfish, aequorea victoria greenfluorescent protein) in tobacco (shivprasad et al., 1999), bean pod mottle virus (bpmv) for expression the gfp in the soybean (zhang et al., 2010), wheat streak mosaic virus (wsmv) for expression the gfp in cereals (tatineni et al., 2011) etc. turnip mosaic virus (tumv) belongs to potyviridae family and infects a wide range of plant species especially cruciferous (brassicaceae family). it is a positive-sense single stranded rna virus with a linear and monopartite genome and average length of 720 nm (brunt et al., 1996). previously beauchemin et al. (2005) strongly expressed gfp and gus (bacterial βglucuronidase) reporters genes in brassica perviridis plants via tumv virus. furthermore, chen et al. (2007) introduced gfp in some brassica hosts such as b. campestris and b. juncea and high levels of the recombinant protein expression were observed. therefore, in this study, to investigate the performance of recombinant protein production, the gfp reporter gene was introduced into the tobacco (nicotiana tabacum and n. benthamiana) plants by using tumv vector. 2. materials and methods plant material and growth conditions nicotiana tabacum cv. xanthi and cv. samsun and n. benthamiana seeds were grown in pots containing autoclaved soil, including 40% farm soil, 30% peat moss and 30% perlite. they were kept at 25°c in a phytotron under a 16-hour photoperiod (16:8 h l: d). plasmid and viral constructs the tumv-gfp construct (fig. 1) was kindly provided by dr. shyi-dong yeh, plant pathology department, national cheng hsing university, taichnug, taiwan. the plasmid contains a cauliflower mosaic virus 35s promoter (camv 35s) and gfp coding sequence between the nib (nuclear inclusion protein b) and cp (coat protein) positions. recombinant viral construct, ptumv-gfp, was transferred into bacterial (escherichia coli dh5α) competent cells (sambrook and russell, 2001). bacteria were grown in 200 ml luria-bertani medium and then, ptumvgfp was extracted (engebrecht et al., 1991). plant rub-inoculation with tumv-derived vector wild-type tumv (for control plants) and ptumvgfp was mechanically inoculated on upper surface of two top leaves (10 µg in 10 µl per leaf), using a cotton stick and carborandum powder according to hosseini et al. (2013). systemically infected (noninoculated leaves) were used for further analysis. total rna extraction and reverse transcription-polymerase chain reaction (rt-pcr) the presence of the gfp gene in inoculated leaves was determined by rt-pcr. total rna was extracted from inoculated and control plant leaves (five independent samples) by qiagene kit (south korea) twelve days after incubation according to the manufacturer’s instruction. rna was extracted from noninoculated leaves for confirming replication and movement of the virus. after treating with dnase i (thermo fisher scientific, usa), cdna was synthesized using the revertaid reverse transcriptase (thermo fisher scientific, usa) and gfp reverse primer (5’ –ttg tac tcc agc ttg tgc cc-3’) according to the producer’s instructions. rt-pcr was conducted using the cdna and the following forward and reverse primers under the following cycling conditions: forward (5’acg acg gca act aca aga cc 3’) and reverse (5’ttg tac tcc agc ttg tgc cc 3’). pcr cycling conditions were as follows: 94°c for 3 min for initial denaturation; 35 cycles of 94°c for 30 s, 51°c for 30s, and 72°c for 30 s; and 72°c for 10 min for a final extension. then pcr products were analyzed by 1% tae agarose gel. fluorescence microscopy leaves from tumv-based vector inoculated and control plants were observed under an olympus fluorescent microscope 6 (version ix71, tokyo, japan). fig. 1 schematic representation of the viral construct containing gfp under the 35s promoter that was used in this expression analysis. the foreign gene insertion site is between nib (nuclear inclusion protein b) and cp (the virus coat protein gene) provided by ncoi and nhei restriction endonuclease enzymes. modarresi et al. heterologous proteins production via viruses 259 the fluorescence photographs were taken using a mounted high-resolutionm7 olympus dp70 dp71 digital camera at 12 days post-inoculation (dpi). protein extraction and gfp analysis proteins were extracted from 0.5g tissues of control and inoculated tobacco leaves (five independent samples) with extraction buffer, including 0.2m trishcl (ph 8.0), 5mm ethylenediaminetetraacetic acid (edta), 100 mm sucrose, and 0.1 mm 2-mercapthoethanol (abdoli-nasab et al., 2013) and the concentration was assessed by bradford’s assays (bradford, 1976). dot-blot (stott, 1989) and indirect enzyme-linked immunosorbent assay (elisa) (wang and gonsalves, 1990) were carried out to quantitative detection of the gfp protein in the inoculated tobacco plants. statistical analysis all experiments were done according to a completely randomized design at five independent samples. data analyses were performed using microsoft excel program software and spss version 22. when significant differences were found least significant difference (lsd) test at p<0.05 was applied to separate means. 3. results and discussion the main benefits of the plant made proteins (pmps) are lower costs and potential to produce a very large scale of recombinant proteins. viral vectors have the ability to express transgenes in hosts and they are suitable and rapid platform for production high-level of recombinant proteins. in this research, we utilized a tumv viral vector (fig. 1) under the control of the camv 35s promoter for transient expression of the gfp in tobacco plants. although systemic symptoms of tumv were not observed on infected plants, gfp was detected by the fluorescence microscopy (fig. 2) twelve days post-inoculation. this research has displayed for the first time that recombinant protein (gfp) can accumulate in tobacco plants via tumv based viral vector with camv 35s promoter. tumv can infect tens different plant species (chen et al., 2007) (to compare common viral vectors which can affected specific plant species), therefore, tumv based viral vector can be economical. in this study, two different tobacco species, n. benthamiana and n. tabacum (two different cultivars xanthi and samsun) were investigated. fluorescence microscopy analysis of gfp (fig. 2), rt-pcr (fig. 3), dot-blot analyses (fig. 4) and elisa assay (fig. 5) indicated that recombinant protein expression in tobacco plants leaves occrued. rt-pcr (fig. 3) showed that, as expected, 160 bp bands were found in infected plants, while not observed in the negative control (wild type). it shows that the tumv virus can infect the plant and replicate its genome. viruses (like tumv from potyviridae family) have developed proteins such as helper component proteinase (hcpro), fig. 2 fluorescence microscopy analysis of gfp expression in tobacco plant's leaves which infected by ptumv-gfp. (a) nicotiana benthamiana, (b) n. tabacum cv. xanthi, (c) n. tabacum cv. samsun (d) negative control (tobacco plant infected with wild-type tumv). green color indicated gfp expression and the red indicated chlorophyll autofluorescence. fig. 3 rt-pcr amplified a 160 bp fragment from the gfp with specific primers in 1% agarose gel. c1= negative control (water template), c2= negative control (rna template), c3= negative control (wild type (non-inoculated plant)), c+= positive control, lane 1= nicotiana benthamiana transformed plants, lane 2= n. tabacum cv. xanthi, lane 3= n. tabacum cv. samsun transformed plants, m= molecular weight marker (1 kb standard generuler). adv. hort. sci., 2019 33(2): 257-262 260 which suppress the plants silencing defense (voinnet, 2001). furthermore, hcpro has protease activity and it is necessary for virus genome replication and viral movement and transmission (klein et al., 1994; chiera et al., 2008). dot-blot assay (fig. 4) indicated that gfp protein was recognized by specific antibody and developed brown color in transformed plants and positive control. elisa assay indicated that expression levels of gfp was estimated approximately ≤0.5% of total soluble protein (tsp) of fresh weight of tobacco leaves. these results show lower accumulation of recombinant proteins compared with a number of previous studies which expressed by viral vectors such as artichoke mottled crinckle virus (lombardi et al., 2009), beet curly top virus (kim et al., 2012) etc. some strategies, such as codon-optimization (love et al., 2012) and the use of improved viral vector elements including strong viral promoters (gleba et al., 2007) can increase the expression of recombinant protein. it seems that, n. benthamiana lacks rna-dependent rna polymerases (rdrps) which required for defense against viruses, therefore n. benthamiana infected plant displays more strong symptoms and its products more than do other tobacco species (yang et al., 2004). in many previous studies (kumar and kirti, 2010; sasaki et al., 2015; vojta et al., 2015; park et al., 2016), rhizobium radiobacter (formerly agrobacterium tumefaciens) delivery systems has been used to express the transient expression of recombinant protein using a viral vector. however, in this study, a direct virus inoculation system via dusted with carborundum has been used. our study indicated that this method is useful to accelerate the production of recombinant proteins in tobacco plants. 4. conclusions in conclusion, our results showed that tumv, as a virus that could infect a wide range of plant species, could be used to produce recombinant proteins in tobacco. in this investigation, all inoculated plants expressed gfp protein. results showed that incubated n. benthamiana has more accumulated recombinant protein compared to the two n. tabacum cultivars. although the level of expression is low and should be optimized for future studies. acknowledgements the authors thank dr. sayed mohsen nassaj hosseini (environmental research institute, acecr, rasht, iran) and mrs. m. azmoodeh (laboratory of biotechnology) for their helpful advice and assistance. we thank dr. shyi-dong yeh for kindly provided tumv-gfp vector. furthermore, we thank the plant breeding and biotechnology department at tarbiat modares university for their support. references abdoli-nasab m., jalali-javaran m., cusidó r., palazón j., baghizadeh a., alizadeh h., 2013 expression of the truncated tissue plasminogen activator (k2s) gene in tobacco chloroplast. mol. biol. rep., 40: 5749-5758. atkinson r.g., gleave l.r., janssen b.j., morris b.a., 1998 post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector. plant j., 15: 593-604. beauchemin c., bougie v., laliberté j.f., 2005 simultaneous production of two foreign proteins from a potyvirus-based vector. virus res., 112: 1-8. boothe j.g., saponja j.a., parmenter d.l., 1997 molecular farming in plants: oilseeds as vehicles 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physiol., 153: 5265. impaginato 333 adv. hort. sci., 2019 33(3): 333-344 doi: 10.13128/ahs-23633 preliminary screening of agricultural feedstocks for anaerobic digestion e. palchetti 1 (*), a. calamai 1, l. verdi 1, a. masoni 1, l. marini 1,, d. chiaramonti 2 1 dipartimento di scienze e tecnologie agrarie, alimentari, ambientali e forestali (dagri), università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. 2 renewable energy consortium for r&d (re‐cord), università degli sudi di firenze, viale morgagni 40, 50134 firenze, italy. key words: agro-industrial residues, amaranthus cruentus, bio methane potential, jatropha curcas l., milk whey, pelargonium graveolens. abstract: the aim of this study is to evaluate the performances in the early stages of biogas production of various unconventional and low inputs crops, such as: kenaf (hibiscus cannabinus l.), amaranthus (amarathus cruentus l.), sorghum (sorghum bicolor l.), and sunflower (helianthus annuus l.). moreover, according to a circular economy approach, that foreseen the re-use of all the materials, a wide range of agro-industrial residues were tested such as: pomace, olive oil cake, cow milk whey, ewe milk whey, beer residues, jatropha (jatropha curcas l.) oil cake and pelargonium (pelargonium graveolens l.) residues after essential oil extraction. the biogas production was estimated starting from the chemical composition of the substrates as well as through tests in bench’s static reactors. the results showed that the use of silage from crops with reduced agronomic requests (kenaf and amaranthus) versus a conventional crop (corn) led to comparable, or even better, biogas production performances during the initial stages. moreover, the performance of some residues from the milk industry allowed to conclude that the ewe milk whey can be considered a booster feedstock for the first phase of digestion. all the tested substrates produced a digestate suitable, according to the italian rules, for soil fertilization or amendment. 1. introduction biogas production from anaerobic digestion (ad) for electricity and heat generation represents a significant and well-established opportunity for farmers in the eu countries thanks to several reasons, such as: large technology availability and versatility, very attractive integration of the system in the agronomic practices and rotations, and the availability of incentives provided by the eu-ms to renewable energy generation. for all these reasons the biogas sector in europe showed a remarkable increase during the last decade, with a global amount of energy produced in 2013 in the eu of 561 x 10-9 gj (eurobserv’er, 2014). in this european context, the italian biogas growth was mostly related (*) corresponding author: enrico.palchetti@unifi.it citation: palchetti e., calamai a., verdi l., masoni a., marini l., chiaramonti d., 2019 preliminary screening of agricultural feedstocks for anaerobic digestion. adv. hort. sci., 33(3): 333-344 copyright: © 2019 palchetti e., calamai a., verdi l., masoni a., marini l., chiaramonti d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 january 2019 accepted for publication 12 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 333-344 334 to the agriculture sector and almost 800 power plants based on agricultural biogas were operating at the end of 2012 with a total capacity of 650 mw (patrizio et al., 2015). the plants are concentrated in the northern part of the country, mainly fed with silage corn and manure, thanks to the livestock sector present there. however the use of corn as feedstock, although it represents one of the most performing biomass in ad (with an average biogas production of 498 m3 t-1 and a content of 53% of methane), exposes the biomass chain to the criticism of food-feed conflict. the largest part of the italian biogas plants are fed with livestock manure and energy crops (62.2%), 17.7% of plants use only livestock manure and 20.1% use only energy crops and cereals (carrosio, 2013). nevertheless, the main constituent of the feedstock recipes is still represented by the dedicated crops (dresseler et al., 2012; bacenetti et al., 2013). for all these reasons, the present work aimed at preliminary testing several unconventional biomass for the production of biogas in comparison with a traditional feedstock such as silage corn. these feedstocks belong to two main groups: dedicated crops, such as corn silage (as control), sorghum silage, kenaf silage, sunflower silage and amaranthus silage; and agro-industrial residues such as pelargonium residues, olive oil cake, jatropha oil cake, pomace, beer thrasher, cow milk whey and ewe milk whey. as regards the agro-industrial residues, some of these have been selected since they are widely available in europe. in fact, they are originated from processes such as olive oil extraction, wine beer and cheese factory, activities very common in this area. concerning the jatropha cake (from jatropha cur‐ cas oil extraction process) and the residues of the essential oil extraction from pelargonium graveolens, these two biomasses, requiring tropical climates, well fit to tropic areas, where short energy chain is often necessary due to the lack of grid connection and where biogas production could represent an interesting energetic chance. the bio-methane potential (bmp) of the selected organic matter can be estimated through chemical analysis followed by the use of algorithms, to approximate the biogas and methane potential yield (buswell and symons, 1993), or assessed by a simulating small scale anaerobic digestion in laboratory, either batch wise or continuous (angelidaki et al., 2009; kowalczyk et al., 2013; edward et al., 2015). the bio-methane potential of a biomass can be estimated from its elemental composition (carbon, oxygen, hydrogen, nitrogen and sulfur content) using the well-known buswell’s formula (1): where represent the molar number of each element. results are calculated under standard conditions, according to eq. (2): where represent the molar number of each element and 22.4 is the coefficient that express the volume of 1 mole of perfect gas at standard conditions (0°c and 1 atm). but several factors influence the estimation of buswell’s formula, such as the particles size, the retention time, the temperature, any unbalance in the biomass recipe, the recalcitrant molecules that might occur in the biomass, etc. and, in order to overcome these difficulties, a wide range of tests has been carried out during the last decades to detect the biomass bio-methane potential production. biomethane potential (bmp) tests are very useful for this scope, as they provide a measure of the maximum amount of biogas or bio-methane produced per gram of volatile solids (vs) contained in the organic feedstock (esposito et al., 2012; thomsena et al., 2014). the bmp methodology requires to control the substrate chemical composition, the operating temperature, the inoculum, the length of the trial and the output characterization (biogas and digestate composition). owens and chynoweth (1993) and angelidaki and sanders (2004) proposed a bmp method based on a batch process, with a good stability due to the equilibrium between the symbiotic growth of the principal metabolic groups of bacteria; the methodology chosen for the present study refers to angelidaki et al. (2009). the use of dedicated crops for the biogas process fuelling is well known and a large amount of data is available in scientific literature since the ‘50s (reinhold and noak, 1956), showing a wide range of methane yield such as: vs 200-450 m3 t-1 ch4 corn, vs 384-426 m3 t-1 ch4 wheat (schievano et al., 2009; wu et al. , 2010), vs 236-428 m3t-1 ch4 sorghum c h o n s n a b c d h o n a b c d       4 2 3 4 2 2 • n a b c d co n a b c d ch c nh d h s 2 8 4 3 8 4 2 8 4 3 8 4 0 2 4 3 2              • • • • ch 4 2 8 4 3 8 4 22 4 12 16 14 32              n a b c d n a b c d • . palchetti et al. ‐ feedstocks for anaerobic digestion 335 (windpassinger et al., 2015), vs 242-324 m3 t-1 ch4 straw, vs 298-467 m3 t-1 ch4 grass (zauner and küntzel, 1986; weiland, 2010; wu et al., 2010), vs 177-400 m3 t-1 ch4 sunflower (schittenhelm, 2010; weiland, 2010) and vs 355-409 m3 t-1 ch4 hemp (heiermann et al., 2009; nges et al., 2012) however, new crops are emerging in recent years (molinuevosalces et al., 2013; mast et al., 2014; corno et al., 2015). examples of crops not yet widely exploited for biogas production are kenaf (hibiscus cannabinus) and amaranthus (amaranthus cruentus). the first one represents a major feedstock for cellulose pulp (nishinoa et al., 2003) or for green building component production (deka et al., 2013) while amaranthus well fit to temperate environments, where short days varieties of tropical origin can enhance the biomass growth rather than the seeds production. there is a common pre-treatment step among different biomass crops: the after harvest silage process. this is essential to simulate the usual practice in actual biogas plants because the process allows the biomass storage for several months. as regards the by-products considered in the present article, no literature is available for geranium (pelargonium graveolens), where leaves remaining after essential oil extraction can be used for ad. similarly, jatropha (jatropha curcas) cake, after oil extraction, could represent an interesting feedstock in some tropical areas. no specific eu regulation or rules are actually available for the use of digestate as fertilizer or amendment, although its potential utilization can also reduce dependence on energy intensive mineral fertilizers, to further mitigate ghg emissions (pöschl et al., 2010). in italy the only limitation is expressed by the decreto del ministero dell’agricoltura 7.4.2006 (mpaf, 2006) stating that if the feedstock is animal manure also the digestate could be considered appropriate as fertilizer, otherwise its utilization is uncertain. recently a new italian rule (legge, 7.10.2012, n. 134) declared that if the digestate is not originated from waste, it can be classified as a byproduct usable in agriculture as soil amendment, but every region applies this rule differently. the use of digestate as fertilizer or soil amendment is validated from several scientific articles (haraldsen et al., 2011; mantovi et al., 2011; chen et al., 2012; garcíasanchez et al., 2015) but only a few works in literature are available regarding the digestate from these still unexploited (in most of the cases) sources. 2. materials and methods feedstock description an experimental trial was conducted using twelve different substrates, some of which were dedicated crop specifically cultivated for this use and silaged after harvest (biomass), while the others residues came from various agricultural activities (agro-industrial by-products). the following table 1 summarizes all the crop type and the origin of the different feedstock tested in the experiment. the crops substrates were corn (zea mays l.), sorghum (sorghum bicolor l.), amaranthus (amaranthus cruentus l.), kenaf (hibiscus cannabinus l.) and sunflower (heliantus annuus l.) cropped in experimental fields in the farm owned by the istituto tecnico agrario (florence, italy) and transformed with the silage technique. a field experiment was carried out in 2012 during april september, in the farm of the university of florence, italy. the seeds for the experimental field were collected from different sources (table 2). the research fields were located at latitude 43°47’07’’ n and longitude 11°13’12’’ e, with an altitude of about 40 m above mean sea level. maximum and minimum temperature were respectively 29.8 and 17°c, with a mean of 24.6°c as recorded by oregon scientific wmr300. the soil texture was sandy-loam (67.6% and 20.7% respectively) with ph of 6.5. no fertilization was planned because the pretable 1 feedstock description no. feedstock name type origin 1 corn green silage italy 2 sorghum green silage italy 3 pelargonium residues from distillation of essential oil, dry madagascar 4 kenaf green silage italy 5 sunflower green silage italy 6 amaranth green silage italy 7 olive cake residues from mechanical oil extraction italy 8 jatropha cake residues from mechanical oil extraction madagascar 9 wine residues residues after fermentation italy 10 cow milk whey residues after cheese production italy 11 ewe milk whey residues after cheese production italy 12 beer residues residues after fermentation italy adv. hort. sci., 2019 33(3): 333-344 336 vious crop insisting on that area was a legume (phaseolus vulgaris l.) and the nitrogen soil content was 1.4%. the experimental scheme adopted was a completely randomized block with 3 repetition and each plot measured 4 x 5 m and was irrigated immediately after seeding, then irrigation plan was not necessary and the rainfall recorded during the cultivation months was 125.47 mm. weed control was done manually in the early stages of plants growing. considering the restricted number of plants, harvest and ensilage were realized manually on 6 september 2012, collected biomass of each crop was chopped by stayer trito 1800 shredder and vigorously compacted into nylon bags. all the bags, hardly sealed, were left for two months in a temperature controlled environment (25°c), to allow the fermentation processes and the conversion of chopped and pressed biomass in silage. after that, samples were collected in vacuum envelopes, by valko favola 310 vacuum packaging machine, with a total amount of 500 g for each crop. the agro-industrial by-products substrates consisted in pomace, olive oil cake, cow and ewe milk whey and beer residues that were obtained directly from agro-industrial districts in the tuscany region, italy (farm, winery, oil mill, dairy). as regards the by products used as feedstock for ad: the ewe whey was collected from “società agricola bacciotti giovanna” an organic farm located in mugello valley into the municipality of scarperia san piero (florence) that produces both the sheep milk and the diary. the ewe whey is the residue of ricotta cheese (ricotta) production, a typical italian unripe cheese variety obtained through heat-induced (85-90°c) coagulation of whey proteins, after addition of acidifying agents. during this phase, coagulated whey proteins are divided by the liquid part that, in this case of study, represents the feedstock used for the biogas production and was collected in 1 dm3 sterile plastic container. the experimental samples of cow milk whey were sampled from “tre p” diary located in mugello valley in the municipality of scarperia san piero (florence) and the milk used was produced by the “emilio sereni” farm located in mugello valley as well, where frisian and pardo-alpina races are grown by organic livestock. the cow milk whey is the liquid residue that separates from the solid mass obtained from the coagulation of casein, during the production of mozzarella cheese. mozzarella cooking is accomplished by melting the curd in hot water (60-85°c) and then working the molten curd by manually stretching and kneading, until the desired texture is achieved. during this step, 1 dm3 of the residual liquid part was collected in sterile plastic container. the olive cake came from the mill “il mandorlo” located in trespiano (florence) where the farm has olives trees grown by organic agriculture. this feedstock represents the ‘cold extraction’ process residue and it is obtained during extra-virgin olive oil production at a temperature below 27°c from mechanical pressing of the olive paste, using a traditional extraction system with hydraulic presses. it is constituted from a mixture of olive endocarp, olive pulp and skin, as well as pomace olive oil plus the water added in the olive mills. the olive varieties used for oil production were frantoio, moraiolo, and leccino. during extra-virgin oil production, 1 kg of olive cake was collected and vacuum-sealed. wine residues came from the “ornina” winery located in castel focognano, arezzo. the winery produces red wine starting from the organic grapes grown in his own vineyards and the varieties cultivated are sangiovese and malvasia nera. winemaking process is carried out in stainless steel tanks during the fermentation step, then, the following maceration of grapes continues for about 10 days. the byproduct of red vinification that was used as feedstock for biogas production, is the “fermented grape marc” composed of skins and seeds: in october 2012, 1 kg of this material was collected and vacuum-sealed. beer residues came from “birra dell’elba” brewery, located in elba island in the municipality of portoferraio. the feedstocks used for the biogas trials is represented by the residues of the mashing step realized to produce unfiltered and not pasteurized beers, with low fermentation temperatures (8-12°c). in october 2012, 1 kg of these residues were collected and vacuum-sealed. pelargonium (pelargonium graveolens) residues after the essential oil extraction and jatropha (jatropha curcas l.) cake were also investigated. both table 2 dedicated crops description species variety name seed provenance zea mays l. cisko syngenta ltd sorghum bicolor l. (moench) bulldozer kws ltd hibiscus cannabinus l. hib 35 ipk seed bank helianthus annuus l. pr64h41 pioneer ltd amaranthus cruentus l. perucho dispaa seed bank palchetti et al. ‐ feedstocks for anaerobic digestion 337 extraction method using a bench komet screw press (model ca59g) with a pressing capacity of 3-5 kg seeds per hour and an electric power of drive motor of 1.1 kw. both jatropha and pelargonium residues were divided in samples of 100 g and the total amount of 500 g for each crop was prepared for the shipping to italy. each sample was collected in a vacuum envelope, by valko favola 310 vacuum packaging machine, to avoid alterations during the transport to the florence laboratory; in addition, the pelargonium residues for ad have been rehydrated adding water 3 days before the beginning of the trial. physical‐chemical and energetic analysis different laboratory equipment and methodologies were utilized for the analysis of substrates that were prepared through a cutting mill (mod. sm 300, retsch). moisture (uni 14774-1:2009) and ash content (uni 14775: 2010) were measured using a leco 701 tga. chnos content was measured using a truespec chn and s (leco) (uni cen/ts 15104; ktbl, 2015). data from these analyses were utilized to calculate the theoretical production of biogas for each substrate according to buswell and symons (1993). the digested sludge was analyzed using the same technologies described above in order to estimate its potential use in agriculture as a soil improver. furthermore the calorific value was measured by isoperibolic calorimeter ac 500, leco. the total solid (ts), the volatile solid (vs) and the ash content of substrates were determined through a stf n-80, falc stove. each sample was weighted and placed in stove for 105°c until a constant weight was reached, after that, the dried material was burnt in a muffle furnace at 550°c and used for the determination of the raw ash content. the organic dry matter was calculated by subtracting the raw ash content from the dry matter. specific biogas and methane yield static reactor description. the static reactor was a 100 cm3 glass vessel hermetically closed and placed on a heating plate connected to a thermocouple to control the constant heating (about 45°c) with a continuous monitoring of the system temperature. 4 repetitions were simultaneously carried out for each substrate. the measurement and temporary storage for the produced biogas consisted in a graduate syringe (30 cm3) placed above the cap with the needle inserted into the rubber membrane and sealed with several layers of parafilm. these crops were produced in the same area of cultivation: south madagascar, ihorombé region, village of satrokala (22°19’39 s, 45°42’54’’ e; altitude 1025 m above sea level), where the research farm is located with a characteristic rainfall of 1200 mm year-1 mostly concentrated between october until march. the samples were collected during the agronomic season of 2012, during that period the mean temperatures followed the rainfall with the higher value during the winter season (max 26.2°c, min 17.2°c) and the lower value during the summer (max 19.7°c, min. 10.1°c). the soil texture is sandy-clay (50.6% and 38.9% respectively) with an average ph value of 5.5 and a general limited mass fraction of dry matter of nitrogen (0.12%) and carbon (1.97%). pelargonium (pelargonium graveolens) was cropped during the agronomic season 2012 2013, from december to april in an open-field plantation in double line with a planting density 50.000 plants ha-1. the cuttings, coming from the farm nursery, were irrigated immediately after transplanting for the proper establishment of the crop in the field and after that the water supply was guaranteed with a dripping irrigation system that supply 4 liters day-1 of water for each plant. only organic manures were applied before the crop installation using cow manure. after 4 months of growing, at the so called balsamic time, the crop was harvested and the essential oil extraction from the biomass was carried out by steam distillation in a 2 tons distillatory plant for 6 hour. the residues coming from this process, roughly 99% of the original biomass (the oil content is approximately 2 g kg-1) is represented by moist leaves and stocks that have been collected and sun-dried for 48 hours before shipping to italy. concerning the jatropha cake, it represents the residual part of the oil extraction process from seeds. the seeds were harvested from a 3 years old plantation of jatropha curcas l. located in the same experimental fields previously described for pelargonium in madagascar. the plantation was established starting from cuttings grown in the farm nursery for 6 months and transplanted in open field during the agronomic season 2009/2010, no irrigation was performed and the amount of 200 g of npk fertilizer (10:10:10) was applied for each plant. the unique agronomical practice performed during the cultivation is the second year pruning at 1 m high. the seed harvest was performed during the dry season of 2012 and the oil extraction was carried out following the mechanical 338 adv. hort. sci., 2019 33(3): 333-344 the ratio between volatile solids (vs) in the substrate and vs in the inoculum was 0.5. the produced amount of biogas was monitored on a daily basis for 10 days and then until there was no more biogas production. regular weekly analysis of inoculum and ph level in the reactors were carried out to evaluate the activity of inoculum and the progress level. the qualitative analysis of biogas production was carried out through samples performed with a glass microsyringe and successive injection in gcms (shimadsu) for the gas chromatography determination. the biogas composition was measured with mass spectrometer gas chromatography gc-ms (gc 2010 and qp 2010, shimadsu). statistical analysis the data generated in the present work belong to three major groups: i) theoretical biogas production with buswell’s formula; ii) biogas production of the different substrates from static digestion; iii) substrate and digestate chemical characterization. each different data was submitted to a specific statistical analysis and therefore: as regards type 1, the accuracy of buswell’s formula was evaluated through the calculation of standard deviation of the delta between the produced and measured biogas, for each substrate. the standard deviation comparison between the substrates was then evaluated with the “chi square” method. anova analysis on type 2 data was performed on the biogas production on three key moments: biogas t1 the biogas production expressed in ml g-1 vs measured at the end of first day of anaerobic digestion; biogas tf the cumulate final biogas production expressed in ml g-1 vs measured at day number 9; ibrt(2-8) increasing biogas rate (ibr) production calculated with the following formula (3): where biogttn represents the biogas production per day. the fixed model of analysis of variance was applied using the statistical software spss ibm, and the significance of the variance was tested with the tukey’s test. regarding the biogas production trend during the digestion process, data were evaluated through the analysis of the regression within each different substrates. in addition, a regression analysis of the whole data set was carried out. the linear and the polynomial regression approximation were calculated and their significance tested according to anova analysis and fisher test. with respect to type 3 data, a simple correlation between the substrate and digestate composition was performed. 3. results and discussion accuracy of buswell estimation of innovative sub‐ strates for ad production based on the chemical composition of the different substrates showed in table 3, the potential biogas yields have been calculated using the buswell’s formula. the buswell estimation was applied to all the feedstock under investigation (table 4), however, it provided acceptable values compared to bibliography (astm, 1998) only for some of these, specifically corn (oslaj et al., 2019), sorghum (wannasek et al., 2017), pelargonium (gamal-el-din et al., 2012, marsili libelli ibr        2 8 1 biogt biogt tn tn table 3 feedstock elemental composition no. feedstock moisture (%) ash (db) (%) c (db) (%) h (db) (%) n (db) (%) s (db) (%) o (db) (%) c/n ratio vs ratio ts (%) 1 corn 81.00 4.31 50.67 6.20 0.83 0.20 37.79 61.05 71.42 19.00 2 sorghum 80.00 7.65 46.46 5.51 0.96 0.17 39.25 48.40 84.61 20.00 3 pelargonium 19.00 10.67 46.46 5.19 1.04 0.20 36.44 44.67 94.00 81.00 4 kenaf 76.01 7.93 57.40 4.06 1.10 0.13 29.38 52.18 97.70 23.99 5 sunflower 78.70 8.50 62.95 3.14 1.10 0.11 24.20 57.23 90.00 21.33 6 amaranth 74.00 5.45 41.40 4.66 1.70 0.20 46.68 24.35 77.80 26.00 7 olive cake 64.60 4.40 63.90 6.88 0.80 0.20 23.82 79.88 95.60 35.40 8 jatropha cake 52.40 1.96 58.55 6.08 3.40 0.20 29.81 17.22 98.00 47.62 9 wine residues 69.02 1.14 32.05 8.13 2.20 0.20 56.29 14.57 96.50 30.98 10 cow milk whey 99.10 0.06 1.13 11.00 0.30 0.00 87.51 3.77 100.00 0.91 11 ewe milk whey 92.50 0.79 3.41 10.70 0.48 0.00 84.62 7.10 93.30 7.56 12 beer residues 72.30 0.91 44.00 6.43 1.80 0.05 46.82 24.44 95.60 27.70 palchetti et al. ‐ feedstocks for anaerobic digestion 339 et al., 2014) and beer residues (maier, 2015; mugodo et al., 2017), with a biogas estimated productions equal to 990.51 m3 t-1, 940.83 m3 t-1, 973.02 m3 t-1 and 829.30 m3 t-1 respectively. for the other silage feedstock, kenaf (saba et al. , 2015), sunflower (dubrovskis et al., 2012; adamovics and dubrovski, 2015; markou et al., 2017) and amaranth (sitkey et al., 2013; minzanova et al., 2018), the buswell’s formula application provides unrealistic values of 1165.39 m3 t-1, 1285.77 m3 t-1 and 818.29 m3 t-1 respectively. similar high values of 1250.31 m3 t-1 and 1117.00 m3 t-1 arise also respectively from the buswell estimation performed among the olive oil cake (tekin and dalgic, 2000; battista et al., 2015; valenti et al., 2017) and jatropha oil cake (staubmann et al., 1997; grimsby et al., 2013; jingura and kamusoko, 2018) while the cow milk whey and ewe milk whey (battista et al., 2015) record limited and unacceptable low value of 21.10 m3 t-1 and 64.16 m3 t-1 respectively. an acceptable (failin and restuccia, 2014; mugodo et al., 2017) level of accuracy could lie on the buswell estimation performed over wine residues with: 829.30 m3 t-1. therefore the reliability of the biogas production methodology among the substrates was evaluated in comparison with the corn silage biogas production observed during the experiment (table 4). a general lower production, when compared to corn silage biogas yield, can be observed among the majority of the feedstock and this reduced production ranges from admissible values of 10.22% and 13.58% of sorghum and olive oil cake respectively, until 81.40% of wine residues. on the contrary, a positive value of 34.60% is observed from the comparison between corn and ewe milk whey, probably due to the booster effect of this special milk whey. this difference is even wider when the ewe milk whey biogas production is compared to the theoretical biogas production of 16.30 m3 t-1 expressed by buswell’s formula application. finally, as regard the kenaf silage, it showed a limited increase of 3.06% in biogas production at day 9, when compared to the corn biogas production. biogas production from static digestion of the differ‐ ent substrates the anova analysis performed on biogas production data showed a significant effect (p<0.001) of the substrate. as regards the biogas(t1) (fig. 1), the substrates that show a significantly higher production at the first day of digestion are sorghum silage, pelargonium residues and beer residues, with a production of 36.75; 35.25 and 35.23 ml g-1 vs biogas respectively. on the opposite, a very limited performance in terms of biogas production was observed for cow milk whey, wine residues, amaranth silage and jatropha oil cake with a production of: 1.73, 9.37, 10.00 and 13.37 ml g-1 vs of biogas respectively. table 4 buswell’s formula estimation, biogas experimental production and comparison with corn’s biogas yield no. feedstock biogas production estimation by buswell’s formula (m3 t-1) trial measurement biogas at day 9 (m3 t-1) biogas yield at day 9 in comparison to corn (%) 1 corn 990.51 359.50 2 sorghum 940.83 322.75 10.22% 3 pelargonium 973.02 217.25 39.57% 4 kenaf 1165.39 370.50 + 3.06% 5 sunflower 1285.77 150.68 58.09% 6 amaranth 818.29 156.14 56.57% 7 olive cake 1250.31 310.67 13.58% 8 jatropha cake 1117.00 222.25 38.18% 9 wine residues 829.3 66.85 81.40% 10 cow milk whey 21.10 245.93 31.59% 11 ewe milk whey 64.16 483.87 + 34.60% 12 beer residues 829.3 248.43 30.90% fig. 1 biogas(t1) experimental production (cm3 g-1 vs) for the different substrates. different letters means significative differences between substrates according to bonferroni multiple comparisons test (p<0.05). adv. hort. sci., 2019 33(3): 333-344 340 among all the remaining substrates, it is possible to identify substrates that produce a large amount of biogas compared to the others such as the corn silage with 31.50 ml g-1 vs of biogas. finally, kenaf silage, olive oil cake, ewe milk whey and sunflower silage generated a reduced quantity of biogas but significantly higher than cow milk whey. the cumulate final biogas production biogas t(f) at the end of day 9 (fig. 2) shows instead a very different behavior. the most productive substrate is the ewe milk whey, with a production of 488.87 ml g-1 vs of biogas, while the smaller amounts of biogas were produced from kenaf silage and corn silage, 370.50 and 359.50 ml g-1 vs biogas respectively. at the opposite, the wine residues were the worst performing substrates with only 66.87 ml g-1 vs biogas, significantly differing from the majority of the substrates tested during the experimental campaign; only amaranth silage and sunflower silage had a statistically similar behavior, with 156.14 and 150.68 ml g-1 vs biogas production respectively. all the other substrates tested show intermediate levels of production: only sorghum silage and olive oil cake were similar to corn silage and kenaf silage. the ibrt(2-8) anova analysis allowed us to identify the cow milk whey (fig. 3) as the substrate having the fastest growing rate of biogas production during the intermediate period of anaerobic digestion (ibr=1.59). a good ibr performance was observed also by ewe milk whey (ibr= 1.48) and jatropha oil cake (ibr=1.41) while the pelargonium residues, with an average value of 1.25, achieve lower ibr value similar to beer and wine residues. observing the overall tendency of cumulate biogas production (fig. 4) with the support of the regression analysis that showed significance for the overall variance of the regression and, when performed within each substrate, for the linear and polynomial regression as shown in table 5, it is possible to make some general assumptions. ewe milk whey substrate showed a cumulate biogas production superior during the entire digestion period, with the exclusion of the first day. the tendency is therefore a fast growth of biogas production during the whole experimental campaign (table 5, figs. 4 and 5). at the opposite, residues showed a limited increase of biogas production during the entire cycle, leading to the lower biogas (fig. 4). kenaf silage, corn silage, sorghum silage and olive oil cake have a similar behavior during the digestion process, with similar final and intermediate biogas yields (fig. 4). more complex trends were observed for the remaining substrates; in effect, it is possible to identify three main behaviors: beer residues and pelargonium residues showed a fig. 2 biogas(t9) cumulate production (cm3 g-1 vs) the end of day 9 for different substrates. different letters means significative differences between substrates according to bonferroni multiple comparisons test (p<0.05). fig. 3 ibrt(2-8) values for different substrates. different letters means significative differences between substrates according to bonferroni multiple comparisons test (p<0.05). fig. 4 overall tendency of biogas production among the different substrates. palchetti et al. ‐ feedstocks for anaerobic digestion 341 common trend until day 5, similar to the other groups, and then diverge, with a reduction in the increasing rate of biogas production. jatropha oil residues and cow milk whey, that generally showed a low level of biogas production, had an initial poor production until day 5 followed by a certain increase during the second period. sunflower silage and amaranth silage showed a limited but continuous increase in biogas production from day 1 to 9. digestate chemical characterization digestate from the ad of different substrates was chemically characterized (table 6). the use of digestate as fertilizer or soil amendment is suggested when an appropriate n content (14%) is linked to a c/n ratio between 10 and 20 (haraldsen et al., 2011; mantovi et al., 2011; chen et al., 2012; garcia-sanchez et al., 2015): for these reasons, all the different tested feedstocks used were reasonably suitable for this purpose. the high c/n ratio of some of them, such as jatropha cake (19.13) and amaranth (17.56), seems very interesting towards microbial processing soil bacteria. 4. conclusions the buswell’s formula effectiveness, estimating the biogas production from various feedstock, was investigated and results of the analysis showed that while it approximates sufficiently well the biogas production rate during the early estimation for conventional feedstock, it does not perform adequately n. substrate linear regression polynomial regression equation r2 (sign) equation r2 (sign) 1 corn silage y = 40.629 x + 7.770 0.976 ** y = 1.57 x2 + 56.35 x 21.03 0.983 ns 2 sorghum silage y = 36.671 x + 8.868 0.919 ** y = 1.33 x2 + 50.03 x 15.63 0.925 ns 3 pelargonium residues y = 23.400 x + 35.389 0.864 ns y = 3.07 x2 + 54.17 x 21.05 0.940 * 4 kenaf silage y = 41.904 x + 22.951 0.954 ns y = 3.82 x2 + 80.18 x 47.25 0.995 ** 5 sunflower silage y = 17.584 x + 4.861 0.947 ns y = 1.33 x2 + 31.15 x 15.52 0.974 * 6 amaranth silage y = 18.43 x + 10.837 0.702 ** y = 0.14 x2 + 16.94 x 8.11 0.702 ns 7 olive oil silage y = 37.249 x + 12.135 0.980 ** y = 1.05 x2 + 47.79 x 31.48 0.984 ns 8 jatropha oil silage y = 29.567 x + 36.483 0.867 ** y = 0.91 x2 + 20.37 x 19.62 0.871 ns 9 wine residues y = 6.833 x + 8.722 0.317 ** y = 0.61 x2 + 12.99 x 2.56 0.330 ns 10 cow milk whey y = 32.300 x + 59.110 0.962 ns y = 2.58 x2 + 6.49 x 11.79 0.990 ** 11 ewe milk whey y = 52.108 x + 76.440 0.843 ns y = 8.36 x2 + 135.72 x 76.83 0.954 ** 12 beer residues y = 27.833 x + 23.194 0.956 ns y = 1.57 x2 + 56.35 x 21.03 0.983 * table 5 linear and polynomial regressions equations. r2 values and significance for the different substrate tested ** significative for p<0.01; * significative for p<0.05; ns= not significative. fig. 5 regression (linear and polynomial) of ewe milk whey, corn silage and pelargonium residues. table 6 digestate elemental composition no. digestate moisture (%) ash (db) (%) c (db) (%) h (db) (%) n (db) (%) s (db) (%) hhv (db) (mj/kg) c/n ratio 1 corn 95.55 0.89 39.23 5.48 3.11 0.33 16.45 12.61 2 sorghum 95.40 0.92 39.25 5.54 2.85 0.31 16.57 13.77 3 pelargonium 95.00 1.00 38.67 5.39 2.73 0.36 16.08 14.16 4 kenaf 95.26 6.40 40.00 5.13 2.72 0.40 16.65 14.71 5 sunflower 94.24 5.20 39.00 5.01 2.61 0.32 16.51 14.94 6 amaranth 94.40 2.00 37.75 5.28 2.15 0.34 15.18 17.56 7 olive cake 94.50 1.80 39.40 5.34 2.87 0.34 16.45 13.73 8 jatropha cake 94.60 4.05 39.60 5.36 2.07 0.31 15.99 19.13 9 wine residues 93.20 4.00 42.97 5.75 2.73 0.32 17.37 15.74 10 cow milk whey 97.30 3.70 34.35 4.75 2.40 0.38 15.10 14.31 11 ewe milk whey 96.90 3.75 36.07 5.03 2.73 0.38 14.87 13.21 12 beer residues 93.50 4.10 41.80 5.55 2.72 0.30 16.87 15.37 adv. hort. sci., 2019 33(3): 333-344 342 when applied to un-conventional substrates, with a general over estimation for the crop silage or crop residue, and with an under estimation for the milk residues. consequently, the application of the buswell formula to test new substrates should be limited to a preliminary survey and coupled with a bmp test. the tests carried out on these new substrates led to several conclusions. the ewe milk whey represents a good booster product in ad and should thus be coupled in a limited percentage with conventional substrates, such as corn silage or similar. the cow milk whey did not perform equally well as booster product, despite it shows the best increasing biogas rate (ibr). corn and sorghum silage showed a common behavior in ad, as expected. kenaf silage gave an interesting and remarkable performance, as this low input crop produces almost the same amount of biogas than corn and sorghum. therefore, kenaf could represent a promising alternative to conventional corn silage. the olive oil cake moreover, with its constant and increasing biogas production over the whole period, could be an alternative to kenaf silage but, due to an expected reduction during the following days after day 9, can be use only in limited percentage. sunflower and amaranth silage, despite a limited biogas production, might represents the most sustainable crops due to their limited requests of water, fertilizers and labor needs. regarding wine and beer residues they both did not perform sufficiently well in anaerobic digestion; 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goiás, goiania, goiás, brazil. 2 federal university of uberlândia, uberlândia, minas gerais, brazil. key words: genetic parameters, processed tomato, solanum lycopersicum. abstract: source of important vitamins, fibers, and minerals, the tomato (solanum lycopersicum l.) stands out in the world agricultural scenario for its economic and social relevance and versatility. the brazilian market is dominat­ ed by multinationals companies, and this market segment obtains cultivars from other countries, with genetics accurate to climatic conditions and cultiva­ tion method very different from those used in brazil. as a result, the local culti­ vation of tomatoes plants becomes dependent on market variations and has required a material that has limited production efficiency. this study aimed to estimate genetic parameters from agronomic traits and to select industrial tomato lines using the selection index. a randomized block experimental design with three replications was used. eighty­five industrial tomato lines from the germplasm bank of the vivati plant breeding ltda were evaluated. each plot had 12 plants. the two central plants of each plot were evaluated. the evaluations were carried out using adapted morphological descriptors described in the guidelines for carrying out the distinguishability, homogeneity, and stability (dhe) tests of the ministry of agriculture, livestock, and supply of brazil (mapa). the genotypic determination coefficient (h2) of the traits related to fruit pericarp thickness, fruit firmness, fruit yield, average cycle, average number of fruits per plant, and soluble solids was high. the base index and the classic index presented the largest gain from selection for the fruit yield trait. rank summation index and genotype­ideotype distance index had the highest total selection gain values. the tomato lines pxt­601 and pxt­610 stood out as superior genotypes by the methods of direct selection and by selection indexes. 1. introduction tomato is grown in different regions of the world and stands out as the most produced vegetable in the world, second only to potatoes in the cultivated area (geraldini et al., 2018). part of the success of tomatoes comes from its diversity in food and nutritional aspects that help human health. the fruit is rich in vitamins a and c and lycopene, substances that help prevent cancer of the gastrointestinal tract (simão and rodriguez, (*) corresponding author: nascimento_mariana1@hotmail.com citation: viera nascimento m., ribeiro ávila m.c., fiori de abreu­tarazi m., oliveia nogueira a.p., cardoso campos l.f., dos reis nasci­ miento a., 2020 ­ identification of promising tomato breeding lines with determinate growth by selection index. ­ adv. hort. sci., 34(3): 337­ 347 copyright: © 2020 viera nascimento m., ribeiro ávila m.c., fiori de abreu­tarazi m., oliveia nogueira a.p., cardoso campos l.f., dos reis nascimiento a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2020 accepted for publication 18 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 337­347 338 2008). brazil is in ninth place in the world tomato pro­ duction ranking. at the top is china, accounting for 31% of production, followed by india with 11%, and the united states with 8% of global production (dossa and fuchs, 2017). in 2017, 36.688 hectares of tomato were grown in brazil, 47.40% of the produc­ tion was destined for fresh consumption, and 52.60% for processing industries (marcomini and molena, 2018). the brazilian national market is dominated by multinationals and acquires imported cultivars, with genetic characteristics adapted to climatic conditions and cultivation systems very different from those found in brazil. as a result, the brazilian cultivation of tomato becomes dependent on market swings. it obtains cultivars with productive potential restricted if compared to the yields reached in the environment that they were developed. also, the plants may suffer losses by climate intolerances and plant diseases, when facing the brazilian growing conditions. due to the economic importance of the crop, tomatoes produced for processing industries have been the focus of research, especially in genetic breeding with the aim of produce cultivars that pos­ sess genes able to assist in the adaptation and toler­ ance to biotic and abiotic stresses, which can result in important contributions to the sector (parmar et al., 2017). in a breeding program, the objective is to enhance the main phenotypic trait and conserve or improve the expression of secondary traits at the same time (nogueira et al., 2012). however, the direct selection of quantitative traits is influenced by the environ­ ment, which may cause unfavorable changes in other traits (vasconcelos et al., 2010). one way to improve this process is to use the simultaneous selection of a group of important agro­ nomic traits, that is, to use the selection indexes. these indexes relate information of different traits and make it possible to perform a selection effective­ ly, which increases the probability of success in a plant breeding program (cruz et al., 2012; vianna et al., 2013; rezende et al., 2014). considering the importance of the industrial pro­ cessing of tomatoes and the market demand for cul­ tivars that meet the requirements of this industrial chain, it is indispensable to know the relationship between agronomic traits and the study of the index­ es. this makes it possible to obtain the best predic­ tion of gains and yields and greater efficiency in the selection process. given the above, this aimed to estimate genetic parameters for agronomic traits and to select industrial tomato lines using the selection index. 2. materials and methods the study was conducted in the experimental area of vivati plant breeding ltda, in abadia de goiás unit, goiás, brazil, at 16°45’26” s, 49°26’15” w, and 898 m of altitude. the climate, according to koppen, is classified as tropical humid, characterized by rainy summer with high temperatures and dry winter, with an average annual rainfall of 1.575 mm. the genotypes analyzed in this study are owned by vivati plant breeding ltda, which use their own selection and maintain methods. the seeds were sown in 450­cell polystyrene trays, filled with a sub­ strate composed of coconut fiber, rice husk, and peat and covered with vermiculite. the trays were kept in a greenhouse for 35 days when the seedlings had from two to three true leaves, and they were able to transplant to the field. the soil preparation was carried out with a tractor and rotary tiller. seedbeds were prepared with 1.0 m wide, 0.20 m high, with 1.0 m spacing between beds. at the transplanting, 1.500 kg ha­1 of the npk formu­ lation 04­30­10 was applied. as topdressing fertiliza­ tion, 20 kg ha­1 of map, 75 kg ha­1 of ammonium sul­ fate, 100 kg ha­1 of ammonium nitrate, and 200 kg ha­ 1 of potassium chloride were divided into four appli­ cations with a 20­day interval after transplanting. the seedlings were manually transplanted to the field 35 days after sowing (das), with 0.40 m between plants and 1.0 m between rows. irrigation was performed by a drip system, supplying the water requirement based on the parameters for crop irriga­ tion management. weed control was performed weekly to avoid competition. insecticide baits were placed through­ out the field to identify the insect infestation rate and help the decision of pesticide application. phytosanitary control was carried out whenever nec­ essary, to maximize fruit production (fao, 2006). the tomato lines were characterized by morpho­ logical traits contemplated in the guidelines for per­ forming the distinguishability, homogeneity, and sta­ bility (dhe) assays by the mapa, which were modi­ fied by the authors. a randomized block experimen­ tal design with three replications was used. eighty­ vieira nascimento et al. ‐ identification of tomato strains in brazil 339 five industrial tomato lines were evaluated. each plot had 12 plants. the two central plants of each plot were evaluated. the descriptors analyzed are shown in table 1. it was estimated the genotypic determination coefficient (h2), according to the estimator below: where: h2 = genotypic determination coefficient; ∅̂ = quadratic genetic component; qmt = mean square of genotypes; qmr = mean square of the residue; and ϒ = number of replications. genotypes were grouped based on the scott­ knott test at the 1% and 5% probability level. subsequently, the selection gains estimates were reached by the aid of the selection index methodolo­ gies cited by cruz (2006): direct and indirect selec­ tion; classic index proposed by smith (1936) and hazel (1943); rank summation index of mulamba and mock (1978); base index of williams (1962); and genotype­ideotype distance index (gid). the selec­ tion criterion applied was to increase the traits: fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), average number of fruits per plant (nfp), and soluble solids (ss). the index proposed by smith (1936) and hazel (1943) was established by the selection index (i) and the genotypic aggregate (h) described below: where: n = number of traits evaluated; b = vector of dimension 1 xn of the selection index weighting coefficients to be estimated; table 1 ­ descriptors for industrial tomatoes (adapted from mapa, 2005) and details on their analysis traits trait description description code comments 01. fruit pericarp thickness slim s the analysis was performed using a digital caliper, measuring the diameter (mm) from the outer wall to the inner wall of the pericarp average a thick t 02. fruit: firmness soft s the analysis was performed by subjecting the fruits to pressure at one point in the middle region, measuring the resistance of the pulp to penetration, using instrutherm model ptr­300 digital penetrometer, and obtaining the values expressed in newton (n) medium m firm f 03. maturation cycle precocious p it was evaluated from the transplanting of seedlings medium m late l 04. yield low l it was determined by the weight and number of fruits per plant average a high h 05. number of fruits per low l it was counted all fruits of each plant, including the green and damaged ones average a high h 06. soluble solids low l the analysis was performed by transferring a drop of the fruit juice to the hanna instruments model hi 96801 digital refractometer prism and then reading it, expressed in °brix average a high h adv. hort. sci., 2020 34(3): 337­347 340 y = nxp dimension matrix (plants) of phenotypic val­ ues of traits; a = is the 1 xn dimension vector of previously estab­ lished economic weights; g = nxp dimension matrix of unknown genetic values of the n traits considered. the vector b = p ­1 ga, where p ­1 is the inverse of the matrix, of dimension nxn of phenotypic variance and covariance between traits. g is the nxn dimen­ sion matrix of genetic variance and covariance between traits. the expected gain for trait j was expressed by: where: ag j(i) = gj (i): expected gain for trait j, with selection based on index i; ds j(i) = selection differential of trait j, with selection based on index i; h2j = heritability of trait j. in the rank summation index of mulamba and mock (1978), the orders of each genotype were summed, resulting in the selection index, as described below: i= r1+r2+....+rn where: i = index value for a given individual or family; rn = an individual’s rank (or rank) from the jth trait; n = number of traits considered in the index. the weights were given by: l=p1r1+p2r2+....+pnrn where: pj = economic weight attributed to the jth trait. for the base index of williams (1962), the follow­ ing index was used as selection criteria: where: y = are the means; a = are the economic weights of the traits studied. for the index of genotype­ideotype distance (cruz, 2006), the mean and maximum and minimum values for each variable were calculated. xij was con­ sidered as the mean phenotypic value of the ith genotype concerning the ith trait. as well, we consid­ ered the value yij representing the transformed mean phenotypic value and cj as a constant relative to the average genotype depreciation. thus, we had: lij as the lower limit to be presented by the geno­ type, relative to the characteristic j, lsj as the upper limit to be presented by the genotype and voj as the optimal value to be presented by the genotype, under selection. if lij <xij <lsj, then yij = xij; if xij <lij, yij = xij + voj ­ lij ­ cj; if xij> lsj, yij = xij + voj ­ lsj + cj. in the methodology, it was considered cj = lsj ­ lij. the cj value ensured that any value of xij within the range of variation around the optimum resulted in a value of yij of magnitude close to the optimal value (voj), as opposed to the values of xij outside this range. thus, the xij transformation was per­ formed to ensure the depreciation of phenotypic val­ ues out of range. the yij values obtained by transfor­ mation were later standardized and weighted by the weights assigned to each characteristic, obtaining the yij values, as described below: where: s (yj) = standard deviation of the mean phenotypic values obtained by the transformation; aj = weight or economic value of the characteristic. then, we calculated the gid index values expressed by the distances between the genotypes and the ideotype, as illustrated: from these indexes, the best genotypes were identified, and the selection gains were calculated. all genetic and statistical analyzes were processed through the computational program in genetics and statistics ­ genes program (cruz, 2016). 3. results and discussion genetic variability was found for all traits by the f­ test at 1% or 5% probability level, which evidenced the ability to perform the selection of superior toma­ to lines. it was verified by values of coefficient of vari­ ation (cv) ranging from 1.36% to 29.03% for mc and nfp, respectively. the highest cv values were observed in trait nfp (29.03%), ss (18.28%), and yld (18.05%) (table 2). the genotypic coefficient of determination (h2) allows us to define the estimate of genetic gain to be achieved and to establish the most appropriate strat­ egy to be used in the breeding program (baldissera et al., 2014). h2 values change according to each charac­ vieira nascimento et al. ‐ identification of tomato strains in brazil 341 teristic and are classified as high when they are high­ er than 0.7 (alvares et al., 2016). the highest h2 values were found for the matura­ tion cycle (71.93%), fruit firmness (58.30%), and yield (51.48%). these values allow us to reach success by the phenotypic selection, which can be proven by the results found in the cvg/cve ratio, which were close to 1.0 for these traits. the lowest h2 values were observed for the number of fruits per plant (29.38%) and soluble solids (33.29%). the medium and high results of the heritability coef­ ficient and coefficient of genetic variation are related to higher selective accuracy, higher genetic variability, and the probability of successfully choosing genotypes with optimal agronomic traits (storck and ribeiro, 2011). the cvg/cve ratio was close to 1.0 only for the table 2 ­ mean square, coefficient of variation, and genetic parameters of agronomic traits and yield of 85 industrial tomato lines fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, mc= maturation cycle, nfp= number of fruits per plant, ss= soluble solids, h2= genotypic coefficient determination, cv= coefficient of variation, cvg= coefficient of genetic variation, cve= coefficient of experi­ mental variation. ** and * significant by f­test at 1% and 5% probability, respectively. source of variation df mean square fpt ff yld cm nfp ss blocks 2 0.65 0.59 488.22 2.89 1663.12 37.75 lines 84 2.08 ** 0.56 ** 698.53 ** 8.16 ** 1664.94 * 0.82 * residue 168 1.35 0.23 338.89 2.29 1175.83 0.55 cv (%) ­ 15.96 16.69 18.05 1.36 29.03 18.28 cvg/cve ­ 0.42 0.68 0.59 0.92 0.37 0.41 h2 ­ 34.84 58.30 51.48 71.93 29.38 33.29 medium cycle. the cvg/cve ratio can be accepted as an indicator of the obtaining of more relevant genet­ ic gains in the selection of superior genotypes (cruz et al., 2012). the constitution of tomato fruits for the industry has been remodeled through genetic improvement, to select cultivars with desirable characteristics for processing. as a general rule, the desired tomato lines are those that combine higher yield with quali­ ty, and that meet the needs of the industry, which currently are firm fruits, with a high content of solu­ ble solids, a shorter cycle, a higher number of fruits per plant and higher fruit pericarp thickness (iglesias et al., 2015; peixoto et al., 2017). fruit pericarp thickness ranged from 5.36 to 9.04 mm (table 3). only 3.7% of the tomato lines had a table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­102 5.5 b 1.79 b 103.79 a 109 b 123.67 a 4.17 a pxt­104 6.55 b 1.98 b 93.96 b 107 b 111 b 4.57 a pxt­106 7.59 a 2.39 b 108.19 a 111 a 130.83 a 3.07 b pxt­107 6.11 b 2.31 b 111.50 a 109 b 109 b 4.07 a pxt­108 7.20 a 2.67 b 123.94 a 110 b 96.33 b 4.13 a pxt­109 7.91 a 2.30 b 85.28 b 110 b 92.33 b 3.77 b pxt­111 7.75 a 2.58 b 68.98 b 107 b 74.83 b 4.43 a pxt­113 7.15 a 3.15 a 85.76 b 112 a 99.17 b 4.77 a pxt­114 7.40 a 2.38 b 72.87 b 110 b 112.83 b 3.03 b pxt­115 8.08 a 2.55 b 86.27 b 107 b 106.00 b 4.20 a pxt­116 7.36 a 1.94 b 117.45 a 113 a 107.17 b 3.67 b pxt­117 8.08 a 2.65 b 119.54 a 112 a 130.83 a 4.40 a pxt­118 6.90 b 2.70 b 110.33 a 110 b 141.17 a 3.77 b pxt­120 7.17 a 2.13 b 114.81 a 113 a 128.17 a 4.73 a pxt­121 6.00 b 2.63 b 115.12 a 112 a 135.17 a 3.17 b pxt­122 7.39 a 2.40 b 106.26 a 113 a 129.33 a 4.10 a pxt­123 8.58 a 2.68 b 103.88 a 112 a 143.33 a 4.30 a pxt­124 6.15 b 2.41 b 113.87 a 113 a 77.67 b 3.87 b pxt­125 5.69 b 3.14 a 93.54 b 113 a 159.00 a 4.80 a pxt­126 6.15 b 3.05 a 115.37 a 109 b 155.83 a 4.83 a to be continued... means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. 342 adv. hort. sci., 2020 34(3): 337­347 table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­401 5.37 b 3.08 a 95.68 b 113 a 173.83 a 3.60 b pxt­402 5.90 b 2.74 b 107.19 a 112 a 139.00 a 4.57 a pxt­403 6.37 b 3.33 a 122.89 a 112 a 146.00 a 4.00 b pxt­404 6.74 b 2.11 b 118.68 a 113 a 93.67 b 3.37 b pxt­405 5.50 b 2.88 b 117.25 a 112 a 115.50 b 4.30 a pxt­406 6.37 b 3.30 a 100.62 a 110 b 156.17 a 4.40 a pxt­407 6.43 b 2.85 b 103.61 a 111 a 99.33 b 3.80 b pxt­408 6.01 b 2.62 b 90.30 b 112 a 126.00 a 4.87 a pxt­409 6.03 b 3.22 a 99.00 b 111 a 100.33 b 4.87 a pxt­410 7.42 a 2.80 b 106.20 a 112 a 112.00 b 4.63 a pxt­411 7.81 a 2.46 b 101.26 a 113 a 96.83 b 4.00 b pxt­412 7.58 a 2.65 b 110.89 a 113 a 80.92 b 4.63 a pxt­413 7.95 a 3.12 a 111.25 a 113 a 116.83 b 3.10 b pxt­501 9.04 a 2.85 b 90.56 b 110 b 120.00 a 4.63 a pxt­502 8.44 a 3.35 a 116.72 a 110 b 101.67 b 3.57 b pxt­503 7.72 a 3.35 a 100.34 a 113 a 124.67 a 4.17 a pxt­504 7.10 a 3.48 a 95.05 b 112 a 122.83 a 4.17 a pxt­505 7.44 a 2.84 b 113.92 a 108 b 127.00 a 4.37 a pxt­506 6.79 b 3.06 a 114.68 a 111 a 110.83 b 3.97 b pxt­551 6.66 b 2.97 a 95.56 b 112 a 128.33 a 4.07 a pxt­552 7.16 a 2.68 b 112.40 a 111 a 109.00 b 3.47 b pxt­553 6.92 b 2.99 a 112.46 a 111 a 146.83 a 3.63 b pxt­554 8.69 a 2.67 b 83.15 b 112 a 69.50 b 4.47 a pxt­555 6.96 b 2.65 b 104.99 a 108 b 154.17 a 4.10 a pxt­556 8.49 b 3.06 a 101.46 a 109 b 91.83 b 4.63 a pxt­557 6.92 b 3.08 a 104.24 a 112 a 96.67 b 3.60 b pxt­558 7.53 a 3.69 a 102.49 a 109 b 164.00 a 4.23 a pxt­559 7.31 a 3.77 a 103.30 a 112 a 98.76 b 3.26 b pxt­560 6.88 b 2.50 b 109.27 a 109 b 93.00 b 4.50 a pxt­561 8.22 a 2.70 b 85.59 b 109 b 102.50 b 3.63 b pxt­562 7.73 a 2.88 b 102.51 a 110 b 94.00 b 5.03 a pxt­563 7.22 a 3.05 a 111.74 a 113 a 90.50 b 4.07 a pxt­564 6.95 b 2.50 b 131.20 a 113 a 98.33 b 3.26 b pxt­565 7.12 a 3.07 a 104.29 a 111 a 114.83 b 4.10 a pxt­566 8.44 a 3.38 a 114.83 a 111 a 128.17 a 4.43 a pxt­567 8.35 a 2.84 b 91.36 b 112 a 148.83 a 3.83 b pxt­568 7.23 a 3.42 a 105.30 a 111 a 122.50 a 3.80 b pxt­569 8.06 a 2.87 b 76.93 b 112 a 121.83 a 4.13 a pxt­570 8.02 a 3.34 a 88.84 b 113 a 139.33 a 3.40 b pxt­571 7.23 a 2.80 b 90.29 b 112 a 94.17 b 3.96 b pxt­572 8.14 a 2.76 b 83.28 b 110 b 104.33 b 4.76 a pxt­601 6.4a b 3.31 a 137.92 a 112 a 154.66 a 4.26 a pxt­602 7.59 a 2.76 b 122.97 a 110 b 114.17 b 5.26 a pxt­603 6.19 b 3.08 a 98.68 b 112 a 136.67 a 3.66 b pxt­604 7.60 a 2.98 a 117.62 a 109 b 105.33 b 3.37 b pxt­605 7.82 a 2.94 a 77.11 b 111 a 116.17 b 3.60 b pxt­606 7.67 a 2.82 b 99.15 b 112 a 144.50 a 4.03 a pxt­608 8.82 a 2.79 b 93.17 b 110 b 84.33 b 3.03 b pxt­609 7.29 a 2.78 b 97.48 b 109 b 104.17 b 3.77 b pxt­610 7.69 a 3.08 a 145.98 a 113 a 132.67 a 4.27 a means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. to be continued... vieira nascimento et al. ‐ identification of tomato strains in brazil 343 high thickness of the pericarp. according to vieira et al. (2019), the thickness of the pericarp, together with the resistance of the epidermis and the texture of the placenta tissue, influences the firmness of the fruit (the relationship between the volume of the pericarp and volume of the locular material). only 3.7% of the tomato lines had high values of fruit firmness. firmer fruits present less degradation of the cell wall and increase the resistance of the fruits during the transport process. the fruit firmness ensures resistance to mechanical damage during mechanized harvesting and bulk transport. fruits that are not firm are more susceptible to the transforma­ tion and breakage of the skin, releasing cellular juice and causing fermentation and deterioration of the fruits before the arrival in the industry (vieira et al., 2019). the fruit firmness is extremely important for the industry, because, between the harvest and the unloading process in the industry, there are many losses, due to a large number of disintegrated fruits, related to excessive compression (moura and golynski, 2018). one of the main characteristics to be used in the selection of the ideal genotype for the tomato pro­ cessing industry and mainly for the producers is fruit yield. among the tomato lines evaluated, again, 3.7% of them obtained high values, above 131 t ha­1. the average yield of the state of goiás, where the tomato lines were evaluated, were 85 and 94 t ha­1 in the 2017 and 2018 harvests, respectively (globo rural, 2018). the average cycle ranged from 106 to 113 days. only 5.88% of tomato lines evaluated had a short cycle. most cultivars marketed by seed companies have a cycle between 95 and 125 days (kelley et al., 2010), which demonstrates that all tomato lines eval­ uated are classified between the short and middle cycles. the use of short­cycle genotypes is desirable in breeding programs, as it allows for a shorter stay in the field, where they will be subject for a shorter time to effects of biotic and abiotic factors such as disease and drought stress (gatut­wahyu et al., 2014). the number of fruits per plant ranged from 69.50 for pxt­554 to 173.83 for pxt­401. cultivars with a low number of fruits per plant are not recommended because they have lower yield during the harvesting process (santos, 2015). high soluble solids content is one of the main characteristics that an industrial tomato material must­have. according to figueiredo et al. (2015), the higher the soluble solids content, the higher the effi­ ciency of industrial production, and the lower the energy expenditure during the pulp concentration procedure. in practice, for each addition of a °brix in the pulp, there is a 20% increase in industrial produc­ tion. values above 4.5°brix are higher than the brazilian average. among 85 tomato lines evaluated, 23.17% is above this value, reaching the maximum value of 5.23 °brix. table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­611 7.94 a 2.84 b 104.42 a 113 a 110.67 b 4.00 b pxt­613 7.75 a 3.03 a 92.45 b 109 b 106.50 b 4.40 a pxt­614 7.46 a 3.31 a 99.55 b 112 a 107.00 b 3.50 b pxt­615 7.37 a 2.67 b 83.69 b 110 b 79.33 b 3.73 b pxt­616 7.69 a 3.91 a 98.75 b 111 a 122.50 a 4.87 a pxt­617 8.27 a 3.04 a 85.38 b 113 a 92.83 b 3.97 b pxt­618 8.68 a 3.04 a 114.23 a 110 b 96.33 b 4.57 a pxt­619 6.61 b 3.57 a 97.11 b 111 a 152.00 a 4.17 a pxt­651 7.45 a 3.37 a 56.63 b 109 b 125.83 b 3.60 b pxt­652 8.16 a 4.10 a 74.64 b 109 b 157.67 a 3.57 b pxt­653 8.39 a 3.31 a 82.29 b 112 a 103.83 b 2.93 b pxt­654 6.84 b 3.43 a 84.09 b 111 a 166.17 a 3.87 b pxt­655 7.55 a 2.35 b 108.04 a 109 b 124.17 a 4.27 a pxt­656 6.90 b 3.43 a 114.19 a 110 b 149.50 a 3.90 b pxt­687 6.70 b 2.95 a 98.99 b 109 b 113.83 b 4.77 a means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. adv. hort. sci., 2020 34(3): 337­347 344 direct selection resulted in higher individual gains (table 4). this selection is directed only for one trait of interest and comprises the obtention of maximum gains of a single trait for which selection is practiced. according to how this trait is associated with others, favorable or unfavorable results may occur in traits of secondary importance (cruz, 2016). direct selection for fpt, nfp, and ss resulted in direct gains for fruit firmness, with values of 1.97%, 8.69%, and 2.93%, respectively. noteworthy was the direct selection for the number of fruits per plant, which resulted in the largest indirect gain for fruit firmness. the indexes of selection consist of an alternative that allows the simultaneous selection to perform effectively by combining different traits (rosado et al., 2012). in general, the index of the rank summa­ tion index of mulamba and mock (1978) showed the largest gain of yield (7.89%) and soluble solids (4.02%), followed by the smith (1936) and hazel (1943) index, with 7.20% of the gain of yield. however, these two indexes had low selection gain values for the other traits (table 5). the rank summation index of mulamba and mock (1978) had the highest gain for all the traits and the highest total gain, with values of 22.92%. the geno­ type­ideotype distance index obtained the second­ highest total gain value, with 22.54%. these indices presented a balanced distribution of selection gains. in the research carried out by rosado et al. (2012), the authors reported that the rank summation index of mulamba and mock (1978) was the most appropri­ ate, allowing for a balanced distribution of selection gains for a larger number of yellow passion fruit progenies. the top ten genotypes, selected by all selection methods used in this study and their values of fruit pericarp thickness (table 6), fruit firmness (table 7), yield (table 8), number of fruits per plant (table 9), and soluble solids (table 10) are shown in the tables below. the lines pxt­601 and pxt­610 were selected in all selection methods applied, verifying the superi­ ority of these genotypes. 4. conclusions the rank summation index of mulamba and mock (1978) and the classical index proposed by smith (1936) and hazel (1943) applied to agronomic traits of eighty­five industrial tomato lines turned out to the largest selection gain for the yield trait. rank summation index of mulamba and mock (1978) has the highest total genetic gain values. the lines of tomato pxt­601 and pxt­610 stand out as superior genotypes by the direct selection method and selection indexes. acknowledgements to vivati plant breeding ltda, for support in con­ ducting the project and providing access to the germplasm bank, and to cnpq, for the master’s scholarship granted to the first author. table 4 ­ genetic gain estimates obtained for five traits evaluat­ ed by direct and indirect selection for 85 industrial tomato lines traits genetic gain (%) fpt ff yld nfp ss fpt 6.21 0.43 ­0.43 ­3.25 ­1.94 ff 1.97 14.41 ­3.34 8.69 2.93 yld ­1.41 ­1.51 12.05 ­0.67 ­1.47 nfp ­2.32 5.05 ­0.05 10.26 0.2 ss ­0.12 ­0.23 ­0.44 1.06 6.81 total 4.33 18.15 7.79 16.09 6.53 fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, nfp= number of fruits per plant, ss= soluble solids. table 5 ­ genetic gain estimates obtained for five traits by selection by the classical index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index for 85 industrial tomato lines fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, nfp= number of fruits per plant, ss= soluble solids. traits genetic gains (%) smith (1936) and hazel (1943) mulamba and mock (1978) williams (1962) genotype­ ideotype distance fpt ­4.55 1.88 ­2.98 2.31 ff 5.34 5.12 5.83 7.07 yld 7.20 7.89 6.71 6.69 nfp 7.22 4.01 8.77 4.12 ss 0.29 4.02 0.57 2.35 total 15.50 22.92 18.90 22.54 vieira nascimento et al. ‐ identification of tomato strains in brazil 345 table 6 ­ fruit pericarp thickness (fpt) in mm from ten superior genotypes selected by direct selection for fruit pericarp thickness, and classic index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index (gid) selection indexes williams (1962) and direct selection of fruit pericarp thickness smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines fpt lines fpt lines fpt lines fpt pxt­601 6.44 pxt­601 6.44 pxt­566 8.44 pxt­566 8.44 pxt­610 7.69 pxt­403 6.37 pxt­610 7.69 pxt­558 7.53 pxt­126 6.15 pxt­610 7.69 pxt­558 7.53 pxt­616 7.69 pxt­403 6.37 pxt­126 6.15 pxt­616 7.69 pxt­601 6.40 pxt­558 7.53 pxt­401 5.37 pxt­601 6.44 pxt­117 8.08 pxt­401 5.37 pxt­656 6.90 pxt­117 8.08 pxt­656 6.90 pxt­656 6.90 pxt­405 5.50 pxt­126 6.15 pxt­610 7.69 pxt­555 6.96 pxt­121 6.00 pxt­602 7.59 pxt­123 8.58 pxt­553 6.92 pxt­406 6.37 pxt­618 8.68 pxt­503 7.72 pxt­406 6.37 pxt­619 6.61 pxt­123 8.58 pxt­618 8.68 table 7 ­ fruit firmness (ff) in newton from ten superior genotypes selected by the direct selection for fruit firmness, and classic index proposed by smith (1936) and hazel (1943), rank index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index (gid) selection indexes williams (1962) and direct selection of fruit firmness smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines ff lines ff lines ff lines ff pxt­601 3.31 pxt­601 3.31 pxt­566 3.38 pxt­566 3.38 pxt­610 3.08 pxt­403 3.33 pxt­610 3.08 pxt­558 3.69 pxt­126 3.05 pxt­610 3.08 pxt­558 3.69 pxt­616 3.91 pxt­403 3.33 pxt­126 3.05 pxt­616 3.91 pxt­601 3.31 pxt­558 3.69 pxt­401 3.08 pxt­601 3.31 pxt­117 2.65 pxt­401 3.08 pxt­656 3.43 pxt­117 2.65 pxt­656 3.43 pxt­656 3.43 pxt­405 2.88 pxt­126 3.05 pxt­610 3.08 pxt­555 2.65 pxt­121 2.63 pxt­602 2.76 pxt­123 2.68 pxt­553 2.99 pxt­406 3.30 pxt­618 3.04 pxt­503 3.35 pxt­406 3.30 pxt­619 3.57 pxt­123 2.68 pxt­618 3.04 table 8 ­ yield (yld), in mg ha­1, of ten superior genotypes selected by direct selection for yield, and classic index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ ideotype distance index (gid) selection indexes williams (1962) and direct selection of yield smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines yld lines yld lines yld lines yld pxt­601 137.92 pxt­601 137.92 pxt­566 114.83 pxt­566 114.83 pxt­610 145.98 pxt­403 122.89 pxt­610 145.98 pxt­558 102.49 pxt­126 115.37 pxt­610 145.98 pxt­558 102.49 pxt­616 98.75 pxt­403 122.89 pxt­126 115.37 pxt­616 98.75 pxt­601 137.92 pxt­558 102.49 pxt­401 95.68 pxt­601 137.92 pxt­117 119.54 pxt­401 95.68 pxt­656 114.19 pxt­117 119.54 pxt­656 114.19 pxt­656 114.19 pxt­405 117.25 pxt­126 115.37 pxt­610 145.98 pxt­555 104.99 pxt­121 115.12 pxt­602 122.97 pxt­123 103.88 pxt­553 112.46 pxt­406 100.62 pxt­618 114.23 pxt­503 100.34 pxt­406 100.62 pxt­619 97.11 pxt­123 103.88 pxt­618 114.23 adv. hort. sci., 2020 34(3): 337­347 346 references alvares r.c., silva f.c., melo l.c., melo p.g.s., pereira h.s., 2016 ­ estimation of genetic parameters and selection of high‐yielding, upright common bean lines with slow seed‐coat darkening. ­ genet. mol. res., 15(4), 1­10. baldissera j.n.c., valentini g., coan m.m.d., guidolin a.f., 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res., 8(30): 4162­4169. vieira d.a.p., caliari m., souza e.r.b., soares júnior m.s., 2019 ­ mechanical resistance, biometric and physicochemical characteristics of tomato cultivars for industrial processing. ­ food sci. technol., 39(2): 363­ 370. williams j.s., 1962 ­ the evaluation of a selection index. ­ biometrics, 18(3): 375­393. impaginato 227 adv. hort. sci., 2019 33(2): 227-233 doi: 10.13128/ahs-23812 foliar application of asparagine and casein on biochemical and morphological attributes of garden cress (lepidium sativum l.) under greenhouse conditions a. jorkesh 1 (*) , m.h. aminifard 2 1 department of horticulture, faculty of agriculture, university of guilan, rasht, iran. 2 department of horticulture science, special plants regional research centre, faculty of agriculture, university of birjand, iran. key words: foliar spray, nitrogen content, phosphorus content, vegetative stage. abstract: in this study, the effect of foliar application of asparagine (asn) and casein (csn) during vegetative stage at four rates (0, 50, 100 and 150 mg l-1) was investigated on garden cress (lepidium sativum l.). the results showed that asparagine application, especially at a high level, could significantly increase the morpho-physiological traits such as plant height, leaf and stem fresh weights and leaf and stem dry weights, leaf pigments (chlorophyll a and chlorophyll b) and leaf nutrients content (nitrogen and phosphorus). also, the results indicated that casein application at 50 mg l-1 rate had the best performance through in stem and root fresh weights, stem dry weight and diameter of main stem traits. casein application at rate 100 mg l-1 had the highest leaf nitrogen and phosphorus content. generally, our findings suggest that the use of asparagine and casein can be considered as an appropriate growth regulator in garden cress cultivation. 1. introduction garden cress (lepidium sativum l.) is an edible herb and a member of the cruciferae (brassicaceae) family. it is commonly cultivated throughout the temperate regions of india and pakistan (nadkarni, 1954). the plant is cultivated as culinary vegetable all over asia (nadkarni, 1976). garden cress is an annual standing plant, growing up to 30 cm. it is a well known cookable herb and the leaves are widely used as a garnish in salads. in addition to its leaves that have medicinal properties, the seeds are aperients, diuretic, tonic, demulcent, aphrodisiac, carminative (chopra et al., 1986). moreover, the seeds, which are used in folk therapies, have many activities like thermogenic, depurative, rubefacient, tonic, aphrodisiac, abortive, ophthalmic, diuretic (gokavi et al., 2004; dugasani et al., 2009). intensive farming practices, which produce high yields and quality, require the extensive use of chemical fertilizers that are both costly and (*) corresponding author: a.jorkesh@gmail.com citation: jorkesh a., aminifard m.h., 2019 foliar application of asparagine and casein on biochemical and morphological attributes of garden cress (lepidium sativum l.) under greenhouse conditions. adv. hort. sci., 33(2): 227-233 copyright: © 2019 jorkesh a., aminifard m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 august 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 227-233 228 create environmental problems. therefore, there has been a recent resurgence of interest in environmentally friendly, sustainable and organic agricultural practice (orhan et al., 2006). thus, it is necessary to supply the plant requirement to nutrient through proper procedure. there are different ways to supply plant nutrient’s requirement such as soil feeding and foliar application. of these, foliar feeding is an effective method for improving soil deficiencies and overcoming the soils inability to transfer nutrients to the plant. it has reported the foliar feeding can be 8 to 10 times more effective than soil feeding and up to 90% of a foliar fed nutrient solution can be found in the smallest root of a plant within 60 minutes of application (garcia and hanway, 1976). amino acids are the major building element for proteins (andrews, 1986). amino acids is a wellknown biostimulant which has positive influence on plant growth, yield and significantly decrease the damages caused by abiotic stresses (kowalczyk and zielony, 2008). asn is widely used as a source of organic nitrogen in the media upon which certain bacteria are grown (long and seibert, 1926). asn is thought to play a clearly important role in the transportation and storage of nitrogen (lehmann and ratajczak, 2008), because of their relatively stable nature and high n⁄c ratio (ireland and lea, 1999; masclaux-daubresse et al., 2006). in plants, asn, one of the most prevalent amides, has been reported to be the primal source of nitrogen for protein synthesis, particularly in actively growing tissues (brouquisse et al., 1992). asn aggregation in plants in response to environmental stress could be an ammonium detoxification mechanism and a means to stock up nitrogen when protein synthesis is impaired in plants due to stressful environments (herrerarodríguez et al., 2007). csn is a very rich source of essential amino acids (sarode et al., 2016). there are four different csn proteins (αs1, αs2, β and k), which are different in their amino acid composition (dalgleish, 1989). among proteins, csn has been vastly used in artificial rearing diets because it contains all the essential amino acids, is soluble in water, and does not coagulate or precipitate after heating (parra, 1979). csn also contains important substances such as fatty acids, cholesterol, sugars, vitamins, and minerals (vanderzant, 1974). csn contains 0.7-0.9% phosphorus. so, csn is known as a phosphoprotein (sarode et al., 2016). several pre-harvest factors like climatic conditions and available nutrients can influence on yield and quality of vegetables (lee and kader, 2000). in spite of wide rang of properties, there are a few study about the effect of amino acids on medicinal plants. so this study was conducted to evaluate the effects of asn and csn foliar application on vegetative and reproductive growth, physiological and biochemical traits of garden cress. 2. materials and methods in order to investigate the response of lepidium sativum to foliar application of asparagine (asn) and casein (csn) two separate pot experiments were conducted at research greenhouse of university of guilan, iran. the experiments were carried out based on two randomized complete design with three replications. the treatments in this study were different levels of asn and csn (0, 50, 100 and 150 mg l-1). foliar application of experimental solutions was started from the four-leaf stage and done once every two weeks in the morning, up to the early may. in each spraying date, 20-25 ml of corresponding solution was applied per pot. garden cress seeds which were prepared from isfahan-pakanbazr company were planted in 6th january. the sowing was done in pots with the 33 cm diameter and 22 cm height. the seed bed was a mixture of soil, cow manure and sand with the ratio of 2:1:1. the main properties of soil used in the pots are shown in table 1. except for cow manure, no fertilizer was used during the plants growth cycle. plants were irrigated every 10 days. plant thinning was performed 15 days after emergence, so that, 6 plants were kept in each pot. during plant growth the greenhouse temperature was 25˚c on the day and 15°c during the nights. in addition, co2 concentration was 350 ppm, relative humidity was 40% and photoperiod adjusted as 16 hours’ light and 8 hours of darkness. organic carbon (%) nitrogen (%) calcium (%) phosphorous (%) potassium (%) ph texture 1.2 1 0.48 0.19 0.55 7.1 loam sandy table 1 some chemical indices of soil used in pots for garden cress cultivation jorkesh and aminifard foliar application on lepidium sativum under greenhouse conditions 229 in 4th may, four plants were lifted form the soil in each pot for determination of some aboveand below-ground vegetative indices. the measured indices were plant height, leaf weight, root weight, stem weight and main stem diameter (by a ruler). the remained two plants were used to measure some reproductive traits including number of florets. the pigments content (carotenoid, chlorophyll a and chlorophylls b) was measured by the method has been explained by minguez-mosquera and prezgalvez (1998). to determine the leaf nitrogen, phosphorous and calcium percentages, the method of jones (2001) was used. the method of bakhshi and arakava (2006) also was used for the extraction of leaf extract to determine phenolic compounds and antioxidant capacity. then, phenols were analyzed by using the method of folin-ciocalteau reported by tavarini et al. (2008). for this purpose, the absorbance at 760 nm was measured using a spectrophotometer (t80+pg instrument uv/vis spectrometer) and the values were expressed as mg gallic acid/100 g fresh weight. antioxidant capacity was determined using dpph free radical scavenging method which has been described by sanchez-moreno et al. (1999). finally, data analysis was done using sas 9.2 software and means were separated via tukey’s (honest significant difference) test at the 5% level of probability. 3. results and discussion vegetative and reproductive growth the analysis of variance of the investigated morphological traits was presented in table 2. the results revealed that both asn and csn significantly affected the plant length, leaf and stem fresh weight, leaf and stem dry weight. so that, the greatest plant height was observed at a rate of 150 mg l -1 of asn. moreover, applying asn at rate of 100 mg l-1 had the highest leaf and stem fresh weights and leaf and stem dry weights. casein application significantly affected the stem and root fresh weights, stem dry weight and diameter of main stem (table 2). so that, the plants grown under csn spray at rate of 50 mg l-1 had the best performance due to stem dry and fresh weight, root fresh weight and main stem’s diameter. asparagine and csn foliar spraying could improve the morphological characteristics in garden cress. these results are in agreement with the previously studies by kaya et al. (2013) on maize, rasmia et al. (2014) on palm, saeed et al. (2005) on soybean, akladious and abbas (2013) and el-desouky et al. (2011) on tomato, shafeek and helmy (2012) on onion. also, el-zohiri and asfour (2009) on potato found that spraying of amino acids at 0.25 ml/l significantly increased vegetative growth expressed as plant height and dry weight of plant. their finding indicated that amino acids could improve the vegetative and reproductive traits in plants. these ability my be due to their important role in plant metabolism and protein assimilation which is necessary for cell formation and consequently increase the fresh and dry matter (fawzy et al., 2012). amino acids contribute to the synthesis of growth hormones; therefore, it can be concluded that an increase in cell division and cell enlargement is the reason behind enhanced growth parameters (shafeek and helmy, 2012). the positive effect of amino acids on growth was stated by goss (1973) who indicated that amino acids can serve as a source table 2 mean squares for the effect of different levels of aspargine (asn) and casein (csn) on vegetative and reproductive indices of garden cress traits source of variation treatment error cv asn csn asn csn asn csn plant height 24.22 * 5.86 ns 3.75 10.41 5.75 10.38 leaf fresh weight 0.66 * 1.008 ns 0.09 0.25 5.09 7.97 leaf dry weight 0.27 ** 0.04 ns 0.03 0.02 11.69 12.28 root fresh weight 0.17 ns 0.2 ** 0.06 2 8.52 5.22 root dry weight 0.002 ns 0.004 ns 0.01 0.005 8.54 5.94 stem fresh weight 2.65 ** 2.001 ** 0.04 0.16 3.62 6.83 stem dry weight 0.5 ** 0.22 * 0.02 0.04 9.66 14.13 diameter of main stem 0.04 ns 0.11 * 0.05 0.01 10.98 5.87 number of florets 0.22 ns 0.75 ns 0.66 0.5 11.66 10.47 ns, *, ** non-significant, significant at 5%, and 1% probability level, respectively. adv. hort. sci., 2019 33(2): 227-233 230 of carbon and energy when carbohydrates become deficient in the plant’s releasing the ammonia and organic acid form which the amino acid was originally formed (table 3). the organic acids then enter kerb’s cycle, to be broken down to release energy through respiration (goss, 1973). serna et al. (2012) found that the spray of pepper plants with a mixture of amino acids led to raise the efficiency of photosynthesis, and thus, give the best vegetative growth. biochemical traits and nutrients content the mean squares of physiological and mineral content are presented in table 4. total phenolic compounds and antioxidants activity were not affected by foliar spraying of asn and csn (table 5). chlorophyll a and b content significantly increased in response to foliar asn treatments (table 4). the highest contents of chlorophyll a and b observed in plants grown under spraying of asn at rate of 100 table 3 means comparison for the effect of different levels of asparagine and casein on some vegetative and reproductive growth parameters of garden cress means with the same letter(s) within a column are not significantly different (p≤0.05) based on tukey's test. table 4 mean squares for the effect of different levels of aspargine (asn) and casein (csn) on leaf nutrient and pigments content and some biochemical parameters in garden cress ns, *, ** non-significant, significant at 5%, and 1% probability level, respectively. traits source of variation treatment error cv asn csn asn csn asn csn chlorophyll a 0.13 ** 31.66 ns 0.006 0.008 4.9 6.75 chlorophyll b 0.05 ** 0.01 * 0.001 0.002 5.83 7.89 carotenoid content 0.0004 ns 0.003 ns 0.001 0.002 8.32 12.96 leaf nitrogen content 0.09 ** 0.18 ** 0.006 0.01 4.79 6.72 leaf phosphorous content 0.01 * 0.03 ** 0.003 0.002 8.86 6.66 leaf calcium content 0.007 ns 0.0007 ns 0.007 0.003 9.45 6.95 total antioxidants 21.41 ns 6.97 ns 12.75 6.16 6.64 4.7 total phenol 31.66 ns 6.97 ns 7.33 17.33 4.99 7.81 treatments (mg l-1) plant height (cm) leaf fresh weight (g plant-1) leaf dry weight (g plant-1) root fresh weight (g plant-1) root dry weight (g plant-1) stem fresh weight (g plant-1) stem dry weight (g plant-1) diameter of main stem (mm) number of florets aspargine 0 29.66 b 5.47 b 1.18 b 2.87 a 1.20 a 4.77 c 1.15 c 1.99 a 7.00 a 50 34.66 ab 6.05 ab 1.81 a 2.97 a 1.25 a 5.73 b 1.44 bc 2.08 a 6.66 a 100 34.00 ab 6.57 a 1.83 a 3.42 a 1.26 a 6.96 a 2.06 a 2.25 a 7.00 a 150 36.33 a 6.31 a 1.62 ab 3.12 a 1.26 a 6.38 ab 1.86 ab 2.02 a 7.33 a casein 0 29.66 a 5.47 a 1.18 a 2.87 b 1.2 a 4.77 b 1.15 b 1.99 b 7.00 a 50 31.00 a 6.75 a 1.45 a 3.39 a 1.22 a 6.64 a 1.80 a 2.37 a 6.33 a 100 30.66 a 6.23 a 1.38 a 2.82 b 1.15 a 6.22 ab 1.58 ab 2.01 b 6.33 a 150 33.00 a 6.65 a 1.43 a 3.14 ab 1.25 a 6.16 ab 1.55 ab 2.3 ab 7.33 a table 5 means comparison for the effect of different levels of asparagine and casein on biochemical parameters of garden cress means with the same letter(s) within a column are not significantly different (p≤0.05) based on tukey's test. treatments (mg l-1) chlorophyll a chlorophyll b carotenoid content leaf nitrogen content leaf phosphorous content leaf calcium content total antioxidants total phenol aspargine 0 1.38 b 0.56 c 0.39 a 1.45 b 0.57 b 0.89 a 51.6 a 50.3 a 50 1.53 b 0.73 b 0.4 a 1.6 ab 0.66 ab 0.83 a 53 a 52.6 a 100 1.87 a 0.89 a 0.37 a 1.81 a 0.71 ab 0.95 a 57.6 a 57.3 a 150 1.64 ab 0.71 b 0.35 a 1.81 a 0.73 a 0.87 a 52.6 a 56.3 a casein 0 1.36 a 0.56 b 0.39 a 1.45 b 0.57 c 0.89 a 51.66 a 50.3 a 50 1.41 a 0.73 a 0.37 a 1.62 ab 0.60 bc 0.86 a 52 a 54.3 a 100 1.45 a 0.70 a 0.44 a 2.01 a 0.78 a 0.82 a 55 a 54.6 a 150 1.38 0.65 ab 0.43 a 1.88 a 0.74 ab 0.87 a 52.3 a 53.6 a jorkesh and aminifard foliar application on lepidium sativum under greenhouse conditions 231 needs of the cells are provided with the lowest losses. results of the current study revealed that foliar application of asn and csn could increase the growth and yield of garden cress. it could be recommended that spraying garden cress plants by asn (100 and 150 mg l-1) increased the vegetative growth traits (plant height, leaf fresh weight, number of florets), nutrients uptake (leaf nitrogen and phosphorus content) and pigments concentration (chlorophyll a and chlorophyll a) and also application of csn at 50 mg l-1 level could improved some morphological and biochemical traits of garden cress such as: root fresh weight, stem fresh weight, chlorophyll b, leaf nitrogen and phosphorus content in comparison control treatment. therefore, it can be concluded that application of asn and csn is a good strategy in garden cress cultivation, but their effectiveness must be also evaluated under environmental stresses, in the future studies. also spraying different nutrient solution in combination with each other may show interaction and contrary behavior in plants in comparison applying them solely, so further research with higher concentrations and also combined spraying of these amino acids, applying on different vegetables in greenhouse and farm conditions is suggested. references abo sedera f., abd el-latif a., bader l., rezk s., 2010 effect of npk mineral fertilizer levels and foliar application with humic and amino acids on yield and quality of strawberry. j. appl. sci., 25: 154-169. akladious s.a., abbas 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massai r., guidi l., 2008 antioxidant capacity, ascorbic acid, total phenol and carotenoids change during harvest and after storage of hayward kiwifruit. food chem., 107: 282-288. vanderzant e.s., 1974 development, significance, and application of artificial diets for insects. annual rev. entom., 19: 139-160. youssefi f., brown p., weinbaum s., 2000 relationship between tree nitrogen status, xylem and phloem sap amino acid concentrations, and apparent soil nitrogen uptake by almond trees (prunus dulics). j. hort. sci. biotec., 75: 62-68. impaginato 41 adv. hort. sci., 2018 32(1): 41-47 doi: 10.13128/ahs-21894 tomato plant growth, leaf nutrient concentrations and fruit quality under nitrogen foliar applications m.k. souri*, s. dehnavard department of horticultural sciences, tarbiat modares university, tehran, iran. key words: ammonium sulfate, calcium nitrate, foliar feeding, plant nutrition, urea, yield. abstract: tomato is a typical plant that has distinct response to different nitrogen forms in hydroponic culture. in addition, it is a well known susceptible plant to ammonium nutrition in hydroponic culture. however, its response to foliar application of nitrogen sources and n-forms has not been well investigated. in the present study, the growth, productivity and fruit quality of tomato was investigated under foliar application of nitrogen from different sources. ammonium sulfate, urea and calcium nitrate with constant concentration of 100 mm n were weekly sprayed during four months under hydroponic culture system. a water spray treatment was considered as control. the results showed that vegetative growth parameters were significantly affected by n sources in different patterns. the factors such as plant height, leaf area, number of lateral shoots and shoot fresh and dry weight, as well as leaf nitrate reductase activity was significantly reduced by foliar application of ammonium sulfate and to less extent by urea, while there was improvement of these traits by foliar application of calcium nitrate compared to control. however, ammonium sulfate treated plants had the highest leaf spad value and leaf n concentrations. plant fruiting pattern was also influenced by treatments, as ammonium sulfate spray reduced the fruit yield, and fruit vitamin c content, while it increased fruit tss and titratable acidity. the highest value of yield and vitamin c was recorded in calcium nitrate sprayed plants. 1. introduction application of different fertilizers play important role in agricultural production of food commodities. supply of adequate essential nutrients can significantly improve plant growth, quality and their nutritional values (marschner, 2011). different sources of each nutrient element can be applied as fertilizer to meet plant’s need of that special element. generally for most nutrients, there is little difference among effects of various sources; however, regarding nitrogen there is significant different effect of n form and sources on many vegetative and reproductive traits of plants (souri and roemheld, 2009). (*) corresponding author: mk.souri@modares.ac.ir citation: souri m.k., dehnavard s., 2018 tomato plant growth, leaf nutrient concentrations and fruit quality under nitrogen foliar applications. adv. hort. sci., 32(1): 41-47 copyright: © 2018 souri m.k., dehnavard s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 october 2017 accepted for publication 22 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 41-47 42 nitrogen fertilizers have important role in improving crop productivity; however low use efficiency rate of n fertilizers threatens sustainable plant production (souri, 2010). on market, various nitrogen sources exist for application under field and hydroponic culture. urea, ammonium sulfate and ammonium nitrate are the main nitrogen fertilizers for soil application, while calcium nitrate and potassium nitrate are the major nitrogen sources commonly are used in hydroponic systems (marschner, 2011). nitrogen forms (ammonium vs. nitrate) can have significant effect on morphology and physiology of plants particularly under hydroponic culture (souri and roemheld, 2009). in addition, ammonium instead of nitrate (smoleń and sady, 2009; souri 2010; marschner, 2011) and foliar complementation (kolota and osinska, 1999; dehnavard et al., 2017) supply of nitrogen can significantly improve n fertilizing efficiency in cropping systems. there may be several potential benefits of providing nitrogen to greenhouse crops via the foliage. these include: reduced nitrogen losses through denitrification and leaching, the ability to supply nitrogen when root activity is impaired e.g. in soil or water saline conditions, and luxury supply of plants with nitrogen. in cereals and some agronomic crops, late foliar application of urea generally results in higher grain protein and n content (fageria et al., 2009). the best quality parameters of plant growth and productivity of cabbage was reported when foliar versus soil application of fertilizers was applied (atanasova et al., 2007). tomato is one of the major vegetable crops that is cultivated in many parts of the world and consumed in many dishes. the application rate of n fertilizers in tomato culture is generally high (souri and roemheld, 2009), with n efficiency of about 30-50% (zotarelli et al., 2009; souri, 2010). in many greenhouses due to continuous cultivation and fertilization, soil salinity level is generally much higher than the threshold. if some levels of required n could be applied on plants as foliar spray, it can improve n use efficiency with less soil salinity buildup and less environmental side effects. in literature the tomato responses to continuous foliar spray of nitrogen sources have not well been established. therefore, the aim of this study was to evaluate response of tomato plants to foliar application of various nitrogen forms and sources under greenhouse and hydroponic culture. 2. materials and methods experimental set up this study was conducted during 2012 under greenhouse conditions at faculty of agriculture, trabiat modares uni., tehran-iran. the experiment was done in hydroponic system with four treatments and four replications arranged in completely randomized design. tomato seeds (lycopersicon esculentum var. money maker) were germinated in quartz sands and after germination (in four-leaf stage) two homogeneous seedlings were transferred to pots containing a mixture of cocopeat and perlite in ratio of 3:1 (v/v). one week later one of them was removed and one week later foliar treatments were applied on plants. the nutrient solution composition was prepared following hoagland formula (dehnavard et al., 2017). black plastic pots as replication, with a volume of nearly 12 liter were used for plants cultivation. for first two weeks after seedling transplanting, plants were supplied with one daily application of 100-200 ml of nutrient solution. thereafter, pots were supplied two times per day with nutrient solution of a final quantity of 250-1500 ml until end of experiment. the amount of applied solution increased with plant size and reached the amount of 1.5 liter per day at full plant size. treatments were foliar spray of three nitrogen sources of ammonium sulfate (as), urea, calcium nitrate (cn) and a no spray control. all three n sources were applied in constant concentration of 100 mm n (equals to 1400 mg l-1 n). sprays were done on weekly basis during 4 months of active growth period from 25 march (first foliar spray) until the end of july 2012. distilled water was sprayed in control plants. spraying treatments were done in the early morning, one hour after sun rise. measurements during plant growth period for four months, various vegetative traits as well as fruit harvesting records were collected. the final harvest of plants was done at the end of july. chlorophyll index was measured two times by spad meter (model 502 plus, illinois, usa), each time with 30 readings on 3 different areas for 10 randomly selected leaves per pot that the average was presented as leaf spad value. spad readings were done at the middle of experiment and before final harvest, at 10 o’clock in the morning. plant height, number of lateral shoots, souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar applications 43 shoots fresh and dry weight were measured at final harvest. cumulative harvest of fruits was recorded as final yield. plant leaf area was measured by leaf area meter and calculated as average area of a single leaf. fruits after harvesting were transferred to laboratory for further quality assessment. fruit firmness was measured by penetrometer (model wagner) after removing fruit skin using a blade. fruit total soluble solids (tss), titratable acidity (ta) and ph were determined in fruit juice squeezed by a squeezer. fruit tss percentage was measured by a portable refractometer (atago, tokyo, japan). fruit ph was determined using a portable ph meter, and titratable acidity was determined with titration of 5 ml of fruit juice with naoh 0.1 n until end ph of 8.1. for determination of vitamin c (l-ascorbic acid), 50 g of fresh fruit tissue was crushed in a porcelain mortar in vicinity of 20 ml metaphosphoric acid 6%, and then the juice transferred into a 50 ml tube, then centrifuged at 4000 rpm for 10 min. five ml of the supernatant transferred into an erlenmeyer flask, and received 20 ml of metaphosphoric acid 3%. then titration of the extract was done by di-chloro phenol indophenols until appearance of a rosa color, which the amount of vitamin c (mg 100-1 g fw) was calculated accordingly and based on a standard curve of lascorbic acid concentrations. from each treatment and replicates 3 fruits were kept in room temperature (25±2°c) for one week, thereafter their weight loss percentage was calculated. total nitrogen of leaves was determined using kejeldahl method and the activity of leaf nitrate reductase enzyme (nr) was determined after grinding and homogenizing of leaf materials in a mortar containing liquid nitrogen. nitrate reductase was extracted in a buffer consisting of 100 mm hepes (ph 7.5), 1 mm edta, 7mm cystein, 3% polyvinyl polypyrolidone (pvpp), 10 μm leupeptin, and 1 mm phenyl methyl sulfonyl fluoride (pmsf). after preparation of extracts sulfanilamide (0.5%) and n-(1-naphthyl)-ethylenediamine dihydrochloride (0.01%) in 1.5 m hydrochloric acid (hcl) were used for color development and the amount of no2 was determined spectrophotometrically at 540 nm and then nitrate reductase activity was calculated accordingly. statistical analysis excel software was used for calculating means and standard deviations and data were analyzed by spss software. comparison of means was performed at 5% by duncan’s multiple range test. 3. results the results of present study showed that plant vegetative growth parameters were significantly affected by nitrogen sources. plant height was significantly higher in calcium nitrate treated plants compared to ammonium sulfate and urea treated plants (table 1). the significant largest area of a single leaf and the highest number of lateral shoots were recorded in those plants which were treated with calcium nitrate, while the significant lowest records were in ammonium sulfate treated plants (table 1). spad value as a chlorophyll concentration index of plants were highest in ammonium sulfate treated plants (table 1), and there was no significant effects among other treatments. plant shoot fresh and dry weights were significantly affected by foliar spray of nitrogen sources. the significant highest shoot fresh and dry weight was obtained from plants treated with calcium nitrate and control plants. the significant lowest shoot fresh foliar spray treatment plant height (m) leaf area (cm2) lateral shoots (no.) spad value control (d-water) 1.98±0.16 ab 74.95±6.2 b 59.25±3.5 b 37.025±1.6 b ammonium sulfate 1.365±0.22 c 66.60±6.0 c 43.25±5.6 c 46.925±3.8 a urea 1.753±0.17 b 71.25±5.6 bc 51.00±9.7 bc 39.825±1.8 b calcium nitrate 2.225±0.22 a 80.60±2.7 a 70.50±5.2 a 39.275±1.8 b table 1 mean values of plant height, leaf area, number of lateral shoots and leaf spad of tomato plants. plants were grown in hoagland nutrient solution for 17 weeks all three n sources were applied in constant concentration of 100 mm n. spad readings were done two times at the middle of experiment and before final harvest, and the average was presented. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. adv. hort. sci., 2018 32(1): 41-47 44 and dry weight was in plants treated with ammonium sulfate and urea (table 2). determination of leaf nitrogen concentration (table 2) revealed that plants treated with ammonium sulfate and urea had significantly higher amounts compared to control and calcium nitrate treated plants. foliar spray of nitrogen sources showed significant effect on leaf nitrate reductase enzyme activity (table 2). nitrate reductase is the key enzyme in nitrate assimilation that its activity depends on several factors including nitrogen and nitrate status of plant tissues. the significant highest activity of this enzyme was in leaf of plants treated with calcium nitrate followed by control plants and those treated with urea. the significant lowest nitrate reductase activity was in ammonium sulfate treated plants. fruiting pattern of plants was also influenced by sprays of nitrogen sources and forms (table 3 and 4). number of fruits per plant was highest in calcium nitrate treated plants; however, they had no significant difference with control and urea treated plants. those plants which were treated with ammonium sulfate produced significant lowest number of fruits (table 3). the amounts of fruit yield per plant was significantly higher in calcium nitrate treated plants (table 3), followed by control, urea and ammonium sulfate treated plants. fruit firmness was not affected by foliar spray of nitrogen sources; however the percentage of fruit weight loss was significantly influenced by n foliar treatments. the highest weight loss, during one week keeping fruits at room temperature, was in fruits of those plants which were treattable 2 mean values of shoot fresh and dry weight, leaf n concentration and nitrate reductase activity of tomato plants plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. foliar spray treatment shoot fw (g) shoot dw (g) leaf n concentration (%) leaf nitrate reductase activity (µ mol no 2 g fw h) control (d-water) 1581±122 ab 13.7±1.6 a 2.3±0.17 b 0.71±0.1 b ammonium sulfate 1213±59 c 11.5±0.8 b 3.2±0.30 a 0.17±0.01 d urea 1469.5±79 b 11.4±1.14 b 2.9±0.32 a 0.30±0.09 c calcium nitrate 1758.3±186 a 14.2±1.7 a 2.5±0.13 b 0.90±0.06 a table 3 mean values of fruit number, fruit yield, fruit firmness and fruit postharvest weight loss in tomato plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. fruit weight loss was measured after one week in room temperature of 25±°c data are average of 4 replications ± sd. in each column means with a common letter have no significant diff erence at 5% of duncan test. foliar spray treatment number of fruits plant-1 fruit yield (g plant-1) fruit firmness (kg cm2) fruit weight loss (%) control (d-water) 19.7±2.7 a 2210.7±253 b 1.24±0.16 a 5.15±0.83 b ammonium sulfate 17.2±2.6 b 1824.5±218 c 1.32±0.10 a 12.25±2.04 a urea 19.0±2.2 a 1896.0±230 c 1.23±0.21 a 6.35±0.79 b calcium nitrate 20.2±2.2 a 2994.5±306 a 1.38±0.11 a 5.40±1.01 b table 4 mean values of fruit tss, fruit ta, fruit ph, and fruit l-ascorbic acid content in tomato under different fertilization plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. foliar spray treatment fruit tss (%) fruit ta (%) fruit ph fruit l-ascorbic acid (mg 100 g fw-1) control (d-water) 5.50±0.16 ab 3.75±0.13 b 3.68±0.1 a 36.8±4.1 a ammonium sulfate 5.98±0.27 a 4.57±0.22 a 3.95±0.3 a 31.0±2.1 b urea 5.48±0.17 ab 3.85±0.21 b 3.90±0.2 a 33.5±2.9 ab calcium nitrate 5.35±0.18 b 3.60±0.18 b 3.78±0.2 a 37.1±3.4 a souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar applications 45 ed with foliar spray of ammonium sulfate (table 3). fruit tss was also highest in ammonium sulfate treated plants and the significant lowest fruit tss was in calcium nitrate treated plants (table 4). similarly, fruit titrateable acidity values (ta) unchanged in urea and calcium nitrate treated plants compared to control, however sprays of ammonium sulfate resulted in significant higher amounts of fruit ta (table 4). fruit juice ph was not affected by foliar sprays of nitrogen sources (table 4). the significant highest l-ascorbic acid concentration was in calcium nitrate treated plants and control plants, while the significant lowest l-ascorbic acid was in ammonium sulfate treated plants (table 4). 4. discussion and conclusions the results showed that many growth and productivity traits of tomato plants were significantly affected by spray of n sources. plant biomass production and its different parameters including plant height, leaf area, number of lateral shoots, fresh and dry weight were significantly reduced by both ammonium sulfate and to less extent by urea treatments, while calcium nitrate sprays resulted in improvement of all these growth parameters compared to as and urea treatments. reduction in growth parameters of tomato due to foliar application of various concentration of ammonium sulfate have been reported by dehnavard et al. (2017). on the other hand, tomato is a distinct sensitive plant to ammonium nutrition particularly under hydroponic culture (loqué and von wirén, 2004; souri and roemheld, 2009). in present study, despite plants were fed by nitrate in nutrient solution; however foliar application of ammonium forms of nitrogen (ammonium sulfate and urea) resulted in reduced growth of plants. foliar absorption of nitrogen cannot be restricted by root medium n status, as there is always plant affinity to absorb nitrogen (marschner, 2011; dehnavard et al., 2017). foliar spray of ammonium sulfate in concentration of 100 and 200 mm with weekly application was resulted in significant growth restriction and less biomass production of tomato plants (dehnavard et al., 2017), while sprays of 50 mm improved tomato plant growth parameters, probably due to the fact that applied ammonium concentration and corresponding absorption was not in stressful level, but rather favored better photosynthesis and plant growth. daily foliar application of urea as the sole n source for tomato seedlings improved seedlings growth (nicouloud and bloom, 1996). despite the tomato tissue concentrations of ammonium increases significantly in 12-24 hour after foliar urea application (nicouloud and bloom, 1996), however from various studies it seems that plants can tolerate urea sprays better than ammonium sulfate (souri and roemheld, 2009). metabolism of malate and excretion of protons play important role in maintaining ph during ammonium assimilation in the shoot following ammonium sprays (peuke et al., 1998). urea in frequent applications and higher levels may have toxicity to plants (bowman and paul, 1992). however, although foliar spray of urea is common in some crops, but its physiological effects varies with season, cultivar and concentration (bowman and paul, 1992; fageria et al., 2009). in addition, it has been reported that foliar spray of urea compared to ammonium and nitrate may has less damage to leaves (bowman and paul, 1992). the absorption rate of urea is generally higher than calcium nitrate and ammonium sulfate in foliar spray (bowman and paul, 1992; fageria et al., 2009). however, within the tissues urea breakdowns to ammonium ions that similar to ammonium uptake can result in some toxicities (but with lesser extent) and restricted plant growth traits. spad values and leaf n concentration in ammonium sulfate treated plants were significantly higher compared to control and calcium nitrate sprayed plants. there are few studies reporting the effects of foliar application of nitrogen sources on vegetable crops. urea has been mainly used in one or limited applications with no negative side effect on plant growth (fageria et al., 2009; zhang et al., 2009). higher leaf spad value by ammonium sprays could be mainly due to higher chlorophyll concentration induced by restricted leaf expansion and higher n concentrations (souri and roemheld, 2009; dehnavard et al., 2017). the effect of foliar applications of ammonium sulfate and urea on chlorophyll readings in this study is in agreement with the results of foliar ammonium application on tomato (dehnavard et al., 2017), and urea spray on broccoli (yildirim et al., 2007) and onion (charbaji et al., 2008). ammonium spray probably by restriction of leaf area expansion has resulted in higher leaf n concentrations. in addition, ammonium absorption can take place by many nutrient specific and unspecific transporters, which results in less plant cell control over ammonium uptake and transport within the tissues (souri and roemheld, 2009). it seems that chlorophyll biosynthesis is less sensitive to ammonium rather than other leaf parameters adv. hort. sci., 2018 32(1): 41-47 46 such as leaf cell expansion and cell division, root and shoots growth and protein biosynthesis. it has been shown that foliar application of urea or soil application of a stabilized ammonium fertilizer (entec) can significantly increase n concentrations of leaves and root in carrot (smoleń and sady, 2009) and tomato (souri and roemheld, 2009) with less nitrate content. three foliar sprays of urea with 3 days interval on onion plants showed that bulb fresh and dry weight were increased by urea levels to 5000 mg l-1 without any damage to leaves (charbaji, et al., 2008). the best quality parameters of dry weight, total soluble sugars, vitamin c and low nitrate content in the cabbage leaves were achieved by foliar versus soil application of fertilizers (atanasova et al., 2007). foliar application of ammonium and then urea significantly reduced nitrate reductase activity of tomato leaves (table 2). this can be a negative factor when nitrate is actively taken up by plant roots and due to low activity of this enzyme most of nitrate accumulate in vacuoles resulting in higher nitrate accumulation in leaves and probably in fruits. tomato plant yield was significantly reduced by ammonium sprays, while it was increased by calcium nitrate treatment. this ammonium effect could be due to restricted vegetative growth, reduced hydraulic conductance, phloem translocation and less fruit set, while calcium nitrate had no effect on fruit number but it increased the average fruit weight resulted in higher yield compared to control. foliar nitrogen application generally increase plant yield. the maximum increase in marketable yield in cabbage, onion, and cucumber using supplementary foliar n fertilization was 20.3%, 10.8% and 7.3%, respectively (kolata and osinska, 1999). foliar fertilization significantly decreased the level of cucumber leaf infestation by downy mildew disease (kolata and osinska, 1999). changes in fruit quality traits are generally observed due to foliar n applications. in this study increase in fruit tss by ammonium spray can be due to higher chlorophyll content of leaves and higher photosynthetic rates. it is probably feasible that foliar sprays of ammonium induced stress signals leading plant to have more fruit sugars and tss content. guvenc et al. (1995) reported that foliar urea application improved some quality (vitamin c and titratable acidity) and growth properties of tomato. in onion plants foliar spray of urea increased n, p, k and ca concentration of leaves that resulted in higher photosynthesis and sugar production; however, other nutrients were not affected (charbaji et al., 2008). similar results in carrot were found by foliar application of nitrogen sources (smoleń and sady, 2009). in present study, ammonium spray reduced vitamin c content of fruits. this can be due to stress conditions induced by foliar ammonium spray and less hydraulic conductance of plant tissues (souri and roemheld, 2009). changes in quality parameters of tomato by foliar nitrogen sprays are in agreement with similar studies on other vegetable crops (guvenc et al., 1995; chaurasia et al., 2005; yildirim et al., 2007; dehnavard et al., 2014), which showed that increasing nitrogen application reduced the vitamin c content. in addition, foliar application of nitrogen compounds can significantly improve plants tolerate to heat stress (zhao et al., 2008). foliar n application can improve plant growth parameters of dry weight, relative water content and nitrate reductase activity under moderate water stress particularly with drought sensitive varieties (zhang et al., 2009). in this experiment, foliar application of ammonium sulfate and to less extent urea reduced normal plant growth and some quality traits, while calcium nitrate generally improved tomato growth and some quality factors. however, calcium nitrate can increase nitrate content of fruits that is not suitable, and therefore is not recommended in repeated applications. spray of ammonium sulfate increased fruit tss and titrateable acidity while it decreased vitamin c and fruit postharvest freshness. so, restricted growth through lower height and less lateral shoots in ammonium foliar sprayed plants may suggest benefits regarding labor requirement for plant pruning-training and management under greenhouse production of tomatoes. in addition, by foliar n application (particularly ammonium sulfate and urea) less soil salinity and less nitrate accumulation occur. nevertheless, it is revealed that tomato still show sensitivity to ammonium nutrition through the foliage similar to root ammonium nutrition via nutrient solution. references atanasova e., mitova i., dimitrov i., stancheva i., 2007 effect of different fertilizer sources on the quality of head cabbage. j. appl. hort., 9(1): 74-76. bowman d.c., paul j.l., 1992 foliar absorption of urea, ammonium, and nitrate by perennial ryegrass turf. j. amer. soc. hort. sci., 117: 75-79. charbaji t., arabi m.i.e., jawhar m., 2008 urea foliar fertilization affects onion weight and nutrient content. inter. j. vegetable sci., 14(3): 198-204. souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar applications 47 chaurasia s.n.s., singh k.p., rsi m., 2005 effect of foliar application of water soluble fertilizers on growth, yield, and quality of tomato (lycopersicon esculentum l.). sri lankan j. agric. sci., 42: 66-70. dehnavard s., souri m.k., mardanlu s., 2014 qualitative parameters of tomato fruits affected by foliar application of nitrogen in hydroponic culture. iranian j. plant and seed, 30(2): 237-240. dehnavard s., souri m.k., mardanlu s., 2017 tomato growth responses to foliar application of ammonium sulfate in hydroponic culture. j. plant nutr., 40(3): 315-323. fageria n.k., barbosafilho m.p., moreira a., guimar c.m., 2009 foliar fertilization of crop plant. j. plant nutr., 32: 1044-1064. guvenc i., padem h., alan r., 1995 effect of foliar application of different levels of urea on yield and yield component of tomatoes. ii. turkey nat. hort. symp., adana, turkey. kolota e., osinska m., 1999 efficiency of foliar nutrition of field vegetables grown at different nitrogen rates. inter. conf. environ. problems associated with nitrogen 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nitrification inhibition in soil. commun. soil sci. plant anal., 41(14): 1769-1778. souri m.k., roemheld v., 2009 split daily application of ammonium cannot ameliorate ammonium toxicity in tomato plants. hort. environ. biotechnol., 50: 384391. yildirim e., guvenc i., turan m., karatas a., 2007 effect of foliar urea application on quality, growth, mineral uptake and yield of broccoli (brassica oleracea l., var. italica). plant soil and environ., 53(3): 120-128. zhang l., li s., liang z., li s., 2009 effect of foliar nitrogen application on nitrogen metabolism, water status, and plant growth in two maize cultivars under shortterm moderate stress. j. plant nutr., 32(11): 18611881. zhao w.y., xu s., li j.l., cui l.j., chen y.n., wang j.z., 2008 effects of foliar application of nitrogen on the photosynthetic performance and growth of two fescue cultivars under heat stress. biol. plant., 52(1): 113116. zotarelli l., scholberg j.m., dukes m.d., muñozcarpena r., icerman j., 2009 tomato yield, biomass accumulation, root distribution and irrigation water use efficiency on a sandy soil, as affected by nitrogen rate and irrigation scheduling. agr. water manag., 96(1): 23-34. impaginato 465 adv. hort. sci., 2019 33(4): 465­474 doi: 10.13128/ahsc­8110 prostrate or upright growth habit in tomato cultivars: contributory roles of stem diameters and fruit weight under fertilizer application s.o. olagunju 1 (*), o.s. sosanya 1, o.a. oguntade 1, k.m. adewusi 1, o.a. odusanya 1, a.l. nassir 1, a.o. joda 1, a.t. adegoke 1, o.b. banjo 2 1 department of crop production, college of agricultural sciences, olabisi onabanjo university, p.m.b. 0012, ayetoro campus, ayetoro, ogun state, nigeria. 2 department of forestry, wildlife, and fisheries, college of agricultural sciences, olabisi onabanjo university, p.m.b. 0012, ayetoro campus, ayetoro, ogun state, nigeria. key words: aerial stem, basal stem, cultivars, growth habit, npk 15:15:15. abstract: for increased production of fresh market tomato fruits, identification of cultivars that combine upright growth habit (ugh) and increased fruit weight under increased fertilizer rates is essential. six tomato cultivars comprising five improved types namely tropimech, buffalo, roma vf, roma savana, uc 82 and a local cultivar kerewa were evaluated to identify cultivars that combined aver­ age fruit weight with ugh under different rates of npk 15:15:15 fertilizer (0, 30, 50, and 80 kg ha­1). aerial and basal stem diameter (asd and bsd), and weight per fruit (wpf) cumulatively accounted for the largest significant variation (47.17 **) in growth habit of the tomato cultivars with asd being the most determinant of all. increased fertilizer rates resulted in increased morphologi­ cal and yield parameters but promoted prostrate growth habit in tomato culti­ vars. at 30 kg ha­1, roma vf and roma savanna combined ugh with ample yield per plant while at 50 and 80 kg ha­1 of fertilizer, uc 82 consistently main­ tained the most ugh with higher yield. growing uc 82 at 50 kg ha­1 of fertilizer is recommended for better ugh and higher yield. consideration should be given to asd, bsd, and wpf in future improvements of tomato for ugh and yield. 1. introduction one of the challenges confronting tomato production is the inability of the crop to maintain an upright growth habit at full fruit formation due to increased fruit weight and weak stem. this attributes manifest more in most improved tomato cultivars that are bred purposely for high yield and do not exempt the local ones. maintenance of upright growth habit in tomato cultivars at maturity is an important attribute in harvesting high percentage of fresh tomato fruits considering the prevalence of diseases (*) corresponding author: solomondwiseman@yahoo.com citation: olagunju s.o., sosanya o.s., oguntade o.a., adewusi k.m., odusanya o.a., nassir a.l., joda a.o., adegoke a.t., banjo o.b., 2019 ­ prostrate or upright growth habit in tomato culti‐ vars: contributory roles of stem diameters and fruit weight under fertilizer application. ­ adv. hort. sci., 33(4): 465­474. copyright: © 2019 olagunju s.o., sosanya o.s., oguntade o.a., adewusi k.m., odusanya o.a., nassir a.l., joda a.o., adegoke a.t., banjo o.b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 december 2018 accepted for publication 8 july 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 465­474 466 that occur with contact of fruits with bare soil (santamaría and toranzos, 2003). tomato fruit is per­ ishable and the contact with soil can promote rotting and scorching and increase disease prevalence that can lead to low quality fruits and greater yield loss (naika et al., 2005). tomato plant are often staked or trellised in order to minimize the contact of fruit with soil however, the cost incurred on staking during pro­ duction of fresh market tomato fruits are among the highest for any vegetable crop (davis and estes, 1993; kemble et al., 1994; frasca et al., 2014). identifying tomato cultivars with most upright growth characteristics can prevent scorching and rot­ tenness often associated with tomato fruit at maturi­ ty and could contribute to increased fruit quality and fruit yield and reduce cost of staking in tomato pro­ duction. the permanent displacement of plant from its upright position otherwise termed ‘lodging’ is one major cause of yield loss in most crops (crook and ennos, 1995). it usually occurs due to inability of plants to withstand strong winds and poor anchorage of plant roots to its substratum. terminal and axillary weights from fruits can contribute to plant inability to stay erect. the height of the plant as well as thick­ ness of the basal stem are some of the contributing factors to lodging in plants (zhang et al., 2014). in tomato, variations in stem diameter occur along the height of the crop. the basal section of the stem which is usually thinner though could be more ligni­ fied is unable to support the uppermost part which is characterized by robust stem and heavy fruit weights at fruiting stage in most cultivars. the variation in stem diameter usually observed along the stem of tomato cultivars could be a factor contributing to lodging in tomato. assessing the extent of this varia­ tion along the height of the crop along with other morphological traits could provide information on morphological traits that should be considered in future improvement of tomato cultivars for upright growth and yield. plant response to fertilizer application occurs through expansion of various plant parts culminating eventually in surface area expansion. application of inorganic fertilizer at high dosage rates can increase yield but may also increase susceptibility of the crop to lodging due to reduced structural carbohydrates content and lignin deposition resulting in weakening of the stem (zhang et al., 2014; zhang et al., 2016). conversely, poor fertilization culminating in nitrogen deficiency can instigate stem diameter variation attributable to reduced transpiration and assimilate loadings which ultimately affects storage tissue con­ centration (de swaef et al., 2015). in tomato, applica­ tion of inorganic fertilizers such as calcium nitrate at different rates increased plant height and fruit yield (montagu and goh, 1990; souri and dehnavard, 2018). this could also induce bioaccumulation of salt in plant tissue at higher rates which often affect growth of tomato (romero­aranda et al., 2006; tuna et al., 2007; caruso et al., 2011). finding a balance in upright growth, increase in dosage rates of nitrogen fertilizer applied and ample fruit yield is therefore a necessity in ensuring maximum gains from tomato cultivation. little studies have explored the contribu­ tion of stem diameter variation and fruit weight on upright growth of improved tomato cultivars. past breeding objectives in tomato has focused mainly on yield, shelf­life, taste and nutritional quality (bai and lindhout, 2007), however evaluation of morphologi­ cal traits that could contribute indirectly to these traits is also necessary. documented findings should assist in identifying cultivars that combine high yield with upright growth under different rates of fertilizer application. the following questions were raised and addressed in this study (i) does variation in stem diameter contribute to growth habit of tomato culti­ vars? (ii) do tomato cultivars maintain similar growth habit at physiological maturity when maximum weight of fruits is achieved and when fruits are turn­ ing red? (iii) do fertilizer rates influence growth habit of tomato cultivars? and (iv) does fruit weight, num­ ber of fruits or both contribute to upright growth habit of tomato cultivars? 2. materials and methods experimental site the experiment was conducted at ilaraa, a tomato production area located in the derived savanna zone of yewa north ogun state, nigeria (7.4°n, 2.7°e, 188 m asl). the area is characterized by erratic rainfall at early and later part of the year which usually remains stable around june to july. the soil is well drained and is characterized by high proportion of sand with relatively lower proportion of clay and silt and hence supports good growth of tomato. the commonly grown cultivar, kerewa, is supplied in commercial quantities to adjoining markets and lagos, a neigh­ bouring state. soil sample collection and preparation prior to establishment of the trial, the experimen­ olagunju et al. ‐ stem diameters contribution to growth habit of tomato cultivars 467 tal field was divided into three rectangular blocks each with 3 m x 6 m in dimension. core soil samples were taken at 0­15 cm along the diagonals of each block after which they were bulked to form compos­ ite samples. the soil samples were air dried and sieved with 2 mm sieve after which they were sub­ jected to laboratory soil analysis. laboratory analysis of soil sample the ph of the soil was determined in 1:2 soil water ratios with a glass electrode ph meter (mclean, 1982). particle size distribution was deter­ mined by hydrometer method (gee and bauder, 1986). exchangeable bases (ca, mg, k and na) in the soil were extracted using ammonium acetate method (thomas, 1982). following extraction of exchange­ able bases, ca and mg were determined with buck scientific 210 vgp model, atomic absorption spectrophotometer (aas), while k and na were read on flame photometer. the exchangeable acidity was determined by titration method (anderson and ingram, 1993). effective cation exchange capacity (ecec) was estimated by the summation of exchangeable bases and exchangeable acidity (anderson and ingram, 1993). base saturation of the soil was calculated as the fraction of the exchange­ able bases and ecec expressed in percentage. total nitrogen was determined by kjeldahl method (bremner, 1996). organic carbon was determined by the wet oxidation method as described by walkley­ black (nelson and sommer, 1996). available phos­ phorus was determined by bray­1 method (bray and kurtz, 1945). experimental methods the experimental protocol was based on the fac­ torial combination of six cultivars and three fertilizer rates plus a non­fertilized control. six tomato culti­ vars comprising five improved types namely roma vf, roma savana, uc 82, tropimech ­with determi­ nate growth habit, buffalo and the local cultivar­ kerewa, which are both an indeterminate cultivars that require staking. buffalo has a large fruit with thick flesh and few seeds. tropimech has an egg­ shaped fruit and high fruit setting. uc 82 in addition to having determinate growth is an early maturing cultivar with square to egg­shaped red fruit. the seeds of the tomato cultivars were all obtained from agro service station and were nursed for 3 weeks. the seedlings were later transplanted onto a pre­ pared field at a spacing of 50x50 cm between plants along and between rows. the plot dimension was 1 m x 1.5 m with six plants. spacing between plots and replicates was 50 cm and 1 m, respectively (fig. 1). fertilizer as n:p:k 15:15:15 in the form of elemental nitrogen (n), phosphate (p2o5) and potash (k2o) respectively, was applied at the rates of 30, 50 and 80 kg ha­1 at 8 weeks after planting while plot with no fertilizer application served as the control. the exper­ imental treatments were distributed in the field according to the randomized complete block design (rcbd) with three replicates. harvesting of tomato fruits commenced at physiological maturity, when assimilates had been fully partitioned into the fruits as confirmed by fully formed fruits, and when fruits were turning red. the fruits were counted and weighed using electronic compact scale (atom a­ 110c), china. data collection at physiological maturity of tomato fruits, plant basal stem diameters was measured at 5 cm above the soil mark while aerial diameter was measured near the last leaf at the top using plant leaf thickness gauge (model yh­1 top instrument). height measure­ ment was performed on the primary stem axis from soil mark to the base of the last leaf. the growth habit of the tomato cultivars was assessed using visu­ al scoring on a scale of 1 to 5:1 represents upright growth, 2 for 30° deviation, 3 for 45° deviation, 4 for 70° deviation and 5 for fully prostrate stem (ozminkowski et al., 1990). fruit production per plant fig. 1 ­ plot layout of the field showing the arrangement of plots within the field. plots in group of six represent the culti­ vars arranged randomly within each group. adv. hort. sci., 2019 33(4): 465­474 468 was obtained by harvesting all fruits on a plant at physiological maturity which were later counted and weighed. total fruit weight per plant was obtained by weighing all fruits harvested within a plot and divided by the total number of plants per plot. stem diame­ ter variation (mm cm­1) was computed using the fol­ lowing formula: stem diameter variation (mm cm­1) = asd ­ bsd x 100 ph where asd = aerial stem diameter; bsd = basal stem diameter; and ph = plant height. these parame­ ters were selected for measuring stem diameter vari­ ation (sdv) based on their ability to measure differ­ ence in terminal stem diameters (asd and bsd) in relation to average distance between the two as measured by plant height (ph). statistical analyses data of growth and yield parameters were sub­ jected to analyses of variance (anova) using genstat 12th edition (payne et al., 2009) package. the means relevant to the variables significantly affected by the experimental treatments were separated using fischer’s protected least significant difference at p≤0.05. correlation and stepwise regression analy­ ses of the morphological and yield parameters were conducted to study the association among the vari­ ables and to identify the traits with largest contribu­ tion to growth habit of tomato at physiological matu­ rity. multivariate analysis was also conducted to identify cultivars that combined an upright growth habit with high yield among the tomato cultivars. in correlation, regression and principal component analyses (pca) carried out, logarithm transformation was conducted on the data before analyses. the mean of the transformed values for each of the vari­ able across fertilizer rates were first computed for each cultivar before conducting pca. the pca was conducted using correlation matrix method. 3. results the routine soil analysis of the experimental site is presented in table 1. the ph of the soil (6.9) is neu­ tral and has higher proportion of sand (886.0 g kg­1 equivalent to 88.6% of 1 kg soil sample) as compared with that of silt and clay (69.33 and 44.67 g kg­1 respectively) amounting to 6.9 and 4.5% respectively of 1 kg soil sample. exchangeable ca and mg con­ tents of 3.72 and 1.48 cmol kg­1, respectively were moderately high. however, potassium (0.08 cmol kg­ 1) level in the soil was very low compared to critical level of 0.15 cmol kg­1. exchangeable sodium (0.10 cmol kg­1) was also low while the ecec of 5.43 cmol kg­1 was lower than critical level of 8.0 cmol kg­1 for low soil fertility in tropical soils. there was low exchangeable acidity (0.05 cmol kg­1) and high base saturation percentage of 99.12 %. total nitrogen (0.70 g kg­1) and organic carbon (6.43 g kg­1) were very low. available phosphorus (26.02 mg kg­1) was very high and in several folds higher than critical level of 8.0 mg kg­1 in non­degraded soils. the effects of fertilizer rates and cultivars on mor­ phological and yield parameters of tomato are pre­ sented in table 2. significant effects (p<0.01) of fertil­ izer rate and cultivar were observed on all the para­ meters with the exception of basal stem diameter (bsd) and weight of fruit per plant (wfpp) which were not significantly affected by cultivar. increased application rate of npk 15:15:15 fertilizer from 30 to 80 kg ha­1 resulted in a significant increase in all mor­ phological parameters of the tomato cultivars. a more enhanced prostrate growth habit (pgh) was however observed in tomato plants with increased rate of fertilizer. the control recorded 2.17 growth habit score (ghs) while fertilizer application rate of 50 kg ha­1 and 80 kg ha­1 recorded 3.33 and 3.17 in ghs, respectively. among the cultivars, the widest aerial stem diameter, asd (8.46 mm) and stem diam­ eter variation (sdv) (5.75 mm cm­1) were observed in buffalo, while kerewa had the highest number of fruit per plant, nfpp (5.61) but the least upright plant with mean ghs of 3.42. the highest weight per fruit table 1 ­ pre­experimental characteristics of soil physicochemical properties soil composition ph 6.92 sand (g kg­1) 886.00 silt (g kg­1) 69.33 clay (g kg­1) 44.67 texture sandy ca (cmol kg­1) 3.72 mg (cmol kg­1) 1.48 k (cmol kg­1) 0.08 na (cmol kg­1) 0.10 al+h (cmol kg­1) 0.05 ecec_(cmol kg­1) 5.43 bs_% 99.12 n (g kg­1) 0.70 oc (g kg­1) 6.43 av_p (mg kg­1) 26.02 olagunju et al. ‐ stem diameters contribution to growth habit of tomato cultivars 469 regression of morphological and yield parameters on growth habit of tomato cultivars across fertilizer application rates are presented in table 4. under each parameter contribution to growth habit i.e. one term, asd accounted for the highest (44.89**) signifi­ cant variation among the growth and yield parame­ ters while wfpp contributed the least (9.82**) signif­ icant variation. the wpf did not solely account for significant variation in growth habit of the tomato cultivars. across the various subsets of regression i.e. two terms and above, asd in most cases, was consis­ tent in its significant contribution to variations in growth habit of tomato cultivars. the highest signifi­ cant variation (47.17**) in gh of tomato cultivars was obtained when three terms consisting asd, bsd, and wpf were included in the regression model while other cumulative inclusions accounted for less­ er significant variation in growth habit of tomato cul­ (wpf 25.65 g) was however observed in uc 82. no significant interaction between fertilizer rate and cul­ tivar was observed for all the parameters studied. table 3 shows the correlation coefficients among the morphological and yield parameters of the toma­ to cultivars. significant correlations were observed among most of the parameters with correlation between nfpp and wfpp being the highest (0.79**) and followed by correlation between asd and sdv (0.76**). the lowest significant correlation was observed between plant height (ph) and sdv (0.24*). the correlation of wpf was significant (0.50**) only with wfpp. among the yield parameters, wpf did not show significant correlation (0.05 ns) with sdv. growth habit score had positive significant correla­ tion with all the morphological and yield parameters with the exception of wpf (­0.02 ns). the percentage variations (adjusted r2) of subsets table 2 ­ effects of fertilizer rate and cultivar on morphological and yield parameters of improved tomato cultivars sources of variation levels of variation (kg) aerial stem diameter (mm) basal stem diameter (mm) plant height (cm) stem diameter variation (mm cm­1) number of fruit plant­1 weight fruit­1 (g) weight of fruit plant­1 (g) growth habit fertilizer 0 5.66 5.32 31.96 0.01 2.78 18.46 49.68 2.17 rates (f) 30 6.89 5.87 41.01 0.03 3.96 22.40 83.35 2.83 (kg ha­1) 50 7.52 5.91 42.68 0.04 4.27 21.70 84.29 3.33 80 7.70 6.23 42.62 0.03 5.20 25.77 126.80 3.17 lsd 0.64 0.50 4.31 0.01 0.14 t 4.43 28.49 0.10 t significance ** ** ** ** * * ** ** cultivars (c) roma vf 6.12 5.68 39.51 0.01 3.03 24.63 76.25 2.33 uc 82 6.82 5.69 36.39 0.03 3.78 25.65 93.99 2.58 roma savana 6.45 5.83 38.73 0.01 2.98 20.72 64.87 2.42 buffalo 8.46 6.08 39.17 0.06 5.58 19.08 107.79 3.33 tropimech 6.64 5.45 43.66 0.03 3.26 25.90 85.36 3.17 kerewa 7.19 6.25 39.96 0.02 5.61 16.53 87.93 3.42 lsd 0.78 0.61 5.28 0.01 0.17 t 5.43 34.90 0.12 t significance ** ns * ** * ** ns ** f x c ns ns ns ns ns ns ns ns parameters aerial stem diameter, asd (mm) basal stem diameter, bsd (mm) plant height (cm) stem diameter variation (mm cm­1) number of fruit plant­1 weight fruit­1 (g) weight of fruit plant­1, (g) growth habit score arial stem diameter basal stem diameter 0.69** plant height 0.52** 0.36** stem diameter variation 0.76** 0.10 ns 0.24* number of fruit per plant­1 0.55** 0.57** 0.34** 0.28* weigh fruit­1 0.12 ns 0.14 ns 0.13 ns 0.05 ns ­0.14 ns weigh of fruit plant­1 0.56** 0.59** 0.38** 0.27* 0.79** 0.5** growth habit score 0.68** 0.35** 0.38** 0.61** 0.40** ­0.02 ns 0.33** table 3 ­ correlation analysis showing the relationships among growth and yield parameters of improved tomato cultivars grown in ilaraa ** and * = significant at p≤0.01 and 0.05 probability level respectively; ns= non­significant. least significant difference (lsd) with ‘t’ are from transformed data. ** and * = significant at p≤0.01 and 0.05 probability level respectively; ns= non­significant. values in bold are the highest and lowest significant correlations values from the parameters involved. 470 adv. hort. sci., 2019 33(4): 465­474 tivars. the comparison of growth habit and wfpp (yield) of tomato cultivars at each fertilizer rate for identifi­ cation of cultivar with best combination of high yield with the most upright growth is presented in figure 2. for the control (0 kg ha­1), the logarithm of wfpp exceeded that of the growth habit for all the tomato cultivars except kerewa. with application of fertilizer, overlapping relationship between growth habit score and wfpp were observed among the cultivars. at 30 kg ha­1 of fertilizer, roma vf and roma savana main­ tained higher yield than their respective growth habit while at higher doses, uc 82 consistently maintained higher yield combined with the most upright growth habit. across the fertilizer rates, growth habit score of buffalo, tropimech and kerewa exceeded their respective wfpp. figure 3 shows the principal component biplot of the cultivars using all morphological parameters. the lines in the biplot represent the direction of increase of the variables with the side of the line with the label having the highest value for the variable. the two axes of the biplot accounted for 79.9% variation among the cultivars. at one side of the plot are tropimech, buffalo and kerewa with the cultivars having the highest sdv, pgh, nfpp, wfpp and ph while uc 82, roma vf and roma savana are at the other side where the values for these traits are lower. the cultivar, uc 82 however falls within the center of polygon while other cultivars are at the ver­ tices position. ** and * = significant at p≤0.01 and 0.05 probability level respectively; ns= non­significant. adjusted r2 presented are the first few highe­ st ranked adjusted r2 from each subset. the bold­italic adjusted r2 in the column for six terms represents the adjusted r2 when all terms are included in the regression model. values in parenthesis represent the parameters and their significant level of contribution to the adjusted r2 in each subset. 1= aerial stem diameter; 2= basal stem diameter; 3= plant height; 4= stem diameter variation; 5= number of fruits per plant; 6= weight per fruit; and 7= weight of fruit per plant. one term two terms three terms four terms five terms six terms 44.89 (1**) 47.00 (1**, 2 ns) 47.17 (1**, 2 ns, 6 ns) 46.88 (1**, 2 ns, 5 ns, 7 ns) 46.24 (1**, 2 ns, 3 ns, 5 ns, 7 ns) 45.46 (1**, 2 ns, 3 ns, 5 ns, 6 ns, 7 ns) 36.13 (4**) 46.21 (1**, 4 ns) 47.09 (1**, 2*, 5 ns) 46.85 (1**, 2*, 5 ns, 6 ns) 46.20 (1**, 2 ns, 3 ns, 5 ns, 6 ns) 45.41 (1 ns, 2 ns, 3 ns, 4 ns, 5 ns, 7 ns) 14.78 (5**) 45.28 (1**, 6 ns) 46.42 (1**, 4 ns, 6 ns) 46.83 (1**, 2*, 6 ns, 7 ns) 46.17 (1**, 2 ns, 3 ns, 6 ns, 7 ns) 45.37 (1 ns, 2 ns, 3 ns, 4 ns, 5 ns, 6 ns) 13.36 (3**) 44.40 (1**, 7 ns) 46.37 (1*, 2 ns, 3 ns) 46.59 (1*, 2 ns, 3 ns, 6 ns) 46.16 (1*, 2 ns, 4 ns, 5 ns, 7 ns) 45.37 (1*, 2 ns, 4 ns, 5 ns, 6 ns, 7 ns) 10.65 (2**) 44.23 (1**, 3 ns) 46.28 (1*, 2 ns, 4 ns) 46.44 (1*, 2 ns, 4 ns, 6 ns) 46.13 (1*, 2 ns, 4 ns, 5 ns, 6 ns) 45.34 (1 ns, 2 ns, 3 ns, 4 ns, 6 ns, 7 ns) 9.82 (7**) 44.21 (1**, 5 ns) 46.22 (1**, 2 ns, 7 ns) 46.40 (1**, 2* ,3 ns, 5 ns) 46.11 (1**, 2 ns, 5 ns, 6 ns, 7 ns) 45.09 (1*, 3 ns, 4 ns, 5 ns, 6 ns, 7 ns) <0.00 (6 ns) 43.52 (2**, 4**) 46.07(1**, 3 ns, 4 ns) 46.38 (1*, 2 ns, 4 ns, 5 ns) 46.10 (1*, 2 ns, 4 ns, 6 ns, 7 ns) 44.08 (2 ns, 3 ns, 4**, 5 ns, 6 ns, 7**) 41.18 (3**, 4**) 46.00 (1*, 4 ns, 5 ns) 46.37 (1**, 3 ns, 4 ns, 6 ns) 45.85 (1*, 3 ns, 4 ns, 5 ns, 7 ns) 44.61 (1 ns, 2 ns, 3 ns, 4 ns, 5 ns, 6 ns, 7 ns) for 7 terms table 4 ­ adjusted r2 of subsets linear regression of morphological and yield parameters against growth habit of tomato cultivars across rates of fertilizer fig. 2 ­ comparison of growth habit and weight of fruit per plant for identification of tomato cultivar with better combina­ tion of traits under different fertilizer rates. fig. 3 ­ principal component’s biplot showing the differential performance of the tomato cultivars for the morphologi­ cal and yield parameters. olagunju et al. ‐ stem diameters contribution to growth habit of tomato cultivars 471 4. discussion and conclusions stem diameter variation measurement is not com­ mon in tomato cultivars assessment but its measure­ ment along the height of the crop provides useful information on the susceptibility of tomato to pros­ trate growth at maturity. the need to improve toma­ to cultivars for upright growth while maintaining higher yield, particularly under the tropical humid condition where fruits get in contact with moist soil due to the weight of fruits, is quite imperative. among the various morphological and yield para­ meters examined, the asd and bsd, and wpf were among the most contributory traits that cumulatively influenced the growth habit of tomato with asd of the tomato cultivars being the most influential on growth habit of the tomato cultivars. the non­signifi­ cant cumulative contribution of nfpp to growth habit of the tomato cultivars underscores the importance of wpf present on a plant. the likely concentration of heavy­weight fruits at one side of the plant could have promoted prostrate growth habit of the tomato cultivars. the mean fruit weight of tomato fruits has been regarded as a strong varietal character in deter­ mining the marketable yield of tomato (moreno and moreno, 2008; de sio et al., 2018). this therefore implied that wpf in addition to stem diameter of both basal and aerial stems but most importantly the aerial stem should be focused on in future improve­ ment of tomato cultivars, particularly where infection and damage by soil borne pathogens are prevalent. improved tomato cultivars though varied in their individual growth responses, are similar in response to increased fertilizer rates as also reported in previ­ ous research (ercolano et al., 2015). virtually all the morphological and yield parameters showed increase response with increase rate of fertilizer with the highest performance observed at 80 kg ha­1. the low level of nitrogen observed in the soils could have elicited the responses of the tomato cultivars to doses of fertilizer applied. the low levels of n and organic carbon in soil with low clay has been attrib­ uted to leaching of nitrates and high rate of mineral­ ization of organic matter that characterize very highly degraded tropical soils such as the type in the experi­ mental site (snakin et al., 1996). nitrate leaching in sandy soils can be prevented by using organic or organo­mineral forms of nitrogen fertilizer, which also reduces the accumulation of this anion in plant edible parts (caruso et al., 2011). the low n content in the soil in the present research can also be attrib­ uted to low ecec of the soil which was a reflection of higher sand particle and low activity clay of the soil which encouraged leaching of cations (udoh et al., 2013). the low exchangeable acidity and high base saturation also observed in the soil is an indication that the soil’s exchange site was dominated by basic cations. the increase in vegetative growth such as asd and bsd, observed with higher dosage rate of fertiliz­ er would have been the cumulative effect of increase nitrogen content of the fertilizer. the increase in basal stem diameter (bsd) observed with increase fertilizer rates did not however translate to upright growth in most of the improved tomato cultivars selected as there were corresponding increases in fruit weight and robustness of the asd. in rice, reduced structural carbohydrates content and lignin deposition is usually associated with increased fertil­ izer application and can contribute to pgh (zhang et al., 2016). this could also have been responsible for pgh exhibited by the tomato cultivars in the present research. increased fruit yield observed in the toma­ to cultivars with increased fertilizer rates can be attributed to increased dosage rate of phosphorus and potassium in the fertilizer that complimented soil available phosphorus, as both stimulate yield increase in crop (vance et al., 2003). in previous investigations (amalfitano et al., 2017; morano et al., 2017), the increase of macronutrients supply to plants led to yield increase up to a crop system dependant threshold. high level of available p in this soil may have been caused by the ph level of the soil. at soil ph of 6.92, phosphorus is more likely to be available than at lower ph of 4 to 4.5 where p is more likely to be fixed (deforest et al., 2012). plotting the log values of traits against their summed log values as obtained in figure 3 can be an alternative way of selecting cultivars of plant with good combination of traits. the plot of log of wfpp and growth habit against their summed log values at each rate of fertilizer applied provided the opportuni­ ty to identify cultivars with better performance for these two traits under a given fertilizer rate. the plots indicated upright growth habit for lower loga­ rithm values of growth habit and higher yield for higher logarithm value of wfpp. it was revealed that under no fertilizer application, higher wfpp of all the cultivars except kerewa combined with upright growth habit whereas this varied among the cultivars under application of fertilizers. with npk 15:15:15 fertilizer, most of the cultivars especially buffalo, tropimech and kerewa maintained pgh in combina­ tion with higher yield. this corroborated the earlier adv. hort. sci., 2019 33(4): 465­474 472 findings that increased fertilizer application rates can increase yield but also increase the susceptibility of plant to lodging (zhang et al., 2014). roma vf and roma savanna were however identified as cultivars that combined average yield with the most upright growth among the cultivars at 30 kg ha­1 of fertilizer. at higher doses i.e. 50 kg ha­1 and 80 kg ha­1, uc 82 consistently maintained the most upright growth combined with higher yield among the cultivars. however, higher rates of chemical fertilizer that can cause increased accumulation of salt in soil and tis­ sue of tomatoes as reported by widders and garton (1992) and tuna et al. (2007) may therefore make it necessary to recommend application of 50 kg ha­1 of the fertilizer for upright growth habit and higher yield and for possible reduction in accumulation of the salt in tissues of tomato. the inclusion of other morphological parameters in the multivariate principal component analyses fur­ ther revealed the cultivars performance for other morphological traits. the selected cultivar in addition to combining high yield with most upright growth was able to maintain a reduced variation in basal and aerial stem diameter with a reduced height coupled with a reduced nfpp. these important traits are to be considered in the breeding of cultivars for upright growth habit and high yield in future breeding pro­ grams for tomato. the positioning of the tomato cul­ tivar uc 82 at the center of the polygon further indi­ cated the average performance of the cultivar for the important traits considered in this study while the polygon with other cultivars positioned at its vertices identified cultivars with exceptional performance for some specific traits. tropimech for example main­ tained a closer relationship with ph and wfpp in the biplot which confirmed the highest value it recorded for these traits. furthermore, the positioning of buffalo and kerewa in the direction of asd and nfpp also indicated their higher performance for these traits while roma vf positioned at opposite end of these vertices had lower performance for the traits. based on the average performance of uc 82 among the cultivars for all the traits, the cultivar is therefore recommended for upright growth habit and high yield under application of 50 kg ha­1 of n:p:k 15:15:15 fertilizer. stem diameter variation along the height of toma­ to plants is important to the upright growth habit of tomato cultivars. improved tomato cultivars however varied in their susceptibility to prostration at maturi­ ty. the robustness of the aerial stem diameter, the thin nature of the basal stem as well as wpf at har­ vest are some of the factors responsible for this growth habit. cultivars with robust basal stem com­ bined with reduced variation in stem diameter and appreciable yield will be a promising cultivar for pro­ ducing fresh tomato fruits in future. application of 50 kg ha­1 of npk 15:15:15 fertilizer can also reduce the susceptibility of the crop to prostrate growth at maturity. studies that consider reducing the spacing of the identified cultivar to achieve optimum yield ha­ 1 in comparison with others with higher yield and pgh is suggested to justify the reduced yield that upright growth of stem could cause. the growing of the tomato cultivars in locations with different agro­ ecology is recommended for a better varietal selec­ tion. acknowledgements the project was funded by tertiary education trust fund (tetfund) with a research code oou/ibr/011. the contribution of late s.k sanwo (ph.d) to the improvement of the proposal and the design of the experiment is very well appreciated. we appreciate our research students for their dedi­ cation to the project. also appreciated are the ilaraa farmers who allowed the experimental field to be sited on their farm. references amalfitano c., del vacchio l., somma s., cuciniello a., caruso g., 2017 ­ effects of cultural cycle and nutrient solution electrical conductivity on plant growth, yield and fruit quality of “friariello” pepper grown in hydroponics. ­ hort. sci., 44 (2): 91­98. anderson j.m., ingram j.s.i., 1993 ­ soil organic matter and organic carbon. pp. 62­70. in: anderson j.m., j.s.i. ingram (eds.), tropical soil biology and fertility: a hand book of methods. 2nd ed. ­ cab international, wallingford, uk., pp.221. bai y., lindhout p., 2007 ­ domestication and breeding of tomatoes: what have we gained and what can we gain in the future? 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(2005) reported that the combination of fresh irrigation water (1.2 ds m-1) and brackish water (7 ds m1) increased the yield level of melon in comparison to that of fresh water plants. in addition to the effects of salinity on crop yield, growth, leaf chlorophyll and mineral content are also affected. in this regard, vines that were grafted showed less clin the leaves compared to vines on their own roots (walker et al., 2004). among the rootstocks studied, p1103 was the best chloride excluder based upon the results of leaf chloride concentrations. on the other hand, despite that own rooted ‘sultana’ vines accumulated more cland na+ in the leaves, they were considered to better tolerate salinity conditions compared to those grafted based upon accumulating more dry matter (fisarakis et al., 2001). there is great debate regarding the tolerance of grapevines to salinity. moreover, contradictions can be found in the literature in terms of the salt tolerance of grapevine rootstocks implying that different factors are involved, which eventually determine grapevine response to salt stress. southey and jooste (1991) found that american hybrids performed poorly in response to salinity when used as rootstocks for the cultivar ‘colombard’. in addition, cavagnaro et al. (2006) concluded that argentinean cultivars performed better than european cultivars in an in vitro salinity evaluation study. regarding differences in ranking rootstocks, dardeniz et al. (2006) indicated that 41b was the most salt resistant rootstock, followed by 140ru and p1103, and the least resistant was 5 bb. on the other hand, a previous study showed that the highest salt resistance was obtained when p1003 was used as a rootstock (walker et al., 2002). our hypothesis is that alternate irrigation with diluted brackish water followed by irrigation with good quality water could result in, on one hand, saving fresh water for other uses and, on the other hand, establishing good vineyards. in addition, american rootstocks could perform differently under diluted brackish irrigation water. therefore, the objective of this study is to evaluate the vigor performance of ‘superior seedless’ cultivar grafted on three pot grown rootstocks: p1103, r110 and 41b under different diluted brackish water irrigation levels to detect a suitable irrigation period with diluted brackish water, without showing adverse effects on growth. 2. materials and methods plant and soil material three grape rootstocks were evaluated in this study: r110 (vitis berlandieri x v. rupestris), 41b (v. berlandieri x v. vinifera) and p1103 (v. berlandieri x v. rupestris). the rootstocks were purchased from les pépiniéristes du comtat, sarrians, france. after being imported, vitis vinifera ‘superior seedless’ was grafted on the rootstocks in a local nursery; (albushra nurseries, may, 2013). grafted plant materials were planted in polyethylene bags filled with peatmoss. the one year old grafted grapevine rootstocks were grown for several months to allow for the formation of a well-developed root system before applying treatments. fertilizers and fungicides were applied as necessary. the soil was brought from the southern jordan valley. the soil was relatively saline with an ec of 3.88 ds m-1. such soils are common in the jordan valley under the agricultural practices applied by farmers. soil was crushed and sieved through 1 cm sieve and plastic pots (the working volume of the pots was 44 l) were filled with 50 kg each in order to roughly have a bulk density of 1.14 g cm-3. a soil sample was taken to be analyzed for texture and some chemical properties (table 1). the pots were placed in a controlled greenhouse. grafted grapevines were transplanted in february and the growth was unified based on the number of buds and root length. the root system was cut back to 15 cm in length and the vegetative system was cut back to eight buds. qrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. i. growth performances 251 irrigation treatments and experimental design brackish water was brought from al-karameh dam located in the jordan valley and stored in a galvanized tank. a water sample was taken to be analyzed for some chemical properties (table 2). three levels of irrigation water salinity, in terms of electrical conductivity (ec), were applied: 1.5, 3.0 and 5.0 ds m-1 in addition to the 0.8 ds m-1 control. the treatments were prepared by mixing the dam water with tap water. a portable conductivity meter (model cond 3210, wtw, germany) was used to measure the ec and to obtain the determined salinity levels. a randomized complete block design was used with three blocks of 12 pots each. the grafted grapevines started to break the dormancy period during spring. composite fertilizer (20: 20: 20), urea and ammonium sulfate were applied to the grapevines and growth was again unified before applying the assigned treatments. irrigation with the assigned treatments started in may. all pots received the same amount of water whenever irrigation was applied. each pot received a total amount of irrigation water equal to 446 mm. irrigation was scheduled according to evaporation readings from free water surface (in mm) taken every 48 hours and corrected using proper grapevine crop coefficient of 0.30 (according to food and agriculture organization of the united nationsfao). chlorophyll content and growth parameters a spad-502 purchased from minolta co., ltd, japan, was used to measure the chlorophyll content of fully expanded matured leaves before irrigation with brackish water on april, 2014 and after irrigation with water on june, 2014 and november, 2014. shoot length, leaf area and number of newly formed leaves were recorded three times during the growing season of 2014 on june, august and october. leaf sampling and analysis fully expanded mature leaf samples were taken in june, august and october, 2014. leaf fresh and dry weights were determined. leaf water percentage was determined gravimetrically. after being dried, the leaf samples (5 leaves/plant) were analyzed for k, p, na, mg and ca. leaf area was also determined in november, 2014 by using a leaf area meter (am300, bioscientific ltd., uk). a cintra 5 spectrophotometer (gbc scientific equipment, australia) was used for analyzing p. flame photometer (jenway, germany) was used for analyzing k and na. ca and mg were analyzed by titration with edta. chloride was analyzed by titration with agno3. digestion and analysis methods followed the procedures of estefan et al. (2013). soil analysis the soil was analyzed for p, k, ca, mg, na, cl, ph and ec (1:1 soil: water extract) using the same previously mentioned instruments and procedures. statistical analysis all statistical analyses were performed using sas/stat version 9.2 and analysis of variance (anova) was conducted by the proc glimmix procedure. 3. results and discussion soil and water analysis one-year old grapevine rootstocks were grown on a calcareous clayey soil (45% caco3). soil chemical properties are presented in table 1. the soil used in the current study is saline with relatively high organic matter. such soils are common under agricultural practices applied in the jordan valley. in the jordan valley, farmers used to annually add organic materials, a practice that contributed to elevated salinity level. plants were regularly irrigated with diluted table 1 chemical properties of the soil brought from the southern jordan valley soil property value soil texture clay ph 8.25 ec 1:1 3.88 ds m-1 organic matter 1.99% ca 761.52 mg kg-1 soil mg 121.52 mg kg-1 soil caco 3 450.00 g kg-1 soil soluble k 29.00 mg kg-1 soil soluble na 85.00 mg kg-1 soil olsen-p 5.99 mg kg-1 soil so 4 27.73 mg kg-1 soil cl 1489.66 mg kg-1 soil table 2 chemical analysis of brackish water used for irrigation in the current research water properties value ph 8.62 ec 15.43 ds m-1 cl 6098.00 mg l-1 ca 801.60 mg l-1 mg 710.53 mg l-1 k 144.45 mg l-1 na 2335.00 mg l-1 adv. hort. sci., 2017 31(4): 249-256 252 brackish water at three salinity levels. the water chemical properties of the brackish water are presented in table (2). brackish water used in the current study is extremely saline with total dissolved solids of approximately 9875 mg l-1. chloride formed approximately 60% of the ionic composition of the brackish water (before dilution) followed by sodium, which formed approximately 23%. such high ec and cl and na concentrations justified the use of diluted brackish water obtained by diluting the latter with tap water in order to prepare irrigation water with three salinity levels as previously mentioned. the maximum salinity level of irrigation water was 5 ds m-1 electrical conductivity to avoid the buildup of salts in the growth medium far beyond the threshold ec of grapevines, which is approximately 2-3 ds m-1. spad chlorophyll reading the analysis of variance of the chlorophyll content of fully expanded mature leaves showed no interaction effect among rootstock, salinity and time. meanwhile, a significant rootstock by salinity interaction at (p≤0.05) was detected only in november as well as an expected salinity by time interaction (p≤0.05). diluted brackish water effects on ‘superior seedless’ chlorophyll content were clearly observed in june and november (table 3). in april, no interaction was noticed among rootstocksalinity combinations due to the fact that the soil used is originally saline and no considerable salt build up was expected yet as a result of irrigation. if nonsaline soil was used, findings would not be of practical significance. in addition, brackish water was diluted before being used for irrigation. salt build up could be expected if irrigation was practiced for longer period of time. however, salt build up is not favorable because it will adversely affect grape yield, which was not measured under the conditions of our experiment. as expected, chlorophyll content levels decreased for all three rootstocks (table 3). however, ‘superior seedless’ showed higher chlorophyll content when grafted on p1103 compared to r110 and 41b in november at salinity level of 5.0 ds m-1 (table 3). the highest reduction in ‘superior seedless’ chlorophyll content was observed when grafted on rootstock 41b, followed by r110 and the least reduction was observed for ‘superior seedless’ grafted on p1103. the influence of salinity levels varied with time and rootstock (table 3). chlorophyll content of r1 (p1103) and r2 (41b) was adversely affected in june particularly at s2 and s3. in november, the adverse effect of salinity levels on r1 and r2 at s2 and s3 was even more obvious. however, r3 (r110) was mainly affected by s3 particularly in november. this might suggest that diluted brackish water can be used for irrigation for a short period of time (i.e. from april to june) followed by irrigation with better quality irrigation water. this can be true mainly for r3 (r110) followed by r2 (41b) and for a longer period for r1 (p1103). additional supply of ca+2 can prevent the toxic effects of na+ on leaf photosynthesis (montesano and van iersel, 2007). it was indicated that the effect of salinity on leaf chlorophyll content is ion-specific and not due to a decrease in the osmotic potential of the nutrient solution. in our study, ‘superior seedless’ showed higher calcium ion content in both plant tissues and soil when grafted on p1103 compared to r110 and 41b. the mean ‘superior seedless’ leaf ca content (mg g-1 dry weight) grafted on the three rootstocks at 5.0 ds m-1 measured in october was 29.40 when grafted on p1103; whereas it was 26.00 and 19.80 when grafted onto 41b and r110, respectively (table 4). meanwhile, the soil ca content (mg g-1 soil) for the three rootstocks at 5.0 ds m-1 measured in october were as follow: 5.2, 4.7 and 4.5 when grafted on p1103, 41b and r110, respectively. this might explain the relatively better performance of p1103 under the conditions of the current study. moreover, ‘superior seedless’ grafted onto 41b showed a higher leaf mg content in october, however, the reduction magnitude in leaf mg content in october compared to june was the least in ‘superior r1 = p1103, r2 = 41b, r3 = r110. c = control 0.8 ds m-1, s1 = 1.5 ds m-1, s2 = 3.0 ds m-1, s3 = 5.0 ds m-1. (z) = the number in parenthesis indicates the reduction in the spad reading compared to the reading before treatment in april. table 3 the effect of brackish water treatments on the average ‘superior seedless’ chlorophyll content measured in june and november for the three rootstocks treatment june november r1 c 28.5±1.0 27±1.0 r2 c 27.6±1.2 27.6±1.1 r3 c 25.8±1.9 24.6±1.5 r1 s1 25.7±1.2 24.1±1.3 r2 s1 26.8±1.3 25.8±1.3 r3 s1 26.6±2.2 23.7±0.6 r1 s2 23.1±1.8 (3.3) (z) 22.9±1.6 (3.5) r2 s2 23±1.3 (4.7) 22.5±1.0 (5.2) r3 s2 25.1±1.1 (3.9) 22.2±1.2 (6.8) r1 s3 20.5±0.7 (6.7) 18.8±0.7 (8.4) r2 s3 23.8±3.2 (4.9) 12.9±3.6 (15.8) r3 s3 23±2.7 (7.4) 18.1±0.5 (12.3) qrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. i. growth performances 253 seedless’ grafted onto p1103 in comparison with r110 and 41b (table 5), which might also explain the relatively better performance of p1103. in addition, ‘superior seedless’ grafted onto p1103 showed a higher mg leaf content at salinity level (s2) in october compared to june. meanwhile, ‘superior seedless’ leaf mg content decreased when grafted on 41b and r110 at the same level of salinity. stem length and number of leaves the analysis of variance of stem length after irrigation with brackish water measured in june, august and october detected no interaction effect. however, a salinity effect was observed slightly in october. at the highest salinity level, ‘superior’ showed longer stem length when grafted on p1103 compared to r110 and 41b (table 6). however, the longer ‘superior seedless’ stem was not significantly different when grafted onto r110 but were significantly different when grafted onto 41b. this indicates that ‘superior seedless’ bud grafted on p1103 showed more growth compared when grafted onto 41b. another indication of the vigorosity and tolerance of p1103 was the total number of leaves counted in june, august and october. according to the analysis of variance, no interaction effect was detected. however, a main rootstock and salinity effect were detected in june, august and october. generally, in the months of august and october and at the levels of s2 (3.0 ds m-1) and s3 (5.0 ds m1), ‘superior seedless’ bud showed more number of leaves when grafted onto p1103 followed by r110 and the least number was when grafted onto 41b (table 7). this also supports the vigor and indication of tolerance of p1103. in addition, the results of the total counting coincide well with the shoot length r1 = p1103, r2 = 41b, r3 = r110. c = control 0.8 ds m-1, s1 = 1.5 ds m-1, s2 = 3.0 ds m-1, s3 = 5.0 ds m-1. table 4 the effect of brackish water treatments on the average ‘superior seedless’ ca leaf content (mg g-1 dry weight) grafted on the three rootstocks measured in april, june and november treatment june august october r1 c 40.6±2.9 50.0±6.7 25.2±4.5 r2 c 54.2±10.0 67.8±3.8 27.9±1.9 r3 c 43.0±4.1 46.8±3.3 19.5±2.0 r1 s1 42.5±1.5 42.5±5.0 24.2±1.3 r2 s1 55.8±4.9 56.4±5.1 21.8±5.0 r3 s1 36.1±0.0 41.7±3.7 24.0±1.4 r1 s2 47.8±6.2 58.3±16.0 21.8±4.0 r2 s2 52.9±2.9 74.5±0.8 23.5±3.4 r3 s2 40.9±3.0 46.5±3.4 23.4±3.0 r1 s3 38.2±6.4 59.9±12.9 29.4±9.7 r2 s3 44.9±12.7 72.2±9.4 26.0±5.0 r3 s3 42.5±1.0 56.9±4.0 19.8±1.7 r1 = p1103, r2 = 41b, r3 = r110. c = control 0.8 ds m-1, s1 = 1.5 ds m-1, s2 = 3.0 ds m-1, s3 = 5.0 ds m-1. (z) = the number in parenthesis indicates the reduction in the spad reading compared to the reading before treatment in april. table 5 the effect of brackish water treatments on the average ‘superior seedless’ mg leaf content (mg g-1 dry weight) grafted on the three rootstocks measured in april, june and november treatment june august october r1 c 18.1±3.0 19.3±1.2 7.6±1.9 r2 c 11.5±0.5 13.5±1.4 6.0±2.5 r3 c 11.5±1.9 18.1±3.5 7.9±0.8 r1 s1 12.8±1.1 12.8±4.9 7.6±2.6 r2 s1 13.6±3.3 12.5±1.1 7.5±0.9 r3 s1 14.6±2.2 14.8±0.5 7.9±1.1 r1 s2 11.5±0.2 13.6±1.4 13.6±3.2 r2 s2 12.5±1.6 11.7±0.7 10.2±0.8 r3 s2 10.2±2.2 15.4±3.8 7.8±0.9 r1 s3 11.0±1.0 14.8±1.9 9.2±2.3 (1.8) r2 s3 15.4±0.4 16.0±0.7 12.8±1.6 (2.6) r3 s3 11.2±1.8 17.3±2.1 8.4±1.1 (2.8) different letters in a column indicate significant differences at p≤0.05 according to fisher’s protected lsd. table 6 the effect 5.0 ds m-1 on average stem length (cm) for the three rootstocks measured in october table 7 effect of treatments on average number of ‘superior seedless’ leaves grafted on the three rootstocks counted in june, august and october rootstock stem length (cm) p1103 110±6.0 a 41b 89±5.0 b r110 100±10.0 ab r1 = p1103, r2 = 41b, r3 = r110. c = control 0.8 ds m-1, s1 = 1.5 ds m-1, s2 = 3.0 ds m-1, s3 = 5.0 ds m-1. treatment june august october r1 c 23.7±3.6 31±3.2 60.3±5.2 r1 s1 17.3±1.5 26.3±0.4 49±5.3 r1 s2 17±0.9 24.3±2.7 36.3±3.3 r1 s3 14.7±1.2 21.7±2.7 31.3±2.5 r2 c 22.3±0.7 27±1.4 51.3±3.6 r2 s1 18.3±1.0 23±1.2 41±5.6 r2 s2 16.7±0.8 20.3±1.8 31±3.9 r2 s3 14.3±0.4 18±0.7 30.7±2.5 r3 c 25.3±3.9 35.3±3.3 62.3±5.7 r3 s1 22.7±2.5 26.7±2.5 49.3±7.8 r3 s2 19.7±1.8 24±2.1 36±3.7 r3 s3 17± 1.2 20.7±1.5 33.7±2.9 adv. hort. sci., 2017 31(4): 249-256 254 results presented in table 6. leaf area regarding the ‘superior seedless’ leaf area, the analysis of variance revealed no interaction or rootstock effect but a salinity effect. the reduction of leaf area by water stress (gomez-del-campo et al., 2002) and by salinity treatments is very well documented on various crops such as tomatoes (montesano and van iersel, 2007), olives (al-absi et al., 2003) and chrysanthemums (lee and van iersel, 2008). the previous studies indicated the effect of salinity on leaf area is mainly through the osmotic effect of the solution which mainly depends on the total amounts of salts in the nutrient solution. our findings are consistent with the previous mentioned studies. leaf area decreased with increasing salinity levels (fig. 1). however, relatively larger ‘superior seedless’ leaves were noticeable when grafted on p1103 compared to 41b and r110. it is worth mentioning that the reduction of chlorophyll spad readings is attributed to the salinity effects, not to the leaf area since both parameters, chlorophyll spad and leaf area, were reduced with increasing the salinity levels. the vigor of p1103 and higher chlorophyll content, longer shoot and more leaves did not significantly increase the leaf fresh and dry weights (figs. 2 and 3). our findings also are in agreement with walker et al. (1997) who found that leaf relative water contents were not affected by rootstock or salinity treatments. figure 3 illustrates the effect of the three rootstocks and salinity levels on ‘superior seedless’ leaf water content. except for salinity level 1 (1.5 ds m-1), ‘superior seedless’ leaves when grafted on p1103 showed relatively higher water content compared when grafted on 41b and r110. the p1103 rootstock seems to activate a certain mechanism at high soil salinity levels (particularly at s2 and s3). on the other side, 41b and r110 were not adversely affected by the salinity levels. our findings are similar to those of walker et al. (1997) who found that the relative water contents were not affected by rootstock or salinity treatments. a correlation analysis has been conducted to figure out the major ions that had the greatest effect on soil salinity and consequently on rootstock performance. results showed that the correlation between soil ec and the concentration of cl, na, ca and mg ions were 0.97, 0.95, 0.83 and 0.5; respectively. this clearly proved that cl, na and ca were the major ions that influenced the response of rootstocks to soil salinity levels. such results reflected well the chemical composition of water used for irrigation (table 2). many researchers focused on cl exclusion, such as walker et al. (2004) who reported that p1103 was the best chloride excluder. our focus will be on na exclusion by reporting the leaf k: na ratio. concerning the leaf k:na ratio, as shown in figure 4, it can be seen that in august, the order of the rootstocks is as follows: r110>41b>p1103. however, na concentration in leaves became significantly higher than that of k (k:na ratio 1) for r110 and 41b, but particularly for 41b. on the other hand, in october, fig. 1 the effect of treatments on average ‘superior seedless’ leaf area (cm2) grafted on the three rootstocks measured on november, 2014. fig. 2 the effect of the three rootstocks and salinity on the ratio of ‘superior’ leaf fresh and dry weights (g) to leaf area (cm2). fig. 3 the effect of the three rootstocks and salinity levels on ‘superior seedless’ leaf water content. qrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. i. growth performances 255 terms of leaves compared to the other two rootstocks (fig. 5). except for the control, there were significant differences between p1103 and the two other rootstocks at the three salinity levels (fig. 5). this might indicate that p1103 was less affected by diluted brackish water irrigation. 4. conclusions scion is dependent on the rootstock for all things coming from the soil and there could be a considerable effect of rootstock on the vine performance (creasy and creasy, 2009). therefore, rootstock choice should be taken with careful consideration. p1103 (v. berlandieri x v. rupestris) rootstock seems to be a suitable choice for irrigating with diluted brackish water. irrigating ‘superior seedless’ vines grafted on p1103 with diluted brackish water, up to 3.0 ds m-1, from april until june could be a practical procedure to be adopted by grape growers in the jordan valley. however, irrigation should be followed by irrigation with fresh water especially that by the end of the experiment the soil ec reached approximately 6.0 ds m-1. in addition, our findings coincide well with the findings of many researchers such as (troncoso et al., 1999; walker et al., 2002). it does seem that grapevine rootstocks that have either v. rupestris or v. berlandieri in their parentage are good candidates for salinity tolerance. acknowledgements this research was funded by the scientific research support fund under the number agr/2/04/2011. we wish especially to acknowledge the help of marrwa atteyat and ayah awad for their technical assistance. references abdel gawad g., ghaibeh a., 2001 use of low quality water for irrigation in the middle east. proc. symp. sustainable management of irrigated land for salinity and toxic elements control, us salinity laboratory riverside, california, usa. al-absi k., qrunfleh m., abu-sharar t., 2003 mechanism of salt tolerance of two olive olea europaea l. cultivars as related to electrolyte concentration and toxicity. acta horticulturae, 618: 281-290. bustan a., cohen s., de malach y., zimmermann p., 41b and r110 rootstocks particularly at salinity levels s2 (3.0 ds m-1) and s3 (5.0 ds m-1) showed higher na concentration in leaves than that of k (very low k:na ratio). this might indicate that none of the investigated rootstocks is na excluder; particularly p1103. p1103, which seems to be na include, took up na in greater amounts than the other rootstocks starting from earlier stages of treatment applications. this mechanism (na uptake) could help the grapevine to relief the osmotic stress, by maintaining the water potential gradient and consequently increase water uptake. jogaiah et al. (2014) found that the maximum osmotic potential was recorded on p1103 rootstock. this might explain the relatively higher leaf water content observed in p1103 as mentioned above. growth after bud break in order to obtain data regarding cumulative effects of the treatments on number of ‘superior seedless’ leaves after bud break, total numbers of leaves/vine were counted on april of the following year. analysis of variance showed that there was no interaction effect however; rootstocks did show such an effect (p≤0.05). p1103 shows vigorous growth in fig. 4 ‘superior seedless’ leaf k:na ratio grafted on the three rootstocks at the different salinity levels measured in august and october. fig. 5 the effect of the salinity level treatments on the total number of leaves per grapevine counted in april of the following year for the three rootstocks. adv. hort. sci., 2017 31(4): 249-256 256 golan r., sagi m., pasternak d., 2005 effects of timing and duration of brackish irrigation water on fruit yield and quality of late summer melons. agric. water manag., 74(2): 123-134. bustan a., sagi m., de malach y., pasternak d., 2004 effects of saline irrigation water and heat waves on potato production in an arid environment. field crops research, 90(2-3): 275-285. cavagnaro j., ponce m., guzmán j., cirrincione m., 2006 argentinean cultivars of vitis vinifera grow better than european ones when cultured in vitro under salinity. biocell, 30(1): 1-7. creasy g., creasy l., 2009 grapes. crop production science in horticulture. cabi, cambridge, ma, usa, pp. 312. dardeniz a., müftüoğlu n., altay h., 2006 determination of salt tolerance of some american grape rootstocks. bangladesh j. bot., 35(2): 143-150. estefan g., sommer r., ryan j., 2013 methods of soil, plant, and water analysis: a manual for the west asia and north africa region. third edition. international center for agricultural research in the dry areas (icarda). fisarakis i., chartzoulakis k., stavrakas d., 2001 response of sultana vines (v. vinifera l.) on six rootstocks to nacl salinity exposure and recovery. agric. water manag., 51(1): 13-27. gomez-del-campo m., ruiz c., lissarrague j., 2002 effect of water stress on leaf area development, photosynthesis, and productivity in chardonnay and airen grapevines. american journal of enology and viticulture, 53(2): 138-143. jogaiah s., ramteke s., sharma j., upadhyay a., 2014 moisture salinity stress induced changes in biochemical constituents and water relations of different grape rootstock cultivars. intern. j. agron., 1-8. lee m.k., van iersel m., 2008 sodium chloride effects on growth, morphology, and physiology of chrysanthemum (chrysanthemum x morifolium). hortscience, 43(6): 1888-1891. mizrahi y., taleisnik e., kagan-zur v., zohar y., offenbach r., matan e., golan r., 1988 a saline irrigation regime for improving tomato fruit quality without reducing yield. j. amer. soc. hort. sci., 113(2): 202-205. montesano f., van iersel m., 2007 calcium can prevent toxic effects of na+ on tomato leaf photosynthesis but does not restore growth. j. amer. soc. hort. sci., 132(3): 310-318. niu g., rodriguez d., aguiniga l., 2008 effect of saline water irrigation on growth and physiological responses of three rose rootstocks. hortscience, 43(5): 14791484. paranychianakis n., angelakis a., 2008 the effect of water stress and rootstock on the development of leaf injuries in grapevines irrigated with saline effluent. agric. water manag., 95(4): 375-382. southey j., jooste j., 1991 the effect of grapevine rootstock on the performance of vitis vinifera l. (cv. colombard) on a relatively saline soil. s. afr. j. enol. vitic., 12(1): 32-41. troncoso a., matte c., lavee s., 1999 evaluation of salt tolerance of in vitro grown grapevine rootstock varieties. vitis, 38(2): 55-60. walker r., blackmore d., clingeleffer p., correll r., 2002 rootstock effects on salt tolerance of irrigated field grown grapevines (vitis vinifera l. cv. sultana). 1. yield and vigor inter-relationships. australian j. grape and wine res., 8: 3-14. walker r., blackmore d., clingeleffer p., correll r., 2004 rootstock effects on salt tolerance of irrigated field grown grapevines (vitis vinifera l. cv. sultana). 2. ion concentrations in leaves and juice. australian j. grape and wine res., 10: 90-99. walker r., blackmore d., clingeleffer p., iacono f., 1997 effect of salinity and ramsey rootstock on ion concentrations and carbon dioxide assimilation in leaves of drip-irrigated, field-grown grapevines (vitis vinifera l. cv. sultana). australian j. grape and wine res., 3: 66-74. impaginato 113 adv. hort. sci., 2019 33(1): 113-122 doi: 10.13128/ahs-24787 a new device to improve the mechanical winter pruning in olive trees hedgerows a. ottanelli 1, e. marone 2 (*), p. fiorino 1 1 dipartimento di scienze e tecnologie agrarie, alimentari, ambientali e forestali, dagri, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, campus coste s. agostino, via r. balzarini, 1, 64100 teramo, italy. key words: branches, fruit bearing, leaves, olea europaea, shoots, superintensive plantations. abstract: the economic success of superintensive olive plantations is mainly due to the full mechanization of the harvesting and pruning. while the advantage of straddling machines is undoubted, winter mechanical pruning determines falls in productions. this is due to the indiscriminate suppression of both fertile leafy shoots destined to fruiting and growth, and exhausted parts of the plant. to reduce this damage, an innovative device has been developed, applied to a pruning machine, able to selectively cut the “aged” parts of vegetation. the selection is achieved by an air’s fluid dynamic action obtained throughout defined and directional air jets able to push the young and flexible shoots upwards and downwards; in this way they are saved by the cut, regenerating at least one year in advance the new fruiting hedge. tests were carried out on the cultivar arbequina, tosca and sikitita, in three superintensive olive groves located in the province of grosseto, rome and latina, assessing the amount of leaves, shoots and branches, as well as fruits present at harvest, preserved from the pruning thanks to the action of the air flow, respectively for the east and west side of the rows. to get a profile of the biomass distribution along the cross section of the tree canopy, in the grosseto farm a trial was also carried out to better assess leaves, shoots and branches distribution in the canopy. the statistical data analyses immediately evidenced two different populations due to the selective pruning. the work highlighted the remarkable effectiveness of the air jet in safeguarding the flexible and leafy vegetation and allowed to quadruple leaf surface and production. 1. introduction the improved knowledge and agronomic techniques during the last decades have allowed the development of olive cultivation beyond the original distribution area (mediterranean basin), in new zones where olive growing represents one of the most promising crops (marone and (*) corresponding author: emarone@unite.it citation: ottanelli a., marone e., fiorino p., 2019 a new device to improve the mechanical winter pruning in olive trees hedgerows. adv. hort. sci., 33(1): 113-122 copyright: © 2019 ottanelli a., marone e., fiorino p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 november 2018 accepted for publication 9 march 2019 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 113-122 114 fiorino, 2012). since the beginning of the 1990s, the introduction of a canopy management and training system defined as “superintensive” contributed to the spread of olive cultivation (tous et al., 1997; tous et al., 2010; rallo et al., 2013), as the high number of trees per hectare (over 1600), with particularly short distances between the plants on the row; the close canopies form, such as in the vine, a single hedge; the system allows the complete mechanization of the harvesting, carried out with straddling machines that detach, intercept and storage the fruits at once, working continuously; the system also allows the mechanical pruning (vivaldi et al., 2015), performed by circular saws fitted on adjustable bars, by eliminating the vegetation along horizontal (topping) or vertical (hedging) planes. the continuous harvesting yards, based on the use of straddle machines, allow strong scale economies and guarantee the greatest efficiency with respect to other systems like shakers and vibrating combs, which can work in traditional or intensive farming systems (fiorino et al., 2010). the mechanical pruning using circular saws fitted on bars, borrowed from viticulture, can be applied to both intensive (individual canopies) and superintensive (hedge) plantations. the comparison between manual and mechanical pruning highlight a greater operative speed of the second (giametta and zimbalatti, 1997), partially compromised by the decreasing of productivity (ferguson et al., 2002; peça et al., 2002). in fact, the mechanical pruning simultaneously eliminates, together with the exhausted, also the young vegetation, influencing the canopy vegetative-productive ratios, as well as the distribution of the natural resources and of the light (cherbiy-hoffmann et al., 2012). in intensive plantations, it needs several years to evaluate the influence of regular winter pruning on plant productivity (dias et al., 2012) and, according to albarracín et al. (2017), depending on the intensity of the intervention, it can take up to two growth seasons to reconstitute a fruiting canopy and three to bring the plants back to full production. while in traditional and intensive plantations the mechanical pruning can be considered an economical and convenient alternative to the manual one, it is the only technique available to manage the canopy of adult plants grown in superintensive plantations. in this case its role changes, from tool to preserve the vegetative-productive plants equilibrium, to tool for constraining the plant dimensions within defined limits of height and width, required to perform the harvesting by straddling machines (vivaldi et al., 2015); as a consequence it is largely applied in these plantations, despite the negative consequences on the productive vegetation, due to the particular growth model of the olive tree. in fact, in the olive tree, the fruiting takes place on the vegetation of the previous year, simultaneously with the elongation of the shoots apex that will take fruits the following year; over the time, these shoot elongations progressively move away from the central leader of the tree and, as the competition between the different sinks (apical shoot growth and fruits energy demand), progressively become weak and less productive. at the same time, from the branch that originated them, some latent buds can sprout, fated to reiterate the cycle, by substituting the fertile vegetation that has already produced fruits (fiorino and marone, 2010). these vegetative structures coexist on the surface of the plants, or along a “wall” in the case of hedgerows, where the renewal vegetation is dragged downwards by the weight of the sprouts, being the wood of olive branches flexible, and due to the weight of olives, which also curve the young branches. the “aged” and exhausted parts are periodically eliminated by the winter pruning (fiorino and marone, 2010). an innovative device has been designed and built, inserted on a bar equipped with counter-rotating circular saws, able to delivering defined and directional air jets that push the shoots upwards and downwards, bending them. in this way the young flexible shoots inserted on the two or three year-old branches escape the action of the pruning machine blades, recovering their natural position after the course of the tool, while the short shoots inserted on the rigid vegetation that produced fruits, depleted and to remove, are cut. the present study would to test the efficiency of a new selective blower device, with the goals: a) to save leaves on the green wall, improving its efficiency in light interception, and b) to save a greater number of fertile buds since the first year after cutting and at least a part of the fruits resulting from the subsequent flowering. moreover, the study would determine the quantity of the biomass produced in olive hedgerows as byproduct of the mechanical pruning, characterized by a remarkable presence of leaves, to verify the possibility of alternative uses, in addition to those already tested for manual pruning residues that, mixed with olive pomace, contribute to the production of pellets (barbanera et al., 2016) and, as already demonstratottanelli et al. improving mechanical winter pruning 115 ed for pomace, to identify a possible use as supplements/components of livestock feed (gonzález et al., 2012; castellani et al., 2017; taticchi et al., 2017) or as a natural source of antioxidant compounds (talhaoui et al., 2015). 2. materials and methods plant material three different pruning trials were carried out in three different localities in high density plantations, on rows of olive trees trained with a central leader, allowing their natural lateral branching. 1) year 2014. the first trial was performed in the “castello di torrimpietra” farm, located in torrimpietra (rome) (41° 53 ‘43’’ north latitude, 12° 14’ 8’’ east longitude, 44 m asl), on trees of the cv. arbequina and tosca, 8 years old, with planting density of 3.8 x 1.6 m (2630 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was about 120 cm, the height of 2.20-2.40 cm. the useful height of the fruiting wall was 180 cm. the soil is sandy (over 60% sand), ph = 7.6. 2) year 2015. the second trial was performed in the “casale san giorgio” farm, located near latina (41° 37’ 7’’ north latitude, 12° 34’ 50’’ est longitude, 64 m asl), on trees of the cv. arbequina and sikitita, 5 years old, with planting density of 3.8 x 1.6 m (2630 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was 120 cm, the height of 2.00-2.20 cm. the useful height of the fruiting wall was 160 cm. the soil is clayey (36% clay), with a high percentage of sand (40%), ph = 6.7. 3) year 2018. the third trial was performed in the “tombolo” farm located in grosseto (42° 44’ 3’’ north latitude, 10° 59’ 10’’ est longitude, 8 m asl), on trees of the cv. arbequina, 10 years old, with planting density of 4.0 x 1.6 m (2500 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was 140-160 cm, the height of 2.00-2.20 cm. the useful height of the fruiting wall was 160 cm. the soil is sandy-silty (over 60% sand), ph = 7.2. the rows in the three farms are oriented northsouth. all the plants were subjected to fertigation, soil management by grassing between rows and weed control on the rows, and fight against spilocaea oleagina and bactrocera (dacus) oleae. in all trials the mechanical pruning was set to leave a maximum thickness of the canopy of the hedge of 80 cm (40 cm on the east side and 40 cm on the west side). to verify the efficiency of the air-jet apparatus, samplings were carried out to quantify leaves, shoots (up to 1 year old) and branches (over 1 year old) saved when cut as pushed by the air of the blower device towards the inside of the canopy and then come back to its original position. during the pruning, the machine proceeded along each row keeping constant the driving speed and the cutting depth of the blades, let the blower device operating for a defined number of spans (15-20 m each, depending on the plantation), alternating with spans where it remained off, and for each row on both sides (east and west orientation of the plants, respectively). the surveys were made at the center of the spans. to perform the surveys on the vegetation, a square frame (1.0 x 1.0 m) was used, placed in correspondence to the central axis of a plant, at a height of 1.0 m from the soil, and positioned at the theoretical cutting blades surface (fig. 1); all the plant material (leaves, shoots, branches) found outside this frame after the cutting was removed. table 1 shows the list of the number of plants chosen for each locality, from which the different samples of vegetal material were taken, used to carry out the experimental surveys, which concerned, for the east side and the west side, respectively: leaves number and surface, shoots length, branches length. in the third test, surveys were also made on the number of fruits present at harvest on pruned plants with and without the presence of the air flow, respectively, to evaluate the difference in the production ability of the plants determined by the two types of techniques, for the two sides of the rows (east and west). fig. 1 sampling units after the pruning in the casale san giorgio farm (2015): a) air flow off; b) air flow on. adv. hort. sci., 2019 33(1): 113-122 116 in the tombolo farm, the day before the winter pruning (march 2018), on 6 plants of the cv. arbequina, the amount of leaves, shoots and branches on unpruned trees was determined, separately for the east and the west side, to obtain a profile of the canopy biomass distribution along the transversal section of the hedgerow. at this aim, the vegetal material present in the selected sampling units (1 m2) was collected, keeping separated the outer 15 cm of the canopy (outer layer), corresponding to a “light” pruning, the next approximately 25 cm (intermediate layer), corresponding to a “severe” pruning, and the remaining 40 cm up to the permanent structures (inner layer) of the hedge. for all the samples the leaves number and the leaf surface, the leaves dry weight, the shoots total length, the branches total length have been determined. the leaf surface was measured by collecting 50100 leaves per sampling unit; for the trials before the pruning of 2018 in the tombolo farm the leaf area was instead determined through a sampling of at least 100 leaves completely developed per sampling unit (total 600 leaves), chosen in the different sections (outer, intermediate and inner) in which the canopy has been subdivided. the leaf area was determined by scanning and analyzing the image (uthscsa image tool it version 2.03), and the dry weight was determined for the same samples. all trials and surveys were carried out in the second half of march, after the winter cold and shortly before the vegetative restart. the selective pruning device the pruning machine (bmv-fl480s), designed for use in intensive olive groves, is equipped with a removable device able to perform selective cutting, unlike commonly adopted solutions, that cut all the shoots and branches which are in a predetermined position. this is achieved by the air’s fluid dynamic action. characteristics: the pruning machine used for the tests consists of a 240 cm cutting bar fitted on a frame that allows the adjustment in lateral height and inclination, composed of six blade disks with a diameter of 500 mm, a fluid dynamic flow generator and four air flow conveyors arranged in front of the cutting bar (fig. 2). in all the trials a vehicle of more than 80 hp was used. statistical analyses the experimental design was chosen to guarantee the uniformity of sampling units, deriving from the same starting population for each locality and cultivar. average and standard deviations of raw data related to the four treatment (east air on, east air off, west air on, and west air off) for each locality and cultivar were compared; since in this experiment authors were interested in the evaluation of the effectiveness in the performances of a pruning machine (i.e. kg of production or cm of elongation tree-1, or ha-1), only raw data were submitted to the parametric test, as logarithmic transformation is most suitable to express magnitude discrepancy; for each data set, tests were carried out to evaluate the normality of the distributions, and levene’s test were performed to evaluate homoscedasticity at 95% confidence level. since in the most of cases the data distribution indicates some significant nonnormality (the standardized skewness and/or kurtosis is outside the range of -2 to +2), and since significant difference amongst the standard deviations at the 95.0% confidence level were evidenced, neither analyses of variance nor non-parametric tests to compare the medians instead of the means could be performed; as a consequence, average values and s.d. were only reported; box and whiskers plot were built up to table 1 list of the sampling units related to different cultivar and locality fig. 2 a) cutting bar equipped with the selective pruning device; b) the pruning machine working along a row. codex samples provenience east air on east air off west air on west air off total 1 cv. arbequina, torrimpietra, roma (2014) 6 6 6 6 24 2 cv. tosca, torrimpietra, roma (2014) 9 9 9 9 36 3 cv. arbequina, casale san giorgio, latina (2015) 12 12 12 12 48 4 cv. sikitita, casale san giorgio, latina (2015) 4 4 4 4 16 5 cv. arbequina, grosseto, (2018) 12 12 12 12 48 total 43 43 43 43 172 ottanelli et al. improving mechanical winter pruning 117 compare the medians, as the effect of the applied air flow to the pruning machine is so high to generate two populations different at all. this aspect is very important to consider, as the standard deviation can play a meaningful role also when it results to high to allow the application of the common descriptive statistical tests (marone et al. , 2017). the same approach was applied to evaluate the differences in the fruit bearing parameters measured in olive trees of cv. arbequina (tombolo farm, 2018), and the same conclusions were drawn. one-way analyses of variance (anova) were performed to compare vegetative growth parameters in olive trees of cv arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2) before carrying out the pruning trials, evaluating the side effect (est/west); separation of means was performed by the fisher’s lsd test (p = 0.05). levene’s test to check the variance, and mood’s median χsquare test to evaluate the medians samples differences, if necessary, were also applied. all the computations were performed on the raw data by statgraphics centurion xv v. 15.0.04. a multivariate partial least squares-discriminant analysis (pls-da, supervised method), based on venetian blinds cross validation procedures, was applied on the data obtained from all the samples of the cv. arbequina, after applying the two alternative pruning methods, coming from the sampling units collected in the three different zones, to highlight the possibility of differentiating the sampling submitted to the pruning with the air flow on, based on the chosen canopy vegetative parameters. for a description of the method, see colzi et al. (2017). data were preprocessed by a logarithmic transformation (log+1) to express magnitude discrepancy. the analysis was performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab® r2015b (mathworks inc., natick, ma, usa). a factor analysis (fa) was performed on (log+1) data obtained from the sampling units of cv. arbequina (farm of tombolo, 2018), considering as factors both vegetative and productive parameters (leaves number and areas, shoots and branches length, fruits number). computations were performed by xlstat 2014.5.03. 3. results vegetative growth and fruiting parameters table 2 shows the data related to the canopy parameters measured on the (residual) vegetation outside the cutting surface: leaves number (n.) and surface (cm2), shoots total length (cm) and branches total length (cm). for each locality and cultivar, the average and standard deviation values of the sampling units (1 m2), obtained with and without air flow, are separately indicated for the two sides of the row (east/west). all the cultivars under study belong to the class of vigor suitable for their use in superintensive plantations. the data shows the great difference between the values of all the “on” tests parameters compared to those of the “off” tests. in particular, the number of leaves (and axillary buds) results in many combinations more than quadrupled in the “on” tests (table 2, fig. 3), and the same proportion is found in the leaf surface present after the cut which, in the west on test of the cv. tosca overcome the coefficient of 1.12 m2 of leaf area per m2 of cutting area. it can be also seen that, in general, the west side of the row is richer in leaves, compared to the east side, except the cv arbequina in casale san giorgio (2015) and tombolo farms (2018), which show a behavior that tends to be opposite. vegetation up to 1 year old shows the same table 2 differences in vegetative growth parameters in olive trees measured as residual vegetation in the survey units (1 m2) due to action of the selective device and the side (east/west) effect (average ± s.d.) samples 1, 3 and 5 refer to the same cv. arbequina. sample 2 to the cv. tosca, sample 4 to the cv. sikitita. treatment leaves (number) leaves (cm2) shoots (cm) branches (cm) 1 east off 99.0±65.2 471.6±350.4 273.8±201.2 21.0±35.3 east on 406.5±75.3 2060.7±359.6 1040.5±361.4 125.0±75.7 west off 179.4±100.8 795.4±434.2 368.3±226.5 27.8±20.9 west on 714.2±322.4 3322.3±1480.7 1134.0±428.5 118.2±56.8 2 east off 660.6±211.6 2542.5±926.3 782.2±220.1 66.3±40.2 east on 2358.1±564.7 8823.2±2098.2 2926.4±840.2 133.8±48.9 west off 855.7±255.9 3287.1±1124.6 855.4±233.6 78.2±33.4 west on 2980.3±665.3 11160.5±2527.3 3479.2±917.9 142.5±47.7 3 east off 115.7±65.1 542.7±305.3 270.2±83.3 5.2±9.0 east on 928.9±379.8 4356.5±1781.5 1453.9±552.6 57.08±35.9 west off 57.1±22.4 268.0±105.0 196.9±67.0 10.2±8.9 west on 596.0±232.4 2795.2±1090.2 1124.1±418.3 69.3±42.6 4 east off 15.0±10.0 46.8±31.0 200.0±92.5 9.2±14.8 east on 79.5±36.1 247.2±112.4 911.7±282.4 65.5±72.8 west off 22.3±10.9 69.5±33.9 256.5± 12.1 8.7±17.5 west on 103.3±21.4 321.3±66.4 1342.0±434.9 80.7±34.6 5 east off 35.6±15.2 167.2±71.3 99.0±34.3 2.4±4.5 east on 285.7±68.6 1335.6±321.9 572.9±223.9 48.3±37.5 west off 35.0±14.0 164.1±65.5 84.0±19.5 0.0±0.0 west on 572.7±223.9 2681.4±1050.1 943.9±295.2 29.2±12.8 118 adv. hort. sci., 2019 33(1): 113-122 trend. the amount of branches is generally negligible, except for the cv. tosca, which gives length values from 66 (east off) to 142 cm (west on). figure 4 shows the score plot and the summary statistics of the pls-da model, that compare the data related to all the samples of the cv. arbequina, coming from the sampling units collected in the 3 localities under test, after applying the two alternative pruning methods, based on the chosen vegetative parameters of the trees canopy. the model statistic indicators and the score plot clearly confirm the presence after pruning performed with and without air flow of two completely different populations. as the model is representative of three distinct agronomic situations (castello di torrimpietra, casale san giorgio and tombolo farms) and of the two sides (east/west) of the rows, this output confirms the validity of the air jet system in safeguarding a significant part of the most important vegetation useful for the fruit production and the subsequent growth of the fruiting shoots, subtracting it from the indiscriminate mechanical cutting inevitable using the current pruning machines without blower device. in the tombolo farm, the number of fruits per m2 of fruiting area (sampling unit) present on the plants of the cv. arbequina was also determined. the numerical differences between air on and air off units related to the two orientations (east and west), respectively, highlight the enormous advantage obtained using the selective blower device, exceeding 100 fruits (with an increase of 107 fruits for the east and 116 for the west) (see table in fig. 5). calculating an average increase of only 100 fruits per m2, multiplied by an useful area of about 8000 m2 ha-1, there would be a productive advantage already on the first yield after the pruning of 800000 fruits; multiplying this value by the average weight of 1 fruit of cv. arbequina in the area (about 2.0 g), gives over 1600 kg of increased product in average per hectare, compared to the production of 284 kg that would have been obtained without the support of the device, with an average increase of 412% in olive production, and a recovery of over 1.7 t of product already in the pruning year. in the figure 6 a biplot from factor analysis is shown, simultaneously representing the relationships amongst the vegetative and productive parameters measured on the cv. arbequina (tombolo farm, 2018). the first axis explains the 97.83% of the total variability in the data. the four populations deriving from the treatments and the orientation are well separated in the four plots of the diagram. the two clusters that represent the trials with the application of the blower device (on the right in the figure) are significantly related to the vegetative-productive parameters of the leaves. it is interesting to note how the parameters related to leaves, shoots and fruits are also influenced by orientation to the west, as shown by the direction and length of the arrows in the graph. fig. 3 box-and-whisker plots related to the leaves number and area as influenced by the flow (on/off) and side (est/west). 1-2: ‘arbequina’, 2014; 3-4: ‘tosca’, 2014; 56: ‘arbequina’, 2015; 7-8: ‘sikitita’, 2015, 9-10: ‘arbequina’, 2018. median notches, and mean (+) ± s.d. notches added to the plot show the estimation error associated with each median; if they do not overlap, highlight which medians are significantly different from which others at 95% confidence level. mean markers (+) indicate the location of samples mean. outliers are indicated (�) if present. ottanelli et al. improving mechanical winter pruning 119 canopy distribution of biomasses in hedgerows trained olive trees table 3 compares the vegetative parameters (leaves number and area, shoots and branches length) per m2 of the three sections in which the canopy has been transversely divided. an initial evaluation seems to indicate that, for the particular area of tombolo farm, the east/west orientation plays an important role in determining the vegetative parameters taken into consideration; in fact, in the west section, regardless of the depth of the evaluation, the leaves number and surface (cm2), and the shoots length (cm) is greater. branches cannot be taken into consideration, since they are absent from the outer part, and almost absent from the intermediate section. this advantage determined by the orientation, whose causes are to better investigate, seems to be present also in the plot of castello di torrimpietra farm for both the cultivar (arbequina and tosca) and in the plot of casale san giorgio farm limited to the cv. sikitita. table 3 partition of components (leaves number and surface, shoots and branches lenght) of the three canopy transversal sections in olive trees of cv arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2), side (est/west) (average ± s.d.). average leaves dry weight for the three sections. taking into account the east/west averaged values, it results that a “light” pruning (taking away the fig. 4 score plot of pls-da model and summary statistics for the cv. arbequina samples in the three different zones after the pruning, based on the measured vegetative growth parameters: 0) air off (red), 1) air on (blue). fig. 5 differences in fruit number in olive trees of cv. arbequina (tombolo farm, 2018). in the table: average ± s.d.; in the box and whisker plot: median notches, and average (+) ± s.d. fig. 6 biplot from factor analysis. relationships among leaves number (ln), leaves area (la), shoots length (sl), branches length (bl), fruits number (fn), and the four different treatment: east air on (e/on), east air off (e/off), west air on (w/on), west air off (w/off); cv. arbequina, tombolo farm, 2018. adv. hort. sci., 2019 33(1): 113-122 120 first 15 cm of the vegetation), removes about 1/3 of the leaves thus reducing 1/3 the leaf surface of the plant, and a “severe” pruning (at a depth of about 40 cm), would remove more than ¾ of the present leaves, leaving the final permanent structure with a highly reduced leaf apparatus. even considering the shoots length (cm) it is possible to evaluate the meaningful effect of the intervention: a “light” pruning would remove about 1/5 of the present shoots, while a “severe” pruning would leave only 35% of the shoots, thus eliminating most of the vegetation potentially productive. figure 7 shows an example of partition between the different components of the canopy for each of the three considered sections. it is also interesting to consider the amount of leaves biomass that, by the pruning, can be made available for other uses: a “light” pruning, limited to a 15 cm layer, could produce, in this typology of rows, 541 kg of leaves (dry weight), and further 692 kg (dry weight) would also be removed from the intermediate section, for a total of 1.2 t of dry weight leaves to be considered an additional resource and not longer as a residual. 4. discussion and conclusions mechanical winter pruning on adult hedges in superintensive plantations, unlike manual pruning, is a useful tool to control the size of the vegetative structure, thus decreasing its traditional role of baltable 3 partition of components (leaves number and surface, shoots and branches length) of the three canopy transversal sections in olive trees of cv. arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2), side (est/west) (average ± s.d.) average leaves dry weight for the three sections *there is more than a 3 to 1 difference between the smallest standard deviation and the largest. since the levene’s test p-value is less than 0.05 (0.0004) there is a statistically significant difference amongst the standard deviations at the 95.0% confidence level. since the mood’s median χ-square test is greater than or equal to 0.05 (0.102467), the medians of the samples are not significantly different at the 95.0% confidence level. section parameter east west average leaves dry weight (g) outer section (15 cm) leaves (no.) 689.1 ± 66.2 a 815.2 ± 86.9 b 67.69 (33.4%) leaves (cm2) 3232.0 ± 310.4 a 3823.3 ± 407.5 b shoots (cm) 1234.7 ± 30.6 a 1121.5 ± 111.3 b branches (cm) 0.0 ± 0.0 0.0 ± 0.0 intermediate section (about 25 cm) leaves (no.) 680.4 ± 39.7 a 1243.4 ± 88.2 b 86.57 (42.7%) leaves (cm2) 3191.3 ± 186.2 a 5831.5 ± 413.5 b shoots (cm) 2132.7 ± 510.7 a 2979.7 ± 232.3 b branches (cm) 214.0 ± 64.1 a 240.3 ± 82.0 a inner section (40 cm from the main tree axis) leaves (n.) 367.8 ± 145.1 a 708.4 ± 206.6 b 48.43 (23.9%) leaves (cm2) 1725.1 ± 680.6 a 3322.6 ± 968.9 b shoots (cm) 2016.2 ± 423.8 a 1817.4 ± 904.2 a branches (cm) 580.2 ± 234.3* 709.2 ± 71.3* fig. 7 example of partition of components (leaves, shoots, and branches) of the canopy transversal sections in a olive tree of cv. arbequina (tombolo farm, 2018) measured in a sampling units (1 unit = 1 m2), side west. a) outer section (15 cm); b) intermediate section (25 cm); c) inner section (40 cm from the main tree axis). ottanelli et al. improving mechanical winter pruning 121 ancer between vegetative growth and production (ferguson et al., 2002). in fact, by manual winter pruning, the aged, shaded and exhausted parts of the plant are selectively eliminated, to leave light and space for the parts destined to growth and to produce the following years (peça et al., 2002). mechanical winter pruning, on the other hand, proceeds by vertical sections, indiscriminately eliminating the part exceeding the size limits imposed by the use of straddling machines; this leads, as immediate result, as pointed out by several authors (vivaldi et al., 2015; albarracín et al., 2017), a sharp decline in productivity, a long unproductive period, due to the need to regenerate a fruiting canopy and, therefore, an economic damage that decreases the benefits offered by the mechanization of the harvesting (peça et al., 2002; albarracín et al., 2017). in this work the authors tried for the first time to overcome the problem by verifying the effectiveness of a tool that combines the action of circular saws with a jet of air which push the shoots away from the cutting area, allowing to reduce the removal of plants young leafy parts. the main effect of the air flow is evidenced by the presence of two population (air on and air off), totally different and distinct. the new air-jet system, compared to traditional pruning machines, preserves over 400% of the leaf surface and relative shoots length in the canopy zones where it has been applied, determining a proportional increase in production, already economically interesting the first year after the pruning. the difference in olive produced resulted higher than 1 t ha-1 using the air jet, demonstrating the true economic advantage of the new device, able to prevent an almost total fall in production in the winter pruning year. this improves the use of agronomic practices, keeping in mind that in superintensive olive groves, fertigation, soil and pests management are always to be applied, without exceptions (vivaldi et al., 2015). at the same time, the preservation of a larger leaf surface on the vegetation of the year, also guarantees a more suitable use of the solar radiation (cherbiy-hoffmann et al., 2012), which allows both the activation and development of new growth points, and the growth of new potentially productive vegetative structures on the shoots elongations, able to guarantee an adequate continuity of the production (fiorino and marone, 2010). further research will be needed to improve the efficiency of this tool, and better understand the mechanisms and the evolution of the different buds present in the different parts of the canopy, which regulate the distribution of resources, also verifying the possible causes determining the growth differences due to the orientation east/west of the row canopy. it is also important to determine the quality of the removed biomass which, in the case of “light” pruning, is exclusively composed of leaves and shoots, and could constitute a considerable amount of vegetal material to be used as a supplement/component of the livestock feed ration, or become an important source for the growing demand for phenolic antioxidants of nutraceutical interest (talhaoui et al., 2015; castellani et al., 2017). acknowledgements the authors are grateful to: dr. ing. stefano borio, bmv di borio valerio & c. s.n.c., alba (cn), italy, for his invaluable technical support and to having provided the pruning machine equipped with the new selective device; the farms: azienda casale san giorgio, latina, azienda castello di torrimpietra, roma, azienda tombolo, grosseto, to have allowed and hosted the trials, and the olive consultant alessandro mersi, for its helpful collaboration in the organization of the tests. references albarracín v., hall a.j., searles p.s., rousseaux m.c., 2017 responses of vegetative growth and fruit yield to winter and summer mechanical pruning in olive trees. sci. hortic., 225: 185-194. barbanera m., lascaro e., stanzione v., esposito a., altieri r., bufacchi m., 2016 characterization of pellets from mixing olive pomace and olive tree pruning. renew. energ., 88: 185-191. castellani f., vitali a., bernardi n., marone e., palazzo f., grotta l., martino g., 2017 dietary supplementation with dried olive pomace in dairy cows modifies the composition of fatty acids and the aromatic profile in milk and related cheese. j. dairy sci., 100(11): 8658-8669. cherbiy-hoffmann s.u., searles p.s., hall 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baiges j., 1997. planting density trial with ‘arbequina’ olive cultivar in catalonia (spain). acta horticulturae, 474(1): 177-180. vivaldi g.a., strippoli g., pascuzzi s., stellacci a.m., camposeo s., 2015 olive genotypes cultivated in an adult high-density orchard respond differently to canopy restraining by mechanical and manual pruning. sci. hortic., 192: 391-399. impaginato 535 adv. hort. sci., 2018 32(4): 535-540 doi: 10.13128/ahs-22474 effect of a novel hydrogel amendment and seedling plugs volume on the quality of ornamental/miniature tomato r.a.c. melo (*), m.h. jorge, n. botrel, l.s. boiteux brazilian agricultural research corporation, embrapa vegetables, brasilia df, brazil. key words: copolymer, pot plant, solanum lycopersicum l., transplants. abstract: the market for ornamental/miniature plants values aesthetic morphological characteristics, which give harmony to the potted plant. these traits depend on the growing media capacity to maintain quality and plant longevity. the use of hydrogels has increased recently in order to achieve visually attractive and long-lasting plants. thus, this study aimed to evaluate the effects of a novel hydrogel (h) amendment and seedling plugs volume (spv) on the quality of ornamental/miniature tomato. seedlings of tomato cv. brs finestra were produced in 200 and 162 plugs plastic trays with 18 cm³ trapezium-shaped plugs and 50 cm³ conically shaped plugs, respectively. 18 cm³ plugs and h amendment presented several significant responses for plant characters height, soluble solids, number of leaves, stem diameter, shoot dry matter and fruits weight per plant; making evident the advantages of using a growing medium of smaller volume and this copolymer amendement. although consistent results were obtained, a combination of both these factors in terms of an optimal aesthetic value and considering all the morphological traits could not be accomplished. therefore it’s necessary to study other elements such as plant nutrition and the use of plant growth regulators to complement them, aiming to promote better quality. 1. introduction miniature tomatoes can be grown singly in small pots, or more plants in larger hanging pots. they are also ideal for window boxes or garden borders because their plant canopy diameter is little (scott and harbaugh, 1995). these plants combine ornamental aspects of a well-proportioned, diminutive, tomato plant with tasting fruits that can be eaten. small plant sizes are ideal for commercial growing, shipping, and retail selling (scott and harbaugh, 1995). the commercial success of the cultivation of miniature/ornamental potted plants such as tomatoes and peppers depends on consumer appeal conferred by the plant beauty, quality, vigor, color, shape, and size of leaves and fruits. in addition to that, the cultivars must present canopy harmony and be able to develop in relatively small pots (costa et al., 2015). (*) corresponding author: raphael.melo@embrapa.br citation: melo r.a.c., jorge m.h., botrel n., boiteux l.s., 2018 effect of a novel hydrogel amendment and seedling plugs volume on the quality of ornamental/miniature tomato. adv. hort. sci., 32(4): 535-540 copyright: © 2018 melo r.a.c., jorge m.h., botrel n., boiteux l.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 january 2018 accepted for publication 28 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 535-540 536 non-miniature tomato cultivars tend to overgrow small containers, and their plant size is restricted by the container size (scott and harbaugh, 1995), producing commercially unviable plants, requiring the use of plant growth regulators (pgr) to achieve attractive compact potted grown plants (moraes et al., 2005). another important feature of ornamental plants is the maintenance of interior quality and longevity, a never-ending effort by producers (wang, 1992). growing medium ability to prevent drying out is desirable, especially in peat-based substrates. some growers report a benefit when plants are watered with a wetting agent prior to shipment, thus making it easier for retail clerks and consumers to thoroughly rewet the medium. interest has increased recently among growers and mass market buyers in using water-absorbing gels (nell, 1991). however, studies investigating the effects of hydrogel (h) application on ornamental plants are scarce and limited (ljubojević et al., 2017). particularly to ornamental/ miniature vegetables, this scenario can be considered negligible. in regard to this matter, a promising nanocomposite hydrogel developed by an innovative technique using calcium montmorillonite showed great swelling degree, higher than 2000 times in water. the formulated h with high calcium montmorillonite content (approximately 50.0% wt) as well as featuring high loading capacity and individual and simultaneous release, denotes an interesting material for agricultural applications (bortolin et al., 2016). thus, the present study has the objective of evaluating the effects of a novel hydrogel amendment and seedling plugs volume (spv) on the quality of ornamental/miniature tomato. 2. materials and methods plant material and growth conditions an experiment was conducted from july 18 (sowing) to october 25 (harvest), 2017 at embrapa vegetables 996 m altitude, 15° 56’ s, and 48° 08’ w brasília-df, brazil, in a glass-glazed greenhouse. a photoselective shading net (aluminet® ic 50 ginegar polysack®) was installed 2 m above the benches. the greenhouse presented an air temperature of 15°c minimum and 44°c maximum, average dli (daily light integral) of ≈ 11 mol.m-2.d-1 considering a 12h period of sunlight and 81% maximum and 11% minimum of humidity. measures were taking after seedlings transplantation 15 minutes apart by sensors connected to a datalogger (watchdog 1000 series micro station spectrum technologies®). seedlings were produced in plastic trays with 200 and 162 plugs (jks® 18 cm³ trapezium shaped plugs and 50 cm³ conically shaped plugs, respectively) using a peat moss based substrate (carolina soil®) (fig. 1 a). solanum lycopersicum l. cv. brs finestra was selected, being the first brazilian ornamental/miniature tomato cultivar released by embrapa as a product for a very specific and demanding market (giordano et al., 2001). they were transplanted at 36 das (days after sowing) to 1 dm³ pots (nutriplan® np14) filled with a pine bark-based substrate (rohbacher®) with the following characteristics: ec 0.4; ph 6.0; water holding capacity 50%; humidity 60%; density 185 kg m³. filling were complemented with 5 g of bokashi compost per pot (korin® garden bokashi). nutrients were supplied weekly during the experiment, with a solution developed for ornamental peppers containing 14.4, 1.95, 12.92, 2.5, 1.0, 2.44 mmol/l of n, p, k, ca, mg, s and 30, 5, 50, 40, 2 and fig. 1 (a) seedling plugs volume and visual comparison 50 cm³ (left) and 18 cm³ (right); (b) plants from 50 cm³ plugs with hydrogel amendment (left) and without (right); (c) plants from 18 cm³ plugs with hydrogel amendment (left) and without (right). melo et al. quality of ornamental, miniature tomato 537 0.1 mol/l of b, cu, fe, mn, zn and mo, respectively, according to costa et al. (2015). watering from seeding to the transplantation was performed twice daily with enough water to start the run off at the bottom of the trays. all other cultural practices were performed using technical recommendations for controlled environment miniature tomato cultivation (schwarz et al., 2014). plant morphological characteristics and fruit quality analysis all plant morphological characteristics were analyzed when a commercial stage was reached, meaning that when 50% of the plant population of each treatment had at least 30% of fully ripe fruits or visually marketable fruits with the maximum size and shape typical of growth for miniature/ornamental tomato. these agronomic characteristics are based on the morphological descriptors suggested by ipgri (1995) and were validated by costa et al. (2015) for ornamental peppers: (p) precocity expressed by the number of days between transplantation and commercial stage; dry matter content of shoot (sdm) parts were dried in oven at 70°c until constant weight and values were obtained by equation: sdm% = {dry weight (g)/fresh weight (g) x 100}; (ph) plant height (cm) measured using a ruler, from the stem bottom until the last fully expanded leaf; (sd) stem diameter (mm) measured above the cotyledon leaves using a digital caliper; (cr) plant canopy ratio obtained from between the longitudinal (ld) and transverse diameters (td), where the closer the value to 1, more circular is the canopy. (nl) number of leaves expressed by counting the number of leaves per plant; number of fruits per plant (nfp); number of fully ripe (nr); (fw) fruit weight per plant (g) expressed by the sum of different fruits ripening stages per plant; (fd) ripe fruits diameter (cm) obtained from the longitudinal diameter of 4 fruits from each treatment. the following fruit quality basic parameters were analyzed by aoac (2010) and mcguire (1992) methodologies, utilizing 4 fruits from each treatment: (ss) soluble solids (°brix); (ac) acidity; ss/ac ratio; color (c) evaluated by means of ripe fruits with the measurement of the colorimetric parameters l*, a*, b* c* and angle hue (h°) in fruits. the l * coordinate expresses the degree of clarity of the measured color (l = 100 = white; l = 0 = black), c* the intensity of the color and h ° the saturation of the color. statistical design and analysis the trial was conducted in a 2x2 factorial design in a complete randomized scheme, with six replications. the presence (amendment) and absence of h represented the first factor. spv18 cm³ plugs and 50 cm³ plugs represented the second factor. each replication was composed of 10 plants. the rate of h per substrate consisted of 2.0% (on volume/volume basis) and followed previous studies recommendations (bortolin et al., 2016). data were subjected to an analysis of variance (anova). all computations were performed with assistat® software (silva and azevedo, 2016). preliminary analysis indicated that ph, and npp presented a skew and overdispersed distribution, and it was required a transformation to normalize data. thus, their means were evaluated after square root transformation. normality of residuals was tested using shapiro-wilk test (alpha 5%) and the distribution presented as normal subsequently. 3. results plants grown utilizing 50 cm³ plugs required a shorter period of time to reach the ideal commercial stage precocity (p). when 18 cm³ plugs were used, this stage was reached at the same time, independently of h amendment (table 1). this differentiates the 50 cm³ plugs in 5 days (earlier) when compared to 18 cm³ plugs. a difference of 9 or 14 days when 50 cm³ plugs were amended or not with h, respectively, was also observed when comparing to 18 cm³ plugs. 50 cm³ plugs use resulted in plants with a very long stem (fig. 1 b). table 1 ornamental/miniature tomato precocity (p) stage reached according to hydrogel amendment and seedlings plugs volume p= when 50% of the plant population had at least 30% of fully ripe fruits or visually marketable fruits with the maximum size and shape typical of growth for miniature/ornamental tomato. plug volume p (days after transplantation) with hydrogel without hydrogel 18 cm³ 57 57 50 cm³ 43 48 adv. hort. sci., 2018 32(4): 535-540 538 ph values were significant for both factors and their interaction (tables 2 and 3), with 18 cm³ plugs and h combination reaching 19.63 cm. the general mean height value achieved in this trial (17.1 cm) is typical of the cv. brs finestra and is within the values quantified by scott and harbaugh (1995) evaluating different miniature tomatoes, ranging from 9 cm with cv. micro-tom to 25 cm with cv. micro-gold. characters of aesthetic significance, such as plant architecture, number, position, and color of fruits, leaves shape and density are some of the reasons that ornamental species of the solanaceae family are admired, being strictly related to plant longevity and to facilitate cultural handlings (neitzke et al., 2016). one of these traits, nl, is consistent with the cultivar and growing conditions, presenting a significant response for all the studied factors. the interaction of 18 cm³ plugs and h enhanced the nl to 51.6 (table 2) which can be perceived by the observation of leaves density in fig. 2 d, but this response was not converted into a greater nfp. cr represents the aspect of the aerial parts of the plant, where the closer the value to 1, more circular it is the canopy. therefore, when 50 cm³ plugs were used, a more circular shape was attained (ld/td = 1.16) (table 2). this format has a greater visual appeal, which is often decisive in the choice of the consumer to become more attractive and makes it easier to handle (costa et al., 2015), although with plants being long-stemmed, a visual aspect uncommon for miniature tomatoes was observed, resembling a palm tree shape (fig. 1 b). sdm and sd values were significant for both treatments (table 3). costa et al. (2015) evaluated the quality of ornamental pepper using two substrates and genotypes, founding distinct sdm content responses, with values ranging from 20.58% to 26.54%, corroborating with the best results here found. sd results presented in this study (5.53 mm with h amendment and 5.55 mm from 18 cm³ plugs) table 2 interaction between hydrogel and seedling plugs volume for plant ornamental/miniature tomato characters means followed by the same lowercase letters in the columns and capital letters in the lines do not differ by tukey test at 5% probability. fig. 2 ornamental/miniature tomato plants visual aspect when precocity stage was reached. (a) from 50 cm³ plugs with hydrogel amendment; (b) from 50 cm³ plugs without hydrogel amendment; (c) from 18 cm³ plugs without hydrogel amendment; (d) from 18 cm³ plugs with hydrogel amendment. table 3 hydrogel amendment and seedlings plug volume effect on ornamental/miniature tomato plant characters means followed by the same lowercase letters in the columns and capital letters in the lines do not differ by tukey test at 5% probability. plug volume 50 cm³ 18 cm³ plant height (cm) with hydrogel 16.49 ab 19.63 aa without hydrogel 16.30 aa 16.02 ba soluble solids (°brix) with hydrogel 4.65 ab 5.35 aa without hydrogel 4.50 ab 5.20 aa number of leaves (per plant) with hydrogel 37.41 ab 51.60 aa without hydrogel 37.45 ab 43.88 ba canopy ratio with hydrogel 1.16 ab 1.32 aa without hydrogel 1.23 aa 1.26 aa plant characters hydrogel plug volume with without 50 cm³ 18 cm³ stem diameter (mm) 5.53 a 5.31 b 5.29 b 5.55 a shoot dry matter (%) 19.49 a 18.37 b 18.31 b 19.55 a fruit weight per plant (g) 68.39 a 67.17 b 77.39 a 58.17 b number of fully ripe fruits 3.08 a 2.43 b number of fruits per plant 9.90 a 6.01 b melo et al. quality of ornamental, miniature tomato 539 (table 3) are inferior but consistent with the ones found in the work of backes et al. (2007) with ornamental pepper. they obtained an sd of 6.42 mm as the best result using controlled release fertilizer mixed to a commercial substrate. even though sd values in this study were significant to h and spv, only a slight increase was observed amongst treatments. nfp, nr, and fw values were superior with the use of 50 cm³ plugs (table 3). with a compact canopy and well-distributed leaves, the photosynthetic process that depends on the interception of light energy most likely was converted into chemical energy in an efficient way, resulting in this positive response. ss and ac values were significant for spv and also for the interaction of 18 cm³ plugs with h. the values of 5.35 and 0.61 achieved for ss and ac, respectively, are comparable to 5.37 and 0.65 from cv. micro-gold bred by scott and harbaugh (1995), ensuring that cv. brs finestra produces mild tasting fruits that can be appreciated/consumed. ni (general mean value of 1.52), fd (general mean value of 3.1 cm), ss/sc ratio (general mean value of 8.38) and c (general mean value of l* 40.67; c* 51.05 and h° 48.51) were not significant (ns) to both treatments. for c values, although being ns, ripe fruits exhibited an intense red coloration (fig. 1b). this color is favored by consumers, a point which is believed to possess the highest carotenoids content such as β-carotene and lycopene (kader et al., 1977). 4. discussion and conclusions plants grown with 50 cm³ plugs, in the presence or absence of h amendment, demanded a shorter period of time to reach the ideal commercial stage precocity (p). precocity is a very important attribute, as it would allow the grower to commercialize plants earlier and to reutilize the spaces emptied in the greenhouse benches. but for its recommendation, the fact that ph in this plug volume resulted in an undesired visual aspect displaying a very long stem, needs to be taking in to account. the 50 cm³ plugs were significant for nfp, nr, ng, and fw characters as well. an efficient interception of light with plants produced using this plugs made most likely converted chemical energy into fruits, being a response of a compact canopy with well-distributed leaves. 18 cm³ plugs were significant for sd, sdm and fw, making evident the advantages of using a smaller volume of substrate, which can be fully employed for the growing of ornamental/miniature tomatoes in 1 dm³ pots. the use of h resulted in higher sd, sdm and fw values, considered fundamental in the aspect of the plant canopy and its longevity. h and spv interaction responded differently to the analyzed plant characteristics, with 18 cm³ plugs and h amendment together showing significant responses only for ph and nl. in conclusion, although several consistent results for plant characters were obtained when 18 cm³ plugs and h were used solely, a combination of both in terms of an optimal aesthetic value and considering all the morphological traits could not be accomplished. therefore it’s necessary to study other elements such as plant nutrition and the use of pgr to complement them, aiming to promote better quality. references aoac, 2010 international official methods of analysis of aoac. 18th ed. association of analytical chemists, gaithersburg, md, usa. backes c., fernandes f.m., krohn n.g., lima c.p., kiihl t.a.m., 2007 produção de pimenta ornamental em função de substratos e doses de adubação com fertilizantes de liberação lenta e tradicional. scientia agraria paranaensis, 6: 67-76. 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(folder in portuguese). ipgri, 1995 descriptors for capsicum (capsicum spp). international plant genetic resources institute, rome, italy, pp. 110. kader a.a., stevens m.a., albright-holton m., morris l.l., algazi m., 1977 effect of fruit ripeness when picked on flavor and composition in fresh market tomatoes. j. amer. soc. hortic. sci., 113: 724-731. ljubojević m., ognjanov v., maksimović i., čukanović j., dulić j., szabò z., szabò s., 2017 effects of hydrogel on growth and visual damage of ornamental salvia species exposed to salinity. clean soil, air, water, 45(2): 1-12. mcguire r.g., 1992 reporting of objective color measurements. hortscience, 27: 1254-1255. moraes p.j., saraiva grossi j.a., de araújo tinoco s., 540 adv. hort. sci., 2018 32(4): 535-540 henriques da silva d.j., cecon p.r., barbosa j.g., 2005 ornamental tomato growth and fruiting response to paclobutrazol. acta horticulturae, 683: 327-332 neitzke r.s., fischer s.z., vasconcelos c.s., barbieri r.l., treptow r.o., 2016 pimentas ornamentais: aceitação e preferências do público consumidor. horticultura brasileira, 34: 102-109. nell t.a., 1991 how to make long-lasting top performers out of your pot mums. grower talks, 54: 67-80. schwarz d., thompson a.j., kläring h.p., 2014 guidelines to use tomato in experiments with a controlled environment. front. plant sci., 5(625): 1-16. scott j.w., harbaugh b.k., 1995 ‘micro-gold’ miniature dwarf tomato. hortscience, 30: 643-644. silva f.a.s., azevedo c.a.v. 2016 the assistat software version 7.7 and its use in the analysis of experimental data. african j. agric. res., 11(39): 3733-3740. wang y.t., 1992 hydrophilic polymer and wetting agent had limited effect on growth and postproduction performance of poinsettia. subtr. plant sci., 45: 32-35. impaginato 183 adv. hort. sci., 2020 34(2): 183­189 doi: 10.13128/ahsc­8306 influence of plant biostimulant and spacing on production and postharvest conservation of watermelons cv. quetzali v.f.s. de paula, j.c. vilvert (*), n.o. de aráujo, i.b. do nascimento, j.f. de medeiros, e.m.m. aroucha federal rural university of the semi‐arid region, mossoró, rio grande do norte, brazil. key words: citrullus lanatus, cold storage, crop set®, physico­chemical quality, plant growth regulators. abstract: the aim of this study was to evaluate the influence of pre­harvest application of plant biostimulant crop set® and different plant spacings on the production attributes and postharvest quality of watermelon ‘quetzali’. the experiment was set up in a completely randomized split­plot (3 × 2 × 4) design, corresponding to three plant spacings (0.40, 0.45 and 0.50 m), application of the plant biostimulant (with and without) and four storage periods at 10°c and rh 90% (0, 14, 21 and 28 days). fruits were assessed after harvest in terms of average mass of fruits, number of fruits per plant and yield, and throughout the storage periods for flesh firmness, soluble solids content (ssc), titratable acidity (ta), ssc/ta ratio, ph and total soluble sugars (tss). the average mass of fruit (4.02 kg) was higher in the larger spacing without application of biostimulant. the pre­harvest application of plant biostimulant negatively influenced ssc of fruits, depending on the plant spacing and storage periods. for ta and tss con­ tent, the effect of this product varies with plant spacing and storage days. the lower plant spacing provided higher tss to the fruits. 1. introduction watermelon [citrullus lanatus (thunb.) matsum. and nakai] is a veg­ etable belonging to the cucurbitaceae family that has great economic and social expression, with a world production of 118,413,465 tonnes in 2017. among the four largest producers are china, turkey, iran and brazil, which together are responsible for 76% of global watermelon production (faostat, 2019). in brazil, the northeast region is an important pole of agricultural crop, with soil and climate conditions favorable for watermelon cultivation throughout the year. cultural practices are always studied to increase yield and quality of the products. watermelon is the second most export­ ed vegetable in brazil and quetzali is an early commercial cultivar, with (*) corresponding author: jcvilvert@gmail.com citation: de paula v.f.s., vilvert j.c., de aráujo n.o., do nascimento i.b., de medeiros j.f., arou­ cha e.m.m., 2020 ­ influence of plant biostimu‐ lant and spacing on production and postharvest conservation of watermelons cv. quetzali. ‐ adv. hort. sci., 34(2): 183­189. copyright: © 2020 de paula v.f.s., vilvert j.c., de aráujo n.o., do nascimento i.b., de medeiros j.f., aroucha e.m.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 march 2020 accepted for publication 21 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 183­189 184 the harvest at 70 days. this cultivar has average mass of 2.5 to 6.0 kg, green skin color with dark and thin streaks, red pulp with few seeds and high soluble solids content, which are desirable characteristics for consumers (dia et al., 2016). during the vegetable development an unable cul­ tural management of plant, in the field, can cause irreversible damage in fruit cells, which can affect their shelf life. the management techniques can mor­ phologically and physiologically alter the plant, inter­ vening in its productive potential and affecting the quality and the conservation of the fruits are plant spacing (gomes et al., 2017) and use of plant bios­ timulants (martins et al., 2013). species grown in high densities, especially cucur­ bits, produce a large number of fruits per area, but with small size, weight and number per plant, which may affect their development, and consequently, the final quality of fruits (sabo et al., 2013; oga and umekwe, 2016). on the other hand, plant biostimulants are sub­ stances applied to plants that enhance their nutrition efficiency, abiotic stress tolerance and/or quality traits (jardin, 2015). they can be defined as mixtures of one or more plant growth regulators with other compounds of a different chemical nature, such as mineral salts (castro and pereira, 2008), which are applied in various species of fruits and vegetables with the aim of increasing its production and quality (leão et al., 2005; costa et al., 2008; martins et al., 2013; aroucha et al., 2018). crop set® (improcrop­kentucky­usa) is a com­ mercial product registered in brazil as foliar fertilizer, containing 1.5% manganese, 1.5% iron and 1% cop­ per, it is a composed of agave (yucca shidigera) extracts and mineral micronutrients with cytokinin­ like action (leão et al., 2005). the use of plant regula­ tors belonging to the cytokinin group can increase the fruit size (tecchio et al., 2006; ainalidou et al., 2016) because inducing cell division and thus stimu­ lating cell growth in plant tissues (taiz et al., 2015). nevertheless, its influence on the yield and water­ melon conservation was not reported yet. the water­ melon shelf life is around 2­3 weeks at 10­15°c (maynard, 2001), depending on cultivar. a good qual­ ity is reached as soluble solid is above 8% (tlili et al., 2011). the aim of this study was to evaluate the influ­ ence of pre­harvest application of plant biostimulant crop set® and different plant spacings on the produc­ tion attributes and postharvest quality of watermel­ on cv. quetzali. 2. materials and methods the experiment was carried out in mossoró, state of rio grande do norte, brazil (4° 39’ 39” s, 37° 23’ 13” w, and 20 m of altitude). the climate of the region according to köppen climate classification is bswh type (hot and dry). the region has average annual temperature of 27°c, average annual precipi­ tation of 673.9 mm, unevenly distributed, and air rel­ ative humidity of 68.9%. the soil of the experimental area is classified as neossolo quartzarenico (santos et al., 2018) and its physical­chemical properties are: ph (h20) = 5.52; organic matter: 5.5%; p (mehlich) = 32 mg dm­3; k = 96.5 cmol dm­3; ca = 1.60 cmol dm­3; mg = 0.43 cmol dm­3; sand = 935.8 g kg­1; silt = 26.5 g kg­1; clay = 37.7 g kg­1; bulk density = 1.48 g cm­3; soil particle density = 2.69 g dm­3; and total porosity = 0.45 m3 m­3. the results of the chemical analysis of the irrigation water are: ph = 7.70; electrical conductivity = 2.11 ds m­1; k+ = 0.12 mmol l­1; na+ = 5.02 mmol l­1; ca2+ = 10.43 mmol l­1; mg2+ = 3.05 mmol l­1; cl­ = 11.48 mmol l­1; co3 ­2 = 0.30 mmol l­1; hco3 ­ = 3.70; sodium adsorp­ tion ratio = 1.9 (mmol­1)0.5; hardness = 5.4 mg l­1; cations = 18.7 mmol l­1; and anions = 15.4 mmol l­1. the experiment was set up in a completely ran­ domized split­plot (3 × 2 × 4) design, with six repli­ cations, each one corresponding to a plant. the plots consisted of plant spacings (2.0 x 0.4 m; 2.0 x 0.45 m and 2.0 x 0.5 m), application of the plant biostimulant crop set® (with and without), and postharvest storage (0, 14, 21 and 28 days) in the subplot (fig. 1). seeds of watermelon cultivar quetzali were used. plant biostimulant was sprayed with a 20 l backpack sprayer, with stainless steel cone nozzle with flow rate of 615 ml/min, at 18 and 25 days after transplanting, applying 8 and 16 ml of crop set®, respectively, regularly on the plants, always in the same way. the dose of the biostimulant was determined according to the manufacturer’s rec­ ommendations. the harvest was realized at 65 days after of seedling transplanting. fruits were transported to laboratory of food technology of the federal rural university of the semi­arid region, where part of the fruits were characterized previously by sam­ pling six fruits per treatment. the other part was stored in a cold chamber at 10±2°c and rh 90±1%, for 14, 21 and 28 days. after each storage periods, the fruit quality was evaluated. fruits were assessed after harvest for production de paula et al. ‐ effects of biostimulant and spacing on watermelon 185 in terms of number of fruits per plant, average mass of fruits and yield. at harvest and during cold stor­ age, the quality characteristics were: flesh firmness (n), measured with a 12­mm tip manual penetrome­ ter model 327 ft (mccormick, usa); soluble solids content (ssc, in °brix), measured with a refractome­ ter (pr ­ 100, palette, atago co., ltd., japan); titrat­ able acidity (ta, in % of malic acid), analyzed by titrimetry; ssc/ta ratio; ph, evaluated using a digital potentiometer with glass membrane, calibrated with buffers of ph 7 and 4, according to the method of the association of official analytical chemists (aoac, 2016); and total soluble sugars (tss), measured by the antrona method, as described by yemn and willis (1954), expressing results in percentage (%). data were subjected to analysis of variance and means of the biostimulant and plant spacings factors were compared by the tukey test (p≤0.05). the effect of storage periods was evaluated by regression analy­ sis. all statistical analysis were carried out using soft­ ware sisvar 5.3 (ferreira, 2014). 3. results there was effect of plant spacing and biostimulant application on the average mass of fruits. while, the production attributes as number of fruits per plant and yield were not affected by plant spacing or appli­ cation of plant biostimulant (table 1). during the fruit storage, there was a significant interaction effect between plant spacing, biostimulant application and storage periods on ssc (fig. 2), ta (fig. 3) and tss (table 2) of fruits. also, there was an isolated effect of the storage periods on the ssc/ta fig. 1 ­ scheme representing the treatments applied to the watermelon fruits. production attribute application of crop set plant spacing 40 cm 45 cm 50 cm average mass (kg) with 3.43 aa 3.51 aa 3.92 aa without 3.41 ba 3.47 ba 4.02 aa number of fruits per plant with 1.16 aa 1.06 aa 1.43 aa without 0.97 aa 1.39 aa 1.58 aa yield (t ha­1) with 11.77 aa 10.20 aa 13.90 aa without 10.19 aa 13.38 aa 14.29 aa table 1 ­ average mass, number of fruits per plant and yield of ‘quetzali’ watermelon depending on crop set ® application means followed by the same letter do not differ by the tukey test (p≤0.05). uppercase letters compare plant spacings and lowercase let­ ters compare application of biostimulant. fig. 2 ­ means followed by the same letter do not differ by the tukey test (p≤0.05). uppercase letters compare the sto­ rage periods within the biostimulant x spacing combina­ tion; lowercase letters compare the presence (a) or absence (b) of the crop set® application within the stora­ ge period x biostimulant combination; italic lowercase letters compare the means of the plant spacings within storage periods x biostimulant combination. dms for sto­ rage periods= 1.811; dms for biostimulant application= 1.374; dms for plant spacing= 1.649. adv. hort. sci., 2020 34(2): 183­189 186 ratio, flesh firmness and ph of fruits (fig. 4). fruits without application of biostimulant had the highest average mass in the larger plant spacing (50 cm) (table 1). the ssc increased, from 0 to 28 days, in fruit from plants on 45 cm spacing, with crop set® application, and non­spayed plants in growth on 40 and 50 cm spacings (fig. 2). the biostimulant application only influenced on this physicochemical parameter at the day of the harvest, when fruits produced on 45 cm fig. 3 ­ means followed by the same letter do not differ by the tukey test (p≤0.05). uppercase letters compare the sto­ rage periods within the combination of biostimulant x spacing; lowercase letters compare the presence (a) or absence (b) of the crop set® application within the stora­ ge period x biostimulant combination; italic lowercase letters compare the means of the plant spacings within storage periods x biostimulant combination. dms for sto­ rage periods= 0.012; dms for biostimulant application= 0.009; dms for plant spacing= 0.011. storage period (days) application of crop set® no application of crop set® 40 cm 45 cm 50 cm 40 cm 45 cm 50 cm 0 7.66 aaa 5.23 bbb 7.80 abaa 6.53 abaa 7.67 aaa 7.66 aaa 14 7.74 aaa 8.30 aaa 8.55 aaa 8.74 aaa 6.91 aaab 6.26 abbb 21 4.52 baa 4.80 baa 4.48 caa 5.14 baa 4.36 baa 4.94 baa 28 4.18 caa 4.48 baa 5.49 bcaa 4.43 baa 5.73 abaa 5.06 baa table 2 ­ ssc/ta ratio (a), flesh firmness (b) and ph (c) of ‘quetzali’ watermelon depending on storage periods means followed by the same letter do not differ by the tukey test (p≤0.05). uppercase letters compare the storage periods within the combination of biostimulant x spacing; lowercase letters compare the presence (a) or absence (b) of the crop set® application within the storage period x biostimulant combination; italic lowercase letters compare the means of the plant spacings within storage periods x bio­ stimulant combination. dms for storage time= 2.499; dms for biostimulant application= 1.896; dms for plant spacing= 2.275. fig. 4 ­ ssc/ta ratio (a), flesh firmness (b) and ph (c) of ‘quetzali’ watermelon depending on storage periods. de paula et al. ‐ effects of biostimulant and spacing on watermelon 187 spacing with crop set® application had lower ssc than fruits by plants without application (fig. 2). the reduction in spacing from 50 to 45 cm possibly increased the competition for water, mineral and luminous resources, leading to a reduction in the size of the fruits and, consequently, to an increase in the ssc due to the concentration effect. besides that, fruits by non­sprayed plants in 45 and 50 cm spacings showed the highest ssc at 0 and 28 days, respective­ ly. it is important to highlight that fruits produced on 40 cm spacing did not differed from ssc values of higher plant spacing, and therefore is the plant spac­ ing with the best effect on ssc of fruits (fig. 2). during the storage, the ta of the fruits decreased as a function of storage periods (fig. 3). when crop set® was applied, there was an effect of plant spac­ ings only at the harvest day, in which fruits of 40 cm spacing shown a lowest ta than fruits of 45 and 50 cm spacings (fig. 3a). no­application of crop set® did not affect the ta of fruits under different spacings (fig. 3b). when comparing fruits with and without applica­ tion, we observed crop set® effect at zero and 21 days of storage. at the harvest day, fruits of plants cultivated in 40 cm spacing had lower ta with crop set® application, while in 45 cm plant spacing, the fruits had higher ta values with application of plant biostimulant. at 21 days of storage, in fruits by 40 cm plant spacing, crop set® application influenced the ta of fruits, increasing this value compared to the fruits without application of the biostimulant (fig. 3). with crop set application, fruits in 40 cm plant spacing showed a highest sugar content at the first 14 days of storage. on 45 and 50 cm spacing, fruits had the highest sugar content at 14 days, with fur­ ther reduction of these values (table 2). in all plant spacings, without crop set application, fruits shown high sugar content until 14 days of storage, followed by the decrease of these values. when comparing fruits with and without crop set® application, we observed that the plant biostimulant reduced sugar content of 45 cm spacing fruits at harvest. in con­ trast, this product shown a positive effect on 50 cm plant spacing fruits at 14 days of storage, elevating their sugar content (table 2). comparing the different spatial arrangements in crop set® sprayed plants, we observed a lowest sugar content in fruits in 45 cm plant spacing, only at harvest. in plants without application, the 40 cm spacing favored sugar accumulation on the fruits, dif­ fering of the highest plant spacing at 14 days of stor­ age (table 2). during storage, ssc/ta ratio had an increase of 65% from zero (106.81) to 28 days (314.61) (fig. 4a). despite the reduction in both parameters, the more pronounced reduction in ta, compared to ssc, increased the ssc/ta ratio. the flesh firmness of fruits varied from 14.6 n to 10.9 n during storage, decreasing 25% (fig. 4b). during storage, we observed a variation of the ph in the fruits, starting in 5.19 at the harvest and end­ ing in 5.02 at 28 days (fig. 4c). 4. discussion and conclusions the highest average mass of fruits by non­sprayed plants produced in the larger plant spacing (50 cm) can be attributed to the lower competition among the plants for soil nutrients, water and solar radia­ tion, due to the lower density, which can lead to greater fruit development. furthermore, a larger plant spacing reduce incidence of diseases, improving the mass of fruits (bastos et al., 2008; ban et al., 2011; jafari et al., 2016). the use of plant growth regulators can affect crop growth and development, stimulating cell division and increasing nutrient and water uptake (castro and vieira, 2001). in this study, the application of the biostimulant may have caused this effect, which reflects the absence of difference between plant spacings in relation to the average mass of fruits by sprayed plants. our results showed an increase of ssc in fruits on 45 cm spacing, with crop set® application, and non­ spayed plants in growth on 40 and 50 cm spacings. according to yau et al. (2010), the ssc of fruits usual­ ly decrease after a few days of storage due to the respiration process of the fruit, which is the oxidative breakdown of sugars into simpler molecules. in this case, the increase on the ssc of watermelon fruits observed during storage can be attributed to the sol­ ubilization of pectins. the sweetness is the most criti­ cal quality trait of watermelon, being mostly influ­ enced by mono­ and di­saccharides found in the fruit juice, and partly on other solutes, being all con­ tributes to the juice ssc (kyriacou et al., 2018). in relation to the crop set® application, martins et al. (2013) observed that this biostimulant raised the ssc of ‘quetzali’ and ‘style’ watermelons. some stud­ ies did not find influence of plant spacing on ssc of watermelon fruits (bastos et al., 2008; gomes et al., 188 adv. hort. sci., 2020 34(2): 183­189 2017). the ssc in an important quality attribute of watermelon fruits, being desirable that they have val­ ues of ssc higher than 8°brix (tlili et al., 2011), reached in this study. watermelon acidity is mainly attributed to the accumulation of malic acid (özdemir et al., 2016) and its content tends to decrease during storage due to its use as a substrate in respiration (silveira et al., 2013). corroborating with the results of the present study, silva et al. (2016) and yau et al. (2010) also observed decreasing of the ta of fruits during the watermelon storage. gomes et al. (2017) and bastos et al. (2008) reported in their works that the plant spacing have not influence on ta of watermelon fruits. in its turn, campagnol et al. (2012) found effect of plant spacing on fruit acidity, with the high­ est values in lowest plant spacing. in relation to crop set® application, martins et al. (2013) did not observe an effect of this plant biostimulant on the ta of ‘quetzali’ watermelons, differently of ‘style’ fruits, which ta had reduction when this product was applied in the plants. aroucha et al. (2018) empha­ size that small variations in acidity levels of water­ melon fruits are little significant, due to the low con­ centration of organic acids. the reduction of sugar content of fruits observed at the last days of storage is related to the respirato­ ry process of the fruits, that involves oxidative degra­ dation of carbohydrates and organic acids. the ssc/ta ratio of the fruits increased over stor­ age. this parameter in an important indicator of fla­ vor of fruits. generally, highest ssc/ta ratio indicates a greater sweetness of fruits. this ratio is used to evaluate maturity and palatability of watermelon fruits. values found in this work are much higher than those pointed by campagnol et al. (2012) in ‘smile’ watermelon, which showed ssc/ta ratio vari­ ation from 75.55 to 81.88. the flesh firmness of the fruits decreased 25% over storage. this attribute is important to detect ripeness of watermelon fruits, being associated with the pectin solubilization and depolymerisation (kyriacou et al., 2018). the same way that this work, martins et al. (2013) did not observe effect of crop set® application on flesh firmness of watermelons of cultivars quetzali and style. besides that, campagnol et al. (2012) also did not appoint influence of plant spacing on flesh firmness of ‘smile’ watermelon fruits. a small variation on ph was observed on the fruits, and it is explained by the buffer capacity of some fruits, which stabilizes ph even when the decrease of ta is high (paulson and stevens, 1974). in conclusion, the pre­harvest application of plant biostimulant crop set® negatively influenced some quality characteristics of ‘quetzali’ watermelon, including the decrease of ssc of fruits, depending on the plant spacing and storage periods. in relation to titratable acidity and total sugar content, the effect of this product varies with plant spacing and storage days. the 40 cm plant spacing provided higher total sugar content to the fruits. still, the 50 cm spacing increased mass of fruit without alter the yield, besides increasing acidity and soluble solids content at the end of storage, being the recommended plant spacing for ‘quetzali’ watermelon plants. references ainalidou a., tanou g., belghazi m., samiotaki m., diamantidis g., molassiotis a., karamanoli k., 2016 ­ integrated analysis of metabolites and proteins reveal aspects of the tissue‐specific function of synthet‐ ic cytokinin in kiwifruit development and ripening. ­ j. proteomics, 143: 318­333. aoac, 2016 ­ official methods of analysis of aoac interna‐ tional ­ 20th edition. association of analytical chemists, gaithersburg, md, usa. aroucha e.m.m., sousa c.m.g., medeiros j.f., gois g.b., nascimento i.b., araujo n.o., 2018 ­ pre‐ harvest application of plant biostimulant on the quality and shelf‐life of yellow melon (cucumis melo l.). ­ j. agric. sci., 10(2): 252­260. ban d., ban s.g., oplanić m., horvat j., novak b., žanić k., žnidarčič d., 2011 ­ growth and yield response of watermelon to in‐row plant spacings and mycorrhiza. ­ chilean j. agric. res., 71(4): 497­502. bastos f.g.c., azevedo b.m., rego j.l., viana t.v.a., d’ávila j.h.t., 2008 ­ efeitos de espaçamentos entre plantas na cultura da melancia na chapada do apodi. ­ rev. ciên. agron., 39(2): 240­244. campagnol r., mello s.c., barbosa j.c., 2012 ­ vertical growth of mini watermelon according to the training height and plant density. ­ hortic. bras., 30(4): 726­732. castro p.r.c., pereira m.a., 2008 ­ bioativadores na agricultura, pp. 118­126. ­ in: gazzoni d.l. 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technology and environment, the university of the south pacific, private mail bag, suva, fiji. 2 school of science and technology, the university of fiji, private mail bag, lautoka, fiji. key words: abundance, aleurodicus dispersus russell, management, south pacific, spiralling whitefly. abstract: a few species of whiteflies are considered a serious insect pest of vegetation and ornamental plants across many countries. the spiralling whiteflies, aleurodicus dispersus russell has been recorded on many different plant species across countries. these whiteflies feed exclusively on leaves and have the ability to spread plant diseases. a great deal of research has been done on whiteflies in relation to which control methods would be most effective in pest management. the management of spiralling whitefly in the south pacific is heavily reliant on biological control (using the parasitic wasps, predators and entomopathogenic fungi). other control methods include physical, botanical, and chemical to keep the whitefly population at lower levels. in this paper, an overview of spiralling whiteflies and its management practices in the south pacific will be reviewed. 1. introduction spiralling whiteflies, aleurodicus dispersus russell, 1965 (hemiptera: aleyrodidae), are polyphagous pest of agricultural and horticultural crops in glasshouses and fields worldwide (oliveira et al., 2001; mani and krishnamoorthy, 2002; stansly and natwick, 2010). it is a native to the caribbean region and central america (waterhouse and norris, 1989). it was first noticed as a pest in hawaii in 1978 and since then has spread to the pacific islands and other continents (kumashiro et al., 1983; waterhouse and norris, 1989). whitefly adults and larvae feed on leaves, stems and fruits by inserting stylets into the plant. some specific plants that are usually attacked include cassava, pepper, papaya, mango, eggplant, citrus, guava, banana, coconut, breadfruit, tropical almond, sea grape, paper bark and rose (russell, 1965; jayma et al., 1993; neuenschwander, 1994; reddy, 2015). when the stylets are in the phloem, the whiteflies ingest large quantities of sap that contains a lot of sugar. they excrete the excess liquids and sugar which is called honeydew. the honeydew is deposited on leaves (*) corresponding author: s11074077p@gmail.com citation: chand r.r., jokhan a.d., kelera r., 2019 spiralling whitefly and its management practices in the south pacific. a review. adv. hort. sci., 33(1): 123-131 copyright: © 2019 chand r.r., jokhan a.d., kelera r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 march 2018 accepted for publication 7 september 2018 ahs advances in horticultural science mini review http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 123-131 124 and fruits, fostering the growth of black sooty mould fungi and falling of premature leaves. in addition, these moulds influence the rate of photosynthesis and transpiration as it hinders the light penetration, vapour movement and exchange, leading plants to exhibit yellowish specks on leaves, to wilt or die off (mcauslane et al., 2004; al-shareef, 2011; reddy, 2015). spiralling whiteflies have caused detrimental effects in the production of crops and ornamental plants. it is one of the most common pest that has the ability to spread diseases and influence the global food production (ecpa, 2015). over 300 plant species from approximately 77 families have been recorded as hosts of a. dispersus worldwide (lambkin, 1999). the spread of the spiralling whitefly continues to increase rapidly due to the general ineffectiveness of chemical control and other pest control measures (mani and krishnamoorthy, 2002). alternative measures for controlling spiralling whiteflies include physical, cultural and biological control. these approaches have been used in the south pacific but there is no data available (except biological control) that supports their practice and effectiveness in terms of whitefly management (waterhouse and norris, 1987). hence, the present review builds information on studies carried in the south pacific on a. dispersus russell alternative control methods, which would be valuable in terms of effective management of the pest. 2. origin, biology and whitefly-plant interactions aleurodicus dispersus is a tropical pest to a variety of horticultural crops, ornamental plants and shade trees (department of agriculture and fisheries, 2015). it was first reported in florida in late 1957 (native area being the caribbean and central america) and since then it has expanded its range to most regions around the world including north america, south america, asia, africa, australia and several pacific islands (russell, 1965; waterhouse and norris, 1987; reddy, 2015). in the south pacific, the pest has been reported in majuro (1986) (marshall is), cook islands (1984), fiji (1985), nauru (1987), papua new guinea (1987), kiribati (june 1988), tokelau (late 1988) and tonga (november 1988) (waterhouse and norris, 1987). the lifecycle stages of a. dispersus are eggs, four larval instars and adults. the eggs (0.3 mm long) are usually smooth surfaced, yellow and tan elliptical in shape (reddy, 2015). these are laid at an angle of 90° with spiralling deposits of white flocculence on underside of the leaves. the first instars are mobile and called crawlers. they can travel to a short distance to select their feeding sites (martin, 1987). they are usually 0.32 mm long and settle near the spiral pattern of the eggs from which they hatched. as the crawler grows, they develop mid-dorsal waxy tufts and the secretion of wax is usually from the narrow band of sub-margin. the second and third instars are 0.5-0.65 mm long and remains feeding at same place. the distinguishing feature about the third instar larvae is the presence of glass-like rods of wax (usually short and evenly-spaced) lined along the body. these cottony secretion is much less abundant than on the fourth instar [russell (1965) cited in the centre for agriculture and bioscience international (cabi, 2015)]. the fourth instar or puparium is 1.06 mm long and covered with numerous amounts of white materials and long glass-like rods (~8 mm in length); due to fragmentation some are shorter (fig. 1). the second to fourth instars are protected by waxy secretions making them sessile and scale-like (martin, 1987; banjo, 2010). the adults are mobile and most active during the morning. the bodies of males are fig. 1 mature puparium of spiralling whitefly glassy wax rods emanating from each compound spores. these glassy rods are whitish in colour, translucent and longer (3-4 times) than the width of the body from the dorusm (extending upwards and outwards) of mature puparium, a copious amount of white cottony substance is secreted chand et al. whitefly management in the south pacific 125 usually 2.28 mm and females are usually 1.74 mm (3-4 times longer than the body width). the adults develop white translucent powder covering on their bodies. these whiteflies also have a pair of antenna. the males have several pores on the abdomen scattered dorsally, laterally and ventrally on the first 2 segments posterior to wax plates while the females are without pores [russell (1965) cited in the centre for agriculture and bioscience international (2015)]. the eye is reddish-brown in colour. the spiralling whiteflies also have two characteristic dark spots on their forewings (fig. 2). after mating adult females lay eggs in irregularly spiralling patterns and it is where whiteflies derived their common name, spiralling whitefly (reddy, 2015). heavy occasional rains and cool temperatures result in a temporary reduction in a. dispersus population (mani, 2010). the mortality rate of immature stages increase between 40-45°c, and for adults the mortality rate increases at 35-40°c. temperature below 10°c also lead to increased mortality (cherry, 1979; waterhouse and norris, 1987). however, population of spiralling whiteflies will increase during droughts and presumably when the natural enemies decline. the spread of crawler and winged adult whiteflies usually occurs with short-distance movements whereby the crawlers are walking on the plant on which it hatched and the plants that touch it and adults fly to other parts of the same plant or to other nearby host plants. instead, the movement to long distance involves the international horticultural trade (pacific pests and pathogens, 2016). whitefly-plant interactions stomatal conductance whiteflies cause damage to plant productivity in terms of photosynthesis, respiration and transpiration performance (shannag and freihat, 2009). the whitefly a. dispersus is considered to be a major pest causing damages to crops (boopathi et al., 2015). these insect pests are reported to alter the chemical processes, growth and photosynthesis (schröder et al., 2005; fatouros et al., 2012). moreover the plants primary metabolism is also altered by this particular infestation. for example, egg deposition by a particular species of moth (podoptera frugiperda) demonstrated inhibitory effect towards the production of herbivore induced plant volatiles in maize plants (fatouros et al., 2012). the egg deposition has also shown to influence the rate of photosynthesis by reducing the amount of carbon dioxide diffusion in the mesophyll cells (fig. 3) (schröder et al., 2005; fatouros et al., 2012). for instance, the study by shannag and freihat (2009) concluded that gas exchange in cucumber plants infested by whiteflies (bemisia tabaci) caused photosynthesis to decline by up to 30%. the feeding behaviour of whiteflies also fig. 2 adult of spiralling whitefly. fig. 3 (a) the stomatal underneath the normal leaf surface and (b) the egg attached on the stomata of infected leaf. presence of dark spot on the forewings white waxy flocculants materials dark reddish-brown eye, where part the compound eye joined by 3 or 4 facets presence of a pair of antenna adv. hort. sci., 2019 33(1): 123-131 126 increased the respiration rate by 24-78% and the rate of water loss from infested leaves was 3-32% greater than that of control leaves. likewise, the increasing number of whiteflies (tobacco whitefly) led to increase in transpiration rate (shannag and freihat, 2009). the artificial infestation of spiralling whiteflies described by pitan (2003) on pepper led to increase in the damage of leaves. this simply means that the chlorophyll, sugar, protein and crude fibre contents of the leaves decreased as with the level of infestation. 3. status and damage in the south pacific the reproduction and dispersal rate of spiralling whiteflies are relatively high, posing great threat to vegetable, tropical fruit tree and ornamental industries around the globe (pacific pests and pathogens, 2016). the host range of these spiralling whiteflies covers at least a range of 27 plant families, 38 genera and more than 100 species (waterhouse and norris, 1989). despite there is no evidence in the measurement of economic loss by these spiralling whiteflies in the south pacific. heavy infestations on plants is more likely to result in economic loss (pacific pests and pathogens, 2016). being a polyphagous, spiralling whitefly has been recorded on many plant species in different countries. according to surveys conducted from 1996 to 1997 (waterhouse and norris, 1989; barro et al., 1997), spiralling whitefly is an exotic pest in american samoa, cook islands, fiji, kosrae (fsm), pohnpei (fsm), truk (fsm), yap (fsm), guam, kiribati, marshall islands, nauru, new caledonia, northern mariana islands, palau, papua new guinea, solomon islands, french polynesia, tonga and western samoa (table 1). the survey also revealed the absence of a. dispersus from niue, tuvalu and vanuatu. 4. management of aleurodicus dispersus russell the management of whitefly has been difficult as a result of its many host plants. perennial plants such as ornamentals, fruit trees and shade trees were probably used successfully throughout the year by this coloniser (whiteflies) which sucks the sap of leaves (kajita et al., 1991). according to chandel et al. (2010), whiteflies must be dealt with a combination of environmental manipulations, natural enemy enhancement and area-wide control programme. this technique is known as integrated pest management (ipm) which uses a combination of different strategies to control pests. the ipm program uses current, comprehensive information on the life cycles of pests and their interaction, combination with pest control methods to manage pest by the most economical means with least hazardous to the environment and the people (boopathi, 2013; epa, 2017). however, based on the literature available in the south pacific, there are no reported studies on chemical and physical or combined (ipm) control practices on a. dispersus. the only data available is on biological control (waterhouse and norris, 1987). there are various methods of biological control of whitefly techniques utilised in the south pacific, such as the use of parasitic wasps, predators (lacewings, big-eyed bugs and minute pirate bugs) and the use of entomopathogenic fungi of the genus aschersonia. biological control natural predators and parasitoid. spiralling whitefly is not regarded as a pest in its native area of caribbean and central america where it is assumed that populations are kept low by natural enemies (prathapan, 1996). biological control is perhaps the safest and most sound approach to pest control. it is an effective tool in programmes of integrated pest table 1 distribution of aleurodicus dispersus whiteflies in the pacific found during the whitefly survey (1996-1997) aleurodicus dispersus ams coi fij kos poh tru yap gua kir mai nau niu nmi pal png si frp ton tuv van wsa distribution in 1996/7 x x x x x x x x x x x x x x x x x x exotic x x x x x x x x x x x x x x x x x x serious pest potential x x x x x x x x x x x x x x x x x x ams= american samoa; coi= cook islands; fij= fiji; kos= kosrae (fsm); poh= pohnpei (fsm); tru= truk (fsm); yap= yap (fsm); gua= guam; kir= kiribati; mai= marshall islands; nau= nauru; nca= new caledonia; niu= niue; nmi= northern mariana islands; pal= palau; png= papua new guinea; si= solomon islands; frp= french polynesia; ton= tonga; tuv= tuvalu, van= vanuatu and wsa= western samoa. the letter ‘x’ indicates a.dispersus indication for presence and concern. information retrieved from barro et al. (1997). chand et al. whitefly management in the south pacific 127 management. utilising natural enemies reduces the risk of pesticide usage that results into environmental pollution increase. the importation of parasitoids of genera encarsia or eretmocerus and of various predators have been successfully used in greenhouses for whitefly control (gerling et al., 2001). the three parasitic species, eretmocerus mundus (mercet), eretmocerus eremicus rose and zolnerowich and encarsia formosa gahan (hymenoptera: aphelinidae) have been used against whiteflies in japan (sugiyama et al., 2011). in fiji, the common predators of a. dispersus are coccinellids megalocarla (= archaioneda tricolor) fijiensis, serangiella and the neuropteran cbrysop [kumar et al. (1987) cited in waterhouse and norris (1989)]. some common predators of whiteflies are lacewings, big-eyed bugs, minute pirate bugs and several lady beetles (for example; scymnus or chilocorus species). a major outbreak of spiralling whiteflies on papaya was reported in samoa (pestnet, 2005). the outbreak of whiteflies usually happens when their natural enemies are disturbed or destroyed by pesticides, dust build-up and other factors. these outbreaks commonly affected guavas, palms, ground orchids, and poinsettias (ornamental). a recent study showed that a. swirskii (mite) is increasingly used for the biological control of thrips and whiteflies in many crops (messelink et al., 2008). three major predators that have been found to be most effective in attacking spiralling whiteflies are megalocaria fijiensis, serangiella and the neuropteran chrysopa species (waterhouse and norris, 1989). according to waterhouse and norris (1989), the establishment of pacific bridgehead in hawaii (1978) led to spread of spiralling whiteflies to many of the pacific nations. parasitic wasps encarsia (?) haitiensis, encarsia species and three coccinellid predators from trinidad were used as biological agents to reduce the damaging populations of whitefly in guam. it was noted in lanai island (hawaii) that e. (?) haitiensis and encarsia species could effectively control the spiralling whitefly. adult whiteflies were observed on buses, cars and near parking areas. by late 1979, a. dispersus was considered to be an economic pest and initiated a search for natural enemies in the caribbean. different species of coccinellid predators and aphelinid parasite were introduced in 1979 and 1980 to reduce the population of a. dispersus. in fiji, a. dispersus spread rapidly and became a serious pest since 1986. the introduction of encarsia (?) haitiensis, n. oculatus and n. bieolor from guam and hawaii became well established up to 2 km from their release sites. the three predators found to attack a.dispersus prior to 1986 were the coccinellids megalocarla fijiensis, serangiella sp. and the neuropteran chrysopa sp. similarly, in american samoa, a. dispersus were found in 1981 on a wide range of plants including ornamentals, citrus and other fruit trees. the introduction of coccinellid predators and the parasite encarsia (?) haitiensis in 1984 from hawaii rapidly reduced the a. dispersus population. table 2 provides detail as where the spiralling whiteflies were discovered, their host range and biological control in hawaii, american samoa, cook islands, fiji, pohnpei, guam, kiribati, palau, papua new guinea, tonga and western samoa. physical control of a. dispersus removal and traps. removal of leaves may be an environmental friendly approach, but it does not completely remove the pest, it rather lessens the level of whitefly population from the plant. a slight infestation can quickly spread to other plants. the removal of leaves is a good approach to get rid of non-mobile nymphal and pupal stages of whiteflies from highly dense leaves. in addition, yellow sticky traps are used to trap adults since whiteflies are attracted to yellow (barbedo, 2014). it is where a trap consisting strips of paper and sticky substances such as petroleum are placed in the greenhouse. the insects are caught as they fly. the drawback of this type of approach is that it only captures specimens that can fly. however it is generally ineffective for the insects that are in their early stages since they are not able to fly (barbedo, 2014). this method is not a full-proof control method for farmers since it does not eliminate damaging population, but aims to reduce the whitefly population (nakamura et al., 2007). botanicals. natural pesticides such as plant essential oils would represent an alternative in crop protection (coats, 1994; isman, 2000; koul et al., 2008). different plants have been used for the control of pests and the research has worked out well (gonzalezcoloma et al., 2010). medicinal plants can be an alternative to a lot of synthetic chemicals for human health and agriculture. however, people in fiji and the rest of the south pacific countries are not very cognizant of the presence of the great plant diversity surrounding them. the only study published in relation to botanical effects against a. dispersus were carried by chand et al. (2016). plant extracts such as essential oils from these medicinal plants could possibly be used in agriculture in the form of pest controls. the study focused 128 adv. hort. sci., 2019 33(1): 123-131 on five common medicinal plants randomly selected and screened for the insecticidal properties (fumigant and repellent test). these medicinal plants were makosoi (cananga odorata), lemon grass (cymbopogon. citratus), curry leaves [murraya. koenigii (l.) spreng] tulsi [ocimum tenuiflorum (l.)] and uci (euodia. hortensis forma hortensis). the results revealed that tulsi essential oils showed strong fumigant toxicity (100% mortality in 3 hours) while lemon grass and curry leaves showed the best repellent effect with lc50 value of 0.004 and 0.113, respectively (chand et al., 2016). table 2 introductions for the biological control of a.dispersus country aleurodicus dispersus discovered place and year plants on which they were found first introduction of biological control hawaii honululu in september 1978 tropical almond coleoptera. delphastus pusillus (1980) from trinidad. nephaspis oculatus (1979) from trinidad. nephaspis oculatus (1979) from honduras. n. bicolor(1979) from trinidad. hymenoptera. e. ? haitiensis (1979) from trinidad. encarsia sp. (1980) from trinidad. american samoa tutuila in 1981 guava, ornamentals, citrus and other fruit trees. infestations were noted on banana leaves and later in vegetable gardens coleoptera. delphastus pusillus (1984) from hawaii. nephaspis oculatus (1984) from hawaii. nephaspis bicolor (1984) from hawaii. hymenoptera. e.? haitiensis (1984) from hawaii. cook islands rarotonga in 1984 frangipani, guava, hibiscus and mango coleoptera. ? nephaspis bicolor (1985) from hawaii. hymenoptera. ?encarsia ? haitiensis (1985) from hawaii. encarsia ? haitiensis (1988) from fiji. fiji suva in april 1986 -coleoptera. nephaspis oculatus (1987) from guam. nephaspis oculatus (1987) from hawaii. n. bicolor (1987) from hawaii. hymenoptera. e. ? haitiensis (1987) from guam. e. ? haitiensis (1987) from hawaii. pohnpei --hymenoptera. e.? haitiensis (1986) from guam. guam guam in 1981 coconut, frangipani, guava and mango coleoptera. nephaspis oculatus (1981) from hawaii. hymenoptera. encarsia ? haitiensis (1981) from hawaii kiribati bikenibeu, tarawa, in june 1988 chillies, bell peppers, tomatoes, pawpaw, guava, breadfruit, banana, ornarnentals -including, frangipani and coleus a biological control project is to be commenced in the near future palau --hymenoptera. e.? haitiensis (1986) from guam. papua new guinea october 1987 in the port moresby guava, mango leaves and coconut palms *coccinellids and spiders were seen preying on them tonga in november, 1988 -*attacked by unidentified wasps. western samoa first recognised in 1985 --chand et al. whitefly management in the south pacific 129 chemical control. the use insecticides are widespread among farmers. for instance the paper by kajita et al. (1991) provides a description of several insecticides against a. dispersus on soya beans in indonesia. however, because the whitefly has wide host-plant ranges in addition to the fact that insecticides also have impact on natural enemies, chemical control is usually considered impractical and uneconomic in the long-term (kajita et al., 1991; lambkin, 1998). carmichael et al. (2008) also discourage the use of chemical control for managing spiralling whiteflies, suggesting that soaps and detergents can provide effective control in small scale planting. in the south pacific, there are no recorded publications dealing with chemical control measures of a. dispersus yet (waterhouse and norris, 1987). according to reddy (2015), the common chemicals used for controlling whiteflies are dimethoate 30 ec at 0.05% and insecticidal soap at 2.5%, which deterred the adults. likewise, the following chemicals imidacloprid, buprofezin and pyridaben are also used to manage whiteflies (bi et al. , 2002). spiromesifen, a novel insecticide inhibited egg hatching in green house by 80% to 100% at the concentrations of 3.1, 3.0, and 10.0 µg ml-1. the insecticide also showed mortality of 100% for the first, second, and third instar nymphs of whiteflies (toscano and bi, 2007). chemical approach mostly kills those that come in contact with the insecticides (chemicals). the use of the chemical approach showed efficiency towards controlling pests in small and in large scale farms. for instance, farmers in colombia intensify the use of insecticides, as the whiteflies reduced the crop yield by 79% (carabalí et al., 2010). although, plant productions may have increased due to pesticidal applications at the same time these chemicals may have raised detrimental concerns for so many (aktar et al., 2009). chemical pollution is a major concern to the environment and to human health due to the bioaccumulation of chemicals through food chains, resulting in severe physiological disorders and diseases (oliva et al., 2001; baldi et al., 2003; briggs, 2003; saiyed et al., 2003; lemaire et al., 2004). the extensive use of synthetic chemicals has led to pests in developing resistance to chemicals and at the same time resulting into the accumulation of harmful chemical pollutants in the environment. these pollutants gradually affect the quality of air and water, on which many organisms rely on. 5. conclusions and future perspective whiteflies are considered serious pests to vegetation and ornamental plants in many countries and as such, spiralling whiteflies are fast becoming a concern for many farmers in the pacific and other parts of the world. in the south pacific the pest has been reported in majuro (1986) (marshall is), cook islands (1984), fiji (1985), nauru (1987), papua new guinea (1987), kiribati (june 1988), tokelau (late 1988) and tonga (november 1988) (waterhouse and norris, 1987). these whiteflies feed exclusively on leaves, which eventually damage the plant leading to diseases or plant death. whiteflies are very difficult to manage as a result of multi host plants that support their lifespan. the most common management practices used for the control of whiteflies are biological control using the parasitic wasps, predators and entomopathogenic fungi, physical method using removal and traps, botanicals such as plant extracts and essential oils, and chemical control. at present there is no available literature in the south pacific that provides data on chemical and physical practices. the only data available were on biological control methods and the use of botanicals (essential oils) for the management of a. dispersus (waterhouse and norris, 1987; chand et al., 2016). the salient findings gathered from this review indicate a general lack of information and research on the current status and the degree of damages that the spiralling whitefly has inflicted on agricultural and ornamental crops in the south pacific. acknowledgements the authors are grateful to mrs reema prakash for the support provided throughout the research journey especially in collecting data on whiteflies. the authors are also thankful to the university of the south pacific for providing research funding in form of graduate assistantship to the corresponding author. references aktar m.w., sengupta d., chowdhury a., 2009 impact of pesticides use in 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3 (*) 1 laboratory of applied biology, st. aloysius college, 575 003 mangalore. 2 plant cell culture technology section, nuclear agriculture and biotechnology division, bhabha atomic research centre, trombay, 400 085 mumbai, india. 3 nitte university center for science education and research, paneer campus, deralakatte, 575 018 mangalore, india. key words: gamma rays, induced mutagenesis mutation, plant breeding, zinnia elegans var. dreamland. abstract: zinnia elegans is a herbaceous annual with diverse flower colours, flower types and plant height. zinnia elegans are popular as pot plants and also for landscape gardening. the commercial value of zinnia can be increased with novel traits such as flower colour and form. one of the techniques to develop plant varieties with superior traits is to induce mutations using gamma radiation. hence, three doses of gamma radiation (75gy, 100gy and 125gy) were utilised to obtain new and novel varieties of zinnia elegans var. dreamland and to study the effect of gamma rays on germination of seeds, growth and survival of the seedlings, height of the plants. all the three gamma ray doses were found to decrease the germination and survival of seeds of zinnia elegans. the higher doses of gamma rays were found to be detrimental for the germination and survival of seeds and height of the seedlings. phenotypical variations such as plant height, the number of flowers and flower diameter of the third generation mutants were highly significant as compared to the control. eight floral variations could be obtained with novel form and colour. 1. introduction in recent years floriculture has become a flourishing industry. one of the requirements of the floricultural industry is diversity in order to introduce new ornamental plants in the market. zinnias are important ornamental plants on the world floral market. zinnia has been reported to be the first flower to be grown in space stations (loff, 2016). zinnia elegans offers a wide range of flower forms and colours which have an immense ornamental value. however there is a continuous demand for ornamental (*) corresponding author: smitha.hegde@nitte.edu.in citation: pallavi b., nivas s.k., d’souza l., ganapathi t.r., hedge s., 2017 gamma rays induced variations in seed germination, growth and phenotypic characteristics of zinnia elegans var. dremland. adv. hort. sci., 31(4): 267-273 copyright: © 2017 pallavi b., nivas s.k., d’souza l., ganapathi t.r., hedge s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 september 2017 accepted for publication 25 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 267-273 268 cultivars with new/novel forms and colours in modern and industrialized horticulture (yunus et al., 2013). induced mutations offer a possibility of obtaining ornamentals with novel forms and colours to meet this demand (tiwari and kumar, 2011). more than 2,200 mutant varieties of crop plants have been released using induced mutagenesis and among them, 566 represent ornamental plants (jain, 2005; barakat and el-sammak, 2011). 76 new mutant ornamental varieties with changed flower colour/shape and chlorophyll variegation in leaves have been developed using gamma rays and released (datta et al., 2009; barakat and el-sammak, 2011). but, no reports of registration of mutant varieties of zinnia under mutant variety database of international atomic energy agency are found. there is only one report of production of four new varieties in zinnia by venkatachalam and jayabalan (1997). radiation with gamma rays has been reported to give rise to a large number of novel mutants in several ornamental species (chrysanthemums, orchids, rose, pelargonium, canna, and carnations). the present study was carried out to utilize the mutagenic effect of gamma rays on zinnia elegans to obtain dwarf varieties and flowers with novel architecture and colours. 2. materials and methods plant materials seeds of zinnia elegans var. dreamland with pink flowers which were certified to be pure breeding were procured from the indo american hybrid seeds, bangalore. gamma irradiation zinnia elegans var. dreamland seeds were treated with gamma radiation at the bhabha atomic research center, mumbai, india. three gamma ray doses (75gy, 100gy and 125gy) were selected after studying the radiosensitivity and lethal dose (ld50) of zinnia seeds (pallavi et al., 2017). the gamma ray doses were given at the rate of 1.7gy/min using 60co source. evaluation of germination of seeds, survivability and growth of seedlings for the evaluation of germination of the irradiated seeds, survivability and growth of the seedlings, a total of 90 seeds were treated in triplicates. 30 nonirradiated seeds were established as controls. the irradiated seeds were maintained as axenic cultures. the germination of the seeds was recorded for the first seven days and the percentage of survival of seedlings on the 15th day after planting. the seedling growth parameters such as plant height and root length were recorded at an interval of 5 days for 15 days. the data obtained was used to calculate the percentage of germination and the percentage of survival for each treatment as follows: germination % = no. of germinated seeds × 100/no. of irradiated seeds planted; survival % = no. of survived seedlings × 100/no. of irradiated seeds. evaluation of phenotypic variations observed in m3 generation to determine the effect of gamma rays on phenotypic characteristics, 500 seeds were irradiated with 75gy, 100gy and 125gy doses of gamma radiation in each treatment. 500 non irradiated seeds were established as control. the irradiated seeds were sown in the green house of st. aloysius college, mangalore and the seedlings were grown up to the flowering stage and self-pollinated. the seeds of selfpollinated flowers were collected and sown and the procedure was repeated till the third mutant generation. the irradiated populations were screened for mutants at the m3 generation. the plant height was recorded after 60 days of germination. the floral characters such as flower diameter, the number of flowers and flower colour were recorded at the flowering stage. statistical analysis the data was recorded and statistically analysed using ibm spss 20 software. one-way anova test was performed to determine significant differences between the variations. tuckey hsd was used to ascertain significant differences among treatments at p= 0.05. 3. results effect of gamma rays on seed germination, survivability and seedling growth the seeds started germinating within 3 to 4 days of sowing in all the treatments and the control. the percentage of germination of control seeds was found to be 68.89±2.22. the percentage of germination of seeds irradiated with 75gy (55.55±2.22) was not significantly different from the control. irradiation with 100gy and 125gy gamma ray doses significantly decreased the germination percentage pallavi et al. gamma ray induced variations in zinnia elegans 269 of seeds as compared to the control (fig. 1). furthermore, the percentage of survival of seedlings obtained from seeds irradiated with 75gy, 100gy and 125gy was significantly lower as compared to the control (fig. 1). no significant difference was observed in the percentage of survival of seedlings obtained from seeds irradiated at 100gy and 125gy. there was no significant variation in plant height and root length of the seedlings up to 10 days as compared to the control. from the 10th day onwards there was a significant decrease in plant height and root length in seedlings obtained from seeds irradiated at 100gy and 125gy as compared to the control (figs. 2 and 3). mutants obtained at m3 generation variation in plant height. variation in plant height was observed in third generation plants obtained from seeds irradiated at all three levels (fig. 4). the lowest plant height was seen in variant v1 (18.33±2.028 cm) obtained from seeds irradiated at the 75gy dose. the variant v3 obtained from seeds irradiated at 100gy dose had the highest mean value of plant height (55.33±1.85 cm). variation in number of flowers. the number of flowers found in the variants is shown in figure 5. the variant v7 gave the highest mean number of flowers fig. 1 effect of gamma radiation on percentage of seed germination and percentage of plant survival in zinnia elegans var. dreamland. the mean values with same alphabets do not differ significantly at p<0.05 level. fig. 2 effect of gamma radiation on plant height of the seedlings at 5 days interval. fig. 3 effect of gamma radiation on the root length of seedlings at 5 days interval. fig. 5 comparison of number of flowers in control and variants of zinnia elegans var. dreamland. the mean values with same alphabets do not differ significantly at p<0.05 level. fig. 4 comparison of plant heights of the variants and control plants of zinnia elegans var. dreamland. the mean values with same alphabets do not differ significantly at p<0.05 level. adv. hort. sci., 2017 31(4): 267-273 270 (7±0.57) at 100gy dose. the lowest number of flowers was given by variant v8 (2.66±0.33) at 125gy as compared to the control (5.33±0.33). the variation found in the number of flowers in all the variants is statistically highly significant. variation in flower diameter. variation in the diameter was found in the flowers of plants of seeds radiated at all three doses (fig. 6). except variant v3 most of the flowers were smaller than the control. the variations in the smaller flowers are not significant except for the variant v7. variation in form and colour of flowers. variations in flower form and colour were observed after treatment with gamma rays of different doses as shown in table 1 and figure 7. in plants irradiated with 75gy, two variants v1 with white coloured flowers and v2 with single whorled light yellow coloured flowers were obtained. in plants of seeds irradiated with 100gy, 5 flower colour variants were observed. v4 and v5 plants showed light yellow coloured ray florets with pink coloured tips. v3 showed single whorl pink flowers, v6 plants had orange yellow coloured flowers. the v7 had only yellowish disc florets without any ray florets. in plants irradiated with 125gy, only one variant with yellow flowers was obtained. 4. discussion and conclusions higher doses of gamma radiation were found in our study to reduce seed germination and survival of seedlings. this is confirmed by hanafiah et al. (2010), who found that higher doses of radiation have an adverse effect on seed germination and survival of soybean seedlings. similarly, kumari et al. (2013) reported that gamma rays significantly reduced plant survival and growth of chrysanthemum morifolium variety ‘otome pink’; reduction in survival increased with increase in dose. significant reduction in survival of plants obtained from seeds irradiated with gamma rays was also observed by jala and bodhipadma (2011) in celosia argentea var. cristata. according to fig. 6 comparison of flower diameter in control and variants of zinnia elegans var. dreamland. the mean values with same alphabets do not differ significantly at p<0.05 level. fig. 7 effect of gamma radiation on flower colour and form of zinnia elegans var. dreamland. c= control, v1-v8 = variants. plant variants gamma ray doses (gy) flower characteristics control pink, double whorled v1 75 white, double whorled v2 75 light yellow, single whorled v3 100 pink, single whorled v4 100 light yellow, double whorled ray florets with pink shaded tips v5 100 light yellow, double whorled ray florets with light pink shaded tips v6 100 orange yellow, double whorled v7 100 no ray florets v8 125 yellow, double whorled table 1 effect of gamma radiation on flower colour and form pallavi et al. gamma ray induced variations in zinnia elegans 271 datta and gupta (1982), banerji and datta (2002), and khan (2003), the decreased survival percentage of plants obtained from seeds treated with higher gamma radiation doses is due to chromosomal aberrations and gene mutation after irradiation treatment. according to tiwari and kumar (2011) many mutations can be lethal due to the inhibition of cell division and induction of cell death. in our study higher doses of radiation were found to reduce height of the plants in zinnia. significant reduction in plant height is also reported with higher doses of radiation in chrysanthemum morifolium variety ‘otome pink’ (kumari et al., 2013). the decrease in plant height and root length observed in our study has also been reported in a number of other crops (thimmaiah et al., 1998; yaqoob and ahmad, 2003; al-salhi et al., 2004; toker et al., 2005; kon et al., 2007; el sheriff et al., 2011). the plant height and root length of seedlings from the seeds treated with 100gy and 125gy was found to be similar to the control seedlings until 10th day in our study but both decreased after the 10th day. similar results have been reported by khalil et al. (1986) in barley and el sheriff et al. (2011) in hibiscus sabdariffa. according to them the probable cause for the reduction in plant height and root length after the 10th day, could be the decrease in activity of mitotic division of meristematic tissues and decrease in the moisture content of the seeds. induction of mutations based on ionising radiations has played a major role in the development of many new and novel flower colour and shape mutants in ornamentals (datta et al., 2005). schum and preil (1998) reported that 55% of the records on induced mutation in ornamental plants concerned changes in flower colour and 15% in flower morphology. yamaguchi et al. (2008) obtained more than 8 types of flower colour mutants from chrysanthemum plants bearing pink flowers. datta and chakrabarty (2009) developed four mutants with flower colour and floret shape variation by irradiating the ray florets of five decorative type chrysanthemums. venkatachalam and jayabalan (1997) induced four types of new flower colour mutations: majenta, yellow, red and red with white spots in zinnia elegans jacq. cv. crimson red using gamma irradiation. in our study, eight zinnia mutants with novel variations in form and colour of flowers were obtained with gamma rays. studies by datta et al. (2009); zalewska et al. (2011) on pigment analysis of florets of chrysanthemum flower colour mutants indicated that changes in flower colour were due to qualitative and quantitative changes in the pigments as a result of mutations induced by gamma rays. flower pigments are composed of flavonoids including anthocyanins, flavones and flavonols (harborne and grayer, 1988; hattori, 1992). mutations in the core structural genes or regulatory loci of anthocyanine biosynthesis pathway result in changes in flower colour according to streisfeld et al. (2013) and nakatsuka et al. (2005). the blockage in the early steps of anthocyanin synthesis leads to the loss of floral anthocyanine pigments ultimately resulting in the formation of white flowers whereas a blockage in the later steps leads to flower colour changes from blue to red because of the accumulation of a particular anthocyanin (mato et al., 2000; lee et al., 2008; tanaka et al., 2008; casimiro-soriguer et al., 2016). a higher frequency of flower colour mutation was observed in 100gy. this could be a suitable dose for obtaining higher number of flower colour mutants in zinnia elegans var dreamland. our studies have resulted in obtaining one dwarf variety (18.33±2.028 cm) and eight varieties with varying floral colours from which desirable variants can be selected and commercially exploited. acknowledgements the authors thank mangalore jesuit educational society for providing the facility to carry out the work and the board of research in nuclear sciences (brns), department of atomic energy, mumbai for a jrf to pallavi b. the authors also thank the staff members of the laboratory of applied biology, st. aloysius college, mangalore for their immense support. references al-salhi m., ghannam m.m., al-ayed m.s., elkameesy s. u., roshdy s., 2004 effect of gamma irradiation on the biophysical and morphological properties of corn. nahrung, 48(2): 95-98. banerji b.k., datta s.k., 2002 induction and analysis of gamma ray-induced flower head shape mutation in ‘lalima ‘chrysanthemum (chrysanthemum morifolium). indian j. agr. sci., 72(1): 6-10. barakat m.n., el-sammak h., 2011 in vitro mutagenesis, plant regeneration and characterization of mutants via rapd analysis in baby’s breath ‘gypsophila paniculata l.’ aust. j. crop sci., 5(2): 214-222. casimiro-soriguer i., narbona e., buid m.l., del vall j.c., whittal j.b., 2016 transcriptome and biochemical analysis of a flower colour polymorphism in silene adv. hort. sci., 2017 31(4): 267-273 272 littorea (caryophyllaceae). front plant sci., 7: 204. datta s.k., chakrabarty d., 2009 management of chimera and in vitro mutagenesis for development of new flower color/shape and chlorophyll variegated mutants in chrysanthemum, pp. 303-305. in: shu q.y (ed.) induced plant mutations in the genomics era. food and agriculture organization of the united nations, rome, italy, pp. 460. datta s.k., datta k., chattergee j., 2009 cyto-palynological, biochemical and molecular characterization of original and induced mutants of garden chrysanthemum, pp. 253-256. in: shu q.y (ed.) induced plant mutations in the genomics era. food and agriculture organization of the united nations, rome, italy, pp. 460. datta s.k., gupta m.m., 1982 effect of gamma irradiation on rooted cuttings of small flowered chrysanthemum. new botanist, 7: 73-85. datta s.k., misra p., mandal a.k.a., 2005 in vitro mutagenesis a quick method for establishment of solid mutant in chrysanthemum. curr. sci., 88(1): 155-158. el sherif f., khattab s., ghoname e., salem n., radwan k., 2011 effect of gamma irradiation on enhancement of some economic traits and molecular changes in hibiscus sabdariffa l. life sci. j., 8(3): 220229. hanafiah d.s., yahya s., wirnas d., 2010 induced mutations by gamma ray irradiation to argomulyo soybean (glycine max) variety. nusantara bioscience, 2(3): 121-125. harborne j.b., grayer r.j., 1988 the anthocyanins, pp. 1-20. in: harborne j.b. 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(jack) and early detection of mutation using rapd markers. turk. j. biol., 37: 716-725. zalewska m., tymoszuk a., miler n., 2011 new chrysanthemum cultivars as a result of in vitro mutagenesis with the application of different explant types. acta sci. pol., hortorum cultus, 10(2): 109-123. impaginato 3 adv. hort. sci., 2019 33(1): 3-11 doi: 10.13128/ahs-22935 the effect of cutting type, leaf area, leaf number, putrescine and indole-3butyric acid on the rooting of ficus cuttings (ficus elastica roxb. ex hornem.) m. ghasemi ghehsareh 1 (*), m. kosh-khui 2 1 department of horticultural sciences, college of agriculture, shahrekord university, shahrekord, iran. 2 department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. key words: auxin, cutting, plant propagation, polyamine, rubber fig. abstract: in order to study the importance of lateral or apical buds and also the possibility of replacing the role of bud and leaf with putrescine (put) and auxin, three experiments were conducted using leaf-bud cuttings with intact leaf blade (full blade) or halved-blade, and terminal cuttings having 1 to 4 leaves of ficus elastica. treatments included iba (0, 2000 mg/l) and put (0, 1000, 2000, 4000 mg/l). comparing the lateral cuttings with intact or halved-blade showed that the rooting of the cuttings with intact leaf blade was better. the longest root length and the best rooting index were observed in cuttings treated with 1000 mg/l put + 2000 mg/l iba. the highest root number resulted from the iba treatment. in cuttings with halved-leaf blade, put along with iba improved the indices of rooting in comparison with the control. the rooting of leaf-bud cutting was better in comparison with apical cuttings and the application of put with iba increased rooting indices in both types of cuttings and there was no significant difference between the different concentrations of put. results showed that terminal cuttings with three and four leaves had the longest, heaviest and most abundant roots, and that the rooting index resulted in the highest value. the rooting of leaf-bud cutting with intact leaf blade was better than that of one-leaf apical cutting. in general, the experiment showed that in the one-leaf terminal cutting, the apical bud has a negative effect on rooting and the increase in the leaf area or the application of put with auxin improves rooting. 1. introduction rooting of cuttings is a method of vegetative propagation and is one of the most important methods of clonal propagation for many plants. stimulating the formation of adventitious roots in stem cuttings with the application of auxin is well-known (altman, 1972; bolat, 1995). however, other factors do also play a role and may be restrictive under certain cir(*) corresponding author: mghasemi1352@gmail.com citation: ghasemi ghehsareh m., kosh-khui m., 2019 the effect of cutting type, leaf area, leaf number, putrescine and indole-3-butyric acid on the rooting of ficus cuttings (ficus elastica roxb. ex hornem.). adv. hort. sci., 33(1): 3-11 copyright: © 2019 ghasemi ghehsareh m., kosh-khui m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 march 2018 accepted for publication 7 august 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 3-11 4 cumstances. inhibitors (hess, 1969), rooting co-factors (hess, 1969; heaser and hess, 1972), auxin antagonists (heaser and hess, 1972; batten and goodwin, 1981) and nutrients (nanda et al., 1971; heaser and hess, 1972) are specified to have a role in regulating the process of rooting. it has been found that cuttings without buds do not produce roots even when they are treated with auxin. this suggests that another factor other than auxin which is likely to be produced in the bud is essential for root formation (hartmann et al., 2011). it has also been specified that the presence of leaves on cuttings has a stimulatory effect on rooting (reuveni and raviv, 1980). the stimulatory effect of the leaves on rooting in stem cuttings has been well-specified in an experiment on avocado (reuveni and raviv, 1980). the cuttings of hard rooting cultivars soon lost their leaves under the mist system, while the leaves remained on rooted cuttings for a long time (9 months). after five weeks, it was observed that the amount of starch found in the planting bed of easy-rooting avocado cuttings was five times more than that of the beginning of the experiment. in hibiscus, the maintenance of leaves on cuttings increased rooting (van overbeek et al., 1946). carbohydrates that are transmitted from the leaves are important for root development. but the effects of improving leaves and bud on rooting are likely to be related to other factors (hartmann et al., 2011). it has been reported that carbohydrates and nitrogen affect the onset of adventitious roots. using tomato cuttings, researchers explained the interactions between carbohydrates and nitrogen in regulating root and shoot growth and adventitious root formation (kraus and kraybill, 1918; starring, 1923; reid, 1924 a, b). cuttings from plants grown under conditions of high carbohydrates availability (i.e., high light intensity) relative to nitrogen availability produced roots that grew vigorously (reid, 1924 a, b; schrader, 1924). van overbeek et al. (1946) reported that the rooting stimulation effect of leaves in red hibiscus cuttings could be replaced with sucrose and nitrogen. in the study of the effect of lateral and apical buds on rooting of avocados under in vitro conditions, it was reported that the apical buds had the highest rooting percentage, root number and root length, but lateral buds had no significant effect on these indices (zulfiqar et al., 2009). the effect of polyamines on the rooting of woody plants varies. put, spermidine (spd), and spermine (spm), along with iba, improved the rooting of hazel cuttings (rey et al., 1994). on the contrary, higher levels of endogenous put, spd and spm were observed at maturity stage (difficult to root) compared to the juvenile stage (easy to root) (ballester et al., 1999). the rooting of micro cuttings of olives was increased with the use of polyamines and naa, but did not increase in almonds, pistachios, chestnut, jujuba, apricots and walnuts (reuveni and raviv, 1980). in vitro studies showed an increase in polyamine levels, especially put, with root formation by culturing leaf explants of the datura innoxia (chriqui et al., 1986) and passiflora alta-caerulea (desai and mehta, 1985). investigating the role of polyamines during adventitious root formation with the evaluation of the de-bladed petiole rooting in juvenile stage (easy-to-root) and maturity stage (difficult-to-root) in hedera helix showed that auxin stimulated the root formation at the juvenile stage (easy to root), but did not have a positive effect on the mature phase. adding put, spd and spm with or without naa, did not affect the rooting in juvenile or mature phase. but a significant increase was observed in endogenous levels of put and spd in cuttings treated with auxin (geneve and kester, 1991). thus, the polyamines may have the role of a secondary messenger for rooting (hartmann et al., 2011). ghasemi ghehsareh and khosh-khui (2016) showed that put along with auxin improved rooting and increased the quality of roots in ficus leaf-bud cuttings. according to previous studies regarding the positive role of buds, leaves, glucose, nitrogen, auxins and polyamines in rooting of cuttings, this experiment was conducted to examine the importance of lateral and apical buds, as well as the possibility of replacing the role of buds and leaves with put and auxin. for this purpose, three separate experiments were carried out using leaf-bud cuttings with intact leaf blade or leaf blade trimmed to 50%, and terminal cuttings of the rubber fig (ficus elastica roxb. ex hornem.) having 1 to 4 leaves with iba and put treatments. 2. materials and methods experiment conditions the experiments were carried out using ficus cuttings in the greenhouse of the department of horticultural sciences of shiraz university. the medium containing washed sand was equipped with a bottom heat system at a temperature of about 22°c. ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 5 an intermittent mist system was employed to provide moisture, and the misting was carried out daily at 10, 13 and 16 hours, and each session lasting 1 minute. the greenhouse temperature was between 21 to 25°c during daytime and 15 to 18°c at night. first experiment effect of leaf size, auxin and put. healthy and uniform leaf-bud cuttings included leaf blade, petiole and a part of the stem with a length of 3 to 4 cm with lateral buds from the mid-section of the annual stems were taken in january. in half of the cuttings, the leaf blade was halved, and the rest remained intact. the cuttings were kept in a benomyl fungicide solution with a concentration of 2000 ppm for five minutes and then were washed with distilled water. after removing the surface moisture, the cuttings were treated with put [zero (distilled water), 1000, 2000, and 4000 mg/l)], indole-3-butyric acid (iba) (zero and 2000 mg/l, the best concentrations resulting from the previous experiment) (ghasemi ghehsareh and khosh-khui, 2016) and their combinations by dipping the stems in the solutions for 10 seconds and then planted in the bed. second experiment effect of leaf-bud or terminal cutting, auxin and put. in this experiment, leaf-bud and terminal cuttings with one leaf were used. growth regulators were used and other conditions were similar to the first experiment. third experiment effect of leaf-bud or terminal cutting, leaf area and leaf number. in this experiment, the rooting of different cuttings including leaf-bud cuttings with halved blade, leaf-bud cuttings with intact blade and terminal cuttings with 1, 2, 3 and 4 leaves were compared. all cuttings were treated with iba at a concentration of 2000 mg/l. other conditions were similar to the first experiment. in all three experiments, after 2 months, the cuttings were removed from the bed and after isolating the medium form the roots, root fresh weight, root length (length of the longest root), root number were measured. to determine the quality of the roots, rooting index was calculated. for this purpose the cuttings were visually grouped into five groups including cuttings with heavy, medium and weak rooting, rootless but alive cuttings and dead cuttings. for each group, a coefficient was considered as the weight of rooting. number 5 was for heavy rooting, 4 for medium rooting and 3 for weak rooting. a coefficient value of 2 was used for rootless but alive cuttings, and a coefficient value of 1 was used for dead cuttings (criley, 2011; ghasemi ghehsareh and khosh-khui, 2016). statistical design and data analysis the first and second experiments were conducted as factorial in a completely randomized design with three replications. each replication included 10 cuttings. the third experiment was conducted in a completely randomized design with three replications, each with 6 cuttings. statistical analysis of the data was performed using sas 9.13 software and the mean values were compared using lsd (p≤0.05). 3. results first experiment the results of analysis of data variance showed that the treatments had a significant effect on the root length, root number, root fresh weight and root quality. maximum root length (8.9 cm) was caused by iba 2000 mg/l + put 1000 mg/l treatment in fullblade cuttings, but did not show any significant difference with iba 2000 mg/l + put 2000 or 4000 mg/l on full-blade cuttings. furthermore, no significant difference was observed between the mentioned results and the result of treatment with iba 2000 mg/l + put 1000 or 2000 mg/l on halved blade leaves. the lowest root length was observed in cuttings with halved leaf blade, and generally, root length decreased with a decrease in leaf area. in terms of the number of main roots, the highest number (13.3) was obtained by iba 2000 mg/l on fullblade, and the lowest number of leaves was obtained with halved blade cuttings, without growth regulators. the application of put did not significantly affect the number of main roots. the highest root fresh weight was observed by the application of put 4000 mg/l with iba treatment in full-blade cuttings, which did not have significant difference with other concentrations in full leaves and put 4000 mg/l with auxin in cuttings with halved blade leaves. in fact, by increasing put concentration, the root fresh weight increased in both types of cuttings and the amount of increase was more in cuttings with halved-blade leaves (table 1). in terms of root quality, the highest rooting index (4.7) was observed by treatment with put (1000 mg/l) + iba (2000 mg/l) in full-blade cuttings. the highest rooting index in halved-blade cuttings (3.8) adv. hort. sci., 2019 33(1): 3-11 6 was observed by put 1000 mg/l + iba treatment. the lowest rooting index (2.3) was observed in control treatment with halved-blade cuttings. in general, cuttings with leaf blade trimmed to 50% had roots with lower quality compared to cuttings with full leaf. with increasing the put concentration, the quality of the roots first increased and then decreased (table 2). second experiment the results showed that the interaction of cutting types (leaf-bud or terminal cutting) with growth regulators had a significant effect on rooting characteristics. the terminal cuttings were not rooted without the application of growth regulators, but rooting improved with the application of rooting growth regulators. the longest root length (9.7 cm) was found in leaf-bud cuttings, and there was not a significant difference between different treatments in this cutting. in the terminal cuttings, the use of put + auxin in comparison with the control treatment and auxin alone significantly increased the root length, but there were no significant differences between different concentrations of put. the highest main root number (14.8) was observed in the leaf-bud cutting treated with put 4000 mg/l + iba 2000 mg/l. the application of iba as well as increased put concentration increased the number of roots. in the terminal cuttings, the highest number of roots (6.4) was observed by treatment with 2000 mg/l of put along with iba, which did not differ significantly in different concentrations (table 3). the highest root fresh weight was observed in leaf-bud cuttings. the highest fresh weight (2.9 g) was obtained by using auxin alone, and by put at 2000 mg/ml + iba, which did not differ significantly with other concentrations of put. in the terminal cuttings, the application of put and its increased concentration increased the fresh weight of the root. the best root quality (4.0) was observed in the leaf-bud cuttings treated with put (2000 mg/l) +iba. in the terminal cuttings, the application and increase of the concentration of put improved the quality of the roots as compared with other treatments (table 4). table 2 effect of leaf size, auxin and put on root quality (rooting index) of ficus leaf bud cutting table 1 effect of leaf size, auxin and put on root length, root number and root fresh weight of ficus leaf bud cutting * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. leaf size treatments mean control aux2000 aux2000+put1000 aux2000+put2000 aux2000+put4000 root length intact leaf blade 3.6 c ∗ 6.5 ab 8.917 a 8.417 ab 6.917 ab 6.87 a halved-blade 0.833 d 5.833 bc 6.75 ab 8.333 ab 5.917 bc 5.5333 b mean 2.2167 c 6.1667 b 7.8333 ab 8.375 a 6.416 7 b root number intact leaf blade 1.333 e 13.333 a 8.167 b-d 8.5 bc 10.333 b 8.3333 a halved-blade 0.5 e 7.167 cd 5.833 d 6.0 d 6.667 cd 5.2333 b mean 0.9167 c 10.25 a 7.0 b 7.25 b 8.5 ab root fresh weight intact leaf blade 0.475 d 1.6533 a 1.5233 ab 1.7533 a 1.93 a 1.467 a halved-blade 0.4833 d 0.7033 cd 0.83 b-d 0.9067 b-d 1.2167 a-c 0.828 b mean 0.4792 b 1.1783 a 1.1767 a 1.33 a 1.5733 a leaf size treatments high (×5) medium (×4) low (×3) alive (×2) sum of weights rooting index intact leaf blade control 0 0 20 10 80 2.7 aux2000 13 13 4 0 129 4.3 aux2000+put1000 20 10 0 0 140 4.7 aux2000+put2000 20 10 0 0 140 4.7 aux2000+put4000 20 5 5 0 135 4.5 halved-blade control 0 0 10 20 70 2.3 aux2000 0 17 13 0 107 3.6 aux2000+put1000 0 25 5 0 115 3.8 aux2000+put2000 5 10 15 0 110 3.7 aux2000+put4000 5 7 18 0 107 3.6 ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 7 third experiment by comparing the rooting of leaf-bud cuttings having either full or halved-leaf blade, and terminal cuttings having 1, 2, 3 or 4 leaves, it was observed that by increasing the leaf area, the rooting was also increased. accordingly, the longest roots were obtained in the terminal cuttings with 4 leaves (13.1 cm), followed by terminal cuttings with 3 leaves (9.75 cm). the shortest roots (1.47 cm) were obtained in leaf-bud cuttings with halved-blade. terminal cuttings with 4 leaves produced the highest main root number (6.4) which did not have a significant difference with two and three leaf cuttings. the highest root fresh weight (1.72 g) was observed in four leaf cuttings. the lowest root number, root fresh weight and rooting index were observed in one-leaf terminal cuttings (table 5). in terms of rooting index, the highest rate (3.6) was observed in three and four leaf cuttings (table 6). generally, the experiment showed that the best rooting was obtained in the three or four leaf terminal cuttings and it was weaker in oneleaf terminal cuttings compared to other cuttings. table 4 effect of leaf bud or terminal cutting, auxin and put on root quality (rooting index) of ficus cutting table 3 effect of leaf bud or terminal cutting, auxin and put on root indices of ficus cutting * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. table 5 effect of cutting type, leaf area and leaf number on root indices of ficus elastica leaf area/cutting type root length root no. root fresh weight half leaf/leaf bud 1.47 c ∗ 3.83 bc 0.49 cd full leaf/leaf bud 2.25 c 4.6 ab 0.83 bc 1 leaf/terminal 1.92 c 2.33 c 0.22 d 2 leaf/terminal 6.92 b 3.33 ab 0.68 bc 3 leaf/terminal 9.75 a 4.25 ab 0.93 b 4 leaf/terminal 13.0 a 6.42 a 1.72 a cutting type treatments mean control aux2000 aux2000+put1000 aux2000+put2000 aux2000+put4000 root length terminal 0.000 c ∗ 0.900 c 4.200 b 4.300 b 4.170 b 2.7140 b leaf bud 9.718 a 8.518 a 8.532 a 9.582 a 8.750 a 9.0200 a mean 4.8590 bc 4.7090 c 6.3660 ab 6.9410 a 6.4600 a root number terminal 0.000 c 1.200 c 4.000 b 6.400 b 5.600 b 3.4400 b leaf bud 4.167 b 10.833 a 11.667 a 13.333 a 14.833 a 10.9666 a mean 2.083 c 6.017 b 7.833 ab 9.867 a 10.217 a root fresh weight terminal 0.0000 c 0.2300 c 0.9460 b 1.3120 b 1.4790 b 0.7934 b leaf bud 1.2000 b 2.9000 a 2.4550 a 2.9032 a 2.4318 a 2.3780 a mean 0.6000 b 1.5650 a 1.7005 a 2.1076 a 1.9554 a cutting type treatments high (×5) medium (×4) low (×3) alive (x2) sum of weights rooting index terminal 0 0 0 0 30 60 2.0 aux2000 2 3 4 21 76 2.5 aux2000+put1000 1 2 24 3 91 3.0 aux2000+put2000 3 7 18 2 101 3.4 aux2000+put4000 2 6 19 3 97 3.2 leaf bud 0 0 2 24 4 88 2.9 aux2000 1 18 11 0 110 3.7 aux2000+put1000 2 22 6 0 116 3.9 aux2000+put2000 6 17 7 0 119 4.0 aux2000+put4000 5 14 11 0 114 3.8 8 adv. hort. sci., 2019 33(1): 3-11 4. discussion and conclusions first experiment our test results showed that the application of put along with auxin increased the main root length and root fresh weight, which is consistent with the results of other researchers (cristofori et al., 2010; birlanga et al., 2015; ghasemi ghehsareh and khoshkhui, 2016). the studies of root formation in the mung bean showed that in the rooting phase stimulated with auxin, the endogenous polyamines increase (friedman et al., 1982; jarvis et al., 1983). desai and mehta (1985) and chriqui et al. (1986) studied the rooting of leaf explants in vitro which showed that at the time of root formation the amount of polyamines, especially put, increases. they showed that the activity of ornithine decarboxylase as a key enzyme in put biosynthesis increased in auxin-treated explants, but the use of polyamines in an auxin-free environment did not lead to rooting. the increase in endogenous polyamines during root formation by in-vitro auxin is reported to stimulate the formation of callus, leaf discs and de-bladed petiole explants (malfatti et al., 1983; desai and mehta, 1985; chriqui et al., 1986; tiburcio et al., 1989; geneve and kester, 1991). this increase in put in the formation and extension of the roots shows that put is a biochemical marker for root differentiation (tiburcio et al., 1989). researchers have shown that put, spm and spd inhibit root formation (friedman et al., 1982; jarvis et al., 1983; palavan-ünsal, 1987). also, schwartz et al. (1986) reported that the activities of ornithine decarboxylase (odc) and arginine de-carboxylase (adc) enzymes of put biosynthesis pathway increase during lateral root formation in zea mays. the biosynthesis of the polyamines is associated with cell division in the organogenesis process. in most cases, polyamines have not been able to stimulate root formation in the absence of auxin (birlanga et al., 2015), and numerous studies have shown that the simultaneous application of auxins and polyamines increases rooting (sankhla and upadhyaya, 1988; birlanga et al., 2015). biondi et al. (1990) showed that the polyamines do not directly influence the induction of adventitious root in the prunus avium micro-cuttings, but do play a role in the later stages of root development and elongation. however, put improved the quality of roots in our experiment. in this experiment, the decrease in leaf area caused a decrease in rooting, which is consistent with akinyele (2010) results in bachholzia coriaria. it was reported that leaf size had a very significant effect on rooting, and that the rooting of full-leaf cuttings was better than half-leaf ones. our results showed that the highest number of main roots was obtained by auxin treatments alone and the combination of put along with auxin reduced the number of main roots, but increased root length and weight. on the other hand, rooting index increased in treatments containing both auxin and putrescine in full-blade cuttings. it seems that put, along with auxin, increases root weight by increasing the number of root branches and also the root length, and in comparison with using auxin alone, it reduced the number of thick roots, thereby improving the quality of the roots. based on our results, the simultaneous application of auxin and put has been shown to improve rooting, and to some extent it has been able to compensate for the decrease in leaf area in cuttings with halved-blades. this was observed in a manner that no significant difference existed among the indices of root length and root weight, even when comparing cuttings with full and half blades treated with put 2000 or 4000 mg/l. this suggests that perhaps a part of the positive role of the leaf blade in rooting is related to polyamines and their derivatives. second experiment results of comparisons between one-leaf terminal and leaf-bud cuttings showed that rooting rate in terminal cuttings was zero without using growth regulatable 6 effect of cutting type, leaf area and leaf number on root quality (rooting index) of ficus elastica leaf area/cutting type high (×5) medium (×4) low (×3) alive (×2) sum of weights rooting index half leaf /leaf bud 0 5 8 5 54 3 full leaf/leaf bud 2 6 5 5 59 3.44 1 leaf/terminal 0 5 4 8 48 2.89 2 leaf/terminal 0 9 6 3 60 3.33 3 leaf/terminal 2 9 5 2 65 3.61 4 leaf/terminal 3 12 1 0 66 3.67 ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 9 tors and treatment of cuttings with put and iba significantly increased rooting in comparison to the use of auxin alone, thereby confirming the results of the first experiment and indicating that part of the positive role of leaf in rooting is related to put. on the other hand, the rooting conditions of the leaf-bud cuttings is better than that of the terminal cuttings, which indicates that the apical buds have had a negative effect on rooting. the results of this experiment are consistent with previous reports by al-zebari and al-brifkany (2015), where it was observed that the rooting of citron (citrus medica) cuttings was mostly successful in cuttings obtained from the middle of the stem and the least rate of rooting was in the apical cuttings. this indicates that the apical bud is not only a strong source of auxin, compared to the lateral bud (thimann and skoog, 1933; thimann et al., 1934), but also a strong consumer which cannot play a positive role in rooting without the presence of a leaf. concerning the inhibitory effect of the apical buds on the growth of lateral buds, it has been reported that the apical bud is a strong consumer of auxin and sucrose. in this regard, thimann (1937) suggested that the optimal auxin concentration for stem elongation is more than the concentration necessary for the development of lateral buds. there is also evidence that the apical bud is a strong consumer which limits access to sugars for lateral buds (taiz et al., 2015). this strong sink effect of the apical bud can also have a negative effect on rooting. a study on in vitro rooting of avocado showed that the apical buds had a better rooting than the lateral buds (zulfiqar et al., 2009). on the other hand, rooting is a highly critical and energy-demanding process that is influenced by the complex interaction between sucrose and hormonal levels (birlanga et al., 2015). roberts and fuchigami (1973) studied the effect of seasonal changes on auxin and rooting of the douglas-fir stem cuttings. they showed that at the time of removing cuttings from the beds, rooted cuttings showed a certain activity in buds, but most of the cuttings with very active buds had not produced roots. lanphear and meahl (1963) observed that rooting occurred more actively at or before the bud break, but it decreased sharply with increasing bud activity. the decline was probably due to the competition for growth factors necessary for rooting. third experiment by comparing the leaf-bud cuttings with half and full blade, and terminal cuttings with 1, 2, 3 and 4 leaves, all of which were treated with iba 2000 mg/l, was observed showed that increasing the leaf area, improves rooting. however, by comparing leaf-bud cuttings and one-leaf terminal cuttings, leaf-bud cuttings had a better rooting, which confirmed the results of the second experiment, indicating that the apical bud probably acted as a strong consumer and reduced rooting. the increase in the number of leaves improved rooting so that the highest rooting was observed in terminal cuttings with 3 and 4 leaves. this suggests that, although the apical bud is a strong source of auxin, it was not able to compensate for the lack of factors necessary for rooting. here, the importance of other roles of the leaf becomes clear. the results of this experiment are consistent with reports by other researchers (leakey et al., 1982; badji et al., 1991; tchoundjeu and leakey, 1996; tchoundjeu et al., 2002; atangana et al., 2006; opuni-frimpong et al., 2008). in an experiment by leakey (leakey, 2004), stem cuttings of the khaya senegalensis showed that rooting was limited to leafy cuttings. the inability of rooting in leafless cuttings was associated with the rapid evacuation of carbohydrates in stem tissues, but their concentration in leafy cuttings increased (leakey et al., 1982). this shows that rooting depends on the formation and consumption of carbohydrates after the cuttings are separated from the mother plant (leakey and coutts, 1989). van overbeek et al. (1946) reported that rooting stimulation by the presence of leaves on red hibiscus cutting could be replaced with sucrose and organic or inorganic nitrogen. welander (1976) showed that in vitro rooting of sugar beet hypocotyls was stimulated by an increase in sucrose and inorganic nitrogen in the presence of high concentrations of iaa, whereas low concentrations of auxin were ineffective. gabryszewska (2011) studied syringa vulgaris under in vitro conditions and showed that increasing the amount of sucrose in the medium causes spontaneous root formation on cultivated plantlets in the presence of low levels of nitrogen salts. the planting of rosa ‘improved blaze’ shoots for the purpose of investigating the effect of sucrose and inorganic nitrogen on adventitious root formation showed that high concentrations of sucrose led to the production of higher and more elongated roots (hyndman et al., 1981). therefore, the role of the leaf in helping rooting can occur by the presence of carbohydrates through photosynthesis and also by polyamines as a source of nitrogen. in general, this study shows that the application of put along with auxin improves rooting and root quality in ficus cuttings. moreover, in terminal cutadv. hort. sci., 2019 33(1): 3-11 10 tings, due to the competition for nutritional and hormonal factors necessary for the simultaneous growth of buds and the occurrence of rooting, a certain amount of leaf area is necessary to provide these factors. the application of put along with auxin can partly satisfy this requirement in cuttings with smaller leaf areas. references akinyele a.o., 2010 effects of growth hormones, rooting media and leaf size on juvenile stem cuttings of buchholzia coriacea engler. ann. for. res., 53(2): 127133. altman a., 1972 role of auxin in root initiation in cuttings. proceedings of international plant propagation society, london, uk, 22: 284-294. al-zebari s.m.k., al-brifkany a.-a.a.m., 2015 effect of cutting type and iba on 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19(1): 43-54. van overbeek j., gordon s.a., gregory l.e., 1946 an analysis of the function of the leaf in the process of root formation in cuttings. am. j. bot., 33(2): 100-107. welander t., 1976 effects of nitrogen, sucrose, iaa and kinetin on explants of beta vulgaris grown in vitro. physiol. plant., 36: 7-10. zulfiqar b., abbasi n.a., ahmad t., hafiz i.a., 2009 effect of explant sources and different concentrations of plant growth regulators on in vitro shoot proliferation and rooting of avocado (persea americana mill.) cv. “fuerte”. pak. j. bot., 41: 2333-2346. impaginato 335 adv. hort. sci., 2018 32(3): 335-341 doi: 10.13128/ahs-22472 ascorbic acid content and senescence in blueberry (vaccinium corymbosum l.) during storage a. spinardi (*), i. mignani dipartimento di scienze agrarie e ambientali (disaa), università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: controlled atmosphere (ca), malondialdehyde, oxidative stress, redox state and quality. abstract: blueberry consumption increases because its health properties linked to antioxidants, easy cultivation and profitability. the ability to preserve fruits in controlled atmosphere (ca) allows extending the marketing calendar. the work evaluates parameters linked to the cellular redox state of blueberry fruits, cv. brigitta, stored at 0°c at different atmosphere regimes (ca1= 10% co2, 4% o2 and ca2= 9% co2, 2% o2, compared to air as control). during storage, quality was assessed by the content of ascorbic acid (aa), antioxidant and index of fruit metabolic status, and of malondialdehyde (mda), cell membranes oxidative stress and senescence marker; soluble solids content, titratable acidity and dry matter were also determined. storage in ca increases the blueberries shelf life, particularly at the intermediate times; after 2 months there is a drastic lowering in aa levels and differences among treatments are no longer detectable. ascorbate is confirmed to be an excellent index of oxidative stress in fruit senescence processes. in control, along with the aa decrease, there is a higher mda content, in particular up to the intermediate dates. ca2 proves to be the most suitable atmosphere for delaying the senescence process. titratable acidity and soluble solids remain constant in all samples throughout storage. 1. introduction the blueberries (vaccinium corymbosum l.) industry is rapidly increasing because of its healthy properties linked to antioxidant content and its meeting the consumer expectation of healthy food (gosch, 2003; kähkönen et al., 2003). therefore the blueberry growing area is rapidly extending in many regions, taking advantage by the adaptability and ease of cultivation of this crop and its profitability. blueberry cultivation is well adapted to mountain and hill soil and climate conditions and to oriented organic or environmental friendly agricultural methods with integrated pest management, endowing the growing areas with a benefit of local production and environmental respect. moreover blueberry, as other small fruit, can be established as small-scale farms giving extra income to family businesses. (*) corresponding author: anna.spinardi@unimi.it citation: spinardi a., mignani i., 2018 ascorbic acid content and senescence in blueberry (vaccinium corymbosum l.) during storage. adv. hort. sci., 32(3): 335-341 copyright: © 2018 spinardi a., mignani i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 january 2018 accepted for publication 17 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 335-341 336 the quality of this fruit and the content of healthpromoting compounds are influenced by many factors, such as environmental conditions, genetic diversity and degree of maturity at harvest (ehlenfeld and prior, 2001; connor et al., 2002). blueberry has often been reported to be a highly perishable fruit, thus its commercial value could be strongly affected by storage conditions. its profitability may take interesting advantage by a storage and shelf life extension that can be achieved by cold storage (0-1°c) and controlled atmosphere (ca) with low oxygen (1-4 kpa o2) and high carbon dioxide (9-12 kpa co2) concentrations (krupa and tomala, 2007). after harvest, blueberry quality and product losses are mainly due to dehydration, weight loss, shrivel and fungal spoilage. to understand better the effects of long-term storage on the antioxidant components of blueberry the present study evaluates some parameters related to the cellular redox state of berries of the late cultivar brigitta, stored at 0°c in different ca regimes. 2. materials and methods full ripe ‘brigitta’ berries were harvested in the valtellina area (northern italy, lat: 46.1653333, long: 9.6461111) from 10-year-old plants. the same day of harvest, fruits were put in polyethylene punnets containing about 200 g of fruits each, labelled, weighed and randomly assigned to each of the different storage conditions. berries were stored at 0°c, 95% relative humidity in the following controlled atmosphere modes: ca1: 4 kpa o2 and 10 kpa co2; ca2: 2 kpa o2 and 9 kpa co2. the control was kept in the air (20.1 kpa o2 and 0.03 kpa co2). samples were taken for quality assessment at the following storage times: 0, 12, 33, 47, 61, 76, 94, 110, 132, 147 days. at each sampling time, 3 punnets per storage condition were removed from the storage and held at -80°c until chemical analysis and quality measurements. each parameter was determined on 3 replicates, obtaining one fruit sample from each of the 3 punnets per storage condition and time. dry matter was determined on 50 g of homogenate placed in crucibles and left in an oven at 70°c for 24 hours. ascorbic acid was extracted in a 6% (w/v) metaphosphoric acid solution. the amount of 7.5 g of blueberries was homogenized in a mortar with 10 ml of cold extraction medium and centrifuged at 10,000 g at 4°c. the supernatant was transferred into a 25ml volumetric flask at 4°c. the pellet obtained by centrifugation was washed with 7 ml of cold metaphosphoric acid solution and centrifuged. the supernatants were combined and brought to a final volume of 25 ml with cold 6% metaphosphoric acid. after filtration through 0.2-µm nylon filter, a 10-µl sample aliquot was injected onto an inertsil ods-3 (5 µm; 4.6 mm × 250 mm) gl science column at 20°c attached to a series 200 lc pump (perkinelmer, norwalk, ct, usa). the column was eluted with 0.02 m orthophosphoric acid at a flow rate of 0.7 ml/min and ascorbic acid was monitored at 254 nm with a uv-975 intelligent uv-vis detector (jasco model 7800, tokyo, japan). ascorbic acid was identified by the retention time and quantification was achieved according to the concentration of a corresponding external standard (sinelli et al., 2008). the determination of the thiobarbituric acid-reactive-substances (tbars) content was carried out using a 5% trichloroacetic acid extract. five grams of mesocarp were homogenized in 25 ml of 5% (w/v) trichloroacetic acid then centrifuged at 4°c at 10,000 g for 30 min. the extract was added to an aqueous solution of 15% (w/v) tca and 0.5% (w/v) 2-thiobarbituric acid. samples were mixed and heated at 95°c for 15 min in a water bath, cooled and centrifuged at 4,000 g for 15 min. samples were then analyzed in a spectrophotometer (jasco, model 7800, tokyo, japan) at 532, 600, and 440 nm. the value of absorbance at 532 nm was purged from the absorbance at 440 nm and at 600 nm due to sucrose and tonon-specific turbidity (cocetta et al., 2016). tbars concentration was expressed in mda equivalent (nmol/g fw) following the equation (du and bramlage, 1992): {[(a 532 a 600 ) (a 440 a 600 )(8.4/147)/157 000]}106 for the determination of the titratable acidity (ta), a 5 g sample of homogenized blueberry puree was diluted with 30 ml of distilled water. the ta was measured after 15 sec stirring by titration with 0.1 n naoh to an end-point of ph 8.3 by a compact titrator d (crison strumenti spa, carpi, italy). the acidity was expressed as meq/100 g fw. total soluble solids (tss), expressed as percent of soluble solids, were determined by a hand refractometer (atago mod., n1, tokyo, japan) on juice obtained from squeezing the berries. analysis of variance was performed by spss software, ibm spss statistics 22 (spss inc., chicago, il), using general linear model univariate analysis. sources of variation were time of storage and atmosphere regimes. significant differences between spinardi and mignani ascorbic acid content and senescence in blueberry 337 means were calculated by tukey’s mean test. differences at p≤0.05 were considered as significant. 3. results blueberry fruit cv. brigitta showed at harvest a percentage of dry matter of 12.52% (fig. 1). within the first 47 day storage period, berries maintained in air and in controlled atmospheres exhibited similar dry matter contents (13.45% in control; 12.55% and 12.14% in ca1 and ca2 stored berries, respectively). thereafter, the level of dry matter increased in control samples until end of storage and was higher than in treated samples. in berries stored in both ca regimes dry matter did not change throughout the entire storage period. at the end of the trial dm accounted for 14.52% of the weight of control fruit and for 11.69% and 11.54% of the weight of ca1 and ca2 stored fruit, respectively. the trend of total soluble solids fairly followed the parallel sample variation in dry matter content during storage (fig. 2). soluble solid content increased in control fruit during the first storage period, from 11.10% at harvest to 12.70% at 47 days storage, and during the trial it was always higher in fruit stored in air than that in berries stored in ca1 and ca2, except at 132 days of storage. during the trial, berries stored in ca showed no differences in this parameter until day 147, when a decrease was recorded. comparing total soluble solids at the end of storage period with respect to the beginning, the amounts were 5% higher in berries maintained in air and 12% and 17% lower in berries under ca1 and ca2, respectively. the titratable acidity (11.91 meq/100 g fw at harvest) remained stable in the first 94 days of storage in all the samples and no differences were detectable among treatments (fig. 3). after this sampling time, ta increased in all samples, reaching a maximum value at day 132 and either maintaining higher values at the last sampling point, in fruit under controlled atmosphere regimes (20.27 meq/100 g fw in ca1 and 19.69 meq/100 g fw in ca2 after 147 days storage), or decreasing, in control, at the end of storage to values not statistically different from those at the beginning of the trial (10.92 meq/100 g fw). fig. 1 changes in blueberry dry matter during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 2 changes in blueberry soluble solid content during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 3 changes in blueberry titratable acidity during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). adv. hort. sci., 2018 32(3): 335-341 338 ascorbic acid content (0.35 mg/100 g fw at harvest) increases after the first 12 days of storage in control fruit by 377% and in ca1 and ca2 stored samples by 218% and 239%, respectively (fig. 4). after this sampling time the levels in the control progressively declined reaching the minimum level of 0.18 mg/100 g fw, while ascorbate levels remained higher in the berries stored in both ca regimes until the day 61 (1.31 mg/100 g fw in ca1 and 1.39 mg/100 g fw in ca2). from that date the ascorbic acid content of treated fruit decreased drastically and after the sampling time at 94 days storage ascorbate content was no longer detectable in any sample. in control fruit tbars levels (197.95 nmol/g fw at harvest) decreased at the first sampling date and then increased markedly (+33%), reaching a maximum after 33 days of storage (fig. 5). tbars decreased over time, starting at 94 days of storage, from 222.10 nmol/g fw to 138.18 nmol/g fw at the end of storage. in ca stored berries, the trend of tbars content progressively decreased, but no significant differences were observed compared to harvest date, except for fruit stored in ca1 for 61 days. at the end of the trial, tbars levels reached values of 170.32 and 143.9 nmol/g fw in ca1 and ca2 stored berries, respectively. 4. discussion and conclusions dry matter content, linked to water and weight loss and to metabolic activity, is an important factor affecting blueberries storage life. as blueberries have a high surface-to-volume ratio, they are prone to water loss. the similar values showed by all the samples during the first 47 day of cold storage are in accordance with the research findings of duarte et al. (2009). they reported a markedly low (0.9%) weight loss found in ‘brigitta’ fruit at 48 d of cold storage, regardless of the gas mixture used, either air or ca. the higher percentages of dry matter determined in control starting from the sampling date after 61 days is due to the greater loss of water sustained by the berries kept in the air with respect to the samples stored in ca1 and ca2. in contrast to our data, alsmairat et al. (2011) reported an effect of ca on moisture loss, yielding a 13-fold difference between the 0% co2/21% o2 treatment (0.25% weight loss) and the 19% co2/2% o2 treatment (3.3% weight loss). the authors reported that the greater weight loss for the highest co2/low o2 treatment could be associated to physical causes, e.g. the greater flux of dry gas through the ca chambers, and secondly because high levels of co2 can stress blueberry fruit. on the other hand, in our trial a slight impact on moisture loss in control fruit may stem from similar physical causes, because the ca chambers are smaller than the storage room of control fruit and also because control storage condition could be more stressful to berries than the ca. blueberry fruit cv. brigitta at harvest showed a total soluble solids content and titratable acidity according to other research data (hancock et al., fig. 4 changes in blueberry ascorbic acid content during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 5 changes in blueberry malondialdehyde level, expressed as tbars content, during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). spinardi and mignani ascorbic acid content and senescence in blueberry 339 2008; gündüz et al., 2015). soluble solids content and titratable acidity level are important quality parameters that account for the flavor of fruit. total soluble solids of control samples increased by 13% during the first storage period of 47 days, according to chiabrando et al. (2009) who recorded changes in soluble solid content in cv. coville, with significantly higher values after 28 days of storage in air at 0°c. this increase in control samples cannot be completely explained on the basis of water loss by transpiration and could be more closely associated to the final events of the ripening process. although blueberry is expected to stop sugar accumulation once the fruit is picked (nonclimacteric behavior) and does not have starch to support soluble sugar synthesis after harvest, an increase in carbohydrate levels may be a consequence of cell wall degradation. in fact, during this storage period of 47 days no significant increase in dry matter linked to a change in moisture content is observed in control fruit. thereafter, soluble solids content did not change until end of the trial. similar research findings were reported on cultivars brigitta (duarte et al., 2009), bluecrop and ivanhoe (beaudry et al. , 1998), burlington (forney et al., 2003). in ca stored fruit, soluble solids content shows similar values throughout the storage period until the last sampling time, when it decreased significantly both in ca1 andca2 stored berries. the steady values of total soluble solids shown by the samples stored in ca starting from the beginning of cold storage can be due to a stronger effect of ca on fruit metabolism compared to air-stored fruit. tritatable acidity showed no changes in all samples stored up to 94 days. this is in accordance with previous studies carried out by chiabrando et al. (2009) on ‘bluecrop’ and ‘coville’ (fruit stored for 35 days in air) and smittle and miller (1988) on rabbiteye blueberry (fruit stored for 42 days at different atmosphere regimes) which showed titratable acidity not to be affected by storage duration or atmosphere. on the other hand, after this storage period of 94 days, the parameter increased markedly at day 132 and then remained stable in ca stored fruit until end of the trial. schotsmans et al. (2007) reported a significant increase in titratable acidity during ca storage (2.5% o2, 15% co2) of rabbiteye blueberry ‘centurion’ fruit from 35 days onwards. in contrast to ca stored fruit, in control fruit titratable acidity decreased after the peak at day 132 to levels similar to that recorded at harvest. according to this trend, harb et al. (2014) found no significant differences in titratable acidity between air-stored and ca stored blueberries cv. duke after 4 weeks storage, but a 8 week storage period resulted in significant higher level of titratable acidity in ca stored fruit respect to control fruit. increased titratable acids contents of blueberries did not correspond proportionately and cannot be explained with declining moisture content of the berry, rather may be linked to the onset of senescence and to the release of additional acids associated with softening and cell wall breakdown (proctor and peng, 1989). the higher levels in titratable acidity at the end of storage in ca stored fruit compared to control could indicate that such fruit still had more reserves remaining whereas the reserves for the airstored fruit were partially depleted. ascorbic acid is an antioxidant which concur to fruit quality and it is a marker of the metabolic status of fruit. growing season, location, agricultural practices, cultivar and crop ripeness affected to varying degrees ascorbic acid levels at harvest. the levels of 1.7 mg/100 g recorded at full ripeness in control fruit of ‘brigitta’ were low compared to some other research data (10.2 mg/100 g reported by golding et al., 2014; 13.6 mg/100 g reported by kozos et al., 2014) but similar to other studies (4.8 mg/100 g reported by spinardi et al., 2009; 2.6 mg/100 g reported by sinelli et al., 2008). during the first 12 days of storage ascorbic acid levels increased markedly and reached a maximum in air-stored fruit, when the ripening process is completed. the significant increment in ascorbic acid content may rather be due to a partial disassembly of cell wall polysaccharides (davey et al., 1999; gilbert et al., 2009; cruz-rus et al., 2011) than to the activation of the ascorbic acid biosynthetic pathway. it could also be related to a more efficient ascorbic acid recycling pathway (e.g. enzymes of the ascorbateglutathione cycle), which plays an important role in the response and adaptation to the stress (stevens et al., 2008). after this storage period, ascorbic acid content in control samples progressively diminished. this steady decrease could be due to less effective recycling of ascorbic acid or the presence of factors that promote oxidation of the ascorbic acid pool such as the enzymatic activity of ascorbate peroxidase (apx) or the direct interaction with reactive oxygen species (ros). zhou et al. (2014) found a progressive increment in apx activity in blueberries until 45 days of cold storage in air and a higher level after 60 days compared to the beginning of storage. throughout 340 adv. hort. sci., 2018 32(3): 335-341 storage, they found also a steady increase in superoxide radical production rate and hydrogen peroxide content. in contrast to the decrease recorded in control fruit after the first sampling time, ascorbic acid content did not change in ca stored fruit up to 61 days, when the levels were significantly higher than in control fruit. this evidence is partially in contrast to a previous report on cv. bluecrop (harb et al., 2010) that indicate that a marked loss in ascorbic acid over the entire storage period of 6 weeks occurred under all storage conditions (ranging from 0% co2, 18% o2 to 24% co2, 2% o2) and stated that increasing co2 and/or decreasing o2 partial pressures within the storage atmosphere did not decisively change this loss. on the other hand, harb et al. (2010) found that storing fruit under low o2 combined with high co2 level (up to 18%) resulted in better preservation of ascorbic acid and that the highest co2 level (24%) was injurious and resulted in lower ascorbic acid content. the malondialdehyde (mda) content (determined as tbars level) of 198 nmol/g fw at harvest is similar to the levels reported on blueberries (zhou et al., 2014), on pears (cocetta et al., 2016) and grapes (xu et al., 2009). the levels of mda were coherent in part with the ascorbic acid levels, and reflected more the differences between the various treatments than the trend of a single treatment during the storage period. in control fruit the decreasing trend of ascorbic acid levels during storage is accompanied with a significant higher malondialdehyde content than that recorded in fruit maintained in ca2, up to 94 days of storage. malondialdehyde is considered a biochemical marker of lipid peroxidation of membranes. this data demonstrates a greater oxidative damage to the cellular components of fruit in a natural atmosphere. the drastic decrease in the ascorbic acid content in the second part of the storage period is not in fact accompanied by an increase in mda levels. this could be due to the onset of senescence processes associated to a loss of cellular integrity and compartimentalisation, as a result of the drastic lipid peroxidation of cell membranes caused by ros accumulation, and therefore not to a recovery of oxidative stress. the analysis of the tbars content shows a positive effect especially of ca2 on the fruit, in which the senescence process is delayed and there is no increase in the levels of this parameter. the present work confirms the ascorbic acid content as a parameter closely associated to the cellular metabolic state and therefore an excellent index of oxidative stress that intervenes in the processes of senescence of the fruits during storage. moreover, the blueberry ‘brigitta’ storage in controlled atmosphere, mainly 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cheng s., wei b., liu x., ji s., 2014 changes in antioxidative metabolism accompanying pitting development in stored blueberry fruit. postharvest biol. techn., 88: 88-95. impaginato 71 adv. hort. sci., 2020 34(1s): 71­80 doi: 10.13128/ahsc­7666 anthocyanin and carotenoid contents assessed by time­resolved reflectance spectroscopy in potato tubers (solanum tuberosum l.) with different flesh colors m. vanoli 1 (*), l. spinelli 2, a. torricelli 2, 3, a. ibrahim 4, b. parisi 5, r. lo scalzo 1, a. rizzolo 1 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (crea‐it), via venezian, 26, 20133 milano, italy. 2 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn‐cnr), piazza leonardo da vinci, 32, 20133 milano, italy. 3 politecnico di milano, dipartimento di fisica, piazza leonardo da vinci, 32, 20133 milano, italy. 4 agricultural engineering research institute (aenri), agricultural research center (arc), nadi el‐seid st., 12311 dokki‐giza, egypt. 5 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca cerealicoltura (crea‐ci), via di corticella, 133, 40128 bologna, italy. key words: absorption spectra, flesh color, partial least square regression, solanum tuberosum l., trs. abstract: this work aimed at studying the relationships between the absorption spectra acquired by time­resolved reflectance spectroscopy (trs) and the carotenoid (car) and/or the anthocyanin (ant) contents in 9 potato genotypes with different flesh color (white, yellow, red, purple). fifty whole and intact tubers/genotype were non­destructively measured by trs in the 540­980 nm range; white­ and yellow­fleshed were ranked according to increasing µa540, the red ones according to µa670 and the purple ones according to µa780. then, 5 tubers/genotype, corresponding to the highest, the lowest and 3 intermediate val­ ues of each μa range, were analyzed for flesh color and car and ant contents. in white­ and yellow­fleshed genotypes, µa540 ranged from 0.078 to 0.207 cm­1, showing the highest value in ‘melrose’ and in ‘isci 133/12­1’ and the lowest ones in ‘romantica’ and in ‘cn 07.16.3’. in red­fleshed tubers, µa670 ranged from 0.049 to 0.146 with no significant differences between genotypes; in purple­fleshed genotypes, µa780 ranged from 0.147 to 0.473, showing the highest values in ‘bleuet’. car content ranged between 0.071 to 5.937 mg kg­1 fw, displaying the highest amounts in the deep yellow genotypes ‘melrose’ and ‘isci 133/12­1’ and the lowest ones in the white ‘cn 07.16.3’ and in the dark purple ‘bleuet’ tubers. ant content ranged from 31.63 to 798.44 mg kg­1 fw in red­purple genotypes, having the highest values in ‘bleuet’. by using trs spectra and pls analysis, it was possible to predict car (r2 cv=0.79, rmsecv=0.89) and ant (r2 cv =0.81, rmsecv=95.53) contents and flesh color (h°) in yellow­fleshed genotypes (r2 cv =0.93, rmsecv=0.67) and purple genotypes (r2 cv =0.82, rmsecv=1.63). (*) corresponding author: maristella.vanoli@crea.gov.it citation: vanoli m., spinelli l., torricelli a., ibrahim a., parisi b., lo scalzo r., rizzolo a., 2020 ­ anthocyanin and carotenoid contents assessed by time‐resolved reflectance spectroscopy in potato tubers (solanum tuberosum l.) with different flesh colors. ­ adv. hort. sci., 34(1s): 71­80 copyright: © 2020 vanoli m., spinelli l., torricelli a., ibrahim a., parisi b., lo scalzo r., rizzolo a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 december 2019 accepted for publication 13 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 71­80 72 1. introduction potatoes are grown throughout the world and are consumed in large quantities. potatoes present wide biodiversity, with approximately 5000 known vari­ eties, most of them developed through man selection (fernandez­orozco et al., 2013). potatoes account for only about 2% of the food energy supply; however, they are the predominant staple for many countries. potato is mainly composed of water (80%) and carbohydrate, with starch being the most abundant; contributes up to 3.3% of dietary fiber, shows low amount of proteins and aminoacids with excellent nutritional value and is also rich in vitamins (ascorbic acid, folic acid, niacin, riboflavin, thiamine, pyridox­ ine) and in minerals such as potassium, phosphorous and calcium (burlingame et al., 2009; fernandez­ orozco et al., 2013; zaheer and akhtar, 2016). in addition to ascorbic acid, potatoes contain several phytochemicals such as polyphenols, anthocyanins, flavonoids, carotenoids, tocopherols, and alpha­ linoleic acid, which have beneficial effects on human health due to their antioxidant activity (ezekiel et al., 2013; zaheer and akhtar, 2016). although other fruits and vegetables have antioxidant content higher than that of potatoes, considering the large quanti­ ties in which potatoes are consumed throughout the world, their contribution to the human diet is very significant. phytochemical content in potatoes is affected by various factors such as genotype, cultiva­ tion conditions and methods (organic vs convention­ al), developmental stage, postharvest storage, cook­ ing and processing conditions (lachman et al., 2012; ezekiel et al., 2013; murniece et al., 2013). generally, the skin and/or the fleshes of potatoes varieties are white, yellow, or deep yellow. however, the intro­ duction and availability of pigmented potatoes in which skin and/or fleshes are red, purple, blue, or orange have attracted consumers over the last two decades due to their high antioxidant content in terms of anthocyanins, carotenoids and total pheno­ lics (tierno et al., 2016). the coloration pattern of the skin and fleshes of colored potatoes is variable, i.e., the skin alone may be pigmented, or the flesh may be partially or entirely pigmented. potato cultivars with white flesh contained fewer carotenoids as compared to cultivars with yellow or orange flesh (ezekiel et al., 2013; fernandez­orozco et al., 2013; kaspar et al., 2013; murniece et al., 2013). carotenoid concentrations in white­ and pur­ ple­fleshed potatoes were similar, while yellow pota­ toes having a 45­fold greater carotenoids concentra­ tion compared to white and purple potatoes (kaspar et al., 2013; hejtmankova et al., 2013). lutein, zeax­ anthin, violaxanthin and neoxanthin are the major carotenoids present in potatoes and β­carotene is present in trace amounts (lu et al., 2001; ezekiel et al., 2013; hejtmankova et al., 2013; kaspar et al., 2013). both total and individual carotenoid contents were positively correlated with tuber yellow intensity (lu et al., 2001; murniece et al., 2013). anthocyanins are present in considerable amounts in purple­red pigmented potatoes and their concentrations are considerably higher in the skin than in the flesh (ezekiel et al., 2013). purple­fleshed potatoes had higher anthocyanins compared to red­ fleshed potatoes, while low or non­detectable amounts were found in yellow and white­fleshed cul­ tivars (nayak et al., 2011, lachman et al., 2012; ezekiel et al., 2013; kaspar et al., 2013; kita et al., 2013; lachman et al., 2013; tierno et al., 2015, 2016; akyol et al., 2016). the most common anthocyanins present in potatoes are pelargonidin, malvidin, petu­ nidin, cyanidin, peonidin and delphinidin (lachman et al., 2012; hejtmankova et al., 2013; akjol et al., 2016). red­fleshed genotypes contain predominantly acylated glycosides of pelargonidin, while purple­ fleshed clones contain predominantly acylated glyco­ sides of petunidin, malvidin and peonidin (lachman et al., 2012; hejtmankova et al., 2013; kita et al., 2013; akyol et al. , 2016; tierno et al. , 2016). especially due to anthocyanins, pigmented potatoes also exhibit higher antioxidant activity in comparison to common yellow­fleshed potatoes (lachman et al., 2009; nayak et al., 2011; lachman et al., 2012). anthocyanin and carotenoid contents are general­ ly determined by analytical methods, such as gas­liq­ uid chromatography (glc), hplc and uv­vis spec­ trophotometry. these techniques, however, are cost­ ly and time­consuming and are not suitable for on­ line applications in the food industry. consequently, rapid, accurate, and non­destructive techniques have been studied to monitor antioxidant amounts in potato tubers. however, most of the published papers concern the estimation of dry matter, starch, proteins and sugars in potatoes and only a few arti­ cles deal with the prediction of anthocyanin and carotenoid contents in raw and processed potatoes (lópez et al., 2013). nir spectroscopy applied on whole tubers was able to accurately identify samples containing different levels of soluble phenolics, anthocyanins and hydrophilic antioxidant capacity vanoli et al. ‐ anthocyanins and carotenoids estimation in pigmented potatoes by time‐resolved reflectance spectroscopy 73 belonging to a collection of 18 purple­ and red­ fleshed potatoes and to predict the total phenolic content in 98 potato varieties (lópez et al., 2014; tierno et al., 2016). total and individual carotenoids, anthocyanins as well as total phenolics and antioxi­ dant activity have been estimated with good/high accuracy by nir, hyperspectral imaging, infrared and raman spectroscopy during drying process, in homogenized potato chips and in lyophilized pota­ toes (shiroma­kian et al., 2008; bonierbale et al., 2009; liu et al., 2017; mazurek et al., 2017; escuredo et al., 2018; sebben et al., 2018). nir was also used to differentiate accessions with low, medium and high concentrations of violaxanthin, antheraxanthin, lutein and b­carotene (bonierbale et al., 2009). among non­destructive optical techniques, time­ resolved reflectance spectroscopy (trs) is gaining increasing interest (nicolai et al., 2014). trs has been mainly applied in postharvest studies for estimating fruit maturity, for discriminating fruit having different texture and sensory characteristics and for the detec­ tion of internal defects in fruits and vegetables (rizzolo and vanoli, 2016). trs, in combination with proper models of photon migration, allows the com­ plete optical characterization of a diffusive medium through the measurements of the absorption (µa) and of the scattering (µs) coefficients by probing flesh at a depth of 1­2 cm with no or limited influence from the skin (cubeddu et al., 2001; rizzolo et al., 2016). while scattering is related to the structure, absorption depends on the chemical composition of the tissue, mainly on the presence of pigments such as chlorophylls, anthocyanins and carotenoids. trs absorption spectra measured in the 540­780 nm range were successfully used to predict total carotenoids content in mangoes in combination with partial least squares regression achieving a r2 cv=0.83 and 0.93 depending on the cultivars (vanoli et al., 2016). in ‘haden’ and ‘palmer’ mangoes, the absorp­ tion coefficient measured by trs at 540 nm (µa540), in correspondence of the tail of carotenoid absorp­ tion, significantly correlated (r=0.78­0.94) with total carotenoids, all‐trans­β­carotene, all‐trans­violaxan­ thin no.3, all‐trans­violaxanthin no.1, no.2, no.6 (‘haden’), and 9­cis­violaxanthin no.2, no.3 (‘palmer’) (vanoli et al., 2018). furthermore, high positive cor­ relations were also found among µa540 and a* and yellowness index (r=0.83­0.98), as well as high but negative correlation between µa540 and h° (r=­0.83­ 0.98) (rizzolo et al., 2016; vanoli et al., 2016, 2018). the absorption coefficient measured in the 500­580 nm range was also related to the presence of antho­ cyanins, as found in plums and in red­fleshed peach­ es (rizzolo and vanoli, 2016). the aim of this work was to investigate the rela­ tionships between trs absorption spectra and carotenoids and/or anthocyanin contents in nine potato genotypes with white, yellow, red and purple flesh color. 2. materials and methods potato tubers the experiment was carried out on 9 potato geno­ types: 7 commercial varieties and 2 belonging to crea­ci breeding programme. the 9 genotypes showed different flesh color: 2 had purple flesh (‘bleuet’, ‘salad blue’); 2 red flesh (‘magenta love’, ‘isci 218/3’), 4 yellow flesh (‘isci 133/12­1’, ‘doribel’, ‘melrose’, ‘romantica’) and 1 white flesh (‘cn 07.16.3’), whose traits are reported in table 1. all the potato genotypes were grown in the experimental field in budrio (bologna province, northern italy), 44°32ʹ14ʺ n ­ 11°32ʹ03ʺ e ­ 28 m a.s.l. in accordance to the emilia­romagna region’s ipm table 1 ­ potato genotype characteristics genotype dealer ploidy skin colour flesh colour weight (g) mean ± sd gmd (mm) mean ± sd cn 07.16.3 bernard sas, france 2n=4x=48 yellow white 203.5 ± 61.9 68.4 ± 4.7 romantica danespo a/s, denmark 2n=4x=48 dark red cream 191.7 ± 42.7 68.4 ± 4.7 doribel pizzoli spa, italy 2n=4x=48 yellow cream 203.8 ± 42.9 68.1 ± 4.8 melrose romagnoli f.lli spa, italy 2n=4x=48 reddish brown deep yellow 187.0 ± 36.5 66.5 ± 4.1 isci 133/12­7 not on the market yet 2n=4x=48 yellow deep yellow 196.0 ± 60.4 67.7 ± 6.5 isci 218/3 not on the market yet 2n=4x=48 red red with yellow pigmentation 104.6 ± 24.5 55.1 ± 4.4 magenta love gm sottotetti srl, italy 2n=4x=48 red red 132.0 ± 36.0 58.4 ± 5.2 salad blue d.t. brown seeds, united kingdom 2n=4x=48 blue parti­coloured purple 88.0 ± 18.8 51.5 ± 3.3 bleuet newstyle potatoes bv, the netherlands 2n=4x=48 blue deep purple 149.3 ± 37,7 62.5 ± 5.1 adv. hort. sci., 2020 34(1s): 71­80 74 guidelines. potatoes were harvested at full maturity on august 13, 2018 by a mechanical potato digger and stored at 4°c, 90% relative humidity, up to february 14, 2019. at storage removal, 50 pota­ toes/genotype without external defects were select­ ed, and the diameters (x=longest axis, y= longest axis normal to x; z= longest axis normal to y) were mea­ sured. geometrical mean diameter (gmd) of each tuber was calculated according to mohsenin (1986) as following: gmd = (xyz)1/3 then each tuber was measured by trs on two opposite sides in the central region in the 540­980 nm range for white­ and yellow­fleshed ones, in the 670­980 nm range for red­fleshed and in the 780­980 nm range for purple­fleshed ones. within each geno­ type, white­ and yellow tubers were ranked accord­ ing to increasing µa540, the red ones according to µa670 and the purple ones according to µa780. then, 5 tubers/genotype, corresponding to the highest, the lowest and 3 intermediate values of µa540 (yellow), µa670 (red) and µa780 (purple) were selected for physical­chemical analyses. each tuber was cut in half and the flesh was measured for color in correspon­ dence of the two trs measurement points; then samples were immediately deep frozen at ­20°c until carotenoids (car) and anthocyanin (ant) analysis. time‐resolved reflectance spectroscopy (trs) a portable compact setup working at discrete wavelengths developed at politecnico di milano (torricelli et al., 2015) was used. the light source is a supercontinuum fiber laser (sc450­6w, fianium, uk) providing white­light picosecond pulses, with the duration of a few tens of picoseconds. a custom­ made filter wheel loaded with 14 band­pass interfer­ ence filters (nt­65 series, edmund optics, new jersey, usa) is used for spectral selection in the range 540­940 nm. light is delivered to and collected from the sample by 1 mm fiber placed at 1.5 cm dis­ tance from the illumination point. a second filter wheel identical to the first one is used for cutting off the fluorescence signal originating from the sample when it is illuminated in the visible spectral region. the light then is detected with a photomultiplier (hpm­100­50, becker&hickl, germany) and the pho­ ton time­of­flight distribution is measured by a time­ correlated single­photon counting board (spc­130, becker&hickl, germany). the instrumental response function has a full width at half maximum of about 260 ps and the typical acquisition time is 1 s per wavelength. a model for photon diffusion in a spheri­ cal turbid medium was used to analyze trs data to assess the bulk optical properties of the samples (martelli et al., 2009) to obtain the estimates of µa and µs at each wavelength. flesh color flesh color was measured with a spectropho­ tometer (cm­2600d, minolta co., japan), using the primary illuminant d65 and 2° observer in the l*, a*, b* color space. from a* and b* values, hue (h°) was computed according to: h° = arctangent (b*/a*) × 360/(2×3.14). carotenoids and anthocyanin analysis car and ant analyses were carried out on indi­ vidually frozen samples by slicing flesh portion after skin removal. for car analysis, 1 g of flesh was extracted with 2 ml of nacl 20% in water and 4 ml of a solution of hexane/acetone/ethyl acetate 2:1:1 v/v/v (picchi et al., 2012). for ant analysis, 1 g of flesh was extracted with 4 ml of ethanol/water 50:50 acidified with hcl, final concentration 0.2 m, ph=1.2 (giusti and wrolstad, 2001). then the mixtures were accurately stirred, mixed, centrifuged at 4890 g for 5 minutes at 4°c, and the supernatants were used for the spec­ trophotometric analysis. the extracts were stored at ­20°c until spectrophotometric analysis (jasco, model v­630, deutschland gmbh, pfungstadt, germany). total carotenoid content (car) was determined by measuring the absorbance at 441 nm and quanti­ fied considering the epsilon value of 2540 g 100 g­1 for zeaxanthin (baurernfeind et al., 1971). car data were expressed as mg of zeaxanthin equivalent (ze) per kilogram of fresh weight (mg ze kg­1 fw). total anthocyanin content (ant) was determined by measuring the absorbance at 503 nm (giusti and wrolstad, 2001) and quantified, considering the epsilon values of 18420 moles cm−1 for pelargonidin (giusti and wrolstad, 2001). ant data were expressed as mg of pelargonidin equivalent (pe) per kilogram fresh weight (mg pe kg −1 fw). statistical analysis data of µa540, µa670 and µa780, car, ant and flesh color (h°) were submitted to anova consider­ ing genotype as factor (means compared by tukey’s test at p≤0.05%) by using the statgraphics v. 5.2 (manugistic inc., rockville, md, usa) software pack­ age. trs absorption spectra were processed by unscrambler x 10.0.1 (camo, norway) in order to build partial least square (pls) regression models for vanoli et al. ‐ anthocyanins and carotenoids estimation in pigmented potatoes by time‐resolved reflectance spectroscopy 75 fleshed tubers, µa670 ranged from 0.049 to 0.146 cm−1, with no significant differences between geno­ types (table 2). in purple genotypes, µa780 ranged from 0.147 to 0.473 cm−1 assuming the highest values in ‘bleuet’ (table 2). carotenoid and anthocyanin contents carotenoid content (car) ranged from 0.071 to 5.937 mg ze kg­1 fw, i.e. values comparable with the data found by ezekiel et al. (2013), hejtmankova et al. (2013) and tierno et al. (2015), on other geno­ types. car was present in white, yellow and also in red and purple (except in ‘salad blue’) genotypes, and showed the highest amounts in the deep yellow genotypes ‘melrose’ and ‘isci 133/12­1’, intermedi­ ate contents in the red­fleshed ‘isci 218/3’ and in ‘magenta love’ and the lowest ones in the white genotype ‘cn 07.16.3’ and in the dark purple ‘bleuet’ tubers (table 3). these data confirmed that deep yel­ low­fleshed genotypes are usually characterized by much higher carotenoid contents than white­ and red­fleshed ones (ezekiel et al., 2013; kaspar et al., 2013; tierno et al., 2015; kotíková et al., 2016; tierno et al., 2016). in contrast, purple­fleshed tubers had either no carotenoids or a carotenoid content similar to that of white genotypes (hejtmankova et al., 2013; kaspar et al., 2013; kotíková et al., 2016). anthocyanins (ant) were present in red and pur­ ple genotypes, with values ranging from 31.63 to 798.44 mg pe kg­1 fw (table 3) in agreement with the findings of ezekiel et al. (2013), hejtmankova et al. (2013), lachman et al. (2012, 2013), kita et al. (2013) and tierno et al. (2015) on other potato cultivars. ant showed the highest amount in ‘bleuet’ tubers, car, ant and h° prediction, without pretreatments of spectral data. 3. results and discussion trs absorption spectra the trs absorption spectra of the 5 selected white, yellow, red and purple tubers are illustrated in figure 1. the absorption spectra of white and yellow potatoes showed a maximum at 980 nm, correspond­ ing to water, and high values at 540 nm, in corre­ spondence to the tail of carotenoid absorption, as previously found by rizzolo et al. (2016) and vanoli et al. (2016, 2018) in mangoes. the absorption spectra of red potatoes showed a peak at 980 nm and high absorption at 670 nm, while in purple potatoes maxi­ ma were observed at 670 nm for ‘salad blue’ tubers and at 780 nm for ‘bleuet’ ones, with a lower water peak. the absorption at 670 and at 780 nm could be linked to the presence of anthocyanins, as the promi­ nent absorbance peaks of anthocyanin were around 500­550 nm (giusti and wolstrad, 2001), but some absorbance was also noticed above 650 nm (laksmiani et al., 2016; noda et al., 2017). contrary to what found in fruit such as apples, pears, peaches, mangoes and plums (rizzolo and vanoli, 2016), µa670 in potatoes was not linked to chlorophyll content, as no greening development was detected in tubers studied in this experiment. in white and yellow genotypes, µa540 ranged from 0.078 to 0.207 cm−1 and showed the highest value in ‘melrose’ and ‘isci 133/12­1’ and the lowest ones in ‘romantica’ and ‘cn 07.16.3’ (table 2). as for red­ fig. 1 ­ absorption spectra of the five selected purple­red­yel­ low­white fleshed tubers. the inset figure shows the variability of the absorption spectra for white­ and yel­ low­fleshed genotypes. table 2 ­ values of the absorption coefficients measured by trs at 540 nm (µa540), 670 nm (µa670) and 780 nm (µa780) used to rank white­yellow, red and purple potatoes, respectively mean min max sd µa540 (cm‐1) cn 07.16.3 0.104 0.078 0.136 0.023 romantica 0.108 0.089 0.132 0.016 doribel 0.141 0.101 0.176 0.029 melrose 0.167 0.139 0.197 0.022 isci 133/12­1 0.161 0.122 0.207 0.033 µa670 (cm‐1) isci 218/3 0.096 0.049 0.146 0.038 magenta love 0.080 0.055 0.107 0.021 µa780 (cm‐1) salad blue 0.256 0.147 0.367 0.086 bleuet 0.937 0.318 1.510 0.473 76 adv. hort. sci., 2020 34(1s): 71­80 while did not significantly differ among the other 3 genotypes (table 3). the highest ant content was usually found in dark purple genotypes (ezekiel et al., 2013; hejtmankova et al., 2013; lachman et al., 2012, 2013; nayak et al., 2011; tierno et al., 2015, 2016). ‘salad blue’, characterized by a parti­coloured purple flesh, showed lower ant content than the deep purple ‘bleuet’ genotype, but similar ant val­ ues to the red­fleshed genotypes, as previously observed by hejtmankova et al. (2013), kita et al. (2013), lachman et al. (2012, 2013). ant was not detected in white and yellow genotypes (table 3) as found by tierno et al. (2015); on the other hand, kaspar et al. (2013) observed that white potatoes had no ant, whereas yellow­fleshed ones showed 20­fold lower ant concentrations than the purple ones. flesh color considering the white and yellow genotypes, ‘cn 07.16.3’ and ‘romantica’ exhibited the highest h° val­ ues, ‘melrose’ and ‘isci 133/12­1’ the lowest ones and ‘doribel’ intermediate values (table 3). ‘cn 07.16.3’ and ‘romantica’ had a pale yellow flesh, even if classified white and cream, respectively (table 1); ‘doribel’ showed a slightly yellower flesh than ‘cn 07.16.3’ and ‘romantica’, even if classified creamy as ‘romantica’ (table 1); ‘melrose’ and ‘isci 133/12­1’ tubers had the yellowest flesh color, even if the yel­ low intensity was higher in ‘melrose’ tubers: both these genotypes were classified as deep yellow­ fleshed (table 1). the flesh color of these 5 geno­ types agreed with the respective carotenoid con­ tents: more intense was the yellow color of the flesh, the higher the car content. considering all the 5 genotypes, a high negative linear (r=­0.83, p<0.001) relationship was found between h° and car content of the flesh, in agreement with lu et al. (2001), reporting a strong relationship between total and individual carotenoids and tuber yellow intensity, and murniece et al. (2013), finding a positive correla­ tion between carotenoid content and the b*coordi­ nate of the flesh in organically and in conventionally cultivated potatoes. as for purple genotypes, a slight but significant difference in the flesh color existed between ‘bleuet’ and ‘salad blue’, as the former showed a lower h°, indicating a darker purple color (table 3). in addition, ‘bleuet’ also had an 11­fold greater ant content compared to ‘blue salad’; this higher ant content was responsible for the deeper purple color as con­ firmed by the negative and significant correlation between ant content and h° (r=­0.89, p<0.001). considering red­fleshed potatoes, ‘magenta love’ showed lower h° than ‘isci 218/3’, confirming that the former had a red color and the latter a deep orange color due to the presence of a slightly higher car and a slightly lower ant contents in the flesh (table 3). a negative and significant correlation was found between ant content and h° (r=−0.87, p<0.001) also for red genotypes. dependence of the flesh coloration of the tubers measured by the cielab scale with phenol flavonoid contents was also observed by escuredo et al. (2018) in 35 potato vari­ eties with different flesh color. partial least square (pls) regression models trs absorption coefficients measured at the dif­ ferent wavelengths were used to develop partial least squares (pls) regression models for predicting car and ant contents and h° color of the potato flesh. for each parameter, the best model was select­ ed considering the lowest root­mean­square error of cross­validation (rmsecv), combined with the lowest number of latent variables (lv) and the highest coef­ ficient of determination in cross­validation (r2 cv). the table 3 ­ carotenoid and anthocyanin contents and pulp color (h°) of white, yellow, red and purple­fleshed potatoes nd= not detected. car (mg ze kg­1 fw) ant (mg pe kg­1 fw) h° pulp mean min max sd mean min max sd mean min max sd cn 07.16.3 0.241 0.071 0.496 0.181 nd nd nd nd 97.8 97.5 98.3 0.4 romantica 0.997 0.323 1.498 0.563 nd nd nd nd 97.4 96.7 98.2 0.6 doribel 1347 0.278 2.244 0.716 nd nd nd nd 96.1 95.9 96.3 0.2 melrose 4540 3.457 5.937 0.955 nd nd nd nd 92.2 91.1 93.8 1.1 isci 133/12­1 4063 3.323 5.291 0.751 nd nd nd nd 94.0 93.4 95.1 0.7 isci 218/3 2.655 2.000 3.882 0.784 88.42 47.37 130.42 29.65 55.8 44.7 65.8 7.9 magenta love 2.285 1.276 3.244 0.812 144.49 80.62 245.84 65.96 38.4 21.3 58.1 15.4 salad blue nd nd nd nd 49.05 31.63 73.05 19.74 337.0 333.6 339.7 2.3 bleuet 0.526 0.331 0.843 0.212 529.38 328.08 798.44 176.58 331.8 328.2 335.4 2.7 vanoli et al. ‐ anthocyanins and carotenoids estimation in pigmented potatoes by time‐resolved reflectance spectroscopy 77 results of pls regressions are reported in table 4 and in figures 2 and 3. a good result was obtained for the prediction of car contents, as the pls model had a r2 cv of 0.79 and an rmsecv of 0.89, being µa540 and µa580 the important variables (fig. 2, top). a slightly better result was achieved for ant prediction, as the perfor­ mance of the pls model showed r2 cv of 0.81 and rmsecv of 95.53 (fig. 2, bottom). the µa780 and µa830 were the important variables. however, figure 2 (bottom) showed that samples are not equally dis­ tributed according to ant content. there are two groups: the larger one with ant content up to 250 mg pe kg­1 fw, including the red genotypes and the purple genotype ‘salad blue’, and a second group with ant content ranging from ⁓300 to 800 mg pe kg­1 fw corresponding to the deep purple­fleshed ‘bleuet’ genotype. probably, the highest ant con­ tent, together with highest variability of ‘bleuet’ tubers, strongly affected the performance of the pls model for ant content prediction. to the best of our knowledge, there are a few papers in literature dealing with the non­destructive determination of antioxidant compounds in whole tubers. tierno et al. (2016) found that nir measure­ ments on unpeeled intact potatoes combined with pls­da allowed to accurately identify samples con­ taining different levels of total phenols, total table 4 ­ performance of pls regression models on original trs absorption spectral data for prediction of total carotenoids (car) and total anthocyanin (ant) contents and of flesh color (h°) r2c= coefficient of determination between predicted and measured values in calibration; r2cv= coefficient of determination between predicted and measured values in cross­validation; rmsec= root mean square error of calibration; rmsecv= root mean square error of cross­validation. fig. 2 ­ measured and predicted car (top) and ant (bottom) contents by pls regression analysis. fig. 3 ­ measured and predicted flesh color of white­yellow (top) and purple (bottom) potato genotypes by pls regression analysis. dependent variables trs parameters variable number calibration validation r2 c rmsec r2 cv rmsecv car µa540­980 5 0.84 0.73 0.79 0.89 ant µa780­980 1 0.81 90.61 0.81 95.53 h° white‐yellow genotypes µa540­980 4 0.94 0.54 0.93 0.67 h° purple genotypes µa780­980 2 0.87 1.24 0.82 1.62 adv. hort. sci., 2020 34(1s): 71­80 78 monomeric anthocyanins and hydrophilic antioxidant capacity belonging to a collection of 18 purple­ and red­fleshed potatoes. regarding total carotenoids content, tierno et al. (2016) found that nirs was only capable of identifying samples with a high con­ tent of these compounds. good models for predict­ ing total phenol content have also been built by lópez et al. (2014) measuring 1157 whole potato tubers with nir, and obtaining coefficients of deter­ mination of 0.88, 0.77 and 0.74 for calibration, cross­ validation and external validation, respectively. however, when nir technology was applied on freeze­dried and milled material (bonierbale et al., 2009; escuredo et al., 2018; liu et al., 2017) it was possible to successfully estimate car and/or ant contents. bonierbale et al. (2009), measuring 152 solanum phureja germplasm accessions by nir, esti­ mated total carotenoids and zeaxanthin concentra­ tions with r2 values ranging from 0.63 to 0.92, and they were able to differentiate accessions with low, medium and high concentrations of violaxanthin, antheraxanthin, lutein or b­carotene. total flavonoid content was predicted by nir with r2=0.82 (escuredo et al., 2018) and total anthocyanin amount by hyper­ spectral imaging in purple­fleshed sweet potato dur­ ing drying process achieving a coefficient of determi­ nation for calibration of 0.868 and a coefficient of determination for prediction of 0.866 by using ten key wavelengths (637, 660, 666, 700, 729, 761, 801, 837, 892, and 957 nm) (liu et al., 2017). the flesh color prediction model for yellow geno­ types (fig. 3, top) showed the best performance, as r2 cv was 0.93 and rmsecv was 0.67 and, as found for car content, the important variables were µa540 and µa580. pls models were separately developed for flesh color prediction of red and purple genotypes considering the very high differences in the h° values, being on average, 47 for red­fleshed tubers and 335 for purple­fleshed potatoes (table 3). a good model was obtained for the prediction of flesh color of pur­ ple genotype with r2 cv = 0.82 and rmsecv = 1.62 (fig. 3, bottom), while no significant model could be developed for red genotypes. by using nir spectra, escuredo et al. (2018) were able to estimate the b* coordinate of the flesh with r2=0.75 by using nir spectra, while poor results were obtained for the a* and l* coordinates in lyophilized creamy, yellow and purple­fleshed potatoes; on the other hand, mazurek et al. (2017), successfully modeled l* parameter (r2=0.992) in potato chips. 4. conclusions trs was able to quantify with a reasonable accu­ racy carotenoid and anthocyanin contents in yellow and in red/purple fleshed­genotypes, respectively. trs also allowed the estimation of flesh color in yel­ low­fleshed­genotypes, without being influenced by the different color of the skin, and in purple­fleshed ones. however, trs was not able to predict flesh color in red­fleshed genotypes. the highly significant correlations between h° color coordinate and car and ant contents can be used for discriminating potato tubers with different concentrations of pig­ ments. the encouraging results of this study indicat­ ed the potential application of trs for the non­ destructive determination of carotenoid and antho­ cyanin contents and for the flesh color estimation in whole and intact potato tubers. however, further studies with a larger set of samples will be advisable in order to obtain better and more reliable models. acknowledgements we are grateful to science and the technology development fund (stdf), ministry of state for scientific research, egypt for financial support to ayman ibrahim (project id: 25329. this research was carried out within the activity of the agrofiliere project funded by the italian ministry of agriculture (d.m. 36503/7305/2018). references akyol h., riciputi y., capanoglu e., caboni m.f., ver­ ardo v., 2016 ­ phenolic compounds in the potato and its byproducts: an overview. ­ int. j. mol. sci., 17: 835. bauernfeind j.c., brubacher g.b., kläui h.m., maru­ sich w.l., 1971. ­ use of carotenoids. ­ in: isler o., h. gutmann, and u. solms (eds.) carotenoids . chemische reihe. lehrbücher und monographien aus dem gebiete der exakten wissenschaften, vol 23, birkhäuser, basel, switzerland. bonierbale m., gruneberg w., amoros w., burgos g., salas e., porras e., zum felde t., 2009 ­ total and individual carotenoid profiles in solanum phureja cultivated potatoes: ii. 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t2: 2.5 µm se; t3: 2.5 mm si; t4: 50 µm cd; t5: 50 µm cd + 2.5 µm se; t6: 50µm cd + 2.5 mm si. the treatments were arranged in completely randomized design, with four replicates for each treatment and six plants per replicate. the plants were collected at 30 and 60 days after application of the treatments. cadmium was highly toxic in all parameters (dry and fresh weight, plant height, leaf number, leaf area, root and photosynthetic parameters), in both assessments. however, se and si were effective in mitigating cd toxicity in all parameters, although si has been shown to be more efficient than se in dry weight and plant height parameters. thus, from data obtained in this study, it is clear that the beneficial elements tested have power to ameliorate cd toxicity. 1. introduction plants differ in their ability to absorb, accumulate and tolerate heavy metals, including cadmium (cd), and toxic levels of heavy metals affect a variety of plant processes (gupta et al., 2013). cadmium is one of most toxic heavy metals, having a high mobility in environment (tang et al., 2015), being absorbed by roots and transported to shoot of many plant species (shi et al., 2005). although cd has no known biological function in plants (pence et al., 2000; pereira et al., 2016), it can be easily absorbed and transported by xylem (lux et al., 2011), since it has an electronic con(*) corresponding author: athos_odin@hotmail.com citation: dorneles a.o.s., soares pereira a., possebom g., peligrinotti tarouco c., rossato l.v., almeri tabaldi l., 2019 ameliorate the cadmium toxicity in solanum tuberosum l. plants with selenium and silicon application. adv. hort. sci., 33(1):49-56 copyright: © 2019 dorneles a.o.s., soares pereira a., possebom g., peligrinotti tarouco c., rossato l.v., almeri tabaldi l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2018 accepted for publication 4 october 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 49-56 50 figuration and state of zinc-like valence (nan et al., 2002). this heavy metal is toxic even at low concentrations, inducing stress responses in plants from 510 μg g-1 soil (while and brown, 2010). several studies have reported significant reductions in biomass accumulation in plants exposed to cd (farooq et al., 2013; said et al., 2014; vaculík et al., 2015), and this inhibition in biomass production can occur in short time of exposure to this heavy metal (han et al., 2015). in contrast to others toxic heavy metals, cd present in soil is easily absorbed by plant roots, particularly in acidic soils (guimarães et al., 2008). the plant development stage and time of exposure to heavy metal affect the absorption and distribution of cd in different parts of plant (gonçalves et al., 2009 a). thus, it is necessary to develop strategies that result in lesser absorption of these toxic elements present in soil by plants, optimizing the use of natural resources and production of safe food, especially when it comes to a plant used in food such as potatoes. potato (solanum tuberosum l.) is one of main foods for mankind, consumed by more than one billion people worldwide, due to its composition, gastronomic and technological versatility, as well as the low market price of tubers (coelho et al., 1999; dorneles et al., 2016). according to birch et al. (2012), potatoes are third most important crop in world, behind only rice and wheat. the potato is susceptible to cd, and this sensitivity can be accentuated by level of cd in soil, time of exposure and cultivar (gonçalves et al., 2009 b). one of options sought to solve this problem with cd in plant growth is use of beneficial elements, which when used in low concentrations can alleviate the damaging cd effects. in this sense, selenium (se) and silicon (si) are recognized as beneficial elements for growth of some plants, and can increase the tolerance of plants to abiotic stresses. these elements are recognized as being capable of mitigating metal toxicity in plants (wu et al., 2017). however, it is necessary to evaluate the potential use of these mitigating elements in the presence of cd, since some studies show that these may not be effective to reduce the toxicity of this element in some species (khattab, 2004; liu et al., 2013). however, si has been shown to be effective in alleviating al toxicity in potato plants (dorneles et al., 2016) and cd in species such as peanuts (shi et al., 2010), chinese cabbage (song et al., 2009), rice (tripathi et al., 2013), maize (lukacová et al., 2013), wheat (khan et al., 2015) and sunflower (said et al., 2014). selenium, however, has stress-alleviating properties that are more focused on biochemical mechanisms activation (kumar et al., 2012; feng et al., 2013; tamaoki and maruyamanakashita, 2017). however, it already has great potential in use to relieve stresses by metals (he et al., 2004; pezzarossa et al., 2012). however, this element, to date, has not been tested as potential to relieve the stress caused by cd toxicity in potato plants. thus, the aim of this work was to test the possibility of using se or si as reliever of cd toxicity in potato plants. 2. materials and methods plants of solanum tuberosum l., asterix genotype, were used for the experiment, which were propagated in vitro from nodal segments for 25 days in ms culture medium (murashige and skoog, 1962). after this period, plants were transferred to plastic vessels containing 1 plant each and 5 kg of sand, being watered daily with complete nutritive solution. this solution had the following composition (mg l-1): 85.31 n; 7.54 p; 11.54 s; 97.64 ca; 23.68 mg; 104.75 k; 176.76 cl; 0.27 de b; 0.05 mo; 0.01 ni; 0.13 zn; 0.03 cu; 0.11 mn and 2.68 fe (feso4/na-edta). after five-day of acclimation period, treatments were applied, which consisted of following combinations: treatment 1: complete nutritional solution and absence of cd, se and si; treatment 2: complete nutritional solution + 2.5 μm se; treatment 3: complete nutritional solution + 2.5 mm si; treatment 4: complete nutritional solution + 50 μm cd; treatment 5: complete nutritional solution + 50 μm cd + 2.5 μm se; treatment 6: complete nutritional solution + 50 μm cd + 2.5 mm si. these solutions were applied daily maintaining 80% of the vessel capacity, which was monitored by daily determining the weight of the vessel. the treatments were arranged in a completely randomized design, with four replicates for each treatment and six plants per replicate. the ph of the solutions was adjusted daily (4.5±0.1). two collects were performed, the first collect being at 30 days of exposure to treatments and the second at 60 days. in both collections, the following evaluations were carried out: photosynthetic parameters were evaluated in fourth fully expanded leaf of four plants per replicate: the photosynthetic rate (a μmol co2 m-2 s-1), the stomatal conductance of water vapors (gs mol h2o m-2 s-1), internal co2 concentration (ci μmol dorneles et al. selenium and silicon ameliorate the cadmium toxicity in potato 51 m-2), water use efficiency (wue mol co2 mol h2o1), and the transpiration rate (tr mmol mmol h2o m-2 s-1) obtained by the ratio between the amount of co2 fixed by photosynthesis and the internal co2 concentration. the evaluations were carried out in period between 8 and 11h with use of irga portable meter, brand li-cor, model li-6400xt. growth parameters: fresh and dry biomass, height, leaf area, number of leaves, and morphological parameters of the root system (length, diameter, volume and number of root branches), according to methodology described by dorneles et al. (2016). for statistical analysis of data, it was verified the normality of error distribution through andersondarling test and homogeneity of error variances through the bartlett test (estatcamp, 2012) for all variables of experiment. the averages were submitted to analysis of variance and compared by the scott-knott test, with 5% significance, using sisvar (ferreira, 2011). the graphic program used was sigmaplot 12.5. 3. results for experimental conditions tested, cadmium (cd) presented great toxicity to potato plants both at 30 and 60 days of growth. this is evidenced by averages of fresh weight accumulation obtained by plants exposed to cd, which did not increase after 30 days of cultivation, remaining unchanged up to 60 days (fig. 1) both being statistically smaller than in the control treatment. besides, in general, when applied in isolation, si and se did not promote greater accumulation of fresh and dry weight in comparison to control plants (figs. 1, 2). however, both elements tested proved to be effective in mitigating cd toxicity from first collect, inducing a higher accumulation of fresh weight in both tissues, compared to the treatment where only cd was applied. the enhancing effects of se and si are more evident in shoot, where plants exposed to cd combined with se or si maintained some growth in second collect (fig. 1 a). the roots of potato plants (fig. 1 b) also show a significant improvement in presence of both beneficial elements. this probably contributed to growth of shoot, considering that roots are the first tissue to come into contact with cd. for dry weight, potato plants showed similar behavior to that observed for fresh weight in both tissues (fig. 2). cadmium showed to be toxic to both tissues for dry weight, inhibiting this accumulation until last collect. however, se and si induced biomass accumulation similar to control plants, even when applied together with cd. silicon induced greater accumulation of shoot dry weight when compared to se (fig. 2 a). in roots, cd prevented the dry weight increase, while plants treated with se and si presented increase in root dry weight in second collect (fig. 2 b), that is, se and si alleviated the toxic effects of cadmium. in addition to affecting the production of fresh and dry biomass, cd inhibited the production and expansion of leaves, as well as the growth in height of potato plants. however, plants that received si and se together with cd obtained higher means even in comparison to control plants (figs. 3 a, b). in addition, the number of leaves and height of plants exposed to cd were higher when applied si, in comparison to plants treated with se as amendment. both se and si were effective in reducing the toxicity of cadmium from first collect, inducing higher height, number of leaves and leaf area than plants with only fig. 1 effect of selenium (2.5 μm) or silicon (2.5 mm) on shoot (a) and roots fresh weight (b) of potato plants grown in presence of cadmium (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different uppercase letters indicate significant differences between collects for same treatment. adv. hort. sci., 2019 33(1): 49-56 52 cd in nutrient solution. there was no statistical difference between treatments for root length and root diameter at 30 days, but plants treated with cd presented root length 45% lower than the control (table 1). at 60 days, plants exposed to cd showed root length 16% and root different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. fig. 2 effect of selenium (2.5 μm) or silicon (2.5 mm) on shoot (a) and roots dry weight (b) of potato plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different uppercase letters indicate significant differences between collects for same treatment. fig. 3 effect of selenium (2.5 μm) or silicon (2.5 mm) on leaves number (a), plants height (b) and leaf area (c) of potato plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. table 1 length, diameter, volume and number of branch roots of solanum tuberosum plants grown in presence of selenium (2.5 μm), silicon (2.5 mm) and cadmium (50 μm) at 30 and 60 days after application of the treatments collect treatments root length (cm) root diameter (mm) root volume (cm3) branch number 30 days control 798±0.24 aa 0.35±0.02 ba 1.64±0.49 aa 715±68 aa se 776±18.3 aa 0.38±0.04 ba 0.91±0.25 ab 394±18 bb si 743±9.24 aa 0.35±0.02 ba 0.84±0.19 ab 401±20 bb cd 438±3.76 ba 0.34±0.02 aa 0.37±0.07 ac 201±13 ad se + cd 650±4.62 aa 0.36±0.00 ba 0.65±0.05 ab 323±35 bc si + cd 711±3.43 aa 0.41±0.03 aa 0.83±0.08 ab 318±38 bc 60 days control 998±0.57 aa 0.45±0.02 aa 1.83±0.21 aa 790±48 aa se 884±21.6 aa 0.45±0.01 aa 0.85±0.03 ab 794±58 aa si 862±25.2 aa 0.45±0.02 aa 0.76±0.08 ab 790±62 aa cd 839±10.11 ab 0.34±0.02 ab 0.34±0.02 ac 253±24 ac se + cd 795±5.50 aa 0.46±0.00 aa 0.75±0.04 ab 423±21 ab si + cd 791±6.11 aa 0.44±0.02 aa 0.85±0.07 ab 452±22 ab dorneles et al. selenium and silicon ameliorate the cadmium toxicity in potato 53 diameter 25% lower, compared to the control. on the other hand, all treatments presented lower root volume compared to control, but this effect was more significant in the treatment containing only cd in the growth medium, both at 30 and 60 days of cultivation. this same behavior was observed for the number of branches at 30 days. at 60 days, branch numbers was lower in the treatments containing cd, se + cd and si + cd, and this effect was more significant in the treatment containing only cd in the growth medium. cadmium showed to be highly toxic, reducing volume of roots and ramifications to less than half of values presented by control plants. however, si and se presented an amendment effect on all root variables, mainly for total length and diameter, both at 30 and 60 days of cultivation. in these variables, plants exposed to cd treated with both si and se were statistically the same as control plants. plants exposed to cd had photosynthetic rate (a) reduced by 37% compared to control plants at first collect, and 42% at second collect (table 2). besides, cd caused significant reductions in stomatal conductance (gs), internal co2 concentration (ci) and transpiratory rate (trmmol) in both collects. the water use efficiency (wue) in both collects was higher only for the treatment with se + cd. however, in both collects si and se were effective in mitigation of cd toxicity for all variables except for wue (table 2). 4. discussion and conclusions in the present study, cd promoted a significant reduction in biomass production in potato plants (fig. 1). the reduction in biomass accumulation caused by cd may be due, in part, to its effect on the inhibition of nutrient uptake by roots (cao et al., 2014; li et al., 2016). the effects of cd on absorption of nutrients may be due to damage caused by this element in the roots. since root tissues are first to come into contact with the cd present in solution, they are also the most affected. several studies have already reported negative effects of cd on cellular level of root tissues (benavides et al., 2005; lux et al., 2011; martinka et al., 2014). the data presented in this work show the significant reduction in root parameters caused by cd. this behavior may be due to effect of cd on degradation of membranes and nucleotides of root cells (han et al., 2015). this degradation of membranes and organelles can be explained by the increase in concentration of reactive oxygen species (ros) induced by cd (chou et al., 2012; farooq et al., 2013; said et al., 2014; han et al., 2015). it is possible that this effect of cd on increase of ros, reported in these studies, may inhibit cell division of roots (said et al., 2014) in addition, the cd has affinity for phosphates and some amino acids components of enzymes and table 2 effect of selenium (2.5 μm) or silicon (2.5 mm) on photosynthetic rate (a-μmol co2 m-2 s-1), stomatal conductance (gs mol h2o m-2 s-1), internal co2 concentration (ci μmol m-2 s-1), transpiration rate (trmmol h2o mmol m-2 s-1),and water use efficiency (wue co2 mol h2o mol-1) of solanum tuberosum plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments collect treatment a gs ci trmmol wue 30 days control 9.47±0.13 ba 0.20±0.07 aa 288±0.48 aa 9.47±0.13 ba 192±4.59 bb se 8.39±0.32 bb 0.16±0.04 aa 295±11.8 ba 8.39±0.32 bb 178±11.8 bb si 8.92±0.31 bb 0.04±0.00 ac 272±2.44 bb 8.92±0.31 bb 200±20.3 bb cd 5.92±0.27 bc 0.10±0.00 ab 257±8.54 ac 5.92±0.27 bc 228±8.63 ab se + cd 8.73±0.07 bb 0.06±0.03 ac 297±5.57 aa 8.73±0.07 bb 308±3.05 aa si + cd 8.26±0.03 bb 0.10±0.00 ab 255±9.29 ac 8.26±0.03 bb 236±12.6 ab 60 days control 12.5±0.14 aa 0.20±0.03 aa 289±0.24 aa 12.5±0.14 aa 205±0.25 ab se 10.3±0.32 ab 0.14±0.01 aa 268±14.6 ab 10.3±0.32 ab 215±0.24 ab si 10.1±0.06 ab 0.06±0.00 ab 298±5.53 aa 10.1±0.06 ab 236±0.02 ab cd 7.41±0.31 ad 0.04±0.00 bc 269±8.13 ab 7.41±0.31 ad 220±0.26 ab se + cd 9.08±0.72 ac 0.08±0.00 ab 257±8.54 bc 9.08±0.72 ac 298±0.14 aa si + cd 8.98±0.61 ac 0.10±0.00 ab 255±9.29 ac 8.98±0.61 ac 245±0.22 ab different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. 54 adv. hort. sci., 2019 33(1): 49-56 proteins, which leads, in addition to damage to membranes, genetic damage and can disrupt oxidative phosphorylation in exposed tissues (hasanuzzaman and fujita, 2012; nahar et al., 2016). the present study also shows the effect of cd under photosynthetic parameters, in which there was a significant reduction in plants exposed to this heavy metal. this reduction in photosynthetic parameters may be a consequence of the degradation of chlorophylls caused by cd (lópez-millán et al., 2009). furthermore, cd causes disorder in arrangement of grana and thylakoids, which limits the efficiency of most photosynthetic parameters (han et al., 2015; bayçu et al., 2016). the potato plants used in present study, treated with si or se as amendments of cd toxicity, showed higher biomass production than plants exposed to only cd. in addition, se and si significantly reduced the damage caused by cd in photosynthetic parameters. these results of biomass and photosynthetic rate are related, since the beneficial effects of si and si on the cd toxicity in photosynthetic parameters are expressed, to a greater or lesser degree, in higher production of biomass by plants. each element used as amendment in this work (se or si) act in different ways in the plant. silicon have been proven to be effective in easing stress by cd in different species such as cucumber (feng et al., 2010), corn (malčovská et al., 2014; vaculík et al., 2015) and cotton (farooq et al., 2016). this effect of si may be due to its deposition on cell wall which increases its plasticity and elasticity (vaculík et al., 2009). the si deposited on cell wall of the plants may increase cd retention in apoplast (lukačová et al., 2013; vaculík et al., 2015), which may reduce the availability and translocation of cd. due to these cell wall modulations, si can inhibit cd uptake (liu et al., 2013). these effects of si can explain the more evident amelioration of cd in shoot than in roots for dry weight in potato plants (fig. 2). in addition, silicon has photoprotective properties when deposited on leaves (tripathi et al., 2017), which may explain the mitigation of cd toxicity in gas exchange parameters. there is a chance that se also has the property of forming complexes with toxic or heavy metals, but were only found complexes of se-mercury (hg) in plants (said et al., 2014). however, there are reports that selenium reduces cd accumulation in tissues (lactuca sativa l.) (he et al., 2004). while si is recognized for increasing the enzymatic antioxidants activity (debona et al., 2017), se has the property of inducing resistance by activating routes of synthesis of hormones linked to stress response and antioxidant activities (freeman et al., 2010; feng et al., 2013; tamaoki and maruyama-nakashita, 2017). thus, the mechanism of mitigation of se may be more directly related to antioxidant system. many studies have reported this effect of se under plant antioxidant enzymes, as well as its effect on direct removal of ros (cartes et al., 2010; zembala et al., 2010). in present study, the cd toxicity was reduced in most of evaluated parameters, mainly under photosynthetic parameters. the possible effect of se under enzyme activity may help to explain its most evident effect under these parameters. selenium was more effective in reducing the effects of cd on photosynthetic parameters than for other parameters evaluated. for potato plants under experimental conditions used in this study, si and se elements proved to be effective in mitigating cd toxicity. thus, these elements have potential for use in fertilizers applied to contaminated soils. future biochemical, histological and molecular analyzes may complement the elucidation of action mechanisms these elements. acknowledgements the authors thank the coordenação e aperfeiçoamento de pessoal de nível superior, conselho nacional de desenvolvimento científico e tecnológico, and fundação de amparo à pesquisa de estado do rio grande do sul for the research fellowships. references bayçu g., gevrek-kürüm n., moustaka j., csatári i., rognes s.e., moustakas m., 2016 cadmium-zinc accumulation and photosystem ii responses of noccaea caerulescens to cd and zn exposure. environ. sci. pollut. res., 24: 2840-2850. benavides m.p., gallego s.m., tomaro m.l., 2005 cadmium toxicity in plants. j. plant physol., 17: 21-34. birch p.r.j., bryan g., fenton b., gilroy e.m., hein i., jones j.t., prashar a., taylor m.a., torrance l., toth i.k., 2012 crops that feed the world 8: potato: are the trends of increased global production sustainable? 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dadashpour 1 (* ), a.r. talaie 1, m.a. askari-sarcheshmeh 1, a. gharaghani 2 1 department of horticulture, university college of agriculture and natural resources, university of tehran, p.o. box 31587‐77871 karaj, iran. 2 department of horticultural science, college of agriculture, shiraz university, p.o. box 65186‐71441 shiraz, iran. key words: intensive planting, v-system, y-system. abstract: this research was carried out to compare several attributes pertaining to the growth, fruit and yield of four apple cultivars, i.e. ‘golab-kohans’, ‘fuji’, ‘starking’ and ‘delbar estival’. these cultivars were grafted onto m.9 rootstocks trained into ‘guttingen v-slender-spindle (or v-system) and ‘geneva y-trellis (or y-system) systems. compared to the y-system, it was observed that the v-system caused the trees to yield more fruits, dry matter, ash and total soluble solids (tss). in contrast, the y-system caused the trees to have broader trunk cross sectional areas (tcsa), along with higher yield, fruit weight, fruit diameter, fruit length and fruit firmness, compared to trees trained with the v-system. in summary, these results showed that both systems can be employed as promising approaches, but the ‘y-system’ appears to be more productive than the ‘v-system’. in addition, among the studied cultivars, it seems that the ‘delbar estival’ and ‘fuji’ were more adaptive to these intensive training systems, especially when considering the fruit traits. 1. introduction intensive training systems are particular layouts that assist orchard managers in improving the productivity of orchards (ferree and warrington, 2003). the need to improve training and pruning methods can better fit the natural growing conditions, and this can be associated with higher fruiting performances by the fruit trees (lauri, 2009). thus, modern apple orchards are planned on the basis of higher tree density than that of traditional planting systems which use dwarfing apple rootstocks (ferree and warrington, 2003). dwarfing rootstocks are increasingly becoming prevalent among the sectors of the fruit industry. they are an important factor that improve orchard productivity due to their significant effects on agro -morphological characteristics such as the yield (*) corresponding author: dadashpour@ut.ac.ir citation: dadashpour a., talaie a.r., askari-sarcheshmeh m.a., gharaghani a., 2019 influence of two training systems on growth, yield and fruit attributes of four apple cultivars grafted onto ‘m.9’rootstocks. adv. hort. sci., 33(3): 313-320 copyright: © 2019 dadashpour a., talaie a.r., askarisarcheshmeh m.a., gharaghani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 june 2018 accepted for publication 22 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 313-320 314 (barritt et al., 1995). the guttingen-v system, the ysystem (tatura), the drilling system, and the mikado system are the most popular v-shaped canopy systems, and are suggested as promising alternatives to high density orchards (robinson, 2000). dwarfing rootstocks, such as m.9 and m.27, are generally employed in v-shaped systems (ferree and warrington, 2003). v systems allow better light penetration than other training-shaped trees (robinson, 2003). the ‘geneva y-trellis’ system is a v-shaped system which uses a y shaped trellis to support the trees. the ‘guttingen v-slender-spindle’ system includes individual conic-shaped trees allowing high tree densities within multiple rows. it has been reported that the guttingen v causes the production of higher yield per hectare and thinner trunks, compared to the drilling system (sosna and czaplicka, 2008). many investigations have shown that there are significant differences between local and foreign apple cultivars in terms of growth and productivity (dadashpour et al., 2010; dadashpour et al., 2011). such reports also indicate the same with regard to apricot (strikic et al., 2007) when trained by intensive training systems. recently, it has been reported that rootstocks and training forms have significant effects on the vegetative growth, yield and fruit traits of apple cultivars (alizadeh and pirmoradiyan, 2016). it has been reported that the efficiency of several parameters can be improved by more production or by the reduction in tree size (fioravanco et al., 2016). when apple scions are grafted onto dwarfing and semi-dwarfing rootstocks, they usually produce larger fruits and more yield, compared to when scions are grafted onto non-dwarfing rootstocks (perry and byler, 2001; gjamovski and kiprijanovski, 2011). negligible differences have been reported in the cumulative yield among ‘slender spindle’, ‘hybrid tree cone’ (‘hytec’) and ‘vertical axis’ (crassweller and smith, 2004). rutkowski et al. (2009) studied nine training systems for apple trees, and reported that the growth and yield of trees may be more dependent on genetic traits, while the shapes of trees can modify the skeletal structure of an orchard. to this end, gonkiewicz (2011) showed that trees having spindle shapes can produce the best yield and fruit weight among the studied pruning systems in sweet cherry. by studying the ‘fuji’ apple, grafted onto the m.9 rootstock under five training systems, ozkan et al. (2016) reported that there were significant differences among the studied training systems in relation to canopy volume, trunk-cross sectional area (tcsa), yield, yield efficiency and fruit size. with 2.8% of the total harvestable area (134,000 ha) and 2.2% of the total production (1.7 million tons) in the world, iran is among the largest producers of apple after china, usa, turkey, poland, india and italy (faostat, 2012). the majority of apple orchards in iran are traditional ones. they are characterized by low tree densities and are commonly grown on seedling rootstocks. however, semi-intensive and intensive apple orchards are recently becoming popular among apple growers. ‘golden delicious’ and ‘red delicious’ are two apple cultivars that are planted in about 90% of cultivated areas. meanwhile, the early ripening cultivar ‘golab-kohans’ is the most prevalent, native apple cultivar in iran. it provides the summer demand for fresh apples in the market. furthermore, ‘granny smith’, ‘fuji’, ‘gala’, ‘jonagold’, and ‘braeburn’ are increasingly becoming popular in the country (gharaghani et al., 2015). as the apple industry in iran is about to shift dramatically from traditional to modern production systems, e.g. semi-intensive and intensive orchard, it is important and necessary to study the performance of popular apple cultivars on different rootstocks, especially within the context of various training systems. accordingly, the objective of this study was to evaluate two training systems, i.e. ‘guttingen v-slenderspindle’ and ‘geneva y-trellis’, and compare their effects on growth characteristics, yield and fruit quality of four apple cultivars. their scions were grafted onto m.9 rootstocks in the alborz province of iran. 2. materials and methods plant materials and experimental design this research was conducted at an experimental field belonging to a horticultural research station, karaj, iran. the duration of the entire experiment took from 2007 to 2010. the average maximum temperature of the region is 13.7°c, with an annual rainfall of 254 mm. the soil in the region is classified as clay-loam. the experiments were arranged as spiltplot (main plot: training system; split-plot: cultivar) according to a randomized complete block design (rcbd) with four replicates. four apple cultivars were used, i.e. ‘delbar estival’, ‘fuji’, ‘golab-kohans’ and ‘starking’, and their scions were grafted onto dwarfing m.9 rootstocks. all trees were planted in march 2005, and trellis systems were established in june 2006. the trees were trained into two training systems, i.e. ‘guttingen v-slender-spindle’ (v-system) (0.9×3.7 m or 3000 trees/ha) and ‘geneva y-trellis’ dadashpour et al. ‐ training systems of apple cultivars 315 (y-system) (1.6×3.7 m or 1680 trees/ha), based on the relevant protocols described by previous research on apples (robinson, 2003). drip-irrigation was scheduled to operate twice a week. the soil was fertilized once in every season and was managed according to the common practice in the region. trees received their first fertilizers in the second year after planting. they were pruned during the winters, but the amount of wood being removed by pruning was not documented. fruit thinning was performed if necessary. the fruits were harvested manually. twenty representative trees within each replicate were selected for sampling and data collection. agro‐morphological and yield traits to calculate the trunk cross sectional area (tcsa), the trunk circumference was measured (20 cm above the graft union) from both sides (north-south) with a hand caliper. this was performed at the end of the growing season in the november of 2007, 2008, 2009 and 2010. the average measurement of the two sides on the trunk were taken to make trunk diameter (r) and “area= πr2”. a formula assisted in calculating the tcsa in cm2. in addition, the cumulative yield per tree and per hectare were recorded at harvest time (kg/tree and kg/ha). the yield efficiency was defined as “yield per tree divided by tcsa (kg/cm2)”. fruit properties all attributes pertaining to fruit traits were measured using 5 randomly-sampled fruits from each test tree. then, their average was recorded. the individual fruit length, the fruit diameter and the ratio of length to diameter (l/d) were calculated by a vernier caliper. the fruits fresh weight was determined using a mettler pc 8000 scale. in addition, fruit firmness was measured using a penetrometer (instron universal machine, model 1011) and recorded as kg.cm-2. total soluble solids (tss) were measured with a bausch and lomb abbe 3l refractometer. moreover, the dry matter content was determined after the fruits were exposed to a process of drying at 70°c for 48 h. one gram of dry matter was burnt to yield ash in a gaallankamp furnace at 550°c for 6 h. titratable acidity (ta) was determined using an aminex hpx-87h column which operated at 65°c, while 4 mm sulfuric acid was used as an eluent. data analysis the data were obtained by field measurements. laboratory observations were processed by analysis of variance (anova) using the sas software and the duncan mean separation test procedure. 3. results agro‐morphological and yield traits in general, all cultivars had developed a sufficient stem diameter (data not shown). the analysis of variance signified substantial differences among the cultivars and training systems. tree vigor was affected substantially by training systems. after four years, there were significant differences in tcsa among the four cultivars. ‘golab-kohans’ exhibited the highest value of tcsa (17.12 cm2) (table 1). the apple trees that were trained by the y-system showed significantly higher tcsa values (16.41 cm2) compared to those trained by the v-system which formed thinner trunks (9.80 cm2) (table 2). the interaction between table 1 means comparison of four apple cultivars about studied characteristics in guttingen v and geneva-y trellis systems during 2007-2010 means with same letters are not significantly different. (p>0.05) using duncan multiple range test. cultivar fruit firmness (kg/cm2) fruit weight (gr) fruit diameter (cm) fruit length (cm) l/d tss ta (%) ash (%) dry matter (%) cumulative yield (kg/tree) yield efficiency (kg/cm2) tcsa (cm) delbar estival 10.00 b 130.15 b 6.57 b 5.81 a 0.86 a 14.53 a 0.45 bc 0.40 b 20.63 bc 16.4 a 0.41 a 9.58 c fuji 14.52 a 148.40 a 6.94 a 5.78 ab 0.83 b 15.33 a 0.68 a 0.35 b 23.89 ab 14.72 ab 0.1 c 14.69 b golab-kohans 8.44 c 79.25 c 5.72 c 5.01 c 0.86 a 11.23 b 0.28 c 0.38 b 19.56 c 7.72 c 0.1 c 17.12 a starking 14.37 a 143.99 a 6.63 b 5.58 b 0.82 b 14.56 a 0.47 b 0.73 a 24.14 a 10.64 b 0.22 b 10.98 c table 2 properties in guttingen v and geneva-y trellis systems during 2007-2010 means with same letters are not significantly different. (p>0.05) using duncan multiple range test. system fruit firmness (kg/cm2) fruit weight (gr) fruit diameter (cm) fruit length (cm) l/d tss ta (%) ash (%) cumulative yield (kg/tree) cumulative yield (t/ha) yield efficiency (kg/cm2) tcsa (cm) guttingen v 10.53 b 122.45 b 6.36 b 5.39 b 0.84 a 14.17 a 0.46 a 0.5 a 7.88 b 23.640 b 0.25 a 9.80 b geneva-y trellis 12.90 a 126.69 a 6.54 a 5.69 a 0.84 a 13.55 a 0.47 a 0.43 a 16.72 a 28.089 a 0.22 b 16.41 a adv. hort. sci., 2019 33(3): 313-320 316 training systems and cultivars showed that ‘fuji’ had the largest trunk diameter and the largest tcsa (19.98 cm2) (fig. 1a). regardless of the training system, ‘delbar estival’ produced the most cumulative yield (16.4 kg/tree) (table 1). table 2 shows that the y-system results in a higher average value of cumulative yield per tree (16.72 kg/tree) and per hectare (28.08 t/ha) than that of the v-system (7.88 kg/tree and 23.64 t/ha, respectively). the v-system contributed to a higher density of trees (3000 tree/ha), compared to the y-system (1680 tree/ha). results show that ‘fuji’ and ‘delbar estival’ exhibited the most cumulative yield per tree and per hectare, under the y-system and the v-system, respectively (figs. 1b and 1c). concerning the yield efficiency, during the four years, regardless of the training system, the ‘delbar estival’ yielded the highest amount of fruit per trunk cross sectional area (table 1). in addition, the v-system showed a higher yield efficiency (0.25 kg/cm2), compared to the y-system (0.22 kg/cm2). a smaller trunk diameter and a higher tree density per hectare can be reasons for the higher yield efficiency (table 2). the interaction between training systems and cultivars functioned mostly in determining the yield efficiency (0.57 kg/cm2) in the ‘delbar estival’ through the v-system (fig. 1d). fruit properties results showed that the ‘fuji’ cultivar yielded the heaviest fruit weight (148.40 gr), whereas ‘golabkohans’ had the lightest fruit (79.25 gr) (table 1). trees trained by the v-system (as a denser system in this study) developed fruits with an average lighter weight (122.45 gr), but the apples obtained from the y-system were slightly heavier (126.69 gr) (table 2). the ‘starking’ cultivar exhibited the heaviest (159.69 gr) and longest fruit (6.1 cm) by the y-system (figs. 2a and 2b). in fact, the y-system caused the ‘starking’ to exhibit the maximum fruit length among the four cultivars. the y-system contributed to the production of fruits that were significantly longer (5.69 cm) than those obtained by the v-system (5.39 cm) (table 2). in addition, the ‘fuji’ yielded the widest fruit (6.94 cm) among the four studied cultivars (table 1). the y-system caused a greater fruit diameter (6.54 cm) than the v-system (6.36 cm) (table 2). figure 2c shows that the maximum width of fruit (7.1 cm) was recorded in the ‘fuji’ by the ysystem. the highest l/d ratio (0.87) belonged to the ‘delbar estival’ by the y-system. in general, the greatest value of fruit firmness was observed in ‘fuji’ (14.52 kg.cm-2) and the lowest was observed in ‘golab-kohans’ (8.44 kg.cm-2) (table 1). also, trees trained by the y-system yielded fruits with the greatest value of firmness (12.90 kg/cm2), compared to the function of the v-system (10.53 kg/cm2) (table 2). ‘fuji’ yielded the firmest fruits (15.96 kg/cm2) by the y-system (fig. 2d). the highest tss (15.33%) and ta (0.68%) were produced by ‘fuji’, whereas the lowest tss and ta were recorded in the fruits of ‘golabkohans’ (table 1). the content of ta also differed because of the training systems. the y-system caused higher ta values in fruits, compared to the vsystem, but this difference was insignificant (table 2) which suggests that the training system had no remarkable influence on the acidity of fruits in this fig. 1 interaction of training systems (v-system and y-system) and four cultivars (delbar estival, fuji, golab-kohans, starking) on fruit properties. dadashpour et al. ‐ training systems of apple cultivars 317 research. the ‘fuji’ yielded fruits with the highest amounts of tss and ta by the y-system and v-system, respectively. when comparing the cultivars, ‘starking’ had the best results regarding the dry matter of fruits (24.14%) and ash (0.73%) (table 1). regardless of the cultivar, the fruits contained more dry matter when the trees were trained by the v-system, compared to training by the y-system (table 2). additionally, ‘starking’ yielded the highest amount of ash by interaction with training systems examined in this study (fig. 2e). 4. discussion and conclusions the results herein suggest that the cultivars and training systems caused differences in the measured characteristics. the occurrence of more tree growth by ‘golab-kohans’ may be due to a higher degree of shading in the canopy than in other cultivars (lo bianco et al., 2007). in addition to the influence of rootstocks, cultivar vigor can be affected by training systems. a lower tcsa was observed in trees of the v-system. this can be attributed to the competition between adjacent trees which, in turn, was a result of shorter spacing between trees (0.9 m) in comparison with the y-system (1.6 m). as reported by other researchers (musacchi et al., 2015; sosna, 2017), planting the trees closer to each other might have negatively affected the stem diameter in this study. these results are in accordance with the latest findings in the available literature (robinson, 2007; ozkan et al., 2016) in which intensive cultivations had remarkable effects on tree growth. the greater yield caused by the y-system might be due to the larger (wider) tree canopy. this result is in agreement with recent reports which suggest that the number of trees per unit area has a great influence on the yield per tree and per hectare (robinson, 2007; ozkan et al., 2016). in general, a more even distribution of fruit-bearing can be observed in apple trees with vshaped canopies, as trained by the yand v-systems, compared to other popular training systems. this has been suggested before by similar research (sosna, 2017). it is known that the yield efficiency depends on the tree’s vegetative vigor and fruit production. when the cultivar has good yield and high tcsa, a lower yield efficiency occurs compared to trees of other cultivars by the same yield and lower tcsa. a lower tree vigor, as caused by the v-system, did not result in a higher yield efficiency. this can be due to a lower yield per tree. in fact, results show that a highfig. 2 interaction of training systems (v-system and y-system) and four apple cultivars (delbar estival, fuji, golabkohans and starking) on fruit properties. 318 adv. hort. sci., 2019 33(3): 313-320 er yield efficiency can be attained by increasing the number of fruits in each tree or by controlling the tree vigor by dwarf rootstocks. significant differences in yield efficiency were also reported in a previous study (fioravanco et al., 2016). it may be assumed that trees on dwarf rootstocks exhibit a weaker vegetative vigor and result in a higher amount of yield (robinson, 2007). nonetheless, the differences among cultivars in this study is likely due to the variations in morphological traits, which is in agreement with previous studies (barritt et al., 1995; dadashpour et al., 2010). no incremental trend was observed in the fruit weight during the four years, even by the influence of training systems. the contradictory effects of planting density on the fruit weight in this study are consistent with earlier reports (ozkan et al. , 2012; sosna, 2017). nonetheless, fruit quality is influenced by many factors such as the specifications of a training system (robinson et al., 1991). therefore, it is natural to expect variations in the type of influence caused by the two different training systems on the measured traits in fruits. the l/d (≥1) is a criterion used for apple marketing, but all cultivars showed l/d <1 in this study. this observation is probably due to warmer nights in the climatic conditions of the experiment, resulting in insufficient cell elongation. this confirms the results of previous research (dadashpour et al., 2011). based on the current discussion, the ‘delbar estival’ probably has the highest marketable value in terms of its visual appearance among the cultivars. the denser cultivation of trees in the v-system contributed to the production of fruits with lower amounts of coloration, but this was not substantially different compared to the other training system. the good quality of apples obtained from the v-system was noticed in previous studies (rutkowski et al., 2009; dadashpour et al., 2012). it seems that the climatic temperature can affect the fruit firmness. in most of the cultivars, the softest fruits were observed in 2008 (as a cool year in this experiment). however, the relation between temperature and fruit firmness is not fully understood. the y-system caused firmer fruits, compared to the v-system (table 2), and this confirms that fruits harvested from the y-system can be transported with less physical damage. significant differences in apple firmness support recent findings (talaie et al., 2011). ‘golabkohans’ was the earliest ripening cultivar and produced the softest fruits (7.25 kg.cm-2) by the v-system (fig. 2d). the ‘fuji’ produced the firmest fruits, probably because of the small fruit size, thereby confirming the findings of previous studies (drake et al., 1988; dadashpour et al., 2010). in addition, differences in fruit firmness might have been due to genetic variations among cultivars. in addition, it has been reported that fruit firmness is the first edible criterion affecting buyer acceptance (harker et al., 2008). considering the fruit sweetness, fruits and leaves that are exposed to higher light intensities may exhibit more tss (tustin et al., 1988). also, the different tss contents among cultivars may result from variations in leaf area, as suggested by previous research (hudina and stamper, 2002) or by a presumably higher canopy shading of cultivars which produce fruits of lower tss (garriz et al., 1996, 1998). although the tss was not significantly affected by the two training systems, the v-system caused slightly higher levels of tss than the y-system (table 2). among the cultivars, the ‘fuji’ produced the sourest fruits. these results show that acidity, in general, varies with cultivar, confirming previous studies (platon, 2007; dadashpour et al., 2010). the highest amount of ta was observed in fruits of the ‘fuji’ cultivar. this may have resulted from less shading in the tree canopy or because of good nutritional conditions. in general, the ‘starking’ cultivar produced the highest amount of dry matter, thereby confirming previous claims regarding the differences among cultivars in this regard (lata, 2007; palmer et al., 2010). in addition, the dry matter content varies among cultivars, and different training systems cause variations in the dry matter. the dry matter can vary from fruit to fruit and from training system to training system, in agreement with a previous study (palmer et al., 2010). in conclusion, the ‘delbar estival’ exhibited better results under intensive training systems, whereas ‘golab-kohans’ and ‘fuji’ showed the best growth characteristics. in general, the y-system was better than the v-system when considering the majority of characteristics. the two cultivars ‘fuji’ and ‘delbar estival’ were more adaptable to intensive training systems in karaj’s climatic conditions. references alizadeh a., pirmoradyian m., 2016 comparison test of training systems and its influence on yield and fruit quality of g. delicious on clonal rootstocks. sci. agri., 14: 289-292. barritt b.h., konishi a.s., dilley m.a., 1995 performance of three apple cultivars with 23 dwarfing rootstocks during 8 seasons in washington. fruit var. dadashpour et al. ‐ training systems of apple cultivars 319 j., 49: 158-170. 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estahban, iran. 4 horticultural science, research institute of biotic technologies of medicinal and aromatic plants, sari agricultural sciences and natural resources university (sanru), iran. key words: abiotic stress, electrolyte leakage, specific leaf area, stem diameter, stem length, stomata conductance, transpiration rate. abstract: the growing demand for both fresh and dry figs worldwide is due to its richness in mineral compounds (i.e. iron and copper) and polyphenols. considering the position of iranian cultivars in global fig market, the present study examined the growth and photosynthetic rate of commercial fig cultivars (i.e. ‘sabzʼ, ‘siyahʼ, ‘shah anjirʼ, ‘atabakiʼ, ‘kashkiʼ, ‘matiʼ and ‘bar anjirʼ) exposed to six salt treatments corresponding to the following electrical conduc­ tivities (ec): 0.5, 2, 4, 6, 8 and 10 ds m­1. the results indicated a decrease trend of stem length, stem diameter and leaf number in salt­exposed plants. the electrolyte leakage and protein content in all cultivars followed an ascending trend. the specific leaf area, relative water content, photosynthetic indices and nitrogen content followed a decreasing trend according with increasing salinity. the ‘siyahʼ and ‘sabzʼ, as the most salt­tolerant cultivars, had the maximum leaf abscission, the lowest transpiration rate and leaf water content under salt condition, compared to all other tested cultivars. moreover, they had the most leaf succulence and leaf dry matter content and the lowest specific leaf area, which related to the balance between growth ratio and osmotic regulation under salt conditions. the ‘shah anjirʼ, as the most salt­sensitive cultivar, could not balance transpiration rate and leaf water content under salt treatment higher than 4 ds m­1. 1. introduction the fig (ficus carica l., 2n= 26) of the moraceae family, is one of the first plants cultivated and consumed by human beings (duenas et al., 2008). according to the fao, the fig is harvested from 36,535 hectares of cultivated land, with an annual production of over one million tons (fao, 2017). iran is the third largest producer of dried figs in the world, as well as the fifth largest fresh fig producer in the world with a cultivated area of 53,101 hectares and a production of 70,730 tons per year of fresh figs (fao, 2017). (*) corresponding author: shamili@ut.ac.ir citation: salimpour a., shamili m., dadkhodai a., zare h., hadadinejad m., 2019 ­ evaluating the salt tolerance of seven fig cultivars (ficus carica l.). ­ adv. hort. sci., 33(4): 553­565. copyright: © 2019 salimpour a., shamili m., dadkhodai a., zare h., hadadinejad m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 june 2019 accepted for publication 5 september 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 553­565 554 the growing demand for the fig worldwide (aksoy, 2005) is due to its richness in mineral com­ pounds (i.e. iron and copper) and considerable amounts of vitamins a and c (flaishman et al., 2008). the fig is consumed in fresh, dried, powdered, canned, chocolate­covered forms, and utilized in the preparation of jam, syrup and muscat­halvah (de masi et al., 2005). significant genetic variation in the fig species, due to obligatory outcrossing, has led to the establish­ ment of new genotypes with desirable properties. according to the latest reports, there are currently more than 600 known fruit­producing cultivars and genotypes, which are distinct in leaf morphology, growth vigor, internal and external color of the fruit, taste and quality index of the fruit, shape and thick­ ness of the fruit, the diameter of the ostiole, and pro­ ductivity period (condit, 1955; toribio and montes, 1996; garcía­ruiz et al., 2013). sabz or verde (green), siyah (black), shah anjir (king fig), atabaki, kashki and mati are considered as the most important and marketable cultivars of iranian figs for local markets or export. the ‘bar anjirʼ is the most commonly­used caprifig (condit, 1955; pourghayoumi et al., 2016). several research centers have focused on differ­ ent aspects of physiology and breeding of fig cultivars and genotypes. for example, some researchers emphasized on genetic diversity of fig using morpho­ logical (khadivi et al., 2018) and molecular (cabrita et al., 2001; khadari et al., 2005; giraldo et al., 2008) markers. some others attempted to study the capri­ figs (dalkılıç et al., 2011), while others have consid­ ered the variety of the fruit and its qualitative fea­ tures (solomon et al., 2006; polat and caliskan, 2008; ercisli et al., 2012). in addition, the variation in the behavior of fig cul­ tivars and genotypes to abiotic stress such as chilling (karami et al., 2018), drought (gholami et al., 2012) and salinity (zarei et al., 2016) have attracted the researchers’ attention. salinity is one of the most important environmen­ tal factors which reduces the growth, development and production of plants (sevengor et al., 2011). while some researchers suggested reducing leaf area as the plants’ responses to the salinity stress, others enumerate reduced stem length, root length, fresh and dry weights, and relative leaf water content (yamasaki and dillenburg, 1999; bolat et al., 2006; najafian et al., 2008; adish et al., 2010; khayyat et al., 2014; khoshbahkt et al., 2014; soliman and abd alhady, 2017). other important processes that are negatively affected by salinity are protein synthesis (taylor et al., 2004; murcute et al., 2010) and nitro­ gen metabolism (owais, 2015; ashraf et al., 2017), whereas in other studies the inhibition of plant growth caused by salinity has been attributed to a decrease in photosynthesis (garcia­sanchez et al., 2006). fig is a moderate salt­tolerant crop (golombek and lüdders, 1990). the available studies on the response of fig commercial cultivars to different lev­ els of salinity indicated a significant variation in mor­ phological characteristics, growth parameters, physi­ ological behavior, photosynthetic efficiency, gas exchange ratio, product quality, and productivity (essam et al., 2013; metwali et al., 2014; alswalmeh et al., 2015; zarei et al., 2016, 2017; soliman and abd alhady, 2017). considering the position of iranian cultivars in global fig market, the present study examined the growth and photosynthetic rate of commercial fig cultivars (i.e. ‘sabzʼ, ‘siyahʼ, ‘shah anjirʼ, ‘atabakiʼ, ‘kashkiʼ, ‘matiʼ and ‘bar anjirʼ) exposed to six salt treatments corresponding to the following electrical conductivities (ec): 0.5, 2, 4, 6, 8 and 10 dsm­1 to identify the most salt­tolerant cultivar. 2. materials and methods the present study was conducted in the plant breeding department, faculty of agriculture, university of shiraz (36° 29’ n and 32° 52’ e), iran during 2016­2018. plant materials the plant materials were included six 20­years­old edible fig cultivars (sabz, siyah, shah anjir, atabaki, kashki, mati), and a caprifig (‘bar anjirʼ) were located at estahban fig research station (36° 29’ n and 32° 52’ e) (table 1). the hard­wood cuttings, 20 cm in length and one cm in diameter, were collected from one­year­old branches on march 25, 2016. the cut­ tings were treated with a fungicide (benomyl 2000 ppm) and a rooting hormone (iba solution 3000 ppm). then, the upper side of the cutting was cov­ ered to prevent the decay, and each cutting was placed in a dark plastic bag (25 x 18 cm2), which was filled by sand. in the next stage, the bags were locat­ ed in the shade­house conditions (temperature: 28±2°c d/18±2°c n, rh=50%, and 50% shade) and were irrigated twice a day. in june 2017, rooted­cut­ tings were transplanted in pots filled with 500 g of gravel and 20 kg of the media described in table 2. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 555 the media included the mixture of soil, leaf compost and sand (1:1:1), which was steam­disinfected. a pressure plate extractor (model adc, by santa barbara, united states) was used to measure the media water capacity. the pots were kept under shade­house condition (temperature: 30±1°c d/18±0.5°c n, rh=50%, and 50% shade). salt treatment the salt treatments were provided through the irrigation water. treatments included low salt treat­ ments (ec= 0.5 and 2 ds m­1, a and b, respectively), intermediate salt treatments (ec= 4 and 5 ds m­1, c and d, respectively) and high salt treatments (ec= 8 and 10 ds m­1, e and f, respectively). in order to avoid osmotic stress, salt treatments were introduced gradually, starting from 1/4 up to the final concentration. then, irrigation frequency was calculated based on the media filed capacity and water requirement (essam et al., 2013; zarei et al., 2016). salt treatment was performed within nine weeks from 23/7/2017­ to 26/9/2017. in addition, all of the plants were irrigated by distilled water for four months (26/1/2018). stem length the length of the stem was recorded at the begin­ ning and end of the experiment. the difference between the two values was recorded as the differ­ ence in stem length (cm). stem diameter the stem diameter was recorded at the beginning and end of the experiment by digital caliper (4 cm above the soil surface). the difference was recorded as the difference in stem diameter (mm). number of leaves the number of expanded leaves was counted at table 1 ­ the growth and bearing habit of studied fig cultivars (sabet sarvestani, 1999; safai 2002; jafari et al., 2016) parameters cultivar ‘sabzʼ ‘siyahʼ ‘shah anjirʼ ‘atabakiʼ ‘kashkiʼ ‘matiʼ ‘bar anjirʼ growth and bearing relatively high high moderate moderate growth and low bearing high growth and moderate bearing moderate growth and low bearing low­ moderate growth and high bearing bud conical terminal buds with curved tip curved and pointed terminal bud terminal pointed bud terminal conical bud terminal conical, with tip terminal conical bud without tip terminal conical bud fruit medium, yellowish, no neck, thick pulp medium, dark purple, no neck, thin skin, flesh uniformly red, low pulp thickness large, necked, yellow, pink pulp, fully seed large, rounded, reddish­purple fruit, reddish pulp, reddish medium, green, , open ostiole necked, white pulp, low seedy large, no neck, open ostiole, dark and thick fruit, white, reddish pulp medium, with long neck, containing blastophagous bees type of consumption dried fruit fresh fruit both fresh and dried fruit fresh fruit fresh and processed fruit fresh fruit caprifig yield very high high high moderate moderate moderate moderate taste excellent sweet­and­sour sweet sweet­and­sour sweet sweet­and­sour sweet bearing period early bearing early bearing mid­season bearing early bearing late bearing early bearing mid­season bearing table 2 ­ physico­chemical properties of the soil ec= electrical conductivity; cec= cation exchange capacity. soil texture san (%) silt (%) clay (%) ec (ds/m) cec (me/100) ph lime (%) sandy clay­loam 58±1.01 26±1 16±0.9 1.45±0.21 10.84±0.81 7.7±0.17 ±351.33 organic c (%) n (%) k (ppm) p (ppm) cu (ppm) mn(ppm) fe (ppm) zn (ppm) 1.17±0.05 0.17±0.002 126±1.3 3.2±0.05 0.26±0.002 3.86±0.04 2.85±0.03 0.056±0.001 adv. hort. sci., 2019 33(4): 553­565 556 the beginning and end of the experiment. the differ­ ence between the two values was recorded as the difference in the number of leaves. specific leaf area (sla), leaf dry matter content (ldmc) and leaf succulence the fourth top leaf was harvested after ending the experiment. the leaf area was recorded using a leaf area meter (ci­202 portable laser leaf area meter). the leaves were then dried in an oven (75°c, 48 hrs.) and the dry weight was recorded (ldw). the specific leaf area (in cm2 g­1) was calculated by using the eq. (1). ldmc and leaf succulence were calculated using eq. (2) and (3). sla = la/ldw (1) where la is leaf area (cm2) and ldw is leaf dry weight (g), according to hunt et al., 2002. ldmc = ldw/lfw (2) where ldw and lfw are leaf dry weight (g) and leaf fresh weight (g) respectively (garnier et al., 2001). leaf succulence = lfw/la (3) where lfw is leaf fresh weight (g) and la is leaf area (cm2), according to agarie et al., 2007. relative water content (rwc) mature leaves were collected nine weeks after salt application at mid­day, and were transferred immediately to the lab. then, five similar leaf discs without any vein were separated from each sample and weighted (w1). further, the disc samples were placed in distilled water (4 hrs) under laboratory con­ ditions (24 ± 1oc). subsequently, the samples were surface dried and re­weighed (w2). furthermore, the discs were placed in an electric furnace (model: memmert, made by karl klob factory, germany) (90°c, 60 min) and reweighted (w3). finally, the rela­ tive water content was calculated using the eq. (4) (barrs and weatherley, 1962). rwc = (w1 ­ w3) x 100 (4) (w2 ­ w3) electrolyte leakage (el) the top expanded leaf was harvested and 5 leaf discs without any vein were prepared. the samples were rinsed three times with distilled water, and incu­ bated in 10 ml of distilled water (at 40°c for 30 min). after cooling, the electrical conductivity was mea­ sured using an electrical conductivity meter (h18633 model) (c1). the samples were then autoclaved (at 120°c for 15 min). after cooling, the electrical conduc­ tivity was re­measured (c2). the electrolyte leakage was calculated via eq. (5) (sairam et al., 1997). el = (c1/c2) x 100 (5) leaf protein content first, the fresh leaf sample (0.2 g) was powdered with liquid nitrogen. then, two ml of potassium phosphate buffer (38.5 ml nah2po4, 68.5 ml of na2hpo4, 0.074 g edta and 1 g of pvp), ph = 7.15 was added and well­homogenized. the extract was cen­ trifuged at 13000 rpm for 15 min at 4°c, and the super­ natant was used to measure protein content. about 20 μl of the extract, 80 μl of potassium phosphate buffer (ph= 7.15) and 5 ml of coomassie brilliant blue (c47h48n3nao7s2) was stirred for 2 min. in addition, the absorbance was read at 595 nm by using a spec­ trophotometer (biowave ii model) after incubating 5 min at room temperature. the extraction buffer was used as blank. the protein content (in mg g­1 fw) in the sample was calculated according to the sample absorp­ tion using the bovine albumin serum (c123h193n35o37) standard curve (bradford, 1976). leaf nitrogen content the kjeldahl method was used to determine the nitrogen content in fresh leaf samples (kjeldahl, 1883). photosynthetic indexes the photosynthetic indices were recorded using a compact­portable­photosynthesis­system (lci, uk). the device was put on attached leaf (third expanded leaf) at midday, then transpiration rate (in mol h2o m­2 s­1) and stomata conductance (in mol co2 m⁻² s⁻¹) were recorded after two min (evans and von caemmerer, 1996). experimental design and data analysis the present experiment was conducted in a complete randomized design. the factors included fig cultivars (seven cultivars) and nacl treatments (six concentrations), with five replications. sas version 9.1.3 (sas®, 1990) was used for the statistical analy­ sis. shapiro­wilks test confirmed the normality of the data. in addition, leven's test confirmed the variance homogeneity. further, tukey test was conducted for mean comparisons (p<0.01). finally, pearson coeffi­ cient was used for analyzing the relationship between the parameters. 3. results the results of variance analysis indicated that the studied cultivars had a significantly different behavior salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 557 3 leaves in the lowest and highest salinity levels in this cultivar. in addition, ‘sabzʼ and ‘atabakiʼ cultivars had the lowest number of leaves under intermediate salt conditions (4 and 6 dsm­1) (table 4). the leaf number of ‘atabakiʼ cultivar under higher salt condi­ tion was significantly lower than 0.5 ds m­1 of ec. specific leaf area, leaf dry matter content and leaf succulence the specific leaf area followed a decreasing trend due to an increase in salt concentration in the ‘sabzʼ, ‘siyahʼ, ‘shah anjirʼ and ‘kashkiʼ cultivars. in ‘atabakiʼ cultivar, under ec of 6 and 10 dsm­1 the reduction was observed. in ‘bar anjirʼ cultivar, there was no sig­ nificant difference between 2­10 dsm­1 salt levels (table 4). except for ‘matiʼ and ‘bar anjirʼ cultivars showing 32.21 and 60.55% increase in sla, the rest of the cultivars had descending trend of sla during the experiment. leaf dry matter content of salt­ exposed fig cultivars followed a descending trend. the difference in ldmc of seven cultivars under low salt concentration (0.5 and 2 dsm­1) was not remark­ able. under moderate salt condition (4 dsm­1), most of the cultivars had similar values but under higher salt (6 dsm­1 and more), ‘shah anjirʼ and ‘siyahʼ culti­ vars showed an ascending trend. leaf succulence dis­ played slight rising trend. ‘bar anjirʼ had the highest value under low salt conditions, while ‘siyahʼ showed the highest value under moderate and high salt con­ dition. the values of this parameter was unchanged under moderate salt conditions and the decrease started when nacl reached 8 dsm­1 and more (table 4). leaf succulence of ‘sabzʼ, ‘shah anjirʼ and ‘siyahʼ under ec of 10 ds m­1 were 1.64, 1.64 and 1.61 times higher than their value under ec of 6 dsm­1. relative water content of leaf salinity had a significant effect on reducing leaf under salt conditions, due to salt concentration and cultivar variation (table 3). stem length with increasing salinity level, stem length decreased significantly in all cultivars. the greatest effect was observed in ‘bar anjirʼ cultivar, which decreased from 78.84 cm (under 0.5 ds m­1 salinity) to 28.44 cm (under 10 ds m­1 salinity). the lowest effect was observed in the ‘siyahʼ cultivar, which reduced from 51.3 cm (in 0.5 ds m­1 salinity) to 35.8 cm (in 10 ds m­1 salinity) (table 4). the decrease in stem length for all the tested cultivars was between low (0.5 ds m­1) and high (10 ds m­1) nacl­treated plant. this decrease for most of the cultivars (includ­ ing ‘sabzʼ, ‘shah anjirʼ, ‘matiʼ and ‘bar anjirʼ) was more than 200%. stem diameter by raising salinity, the stem diameter decreased significantly in all cultivars. the highest reduction in stem diameter was observed in the ‘matiʼ cultivar, which decreased from 6.35 mm (under 0.5 dsm­1 salinity level) to 3.41 mm (under 10 dsm­1). the low­ est effect was observed in ‘siyahʼ cultivar, reducing from 5.37 mm at the lowest salinity level to 3.2 mm at the highest salinity level (table 4). in addition, the decrease in stem diameter of low and high salt treat­ ed plants varied between 157.93 to 219.89% (‘kashkiʼ and ‘bar anjirʼ cultivars, respectively). number of leaves with an increase in salinity levels, the number of leaves in all cultivars followed a decreasing trend. the greatest effect of salinity was observed in ‘bar anjirʼ, which difference in the number of the leaves in the lowest and highest salinity levels was 10.2 leaves. the lowest effect of salinity on leaf number was observed in ‘shah anjirʼ, and the difference was table 3 ­ the interaction of cultivar and salinity on growth and physiological parameters of seven fig (the mean square value is given) ns, * and **= not significant, significant at 5 and 1% respectively (by tukey mean comparison test). physiological parameters cultivar salinity cultivar x salinity difference in stem length 1273.11 * 5916.23 * 223.68 ** difference in stem diameter 9.61 ** 30.78 * 1.34 ** difference in leaf number 107.80 ** 220.38 * 11.25 ** specific leaf area 174.37 ** 27.628 ns 11.23 ** relative water content 1540.57 ** 264.34 ** 289.57 ** electrolyte leakage 21.31 ** 239.41 ** 11.23 ** leaf protein 37007542 ** 268172052 ** 4586351 ** leaf nitrogen 0.93 ** 5.94 ** 0.071 ** transpiration rate 4.46 ** 90.58 ** 3.89 ** stomata conductance 0.099 ** 1.025 ** 0.074 ** 558 adv. hort. sci., 2019 33(4): 553­565 relative water content in all cultivars. the highest decrease was observed in the ‘siyahʼ cultivar, which reduced from 90.83% in the lowest salinity level to 53.03% in the highest salinity level. the lowest effect was observed on the ‘sabzʼ cultivar. the intermediate salt condition of 4 dsm­1 did not make a significant difference from 2 dsm­1, except in ‘sabzʼ (table 5). the ‘kashkiʼ and ‘bar anjirʼ cultivars had the lowest decline of rwc during the experiment (15.15 and 15.33%, respectively), while ‘siyahʼ showed the stronger decrease in rwc (41.62%). electrolyte leakage salinity had a significant ascending effect on elec­ trolyte leakage in seven fig cultivars. the highest salini­ ty increased the ionic leakage in ‘siyahʼ and ‘atabakiʼ table 4 ­ the influence of saline water on stem length, stem diameter, leaf number, specific leaf area, ldmc and leaf succulence of fig cultivars genotype treatments stem length (cm) stem diameter (mm) leaf number specific leaf area (cm2 g­1) ldmc leaf succulence sabz a 59.72 b 4.53 c 8.20 c 11.53 b 0.68 b 0.13 i b 48.00 c 3.19 d 6.80 d 13.38 a 0.70 b 0.11 i c 38.64 c 3.29 d 6.00 d 11.60 b 0.52 d 0.17 h d 27.90 d 2.34 e 4.00 e 11.76 b 0.57 c 0.15 h e 30.75 d 2.51 e 3.20 f 10.22 c 0.57 c 0.17 h f 27.30 d 2.39 e 2.60 f 7.85 e 0.52 d 0.24 g siyah a 51.30 c 5.37 b 5.80 d 4.79 f 0.74 a 0.28 e b 38.80 c 4.91 c 4.60 d 3.64 g 0.73 a 0.38 d c 30.24 d 3.24 d 2.50 f 3.94 g 0.61 c 0.41 c d 36.50 d 3.87 d 3.40 f 3.76 g 0.60 c 0.44 c e 28.66 d 3.49 d 1.00 g 3.77 g 0.60 c 0.44 c f 35.80 d 3.20 d 2.50 f 2.04 g 0.69 b 0.71 a shah anjir a 49.72 c 4.72 c 4.00 d 11.27 b 0.70 b 0.13 i b 52.40 c 5.27 b 6.40 d 11.04 b 0.54 d 0.17 h c 29.30 d 3.68 d 5.40 d 10.35 c 0.62 c 0.16 h d 32.90 d 3.60 d 2.60 f 9.73 c 0.74 a 0.14 h e 33.04 d 3.15 d 1.60 f 9.52 c 0.76 a 0.14 h f 23.06 d 2.54 e 1.00 g 6.40 e 0.69 b 0.23 g atabaki a 76.50 a 4.75 c 7.60 c 11.89 b 0.71 b 0.12 i b 59.20 b 5.17 b 7.20 c 8.49 d 0.68 b 0.17 h c 53.74 c 4.56 c 6.60 d 7.76 e 0.61 c 0.21 g d 40.50 c 3.99 d 5.00 e 5.64 f 0.49 d 0.36 e e 25.30 d 1.45 f 3.00 f 8.82 d 0.44 e 0.26 g f 44.08 c 2.46 e 3.60 f 6.01 e 0.49 e 0.34 e kashki a 78.00 a 5.18 b 12.40 a 9.55 c 0.61 c 0.17 h b 53.62 b 5.50 b 7.00 c 9.23 d 0.62 c 0.18 h c 50.14 c 3.77 d 8.00 c 9.08 d 0.52 d 0.21 g d 38.88 c 2.97 d 4.40 e 7.88 e 0.57 c 0.22 f e 46.02 c 3.26 d 3.80 f 6.75 e 0.57 c 0.26 g f 45.46 c 3.28 d 6.60 d 6.65 e 0.52 d 0.29 f mati a 68.22 b 6.35 a 9.80 b 5.95 f 0.68 b 0.25 g b 64.80 b 5.80 b 9.60 b 8.55 d 0.62 c 0.19 h c 52.10 c 4.65 c 5.80 d 5.21 f 0.45 e 0.43 c d 38.30 c 4.19 c 3.20 f 4.64 f 0.45 e 0.48 c e 33.80 d 4.24 c 5.20 e 4.20 f 0.45 e 0.53 b f 30.90 d 3.41 d 2.40 f 7.86 e 0.44 e 0.29 f bar anjir a 78.84 a 3.98 c 13.60 a 5.60 f 0.35 f 0.51 b b 58.00 b 4.02 c 11.00 b 8.36 d 0.29 f 0.41 c c 48.00 c 3.67 d 8.40 c 8.74 d 0.30 f 0.39 d d 43.56 c 3.19 d 7.40 c 8.98 d 0.32 f 0.35 e e 32.20 d 2.90 d 4.20 e 8.68 d 0.28 f 0.42 c f 28.44 d 1.81 e 3.40 f 8.99 d 0.30 f 0.38 e data are average of five replications. in each column means with a common letter have no significant difference at 1% of tukey test. treatments a, b, c, d, e and f are 0.5, 2, 4, 6, 8, and 19 ds m­1 of ec, respectively. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 559 cultivars (table 5). the electrolyte leakage of ‘atabakiʼ, ‘siyahʼ and ‘bar anjirʼ cultivars showed the highest dif­ ference between the moderate and high salt condi­ tions (39.19, 32.89 and 31.65% increases, respectively). leaf protein the results indicated that salinity had a significant effect on leaf protein of studied fig cultivars. increasing the stress to 4 ds­1 of ec increased the table 5 ­ the influence of saline water on electrolyte leakage, protein, nitrogen, transpiration rate, stomata conductance and rwc of fig cultivars data are average of five replications. in each column means with a common letter have no significant difference at 1% of tukey test. treatments a, b, c, d, e and f are 0.5, 2, 4, 6, 8, and 19 ds m­1 of ec, respectively. genotype treatments rwc (%) electrolyte leakage (%) protein (mg. gˉ¹. fw) nitrogen content (%) transpiration rate (mol h2o m­2s­1) stomata conductance (mol co2 m⁻² s⁻¹) sabz a 76.20 c 19.53 c 0.90 c 3.73 a 59.72 b 0.46 d b 74.80 c 20.75 c 0.93 c 3.55 b 48.00 d 0.13 g c 68.20 d 18.76 c 1.21 b 3.28 b 38.64 c 0.20 f d 62.55 d 17.80 d 1.79 b 3.13 b 27.90 c 0.20 f e 62.22 d 19.20 c 2.20 a 2.47 c 30.75 g 0.06 h f 61.88 d 22.01 c 2.45 a 2.31 c 27.30 g 0.05 h siyah a 90.83 a 15.77 e 0.86 c 3.55 b 51.30 b 0.53 c b 76.78 c 16.42 d 0.86 c 3.59 b 38.80 c 0.78 a c 70.15 c 18.61 c 1.22 b 3.43 b 30.24 e 0.16 g d 67.55 d 19.91 c 2.17 a 3.21 b 36.50 e 0.14 g e 65.09 d 22.31 b 2.35 a 2.98 c 28.66 g 0.07 h f 53.03 e 24.73 a 2.60 a 2.97 b 35.80 g 0.04 h shah anjir a 89.23 b 16.23 d 0.76 c 3.28 b 49.72 b 0.45 d b 88.79 b 15.17 e 0.92 c 3.28 b 52.40 b 0.48 d c 81.68 b 18.67 c 0.99 c 3.10 b 29.30 d 0.30 d d 77.66 c 21.24 b 1.01 b 2.50 c 32.90 d 0.15 g e 77.56 c 20.61 c 1.56 b 3.15 b 33.04 f 0.08 h f 72.15 c 22.22 c 1.36 b 2.92 c 23.06 e 0.11 g atabaki a 81.16 b 14.37 e 0.91 c 3.13 b 76.50 a 0.67 b b 77.22 c 15.39 e 1.01 b 3.34 b 59.20 c 0.36 e c 75.33 c 16.56 d 1.15 b 3.16 b 53.74 d 0.38 d d 73.47 c 19.29 c 1.62 b 2.97 c 40.50 d 0.40 d e 71.09 c 22.75 b 2.04 a 2.43 c 25.30 g 0.06 h f 64.65 d 23.05 b 2.31 b 2.29 c 44.08 h 0.03 h kashki a 85.34 b 16.51 d 0.74 c 2.98 c 78.00 b 0.40 a b 78.92 c 20.10 c 0.85 c 3.31 b 53.62 c 0.27 f c 75.33 c 20.04 c 1.08 b 3.13 b 50.14 e 0.13 g d 73.48 c 22.49 b 1.31 b 2.89 c 38.88 e 0.19 g e 73.29 c 23.20 b 1.84 b 2.75 c 46.02 e 0.14 g f 72.41 c 22.75 c 2.19 a 2.33 c 45.46 e 0.11 g mati a 96.32 a 14.46 e 0.84 c 2.97 c 68.22 a 0.75 a b 88.58 b 17.27 d 0.94 c 3.50 b 64.80 b 0.43 d c 83.88 b 19.24 c 1.09 b 3.27 b 52.10 e 0.13 g d 78.39 c 21.22 b 1.23 b 3.09 b 38.30 e 0.23 f e 74.58 c 21.65 c 1.71 b 2.66 c 33.80 f 0.15 g f 70.23 c 22.38 c 2.01 a 2.60 c 30.90 g 0.04 h bar anjir a 95.00 a 14.83 e 0.71 c 3.78 a 78.84 d 0.20 f b 93.68 a 15.30 e 0.75 c 3.32 b 58.00 d 0.34 e c 91.82 a 16.68 d 0.81 c 3.14 b 48.00 b 0.26 f d 88.52 b 19.72 c 1.04 b 2.82 c 43.56 b 0.09 h e 82.81 b 21.26 b 1.76 b 2.75 c 32.20 f 0.08 h f 80.44 b 21.96 c 1.96 b 2.30 c 28.44 g 0.04 h adv. hort. sci., 2019 33(4): 553­565 560 total protein content gradually. the highest amounts were observed in ‘siyahʼ (2.60 mg g­1 fw) and ‘sabzʼ (2.45 mg g­1 fw) cultivars and the lowest in ‘shah anjirʼ (1.36 mg g­1 fw) and ‘bar anjirʼ (1.96 mg g­1 fw) cultivars (table 5). the increase in protein content was expected between moderate (4 ds­1) and high (10 ds­1) nacl treated plant. this increase for most of the cultivars (including ‘sabzʼ, ‘siyahʼ, ‘atabakiʼ, ‘kashkiʼ and ‘bar anjirʼ) was more than 200%. leaf nitrogen by increasing salinity levels in all seven fig culti­ vars, leaf nitrogen content decreased. the highest reduction was observed in ‘bar anjirʼ cultivar at ec of 10 ds­1 (2.30%) and the lowest reduction was observed in ‘siyahʼ cultivar, which decreased from 3.55% at the lowest salinity level to 2.99% at the ec of 10 dsm­1 (table 5). the ‘matiʼ, ‘kashkiʼ and ‘atabakiʼ showed 10.03, 5.04 and 0.88% increase in n content under moderate salt condition (4 dsm­1), while the rest cultivars exhibited a decrease in n con­ tent (table 5). photosynthetic indices the interaction of salinity stress and cultivar on photosynthetic indices (transpiration rate and stoma­ ta conductance) was significant. with increasing nacl levels, the rate of transpiration decreased in seven fig cultivars. the highest reduction was observed in ‘atabakiʼ cultivar (0.85 mol h2o m­2 s­1 at 10 dsm­1 of ec). the lowest influence was observed in ‘kashkiʼ (3.41 mol h2o m­2 s­1 at 10 dsm­1 of ec). for all the studied cultivars this parameter did not differ signifi­ cantly between 4 and 6 dsm­1 (intermediate salt con­ ditions). high salt concentrations (8 and 10 dsm­1) caused a noticeable difference among the genotypes, where ‘atabakiʼ, ‘siyahʼ, ‘matiʼ and ‘sabzʼ were fall to 12.18, 19.63, 22.16 and 23.40% of their initial values (table 5). stomata conductance decreased by increasing salt concentration in all cultivars, and reached its lowest level at the highest salinity level (10 dsm­1). the lowest stomata conductance at of ec 10 dsm­1 was observed in ‘atabakiʼ (0.03 mol co2 m⁻² s⁻¹) and the lowest in ‘kashkiʼ (0.11 mol co2 m⁻² s⁻¹) (table 5). the ‘atabakiʼ, ‘matiʼ and ‘siyahʼ cultivars displayed the highest decrease of stomata conduc­ tance from 05. to 10 dsm­1of ec (3.86, 5.91 and 7.95% decrease of their initial values, respectively) correlation analysis table 4 indicates the bivariate pearson correla­ tions among the parameters. the bold faces values indicate high correlated values (higher than 0.5). difference in the number of leaves had high correla­ tion with relative water content (0.583**), transpiration rate (0.899**) and stomata conduc­ tance (0.915**). specific leaf area and relative water content were positively correlated (0.680**). in addi­ tion, both photosynthetic indices showed high corre­ lation (0.877**) (table 6). 4. discussion and conclusions the negative effect of salinity leads to the changes in soil structure, competition in nutrients uptake in different parts of the plant and eventually inhibition of nutrients absorption (gholami et al., 2012). na+ reduces plant biomass by disrupting the protein syn­ thesis, destroying chlorophyll, and decreasing the activity of the enzymes which are involved in biosyn­ physiological parameters leaf nitrogen leaf protein electrolyte leakage difference in stem length difference in the number of leaves difference in stem diameters specific leaf area relative water content transpiration rate stomata conduc­ tance leaf nitrogen 1 leaf protein ­0.170 * 1 electrolyte leakage ­0.225 ** 0.017 1 difference in stem length ­0.375 ** ­0.006 0.068 1 difference in the number of leaves 0.331 ** ­0.009 ­0.055 ­0.108 1 difference in stem diameters ­0.415 ** 0.323 ** 0.179 ** 0.093 ­0.287 ** 1 specific leaf area 0.393 ** ­0.239 ** ­0.103 ­0.079 0.199 ** ­0.480 ** 1 relative water content 0.351 ** ­0.124 ­0.122 ­0.077 0.583 ** ­0.499 ** 0.680 ** 1 transpiration rate 0.362 ** ­0.111 ­0.098 ­0.079 0.899 ** ­0.435 ** 0.227 ** 0.581 ** 1 stomata conductance 0.195 ** 0.025 ­0.033 ­0.036 0.915 ** ­0.149* ­0.020 0.463 ** 0.877 ** 1 table 6 ­ the correlation analysis of physiological parameters of fig cultivars * and **= correlation is significant at the 5 and 1% levels, respectively. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 561 thesis (phosphoenolpyruvate carboxylase, ribulose­ l,5­bisphosphate carboxylase, pentose phosphate pathway enzymes and glycolysis pathway enzymes) (demiral, 2005). the major strategies of plants to overwhelm this stress included: reduction in cl­ and na+ uptake, leaf loss, decrease in leaf specific area and relative leaf water content, synthesis of osmotic compounds, exclusion of toxic ions into vacuole, change in mem­ brane stability, and increase in the activity of antioxi­ dant enzyme (mutsushita and matoch, 1992; sato et al., 2006). based on the results of the present study, the stem length in ‘siyahʼ and ‘sabzʼ was less affected by salt stress. in addition, under intermediate salt condi­ tions ‘shah anjirʼ, ‘siyahʼ and ‘sabzʼ had the lowest stem length. it has already reported that salt­sensi­ tive fig cultivars such as ‘brown turkiʼ and ‘piusʼ dis­ played reduction in stem length under salinity condi­ tions (alswalmeh et al., 2015; zarei et al., 2016). a similar decrease in stem length under salt conditions was reported in almond (najafian et al., 2008) and pistachio (adish et al., 2010). the findings of soliman and abd alhady (2017) and zarei et al. (2016), indicated that the stem diam­ eter in salt­exposed fig cultivars had a l inear decrease. furthermore, the decrease in stem diame­ ter in salt­treated plums (bolat et al., 2006), citrus (khoshbahkt et al., 2014) and pomegranate (khayyat et al., 2014) showed similar pattern. in the present study, stem diameter of ‘siyahʼ cultivar was less affected by salt. reducing the number of leaves in salt­exposed plants is due to limit leaf production or early leaf aging (yeo et al., 1991; munns and tester, 2008). based on the results, the highest leaf loss was observed in ‘bar anjirʼ cultivar. similar results are available in almond (momenpour et al., 2018), pista­ chio (adish et al., 2010) and fig cultivars (essam et al., 2013; alswalmeh et al., 2015; zarei et al., 2016; soliman and abd alhady, 2017). reduction in the relative leaf water content under salinity stress indicates lower water uptake by plants. limited access to water due to increase in osmotic potential reduces the cell development and decreas­ es turgor pressure of the cells (yamasaki and dillenburg, 1999). in the present study, salinity reduced the relative content of leaf water in fig culti­ vars, except ‘bar anjirʼ. in ‘siyahʼ cultivar, a dramatic decrease was observed in the relative leaf water con­ tent. under various salt concentrations, the trend of this parameter stayed unchanged. under intermediate salt conditions the specific leaf area of ‘siyahʼ and ‘sabzʼ had not evident changes. according to owais (2015), with increasing salt levels, the relative leaf water content in grape genotypes decreased, but this decrease was lower in tolerant cultivars. a similar decrease was observed in the relative leaf water content under salinity stress in fig (zarei et al., 2016; soliman and abd alhady, 2017) and pomegranate (khayyat et al., 2014). the values of sla and ldmc reveal an important exchange in plant function between high sla, low ldmc (cultivars with rapid production of biomass) and low sla, high ldmc (cultivars with an efficient conservation of nutrients) (poorter and garnier, 1999). according to our results, ‘siyahʼ and ‘sabzʼ cul­ tivars had the lowest sla and the highest ldmc value, confirmed its production efficiency under vari­ ous salt concentration. in addition, ‘bar anjirʼ and ‘shah anjirʼ had the highest sla and the lowest lsdm under different nacl concentration, due to their lim­ ited production efficiency under salt conditions. rising the salinity level, increased leaf succulence. this ability characterizes a balance between growth rate and the necessity of osmotic adjustments (flowers and yeo, 1986), which regulate low external water potential encouraged by salinity stress (flowers and colmer, 2008). moreover, it explains the better carbon assimilation capacity per unit area (de vos et al., 2013). based on our findings ‘siyahʼ and ‘sabzʼ cultivars had the highest leaf succulence, which related to the balance between growth ratio and osmotic regulation under salt conditions. peroxidation of lipids is regarded as an extra effect of salinity on plants (demidehik et al., 2002). the findings of the previous researchers on increas­ ing ion leakage under salinity stress in fig (abdoli nejad and shekhafandeh, 2014; zarei et al., 2016) and pomegranate (khayyat et al., 2014) confirms our results. proteins biosynthesis is an important biochemical process which is affected by salt stress. expression of specific genes under nacl stress assistances the plant to adapt to adverse conditions (murcute et al., 2010). according to our findings, protein content increased under high salt condition, but this increase was greater under intermediate salt for ‘siyahʼ and ‘sabzʼ cultivars. it has reported that sodium chloride treatment reduced leaf protein content in salt­sensitive grape (alizadeh et al., 2010) and figs (abdoli nejad and shekhafandeh, 2014). in fact, salt inhibits the synthesis of nitrate reduc­ adv. hort. sci., 2019 33(4): 553­565 562 tase, glutamine synthase and glutamate synthase (which are involved in nitrogen metabolism), decreases nitrogen metabolism (hossain et al., 2012), changes active forms of nitrogen, reduces amino acids synthesis, and finally increases the activity of degrading enzymes (de souza et al., 2016). in saline conditions, cl­ competes with nitrate (abdelgadir et al., 2005), leading to the decline of nitrogen in differ­ ent parts of the plant (yu et al., 2016; hasan and miyake, 2017). according to the owais (2015) and doulati baneh et al. (2014), salinity had a decreasing effect on the leaf nitrogen content in fruit crops. although the relationship between salinity and nitro­ gen metabolism is very complex, balanced nitrogen metabolism significantly affects salinity tolerance in plants (teh et al., 2016). in the present study, the impact of salinity on leaf nitrogen content of differ­ ent fig cultivars was not the same. photosynthesis is another important plant phe­ nomenon, significantly affected by abiotic stress. reduction of photosynthesis under salinity stress is attributed to stomata factors (reduced co2 perme­ ability, stomata closure, decrease in plants transpira­ tion, stomata conductance reduction), and non­stom­ ata factors (cell membrane dehydration, structural changes in the cytoplasm and chlorophyll degrada­ tion) (brugnoli and lauteri, 1991; reza et al., 2006; tabatabaei, 2006). salinity reduces stomata conduc­ tance in hybrids of fig, by decreasing the photo­con­ ductivity of leaf cells (golombek and lüdders, 1990; zarei et al., 2016), which is in agreement with the results of the present study. similar results were reported in pistachio (adish et al., 2010). according to the results of the present study, ‘atabakiʼ and ‘kashkiʼ cultivars showed the lowest and the highest stomata conductance under high salt condition, respectively. salinity reduces evaporation and transpiration in plants (bhantana and lazarovitch, 2010; dudley et al., 2008). the linear relationship among reduction of plant evapotranspiration, transpiration and increase in salinity levels was reported in pomegranate (shani and ben­gal, 2005; mohamed ibrahim and abd el­samad, 2018) and date palm (tripler et al., 2007), which is coordinated with the results of the current study. in addition, the maximum quantum efficiency of photosystem ii, electron transfer, gas exchange, and carbon dioxide assimilation decrease under salt con­ ditions (joao­correia et al., 2006). in the present study, atabaki cultivar had the highest transpiration rate at the low and mild salinity levels, and with increasing salinity, a greater decrease was observed (table 5). the lowest effect of salinity on transpira­ tion rate was observed in ‘kashkiʼ (table 5), the culti­ var which was able to balance the transpiration level at mild and severe stress levels, probably due to hav­ ing thicker cuticle. the most important physiological process affected by this stress is photosynthesis (sudhir and murthy, 2004; acosta­motos et al., 2017). reduction in pho­ tosynthesis efficiency is followed by a series of mole­ cular events including cell membrane dehydration, stomata closure, decreased co2 entry, reduction in leaf permeability to co2, structural changes in the cytoplasm and subsequent alteration in the activity of enzymes (tabatabaei, 2006). on the other hand, nitrogen plays effective role in plant growth and construction of vital plant struc­ tures such as amino acids and proteins (arghavani et al., 2017). nitrogen is necessary to generate cellular components such as rubisco, which is responsible for assimilating carbon dioxide. therefore, by limiting nitrogen uptake, salinity stress affects the photosyn­ thesis efficiency, leading to a decrease in vegetative and reproductive growth (coruzzi and bush, 2001; marschner, 2012; zarata­valdez et al., 2015). in the present research, salinity had a significant effect on growth parameters and photosynthetic indices in seven cultivars of fig. the differences in stem length, stem diameter and leaf number in all cultivars followed a downward trend. the stomata conductance of all fig cultivars was same up to 4 ds m­1 nacl. moreover, the transpiration rate did not exhibited variation unless in salt concentration higher than 8 dsm­1. the ‘siyahʼ and ‘sabzʼ cultivars had the lowest decreases in stem length and diameter, the lowest leaf water content and transpiration rate, and the maximum leaf abscission. additionally, ‘siyahʼ and ‘sabzʼ cultivars had the highest leaf succulence and ldmc and the lowest sla, which related to the balance between growth ratio and osmotic regula­ tion under salt conditions. the ‘matiʼ, as an interme­ diate salt­tolerance cultivar, had the lowest leaf abscission under severe salinity levels. the ‘shah anjirʼ, as the most salt­sensitive cultivar, could not balance transpiration rate and leaf water content under salt treatment higher than 4 dsm­1. acknowledgements the project was supported by the head of research and technology center, university of hormozgan; plant breeding department, faculty of salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 563 agriculture, shiraz university; and estahban fig research station. references abdelgadir e.m., oka m., fujiyama h., 2005 ­ characteristics of nitrate uptake by plants under salini‐ ty. ­ j. plant nut., 28: 33­46. abdoli nejad r., shekafandeh a., 2014 ­ salt stress‐ induced changes in leaf antioxidant activity, proline and protein content in shah anjir and 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department of plant production, university of torbat heydarieh, torbat heydarieh, iran. key words: abiotic stress, germination percentage, medicinal plant, salt. abstract: this study aimed to investigate the effect of nanoparticles and non­ nanoparticles of titanium dioxide on germination indices of echinacea under salinity stress. experimental treatments included nano and bulk particles of titanium dioxide at concentrations of 0, 10, 50, 100 and 150 mg/l and salinity stress from nacl at levels of 0, ­3, ­6 and ­9 bar. the results showed that echinacea is sensitive to high salinity stress levels (­6 and ­9 bar). the use of nano and non­nano titanium dioxide treatment improved some traits under severe salinity stress. the germination percentage did not occur at salinity lev­ els of ­6 and ­9 bar, but the addition of nano titanium dioxide with a concentra­ tion of 150 mg/l and 50 mg/l non­nano increased germination by 50.6%. application of nano titanium dioxide increased the seedling weight in control by 1.28 mg to 4.26 mg in the treatment of 150 mg/l nanoparticles. the applica­ tion of nano and bulk titanium dioxide could significantly reduce the negative effect of high salinity stress levels. this can be a valuable and hopeful solution to solve the problem of salinity stress in echinacea. 1. introduction echinacea (echinacea purpurea) is a perennial herbaceous plant of the chicory family (asteraceae) and is native to the rocky areas, highlands and atlantic plains of north america and canada (raman et al., 2004). the most important medicinal property of this plant, a selected plant of the world health organization, is to strengthen the immune system (sun et al., 1999). this plant contains valuable active ingredients such as flavonoid compounds, alkaloids and chicoric acid (sandra, 2004). the most important substances in echinacea are essential oils of borneol and alpha­pinene (faravani et al., 2016). in recent years, low rainfall and uncontrolled withdrawal of groundwater resources in the country, fol­ lowed by an increase in groundwater salinity, has become a significant problem for agriculture, which in addition to reducing fresh water resources, has also increased soil salinity. therefore, using new technolo­ (*) corresponding author: h.feizi@torbath.ac.ir hasanfeizi@yahoo.com citation: behnam h., feizi h., alipanah m., 2021 ­ alleviation the effects of salinity stress using tita‐ nium dioxide nano and bulk particles in echinacea seeds and seedlings. ­ adv. hort. sci., 35(4): 351­ 360. copyright: © 2021 behnam h., feizi h., alipanah m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 may 2021 accepted for publication 29 october 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10850 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 351­360 352 gies to eliminate and reduce the effects of salinity stress on plants is inevitable. one of the advanced technologies that can be used in this field is the use of nanoparticles. undoubtedly, by taking advantage of nanotechnology as an emerging advanced technol­ ogy in the agricultural sector, desirable results can be achieved, including ensuring food security and the development of sustainable and environmentally friendly agriculture in developing countries and regions of the world (kamali et al., 2018). one of the most important nanoparticles that has been widely used in various sciences is titanium diox­ ide nanoparticles. titanium dioxide nanoparticles appear to stimulate plant root and shoot growth by stimulating plant metabolism and increasing cell divi­ sion. a study by karami and sepehri (2018) stated that the application of titanium dioxide nanoparticles improved the growth and photosynthetic perfor­ mance of barley under salinity stress. this improve­ ment was reported due to increased antioxidant activity in the presence of nanoparticles. navarro et al. (2008) stated that nanoparticles might create new larger pores in the seed coat that facilitate the entry of water and oxygen and increase seed germination. faraji and sepehri (2019) showed that the application of titanium dioxide nanoparticles increased germina­ tion and morphological traits of wheat seedlings under drought stress. under moderate and severe stress conditions, the application of titanium dioxide and nitroprusside nanoparticles alone or in combina­ tion improved the average germination time of wheat seeds by 56%. the positive effects of titanium dioxide nanoparticles on increasing plant growth, antioxidant enzyme activity, soluble sugars, amino acid and pro­ line content and reduction of h2o2 and melonic dihy­ drogenase in beans under salinity stress have been reported (abdel latef et al., 2018). khan (2016) reported a decrease in salinity stress in tomatoes by foliar application of titanium dioxide nanoparticles at a concentration of 20 ppm by improving agronomic traits, leaf chlorophyll content, phenolic and antioxi­ dant capacity, antioxidant enzyme activity and fruit yield. titanium can act as a stimulant for the plant, activating the immune system against stress. feizi et al. (2020) showed that application of 300 ppm of tita­ nium dioxide nanoparticles improved the mean ger­ mination time (mgt) and seed germination rate of lentil by 39% and 62%, respectively. nasir khan (2016) reported using 20 mg/lit nano­tio2 on tomato plant improved activities of carbonic anhydrase, nitrate reductase, sod and pox and accumulation of proline and glycine betaine in salinity stress condition. gohari et al. (2020) showed that the application of titanium dioxide nanoparticles offset the adverse effects of salinity stress on agronomic traits of dracocephalum moldavica. application of 100 mg/l titanium dioxide nanoparticles under salinity stress of 50 and 100 mm sodium chloride increased the activi­ ty of antioxidant enzymes and decreased h2o2 con­ centration. the results of a study on the effect of osmotic and salinity stress on germination and seedling growth indices of echinacea purpurea and cynara scoolymus showed that echinacea is sensitive to low and medium levels of salinity stress (amiri et al., 2010). also, with increasing the intensity of osmotic stress, the root length of echinacea and shoot length of both plants decreased until it reached zero at the levels of ­10 and ­14 bar for echinacea and artichoke, respectively (amiri et al., 2010). in a study on the effect of different osmotic potentials of sodium chloride and calcium chloride salts on the germination characteristics of echinacea seeds, it was observed that with increasing salt con­ centration, all germination traits significantly (p≤5%) are reduced, so that in the potential of ­9 bar, the percentage and rate of germination decreased by 50% and seed vigor by 83% (ebrahimi anjeshshi et al., 2011). lyu et al. (2017) stated that seeds soaked in titanium dioxide nanoparticles showed a higher germination rate, more root growth and improved seedling growth. nanoparticles can enter the seed coat and increase the entry of water and nutrients and improve seed growth. but their toxic effects also occur in seeds. younes et al. (2020) reported that the application of 100 ppm of tio2 and zno nanoparticles on three species of the solanaceae family significant­ ly improved their germination traits and reduced their average germination time. kamali et al. (2018) showed that at 75 mm salinity, foliar application of titanium dioxide nanoparticles in 15 ppm treatment increased the number of flowers in petunia hybrida plant from 5.6 to 9.3. also, the highest shoot fresh weight was observed in the treatment of 15, 20 and 40 ppm foliar application of titanium dioxide nanoparticles. according to research, it seems that the use of nanotechnology can reduce the adverse effects of salinity stress on seeds and plant growth. therefore, the present study was conducted to investigate the effect of titanium dioxide nano and bulk particles on seed germination and seedling growth of echinacea in modulating salinity stress conditions. behnam et al. ‐ alleviation of salinity stress by titanium dioxide in echinacea 353 2. materials and methods in order to investigate the effect of nanoparticles and non­nanoparticles of titanium dioxide on germi­ nation indices of echinacea under experimental salin­ ity stress, an experimental study was performed in the laboratory of medicinal plants of the university of torbat heydarieh, iran. to perform the study, 100 grams of echinacea seeds were purchased from the agricultural and natural resources research center of isfahan province. the seeds were carefully threshed and 2700 seeds were isolated for testing. this experiment was conducted as a factorial layout based on a completely randomized design with four replicates. each of the experimental steps has 36 integrated treatments, including nine levels of zero (control) titanium dioxide concentration, 10, 50, 100 and 150 mg/l of nanoparticles and 10, 50, 100 and 150 mg/l of bulk particles and four levels of salinity stress were zero (control), ­3, ­6 and ­9 bar in three replications. in these experiments, sterile petri dishes and filter paper with a diameter of nine cm were used as the culture medium (top paper culture method or tp). exert treatment the filter papers were wrapped in aluminum foil for each stage of culture and disinfected in an auto­ clave at 120°c for 20 minutes. seeds were disinfected using 10­14% sodium hypochlorite for 30 seconds and then washed thoroughly with distilled water three times each time for three minutes until the dis­ infectant was completely removed from the seed sur­ face. the work surface and all utensils and utensils used were disinfected using 70% ethanol. in each petri dish, 25 disinfected seeds were placed at a suitable distance from the bed of filter paper. then 5 ml of the prepared solutions were added and the lid of the petri dishes was closed using para film to prevent evaporation of the material and the solution was not added until the end of the test period. petri dishes were placed in a germinator with a temperature of 25°c and a humidity of 60% at 16/8 hours length in day/night. preparing saline and tio2 solution to prepare the final solution of the treatments, each level of titanium dioxide factor must be mixed separately with each of the salinity stress levels in a ratio of 1:1 to obtain a homogeneous solution and then applied to the seeds. for this purpose, titanium dioxide levels, as well as salinity stress, should be prepared in double concentration to achieve the desired concentration after mixing them; therefore, to prepare 100 ml of each of the concentrations of 20, 100, 200 and 300 mg/l of nanoparticles and non­ nano titanium dioxide, the amounts of 5, 25, 50 and 75 ml of stock solution, respectively. it was poured separately and each of them was brought to a vol­ ume of 100 ml with the help of distilled water. to prepare different concentrations of salinity, nacl salt made in germany (merck) was used based on richards method (richards, 1954). different salinity levels were prepared with double concentration. then, 10 ml of titanium dioxide solution was mixed with 10 ml of saline solution at the desired stress level in a beaker and homogenized. nanosized tio2 powder was aeroxide® tio2 p25, supplied by degussa gmbh company. specific sur­ face area of nanosized tio2 was 50 m2 g­1, average primary particle size was 21 nm and purity was >99.5%. the size of tio2 nanoparticles (fig. 1) and bulk particles (fig. 2) were determined. in order to gain accurately dispersed and stable tio2 suspensions of each concentration, an ultra­sonication treatment was applied to bulk and nanoparticles tio2 powders dispersed in wa ter for 15 minutes. fig. 1 ­ images of nanosized tio2 by scanning tunneling microscope (stm). fig. 2 ­ image of bulk tio2 particles by scanning electron microscope (sem). adv. hort. sci., 2021 35(4): 351­360 354 measurement methods daily counting started from the day after planting at a specific time and continued until the 21st day (ista, 2009). seeds with a root length of more than two millimeters were counted as germinated seeds (ista, 2009). at the end of the day, 10 seedlings were randomly selected from each experimental unit and the length of roots, stems and seedlings were mea­ sured and recorded using millimeter paper. the stems and roots were separated and each was taken in separate paper bags in an oven at 70°c for 24 hours and then weighed with a digital scale to the accuracy 0.0001 g. to determine the germination rate of maguire formula (maguire, 1982), the mean germination time (mgt) (matthews and khajeh­ hosseini, 2007) and the mean daily germination (mdg) (azimi et al., 2013) from the following equa­ tions used: germination rate (gr) = (a/1) + (b­a)/2 + (c­b)/3 + …. [n­ (n­1)]/n (1) where gr indicates the germination rate in terms of germinated seeds per day, a, b, c ... n indicates the number of germinated seeds after n ... 3, 2, 1 day after dewatering. mgt = [∑(f∙x)]/(∑ f) (2) mdg = germination% / total experiment days (3) in equation (2), mgt: mean germination time (days), f: the number of new germinated seeds per day of count x and x days of counting. equation (4) and (5) were used to calculate the seed vigor index (vashith and nagarajan, 2010): vigor index i = germination % x seedling length in cm (shoot + seminal root) (4) vigor index ii = germination % x seedling dry mass in mg (shoot + semi­ nal root) (5) data related to excel software were sorted and processed and then statistical analysis of the data was performed by sas jmp software and compar­ isons of means were performed by tukey test at 5% probability level. 3. results and discussion the results of data analysis of variance are report­ ed in table 1. application of titanium dioxide treat­ ment had a significant effect on all studied traits except seed vigor index i and shoot length. the effect of salinity stress was significant on all traits except shoot, root and seedling weight. the results also showed that all traits except shoot and seedling weight, seed vigor index i were significantly affected by the interaction of titanium dioxide and salinity stress. effect of titanium dioxide on germination indices of echinacea under salinity stress as shown in table 2, the application of titanium dioxide treatment improved the germination per­ centage and rate and mean daily germination com­ pared to the control. application of titanium dioxide nanoparticle increased the seedling weight from the control with a value of 1.28 mg to 4.26 mg in the treatment of 150 ppm nanoparticles. the application of titanium dioxide had no effect on the shoot weight of echinacea. the results of this test are the same as the experimental results performed by tokalloo et al. (2013) on barley and feizi et al. (2013) on sage. all experimental treatments significantly positively affected shoot length, seedling weight, and root weight compared to the control. the lowest root length was related to the control and the concentra­ tion of 150 mg/l of non­nanoparticles had the best performance, so that it increased the length of root, stem and seedling about three times compared to the control. table 1 ­ analysis of variation of nano and bulk titanium dioxide particles on germination and seedling traits of echinacea source of variation df seed vigor ii seed vigor i seedling length root length shoot length seedling weight root weight shoot weight mean daily germination mean germination time germination rate germination tio2 8 117638 ** 25137.69 ns 158.72 ** 50.52 ** 58.85 ** 6.99 ns 0.72 ** 5.53 ns 1.77 ** 21.47 ** 1.32 ** 781.92 ** salinity 3 4829442 ** 84607.21 ** 829.73 ** 508.05 ** 94.85 ** 5.74 ns 0.52 ns 8.74 ns 17.50 ** 20.06 ** 48.48 ** 7542.51 ** tio2× salinity 24 434076 ** 26985.32 ns 43.58 ** 35.76 ** 13.92 ** 5.63 ns 0.44 ** 4.34 ns 1.31 ** 11.50 ** 0.84 ** 587.40 ** error 72 36544 24260.7 8.85 6.47 2.82 4.35 0.23 4.13 0.27 0.85 0.21 121.93 total 107 ns, *, and **: no significant, significant at 5 and 1% probability, respectively. behnam et al. ‐ alleviation of salinity stress by titanium dioxide in echinacea 355 the effect of salinity stress on germination indices of echinacea the results reported in table 3 show that echinacea had the highest germination percentage at the level of zero and salinity stress ­3 bar, but with increasing salinity stress, the germination percentage decreased significantly. at the stress level of ­9 bar, it decreased by 37.5% compared to the control. the highest germination percentage was related to the control treatment and decreased significantly with increasing salinity stress levels, so that it decreased by 2.5 times compared to the control at ­9 bar. miri and mirjalili (2013) stated that one of the ways to priming seed is to use nacl salt solution with salinity 1 ds m­1, which in saline areas improves seedling growth rate by up to 20%. they stated that at 1 ds m­1 salinity the germination rate of echinacea is 57.7% but with increasing the amount of nacl to 6 ds m­1 salinity the germination rate decreases to about 9.8%. the highest germination rate was observed in the control treatment and with increasing the intensity table 3 ­ effect of salinity stress on germination traits of echinacea means, in each column, followed by same letter are not significantly different at the 5 % probability level­ using tukey test. n=nano, b=bulk. table 2 ­ effect of titanium dioxide particles on germination traits of echinacea under salinity stress means, in each column, followed by same letter are not significantly different at the 5% probability level, using tukey test. n=nano, b=bulk tio2 concentration mg/l vigor index ii vigor index i seedling length (mm) root length (mm) shoot length (mm) seedling weight (mg) root weight (mg) shoot weight (mg) mean daily germination (seed) germination rate (seed/day) germinat­ ion (%) 0 601.46 ab 107.05 a 7.16 c 3.19 c 3.97 b 1.28 b 0.16 b 1.12 a 2.03 b 1.75 b 42.66 b n10 826.53 a 181.13 a 16.79 ab 6.83 ab 9.95 a 2.82 ab 0.84 a 1.98 a 3.19 a 2.44 a 67.00 a n50 648.57 ab 171.00 a 15.50 b 5.24 bc 10.25 a 2.97 ab 0.89 a 2.08 a 2.79 a 2.59 a 58.33 a n100 574.85 b 161.40 a 14.00 b 4.09 bc 9.89 a 2.60 ab 0.44 ab 2.15 a 2.93 a 2.75 a 61.66 a n150 605.16 ab 282.37 a 15.05 b 5.29 bc 9.91 a 4.26 a 0.49 ab 3.76 a 2.96 a 2.56 a 62.00 a b10 811.69 ab 195.43 a 16.97 ab 6.67 b 10.31 a 2.95 ab 0.80 a 2.15 a 3.19 a 2.75 a 64.33 a b50 762.01 ab 167.62 a 17.82 ab 5.97 bc 11.93 a 2.57 ab 0.40 ab 2.17 a 3.11 a 2.60 a 65.33 a b100 769.84 ab 181.81 a 14.17 b 3.89 bc 10.27 a 2.56 ab 0.43 ab 2.13 a 3.33 a 2.81 a 70.00 a b150 768.81 ab 168.33 a 20.32 a 10.04 a 10.27 a 2.54 ab 0.42 ab 2.12 a 3.20 a 2.82 a 67.33 a of salinity stress, the germination rate decreased sig­ nificantly. seeds that are exposed to salinity stress face water shortage, resulting in a decrease in germi­ nation rate and percentage under the influence of salinity (kafi et al., 2005). seed and impaired storage protein synthesis reduce seed germination (vigot, 2009). in addition, the toxicity of sodium and chlo­ rine ions in salinity stress play an important role in reducing seed germination (hanslin and eggen, 2005). in a study on the effect of different osmotic potentials of sodium chloride and calcium chloride salts on the germination characteristics of echinacea seeds, it was observed that with increasing salt con­ centration, all germination traits significantly (p≤ 5%) are reduced, so that in the potential of ­9 bar, the percentage and rate of germination decreased by 50% and seed vigor by 83% (ebrahimi anjeshshi et al., 2011). the inhibitory effects of sodium chloride on seed germination may be due to its direct effect on embryo growth. the researchers found that fetal axis elongation was severely inhibited by high levels of sodium chloride in the irrigation solution. on the salinity (bar) vigor index ii vigor index i seedling length (mm) root length (mm) shoot length (mm) seedling length (mg) root length (mg) shoot length (mg) mean daily germination (seed) germination rate (seed/day) germination (%) 0 335.32 c 198.97 ab 21.76 a 11.71 a 10.05 b 2.53 a 0.71 a 1.81 a 3.76 a 4.31 a 78.37 a ­3 1288.88 a 245.66 a 18.04 b 6.01 b 12.06 a 3.42 a 0.39 a 3.03 a 3.43 a 2.80 b 72.14 a ­6 741.65 b 159.21 ab 11.41 c 2.68 c 8.73 c 2.49 a 0.49 a 1.99 a 2.70 b 1.97 c 56.74 b ­9 464.78 c 114.43 b 10.02 c 2.36 c 7.72 c 2.47 a 0.57 a 1.90 a 1.95 c 1.17 d 41.03 c 356 adv. hort. sci., 2021 35(4): 351­360 other hand, sodium chloride, due to inhibition of water uptake by seeds, slows down vital activities in the seed and increases rooting time (mohammadi et al., 2011). the mean daily germination trait had the best performance at zero and ­3 bar the salinity stress level, but decreased significantly with increasing the stress level and reached the lowest level at ­9 bar. no significant difference was observed between stem levels in shoot weight, root weight and seedling weight. however, numerically, yield in shoot weight and seedling weight at the level of 3­bar stress showed a slight increase compared to the control, which decreased with increasing stress intensity. the highest shoot length was observed at the level of ­3 bar salinity stress, which was higher than the control. the root length in control had the highest value and decreased sharply with increasing the stress level, so that the root length at the level of ­9 bar drought load was reduced about 10 times compared to the control. seedling length was highest at zero stress level, but with increased stress level to ­9 bar, seedling length was reduced by half. the results of research on sage showed a decrease in root and stem length with increasing osmotic stress (stephanie et al., 2005). the results of studies on sage and ten species of medicinal plants showed the negative effect of salinity stress on plant length (fallahi et al., 2009). the best seed vigor index i was seen at the stress level of ­3 bar, which was not significantly different from the control level and ­6 bar. but compared to the ­9 bar stress level, it was about 2 times higher. seed vigor index ii had the best performance at ­3 bar stress and a significant decrease was seen at ­6 bar level. seed vigor index ii in control and ­9 bar stress had the lowest value (table 3). interaction of titanium dioxide and salinity stress on germination indices of echinacea as can be seen from the results reported in table 4, the use of titanium dioxide treatment increased the germination percentage at high salinity stress condition. in the absence of nano titanium dioxide at ­6 and ­9 bar level salinity stress, no germination occurred at all, but with the application of titanium dioxide, the germination percentage increased signif­ icantly in the mentioned stress intensities. so that in the control treatment with salinity stress ­9 bar ger­ mination was zero; however, in the treatments of 50 mg/l non­nanoparticles and 150 mg/l nanoparticles at the same stress level, germination was observed 50.66%. contrary to the results of the present experiment, zheng et al. (2005) reported that titanium dioxide nanoparticles absorb more water in spinach seeds, thus accelerating seed germination. khot et al. (2012) pointed out that the main reason for the increase in plant growth rate in response to titanium dioxide nanoparticles is the production of sterile radiation oxygen, which increases seed resistance to stress and improves water and oxygen penetration in accelerat­ ing germination. in terms of germination rate, the application of titanium dioxide increased the germination rate at high levels of salinity stress compared to the control and the treatment of 10 mg/l of non­nanoparticles maintained the germination rate during increasing salinity stress. however, under non­stress conditions, the concentration of 150 mg/l of non­nanoparticles had a higher performance than all experimental treatments. the mean germination time in echinacea seeds decreased with increasing salinity stress level. due to this, a limited number of seeds germinated at high salinity stress levels in the first days of the experiment and during the counting days, due to severe salinity, the germination process stopped; however, at low stress levels, the germination process continued until the last days of counting, which increased the mean germination time at low salinity stress levels. the application of titanium dioxide caused the mean daily germination during the increasing process of salinity stress to be higher than the control. in gen­ eral, increasing the salinity stress level from zero to ­ 9 bar reduced the mean daily germination; however, among the experimental treatments, 150 mg/l nanoparticles and 50 mg/l non­nanoparticles were able to better inhibit the decrease in germination mean due to increased salinity stress. according to the results reported in table 5, the best treatment for stem weight was 150 mg/l nanoparticles at a stress level of ­3 bar. no significant difference was observed between other experimen­ tal treatments; however, treatments of 100 mg/l nanoparticles and 150 mg/l of non­nanoparticles had 6 times higher shoot weight at the stress level. application of titanium dioxide treatment increased root weight compared to control at high stress levels. there was no significant difference in non­nano and control treatments at different levels of salinity stress in seedling weight. behnam et al. ‐ alleviation of salinity stress by titanium dioxide in echinacea 357 feizi et al. (2012) stated that titanium dioxide treatment had no significant effect on shoot dry weight, seedling, vigor index i and ii of wheat; however, the application of titanium dioxide at all levels caused a significant increase in root dry weight and the highest root dry weight was observed in the treatments of 2 and 500 mg/l non­nanoparticles and 100 mg/l nanoparticles. there was a significant difference between con­ trol treatment and different concentrations of titani­ um dioxide in shoot length, especially at high salinity stress levels. in general, in all treatments, ­3 bar level salinity stress increased stem length. studies on seedling length also showed that the application of titanium dioxide had a significant posi­ tive effect on non­use at high stress levels and in most experimental treatments at the level of ­3 bar the salinity stress compared to the non­stress state, seedling length increased shows. treatment of 150 mg/l non­nanoparticles in the process of increasing salinity stress from zero to ­9 bar was able to main­ tain the root length to a higher value. the results of paravar and omidi (2014) and motevasel et al. (2014) showed that with increasing salinity stress, seedling means, in each column, followed by same letter are not significantly different at the 5 % probability level, using tukey test. n=nano, b=bulk table 4 ­ interaction effect of titanium dioxide particles and salinity on germination traits of echinacea tio2 (mg/l) salinity (bar) mean daily germination (seed) mean germination time (day) germination rate (seed/day) germination (%) 0 0 4.06 ab 6.46 c­g 4.31 abc 85.33 ab ­3 3.87 a­d 9.04 abc 2.57 d­i 81.33 a­d ­6 0.19 fg 0.42 h 0.11 jk 4.00 fg ­9 g 0 h 0 k 0 g 0 10n 0 3.55 a­e 7.28 a­g 3.04 c­g 74.66 a­e ­3 3.49 a­e 7.36 a­g 2.92 c­h 73.33 a­e ­6 3.36 a­e 8.95 a­d 2.39 f­i 70.66 a­e ­9 2.34 b­e 9.48 ab 1.41 h­k 49.33 b­e 50n 0 3.87 a­d 5.39 fg 4.57 ab 80 a­d ­3 2.98 a­e 7.06 b­g 2.54 e­i 62.66 a­e ­6 2.41 a­e 6.99 b­g 2.26 f­i 50.66 a­e ­9 1.90 ef 8.90 a­d 1.23 ijk 40.00 ef 100n 0 4.12 a 6.43 c­g 4.92 a 86.66 a ­3 3.49 a­e 6.42 c­g 3.19 b­f 73.33 a­e ­6 2.22 de 8.43 a­e 1.63 g­j 46.66 de ­9 1.90 ef 8.39 a­f 1.26 ijk 40.00 ef 150n 0 3.36 a­e 4.84 g 4.09 a­d 69.33 a­e ­3 3.11 a­e 7.34 a­g 2.58 d­i 65.33 a­e ­6 2.98 a­e 7.58 a­g 2.26 f­i 62.66 a­e ­9 2.41 a­e 10.18 a 1.31 ijk 50.66 a­e b10 0 3.49 a­e 5.72 efg 4.33 abc 70.66 a­e ­3 3.55 a­e 6.98 b­g 3.02 c­g 74.66 a­e ­6 3.17 a­e 8.63 a­e 2.32 f­i 66.66 a­e ­9 2.15 de 9.08 abc 1.31 ijk 45.33 d­e b50 0 3.80 a­d 7.13 b­g 4.56 ab 80.00 a­d ­3 2.85 a­e 8.53 a­e 2.045 f­i 60.00 a­e ­6 3.36 a­e 8.60 a­e 2.36 f­i 70.66 a­e ­9 2.41 a­e 9.40 abc 1.41 h­k 50.66 a­e 100b 0 3.42 a­e 5.96 d­g 3.93 a­e 72.00 a­e ­3 4.00 abc 7.37 a­g 3.36 b­f 84.00 abc ­6 3.61 a­e 8.20 a­f 2.66 d­i 76.00 a­e ­9 2.28 cde 10.18 a 1.30 ijk 48.00 cde 150b 0 4.12 a 6.67 b­g 5.03 a 86.66 a ­3 3.55 a­e 7.37 a­g 2.95 c­g 74.66 a­e ­6 2.98 a­e 9.10 abc 1.99 f­i 62.66 de ­9 2.15 de 9.06 abc 1.32 ijk 45.33 de adv. hort. sci., 2021 35(4): 351­360 358 length, root length and seedling dry weight decreased. in seed vigor index ii, all treatments containing titanium dioxide, salinity stress ­3 bar made a signifi­ cant positive difference compared to the control, but in the conditions without stress, the control had bet­ ter performance. at high salinity stress levels, the application of titanium dioxide significantly increased the seed vigor index ii compared to the time of non­ use. 4. conclusions investigation of the main effect of titanium diox­ ide treatment in testing the effect of nano and non­ nano titanium dioxide on germination of echinacea under salinity stress showed that the use of this treatment in all concentrations, measured indices (except increased seed vigor indices) increased com­ pared to the control treatment. echinacea tolerated salinity stress up to ­3 bar in germination percentage, table 5 ­ interaction effect of titanium dioxide particles and salinity on seedling traits of echinacea tio2 (mg/l) salinity (bar) vigor index ii vigor index i shoot length (mm) root length (mm) seedling length (mm) seedling weight (mg) root weight (mg) shoot weight (mg) 0 0 1622.13 ab 215.28 ab 19.03 b­g 10.03 b­e 9.00 b­g 2.52 b 0.45 ab 2.07 b ­3 783.73 d­i 212.92 ab 9.63 ghi 2.73 d­g 6.90 fg 2.61 b 0.20 ab 2.41 ab ­6 k 0 b 0 i 0 g 0 h 0 b 0 0.00 b b 0 ­9 k 0 b 0 i 0 g 0 h 0 b 0 0.00 b b 0 10n 0 149.85 jk 174.94 ab 19.70 b­f 11.70 b 8.00 c­g 2.30 b 0.81 ab 1.49 b ­3 1663.06 a 188.41 ab 22.70 bcd 9.30 b­f 13.40 abc 2.56 b ab0.41 2.15 b ­6 923.46 c­h 166.25 b 13.13 d­h 4.16 b­g 8.96 b­g 2.34 b ab0.38 1.95 b ­9 569.73 f­k 194.92 ab 11.64 fgh 2.18 efg 9.45 b­g 3.08 ab 0.75 ab 2.33 b 50n 0 135.36 jk 197.22 ab 16.93 b­h 7.23 b­g 9.70 a­g 2.46 b 0.85 ab 1.61 b ­3 1414.00 abc 183.61 ab 22.76 bcd 7.80 b­g 14.96 a 2.91 ab 0.38 ab 2.53 ab ­6 597.13 f­k 157.61 b 11.75 fgh 2.90 d­g 8.85 b­g 3.06 ab 0.88 ab 2.18 b ­9 447.80 g­k 145.54 b 10.55 fgh 3.03 c­g 7.51 efg 2.45 ab 0.45 ab 2.00 b 100n 0 169.84 ijk 219.93 ab 19.60 b­f 7.03 b­g 12.56 a­e 2.54 b 0.65 ab 1.88 b ­3 1243.73 a­e 195.29 ab 17.00 b­h 4.23 b­g 12.76 a­e 2.67 b 0.30 ab 2.36 ab ­6 437.20 g­k 133.38 b 8.10 hi 1.33 fg 6.73 g 2.77 b 0.33 ab 2.44 ab ­9 448.64 g­k 97.02 b 11.31 fgh 3.78 b­g 7.52 efg 2.42 b 0.50 ab 1.92 b 150n 0 160.93 ijk 173.66 ab 23.16 bc 11.23 bc 11.93 a­g 2.48 b 0.71 ab 1.77 b ­3 944.00 c­h 681.26 a 14.43 c­h 4.86 b­g 9.56 a­g 9.71 a 0.70 ab 9.01 a ­6 840.40 c­h 154.45 b 13.36 d­h 2.86 d­g 10.50 a­g 2.44 b 0.25 ab 2.19 b ­9 475.33 g­k 120.09 b 9.26 hi 2.20 efg 7.66 d­g 2.40 b 0.32 ab 2.08 b b10 0 139.44 jk 188.77 ab 19.63 b­f 10.86 bcd 8.77 b­g 2.67 b 0.83 ab 1.84 b ­3 1632.40 a 202.61 ab 21.86 b­e 9.13 b­f 12.73 a­e 2.70 b 0.48 ab 2.22 b ­6 896.00 c­h 282.26 ab 13.60 c­h 4.60 b­g 9.04 b­g 3.04 ab 0.78ab 2.25 b ­9 579.04 f­k 108.09 b 12.81 e­h 2.10 efg 10.70 a­g 2.41 b 0.12 b 2.28 b b50 0 200.85 ijk 213.36 ab 24.83 b 10.80 bcd 14.03 ab 2.66 b 0.66 ab 1.99 b ­3 1166.80 a­f 155.04 b 19.33 e­g 6.83 b­g 12.83 a­e 2.68 b 0.37 ab 2.31 b ­6 1044.00 a­g 173.58 ab 14.93 c­h 2.80 d­g 12.13 a­g 2.49 b 0.21 ab 2.27 b ­9 636.40 e­j 128.49 b 12.20 e­h 3.46 b­g 8.73 b­g 2.46 b 0.35 ab 2.11 b 100b 0 114.96 jk 198.16 ab 15.93 b­h 7.80 b­g 8.13 c­g 2.74 b 0.78 ab 1.96 b ­3 1451.60 abc 215.37 ab 17.30 b­h 4.20 b­g 13.10 a­d 2.56 b 0.34 ab 2.22 b ­6 1003.06 b­g 199.90 ab 12.96 e­h 2.13 efg 10.83 a­g 2.61 b 0.30 ab 2.31 b ­9 509.74 g­k 113.80 b 10.50 fgh 1.43 fg 9.03 b­g 2.34 b 0.29 ab 2.04 b 150b 0 324.68 h­k 209.42 ab 37.06 a 28.70 a 8.36 c­g 2.41 b 0.70 ab 1.71 b ­3 1300.66 a­d 176.44 ab 17.33 b­h 5.03 b­g 12.30 a­f 2.38 b 0.30 ab 2.07 b ­6 933.60 c­h 165.46 ab 14.90 c­h 3.33 c­g 11.56 a­g 2.66 b 0.29 ab 2.37 ab ­9 516.30 g­k 122.01 b 11.98 fgh 3.10 c­g 8.87 b­g 2.71 b 0.38 ab 2.32 b means, in each column, followed by same letter are not significantly different at the 5 % probability level­ using tukey test. n=nano, b=bulk behnam et al. ‐ alleviation of salinity stress by titanium dioxide in echinacea 359 mean germination time, shoot length, seed germina­ tion index i and ii, and sometimes even at ­3 bar stress observed better yield than the control. but ger­ mination rate, root and seedling length were strongly affected by salinity stress. the interaction of titanium dioxide and salinity stress treatments showed that although in the increasing trend of salinity stress intensity all studied traits were significantly reduced compared to non­stress conditions, but the applica­ tion of titanium dioxide treatment in nano and non­ nano state in ­6 and ­9 bar salinity improved signifi­ cant yield of echinacea seeds and seedlings in all traits compared to the control. therefore, the posi­ tive effects of using titanium dioxide in mitigating the negative effects of salinity stress on the seeds and seedlings of echinacea can be a useful and promising solution to solve this problem. further research is needed to determine the physiological and molecular effects of this substance on the metabolism of plant resistance to salinity stress. references abdel latef a.a., srivastava a.k., abd el­sadek m.s., kordrostami m., 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and is even higher in sub­saharan africa (ssa) (kitinoja and kader, 2015). in bell pepper (capsicum annuum l.), losses of 28.6% and 38.7% have been reported during dry and wet seasons, respectively in nigeria (tsegay et al., 2013). a short shelf life, even under the most favourable conditions is a major postharvest limiting factor in bell pepper handling (ilić et al., 2017). since bell pepper is a non­climacteric fruit, its senes­ cence is mainly accelerated by excessive water loss through respiration. there is increasing interest in edible fruit and vegetable coatings to extend postharvest life. cactus [opuntia ficus‐indica (l.) mill.] mucilage has potential in postharvest preservation of horticultural commodities such as minimally processed cactus pear fruits (liguori et al., 2021), (*) corresponding author: aloomaurine@gmail.com citation: aloo m.a., opiyo a.m., saidi m., 2022 ­ maintaining postharvest quality of bell pepper (capsicum annum l. cv. california wonder) using cactus (opuntia stricta l.) mucilage coating. ­ adv. hort. sci., 36(3): 193­200. copyright: © 2022 aloo m.a., opiyo a.m., saidi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 november 2021 accepted for publication 1 july 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12275 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 193­200 194 mango (mangifera indica l.) (abera et al., 2019) and papaya (oluwaseun et al., 2014). according to oluwaseun et al. (2014), papaya fruits that were dipped for 30 seconds in cactus mucilage + glycerol and in pure cactus mucilage recorded a significantly lower weight loss, lower increase in fruits’ tss con­ tent and higher ascorbic acid content as compared to uncoated fruits at the end of storage period at a tem­ perature 27 ± 2°c and rh of 55­60%. low temperature storage and modified atmosphere packaging (map) have been successfully used in maintaining quality and extending shelf life of bell pepper (manolopoulou et al., 2012; bayogan et al., 2017). the most effective method has been rapid cooling after harvest followed by storage at low tem­ perature and high relative humidity (bayogan et al., 2017). bell pepper being a tropical fruit, suffers chill­ ing injury at temperatures below 7°c, which favors development of fungal diseases (ilić et al., 2017). the cost of purchasing, installing and running a cold stor­ age facility is also high and unaffordable for most small­scale bell pepper growers, retailers and con­ sumers in developing countries hence rendering the technology untenable. modified atmosphere packaging (map) using plastic bags has also been used for a long time for maintenance of quality of bell pepper. however, their use may trigger development of anaerobic microorganisms (manolopoulou et al., 2012). these together with the restricted use of plastics in several countries due to environmental pollution has made the technology unreliable. therefore, the objective of this study was to determine the effects of cactus mucilage coating, an alternative postharvest treat­ ment, on postharvest quality and shelf life of bell pepper. 2. materials and methods experimental materials bell pepper, cv. california wonder, fruit was pro­ duced at the horticulture teaching and research field of egerton university, njoro, kenya under white agro net covers. the university lies at a latitude 0°23’ south, longitude 35°56’east; and is 2,227 m above sea level (jaetzold et al., 2006). fruit was harvested at mature green stage (díaz­pérez et al., 2007), packed in plastic buckets, and taken to the laborato­ ry. average minimum temperature, maximum tem­ perature and relative humidity of the laboratory site was 11°c, 24.5°c and 64.7%, respectively (egerton meteorological weather station, 2020), where fruit free from bruises and blemishes were selected and used for the study. cactus (opuntia stricta l.) stems were also harvested from the field at egerton university, packed in plastic buckets and transported to the laboratory. extraction of cactus mucilage and preparation of cac‐ tus mucilage treatments cactus mucilage was extracted at room tempera­ ture (25 ± 2°c) using the method described by sepulveda et al. (2007). cactus stems were washed using 2% volume per volume (v/v) sodium hypochlo­ rite (naclo) to remove dirt and for disinfection. stems were peeled and chopped into small pieces using a sharp knife. distilled water was added to the chopped pieces in a ratio of 1:1 (w/v) (200 g of the chopped pieces in 200 ml distilled water) and blend­ ed for 3 min using a blender (pps sb­4171, sayona, china) to obtain slurry which was gravity filtered through muslin cloth. the filtrate was precipitated using 20% isopropyl alcohol in a ratio of 1:1 (v/v) (1 l of the filtrate in 1 l of 20% isopropyl alcohol). the precipitated filtrate was centrifuged for 10 min at 2,683 × g using a centrifuge (dl­5­d). the super­ natant was drained off and precipitates at the bot­ tom of the eppendorf tubes dried in a forced air oven at 70°c for 4 h to obtain dried cactus mucilage. to obtain 1, 2 or 3% mucilage solution, 1, 2 or 3 g, respectively, of the dried cactus mucilage was weighed using an electronic weighing balance (denver instrument xl­1810) and dissolved in 80 ml of distilled water. to each solution, 2 ml of glycerol plasticizer was added, volume made to 100 ml mark using distilled water and blended for 3 min to obtain complete dispersion. the solutions were centrifuged for 10 min at 2,683 × g using a centrifuge (dl­5­d) to obtain a supernatant; which was used to coat fruit. the different concentrations of cactus mucilage coat­ ing (1%, 2% and 3%) were chosen for the current study based on past research that were done on effects of cactus mucilage on other fruits and fruit vegetables (alikhani, 2014; zegbe et al., 2013) treatments application before treatment application, all fruits were disin­ fected by washing for 5 min using 0.5% (v/v) naclo (lerdthanangkul and krochta, 1996). this was fol­ lowed by air drying of fruit at room temperature (25 ± 2°c) until the disinfecting solution on fruit skin was completely dry. the fruit were dipped in 1 litre of aloo et al. ‐ postharvest handling of bell pepper 195 cactus mucilage solutions for 5 min based on the treatments (alikhani, 2014) after which the excess coating was allowed to drain off. the fruits were air dried until the cactus mucilage on skin was complete­ ly dry allowing formation of a layer of coating on the fruit surface. control fruit were dipped in distilled water for 5 min, removed and allowed to air­dry at room temperature (25±2°c) until distilled water on fruit skin was completely dry. after treatments appli­ cation, all fruit were stored on plastic trays under ambient conditions (25 ± 2°c temperature and 65 ± 2% relative humidity) until they senesced. experimental design the experiment was a single factor experiment arranged in a randomized complete block design, with 3 replications. blocking was done against differ­ ent harvesting times; harvesting of the 3 blocks was done at 1 month interval. in total, there were 12 experimental units with each experimental unit rep­ resented by a plastic tray containing 30 fruits. data collection data collection commenced immediately after treatments application and continued until fruit lost 25% of their initial weight (sibomana et al., 2015). data collection was done on fresh weight loss and total soluble solids (tss) at 3 days intervals; and iron and ascorbic acid content at 4 days intervals. three fruits per experimental unit, were selected at ran­ dom at the onset of the study, marked and used for data collection throughout the study for non­destruc­ tive variables which were fresh weight loss and shelf life. on the other hand, three fruits per experimental unit were also randomly selected from the remaining fruits and used to collect data for the destructive variables (tss, iron and ascorbic acid content). data for each destructive variable was collected from the three fruits with a new set of fruits used on each sampling date. the variables were determined as described below. fresh weight loss the fresh weight (g) of the three selected fruits per experimental unit was measured using an elec­ tronic weighing balance (denver instrument xl­1810) immediately after treatment application (before stor­ age). the same fruits were thereafter weighed at 3 days intervals until they lost 25% of their initial total weight. progressive % fresh weight loss was deter­ mined using the formula by moneruzzaman et al. (2008). average % fresh weight loss of the 3 fruits was calculated and recorded as average % weight loss per fruit for the time period (moneruzzaman et al., 2008). the shelf life of the fruit on the other hand was determined by counting the number of days the fruit took from harvesting to lose 25% of their initial weight (sibomana et al., 2015). total soluble solids content total soluble solids (%tss) content was deter­ mined using a portable hand­held refractometer rhb­32/atc (yhequipment co., limited, shenzhen city, china) as described by opiyo and ying (2005). a small piece of pepper fruit was cut, squeezed and the juice obtained dropped onto a refractometer and readings taken. average %tss of the 3 fruits was cal­ culated and recorded as average %tss per experi­ mental unit for the time period. iron content iron (fe) content was determined using an atomic absorption spectrophotometer (model 210 vgp, buck scientific, norwalk, ct) following jones and case (1990). dried ground sample (1 g) was weighed into crucibles and ashes were obtained in a furnace at a temperature of 550°c for 2 h. the ash was cooled to room temperature (25 ± 2°c), transferred into a 100 ml beaker and 10 ml of the digestion mix added. distilled water (50 ml) was added. activated charcoal (1 g) was added to obtain a clear sample and stirred. the contents were gravity filtered through whatman no.5 filter paper into a 100 ml volumetric flask. the filtrate was filled to the mark with distilled water. into a cuvette, 10 ml of filtrate was pipetted and absorbance read at 248 nm. iron standard solutions of 0, 5, 10, 15, 20 and 25 µg/g were prepared from iron sulphate. into a cuvette 10 ml of each standard was pipetted and absorbance read at 248 nm, and a standard curve developed. the amount of iron was calculated against the standards, converted to µg/g and expressed using the formula of okalebo et al. (2002). ascorbic acid content ascorbic acid (vitamin c) was determined by titra­ tion with 2, 6­dichloro­phenol­indophenol dye fol­ lowing a standard procedure (aoac, 1990). using an electronic weighing balance (denver instrument xl­ 1810, usa), 10 g of fruit sample was weighed. the weighed fruit sample was extracted in 20 ml 5% oxal­ ic acid using a mortar and pestle, and then gravity fil­ tered through cotton wool. ascorbic acid standard solution was prepared by dissolving 0.05 g of pure ascorbic acid in a small volume of 5% oxalic acid solu­ tion and then diluting to 250 ml with the same oxalic adv. hort. sci., 2022 36(3): 193­200 196 acid solution. ascorbic acid standard solution (10 ml) was titrated with 0.005% indophenol solution to a persistent slight pink colour end point and 10 ml of oxalic acid as a blank. the amount of ascorbic acid corresponding to 1 ml of indophenol solution was calculated. into a 50 ml flask, 10 ml of the gravity fil­ tered sample extract was pipetted and made to the mark with the 5% oxalic acid solution. the standard indophenol solution was used to titrate 10 ml of the filtrate to a slight pink end point. vitamin c content was calculated following obel et al. (2019). data analysis all the data were subjected to analysis of variance (anova) in sas (ver. 9.0, sas institute inc., cary, nc). significant means at f­test were separated using tukey’s honestly significant difference (sas, 2010). 3. results effect of cactus mucilage coating on fresh weight loss and shelf life of bell pepper fruit cactus mucilage treatments significantly reduced fresh weight loss of bell pepper fruits from 3 dah (days after harvest) to the end of storage (fig. 1). at 3 dah, fruits coated with 1, 2 and 3% cactus mucilage recorded a significantly lower weight loss as com­ pared to a higher weight loss observed for the con­ trol treatment (distilled water) (fig. 1). a similar trend was observed at 6 dah and 9 dah. at 12 dah, weight loss of fruits coated with 1% cactus mucilage was significantly lower as compared to weight loss of fruits coated with 0, 2 and 3% cactus mucilage (fig. 1). fruits coated with 1% cactus mucilage treatment recorded a significantly lower weight loss as com­ pared to the control fruits at 15 dah (fig. 1) application of cactus mucilage treatments signifi­ cantly extended shelf life of bell pepper fruit by up to 6 days during storage (fig. 2). fruits coated with 1% cactus mucilage recorded a significantly longer shelf life, followed by a shelf life recorded for the control fruits with the shortest shelf life recorded for the fruits coated with 2 and 3% cactus mucilage (fig. 2). effect of cactus mucilage coating on total soluble solids of bell pepper fruit cactus mucilage coating had a significant effect on total soluble solids (tss) content of bell pepper fruit from 3 dah through the end of storage (fig. 3). in addition, there was an increase in tss content of bell pepper fruit as storage duration progressed except for fruits coated with 2 and 3% cactus mucilage where a decrease was observed from 9 dah (fig. 3). at 3 dah, fruits coated with 2 and 3% cactus mucilage treatments recorded a significantly lower tss content, followed by tss content recorded for fruits coated with 1% cactus mucilage with the high­ est tss content recorded for the control treatment (fig. 3). the trend was the same at 6, 9 and 12 dah of storage. at 15 dah, fruit coated with 1% cactus mucilage recorded a significantly lower tss content as compared to a higher tss content recorded for the control treatment (fig. 3). fig. 2 ­ effect of cactus mucilage coating on shelf life of bell pep­ per. error bars indicate ±se of the mean. cm0 is fruit coa­ ted with 0% cactus mucilage, cm1 is fruit coated with 1% cactus mucilage, cm2 is fruit coated with 2% cactus mucilage and cm3 is fruit coated with 3% cactus mucila­ ge. fig. 1 ­ effect of cactus mucilage coating on weight loss of bell pepper. error bars indicate ±se of the mean. cm0 is fruit coated with 0% cactus mucilage, cm1 is fruit coated with 1% cactus mucilage, cm2 is fruit coated with 2% cactus mucilage and cm3 is fruit coated with 3% cactus mucila­ ge. aloo et al. ‐ postharvest handling of bell pepper 197 effect of cactus mucilage coating on iron content of bell pepper fruit cactus mucilage coating also had a significant effect on iron content of bell pepper fruit from 4 dah until the end of storage (fig. 4). at 4 dah, fruit coated with 1, 2 and 3% cactus mucilage recorded a signifi­ cantly higher iron content as compared to a lower iron content recorded for 0% cactus mucilage treat­ ment (fig. 4). a similar trend was observed at 8 dah (fig. 4). at 12 dah, a significantly higher iron content was recorded for fruits coated with 1% cactus mucilage as compared to a lower content recorded for 0, 2 and 3% cactus mucilage treatments (fig. 4). fruits coated with 1% cactus mucilage recorded a sig­ nificantly higher iron content as compared to a lower content recorded under the control treatment (fig. 4). effect of cactus mucilage coating on ascorbic acid content of bell pepper fruit ascorbic acid content in bell pepper fruit was influenced by cactus mucilage treatments during storage from 4 dah through 16 dah (fig. 5). at 4 dah, fruits coated with 1, 2 and 3% cactus mucilage recorded a significantly higher ascorbic acid content as compared to a lower ascorbic acid content record­ ed for the control treatment (fig. 5). a similar trend was observed at 8 dah (fig. 5). a significantly higher ascorbic acid content was recorded for fruit coated with 1% cactus mucilage as compared to a lower ascorbic acid content recorded for 0 2 and 3% cactus mucilage treatments at 12 dah (fig. 5). at 16 dah, fruit coated with 1% cactus mucilage recorded a higher ascorbic acid content as compared to a lower ascorbic acid content recorded under the control treatment (fig. 5). 4. discussion and conclusions effect of cactus mucilage coating on fresh weight loss and shelf life of bell pepper fruit fresh weight loss is an important index in deter­ mining postharvest quality and shelf life of pepper. weight loss in harvested fruit is normally caused by continuous loss of water and stored starch as a result of respiration and evaporation leading to increase in weight loss as storage duration progresses. cactus mucilage coating forms a film on the fruit’s skin/cuti­ cle which acts as a semi­permeable barrier against moisture, oxygen, carbon (iv) oxide, and solute fig. 4 ­ effect of cactus mucilage coating on iron content of bell pepper. error bars indicate ±se of the mean. cm0 is fruit coated with 0% cactus mucilage, cm1 is fruit coated with 1% cactus mucilage, cm2 is fruit coated with 2% cactus mucilage and cm3 is fruit coated with 3% cactus mucila­ ge. fig. 3 ­ effect of cactus mucilage coating on total soluble solids content of bell pepper. error bars indicate ±se of the mean. cm0 is fruit coated with 0% cactus mucilage, cm1 is fruit coated with 1% cactus mucilage, cm2 is fruit coa­ ted with 2% cactus mucilage and cm3 is fruit coated with 3% cactus mucilage. fig. 5 ­ effect of cactus mucilage coating on ascorbic acid con­ tent of bell pepper. error bars indicate ±se of the mean. cm0 is fruit coated with 0% cactus mucilage, cm1 is fruit coated with 1% cactus mucilage, cm2 is fruit coated with 2% cactus mucilage and cm3 is fruit coated with 3% cac­ tus mucilage. 198 adv. hort. sci., 2022 36(3): 193­200 movement in the produce or between the produce and its environment. this leads to reduced rate of respiration, reduced water loss, starch or sugar loss, weight loss and extended shelf life. this could explain the reduced weight loss and extended shelf life recorded for fruit coated with 1% cactus mucilage compared to control fruit in the current study. many studies have also reported reduced weight loss and extended shelf life in fruits and vegetables as a result of polysaccharide­based edible coatings (menezes and athmaselvi, 2016; vishwasrao and anantha­ narayan, 2016). a thick layer of fruit coating blocks pores on the fruit’s skin, decrease oxygen concentra­ tion in the fruit’s tissues since oxygen in the fruit’s environment cannot get inside and the respiration products cannot also get outside the fruit’s tissues. anoxic conditions initiated leads to ethanol fermen­ tation in which stored carbohydrates and sugars are broken down to lactic acid, ethanol, acetaldehyde and carbon (iv) oxide which explains reduced fresh weight loss observed for fruits coated with 2 and 3% cactus mucilage during storage in the current study. increased weight loss caused by anaerobic conditions led to a shorter shelf life of fruits. fermentation bac­ teria and yeast proliferate the bell pepper tissues and breaks down stored carbohydrate, sugar, water and minerals for their growth and other metabolic activi­ ties. this explains the increased rate of fresh weight loss observed after 9 dah of storage for fruits coated with 2 and 3% cactus mucilage. according to kareem et al. (2017), anaerobic respiration leads to a decrease in stored carbohydrate content in fruits due to the utilization of some of the sugars by the fer­ menting organisms such as lactic acid bacteria for their growth and other metabolic activities. effect of cactus mucilage coating on total soluble solids of bell pepper fruit fruit tss content tends to increase during stor­ age due to biosynthesis of polysaccharides and accu­ mulation of sugars during ripening (ullah et al., 2017) and volatilization of soluble compounds and water. at advanced stages of ripening, disassociation of some molecules and structural enzymes in soluble compounds results in increased levels of tss. a slower rate of increase in tss content in cactus mucilage coated fruit observed in the current study could be attributed to the role of fruit coatings act­ ing as a barrier against oxygen, carbon iv oxide and ethylene, slowing down the rate of respiration and ripening leading to reduction in accumulated sugars and polysaccharides. increased tss content in con­ trol (uncoated) bell pepper fruit could be due to volatility of soluble compounds and water at a faster rate due to lack of a protective barrier on the sur­ face of such fruit. in addition, possible accumulation of sugars and polysaccharides as a result of increased rate of hydrolysis could have led to increased tss content in control fruits. these results are consistent with that of menezes and athmaselvi (2016) in sapota (manilkara zapota). ethanol fer­ mentation lowers tss content in fruits due to devel­ opment of off­flavours as observed for fruits coated with 2 and 3% cactus mucilage. ethanol fermenta­ tion is a two­step process in which pyruvate is first carboxylated to acetaldehyde by pyruvate decarboxylase and acetaldehyde is subsequently converted to ethanol by alcohol dehydrogenase. this explains the lower tss content observed for fruits coated with 2% and 3% cactus mucilage as compared to those coated with 1% and dipped in distilled water during storage. effect of cactus mucilage coating on iron content of bell pepper fruit results of this study indicate that coating bell pep­ per fruit with cactus mucilage may preserve the fruit’s iron content. iron is stored in fruit’s tissues in the chloroplast where 80% of the iron is located as iron­ protein complexes known as fe­phytoferritin, fe­cit­ rate, fe­phytosiderophore and fe­nicotianamine (maathuis and diatloff, 2013). iron is necessary for the synthesis of many proteins (ferredoxin and cytochromes) that carry electrons during respiration in which most iron ions are used to biosynthesize pro­ teins that carry electrons (bhatla and lal, 2018). cactus mucilage coating acts as a barrier against o2 and co2 inside and out of the fruit, thus reducing the rate of respiration and therefore reducing the amount of iron ions used to synthesize proteins that carry electrons during respiration. this could explain the high amount of iron in fruit coated with cactus mucilage during the current study. rapid decline of iron content in fruit that were dipped in distilled water could possibly have been due to increased res­ piration rate as a result of increase in o2 and decreased co2 inside and out of the fruit. results of the current study are consistent with those of amirthaveni and daga (2016) who recorded higher iron content in bell pepper coated with aloe vera gel and gum arabic. ethanol fermentation causes decline in minerals such as iron in the fruit. the nutrients are utilised by yeasts and lactic acid bacteria as they carry out their metabolism and fermentation activity. in aloo et al. ‐ postharvest handling of bell pepper 199 addition, their growth is supported by the existence of basic compounds such as fermentable minerals. this could offer an explanation for the result observed on fruits coated with 2 and 3% cactus mucilage during storage. these findings are supported by kareem et al. (2017) and maicas (2020) who reported utilization of minerals by lactic acid bacteria for growth and other metabolic activities during fermentation of fruits. effect of cactus mucilage coating on ascorbic acid content of bell pepper fruit ascorbic acid is commonly used as a quality indi­ cator of fruits and vegetables since it is very sensitive to degradation due to its oxidation compared to other nutrients during food processing and storage. plants biosynthesize ascorbic acid mainly through the smirnoff­wheeler pathway. in the final step of ascor­ bic acid synthesis, galactono­1,4­lactone is oxidized by galactono­1,4­lactone dehydrogenase (gldh) to produce ascorbic acid. ascorbic acid produced reduces during storage due to degradation mainly through the direct oxidation of dehydroascorbate (dha) or 4­o­oxalyl­l­threonic acid to produce both oxalic acid and l­threonic acid. in the current study, cactus mucilage coating could have acted as a barrier against oxygen gas that enters the fruit thereby reducing oxidation of dehydroascorbate (dha) or 4­ o­oxalyl­l­threonic acid resulting in higher amounts of ascorbic acid in the fruit during storage. a rapid decrease in ascorbic acid in uncoated fruits could be attributed to increased oxidation of dehydroascor­ bate (dha) or 4­o­oxalyl­l­threonic acid due to increased oxygen concentration in the fruit tissues. results of this study are in agreement with those of a number of scholars who also reported oxidation reactions in the presence of oxygen in uncoated fruits during storage leading to reduction of ascorbic acid (menezes and athmaselvi, 2016; ullah et al., 2017). a rapid decrease in ascorbic acid content observed for fruits coated with 2 and 3% cactus mucilage during storage was attributed to ethanol fermentation of fruits as a result of low oxygen con­ centration in the fruit tissues. during fermentation, microorganisms such as yeasts and lactic acid bacte­ ria uses nutrients such as minerals like ascorbic acid for their growth, reproduction and other metabolic activities leading to a decrease in ascorbic acid con­ tent in fermenting fruits. based on the objective and findings of this study, it can be concluded that cactus mucilage coating sig­ nificantly influenced postharvest quality and shelf life of bell pepper. one % cactus mucilage coating was the best treatment in terms of fresh weight loss reduction, maintenance of total soluble solids, iron, ascorbic acid content, and extension of shelf life of bell pepper fruit. acknowledgements sincere gratitude is extended to world bank through the centre of excellence in sustainable agriculture and agribusiness management (cesaam), egerton university, kenya for financial support, which greatly contributed to the success of this research. references abera n.g., kebede w., wassu m., 2019 ­ effect of aloe gel and cactus mucilage coating on chemical quality and sensory attributes of mango (mangifera indica l.). ‐ j. postharvest technol., 7(2): 31­43. alikhani m., 2014 ­ enhancing safety and shelf life of fresh‐cut mango by application of edible coatings and microencapsulation technique. ­ food sci. nutr., 2(3): 210­217. amirthaveni m., daga p., 2016 ­ effect of aloe vera gel and gum arabic coating on shelf life and nutrient con‐ tent of green capsicum (capsicum annuum l.). ­ indian j. res. food sci. nutrition, 3(2): 37­43. aoac, 1990 ­ official method of analysis (15th edition). ­ association official analytical chemists, washington dc, usa, pp. 123­126. bayogan e.r., salvilla r., majomot a.m.c., acosta j., 2017 ­ shelf life of two sweet pepper (capsicum annu­ um) cultivars stored at ambient and evaporative cool‐ ing conditions. ­ south­west. j. hortic. biol. environ., 8(1): 1­15. bhatla s.c., lal m.a., 2018 ­ plant physiology, develop‐ ment and metabolism. ­ springer nature singapore pte ltd, pp. 1237. díaz­pérez j.c., muy­rangel m.d., mascorro a.g., 2007 ­ fruit size and stage of ripeness affect posthar‐ vest water loss in bell pepper fruit (capsicum annuum l.). ‐ j. sci. food agric., 87(1): 68­73. egerton meterological weather station, 2016 ­ egerton university weather statistics. ­ department of agricultural engineering, egerton university, njoro, kenia. ilić z.s., šunić l.j., fallik e., 2017 ­ quality evaluation and antioxidant activity of mini sweet pepper cultivars during storage in modified atmosphere packaging (map). ­ rom. biotechnol. lett., 22(1): 12214­12223. adv. hort. sci., 2022 36(3): 193­200 200 jaetzold r., schmidt h., hornetz b., chisanya c., 2006 ­ farm management handbook of kenya. natural conditions and farm management information. ­ ministry of agriculture, kenya. jones j.b., case v.w., 1990 ­ sampling, handling and ana‐ lyzing plant tissue samples, 389­427. ­ in: r.l. wester­ man (ed.) soil testing and plant analysis. soil science society of america, inc., book series no. 3, madison, wisconsin, usa, pp. 784. kareem k.a., ojokoh j.b., baba j., 2017 ­ the effects of fermentation on the nutritional and anti‐nutritional constituents of irish potato peels. ­ j. appl. res. child., 8(2): 680­685. kitinoja l., kader a.a., 2015 ­ measuring postharvest losses of fresh fruits and vegetables in developing coun‐ tries. ­ pef white paper, 15: 26. lerdthanangkul s., krochta j.m., 1996 ­ edible coat‐ ing effects on postharvest quality of green bell peppers. ­ j. food sci., 61(1): 176­179. liguori g., gaglio r., greco g., gentile c., settanni l., inglese p., 2021 ­ effect of opuntia ficus­indica mucilage edible coating on quality, nutraceutical, and sensorial parameters of minimally processed cactus pear fruits ­ agron. j., 11(10): 1963. maathuis f.j.m., diatloff e., 2013 ­ roles and functions of plant mineral nutrients, pp. 1­21. ­ in: maathuis f.j.m. 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krishnamurthy et al., 2013). water deficit is a multidimensional stress affecting plants at morpho-physiological, biochemical and molecular levels including inhibition of growth, accumulation of compatible organic solutes, changes in phytohormones endogenous contents, modifications in expression of stress responsive-genes among others (vasanthaiah and kambiranda, 2011). in the semi-arid areas of the mediterranean region, this grain legume is cultivated on a large scale under rain-fed conditions, where water stress occurring during the post-flowering period is considered the major limiting abiotic stress, reducing growth and n2-fixation (kurdali, 1996; kurdali et al., 2002). thus, increasing n2-fixation is considered of a (*) corresponding author: ascientific5@aec.org.sy citation: al-chammaa m., al-ain f., kurdali f., 2019 effect of salicylic acid on growth, nodulation and n2‐fixation in water stressed chickpeas using 15n and 13c. adv. hort. sci., 33(3): 391-401 copyright: © 2019 al-chammaa m., al-ain f., kurdali f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 october 2018 accepted for publication 27 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 391-401 392 great importance to improve yield and performance of chikpea in the the agricultural systems. increasing the efficiency of legumes to fix n2 may be addressed by several approaches including selection of the best plant-microbial combinations and appropriate agricultural practices and managements (hardarson, 1993; kurdali et al., 2005). a better plant nutrition (e.g., k, p, si, etc. ) can effectively alleviate the adverse effects of drought (waraich et al., 2011) and hence enhance n2-fixation (kurdali and al-shammaa, 2010; kurdali et al., 2013). salicylic acid (sa) is one of the endogenous growth regulators that are involved in a range of physiological and metabolic responses in plants (hayat et al., 2010). it coordinates growth and development with plant responses to the environment in a complex signal-transduction network (aimar et al., 2011). in recent years, sa has been the focus of intensive research due to its function as an endogenous signal mediating local and systemic plant defense responses against pathogens (rivas-san vicente and plasencia, 2011). moreover, it has, also, been reported that sa is a potential tool in reducing or alleviating the adverse effects of abiotic stress in plants (khan et al., 2015; amirinejad et al., 2017). exogenous application of sa has been shown to be beneficial for plants either in optimal or stress environments. salicylic acid is involved in regulatating various plant metabolic processes and modulating the production of varied osmolytes and secondary metabolites, as well as maintaining plant-nutrient status. hence, it protects plants under abiotic stress conditions (khan et al., 2015). the effectiveness of sa in inducing stress tolerance depends upon plant species, method of addition, time of application and the concentration (hayat et al., 2010; gharbi et al., 2018). low concentrations of exogenous sa provides tolerance against damaging effects of stresses on plants, whereas, higher concentrations of sa did not show the same effects (senaratna et al., 2000). recent evidence highlighted the importance of sa as a regulator of photosynthesis due to its effect on stomatal conductance and the activity of enzymes such as rubisco and carbonic anhydrase (rivas-san vicente and plasencia, 2011). extensive studies have been conducted on the effect of sa application on physiological and biochemical parameters (e.g., gas exchange, stomatal conductance, chlorophyll, photosynthetic rate measurements and enzyme activity estimations) in relation to abiotic stresses (khan et al., 2003; khodary, 2004; hayat et al., 2010; ghasemzadeh and jaafar, 2013; lee et al., 2014; miura and tada, 2014). during photosynthesis, c3 plants discriminate against the heavy isotope of carbon (13c) leading to a depletion of the plant dry matter in 13c. carbon isotope discrimination (δ13c) positively correlates with ci/ca (i.e., the ratio of internal leaf co2 concentration to ambient co2 concentration) and thus provides an integrated measurement of the photosynthesis efficiency in response to environmental conditions prevailing during the plant growth cycle (farquhar et al., 1989). δ13c has been intensively studied as a selection criterion for drought tolerance in several c3 species (farquhar et al., 1989). water stress can alter δ13c as a result of its effects on the balance between stomatal conductance and carboxilation (i.e. rubisco), (farquhar et al., 1989). because of the correlation between δ13c and gas exchange values (i.e., ci/ca), the isotopic methods represent an alternative to gas exchange measurements. (farquhar et al., 1989). carbon isotope discrimination was also used for studying the impact of agricultural practices including fertilizer applications such as nitrogen (iqbal et al., 2005), potassium (kurdali and al-shammaa, 2010) and silicon (kurdali et al., 2013) on crop performance enhancements under water stress conditions. accordingly, we hypothesize that δ13c can be affected by sa application. available l iterature on the relationships between sa and carbon isotope discrimination is very scarce and, to our knowledge, only in one case, the effect of sa on plant water relationships was studied (barkosky and einhellig, 1993). on the other hand, the 15n isotope dilution is one amongst several available methods to quantify plant-associated n2-fixation and provides a valuable mean for evaluating factors affecting n2-fixation such as drought (kurdali et al., 2002) and salinity (kurdali and al-ain, 2002). therefore, the objective of this study was to determine the effect of sa on the performance of chickpea plants (growth, nitrogen uptake and n2-fixation) grown under various soil moisture levels using stable isotopes (i.e., 15n isotopic dilution and 13c isotope discrimination). 2. materials and methods soil properties and plant materials the experiment was conducted in pots, each one containing 5 kg of thoroughly mixed soil collected from deir al-hajar agricultural experiment station, located south east of damascus, syria (36° 28’e, 33° 21’ n; altitude 617 m). some climatic data of the al‐chammaa et al. ‐ salicylic acid and water stress effects on chickpea 393 experimental site during the growing period is shown in table 1. the main physical and chemical soil properties were: ph 7.80, ece 0.31 dsm-1, soil bulk density was 1.20 g cm-3, organic matter 1.25 per cent, cations (ca++ 2.25, mg++ 0.97, k+ 0.14 and na+ 1.27 mmol l-1), anions (so4 — 1.27, hco3 -1.07 and cl0.55 mmol l-1), available p (olsen) 13.40 mg g-1; total n 0.12 per cent, no3 33.6 mg g-1, nh4 +28.1 mg g-1. the soil is classified as a clay loam, with an average 57.89% clay, 39.47% silt, and 2.63% sand. seeds of chickpea (cicer arietinum l.), and barley as a non-fixing plant were sown. after germination, plants were thinned to two plants per pot. the pots were set outdoors under natural climatic conditions. all pots were protected from rainfall by manually operated shelter equipped with movable sheet of transparent flexible plastic. since abundant nodules had already been observed on the roots of chickpea plants grown in the area, from which the soil was collected and used for this experiment, the seeds were not inoculated. experiment design and treatments the pots were arranged in a split plot design, with salicylic acid treatments (sa) being the main plots and the irrigation regimes are the sub-main. salicylic acid c6h4(oh).cooh had the following specification: assay min. 99%, melting point 157-162°c, maximum limits of impurities: chloride 0.01%, sulphate 0.03%, iron 0.002%, heavy metals 0.001%. two sa treatments were used: (sa-, control without sa and sa+,10-5mol l-1). within each of the sa treatments, three irrigation regimes, expressed as percent of field capacity(fc), were applied (fc1, high stress 45-50%; fc2, mild stress 55-60% and fc3, wellwatering 75-80%). all treatments were replicated four times. soil water content in all pots was maintained at around 75% of field capacity from planting up to bud flower initiation (5 weeks after planting). thereafter, plants were subjected to the above-mentioned soil moisture regimes. foliar spraying of the plants with salicylic acid (sa) was initiated at the same time of applying water regimes and performed 6 times at 10 days intervals. for non-sa treated chickpeas, plants were sprayed with distilled water set as control (sa-). pots were weighed every three days, and water was added to maintain the soil moisture regimes as previously described. the pots were kept weed-free and any drainage was prevented. 15n‐application an equivalent rate of 25 kg n ha-1 of 15n labeled urea (5% 15n atom excess) was applied to chickpea and barley plants to estimate the fractional contribution of nitrogen derived from air (ndfa, i.e n2-fixation), soil (ndfs) and from fertilizer (ndff), using the isotopic dilution method (fried and middelboe 1977). two equally split applications of n fertilizer (12.5 kg n ha-1 for each application) were applied at 2-week intervals starting from complete seedling emergence. this procedure was followed to stabilize the 15n enrichment of the n pool and to minimize n immobilization. barley was used as a non-fixing reference crop for estimating the n fraction derived from the atmosphere (%ndfa) in chikpeas, and was similarly treated with the above mentioned treatments (i.e., both of watering regimes and sa application). plant sampling and isotopic composition analysis plants were harvested twelve weeks after planting. shoots and nodules were dried at 70°c for 72 h, weighed for dry matter determinations. shoots were then ground to a fine powder. total nitrogen was determined by kjeldahl procedure, and 15n/14n isotope ratio was measured using an emission spectrometry (jasco-150, japan). the nitrogen fraction derived from the atmosphere (%ndfa) was calculated using the equation of fried and middelboe (1977): %ndfa= (1 atom % 15n excess chikpea ) x 100 atom % 15n excess barley the percent n derived from fertilizer (%ndff) was calculated using the following equations: %ndff= atom % 15n excess plant x 100 atom % 15n excess fertilizer the percent n derived from soil (%ndfs) was calculated as follows: %ndfs = 100 (%ndfa + %ndff) amounts of nitrogen (mg n plant-1) derived from n2-fixation (ndfa), soil (ndfs) and from fertilizer (ndff) were calculate by multiplying the fractional contribution of each source (%) by nitrogen yield. the 13c/12c ratio (13c‰) was determined on subsample of shoots using the continuous-flow isotope table 1 some climatic data during the growing season of the experimental site variable february march april may minimum temperature (oc) 4.9 7.7 11.5 14.8 maximum temperature (oc) 19.5 21.2 29.2 30.5 relative air humidity (%) 74 67 59 58 et0 (mm day-1) 2.7 3.2 6.3 8.4 adv. hort. sci., 2019 33(3): 391-401 394 ratio mass spectrometry (integra-cn, pdz europea scientific instrument, uk). carbon isotope discrimination (δ13c‰) values were estimated using the equation of farquhar et al. (1982): δ13c = ( 13cair  13csample)/(1 13csample/1000) where 13cair is the 13c value in air (-8‰) and 13csample is the measured value in the plant. statistical analysis the data were subjected to analysis of variance (anova) test, and means were compared using the least significant difference (fisher’s plsd) test at the 0.05 level of probability (p<0.05). moreover, correlation coefficients (r) between δ13c and the studied parameters (e.g., dm, ny, per cents & amounts of ndfa, ndff and ndfs ) were estimated. 3. results effect of salicylic acid on dry matter and nitrogen yield dry matter yield (dm) of chikpea plants was significantly affected by water stress (table 2). the lowest dry matter value (5.57 g pot-1) was observed in nonsa treated plants grown under the highest water stress level (fc1). increasing soil moisture from fc1 to fc2, from fc1 to fc3, and from fc2 tofc3 resulted in significant increases in dm by 40%, 64%, and 17%, respectively. the foliar spray of sa had produced appreciable results by increasing dry matter yield under stress conditions and the effect was more pronounced in plants subjected to a mild water stress. at each irrigation regime, the sa increased dm by 13%, 17%, and 6% in fc1, fc2, and fc3, respectively. it is worth mentioning, also, that dm in sa-treated plants grown under moderate water stress (fc2, 9.06 g pot1) was relatively similar to that of well-watered plants without sa treatment (fc3, 9.12 g pot-1). however, dm of well-plants (fc3) was not significantly enhanced by sa as compared to non-treated sa plants. the pattern of total nitrogen yield (ny) was relatively similar to that of dry matter yield (table 2). water stress significantly reduced the ny and the highest reduction was recorded under the highest stress level (fc1). in non-sa treated plants, the lowest value of ny (129 mg n pot-1) was in fc1. increasing soil moisture from fc1to fc2, from fc 1to fc3 and from fc2 to fc3 resulted in significant increases in ny by 38%, 58%, and 15%, respectively. the exogenous supply of sa significantly enhanced nitrogen accumulation by 14%, 20%, and 10% in fc1, fc2, and fc3, respectively. the highest amount of tn was observed in well-watered plants (fc3) treated with sa application (223.4 mg n pot-1) representing a 73% increment over the control (i.e., non-treated sa plants in fc1). it is evident from dm and ny data that water stress adversely affected the production of dry matter and nitrogen yield of chikpeas, and exogenous application of sa at 5 mm l-1 was successful in alleviating the adverse effect of water stress. effect of salicylic acid on nodule dry matter effects of sa on nodule dry weight of chickpea plants grown under different water stress conditions are given in figure 1. the lowest nodule dry weight was noted in the highest water stressed plants (fc1). for the non-sa treated plants, increasing soil moisture from fc1 to fc2, from fc1 to fc3, and from fc2 to fc3, resulted in increases in nodule dry weight by 31%, 62%, and 24%, respectively. the exogenous supply of sa significantly enhanced nitrogen accumutable 2 total dry matter yield (g pot-1) and nitrogen yield (mg n pot-1) of chickpea plants grown under different water regimes as affected by salicylic acid (sa) means ±sd within a column (capital letter) and within a row (small letter) followed by the same letter are not significantly different (p<0.05). fc: water regimes, expressed as % of field capacity (fc1, high stress 45-50%; fc2, mild stress 55–60% and fc3, well-watering 75–80%). sa-: control without sa, sa+ 10-5 mol l-1. salicylic acid irrigation treatments fc1 fc2 fc3 lsd 0.05 dm (g pot-1) sa5.57±0.12 b c 7.77±0.41 b b 9.12±0.31 a a 0.48 sa+ 6.29±0.41 a b 9.06±0.47 a a 9.68±0.40 a a 0.68 lsd 0.05 0.52 0.76 0.62 n-uptake (mg n pot-1) sa129.06±4.68 b c 177.45±2.95 b b 203.33±10.53 b a 10.98 sa+ 146.78±5.11 a c 213.09±4.51 a b 223.41±4.17 a a 7.37 lsd 0.05 8.47 6.59 13.85 al‐chammaa et al. ‐ salicylic acid and water stress effects on chickpea 395 sa treated plants, increasing soil moisture from fc1 to fc2, from fc1 to fc3, and from fc2 to fc3, resulted in increases in nodules dry weight by 71%, 93%, and 13%, respectively. these results illustrate the importance of foliar spray of sa in enhancing nodules dry weight of chickpea grown under the different water regimes. effect of salicylic acid on nitrogen uptake from the available sources nitrogen derived from fertilizer (ndff), soil (ndfs) and atmosphere (ndfa, i.e., n2-fixation) in chickpeas grown under various watering regimes as affected by sa application are given in table 3. regardless of sa application, the proportions of ndff and ndfs (%) in chickpea plants significantly decreased as soil field capacity increased. however, the opposite was true regarding the %ndfa which showed a higher value under optimal irrigation conditions (fc3) compared to water stressed treatments (fc1 and fc2). for the non-sa treated plants, the observed values of %ndfa were 33, 40, and 43.8% in fc1, fc2, and fc3, respectively. chickpea plants significantly enhanced their nitrogen fixation (i.e., 39, 43.3 and 47.3% in fc1, fc2, and fc3, respectively) in response to sa application lation by 41% in fc2 and 29% in fc3 watering treatments. however, the enhancement of nodule dry weight in fc1 by sa was not significant (8%). for the fig. 1 nodule dry matter (g pot-1) of chickpea plants grown under different water regimes as affected by salicylic acid (sa). columns followed by the same letter are not significantly different (p<0.05); capital letters (effect of sa in each irrigation regime); small letters within a row (comparison among irrigation regimes either for sa+ or sa-). table 3 proportions (%) and amounts (mg n pot-1) of nitrogen derived from fertilizer (ndff), soil (ndfs) and atmosphere, i.e. n2-fixation (ndfa) in chickpea plants grown under different water regimes as affected by salicylic acid (sa) means ±sd within a column (capital letter) and within a row (small letter) followed by the same letter are not significantly different (p<0.05). fc: water regimes, expressed as % of field capacity (fc1, high stress 45-50%; fc2, mild stress 55-60% and fc3, well-watering 75–80%). sa-: control without sa, sa+ 10-5 mol l-1. salicylic acid irrigation treatments lsd0.05 fc1 fc2 fc3 %ndff sa12.62±0.22 a a 11.40±0.18 a b 10.69±0.27 a c 0.36 sa+ 11.59±0.08 b a 10.77±0.21 b b 10.02±0.19 b c 0.27 lsd 0.05 0.29 0.34 0.40 ndff (mg n pot‐¹) sa16.30±0.74 a b 20.22±0.24 b a 21.73±1.45 a a 1.52 sa+ 17.01±0.67 a b 22.96±0.84 a a 22.38±0.82 a a 1.25 lsd 0.05 1.22 1.07 2.04 %ndfs sa53.83±0.94 a a 48.60±0.77 a b 45.55±1.14 a c 1.54 sa+ 49.42±0.36 b a 45.94±0.90 b b 42.71±0.82 b c 1.17 lsd 0.05 1.24 1.45 1.72 ndfs (mg n pot‐¹) sa69.48±3.1 a b 86.22±1.0 b a 92.66±6.2 a a 6.49 sa+ 72.54±2.9 a b 97.91±3.6 a a 95.44±3.5 a a 5.31 lsd 0.05 5.18 4.56 8.71 %ndfa sa33.55±1.2 b c 40.01±0.8 b b 43.76±1.4 b a 1.90 sa+ 39.00±0.5 a c 43.29±1.1 a b 47.28±1.0 a a 1.45 lsd 0.05 1.53 1.79 2.13 ndfa (mg n pot‐¹) sa43.29±1.8 b c 71.01±2.6 b b 88.94±4.2 b a 4.92 sa+ 57.22±1.7 a c 92.23±1.9 a b 105.6±1.2 a a 2.58 lsd 0.05 3.06 3.97 5.38 396 adv. hort. sci., 2019 33(3): 391-401 in all watering regimes. inversely, both %ndff and %ndfs values were decreased by sa treatments. however, amounts of ndff and ndfs (mg) increased due to the foliar application of sa in all watering regimes. likewise, the exogenous supply of sa significantly enhanced the amounts of fixed n2 by 32%, 30%, and 19% in fc1, fc2, and fc3, respectively. regardless of sa application, it can be noticed that the amount of fixed n2 in the well-watered plants (fc3) was almost doubled as compared to those subjected to high stress (fc1). it is, also, worth mentioning that amount of ndfa in sa-treated plants grown under moderate water stress (fc2) was close or even higher than that of well-watered (fc3) chickpea without sa. these results may illustrate the importance of sa in saving irrigation water and alleviating water stress influences to ensure appropriate yield and n2fixation. effect of salicylic acid on carbon isotope discrimina‐ tion effect of sa on carbon isotope discrimination (‰δ13c) in shoots of chickpea plants subjected to different water stress conditions is given in figure 2. water stress caused a considerable decrease in δ13c compared to well irrigated conditions (fig. 2). for the non-sa treated plants, the mean δ13c value in wellwatered chickpea (fc3) was 21.29‰, while it significantly decreased to 20.41‰ and 19.88‰ in mild (fc2) and high water stressed (fc1) plants, respectively. the exogenous application of sa increased δ13c particularly in high and mild water stressed plants. the lowest δ13c value was obtained in fc1 (20.43‰) and increased to 21.34‰ in fc2 and 21.50‰ in fc3, with no significant difference being obtained between the latter two values. on the other hand, correlations between δ13c and the studied parameters showed positive relationships with dm (0.953**), ny (0.949**), nodule dry weight (0.92*), %ndfa (0.948**), and amounts of ndfa, ndfs and ndff (0.957**, 0.91* and 0.91*, respectively). however, negative relationships between δ13c and %ndff and %ndfs (-0.948**) were observed. 4. discussion and conclusions the results of this study showed that water stress occurring during the flowering period of chickpea plants considerably affected their growth and n2-fixation. total dry weight and nitrogen yield decreased significantly in plants as field capacity decreased. salicylic acid-treated plants exhibited an increase in tolerance to water stress, particularly under mildstress conditions where the measured growth criteria (i.e., dry matter and nitrogen yield) were relatively similar to those of the non-stressed plants regardless of whether the plants treated with or without sa applications. gutierrez-coronado et al. (1998) reported a similar increase in the growth of soybean plants in response to salicylic acid treatment. afshari et al. (2013), also, indicated that sa increased the growth and physiological attributes of cowpea under water stress. moreover, foliar application of sa in drought stressed chickpeas significantly enhanced plant biomass through increasing proline content of leaves (farjam et al., 2014). correspondingly, the beneficial role of sa to adverse effects of other abiotic stresses (e.g., heavy metals, salinity and cold..) has been examined in various legumes such as chickpea (hayat et al., 2014), medic (palma et al., 2013), lentil (misra and saxena, 2009), and common bean (torquato de agostini et al., 2013). water stress is known to decrease nodulation by affecting the establishment of the symbiosis between the host and rhizobia, thereby decreasing the number and the mass of nodules and reducing n2-fixation (kurdali et al., 2002). the undertaken study showed that nodulation and n2-fixation were reduced as soil water content decreased. such reductions could be attributed to the limitation of photosynthesis and the decrease of photosynthates supply to nodules (kurdali, 1996). other physiological factors such as o2 availability in nodules could be involved with the decline of n2fixation. guérin et al. (1991) suggested fig. 2 carbon isotope discrimination (δ13c) in chickpea plants grown under different water regimes as affected by salicylic acid (sa). columns followed by the same letter are not significantly different (p<0.05); capital letters (effect of sa in each irrigation regime); small letters within a row (comparison among irrigation regimes either for sa+ or sa-). al‐chammaa et al. ‐ salicylic acid and water stress effects on chickpea 397 that o2 supply to bacteroids, in water stressed nodules, is restricted either by a limitation of diffusion or by a degradation of leghaemoglobin, and therefore hinders respiration and atp production, with the final result of reduction of n2-fixation (patterson and hudak, 1996). however, application of sa is found to be beneficial for nodulation and n2-fixation in plants either grown under optimal or water stress conditions. several factors are well known to be involved in nodulation and n2-fixation of grain legumes including the occurrence of effective rhizobium strains, the availability of photosynthesis, nitrogenas activity and its protection against o2 via increasing of leghemoglobin contents, and the effectiveness of enzymes mediating the export of fixed nitrogen from nodules to plant (e,g., gs, gogat, and gdh). the occurrence of n2-fixation under various soil water regimes could be explained by the presence of effective indigenous rhizobia strains. on the other hand, it is well known that the high dry matter yield of a given plant species may imply a higher photosynthetic rate (kurdali and al-shammaa, 2010). in a study carried out on soybean, foliar application of salicylic acid enhanced the water use efficiency (wue) and photosynthetic rate (kumar et al., 2000). also, hayat et al. (2010, 2012) reported that the exogenous application of sa to chickpea increased the net photosynthetic rate, favoring the production of photosynthates and the nodules obtained a significant proportion of the carbohydrates. the development of healthy nodules in sa-treated plants, as expressed in terms of an increase in nodule dry matter (fig. 1), is expected to be accompanied by leghemoglobin content increment and consequently increased nitrogen fixation via stimulation of nitrogenase activity (hayat et al., 2012). moreover, higher leghemoglobin content in sa-treated plants (balestrasse et al., 2004; hayat et al., 2014) might be a consequence of the decrease generation of ros. thereby preventing oxidative damage to plants in general and nodules in particular, for cadmium (cd) stressed chickpea (hayat et al., 2014) and soybean plants (balestrasse et al., 2004). in this context, palma et al. (2013) reported that sa alleviated the negative effect of salt stress in the medicago sativa sinorhizobium meliloti symbiosis through the increased level of nodule biomass and the induction of the nodular antioxidant metabolism under salt stress. similarly, misra and saxena (2009) showed that sa could be used as a potential growth regulator to improve salinity tolerance of lentil plants by enhancing proline metabolizing system. hayat et al. (2012, 2014) reported that, the exogenous application of sa to chickpea plants enhanced the activities of the enzymes involved in nitrogen fixation and assimilation (e,g., nr, gs, gogat, gdh and nitrogenase) regardless of whether the plants are grown in the presence or absence of cadmium. on the light of the aforementioned studies, it can be suggested that the beneficial effect of sa on n2-fixation in chickpea plants might be resulted from enhancing nodulation, leghemoglobin contents and the activity of enzymes involved in nitrogen fixation and assimilation. in a review paper, hayat et al. (2010) reported that, during the early stages of nodulation, exogenous sa inhibited the growth of rhizobia and the production of nod factors by them and also delayed the nodule formation, particularly in plants producing indeterminate nodules, thereby decreasing the number of nodules per plant (van-spronsen et al., 2003; mabood and smith, 2007). in this study, however, since sa was supplied prior to flowering stage of chickpeas (i.e. indeterminate nodule type), it is most l ikely that nodule formation was established. therefore, the further benefits of sa were most probably resulted from nodule growth development (dm) and functioning (n2-fixation). carbon isotope discrimination values (δ13c) in chickpea’s shoots were affected by soil water content and sa applications. water stress significantly decreased δ13c values as field capacity decreased. however, the exogenous application of sa increased δ13c particularly in high and mild water stressed plants. it has been reported that δ13c can reflect the integrated response of physiological processes to environment. water stress can alter δ13c as a result of effects on the balance between stomatal conductance and carboxilation (farquhar et al., 1989). the lower δ13c value in the stressed plants compared to the non-stressed plants implies that ci/ca ratios were lower under stress. a lower ci/ca ratio could result either from stomatal closure induced by stress or from higher rates of photosynthetic capacity or a combination of both (condon et al., 2002; kurdali et al., 2013). because of the lower dry matter yield in the water stressed plants (fc1-sa-) compared with fc2-saor with fc3-sa-, it was unlikely that a higher photosynthetic capacity occurred in fc1. therefore, the lower δ13c value in the high and mild water stressed chickpeas compared to well-watered plants (i.e. sa-)resulted mainly from stomatal closure induced by stress. the principal components of photosynthesis that influence discrimination are diffuadv. hort. sci., 2019 33(3): 391-401 398 sion of co2 through stomata and the carboxylation process mediated by rubisco (o’leary, 1988). in sa treated plants, the higher ‰δ13c value in high and mild water stressed plants comparing with non-sa treated plants were associated with higher dry matter yield (i.e. higher photosynthetic activity). therefore, the higher δ13c following sa applications imply that a maximization of yield may occur via a maximization of co2 uptake activity due to stomatal opening (condon et al., 1987). janda et al. (2014) concluded that the effect of sa on the photosynthetic machinery is indirect, originating from its influence on stomatal conductivity. moreover, it has been reported that the increases in photosynthetic rates of soybean (khan et al., 2003) and ginger (ghasemzadeh and jaafar, 2013) plants following sa applications were the result of increased co2 uptake activity at the chloroplast level (i.e. rubisco activity), rather than simple increase in stomatal opening, i.e., reduced the resistance to entry co2 in the leaves. in stressed plants, lee et al. (2014) reported that the content of rubisco was increased by sa in tobacco plants treated with nacl. likewise, khodary (2004) concluded that sa treatment of salt stressed maize could stimulate their salt tolerance via accelerating their photosynthesis performance (i.e. rubisco activity) and carbohydrate metabolism. recently, gharbi et al. (2018) reported that combination of applied sa as a priming agent or concomitantly with nacl were required to maintain a good water use efficiency in salt-treated tomato plants using carbon isotope discrimination. according to farquhar et al. (1982), a higher δ13c values is caused by a higher ci/ca ratio due to higher stomatal conductance which leads to higher photosynthetic rate and hence higher biomass, (i.e, positive correlation between δ13c and dm, r=0.95**). accordingly, it can be suggested that the beneficial effect of sa in enhancing dry matter yield of high and mild water stressed chickpeas could be resulted from higher co2 uptake activity by rubisco (i.e., photosynthetic activity) in addition to higher co2 uptake at stomatal level (i.e., higher ci). on the other hand, in fc3, no significant effects of sa were obtained neither on δ13c nor on dry matter yield, indicating that the field soil capacity (75-80%) is an optimal watering regime to ensure a good biomass production. consequently, it can be concluded that sa application can alter δ13cin water stressed plants (e.g., fc1 and fc2) as a result of effects on the balance between stomatal conductance and carboxylation. in addition to the positive relationship between δ13c and dry matter yield of chickpeas, higher δ13c values were also associated with enhancements of %ndfa (0.948**), amount of n2-fixed (0.957**) and nodule dry matter yield (0.92*). hence, it can be suggested that carbon isotope discrimination could be used as an indicator for nodulation and n2-fixation efficiency. this observation is in harmony with that of knight et al. (1993) who reported a positive correlation between δ13c and the amount of n2-fixed in lentil inoculated with different strains of rhizobia, and with results of kurdali and al-shammaa (2010) in potassium fed lentil grown under water stress conditions. salicylic acid can be involved in the regulation of uptake of several plant-beneficial elements (khan et al., 2015). exogenously supplied sa can improve plant growth under stresses by stimulating the accumulation of mineral elements including nitrogen (gunes et al., 2005; yildirim et al., 2008). in this study, exogenously applied sa to mild water-stressed chickpea resulted in increments of soil nitrogen uptake (ndfs) as well as amount of n derived from fertilizer (ndff) and its use efficiency (%nue). such increments may support the idea that low sa concentrations (10–5 m) can induce nitrite reductase synthesis, which plays a key role in nitrogen metabolism, by mobilizing intracellular no3− and can provide protection to nitrite reductase degradation (ghasemzadeh and jaafar, 2013). consequently, our finding indicates that the application of sa is beneficial to get improvement in nitrogen uptake and metabolism in plants grown under stressed conditions. such improvements enhance the protein/enzyme/growth hormone synthesis, developing the metabolic activity and resulting in increasingthe growth and plant productivity (akhtar et al., 2013). on the other hand, the positive correlations between δ13c and total n uptake, amounts of ndfs or ndff may illustrate a relationship between photosynthesis and nitrogen metabolism. in addition, the negative relationship between δ13c with %ndfs or %ndff (0.91*) is also reported by iqbal et al. (2005) in wheat plants grown under rain-fed conditions implying that δ13c could be used to predict these parameters. consequently, it can be suggested that carbon isotope discrimination could be used as an indicator for nitrogen uptake from the available sources. a priority for future research should be elucidation of relationships between carbon isotope discrimination and nutrient use efficiency in various plant species and genotypes, and how these vary in response to environmental conditions and agricultural practices. al‐chammaa et al. ‐ salicylic acid and water stress effects on chickpea 399 the ability of sa to increase chickpea growth, nitrogen uptake and n2fixation, ameliorating the adverse effect of water stress, may have significant implications in improving the plant performance and overcoming the growth barrier arising from conditions of limited water availability. a simplified scheme representing the beneficial effects of exogenous application of salicylic acid on growth and n2fixation as reported in this study and in the literatures (i.e. hayat et al., 2010, 2012 and 2014) is shown in figure 3. further field investigations are required to illustrate the role of salicylic acid in terms of growth, yield, and the time course of n2 fixation of legumes grown under rain-fed conditions in the semi-arid areas. to the best of our knowledge, the present study is the first report on the relationship between sa application and carbon isotope discrimination (δ13c) in n2 fixing plants (e.g., chickpea). salicylic acid increased δ13c values in water stressed plants, implying that a maximization of dry matter yield may occur via an enhancement of co2 uptake due to stomatal opening and carboxylation activity. moreover, δ13c could be used as an indicator for nitrogen uptake, nodulation and n2 fixation. although application of sa is found to be beneficial for plants either in optimal or water stress conditions, its main effect in enhancing plant performance (growth and n-uptake from the available sources) was affected by soil water content as follows: under high water stress conditions (fc1), the main effect of sa was on plant growth, nodulation fig. 3 simplified schemes representing the beneficial effects of exogenous application of salicylic acid on growth and n2-fixation as presented in this study and in the literatures (i.e. hayat et al., 2010, 2012 and 2014). adv. hort. sci., 2019 33(3): 391-401 400 and n2 fixation. under mild water-stressed conditions (fc2), sa ameliorated plant growth, nodulation, soil and fertilizer n uptake in addition to fixed n2. under optimal watering conditions (fc3), the beneficial effect of sa was on nodulation and n2 fixation. overall, sa application may be considered as an important agricultural practice for the symbiotic performance (i.e., nodulation and n2 fixation) in water stressed as well as in well-watered chickpeas plants. acknowledgements we would like to thank professor i. othman, director general of aecs, 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of crop, soil and pest management technology, rufus giwa polytechnic, p.m.b. 1019, owo, ondo state, nigeria. 2 insect chemical ecology laboratory, institute of bioresources and sustainable development, department of biotechnology, government of india, takyelpat, imphal 795001, manipur, india. key words: bryscocarpus coccineus, calotrophus procera, canarium schweinfurthii, insecticidal activity, phytochemical screening. abstract: foliar application of 25% w/v crude aqueous extracts of calotrophus procera (aiton) w.t. aiton, canarium schweinfurthii (engl.) and bryscocarpus coccineus (schum. & thonn.) were evaluated for their insecticidal activity in reducing podagrica infestation on okra. results showed that the plants extracts significantly suppress podagrica spp. infestation and protect okra plant from severe leaves defoliation, with c. schweinfurthii (21.67 and 20.14) and b. coccineus (23.07 and 24.55) showing promising insecticidal activity for both cropping seasons. the yield attributes from okra sprayed with lambda cyhalothrin did not differ significantly compared to those sprayed with botanical insecticide despite having highest yield attributes. qualitative phytochemical screening revealed the presence of triterpenoids, steroids, flavonoids, phlobatanins, saponins, tannins, cardiac glycoside and anthraquinones. alkaloids and anthraquinones were not detected in c. procera and c. schweinfurthii while triterpenoids and phlobatanins were absent in c. schweinfurthii. the presence of these phytochemicals indicates that the plants possess insecticidal properties responsible for significant reduction in podagrica spp. infestation, severity of leaves damaged and improved okra yields. performance of the treatments is rated in the following order: lambda cyhalothrin > b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means in all the parameters evaluated. in light of the foregoing, crude extracts of b. coccineus and c. schweinfurthii could be utilized as suitable alternative to synthetic insecticide in sustainable okra production. 1. introduction okra (abelmoschus esculentus l. moench) is an important fruit veg(*) corresponding author: moboladesina@yahoo.com citation: adesina j.m., rajashekar y., 2018 phytochemical composition and insecticidal potentials of some plant aqueous extracts in suppressing podagrica spp. (coleoptera: chysomelidae) infestation on okra (abelmoschus esculentus l. moench) adv. hort. sci., 32(1): 7178 copyright: © 2018 adesina j.m., rajashekar y. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 september 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 71-78 72 etable crop in the diets of most people in the tropics and subtropical countries. it is grown mainly for its freshly immature pods and ranks fourth worldwide after pepper, tomato and onion on the basis of land area, production and value. despite the great demand for okra due to its nutritional and economic importance, okra production is being hampered by array of insect pest infestation resulting in poor yield and low market value. different growth stages of okra are generally attacked by different insect pests (adesina and afolabi, 2014; kedar et al., 2014) and podagrica species (coleoptera: chrysomelidae) is one of the most prevalent and damaging insect pests considered as major constraint to cultivation of okra which defoliate or damage the plant leaves and flowers; thereby results in reduction of the photosynthetic capability of the crop (odebiyi, 1980). the insects equally act as vectors in transmitting mosaic viral diseases and ultimately reduce yield (fasunwon and banjo, 2010), mainly if control measures are not undertaken. important yield losses of 20-50% are reported in nigerian and ghana (obeng-ofori and sackey, 2003; ahmed et al., 2007; fajinmi and fajinmi, 2010). currently, synthetic insecticide is being used across the globe to control agriculturally important insect pests due to their quick action and lasting effect (alao et al., 2011). the failure of synthetic insecticides to ensure total insect pest control due to development of resistant and resurgence and couple with increasing concern over environmental pollution, carcinogenic effect and destruction of beneficiary insects (isman, 2008), level of pesticide residues in food had compelled the scientific community to explore the abandoned and neglected traditional and indigenous products that are cheap, environmental safe and easily biodegradable products as alternatives to synthetic insecticides for tackling agricultural insect pests (antonio, 2009; alao et al., 2011; aetiba and osekre, 2016). presently, in many parts of the world, attention has been focused on the utilization of plant products that possess both medicinal and aromatic properties which are abound in various agro-ecological zones of the world as novel chemotherapeutants in plant protection (dubey et al., 2010). the popularity of botanical pesticides is once again increasing and some plant products either in crude form or by processing into different formulations are being used globally as green pesticides (dubey et al., 2008). the shortcomings associated with the use of synthetic chemicals, necessitated the idea of developing effective, cheap and easily biodegradable alternative products. therefore, this study aims to screen and report the efficacy of aqueous extracts of calotrophus procera (ait.) ait., canarium schweinfurthii (engl.) and bryscocarpus coccineus (shum and thonn.) in suppressing flea beetles podagrica spp. (coleoptera: chrysomelidae) infestation on okra. 2. materials and methods experimental location, designs and treatments the study was carried out on 168 m2 area of land manually cleared and prepared at the teaching and research farm, rufus giwa polytechnic (rugipo), owo, ondo state located in south western nigeria and lies on latitude 7° 12’ n and longitude 7° 35’ e. the experiment was laid out in a randomized complete block design (rcbd) with three replications. the experimental land was divided into 4 blocks of 4x4 m to produce a total of 12 plots. the plot size was 2x1.5 m with 0.5 m between plots and 1x1 m between blocks to prevent pesticide drift and inter-plot interference, respectively. an early maturity okra cultivar (nh-47) obtained from ondo state agricultural development project (adp), akure, ondo state, nigeria was sown with two to three seeds per hole at a depth of 2-3 cm and spaced 50x60 cm and later thinned to one plant per stand. weeding was carried out manually as at when due to ensure clean plots free from weed competition. the treatments applied were 25% w/v b. coccineus, c. schweinfurthii, c. procera and 0.8 l/ha synthetic insecticide (lambda cyhalothrin) as control. application of treatments commenced four weeks after planting (wap) with a 2-litre capacity hand-held sprayer. the spraying was carried out weekly till fruiting early in the morning to avoid photo decomposition and drifting of the extracts. synthetic insecticide was applied every two weeks. collection and preparation of plant extracts leaves of b. coccineus and c. schweinfurthii were collected from owo, ondo state, nigeria and c. procera were collected from ogbagi akoko ondo state, nigeria (7° 35’ n, 5° 43’ e) at full blooming and 25 kg were weighed separately using electronic balance (table 1). thereafter, the weighed plants were washed with water to remove dirt or other contaminants and each of the plant materials was pounded separately in a mortar into a fine soft paste form. the adesina and rajashekar plant aqueous extracts against podagrica spp. 73 paste (crushed plant leaves) was then put into a 10litre plastic bucket and the appropriate volume of distilled water added to make a 25% w/v crude aqueous extract solution (25% w/v represents an extract made with 25 g of crushed plant leaves per every 100 ml of water). the pastes were soaked overnight (approximately 14 hours) in a covered bucket with occasional stirring and filtered using muslin cloth with the filtrates stored in a 10 l keg in a cool dry place till use. phytochemical screening the qualitative phytochemical tests were performed on the crude aqueous extract to detect the presence of bioactive secondary metabolites in tested plant materials using standard laboratory methods following the procedure described by odebiyi and sofowora, 1978; sofowora, 1982; williamson et al., 1996; banso and ngbede, 2006; ngbede et al., 2008. data collection and statistical analysis all data were collected from five plants randomly tagged per treatment in the two middle rows of each plot. leaves damage index was determined by visual counting of the number of holes caused by the insect feeding activities. while insect count for estimation of the population densities of p. uniformis and p. sjostedti was made 4 wap between the hours 6-7 am when the insects are still inactive through visual counting. the fruits were harvested two months after planting (map), when the fruits were still fresh at the interval of 4 days. weighing balance was used to determine the weight of the fruits and the fruits length measured using ruler. data collected were analyzed by analysis of variance (anova) using gester version 1.2, insect count were subjected to square root (y =√) transformation before analysis to normalize the data. significant treatment means were compared using tukey test at 5% probability. 3. results podagrica spp. population before application of some plant aqueous extracts table 2 shows podagrica spp. population on okra before the application of the aqueous plant extracts treatments. the results showed that the insect populations were not significantly different. the population ranges between 4.07-4.93/plant for 2015 cropping season and 4.10-4.98/plant for 2016 cropping season. toxicity effect of some plant aqueous extracts in suppressing podagrica spp. infestation the utilization of plant aqueous extracts to suppress podagrica spp. infestation on okra was presented in tables 3 and 4 for 2015 and 2016 cropping season, respectively. application of the various treatscientific name common name family part used calotropis procera giant swallow wort or milkweed asclepiadaceae leaves canarium schweinfurthii african elemi or canarium burseraceae leaves byrsocarpus coccineus short-pod connaraceae leaves table 1 plant materials used for the experiment table 2 podagrica spp. population before application of some plant aqueous extracts treatments insect population 2015 2016 c. procera 4.07±0.77 a 4.98±0.15 a c. schweinfurthii 4.08±0.66 a 4.46±0.34 a b. coccineus 4.27±0.38 a 4.10±0.07 a lambda cyhalothrin 4.93±0.66 a 4.47±0.28 a treatments with the same letter in column are not statistically significant different from each other. treatments 4 wap 5 wap 6 wap 7 wap 8 wap 9 wap 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das c. procera 2.93±1.86 a 0.33±0.31 ab 0.80±0.60 ab 0.87±1.17 a 1.80±1.51 a 3.27±1.36 b 1.33±1.03 a 0.67±0.12 a 0.20±0.20 b 0.60±0.35 a 0.33±0.31 a 0.80±0.31 ab c. schweinfurthii 1.13±1.14 ab 0.07±0.12 c 0.27±0.23 c 0.07±0.12 b 0.37±0.35 b 1.13±0.70 a 0.27±0.35 ab 0.20±0.35 a 0.00 0.53±0.61 a 0.18±0.12 a 0.73±0.51 ab b. coccineus 1.40±1.22 ab 0.6±0.87 a 1.0±1.40 a 0.40±0.53 a 0.22±0.06 b 0.47±0.53 a 0.4±0.69 ab 0.80±1.22 a 0.82±1.05 a 0.6±0.53 a 0.8±1.22 a 0.93±0.52 a lambda cyhalothrin 0.13±0.23 c 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00a 0.00 table 3 toxicity effect some plant aqueous extracts in suppressing podagrica spp. infestation 2015 wap = weeks after planting; das = days after spray. treatments with the same letters in columns are not statistically significant different from each other. adv. hort. sci., 2018 32(1): 71-78 74 ments resulted in significant reduction in the insect population, while synthetic insecticides completely suppressed the insect pest infestation. the results clearly indicate that insect population reduced in relation to the number of spraying regimes, with the exception of okra plants sprayed with c. procera aqueous extract, which slightly increased or resurgence at 6 wap for both seasons and 3 days after spraying (das) at 7 wap in 2015 cropping season only. in 2015 nonsignificant difference was observed at 3 das and 7 das in all the treatments at 7, 8 and 9 wap, while in 2016 non-significant difference was recorded 7 das at 6, 8 and 9 wap in 2016 cropping season, respectively. the insecticidal activity of the aqueous plant extracts treatments is rated in the following decreasing order: b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means which is not significantly different in most instances but significantly differ compared to c. procera in both seasons. toxicity effect of some plant aqueous extracts on okra yield attributes the yield of okra plant in response to various plant aqueous extracts evaluated in reducing podagrica spp. infestation was presented in table 5. from the results, fruit weight and fruit diameter were not significantly different in both cropping seasons for all the treatments sprayed on the okra against podagrica spp. infestation. also non-significant difference equally observed between okra plants sprayed with b. coccineus and c. schweinfurthii compared to lambda cyhalothrin for number of harvested fruit and fruit lengths for both seasons except for c. schweinfurthii in 2015 season. although, there was significant difference in the number of harvested fruits and fruit lengths between okra sprayed with b. coccineus , c. schweinfurthii and lambda cyhalothrin compared to c. procera in both seasons. the yield attribute performance of the treatments is rated in the following decreasing order: lambda cyhalothrin > b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means. effects of plants aqueous extracts on severity of leaves damage on okra by podagrica spp. the utilization of plants aqueous extract on severity of leaves damage on okra (a. esculentus l.) by podagrica spp. was presented in table 6. significant table 5 toxicity effect some plant aqueous extracts okra yield attributes treatments 4 wap 5 wap 6 wap 7wap 8 wap 9 wap 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das c. procera 2.05±0.62 a 0.73±0.31 a 0.98±0.56 a 0.53±1.04 a 1.18±1.51 a 1.02±0.16 a 0.88±1.06 a 0.63±0.14 b 0.42±0.20 b 0.6±0.35 a 0.55±0.24 a 0.56±0.31 a c. schweinfurthii 1.89±0.14 ab 0.28±1.52 b 0.67±0.23 a 0.18±0.32 a 0.37±0.35 ab 0.63±1.08 a 0.41±0.05 ab 0.4±0.33 a 0.20±0.07 b 0.53±0.61 a 0.17±0.16 b 0.41±1.61 a b. coccineus 1.44±0.21 ab 0.92±0.87 a 0.82±1.03 a 0.46±1.07 a 0.08±0.11 b 0.82±0.12 a 0.2±0.09 ab 0.87±0.22 a 1.0±0.35 a 0.6±0.53 a 0.46±1.42 a 0.57±0.25 a lambda cyhalothrin 0.10±0.23 c 0.22±1.61 b 0.00 0.00 0.00 0.00 0.00 0.00a 0.00 12:00 0.00 0.00 wap = weeks after planting; das = days after spray. treatments with the same letters in columns are not statistically significant different from each other. table 4 toxicity effect some plant aqueous extracts in suppressing podagrica spp. infestation 2016 parameters c. procera c. schweinfurthii b. coccineus lambda cyhalothrin 2015 2016 2015 2016 2015 2016 2015 2016 number of pod 3.75±1.13 b 3.98 ±0.19 b 4.69±1.59 a 4.51±1.79 a 4.75±1.79 a 4.93±2.41 a 5.12±0.84 a 5.45±1.79 a fruit length 3.02±0.88 ab 3.47±1.66 ab 3.93±2.71 ab 4.00±1.34 a 4.12±1.76 a 4.70±0.28 a 4.83±2.02 a 4.67±1.17 a fruit weight 0.08±0.06 a 0.05±0.02 a 0.05±0.03 a 0.09±0.11 a 0.05±0.06 a 0.07±0.12 a 0.04±0.03 a 0.06±0.02 a diameter 5.51±1.89 a 4.80±0.43 a 4.9±1.45 a 4.64±0.93 a 4.44±1.72 a 4.03±1.06 a 4.72±0.69 a 4.46±0.82 a severity of leaf damage c. procera c. schweinfurthii b. coccineus lambda cyhalothrin 2015 2016 2015 2016 2015 2016 2015 2016 4 wap 3.47±1.08 e 4.51±0.71 e 4.73±0.43 e 4.02±0.66 b 3.40 ±1.83 c 3.27±1.10 d 1.67±0.76 a 1.44 ±0.83 a 5 wap 6.27±0.73 e 8.12±2.46 e 5.82±1.18 e 6.83±0.37 b 4.00±1.95 c 4.17±1.38 d 3.20 ±1.51 a 4.27 ±0.73 a 6 wap 12.23±1.09 d 12. 85±2.03 d 9.50±1.76 d 8.91±1.92 b 10.53±1.68 b 11.48±2.06 c 4.07 ±0.69 a 4.23 ±1.09 a 7 wap 19.93±1.93 c 20.14±1.17 c 11.97±0.86 c 13.25±1.81 ab 16.27 ±2.68 ab 17.04±1.39 b 4.07 ±0.39 a 4.93 ±2.93 a 8 wap 29.60±1.55 b 26.92±2.41 b 15.53±1.78 b 17.09±2.01 a 20.67±2.61 a 19.85±2.14 ab 4.80 ±1.70 a 5.60 ±1.55 a 9 wap 32.20±0.66 a 33.32±1.82 a 21.67±2.90 a 20.14±1.26 a 23.07±1.32 a 24.55±2.82 a 5.20 ±1.30 a 6.33 ±1.75 a table 6 effects of plants aqueous extracts on severity of leaves damage on okra by podagrica spp. treatments with the same letters in columns are not statistically significant different from each other. treatments with the same letters in columns are not statistically significant different from each other. adesina and rajashekar plant aqueous extracts against podagrica spp. 75 difference was not recorded between okra plant sprayed with the extracts at 4, 5, 6, 8 and 9 wap. however, there was significant difference in the severity of leaves recorded at 7 wap. significant difference was also recorded between c. procera and chemical insect controlled okra but c. schweinfurthii and b. coccineus were not statistically significant from each other. the highest severity was recorded at 9 wap followed by 7 wap which could be due to lack of rainfall in these periods. severity of leaves damage by podagrica spp. is presented in table 6. the least percentage leaves defoliation was observed from the plants treated with synthetic insecticide and c. procera treated plant recorded highest percentage of defoliation among the botanical insecticides. c. schweinfurthii and b. coccineus showed significantly promising effect in reducing severity of leaves damage. however, all botanical treated okra plants prevented the leaves from being severely defoliated as all the plants suffered below 33.32% defoliation at 9 wap. in general term, leaves damage increased with the increasing age of okra plants. phytochemical screening the result of phytochemical screening of crude leaf extracts revealed the presence of triterpenoids, steroids, flavonoids, phlobatanins, saponins, tannins, cardiac glycoside and anthraquinones in the plants (table 7). alkaloids and anthraquinones were not detected in both c. procera and c. schweinfurthii while triterpenoids and phlobatanins were absent in c. schweinfurthii. the presence of these bioactive compounds in the plant materials is an indication that it may have some insecticidal potential against agriculturally important insect pest. 4. discussion and conclusions plants are rich sources of natural substances that can be utilized in the development of environmentally safe methods for insect pests control as veritable alternative to synthetic insecticide (sadek, 2003). moreover, growing awareness of health and environmental issues accompanied by the intensive use of chemical inputs has led to increased concerns and the need for alternate forms of crop protection in the world (adesina, 2013). the crude aqueous extracts evaluated in this study have been reported to contain insecticidal, termiticidal, antifeedant, ovacidal and larvacidal properties (ahmed, 1993; shaaya et al., 1977; david, 1989; jahan et al., 1991; abayeh et al., 1999; abbassi et al., 2003; umsalama et al., 2006; bakavathiappan et al., 2012; katunku et al., 2014; nagawa et al., 2015; okoli et al., 2016). the result of the study showed that the crude plant extracts were able to reduce insect population, but not significantly, compared to the synthetic insecticide, but were able to show some suppressing effect on the rate of feeding of the insects as the severity of the leaves damaged by the insects was not enough to cause significant reduction in yield compared to results obtained from okra plants sprayed with chemical. the target insects are notorious for leaves defoliation owing to their biting and chewing mouthparts resulting in reduction of photosynthetic ability of the infested plants which invariably affect the plant growth and yield (dent, 1999). the aqueous extracts were not effective at first two weeks of application. synthetic insecticide recorded a higher efficacy compared to aqueous extract. this might be as a result of active ingredients of aqueous extracts being easily volatilized especially in the sun, thereby leading to their limited efficacy (ware, 2000) and the delayed effect of aqueous extracts is reported to be one of the major problems of botanical insecticides (oparaeke, 2006; isman, 2008). since the beetles live and feed on the vegetative parts, any chemical that shows a remarkable efficacy against them must have contact toxicity, repellent or anti-feeding action. several authors have reported that, the deleterious effects of crude plant extracts on insects are manifested in several ways, including toxicity (hiremath et al., 1997) and feeding inhibition (klepzig and schlyter, 1999; wheeler and isman, 2001). thus, reducing the level of beetle infestation and increasing okra pod carrying capacity; this confirmed the earlier report of thirumalai et al. (2003)+ = present, = absent. table 7 phytochemical screening of crude aqueous extract of c. procera, c. schweinfurthii and b. coccineus chemical constituents calotrophus procera canarium schweinfurthii bryscocarpus coccineus alkaloids + terpenoids + + flavonoids + + + anthraquinones + tannins + + + phlobatanins + + saponins + + + cardiac glycosides + + + steroids + + + adv. hort. sci., 2018 32(1): 71-78 76 who observed the effective reduction of mite population with application of neem seed kernel extract. the reduction in podagrica beetle population and increase in yield caused by the plant extracts treatment was comparable with those caused by synthetic insecticide. this supports the findings of katunku et al. (2014) and those of yusuf and mohammed (2009) who reported that c. schweinfurthii and monordica balsamina powder treatments were as effective as and comparable to permethrin and pirimiphos methyl in suppressing c. maculatus population and growth in cowpea storage. the high rate of podagrica spp. population reduction on exposure to the aqueous extracts treatments may be attributed to the chemical composition of the products. plant extracts often consist of complex mixtures of bioactive constituents which may act as antifeedants, disturb insect growth, development and inhibit oviposition (gerard and ruf, 1991; emimal victoria, 2010). thus, suggesting that most of the secondary metabolites such as terpenoids and alkaloids can be used as active insecticidal compounds that could be an effective alternative to synthetic insecticides for insect pest management. the toxicity of alkaloids was reported by abbassi et al. (2003) and david (1989) reported that tannic acid acts as toxin and feeding deterrent to insects. bernhoft (2010) reported that saponnins is found to affect the respiratory system of insects and causes emetic effect due to their detergent action. in the same vein, philip et al. (2009) reported that plant products have repellent properties and toxic effect on the heart muscles in insects. frazier (1986) reported that antifeedants can be found amongst all major classes of secondary metabolites (alkaloids, flavonoids, terpenoids and phenolics). plant metabolites may produce toxic effects if ingested leading to rejection of the host plant (russel and lane, 1993). the utilization of c. schweinfurthii and b. coccineus aqueous extracts had positive toxicity impact in decreasing the rate of podagrica spp. infestation and also increased the yield of okra plant thus could serve as alternative to synthetic insecticide to protect okra plant against podagrica spp. infestation to maximize the yield and income of the resource poor farmers. acknowledgements the authors are grateful to omowaye, iyanuoluwa daniel and umaru, muritala oluwateru of horticultural technology programme, department of crop, soil and pest management technology, rufus giwa polytechnic, owo, ondo state, nigeria for the support received from them in collecting the plant materials used for the study. references abayeh o.j., abdulrazaq a.k., olaogun r., 1999 quality characteristics of canarium schweinfurthii engl. oil. plant foods hum. nutr., 54(1): 43-48. abbassi k., atay-kadiri z., ghaout s., 2003 biological effects of alkaloids extracted from three plants of moroccan arid areas on the desert locust. physiol. entomol., 28: 232-236. adesina j.m., 2013 exploration of erythrina excelsa baker and aneilema beniniense (p. beauv.) kunth aqueous extracts for the management of flea beetles (podagrica spp) on okra (abelmoschus esculentus). nat. sci., 11(12): 210-215. adesina j.m., afolabi l.a., 2014 comparative bio-efficacy of aqueous extracts of loncarpous cyanescens and trema orientalis against flea beetle (podagrica spp.) (coleoptera: chrysomelidae) infestation and yield of okra. int. j. hortic., 4(2): 4-9. aetiba j.p.n., osekre e.a., 2016 management of insect pests of okra (abelmoschus 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of trichilia americana extract against the larvae of spodoptera litura. entomol. exp. appl., 98(1): 9-16. williamson e.m., okpako d.g., evan f.j., 1996 pharmacological methods in phytotherapy research. vol. 1. selection, preparation and pharmacological evaluation of plant materials. john wiley and sons ltd. chichester, uk, pp. 9-13. yusuf s.r., mohammed y.s., 2009 potentials of bitter mellon monordica balsamina (l.) and asthma plant [mitracarpus villosus (l.) walp] against cowpea bruchid (callosobruchus maculatus (f.) damage. savanah j. agric., 4: 54-61. impaginato 441 adv. hort. sci., 2020 34(4): 441­448 doi: 10.13128/ahsc­7725 variations in tree and fruit characteris­ tics revealed potential dwarfing geno­ types within iran’s pomegranate germplasm a.a. ghasemi soloklui 1, a. gharaghani 1, 2 (*), a. sarkhosh 3 1 department of horticultural sciences, faculty of agricultural sciences, shiraz university, shiraz, iran. 2 drought research centre, faculty of agricultural sciences, shiraz university, shiraz, iran. 3 horticultural sciences department, university of florida, gainesville, fl 32611, florida, usa. key words: internodes length, rootstocks, stomatal density, sucker. abstract: this study aimed to explore diversity in dwarfing tendencies, deter­ mine the correlation of measured traits with dwarfing, and identify and select promising dwarf candidates as potential scions or rootstock cultivars. growth habit, vegetative attributes, fruit physicochemical characteristics,and leaf stom­ atal densityof 19 iranian pomegranate cultivars, which have been collected across the country and established in the yazd pomegranate germplasm, were assessed. results showed that the cultivars differed in almost all measuredtraits. the tree height and canopy width, current year’s shoot, and internode length were within the range of 1.97­4.6 m, 1.53­4 m, 15­41.5 cm 1.96­3.39 cm, respec­ tively. moreover, a positive correlation was observed between tree height and internode length (r= 0.55), whereas a negative correlation was obtained between stomatal density and tree height (r= ­0.44). based on characteristics measured for the vegetative growth, ‘malas no. 1 saravan’ and ‘torosh nar riz zirab’ proved dwarfing habit. ‘rabab poost ghermez neyriz’,a commercial culti­ var, showed semi­dwarfing growth and ‘khajei ghasrodasht fars’, ‘shahsavar seydan marvdasht’, ‘bihaste ravar’, ‘bihaste sangan khash’, ‘torosh goli naz behshahr’ and ‘anar siah’ resulted in vigorous trees. this preliminary study found promising dwarf and semi­dwarf genotypes at iran’s pomegranate germplasm. 1. introduction according to historical documents, pomegranates originated in central asia, especially in parts of iran, and believed to have spread to nearby areas due to traveling and incursion (harlan, 1975; levin, 1994; verma et al., 2010). the main iranian collection of pomegranate in yazd contains 762 accessions, including wild, semi­wild, and commercial types (behzadi shahrbabaki, 1998; zamani et al., 2007). these diverse and valuable (*) corresponding author: agharghani@shirazu.ac.ir citation: ghasemi soloklui a.a., gharaghani a., sarkhosh a., 2020 ­ variations in tree and fruit characteristics revealed potential dwarfing genotypes within iran’s pomegranate germplasm. adv. hort. sci., 34(4): 441­448 copyright: © 2020 ghasemi soloklui a.a., gharaghani a., sarkhosh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 december 2019 accepted for publication 30 november 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 441­448 442 genetic resources would benefit from a further genetic improvement to develop dwarfing scion and rootstock genotypes to establish modern high­density pomegranate orchards. ingels et al. (2002) suggested that the term ‘dwarf tree’ applies to a tree that appears smaller than usual owing to selection of dwarf genotypes, specific training or pruning meth­ ods, or grafting on dwarfing rootstocks. besides, in classifying trees according to size, dwarf trees are approximately 2.5 m or less when they mature (castle, 1992). dwarf trees have many benefits com­ pared to larger vigorous ones, such as being able to be spaced closer together without suffering from excessive crowding or the need for frequent, severe pruning. moreover, dwarf trees allow for ease of pruning, pest control, fruit thinning, spraying, harvest­ ing, and increased production of high­grade fruit, higher fruit quality and decreased production costs (tukey, 1964). the advantages of dwarf and semi­ dwarf genotype trees have been demonstrated in the fruit industry resulting in the widespread use of dwarfing rootstocks in tree crops such as apple (looney and lane, 1983), cashew (moura, 2001), and peach (dejong et al., 2005). lately, breeding efforts have resulted in the selection of dwarfing rootstock or scion cultivars in almost all temperate and tropical fruit tree crops (busov et al., 2003). the primary iden­ tification of dwarf genotypes should be based on field evaluations of fruit tree cultivars and genotypes. genetic dwarfism often manifests itself in distinctive morphological characteristics, easily identified, and often appears to a casual observer (castle, 1992). analysis of morphological diversity as tree height, canopy shape, internode, and branching pattern is a useful method for detecting dwarfing phenotypes in a population of many genotypes. although some pome­ granate cultivars, such as nana, are considered dwarf­ ing (terakami et al., 2007), these are ornamentals, and there are no published reports on new dwarfing pomegranates. the objectives of this study were: 1) evaluate tree growth habits, vegetative and fruit char­ acteristics of 19 iranian pomegranate cultivars, 2) to examine diversity in dwarfing potential, 3) determine inter­correlations among measured traits with dwarfism, and 4) identify promising candidates with dwarfing potential as pomegranate scions and root­ stocks. 2. materials and methods plant materials the experiment was conducted in 2015, at the agriculture and natural resources research centre table 1 ­ name, origin, fruit and tree characteristics of pomegranate cultivars used in this study cultivars provinces cities skin color aril color taste uses anar siah esfahan esfahan black dark red sweet medicinal bihaste ravar kerman ravar yellow­pink white sweet local bihaste sangan khash sistan baluchistan khash white white sweet local jangali poost ghermez roodbar gilan roodbar red red sweet­sour local khajei ghasrodasht fars fars shiraz pink white sweet local malas pishva varamin tehran varamin yellow white sweet local malasyazdi yazd yazd red red sweet­sour commercial makhmal malas shahreza esfahan shahreza red red sweet­sour local malas no. 1 saravan sistan baluchistan saravan yellow white sweet­sour local poost nazok torosh abarkuh yazd abarkuh red red sweet­sour local poost sefid dezfoul khuzestan dezfoul yellow­white white sweet­sour local rabab poost ghermez neyriz fars neyriz red red sweet­sour commercial rabab poost ghermez kazeroon fars kazeroon red pink sweet­sour local sefid biardal borujen chahar mahal­e bakhtiar borujen yellwo pink sour local shirin jangal sisangan mazandaran sisangan red­yellow pink sweet­sour local shirin semnan semnan semnan green­yellow white sweet local shahsavar seydan marvdasht fars marvdasht white­yellow white sweet­ local torosh goli naz behshahr mazandaran behshahr white­yellow white sour local torosh nar riz zirab fars darab green­yellow white very sour wild ghasemi soloklui et al. ‐ potential dwarfing within iran’s pomegranate germplasm 443 (anrrc), yazd province, iran. nineteen pomegranate cultivars were used in the study (table 1). two of the cultivars, ‘rabab poost ghermez neyriz’ and ‘malas yazdi’, are commercial cultivars widely cultivated in the country. the rest are of local importance in dif­ ferent provinces, except ‘torosh nar riz zirab’, a non­commercial (semi­wild type) from the darab region. the pomegranate cultivars have been planted in a randomized complete block design (rcbd) with three replications per cultivar. trees were 26­year­ old at the time of the experiment and managed fol­ lowing the region’s recommended orchard practices. measurement of vegetative characteristics a total of 6 trees per cultivar (3 trees × 3 repli­ cates) was used for vegetative traits measurements. since the pomegranate trees are trained to the multi­ ple trunk (3­4 trunks) system (the common practice in iran’s commercial orchards), individual trunks diameters were measured at 30 cm above the soil surface and then averaged to get the value for the single trunk diameter. bark thickness was measured in a small­detached section of bark at 30 cm above the soil surface. tree height (from the ground level up to the tree peak) and canopy width (in the widest point). numbers of suckers were simply counted on trees and for shoot angle (o), the insertion of shoots that came directly from the scaffold were measured. in addition, the current year’s shoot length (cm) was evaluated after shoot growth stopped (in november) on three scaffolds of trees. moreover, internode length (cm) was calculated by dividing the current year’s shoot length by its corresponded node num­ ber. measurement of fruit quality attributes physical properties. at harvest (which was varied for each cultivar), measurements of fruit physical properties were done on 90 randomly selected fruits per cultivar (10 fruits per tree × 3 trees per replicate × 3 replicate = 90 fruits for each cultivar). fruits were weighed using a digital balance. peel and arils were carefully separated manually from the fruit to mea­ sure the edible portion. the extracted arils were col­ lected in a tray and mixed thoroughly to assure uni formity.the edible portion of the fruit was deter­ mined using the following formula (ghasemi soloklui et al., 2019): edible portion of fruit (%)= fresh weight ­ peel weight ­ capillary membranes x 100 fruit weight chemicals properties. at harvest, total soluble solids content (tss) and total acidity (ta) were mea­ sured in juice extracted from 90 fruits per cultivar (10 fruits per tree × 3 trees per replicate × 3 replicates = 90 fruits for each cultivar). tss (in °brix) was deter­ mined using a digital refractometer (model pr­1, atago, japan) and ta by titration to ph end­point 8.2 with 0.1 n naoh and expressed as citric acid equiva­ lent (g cae100 ml­1) (horwitz, 1980). measurement of stomatal density in late summer, fully expanded leaves (15 leaves per replication) were collected randomly from the midpoint of the current season’s shoots. stomata numbers were determined using the replica method (soleimani et al., 2002). the stellate hairs were removed from the lower surface of each leaf using an adhesive tape. a thin film of cellulose acetate was painted directly onto the lower epidermis of the leaf. the cellulose acetate was allowed to dry at room temperature before being peeled from the leaf. sections were taken from the middle of each leaf. the slides were coded, and a binocular microscope was used for stomatal counts at x 40 magnification. stomatal density was counted in a field area of one mm2. statistical analysis analysis of variances (anova) was performed using sas version 9.1 (sas, 2003.). the means were carried out at p<0.05 using duncan’s multiple range tests. correlation between pairs of traits was deter­ mined using pearson’s correlation coefficient. 3. results vegetative characteristics measured vegetative traits are presented in table 2 and figure 1. tree height varied between 1.97 to 4.6 m (fig. 1). the highest tree height value was observed in ‘khajei ghasrodasht fars’ (4.6 m), fol­ lowed by ‘bihaste ravar’ (4.3 m), ‘bihaste sangan khash’ (4.26 m), and ‘shahsavar seydan marvdasht’ (4.26 m). ‘malas no. 1 saravan’ showed the smallest tree height (1.97 m) (fig. 2), some cultivars such as ‘rabab poost ghermez neyriz’ and ‘shirin semnan’ categorized as medium height (fig. 1). among the 19 cultivars, the greatest canopy width (4 m) was observed in ‘shahsavar seydan marvdasht’, while ‘makhmal malas shahreza’showed the smallest canopy width (1.50 m) (fig. 1). ‘khajei adv. hort. sci., 2020 34(4): 441­448 444 ghasrodasht fars’ had the greatest single trunk diam­ eter (121.33 mm), whereas ‘malas no.1 saravan’, had the lowest single trunk diameter (61 mm) (table 2). the longest current year’s shoot length (41.50 cm) was recorded in ‘poost nazok torosh abarkuh’, while the shortest (15 cm) obtained in ‘poost sefid dezfoul’ (table 2). internode length varied from 1.96 cm (‘malas no. 1 saravan’) to 3.39 cm (‘khajei ghasrodasht fars’) (table 2). the correlation between internode length and tree height was also statistically significant (r=0.55; p=0.0001) (fig. 3). shoot angle was within the range of 46.6 to 90 (table 2). significant differences were observed in the num­ ber of suckers among the pomegranate cultivars. the highest number of suckers per tree was recorded in ‘sefid biardal borujen’ (215), while the lowest num­ ber was counted in ‘rabab poost ghermez kazeroon’. in addition, the highest (3.24 mm) and lowest (1.55 mm) bark thickness were recorded in ‘poost nazok torosh abarkuh’ and ‘sefid biardal borujen’, respec­ tively (table 2). considering all vegetative characteristics, in par­ ticular, tree height, trunk diameter and internode length; cultivars could be classified into four groups, fig. 1 ­ tree height and canopy width in 19 iranian pomegranate cultivars. similar letters indicate non­significant differen­ ces among cultivars (p≤0.05). fig. 2 ­ comparison of tree height between a vigorous pomegra­ nate cultivar (a) and ‘malas no. 1 saravan’ (b), the most dwarf cultivar in the pomegranate genotypes studied. table 2 ­ vegetative tree characteristics of the pomegranate cultivars used in the experiment similar letters in each column indicate non­significant differences among cultivars at p≤0.05. cultivars trunk diameter (mm) current year shoot length (cm) internode length (cm) shoot angle (o) number of suckers bark thickness (mm) anar siah 114 ab 21 bcdefg 3.11 ab 57 defg 18 gh 2.03 b bihaste ravar 98 ab 24.16 b 3.17 ab 60.16 defg 55 bc 2.60 b bihaste sangan khash 99 ab 17 defg 2.83 abc 53.33 fg 90 cd 2.22 b jangali poost ghermez roodbar 78 ab 15.66 fg 2.57 abc 63.33 defg 191 bcd 2.83 b khajei ghasrodasht fars 121.33 a 22 bcde 3.39 a 65 def 8.33 h 2.56 b malas pishva varamin 97.67 ab 16.33 efg 3.17 ab 65 def 161 abc 1.82 b malas yazdi 74.67 ab 23 bcd 2.14 c 46.66 g 70 efg 2.46 b makhmal malas shahreza 77 ab 26.50 b 2.05 c 86.66 ab 180 abc 1.77 b malas no. 1 saravan 61.0 b 17.6 cdefg 1.96 c 60 defg 22 gh 1.70 b poost nazok torosh abarkuh 93.83 ab 41.50 a 3.39 a 63.33 defg 70 efg 3.24 a poost sefid dezfoul 89 ab 15 g 2.39 bc 73.33 abcde 93 def 1.64 g rabab poost ghermez neyriz 110 ab 21 bcdefg 2.52 abc 56.66 efg 143 bcd 2.23 bcdefg rabab poost ghermez kazeroon 107.17 ab 16.33 efg 2.33 bc 83.33 abc 1 h 1.62 g sefid biardal borujen 111.33 ab 23.50 bc 2.61 abc 86.66 ab 215 a 1.55 g shirin jangal sisangan 73.76 ab 17.6 cdefg 2.01 c 68.33 cdef 89 def 2.10 cdefg shirin semnan 99 ab 16.83 defg 2.80 abc 75 abcd 126 def 2.92 ab shahsavar seydan marvdasht 114 ab 21.3 bcdef 3.17 ab 70 bcdef 35 fgh 2.64 abcd torosh goli naz behshahr 67.75 ab 17.6 cdefg 2.33 bc 90 a 35 fgh 2.40 bcdef torosh nar riz zirab 62.17 b 21.3 bcdef 2.52 abc 66.66 cdef 69 efg 2.42 bcdef ghasemi soloklui et al. ‐ potential dwarfing within iran’s pomegranate germplasm 445 1) vigorous cultivars: ‘khajei ghasrodasht fars’, ‘shahsavar seydan marvdasht’, ‘bihaste ravar’, ‘bihaste sangan khash’, ‘torosh goli naz behshahr’ and ‘anar siah’, 2) semi­vigorous cultivars: ‘malas pishva varamin’, ‘rabab poost ghermez kazeroon’, ‘poost nazok torosh abarkuh’, ‘jangali poost ghermez roodbar’, ‘sefid biardal borujen’, ‘malas yazdi’ and ‘shirin jangal sisangan’, 3) semi­dwarf cul­ tivars: ‘rabab poost ghermez neyriz’, ‘shirin semnan’, ‘makhmal malas shahreza’ and ‘poost sefid dezfoul’, and 4) dwarf cultivars: ‘torosh nar riz zirab’ and ‘malas no. 1 saravan’. stomata density as shown in figure 4, a large variation in stomatal density (from 46.91 to 108.91 stomata per mm2) was observed among studied cultivars. ‘shirin semnan’, showed the highest stomatal density, while ‘shahsavar seydan marvdasht’ had the lowest stom­ atal density. results of pearson correlation analysis provide significant negative correlations between stomatal density and tree height (r = ­0.44; p =0.0005) (fig. 5). fig. 3 ­ pearson’s correlation coefficients between internode length and tree height in 19 iranian pomegranate culti­ vars. fruit quality traits the highest fruit weight was perceived in ‘shahsavar seydan marvdasht’ (378.17 g), followed by ‘malas yazdi’ (230.83 g) and ‘jangali poost ghermez roodbar’ (214.67 g), while the smallest fruit (62.17 g) was observed in ‘torosh nar riz zirab’ (a semi­wild cultivar) (table 3). the percentage of the edible portion of the fruit ranged from 49.80 (in ‘torosh nar riz zirab’) to 71.98% (in ‘biardal borujen’) (table 3). the highest (18.7°brix) and low­ est (13.0°brix) tss was measured in ‘sefid biardal borujen’ and ‘shahsavar seydan marvdasht’, respec­ tively (table 3). fruit juice ph varied from 3.13 to 4.43 among the studied pomegranate cultivars, with the minimum and maximum ph measured respec­ tively in ‘torosh nar rizzirab’, and ‘anar siah’. moreover, the highest and lowest ta were observed in ‘torosh nar riz zirab’ (8.47 g cae 100 ml­1) and ‘bihaste sangan khash’ (0.50 g cae 100 ml­1), respec­ tively. 4. discussion and conclusions the results of this research present a wide range of vigor and dwarfing potential in iranian pomegran­ ates. in high­density orchards, controlling tree vigor and canopy size is important for enhancing the orchard efficiency and productivity (umar and sharma, 2008). vegetative growth can be defined by several parameters such as; total shoot length, internode length, number of terminals and lateral shoot, and trunk cross­sectional area (weibel et al., 2003). in classifying trees according to size, dwarf trees are approximately 2.5 m or less in height when mature (castle, 1992). considering the above men­fig. 4 ­ frequency distribution of stomatal density in 19 iranian pomegranate cultivars. bars indicate se (n=45). fig. 5 ­ pearson’s correlation coefficients between stomatal den­ sity and tree height in 19 iranian pomegranate cultivars. ous rootstock. the results of the current study also showed a wide variation in sucker production among cultivars. in this regard, cultivars such as ‘rabab poost ghermez kazeroon’ and ‘khajei ghasrodasht fars’ with the lowest number of suckers have the advantage of easy management and also may be suitable for preferred single trunk training system in modern fruit orchards. stomata are directly responsible for the trade­off between water loss and carbon acquisition (raven, 2002). stomatal density as a quantitative attribute is genetically determined (gailing et al., 2008). some plant species have been reported as possessing gen­ erally high heritability (i.e., less dependence on envi­ ronmental conditions) in their stomatal traits (sharma and dunn, 1969; orlovic et al., 1998). drogoudi et al. (2012) reported stomatal density among four pomegranate cultivars ranging from 68 to 149.9 stomata mm­2. also, meena et al. (2011) reported astomatal density of 130.67 stomata mm­2 for pomegranate. these results are following the findings of the current study, and minor differences in the results could be due to cultivar or climate dif­ ferences. interestingly, some of the vigorous cultivars such as ‘bihaste ravar’, ‘shahsavar seydan marvdasht’ and ‘bihaste sangan khash’ had low 446 adv. hort. sci., 2020 34(4): 441­448 tioned vegetative attributes and keeping in mind the castle (1992) scale, ‘malas no. 1 saravan’ and ‘torosh nar riz zirab’ were the most dwarf size, about half that of vigorous cultivars. thus, this culti­ var has potential to be used directly as dwarfing pomegranate rootstocks, although a more detailed study on propagation, graft compatibility, and toler­ ance to biotic and abiotic stress will shed more light on the suitability of these cultivars as rootstock. as a dwarfing source, these cultivars could be utilized as parents in breeding programs to develop superior dwarf scion and rootstock cultivars. this study demonstrated that internode length was associated with tree size. in general, some culti­ vars such as ‘makhmal malas shahreza’, ‘poost sefid dezfoul’ and ‘malas no. 1 saravan’ have the smallest tree sizes and shorter internodes lengths than other cultivars. the average internode length depended on the number of nodes per extension unit (costes and garcia­villanueva, 2007). dwarf trees usually pro­ duce very short internodes length, resulting in branches more compact than vigorous trees (ingels et al., 2002). obtained results for internode length are in agreement with those of murase et al. (1990), on peach trees grafted on dwarfing rootstocks that had shorter internodes than trees grafted on vigor­ table 3 ­ fruit characteristics of the pomegranate cultivars used in the experiment similar letters in each column indicate non­significant differences among cultivars at p≤0.05. cultivars fruit weight (g) edible portion (%) tss (°brix) ph ta (%) anar siah 124.0 ef 53.31 cd 13.8 hi 4.43 a 0.70 hi bihaste ravar 140.32 e 54.30 cd 14.0 fghi 4.06 cd 0.55 i bihaste sangan khash 156.0 de 60.39 abcd 13.83 ghi 4.38 ab 0.50 i jangali poost ghermez roodbar 214.67 b 61.88 abcd 18.33 ab 3.49 hij 2.17 efg khajei ghasrodasht fars 207.50 bc 59.53 abcd 14.66 efghi 3.65 gh 2.51 def malas pishva varamin 131.0 ef 67.81 ab 15.5 defgh 3.89 def 1.41 ghi malasyazdi 230.83 b 49.80 d 16.0 cdef 3.70 fgh 1.85 fg makhmal malas shahreza 133.50 ef 60.32 ab 17.66 abc 4.2 bc 0.6 i malas no. 1 saravan 125.83 ef 61.52 abcd 15.33 defgh 3.34 ijk 3.30 cd poost nazok torosh abarkuh 129.50 ef 62.66 abcd 16.0 cdef 3.49 hij 2.8 de poost sefid dezfoul 88.0 fg 57.94 bcd 16.66 bcde 4.16 bc 0.88 hi rabab poost ghermez neyriz 190.0 bcd 56.61 bcd 14.83 efghi 3.53 hi 2.57 def rabab poost ghermez kazeroon 161.67 cde 54.16 cd 15.8 cdefg 3.84 efg 1.55 k sefid biardal borujen 159.33 de 71.98 a 18.66 a 3.28 jk 3.97 c shirin jangal sisangan 192.67 bcd 55.10 bcd 15.5 defgh 4.04 cde 1.35 ghi shirin semnan 158.0 de 62.01 abcd 14.0 fghi 4.16 bc 0.70 hi shahsavar seydan marvdasht 378.17 a 56.52 bcd 13 i 4.16 sm 0.61 i torosh goli naz behshahr 126.83 ef 64.67 abc 17.16 abcd 3.14 k 5.47 b torosh nar riz zirab 62.17 g 60.77 abcd 16.66 bcde 3.13 k 8.47 a ghasemi soloklui et al. ‐ potential dwarfing within iran’s pomegranate germplasm 447 stomatal density (between 46 to 52 stomata mm­2), whereas dwarf and semi­dwarf cultivars, including ‘shirin semnan’, ‘torosh nar riz zirab’ and ‘poost sefid dezfoul’ possess very high stomatal density (108, 96 and 90 stomata mm­2, respectively). these results are in line with the findings of barrientos­ pérez and sanchez­colín (1982), who reported that stomatal density could be a good method to classify the growth habit in avocado trees (barrientos­pérez and sánchez­colín, 1982). thus, the data on fruit attributes would provide useful information for selecting the best dwarf culti­ vars to be used directly as scion cultivars on their root or as parent materials in scion cultivars breeding programs. the evaluation of pomegranate fruit quali­ ty (physical and chemical) in the local material has previously been carried out in iran (akbarpour et al., 2009), turkey (özkan, 2001), italy (barone et al., 2001), and greece (drogoudi et al., 2005). tehranifar et al. (2010) described important fruit traits of 20 pomegranate cultivars from different regions in iran. they found that the fruit weight, peel percentage, aril percentage, and juice percentage were within the range of 196.89­315.28 g, 32.28­59.82%, 37.59­65% and 26.95­46.55%, respectively, which are in line with the results of the current study. moreover, yıldız et al. (2003) reported that promising pomegranate genotypes, selected from hizan (bitlis) in turkey, had 192.3­388.3 g fruit weight, 28­55% juice percentage, 0.33­4.03% juice acidity and 10.0­17.0% juice soluble solids content. on the other hand, mars and marrakchi (1999) defined fruit characteristics of 30 pomegranate genotypes from tunisia. they reported fruit weights ranging from 196.1 to 673.6 g, ph from 2.9 to 4.6, soluble solid contents from 13.3 to 16.9°brix, and acidity from 0.2 to 3.1 g cae 100 ml­1. consequently, the pomegranate studied herein had many similarities to those described in other studies concerning fruit traits such as fruit weight, soluble solids content, ph and acidity. minor differences in these traits across the studies could arise from differ­ ent plant materials and varied climatic conditions. in this study edible portion was between 49.80 to 71.98%; whereas, al­maiman and ahmad (2002) reported an edible portion of about 55­60% of the total fruit weight. this study showed that most culti­ vars except ‘torosh nar riz zirab’ and ‘poost sefid dezfoul’ have big and medium sized fruits. in general, considering vegetative characteristics and fruit quality attributes, ‘rabab poost ghermez neyriz’ a commercial cultivar with semi­dwarfing growth habit and good fruit quality (have big fruits, with high tss contents and low acidity) is a promising candidate for establishing high­density orchards on its roots. moreover, some cultivars such as ‘shirin semnan’, ‘makhmal malas shahreza’ and ‘malas no. 1 saravan’, which categorized as dwarfing or semi­ dwarfing cultivars and possessed quite good fruit quality, have the potential to be used as a parent in breeding programs to develop dwarf pomegranate cultivars or dwarfing rootstocks. ‘torosh nar riz zirab’ is a semi­wild cultivar with small tree size (dwarf cultivar) but represents poor fruit quality attributes. thus, this cultivar can be considered as a dwarfing rootstock in pomegranate production. 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and research farm of agricultural faculty, university of jambi, indonesia, from april through to september 2019. the aim of this study was to investigate the effect of biostim­ ulants and media compositions on the growth and yield of chili pepper during restricted soil water content. the study was arranged in a split plot design with 3 replicates (groups). different types of biostimulants (citorin®, hantu®, and a control) were designated as main plot, whereas media compositions (2:2:1, 2:1:1, 1:2:1 and 1:1:2) made of soil+trichocompost+rice husk charcoal were employed as sub plot. at the time of transplanting, soil water content was set to approximately 75% of field capacity to create stress conditions. the results showed that the proper choice of biostimulant and medium composition could increase nutrient status, total sugar and chlorophyll contents, and reduce pro­ line level in plants grown under restricted water availability. citorin® applica­ tion on chili plants grown on organic media (soil+trichocompost+rice husk char­ coal) with ratio of 2:1:1 could be recommended to support plant growth and production under drought stress conditions. 1. introduction chili pepper (capsicum annuum l.) is one of important vegetable crops in indonesia which is cultivated almost throughout the country. the demand of this commodity has significantly increased, but the production is not yet able to meet the requirement. current average yield of chili pepper is about 8.47 ton ha­1, and this was far lower than its potential yield that may reach 15­20 ton ha­1 (statistics indonesia, 2019). therefore, efforts should be done to promote chili pepper production through the improvement of cultivation techniques and expansion of planting area. the improvement of chili production through expansion of planting area both in dry and rainy season is restricted by uncertain condition of growing environment, resulting in poor plant growth and development. (*) corresponding author: dr.zulkarnain@yahoo.com citation: ichwan b., eliyanti e., zulkarnain z., 2021 ­ effect of biostimulants and media compositions on growth and yield of capsicum annuum l. under under drought stress conditions. ­ adv. hort. sci., 35(2): 129­137 copyright: © 2021 ichwan b., eliyanti e., zulkarnain z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 march 2020 accepted for publication 19 march 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8354 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 129­137 130 chili pepper growing during dry season is constrained by limited soil water availability, causing environ­ mental stress to the plants. study by ichwan et al. (2017) revealed that chili pepper grown under 50% field capacity showed slow growth, reduced yield, high proline content, and low sugar content. efforts to improve the ability of chili plants to sur­ vive drought stress and to be able to grow and result in maximum yield can be done by applying effective biostimulants to plants grown under a mixture of soil, trichocompost and rice husk charcoal. the effect of biostimulant in enhancing plant growth and produc­ tion had been reported by many authors (lópez­ bucio et al., 2015; rady and ur rehman, 2016; niyokuri et al., 2018; drobek et al., 2019). also, the use of trichocompost or trichoderma­enriched com­ post in supporting plant growth and development under limited soil water content had been studied with positive results (bae et al., 2009; mastouri et al., 2010; khoshmanzar et al., 2019). the use of organic materials may increase plant tolerance to drought stress due to their ability to improve soil structure, increase aeration in root zone, reduce mass density, increase cation exchange capacity, and maintain primary nutrients such as n and p, in addition to increasing soil water holding capacity. trichocompost (trichoderma in compost) is one of organic materials that can be used to improve plant resistance to drought stress. in addition, the application of husk charcoal in growing media is another way to enhance soil organic matter and improve chemical, physical and biological properties of soil. husk charcoal contains 0.32% n, 15% po, 31% ko, 0.95% ca, 180 ppm fe, 80 ppm mn, 14.1 ppm zn and ph 6.8. another characteristic of husk charcoal is light (specific gravity of 0.2 kg l­1). rice husk charcoal also increased field capacity and water content by increasing the porosity of the amended soil, and reduced the acidity of soil (mishra et al., 2017). rice husk charcoal treatments on pot grown let­ tuce (lactuca sativa) and cabbage (brassica chinen‐ sis) were found to increase the final biomass, root biomass, plant height and number of leaves in all the cropping cycles in comparison to charcoal free treat­ ments (carter et al., 2013). mannan and shashi (2019) reported that the application of rice husk charcoal increased plant height, days to maturity, total dry weight, cob diameter, cob length, 100­grain weight, and yield of maize under drought conditions. chlorophyll content was also found to increase, but proline content decreased due to rice husk charcoal application. meanwhile, imanda and ketty (2018) claimed that rice husk charcoal along with cow manure was best composition for the growth of papaya plant. in chili pepper, however, study on the use of mixture of soil, trichocompost and rice husk charcoal, particularly under drought stress, is not yet well documented. the application of biostimulant along with the improvement of growing media is expected to be synergistically improve the resistance of chili pepper against drought stress while sustaining best growth and yield. the purpose of this study was to obtain a proper combination of biostimulant and growing media compositions that enhance the growth and yield of chili pepper during drought stress. 2. materials and methods experimental design and plant handling the study was conducted at the teaching and research farm, faculty of agriculture, university of jambi from april 2019 through to september 2019. a split plot design with three replications was employed in this study. the main plot consisted of different types of commercial mixed biostimulants (citorin® and hantu®) and a control, while the sub­ plot consisted of different ratio of soil+trichocom­ post+rice husk charcoal (2:2:1, 2:1:1, 1:2:1, and 1:1:2). the biostimulant citorin® contained gibberellic acid, p2o5, k2o, mgo, mn, antioxidant and vitamins (amanah and putra, 2018), and hantu® contained gibberellic acid, indoleacetic acid, kinetin, zeatin, n, p, na, mg, cu, fe, mn, zn, co, cd, and pb (lidar and mutryarny, 2017). seeds of chili pepper cv. lado were sown on media consisted of soil+trichocompost+rice husk charcoal (2:1:1) on a seedbed. seven days later seedlings were transferred to nursery, and left for 21 days before transplanting on individual pots with dif­ ferent media compositions according to the treat­ ment. soil water content in the media was set to 75% of field capacity to create drought condition, except those controls. this is in accordance with our previ­ ous investigation (ichwan et al., 2017). biostimulants were applied to the plants on week­ ly basis from week 2nd to week 12th after transplant­ ing by foliar spraying. fertilization and maintenance of plants were carried out in accordance with the standard of chili pepper cultivation (zulkarnain, 2013). ichwan et al. ‐ biostimulants and media compositions influence on growth and yield of capsicum annuum l. 131 variables observed data on plant growth and yield were collected 10 weeks after transplanting (the time of fruit forma­ tion) and 14 weeks after transplanting (the time of first harvest). variables observed were plant height, number of productive branches, total leaf area, dry weight (total and above­ground parts), fruit number, and total weight of fruits per plant. chemical analysis data on n, p, k+, ca2+ and mg2+ content in plant tissues were also recorded as well as total sugar, chlorophyll content, and proline content within leaf tissues. composite leaf samples were made by physi­ cally mixing individual leaves taken from 3 sample plants of 3 replicates into one homogenous sample. compositing reduced the number of analyses to be performed and was designed to provide a represen­ tative sample of the treatment. ten youngest mature leaves on main stem were collected at 10 weeks after transplanting (wat). dry and clean leaf samples were placed in a sample bag prior to laboratory analysis. total nitrogen content was determined by kjeldahl method (labconco corporation, 1998), phos­ phorus concentration was determined by vanadium molybdate yellow colorimetric method (karlberg and pacey, 1989), whereas k+, ca2+ and mg2+ were anal­ ysed using atomic absorption spectrometry (aas) method (the perkin­elmer corporation, 1996). total sugar was determined according to irigoyen et al. (1992), chlorophyll content was determined accord­ ing to hall and rao (1986), and proline content was analysed according to bates et al. (1973). statistical analysis data were analysed statistically using analysis of variance (anova) module (petersen, 1985) to judge the significance of the effect of biostimulants and growing media compositions. if the result from anova is significant (p<0.05), then the fisher’s least significant different (flsd) is applied to see the dif­ ference between two treatment means. any differ­ ence larger than flsd is considered a significant result. 3. results there was no significant effect of biostimulants on plant height at 10 weeks after transplanting (wat). however, significant response was shown by plants grown on different media compositions. in the absence of biostimulant, the composition of 2:1:1, 1:2:1 and 1:1:2 were found to enhance plant height. when citorin® was used as plant biostimulant, the medium composition of 1:1:2 was the best. however, with the use of biostimulant hantu® none of medium composition showed significant effect on plant height (table 1). the interaction between biostimulants and media compositions showed significant effect on the num­ ber of productive branches at 10 wat (table 2). the application of citorin® on plant grown on medium composition of 1:1:2 resulted in the highest number of productive branches, followed by those grown on 2:2:1 medium composition, and they differed signifi­ cantly from those grown on 1:2:1 medium composi­ tion. while in the use of hantu®, medium composi­ tion of 2:2:1 was the best and the effect was signifi­ cantly different from 1:2:1 and 1:1:2 compositions, but not 2:1:1 composition. in the absence of biostim­ ulant, however, there was no significant difference in the number of productive branches on plants grown on different media compositions (table 2). table 1 ­ the effect of biostimulants on the height of chili pep­ per (cm) grown on different media compositions with limited soil water content (10 wat) (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows are not significantly different according to fisher’s least significant different test (α = 0.05). biostimulants plant height media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 88.00 b 105.33 a 106.67 a 102.33 a citorin® 98.00 b 114.33 ab 99.00 b 118.00 a hantu® 102.00 a 108.00 a 95.33 a 104.67 a table 2 ­ the effect of biostimulants on the number of produc­ tive branches of chili pepper grown on different media compositions with limited soil water content (10 wat) (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows are not sig­ nificantly different according to fisher’s least significant different test (α = 0.05). biostimulants number of productive branches media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 88.00 aa 94.00 aa 90.66 aa 71.33 ab citorin® 106.66 aa 96.00 aba 78.66 ba 121.33 hantu® 112.66 aa 100.00 aba 83.00 ba 79.00 bb adv. hort. sci., 2021 35(2): 129­137 132 at 14 wat, the number of productive branches was not affected by biostimulant application, but the composition of the media was found to affect this parameter significantly (table 3). hantu® was effec­ tive in promoting the number of productive branches on plants grown on medium composition of 2:1:1, 1:1:2 and 2:2:1, which were significantly different from those grown on 1:2:1 medium composition. with citorin® application as well as in the absence of biostimulant, the effect of media compositions did not show any difference on the number of productive branches (table 3). the interaction between biostimulants and media compositions did not significantly affect total leaf area and total dry weight (tables 4 and 5). the largest absolute total leaf area was found in the com­ bination of hantu® and 2:1:1 medium composition (2,644.66 cm2) (table 4). in addition, the greatest total dry weight was obtained in the combination of citorin® and 1:1:2 medium composition (table 5). significant effect of the interaction between bios­ timulants and media compositions was noted on dry weight of above­ground parts (table 6), fruit number (table 7) and fruit weight (table 8). data presented in table 6 show that the dry weight of above­ground parts of plants treated with citorin® on medium com­ position of 1:1:2 was significantly higher than those without biostimulant. in the absence of biostimulant, media composition of 2:1:1 and 1:2:1 were better than 2:2:1 or 1:1:2, while in the application of citorin®, the composition of 1:1:2 and 2:1:1 were better than 2:2:1 or 1:2:1. however, when hantu® was applied there was significant difference in the dry weight of above­ground parts of chili peppers grown on all media compositions (table 6). data on fruit number presented in table 7 show that plants grown on media with composition of 2:2:1 and treated with biostimulants produce more fruits than those without biostimulants. on 2:1:1 media composition, the application of hantu® resulted in the greatest number of fruit, which was significantly different from either the application of citorin® or no biostimulant treatment. on 1:1:2 media composition, however, the effect of hantu® was significantly differ­ ent to those plants grown in the absence of biostimu­ lant only. on media composition of 1:2:1, the control treatment produced more fruits than those plants treated with citorin® (table 7). table 3 ­ the effect of biostimulants on the number of produc­ tive branches of chili pepper grown on different media compositions with limited soil water content (14 wat) biostimulants number of productive branches media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 161.00 a 176.67 a 129.00 a 170.67 a citorin ® 195.67 a 211.00 a 135.33 a 186.67 a (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows are not sig­ nificantly different according to fisher’s least significant different test (α = 0.05). table 4 ­ the effect of biostimulants on total leaf area of chili pepper (cm2) grown on different media compositions with limited soil water content biostimulants total leaf area (cm2) media compositions 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 1497.46 1930.62 1827.07 1784.26 citorin® 2115.81 2285.40 1531.72 2361.31 hantu® 1997.24 2644.66 1556.42 2413.54 table 5 ­ the effect of biostimulants on total dry weight of chili pepper (g) grown under drought stress condition on different media compositions with limited soil water content biostimulants total dry weight media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 10.11 13.68 13.34 10.41 citorin® 11.38 14.31 11.51 16.44 hantu® 13.58 15.38 12.48 13.04 (z) media compositions= soil : trichocompost : rice husk charcoal. (z) media compositions= soil : trichocompost : rice husk charcoal. table 6 ­ the effect of biostimulants on dry weight of above­ ground parts of chili pepper (g) grown on different media compositions with limited soil water content (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows and the same uppercase in the columns are not significantly different according to fisher’s least significant different test (α = 0.05). biostimulants dry weight media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 4.73 ba 7.13 aa 6.93 aa 4.83 bb citorin® 5.03 ba 7.86 aa 5.93 ba 8.23 aa hantu® 6.60 aa 7.86 aa 6.46 aa 6.50 aab ichwan et al. ‐ biostimulants and media compositions influence on growth and yield of capsicum annuum l. 133 data presented in table 8 show that biostimulant citorin® significantly increased fruit weight when applied on plants grown on medium with 2:2:1 com­ position. on medium with 1:2:1 composition, the application of either citorin® or hantu® was found to result in less fruit weight significantly. in the absence of biostimulant, the composition of 2:1:1 significantly increased fruit weight than 2:2:1 and 1:1:2 media compositions. also, in the application of citorin®, the fruit weight of plants grown on medium with the composition of 2:1:1 was significantly heavier than of those grown on medium composition of 1:2:1. further, in the application of hantu® the fruit weight of plants grown on the media composition of 2:1:1 was significantly heavier than the weight fruits pro­ duced by plants grown on any other media composi­ tions (table 8). the use of biostimulants and growing media con­ taining soil+trichocompost+rice husk charcoal was able to maintain nutrient status within plant tissues to remain at optimal level during limited soil water availability, except for potassium which was at luxury consumption level and calcium which was below crit­ ical limit. nutrient content of chili leaf tissues was measured when the plant was 14 weeks old after transplanting. the results of leaf tissue nutrient mea­ surement is presented in table 9. plants grown on different media compositions and treated with both citorin® and hantu® showed a higher total sugar and chlorophyll contents and lower proline level compared to those without biostimulant application (control). in spite of media compositions, plants treated with citorin® show a higher total sugar and lower proline content in comparison to those table 7 ­ the effect of biostimulants on the number of fruit of chili pepper grown on different media compositions with limited soil water content (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows and the same uppercase in the columns are not significantly different according to fisher’s least significant different test (α = 0.05). biostimulants fruit number media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no biostimulant 25.00 bb 38.66 ab 41.66 aa 33.00 abb citorin® 42.00 aa 45.33 ab 24.66 bb 40.33 aab hantu® 54.00 ba 73.66 aa 29.66 cab 61.33 aba table 8 ­ the effect of biostimulants on the weight of fruit of chili pepper (g) grown on different media compositions with limited soil water content (z) media compositions= soil : trichocompost : rice husk charcoal. numbers followed by the same lowercase in the rows and the same uppercase in the columns are not significantly different according to fisher’s least significant different test (α = 0.05). biostimulants fruit weight media compositions (z) 2:02:01 2:01:01 1:02:01 1:01:02 no 54.89 cb 96.89 aa 81.08 aba 67.61 bca citorin® 99.03 aba 107.75 aa 31.37 cb 76.77 ba hantu® 67.58 bb 106.95 aa 28.70 cb 71.47 ba table 9 ­ leaf nutrient contents of chili pepper as affected by biostimulants and different media compositions during limited soil water availability (z) media compositions= soil : trichocompost : rice husk charcoal. (y) leaf nutrient content was determined compositely by physically mixing individual leaves taken from each 3 sample plants of 3 repli­ biostimulants media (z) compositions leaf tissue nutrient content (%) (y) n p k+ ca2+ mg2+ no biostimulant 2:02:01 2.46 0.31 7.22 0.88 0.56 2:01:01 1.94 0.37 6.22 1.18 0.73 1:02:01 2.55 0.32 6.53 1.02 1.05 1:01:02 3.01 0.26 6.13 1.03 1.02 citorin® 2:02:01 3.21 0.33 4.37 1.01 0.86 2:01:01 2.83 0.31 5.99 1.12 0.89 1:02:01 3.12 1.34 8.24 0.85 0.44 1:01:02 3.11 0.30 7.03 0.85 0.99 hantu® 2:02:01 3.44 0.29 7.78 1.05 0.72 2:01:01 3.65 0.31 5.83 1.08 0.61 1:02:01 3.14 0.34 7.47 0.65 0.48 1:01:02 3.02 0.30 7.67 0.86 0.93 134 adv. hort. sci., 2021 35(2): 129­137 treated with hantu®. however, plants grown on medium composition of 1:1:2 and treated with hantu® produced the highest chlorophyll content. total sugar, proline and chlorophyll content of leaves were measured when the plants were 14 weeks after planting, and the results are presented in table 10. 4. discussion and conclusions the application of biostimulants on chili pepper grown on different growing media compositions with limited soil water content was found to increased plant growth and yield. our results proved that biostimulant application was important for chili pepper grown on media consisting of soil+trichocom­ post+rice husk charcoal but with limited water sup­ ply. plants treated with biostimulants grew better than those without biostimulant on all growing media. plants treated with either citorin® or hantu® and grown on soil+trichocompost+rice husk charcoal with ratio of 2:1:1 were taller and bigger than those grown on other media compositions without biostim­ ulant application (fig. 1 and fig. 2). this increased in pepper growth and yield is presumably due to hor­ mones, organic acids, and macro and micro nutrients contained within the biostimulants. citorin® contains gibberellic acid (ga3) along with nutrients such as p, k, mg, mn, antioxidants and vitamins (amanah and putra, 2018). meanwhile, hantu® contains the gib­ berellic acids (ga3, ga5, ga7), iaa, kinetin and zeatin along with nutrients such as n, p, na, mg, cu, fe, mn, zn, co, cd, and pb (lidar and mutryarny, 2017). hedden and thomas (2012) claimed that ga phys­ iologically acted as growth stimulant of plant organs through cell division and elongation. further, gupta and chakrabarty (2013) suggested that gibberellic fig. 1 ­ the effect of biostimulants on the growth of chili pepper during limited water supply (a = no biostimulant; b = citorin®; c = hantu®). table 10 ­ total sugar, proline and chlorophyll contents as affected by biostimulants and different media compositions during limited soil water availability (z) media compositions= soil : trichocompost : rice husk charcoal. (y) total sugar, proline and chlorophyll contents was determined compositely by physically mixing individual leaves taken from each 3 sample plants of 3 replicates into one homogenous sample at 10 weeks after transplanting. biostimulants media (z) compositions total sugar (y) (mg g­1) proline (y) (mm g­1) chlorophyll (y) (cm2 ml­1) a b total no biostimulant 2:02:01 3.045 0.925 6.096 9.523 15.619 2:01:01 3.136 0.592 6.688 10.175 16.863 1:02:01 2.091 1.048 7.386 11.380 18.766 1:01:02 3.091 0.304 7.527 10.695 18.222 citorin® 2:02:01 3.136 0.439 7.731 11.723 19.455 2:01:01 4.841 0.254 8.366 11.325 19.691 1:02:01 2.614 0.921 5.252 7.109 12.361 1:01:02 3.909 0.347 7.585 10.568 18.154 hantu® 2:02:01 4.091 1.209 7.430 9.888 17.318 2:01:01 3.727 0.803 6.681 9.217 15.899 1:02:01 3.864 1.141 3.723 5.076 8.799 1:01:02 2.455 0.566 8.695 12.011 20.706 ichwan et al. ‐ biostimulants and media compositions influence on growth and yield of capsicum annuum l. 135 acids in plants played an important role in triggering the transition from meristem to shoot growth until mature organs. various recent studies on the use of gibberellic acids indicated that these plant hormones could increase plant growth and development, improved yield, and increased tolerance to abiotic stresses such as drought, heat and salinity (pal et al., 2016; sarwar et al., 2017; miceli et al., 2019; zhu et al., 2019). growing media is one of important elements sup­ porting plant growth and development. a good media should have good aeration, be able to hold water, and capable to store nutrients for plants. the mixture of soil+trichocompost+rice husk charcoal with the ratio of 2:1:1 or 1:1:2 produced better plant growth compared to others. the greatest plant height, total leaf area and dry weight of above­ ground parts were achieved on plants grown on media with the ratio of 2:1:1 on all biostimulant applications, except citorin® on 1:1:2 medium com­ position. meanwhile, the highest number of produc­ tive branches (aged 10 and 14 wat) was obtained on medium composition of 2:1:1 in all biostimulant applications. based on these results it can be seen that media with more soil or more rice husk charcoal were preferable to produce better growth of chili pepper treated with biostimulants. trichocompost is trichoderma­based fertilizer that function to enhance plant’s drought tolerance by improving root development (shukla et al., 2012), activating antioxidant protection to prevent damage caused by dehydration (brotman et al., 2013), and delaying changes in stomatal opening, photosynthe­ sis and chlorophyll content due to drought (lópez­ bucio et al., 2015). trichoderma sp. help plants better resist environmental stress such as drought via rein­ forcing plant growth and reprograming gene expres­ sion in roots and shoots. the tolerance to water deficit was attributed to activation of antioxidant responses and higher activity of ascorbate and glu­ tathione­recycling enzymes (mastouri et al., 2012). the fungal mycelium secreted different compounds that increase the branching capacity of the root sys­ tem, thus improving nutrient and water acquisition (lópez­bucio et al., 2015). good growth performance of chili pepper grown on soil+trichocompost+rice husk charcoal and sprayed with biostimulants was followed by good production in term of fruit number and weight. these result was the consequence of a significant interac­ tion of the two factors. moreover, plants grown in medium with ratio of 2:1:1 and sprayed with citorin® produced higher total sugar and chlorophyll content and lower in proline compared to those grown on the same medium but in the absence of biostimulant, as well as plants grown on other media ratios but treat­ ed with hantu®. total sugar content in chili pepper grown on soil+trichocompost+rice husk charcoal with ratio of 2:1:1 and treated with biostimulants was higher than those grown on other media but in the absence of biostimulant. the results of this study are in line with study conducted by martim et al. (2009) on grapevines which showed that drought stress could increase respiration rate of plants. increased respira­ tion rate will lower plant carbohydrates and promote total sugar content which also function as an osmotic adjustment. chloroplast contains chlorophyll which is a major component involving in photosynthesis. decrease in chlorophyll content during drought was an indication of oxidative stress caused by photo­oxidative pig­ ment and chlorophyll degradation (farooq et al., 2009; anjum et al., 2011). the increase of chlorophyll content in chili peppers grown on different ratios of soil+trichocompost+rice husk charcoal indicates that the plants were able to survive drought stress condi­ tion. the application of biostimulants may thus improve plant physio­biochemical attributes under drought stress. this is in accordance with the results noted on triticum aestivum and solanum lycoper‐ sicum (yasmeen et al., 2013) and phaseolus vulgaris (rady and mohamed, 2015; elzaawely et al., 2017). el­mageed et al. (2017) claimed that the improve­ ment of chlorophyll content due to biostimulant application under drought stress may be attributed to the protection impacts on the photosynthetic sys­ tems. proline is one of dissolved compounds produced fig. 2 ­ the effect of the ratio of soil+trichocompost+rice husk charcoal on the growth of chili pepper during limited water supply (a = 2:1:1; b = 2:2:1; c = 1:2:1; d = 1:1:2). adv. hort. sci., 2021 35(2): 129­137 136 by plants in drought stress condition, which acts as an osmotic adjustment in addition to other com­ pounds such as fructan, trehalose, polyol, polyamine and glycinbetain (mitra, 2001). as an osmotic adjust­ ment, proline keeps plants to continue to grow even in a low water potential condition. low proline con­ tent in plants grown on media ratio of 2:1:1 and treated with citorin® indicates that they do not expe­ rience stress due to drought. this is in accordance with report by goñi et al. (2018) on tomato grown on limited soil water content and treated with ascophyllum nodosum extract which showed a lower leaf proline content in comparison to untreated plants. biostimulants containing bioactive compounds are desirable in today’s agriculture because of their capa­ bility to enhance nutrient uptake which positively affect overall plant vigor resulting in high quantity and quality of harvest (parađiković et al., 2017). in our study biostimulant application on chili pepper grown on soil+trichocompost+rice husk charcoal could improve growth, increase nutrient status as well as total sugar and chlorophyll contents, and reduce proline level in leaves. in addition, plant height, number of productive branches, total leaf area, and dry weight of above­ground parts were higher in biostimulant­treated plants. biostimulant citorin® might be used to ensure the production of chili pepper by overcoming drought stress and pro­ viding good nutrient uptake on medium consists of 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(2) anthesis (perfect blooming flow­ ers), 0­7 days after anthesis. (3) fruit set, 7­14 days after anthesis. (4) fruit development, 14­28 days after anthesis. (5) fruit maturation, 28­35 days after anthesis. (6) fruit ripening, 35­50 days after anthesis. the results showed that the content of iaa, zeatin, ga3, and total sugar of flowers and fruit of wax apple at 6 stages that would fall smaller than those of retention and acc con­ tent and starch was higher in flower and fruits that drop easily than retention. the kinetin content in the flower development that drop easily is smaller than the retention but in the fruit development the kinetin content is not significant­ ly different between those that drop easily and retention. 1. introduction water apple originates from the southeast asian region (indonesia and malaysia), then spreads to the islands of the pacific and north and central america. generally, wax apple fruit is consumed as fresh fruit, but it can also be made for salad and preparations such as pickles, syrups, jel­ lies, and cocktails. water apple fruit is not only sweet and refreshing but has diversity in appearance. types of water apple are syzygium aqueum (water apple) and syzygium samarangense (wax apple). the varieties of syzygium samarangense include delima, lilin, camplong, cincalo, citra, kesuma, and madu, (kuswandi, 2008). corona virus disease (covid­19) is originating from wuhan, china began to spread throughout the world from january 2020 to 31 december 2020, so there were 83,264,353 cases, with 1,816,164 deaths from 218 affected countries (world health organization, 2020). no vac­ cine has been found to treat patients with the covid­19 virus. according (*) corresponding author: etik.unsoed@gmail.com citation: wukir tini e., dwi haryanto t.a., sakhidin, saparso, 2021 ­ endogenous hormone causes flower and fruit drop of wax apple (syzygium samarangense cv. citra). ­ adv. hort. sci., 35(1): 53­60 copyright: © 2021 wukir tini e., dwi haryanto t.a., sakhidin, saparso. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 september 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9663 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 53­60 54 to the ministry of health of the republic of indonesia (2020), prevention of viruses can be done by fre­ quently washing hands, wearing masks, consuming vegetables and fruit, exercise, and adequate rest. one of the prevention efforts that can be done by consuming fruits that contain vitamin c to enhance immunity. every 100 g of wax apple fruit contains 22.3 µg of vitamin c (asia­pacific association of agricultural research institutions, 2014). therefore wax apple is a tropical fruit that is cheap, easily obtained, and contains vitamin c which can be used as an alternative to increasing body immunity as a preventative measure against the covid­19 virus. the main problem in the cultivation of wax apple plants is the high drop of flowers and fruit. the high rate drop of flowers and fruit causes a few numbers of wax apple fruit that can be produced. wax apple drop fruit rate reaches 52% (khandaker et al., 2016). fruit drop also occurs in other fruits, such as durian flowers reach 95 to 100% (suparto and sakhidin, 2013), lychee fruit is 90 to 97% (stern et al., 1995), star fruit is 25 to 30% (kurniawati and hamim, 2009), mangosteen flower is 14.1% and mangosteen fruit is 70.1% (rai et al., 2008). dropped fruit is a natural phenomenon that occurs in almost all types of fruit. drop fruit, especially at the beginning of fruit growth, is a mechanism for regulat­ ing autoregulation in each plant (davarynejad et al., 2009). physiologically the loss of flowers and fruit cor­ relates with the limited supply of photosynthate and nutrient adequacy (iglesias et al., 2007), as well as hormonal regulation in the abscission zone (bangerth, 2000). flower and fruit drop are also influ­ enced by endogenous hormone content in plants due to high ethylene concentrations and low concentra­ tions of auxin and gibberellins. auxin, gibberellins, and ethylene are hormones that are directly related to the process of flowers and fruit drop (bangerth, 2000). complex hormonal interactions occur during fruit development. gibberellins and cytokines gener­ ally stimulate fruit growth and auxin as a growth stim­ ulator and also as a fruit drop agent. abscisic acid (aba) and ethylene are also involved in the process of loss (sakamoto et al., 2008). high ethylene concentra­ tions, low concentrations of auxin and gibberellins, and high aba concentrations in plants are the main causes of drop fruit (iglesias et al., 2007). the causes of flower and fruit drop of the wax apple due to imbalance of plant growth regulator as well as cultivation and environmental engineering factors, including pollination, fertilization, fruits set, lack of water and nutrition supply, pest attack, rain­ fall and wind (khandaker et al., 2016). research on the use of synthetic growth plant regulators (gpr) to reduce the fruit drop of wax apple has been conduct­ ed by khandaker et al. (2013), khandaker et al. (2016), 50 mg l­1 gibberellins (ga3), ga3 produces the lowest flower and fruit buds loss of 29% compared to controls (not given ga3 36%). ga3 20 mg l­1 reduced fruit loss 32% compared to 52% control. 2­4­ dichlorophenoxyacetid (2,4 d) 5 mg l­1 reduces flower and fruit bud loss by at least 30% and 18% compared to 35% and 40% controls. naphthaleneacetid acid (naa) 5 mg l­1 reduced the flower buds and fruits drop as lower as 28% and 30% compared to 30% and 52% controls. the causes of flowers and fruit drop of wax apple are because the content of endogenous hormones is not yet known, so the application of growth plant regulator given is not appropriate to reduce the flowers and fruit drop. singh et al. (2017) the results of research on the application of growth plant regulator on khasi man­ darin (citrus reticulata blanco) showed an increase in fruit retention and the number of fruit with the appli­ cation of urea 1% + 2, 4­d 15 ppm (45.4% and 244, 3 fruits/tree), application of naa 5 ppm + 2,4­d 10 ppm (44.3% and 241.6 fruit/tree) compared to con­ trols (17.3% fruit retention and 181 fruit/tree). naa treatment 15 ppm increased the proportion of cape gooseberry cv. aligarh is retention (71.40%) (kaur and kaur, 2016). the purpose of this study was to obtain informa­ tion on hormones that causes flower and fruit drop at several stages of the development of wax apple fruit and to obtain differences in the hormone con­ tent at how many stages of flower and fruit develop­ ment are easily dropped and retention. 2. materials and methods the research used an experimental method from may 2019 to april 2020. the experimental design used was a completely randomized design (crd) with the treatment being tried, namely flower and fruit retention and drop easily of wax apple at six stages fruit development: (1) bud development (from 0 to 7 days before anthesis); (2) anthesis (full bloom), from 0 to 7 days after anthesis; (3) fruit set (the for­ mation of finished fruit), from 7 to 14 days after anthesis; (4) fruit development, from 14 to 28 days after anthesis; (5) fruit maturation, from 28 to 35 wukir tini et al. ‐ hormone endogenous causes flower and fruits drop 55 days after anthesis; (6) fruit ripening (from 35 to 50 days after anthesis). experiment with 4 replications and data analysis using t­test and advanced test dmrt (duncan multiple range test). flower and fruit samples were taken from 46 wax apple plants, kajongan village, bojongsari district, purbalingga regency, central java. the variables observed were auxin content (iaa), cytokinins (zeatin and kinentin), gibberellin (ga3), acc (1­aminocyclopropane 1­ carboxylic acid), total sugar, and starch. the samples used for the study were flowers and fruits from 6 stages of development of flowers and water guava. plants are 5 years old, plant spacing 8 m x 9 m, with a height of ± 7 m and have been fruiting for the last 4 years. temperature 25.8°c with 76% humidity, sunlight intensity 571 lux outside the canopy, and under the canopy 529 lux and soil ph 6.8. organic cultivation of wax apple uses goat manure at a dose of 100 kg per plant per year. irrigation in the dry season with springs that are channeled from a hose from a pipe made by the gar­ den. the results of soil analysis conducted at the postharvest center of the ministry of agriculture, inceptisol soil type, ph 6.14, organic c 0.769%, total n 0.082%, available n 0.025%, total k 55.960 ppm, k available 30.277 ppm, total p 45.189 ppm, and 1.321% organic matter. the samples drop easily are taken from the flower and fruit which when the branches or twigs are shak­ en fall out and after the fall there is a black abscission layer on the flower stalk or fruit. the retention sam­ ples are wax apple flower and fruit which if the branches or twigs are shaken not fall off, are still attached to the plant, and usually, the flower stalk or fruit is still present. the sample is placed in an icebox containing blue ice and then taken to the integrated research laboratory, jenderal soedirman university to be dried in a 2kxc bench top vacuum freeze dryer. wax apple samples for bud development, anthesis, and fruit set for 25 hours, and fruit development, fruit maturation, fruit ripening for 55 hours with a temperature of ­70°c, and a pressure of 13,332.2 pa the contents of auxin (iaa), ga3, zeatin, kinetin, acc, total sugar, and starch were carried out at the chemical laboratory, center for postharvest, ministry of agriculture, bogor, west java, indonesia. procedure for measuring hormone content (auxin, cytokinin, and gibberellins) using hplc (high­ pressure liquid chromatography) at a wavelength of 214 nm, sample temperature of 10°c and column temperature of 25°c, stationary phase using c­18. the formula for hormone content by hplc is sample area divided by standard area multiplied by standard concentration (harborne, 1973). measurement of acc content according to lizada and yang (1979), using gc (gas chromatography). the formula for acc content in gc is sample area divided by standard area multiplied by standard concentration. measurement of total sugar according to the indonesian national standard (1992) using the luff schoorl method. starch calculation according to horwitz and latimer (2006). 3. results table 1 shows that the fruit drop easily having lower iaa, zeatin, and ga3 content than fruit reten­ tion in all phases of fruit development. the kinetin content at the bud development, anthesis, and fruit set stages that drop easily is lower than the reten­ tion, but the kinetin content at the fruit develop­ ment, fruit maturation and fruit ripening stages is not significantly different in the fruit that drop easily dan retention. while the acc content of fruit that drops easily is greater than that of retention. table 2 shows that the total sugar content at vari­ ous stages of flower and wax apple fruit develop­ ment in flower and fruit retention is higher than that of drop easily. the content of starch in fruit retention of various stages of development of the flower and fruit is lower than those that are of drop easily. 4. discussion and conclusions the iaa content in wax apple flower and fruit which drops easily on bud development, anthesis, fruit set, fruit development, fruit maturation, and fruit ripening is lower than the retention flower and fruit. this is according to rai et al. (2008). the iaa content of mangosteen flowers falling 3.37 ng g­1 is lower than the retention rate of 8.80 ng g­1 dry weight and iaa content in mangosteen fruit falling 0.83 ng g­1 is lower from the iaa content of 6.43 ng g­1 retention fruit. sakhidin et al. (2011), the content of iaa in mango fruit loss is lower than that of reten­ tion. gadung 21 and lali jowo cultivars that will fall age 6 and 9 days after anthesis are 5.96 and 3.86 10­1 µg g­1 sample fresh weight and 4.48 and 4.12 10­1 µg g­1 sample fresh weight. mango fruit retention of 10.32 and 8.12 (10­1 µg g­1 sample fresh weight and adv. hort. sci., 2021 35(1): 53­60 56 10.08 and 7.98 10­1 µg g­1 sample fresh weight. according to kurniawati and hamim (2009), star fruit with an application of 15 ppm 2, 4­dichlorophenoxy­ acetic acid (2,4­d) contains 227 ppm iaa and 60 ppm ga3 applications have 221 ppm iaa. the formation of the absicission layer at the stem point that causes fruit drop is an imbalance of auxin, cytokinin, and gibberellins (chen et al., 2006). flower and fruit loss is also influenced by high ethylene con­ centrations and low iaa type auxin concentrations and low gibberellins. auxin and ethylene are hor­ mones that are directly related to the process of a drop of flowers and fruit (bangerth, 2000). auxin is often reported to delay or to induce fruit, it increases cell enlargement rather than cell division. this observation might indicate that auxin is related to cell enlargement, an essential factor controlling fruit size during the rapid fruit growth phase. enlargement of fruits treated with auxin seems to be due to cell expansion rather than cell division (iglesias et al., 2007). table 1 shows that the content of zeatin type cytokinins and fruit kinetin that drop easily is smaller than retention. this is because cytokinins function to inhibit aging (kieber and schaller, 2018) so that the flowers and fruits of wax apple which will fall at sev­ eral stages of development have a smaller zeatin content compared to the retention seen in table 1. the cytokinin content of both zeatin and kinetin at the stage of development flowers and fruit of wax apple was the highest at the fruit set stage. according to chen (1983), the cytokinin content table 1 ­ the content of several hormones at the stage of development fruit of wax apple var. citra the number followed by the same letter in the same column for each fruit development is not significantly different from the t­test at p<0.05. treatment flower and fruit development bud development anthesis fruit set fruit development fruit maturation fruit ripening iaa (ppm) retention 69.50 a 33.72 a 47.40 a 57.02 a 42.86 a 42.10 a drop easily 1.42 b 0.73 b 0.68 b 1.22 b 0.78 b 0.41 b zeatin (ppm) retention 2.18 a 2.25 a 3.29 a 2.19 a 2.59 a 2.04 a drop easily 0.17 b 0.50 b 0.66 b 0.36 b 0.03 b 0.46 b kinetin (ppm) retention 4.36 a 2.46 a 11.31 a 3.17 a 3.30 a 3.93 a drop easily 2.16 b 1.38 b 2.96 b 3.05 a 2.75 a 2.39 a ga3 (ppm) retention 79.65 a 68.08 a 90.47 a 68.25 a 63.45 a 57.72 a drop easily 28.63 b 16.13 b 20.14 b 26.69 b 17.57 b 8.97 b acc (ppm) retention 19.18 b 17.26 b 14.21 b 14.13 b 13.31 b 12.14 b drop easily 48.39 a 38.09 a 37.99 a 35.75 a 34.14 a 30.92 a table 2 ­ total sugar content and starch content on stages of wax apple flower and fruit development the number followed by the same letter in the same column each fruit development is not significantly different in the t­test at the level of p<0.05. treatment flower and fruit development stage bud development anthesis fruit set fruit development fruit maturation fruit ripening total sugar content (%) retention 6.87 a 8.72 a 11.38 a 23.35 a 28.93 a 32.59 a drop easily 5.80 b 7.60 b 8.60 b 20.61 b 26.36 b 28.66 b starch content (%) retention 17.23 b 16.29 a 14.52 b 13.11 b 11.02 b 8.77 b drop easily 17.90 a 16.40 a 15.80 a 13.89 a 12.62 a 11.21 a wukir tini et al. ‐ hormone endogenous causes flower and fruits drop 57 increased in mango flowers maximum at 10 days after anthesis and decreased gradually up to 50 days after anthesis. this is consistent with trueman (2011), that high cytokinin concentrations in macadamia fruit are retention. cytokinins are pro­ duced in roots and young fruit (pratima and chawla, 2019). table 1 shows that the ga3 content in several stages of flower and fruit development will fall lower than the retention of flowers and fruit. the ga3 con­ tent in fallen fruit was lower than that in retention (bains et al., 1997). the role of ga3 is able to stimu­ late plant growth and flowering, increase flowering and reduce drop flower (budiarto and wuryaningsih, 2007). according to kurniawati and hamim (2009), gibberellin and auxin can support fruit retention by inducing the enzyme α­amylase to hydrolyze starch to sugar that is needed for fruit growth and develop­ ment (subiyanto, 1991). this can be seen in table 1 that the increased auxin content also increases the gibberellin content in retention flowers and fruits compared to drop easily and in table 2 flowers and fruits retention have a higher total sugar content than drop easily. the acc content of wax apple flowers and fruits that drop easily is higher than the retention of flow­ ers and fruits are seen in table 1. this is because the acc which is an ethylene precursor makes the drop of flowers or fruits become higher (wang et al., 2002). the mango acc content that drops easily is higher than that of retention (sakidin et al., 2011). the acc content of gadung 21 and lali jiwo cultivars which drop easily higher at the age of 3, 9, 12 days after anthesis are 26.81, 35.78, 43.41 (10­1 mg g­1) sample fresh weights and 29.13, 29.35, 35.79 (10­1 mg g­1) sample fresh weight. the acc content of gadung 21 and lali jiwo cultivars with age retention of 3, 9, 12 days after anthesis is 26.81; 35.78; 43.41 (10­1 mg g­1) fresh sample weights and 10.13; 13.66; 11.94 (10­1 mg g­1) sample fresh weights (sakhidin et al., 2006). flower and ovary abscission occur in az­a (abscission zone) located between branches and flower stalks. absence in the az­a zone starts from the fruit set period and this negative effect is regulat­ ed by the content of gibberellins in the ovaries. fruitlet abscission during fruit drop in june at the end of the fruitset period on az­c which is located in the petals, between the flower disks and the ovarian wall, and highly dependent on carbohydrate avail­ ability. sugar in mature leaves is transported for fruit­ let growth and activates az­c (iglesias et al., 2007). according to iglesias et al. (2007), the lack of car­ bon in fruitlets induces an increase in abscisic acid (aba) and acc (1­aminocyclopropane 1­carboxylic acid) which are ethylene precursors. these precur­ sors are then oxidized to ethylene (c2h4) and release of gases that cause fruit abscission. in contrast to the development of fruitlets, in the process of cooking the fruit accumulation of sugar in ripe fruit has a role to induce the activation of abscission before harvest. the balance between c2h4 as a process accelerator, and auxin (aux) as an inhibitor, is one of the main factors in the regulation of ripe fruit abscission. synthesis of auxin in young leaves and transported to adult fruit as an inhibition inhibitor that protects az from high c2h4 content. the role of the regulation of jasmonic acid (ja) in fruit reduction is thought to be mediated through the stimulation of c2h4 biosynthe­ sis. the balance between aux and aba in mature fruits is also important in determining the sensitivity of az­c for abscission stimulus because acc is an ethylene precursor (wang et al., 2002). fruit abscission that occur during fruit develop­ ment due to the active abscission zone. the process is induced by several environmental factors, competi­ tion in the use of assimilates, and internal hormone content. the abscission zone on the mango is located on the fruit stalk with a distance of several mm from the fruit concave (where the fruit is attached to the fruit stalk). from the biochemical and molecular aspects, abscission occurs due to the active enzyme ß­1,4­endoglucanase (eg) and polygalacturonase (pg). the two hydrolase enzymes are involved in damage to plant cell walls that are responsible for the drop of flowers and fruits. the specificity of the abscission zone in responding to organ drop depends on the sensitivity of the layer to ethylene (bonghi et al., 2000). control of fruit growth and abscission in oranges is due to three regulatory factors: genetic, metabolic, and environmental. all three of these affect hormon­ al signals in citrus plants (iglesias et al., 2007). overall, these studies show that a complex set of hormonal interactions occur during fruit develop­ ment. thus, gibberellins (gas) and cytokinins are generally considered positive fruit growth regulators while auxin has been reported to act as a growth stimulator and also as an abscess agent. abscisic acid (aba) and ethylene have been involved in several ways in an abscess. concentrations of iaa and ga3 in fruit and fruit stalks that drop easily are lower than 58 adv. hort. sci., 2021 35(1): 53­60 those in fruit retention and fruit stalks, fruits that will fall out have high abscisic acid content. drop fruit is also caused by an increase in ethylene production. table 2 shows that the flower and fruit drop had a lower total sugar content but a higher starch content than the retention. this is following stopar et al. (2001), apples that drop easily have higher starch content and lower sugar content compared to reten­ tion. mangoes that pre­abscission have a lower total sugar content and a higher starch than retention (sakhidin et al., 2011). total sugar content in flower and fruit develop­ ment of wax apple var. citra of bud development, anthesis, fruit set, fruit development, fruit matura­ tion, and fruit ripening stages 5.8, 7.6, 8.6, 20.16, 26.36, 28.66% lower in fruits that drop easily with fruit retention of 6.87, 8.72, 11.38, 23.35, 28.93, 32.59%. this is in accordance with rai et al., (2008), carbohydrate content (total sugar) in leaves in flower branches and mangosteen fruit which is lower (40.3 and 52.3 mg g­1 leaf dry weight) compared to leaves in branches that are flower and fruit retention (41.2 and 59.1 mg g­1 leaf dry weight). the retention of glu­ cose, fructose, and inverted sugar of wax apple fruit was as follows: 8.9%, 8.9%, 8.7% var. giant green, 9.83%, 9.9%, 9.6% var. masam manis pink, and 9.61%, 9.6%, 9.3% var madu red (khandaker et al., 2011). this shows that the drop of flowers and fruit is associated with a low supply of carbohydrates. limited carbohydrate sources affect the formation and development of fruit (pawar and rana, 2019). increasing the rate of photosynthesis is very important to produce carbohydrates during the development of fruit sets (iglesias et al., 2002). the limited supply of photosynthate and nutritional sta­ tus of plants can limit the number of flowers that develop into fruit that can be harvested (rai et al., 2008). in oranges, carbohydrate deficiencies produce young fruit abscess by triggering an increase in acc levels (gómez­cadenas et al., 2000). auxin applied to trees that have yellow leaves due to low carbohy­ drate cannot prevent pre­harvest loss (sakhidin et al., 2006). table 2 shows that the starch content in flower and fruit development stages of bud development, anthesis, fruit set, fruit development, fruit matura­ tion, and fruit ripening 16.29, 14.52, 13.11, 11.02, 8.77% in fruit retention. the starch content in wax apple is getting smaller as the fruit develops. according to mureşan et al. (2015), the starch con­ tent was reduced in the early stages of fruit develop­ ment in the 3 apple varieties studied, then the starch concentration increased significantly from 35 to 65 days after anthesis. starch concentration gradually decreases as the fruit ripens. this can be seen in table 2 that the starch content during the develop­ ment of the wax apple fruit is seen to decrease from flower buds to fruit maturity, and in fruits that drop easily the starch content is higher than that of reten­ tion. it was concluded the content of iaa, zeatin, kinetin, ga3, and total sugars at several stages of the flower and fruit wax apple which drop easily lower than that of retention and the acc content and starch are higher in fruit which drops easily than retention. the endogenous hormone content of wax apple flower and fruit that drop easily are as follows iaa 0.41 to 1.42 ppm, zeatin 0.03 to 0.66 ppm, kinetin 1.38 to 2.96 ppm, ga3 8.97 to 28.63 ppm, and acc 30.92 to 48.39 ppm. the wax apple fruit that retention iaa content of 30.92 to 48.39 ppm, zeatin 2.04 to 3.29 ppm, kinetin 2.46 to 11.31 ppm, ga3 57.72 to 79.65 ppm, and acc 30.92 to 48.39 ppm. the total sugar content of wax apple flower and fruit which drop easily from 5.8 to 28.66% is lower than the retention rate of 6.87 to 32.59%. the content of fruit starch that drops easily is 11.21 to 17.9% higher than the retention of 8.77 to 17.23%. actions that can be taken to prevent the drop of wax apple fruit by giving natural endogenous hormones contained in other plant materials. this can be done after knowing the content of auxin, zeatin, kinetin, gibberellins, and acc in wax apple fruit that drop easily or retention. acknowledgements the research was supported by research funds from the directorate of research and community service, ministry of education and culture doctoral dissertation research scheme for the 2020 budget year through a decree of the chairperson of the research and community service institute, 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and drought tolerant rootstocks m. tatari 1 (*) h. abdollahi 2, m. henareh 3, m. dehqani 4 1 horticulture crops research department, isfahan agricultural and natural resources research and education centre, areeo, isfahan, iran. 2 temperate fruits research center, horticultural scinces research institue, areeo, karaj, iran. 3 horticulture crops research department, west azarbaijan agricultural and natural resources research and education center, areeo, urmia, iran 2 soil and water research department, isfahan agricultural and natural resources research and education center, areeo, isfahan, iran. key words: drought stress, genotypes, growth vigor, pyrus spp., seedlings. abstract: one of the important products in iran and isfahan province is the pear that its cultivation has been limited by drought stress and global warming in recent years. the use of drought-tolerant rootstocks is one of the available solutions for pear orchards in semi-arid regions. in addition, the lack of dwarf or semi-dwarf rootstocks, which are appropriate and compatible with iran climatic conditions, limited high density pear orchards. in order to obtain drought tolerant rootstocks, in this research fruit of pyrus glabra, pyrus syriaca, and pyrus salicifolia, along with p. communis cv. spadona, dargazi, as well as khoj n. 1 and n. 2 species were collected from different regions of iran in august and september of 2016. the seeds were separated from the flesh and dried at room temperature. the seeds were cultivated in uniform and light texture of soil in november in the field condition to break the seed dormancy. seedlings were irrigated regularly for three months in order to establish in the soil; drought stress began in july. in order to apply drought stress, irrigation time was considered based on 80% of allowed water depletion. morphological traits of seedlings were recorded before stress and at the end of the stress period (late september). the viability percentage of seedlings after drought stress was between 14.28% (p. communis cv. dargazi progenies) to 82.55% for p. salicifolia. comparison of the means and cluster analysis, among populations showed that the three populations of p. salicifola, p. glabra and p. communis cv. khoj n. 2 had the lowest height and were placed in the same group. after studying single genotypes in these three populations, genotypes no. 31, 32, 41, 57 and 12 from p. salicifolia, genotypes 10, 11, 7, 3 and 9 of p. glabra and genotype 4 of p. communis cv. khoj n. 2 populations were selected as drought tolerant and dwarf genotypes and were taken to the propagation phase for future evaluation. 1. introduction pear is from the rosaceae family and the pyrus genus. this species has been cultivated in iran since ancient times, and iran is one of the earliest (*) corresponding author: mtatari1@gmail.com citation: tatari m., 2019 selection of open pollination progenies in some pear species in order to achieve dwarf and drought tolerant rootstocks. adv. hort. sci., 33(2): 245-255 copyright: © 2019 tatari m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 25 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 245-255 246 areas of pear distribution and variation in the world (abdollahi, 2011). drought stress is the most important environmental stress that occurs annually with extreme damage to crops, especially in arid and semi-arid regions (xoconostle-cazares et al., 2010). in dry and semi-arid regions of iran, the amount of annual evaporation is higher than precipitation. in recent years, the cultivation of fruit trees, such as pears, has been limited due to climate change and reduce rainfall. considering that the drought tolerance of the rootstocks is transmitted to the scion (landsberg and jones, 1981), the choice of drought tolerant rootstocks with a low water requirement is one of the solutions to drought problem of pear orchards in arid and semi-arid regions such as iran (cheruth et al., 2009). another problem in the cultivation of pears in iran is the lack of suitable dwarf or semi-dwarf rootstocks that are compatible with cultivars and climatic conditions of the country for high density orchards. the pear and quince are used as rootstocks for pear trees. the quince rootstocks are very dwarf (em-c) and semi-dwarf (ba29), but these rootstocks could not overcome the problem of graft incompatibility with some pear cultivars. these rootstocks are not also tolerant to winter frosts and chlorosis caused by iron deficiency in the calcareous soils (harotko, 2007). in recent years, a number of clonal rootstocks have been introduced into iran such as pyrodwarf, fox-11 and some of the american rootstocks of the old home × farmingdale series (abdollahi, 2011). the use of native pear species as a seed or clonal rootstocks can be a solution for access to proper rootstocks with good adaptability to the climatic conditions of the country. some of these species are dwarf and have a high tolerance to unfavorable environmental conditions that can be used to produce clonal rootstocks (abdollahi et al., 2012). in the pear genus, 22 species have been identified in the world that are mainly native to europe, africa and asia. of the 22 identified species, there are 12 species in iran (sabeti, 1995). in some parts of the world, these species are used as the rootstock for pear commercial cultivars. for example, a large number of pear cultivars in turkey are grafted on p. elaeagrifolia, in syria and lebanon on p. syriaca, in ancient yugoslavia, turkey and greece on p. amygdaliformis, in the south of russia on p. salicifolia and in algeria and morocco are grafted on p. longipes (henareh, 2015). wild germplasm has evolved in natural dry ecosystems for tolerance of stress conditions such as high temperatures, drought and salinity. identification of wild pear germplasm is very important to use as a rootstock in semi-arid regions (zarafshar et al., 2014). according to this, three wild pear genotypes from p. syriaca were exposed to four irrigation treatments. the results showed that the coile wild genotype (p. syriaca) was more tolerant to drought conditions compared with other genotypes due to its high relative water content during drought stress and non-decreasing dry weight. drought stress reduced leaf photosynthesis, stomatal conductance and transpiration in a number of pear species (javadi and bahramnejad, 2011). in another study, drought tolerance was evaluated in three populations of wild pear germplasm (p. boisseriana) in greenhouse conditions. among the three populations studied, the collected population from semi-arid regions showed higher drought tolerance than the other two populations that were collected from semi-humid areas, and recovered more rapidly after irrigation. these seedlings were introduced as promising sources for use as rootstock for commercial pear cultivars in drought conditions (zarafshar et al., 2014). in a research carried out by ghasemi et al. (2014), chlorophyll index was significantly different between studied pistachio rootstocks. in these rootstocks, chlorophyll index in stress conditions was lower than drought conditions. so, the reduction of chlorophyll content can be caused by chlorophyll degradation under drought stress conditions, which leads to reduced pure photosynthesis. the rootstocks of european pear species are from very vigor to relatively dwarf. these rootstocks are compatible with all the pear tree cultivars and have considerable tolerance to the adverse conditions of soil and fire blight disease. other species of pyrus genus, such as p. syriaca boiss is compatible with commercial pear cultivars and is currently used in some countries (radnia, 1996). seed cultivation after cross or open pollination is one of the breeding methods that is frequently used in breeding programs. in this way, it is possible to achieve a wide variation for choice of new rootstocks and cultivars. for example, the manon cultivar was obtained from open pollination of beurre bosc cultivar. the pib-bu3 dwarf pear rootstock was obtained from open pollination of p. longipes, while pi-bu4 and pi-bu7 rootstocks were obtained from open pollination of the p. pyrifolia species (mohan jain and priyadarshan, 2009). currently, most pear cultivars in iran are grafted on different seedling rootstocks of the p. communis tatari selection of drought tolerant pear rootstocks 247 species. considering that wild pear genotypes grow on rocks and dry or low moisture soils, as well as some of them have a small growth in the form of shrubs, so some of them can be used as drought tolerant and dwarf rootstocks for commercial pear cultivars (ashraf and karimi, 1991; henareh, 2015). the aim of this research was an evaluation of drought stress tolerance in some wild pear species, and then selection of the dwarf genotypes among the drought tolerant genotypes. 2. materials and methods plant materials in this research, seeds from open pollination of pyrus syriaca boiss., pyrus salicifolia pall. and pyrus. glabra boiss. along with p. communis l. cv. spadona and dargazi as well khoj including large fruit (khoj no. 1) and small fruit (khoj no. 2) were evaluated. p. syriaca boiss. is distributed from west azarbaijan to fars in the zagros mountains and northwest of iran (abdollahi, 2011). this species has shown compatibility with spadona and kochia pear cultivars and have improved the growth of the scion in calcareous soils. it is currently used as a rootstock in some countries (fallouh et al., 2008). p. glabra species is known as anchuchek in iran, and has spread mainly in the zagros mountains. the seeds of this species are large and are consumed as snacks in fars province (abdollahi, 2011). p. communis is more commonly known as khoj and is scattered in the forests of the north, west azarbaijan, sardasht and baneh in kordestan province. the fruit of this species is very diverse. two types of native khoj including large fruit (khoj n. 1) and small fruit (khoj n. 2) were studied in this research (moazedi et al., 2014). p. communis cv. dargazi is native commercial cultivar of iran that has been introduced as tolerant rootstock (mansouryar et al., 2017). p. communis cv. spadona is high yielding and has high resistance to chlorosis due to iron deficiency. it is also relatively tolerant to psylla and fire blight. p. salicifolia species spread in the northwest of iran, including west and east azerbaijan provinces (abdollahi, 2011). cultivation of seeds fruits of studied species were collected in august and september of 2016 from different regions of west azarbaijan and isfahan provinces as well as northern regions of iran and transferred to the laboratory. the seeds were separated from the flesh and kept in a cool and dry place in paper bags after washing and drying, in order to eliminate the chilling requirement, seeds were cultivated in separate rows in the nursery soil with a sandy loam texture at isfahan agricultural and natural resources research center, in december. from each of these species, 500 seeds were cultivated at intervals of 2-3 cm in the nursery and each produced seedlings were studied as a genotype. during chilling period, adequate moisture was provided and prevented from drying of the culture bed. irrigation of seedlings after emergence of seeds, the seedlings were irrigated for three months in order to establish in the nursery soil. determination of irrigation time based on allowed water depletion of pear trees is after a 50% decrease in humidity, but because of the seedling roots had not yet been sufficiently developed, and needed enough moisture for better growth, so allowed water depletion was considered 35%. in the irrigation intervals, soil moisture at various depths of the soil was measured up to a depth of 100 cm by the time domain reflectometry device (tdr, trase6050x1) (doorenbos and pruitt, 1977; alizadeh, 2006). to obtain the amount of irrigation (irrigation volume), first, net irrigation depth was calculated according to formula 1. in= ∑ [(θfci θbli)×di] (1) in this formula, in is the net irrigation depth (mm), θfci is the moisture content of the field capacity for each layer, θbli is the soil moisture before irrigation for each layer, di is the root development depth (mm), and i is the number of each soil layer. then, according to formula 2, gross irrigation depth was calculated. ig = in/(1-lr)×ei (2) in this formula, ig is the gross irrigation depth (mm), in is the net irrigation depth (mm), (1-lr) is the amount of leaching and ei is the irrigation efficiency (usually 80-90% for drip irrigation). irrigation volume was obtained from the multiplication of gross irrigation depth in the irrigation plot area. this volume was controlled by the installed counter on the pipe before irrigation plot. for irrigation of seedlings leaked tubes with drippler at 10 cm intervals were used. the outlet flow of each drippler was measured in one liter per hour at an appropriate pressure. in this study, the efficiency of the drip system and the leaching requirement was considered 90% and 10% respectively. adv. hort. sci., 2019 33(2): 245-255 248 drought stress drought stress began in july, which coincided with the beginning of the stress period in isfahan region. irrigation frequency was changed in order to apply drought stress, and irrigation time was considered based on 80% of allowed water depletion. table 1 shows the volume of pure and impure irrigation water and number of irrigation per month. evaluated traits the morphological traits of seedlings were recorded separately for each genotype at the end of the stress period (late september). these traits included height and diameter of seedlings, number and length of internode, crown width, number of suckers and branches, chlorophyll index and leaf dimensions. before applying stress, seedling height and diameter at 5 cm above the soil surface were recorded. the difference in both seedling height and diameter before and after stress were also calculated. qualitative traits, including leaf chlorosis and trichome, as well as seedling growth vigor were recorded after stress using national guideline for distinctness, uniformity and stability in pear (dus guileline) in pears. the abbreviation and measurement method of evaluated traits are given in table 2. data analysis mean comparison was calculated with sas software (version 9.1). descriptive statistics including mean, minimum, maximum and coefficient of variation and also cluster analysis were performed by ward method based on squared euclidean distance with spss software (version 15). 3. results viability percent viability percent of seedlings of the studied pear species after the stress was shown in table 3. according to the results, p. salicifolia showed the highest survival in drought stress conditions. after that, p. communis cv. khoj n. 1 and 2 were placed in the next rank. the lowest percentage of seedling viatable 2 symbol and measurement method for recorded traits (based on dus guideline) table 1 number of irrigation, net and gross volume of irrigation water irrigation february march april may june july august september october november total number of irrigation 3 3 5 7 8 1 1 1 1 1 32 net volume of irrigation water (m3.ha-1) 540 540 900 1260 1440 180 180 180 180 180 5580 gross volume of irrigation water (m3.ha-1) 666.6 666.6 1111.1 1555.5 1777.7 222.2 222.2 222.2 222.2 222.2 6888.2 characteristic symbol unit measurement method leaf chlorosis lc code no chlorosis (1), low chlorosis (3), medium chlorosis (5), high chlorosis (7), very high chlorosis (9) leaf trichome lt code no trichome (1), low trichome (3), medium trichome (5), high trichome (7), very high trichome (9) seedling growth vigor gv code very low (1), low (3), medium (5), high (7), very high (9) seedling height se cm ruler seedling diameter sd mm caliper height difference hd cm calculation diameter difference dd cm calculation internode number in number counting internode length il cm ruler (average of internodes in branches) crown width cw cm meter number of secondary branches nsb number counting number of suckers ns number counting chlorophyll index ci chlorophyll meter (spad) leaf length ll cm ruler (average of 10 leaves) leaf width lw cm ruler (average of 10 leaves) table 3 seedlings viability percent of pear cultivars and species after drought stress species viability percent pyrus communis cv. spadona 14.77 pyrus communis cv. dargazi 14.28 pyrus communis cv. khoj n. 1 55.17 pyrus communis cv. khoj n. 2 43.79 pyrus syriaca 20.33 pyrus salicifolia 82.55 pyrus glabra 17.74 tatari selection of drought tolerant pear rootstocks 249 were lower than other species. similarly, p. communis cv. spadona showed the highest height difference before and after applying stress. the seedling diameter of p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca was more than the other seedling diameter, so that p. glabra and p. communis cv. khoj n. 2 had the lowest seedling diameter. no significant difference was observed among the seedling diameter of the species before and after drought stress. p. communis cv. spadona had the highest number of internode after the end of drought stress. the lowest number of internode belonged to p. glabra and p. communis cv. khoj n. 2. p. salicifolia produced the crown with the widest (24.71 cm) width, compared to other species. it should be noted that p. communis cv. spadona, khoj n. 1 and p. syriaca did not show any significant difference with pyrus salicifolia. p. communis cv. khoj n. 2 had the lowest crown width (4.75 cm). p. salicifolia did not produce a secondary branch. there were no significant differences in the number of secondary branches among the other species. p. salicifolia showed the highest chlorophyll index, but did not show significant differences with p. communis cv. spadona, khoj n. 2, p. syriaca and p. glabra. the lowest chlorophyll index belonged to p. communis cv. dargazi (table 5). p. communis cv. spadona , with a length of 6.06 cm and a width of 2.7 cm had the highest leaf length and width compared with other species. after p. communis cv. spadona, p. communis cv. khoj n. 1 had the highest leaf length and width. the lowest leaf length was related to p. communis cv. khoj n. 2 with an average of 2.01 cm. p. syriaca also produced the leaves with the lowest width (1.37 cm). p. communis cv. spadona and dargazi had the longest internode, but did not show significant differences with p. communis cv. khoj n. 1 and p. syriaca. bility during drought stress belonged to p. communis cv. spadona and dargazi. traits before applying stress the mean comparison of recorded traits among populations before applying drought stress is presented in table 4. according to the results, p. communis cv. spadona had the highest seedling height. the seedling height in the wild species of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 was the lowest. p. communis cv. khoj n. 2 and p. glabra produced seedlings with the lowest diameter. the largest seedling diameter belonged to p. communis cv. spadona and p. syriaca (table 4). generally, without drought stress conditions, growth of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 were lower than p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca. traits after applying stress the mean comparison of recorded traits among populations after stress is presented in table 5. p. communis cv. spadona had the highest seedling height (36.73 cm) after stress. the seedling height of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 table 4 the mean comparison of the seedling height and diameter among populations of pear species before applying stress similar letters in each column indicate no significant difference (lsd). table 5 mean comparison of some traits in the seedling populations of pear species after applying stress similar letters in each column indicate no significant difference (lsd). species seedling height (cm) seedling diameter (mm) pyrus communis cv. spadona 33.52 a 3.44 a pyrus communis cv. khoj n. 1 23.38 b 3.15 ab pyrus communis cv. dargazi 24.23 b 3.15 ab pyrus syriaca 20.92 b 3.31 a pyrus salicifolia 12.80 c 2.62 bc pyrus communis cv. khoj n. 2 11.83 c 1.74 d pyrus glabra 7.54 c 2.25 cd species seedling height (cm) height difference (cm) diameter (mm) diameter difference (mm) internode number crown width (cm) number of secondary branches chlorophyll index leaf length (cm) leaf width (cm) internode length (cm) pyrus communis cv. spadona 36.73 a 3.20 a 4.34 a 0.89 a 20.23 a 10.03 ab 0.23 ab 44.99 ab 6.06 a 2.70 a 1.91 a pyrus communis cv. khoj n. 1 26.65 b 2.80 ab 4.00 a 0.84 a 14.56 b 10.62 ab 0.68 a 42.48 b 5.45ab 2.23 b 1.21 ab pyrus communis cv. dargazi 25.83 b 1.60 ab 3.81 a 0.66 a 14.66 b 7.83 b 0.66 a 42.02 b 4.73 bc 2.10 bc 1.93 a pyrus syriaca 21.60 b 1.41 ab 4.02 a 0.70 a 12.91 bc 9.16 ab 0.25 ab 49.77 ab 4.50 c 1.37 d 1.34 ab pyrus salicifolia 14.21 c 0.67 b 3.56 ab 0.94 a 10.05 cd 11.24 a 0.00 b 53.83 a 4.30 c 1.73 c 0.94 b pyrus communis cv. khoj n. 2 13.67 c 0.84 b 2.63 c 0.88 a 8.31 de 4.75 c 0.21 ab 46.53 ab 2.01 d 1.97 bc 0.71 b pyrus glabra 10.22 c 0.68 b 2.92 bc 0.66 a 5.27 e 8.31 b 0.36 ab 48.85 ab 3.98 c 2.10 bc 0.73 b 250 adv. hort. sci., 2019 33(2): 245-255 the lowest internode length belonged to p. salicifolia, p. glabra, and p. communis cv. khoj n. 2 (table 5). cluster analysis of populations according to the results of cluster analysis, species were classified into three groups at five squared euclidean distance basis of the ward method (fig. 1). p. communis cv. spadona and dargazi were placed in the first group. p. syriaca and p. communis cv. khoj n. 1 in the second group, while p. communis cv. khoj n. 2, p. salicifolia and p. glabra species in the third group. study of single genotypes the mean and the range of traits for each of the examined genotypes, as well as the coefficient of variation in each trait are presented in table 6. each genotype was identified by a number and genotypes with minimum and maximum values were presented. internode length (66.12%), seedling height before stress (60.45%) and seedling height after stress (56.85%) had the highest and chlorophyll index (14.34%) had the lowest coefficient of variation. in order to achieve dwarfing genotypes, the trait of seedling height was considered. the three pear populations, including p. salicifolia, p. glabra and p. communis cv. khoj n. 2 had a lower mean seedling height than other populations, so the height of single genotypes of them after stress was shown in figures 2, 3 and 4, respectively. in p. salicifolia, genotypes no. 31 (4.5 cm), 32 (5 cm), 41 (5.5 cm), 57 (6 cm) and 12 (6 cm) had the lowest seedling height. genotypes fig. 2 height of single genotypes in p. salicifolia after stress. table 6 mean, range and coefficient of variation in studied traits fig. 1 grouping of pear species based on measured characteristics by ward method. characteristics cv (%) standard deviation mean minimum maximum genotype (number of genotypes) rate genotype (number of genotypes) rate seedling height before stress 60.45 9.83 16.26 p. glabra (42) 1.4 p. communis cv. spadona (23) 53.9 seedling diameter before stress 28.35 0.76 2.68 py. communis cv. khoj n. 2 (33) 0.75 p. communis cv. spadona (64) 6.3 seedling height after stress 56.85 10.28 18.08 p. glabra (10 & 11) 3 p. communis cv. spadona (2) 55 seedling diameter after stress 26.47 0.94 3.55 p. communis cv. khoj n. 2 (8) 1.6 p. salicifolia (8) 7.28 height difference 55.32 2.19 1.82 p. salicifolia (24 & 28) 0 p. communis cv. khoj n. 1 (9) & p. syriaca (3, 4,5,6 & 7) 19 diameter difference 55.17 0.48 0.87 p. salicifolia (47) 0.04 p. salicifolia (66) 3.06 internode number 44.65 5.00 11.20 p. glabra (7,10 & 11) 3 p. communis cv. khoj n. 1 (14) 26 crown width 30.67 3.11 10.14 p. communis cv. khoj n. 1 (5) & p. glabra (10) 4 p. communis cv. khoj n. 1 (8) 30 number of secondary branches 37.22 0.67 0.18 many genotypes 0 p. communis cv. khoj n. 2 (4) 4 chlorophyll index 14.34 7.81 54.46 p. communis cv. khoj n. 1 (8) 23.2 p. salicifolia (36) 69.8 leaf length 31.93 1.37 4.29 p. communis cv. khoj n. 2 (15) 1 p. communis cv. spadona (13) 7.2 leaf width 26.70 0.51 1.91 p. syriaca (5) 1 p. communis cv. spadona (2) 3.3 internode length 66.12 0.82 1.24 p.salicifolia (30) 0.3 p. communis cv. spadona (1) 10 tatari selection of drought tolerant pear rootstocks 251 no. 10 (3 cm), 11 (3 cm), 3 (6 cm), 9 (6 cm) and 7 (6 cm) showed the lowest seedling height in the p. glabra species. genotype no. 4 had the lowest height from p. communis cv. khoj n. 2 population. cluster analysis of single genotypes based on all studied traits cluster analysis among the single genotypes of the p. salicifolia, p. glabra and p. communis cv. khoj n. 2 populations was shown in figures 5, 6 and 7. genotypes no. 12, 31, 32, 41 and 57 of p. salicifolia (fig. 5) and genotypes no. 10, 11, 3, 9 and 7 of p. glabra (fig. 6) were placed in the groups close to each other. qualitative traits for selected genotypes were presented in table 7. all genotypes had a small trichome and had low or very low growth potentials. leaf chlorosis was not observed in genotypes except for the genotype no. 31 from p. salicifolia and genotype no. 3 from p. glabra that had low chlorosis. genotypes no. 12 and 32 of p. salicifolia were green and very green, respectively. genotypes no. 9, 7, 10 and 11 of p. glabra and genotype no. 4 of the p. communis cv. khoj n. 2 preserved their green color after applying drought stress. 4. discussion and conclusions viability percent after the drought stress period, 35.51% of the genotypes survived, and the rest of them were dried. most surviving genotypes belonged to p. salicifolia species. p. communis cv. spadona and dargazi fig. 3 height of single genotypes in p. glabra after stress. fig. 4 height of single genotypes in p. communis cv. khoj n. 2 after stress. fig. 5 grouping of drought tolerant genotypes in p. salicifolia species based on measured traits by ward method. fig. 6 grouping of drought tolerant genotypes in p. glabra species based on traits measured by ward method. adv. hort. sci., 2019 33(2): 245-255 252 showed the lowest percentage of survival (table 3). for a long time, wild pear genotypes have been considered in iran’s plateau due to the tolerance to biotic and abiotic stresses (javadi et al., 2005). the adaptation of wild pears with rocky areas and dry or low moisture soils can lead to more tolerance of them under drought stress conditions compared with domestic and commercial rootstocks (henareh, 2015). traits before and after applying stress the analysis of variance showed that the investigated species had a significant difference in most of the studied traits, which is due to the diversity among populations, so it is possible to select species for different values of a trait. according to table 4, in normal conditions, seedling height and diameter of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 were less than p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca. similarly, after applying drought stress, the seedling height and diameter of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 species were lower than other populations (table 5). morphological adaptations in plants can be one of the adaptive mechanisms under drought stress (pire et al., 2007). the first reaction of plants against drought stress is a reduction in their vegetative growth. drought stress affects the vegetative characteristics of trees, including their height (higgs and jones, 1990). due to height difference among populations before and after stress, it seems that the effect of drought stress on the seedling height trend of these populations before and after applying the stress is almost same. before drought stress, the seedling diameter of p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca was higher than the seedling diameter of p. salicifolia, p. glabra and p. communis cv. khoj n. 2, while the diameter difference before and after drought stress among compared species did not show a significant difference, therefore, it can be concluded that the effect of drought on the seedling diameter among populations was not the same. other results also showed that the negative effect of drought stress on seedling diameter was less than its effect on seedling height (haghighatnia et al., 2013). growth of branch and internode length is an appropriate index for detecting the effect of drought stress on the plants, so that the occurrence of drought stress can be observed even before the change in the water potential of the leaves (grimplet et al., 2007). among the remaining genotypes after the stress, p. communis cv. spadona had the highest and p. glabra and p. communis cv. khoj n. 2 had the lowest number and length of internode. the chlorophyll meter indicates the relative fig. 7 grouping of drought tolerant genotypes in p. communis cv. khoj n. 2 based on the measured traits by the ward method. table 7 growth vigor, trichome and chlorosis of leaf in selected genotypes species no. genotype leaf chlorosis leaf trichome seedling growth vigor pyrus salicifolia 12 no chlorosis low trichome low 31 low chlorosis low trichome low 32 no chlorosis low trichome low 41 no chlorosis low trichome low 57 no chlorosis low trichome low pyrus glabra 3 low chlorosis low trichome low 9 no chlorosis low trichome low 7 no chlorosis low trichome low 10 no chlorosis low trichome very low 11 no chlorosis low trichome very low pyrus communis cv. khoj n. 2 4 no chlorosis low trichome low tatari selection of drought tolerant pear rootstocks 253 chlorophyll concentration, based on the difference between the light transmittance in two red and infrared wavelengths, which correlates with the chlorophyll content of the leaves (hoel and solhaug, 1998). in the present study, p. salicifolia and p. communis cv. dargazi showed the highest and the lowest chlorophyll index, respectively. preservation and not decomposition of chlorophyll in p. salicifolia during drought stress indicates the tolerance of that species to this stress (tarahomi et al., 2010). cluster analysis of populations drought tolerance in plants has a direct or indirect relationship with a complex of traits, therefore, all the traits should be considered for selection of tolerant plants. for this reason, in this research cluster analysis was used to facilitate the selection of drought tolerant species. cluster analysis classified species into three groups at five squared euclidean distance (fig. 1). p. communis cv. spadona and dargazi were placed in the first group that had the lowest survival rate after drought stress. the highest seedling diameter belonged to the plants of this group. in the second group, p. syriaca and p. communis cv. khoj n. 1 were placed, which showed low to moderate survival rate after drought stress. in total, the most crown width, primary and secondary seedling height and diameter, as well as the highest number of internode were observed in the species of the first and second groups. p. communis cv. khoj n. 2, p. salicifolia and p. glabra species formed the third group. these populations had the lowest seedling diameter and height. the lowest seedling growth vigor was also found in these species; therefore, selection of dwarf and drought tolerant genotypes in this group was more possible than other groups. it seemed that traits such as seedling height and diameter were the most effective grouping traits. in the research carried out by aran et al. (2012), seedling height was also one of the important traits in seedling grouping. of course, different values of some traits were seen in this group. for example, the crown width and viability percentage were observed at low, medium and high levels in this group. study of single genotypes the mean, the range of changes, and the coefficient of variation of each trait were shown in table 6. coefficient of variation shows the extent of variability in relation to the mean of the population. in the traits with a high coefficient of variation has provided a higher selection range. genetic variation helps the plant to overcome environmental changes and also provides more chance for selection of new cultivars (liu, 2006). in this research, high variation was observed for internode length and seedling height before and after stress. the variability of some traits in 15 cultivars of vitis vinifera l. was previously investigated by mousazadeh et al. (2014). they reported that leaf traits had the highest diversity among the studied traits. doulati baneh et al. (2013) and tahzibihagh et al. (2012) also observed a high variation in the morphological characteristics of the leaves of grape and pear cultivars. in the current research, the coefficient of variation in leaf dimensions was not high compared to other traits, because of less scatter in relation to the mean of the population. the high diversity coefficient for internode length and seedling height before and after stress indicates the high range of variation in these traits among the studied seedlings, so it is possible to select genotypes based on these two traits. survived genotypes after drought stress were selected in order to dwarfing. for this purpose, seedling height of each genotype was investigated separately. considering that the average height of three populations including p. salicifolia, p. glabra and p. communis cv. khoj n. 2 were lower than other populations, therefore selection was carried out among genotypes of these populations. the highest number of genotypes was selected from p. salicifolia. the least seedlings height was belonged to p. glabra. only a dwarf genotype was selected from the p. communis cv. khoj n. 2 population (figs. 2, 3 and 4). finally, 11 dwarf genotypes were selected from these species. cluster analysis of single genotypes cluster analysis was performed for three populations with a lower seedling height. according to this, five selected genotypes of p. salicifolia (fig. 5), five genotypes of p. glabra (fig. 6) and a genotype of the p. communis cv. khoj n. 2 population (fig. 7) were classified in the same subgroups. these genotypes had lower seedling height and internode number. leaf width was also lower in these genotypes. qualitative traits of 11 selected genotypes showed that all genotypes had low trichome. the survival and tolerance of these genotypes to drought stress was not along with the increase of leaf trichome. the growth vigor of genotypes was low, and two genotypes 10 and 11 from p. glabra had very low growth vigor. these genotypes also had the lowest seedling height. in fact, their growth vigor was correlated with the seedling height. this positive correlation was observed between seedling height and adv. hort. sci., 2019 33(2): 245-255 254 growth vigor in bitter cherry seedlings (mahlab) (ganji-moghadam and khalighi, 2006). under drought stress, studied genotypes did not show chlorosis, only genotypes 3 of p. salicifolia and 31 of p. glabra were greenish-yellow after drought stress. selected genotypes were transferred to a propagation phase through cutting and layering. after studying single genotypes in these three populations, genotypes no. 31, 32, 41, 57 and 12 from p. salicifolia, genotypes 10, 11, 7, 3 and 9 of p. glabra and genotype no. 4 of p. communis cv. khoj n. 2 populations were selected as drought tolerant and dwarf genotypes and were taken to the propagation phase for future evaluation. references abdollahi h., 2011 pear, butany, cultivars and rootstocks. publication of agricultural education, iranian ministry 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however, some obstacles exist. an extreme decline in tree vigour, so called “soil sickness”, has long been a serious obstacle for fig culture in japan (hirai and nishitani, 1953; hosomi, 2011). although the exact cause of soil sickness is still unclear, there exist several causal hypotheses, e.g., toxic chemicals (hatsuda et al., 1960), nematode pests (condit, 1947; sato and shichijo, 1953), and some soil­borne diseases (hosomi and uchiyama, 1998). it is a common characteristic that some trees in a field will begin to weaken within a few years after planting and that normal growth cannot be expected merely by replanting (hirai and nishitani, 1953). however, it is unknown whether soil sickness damage increases year by year and finally kills the trees, or if the soil does not degenerate further and sometimes recovers. in other fruit, it is known that rooted (*) corresponding author: vividlyoceanly@gmail.com citation: hosomi a., 2020 ­ stable growth inhibition of potted fig (ficus carica l.) trees by soil sicknes. ­ adv. hort. sci., 34(4): 449­453 copyright: © 2020 hosomi a.this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2020 accepted for publication 18 february 2020 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 449­453 450 cutting size influences growth inhibition by sick soil (iwasaki, 1962; hirano, 1968), but it is also unknown for fig trees whether such initial planting condition influences subsequent inhibition by sick soil for long periods. in the present study, yearly changes in shoot growth were investigated using potted ‘masui dauphine’ fig trees with sick soil. whether the timing of planting, physical disturbing of rooting or the size of rooted cuttings influence growth inhibition by sick soil, have also been investigated. the results will aid in recognition of the infestation process of soil sick­ ness, and give information for a replanting plan of cultivated fig trees. 2. materials and methods yearly shoot growth of potted fig trees with sick soil (exp. 1) every april from 1990 to 1994, the rooted cuttings of ‘masui dauphine’ figs were planted in 10­liter unglazed pots filled with a mixture of sandy soil, kanuma soil and vermiculite (1:1:1, v/v/v). these were grown for 9 years in the open. in each june of the 1st planting year, 500 milliliter (ml) of the stored sick soil, which had been collected from fig orchards exhibiting the symptoms of soil sickness (hosomi and uchiyama, 1998), were added to the bed surface of 4 to 8 trees (28 trees in total). four to 5 trees in each planting year (23 trees in total) were controls. each surface of the bed (including control) was mulched with 500 ml of peat moss. fifty ml of slow­release fertilizer (100 days type n: p2o5: k2o=16: 5:10 plus micronutrients) was applied each june. irrigation was applied automatically to prevent soil drying. one shoot per tree was elongated and the other shoots were disbudded. each march, the shoots were pruned above their 2nd or 3rd node from the base and the dry weight was measured. growth inhibition by soil sickness under conditions of planting timing with/without physical barriers to rooting (exp. 2) in december 1999, 18 rooted cuttings of ‘masui dauphine’ figs was raised in individual bag (22×5 cm) with vermiculite under the greenhouse heated at 15­ 25°c. the bags were made of non­woven fabric, which had been treated by cupric hydroxide (spin out; griffin co.) to prevent root spiraling. two shoots per rooted cutting were elongated. in february 2000 the rooted cuttings were removed from the bag and planted in 25­liter plastic pots filled with a mixture of kanuma soil and vermiculite (1:1, v/v). each 2.3­liter root area in 6 rooted cuttings was enclosed with non­ woven fabric (polylactic acid span­bond 100 g/m2) at planting. two­liter of the stored sick soil in experi­ ment 1, was added to each bed surface at 25 days after planting. the sick soil was also added to each bed of other 6 rooted cuttings without enclosing the roots. the remaining 6 rooted cuttings were controls. soon after the sick soil addition, each surface of the beds (including controls) was mulched with 1.5­ to 2­ liter of peat moss. the other 36 rooted cuttings were likewise raised by cuttings from april 2000, and were planted in 25­liter plastic pots in june 2000. at that time 12 rooted cuttings had the root areas enclosed and were added with sick soil, a further 12 rooted cuttings were added with sick soil alone, and the other 12 were controls. the trees were grown for 3 years in unheated greenhouse ventilated by roof and side vents. one hundred ml of slow­release fertilizer in experiment 1 was applied to each tree in march (february planti­ ng) or july (june planting) in the 1st year, and in june in the 2nd and 3rd year. irrigation was applied auto­ matically to prevent soil drying. only 2 shoots per tree were elongated, and all other shoots were dis­ budded each year. each march from 2001, the shoots were pruned above their 2nd or 3rd node from the base and the dry weight was measured. growth inhibition by soil sickness under conditions of different rooted cutting size (exp. 3) from april 1998, the rooted cuttings of ‘masui dauphine’ figs were raised by cuttings in 1.3­liter, 10­ liter and 18­liter plastic pots filled with vermiculite with 6.5 ml, 50 ml and 90 ml respectively of slow­ release fertilizer in experiment 1. the inner walls of these pots had previously been painted with cupric hydroxide (spin out; griffin co.) to prevent root spi­ raling. one shoot per tree was elongated and all other shoots were disbudded. in june 1999, the shoots were pruned and their length, basal diameter and dry weight was measured. i also took the rooted cuttings from the pots, washed off the soil, and calcu­ lated the root volume from the loss in weight when the roots were dipped in water. thus, each of 10 rooted cuttings in 3 size categories (s, m and l) were prepared as in table 1 and were planted to 60­liter plastic pots filled with a mixture of kanuma soil and vermiculite (1:1, v/v). two weeks after planting, 5­ liter of the stored sick soil in experiment 1 was added to the bed surface of 6 trees in each category. four hosomi ‐ damage stability of fig soil sickness 451 trees in each category were controls. each surface of the beds (including controls) was mulched with 1.5­ liter of peat moss, and all trees were grown for 4 years in the open. three hundred ml of slow­release fertilizer in experiment 1 was applied each june. irrigation was applied automatically to prevent soil drying. three shoots per tree in the 1st year, and 6 shoots in subsequent years, were elongated and all other shoots were disbudded. each march the shoots were pruned above their 2nd or 3rd node from the base and dry weight was measured. 3. results and discussion yearly shoot growth of potted fig trees with sick soil the results are shown in figure 1. for the control trees, the average dry weight of shoot (shoot weight) varied in the range of 48 to 80 g. for trees with sick soil, the shoot weight was 49 g (81% of control) in the 1st year, decrease in the 2nd year and was main­ tained the range of 24 to 29 g (32 to 50% of control) in subsequent 8 years. only 2 trees died in the sick soil during the testing period. in other word, the sick soil, added to bed soil, inhibit growth of 10­liter pot­ ted fig trees from the 1st planting year. it takes 1 year to confirm the growth inhibition, after which sick soil cause stable dwarf growth for many years without trees dying. growth inhibition by soil sickness under conditions of planting timing with/without physical barriers to rooting the results are shown in figure 2. for control trees, the average dry weight of total shoot per tree table 1 ­ rooted cuttings used in the test examining the effect of its size on sick soil damage z yes (+) or no (­) of scheduled sick soil inoculation. y only one shoot per rooted cutting was elongated, pruned before planting and its size was measured. x estimated from the loss in rooted cutting weights when dipping roots in water. w mean±se. size categories silck soil inoculation z shoot y root volume x length (cm) basal diameter (mm) dry weight (g) in each group (cm3) in each size (cm3) large (l) + 116.5±5.5 w 15.8 ± 0.4 43.5±2.4 369.7±63.4 357.6 ­ 126.5±6.9 15.3 ±0.7 40.7±5.8 339.5±65.9 medium (m) + 107.8±5.0 14.4±0.5 33.4±3.2 296.8±32.9 280.2 ­ 116.8±4.8 14.0±1.0 33.8±4.4 255.3±53.2 small (s) + 65.5±2.1 11.4±0.2 11.1±0.4 148.5±26.1 168.2 ­ 66.5±4.3 12.8±0.3 13.0±1.3 197.8±20.9 fig. 1 ­ yearly change in dry weight of shoot per ‘masui dauphine’ fig trees grown in 10­liter potted with/without sick soil. vertical bars indicate se. fig. 2 ­ dry weight of total shoot per ‘masui dauphine’ fig trees grown in 25­liter pots with/without sick soil under condi­ tions of planting timing (february and june) with/with­ out root­enclosing by non­woven fabric. vertical bars indicate se. different letters indicate significance among conditions in each age at 5% level by tukey­hsd test using r project 3.6.1. adv. hort. sci., 2020 34(4): 449­453 452 (total shoot weight) varied in the range of 88 to 445 g, and the values of the trees planted in june was apparently less than those planted in february in the 1st and 2nd years, and equalized with them in the 3rd year. for trees with sick soil, the total shoot weight var­ ied in the range of 89 to 223 g, less than in controls. these values for june planted trees were less than for february planted ones in the 1st year, but were equalized form the 2nd to the 3rd year. for trees with sick soil plus root­enclosing by non­woven fab­ ric, the total shoot weight varied in the range of 66 to 149 g. in early years for february planting trees, inhibited growth by sick soil plus root­enclosing tend­ ed to be less than those with sick soil alone. uchida et al. (1998) reported that root wrapping materials such as woven flax disturbed the initial root growth of deciduous trees (magnolia kobus and quercus acutissima) for up to 10 months after planting. in this experiment the fabric may also disturb root elonga­ tion before rooting out, and may act as a growth inhibitor in the early growing period. for june planting trees, however, no effect to the corresponding control was detected in sick soil appli­ cation in the 1st year, and in root­enclosing in all tested years. hirano (1968) reported that, in peach prunus persica batsch trees, growth inhibition by sick soil infestation was greater when vigorous growth was expected. it seems that the damage of sick soil and root­enclosing of this study were masked under delayed growth conditions due to the late planting. growth inhibition by soil sickness under conditions of different rooted cutting size the results are shown in figure 3. for control trees, the dry weight of total shoot per tree (total shoot weight) was about 125 g in the 1st year and did not differ between the rooted cutting size (l, m and s). after the 2nd year the values increased, and var­ ied in the rage of 230 to 336 g with a tendency to be greater in size­s. ishimaru et al. (2003) reported that a smaller size of the rooted cuttings cause high rela­ tive initial growth rate in 3 species of broad­leaved tree (quercus serrata, q. glauca and myrica rubra). the smaller rooted cuttings in this experiment also increase shoot vigour in the early period, and over­ come the initial handicap of the tree biomass. for trees with sick soil, the total shoot weight was about 107 g (86% of control) in rooted cutting size­l and m, and 70 g (56% of control) in size­s in the 1st year. values were about 73 g in the 2nd year, 169 g in the 3rd year and 196 g in the 4th year, and appar­ ently less than in controls, but did not differ between the size categories. in other word, the smallness of the rooted cutting intensified the sick soil damage in the planting year. however after the 2nd year from planting, that damage intensification and the vigour of small rooted cuttings compensate for each other and the shoot growth of every group with sick soil equalized. it had been reported that growth inhibi­ tion by sick soil is severe in smaller rooted cuttings in satsuma mandarin citrus unshiu (iwasaki, 1962) and peach (hirano, 1968). my results are not inconsistent with these reports, because the phenomena they report were observed only in the 1st planting year. the results of my 3 experiments suggest that tree growth with sick soil may stabilize to a particular level determined by the pot size. longer times may be required for stabilization in larger pots with sick soil: one year in experiment 1 (10­liter pots), over 2 years in experiment 2 (25­liter pot) and over 3 years in experiment 3 (60­liter pot). probably the longer terms for stabilization are because larger pots take longer to full with roots. the initial conditions, such as the timing of planting, physical barriers to rooting, and rooted cutting size, affect tree growth and observed damage due to soil sickness for the early planting period. however, the initial delays in growth made up, and did not determine future growth inhi­ bition by sick soil. hosomi and uchiyama (1998) reported that parasitization by some microorganisms are most important factors in sick soil in fig orchards. stable inhibition of soil sickness may be because root regeneration activity in certain restricted root zone balances the damage caused by these parasites. in fig. 3 ­ dry weight of total shoot per ‘masui dauphine’ fig trees grown in 60­liter pots with/without sick soil under condi­ tions of different rooted cutting size. the rooted cuttings in 3 size categories (s, m and l) were prepared as in table 1. vertical bars indicate se. different letters indi­ cate significance among conditions in each age at 5% level by tukey­hsd test using r project 3.6.1. hosomi ‐ damage stability of fig soil sickness 453 peach orchards, yamada and ono (1970) observed that the trees grew for 7 years with a constant differ­ ence between with and without sick soil. in fig orchards, it is also estimated that a stable dwarfish growth is maintained for many years in sick soil. i can expect an effect to overcome the problem in a meth­ ods to accelerate tree growth permanently, for example a vigour root stock (hosomi et al., 2002), and are unable to expected it in devising the meth­ ods of planting. all results in present study use pot­ ted trees in which rooting is restricted physically by the pot wall. field tests with free rooting and sick soil are needed to learn more about these competing effects. 4. conclusions dwarfish growth by sick soil inoculation in potted ‘masui dauphine’ figs was maintained form the 2nd to 9th years after inoculation, resulting in few deaths. the initial planting conditions, such as planting timing (february and june), non­woven fabric as physical barriers to rooting, and rooted cutting size, all influ­ ence the observed damage caused by sick soil in the initial growth of those potted trees. however, in sub­ sequent years, their growth converged to similar lev­ els of weakness. a certain and stable growth inhibi­ tion on figs over many years, irrespective of planting condition, seems to be a basic characteristic of soil sickness in fig culture. references condit i.j., 1947 ­ insects and other pests, pp. 177­187. ­ in: condit i.j. (ed.) the fig. chronica botanica co., waltham, mass., usa, pp. 222. condit i.j., 1955 ­ fig varieties: a monograph. ­ hilgardia, 23(11): 323­538. hatsuda y., murao s., terashima n., yokota t., 1960 ­ biochemical studies on the soil sickness. part 1. on the toxic substance in fig roots. ­ nippon nogeikagaku kaishi, 34(6): 484­486. hirai j., nishitani k., 1953 ­ studies on the fig sick soil. (iv) effect of successive planting upon the growth. ­ studies from the institute of horticulture, kyoto university, 6: 32­34. hirano s., 1968 ‐ studies on peach sick soil. vi. some fac‐ tors affecting the growth of replants in old peach soil. ­ j. japan. soc. hort. sci., 37(3): 192­198. hosomi a., 2011 ­ incidence of soil sickness in areas producing ’masui dauphine’ figs. ­ bun. res lnst. env. agr. fish. osaka, 4: 9­13. hosomi a., dan m., kato a., 2002 ­ screening of fig vari‐ eties for rootstocks resistant to soil sickness. ­ j. japan. soc. hort. sci., 71(2): 171­176. hosomi a., uchiyama t., 1998 ­ growth inhibiting factors in sick soil of fig orchards. ­ j. japan. soc. hort. sci., 67(1): 44­50. ishimaru k., iwama t., osawa n., takeda h., 2003 ­ effect of sapling size on erosion control plantation. ­ j. jpn. soc. reveget. tech., 29(1): 39­44. iwasaki t., 1962 ­ growth inhibiting factors and counter‐ measure in sick soil of mandarin orange orchards. ­ kajitunihon, 17(1): 41­43. sato k., shichijo t., 1953 ­ growth of young fig trees in old fig orchard soils. ­ j. japan. soc. hort. sci., 22(3): 163­166. uchida h., kato m., muramoto j., hagiwara n., 1998 ­ evaluation of the above‐grand and root growth of deciduous trees when different root‐wrapping materials are used. ­ j. jila 61(5): 487­492. yamada y., ono t., 1970 ­ study on the replanting prob‐ lem of peach 1. on the growth of young peach trees grown in old peach orchards. ­ bull. yamanashi. fruit tree exp. stn., 2: 1­14. 301 adv. hort. sci., 2019 33(3): 301-312 doi: 10.13128/ahs-23731 descriptions of okra seed longevity loss behavior using nonlinear regression models g. parmoon 1, s.a. moosavi 2 (*), s. ataollah siadat 2 1 department of agronomy, faculty of agriculture, university of mohaghegh ardabili, ardabil, iran. 2 department of plant production and genetics, faculty of agriculture, agricultural sciences and natural resources, university of khuzestan, khuzestan, iran. key words: deteriorations, gompertz, logistic, regression, viability. abstract: seeds longevity loss is an inevitable problem of seed storage. applications of nonlinear regression models to describe and predict the aging damages to seed germination would be reliable and helpful for understanding the relationship between seed quality and storage condition. in this study, various nonlinear models, including logistic, hill, weibull, sigmoid, gompertz and probit, were applied on seed germination data, obtained from the accelerated aging test of three iranian okra landraces. results revealed that the weibull 4 parameter and probit 4 parameter functions failed to describe cumulative germination of ahwaz ecotype in contrast to sigmoid models. the best three parameters sigmoid model to describe germination data of isfahan ecotype was hill 3p (aicc=26.89) while there was a failure to fit germination data using weibull 4p and probit 4p. mashhad germination and vigor was well described using hill 3p (aicc=33.72 and 32.22). it is suggested that the use of the hill, gompertz and weibull parameters provided more information of viability and vigor loss of okra seeds during deterioration conditions. 1. introduction okra is a crop belonging to malvaceae family, largely cultivated in africa and asia (düzyaman, 2005; sorapong, 2012). it is mainly planted for its fresh, tender and tasty pods as food purposes alongside high generic medical or industrial potential applications (dhankhar and singh, 2009). recent researches revealed that okra seed oil has a great potential use as industrial products and liquid biofuels (anwar et al., 2010; moosavi et al., 2018). good seed quality assures rapid, uniform seed germination and healthy seedling establishment. seed germination as the complex physiologically active process is initiated with water uptake by dry seeds and completed by radicle protrusion from the seed coat (bewley and black, 1994; mcdonald and kwong, 2005). germination characteristics of okra seeds depend at least partly on the (*) corresponding author: amirmoosavi@asnrukh.ac.ir citation: parmoon g., moosavi s.a., ataollah siadat s., 2019 descriptions of okra seed longevity loss behavior using nonlinear regression models. adv. hort. sci., 33(3): 301-312 copyright: © 2019 parmoon g., moosavi s.a., ataollah siadat s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 july 2018 accepted for publication 21 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 301-312 302 duration, temperature and relative humidity of storage condition. seeds from different geographical locations commonly react differently to germination conditions (campbell and sorensen, 1979). okra is a crop of warm and humid climate and when it comes to seed production, one of the main concerns would be storage damages to see due to storage conditions (george, 2009). understanding the pattern of seed quality loss under various storage conditions would be very useful for okra seed producers to estimate their seed loss and find solutions for better seed storage condition. the conventional method of seed germination data analysis depends on the single-value indices or descriptive statistics such as mean, variance, final germination, mean germination time to represent the results of experimental treatment (shafii et al., 1991). however, such indices retain no information on either the initiation or rate of germination (brown and mayer, 1988; mcnair et al., 2012). moreover, there is no room to put information of lag, speed, and extent phase of germination into the single value data. another concern of statistics in seed germination is the skewness of germination frequency (soltani et al., 2016). therefore, cumulative germination will not only provide ambiguous information about seed germination but also it will make much easier to understand and amenable to statistical analysis (brown and mayer, 1988). regression analysis studies the relationship between dependent variable y and one or more explanatory or independent variables x i . basic model is: y i = h (x i (1), x i (2), ….., x i (n); θ 1 , θ 2 , . . . , θ pi ) + e i . where h is an appropriate function that depends on the explanatory variables and parameters. application of nonlinear model in cumulative data analysis is a powerful tool to predict and understand the changes in cumulative germination data (yin et al., 1995; aparecida guedes et al., 2014). the logistic model is a nonlinear regression method, which developed for the sigmoid models. it is symmetric about the inflection point but its application to fit germination data needs special care due to the skewed nature of such data (shafii et al., 1991). in contrast to logistic, gompertz is asymmetric sigmoidal function. the gompertz model was first introduced to fit the relationship between increasing death rate and age by the mr. benjamin gompertz and now it is one of the most useful sigmoid models fitted to different data types including growth data (gompertz, 1825; tjørve and tjørve, 2017). the weibull function has widely been used by seed researchers to explain germination data (scott et al., 1984; dumur et al., 1990; akbari et al., 2016). this regression model works well with small sample data and provides reasonable, simple and useful failure graph and analysis (abernethy, 2006). the four-parameter hill function was useful to explain seed germination variations among different seed lots of coniferous by incorporating germination rate, germination onset and cumulative germination data (el-kassaby et al., 2008). probit model analysis has been widely applied to seed longevity experiments to estimate the probabilities of germination at each level of treatment (probert et al., 2009; gazola et al., 2015). large amounts of naturally existing plants produce seed populations with a different degree of germinability (meyer and kitchen, 1994; gesch et al., 2016). prediction of germination behavior of such germplasm will help us to provide a better genetic pool to breed better crops. the aim is also to compare different methods of nonlinear regression models for the description of okra seed germination behavior after exposure to the accelerated aging condition. 2. materials and methods seed materials the okra seeds were purchased in dry state form local seed stores of ahwaz, isfahan and mashhad. seeds were then cleaned from the white pappus and sent immediately to seed technology laboratory of the department of plat production and genetics. all seed germination measurements were performed based on the ista rules for seed testing (ista, 2013). accelerated aging test for the accelerated aging test, 200 ml of distilled water were added to each plastic box (20*15*10 cm) and 200 seeds were placed on a wired mesh tray (19*14*10 cm) inside the box. to avoid the direct content of seed with water, they were placed in the middle part of the tray. seeds were aged at 40°c and 99% humidity for 24, 48, 72 and 96 h using one box for each aging/time combination (demir et al., 2004; souza et al., 2017). seeds were disinfected before standard germination test using 3% solution of naocl (sauer and burroughs, 1986). germination test to test the germination of okra seeds, 50 seeds parmoon et al. ‐ okra seed loss longevity 303 were disinfected with 3% sodium hypochlorite for 5 minutes, then cultured by sandwiching between two layered filter papers. the counting of germinated seeds was done regularly after every 24 h and the appearance of 2 mm or more of radicle was considered as germination. germination test was ended after 14 days when the number of germinated seeds was equal in two sequential counting. the seedling size was measured on the last day of the germination test. final germination percentage (fgp) was calculated using the following formula (sumithra et al., 2006): fgp (%) = number of total geminated seeds x 100 total number of seeds tested seedling vigor seedlings vigor was determined when the number of germinated seeds at the two subsequent counts remained constant (day 14th). all seedlings that had completed morphological parts without lesions or defects, were selected and computed as vigorous seedlings and average seedling length and weight of 10 seedlings were measured for calculating seedling vigor index (svi) by a modified formula of (abdulbaki and anderson, 1973; will iamson and richardson, 1988): svi = fgp (%) x means of seedling length (cm) 100 statistics the experimental design was a factorial experiment fitted into the randomized complete design with three replications. treatments were three okra ecotypes and five durations (0, 24, 48, 72 and 96 h) of accelerated aging conditions arranged as the first and second factor, respectively. all acquired experimental data were subjected to fit with non-linear regression models to evaluate model parameters and their performance of data explanations. models applied were: i) sigmoid 3 parameter: y= and sigmomoid 4 parameter: y=y 0 ii) standard logistic 3 parameter: y= and logistic 4 parameter: y=y 0 iii) gompertz 3 parameter: y =ae and gompertz 4 parameter: y =y 0 +ae iv) hill 3 parameter: y= and hill 4 parameter: y=y0+ where a is y max or upper asymptote, b was a slope, x 0 critical point or the x that reached 50% of y max and y 0 is a lower asymptote. variable “y” corresponds to the germination percentage and “x” to the time of accelerated aging, respectively. also, other non-linear regression models included: vi) weibull 4 parameter: y= vii) probit 4 parameter: y= y0+ (a-y0) × normal distribution where a is y max or upper asymptote, b is the slope, x 0 is the critical point or the x that reached 50% of y max and y 0 is a lower asymptote. sigma plot v. 11 was used for calculating the type of regression equation. r2, aicc, and rmse was applied to determine the best estimates of the parameters. r2 was calculated using the following formula: r2= ssr/sst where ssr denotes the sum of squares (ss) for regression (∑n i=1 l-ḹ) and sst the total ss (∑n i=1 li-ḹ). li is the observed value and ḹ is the corresponding estimated value. in addition, root mean square error (rmse) calculated using following the formulae: rmse =√(1/n) ∑ (y obs -y pred )2 where y obs denotes observed value, y pred predicted value, and n is the number of samples. to identify the best model for estimating, the akaike information criterion corrected (aicc) was used. this statistic incorporates the amount of reduction of rss and the model complexity (butler and king, 2004; kamkar et al., 2012). aicc=n ln + (rss/n) + 2k [2k (k-1)]/(n-k-1) where n is number of data points, and k is the number of parameters in the model. 3. results and discussion the data subjected to model data analysis were from an experiment to study seed longevity loss during accelerated aging conditions. main investigated traits were cumulative seed germination and seedling vigor. results of fitted parameters with both three and four parameters nonlinear regression models are presented in table 1. among investigated ecotypes, a e x x bln b c 1 0 2 1 2                             ฀ x x b        0 a e x x b1 0        a e x x b1 0         a x x b 1 0        a x x b 1 0                e x x b 0         e x x b 0 ax c x b b b ฀ax c x b b b ฀ adv. hort. sci., 2019 33(3): 301-312 304 ta b le 1 c o m p ar at iv e in d ic es o f m o d el s p er fo rm an ce t o d es cr ib e cu m u la ti ve g er m in at io n d at a o f th re e o kr a ec o ty p es ( a h w az , is fa h an a n d m as h h ad ) u n d er d et er io ra ti o n c o n d iti o n s m o d el n o rm al 2 4 h 4 8 h 9 6 h 1 9 2 h a h w az is fa h an m as h h ad a h w az is fa h an m as h h ad a h w az is fa h an m as h h ad a h w az is fa h an m as h h ad a h w az is fa h an m as h h ad si gm o id 3 p r 2 0 .9 9 8 0 .9 9 6 0 .9 9 4 1 .0 0 0 .9 9 7 0 .9 9 8 0 .9 9 9 0 .9 9 2 0 .9 6 1 0 .9 8 9 0 .9 7 8 0 .9 6 8 1 .0 0 0 .9 8 3 0 .9 8 2 r m se 1 .7 4 2 .7 0 3 .3 1 0 .3 1 1 .9 8 1 .3 8 0 .4 9 3 .2 5 7 .0 3 4 .7 4 6 .4 5 6 .9 0 0 .1 4 4 .0 1 1 .4 2 a ic c 2 5 .2 9 2 9 .0 8 3 0 .8 5 1 0 .5 5 2 6 .3 9 2 3 .2 8 1 4 .4 1 3 0 .7 0 3 7 .4 0 3 3 .9 7 3 6 .6 4 3 7 .2 3 3 .3 8 3 2 .5 2 2 3 .5 2 lo gi st ic 3 p r 2 0 .9 9 6 0 .9 9 9 0 .9 9 7 0 .9 9 9 0 .9 9 2 0 .9 9 9 0 .9 9 7 0 .9 9 4 0 .9 9 4 0 .9 8 8 0 .9 7 2 0 .9 6 4 0 .9 9 9 0 .9 9 3 0 .9 8 3 r m se 2 .9 3 1 .5 4 2 .2 0 0 .4 3 3 .6 1 0 .5 2 2 .1 1 2 .7 6 2 .7 6 4 .7 9 6 .5 5 7 .3 1 0 .2 3 2 .4 6 1 .4 1 a ic c 2 9 .8 0 2 4 .2 4 2 7 .3 0 1 3 .2 0 3 1 .6 0 1 4 .7 8 2 6 .9 6 2 9 .2 6 2 9 .2 9 3 4 .0 7 3 6 .7 7 3 7 .7 3 8 .0 0 2 8 .2 9 2 3 .4 8 g o m p er tz 3 p r 2 0 .9 9 3 0 .9 9 9 0 .9 9 8 0 .9 9 9 0 .9 9 6 1 .0 0 0 .9 9 6 0 .9 9 6 0 .9 7 8 0 .9 8 8 0 .9 7 8 0 .9 6 6 1 .0 0 0 .9 9 2 0 .9 8 4 r m se 3 .7 5 0 .9 7 1 .9 4 0 .5 0 2 .6 3 0 .1 2 2 .4 8 2 .0 6 5 .2 0 4 .8 1 6 .5 7 7 .1 2 0 .2 1 2 .7 0 1 .4 0 a ic c 3 1 .9 4 2 0 .2 0 2 6 .2 2 1 4 .5 3 2 8 .5 8 2 .5 3 2 8 .3 4 2 6 .7 3 3 4 .7 3 4 .0 9 3 6 .8 1 3 7 .5 0 7 .3 1 2 9 .1 0 2 3 .3 7 h ill 3 p r 2 0 .9 9 6 0 .9 9 0 0 .9 9 7 0 .9 9 9 0 .9 9 2 0 .9 9 9 0 .9 9 7 0 .9 9 4 0 .9 9 4 0 .9 8 8 0 .9 7 8 0 .9 6 4 0 .9 9 9 0 .9 9 3 0 .9 8 3 r m se 2 .9 3 1 .5 4 2 .2 0 0 .4 3 3 .6 1 0 .5 2 2 .1 1 2 .7 6 2 .4 6 4 .7 9 6 .5 5 7 .3 1 0 .2 3 2 .4 6 1 .4 1 a ic c 2 9 .8 0 2 4 .2 4 2 7 .3 2 1 3 .2 0 3 1 .6 0 1 4 .7 8 2 6 .9 6 2 9 .2 6 2 9 .2 9 3 4 .0 7 3 6 .7 7 3 7 .7 3 8 .0 0 2 8 .2 9 2 3 .4 8 si gm o id 4 p r 2 0 .9 9 8 0 .9 9 7 0 .9 9 6 1 .0 0 0 .9 9 8 0 .9 9 9 0 .9 9 9 0 .9 9 2 0 .9 6 1 0 .9 9 3 0 .9 8 7 0 .9 6 8 1 .0 0 0 .9 8 7 0 .9 8 2 r m se 1 .8 3 2 .6 5 3 .1 0 0 .2 7 1 .8 1 1 .3 5 0 .5 3 3 .5 1 7 .6 0 3 .9 0 5 .4 4 7 .4 2 0 .1 2 6 3 .6 6 1 .5 3 a ic c 3 0 .5 4 3 3 .7 6 3 5 .1 2 1 4 .2 0 3 0 .4 5 2 7 .9 3 1 9 .8 6 3 6 .2 0 4 2 .9 0 3 7 .1 2 4 0 .0 4 2 .6 9 7 .3 2 8 0 .4 9 2 9 .0 2 lo gi st ic 4 p r 2 0 .9 9 7 0 .9 9 9 0 .9 9 7 1 .0 0 0 .9 9 2 0 .9 9 9 0 .9 9 8 0 .9 9 4 0 .9 9 4 0 .9 9 3 0 .9 8 7 0 .9 6 9 0 .9 9 9 0 .9 9 4 0 .9 8 3 r m se 2 .8 4 1 .6 0 2 .3 5 0 .3 5 3 .9 0 0 .5 6 1 .6 9 2 .9 8 2 .9 8 3 .8 8 5 .4 1 7 .3 4 0 .2 5 2 .5 2 1 .5 2 a ic c 3 4 .3 5 2 9 .3 9 3 2 .7 2 1 6 .2 3 3 7 .1 0 2 0 .2 8 2 9 .8 5 3 4 .7 6 3 4 .7 9 3 7 .0 7 3 9 .9 5 4 2 .6 0 1 3 .5 0 3 3 .3 2 2 8 .9 7 g o m p er tz 4 p r 2 0 .9 9 6 0 .9 9 9 0 .9 9 8 0 .9 9 9 0 .9 9 6 1 .0 0 0 .9 9 8 0 .9 9 7 0 .9 7 8 0 .9 9 3 0 .9 8 7 0 .9 6 8 1 .0 0 0 .9 9 2 0 .9 8 3 r m se 3 .1 1 1 .0 4 2 .0 8 9 0 .3 8 2 .8 3 0 .1 3 6 1 .9 1 2 .1 4 5 .6 1 3 .8 8 5 .4 1 7 .4 8 0 .2 3 5 2 .8 0 1 .5 1 1 a ic c 3 5 .1 8 2 5 .6 6 3 1 .6 8 1 7 .0 6 3 4 .3 4 7 .9 9 3 0 .9 1 3 1 .9 4 0 .2 7 3 7 .0 7 3 9 .9 5 4 2 .7 6 1 2 .7 3 3 4 .2 4 2 8 .8 6 h ill 4 p r 2 0 .9 9 7 0 .9 9 9 0 .9 9 7 1 .0 0 0 .9 9 2 0 .9 9 9 0 .9 9 8 0 .9 9 4 0 .9 9 4 0 .9 9 3 0 .9 8 7 0 .9 6 9 0 .9 9 9 0 .9 9 4 0 .9 8 3 r m se 2 .8 4 1 .6 0 2 .3 5 0 .3 5 3 .9 0 0 .5 6 1 .6 9 2 .9 8 2 .9 8 3 .8 8 5 .4 1 7 .3 4 0 .2 5 2 .5 2 1 .5 2 a ic c 3 4 .3 5 2 9 .3 9 3 2 .7 2 1 6 .2 3 3 7 .1 0 2 0 .2 8 2 9 .8 5 5 3 .3 3 4 .7 9 3 7 .0 7 3 9 .9 5 4 2 .6 0 1 3 .5 0 3 3 .3 2 2 8 .9 7 w ei b u ll 4 p r 2 0 .9 9 9 0 .9 9 9 0 .9 9 8 0 .9 9 9 0 .9 9 9 1 .0 0 0 .9 9 9 0 .9 9 8 0 .9 9 5 0 .9 8 9 0 .9 8 0 0 .9 6 6 1 .0 0 0 .9 9 6 0 .9 8 5 r m se 0 .4 0 0 .6 7 1 .7 7 0 .5 4 1 .2 0 0 .1 3 3 0 .4 7 1 .6 7 2 .6 0 4 .9 3 6 .7 6 7 .7 0 0 .0 0 2 2 .0 0 2 1 .5 3 a ic c 1 7 .4 8 2 1 .9 2 3 0 .2 5 2 0 .0 2 2 6 .8 9 7 .7 7 1 8 .8 7 2 9 .7 8 3 3 .6 3 9 .1 4 4 1 .8 9 4 3 .0 1 0 .0 0 1 3 1 .3 1 2 8 .1 9 p ro b it 4 p r 2 0 .9 9 8 0 .9 9 7 0 .9 9 6 0 .9 9 9 0 .9 9 9 0 .9 9 8 0 .9 9 9 0 .9 9 8 0 .9 9 4 0 .9 9 3 0 .9 8 6 0 .9 6 7 1 .0 0 0 .9 8 5 0 .9 8 2 r m se 1 .7 1 2 .5 1 3 .0 3 0 .4 4 1 .2 3 1 .8 3 0 .7 7 1 .3 8 2 .7 7 3 .9 4 5 .4 9 7 .5 7 0 .0 1 7 3 .8 5 1 .5 3 a ic c 5 9 .0 3 6 6 .7 4 7 0 .4 9 3 2 .0 8 5 2 .5 3 6 0 .3 4 3 .0 4 5 4 .7 7 6 8 .6 9 7 5 .7 0 8 2 .3 5 8 8 .7 5 0 .0 0 7 5 .2 7 5 6 .9 parmoon et al. ‐ okra seed loss longevity 305 mashhad ecotype was more susceptible to longevity loss, especially at higher aging stress durations (fig. 1, a 1 -a 5 ). among four parameter models, weibull and probit functions were produced the best curve fit as illustrated in figure 2 (e 1 -e 5 ; f 1 -f 5 ). our results revealed that at most aging treatments, the best-fitted data obtained from three parameter nonlinear regression models (fig. 3). the differences in parameter estimates among aging treatments had biological significance. the maximum germination, estimated by the parameter a (asymptotic part of the model), decreased as aging duration increased meaning that aging damages decrease the proportion of seeds which are able to survive and complete germination by radicle protrusion. the estimates of parameter b, which is the rate of seed germination were also reduced due to the increase in aging durations (fig. 3). deterioration damages due to oxidative stress caused by aging resulted in a reduction of seed germination properties and aged seeds germinated slower than no aged seeds (anderson and baker, 1983; goel et al., 2003; balešević-tubić et al., 2007; parmoon et al., 2015). under no aging conditions, there was a significant difference in the estimated parameter for all three okra ecotypes. using sigmoid 3p, weibull 4p models parameter a, was well predicted and as shown in figure 3 it was very close to actual observed value (dash red line). our results revealed that only gompertz model did not provide precise estimation for parameter x 0 (the time required for 50% of viable seeds to germinate) while other 3 parameter models performed well. parameter x 0 in ahwaz and isfahan ecotypes well fitted using 4p weibull and hill while in mashhad ecotype probit model was closer to the observed value. fig. 1 model fit function 3 parameter of germination ecotypes okra (abelmoschus esculentus l.) under deterioration condition. ● ahwaz, ○ isfahan and ▲ mashhad ecotypes. 306 adv. hort. sci., 2019 33(3): 301-312 accelerated aging (deterioration) after 24h, resulted in a reduction of x 0 value compared to no aged seeds (normal condition). results revealed that an increase in deterioration treatments led to a decrease in observed maximum germination and predicted parameter. model output for parameter a (the maximum germination) of ahwaz ecotype using sigmoid 3p model was less than the observed value. the result of the weibull 4p function showed that the predicted parameter a, was the same as the maximum value of observed germination. there was no difference in estimated parameter a (asymptote), for isfahan ecotype using gompertz 3p and weibull 4p. model performance to predict germination of okra ecotypes under deterioration conditions varied among regression models (table 1). for instance, at no aged treatment (normal condition), the best model to predict cumulative germination of ahwaz ecotype were weibull 4p and sigmoid 3p with aicc values of 17.48 and 25.29 respectively. results showed that, at the 96 h of accelerated aging conditions, the best fit and the lowest aicc values were obtained from three parameter growth models. in the case of mashhad ecotype, the gompertz 3p provided the best model fit to estimate germination loss at both no aged and severe aging treatments with the aicc values of 23.37 and 26.22, respectively. application of regression models will result in various measures of the asymptotic point of cumulative seed germination curves (aparecida guedes et al., 2014). to compare the model performance of parameter estimation, aicc was a well discriminant index (hurvich and tsai, 1989; spiess and neumeyer, 2010). the lower aicc, fig. 2 model fit function 4 parameter of germination ecotypes okra (abelmoschus esculentus l.) under deterioration condition. ● ahwaz, ○ isfahan and ▲ mashhad ecotypes. parmoon et al. ‐ okra seed loss longevity 307 the better parameter estimation of the model is expected (sandvik, 2008). under 48 and 96 hours of deterioration, the estimated parameter a using sigmoid 4p, logistic 4p and hill 4p, was less than the maximum observed germination. our results showed that the sigmoid 3p model predicts lower values for actual maximum germination, while sigmoid 4p and logistic 4p estimated higher values especially in isfahan ecotype seeds. weibull was the best model to show the alteration of the parameter b (rate of increase in seed germination), at different deterioration durations. aging reduced the value of parameter b, which mean that deterioration because the reduction of seed germination rate of seed lot compared to no aged (normal condition) seeds (fig. 3). results of experiments suggested that the weibull model was the best among all of four parameter models for cumulative germinating data of ahwaz and mashhad ecotypes while gompertz 4 parameter performed well for the fitting of isfahan data. the study of the germination data of deterioration seeds after 24 hours showed that the best three parameter model to fitted data of ahwaz and isfahan ecotypes was sigmoid 3p (aicc= 10.55 and 26.39) while gompertz exhibited the best fit for mashhad ecotype (aicc= 2.53) (table 1). germination data of deterioration seeds for 48 hours were best-fitted using sigmoid 3p, gompertz 3p and hill 3p in ahwaz, isfahan and mashhad ecotypes respectively. among four parameter models, the best fit data was weibull 4p for all ecotypes. results of fitting cumulative gerfig. 3 estimate parameter of fit models germination ecotypes okra (abelmoschus esculentus l.) under deterioration condition. dash red line indicated observed parameter value. parameter a= asymptote (theoretical maximum for y); parameter b=rate of increase; x0= time required to completion of germination in 50% of seed population or d50; y0= initial seed germination. adv. hort. sci., 2019 33(3): 301-312 308 mination data of deterioration seeds for 96 hours showed that the sigmoid 3p model was the best model to describe data. among four parameter models, gompertz and hill both performed well to describe germination data of ahwaz and isfahan ecotypes having similar aicc, while mashhad ecotype was well-fitted using logistic 4p and hill 4p. the study performed at the 192 hours of deterioration showed that the sigmoid 3p was the best-fit model for ahwaz, isfahan while for the mashhad ecotype gompertz was the best model (table 1). it is concluded that the weibull 4p was the best-fit model to describe germination data of okra seeds. seed vigor is an important index of seed quality and any change in this trait resulted in a reduction of seedling emergence. among all three parameter models, hill 3p well-illustrated changes of seed germination and vigor loss for mashhad ecotype (fig. 4). model comparisons to describe germination and vigor of okra ecotypes during deterioration conditions revealed that weibull 4p and probit 4p functions failed to describe cumulative germination of ahwaz ecotype in contrast to sigmoid models. the best 3p model to describe germination data of isfahan ecotype was hill 3p (aicc=26.89) while there was a failure to fit germination data using weibull 4p and probit 4p. mashhad germination and vigor was well described using hill 3p (aicc=33.72 and 32.22) (table 2). non-linear model fit to germination of vigor data of okra ecotypes showed that in almost all treatments decline of seed vigor initiated earlier than germination loss. however, predictions of vigor loss with an increase in deterioration treatments was not well described by four parameter models (fig. 5). application of logistic and weibull functions to describe the germination and vigor of alfalfa seeds fig. 4 model fit function 3 parameter of process deterioration germination and vigor ecotypes okra (abelmoschus esculentus l.). points are the percentage of improvements in seed properties compared with control treatment (no aged seeds ) (● germination percentage, ○ seedling vigor) and lines are predicted values. parmoon et al. ‐ okra seed loss longevity 309 showed that the weibull parameter was more informative than the logistic (bahler et al., 1989). among ecotypes, mashhad ecotype vigor and germination data were fitted more uniformly to four parameter models. the hill 4p was not capable of fitting germination data of the isfahan ecotype. we found that none of the four-parameter models is suitable to describe seedling vigor loss of okra seeds due to accelerated aging condition. at high levels of aging treatments, seed germination was declined to zero and so there is no lower limit to produce y 0 parameter, and like the results, model equations could not predict satisfied result (fig. 6). interestingly, results showed that aging condition did not exceed to deteriorative level before 24 hours and instead it might improve seed germination by providing after-ripening requirements. 4. conclusions in this study, we test the moments and indices of seed germination using growth models with three to four parameter. this method of data description provides sufficient information about the dispersion of germination rate in time and extent of germination due to aging conditions. the hill and weibull models were capable of good predictions across cumulative germination and seedling vigor data of three okra ecotypes. we recommend non-linear regression table 2 comparative indices of models performance to describe seed germination and vigor of okra ecotypes model ahwaz isfahan mashhad germination vigor germination vigor germination vigor sigmoid 3p r2 0.990 0.987 0.976 0.968 0.992 0.998 rmse 2.23 3.11 3.18 4.29 4.28 1.79 aicc 35.33 37.05 37.17 38.72 38.72 34.17 logistic 3p r2 0.990 0.981 0.979 0.977 0.9942 0.999 rmse 2.21 3.80 3.02 3.59 3.80 1.23 aicc 35.28 38.10 36.89 37.80 38.10 32.22 gompertz 3p r2 0.990 0.988 0.976 0.967 0.991 0.998 rmse 2.23 30.046 3.20 4.37 4.49 2.18 aicc 35.33 36.87 37.20 38.82 38.96 35.20 hill 3p r2 0.991 0.984 0.999 0.984 0.998 0.999 rmse 0.68 3.34 0.44 2.97 1.64 1.23 aicc 29.12 37.34 26.89 36.81 33.72 32.22 sigmoid 4p r2 0.990 0.989 0.979 0.987 0.994 0.999 rmse 3.13 4.11 4.20 3.79 5.23 1.56 aicc 25.74 30.17 30.36 28.77 33.78 14.92 logistic 4p r2 0.990 0.985 0.979 0.987 0.994 0.999 rmse 3.13 4.87 4.20 3.79 5.23 1.56 aicc 25.74 32.68 30.36 28.77 33.78 14.92 gompertz 4p r2 0.990 0.977 0.979 0.987 0.994 0.999 rmse 3.13 6.04 4.20 3.70 5.23 1.56 aicc 25.74 36.04 30.36 28.77 33.73 14.92 hill 4p r2 0.998 0.981 0.999 0.992 0.999 0.999 rmse 1.02 5.09 0.49 2.84 2.07 1.55 aicc 8.28 33.35 0.00 24.25 19.35 14.85 weibull 4p r2 0.00 0.00 0.00 rmse 31.9 191.41 29.51 34.16 70.6 75.3 aicc 62.05 90.05 60.82 63.11 74.46 75.48 probit 4p r2 0.731 0.938 0.987 0.993 0.963 rmse 16.53 9.86 29.51 3.79 5.65 14.49 aicc 51.76 43.68 60.82 27.77 35.02 49.7 adv. hort. sci., 2019 33(3): 301-312 310 models as an unambiguous and strong approach to predict okra seed germination and longevity loss during aging conditions. acknowledgements authors wish to thank iranian ministry of science, fig. 5 model fit function 4 parameter of process deterioration germination and vigor ecotypes okra (abelmoschus esculentus l.). points are the percentage of improvements in seed properties compared with control treatment (no aged seeds ) (● germination percentage, ○ seedling vigor) and lines are predicted values. fig. 6 estimate parameter of fit models process deterioration germination and vigor ecotypes okra 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solani, f. verticillioides and f. equiesti) was studied on six syrian wheat cultivars under controlled conditions. two aggressiveness criteria: diseased-head severity (dhs, fusarium infection) and disease development (dd, fusarium spread) were visually estimated as percentage of heads showing fusarium symptoms in wheat cultivars at the soft dough stage. results showed significant differences among fungal isolates and wheat cultivars for the two tested criteria. the mean values of dhs evaluations ranged from 33.27 to 45.49% among fungal isolates, and from 29.62 to 42.22% among tested cultivars. the mean dd rating varied from 25.58 to 35.43% among fungal isolates, and from 25.33 to 34.01% among tested cultivars. results in the current research highlighted that the level of resistance in syrian cultivars to fusarium species is characterized with low to moderate dhs and dd evaluations (%). also, the results were comparable with those previously obtained using the same fungal isolates and wheat cultivars in vitro. the current study confirmed the suitability of in vitro method to be used as fast and reliable test to analyze aggressiveness in fusarium species. 1. introduction fusarium head blight is one of the most destructive global diseases of wheat. in infected plants, it leads to kill the developing seed (prematurely bleached spikes) within moist conditions and moderate temperatures prevail during flowering. since it was identified in 1884, severe epidemic outbreaks caused quantitative losses in yield of up to 50-75% (parry et al., 1995; mcmullen et al., 2012). it also reduces grain quality due to contamination of harvest with large amount of mycotoxins that cause toxicities to human and livestock (maresca, 2013). at least seventeen fusarium species with several habitats and types of mycotoxins produced have been associated with fusarium species (parry et al., 1995). fusarium graminearum is the main causal agent of this disease and has been subdivided into at least 11 cryptic species (o’donnell et al., 2004). other species can cause fusarium disease on a lesser scale such as f. avenaceum, f. culmorum, f. solani, f. equiseti, f. verticillioides and f. poae (*) corresponding author: ascientific@aec.org.sy citation: sakr n., 2017 aggressiveness of four fusarium head blight species on wheat cultivars. adv. hort. sci., 31(3): 199-203. copyright: © 2017 sakr n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 april 2017 accepted for publication 6 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 199-203 200 (xu et al., 2008). understanding the interaction between wheat plants and fusarium populations requires more detailed knowledge about the variation of aggressiveness (wu et al., 2005). van der plank (1968) defined aggressiveness as a quantitative ability of an isolate to cause disease on a susceptible host plant in a non-race-specific pathosystem. aggressiveness is an important factor determining the potential ability of fusarium isolates to cause fusarium epidemics. variability of quantitative component of pathogenicity in f. graminearum has been the subject of several studies (parry et al., 1995; leonard and bushnell, 2003; wu et al., 2005; mcmullen et al., 2012). however, other fusarium species have attracted less pathogenic analyses (xu et al., 2008; bakri et al., 2012; sakr, 2017). fusarium resistance in wheat plants is conferred by quantitative trait loci (qtl) detected on all chromosomes (loffler et al., 2009). it is necessary to combine type i (resistance to initial infection) and type ii (resistance to spreading) to get fusarium resistant wheat plants (loffler et al., 2009). in syria, about 1.7 million hectares were sown to wheat, with an annual production of 3.9 million tons in 2011. host-pathogen interactions were evaluated for several local wheat cultivars inoculated with fungal isolates of different species associated with fusarium species, and differential reactions on cultivars were detected (alazem, 2007; talas et al., 2011; bakri et al., 2012). recently, sakr (2017) analyzed aggressiveness of four fusarium species in vitro, and significant differences were detected between pathogen isolates and wheat genotype. in order to underline pathogenic variation for fusarium species collected from ghab plain, one of the principal syrian wheat production areas, the objectives of the current study were to (1) evaluate aggressiveness of four isolates [f2 (f. culmorum), f27 (f. verticillioides), f35 (f. solani), and f43 (f. equiesti)] on six wheat syrian cultivars under controlled conditions, and (2) compare results previously obtained by sakr (2017) in vitro with the current data from floret inoculation under controlled conditions. 2. materials and methods fungal isolates and inoculum production the fungal isolates of four fusarium species [f. culmorum (f2), f. verticillioides (f27), f. solani (f35), and f. equiesti (f43)] were collected in 2015 from naturally wheat spikes exhibiting fusarium symptoms from different locations of ghab plain in 2015. isolates were identified morphologically according to nelson et al., (1983). the cultures were maintained in sterile distilled water at 4°c and freezing at -16°c until needed. for inoculum preparation, four to six agar plugs out of the stored isolates were put over the surface of pda in 9-cm petri dishes and incubated for 10 days, at 22°c in the dark to allow mycelial growth and sporulation. ten ml of sterile distilled water were added to each dish, and the resulting spore suspensions were adjusted to 5 × 104 spores/ml for inoculation following a count in a hemacytometer (bakri et al., 2012). wheat cultivars and growth chamber conditions in the current study, aggressiveness for the four fusarium head blight isolates was measured on six wheat cultivars previously analyzed in vitro (‘cham1’, ‘cham7’, ‘acsad65’, ‘cham4’, ‘cham6’ and ‘douma4’, most cultivated in different syrian areas) under controlled conditions. wheat seeds were surface-sterilized with 5% sodium hypochlorite solution for 8 min and then washed six times in sterile distilled water (purahong et al., 2012). they were sown into plastic pots (15-cm) filled with 2 kg of sterilized soil (ten seeds per pot), and arranged in a complete randomized design with three replicates. three plots per replicate were left non-inoculated as control treatment. pots were placed in a growth chamber operated at 20°c during day and night with an 16-h photoperiod. following emergence, plants were thinned to three per pot and nitrogen fertilizer was applied twice at two dates: emergence and tillering. aggressiveness tests at 10-14 days after heading, spore suspensions of the four fusarium head blight isolates or sterile distilled water (control) were sprayed one time into flowering spikes. six flowering spikes were randomly selected within each replicate of the six cultivars. after the inoculum dried for 30 min, inoculated spikes were then kept covered for 48 h using polythene bags to ensure 100% rh. head blight symptoms were evaluated as percentage of spikes showing fusarium symptoms after 7, 14, and 21 days, when plants were at the soft dough stage. fusarium disease severity was visually estimated in situ for each inoculated spike using the xue’s et al., (2004) scale. this scale includes nine levels of incidence expressed in percentage of bleached spike area on a head: 0 (no visible fusarium symptoms) to sakr aggressiveness of four fusarium head blight species on wheat cultivars 201 9 (severely diseased, spike dead). each head was assessed separately in all experiments. the values of diseased-head severity as percentage of infected spikes measured 21 days after inoculation (dai) were considered, for each cultivar, a parameter to determine initial infection. the values of disease development calculated by the means of each evaluation; 7, 14 and 21 dai over the estimation time were considered, for each cultivar, a parameter to determine pathogen spreading. statistical analyses statistical analyses of aggressiveness data were performed using statview, 4.57® abacus concepts, berkley, canada. before statistical analysis, the percentages were transformed using the arcsines function. a complete randomized design with two factors (fusarium isolate and wheat genotype) and 3 replications was used for aggressiveness analysis. fisher’s lsd test was used to compare the means at p <0.05. 3. results and discussion understanding the interaction between fusarium head blight species and wheat plants requires knowledge of the variation of quantitative component of pathogenicity (wu et al., 2005). with this in mind, aggressiveness variability for four local fusarium species was analyzed by using a floret inoculation in a growth chamber on six wheat cultivars most cultivated in different syrian areas. differences in aggressiveness of four fusarium species (f. culmorum, f. verticillioides, f. solani, and f. equiesti) are indicated when isolates vary in the amount of damage that they cause in wheat plants. the results demonstrated that none of the six tested cultivars was immune from disease. however, typical fusarium symptoms induced by the four isolates (f2, f27, f35 and f43) were clear and easy to score in the inoculated spikes, while no symptoms were present in the control (fig. 1). the mean values of diseasedhead severity (dhs) ranged from 33.27% to 45.49% as compared with 0% for the control treatment (table 1). there were significant differences among four isolates (f isolates=4.376; probability=0.0084). the mean values of disease development (dd) varied from 25.58% to 35.43% (table 1). significant differences among four isolates (f isolates=4.257; probability=0.0096) were detected. results in the current study showed that there was no interaction between fusarium isolates and host plant for the two parameters. this indicates to non-race-specific interaction described for this pathosystem (loffler et al., 2009). high diseased-head severity and disease development values represent high aggressiveness (parry et al., 1995; wu et al., 2005; xu et al., 2008). results shown in table 1 indicate that the isolate f35 of f. solani was the most aggressive with a mean value of dhs of 45.49% and dd of 35.43%; followed by other tested isolates. these results are in accordance with previous analysis on the aggressiveness of theses isolates in vitro; sakr (2017) observed that the isolate f35 was the most aggressive one, followed by other analyzed isolates. the current study confirmed the suitability of in vitro modified petri-dish method to be used as fast and reliable test to analyze aggressiveness in fusarium species. results in the current study are comparable with those found by alazem (2007) and bakri et al., (2012) for f. culmorum, f. solani, f. verticillioides and f. equiesti in which significant differences were detected for aggressiveness among fungal isolates in each fusarium head blight species in a growth chamber. the mean value of dhs and dd rating for six fig. 1 fusarium head blight symptoms on spike of syrian wheat cultivar cham4 inoculated with isolate f35 (fusarium solani) compare with control (water). adv. hort. sci., 2017 31(3): 199-203 202 wheat cultivars (table 1) reflects the ability of the same isolate of the pathogen (f2, f27, f35 and f43) to distinguish different levels of resistance as observed for the same pathosystem (alazem, 2007; talas et al., 2011). also, the resistance of a given wheat cultivar is not related to a certain fusarium species (table 1). significant differences were underlined for dhs (f cultivars=2.907; probability=0.0226) and dd (f cultivars=1.870; probability=0.1171) criteria among wheat cultivars (table 1). the mean values of dhs evaluations ranged from 29.62 to 42.22% among tested cultivars. the mean dd rating varied from 25.33 to 34.01% among tested cultivars. quantitative resistant wheat cultivars are identified by low dhs and dd values of the fungus compared with the susceptible one (parry et al., 1995). results in the current research highlighted that the level of resistance in syrian cultivars to fusarium species is characterized with low to moderate dhs and dd evaluations (%). these results are in accordance with previous analysis on the comportment of local wheat cultivars in which differential reactions on cultivars were detected (alazem, 2007; bakri et al., 2012; talas et al., 2011). however, fusarium resistance scores ranged one fold and half between resistant and susceptible cultivars for the two tested parameters (table 1). thus our observation suggests that in resistant wheat cultivars, the development of the pathogen was slowed, and may be due to resistance mechanisms expressed by accumulation of qtl in host cultivars (alazem, 2007; talas et al., 2011). results in the current study showed that the level of quantitative resistance in the six wheat cultivars made it possible to detect significant differences between isolates of four fusarium species. these results are in accordance with our previous analysis on the behavior of theses cultivars in vitro (sakr, 2017). the variability of resistance for the syrian cultivars is interesting and promising for ecological framing/breeding and also for improving resistance of wheat cultivars. for f. graminearum, purahong et al., (2012) validated the modified petri-dish method (used by sakr, 2017) by highly significant correlation with the data from floret inoculation in adult plants in a growth chamber. results indicted that the petri-dish aggressiveness test conducted on other fusarium species (f. culmorum, f. verticillioides, f. solani, and f. equiesti) is repeatable and stable with the six wheat cultivars in a growth chamber (table 1). it will be necessary to analyze the pathogenic variation in a large number of fusarium isolates on several wheat cultivars under controlled and field conditions to screen fusarium resistance in syrian wheat cultivars. acknowledgements the author would like to thank director general of aecs, and the head of the agriculture department fusarium head blight incidence scores were evaluated as percentage of spikes showing fusarium species symptoms using the xue’s et al., (2004) scale. f tests (p<0.05), ns= not significant. table 1 diseased-head severity and disease development scores in % among isolates of four fusarium head blight species measured on six syrian wheat cultivars isolate ‘cham1’ cham7' acsad65' cham4' cham6' douma4' mean diseased-head severity scores (%) f2 25.92 25.92 29.62 37.03 44.44 44.44 35.24 f27 29.62 29.62 37.03 33.33 37.04 36.66 33.27 f35 33.33 33.33 40.74 58.88 47.77 47.77 45.49 f43 29.62 29.62 44.44 40.00 37.04 40 37.40 mean 29.62 29.62 37.96 42.31 41.57 42.22 f isolates=4.376; probability=0.0084 f cultivars=2.907; probability=0.0226 f interactions=0.601 ns; probability=0.8594 diseased development scores (%) f2 23.56 24.69 21.16 30.86 40.4 29.62 28.38 f27 24.69 23.56 28.49 25.64 24.69 26.45 25.58 f35 30.30 31.74 29.10 45.58 32.09 43.77 35.43 f43 22.79 25.64 37.03 33.95 26.45 29.10 29.16 mean 25.33 26.41 28.94 34.01 30.91 32.23 f isolates=4.257; probability=0.0096 f cultivars=1.870; probability=0.1171 f interactions=1.199 ns; probability=0.3051 sakr aggressiveness of four fusarium head blight species on wheat cultivars 203 for their support. references alazem m., 2007 evaluating genetic variation of fusarium head blight by molecular markers. master, faculty of agriculture, university of damascus, syria, pp. 72. bakri y., jawhar m., arabi m.i.e., 2012 correlative analysis of fusarium species pathogenicity and in vitro xylanase activity. j. plant biol. res., 1(2): 86-92. leonard k.j., bushnell w.r., 2003 fusarium head blight of wheat and barley. vol. i. the american phytopathological society, minnesota, usa, pp. 530. loffler m., schon c.c., miedaner t., 2009 revealing the genetic architecture of fhb resistance in hexaploid wheat (triticum aestivum l.) by qtl meta-analysis. mol. breed., 23(3): 473-488. maresca m., 2013 from the gut to the brain: journey and pathophysiological effects of the food-associated trichothecene mycotoxin deoxynivalenol. toxins, 5(4): 784-820. mcmullen m., bergstrom g., de wolf e., dill-macky r., hershman d., shaner g., van sanford d., 2012 a unified effort to fight an enemy of wheat and barley: fusarium head blight. plant dis. 96(12): 17121728. nelson p.e., toussoun t.a., marasas w.f.o., 1983 fusarium species: an il lustrated manual for identification. the pennsylvania state univ. press, university park, usa, pp. 226. o’donnell k., ward t.j., geiser d.m., kistler h.c., aoki t., 2004 genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the fusarium graminearum clade. fungal genet. biol., 41(6): 600-623. parry d.w., jekinson p., mcleod l., 1995 fusarium ear blight (scab) in small grain cereals-a review. plant pathol., 44(2): 207-238. purahong w., alkadri d., nipoti p., pisi a., lemmens m., prodi a., 2012 validation of a modified petri-dish test to quantify aggressiveness of fusarium graminearum in durum wheat. eur. j. plant pathol., 132(3): 381-391. sakr n., 2017 in vitro assessment of fusarium head blight spp. on wheat cultivars. arch. phytopathol. plant protect., 50(5-6): 254-261. talas f., longin f., miedaner t., 2011 sources of resistance to fusarium head blight within syrian durum wheat landraces. plant breed., 130(3): 398-400. van der plank j.e., 1968 disease resistance in plants. vol. i. academic press, new york and london, pp. 206. wu a.b., li h.p., zhao c.s., liao y.c., 2005 comparative pathogenicity of fusarium graminearum isolates from china revealed by wheat coleoptile and floret inoculations. mycopathologia, 160(1): 75-83. xu x.m., parry d.w., nicholson p., thomsett m.a., simpson d., edwards s.g., cooke b., mdoohan f.m., monaghan s., moretti a., tocco g., mule g., hornok l., béki e., tantnell j., ritieni a., 2008 within field variability of fusarium head blight pathogens and their associated mycotoxins. eur. j. plant pathol., 120(1): 21-34. xue a.g., armstrong k.c., voldeng h.d., fedak g., babcock c., 2004 comparative aggressiveness of isolates of fusarium species causing head blight on wheat in canada. can. j. plant pathol., 26: 81-88. impaginato 3 adv. hort. sci., 2020 34(1s): 3­12 doi: 10.13128/ahsc­7760 watercore in ‘pomella genovese’ apples: quality characteristics and antioxidants m. buccheri 1 (*), r. caramanico 1, v. ughini 2, m. grassi 1, m. vanoli 1 1 crea ‐ centro di ricerca ingegneria e trasformazioni agroalimentari, via venezian 26, 20133 milano, italy. 2 dipartimento di scienze delle produzioni vegetali sostenibili‐facoltà di scienze agrarie alimentari ed ambientali‐università cattolica s.c., piacenza, italy. key words: antioxidant activity, ascorbic acid, dehydroascorbic acid, malus x domestica borkh., polyphenols, texture. abstract: the study aimed to evaluate apple fruit affected by watercore by a physical and biochemical point of view and, at the same time, to gain an insight into the mechanisms of the watercore­related oxidative stress and browning. fruit of the cv. pomella genovese (malus x domestica borkh.) were harvested in three different orchards and stored at 1°c (85­90% rh) for 4 months. the fol­ lowing analysis were performed on the fruit flesh: density, mechanical (firm­ ness and stiffness) and acoustic (crispness) parameters, soluble solids content (ssc), titratable acidity, ascorbic acid (aa), dehydroascorbic acid (dha), total phenols and antioxidant activity (dpph). in all the three orchards, fruit affected by watercore (w­fruit) had a higher density and ssc than watercore­free ones (wf­fruit), probably because of the sugar­rich liquid that accumulates in the intercellular spaces. the peel colour of the w­fruit was darker, their flesh was firmer and crispier and the content in total phenols increased with respect to the wf samples. watercore led to a decrease of aa and to an increase of dha, probably caused by an imbalance of the ascorbic­glutathione cycle. the altered aa/dha ratio can indicate an oxidative stress status of the fruit. dpph was higher in w fruit and was related to the phenol content (r=0.83) but not with aa. 1. introduction watercore is a serious disorder that occurs in apple fruit when still on the tree. it is characterized by water­soaked and translucent areas which are often associated with the vascular bundles of the core line, but in some cases it can affect the entire fruit (williams, 1966). a very long list of watercore­susceptible varieties is reported by marlow and loescher (1984). some of the most well­known cultivar are ‘fuji’, ‘gloster’ (zupan et al., 2016) and ‘delicious’ but this disorder affects also local “old” culti­ vars, such as ‘pomella genovese’. ‘pomella genovese’ is a typical cultivar of the pavia territory (italy), (*) corresponding author: marina.buccheri@crea.gov.it citation: buccheri m., caramanico r., ughini v., grassi m., vanoli m., 2020 ­ watercore in ‘pomella genovese’ apples: quality characteristics and antioxidants. ­ adv. hort. sci., 34(1s): 3­12 copyright: © 2020 buccheri m., caramanico r., ughini v., grassi m., vanoli m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2019 accepted for publication 26 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 3­12 4 where the production is around 500­1000 q. it is a very rustic scab­resistant variety that, in december 2019, was included in the list of the traditional food products of the lombardy region (regione lombardia, 2019). it has a white, firm, juicy, sweet and very aro­ matic flesh; it is usually harvested in october and can bestored in air until april. unfortunately, ‘pomella genovese’ is highly susceptible to watercore, which affects around 20­30% of the total production pre­ venting the diffusion of this cultivar. the onset of watercore can be affected by many factors (high or very low fruit temperatures, high day/night fluctuation, high nitrogen levels, high source­to­sink ratio, advanced maturity stage) (marlow and loescher, 1984) and the disorder is not visible from outside the fruit. for this reason, a high number of fruits are often needed to carry out stud­ ies about watercore. though watercore was described several years ago (faust et al., 1969) and many studies were car­ ried out on the subject, its origin is still not complete­ ly clear. the cause of the disorder is the accumula­ tion of sugar­rich liquid in the intercellular spaces. there have been proposed different theories to explain this phenomenon and the most reliable hypothesis seems to be related to an accumulation of sorbitol, which is the main transport sugar in the plant (loescher et al., 2005). in tissues affected by watercore, sorbitol, that is unloaded from the phloem, is inhibited from being absorbed by the fruit cells (gao et al., 2005) and accumulates in the inter­ cellular spaces, causing an increase of the osmotic potential that promotes water retention (williams, 1966). watercore can regress in the first months of stor­ age (brackmann et al., 2001; kasai and arakawa, 2010; neuwald et al., 2012) but often several disor­ ders, including browning and brown core, have been observed in affected fruits (argenta et al., 2002). ‘delicious’ apples can develop brown core while fruit with medium to slight watercore tends to develop flesh browning (fukuda, 1984). however, the mecha­ nism of browning remains unclear. lee et al. (2012) suggested that the accumulation of amino acids, acetaldehyde and ethanol, which increase under anaerobic conditions, can cause flesh browning. other authors (hulme, 1956) indicate that the accu­ mulation of succinic acid, due to the inhibition of the succinate dehydrogenase activity under high co2 conditions, might account for the development of brown heart in apples. another hypothesis is linked to oxidative stress. watercored tissue has a lower intercellular air space volume, reduced permeance to gas diffusion and increased internal co2 level (argenta et al., 2002). under low o2 or high co2 pressure, reactive oxygen species (ros) like h2o2 or o2 ­are produced in the fruit. if the ros production exceeds the scavenging capacity of the system, fruit undergoes oxidative stress, cell membrane are damaged and phenolic compounds oxidized (zupan, 2016). the ascorbate­glutathione cycle is one of the main scavenger systems in plants (arora et al., 2002). during the reduction reaction of h2o2 to h2o, ascor­ bic acid (aa) is oxidized by ascorbate peroxidase (apx) to monodehydroascorbic acid (mdha) which is an unstable compound and, if not reduced rapidly again to aa (by mdha reductase), it can be convert­ ed to dehydroascorbic acid (dha). dha is reduced to aa by dha reductase using glutathione as electron donor, while the oxidized form of glutathione (gssg) is reduced back to glutathione by the glutathione reductase (lum et al., 2016). under stress conditions caused by low oxygen pressure, the ascorbate­glu­ tathione recycling pathway is dysfunctional and aa content decreases while there is an increase in dha. kasai and arakawa (2010), in fuji apples affected by watercore, observed a decrease in aa and in apx activity but no increase in dha, and hypothesized that dha was hydrolysed in the tissue. this study aimed to characterize watercore­affect­ ed fruit through biochemical and physical analyses and, at the same time, to gain an insight into the mechanisms of watercore­related oxidative stress that leads to tissue browning. the study was carried out on ‘pomella genovese’ apples since this cultivar showed a high percentage of fruit strongly affected by watercore, thus providing a suitable material for the analyses. in order to figure out if the properties of watercore­fruit where somewhat affected by the growing conditions or were, instead, mainly due to the onset of the disorder, apples were harvested from three experimental orchards characterized by different pedoclimatic conditions. since this disorder can dissipate in the first months after harvest (neuwald et al., 2012), fruit analysed too early could be only affected by a “tem­ porary” form of watercore. to overcome this prob­ lem, the analyses on the fruit were carried out after 4 months of cold storage, when the disorder was in its stable form and browning symptoms were still at an early stage. buccheri et al. ‐ watercore in pomella genovese apples 5 2. materials and methods raw material fruit of the cv. pomella genovese (malus x domestica, borkh.) were harvested in the staffora valley (pavia­italy) in three orchards with different characteristics (table 1). immediately after harvest, apples were transported to the crea­it laboratory in milan where a sample of 30 fruit from each orchard was analysed for the standard maturity indices (firm­ ness, soluble solids content, acidity) (table 2). apples were then stored at 1°c (90% r.h.) for 4 months and analysed after 1 day at 20°c. fifty fruit watercore­free (wf) and 50 fruit affected by differ­ ent levels of watercore symptoms (w) (fig. 1) per each orchard, were assessed for quality parameters (skin colour, texture, ssc, acidity), antioxidant com­ pounds (total phenols, ascorbic and dehydroascorbic acid) and antioxidant activity. fruit were first subjected to skin colour and tex­ ture analysis, then equatorially cut and assigned to a treatment (watercore or watercore­free); finally, the fruit flesh was sampled (avoiding core and seeds), rapidly frozen and lyophilized for the subsequent analysis. quality analyses were carried out on individ­ ual fruit while antioxidant compounds were assessed on pools of 3 lyophilized fruit. quality analyses acidity was assessed according to the aoac offi­ cial methods of analysis (aoac, 1985). soluble solids content (ssc) was measured by a multiscale auto­ matic refractometer (mod. rfm91, bs, uk) on few drops of apple juice. a spectrophotometer cm­2600­ d minolta was used to perform the colour measure­ ments : l* (lightness) a* (green­red), b* (yellow­blue) were assessed on the peel on 2 opposite sides of each fruit, avoiding the red blush and any external fig. 1 ­ fruit of ‘pomella genovese’ watercore­free (left) or affected by different levels of watercore (right). table 1 ­ characteristics and average climatic data of the growing season (april­october) for the three orchards orchard altitude (m asl) training system planting distance (m) row orientan­ tion slope rootstock plants age (yrs) irrigation fertilization average temp. (max/min. °c) total rainfall (mm) a 342 palmette 4 x 2 sw­ne slight (<15%) mm106 25 emergency irrigation manure (winter) + npk (spring) 14/25 533 b 392 palmette 4 x 2 sw­ne slight (<15%) seedling 17 emergency irrigation manure (winter) + npk (spring) 15/23 610 c 579 vase one row n­s slight (<15%) seedling 11 emergency irrigation burial of crop residues 15/24 547 table 2 ­ maturity indices of ‘pomella genovese’ apples at har­ vest different letters indicate significant differences (lsd test, p<0.01). orchard firmnes (n) ssc (%) acidity (g/l malic acid) a 91.0 a 12.5 a 2.51 a b 88.2 a 12.5 a 2.45 a c 90.6 a 13.6 b 2.86 b symptom of watercore, if present. the hue (h°) was calculated as arctangent (b*/a*). the specific weight was calculated on each fruit as the ratio between the fruit weight (g) and its vol­ ume (cm3) measured underwater. fruit mechanical and acoustic parameters were measured on two peeled areas of each fruit using a ta­xt plus texture analyzer (stable micro systems, godalming, uk) coupled with an acoustic emission detector (aed, stable micro systems). by means of this system, it was possible to simultaneously evalu­ ate the mechanical force­displacement and the cor­ responding acoustic response. a puncture test with an 11 mm diameter cylindrical probe at the cross­ head speed of 3.33 mm/s to a depth of 8 mm, allowed to obtain the mechanical profile; at the same time, the aed device, with a frequency cut­off set of 3.125 khz, gave the acoustic response of the sample. the microphone was placed 10 mm far from the apple at the mid­height of the fruit. from the adv. hort. sci., 2020 34(1s): 3­12 6 fig. 2 ­ force/deformation curve and acoustic profile of ‘pomella genovese’ apples. fbk= force at the breaking point; wr = work; st= stiffness. combined mechanical/acoustic profile, a total of five parameters were chosen to describe the texture properties of ‘pomella genovese’ apples. three mechanical parameters (fbk= the force measured at the breaking point of the flesh; st= the stiffness given by the ratio between the force applied and the defor­ mation before the breaking point, wr= work, the energy related to flesh penetration up to 8 mm, given by the area under the force/deformation curve) and two acoustic parameters related with the flesh crispness (pk= the number of the acoustic peaks; σpk= the sum of all the peak values) were extracted (fig. 2). equivalent (gae) per 100 g of fresh weight (fw). samples were also subjected to one of the most used free radical scavenging assays, which is relative­ ly easy and cheap in execution and is strongly corre­ lated with the sample’s nutraceutical content: the 2,2­diphenyl­1­pycrilidrazyl (dpph.) quenching (brand­williams et al., 1995). 600 µl of a 0.5 mm 2,2­ diphenyl­1­picrylhydrazyl radical (dpph) ethanol solution, 2.0 ml ethanol and 200 µl of sample were put in a 1­cm path cuvette, and the absorbance at 517 nm against blank (2.5 ml ethanol and 100 µl of sample) was recorded at 0 s and 180 s. the inhibition percentage of dpph was calculated as: dpph %= [(at0 ­ at180) / at0] × 100 a calibration curve was built by using different dilutions of trolox and the results were expressed as mg trolox equivalents (te) in 100 g fw. ascorbic (aa) and dehydroascorbic acid (dha) were determined by hplc according to lo scalzo et al. (2007) and wechtersbach and cigić (2007). briefly, 50 mg of lyophilized powder were extracted with 1 ml of 3% metaphosphoric acid, vortexed for 30 s, centrifuged at 12,000 rpm for 20 min at 4°c and immediately analysed for aa content. for the deter­ mination of total aa (aa + dha), the extracts were added with 100 mmol l­1 in hcl 1m of tris­car­ boxyethyl phosphine (tcep) as a reducing agent for dha. 400 µl of extracts were, then, diluted 1:2.5 in a 0.02m ortophosphoric acid solution. aa was deter­ mined using a jasco (tokyo, japan) hplc system con­ sisting of a pu­980 liquid chromatographic pump, a model as 1055­10 auto sampler and an uv­vis 15770 detector set at 254 nm, coupled to an inertsil ods­3 column (4.6 mm i.d. × 250 mm length, particle diam­ eter 5 μm, gl science) at the temperature of 30°c, and was eluted with 0.02 m orthophosphoric acid at a flow rate of 0.7 ml min­1.aa was estimated from a standard curve of l­ascorbic acid in 3% mpa. dha content was calculated by subtracting the aa content from the total aa content. data were expressed as mg per 100 g of fresh weight (mg aa or dha 100 g fw­1). statistical analysis statistical analyses were carried out with the statgraphics software v.5.1 package (manugistics, rockwell md). data were submitted to multifactor anova evaluating the main effects of the factors “watercore” and “orchard” as well as their interac­ tion. differences were determined by the least analysis of antioxidants total phenols and antioxidant activity were assessed on the following extract: 50 mg of powder from lyophilized apple flesh were extracted with 1.5 ml of an ethanol 96%/hcl 0.06n solution (1:1 v/v), vortexed for 30sec and centrifuged (12 min, 4°c, 10000 x g).the supernatant was recovered and stored a few days at ­20°c until the analyses were carried out. total phenols content (tpc) was measured by the folin­ciocalteu method, as described by singleton et al. (1999), 100 µl of fruit extract, 3 ml of distilled water and 500 ml folin­ciocalteu reagent were incu­ bated for 3 min, followed by the addition of 2 ml 10% sodium carbonate. after 2 h in the dark, the absorbance was measured at 730 nm against blank with a v­630 spectrophotometer (jasco, japan). a calibration curve was built with different concentra­ tion of gallic acid and the polyphenols concentration of the samples was expressed as mg of gallic acid buccheri et al. ‐ watercore in pomella genovese apples 7 crispness also showed important differences among the treatments. w­fruit always had more acoustic peaks and a higher sum of the peak value without any differences among orchards. in fruit affected by watercore, the concentration of some antioxidant compounds changed. regardless of the orchard, tpc increased, in average, 11.5% in w significant distance (lsd) test. 3. results watercore­free and watercore­affected fruit revealed different quality characteristics. w­fruit always showed a darker (lower l*) and less yellow (lower b*) peel colour with respect to wf samples, while the parameters a* and h° were mostly affected by the orchard (table 3). fruit harvested in the orchard c had a less green (lower a*) and more yel­ low (lower h°) peel colour. in all the three orchards, fruit density (fig. 3a) was higher in the w­fruit than in the wf ones. fruit from the orchard a showed, in average, the highest value, followed by those harvested in the orchard c and b. this difference was mainly due to the variation in the fruit density among the wf fruit, while the values of w­samples were similar among each other. the soluble solids content (fig. 3b) was always higher in the w­fruit that showed, on average, an increase of 12% with respect to wf apples. fruit har­ vested in the orchard c had the highest ssc, while those of a and b showed lower values without signifi­ cant differences among each other. no differences among treatments were found for acidity (data not shown). the mechanical parameters of the fruit were sig­ nificantly affected by watercore (table 4). w­fruit needed more energy (wr) to penetrate the fruit flesh up to 8 mm and showed a higher value of fbk with­ out any significant difference among fruit harvested in the three orchards. stiffness, which measure the “rigidity” of the flesh, was markedly higher in w fruit than in the wf ones. the acoustic parameters related to the flesh table 3 ­ color parameters of watercore­free (wf) and watercore (w) fruit of ‘pomella genovese’ lsd values for the two main factors and their interaction are reported at the bottom (**=p<0.01). orchard l* a* b* hue (h*) wf w wf w wf w wf w a 70.8 63.7 ­10.9 ­9.1 44.7 38.9 103.1 102.7 b 70.6 64.1 ­13.7 ­12.3 46.2 40.8 106.3 106.8 c 67.7 64.7 ­8.1 ­9.9 44.4 41.6 97.6 102.1 mean 69.7 64.2 ‐10.9 ‐10.4 45.1 40.4 102.4 103.9 main factors watercore 1.41 ** ns 1.23 ** ns orchard ns 2.22 ** ns 3.43 ** orchard x watercore 2.94 ** 3.99 ** 2.93 ** 5.11 ** fig. 3 ­ fruit density (a) and soluble solids content (b) of water­ core­free (wf) and watercore (w) fruit of ‘pomella genovese’ from three different orchards. average ± stan­ dard error. lsd is reported in each figure (p>0.01). in the box at the bottom the average and lsd values are indica­ ted for each of the main factors (*=p<0.05; **=p<0.01). 8 adv. hort. sci., 2020 34(1s): 3­12 fruit with respect to the wf samples (fig. 4a). fruit from the orchard c showed the highest value while no difference was found between a and b. the average aa content (fig. 5a) decreased by 30% in w­fruit with respect to wf ones. this effect was found, above all, in fruit from orchards a and b, while the decrease was not significant in the orchard c. the dha content (fig. 5b) increased markedly in fruit affected by watercore. w­fruit showed, on aver­ age, an increase in dehydroascorbic acid of about 40% with respect to wf­fruit. the ratio between ascorbic and dehydroascorbic acid was, on average, 1.2 for the wf­fruit and decreased by half (0.6) in the fruit affected by watercore. antioxidant activity (fig. 4b) was always higher in watercore­affected fruit, regardless of the orchard. a high correlation coefficient was found between antioxidant activity and total polyphenol content (r=0.83) while no relationship was observed between antioxidant activity and ascorbic acid content. 4. discussion and conclusion despite the different field characteristics and plants training systems, w­fruit had always a lower lightness and were less yellow than wf apples, even if the measurement were taken avoiding any external symptom of watercore on the peel. fruit from plants grown in the field c, had a more yellowish colour, probably because of the change in the light intercep­ tion inside the canopy due to the different training systems and planting distances (table 1). however, also in this case, peel colour (h° and b*) was signifi­ cantly different between wf and w­fruit. according to the results of watkins et al. (1993) on fuji apples, background peel colour could be considered a possi­ ble indicator for watercore anyway, further studies are needed to confirm this hypothesis. fruit density of ‘pomella genovese’ w­samples was increased by the fluid accumulation in the inter­ cellular spaces, which is considered a distinctive fea­ table 4 ­ mechanical and acoustic parameters of watercore­free (wf) and watercore (w) fruit of ‘pomella genovese’ fbk= force at the breaking point; wr= work; st= stiffness; pk= peak number; σpk= sum of peak values. lsd values for the two main factors and their interaction are reported at the bottom (*=p<0.05; **=p<0.01). orchard mechanic acoustic fbk (n) wr (n x mm) st (n/mm) pk (n*) ∑pk (mv) wf w wf w wf w wf w wf w a 44.3 57.4 79.6 109.6 59.7 72.8 2.8 5.4 0.09 0.21 b 42.6 60.3 76.6 113.2 61.2 77.9 2.4 5.1 0.08 0.19 c 46.0 65.7 83.6 125.3 61.5 79.0 3.0 5.4 0.09 0.18 mean 44.3 61.1 79.9 116.0 60.8 76.6 2.7 5.3 0.08 0.19 main factors watercore 3.1 ** 7.2 ** 6.6 ** 0.75 ** 0.03 ** orchard ns ns ns ns ns orchard x watercore 6.5 ** 19.4 3.3 ** 1.56 ** 0.06 ** fig. 4 ­ total phenols content (a) and antioxidant activity (b) of watercore­free (wf) and watercore (w) fruit of ‘pomella genovese’from three different orchards. average ± stan­ dard error. lsd is reported in each figure (p>0.01). in the box at the bottom the averages and lsd values are indica­ ted for each of the main factors (*=p<0.05; **=p<0.01). buccheri et al. ‐ watercore in pomella genovese apples 9 ture of watercore fruit (bowen and watkins, 1997; suzuki et al., 2002; zupan et al., 2016). the fruit den­ sity is usually cultivar­dependent (yamada and kobayashi, 1999) but, in this case, it was also depen­ dent on the orchard. the variability in the fruit densi­ ty makes it difficult to use some watercore detection methods which have been developed based on this fruit parameter (cavalieri et al., 1998). the liquid that accumulates in the intercellular spaces of watercore fruit is rich in sugars, above all sorbitol, which is the primary transport carbohydrate in apple (loescher et al., 2005). sorbitol is actively unloaded from the phloem, but a decreased ability to transport sugars into the fruit can lead to sorbitol accumulation in the intercellular spaces (gao et al., 2005) that promotes water retention (williams, 1966). yamada and kobayashi (1999) found an increase in sorbitol, as well as glucose, fructose and ssc in any cellular compartment (vacuole, cytoplasm, free spaces) of watercore­affected fruit, while other authors (zupan et al., 2016) reported only an increase in sorbitol and a decrease in other sugars and in soluble solids content. in ‘pomella genovese’ apples, the ssc of w­fruit was well above the value of the wf samples and this may indicate an increase in all the main sugars of the fruit. fruit from the orchard c had, on average, higher soluble solids con­ tent than a and b, probably because of the different training systems and planting distances that allowed a higher light interception efficiency. in any case, the higher ssc value shown by w­fruit with respect to wf­treatments was not influenced by the orchard characteristics. the flooding of the intercellular spaces affected the texture properties of watercore fruit. since the standard method to assess firmness (usually the max­ imum force tested by a penetrometer), is limited to a data/point measurement and can be considered inappropriate to describe the complex texture of an apple (ioannides et al., 2007), we included in the analysis different mechanical and acoustic parame­ ters related with fruit firmness, stiffness and crisp­ ness. the results of the mechanical/acoustic profile indicate that the watercore fruit had a firmer texture and were crispier than the wf­fruit. apple texture properties depend on the morphological features of the cells, with a firmer tissue having smaller and compact intercellular spaces than a softer one (ting et al., 2013). the higher flesh firmness of the w­ apples could, then, be due to the greater turgidity of the fruit and to the increased density and compact­ ness of the tissue, associated with fluid­filled instead of air­filled intercellular spaces (bowen and watkins, 1997). different authors reported this watercore effect on “fuji” and “himerami” apples (bowen and watkins, 1997; yamada and kobayashi, 1999). in the plant cells, the electron transport chain (etc) is the major producer of reactive oxygen species (ros). in apples (cv. fuji) affected by water­ core, the lower intercellular air volume reduces the permeance to gas diffusion, and causes an increase of the co2 pressure inside the fruit, while the o2 con­ centration decreases (argenta et al., 2002; kasai and arakawa, 2010). the anoxia in watercore­fruit led to the inactivation of the two terminal oxidases of the etc (cytochrome c oxidase and alternative oxidases), causing an increase of ros, as well as of other enzymes involved in the oxidation process (kasai and arakawa, 2010; zupan et al., 2016). this increased ros level in the mitochondria can act as a stress sig­ nal, affecting the nuclear gene expression and modi­ fig. 5 ­ ascorbic (a) and dehydroascorbic (b) acid content of watercore­free (wf) and watercore (w) fruit of ‘pomella genovese’ apple from three different orchards. average ± standard error. lsd is reported in each figure (p>0.01). in the box at the bottom the average and lsd values are indicated for each of the main factors (*=p<0.05; **=p<0.01). adv. hort. sci., 2020 34(1s): 3­12 10 fying the level of some stress­related compounds as, for example, ascorbic acid or phenols. phenols are plant secondary metabolites and are synthesized through the shikimic acid pathway. the key enzyme in the biosynthesis of phenols is the phenylalanine ammonia lyase (pal) which catalyses the reaction converting l­phenylalanine to ammonia and trans­cinnamic acid (tomás­barberán and espín, 2001). through the regulation of pal activity, the plant can modulate all the phenylpropanoid biosyn­ thesis. the activity of phenylalanine ammonia lyase can be stimulated by a variety of environmental stresses, including tissue wounding, uv irradiation, low temperatures (zhang and liu, 2015). tissue expo­ sure at high levels of ethylene can cause an increase in pal activity (tomás­barberán and espín, 2001) and in ‘himerami’ and ‘fuji’ apples severely affected by watercore an internal accumulation of ethylene was reported (bowen and watkins, 1997; yamada and kobayashi, 1999), because of the osmotic stress and of the reduction of permeance to ethylene (argenta et al., 2002). in all the three orchards assessed in this study, fruit affected by watercore had a higher amount of polyphenols with respect of the watercore­free sam­ ples. an increase of ros and ethylene production, caused by the reduced gas permeability inside the fruit, could have enhanced pal activity and, conse­ quently, induced the increase of the polyphenol con­ tent in the watercored fruit. some authors (zupan et al., 2016) found a lower phenols content in watercore fruit of the cultivars ‘delicious’, ‘gloster’ and ‘fuji’, which were analysed immediately after harvest. the different results observed in ‘pomella genovese’ apples could be due to a different cultivar behavior but also to the differ­ ent watercore intensity of the fruit. pomella genovese fruit were analyzed after 4 months of stor­ age, when the “temporary watercore” had already dissipated (neuwald et al., 2012). the fruit analysed were, then, only those severely affected by water­ core, in which the higher ethylene content (bowen and watkins, 1997; yamada and kobayashi, 1999) could have induced the increase in polyphenol con­ tent. in plants, the ascorbate­glutathione cycle oper­ ates to detoxify the hydrogen peroxide in order to avoid its reaction with the superoxide anion that pro­ duces the highly reactive hydroxyl radical (hodges et al., 2004). ascorbate acts as a scavenger of peroxide and it is oxidized by the ascorbate peroxidase (apx) to dehydroascorbic acid, which is recycled to ascor­ bate by means of the dehydroascorbate reductase (mittler, 2002). however, when the production of ros exceeds the scavenger capacity of the system, the ascorbic acid starts to decrease, so that this com­ pound is often used to assess oxidative stress in postharvest studies (hodges et al., 2004). in our study, we found, on average, lower ascor­ bic acid and higher dehydroascorbic acid content in w­fruit. the higher ros production due to the hypox­ ia caused by the flooding of intercellular spaces can affect the ascorbic­glutathione cycle. kasai and arakawa (2010) found lower ascorbic acid content and higher apx activity in watercore­affected ’fuji’ apples, probably caused by the higher h2o2 levels in the fruit flesh. the decrease of the ascorbic/dehy­ droascorbic ratio in our experiment can indicate a shift in the reduction state of ascorbate under oxy­ gen deprivation (blokhina et al., 2003) and could be considered a signal of an oxidative stress status. total antioxidant activity of the fruit followed the trend of polyphenol content and seems to be not related to ascorbic acid. as reported by different authors (miller and rice­evans, 1997; szeto et al., 2002), ascorbic acid activity represents, in fact, a minimal fraction of the total antioxidant activity of apple fruit while some phenolic compounds, as the hydroxycinnamate chlorogenic acid, are the major contributors. in conclusion, in this work different aspects of watercore­affected apples have been studied. the flooding of intercellular spaces influenced different quality characteristics of watercore­apples. fruit den­ sity and ssc increased, the texture was firmer and crispier, and the peel background colour was darker. the anoxic conditions caused by watercore lead to oxidative stress, as shown by the decrease in the aa/dha ratio in the watercore­fruit. on the other hand, the probable increase in ethylene content in fruit severely affected by watercore could have enhanced the activity of the pal enzyme, leading to an increase in the polyphenol content and antioxi­ dant activity. antioxidant compounds showed roughly the same differences between watercore and watercore free­fruit regardless to the orchard, suggesting that these compounds were mostly affected by the onset of the disorder, while they were only slightly influ­ enced by the growing conditions. the findings of this work support the hypothesis that, in watercore­affected fruit, browning disorders buccheri et al. ‐ watercore in pomella genovese apples 11 could be due to oxidative stress that causes the los­ ing of membrane integrity and disrupt cell compart­ mentation. polyphenols are then oxidized by polyphenol oxidase to mono­diphenolic compounds which impart a brown colour to the fruit. references aoac, 1985 ­ official methods of analysis, 16th ed. ­ association of official analytical chemists, washington, usa. argenta l., 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afghanistan m. gori 1, s. pecchioli 1, e. giordani 1, m.a. saeedi 2, f.h. wafa 2,, s. biricolti 1 (*) 1 dipartimento di scienze e tecnologie agrarie, alimentari, ambientali e forestali, sezione colture arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino, italy. 2 afghanistan national horticulture development organization (anhdo), taimani street, 9, kabul, afghanistan. keywords: afghanistan, citrus, psba-trnh spacer. abstract: the establishment of a modern fruit culture in developing countries requests an accurate evaluation of the preexisting germplasm and its health status. this to prevent the possibility to introduce new germplasm which can be easily prey of the endemic diseases carried by asymptomatic host plants. therefore, after the identification of cases of citrus plants affected by tristeza virus, a survey of the germplasm cultivated in the nangarhar valley and some nearby regions was run. the survey was focused on the identification of the main citrus species widely cultivated using barcoding analysis of conserved sequences located in the plastid dna. the sequences of matk and rbcl genes did not show any discriminatory ability while the analysis of the the non-coding psba-trnh intergenic spacer (psba‐trnh) showed a robust single nucleotide polymorphism (snp) discriminating c. aurantium from c. sinensis in all analysed samples. these non-coding regions have no known function; thus, much of the variation may result from the spread of mutations unconstrained by selection. because nucleotide variation in the psba‐trnh spacer region is high, mutational hot spots may be useful to detect species-level variations. according to our study, the afghan citrus germplasm belong to the c. aurantium species which is commonly used in citrus culture as a rootstock. in afghanistan it is widely cultivated for fresh consumption, without topworking with selected varieties and this may be the reason why symptoms are often mild and cultivation can be anyhow carried on. 1. introduction in developing countries, farmers are growing landraces or older improved varieties that are not optimized for today’s climate or production systems. therefore, the replacement of the local germplasm traditionally cultivated for long should be carried on by introducing elite varieties in order to complying with the rules of a modern cropping system. however, this change rises concerns about the adaptation of the elite (*) corresponding author: stefano.biricolti@unifi.it citation: gori m., pecchioli s., giordani e., saeedi m.a., wafa f.h., biricolti s., 2019 barcoding assessment of the citrus species cultivated in eastern afghanistan. adv. hort. sci., 33(3): 403408 copyright: © 2019 gori m., pecchioli s., giordani e., saeedi m.a., wafa f.h., biricolti s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 june 2019 accepted for publication 1 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 403-408 404 germplasm to the new conditions which may poses the farmers revenue at risk. the choices to be undertaken are very delicate and consequences may be devastating if not based on preliminary scientific investigations. nangarhār (pashto: راهرګنن ; persian: راهرگنن ), laghman and kunar are three of the 34 provinces of afghanistan, located in the eastern part of the country. the lowlands in those regions benefit from a semi-tropical climate and have the highest proportion of high cropping intensity irrigated land in the country. the riverine farms, situated along valley bottoms of varying widths, produce a range of crops throughout the year. semitropical crops such as citrus, sugar canes and henna are produced around jalalabad. high potential for fruits production occurs in those regions despite the still ongoing conflict. promoting commercial orchards, establishment through professional nurseries, farmers investment, extension service with technical input from neighboring countries and research are therefore welcome to develop the potential for fruit production and marketing in the afghan eastern region (giordani et al., 2014). a screening of the health status of the afghan germplasm of citrus was undertaken to ensure multiplication of not only the best-selected varieties or ecotypes but also to avoid reproduction and distribution of virus-infected fruit tree. in 2012 a first report showed the occurrence of citrus tristeza virus (ctv) in plants sampled in the national collection experimental farm in jalalabad (nangarhar province) (rehman et al., 2012). a successive investigation showed that ctv was detected in several samples collected in some farms located in the nangarhar valley, a warning that ctv could spread rapidly if sensitive citrus cultivars would be introduced in the area. however, citrus cultivation is still thriving in nangarhar valley despite the widespread presence of ctv. different possibilities are therefore to be considered: 1) the local germplasm is made up of low reactive host species which do not heavily show the symptoms of the disease; 2) mild ctv isolates established in the area; 3) an unknown source of resistance to ctv is occurring in the area. before introducing new rootstocks resistant to such disease, another survey was conducted in order to identify the main species of citrus. this is to address the choices to be undertaken for the establishment of a modern cropping system according to the market demand and to prevent the diffusion of quarantine diseases. usually, identification of the cultivated species relies on the use of phenotypic descriptors (upov). phenotypic descriptors are often subjected to the observer’s opinion and the environment can deeply affect the behavior of the plant, determining errors in the species attribution. a more reliable system to identify species is based on the genetic analysis and particularly on the dna barcoding procedure which consists in the comparison of highly conserved sequences located in the ribosomal nuclear (sun et al., 2015) or plastid dna (taberlet et al., 1991; penjor et al., 2010, 2013). being highly conserved, such fragments accumulate mutations slowly and it is possible to design universal primers which can be used to distinguish plenty of species. furthermore, many databases (pubmed, genbank, omim) collect and store millions of sequences, which can be compared by means of specific software (blast) with unknown sequences (zhang et al., 2000). when properly queried a dna database provides, along with the alignment, a similarity index which can be useful for identifying species or even sub-species, depending on the proximity of the taxonomic entities. therefore, due to the restrictions to accessibility to a conflicting area, a barcoding analysis has been carried out in order to identify the species of citrus which are commonly grown in the eastern area of afghanistan in order to better address the choices for a modern fruit culture. 2. materials and methods plant material afghan citrus seeds have been collected in three areas where citrus fruit trees are intensively cultivated (laghman, kunar and nangarhar) in eastern afghanistan (fig. 1). we used seeds because they can fig. 1 map of afghanistan showing the provinces where citrus production is located and where the survey was carried on. gori et al. ‐ barcoding assessment of the afghan citrus population 405 be easily transported. furthermore, most citrus species are characterized by adventitious embryony and seeds originate individuals which are true clones of the mother plant because rarely zygotic embryo survives. most sequences for dna barcoding, being located in the plastid or mitochondrial dna, are inherited only from the maternal line, therefore germinating seeds produce plants whose organelle genomes are not affected by the pollen donor plant, but are expected to be exactly the same as the seed donor plant. in each area, some plants were selected and batch of seeds have been collected from single fruit. in table 1 the origin of the seeds is reported. each batch was labeled and delivered to the plant pathology department of the university of bologna. the seeds were germinated in pots and leaf samples were collected. for each seed batch, a single seedling has been selected for dna barcoding analysis. before starting the present work, citrus barcoding sequences (its 1 and 2, matk, rbcl, psba‐trnh intergenic spacer) were retrieved from genbank. the dataset was used to compare with the results of sequencing and to assign the analysed samples to a citrus species. since the available sequences are few and showed heavy discrepancies, casting doubts about the reliability of the data retrieved online (bengtsson-palme et al., 2016), we decided to provide robust references by analyzing accessions whose origin was absolutely certain. therefore we have sampled sour orange along with other citrus species from private and public collections (vivai oscar tintori, orto botanico “giardino dei semplici” of the university of florence, istituto agronomico per l’oltremare) in order to be compared with the samples coming from afghanistan (table 2). pcr amplification and primers total dna was extracted from the selected plants following the guidelines of the dna invisorb dna extraction kit producer (stratec italy). dna has been electrophoresed on an agarose gel to validate quality and quantity. universal primers for pcr amplification, used in the present study have been retrieved in literature (chen et al., 2010; luo et al 2010; penjor et al., 2013; mahadani and ghosh, 2014; uchoi et al., 2016; wattoo et al., 2016; bailey et al., 2018; zhao et table 1 list of the samples coming from afghanistan table 2 citrus species used as control, coming from public and private collections in tuscany sample province district village variety af-1 a laghman mehtarlam chardehi local af-2 a laghman mehtarlam chardehi local af-5 a laghman mehtarlam chardehi local af-7 a kunar asadabad landi tesha local af-8 a kunar asadabad landi tesha local af-9 a kunar asadabad landi tesha local af-11 a kunar asadabad landi tesha local af-18 a nangarhar surkhroad naghrak local af-19 a nangarhar surkhroad naghrak local af-21 a nangarhar surkhroad sabzabad local af-22 a nangarhar surkhroad sabzabad local af-limon nangarhar phdc-center citrus volkamar sample no. species or cultivar province origin of the accession 1 citrus sinensis (washington navel) pescia (italy) oscar tintori 2 citrus sinensis (ovale calabrese) pescia (italy) oscar tintori 3 citrus sinensis (tarocco) pescia (italy) oscar tintori 4 citrus aurantium lucca (italy) botanical garden 5 citrus aurantium firenze (italy) iao 6 citrus aurantium (foetifera) pescia (italy) oscar tintori 7 citrus aurantium (tipo) pescia (italy) oscar tintori 8 citrus aurantium (dolce del gargano) pescia (italy) oscar tintori 9 citrus aurantiifolia (philippines red lime) pescia (italy) oscar tintori 10 citrus aurantiifolia (mexico) pescia (italy) oscar tintori 11 citrus aurantium firenze (italy) botanical garden 12 citrus limon firenze (italy) botanical garden 13 citrus mitis firenze (italy) botanical garden 14 citrus histrix firenze (italy) botanical garden 15 citrus decumana firenze (italy) botanical garden 16 citrus grandis firenze (italy) botanical garden 17 citrus reticulata firenze (italy) botanical garden 18 citrus lumia firenze (italy) botanical garden 19 citrus medica firenze (italy) botanical garden adv. hort. sci., 2019 33(3): 403-408 406 al., 2018) and are designed on the sequence of the internal transcribed sequence 1 (its1) and 2 (its2) of the nuclear ribosomal dna, of the maturase k (matk), the rubisco large chain unit (rbcl) and of the predominantly non-coding psba-trnh intergenic spacer (table 3). pcr analyses were performed with a thermal cycler primus 96 (peqlab) in a 25 µl volume containing 25 ng total dna, 1x taq buffer, 1,5 mm mgcl2, 1 unit gotaq (promega) and 200 nm of each primer. pcr conditions were 95°c for 5 min, then 35 cycles at 95°c for 30 seconds followed by 30 seconds at a temperature ranging from 50 to 60°c depending on the chosen primer pair and an extension cycle at 72°c for 50 sec. a final extension at 72°c for 5 minutes was carried on. the amplification products were purified with the qiaquick pcr purification tubes (qiagen) and then sequenced. even though the primers for barcoding are called “universal”, the presence of conserved flanking sites complementary to the primers sequence near the variable part and the locus copy number have been shown to account for much of the variability of amplification success. therefore, the all primers have been tested by pcr, amplifying and sequencing the amplicons using a set of the samples as templates. to perform a phylogenetic analysis we have used the mega7 software package (tamura et al., 2013) which compares the sequence calculating a similarity matrix, transforms similarity coefficients into distances and makes a clustering using the unweighted pair group method with arithmetic mean (upgma) algorithm. the final output is represented by a dendrogram which clusters the samples. 3. results the primers constructed on the sequence of the internal transcribed sequence 1 (its1) and 2 (its2) resulted in a faint amplification and were therefore discarded, while primers designed on the sequence of the maturase k (matk) and rubisco large chain unit (rbcl) genes were correctly amplified and sequenced. unfortunately, such sequences did not show any discriminatory ability being completely overlapping for most analysed samples (mahadani and ghosh, 2014). on the contrary, the primers of the non-coding psba-trnh intergenic spacer showed either good amplification (a fragment of about 525 bp) and bidirectional sequencing output either a good discriminatory capacity. we found a robust single nucleotide polymorphism (snp) in the psba‐trnh spacer (fig. 2) which is capable to discriminating c. aurantium (sour orange) from c. sinensis showing this mutation in all analysed samples independently from the origin area. despite the limited region of the genome analysed containing, as expected, a low number of mutations, all the samples of c. aurantium, independently from their origin, clustered together with a reasonable certainty (fig. 3). the same occurs for the three c. sinensis samples which resulted in a welltable 3 universal primers sequence for barcoding used in this study name 5’ → 3’ primer sequence references matk f cgtacagtacttttgtgtttacgag jeanson et al., 2011 matk r acccagtccatctggaaatcttggttc jeanson et al., 2011 rbcl_f1 atgtcaccacaaacagagactaaagc uchoi et al., 2016 rbcl_r634 gaaacggtccctccaacgcat jeanson et al., 2011 rbcl_r724 tcgcatgtccctgcagtagc kress et al., 2005 its1_5f_746 ggaagtaaaagtcgtaacaagg cheng et al., 2016 its1_4r_746 tcctccgcttattgatatgc cheng et al., 2016 its2_s2_f497 atgcgatacttggtgtgaat chen et al., 2010 its2_s3r_497 gacgcttctccagactacaat chen et al., 2010 psba‐trnh_fw cgcgcatggtggattcacaatcc zhao et al., 2018 psba‐trnh_rev gttatgcatgaacgtaatgctc zhao et al., 2018 matk1f accgtatcgcactatgtatc penjor et al., 2013 matk1r gaactagtcggatggagtag penjor et al., 2013 matk2f acggttctttctccacgagt penjor et al., 2013 matk3f ggtccgatttctctgattct penjor et al., 2013 matk2r agaatcagagaaatcggacc penjor et al., 2013 matk3r actcgtggagaaagaaccgt penjor et al., 2013 gori et al. ‐ barcoding assessment of the afghan citrus population 407 separated group while the sample c. limon need to be better characterized with additional sequence analysis (penjor et al., 2010, 2013). 4. discussion and conclusions the barcoding analysis of the afghan germplasm and citrus species has been carried out with a set of primers targeting nuclear ribosomal dna or chloroplast genes (its1 and 2, matk, rbcl and psba-trnh). apart from its 1 and 2, all the primers enabled amplification and sequencing of all the samples. most of the citrus species were correctly identified even if the comparison of the analysed dna fragments with the data available in the databases showed several discrepancies. this observation suggested to introduce samples of certain origin in barcoding analysis in order to avoid biases due to errors occurring in the sequences uploaded on the database. according to the results of our analysis, we can state with reasonable certainty that all the afghan citrus samples are sour orange, which is commonly used as a rootstock. the wide diffusion of sour orange in those regions of afghanistan is due to the fact that its fruits are consumed fresh (http://anhdo.org.af/wpcontent/uploads/2017/06/citrus-market-trend.pdf). the plants are not topworked, as usual, with selected c. sinensis varieties. grafting would have shown heavily the symptoms of tristeza disease, while this does not occur in ungrafted c. aurantium (gómezmuñoz et al., 2017). in conclusion, stepwise replacement of the orange germplasm in afghanistan with ctv resistant rootstocks is advisable before grafting with selected orange varieties to prevent ctv spreading. however, the replacement of sour orange, a rootstock characterized by highly desirable agronomic features, with ctv resistant rootstocks should be carried out after devising the whole production chain, starting from the adoption of proper cropping fig. 2 two haplotypes of c. aurantium and c. sinensis originated by a single nucleotide polymorphism getting from the sequencing of psba‐trnh intergenic spacer (psba‐trnh). fig. 3 upgma dendrogram representing the genetic distances among the analysed samples. http://anhdo.org.af/wp-content/uploads/2017/06/citrus-market-trend.pdf http://anhdo.org.af/wp-content/uploads/2017/06/citrus-market-trend.pdf 408 adv. hort. sci., 2019 33(3): 403-408 system (irrigation, fertilization, soil management, etc.) in order to prevent that other endemic diseases can affect the newly introduced germplasm. references bailey j.b., lamb m., walker m., weed c., craven k.s., 2018 detection of potential fungal pathogens fusarium falciforme and f. keratoplasticum in unhatched loggerhead turtle eggs using a molecular approach. ‐ endang. species res., 36: 111-119. bengtsson-palme j., boulund f., edström r., feizi a., johnning a., jonsson v.a., karlsson f.h., pal c., buongermino pereira m., rehammar a., sanchez j., sanli k., thorell k., 2016 strategies to improve usability and 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pisano, 12, 56126 pisa, italy. 3 crea centro di ricerca orticoltura e florovivaismo, corso inglesi, 508, 18038 sanremo (im), italy. 4 centro interdipartimentale di ricerca nutraceutica e alimentazione per la salute (nutrafood), università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: antioxidant activity, enzyme activity, low temperature, postharvest, secondary metabolites, sweet wild garlic, volatile organic com­ pounds. abstract: edible flowers are appreciated due to their aesthetic features, nutri­ tional value and antioxidant properties. tulbaghia simmleri beauv. (amaryllidaceae family) flowers are characterized by a pleasant garlic taste and are consumed both as fresh and dried products. the aim of this work was to assess the effect of chilling temperature (+4°c) on the visual quality, nutritional content, and aroma profile of t. simmleri flowers after two (t2) and six (t6) days of storage. colorimetric analysis highlighted a reduction in petal bright­ ness at t6 and hence their darkening, due to a significant increase in a* coordi­ nate and the decrease in the b* one. total polyphenols and flavonoids content remained unchanged until the end of the experiment, while total anthocyanins increased at t2. flowers antioxidant activity (dpph assay) decreased progres­ sively during cold storage, while catalase (cat) and ascorbate peroxidase (apx) activities increased. the aroma profile was analyzed by hs­spme associated with gc­ms, underlining that fresh flowers were dominated by high content in monoterpenes (around 80%), with 1,8­cineol as main compound (53.1%). cold storage reduced this class of volatiles while sesquiterpenes and non­terpenes increased; between them, benzyl benzoate reached 12%. 1. introduction edible flowers (efs) are traditionally consumed since ancient times (mlcek and rop, 2011). some of them are commonly recognised as veg­ etables (e.g. artichokes, broccoli, capers), while others are still considered (*) corresponding author: basmanajar@hotmail.fr citation: marchioni i., najar b., copetta a., ferri b., ruffoni b., pistelli l., pistelli l., 2023 ­ phytonutritional and aromatic profiles of tulbaghia simmleri beauv. edible flowers during cold storage. ­ adv. hort. sci., 37(1): 25­32. copyright: © 2023 marchioni i., najar b., copetta a., ferri b., ruffoni b., pistelli l., pistelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 october 2022 accepted for publication 13 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13857 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 25­32 26 “unusual food” (reviewed in pires et al., 2019). efs straights rely on their colours, shapes, flavours, tastes, and nutrients (e.g. carbohydrates, proteins, vitamins, phytochemical compounds with antioxidant and healthy properties) (fernades et al., 2017). their market is constantly expanding, and new species with attractive sensorial features and good storage attitude are required. tulbaghia (common name: wild garlic) is a genus of monocotyledonous plants (amaryllidaceae family) indigenous to south africa (lyantagaye, 2011). herbaceous perennial bulbs, corms or rhizomes char­ acterize its species. tulbaghia spp. flowers, held in umbels in groups of ten or more, are strongly fra­ grant and characterised by tubular shape (zschocke and van staden, 2000). a raised crown­like structure or a fleshy ring at the centre of the flower tube are distinctive features of this genus (vosa, 2000). the colours are different, mainly white, pink or mauve. flowers and rhizomes produce cysteine­derived sul­ phur compounds (e.g. marasmicin), which confer to this organs a pleasant alliaceous smell, especially when bruised or during senescence (aremu and van staden 2013; kubec et al., 2013). the peculiar aroma and the pungent garlicky taste of flowers make sever­ al tulbaghia spp. interesting for the food industry (kubec et al., 2013). t. simmleri beauv. is mainly known as ornamental plant, which flowers consist of six tepals and a cen­ tral crown of six lobes, fused for more than a third of their length to form a tube. the lobes have pointed tips, giving the crown a fringed edge (vosa, 2000). in the southern hemisphere, its period of blooming ranges between april to october, even though, with particular climate conditions, it could be extended until early spring (zschocke and van staden, 2000). in the northern hemisphere, however, its period of blooming ranges between october to april. several bioactive compounds characterize this plant, since it is used to treat fever, colds, headaches, asthma, and tuberculosis in south african traditional medicine (zschocke and van staden, 2000). t. simmleri has been severely neglected when compared to the most common t. violacea, for which several culinary uses are known, also concerning flowers (aremu and van staden, 2013; rivas­garcía et al., 2022). further investigation on t. simmleri worth to be performed, since this species produce deep mauve, long lasting edible flowers, which period of bloom does not over­ lap the one of t. violacea (not available in autumn and winter). this will ensure the availability of efs with garlic taste for most of the year. moreover, takaidza et al. (2018) highlighted good total polyphe­ nolic and flavonoid content, and hence good antioxi­ dant activity, in t. simmleri plants, in comparison with other seven tulbaghia species, t. violacea included. postharvest technologies are common methods to extend efs shelf­life, as it is generally rather short (2­ 10 days) (fernandes et al., 2019, 2020). flowers are high value products, which must be picked with care, packaged properly to protect them from any mechanical damage, and stored at proper tempera­ ture until consumption (fernandes et al., 2020). improperly handled/stored edible flowers suffer tis­ sue browning, flower wilt, dehydration, petal discol­ oration, and abscission. the senescence process is associated with physiological changes and catabolism, which are linked to accelerated respira­ tory levels, weight reduction, and/or plant hormone response (kou et al., 2012; landi et al., 2018). to address these concerns, fresh edible flowers are often stored under low temperatures, generally at chilling ones (4­5°c) (fernandes et al., 2020). since different efs species showed different behaviour at cold storage (landi et al., 2018; marchioni et al., 2020 a, 2020 b), postharvest studies should be per­ formed for each flower, in order to elucidate their physiological response to low temperature and hence their shelf­life. the aim of this work was to evaluate the phytonu­ tritional and aromatic profile of t. simmleri efs stored at 4°c for 0, 2 and 6 postharvest days. spectrophotometric and chromatographic analyses were performed in order to highlight any changes in polyphenolic content (flavonoids and anthocyanins included), antioxidant activity, and volatile organic compounds (vocs) during cold storage. 2. materials and methods plant material and postharvest conditions tulbaghia simmleri plants were provided by the chambre d’agriculture des alpes­maritimes (cream, nice, france) and were grown at research centre for vegetable and ornamental crops (crea, sanremo, imperia, italy, gps: 43.816887, 7.758900). details on plant cultivation is reported in najar et al. (2019). full open flowers were picked in april, weighed and cold stored as described in marchioni et al. (2020 b), for two (t2) and six (t6) postharvest days. fresh flowers marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 27 were considered as control (t0). weight loss and colour determination flowers weigh was measured (ohaus® analytical standard series™ model as60s, ohaus corporation, florham park, n.j. usa) before cold storage (t0) and at the end of each experimental point (t2 and t6) to calculate their weight loss (formula reported in fernandes et al., 2018). once flowers had been weighed, their colour was evaluated with a spec­ trophotometer sp60 series (x­rite incorporated, michigan, usa). l* (lightness), a* (redness) and b* (yellowness) colour coordinates (cielab scale, cie 1976) were measured in different point of at least ten flowers, in order to best describe their colour variations. biochemical analyses biochemical analyses were performed using frozen samples. total phenolic, flavonoid and antho­ cyanins content were determined as reported by marchioni et al. (2020 b). data were reported as mg gallic acid equivalents (gaeq)/g fresh weight (fw) (polyphenols), mg catechin equivalents (ceq)/g fw (flavonoids), and mg malvin chloride equivalents (meq)/g fw (anthocyanins). radical scavenging activ­ ity (dpph assay) of each sample was determined as described by brand­williams et al. (1995). data was expressed in ic50, which represent the concentration of the sample able to inhibit by 50% the radical dpph. all absorbance were read in a uv­1800 spec­ trophotometer (shimadzu corp., kyoto, japan). enzymatic activities frozen flowers (200 mg) were pulverized and homogenized in 2 ml of extraction buffer, consisting of 50 mm sodium phosphate buffer (ph 7.0), 1 mm edta, 1 mm phenylmethylsulfonyl fluoride (pmsf), and 2% (w/v) insoluble polyvinylpolypyrrolidone (pvpp), as reported by pistelli et al. (2017). samples were centrifuged at maximum speed for 30 min at 4°c and the supernatant was used for enzyme activi­ ties. the soluble protein content was determined according to bradford (1976) using bovine serum albumin as standard. catalase (cat, ec 1.11.1.6) activity was measured by monitoring the decomposition of hydrogen perox­ ide (h2o2), recording the decline in absorbance per minute at 240 nm (zhang and kirkham, 1996). the reaction started by adding 20 μl of extract to 980 μl of 8.8 mm h2o2 solution in 50 mm sodium phosphate buffer. one unit of cat is determined as the amount of enzyme required to detoxify 1 μmole of h₂o₂ (ε= 394 m­1 cm­1) per minute. data were expressed as unit of cat per mg of soluble proteins (μmol min­1 mg­1). ascorbate peroxidase (apx, ec 1.11.1.11) activity was determined by following the decrease in absorbance at 290 nm (ε = 2.7 mm­1 x cm­1) due to enzymatic ascorbate oxidation (nakano and asada, 1981). the reaction started by the addition of 50 mm h2o2 solution to the reaction mixture (20 μl of extract, 0.15 mm disodium edta and 0.37 mm ascor­ bic acid in 50 mm sodium phosphate buffer). a unit of apx is defined as the amount needed to oxidize 1 μmole of ascorbic acid per minute. data were expressed as unit of apx per mg of soluble proteins (μmol min­1 mg­1). spontaneous emission analysis the spontaneous emission analysis was per­ formed as reported in our previous work (marchioni et al., 2020 b). briefly, and after the chosen storage time had elapsed (0, 2 and 6 days at 4°c), 1g of t. simmleri was properly weighted to be sealed in a 25 ml glass flask and kept at laboratory temperature (around 21°c) for 15 min (equilibration time). once the time expired, the 100 μm polydimethylsiloxane pdms fiber (supelco, bellefonte, pa, usa), was exposed to the flask headspace for 10 min, to be than transferred into the gc­ms instrument. statistical analysis the normal distribution of the residuals and the homogeneity of variance was determined and then data were statistically analyzed by one­way analysis of variance (anova) (past3, version 3.15), using tukey honestly significant difference (hsd) with a cut­off significance of p<0.05 (letters). 3. results and discussion weight loss and chromatic changes during cold stor‐ age the visual quality of t. simmleri flowers has been almost entirely maintained up to the sixth days of cold storage (t6) (fig. 1, table 1). the main changes observed during postharvest treatment were the decrease in flowers fresh weight, brightness (l*) and bluish parameter (b*), along with the increase in the reddish parameter (a*) (table 1). taken together, these variations resulted in a slight darkening of the petals at the end of the experiment, without any evi­ adv. hort. sci., 2023 37(1): 25­32 28 dent loss of flower firmness. the decrease in fresh weight is due to the loss of cell turgor, which is correlated to flower shape. significant water loss can determine decreased floral diameter, as well as petals curling and crumpling (kou et al., 2012; ahmad and thair, 2016; marchioni et al., 2020 b). nevertheless, the weight loss in t. simmleri flowers was very limited (around 7%), show­ ing, therefore, a good aptitude to cold storage. moreover, the latter was observed to reduce the brightness of seven different efs (landi et al., 2018), as well as t. simmleri flowers (table 1). this decrease in l* values is indicative of tissue darkening, com­ monly associated with the oxidation of phenolics and their polymerization into dark brown pigments, as a result of the activities of polyphenol oxidase (ppo), peroxidase and phenylalanine ammonia lyase (pal) (landi et al., 2018; hu and shen, 2021). the same process could also be responsible for the changes in the color coordinates a* and b*, which turn towards darker hues (table 1). antioxidant compound and enzyme activities polyphenols are considered as the most important and widest natural compounds with antioxidant activity (cavaiuolo et al., 2013). thanks to their bioactive potential, these molecules can help to pre­ vent chronic degenerative diseases, cardiovascular disorders, and different types of cancer (pires et al., 2019; skrajda­brdak et al., 2020). postharvest treat­ ment should maintain unaltered flowers polyphenols concentration to guarantee health benefit until flow­ ers consumption. our results satisfied this statement, because no changes were observed up to t6 for polyphenol and flavonoids amounts (table 2). indeed, a short increase in the total anthocyanins content was quantified already after 2 days (t2) that could be correlated to the interchange between bluish and reddish parameters (table 1). despite this positive trend, it should be noted that t. simmleri fresh flowers are characterized by low amount of phenolic compound than other well­known and cur­ rently consumed efs (li et al., 2014; chen et al., 2018). moreover, higher quantities of polyphenols and flavonoids were also reported in other species of the same genus, such as t. cominsii and t. violacea, probably connected to the use of different extraction methods (landi et al., 2018; rivas­garcía et al., 2022). nevertheless, maintaining the levels of pheno­ lic compounds in t. simmleri flowers could indicate that this species did not show substantial signs of decay up to the end of the experiment. as regards total anthocyanins content, their increase was previ­ table 1 ­ weight loss and chromatic changes of t. simmleri flowers at 0 (t0), 2 (t2), and 6 (t6) postharvest days (storage at 4°c) data are reported as mean ± standard error (weight loss, n = 4; l*, a*, b*, n = 15). different letters indicate statistically signifi­ cant differences (p<0.05; tukey’s hsd test). table 2 ­ antioxidant compounds, radical scavenger activity (dpph assay), catalase (cat) and ascorbate peroxidase (apx) activities of t. simmleri flowers at 0 (t0), 2 (t2), and 6 (t6) postharvest days (storage at 4°c) data are reported as mean ± standard error (n = 6). different letters indicate statistically significant differences (p<0.05; tukey’s hsd test). parameters days 0 2 6 weight loss (%) 0 c 3.21 ± 0.04 b 7.34 ± 0.69 a l* 56.01± 1.25 a 55.54 ± 1.19 a 49.45 ± 0.68 b a* 22.71 ± 0.64 c 25.78 ± 0.65 b 27.84 ± 0.46 a b* ­14.16 ± 1.08 a ­19.73 ± 0.69 b ­20.02 ± 0.51 b parameters days 0 2 6 total polyphenols (mg gaeq/g fw) 1.22 ± 0.01 a 1.30 ± 0.04 a 1.32 ± 0.03 a total flavonoids (mg ceq/g fw 0.30 ± 0.01 a 0.32 ± 0.01 a 0.29 ± 0.01 a total anthocyanins (mg meq/g fw) 0.21 ± 0.02 b 0.29 ± 0.01 a 0.24 ± 0.02 a dpph assay (ic50 mg/ml) 4.20 ± 0.28 a 3.46 ± 0.19 a 5.22 ± 0.09 b cat activity (µmol min­1 mg­1) 12.68 ± 0.41 b 9.05 ± 0.24 c 21.25 ± 0.27 a apx activity (µmol min­1 mg­1) 0.66 ± 0.04 b 0.64 ± 0.04 b 0.83 ± 0.04 a fig. 1 ­ visual appearance of t. simmleri flowers after different times of cold storage (4°c): freshly picked flowers (a); after 2 days of cold storage (t2) (b); and after 6 days of cold storage (t6) (c). bar scale: 1 cm. marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 29 ously observed also in other efs stored at low tem­ perature, but the regulatory mechanisms in flowers are still under debate (shvarts et al., 1997; landi et al., 2015; marchioni et al., 2020 b). senescence and flowers exposure to low tempera­ tures are tightly associated with a rise in reactive oxy­ gen species (ros) level in the cells, whose production is accompanied by the activation of several enzymes involved in ros scavenging (cavaiuolo et al., 2013; darras, 2020). polyphenolic compounds also take part to this process, as demonstrated by the reduc­ tion of flowers antioxidant activity observed at t6 (table 2). in this work, the attention was paid to the ros scavenging enzymes that use hydrogen peroxide (h2o2) as substrate, namely catalase (cat) and ascor­ bate peroxidase (apx). t. simmleri flowers showed that cat activity is higher than the one of apx (table 2), suggesting a greater involvement of cat in h2o2 inactivation. moreover, both the enzymes increased their activity at t6 (table 2). to the best of our knowledge, very few papers investigated ros scav­ enging enzymes activity in efs stored at chilling tem­ perature as single postharvest treatment. in fact, chrysargyris et al. (2018, 2019) combined the conser­ vation at 5°c with preharvest salinity treatment and modified atmosphere packaging to observe the stor­ age aptitude of tagetes patula and petunia × hybrida flowers. nevertheless, in agreement with our results, apx activity was lower than the one of cat in t. pat‐ ula flowers, after both 7 and 14 postharvest days (chrysargyris et al., 2018). cat activity was also investigated by rizzo et al. (2019), highlighting differ­ ent trend depending on the species and the polypropylene (pp) film used. in the control thesis (comparable with our experiment), cat activity increases significantly after 6 days of cold storage only in half out of the four studied flowers (malva sylvestris and papaver rhoeas), similarly to what we observed for t. simmleri. aroma profile monoterpenes were the main class of compounds, regardless the storage time and their percentage, that represented at least 50% of the identified fraction (table 3). interesting to note is the drastically decrease in oxygenated hydrocarbons content which was of 77% (passing from 0­ to 2­day conservation) and 60% (passing from 0­ to 6­day conservation) respectively. on the contrary, this decrease was some­ how compensated by the increase in the monoter­ pene hydrocarbons after 2­day storage (an increase of about 2­folds) and by non­terpene compounds after 6­day storage (an increase of about 2.5­folds). in detail of composition, the fresh flower (t0) was rich in linalool and 1,8­cineol and these compounds almost completely disappear after 2 days of storage. a decrease of linalool content was observed also in papaya “golden” fruit stored at low temperature (gomes et al., 2016). interestingly is also the increase of limonene content, about 5­folds, from t0 and t2 (3.01% vs 14.78%, respectively), the same compound conserved the latter percentage even at t6. worthy to note, the presence of benzyl­benzoate in the flow­ ers is only noticeable after 2­ and 6­days of refrigera­ tion, and its quantity is tripled during this time. this work reported for the first time the chemical composition of spontaneous emission of the studied species. also noteworthy is the absence of sulfur compounds. almost similar behavior has been seen in t. violacea, where such compounds were present in a negligible amount, which were around 1.2% in leaves and do not exceed 4% in roots detected using the same analysis technique (hs­spme) (staffa et al., 2020). rhizomes’ essential oil (eo) of a south african species of t. violacea was also reported to be rich in 2,4­dithiapentne, which represent more than the half of the identified fraction (soyingbe et al., 2013). hydrocarbons were the major compounds in the hexane extract of t. violacea calli from cairo (egypt) (55.0%), while the flowers were rich in oxygenated compounds (74.6%) (eid and metwally, 2017). on the contrary, the eo from the same species studied by the same team but published two year before under­ line the prevalence of sulfur compounds in both leaves and flowers and represented 79.7% and 57.5%, respectively (eid, 2015). 4. conclusions cold storage can reduce some biochemical reac­ tions, although stress conditions increase the reactive species of oxygen (ros) inside plant tissues. tulbaghia simmleri flowers maintain almost unal­ tered their visual quality, and their content in antioxi­ dant compounds, up to 6 postharvest days. moreover, cells counteract ros production increas­ ing cat and apx activity. the aroma profiles changed during the cold treatment, even if monoterpenes remained the most represented class of volatile com­ pounds. looking at the main characteristics of the flowers we can conclude that t. simmleri showed a good aptitude to chilling temperature, suggesting the need to test longer period of storage. 30 adv. hort. sci., 2023 37(1): 25­32 table 3 ­ aroma profile of t. simmleri flowers detected by headspace solid phase microextraction (hs­spme) at 0 (t0), 2 (t2), and 6 (t6) postharvest days n° class component l.r.i days 0 2 6 1 nt (e)­3­hexen­1­ol 866 2.37 ± 0.10 2 mh α­thujene 932 0.20 ± 0.00 tr 3 mh α­pinene 939 0.19 ± 0.02 3.36 ± 0.83 1.78 ± 0.12 4 mh camphene 953 0.38 ± 0.08 0.19 ± 0.01 5 nt benzaldehyde 961 0.93 ± 0.18 6 mh sabinene 976 0.53 ± 0.11 0.26 ± 0.00 7 nt 1­octen­3­ol 978 4.89 ± 0.16 8 mh β­pinene 980 1.18 ± 0.27 0.59 ± 0.02 9 nt 3­octanone 988 2.90 ± 0.16 10 om 2,3­dehydro­1,8­cineole 991 0.96 ± 0.08 11 mh myrcene 992 1.48 ± 0.61 12 nt 3­octanol 993 2.09 ± 0.13 0.80 ± 0.21 13 mh δ­3­carene 1011 0.44 ± 0.00 14 mh α­terpinene 1018 0.18 ± 0.04 1.03 ± 0.37 0.84 ± 0.08 15 mh p­cymene 1026 0.12 ± 0.01 7.26 ± 2.92 4.72 ± 0.70 16 mh limonene 1031 3.10 ± 0.08 14.74 ± 6.42 14.80 ± 1.13 17 om 1,8­cineole 1033 53.10 ± 0.08 10.38 ± 0.25 18 om (z)­β­ocimene 1033 0.20 ± 0.06 0.14 ± 0.03 19 mh (e)­β­ocimene 1040 0.62 ± 0.00 0.38 ± 0.08 20 nt phenyl acetaldeyde 1043 1.30 ± 0.04 21 mh γ­terpinene 1062 0.63 ± 0.08 5.74 ± 0.12 3.82 ± 0.02 22 om cis­sabinene hydrato 1068 0.82 ± 0.16 23 mh terpinolene 1088 0.32 ± 0.15 1.23 ± 0.39 1.05 ± 0.02 24 mh linalool 1098 15.51 ± 0.10 1.32 ± 0.68 0.92 ± 0.04 25 nt phenyl ethyl alcohol 1110 1.31 ± 0.03 26 om trans­limonene oxide 1139 1.24 ± 0.06 27 om trans­pinocarveol 1140 0.83 ± 0.10 28 om camphor 1143 1.89 ± 0.44 1.95 ± 0.14 29 om menthone 1154 0.46 ± 0.03 0.51 ± 0.01 30 om isomenthone 1164 0.40 ± 0.18 0.22 ± 0.01 31 om borneol 1165 0.76 ± 0.06 0.59 ± 0.02 32 om δ­terpineol 1167 tr 33 om trans­linalool oxide 1172 0.47 ± 0.05 0.32 ± 0.02 34 om neo­menthol 1174 0.76 ± 0.14 35 om cis­pinocamphone 0.71 ± 0.06 36 om 4­terpineol 1177 0.24 ± 0.08 1.57 ± 0.40 1.19 ± 0.09 37 om α­terpineol 1189 5.27 ± 0.08 0.79 ± 0.15 0.26 ± 0.04 38 nt decanal 1204 0.64 ± 0.06 39 om verbenone 1205 0.30 ± 0.05 40 om lilac alcohol b 1210 1.08 ± 0.11 41 nt methyl 4­nonenoate 0.36 ± 0.14 42 om trans­carveol 1217 0.32 ± 0.08 43 om methyl carvacrol 1244 0.68 ± 0.30 0.68 ± 0.12 44 om linalyl acetate 1257 2.14 ± 0.33 1.95 ± 0.35 45 om isobornyl acetate 1285 2.11 ± 0.49 1.98 ± 0.35 46 om myrtenyl acetate 1325 1.43 ± 0.16 0.30 ± 0.00 47 om methyl perillate 0.15 ± 0.07 48 sh α­cubebene 1351 0.23 ± 0.00 49 sh α­longipinene 1352 1.98 ± 0.35 50 sh α­copaene 1376 0.63 ± 0.11 0.27 ± 0.07 51 sh β­caryophyllene 1418 0.99 ± 0.06 2.48 ± 0.49 2.89 ± 0.21 52 sh α­guaiene 1439 0.35 ± 0.04 53 sh aromandrene 1442 0.12 ± 0.00 0.17 ± 0.02 54 ac­12 (e)­geranyl acetone 1453 tr data are reported as mean ± standard deviation (sd) (n=2). ... to be continued marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 31 acknowledgements this work was supported by a grant from european union in the frame of interreg alcotra v­a france­italy antea project n.1139 ­ attività inno­ vative per lo sviluppo della filiera del fiore edule/ fleurs comestibles: innovations pour le development d’une filière transfrontalière, and interreg alco­ tra v­a france­italy antes project n. 8336 ­ capitalizzazione di progetti antea e essica. the authors want to thank mrs. arianna cassetti for the technical support of the preparation of figure 1. references ahmad s.s., tahir i., 2016 ­ increased oxidative stress, lipid peroxidation and protein degradation trigger senescence in iris versicolor l. flowers. ‐ physiol. mol. biol. plants, 22(4): 507­514. aremu a.o., van staden j., 2013 ­ the genus tulbaghia (alliaceae) ‐ a review of its ethnobotany, pharmacolo‐ gy, 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l.r.i days 0 2 6 55 sh α­humulene 1454 0.21 ± 0.00 0.22 ± 0.01 56 sh alloaromandrene 1461 0.30 ± 0.06 0.43 ± 0.01 57 sh viridiflorene 1493 0.57 ± 0.06 0.66 ± 0.06 58 sh (e,e)­α­farnesene 1508 tr 1.72 ± 0.10 59 sh trans­γ­cadinene 1513 0.55 ± 0.02 60 om geranyl isobutyrate 1514 tr 61 sh trans­calamenene 1532 1.35 ± 0.14 62 os caryophellene oxide 1581 0.15 ± 0.00 2.36 ± 0.02 63 sh cadalene 1674 0.30 ± 0.07 0.59 ± 0.08 0.44 ± 0.06 64 nt benzyl benzoate 1762 12.58 ± 2.85 35.30 ± 0.02 monoterpene hydrocarbons (mh) 18.95 ± 0.02 37.55 ± 0.67 24.80 ± 1.83 oxygenated monoterpenes (om) 63.82 ± 0.01 14.46 ± 3.01 25.32 ± 0.66 total monoterpenes 82.77 ± 0.01 52.01 ± 3.68 50.12 ± 0.17 sesquiterpenes hydrocarbons (sh) 1.29 ± 0.13 7.90 ± 0.64 9.67 ± 0.52 oxygenated sesquiterpenes (os) ­ 0.15 ± 0.00 0.55 ± 0.02 total sesquiterpenes 1.29 ± 0.13 7.97 ± 0.75 10.22 ± 0.54 non terpenes (nt) 15.54 ± 0.39 14.81 ± 3.00 35.73 ± 0.08 total identified 98.59 ± 0.26 74.79 ± 0.06 96.06 ± 0.71 data are reported as 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ambientali, 06121 perugia, italy. key words: cuttings, germplasm, naa, nad, olea europaea l., propagation. abstract: the effects of two different types of auxins (660 ppm alpha-naphthaleneacetic acid naa in liquid solution or 750 ppm alpha-naphthaleneacetamide nad dispersed in a talcum powder) and cuttings from three different portions of the shoots (basal, middle and apical) on the rooting ability of twenty autochthonous olive cultivars were investigated in two growing seasons (spring and autumn). the results showed that the autochthonous olive cultivars of the campania region are characterized by a wide variability in the potential rhizogenic ability. the two periods of cutting collection (march and september) significantly affected the rooting aptitude of the cultivars, indicating that in some cultivars the cuttings collected in autumn had a higher rooting rate than those collected in spring. the effects of naa and nad on rooting strongly depended on interaction with the cultivar, time of collection (autumn or spring) and type of cuttings (basal, medium or apical). among the twenty cultivars tested, we found only eight cultivars with a satisfactory rooting ability after hormonal applications (ortolana, racioppella, tenacella, tonda, biancolilla, carpellese, cornia and pisciottana). in general, the apical and the median portions of the shoots gave the best rooting results. 1. introduction among the vegetative propagation methods of olive cultivars (olea europaea l.) the use of semi-hardwood cuttings is the most common, since rootings are easy to prepare and the requirement in special equipment is negligible and cheap (cimato, 1999; ismaili et al., 2011). this is why in the mediterranean basin olive is mainly propagated by cuttings, a propagation method that relies on the ability of the cuttings to form adventitious roots (fabbri et al., 2004). while some cultivars are easily propagated by this technique, others are difficult-to-root and this poses a challenge for their preservation and commercialization (hartmann and kester, 1975; hartmann et al., 1990; carfi et al., 1994; porfírio et al., 2016). rooting aptitude of the different olive cuttings depends on both intrinsic and extrinsic factors (wiesman and lavee, 1994; hechmi et al., (*) corresponding author: divaio@unina.it citation: cirillo c., russo r., famiani f., di vaio c., 2017 investigation on rooting ability of twenty olive cultivars from southern italy. adv. hort. sci., 31(4): 311-317 copyright: © 2017 cirillo c., russo r., famiani f., di vaio c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 july 2017 accepted for publication 26 october 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 311-317 312 2013), such as the genotype (avidan and lavee, 1978; fabbri et al., 2004; chiancone et al., 2011), the age of the mother tree, the timing of cutting collection as well as the type of cuttings (fontanazza and jacoboni, 1975; del rio et al., 1991; khabou and trigui, 1999). taking this background into consideration, the main aim of this study was to assess the rooting ability of olive cuttings obtained from twenty autochthonous olive cultivars of the campania region, by using different combinations of auxin treatments (naa and nad), times of collection of cuttings (spring and autumn) and portions of shoots to prepare the cuttings (basal, middle or apical). the cultivars considered in this study have been previously evaluated in terms of vegetative-productive characteristics and oil quality, and some of them present agronomical behaviors and quality of the oils which make them interesting for the use in new olive orchards, also for the production of oils with a strong yet greatly diverse typicality (di vaio et al., 2013). this makes very important to know their intrinsic ability to root and the best combination of factors (hormones, time of cutting collection and portion of shoots to use) to utilize in order to obtain the best rooting results from each cultivar. 2. materials and methods experimental site and plant material the trial was carried out in 2014/2015 at the experimental station “improsta” of the campania region, located in battipaglia (40°37′ 00’’ n lat, 15°03′ 23’’ e long, 72 m above sea level) sele plain (salerno, south italy). the average annual rainfall and the average minimum and maximum temperature of the area were 988 mm, 10.9°c and 21.0°c, respectively. the experimental station hosts a germplasm collection orchard, established in 2001, which includes all the main autochthonous olive cultivars of the region. trees were trained at central leader and spaced 6 × 3 m. among the available germplasm, 20 cultivars were selected for their economic importance and potential to be used in new orchards considering their good agronomical characteristics (productivity and resistance to biotic and abiotic stresses) and high quality/typicality of the oils, since some of them also allow the production of certified oils under the protected designation of origin. the cultivars were grouped according to their origin (main province of cultivation). therefore, the selected olive cultivars were four from the province of avellino (‘ogliarola campana’, ‘ravece’, ‘ritonnella’ and ‘ruveia’), five from the province of benevento (‘femminella’, ‘ortice’, ‘ortolana’, ‘pampagliosa’ and ‘racioppella’), four from the province of caserta (‘asprinia’, ‘caiazzana’, ‘tonda’ and ‘tenacella’) and seven from the province of salerno (‘biancolilla’, ‘carpellese’, ‘cornia’, ‘oliva bianca’, ‘pisciottana’, ‘rotondella’ and ‘salella’) (di vaio et al., 2013). cutting collection and preparation the cuttings were collectioned in the autumn (end of september) and in the next spring (second decade of march). for each cultivar, three trees were selected by following homogeneity criteria for developmental stage and productivity. the semi-hardwood cuttings were obtained in autumn (a) and spring (s) from 30 cm long one-year shoots/season. each shoot was divided in three different portions (basal, middle and apical) (180 cuttings/cultivar/season). cuttings were 10 cm long and presented 4 nodes and 2 pairs of terminal leaves. rhizogenic treatments for each combination of cultivar, collection time, portion of shoot and hormonal treatment three groups of ten cuttings each were used. the trial was conducted under mist system in cold greenhouse conditions. two different commercial formulations of auxins, alpha-naphthaleneacetic acid (naa) in liquid formulation (hydroalcoholic solution at 30% alcohol) at the concentration of 660 ppm (t1) and alphanaphthaleneacetamide (nad) at the concentration of 750 ppm (t2) in talcum powder formulation (0.075/100 w.w.) were compared to untreated control (t0). these concentrations were used considering the good results obtained with naa 500-1000 ppm in other cultivars (denaxa et al., 2011). the t1 treatment was performed by dipping 2 cm basal part of cuttings in the hydroalcoholic solution for 5 seconds, whereas the t2 treatment by dipping 2 cm basal part of cuttings in distilled water first and then in the powder. the t0 (control) was obtained by dipping 2 cm basal part of cuttings in distilled water for 5 seconds. cuttings were then placed in perlite filled rooting benches provided with basal heating (substrate temperature 22-24°c) and with mist system to get periodically wet the cuttings avoiding their dehydration (air humidity about 90-95%). rooting sampling and score semi-hardwood cuttings were evaluated 70 days after the rooting treatments and each cutting was cirillo et al. rooting ability of twenty olive cultivars from southern italy 313 scored for the rooting rate, the number of roots/cutting (primary and secondary roots), and the length of roots/cutting (the length of the different roots of each cutting was summed). the percentage of rooting was calculated as the number of rooted cuttings with respect to the total number of cuttings per treatment. statistical analysis all data were statistically analyzed by three-way analysis of variance (anova) using the spss 13 software package (spss 13.0 for windows; spss inc., chicago, il). whenever the two-way interaction was significant, a one-way anova was performed. to separate treatment means for each measured parameter, duncan’s multiple range test was performed at a significance level of p≤0.05. 3. results and discussion adventitious rooting process in cuttings is still to be unraveled under the genetic point of view, however, as referred in studies on italian and international olive cultivars (fontanazza and baldoni, 1989; chiancone et al., 2011), the capability of cuttings of forming adventitious roots is mainly affected by the cultivar. among the wide number of local and worldwide grown cultivars, the main part of them displays a poor aptitude for rooting of cuttings, whereas few cultivars are known to be well rooting cultivars. a dataset of the international olive council (ioc, 2005) reported the results of a screening on rooting rate of 426 cultivars: 59 cultivars showed an average rooting rate of 1.5%, 213 cultivars reached an average rate of 21.3%, whereas the rooting rate recorded in 86 cultivars was approximately 54% and only 68 (=16% of the total) showed a rate of rooting higher than 70%. in the present study, rooting rate of the cuttings was significantly affected by the cultivar (c), by the interaction between the cultivar and the time of sampling (c × ts) and by the interaction between the hormonal treatment and the time of sampling (t × ts) (table 1). the highest percentage of rooted cuttings, over the rooting treatment and the time of sampling, was recorded in cv. ortolana (66%), followed by cv. racioppella (54.6%) and cv. biancolilla (51%) (fig. 1). on the contrary, cv. ogliarola campana, cv. ortice and cv. salella showed the lowest rooting rate (14.9%, 15.8% and 11.2%, respectively). timing of cutting collection, according to the literature, is the second pivotal element, following the cultivar, to be concerned about, since very relevant differences in the success of rooting process may depend on this factor (hartmann and loreti, 1965). thus, the definition of the most suitable season for cutting collection from mother plants has been a critical item for researchers dealing with fruit trees species, although the fine tuning of protocols ready to use in the nurseries is still lacking, especially if single cultivars are considered. the c × ts interaction highlighted that the cultivars considered in this study responded differentially to the time of cutting sampling (fig. 2). for instance, among the three cultivars that showed the highest rooting rate, cv. ortolana and cv. raciopella reached higher percentages of rooting when the cuttings were collected during spring season, whereas the rooting rate of cv. biancolilla was significantly increased by using the cuttings collected in autumn (fig. 2). on the contrary, the cultivars that showed the lowest rooting rate (cvs. ogliarola campana, ortice and salella) did not ns= non significant; *, **; ***= significant at p≤ 0.05, ≤ 0.01, 0.001, respectively. fig. 1 average rooting rate (mean ± standard error) of semihardwood cuttings of the olive cultivars of the campania region. bars with the same colour correspond to cultivars of the same province. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = tenacella, ton = tonda, bia = biancolil la, car = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. means followed by different letters are significantly different for p ≤ 0.001. source of variance rooting rate (%) cultivar (c) *** hormonal treatment (t) ns time of sampling (ts) ns c × t ns c × ts *** t × ts * c × t × ts ns table 1 analysis of variance for cultivars, hormonal rooting treatments, time of sampling and their interactions on rooting rate of olive cuttings adv. hort. sci., 2017 31(4): 311-317 314 perform differently comparing the two times of sampling (fig. 2). the overall influence of hormonal treatments (naa or nad) and time of sampling (t × ts) on cutting rooting rate indicated that the effect of treatments was significantly different on the cuttings collected in spring (fig. 3). in particular, the lowest rooting rate was observed on t0 cuttings collected in spring (fig. 3). on the base of cutting rooting rate, the international olive council classified the cultivars in four ranks: 0-5%; 5-40%; 40-70% and 70-100% (ioc, 2005). according to this classification, the twenty cultivars tested in the present experiment resulted to be distributed in three different groups: twelve between 5 and 40% (‘ogliarola campana’, ‘ravece’, ‘ritonnella’, ‘ruveia’, ‘femminella’, ‘ortice’, ‘pampagliosa’, ‘asprinia’, ‘caiazzana’, ‘oliva bianca’, ‘rotondella’, ‘salella’); seven between 40 and 70% (‘racioppella’, ‘tenacella’, ‘tonda’, ‘biancolilla’, ‘carpellese’, ‘cornia’ and ‘pisciottana’); one between 70 and 100% (‘ortolana’). as far as root number and length are concerned, the effects of cultivar, hormonal treatments and portion of the shoot and their first level interactions were all significant (table 2). the twenty cultivars of olive exhibited a high variability in the number and length of roots produced both in autumn and spring experiments. the root number and length were increased by the rhizogenic treatments. generally, both the liquid and powdery formulations used for the rooting stimulation of the cuttings were effective in improving rooting emission and growth. with regard to the number of roots, our findings indicated that the liquid treatment with 660 ppm naa was the most effective, showing, with respect to the control, an average increase of about 266% in cuttings from the basal, median and apical portions of the shoots. the same treatment was particularly effective in stimulating the length growth of the roots, with an average increase of 236% compared to the control. furthermore, the root number and length of cuttings collected in autumn and spring were significantly affected by the interaction between the cultivar (c) and hormonal rooting treatment (t) (table 2). for instance, the average root number in autumn cuttings was 2.7 roots per cutting, and the application of the rhizogenic treatment t2, compared to t0, induced a significant increase of the number of roots in nine cultivars: ortolana (+245%), cornia (+169%), tonda (+160%), ruveia (+165%), tenacella (+192%), asprinia (+312%), biancolilla (+320%), pisciottana fig. 3 average rooting rate (mean ± standard error) of semihardwood cuttings as affected by the interaction between the hormonal rhizogenic treatments (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) and the time of sampling. a= autumn; s= spring. means followed by different letters are significantly different for p ≤ 0.05. fig. 2 average rooting rate (mean ± standard error) of semihardwood cuttings as affected by the interaction between the cultivar and the time of sampling. a= autumn; s= spring. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = tenacella, ton = tonda, bia = biancolil la, car = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. means followed by different letters are significantly different for p≤0.001. source of variance root number root length (cm/cutting) autumn spring autumn spring cultivar (c) *** *** *** *** hormonal treatment (t) *** *** *** *** portion (p) *** ** *** *** c × t ** *** *** ** c × p * *** * *** t × p * ns ns * c × t × p ns *** ns * ns= non significant; *, **; ***= significant at p≤ 0.05, ≤ 0.01, 0.001, respectively. table 2 analysis of variance for cultivars, hormonal rooting treatments, shoot portion and their interactions on number and length of roots of olive cuttings obtained in autumn or spring cirillo et al. rooting ability of twenty olive cultivars from southern italy 315 (+610 %) and carpellese (+236 %) (fig. 4a). the average root length in autumn cuttings was 4.2 cm per cutting, and, similarly to the root number, it was also increased by the rhizogenic treatment t2 in ten cultivars: ortolana, ravece, cornia, tonda, ruveia, tenacella, asprinia, biancolil la and pisciottana (fig. 4 b). a similar pattern was observed in the spring cuttings (fig. 5 a, b), even though the fig. 4 effects of the interaction of the cultivars and the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) on root number/cutting (a) and root length/cutting (b) of semi-hardwood cuttings collected in autumn. the dotted line shows the average value of all cultivars under observation. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = tenacella, ton = tonda, bia = biancolil la, car = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. fig. 5 effects of the interaction of the cultivars and the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) on root number/cutting (a) and root length/cutting (b) of semi-hardwood cuttings collected in spring. the dotted line shows the average value of all cultivars under observation. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = tenacella, ton = tonda, bia = biancolilla, car = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. adv. hort. sci., 2017 31(4): 311-317 316 values of both the average number and length of roots were higher than those recorded in autumn. the root number of autumn cuttings was also affected by the interaction between the hormonal treatment (t) and the shoot portion (p). indeed, the number of roots per cutting increased from the bottom (b), to the middle (m), and then to the apical (a) portion of collected shoots with the highest value recorded in the apical cuttings treated with t1 (4.5 roots/cutting) followed by t2 treated apical and middle cuttings (3.9 and 3.4 roots/cutting, respectively) (fig. 6 a), whereas the lowest number of roots was observed in the basal cuttings of untreated control (t0) and t1 (1.5 roots/cuttings) (fig. 6 a). with respect to the control and t1, a significant increase of the root number was observed in the basal cutting treated with t2 (2.8 roots/cuttings). the capability of olive cuttings on forming adventitious roots is known to be related to the portion (basal, middle or apical) of the shoots, even though different cultivars can respond differentially. a general hypothesis has been formulated on the higher aptitude for rooting of subapical cuttings that seems to be related to a possible higher content in auxinic compounds driven basipetally from the young leaves of cuttings (fabbri et al., 2004). on the other hand, only the root length of spring cuttings was significantly affected by the interaction between the hormonal treatment (t) and the shoot portion (p) (table 2, fig. 6 b), with the highest values (11.4 cm) recorded in the apical cuttings treated with naa in liquid formulation (t1) and in basal, middle and apical cuttings (10.3, 10.9, and 12.2 cm, respectively) treated with nad in powdery formulation (t2). 4. conclusions this study highlighted that the olive cultivars belonging to autochthonous germplasm of the campania region are characterized by a wide variability in the potential rhizogenic activity. overall the two sampling periods of cutting collection (march and september) significantly affected the rooting aptitude of several cultivars, indicating that in some cultivars the cuttings collected in autumn may have a higher rooting rate than the spring collected ones. moreover, it was possible to evaluate the rhizogenic aptitude of the cultivars belonging to the olive campanian germplasm under the influence of rhizogenic treatments. the results showed that the effects of naa and nad on rooting strongly depended on interaction with the cultivar, time of collection (autumn or spring) and type of cuttings (basal, medium or apical). in general, the apical and the median portions of shoots were confirmed to be the most suitable for improving the rooting of cuttings. references avidan b., lavee s., 1978 physiological aspects of the rooting ability of olive cultivars. acta horticulturae, 79: 93-101. carfi c.h., ben hadj salah h., msallem m., haddar t., hellali r., 1994 effet des doses d’aib et des dates de prélévement sur la rhizogenése des boutures de 6 variétés d’olivier (olea europaea l.) “chetoui”, “meski”, “picholine”, “besbessi”, “chemlali”, “arbequina”. revue inat, vol. 9, no. 1-2. chiancone b., macaluso l., germanà m.a., 2011 prove sulla radicazione di talee di cultivar siciliane di olea europaea l. acta italus hortus, 1: 370-375. cimato a., 1999 l’élevage des plants d’olivier en pèpinière. innovations technologiques et leur application en oléiculture et oléotechnie. séminaire international, firenze (italia). del rio c., rallo r., caballero j.m., 1991 effect of carbohydrate content on the seasonal rooting of vegetative and reproductive cuttings of olive. j. hortic. sci., fig. 6 effects of the interaction of the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) and shoot portion used to make the cuttings (b= basal; m= medium; a= apical) on root number/cutting (a) of semi-hardwood cuttings collected in autumn and on root length/cutting (b) of semi-hardwood cuttings collected in spring. means followed by different letters are significantly different for p≤0.05 (root number) and p≤0.05 (root length). cirillo et al. rooting ability of twenty olive cultivars from southern italy 317 66(3): 301-309. denaxa n.k., vemmos s.n., roussos p.a., kostelenos g., 2011 the effect of iba, naa and carbohydrates on rooting capacity of leafy cuttings in three olive cultivars (olea europaea l.). acta horticulturae, 924: 101-109. di vaio c., nocerino s., paduano a., sacchi r., 2013 characterization and evaluation of olive germplasm in southern italy. j. sci. food agric., 93(10): 2458-2462. fabbri a., bartolini g., lombardi m., kailis s.g., 2004 olive propagation manual, pp. 2-7. in: fabbri a., g. bartolini, m. lombardi, and s.g. kailis. olive propagation manual. csiro publishing, landlinks press, collingwood vic, australia, pp. 145. fontanazza g., baldoni l., 1989 innovazioni tecnologiche in olivicoltura. olivicoltura: innovazioni tecnologiche e valutazione dei risultati economici in alcune realtà aziendali. formez, archivio dei corsi di formazione, pp. 41-54. fontanazza g., jacoboni n., 1975 indagine sugli effetti della defogliazione e della degemmazione di talee di olivo sulla rizogenesi in camera di nebulizzazione. frutticoltura, 9: 25-30. hartmann h.t., kester d.e., 1975 plant propagation: principles and practices. prentice-hall inc., englewood cliffs, nj, usa, pp. 662. hartmann h.t., kester d.e., davies f.t., 1990 plant propagation principles and practices. prentice-hall inc., englewood cliffs, nj, usa, pp. 547. hartmann h.t., loreti f., 1965 seasonal variation in rooting leafy olive cuttings under mist. proc. amer. soc. hort. sci., 87: 194-198. hechmi m., khaled m., abed s., el-hassen a., faiez r., m’hamed a., 2013 performance of olive cuttings (olea europaea l.) of different cultivars growing in the agro-climatic conditions of al-jouf (saudi arabia). am. j. plant physiol., 8(1): 41-49. ioc, 2005 olive nursery production and plant production techniques. international olive council http://www. internationaloliveoil.org/projects/paginas/sectionb.htm. ismaili h., ianni g., dervishi a., 2011 study of main factors influencing olive propagation. j. int. environ. appl. & sci., 6(4): 623-629. khabou w., trigui a., 1999 optimisation of the hardwood cutting as a method of olive tree multiplication. acta horticulturae, 474: 55-58. porfírio s., sonon r., gomes da silva m.d.r., peixe a., cabrita m.j., azadi p., 2016 quantification of free auxins in semi-hardwood plant cuttings and microshoots by dispersive liquid-liquid microextraction/ microwave derivatization and gc/ms analysis. anal. methods, 8: 6089-6098. wiesman z., lavee s., 1994 vegetative growth retardation, improved rooting and variability of olive cuttings in response to application of growth retardants. plant growth regul., 14: 83-90. impaginato 485 adv. hort. sci., 2019 33(4): 485­493 doi: 10.13128/ahsc­8115 micropropagation of two near threat­ ened orchid. part 2: phalaenopsis amabilis blume var. grandiflora m. mohammadi, b. kaviani (*), sh. sedaghathoor department of horticultural science, rasht branch, islamic azad university, rasht, iran. key words: in vitro multiplication, orchid propagation, ornamentals, plant growth regulators. abstract: phalaenopsis is one of the most popular orchids in the world, through the development of many artificial hybrids. in this research, a reliable and effi­ cient protocol is presented for in vitro proliferation of phalaenopsis amabilis blume cv. grandiflora. protocorm­like bodies (plbs) were cultured on murashige and skoog (ms) medium containing different concentrations of kinetin (kn; 0.00, 0.50, 1.00, 2.00 and 3.00 mg l­1) and indole­3­butyric acid (iba; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination and activated charcoal (ac; 0.00, 0.50 and 1.00 g l­1). a combination of 0.20 mg l−1 iba and 2.00 mg l­1 kn on medium containing 1.00 g l­1 ac was found to be suit­ able for maximum leaf number (6.16±0.503 per explant). the highest rooting frequency with 7.13±0.153 roots per explant was achieved on medium enriched with 0.50 mg l­1 iba and 0.50 mg l­1 kn on medium containing 1.00 g l­1 ac. the largest number of callus (9.10±0.611) was induced on explants cultured in medium containing 0.20 mg l−1 iba and 0.50 mg l­1 kn on medium without ac. the plantlets were successfully acclimatized in the greenhouse with a survival rate of 95% exhibiting normal developmental patterns. 1. introduction phalaenopsis blume, known as moth orchid, is a genus of approxi­ mately 60 species native to tropical rainforests of south and south­east asia, australia and new guinea (winkelmann et al., 2006). phalaenopsis as a cut and pot flowering plant is one of the most popular orchids in the trade and hobbyists through the development of many artificial hybrids. they are epiphytic plants, and consist of only a few leathery leaves (sinha et al., 2010). large scale natural clonal propagation is not possible in phalaenopsis. therefore, the establishment of protocols for in vitro proliferation of orchids is the only method for high frequency regeneration of these plants. in vitro techniques can be used for storage of rare and endangered plant species and production of large number of plantlets in short period of time (engelmann, 2011). in vitro multiplication of orchids deals with some problems such as high cost of production, low rate of shoot prolifer­ (*) corresponding author: kaviani@iaurasht.ac.ir citation: mohammadi m., kaviani b., sedaghathoor sh., 2019 ­ micropropagation of two near threa‐ tened orchid. part 2: phalaenopsis amabilis blume var. grandiflor. ­ adv. hort. sci., 33(4): 485­493. copyright: © 2019 mohammadi m., kaviani b., sedaghathoor sh. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 december 2018 accepted for publication 8 july 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 485­493 486 ation, poor rooting frequency and genetic variations (bhattacharyya et al., 2016). several methods for in vitro propagation of phalaenopsis through callus induction and cell suspension culture were developed (tanaka, 1992; arditti and ernst, 1993; tokuhara and mii, 2001; sinha et al., 2010). park et al. (2002) devel­ oped an efficient in vitro propagation method for phalaenopsis by using protocorm­like bodies (plbs) derived from leaf explants. medium composition for in vitro culture of orchids by plbs is species­specific and depends on several factors (luo et al., 2009). kuo et al. (2005) reported a protocol for regenerating a phalaenopsis cultivar by direct somatic embryogene­ sis. this method was not so efficient and feasible for commercial propagation because of low frequency regeneration of different cultivars of phalaenopsis hybrid. on the other hand, many protocols for in vitro propagation of orchids, especially those in danger of extinction, using plbs as explants and various pgrs, have been reported (sinha et al., 2010; teixeira da silva, 2006; baker et al., 2014; kaviani et al., 2017). various pgrs like α­naphthaleneacetic acid (naa), indole­3­butyric acid (iba), 1­phenyl­3­(1,2,3­thiadia­ zol­5­yl)­urea (tdz), 6­benzyle amino purine (bap), 6­ benzyladenine (ba) and kinetin (kn) have been applied for micropropagation of rare and endangered orchids (roy et al., 2011; panwar et al., 2012; zeng et al., 2012; bhattacharyya et al., 2016; kaviani et al., 2017). many explants such as seed, leaf, node section, protocorm, plb, tuber, shoot tip and inflorescence have been used for in vitro proliferation of endan­ gered orchids (sinha et al., 2010; roy et al., 2011; panwar et al., 2012; zeng et al., 2012; baker et al., 2014; chen et al., 2015; bhattacharyya et al., 2016). plb is more efficient because of maximum multiplica­ tion in a short period of time (luo et al. 2003). this study describes an efficient and reliable protocol for high frequency regeneration and callus induction of phalaenopsis amabilis blume var. grandiflora, a rare and near endangered orchid species by plbs. 2. materials and methods source of explant leaves (0.5­1 cm long) were excised from young phalaenopsis amabilis blume var. grandiflora plants growing in the greenhouse of hyrcan agricultural sciences and biotechnology research institute, amol, iran. the leaves were washed under running tap water for 15­20 min and rinsed thoroughly with dis­ tilled water. these were surface sterilized with hgcl2 (0.1% w/v) for 10 min followed by naocl (20%) for 15 min with 1 drop of tween 20, then rinsed with sterile distilled water. finally, leaves were sterilized in ethanol 75% for 1 min and washed 3­4 times with sterilized distilled water and finally excised to seg­ ments of 5­7 mm as primary explants for culture in murashige and skoog (ms) medium (murashige and skoog, 1962) containing 3% sucrose and 0.8% agar. the medium was supplemented with 0.20 mg l­1 naa along with 3.00 mg l­1 bap (appropriate types and concentrations of pgrs obtained before for maxi­ mum production of plbs; data not shown). healthy and sterilized plbs (fig. 1a) produced in the plant biotechnology laboratory, hyrcan agricultural sciences and biotechnology research institute, amol, iran, were excised and used as secondary explants for in vitro propagation. culture medium and growth conditions the explants (plbs) were cultured in ms medium containing 3% sucrose and 0.8% agar. the medium was enriched with various pgrs and activated char­ coal (ac). the ph of the medium was adjusted to 5.8 with 0.1 n naoh or hcl prior to autoclaving. all media contained in culture bottles were autoclaved at 104 kpa and 121°c for 20 min. to evaluate the effect of pgrs and ac on shoot multiplication (i.e. increasing the number of leaves and their development) and root induction, the explants were cultured on ms medium containing different concentrations of kn (0.00, 0.50, 1.00, 2.00 and 3.00 mg l­1) and iba (0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination. medium was supplemented with or without activat­ ed charcoal (ac; 0.00, 0.50 and 1.00 g l­1). for each treatment, three replicates and for each replicate, three specimens (explants) were taken (totally; 75 treatments, 225 replicates and 675 specimens or explants). following establishment, cultures were maintained at 24±2°c, 70­80% rh, and 16­h photope­ riod of 50­60 μmol m­2 s−1 irradiance provided by cool­white fluorescent tubes. after 60 days, the effect of pgrs and ac on advanced plbs development was assessed by measur­ ing number of leaves per explant, leaf length, leaf width, number of roots per explant, root length, num­ ber of explants with callus and viability percentage. plant development and acclimatization in vitro rooted plantlets were taken out from cul­ ture vessels and washed thoroughly under running tap water to remove adherent nutrient and transplanted to plastic pots (18 cm height × 12 cm diameter) filled kaviani et al. ‐ micropropagation of phalaenopsis amabilis var. grandiflora 487 with a potting mixture of leca (light expanded clay aggregate), peat moss and perlite (1:1:1). all the pots were then transferred to the greenhouse with tem­ perature of 24±2°c to 20±2°c day/night (light intensity of 3500 lux, rh of 80­90% and 14­h photoperiod) for acclimatization. the pots were covered with polyeth­ ylene bags to retain moisture inside and were opened gradually during 2 weeks. plantlets were initially cov­ ered with a polythene sheet to maintain relative humidity (90%). the number of surviving plants was recorded after 12 weeks of transfer. experimental design and data analysis the experiments were established in a completely randomized design with three replicates per treat­ ment (totally 675 explants). pgrs­free ms medium was used as control in the experiments. the results were expressed as mean±sd. data were subjected to analysis of variance (anova) (except for acclimatiza­ tion records) and means were compared by the lsd test at p<0.05 using the spss ver. 17 (spss inc., usa). 3. results the effect of pgrs and ac on the leaf growth (number, length and diameter) and root growth (number and length) of phalaenopsis amabilis blume var. grandiflora is shown in tables 1­4 and figure 1. plbs produced on ms medium containing 0.20 mg l­1 naa + 3.00 mg l­1 bap (appropriate types and con­ centrations of pgrs obtained before for maximum production of plbs; data not shown), were used as primary explants (fig. 2a). these plbs were produced after 60 days of culture of leaf explant on this medi­ um (fig. 2b). these plbs were used as secondary explants and cultured on media supplemented with fig. 1 ­ micropropagation of phalaenopsis amabilis blume cv. grandiflora through protocorm­like bodies (plbs). (a) plbs produced on ms medium containing 0.20 mg l­1 naa + 3.00 mg l­1 bap. (b) micropropagated shoots from plbs on medium containing 0.50 mg l­1 iba + 2.00 mg l­1 kn. (c) plantlets produced on medium supplemented with 0.20 mg l­1 iba + 2.00 mg l­1 kn + 1.00 g l­1 ac. (d) length of leaf obtained on medium containing 0.50 mg l­1 iba together with 2.00 mg l­1 kn and 1.00 g l­1 ac. (e) width of leaf obtained on medium containing 0.50 mg l­1 iba together with 1.00 mg l­1 kn 1.00 g l­1 ac. (f) plantlets produced on media contai­ ning 1.00 g l−1 ac together with different concentrations of iba and kn. from left to right: 0.10 mg l−1 iba + 0.50 mg l−1 kn, 0.50 mg l−1 iba + 0.50 mg l−1 kn, 0.20 mg l−1 iba + 1.00 mg l−1 kn, 0.10 mg l−1 iba + 1.00 mg l−1 kn and 0.50 mg l−1 kn without iba. (g) number and length of roots obtained on medium enriched with 0.50 mg l−1 iba plus 0.50 mg l−1 kn and 1.00 g l−1. (h) greenhouse acclimatized plantlets in pots filled with leca, peat moss and perlite (in ratio of 1:1:1). fig. 2 ­ (a) plbs and callus formation. (b) plbs development and multiplication in ms medium containing 0.20 mg l­1 naa + 3.00 mg l­1 bap. (c) callus formation from the explants cultured in media enriched with 0.50 mg l­1 iba + 3.00 mg l­1 kn (left) and 0.20 mg l­1 iba and 0.50 mg l­1 kn (right). (d) callus formation from the explants cultured in media enriched with different concentrations of iba and kn (from left to right: 0.50 mg l­1 iba + 3.00 mg l­1 kn, con­ trol, 1.00 mg l­1 iba + 1.00 mg l­1 kn and 0.20 mg l­1 iba and 0.50 mg l­1 kn). adv. hort. sci., 2019 33(4): 485­493 488 different concentrations of pgrs and ac. anova showed significant differences between various con­ centrations of pgrs and ac on most measured para­ meters (table 1). effect of pgrs and ac on multiplication parameters advanced shoot development was significantly affected by the composition of the medium. the media containing 0.20 mg l­1 iba and 2.00 mg l­1 kn along with 0.50 g l­1 ac, and without ac were suitable for leaf number (tables 2, 3, figs. 1b, c). the highest leaf length (4.66±0.702 cm per explant) and leaf width (3.13±0.603 cm per explant) were obtained in media enriched with 0.50 mg l−1 iba along with 2.00 mg l­1 kn and 0.50 mg l­1 iba along with 1.00 mg l­1 kn, respectively (table 4, figs. 1d, e). ms medium enriched with 0.20 mg l­1 iba and 2.00 mg l­1 kn along with 1.00 g l­1 ac was the most appropriate medium for leaf number (6.16±0.503 per explant) (table 4). among all concentrations of iba, kn and ac used table 2 ­ effect of different concentrations of kn and iba without ac on the studied parameters of in vitro grown phalaenopsis amabilis blume cv. grandiflora means with different letters on the same column are significantly different (p<0.05) based on lsd test. table 1 ­ effect of different concentrations of kn, iba and ac on the studied parameters of in vitro grown phalaenopsis amabilis blume cv. grandiflora *, **: significant at the 0.05 and 0.01 probability level, respectively, ns: not significant at p=0.05. source of variations df mean of squares leaf number leaf length leaf width root number root length callus number viability percentage ac 2 6.526 ** 5.563 ** 6.881 ** 6.38 ** 5.87 ** 3.402 ns 76.00 ns iba 4 14.71 ** 8.818 ** 4.708 ** 23.50 ** 48.60 ** 5.224 ** 1000 ** kn 4 48.81 ** 9.746 ** 9.030 ** 5.54 ** 11.13 ** 49.2 ** 1026 ** ac × iba 8 0.206 ns 1.139 ** 0.417 * 3.29 ** 2.35 ** 9.66 ** 291 ** ac × kn 8 1.238 ** 1.560 ** 0.3782 ns 1.63 ** 1.434 * 12.87 ** 297 ** iba × kn 16 7.043 ** 4.208 ** 2.805 ** 6.35 ** 4.43 ** 20.60 ** 651 ** ac × iba × kn 32 1.326 ** 1.587 ** 0.697 ** 2.89 ** 2.066 ** 10.90 ** 662 ** error 150 0.453 0.304 0.197 0.597 0.602 1.517 99.66 cv − 19.09 20.83 25.01 15.79 17.6 25.47 12.47 pgrs (mg l­1) leaf number leaf length (cm) leaf width (cm) root number root length (cm) callus number viability percentageiba kn 0.00 0.00 2.66±0.681 d­g 1.73±0.153 h 1.16±0.306 fg 4.03±0.513 bc 3.33±0.603 hi 4.76±2.454 cd 70.00±10.000 bc 0.00 0.50 3.76±0.624 bcd 2.03±0.208 e­h 0.93±0.153 g 4.33±0.929 a­c 3.10±0.624 i 5.03±0.854 cd 63.30±10.000 c 0.00 1.00 2.93±0.265 c­g 2.13±0.520 d­h 1.20±0.265 e­g 4.00±0.529 bc 3.53±1.206 f­i 5.10±1.539 cd 73.30±10.000 a­c 0.00 2.00 2.20±0.493 fg 2.86±0.503 b­e 1.56±0.153 d­g 4.86±1.358 a­c 4.73±0.400 bcdef 4.53±0.737 cd 90.00±15.275 a 0.00 3.00 2.83±0.208 d­g 2.40±0.208 c­h 2.00±0.100 c­e 3.76±0.416 c 3.30±0.231 hi 7.96±2.166 ab 80.00±10.000 ab 0.10 0.00 2.06±0.200 g 1.83±0.321 f­h 1.66±0.100 c­g 4.80±0.889 a­c 4.20±0.794 b­i 3.63±1.332 cd 96.60±15.275 a 0.10 0.50 3.20±0.929 c­g 2.30±0.802 c­h 1.56±0.361 d­g 4.56±0.100 a­c 3.93±0.493 c­i 3.16±0.954 d 80.00±15.275 ab 0.10 1.00 3.10± 0.643 c­g 2.43±0.781 c­h 2.43±0.265 a­c 5.16±0.800 a­c 5.13±0.702 bcd 5.73±2.452 bc 70.00±10.000 bc 0.10 2.00 4.10±0.624 a­c 2.26±0.153 c­h 2.86±0.794 ab 4.53±1.415 a­c 4.43±0.451 b­h 3.76±3.694 cd 73.30±20.000 a­c 0.10 3.00 3.00±0.300 c­g 1.80±0.153 gh 1.26±0.351 d­g 4.06±0.493 bc 4.20±1.012 b­i 4.60±1.193 cd 83.30±10.000 ab 0.20 0.00 2.56±0.300 efg 2.40±0.854 c­h 1.16±0.361 fg 4.96±1.450 a­c 5.30±0.351 b 3.56±0.781 cd 73.30±10.000 a­c 0.20 0.50 3.76±0.306 bcd 2.43±0.321 c­h 1.80±0.529 c­f 5.40±0.700 ab 4.20±0.462 b­i 9.10±0.611 a 73.30±10.000 a­c 0.20 1.00 3.73±0.723 b­e 3.10±0.700 a­d 2.03±0.500 cd 5.40±0.493 ab 6.60±0.458 a 4.76±1.858 cd 83.30±10.000 ab 0.20 2.00 5.10±0.889 a 2.76±0.493 b­g 3.06±0.153 a 5.63±1.015 a 5.10±0.586 bcd 3.40±0.473 d 80.00±10.000 ab 0.20 3.00 3.73±0.153 b­e 3.66±0.794 ab 1.96±0.529 c­f 4.90±0.651 a­c 4.96±1.365 bcde 4.60±0.651 cd 80.00±15.275 ab 0.50 0.00 2.83±0.153 d­g 2.16±0.306 d­h 1.33±0.814 d­g 4.90±0.954 a­c 5.23±0.361 b 4.73±0.896 cd 90.00±5.774 a 0.50 0.50 3.46±0.300 b­e 2.33±0.153 c­h 1.70±0.058 c­g 5.20±0.721 a­c 4.40±1.250 b­h 3.93±0.404 cd 80.00±10.000 ab 0.50 1.00 3.66±0.361 b­e 2.90±0.854 a­e 2.06±0.200 b­d 4.50±0.971 a­c 5.20±1.552 bc 4.86±1.973 cd 80.00±10.000 ab 0.50 2.00 4.60±0.361 ab 3.63±0.721 ab 1.33±0.351 d­g 4.56±0.635 a­c 4.66±0.961 b­g 3.40±0.833 d 80.00±10.000 ab 0.50 3.00 3.20±0.503 c­g 2.20±0.458 c­h 1.30±0.265 d­g 5.06±1.250 a­c 3.40±0.513 g­i 4.73±1.249 cd 80.00±10.000 ab 1.00 0.00 2.86±0.503 d­g 2.26±0.306 c­h 1.56±0.100 d­g 4.56±1.457 a­c 3.73±1.350 e­i 4.43±1.058 cd 80.00±10.000 ab 1.00 0.50 3.13±0.458 c­g 2.13±0.764 d­h 1.66±0.208 c­g 4.63±1.358 a­c 4.20±1.286 b­i 5.73±3.134 bc 90.00±10.000 a 1.00 1.00 3.03±0.833 c­g 2.83±0.306 b­f 1.96±0.751 c­f 4.33±0.681 a­c 3.86±0.987 d­i 4.93±1.102 cd 73.30±10.000 a­c 1.00 2.00 4.56±0.723 ab 3.20±0.351 a­c 1.63±0.800 c­g 4.70±0.651 a­c 3.86±1.845 d­i 4.03±2.364 cd 80.00±5.774 ab 1.00 3.00 3.26±0.854 c­e 3.90±0.208 a 1.40±0.850 d­g 4.56±0.404 a­c 4.06±0.416 b­i 4.20±0.513 cd 90.00±15.275 a kaviani et al. ‐ micropropagation of phalaenopsis amabilis var. grandiflora 489 half­concentration of iba and kn (fig. 1f). all other media differed significantly and gave lower root length growth rates. among all treatments, 0.50 mg l­ 1 iba plus 0.50 mg l­1 kn and 1.00 g l­1 ac was found to be the most effective for root formation (7.13±0.153 per explant) (table 4, fig. 1g). however, the root number (7.06±0.777 per explant) produced in medi­ um containing 0.20 mg l­1 iba plus 1.00 mg l­1 kn and 0.50 g l­1 ac was noticeable (table 3). in most cases, minimum root number was recorded in media with­ out iba. among all concentrations of iba, kn and ac used individually, maximum root number (5.28±0.564 per explant) was produced in medium containing 0.20 mg l­1 iba (data not shown). the in vitro rooted plantlets were successfully acclimatized in the greenhouse (fig. 1h). pots were filled with leca, peat moss and perlite (in ratio of 1:1:1). acclimatization was achieved in 4­6 weeks, and at this stage plants attain the height of about 12­ 16 cm. acclimatization of micropropagated plantlets to the natural conditions requires several anatomical, morphological and physiological changes especially in xylem, leaves and photosynthesis. the hardened plantlets were maintained in the hyrcan agricultural individually, maximum leaf number (4.52±0.33 per explant) was produced in medium containing 2.00 mg l­1 kn (data not shown). production of leaf was relatively high by all plbs grown on medium contain­ ing 2.00 mg l­1 kn in combination with all concentra­ tions of iba with or without ac (tables 2, 3, 4). thus, the optimal concentration of kn was 2.00 mg l­1. also, the optimal concentrations of iba were 0.20 and 0.50 mg l­1. these concentrations in combination with each other recorded maximum shoot and root pro­ duction. media supplemented with 1.00 g l­1 ac was most suitable for in vitro leaf growth since it resulted in the largest leaf number and development (table 4, figs. 1d, e, f). advanced root development was significantly affected by the composition of the medium, when measured through root length and root number. all treatments of pgrs and ac, individually and in com­ bination had significant effects (p<0.01) on root growth (table 1). root length was highest (6.66±0.709 cm per explant) in presence of 0.20 mg l­ 1 iba plus 2.00 mg l­1 kn and 1.00 g l­1 ac medium (table 4). however, no statistically significant differ­ ence in root length was detected between this and table 3 ­ effect of different concentrations of kn and iba along with 0.50 mg l­1 ac on the studied parameters of in vitro grown phalaenopsis amabilis blume cv. grandiflora means with different letters on the same column are significantly different (p<0.05) based on lsd test. pgrs (mg l­1) leaf number leaf length (cm) leaf width (cm) root number root length (cm) callus number viability percentageiba kn 0.00 0.00 3.40±0.950 cd 1.66±0.872 f 1.73±1.210 b­e 3.86±0.907 gh 3.20±0.493 g 4.00±0.624d­g 90.00±10.000 a 0.00 0.50 2.66±0.907 d 1.76±0.252 ef 0.80±1.250 f 3.23±1.286 h 3.33±0.351 efg 5.60±1.054 bcd 80.00± 0.000 ab 0.00 1.00 3.63±0.819 bcd 2.60±0.265 a­f 1.30±0.208 c­f 4.10±0.751 f­h 3.16±0.346 g 6.70±1.136 abc 80.00±20.817 ab 0.00 2.00 2.90±1.106 d 3.10±0.306 abc 1.60±0.513 b­e 5.13±0.889 b­g 3.23±0.458 fg 3.00±0.666 g 70.00±15.275 b 0.00 3.00 2.90±0.917 d 2.33±0.961 b­f 1.33±0.513 c­f 4.80±0.907 b­g 3.33±0.954 efg 3.73±1.159 d­g 70.00±10.000 b 0.10 0.00 3.20±0.351 d 2.33±0.265 b­f 1.13±0.115 ef 5.80±1.002 a­e 5.06±1.212 a­d 3.26±1.332 fg 90.00±5.774 a 0.10 0.50 2.66±0.777 d 2.10±0.265 def 1.60±0.100 b­e 6.16±0.473 abc 3.76±0.751 d­g 5.50±0.721 bcd 90.00±10.000 a 0.10 1.00 2.86±0.702 d 2.43±0.721 b­f 2.06±0.208 abc 5.83±0.200 a­e 4.23±0.557 c­g 6.56±0.656 abc 90.00±10.000 a 0.10 2.00 5.13±0.666 a 2.10±0.611 def 2.30±0.265 a 5.96±1.682 a­d 3.86±0.100 c­g 3.40±1.353 efg 90.00±15.275 a 0.10 3.00 3.33±1.193 d 2.53±0.666 b­f 1.56±0.306 b­f 4.90±0.917 b­g 4.70±1.266 a­e 3.53±1.159 d­g 73.30±10.000 ab 0.20 0.00 2.96±1.277 d 2.90±0.458 a­d 1.20±0.265 def 3.73±0.520 gh 5.96±1.106 ab 3.90±0.656 d­g 70.00±10.000 b 0.20 0.50 3.26±0.473 d 2.63±0.436 a­f 1.93±0.451 a­d 6.10±1.411 abc 4.63±1.229 b­f 5.36±0.666 b­e 70.00±15.275 b 0.20 1.00 3.80±1.044 bcd 2.23±0.351 c­f 2.26±0.289 ab 7.06±0.777 a 6.06±1.710 a 5.43±1.514 b­e 76.60±20.817 ab 0.20 2.00 5.80±1.002 a 2.16±0.557 c­f 1.83±0.814 b­e 5.56±0.557 a­f 5.26±0.902 abc 8.56±3.029 a 70.00±15.275 b 0.20 3.00 3.83±0.800 bcd 3.23±0.611 ab 1.73±1.150 b­e 4.50±1.436 d­h 4.00±0.800 c­g 3.16±0.624 fg 90.00±10.000 a 0.50 0.00 3.10±1.539 d 2.10± 0.950 def 1.36±0.681 c­f 5.40±1.234 b­f 4.40±1.290 c­g 7.30±0.702 ab 70.00±10.000 b 0.50 0.50 4.63±1.629 abc 2.70±0.153 a­e 1.63±0.586 b­e 6.23±1.320 ab 3.80±2.022 d­g 4.33±0.643 d­g 73.30±10.000 ab 0.50 1.00 2.86±0.709 d 2.73±1.210 a­e 1.90±0.611 a­f 4.93±0.700 b­g 4.53±0.709 c­g 5.16±1.229 c­f 80.00±10.000 ab 0.50 2.00 4.76±0.473 ab 3.56±0.709 a 1.46±0.709 c­f 4.66±0.624 c­h 4.00±0.954 c­g 4.76±0.833 c­g 86.60±10.000 ab 0.50 3.00 3.53±0.361 bcd 2.93±1.595 a­d 1.70±0.458 b­e 4.13±1.007 f­h 4.56±0.900 b­g 4.90±0.436 c­g 86.6±10.000 ab 1.00 0.00 2.86±0.681 d 3.03±0.306 a­d 1.56±0.252 b­f 4.73±0.794 b­h 3.66±1.234 d­g 3.20±2.219 fg 70.00±10.000 b 1.00 0.50 3.36±0.900 cd 2.36±0.755 b­f 1.66±0.208 b­e 4.53±0.361 d­h 4.56±0.436 b­g 5.60±3.005 bcd 90.00±10.000 a 1.00 1.00 3.40±0.794 cd 2.93±0.300 a­d 1.93±0.755 a­d 4.43±0.987 e­h 4.53±1.405 c­g 4.63±3.233 c­g 70.00±15.275 b 1.00 2.00 4.73±0.379 ab 2.90±0.153 a­d 1.46±0.200 c­f 4.13±1.493 f­h 4.26±0.624 c­g 3.93±0.666 d­g 73.30±10.000 ab 1.00 3.00 3.76±0.907 bcd 2.73±0.100 a­e 1.63±0.451 b­e 4.20±1.795 f­h 4.20±0.755 c­g 3.93±0.153 d­g 90.00±11.547 a 490 adv. hort. sci., 2019 33(4): 485­493 sciences and biotechnology research institute, amol, iran with 95% field establishment rate. effect of pgrs and ac on viability percentage significant differences were found in viability per­ centage among the different concentrations of pgrs alone and in combination with each other, also with ac concentrations. the rate of produced plantlets was highest when iba at 0.10 mg l­1 alone was added to the media (table 2). least viability percentage (63.30±10.00) was observed in plbs cultured on media containing 0.50 mg l­1 kn without ac and 1.00 mg l­1 iba along with 1.00 mg l­1 kn with 1.00 g l­1 ac (tables 2, 4). effect of pgrs and ac on callus production lsd test did not show significant differences among different concentrations of ac for callus pro­ duction. a combination of 0.20 mg l­1 iba and 0.50 mg l ­1 kn induced highest callus production (9.10±0.611) (figs. 2c, d), which differed significantly from the other tested combinations, being this rate two or three­fold higher than in the other treatments (tables 2, 3, 4). there was no any direct correlation between increasing pgrs and ac concentrations and increase in callus production. in most cases, mini­ mum callus formation was observed in the explants cultured on media without iba or kn with or without ac (tables 2, 3, 4, figs. 2c, d). 4. discussion and conclusions the present investigation demonstrated that the addition of external pgrs in proper concentrations induced leaf formation from the plbs explants cul­ tured in the ms medium. the regeneration of multi­ ple shoots (leaves in some orchids like phalaenopsis amabilis) has been reported to be closely related with the type and concentration of cytokinins used (amoo et al., 2014). development of multiple shoots from plbs has been successfully achieved in some orchids such as cymbidium, dendrobium, catasetum, phalanoepsis, habeneria and satyrium (talukdar, 2001; sheelavanthmath and murthy, 2001; mahendran and bai, 2009; baker et al., 2014; kaviani et al., 2017). in dendrobium huoshanense, kn was reported to be more effective for plantlet regenera­ tion from plbs than other cytokinins (luo et al., 2009). kn was also used for shoot multiplication of means with different letters on the same column are significantly different (p<0.05) based on lsd test. table 4 ­ mean comparison of the effect of different concentrations of kn and iba on measured characters of catasetum pileatum alba grown in vitro condition pgrs (mg l­1) leaf number leaf length (cm) leaf width (cm) root number root length (cm) callus number viability percentageiba kn 0.00 0.00 3.30±0.896 e­h 2.30±0.529 ef 1.40±0.462 d 4.50±0.802 c­f 3.46±0.436 de 5.80±0.208 a­c 90.00±15.275 a 0.00 0.50 3.10±0.493 e­h 2.56cdef ± 0.656 1.60±0.451 d 3.90±0.529 f 3.20±1.153 e 4.56±1.422 bc 73.30±10.000 a­c 0.00 1.00 3.40±0.666 d­g 2.73±0.802 cdef 2.20±0.608 a­d 4.83±1.464 b­f 4.16±1.210 c­e 5.66±0.451 a­c 80.00±10.000 a­c 0.00 2.00 3.43±0.473 d­g 2.33±0.361 def 1.90±0.737 bcd 4.90±0.917 b­f 3.73±1.069 c­e 4.43±1.779 bc 70.00±11.547 bc 0.00 3.00 3.06±1.453 e­h 2.20±1.405 ef 2.30±0.153 a­d 4.33±0.321 def 4.53±0.208 b­e 3.86±0.265 c 73.30±10.000 a­c 0.10 0.00 3.00±0.611 e­h 2.30±0.416 ef 1.50±0.321 d 5.36±0.493 b­e 5.10±0.458 a­d 3.83±0.666 c 80.00±15.275 abc 0.10 0.50 2.83±0.945 gh 3.16±1.401 cde 2.00±0.208 a­d 4.73±0.764 b­f 4.53±0.643 b­e 6.90±1.007 ab 90.00±10.000 a 0.10 1.00 3.26±1.277 e­h 2.23±0.794 ef 2.10±0.200 a­d 5.86±0.451 abc 6.13±0.436 ab 5.80±1.097 a­c 73.30±10.000 a­c 0.10 2.00 5.03±1.290 ab 3.46±0.551 bc 2.63±0.400 ab 5.76±0.208 abcd 5.13±1.168 a­d 5.20±0.889 a­c 90.00±5.774 a 0.10 3.00 2.90±1.650 fgh 3.06±0.200 cdef 2.06±0.513 a­d 4.73±0.200 b­f 4.50±0.624 b­e 4.26±1.217 bc 90.00±10.000 a 0.20 0.00 2.26±0.850 h 2.90±0.208 cdef 1.43±0.351 d 6.10±0.950 ab 4.70±0.473 b­e 3.80±1.332 c 90.00±10.000 a 0.20 0.50 3.80±0.929 b­g 3.20±0.416 cde 1.90±0.416 bcd 4.40±0.473 def 4.06±0.666 c­e 6.90±0.300 ab 80.00±10.000 a­c 0.20 1.00 3.23±0.513 e­h 2.60±0.794 cdef 2.56±0.896 abc 6.10±0.700 ab 6.66±0.709 a 5.03±1.350 bc 76.60±10.000 a­c 0.20 2.00 6.16±0.503 a 2.73±0.907 cdef 2.86±0.058 b 4.46±0.643 c­f 5.40±0.361 a­c 4.36±1.790 bc 70.00±10.000 bc 0.20 3.00 4.20±0.208 b­f 3.43±0.666 bcd 2.00±0.208 a­d 4.93±0.850 b­f 5.20±0.850 a­c 3.80±1.836 c 90.00±10.000 a 0.50 0.00 3.00±0.153 e­h 2.20±0.651 ef 1.80±0.058 bcd 5.36±0.252 bcde 4.66±0.850 b­e 6.16±1.159 a­c 76.60±10.000 ac 0.50 0.50 4.63±0.702 bcd 2.70±0.306 cdef 1.56±0.987 d 7.13±0.153 a 4.40±1.124 c­e 5.26±0.586 a­c 86.60±11.547 ab 0.50 1.00 3.73±0.436 b­g 2.80±0.737 cdef 3.13±0.603 a 4.10±0.416 ef 3.86±0.493 c­e 4.46±1.909 bc 73.30±10.000 a­c 0.50 2.00 4.93±0.814 abc 4.66±0.702 a 1.43±0.551 d 4.76±0.681 b­f 4.36±0.751 c­e 4.03±0.889 c 80.00±5.774 a­c 0.50 3.00 3.66±0.404 c­g 2.90±0.300 cdef 1.76±0.306 c 4.50±0.854 c­f 4.40±0.252 cde 5.10±0.751 a­c 90.00±10.000 a 1.00 0.00 3.06±0.569 e­h 3.00±0.300 cdef 2.06±0.231 a­d 5.00±0.971 b­f 3.96±0.862 c­e 4.13±0.751 c 80.00±15.275 a­c 1.00 0.50 3.33±1.114 d­g 2.53±1.124 cdef 1.66±0.709 cd 4.86±0.757 b­f 5.06±0.529 a­d 7.73±1.210 a 70.00±10.000 bc 1.00 1.00 3.86±0.404 b­g 1.96±0.700 f 2.06±0.265 a­d 4.96±0.451 b­f 4.86±0.850 b­e 4.90± 1.253 bc 63.30±10.000 c 1.00 2.00 4.30±0.416 bcde 2.26±0.777 ef 1.86±0.115 bcd 4.76±0.557 b­f 4.30±0.361 c­e 4.56±0.929 bc 90.00±10.000 a 1.00 3.00 4.10±0.458 b­g 4.46±0.321 ab 1.70±0.458 bcd 4.83±0.651 b­f 3.80±0.624 c­e 3.80±0.458 c 90.00±10.000 a kaviani et al. ‐ micropropagation of phalaenopsis amabilis var. grandiflora 491 some other orchids (saiprasad et al., 2004; malabadi et al., 2005; panwar et al., 2012). in satyrium nepalense, protocorm developed multiple shoots directly on the medium supplemented with cytokinins. in most of the orchids the presence of cytokinins alone promoted optimal shoot prolifera­ tion (mahendran and bai, 2009). ba is known to pro­ mote seedling leaf formation in paphiopedilum (huang et al., 2001; chen et al., 2015). bhattacharyya et al. (2016) reported that when the explants were grown in medium containing cytokinin and auxin, a higher rate of response frequency of shoot buds and plbs was observed in all pgrs combinations. also according to seeni and latha (2000) and roy et al. (2011), pgrs in orchids act more efficiently when used in combination. therefore, cytokinin and auxin are supposed to act synergistically. effectiveness of ac on shoot multiplication and leaf growth has been demonstrated in some orchids (george and ravishankar, 1997; thomas and michael, 2007; hossain et al., 2010; roy et al., 2011; zeng et al., 2012; panwar et al., 2012). study on paphiopedilum wardii evidenced that the plantlet growth in vitro was significantly affected by ac along with pgrs (zeng et al., 2012). roy et al. (2011) showed that healthy plantlets of vanda coerulea were induced from plbs when cultured on medium fortified with 3.00 g l­1 ac, 5.36 µm naa and 3.80 µm bap. in our study, there was no difference between 0.50 and 1.00 g l­1 ac for leaf growth (tables 3, 4). supplementation of ac in the media significantly influenced plantlet growth (shoot multiplication and root growth) over the control (roy et al., 2011). this finding confirmed our results on the effect of ac on leaf growth parameters. in fact, a combination of 0.20 mg l­1 iba and 2.00 mg l­1 kn on medium contain­ ing 0.50 and 1.00 g l­1 ac was found to be suitable for maximum leaf number. this positive effect of ac on shoot multiplication has been attributed to the ability of ac to absorb phenolic compounds released by the plantlets into the media and regulate the ph level (pan and van staden, 1998; eymar et al., 2000). a positive linear relationship was found between ac concentration and plantlet growth of vanda coerulea griff ex. lindl. (blue vanda) (roy et al., 2011). in paphiopedilum spicerianum , 1.0 mg l ­1 naa, 10% banana homogenate and 0.50 g l­1 ac was the most effective to promote seedling formation (chen et al., 2015). ac might also act as a growth promoter that inhibits harmful effects of some compounds produced during seedling formation (roy et al., 2011). our study showed the positive effect of iba on root formation. in satyrium nepalense and dendrobium nobile, iba resulted in a better rooting efficiency over naa in terms of rooting frequency and root number (mahendran and bai, 2009; bhattacharyya et al., 2016). according to these authors, maximum rooting efficiency (86% or 5.4 roots/shoot) was obtained in medium fortified with 2.00 mg l­1 iba in dendrobium nobile (bhattacharyya et al., 2016), while the highest number of roots per shoot (6.40) was achieved at 9.84 mm iba in satyrium nepalense d. don. (mahendran and bai, 2009). the effectiveness of iba in rooting has been shown for some other orchids like vanilla planifolia (giridhar et al., 2001), cymbidium aloifolium (l.) sw. and dendrobium nobile lindl. (nayak et al., 2002), cymbidium pendulum (nongdam et al., 2006), satyrium nepalense (mahendran and bai, 2009), vanda teres (firoz alam et al., 2010) and eulophia nuda lindl. (panwar et al., 2012). a maximum 90% response for root formation and highest number of roots (5.50) with length of 5.30 cm per shoot was obtained on iba (2.46 mm) treated shoots of eulophia nuda lindl. (panwar et al., 2012). study of baker et al. (2014) on micropropagation of catasetum demonstrated that the largest number of root (7.16) and root length (193.40 mm) were obtained on ms medium supplemented with 0.50 mg l−1 ba along with 0.50 mg l­1 naa. our results are in line with previous findings, as maximum root length and root number were obtained in medium contain­ ing both iba and kn. our study showed the positive effect of ac on root growth. similarly, roy et al. (2011) evidenced a positive influence of ac on root growth of vanda coerulea. some other researches demonstrated that the presence of ac in the media stimulated rooting in vanilla planifolia (george and ravishankar, 1997), cymbidium sinense (chang and chang, 2000) and paphiopedilum spicerianum (chen et al., 2015). addition of ac in rooting medium maintained the ph level, increased the nitrogen uptake and stimulated the rooting of in vitro shoots (eymar et al., 2000; panwar et al., 2012). in the present work, minimum callus formation was obtained frequently on media without iba or kn with or without ac. in eulophia nuda and on medium containing higher concentration of ba, the explants produced callus at the base of shoots while lesser number of shoots were differentiated on medium with lower ba concentration (panwar et al., 2012). also, in medium with higher concentration of ba adv. hort. sci., 2019 33(4): 485­493 492 combined with kn lower shoot production with more callus induction 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319­327. zeng s., wua k., teixeira da silva j.a., zhanga j., chena z., xiaa n., duan j., 2012. asymbiotic seed ger‐ mination, seedling development and reintroduction of paphiopedilum wardii sumerh., an endangered terres‐ trial orchid. ­ sci. hortic., 138: 198­209. impaginato 197 adv. hort. sci., 2019 33(2): 197-204 doi: 10.13128/ahs-23599 water retention of substrates potentiates the quality of lettuce seedlings j.l.t. chiomento ¹ (*), p. frizon ¹, r.c. costa ¹, n.s. trentin ², f.s. nardi ¹, e.o. calvete ¹ ¹ programa de pós-graduação em agronomia, faculdade de agronomia e medicina veterinária, universidade de passo fundo, passo fundo, rs, brazil. ² curso de agronomia, faculdade de agronomia e medicina veterinária, universidade de passo fundo, passo fundo, rs, brazil. key words: chemical properties, lactuca sativa l., physical properties, root system morphology, shoot morphology. abstract: a difficulty in the production of lettuce seedlings in containers is to assure the production of shoot biomass with limited portion of roots, restricted to a small volume of substrate. therefore, we investigate if substrates associated to lettuce cultivars interfere in the seedling quality. the treatments, outlined in a two-factorial scheme, were two cultivars of lettuce and four substrates, arranged in a randomized complete block design, with three replications. the results showed that seedlings produced in the substrate with higher water retention capacity had higher performance in relation to shoot morphology and root system morphology. in conclusion, the data show that the seedlings quality of lettuce cultivars associate with the types of substrates studied and that seedlings produced in substrate with higher water retention have better quality. 1. introduction in horticultural crops the supply of quality seedlings to the producers is important to obtain high production after the establishment of the plants in their growth medium. such quality is related to the plants resistance to biotic and abiotic stresses (zhao et al., 2016). among vegetables, lettuce (lactuca sativa l.) is one of the most cultivated (kim et al., 2016), with a world production of approximately 25 million tons (fao, 2014). in order to maintain this production in an upward manner, quality seedlings must be provided to producers. thus, the production of seedlings is one of the most important stages in lettuce cultivation, because this process reflects on the productive performance of the plants (auler et al., 2015). however, a difficulty in the production of seedlings in containers is to ensure the production of shoot biomass with limited portion of root (lemaire, 1995), restricted to a small volume of substrate, in response to the species/cultivars used. as the substrates have a wide variation in their physicochemical pro(*) corresponding author: jose-trevizan@hotmail.com citation: chiomento j.l.t., frizon p., costa r.c., trentin n.s., nardi f.s., calvete e.o., 2019 water retention of substrates potentiates the quality of lettuce seedlings. adv. hort. sci., 33(2): 197-204 copyright: © 2019 chiomento j.l.t., frizon p., costa r.c., trentin n.s., nardi f.s., calvete e.o. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 july 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 197-204 198 perties (fermino and kämpf, 2012), choosing a suitable material is essential to the development of the plants (mondragón-valero et al., 2017). the substrates used must be low cost and easy to handle (noya et al., 2017), to have porosity around 85% (kämpf et al., 2009) and water retention capacity (graceson et al., 2013). for lettuce, it is necessary to choose material that ensures these physical characteristics, since it is a species with high water demand (nunes et al., 2017). moreover, the choice of the cultivar is also important, because the genotypes can interact with biotic and abiotic factors, influencing the quality of the seedlings produced (martins et al., 2017). knowing that substrates used in seedlings production are essential to plants germination and establishment (auler et al., 2015) and that crop productivity is linked to this input (smiderle et al., 2001), this question arises: how substrates associated to lettuce cultivars affect the seedlings quality? therefore, based on the hypothesis that the quality of lettuce cultivars is dependent of the water retention capacity of the substrate, the objective of the present study was to evaluate if substrates associated to lettuce cultivars interfere in the seedlings quality. this study provides a view of the development of lettuce seedlings using different substrates to improve the seedlings quality (e.g., increase the growth of shoot biomass and root system) grown in greenhouses. 2. materials and methods plant material, treatments description and experiment site the seeds of lettuce used in the work were of the cultivars mimosa roxa salad bowl (purple), of bright greenish purple color, and mônica sf 31 (green), of medium green color, both of the group crisphead. the materials used as substrates were carbonized rice husk (crh), horta 2® (hor), tn gold® (tng) and a mixture (mix) composed of 40% crh, 40% hor and 20% tng. the composition of hor consists of pine bark, vermiculite, acid correction and fertilizers (nitrogen, phosphorus and potassium) in quantities not supplied by the manufacturer. the composition of tng consists of sphagnum peat, expanded vermiculite, dolomitic limestone, agricultural gypsum and fertilizers (nitrogen, phosphorus and potassium) in quantities not supplied by the manufacturer. no fertilizer was added to the substrates. the rice husk used in the work was carbonized (kämpf et al., 2006). the experiment was developed in the brazilian subtropics, in the city of passo fundo/rs (28° 15’ 46” s, 52° 24’ 24” w), from april to may (fall) of 2017. the trial took place on trays kept on metal benches, 1.2 m above the soil surface, in a agricultural greenhouse of 90 m², with semicircular roof, installed in the northeast-southeast direction. the galvanized steel frame was covered with low density polyethylene film, with anti-ultraviolet additive and with a thickness of 150 microns, and the sides were covered with anti-aphid screen. the irrigation used was with sprinklers, in the mechanized system, with a flow rate of 2 l min-¹ per unit. the irrigation regime consisted of four sprinklers per day, with total wetting of seven minutes. the water blade supplied to the seedlings was 4.35 mm day-1. during the execution of the experiment, the photosynthetically active radiation (par) and the mean air temperature inside the greenhouse were monitored, with mean values of 110.5 μmol m-² s-¹ and 17.4°c, respectively. experimental design the treatments, outlined in a two-factorial scheme, consisted of two lettuce cultivars (purple and green) and four substrates (crh, hor, tng and mix). the production of the seedlings was carried out in trays of expanded polystyrene, with dimensions of 0.34 m of width and 0.68 m of length. each tray had 128 cells, with a volume of 35 cm³. the experimental design was randomized blocks with three replicates (n= 3; one replicate per tray, i.e. three trays were used in the experiment). on april 20, the trays were filled with the substrates crh, hor, tng and mix, and after that, five seeds of the lettuce cultivars were sown in each cell. in each tray each treatment was composed of 16 seedlings, that is, a total of 48 seedlings per treatment (16 seedlings/treatment x 3 replicates). considering that we used 8 treatments, our experiment consisted of a total of 384 seedlings (16 seedlings/treatment x 3 replicates x 8 treatments). determination of physicochemical properties of substrates a sample of 1 l of each substrate was collected and analyzed to obtain physicochemical attributes of the materials. the physical attributes determined in the substrates were: density (d), total porosity (tp), aeration space (as), readily available water (raw) and buffer water (bw). the chemical attributes determined in the subschiomento et al. quality of lettuce seedlings 199 trates were nitrogen (n), phosphorus pentoxide (p2o5), potassium oxide (k2o), organic carbon (oc), hydrogenionic potential (ph), electrical conductivity (ec) and cation exchange capacity (cec) (mapa, 2014). regarding the seedlings, the evaluations began one week after sowing. morphological attributes of the shoot and the root system of the seedlings were evaluated. determination of shoot morphology in relation to the shoot morphology, forty-eight seedlings per treatment were evaluated. four days after sowing the percentage of seed germination was evaluated by means of the equation: germination (%)= (seed germinated/total number of seeds) x 100 (1) after the germination, thinning was performed, leaving one plant per cell in each tray. in addition, the date of emergence of the cotyledons and the issuance of the first, second and third leaves were noted. thirty-three days after sowing, the stem base diameter (sbd) and the shoot height (sh) of the seedlings were measured with a digital caliper. the fresh (sfw) and dry (sdw) weight of the shoot was also evaluated. in order to obtain the dry weight, the plants were kept in a drying oven with forced air circulation, at 65°c for 48 hours, until constant weight, and weighed in an electronic analytical balance. determination of root system morphology regarding to the root system morphology, fortyeight seedlings per treatment were evaluated. the roots were collected and washed in water to eliminate the substrate fragments. thus, the roots were scanned and then the images obtained were analyzed by winrhizo® software. the attributes evaluated were the total root length (tl, cm), root surface area (sa, cm²) and root volume (rv, cm³). the roots were grouped by software in different diameter classes in relation to their total length (böhm, 1979): very thin roots (vtr, ø <0.5 mm), fine roots (fr, ø 0.5 to 2 mm) and thick roots (tr, ø > 2 mm). the fresh (rfw) and dry (rdw) weight of the root system was also evaluated, following the methodology described previously. determination of seedlings quality the seedlings quality was obtained by models of plant development. these development models are mathematical models that consider plant growth variables (cournède et al., 2013), such as shoot morphology and root system morphology. thus, the seedling vigor index (svi) was determined according to abdul-baki and anderson (1973), by the equation: svi= germination (%) x (shoot length + root length) (2) it was determined, also, the dickson quality index (dqi), proposed by dickson et al. (1960), by the equation: dqi= (tdw) / (h/sbd + sdw/rdw) (3) where tdw = total dry weight (g); h = shoot height (cm); sbd = stem base diameter (cm); sdw = shoot dry weight (g); rdw = root dry weight (g). in addition, dry matter accumulation (dma) of the shoot and of the root system was determined, according to atif et al. (2016), by the equation: dma = (dw/fw) x 100 (4) where dw = dry weight (g); fw = fresh weight (g). statistical analysis the data were submitted to analysis of variance and the means of the treatments were compared by tukey test, at 5% probability of error, with the aid of the assistat® program (silva and azevedo, 2016). 3. results physicochemical properties of substrates the results of the physical characterization of the substrates used in this experiment (table 1) showed substrates density (kg m-3) total porosity (m3 m-3) aeration space (m3 m-3) readily available water (m3 m-3) buffer water (m3 m-3) carbonized rice husk 170±12.33 c 0.879±0.11 b 0.365±0.02 b 0.395±0.10 a 0.009±0.003 horta 2® 241±05.26 a 0.837±0.10 d 0.303±0.01 d 0.149±0.02 d 0.020±0.001 tn gold® 088±10.98 d 0.916±0.13 a 0.519±0.11 a 0.202±0.08 c 0.007±0.002 mix 183±07.47 b 0.869±0.14 c 0.325±0.08 c 0.259±0.09 b 0.030±0.001 mean 170.50 0.87 0.378 0.251 0.016 coefficient of variation (%) 13.58 14.11 15.26 14.45 12.25 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 1 physical properties of the substrates used in the study adv. hort. sci., 2019 33(2): 197-204 200 that, considering the density of the materials, the tng substrate is the lightest. with the values of tp, as, raw and bw of table 1 we elaborated a graph to visualize the relation between air and water in each substrate (fig. 1). we observed that tng material showed an unbalanced air-water relation (fig. 1). in addition, we observed a better balance between airwater in the mix substrate, that is, when the other materials were combined (40% crh, 40% hor and 20% tng). in addition, also with the values of tp, as, raw and bw of table 1 we elaborated a graph to visualize the water retention curve of each substrate (fig. 2), according to de boodt and verdonck (1972). the hor and mix substrates presented higher water retention, requiring volumes of 0.385 m3 m-3 and 0.285 m3 m-3, respectively, to remain in the range of water easily available to plants (10-50 -cm h2o) (fig. 2). on the other hand, the crh substrate had greater drainage of water (fig. 2). the four materials showed availability of nutrients, except for k2o. among the substrates, crh presented 23% more ph than tng. the opposite was obtained for ec and cec, with crh being 58% lower than the hor for ec and 85% lower than tng for cec (table 2). shoot morphology in all treatments there was 100% germination of the seeds. up to the first true leaf, the two cultivars took the same period (11 days) to differentiate. the differences began to be identified from the second true leaf. for this attribute, the two cultivars produced in the crh and the green cultivar produced in the tng took longer to emit the third true leaf (fig. 3). as a consequence, at the time of transplantation the third leaf was poorly expanded. we did not observe effects of the cultivars on the shoot morphology of the seedlings. this means that the seedlings of both cultivars can be produced on any of the substrates. however, we observed only effect in relation to the substrates for the sh, sfw and sdw. the seedlings produced on the hor substrate had higher sh (5.86 cm ± 1.82) than those produced on the crh substrate (3.24 cm ± 0.77), but did not differ fig. 2 water retention curve of the substrates. crh: carbonized rice hull; hor: horta 2®; tng: tn gold®; mix: mixture. different letters indicate significant differences by the tukey test (p≤0.05, n = 3). fig. 1 physical characterization of the substrates used in the study; n= 3 crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. table 2 chemical properties of four substrates substrates n % (m/m) p2o5 % (m/m) k2o % (m/m) oc % (m/m) ph ec (ms cm-1) cec (mmolc kg-1) carbonized rice husk 0.69±0.01 a 1.71±0.10 a 0 07.21±03.03 d 7.2±1.0 a 0.19±0.01 c 134.60±12.2 d horta 2® 0.36±0.09 c 0.39±0.06 d 0 12.60±06.22 b 6.1±2.2 c 0.45±0.01 a 278.60±15.4 b tn gold® 0.65±0.01 b 1.37±0.11 c 0 31.16±11.02 a 5.6±1.3 d 0.36±.0.2 b 892.98±13.7 a mix 0.35±0.02 c 1.48±0.03 b 0 10.38±04.33 c 6.4±3.1 b 0.37±0.03 b 230.05±11.2 c mean 0.51 1.23 0 15.33 6.32 0.34 384.05 cofficient of variation (%) 12.25 15.90 0 16.30 11.51 17.91 14.15 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). chiomento et al. quality of lettuce seedlings 201 root system morphology regarding the root system morphology, we observed significant differences for the interaction between substrates and cultivars in relation to rv and tr (table 3). for the interaction between the factors, the best combination corresponded to the seedlings of the green cultivar produced on the hor substrate, both for root volume and for thick roots (table 3). we also observed significant differences for substrates only for rfw, rdw, sa and fr (table 4). in this respect, we observed that the seedlings produced in hor presented a larger sa, but did not differ statistically from the seedlings produced in mix (table 4). the seedlings developed in crh had smaller amount of fr in relation to those obtained in the other substrates (table 4). regarding the rfw, seedlings produced in hor were superior to those obtained in crh, but did not differ from the seedlings produced in tng and mix (table 4). in addition, the seedlings obtained from hor and mix had a higher rdw in comparison to those developed in crh and tng (table 4). in relation to lettuce cultivars the significant differences occurred for rfw, rdw, tl, sa and fr (table 4). thus, we observed that the seedlings of the green cultivar presented superior performance of the root statistically from the seedlings produced in tng and mix (fig. 4 a). in addition, the seedlings produced on the hor substrate showed higher sfw (0.27 g ± 0.17) and sdw (0.012 g ± 0.0033) than those produced on other substrates (fig. 4 b and c, respectively). in general, seedlings obtained on the hor substrate produced 71% more shoot biomass than those grown in crh (fig. 4). fig. 3 combination of two lettuce cultivars produced in four substrates in relation to the period of emergence and expansion of the first true leaves. crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture; purple=mimosa roxa salad bowl cultivar; green= mônica sf 31 cultivar. fig. 4 shoot morphology of lettuce seedlings produced in substrates. (a) shoot height (sh, cm); (b) shoot fresh weight (sfw, g); (c) shoot dry weight (sdw, g). values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n = 3). crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. substrates root volume (cm³) thick root (cm) purple cv. green cv. purple cv. green cv. carbonized rice husk 0.019 ±0.004 a b 0.043±0.017 a c 0.090±0.15 a a 0.078±0.10 a c horta 2® 0.074 ±0.029 b a 0.185±0.023 a a 0.501±0.41 b a 3.021±0.51 a a tn gold® 0.053 ±0.014 a ab 0.079±0.027 a bc 0.209±0.17 a a 0.660±0.61 a bc mix 0.066 ±0.007 b a 0.111±0.019 a b 0.420±0.22 b a 1.440±0.78 a b mean 0.07 0.80 cofficient of variation (%) 21.77 53.88 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 3 root system morphology of two lettuce cultivars (lactuca sativa l.) produced in four substrates 202 adv. hort. sci., 2019 33(2): 197-204 system in relation to the seedlings of the purple cultivar (table 4). seedlings quality regarding the seedlings quality, we observed statistical diferences for the substrates and for the cultivars. analyzing only the substrates, there were significant differences for the dqi. as for the cultivars, the significant differences were observed regarding the svi and dqi. seedlings produced on the hor substrate presented higher dqi (0.000249 ± 0.000089) than those produced in chr (0.00011 ± 0.000071), but did not differ statistically from seedlings produced in mix (fig. 5). this higher quality of the seedlings produced on hor increased by 56% in relation to those produced on chr material (fig. 5). the green cultivar, regardless of the substrate, presented superiority regarding the svi (fig. 6 a) and dqi (fig. 6 b) in relation to the purple cultivar. 4. discussion and conclusions in general, the research showed that the quality of lettuce cultivars was associated with the types of substrates studied. however, our study showed that fig. 5 quality of the development of lettuce seedlings produced in four substrates. values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n= 3). crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 4 root system morphology of two lettuce cultivars (lactuca sativa l.) produced in four substrates total root length (cm) root surface area (cm²) fine root (cm) root fresh weight (g) root dry weight (g) substrates carbonized rice husk 21.18±05.89 2.81±1.13 c 5.01±2.52 b 0.030±0.01 b 0.0017±0.0008 b horta 2® 28.42±10.55 6.54±2.48 a 9.87±2.30 a 0.087±0.04 a 0.0036±0.0010 a tn gold® 24.44±13.36 4.39±1.78 bc 8.61±2.58 a 0.060±0.04 ab 0.0023±0.0006 ab mix 28.73±09.55 5.54±1.53 ab 9.09±1.93 a 0.061±0.02 ab 0.0032±0.0006 ab cultivars purple 21.92±08.13 b 3.70±1.54 b 7.20±3.07 b 0.038±0.01 b 0.0021±0.0008 b green 29.46±10.54 a 5.94±2.23 a 9.55±2.52 a 0.080±0.04 a 0.0033±0.0010 a mean 25.69 04.82 08.37 00.05 0.0027 cv (%) 26.28 21.32 20.07 46.77 35.41 fig. 6 development of seedlings of two lettuce cultivars produced in four substrates. (a) seedling vigor index (svi); (b) dickson quality index (dqi). values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n = 3). purple: mimosa roxa salad bowl cultivar; green: mônica sf 31 cultivar. chiomento et al. quality of lettuce seedlings 203 substrates with higher water retention promoted greater development of seedlings, through models of plant development. in this way, the physical characterization of the substrates used in the production of seedlings allows to select materials with greater water availability, in order to increase the seedlings quality. the higher quality of the seedlings produced in the hor substrate was attribute to the higher availability of water of this material, because the water retention capacity of the substrates influences the growth and development of the seedlings (graceson et al., 2013), covering the shoot morphology (prevedello and armindo, 2015) and the root system morphology (ferraz et al., 2005). in practice, the data referring to the root system morphology indicated that seedlings of the green cultivar produced in the hor substrate have a more structured lump, which improves seedling sustainability after transplanting and increases plant survival. as in crh there is predominance of large particle sizes, this impairs water retention by the material (zorzeto et al., 2014), which explained the lower quality of the seedlings produced in this substrate. in practice, the expansion data of the first true leaves showed that seedlings produced in the crh would not be suitable for transplant because, in addition to lower water retention, this substrate has ph above the ideal range (5.0 to 6.5) (bunt, 1988), which reduces the availability of nutrients to plants (lemaire, 1995). the higher the seedlings quality delivered to the producers the better the development of the plants in their growth medium and the lower their susceptibility to stresses after transplantation. through the dqi, we verified that more robust seedlings were produced in the hor substrate. during the production of the seedlings we observed that the par mean (110.5 μmol m-2 s-1) was below ideal for the lettuce, corresponding to 196.7 μmol m-2 s-1 (ferentinos et al., 2000). this may have reduced the photosynthetic rate of the plants, reducing accumulations of biomass and, therefore, dqi values. as in our study, other studies showed higher growth and development of seedlings produced in substrates with higher water retention (smiderle et al., 2001; costa et al., 2007), as verified in the hor, and lower performance for seedlings produced in substrates with low water availability (freitas et al., 2013), as observed in the crh. considering the physicochemical characterization of the substrates, the chemical properties are less relevant than the physical properties (belda et al., 2016). this is because fertirrigation is provided to the plants, according to the need of the cultivated species. however, the physical quality of the substrates must still be weighed in the choice of materials. the density of the substrates, for example, is linked to plant stability (noya et al., 2017; wisdom et al., 2017). very light substrates (< 100 kg m-3) do not sustain plants and very dense substrates (>300 kg m-3) impair the root growth of seedlings due to mechanical impediment (de boodt and verdonck, 1972; fermino and kämpf, 2012). in addition, a common problem in substrates is insufficient aeration (nemati et al., 2002) and, therefore, the nurseryman should choose materials with higher aeration levels to improve root growth and increase the acquisition of water and nutrients by seedlings (jones and dolan, 2012). thus, in order to maximize the quality of the seedlings produced, nurserymen must obtain the physical characterization of the substrates, selecting materials with greater water retention capacity. in addition, seedling development models, such as svi and dqi, can be used as indicators of the quality of seedlings produced, as we have verified in our study. in conclusion, the data show that the seedlings quality of lettuce cultivars associate with the types of substrates studied. in addition, we proves that substrates with greater water retention promote greater development of the seedlings. we emphasize that the use of development models can be an alternative to analyze the seedlings quality provided to producers in order to increase lettuce production. we suggest to substrate producers to sell materials with a clear label informing the physicochemical characteristics of the substrate so that nurserymen and producers can establish an adequate management to potentiate the lettuce production chain. acknowledgements to the programa de suporte à pós-graduação de instituições comunitárias de ensino particulares (prosuc) of the coordenação de aperfeiçoamento de pessoal de nível superior (capes), for granting the scholarship, and to the 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2 department of plant sciences, university of tennessee, usa. key words: bio-fertilizer, chemical fertilizer, dpph test, inoculation, organic fertilizer, perilla, rosmarinic acid. abstract: medicinal plants are one of the main natural resources of iran from ancient times. perilla is one of the most important medicinal plants of the mint family lamiaceae, since there is no study about adaptability of perilla in iran climate conditions and different fertilizer systems, this experiment was conducted in two experimental sites. the experiment was conducted as split-plot factorial based on a randomized complete block design with three replications at two experimental regions. the main factor was three chemical fertilizer levels (control, 50, 100, 200 kg/ha) and subplots were different kinds of organic fertilizer (control, humic acid, and compost application) and inoculation with piriform osporaindica (inoculation and without). among levels of chemical treatments, 50 and 100 kg/ha lead to a better result. also, humic acid allows to achieve the highest amount of measured traits between different treatments of organic fertilizer. the highest plant yield (147.2 g/m2) and rosmarinic acid yield per area (3.432 g/m2) was achieved in 100 kg/ha at chemical fertilizer with humic acid and biological fertilizer application and the lowest plant yield (89.86 g/m2) and rosmarinic acid yield per area (1.253 g/m2) was observed in control. also, the highest stomatal conductance was obtained with application of compost fertilizer (67.67 mmol h2o m-2s-1). integrated application of the studied fertilizers showed the more positive effect on yield and quality of perilla than individual application of those fertilizers. 1. introduction the perilla is a medicinal plant belonging to the labiatae family and is widely cultivated in southeast asian countries (igarashi and miyazaki, 2013). a number of studies have shown that advantages of perilla are related to the metabolites contained therein (ghimire et al., 2017). to date, very limited information exists regarding the adaptability and management of chemical, organic and bio-fertilizer of perilla in iran. organic matter effects on physicochemical properties and health of soil. it also affects the efficiency of fertilizer application, pesticides and herbicides. one of the most effective organic fertilizers on the growth of (*) corresponding author: mmohamm9@utk.edu citation: ghane m., mohammadi m., pirdashti h., 2019 yield and physiological response of perilla (perilla frutescens) under different soil fertility treatments. adv. hort. sci., 33(2): 205-214 copyright: © 2019 ghane m., mohammadi m., pirdashti h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 august 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 205-214 206 plants and improvement of soil status is humic acid. humic acid can be obtained from any material such as organic matter, coal, or well-decomposed compost. this material plays an important role in increasing soil moisture, absorbing micronutrients and contributing to carbon sequestration (spaccini et al., 2002). another organic material that plays an important role in soil fertility is compost and it comes from plant and food residues. some experimental researches have shown the important role of compost in crop production (adugna, 2016). compost retains moisture in the soil, slow release nutrients to crops and finally increase the crop yield. application of compost obtained from plant remains leads to increased fertility, soil nutrients and increases water retention in the soil. zemanek (2011) also confirmed that application of 50 t/ha and 100 t/ha compost has a positive effect on soil moisture retention. moreover, biologic fertilizers assists well in mineralization and channelization of nutrients leading to enhanced plant productivity (fischer et al., 2007; ansari et al., 2017). biologic fertilizers adopt various possible ways to accelerate the rate of crop production (rizvi et al., 2015; ansari et al., 2017). they increase and improve plant growth by increasing access to nutrients in the root rhizosphere. these fertilizers provide nutrients through biological processes such as biological nitrogen fixation, phosphorus solubilization, and plant growth stimulation. also, they help to natural nutrient cycle and build soil organic matter (kapoor et al., 2015). use of biologic fertilizers ensures healthy plants growth, while enhancing the sustainability and the vigor of the soil. these biologic fertilizers play a special role in increasing plant nutrition and fertility of soils (vessey, 2003). a number of studies have shown that the chemical composition of secondary metabolites in the perilla plant is influenced by various factors such as soil conditions, temperature, growth season (kiazolu et al., 2016), geographic region (ruberto et al., 2002), and phenological stages (saeb and gholamrezaee, 2012). therefore, this study was carried out to evaluate the yield and physiological traits of perilla cultivars under different fertilizer treatments in two regions. based on the work, attempt was also made to provide amicable solutions to address the challenges of organic farming with the help of perilla cultivation in two different locations in dry region of iran. 2. materials and methods the field experiments were conducted at two locations in esfahan province: 1-mashhad ardehal (latitude 34° north, longitude 51° east, altitude 1800 m above mean sea level) 2sensen, iran (latitude 33°, longitude 51°, altitude 945 m above mean sea level) during 2015. mashhad ardehal is located in warm and dry condition and sensen is located in the mountainous and dry region. the meteorological data recorded during the period of plant cultivation are given in table 1. in order to determine the physical and chemical characteristics of experimental fields, two weeks before planting, the soil samples were taken. physicochemical properties of experimental field soil are presented in table 2. this experiment was conducted as split-plot factorial based on a randomized complete block design with three replications at two experimental sites in the year 2015. the main factor was three chemical fertilizer levels (50, 100, 200 kg/ha) plus control, and sub plots were different kinds of organic fertilizer (humic acid, and compost application), plus control; inoculation with piriformospora indica (inoculation and without inoculation) was also evaluated. table 1 weather characteristics in mashhad ardehal and sensen year temperature mean (°c) precipitation mean (mm) humidity mean (%) sensen mashhad ardehal sensen mashhad ardehal sensen mashhad ardehal 2015 19.8 18.4 89.3 90.8 42 49 average of 5 years 20.86 19.88 143.36 133.42 40.6 46.6 parameter location sensen mashhad ardehal total nitrogen (%) 0.09 0.28 phosphorous availability (mg/kg) 15.56 12.18 potassium availability (mg/kg) 245.6 209.7 ph 7.93 7.83 ec (ds.m-1) 2.82 0.89 organic carbon (%) 0.53 1.63 clay (%) 14.3 10.3 silt (%) 33.3 43.4 sandy (%) 52.4 46.3 table 2 soil analysis of the experimental site ghane et al. yield and physiological response of perilla 207 foliar-applied humic acid (95% purity) was obtained from humic strong company (70% w/w, ph 5.17, ec: 4.80 ms/cm) and added to the plots at 4 stages of the plant growth. compost fertilizer was prepared from barij essence company (including 1.5% nitrogen, 1.1% phosphorous, 0.9% potassium, 50% organic matter). compost fertilizer was used as a strip one week after plantation. perilla seeds were purchased from barij essence company, isfahan, iran. first of all, the seeds were germinated in the laboratory and then transplanted to the pots with 10 cm diameter and 15 cm height at the 4-leaf stage. the mycorrhizal-l ike fungus piriformospora indica was prepared in mycology laboratory of sari university, iran and then prepared biologic fertilizer was placed two weeks in incubator with 20-25˚c and 50 rpm under dark condition. before planting of perilla plants in the field, half of the plants in pots were inoculated with biologic fertilizers and then translocated to the field. each plot was 2×2 meter and had 4 furrows with 25 plant per plot. the recommended dose of chemical fertilizer (050-100 and 200 kg/ha) in the form of urea, triple super phosphate and sulfate potassium was applied to grow the crop. nitrogen was applied in three splits, the first along with phosphorus and potassium fertilizer at the time of soil preparation while the second part at the time of transplanting of plants and the third part at the flowering of crops. date of transplanting of both experimental sites was 20 of march. after plant growth, different traits were studied at the suitable stage. a furrow irrigation system was applied for both experimental sites. hand weeding of the experimental area was performed as required. in both experimental sites, after flowering five plants from each plot were harvested from two central rows and then plant height, fresh weight and dry weight of plants were measured. samples dried in an oven at 80°c for 24 hours and the mean of dry weight for each treatment at each replicate was determined. rosmarinic acid determination by hplc the dried seeds of perilla were pulverized (60 mesh) for 3 min using an hr 2860 coffee grinder (philips, drachten, netherlands), and each sample (1.0 g) extracted in 30 ml of 80% methanol for 6 h at room temperature in a shaking incubator. the supernatant was centrifuged at 3000g for 3 min and then filtered through a 0.45 lm syringe filter (whatman inc., maidstone, uk) prior to hplc analysis. for quantification, the peak areas of the isolated compounds were integrated from the hplc chromatogram at 330 nm using the dionex software. the stock solutions were prepared by dissolving in methanol to obtain a 1 mg/ml concentration. calibration curves were obtained with methanol at eight different concentrations (0.5, 1, 2, 5, 10, 25, 50, and 100 g/ml). all calibration curves had coefficients of linear correlation r2>0.998. stomatal conductance twenty days after plantation stomatal conductance was measured in the shade-enclosure with saturated light using promoter (model kr1301, korea tech) stomatal conductance (mmol h2o m-2 s-1) was measured at 12:00-14:00 hours on a clear, cloud-less day in fully expanded, healthy, turgid, three flat and uniform in color and size leaves of each samples (barbieri et al., 2012). dpph radical-scavenging activity this test was used for the determination of the free radical-scavenging activity of the extracts (ebrahimzadeh et al., 2008). dpph test was performed following the method proposed by ebrahimzadeh et al. (2008). three young fully developed leaves were selected from each replication. polyphenoloxidase (ppo) activity crude extract was prepared by homogenization of frozen plant sample in buffer medium. leaves of perilla plant which were stored at -20°c was used for the enzyme extraction. 10 g of the sample were cut quickly into thin slices and homogenized in 50 ml of 100 mm sodium phosphate buffer (ph 7.0) containing 1 mm ascorbic acid and 0.5% (w/v) polyvinylpyrrolidone for 5 min at 4°c. the homogenate was filtered through three layers of cheesecloth and then the filtrate was centrifuged at 5,000 x g for 15 min, and the supernatant was collected. enzymatic activity was assayed by determining the rate of increase in absorbance at 420 nm and 25˚c in a perkin-elmer lambda 15 uv/vis spectrophotometer (shimadzu corp., tokyo, japan). the reaction mixture contained 3.0 ml of catechol substrate, the solution freshly prepared in 0.05 m sodium phosphate buffer at ph 6.5 and a fixed quantity of ppo. the reference cuvette contained only the catechol substrate solution. the reaction was conducted at 25°c. the ppo activity was defined as a change of 0.001 in absorbance at the conditions of the assay (pizzocaro et al., 1993). catalase activity assay cat activity was measured by monitoring the h2o2 decomposition at 240 nm in 3 ml of reaction adv. hort. sci., 2019 33(2): 205-214 208 mixture containing 50 mmol/l phosphate buffer (ph 7.0), 15 mmol/l h2o2, 100 ml enzyme extract and 0.1% (v/v) triton x-100 (aebi, 1984). the activity was expressed in terms of mmol h2o2 reduced min/mg/protein. statistical analysis the data were tested for homogeneity and normality of residuals using the bartlett tests and kolmogorov-smirnov, respectively. a combined anova was used to compare treatments for 2 location using proc glm of sas 9.1 software. means were separated by application of lsd test when the f test proved significant at p≤0.05 and 0.01. 3. results and discussion combined statistical analysis of two studied location is presented in table 3. also, the analysis of variance of mashhad ardehal and sensen locations are shown separately in table 4 and 5. in mashhad ardehal location, the maximum plant yield (157.6 g/m2) was achieved in 100 kg/ha chemical fertilizer plus humic acid treatment and the minimum amount (90.68 g/m2) was produced in zero levels of chemical fertilizer and without application of organic treatment (fig. 1, table 3). while in sensen location, the maximum plant yield (126.9 g/m2) was achieved with the application of 50 kg/ha chemical fertilizer plus humic acid (fig. 1). between treatments of chemical × organic × biologic fertilizer interaction, the maximum plant yield (147.2 g/m2) was produced in 100 kg/ha along with humic acid and application of biologic fertilizer (table 6). differences between plant yield (147.2 g/m2 and 89.86 g/m2) showed the importance of fertilizer application in improving yield in this plant. in all of the chemical fertilizer treatments, the plant yield was increased with application of organic and biologic fertilizer, especially with organic fertilizer. so, the integrated fertilizer application was very positive. in the present investigation, application of organic fertilizer especially humic acid significantly increased plant fig. 1 effect of chemical and organic fertilizers on plant yield of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). *, ** significant at 5% and 1% probability levels, respectively. table 3 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen and mashhad ardehal in kashan regions s.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase location (l) 1 14012.64 ** 13.80 ** 8.87 ** 248.11 ** 224.60 ** 0.00357 * 0.000935 ** replication (location) 4 56.75 0.70 0.01 4.02 18.56 0.00047 0.000021 chemical fertilizer (c) 3 10188.25 ** 244.44 ** 13.92** 1504.81 ** 99.75 ** 0.03396 ** 0.010834 ** l×c 3 1899.70 ** 27.75 ** 2.53** 184.57 ** 19.74 0.01396 ** 0.000478 ** ea 12 28.48 1.18 0.04 5.74 44.51 0.00063 0.000072 organic fertilizer (o) 2 1435.58 ** 27.93 ** 1.69** 76.66 ** 75.18 * 0.00178 0.000454** biologic fertilizer (b) 1 308.17 ** 8.41 ** 0.53** 213.84** 0.24 0.00003 0.000146 * c × o 6 73.04 4.75 ** 0.06* 8.46 47.80 * 0.00170 * 0.000217 ** c × b 3 130.19 * 1.05 0.06 2.01 2.82 0.00257 ** 0.000022 o × b 2 236.97 ** 1.31 0.09* 2.47 19.55 0.00014 0.000008 l × o 2 71.26 4.79 ** 0.17** 13.49 2.83 0.00253 * 0.000275 ** l × b 1 88.64 4.42 * 0.22** 1.92 22.75 0.00026 0.000003 l × c × o 6 83.62 * 2.89 ** 0.03 10.55 5.98 0.00244 ** 0.000180 ** l × c × b 3 18.09 0.16 0.001 3.44 0.20 0.00178 * 0.000009 l × o × b 2 95.96 0.77 0.11* 9.61 0.13 0.00075 0.000010 c × o × b 6 191.38 ** 2.38 * 0.11 ** 2.76 18.59 0.00068 0.000004 l × c × o × b 6 66.33 1.42 0.04 3.05 6.50 0.00009 0.000006 eb 80 37.44 0.79 0.02 5.84 19.15 0.00059 0.0000224 cv (%) 5.11 4.69 6.66 3.64 2.42 5.48 3.06 ghane et al. yield and physiological response of perilla 209 tion in perilla. the highest rosmarinic acid (23.48 mg.g dm) was acheved in 100 kg/ha chemical fertilizer plus humic acid and without application of biologic fertilizer (table 6). similarly, with plant yield, rosmarinic acid concentration was affected by fertilizer application. these results are in agreement with hendawy et al. (2015) on mentha piperita. they reported foliar application of humic acid increased growth characteristics and finally possessed the best oil percentage and yield in mint plant. zaghloul et al. (2009) reported also the application of humic acid increased oil content of thuja orientalis. at both of studied location and with/without application of biologic fertilizer, the maximum rosmarinic acid yield was achieved by using humic acid. the maximum rosmarinic acid yield (2.967 g/m2) was observed in mashhad ardehal and application of yield in all levels of chemical fertilizer. however, there was not clear trend about application of biologic fertilizer. previously, researchers (ciarkowska et al., 2017) had highlighted the important role of humic acid, especially in root formation. rosmarinic acid concentrations in perilla were significantly affected by location, chemical and organic fertilization (table 3) and, at both locations and all chemical fertilization levels, the humic acid application increased rosmarinic acid content in perilla. this fact exhibited the importance of humic acid on rosmarinic acid content in perilla. the maximum rosmarinic acid (25.01 mg.g dm) was achieved in mashhad ardehal along with using 100 kg/ha chemical fertilizer and application of humic acid (fig. 2). application of organic fertilizer especially humic acid with/without biologic fertilizer in all chemical fertilizer levels increased rosmarinic acid concentras.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase replication 2 61.95 0.46 0.01 5.24 24.06 0.0006 0.000040 chemical fertilizer (c) 3 10082.10 ** 195.60 ** 13.42** 1256.35 ** 540.52 ** 0.0411 ** 0.00749 ** ea 6 10.21 1.61 0.04 5.48 58.46 ** 0.0003 0.00013 ** organic fertilizer(o) 2 1049.35 ** 27.60 ** 1.46** 42.86 ** 581.28 ** 0.0012 0.00071** biologic fertilizer (b) 1 363.69 ** 12.52 ** 0.71** 87.60 ** 13.86 0.0002 0.00005 c × o 6 50.03 * 3.84 ** 0.07** 13.03 ** 11.17 0.0006 0.00037 ** c × b 3 42.09 0.63 0.03 2.73 1.39 0.0027 ** 0.00001 o × b 2 240.40 ** 1.60 0.18** 1.28 8.78 0.0006 0.00001 c × o × b 6 89.83 ** 1.67 0.08 ** 5.60 5.38 0.0009 * 0.000005 eb 40 21.24 0.86 0.02 3.16 14.71 0.0004 0.00003 cv (%) 3.56 4.79 5.65 2.63 2.32 4.48 3.64 s.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase replication 2 51.56 0.96 0.01 2.80 13.07 0.00030 0.000003 chemical fertilizer (c) 3 2005.86 ** 76.59 ** 3.03** 433.02 ** 326.18 ** 0.00677 ** 0.003819 ** ea 6 46.75 0.76 0.04 5.99 30.56 0.00090 0.000009 organic fertilizer(o) 2 457.49 ** 5.13 ** 0.39** 47.28 ** 521.70 ** 0.00307 * 0.000019 biologic fertilizer (b) 1 33.13 0.31 0.03 128.16** 9.14 0.00005 0.000095 ** c × o 6 106.63 3.80 ** 0.01 5.98 36.55 0.00351 ** 0.000025 c × b 3 106.20 0.58 0.02 2.72 1.62 0.00156 0.000019 o × b 2 92.53 0.48 0.02 10.79 10.91 0.00030 0.000006 c × o × b 6 167.87 * 2.15 * 0.06 ** 0.21 19.72 0.00065 0.000006 eb 40 53.64 0.74 0.02 8.51 23.59 0.00077 0.000012 cv (%) 6.67 4.59 7.88 4.48 2.98 6.37 2.28 table 4 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in mashhad ardehal in kashan regions *, ** significant at 5% and 1% probability levels, respectively. *, ** significant at 5% and 1% probability levels, respectively. table 5 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen in kashan regions 210 adv. hort. sci., 2019 33(2): 205-214 humic acid beyond biologic fertilizer (fig. 3). also, between chemical × organic × biologic fertilizer treatments, the maximum rosmarinic acid yield (3.432 g/m2) was observed in 100 kg/ha along with humic acid and biologic fertilizer application (table 6). integrated application of chemical, organic and biological fertilizers improved soil physical and chemical properties and resulted in the increase in availability of nutrients and ultimately the yield and quality of plants. darzi et al. (2007) reported the application of mycorrhiza and vermicompost and phosphate solubilizing biologic fertilizers determined increased essential oil yield in fennel. stomatal conductance stomatal conductance is one of the most important factor in determining the photosynthesis amount. the study of stomatal conductance between location and chemical fertilizer treatments showed the highest stomatal conductance (75.88 mmol h2o m-2 s-1) in mashhad ardehal with application of 100 kg/ha chemical fertilizer (fig. 4). at both of studied location especially in mashhad ardehal, application of chemical fertilizer increased the stomatal conductable 6 means comparison of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen and mashhad ardehal in kashan regions chemical fertilizer organic fertilizer biologic fertilizer (inoculation) plant yield (g/m2) rosmarinic acid content (mg/g d.m.) rosmarinic acid yield (g/m2) 0 kg/ha control (-) 93.12 lm 14.40 lm 1.34 l (+) 89.86 m 13.94 m 1.25 l compost (-) 99.40 kl 15.31 l 1.52 k (+) 103.40 jk 17.29 jk 1.67 ij humic acid (-) 99.91 kl 16.44 k 1.64 jk (+) 95.17 lm 17.31 jk 1.65 jk 50 kg/ha control (-) 115.70 gh 19.52 def 2.25 gh (+) 128.40 de 19.38 defg 2.54 ef compost (-) 129.50 de 19.56 def 2.52 ef (+) 126.90 de 20.11 ced 2.54 e humic acid (-) 127.90 de 18.92 fgh 2.42 efg (+) 141.20 ab 20.37 cd 2.89 bc 100 kg/ha control (-) 131.40 cd 21.05 bc 2.79 d (+) 126.70 de 21.58 b 2.75 d compost (-) 138.00 bc 21.7 b 3.02 bc (+) 137.70 bc 21.63 b 3.00 bc humic acid (-) 131.70 cd 23.48 a 3.11 b (+) 147.20 a 23.02 a 3.43 a 200 kg/ha control (-) 107.20 ij 17.52 ij 1.88 i (+) 113.50 hi 18.4 ghi 2.09 h compost (-) 126.20 def 18.2 hij 2.30 g (+) 119.40 fgh 19.13 egh 2.29 g humic acid (-) 116.90 gh 19.33 efg 2.27 gh (+) 122.70 efg 19.07 fgh 2.35 fg different letters in each column denote a significant differences at p≤0.05 (lsd test). fig. 2 effect of chemical and organic fertilizer on rosmarinic acid content of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 3 effect of organic and biologic fertilizer on rosmarinic acid yield of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). ghane et al. yield and physiological response of perilla 211 tance of perilla leaves. increasing of stomatal conductance increases the photosynthesis activities and finally improved the plant yield (atteya, 2003). the results of stomatal conductance are agreement with result of plant yield. this result showed the increasing of stomatal conductance can be useful to increasing of plant yield especially with 100 kg/ha application of chemical fertilizer. the highest stomatal conductance was obtained with application of compost fertilizer (67.67 mmol h2o m-2 s-1) (table 7). organic fertilizers can provide better moisture conditions by increasing the absorption and preservation of water and the availability of water and food, and this can contribute to the favorable conditions for photosynthesis in the plant. if there is enough stomatal conductance, co2 gas will enter the stomata more easily and sufficient photosynthesis will be performed. also means comparison showed inoculation with mycorrhiza improved stomatal conductance as compared to without inoculation (table 7). the physiological effects of mycorrhizal fungi symbiosis include aboveground modifications of water relations and physiological status in terms of leaf water potential, relative water content, stomatal conductance, co2 assimilation, and efficiency of photosystem ii as compared to non-mycorrhizal plants (barzana et al., 2012). mycorrhizal fungi increase the contact surface with soil and moisture around the plant roots by 10 to 1,000 times, thus increasing the plant’s ability to use the resources in its surroundings (sharma, 2002). dpph test many researchers have used dpph test to express the antioxidant status of plants (dasgupta and de, 2007; sahu et al., 2013). the results showed the antioxidant activity of perilla plant in mashhad ardehal (165.29 ug/ml) was higher than sensen (162.79 ug/ml) location (table 7). perilla is very sensitive to free radicals productions. in a study in korea on different species of perilla, different species for dpph test showed a significant difference (choi et al., 2002). increasing levels of chemical fertilizer reduced the plant’s efficiency in inhibiting free radicals. the highest amount of dpph test was obtained (169.47 μg/ml) under control condition and the lowest (55.85 μg/ml) was obtained from the highest level of fertilizer application (200 kg/ha) (table 7). omar et al., (2012) reported the antioxidant activity determined using the dpph was high with the application of organic fertilizer compared to chemical fertilizer in cassava tubers. organic fertilizers, especially compost (277.17 μg/ml), increased the antioxidant activity and inhibited free radicals (table 7). uthairatanakij et al. (2017) reported an increase in antioxidant activity and inhibition of free radicals due to the application of organic fertilizers. organic fertilizers with plant sources activated defense mechanisms against pests, diseases and other tensions (brandt and molgaard, 2001). catalase activity the highest catalase activity (0.5332 nmol h2o2/min) was observed in mashhad ardehal region with the application of 100 kg/ha fertilizer and withfig. 4 effect of chemical fertilizer on stomatal conductance of peril la in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). treatments stomatal conductance (mmol h 2 o m−2s−1) dpph (ug/ml) polyphenol oxidase (μmol/min) location mashhad ardehal 67.61 a 165.29 a 0.156 a sensen 64.98 b 162.79 b 0.152 b chemical fertilizer 0 kg/ha 57. 36 d 169.48 a 0.130 c 50 kg/ha 65.99 c 161.73 c 0.152 b 100 kg/ha 72.05 a 166.41 b 0.167 a 200 kg/ha 69.80 b 158.55 d 0.168 a organic fertilizer control 66.03 b 158.54 b 0.152 b compost 67.68 a 167.27 a 0.158 a humic acid 65.19 b 166.31 a 0.153 b biologic fertilizer (inoculation) (-) 65.08 a 164.08 a 0.153 b (+) 67.52 b 164.00 a 0.155 a table 1 weather characteristics in mashhad ardehal and sensen different letters in each column denote a significant differences at p≤0.05 (lsd test). adv. hort. sci., 2019 33(2): 205-214 212 out application of biologic fertilizer. the lowest amount was obtained from mashhad ardeal area without using of chemical and biologic fertilizer. also, in the sansen location, the highest catalase activity was obtained from 200 kg/ha chemical fertilizer and biologic fertilizer application (fig. 5). among the chemical and organic fertilizer treatments in two studied locations, the highest catalase activity was obtained from 100 kg/ha of chemical fertilizer with compost application in mashhad ardehal region (0.5182 nmol h2o2/ min) and the lowest amount (0.338 nmol h2o2/ min) was obtained without using of chemical and organic fertilizer control in mashhad ardehal location (fig. 6). logan et al. (1999) stated that nitrogen had a significant effect on the activity of enzymes involved in photosynthesis, such as ribulose-1,5-bisphosphate. in another study the researchers reported increased nitrogen levels caused to production of antioxidant enzymes like apx, sod, cat, and pod in the populus yunnanensis plants (lin et al., 2012). our result indicated that sources of fertilizer had a significant influence on the level of catalase activity in field grown perilla. polyphenol oxidase activity among the treatments of chemical and organic fertilizer in different locations, similar to catalase activity, the highest polyphenol oxidase activity was obtained from 100 kg/ha with compost application in mashhad ardeal location (0.193 μmol/min) and the lowest amount (0.129 μmol/min) was obtained from without application of chemical and organic fertilizer in mashhad ardehal area (fig. 7). at all levels of chemical fertilizer in both locations, application of organic fertilizers increased the activity of polyphenol oxidase activity, and in most cases, organic matter compost was superior to humic acid. also, the application of biologic fertilizer led to an increase in polyphenol oxidase activity (table 7). similar to our results, the application of biologic fertilizer increased the amount of polyphenol oxidase activity in triticale (kheirizadeh-arough et al., 2016). 4. conclusions in the present experiment, the results showed that proper nutritional management of perilla medicinal plant has a special role in improving quantitative and qualitative traits. among individual fertilizer treatments, 50 and 100 kg/ha of fertilizer and among organic fertilizers, humic acid played a more effective role in improving the studied indices, but the application of biologic fertilizer separately did not have a very significant effect on this plant. integration with other fertilizers has been shown to be more effective. mashhad ardehal area has better conditions for cultivating perilla due to its soil characteristics and climatic characteristics. also, the combined application of different fertilizer sources in comparison with the single application of each of them in both studied regions significantly improved the growth characteristics, as well as essential oil yield and biochemical fig. 5 effect of chemical and biologic fertilizer on catalase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 6 effect of chemical and organic fertilizer on catalase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 7 effect of chemical and organic fertilizer on polyphenol oxidase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). ghane et al. yield and physiological response of perilla 213 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horticulturae, 3(1): 21. vessey j.k., 2003 plant growth promoting rhizobacteria as biofertilizer. plant soil, 255: 271-286. zaghloul s.m., el-quesni f.e.m., mazhar a.a.m., 2009 influence of potassium humate on growth and chemical constituents of thuja orientalis l. seedlings. ozean j. applied sci., 2: 73-78. zemanek p., 2011 evaluation of compost influence on soil water retention. acta univ. agric. silvic. mendel. brun., 59 (3): 227-232. impaginato 133 adv. hort. sci., 2020 34(2): 133­145 doi: 10.13128/ahsc­7491 comparative postharvest responses of carnation and chrysanthemum to syn­ thesized silver nanoparticles (agnps) n. rashidiani 1, f. nazari 1 (*), t. javadi 1, s. samadi 2 1 department of horticultural science, college of agriculture, university of kurdistan, sanandaj, iran. 2 department of chemistry, faculty of science, univesity of kurdistan, sanandaj, iran. key words: cut flowers, ethylene, microbial growth, silver nanoparticles, vase life, water relations. abstract: carnation (dianthus caryophyllus l.) and chrysanthemum [dendranthema grandiflorum (ramat.) kitam.] cut flowers are among the most important commodities that dominate flower markets throughout the world. two major problems in the transportation and marketing of these flowers are their relatively short vase life and the rapid decline of their aesthetic value. in this respect, the current study investigates the effects of silver nanoparticles (agnps) on ethylene­sensitive (carnation) and ethylene­insensitive (chrysanthe­ mum) cut flowers. specifically, this research examines their morpho­physico­ chemical characteristics, antioxidant enzyme activities and vase life. here, the agnps were synthesized by chemical methods and then applied on both flow­ ers by a pulsing method. the treatments involved two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water), and the duration of exposure lasted for 24 h. then, the flower stems were placed in an aqueous sucrose solution (4%) until the end of the experiment. all traits, except the vase life, were evaluated after 0, 3, 6 and 9 d following the treatments during the vase period. during this time, the control groups of both flowers showed con­ siderable amounts of decrease in the relative fresh weight (rfw), vase solution uptake (vsu), flower diameter, membrane stability index (msi) and total solu­ ble carbohydrate (tsc). meanwhile, there were increases in hydrogen peroxide content (h2o2) and peroxidase (pod) activity. the bacterial population of the stem end and total soluble protein (tsp) increased in carnation petals, but decreased in chrysanthemum petals. the activity of superoxide dismutase (sod) dropped in carnation petals, whereas it rose in chrysanthemum petals. using agnps at concentrations of 0.08 and 0.04 g l­1 can optimally extend the vase life of carnation and chrysanthemum, respectively. 1. introduction nowadays, the floriculture industry is one of the most profitable sec­ tors in horticulture. its financial turnover in all respects amounts to over 300 billion dollars, and one­third of which is related to cut flowers (chandler and sanchez, 2012).consumer demand for cut flowers and (*) corresponding author: f.nazari@uok.ac.ir citation: rashidiani n., nazari f., javadi t., samadi s., 2020 ­ comparative postharvest responses of car‐ nation and chrysanthemum to synthesized silver nanoparticles (agnps). ­ adv. hort. sci., 34(2): 133­145 copyright: © 2020 rashidiani n., nazari f., javadi t., samadi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 november 2019 accepted for publication 1 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 133­145 134 ornamental plants are increasing, and the value of production is inevitably becoming higher. in this regard, flower quality is one of the most important indices in the sale and marketing of cut flowers. this makes flower quality an important factor in attracting customers over and over again (scariot et al., 2014). therefore, the advancement of post­harvest pro­ grams is a definite requirement within the cut flower market. even after their separation from mother plants, cut flowers continue to be metabolically active and proceed with all of their vital processes by consuming the available food in their tissues. carbohydrates, proteins and fat can be used by cut flowers to meet their metabolic demands. the increase in vase life and the delay in flower senescence can be achieved by upholding the normal rate of water uptake, pre­ venting the depletion of carbohydrate storage and limiting the exposure of flowers to ethylene (halevy and mayak, 1981). water balance is an essential factor in determin­ ing the quality and vase life of flowers. it should be maintained between water uptake and transpiration (lu et al., 2010). water uptake can be reduced due to the occlusion phenomenon generated by bacterial accumulation, physiological factors or air embolism (damunupola and joyce, 2008). post­harvest senes­ cence occurs within a few days, and is a major limita­ tion in the marketing of cut carnation and chrysan­ themum flowers. stem end blockage is one of the important factors in early wilting of leaves and inflo­ rescences in some cut flowers. van doorn and vaslier (2002) reported stem blockage is a major factor caus­ ing severe leaf wilting of chrysanthemum. the in­rolling of petal margin and wilting of whole petals in senescence process in some cut flowers (e.g. carnation), is associated with the self­regulation of ethylene production (yang and hoffman, 1984), as a hormone, implying that the flowers are sensitive to ethylene. on the other hand, there are flowers such as chrysanthemum, pericallis and narcissus that initi­ ate senescence without being influenced by ethyl­ ene, and thus are considered insensitive to ethylene (dole and wilkins, 2005; jones 2013). therefore, the short vase life and early wilting of inflorescences of carnation and chrysanthemum may be due to sensi­ tivity of ethylene and stem end blockage, respective­ ly. in fact, in ethylene­sensitive flowers, ethylene is a necessary requirement for the initiation and suste­ nance of senescence­based processes. the mecha­ nisms by which ethylene can stimulate senescence involve changing the cell structure and increasing the concentration of reactive oxygen species (ross) such as superoxide radicals, hydroxyl radicals and hydro­ gen peroxide (abeles et al., 1992). studies have also shown that physiological changes occur during senescence. these include chlorophyll decomposition, decreased activity of antioxidant enzymes, increased productions of ethyl­ ene and ross, along with membrane damage to the cells of cut flowers (prochazkova and wilhelmova, 2007). advancements in nanotechnology, especially the development of silver nanoparticles (agnps), have led to a wide range of nanocomposites with antimi­ crobial properties. the high surface­to­volume ratio of these particles makes them biologically more active and increases their contact with fungi and bac­ teria (chau et al., 2007). agnps are becoming popular in the flower industry for their ability to inhibit the synthesis of ethylene and delay the senescence in cli­ macteric flowers (kim et al., 2005). in addition, agnps tend to regulate the stomatal aperture, main­ tain the chlorophyll content, preserve the relative fresh weight (rfw) and membrane stability index (msi), reduce the transpiration rate, weight loss, hydrogen peroxide(h2o2), malondialdehyde (mda) and ross, and increase the activity of antioxidant enzymes along with their effects on the vase life of different cut flowers such as gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2017), rose (lu et al., 2010; nazemi rafi and ramezanian, 2013; hassan et al., 2014), chrysanthemum (carrillo­ lópez et al., 2016), carnation (naing et al., 2017; lin et al., 2019 b), gladiolus (li et al., 2017), peony (zhao et al., 2018) and cut gardenia foliage (lin et al., 2019 a). the application of agnps in vase solution of carna­ tion alleviated vascular occlusion by inhibiting bacter­ ial colonisation and biofilm formation on stem­end cut surfaces and in the xylem vessels (lin et al., 2019 b). maity et al. (2019) also showed that the piper betle silver nanoparticles (pbsnps) in vase solution of gladiolus have played an important role for scaveng­ ing ross by enhancing antioxidant enzyme activities that led to decrease in mda and increased the msi. nanosponges are new nano­sized colloidal carri­ ers synthesized from ß­cyclodextrins that have been prepared for delivering preservative and anti­ethyl­ ene compounds (devecchi et al., 2009). devecchi et al. (2009) evaluated the effect of nanosponges including anti­ethylene molecules, such as 1­methyl­ cyclopropene (1­mcp), 1­methylcyclopentene (1­ mcpt), 2,5­norbornadiene and agno3 on vase life of carnation flowers. they concluded that, 1­mcp­ rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowers 135 nanosponge complex outperformed the other treat­ ments in extending the vase life. in addition, seglie et al. (2011) reported that 1­mcp in cyclodextrin­based nanosponges improved the vase life of carnation cut flowers. nonetheless, no report has so far described com­ parisons between the responses of ethylene­insensi­ tive and ethylene­sensitive flowers to agnps. therefore, the current research is aimed an evaluat­ ing how the cut flowers of carnation (highly sensitive to ethylene) and chrysanthemum (insensitive or slightly sensitive) respond to the application of agnps by the pulsing method. comparisons are made between the two types of cut flowers by measuring their morphological, physiological and biochemical properties, as well as their enzymatic activities. 2. materials and methods plant materials and application of treatments the cut flowers of carnation (dianthus caryophyl‐ lus cv. yellow viana) and chrysanthemum (dendranthema grandiflorum cv. boris becker sunny) were harvested at their commercial harvesting stage from a soilless­cultured greenhouse. they were taken to the laboratory and the basal ends of the stems were immediately cut to reduce the stem length to 40 cm. apart from 3­4 leaves on the top of each stem, all other leaves were removed. then, agnps were applied at two concentrations (0.04 and 0.08 g l­1) by the pulsing method for 24 h. the flowering stems were individually put in a bottle vase contain­ ing 300 ml deionized water. this also contained 4% (w/v) sucrose until the end of the experiment. to prevent the evaporation and contamination of the vase solution, the vase opening was covered with aluminum foil. all traits, except for vase life, were evaluated after 0, 3, 6 and 9 d following the applica­ tion of treatments. the ph of vase solution at the first day (d 0) was 7.82 and 8.76 for carnation and chrysanthemum, respectively. on d 9 of the vase period the ph of vase solution was 3.95 for carnation and 4.06 for chrysanthemum. the pulsing treatments and vase life were evaluated at 23±2°c, 50%±10% rh and 12 h of 15­20µmol m­2s­1irradiance from cool­ white fluorescence lamps. synthesis of agnps to a solution of 0.265 mm (0.045 g) of silver nitrate in 100 ml distilled water, 10 ml of a trisodium citrate aqueous solution (1%) was added slowly at room temperature. after 10 minutes, 0.2 ml of ascorbic acid (0.005 m) was added to the mixture of reaction and stirred for 1 h until a yellow­green agnps colloid was formed (tavallali and pouresmaeil, 2012). the surface morphology of agnps was indicat­ ed by scanning electron microscopy (sem) (tscan, czech republic) and showed in figure 1, it is exhibited that agnps have a size of about 8­80 nm. measurement of flower and stem diameter the flower diameter was measured in two direc­ tions, and the stem diameter was measured in three parts (i.e. under peduncle, middle, and end of the stem). these measurements were repeated every three days and the average of values were reported. measurement of relative fresh weight the relative fresh weight (rfw) of flowering stems in both types of cut flower was calculated every three days by the following formula: rfw (%) = (wt/wt0)×100 fig. 1 ­ the sem image of agnps. adv. hort. sci., 2020 34(2): 133­145 136 where wt is the weight of the stem (g) at t = d 0, 3, 6, and 9,while the wt0is the weight of the same stem (g) at t = d 0 (he et al., 2006). measurement of vase life the vase life of both types of cut flower was mea­ sured by counting the number of days from the beginning of the experiment until 50% of the flower had wilted. in carnations, the occurrence of in­rolling and browning petals by more than one­third was considered as the end of the vase life (naing et al., 2017). in chrysanthemum, however, the vase life ended when 50% of petals had wilted and the leaves had yellowed (carrillo­lópez et al., 2016). measurement of vase solution uptake the weight of the vase with and without the flower shoots was recorded daily, and the following formula was used for calculating the vase solution uptake (vsu): vsu (mg g­1 stem f. w.) = (st−1­st); where st is the weight of vase solution (g) at t = d 0, 3, 6 and 9, st−1 is the weight of vase solution (g) on the previous day (he et al., 2006; lu et al., 2010). measurement of membrane stability index to measure the membrane stability index (msi) of petals, this method involved the preparation of petal discs (measuring 1 cm in diameter) which were placed in falcons containing 20 ml distilled water. after this, a series of falcons were placed in a warm bath (40°c) for 30 min and their electrical conductivi­ ty (ec) was read by a conductivity meter (c1) after the falcons had cooled down to 25°c. then, the sec­ ond series of falcons was placed in a warm bath of 100°c for 20 min and their ec was read after having cooled down to 25°c (c2). in the end, the msi was calculated using the following equation (sairam et al., 2002): msi= [1­(c1/c2)] 100 c1 = ec after exposure to 40°c and c2= ec after expo­ sure to 100°c. measurement of bacterial population of stem end to measure the bacterial population on the stem end, one gram of the stem end was homogenized and diluted with peptone water until a concentration of 10­3was reached. subsequently, 1 ml of this solu­ tion was transferred to petri dishes and then a vol­ ume of 10 ml sterilized plate count agar medium was added to each petri dish. these were slowly mixed for 5 to 10 s. the cultured petri dishes were then kept in an incubator at 32°c for 2 d and, after count­ ing the bacterial colonies, the results were reported as log cfu g­1 (balestra et al., 2005; liu et al., 2009). measurement of total soluble protein according to the bradford method (1976), 0.5 g of petal tissue was powdered with liquid nitrogen, and then 0.25 g of polyvinylpyrrolidone (pvp) was added to the solution when stirring the 1.5 ml of potassium phosphate buffer containing sodium metabisulfite (0.019 g per 100 ml buffer). the homogenized sam­ ples were centrifuged (hettch, germany) at 4°c for 20 min at 15,000 g. then, 50 μl of supernatant was mixed with 950 μl of bradford solution and, after 15 min, the light absorption was read at 595 nm by a spectrophotometer (unico 2100, usa). measurement of peroxidase activity to measure the peroxidase (pod) enzyme activity, according to a method reported by hemeda and klein (1990), first 400 μl of 50 mm potassium phos­ phate buffer (ph 7) was mixed with 40 μl of 1% gly­ col and 40 μl of 0.3% hydrogen peroxide in an ice bed. then immediately, 65 μl of protein extract was added to the mentioned composition. the changes in light absorption were read at 120 nm by a spec­ trophotometer within a wavelength of 470 nm. measurement of superoxide dismutase activity the activity of superoxide dismutase enzyme (sod) was measured by the beyer and fridovich (1987) method. according to this method, the solu­ tion used for the reaction was prepared by mixing 25 ml of 50 mm phosphate buffer (ph 7) with 0.0035 g l­methionine (9.9 mm), 0.004 g nbt (57 μm) and 7.5 μl triton x­100. one ml of the reaction mixture was blended with 10 μm riboflavin and 20 μl of protein extract. the mixture was placed on a shaker at a dis­ tance of 30 cm from a 20­watt fluorescent lamp for 10 min. then, the light absorption was measured at 560 nm by a spectrophotometer. measurement of total soluble carbohydrate to measure total soluble carbohydrates (tsc), 0.5 g of petal tissue was crushed using liquid nitrogen along with 5 ml of 95% ethanol which helped obtain uniform extracts. the supernatant extract was cen­ trifuged for 10 min at 3,500 g. then, 1 ml of this extract was combined with 3 ml of anthrone before being transferred to a warm bath of 100°c. the heat caused the appearance of a colored phase after 10 min. subsequently, the samples were removed from the warm bath and were allowed to cool down at room temperature. their light absorption was read at 625 nm. the tsc content of petals was determined by creating a standard curve using standard glucose. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 137 the results were expressed as mg g­1 f.w. (irigoyen et al., 1992). measurement of hydrogen peroxide in order to measure the amount of hydrogen per­ oxide (h2o2), the method was similar to the one used by alexieva et al. (2001). accordingly, 0.2 g of petal tissue was completely ground with 5 ml trichloroacetic acid (tca). the extract was obtained and centrifuged at 10,000 g for 5 min. then, 250 μl of the supernatant was mixed with 250 μl of 100 mm phosphate­potassium buffer (ph7) and 500 μl of 1 m of potassium iodide (ki). the absorbance of each sample was read by a spectrophotometer within a wavelength of 390 nm. statistical analysis this research was conducted as a factorial based on a completely randomized design (crd), it had three factors that the first factor was the type of flower at 2 levels (carnation and chrysanthemum), second factor was agnps at 3 levels (0.04 and 0.08 g l­1 along with deionized water) and the third factor was the sampling time at 4 levels (0, 3, 6 and 9 d of the vase period) with 3 replications, each of which included 8cut flower stems. for measuring the vase life, two factors of the type of flower and the sam­ pling time were not considered and its design was as a crd with three treatments (agnps at 0.04 and 0.08 g l­1 along with deionized water). data were analyzed using sas software and a comparison of mean values was made by the lsd test at 5% probability level. 3. results relative fresh weight based on the comparison of mean values, the rel­ ative fresh weight (rfw) of both cut flowers gradual­ ly decreased in the control treatment during the vase period. a faster rate of this decline was observed in carnations, as compared to the chrysanthemum. from the initial days to the ninth day, the decrease of rfw in carnations was about twice as much as the decrease in chrysanthemum. the application of0.04 and 0.08 g l­1 agnps caused the percentage of rfw to remain relatively constant in both cut flowers until the third days, but then the rfw gradually decreased. furthermore, treating the cut flowers with agnps caused a better preservation of their rfw from d 3 to d 9 of the vase period, as compared to the control, but the difference between the two concentrations was no significant. on d 9 of the vase fig. 3 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on vsu of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. period, the rfw of chrysanthemum was heavier than that of carnations, and the differences were signifi­ cant (fig. 2). vase solution uptake there was an increase in the rate of vase solution uptake (vsu) by both flowers until the third day of the vase period, by which time the vsu in carnations was almost twice as much as that in chrysanthemum. from the third day onward, the vsu in both flowers decreased. the application of agnps caused an increase in the vsu in both flowers compared to the control. on d 9 of the vase period, the vsu in carna­ tions had been significantly affected by both concen­ trations of agnps, whereas the chrysanthemum was only affected by 0.08 g l­1 agnps to a substantial degree (fig. 3). fig. 2 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on rfw of two cut flowers during the vase period. values represent means ± standard error (n=3). least significant differen­ ce (lsd) at p≤0.05 was used for means comparison. 138 adv. hort. sci., 2020 34(2): 133­145 bacterial population of stem end the bacterial population of the stem end in carna­ tions increased throughout the vase period. in chrysanthemum, however, the same trend continued until the third days before declining. both concentra­ tions of agnps reduced the bacterial population of the stem end in chrysanthemum during the vase period, making it significantly different when com­ pared to the control on d 9. on the other hand, this trait in carnations was only affected by 0.08 g l­1 agnps to make a significant difference compared to the control. in general, in both flowers, 0.08 g l­1 agnps was more effective than the 0.04 g l­1 in reducing the bacterial population of the stem end. the highest of this trait among both cut flowers was observed in the control group of chrysanthemum on d 3, whereas the lowest of this value was caused by 0.08 g l­1 agnps and measured on d 9 (fig. 4). changes in the stem and flower diameter the stem diameter was one of the traits which was not significantly affected by agnps in both flow­ ers. even a comparison between d 0 and 9 of the control groups showed no significant difference. despite the fact that 0.08 g l­1 agnps in both flowers caused the stem diameter to become thicker in com­ parison with the stems of plants treated by 0.04 g l­1 and the control, there were no significant differences between these groups (data not shown). from d 3 onward, the diameter of both flowers decreased. however, it occurred more dramatically in carnations which shrank twice as much as the chrysanthemum. the effect of agnps on changing the flower diameter was significant only in carnations, during their vase period. although agnps increased the diameter of flowers in both species, as compared with their respective control groups, the diameters had finally decreased by d 9 of the vase period. on the mentioned day, these treatments had made a significant difference in carnations only, as compared to the carnations control group. in general, the biggest flower diameter was observed in chrysanthe­ mum on d 0 after being treated with 0.04 g l­1 agnps, and the smallest of all diameters was obtained in the control group of carnations on d 9 (data not shown). membrane stability index the decline in values of the membrane stability index (msi) occurred in both flowers during the vase period, and no significant differences were observed between the two flowers in this respect. in the begin­ ning of the vase period (d 0), the msi in carnations was higher than in chrysanthemum, but the value of this trait decreased in both flowers through time. on d 9, this decrease was twice as much in carnation when compared to chrysanthemum. in both flowers, agnps caused the msi to increase during the vase period, as compared to the control, but chrysanthe­ mum responded more strongly to the treatment than the extent to which carnations did. in response to agnps, chrysanthemum showed a gradual increase in the msi value ­ so much so that it became slightly ­1 agnps can be a successful treatment for increasing the msi in carnations. in contrast, however, the con­ centration of 0.04 g l­1 worked optimally on chrysan­ themum. in general, the highest value of msi was observed in carnations on d 0 when treated with 0.08 g l­1 agnps, whereas the lowest value occurred in the control on d 9 (fig. 5). total soluble protein a comparison of the mean values showed that the fig. 4 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on bacterial population of the stem end of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 5 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on msi of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 139 content of total soluble protein (tsp) in chrysanthe­ mum petals was about 4 times higher than that of carnation petals on d 0. in carnations, the tsp con­ tent of the petals increased during the vase period. meanwhile, chrysanthemum underwent a different pattern of change, whereby the content of tsp increased from d 0 to d 3, but then decreased sharply until d 6, and continued to decline at a gradual rate until d 9. the agnps increased the tsp in both flow­ ers during the vase period, as compared to the con­ trol. generally, the highest value of tsp was observed in chrysanthemum on d 3 in the treatment group of 0.04 g l­1 agnps, whereas the lowest value was observed in the control on d 9 (fig. 6). total soluble carbohydrate the total soluble carbohydrate (tsc) content in carnation petals was more than the content in chrysanthemum petals. in the control groups of both flowers, the content of tsc decreased. the use of agnps in both flowers did not cause a significant dif­ ference in tsc on d 0, as compared with the control, but thereafter the difference gradually became signif­ icant until d9. applying the agnp at 0.08 g l­1 on car­ nations and at 0.04 g l­1 on chrysanthemum signifi­ cantly increased the tsc in comparison with their respective control groups. in general, carnations responded more strongly to the use of agnps, and the increase in their tsc content was much greater than in the case of chrysanthemum. the highest tsc content was measured in carnations on d 3 of the vase period after being treated with 0.08 g l­1 agnps. the lowest content was measured in the control group of chrysanthemum on d 9 (fig. 7). hydrogen peroxide content the petals of both flowers initially contained simi­ lar amounts of hydrogen peroxide (h2o2) which grad­ ually increased during the vase period. the rate of this increase was higher in carnations compared to the chrysanthemum. when comparing the h2o2 con­ tent between d 0 and d 9, its increase in carnations was about three times more than the increase mea­ sured in chrysanthemum. the application of agnps on both flowers reduced the h2o2 content in their petals, as compared with the control, but the increase was not prevented completely. on the last day of the vase period, the application of agnps on carnations led to a significant difference in h2o2 con­ tent when compared with the control. however, this was not the case in chrysanthemum. in general, the highest amount of h2o2 was measured in the control group of carnations on d 9 of the vase period. its low­ est amount was obtained in chrysanthemum on d 6 by 0.08 g l­1 agnps (fig. 8). fig. 6 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on tsp of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 7 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on tsc of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 8 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on h2o2 con­ tent of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. adv. hort. sci., 2020 34(2): 133­145 140 peroxidase activity based on the results, peroxidase (pod) activity in both flowers increased until d 3 of the vase period and then decreased until d 6 before increasing again thereafter. using agnps on chrysanthemum, unlike carnation, significantly increased the activity of pod in comparison with the control. when treated with 0.04 g l­1 agnps, the chrysanthemum showed a level of pod activity on d 9 that was about 2.5 times greater than the activity in carnations on the same day. in general, the highest level of pod activity was measured in chrysanthemum on d 9 after the treat­ ment with 0.04 g l­1 agnps, whereas the lowest level of activity was measured on d 6 in the control (fig. 9). superoxide dismutase activity based on the results, that the superoxide dismu­ tase (sod) activity in carnation petals was about 2.5 times higher than that of chrysanthemum petals on d 0, but on d 9 it was completely different, and the activity of this enzyme in chrysanthemum was more than 4.5 times that of carnation. generally the activi­ ty of sod dropped in carnation petals, whereas it rose in chrysanthemum petals. the agnps decreased the sod in both flowers during the vase period, as compared to the control. generally, the highest value of sod was observed in chrysanthemum on d 9 in the control treatment, whereas the lowest value was observed in carnation on this day of vase period in the treatment group of 0.08 g l­1 agnps (fig. 10). vase life clearly, agnps caused the vase life of both flowers to increase. both concentrations had a significant effect on carnations, while chrysanthemum was sig­ nificantly affected by the 0.04 g l­1 only. in carna­ tions, the vase life increased parallel to the increase in applied concentrations of agnps from 0.04 to 0.08 g l­1, the effects of which were significantly different compared to each other. using 0.08 g l­1 agnps yield­ ed more appropriate results in carnations. on the other hand, the vase life of chrysanthemum was slightly affected in a negative manner as the concen­ tration of agnps rose from 0.04 to 0.08 g l­1, but the difference between the two concentrations was insignificant. nonetheless, both caused significant differences in comparison with the control (fig. 11a and b). 4. discussion and conclusions in this study, the cut flowers of carnation and chrysanthemum showed various levels of decrease in rfw (fig. 2), vsu (fig. 3) and flower diameter throughout the vase period. similar results have been reported after assessing the rfw and vsu of gerbera cut flowers (liu et al., 2009), rose (chamani et al., 2005) and cut gardenia foliage (lin et al., 2019 a) dur­ ing the vase period. the current study showed that using the agnps reduced the bacterial population of the stem end in both flowers, while the rfw was improved as compared to the control. previous stud­ ies confirm such findings on the use of agnps in cut flowers such as gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2017) and rose (lu et al., 2010; nazemi rafi and ramezanian, 2013; hassan et al., 2014). these studies showed that the vase life improves when the bacterial population of the stem end decreases, besides when the vsu and rfw increase. fig. 9 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on pod acti­ vity of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 10 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on sod activity of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 141 the decrease in the rfw of cut flowers marks the beginning of senescence in flowers, and the more the flowers become closer to the process of senescence, their ability to take up water from the vase is reduced. the imbalance between water uptake and transpiration distorts cell turgor, thereby causing the flowers to wither (reid and jiang, 2012). the survival of cut flowers depends largely on a positive water balance: an increase in water uptake and a decrease in water loss (halevy and mayak, 1981; van doorn, 1997). after being placed in the vase, the flower loses rfw in part because of vascular occlusion and the growth of microorganisms which grow in the vase solution. they reduce the water uptake by blocking the stem end and vessels, thereby causing water stress which is a main factor in the reduction of vase life (van doorn, 1997; macnish et al., 2005). adding a germicide to the vase solution may control the activi­ ty of microbes. it has been reported that adding antimicrobial compounds containing ag+ ions to the vase solution improves the vase life of cut flowers (hassan et al., 2014). agnps nanoparticles function effectively because of their high surface­to­volume ratio and their crystallographic surface structure (rai et al., 2009). furthermore, ag+ ions cause bacterial cell death by affecting membrane structure and per­ meability, inhibiting dna transcription, disrupting transport activity and changing cellular content and atp (feng et al., 2000; sondi and salopek­sondi, 2004; rai et al., 2009; dakal et al., 2016). this difference in the bacterial population of the stem end of the two cut flowers may depend on the genotype of the plant, as well as the extract and metabolites that are secreted from the stem end and are released into the vase solution, thereby affecting the growth of bacteria. phenolic compounds are sec­ ondary metabolites that are involved in plant defense against pathogens (naczk and shahidi, 2004). in tradi­ tional chinese medicine, the flowering heads of chrysanthemum indicum are used as a source of bac­ tericide, with additional antifungal and antiviral prop­ erties (shunying et al., 2005). in agreement with our results, shunying et al. (2005) showed that the chemi­ cal composition and secondary metabolites of ch. indicum possess antimicrobial activity. flower diameters in this study were significantly influenced by agnps in carnation flowers, butthe effect on chrysanthemum was not significant. dar et al. (2014) reported that the flower diameter in dianthus chinensis first increased and then finally decreased during post­harvest development and senescence, respectively. the current research showed that the flower diameter of carnations tends to increase after being treated with agnps. this accords closely witha previous report on polianthes tuberosa which was treated with 0.015 g l­1 agnps and led to a significant increase in the diameter of flowers (bahrehmand et al., 2014). it is well known that the msi gradually decreases from the time when the flowers open to the time of their senescence. such a trend occurs evidently in certain flowers such as lilium (bieleski and reid, 1992), rose (hassan et al., 2014) and iris (ahmad and tahir, 2016). the senescence process is mainly asso­ ciated with protein loss, increased lipid peroxidation, membrane leakage, cell wall component degradation and cellular membrane disruption (buchanan­ wollaston, 1997). agnps may preserve the mem­ brane stability by curbing the peroxidation of lipids (hatami and ghorbanpour, 2013). there was a lower percentage of msi in carnation petals, compared to chrysanthemum, which may be due to the fact that carnations are highly sensitive to ethylene. the rea­ son becomes clear when knowing that ethylene fig. 11 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on vase life of carnation (a) and chrysanthemum (b). values repre­ sent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. adv. hort. sci., 2020 34(2): 133­145 142 degrades the cell membrane and increases its leak­ age (van doorn and woltering, 2008), thereby result­ ing in a lower value of msi. however, agnps acts against the production of ethylene (hassan et al., 2014). in fact, agnps induce an efficient cellular elec­ tron exchange mechanism which reduces electron leakage and, subsequently, limits the creation of ross. agnps also dys regulate lipid peroxidation and have a propensity to maintain the msi (lu et al., 2010; hassan et al., 2014). previous studies have reported a decrease in tsp content and an increase in protease activity during the vase life of cut flowers (wagstaff et al., 2005; dar et al., 2014; zhao et al., 2018). quite differently, however, we concluded that changes in the tsp con­ tent may vary depending on the type of flower, species and cultivar. still, contrary to our results, dar et al. (2014) showed that protein degradation in dianthus chinensis is a key factor in regulating the senescence process of flowers. shahri et al. (2011) also reported that tsp decreased during the senes­ cence process of helleborus orientalis cv. ‘olympicus’, while there was an increase in its low­ molecular­weight proteins. accordingly, realizing an increase of tsp content in carnation petals could be a result of these low­molecular­weight proteins. it has been shown that in flowers such as helleborus orientalis and dianthus chinensis, the tsc is reduced during the senescence process (shahri et al., 2011; dar et al., 2014).there was a lower content of tsc in chrysanthemum petals, as compared with carnations, which may be due to the genetic differ­ ences between the two plants, as well as the differ­ ence in the rate of polysaccharide decomposition during the opening and development of flowers and petals. in agreement with our results, it has been found that the starch and fructan polysaccharides are degraded and reduced during the development of flowers and the expansion of petals in chrysanthe­ mum (trustyl and miller, 1991). therefore, in this study, agnps in both flowers may have benefited the vase life by maintaining the content of tsc in petals and by promoting the mechanisms through which carbohydrates stabilize the cell membrane (ashraf et al., 2010). furthermore, agnps regulate and protect the cellular osmotic potential, inhibit the formation of free radicals (parida and das, 2005) and regulate the expression of genes (rahdari et al., 2012). usually, the amount of h2o2 increases in plant cells during senescence (ezhilmathi et al., 2007; saeed et al., 2014). it can be suggested that high lev­ els of h2o2 in carnation petals occur because of the plants sensitivity to ethylene. this can be compared with chrysanthemum which is not sensitive to the hormone. our results showed that the use of agnps on both flowers reduced the production of h2o2 in petals, as compared with the control. these results accord closely with a previous report by hassan et al. (2014) where the production of h2o2 became signifi­ cantly limited in roses because agnps was used. senescence is an oxidation process in which ros and antioxidant systems are involved (buchanan­ wollaston, 1997). plant cells have developed a series of antioxidant mechanisms for defense to prevent the production of ross and to limit their destructive effects on proteins, fats and nucleic acids (arora et al., 2002). the enzymatic part of this system consists of antioxidant enzymes such as sod, pod, catalase (cat) and ascorbate peroxidase (apx) which degrade all types of ross (balakhnina and borkowska, 2013). depending on the species or type of cultivars, the pod activity and sod enzymes can exhibit different patterns of change in cut flowers during the senes­ cence process (hassan et al., 2014). as the produc­ tion of h2o2 increased in the petals of both flowers during the vase period, the pod enzyme likewise increased its activity to scavenge the higher amounts of h2o2. furthermore, hassan et al. (2014) reported similar results which indicate that petals of the rose cut flower cv. ‘first red’ ­ which is sensitive to ethyl­ ene (chamani et al., 2005) ­ showed declining levels of sod activity during the vase period. however, con­ trary to our results on the incremental trend of pod in carnations and chrysanthemum during the vase period, hassan et al. (2014) reported that the pod activity in the rose cultivar decreases. it has also been reported that sod activity in ethylene­sensitive flowers such as carnation is relatively lower com­ pared to ethylene­insensitive flowers such as chrysanthemum. this comprises a major factor in accelerating the senescence (bartoli et al., 1995). in addition, it may be suggested that in ethylene­ insensitive flowers such as chrysanthemum, ros cause the greatest amount of damage to the cell components, thereby leading to a shorter vase life. for this reason, the sod activity has to increase so as to create a parallel level of scavenging. in certain flowers such as carnations, which are sensitive to ethylene, the level of sod activity may not be pro­ nounced as much. as previously mentioned, agnps cause the plants to acquire an efficient cellular elec­ tron exchange mechanism, whereby the electron leakage and ros production are reduced (hassan et al., 2014). perhaps, the activity of sod does not rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 143 increase in carnations even when agnps are applied. agnps increased the vase life of chrysanthemum and carnation (figs. 11a and b) by increasing the rfw (fig. 2) and reducing the bacterial population of the stem end (fig. 4). the scientific literature con­ tains several reports that mention the efficiency of agnps in increasing the vase life of gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2017), rose (lu et al. , 2010; nazemi rafi and ramezanian, 2013; hassan et al., 2014), chrysanthe­ mum (carrillo­lópez et al., 2016), carnation (naing et al., 2017) and gladiolus (li et al., 2017). in conclusion, agnps improved the vase life of both cut flowers by contributing to the values of their rfw, vsu, flower diameter, msi, tsc, tsp and pod activity and by limiting their bacterial population of the stem end and h2o2 levels, as compared to the control. it is highly probable that agnps are capable of reducing the expression of genes responsible for the production of ethylene, as well as limiting the rate of transpiration and the opening of stoma. furthermore, agnps have roles in regulating the aperture of the stoma and in reducing mda produc­ tion, thereby prolonging the vase life of cut flowers. ultimately, this research revealed that the use of agnps at 0.04 g l­1and 0.08 g l­1 can extend the vase life of at least two cultivars belonging to chrysanthe­ mum and carnation, respectively. acknowledgements the authors wish to thank the university of kurdistan for providing facilities and financial support (grant no. crc97­00242­1) of this research. references abeles f., morgan p., saltveit j.r.m., 1992 ­ ethylene in plant biology. ­ academic press, san diego, ca, usa, pp. 399. ahmad s.s., tahir i., 2016 ­ increased oxidative stress, lipid peroxidation and protein degradation trigger senescence in iris versicolor l. flowers. ­ physiol. mol. biol. 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annu. rev. plant biol., 35: 155­189. zhao d., cheng m., tang w., liu d., zhou s., meng j., tao j., 2018 ­ nano‐silver modifies the vase life of cut herbaceous peony (paeonia lactiflora pall.) flowers. ­ protoplasma, 1: 1­13. impaginato 255 adv. hort. sci., 2020 34(3): 255­263 doi: 10.13128/ahsc­7767 enhancement of pentacyclic triterpenoids (betulinic and oleanolic acids) production from callus cultures of lantana camara l. a.m. al­ashoush 1, r.a. shibli 1 (*), r.w. tahtamouni 2, t.s. al­qudah 3, b.b. abu­irarmaileh 3 1 department of horticulture and agronomy, faculty of agriculture, university of jordan, amman, jordan. 2 department of biotechnology, faculty of agricultural technology, al‐ balqa applied university, amman, jordan. 3 hamdi mango center for scientific research (hmcsr), university of jordan, amman, jordan. key words: elicitation, growth regulators, heavy metals, in vitro, lantana, lantana camara l., sugars. abstract: lantana camara l. is an ornamental plant with high medicinal value. this study aimed to investigate the possibility of enhancing the production of betulinic and oleanolic acids in lantana callus by adding different types and lev­ els of chemical elicitors (nacl, sugars, growth regulators and heavy metals) to murashige and skoog (ms) medium. data revealed that, adding nacl to the cul­ ture medium affected callus fresh weight and color negatively, but it increased the extracted amounts of oleanolic and betulinic acids significantly to reach maximum levels of 0.702 and 0.051 mg/g dw at 120 mm nacl compared to 0.659 mg/g dw and 0.014 mg/g dw obtained in the control. meanwhile, increasing glucose level to 36.02 g/l in the medium maximized oleanolic acid accumulation to 0.829 mg/g dw, while betulinic acid accumulation reached 0.038 mg/g dw at 54.03 g/l glucose. in growth regulators experiment, highest callus fresh weight was observed in the control medium, while it declined to the minimum at 0.50 mg/l of thiadiazuron (tdz). maximum values of both acids (0.685 and 0.033 mg/g dw) were recorded in ms medium plus 1.0 mg/l tdz. callus fresh weight decreased significantly in response to heavy metals addition, while adding chromium at 0.08 mg/l improved production of oleano­ lic acid to reach the maximum of 0.676 mg/g dw. meanwhile betulinic acid was maximized at 0.057 mg/g dw in callus cultures exposed to 0.08 mg/l cobalt. 1. introduction lantana camara l. is a popular flowering ornamental plant (charitha and ranwala, 2018), belonging to the family verbenaceae (mishra, 2015). it is native to tropical and sub­tropical areas of america (singh and saxena, 2016), west africa (waoo et al., 2015 a) and tropical asia (*) corresponding author: r.shibli@ju.edu.jo citation: al­ashoush a.m., shibli r.a., tahtamouni r.w., al­qudah t.s., abu­irarmaileh b.b., 2020 ­ enhancement of pentacyclic triterpenoids (betulinic and oleanolic acids) production from callus cultures of lantana camara l. ­ adv. hort. sci., 34(3): 255­263 copyright: © 2020 al­ashoush a.m., shibli r.a., tahtamouni r.w., al­qudah t.s., abu­irarmaileh b.b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 december 2019 accepted for publication 26 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 255­263 256 (ghisalberti, 2000). this plant is routinely used as an evergreen aromatic ornamental or hedge shrub (bhakta and ganjewala , 2009; zoubiri and baaliouamer, 2012). it’s commonly named as lantana, red sage, surinam tea plant, spanish flag and west indian lantana (kalita et al., 2012). lantana camara l. is also listed as one of the most important medicinal plants, as it possesses many dis­ tinguished medicinal properties (waoo et al., 2014). the interest in lantana camara l. has recently, increased because it is an excellent source of many chemical compounds of medicinal potential (srivastava et al., 2011; saxena et al., 2012; waoo et al., 2014) like pentacyclic triterpenoids which includes many active compounds such as betulinic and oleanolic acids (kensa, 2011; venkatachalam et al., 2011; kazmi et al., 2012; mariajancyrani et al., 2014). betulinic acid has recently gained increased atten­ tion as it possesses a remarkable variety of biological and medicinal properties (moghaddam et al., 2012; pandey et al., 2015). oleanolic acid was also reported to possess many important biological activities (ghosh et al., 2010; xia et al., 2011; singh et al., 2012). so producing such valuable compounds in commercial amounts is of great importance. in vitro culture was utilized as an efficient approach for production of secondary metabolites. currently, in vitro culture techniques are used to facilitate the pos­ sibility of making quantitative and qualitative elicita­ tion of the production of plant secondary metabolites by changing the culturing media composition (affonso et al., 2007; alenizi et al., 2020). according to taiz and zeiger (2002), adding elevated levels of growth regula­ tors, sugars and heavy metals was found to drive the plant cell machinery to produce more secondary metabolites rather than cell division. for example, pasqua et al. (2005) reported successful enhancement of anthocyanin production in camptotheca acuminata cell cultures by adding different types and levels of growth regulators and sugars to the growth media. in addition, lee et al. (2011) were able to produce more rutin from callus and adventitious roots of white mul­ berry tree by adding auxins, cytokinins, and nitrogen to the growth medium. heavy metals were also found to increase the pro­ duction of bioactive compounds in many plants (verpoorte et al., 2002). so, this study was carried out to enhance the possibility of production of betulinic and oleanolic acids in lantana callus cul­ tures by modifying the culture medium using differ­ ent types and levels of growth regulators, sugars, nacl, and heavy metals in callus culture media. 2. materials and methods mother stock culture establishment and maintenance in vitro grown callus cultures of lantana camara l. were implemented by the plant tissue culture and microbiology laboratories at hamdi mango center for scientific research (hmcsr). the cultures were subcultured on callus maintenance semi solid medi­ um consisting of 4.4 g/l of murashige and skoog (murashige and skoog, 1962) ms premix (duchefa biochemie) plus 34.2 g/l sucrose in addition to 1.0 mg/l kinetin and 2.0 mg/l 2,4­d. cultures were main­ tained in the growth room under dark conditions at 24± 1°c. subculturing of callus was performed every 3­4 weeks by subdividing callus under sterile condi­ tions. enhancement of betulinic and oleanolic acids produc‐ tion to study the possibility of enhancing production of betulinic and oleanolic acids in lantana camara l. callus cultures, different types and levels of chemical elicitors (nacl, sugars, growth regulators, heavy met­ als) were added to the culture medium. in nacl experiment, callus clumps of 1.0 g were subcultured onto fresh ms callus maintenance medi­ um described earlier and supplemented with nacl at different concentrations (0, 40, 80, 120 and 160 mm). meanwhile, the effect of sugars on betulinic and oleanolic acids was experimented by transferring 1.0 g of callus clumps onto maintenance medium supple­ mented with different types and levels of sugars: sucrose (34.2, 68.4 and 102.6 g/l); glucose or fructose (18.01, 36.02 and 54.03 g/l). in heavy metals experiment, lead (pb), cobalt (co) or chromium (cr) were prepared from their salts; lead nitrate pb (no3)2, potassium chromate (k2cro4), and cobalt (ii) chloride (cocl2.6h2o) respectively. a stock solution of 100 mg/l was prepared from each salt. after that the requested amount of each heavy metal (0.08, 0.16 and 0.24 mg/l) was added to the callus medium prepared as described earlier. to test the effect of plant growth regulators thidi­ azuron (tdz) and kinetin on betulinic and oleanolic acids production, the explants were transferred onto hormone free ms medium for one week to remove any carry over effect of callus maintenance media. next, callus clumps of 1.0 g were subcultured onto full strength ms solid medium supplemented with different levels (0.5, 1.0 and 1.5 mg/l) of either tdz al‐ashoush et al. ‐ enhancement of pentacyclic triterpenoids production in lantana camara l. 257 or kinetin in combination with 2.0 mg/l 2,4­d. all cultures were maintained in the growth room under dark conditions at 24±1°c, and data were col­ lected after 6 weeks for callus fresh weight and color before oven drying. extraction calluses from each treatment were oven dried at 50oc for 2 days. then, the dried callus was ground using liquid nitrogen. next, 100 mg were taken from the dried matter of each treatment and soaked in 10 ml of methanol and water in a ratio of 1:1. the sam­ ples were then placed on a shaker at slow speed for at least 72 hours at room temperature (hussain et al., 2012). the plant extract was filtered and cen­ trifuged at 5000 g for 10 min. the resulting super­ natant was dried in a rotary evaporator at 40oc. next, the dried extract was resuspended in the mobile phase for hplc analysis. preparation of betulinic and oleanolic acids stock solutions and working standards betulinic and oleanolic acid stock solutions at con­ centration of 1000 mg/l were prepared by weighing 5 mg of each in 5 ml volumetric flask, dissolved and brought to volume by methanol hplc grade. both stock solutions were stored at 4oc in dark. working solutions were prepared by serially diluting stock solutions using the mobile phase at concentrations of 800, 400, 200, 100, 50, 25 and 12.5 mg/l. fresh work­ ing standards were prepared daily. betulinic and oleanolic acids were eluted at 15.8 and 19 min, respectively. calibration curves were constructed for betulinic and oleanolic acids before starting chemical analysis (r²= 0.9999). hplc instrumentation and conditions hplc analysis was performed using a shimadzu­lc system (shimadzu corporation, kyoto, japan) equipped with a cbm­20a controller, lc­20at pump, and spd­20av uv/vis detector chromatographic separations were achieved using an agilent eclipse plus c18 column and were carried out using an iso­ cratic flow rate of 1 ml/min, a column temperature of 25o c, a mobile phase of acetonitrile: methanol: acidi­ fied water (70:20:10 v/v), with ph adjusted to 2.8 using 85% phosphoric acid. the ultraviolet (uv) detection was set at 210 nm. the injection volume was 20 μl of sample solution. total run time was 19 min for each injection. data were acquired and processed with lc­solution software (shimadzu corporation, kyoto, japan). statistical analysis each treatment was arranged in a complete ran­ domized design (crd) and replicated ten times with two callus clumps/replicate. hplc analysis was con­ ducted on three replicates for each treatment with two samples/replicate. the collected data were sta­ tistically analyzed using spss analysis system. the analysis of variance (anova) was used and mean separation was done according to the tukey’s hsd at probability level of 0.05. 3. results effect of nacl after 6 weeks of incubation under different levels of nacl, the obtained results indicated that, callus fresh weight and color (callus quality) declined in response to nacl and the effects became more severe as nacl level increased (table 1). the maxi­ mum fresh weight 8.6 g was recorded in the control, while brownish yellow callus with minimal fresh weight value of (2.6 g) was obtained on the medium supplemented with 160 mm nacl (table 1). on the other hand, adding nacl to the growth medium enhanced the production of oleanolic acid in the callus cultures in response to nacl level reaching the maximum level of 0.702 mg/g dw at 120 mm nacl (mm) weight (g) color oleanolic acid (mg/g dw) betulinic acid (mg/g dw) control 8.60 ± 0.36 a * white 0.659 ± 0.011 d 0.014 ± 0.0003 e 40 5.90 ± 0.35 b white 0.668± 0.0028 c 0.021± 0.0531 d 80 4.70 ± 0.59 bc yellow 0.674± 0.0124 b 0.043 ± 0.0081 b 120 4.11 ± 0.32 c yellow 0.702 ± 0.0088 a 0.051 ± 0.0083 a 160 2.62 ± 0.53 d yellow to brown 0.656± 0.0096 d 0.035± 0.0057 c table 1 ­ effect of nacl on callus fresh weight, color and levels of oleanolic and betulinic acids in callus culture of lantana camara * values represent means ± standard error. control: represents callus maintenance medium consisting of solid ms medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­d + 1.0 mg/l kinetin. means with different letters are significantly different according to tukey hsd range test at p≤0.05. adv. hort. sci., 2020 34(3): 255­263 258 nacl compared to 0.659 mg/g dw extracted from the control treatment (table 1). also, the production of betulinic acid was enhanced by adding nacl. the maximum value 0.051 mg/g dw was obtained at 120 mm nacl compared to 0.014 mg/g dw extracted from the control (table 1). effect of sugars sucrose. our results revealed that, increasing sucrose concentration in the ms media led to a dra­ matic decline in fresh weight and quality of lantana camara l. callus cultures (table 2). fresh weight of the white callus decreased significantly from 8.6 g in con­ trol treatment to reach the minimum (3.1 g) at 102.6 g/l sucrose where it turned dark brown (table 2). meanwhile, in response to level of sucrose, we observed significant increases in oleanolic and betulin­ ic acids production. the maximum amounts of oleano­ lic acid (0.97 mg/g dw) and betulinic acid (0.035 mg/g dw) were recorded in media with 102.6 g/l of sucrose. this level of sucrose enhanced the production of betulinic and oleanolic acids in callus cultures to levels that exceeded values of those extracted from naturally growing plants (table 2). on the other hand, callus was not healthy and the growth was very limited in all treatments compared to the control (table 2). glucose. adding glucose resulted in a significant decline in callus growth and quality as shown in table 2. instead increasing glucose level in the medium resulted in increasing level of oleanolic acid to reach the maximum at 36.02 g/l glucose (0.829 mg/g dw) compared to control (0.659 mg/g dw). meanwhile, betulinic acid accumulation increased to 2.5 times when 54.03 g/l glucose was added to the media com­ pared to the control (0.014 mg/g dw) as shown in table 2. fructose. the obtained data indicated that, fruc­ tose was not a good choice for lantana camara l. callus cultures in terms of growth, color and sec­ ondary metabolite production. callus fresh weight decreased gradually with increasing fructose levels in the medium and minimum fresh weight of the brown callus (1.55 g) was obtained on 54.03 g/l fructose (table 2). meanwhile, adding fructose to the culture medium at levels higher than 18.01 g/l negatively affected oleanolic accumulation compared to the control, and the same for betulinic acid (table 2). effect of plant growth regulators effect of kinetin. data obtained revealed a signifi­ cant increase in fresh weight (8.6 g) at kinetin con­ centration of 1.0 mg/l (table 3). meanwhile maxi­ mum amounts of oleanolic acid and betulinic acids (0.67 and 0.021 mg/g dw) were extracted from cal­ lus cultures grown on media supplemented with low rate of kinetin (0.5 mg/l), while the amount of fresh weight and both acids declined significantly as kinetin level increased in the media (table 3). * values represent means ± standard error. control: represents callus maintenance medium consisting of solid ms medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­d + 1.0 mg/l kinetin. means with different letters are significantly different according to tukey hsd range test at p≤0.05. table 2 ­ effect of sucrose; glucose and fructose on callus fresh weight, color and production of betulinic acid and oleanolic acid in callus culture of lantana camara carbohydrate (g/l) weight (g) color oleanolic acid (mg/g dw) betulinic acid (mg/g dw) sucrose control (34.2) 8.60 ± 0.36 a* white 0.659± 0.011 c 0. 014 ± 0.0003 c 68.4 3.21 ± 0.16 b yellow 0.824 ± 0.0082 b 0.027± 0.013 b 102.6 3.11 ± 0.32 b dark brown 0.970± 0.0081 a 0.035± 0.0291 a glucose control 8.60 ± 0.36 a white 0.659± 0.011 c 0.014 ± 0.0003 c 18.01 4.49 ± 0.197 b yellow 0.765± 0.0093 b 0.025 ± 0.0084 b 36.02 3.80 ± 0.56 bc yellow start to be brown 0.829± 0.0089 a 0.036± 0.0189 a 54.03 3.00 ± 0.46 c yellow start to be brown 0.764± 0.0077 b 0. 038 ± 0.0095 a fructose control 8.60± 0.36 a white 0.659 ± 0.011 a 0.014 ± 0.0003 a 18.01 4.40 ± 0.22 b yellow 0.661 ± 0.0035 a 0.012 ± 0.0066 ab 36.02 2.34 ± 0.47 c yellow 0.514 ± 0.0092 b 0.010 ± 0.0090 b 54.03 1.55 ± 0.13 d brown 0.380 ± 0.0060 c 0.010± 0.0089 b al‐ashoush et al. ‐ enhancement of pentacyclic triterpenoids production in lantana camara l. 259 effect of tdz. the highest fresh weight of callus (8.6 g) was obtained in the control treatment fol­ lowed by that obtained on 1.5 mg/l tdz in combina­ tion with 2.0 mg/l 2,4­d (table 3). however, the resulted callus was of good quality as it remained white in all treatments levels (table 3). moreover, a significant increase in oleanolic acid and betulinic acids was obtained in response to tdz, and maxi­ mum values of both acids 0.685 and 0.033 mg/g dw were recorded in callus cultures grown in ms medi­ um plus 1.0 mg/l tdz, while adding higher levels of tdz negatively impacted on oleanolic and betulinic acids (table 3). * values represent means ± standard error. control: represents callus maintenance medium consisting of solid ms medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­d + 1.0 mg/l kinetin. means with different letters are significantly different according to tukey hsd range test at p≤0.05. table 3 ­ effect of different growth regulators in combination with 2.0 mg/l of 2, 4­d on callus fresh weight, color and production of betulinic and oleanolic acids in callus culture of lantana camara growth regulator (mg/l) weight (g) color oleanolic acid (mg/g dw) betulinic acid (mg/g dw) kinetin 0.5 4.63 ± 0.55 b* white 0.672 ± 0.0091 a 0.021 ± 0.0067 a 1.0 (control) 8.60 ± 0.36 a white 0. 659 ± 0.011 b 0.014 ± 0.0003 b 1.5 4.72 ± 0.44 b yellow 0. 621 ± 0.0001 c 0.014 ± 0.0010 b tdz control 8.60 ± 0.36 a white 0.659 ± 0.011 c 0.014 ± 0.0003 c 0.5 1.40 ± 0.14 c yellow 0. 651 ± 0.0088 d 0.025±0.023 b 1.0 3.44 ± 0.63 b white 0.685 ± 0.0082 a 0.033±0.009 a 1.5 3.68 ± 0.23 b white 0.668±0.0045 b 0.012± 0.0072 c effect of heavy metals effect of cobalt. callus fresh weight and color were negatively affected by adding cobalt to the media at levels higher than 0.08 mg/l. minimum weight of 5.32 g was recorded at co level of 0.24 mg/l (table 4). on the other hand, callus remained healthy white despite of co addition at all levels (table 4, fig. 1). meanwhile, amounts of oleanolic acid were negatively affected by adding co to the media at all levels compared to the quantity extract­ ed from the control (table 4). on the other hand, production of betulinic acid was significantly improved by adding co to the medium. at 0.24 mg/l * values represent means ± standard error. control: represents callus maintenance medium consisting of solid ms medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­d + 1.0 mg/l kinetin. means with different letters are significantly different according to tukey hsd range test at p≤0.05. table 4 ­ effect of heavy metals on callus fresh weight, color and production of betulinic and oleanolic acids in callus culture of lantana camara heavy metal (mg/l) weight (g) color oleanolic acid (mg/g dw) betulinic acid (mg/g dw) cobalt control 8.60 ±0.36 a * white 0.659 ± 0.011 a 0.014 ± 0.0003 d 0.08 6.87 ± 0.59 ab white 0.501 ± 0.0066 b 0.020 ± 0.0003 c 0.16 5.41 ± 0.71 b white 0.472 ± 0.0046 c 0.033± 0.0072 b 0.24 5.32 ± 0.75 b white 0.215 ± 0.0082 d 0.057± 0.0102 a lead control 8.60± 0.36 a white 0.659 ±0.011 a 0.014 ± 0.0003 b 0.08 3.80 ± 0.76 b white with red spots 0.344 ± 0.001b 0.020 ± 0.009 a 0.16 3.42 ± 0.90 b yellow with red spots 0.343 ± 0.006 b 0.021 ± .0001 a 0.24 1.57 ± 0.10 b yellow with red spots 0.117± 0.0085 c 0. 016 ± 0.008 b chromium control 8.6 ±0.36 a white 0.659 ± 0.011 c 0.014 ± 0.0003 a 0.08 3.60 ± 0.54 b yellow with brown spots 0.676 ± 0.001 a 0.013 ± 0.0003 a 0.16 2.81 ± 0.66 b yellow with brown spots 0.665 ± 0.0083 b 0.010 ± 0.0085 b 0.24 0.99 ± 0.13 c yellow with brown spots 0.435 ± 0.0071 d 0.010± .0069 b 260 adv. hort. sci., 2020 34(3): 255­263 it was four times higher than the control as shown in table 4. effect of lead. data revealed that, callus growth, color and oleanolic acid production decreased dra­ matically with increasing pb level in the media (table 4, fig. 2). the minimal value for fresh weight (1.57 g) was recorded in cultures grown in media supple­ mented with 0.24 mg/l pb. furthermore, adding pb to the medium determined the appearance of red spots on the white callus at all pb levels (table 4, fig. 2). meanwhile, production of betulinic acid was improved significantly by adding either 0.08 or 0.16 mg/l pb while it tended to decline at 0.24 mg/l pb (table 4). effect of chromium. the results obtained showed that callus growth was negatively affected by adding cr in the medium (table 4). furthermore, adding cr to the medium resulted in development of brown spots on the white callus at all levels (table 4). moreover, chromium had improved the production of oleanolic acid to reach the maximum (0.676 mg/g dw) at 0.08 mg/l, while it tended to decline at higher cr levels (table 4). meanwhile, amounts of betulinic acid decreased in response to cr to reach minimum level of (0.010 mg/g dw) at either 0.16 or 0.24 mg/l, respectively (table 4). 4. discussion and conclusions effect of nacl adding nacl to the culture medium resulted in reduction in most growth parameters in callus cul­ tures of lantana camara which agrees with nikman, et al. (2004) and nikman et al. (2006) studies on nicotiana seedlings and on calli and seedlings of two trigonella species on culture media supplemented with elevated levels of nacl, where growth and quali­ ty of the cultures declined sharply at levels high lev­ els. generally, quality parameters including color, declined in tissue cultured plant material of many plant species when exposed to osmotic stress due to addition of salts or sugars to the media (taiz and zeiger, 2002). this could be due to the continuous accumulation of osmotica, dehydration and phenolic compounds accumulation that results in a shift in color toward brown which might determine, with time, cell death. meanwhile, enhanced accumulation of oleanolic and betulinic acids in lantana camara callus cultures was obtained at high nacl levels agrees with parida and das (2005) who reported that salt stress might induce the production of secondary metabolites to maintain cell turgidity and to reduce dehydration. also, wang et al. (2015) confirmed in their study on cotton that salinity stress resulted from adding nacl to the growth medium induced the production of some secondary metabolites such as gossypol, flavonoids and tannin. effect of sugars it was clear from our data, that increasing sugar concentration in the medium inversely affected callus fresh weight and color. elevated levels of sugars would make them act as osmotic agents instead of energy sources, and this would hinder growth rate of the explants (tahtamouni et al., 2016). consequently cell volume would decrease as a result of low turgor pressure which would be translated in a form of growth reduction (taiz and zeiger, 2002). meanwhile, according to our results increasing sucrose level in the medium led to significant increase in production of oleolinic acid and betulinic acid. generally, high levels of sucrose were reported to increase the secondary metabolite production in plant tissue cultures (bandhakavi and kamarapu, 2016). similar results were also recorded in different species such as, grape berries (dai et al., 2014), ginkgo biloba (park et al., 2004), and clematis pitcher (kawa­miszczak et al., 2009). fig. 2 ­ effect of lead on callus fresh weight and color in callus culture of lantana camara l. after 6 weeks of incubation in a medium containing 0.08; b medium containing 0.16 and c medium containing 0.24 mg/l lead. fig. 1 ­ effect of cobalt on callus fresh weight and color in callus culture of lantana camara l. after 6 weeks of incubation in a medium containing 0.08; b medium containing 0.16 and c medium containing 0.24 mg/l cobalt. al‐ashoush et al. ‐ enhancement of pentacyclic triterpenoids production in lantana camara l. 261 enhancement of secondary metabolites production in response to glucose was also obtained by verma et al. (2012) in their study about improving alkaloid con­ tent in callus culture of catharanthus roseus. effect of plant growth regulators callus fresh weight was influenced by the level of kinetin in the medium. this was in full agreement with singh and saxena findings (2016) where maxi­ mum callus growth was obtained when 2, 4­d was used in combination with kinetin to induce callusing in yellow variety of lantana camara l. effect of heavy metals our results showed that, increasing levels of the experimented heavy metals in the culture medium led to a gradual decline in callus fresh weight and color. a decline in cultures growth in response to addition of chromium to the medium was reported by waoo et al. (2015 a) who observed that increasing chromium in the medium decreased shoot length and percentage of survival in lantana camara l. similar response was also reported in waoo et al. (2015 b) study about the toxic effects of different lead concentrations on in‐ vitro grown shoot cultures of lantana camara. our results had also indicated that production of oleanolic and betulinic acids varied with type and level of the added heavy metal. chemical elicitation was commonly combined with osmotic stress that usually reduces growth (taiz and zeiger, 2002). in many studies, increasing biosynthesis of secondary metabolites by stressing the plant using chemical and/ or physical elicitation agents was possible. but adding such elicitors usually resulted in decreasing biomass of the plant, and consequently the levels of secondary metabolites was unchanged or declined, although the concentration is strongly enhanced at tissue or cell level (gershenzon, 1984; selmar and kleinwächter, 2013; paulsen and selmar, 2016). therefore, although biosynthesis of these com­ pounds increased significantly in plant material like callus cultures or microshoots (paulsen and selmar, 2016) due to chemical elicitation, the total biomass sharply decline because of the negative effects of the elicitors. so, the total biosynthesis of secondary metabolites is dramatically decreased. it can be concluded from our study that enhance­ ment of oleanolic and betulinic acids production in lantana camara callus cultures is possible by adding some types of chemical elicitors to the culture medi­ um. meanwhile, obtaining enough plant material is crucial to guarantee sustainable production of sec­ ondary metabolites in commercial amounts. this would open the gate for conducting research to improve our methodology to maintains proper amounts of plant material with enhanced levels of both acids. acknowledgements authors would like to thank the university of jordan, the deanship of academic research, for grant­ ing this research fund. further thanks are extended to hamdi mango center for scientific research, the faculty of agriculture: the department of horticulture and crop sciences at the university of jordan. references affonso v.r., bizzo h.r., lima s.s., esquibel m.a., sato a., 2007 ­ solid phase microextraction (spme) analysis of volatile compounds produced by in vitro shoots of lantana camara l. under the influence of aux‐ ins and cytokinins. ­ j. braz. chem. soc., 18: 1504­1508. alenizi a., shibli r.a., tahtamouni r.w., al­qudah t.s., remeilh b.a., 2020 ­ in vitro propagation and enhancement of quercetins and isorhamnetin produc‐ tion in wild paronychia argentea l. ­ jordan j. pharm. sci. 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research corporation, embrapa roraima, caixa postal 133, 69301‐970 boa vista, rr, brazil. key words: biometric characterization, camu-camu, class size, fruit growing, rate of emergency, water content. abstract: camu-camu has aroused the interest of various industries like natural preservatives, ice creams, juices, jellies, wines, natural dyes, but there is little technical information about the seeds. the objective of this work was to determine the biometric, physical and vigor characteristics of camu-camu seeds. seeds originating from native populations of roraima were used. the biometry was determined and the data were analyzed in excel spreadsheet and calculated the mean, median, variance, standard deviation and seeds classified as small, medium and large, based on mass. the vigor was determined by electrical conductivity, seedling emergence, emergence velocity, plant height, stem diameter, in a completely causal design, with four replicates of 25 seeds. the average results for width, thickness, length, individual mass, volume, weight of one thousand seeds and number of seeds per kilogram showed large variability. the size of the seed has direct correlation with vigor, large seeds have greater vigor. 1. introduction the camu-camu, myrciaria dubia (kunth) mcvaugh, is a small fruit tree of the myrtaceae family, popularly known as araçá-dágua, azedinho, camocamo and caçari (smiderle and sousa, 2008). it occurs naturally in the amazon region, growing naturally along the banks of rivers, streams, channels and lakes (chagas et al., 2012; souza et al., 2017). the fruit of the camu-camu is used in the preparation of juices, jellies, alcoholic drinks and ice creams, among others (yuyama, 2011; sousa et al., 2013). the main property of the camu-camu is the high vitamin c content, around 6,112 ± 137.5 ascorbic acid 100 g-1 of pulp, better than most cultivated plants (yuyama, 2011). the skin of the camu-camu when fresh contains high levels of anthocyanin and ascorbic acid (villanueva-tiburcio et al., 2010). the interest aroused by the camu-camu is due to its bioactive compounds and biological activity of antioxidant action, which reduce lipid peroxidation, revert high levels of total cholesterol and triacylglycerols, and increase the levels of hdl-cholesterol (gonçalves, 2012). (*) corresponding author: nascimentocr0@gmail.com citation: do nascimento c.r., chagas e.a., smiderle o.j., de andrade souza a., cardoso chagas p., 2019 biometric characteristics of camu‐camu seeds from native populations in the state of roraima, classified by size class. adv. hort. sci., 33(3): 359-364 copyright: © 2019 do nascimento c.r., chagas e.a., smiderle o.j., de andrade souza a., cardoso chagas p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 23 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 359-364 360 seed characteristics are important in the study of any species, as they give an understanding of the dispersion and establishment of seedlings. the classification of seeds by size or weight is a strategy to be adopted in the standardisation and emergence of seedlings, and to obtain seedlings of similar size or greater vigour (carvalho and nakagawa, 2012). therefore, the biometry of fruits and seeds have a diagnostic value in differentiating species within a genus, and can contribute to their recognition (pereira and ferreira, 2017). in order to find the ideal size class for the multiplication of different plant species, biometry is used to determine physiological quality, but the results have differed considerably, even when the seeds are from the same species. biruel et al. (2010) found that larger seeds of caesalpinia leiostachya displayed a greater percentage and speed of germination; different results to those reported by queiroga et al. (2011), who studied peanut seeds and found that smaller seeds had a higher germination percentage. seeds of different sizes showed the same high germination percentage, with small seeds being the most viable, and medium and large seeds yielding seedlings that were more vigorous (vendramin and carvalho, 2013; smiderle et al., 2016). this ambiguous situation may be one of the reasons for two or more seed sizes being produced by the same species. variations in size were seen in seeds collected in an area of natural occurrence in the state of roraima, brazil. in view of the above, the aim of this work was to determine the biometry, physical characteristics and vigour of seeds of the camu-camu. 2. materials and methods the experiment was carried out at the seed analysis laboratory of embrapa roraima. the camucamu fruit used in the experiment were harvested from native plants located on the banks of the jatapú river (0° 41.072” n and 59° 18.046” w) at an altitude of 144 m. here the plants develop in rocky soil in the district of caroebe in the state of roraima, and are distributed along the river in various populations and subpopulations. after harvesting, the fruits were packed in plastic bags to prevent damage from crushing, and carefully transported to the laboratory, where they were selected for lack of damage, and then homogenised and sanitised. the mature selected fruit were then depulped and washed in running water, using a sieve for the complete removal of any pulp adhering to the seeds. physical characterisation of the seeds the seeds were evaluated for their physical characteristics. seed biometry. the individual thickness, length and width of 600 seeds were measured using a calliper, with the results expressed in mm. the length was measured along the longitudinal axis of the seed; the width was measured perpendicular to the length, considering the broad face of the seed, which corresponds to the median line; and the thickness, by again measuring perpendicular to the length, but on the smaller seed face, corresponding to the median line including the two cotyledons. the following were then obtained: seed volume (mm3): multiplying the three dimensions (width, length and thickness), without considering the actual shape of the seed. individual seed weight: obtained by weighing each seed. based on these measurements, the seeds were separated into three size classes (large, medium and small). 1000 seed weight (g). six samples of 100 seeds were used for each size class. the samples were weighed on a 0.001 g precision balance and the weight determined by multiplying the result by 10. number of seeds per kilogram. after determining the thousand-seed weight (tsw), the number of seeds per kilogram was calculated using the “rule of three”. water content (%). carried out using the standard oven method at 105±3°c for 24 hours. four replications of 10 seeds were used, where each sample was wrapped in aluminium and placed in an oven. after 24 hours, the samples were removed from the oven and placed in a desiccator for cooling, and then weighed on a 0.001 g precision balance. the water content was calculated based on the difference between the wet and dry weights, applying the formula proposed in the rules for seed analysis (mapa, 2009), with the result expressed as a percentage. the biometric data were analysed in an excel spreadsheet. the mean, median, variance and standard deviation were all calculated. determining the vigour of small, medium and large seeds of the camu‐camu the experiment was carried out in a greenhouse at embrapa roraima, in boa vista. the experimental design was completely randomised (crd), with three treatments comprising the size classes (small, medido nascimento et al. ‐ seeds biometry and vigor of camu‐camu 361 um and large), and four replications of 25 seeds. seeds with a weight between 0.66 g and 0.84 g were considered small, those with a weight between 0.92 g and 1.20 g were considered medium, and those with a weight between 1.27 g and 1.60 g considered large. after classification, the following analyses were carried out: electric conductivity (ec). the seeds were weighed on a 0.0001 g precision balance and then immersed in 75 ml of water for 24 hours at a constant temperature of 25°c. the electrical conductivity was then measured using a digital conductivimeter. the results were expressed in μs cm-1 g-1 of seed. seedling emergence. the test was carried out following procedures described in the rules for seed analysis (mapa, 2009). the tests were conducted in trays containing 50% sand + 50% sawdust as substrate moistened with water up to 60% of its retention capacity, using four replications of 25 seeds distributed at a depth of 2.0 cm. the test was performed in a nursery of 50% sombrite shade screen. to evaluate the non-germinated seeds, they were removed from the substrate, cut down the middle with pruning shears and identified for viability and senescence. the formula used for calculation was: e = (n/a) x 100: where e = percentage emergence; n = number of emerged seedlings; a = total number of seeds placed for emergence. speed of seedling emergence. established by means of a daily count of emerged seedlings, the index being calculated from the expression: se = (e1/n1) + (e2/n2) + ... + (en/nn), where: se = speed of seedling emergence; e1 = number of seedlings emerged at the first count; n1 = number of days elapsed until the first count; e2 = number of seedlings emerged at the second count; n2 = number of days elapsed until the second count; n = last count. plant height. evaluated four months after sowing with the aid of a graduated rule (cm). measured from the cotyledon node to the end of the first pair of leaves in normal seedlings identified at the end of the test for seedling emergence. stem diameter. evaluated four months after sowing using a digital calliper (mm). determined at the insertion of the cotyledon. the data for water content, electrical conductivity, seedling emergence, speed of emergence, plant height and stem diameter were submitted to the lilliefors test for normality. they were then submitted to analysis of variance (anova), using a completely randomised design with four replications, and the mean values compared by tukey’s test at 5% probability using the sisvar software (ferreira, 2014). 3. results and discussion from the results, it was seen that there was a significant difference in the biometric variables of the analysed seeds, and it was possible to separate them into three classes according to size: small, medium and large. the data for variance, standard deviation and coefficient of variation, and the mean results for individual weight, width, thickness, length and volume of the camu-camu seeds are shown in table 1. the biometric data shown indicates that the studied population was accurately sampled, since the values for variance were low (<1) for each of the characteristics under study. the values for standard deviation shown in table 1 indicate a low sample variation for each of the characteristics under evaluation. the values for the coefficient of variation demonstrate the low variation in the variables, considering the mean value of the characteristics. however, variation can be seen when the seeds are sorted by size. table 1 biometric characteristics of camu-camu seeds from native populations in the state of roraima, classified by size class class mean variance standard deviation coefficient of variation (%) weight (g) small 0.79 0.002 0.04 5.65 medium 1.11 0.004 0.06 5.90 large 1.45 0.008 0.08 6.12 width (mm) small 12.04 0.541 0.73 6.11 medium 12.90 0.147 0.38 2.97 large 14.42 0.233 0.48 3.34 thickness (mm) small 5.54 0.065 0.25 4.59 medium 6.11 0.213 0.46 7.56 large 6.74 0.329 0.57 8.51 length (mm) small 15.54 0.620 0.79 5.07 medium 17.42 0.206 0.45 2.61 large 19.14 0.342 0.58 3.05 volume (mm3) small 1,035.77 6,144.378 78.38 7.57 medium 1,371.98 6,104.830 78.13 5.69 large 1,859.47 20,759.298 144.08 7.75 adv. hort. sci., 2019 33(3): 359-364 362 similar results were found in camu-camu seeds from the banks of the anauá river in roraima, with a weight which ranged from 0.80 g to 1.46 g, and greater results than the seeds of populations from the rio urubu, which ranged from 0.56 to 0.78 g (souza et al., 2017). according to gonçalves et al. (2008), species with wide geographic distribution may present differences in their characteristics due to the effects of adaptation and to origin. the variation in seed size may interfere with their physiological quality, still poorly researched in forest species (oliveira et al., 2009; smiderle et al., 2016). the three classes of seed size displayed significant differences for thousand-seed weight (tsw) and the number of seeds per kilo (nsk) with a low coefficient of variation, showing little variation within each size (table 2). the initial water content of the seeds in the different size classes at the time the experiment was set up was greater than 35%, with significant differences between the classes (table 3), however it was lower than that reported by yuyama et al. (2011), who obtained a value for moisture between 45 and 56%, with no effect on germination. according to braga et al. (2012), hydration of the seeds can favour test performance, because seeds that are more humid, within certain limits, germinate more quickly. the different classes of seed displayed variations in the variables under analysis. it was found that values for electrical conductivity depend on the size of the seed, where the large, small and medium seeds presented higher, lower and intermediate physiological quality respectively (table 3). according to vieira and krzyzanowski (1999), the lower physiological potential in small seeds is probably due to the lower organisational intensity of the cell-membrane systems. the data for electrical conductivity in the small seeds showed a negative correlation with the other variables under study, demonstrating that the highest value for electrical conductivity corresponded to reductions in the percentage and speed of seedling emergence. these results confirm those reported by vinhal-freitas et al. (2011), and demonstrate that tests of vigour differentiate between seed classes, indicating significant differences between the larger and smaller size classes, where larger seeds showed greater vigour with the smallest values for electrical conductivity. with the large seeds, the lower value for electrical conductivity was due to a greater organisation of the cell components, since, despite field emergence not differing statistically from that of the small seeds, they showed greater speed of emergence in addition to viability, even when not germinated, as the small seeds that did not emerge had all died. it is important to point out that despite the differences seen in electrical conductivity between the seed sizes, each seed class had low values for ec, and values for emergence that were higher than the minitable 2 summary of the analysis of variance and comparison of the mean values for thousand-seed weight (tsw) and number of seeds per kilo (nsk) in camu-camu seeds from native populations in the state of roraima, classified by size * significant at 5% probability by f-test. mean values followed by the same letter do not differ by tukey’s test at 5% probability. * significant at 5% probability by f-test. table 3 summary of the analysis of variance and comparison of mean values for water content (wc, %), electrical conductivity (ec, μs cm-1 g-1), emergence (emerg, %), speed of emergence, (se, index), seedling height (ht s, cm) and stem diameter (diam s, mm) in camu-camu seeds classified by size source of variation df mean square tsw nsk treatment 2 346,622.68* 435,142.53* replication 5 633.44 672.57 error 10 1,610,651.55 1,318.23 cv (%) 4.16 3.35 overall mean value 963.88 1,082.76 class mean value small 734.27g 1.362 medium 943.67 g 1.061 large 1,213.70 g 825 source of variation df mean square wc (%) ec μs cm-1 g-1 emerg (%) se ht s (cm) diam s (mm) treatment 2 14.67* 1.83* 433.33* 0.021* 79.12* 0.599* replication 3 0.08 0.002 5.55 0.000008 0.131 0.0012 error 6 0.29 0.001 5.55 0.000008 0.049 0.0008 mean 37.17 3.27 88.33 0.056 15.61 2.13 cv (%) 1.46 1.11 2.67 5.17 1.42 1.36 class mean values small 35.35 c 3.95 c 80 b 0.01 c 11.30 c 1.71 c medium 36.99 b 3.22 b 100 a 0.03 b 15.34 b 2.20 b large 39.17 a 2.61 a 85 b 0.14 a 20.18 a 2.48 a do nascimento et al. ‐ seeds biometry and vigor of camu‐camu 363 dência de sementes de ‘mimosa caesalpiniifolia’ benth., sobre a germinação e vigor. ‐ revista árvore, 29(6): 877-885. biruel r.p., paula r.c., aguiar i.b., 2010 germinação de sementes de ‘caesalpinia leiostachya’ (benth.) ducke (pau‐ferro) classificadas pelo tamanho e pela forma. ‐ revista árvore, 34(2): 197-204. braga n.s., morais c.s.b., rossetto c.a.v., 2012 hidratação controlada de sementes de pinhão manso. revista ciência agronômica, 43(3): 589-597. carvalho n.m., nakagawa j., 2012 sementes: ciência, tecnologia e produção. 5th edition. ‐ jaboticabal: funep, pp. 590. chagas e.a., lima c.g.b., carvalho a.s., ribeiro m.i.g., sakazaki r.t., neves l.c., 2012 propagação do camu‐camu (‘myrciaria dubia’) (h.b.k.) mcvaugh). revista agro@mbiente on-line, 6(1): 67-72. ferreira d.f., 2014 sisvar: a guide for its bootstrap pro‐ cedures in multiple comparisons. ‐ ciência e agrotecnologia, 38(2): 109-112. gonçalves a.e.s.s., 2012 compostos bioativos do camu‐ camu (‘myrciaria dubia’ mcvaugh): caracterização e atividade biologica. tese doutorado, universidade de são paulo, brazil, pp. 114. gonçalves j.v.s., albrecht j.m.f., soares t.s., titon m., 2008 caracterização física e avaliação da pré‐ embebição na germinação de sementes de sucupira‐ preta (‘bowdichia virgilioides’ kunth). ‐ cerne, 14(4): 330-334. mapa, 2009 regras para análises de sementes. ministério da agricultura, pecuária e abastecimento, secretaria de defesa agropecuária, brasília, df, brazil, pp. 399. oliveira a.b., medeiros-filho s., bezerra a.m.e., bruno r.l.a., 2009 emergência de plântulas de ‘copernicia hospita’ martius em função do tamanho da semente, do substrato e do ambiente. ‐ revista brasileira de sementes, 31(1): 281-287. pereira s.a., ferreira s.a.n., 2017 fruit and seed biom‐ etry and seedling morphology of ‘parkia discolor’ (spruce ex benth.). ‐ revista árvore, 41(2): 1-8. queiroga v p., freire r.m.m., araujo m.e.r., lima v.i., queiroga d.a.n., 2011 influência do tamanho da semente de amendoim sobre sua qualidade fisiológica. ‐ revista agro@mbiente on-line, 5(1): 30-34. silva a.c.d., smiderle o.j., oliveira j.m.f., silva t.j., 2017 tamanho da semente e substratos na produção de mudas de açaí. adv. for. sci., 4: 151-156. smiderle o.j., sousa r.c.p., 2008 teor de vitamina c e características físicas do camu‐camu em dois estádios de maturação ‐ revista agro@mbiente on-line, 2(2): 61-63. smiderle o.j., souza a.g., almeida m.s., souza a.a., 2016 caracterização biometrica e superação de dor‐ mencia de sementes de biribá no crescimento inicial de seedlings. revista congrega urcamp (cd-rom), 1: 23-34 mum established by the seed standards (mapa, 2009). as for plant height and stem diameter, the larger seeds promoted the best results, followed by the medium and small seeds respectively. there was a positive correlation between seed size and plant height and stem diameter, i.e. larger seeds gave rise to larger, more vigorous plants. these results differ from those obtained by souza et al. (2017), who reported that seeds from a population of the anauá river considered small, displayed better results for these characteristics. wagner junior et al. (2011) demonstrated that seed size has an effect on the emergence and initial development of jabuticaba seedlings (plinia cauliflo‐ ra), and that large seeds gave seedlings that were more vigorous. in açaí silva et al. (2017) in organic substrate obtained higher values for plant height and stem diameter when large seeds were used, as well as. large seeds produce more vigorous plants independent of the substrate in euterpe oleracea. alves et al. (2005) pointed out that in general, larger seeds are correlated with higher rates of initial seedling growth, which increases the probability of success during their establishment, since the rapid growth of roots and shoots allows the plant to take advantage of the nutrient and water reserves of the soil and carry out photosynthesis. wagner junior et al. (2011) stated that the germination process in many species is influenced by seed size. consequently, within the same batch, small seeds present lower seedling emergence and less vigour than the medium and large seeds. 4. conclusions the physical characterisation of camu-camu seeds shows great variability in weight, width, thickness, length and volume. the thousand-seed weight and the water content are influenced by size, with values increasing in direct proportion to the size of the seed, while the number of seeds per kilo decreases in inverse proportion. medium and large seeds give rise to plants that are more vigorous. references alves e.u., bruno r.l.a., oliveira a.p., alves a.u., paula r.c., 2005 influência do tamanho e da proce‐ 364 adv. hort. sci., 2019 33(3): 359-364 sousa r.c.p., santos d.c., neves l.t.b.c., chagas e.a., 2013 tecnologia de bioprocesso para produção de ali‐ mentos funcionais. revista agro@mbiente on-line, 7(3): 366-372. souza o.m., smiderle o.j., souza a.g., chagas e.a., chagas p.c., bacelarlima c.g., morais b.s., 2017 influência do tamanho da semente na germinação e vigor de plântulas de populações de camu‐camu ‐ scientia agropecuaria, 8(2): 119-125. vendramin d.w., carvalho r.i.n., 2013 qualidade fisiológica de sementes de pitangueira (‘eugenia uniflora’ (l.) (myrtaceae). ‐ estudos biologicos, 35(84): 59-65. vieira r.d., krzyzanoswski f.c., 1999 teste de conduti‐ vidade elétrica, pp. 1-26. in: krzyzanoswski f.c., r.d. vieira, and j.b. frança neto, (eds.) vigor de sementes: conceitos e testes. abrates, londrina, brasil, pp. 218. villanueva-tiburcio j.e., condezo-hoyos l.a., asquieri e.r., 2010 antocianinas, ácido ascórbico, polifenoles totales y actividad antioxidante, en la cás‐ cara de camu‐camu (‘myrciaria dubia’ (h.b.k) mcvaugh). ciências e tecnologia de alimentos, 30(supl.1): 151-160. vinhal-freitas i.c., garcia junior j.e., segundo j.p., vilarinho m.s., 2011 germinação e vigor de semen‐ tes de soja classificadas em diferentes tamanhos. agropecuaria técnica, 32(1): 108-114 wagner júnior a., silva j.o.c., pimentel l.d., santos c.e.m., bruckner c.h., 2011 germinação e desenvol‐ vimento inicial de duas espécies de jabuticabeira em função do tamanho de sementes. acta scientiarum agronomy, 33(1): 105-109. yuyama k., 2011 a cultura de camu‐camu no brasil. ‐ revista brasileira de fruticultura, 33(2): 335-690. yuyama k., mendes n.b., valente j.p., 2011 longevidade de sementes de camu‐camu submetidas a diferentes ambientes e formas de conservação. ‐ revista brasileira de fruticultura, 33(2): 601-607. impaginato 479 adv. hort. sci., 2018 32(4): 479-485 doi: 10.13128/ahs-21883 factors affecting in vitro propagation of some genotypes of himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.] z. nazemi rafi, h. salehi (*) department of horticultural science, school of agriculture, shiraz university, shiraz, iran. key words: auxin, conifers, cytokinins, micropropagation, pinaceae. abstract: four genotypes of himalayan cedar were grown in vitro for assessing shoot proliferation. this experiment consist three parts. initially, the explants (leafy and defoliated shoot-tips) of mature plants were disinfected and cultured on different basal media (lp, ms and wpm) that supplemented with benzyladenine (ba) at different concentrations for 6 weeks. leafless explants produced the highest number of shoots and the longest shoots for four genotypes. there was no significant difference between the culture mediums and benzyladenine concentrations. in second phase, the influence of benzyladenine (2.5, 5, 10, 20 µm) and thidiazuron (tdz) (0.4, 0.8, 1.6 µm) with combination of different auxin (naa) concentrations (0, 1, 2, 3 µm) was determined on axillary shoot proliferation of the leafless explants of four genotypes grown on wpm. for all thidiazuron concentrations, significant differences between genotypes were detected. in general, with all genotypes, the use of 0.8 μm thidiazuron in the absence or presence of auxin (2 μm) led to the highest length and number of axillary shoots per explant, respectively. finally, in another experiment, the following cytokinin treatments were investigated for axillary shoot multiplication of the cd1 genotype: thidiazuron (0, 0.1, 0.2, 0.4, 0.8 μm) and n6-[2isopentenyl] adenine (2ip) (0, 0.1, 0.2, 0.3, 0.4, 0.5 μm) in combination with benzyladenine (2.5 µm). the best results were obtained in thidiazuron (0.8 µm) with combination of benzyladenine (2.5 µm). this protocol is considered as the first successful report on culture establishment of some genotypes of mature c. deodara trees. 1. introduction the himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.], which belongs to the pinaceae family, is a beautiful, evergreen, and ornamental tree growing widely on the gradient of the western himalayas (champion et al., 1965). commercial seed bearing of c. deodara begins about 30 to 45 years of age, and good seed crops are borne every 3 years (tewari, 1994). regeneration through seeds in cedrus deodara is quite slow and undependable. generally, there is a preference for propagation of mature trees, which helps improve afforestation management, breeding projects and production of elite tree genotypes. however, maturation of most tree species is a major limiting factor for the use of micropropagation in (*) corresponding author: hsalehi@shirazu.ac.ir citation: nazemi rafi z., salehi h., 2018 factors affecting in vitro propagation of some genotypes of himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.]. adv. hort. sci., 32(4): 479-485 copyright: © 2018 nazemi rafi z., salehi h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2017 accepted for publication 18 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 479-485 480 afforestation projects (lin et al., 1991). there are a few reports on in vitro propagation of mature conifers in the last 20 years (gupta and durzan, 1985; dumas and monteuuis, 1995; parasharami et al., 2003; andersone and ievinsh, 2005; malabadi and van staden, 2005; cortizo et al., 2009; de diego et al., 2010). few reports on in vitro propagation of cedars (cedrus libani a. rich. and c. atlantica manetti) are available (piola and rohr, 1996; piola et al., 1998, 1999; renau-morata et al., 2005). there are only two reports on the in vitro culture of deodar cedar with the sole use of the seeds (bhatnagar et al., 1983; tamta and palni, 2004). among regeneration methods, axillary bud induction is preferable for most maintenance of genetic stability and less mutation risk (vasil and vasil, 1980; pierik, 1987). the enhancement of reliability of tissue culture system is being achieved through improving of medium component or correction of the conditions of environment, or both. however, the genotype has a significant impact on the accuracy and repeatability of tissue culture and its effect must be evaluated (sul and korban, 1994). in this domain, cytokinins and auxins play important roles for maintenance and acceptable growth of cultures. during the recent decades, several synthetic compounds have been introduced for induction of regeneration potential of plants (guo et al., 2011). among the compounds, tdz (tang and newton, 2005) and ba (datta et al., 2006) are highly effective on pinaceae family such as eastern white pine and lodgepole pine, virginia pine, red spruce, and taxus wallichiana zucc., respectively. to our best knowledge, no report document the in vitro vegetative regeneration of this species from mature tissues. our goal was to build methods for the in vitro propagation of c. deodara from adult trees, and to show the effect of genotype on its micropropagation. 2. materials and methods experiment 1. plant materials actively growing shoots (4-6 cm long) were collected from mature (20-25 years old) c. deodara trees (genotype not specified) in a seed orchard near the school of agriculture, shiraz university, iran. this was done from september 2015 to november 2016. the shoots were wrapped with wet paper toweling, enclosed in plastic bags and then kept at 4°c until 1 day before use. surface sterilization of explants in this experiment, two kinds of explants with the similar length were used. the first type of explants retained their needles (leaves) to full length (fig. 1 a). in the second type of explants, the leaves were trimmed to a quarter of their initial length (fig. 1 a). both types of explants were soaked in tap-water for at least 2 h. then, they were submerged for 30 minutes in an aqueous solution of 2% benomyl to reduce fungal contamination. afterwards, they were treated with 70% ethanol for 2 minutes followed by 15% clorox (containing 5.25% sodium hypochlorite) with 0.2% ‘tween-20’ for 15 minutes for surface sterilization. finally, they were rinsed three times with sterile distilled water. both kinds of explants were cut into 1-2 cm pieces under sterile conditions and subsequently cultured on different nutrient media. culture establishment the explants were cultured on three different culture media: lp (quoirin and lepoivre, 1977), woody plant medium [wpm] (lloyd and mccown, 1980) and ms (murashige and skoog, 1962), that were supplemented with benzyladenine (ba) at different concentrations (0, 0.63, 1.25, 2.5, 5 µm). then, the cultures were placed in a growth chamber at 25±2°c under a 16 h photoperiod provided by cool white fluorescent lamps (30 μm m-2 s-1) for 6 weeks. at the end, the number and length of proliferated shoots were measured. experiment 2. plant materials and aseptic culture this experiment consisted of two phases. in the first phase, the effect of different growth regulators on axillary shoot proliferation of four genotypes was fig. 1 in vitro proliferation of cedrus deodara (roxb. ex lamb) g. don. (a) different types of explants: explants retained their needles (leaves) and, defoliated explants; (b) axillary shoot proliferation on wpm medium containing 0.8 μm tdz and 2 μm naa (after 4 weeks); (c) elongation of axillary bud on em medium after 90 days. nazemi rafi and salehi in vitro propagation of himalayan cedar 481 studied. in the second phase, axillary shoot multiplication of genotype cd1 was studied. to examine the effect of genotype on proliferation rate, 110 to 130 actively growing shoots with 4-6 cm length were collected from four mature 20-25 year-old c. deodara trees (genotypes cd1 to cd4) in a seed orchard near the school of agriculture, shiraz university, iran. the shoots were defoliated, cut into 1-2 cm in length pieces and used as initial explants (n=114 per genotype). surface sterilization was carried out in the same manner as in experiment 1. for culture establishment, the explants were cultured on wpm medium supplemented with growth regulators [ba, naa and thidiazuron (tdz)] for shoot induction and proliferation. the medium was supplemented with ba (0, 2.5, 5, 10, 20 µm) and tdz (0, 0.4, 0.8, 1.6 µm) alone or in combination with naa (1, 2, 3 µm). the effect of tdz and ba on shoot proliferation of four genotypes (cd1 to cd4) was investigated. the conditions, under which the cultures were incubated, were same to those of experiment 1. elongation of induced shoots axillary shoots formed on explants (from genotype cd1) were isolated and transferred to elongation media (em). the em was growth regulator-free half strength wpm, and it was supplemented with 3 g l-1 activated charcoal (ac), 15 g l-1 sucrose and 8 g l-1 agar. the explants were maintained in the culture medium, and then subcultured into glass jars (150 ml) containing 40 ml of the fresh em. the environmental conditions were same as that in culture establishment. the subculturing procedure was repeated every 4 weeks for 3 months. the culture conditions (temperature and light) were same as those used for culture establishment experiment. shoot multiplication after 3 month, the shoots grown on em were cut to the same length and then transferred to shoot multiplication medium containing tdz (0, 0.1, 0.2, 0.4, 0.8 µm) and 2ip (0, 0.1, 0.2, 0.3, 0.4, 0.5 µm) in combination with 2.5 µm ba for 6 weeks. statistical analysis all experiments were conducted as factorial based on a completely randomized design with four replications, each replicate comprised of four jam glasses, four explants per glass. all experiments were repeated twice. shoot proliferation rate and shoot length were recorded at the end of the fourth week. spss statistical software was used for analyzing data, and the one-way anova with tukey’s test (p<0.05) was used for comparing means. three-way anova was applied to examine the interactions of tdz, ba and genotype. 3. results experiment 1 the effects of different culture media (lp, ms and wpm) on two types of explants were investigated. the defoliated explants showed the best results, and there was a significant difference for shoot proliferation between defoliated and leafy explants, in all three culture media. the lowest frequency in shoot proliferation belonged to the leafy explants in ms medium (0.06). according to the results, wpm medium showed the highest number and length of proliferated shoots, but without significant difference as compared with lp. moreover, leafless explants produced higher and longer proliferated shoots than leafy explants. there was no significant difference between different amounts of ba on length and number of proliferated shoots (data not shown). experiment 2 when all four genotypes (cd1, cd2, cd3, cd4) were grown at different levels of cytokinin treatments, shoot proliferation from leafless explants was observed. the highest mean number of shoots per explant was observed at 0.8 μm thidiazuron for genotypes cd1 (4.6), cd2 (4.4), cd3 (2.4), and at 0.4 μm thidiazuron for cd4 (2.4) (fig. 2). genotypes cd1 and cd2 were the most responsive genotypes over all cytokinin treatments. furthermore, the highest fig. 2 effect of tdz concentrations on shoot proliferation of four genotypes of cedrus deodara. bars with the same letters indicate no significant difference at tukey test (p=0.05). adv. hort. sci., 2018 32(4): 479-485 482 mean number of axillary shoots per explant of all genotypes (4.31) was obtained when 0.8 μm tdz was used in combination with 2 μm naa and the highest mean length of axillary shoots of all genotypes (11.2 mm) was obtained when 0.8 μm tdz was applied alone (tables 1, 2, fig. 1 b). naa had a positive effect on quality attributes (like color, vitality, and size). however, increasing the concentration of naa to 3 μm reduced the number and length of proliferated shoots (tables 1, 2). then, axillary shoot multiplication of genotype cd1 was studied (fig. 3). the highest mean number of axillary shoots per explant (4.13) and mean length of axillary shoots (10.38 mm) were obtained when 0.8 μm tdz was applied (table 3). the best result of shoot proliferation obtained for cd1 genotype on wpm medium supplemented with 0.8 μm tdz (fig. 2). the same result was obtained for cd1 genotype on wpm medium supplemented with 2.5 μm ba (fig. 4). 4. discussion and conclusions propagation of conifers from mature explants has always been difficult. in general, most studies on the induction of embryogenesis and/or organogenesis in conifers includes the culture of seed or zygotic tismeans with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at tukey’s test (p=0.05). table 1 effects of different concentrations of tdz, ba and naa on the number of proliferated shoots in cedrus deodara treatments naa mean values 0 1 2 3 control 2.41± 0.02 lm† 2.16±0.02 pq 2.31±0.05 mno 1.97±0.01 r 2.21 f tdz (µm) 0.4 3.75±0.00 b 3.31±0.03 e 3.44±0.03 de 3.06±0.03 f 3.39 b 0.8 3.38±0.03 de 3.50±0.00 cd 4.31±0.03 a 3.63±0.03 bc 3.70 a 1.6 2.25±0.00 nop 2.50±0.00 jkl 2.63±0.03 ij 2.00±0.00 r 2.34 ef ba (µm) 2.5 2.69±0.03 hi 3.06±0.03 f 3.40±0.03 de 2.50±0.00 jkl 2.91 c 5 2.38±0.03 lmn 2.44±0.03 klm 2.44±0.03 klm 2.56±0.03 ijk 2.45 e 10 2.44±0.03 klm 2.88±0.03 g 2.81±0.03 gh 2.31±0.03 mno 2.61 d 20 2.25±0.00 nop 2.38±0.03 lmn 2.19±0.03 opq 2.06±0.03 qr 2.22 f mean values 2.66 b 2.71 ab 2.87 a 2.45 c 2.67 means with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at tukey’s test (p=0.05). table 2 effects of different concentrations of tdz, ba and naa on the length of proliferated shoots in cedrus deodara treatments naa mean values 0 1 2 3 control 0.0 5.80±0.06 h 5.55±0.06 hij 4.88±0.04 klm 4.50±0.09 mn 5.18 d tdz (µm) 0.4 8.75±0.05 c 6.92±0.01 fg 8.12±0.09 d 7.12±0.12 fg 7.73 b 0.8 11.20±0.14 a 7.75±0.07 de 9.56±0.05 b 6.81±0.22 g 8.83 a 1.6 4.96±0.07 klm 5.24±0.08 ijk 5.20±0.08 i-l 4.67±0.02 lm 5.02 d ba (µm) 2.5 6.82±0.16 g 7.32±0.10 efg 5.57±0.10 hij 5.08±0.08 jkl 6.20 c 5 7.15±0.19 fg 7.43±0.10 ef 5.93±0.11 h 5.18±0.15 i-l 6.42 c 10 5.65±0.16 hi 5.93±0.08 h 4.07±0.09 no 3.12±0.08 q 4.69 d 20 3.44±0.12 pq 3.84±0.17 op 3.90±0.13 op 2.16±0.07 r 3.34 e mean values 6.62 a 6.17 ab 5.79 b 4.79 c 5.84 fig. 3 multiple shoot formation on subculture of cedrus deodara on wpm with 0.8 μm tdz in combination with 2.5 μm ba. nazemi rafi and salehi in vitro propagation of himalayan cedar 483 sues (tang et al., 2006; humánez et al., 2011). to our best knowledge, this is the first report of successful induction of axillary shoots on explants taken from adult c. deodara. there have been few reports on the effect of different kinds of basal media on the axillary shoot proliferation in cultures of explants from mature conifer trees (andersone and ievinsh, 2002; de diego et al., 2010; renau-morata et al., 2005). in the present investigation, shoot proliferation rates were generally greater on basal wpm than on basal lp and ms media. shoot buds cultured on ms medium presented the lowest organogenic response, and this was perhaps as a result of the comparatively high nitrate concentration as compared to the lp medium or wpm. tuskan et al. (1990) showed that the extra nitrate had a negative effect on the organogenic response during micropropagation. therefore, low nitrogen content of the medium is a major factor for promoting organogenesis in conifer species (tang et al., 2001; schestibratov et al., 2003). explant type was an important factor affecting axillary bud proliferation in in vitro culture. piola et al. (1998) demonstrated that the accumulation of aba in needles seems to be the major cause of bud dormancy in micropropagation of c. libani. in this experiment, defoliated explants of c. deodara also showed better response on proliferation medium. in in vitro bicentennial cedar micropropagation, it was found that the accomplishment of the protocol depends on genotype (renau-morata et al., 2005). among two cytokinin treatments examined, tdz induced the highest number of proliferated shoots for all genotypes. after investigating the effect of three tdz concentrations on shoot proliferation of four genotypes, the significant difference for their interaction was found. however, in our investigation, ba showed no significant genotypic differences for shoot proliferation. tdz has gotten more attention in recent years due to its ability to assist in vitro regeneration of conifers (mathur and nadgauda, 1999; sul and korban, 2004; renau-morata et al., 2005; tang and newton, 2005; cortizo et al., 2009; de diego et al., 2010; humánez et al., 2011). tdz can decrease the enzyme activity related to oxidative stress during formation of adventitious shoots (tang and newton 2005). recent reports on mature stone pine displayed the superiority of tdz over other cytokinins in advancing axillary shoot proliferation (cortizo et al., 2009). it was shown that high concentrations of cytokinin in the medium, particularly ba led to the low regeneration response, which may be attributed to the toxic effects of high concentrations of cytokinins (sarmast et al., 2012). in this report, the presence of naa with either tdz or ba improved the incidence of shoot organogenesis in adult tissues of c. deodara. this has been also observed in other conifer species (sul and korban, 2004; zhu et al., 2010). as this study shows, the type of explants and culture media has a very important role in success of culture establishment of cedrus deodara. it was proven that defoliation of the explants has positive effect on shoot proliferation. the effects of genotype and growth regulators on proliferation of axillary in each column, means with the same letters indicate no significant difference at tukey’s test (p=0.05). table 3 effects of different concentrations of tdz and 2ip in combination with 2.5 µm ba on shoot multiplication of cedrus deodara treatments number of proliferated shoots length control 0.0 2.44± 0.07 e 6.69± 0.12 e tdz (µm) 0.1 3.38± 0.16 bc 7.26± 0.16 de 0.2 3.44± 0.21 bc 7.80± 0.27 cd 0.4 3.88± 0.16 ab 9.93± 0.24 a 0.8 4.13± 0.13 a 10.38± 0.11 a 2ip (µm) 0.1 2.50± 0.20 e 6.58± 0.11 e 0.2 2.69± 0.12 de 7.38± 0.18 de 0.3 3.00± 0.10 cde 7.93± 0.21 cd 0.4 3.25± 0.18 bcd 8.38± 0.15 bc 0.5 3.56± 0.12 abc 8.88± 0.14 b fig. 4 effect of ba concentrations on shoot proliferation of four genotypes of cedrus deodara. bars with the same letters indicate no significant difference at tukey test (p=0.05). 484 adv. hort. sci., 2018 32(4): 479-485 shoots have been studied and low concentration of tdz combined with naa has positive effects on the number of proliferated axillary shoots. furthermore, two of the four genotypes showed better response to cytokinins. acknowledgements we are grateful to shiraz university for supporting this study. also, we would like to thank prof. morteza khosh-khui for his guidance and advice. references andersone u., ievinsh g., 2002 changes of morphogenic competence in mature pinus sylvestris l. buds in vitro. ann. bot., 90: 293-298. andersone u., ievinsh g., 2005 in vitro regeneration of mature pinus sylvestris buds stored at freezing temperatures. biol. plant., 49: 281-284. bhatnagar s., singh m., kapur n., 1983 preliminary investigations on organ differentiation in tissue cultures of cedrus deodara and pinus roxburghii. indian j. exp. biol., 21: 524-526. champion s.h., seth s.k., khattak g., 1965 forest types of pakistan. pakistan forest institute, peshawar, pakistan, pp. 238. cortizo m., de diego n., moncaleán p., ordás r.j., 2009 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cult., 20: 47-52. vasil i.k., vasil v., 1980 clonal propagation. int. rev. cytol. suppl., 11a: 146-173. zhu l.h., wu x.q., qu h.y., ji j., ye j.r., 2010 micropropagation of pinus massoniana and mycorrhiza formation in vitro. plant cell, tiss. org. cult., 102: 121-128. impaginato 205 adv. hort. sci., 2020 34(2): 205­212 doi: 10.13128/ahsc­8255 aloe vera coatings maintain antioxi­ dants of fig (ficus carica l.) fruit during storage a. mirshekari 1 (*), b. madani 2, m. wall 3, a.r. biggs 4 1 department of agronomy and plant breeding, yasouj university, yasouj, iran. 2 horticultural crops research department, natural resources research and education center of hormozgan, areeo, bandar abbas, iran. 3 u.s. department of agriculture, agricultural research service, hilo, hi, usa. 4 west virginia university, tree fruit research and education center, kearneysville, wv, usa. key words: coating, ficus carica, phytochemicals, quality. abstract: demand for fig fruit is increasing because of its high antioxidant capacity and health benefit for human. so, interest in maintaining bioactive components of fruit has been increased. however, antioxidant capacity of fruits decreases during storage. fresh fruits of the well­known fig (ficus carica l.) cul­ tivar ‘siyah’ were grown in fars province, iran, treated with different concen­ trations of aloe vera gel prior to being placed into cold storage. we examined fruit for firmness, soluble solids contents, anthocyanin concentrations, total phenolic compounds, flavonoid concentrations, ascorbic acid content, antioxi­ dant activity, phenylalanine ammonia­lyase and superoxide dismutase activi­ ties. figs coated with aloe vera gel maintained higher firmness, anthocyanin concentrations, total phenolic compounds, flavonoid concentrations, ascorbic acid content, and antioxidant capacity than the control. phenylalanine ammo­ nia­lyase and superoxide dismutase activities were enhanced with aloe vera gel treatments. the results showed that aloe vera gel treatments could be an alter­ native to the current chemical protocols for preserving nutraceutical traits of fig fruit by maintaining antioxidant capacity during storage. 1. introduction fig fruit is an important part of the mediterranean food and is rich in fibers and antioxidant compounds (arvaniti et al., 2019). antioxidant compounds are rich in fruits like fig and can prevent free radical forma­ tion. foods which are rich in phytochemicals decrease incidence of dis­ ease and maintain body healthful (singh, 2016). for this reason, it is important to use these phytochemicals in our diet (singh, 2016). it has been detected that anthocyanin intake can prevent heart diseases and promote anti­carcinogenic and hypoglycemic activities. thus, high con­ tent of anthocyanin has made fig attractive for consumers. also, figs con­ (*) corresponding author: a_mirshekari@yu.ac.ir citation: mirshekari a., madani b., wall m., biggs a.r., 2020 ­ aloe vera coatings maintain antioxi‐ dants of fig (ficus carica l.) fruit during storage. ‐ adv. hort. sci., 34(2): 205­212. copyright: © 2020 mirshekari a., madani b., wall m., biggs a.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 may 2019 accepted for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 205­212 206 tain high level of flavonoid which reduces oxidative stress (reyes­avalos et al., 2016). delicate epidermal tissue, climacteric behavior and extensive softening make fig fruit susceptible to the wounding and spoilage which limit shelf life of fruit (bahar and lichter, 2018). also, it has been confirmed that antioxidant activity of fruits decreases during storage (madani et al., 2016). therefore, maintenance of antioxidants of fig fruit during storage is important, because of their health benefits for human. several strategies have been implemented to maintain bioactive compounds of fruit during stor­ age. among them, coatings are popular because of their biodegradable and non­toxic materials (reyes­ avalos et al., 2016; allegra et al., 2017). they provide a barrier against gas transfer, which delays ripening and improves taste and texture (khaliq et al., 2019). the aloe vera gel compounds are mainly polysaccha­ rides, minerals, sugars, vitamins, and antioxidant agents like phenolic compounds (rasouli et al., 2019). application of aloe vera gel has received increased attention by the food industry due to its effectiveness for increasing shelf­life of fresh prod­ ucts and increasing antioxidant activity (sogvar et al., 2016). moreover, aloe vera gel maintained antioxi­ dant activity of button mushrooms (mirshekari et al., 2019) and sapota fruit (khaliq et al., 2019). however, few studies have addressed the effects of edible coatings on quality of fig under cold storage condi­ tions. application of alginate­chitosan decreased fun­ gal contamination and increased firmness of fig fruit under cold storage (reyes­avalos et al., 2016). thus, the objective of this study was to determine the effects of aloe vera gel application at different concentrations for maintaining phytochemicals of fig fruit during cold storage. we examined several bene­ ficial phytochemicals (anthocyanin, total phenolic compound, total flavonoid concentration, and ascor­ bic acid), antioxidant activity, and phenylalanine ammonia­lyase and superoxide dismutase activities. 2. materials and methods plant materials and treatments mature harvested figs (ficus carica l.), cv. siyah (black, firm with 13% soluble solids content) grown in fars province, iran, were transported to the faculty of agriculture of yasouj university. mature leaves of greenhouse­grown aloe vera plants were excised. the leaf matrix was detached from the outer cortex, and then the hydroparenchyma was mixed in a blender. the mixture was filtered through cheese­ cloth to remove the fibers, and the filtrate constitut­ ed fresh aloe vera gel (sogvar et al., 2016). defect­ free fruits with uniform size and color were divided into four groups, each group received one of four treatments: 1) control (0%), 2) aloe vera gel 1:3 (25%), 3) 1:1 (50%), and 4) 3:1 (75%). after that, all fruits were placed into plastic trays (285 x 125 x 65 mm) over­wrapped with plastic films [0.02 mm­thick polyvinyl chloride (pvc)]. trays with fruit were stored at 2±1°c and 85­90% rh for 15 days (cold storage). analyses were performed after 0, 3, 6, 9, 12, and 15 days of cold storage. firmness, soluble solids (ssc) and ascorbic acid con‐ tent a digital fruit hardness tester (step systems gmbh, germany) with a 5­mm diameter probe was used to determine fruit firmness (expressed in newtons (n). five g of the homogenate of a compos­ ite sample using a kitchen blender (nu­777, nautiunl, japan) with 40 ml of distilled water was used for analyses of chemical parameters (ranganna, 1986). then samples filtered through cotton wool. ssc was measured with a handheld refractometer (atago­ pal1, tokyo, japan) and expressed as percent. ascorbic acid content was determined using the 2, 6­ dichlorophenolindophenol dye titration method described by mirshekari et al. (2017). five g of fruit tissues homogenized in 90 ml of 3.0% metaphosphor­ ic acid solution. an aliquot of the sample (10 ml) was titrated against 2, 6­dichlorophenolindophenol dye until a pink color persisted for 15 s and results expressed as mg ascorbic acid per 100 g of fresh weight (fw). anthocyanin and flavonoid concentration (fc) anthocyanin content in fruit samples was mea­ sured by the ph differential method described by hassanpour (2015). two g of samples was added to the 20 ml of methanol containing hcl (1%). the mix­ ture was centrifuged at 17,000 g for 15 min at 4 ◦c. absorbance of supernatant was measured in a spec­ trophotometer (shimadzu, usa) at 530 and 700 nm in buffers at ph 1.0 and 4.5, using following formula: a = [(a530­a700) ph1.0 ­ (a530 ­a700) ph4.5] results were expressed as mg of cyanidin­3­o­glu­ coside equivalents per 100 g of fw. fc was measured according to the method described by saba and sogvar (2016). extraction from four g of the sample was done using 50 ml methanol. one ml aliquot of catechin standard solution (0­100 mirshekari et al. ‐ aloe vera gel maintains antioxidants of fig fruit 207 mg l­1) or samples were added to 10 ml volumetric flasks containing 4 ml water. initially 0.3 ml of 5% nano2 was added to the flask, following 0.3 ml of 10% alcl3 was added after 5 min, and then 2 ml of 1 m naoh was added to the mixture. immediately, the solution was diluted to a final volume of 10 ml with water and mixed thoroughly. the absorbance was measured at 510 nm, using a spectrophotometer (uv/vis perkin elmer, lambda ez201, usa) and result was expressed as mg catechin equivalents per 100 g of fw. total phenolic compounds (tpc) and antioxidant activity fruit extract was prepared using the method described by ong et al. (2013). fruit tissue (2 g) was homogenized in a glass tube with 10ml of methanol (80%). the mixture was then incubated at 45∘c for 1 h. for tpc measurement 0.1 ml of the crude extract solution was placed in a test tube and 0.1 ml distilled water in a test tube served as the control (blank). then six millilitres of water was added to the sample and blank. after that, 0.5 ml undiluted folin­ ciocalteu reagent was added to the mixtures. between 30 s and 8 min later, 1.5 ml saturated sodi­ um carbonate was added. then 1.9 ml water was added to the solutions to give a final volume of 10 ml and the mixture vortexes and incubated for 2 h at 35∘c. the absorption of tpc was determined at 765 nm using a spectrophotometer. a calibration stan­ dard curve was established using gallic acid. tpc was determined against the standard gallic acid calibra­ tion curve and the absorbance value was converted to mg of gallic acid equivalents (gae) per 100 gram of fresh weight (mg gae 100 g­1 fw) (ong et al., 2013). antioxidant capacity (ferric reducing antioxidant power (frap) and trolox equivalent antioxidant capacity (teac) assays were determined spectropho­ tometrically, according to benzie and strain (1996) and kerem et al. (2006), respectively. the results were expressed as fe2+ equivalents mm kg­1 for frap and μm trolox equivalents for teac in 100 g of fw. phenylalanine ammonia‐lyase (pal) and superoxide dismutase (sod) activities fig samples were frozen in liquid nitrogen and then stored at ­80°c until analysis. each frozen sam­ ple (10 g) was ground with a mortar and pestle and used to determine pal and sod activity. two g of samples was homogenized in a 4 ml solution con­ taining 0.05 mol l­1 tris­hcl buffer (ph =7.5), 3 mmol l­1 mgcl2 and 1 mmol l­1 edta at 4◦c. the homogenate was then centrifuged at 25,000 g for 20 min at 4°c and the supernatant was used as the crude extract for sod and pal assays (maghoumi et al., 2013). measurement of pal activity was per­ formed at 290 nm, according to the method described by aghdam et al. (2012). pal activity was evaluated as nm cinnamic acid h­1 mg­1 protein. the procedure for assay of sod was performed using methods described by (maghoumi et al., 2013). sod activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazoli­ um. the absorbance by the reaction mixture was read at 560 nm. concentration of protein of the extracts was determined according to bradford (1976) with bovine serum albumin as a standard. enzyme activity was expressed as unit mg­1 protein. sensory evaluation texture, taste and overall quality of samples ana­ lyzed by ten trained panelist after 15 days at cold storage (2±1°c) and transferring fruits to the room temperature for sensory analysis. the sensory were evaluated using a hedonic scale 1­5, where 1= very poor, 2= poor, 3= fair, 4= good and 5= excellent. experimental design and statistical analysis all experiments were conducted within a com­ pletely randomized design (crd). data were pooled before analysis and the whole experiment was repeated three times. in each biological and technical experiment 328 fruit were used. there were four replicates per treatment in each experiment. data were subjected to analysis of variance using the statistical analysis system (sas, anova procedure) version 8.2 (sas institute inc., cary, nc, usa). the means were compared with the duncan’s multiple range test (dmrt) at significance level of 0.05. 3. results and discussion firmness, ssc and ascorbic acid content firmness is the key factor of quality and which changes during ripening (madani et al., 2014). fruit firmness was reduced during storage irrespective of treatment, but aloe vera gel treated fruits softened more slowly (fig. 1a). short postharvest life of fig fruit is because of softening and epidermal cracking (villalobos et al., 2016). aloe vera film acts as a barri­ er which prevents o2 uptake on fruit, thereby decreases softening and ripening processes (hassanpour, 2015). moreover, polygalacturonase and pectin methylesterase activity increases during ripening, and this causes fruit softening (madani et adv. hort. sci., 2020 34(2): 205­212 208 al., 2014). the results are comparable with reyes­ avalos et al. (2016) who indicated that alginate­chi­ tosan coating could maintain firmness of fig fruit dur­ ing storage. aloe vera gel might decrease polygalac­ turonase and pectin methylesterase activity and thereby maintain firmness of fig fruit during cold storage. there were significant differences in the ssc among treatments. ssc of control fruits increased during storage (fig. 1b). however, aloe vera gel treated fruits had the lowest ssc compared to the control during cold storage. the increase in ssc might be related to the solubilization of polyuronides and hemicelluloses of fruit cell walls and hydrolysis of insoluble polysaccharide into simple sugars (tanada­ palmu and grosso, 2005). these results are compara­ ble with rasouli et al. (2019) and (martínez­romero et al., 2017) who mentioned that aloe vera gel reduced the ssc of orange and plum fruit, respective­ ly. aloe vera gel might decrease respiration rate and ssc in fig fruit. during cold storage, ascorbic acid content of con­ trol fruit decreased from 24.75 mg per 100 g of fw to 7 mg per 100 g of fw (fig. 1c). however, aloe vera gel treated fruits maintained ascorbic acid content relative to the control. ascorbic acid is the most important antioxidant which decreases the damage of ros (rasouli et al., 2019). autoxidation causes ascorbic acid losses during storage when combines with oxygen in the air (baraiya et al., 2015). aloe vera gel might causes a barrier layer for gas and decreases oxidation of ascorbic acid which caused by ascorbate oxidase enzyme in the presence of oxygen (sogvar et al., 2016). since ascorbic acid has beneficial effect on human health, the positive effects of aloe vera gel on maintaining ascorbic acid content of fig fruit can be interested for nutraceutical purposes. anthocyanin, fc, tpc and antioxidant capacity fig fruit is rich in phenolic compounds, which are responsible for antioxidant activity (ercisli et al., 2012). anthocyanin of non­treated fruits at harvest and after 15 days of cold storage was 11.12 and 21.45 of mg cyanidin­3­o­glucoside equivalents per 100 g fw, respectively. (fig. 2a). aloe vera gel 50% and 75% treatments increased anthocyanin after 15 days at cold storage relative to the control. (fig. 2a). moreover, fc of aloe vera gel treatments was signifi­ cantly higher than control during cold storage (fig. 2b). tpc decreased during cold storage regardless of treatments; but tpc of treated fruits were significant­ ly higher than that of non­treated fruits (fig. 2c). from a biological and nutritional perspective, the antioxidant capacity of anthocyanin is important (wang et al., 1996); therefore, maintaining antho­ cyanin is potentially beneficial. it has been also reported that strawberries, and bush blueberry treat­ ed with chitosan maintained higher levels of antho­ cyanin (wang and gao, 2013; chiabrando and giacalone, 2015). flavonoids are water soluble polyphenolic mole­ cules which have health promoting effects like antioxidants, radical scavengers, anti­mutagenic, anti­inflammatory, anti­carcinogen, and anti­depres­ sant (singh, 2016). moreover, with their antioxidant activity, they increase shelf life of fruits and vegeta­ bles (ververidis et al., 2007). nair et al. (2018) showed higher fc in guava fruit treated with chitosan coatings. also, (khaliq et al., 2019) indicated that fig. 1 ­ firmness (a), soluble solid concentration (ssc) (b) and ascorbic acid content (c) values in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%). fruit were stored at 2°c. vertical bars represent standard error of means of three experiments with four replicates per experiment. mirshekari et al. ‐ aloe vera gel maintains antioxidants of fig fruit 209 aloe vera gel treated sapodilla fruits had higher fc. phenols are one of the most important antioxi­ dant compounds of fruits and vegetables. the appli­ cation of coatings like aloe vera gel might delay senescence and decrease tpc loss during storage (rasouli et al., 2019). increase in anthocyanin, fc and tpc of fruit treated with aloe vera gel is comparable to those reported previously by hassanpour (2015) for raspberry fruit treated with aloe vera gel. this may be related to the persistent biosynthesis of anthocyanin, flavonoids and tpc after harvesting. also, enzymes which are involved in biosynthesis process of tpc, flavonoids and anthocyanin like pal might be up regulated with aloe vera gel treatment. the higher nutraceutical compounds detected in loquat fruits coated with chitosan could be related to fig. 3 ­ antioxidant capacity with trolox equivalent antioxidant capacity (teac) (a) and ferric reducing antioxidant power (frap) (b) in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%) during cold storage at 2°c. vertical bars represent standard error of means of three experiments with four replicates per experiment. the lower ros due to the long­time physiological stress of storage (petriccione et al., 2015). therefore, aloe vera treated fruit might have a protective effect on nutraceutical compounds in delaying their oxida­ tive processes and bio­transformation during stor­ age. figs treated with aloe vera gel had higher antioxi­ dant activity during cold storage, which indicated by teac and frap. however, antioxidant activity of con­ trol fruit decreased during storage (fig. 3a­b). serrano et al. (2006) observed that aloe vera gel treatment increased antioxidant activity in grape. it was supposed that antioxidant activity of aloe vera gel is related to aloe­emodin, a hydroxyan­ thraquinone present in aloe vera gel leaves and extracts (serrano et al., 2006). therefore, higher antioxidant capacity observed in aloe vera gel treat­ ed fig fruit could be related to the biochemical com­ ponents of aloe vera extract. association was stated between the bioactive compounds and antioxidant activity, with the coated samples recording higher antioxidant activity (anraku et al., 2011). fig. 2 ­ anthocyanin (a), flavonoid (b) and total phenolic concen­ trations (tpc) (c) values in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%). fruit were stored at 2°c. vertical bars represent standard error of means of three experiments with four replicates per experiment. 210 adv. hort. sci., 2020 34(2): 205­212 pal and sod pal activity in treated fruits increased during stor­ age when compared with the control (fig. 4). this indicates that aloe vera gel treatment activated enzymes that are important in biosynthetic pathways of secondary metabolites of fruit. pal is the first enzyme in phenylpropanoid pathway which catalyzes conversion of phenylalanine to trans­cinnamic acid and plays an important role in phenolic compounds biosynthesis (hassanpour, 2015). pal connects pri­ mary metabolism (shikimic acid pathway) to sec­ ondary metabolism (phenylpropanoid pathway) (razavi and hajilou, 2016). the results are compara­ ble with hassanpour (2015) who demonstrated that pal activity in raspberry fruit increased when treated with aloe vera gel. we suggest that aloe vera gel treatment might be an efficient strategy for main­ taining phenolic content in fig fruit via activation of pal. sod activity of non­treated figs was reduced dur­ ing storage. however, sod activity increased from day 3 to 6 in cold storage and decreased afterward when treated with aloe vera gel (fig. 5). superoxide dismutases (sods) which are metalloenzymes, are believed to play a crucial role in antioxidant defense because they catalyze the dismutation of o2 ­ to h2o2. however, defensive action of sod against o2 ­shows age­related changes. higher sod activity of aloe vera coating treatments have been associated with cold storage stress tolerance fruit because it neutralizes the reactivity of the superoxide radical, which is over produced under stress (bowler et al., 1992). these results are comparable with sun et al. (2010) who reported that sod activity in litchi fruit treated with chitosan was higher than control fruit. these results have suggested that aloe vera gel treatment might maintain tpc, fc and anthocyanin of fig fruit by increasing activity pal and sod enzymes. sensory evaluation according to the judges, postharvest aloe vera application did not have any negative effect on tex­ ture, taste and overall quality of fig (fig. 6). the posi­ tive effect has an important role for consumers to buy the fruit. these results are in agreement with song et al., 2013 in which aloe vera gel coated fresh cut apple had higher score than un­coated fruits. fig. 4 ­ phenylalanine ammonia­lyase (pal) activity in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%) during cold storage at 2°c. vertical bars repre­ sent standard error of means of three experiments with four replicates determinations per experiment. fig. 5 ­ superoxide dismutase (sod) activity in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%) during cold storage at 2°c. vertical bars represent standard error of means of three experiments with four replicates determinations per experiment. fig. 6 ­ sensory evaluation in figs (ficus carica l.) ‘siyah’ fruit treated with aloe vera gel (0, 25, 50 and 75%) after tran­ sferring from 15 days in cold storage to the ambient tem­ perature. mirshekari et al. ‐ aloe vera gel maintains antioxidants of fig fruit 211 thus, these results revealed that aloe vera coting could decrease the loss of the sensory characteristics of fruit. 4. conclusions this research indicated that aloe vera gel plays a positive role in maintaining tpc, anthocyanins, fc and antioxidant than control fruit. we conclude that aloe vera gel treatment could be a useful alternative to the current chemical protocols for preserving nutraceutical traits of fig fruit by maintaining antioxi­ dant capacity during storage. acknowledgements the authors 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antioxidant enzyme system, and postharvest fruit quality of strawberries (fragaria x ananassa duch.). ­ lwt­food sci. technol., 52: 71­79. impaginato 127 adv. hort. sci., 2022 36(2): 127­134 doi: 10.36253/ahsc­12801 effect of different packaging materials on shelf life and postharvest quality of tomato (lycopersicum esculentum var. srijana) s. poudel 1 (*), p. aryal 1 , m. basnet 2 1 tribhuvan university, institute of agriculture and animal science, lamjung campus, sundarbazar‐07, lamjung, nepal. 2 tribhuwan university, institute of agriculture and animal science, paklihawa campus, siddharthanagar‐1, paklihawa, rupandehi, nepal. key words: hdpe, ldpe, modified atmosphere, permeability. abstract: tomatoes, being highly perishable, experience extreme post­harvest losses due to improper packaging materials. experimentation was done to investigate the effect of different packaging materials on shelf life and quality traits of tomato var. srijana at the horticulture laboratory of the institute of agriculture and animal science, lamjung campus under a completely random­ ized design. seven treatments viz. no packaging (control), unperforated low­ density polyethylene (ldpe) bag, perforated (4 holes of 2 mm) ldpe bag, unper­ forated high­density polyethylene (hdpe) bag, perforated hdpe bag, unperfo­ rated non­woven fabric bag, and perforated non­woven fabric bag with 3 repli­ cations were used. tomatoes were evaluated for weight loss, color develop­ ment, total soluble solids, titratable acidity, ph, and shelf life. among the treat­ ments, the lowest percentage of weight loss (0.66%) was observed on toma­ toes packed in an unperforated hdpe bag, however, it had a higher fungus attack. no packaging group showed rapid shriveling of fruits with the highest percentage of weight loss (14.70%). although packaging in a non­woven fabric bag was better than control, it showed a higher percentage of weight loss than plastic packaging due to its high permeability to gases and water vapor. the tss and ph values were found to be higher and ta to be lower in no packaging compared to other packagings. the longest shelf life of tomatoes was observed in perforated ldpe (24 days), followed by hdpe (23 days) whereas the lowest was observed in control (16 days). overall, the perforated plastic packaging was found best among all treatments with no significant variation among perforat­ ed hdpe and perforated ldpe for maintaining qualities of tomatoes and longer shelf life. 1. introduction tomato (lycopersicon esculentum mill.) is considered as one of the widely grown horticultural crops across the world that ranks second in importance to potato. (fao, 1989). the area under tomato cultivation in (*) corresponding author: saritapaudel12345@gmail.com citation: poudel s., aryal p., basnet m., 2022 ­ effect of different packaging materials on shelf life and postharvest quality of tomato (lycopersicum esculentum var. srijana). ­ adv. hort. sci., 36(2): 127­134. copyright: © 2022 poudel s., aryal p., basnet m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 february 2022 accepted for publication 4 april 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12801 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 127­134 128 nepal occupies about 21,747 ha giving a total pro­ duction of 413,761 mt (moald, 2019/2020). despite this production, post­harvest loss in tomatoes accounts for about 30­33% of total production. (tiwari et al., 2020). this loss has been attributed to a broad number of factors among which improper packaging, storage facilities, and poor means of transportation and roads constitute a major part. reduction of post­harvest losses of any perishables is of utmost importance as it is hard to increase a 10% production than to reduce a 10% loss without laying additional land for cultivation (bhattarai and gautam, 2006). post­harvest quality maintenance is of great chal­ lenge in developing countries like nepal where post­ harvest technologies are not so far developed or available in every part of the country. even the avail­ abilities of some costly technologies are not afford­ able to smallholder consumers, sellers and farmers. so, there is a need to explore every possible cost­ effective way to minimize the prevailing post­harvest losses. one of the better alternative and viable options for improving shelf life and reducing quality degradation of the produce inexpensively is modified atmospheric packaging (map) (kader et al., 1989). map is achieved by using various packaging materials like ldpe, hdpe that result in alteration of the gaseous environment inside the packages by manipu­ lating the levels of o2, co2, n2, and c2h4. the perme­ ability of film decides the level of o2 and co2 inside it. if the film is of correct permeability, a preferable equilibrium modified atmosphere can be entrenched where the o2 and co2 transmission rate via package can balance the product’s respiration (day, 2001). reduced o2 and/or elevated co2 levels can reduce respiration, retard ethylene production and ripening, impede textural softening and slow down biochemi­ cal changes associated with ripening and thus ulti­ mately resulting in an extension of shelf life (farber, 1991). low­density polyethylene (ldpe) and high­density polyethylene (hdpe) are the materials that are com­ monly used for map. the non­woven fabric bag is the packaging material that has recently evolved in the nepalese market and has been replacing plastic pack­ ages. these packaging materials are easily available and purchasable in the nepalese market. different studies suggest that the use of improper packaging and storage has significantly shortened the shelf­life of tomatoes. hence, to extend the storage life cou­ pled with quality maintenance cost­effectively, this research is focused on identifying the most suitable and effective packaging for tomatoes. 2. materials and methods description of the study area the research was conducted in the horticultural laboratory of the institute of agriculture and animal science (iaas), lamjung campus located in lamjung, nepal. it lies at an elevation of 800 meters with 28.127°n latitude and 84.4167°e longitude. the mean annual rainfall of the area is 700 mm. within a year, the average maximum temperature occurs dur­ ing june at around 35.7°c whereas the average mini­ mum temperature occurs during january at around 14.5°c. experimental materials breaker stage defect­free tomato fruits of srijana variety were harvested from the field of lamjung campus during morning time and taken to the horti­ cultural lab of lamjung campus. the harvesting was done by handpicking with leaving a small pedicel above the fruit. the fruits were free from defects such as sun scorch, bruises, and pest or disease dam­ age. then, these tomatoes were cleaned, washed, and dried before preparing each sample. environmental parameters the maximum temperature of the experimental lab varied from 33­28°c, minimum temperature from 17­22°c, and relative humidity from 48­75% (fig. 1). treatments and experimental design the experiment was carried out with seven treat­ ments elaborated be laid in a completely randomized fig. 1 ­ graphical representation of temperature and relative humidity during storage period of tomato. poudel et al. ‐ extended shelf life of tomato on packaging 129 design with three replications. thus, the overall experimental units were 21. the washed tomato fruits were divided into 21 groups each with a half kilogram then subjected to various treatments and kept in the ambient environment for its post­harvest quality assessment and physiological weight loss. each plastic bag was of 25­micron thickness and 20 x 15 cm size while the non­woven fabric bag was sin­ gle­layered spun­bond polypropylene non­woven fabric with 50 gsm and 0.520 mm thickness. the rub­ ber band was used for sealing the mouth of packages each of the same size. 4 perforations of 2 mm diame­ ter were made in perforated packages with a heated wall nail. treatments detail: t1= no packaging or control; t2= unperforated low­density polyethylene (ldpe); t3= perforated low­density polyethylene; t4= unperforated high­density polyethylene (hdpe); t5= perforated high­density polyethylene; t6= unperforated non­woven fabric bag (nw fabric); t7= perforated non­woven fabric bag. data collection data for physiological weight loss was observed at every three days of storage whereas the qualitative data was observed every six days. weight loss (%) the fruit was weighed using the electronic digital balance on successive intervals and the loss in weight at each interval was calculated by using the following formula: weight loss, % = [(initial weight ­ final weight )/initial weight] x 100 color development the color development was observed visually. usda standard color chart (fig. 2) was used for observation of various maturity stages of tomatoes. mature green = complete green color on the surface of the tomato; breaker = distinct break in color from green to tar­ nish­yellow, pink or red on not more than 10% of the surface; turning = change in color from green to tannish­yel­ low, pink, red up to 10­30% of the surface; pink= pink or red color on 30% to 60% of the surface. light red = pinkish red or red color on 60% to 90% of the surface; red = red color on more than 90% of the surface. the color changes were determined by using a numerical rating scale from 1­7 where 1= green, 2= breaker, 3= pink, 4= turning, 5= light red, 6=red, and 7= deep red. total soluble solids (°brix) to determine the total soluble solids, tomato fruit was squeezed and a few drops of juice were added onto the prism plate of the refractometer (erma inc­ japan) and tss (°brix) was recorded. the prism plate of the refractometer was cleaned after each test with distilled water and soaked up with soft cotton. titratable acidity (%) ta was determined by titrating the 10 ml of toma­ to juice with 0.1% naoh which was placed in the burette. a few drops of phenolphthalein were added to the juice as the indicator. the mouth of the burette was opened to allow naoh to drop down until the color of the juice changed to pink for about 10 seconds. the volume of naoh required was recorded and titratable acidity was calculated by using the following formula that is expressed as % of citric acid. ta (%) = [(nb x vb x meq. of acid)/volume of sample] x d.f. x 100 where, nb = normality of base (naoh), vb= volume of the base, d.f. = dilution factor, and meq.= milliequivalent weight of predominant acid i.e. citric acid = 0.064. the ph of the juice the ph reading was measured by using a digital ph meter from the juice extracted for titration. the ph meter was placed in the juice and left for a certain time until the reading become stable and the stable ph value was noted. shelf life the shelf life of fruits was recorded by judging the non­marketability parameters such as damage by fungus attack, shriveling, etc. it was detected when 50% of tomatoes of each treatment were non­mar­ ketable. fig. 2 ­ standard tomato ripening color chart. adv. hort. sci., 2022 36(2): 127­134 130 data analysis the collected data entry and analysis on various parameters were done using the computer software package, microsoft excel (2016) and r (agricolae v.1.3­2). the analyzed data were subjected to lsd for mean comparison. 3. results weight loss statistically, significant variation was found con­ cerning the weight loss of tomatoes among different treatments. the higher rate of weight loss was observed in no packaging (open) and the lower was in unperforated hdpe that was statistically at par with unperforated ldpe at all days of the storage. the perforated packages showed a higher weight loss than any unperforated packaging material (table 1). one of the important factors that determine the shelf life of the tomato is weight loss and it was found to increase with the increase in permeability of the packaging material. the non­woven fabric bag has a high permeability to gases and water vapor as compared to the plastic film resulting in higher weight loss. color development the color development was observed to be rapid with the increase in permeability of the packaging material. extremely slow development of the color was observed in unperforated polyethylene storage and rapid in open tray storage (table 2). total soluble solids the tss of the tomato increased under all the packaging material up to 18 days of storage. there was no significant difference in the tss value of the tomato under different packaging materials on the 6th day of storage. however, the significant difference among the treatments began to appear from the 12th day of storage. the highest tss was observed in no table 1 ­ effect of packaging materials on physiological weight loss (%) means in the column followed by similar letters are not statistically different at p=0.05 by lsd. das= days after storage, lsd = least significant difference, cv = coefficient of variance, * significant at 5%, ** significant at 1%, *** sig­ nificant at 0.1% level of significance. table 2 ­ effect of packaging materials on color development means in the column followed by similar letters are not statisti­ cally different at p=0.05 by lsd. color score (1 green, 2 breakers, 3 turning, 4 pink, 5 light red, 6 red, 7 deep red). das=days after storage, lsd=least significant difference, cv=coefficient of variance, ns=non significant, * significant at 5%, ** significant at 1%, ***significant at 0.1% level of significan­ ce. treatments weight loss (%) 3das 6das 9das 12das 15das 18das t1 2.67 a 3.08 a 4.95 a 8.12 a 12.18 a 14.70 a t2 0.09 d 0.21 e 0.30 e 0.37 e 0.50 e 0.81 e t3 0.41 c 0.82 d 1.29 d 1.62 d 2.07 d 2.77 d t4 0.04 d 0.19 e 0.26 e 0.30 e 0.40 e 0.66 e t5 0.038 c 0.78 d 1.22 d 1.37 d 1.85 d 2.58 d t6 1.84 b 2.34 c 3.47 c 6.08 c 9.07 c 11.54 c 7 1.94 b 2.62 b 3.86 b 6.55 b 9.87 b 12.37 b grand mean 0.994 1.435 2.198 3.488 5.134 6.49 lsd 0.1397 *** 0.2256 *** 0.3190 *** 0.3588 *** 0.4662 *** 0.7020 *** cv% 8.02% 8.97% 8.32% 5.87% 5.18% 6.18% treatments color development 6das 12das 18das t1 4.17 a 6.33 a 6.80 a t2 2.17 a 3.00 d 4.33 d t3 3.33 cd 5.33 c 6.10 c t4 2.10 e 3.16 d 4.16 d t5 3.17 d 5.10c 6.13 c t6 3.5 c 5.76 b 6.46 b t7 3.93 b 6.03 b 6.60 ab grand mean 3.19 4.96 5.80 lsd 0.17 *** 0.29 *** 0.31 *** cv 3.05% 3.38% 3.01% poudel et al. ‐ extended shelf life of tomato on packaging 131 packaging on all days of the storage and the lowest was in unperforated hdpe (table 3). titratable acidity (ta) ta of the tomato decreased with an increase in the period of storage in all the packaging materials used. the lowest ta was observed in no packaging storage while the unperforated plastic packaging showed significantly higher ta (table 4). fig. 3 ­ effect of different packaging material on shelf life of tomato. the ph of the juice the ph of the tomato increased with the increase in the storage days for all the packaging material. the ph was found more in open tray conditions and low in unperforated polyethylene. the higher the barrier to the gases through the packaging film, the lower was the ph (table 5). shelf life the longest shelf life was observed in perforated ldpe (24 days) and the shortest in no packaging (16 days) (fig. 3). 4. discussion and conclusions the weight loss of the tomatoes is primarily due to the loss of water from transpiration and respira­ tion (singh, 2010). lower weight loss in all the pack­ table 3 ­ effect of different packaging materials on tss content of tomato means in the column followed by similar letters are not signifi­ cantly different at p=0.05 by lsd. das=days after storage, lsd=least significant difference, cv=coefficient of variance, ns= non significant, * significant at 5%, ** significant at 1%, ***significant at 0.1% level of significan­ ce. treatments tss of tomato 6das 12das 18das t1 4.016 a 4.533 a 5.100 a t2 3.850 a 4.067 d 4.301 d t3 3.867 a 4.183 bc 4.550 c t4 3.850 a 4.083 cd 4.301 d t5 3.867 a 4.177 bcd 4.533 c t6 3.867 a 4.233 b 4.750 b t7 3.933 a 4.277 b 4.77 b grand mean 3.893 4.222 4.614 lsd ns 0.1106 *** 0.1079 *** cv(%) 2.64% 1.49% 1.33% table 4 ­ effect of different packaging materials on ta content of tomato means in the column followed by similar letters are not statisti­ cally different at p=0.05 by lsd. das=days after storage, lsd = least significant difference, cv = coefficient of variance, * significant at 5%, ** significant at 1%, ***significant at 0.1% level of significance. treatments ta of tomato 6 das 12das 18das t1 0.697 e 0.613 e 0.303 d t2 0.987 a 0.863 a 0.563 a t3 0.750 c 0.640 cd 0.367 b t4 0.990 a 0.853 a 0.550 a t5 0.767 b 0.670 b 0.353 b t6 0.713 d 0.647 c 0.326 c t7 0.703 de 0.627 de 0.323 c grand mean 0.80 0.701 0.398 lsd 0.013 *** 0.016 *** 0.016 *** cv(%) 0.9% 1.28% 2.33% table 5 ­ effect of different packaging materials on ph content of tomato means in the column followed by similar letters are not statisti­ cally different at p=0.05 by lsd. das= days after storage, lsd= least significant difference, cv= coefficient of variance, * significant at 5%, ** significant at 1%, *** significant at 0.1% level of significance. treatments ph of tomato 6das 12das 18das t1 4.13 a 4.30 a 4.53 a t2 3.80 d 4.03 c 4.20 d t3 3.95 b 4.17 b 4.30 bc t4 3.83 cd 4.00 c 4.20 d t5 3.93 b 4.17 b 4.26 cd t6 3.90 bc 4.00 c 4.33 bc t7 4.10 a 4.12 ab 4.37 b grand mean 3.95 4.12 4.31 lsd 0.074 *** 0.114 *** 0.076 *** cv(%) 1.07% 1.59% 1.01% 132 adv. hort. sci., 2022 36(2): 127­134 aged fruits as compared to control could be due to a lower rate of transpiration and counteraction of excessive moisture loss which was similar to the results presented by gonzalez et al. (1990) and nath et al. (2011). the difference in weight losses of fruits among different films could also be largely due to dif­ ferences in transmission rates of water vapor through the packaging film (batu and thompson, 1998). here the lower transmission rate of polyethyl­ ene packages might contribute to the development of higher relative humidity inside the package there­ by reducing weight loss (thompson, 2001). batu and thompson (1998) also found less weight loss in sealed packed tomatoes after 60 days of storage at 13°c. a modified atmosphere is created around the fruits due to the permeable nature of the film. during the storage, the co2 concentration accumulates inside map restricting the respiration of the produces thereby reducing weight loss and prolonging the shelf life (selçuk et al., 2020). similarly, higher weight loss was observed in macro­perforated packages than any of the unperforated packages due to higher permeability to gases and water vapor (van der steen et al., 2002). similar might be the case for non­ woven packages where the bag itself has a low barri­ er to the exchange of gases even without any perfo­ rations resulting the higher weight loss. lycopene pigment is the primary reason for the development of the color in tomatoes. as the ripen­ ing proceeds, lycopene content increases. however, in presence of low oxygen, the formation of lycopene is inhibited resulting in the slow development of color. due to low o2, high co2, or the suitable combi­ nation of these two gases, there could be retardation of color change in tomato fruits (kidd and west, 1930). yang and chinnan (1987) also found that there is less accumulation of lycopene in sealed packaged tomatoes with fewer chroma values than that of con­ trol treatment. lycopene formation was found to be completely inhibited at 1% o2 and 99% n2 for 50 days of storage (yang et al., 1987). ethylene is responsible for triggering the ripening of tomatoes and it is asso­ ciated with a sudden change in the physiology of tomatoes at the onset of ripening. co2 concentration affected the development of color in tomatoes by suppression of ethylene production (kubo and inaba, 1989). while exposing the tomatoes to high levels (20, 40, and 60%) of co2, color development was inhibited in tomatoes (buescher, 1979). the tss content of fruit determines its overall taste (baldwin et al., 1998). getinet et al. (2008) have report­ ed a low total soluble solid at the color breaker stage but higher when tomato fruits were harvested at the pink mature stage. the increase in tss with the increase in maturity could be attributed to the breakdown of starch to simple sugars or the hydrolysis of cell wall polysaccha­ rides (crouch, 2003). the lower tss in packed tomatoes as compared to the open storage could be due to a slow­ er rate of respiration and metabolic activities that slow down the ripening process in packed tomatoes (gharezi et al., 2012). however, the higher tss observed in non­ woven treatment might be attributed to the relatively faster rate of respiration resulting by the comparatively higher air permeability of the bag. the more tss is relat­ ed to more ripen of fruit (dhakal et al., 2020). however, the rapid increment in tss is not desirable as it causes rapid shriveling and decreases the shelf life. priyankara et al. (2017) also found the titratable acidity reaching the peak at the color breaker stage and decreasing along with the advancement of fruit ripening. since the acidity of the fruit is due to the presence of various organic acids, the amount of organic acid is usually found decreasing during matu­ rity as being a substrate of respiration (albertini et al., 2006). the slow rate of decrement of ta in unper­ forated plastic packaging could be attributed to the reduced o2 and increased co2 inside the packages that result in the slow rate of respiration (mathooko, 2003); thus, it may impede the loss of organic acids (wang, 1990). even so, the rapid decrement of ta in non­woven packages could be ascribed to the exchange of gases good enough to deplete the organic acid. every factor that is responsible for reducing cellular respiration and catabolism prevent the reduction of organic acid in the product (feizi et al., 2020). de castro et al. (2005) also reported the decrement in acidity with maturity evolution. the difference in the ph and ta in different pack­ ages is attributed to the variations in respiration rate and enzyme activities (feizi et al., 2020). organic acid being an intermediate of carbon metabolism increase the ph of the produce. the higher ph under the open tray could be associated with the faster utilization of acids for sugar catabolism. for a similar reason, ph is higher in the non­woven bag as compared to others owing to increased o2 inside the packages engen­ dered by the air enterable nature of the bag. the sig­ nificantly lower ph values of unperforated packaged fruits could be explained by the relatively reduced respiration rate due to reduced o2 inside the pack­ ages. the increase in the ph of the fruits during stor­ age was also observed by batu and thompson poudel et al. ‐ extended shelf life of tomato on packaging 133 (1998). the difference in the shelf life was due to the dif­ ference in marketability of fruits due to the decaying of fruits by fungus or shriveling of the fruits unac­ ceptably. unperforated plastic packaging showed the highest percentage of fungus attacks that could be probably due to higher relative humidity inside the packages. the non­woven fabric bag allows access to air sufficient enough to escape the modified atmos­ pheric condition resulting relatively higher shriveling and weight loss ensued from faster ripening and transpiration and ultimately over­ripening. the beneficial effect of perforated plastic packag­ ing could be attributed to the well­modified atmos­ phere created inside the package along with the reduction in water loss. lower rate of respiration and ethylene production inhibited ethylene action, delayed ripening and senescence, impeded growth of decay­causing pathogens and insects due to gaseous modification inside the package could be the proba­ ble reason to extend the shelf life of fruits (kader and rolle, 2004). ben­yenonshuna (1985) also reported the delayed ripening and softening in the case of packaging of climacteric fruits in low­density polyeth­ ylene bags and hence improving marketability. thus, packaging significantly affected various quantitative and qualitative post­harvest properties. all the packaging system was found to be better than open tray storage. the permeability of packaging material had a huge influence on the composition of the internal atmosphere and the creation of optimal storage conditions. very low permeability would induce fungal growth due to high relative humidity and high permeability would result in higher weight loss due to faster respiration. therefore, it is neces­ sary to establish an optimal condition that may be an equilibrium­modified atmosphere that avoids both of these problems. based on the result of this experi­ ment, perforated plastic bag (both hdpe and ldpe) was discovered to be best among all packaging treat­ ments for reducing weight loss, avoiding fungal 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sterilized potato tuber moth males. mating experiments were carried out to obtain partially sterilized males and their descending offspring. then, 316 aflp bands were amplified using eight primer combinations of which 33.8 were polymorphic 85.5%, which varied from 68.57% to 100%. the upgma dendrogram generated for the aflp data revealed that irradiated and unirradiated male samples were clustered into two groups, and the offspring of f1 and f2 of unirradiated parents were clustered into one group. moreover, the progeny of f1 and f2 of irradiated parents clustered into three groups. no specific dna marker could identify the irradiated males; however, there was a clear genetic variability between examined individuals. thus, the aflp technique could be utilized to study genetic variations among individuals of the same line. the aflp markers could enhance the monitoring system of mass-released insects program when inherited sterility technique is applied against potato tuber moth. 1. introduction the potato tuber moth phthorimaea operculella zeller (lepidoptera: gelechiidae) is a cosmopolitan pest on potato crop, causing an annual yield reduction of 50 to 100% in some country around the world (ahmed et al., 2013). insecticides are widely used to control this pest, but these methods have many drawbacks like high cost, nonselective and environmentally unfriendly. moreover, insects could develop resistance to insecticides (harba and idris, 2018). therefore, more environmentally friendly methods are required. the inherited sterility technique (ist) was suggested as an alternative control method to compact p. operculella (makee and saour, 1997; larraín et al., 2009). because of no-practical methods are available to separate the adult moths by gender, the males and females are mass-reared, irradiated with low sterilizing doses of gamma radiation, then released within the targeted area (eyidozehi et al., 2015). moths irradiated with low doses live longer, stronger fliers and mate more frequently than moths irradiated with higher radiation doses (*) corresponding author: eidris1972@yahoo.com citation: idris i., shoaib a., 2019 efficiency of aflp markers to detect genetic variation in phthorimaea operculella (lepidoptera: gelechiidae) offspring irradiated males. adv. hort. sci., 33(3): 321-326 copyright: © 2019 idris i., shoaib a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 june 2018 accepted for publication 22 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 321-326 322 (vreysen et al., 2016). however, a dose of 400 gy induced almost 90% sterility in irradiated males while, a complete sterility in p. operculella females was achieved by 200 gy dose (makee and saour, 2004). furthermore, the costs of using ist program are likely to be more acceptable in terms of monetary expenditures and efficacy, as reported by edgington and alphey (2017), when they released dominant-lethal strain aedes aegypti (l.) (diptera: culicidae) mosquitoes. the cost-effective improvements to the ist programs are required by applying modern genetic methods (leftwich et al., 2018). rapd, aflp, microsatellites and ests are popular dna marker systems used in insect genetic research (singh et al., 2017). they are used as monitoring systems of insects mass-release programs to improve the application of ist against insects (oliva et al., 2012; edgington and alphey, 2018). in this study, aflp technique was employed to investigate the genetic variation among the offspring of partially sterilized males of p. operculella. 2. materials and methods inherited sterility experiment p. operculella insects used in this study were obtained from our laboratory stock cultures. they were reared on wax coated potato slices, maintained at a constant temperature of 25±1˚c, with 70±5% relative humidity, and 12 hour light-darkness cycle as described by makee and saour (2004). fifty couples of females and males were placed in 350 ml transparent plastic boxes with filter papers as an oviposition site. a 10% sucrose solution was provided as food source. both females and males were kept together until death. the eggs were removed daily, counted, and left until hatching. from the 50 reared couples only two were chosen depending on their fecundity (number of eggs per female), and fertility (percentage egg hatch). all the newly hatched larvae of two couples choosing were reared on small-waxed potato pieces, and the pupae were collected. the couple, with most pupae, was chosen to be the first family for tracking to the f1 and f2 progeny. males were divided into two groups, the first male group was used as a control (♂ n x n ♀), and the second group was irradiated with a 150 gy in a gamma cell supplied with a co-60 source rounded the cylindrical (15x25 cm2) irradiation chamber (isslcdo-vatel gamma irradiator, techsnabexport co. ltd. usr). the average dose rate at the time of irradiation was approximately 40.12 gy/min with a factor of homogeny (max:min dose ratio) of about 1.05 and the absorbed dose was calibrated with fricke solution. during this treatment, adult females were kept individually in small plastic tubes inside the irradiation source. the second males group was individually mated with normal virgin females (♂ t x n ♀). all f1 and f2 generations were reared on small waxed potato pieces as mentioned above. fecundity and fertility of the f1 and f2 generation were recorded. adult male parents were kept for dna extraction and aflp analysis. dna extraction and aflp analysis six dna isolation protocols of p. operculella males from adult stage were used to obtain a good quality and quantity of dna for aflp analysis (m1: beye and raeder, 1993; m2: blanchetot, 1991; m3: favia et al., 1994; m4: harrison et al., 1987; m5: marchant, 1988; m6: moeller et al., 1992) (reineke et al., 1998). the m5-modified protocol was the most appropriate to produce a high quality and quantity of dna from one adult moth. from each adult moth of 4-5 mg, an 8 to 12 µg pure genomic dna was obtained. the aflp protocol was carried out as reported by shoaib et al. (2008). dna from all samples was digested with ecor1 and msei restriction enzymes (0.125 u/µl). selective amplification reactions were performed using eight primer combinations and the amplified fragments were separated by gel electrophoresis. the sequences of eight primers combinations and adapters used in this study are presented in table 1. aflp data analysis for each primer pair, the numbers of polymorphic and monomorphic bands were determined. each gel from the aflp experiments was scored as presence (1) or absence (0) of a specific band for every sample. percentage of polymorphism was calculated as the proportion of polymorphic bands over the total number of bands. allelic polymorphic information content (pic) was calculated using the formula of botstein et al. (1980). data for all the 8 primer combinations were used to estimate the genetic distances among analyzed individuals on the basis of the number of shared amplification products by using the nei and li, (1979) method. a dendrogram was generated using the unweighted pair group of arithmetic means (upgma) by statsoft program (2003). 3. results the data revealed that the first and the second idris and shoaib ‐ genetic variations of potato tuber moths by aflp 323 couples were the best. the fecundity and fertility of the two couples were (111/103) and (95/88) (total eggs/ hatched eggs), respectively (table 2). the first couple (89 pupae, no. of males and females 37 ♂/ 35 ♀) was selected to be the first family. table 2, 3 show the f1 and f2 generations of irradiated and unirradiated males that resulted from seventeen males of this family, which were irradiated with 150 gy dose and seven males were kept as a control. table 2, 3 show the families of irradiated (t) and unirradiated (n) males which were selected based on the fecundity and fertility of f1 and f2 generations, and presenting in a marker (*). all purified genomic dna of p. oper‐ culella samples submitted to aflp analysis (table 4). eight primer pairs successfully amplified dna fragments from the genomic of 17 samples. however, 316 fragments were scored with an average of 85.5% polymorphic bands per primer combination. the percentage of polymorphism detected by individual primer combination ranged from 68.57% for e-aag/ m-cta primer combination to 100% for e-aac / mctg primer combination (table 5). the ratio of number of fragments produced by primer pairs were 39.5. the upgma dendrogram generated for the aflp data shows that irradiated and unirradiated males samples were clustered into two groups. hence, the offspring of f1, and f2 of unirradiated parent clustered into one group. while, the progeny of f1 and f2 of irradiated parent clustered into three groups. the first group include female parent, the second include the male parent, and the third one include all f2 progeny that were produced from irradiated male parents (fig. 1). table 1 sequences of oligonucleotide adapters and primers used in the pre amplification step and the selective aflp primers combinations table 2 inherited sterility technique experiments and the families of f1 generations selected for aflp analysis * families selected for aflp analysis. name reaction code sequence ecori adapter ligation 5¢-aattggtacgcagtctac3¢ 3¢ccatgcgtcagatgctc-5¢ msei adapter ligation 5¢-tactcaggactcat-3¢ 3¢-gagtcctgagtagcag-5¢ ecori preamplification e 5¢-gactgcgtaccaattc3¢ msei m 5¢-gatgagtcctgagtaa3¢ ecori +a selective amplification e-a 5¢-gactgcgtaccaattca-3¢ ecori +g e-g 5¢-gactgcgtaccaattcg-3¢ ecori+ c e-c 5¢-gactgcgtaccaattcc-3¢ ecori+ t e-t 5¢-gactgcgtaccaattct-3¢ msei + c m-c 5¢-gatgagtcctgagtaac-3' msei + t m-t 5¢-gatgagtcctgagtaat-3' msei + a m-a 5¢-gatgagtcctgagtaaa-3' msei + g m-g 5¢-gatgagtcctgagtaag-3' * families selected for aflp analysis. tow couples were chosen from 50 no. of families no. of eggs eggs hatching no. of pupea *1 111 103 89 2 95 88 77 irradiated f1 males (♂ n/ ♀ n) no. of families no. of eggs eggs hatching no. of ♂\♀ 1 41 25 1\6 2 death 3 4 0 0/0 4 29 14 2\1 5 11 2 1\1\ 6 48 37 8\1 7 death 8 6 3 1\1 9 48 20 5\1 10 38 26 14\1 11 3 3 2\1 12 204 140 45\12 *13 131 70 22\8 14 25 4 2\1 15 5 3 0\1 *16 206 146 61\17 *17 45 14 6\2 unirradiated f1 males (♂ n/ ♀ n) *18 127 67 8\11 19 death 20 34 18 2\5 21 26 19 0\0 22 23 5 0\0 *23 138 92 38\47 *24 153 153 22\18 adv. hort. sci., 2019 33(3): 321-326 324 4. discussion and conclusions potato tuber moth, like most of lepidoptera moths, when exposed to substerilizing doses of gamma rays undergo several physiological, biochemical and genetic changes (makee and saour, 2004; hallman et al., 2013, sachdev et al., 2017 ). however, some of the dna damages due to irradiated male parents are inherited by their progeny (steinitz et al., 2015). although, inherited sterility did not occur in p. operculella females but infertility of irradiated males and females is irreversible (makee and saour, 1997, 1999; idris et al., 2019). thus, the sterility in f1 progeny was more than in its irradiated male parents when ist applied against p. operculella (makee and saour, fig. 1 upgma dendrogram showing genetic relationships among 17 dna samples of unirradiation and irradiation of p. operculella. samples are: 1. female, 2. male, 3-4. f1 irradiated males, 5-6. f1 unirradiated males, 8-12. f2 unirradiated males, 7. mix dna samples of f2 irradiated males 8-12, 18-14, 13. mix dna samples of f2 irradiated males 14-18. table 3 f1 progressed studied families and the families of f2 generations selected for aflp analysis * families selected for aflp analysis. the f1 progressed studied families no. of f1 families no. of couples studies no. of couples sustained 12 6 1 13 1 0 *16 8 6 18 6 4 *23 11 8 24 5 2 irradiated f2 males no. of f1 families no. of cross no. of eggs eggs hatching *16 2 2 0 3 52 0 4 7 0 5 7 0 7 7 0 8 3 0 12 6 23 0 unirradiated f2 males no. of f1 families no. of cross no. of eggs eggs hatching 18 1 17 12 2 11 45 3 1 0 6 8 0 *23 1 23 1 2 35 29 3 44 35 4 28 11 7 153 63 8 8 7 9 166 65 11 58 27 24 3 80 37 5 121 67 table 4 dna samples for aflp analysis extraction from no. of samples female 1 male 2 irradiated males of f1 3-apr unirradiated males of f1 5-giu mix samples of dna 8-9-11-12 7 unirradiated males of f2 8-9-11-12 mix samples of dna 14-15-16-17-18 13 irradiated males of f2 14-15-16-17-18 table 5 percent polymorphism, band numbers and polymorphic bands produced by eight primer combinations no. primers combination total no. of bands polymorphic bands polymorphism % 1 e-act x m-ctg 49 46 93.87 2 e-aag x m-cta 35 24 68.57 3 e-acg x m-cac 51 42 82.35 4 e-acg x m-cta 47 42 89.36 5 e-aca x m-cat 45 36 80 6 e-aac x m-cac 41 34 82.92 7 e-agg x m-ctc 24 23 95.83 8 e-aac x m-ctg 24 24 100 total 316 271 average 39.5 33.8 85.5 idris and 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moth (medina et al., 2010). our aflp data that were obtained from this study shown that no specific dna marker could distinguish irradiated males from the unirradiated ones, but there was a clear dna polymorphism between in f1 and f2 generations of partially sterilized males of irradiated and unirradiated male parents. induced dna damage could have significantly begun at 20 gy and higher doses as reported by hambarde et al., 2013 on sf9 lepidoptera cells. consequently, it is known, that dna damages caused by irradiated males at 150 gy are irreversible and randomly inherited to their offspring (makee and saour, 2004; vreysen et al., 2016). thus, the dna damages inherited randomly in f1 and f2 generation are not stable when the males exposed to the partially sterility irradiation doses (sauor, 2014; kheirallah et al., 2017). based on these facts, we suggest that dna changes in f1 and f2 generations between irradiated and unirradiated were adequate to be detected by aflp technique. 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a., kliot a., harari a., 2015 effects of radiation on inherited sterility in the european grapevine moth (lobesia botrana). pest. manag. sci., 71(1): 24-31. vreysen m.j., klassen w., carpenter j.e., 2016 overview of technological advances toward greater efficiency and efficacy in sterile insect‐inherited sterility programs against moth pests. fla. entomol., 99(1): 112. impaginato 285 adv. hort. sci., 2021 35(3): 285­292 doi: 10.36253/ahsc­10536 investigation of modified wpm medium for the best meristem proliferation of corylus avellana l. s. kıvrak kiran, s. galatali, s. yeniocak, d.e. ozkaya, t. mercan, s. guldag, o. celik, n. abdul ghafoor, e. kaya (*) molecular biology and genetics department, faculty of science, mugla sitki kocman university, 48000 mugla, turkey. key words: charcoal, fe­eddha, hazelnut, issr, micropropagation. abstract: cultivation of corylus avellana l. in turkey is performed generally in the northern regions where it is an important source of livelihood for the local farmers. more than 70% of world hazelnut production is supplied by turkey, but compared with other countries, turkey’s hazelnut production area is quite narrow. in this study was aimed to develop an effective in vitro production for seven local cultivars of c. avellana. therefore, wpm medium supplemented with 6­benzylaminopurine (bap) was modified by using single or in combinati­ on of fe­eddha, agno3, h3bo3, charcoal and gibberellic acid. in all varieties, the best regeneration rates varying between 68% and 94% were obtained from wpm medium supplemented with 4.4 µm bap, 27.8 µm fe­eddha and 10g/l charcoal. genetic stability of shoots derived from meristem culture using the best medium was analysed using issr primers, when the gel images of the pcr products were examined, no polymorphic band was observed in samples col­ lected from seven provinces, and the genetic stability was determined as 100%. 1. introduction corylus avellana l. plants are grown at altitudes in the range of 20­ 1500 m in the northern parts of the turkey, they can also be grown in the southern and eastern creeks, and rarely in the western regions (aydinoglu, 2010). annual rainfall is balanced according to the seasons and temperatures, above 700 mm rainfall and temperatures ranging from ­8°c to ­10°c with the highest between 33°c and 36°c are ideal conditions for the production of c. avellana l. (koksal et al., 2006). more than 70% of world hazelnut production is produced by turkey (zhao et al., 2015). however, turkey’s hazelnut production efficiency is very low compared to other countries. the average yield is 179.2 kg/ha in the us, 168.6 kg/ha in italy whereas in turkey it is 91.7 kg/ha (mennan et al., 2020; sen and kahveci, 2020). the in vitro propagation procedures for clonal multiplication provide the useful alternative tools to traditional propagation of hazelnut culti­ vars. development of an efficient procedure for hazelnut micropropagati­ (*) corresponding author: ergunkaya@mu.edu.tr citation: kivrak ki̇ran s., galatali̇ s., yeni̇ocak s., ozkaya d.e., mercan t., guldag s., celi̇k o., abdul ghafoor n., kaya e., 2021 ­ investigation of modified wpm medium for the best meristem proliferation of corylus avellana l. ­ adv. hort. sci., 35(3): 285­292 copyright: © 2021 kıvrak kiran s., galatali s., yeniocak s., ozkaya d.e., mercan t., guldag s., celik o., abdul ghafoor n., kaya e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 february 2021 accepted for publication 29 june 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10536 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 285­292 286 on can also be beneficial for increase of clones, culti­ vars and varieties resistant to different pathogens such as hazelnut worm (curculio nucum), eastern fil­ bert blight (anisogramma anomola) and powdery mildew (erysiphe corylacearum) (bassil et al., 1992; lucas et al., 2018). the transfer of parts of the plant such as tissue, cell, organ, embryo to in vitro environment under aseptic conditions in an artificial nutrient medium for clonal propagation is important to produce a patho­ gen­free plant with protected germplasms and/or to maintain its secondary metabolites production which otherwise is limited for various reasons is called plant tissue culture (hayta et al., 2017; espinosa­leal et al., 2018). in addition to the continuous production thro­ ughout the year by obtaining thousands of plants with the same form and characteristics as the mother plant in a short time by micropropagation method from plant tissue cultures; superior species resistant to factors such as drought, salinity, acidity, and/or cold can also be produced. micropropagation invol­ ves plant selection, sterilization, in vitro transfer, clo­ nal propagation, rooting, conditioning, and transfer to soil. due to these advantages, micropropagation is one of the most effective methods when compared with other production techniques (ozudogru et al., 2011; ozudogru and kaya, 2012). different techniques in plant tissue culture can be used such as shoot tip culture by transferring the shoot tips with growth cone including meristematic doom to in vitro environment, bud node culture by transferring the axillary or apical buds of the shoots to aseptic conditions, meristem culture by transfer­ ring meristem from the meristematic region to the nutrient medium with the help of a stereomicrosco­ pe, embryo culture by taking the embryo from the seed template or from the seed and transferring it to the germination medium (ahmad and anis, 2007; usha et al., 2007). clonal production of plants in a short time is one of the main purposes of plant tissue culture, howe­ ver, changes may occur in micropropagated plants, all the changes that may occur in the hereditary material are called somaclonal variation, somaclonal variations can be detected by various molecular tests (samir, 2004; lattoo et al., 2006; ozudogru et al., 2011). nowadays, genetic differences in the germ­ plasms of many plant species can be detected using molecular markers, non­specific primers are often preferred in determining the differences between germplasms in methods based on the polymerase chain reaction, these primers are particularly advan­ tageous for plant species that have not been previ­ ously studied or have a limited number of researches performed on them (devarumath et al., 2002; carvalho et al. , 2004; kaya and souza, 2017). although there are a wide variety of molecular mar­ ker techniques used nowadays, aflp (amplified fragment length polymorphism), rapd (random amplification of polymorphic dna), ssrs (simple sequence repeats), issrs (inter simple sequence repeats) techniques are preferred. the method to be used is selected based on the technical facilities accessible and the purpose of the study (belaj et al., 2003; kaya, 2015). the issr technique used in many plants is a sim­ ple, fast, and low­cost technique, it also stands out for its ability to work with a small amount of templa­ te dna and provides a high level of information. with all these advantages, the issr technique is often pre­ ferred in determining the difference in germplasm collections (gilbert et al., 1999; kaya and souza, 2017). when there are some limitations (shoot tip necro­ sis, big contaminations, sensitivity for surface sterili­ zation treatments) causing from plant material such as shoot tips, nodal segments, buds in in vitro propa­ gation studies, seeds can be used as starting material and there are many similar studies in the literature such as micropropagation of thymus vulgaris (ozudogru et al., 2011), citrus spp. (kaya et al., 2016), musa spp. (kaya et al., 2020 b). similarly, embryos isolated from hazelnut seeds were used as starting material in the current study, as there was a major contamination problem. thus, the optimal growth medium developed for hazelnut these local varieties in the current study will be a model growth medium for future in vitro studies such as cryopre­ servation. in this study, wpm nutrient medium developed by lloyd and mccown which commonly used in mic­ ropropagation of woody plants in order to develop an effective environment for in vitro meristem repro­ duction of hazelnut plant, which is relatively difficult to micro­propagate due to some limitations was used (lloyd and mccown, 1980). the main aim of this study was to determine optimal medium combinati­ on by using wpm. seven different cultivars of hazel­ nuts grown in seven different provinces of turkey were examined with different combinations of benzyl amino purine and gibberellic acid growth regulators, ethylene diamine di­2­hydroxyphenyl acetate ferric kivrak kiran et al. ‐ the meristem proliferation of corylus avellana l. 287 (fe­eddha), silver nitrate (agno3), boric acid (h3bo3) and activated charcoal. in addition, the another aim was to confirmation genetic stability of in vitro grown plants. for this reason, the genetic stability of the plants growing in in vitro conditions that gave the best results in the study was analysed by the issr technique. 2. materials and methods in vitro culture initiation and micropropagation c. avellana l. plant samples (fruits) from turkey’s; bartin (guzelcehisar village, inkumu location), düzce (akcakoca, yesilkoy village), trabzon (ortahisar, yanyamac location), ordu (ulubey, inonu location), samsun (carsamba, ordubasi district), giresun (eriklimanı village), and zonguldak (eregli, sarikaya village) (seven different) provinces were obtained from their respective hazelnut breeders. the fruits were surface sterilized to remove pathogens before transfer to the in vitro medium using 70% ethanol (etoh) and 30% commercial bleach (domestos, unilever©). this step was followed by 3­stage internal steril ization (70% etoh, 10% h2o2, and 10% commercial bleach) processes applied to the materi­ als separated from their peels with the help of sterile forceps and pliers (ozudogru et al., 2011; kaya et al., 2013, 2016). after the sterilization processes, embryo isolation was performed from c. avellana l. samples under a stereomicroscope in a laminar flow cabinet (fig. 1 a), the isolated embryos were then transferred to wpm semi­solid medium containing 4.44 µm 6­ benzilaminopurin (ba), 30 g/l sucrose and 7 g/l agar for germination (ph 5.8). the germinated embryos were taken into wpm medium supplemented with 4.44 µm ba 30 g l­1 sucrose, 7 g l­1 agar and 27.8 µm fe­eddha [ethylenediamine­n, n’­bis (2­hydroxyp­ henylacetic acid) iron sodium complex] (fig. 1 b). in order to determine the suitable medium for the hazelnut plant, the meristematic regions of 0.3 ­ 0.8 mm length of shoots of the seven different hazelnut varieties grown in vitro were isolated (fig. 1c) and transferred to eight different modified wpm medi­ ums (table 1). the all in vitro materials were incuba­ ted at standard culture conditions (27±2°c, 16/8 h photoperiod, with cool daylight fluorescent lamps rated at 50 μmol−1 m−2 s−1). determination of genetic stability in order to determine the effects of the modified wpm nutrient medium that gives the best regenera­ tion with the meristem culture method on the gene­ tic stability of the plant, dna isolation was performed manually following the 2× ctab protocol developed by ferdous et al. (2012). following the dna isolation, the protocol developed by williams et al. (1990) was used with the issr primers developed by martins­ lopes et al. (2007) [(tc) 8c ubc823; (ga) 8c ubc811; (ag) 8g ubc809; (ca) 8a ubc817; (ag) 8t ubc807; (ga) 8t ubc810] to perform an issr pcr reaction. the products obtained from the issr pcr were run on an agarose gel electrophoresis containing 1.5% agarose and the bands profiles were visualized under uv­transilluminator. the “similarity ratio” formula developed by sokal and sneath (1963) was used to medium plant growth regulator chemical wpm 1 4.44 µm ba 27.8 µm fe­eddha wpm 2 4.44 µm ba 27.8 µm fe­eddha 2.89 µm ga3 wpm 3 4.44 µm ba 27.8 µm fe­eddha 5.89 µm agno3 wpm 4 4.44 µm ba 27.8 µm fe­eddha 16.17 µm h3bo3 wpm 5 4.44 µm ba 27.8 µm fe­eddha 10 g l­1 charcoal wpm 6 4.44 µm ba 27.8 µm fe­eddha 2.89 µm ga3 10 g l­1 charcoal wpm 7 4.44 µm ba 27.8 µm fe­eddha 5.89 µm agno3 10 g l­1 charcoal wpm 8 4.44 µm ba 27.8 µm fe­eddha 16.17 µm h3bo3 10 g l­1 charcoal table 1 ­ eight different modified wpm mediums for in vitro meristem proliferation of seven corylus avellana l. cul­ tivars fig. 1 ­ the embryo was isolated from fruit of c. avellana l. cv. duzce­akcakoca (a), the embryo germination on wpm semi­solid medium containing 4.44 µm bap 30 g l­1 sucrose and 7 g l­1 agar 27.8 µm fe­eddha (b), the meri­ stem was isolated from in vitro grown shoot of c. avella‐ na l. cv. duzce­akcakoca (c) (bars 1 mm). adv. hort. sci., 2021 35(3): 285­292 288 calculate the similarities between the samples in determining the genetic stability or differences as a result of data analysis by scoring the band profiles displayed as a result of issr pcr according to the 1/0 (yes/no) principle. similarity ratio= homologous bands/(homologous bands + non homologous bands) the dna of the samples were obtained from in vitro embryo­grown leaves (in wpm medium supple­ mented with 4.44 µm ba), leaves of shoots that developed after subculture (in wpm medium contai­ ning 4.44 µm ba and 27.8 µm fe­eddha), and post­ meristem culture grown shoot leaves (in wpm medi­ um containing 4.44 µm ba, 27.8 µm fe­eddha and 10 g l­1 charcoal) were used. experimental design, data collection, and statistical analysis for each cultivar, more than 100 embryos of seeds were used for in vitro culture establishment. three petri dishes (replicates), each containing 10 shoot tips, were used for each in vitro proliferation treatment, and each experiment was repeated at least three times. data of in vitro proliferation were recorded 4 weeks after culture initiation and consis­ ted of the percentage of shoot tips that generated at least one elongated shoot. regeneration percentage was compared by multiple x2 test by the spss prog­ ram (ibm spss statistics 21.0) and statistical analysis was also performed with anova, followed by the lsd test at p≤0.05 (marascuilo and mcsweeney, 1977). 3. results after surface sterilization, the embryos germina­ ted in wpm medium containing 4.44 µm bap were transferred to the regeneration medium, from the samples that were used as starting material, 71% to 85% sterile material was obtained as a result of surfa­ ce sterilization. the obtained clean materials showed successful regeneration between 81.1% and 100%. shoot tips grown in vitro were subcultured, and rep­ roduced in 30­day periods, and used as the material in meristem culture study. the desired amount of in vitro material was obtained from regenerated plants for use in meristem culture applications. the best regeneration rate from eight different wpm media in different combinations tested for meristem regeneration was obtained from wpm media containing 4.44 µm bap, 27.8 µm fe­eddha, and 10 gl­1 charcoal. the regeneration rate obtained on this medium ranged from 68% to 94% for the seven hazelnut local cultivars tested (fig. 2). polymerase chain reactions were carried out using six different primers to determine the genetic stabi­ lity of regenerated hazelnut varieties. in order to determine genetic stability, clones of each sample developed from a single embryo were used. pcr pro­ ducts obtained from the pcr using six different issr primers were run on an agarose gel and visualized. a total of 324 bands with a length of 120 bp and 2800 bp were scored from the band profiles obtained. in the light of the band profiles obtained, no polymorp­ hic bands were observed in samples collected from the seven provinces, and the genetic stability was determined as 100% (fig. 3). 4. discussion and conclusions in turkey, for hazelnut production, the certified seedlings have not still been studied and conventio­ nal production does not meet all requirements of hazelnut plants. because these plant cultivars can be affected very quickly by climatic conditions and fruit fig. 2 ­ the meristem regeneration percentages of seven diffe­ rent cultivars of c. avellana l. on eight different modified wpm mediums. kivrak kiran et al. ‐ the meristem proliferation of corylus avellana l. 289 yield may decrease accordingly, physical factors such as the reduction in the mineral and organic matter content of the soil, and biological factors such as hazelnut pests and diseases can further decrease its yield. in this case, it is extremely important to exami­ ne the factors that cause low productivity and to determine the measures that could eliminate these factors or reduce their effect (amaral et al., 2006; aydinoglu, 2010; ciarmiello et al., 2014). increasing trends in areas such as the develop­ ment of systems for densely planting tree seedlings, selection of new parents for nutrition programs, development of new cultures in order to better eva­ luate the existing limited areas, increased the need for modern techniques in fruit tree cultivation. traditional methods, which were carried out to meet the demand for more high­quality plant material are insufficient for they are both time consuming and insufficient for standard and healthy production, the­ refore, the application of in vitro techniques in fruit growing may be a suitable alternative way to overco­ me these problems. developments in plant biotech­ nology, particularly in the fields of in vitro cultures and molecular biology, have been a powerful tool in the control and conservation of plant biodiversity (withers, 1995). nowadays, biotechnological met­ hods comprise the most suitable methods for patho­ gen­free short, medium, and long­term protection of ornamental, medicinal, and aromatic plants as well as woody plant species, which are in danger of extincti­ on and poses rare economical value. in vitro conser­ vation methods are especially important for the pro­ tection of vegetatively propagated plant species with seeds that are intolerant to desiccation (engelmann, 2011). in addition to the advantages mentioned above, in vitro techniques provide a reliable platform for international plant material exchange, enable large collections using minimal space, provide valu­ able materials for species recovery, and facilitate molecular research and ecological studies (tandon et al., 2005). traditional propagation procedures for hazelnut through shoots is time consuming, limited and ineffi­ cient for large scale production. these days, many hazelnut cultivars have been identified for plantation in turkey and it immediately needs to develop effici­ ent procedures for rapid clonal propagation. the plant tissue culture systems offer an useful tools for large scale production of plantlets (damiano et al., 2005; bacchetta et al., 2008). the current study aimed to develop and integrated procedure to clonal propagation of turkish local hazelnut cultivars with optimal combination of growth media supplemented with plant growth regulators/additives in tissue cul­ ture techniques. meristem culture has been used for the purificati­ on of pathogens from plants since the 1930s (dodds and roberts, 1986; nehra and kartha, 1994). in this technique, the difference in the amount of patho­ gens in different plant cells is utilized such that the apical and root meristem cells of the plant are very small due to their continuous division characteristics hence the probability of viruses in these cells is very low, therefore, these tissues are highly preferred in the production of pathogen­free and/or disease­free plants (kaya et al., 2020 a). although it cannot be sta­ ted that these parts of the plant are completely free from viruses, the probability can be increased when applying different treatment combinations together (sherwood, 1993). in this study, meristem culture examinations were carried out using meristematic regions of length between 0.1­0.8 mm extracted from axenic (virus­free) shoots of plants belonging to hazelnut varieties collected from seven different pro­ vinces that were grown in vitro using clonal propaga­ tion. the micro­shoots regenerated in vitro were sub­ cultured in 4­6 week periods at standard conditions and the best regeneration rate among the eight diffe­ rent wpm media containing different ingredient combinations tested for meristem regeneration was obtained from wpm medium containing 4.44 µm bap, 27.8 µm fe­eddha and activated charcoal, and the regeneration rate obtained on this medium was between 68% and 94% for seven different hazelnut local cultivars tested. the iron component of medium is an essential micronutrient for hazelnut in vitro cultures and these cultures can also be very sensitive to the iron source and concentration. in in vitro cultures of hazelnut, beneficial effect of fe­eddha as source of iron, have fig. 3 ­ the interaction effect of different rootstocks and no3:nh4 ratio on a) number of leaves and b) number of nodes. acknowledgements this study was supported by mugla sitki kocman university, scientific research projects coordination unit (mugla, turkey, msku­bap, project number: 17­ 057). references ahmad n., anis m., 2007 ­ rapid clonal multiplication of a woody tree, vitex negundo l. through axillary shoots proliferation. ­ agro syst., 71: 195­200. amaral j.s., cunha s.c., santos a., 2006 ‐ influence of cultivar and environmental 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stormwater bioretention system a. russo 1 (*), a. speak 2, c. dadea 2, a. fini3, l. borruso 2, f. ferrini 4, s. zerbe 2 1 school of arts, university of gloucestershire, francis close hall campus, swindon road, cheltenham, gl50 4az, united kingdom. 2 faculty of science and technology, free university of bozen‐bolzano, piazza università, 5, 39100 bolzano, italy. 3 department of agricultural and environmental sciences, production, landscape, agroenergy, university of milan, milan, italy. 4 department of agrifood production and environmental sciences, section woody plants, university of florence, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: blue­green infrastructure, rain gardens, urban stormwater runoff, water sensitive urban design. abstract: several laboratory studies have shown the ability of bioretention sys­ tems to remove pollutants from stormwater. however, to our knowledge, no existing research has addressed the use of ornamental shrubs for improving water quality in bioretention systems in italian cities. in this short note, we evaluated the potential of three ornamental shrub species (lonicera pileata oliver, cotoneaster horizontalis decne., hypericum hidcoteense ‘hidcote’) for the removal of heavy metals in a stormwater bioretention system. pot experi­ ments in “pot prototypes” using an alternative bioretention system filter media have been carried out under controlled conditions. the ornamental shrubs were irrigated with semisynthetic stormwater with known heavy­metal con­ centrations. experimental results indicate that the removal of heavy metals by the system is very efficient. however, there was not a significant effect of the plant on the system’s retention efficiency. the removal of lead and cadmium by the system was over 87%. in order to provide accurate information for biore­ tention design, future research should comparatively assess plant species in a laboratory­scale filter column and in situ. 1. introduction urban stormwater runoff contains pollutants which can impact the quality of surface, seepage, and ground water (eckley and branfireun 2009; göbel et al., 2007). stormwater carries different pollutants, both organic and inorganic (barbosa et al., 2012), including copper, zinc, lead, cadmium, sediments, polycyclic aromatic hydrocarbons, and de­icing salts (muthanna et al., 2007) so that its quality management is of crucial (*) corresponding author: arusso@glos.ac.uk citation: russo a., speak a., dadea c., fini a., borruso l., ferrini f., zerbe s., 2019 ­ influence of diffe‐ rent ornamental shrubs on the removal of heavy metals in a stormwater bioretention systems ­ adv. hort. sci., 33(4): 605­612. copyright: © 2019 russo a., speak a., dadea c., fini a., borruso l., ferrini f., zerbe s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 july 2019 accepted for publication 20 september 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 605­612 606 importance to urban development and water resource planning (zgheib et al., 2012). in particular, cadmium has become an increasing problem because of its toxic effects on biological systems (mishra and tripathi, 2008). additionally, contaminated soils and waters represent an environmental and human health problem, which may be partially solved by the phytoremediation technology ( mojiri 2012; dadea et al., 2017). new approaches to improve water quality as well as water cycle in urban areas have been proposed, for example with best management practices (bmp), low impact design (lid), sustainable urban drainage system (suds), water sensitive urban drainage systems (wsud) and sponge cities ( (pompêo 1999; raja segaran et al., 2014, fletcher et al., 2015; griffiths 2017). these systems have been implement­ ed around the world because they provide important environmental, economic and health benefits such as improving water quality, reducing flood risk, increas­ ing amenity and increasing biodiversity in cities (griffiths, 2017). retention and degradation of stormwater pollutants using the above systems are becoming an important ecosystem service in urban environments (kabir et al., 2014). according to kabir et al. (2014), more than 75% of metals, such as pb, zn, cu, and cd is retained by blue­green infrastruc­ ture. in particular, bioretention systems, also known as biofilters or rain gardens, have been used to remove a wide range of pollutants, such as suspended solids, nutrients, metals, hydrocarbons, and microorganisms from stormwater runoff (muthanna et al., 2007; sun and davis 2007; hatt et al., 2009; blecken et al., 2010; megharaj et al., 2011; trowsdale and simcock 2011; weerasundara et al., 2016). well­designed bioretention systems can remove several pollutants from the urban runoff via physical, chemical, and bio­ logical processes, including plant uptake, sedimenta­ tion, filtration, and sorption on mulch and soil layers, and biodegradation by soil microorganisms (weerasundara et al., 2016). a bioretention system consists of several layers of filter media, normally a soil/sand/organic media matrix (approximately 0.7 ­ 1 m deep), a mulch layer and both woody and herba­ ceous plants (sun and davis 2007; davis et al., 2009; liu et al., 2014). plants not only assimilate pollutants directly from wastewater and rooting media into their tissues, but also act as catalysts for purification reactions by increasing the environmental diversity in the rhizos­ phere and promoting a variety of chemical and bio­ logical reactions that enhance pollutant removal (zhang et al., 2011). the benefits of bioretention by vegetation have not been well quantified (davis et al., 2009) and the majority of studies have focused on herbaceous plants in bioretention systems (sun and davis 2007; read et al., 2008; feng et al., 2012; barrett et al., 2013; payne et al., 2014). woody shrubs may also provide low maintenance and might be an attractive cover for stormwater systems (environmental services division, 2009). feng et al. (2012), conducted a large­scale stormwater biofilter column study and found that vegetation and the type of filter are significant fac­ tors for the treatment of metals. while most studies evaluated individual plant performance for metal uptake, some plant species have been shown to improve the performance of stormwater biofiltration systems (read et al., 2008; houdeshel et al., 2012). therefore, the assemblage of different species may be suitable for increasing biofilter efficiency and max­ imizing the spectrum of removed pollutants, but this topic remains largely unexplored. species mixes might also be preferred for aesthet­ ic and ecological reasons (read et al., 2008). however, higher concentration of heavy metals can cause damage to plants by reducing growth and the rates of photosynthesis and respiration, so that fur­ ther understanding on species’ tolerance to pollution is needed (hossain et al., 2012; ovečka and takáč 2014). plant species suitable for the use in bioreten­ tion systems are provided by north american and australian bioretention design guidelines (environmental services division, 2009; houdeshel et al., 2012). however, this information is not based on data from replicated experiments (dylewski et al., 2011) and little is known about the most suitable type of plant for bioretention systems in terms of survival and performance for italian cities. therefore, the objectives of our study were: i) to evaluate an alternative bioretention filter media; and ii) to test the hypothesis that species association may increase heavy­metal retention by the system constituted by different plant combinations and substrates; and iii) to understand the heavy­metal effect on chlorophyll and root/shoot ratios. 2. materials and methods experimental setup and planting material three species potentially suitable for planting in bioretention systems were chosen across a range of russo et al. ‐ influence of ornamental shrubs on the removal of heavy metals 607 evergreen ornamental shrubs commonly grown in urban areas in central­northern italy. 70 plastic pot prototypes (fig. 1) with a truncated pyramid shape (418 x 310 mm, 347 x 245 mm base, and 575 mm height) with lateral taps at the bottom, were put in a greenhouse facility at the university of florence in sesto fiorentino, italy, in october 2013 (fig. 2). the pots consisted of four layers: (1) the drainage layer at the bottom of the pot was filled with 150 mm of per­ lite (agrilit 2, perlite italiana) and (2) a filter sheet (drenalit f130, perlite italiana) was placed to sepa­ rate the 300 mm substrate layer (3) (agriterram tv, perlite italiana) from the drainage layer, followed by a 50 mm mulch layer (4) (geobark pine bark) to cover the soil and improve pollutant retention (muthanna et al., 2007). the substrate basic properties were ph 6­7, ec <40 ms/m, cation­exchange capacity (cec) 55­60 meq/100 g, total organic content <20­25%, bulk densi­ ty 400 kg/m3 ± 5%, and vertical permeability >13 mm/min. the system consisting of agrilit 2 and agriterram tv (perlite italiana), known as per­ liround™, is used for the greening of roundabouts and traffic islands (perlite italiana, 2011). three­year­ old plants of lonicera pileata oliver, cotoneaster hori‐ zontalis decne., and hypericum hidcoteense ‘hidcote’ were potted in the containers. each pot contained 2 plants of the same species, namely lonicera pileata (lp), cotoneaster horizontalis (ch), and hypericum hid‐ coteense ‘hidcote’ (hh), or plants of two species, in all possible combinations (lp + ch, lp + hh, and ch + hh). 5 additional pots were prepared as previously described but left unplanted. the experiment was car­ ried out from october 2013 until june 2014. plants were grown at 28/18°c day/night temperatures and exposed to natural daylight, and the light transmission was of 90%. relative humidity was always above 60%. measurement of pollutants and plant growth synthetic stormwater runoff was prepared using tap water that was left to stand at room temperature in 200­l plastic water storage tank for 24 h to dechlo­ rinate and thermally equilibrate (fig. 2) (sun and davis, 2007). the first irrigation with synthetic stormwater started on april, 3rd 2014 after approxi­ mately 6 months of plant growth in the pots. plants were irrigated with synthetic stormwater with heavy metal concentrations (pb and cd) once per week for 3 weeks. the total volume of runoff applied to each pot was 5 l, this amount was based on rainfall pre­ cipitation in florence (vijaya kumar et al., 2013). the concentrations (mg l­1) of pollutants in our synthetic stormwater were 2.02 (mg l­1) in the first irrigation and 1.97 in the successive irrigations for pb and 0.37 (mg l­1) in the first irrigation and 0.39 mg l­1 in the suc­ cessive irrigations for cd. these values are the highest concentrations of highway runoff reported in the liter­ ature (kayhanian et al., 2012). to determine the effect of plants on pollutant removal from stormwater, the water that drained from the tap (outflow) was collect­ ed during the first and second irrigations. we collected 60 samples from the “stormwater plants’’ and 10 from the unplanted containers “stormwater soil”. we also collected stormwater (inflow) in order to assess its quality, before each irrigation. furthermore, ph was measured immediately after each sampling using a ph electrode le407. samples were filtered through 0.45 µm membrane filter (swinnex filter holder) and acidi­ fied with 1% of nitric acid. the samples were sent to an accredited analytical chemistry laboratory (research centre for agriculture and forestry, laimburg, italy) and analyzed according to standard methods for pb and cd using icp. the removal efficien­ cy was calculated as percentage of inflow concentra­ tions. a minolta spad­502 leaf chlorophyll meter was fig. 1 ­ schematic drawing of the bioretention pot prototype. not to scale. fig. 2 ­ photo of the greenhouse experiment at the university of florence, italy: (a) bioretention pot prototypes, (b) 200­l plastic water storage tanks. adv. hort. sci., 2019 33(4): 605­612 608 used for non­destructive data collection. the instru­ ment is able to provide a rapid and reasonably accu­ rate estimate of leaf chl. measurements were made before the first irrigation and after the second irriga­ tion. spad readings were recorded for 3 positions on each leaf and for 3 different leaves on a single shrub (table 1). at the end of the experiment, dry weight (dw) of roots, stems and leaves was determined in 36 treated plants and in 36 control plants. the total plant dw and shoot/root ratio were calculated. experimental design and statistics the experiment was a randomized complete block with five blocks (rao, 2007). the outflow data were checked for normality using kolmogorov­smirnov and ryan­joiner tests using minitab 17. the data did not fit a normal distribution and we used a non­parametric kruskal­wallis test to analyse statistical differences among treatments. in order to determine whether there was a statistically significant effect between treatments on the plant­growth parameters, including stem, roots and leaves, a post­ hoc comparison on means was conducted by duncan’s test (spss statistics) with p<0.05. 3. results and discussion mean outflow concentrations and reduction are shown in table 2. outflow pb concentrations ranged in the first irrigation from 4.13 µg/l in lonicera + cotoneaster to 9.37 µg/l in lonicera pileata + hypericum hidcoteense ‘hidcote’. cd concentrations ranged in the first irrigation from 1.57 µg/l in lonicera and cotoneaster to 3.23 µg/l in cotoneaster + hypericum. however, pb concentrations ranged in the second irrigation from 5.88 µg/l in soil to 237.80 µg/l in lonicera + lonicera. cd concentrations ranged in the second irrigation from 1.44 µg/l in soil to 8.34 µg/l in cotoneaster as single species. we found that the different shrub species did not affect the reduction and there was no significant dif­ ference in metal concentration between the effluent from soil­only controls and shrubs or mix of species. based on the results above, heavy metals are mainly retained by physical processes (i.e., sedimentation and chelation) within the perliround substrate and we were unable to determine removal by vegetation uptake. however, previous studies have highlighted the limited role of plant uptake in the removal of metals from storm water in bioretention systems (read et al., 2008; lefevre et al., 2015). several fac­ tors could interact with the cd uptake, for example the interaction of soil composition, ph, organic mat­ ter, and available mineral elements may decrease or increase the plant availability of cd (chizzola and lukas, 2006). furthermore, effective vegetation metal removal performance in bioretention has been attributed to species (i.e. hyperaccumulating plants), root architecture, plant age, and leaf area and the species chosen may not be metal accumulators or alter the soil chemistry/ecology to enhance metal retention (muerdter et al., 2018). based on the aver­ age effluent concentrations, reduction efficiency for pb and cd was more than 87%. removal was very high in non­vegetated bioretention containers >99.4%, this is due to the absence of roots and soil compaction (rycewicz­borecki et al., 2016). similarly, table 1 ­ effects of cd and pb on the spad clorophyll in three ornamental shrubs lp= lonicera pileata, hh= hypericum ‘hidcote’, ch= cotoneaster horizontalis. standard deviation in brackets. spad readings were recorded for 3 positions on each leaf and for 3 different leaves on a single shrub. treatments were at 2 plants per pot, each pot contained 2 plants of the same species (column a, b and c) and plant mix (2 species, column d, e and f). treatments a b c d e f lp lp hh hh ch ch lp hh lp ch ch hh control ­ without heavy metals mean 69.03 68.10 38.90 38.23 49.70 53.80 42.67 36.93 58.60 67.17 41.53 47.43 (6.55) (3.73) (1.54) (0.29) (1.39) (1.41) (7.61) (4.83) (0.10) (3.10) (0.58) (3.47) mean 44.37 56.17 43.50 44.60 56.13 60.70 49.40 38.67 54.50 61.43 63.43 47.03 (4.08) (2.76) (8.44) (3.75) (4.22) (5.47) (4.76) (4.36) (5.60) (5.75) (8.13) (5.55) mean 51.27 52.80 42.83 42.17 62.93 61.67 54.43 38.83 53.70 66.30 66.20 45.50 treatment with heavy metals (1.58) (1.57) (1.81) (1.29) (6.37) (3.21) (1.66) (3.97) (4.47) (2.41) (4.22) (3.74) mean 75.40 66.53 38.57 41.70 60.57 69.70 66.30 43.57 77.03 65.53 62.70 44.50 (8.59) (9.37) (4.40) (2.17) (4.30) (4.25) (8.83) (2.61) (4.74) (6.33) (2.98) (2.85) mean 70.90 63.73 43.27 42.33 61.57 61.53 52.97 43.80 69.20 60.23 60.93 40.33 (10.62) (14.17) (5.43) (2.81) (7.09) (5.89) (6.75) (5.16) (12.33) (2.97) (4.34) (4.44) mean 60.87 70.57 41.23 46.30 44.77 44.40 55.20 41.43 65.77 65.00 65.63 40.37 (13.55) (9.64) (3.09) (3.64) (3.49) (1.75) (2.67) (2.61) (7.16) (2.31) (1.91) (1.75) russo et al. ‐ influence of ornamental shrubs on the removal of heavy metals 609 tention systems in laboratory (davis et al., 2003; kabir et al., 2014; wang et al., 2017; muerdter et al., 2018). the results suggested that plant growth was not influenced by heavy­metal treatments for the majority of species. it is likely that the heavy metal concentra­ tions were below the tolerance limits of these species or the length of exposure time was not long enough. however, we found statistically significant differ­ ences (duncan multiple range test; p<0.05) in root/shoot weight ratios for hypericum sp. the addi­ tion of heavy metals appeared to increase the root/shoot ratio (table 3). this observation may be due to the fact that low and moderate doses of cd could stimulate multiplication, rooting, and biomass rycewicz­borecki et al. (2016), found that compacted soil conditions of unplanted controls retained signifi­ cantly more cu, pb, and zn than carex praegracilis, and carex microptera treatments. the outflow concentrations changed over time and the removal efficiency was lower in the second irrigation for the majority of planted pots and not for the unplanted ones. this may be due to soil com­ paction. the lower removal rate could be attributed to leaching of pb and cd from the bioretention media as the concentration of heavy metals in the bottom layer increases (muthanna et al., 2007). reduction rates in this study agree with the rates observed in previous experiments carried out on biore­ standard deviation in brackets. duncan multiple range test; significant at p<0.05. ns=not significant. table 3 ­ effect of heavy metals on stem dry weight (sdw), root dry weight (rdw) leaf dry weight (ldw), total dry weight (tdw) and root/shoot table 2 ­ outflow concentrations and reduction efficiencies for pb and cd standard deviation in brackets. kruskal­wallis test; significant at p<0.05. ns= not significant. soil (unplanted pots) lonicera sp. & lonicera sp. hypericum sp. & hypericum sp. cotoneaster sp. & cotoneaster sp. lonicera sp. & hypericum sp. lonicera sp. & cotoneaster sp. cotoneaster sp. & hypericum sp. outflow concentration (pb) (µg/l) 1st irrigation 7.36 (8.97) 8.88 (8.54) 4.17 (1.84) 7.03 (11.02) 9.37 (5.67) 4.13 (1.58) 7.07 (12.17) p value (kruskal­wallis test) ns ns ns ns ns ns ns reduction % (pb) 99.6 99.6 99.8 99.7 99.5 99.8 99.7 outflow concentration (pb) (µg/l) 2nd irrigation 5.88 (1.87) 237.80 (313.60) 53.04 (79.04) 49.42 (31.49) 20.52 (4.69) 13.94 (6.52) 80.32 (107.64) p value (kruskal­wallis test) ns ns ns ns ns ns ns reduction % (pb) 99.7 87.9 97.3 97.5 99.0 99.3 95.9 outflow concentration (cd) (µg/l) 1st irrigation 2.08 (2.32) 1.45 (0.88) 1.77 (1.05) 2.38 (4.28) 2.68 (3.41) 1.57 (1.82) 3.23 (3.78) p value (kruskal­wallis test) ns ns ns ns ns ns ns reduction % (cd) 99.4 99.6 99.5 99.4 99.3 99.6 99.1 outflow concentration (cd) (µg/l) 2nd irrigation 1.44 (0.93) 3.78 (2.28) 2.54 (1.52) 8.34 (6.97) 2.22 (1.69) 2.04 (1.72) 7.34 (10.42) p value (kruskal­wallis test) ns ns ns ns ns ns ns reduction % (cd) 99.6 99.0 99.3 97.9 99.4 99.5 98.1 treatments sdw (g) rdw (g) ldw (g) tdw (g) root/shoot (g) lonicera sp. & lonicera sp. without heavy metals 22.93 (4.07) 10.37 (3.73) 19.53 (3.71) 52.83 (7.93) 0.63 (0.26) lonicera sp. & lonicera sp. with heavy metals 27.97 (4.04) 11.85 (1.64) 24.13 (5.11) 63.95 (10.28) 0.60 (0.06) p value (duncan multiple range test) ns ns ns ns ns hypericum sp. & hypericum sp. without heavy metals 19.92 (8.43) 9.32 (4.51) 13.38 (7.41) 42.62 (19.70) 0.46 (0.21) hypericum sp. & hypericum sp. with heavy metals 22.40 (5.70) 6.75 (4.14) 20.48 (7.61) 49.63 (16.22) 0.71 (0.14) p value (duncan multiple range test) ns ns ns ns <0.01 cotoneaster sp. & cotoneaster sp. without heavy metals 55.03 (11.37) 11.45 (7.62) 16.73 (7.01) 83.22 (24.79) 0.24 (0.06) cotoneaster sp. & cotoneaster sp. with heavy metals 65.87 (6.79) 16.35 (3.19) 18.00 (2.92) 100.22 (10.72) 0.22 (0.03) p value (duncan multiple range test) ns ns ns ns ns lonicera sp. & hypericum sp. without heavy metals 28.28 (7.68) 10.73 (4.87) 20.50 (6.45) 59.52 (15.85) 0.54 (0.14) lonicera sp. & hypericum sp. with heavy metals 26.65 (4.89) 8.92 (3.01) 16.50 (2.75) 52.07 (9.38) 0.47 (0.07) p value (duncan multiple range test) ns ns ns ns ns cotoneaster sp. & hypericum sp. without heavy metals 43.07 (18.61) 11.62 (6.98) 17.28 (4.23) 71.97 (23.38) 0.37 (0.21) cotoneaster sp. & hypericum sp. with heavy metals 45.90 (24.22) 10.23 (5.04) 18.75 (6.51) 74.88 (33.52) 0.38 (0.18) p value (duncan multiple range test) ns ns ns ns ns lonicera sp. & cotoneaster sp. without heavy metals 45.38 (21.95) 13.35 (4.47) 19.07 (6.50) 77.80 (28.10) 0.35 (0.11) lonicera sp. & cotoneaster sp. with heavy metals 49.18 (17.28) 16.23 (5.78) 18.95 (7.81) 84.37 (16.94) 0.31 (0.15) p value (duncan multiple range test) ns ns ns ns ns 610 adv. hort. sci., 2019 33(4): 605­612 production in heavy metal­tolerant shrubs (wiszniewska et al., 2017). furthermore, the genus hypericum l. has been described as a cadmium hyperaccumulator (gardea­torresdey et al., 2005). spad readings ranged from 36.93 hypericum sp. to 77.03 in lonicera. differences in chlorophyll con­ tent (table 1) were statistically significant (one­way anova test; p<0.05) in mono­specific pots between hypericum, lonicera and cotoneaster (table 1, columns a,b,c) as well as in mixed pots containing, respectively, hypericum and lonicera, and lonicera and cotoneaster plants (table 1, columns d and f). this result agrees with previous studies that found that mixed heavy metals decrease the chlorophyll content in various plants (chandra and kang, 2016). the concentration of non­essential metals like pb and cd may be the cause of low chlorophyll content and could also have several negative impacts via oxida­ tive stress (nadgórska­socha et al., 2013). recent studies have suggested that laboratory­ scale filter columns do not satisfactorily replicate field­scale conditions leading to calls for in situ evalu­ ation of bioretention systems (trowsdale and simcock, 2011; liu et al., 2014). furthermore, previ­ ous studies conducted in greenhouses in which plants were grown in pots have shown that pot size can have a limiting effect on plant growth, nutrient efficiency and photosynthesis rates (ray and sinclair, 1998). future research should comparatively assess plant species in a laboratory­scale filter column and in situ. 4. conclusions this study tested an alternative bioretention sys­ tem filter media and species design. the reduction of cd and pb concentrations was over 87% similar to other studies, however there were no differences between replicates with plants and the soil­only con­ trol. therefore, the presence of vegetation did not significantly affect heavy metal removal. some species appeared cd and pb tolerant 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iran. key words: abiotic stress, membrane integrity, tropical fruit. abstract: guava fruit is a tropical fruit thus sensitive to the chilling injury. in this study the effects of melatonin (known to protect membrane integrity and to help to face abiotic and biotic stress) is evaluated for reduction of chilling injury during postharvest. guava fruits were dipped into 10, 100 and 1000 μmol l­1 melatonin solutions, then kept at cold storage (10±1°c and 90% relative humidity) for 21 days. several parameters including chilling injury, malondi­ aldehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity, phospholipase d and lipoxygenase activity were mea­ sured after treatment. measurements were made every 7 days during the stor­ age. results showed that melatonin decreased chilling injury, malondialdehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity compared to the control. also, results indicated that chill­ ing injury of guava fruit by using melatonin decreased through increasing integrity of membrane and reducing phospholipase d and lipoxygenase activity. thus, melatonin can be a useful treatment for decreasing postharvest chilling disorder of guava fruit. 1. introduction guava (psidium guajava l.) is one of the most important fruits of tropical and sub­tropical regions in the world. the fruits are delicious, rich in vitamin c and minerals (deepthi et al., 2016). there is a great demand of guava fruits in both domestic and international markets for fresh and processing purposes. cold storage is one of postharvest technologies for maintaining quality of horticultural crops until human consumption. however, guava is sensitive to chilling disorder of cold storage (temperature of below 12°c). signs of chilling injury include irregular ripening and surface pitting on the fruit which decreases quality of fruit (etemadipoor et al., 2020). resistance to the chilling temperature related to several factors. one of the most important factors is maintaining membrane integrity (*) corresponding author: a_mirshekari@yu.ac.ir citation: mirshekari a., madani b., 2022 ­ decreasing postharvest chilling injury of guava fruit by using melatonin treatment. ­ adv. hort. sci., 36(1): 37­ 42. copyright: © 2022 mirshekari a., madani b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 october 2021 accepted for publication 13 february 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12222 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 37­42 38 (wongsheree et al., 2009). membrane integrity can be measured using leakage, malondialdehyde con­ tent, lipoxygenase and phospholipase d (aghdam et al., 2014). several methods used to decrease chilling injury symptoms of fruit rely on the use of hot water and uv­c (pongprasert et al., 2011). melatonin plays in fruit ripening and senescence and membrane integrity and protection against abiotic and biotic stresses (rastegar et al., 2020). melatonin treatment maintained quality of in pear (liu et al., 2019), peach (gao et al., 2016), and grape (xu et al., 2018) and tomato (aghdam et al., 2019) fruits during cold storage. however, melatonin effects on reducing chilling injury of guava fruit have not been evaluated during cold storage. therefore, the purpose of this study was to investigate melatonin effects on chilling injury reduction. 2. materials and methods the guava fruits (green stage maturity) were bought from a commercial orchard in hormozgan province, iran and the uniform sized fruits were transferred to the laboratory. twelve fruits per four replications were dipped into 10 (t2), 100 (t3) and 1000 (t4) μmol l−1 melatonin solutions for 10 min. distilled water was used as the control (t1). fruit were kept at cold storage (10±1°c and 90% relative humidity) for 21 days. several parameters were mea­ sured soon after treatment, and then the measure­ ments were made every 7 days during the storage. finally, the parameters were checked again one day after exposing to the ambient temperature (25±1°c). the measurements included chilling injury index assessment, percentage of ion leakage, malondialde­ hyde content, weight loss, titratable acidity, soluble solids concentration (ssc), ascorbic acid, total pheno­ lic content (tpc), antioxidant activity, phospholipase d and lipoxygenase activity. chilling injury (ci) index assessment, percentage of ion leakage (el) and malondialdehyde (mda) content ci index was assessed subjectively with a scale from 1 to 5, where 5= >50% surface pitting area, 4= 31­50% surface pitting area, 3= 16­30% surface pit­ ting area, 2= 1­15% surface pitting area, 1= 0% no chilling symptoms. el was measured by using method described by madani et al. (2016) and results expressed as per­ centage. the mda content was determined based on the method described by wang et al. (2015). the content of mda was expressed as nmol g­1 fw. weight loss, soluble solids concentration (ssc), titratable acidity (ta), ascorbic acid, total phenolic content (tpc) and total antioxidant activity weight loss was measured based on initial and final experiment at 7­day intervals during storage using a digital balance, and results were expressed as percentage. soluble solids concentration (ssc) and ta of pulp tissues were measured by using the method of ali et al. (2011) and the results were expressed as %ssc and %ta, respectively. ascorbic acid was measured using dye method described by ranggana (1986) and results were expressed as mg 100 g­1 fresh weight (fw). tpc were assayed using folin­ciocalteu procedure (singleton and rossi, 1965). results were expressed as mg of gallic acid equivalents (gae) per gram of fresh weight (mg gae g­1 fw). the dpph assay was measured according to the method described by mirshekari et al. (2019). results were expressed as percentage. phospholipase d and lipoxygenase activity phospholipase d and lipoxygenase assay was determined based on the method described by aghdam and mohammadkhani (2014). one unit of phospholipase d was defined as the amount of enzyme that catalyzed the formation of 1 nmol d­ nitrophenol h­1. one unit of lipoxygenase was defined as the amount of enzyme which causes an increase in absorption of 0.01 min­1 at 234 nm and 25°c when linoleic acid was used as the substrate. protein con­ tent was estimated according to bradford (1976). enzymes activities were expressed as units per mil­ ligram of protein. statistical analysis experiments were carried out using completely randomized design. four replications per treatment used for this study. data were analyzed using (sas) version 8.2 (sas institute inc., cary, nc, usa). variation sources were storage days and treatments and means were compared with duncan’s multiple range test (dmrt) at significance level of 0.05. mirshekari and madani ‐ decreasing chilling injury of guava by melatonin 39 3. results and discussion chilling injury (ci), electrolyte leakage (el), and malondialdehyde (mda) in the present study, melatonin treatment reduced chilling injury index of guava fruit after 7 days of chilling storage when compared with the con­ trol (table1). moreover, ci increased with storage time (table 1). at the end of storage day control (t1) had the severe chilling injury index (4.8) with highest pitting signs, while t4 had the lowest chilling injury index of 3.2. usually, ci happens at the cell mem­ brane, and maintaining its integrity reduces ci (mirdehghan et al., 2007; mirshekari et al., 2020). accordingly, electrolyte leakage has been used as an indicator of membrane damage. in this study, el increased during storage for control and treated melatonin fruits (table 1). however, at the end of storage day el was lower in t3 and t4 compared to the t2 and t1 (table 1). these results showed the role of melatonin in maintenance of membrane integrity. comparable results have been stated for sapota fruit by mirshekari et al. (2020). researches have shown that melatonin treatments affect elec­ tron flow acceleration in mitochondria to maintain membrane integrity (tan et al., 2013). as shown in table 1, mda of control fruit increased during storage. also, lower mda content were observed in all melatonin treated fruits com­ pared to the (t1) after 21 days of chilling storage (table 1). one of the first events in the ci is mem­ brane lipid peroxidation. mda is the final product of lipid peroxidation (imahori et al., 2008). lower tem­ peratures are the main inducers of oxidative damage which produce higher ros and change the ratio of unsaturated fatty acids to saturated forms (antunes and sfakiotakis, 2008). melatonin treatments low­ ered mda accumulation of sapota fruit (mirshekari et al., 2020). weight loss, ssc, ta, ascorbic acid, tpc and total antioxidant activity weight loss was at the highest rate (14.3 %) in the control fruits (t1) after 21 days. treated fruit (t4) showed lower weight loss (6.7%) compared to the t1 and t2 at the end of storage day (table 2). weight loss is an index for assessing quality of fruits during storage (yaman and bayonidirli, 2002). skin strength properties of fruit by using melatonin treatment might lower weight loss. our results are comparable with rastegar et al. (2020) who indicated that the weight loss was decreased by using melatonin treatment in mango. ssc and ta concentration are main factors for fruit quality judgment. the initial ssc value of this study was 5.2% in control fruits (t1) and increased table 1 ­ melatonin treatments (0, 10, 100 and 1000 μmol l−1) effects on chilling injury index (ci), electrolyte leakage (el) and malondi­ aldehyde (mda) content in guava fruit stored at 10°c for up to 21 days (z) small and capital letters show significant differences by dmrt at p= 0.05 between treatments in columns, and storage time for each parameter, respectively. treatment (μmol l­1) storage (day) 0 7 14 21 ci 0 (t1) 0 a d z 2.3 a c 3.5 a b 4.8 a a 10 (t2) 0 a d 2.1 a c 3.2 ab b 4.1 b a 100 (t3) 0 a d 1.1 b c 2.5 bc b 3.8 b a 1000 (t4) 0 a d 1.3 b c 2.1 c b 3.2 c a el (%) 0 (t1) 6.5 a d 18.0 a c 32.3 a b 49 a a 10 (t2) 6.5 a d 16.5 a c 30.2 a b 47 a a 100 (t3) 6.5 a d 12.5 b c 22.2 b b 31.7 b a 1000 (t4) 6.4 a d 10.5 b c 20.5 b b 32.3 b a mda (nmol g‐1 fw) 0 (t1) 5.5 a d 8.3 a c 12.3 a b 15.6 a a 10 (t2) 5.3 a d 7.2 b c 11.6 a b 13.1 b a 100 (t3) 5.0 a b 5.6 c b 7.6 b a 8.5 c a 1000 (t4) 5.6 a b 5.9 c b 8.2 b a 9.1 c a adv. hort. sci., 2022 36(1): 37­42 40 ta content compared to the t1 and t2 (table 2). the higher amounts of ta in melatonin treated fruit can be related to the reduction of respiration rate during storage (han et al., 2004). ascorbic acid decreased during storage. after 21 days of storage, ascorbic acid was higher in t3 and t4 compared to the t1 (table 2). one of the most signifi­ cant signs of the nutrient value of fruits is ascorbic acid. the ascorbic acid reduction during storage can be related ascorbic acid oxidase (choudhary et al., 2016). melatonin treatment increases oxidative stress resistance by increasing ascorbic acid (gao et al., 2016). our results are comparable with gao et al. (2016) who stated that melatonin treatment main­ (z) small and capital letters show significant differences by dmrt at p= 0.05 between treatments in columns, and storage time for each parameter, respectively. table 2 ­ melatonin treatments (0, 10, 100 and 1000 μmol l­1) effects on weight loss, soluble solids content (ssc), titratable acidity (ta), ascorbic acid, total phenolic content (tpc) and total antioxidant activity (taa) in guava fruit stored at 10°c for up to 2 treatment (μmol l−1) storage (day) 0 7 14 21 weight loss (%) 0 (t1) 0 a d z 4.5 a c 9.5 a b 14.3 a a 10 (t2) 0 a d 3.2 b c 8.3 b b 13.4 a a 100 (t3) 0 a d 2.6 c c 5.2 c b 8.6 b a 1000 (t4) 0 a d 2.1 c c 4.2 d b 6.7 c a ssc (%) 0 (t1) 5.2 a d 8 a c 12.1 a b 13.4 a a 10 (t2) 5..1 a c 7.3 ab b 11.7 a a 12.6 a a 100 (t3) 5.2 a d 6.6 bc c 8.2 b b 9.2 b a 1000 (t4) 5.2 a c 5.8 c bc 6.9 c ab 7.5 c a ta (%) 0 (t1) 0.9 a a 0.6 a b 0.4 b c 0.3 bc c 10 (t2) 0.9 a a 0.7 a b 0.4 b c 0.2 c c 100 (t3) 0.9 a a 0.8 a a 0.7 a a 0.5 ab b 1000 (t4) 0.9 a a 0.7 a ab 0.6 a bc 0.5 a c ascorbic acid (mg 100 g‐1 fw) 0 (t1) 135.5 a b 145.6 b a 121.5 b c 118.3 c c 10 (t2) 136.4 a b 149.2 ab a 126.5 b c 122.4 bc c 100 (t3) 136.2 a b 151.2 a a 132.7 a c 127.2 ab d 1000 (t4) 134.5 a b 150.3 ab a 133.8 a b 129.1 a c tpc (mg gae g−1 fw) 0 (t1) 175 a d 210.6 c a 189.7 b b 181.5 d c 10 (t2) 172 a c 201.2 d a 191 b b 188.5 c b 100 (t3) 173.2 a d 233 a a 213.2 a b 200.7 b c 100 (t4) 174 a d 224.2 b a 218.7 a b 214.2 a c taa (%) 0 (t1) 52.3 a c 62..1 b b 70 c a 65.2 b ab 10 (t2) 52 a c 64.2 b b 72.7 c a 67 b b 100 (t3) 53.2 a c 72.4 a b 78.5 a a 74.2 a b 1000 (t4) 52.7 a b 75 a a 77.2 ab a 75.5 a a during storage (table 2). however, at the end of stor­ age, t3 and t4 melatonin treatments decreased ssc content compared to untreated (t1) and t2 treat­ ments. lower ssc content in this research is in agree­ ment with results of liu et al. (2018) who showed that ssc decreased with melatonin treatments during storage. lower amounts of ssc might be due to the slower respiration rate and a weaker metabolic activ­ ity due to the reduced rate of carbohydrate hydroly­ sis. table 2 shows ta content of treated and control fruits. ta content was the highest at harvest day (0.9%). however, ta content decreased during stor­ age. at the end of storage t3 and t4 showed higher mirshekari and madani ‐ decreasing chilling injury of guava by melatonin 41 tained ascorbic acid content of peach. tpc and antioxidant activity decreased during chilling storage. however, melatonin treated fruit had higher tpc and antioxidant capacity compared with control (table 2). it has been shown that mela­ tonin treatment increased tpc by regulating gene expression in phenyl propanoid pathway (zhang et al., 2016). moreover, liu et al. (2018) stated that melatonin treatment increased tpc and dpph scav­ enging capacity of strawberry. this indicated that melatonin showed positive effect on antioxidant activity of guava fruit. phospholipase d and lipoxygenase activity phospholipase d and lipoxygenase activity increased during chilling storage. however, mela­ tonin treatment decreased their activities during storage (table 3). similarly, melatonin treatment decreased ci signs and inhibited phospholipase d and lipoxygenase activity of sapota fruit (mirshekari et al., 2020). studies indicated that ci was achieved by the activities of membranous lipolytic enzymes like phospholipase d and lipoxygenase which catalyse peroxidation of polyunsaturated fatty acids and are believed to be major contributors to ci in plant tissue (aghdam and mohammadkhani, 2014). 4. conclusions results of this study showed that melatonin treat­ ments during cold storage of guava fruit decreased chilling injury, soluble solids concentration, malondi­ aldehyde content, electrolyte leakage, phospholipase d and lipoxygenase activity and increased titratable acidity, ascorbic acid, total phenolic compounds and antioxidant activity compared to the control. accordingly, we found that melatonin application reduced ci of guava fruit with enhancing membrane integrity and decreasing phospholipase d and lipoxy­ genase activity. concerning the different treatments, we found out that while 10 μmol l­1 produced results which were not significantly different from control treat­ ment, 100 μmol l­1 and 1000 μmol l­1 were signifi­ cantly different for ci, weight loss, soluble solids con­ centration, total phenolic compounds and phospholi­ pase d activity at the end of storage. in conclusion, melatonin application at 1000 μmol l­1 can be recom­ mended to be used to decrease ci in guava fruit under cold storage. references aghdam m.s., mohammadkhani n., 2014 ­ enhancement of chilling stress tolerance of tomato fruit by postharvest brassinolide 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institute for the bioeconomy (ibe), italian national research council (cnr), 50019 sesto fiorentino, italy. 3 department of chemistry, life sciences and envinronmental sustainability, university of parma. parco area delle scienze, 11/a, 43124 parma, italy. key words: genetic diversity, phenotype, pomegranate, rapds, ssrs, varieties. abstract: the italian research on p. granatum l. is still limited, although the study of local germplasm is extremely important in order to preserve the exist­ ing biodiversity and to identify potential useful characters for a renewed indus­ try. the study aimed at characterizing for the first time ancient pomegranates, grown in emilia romagna (italy), through 38 quantitative morphometric descriptors related to leaf, flower, fruit and seed, 42 rapd and 12 ssr markers. morphological analyses showed large variation of traits among accessions and the descriptors related to fruit and seed had the highest power of discrimina­ tion. the considerable variation found was consistent with anova and pca results. among all rapds tested, 7 were selected for their polymorphism; whereas among selected ssrs, 8 presented differences in the genetic profiles allowing a good discrimination of the local pomegranate accessions. the genet­ ic relationships among pomegranates were studied by upgma cluster analysis and the accessions were clearly regrouped in four different genotypes. the study has highlighted significant differences and interesting pomological char­ acteristics in the local pomegranates, which confirmed the good potential of this germplasm for the pomegranate industry. 1. introduction pomegranate (punica granatum l.) is one of the world most ancient domesticated fruit crops and it is believed to have been first grown in the region between the caspian sea and the caucasus (zohary and spiegel­ roy, 1975). its diffusion occurred across the millennia due to man’s activi­ ties or gene flow in quite varied environments as concerns climatic condi­ tions, has produced a rich and diversified germplasm. p. granatum l. has (*) corresponding author: deborah.beghe@unipr.it citation: beghè d., fabbri a., petruccelli r., marieschi m., torelli a., ganino t., 2019 ­ morphological and molecular characterization of ancient pome‐ granate (punica granatum l.) accessions in northern italy. ­ adv. hort. sci., 33(4): 581­592. copyright: © 2019 beghè d. , fabbri a., petruccelli r., marieschi m., torelli a., ganino t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 june 2019 accepted for publication 22 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 581­592 582 a large genetic pool, represented by over 500 described cultivars throughout the world, and by a wide amount of wild plants, and its germplasm is so far only partially explored (beghè et al., 2016). in spite of the wide genetic diversity, only 50 cultivars were widely cultivated in the main growing areas at the time of the last official survey made by interna­ tional institutions (ipgri, 2001). consequently, the risk of a drastic loss of the existing biodiversity is high. in the last decade the interest on pomegranate has grown and the world production (estimated to be around 1.5 million tonnes, of which 90% provided by the main producers: india, china, iran) has rapidly increased (da silva et al., 2013). the renewed interest in this crop is to be ascribed to socio­economical and cultural factors, which led to a change in food habits in the west, with a growing attention to the nutrition­ al quality of foods (negri, 2003). pomegranate, in this respect, is considered one the fruits with the most valuable nutritional properties (calani et al., 2013). pomegranate cultivation is also growing in italy, and in a ten­year period (since 2008) the surface cov­ ered, from a mere 7 ha (2 ha in calabria and 5 in sicily), passed to 1142 ha (mainly in southern italy, sicily (364 ha) and apulia (363 ha) and, with minor productions, also in venetia (152 ha), latium (101 ha), emilia romagna (44 ha), tuscany (21 ha) and lombardy (20 ha). productions rose from only 69 tons to over 12531 tons (agristat, 2017). one of the trends of recent years has been to import from israel pomegranate cultivars created and patented by israel breeders, to be utilised in italian orchards. although this strategy has led to an increase in production, it hinders any attempt at exploiting italian cultivars, which have been known for centuries but are not utilised in commercial culti­ vation. in italy, there are several local cultivars which are little known and scarcely diffused in the country. scarce research, mostly confined to southern italy, has been devoted to the characterization of p. grana‐ tum l. italian diversity, by means of molecular, mor­ phological and biochemical markers (adiletta et al., 2018). a research on the whole national territory is unavoidable to select autochthonous genotypes suit­ ed to the different environments, and to promote a pomegranate industry able to satisfy the internal demand and to compete with the international product. the best suited italian areas for pomegranate growing are the central and southern regions, char­ acterized by a mediterranean climate, unlike the northern regions, which have a continental climate, with cold and snowy winters. however, in some zones of emilia­romagna (44°­45° n and 11°­12° e, northern italy) microclimate conditions are such as to allow the cultivation of this species and of other mediterranean species like olive (lona et al., 1981; calani et al., 2013). ancient pomegranate trees, survived for hun­ dreds of years and adapted to local conditions have been retrieved in these areas. this local germplasm is of particular interest, being the result of a selection process occurred during many centuries in the unfa­ vorable conditions of this territory. the present research is part of a multidisciplinary project aiming at valorising ancient pomegranate cul­ tivars of northern italy. we utilized morphological and molecular markers (random amplified polymorphic dna (rapds) and simple sequence repeat (ssrs)) to characterize ancient pomegranate accessions present in emilia­romagna region. the pomological comparisons also had the purpose of determining the peculiar features and the potential of these plants, for a possible introduction in commer­ cial plantings or for their use in breeding programs. 2. materials and methods plant materials the pomegranate germplasm subject of this study was represented by very ancient trees located in a small hill area of parma province (emilia­romagna) (44°69 n, 10°02 e), at an altitude from 150 to 250 m a.s.l. eight accessions of p. granatum l., tagged with an alpha­numerical code (id): me1, me2, me3, me4, me5, me6, me7 and me8 (table 1), were studied during two seasons, 2014 and 2015. the selected plant material was maintained at pomegranate germplasm collection field in the low hills of the emilia appennins, where each accession was repli­ cated 4 times. the pomegranate trees were planted at a spacing of 5 x 5 m and trained to form a bush. morphological characterization the accessions were characterized according to the guidelines proposed by the project ec project genres 29 “conservation, evaluation, exploitation and collection of minor fruit tree species” (bellini and giordani, 1998), integrated by the list of characters proposed by bellini et al. (2007) and by the international union for the protection of new varieties of plants (upov, 2012). plant material was randomly sampled from beghè et al. ‐ characterization of ancient pomegranates in northern italy 583 around the canopy of four plants per each accession, by collecting 40 flowers (20 hermaphrodite also called “long­styled” and 20 male also called “short­ styled”) at full bloom, on june 1st, 40 adult leaves, from the middle part of the shoot in summer and 12 fruits, at ripening, in the first decade of october. all seeds were extracted from each fruits and 25 of them were randomly selected; arils (the seed fleshy coats, containing edible juice, that represent the seed outer integument or testa) were hand removed to analyze also the tegmen (seed lignified inner integument). the morphological characters evaluat­ ed included 38 quantitative traits (table 2). the linear dimensions were determined with a caliper, and the weight was measured using a semi­analytic electronic scale. from these values other indices have been cal­ culated as indicated in table 2. furthermore, some qualitative characters were observed; these traits are reported in table 1. dna extraction and molecular characterization total cellular dna was extracted from young leaves following the ctab (cetyl trimethylammonium bromide) as reported in ganino et al. (2008). forty­two decamer oligonucleotide primers belonging to the ai, ah, opa, opc, opx and opk series (table s1 of supplementary data) and twelve couples of ssr primers belonging to the pgaer (çalişkan et al., 2017), pgkvr (ravishankar et al., 2015), pom (hasnaoui et al., 2012), pom­agc (currò et al., 2010) and pg (ebrahimi et al., 2010) were used for polymorphism detection on the samples. rapd amplifications were performed as reported in marieschi et al. (2016). the rapd profiles obtained with each utilized primer were analyzed by comparison with gene ruler 100 pb dna ladder plus marker (m­medical, milano, it), with the kodak digital sciences 1 d images analysis software, calculating the size in base pairs (bp) of each amplicone present in the elec­ trophoretic run of each sample. ssr amplification reaction was performed as reported in ganino et al. (2008). the amplification condition, for the pgkvr, pgaer, pom­agc and pg series, were: a first step at 95°c for 5 min followed by 35 cycles of 45 s at 94°c, 45 s at 57°c, 45 s at 72°c, for denaturation, annealing, and primer extension; the last step included 8 min of incubation at 72°c. for the “pom” serie, the following thermal cycling proto­ col was used: a first step at 95°c for 3 min followed by 10 touchdown cycles of 30 s at 94°c, 40 s at 65°c (­1°c per cycle), 30 s at 72 and 25 cycles of 30 s at 94°c, 30 s at 55°c, 40 s at 72°c with final extension time of 8 min at 72°c. the amplification products were separated with a ceq 2000 genetic analysis system (beckman coulter, inc.) sequencer on acry­ lamide gel ceq separation gel lpa­1 (beckman coulter, inc.). a marker ceq dna size standard kit 400 (beckman coulter, inc.) was used to estimate the molecular weight of the amplified products. data analysis the quantitative morphological characters were evaluated: means, minimum and maximum, standard deviation. the coefficient of variation (cv) was calcu­ lated as indicator of variability. all data were subject­ ed to one way analysis of variance (anova) followed by tukey test to determine the statistically significant differences (p≤0.05). correlation analyses between descriptors to reveal possible relationships were car­ table 1 ­ punica granatum l. accessions studied, coded (id) and main qualitative characteristics of their fruit, leaf and flower id fruit shape size epicarp colour calyx type leaf shape petiol colour mucro blade colour flower petal colour shape short­ stiled shape long­ stiled me1 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic yellow no yellow red/orange medium bell sinuolate jug me2 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me3 oblate/rounded­ spheroid large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me4 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me5 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me6 oblate/rounded­ spheroid large reddish­yellow semi­ closed/ open elliptic red no yellow red/orange broad bell jug with base me7 oblate/rounded­ spheroid very small reddish­yel­ low/red open elliptic yellow no yellow red/orange narrow bell sinuolate jug me8 oblate/rounded­ spheroid very small reddish­yel­ low/red open elliptic yellow no yellow red/orange narrow bell sinuolate jug adv. hort. sci., 2019 33(4): 581­592 584 ried out using a bilateral pearson correlation. the same characters were also submitted to a principal component analysis (pca) to evaluate the relation­ ship between pomegranate accessions. the analysis was performed using xlstat 2009 software (addinsofttm1995­2009). rapd bands were treated as binary characters (present = 1 or absent = 0), xlstat 2009 software was used to estimate genetic similarities/dissimilari­ ties using jaccard’s similarity coefficient and cluster analysis by using the unweighted pair­group method with arithmetic mean (upgma) algorithm. the size of ssr fragments was determined using a conservative binning approach (kirby, 1990) through the statistical r software. the information content of the ssr markers under study was evaluated according to number of alleles per locus (na), observed (ho) and expected (he) heterozygosity, and polymorphic infor­ mation content (pic) (botstein et al., 1980) using the cervus 3.0 software (kalinowski et al., 2007). the level table 2 ­ quantitative traits used for characterizing pomegranate accessions and their descriptive statistics analysis using the mean, minimum, maximum, standard deviation (sd) and coefficient of variation (cv) (z) aril weight were calculated by subtracting tegmen fresh weight from whole seed fresh weight. trait trait code mean minimum maximum sd cv (%) leaf leaf fresh weight (g) lfw 0.10 0.070 0.160 0.029 29.13 leaf length (cm) ll 5.44 4.450 6.920 0.828 15.21 leaf width (cm) lw 1.62 1.260 2.150 0.278 17.21 leaf shape (length/diameter) ls 3.50 2.970 3.910 0.341 9.76 flower flower diameter long­styled (cm) fdl 1.51 1.220 1.810 0.221 14.57 flower length long­styled (cm) fll 4.75 2.900 5.950 1.193 25.11 petal number long­styled (cm) pnl 6.52 5.670 7.500 0.713 10.93 pistil length long­styled (cm) pll 1.75 1.500 2.030 0.231 13.20 flower diameter short­styled (cm) fds 1.48 1.200 1.770 0.163 11.06 flower length short­styled (cm) fls 3.73 2.840 4.490 0.564 15.10 petal number short­styled pns 6.50 6.000 7.400 0.510 7.86 pistil length short­styled (cm) pls 0.43 0.310 0.600 0.105 24.43 fruit fruit weight (g) fw 274.02 87.010 388.730 120.303 43.90 fruit diameter equatorial (cm) fd 8.22 5.070 9.600 1.799 21.88 calyx diameter equatorial (cm) cd 2.14 1.430 2.840 0.545 25.50 fruit height without calyx (cm) fl1 6.64 4.400 8.120 1.380 20.80 total fruit length (cm) fl2 8.11 5.570 9.530 1.568 19.34 calyx height (cm) cl 1.56 1.170 2.220 0.323 20.67 fruit skin thickness equatorial (mm) ft 0.42 0.300 0.550 0.096 22.69 fruit skin and carpellary membranes weight (g) scw 129.94 28.980 191.810 66.526 51.20 number of carpel in equatorial section nc 6.88 5.330 8.000 0.993 14.43 fruit shape index (height/diameter) fsi 0.81 0.730 0.890 0.059 7.21 calyx shape index (height/diameter) csi 0.80 0.610 0.880 0.095 11.93 % skin and carpellary membranes sc (%) 45.10 33.300 53.400 7.499 16.63 % seeds s (%) 54.92 46.600 66.700 7.491 13.64 total seeds weight (g) stw 141.918 58.076 202.556 53.394 37.62 seed weight (g) sw 0.27 0.144 0.381 0.080 29.57 seed length (cm) sl 0.96 0.786 1.080 0.113 11.73 seed diameter (cm) sd 0.69 0.547 0.804 0.090 13.04 tegmen weight (g) tw 0.02 0.016 0.029 0.005 19.95 tegmen length (cm) tl 0.65 0.544 0.769 0.074 11.35 tegmen diameter (cm) td 0.31 0.253 0.416 0.052 16.88 woody portion index (tegment weight/aril weight ) wpi 0.09 0.071 0.121 0.021 22.12 seed shape (length/diameter ) sl/sd 1.39 1.310 1.540 0.083 5.93 tegment shape (length/diameter) tl/td 2.15 1.880 2.410 0.176 8.19 aril weight (g) z aw 0.25 0.120 0.350 0.080 32.49 aril weight/tegmen weight aw/tw 10.04 7.400 12.390 2.162 21.53 % aril a (%) 90.35 88.200 92.600 1.653 1.83 beghè et al. ‐ characterization of ancient pomegranates in northern italy 585 wpi, aw, aw/tw). these results are consistent with previous studies (zamani et al., 2007, mansour et al., 2011). the mean leaf quantitative values are reported in table s2 and the traits values presented significant differences between the accessions. moreover, leaf blade margin color and petiole color next to the shoot was red for accessions me1, me7 and me8, and yellow for the others. all accessions have an elliptic shape and absence of mucro (table 1). the flower characteristics are reported in table 1 and table s3 and the observed values are comparable with those of lebanese genotypes studied by dandachi et al. (2017). the flowers of me7 and me8 accessions showed a much smaller size than that of the flowers of the other, but presented a similar style length, a feature that favors pistil pollination. moreover, the long­styled flowers presented a “sinu­ ate jug”, except the flowers of me6 accession that pre­ sented “jug with base”. me7 and me8 presented a “narrow bell” shape in the short­styled flowers where­ as me6 presented “broad bell” shape and other acces­ sions presented flowers with “medium bell” shape. the mean values of quantitative fruit and seed traits are reported in tables 3 and s4. significant vari­ ability was observed in total fruit weight (fw), in maximum equatorial diameter (fd) and in fruit length, with calyx (fl2) and without calyx (fl1). in particular, these characters have lower values for me7 and me8. all accessions showed fruits with shape “oblate or rounded­spheroid” and “closed or semi­ closed calyx”, except me7 and me8 that have a majori­ ty of fruits with “open calyx”. the number of locules (nc) was higher in fruits of higher total weight. of similarity/dissimilarity among examined accessions was obtained through the genetic similarity matrix uti­ lizing manhattan distance and cluster analysis (upgma) algorithm, with xlstat 2009 software. finally, to test the correlations between genetic distance matrices and between the morphological and genetic distance matrices among accessions, mantel tests were performed (mantel, 1967). each matrix dis­ tance was obtained by calculating pearson’s index. mantel tests were performed with 100,000 permuta­ tions (p = 0.05). pearson’s r­value was used to mea­ sure linear correlation between two matrices. 3. results and discussion morphological characterization the data resulting from the 2­year study were grouped and the average values were used for statis­ tical analysis. the accessions showed significant vari­ ability in many of the characters analyzed. descriptive values for each quantitative trait are recorded in table 2. the coefficient of variation (cv) was used to determine the total variability present in each trait. the cv varied from 1.83% (a%) to 51.20% (scw%), with seven traits having cv between 15 and 20% and fourteen traits with cv value higher than 20%. according to audergon (1987), the descriptors with a high cv are more discriminating than the other ones, and can be reliable markers for the char­ acterization of pomegranate accessions. the highest cv values were evident in traits involving fruits and the lowest were in flowers (except fll and pls), leaves (except lfw) and seeds (except stw, sw, table 3 ­ mean values, standard deviation and anova analysis for fruit characteristics the same letter show no statistically significant differences (p<0.05). (z) for explanation of character symbols, see table 2. id fw (z) fd cd fl1 fl2 cl ft scw nc fsi csi sc% s% me1 373.48 ±49.78 a 9.60 ±0.62 a 2.68 ±0.51 a 7.00 ±1.14 a 9.22 ±1.28 a 2.22 ±0.62 a 0.55 ±0.10 a 191.81 ±19.64 a 8.00 ±1.58 a 0.73 ±0.12 a 0.84 ±0.27 a 53.4 ±0.03 a 46.6 ±0.03 d me2 303.53 ±93.46 a 8.83 ±1.17 a 2.40 ±0.62 a 7.83 ±0.72 a 9.53 ±1.06 a 1.70 ±0.43 a 0.53 ±0.11 ab 144.42 ±51.87 a 6.67 ±0.58 ab 0.89 ±0.07 a 0.70 ±0.03 a 47.2 ±0.03 ab 52.8 ±0.03 cd me3 335.73 ±13.03 a 9.10 ±0.52 a 2.43 ±0.55 a 7.10 ±0.17 a 8.53 ±0.61 ab 1.43 ±0.49 a 0.44 ±0.10 bc 163.25 ±11.59 a 7.67 ±0.58 a 0.78 ±0.06 a 0.61 ±0.23 a 48.6 ±0.02 ab 51.4 ±0.02 cd me4 388.73 ±89.49 a 9.40 ±0.84 a 2.15 ±0.78 a 8.12 ±1.03 a 9.00 ±1.27 ab 1.61 ±0.41 a 0.48 ±0.11 abc 185.73 ±46.34 a 7.60 ±0.55 a 0.86 ±0.06 a 0.82 ±0.31 a 48.3 ±0.03 ab 51.7 ±0.02 cd me5 345.68 ±87.85 a 9.50 ±1.14 a 2.84 ±0. 94 a 7.47 ±0.67 a 9.24 ±0.81 a 1.63 ±0.55 a 0.41 ±0.09 bc 181.74 ±47.63 a 7.25 ±0.58 ab 0.80 ±0.05 a 0.85 ±0.27 a 52.5 ±0.01 a 47.5 ±0.01 d me6 266.81 ±23.78 ab 8.60 ±0.71 a 1.70 ±0.14 a 6.45 ±0.64 ab 7.95 ±0.78 ab 1.50 ±0.14 a 0.37 ±0.08 bcd 111.03 ±6.31 ab 7.00 ±0.45 ab 0.75 ±0.01 a 0.88 ±0.01 a 41.9 ±0.06 bc 58.2 ±0.06 bc me7 87.01 ±21.77 b 5.67 ±0.57 b 1.43 ±0.06 a 4.73 ±0.38 b 5.82 ±0.58 b 1.25 ±0.11 a 0.30 ±0.09 d 28.98 ±7.48 b 5.33 ±0.58 b 0.84 ±0.02 a 0.88 ±0.19 a 33.3 ±0.01 d 66.7 ±0.01 a me8 91.21 ±27.45 b 5.07 ±0.11b 1.47 ±0.06 a 4.40 ±0.10 b 5.57 ±0.23 b 1.17 ±0.15 a 0.30 ±0.79 d 32.54 ±10.48 b 5.50 ±0.55 b 0.87 ±0.03 a 0.80 ±0.13 a 35.5 ±0.02 cd 64.5 ±0.02 ab 586 adv. hort. sci., 2019 33(4): 581­592 according to the list of “pomegranate descriptors” of bellini et al. (2007), the fruits were classified as “large or very large” (me1, me2, me3, me4, me5, me6) and “very small” (me7, me8). the first group had a total weight mean values of about 335 g, comparable to those of the fruits of many italian and spanish cultivars (martinez et al., 2006 and ferrara et al., 2014). moreover, the epicarp (or “skin”) of the local fruits has presented different colors, ranging from reddish­yel­ low to red. the size of the fruit and the color of the epicarp are two important parameters considered in the international market as concerns the quality of the fresh product (mansour et al., 2011). another impor­ tant parameter for fruit quality was the descriptor “skin thickness”. me7 and me8 fruits have a thinner “skin thickness” than other accessions, and in field their fruits were more subjected to cracking at the first rainfall in october. significant variability was observed in seeds total weight (stw), skin and carpellary membranes weight (scw) and seeds percentage (s%) (tables s4 and 3). the mean s% found was of 54.91% and it was similar to that reported in another italian study (cristofori et al., 2011). furthermore, our mean values were lower than those reported on italian and iranian cultivars (ferrara et al., 2014). the seed descriptors showed, for the majority of traits, significant differences between two groups of accessions: me1, me2, me3, me4, me5, me6 and me7, me8; the former had sl, sd, sw, tl, tw greater than the latter (table s4). tegmen index (wpi), aril percentage (a%), aril weight (aw) and aril/tegmen ratio (aw/tw) are very important parameters from a qualitative point of view. the wpi is a parameter that refers to the quantity of lignified tissue contained in the seed compared to total seed weight, and consumers greatly appreciate seeds with a limited amount of lignified tissue (martinez et al., 2006). accessions me1, me2, me3, me4, me5 had a higher quantity of aril (aw) and a lower percentage of the tegmen index (wpi) than me6, me7 and me8. the wpi presented an average value of 7.7% in the first group of accessions, and an average value of 11.9% in the accessions me6, me7 and me8. these values were in agreement with those of other italian (5.4 to 10%), spanish (7.4 to 9.7%), moroccan (6.1 to 10.7%) and iranian (5.4 to 7.5%) accessions (martinez et al., 2006; 2012; sarkhosh et al., 2009). the aril is a tissue valued for the high pro­ duction of juice; aw and a%, with reference to the individual seed, were high in all accessions, and a% showed an average value of about 90%; a value com­ parable and higher than that of other italian and spanish genotypes (la malfa et al., 2009). correlation among morphometric traits the correlations found between the quantitative variables, significant at p < 0.05, are reported in fig­ ure 1. the correlation coefficient can provide infor­ fig. 1 ­ pearson's correlation matrix of the quantitative traits in pomegranate accessions, visualized as a heat map plot. for explanation of character symbols, see table 2. beghè et al. ‐ characterization of ancient pomegranates in northern italy 587 mation on the traits that are most important in assessing accessions (norman et al., 2011). skinner et al. (1999) recommended analyzing correlation coeffi­ cients close to 0.7: in these conditions, the variance of one trait is strongly dependent on the others. according to this criterion, we estimated two hun­ dred and fifteen valid correlations. most of the signif­ icant correlations among traits coincide with those from the same plant organ, in particular the variables relative to fruits and seeds. a strong correlation value was found for fruit and seed descriptors, and these were also positively correlated with each other (e.g. fw was positively correlated with other fruit descrip­ tors, fd, fl1, fl2, cl, scw, nc, sc%, s% and seed descriptors, stw, sl, sd, sw, tw, a%, aw, aw/tw). the character wpi was negatively correlated with fruit traits relative to dimensions and with sw, sl, sd. the same results were observed in several for­ mer works that analyzed accessions from different countries (martinez et al., 2006; zarei et al., 2013). moreover, the trait wpi could be an index of seed hardness, because this feature is strongly dependent on the trait wpi, as reported in martinez et al. 2012 (r= 0.63 p ≤ 0.01). in flowers, positive correlations were found among the variables relative to dimen­ sions: between fls and fds and between fll and fdl. in leaves, a positive correlation was between ld and ll, actually all accessions showed an elliptic leaf shape. finally, some correlations were observed between seed (sw, a%, aw, tl, tw) and flower char­ acteristics (fdl, fll, fds, fls, pns); we noted that accessions with small flowers (me7­me8) presented fruits and seeds of smaller dimensions. in fact, fruit growth potential is largely determined genetically through the ovary size in several species (rosati et al., 2009). these correlations between different traits could be due to genetic linkage or to a pleiotropic effect (i.e., when one gene influences two or more seemingly unrelated phenotypic traits) (iezzoni and pritts, 1991). principal component analysis the results of pca revealed the existence of large variability among accessions. the total variance explained by the first three principal components (pcs) in the model was 83.53%. a plot of the percent­ age of variance explained by seven pcs and eigenval­ ues associated with the first seven pcs for each quan­ titative trait are reported in supplementary material (fig. s1 and table s5), respectively. the pc1 explained the 54.60 % of total variance and the traits with the greatest weight on this component were related to fruit (fw, fd, cd, fl1, fl2, cl, ft, scw, nc, sc%), seed (stw, sl, sd, sw, tw, wpi, a%, s%) and some flower traits (fdl, fll, fls). the pc2 explained 15.82% of the variability, and showed a strong negative load for td, ld and pns, whereas a strong positive load was present for sl/sd ,tl/td, fsi, and ls. finally, ld, ll relating to the leaf and pls relating to the flower showed the highest contribu­ tion to pc3 (13.11% of the variability). the compari­ son of plot scores for pc1, pc2 and pc3 in figure 2 permits to obtain a view of accession dispersion and their clustering based on morphological traits. the accessions, for the first two pcs, were grouped into two main groups highly dissimilar: the first group consists of me7 and me8, the second group consists of three sub­groups (sub­group me6; sub­group me2, me3, me4, me5 and sub­group me1). the groups plotted for the pc1 and pc3 were very similar to those on pc1­pc2 plot, though accession me6 showed less differences with the sub­group me2, me3, me4, me5. as already reported in the literature (martinez­nicolas et al., 2016), fruit characteristics fig. 2 ­ loading plots of the first, second and third principal component showing the position of accessions. adv. hort. sci., 2019 33(4): 581­592 588 had the highest loading values for the first compo­ nent in principal component analysis. our results confirmed that the traits related to fruit and seed had the highest power of discrimination, and were, therefore, the most useful for characterization of this local germplasm. rapds and ssrs characterization and genetic rela‐ tionships only 7 rapd oligonucleotides (ai08, ai12, ai105, ah17, opa19, opb08, opc16) out of 42 belonging to the ai, ah, opa, opc, opx and opk series, showed polymorphism in two or more accessions, by produc­ ing polymorphic and reproducible amplification pat­ terns. the 7 oligonucleotides amplified a total of 84 rapd fragments, 14 of which were polymorphic, making 16.67% polymorphism (table 4). the number of polymorphic fragments found per primer was between 1 (opb8, ai05 and ah17) and 3 (ai12, opc16, ai08), with a mean of 2 (table 4) and their size ranged from 250 to 3000 bp. the rapd markers have been applied in many investigations aimed at the study of polymorphism in pomegranate, for their simplicity and low cost (kathuria et al., 2017), but these markers have often shown low polymorphism in this species. as reported in literature it is neces­ sary an initial screening by a high number of rapd primers to detect a discrete number of discriminating markers (zamani et al., 2010). the level of polymor­ phism detected in our study is lower than that reported in other works (sarkhosh et al., 2006; zamani et al., 2007). however, a percentage of poly­ morphism similar to ours was detected by hasnaoui et al. (2010). in agreement with these authors we hypothesized that the slightly lower percentage of polymorphism detected could be due to the reduced dimension of the sample collection and to the limited variability in terms of geographical origin. relationships among accessions were studied by clus­ ter analysis (upgma) based on jaccard’s coefficient, and following statistical analysis a dendrogram was produced (fig. 3a). the genetic distance among the accessions ranged from 0 to 0.8, showing genetic diversity among the pomegranate accessions. in the dendrogram, two main clusters could be identified: the first cluster included only two accessions (me7 and me8) at a 0 dissimilarity level (genetic identity). the second cluster comprised all other accessions which presented a level of dissimilarity that varied between 0 and 0.6. this last cluster presented three subgroups: me1, me6 and a subgroup that included accessions with genetic identity or with a very little (0.10) dissimilarity’s distance (me2, me3, me4, me5). the ssr molecular technique was utilized as a sec­ ond molecular method to discriminate the pome­ granates and to characterize their genetic profile. although for p. granatum there is not yet a set of the best ssr primers with validity recognized at the inter­ national level, these markers have been successfully employed by several researchers to characterize the pomegranate germplasm (beghè et al., 2016). ssrs utilized in this study have been chosen between the primers which had shown a high discriminating capacity and yielded a total number of 31 repro­ ducible fragments, which allowed a good discrimina­ fig. 3 ­ upgma clusters of 8 pomegranate accessions generated by rapd markers using the jaccard similarity coefficient (a) and by ssr markers using manhattan distance (b). table 4 ­ primer sequence of the most informative primers and level of polymorphism found by the rapd analysis rapds sequence total n° of bands n° of polymorphic bands opa19 5ʹ­d[caaacgtcgg]­3ʹ 13 2 opb08 5ʹ­d[gtccacacgg]­3ʹ 12 1 opc16 5ʹ­d[cacactccag]­3ʹ 13 3 ah17 5ʹ­d[cagtggggag]­3ʹ 13 1 ai08 5ʹ­d[aagcccccca]­3ʹ 15 3 ai12 5ʹ­d[gacgcgaacc]­3ʹ 15 3 ai105 5ʹ­d[gtcgtagcgg]­3ʹ 3 1 tot 84 14 mean 12 2 beghè et al. ‐ characterization of ancient pomegranates in northern italy 589 tion of the local accessions (table 5). among 12 selected microsatellites, 10 showed polymorphism and 8 showed differences in the pomegranates genetic profiles. the alleles obtained by amplification of ssrs loci produced four different genetic profiles from eight ancient accessions analyzed (table s6 of supplementary data). the number of alleles at each locus (na) varied between 1, for loci pgkur114 and pgkur127, and 5, for loci ssroeua­pg6 and pom021, with an average value of 2.58 and their size ranged from 155 to 319 bp. the values of expected (he) and observed (ho) heterozygosity were always above 0.500, except for the locus pgkvr027, where was not observed heterozigosity, and obviously for the two monomorphic loci (pgkvr114 and pgkvr127). it is important to underline that four primers (pg6, pom021, pom045, pgaer154) were highly polymor­ phic, showing a pic> 0.5, as defined by botstein et al. (1980) (table 5). these last markers showed genetic parameters (ho, he, pic) higher (or similar) to those reported in previous studies where they have been developed (ebrahimi et al., 2010; hasnaoui et al., 2012; caliskan et al., 2017). instead, the pgkvr­ primers presented genetic parameters lower to these reported in literature (ravishankar et al., 2015); these primers had low polymorphism; of 6 primers 2 resulted monomorphic markers and only 3 (pgkvr027, pgkvr064 and pgkvr165) showed dif­ ferences in the studied accessions. similarly, the na, he, ho and pic values in other studies of pomegranate varieties also varied according to the primers tested and the geographic origin of population analyzed (caliskan et al., 2017). these results confirm the necessity to test different series of ssrs to obtain the best set of markers for each local germplasm. the upgma cluster based on ssr data divided the set of pomegranate accessions into two main cluster at a dissimilarity level of 45 (fig. 3b). in the ssrs den­ drogram, the first cluster included only two acces­ sions (me7 and me8) with genetic identity. the sec­ ond cluster instead presented all other accessions which a level of dissimilarity that varied between 0 and 38%. this last cluster comprised three genetic subgroups: me1, me6 and a subgroup that included accessions with genetic identity (me2, me3, me4, me5). as confirmed by mantel’s test (r = 0.399; p ≤ 0.033), the ssrs clustering was very similar to that performed by rapd markers. in fact, it showed the distinction of the same four genetic groups (fig. 3a and b). comparison between morphological and molecular based clusters and potential use of pomegranate genetic resources it is known that rapd fragments derived from any region of the genome and that ssr fragments derived only from non­transcribed regions, therefore post­transcriptional modifications and non­nuclear inheritance of some characteristics can’t be detected by these markers (sarkhosh et al., 2006). for these reasons, in literature there are contrasting results about the correlation between these molecular and morphological descriptors (sarkhosh et al., 2009; zamani et al., 2010; basaki et al., 2013). however, researchers agreed that the combination of morpho­ logical and molecular techniques are essential for a proper and complete characterization of the germplasm of this species (beghè et al., 2016). in this study, the rapd and ssr analysis reflected the main morphological differences observed among the local accessions studied; the molecular cluster analyses confirmed the same two main clusters detected with pca analysis using quantitative mor­ phological traits. moreover, molecular analyses allowed to detect clearly four different genotypes. analysis of correlation between distance matrices (morphological traits and molecular markers) by mantel’s test confirmed a high statistical significance (r = 0.412; p ≤ 0.034 and r = 0.583; p ≤ 0.002 for ssrs and rapds, respectively). it is important to stress that in populations adapt­ ed to difficult ecological conditions, as the germo­ plasm in study, the polyphenolic content was high (calani et al., 2013). in this previous study the me1, me3, me5 and me8 accessions were subjected to phytochemical discrimination fingerprinting in pome­ ssrs na size he ho pic pgkvr027 2 236­242 0.429 0 0.305 pgkvr064 2 239­241 0.536 0.750 0.359 pgkvr065 2 202­204 0.571 0 0.375 pgkvr114 1 258 ­ ­ ­ pgkvr127 1 246 ­ ­ ­ pgkvr165 2 307­319 0.571 1.000 0.375 pom­agc11 2 183­185 0.536 0.250 0.359 pg4 2 198­244 0.571 1.000 0.375 pg6 5 191­199 0.786 0.750 0.653 pom021 5 203­211 0.857 1.000 0.712 pom045 3 155­163 0.750 0.750 0.581 pgaer154 4 262­300 0.786 1.000 0.630 tot 31 mean 2.58 0.533 0.542 0.394 table 5 ­ number of alleles (na), size (bp) expected (he) and observed (ho) heterozigosity, polymorphic information content (pic) at 12 loci in pomegranate accessions adv. hort. sci., 2019 33(4): 581­592 590 granate juices. the emilian pomegranates have pre­ sented interesting and peculiar phytochemical pro­ files. moreover, the juices were rich in ellagitannins and had high total phenol content and total antioxi­ dant capacity, especially me8 pomegranate. for these reasons, the local germplasm studied could be considered a source of useful traits (e.g. resistance to diseases, frost tolerance, polyphenol synthesis) for genetic improvement of this species. according to pomological descriptors and phytochemical charac­ teristics, we could appreciate peculiar features of these plants. indeed, the me7 and me8 accessions showed some characteristics (e.g. small size of fruits, high woody portion in seeds, low ph) that make the fruits unlikely to be used for direct consumption, but have a juice with high antioxidant capacity, and could be successfully employed for the preparation of nutraceutical products or for industrial blending of juices. the other pomegranates (except me6 acces­ sion that presented a high woody portion in seeds) presented good pomological characteristics for which a fresh use of the fruit could also be expected. 4. conclusions the present work is a first contribution to the genetic and morphological characterization of the pomegranate germplasm still present in emilia romagna region. the morphological traits, in particu­ lar those related to fruit and seed, seven rapds and eight ssrs have allowed to characterise the genetic diversity of ancient pomegranate accessions. the study, although preliminary and limited to a restrict­ ed area, highlighted significant differences and inter­ esting pomological traits in local pomegranates. these results, presented in association with the study of calani et al. 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schneider 1 (*), l.a. thiesen 2, t.d. engroff 3, e. holz 2, b.s. altíssimo 2 1 faculty of agronomy and veterinary medicine, university of passo fundo, passo fundo, rs, brazil. 2 department of agriculture and environment sciences, federal university of santa maria, frederico westphalen, rs, brazil. 3 department of plant production, superior school of agriculture “luiz de queiroz”, university of são paulo, são paulo, sp, brazil. key words: lactuca sativa l., leaf area, solar radiation, substrate, temperature. abstract: the objective of this work was to evaluate lettuce growth in greenhouse under different types of substrates. the experiment was conducted in a greenhouse, under randomized block design, with six treatments and three replicates. the compositions of the substrates were: t1= 100% organic compound; t2= 75% organic compound plus 25% substrate plantmax®; t3= 50% organic compound plus 50% substrate plantmax®; t4= 25% organic compound plus 75% substrate plantmax®; t5= 100% substrate plantmax®; t6= vermiculite. the number of leaves, dry mass, leaf area index, culture growth rate, relative growth rate, net assimilation rate, specific foliar area, foliar area ratio and foliar weight ratio were evaluated. higher growth of lettuce plants are produced by mixture of organic compound and substrate plantmax®. the isolated use of vermiculite does not give good results for the growth of lettuce plants, but is an alternative for mixing with other substrates. 1. introduction lettuce (lactuca sativa l.) is one of the most consumed vegetables in the world (gomes et al., 2008), due to its taste, nutritional quality and low price (teodoro et al., 2016). its adaptability to different climatic conditions that allows successive crops during the year, low cost of production and safe marketing, makes it a crop preferred by small producers, adding economic and social value to its cultivation (medeiros et al., 2007). lettuce is responsive to organic fertilization, varying according to the cultivar and source of nutrients used (teodoro et al., 2016). thus, the use of substrates must ensure that lettuce presents characteristics appropriate to plant growth, with adequate physical and chemical compositions (lima et al., 2006). it should be well structured and with good texture, adequate ph, good fertility and pathogen free (araújo et al., 2013). the different substrates compositions have an effect on the biomass production of the plants, since they are able to comply with the species’ (*) corresponding author: juliaschneider07@hotmail.com citation: schneider j.r., thiesen l.a. engroff t.d., holz e., altíssimo b.s., 2018 growth analysis of lettuce under different substrate compositions. adv. hort. sci., 32(2): 221-227 copyright: © 2018 schneider j.r., thiesen l.a., engroff t.d., holz e., altíssimo b.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 october 2017 accepted for publication 23 february 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 221-227 222 requirements (afonso et al., 2012). there is a trend toward using organic compounds because they provide nutrients and contribute to good development of the root system (lima et al., 2006). a lettuce crop has high production potential with organic fertilizers (santos et al., 2001). the organic compounds, resulting from composting, vermicomposting or other sources, have good aeration, structure, water retention capacity, ability to regulate the temperature of the substrate, and are sources of several nutrients that may be readily available (trindade et al., 2001). organic compounds are also stabilized products, rich in nutrients and derived from vegetable and animal waste (souza and alcântara, 2008). moreover, the use of organic substrates reduces cultivation time and consumption of chemical inputs (medeiros et al., 2015), modifying the microbial populations that improve substrate quality and plant production. these microorganisms decompose the residues, releasing nutrients and substances that stimulate plant growth (medeiros et al., 2015). growth analysis is still the most accessible and accurate way to evaluate plant growth and the contribution of physiological processes to plant behavior. it allows differentiating the behavior of the same cultivar under different cultivation conditions (benincasa, 2003). due to the influence of factors on plant production and the importance of knowing the growth and development of the plants, the objective of this work was to evaluate lettuce growth in a greenhouse under different types of substrates. 2. materials and methods location of experiment, plant material and cultivation the experiment was conducted in a greenhouse located on the experimental area of the federal university of santa maria, campus of frederico westphalen, rs, brazil, with geographic location 27° 23’ s, 53° 25’ o and altitude of 490 m. according to köppen’s classification, the climate of the region is humid cfa-temperate with hot summer, with maximum air temperatures in warmer months over 22°c (alvares et al., 2013). the average temperature inside the greenhouse were 22±3 °c during the day and 18±3°c in the night. the lettuce seedlings, cultivar pira verde, were transplanted to the wooden benches on 3 days in october 2013, arranged in spacing of 20 cm between plants and 30 cm between rows. the plants were cultivated for 49 days until november 21, when all had reached the harvest point. a 150-micron doublesided canvas was placed on the substrate to reduce water loss through soil evaporation. before transplantation, small holes were opened to introduce the seedlings into the substrates. the irrigation method used was drip irrigation, allowing a distribution of the same volume of water for all plants. water was supplied depending on environmental conditions, taking into consideration temperature, relative humidity, and other factors. along with water, nutrients were supplied through nutrient irrigation. the nutrients were chosen according the recommendations of furlani (2009), obtaining electrical conductivity around 1.2 μs cm-2, by use of hidrogoodfert, calcinit and chelated iron, which have soluble nutrients in water. hidrogoodfert is composed of the macronutrients nitrogen, phosphorus, potassium, magnesium, sulfur and the micronutrients boron, copper, molybdenum and zinc. calcinit is composed of calcium nitrate. the solution was replaced according to the evaporation and absorption of the plants. experimental design and treatments the experiment was conducted on wooden benches inside the greenhouse, with a randomized block design, with six treatments and three replications per treatment. each replication have had 40 plants. the compositions of the substrates were t1= 100% organic compound; t2= 75% organic compound plus 25% substrate plantmax®; t3= 50% organic compound plus 50% substrate plantmax®; t4= 25% organic compound plus 75% substrate plantmax®; t5= 100% substrate plantmax®; and t6= vermiculite. the organic substrates were obtained by mechanized and automated composting unit (umac), a project of the company lpc-ambiental technology, located in the municipality of concórdiasc. this residue is generated by mixing pig slurry with compost shavings and storing it in retention rails. the commercial substrate plantmax® has excellent physical properties and high water retention capacity. this substrate is composed mainly of pine bark and vermiculite, and presents chemical properties constituted of macronutrients and micronutrients. vermiculite was used for application of nutrients by means of irrigation, since it is a non-nutritive substrate with high water retention capacity. schneider et al. lettuce under different substrate compositions 223 evaluations and analysis the evaluations of the plants were conducted every week, when 3 plants of each treatment were sampled by the destructive method, from the transplant to the beginning of the stem elongation for posterior emission of the floral tassel. the evaluations were made by counting the number of leaves and separating the morphological parts of the plant, which was put into the drying oven at 60°c until it reached constant weight for determination of the dry mass. the analyzed variables were number of leaves (nl), dry mass (dm) and leaf area index (lai). the number of leaves was determined by counting all the leaves of the plants, and dry mass was obtained by measuring the mass of the aerial part of the plant. leaf area (la) was determined by the disc method, in which 10 discs were removed from the leaves, obtaining its dry mass, and estimating the leaf area of the plant through the equation: la = disc number x nozzle number x total leaf dry mass dry mass of the discs from the leaf area, the leaf area index was calculated by the following equation: lai = la sa where sa is the area of soil occupied by a plant. in addition, culture growth rate (cgr), relative growth rate (rgr), net assimilation rate (nar), specific foliar area (sfa), foliar area ratio (far) and foliar weight ratio (fwr) were calculated according to the methodology presented by barbero et al. (2013). the cgr was determined by: cgr = dm sa xδt where δt is the time interval of the evaluations. this variable is an indicator of productivity, since it represents the increase of dry matter in a time interval, considering the area of soil occupied by the plant. the equation to obtain the rgr is: rgr = log δdma δt where log δdm is the logarithm of the increment of dm, so rgr is an increment of productivity of dry mass in a time interval. nar was calculated as: nar = cgr lai this variable indicates the rate of increase in dry matter per unit of time and per unit of leaf area. according to benincasa (2003), nar demonstrates the photosynthetic efficiency of the leaves. far is the ratio between leaf area and dry mass and expresses the foliar area useful for photosynthesis; it is the measure of the size of the assimilating apparatus. it is obtained by: far = la dm fwr relates to how much of the dry matter accumulated by the plant is composed by leaves, which, besides being the organ responsible for photosynthesis, is the organ of commercial interest. it is given by: fwr = dm leaves dm plant sfa were determined by the equation: sfa = la dm leaves this variable indicates the accumulation of photoassimilates in the leaves or the translocation to the other organs, that is, differences in leaf thickening (taiz and zeiger, 2013). the data were submitted to analysis of variance and comparison of means by the tukey’s test, with a 5% probability of error. the meteorological data, like air temperature and global solar radiation, were collected at the mobile weather station installed inside the greenhouse. 3. results and discussion weather conditions during the experiment, minimum and maximum average temperatures were 15-27±3°c, respectively. these values are in agreement with the values considered optimal for the culture between 15-24°c (knott, 1962) and 15-20°c (santana et al., 2009). the global solar radiation presented variations between 653 and 1.090 kj m-2. these values are considered adequate for the growth of the culture. a reduction of solar radiation at the beginning of the crop cycle can be observed, but it did not have any influence, since the plants were in establishment period and had little leaf area (fig. 1). thus, air temperature and incident solar radiation were the main environmental factors that affected lettuce growth, development and yield. growth variables during the growth and development of the plants, adv. hort. sci., 2018 32(2): 221-227 224 small burns were observed in the border of the new leaves. these were symptoms of calcium deficiency, because it is a little mobile element in the plant and then wasn’t available to the plants. however, by harvest, the plants had recovered with no apparent symptoms. the leaves are the main component of interest of lettuce. thus, it is important that the plant produces the maximum number of leaves, dry matter and leaf area for good production. figure 2 shows that the number of lettuce leaves increased during the crop cycle for the different substrate compositions. therefore, it can be observed that the t1 treatment (100% organic compound) showed a smaller number of leaves at 28 days after transplantation. however, at 35 days after transplant, the treatment composed of 100% substrate plantmax® (t5) was less effective than the others, with approximately 12 leaves. at the end of the cycle of the culture, the substrate with the lowest number of leaves was vermiculite, with approximately 20 leaves, but that did not differ statistically from the others. this demonstrates that the combination of different substrates such as organic compost and commercial substrate present better conditions for the development of plants than when used alone. the results obtained show that the analyzed growth variables presented different behaviors in relationship to the types of substrates used in the lettuce crop. a decline was also observed on the growth variables toward the end of the crop cycle (fig. 3), which is considered normal because they are relational to growth of this culture. figure 3a shows the dry mass accumulated by the plants during the cycle. slow initial growth due to the small size of the plants and small leaf area makes a lower nutrient absorption and a low solar radiation, causing little growth of these plants. from 21 days after transplant, an increase in the dm for all the substrates was observed, with emphasis on mixtures of substrates. the substrate composed of 50% organic compound plus 50% substrate plantmax® (t3), was outstanding for presenting higher values in most of the crop cycle, reaching maximum increment at 49 days after the transplant, with approximately 26 g. the worst results were obtained with vermiculite, reaching approximately 11 g (fig. 3a). the leaf area index presented a similar behavior to dm, with the most accentuated and perceptible increase at 21 days after transplant, when the plant presented an increase in dry matter (fig. 3b). lai falls at certain points in the crop cycle may be related to the abscission of older leaves. lai is important for studying crop growth, development and productivity. the leaf area will depend in addition to the number and size of the leaves of the plant and the period in which the leaves remain on the plant (monteiro et al., 2005). this demonstrates that the increase of leaf area provides greater interception and absorption of the available solar radiation, and, consequently, higher photo assimilates production, which results in higher growth and dry matter of the plants. the best results for these variables were obtained by substrate mixtures. since cgr represents the increment of dry matter per unit of soil area occupied over a given period of time, the pattern of the graph was very similar to that for dry matter. at 49 days after transplant, the substrate, composed of 50% organic compound plus 50% substrate plantmax®, was higher than the others, differing statistically just from vermiculite (t6). at this point, t3 reached approximately 60 g m-2 day-1 (fig. 3c). according to beckmann-cavalcante et al. (2009), growth rate of the crop is mainly determined fig. 1 solar global radiation (kj m -2 ), maximum temperature and minimum temperature (°c) during the lettuce crop cycle. frederico westphalen, rs, brazil, 2013. fig. 2 number of leaves of lettuce growth under different substrates. frederico westphalen, rs, brazil, 2013. schneider et al. lettuce under different substrate compositions 225 by the air temperature. thus, from 21 day after transplant (dat), cgr also increased as a function of changes in air temperature. the highest rates of rgr were observed at 14 and 21 days after transplant (fig. 3d). substrate with 75% organic compound plus 25% substrate plantmax® (t2) reached maximum rgr-approximately 0.25 g m-2. this may be because the plants were in a phase of high photosynthetic rate due to the elevation of lai and high growth. this same treatment has achieved the highest rgr at 49 dat, statistically differing from the others. at 28 days after transplant, the growth rate tended to decrease. according to zuffo et al. (2016), this is mainly due to the shading between the plants and the increase in the respiratory rate. this shows that with shading and high respiration of the plants, the growth promotes a reduction of the rgr of the crop. the photosynthetic efficiency of the leaves presented variations along the cycle and is represented by nar. this rate tends to be higher at the beginning of the development of the crop due to lower selfshading (gondim et al., 2008). however, in this study the nar values increased at the end of the cycle because nar depends on available leaf area, leaf distribution, leaf angle, and translocation and assimilation partition (pedó et al., 2010; aumonde et al., 2011). some statistical differences were observed between the treatments on 7, 14 and 42 dat (fig. 3e). the highest rates of far were obtained at 7, 14 and 21 dat (fig. 3f). after this period, the ratio steadily decreased for all treatments, corroborating the results of pedó et al. (2013) for pepper. however, fig. 3 dry mass (a), leaf area index (b), culture growth rate (c), relative growth rate (d), net assimilation rate (e), foliar area ratio (f), foliar weight ratio (g) specific foliar area (h) for lettuce culture growth under different substrates. frederico westphalen rs, ufsm , 2013. lowercase letters differ by tukey means test at 5% error probability. adv. hort. sci., 2018 32(2): 221-227 226 these authors obtained results in the higher far, providing a greater area useful for photosynthesis, which produced higher rates of nar. as for caron et al. (2007), the decrease indicates that at this stage, most of the photosynthesized material is accumulated in the aerial biomass of the lettuce to increase available solar radiation. this shows that the decrease may be due to self-shading and leaf fall resulting from the age of plants, or to energy demand for the development of other organs, such as flowers. statistical differences were found only at 7, 14 and 42 dat. foliar weight ratio remained similar between the treatments, with values ranging from 0.7 to 0.9 g g-1 during a large part of the crop cycle, with decreases at 42 and 49 dat (fig. 3g). this may be due to the appearance of preferential metabolic sinks by the formation of the reproductive organs. t1 and t2 were superior and differed statistically to t6 at 42 dat. specific foliar area reached differences between the treatments at 7 and 14 dat. at 7 dat, the major sfa were obtained by t5, which was statistically equal to t3 and t6 treatments. at 14 dat, the higher sfa was observed by t2, t3 and t5. at 21 dat, the values were similar to those obtained at 7 and 14 days. in the rest of the cycle, sfa remained around 0.04 m2 g-1. after the development of the plants, there were an increase in leaf area and dry mass of the leaves, causing decreases in sfa (benincasa, 2003). table 1 shows the means of fresh mass at 42 and 49 dat. the values show that substrates mixtures obtained the better results. although they had not shown a significant difference from the others at 49 dat with the exception of vermiculite there were differences that could bring greater profits. however, from the values observed at 42 dat, it can be concluded that the treatments with mixtures of substrates were superior to the others. in addition, the use of mixtures such as t2 (75% organic compound plus 25% substrate plantmax®) and t4 (25% organic compound plus 75% substrate plantmax®) anticipate the harvest, because the fresh mass was higher at 42 dat compared to 49 dat. it is important to emphasize that vermiculite was used like a control treatment, since it is a inert substrate used in hydroponics cultivation or mixed with soil; it led to worse results for all variables evaluated and reduced the growth of plants cultivated under this substrate. 4. conclusions higher growth of lettuce plants are produced by mixture of organic compound and substrate plantmax®. the isolated use of vermiculite does not give good results for the growth of lettuce plants, but is an alternative for mixing with other substrates. references afonso m.v., martinazzo e.g., aumonde t.z., villela f.a., 2012 composição do substrato, vigor e parâmetros fisiológicos de mudas de timbaúva (enterolobium contortisiliquum (vell.) morong). rev. árvore, 36(6): 1019-1026. alvares c.a., stape j.l., sentelhas p.c., gonçalves j.l.m., sparovek g., 2013 köppen’s climate classification map for brazil. meteorol. z., 22(6): 711-728. araújo a.c., araújo a.c., dantas m.k.l., pereira w.e., aloufa m.a.i., 2013 utilização de substratos orgânicos na produção de mudas de mamoeiro formosa. rev. bras. de agroecologia, 8(1): 210-216. aumonde t.z., lopes n.f., moraes d.m., peil r.m.n., pedó t., 2011 análise de crescimento do híbrido de mini melancia smile® enxertada e não enxertada. interciencia, 36(9): 677-681. barbero l.m., prado t.f., basso k.c., lima l.a., motta k.m., krüger b.c., neto l.r.m., silva g.a.s., 2013 análise de crescimento em plantas forrageiras aplicada ao manejo de pastagens. vet. not., 19(2): 71-85. beckmann-cavalcante m.z., pivetta k.f.l., cavalcante í.h.l., cavalcante l.f., bellingieri p.a., 2009 soluções nutritivas no desenvolvimento do crisântemo cultivado em vaso. irriga, 14(2): 205-219. benincasa m.m.p., 2003 análise de crescimento de plantas (noções básicas). jaboticabal, funep, pp. 41. caron b.o., manfron p.a., lúcio a.d., schmidt s., medeiros s.l.p., bonnecarrére r.a.g., neto d.d., 2007 equações de estimativa da fitomassa da parte aérea da alface. cienc. rural, 37(5):1248-1254. furlani p.r., 2009 cultivo de frutas e hortaliças em ambiente protegido. instituto frutal, fortaleza, brazil, pp. 37. treatment fresh mass (g) 42 dat 49 dat 100% organic compounds 153.20 b 160.67 ab 75% organic compounds + 25% plantmax ® 220.03 a 198.40 a 50% organic compounds + 50% plantmax ® 199.03 a 270.00 a 25% organic compounds + 75% plantmax ® 199.70 a 163.43 ab 100% substrate plantmax ® 110.40 bc 161.43 ab vermiculite 73.07 c 71.17 b table 1lettuce fresh mass at different substrates mixtures dat = day after transplant. schneider et al. lettuce under different substrate compositions 227 gomes l.a.a., rodrigues a.c., collier l.s., feitosa s.s., 2008 produção de mudas de alface em substrato alternativo com adubação. hortic. bras., 26(3): 359363. gondim a.r.o., puiatti m., ventrella m.c., cecon p.r., 2008 plasticidade anatômica da folha de taro cultivado sob diferentes condições de sombreamento. bragantia, 67(4): 1037-1045. knott j.e., 1962 handbook for vegetable 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comparativa de crescimento entre genótipos de pimenta cultivados em casa de vegetação. biosci. j., 29(1): 125-131. pedó t., lopes n.f., moraes d.m., aumonde t.z., sacarro e.l., 2010 crescimento de três cultivares de rabanete (raphanus sativus) ao longo da ontogenia das plantas. rev. tecnol. ciên. agropec., 4(3): 17-21. santana c.v.s., almeida a.c., turco s.h.n., 2009 produção de alface roxa em ambientes sombreados na região do submédio são francisco ba. revista verde, 4(3): 1-6. santos r.h.s., silva f., casali v.w.d., conde a.r., 2001 efeito residual da adubação com composto orgânico sobre o crescimento e produção de alface. pesqui. agropecu. bras., 36(11): 1395-1398. souza r.b., alcântara f.a., 2008 adubação no sistema orgânico de produção de hortaliças. circular técnica, embrapa hortaliças, 65: 1-8. taiz l., zeiger e., 2013 fisiologia vegetal. artmed, porto alegre, brazil, pp. 918. teodoro m.s., seixas f.j.s., lacerda m.n., araújo l.m.s., 2016 produção de alface (lactuca sativa l.) sob diferentes doses de vermicomposto. revista verde, 11(1): 18-22. trindade a.v., muchovej r.m.c., neves j.c.l., barros n.b., 2001 crescimento e nutrição de mudas de eucalyptus grandis em resposta a composto orgânico ou adubação mineral. revista ceres, 48(276): 181194. zuffo a.m., zuffo júnior j.m., silva l.m.a., silva r.l., menezes k.o., 2016 análise de crescimento em cultivares de alface nas condições do sul do piauí. revista ceres, 63(2): 145-153. impaginato 89 adv. hort. sci., 2020 34(1s): 89­96 doi: 10.13128/ahsc­7872 effects of 1­mcp and ethylene on preservation of quality and vase life of alstroemeria (cvs. hercules and mayfair) cut flowers a. nasiri, n. ahmadi (*), g. movahed department of horticultural sciences, tarbiat modares university, tehran, iran. key words: aging, catalase, chlorophyll, climacteric, leaf yellowing, vase life. abstract: in order to improve the vase life characteristics and postharvest quali­ ty of alstroemeria cut flowers cv. hercules and mayfair, the effects of 1­methyl­ cyclopropene (1­mcp) and ethylene have been investigated in a completely randomized design with three replications. first, cut flowers were fumigated at 0, 0.5, 1 and 1.5 µl l­1 of 1­mcp concentrations for 24 h and then exposed to 1 µl l­1 of ethylene for 18 h. this experiment was conducted at 20 ± 2°c, 60­65% rh, and a 12/12 h light/dark photoperiod. the results showed that 1­mcp treatment significantly increased postharvest durability in both cultivars, com­ pared to the control. also, results of 1­mcp treatment on leaf chlorophyll index in both cultivars, confirmed the role of 1­mcp in preventing external ethylene action as the main factor of alstroemeria leaf yellowness. based on data, mayfair cultivar was more sensitive to ethylene in comparing to hercules culti­ var, although 1­mcp treatment reduced the active oxygen species in both culti­ vars by reducing biosynthesis and ethylene action or by direct increase in antioxidant enzyme activity. 1. introduction alstroemeria cultivars are consumed as cut flower due to their varia­ tion in the flower pattern and color which spotted with dark colors, and this caused an increase in their global trade during last decades (ferrante et al., 2002). alstroemeria cut flower quality is often decreased by pre­ maturity yellowing of leaves which reduce viability of leaves before appearing the secondary florets (ferrante et al., 2002). from commercially point of view, the leaf green color preservation is one of the most important qualitative characteristics playing a role in aes­ thetic valuation of this ornamental plant (mutui et al., 2006). the leaf yel­ lowing is affected by various factors such as poor storage conditions, defi­ ciency of internal cytokinin, exposure to internal and/or external ethyl­ ene, darkness, accumulation of abscisic acid, leaf aging and damage (ferrante et al., 2009). ethylene is one of the most important limiting factors in maintaining (*) corresponding author: ahmadin@modares.ac.ir citation: nasiri a., ahmadi n., movahed g., 2020 ­ effects of 1‐mcp and ethylene on preservation of quality and vase life of alstroemeria (cvs. hercules and mayfair) cut flowers. ­ adv. hort. sci., 34(1s): 89­96 copyright: © 2020 nasiri a., ahmadi n., movahed g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2019 accepted for publication 26 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 89­96 90 the quality and life of many cut flowers due to acceler­ ating aging in the post­harvest stage. alstroemeria cul­ tivars showed varied range of sensitivity to ethylene. although they produce very little ethylene, they are sensitive to external ethylene (chanasut et al., 2003) so that low concentration of this gaseous hormone causes leaf yellowing, abscission of petals and accelerating flower aging (reid, 1989). for these reasons, many efforts are being made to gain more information about compounds inhibiting and blocking ethylene biosynthe­ sis and action. 1­mcp patented by edward sisler, acts as a blocking agent of ethylene action by binding to ethylene receptors. in other words, 1­mcp binds com­ petitively to ethylene binding site with affinity higher than that of ethylene and blocks downstream signal transduction, thereby prevents the expression of genes induced by ethylene, determining an extension of the shelf life of many climacteric cut flowers (serek et al., 1994; ahmadi et al., 2008, 2009; daneshi nergi and ahmadi, 2014). although some silver­containing com­ pounds could inhibit ethylene action, they have toxicity effects on human health and the environment, limiting their application on crops. moreover, to reduce the people concerns on these harmful and hazardous com­ pounds, the demand of using safe compound to extend post­harvest life in crops especially edible products is also increasing. the purpose of this study was to evaluate the effi­ cacy of 1­mcp on extending the longevity of cut flow­ ers and on reducing leaf yellowing in alstroemeria cvs. mayfair and hercules. in most alstroemeria culti­ vars the first aging symptom of cut flowers is the onset of yellowness in the leaves which occurs earlier than petals aging or abscission. the activity of antiox­ idant enzymes in petals and ethylene production of both cultivars were also evaluated. 2. materials and methods in this experiment, cut flowers of alstroemeria cvs. mayfair and hercules were harvested based on commercial indices from a greenhouse located in pakdasht, tehran, iran. after harvesting, cut flowers were immediately transported dry to the postharvest laboratory at tarbiat modarres university. stems were re­cut under water to a length of 45 cm and held in 500 ml vase solution containing 200 mg/l 8­ hydroxyquinoline sulfate (8­hqs), and 3% sucrose. (rasouli et al., 2015). then cut flower stems were placed in 200 l glass chambers and treated with 1­ mcp at concentrations of 0, 0.5, 1, and 1.5 µl l­1 for 24 hours (daneshi nergi and ahmadi, 2014). ethyl bloc powder prepared from us agrofresh was used for applying 1­mcp treatment. considering the given concentrations, certain amounts of ethyl bloc were weighed and placed in petri dishes, then warm water was added to the petri dishes inside glass chambers. immediately, the lids of glass cham­ bers were hermetically sealed by adhesive tape (daneshi nergi and ahmadi, 2014). after 24 hours of treatment with 1­mcp, the lids of the chambers were removed and cut flowers were ventilated with fresh air for one hour. then, by closing and sealing the lids again, ethylene was injected into each chamber, to expose all flowers to 1 µll­1 exogenous ethylene for 18 h. when the ethylene treatment was completed, the lid of glass containers was opened and the vases containing flowers were placed on the laboratory table. the experiment was conducted at 20± 2°c, 60­ 65% rh and a 12/12 h light/dark photoperiod at an illumination of 15 µmol m­2 s­1. vase life one of the most important criteria for evaluation of postharvest quality is the viability of cut flowers. in this study, the flower life is the time interval between the beginning of treatment until the cut flowers lose their ornamental value. when 50% of petals falling down in cut alstroemeria, it was considered as the end of vase life quality (mutui et al., 2006). relative fresh weight and chlorophyll content in order to measure this trait, the weight of flower was measured using digital scale (kern model) with an accuracy of ± 0.001 g. this trait was calculated during the evaluation period using equation: relative fresh weight (percent) equal to (wt/wt = 0) in this regard wt: flower weight (g) on day 0, 3, 6, 9 and 12, wt = 0: flower weight g per day (he et al., 2006). to measure the chlorophyll content, 0.2 g of leaf sample was used to extract its chlorophyll in 80% acetone. then, in a volumetric flask, 25 ml of the extract was filtered, acetone reached 25 ml volume and chloro­ phyll was completely extracted (arnon, 1949). evaluation of catalase activity about 200 mg of frozen petal cell mass, sampled on day 6 of the experiment, was extracted in 3 ml of 25 mm sodium phosphate buffer (ph = 6.8) and cen­ trifuged for 4 min at 15°c. the enzyme solution was used to measure enzyme activity. then, the reaction mixture was prepared including 25 mm of sodium phosphate buffer (ph = 6.1), 10 mm of hydrogen per­ oxide and enzymatic extract. catalase activity was nasiri et al. ‐ effects of 1‐mcp and ethylene on cut alstroemeria flowers 91 measured at the wavelength of 240 nm and calculat­ ed as a delta of absorbance at 240 nm per mg pro­ tein. all enzymatic extraction stages were performed on ice (cakmak and horst, 1991). cat activity = (final abs­initial abs)/protein evaluation of superoxide dismutase activity about 200 mg of petal tissue, sampled on day 6 of experiment, was extracted in 3 ml of hepes­koh buffer containing 0.1 mm of sodium edta (ph = 7.8). the resulting homogenate was centrifuged for 30 minutes at 12000 rpm at 4°c. the resulting super­ natant was used to measure superoxide dismutase activity. the reaction composition in the final volume of three ml is as follows: hepes­koh buffer (50 mm) containing 0.1 mm of sodium edta at ph = 7.8, sodium carbonate (50 mm) at ph = 10.2, l­methionine (12 mm), nitro blue tetrazolium (nbt) (75 mm), riboflavin (1 mm), and the enzyme extract were appropriately considered as one unit of superoxide dismutase activity as an enzyme that results in 50% inhibition of nitrobutetra­ zolium at 560 nm (swanson, 1955). adsorption of the reaction mixture was measured using bio­rad spec­ trophotometer. measurement of ethylene after finishing ethylene treatment three flowers from each treatment were placed in a 1.8 l sealed glass bottle and kept at 20 ± 2°c for 48 h, same as experimental condition. by a gas­tight syringe, gas samples were taken through head spaces and were injected into a gc manufactured by agilent america co. model gc­6890n fitted with a capillary column and a flame ionization (fid) detector. the carrier gas was helium at 6.5 ml min−1, injection temperature was 180°c and column temperature was 60°c (daneshi nergi and ahmadi, 2014). statistical analysis of the data the experiment was conducted in a completely randomized design (crd) with four treatments and three replications. the data were analyzed using sta­ tistical software sas version 9 and the means were compared by the least significant difference (lsd) test (p=0.05). the figures were drawn using excel software. 3. results vase life this experiment showed that the vase life of cut flowers cv. hercules was longer than cv. mayfair, although vase life of both cultivars was affected by 1­ mcp application. maximum plant longevity was 12 days for cv. hercules under 1.5 µl 1­mcp treatment and the longevity of cv. mayfair was extended to 10.8 days under 1 µl l­1 of 1­mcp, which were signifi­ cantly different at level as indicated by least signifi­ cant difference (lsd), comparing to the control. no significant differences were observed between 1­ mcp treatment levels in any of cultivars at 1% level by an lsd (fig. 1). fig. 1 ­ vase life of alstroemeria treated with concentrations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1. vertical bars represent standard deviation. relative fresh weight the results of analysis of variance and mean com­ parison of relative fresh weight of cut flower of alstroemeria cv. hercules showed that the highest relative fresh weight (119.5%) was obtained on the sixth day after applying treatment, which was statis­ tically significant at 1% probability level. the lowest relative fresh weight (90.2%) was obtained on the twelfth day after applying treatment, which was not significantly different from the relative fresh weight on day 0, but it was significantly different with rela­ tive fresh weight at other times, at probability level 1% (fig. 2). in cv. mayfair, the highest relative fresh weight (106.32%) was showed at 1 µl l­1 of 1­mcp treatment, which was statistically significant at 1% probability level with 0.5 and 1.5 µl l­1 of 1­mcp treatments. control samples revealed the lowest rel­ ative fresh weight (62.9%) (fig. 3). chlorophyll content data showed that 1­mcp treatments affected sig­ nificantly the chlorophyll content in cv. hercules. the highest content of chlorophyll (1.13 mg) was revealed at 1 µl l­1 of 1­mcp concentration which adv. hort. sci., 2020 34(1s): 89­96 92 fig. 2 ­ relative fresh weight of cut flower alstroemeria cv. hercules treated with concentrations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1. vertical bars represent standard devia­ tion. application, showing significant differences in respect to other treatments (fig. 7). data showed that cv. hercules produced higher content of ethylene in comparison to cv. mayfair. was not significantly different from 1.5 µl l­1 of 1­ mcp. the lowest chlorophyll content (0.94 mg) was measured in leaves treated without any 1mpc (fig. 4). in mayfair cultivar, the effect of treatment on chlorophyll content was significant at 5% probability level so that the maximum chlorophyll content (1.63 mg) was revealed at 1.5 µl l­1 of 1­mcp treatment which was statistically significant at 5% probability level, comparing to control (fig. 5). endogenous ethylene production ethylene production was measured 48 hours after ethylene treatments. cut flowers cv. hercules showed the lowest endogenous ethylene production at 1 µl l­1 of 1­mcp, which was not significantly dif­ ferent to ethylene biosynthesized under 1.5 µl l­1 of 1­mcp (fig. 6). cut flower cv. mayfair revealed low­ est ethylene production under 1.5 µl l­1 of 1­mcp fig. 3 ­ relative fresh weight of cut flower alstroemeria cv. mayfair treated with concentrations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1. vertical bars represent standard deviation. fig. 5 ­ relative chlorophyll content on days 0, 3, 6 and 9 of cut flower of alstroemeria cv. mayfair treated with concen­ trations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1. vertical bars represent standard deviation. fig. 6 ­ ethylene production of cut flower of alstroemeria cv. hercules 48 h after finishing ethylene treatment. vertical bars represent standard deviation. fig. 4 ­ relative chlorophyll content on days 0, 4, 8 and 12 of cut flower of alstroemeria cv. hercules treated with concen­ trations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1. vertical bars represent standard deviation. nasiri et al. ‐ effects of 1‐mcp and ethylene on cut alstroemeria flowers 93 in the cut flower of alstroemeria cv. mayfair, the maximum activity of superoxide dismutase was obtained from the treatment of 1­mcp at a concen­ tration of 1 µl l­1 (90.97 units of enzyme/mg protein) which was no significantly different from other 1­ mcp concentrations according to lsd test (fig. 9). the lowest level of superoxide dismutase activity was related to control samples, which had a significant difference at 1% probability level with other treat­ ments. 4. discussion and conclusions vase life is one of the most important factors for evaluating the quality of cut flowers. the role of eth­ ylene as the most important accelerating factor in aging of plant organs has been entirely investigated, especially in climacteric crops. in this experiment, 1­ mcp was used to reduce the deleterious effects of ethylene (fig. 1). based on the results, ethylene accelerated quality deterioration and leaf senescence in the flowers of two alstroemeria cultivars, while 1­ mcp increased vase life of both alstroemeria cvs. by alleviation of the harmful effect of ethylene. 1­mcp treatment preserves quality and prolongs post­har­ vest life by preventing ethylene action and subse­ quently preventing endogenous ethylene production. 1­mcp compound, like other ethylene action inhibitors such as silver thiosulfate, delayed flower aging when the flowers were exposed to 1 µll­1 of ethylene (serek et al., 1995). this experiment showed that 1­mcp treatment decreased the indige­ nous ethylene biosynthesis in cut flowers of alstroemeria (fig. 6 and 7). accordingly, it seems that the prolonged vase life of cut flowers of alstroemeria catalase activity data on cut flowers alstroemeria cv. hercules showed that 1­mcp had a positive effect on catalase activity, so that the highest activity was evaluated under 1 µl l­1 (55/98 absorption delta/mg protein) concentration, which was significantly different at 1% probability level, comparing to other treatment based on lsd test (fig. 8). flower petals of cv. mayfair showed the highest level of catalase activity under 1 and 1.5 µl l­1 of 1­ mcp concentration, without any significant differ­ ence. the lowest catalase activity was revealed in control sample which did not receive any 1­mcp (27.79 absorption delta / mg protein) (fig. 8). superoxide dismutase activity the analysis of data in cut flowers alstroemeria cv. hercules showed that the maximum activity of superoxide dismutase was appeared in 1­mcp at con­ centration of 1.5 µl l­1 (82.34 enzyme units/mg pro­ tein) according to lsd test, without any significant difference with 1 µl l­1 concentration (fig. 9). fig. 7 ­ ethylene production of cut flower of alstroemeria cv. mayfair 48 h after finishing ethylene treatment. vertical bars represent standard deviation. fig. 9 ­ superoxide dismutase activity in alstroemeria flowers treated with concentrations of 1­mcp at 0, 0.5, 1 and 1.5 µl l−1. vertical bars represent standard deviation. fig. 8 ­ catalase activity in alstroemeria flowers treated with concentrations of 1­mcp at 0, 0.5, 1 and 1.5 µl l­1). vertical bars represent standard deviation. 94 adv. hort. sci., 2020 34(1s): 89­96 by application of 1­mcp could be related to the role of 1­mcp in inhibition of internal ethylene produc­ tion. similar to our results, chutichudet et al. (2011) reported that 1­mcp can prevent ethylene produc­ tion and increase the life of cut flowers. in carnation cut flower cv. fortune, it was shown that vase life increased with increasing concentration of 1­mcp (ranjbar and ahmadi, 2015). the results of the present study showed that the relative fresh weight of cut flowers increased and then reduced over time, which was higher in control samples, comparing to 1­mcp­treated cut flowers (fig. 2 and 3). the reduction in flowering stem weight of flowers treated with 1­mcp during storage may be affected by the interference of 1­mcp in ethylene self­production system (porat et al., 1995). the report of daneshi nergi and ahmadi (2014) on rose cut flowers cv. sparkle treated with 1­mcp and ethyl­ ene showed that the effects of treatment on the rela­ tive fresh weight were significant over time (daneshi nergi and ahmadi, 2014). according to the present results, 1­mcp at 1 and 1.5 µl l­1 concentration had the better yield on chlorophyll content in cv. hercules (fig. 4). also, in cv. mayfair, 1.5 µl l­1 of 1­mcp treatment performed better than control samples (fig. 5). chlorophyll, as the main pigment involved in light harvesting, plays essential role in terms of absorption and utilization of light energy in photosynthesis. basically, the leaf aging is associated with a reduction in chlorophyll content. it was reported that reduction of chlorophyll content of alstroemeria leaves coincided with leaves aging (ferrante et al., 2002). ethylene accelerates chlorophyll degradation in the leaves of many cut flowers. the results of both cultivars showed that 0.5 µl l­1 of 1­mcp could not prevent chlorophyll degra­ dation, which was consistent with some reports (çelikel et al., 2002; seglie et al., 2010). it seems that protection of chlorophyll in leaves of cut flowers alstroemeria under 1­mcp treatment could be relat­ ed to preventing the action of external ethylene, known as the main cue of leaf yellowing in ornamen­ tal plants. in asian pear, 1­mcp treatment delayed chlorophyll degradation, which was attributed to the effect of 1­mcp treatment on suppression of enzymes involved in on ethylene production pathway (cheng et al., 2012). the process of leaves yellowing in cut alstroemeria is under the control of genetic background and environmental conditions. data of hercules cultivar showed that 1 µl l­1 of 1­mcp resulted in lower ethylene synthesis than other treat­ ments (fig. 6). at the concentration of 1.5 µl l­1 in mayfair cultivar, 1­mcp resulted in lower ethylene synthesis than other treatments (fig. 7). in both culti­ vars, 1­mcp treatments prevented ethylene produc­ tion. the pre­treatment with 1­mcp for preventing ethylene production is consistent with the study results of rose cv. samantha (xue et al., 2008) and clove (seglie et al., 2010). catalase is considered an important biological fac­ tor and its major function is in the decomposition of hydrogen peroxide to water and oxygen and prevent­ ing creation of hydroxyl radicals (spanou et al., 2012). superoxide dismutase like cu­zn superoxide dismutase, mn superoxide dismutase and outside cell superoxide dismutase play a critical role in inhibition of superoxide (miao and st clair, 2009). in fact, catalase and superoxide dismutase play roles in protecting the metabolism balance of oxygen in plant tissues (xie et al., 2003). superoxide causes lipid peroxidation, cell membrane damage and finally senescence; 1­mcp can affect enzyme activities, which remove superoxide (li et al., 2007). in the cut flower of hercules, no significant differ­ ence was found between 1­mcp treatments, but the activity of catalase was significantly increased com­ pared to control (fig. 8). the results of mayfair culti­ var cut flower showed that no significant difference was found between 1 and 1.5 µl of 1­mcp but cata­ lase activity increased significantly compared to con­ trol (fig. 8). ethylene­stimulated aging could change the cell structure and increased the concentration of reactive oxygen species such as superoxide radicals, hydroxyl radicals and hydrogen peroxide. therefore, 1­mcp treatment reduced the reactive oxygen species and consequently delayed aging by reducing the biosynthesis and action of ethylene or by directly increasing the activity of antioxidant enzymes (fig. 9). consistent with the results of this experiment, an increase was observed in antioxidant enzymes activi­ ty of 1­mcp ­treated gladiolus flowers (hassan and ali, 2014). it seems that 1­mcp treatment reduced the oxidative stress in cut flowers. in other words, activity of these enzymes is a factor for the protec­ tion of cells against oxidative stresses (zhou et al., 2014). in asparagus, 1­mcp hindered the ethylene signal transduction and resulted in a delay by affect­ ing ethylene biosynthesis, and enhancing superoxide dismutase activity (zhang et al., 2012). in addition, it has been found that applying 1­mcp delayed aging, but its effect varied depending on the flower organ, cultivar, and development stage and treatment con­ centration. it seems that catalase activity depends on the plant species, plant type and experimental condi­ https://en.wikipedia.org/wiki/hydrogen_peroxide https://en.wikipedia.org/wiki/water https://en.wikipedia.org/wiki/oxygen nasiri et al. ‐ effects of 1‐mcp and ethylene on cut alstroemeria flowers 95 tions during aging. finally, it can be concluded that increasing the activity of antioxidant enzymes reduces the aging of flowers. in general, 1­mcp treatment had a positive effect on improving physiological and biochemical charac­ teristics resulted in an extended postharvest longevi­ ty of alstroemeria cultivars. the higher concentra­ tions of 1­mcp revealed better effect in comparing to low concentrations, especially on ethylene produc­ tion. according to the results, 1­mcp treatments, by increasing the protein content and the activities of catalase and superoxide dismutase, improved the 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spread and shallow irrigation wetted area and application of organic mulch on citrus decline amelioration m.s. tadayon (*), s.m. hosseini soil and water research department, agricultural research, education and extension organization (areeo), shiraz, iran. key words: citrus decline, fibrous root, irrigation, orange, root decay. abstract: citrus decline threatens the orchards in the southern part of iran. roots begin to die­out before citrus decline deterioration. in this study, the effect of expanding the irrigation wetted area and decreasing the irrigation depth, and application of compost as organic mulch on root development and amelioration of citrus decline in valencia orange trees (citrus sinensis l. osbeck) was investigated. experimental factors contained the different per­ centage of irrigation wetted area and decreasing irrigation effective root depth by drip irrigation system at three levels ­ w0 (control): 30­40% with 60 cm effective root depth; w1: 50­60% with 45 cm effective root depth; w2: 70­80% with 30 cm effective root depth under tree canopy area and the factor of annu­ al application of compost as organic mulch at two levels ­ m0 (control): means no application of compost and m1: application of 80 kg compost under tree canopy area with 10 cm thickness as ground cover, after rotating the top soil at 10 cm depth for all treatments. the results showed that, annual application of compost as organic mulch under tree canopy area and expanding the irrigation wetted area with decreasing irrigation effective root depth, significantly improved fibrous root length and density at a lower soil depth and decreased the indices of citrus decline such as root rot percentage, leaf and fruit drop and shoot die back. also these treatments increased the water productivity and fruit quality of in declining valencia orange trees. 1. introduction citrus decline, commonly known as ‘dieback’, ‘chlorosis’ or neglecto­ sis, is not a specific disease but a syndrome expressing many disorders in the plant. such syndrome leads to decline in productivity, reduced pro­ ductive life and poor fruit quality. the symptoms of citrus decline contain root rot and blackening, shoot die back, growth stunt, fruit drop, reduc­ tion of canopy, smaller leaf number and size and leaf blotchy mottle (meena et al., 2018). stress­sensitive trees fail to maintain sufficient car­ bohydrate availability resulting in the dieback of the stressed tissues (kreuzwieser and rennenberg, 2014). also, the percentage of total solu­ ble solids and fruit juices in healthy trees is more than declined trees (mauk and shea, 2002). declining trees have more water stress and more (*) corresponding author: m.tadayon@areeo.ac.ir citation: tadayon m.s., hosseini s.m., 2020 ­ effect of spread and shallow irrigation wetted area and application of organic mulch on citrus decline amelioration. ‐ adv. hort. sci., 34(2): 213­221. copyright: © 2020 tadayon m.s., hosseini s.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 january 2020 accepted for publication 5 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 213­221 214 affected trees tend to have higher percentage leaf and fruit drop rates than healthy appearing trees. soil condition and water status (water stress and water logging) significantly reduce the citrus fibrous root density and increase the severity of citrus decline (kozlowski, 1997; graham et al., 2013; morgan, 2015; graham, 2017). citrus decline is an issue that threatens the econ­ omy of various regions of the world, including south­ ern iran. there are several factors that affect the inci­ dence of this complication. the most important influ­ ential factors on citrus decline are environmental stresses such as soil physicochemical conditions (compaction, high ph, bicarbonate, salinity) and soil nutritional status (srivastava and singh, 2009), soil moisture content (water stress and water logging with deficit or over irrigation), and biological stresses including rootstock, nematodes, greening disease (huanglongbing), tristesa virus disease, phytophtho­ ra fungal infection and root fusarium (johnson and graham, 2015; graham, 2017; meena et al., 2018; dewdney et al., 2019). according to the usda, since 2004­2005 total florida citrus production declined by greening disease from 169.1 to 94.2 million boxes in 2015­2016, down 44.4% (usda, 2017). the ideal environment for citrus root develop­ ment is a porous, medium­textured, well­drained soil, where water is easily available but not in excess (dewdney et al., 2019). mulching with organic matter helps retain moisture in the top soil by reducing sur­ face evaporation, as well as moderating soil surface temperatures. it also enhances the decomposition process of soil organic matter and improve the soil aeration and availability of nutrients in the soil (gong et al., 2006; fao, 2011). increasing root water uptake efficiency and life span is possible by irrigation man­ agement such as time and duration (decrease water stress and water logging), also improving the growth environment and drainage, soil compaction, soil ther­ mal stress, bicarbonate and osmosis stress (huber and haneklaus, 2007; dewdney et al. , 2019). increased root density also increases water uptake (morgan et al., 2006). in arid and semi­arid regions, the application of drip irrigation system results in root accumulation under emitter (fernandez et al., 1991; tanasescu and paltineanu, 2004; ruiz­sanchez et al., 2005), so the expansion of irrigation wetted area could enhance the fibrous root density under declining condition. increasing irrigation frequency and decreasing irriga­ tion depth stimulate root length density and increase water uptake and under these conditions, irrigation at the field capacity increases root density by 50% (kadyampakeni et al., 2014 a). in citrus decline new root growth did not stop, but the root survival time was reduced from 9 to 12 months to 4 months and the root decay and dieback were increased (dewdney et al., 2019). citrus decline cause water stress and under such condition root growth is preferable to shoot growth (hsiao and xu, 2000). root development dependent on soil aeration and moisture as two fundamental parameters for root healthiness, growth and distribution. root growth in sandy and loamy soils with higher organic matter content is stronger, and increment in soil clay content has a negative relationship with the density of citrus roots (koudounas, 1994) and the slope of clay content in soil profile is directly related to the effect of citrus decline deterioration (srivastava and singh, 2009). in citrus, the highest fibrous length den­ sities were observed in the humid part of the soil at a depth of 0 to 15 cm with higher root activity until 2 m horizontal distance from the trunk (alves et al., 2012). the rate of soil water depletion is directly related to the abundance of fibrous roots and in gen­ eral pattern of water uptake in citrus fruits indicates that water is drained by surface roots with higher amounts of available soil water (noling, 2003). lack of soil aeration and excessive soil moisture causes more severe damage than lack of moisture in declining trees, due to stop breathing and the break­ down of root cells. there is also the growth of anaero­ bic microenvironments and the production of sub­ stances such as nitrite that is toxic for the roots. centralize wetted area and excessive irrigation results in water logging, oxygen deficiency and root fungal infestationin the root zone (johnson and graham, 2015). deficit irrigation and soil water stress stimu­ lates longitudinal and singular growth without lateral roots and repeated moisture stress causes thicker fibrous roots in citrus that could decrease the water uptake efficiency by the citrus roots (castle, 1978). increment of soil aeration and soil moisture distribu­ tion and retention could provide the better condition for root development and amelioration of citrus decline. so in this experiment, the effects of spreading irrigation wetted area and decreasing irrigation depth, and annual application of compost as organic mulch on root development and diminishing of citrus decline indices in valencia orange was investigated. tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 215 2. materials and methods the experiment was conducted in a commercial orchard (28°38’13.12” n, 54°40’29.69” e and altitude 1138 m) of darab region,with very hot and dry cli­ mate (table 1), located in south west of fars province in iran, over three consecutive years 2016 to 2018. ninty­six uniform 12­year­old valencia orange trees (citrus sinensis l. osbeck), on lime (citrus aurantifo‐ lia) rootstock with citrus decline symptoms (sparse foliage, chlorotic leaves, twig drying, premature leaf fall, reduced productivity and fruit size and die­back canopy complication) were selected based on canopy diameter and labeled based on experimental plan. the distance between the trees was 4×5 m and irri­ gated by drip irrigation system with a loop contained 6 emitters (4 liters per hour). before the application of experimental treat­ ments, a composite soil sample were taken from 0­30 and 30­60 cm depths in oct. 2014 (table 2). soil sam­ ples characteristics were determined at analytical laboratory of soil and water research department, fars agricultural and natural resources research and education center, zarghan, iran. the soil of the experimental field site was calcareous with high ph, high total neutralizing value, and low amount of organic carbon, available p and mn (table 2). the fer­ tilizer application was conducted based on soil test and contained fertigation of ammonium sulfate (21­ 0­0­24s, 450 g tree­1), potassium sulfate (0­0­53+17s, 150 g tree­1) and triple superphosphate (450 g tree­1), manganese sulfate (32mn, 18s, 250 g tree­1) and iron chelate (200 g tree­1 sequestrene 138­fe eddha 6%, 150 g tree­1). a factorial (3×2) experiment was conducted in a randomized complete block design with four replica­ tions and each plot with four declining trees over three years. experimental factors contained the dif­ ferent percentage of irrigation wetted area and decreasing irrigation effective root depth by drip irri­ gation system at three levels as follows: • w0 (control)= 30­40% with 60 cm effective root depth; • w1= 50­60% with 45 cm effective root depth; • w2= 70­80% with 30 cm effective root depth under tree canopy area. also, the factor of annual application of compost as organic mulch at two levels: • m0 (control)= means no application of compost; • m1= application of 80 kg compost with 10 cm thickness as ground cover under tree canopy area. table 1 ­ long­term mean of climatic elements of the study area (darab) synoptic meteorological station statistical period (1997­2018) table 2 ­ soil characteristics in the experimental orchard soil depth bulk density (g cm­³) field capacity (%) wilting point (%) ec (ds.m­1) ph tnv (%) oc (%) p (mg kg­1) k (mg kg­1) zn (mg kg­1) mn (mg kg­1) cu (mg kg­¹) soil texture 0­30 1.25 19.5 10.3 1.32 8.15 35.2 0.76 7.3 264.5 0.76 3.12 0.57 sandy loam 30­60 1.28 19.3 10.4 1.36 8.16 37.7 0.62 7.2 252.4 0.62 3.19 0.56 sandy loam temperature (°c) relative humidity (%) precipitation (mm) evapo. (mm) sunny day no. day max wind (m s­¹) min max ave. abs. min abs. max min max ave. amount no. rainy max daily (montly sum) (montly sum) frost direc. degree velo. m/s april 11.5 26.4 19.0 3.8 34.7 26 70 48 32.50 6 43.70 182.0 276.0 0 253 13 may 16.7 33.6 25.2 9.4 41.6 16 51 34 6.46 2 24.80 286.6 331.4 0 240 15 june 21.6 39.3 30.4 15.6 44.4 11 37 24 1.10 1 9.60 382.8 356.6 0 200 14 july 25.5 41.7 33.6 15.4 46.5 12 38 25 0.92 1 5.80 428.3 341.3 0 171 15 august 26.2 40.9 33.6 19.2 45.2 14 40 27 5.76 2 20.50 422.3 339.8 0 207 16 september 21.9 38.5 30.2 16.2 42.6 14 44 29 0.82 1 4.70 336.0 327.5 0 180 10 october 16.0 33.6 24.8 7.6 38.8 16 48 32 0.32 1 2.60 228.4 308.0 0 226 10 november 10.4 26.5 18.5 2.0 33.5 22 62 42 8.48 2 50.20 138.2 269.6 0 249 8 december 5.7 20.1 12.9 ­2.2 30.0 32 76 54 40.25 5 51.80 79.1 234.9 0 207 7 january 3.8 17.0 10.4 ­2.6 25.6 36 81 58 64.77 6 65.40 64.4 226.1 3 205 8 february 5.0 17.7 11.3 ­2.6 25.8 35 81 58 49.88 6 68.20 79.3 238.4 1 240 10 march 8.0 21.8 14.9 0.2 31.6 30 74 52 37.26 7 63.00 116.7 241.1 0 238 12 adv. hort. sci., 2020 34(2): 213­221 216 compost applied 50 cm away from the trunk of the tree and over the soil surface, after rotating the soil at 10 cm depth for all treatments in march of each year. the physico­chemical properties of the applied compost contained organic carbon (22%), total nitrogen content (1.5%), c/n ratio (15), p2o5 (0.8%), k2o (0.7%), electrical conductivity (6.4 ds. m­1), ph (7.6), moisture content (12%), density (380 kg.m­3), particle diameter (6 mm). the amount of irri­ gation wetted area was adjusted by increasing the number of emitters (6, 9 and 12 per loop with 4 liters per hour discharge) and the irrigation interval was determined by placing tensiometers (irrometer tensiometer model sr manual gauge 12 in) deep down at three irrigations effective root depth (60, 45 and 30 cm, respectively) according to the irrigation experimental factor. each year in february, the com­ posite soil sample were taken from 0­10 cm and the percentage of soil organic matter, porosity and bulk density (g cm­3) were determined. the amount of organic matter was measured by weight loss of oven­ dried (105°c) soil sample after loss on ignition at 400°c. to determine the porosity of the samples, divide the pore space volume by the total volume and multiply the result by 100. soil bulk density was deter­ mined by the weight of dry soil divided by the total soil volume. the amount of consumed irrigation water was measured by contours in each treatment. over the three seasons, the indices of citrus decline were determined on tagged main branches on each tree quadrates. at the late spring, new flush lengths and the percentage of shoot dieback were measured. leaf and fruit drop percentage were determined by counting their number at two separate time (after june drops and pre­harvest time). leaf samples (con­ tain 100 leaves) were collected during july­august from fully expanded new flush sub­terminal leaves from non­fruiting from tagged branches. total chloro­ phyll content was determined by the method worked out by lichtenthaler (1987). leaf fresh weight (wf) were recorded quickly using an analytical balance (mettler toledo al104, switzerland). then, they were dried at 120°c in a circulation oven for 20 minutes, and the temperature dropped to 80°c until the con­ stant weight (dry weight, wd) was reached. then the leaf relative water content (rwc) was calculated as: leaf relative water content (%) = [(wf ­ wd)/wf] x 100 individualleaf areas (la) were determined by li­ cor leaf area meter, and sample were dried and spe­ cific leaf weights, slw (mg cm­2) were measured as: specific leaf weight (mg cm­²) = wd/la root sampling was carried out under emitters from depth of 30, 45 and 60 cm soil depth, based on the effective root depth considered in the irrigation experimental factor, by auger (diameter 9 cm and height 25 cm), in early­august (alves et al., 2012). after washing the root samples, length, weight and number of fibrous root with less than 0.2 cm diame­ ter was determined by digital ruler and weight scale, and length density (cm cm­3), density (mg cm­3) and the percentage of decayed fibrous root were deter­ mined in the soil sample volume harvested by auger (1589.6 cm3) (alves et al., 2012). at the harvesting time total fruit yield per trees was determined by the scale. a random sample of 50 fruits per plot was provided to determine fruit diam­ eter (average of two perpendicular diameters), total soluble solids, ph and titratable acidity of the juice. total soluble solids (brix) were determined by using hand refractometer (wyt portable model) and juice total acid was measured by titration method with 0.1 n sodium hydroxide until ph meter reads 8.2 (graham, 2017). fruit juice percentage was calculat­ ed by fruit juice weight divided by fruit total weight. water productivity (kg m­3) was calculated based on the yield of trees to the amount of consumed irriga­ tion water. the data set was auto scaled before analysis. all the parameters for three years were sub­ jected to combine analysis of variance (anova) by mstat­c software. means were compared by duncan’s multiple range test and pearson correlation were determined by spss software. 3. results the results of the combine analysis of variance (anova) over three consecutive seasonal growths from 2016 to 2018, indicated that there was a signifi­ cant effect of experimental factors on improvement of citrus decline deterioration indices in valencia orange (tables 4­8). annual application of compost as organic mulch and adding them to 10 cm top soil in the following years had significant effect on the increment of soil organic matter and soil porosity up to 170.45, 13.18% respectively, and decreasing soil bulk density by 11.98% at 10 cm soil depth in com­ parison with control (without application of compost as mulch) (table 3). annual application of compost as organic mulch significantly increased the fibrous roots length densi­ ty and fibrous roots density of valencia orange trees by 34.63 and 38.27% respectively. also the interac­ tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 217 tion between expansion the percentage of irrigation wetted area along with and decreasing irrigation effective root depth with annual application of com­ post as organic mulch (w1m1 and w2m1), signifi­ cantly increased the fibrous root density up to 87.09 and 112.9% and decreased the root decay by 43.23 and 46.48% at a lower soil depth respectively in com­ parison with control (w0m0) (table 4). mean separation (± sd) within columns followed by different let­ ters are significantly different at p≤0.05 using duncan’s new mul­ tiple range tests. table 3 ­ effects of experimental treatments on 10 cm top soil organic matter, porosity and bulk density over three consecutive seasonal growths from 2016 to 2018 treatments soil organic matter (%) soil porosity (%) bulk density (g cm­³) w0m0 1.42 ± 0.12 b 42.05 ± 1.21 b 1.49 ± 0.04 a w1m0 1.24 ± 0.14 b 41.86 ± 1.32 b 1.43 ± 0.04 a w2m0 1.30 ± 0.10 b 42.42 ± 1.15 b 1.42 ± 0.05 a w0m1 3.38 ± 0.07 a 47.08 ± 1.01 a 1.31 ± 0.06 b w1m1 3.84 ± 0.08 a 48.51 ± 1.02 a 1.27 ± 0.07 b w2m1 3.50 ± 0.07 a 47.39 ± 0.93 a 1.24 ± 0.06 b mean separation (± sd) within columns followed by different let­ ters are significantly different at p≤0.05 using duncan’s new mul­ tiple range tests. table 4 ­ effects of experimental treatments on root characteri­ stics of declining valencia orange over three consecuti­ ve seasonal growths from 2016 to 2018 treatments fibrous roots length density (cm cm ­³) fibrous root density (mg cm­³) root decay (%) w0m0 0.043 ± 0.003 d 0.31 ± 0.08 d 66.24 ± 6.21 a w1m0 0.054 ± 0.002 c 0.48 ± 0.06 c 53.72 ± 8.60 b w2m0 0.056 ± 0.002 c 0.49 ± 0.06 c 54.25 ± 7.32 b w0m1 0.066 ± 0.003 b 0.53 ± 0.04 bc 48.07 ± 7.56 b w1m1 0.065 ± 0.004 b 0.58 ± 0.04 b 37.60 ± 8.14 c w2m1 0.075 ± 0.003 a 0.66 ± 0.03 a 35.45 ± 8.81 c mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 5 ­ effects of experimental treatments on vegetative growth indices of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments total leaf chlorophyll content (mg g­¹ fw) flush length (cm) leaf drop (%) shoot dieback (%) w0m0 0.29 ± 0.03 d 24.82 ± 2.31 d 35.30 ± 1.12 a 35.70 ± 2.11 a w1m0 0.34 ± 0.02 c 35.53 ± 3.78 c 27.63 ± 1.53 b 29.62 ± 3.10 b w2m0 0.36 ± 0.01 c 38.35 ± 3.81 b 21.30 ± 1.71 c 17.60 ± 2.27 d w0m1 0.42 ± 0.03 b 39.47 ± 3.15 b 24.70 ± 1.92 bc 25.25 ± 2.15 c w1m1 0.46 ± 0.02 a 43.63 ± 2.80 a 15.45 ± 1.65 d 15.60 ± 2.84 d w2m1 0.47 ± 0.02 a 44.90 ± 2.66 a 10.20± 1.86 e 8.41 ± 1.90 e in comparison with control (without compost as mulch), application of compost as organic mulchsig­ nificantly increased the total leaf chlorophyll content and new flushes length up to 36.3 and 29.67% respectively and decreased leaf and fruit drop (table 5, 6) and shoot dieback by 40.21, 38.18 and 40.59%, respectively (table 5), in declining valencia orange trees, respectively. increment the irrigation wetted area percentageand decreasing the effective root depth for irrigation, significantly increased leaf chlorophyll contentand flush lengthand reduced leaf and fruit drop and shoot dieback. the interaction between the expansionof irrigation wetted area with annual application of compost as organic mulch (w1m1 and w2m1) had the highest impact on the increment of total leaf chlorophyll contentup to 58.62 and 62% and flush length by 75.78 and 80.9% and decreasing leaf drop 56.17 and 71.1%, fruit drop by 44.62 and 63.3% and shoot dieback by 56.3, 76.44%, respectively, in comparison with control (w0m0). leaf relative water content, specific leaf weight, fruit diameter and tree yield increased with annual application of compost as organic mulchanddevelop­ ing the irrigation wetted area (table 6). the interac­ tion between irrigation wettedarea and annual appli­ cation of compost as organic mulch (w1m1 and w2m1) had the highest impact on the increment of leaf relative water content up to 11.91 and 14.25% and specific leaf weight up to 18.75 and 22.66%, respectively, in comparison with control (w0m0) (table 6). also fruit diameter significantly increased by increment of wetted area and application of mulch by 15.9 and 14.11%, respectively (table 6). the highest yield of valencia orange trees was belonged to the interaction between irrigation wet­ ted area of 70­80% with 30 cm effective root depth for irrigation and application of compost mulch 218 adv. hort. sci., 2020 34(2): 213­221 (table 7). annual application of compost as organic mulch had significant effect on the reduction of con­ sumed irrigation water by 17.39% in comparison with control (without mulch). the interaction between irrigation wetted area and annual application of com­ post as organic mulch (w1m1 and w2m1) had the highest impact on the increment of water productivi­ ty up to 53.06 and 49.79% respectively, in compari­ son with control (w0m0), and there was no signifi­ cant difference between them (table 7). expansion of irrigation wetted area and decrease effective root depth for irrigation under the condition of compost mulch applicationhad the greatest effect on water productivity in valencia orange. annual application of compost as organic mulch sig­ nificantlyincreased the fruit juice content by 11.98%, total soluble solids (brix) by 10.96%, brix/titratable acidity ratio by 24.52% in declining valencia orange trees. fruit acidity was significantly reduced by annual application of mulch. increment of irrigation wetted area percentage significantly, increased fruit juice and brix/titratable acidity ratio. the interaction between increased irrigation wetted area with mulch application (w1m1 and w2m1), resulted in the highest increase in fruit juice percentage by 35.43 and 34%, total soluble solidsby 23 and 26.36% and brix/titratable acidity ratio by 49.6 and 57.6%, respectively in comparison with control (w0m0) (table 8). mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 6 ­ effects of experimental treatments on the leaf relative water content, specific leaf weight, fruit drop and fruit diameter of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments leaf relative water content (%) specific leaf weight (mg cm­²) fruit drop (%) fruit diameter cm) w0m0 83.50 ± 5.30 d 3.84 ± 0.12 d 31.15 ± 1.10 a 7.23 ± 0.10d w1m0 89.64 ± 5.24 c 4.12 ± 0.08 c 25.12 ± 1.32 b 7.58 ± 0.08 c w2m0 91.25 ± 4.22 bc 4.36 ± 0.07 bc 21.84 ± 1.25 bc 8.20 ± 0.07 b w0m1 88.36 ± 5.75 c 4.04 ± 0.09 cd 19.61 ± 1.74 c 7.71 ± 0.08 c w1m1 93.45 ± 4.12 ab 4.56 ± 0.06 ab 17.25 ± 2.11 c 8.38 ± 0.06 a w2m1 95.48 ± 4.53 a 4.71 ± 0.06 a 11.43 ± 2.73 d 8.25 ± 0.07 ab mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 7 ­ effects of experimental treatments on the leaf characteristics, fruit quantity and water productivity of declining valencia oran­ ge over three consecutive seasonal growths from 2016 to 2018 mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 8 ­ effects of experimental treatments on the fruit quality indices of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments total tree yield (kg) consumed irrigation water (m³ ha­¹ year­¹) water productivity (kg m­³) w0m0 62.33 ± 2.14 e 11625.28 ± 136.58 c 2.45 ± 0.11 c w1m0 71.05 ± 2.71 d 13857.14 ± 122.61 b 2.39 ± 0.16 c w2m0 83.10± 1.50 c 14631.67 ± 121.25 a 2.66 ± 0.14 bc w0m1 68.47 ± 2.43 d 10363.64 ±136.84 d 3.01 ± 0.12 b w1m1 89.40± 1.37 b 10904.76 ± 132.70 d 3.75 ± 0.11 a w2m1 94.40± 1.15 a 11869.57 ± 126.39 c 3.67 ± 0.12 a treatments fruit juice (%) titratable acidity (g 100­¹ ml) total soluble solids (brix) brix/titratable acidity ratio w0m0 42.64 ± 2.02 d 1.16 ± 0.01 a 9.56 ± 0.61 d 8.94 ± 0.22 f w1m0 48.80 ± 1.81 c 0.94 ± 0.03 b 11.13 ± 0.35 c 10.26 ± 0.19 e w2m0 53.71 ± 1.60 b 0.95 ± 0.02 b 11.87 ± 0.24 b 11.87 ± 0.17 c w0m1 47.65 ± 2.17 c 0.82 ± 0.02 c 12.29 ± 0.73 a 11.23 ± 0.18 d w1m1 57.75 ± 1.61 a 0.74 ± 0.03 d 11.76 ± 0.40 b 13.37 ± 0.15 b w2m1 57.14 ± 1.55 a 0.84 ± 0.03 c 12.08 ± 0.37 b 14.09 ± 0.14 a tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 219 results showed that fibrous root density had a significant negative correlation with leaf drops (r= ­ 0.791) and shoot dieback (r= ­0.612) (table 9). also fibrous root density had positive and significant cor­ relation with flush length (r= 0.624), leaf water con­ tent (r= 0.732) and specific leaf weight (r= 0.631). the root decay percentage had significant negative corre­ lation with leaf chlorophyll content (r= ­0.643), flush length (r= ­0.632), leaf water content (r= ­0.603), leaf specific weight (r= ­0.638), yield (r= ­0.691), water productivity (r= ­0.602), and brix/ta (r= ­0.684). also there was a positive and significant correlation between the percentage of root decay with leaf drops (r= 0.784), shoot dieback (r= 0.692), and fruit drops (r= 0.704) (table 9). specific leaf weight had positive and significant correlation with leaf chloro­ phyll content (r= 0.482) (table 9). there was a nega­ tive correlation between fruit drops and relative water content of leaves (r= ­0.774). tree water pro­ ductivity had a positive and significant correlation with the leaf chlorophyll content (r= 0.612), leaf rela­ tive water content (r= 0.766), and specific leaf weight (r= 0.677) (table 9). 4. discussion and conclusions citrus decline was directly related to root decay and decreasing of fibrous root density and root expansion in valencia orange trees. tree perfor­ mance is a function of how the root system is distrib­ uted over a large volume of soil to absorb water and nutrients (lehmann, 2003). in arid and semi­arid regions, the highest density of fibrous roots in the drip irrigation system is located under the emitters and soil wetted area (ciancio and mukerji, 2008; alves et al., 2012). the rate of water adsorption by the tree is reduced when the soil oxygen level is low (levy, 1998; boman et al., 1999) and the most influ­ ential soil condition on citrus decline are soil mois­ ture condition (meena et al., 2018), and soil com­ paction (srivastava and singh, 2009). root develop­ ment is related to the hydrophilicity of roots, soil aer­ ationand distribution of soil moisture (ruiz­sanchez et al., 2005). our results showed that application of compost as organic mulch and rotating them with 10 cm top­ soil improved soil physical properties such as soil organic matter, porosity and bulk density which could provide the best conditions for root development. this treatment could increase the soil aeration and soil moisture retention at topsoil, as two key factors for root health and growth (boman and parsons, 2002; nelson et al., 2008; johnson and graham, 2015). meanwhile, it could reduce surface evapora­ tion, moderate soil surface temperatures and provide the best condition for root development for declining valencia orange trees. application of organic mulches on eureka lemon (citrus limon burm) significantly increased the soil moisture status in various soil table 9 ­ pearson correlation coefficients between the means of citrus decline indices of valencia orange over three consecutive seaso­ nal growths from 2016 to 2018 chlorophyll content flush length leaf drop shoot dieback lrwc 1 slw 2 fruit drop fruit diameter yield water productivity brix/ta. fibrous roots density fibrous roots length density root decay chlorophyll content 1.00 flush length 0.64 ** 1.00 leaf drop ­0.51 * ­0.64 ** 1.00 shoot dieback ­0.55 * ­0.74 ** 0.78 ** 1.00 lrwc 0.54 * 0.64 ** ­0.77 ** ­0.75 ** 1.00 fruit drop ­0.66 ** ­0.25 ns 0.67 ** 0.64 ** ­0.71 ** ­0.69 ** 1.00 fruit diameter 0.43 * 0.49 * ­0.52 * ­0.51 * 0.52 * 0.53 * ­0.53 * 1.00 yield 0.59 ** 0.65 ** ­0.76 ** ­0.67 ** 0.66 ** 0.60 ** 0.26 ns 0.75 ** 1.00 water 0.61 ** 0.50 * ­0.75 ** ­0.65 ** 0.77 ** 0.68 ** ­0.75 ** 0.78 ** 0.78 ** 1.00 productivity brix/t.a. 0.63 ** 0.52 * ­0.70 ** ­0.68 ** 0.15 ns 0.57 ** ­0.66 ** 0.13 ns 0.57 ** 0.46 * 1.00 fibrous roots density 0.71 ** 0.62 ** ­0.79 ** ­0.61 ** 0.73 ** 0.63 ** ­0.71 ** 0.64 ** 0.70 ** 0.71 ** 0.62 ** 1.00 fibrous roots length density 0.58 * 0.48 * ­0.59 ** ­0.54 * 0.68 ** 0.59 ** ­0.54 * 0.61 ** 0.62 ** 0.59 ** 0.52 * 0.78 ** 1.00 root decay ­0.64 ** 0.63 ** 0.78 ** 0.69 ** ­0.60 ** ­0.64 ** 0.70 ** ­0.66 ** ­0.69 ** ­0.60 ** ­0.68 ** ­0.64 ** ­0.71 ** 1.00 1 leaf relative water content. 2 specific leaf weight. *, ** significant at 5 and 1% statistical levels respectively; ns = not significant. adv. hort. sci., 2020 34(2): 213­221 220 depths and farmyard manure were found to be more effective in producing maximum growth extension (kumar et al., 2015). the irrigation wetted area, also affect citrus root system development and distribu­ tion (alves et al., 2012). in our experiment, the expansion of irrigation wetted area and reduction the effective root depth for irrigation with annual appli­ cation of compost as organic mulchand its rotation in the 10 cm soil depth, increased the fibrous root length and root densityat lower soil depths and decreased root decay in valencia orange trees. also, leaf relative water content, specific leaf weight and fruit diameter in valencia orange significantly increased with developing the irrigation wetted area. increment of fibrous root length and root density at lower soil depths with decreasing the irrigation depth and consequent improvement of tree water status were in agreement with the results that showed more root distribution in the drip irrigation method was at the depth of 15 cm, with high water uptake efficiency (kadyampakeni et al., 2014 b). annual application of compost as organic mulch significantly decreased the consumed irrigation water in valencia orange trees. increasing soil organic carbon improve the healthy root system of citrus (sharma et al., 1986). there was a positive and significant correla­ tion between leaf relative water content and leaf chlorophyll content (r= 0.540), and flush lengths (r= 0.643) and a significant negative correlation between leaf water content and leaf drops (r= ­0.771), and shoot dieback (r= ­0.748) (table 8). it has been shown that clementine ‘nules’ vegetative growth and fruit size was higher with increasing numbers of emit­ ters on the double drip­lines treatments (abouatallah et al., 2012). we conclude that annual application of compost as organic mulch under tree canopy area and its rota­ tion at 10 cm soil depth at following years and expanding the irrigation wetted area and decreasing the irrigation effective root depth, significantly improved the indices of citrus decline and increased the water productivity and fruit quality in valencia orange trees. acknowledgements the authors greatly appreciate mr. mojtaba dadgar who provided the commercial orchard for this experiment and fars agricultural research, education and extension center. references abouatallah a., salghi r., el­fadl a., affi n., ghnizar y., zarrouk a., hammouti b., 2012 ­ impact assessment of drippers distribution around the tree on soil moisture, roots and fruits growth of citrus. ­ der pharma chemica., 4.5: 1969­1981. alves j. jr., bandaranayake w., parsons l.r., evange­ lista a.w., 2012 ­ citrus root distribution under water stress grown in sandy soil of central florida. ­ engenharia agrícola, 32(6): 1109­1115. boman b., levy y., parsons l., 1999 ­ water manage‐ ment, pp. 72­81. ­ in: timmer l.w., and l.w. duncan (eds.) citrus health 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systems within the hilly region of romania. ­ inter. agrophysics, 18(2): 175­180. usda, 2017 ­ florida citrus statistics. ­ united states department of agriculture, fl, usa. https://www.nass.usda.gov/statistics_by_state/florida/publications/citrus/citrus_statistics/2015-16/fcs1516.pdf impaginato 25 adv. hort. sci., 2020 34(1): 25­33 doi: 10.13128/ahsc­8253 the effect of different colored netting on quantitative and qualitative traits of two foliage plant species (codiaeum variega‐ tum and aglaonema commutatum) s.k. abbasnia zare 1, s. sedaghathoor 1 (*), m.n. padasht dahkaei 2, d. hashemabadi 1 1 rasht branch, islamic azad university, rasht, iran. 2 agricultural and natural resources research, center of guilan, agricultural research, education, and extension organization, rasht, iran. key words: anthocyanin, brix°, croton, photoselective, yellow netting. abstract: photoselective netting is a new group of colored netting developing in the past few decades. the effect of colored (red, green, and yellow) netting was studied on physiological traits of codiaeum and aglaonema in a trial in flowers and ornamental plants research station of lahijan, north of iran. the trial was based on a split­plot experiment with two factors. the first factor was devoted to colored netting at four levels (no netting, green, yellow, and red) and the second factor was devoted to plant species at two levels of codiaeum and aglaonema based on a randomized complete block design with three replica­ tions. the results showed the positive effect of yellow netting on improving the vegetative capability of the plants, so that the highest plant height, shoot and root fresh and dry weight, and leaf area were observed in the plants grown under the yellow netting. also, the highest anthocyanin, carotenoid contents, and catalase activity were obtained from the red netting and the highest brix° and total chlorophyll from the red and yellow netting. according to the results, the highest vegetative growth rate was related to codiaeum. the application of the colored nets provided the plants with more optimal growth conditions. 1. introduction the average number of sunny days in iran annually amounts to a sig­ nificant value of 280 days, making it unavoidable to use shading to control incident radiation during sunlight exposure (forghani and kiani abri, 2005). the concept of photo­selective shade netting was first devised in israel and was tested on ornamental plants, vegetables, and fruit trees. then, the idea gradually spread to the whole world to be applied to vari­ ous plants, climatic regions, and agronomic practices (shahak, 2008). so far, black netting has mostly been used for shading and transparent net­ ting has been employed for protection against environmental or pest damages. a whole new group of protective nettings has been developed (*) corresponding author: sedaghathoor@yahoo.com citation: abbasnia zare s.k., sedaghathoor s., pada­ sht dahkaei m.n., hashemabadi d., 2020 ­ the effect of different colored netting on quantitative and qualitative traits of two foliage plant species (codiaeum variegatum and aglaonema commu‐ tatum) ­ adv. hort. sci., 34(1): 25­33. copyright: © 2020 abbasnia zare s.k., sedaghathoor s., padasht dahkaei m.n., hashemabadi d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 june 2019 accepted for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 25­33 26 to manipulate both the quality and quantity of radia­ tion intercepted by plants and simultaneously pro­ tect them optimally (shahak et al., 2004 a, b). the technology of colored nets that selectively fil­ ters sunlight and physically protects crops is based on specific nets in which color elements are used during fabrication. this technology seeks to absorb ultravio­ let, blue, green, yellow, red, far­red, or near­infrared spectra. the direct light is intended to change into scattered light too. colored nets can only alter the components of sunlight that penetrates through their plastic strings whereas solar rays that go across the holes are not manipulated (shahak et al., 2016). it has been documented that yellow and red, as well as grey, netting can significantly increase the productivi­ ty of capsicum annuum versus black netting. these results were attributed to the increased number of fruits in a season in plants subjected to the netting (shahak et al., 2008). in another study by shahak et al. (2016), it was revealed that yellow netting outper­ formed red netting in stimulating the vegetative growth of pittosporum and among the studied nets, yellow netting exhibited much stronger strengthen­ ing effects than red netting. other studies have also shown that blue netting reduced vegetative growth and induced dwarfness in leafy ornamental plants and cut flowers whereas red and yellow nets that reduced the intensity of blue light induced vegetative growth in plants (shahak et al., 2016). photoselective nets can, according to studies, alter shade quality by scattering light and changing its spectral composition (shahak et al., 2008). wang and folta (2013) report­ ed that the colored nets had a significant effect on increasing vegetative growth rate in foliage plants as compared to the black nets with a similar coefficient of shading (oren­shamir et al., 2001). the results showed that the red and white nets increased the yield of cordyline with respect to plant height, leaf number, biomass, leaf area, photosynthesis rate, and harvest index as compared to control. also, plants under the colored nets exhibited a longer vase life than those in the open air, but there was no signifi­ cant difference between nets with different colors. according to the results, white and red nets were better for the growth of cordyline (kumar gaurav et al., 2016). since few studies have addressed the effect of colored netting on vegetative and physiolog­ ical traits of foliage plants, the present paper explores the impact of color netting on vegetative traits of ornamental codiaeum variegatum and aglaonema commutatum plants in flower and ornamental plant research station of lahijan, iran. 2. materials and methods the effect of colored (red, green, and yellow) net­ ting was studied on vegetative traits of c. variegatum and aglaonema commutatum in flowers and ornamental plants research station of lahijan, north of iran in the spring and of 2018. the trial was based on a split­plot experiment with two factors. the first factor was devoted to colored netting at four levels (no netting, green, yellow, and red) and the second factor was devoted to plant species at two levels of c. variegatum and a. commutatum based on a rcbd with three replications. each experimental plot was composed of four plants. the light intensity in cloudy days was in the range of 4000­5000 lux, no shade net of 6000 lux and on sunny days in the range of 20,000­ 28,000 lux and no shade net of 30,000­35000 lux. the recorded vegetative parameters included plant growth rate, plant height, shoot and root fresh and dry weight and leaf area. dry weight was obtained oven­dried at 105°c for 24 h. to determine leaf area, the leaves length (l) and widest width (w) were measured, and the leaf area (a) was calculated by the following equation (moll and kamparth, 1997): a= l x w x 0.75 the physiological traits included °brix, chlorophyll a, b and total, carotenoid, anthocyanin, flavonoid, antioxidant capacity, catalase and peroxidase. the brix° of the leaves was measured with an n­1α hand­ held refractometer (atago co., japan). to measure chlorophyll contents of the treatments, 0.5 g of sam­ ple was weighed and ground in a mortar containing 50 ml 80% acetone. then, the extract was infiltrated, adjusted to 50 ml, and poured into cuvettes. to determine chlorophyll content, it was read at 643 and 660 nm with a spectrophotometer. chlorophyll a and b and total chlorophyll contents were estimated by the following equations (mazumdar and majumder, 2003): total chlorophyll (mg/ml) = 7.12(a660) + 16.8 (a643) chlorophyll a (mg/ml) = 993(a660) ­ 0.777(a643) chlorophyll b (mg/ml) = 17.6(a643) ­ 2.81(a660) to measure the carotenoid level, the treatments were sampled. then, 0.5 g was weighed from the sample and was ground in a mortar containing 50 ml 80% acetone. then, it was infiltrated, adjusted to 50 ml , and poured into cuvettes. the extracts were read at 645, 663, and 660 nm and were placed in the fol­ lowing equation, denoted by a , to determine abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 27 carotenoid levels of the treatments (mazumdar and majumder, 2003): carotenoid level = 4.69(a660) ­ 0.268(a643) + 8.02(a663) to measure anthocyanin content, 0.5 of the sam­ ple was taken and ground in a chinese mortar con­ taining 50 ml of hydrochloric­ethanol acid (85% ethanol 95% + 15% hydrochloric acid). then, it was infiltrated, adjusted to 50 ml , and poured into cuvettes. they were placed in a refrigerator at 4°c for 24 hours followed by 2 hours in darkness. the extract was read at 535 nm with a spectrophotometer and was placed in the following equation to determine anthocyanin content (mazumdar and majumder, 2003): total absorption = e x b x c d x a where a = sample weight (0.5 g), b = the volume taken for the measurement (5 ml ), c = total volume (50 ml ), d = the fraction taken for the sample 0.1, and e = absorption read at 535 nm. total anthocyanin content of sample = total absorption of sample 98.2 to measure flavonoid content, 0.1 g of the sample was taken and ground in a mortar containing 85% methanol and was centrifuged at 8000 rpm for 5 minutes. then, it was infiltrated and the supernatant was placed in a hot bath at 80°c for 10 minutes. then, it was cooled down and its absor was read at 270, 300, and 330 nm with a spectrophotometer (humadi and istudor, 2008). a=εbc where: a= absorption rate, ε= extinction coefficient 33000 m.cm­1, b= width of the cuvette (1 cm) and c= total flavonoid content. in order to estimate antioxidant capacity, 1 g of the plant was wrapped in foil and was placed in liquid nitrogen for 2­3 minutes. then, it was ground with 10 ml methanol 85% and the samples were placed in room temperature for one hour. next, their extract was infiltrated and centrifuged for five minutes. then, 150 ml was taken from it and was added with 850 μl dpph. the solution was stirred fast and was kept in room temperature at the dark for 20 minutes. after placing the blank and resetting the instrument, first only dpph was poured into the cuvette and it was read. then, the sample was read at 517 nm by a spectrophotometer. the antioxidant capacity of the extracts was calculated by the following equation in terms of % inhibition in dpph (ramandeep and savage, 2005): %dpph = acont ­ asamp x 100 acont where %dpph = percent inhibition, acont = absorption rate of dpph, and asamp = absorption rate (sample + dpph). the activity of catalase (cat) was measured through the following stages (dazy et al., 2008): 1 g of plant tissue that had been ground in 4 ml ethanol was added with (i) 0.01 mol phosphate buffer (ph = 7), (ii) 0.5 ml h2o2 0.2 mol, and (iii) 2 ml acid reagent (dichromate/acetic acid mixture). then, its absorp­ tion was read at 610 nm with a spectrophotometer. to measure the enzymatic activity of peroxidase (pod), the extract was prepared as described above. then, the variations of od at 430 nm were read with a spectrophotometer once thirty seconds for two minutes (chance and maehly, 1955). data were sta­ tistically analyzed with mstatc software package, and the means were compared with the lsd test. 3. results according to the results of analysis of variance (anova; tables 1, 2), the simple effect of netting type and plant species and their interactions were significant (p<0.01) on plant height. the tallest plants were obtained from the yellow netting and the short­ est from no net exposure. also, means comparison for the interaction of ‘netting type × plant species’ revealed that the highest plant height was related to ‘yellow net × a. commutatum’ and the lowest to ‘no net × a. commutatum’. according to anova (table 1), plant growth rate was significantly (p<0.01) affected by shade netting, plant species, and ‘shade netting × plant species’. means comparison revealed that the highest growth rate was obtained from the yellow netting (table 3) and the lowest from no­netting treatment. means comparison for the interactive effect of ‘shade net­ ting × plant species’ on plant growth rate showed that ‘yellow netting × c. variegatum’ had the utmost plant growth rate and ‘no netting × a. commutatum’ had the lowest one. according to anova, leaf num­ ber was significantly influenced by netting type (p<0.05) and plant species (p<0.01), but the interac­ tion of these two parameters was insignificant for this trait (table 1). means comparison revealed that the highest number of leaves was observed in the plants treated with the yellow net and the lowest in adv. hort. sci., 2020 34(1): 25­33 28 the plants not treated with the nets (table 3). the effect of netting type and plant species was significant (p<0.01) on leaf size (leaf length and width), but the interaction of ‘net type × plant species’ was insignificant (table 1). the yellow net was related to the highest leaf length and width and the control to the lowest leaf size (table 3). as well, means comparison for the effect of plant species on leaf size indicated that c. variegatum produced the largest leaves (table 4). anova shows the significant (p<0.01) influence of netting type and plant species on leaf area, but the insignificant effect of ‘netting type × plant species’ on this trait (table 1). the high­ est leaf area of 138.4 cm2 was associated with the application of the yellow net, but control treatment (in which no net was applied) showed the lowest leaf area of 72.19 cm2 (table 3). means comparison for the effect of plant species on leaf area revealed that c. variegatum had the highest leaf area (table 4). anova revealed the significant (p<0.01) effect of shade netting type and plant species on shoot fresh and dry weight, but their interaction was significant table 1 ­ analysis of variance for the effect of experimental factors on the studied morphological traits ns= insignificant difference; **= significant difference at the p<0.01 level; *= significant difference at the p<0.05 level. source of variables df means of squares final height growth initial leaf no. final leaf no. leaf no. increase leaf length leaf width leaf area shoot fresh weight shoot dry weight root fresh weight root dry weight replication 2 4.76 ns 6.74 * 0.58 ns 21.15 ns 14.86 ns 0.97 ns 0.51 ns 264.38 ns 25.99 ns 0.13 ns 1.26 ns 0.006 ns netting (a) 3 202.24 ** 160.65 ** 4.20 ns 189.84 * 153.79 ** 41.01 ** 7.09 ** 4813.22 ** 3492.35 ** 17.12 ** 35.24 ** 0.173 ** error 6 5.54 0.97 5.57 37.95 15.81 0.76 0.58 226.36 85.45 0.42 1.32 0.006 plant (b) 1 1536.00 ** 997.82 ** 348.84 ** 2095.34 ** 734.27 ** 173.35 ** 19.26 ** 16949.00 ** 28295.47 ** 138.67 ** 0.39 ns 0.002 ns ab 3 42.36 ** 50.11 ** 3.75 ns 22.95 ns 11.98 ns 13.57 ns 1.89 ns 1448.57 ns 529.42 * 2.59 * 6.80 * 0.032 * error 8 1.43 2.28 4.95 25.25 10.24 5.60 0.69 517.91 82.94 0.41 1.01 0.005 c.v. (%) 3.62 14.13 26.23 25.40 28.32 12.93 11.71 22.42 18.07 18.06 21.51 21.79 table 2 ­ analysis of variance for the effect of experimental factors on the studied morpho­chemical traits ns= insignificant difference; **= significant difference at the p<0.01 level; *= significant difference at the p<0.05 level. source of variables df means of squares brix catalase peroxidase anthocyanin chlorophyll a chlorophyll b total chlorophyll carotenoid flavanoid antioxidant capacity replication 2 0.29 ns 0.001 ns 0.015 ns 1170.53 ns 1.81 ns 0.31 ns 1.62 ns 0.45 ns 0.001 ns 0.003 ns netting (a) 3 15.49 * 0.005 ** 0.014 ns 16243.31 ** 2.63 ns 0.95 ns 6.56 * 5.68 ** 0.000 ns 0.008 ** error 6 1.74 0.000 0.005 721.97 1.10 0.39 1.04 0.41 0.000 0.001 plant (b) 1 532.04 ** 0.000 ns 0.032 ns 6699.06 ** 3.06 ns 0.06 ns 1.66 ns 0.36 ns 0.000 ns 0.003 * ab 3 0.49 ns 0.000 ns 0.037 ** 48512.77 ** 2.80 ns 0.28 ns 2.13 ns 1.09 ns 0.001 ** 0.007 ** error 8 0.37 0.001 0.003 5179.48 1.19 0.27 1.92 0.47 0.000 0.000 c.v. (%) 6.97 48.63 13.11 28.83 75.90 56.85 57.60 31.23 10.53 36.02 table 3 ­ means comparison for the effect of netting type on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. 1 peroxidase enzyme unit in μm /h2o2 consumed/min/mg. netting final height (cm) growth (cm) leaf no. shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) leaf length (cm) leaf width (cm2) leaf area sugar content (%) catalase (unit)1 anthocyanin (mg/100 g) total chlorophyll (mg/g) carotenoid (mg/l) antioxidant capacity (dpph%) no netting 25.25 c 3.92 c 13.50 b 23.52 c 1.65 c 2.11 c 0.14 c 14.92 c 6.04 b 72.19 c 6.66 b 0.04 b 1.37 b 0.85 b 0.77 b 9 a yellow 39.33 a 16.50 a 27.17 a 82.05 a 5.75 a 7.89 a 0.55 a 21.17 a 8.50 a 138.4 a 9.68 a 0.04 b 2.62 b 2.90 a 2.60 a 2 b red 33.92 b 11.50 b 19.63 ab 50.25 b 3.52 b 4.82 b 0.34 b 19.17 b 7.33 ab 106.5 b 10.33 a 0.10 a 4.77 a 3.09 a 2.94 a 5 ab green 33.33 b 10.88 b 18.83 ab 45.74 b 3.20 b 3.84 bc 0.27 bc 18.00 b 6.46 b 88.92 bc 8.50 ab 0.03 b 1.20 b 2.77 ab 2.50 a 9 a abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 29 icantly influenced by net type at the p<0.05 level and by plant species at the p<0.01 level, but the interac­ tion of ‘net type × plant species’ was insignificant for this trait (table 2). the highest degree brix was relat­ ed to the red net (10.33%) and yellow net (9.68%) and the lowest to control (6.66%) (table 3). means comparison for the effect of plant species indicated that c. variegatum had higher brix of 13.50% (table 4). the effect of net type was significant (p<0.01) on catalase enzyme, but this enzyme content was not influenced by plant species and ‘net type × plant species’ (table 2). the highest catalase enzyme con­ tent was obtained from the red netting showing insignificant differences from other treatments (table 4). anova showed that the interactive effect of ‘net type × plant species’ was significant (p<0.01) on peroxidase enzyme, but the effect of shading net and plant species was not significant (table 1). the highest peroxidase enzyme content was obtained from ‘no net × a. commutatum’ and the lowest from ‘red net × c. variegatum’, ‘green net × a. commuta‐ tum’, ‘green net × c. variegatum’, ‘yellow net × a. commutatum’, and ‘no net × c. variegatum’ (table 5). anthocyanin content was significantly (p<0.01) influenced by net type, plant species, and ‘net type × plant species’ (table 2). means comparison indicated for this trait (p<0.05) (table 1). the highest shoot fresh and dry weight was obtained from the yellow netting (table 3) as means comparison for the effect of shade net type indicated. likewise, means com­ parison for the interaction of netting type and plant species showed that ‘yellow netting × c. variegatum’ produced the highest shoot fresh and dry weight without any significant differences from that of ‘red netting × c. variegatum’ and ‘green netting × c. varie­ gatum’. the lowest shoot fresh and dry weight was obtained from ‘no netting × a. commutatum’ and ‘green netting × a. commutatum’ (table 5). the effect of net type was significant on root fresh and dry weight at the p<0.01 level and the interaction of ‘net type × plant species’ was significant for these traits at the p<0.05 level (table 1). means comparison indicat­ ed that the highest root fresh and dry weights were obtained from the yellow netting and the lowest were obtained when the plants were barely exposed to radiation (table 3). according to means compari­ son for the interactive effect of the treatments, ‘yel­ low net × a. commutatum’ produced the highest root fresh and dry weight whilst the lowest root fresh and dry weights were obtained from ‘no net × c. variega‐ tum’, ‘no net × a. commutatum’, and ‘green net × a. commutatum’ (table 5). anova indicated that degree brix (°bx) was signif­ table 4 ­ means comparison for the effect of plant species on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. plant species final height (cm) growth (cm) leaf no. shoot fresh weight (g) shoot dry weight (g) leaf length (cm) leaf width (cm) leaf area (cm2) sugar content (%) antho­ cyanin (mg/100 g) antioxidant capacity (dpph%) codaeum variegatum 40.96 a 17.15 a 29.13 a 84.72 a 5.93 a 21 a 8.98 a 128.08 a 13.50 a 3.02 a 5 b aglaonema commutatum 24.96 b 4.25 b 10.44 b 16.05 b 1.12 b 15.63 b 6.19 b 74.93 b 4.08 b 1.97 b 7 a table 5 ­ means comparison for the interactive effect of ‘netting type × plant species’ on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. 1 peroxidase enzyme unit in μm /h2o2 consumed/min/mg. plant species final height (cm) growth (cm) shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) peroxidase (unit)1 ‘no netting × codaeum variegatum’ 29.33 c 6.33 cd 44.32 bc 3.10 b 2.09 c 0.15 c 0.34 b ‘no netting × aglaonema commutatum’ 21.17 d 1.50 d 2.72 d 0.19 c 2.14 c 0.15 c 0.61 a ‘yellow netting × c. variegatum’ 49.33 a 25.33 a 118.2 a 8.27 a 6.68 ab 0.47 ab 0.43 ab ‘yellow netting × a. commutatum’ 29.33 c 7.67 c 45.93 b 3.22 b 9.10 a 0.64 a 0.40 b ‘red netting × c. variegatum’ 42.83 b 17.83 b 88.31 a 6.18 a 5.20 bc 0.36 bc 0.33 b ‘red netting × a. commutatum’ 25.00 d 5.17 cd 12.19 cd 0.85 c 4.43 bc 0.31 bc 0.45 ab ‘green netting × c. variegatum’ 42.33 b 19.08 b 88.10 a 6.17 a 5.21 bc 0.36 bc 0.39 b ‘green netting × a. commutatum’ 24.33 d 2.67 cd 3.38 d 0.24 c 2.49 c 0.18 c 0.33 b 30 adv. hort. sci., 2020 34(1): 25­33 that the highest anthocyanin content was obtained from the red net (table 3), but the other treatments did not differ significantly to one another. according to means comparison for the effect of plant species on anthocyanin content, the highest content was observed in c. variegatum (table 4). also, means comparison for the interactive effect of ‘net type × plant species’ on anthocyanin content indicated that ‘red net × a. commutatum’ had the highest antho­ cyanin content and ‘green net × a. commutatum’ and ‘no net × a. commutatum’ exhibited the lowest one (fig. 1). the results of anova showed that the effect of net type was significant (p<0.05) on total chlorophyll content, but the effect of plant species, net type, and ‘net type × plant species’ was insignificant on chloro­ phyll a and b and so was the effect of plant species and ‘net type × plant species’ on total chlorophyll (table 2). means comparison for the effect of net type on total chlorophyll (table 3) indicated that the highest total chlorophyll was related to the red and yellow nets and the lowest to no­net treatment. shading net type influenced carotenoid content of plants significantly (p<0.01), but the trait was not sig­ nificantly affected by plant species and ‘net type × plant species’ (table 1). means comparison showed that the plants grown under the red nets had a high­ er carotenoid content but without any significant dif­ ferences from those grown under the yellow and green nets. the lowest carotenoid content was relat­ ed to control (table 3). the results of anova showed that the interaction of ‘net type × plant species’ was significant (p<0.01) for flavonoid content, but the effect of net type and plant species was insignificant on this trait (table 2). the highest flavonoid content was obtained from ‘green net × c. variegatum’ and the lowest from ‘red net × c. variegatum’ (fig. 2). antioxidant capacity was significantly influenced by netting type and ‘netting type × plant species’ at the p<0.01 level and by plant species at the p<0.05 level (table 2). a. commutatum plants grown under the yellow net and control plants had the highest antioxidant capacity, while the lowest capacity was related to the yellow net (table 3). also, it was found that a. commutatum had a higher antioxidant capaci­ ty than c. variegatum (table 4). means comparison for ‘net type × plant species’ revealed that the high­ est antioxidant capacity was related to ‘no net × a. commutatum’ and the lowest to ‘yellow net × c. var‐ iegatum’ and ‘yellow net × a. commutatum’ (fig. 3). 4. discussion and conclusions based on our results the highest plant height was related to ‘yellow net × a. commutatum’. in a study on the effect of colored nets on cordyline, kumar fig. 1 ­ the effect of ‘netting type × plant species’ on anthocya­ nin content. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. fig. 2 ­ the effect of ‘netting type × plant species’ on flavonoid content. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. fig. 3 ­ the effect of ‘netting type × plant species’ on antioxidant capacity. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 31 gaurav et al. (2016) stated that plants grown under colored shade nets exhibited variable growth due to the spectral effect on plant growth. they reported that the cordyline plants grown under white and red shade nets were taller than the control plants (not exposure to shade nets). it has been reported that both red and yellow shade nets enhanced the vege­ tative traits of aralia including stem length and thick­ ness, petals, and leaf dimensions and generally increased the yield of commercial cut flowers (oren­ shamir et al., 2001; shahak, 2008). shahak et al. (2016), also, found that the yellow shading net induced vegetative growth of pittosporum. the high­ est growth rate was observed in the plants exposed to the yellow nets. it has been argued that yellow nets outperform red nets in inducing vegetative growth, probably because of the inductive effect of supplementary artificial green light (oren­shamir et al., 2003; kim et al., 2004). in a four­year research study on cut flowers in besor research station (2000­ 2003), an increase was reported in stimulatory capac­ ity and in growth under yellow and red nets (shahak, 2008; ovadia et al., 2009). there is a report that let­ tuce produced the highest number of leaves under colored nets (pearl and red nets) and the lowest number in control (no net application) (ilić et al., 2017). it can be concluded that different plant species differ in their growth and development responses to the spectra generated by different col­ ored nets. leaf area is a crucial parameter for growth. it is defined as a plant’s capacity to synthe­ size dry matter in terms of radiation use rate and photosynthesis rate. kumar gaurav et al. (2016) showed that leaf area of cordyline was higher under colored shade netting than control (no shade net application). colored nets influenced both the length of growth period and morphological traits of lettuce so that they enhanced leaf area index and shortened the length of growth period significantly (ilić et al., 2017). in the present study, the yellow net resulted in higher fresh weight than the other nets and control. in addition, the highest plant growth was related to the yellow net, implying the impact of yellow nets on quantitative and qualitative traits of plants. this leads us to the conclusion that colored shade nets, espe­ cially yellow nets, can enhance plant biomass. in kumar gaurav et al.’s study (2016), the highest leaf fresh weight (85.88 percent higher in the red net than in the control) and leaf dry weight were obtained from the red net and overall, the colored nets had the strongest impact on this trait when compared to control (no net application). brix is a measure of sugar content of the solution and depends on radiation diffraction. it represents the percentage of solid material weight of a solution to the total weight of the solution. our results revealed that flowers grown under the red and yel­ low shade nets had higher degree brix and this may be related to the stimulatory effect of artificial green light under the yellow nets (kim et al., 2004). hydrogen peroxide is the most stable form of reac­ tive oxygen species. it has been suggested that hydrogen peroxide is toxic to cells. catalase and per­ oxidase play a crucial role in inhibiting the accumula­ tion of hydrogen peroxide. these enzymes are abun­ dant in aerobic microbes, but anaerobic microbes lack them (singh, 2003). it has been documented that peroxidases are involved in many cell processes such as auxin metabolism, wood formation, traverse link­ ages in plant cell walls, response to environmental stresses, and so on (yamasaki et al., 1997). thus, the higher catalase and peroxidase enzyme content in plant species plays a considerable role in inhibiting the accumulation of hydrogen peroxide in plants. a laboratory trial has shown that three properties of light color, intensity and duration affect plant growth so that red/infra­red ratio is dictated by the duration and photo flux of radiation treatments and influences anthocyanin development and synthesis significantly (mancinelli, 1990). lefsrud et al. (2008) reported that anthocyanin content of lettuce was increased in plants exposed to red led light. in the present study, plants grown under the red shade nets exhibited the highest anthocyanin content. at intense radiation, chlorophyll degradation rate in plant leaf exceeds its synthesis rate, resulting in the loss of chlorophyll content due to the inhibition of chloroplast formation (gonçalves et al., 2001; fu et al., 2012). it has been documented that colored nets increase chlorophyll content in plants. for exam­ ple, alkalai­tuvia et al. (2014) studied the effect of colored nets on peppers and reported that the chlorophyll content of the peppers grown under the pearl nets was significant higher than that of the pep­ pers grown under the black nets. the plants grown under the red nets had a higher carotenoid. it has been reported by tinyane et al. (2013) and selahle et al. (2014) that carotenoid con­ tent of tomatoes was increased under red and pearl shade nets. kong et al. (2012) reported that the yel­ low net resulted in morphological changes and leaf carotenoid increase in peppers versus the red net, which may relate to the increase in green light con­ adv. hort. sci., 2020 34(1): 25­33 32 tent under the yellow net. leaf carotenoid content was higher in lettuces grown under colored shade nets than control (no net) in ilić et al.’s study (2017), which is consistent with our findings. phytochemical biosynthesis mostly depends on light quantity and quality as was observed in lettuce plants grown under black nets. in a study, plants grown under pearl nets had significantly higher total phenol content, flavonoids, and antioxidant proper­ ties than those grown under other nets (ilić et al., 2017). in another study, an increase was observed in post­harvest flavonoid content in oregano, marjo­ ram, and coriander under pearl nets (buthelezi et al., 2016). although phytochemical content decreases slightly after harvest, a high level of post­harvest phy­ tochemical accumulation enables plants to maintain phytochemical quality in post­harvest period (buthelezi et al., 2016). the accumulation of antioxi­ dant compounds in green plants depends on many parameters such as temperature, light quantity and quality, cultivar, growing season, and metabolic fac­ tors (miller et al., 2010). the control of radiation quality by the red and pearl photo­selective netting resulted in keeping post­flowering antioxidant activi­ ty in vegetables (kong et al., 2013). kong et al. (2013) reported that peppers exhibited an elevated level of antioxidants under yellow nets implying that yellow and pearl nets were likely to enhance plant resis­ tance to biotic stresses. it seems that plants respond differently to different light spectra and plant response to the elevated level of antioxidant capacity depends on the type of colored nets. the results show that among the studied colored shade nets, the yellow net outperformed the other nets in improving the vegetative capacity of the stud­ ied plants. also, the highest anthocyanin, carotenoid, and catalase contents were obtained from the red net and the highest degree brix and total chlorophyll from the red and yellow nets. overall, the application of the colored nets was more desirable for the plants than their non­application. it has been documented that the absorption rate of yellow­colored glass is higher in 360­200 nm (blue and violet) range that is lowly important radiation for photosynthesis than in 560­760 nm range that is photosynthetically impor­ tant (haghshenas and ghiabaklou, 2009) because the latter range is severely intercepted by chlorophyll and increases photosynthesis. this can be a reason for the higher efficiency of the yellow netting. the absorption rate of violet to yellow range of visible light (360­600 nm) by red glass is very similar to yel­ low glass (haghshenas and ghiabaklou, 2009). this explains the change in plants’ behavior to the radia­ tion passing through the red net because it seems that the penetration of photosynthetically active radiation through red nets (i.e. red and orange por­ tions of visible light) contributes to important plant functions such as photosynthesis and increases bio­ mass. references alkalai­tuvia s., goren a., perzelan y., weinberg t., fallik e., 2014 ­ the influence of colored shade nets on pepper quality after harvest ‐ a possible mode‐of‐ action. ­ agric. forest., 60: 7­18. buthelezi m.n.d., soundy p., jifon j., sivakumar d., 2016 ­ spectral quality of photo‐selective nets improves phytochemicals and aroma volatiles in coriander leaves (coriandrum sativum l.) after postharvest storage. ­ j. photochem. photobiol. b, biol., 161: 328­334. chance b., maehly a.c., 1955 ­ assay of catalase and peroxidase, pp. 764­775. ­ in: horecker b.l. 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of horticultural science, college of agriculture, shiraz university, p.o.box 65186‐71441 shiraz, iran. key words: citrus aurantifolia, citrus aurantium, germination, in vitro, rootstock. abstract: the aim of this research was to determine the best time to harvest the fruits for seed production which would ultimately lead to the production of citrus rootstocks of optimum quality. the sour orange and mexican lime fruits were harvested on 7 and 5 occasions, respectively. the very first fruits were harvested 80 days after flowering and subsequent harvests were gathered every 30 days. an in vitro experiment was carried out in a completely random­ ized design, with four replications and 20 seeds in each replication. based on fruit growth curve the time of fruit harvest affected seed germination (percent­ age and rate) and seedling growth (stem and root length, fresh and dry weight of stems, roots and leaves). the results showed that the best time to harvest the fruits of sour orange and mexican lime was 230 and 170 days after flower­ ing, respectively, which led to maximum seed germination (mexican lime 100% and sour orange 85%) and seedling growth. the highest root, stem and leaf fresh and dry weight was also obtained at 230 and 170 days after flowering in sour orange and mexican lime respectively. 1. introduction citrus is an important genus of subtropical fruit trees, with substantial roles in the economy of many countries (iglesias et al., 2007; tercan and dereli, 2020). among different citrus species, the fruits reach maturity at different times of the season and as a result, the harvest time of fruits usually lasts several months (orbović et al., 2011; deterre et al., 2021). it has been reported that a variety of physiological factors in citrus fruits, including fruit color change, sugar concentration and acid content affect the quality and marketability of fruits. however, there is insufficient infor­ mation about the effects of seasonal changes on the fruit seeds, their ger­ mination potential and seedling vigor (moulehi et al., 2012; orbović et al., 2013). many citrus cultivars that are selected for the production of high quality fruits do not have suitable root systems and, thus, it is highly rec­ (*) corresponding author: shekafan@shirazu.ac.ir citation: jokari s., shekafandeh a., 2021 ­ effect of har‐ vest time on seed germination and seedlings growth of sour orange and mexican lime under in vitro conditions. ­ adv. hort. sci., 35(2): 175­ 182 copyright: © 2021 jokari s., shekafandeh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2021 accepted for publication 5 may 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10628 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 175­182 176 ommended that these cultivars be grafted onto desir­ able rootstocks (zhu et al., 2020). previous reports suggest that more than 20 traits of a grafted plant are affected by the rootstock, including drought tol­ erance, nutrient uptake, growth vigor, tree size, root penetration depth, tolerance to disease, amount of yield, fruit size and quality (zhu et al., 2013; khoshbakht et al., 2015). in southern regions of iran, among the various citrus rootstocks, mexican lime (citrus aurantifolia l.) and sour orange (citrus auran‐ tium l.) are the most widely used due to their special characteristics. mexican lime has high growth vigor and yield (haji vand and lee abdullah, 2012) and sour orange is resistance to root rot, tolerance to cal­ careous and salinity soils and has deep root system (louzada et al., 2008; etehadpour et al., 2020). citrus rootstocks are mostly propagated by seed. seed germination is a vital stage in the plant life cycle (bakhshandeh et al., 2017). citrus growers often face many problems such as poor seed germi­ nation and great mortality rate of seedlings during the nursery stage (dilip et al., 2017; chaudhary et al., 2019). citrus seeds usually do not have dormancy, they can germinate quickly and are considered as short­lived seeds (khopkar et al., 2017), their germi­ nation rate decreases as the seeds lose moisture (hassanein and azooz, 2003). therefore, the seeds have low capacity of storability and should be sown quickly after extraction from the fruit (khopkar et al., 2017). nursery operations to establishment of plant are very dependent on the high germination rate and growth of seedlings (alouani and bani­aameur, 2004) so that they will reach the proper size for a short time and be ready for grafting, which can ultimately reduce the cost of growing grafted citrus plants (girardi et al., 2005). an important factor that determines seed quality is the physiological maturity of seeds. maximum ger­ mination (%) is reached when seeds are at their phys­ iological maturity stage, which is associated with an optimum presence of nutrients in seeds to support the growth of seedlings with good vigor (murrinie et al., 2019). it has been also reported that the growth and maturity of fruits can affect seed germination percentage (abbasi and heidari, 2011; mombeini et al., 2011; bareke, 2018). according to our knowledge, there is a lack of information about the best stage and time to harvest the fruits of sour orange and mexican lime for producing seedling rootstocks. the aim of this research was to assess the effects of fruit harvest time, taking into account the fruit growth curve of sour orange and mexican lime, on seed ger­ mination and seedling growth vigor under in vitro condition. 2. materials and methods plant materials and research site this research was carried out in the laboratory of plant tissue culture and biotechnology, faculty of agriculture, shiraz university. the seeds of two citrus species mexican lime and sour orange were collected from an orchard belonging to the citrus research institute, larestan (27.66° n, 54.38° e, altitude 900 meters above sea level). the maximum and minimum temperatures and rainfall on an average of ten years in the region are 43.5°c, 4°c and 203 mm, respective­ ly. the climate is characterized by mild winters and warm summers. seed samples were taken from fruits harvested at different times during the growing season. sampling began from 80 days after flowering (june 1) when the seeds formed in fruits. subsequent samples were taken on a monthly interval. the total time span con­ sidered for the harvest of sour orange and mexican lime were 260 and 200 days after flowering, respec­ tively. all sour orange fruits were harvested from a 10­year­old tree and all mexican lime fruits were from an 8­year­old tree. the fruits were harvested from the same tree throughout the experiment. after harvesting the fruits, they were transferred to the laboratory in order to measure their diameter, length, and weight. data were reported as the mean value of 10 fruits. effect of harvest time on in vitro seed germination the treatments included 7 and 5 harvest times for sour orange (80, 110, 140, 170, 200, 230 and 260 days after flowering) and mexican lime (80, 110, 140, 170 and 200 days after flowering) species, respective­ ly. after separating from the fruits, the seeds were soaked in water for 12 hours and then washed with water and a few drops of dishwashing liquid for a few minutes to remove the gelatin­like material around the seeds. then, the protective layer of the seeds was removed. the seeds were placed in vials contain­ ing water and a few drops of dishwashing liquid for 15 minutes to remove surface contaminants. after that, they were disinfected under sterile conditions by immersing in 70% alcohol for 30 seconds and then in 15% common bleach (containing 5.25% sodium jokari and shekafandeh ‐ effect of harvest time on in vitro seed germination and growth 177 hypochlorite) for 15 minutes. then, the seeds were washed three times in sterile distilled water. the seeds were then cultured on liquid ms medium (murashige and skoog, 1962) without any plant growth regulator and a filter paper was used to pre­ vent them from being submerged. the mentioned medium was fortified with 30 g/l of sucrose, and the ph of the medium, before autoclaving (at 121 ° c and 15 psi) was regulated on 5.8. the jars containing the cultured seeds were taken to the growth chamber in dark conditions (25±2°c). within an initial period of 30 days, the percentage and rate of germination were measured by the maguire (1962) method. germination (%) = snumber of germinated seeds x 100 total number of seeds) germination rate = number of seeds until n­1 day number of days in vitro plantlets growth after 30 days in darkness, all cultures were main­ tained in a growth room at 25±1°c under a 16/8 h (light/dark) photoperiod of 45­50 μmol m­2 s­1 irradi­ ance provided by cool white fluorescent tubes and with 55­60% relative humidity. after 4 weeks, we measured growth indices such as stem and root length, number of leaves, fresh and dry weight of stems, roots and leaves experimental design and data analysis the experiment was performed in a completely randomized design with 4 replications. in each repli­ cation, 20 seeds were checked for the percentage and rate of germination. then, within the germinated seeds, 5 seedlings per replication were used for mea­ suring growth characteristics. statistical analyzes of the data were carried out using sas 9.4 software and mean comparison was performed using lsd (p≤ 0.05). microsoft excel 2013 was used to draw the fig­ ures. 3. results the analysis of variance (tables 1 and 2) showed that the fruit harvest stage significantly affected seed germination indices (germination rate and percent­ age) and seedling growth (root and stem length, number of leaves, fresh and dry weight of leaves, table 1 ­ analysis of variance of the effect of harvest time on sour orange and mexican lime on seed germination and seedling growth in vitro condition **, significant at the level of 1 % probability using lsd. source of variance df germination percentage germination rate root length stem length number of leaves sour orange harvest time 6 5167 ** 0.01 ** 84.17 ** 40.62 ** 30.12 ** error 21 72.32 0.00006 3.82 1.49 0.87 cv (%) ­ 10.18 17.34 15.98 17.15 6.76 mexican lime harvest time 4 3870 ** 0.004 ** 30.62 ** 41.82 ** 19.17 ** error 15 120 0.00008 3.55 1.17 1.88 cv ­ 10.12 10.86 9.32 14.59 11.28 table 2 ­ analysis of variance of the effect of harvest time on growth characteristics of sour orange and mexican lime seedlings in vitro condition **, significant at the level of 1 % probability using lsd. source of variance df root fresh weight stem fresh weight leaf fresh weight root dry weight stem dry weight leaf dry weight sour orange harvest time 6 1.04** 0.214** 0.926** 0.0796** 0.102** 0.100** error 21 0.0003 0.0002 0.0004 0.00009 0.00005 0.0002 cv (%) ­ 10.21 2.4 5.85 12.95 3.38 4.25 mexican lime harvest time 4 1.736** 0.268** 1.176** 0.141** 0.031** 0.055** error 15 0.0004 0.0004 0.0005 0.0002 0.0002 0.0002 cv ­ 8.19 7.82 7.37 4.35 5.48 7.01 adv. hort. sci., 2021 35(2): 175­182 178 roots and stems) in both studied species (p≤0.01). fruit growth characteristics both citrus fruits species exhibited a simple sig­ moid growth curve based on fruit dimensions and weight (fig 1 a and b respectively). the growth curve in the first stage, i.e. 140 and 110 days after flower­ ing showed a slow growth in sour orange and mexican lime, respectively. fruit growth at this stage is mostly a manifestation of cell division. the fruits in the second stage, from 140 to 200 days after flower­ ing in sour orange and from 110 to 170 days after flowering in mexican lime showed rapid growth and cell enlargement and water accumulation in fruit tis­ sues. in the third stage, from 200 to 260 after flower­ ing in sour orange and from 170 to 200 after flower­ ing in mexican lime, fruit growth had reduced growth rate and, accordingly, the process of non­climacteric ripening began in fruits. in vitro germination characteristics the highest percentage of seed germination was observed when sour orange harvested at 230 and mexican lime at 170 days after flowering (fig. 2 a and d), both of which are significantly higher than final stage of harvest. the lowest germination percentage of sour orange and mexican lime was observed in the seeds of fruits harvested at 170 and 80 days after flowering, respectively (fig. 3 a and c). it is also note­ worthy that the seeds obtained from sour orange in the first three stages, namely 80, 110 and 140 days after flowering, were not able to germinate. by increasing the harvest time, the germination rate increased in both species. in sour orange, the highest germination rate occurred at 230 days which was significantly 3.13 times higher than 170 days after flowering. in mexican limes, the maximum seed germination rate occurred at 170 days which was sig­ nificantly increased 1.57 times compared to 80 days after flowering. the lowest germination rate was observed in seeds of fruits harvested 170 days after flowering in sour orange and 80 days after flowering in mexican limes (fig. 3 b and d) in vitro seedling growth indices fruit harvest time in both citrus species had a sig­ nificant effect on seed growth indices (stem length, fig. 1 ­ fruit growth curve based on fruit weight, diameter and length of sour orange (a) and mexican lime (b). fig. 2 ­ in vitro seed germination and plantlet growth. germinated seeds of mexican lime (a) and sour orange (d) in darkness, 170 and 230 days after flowering respec­ tively. plantlets growth of mexican lime (b, c) and sour orange (e, f) after 4 weeks in light, 140 and 230 days after flowering respectively. jokari and shekafandeh ‐ effect of harvest time on in vitro seed germination and growth 179 root length and number of leaves) (fig. 4). in sour orang, the uppermost number of leaves were observed in the produced seedlings from the seeds of fruits that had been harvested 260 days after flow­ ering which was significantly different from the 170 and 200 days (fig. 4 c). however, there was no signif­ icant difference between the two treatments of 260 and 230 days after flowering. in mexican lime, the produced seedlings from the seeds of the fruits harvested 170 days after flowering indicated the higher number of leaves (9 leaves/ seedling) which was significantly different compared to the produced seedlings from 80 (3.75 leaves /seedling) and 200 (6.75 leaves/seedling) days after flowering (fig 4 f). in sour orange, the in vitro seedlings of the 230 day after flowering showed the highest root length (10.75 cm) which did not show significant different compared to 260 days after flowering. regarding mexican lime, the highest root length occurred in the seedlings of 170 days after flowering (12.5 cm) which was significantly higher than those from 80 days after flowering. (fig. 4 a and d). in both sour orange and mexican lime seedlings, stem length increased with increasing the harvest time. in sour orange, stems of the seedlings related to 260 days after flowering showed the highest length of 7.25 cm which was not significantly different compared to 230 days after flowering (fig 2 b and c). in mexican lime, the seedlings of the 170­days harvest treatment indicat­ ed the maximum stem length of 11.75 cm which was significantly higher than the other harvest times (fig 2 e and f) (fig. 4 b and e). the results showed that a significant increase occurred in the fresh and dry weight of seedlings by increasing the harvest time. in sour orange, the pro­ duced seedlings in 230­day harvest treatment time indicated the highest fresh and dry weights of roots, stems and leaves which were significantly greater than other treatments (except in root fresh weight and leaf dry weight that was not significant different between 230 and 260 days after flowering) (fig. 5 a­ f). regarding mexican lime seedlings, with increasing the harvest time the fresh and dry weight of all organs increased. the highest fresh and dry weights of roots, stems and leaves were occurred in the seedlings of the 170­day harvest period, however with increasing harvest time to 200 days all mea­ sured traits decreased (fig. 6 a­f). 4. discussion and conclusions in this research, different stages of fruit harvest affected seed and rate of germination and seedling fig. 3 ­ effect of harvest time on germination percentage and germination rate of sour orange (a, b and mexican lime (c d), under in vitro conditions. means with the same let­ ter are not significantly different at 1% probability using lsd test. fig. 4 ­ effect of harvest time on seedlings root length, stem length and leaf number of sour orange (a­c) and mexican lime d­f) under in vitro conditions. means with the same letter are not significantly different at 1% pro­ bability using lsd test. 180 adv. hort. sci., 2021 35(2): 175­182 growth in both species (sour orange and mexican lime). it has been demonstrated that by increasing the number of days after flowering to 230 days in sour orange and 170 days in mexican lime, germina­ tion and seedling growth factors improved (murti and upreti, 2003; kondo et al., 2004). the results showed that the fruit growth of sour orange and mexican lime based on fruit weight and dimensions have a single sigmoid growth, which is divided into three stages (tadeo et al., 2008). in the first phase of fruit growth, which is approximately between the flowering onset and june­drop, the rate of fruit growth is slow but cell division is high. the second is a period of rapid growth in which the size of the fruit increases through cell enlargement and accumulation of water. as these two growth stages end, the growing fruits change from the consumption phase to the storage stage (mehouachi et al., 1995). in the third stage, growth stops and the fruits under­ go a non­climacteric ripening process (mehouachi et al., 1995; tadeo et al., 2008). regarding fruit growth curve, our results showed that in order to achieve quality seeds and produce strong seedlings, the best time to harvest sour orange and mexican lime fruits was in the beginning of third stage of fruit growth, about 230 and 170 days after flowering, respectively. early harvest may reduce seed quality due to the par­ tial development of basic seed structures, while late harvest may lead to reduced seed quality because of aging. mombeini et al. (2011) reported that the high­ est seed germination occurred in sour orange at 250 days after flowering before full fruit ripening and then reduced until ripening, which is in line with our results. it has been documented that one of the possible reasons for the differences in the physiological potential of seeds is related to changes in the embryo and endosperm of seeds at different stages of growth and development (tekrony, 2003). the seed reaches its maximum potential for germination at the stage of physiological maturity, when more nutrients are available to support seedling growth and vigor (murniati et al., 2008). however, murniati et al. (2008) also reported that papaya seeds extract­ ed from fruits before full ripening (30­40% yellow color of the fruit skin) had maximum germination and growth. another study indicated that physiological maturi­ ty is a genotypic trait that is influenced by environ­ mental factors. environmental conditions during seed growth and maturity, including temperature, envi­ fig. 5 ­ effect of harvest time on seedlings root, stem and leaf fresh weight (a­c) and dry weight (d­f) of sour orange under in vitro conditions. means with the same letter are not significantly different at 1% probability using lsd. fig. 6 ­ effect of harvest time on seedlings root, stem, and leaf fresh weight a­c and dry weight (d­f) of mexican lime. means with the same letter are not significantly different at 1% probability using lsd test. jokari and shekafandeh ‐ effect of harvest time on in vitro seed germination and growth 181 ronmental stresses, and nutrient deficiencies, affect seed quality (mahesha et al., 2001). in the process of seed development, various mechanisms occur from fertilization to physiological maturity and into the phases of cell division, development and then the phase of nutritional storage in seeds. there is usually an increase in the dry weight of seeds and finally a decrease in seed moisture due to changes in cell membrane structure and enhanced levels of enzyme synthesis, necessary for successful seed germination (bareke, 2018). theoretically, it can be said that dur­ ing physiological maturity, the germination percent­ age of seeds increases and reaches a maximum when the seeds reach their maximum dry weight (orbović et al., 2013). in another study, the relationship between the germination percentage of grapefruit seeds and sour orange were evaluated from fruits harvested at the beginning of the season. studies have shown that when a seed reaches physiological maturity, the seed vigor becomes consistently high throughout the harvest season (fucik, 1978). the produced seedlings from the seeds extracted from fruits at 230 and 170 days after flowering in sour orange and mexican lime, respectively, had bet­ ter growth than the seedlings produced from the har­ vested seeds at the final stage of fruit maturity. accordingly, seedling growth indices such as fresh and dry weight stems, root and leaves were signifi­ cantly higher at a stage before the last stage of har­ vest. it has also been reported that structural and chemical changes in fruits and seeds are associated with germination vigor and seedling growth indices (abbasi and heidari, 2010; mombeini et al., 2011). orbović et al. (2013) stated that the ability of grape­ fruit seeds to germinate at the end of the season is a physiological manifestation of a change in hormonal balance in the fruits, which is largely associated with a slight decrease in abscisic acid levels in the seed. it has been also reported that valencia orange seeds showed a great peak in aba (abscisic acid) concen­ tration at 150 days after flowering (in stage ii) and this increment obviously decreased at 188 days after flowering while, amount of iaa (indole­3­acetic acid) increased (kojima 1995). it is possible the better seedlings growth in the beginning of third stage of growth be due to the increase of iaa content of the seeds. in conclusion, we found that the fruit harvest stage can have a significant effect on seed quality for rootstock production in citrus. fruit growth (length, diameter and weight) was affected by harvest time in sour oranges and mexican lime. the best seed germi­ nation characteristics were obtained at the onset of 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doi: 10.36253/ahsc­12938 effect of different nitrogen forms and bio­treatments on the growth and seed yield of downy safflower (carthamus lanatus) h.a. ashour (*), s.e.a. esmail, a.b. el­attar department of ornamental horticulture, faculty of agriculture, cairo university, giza, egypt. key words: arbuscular mycorrhiza fungi, downy safflower, nitrogen fertilization, trichoderma viride, vermicompost. abstract: a field experiment was carried out to investigate the effect of differ­ ent nitrogen forms and some biotreatments (trichoderma viride, vermicompost and arbuscular mycorrhiza fungi) alone or in combination on vegetative growth, seed yield and some chemical traits of downy safflower (carthamus lanatus l.). nitrogen was supplied as ammonium sulfate, ammonium nitrate and urea at the rates (5, 3 and 2 g/plant, respectively). bio treatments included trichoderma viride, vermicompost and arbuscular mycorrhiza fungi. the results showed that all nitrogen forms significantly increased the plant growth and yield, pigments content, and total carbohydrates in leaves and seeds, as well as n, p and k%, total phenols and oil content in seeds. all bio treatments signifi­ cantly increased the tested parameters compared to control. the integration of ammonium sulfate with t. viride was the most effective treatment since deter­ mined the highest increases of the tested traits. results showed that for enhancing downy safflower plant growth, and nutritional values of seed, the combined treatment of t. viride at 5 ml/plant and ammonium sulfate at 5 g/plant is recommended. 1. introduction carthamus lanatus l. (also called downy safflower, woolly distaff this­ tle or saffron thistle) is an erect spiny biennial plant native of the mediterranean region. it is closely related to safflower, which is in the same genus. downy safflower is reported to be sudorific (sweat inducing), fever­reducing and anthelmintic (hellwig, 2004; ditomaso et al., 2017; adel el­gazzar et al., 2019), previous studies revealed its importance due to different components of diverse chemical nature such as flavonoids, sesquiterpenes glycosides, lipids, aromatic acids, sterols, triterpenes , volatiles alkaloids, tannins and saponins (abu el­khair, 2020). plant nutrition is one of the most essential factors which increase plant production. nitrogen (n) is the most recognized in plant as it is pre­ sent in the structure of the protein molecule and plays a vital role in syn­ (*) corresponding author: hossam.ahmed@agr.cu.edu.eg citation: ashour h.a., esmail s.e.a., el­attar a.b., 2023 ­ effect of different nitrogen forms and bio‐treat‐ ments on the growth and seed yield of downy saf‐ flower (carthamus lanatus). ­ adv. hort. sci., 37(2): 185­196. copyright: © 2023 ashour h.a., esmail s.e.a., el­attar a.b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 march 2022 accepted for publication 23 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12938 http://en.wikipedia.org/wiki/safflower http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 185­196 186 thesis of plant compositions via the action of various enzymes activities and protein synthesis (taiz and zeiger, 2002). nitrogen has an important role in plant metabolism that impacts quantitative and qualitative plant production by enhancing the growth and stimu­ lating the essential processes which leads to increase the active substances. ammonium sulfate (as), ammonium nitrate (an) and urea are the main forms of inorganic n fertilizers and are extensively utilized in modern agriculture. as application was better than an and urea for increasing vegetative growth yield parameters, chlorophyll content, npk % in seeds and seed oil % of sunflower and jojoba (el mantawy, 2017; el sayed, 2020; hegab et al., 2021). trichoderma is a genus of saprotrophic fungi and a widespread component of the soil rhizosphere; it has been reported to enhance plant growth and to control many of plant diseases (colla et al., 2014). one of the well­known stimulatory effects of trichoderma on plants is the ability to dissolve phos­ phate through acidification, chelation or redox activi­ ty to improving the utilization by plants (mansour et al., 2021). the benefits of trichoderma species in stimulating plant growth can be realized via various mechanisms including boost nutrient uptake, solubi­ lization, sequestration of inorganic nutrients and enhancement of root hair development (harman, 2006; lorito et al., 2010). trichoderma spp. promote plant hormone synthesis that improve root growth and root hair formation which lead to more efficient use of nitrogen, phosphorus, potassium and micronu­ trient (mastouri et al., 2010). moreover, trichoderma is able to produce metabolites with hormonal activi­ ties such as indole­3­acetic acid (contreras­cornejo et al., 2011). the positive impact of t. viride inocula­ tion on physiological and biochemical features of plants has been reported by many authors. the fun­ gus is able to improve growth and yield parameter (ghoneem et al., 2019), promote photosynthetic pig­ ments (kumar et al., 2015; ghoneem et al., 2019), enhance nutrient status in leaves and roots (metwally, 2020), increase essential oil and total phe­ nol content (shaikh et al., 2019; hassanin et al., 2020; sanei and razavi, 2018; ghoneem et al., 2019) and promote peroxidase activity. vermicompost is an organic product that is obtained from biodegradation and stabilization of organic waste via the interaction between earth­ worms and microorganisms, lead to break up organic matter residues into fine particles (ndegwa and thompson, 2001; campitelli and ceppi, 2008). it has a favorable effect on the physical and chemical struc­ ture of soil as well as plant growth (bachmana and metzger, 2008). additionally, it induces and boosts the absorption of nutrients by plants and favors a biological control of bacterial and fungal plant pathogens (rivera and wright, 2009). it has high microbial and enzymatic activity and contains large amounts of plant growth regulators like auxins, gib­ berellins cytokinins, macronutrients and micronutri­ ents (atiyeh et al., 2002). the favorable effect of ver­ micompost application on the growth and yield of many plants has been reported by previous studies (adamipour et al., 2019; levinsh, 2020; abd el­ hamed et al., 2021). arbuscular mycorrhizal fungi (amf) are soil fungi which are prevalent in most agricultural ecosystems to associate with more than 80% of plant species (wang and qiu, 2006). previous studies have shown that plant inoculation with amf improves growth, seeds yield, promotes photosynthetic pigments and carbohydrates content, enhances accumulation of macro­ and micronutrients in leaves (amiri et al., 2017; gashgaril et al., 2020; mohamed, 2020), as well as increases the nutritional values of seeds like proteins and oil percentage (ashour et al., 2021). although the beneficial roles of nitrogen and bio treatments on medicinal and aromatic crops and their valuable effect on improving growth and pro­ duction, there are no sufficient available data about their effectiveness on the growth and yield of downy safflower plants. therefore, this research is aimed to evaluate the influence of different nitrogen forms (ammonium sulfate, ammonium nitrate and urea) and some biotreatments (t. viride, vermicompost or arbuscular mycorrhiza fungi) on vegetative growth, seed yield and some chemical parameters of downy safflower (carthamus lanatus) plant. 2. materials and methods the field experiment was conducted at the experimental area of the ornamental horticulture department, faculty of agriculture, cairo university, giza governorate during the two successive seasons of 2019/2020 and 2020/2021. the latitude, longitude and altitude of the experimental site was 0°01’92.70” n, 31°20’68.08” e and 22 m above sea level, respectively. experimental procedure seeds of carthamus lanatus plants were acquired ashour et al. ‐ effect of different nitrogen forms and biotreatments on carthamus lanatus 187 from experimental farm of faculty of pharmacy, cairo university. on 1st november (of the two con­ secutive years), seeds were sown in a seedling trays (50 x 90 cm diameter) at saran house with 42% shad­ ing, 28/18°c (day/night) temperature, 14 h light con­ ditions, and 30­35% relative humidity. after 30 days from seeds sowing, uniform seedlings, with an aver­ age height of 18­20 cm, were transplanted in the experimental open field in plots (3×3 m), with a dis­ tance of 50 cm among rows, 70 cm between plants. some physical and chemical properties of the experi­ mental soil (average value of the two seasons) were determined according to jackson (1973), and the results are presented in table 1. nitrogen fertilization included ammonium sulfate (21 %n and 23­24%s) at 5 g/plant, ammonium nitrate (33 %) at 3 g/plant and urea (46% n) at 2 g/plant. nitrogen forms were applied as two separate doses. the first addition was before transplanting and the second was before flowering. plants treated with nitrogen forms were also inoc­ ulated with t. viride, vermicompost and arbuscular mycorrhiza fungi (amf), the control plants were not treated. t. viride, were obtained from pest rearing department, central agricultural pesticides laboratory, agricultural research centre, dokki, giza, egypt. 109cfu/ml conidial suspension of t. viride was diluted in 5 liters of water so as to prepare solution strength of 2x105cfu/ml. for each seedling, 100 ml of solution was used which accounted 2x107cfu of trichoderma per seedlings. 100 ml of the solution was used to drench the soil per seedlings (mastouri et al., 2010; chirino­valle et al., 2016). vermicompost was acquired from central laboratory for agricultural climate (clac), agricul­ ture research center, ministry of agriculture, giza, egypt. it was applied at 5 g/seedling. chemical analy­ ses of vermicompost used in this work (average value of the two seasons) are shown in table 2. amf inoculum contained roots, hyphae, spores colonized by glomus mosseae nrc31 and glomus fasciculatum nrc15 obtained from agricultural microbiology department, national research center, dokki, giza, egypt. inoculum material consisted of 275 spores g­1 (the infectivity 104 propagola). amf inoculation treatments were carried out by injecting 5 g/seedling of the inoculum. the three bio treatments were applied as two doses, the first addition was after 3 weeks from trans­ planting (21th december in both seasons, respectively) and the second was after 2 months from transplanti­ ng at branching start (21th february in two seasons, respectively). irrigation, manual weeding, pest and diseases control were done when needed. the layout of the experiment was factorial 4x4 in randomized complete blocks design with 16 treat­ ments. the first factor was 4 nitrogen forms (includ­ ing the control). the second factor was 4 biotreat­ ments (including the control) with 3 replicates, each replicate consisting of 32 plants (2 plants from each treatment). vegetative growth and yield parameters measure‐ ment vegetative growth parameters were registered after 120 days from transplanting (on 1st april). two soil characteristics data physicical characteristics soil texture clay clay (%) 43.30 coarse sand (%) 4.20 fine sand (%) 21.70 silt (%) 30.80 field capacity (v %) 67.85 chemical characteristics macro­nutrients (%) n 94.19 p 21.29 k 59.64 organic matter (%) 1.76 caco3 (%) 1.54 electrical conductivity (ds/m) 1.54 cation exchange capacity (meq/100 g) 40.22 ph 7.27 table 1 ­ physical and chemical properties of experimental soil (mean of two seasons) caco3= calcium carbonate, ph= soil acidity. properties ph ec (ds/m) organic matter (%) n (%) p (%) k (%) fe ppm zn ppm mn ppm vermicompost 8.41 6.6 42.9 1.65 1.14 1.69 166 109 96 table 2 ­ chemical analysis of vermicompost used in this work (mean of two seasons) adv. hort. sci., 2023 37(2): 185­196 188 samples of plants were taken and used to measure growth parameters including plant height (cm), num­ ber of branches/plant, stem diameter (cm, at 5 cm above the soil surface), fresh and dry weights of leaves, stems and roots as well as leaf area (cm2). at the harvesting stage (on 1st to15th may) yield parame­ ter were measured: number of flower heads/plant, weight of flower heads/plant, weight of seeds/plant and weight of 100 seeds (gr). the seed content of total carbohydrates, (n, p and k), total phenols and oil were also determined. chemical analysis the chemical analysis were performed at the end of each season (on 1st to15th may). chlorophyll and carotenoid contents. chlorophyll pigments including chl a, chl b and carotenoid con­ tents (mg g­1) were determined according to lichtenthaler and buschmann (2005), leaves extract­ ed by suspending them in 5 ml of 95% aqueous ace­ tone at 60 ̊c then the total volume completed to 10 ml with 95% aqueous aceton. the aqueous acetone supernatant was then taken for spectrophotometric measurement. a blank of acetone was taken at wavelengths of 663, 645 and 452.5 nm respectively, and data were then calculated using the following equations: chlorophyll a (mg g −1) = 0.0127 a663 − 0.00269a645 chlorophyll b (mg g −1)= 0.0029a663 − 0.00468a645 carotenoids (mg g −1) = 4.2 e 452.5 − 0.0264 total carbohydrates. total carbohydrates content in leaves and seeds (percentage of dry matter) was determined in dried samples according to dubois et al. (1956). a known weight (0.1 g) of the dried sam­ ples was completely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. the solution was decolorized and the fil­ trate was diluted to 100 ml with distilled water. a known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. the optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. the standard curve of glu­ cose was used to calculate the total carbohydrates concentration in the extract. n, p and k content of seeds. half gram of dried seeds samples was digested using tertiary acid mixture (hclo4 + hno3 + h2so4) and the extract was analyzed to determine concentrations of n, p and k (as percent­ age of dry seeds) according to estefan et al. (2013). nitrogen concentration was determined by using the micro­kjeldahl method. phosphorus was determined calorimetrically by using the chlorostannous molyb­ dophosphoric blue colour method in sulphuric acid. potassium was determined by using the flame pho­ tometer apparatus (corning m 410, germany). total phenolics content. total phelolics content was determined in the seeds extract by using the folin ciocalteau’s reagent colorimetric method and results are expressed as milligram of gallic acid equiv­ alent per gram of seeds dry weight extract (mg gae/g dw) (john et al., 2014). briefly, 1 ml of seed extract was mixed with 2.5 ml of 10% (w/v) folin­ciocalteu reagent. after 5 min, 2.0 ml of na2co3 (75%) was subsequently added to the mixture and incubated at 50°c for 10 min with intermittent agitation. afterwards, the sample was cooled and the absorbance was measured utilizing a uv spectrophotometer (shimazu, uv­1800) at 765 nm against a blank without extract. the outcome data were expressed as mg/g of gallic acid equivalents in milligrams per gram (mg gae/g) of dry extract. seed oil (%). the oil content of seeds was deter­ mined according to aoac (1995) using soxhlet appa­ ratus using petroleum ether as a solvent. the clean air dried seeds were separately crushed in a willey mill, then extracted in soxhlet apparatus, samples of 10 g of seeds were moved into soxhlet apparatus in 100 ml of n­hexane and the extraction period extended to 6 hours (30­36 syphon cycle approx.). the n­hexane extract was dried over anhydrous sodi­ um sulfate, then filtered and the oil was obtained by distillation under vacuum. oil % was calculated according to the equation: oil % = extracted oil weight (g) ̸ seeds sample weight (g) ̸ × 100. statistical analysis results of the two field trials performed in two different growth seasons were combined in order to obtain an average value for each parameter. the means of all results were subjected to two­ways analysis of variance (anova) in randomized com­ plete blocks design. means of data were compared by using duncan’s multiple range tests at p = 5% (snedecor and cochran, 1989). 3. results and discussion vegetative growth parameters the data in table 3, 4 and figure 1 revealed that ashour et al. ‐ effect of different nitrogen forms and biotreatments on carthamus lanatus 189 under the same level of n forms, application of t. viride, vermicompost or amf treatments resulted in significant increase of tested vegetative growth para­ meters (viz., plant height, number of branches/plant, stem diameter, fresh and dry weights of leaves, stems and roots and leaf area) compared to control. among the tested treatments, application of t. viride appeared to be the most effective treatment since *, **, *** significant at p≤0.05, p≤0.01, p≤0.001 respectively, (n=3). table 4 ­ plant height, no. of branches/plant, stem diameter, fresh and dry weights of leavesof carthamus lanatus as affected by the interaction between nitrogen forms and biotreatments (mean of two seasons) amf= arbuscular mycorrhiza fungi, as= ammonium sulfate, an= ammonium nitrate. data represent the mean value ±s.e. the mean of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan mul­ tiple rang test. table 3 ­ mean square for the effect of nitrogen forms and biotreatments and their interaction on vegetative growth, yield parameters of carthamus lanatus traits source of variation treatment error cv biotreatments (a) nitrogen sources (b) (a × b) plant height (cm) 4102.63 *** 3164.686 *** 133.843 *** 2.236 0.926 no. of branches/plant 123.894 *** 140.852 *** 18.727 *** 0.785 6.276 stem diameter (cm) 0.779 *** 0.888 *** 0.058 *** 0.011 8.215 fresh weight of leaves (g/plant) 3539.035 *** 2397.09 *** 484.993 *** 3.234 1.495 dry weight of leaves (g/plant) 345.389 *** 253.726 *** 47.235 *** 1.86 3.728 fresh weight of stems (g/plant) 56027.069 *** 16413.722 *** 1476.819 *** 9.154 1.383 dry weight of stems (g/plant) 3390.971 *** 989.016 *** 74.53 *** 0.902 1.799 fresh weight of roots (g/plant) 1451.436 *** 1788.839 *** 476.746 *** 5.688 4.887 dry weight of roots (g/plant) 117.628 *** 128.286 *** 25.247 *** 0.611 6.305 leaf area (cm2) 870.237 *** 1304.444 *** 115.687 *** 0.734 2.09 no. of flower heads/ plant 453.436 *** 382.616 *** 46.209 *** 1.077 6.178 weight of flower heads/ plant 388.412 *** 606.905 *** 45.35 *** 1.783 3.706 weight of seeds/ plant 964.929 *** 1057.951 *** 102.45 *** 0.736 3.534 weight of 100 seeds/ plant 0.549 *** 0.799 *** 0.072 *** 0.002 1.993 biotreatments (a) nitrogen forms (b) plant height (cm) no. of branches/plant stem diameter (cm) fresh weight of leaves (g/plant) dry weight of leaves (g/plant) control control 124.33±0.60 k 7.00±0.58 g 0.70±0.05 e 86.50±2.18 i 26.07±0.81 h as 140.83±1.17 gh 11.33±0.33e 1.15±0.06 c 101.83±0.67 g 30.97±0.48 fg an 130.83±0.44 j 9.33±0.44 f 0.82±0.06 dc 96.83±1.17 h 28.20±0.9 h n­urea 139.67±1.59 hi 11.50±0.29 e 1.17±0.03 c 107.50±0.76 f 34.03±0.12 e t. viride control 148.67±0.88 f 11.00±0.29 e 0.93±0.07 d 109.17±1.01 f 34.07±0.62 e as 189.00±0.76 a 23.17±0.17 a 1.87±0.03 a 178.33±1.42 a 54.57±1.4 a an 175.50±1.50 d 15.50±0.29 c 1.53±0.09 b 136.17±2.6 c 41.42±0.86 c n­urea 179.50±0.29 c 17.83±0.44 b 1.53±0.06 b 136.67±1.86 c 41.63±0.78 bc vermicompost control 137.83±1.17 i 9.17±0.33 f 1.30±0.06 c 110.17±0.44 f 30.63±1.12 g as 184.17±0.60 b 13.33±0.73 d 1.62±0.09 b 142.67±1.09 b 43.65±0.71 b an 175.83±1.09 d 15.33±0.93 c 1.52±0.01 b 107.5±1.61 f 32.98±0.61 ef n­urea 179.67±0.88 c 16.50±0.87 bc 1.65±0.01 b 128.17±0.83 d 38.37±0.59 d amf control 142.67±1.17 g 11.17±0.33 e 0.82±0.03 de 108.33±0.88 f 32.70±0.73 e­g as 187.00±0.76 a 21.83±0.93 a 1.52±0.07 b 121.17±0.67 e 36.48±0.81 d an 169.50±1.04 e 15.17±0.44 c 1.15±0.01 c 118.67±0.44 e 38.02±0.46 d n­urea 179.50±0.76 c 15.33±0.33 c 1.57±0.02 b 134.67±0.83 c 41.55±0.42 bc 190 adv. hort. sci., 2023 37(2): 185­196 registered the highest values. these results are in line with those findings of prior authors (lakshman and ghodke, 2018; shaikh and mokat, 2018; ghoneem et al., 2019; guo et al., 2020; hassanin et al., 2020; el­ dabaa et al., 2021), who reported increases in vege­ tative growth parameters due to t. viride inoculation. the useful effect of t. viride on vegetative growth parameters may be related to participation of such microorganisms in biotransformation of cellulose, increasing cell reproduction, nitrogen mineralization and phosphorus solubilization. they also increase the volume of roots which in turn, increases absorption of water and nutrients, consequently increasing both growth and yield of the crops (nepali et al., 2020). the data in table 4 and figure 1 also revealed that under the same rate of bio treatment (t. viride, ver­ micompost or amf) treating the plants with different nitrogen forms resulted in significant increase in veg­ etative growth parameters compared to control and among the tested nitrogen forms, with the applica­ tion of ammonium sulfate leading to superior growth compared with ammonium nitrate or urea. the increases in vegetative growth parameters due to ammonium sulfate treatments are in agreement with the findings of several studies on different plants including cynara cardunculus (sarhan et al., 2014), urtica pilulifera (wahba et al., 2014), nigella sativa (khalid and shedeed, 2015), sunflower (el mantawy, 2017; el sayed, 2020), thymus vulgaris (basal et al., 2019) and jojoba (hegab et al., 2021). the positive effect of ammonium sulfate may be attributed to the role played by the acidic component that decrease the values of soil ph and thus simplify the uptake of nutrients by the plant roots (fouda, 2017). yield parameters results of figure 2 indicated that within each level of n forms, in most cases, application t. viride, vermi­ compost or amf treatments caused a significant increase in yield parameters (namely, no. of flower heads/plant, weight of flower heads/plant, weight of seeds/plant, weight of 100 seeds) compared to con­ trol. t. viride treatment appeared to be the most effective one since recorded the highest values. increases in yield parameters due to t. viride treat­ ments are matched well with those of previous stud­ ies on different crops including coriandrum sativum (khan and parveen, 2018), triticum aestivum (mahato et al., 2018), cuminum cyminum (ghoneem et al., 2019). fig. 1 ­ fresh weight of stems (g/plant) (a), dry weight of stems (g/plant) (b), fresh weight of roots (c), dry weight of roots (d), leaf area (e) of carthamus lanatus as affected by the interaction between nitrogen forms and bio treat­ ments (mean of two seasons). column with different let­ ters indicate a significant difference at 5% level. data represent the mean values ± se the mean of three repli­ cates. ashour et al. ‐ effect of different nitrogen forms and biotreatments on carthamus lanatus 191 most effective one was ammonium sulfate, for which the highest mean value was found. the increases in yield parameters due to ammonium sulfate treat­ ments are the same as the results of sarhan et al., 2014 on cynara cardunculus, wahba et al. (2014) on urtica pilulifera, khalid and shedeed (2015) on nigella sativa, el mantawy (2017); el sayed (2020) on helianthus annuus, prinsloo and plooy (2017) on sutherlandia frutescens, hegab et al. (2021) on simmondsia chinensis. contents of pigments and total carbohydrates in leaves as shown in table 5 and 6 within each rate of n forms, in most cases, application of t. viride, vermi­ compost or amf rates resulted in significant increase in the mean values of pigments content (chlorophyll a, b and carotenoids) and total carbohydrates in leaves compared to control. the highest mean values were found for the plants treated with t. viride. such increase in pigments content or total carbohydrates in leaves due to t. viride inoculation is in accordance with those obtained by previous reports on salvia officinalis (kumar et al., 2015), cuminum cyminum (ghoneem et al., 2019), allium cepa (metwally, 2020). chlorophyll is used by plants for light­trapping and energy transduction during the anabolic process of photosynthesis. a higher content of photosynthetic pigments can be correlated to the augmentation in carbohydrates content of leaves. results of table 6 also pointed out that within biotreatments (t. viride, vermicompost or amf), in most cases, the mean values of pigments content and total carbohydrates in leaves of the plants treat­ ed with various nitrogen forms were significantly higher than the control. nitrogen in the form of ammonium sulfate was superior to the other two nitrogen sources for enhancing the value of the para­ meters considered. the results are analogy with that recorded by earlier research (sarhan et al., 2014; wahba et al., 2014; el mantawy, 2017), they report­ ed increase in pigments content or total carbohy­ drates in leaves due to application of ammonium sul­ fate. the superior effect of ammonium sulfate in increasing pigments contents may be related to sul­ fur element that is a constituent of succinyl co­a which involved in chlorophyll synthesis in leaves and its activation at cellular level enhances photosynthe­ sis that eventually boost vegetative growth. fig. 2 ­ evolution of total phenolic content in season 2018 (mg gallic acid/100 g of fresh fruit) of four selected farms (131, 272, 351, 432) and their average trend. bars repre­ sent standard error of the mean (±sem). values followed by the same letter in every sampling point are not signifi­ cantly different from each other. mean separation by lsd test (p≤0.05). data in figure 2 also exhibited that within each rate of t. viride, vermicompost or amf, the plants treated with nitrogen forms had significantly higher values of yield parameters, in most cases, than those of control. among the tested nitrogen sources, the adv. hort. sci., 2023 37(2): 185­196 192 moreover, it is known the function of sulfur in the synthesis of proteins, oils, vitamins, and flavored compounds in plants since, it is a constituent of the three amino acids methionine (21% s), cysteine (26% s) and cystine (27% s), that are the building blocks of protein (el mantawy, 2017). contents of total carbohydrates, n, p and k in seeds it is clear from data reported in figure 3 that with­ in each rate of n forms, in most cases, application of t. viride, vermicompost or amf caused significant increase in total carbohydrates, macronutrients (n, p and k %) in seeds compared to control. among the table 5 ­ mean square for the effect of nitrogen forms and bio treatments and their interaction on some chemical constituents of carthamus lanatus traits source of variation treatment error cv bio treatments (a) nitrogen sources (b) (a × b) chlorophylls a content (mg/g f.w) 14.545 *** 9.932 *** 0.801 *** 0.002 2.18 chlorophylls b content (mg/g f.w) 8.327 *** 71.103 *** 3.727 *** 0.112 2.932 carotenoids content (mg/g f.w) 0.288 * 0.268 * 0.148 ** 0.073 7.57 total carbohydrates [%] in leaves 35.699 *** 55.094 *** 2.029 * 0.771 4.30 total carbohydrates [%] in seeds 22.491 *** 47.094 *** 2.901 * 1.188 4.68 n% in seeds 1.682 ** 1.761 ** 0.102 * 0.158 17.71 p% in seeds 0.036 *** 0.029 *** 0.004 ** 0.002 9.91 k% in seeds 0.078 *** 0.161 *** 0.01 * 0.004 4.58 total phenols [%] in seeds 0.568 *** 0.795 *** 0.068 *** 0.003 1.79 oil [%] in seeds 29.47 *** 48.638 ** 0.423 * 1.498 4.63 *, **, *** significant at p≤0.05, p≤0.01, p≤0.001 respectively, (n=3). table 6 ­ pigments, total carbohydrates in leaves as affected by the interaction between nitrogen forms and bio treatments (mean of two seasons) amf= arbuscular mycorrhiza fungi, as= ammonium sulfate, an= ammonium nitrate. data represent the mean value ±s.e. the mean of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan mul­ tiple rang test. bio treatments (a) nitrogen forms (b) chlorophylls a content (mg/g f.w.) chlorophylls b content (mg/g f.w.) carotenoids content (mg/g f.w.) total carbohydrates [%] in leaves control control 3.68±0.03 k 7.04±0.12 i 2.64±0.57 b 15.44±0.59 g as 5.06±0.01 i 13.74±0.07 c 3.55±0.01 a 19.88±1.15 d­f an 4.09±0.11 j 11.06±0.33 f 3.58±0.01 a 16.22±0.58 g n­urea 5.88±0.04 g 11.27±0.1 ef 3.58±0.01 a 20.18±0.52 c­e t. viride control 5.88±0.04 g 7.28±0.05 i 3.60±0.03 a 18.90±0.58 ef as 8.24±0.09 a 15.26±0.14 a 3.88±0.06 a 24.37±1.15 a an 6.11±0.09 f 12.13±0.34 d 3.63±0.02 a 21.32±0.57 b­e n­urea 6.74±0.02 d 12.26±0.21 d 3.65±0.02 a 23.02±1.73 ab vermicompost control 6.62±0.07 de 8.95±0.02 h 3.57±0.08 a 17.28±1.15 fg as 7.8±0.14 b 13.78±0.04 bc 3.59±0.11 a 22.58±1.21 a­c an 5.92±0.11 fg 11.79±0.02 de 3.63±0.09 a 21.21±0.64 bc­e n­urea 7.83±0.10 b 14.31±0.06 b 3.57±0.07 a 22.42±0.59 a­d amf control 5.50±0.02 h 8.78±0.35 h 3.55±0.06 a 18.90±0.58 ef as 7.30±0.05 c 11.11±0.05 f 3.64±0.13 a 21.55±0.55 bcd an 6.45±0.12 e 10.11±0.31 g 3.70±0.04 a 20.67±0.57 b­e n­urea 7.95±0.03 b 13.37±0.13 c 3.62±0.03 a 22.83±1.17 ab ashour et al. ‐ effect of different nitrogen forms and biotreatments on carthamus lanatus 193 tested treatment t. viride appeared to be the most effective one. in this regard, kumar et al. (2015) on salvia officinalis stated that the plants inoculated with t. viride had higher phosphorus content in shoot and root as compared with control. also, metwally (2020) on allium cepa declared that the plants inocu­ lated with t. viride improved total carbohydrates and n, p or k% in plant organs. data in figure 3 also exhibited that within t. viride, vermicompost or amf treatmens, the plants treated with nitrogen forms had significantly higher values of total carbohydrates, n, p and k in their seeds than the control. among the tested nitrogen forms ammo­ nium sulfate was superior in its effect than the other two nitrogen sources. such results confirmed the reports of prior works (wahba et al., 2014; khalid and shedeed, 2015; el sayed, 2020; hegab et al., 2021) who showed increase in total carbohydrates or n, p and k% in seeds as result of ammonium sulfate application. the increased content of total carbohydrate in seeds may be related to the increase in chlorophyll content of plants, corresponding to improved photo­ synthesis efficiency (khalid and shedeed, 2015). total phenols in seeds data in figure 4 a displayed that within each rate of n forms, in most cases, application of t. viride, ver­ micompost or amf treatments caused a significant increase in total phenols in seeds compared to con­ trol. the highest mean values were found to be asso­ ciated with the plants treated with vermicompost. similar results were reported by abd el­hamed et al. (2021) who found that vermicompost caused increase in total phenols in leaves of dracocephalum moldavica. within each treatment of t. viride, vermicompost or amf, the plants fertilized with different rates of nitrogen forms had significantly higher values of total phenols in seeds than those of control. nitrogen as ammonium sulfate was superior to the other nitro­ gen forms in augmentation total phenols in seeds. these results confirmed the reports of earlier researches (munene et al., 2017; petropoulos et al., 2018; prinsi et al., 2020; machado et al., 2022) that reported increase in total phenols in plant organs due to ammonium sulfate treatments. seed oil (%) it is evident from data figure 4 (b) that within each rate of n forms application of t. viride, vermicompost fig. 3 ­ evolution of superficial ss index of four selected farms (131, 272, 351, 432) and their average trend during stor­ age in 2018. bars represent standard error of the mean (±sem). values followed by the same letter in every sam­ pling point after harvest are not significantly different from each other. mean separation by lsd test (p≤0.05). results are in agreement with those reported by pre­ vious researches on different plants including cynara cardunculus (sarhan et al., 2014), helianthus annuus (el mantawy, 2017) and simmondsia chinensis (hegab et al., 2021), they indicated that ammonium sulfate treatments caused increase in oil percentage in seeds. the increments in oil content due to ammonium sulfate application may be attributed to its promoting role in the formation of amino acids methionine (21% s) and cysteine (27% s); synthesis of proteins and oil content of seeds. also, sulfur is an important element for oil crops which a constituent of acetyl co­a, which converted into malonyl co­a to synthesis of fatty acid (el mantawy, 2017). 4. conclusions summing up the results, it can be concluded that for enhancing growth, nutritional values of seeds, the interacted treatment of t. viride 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however, internal bac­ terial contaminations are an obstacle in tissue culture of fruit tree species. for this purpose, this investigation was conducted with several experiments to manage bacterial contamination. first, gram test for bacterial contamination related to pyrus shoots proliferating was conducted. gram test revealed that bacteria contaminating pear shoots were negative gram. then, we investigated the application of cefixime (0, 100, 300 and 500 mg l­1) or ampicillin (0, 100, 300 and 500 mg l­1) for managing bacterial contaminations. it was found that the contaminated area on medium containing 500 mg l­1 cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). therefore, cefixime at 500 mg l­1 was selected to control the bacterial contamination. next, different proce­ dures were used included shaking with (1: sterile distilled water, 2: 500 mg l­1 cefixime and culturing in media with 500 mg l­1 cefixime, 3: 500 mg l­1 cefixime, culturing and subculturing in media with 500 mg l­1 cefixime 4: disinfection). the third procedure was known the best due to the low bacterial contamina­ tion percentage and rate also the healthy growth of plants. finally, the effect of gibberellic acid at 0 and 1 mg l­1 was investigated to compensate for shoot growth reducing in the presence of cefixime. 1 mg l­1 gibberellic acid improved the growth indices in the presence of cefixime. 1. introduction fire blight, the most devastating disease of pear, leads to the death of the whole pear tree through the systematical infection in all underground and aerial parts of the tree (vanneste, 2000; evrenosoğlu et al., 2019). from the horticultural science perspective, the revival of the pear orchards is dependent on large­scale propagation. the majority of culti­ vated pear is pyrus communis (morgan et al., 1994) and p. communis cv. williams is sensitive to fire blight (abdollahi et al., 2010). however, pyrus communis is difficult­to­root (zhu et al., 2003; sun et al., 2011); therefore, the tissue­culture technique can support large­scale propagation of pear. (*) corresponding author: davarynej@um.ac.ir citation: ameri a., davarynejad g.h., tehranifar a., moshtaghi n., 2020 ­ cefixime manages inter‐ nal bacterial contamination during tissue culture operation ­ adv. hort. sci., 34(1): 43­48 copyright: © 2020 ameri a., davarynejad g.h., tehranifar a., moshtaghi n.. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 october 2018 accepted for publication 6 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 43­48 44 one of the most important factors in tissue culture is the control of microbial contamination. plant immune system acts against pathogens. there are three steps of plant defense responses included the response in the step of entry, establishment, and spread of pathogens. overall, plant immune systems can be classified into cell wall reinforcement and pro­ grammed cell death. in pathogen entry condition, cell wall reinforcement is efficient and in pathogen estab­ lishment and spread conditions programmed cell death can be restrictive (abramovitch and martin, 2004). when pathogens suppress the plant defenses responses, the plant was invaded by pathogens and contaminated. one of the phytopathogens is bacte­ ria; they cause serious troubles in vitro conditions. some of the modes of bacteria actions to suppress the host defenses refer to the use of type iii effector proteins and toxins (abramovitch and martin, 2004) as well as, type iv secretion systems to inject effector proteins into cells (angot et al., 2007). tissue culture technique is very sensitive to special pathogenic fac­ tors and all of the microbes in air condition and equipment. in other words, all of the microbes infect cultures with the aim of nutrition (nadha et al., 2012). internal infections in plant cultures had often harmful effects for shoot proliferation, shoot rooting and quality of plant growth (nadha et al., 2012). in tissue culture, the selection of the appropriate antibi­ otic is important. there are several reports about the use of antibiotics to manage the bacterial contamina­ tion (phillips et al., 1981; falkiner, 1990; kneifel and leonhardt, 1992; barrett and cassells, 1994; falkiner, 1997; nadha et al., 2012). cefixime is an antibiotic belonging to cephalosporin class. the mode of action of cephalosporins is related to inhibiting the cell wall biosynthesis so that this class arrests the formation of peptide bonds (kohanski et al., 2010). affect sites of various antibiotics are different; therefore the efficiency of different antibiotics is dif­ ferent in the removal of bacteria. there are some observations for different antibiotics affect sites in the previous studies, such as, inhibiting of cell wall synthesis that is related to benzylpenicillin and phos­ phomycin, inhibiting protein synthesis related to chloramphenicol and streptomycin, inhibiting of rna and dna synthesis related to rifampicin and nalidixic acid (phillips et al., 1981). one of the side effects of antibiotic application in plant tissue culture is the reduction of growth. however, in control of bacterial infection in guadua angustifolia, streptomycin sul­ fate decreased growth shoot, but kanamycin caused intensive growth with high­quality; therefore, the effects of antibiotics are different on plant growth (nadha et al., 2012). the management of contamina­ tion in tissue culture leads to the prevention of waste of time and energy. in this investigation, we examine the use of antibiotics to manage the bacterial conta­ minations coupled with the use of ga3 to compen­ sate for the poor growth of the plant in the presence of antibiotic in the media. 2. materials and methods plant materials three months old proliferating micro­shoots of pyrus communis cv. williams, as the most common cultivar in the world, exhibited bacterial contamina­ tion. contaminated micro­shoots were picked to investigate the experiments of bacterial control for the large­scale production of p. communis. these micro­shoots were maintained in ms medium (murashige and skoog, 1962) supplemented with 1.5 mg l­1 ba, 0.1 mg l­1 naa and 3% sucrose. gram test of bacteria two drops, approximately 50 µl, of a 3% (w/v) solution of potassium hydroxide were placed on a clean glass slide as outlined by ryu (1940). bacterial cells were transferred from culture media aseptically with a flat wooden toothpick and placed into the drop of koh with rapid, circular agitation. after 5­8 sec, the toothpick was alternately raised and lowered just off the slid surface to detect a stringing effect. it was considered gram­negative bacteria if drop vis­ cosity increased within 15 sec (suslow et al., 1982; schaad et al., 2001). antibiotic selection test contaminated shoots were cultured on ms medi­ um (murashige and skoog, 1962) supplemented with either ampicillin 0, 100, 300 and 500 mg l­1 or cefixime at 0, 100, 300 and 500 mg l­1. vessel cul­ tures with 32 mm inner diameter were maintained at a constant temperature of 25±1°c and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) using cool white fluorescent lamps (sylvania, germany). after a week, the contaminated area was measured in each treatment. bacterial contamination removing in the before step, we selected the proper antibi­ otic (cefixime at 500 mg l­1). then, we used four dif­ ferent procedures using cefixime at 500 mg l­1 to control any eventual bacterial contamination. for each procedure, 3 micro­shoots were cultured in a ameri et al. ‐ in vitro plant rescue by cefixime 45 candle jar as a replicate. 1) shaking of contaminated shoots with sterile dis­ tilled water (control); 2) the first, shaking of contaminated shoots with cefixime at 500 mg l­1 then, cultured in media with cefixime at 500 mg l­1. finally, sub­culturing in free antibiotic media; 3) the first, shaking of contaminated shoots with cefixime at 500 mg l­1, then, cultured in media with cefixime at 500 mg l­1. finally, sub­culturing in media with cefixime at 500 mg l­1; 4) disinfection of contaminated shoots (immersing in 1% hypochlorite sodium for 5 min then rinsed with sterile water three times). ms medium (murashige and skoog, 1962) supple­ mented with 1.5 mg l­1 ba, 0.1 mg l­1 naa and 3% sucrose were used for each procedure. cultures were maintained at a constant temperature of 25±1°c and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) using cool white fluorescent lamps (sylvania, germany). after 30 days, several traits were evaluat­ ed: percentage of fungal contamination and bacterial contamination (bc), bacterial contamination rate (bcr) and general health. based on the following equation (e1) bacterial contamination rate was counted per each micro­ shoot in each candle jar: bcr =∑ ni di where bcr= bacterial contamination rate, ni = number of the contaminated shoot in each day, di = day number. rescued shoot improvement micro­shoots related to the best procedure were transferred to ms medium (murashige and skoog, 1962) supplemented with 1.5 mg l­1 ba, 0.1 mg l­1 naa, 3% sucrose, 500 mg l­1 cefixime and gibberellic acid (ga3) treatments. the concentrations of ga3 were 0 and 1 mg l­1. ph was adjusted at 5.8 with naoh prior to autoclaving at 98 kpa and 121°c, and the media were solidified using 0.8% agar. cefixime antibiotic and ga3 added to the media after autoclav­ ing by filtering. related traits of this experiment were included: the percentage of new growth, the per­ centage of proliferation, the average number of bud and leaf, as well as, the average shoot length. the evaluation of declined antibiotic dose after the six months using cefixime at 500 mg l­1, proliferated shoots were divided into two groups. each group of plants was cultured in media with cefixime at either 500 mg l­1 or 250 mg l­1. ph was adjusted at 5.8 with naoh prior to autoclaving at 98 kpa and 121°c, and the media were solidified using 0.8% agar. cefixime antibiotic and ga3 were added to the media after autoclaving with the syringe filter (pore size: 0.22 µm). after 10 days, the percentage of bacterial contaminations and bacterial contamination rate were measured per each micro­shoot in each candle jar based on (e1). rooting micro‐shoots after six months, micro­shoots were transferred to ½­strength ql (quoirin and lepoivre, 1977) medi­ um supplemented with 1.5 mg l­1 naphthaleneacetic acid (naa) and 500 mg l­1 cefixime. cultures were maintained a week in dark conditions, then, trans­ ferred to 16/8 h (light/dark) photoperiod and light intensity of approximately 45 µmol m­2 s­1 photosyn­ thetic photon flux density (ppfd) emitted by cool­ white fluorescent tubes in 35% relative humidity. statistical analysis these experiments were arranged as a completely randomized design with three replications. the data were statistically analyzed using a one way anova test and means were compared with the duncan test at the 5% level of confidence. all of the statistical tests were performed using sas (statistical analysis system) software v9.1. all of the percentage data were transformed to arcsin √x. 3. results and discussion the result of the gram type detection showed contamination of this investigation is related to gram­negative bacteria. in the antibiotic selection step, we compared ampicillin and cefixime. we used 0, 100, 300 and 500 mg l­1concentrations of each antibiotic. ampicillin is a common antibiotic in tissue culture, and its activity spectrum is related to gram­ positive and gram­negative bacteria, whereas the cefixime is antibiotic acting against gram­negative bacteria. in antibiotic selection test, results revealed 500 mg l­1cefixime could overcome contamination better than other treatments. the contaminated area on medium containing 500 mg l­1cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). therefore, we selected 500 mg l­1 cefixime to control the contamination for later experiments. anova revealed that the difference between the four procedures was significant (p≤0.01) for bacterial contamination percentage and the rate of bacterial adv. hort. sci., 2020 34(1): 43­48 46 contamination (table 1). four procedures were used for the survival and rescuing of shoot from bacterial contamination. the third procedure (the first, shak­ ing of contaminated shoots with 500 mg l­1 cefixime; then, cultured in media with 500 mg l­1 cefixime. finally, sub­culturing in media with 500 mg l­1 cefixime) was known the best due to the low bacteri­ al contamination percentage, rate and finally the healthy and fresh growth of plants. these indices in other procedures were not desirable; as the highest percentage of bacterial contamination was observed in the first procedure (shaking with sterile distilled water). all of the procedures led to necrotic plants except for procedure 3 (table 2). the presence of antibiotic in media effected on plant growth and weakened their growth; therefore, we used ga3 and its effect evaluated on plants growth. ga3 application in media containing 500 mg l­1 cefixime had a signifi­ cant effect on the percentage of new growth, the average of bud number, the average shoot length and the average of leaf number (p<0.05) (table 3). without the application of ga3 were not observed any proliferation and new bud formation; while in media containing ga3, 22.53% proliferation and 1.46 the average number of bud were observed (table 4). as well as, the results showed the decrease in the antibiotic dose to 250 mg l­1 cefixime increased bcp table 4 ­ evaluation of gibberellic acid in ms medium along with pgrs and 500 mg/l cefixime on secondary growth traits values are mean ± standard error. table 1 ­ analysis of variance traits under study table 2 ­ evaluation of different procedure to come over bacterial contamination during the culture ns= no significant; **= significant at the 0.01 level of probability according to duncan test. **= significant at the 0.01 level of probability according to duncan test. *= significant at the 0.05 level of probability according to duncan test. source of variance df means square bacterial contamination rate of bacterial contamination treatment 3 2963.807 ** 45.48649 ** error 8 7.350.308 0.5436 procedure fungal contamination (%) bacterial contamination (%) bacterial contamination rate general health after 30 days 1 0 90 ± 0 9.47 ± 0.77 necrotic leaves 2 0 74.55 ± 2.42 4.93 ± 0.119 necrotic leaves 3 0 17.01 ± 1.22 0.45 ± 0.053 green leaves and healthy 4 0 57.85 ± 1.56 7.47 ± 0.323 necrotic leaves 1= shaking with sterile water. 2= the first, shaking of contaminated shoots with cefixime (500 mg/l) then, culturing in media with antibiotic. finally, sub­culturing in free antibiotic media. 3= the first, shaking of contaminated shoots with cefixime (500 mg/l), then, culturing in media with antibiotic. finally, sub­culturing in with antibiotic media. 4= disinfection (immersing in 1% hypochlorite sodium for 5 min then shaking with sterile water for three times. values are mean ± standard error. table 3 ­ analysis of variance of traits under study source of variance df means square percentage of new growth percentage of proliferation average of bud number average shoot height average of leaf number treatment 1 514.20 ** 761.53 ** 3.22 ** 60.16 * 54.0 * error 4 6.022 4.53 0.0066 7.33 0.33 concentration (mg/l) new growth (%) proliferation (%) average bud number average shoot height (mm) average leaf number 1 43.93 ± 1.23 22.53 ± 1.74 1.46 ± 0.07 9.66 ± 2.03 10.33 ± 0.33 0 25.41 ± 1.58 0 ± 0 0 ± 0 3.33 ± 0.88 4.20 ± 0.33 ameri et al. ‐ in vitro plant rescue by cefixime 47 leifert and cassells (2001) mentioned alternatives for the antibiotic in their review. these alternatives included medium acidification and autotrophic cul­ ture (e.g. culture without carbohydrate) (leifert and cassells, 2001). in other literature were noted to acti­ vating of endogenous bacteria as a result of sub­cul­ turing in media with cytokinins (kneifel and leonhardt, 1992). however, plant tissue culture with­ out cytokinins, carbohydrates and with the modifica­ tion in media acidity is impossible. in this regards, this investigation showed with the presence of cefixime in media containing cytokinins, carbohy­ drates could manage the bacterial contamination. based on the results of this investigation, cefixime at 500 mg l­1 had not any toxicity effect on growth and proliferation. cefixime is an antibiotic belonging to cephalosporins class. the cephalosporin antibiotics have been introduced as the appropriate antibiotic plant tissue culture since they have low eukaryote toxicity (mathias and boyd, 1986) which our results emphasize this point. 4. conclusions bacterial contamination incidence is common and unavoidable during the in vitro propagation of fruit tree species. this investigation presented a proce­ dure to manage the bacterial contamination of p. communis cv. williams during the in vitro culture. based on the results of this investigation, cefixime at 500 mg l­1 could control the bacterial contamination. the use of antibiotic in a medium is associated with a decrease in the growth of plants. this side effect of antibiotic was managed with the application of ga3 at 1 mg l­1. therefore, we suggest cefixime at 500 mg l­1 for in vitro propagation of fruit trees. references abdollahi h., tahzibi f., ghahremani z., 2010 ­ correlation between fire blight resistance and morpho‐ logical characteristics of pear (pyrus communis l.). ­ acta horticulturae, 896: 339­345 abramovitch r.b., martin g.b., 2004 ‐ strategies used by bacterial pathogens to suppress plant defenses. ­ curr. opin. plant biol., 7(4): 356­364. angot a., annette v., stéphane g., nemo p., 2007 ­ exploitation of eukaryotic ubiquitin signaling pathways by effectors translocated by bacterial type iii and type iv secretion systems. ­ plos pathog., 3(1): e3. barrett c., cassells a.c., 1994 ‐ an evaluation of antibi‐ to 41% and bcr to 1.2 (table 5). therefore, using the antibiotics at 500 mg l­1 should continue because the plants grow without bacterial contamination only in the presence of 500 mg l­1 cefixime (fig. 1). nadha et al. (2012) stated the removal of kanamycin from the medium did not result in resumption contamination after 10 days (nadha et al., 2012); while other litera­ ture mentioned that the usage of antibiotics for inhibiting the bacteria growth has impermanent impact and removal of antibiotics has accompanied by resumption contamination (falkiner, 1990; barrett and cassells, 1994; falkiner, 1997; leifert and cassells, 2001) confirming the results of this experi­ ment. in the consumption of antibiotic, resistant­bac­ teria theory is undeniable. despite long­term using of cefixime, about six months, it could not only remove bacterial contamination, but also act without any resistant­bacteria. finally, rescued shoots were able to produce healthy roots. table 5 ­ evaluation of decreasing of cefixime on bcr and bcp after ten days fig. 1 ­ contaminated shoots in vitro culture (a). contaminated shoot after sub­culturing on media without cefixime (b). new growth after using ga3 treatment (c, d) different letters in columns indicate significant difference between treatments at 5% level. traits cefixime concentration (mg/l) 250 500 bacterial contamination rate 41 a 0 b bacterial contamination percentage 1.2 a 0 b 48 adv. hort. sci., 2020 34(1): 43­48 otics for the elimination of xanthomonas campestris pv. pelargonii (brown) from pelargonium x domesticum cv. ‘grand slam’explants in vitro. ­ plant cell tissue organ cult., 36(2): 169­175. evrenosoğlu y., mertoğlu k., bilgin n.a., misirli a., özsoy a.n., 2019 ­ inheritance pattern of fire blight resistance in pear. ­ sci. hortic., 246: 887­892. falkiner f.r., 1990 ­ the criteria for choosing an antibiot‐ ic for control of bacteriain plant tissue culture. ‐ int. soc. plant tiss. cult. newsletter, 60:13­23. falkiner f.r., 1997 ­ antibiotics in plant tissue culture and micropropagation. ‐ what are we aiming at?, pp. 155­ 160. ­ in: cassells a.c. 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edition. ­ am. phytopathol. soc. press (aps), st. paul, mn, usa, pp. 373. sun q., sun h., bell r.l., li h., xin l., 2011 ‐ variation of phenotype, ploidy level and organogenic potential of in vitro regenerated polyploids of pyrus communis. ‐ plant cell tissue organ cult., 107: 131­140. suslow t.v., schroth m.n., isaka m., 1982 ­ application of a rapid method for gram differentiation of plant pathogenic and saprophytic bacteria without staining. ­ phytopathology, 72(7): 917­918. vanneste j.l., 2000 ­ fire blight: the disease and its causative agent, erwinia amylovora. ­ cabi publishing, wallingford, uk, pp. 370. zhu l.h., li x.y., ahlman a., welander m., 2003 ‐ the rooting ability of the dwarfing pear rootstock bp10030 (pyrus communis) was significantly increased by intro‐ duction of the rolb gene. ‐ plant sci., 165: 829­835. impaginato 49 adv. hort. sci., 2018 32(1): 49-59 doi: 10.13128/ahs-21360 partial root-zone irrigation effects on growth, metabolism and calcium status of mangosteen seedling (garcinia mangostana l.) d.p. hapsari, r. poerwanto(*), d. sopandie, e. santosa department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, bogor, indonesia. key words: drought, low soil moisture, photosynthetic rate, tropical plant, water management. abstract: efficient irrigation technique for mangosteen seedling was evaluated, from october 2016 to may 2017, in order to determine the growth and morphophysiology of both the conventional deficit irrigation (cd) and partial rootzone irrigation (pr). a set of randomized block design, with 4 replicates each, was applied on 100% field capacity (control), 50% field capacity (cd1), 30% field capacity (cd2), and ratios of 100:50% field capacity (pr1), 100%:30% field capacity (pr2) and 50:30% field capacity (pr3). the results showed a restriction in mangosteen growth, except control, as indicated by decrease in total dry mass, which due to decrement in leaf number, photosynthetic rate and root growth. malondialdehyde (mda) level and glutathione peroxidase (gpx) activity was higher while proline accumulation was lower in pr compared to those of both cd and control treatments. secondary metabolites content changes in treatments, such as octacosane, cysteamine sulfonic acid, propyl oleate, 1-nanodecene, and 2-butyn-1-ol-4metoxy were synthesized in the low soil moisture conditions. leaf ca-pectate, ca-phosphate and dissolved ca tended to increase in low soil moisture. the pr1 treated plant had the highest water use efficiency. therefore, pr technique could be applied when the soil moisture level represents 50% (or more) of the field capacity. 1. introduction mangosteen (garcinia mangostana l.) is a tropical perennial crop that plays an ecologically important role in agroforestry system (wijayanto and hartoyo, 2015). it produces an exotic fruit with high antioxidant level (kurniawati et al., 2010). however, many producing countries, such indonesia, malaysia, thailand and india (osman and milan, 2006), are facing both fluctuations in production and yellow latex matter in mangosteen fruits production (sdoodee and limpun-udom, 2002; poerwanto et al., 2010). matra et al. (2016) reported that mangosteen exhibits moderate genetic variation within a population. on the other hand, according to (*) corresponding author: roedhy8@yahoo.co.id citation: hapsari d.p., poerwanto r., sopandie d., santosa e., 2018 partial root-zone irrigation effects on growth, metabolism and calcium status of mangosteeen seedling (garcinia mangostana l.). adv. hort. sci., 32(1): 49-59 copyright: © 2018 hapsari d.p., poerwanto r., sopandie d., santosa e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 october 2017 accepted for publication 13 december 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 49-59 50 martias and mansyah (2014), the variations in mangosteen quality were affected by seasonal variation, water availability, and cultivation techniques. furthermore, poerwanto et al. (2010) stated that high water fluctuation in the soil will affect the turgor pressure so that the duct secretory of yellow latex will break out and contaminate the fruit. as other tropical fruits, mangosteen requires low soil moisture root zone to promote flowering (paull and nakasone, 1998). however, extended low water status might adversely affect the plant growth (mustaha, 2012). preventing water fluctuation on mangosteen root zone is not easily carried out due to the fact that mangosteen is usually planted in an arid area (and depend on rainfall). on the other hand, mangosteen was spread in hills area with agroforestry system, making the irrigation setting difficult. thus, the management of irrigation becomes an important factor. however, mangosteen is allegedly non-responsive to irrigation due to the uniqueness of its root morphology (wiebel et al., 1994). indeed, irrigation was observed to be ineffective in decreasing the water fluctuation (sdoodee and chiarawipa, 2005). besides, the root growth of mangosteen is both slow and seasonal and it grows faster before the appearance of new leaves, steadily decreases during the leaves development, and stops post-dormancy period (hidayat, 2005). thus, an efficient irrigation technique is needed to decrease water fluctuation. partial root-zone irrigation (pri), often referred to as partial root-zone drying (prd), is a well-known irrigation method which alternately irrigates the root zone (sepaskhah and ahmadi, 2010). adwirman (2006) has already applied the prd technique on mangosteen but further studies are still needed on both physiological responses and nutrient status of the plant. in present study, the calcium status was also analyzed, either in the form of dissolved, pectate, phosphate and oxalate. in fact, calcium is one of important mineral in mangosteen, especially in relation to yellow latex (dorly et al., 2011; kurniadinata, 2015) and plays an important role in the mechanism of adaptation to stress condition (liu et al., 1998; chen et al., 2002), especially on signal transduction in the responses to water deficit (hong-bo et al., 2008). its status was analyzed in the present research to determine the correlation between the plant root water status and the occurrence of yellow latex, especially ca-complex such as ca-oxalate (korth et al., 2006; setyaningrum, 2011), ca-dissolved, phosphate, and pectate (saure, 2005). therefore, this study aims to: (1) determine the growth and morpho-physiological responses of mangosteen seedlings, (2) determine the role of calcium, and (3) evaluate the level of secondary metabolites in different water status conditions. 2. materials and methods orchard and plants two-years old mangosteen seedlings at averages height of 30±2.02 cm and leaves number, ranged from 17 to 20, were planted on pasir kuda experimental field (±260 m asl), bogor, indonesia between october 2016 and may 2017. a set of randomized block design, with 4 replicates, was used for field capacities of both conventional deficit irrigation (cd) and partial root-zone irrigation (pr) methods, comprised of 100% (control), 50% (cd1), 30% (cd2), 100% a: 50% b (pr1), 100% a: 30% b (pr2) and 50% a: 30% b (pr3). mangosteen seedlings were planted in root-boxes (50 x 40 x 20 cm) in accordance with each treatment. one side of the root-box was made of glass and covered with a black thick cloth to observe the root growth, in a non-destructive way. the root-box was divided into two parts (fig. 1) in the pr treatment and the partitions were layered by hydrophobic plastic material, in order to avoid water flow from one side to the other side. both sides of the root-box were filled with soil and compost at a ratio of 1:1 (w/w). mangosteen roots were carefully cleaned and divided into two symmetrical parts and planted on both side a and side b of the root-box, respectively. the planted mangosteen seedlings were acclimatized and watered within the field capacity conditions for four weeks. in the last week of the acclimatization period 100 g dolomite [camg(co3)2; ±30% cao] was applied in each root-box. fig. 1 root-box illustration in conventional deficit (a) and partial root-zone (b) irrigation treatment. in the pr treatment, the root was planted on 2 sides, side a and side b. hapsari et al. partial root-zone irrigation 51 field capacity determination soil water content (wc) and humidity (rh) were measured to quickly determine the field capacity (fc). soil water content was determined by gravimetric method (abdurachman et al., 2006). a hundred grams of soil was weighed (fw) and heated at 105oc for 24 hours (dw) and the soil water content calculated with the following equation: fc = (fw dw) / dw x 100% soil rh was measured by means of a soil moisture meter (ht5213, china). soil water content and rh were measured every 3 days for the optimization (table 1). water treatment was applied based on table 1. the 100% field capacity occurred when the condition was 64.39% for the soil water content and 10 for the rh. the 50% and 30% field capacity treatments were obtained at a soil water content of 28.16% (day 13) and 20.06% (day 25), respectively. in order to simplify the water availability application, the watering syllabus was set for every 2 days, 2 weeks and 3 weeks for the 100%, 50% and 30% field capacity treatments. soil moisture meter was installed on root-box to control the soil condition. measurements leaf water potential and relative water content. healthy mature leave samples were taken at 7.00 a.m., placed into sealed plastic, and kept in a cooler box for further observation in laboratories. the leaves were cut in a cup and measured in a wp4 chamber and leaf water potential was measured using a wp4 dewpoint potential meter (decagon devices inc, usa). the relative water content (rwc) measurement of leaves was carried out in accordance with the leaf water potential. the samples used in the leaf water potential measurement (the fresh weight/fw measured) were soaked in the cup containing distilled water. the surface of the cup was covered with filtering paper so that the leaves do not float. afterwards, the cup was kept in a cool storage for 24 hours, then drained and weighed to determine the turgid weight (tw). leaves samples were dried at 70°c for 3 days and weighed to determine the dry weight (dw). the rwc was calculated by means of the following equation: rwc = [(fw-dw) / (tw-dw)] x 100 photosynthesis and transpiration rates . photosynthesis and transpiration rate were measured prior to treatment and 2 months post-treatment using li-cor 6400 (li-cor inc, usa). calcium content. calcium content in dissolved form was determined following the method developed by suwwan and poovaiah (1978), while calcium in complexed forms was measured gradually according to chen and uetomo (1976) procedure. malondialdehyde (mda) level. lipid peroxidation activity was determined from mda content that was measured by mean of wang et al. (2013) procedure. briefly, 0.4 g leaves sample was homogenized with 10 ml tca. homogenate was centrifuged at 4oc for 10 min at 3000 g. then, 2.5 ml supernatant was added to the reagent which is containing of 0.5% tba and 20% tca to be incubated at 80°c for 25 min. the absorbance was read at 440, 532 and 600 nm. mda level was calculated with the following equation: mda = 6.45 (a532-a600) (0.56 x a440) proline content. proline content was measured following the method developed by bates (1973). in brief, 0.5 g leaves sample was homogenized with 3% sulfosalicylic acid. the homogenate was centrifuged at 12.000 g for 10 min. the supernatant was mixed with reagent which contains ninhydrin and glacial acetic acid. the mixture was incubated at 100°c for 60 min and transferred into an ice bath immediately. afterwards, sample was extracted with 4 ml toluene and stirred in vortex. the absorbance was read at 520 nm. proline concentration was calculated using proline standard curve. glutathione peroxidase (gpx) activity. gpx activity was analyzed following the method developed by urbanek et al. (1991). in brief, leaf were extracted in phosphate buffer. the reaction mixture containing table 1 optimization of soil water content and humidity of mangosteen root zone during drought stress to determine the watering syllabus numbers, within the same row, followed by the same letters showed no significant differences based on dmrt at a probability level of 5%. bold numbers indicating 100%, 50% and 30% field capacity, consecutively. variables period of drought stress (days) 1 4 7 10 13 16 19 22 25 water content (%) 64.39 a 43.06 b 44.24b 41.37 b 28.16 c 27.43 c 24.19 cd 21.12 d 20.06 d relative humidity (1-10) 10.00 a 8.68 b 8.41 bc 8.04 bc 7.60 c 7.60 c 5.71 d 5.71 d 5.53 d adv. hort. sci., 2018 32(1): 49-59 52 phosphate buffer (ph 7.0), edta, guaiacol, h2o2 and 50 l enzyme extract. the enzymatic reaction was initiated by addition of extract and the increase in absorbance recorded at 470 nm for 1 min. enzyme activity was quantified by the amount of tetraguaiacol formed using its molar extinction coefficient (26.6 mm-1 cm-1). secondary metabolites content. secondary metabolites was analyzed in jakarta regional health laboratory using gcms on fresh mature leaves from control and the most severe (pr3) treatment. plant growth and biomass. the length and volume of the root, the fresh and dry weight of plant were observed 2 months post treatments. plants were cleaned prior to observation. the root variable observation, in pr treatment, was carried out by merging both side a and side b. the length of root was measured from the boundary between root and main stem. the volume of root was measured based on archimedes principle. plant was weighed to measure the fresh weight then heated at 80oc for 72 h to determine the dry weight. statistical analysis data were analyzed with f test and duncan multiple range test (dmrt) at a probability level of 5% using statistical analysis system 9.4 (sas 9.4m4) software. 3. results the leaves in all treatments, except control, withered on three weeks post-treatment (fig. 2). however, the plants became fresh again after rewatered, except in cd2 and pr3 treatments. besides, abortion and drying leaves were also observed in mangosteen seedlings. abortion of old leaves was more severe in pr2 and pr3 treatments. in fact, stressed mangosteen leaf has a unique dried pattern. the whole leaves of the stressed mangosteen seedling did not dry entirely, indeed, it dried step by step starting from both the edge and tip of the leaf blade (fig. 2g). the reduction in leaves number was observed since the first month of the treatment; the decrease was more severe during the second month, especially in cd2, pr2, and pr3 treatments (average 9.1, 8.7, 7.2 leaves per plant, respectively) compared to control which had 14.9 leaves (table 2). pr1 treatment did not indicate any severe leaves abortion. thus, there were no significant differences in terms of leaves number between control and pr1 treatments from the beginning until the end of the treatment period (fig. 2 and table 2). fig. 2 mangosteen seedling canopy in different water availability treatments (2 months post treatment). a= control, b= conventional deficit irrigation, 50%, c =conventional deficit irrigation, 30%, d= partial root-zone irrigation 100% side a: 50% side b, e= partial root-zone irrigation 100% side a: 30% side b, f= partial root-zone irrigation 50% side a: 30% side b, g= dried pattern on mangosteen leaf). field capacity number of leaves 0 mat 1 mat 2 mat control (100%) 18.2 16.6 a 14.9 a cd1 (50%) 18.2 15.2 abc 10.8 b cd2 (30%) 19.2 14.7 bc 9.1 c pr1 (100%a:50%b) 19 16.0 ab 13.6 a pr2 (100%a:30%b) 18.3 14.0 c 8.7 c pr3 (50%a:30%b) 18.4 14.6 bc 7.2 d f-test ns * * table 2 decreasing in mangosteen leaves number in different water availability treatments numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. mat= month after treatment. mangosteen leaf water potential ranged from -2.95 to -3.59 mpa before the treatments (table 3). in present research, the leaf water potential above -3.59 mpa in two months after treatment was classified as a stress condition for seedlings, coincide with the leaf morphological characteristics which showed withered condition (fig. 2). leaf water content decreased post-drought treatment in both cd and pr conditions (table 3). the lowest water potential was observed in pr3 treatment (-4.72 mpa) which had a field capacity of 50% on side a and 30% on side b. on the other hand, leaf water content also decreased in hapsari et al. partial root-zone irrigation 53 all treatment compared to control, although the decreasing was not significant (α 5%), except for pr3 treatment which had the lowest leaf water content (table 3). drought treatment decreased both photosynthesis and transpiration rates of mangosteen seedlings two months after treatment (table 4). pr treatments showed the lowest photosynthesis and transpiration rate and, among pr treatments, pr3 recorded the worst performances in terms of photosynthesis and transpiration rate (6.06 µmol co2 m-2 s-1 and 0.02 mmol h2o m-2 s-1, respectively). the dry weights of canopies and roots were significantly lower in cd and pr treated plants than control (table 5). unexpectedly, pr1 treatment tended to have the lowest decrement in dry weights of the canopy, roots and total plant. on the other hand, the highest decrements were observed in cd2 and pr2 treatments on both canopy and total plant dry weight, although statistically similiar. control and pr1 treatments had the longest root apparatus (fig. 3), although they did not differ significantly (table 6). drought stress significantly restricted the root growth in cd1, cd2 and pr3, excepted pr1 and pr2 treatments. meanwhile the root volume did not show any differences among treatments. mda content in cd1, cd2 and pr1 treatments did not differ significantly to that of the control treatment (fig. 4). pr2 and pr3 treatments had the hightable 3 water potential and content of mangosteen seedling leaf in different water availability treatments at the beginning (0 months) and 2 months post-treatment numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. mat= month after treatment. treatment (fc) leaf water potential (mpa) leaf water content (%) 0 mat 2 mat 0 mat 2 mat control (100%) -2.95 -3.09 a 81.71 97.57 a cd1 (50%) -2.96 -4.00 b 77.5 71.62 ab cd2 (30%) -3.00 -3.99 b 73.93 53.55 ab pr1 (100%a:50%b) -3.59 -4.12 b 69.51 56.89 ab pr2 (100%a:30%b) -3.37 -4.51 b 68.08 58.81 ab pr3 (50%a:30%b) -3.53 -4.72 b 68.04 31.68 b f-test ns * ns * table 4 photosynthesis and transpiration rates of mangosteen seedling in different water availability treatments at the beginning (0 months) and 2 months post-treatment numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. mat= month after treatment. treatment photosynthesis rate (µmol co 2 m-2 s-1) transpiration rate (mmol h 2 o m-2 s-1) 0 mat 2 mat 0 mat 2 mat control (100%) 16.23 16.20 a 0.52 0.21 a cd1 (50%) 15.49 11.76 ab 0.49 0.11 ab cd2 (30%) 18.27 12.05 b 0.56 0.05 ab pr1 (100%a:50%b) 15.51 8.35 cd 0.62 0.07 ab pr2 (100%a:30%b) 15.56 10.42 bc 0.55 0.05 ab pr3 (50%a:30%b) 15.33 6.06 d 0.61 0.02 b f-test ns * ns * table 5 dry weight of mangosteen seedling canopy, root and total plant in different water availability treatments at 2 months posttreatment numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. treatment dry weight (g) relative decrease to control (%) canopy root total canopy dry weight root dry weight total dry weight control (100%) 12.84 a 5.15 a 16.43 a cd1 (50%) 9.20 b 3.08 b 12.28 b 28.35± 0.15 40.19±1.02 39.80±0.11 cd2 (30%) 7.44 b 3.33 b 10.77 b 42.06±0.11 35.34±1.00 47.21±0.10 pr1 (100%a:50%b) 9.65 b 4.83 ab 13.98 ab 24.84±0.11 6.21±0.73 31.47±0.13 pr2 (100%a:30%b) 6.99 b 3.87 ab 10.49 b 45.56±0.07 24.85±1.14 48.58±0.01 pr3 (50%a:30%b) 8.30 b 3.49 b 12.17 b 35.36±0.08 32.23±2.05 40.34±0.03 f-test * * * fig. 3 the root system of mangosteen seedling in different water availability treatments (2 months post treatment). c= control), cd1= conventional deficit irrigation 50%, cd2= conventional deficit irrigation 30%, pr1= partial root-zone irrigation 100% side a:50% side b, pr2= partial root-zone irrigation 100% side a:30% side b, pr3= partial root-zone irrigation 50% side a:30% side b. adv. hort. sci., 2018 32(1): 49-59 54 est mda content, being 1.033 and 1.501 µmol/ml respectively, which were significantly different to that of the control treatment. proline accumulation was in accordance with that of mda content with the highest value in pr3 treatment followed by cd1 and cd2 (fig. 4). proline content in pr1 and pr2 treatments did not show any significant different to that of the control treatment. glutathione peroxidase (gpx) activity showed different result compared to proline and mda. in fact, the control treatment had the highest gpx activity showed not significantly differences among treatments, except pr3 treatment (fig. 4), as a result of severe stress. mangosteen seedling, on limited water condition, produced more diverse secondary metabolites than the control, especially terpenoid and fatty acid (table 7). five secondary metabolites were found in the leaves of control plant, i.e. oleic and hexadecenoic acid (from the fatty acid group), squalene, vitamin e and neophytadiene (from terpenoid group). vitamin e and squalene content increased in stressed mangosteen seedling by 28% and 62% respectively, while oleic acid content decreased by 15%. on the other hand, hexadecenoic acid and neophytadiene were not detected in stress plant. there were 2 kinds of unknown compounds produced in severe stress, i.e. 1-nonadecene and 2-butyn-1-ol 4 metoxy. dissolved ca was mostly found in mature leaves, young leaves, roots, and branch, while ca-pectate and ca-phosphate were mostly found in mature leaves, roots, young leaves and branch (table 8). table 8 shows that both dissolved and pectate calcium contents were high in severely stressed plants, especially in young and mature leaves. furthermore, the leaves calcium content in dissolved and pectate form of cd1 treatment were significantly higher than that of control. in cd2 treatment, calcium, in dissolved and pectate form, was tended to be lower compared to cd1 treatment but not significantly different from the control. a similar result was observed in dissolved calcium content of mangosteen seedling roots, while no differences in calcium content in branch, was observed. table 8 also shows that ca-phosphate and ca-oxalate in branch were significantly higher in all pr treatments compared to control, while there were no differences in ca-oxalate content in leaves and roots. table 6 root length and volume of mangosteen seedling in different water availability treatments at 2 months posttreatment numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at probability level of 5%. treatment root length (cm) root volume (ml) control (100%) 35.00 ab 11.67 cd1 (50%) 32.70 bc 8.33 cd2 (30%) 29.45 cd 6.33 pr1 (100%a:50%b) 37.75 ab 10.33 pr2 (100%a:30%b) 33.30 abc 7.67 pr3 (50%a:30%b) 28.00 d 7.5 f-test * ns fig. 4 mda (a), proline (b) content and gpx activity (c) of mangosteen seedling in different water availability treatments (2 months post-treatment). numbers followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. table 7 secondary metabolites content in control and pr3 treatments of mangosteen seedling 2 months posttreatment secondary metabolite compound group of compounds the existence control pr3 oleic acid unsaturated fatty acid ++ ++ iliadic acid unsaturated fatty acid + hexadecenoic acid saturated fatty acid + squalene terpenoid + + vitamin e terpenoid + + neophytadiene terpenoid + octacaine acyclic hydrocarbon + cysteaminesulfonic acid amino acid + propyl oleate ester fatty acid + 1-nonadecene unknown + 2-butyn-1-ol, 4 methoxy unknown + not detected, + peak area below 20%, ++ peak area between 20-50%, pr3= partial root-zone irrigation 50% side a:30% side b. hapsari et al. partial root-zone irrigation 55 4. discussion and conclusions in the present study, mangosteen leaves changes its morphology and aborts as a consequence of drought stress in different water treatments. leaves abortion was more marked in cd2, pr2 and pr3 treatments, a common symptom of plants under drought stress (munne-bosch and alegre, 2004). it was expected that the leaves of cd2, pr2 and pr3 treatments would accumulate more aba and trigger the abscission process (wingler and roitsch, 2008; peleg and blumewald, 2011). on the other hand, leaves abortion in control and pr1 treatments were not significantly different from the beginning until the end of the treatment period, indicating that pr1 were not severely water stressed. morphological changes and leaves abortion suggest that mangosteen seedlings were less tolerant to drought stress. however, mangosteen showed a low response to drought stress since the withered leaves occurred on 3 weeks post-treatment. the low response in mangosteen was expected to be a consequence of mangosteen seedling low growth as stated by ramlan et al. (1992). an high relation between leaf morphology and changes in water potential due to the variations in terms of water treatments was observed. it is expected to be the mechanism of mangosteen adjustment by decreasing water potential in tissues in order to absorb the water in the soil. zimmerman (1978) stated that turgor potential is partially or fully maintained by osmoregulation during water stress by a reduction in the outflow of water from the cell. in previous study, the decrement of the leaf water table 8 calcium content of mangosteen seedling in different water availability treatments at 2 months post-treatment numbers, in the same column, followed by the same letter indicates no significant differences based on dmrt at a probability level of 5%. treatment calcium (%) dissolved pectate phosphate oxalate total young leaves control (100%) 0.133 c 0.134 c 0.084 c 0.405 0.756 c cd (50%) 0.396 bc 0.247 bc 0.096 bc 0.547 1.287 bc cd (30%) 0.349 c 0.227 bc 0.091 c 0.428 1.096 bc pr1 (100%a:50%b) 0.473 bc 0.290 abc 0.135 ab 0.668 1.568 ab pr2 (100%a:30%b) 0.826 ab 0.429 ab 0.135 ab 0.372 1.763 ab pr3 (50%a:30%b) 1.065 a 0.488 a 0.157 a 0.425 2.136 a mature leaves control (100%) 0.322 b 0.267 b 0.124 b 0.611 1.326 b cd (50%) 0.741 a 0.462 a 0.319 a 0.814 2.337 a cd (30%) 0.600 ab 0.362 ab 0.187 ab 0.501 1.651 ab pr1 (100%a:50%b) 0.690 a 0.428 a 0.185 ab 0.743 2.047 ab pr2 (100%a:30%b) 0.814 a 0.346 ab 0.209 ab 0.68 2.051 ab pr3 (50%a:30%b) 0.763 a 0.441 a 0.174 ab 0.473 1.851 ab branch control (100%) 0.078 0.171 0.086 b 0.763 bc 1.099 bc cd (50%) 0.134 0.198 0.107 ab 0.923 abc 1.364 abc cd (30%) 0.144 0.159 0.104 ab 0.555 c 0.963 c pr1 (100%a:50%b) 0.156 0.247 0.127 ab 1.266 a 1.798 a pr2 (100%a:30%b) 0.142 0.236 0.139 a 1.108 ab 1.627 ab pr3 (50%a:30%b) 0.148 0.227 0.123 ab 1.335 a 1.835 a root control (100%) 0.202 b 0.373 0.148 0.675 1.399 cd (50%) 0.342 a 0.307 0.219 0.971 1.84 cd (30%) 0.231 ab 0.317 0.192 0.862 1.604 pr1 (100%a:50%b) 0.185 b 0.232 0.167 1.443 2.052 pr2 (100%a:30%b) 0.208 b 0.247 0.17 1.296 1.904 pr3 (50%a:30%b) 0.273 ab 0.287 0.198 1.316 2.075 adv. hort. sci., 2018 32(1): 49-59 56 potential and relative water content were occurred in stressed wheat (siddique et al., 2001) and hibiscus rosa-sinensis (egilla et al., 2005). the decreasing of photosynthesis and transpiration rate in stress mangosteen seedlings indicated that both moderate (50% fc) and severe (30% fc) stress conditions greatly affect mangosteen gas exchange. purwanto and agustono (2010) reported that photosynthesis rate in soybean, which was watered at a condition of 60% fc, decreased by 50%, while no significant fall in transpiration rate was noticed. as response to drought stress, stomata respond by reducing aperture, thereby restricting water loss, however, an inevitable consequence is the photosynthesis and canopy transpiration (loveys et al., 1999) reduction. as mentioned in table 6, control and pr1 treatments showed the longest roots compared to the other treatments, while the root volume did not show any differences among treatments. hidayat (2005) reported in his research that mangosteen root has a seasonal growth where roots alternately grow with shoots. it expected lead to the slow response of mangosteen roots to drought stress. on the other hand, no significant differences were noticed in pr1 treatment in terms of root dry weight compared to control (table 5). this was in agreement with liu et al. (2006) which reported that prd increased biomass allocation to roots. promoting root growth under prd has been reported in grapevine (dry et al., 2000), therefore this has been considered as an advantage of prd irrigation. drought stress condition lead to high production of mda which used as a stress indicator in the plant. high mda content indicated that the lipid peroxidation rate, as the main effect of oxidative damage (gill and tuteja, 2010), was also high. sofo et al. (2005) conclude that there is a direct correlation between mda and drought stress, particularly at severe degrees of stress. in present research, the most severe case was observed in pr3 treatment which had highly mda level that coincided with decrements in leaf water potential, photosynthesis and transpiration rates. besides mangosteen, lipid peroxidation was also noticed in cucumber (kubis et al., 2014), bean (svetleva et al., 2012) and maize (ti-da et al., 2006) within a stress condition. the accumulation of proline is a common response in plants to abiotic stress. increasing proline is a plant response to adjust its osmotic potential (slama et al., 2006) which has a strong relation with plant water potential. in addition to its role as an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures, scavenging free radicals, and buffering cellular redox potential under stress conditions (ashraf and foolad, 2007). present research was in agreement with omidi (2010) which stated that in canola plants, proline content increased twofold as a result of drought stress treatment. in the present study, the control treatment had the high gpx activity but did not show significantly differences among treatments, except pr3. it indicates that in normal condition, mangosteen seedlings have the high antioxidant activity, then became higher when the stress condition occurs, such as pr3. halušková et al. (2009) stated that different abiotic stresses may cause differences in the gpx activation. miller et al. (2010) noticed that gpx is plant protector against free radical. previous studies by sofo et al. (2005) and aganchich et al. (2007) have reported upregulation of the antioxidant defense system in young olive plants subjected to different degrees of water stress. mangosteen seedlings in limited water media produced secondary metabolites more than control, especially terpenoid and fatty acid groups. varied antioxidant profile in different plant species is one of the principle reasons for the different adaptability and abiotic stress tolerance in plants (jamali et al., 2016). changes in composition and synthesis in drought stress condition were reported for group of terpenoid such as vitamin e (gershenzon et al., 1978), an important antioxidant that protects the cell from free radical effects (serbinova and packer, 1994) and photosynthetic apparatus (fryer, 1992) from oxidative damages. in the present research, the increasing in vitamin e content by 3.2%, in stress mangosteen seedlings, was a response to the stress condition. abiotic stress-induced changes in the fatty acid composition of plant membrane lipids mainly occur through the regulated activities of fatty acid desaturases (upchurch, 2008). arabidopsis thaliana shows remarkable tolerance to drought stress and has capacities to maintain polar lipid content and stable lipid composition, and increase the fatty acid unsaturation (gigon et al., 2004). mangosteen seedlings under pr1 treatment had the highest calcium level. according to bell and biddulph (1963) some plants absorb calcium based on their physiological demand which is sometimes not comparable to the transpiration rate. as menhapsari et al. partial root-zone irrigation 57 tioned in table 8, calcium contents were higher in drought stress plants in treatments of cd1, pr1, pr2 and pr3. allegedly, there was a relationship between the response of mangosteen seedlings and both long watering interval time and calcium metabolism. cd2 treatment had a longer watering interval time compared to cd1 so that when the drought signaling occurred the water was not available, leading to a fail in calcium absorption. the increment in calcium levels in cd1, pr1, pr2 and pr3 treatments occurred in the forms of ca-pectate and ca-dissolved, both of which play a role in cell wall component (peaucelle et al., 2012) and cytoplasmic transduction signaling (klimecka and muszyńska, 2007), respectively. this was in agreement with jin et al. (2016) which reported that increment in calcium levels in a salinity stress condition occurred in ziziphus jujuba species. mangosteen seedlings in pr1 treatment had higher water use efficiency (wue) leading the possibility of calcium absorption in stress condition. in pr treatment, improvement in wue was a results from partial stomatal closure. however, an inevitable consequence is the photosynthesis and canopy transpiration reduction (mccarthy et al., 2002). hu et al. (2008) showed that pr method in maize able to preserve 29.5-33% water and increased wue. the use of partial root-zone or deficit irrigation in grapevine (vitis vinifera) increased wue by about 40% while only decreasing yield by 15% when compared with full irrigation (dos santos et al., 2003). mangosteen seedlings had a low response and different mechanisms in facing drought, by increasing mda, proline, ca-pectate and ca-dissolved, gpx activity and synthetizing various secondary metabolites in mature leaves. in the present study, there were no significant differences between control and pr1 treatments in leaf morphology, proline, mda and gpx activity, indicating that pr1 treatments were not severely water stressed. therefore, present research concluded that irrigation of mangosteen through pr method was a promising method for implementation, especially in limited water areas, when the soil moisture level represents 50% (or more) of the field capacity. the implementation of pr method can reduce water requirement without significantly affecting the plant growth. it can reduce both time and labor requirements. however, improvements in the technical application to develop an effective procedure for field implementation and its relation to flowering and yellow latex of mangosteen, are still needed as further studies. acknowledgements the authors would like to thank the ministry of research, technology and higher education for funding and supporting the present research through pmdsu program, batch ii, in the fiscal year of 2016. references abdurachman a., haryati u., juarsah i., 2006 determination of soil water content by gravimetric method, pp. 131-142. in: kurnia u., f. agus, a. adimihardja, and a. dariah (eds.) soil physical properties and its analysis method . department of agriculture, jakarta, indonesia, pp 282. 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[in indonesian]. wingler a., roitsch t., 2008 metabolic regulation of leaf senescence: interaction of sugar signaling with biotic and abiotic responses. plant biology, 10(1): 50-62. zimmermann u., 1978 physics of turgor and osmoregulation. ann. rev. plant physiol., 29: 121-148. impaginato 407 adv. hort. sci., 2018 32(3): 407-420 doi: 10.13128/ahs-22856 quality and nutraceutical properties of mango fruit: influence of cultivar and biological age assessed by time‐resolved reflectance spectroscopy m. vanoli 1, m. grassi 1, l. spinelli 2, a. torricelli 2, 3, a. rizzolo 1 (*) 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (creait), sede di milano, via g. venezian, 26, 20133 milano, italy. 2 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn-cnr), piazza l. da vinci, 32, 20133 milano, italy. 3 politecnico di milano, dipartimento di fisica, piazza l. da vinci, 32, 20133 milano, italy. key words: absorption coefficient, ascorbic acid, carotenoid composition, mangifera indica l., total antioxidant capacity, total phenols. abstract: the content and composition of the main antioxidants in the pulp of mangoes depend also on cultivar and maturity degree, the latter being non‐ destructively evaluated by the absorption coefficient measured by time‐ resolved reflectance spectroscopy (trs) at 540 nm (µa540). aiming at evaluat‐ ing the levels of antioxidants [carotenoids (car), phenols (tpc), ascorbic acid (aa)] and antioxidant capacity (tac) in relation to µa540 maturity class, select‐ ed ‘haden’ and ‘palmer’ mangoes were measured for µa540 by trs, classified based on µa540 value as less (lem), medium (mem) and more (mom) mature and analyzed for pulp firmness, pulp color (a*, h°, yellowness index), car (total and composition by hplc‐dad), tpc, aa and tac. ‘palmer’ fruit had higher tpc, aa and tac than ‘haden’ mangoes. on average mom fruit showed higher tpc, total car, total all‐trans‐violaxanthin esters and all‐trans‐β‐carotene than mem and lem fruit. lem fruit did not have compounds belonging to the 9‐cis‐violaxanthin group, while cis‐β‐cryptoxanthin was approx. 19% of total carotenoids. in mom mangoes the main carotenoid was all‐trans‐β‐carotene (53%), followed by total all‐trans‐violaxanthin esters (30%), 9‐cis‐violaxanthin group (8%) and cis‐β‐cryptoxanthin (6%). the µa540 significantly correlated (r=0.78‐0.94) with total car, all‐trans‐β‐carotene, all‐trans‐violaxanthin no.3 (both cultivars), tpc, all‐trans‐violaxanthin no.1, no.2, no.6 (‘haden’), and 9‐cis‐violaxanthin no.2, no.3 (‘palmer’). our results indicate that trs is suitable to non‐destructively measure the pulp color of mangoes and to sort fruit with different ripening degree and nutraceutical properties. 1. introduction mango (mangifera indica l.) is a climacteric fruit belonging to the family of anacardiaceae grown particularly in tropical and subtropical coun(*) corresponding author: anna.rizzolo@crea.gov.it citation: vanoli m., grassi m., spinelli l., torricelli a., rizzolo a., 2018 quality and nutraceutical properties of mango fruit: influence of cultivar and biological age assessed by time-resolved reflectance spectroscopy. adv. hort. sci., 32(3): 407-420 copyright: © 2018 vanoli m., grassi m., spinelli l., torricelli a., rizzolo a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 march 2018 accepted for publication 13 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 407-420 408 tries, with an estimated world production of 42 million tons per year (fao, 2015). brazil is one of the first ten largest mango producers, and more than 25% of its production is exported to europe (mitra, 2016). appreciated for its excellent eating quality due to attractive flesh color, juicy texture and sweet flavor, mango provides high contents of bioactive compounds including carotenoids, phenolic compounds, ascorbic acid and reducing sugars (rocha ribeiro et al., 2007; manthey and perkins-veazie, 2009). carotenoids are responsible for the yellow-orange color of mango pulp and their content and composition depend mainly on cultivar and maturity degree, along with edaphic and climatic factors and postharvest handling, processing and storage conditions (ornelas-paz et al. , 2007, 2008; manthey and perkins-veazie, 2009; hewavitharana et al., 2013; liu et al., 2013; vásquez-caicedo et al., 2006; vanoli et al., 2016). during mango fruit ripening, biosynthesis of carotenoids occurs, due to chloroplasts differentiation into chromoplasts by disintegration of the thylacoid membranes and by the development of new pigment-bearing structures (vásquez-caicedo et al., 2006). this process leads to carotenoids accumulation, which is usually accompanied by color changes of the pulp turning from white to yellow-orange (ornelas-paz et al., 2008; vásquez-caicedo et al., 2006). the accumulation of carotenoids in the mesocarp shows an exponential behavior during fruit ripening and cultivar-specific relationships between total or individual carotenoid (all-trans-β-carotene, all-trans-violaxanthin and 9-cis-violaxanthin) contents and mesocarp color (a*, h°) were established in different mango cultivars (vásquez-caceido et al., 2006; ornelas-paz et al. , 2008). similarly to carotenes, ascorbic acid content and total phenolic content vary according to the cultivars, maturity stage and cultural practices (rocha ribeiro et al., 2007; valente et al., 2011; liu et al., 2013; oliveira et al., 2016; septembre-malaterre et al., 2016). in order to withstand shipping to distant markets and at the same time to have the optimum eating quality when ripe, mango fruit are harvested at the hard green stage, after having reached the physiological maturity, but before the onset of the climacteric rise. if mango fruit are immature at harvest, they do not reach an eating quality when ripe and, hence, the discrimination between mature and immature fruit at harvest is very important from the marketing point of view in order to minimize qualitative losses during the supply chain. fruit shape (“shoulders” should be full), pulp color and firmness are the most used maturity indices for mangoes, but differences among cultivars and growing conditions have precluded universal maturity indices. on the other hand, the current industry measurements of firmness and pulp color have the disadvantage of being destructive of fruit; hence, the development of a non-destructive technique could help the growers to pick fruit at the proper maturity degree for the different market destinations. among the non-destructive techniques able to assess the maturity degree of fruit, time-resolved reflectance spectroscopy (trs) is gaining increasing interest (nicolai et al., 2014). trs is a non-destructive optical technique which can separate the effect of light absorption, due to chemical compounds such as pigments and water, and light scattering, due to microscopic changes in refractive index caused by membranes, vacuoles, starch granules, organelles, and air. by measuring photon time-of-flight distribution with picoseconds temporal resolution, the absorption (µa) and reduced scattering (µs) coefficients in the vis-nir wavelength range are quantified, by probing pulp at a depth of 1-2 cm with no or limited influence from the skin (cubeddu et al., 2001, torricelli et al., 2008). trs has been used to study the internal fruit attributes related to maturity in apples, peaches, nectarines and pears (rizzolo and vanoli, 2016). in nectarines, the µa measured at harvest at 670 nm (µa670), near the chlorophyll-a peak, can be considered an effective maturity index as it is linked to the biological age of the fruit (tijskens et al., 2007) and has been successfully used to predict the softening rate during shelf life in nectarines and to select fruit for different market destinations (eccher zerbini et al., 2009). previous studies carried out on mango fruit have demonstrated the potential of trs for the nondestructive determination of pulp color (vanoli et al., 2013; rizzolo et al., 2016; vanoli et al., 2016), as well as the possibility of using trs absorption in both the carotenoids (540 nm) and chlorophyll-a (670 nm) spectral regions to classify mango fruit according to maturity and to predict the ripening of individual fruit (eccher zerbini et al., 2015). the present work aimed at evaluating the levels of antioxidants and antioxidant capacity in the pulp of two cultivars of brazilian mangoes in relation to fruit maturity class assigned according to the µa540 value, along with selected quality parameters. the relationships between µa540 maturity index and total carotenoid content, total phenolic compounds, ascorbic acid and the fourteen carotenoid comvanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 409 pounds identified were also studied. 2. materials and methods mango fruit on november 2011, ‘haden’ and ‘palmer’ mangoes were picked in experimental orchards of epamig (empresa de pesquisa agropecuária de minas gerais) in minas gerais state (brazil) and transported to milan (italy) by plane soon after harvest. on arrival at crea-it lab (about 5-7 days from harvest), 60 fruits of ‘haden’ and 90 fruits of ‘palmer’ without defects were selected and measured by trs at 650 nm (‘haden’) or at 690 nm (‘palmer’) on two opposite sides in the fruit equatorial region and ranked by decreasing µa averaged over the two sides, that is from less (high µa) to more mature fruit (low µa). ‘haden’ fruit were put at 20°c for 2 days, while ‘palmer’ mangoes were randomized into 3 batches of 30 fruits, corresponding to 0, 4 and 11 days of shelf life at 20°c, in order to have the whole range of µa690 in each batch. sub-samples of 10 fruits, covering the whole range of µa, were selected for ‘haden’ and for ‘palmer’ batch held for 4 days at 20°c. each selected fruit was measured by trs at 540 nm on two opposite sides in the equatorial region; in the same positions of trs measurements, skin was removed by a slicer, and, after measuring pulp color and firmness, the whole fruit were immediately deep frozen and kept at -30°c until carotenoid, ascorbic acid and total phenolic extractions. time-resolved reflectance spectroscopy (trs) a portable compact setup working at discrete wavelengths developed at politecnico di milano (spinelli et al., 2012) was used. the light source is a supercontinuum fiber laser (sc450-6w, fianium, uk) providing white-light picoseconds pulses, with duration of few tens of picoseconds. a custom-made filter wheel loaded with 14 band-pass interference filters (nt-65 series; edmund optics) is used for spectral selection in the range 540-940 nm. light is delivered to the sample by means of a multimode graded-index fiber and diffuse remitted light is collected by 1 mm fiber placed at 1.5 cm distance from the illumination point. a second filter wheel identical to the first one is used for cutting off the fluorescence signal originated when illuminating the fruit in the visible spectral region. the light then is detected with a photomultiplier (hpm-100-50, becker & hickl, germany) and the photon time-of-flight distribution was measured by a time-correlated single-photon counting board (spc-130, becker & hickl, germany). the instrumental response function has a full width at half maximum of about 260 ps and the typical acquisition time is 1 s per wavelength. a model for photon diffusion in turbid media was used to analyze trs data to assess the bulk optical properties of the samples (martelli et al., 2009) to obtain the estimates of µa and µs at each wavelength. firmness and pulp color flesh firmness was measured using a instron utm model 4301 penetrometer (crosshead speed 200 mm min-1, 8 mm diameter plunger). data were averaged per fruit. pulp color was measured with a spectrophotometer (cm-2600d, minolta co., japan), using the primary illuminant d65 and 2° observer in the l*, a*, b* color space. hue (h°) was computed from a* and b* values according to: h°= arctangent (b*/a*) x 360/(23 x 14) yellowness index (i y ) was computed as: i y =[81.2746x−1.0574z)/y]×100 after converting l*a*b* parameters into the xyz color space (jha et al., 2006). data were averaged per fruit. carotenoids, ascorbic acid, total phenols and total antioxidant capacity analysis carotenoids, ascorbic acid, total phenols and total antioxidant capacity analysis were carried out on frozen fruit after 30 min thawing at ambient temperature, by slicing pulp portions without peel near the positions of trs measurements and pooling the slices coming from the two fruit sides. carotenoids were extracted following the procedure described by picchi et al. (2012) with slight modifications. briefly, on individual fruit, 2 g of pulp (two replicates) was extracted with 10 ml of a solution of hexane:acetone:ethyl acetate (2:1:1 v/v/v) containing 100 µl of 1% butylhydroxytoluol (bht) in methanol, to prevent carotenoid oxidative degradation, and centrifuged at 4°c at 15,000 rpm for 20 min. the extracts were stored at -80°c until spectrophotometric and high-performance liquid chromatographic (hplc) analyses. total carotenoid content (car) was determined measuring absorbance at 450 nm using a spectrophotometer (uv-uvidec 320, jasco, japan). the hexane:acetone:ethyl acetate solution was used as the blank. total carotenoid content was estimated from a standard curve of all-trans-β-carotene and data were expressed as mill igrams of β-carotene adv. hort. sci., 2018 32(3): 407-420 410 equivalent (β-car) per kilogram of fresh weight (mg βcar kg fw-1). carotenoid composition was determined on extracts according to azevedo-meleiro and rodriguez-amaya (2004) with some modifications. a jasco (tokio, japan) hplc system consisting of a pu1580 liquid chromatographic pump coupled to lg 1580-04 quaternary gradient unit, a model as 2055plus autosampler and an md 2010-plus multi-wavelength detector was used. separations were performed on an inertsil ods-3 column (4.6 mm i.d × 250 mm length, particle diameter 5 µm, gl science) at the temperature of 40°c which was maintained using a jasco co-1560 intelligent column thermostat. the sample injection volume was 80 µl. the column was eluted with 20% methanol and 80% of a gradient mixture of acetonitrile (a) and ethyl acetate (b) at the flow rate of 0.6 ml min-1, with 10% b at 0-25 min, 10-20% b at 25-35 min, 20-50% b at 35-40 min, 50% b at 40-45 min, 50-10% b at 45-50 min. spectra of all peaks were recorded in the 200-600 nm wavelength range, and peak areas were monitored at 450 nm. carotenoids (table 1) were identified by comparing their retention times and spectral characteristics with those of standards (all-trans-β-carotene and violaxanthin, obtained by pansy petals) and with those reported in the literature (ornelas-paz et al., 2007, 2008), considering the three maximum absorbance wavelengths (λmax) and the spectral fine structure (% iii/ii), which is the percentage of the peak height of the longest wavelength absorption band (λmax iii) to that of the middle absorption band (λmax ii), taking the maximum of the valley between peak ii and peak iii as the baseline (sajilata et al., 2008). carotenoids were quantified referring to the total carotenoid content estimated spectrophotometrically on the same extract in conjunction with the chromatogram percent composition and data were expressed as milligrams of β-carotene equivalent (β-car) per kilogram of fresh weight (mg β-car kg fw-1). all the measurements were carried out in triplicate. the vitamin a value, expressed as retinol equivalent (re) was estimated from all-trans-β-carotene and cis-β-cryptoxanthin amounts using as conversion figures 6 µg for car and 12 µg for crypt (capra, 2006). ascorbic acid was extracted following the procedure described by robles-sánchez et al. (2009 a) with slight modifications. briefly, on individual fruit, 2 g of pulp (two replicates) was homogenized with 10 ml of 6% (w/v) aqueous solution of metaphosphoric acid (mpa), vortexed for 30 s, and centrifuged at 4°c at 15,000 rpm for 20 min and the extracts were kept at -20°c till hplc analysis. ascorbic acid was determined on just thawed extracts according to the conditions reported by rizzolo et al. (2002), using a jasco (tokio, japan) hplc system consisting of a pu-980 liquid chromatographic pump, a model as 1055-10 autosampler and an uv-vis 15770 detector set at 254 nm, coupled to an inertsil ods-3 column (4.6 mm i.d. × 250 mm length, particle diameter 5 µm, gl science) at the temperature of 30°c, which was eluted with 0.02 m orthophosphoric acid at a flow rate of 0.7 ml min-1. ascorbic acid was estimated from a standard curve of l-ascorbic acid in 6% mpa and data were table 1 retention time (rt, min), spectra characteristics [λmax (nm) in the mobile phase, obtained by dad, spectral fine structure (% iii/ii)] and name abbreviation of tentatively identified compounds according to ornelas-paz et al. (2007, 2008) peak no. r t λmax i λmax ii λmax iii % iii/ii tentative identification abbreviation 1 5.24-5.89 419 439 471 82 unknown unk 2 5.92-5.97 415 439 471 82 all-trans-violaxanthin various esters viol no. 1 3 6.03-6.09 415 439 471 100 all-trans-violaxanthin various esters viol no. 2 4 6.11-6.19 415 443 471 90 cis-β-cryptoxanthin crypt 5 6.23-7.29 415 439 471 93 all-trans-violaxanthin various esters viol no. 3 6 7.40-7.83 411 435 463 75 9-cis-violaxanthin 9-viol no. 1 7 8.03-8.77 415 435 467 83 9-cis-violaxanthin 9-viol no. 2 8 9.40-10.31 415 439 467 84 all-trans-violaxanthin various esters viol no. 4 9 10.32-11.15 411 435 463 80 9-cis-violaxanthin 9-viol no. 3 10 33.48-36.19 419 439 471 n.c. all-trans-violaxanthin various esters viol no. 5 11 37.97-40.61 451 479 23 all-trans-β-carotene car 12 40.90-43.60 419 439 471 100 all-trans-violaxanthin various esters viol no. 6 13 43.71-43.99 415 435 467 100 9-cis-violaxanthin 9-viol no. 4 14 44.00-44.60 415 439 467 100 all-trans-violaxanthin various esters viol no. 7 vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 411 3. results trs optical properties in ‘palmer’ fruit, µa690 at harvest ranged from 0.074 cm-1 for the less mature fruit to 0.021 cm-1 for the more mature ones and decreased to 0.061 cm-1 and 0.019 cm-1, respectively, after 4 days of shelf life at 20°c; concomitantly, after shelf life, µa540 ranged from 0.117 cm-1 for the least mature fruit to about 0.33 cm-1 for the most mature ones. in ‘haden’ mangoes, µa650 ranged at harvest from 0.231 cm-1 to 0.030 cm-1, with the majority of the fruit in the 0.0300.065 cm-1 range; after 2 days of shelf life at 20°c, µa650 decreased only in less mature fruit, whereas in all the other mango fruit it increased to values ranging from 0.036 cm-1 to 0.053 cm-1, while the µa540 values after shelf life ranged from 0.157 cm-1 for the least mature fruit to 0.835 cm-1 for the most mature ones. the µa540 maturity index, related to carotenoids content, was then used to classify the selected fruit within each cultivar in three trs maturity classes: less mature (lem) with low µa540, more mature (mom) with high µa540 and medium mature (mem) with intermediate values of µa540. cultivar and trs maturity class influenced the value of µa540 maturity index (table 2); on average µa540 was higher in cv. haden (‘haden’: 0.400±0.025 cm-1; ‘palmer’: 0.248±0.025 cm-1) and in the mom class in both cultivars, with mom ‘haden’ fruit being characterized by the highest µa540 value. quality parameters trs maturity class and cultivar greatly affected a* and h° pulp color parameters and had only a slight influence on firmness, probably due to the high standard error values, whereas iy depended only on maturity class (table 2). on average, ‘palmer’ fruit had lower firmness and a* value, and higher h° than ‘haden’ fruit. in ‘palmer’ mangoes firmness did not vary with maturity class, while in ‘haden’ firmness showed the highest values in lem fruit and the lowest in mem and in mom ones. mom ‘palmer’ fruit had higher a* and iy and lower h° than lem and mem maturity classes, whereas lem ‘haden’ fruit had lower iy than mom fruit, and a* increased and h° decreased from lem to mem and mom maturity classes. ascorbic acid, total phenolic content and total antioxidant capacity aa content and tac were significantly influenced only by cultivar, while tpc depended by both cultivar expressed as milligram per kilogram of fresh weight (mg kg fw-1). all the measurements were carried out in triplicate. total phenol content (tpc) and total antioxidant capacity (tac) were determined on the same extract (two replicates/fruit) obtained by homogenizing 2 g of pulp with 10 ml of acidic ethanol (ethanol:0.04 m hcl, 1:1 v/v), vortexed for 30 s and centrifuged at 4°c at 15,000 rpm for 20 min. extracts were kept at -20°c till total phenol content and antioxidant capacity determinations. tpc was determined using the folinciocalteau method (singleton et al., 1999) based on the reduction of a phosphowolframate phosphomolibdate complex by phenolics to blue reaction products, and measuring absorbance at 730 nm using a spectrophotometer (uv-uvidec 320, jasco, japan). the tpc was estimated from a standard curve of gallic acid and data were expressed as milligrams of gallic acid equivalents (gae) per kilogram of fresh weight (mg gae kg fw-1). all the measurements were performed in triplicate. tac was evaluated using the free radical 1,1,-dyphenyl-2-picrylhydrazil (dpph•) according to brand-williams et al. (1995) with modifications. fifty microlitres of sample extract or trolox standard solution (0.02-0.8 mm) were added to 2 ml of ethanol and 550 μl of dpph• solution (0.05 mm in ethanol) and, during 5 min of incubation, the absorbance at 517 nm was measured with a jasco 7800 uv/vis spectrophotometer (jasco europe s.r.l., cremella, lc, italy). the dpph scavenging capacity of the samples was calculated using a standard curve of trolox, and expressed as micromoles of trolox equivalents (te) per kilogram of fresh weight (µmol te kg fw-1). all the measurements were performed in triplicate. statistical analysis the statgraphics v. 5.2 (manugistic inc., rockville, md, usa) software package was used. data were submitted to multifactor analysis of variance (anova) considering cultivar, trs maturation class and their interaction as source of variation. in addition, one-way anova was used to study the main factors (cultivar, trs maturity class), and the trs maturity class within each cultivar. percentage data of carotenoids were analyzed after arcsine transformation. means were compared by 95 percent bonferroni’s test. relationships between µa540 and pulp color parameters and between µa540, a*, h°, iy and ascorbic acid, tpc, tac and carotenoids were studied using regression analysis. for each parameter, the model with the higher performance was considered. 412 adv. hort. sci., 2018 32(3): 407-420 and maturity class (table 3). on average, ‘palmer’ mangoes had higher aa, tpc and tac than ‘haden’ fruit, and lem fruit had lower tpc than mom mangoes (fig. 1). the average aa values were approx. 190 mg kg fw-1 for ‘haden’ and 390 mg kg fw-1 for ‘palmer’ and aa content did not change with maturity class in ‘palmer’ mangoes while in ‘haden’ showed the highest values in mem fruit (fig. 1). tpc was higher in ‘palmer’ than in ‘haden’ cv., being on average approx. 316 and 264 mg kg fw-1 in ‘palmer’ and ‘haden’ fruit, respectively, as well as it was higher in mom fruit from both cultivars. ‘palmer’ fruit were characterized by higher tac showing on average 2.59 times greater than that from ‘haden’. tac had significant positive correlations with tpc (r= 0.66, p= 0.002) and aa (r= 0.89, p<0.0001). carotenoids total carotenoids (car) depended only by maturity class (table 3), with lem fruit having less car than mom ones (fig. 1). the chromatographic carotenoid patterns of lem and mom maturity classes in both cultivars showed 9 common carotenoids (fig. 2) out of 14 peaks tentatively identified by comparing spectral characteristics with those previously reported using a similar mobile phase (table 1). the carotenoid pattern includes seven all-trans-violaxanthin (viol) and four 9-cis-violaxanthin (9-viol) containing compounds, cis-β-cryptoxanthin and all-transβ-carotene. the most abundant carotenoid in both cultivars was all-trans-β-carotene (tables 4 and 5), representing 49-56% of the total carotenoid content, followed fig. 1 (a) ascorbic acid (aa), (b) total carotenoids (car), total phenol content (tpc) and total antioxidant capacity (tac) in ‘palmer’ and ‘haden’ mangoes in function of µa540 maturity class (lem, less mature; mem, medium mature; mom, more mature). bars refer to se. within each cultivar anova results are indicated as follows: *, **, ***: significant at p≤0.05, 0.01, 0.001, respectively; ns, not significant. mean±se. within each cultivar, means followed by different letters are statistically different (bonferroni's test, p≤0.05). p-value of f-ratio: ns=not significant; *p<0.05; **p<0.01; ***p<0.001. table 2 absorption coefficient at 540 nm (µa540, cm-1), flesh firmness (n) and pulp colour parameters (a*; hue, h°; yellowness index, iy) of 'palmer' and 'haden' mangoes of less mature (lem), medium mature (mem) and more mature (mom) trs maturity classes and results of multifactor anova (f-ratio value and p-value) table 3 multifactor analysis of variance (f-ratio and p-value) for ascorbic acid (aa), total carotenoids (car), total plenolic content (tpc) and total antioxidant capacity (tac) *p<0.05; **p<0.01; ***p<0.001; ns=not significant. cultivar maturity class µ a 540 firmness a* h° i y palmer lem 0.196±0.027 b 8.33 ±0.55 a 0.86±0.79 b 89.13±0.75 a 130.7±1.7 b mem 0.241±0.006 ab 7.70±0.45 a 3.64±1.03 b 86.85±0.89 a 153.3±1.6 ab mom 0.310±0.017 a 6.12±0.63 a 9.84±1.75 a 81.72±1.30 b 165.7±9.5 a haden lem 0.191±0.011 b 37.29±9.89 a 2.98±1.62 c 87.18±1.55 a 130.0±6.0 b mem 0.336±0.033 b 10.47±3.62 a 10.60±1.44 b 80.32±1.15 b 159.7±6.6 a mom 0.677±0.158 a 5.85±0.65 b 20.46±1.12 a 72.37±0.84 c 191.2±6.1 a anova a: cultivar 18.88 *** 4.81* 28.85 *** 32.20 *** 3.94 ns b: maturity class 23.52 *** 4.85 * 36.79 *** 32.25 *** 27.18 *** a × b 9.21 ** 3.98 * 3.94 * 4.16 * 2.01 ns factors aa car tpc tac main factors cultivar (a) 38.79 *** 0.48 ns 5.12 * 32.75 *** maturity class (b) 0.75 ns 7.28 ** 4.20 * 1.85 ns interaction a × b 0.68 ns 0.98 ns 0.81 ns 0.53 ns 413 vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class by cis-β-cryptoxanthin (6-18%) and viol no.3 (1116%). the content of all-trans-violaxanthins was higher than that of 9-cis-violaxanthins in both cultivars. the content of viol no.3, no.4 and no.6, 9-viol no.4 and all-trans-β-carotene, as well as the sums of all-trans-violaxanthins (∑viol) and of 9-cisviolaxanthins (∑9-viol) depended only on maturity class, that of 9-viol no.1 only on cultivar, whereas those of 9-viol no.2 and no.3 on both cultivar and maturity class (table 4). in fact ‘haden’ mangoes had higher amounts of 9-viol no.3 (table 4) and had lower proportion of 9-viol no.4 than ‘palmer’ fruit (table 5). viol no. 1 and 9-viol no.1 were present only in ‘haden’ and 9-viol no. 2 only in ‘palmer’ fruit. fig. 2 typical chromatographic patterns at 450 nm of carotenoid extracts of (a) lem ‘palmer’, (b) mom ‘palmer’, (c) lem ‘haden’ and (d) mom ‘haden’ mangoes. for peak assignment see table 1. table 4 carotenoid compounds of 'palmer' and 'haden' mangoes (mg β-care kg fw-1) and vitamin a value (re 100 g fw-1) influence of cultivar and of trs maturity class and results of multifactor anova (f-ratio value and p-value). for identification data of each carotenoid see table 1 mean±se; 0=not detected. within cultivar and within trs maturity class means followed by different letters are statistically different (bonferroni's test, (*) p<0.10; * p<0.05; **p<0.01; ns =not significant). ∑viol= total all-trans-violaxanthin esters; ∑9-viol= total 9-cis-violaxanthin. cultivar maturity class anova palmer haden less mature medium mature more mature cultivar (a) maturity class (b) a × b viol no. 1 0±0 a 0.49±0.49 a 0 ±0 a 0±0 a 0.99±0.99 a 3.25 (*) 2.89 (*) 2.89 (*) viol no. 2 1.14±0.46 a 0.87±0.68 a 0.68 ±0.40 a 0.49±0.38 a 2.15±1.33 a 0.08 ns 2.11 ns 2.48 ns crypt 2.58±0.55 a 1.82±0.38 a 1.66 ±0.37 a 2.69±0.47 a 2.18 ±0.97 a 1.53 ns 0.89 ns 0.39 ns viol no. 3 2.43±0.31 a 2.49±0.55 a 1.53±0.28 b 2.82±0.67 ab 3.26±0.30 a 0.07 ns 3.08 (*) 2.37 ns 9-viol no. 1 0±0 b 0.81±0.42 a 0±0 a 0.67±0.42 a 0.68±0.68 a 4. 91 * 1.36 ns 1.36 ns 9-viol no. 2 0.33±0.16 a 0±0 b 0±0 b 0.06±0.06 ab 0.52±0.26 a 10.91 ** 6.23 * 6.23 * viol no. 4 0.84±0.16 a 0.91±0.40 a 0.27±0.13 b 0.88±0.40 ab 1.72±0.37 a 0.65 ns 4.90 * 0.89 ns 9-viol no. 3 0.09±0.06 b 0.35±0.15 a 0±0 b 0.21±0.11 ab 0.56±0.25 a 7.62 * 7.57 ** 2.35 ns car 9.36±1.14 a 9.63±2.72 a 5.05±0.67 b 8.85±1.81 ab 16.65±3.47 a 0.81 ns 8.37 ** 1.03 ns viol no. 6 0.64±0.14 a 0.56±0.23 a 0.08±0.08 b 0.74±0.16 ab 1.12±0.25 a 0.12 ns 10.19 ** 1.66 ns 9-viol no. 4 0.55±0.19 a 0.22±0.15 a 0±0 b 0.55± 0.21 a 0.67±0.28 a 1.58 ns 4.15 * 0.99 ns viol no. 7 0.15±0.10 a 0.19±0.10 a 0.07±0.07 a 0.17±0.11 a 0.30±0.18 a 0.19 ns 0.72 ns 0.97 ns ∑viol 5.20±0.76 a 5.51±1.95 a 2.64±0.74 b 5.11±1.22 ab 9.54±2.82 a 0.88 ns 5.69 * 2.26 ns ∑9-viol 0.98±0.35 a 1.38±0.67 a 0±0 b 1.49±0.48 ab 2.43±1.06 a 1.06 ns 4.39 ** 0.32 ns vitamin a value 258.2±22.3 a 162.0±45.3 a 85.6±11.4 b 149.7±30.5 ab 279.3±57.2 a 0.40 ns 8.38 ** 1.01 ns 414 adv. hort. sci., 2018 32(3): 407-420 lem mangoes had no 9-cis-violaxanthins and were characterized by lower contents of viol no.3, viol no.4, viol no.6 and all-trans-β-carotene than mom fruit, but higher proportion of all-trans-β-carotene than mem ones (tables 4 and 5). these carotenoids increased with advancing maturity degree, showing the highest contents in mom mangoes. on average all-trans-β-carotene corresponded to 53% of total carotenoids; the all-trans-β-carotene proportion was not significantly affected by cultivar, whereas on average was lower in mem fruit than in lem ones, while mom mangoes were not statistically different from fruit of the other two maturity classes (table 5). the vitamin a value did not differ between cultivar, but significantly increased with maturity class from 86 of lem fruit to 279 re 100 g-1 of mom mangoes (table 4). regression analysis the results of regression analysis between µa540 and pulp color parameters and between µa540, a*, h°, iy and ascorbic acid, tpc, tac and carotenoids differed for the two cultivars and data are summarized in tables 6 and 7, reporting the type of the model having the best performance. the μa540 was positively related to a* and iy and negatively to h° (table 6) with r ranging from 0.83 to 0.87 for ‘palmer’ fruit and approx. 0.98 for ‘haden’ cultivar. in ‘palmer’ mangoes (table 7) µa540, a*, h° and iy were related to total carotenoids, viol no.3 and no.4, 9-viol no.2 and no.3, all-trans-β-carotene, ∑9-viol and vitamin a value with lower r values (0.620.84) for µa540 respect to those found for pulp color parameters (0.74-0.96). only iy was related to viol no.6, 9-viol no.4 and ∑viol with r≥0.7, and only a* was related to tpc, but with r<0.6. in contrast, in ‘haden’ fruit µ a 540, a*, h° and i y were related to total table 5 carotenoid composition (percent to total carotenoids) of 'palmer' and 'haden' mangoes: influence of cultivar and of trs maturity class and results of anova (f-ratio value and p-value). for identification data of each carotenoid see table 1 table 6 results of regression analysis between absorption coefficient at 540 nm (μa540) and pulp color parameters for each regression, the following data are given: r = correlation coefficient, p = significance of the model (***, p<0.001; **, p< 0.01) and mt= model type (dr= doble reciprocal, e= exponential, l= linear, ln= logarithmic-x, m= multiplicative, rx= reciprocal-x, sc = s-curve). cultivar maturity class anova palmer haden less mature medium mature more mature cultivar (a) maturity class (b) a × b viol no. 1 0±0 a 0.87±0.87 a 0±0 a 0±0 a 0.36±0.36 a 3.25 (*) 2.89 (*) 2.89 (*) viol no. 2 6.74±2.84 a 3.13±1.49 a 2.51±0.64 a 1.04±0.43 a 2.89±0.58 a 0.21 ns 0.36 ns 1.65 ns crypt 14.87±3.01 a 15.40±2.77 a 18.18±0.53 a 15.31±0.07 a 5.88±1.04 a 0.01 ns 1.80 ns 1.40 ns viol no. 3 14.32±2.05 a 16.30±2.07 a 16.33±0.18 a 15.96±0.09 a 11.31±0.07 a 0.04 ns 1.51 ns 2.63 ns 9-viol no. 1 0±0 b 2.44±1.30 a 0±0 a 1.122±0.26 a 0.25±0.25 a 7.84 * 2.48 ns 2. 48 ns 9-viol no. 2 1.36±0.60 a 0±0 b 0±0 b 0.06±0.06 ab 1.08±0.20 a 14.55 ** 5.95 * 5.95 * viol no. 4 4.23±0.58 a 3.17±0.95 a 1.27±0.17 a 3.07±0.12 a 5.51±0.01 a 0.54 ns 2.49 ns 0.40 ns 9-viol no. 3 0.33±0.22 b 1.24±0.46 a 0±0 b 0.46±0.11 ab 1.21±0.08 a 7.51 * 8.04 ** 2.49 ns car 50.62±1.03 a 54.25±2.31 a 55.65±0.08 a 48.86±0.02 b 53.26±0.02 ab 3.22 (*) 3.28 (*) 3.38 (*) viol no. 6 3.20±0.71 a 1.98±0.68 a 0.07±0.07 b 3.27±0.10 a 3.57±0.005 a 1.66 ns 11.66 ** 0.62 ns 9-viol no. 4 2.63±0.91 a 0.71±0.52 b 0±0 b 1.73±0.23 a 1.14±0.20 ab 4.52 * 6.20 ** 2.26 ns viol no. 7 0.77±0.51 a 0.95±0.51 a 0.06±0.06 a 0.25±0.11 a 0.56±0.21 a 0.19 ns 0.46 ns 1.24 ns ∑viol 29.25±3.30 a 26.41±3.04 a 25.030±0.42 a 28.13±0.09 a 28.76±0.07 a 0.22 ns 0.17 ns 4.27 * ∑9-viol 4.31±1.33 a 4.39±1.71 a 0±0 b 6.07±0.19 a 5.79±0.21 a 0.01 ns 13.50 ** 0.00 ns mean±se; 0=not detected. within cultivar and within trs maturity class means followed by different letters are statistically different (bonferroni's test, (*) p<0.10; * p<0.05; **p<0.01; ns =not significant). ∑viol= total all-trans-violaxanthin esters; ∑9-viol= total 9-cis-violaxanthin. µa540 ‘palmer’ ‘haden’ r p mt r p mt a* 0.831 ** l 0.975 *** e h° 0.826 ** rx 0.976 *** sc i y 0.872 ** ln 0.977 *** dr vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 415 carotenoids, viol no.2, no.3, no.4 and no.6, 9-viol no.1 and no.3, all-trans-β-carotene, ∑viol, ∑9-viol, vitamin a value and tpc, with higher r values (0.720.95) for µa540 than for pulp color parameters. in addition, only µa540 was related to 9-viol no. 4. no significant relationships were found between µa540, a*, h° and iy and ascorbic acid and tac, whatever the cultivar, suggesting that μa540 was able to reveal the carotenoids content in the pulp, as this wavelength corresponds to the tail of carotenoid absorption. independently from cultivar. rizzolo et al. (2016) also showed that µa540 maturity index, related to carotenoids content, successfully classified ‘tommy atkins’ mangoes at harvest. as for quality parameters, ‘palmer’ mangoes had firmness values typical of fully ripe fruit (beaulieu and lea, 2003), independently from maturity class, whereas in ‘haden’ fruit lem class showed firmness values typical of firm-ripe stage and mem and mom classes values typical of ready-to-eat or ripe fruit (eccher zerbini et al., 2015). pulp color parameters table 7 results of regression analysis between absorption coefficient at 540 nm (μa540), pulp color parameters and total carotenoid (car), total phenolic compounds (tpc), carotenoid compounds (for identification data see table 1) and vitamin a value µa540 a* h° iy r p mt r p mt r p mt r p mt 'palmer' car 0.78 * l 0.909 *** l 0.902 *** rx 0.897 *** l viol no.3 0.814 ** l 0.789 * l 0.783 * rx 0.735 * l 9-viol no.2 0.839 ** l 0.959 *** l 0.958 *** rx 0.823 ** l viol no.4 0.625 (*) l 0.908 *** l -0.912 *** l 0.784 * l 9-viol no.3 0.765 * l 0.871 ** l 0.871 ** rx 0.778 * l car 0.793 ** l 0.913 *** sy -0.909 *** sy 0.888 ** sy viol no.6 − − − -0.745 * rx 9-viol no.4 − − − -0.847 ** rx ∑viol 0.576 (*) log − − 0.693 * log ∑9-viol 0.812 ** l 0.873 ** l 0.86 ** rx 0.954 *** l vitamin a value 0.792 ** l 0.915 *** sy -0.912 *** sy 0.887 ** l tpc − 0.575 (*) sy − − 'haden' car 0.912 *** l 0.855 ** e 0.854 ** sc 0.876 *** e viol no.2 0.853 ** l 0.618 (*) l 0.634 * rx 0.601 (*) l viol no.3 -0.824 ** sc 0.723 * e -0.742 * e -0.805 ** sc 9-viol no.1 0.756 * l 0.666 * l 0.661 * rx 0.718 * l viol no.4 0.815 ** sx 0.789 ** l 0.79 ** rx 0.823 ** l 9-viol no.3 0.95 *** l 0.83 ** l 0.834 ** rx 0.848 ** l car 0.924 *** l 0.823 ** sy 0.817 ** sc 0.837 ** sy viol no.6 0.944 *** l 0.862 ** l 0.862 ** rx 0.873 *** l 9-viol no.4 0.700 * l − − − ∑viol 0.937 *** l 0.885 *** sy -0.886 *** e 0.905 *** sy ∑9-viol 0.848 ** l 0.723 * l 0.721 * rx 0.756 * l vitamin a value 0.923 *** l 0.823 ** sy 0.817 ** sc 0.838 ** sy tpc 0.867 ** l 0.758 * l 0.761 ** rx 0.766 ** l for each significant regression, the following data are given: r= correlation coefficient, p= significance of the model (***, p< 0.001; **, p <0.01; *, p< 0.05; (*), p<0.10) and mt= model type (dr= doble reciprocal, e= exponential, l= linear, ln= logarithmic-x, log= logistic, rx= reciprocal-x, sc= s-curve, sx= square-root-x, sy= square-root-y). 4. discussion and conclusions the absorption coefficient measured at 540 nm (µa540) showed different value ranges between ‘haden’ and ‘palmer’ mangoes and it was able to distinguish more mature fruit from less mature ones differed among maturity classes, confirming previous results obtained for ‘tommy atkins’ cultivar. in fact rizzolo et al. (2011) and vanoli et al. (2011) found that lem ‘tommy atkins’ mangoes were characterized by higher h° and lower a* and i y than mom fruit. moreover, vanoli et al. (2011) found that with fruit adv. hort. sci., 2018 32(3): 407-420 416 ripening at 20°c h° decreased and iy increased, confirming that the trend of pulp color observed in our work with the trs maturity classes was actually due to a different ripening degree. the average aa values found for ‘palmer’ and ‘haden’ fruit are comparable with the data by rocharibeiro et al. (2007) for the same cultivar, and with aa content reported for other cultivars by liu et al. (2013) and elsheshetawy et al. (2016). however, within the same variety, aa content may vary according to climatic conditions, cultural practices, maturity stage and postharvest factors. for ‘keitt’ cultivar ibarra-garza et al. (2015) found that aa content varied from about 1300 mg kg fw-1 in fruit soon after harvest, to about 2500 mg kg fw-1 till 8 days of ripening at room temperature, followed by a 54% decrease in fully-ripe fruit. similarly, robles-sánchez et al. (2009 b) reported for ‘ataulfo’ fruit stored for 15 days at 12°c a 50% decrease of aa content at the end of shelf-life. the tpc contents found in this work are in agreement with rocha-ribeiro et al. (2007), even if these authors reported lower aa contents than those found in our work for the same cultivars. no data on tpc content in mangoes in relation to trs maturity classes, having same harvest time and same postharvest management, are available in the literature. however, the tpc increasing trend from less to more mature fruit class found in this work is in agreement with the results for the final period of shelf life/storage reported by ibarra-garza et al. (2015) and robles-sánchez et al. (2009 b) when fruit are becoming softer and with a yellower pulp color. ibarragarza et al. (2015) found higher tpc in ‘keitt’ fruit at the beginning of a 10 d shelf life period at room temperature, with a sharp decrease from 2 to 4 days of shelf life, followed by a slight but significant tpc increase till the end of shelf life; a similar trend was also found by robles-sánchez et al. (2009 b) for whole and fresh-cut ‘ataulfo’ mangoes stored at 5°c for 15 days. in agreement with data obtained for tpc and aa, ‘palmer’ fruit showed higher tac than ‘haden’ ones. the positive correlations of tac with tpc and aa found in this work are in agreement with literature data. in fact silva and sirasa (2018) reported significant correlations between ascorbic acid and tpc and frap and dpph scavenging activity measured for several fruit species, and palafox-carlos et al. (2012) between dpph scavenging activity and tpc in ‘ataulfo’ mangoes; on the other hand, liu et al. (2013) and ibarra-garza et al. (2015) found in mangoes correlation between phenolic concentration and antioxidant activity measured with other methods (frap, orac), but not between antioxidant activity and ascorbic acid content; in contrast rocharibeiro et al. (2007) reported that dpph radical scavenging activity of mango extracts was strongly correlated with ascorbic acid content, but not with phenolic content. liu et al. (2013) suggested that the difference in relationships between antioxidant activity and ascorbic acid and phenolic compound contents found among authors could be due to a masking effect of phenolics present in far higher concentration than ascorbic acid. our results suggest that in this work the antioxidant activity can be attributed more to ascorbic acid than to total phenols. no data on total carotenoids in relation to trs maturity classes are available in the literature. vanoli et al. (2016) for ‘palmer’ and ‘haden’ mangoes reported a wide fruit-to-fruit variability in car content for both cultivars and that car content had an increasing trend with µa540 value measured on fruit belonging to the same harvest date, i.e. that car content increases with advancing maturity degree. similarly, an exponential increase in the carotenoid content with fruit ripening has been reported for ‘ataulfo’ mangoes (ornelas-paz et al., 2008) and nine thai cultivars (vásquez-caicedo et al., 2005). as for the chromatographic carotenoid patterns, the tentative identification of peaks was carried out by comparing spectral characteristics with those previously reported using a similar mobile phase. seven peaks were tentatively identified as all-trans-violaxanthin (439 nm maximum absorption wavelength) and four as 9-cis-violaxanthin (435 nm maximum absorption wavelength) containing compounds. the spectral maximum for peak 4 was similar to that reported for cis-β-cryptoxanthin. a standard mixture of all-transβ-carotene was used for the identification of peak 11; the retention time and the spectroscopic characteristics of reference material were identical to those observed for peak 11 in all the samples. in general, the spectral fine structures (% iii/ii values in table 1) found in this work are in agreement with the values reported in the literature (ornelas-paz et al., 2007, 2008). in both cultivars the proportions of all-transβ-carotene and cis-β-cryptoxanthin to total carotenoid content, as well as the higher content of all-trans-violaxanthins than that of 9-cis-violaxanthins are very similar to findings reported in ‘ataulfo’, ‘keitt’, ‘tommy atkins’ and ‘kent’ mangoes (mercadante and rodriguez-amaya, 1998; pott et al., 2003; ornelas-paz et al., 2008). the differences in vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 417 carotenoid composition among the maturity classes found in this research are consistent with literature data. previous researches on carotenoid composition of various mango cultivars carried out by godoy and rodriguez-amaya (1989), mercadante and rodriguez-amaya (1998), yahia et al. (2006), and ornelas-paz et al. (2007) have shown that generally the most important carotenoid in mango is all-transβ-carotene and its proportion to total carotenoids depends on cultivar and fruit maturity stage. ibarragarza et al. (2015) reported for ‘keitt’ fruit that alltrans-β-carotene corresponded to 33% of total carotenoids in unripe fruit at harvest, ranged from 37 to 44% during the first 6 day-period of ripening at room temperature and reached 61% in fully ripe fruit. the contents of all-trans-β-carotene in ‘haden’ and ‘palmer’ mangoes were similar to those reported by rocha-ribeiro et al. (2007) for the same cultivars and by mercadante and rodriguez-amaya (1998) for ‘keitt’ and ‘tommy atkins’ fruit, but lower than those found for ‘haden’ and other cultivars by ornelas-paz et al. (2007). also the amounts of all-transand 9-cisviolaxanthins in both cultivars were lower than those found by ornelas-paz et al. (2007) for ‘haden’ and other cultivars, but similar to those reported by low et al. (2015) for ‘kensington pride’ mangoes. the amounts of cis-β-cryptoxanthin in both cultivars were far higher than the maximum of 0.1 µg g-1 reported by mercadante and rodriduez-amaya (1998) for ‘keitt’ and ‘tommy atkins’ mangoes from maturegreen to ripe stages, indicating that in the fruit of this experiment, not only β-carotene, but also cis-β-cryptoxanthin was a contributor to the vitamin a value for these fruit. the differences in the single carotenoid concentrations respect to literature data for the same cultivar could be due to the different maturity degree of mango fruit. in fact, referring to pulp color parameters, the fruit used in our experiment had pulp a* values similar to those reported by rocha-ribeiro et al. (2007) for the same cultivars and in this case the carotenoid amounts were similar, whereas pulp h° values for ‘haden’ fruit were higher than those reported by ornelas-paz et al. (2007), indicating a less advanced ripening degree consistent with the lower carotenoid content of our results. the results of regression analysis showed that µa540 was positively related to a* and iy and negatively to h° pulp color parameters, confirming the results obtained by spinelli et al. (2012, 2013) and vanoli et al. (2016) for the same cultivars and by vanoli et al. (2013) for ‘tommy atkins’ mangoes. color changes in the pulp of mango fruit are usually accompanied by carotenoid accumulation. in this work significant correlations between µa540, pulp color parameters a*, h°, iy and carotenoids were found for both cultivars. vanoli et al. (2016) found an increasing trend of total carotenoids content with µa540 in ‘palmer’ and ‘haden’ mangoes; they also found high positive correlations between total carotenoids and a* and iy and a higher negative correlation with h° following a logarithmic-law function with higher correlation in ‘palmer’ than in ‘haden’ cv., confirming the better relationships of pulp color in ‘palmer’ than in ‘haden’ fruits. high correlations between pulp color and all-trans-β-carotene, alltrans-violaxanthin and 9-cis-violaxanthin were also observed in ‘ataulfo’ and in ‘manila’ mangoes (ornelas-paz et al., 2008) with the highest correlation coefficients for a* and h° parameters; in ‘manila’ mangoes the best results were associated with the concentrations of all-trans-violaxanthin and 9-cis-violaxhantin, while in ‘ataulfo’ with all-trans-β-carotene, confirming that there is a cultivar specific relationship between pulp color and carotenoids content. similar correlations were also found by vasquezcaceido et al. (2005) in 9 thai mango cultivars (power law functions) and by bicanic et al. (2010) in 21 mango homogenates (second order polynomial dependence). differently from carotenoids, ascorbic acid content and total antioxidant capacity for both cultivars and tpc for ‘palmer’ were not related to pulp color, measured both by a*, iy and h° color parameters and µa540; this is not surprising as literature reported that aa and tpc contents with shelf life does not follow a well-defined increasing or decreasing trend (roblessánchez et al., 2009 b; ibarra-garza et al., 2015). in conclusion our results confirmed that the absorption coefficient at 540 nm (µa540) can be used as a non-destructive maturity index for mangoes. in fact it was able to classify intact fruit of two mango cultivars according to pulp color, the destructive maturity index commonly used for mangoes, as well as according to the contents of total carotenoids and of individual carotenoid compounds and vitamin a value. the good correlations between µa540, pulp color parameters and carotenoids indicate that trs is a suitable tool to sort fruit with different ripening degree, having specific carotenoid pattern. adv. hort. sci., 2018 32(3): 407-420 418 acknowledgements this work was funded by lombardia region (italy) and minas gerais region (brazil) (progetto di cooperazione scientifica e tecnologica “approccio multidisciplinare per l’innovazione della filiera di frutti tropicali tropico” id 17077, rif. n° agro-16). thanks to r.m.a. pimentel, epamig (minas gerais, brazil) for ‘palmer’ and ‘haden’ mangoes supply from experimental orchards. references azevedo-meleiro c.h., rodriguez-amaya d.b., 2004 confirmation of the identity of the carotenoids of tropical fruits by hplc-dad and hplc-ms. j. food comp. anal., 17: 385-396. beaulieu j.c., lea j.m., 2003 volatile and quality changes in fresh-cut mangos prepared from firm-ripe and soft-ripe fruit, stored in clamshell containers and passive map. postharvest biol. technol., 30: 15-28. bicanic d., dimitrovski d., luterotti s., twisk c., buijnsters j.p., dóka o., 2010 estimating rapidly and precisely the concentration of beta carotene in mango homogenates by measuring the amplitude of optothermal signals, chromaticity indices and the intensities of raman peaks. food chem., 121: 832-838. brand-williams w., cuvelier m.e., berset c., 1995 use of free radical method to evaluate antioxidant activity. lwt, 28: 25-30. capra s., 2006 vitamin a, pp. 55-65. in: baghurst k. 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partial identification of ‘ataulfo’ mango fruit carotenoids. acta horticulturae, 712: 333-338. impaginato 123 adv. hort. sci., 2023 37(1): 123­132 doi: 10.36253/ahsc­13899 sustaining low­impact practices in hor­ ticulture through non­destructive approach to provide more information on fresh produce history and quality: the sus&low project m. amodio 1, g. attolico 2, l. bonelli 3, m. cefola 4, h. fazayeli 1, f.f. montesano 3, 5, b. pace 4, m. palumbo 1, 4, f. serio 3, a. stasi 1, g. colelli 1 (*) 1 department of science of agriculture, food and environment, university of foggia, via napoli, 25, 71122 foggia, italy. 2 institute on intelligent industrial systems and technologies for advanced manufacturing, national research council (cnr), via g. amendola, 122/o, 70126 bari, italy. 3 institute of sciences of food production, national research council (cnr), via giovanni amendola, 122/o, 70125 bari, italy. 4 institute of sciences of food production, national research council of italy (cnr), c/o cs‐dat, via michele protano, 71121 foggia, italy. 5 department of soil, plant and food sciences, university of bari, via aldo moro, 70126 bari, italy. key words: marketing strategies sustainability, non­destructive assessment, quality, shelf­life. abstract: the general aim of the project sus&low is to increase the sustainabil­ ity of fresh produce by testing and implementing low­input agricultural practices (lip) with positive impact on product quality with the support of non­destructive (nd) tools for real­time quality assessment and for product discrimination. additionally, new marketing strategies are generated to better support the added value of the products and to satisfy the final consumers’ preferences. the sus&low project consists of three work packages (wp) and the adopted methodology used two model crops: rocket salad and tomato. the wp1, focused on the reduction of agricultural inputs, showed that sensor­based fertigation management might improve sustainability of soilless cultivation. results coming from wp2, aimed to the evaluation of nd techniques, outlined the high poten­ tiality of hyperspectral imaging (hsi) and fourier transformed­near infrared (ft­ nir) techniques for the authentication of sustainable growing methods. moreover, project activities’ proved computer vision system (cvs) as an effec­ tive tool for evaluating the product quality also through the bag. the wp3, deal­ ing with marketing strategies, indicated a positive approach of consumers com­ pared to lip products certified through a visual storytelling platform. 1. introduction production of vegetable crops under controlled environments (i.e. greenhouses) has expanded considerably over recent decades in (*) corresponding author: giancarlo.colelli@unifg.it citation: amodio m., attolico g., bonelli l., cefola m., fazayeli h., montesano f.f., pace b., palumbo m., serio f., stasi a., colelli g., 2023 ­ sustaining low‐impact practices in horticul‐ ture through non‐destructive approach to provide more information on fresh produce history and quality: the sus&low project. ­ adv. hort. sci., 37(1): 123­132. copyright: © 2023 amodio m., attolico g., bonelli l., cefola m., fazayeli h., montesano f.f., pace b., palumbo m., serio f., stasi a., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2022 accepted for publication 10 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13899 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 123­132 124 mediterranean areas (fao, 2013). initially, research efforts and the related introduction of technical inno­ vations focused on high­quality, healthy products. however, concern with environmentally­sustainable production has risen in the last decade as industrial greenhouse crops are usually seen as entailing high environmental impact (torrellas et al., 2012). on the other hand, there is also plenty of evidence that greenhouse vegetable production may decrease the environmental impact compared to the field cultiva­ tion (stanghellini, 2014). the efficient use of resources (water and fertiliz­ ers), in irrigated greenhouse agriculture, is a promis­ ing and increasingly adopted strategy to achieve bet­ ter crop performance, improved nutritional and sen­ sorial quality (montesano et al., 2015; montesano et al., 2018). with respect to traditional systems, soil­ less cultivation and, particularly, closed­cycle with recycling of nutrient solution (ns) produce a number of benefits, including the possibility to standardize the production process, to improve plant growth and yield, and to obtain higher efficiency in water and nutrients use. in addition, it is also possible to modu­ late the regulation of the secondary metabolism of plants through an optimal control of the nutrient solution composition, or by imposing controlled stresses, or through biofortification treatments, gen­ erally leading to an improvement in the nutritional value of products (rouphael and kyriacon, 2018; renna et al., 2022). innovative technologies based on the use of sensor networks for fertigation manage­ ment may considerably reduce water and fertilizers consumption and increase the overall use efficiency of those inputs, and may lead to qualitative and quantitative improvements while preventing both under­ and over­irrigation. the most used instrumental techniques to mea­ sure quality attributes of fruits and vegetables are destructive and involve a considerable amount of manual work, primarily due to sample preparation. in addition, most of these analytical techniques are time consuming and sometimes may require sophisti­ cated equipment. finally, they can be performed only on a limited number of specimens (samples) and therefore their statistical relevance may be limited (amodio et al., 2017 a). research has been focused on developing non­contact, rapid, environmental­ friendly, and accurate methods for non­invasive eval­ uation of quality in fruits and vegetables. nowadays, there are a few emerging non­destructive analytical instruments and approaches for this task, including spectroscopy, hyperspectral imaging, and computer vision (liu et al., 2017). near infrared spectroscopy has gained wide atten­ tion in the food sector due to its capacity of providing fingerprints of different products on the base of the interaction between their molecular structure and the incident light (workman and shenk, 2004) which is the result of different pre­harvest factors that also affect the final composition and quality. the feasibili­ ty of nirs­based analysis to evaluate quality attributes of fresh fruits for commercial application have been reported by numerous authors (arendse et al., 2018; palumbo et al., 2022 a). hyperspectral imaging (hsi) combines the princi­ ples of spectroscopy and conventional imaging or computer vision. it is mainly used for internal bruise and defect detection in fruits and vegetables (ariana and lu, 2010; babellahi et al., 2020; tsouvaltzis et al., 2020) but also to predict the internal composition (piazzolla et al., 2013; yang et al., 2015; liu et al., 2017; piazzolla et al., 2017). amodio et al. (2017 a) showed the potentiality of hyperspectral imaging in the vis­nir spectral range to predict internal content of soluble solids, phenols, and antioxidant activity of fennel heads. in addition, this technique provided important information about the maturity of fennel heads which may be used to determine the optimal harvest time. some studies successfully applied these methods for the discrimination of production origin and agricultural practices, as revised in amodio et al. (2020). nir and his were in fact used for the classifi­ cation of apples (guo et al., 2013), persimmon (khanmohammadi et al., 2014), and arabica coffee (bona et al., 2017) from different origins. as for pro­ duction systems (sánchez et al., 2013) investigated the potentiality of nirs technologies to discriminate green asparagus grown under organic and conven­ tional methods. more recently, amodio et al. (2017 b) successfully discriminated conventionally and organically grown strawberries, being also able to identify two different types of organic production systems applied to the same genetic material on the same site, soil, unheated tunnel. all these studies have suggested multispectral and hyperspectral systems as valid tools to evaluate qual­ ity of different agricultural products and, more inter­ estingly, as tools for product authentication. finally, computer vision systems (cvs) may be applied to extend quality prediction and discrimina­ tion along the whole supply chain from harvesting up to consumers. cvs combine mechanics, optical amodio et al. ‐ the sus&low project 125 instrumentation, electromagnetic sensing, and digital image processing technology (patel et al., 2012). recently, cvss have been used to assess quality and marketability of tomatoes (arias et al., 2000), arti­ chokes (amodio et al., 2011), fresh­cut nectarines (pace et al., 2011), fresh­cut lettuce (pace et al., 2014), fresh­cut radicchio (pace et al., 2015), and rocket leaves (cavallo et al., 2017). moreover, they have been applied for the prediction of internal qual­ ity of colored carrots (pace et al., 2013). even more interesting is the application of these systems during the post­packaging phase and along the whole distri­ bution chain. despite the relevance of quality evalua­ tion of packaged products, few investigations were reported in literature. multi­spectral reflective image analysis has been applied to monitor the evolution and spoilage of leafy spinach covered by plastic materials (lara et al., 2013); more recently, cavallo et al. (2018) have proposed an application of image analysis by cvs for non­destructive and contactless evaluation of quality of packaged fresh­cut lettuce. therefore, the interest of investigating the applica­ tion of cvs to detect quality and shelf­life of pack­ aged products. finally, the possibility of using non­destructive technique for increasing the information on product history (e.g. growing location and agricultural prac­ tices) may be considered as baseline to develop mar­ keting tools to promote the diffusion of sustainable production system. cost barrier is an obstacle for choosing low input products instead of the conven­ tional, even if environment is mentioned as a strong commitment (krystallis and chryssohoidis, 2005). therefore, the knowledge about consumer prefer­ ences for the adoption of lip is still matter of debate. the general aim of the project is to increase the amount of sustainably­produced food by testing and implementing low­input agricultural practices with positive impact on product quality with the support of non­destructive tools for real­time quality assess­ ment and product discrimination, which may inspire new marketing strategies to better support the added value of the products and increase incomes of potential users. 2. project activities and main results the sus&low project structure consists of three work packages (wp). wp1 focused on research activi­ ties aimed to reduce agricultural inputs (water and fertilizers) in greenhouse cultivation, chosen as a strategic high­value sector for mediterranean agricul­ ture. this wp was also in charge of making available to the project team vegetables products (rocket and tomato) different for the level of sustainability char­ acterizing the cropping system adopted, to be used in other wps for the related investigations. then, wp2 was aimed to the quality assessment and to the implementation of new tools to acquire information about quality and history of fresh produce obtained with lip (wp1). non­destructive methods (including nir, hyperspectral imaging and image analysis by cvs) have been used for food authentication, show­ ing interesting and promising results. finally, wp3 realized ad hoc survey to analyze the consumer behaviour with respect to the possibility of purchas­ ing fruit and vegetables lip certified (wp1) and iden­ tified by nd technologies (wp2) with the aim to implement adequate marketing strategies. in this section, an overview of the research strategies and approaches adopted in the three wps is provided. the main results are reported and discussed. wp1: quality crops through low‐impact practices based on the overall project structure, this wp was focused on soilless cultivation, since it has the potentiality to achieve extremely high water and fer­ tilizers use efficiency, beside high yield and quality, in intensive cropping systems. however, the adoption of free­drain open cycle with empiric fertigation schedule management operated by timers (the pre­ dominant case in mediterranean area), may compro­ mise the sustainability of soilless culture. therefore, the adoption of strategies aimed to rational use of water and fertilizers and excess leaching prevention is a key­factor for increased sustainability and reduced environmental impact of soilless culture (massa et al., 2020). in this context, substrate mois­ ture/ec (electrical conductivity) sensor­based irriga­ tion is a promising and increasingly adopted strategy to reduce water and fertilizers consumption and loss­ es, and to improve the overall crop performance, product quality and production process sustainability in soilless greenhouse cultivation (palumbo et al., 2021 a). several experiments were carried out at the experimental farm la noria (mola di bari, ba) of the cnr­ispa (bari), with the common approach to com­ pare treatments providing traditionally adopted empirical fertigation management techniques with treatments in which advanced sensor­based fertiga­ adv. hort. sci., 2023 37(1): 123­132 126 tion management was implemented. the main results of selected experiments carried out during the project are reported hereafter. the research activities focused on two model species [rocket salad (diplotaxis tenuifolia l.) and tomato (solanum lycopersicum l.) selected for their relevance in mediterranean greenhouse vegetable production. in particular, rocket is reported as an emerging leaf vegetable which cultivation is widespread and in further expansion (schiattone et al., 2017), while tomato is the most important green­ house crop grown in soilless cultivation systems (montesano et al., 2015). a study was carried out to test two irrigation scheduling approaches (timer­ or sensor­based) and two fertilization levels (high or low, with reference to the standard dosage range recommended for the specific fertilizers used) of open­cycle soilless rocket in mediterranean autumn­winter unheated green­ house conditions (montesano et al., 2021). rocket plants (cv. dallas, isi sementi) were grown in a peat:perlite (3:1) mixture in 4.5 l plastic pots. four treatments were compared: timer with high or low fertilization (t­hf, t­lf), and sensor­based with high or low fertilization (s­hf, s­lf). in timer­based treat­ ments, irrigation schedule was periodically adjusted based on leaching fraction measurements (≈35% was set as a target, according to common practice). in sensor­based treatments, on­demand irrigation was operated based on substrate ec/temperature/mois­ ture sensors (gs3, decagon devices). these were connected to a cr1000 datalogger programmed to automatically open irrigation valves and supply water enough to constantly maintain volumetric water con­ tent to a pre­defined set­point (0.35 m3 m­3, close to maximum water holding capacity), with no leaching. slow­release fertilizers (osmocote exact and calmag, icl) were mixed with the substrate at high (3.75 and 1 g l­1, respectively) or low dosage (2.25 and 0.6 g l­ 1). yield, quality, water use and substrate parameters trends were evaluated. sensors improved water use efficiency compared to timer (34.4 vs 21.4 g fw l­1, on average) matching water supply with plant needs, and preventing leaching (fig. 1) (no interactive effects of fertilization treatments were observed on those parameters). sensor­based irrigation also pro­ vided the best plant growth conditions, with interest­ ing interactive effects with fertilization rate. in partic­ ular, the highest and the lowest cumulative (three harvests) yield values were obtained in s­hf and t­lf respectively (144.8 and 102.2 g fw pot­1), while simi­ lar values were observed in s­lf and t­hf (131.4 g fw pot­1, on average) (fig. 1). the partial fertilizer factor productivity (g product fresh weight / g fertiliz­ ers applied) was higher at low dosage, and, with the same dosage, when the sensors were used (fig. 1). after each harvest time the fresh­cut rocket leaves were immediately transported in refrigerate condi­ tions to the postharvest laboratory (see wp2 section below) (palumbo et al., 2021 b). in another set of studies, we aimed to apply approaches for the sustainable fertigation manage­ ment of soilless tomato (semi­closed cycle recircula­ tion; sensor­based nutrient solution supply manage­ ment) in comparison with a traditional open cycle free­drain nutrient solution management providing the use timer for fertigation schedule. experiments were conducted with different tomato types (cherry ­ cv. carminio, seminis­bayer, and intermediate type ­ cv. mose, syngenta), and in different environmental conditions typical of mediterranean areas (including the use of brackish water for nutrient solution prepa­ ration). in general, both approaches (semiclosed­ cycle cultivation and open cycle with sensor­based fertigation management) reduced the environmental impact of the production process (reduced water/fer­ fig. 1 ­ water use efficiency (wue), leaching rate, total yield, and partial fertilizer factor productivity of rocket (diplotaxis tenuifolia) grown in open free drain soilless system with timer­ (t) or sensor­based (s) irrigation man­ agement, and subjected to high (hf) or low (lf) fertiliza­ tion rate. amodio et al. ‐ the sus&low project 127 tilizers usage; less nutrient solution released into the environment, increased water use efficiency) and positively affected tomato quality traits, compared to empirically management open­free drain cultivation. wp2: non‐destructive discrimination for low‐impact practices and non‐destructive quality assessment nir spectroscopy and hiperspectral imaging. in this wp, the objective of the tasks was to assess the potentiality of fourier transformed­near infrared (ft­ nir) spectrometry and hyperspectral imaging (hsi) to discriminate tomatoes and rocket leaves produced with different level of input as described in wp1, tak­ ing also into account the degree of efficiency in water and fertilizers used efficiency (wue and fue indexes). a hyperspectral line­scan scanner (version 1.4, dv srl, padova, italy) equipped with two spectrographs, one in the vis­nir range, and the second in the nir range, was used to obtain the hs images. the vis­nir spec­ trograph (400­1000 nm) has a spatial resolution of 1000 × 2000 pixels with a spectral resolution of 5 nm and was connected to a ccd camera. as for the nir spectrograph (900­1700 nm), the spatial resolution was 600 × 320 pixels with a spectral resolution of 5 nm; and a cmos (specim spectral imaging ltd., oulu, finland) with 50 frames per second equipped with c­ mount lenses was used. as for ft­nir spectrometry an mpa multi­purpose (ft­nir analyzer, bruker optics, ettlingen, germany), was used during spectral acquisition over the range of 800­2777 nm (sphere macrosample re­solution 1.71 nm, scanner velocity 10 khz, sample scan time 64 scans, background scan time 64 scans). after image processing and spectra extraction for the hsi, all spectra belonging to hsi and ft­nir were tested in discrimination using the agronomic treatments as discriminant classes and partial least squares­discriminant analysis (pls­da) as classification technique. as for rocket leaves, pls­ da was conducted with the 4 classes (t­hf; t­lf; s­ hf, s­lf) described in the paragraph related to wp1, using 70 percent of samples for calibration purpose and the remaining 30% for the external validation. the model performance was evaluated based on the accuracy, which is an average of the sensitivity calcu­ lated over the various classes, and gives an overall idea of the goodness of the classification. results indicated hsi as a promising technique for the dis­ crimination of rocket produced with different cultural techniques, with an accuracy of classification in the prediction phase of 97.2% in vis­nir and 99.5% in nir range. in figure 2, the results of the discrimination models can be observed. regarding tomato, where 2 experiments with 2 different varieties were conducted (wp1), for each trial a first pls­da was aimed to discriminate the three treatments of cultivation and a second discrimi­ nation was performed for different levels of wue and fue. according to the efficiency of use of water and fertilizers we could individuate 2 levels (high and low) in each experiment and 3 levels (high, medium, and low) merging the data of both experiments. therefore, a pls­da with 3 levels of wue (and fue) was also generated with the full dataset. among the different non­destructive techniques, ft­nir and his in the vis­nir range gave comparable performances in discriminating tomato according to cultural prac­ tices and different use of sources. discrimination for wue for each variety improved the classification results, respect to the individual treatments, but the highest accuracy was obtained when the discrimina­ tion was based on 3 levels of wue merging the 2 datasets, reaching 92.1%. in literature there are no studies aimed to discriminate crops for wue or fue, while we may find the application of hsi for the clas­ sification of water­stressed plants, as for the case of tomatoes (rinaldi et al., 2015). in comparison to this study, reporting a mean accuracy of around 77% for discrimination of the two differently irrigated areas, our findings showed higher accuracy, exploring new area of the application for these techniques. application of cvs for non‐destructive quality evalua‐ tion on packaged products a research activity was carried out to develop and validate an innovative cvs integrating a random forest model for classification: this model automati­ cally selects from the image the most relevant colour features for the task of interest. the developed cvs was applied to digital images of fresh­cut rocket fig. 2 ­ estimated class index values in the calibration and in the prediction process for the classification based on pls­da modes shown on table 2 in a) vnir range (left) b) nir range (right). f d d ld h 128 adv. hort. sci., 2023 37(1): 123­132 leaves cultivated with lip (wp1) to objectively esti­ mate the evolution of their quality levels (ql) during storage and to discriminate the cultivation approach applied on field. at harvest, rocket leaves were stored at 10°c in open polypropylene (pp) bags for a number of days required to reach the lowest ql, according to the rating scale from 5 (very good) to 1 (very poor), as reported in figure 3. at each ql, all the samples were subjected to postharvest quality evaluation, detecting colour parameters by a traditional colorimeter (cr400, konica minolta, osaka, japan) and physical and chem­ ical parameters, in detail respiration rate (kader, 2002), electrolyte leakage (kim et al., 2005) and total chlorophyll content (cefola and pace, 2015). then, images of the same samples were acquired by the cvs for non­destructive quality assessment and for recognizing traits related to the sustainability of the cultivation management used on the field, with spe­ cific reference to water and nutrients use (wp1). image pre­processing was applied: to separate the product from the background; to identify the colour­ chart placed in the scene to estimate the effects of lights and of the sensors and to correct colours to minimize these effects. three colour correction meth­ ods (white balance, linear correction, and polynomial correction) with increasing level of complexity were evaluated and compared in terms of consistency of colour measurements and of classification perfor­ mance. linear colour correction proved to be the best trade­off between efficacy and efficiency providing a slightly lower performance than polynomial correc­ tion with significantly simpler computation. finally, a random forest model was used to train classifiers to assess the ql of rocket leaves and to identify the treatments used during the cultivation. all the postharvest quality parameters measured by traditional destructive methods were significant in ql assessment of fresh­cut rocket leaves. the pro­ posed classifier based on the random forest model was able to identify and select the most relevant colour traits for both the tasks (ql assessment and treatment identification) without human interven­ tion. the accuracy achieved in evaluating qls of rock­ et leaves during storage was high (about 95%), while the performance in discriminating the cultivation approach was lower and not sufficient for practical applications (about 65­70%). indeed, the different cultivation approaches did not significantly affect the visual characteristics of the product and the destruc­ tive measures: this task needs further investigations. another research activity was carried out to develop and validate the capability of the non­destructive and contactless cvs to assess the visual quality changes during the cold storage of fresh­cut rocket leaves coming from soil and soilless growing systems (wp1) and to estimate some internal quality attributes (chlorophyll and ammonia content) also through the packaging material. evaluating quality through the package is critical to identify the regions of the bag where the product is visible without shadows or highlights created by illumination: this is mandatory to measure colour properties in a reliable and mean­ ingful way. at harvest, rocket leaves, cultivated on soil or soilless system (wp1), were packed in open pp bags and stored at 10°c for about 18 d. during stor­ age, all samples were observed to attribute the ql according to the rating scale reported in figure 3 and the postharvest quality traits were evaluated by destructive conventional methods [colour parame­ ters, chlorophyll content, ammonia content (fadda et al., 2016) and electrolyte leakage]. then, images of unpackaged and packaged samples were acquired by the cvs. during image acquisition, no constraints were imposed on the position of the product in the bag, on the position of the bag in the scene or on the highlights created by the illumination on the surface of the bag: this was necessary to demonstrate the applicability of this technology into a real industrial line. colour correction was performed by the linear model, identified as the best trade­off between effec­ tiveness and computational complexity in the previ­ ous research activity. packed and unpacked products were processed using exactly the same phases apart from the artefacts’ elimination step applied to the images of packaged products to select the regions where the colour information was meaningful, with­ out interference from light artefacts and reflections. at last, the random forest model was used to solve fig. 3 ­ changes in the sensory quality level (ql) of fresh­cut rocket leaves during the storage at 10°c according to the 5 to 1 rating scale reported by palumbo et al. (2021 b). in detail, ql5= very good; ql4= good; ql3= fair; ql2= poor; ql1= very poor. amodio et al. ‐ the sus&low project 129 both the classification problem (assessment of the qls) and the regression problems (estimation of quality marker parameters such as chlorophyll and ammonia contents). the same architecture was used for all the tasks, by simply changing the training data. the histogram of the image, evaluated in the a­b plane of the cielab colour space, was used as the set of features. the random forest model was able to automatically select the subset of values more suit­ able for solving each task. all the postharvest quality parameters detected by conventional analysis during the storage of fresh­ cut rocket leaves were significant in the ql assess­ ment and, among them, chlorophyll and ammonia contents proved to be useful marker parameters for the objective separation of each ql considered, both on soil and soilless cultivation approach. the cvs was able to operate without relevant dif­ ferences on unpackaged and packaged products. the test was done joining all the samples, regardless of the cultivation approach: the results showed a not significant performance loss on packaged leaves (pearson’s linear correlation coefficient of 0.84 for chlorophyll and 0.91 for ammonia) with respect to unpackaged ones (0.86 for chlorophyll and 0.92 for ammonia) (fig. 4). finally, three partial least square (pls) models were performed to predict the ql using as predictors chlorophyll and ammonia contents obtained by destructive methods (model i), by cvs on packaged products (model ii) and by cvs on unpackaged ones (model iii) (table 1). the results showed high performances in terms of r2 and the model obtained by predictors estimated non­destructively by the cvs (model ii and iii) provid­ ed better performances in the ql prediction than one obtained by destructive analysis, in both calibration and validation. wp3: marketing strategies to support the added value of the products lip and nd certified implementing a marketing strategy, based on often intangible characteristics to consumers such as lip and nd, it is not an easy task. low impact prac­ tices do not have a highly distinctive impact on prod­ uct characteristics nor determine unique taste, flavour, or look elements to consumers. however, certifications could be used to signal quality through the application of standards of quality and practices. whether certifications could be effective in terms of marketing in the case of products lip and nd, or for signalling quality in general is matter of discussion. vecchio and annunziata (2011), for instance, in their work question the possibility of effective understand­ ing of certification by consumers. at this purpose the research team of wp3 decided to implement a differ­ ent strategy and test it on the market. visual story­ telling certifying lip and nd has been then hypothe­ sized to better communicate the importance and the impact of those practices on food. the research activity, therefore, has been orga­ nized in three steps: identifying the communication fig. 4 ­ values estimated by the cvs (abscissa) vs. values mea­ sured in the laboratory (ordinate) for ammonia content on unpackaged (a) and packaged (b) rocket leaves and for total chlorophyll content on unpackaged (c) and packaged (d) samples (palumbo et al., 2022 b). table 1 ­ root mean square error (rmse) and the coefficient of determination (r2) in calibration (c) or validation (v) of the partial least square (pls) models predicting visual quality of rocket leaves (palumbo et al., 2022 b) pls models predictors rmsec r2 c rmsev r2 v i total chlorophyll and ammonia obtained by destructive methods 0.45 0.9 0.86 0.70 ii total chlorophyll and ammonia obtained by cvs on packaged rocket leaves 0.46 0.89 0.75 0.77 iii total chlorophyll and ammonia obtained by cvs on unpackaged rocket leaves 0.46 0.89 0.7 0.8 adv. hort. sci., 2023 37(1): 123­132 130 strategy and set­up; testing through focus­groups the opportunity conditions for farms and companies; testing though a survey and an econometric analysis the consumers’ preference and their willingness to pay for products with lip and nd. therefore, a draft platform has been developed containing basic com­ munication rules in order to highlight sustainability attributes of products through storytelling. workflow has been established and a simulation has been con­ ducted (fig. 5). focus group with producers has allowed verifying the general appreciation for the marketing approach and allowed a better set­up of the strategy. finally, a picture­based simulation has been produced for the final test and the survey to consumers (fig. 6). as last activity, a questionnaire based survey has been prepared and administered to 467 consumers and an econometric model to estimate willingness to pay and consumers orientation has been set up and then estimated. the whole set of activties within the research project allowed understanding how impor­ tant is a correct communication of products and how different could be the perception of a product based on how you certify or narrate the production method. result allow understanding that older con­ umsers are more aware of sustainability and are more willing to pay for lip products. psicological pro­ file such as traditionalism and benevolence identify the consumer that, more than other profiles, would be willing to pay a higher price. 3. conclusions sensor­based fertigation management applied to rocket leaves and tomato confirmed to be a feasible approach to improve sustainability of soilless cultiva­ tion, also in cases where the complete and rapid switch to closed cycle recirculation systems is still impaired by economic, social, and environmental fac­ tors such as in mediterranean area. the results of this project related to non­destruc­ tive discrimination of tomatoes and rocket leaves, according to cultural practices using different levels of inputs (water and fertilizers), indicated the high potentiality of hsi and ft­nir techniques for the authentication of sustainable growing methods. moreover, project activities’ proved cvs as an effec­ tive tool for evaluating the product quality also through the bag, even working only on the regions of the image that provide meaningful colour information about the product’s surface. the integration of machine learning modules inside the cvs confirmed to be useful to simplify the design and tuning, done mostly automatically without human intervention. moreover, the flexibility introduced by machine learning makes the resulting architecture more flexi­ ble in adapting to different products and applications. as regards the marketing approach, consumers resulted willing to pay a higher price for lip products certified through a visual storytelling platform. in the next future, there could be a good chance that sus­ tainability­oriented practices coupled with a visual storytelling certification style could gain shares on food markets. acknowledgements this research was funded by the project prin 2017 “sus&low­sustaining low­impact practices in horti­ culture through non­destructive approach to provide more information on fresh produce history and quali­ ty” (grant number: 201785z5h9) from the italian ministry of education university. references amodio m.l., cabezas­serrano a.b., peri g., colelli g., 2011 ­ post‐cutting quality changes of fresh‐cut arti‐ chokes treated with different anti‐browning agents as evaluated by image analysis. ­ postharvest biol. technol., 62: 213­220. fig. 5 ­ workflow for products lip and nd certified platform. fig. 6 ­ picture based simulation of visual storytelling certifica­ tion for lip producs and nd. amodio et al. ‐ the sus&low project 131 amodio m.l., capotorto i., chaudhry m.m.a., colel­ li g., 2017 a ­ the use of hyperspectral imaging to pre‐ dict the distribution of internal constituents and to clas‐ sify edible fennel heads based on the harvest time. ­ comput. electron. agric., 134: 1­10. amodio m.l., chaudhry m.m.a., colelli g., 2017 b ­ the use of non‐destructive techniques to assess the nutritional content of fruits and vegetable, pp. 763­780. ­ in: yahia e.m. 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nb= naderi badrood. abstract: the present study was carried out as bifactorial in a completely ran­ domized block design to compare seasonal changes of antioxidant response (enzymatic and non­enzymatic) in seven iranian pomegranate cultivars (‘malas mommtaz saveh’, ‘shishe kab’, ‘zagh aghda’, ‘naderi badroud’, ‘malas daneh ghermez’, ‘shirin shahvar fars’ and ‘zard anar arsenjan’) for a deeper under­ standing of their physiological differences and selecting probable more tolerant and adaptable cultivars to environmental conditions. uniform and healthy rooted (one­year) plants of seven iranian commercial pomegranate cultivars were purchased from a commercial nursery and planted in an orchard site in arsenjan region (one of the main hubs of pomegranate growing), fars province, iran. after full establishment of the trees, three rounds of sampling (fresh leaves) were conducted in spring, summer and fall. results indicated that in summer, activity of enzymatic antioxidants and leaf content of non­enzymatic antioxidants (glutathione, α­tocopherol and total polyphenols) rose in compari­ son to the contents measured in the other seasons in all studied cultivars. significant differences were observed among pomegranate cultivars for these parameters and also content of leaf pigments. ‘shishe kab’ was evaluated as a potential tolerant cultivar with high accumulation to changing environmental conditions, since this cultivar demonstrated the highest leaf content of non­ enzymatic antioxidants, chlorophyll a/b ratio and lowest level of lipid peroxida­ tion in warmest days of summer. similarly, ‘zard anar arsenjan’, ‘shirin shahvar fars’ and ‘malas daneh ghermez’ were evaluated as adaptable culti­ vars to regional conditions. 1. introduction pomegranate (punica granatum l.) is an ancient fruit­bearing decidu­ (*) corresponding author: arbonyanpour@yahoo.com citation: bonyanpour a.r., jamali b., 2020 ­ seasonal enzymatic and non‐enzymatic antioxi‐ dant responses in seven iranian pomegranate cul‐ tivars. ­ adv. hort. sci., 34(3): 265­276 copyright: © 2020 bonyanpour a.r., jamali b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. authors’ contributions: conducting experimental works, statistical analy­ sis of data, drafting of the manuscript, supervi­ sing the works, technical and material supports, critical revision of the manuscript received for publication 2 march 2020 accepted for publication 20 august 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 265­276 266 ous shrub or small tree native of iran (ebtedaie and shekafandeh, 2016). historical evidence reveals that the primary origin of pomegranate is iran and that it has been spread from this region to other areas. a large number of pomegranate varieties can be found in iran, more than 760 original, wild and decorative cultivars (mousavinejad et al., 2009). with a produc­ tion of 700,000 tons/year, iran is the world’s leading producer (sarkhosh et al., 2009). because of its high acclimation, pomegranate tree grows well in wide range of climates and soil conditions (galindo et al., 2014). iran, with an annual precipitation of 200 mm, is considered as a dry country and due to global warm­ ing and climate change, abiotic stresses such as drought and salinity are predicted to intensify in near future (ebtedaie and shekafandeh, 2016). planting fruit trees which are low water consumers such as pomegranate can be a suitable strategy for cultivat­ ing arid and semiarid regions (greenwood et al., 2010; jiménez et al., 2010). one of the main steps in orchard establishment is selection of suitable cultivars. previous investigations indicate varied levels of acclimation potential and tol­ erance to abiotic stress conditions among different pomegranate cultivars (tabatabaei and sarkhosh, 2006; okhovatian­ardakani et al., 2010; ibrahim, 2016). that would be attributed to their varied enzy­ matic and non­enzymatic antioxidant potential in response to seasonal changes in environmental con­ ditions (jamali et al., 2016). maintenance of a high antioxidant capacity to scavenge the toxic reactive oxygen species (ross) has been linked to increased acclimation of plants to environmental stresses (sharma et al., 2012). ross are produced in plants as byproducts during many physiological and biochemi­ cal processes such as photosynthesis and respiration. generation of ross causes rapid cell damage by trig­ gering a chain reaction (ahmad et al. , 2010). naturally occurring antioxidants in plant cells include: enzymatic and peptide defense mechanisms, non­ enzymatic mechanisms, phenolic defense com­ pounds, nitrogen compounds, carotenoids and chlorophyll derivatives. both the enzymatic and non­ enzymatic antioxidants play an important role as nat­ ural antioxidants (el­missiry, 2012) previous literatures focused on comparison between differences in enzymatic and non­enzymatic antioxidants in various iranian pomegranate cultivars are limited and more investigations seem necessary. the goal of present study was to compare seasonal changes in enzymatic and non­enzymatic antioxidant responses of seven iranian commercial pomegranate cultivars for a deeper understanding of their acclima­ tion to environmental and regional conditions. 2. materials and methods uniform and healthy rooted plants of seven iranian cultivars of punica granatum l. were pur­ chased from a commercial nursery and planted in a completely randomized block design with 3 replica­ tions (each replication had 3 plants) with 3­meter dis­ tance in rows and 5­meter distance between rows in an orchard site in arsenjan region (hub of pomegran­ ate growing and production in fars), fars province, southern iran. average annual climate parameters in the experimental region were: precipitation (200 mm), relative humidity (max: 55%, min: 23%), tem­ perature (max: 42°c, min: 4°c). all the cultivars were growing in same soil conditions and were irrigated in a similar way (drip irrigation). routine cultural prac­ tices suitable for commercial fruit production were carried out during the experimental period. cultivars included: malas momtaz saveh (mms), naderi badroud (nb), malas daneh ghermez (mdg), shirin shahvar fars (ssf), zagh aghda (za), shishe kab (sk) and zard anar arsenjan (zaa). after 4 years and full establishment of the plants, samples (fresh leaves) were taken from the trees. leaf samples were taken from different orientations of the trees (north, south, west and east); 25 fully expanded mature leaves from each side of all trees (100 leaves per tree as bulk samples) were samples in liquid n2 and trans­ ported to laboratory. leaves were taken from shoots without terminal fruit. leaves with abnormal symp­ toms such as chlorosis and mechanical lesions caused by pests or diseases were avoided. leaf samples were taken at 3 times (june 2nd, august 10th and october 10th) during the growing season; tempera­ ture and humidity of experimental region at the time of leaf sampling are given in table 1. table 1 ­ average day and night temperature and relative humidity in the experiment region at the time of sam­ pling date of sampling average day/night temperature (°c) relative humidity (%) june 2nd 27/14 45 august 10th 38/25 23 october 10th 27/15 27 bonyanpour and jamali ‐ seasonal antioxidant responses in iranian pomegranate cultivars 267 the following parameters were measured in stud­ ied cultivars for two consecutive years and an aver­ age was reported. enzymes extraction for enzymes extraction, leaves (0.5 g) were ground to fine powder in liquid nitrogen with mortar and pestle and then homogenized in 2 ml extraction buffer (50 mm potassium­phosphate buffer, ph 8.0), 10% (w/v) polyvinylpyrrolidone (pvp), 0.1 mm ethyl­ enediaminetetra acetic acid (edta), 1 mm dithiothre­ itol (dtt). the homogenate was centrifuged (15000 × g) at 4°c for 30 min. then, the supernatants were collected. superoxide dismutase activity the activity of sod was determined by adding 0.1 ml of the enzymatic extract to a tube containing 13 mm l­methionine, 25 mm nitro­blue tetrazolium chlo­ ride (nbt), 0.1 mm edta, 50 mm sodium carbonate and 2 mm riboflavin in a 50 mm phosphate buffer in ph 7.8 (dhindsa et al., 1980). tube was placed under two 15 w fluorescent lamps for 15 min. a complete reaction mixture without enzyme, which gave the maximal color, served as control. reaction was stopped by switching off the lights and keeping the tubes in dark. a non­irradiated complete reaction mixture served as a blank. the absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as that amount of enzyme that reduced the absorbance reading to 50% in comparison with tubes lacking enzyme. sod activity was expressed as units per milligram of protein per minute. catalase activity catalase (cat, ec 1.11.1.6) activity was measured spectrophotometrically according to the method of chance and maehly (1955), by monitoring the decline in absorbance at 240 nm due to h2o2 consumption. one milliliter of reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0) and 15 mm h2o2. the reaction was initiated by adding 50 μl of crude extract to this solution. cat activity was expressed as units (μmol of h2o2 consumed per minute) per milligram of protein. peroxidase activity the activity of guaiacol peroxidase (pox) was determined by adding 50 µl of the crude enzyme preparation to 2 ml of a solution containing 50 mm potassium phosphate buffer (ph 7.0)., 13 mm guaia­ col and 5 mm h2o2. increase in absorbance due to oxidation of guaia­ col (extinction coefficient: 26.6 mm­1 · cm­1) was mon­ itored at 470 nm for a minute. peroxidase activity was expressed as units (μmol guaiacol oxidised per minute) per milligram of protein. ascorbate peroxidase activity ascorbate peroxidase (apx, ec 1.11.1.11) activity was measured spectrophotometrically according to nakano and asada (1981) by following the decline in absorbance at 290 nm due to ascorbate oxidation. the oxidation rate of ascorbate was estimated between 1 and 60s after starting the reaction with the addition of h2o2. one milliliter of reaction mix­ ture contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbate, 0.15 mm h2o2, 0.1 mm edta and 50 μl of enzyme extract. apx activity was expressed as units (μmol of ascorbate oxidized per minute) per milligram of protein. protein content protein content was determined according to bradford (1976) by using bovine serum albumin as a standard. for preparation of bradford reagent, 100 mg of coomassie brilliant blue g­250 weight and then dissolved in 50 ml 0f 95% ethanol. then, 100 ml of 85% orthophosphoric acid (h3p04) added to afore­ said solution and volume reached into1000 ml vol­ ume with distilled water. bradford reagent filtered using whatman paper to remove precipitates before use. for assessment the protein content, 5 ml of bradford reagent and 100 μl protein extraction added in the test tube and shaken vigorously for a few seconds. reaction mixture remained at ambient temperature for 5 min and absorbance was read using spectrophotometer at 595 nm. bovine serum albumin (bsa) was used to elaborate a standard curve. the protein content was calculated according to the obtained equation. proline content proline was extracted and its concentration deter­ mined by the method of bates et al. (1973). leaf seg­ ments were homogenized with 3% sulfosalicylic acid and the homogenate was centrifuged (3000 × g) for 20 min. the supernatant was treated with acetic acid and acid ninhydrin, boiled for one hour and then absorbance at 520 nm was determined. contents of proline are expressed as μmol·g­1 fresh weight. leaf chlorophyll and carotenoids content leaf discs (0.5 g) were extracted in 5 ml of ace­ tone (80%), then centrifuged (8000 × g) for 10 min­ utes. the supernatant was used to make a final vol­ ume of 100 ml of the leaf extract. extraction of leaf tissue with the buffer continued until decoloration. adv. hort. sci., 2020 34(3): 265­276 268 absorbance of the extract was read at 470, 645 and 663 nm with a spectrophotometer and 80% acetone was used as a blank. finally, chlorophyll (a and b) and carotenoids contents were calculated according to the following equations (lichtenthaler, 1987): chl a (mg. g­1 fresh weight): [(12.25a663 ­ 2.79a645) × v / 1000 × w] chl b (mg. g­1 fresh weight): [(21.50a645­ 5.10a663) × v / 1000 × w] chla + chlb (mg. g­1 f.w.): [(7.15a663 + 18.71a645) × v /1000 × w] carotenoids (mg. g­1 f.w.): 1000a470­1.82chla­ 85.02chlb / 198 where chla = chlorophyll a; chlb = chlorophyll b; chla+chlb = total chlorophyll; a = absorbance at ƛ (nm). leaf anthocyanins content total leaf anthocyanins were measured spec­ trophotometrically by ph differential method (lee et al., 2005) with two buffer systems: potassium chlo­ ride buffer (ph 1.0,0.025 m) and sodium acetate buffer (ph 4.5, 0.4 m). leaf samples (0.5 g) were extracted with 2 ml methanol: water: concentrated hcl solution (80:20:1 v/v/v). 0.4 ml of leaf extract was mixed with 3.6 ml of corresponding buffers and read against water as blank at 510 and 700 nm. absorbance (a) was calculated as a = (a510 ­ a700) ph 1.0 ­ (a510 ‐ a700) ph 4.5 then total anthocyanins content was calculated using the equation: anthocyanin (µg. g­1 fresh weight) = (a × mw × df × 1000) / e where a is the absorbance of the diluted sample and df is the dilution factor (10), mw is molecular weight of cyanidin­3­glucoside (449.2) and e= 26,900 l/mol.cm, the molar extinction coefficient of cyani­ din­3­glucoside. glutathione content glutathione (gsh) was estimated by the method of moron et al. (1979). two hundred mg of leaf tissue was homogenized in 2 ml of ice­cold 5% trichloroacetic acid. the homogenate was then cen­ trifuged at 4°c at 17000 × g for 30 min. a volume of 75 µl of the clear supernatant was added to a cuvette containing 300 µl of phosphate buffer (0.2 m, ph 8.0) and 750 µl of 0.6 mm dtnb (5, 5­dithiobis­2­ nitrobenzoic acid) in phosphate buffer. the absorbance at 412 nm was read and glutathione con­ tent was derived against a standard curve prepared with known amounts of gsh in 5% trichloroacetic acid. malondialdehyde content malondialdehyde (mda) content was determined by the thiobarbituric acid (tba) reaction as described by ali et al. (2005), with slight modifications. two hundred mg leaf samples were homogenized with 2 ml of 0.1% trichloroacetic acid and centrifuged at 10000 × g for 15 min. one ml of the supernatant was mixed with 2.5 ml 0.5% thiobarbituric acid in 20% trichloroacetic acid and incubated in hot water (95°c) for 30 min. thereafter, it was cooled immediately on ice to stop the reaction and centrifuged at 10000 × g for 30 min. absorbance at 532 and 600 nm was determined, and mda concentration was estimated by subtracting the non­specific absorption at 600 nm from the absorption at 532 nm, using an absorbance coefficient of extinction (155 mm­1 · cm­1). leaf total polyphenols content leaf polyphenols content was determined with folin­ciocalteu reagent using gallic acid as a standard phenolic compound. in brief, 1 g of lyophilized leaf samples were placed in an eppendorf tube, with 1 ml of methanol (80%), grinded at 4°c and centrifuged at 10000 × g for 15 min. the extract was mixed with 0.5 ml of folin­ciocalteu reagent (diluted 1:1 with water) then 1 ml of a 5% sodium carbonate solution was also added. after 30 min, absorbance was measured at 725 nm and expressed as mg · g­1 fresh weight. leaf α‐tocopherol content α­tocopherol was extracted according to chong et al. (2004). two hundred mg lyophilized sample was homogenized in 1 ml acetone with a prechilled mor­ tar and pestle at 4°c. following the addition of 0.5 ml hexane, the homogenate was first vortexed for 30 s, then centrifuged at 1000 × g for 10 min. the upper hexane layer was removed while the acetone layer containing vitamin e remained in the vial. a second aliquot of 0.5 ml hexane was added, and the extrac­ tion process was repeated at least twice. α­ tocopherol was estimated by the method of kanno and yamauchi (1997). a 0.4­ml aliquot of 0.1% (w/v) 3­(2­pyridyl)­5, 6­diphenyl­1, 2, 4­triazine was added to 0.2 ml of pooled extract. the volume was made up to 3 ml with absolute ethanol, 0.4 ml 0.1% (w/v) ferric chloride (fecl3·6h2o) was added, and the con­ tent was gently mixed under dim light in a dark room to avoid photochemical reduction. after a 4 minutes reaction at room temperature, 0.2 ml 0.2 m orthophosphoric acid was added and the mixture left for another 30 min. absorbance was determined at 554 nm spectrophotometrically and reported as µg. g­1 fresh weight. the blank was prepared in the same manner except that absolute ethanol was used instead of the sample. α­tocopherol (sigma chemical) was used as a standard. bonyanpour and jamali ‐ seasonal antioxidant responses in iranian pomegranate cultivars 269 leaf ascorbic acid content ascorbic acid was estimated by the method of omaye et al. (1979). briefly, to 1 g of lyophilized leaf sample, 10% ice­cold trichloroacetic acid was added and centrifuged for 20 min at 3500 × g in room tem­ perature. one ml of the supernatant was mixed with 0.2 ml of dtc reagent and incubated for 3 hours at 37°c. then 1.5 ml of ice­cold 65% h2so4 was added, mixed well and the solutions were allowed to stand at room temperature for an additional 30 minutes. the color developed was read at 520 nm spectropho­ tometrically and leaf ascorbic acid content reported as µg. g­1 fresh weight. statistical analysis the experiment was carried out as a 7×3 bifactori­ al in a completely randomized block design (seven pomegranate cultivars as first factor and three sam­ pling dates as second factor). data were analyzed by sas and means were compared using duncan’s multi­ ple range tests at 5% probability level. table 2 ­ sod, cat, apx and pod activity in the leaves of different pomegranate cultivars cultivars sod (mg­1 protein min­1) cat (mg­1 protein) apx (mg­1 protein) pod (mg­1 protein) mms 76.72± 6.08 a 23.37± 2.54 ab 7.02± 0.66 d 38.56± 6.74b nb 67.25± 8.25 bc 26.15± 1.34 ab 7.25± 0.66 d 50.41± 1.52a mdg 63.02± 7.96 cd 19.07± 1 c 11.29± 1.20 b 37.83± 5.78b sk 76.17± 3.48 a 22.76± 2.02 bc 11.19± 1.45 b 36.60±2.02 b ssf 68.55± 6.56 bc 27.18± 2.90 a 9.50± 1.45 c 28.38± 4.58d za 58.82± 8.76 d 19.98± 2.72 c 7.28± 0.66 d 24.30± 2.72e zaa 70.21 ± 7.02 ab 24.04± 2.40 ab 13.90± 0.88a 32.58± 1.15 c 3. results the activity of sod, cat, pod and apx in the studied pomegranate cultivars is reported in table 2. sk and mms showed the highest sod activity. no sig­ nificant difference was observed among nb, ssf and zaa. the maximum pod activity (50.41 units mg­1 protein) was found in nb which was significantly higher than other cultivars. activity of this enzymatic antioxidant was not different in mms, sk and mdg. the highest cat activity was detected in ssf (27.18 units mg­1 protein), however zaa, nb and mms were not statistically different. activity of cat was not sig­ nificantly different in sk and za compared to mdg. apx activity in zaa was higher in comparison to the other studied cultivars. activity of apx was not statis­ tically different in sk and mdg. leaf contents of some non­enzymatic antioxidants in studied pomegranate cultivars are presented in table 3. the highest leaf glutathione content (158.57 means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated mean ± standard error (n = 3). table 3 ­ leaf concentration of some non­enzymatic antioxidants and malondialdehyde (mda) in seven iranian cultivars cultivars glutathione (µg g­1 f.w.) proline (µg g­1 f.w.) ascorbic acid (µg g­1 f.w.) α­tocopherol (µg g­1 f.w.) polyphenol (mg g­1f.w.) mda (µg g­1 f.w.) mms 142.2±2.8 c 6.04±0.5 b 3.07±0.84 b 210.66±14.8 d 26.84±0.5 ab 32.99±1.15 a nb 147.5±4.61 b 3.26±0.1 d 2.55±0.2 c 199.3±8.3 f 18.77±0.77 e 29.01±1.63 b mdg 138.74±10.1 d 4±0.1 cd 2.48±0.13 c 172.6±19.47 g 22.42±1.0 d 26.01±0.87 c sk 158.57±4.0 a 7.35±0.3 a 3.58±6.26 a 269.66±7.8 a 27.98±0.66 a 23.23±0.82 d ssf 140.19±11.5 cd 5.92±0.08 b 2.48±0.13 c 172.6±19.47 g 23.55±1.11 cd 23.99±0.72 cd za 128.36±5.7 f 4.62±0.3 c 3.01±0.2 b 204±10.12 e 18.77±0.77 e 30.32±0.54 ab zaa 134.28±4.5 e 6.98±0.32 ab 2.92±0.3 b 229.33±12.34 b 25.31±0.5 bc 24.42±2.08 cd means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated mean ± standard error (n = 3). 270 adv. hort. sci., 2020 34(3): 265­276 µg g­1 fresh weight) was observed in sk which was significantly higher than other cultivars. no signifi­ cant difference was detected between mdg and ssf. leaf total polyphenols in sk was 32% higher com­ pared to nb, mms was not significantly different in comparison to sk. also, za, ssf and mdg were not different. sk demonstrated the highest leaf proline content (7.35 µmol g­1 fresh weight), however zaa was not statistically different in comparison to this cultivar. leaf ascorbic acid content was significantly higher in sk compared to other studied cultivars. this parameter was not statistically different in mms, za, ssf and zaa. leaf α­tocopherol content in sk (269.66 µg g­1 fresh weight) was the highest among the stud­ ied cultivars. the highest mda content was found in mms (32.99 µmol g­1 fresh weight), however, za was not statistically different. seasonal changes of sod, cat, pod and apx in studied pomegranate cultivars are presented in fig­ ure 1 (a­d). sod activity rose in summer in mms, za, nb, ssf and zaa. in fall, activity of this antioxidant enzyme decreased significantly in zaa, ssf and za compared to summer. higher activity of pod was observed in mms, sk, za, nb, mdg and zaa in sum­ mer in comparison to spring. this characteristic was not statistically different in summer compared to spring in ssf. a significant decline in pod activity in fall compared to summer was detected in mms, sk, za and nb. cat activity did not change significantly during spring, summer and fall in mms, za, nb, mdg, ssf and zaa. activity of this enzyme decreased signif­ icantly in sk (about 37%) in summer. rise in activity of apx was observed in mms, sk and zaa in summer. this parameter was not different in mms, za, nb, mdg, ssf and zaa in fall compared to summer. changes in content of some non­enzymatic antioxidants in studied pomegranate cultivars in spring, summer and fall are demonstrated in fig. 2 (a­ e). in all studied pomegranate cultivars leaf content of glutathione rose significantly in summer, and then declined in fall. the highest content of this non­enzy­ matic antioxidant (182.21 µg g­1 fresh weight) was detected in sk in summer. leaf total polyphenols increased significantly in all studied cultivars in sum­ mer, whereas the content decreased in fall in mms, sk, za, mdg, ssf and zaa this characteristic decreased in fall. proline in mms, sk and za was higher in summer compared to spring and fall in nb, mdg, ssf and zaa did not change significantly during spring, summer and fall. ascorbic acid content increased (about 56%) in mdg in summer compared to spring then decreased significantly in fall. in con­ trary in mms ascorbate content declined (34%) in summer then rose in fall. other cultivars showed no significantly changes during these seasons. in all culti­ vars leaf α­tocopherol increased in summer then fig. 1 ­ seasonal activity changes of sod (a), cat (b), pod (c) and apx (d) in studied cultivars. columns with different let­ ters represent significant differences at 5% probability using duncan’s multiple range test, those are valid for all columns. bonyanpour and jamali ‐ seasonal antioxidant responses in iranian pomegranate cultivars 271 decreased in fall. this characteristic in mms, za, nb, mdg and ssf was higher in fall in comparison to spring. zaa and sk leaf α­tocopherol content was not statistically different in spring and fall. the highest mda content was detected in sum­ mer, this parameter was higher in fall compared to spring. leaf total proteins decreased significantly in summer (fig. 3). leaf mda content increased in sum­ mer in all studied cultivars, and the rise was more pronounced in za, nb and mms compared to sk, ssf and zaa. in the former three cultivars mda was high­ er than spring (fig. 3). leaf pigments content in studied cultivars are indicated in table 4. the highest total chlorophyll content was observed in ssf, but sk was not statisti­ cally different. also no significant difference was observed between mdg and zaa. the highest leaf anthocyanin (0.31 µg g­1 fresh weight) and carotenoid (0.34 mg g­1 fresh weight) were obtained from sk. no significant difference was observed between zaa and ssf for these two parameters. chlorophyll a/b ratio was significantly higher in sk and zaa compared to other cultivars. nb and zaa had significantly higher total leaf proteins in comparison to other cultivars. no significant difference was observed between sk, za, mdg and ssf. leaf total chlorophyll content was higher in spring in comparison to summer and fall. in summer the highest levels of anthocyanin and carotenoid con­ tents were detected. chlorophyll a/b ratio was signif­ icantly higher in summer or fall compared to spring (fig. 4 a­d). changes in leaf pigments in spring, summer and fall in studied cultivars are shown in fig. 4 (a­d). leaf total chlorophyll content decreased in summer then rose in fall in all cultivars. in sk, za, nb, mdg, ssf and zaa leaf total chlorophyll was not statistically different in fall compared to spring. leaf anthocyanin fig. 2 ­ seasonal changes in content of glutathione (a), polyphe­ nols (b), proline (c), α­tocopherol (d) and ascorbic acid (e) in studied cultivars. columns with different letters represent significant differences at 5% probability using duncan’s multiple range test, those are valid for all columns. fig. 3 ­ seasonal changes in mda content in studied cultivars. columns with different letters represent significant diffe­ rences at 5% probability using duncan’s multiple range test, those are valid for all columns. adv. hort. sci., 2020 34(3): 265­276 272 and carotenoid content rose in all studied cultivars in summer. in fall leaf anthocyanin decreased in sk and za, similar decline in leaf carotenoid was observed in mms, sk, za, nb and zaa. chlorophyll a/b ratio increased in mm, za and nb, decreased in zaa and did not change in sk, mdg and ssf in summer. this parameter was not different in fall compared to spring in all cultivars. 4. discussion and conclusions in present study mean activity of sod, pod and apx rose significantly in summer similar to the con­ centration of non­enzymatic antioxidants. during summer months solar irradiance intensity and regional mean day temperature rise. drought, salini­ ty, high temperatures and uv­b radiation lead to enhanced generation of ross in plants due to disrup­ tion of cellular homeostasis (mittler, 2002). scavenging or detoxification of excess ross is achieved by an efficient antioxidative system com­ prising of the non­enzymatic as well as enzymatic antioxidants (noctor and foyer, 1998). rise in activity of sod, pod and apx as well as increase in concen­ tration of non­enzymatic antioxidants in summer were a part of protective responses of studied plants. however, significant differences were observed fig. 4 ­ seasonal changes in content of chlorophyll (a), carotenoid (b), anthocyanin (c), chlorophyll a/b ratio (d) in studied cultivars. columns with different letters represent significant differences at 5% probability using duncan’s multiple range test, those are valid for all columns. means followed by the same letters within columns are not different at 5% probability using duncan’s test. all data indicated mean ± standard error (n = 3). table 4 ­ leaf pigments and protein concentration in seven iranian cultivars cultivars anthocyanin (µg g­1 f.w.) chlorophyll (mg g­1 f.w.) chlorophyll a/b ratio carotenoid (mg g­1 f.w.) proteins (mg g­1 f.w.) mms 0.29±0.01 b 1.17±0.59 c 1±0.02 d 0.24±0.006 d 19.62±0.95 c nb 0.26±0.007 c 1.09±0.07 d 1.10±0.03 c 0.23±0.007 de 21.35±0.25 a mdg 0.26±0.002 c 1.21±0.17 bc 1.17±0.01 b 0.27±0.009 c 20.32±1.25 b sk 0.31± 0.002 a 1.34±0.06 a 1.23±0.01 a 0.34±0.008 a 20.48±0.98 b ssf 0.29±0.01 b 1.36±0.04 a 1.15±0.008 b 0.31±0.004 b 20.62±1.59 b za 0.25±0.006 c 1.03±0.06 e 1.07±0.03 c 0.22±0.005 e 20.48±0.40 b zaa 0.29±0.01 b 1.27±0.1 b 1.22±0.038 a 0.30±0.006 b 21.27±0.68 a bonyanpour and jamali ‐ seasonal antioxidant responses in iranian pomegranate cultivars 273 between cultivars. increased activity of sod is often correlated with increased adaptability of the plants to environmental conditions (zaefyzadeh et al., 2009; sharma et al., 2012). high temperatures or drought cause either enhancement or depletion of cat activity depending on the plant species (sharma and dubey, 2005; han et al., 2009). cat activity decreased in sk and did not change significantly in other cultivars in summer; similar results have been reported by gholami et al. (2012). apx scavenges the h2o2 produced by sod using ascorbate as the electron donor (noctor and foyer, 1998). decline in ascorbate level in mms in summer can be contributed to this process. however, this parameter did not change in sk in summer which can be due to higher tolerance of this cultivar to high temperature. overexpression of a cytosolic apx­gene derived from pea in transgenic tomato plants amelio­ rated oxidative injury induced by chilling and salt stress (wang et al., 2005). similarly, over expression of the tapx gene in either tobacco or in arabidopsis increased tolerance to oxidative stress (yabuta, 2002). most of non­enzymatic antioxidants such as polyphenols, glutathione, proline and α­tocopherol rose in summer in our study concomitant with increase in mean day temperature and solar irradi­ ance. this was in agreement with previous works. (ghorbanali et al., 2012; sivaci and duman, 2014) glutathione is one of the most important cellular antioxidants (sharma et al., 2012). it also plays an indirect role in protecting membranes by maintaining α­tocopherol and zeaxanthin in the reduced state (hasanuzzaman et al., 2013). glutathione accumu­ lates to high concentrations, especially under stress conditions. increase in glutathione concentrations during stress offsets stress initiated oxidation of glu­ tathione and causes changes in gene expression directly or through interaction with regulatory pro­ teins and/or transcription factors. this increase is equally important in signal transduction and defense against ross (hasanuzzaman et al. , 2013). glutathione is a potential scavenger of 1o2, h2o2 and •oh (gholami et al., 2012). additionally, this tripep­ tide plays a key role in the antioxidative defense sys­ tem by regenerating another potential water­soluble antioxidant like ascorbic acid (foyer and halliwell, 1976). endogenous glutathione concentration has been reported to be associated with salt stress toler­ ance (sumithra et al., 2006). kattab (2007) reported that brassica napus seed priming with glutathione improved seedling resistance probably by enhancing the activities of antioxidant enzymes. polyphenols have strong antioxidant properties and their presence at an elevated level is associated with increased abiotic stress tolerance. increase in concentration of polyphenolic compounds following abiotic stress conditions such as high temperature has been reported by various authors in different species (jamali et al., 2016). bautista et al. (2016) compared the levels of total phenolic compounds and antioxidant flavonoids in a relatively large num­ ber of plant species from different families growing under varied environmental conditions. their data strongly support a general and relevant role of these compounds in mechanisms of acclimation to environ­ mental and regional conditions. proline accumulation can serve as a selection cri­ terion for the tolerance and adaptability of most species (parida and das, 2005; ashraf and foolad, 2007; ahmad et al., 2009). in addition to its role as a compatible osmolyte and osmoprotectant, several studies have attributed an antioxidant feature to this amino acid, suggesting ross scavenging activity and proline acting as a 1o2 quencher (smirnoff and cumbes, 1989; matysik et al. 2002). it has been reported that proline protects higher plants against osmotic stresses not only by adjusting osmotic pres­ sure but also by stabilizing many functional units such as complex ii electron transport, membranes, and proteins and enzymes such as rubisco (hamilton and heckathorn, 2001). several lines of evidence indicate that α­toco­ pherol plays a major role in plant stress tolerance, keeping an adequate redox state in chloroplasts (munne­bosch, 2005). deficiency of this antioxidant leads to a slightly increased susceptibility to photo oxidative stress (kanwischer et al., 2005). other mechanisms such as increases in abscisic acid con­ centration under stress conditions are also known to enhance α­tocopherol (singh et al., 2011). in our study, ascorbic acid did not change signifi­ cantly during spring, summer and fall. the increase of glutathione pool during summer could be necessary to regulate the levels of ascorbate (foyer and theodoulou, 2001). significant difference in concentration of leaf pig­ ments was observed between cultivars and seasons in the present study. a decrease in the chlorophyll concentration in summer is a typical symptom of oxidative stress (egert and tevini, 2002). in addition to harvesting solar energy, carotenoids play protec­ tion roles keeping integrity of photosynthesis appara­ tus against photo oxidative damages by scavenging adv. hort. sci., 2020 34(3): 265­276 274 free radicals (andrade­souza et al. , 2011). carotenoids are precursor of aba which is an impor­ tant phyto­hormone regulating plant responses to stresses. presence of higher carotenoid concentra­ tion leads to lower photo­oxidative damage and high­ er potential for regulating plant growth under stress conditions (han et al., 2008). our results were in accordance with previous works (christie et al., 1994; chalker­scott, 1999; parida and das, 2005). plant tis­ sues with a higher content of anthocyanins usually have a higher resistance to drought. the purple culti­ var of pepper is more tolerant to water stress than the green cultivar (bahler et al., 1991). the higher ratio of chlorophyll a/b which was observed in some of cultivars was considered to be the result of a decreased emphasis on light collection in relation to the rates of psii photochemistry (demmig­adams and adams, 1996). it was suggested that sod can be used as an indi­ rect selection criterion for screening drought­resistant plant materials (zaefyzadeh et al., 2009). significant differences were observed for the measured parame­ ters between studied pomegranate cultivars. sk culti­ var is well adapted to changing environmental condi­ tions since this cultivar had higher activity of sod and elevated levels of non­enzymatic antioxidants com­ pared to other cultivars. the presence of these com­ pounds at higher concentration probably mitigated overproduction of ross in summer. this was in accor­ dance with previous studies. khayyat et al. (2014) compared salt tolerance in mms and sk. they evalu­ ated sk as more salt tolerant cultivar. in various studies the chlorophyll concentration was used as a sensitive indicator of the cellular meta­ bolic state (chutipaijit et al., 2011). higher chloro­ phyll concentration is related to elevated tolerance against abiotic stresses such as drought and salinity (hasanuzzamn et al., 2013). chlorophyll a/b ratio did not change in sk in summer which could be explained as no decrease of peripheral light­harvesting com­ plexes and a higher stress tolerance in this cultivar (liu et al., 2011). mda concentration which is an index for lipid peroxidation was the lowest in sk. zaa, ssf and mdg could be considered as cultivars with high tolerance capacity to abiotic stresses as well, and mys, za and nb as cultivars with intermedi­ ate abiotic stress tolerance. other important charac­ teristics such as macro and micronutrients absorp­ tion (data not shown), endogenous plant growth reg­ ulators such as auxins, cytokinins and aba (data not shown) and trees productivity and fruits quality (data not shown) were considered for this evaluation. however, as it was mentioned before, pomegranate cultivars have high compatibility capacity and they grow well in wide range of climates and soil condi­ tions. this is the reason why ghasemi soloklui et al. 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between genotypes and drought stress on the superoxide dismu‐ tase and chlorophyll content in durum wheat landraces. ­ turk j. biology, 33(1): 1­7. impaginato 91 adv. hort. sci., 2021 35(1): 91­96 doi: 10.13128/ahsc­9681 warm stratification improves embryos development and seed germination of cycas revoluta j. benjelloun, s. bouzroud (*), z.e. triqui, q. lahlimi alami, r. layachi, a. smouni, a. guedira laboratoire de biotechnologie et physiologie végétales, centre de biotechnologie végétale et microbienne biodiversité et environnement, faculté des sciences, université mohammed v de rabat, morocco. key words: cycas revoluta, development, germination, treatment, warm strati­ fication, zygotic embryos. abstract: the broad objective of this research is to study the effect of warm stratification on cycas revoluta zygotic embryos length, seed germination and plant development. four warm treatments were applied to seeds. our results showed that seeds storage at room temperature or 30°c improved significantly zygotic embryos length. moreover, time of germination was significantly reduced with the warm stratification. the highest percentage of germination was obtained with seeds warm treated at 30°c for 2 months while only 25% of seeds were able to germinate in the control. regarding seedlings development, our results demonstrated that warm stratification did not affect plant develop­ ment. no significant differences have been recorded in all the evaluated para­ meters except for root length. taken together, these results underlined the beneficial effect of warm stratification on cycas revoluta seed germination and plant development and proposed a new method to improve seed germination of cycas revoluta. 1. introduction the sago palm (cycas revoluta l.) is one of the widespread ornamental trees, grown in temperate, subtropical and tropical regions more precise­ ly in miyazaki and kagoshima prefectures in kyushu district down to the ryukyu islands, okinawa prefecture in japan (zarchini et al., 2011). described as one of the most primitive species among the living cycads, cycas revoluta has been used as an indoor and outdoor landscape plant for centuries (stevenson, 1990; jones, 1994). cycas revoluta is propagated either from seeds, which remain viable for only a short time, or from vegetative offshoots (demiray et al., 2017). germination of seed of cycas revoluta is hard and time consuming (zarchini et al., 2011). seeds can take 3 to 9 months to initiate germina­ tion before they can continue to germinate for periods of a year or more. c. revoluta seeds also demonstrates rapid loss of viability and low mor­ (*) corresponding author: sarah.bouzroud@gmail.com citation: benjelloun j., bouzroud s., triqui z.e., lah­ limi alami q., layachi r., smouni a., guedira a., 2021 ­ warm stratification improves embryos development and seed germination of cycas revo‐ luta. ­ adv. hort. sci., 35(1): 91­96 copyright: © 2021 benjelloun j., bouzroud s., triqui z.e., lahlimi alami q., layachi r., smouni a., guedira a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. author contribution statement j.b, a.s and a.g.conceived and designed the experiment. j.b., s.b. performed experiments and collected the data. j.b., s.b., z.t., q.l.a., and r.l. contributed reagents/materials/analysis tools. j.b., s.b., a.s. and a.g. analyzed data and wrote the manuscript. competing interests: the authors declare no competing interests. received for publication 6 september 2020 accepted for publication 12 february 2020 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-9681 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 91­96 92 phogenic potential, which hinder its conservation (naderi et al., 2015). breaking dormancy is the main problem faced by all cycas revoluta (frett, 1987). several attempts have been made to overcome with cycas revoluta seed dormancy problem. priming treatments of seeds seems to offer a new way to increase seeds germination percentage. mechanical and chemical scarification has been described widely as an efficient way to improve germination of the hard­seeds species of cycas and some other species known for their hard­coated seeds (frett, 1987; rouhi et al., 2010). indeed, several studies have reported a great responsiveness of cycads seeds to various pretreat­ ments, namely, scarification, depulping, exposure to chemical substances like potassium nitrate, gibberel­ lic acid or sulfuric acid or soaking in hot water for specific period of time (zarchini et al., 2011; millaku et al., 2012). warm stratification was also used to improve seed germination of many species such as sambucus and symphoricarpos (baskin et al., 2002). the present study aimed to improve the germination of cycas revoluta seeds through different warm strat­ ification treatments as a way to develop an efficient in vivo germination protocol for this ornamental species. 2. materials and methods plant material freshly harvested seeds collected from 50 years old female mature plants grown in faculty of sciences garden, university mohammed v in rabat (morocco) were used in this study. zygotic embryos length measurement seeds were soaked in water for 48 hours in order to soften the sacrotesta; the orange external layer. the sacrotesta was then removed mechanically with a knife. seeds were then flamed with ethanol for 2 minutes. sclerotesta layer was mechanically eliminat­ ed. the megagametophytes were surface sterilized for 20 minutes by soaking in 30% dilution of naocl containing 2­3 drops of tween­20, followed by 3­4 rinses with sterile distilled water. after surface steril­ ization, megagametophytes were pooled, longitudi­ nally bisected and the zygotic embryo (ze) was excised from each megagametophyte. zes length was measured and the mean was calculated from at least 20 biological replicates. warm stratification treatments and seeds cultivation after removing the sacrotesta mechanically, equal samples of seeds were subjected to different treat­ ments. treatments consisted in seed storage at room temperature (18­20°c) or 30°c for 2 or 4 months depending on the treatment (table 1). the warm stratification temperatures were chosen based on previous reports that underlined the beneficial effect of seed storage at 18°c and 30°c on seed’s germina­ tion (roh et al., 2004; baldos et al., 2014; keun et al., 2016). untreated seeds were cultivated immediately and referred as control treatment (t0). seeds of cycas revoluta were then planted in bins containing steril­ ized soil at 2­5 cm depth. cultures were incubated at 25±2°c, with a photoperiod of 16 hours of light and 8 hours of darkness and watered daily depending on soil moisture. germination and plant growth parameters recording weekly observations were performed and seed emergence was recorded after 10 months of cultiva­ tion. the data for the kinetic of germination and time of germination (days) were recorded. percentage of germination was calculated after ten months of cul­ ture. number of leaves, stip height and width, root length and the length of the most developed leaf were determined at the end of the experiment. statistical analysis zygotic embryos length, time of germination, number of leaves, stip height and width, root length and the length of the most developed leaf were com­ pared using a fixed model of analysis of variance (anova). for each parameter and condition, means were calculated based on at least thirty biological replicates. in case of significant difference between groups, a tukey test was used for means separation, at risk of 0.05. table 1 ­ different heat treatments used to enhance seed ger­ mination of cycas revoluta treatment type of treatment time of application t0 control/untreated 0 hours t1 room temperature (18­20°c) 2 months t2 30°c 2 months t3 room temperature (18­20°c) 4 months t4 30°c 4 months benjelloun et al. ‐ warm stratification and cycas revoluta germination 93 3. results effect of warm stratification on zygotic embryos length zygotic embryos (zes) length was investigated in the different warm pre­treatments. our results showed that all the applied treatments (t1, t2, t3 and t4) influenced zes length compared with the untreat­ ed seeds (t0) (fig. 1). zes length of seeds subjected to t1, t2, t3 and t4 was significantly enhanced, but no significant difference was observed between t3 and t4 seeds. indeed, zes length increased by 134%, 276%, 300% and 342% in t1, t2, t3 and t4 warm treat­ ed seeds respectively when compared to untreated seeds. effect of warm stratification on seed’s germination as mean to gain more insight on the effect of warm storage on seed germination, time of germina­ tion (calculated starting from the first day of seeds cultivation), percentage of germination and its kinetic were determined for each treatment. the results were summarized in figures 2, 3 and 4. effect of warm stratification on the time of germina‐ tion minimum time of germination (163.33 days) was recorded in seeds stored at 30°c for 4 months fol­ lowed by those stored at 30°c for 2 months (198.19 days) and those stored at room temperature for 4 months (202 days). note that the difference between these three treatments was statistically insignificant. untreated seeds (t0) and those stored at room tem­ perature for 2 months (t1) took the maximum time duration for germination with an average of 294.4 days and 256.75 days respectively (fig. 2). effect of warm stratification on the percentage of germination investigating the percentage of germination of seeds from the four different treatments and the untreated ones revealed that seed germination response varied among the different warm treat­ ments. the highest percentage of germination (49.33%) was recorded in seeds stored for 2 months at 30°c while the lowest value of 8% was observed for seeds stored at room temperature for 4 months (fig. 3). untreated seeds showed although a percent­ age of germination around 25%. effect of warm stratification on the kinetic of germi‐ nation investigating the kinetic of germination revealed a high variability between the different treatments applied (fig. 4). our data showed that seeds storage at 30°c for two or four months (t2) allowed seeds to germinate faster. indeed, seeds started to germinate fig. 1 ­ effect of warm stratification treatments on cycas revolu‐ ta embryos length. (a) zygotic embryos isolated from seeds subjected to t0, t1, t2, t3 and t4. scale bars=1 cm. (b) zygotic embryos length in the different treat­ ments. bars are mean ± sd of at least thirty biological replicates. asterisks indicate statistical difference accor­ ding to tukey test (p<0.05). fig. 2 ­ effect of different warm stratification treatments on the time of germination of cycas revoluta embryos bars are mean ± sd of at least thirty biological replicates. asterisks indicate statistical difference according to tukey test (p<0.05). adv. hort. sci., 2021 35(1): 91­96 94 after four months of culture whereas, the first germi­ nated seeds appeared after five months and 6 months respectively for t1 and t0. seeds storage at room temperature for four months (t3) delayed the germination by one month compared to untreated seeds (t0). these data joined those related to time of germination. effect of warm stratification on plant’s growth and development seeds pre­treatment with temperature affected zygotic embryos length and their germination. these results prompted us to see whether the pre­treat­ ment can influence seedlings growth and develop­ ment. several growth parameters namely, number of leaves per plant, stip height and width, root length and the length of the most developed leaf were eval­ uated. table 2 summarized the results. data analysis had shown that seeds pre­treatment did not affect the number of leaves per plant. seeds storage for 2 months at 30°c gave a maximum num­ ber of leaves per plant (1.50±0.59) while the lowest value was obtained with t4 treatment (1.14±0.37). note that these differences remained insignificant. leaf length was also not significantly affected by the priming treatments. it was found that maximum leaf length was 57.33±3.05 cm in seedlings subjected to t3 treatment while a minimum of 48.22±4.23 cm was recorded with t1 treatment. stip height showed no significant difference between the different treatments. stip width, on the other hand, displayed significant variations between the different treatments. a significant increase in stip width was observed in seedlings subjected to t1 and t2 as compared to the control (t0) while non­signifi­ cant changes was observed between the remaining treatments(t3 and t4). regarding root development, root length showed significant variations between the different treat­ ments. seeds storage for 2 months at 30°c resulted in a significant increase in root length compared to untreated seeds while a decrease in root length was observed with t3 and t4 treatments. fig. 3 ­ effect of different warm stratification treatments on the percentage of germination of cycas revoluta embryos. fig. 4 ­ effect of different warm stratification treatments on the kinetic of germination of cycas revoluta embryos. table 2 ­ effect of different priming treatments on growth parameters of cycas revoluta seedlings values are mean ± sd of at least thirty biological replicates. values with different letters indicate the statistical significance (p<0.05) according to tukey test. treatments number of leaves/plant stip height (cm) stip width (cm) leaf length (cm) root length (cm) t0 1.31±0.47 a 3±0.47 a 1.81±0.19 b 50.63±6.24 abc 26.62±3.15 bc t1 1.20±0.42 a 3.92±0.32 a 2.10±0.24 a 48.22±4.23 bc 21.60±2.07 cd t2 1.50±0.59 a 3.77±0.45 a 2.15±0.22 a 55.91±4.25 ab 35.42±2.69 a t3 1.33±0.57 a 3.5±0.86 a 1.86±0.30 ab 57.33±3.05 a 31.66±2.31 ab t4 1.14±0.37 a 3.50±0.47 a 1.58±0.15 b 49±5 c 21±3 d benjelloun et al. ‐ warm stratification and cycas revoluta germination 95 4. discussion and conclusions cycas revoluta is an ornamental tree that has been widely used as an indoor and outdoor land­ scape. however, despite its importance in landscape design, it is facing problems regarding its germination mainly due to the hard­coat (ullah et al., 2020). different methods have been tested to overcome hardseededness. these include mechanical, chemi­ cal, and physical scarification treatments to make the seed coat permeable for water uptake. several reports have shown that seeds pre­treatment with sulphuric acid (h2so4), potassium nitrate (kno3) or ga3 results in a better germination results. indeed, zamia furfuracea and cycas revoluta germinations were assessed by schutzman, using a chemical scari­ fication with concentrated h2so4 and then soaking them in gibberellic acid (ga3) (schutzman, 1984, 1989). however, these pretreatments are not always reliable with cycads (dehgan and yuen, 1983). for instance, zarchini et al. (2011) have found that the use of sulphuric acid even at lower concentrations seems to affect negatively seed’s germination while combined with hot water seems to improve cycas revoluta seed germination. it is well admitted that seeds storage in a warm environment usually results in relatively rapid embryos development (baskin et al., 2002; merritt et al., 2007; razavi and hajiboland, 2009). however, no previous work has been conducted to study the effect of seed storage in warm conditions on cycas revoluta germination and seedlings development. thus, we investigated the effect of warm storage on zygotic embryos development, seeds germination and growth of young seedlings of cycas revoluta. our results showed that warm storage at 30°c for 2 months or 4 months speeded the germination when compared to the untreated plot (fig. 2). seeds stor­ age at 30°c for 2 months improves also the germina­ tion percentage. meanwhile, the prolonged storage at 30°c for 4 months reduced the germination per­ centage by half compared to the untreated plot which suggest that prolonged storage have an inhibi­ tive effect rather than stimulating the germination (fig. 3). chen et al. (2007) have found that prunus campanulata seeds required 4­6 weeks of warm fol­ lowed by 8 weeks of cold stratification for maximum germination percentage. this finding was explained by the accumulation of high amounts of ga as a result of the cold stratification while gas in warm stratified embryos were significantly low. thus, prunus warm stratified embryos failed to germinate since gas content was very low. in h. salicornicum and s. imbricate, seeds germination was significantly improved when seeds were stored at 40±2°c for three months (el­keblawy, 2013). warm stratification for at least 1 month appeared to be essential for the germination of japanese snowbell (styrax japonicus) (baskin, 2009). indeed, roh and bentz (2003) found that without warm stratification, seeds were not able to germinate. seed’s dormancy in some orchids, mainly epipactis palustris and goodyera pubescens, could be overcame by a warm incubation of seeds followed by cold storage. this was explained by the fact that warm and cold stratification increased seeds permeability to water thereby softening the testa (roh and bentz, 2003). thus, the increase observed in germination percentage could be attributed to the increase of seed’s hard coat permeability caused by the warm storage which allow the removal of the physical barrier to water absorption. besides the improvement of seeds germination percentage and the reduction of the germination time, warm storage improved significantly zygotic embryos length which suggest that warm scarification result in a better development of zygotic embryos. regarding seedlings development, our data showed that warm scarification did not affect plant development (fig. 5). indeed, no significant differences have been record­ ed in all the evaluated parameters except for root length. taken together, these results suggest that warm scarification improved seeds development and germination by prompting zygotic embryos length, increasing germination percentage and reducing time of germination. fig. 5 ­ cycas revoluta plants obtained from pre­treated seeds with temperature (t1, t2, t3 and t4) and untreated seeds (t0). 96 adv. hort. sci., 2021 35(1): 91­96 this study aims to study the effect of seeds stor­ age at different temperatures on cycas revoluta seeds germination and development in order to develop an efficient in vivo germination protocol that can be used for mass production of this ornamental tree. based on our results, we found that seeds storage at 30°c for 2 months or 4 months reduced time of germina­ tion. however, the highest percentage of germination was only assessed when seeds were stored at 30°c for 2 months. taken together, this protocol repre­ sents a useful and potential method to improve com­ mercial mass propagation of cycas revoluta. references baldos o.c., defrank j., framer m., sakamoto g.s., 2014 ­ storage humidity and temperature affect dor‐ mancy loss and viability of tanglehead (heteropogon contortus) seeds. ­ hortsci., 49: 1328­1334. baskin c.c., zackrisson o., baskin j.m., 2002 ­ role of warm stratification in promoting germination of seeds of empetrum hermaphroditum (empetraceae), a cir‐ cumboreal species with a stony endocarp. ­ am. j. bot., 89: 486­493. baskin j.m., 2009 ­ the woody plant seed manual. ­ nativ. plants j., 10(3): 300­301. chen s.y., chien c.t., chung j.d., yangy.s., kuo s.r., 2007 ­ dormancy‐break and germination in seeds of prunus campanulata (rosaceae): role of covering layers and changes in concentration of abscisic acid and gib‐ berellins. ­ seed sci. res., 17(1): 21­32. dehgan b., yuen c.k.k.h., 1983 ­ seed morphology in relation to dispersal, evolution, and propagation of cycas l. ­ bot. gaz., 144(3): 412­418. demiray h., dereboylu a.e., yazici z.i., bildik s., bul­ bul k., senol s.g., pirhan a.f., 2017 ­ in vitro seed germination of cycas revoluta thunb. ­ bangladesh j. bot., 46(2): 559­564. el­keblawy a., 2013 ­ effects of seed storage on germina‐ tion of two succulent desert halophytes with little dor‐ mancy and transient seed bank. ­ acta ecol. sin., 33(6): 338­343. frett j.j., 1987 ­ seed germination of cycas revoluta. ­ j. environ. hortic., 5: 105­106. jones d.l., 1994 ­ cycads of the world. ­ smithsonian inst. press, washington, dc, usa, pp. 312. keun s.j., hee k.j., kyung l.a., 2016 ­ effect of warm and cold stratification, and ethanol treatment on germina‐ tion of corylopsis seeds. ­ hortic. sci., 43: 84­91. merritt d.j., turner s.r., clarke s., dixon k.w., 2007 ­ seed dormancy and germination stimulation syn‐ dromes for australian temperate species. ­ aust. j. bot., 55: 336­344. millaku f., gashi b., abdullai k., aliu s., osmani m., krasniqi e., mata v., rysha a., 2012 ­ effects of cold‐ stratification, gibberellic acid and potassium nitrate on seed germination of yellow gentian (gentiana lutea l.). ­ african j. biotechnol., 11: 13173­13178. naderi r., mohaiseni k., da silva j.a.t., omidi m., naderi b., 2015 ­ simplified 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(zamiaceae, cycadales) from chiapas, mexico. ­ phytologia, 55: 299­304. schutzman b., 1989 ­ a new species of zamia from honduras. ­ syst. bot., 14(2): 214­219. stevenson d.w., 1990 ­ morphology and systematics of the cycadales. ­ mem. new york bot. gard., 57: 8­55. ullah z., hassan i., hafiz i.a., abbasi n.a., 2020 ­ effect of different priming treatments on seed germination of sago palm (cycas revoluta l.). ­ world j. biol. biotechnol., 5: 1­3. zarchini m., hashemabadi d., kaviani b., fallahaba­ di p.r., negahdar n., 2011 ­ improved germination conditions in cycas revoluta l. by using sulfuric acid and hot water. ­ plant omics., 4(7): 350­353. impaginato 325 adv. hort. sci., 2020 34(3): 325­335 doi: 10.13128/ahsc­7411 the salinity tolerance of pomegranate cultivars: effects of salt stress on root and leaf mineral content e. jadidi 1, m. tatari 2 (*), m. ghasemnezhad, h.r. salemi 1 department of horticultural science, university campus 2, university of guilan, rasht, iran. 2 horticulture crops research department, isfahan agricultural and natural resources research and education center, areeo, isfahan, iran. 3 department of horticultural science, university of guilan, rasht, iran. 4 agricultural engineering research department, isfahan agricultural and natural resouces research and education center, areeo, isfahan, iran. key words: dry and fresh weight, mineral composition, nacl, salt stress. abstract: in this study, the effect of irrigation water salinity on rooted cuttings of eight pomegranate cultivars namely ‘malase saveh’ (‘m­saveh’), ‘malase isfahan’ (‘m­isfahan’), ‘robabe ghermeze shiraz’ (‘robab’), ‘gabrie yazd’ (‘g­ yazd’), gabrie torshe yazd’ (‘gt­yazd’), ‘zaghe sefide yazd’ (‘zs­yazd’), ‘zaghe torshe yazd’ (‘zt­yazd’) and ‘malase torshe pishva’ (‘m­pishva’) was studied. sodium chloride was added to irrigation water to get final concentrations of 3, 6, 9 and 12 ds/m. leaf and root mineral content, leaf abscission and root char­ acteristics were determined at the end of the experiment. the results showed that the salinity reduced significantly fresh and dry weight of root in all pome­ granate cultivars. water salinity up to 3 ds/m increased slightly roots fresh and dry weight of all cultivars and thereafter decreased. with increasing water salinity to 12 ds/m, accumulation of sodium and chloride both in roots and leaves increased, but nitrogen, phosphorus, magnesium and calcium contents decreased. the change of leaf potassium content was dependent to pomegran­ ate cultivar. the lowest sodium and chloride accumulation in root was observed in ‘zt­yazd’, but ‘m­pishva’ translocated the lowest sodium and chlo­ ride to leaf. the low ability of nitrogen absorption was found in ‘m­saveh’, whereas ‘m­pishva’ maintained the highest leaf nitrogen under salt stress con­ ditions. the most potassium content of root was observed in gt­yazd’, while ‘zs­yazd’ and ‘g­yazd’ translocated the highest potassium to leaves. generally, the responses of pomegranate to absorption and translocation of elements to leaves under salinity conditions were completely dependent to cultivar. ‘m­ pishva’ and yazd cultivars showed higher tolerance to salinity stress. 1. introduction high salt concentration in the soil is responsible for decreasing productivi­ (*) corresponding author: mtatari1@gmail.com citation: jadidi e., tatari m., ghasemnezhad m., sale­ mi h.r., 2020 ­ the salinity tolerance of pomegra‐ nate cultivars: effects of salt stress on root and leaf mineral content. ­ adv. hort. sci., 34(3): 325­ 335 copyright: © 2020 jadidi e., tatari m., ghasemnezhad m., salemi h.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2020 accepted for publication 18 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 325­335 326 ty in a wide range of agricultural crops in the world (fao, 2008). soil salinity also is one of the serious environmental stress that limits growth and produc­ tivity of horticultural crops. different factors include the excessive application of chemical fertilizers, the use of saline water for irrigation and the high water levels led to soil salinity (mastrogiannidou et al., 2016). irrigation with saline water affects the absorption of nutrient elements. the adverse effects depend on the salinity level, the type of salt, the plant species and the presence of other stress (grattan and grieve, 1999). therefore, differences in adaptability potential in plant species to prevailing abiotic stress conditions can be attributed to their varied ability for nutrient uptake and consequently different content of macro and micronutrients in plant organs and tissues (jamali et al., 2016). soil salinity indirectly decreases the absorption of nutrients in the plant by reducing root growth (kiani and abbasi, 2010). karimi and nasrollahpour­ moghaddam (2016) also found that salinity affected the accumulation of potassium content in the root and the transfer rate of sodium and calcium to shoot in pistachio plants. in other research, it was found that pistachio rootstocks under salinity stress have restriction mechanisms for absorbing and transfer­ ring of sodium and chloride ions (karimi and maleki kuhbanani, 2015). in olive, rossi et al. (2015) found that roots distinctly play an important role in the reg­ ulation of apoplast to reduce permeation and ion transfer to shoot, which depends on plant genotype and sodium concentration. pomegranate is one of important commercial fruit in tropical and subtropical regions of the world and also in mediterranean climate, which is enriched of nutritional and antioxidant compounds (parvizi and sepaskhah, 2015). other studies indicated that there is different levels of tolerance to abiotic stress condi­ tions such as drought and salinity among different pomegranate cultivars (tabatabaei and sarkhosh, 2006; okhovatian­ardakani et al., 2010; ibrahim, 2016). in general, tolerance to abiotic stress is com­ plex and depends on both genetic and physiological properties. karimi and hasanpour (2014) showed that with increasing salinity level, the sodium, chlo­ rine and potassium contents in roots and shoots of ‘robab’ and ‘shishe gap’ pomegranate cultivars increased. ‘shishe gap’ cultivar showed good growth by restricting the absorption and transfer of chlorine under salt stress conditions (karimi and hasanpour, 2014). it was also reported that the use of saline water reduced stem length, length and number of intern­ ode, leaf area and root development in pomegranate cultivars (amiri et al., 2011). in contrast, bhantana and lazarovitch (2010) observed that there were no differences between ‘vanderfol’ and ‘sp­2’ pome­ granate cultivars in response to salt water stress. some results showed a significant change in the anatomical structure of roots and leaves of the pomegranate, when exposed to salt levels of 800 to 4000 ds/m. these changes included an increase in the thickness of the cuticle layer, the formation of parenchymal cells, and an increase in the number and density of crystals in the parenchymal cells of the leaves and roots in pomegranates (zarinkamar and esfah, 2005). therefore, the aim of this study was the evalua­ tion of the effect of irrigation water salinity on some root growth characteristics and absorption and trans­ fer of root nutrient elements to shoot in eight pome­ granate commercial cultivars. 2. materials and methods plant material and cultivation this study was carried out during 2016­2017 in isfahan agricultural and natural resources research and education center, iran. uniform rooted cuttings of eight pomegranate commercial cultivars including ‘malase saveh’ (‘m­saveh’), ‘malase isfahan’ (‘m­ isfahan’), ‘robabe ghermeze shiraz’ (‘robab’), ‘gabrie yazd’ (‘g­yazd’), gabrie torshe yazd’ (‘gt­ yazd’), ‘zaghe sefide yazd’ (‘zs­yazd’), ‘zaghe torshe yazd’ (‘zt­yazd’) and ‘malase torshe pishva’ (‘m­ pishva’) were used as plant materials. rooted cut­ tings were planted in 20­liter plastic pots (35×30cm). the soil mixture was clay loam and cow manure with 1:1 ratio (v/v). physical and chemical properties of the soil mixture were analyzed at the beginning of the experiment (table 1). salinity treatments four levels of water salinity (control, 3, 6, 9 and 12 ds/m) were used for irrigation of different pome­ granate cultivars (table 2). the source of sodium chloride used in this experiment was from the lake salt that was analyzed before application (table 3). the salinity treatments were carried out from two months after planting in the pots for four months. jadidi et al. ‐ salinity tolerance of pomegranate cultivars 327 in order to avoid salt stress shocks, initially, pome­ granate plants were irrigated with water of 3ds/m electrical conductivity (ec). according to the climatic conditions of the region, the irrigation interval was applied in a three­day interval which reduced to two days at very hot months. the ratio of electrical con­ ductivity of the irrigation water and leach off were measured and the mean value was recorded as the leaching fraction (lf). the leaching coefficient was dependence to salinity level. the amount of the consumed water by each pot during the growing season was measured. in order to determine the irrigation intervals and net irrigation requirement (in), the irrigation depth parameter (dn) was calculated. the depth of stored water in the soil and readily available water (raw) were determined using the depth of root development and bulk densi­ ty (pb). in this way, soil moisture content at field capacity (fc) and wilting point (pwp) levels was mea­ sured. the obtained gross water requirement (ig) depends on irrigation system efficiency. in order to prevent of the salt accumulation, over­irrigation was implemented to the pots (in the amount of 15% of the field capacity), so that the water was drained from the bottom of the pots. evaluated traits at the end of the experiment, the plants were removed from the pot and the roots weighed after washing (root fresh weight). to determine root dry weight, samples were dried for 48 hours at 75°c and thereafter weighed using a digital scale (smit et al., 2000). in the end, the percentage of the root dry matter was obtained from the following formula: root dry matter = [(fresh weight ­ dry weight)/dry weight] x 100 the leaf abscission was counted at the end of the experiment (late september). the necrotic percent­ age of leaves at the end of the stress was visibly eval­ uated and recorded using the scoring method from 0 table 1 ­ some physical and chemical properties of soil mixture property unit value ph ­ 7.75 saturation percentage % 17 field capacity % 30 permanent wilting point % 7.25 organic carbon % 10.14 nitrogen % 0.096 total neutralizing value % 32.5 silt % 35 clay % 35 sand % 30 tissue ­ clay loam k mg/kg 709 p mg/kg 2.26 zn mg/kg 0.64 mg mg/kg 10.23 fe mg/kg 5.01 cu mg/kg 1.23 b mg/kg 2.66 na meq/l 4.53 cl meq/l 7.19 salinity ds/m 3.24 table 2 ­ the chemical analysis of used water for salt stress treatment ec ds/m ph k mg /l mg (mg /l) ca (mg /l) cl (mg /l) na (mg /l) control (0.38) 6.90 3.80 1.2 2.80 3 2.8 3 7.45 5.09 66 1.98 1320 770 6 7.84 12.49 162 4.86 3240 1890 9 7.90 19.66 255 7.65 5100 2975 12 8.20 25.45 330 9.9 6600 3850 table 3 ­ some chemical properties of used lake salt for salinity treatments co (mg/kg) cd (mg/kg) cr (mg/kg) ni (mg/kg) pb (mg/kg) fe (mg/kg) cl (%) na (%) mg (%) ca (%) k (%) p (%) 3 2 0 8 15 0 60 35 3 0.09 0.23 0 adv. hort. sci., 2020 34(3): 325­335 328 to 9. zero means non­necrosis leaves, one for very low necrosis (between 0 to 10%), three for low necrosis (between 10 to 30%), five for moderate necrosis (between 30 to 50%), and seven for sever (between 50­70%), and nine means very severs necrosis (between 70­90%). the content of mineral elements such as nitrogen, phosphorus, potassium, calcium, magnesium, chlo­ ride and sodium in leaf and root of pomegranate cul­ tivars was determined just in control (without salt additions) and 12 ds/m salinity stress treatment at the end of the experiment. the kjeldahl method is used to determine the nitrogen content in leaf and root samples (jones, 2001). briefly, 0.3g of fine dry powder was digested in concentrated h2so4 and distilled with naoh (40%), and ammonium nitrogen was fixed in h3bo3 (2%) and titrated with 0.1n h2so4. the p content of samples was determined by the vanadate­molybdate colorimetric method (chapman and pratt, 1982). the absorbance of samples was measured at 470 nm in a uv/visible spectrophotome­ ter (model pg instrument+80, leicester, uk). potassium (k) and sodium (na) content was deter­ mined by the flame photometric method as described by jones (2001). the digested extract was diluted by calcium chloride (cacl2) at 1:9 ratios (v/v) and the absorbance was measured at 766.5 nm (jones, 2001). calcium (ca) and magnesium (mg) were measured using atomic absorption spectroscopy. briefly, digest­ ed extracts were diluted with distilled water (1:9 v/v), then 4.75 ml of lanthanum nitrate [la (no3)3] was added to 250 ml of the diluted extract. finally, the absorbance was measured at 422.7 nm for ca and 285.2 nm for mg by atomic absorption (jones, 2001). chloride ion content was determined by titra­ tion. statistical analysis this experiment was conducted as the factorial experiment based on a randomized complete block design with two factors. to evaluate each trait, three replications and five observations in each replication were considered. the first factor was eight pome­ granate cultivars and the second factor was salinity treatment at five levels. analysis of data was per­ formed by anova method using statistical software sas (version 9.1) and means comparison using tukey test. 3. results fresh and dry weight of root the results showed that with increasing salinity levels from 3 ds/m to 12 ds/m in irrigation water, both fresh and dry weight of roots in all pomegranate cultivars significantly decreased (fig. 1). the lowest fresh and dry weight was found when plants irrigated with 12 ds/m, however, there was no significant dif­ ference between 12 and 9 ds/m. overall, fresh weight of root in pomegranate cultivars in 6, 9 and 12 ds/m treatments decreased by 46.3, 57.4 and 66%, respectively, compared with control treatment (fig. 1a), as well, root dry weight decreased by 45.4, 52.5 and 59 %, respectively (fig. 1b). the decreasing rate of fresh weight in the studied cultivars was completely dependent on the cultivar. the highest fresh weight of root was found in ‘gt­ yazd’, ‘robab’ and ‘m­saveh’ cultivars, respectively. the lowest fresh weight of root was observed in ‘zs­ fig. 1 ­ effects of different levels of water salinity on fresh (a) and dry (b) weight of root in pomegranate cultivars. jadidi et al. ‐ salinity tolerance of pomegranate cultivars 329 yazd’ (fig. 2). root dry matter also significantly decreased when pomegranate cultivars irrigated with 9 and 12 ds/m water (26.69 and 46.17%, respectively). in contrast, this trait slightly increased when plants irrigated with 3 ds/m saline water (fig. 3). leaf abscission leaf abscission was significantly affected by water salinity levels and pomegranate cultivars. overall, with increasing salinity level, leaf abscission was increased in all studied pomegranate cultivars (fig. 4). the highest leaf abscission was recorded when plants irrigated with 9 and 12 ds/m water. in 3 ds/m, ‘zt­yazd’ and ‘m­pishva’ had the lowest percentage of leaf abscission. the lowest percentage of leaf fig. 2 ­ effect of different pomegranate cultivars on root fresh weight. fig. 3 ­ effects of water salinity on root dry matter percentage of pomegranate cultivar. abscission in salinity of 6 ds/m was observed in ‘m­ isfahan’ and ‘m­pishva’. in 9 ds/m, ‘g­yazd’ and ‘robab’ showed the lowest leaf abscission. the low­ est percentage of leaf abscission in salinity of 12 ds/m was observed in ‘g­yazd’ and ‘zt­yazd’. in this salinity level, leaf abscission was 100% in ‘m­saveh’. leaf necrosis the leaf necrosis also affected by salinity level and pomegranate cultivars (fig. 5). there was no differ­ ence in leaf necrosis between 3 ds/m and control treatments in ‘zt­yazd’ and ‘m­isfahan’ cultivars. in 6 ds/m, the lowest leaf necrosis were observed in ‘m­ pishva’ and ‘zt­yazd’ with 1.6 and 1.8%, respectively. ‘zt­yazd’ and ‘m­pishva’ had the least leaf necrosis in 12 ds/m with the average of 2.3% (fig. 5). root and leaf cl and na content chloride and sodium contents of the root increased significantly when irrigated with 12ds/m saline water in compared with the control (table 4). the lowest root chloride content was found in ‘zt­ fig. 4 ­ the interaction effects of salinity levels and cultivars on leaf abscission. fig. 5 ­ the interaction effects of salinity levels and cultivars on leaf necrosis. similar letters in each column indicate no significant difference at the 5% level of tukey test. salinity (ds/m) cl (mg/kg) na (mg/kg) mg (%) ca (%) p (%) n (%) control 16.325 b 24.798 b 0.228 a 1.071 a 0.173 a 1.213 a 12 51.430 a 90.434 a 0.198 b 0.721 b 0.140 b 0.962 b table 4 ­ effects of water salinity (12 ds/m) on root mineral elements of pomegranate 330 similar letters in each column indicate no significant difference at the 5% level of tukey test. adv. hort. sci., 2020 34(3): 325­335 yazd’ with 22.752 mg/kg. there was no significant difference among other pomegranate cultivars (table 5). in this study, pomegranate cultivars showed dif­ ferent levels of root sodium accumulation. the least sodium content was observed in ‘zt­yazd’ with 39.308 mg/kg. ‘m­saveh’ and ‘zs­yazd’ showed the highest sodium accumulation of roots (74.118 and 62.947 mg/kg, respectively). the interaction effect of saline water treatment and pomegranate cultivars on leaf sodium and chlo­ ride contents was significant. the highest leaf chlo­ ride content was found in ‘m­saveh’ with 12 ds/m saline water (112.8 mg/kg) and the lowest ones was observed in ‘m­pishva’ with an average of 46.72 mg/kg (fig. 6). the highest leaf sodium content was observed in ‘m­saveh’ and ‘gt­yazd’ with averages of 427 and 383.5 mg/kg, respectively (fig. 7). in contrast, ‘m­ pishva’ showed the lowest leaf sodium content (141.5 mg/kg). root and leaf n content the results showed that when pomegranate culti­ vars irrigated with 12 ds/m water, the nitrogen con­ tent of the roots reduced significantly to 20.69% compared with the control (table 4). the response of pomegranate cultivars was different, when exposed to salinity stress (table 5). the lowest roots nitrogen content was found in ‘m­saveh’ (1%) but, no any sig­ nificant difference was found among others cultivars (table 5). the leaf nitrogen content in pomegranate culti­ vars also decreased when irrigated with 12 ds/m saline water. the highest and the lowest leaf nitro­ gen content was found in ‘m­pishva’ and ‘m­saveh’ (in 12ds/m), respectively (fig. 8). fig. 6 ­ the interaction effects of water salinity and pomegrana­ te cultivars on leaf chloride accumulation. table 5 ­ the comparison of root mineral elements in eight pomegranate cultivars cultivars cl (mg/kg) na (mg/kg) mg (%) ca (%) p (%) n (%) 'zs­yazd' 36.178 a 62.947 ab 0.230 ab 0.883 ab 0.153 a 1.108 a 'm­pishva'' 38.848 a 58.632 b 0.213 b 0.910 ab 0.155 a 1.105 a 'm­isfahan' 33.678 a 55.745 b 0.196 b 0.861 ab 0.163 a 1.111 a 'zt­yazd' 22.752 b 39.308 c 0.205 b 0.865 ab 0.151 a 1.106 a 'g­yazd' 31.427 a 55.317 b 0.201 b 0.843 b 0.166 a 1.088 a 'm­saveh' 39.583 a 74.118 a 0.256 a 0.906 ab 0.151 a 1.00 b 'robab' 32.610 a 53.788 b 0.198 b 0.960 a 0.158 a 1.063 ab 'gt­yazd' 35.943 a 61.072 b 0.205 b 0.940 a 0.156 a 1.123 a fig. 7 ­ the interaction effects of water salinity and pomegrana­ te cultivars on leaf sodium accumulation. fig. 8 ­ the interaction effects of water salinity and pomegrana­ te cultivars on leaf nitrogen content. jadidi et al. ‐ salinity tolerance of pomegranate cultivars 331 root and leaf p content the root phosphorus content significantly decreased in 12 ds/m salinity (19.07%) compared to control treatment. however, there was no significant difference among pomegranate cultivars in root phosphorus (tables 4 and 5). in the salinity of 12 ds/m, the leaf p content decreased compared with the control treatment. however, no significant difference was found among pomegranate cultivars for leaf phosphorus in the control treatment (fig. 9). root and leaf k content the response of pomegranate cultivars to potassi­ um accumulation of root was significantly different when irrigated with 12 ds/m water. ‘zs­yazd’, ‘g­ yazd’, ‘m­pishva’ and ‘gt­’yazd showed increase potassium content of root while, potassium contents of ‘robab’, ‘m­isfahan’, ‘m­saveh’ and ‘zt­yazd’ was decreased compared with the control. the lowest and the most potassium content of root were found in ‘gt­yazd’ and ‘m­isfahan’, respectively in salinity of 12ds/m (fig. 10). in contrast, leaf potassium content of all pome­ granate cultivars decreased significantly when irrigat­ ed with 12 ds/m saline water (fig. 11). the lowest content of leaf potassium was observed in ‘m­saveh’, ‘gt­yazd’ and ‘zt­yazd’ with 12 ds/m saline water treatment. the highest leaf potassium content in salinity conditions was found in ‘zs­yazd’ and ‘g­ yazd’. root and leaf ca content root calcium content showed a significant reduc­ tion of 32.67% compared with the control when treated with saline water (table 4). the lowest root calcium content was observed in ‘g­yazd’ with an average of 0.84% (table 5). in contrast, ‘robab’ and ‘gt­yazd’ cultivars showed the highest root calcium with averages of 0.96 and 0.94 %, respectively. the leaf calcium content also decreased under salinity conditions (fig. 12). the lowest leaf calcium content was recorded in ‘g­yazd’ cultivar with an average of 0.843% and the highest ones were found in ‘robab’ with 0.96% (table 6). root and leaf mg content the magnesium content of root also decreased when pomegranate plants irrigated with 12 ds/m saline water (13.15% compared with the control). the lowest of root magnesium content was found in ‘m­isfahan’ and ‘robab’ with averages of 0.196 and 0.198 %, respectively (table 4 and 5). in contrast, ‘m­ saveh’ was able to absorb the most magnesium under salt stress condition compared with other cul­ tivars. leaf magnesium content also decreased when treated with 12 ds/m irrigation water (fig. 12). the least leaf magnesium content was found in ‘m­ isfahan’ with 0.196% (table 6). 4. discussion and conclusions salinity reduces the ability of plants to water absorption and reduces the plant growth rate. the fig. 9 ­ the interaction effects of water salinity and pomegrana­ te cultivars on leaf phosphor content. fig. 10 ­ the interaction effects of water salinity and pomegra­ nate cultivars on root potassium content. fig. 11 ­ the interaction effects of water salinity and pomegra­ nate cultivars on leaf potassium content. adv. hort. sci., 2020 34(3): 325­335 332 salt will eventually rise to a toxic level via transpira­ tion of leaves, causing leaf senescence and abscission (munns, 2002). previous studies also showed that salinity changed growth parameters in pomegranate (karimi et al., 2011; mastrogiannidou et al., 2016), and pistachio (picchioni et al., 1990; naieni et al., 2004; saadatmand et al., 2007). the results of the current study showed that salini­ ty affected the growth characteristics in pomegranate cultivars. fresh and dry weight of roots as well as dry matter of root was decreased with increasing salinity levels. reduction of fresh weight of root was com­ pletely dependent on pomegranate cultivar. for example ‘gt­yazd’, ‘robab’ and ‘m­saveh’ cultivars maintained their growth characteristics more than other cultivars. munns and tester (2008) reported that in ‘malase shirin’ that was the salt tolerant culti­ similar letters in each column indicate no significant difference at the 5% level of tukey test. var, the growth rate increased after an increase in the salinity up to 40 mm, and the more. but in salt sensi­ tive cultivars, increase in salinity decreased the plant growth characteristics (munns and tester, 2008). according to the previous studies, salinity tolerant plants, especially permanent species, have more potential to survive and maintain growth rate under salt stress conditions (ferreira­silva et al., 2008). the results showed that fresh and dry weight of roots as well as dry matter percentage in mild salinity (3 ds/m) slightly increased but thereafter decreased. high levels of salinity reduced the rate of these traits. amiri et al. (2011) showed that salinity levels of 40, 80 and 120 mm of sodium chloride in irrigation water of ‘robab’ cultivar significantly reduced root fresh and dry weight. also, khoshbahkt et al. (2014) found that sodium chloride with 20, 40 and 60 mm concen­ tration in irrigation water caused a reduction in root fresh weight, and root dry weight in citrus. we also found that fresh weight of root was significantly affected by pomegranate cultivars. momenpour et al. (2015) also found a significant increase in almond leaf abscission when irrigated with saline water. khoshbahkt et al. (2014) reported that with increasing sodium chloride in irrigation water, a significant increase was observed in the leaf abscission percentage of two citrus rootstocks. these results are consistent with the findings of other researchers, which the increase in salinity levels of irrigation water, led to an increase in necrotic leaflet growth (momenpour et al., 2015). the result of the current study also showed that by applying salinity stress and increasing its concentration, in all studied cultivars, the percentage of leaf abscission increased. the least leaf abscission was observed in ‘g­yazd’ and ‘zs­yazd’. reducing the number of leaves in the cultivars significantly reduces the plant photosynthe­ sis level, which could be another reason for reducing plant growth characteristics in sensitive salinity culti­ vars. according to the results, with increasing concen­ tration of sodium chloride in irrigation water, leaf necrosis increased in the middle and the end of the stress period that are consistent with the findings of other researchers (momenpour et al., 2015). in the concentrations of 6, 9 and 12 ds/m, ‘m­pishva’, ‘zs­ yazd’ and ‘zt­yazd’ showed the lowest percentage of leaf necrosis, respectively. previous studies showed that with increasing salinity level in irrigation water, accumulation of chlo­ ride and sodium ions increased in the pomegranate fig. 12 ­ effect of 12ds/m saline water treatment on leaf mg and ca content. table 6 ­ the comparison of root mineral elements in eight pomegranate cultivars cultivars leaf mg (%) leaf ca (%) 'zs­yazd' 0.230 ab 0.883 ab m­pishva' 0.213 b 0.910 ab 'm­isfahan' 0.196 b 0.861 ab 'zt­yazd' 0.205 b 0.865 ab 'g­yazd' 0.201 b 0.843 b 'm­saveh' 0.256 a 0.906 ab 'robab' 0.198 b 0.960 a 'gt­yazd' 0.205 b 0.940 ab jadidi et al. ‐ salinity tolerance of pomegranate cultivars 333 root and leaf (karimi and hasanpour, 2014; mastrogiannidou et al., 2016). zarei et al. (2016) reported that salinity stress, increased the amount of root and leaf chlorine and sodium in fig cultivars. khayyat et al. (2014) also reported that with increas­ ing salinity level from 4.61 to 7.46 ds/m, shoot cl content in pomegranate cv. ‘malas mommtaz’ increased, but it decreased in ‘shishe kab’. in fact, ‘shishe kab’ could manage sodium transport into leaves better than ‘malas mommtaz’ cultivar. salinity­tolerant plants transfer less sodium and chlo­ rine to shoot than sensitive plants (fernandez, 2014; munns, 2002). we also find the significant difference in pomegranate cultivars for absorption of sodium and chloride ions and translocation to shoot. ‘zt­ yazd’ showed the lowest content of root chloride and sodium in 12 ds/m. at the same concentration of salinity, the lowest leaf sodium and chloride content was observed in ‘m­pishva’. the results showed that by increasing sodium chloride in irrigation water, nitrogen content of root and leaf reduced, which is in agreement with finding of naeini et al. (2004) on ‘alak torsh’, ‘malas torsh’ and ‘malas shirin’ pomegranate cultivars. karimi and hassanpour (2014) reported that under salinity stress, the root and leaf phosphorus content in ‘shisheh gap’ cultivar decreased. in the present study, there was a decrease in the content of root phosphorus under salinity. momenpour et al. (2015) no significant difference was observed among almond pomegranate cultivars in the root and leaf phosphorus under salt stress conditions that were similar to the results of this study. mastrogiannidou et al. (2016) showed that increasing of sodium chloride concentration in irriga­ tion water decreased root and leaf potassium con­ tent in ‘wonderful’ pomegranate cultivar. however, karimi and hasanpour (2014) reported that with increasing sodium chloride in irrigation water, root and leaf potassium content in pomegranate increased. in our study, salinity reduced leaf potassi­ um, but potassium changes of root were affected by pomegranate cultivar. gt­yazd’ and ‘zs­yazd’ showed the highest root and leaf potassium, respectively. salinity stress led to the reduction of leaf and root magnesium. naeini et al. (2004) also reported that with increasing sodium chloride concentration in irri­ gation water, content of root and leaf magnesium decreased in ‘alake torsh’, ‘malase torsh’ and ‘malase shirin’ cultivars. in the current study, m­ isfahan showed the lowest root and leaf magnesium. the highest leaf and root magnesium was observed in ‘m­saveh’. increasing salinity was associated with a decrease in leaf and root calcium. aboutalebi et al. (2008) reported that by increasing sodium chloride, the root calcium in four citrus species decreased and in one of them increased. in the present study, ‘robab’ had the highest leaf and root calcium in salinity condi­ tions. sarafi et al. (2017) indicated that ‘wonderful’ and ‘ermioni’ cultivars had the different abilities for p, k, ca and mg uptake under salt stress conditions. differences in the absorption of elements were reported between ‘robab’ and ‘shishe kab’ by hasanpour et al. (2015). ‘zt­yazd’ showed the lowest leaf abscission and necrosis. ‘m­pishva’ showed less leaf necrosis than other cultivars in salinity conditions. ‘zt­yazd’ and ‘m­pishva’ showed the least amount of chlorine and sodium accumulation in root and leaf, respectively. it seems that the mechanism of these two cultivars is different to salinity tolerance, so ‘zt­yazd’ transmits excess chlorine and sodium to the leaves, but ‘m­ pishva’ transmits less chlorine and sodium to the leaves and shows the accumulation of these ele­ ments in the root. perhaps for this reason, the amount of leaf nitrogen in ‘m­pishva’ was higher than other cultivars in salinity conditions. however, in severe salinity conditions, in some cultivars, the potassium content of root decreased, but the potas­ sium content of root in these two cultivars, along with ‘g­yazd’ and ‘gt­yazd’ increased. it seems that there is a wide variation among the studied cultivars that leads to different reactions to salinity. in gener­ al, ‘m­pishva’ and yazd cultivars showed higher toler­ ance to salinity stress. acknowledgements the authors are grateful university of guilan and isfahan agricultural and natural resources research and education center for supporting this work. references aboutalebi a., hassanzadeh v., arabzadegan m.s., 2008 ­ effect of salinity on macro‐nutrients and sodium concentrations in five species of citrus root. ­ j. agric. sci. nat. resour., 15 (1): 1­10. amiri e., mirzaei m., moradi m., zare k., 2011 ­ the effects of spermidine and putrescine poly‐amines on growth of pomegranate (punica granatum l. cv. adv. hort. sci., 2020 34(3): 325­335 334 rabbab‘) in salinity circumstance. ­ int. j. plant physiol. biochem., 3(3): 43­49. bhantana p., lazarovitch n., 2010 ­ evapo‐transpira‐ tion, crop coefficient and growth of two young pome‐ granates (punica granatum l.) varieties under salt stress. ­ agric. water manag., 97(5): 715­722. chapman h.d., pratt p.e., 1982 ­ methods of analysis for soil, plants and waters. ­ university of california, berkeley, ca, usa, publication no. 4034. fao, 2008 ­ land and plant nutrition management service. ­ www.fao.org. fernandez j., 2014 ­ understanding olive adaptation to abiotic stresses as a tool to increase crop performance. ­ environ. exp. bot., 103: 158­179. ferreira­silva s.l., silveira j., voigt e., soares l., vie­ gas r., 2008 ­ changes in physiological indicators asso‐ ciated with salt tolerance in two contrasting cashew rootstocks. ­ braz. j. plant physiol., 20(1): 51­59. grattan s.r., grieve c.m., 1999 ­ mineral nutrient acqui‐ sition and response by plants growth in saline environ‐ ment, pp. 203­229. ­ in: pessarakli m. 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(in persian). zarinkamar f., esfah a., 2005 ­ the effect of salinity on anatomical structure and alkaloid production in pome‐ granate. ­ bot. j. iran, 6: 97­107. s impaginato 255 adv. hort. sci., 2021 35(3): 255­267 doi: 10.36253/ahsc­9689 genetic diversity in colocasia esculenta and xanthosoma mafaffa in togo, west africa d. bammite 1 (*), p.j. matthews 2, d.y. dagnon 1, a. agbogan 1, p. agre 3, t.o. akintayo 3, k. odah 1, a. dansi 4, m. abberton 3, k. tozo 1 1 laboratoire de physiologie et biotechnologie végétale, faculté des sciences, université de lomé, boulevard gnassingbé eyadema, 01bp1515 lomé, togo. 2 national museum of ethnology, senri expo park 10‐1, osaka 565‐8511, japan. 3 international institute of tropical agriculture (iita), bioscience center, pmb 5320, oyo road, ibadan, nigeria. 4 université polytechnique d’abomey, faculté des sciences et techniques de dassa, laboratoire de biotechnique, ressources génétiques et amélioration des espèces animales et végétales (biorave), bénin. key words: crop diversity, new cocoyam, ssr, taro, togo. abstract: taro and new cocoyam are root and leaf crops commonly grown in tropical to warm temperate regions. in togo, they are neglected and underuti­ lized. here we report the genetic diversity of 26 accessions of taro and 101 accessions of new cocoyam. analysis of simple sequence repeats revealed low polymorphic information content of 0.43 and 0.25 in taro and new cocoyam, respectively. pca scatterplots and neighbour joining dendrograms based on the ssr data clustered accessions into groups that more­or­less correspond to morphological diversity in both species. amova within and between morpho­ logical groups revealed greater variances within groups than between. this indicates weak genetic differentiation between morphological groups, particu­ larly for taro. genetic diversity was greater among taro cultivars. taro has a longer history of introduction and dispersal in africa, and has had more oppor­ tunity for multiple introduction and local cultivar development. different strategies are suggested for future development of these crops in togo and africa. for taro, further studies of existing diversity and recent experimental introductions to africa are likely to be rewarding. new cocoyam, a modern his­ torical introduction, has spread widely in africa with little genetic diversity. for this crop, international collaboration is needed to clarify taxonomy, and to introduce further cultivars for evaluation under local conditions in africa. 1. introduction root and tuber crops are important sources of food and income for household in rural areas of africa. in sub­saharan africa, they provide (*) corresponding author: bdamigou@gmail.com citation: bammite d., matthews p.j., dagnon d.y., agbogan a., agre p., akintayo t.o., odah k., dansi a., abberton m., tozo k., 2021 ­ genetic and morphological diversity in colocasia esculen‐ ta and xanthosoma maffafa in togo, west africa. ­ adv. hort. sci., 35(3): 255­267 copyright: © 2021 bammite d., matthews p.j., dagnon d.y., agbogan a., agre p., akintayo t.o., odah k., dansi a., abberton m., tozo k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 september 2020 accepted for publication 9 july 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9689 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 255­267 256 about 20% of calories (pinstrup­andersen et al., 1999). taro, colocasia esculenta (l.) schott and new cocoyam, xanthosoma spp., are grown for food and income generation at the household level. both crops are grown in tropical regions of africa, asia, oceania and america, and taro is also common in temperate regions of africa, asia, and oceania (gonçalves, 2011; matthews, 2014; onyeka, 2014; grimaldi, 2016; matthews and ghanem, 2021). taro is considered an ancient crop in africa, with multiple likely routes of introduction from asia (matthews, 2006; fuller et al., 2011; chaïr et al., 2016; grimaldi, 2016). new cocoy­ am is known to have been introduced to ghana in 1843 (karikari, 1971), though earlier introduction fol­ lowing european contact with america has been sug­ gested (bown, 2000). most parts of these plants (corms, side­corms, stolons, petioles, leaf blades, and floral spathes) are edible, the leaves and corms are also commonly used as animal fodder in asia (coursey, 1984; matthews, 2010; mwenye et al., 2010; masuno et al., 2012; matthews, 2014; wada et al., 2017), and medicinal uses are also known (plowman, 1969; ribeiro pereira et al., 2021). the specific parts eaten vary according to cultivar attributes, local food knowledge, and cul­ tural or personal preferences. both crops have great potential for development in africa and globally (okereke, 2020). under cultivation, clonal propagation is universal for both taro and new cocoyam. in a global survey of taro, chaïr et al. (2016) found the greatest genetic diversity and the largest number of private alleles in asian cultivars, especially in india. low genetic diver­ sity was found in western africa, among diploid and triploid cultivars, and also in southern africa, where triploid cultivars were dominant. their observations correspond broadly to what is known about the breeding of taro: flowering, fruiting and seed produc­ tion by wild and cultivated taros are common in trop­ ical regions of asia and oceania (matthews, 2014), but have not been observed in africa despite occa­ sional reports of flowering (e.g. traore, 2013). natural fruiting and seed production by new cocoy­ am has not been reported outside south america, but sterile inflorescences are often produced (obser­ vation by authors), and induced flowering and experi­ mental crosses have been reported in cameroon (onokpise et al., 1992). globally, taro has undergone genetic erosion due to changes in cropping patterns, the spread of improved varieties, and replacement by other crops (lakhanpaul et al., 2003; matthews and ghanem, 2021), including new cocoyam (coursey, 1984). the acridity of taro (and resulting special care needed for cooking) (matthews, 2010), spread of taro leaf blight (tlb) (singh et al., 2012), and shortages of planting materials are contributing factors. in order to identify and preserve cultivars of economic value, maintain living germplasm collections efficiently (without excessive duplication), and provide baseline data for future breeding programs, genetic diversity and mor­ phology must be assessed in cultivars of both species. the taxonomy of cultivated xanthosoma species and relationships with wild species are uncertain, and historically there has been a tendency to use the name x. sagittifolium for all cultivated xanthosoma (giacometti and leon, 1994; castro, 2006; quero­ garcia et al., 2010; doungous et al., 2015). although x. sagittifolium is the name used in many previous studies in africa, the plant is most likely to be x. mafaffa (gonçalves, 2011). the taxonomy of cultivat­ ed xanthosoma spp. in tropical america has been revised by croat and delannay (2017). various culti­ vated species of xanthosoma are also circulating internationally and may have reached africa in the modern historical period. these include x. atrovirens c. koch & bouché (blackish green blades, and “blue” wax on dark green petioles creating a dark purple or black appearance), x. robustum (which can reach 4 m in height, with tall above­ground stems) and x. vio‐ laceum (with violet petiole tissue below a waxy sur­ face) (gonçalves, 2011). in addition to the 1843 intro­ duction of xanthosoma (karikari, 1971), an unsuc­ cessful attempt was made to introduce x. brasiliensis from puerto rico in 1974 (karikari, 1979). in central america, x. mafaffa cultivars vary in corm parenchy­ ma color (red or white), and those with red corms are also tinged with red in the petiole, leaf sheath and spathe (gonçalves, 2011). in togo, a collection of taro and new cocoyam cul­ tivars from throughout the country was assembled, and morphological groups were described in both species (bammite, 2018; bammite et al., 2018 a, b). however, data related to genetic diversity among these crops, based on molecular tools such as ssr, are lacking to enhance effective usage and conserva­ tion of these neglected species and develop a breed­ ing program to improve the quality of their germplasm. this study aimed to assess simple sequence repeat (ssr) diversity in the same collec­ tion of taro and new cocoyam. polymorphic ssr loci have codominant alleles (repeat sequences of vary­ ing length, detected by pcr amplification), and been bammite et al. ‐ genetic diversity of taro and cocoyam 257 used in many studies of taro (devi, 2012; you et al., 2015) and other edible aroids (suppl. table s1), and in genetic linkage mapping of the taro nuclear genome (quero­garcía et al., 2010; soulard et al., 2017). 2. materials and methods plant materials in 2016, cultivars of taro and new cocoyam were collected from 42 localities randomly selected across the five ecological zones of togo (fig. 1). at each vil­ lage, farmers were invited to bring corms of different cultivars grown in their village. for each distinct culti­ var recongnized in group discussions, the local name was recorded and collected corm samples were planted at the centre de recherche agronomique du litoral (cral), an experimental farm of the institut togolais de recherche agronomique (itra) located at davié, at latitude n 6°23’ and longitude e 1°12’ and at 88 m above sea level (table s2). accessions of both species were classified in a binary manner based on morphological characters that are easily observed in the field: taro accessions were identified as either dasheen (with large mother corms, and either stolons or side­corms; pop1), or eddoe (with small mother corms, and few to many side­corms; pop2); new cocoyam accessions were identified as either green (leaves entirely green; pop1) or purple (petioles purple or pink to some extent; pop2). morpological diversity in the same collection was previously analysed with reference to a wide range of agronomic and morphological characters (bammite et al., 2018 b; figs. s1­s3). young leaf tissue from one plant from each of 26 accessions of taro and 101 accessions of new cocoyam was dried on silica gel and taken to the international institute of tropical agriculture (iita) bioscience centre, ibadan, nigeria for dna extraction and genotyping. dna isolation and quantification dna was extracted using an optimized sds proto­ col recommended by iita bioscience centre (2017). about 100 mg of dry leaf tissue was put in a tube with two steel balls and reduced to powder using a spex genogrinder­2000. pre­heated extraction buffer (450 µl of 1m tris­hcl, 0,5m edta, 5m nacl, 20% sds and 1% pvp) was added. tubes were incubated at 65°c for 20 mins and inverted occasionally to homog­ enize each sample. tubes were removed from bath, allowed to cool for two mins, then 200 µl of ice­cold 5m potassium acetate was added and the mixture incubated on ice for 20 mins to precipitate proteins. tubes were then centrifuged at 3500 rpm for 10 mins, and each supernatant was transferred to a new labeled tube. a volume of 200 µl of 4% polyvinylpyrrolidone (pvp) was added to the super­ natant and gently mixed. to precipitate and remove proteins and lipids, 45 µl of chloroform isoamylal­ chohol (24:1) was added, mixed gently and tubes were centrifuged at 3500 rpm for 15 mins. each supernatant was transferred to a new tube, and a 2/3 volume of ice­cold isopropanol was added, mixed, and incubated in ­80°c for 15 mins to precipi­ tate the dna. after centrifuging at 3500 rpm for 15 mins, the dna pellet was washed by adding 400 µl of 70% ethanol, centrifugation at 3500 rpm for 15 mins, and decanting the supernatant until the last drop. the dna pellet was air­dried then resuspended in 100 µl low salt te buffer (10 mm tris­hcl, 1 mm fig. 1 ­ map of togo showing sampling localities in 2016 and ecological zones: zone i, northern lowlands; zone ii, northern togo mountains; zone iii, central lowlands; zone iv, southern togo mountains; zone v, coastal plains of southern togo. figure adopted from bammite et al., 2018 b; zones originally described by ern (1979); base map from ign france, 1990. adv. hort. sci., 2021 35(3): 255­267 258 edta). a volume of 2 µl of rnase a (10 µg/ ml) was added and incubated at 37°c for 40 mins. the quanti­ ty and quality of extracted dna was checked using electrophoresis with 1% agarose gel, and a nanodrop 8000 spectrophotometer, and the extracts were stored at ­20°c until use. pcr amplification initial testing was carried out with 47 primer pairs designed in previous ssr studies: 19 for for amorphophallus paeoniifolius (santosa et al., 2007), 11 for c. esculenta (hu et al., 2009.) and 17 for x. sagittifolium (cathebras et al., 2014) (table s3). the 47 primer pairs were tested first with five samples from each target species to determine which pairs could amplify scorable dna products in each species. pcr amplification was performed in pcr mixture (25 µl) containing 2.5 µl of template dna (20ng/µl), 2.5 µl of 10x nh4 pcr reaction buffer, 1 µl of 50mm mgcl2), 1 µl of 5 µm forward primer, 1 µl of 5 µm reverse primer, 0.2 µl of 5mm each dntp, 0.1 µl of biotaq dna polymerase and 16.1 µl of water. the pcr program consisted of initial denaturation (94°c, 5 mins), 42 cycles each consisting of 20 s denatura­ tion (93°c), 1 min annealing at temperatures ranging from 47 to 59°c (as recommended by the authors above; table s3), and 2 mins elongation (72°c). finally, an extension period of 10 mins was included. after pcr completion, the products were stored at 4°c until gel electrophoresis. ten µl of each pcr product was electrophoresed alongside a 50 bp dna ladder (new england biolab) in polyacrylamide gel (10% instapage gel) at 110 v for one hour, and bands were visualized by silver staining (1l tbe 0,5x buffer + 500 µl of safeview) for 3 mins. amplified fragment sizes were determined by comparison to the 50 bp ladder, and bands were examined and recorded using the endurotm gel documentation system. data analysis recorded gel images with pcr products were analysed the the image studio lite ver 5.2 software, generating binary matrix data for all accessions based on the band patterns observed at each ssr locus: presence of an amplified band was scored as “1”; absence was scored as “0”. summary statistics for each locus were estimated using powermarker 3.25 software. for statistical comparisons within each species, the binary classifications of morphotypes were used: taro ­ dasheen = pop 1, eddoe = pop2; new cocoyam ­ green = pop1, purple = pop2. genalex 6.5 software (peakall and smouse, 2006) was used to calculate the number of polymorphic loci (no. pl), the percentage of poly­ morphic loci (% pl), the observed number of alleles (na), the effective number of alleles (ne), average expected heterozygosity (he) (also known as nei’s gene diversity, nei, 1973), and shannon’s information index (i) (a measure of genetic diversity suitable for codominant data). analysis of molecular variance (amova) was performed to evaluate the genetic varia­ tion within and among morphotype populations by using genalex version 6.503 (peakall and smouse, 2006), and phipt (the proportion of total genetic vari­ ance derived from variance between individuals among populations, i.e. an estimate of population genetic dif­ ferentiation). here, our h0 = no genetic difference among populations, h1 = there is genetic difference among populations, and p= probability of an observed phipt value =/> than that observed by chance, if the null hypothesis (no genetic difference) is true. principal coordinate analysis (pca) was carried out using the genalex version 6.503 (peakall and smouse, 2006). the genetic distance matrix was con­ structed by calculating the shared allele distance for each pair of individuals in powermarker version 3.25. from this matrix, a neighbour joining (nj) tree was constructed using nei’s genetic distances (nei et al., 1983) in the same software. 3. results in the initial test with five samples of each target species, only 27 of 47 ssr primer pairs, amplified and gave polymorphic, scorable bands (fig. 2; table s3): fig. 2 ­ electrophoresis of pcr amplification products to show ssr polymorphism. above: taro tested with primer pair hk29; empty lanes at right are null results for new cocoyam tested with hk29. below: new cocoyam tested with primer pair mxscir10. bammite et al. ‐ genetic diversity of taro and cocoyam 259 two from amorphophallus amplified both target species, 11 from c. esculenta amplified only c. escu‐ lenta and 14 from x. sagittifolium amplified only x. mafaffa. the resulting measures of diversity for each species are shown in tables 1 and 4, and are sum­ marised below. among 26 accessions of taro, 33 alleles were observed at 13 loci, an average of 3.15 alleles per locus (loci are hereafter identified by the primer code names). the frequency of major alleles ranged from 0.38 for locus hk7 to 0.88 for hk5, with an average of 0.62. nei’s gene diversity ranged from 0.21 for hk7 to 0.71 for hk5, with an average of 0.49. polymorphic information content (pic) values ranged from 0.20 to 0.65 with an average of 0.43. the primers hk35, hk26, hk38, ampa9, hk7 with pic values >/= 0.5 were most discriminating (table 1). the percentages of polymorphic loci for each morphotype of taro were 82% (dasheen, pop1) and 58% (eddoe, pop2), with an average of 70% (rounded figures). the num­ bers of different (na) and effective (ne) alleles, shanons information index (i), and nei’s gene diversi­ ty (he) were all higher in dasheen and lower in eddoe (table 2). df = degrees of freedom, ss = sum of squares, ms = mean sum of squares, est. var. = estimated variance, % var = percentage of variation, phipt = proportion of total genetic variance derived from variance between individuals among populations, p = probability value for phipt. table 1 ­ taro: frequency of major alleles, number of alleles, nei's genetic diversity, and polymorphism information content (pic) for 13 primers applied to 26 accessions (togo collection) table 2a ­ taro: statistical measures of genetic diversity in the dasheen and eddoe populations (togo collection) n = no. accessions (test population), na = no. of different alleles, ne = no. of effective alleles, i = shannon's information index, he = nei’s gene diversity, %p = percentage of polymorphic loci (rounded figures) locus freq. major alleles no. alleles genetic diversity pic hk5 0.88 3 0.21 0.2 ampa15 0.85 3 0.27 0.26 hk25 0.77 2 0.36 0.29 hk31 0.65 2 0.45 0.35 hk29 0.65 3 0.48 0.39 ac3 0.5 3 0.54 0.43 hk22 0.65 3 0.51 0.45 hk34 0.65 3 0.51 0.45 hk35 0.65 4 0.52 0.48 hk26 0.54 3 0.59 0.52 hk38 0.42 3 0.64 0.56 ampa9 0.42 5 0.66 0.59 hk7 0.38 4 0.71 0.65 mean 0.62 3.15 0.49 0.43 n na ne i he %p pop1 (dasheen) 15 1.758 1.546 0.451 0.307 82 pop2 (eddoe) 11 1.364 1.234 0.251 0.156 58 mean 1.561 1.39 0.351 0.231 70 se 0.089 0.045 0.033 0.024 12 table 2b ­ taro: summary analysis of molecular variation (amova) in the dasheen and eddoe populations (togo collection) df ss ms est. var. % var. phipt p value between pops 1 15.962 15.962 0.915 17% 0.174 0.014 within pops 24 104.23 4.343 4.343 83% total 25 120.192 ­ 5.258 100% 260 adv. hort. sci., 2021 35(3): 255­267 among the 101 accessions of new cocoyam, 48 alleles were observed at 16 loci, an average of 3.0 alleles per locus. the frequency of major alleles ranged from 0.47 for ampa9 to 0.97 for ampa15, with an average of 0.83. nei’s gene diversity (he) ranged from 0.06 for ampa9 to 0.67 for ampa15, with an average of 0.28. pic values ranged from 0.06 to 0.62, with an average of 0.25. only the ampa9 locus gave a pic value greater than 0.5 (table 3). the percentages of polymorphic loci for each morphotype of new cocoyam were 74% (green, pop1) and 94% (purple, pop2) and with an average of 84% (rounded figures). a lower number of different alleles was recorded in the green population (na = 1.74, n = 23) and a higher number in the purple pop­ ulation (na = 1.87, n = 78), but the number of effec­ tive alleles (ne) and other measures of diversity (i, he) were higher in the green population (table 4). cluster analysis and structuring of genetic diversity analysis of molecular variance (amova) gave per­ centages of molecular variance of 83% within and 17% between the dasheen and eddoe populations of taro, indicating weak differentiation overall (table 2). for new cocoyam, the percentages of molecular vari­ ance were 64% within and 36% between the green and purple population (table 4). the probability value (p) for phipt is higher in taro (0.014) than in new cocoyam (0.001) providing for taro a weaker rejection of the h 0 of no genetic difference between populations. the first and second coordinates of the pca scat­ ter plot (fig. 3) represent, respectively, 48% and 16% (in total 64%; rounded figures) of the detected vari­ ability among taro accessions. for cocoyam acces­ sions, the coordinates represented 41% and 13% (in total 54%) (fig. 4). some dasheen taros formed a dis­ tinct group along the first coordinate, but apart from table 3 ­ new cocoyam: frequency of major alleles, number of alleles, nei's genetic diversity, and polymorphism infor­ mation content (pic) for 16 primers applied to 101 accessions (togo collection) # locus freq. major alleles no. of alleles genetic diversity pic 1 ampa15 0.97 3 0.06 0.06 2 mxscir1 0.95 2 0.09 0.09 3 mxscir1 0.94 2 0.11 0.11 4 mxscir0 0.89 2 0.19 0.18 5 mxscir1 0.87 3 0.23 0.21 6 mxscir1 0.87 3 0.23 0.21 7 mxscir1 0.85 3 0.26 0.23 8 mxscir2 0.86 3 0.25 0.23 9 mxscir2 0.85 3 0.26 0.23 10 mxscir2 0.84 3 0.27 0.24 11 mxscir1 0.83 3 0.29 0.27 12 mxscir2 0.79 3 0.35 0.32 13 mxscir0 0.76 4 0.39 0.35 14 mxscir2 0.77 4 0.38 0.35 15 mxscir1 0.76 3 0.39 0.35 16 ampa9 0.47 4 0.67 0.62 means 0.83 3 0.28 0.25 table 4a ­ new cocoyam: statistical measures of genetic diversity in the green and purple populations (togo collection) n = no. accessions tested, na = average no. of alleles observed per locus, ne = no. of effective alleles, i = shannon's information index, he = nei’s gene diversity, %p = percentage of polymorphic loci (rounded figures). n na ne i he %p pop1 (green) 23 1.742 1.532 0.418 0.287 74 pop2 (purple) 78 1.871 1.176 0.23 0.128 94 mean 1.806 1.354 0.324 0.208 84 se 0.06 0.05 0.033 0.024 10 table 4b ­ new cocoyam: summary analysis of molecular variation (amova) in the green and purple populations (togo collection) source df ss ms est. var. % var. phipt p value between pops 1 54.566 54.566 1.463 36% 0.36 0.001 within pops 99 257.255 2.599 2.599 64% total 100 311.822 ­ 4.061 100% df = degrees of freedom, ss = sum of squares, ms = mean sum of squares, est. var. = estimated variance, % var = percentage of variation, phipt = proportion of total genetic variance derived from variance between individuals among populations, p = probability value for phipt. bammite et al. ‐ genetic diversity of taro and cocoyam 261 this, there is no clear separation of dasheen and eddoe accessions overall in the ssr data. in contrast, the green and purple new cocoyam formed very dis­ tinct groups along the first pca coordinate. these relationships between ssr diversity in pca scatter­ plot and simple morphotype classification are mir­ rored in the nj dendrograms. the nj dendrogram of ssr diversity in taro (fig. 5) revealed one larger cluster (c1) that includes a mix of dasheen (7 accessions) and eddoe types (10 acces­ sions), and a two clusters (c2, c3) that include dasheen types only (9 accessions). the accessions in c3 were all dasheen types, from wet, flooded envi­ ronments, with purple petiole and petiole junction, and generally producing many stolons (see discussion and conclusions) (fig. s1a, fig. s2). the nj dendrogram of ssr diversity in new cocoy­ am revealed two large clusters (c1 and c2) (fig. 6) that largely correspond to the green and purple mor­ photypes of this species (13 and 88 accessions in each category; and many identical haplotypes). with regard to the more specific morphological groups previously reported, c1 includes mostly g1, and c2 includes a mix of mostly g2 and g3 (purple morphotypes; fig. s3). 4. discussion and conclusions the present ssr results indicate few duplicate accessions in the smaller collection of taro (fig. 5) and many apparent duplicates in the larger collection of x. mafaffa (fig. 6). qualitative and quantitative traits for morphological and agronomic characters of taro and new cocoyam were previously recorded and analysed by bammite et al. (2018 b). thirty­eight characters were selected from the descriptor list of ipgri (1999) for c. esculenta and 28 from the descriptor list of ibpgr (1989) for x. saggitifolium. based on these detailed observations, morphological groups within each species were identified using upgma analysis (fig. s1). although these groups (g1­ fig. 3 ­ principal coordinate analysis (pca) of taro accessions classified as dasheen or eddoe. fig. 4 ­ principal coordinate analysis (pca) of new cocoyam accessions classified as green or purple. fig. 5 ­ taro: neighbour joining tree based on ssr data from 13 loci in 26 accessions. cluster 1 includes both dasheen (closed circles) and eddoe (open circles) morphotypes. clusters 2 and 3 include only dasheen morphotypes. morphological groups identified by bammite et al. (2018 b) (figs. s1a and s2) are mixed in cluster 1 (mostly g3, g4 and a few g2) and not mixed in clusters 2 and 3. tgul (togo, university of lomé) accession numbers are shown. adv. hort. sci., 2021 35(3): 255­267 262 g4 in taro, g1­g3 in new cocoyam) do not always correspond as expected to the single­character cate­ gories used in the present study (corm size and shape in taro, plant colour in new cocoyam) (figs. s2 and s3), congruences are apparent between ssr genetic diversity and morphological diversity, however the latter is defined. complete correspondence between single­character and multi­character classifications is not expected, but future studies of morphological diversity can be improved by ensuring greater unifor­ mity in the planting materials used. ahmed et al. (2020) found that the dasheen/ eddoe classification corresponds to two distinct evo­ lutionary lineages within c. esculenta, and suggested that the existence of many intermediate or mixed morphotypes may reflect hybridisation between these lineages. in togo, ssr cluster c1 includes both dasheen and eddoe forms, consistent with the sug­ gestion of mixing (hybridisation) between eddoe and dasheen lineages (see also lakhanpaul et al., 2003). clusters c2 and c3 corresponded entirely to dasheen forms with large mother corms and either stolons or side­corms. the three accessions in c2 had morpho­ logical above­ground traits similar to those typical of eddoe in the togo collection (large pendant or drooping leaves) and underground morphological traits of dasheen type (g2, with large central corm with small side­corms). these accessions were col­ lected in home gardens of farmers from kabyè ethnic group in the ecologic zone iv (southern togo mountains) (fig. 1). they reported having introduced them from ecological zone ii (northern togo mountains), which is consistent with the report by ern (1979) of an expansion of banana, cassava and taro production on steep slopes in zone iv by kabyè settlers from the north. the observation of two major ssr clusters in new cocoyam (c1­c2) suggests that more than one species of xanthosoma is present in togo, not just x. mafaffa (bammite et al., 2018 a, b). a survey of amplified fragment length polymorphism (aflp) in new cocoyam in ethiopia also revealed two major clusters (wada et al., 2018). the existence of such distinct lineages and the general uncertainty of iden­ tification of xanthosoma species suggest an urgent need for direct and detailed comparisons between cultivated xanthosoma spp. in africa, and the wild and cultivated species of xanthosoma in tropical america. for both species, the neighbour joining analysis of ssr allelic diversity at a small number of loci pro­ vides tree diagrams in which terminal branching (near tree tips) is not a reliable indicator of phyloge­ ny. much of the “within population” variation in both species may reflect somatic mutation within clonal cultivar lineages. the small numbers of loci analysed here (13 in taro, 14 in new cocoyam) make it inher­ ently difficult to distinguish clones with certainty, as there are 14 chromosomes in the haploid comple­ ment of c. esculenta (coates et al., 1988; cusimano et al., 2012), and 13 in xanthosoma spp. (cusimano et al., 2012; wada et al., 2018) giving rounded aver­ ages of just 0.9 loci (taro) and 1.1 loci (new cocoyam) fig. 6 ­ new cocoyam: neighbour joining tree based on ssr data from 16 loci in 101 accessions collected in togo. cluster 1 includes green morphotypes (open circles) and cluster 2 includes purple morphotypes (closed circles). morphological groups identified by bammite et al. (2018b) (figs. s1b and s3) are slightly mixed in both clu­ sters (a few g2 and g3 in cluster 1, and a few g1 in cluster 2). tgul (togo, university of lomé) accession numbers are shown. bammite et al. ‐ genetic diversity of taro and cocoyam 263 sampled per chromosome. for taro, actual coverage is less than 0.9 loci/chromosome, as two hk loci have been mapped to one linkage group and chromosome (table s4). although chaïr et al. (2016) introduced a method to estimate clonality based on just 11 loci, the same research group also employed a much more robust method using diversity array technology (darttm) to screen polymorphic loci (pos­ sibly thousands) across the entire taro genome (vandenbrouke et al., 2016). by combining the latter method with a detailed survey of morphological diversity in an assemblage of vanuatu cultivars, vandenbrouke et al. (2016) could unequivocally iden­ tify clonal lineages within which somatic mutation has produced distinct phenotypes that are recog­ nised, selected, and maintained by farmers. most recently, soulard et al. (2017) have mapped polymor­ phic snp and ssr loci across the entire taro nuclear genome, while yin et al. (2021) have published nearly complete sequences for all 14 chromosomes in taro. somatic mutation may explain some of the mor­ phological and genetic diversity found in taro in togo and africa generally, but spontaneous breeding among diploid cultivars in africa may also be involved. the chromosome numbers of togo cultivars have not been studied, but triploid taros are widespread and common in africa (chaïr et al., 2016) and the neighbouring country of burkina faso (traore, 2013), and can be assumed to be inherently sterile because they are triploids. the eddoe­type taros (c. esculenta var. antiquorum) in togo are likely to be triploids, as this morphotype is generally triploid in neighbouring burkina faso (traore, 2013) and eastern asia (plucknett, 1983; matthews, 2014; wang et al., 2020), but this cannot be assumed ­ if the diploid (fertile) progenitors of triploid eddoe cul­ tivars still exist, some might share the eddoe mor­ photype. it also cannot be assumed that the dasheen types are diploid. there are multiple triploid lineages in taro, and some dasheen and intermediate mor­ photypes are also triploid (kreike et al., 2004). the results of our initial primer screening corrobo­ rate those of traore (2013), who found that primers designed for c. esculenta are not transferable to xanthosoma spp. accessions. we also found that, conversely, the primers designed for xanthosoma do not amplify c. esculenta accessions. in their original report of the hk primers, hu et al. (2009) surveyed 30 plants from several provinces of china. chaïr et al. (2016) screened 64 primer pairs developed from c. esculenta and a. paeoniifolius, and selected 11 from c. esculenta, of which three were from the hk primer series. this study (the largest survey of ssr diversity in taro) included 321 cultivars from 19 countries in asia, africa, america and the pacific. several hk primers were also used by hunt et al. (2013). including the present togo survey, results for hk7, hk22 and hk26 can now be compared across four studies (table 5). the largest number of alleles was found in the largest sample set representing many countries (chaïr et al., 2016), which is not surprising. the surprise here is that togo, a relatively small country far from asia, displayed only slightly fewer alleles than a similar number of plants from across china (hu et al., 2009), a much larger country that is also a candidate region for the origin of triploid taros (matthews, 2014; wang et al., 2020; zhu et al., 2000). the number of alleles in two wild breeding populations in papua new guinea and an adjacent region of northern australia was larger than in the china and togo cultivars, but also much less than in the large survey by chaïr et al. (2016). the relatively low number of alleles detected in togo presumably reflects the small number of plants table 5 ­ sample size (n) and number of alleles at ssr loci in taro, in four different studies using the hk primer series designed by hu et al. (2009) (­) = loci not studied locus number of alleles hu et al. (2009), n=30, china hunt et al. (2013), n=42–49, australia & png (two wild populations) chaïr et al. (2016), n= 321 (19 countries excl. china) present study n= 26, togo hk5 6 10 ­ 3 hk7 4 2 12 4 hk22 3 ­ 18 3 hk26 5 8 28 3 hk31 3 4 ­ 2 hk34 3 9 ­ 3 hk35 3 11 ­ 4 adv. hort. sci., 2021 35(3): 255­267 264 tested, and the relatively low genetic diversity of taro in africa generally (chaïr et al. 2016). nevertheless, the overall diversity of taro in togo, and in the neigh­ bouring countries of ghana and burkina faso (traore, 2013) does suggest a complex history of the crop in the region, and in africa. among published studies of ssr diversity in taro (table s1), no two studies have used the same meth­ ods to collect, maintain and test plants, and no stan­ dard set of primer pairs and target loci has emerged. crucially, different sample sets differ in whether they represent initial collections created to assess diversi­ ty in possibly­identical cultivars from different loca­ tions (as in the present study), or later­stage collec­ tions in which apparent duplicates have been removed. observed diversity depends on how plants are collected, how many are collected, and how each collection is maintained over time. taro and new cocoyam collections are constructed “populations” of clones, not random samples from freely breeding populations. for all of these reasons, we do not com­ pare our statistical estimates (calculated data) with those of other small­scale studies. in the near future, new techniques for large­scale and low­cost dna sequencing may allow more accurate, comprehen­ sive and direct comparison of genotypes in different cultivar assemblages. already for taro, public databases contain records of thousands of ssr and snp (single nucleotide polymorphism) loci revealed by whole­genome and transcriptome studies (liu et al., 2015; you et al., 2015; helmkampf et al., 2017; soulard et al., 2017; wang et al., 2020), and a draft sequence for all 14 chromosomes has been published (yin et al., 2021). in the togo collection, flowering occurred among accessions of both species, but fruiting and seed pro­ duction were not observed. togo has a tropical savannah climate, with distinct wet and dry seasons, and annual rainfall ranging from around 800 mm to 1,600 mm (djaman et al., 2017). in burkina faso, in the same general climate zone, but further northwest, most taro is mostly grown in provinces with annual rainfall ranging from around 700 mm to 1,100 mm, near togo (traore, 2013). although condi­ tions during the wet season in togo (april to october) might be suitable for breeding by taro, dry and windy conditions during the winter harmattan (ern, 1979) would be fatal for unprotected seedlings. these are very different circumstances from those in the natural range of taro, in the tropical rainforest zone of asia and the western pacific, where wild breeding populations are found (matthews, 1991; hunt et al., 2013; matthews, 2014). togo itself lies in a dry savannah corridor (the dahomey gap) flanked by tropical rainforest (the upper and lower guinean forests) where spontaneous breeding by taro may be possible. breeding and selection of taro cultivars in these nearby forest regions might have contributed to some of the diversity found in togo. different strategies are suggested here for future development of taro and new cocoyam in togo. for taro, it will be rewarding to study the existing range of eddoe and dasheen cultivars further, and to make experimental introductions of new cultivars from outside africa, following the example of ouedraogo et al. (2018). efforts will be needed to produce dis­ ease­free stocks of existing cultivars so that fair com­ parisons can be made with newly­introduced plants that are disease free. new cocoyam, a relatively modern historical introduction, has spread widely in africa, and a lack of diversity is clear among the accessions collected in togo. international collabora­ tion is needed to identify and introduce new cultivars for evaluation under local conditions. this will be dif­ ficult, as there are no international breeding pro­ grammes for the crop, and little is known about the origins and diversity of cultivated xanthosoma species in central and south america. xanthosoma spp. are even more neglected as orphan crops than taro (matthews and ghanem, 2021). together, taro and new cocoyam offer a range of cultivars suitable for cultivation in wetland to dryland environments. agriculture in togo is predominantly rainfed and often experiences both flooding and drought (djaman et al., 2017). in seasons and loca­ tions when water is abundant, the flooding tolerance of dasheen taro (onwueme, 1999) is a positive attribute that can enhance the food security of farm­ ers working in riverine flood plains. when irrigation is provided, very good yields of taro can be expected in otherwise dry environments: this is shown by the success of taro as an irrigated summer crop in the eastern mediterranean (matthews, 2006), a region with long dry summers and relatively little annual rainfall (approx. 400 mm per year in the main agricul­ tural districts of cyprus). over the last 50 years, cli­ mate change has been very obvious throughout togo, with the wet season becoming 1­2 months shorter (djaman et al., 2017; gadédjisso­tossou, 2018). during the period 1961­2001, for example, annual precipitation decreased at 80% of weather stations across the country (djaman et al., 2017). bammite et al. ‐ genetic diversity of taro and cocoyam 265 under these circumstances, maintaining or expand­ ing the cultivation of taro and new cocoyam may come to depend on the success or otherwise of efforts to improve methods for water storage, con­ servation and irrigation in togo. acknowledgements the authors greatly appreciate the international foundation for science (ifs research grant no. c/5866­2, 2020­2023) for providing research funds. the contribution by matthews was partly funded by jsps kakenhi no. 17h04614 (2017–2020). thanks also to e. tabuchi, osaka, for assistance with figures and tables. references ahmed i., lockhart p.j., agoo e.m.g., naing k.w., nguyen d.v., medhi d.k., matthews p.j., 2020 ­ evolutionary origins of taro (colocasia esculenta) in southeast asia. ­ ecology and evolution, 10: 13530­ 13543. bammite d., 2018 ­ assessment of agromorphological and molecular diversity of colocasia esculenta (l.) schott and xanthosoma mafaffa (l.) schott in togo. ­ phd thesis. university of lomé, togo, pp. 162. bammite d., matthews p.j., dagnon d.y., agbogan a., odah k., dansi a., tozo k., 2018 a ­ constraints to production and preferred traits for taro (colocasia esculenta) and new cocoyam (xanthosoma mafaffa) in togo, west africa. ­ african journal of food, agriculture, nutrition and development, 18: 13388­ 13405. bammite d., matthews p.j., dagnon d.y., agbogan a., odah k., dansi a., tozo k., 2018 b ­ agro morpho‐ logical characterization of taro (colocasia esculenta) and yautia (xanthosoma mafaffa) in togo, west africa. ­ african journal of agricultural research, 13: 934­945. bown d., 2000 ­ aroids: plants of the arum family. second edition. ­ timber press, portland, usa, pp. 392. castro g.r., 2006 ­ studies on cocoyam (xanthosoma spp.) in nicaragua, with emphasis on dasheen mosaic virus. ­ doctoral thesis. swedish university of agricultural sciences, uppsula, sweden. cathebras c., traore r., malapa r., risterucci a.m., chaïr h., 2014 ­ characterization of microsatellites in xanthosoma sagittifolium (araceae) and cross‐amplifi‐ cation in related species. ­ applications in plant sciences, 2: 1­5. chaïr h., traore r.e., duval m.f., rivallan r., mukherjee a., aboagye l.m., rensburg w.j., andrianavalona v.v., de carvalho m. a.a.p., saborio f., prana m.s., komolong b., lawac f., lebot v., 2016 ­ genetic diversification and dispersal of taro (colocasia esculenta (l.) schott). ­ plos one, 11: 1­19. coates d.j., yen d.e., gaffey p.m., 1988 ­ chromosome variatin in taro, colocasia esculenta: implications for origin in the pacific.­ cytologia, 53(3): 551­560. coursey d.g., 1984 ­ potential utilization of major root crops, with special emphasis on human, animal, and industrial uses, pp. 25­35. ­ in: terry e.r., e.v. doku, o.b. arene, and n.m. mahungu (eds.) tropical root crops: production and uses in africa. international society for tropical root crops, douala, cameroon, pp. 232. croat t.b., delannay x., 2017 ­ a revision of xanthosoma (araceae). part 3: guianas. ­ aroideana, 40: 582­649. cusimano n., sousa a., renner s.s., 2012 ­ maximum likelihood inference implies a high, not a low, ancestral haploid chromosome number in araceae, with a cri‐ tique of the bias introduced by ‘x’. ­ ann. bot., 109(4): 681­692. devi a.a., 2012 ­ genetic diversity analysis in taro using molecular markers ‐ an overview. ­ j. root crops, 38(1): 15­25. djaman k., sharma v., rudnick d.r., koudahe k., irmak s., amouzou k.a., sogbedji j.m., 2017 ­ spatial and temporal variation in precipitation in togo. ­ int. j. hydro., 1(4): 97­105. doungous o., kalendar r., adiobo a., schulman a. h., 2015 ­ retrotransposon molecular markers resolve cocoyam (xanthosoma sagittifolium) and taro (colocasia esculenta) by type and variety. euphytica, 206: 541­554. ern h., 1979 ­ die vegetation togos. gliederung, gefährdung, erhaltung. ­ willdenowia, 1: 295­312. fuller d.q., boivin n., hoogervorst t., allaby r., 2011 ­ across the indian ocean: the prehistoric move‐ ment of plants and animals. ­ antiquity, 85: 544­558. gadédjisso­tossou a., avellán t., schütze n., 2018 ­ potential of deficit and supplemental irrigation under climate variability in northern togo, west africa. ­ water, 10: 1803. giacometti d.c., leon j., 1994 ­ tannia, yautia (xanthosoma sagittifolium). ­ in: hernando bermejo j.e., and j. leon (eds.) neglected crops: 1492 from a different perspective. fao plant production and protection service, rome, italy. gonçalves e.g., 2011 ­ the commonly cultivated species of xanthosoma schott (araceae), including four new species. ­ aroideana, 34: 3­23. grimaldi i.m., 2016 ­ taro across the oceans, journeys of one of our oldest crops, pp. 67­81. ­ in: thanheiser u. 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75% humic acid + 25% npk; 50% humic acid + 50% npk; 25% humic acid + 75% npk; and 100% npk. data on plant growth and yield were processed by analysis of variance, and means were compared using fisher’s least significant difference test. in addition, data on plant biochemical and soil chemical parameters were determined compositely by mixing leaves taken from sample plants or soil samples into one homogenous sample. results showed that there was no significant difference in growth and yield of plants treated with 100% humic acid in comparison with those plants treated with 100% npk. however, in comparison with 100% humic acid, the application of different ratios of humic acid/npk increased plant chlorophyll contents by 65% ­ 82% and total sugar by 28% ­ 71%. the application of humic acid/npk increased soil fertility by improving soil ph as well as n, p and k. in the combi­ nation of humic acid/npk, the best growth and yield were obtained with the application of 25% humic acid + 75% npk fertilizers. therefore, for the sustain­ ability of chili cultivation, the use of humic acid needs to be accompanied with npk fertilizers at a reduced amount, along with the increase in the dose of humic acid. 1. introduction in indonesia chili (capsicum annuum l.) is one of vegetable crops with high economic value due to its large domestic and export demaind and, from time to time, price fluctuations may cause consumer unrest. the national inflation rate is significantly influenced by the increase in chili prices in certain seasons. to control the price fluctuations the indonesian government is increasing the planting area in rainy season, and control­ ling the planting area as well as production during dry season. (*) corresponding author: dr.zulkarnain@yahoo.com citation: ichwan b., eliyanti e., irianto i., zulkarnain z., 2022 ­ combining humic acid with npk fertili‐ zer improved growth and yield of chili pepper in dry season. ­ adv. hort. sci., 36(4): 275­281. copyright: © 2022 ichwan b., eliyanti e., irianto i., zulkarnain z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 february 2022 accepted for publication 3 october 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12816 adv. hort. sci., 2022 36(4): 275­281 276 increasing production in the dry season normally is managed by application of inorganic fertilizers and use of chemicals to control pests and diseases. however, this practice has a negative impact on agri­ cultural ecosystem and the environment. additionally, implementing principles of sustainable agriculture such as the application of humic acid, a soil conditioner, has the potential to improve this conventional agricultural practice. humic acid has a complex molecular structure and capability to stimu­ late and activate biological and physiological process­ es in soil organisms. the study of abdellatif et al. (2017) showed that the application of 14.4 kg ha­1 humic acid to chili plants grown under heat stress could increase plant’s average height, number of flower buds, number of flowers, number of fruits per plant, as well as fruit weight. the application of 7.5­12 ml of humic acid into 10 kg of sterile soil increased the population of soil microorganisms azotobacter beijerinckii and aspergillus niger. foliar spray of 50 ml l­1 humic acid significantly increased leaf area, yield and total solu­ ble solids in grapes (popescu and popescu, 2018). onion plants sprayed with 1000 mg l­1 humic acid produced the highest growth rate and yield (al­ fraihat et al., 2018). in addition to the improvement in cultivation, chili production can be increased by growing them during the dry season. whilst a lot of land is available for cropping during the dry season the production is con­ strained by the limited availability of water. application of humic acid as a soil conditioner could remediate water shortage in plants due to increasing leaf water content, increasing photosynthesis, antioxidant metabolism and enzyme activity; thus improving plant tolerance to stress (al­shareef et al., 2018). previous studies have demonstrated that humic acid improved plant growth and yield in a number of plant species. hermanto et al. (2013) reported that application of 20 kg ha­1 humic acid along with 100% dose of npk fertilizer resulted in maximum growth and yield as well as increased the availability and uptake of n, p, k, zn and fe in maize. furthermore, moraditochaee (2012) showed that the application of humic acid at 40 mg l­1 and 75 kg ha­1 n produced the highest harvest index in peanut. sustainable production of plants can be achieved by reducing the use of inorganic fertilizers. application of humic acid could help achieve this important goal. whilst humic acid has been widely applied to various plant species; the search of litera­ ture showed that no research has focused on the effect of humic acid in combination with npk fertiliz­ er on growth and yield of chili pepper. therefore, this study aimed at assessing the effect of humic acid in combination with npk fertilizer on growth and yield of red chili, and to obtain the most suitable ratio of humic acid/npk for best growth and yield in chili plants grown in dry season in indonesia. 2. materials and methods the trial was conducted at the teaching and research farm within the faculty of agriculture at the university of jambi in indonesia during the 2020 dry season. the study used commercially available humic acid (ah­90, with 90% humic acid content) and inorganic npk fertilizer (15:15:15). there were 5 combinations tested: 100% humic acid; 75% humic acid + 25% npk; 50% humic acid + 50% npk; 25% humic acid + 75% npk and 100% npk. a randomized block design with 5 replications was used in this study. humic acid was applied at the recommended dose (5 kg ha­1) and so was npk fertilizer (700 kg ha­1). the humic acid and npk were applied one week before planting in between rows. seeds were sown on a standard seedling medium, then transferred to nursery at 7 days after sowing and left in the nursery for 21 days. the medium used during the nursery growth was a mixture of soil, com­ post, and sand (2:1:1). seedlings were sprayed every 7 days with foliar fertilizer bayfolan­d at 2 ml l­1 and insecticide (active ingredient amabektin and mesurol) at 2 ml l­1. the one­month­old seedlings were transplanted in the field onto the growing media supplemented with humic acid and npk fertilizer at different rates as per treatments described above. a silvercoated black plastic was placed on the beds before trans­ planting the seedlings. prior to planting, a fungicide (carbofuran) was applied into planting holes at 1.5 g per plant. the planting space was 50 cm x 50 cm. a liquid npk fertilizer (15:15:15) was applied at a con­ centration of 2 g l­1 with a volume of 100 ml per plant at 6, 8 and 10 weeks after transplanting. harvesting was carried out during the first flower­ ing period (10 to 13 weeks after transplanting), with the criterion that 80% of fruits becoming red. observation of vegetative growth was carried out during fruit enlargement period (9 weeks after trans­ ichwan et al. ‐ humic acid and npk fertilizers improved growth and yield of chili pepper 277 planting) on the following parameters: plant height, number of productive branches, leaf area, and plant total dry weight. the leaf area was measured using gravimetric method with the following formulae (sitompul and guritno, 1995): leaf area (cm2) = (x/y)·z where x = total leaf dry weight, y = sub sample leaf dry weight, and z = sub sample leaf area. observation of plant biochemical measurements were made on chlorophyll content, total sugar, and relative leaf water content (rlwc). composite leaf samples were made by physically mixing individual leaves taken from 3 sample plants of 3 replicates into one homogenous sample. compositing reduced the number of analyses to be performed and was designed to provide a representative sample of the treatment. ten youngest mature leaves on main stem were collected at 9 weeks after transplanting (wat). clean and dry leaf samples were placed in a sample bag prior to laboratory analyses. chlorophyll analysis was carried out using acetone method (arnon, 1949), and total sugar was analyzed using anthrone method (yoshida et al., 1976). relative leaf water content (rlwc) was determined according to gonzález and gonzález­vilar (2001) method. data on yields in terms of number of the fruits and fruit weight per plant were collected at harvest time. in addition, soil chemical analyses were per­ formed before transplanting and after the last har­ vest. soil analyses included ph, moisture content, organic c based on walkley­black method (walkley and black, 1934), total n using kjeldahl method (kjeldahl, 1883), available p applying bray­i method (bray and kurtz, 1945) and exchangeable k (nh4oac 1n extraction at ph 7) using method as described in (warncke and brown, 1998). the data were scrutinized employing an analysis of variance (anova) from microsoft excel application (version 16.63.1, 2022, microsoft corporation, usa). fisher’s least significant difference (flsd) at 5% level of probability was used to compare means of treat­ ments. 3. results analysis of variance showed that the application of humic acid (ha) and npk fertilizer did not signifi­ cantly affect plant height, number of branches, total leaf area, and dry weight. however, application of 25% ha in conjunction with 75% npk fertilizer result­ ed in enhanced growth compared to other treat­ ments (fig. 1). in addition, the application of 25% ha + 75% npk improved growth compared with 100% ha application whereby plant height, number of branches, leaf area and plant dry matter increased by 29.46%, 21.96%, 23.59% and 28.42% respectively. the measurement of biochemical parameters showed that the application of 100% ha resulted in the lowest chlorophyll content (2.40 mg l­1) and total sugar (0.07%). furthermore, the lowest relative leaf water content (rlwc) was found on 100% npk appli­ cation (table 1). fig. 1 ­ growth of chili on various ratios of humic acid (ha)/npk at 9 weeks after transplantation (a = plant height, b = number of branches, c = leaf area, d = total dry weight). error bars indicate the standard error (n=5). humic acid/npk chlorophyll content (mg·l­1) total sugar (%) rlwc (%) 100% ha 2.40 0.07 92.6 75% ha/25% npk 3.97 0.12 93.3 50% ha/50% npk 4.15 0.12 93.7 25% ha/75% npk 4.37 0.09 93.0 100% npk 4.30 0.11 91.3 table 1 ­ effect of different ratios of humic acid) ha / npk on chlorophyll content (*), total sugar, and relative leaf water content (rlwc) at 9 weeks after transplantation (*) chlorophyll content and total sugar were determined compo­ sitely by physically mixing individual leaves taken from each 3 sample plants of 3 replicates into one homogenous sample. adv. hort. sci., 2022 36(4): 275­281 278 analysis of variance showed that the application of different ratios of ha/npk had no significant effect on the number and weight of fruits per plant. however, among the combinations tested the 25% ha + 75% npk resulted in the highest number of fruits, and the 50% ha + 50% npk produced the largest fruit weight (fig. 2). the results of soil chemical analyses (table 2) showed that the application of various ratios of ha/npk had altered soil chemical properties such as ph, organic c, total n, p, k, and soil water content. generally, the application of ha along with npk fer­ tilizer improved n, p and k content. whereas soil ph and organic carbon are highest when 100% ha is applied (table 2). 4. discussion and conclusions the application of different humic acid/npk ratios had no significant effect on growth and yield of chili as measured by plant height, total leaf area, number of branches and total dry weight, as well as number of fruits and average fruit weight. this means that humic acid can substitute npk fertilizer. however, when compared to the use of only humic acid or npk fertilizer, the application of different ratios of humic acid/npk improved plant height, total leaf area, num­ ber of branches, total dry weight, fruit numbers and fruit weight. npk is widely used to improve plant growth and yield of many horticultural crops (nafiu et al., 2011; agbede et al., 2017; achiri et al., 2018; adekiya et al., 2019; nwokwu et al., 2020). however, the use of inorganic fertilizers such as npk to help increase plant growth and yield need to be carefully consid­ ered, due to their detrimental impact on soil struc­ ture and soil biota. according to adekiya et al. (2019) npk fertilizer did not increase soil organic matter, nor reduced soil bulk density. krestini et al. (2020) claimed that npk fertilizer did not provide carbon compounds that contributed to the improvement of soil physical and biological properties. in addition, agbede et al. (2017) reported that continued applica­ tion of inorganic fertilizers can increase soil acidity and soil degradation because inorganic fertilizers release nutrients more quickly. therefore, balanced fertilization is recommended to increase the efficien­ cy of fertilizer use and help improve physical, chemi­ cal, and biological properties of soil. the results obtained here show that application of 100% humic acid provided less growth and yield in fig. 2 ­ effect of different ratios of humic acid (ha)/npk on num­ ber of fruits (a) and fruit weight (b). error bars indicate the standard error (n=5). humic acid/npk ph c­organic (%) n­total (%) p (ppm) k (cmol+/kg) water content (%) 100% ha 6.09 1.55 0.22 104.50 0.06 7.70 75% ha/25% npk 6.02 1.45 0.29 124.80 0.19 7.56 50% ha/50% npk 5.47 1.28 0.28 152.30 0.47 7.61 25% ha/75% npk 5.52 1.20 0.24 144.70 0.62 7.67 100% npk 5.32 1.22 0.23 227.30 1.07 7.50 table 2 ­ the effect of different ratios of humic acid (ha)/npk on soil chemical properties ichwan et al. ‐ humic acid and npk fertilizers improved growth and yield of chili pepper 279 chili plants compared with mixture of humic acid/npk during the one growth season of this research. however in a longitudinal experiment on peanut li et al. (2019) demonstrated that significant increase in n, p and k content of soil was found after three years continuous application of humic acid. the reason for this increase is due to humic com­ pounds high stability rate, and therefore the mineral­ ization process can occur very slowly. as a conse­ quence, humic acid cannot be used as a source of plant nutrition (canellas et al., 2015). in research reported here the chemical analyses indicated soil that was treated with 100% humic acid was lower in n, p and k content, but higher in organic carbon and soil moisture content in comparison with those treat­ ed with different humic acid/npk ratios (table 2). this was in accordance with (li et al., 2019; 2020) findings that the application of humic acid­based fer­ tilizer could increase soil nutrient contents, including the total soil nitrogen, phosphorus, and potassium. increase in soil water content was aligned with increase in relative leaf water content (rlwc) in chili plants (table 1). al­shareef et al. (2018) reported that the application of humic acid as a soil enhancer could overcome water shortage in plants by increas­ ing rlwc, thereby increasing plant tolerance to stress. further, xu et al. (2015) stated that the appli­ cation of soil enhancers such as humic acid could increase plant growth by retaining available water and nutrients, therefore reducing the impact of drought stress and nutrient loss. the application of 100% humic acid also increased soil ph from 4.43 to 6.09, resulting in better plant growth and development due to improved availabili­ ty of various nutrients in the soil at ph of 6.09. wahyudi (2007) reported that the application of humic acid might reduce the amount of exchange­ able aluminum and increase chelating aluminum in soil, thereby increasing soil ph. hasanudin (2003) suggested that the reduction in exchangeable alu­ minum was due to the formation of complex organo­ metal compound (al­chelate). increase in soil ph will increase the availability of soil nutrients needed by plants (gardner et al., 1985). in peanut, li et al. (2019) found that compared with control, humic acid significantly increased the yield and quality of peanut in continuous cropping. their results showed that application of humid acid significantly increased the soil total nitrogen, total phosphorus, total potassium, available n, available p, available k, and organic mat­ ter from second year, with maximum effect displayed in third year. chili plants treated with different humic acid/npk ratios showed better growth and yields in compari­ son to those plants treated with humic acid or npk alone. the composition of 25% humic acid + 75% npk fertilizer produced the highest growth (plant height, leaf area, number of branches and plant dry matter), as well as the highest yield (number of fruits) com­ pared to other compositions. furthermore, applica­ tion of 50% humic acid + 50% npk fertilizer resulted in the highest fruit weight. this result is in line with the increase in plant chlorophyll content, where the composition of 25% humic acid + 75% npk fertilizer caused higher plant chlorophyll content. an increase in chlorophyll content intensifies the ability of plants to carry out photosynthesis, thereby increasing plant growth (ferrara and brunetti, 2010). increase in soil k content due to humic acid / npk application caused better k uptake, increasing plant growth and yield in potato (xu et al. , 2015). potassium is crucial in plant photosynthetic activity (marschner, 2012) and assimilate translocation effi­ ciency (sawan, 2018). this is in line with our results where application of humic acid and npk (75% ha and 25% npk or 50% ha and 50% npk) increased sugar content in plant tissue (table 1). the composition of 50% humic acid + 50% npk and 25% humic acid + 75% npk in this study provided better growth and yield of chili plants than 100% humic acid or 100% npk applications. this indicates a positive effect of the use of humic acid in partially replacing the role of npk fertilizer. the ability of humic acid to replace npk fertilizer is probably due to the ability of humic acid to modify the plant root sys­ tem architecture as well as improving the capacity of soil to bind water, thus enabling plants to acquire water and nutrients more easily (suwahyono, 2011; canellas and olivares, 2014), reducing plant stress in dry land cultivation during periods of drought (xu et al., 2015). the analysis of soil water content for different ratios of humic acid/npk showed medium treated with humic acid has a better water content than those treated with 100% npk (7.56­7.7 versus 7.5). in addition, the relative leaf water content (rlwc) at various ratios of humic acid / npk was higher than that of 100% npk application (table 1). our findings are consistent with a number of pre­ vious studies indicating that the application of humic acid needs to be accompanied by npk fertilizer to help improve plant productivity (for example in properties and growth and yield of carrot. ­ j. hortic. res., 25(1): 81­88. al­fraihat a.h., al­tabbal j.a., abu­darwish m.s., alhrout h.h., hasan h.s., 2018 ­ response of onion (allium cepa) to foliar application of humic acid under rain‐fed conditions. ­ int. j. agric. biol., 20(5): 1235­ 1241. al­shareef a.r., el­nakhlawy f.s., ismail s.m., 2018 ­ enhanced mungbean and water productivity under full irrigation and stress using humic acid in arid regions. ­ legume res., 41(3): 428­431. arnon d.i., 1949 ­ copper enzymes in isolated chloroplas‐ ts. polyphenoloxidases in beta vulgaris. ­ plant physiology, 24(1): 1­15. bray r.h., kurtz l.t., 1945 ­ determination of total, organic, and available forms of phosphorus in soils. ­ soil sci., 59(1): 39­46. canellas l.p., olivares f.l., 2014 ­ physiological responses to humic substances as plant growth pro‐ moter. ­ chem. biol. technol. agric., 1(3): 1­11. canellas l.p., olivares f.l., aguiar n.o., jones d.l., nebbioso a., mazzei p., piccolo a., 2015 ­ humic and fulvic acids as biostimulants in horticulture. ­ sci. hortic., 196: 15­27. ferrara g., brunetti g., 2010 ­ effects of the times of application of a soil humic acid on berry quality of table grape (vitis vinifera l.) cv italia. ­ span. j. agric. res., 8(3): 817­822. gardner f.p., pearce b., mitchell r., 1985 ­ physiology of crop plants (1st edition). ­ iowa state university press, ames, ia, usa, pp. 327. gonzález l., gonzález­vilar m., 2001 ­ determination of relative water content, pp. 207­212. ­ in: roger m.j.r. (ed.) handbook of plant ecophysiology tech‐ niques. ­ kluwer academic publishers, the netherlands, pp. 468. hasanudin, 2003 ­ the increasing of nutrient availability and uptake of n and p and yield of corn through innoc‐ ulation of mycorrhiza, azotobacter, and organic matter on ultisol. ­ j. ilmu pert. indon., 5(2): 83­89. hermanto d., dharmayani n.k.t., kurnianingsih r., kamali s.r., 2013 ­ the influence of humic acid as fer‐ tilizer supplement to nutrient availability and uptake on maize plant in unirrigated land of kec. bayan‐ntb. ­ ilmu pertanian, 16(2): 28­41. kjeldahl j., 1883 ­ neue methode zur bestimmung des stickstoffs in organischen körpern. ­ zeitschrift für analytische chemie, 22(1): 366­383. krestini e.h., susilawati a., hermanto c., 2020 ­ effect of npk fertilizer and microbial consortium to growth and production of garlic (allium sativum l.), pp. 03010. ­ in: mursyidin d.h., and e. badruzsaufari (eds.) first international conference on tropical wetland biodiversity and conservation (icweb 2019). edp sciences, web of conferences, les ulis cedex, france, 1­2 november, 2019. 280 adv. hort. sci., 2022 36(4): 275­281 peanut (moraditochaee, 2012), tomato (sarno et al., 2015) and cocoa (santi, 2016)). the slow rate of humic acid mineralization needs to be followed by the application of npk fertilizers. this is aimed to increase the availability of nutrients for plants, espe­ cially in relation to the relatively short lifespan of the chili plant which is only 3 ­ 4 months. in addition, it is also necessary to consider the amount of humic acid and the time of application, so that the use of npk fertilizer as a source of plant nutrition can be further reduced. data presented here indicate that the application of different ratios of humic acid / npk could greatly increase the growth and yield of chili pepper grown in dry land cultivating, where the composition of 25% humic acid + 75% npk fertilizer resulted in improved plant growth and yield. it was found that the applica­ tion of humic acid could reduce the use of npk fertil­ izers by up to 50%. thus, for sustainable chili produc­ tion in dry land cultivation, application of humic acid in conjunction with npk fertilizer is recommended at a reduced amount. acknowledgements this research was supported by university of jambi through internal competitive research grant (contract no. 410/un.21.18/pg/spk/2020 dated 20 april 2020). the authors would like to thank the rector of university of jambi for financial support provided to conduct this research. references abdellatif i.m.y., abdel­ati y.y., abdel­mageed y.t., hassan m.a.­m.m., 2017 ­ effect of humic acid on growth and productivity of tomato plants under heat stress. ­ j. hortic. res., 25(2): 59­66. achiri t.d., michele t.z.s., konje c.n., njualem d.k., 2018 ­ effect of npk fertilizer and animal manure on some biometric parameters of irish potato (solanum tuberosum l.) in bougham, west region cameroon. ­ asian j. crop sci., 2(1): 1­10. 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(ed.) recommended chemical soil test procedures for the north central region. ­ missouri agricultural experiment station sb 1001, university of missouri, columbia, usa. xu s., zhang l., mclaughlin n., mi j., chen q., liu j., 2015 ­ effect of synthetic and natural water‐absorbing soil amendments on photosynthesis characteristics and tuber nutritional quality of potato in a semi‐arid region. ­ j. sci. food agric., 96: 1010­1017. yoshida s., forno d.a., cock j.h., gomez k.a., 1976 ­ laboratory manual for physiological studies of rice (3rd edition). ­ the international rice research institute, los baños, laguna,the philippines, pp. 83. impaginato 239 adv. hort. sci., 2018 32(2): 239-248 doi: 10.13128/ahs-22287 somatic embryogenesis, biochemical alterations and synthetic seed development in two varieties of coriander (coriandrum sativum l.) m. ali, a. mujib (*), n. zafar, d. tonk cellular differentiation and molecular genetics section, department of botany, jamia hamdard university, new delhi 110062, india. key words: biochemical attributes, conversion frequency, coriandrum sativum l., somatic embryogenesis, synthetic seeds. abstract: somatic embryogenesis (se), biochemical alterations and synthetic seed formation were carried out in two coriandrum sativum l. varieties (rajendra swathi ‘rs’ and co-1). callus was induced profusely in 1.0 mg/l 2,4dichlorophenoxy acetic acid (2,4-d) added ms medium but co-1 had more callus induction frequency (96.0%) compared to rs (89.3%). the callus turned into embryogenic tissue and variable embryogenic frequency (77.6% in rs and 72.8% in co-1) was noted. somatic embryos started to differentiate on the same 2,4-d added medium but the numbers of somatic embryos were more in rs (63.0 embryos per culture) compared to co-1 (51.0 embryos per culture). these somatic embryos progressed well and showed maximum maturation in rs (78.7%) in 0.25 mg/l 6-benzyladenine (ba) + 0.5 mg/l α-naphthalene acetic acid (naa) added medium. the biochemical analyses of non-embryogenic-, embryogenic-callus and different stages of embryos were conducted in order to know the changes of physiology in different tissues. sugar and proline content were noted to be high at embryo induction stage while protein level was higher at embryo maturation stage. biochemical analysis also revealed that the catalase (cat) and superoxide dismutase (sod) activities were higher at maturation stage of embryos compared to other embryogenic stages. matured somatic embryos were germinated in ms added with 1.0 mg/l ba + 0.5 mg/l gibberellic acid (ga3) in which 83.3% and 76.7% plantlet regeneration were noticed in rs and co-1 respectively. somatic embryos were encapsulated in various alginate and calcium chloride (cacl2) solutions and were kept in different temperature regimes for varied periods. on regeneration medium, the encapsulated embryos germinated into plantlets; in 3% sodium alginate + 100 mm cacl2, maximum plant regeneration (74.0% in rs and 70.6% in co-1) was noted. the influence of low temperature on storage of synthetic seeds and their conversion into plantlets were also studied and we noted that the 4°c was the optimum temperature for synthetic seed conservation and plantlet regeneration compared to -20°c and 25oc temperature conditions. (*) corresponding author: amujib3@yahoo.co.in citation: ali m., mujib a., zafar n., tonk d., 2018 somatic embryogenesis, biochemical alterations and synthetic seed development in two varieties of coriander (coriandrum sativum l.). adv. hort. sci., 32(2): 239-248 copyright: © 2018 ali m., mujib a., zafar n., tonk d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 239-248 240 1. introduction coriandrum sativum l. is an annual herb and belongs to the family apiaceae. the plant is used as a spice and flavouring compound (burdock and carabin, 2009). the plant has potential in pharmaceutical industry as it exhibits antimicrobial (cao et al., 2012), antioxidant (hashim et al., 2005), antidiabetic (eidi et al., 2009), hepatoprotective (samojlik et al., 2010) and antiarthritic properties (rajeshwari et al., 2012). plant tissue culture technology is widely used for large scale plant propagation, beside being used as an in vitro experimental model for studying cellular processes like cell division, differentiation and morphogenesis, all play important key role in somatic embryogenesis (se) and plant development (zimmerman, 1993). se is a process in which somatic cells undergo morphological and metabolic changes under in vitro cultural conditions, acquire embryogenic potency and later develop into somatic embryos (feher et al., 2003). somatic embryos mimic zygotic embryos in various ways and hence it has proved to be a good model for studying morphophysiological, biochemical and molecular events during the course of embryogenesis (dodeman et al., 1997). the fast identification of embryogenic tissue is very important as it plays a central role in markerassisted selection (dodeman et al., 1997). beside morphology, the embryogenic tissues are often differentiated from non-embryogenic callus by various biochemical markers and these markers based selection would be of immense value of se based micropropagation (samar et al., 2011). embryogenic tissues have the ability of producing embryos for an extended period of time without any genetic alteration, which requires efficient conservation protocol for long-term preservation of coriander (murthy et al., 2008). somatic embryos also have an important application in the formation of synthetic seeds and this artificial seed (an alternative to natural seed) technology may be exploited as a complementary method for in vitro production of plantlets (reddy et al., 2012). it also promotes fast plantlet regeneration, facilitates germplasm exchange and conservation (palanyandy et al., 2015). the synthetic seed technology with advantages like easy handling, conservation with germplasm exchange possibility is being widely used between national and international laboratories (rai et al., 2009). beside bipolar somatic embryos, other explants like unipolar micro-bulbs, rhizomes, protocorms, nodal cuttings and shoot buds are used for synthetic seed (rihan et al., 2011; sharma and shahzad, 2012). among the various natural and synthetic polymers available for encapsulation, sodium alginate is used more frequently because of its easy gelling properties, non-toxicity and low cost (saiprasad, 2001). different concentrations of sodium alginate ranging from 1.5 to 6.0% are used in encapsulation in different plant species cacicus carota (latif et al., 2007), spartina alterniflora (utomo et al., 2008), catharanthus roreus (maqsood et al., 2012), rinacanthus nasutus (meena et al., 2013). in the present study, an efficient synthetic seed formation protocol was optimized in c. sativum and their regeneration potential was described after in vitro storage at different storage conditions. we also discussed se and associated biochemical alterations in two varieties of c. sativum. 2. materials and methods seed germination and cultural conditions seeds of two varieties of c. sativum, rajenda swathi (rs) and co-1 were obtained from national research centre for seed spices (nrcss) ajmer, rajasthan, india for this experimental study. the seeds were washed thoroughly under running tap water using cetrimide as detergent and surface disinfection was made with 0.1% (w/v) hgcl2 for 2 min. the seeds were rinsed three times with sterilized double-distilled water before allowed to germinate on half strength ms medium (murashige and skoog, 1962) without plant growth regulators (pgrs). the basal medium was solidified by adding 8.0 g l-1 agar. the ph of the medium was adjusted to 5.7 using 1.0 m naoh and 0.1 n hcl and was sterilised in an autoclave for 15 min at 121°c. all the reagents were prepared using water supplied by a milli-q system (billerica, massachusetts, usa). the cultures were incubated at 25±2°c under 12-h photoperiod provided by cool white fluorescent lamps (100 µmol m-2 s-1 pfd). callus induction and somatic embryogenesis (se) callus was induced from hypocotyl explants of 10day old germinated seedlings. different concentrations (0.5-2.0 mg/l) of 2,4-dichlorophenoxy acetic acid (2,4-d) and 0.5-1.0 mg/l αnaphthalene acetic acid (naa) were used for callus induction. after 4 weeks in induction medium, the callus was transferred in fresh medium for induction of somatic muzamil et al. somatic embryogenesis of two coriander varieties 241 embryos. somatic embryos were induced on same 2,4-d added callus-induction medium. for embryo maturation, ms was supplemented with naa (0.5-1.5 mg/l) and 6-benzyladenine, ba (0.25-0.5 mg/l). the germination of somatic embryos was made by transferring matured green embryos into ms, added with 0.5-2.0 mg/l ba and 0.2, 0.5 mg/l gibberellic acid (ga3). biochemical analysis proline, protein and sugar assay. proline estimation was made according to bates et al. (1973). about 0.05 g of callus was homogenized in 2.0 ml of 3.0% aqueous sulphosalicylic acid under cold conditions; the homogenate was filtered with whatman filter paper (no. 1). the filtrate (1.0 ml) was added with 1.0 ml ninhydrin and 1.0 ml glacial acetic acid; the reaction mixture was incubated for 1 h at 100°c. the reaction was terminated in ice bath and 2.0 ml toluene was added to it. proline content was measured by spectrophotometric assay at 520 nm. the concentration of proline is expressed in mg per 1 g fresh weight. protein estimation was made following bradford et al. (1976). the homogenate of 0.25 g callus in 1.5 ml phosphate buffer (0.1 m, ph 7.0) under pre-cold condition was centrifuged at 104 rpm for 10 min and 1.0 ml supernatant was added with 0.5 ml trichloroacetic acid (10%). after centrifugation, the pellets were washed with acetone and dissolved in 1.0 ml of naoh (0.1 n). to 1.0 ml of aliquot, 1.0 ml of bradford reagent was added and optical density was measured at 595 nm the protein concentration was expressed in mg per 1 g fresh weight. dey (1990) method was used for sugar estimation. callus tissue 0.1 g was extracted twice with 90% alcohol at 60°c. final volume of extract was made up to 10 ml by adding ddw. after mixing 0.5 ml of aliquot with 0.5 ml of 5% phenol, 1.0 ml of concentrated sulphuric acid was added and cooled in air. the optical density was measured at 485 nm. the sugar concentration was expressed mg per 1 g fresh weight. analysis of catalase (cat), ascorbate peroxidise (apx) and superoxide dismutase (sod) activity. calli at different stages such as non-embryogenic, embryogenic tissue and different embryo stages (induction, proliferation and maturation) were homogenized in 2.0 ml of 0.1 m extraction buffer (0.1 m k-phosphate, 0.5 mm edta, 1.0 mm ascorbic acid, ph 7.5). after centrifugation at 104 rpm for 20 min, the supernatant was used for enzyme analysis. cat activity was determined according to aebi (1984) method by measuring a decrease in the absorbance at 240 nm of reaction mixture containing 1.0 ml of 0.5 m reaction phosphate buffer (na-phosphates, ph 7.5), 0.1 ml edta, 0.2 ml enzyme extract and 0.1 ml h2o2. the reaction was run for 3 min. one unit of enzyme determines the amount necessary to decompose 1.0 μm of h2o2 per min. cat activity was calculated by using the co-efficient of absorbance at 0.036 mm-1 cm-1. the activity of cat was expressed in eu mg-1 protein. the method developed by nakano and asada (1981) was used for apx activity. to the mixture of 1.0 ml sodium buffer (0.1 m, ph 7.2), 0.1 ml of edta and 0.1 ml of enzyme extract, 1.0 ml of 0.5 mm ascorbate were added and the reaction was run for 3 min at 25°c. the apx activity was estimated by monitoring the decrease in absorbance due to the breakdown of ascorbate by apx and was calculated by using the coefficient of absorbance 2.81 mm-1 cm-1. the activity of apx was expressed in eu mg-1 protein. the sod activity was estimated following dhindsa et al. (1981) method with slight modifications. callus tissue (0.1 g) was homogenised in 2.0 ml of extraction mixture (0.5 m phosphate buffer (ph 7.3), 3.0 mm edta, 1.0% (w/v) polyvinylpyrollidone (pvp), 1.0% (v/v) triton x100) and centrifuged at 104 for 10 min. the sod activity in the supernatant was assayed by adding 0.1 ml enzyme extract with 1.5 ml reaction buffer, 0.2 ml methionine, 0.1 ml each of 1.0 m naco3, 2.25 mm nitro blue tetrazolium (nbt) solution, 3.0 mm edta, riboflavin and 1.0 ml of millipore h2o was taken in test tubes and was incubated under light for 10 min at 25°c. the absorbance at 560 nm, 50% reduction in colour is 1.0 unit and the enzyme activity was expressed in eu mg-1 protein. synthetic seed preparation mature green somatic embryos at the cotyledonary stage were collected and suspended in a solution of ms, added with different concentrations of sodium alginate (2%, 3% and 4%) for a few seconds and then dropped into the sterile aqueous solution of calcium chloride (cacl2:75 mm, 10 mm and 125 mm) in order to encapsulate the embryo. encapsulated embryos in cacl2 solution were shaken in an orbital shaker at 60 rpm. cacl2 solution was poured off and the beads were washed twice with sterilized water and placed on sterilized filter paper to remove excess water. adv. hort. sci., 2018 32(2): 239-248 242 storage and conversion of synthetic seeds encapsulated somatic embryos were stored at 4°c (in refrigerator), 25°c (incubation room temperature) and at -20°c for varied periods (weeks) in order to evaluate the viability and regeneration/conversion potential of stored synthetic seeds. the synthetic seeds were stored using airtight dark 100 ml conical flasks. the synthetic seeds were periodically removed from the respective storage temperatures and cultured in ms added with various plant growth regulators (pgrs). the conversion rate was recorded by observing the development of shoots in 1.0 mg/l ba and 0.5 mg/l ga 3 added ms after 2 and 4 weeks of culture. statistical analysis for callus induction, hypocotyls explants were cultured in test tubes with a minimum of 30 explants per experiment. in case of somatic embryo, induction each experiment was replicated three times. data on frequency of response and numbers of somatic embryos induced per 500 mg of callus were recorded after 4 weeks of culture. the data presented as mean and its standard deviation (mean ± sd). the significance of differences among means was carried out using duncan’s multiple range test, dmrt (duncan, 1995) at p= 0.05. 3. results callus induction and somatic embryogenesis on 2,4-d (0.5-2.0 mg/l) supplemented ms medium, prolific callus was induced from hypocotyl explants. the optimum callus induction frequency of 96.0% and 89.3% was noticed in co-1 and rs respectively with 1.0 mg/l 2,4-d added medium. the callus induction frequency decreased as the concentration of 2,4-d above 1.0 mg/l was used (table 1). callus induction was also observed on naa at 0.5 mg/l added ms but the induced callus was non-embryogenic in nature. the 2,4-d induced embryogenic callus was white and friable, which produced globular embryos on the same induction medium (fig. 1 a). of the different 2,4-d concentrations used, 1.0 mg/l exhibited higher embryogenic ability (77.6%) in rs with 63.3 somatic embryos; in co-1, the embryogenic ability was also equally high (72.8%) with relatively lower numbers of embryos (51.0) (fig. 1 b). beside 2,4-d, other pgr combinations were also tested for embryo differentiation. in 0.5 mg/l naa + 0.25 mg/l ba amended medium, maximum differentiation of embryos (78.7% in rs and 74.0% in co-1) was noticed (fig. 1 c-d) and in increasing naa concentrations the embryo differentiation decreased. the somatic table 1 effect of different concentrations of 2,4-d on callus induction and somatic embryogenesis from hypocotyl explants of ‘rajendra swathi’ and ‘co-1’ varieties of coriandrum sativum values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according duncan’s multiple range test. 2,4-d (mg/l) rajendra swathi co-1 callus induction (%) embryogenic callus induction frequency no. of embryos formed/culture (0.5 g) callus induction (%) embryogenic callus induction frequency no. of embryos formed/culture (0.5 g) 0.5 86.0±4.0 a 55.3±2.5 b 39.3±2.1 b 89.3±3.0 b 52.0±2.64 b 36.3±2.08 c 1 89.3±4.2 a 77.6±3.2 a 63.0±4.5 a 96.0±2.3 a 72.8±3.0 a 51.0±2.64 d 1.5 74.0±4.0 b 58.0±2.6 b 44.5±2.5 b 80.7±4.1 c 54.2±2.51 b 41.5±2.0 b 2 47.3±1.1 c 45.2±2.3 c 31.0±3.0 c 64.0±2.0 d 42.0±3.0 c 27.0±3.0 d fig. 1 somatic embryo formation in coriandrum sativum (rs). a) globular embryos at induction stage; b) somatic embryos at proliferation stage; c and d) somatic embryos at maturation stage (scale bar, a-2 mm; b-c= 3 mm; d=1 cm). muzamil et al. somatic embryogenesis of two coriander varieties 243 embryo germination was also influenced by pgrs combination; 1.0 mg/l ba + 0.5 mg/l ga3 showed maximum germination of embryos in both varieties (83.3% in rs and 76.7% in co-1). the increased levels of ba reduced plantlet conversion rate (table 2). biochemical analysis the embryogenic callus is often biochemically different from non-embryogenic tissues which could be used in marker assisted early selection. protein, proline and sugar level were noted to be high in early embryogenic tissue compared to non-embryogenic callus (fig. 2 a-c). the sugar content was also noted high at induction stage of embryogenesis (37.2 mg g m-1 fresh weight in ‘rs’ and 33.4 mg g m-1 fw in ‘co1’) as compared to other stages of embryo (matured) and non-embryogenic callus. the same was the case with proline where induction stage had more levels of proline in both the varieties (2.78 mg gm-1 fw in rs and 2.54 mg g m-1 fresh weight in co-1). protein content was higher at maturation stage of embryos showing 7.3 mg gm-1 fw in ‘rs’ and 7.0 mg gm-1fw in co-1compared to other stages of tissues. antioxidant enzymes activity behaved differently in different cultivating tissues (fig. 2 d-f). the highest cat activity was observed in rs with 5.4 eu mg-1 protein min-1 compared to co-1 with 4.7 eu mg-1 protein min-1 at matured stage of somatic embryos; so was sod activity (4.8 eumg-1protein min-1 and 4.3 eu mg-1 protein min-1 in rs and co-1 respectively). the apx activity was highest at induction stage of embryos with 2.1 eu mg-1 protein min-1 in rs and 1.8 eu mg-1 protein min-1 in co-1 compared to other tissues. synthetic seed preparation and plantlet conversion green and matured somatic embryos were encapsulated in different gelling conditions. good synthetic seeds were produced in combination of sodium alginate and calcium chloride mix, which later responded differently in germination medium (fig. 3 a-c). the study revealed that 3% sodium alginate + 100 mm cacl2 was the ideal mixture condition that had fairly good conversion rate (74.0% in rs and 70.6% in co-1) compared to 2% (48.6% in rs and 43.3% in co-1) and 4% (30.0% in rs and 26.6% in co-1) sodium alginate solution (table 3). the higher sodium alginate level (4%) reduced synthetic seed conversion ability. similarly, the level of cacl2 was also equally important in making synthetic seeds and later in obtaining table 2 somatic embryo differentiation and germination frequency in ‘rs’ and ‘co-1’ on different concentrations of naa, ba and ga3 supplemented ms medium values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p = 0.05) according duncan’s multiple range test. naa (mg/l) ba (mg/l) ga 3 (mg/l) rajendra swathi co-1 embryo differentiation conversion rate embryo differentiation conversion rate 0 0.5 0.25 0 54.6±3.0 c 0 51.3±3.0 c 0 1 0.25 0 68.0±3.4 b 0 64.0±3.4 b 0 1 0.5 0 83.3±4.6 a 0 76.7±4.1 a 0 1.5 0.5 0 63.6±3.0 b 0 54.0±2.0 c 0.5 0.25 0 78.7±4.1 a 0 74.0±4.0 a 0 1 0.25 0 61.3±3.0 b 0 55.3±3.0 b 0 1 0.5 0 56.0±3.4 b 0 47.2±2.3 c 0 1.5 0.5 0 39.3±3.0 c 0 34.0±2.0 d 0 fig. 2 biochemical and enzyme activities in two varieties of coriandrum sativum (rs and co-1). a) proline content; b) protein content; c) sugar content; d) catalase activity; e) ascorbate peroxidase activity and f) superoxide dismutase activity. adv. hort. sci., 2018 32(2): 239-248 244 plantlets; 100 mm cacl2 had a high plantlet conversion, followed by 75 mm and 125 mm. the synthetic seeds prepared at low (2%) sodium alginate +75 mm cacl2 were soft, fragile and showed poor conversion; while high (4%) level of sodium alginate and cacl2 (125 mm) produced hard beads, reducing conversion ability. the duration of cacl2 exposure also had an influence on conversion; 20 min exposure resulted in higher rate of germination compared to 10 and 30 min exposure (data not shown). the synthetic seeds containing somatic embryos, were kept in three different storage temperatures for varying periods in order to find the right temperature for preservation. it was observed that the storage temperature and duration had a direct influence on synthetic seed viability and final conversion rate (table 4). the encapsulated somatic embryos stored at 4°c for a week, showed higher regeneration ability (62.0% in rs and 58.6% in co-1) compared to incubation at room temperature (25oc) and -20oc where the conversion rate was 42.6% and 13.3% in rs and 34.6% and 9.3% in co-1 respectively. the conversion rate decreased as the storage duration increased as was observed in rs (44.0%) and co-1 (37.3%) after 5 weeks of 4oc storage (table 4). the synthetic seed’s viability was lost in both the varieties when stored at ultralow temperature (-20oc) for extended period (3 weeks). the regeneration ability gradually declined with increasing storage time in all tested conditions. the plantlets derived from synthetic seeds had a prominent shoot primordia without much root growth and these shoots were transferred to root induction medium, added with 0.5 mg/l iba. well rooted plants (fig. 3 d) were taken out from the test tube, washed with water and transferred in paper cup (fig. 3 e). after 2 weeks of hardening, the cups were transferred to the field. out of 80 plants transferred to soil, 43 (55%) survived in the field. the survived plants in outdoor looked healthy and morphologically similar to mother plants. 4. discussion and conclusions in these two varieties of coriander, callus was vigorously induced from hypocotyl in 1.0 mg/l 2,4-d added ms medium, consistent with previous studies in which 2,4-d played a crucial role in callusing in varfig. 3 synthetic seed development and plantlet formation in coriandrum sativum (rs). a) encapsulated somatic embryos; b) synthetic seeds on the germination medium; c) germinating synthetic seeds; d) rooted plantlet and e) synthetic seed derived plant, grown in outdoor condition (scale bar, a= 1.5 cm, b-d= 1 cm, e=2 cm). table 3 effect of different concentrations of sodium alginate and calcium chloride on the conversion rate of encapsulated somatic embryos on 1.0 mg/l ba and 0.5 mg/l ga3 ms medium values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according duncan’s multiple range test. alginate (%) calcium chloride (mm) conversion rate (%) rajendra swathi co-1 2 weeks 4 weeks 2 weeks 4 weeks 2 75 36.7±2.3 d 45.3±3.0 d 29.3±2.3 d 38.0±3.5 d 100 42.6±3.0 c 48.6±2.3 cd 36.6±3.0 c 43.3±3.0 c 125 30.0±2.0 e 37.3±3.0 e 24.0±2.0 e 32.6±2.3 e 3 75 46.0±3.4 c 52.0±3.4 c 41.3±3.0 e 51.3±3.0 b 100 63.3±4.2 a 74.0±4.0 a 64.0±3.4 a 70.6±4.1 a 125 52.0±3.4 b 57.3±2.3 b 45.3±2.3 b 54.7±3.0 b 4 75 21.3±2.3 f 24.6±3.0 g 18.0±2.0 f 24.0±2.0 f 100 25.3±3.0 ef 30.0±2.0 f 21.3±2.3 e 26.6±2.5 f 125 12.0±2.0 g 20.6±2.3 g 10.6±1.15 f 19.3±1.1 f muzamil et al. somatic embryogenesis of two coriander varieties 245 ious investigated plants (kim et al., 2011). in the present study, embryogenic callus started to differentiate somatic embryos in 2,4-d added medium but the continuous presence of 2,4-d inhibited somatic embryo development beyond globular/heart shaped stage. the formation of somatic embryos on callus induction medium was earlier reported in several other species (kim et al., 2011). the hindrance of embryo differentiation and progression in continuous presence of 2,4-d has earlier been reported in a number studied cases and the transfer of embryogenic tissues or embryos into ‘2,4-d-free’ or less pgr concentrated medium has been suggested for embryo development (junaid et al., 2006). the embryogenic tissues with developing somatic embryos were later cultured on naa and ba added medium in which fast embryo development and maturation were observed. somatic embryo differentiation and maturation in naa and ba added medium was earlier reported in several investigated plants (junaid et al., 2006; puhan and rath, 2012). the use of sucrose, aba and ga3 has also been noted to be very efficient in promoting somatic embryo development and maturation (yang et al., 2013; mujib et al., 2014). as the non-embryogenic callus transforms into embryogenic tissues, biochemical profiles also alter simultaneously. in this study, we noted differences in biochemical and enzyme activities in embryogenic and non-embryogenic tissues. here, we noted increased protein and decreased soluble sugar levels in maturation stage of somatic embryo, which is in confirmatory with kumar and kumari (2010). enhanced protein content was also observed here during maturation stage of embryo and it could be used as a good tissuespecific indicator. the increase in biochemical attributes in embryogenic callus over non-embryogenic tissue was previously reported in several plants (nieves et al., 2003). the extra proline accumulation has been noted in response to stress, which is believed to act as osmo-regulator in protecting cells against osmotic perturbation (elmaghrabi et al., 2013). similar changes in biochemical attributes were earlier observed during transition to differentiated embryogenic state from meristematic tissue under in vitro cultural conditions (samar et al., 2011). kumar et al. (2010) observed alteration of enzyme activities during cellular differentiation, this and similar other findings are important in understanding the metabolic changes in developmental events (santos et al., 2011). here, the enzyme activities were lower in non-embryogenic callus compared to embryogenic one, but it was higher at specific embryo stages. in hevea brasiliensis similar increased peroxidase activity was noted during embryo organization (blanc et al., 2002). increased apx and cat activity in embryogenic tissue, designates its higher efficiency in scavenging h2o2 during sod metabolism, prevents membrane lipids peroxidation process (niknam et al., 2006). beside distinguishing morphogenic events, cat facilitates in forming resistant cell walls and helps in defense mechanism against reactive oxygen species (gaspar et al., 2002). the induced mature somatic embryos were encapsulated in gelling mix and the conversion of synthetic seeds into plantlets was monitored. singh et al. (2010) observed that the quality bead formation and germination ability of synthetic seeds are partly dependent on concentrations of sodium alginate and calcium chloride solution and on duration of exposure. our observation indicated that the somatic embryos, encapsulated in 3% sodium alginate + 100 storage temperature storage duration (weeks) regeneration (%) rajendra swathi co-1 2 weeks 4 weeks 2 weeks 4 weeks -20°c 1 12.6±2.3 f 13.3±1.1 f 6.6±1.1 f 9.3±1.15 e 3 0 0 0 0 1 57.3±4.1 a 62.0±4.0 a 54.0±3.5 a 58.6±3.0 a 4oc 3 38.0±2.0 b 44.0±3.4 b 31.3±3.0 b 37.3±3.0 b 5 24.0±2.0 d 32.0±2.0 c 19.3±2.3 d 27.3±3.0 c 7 10.6±1.1 f 18.6±1.1 e 09.3±1.1 f 16.0±2.0 d 25°c 1 33.3±3.0 c 42.6±3.0 b 27.3±2.3 c 34.6±2.3 b 3 18.0±2.0 e 27.3±2.3 d 13.3±1.15 e 19.3±1.15 d table 4 conversion rate at temperature conditions of somatic embryos encapsulated in 3% sodium alginate and 100 mm cacl2, after storage. ms was added with 1.0 mg/l ba and 0.5 mg/l ga3 values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according duncan’s multiple range test. adv. hort. sci., 2018 32(2): 239-248 246 mm cacl2 produced quality bead with more conversion as compared to the use of lower (2%) and higher level (4%) of sodium alginate. the earlier reports suggested that somatic embryos encapsulated with 3% sodium alginate and 100 mm cacl2 produced uniform and moderately hard beads with optimum regeneration potential (sarmah et al., 2010). the maximum plantlet generation frequency with 3% sodium alginate was found in paulownia elongata (gozukirmizi, 2003), coelogyne breviscapa (mohanraj et al., 2009), stevia rebaudiana (ali et al., 2012). in stevia rebaudiana, hard beads were formed at 4% sodium alginate, which adversely affected germination rate (andlib et al., 2011). the 20 min exposure in cacl2 solution improved conversion of plantlets, the conversion rate however, declined with increasing exposure time and this observation corroborates with similar other previous studies (malabadi and van staden, 2005). over duration of exposure affects germination ability of the synthetic seeds and this decline of conversion may be due to growth inhibition caused by over absorption and penetration of calcium chloride (malabadi and van staden, 2005). the use of liquid ms for preparing gel matrix was found to be beneficial in emerging shoot and this may be attributed to the presence of nutrients in gel matrix, which apparently serves as a nutrient bed around the propagules and facilitates synthetic seed survival and germination (ara et al., 1999). matrix of synthetic seed mimics endosperm of natural seed and this nutrient rich artificial endosperm is essential for maintaining survival of germplasm (antonietta et al., 1999). the storage conditions also affect the germination potential of synthetic seeds. our observation indicated that the viability and regeneration ability of synthetic seeds decreased with increasing storage duration. the same view i.e. the longer storage of synthetic seeds reduced conversion ability was reported in earlier few cases (parveen and shahzad, 2014). during long storage, carbohydrate reserves decrease gradually, which may be responsible for the reduced conversion ability of synthetic seeds (ding et al., 1998). the storage of in vitro grown tissues at -20°c showed a very poor regenerative ability and loss of tissue viability. it is believed that the short spell of low temperature exposure helps forming ice crystals in tissues, prevents food exchange, causes frost injury/stress and reduces tissue viability (mittler, 2006). the successful regeneration system requires germination of synthetic seeds/mature somatic embryos into plantlets at fast pace. the encapsulated somatic embryos were germinated into plantlets on ba and ga3 added ms. the combination of ba and ga3 was found to be very responsive for somatic embryo germination in solanum lycopersicum (godishala et al., 2011). the well-developed plantlets were transferred to iba added half ms for promoting root growth. the positive effect of iba on root induction was reported in several investigated plants like camellia nitidissima (lu et al., 2013), talinum triangulare (swarna and ravindhran, 2013). the synthetic seed derived plants were healthy and morphologically very similar to parent plants. the developed plant regeneration protocol from synthetic seeds will be very important in preserving coriander germplasm for short and medium term basis. in summary, synthetic seed development and plant regeneration after storage may provide a good alternative strategy for coriander germplasm. the ‘short and medium term’ storage can open up and enable conservation possibility of elite important coriander. observed biochemical and enzyme activity differences in various in vitro grown tissues may play an important role in marker based selection of tissues in cellular differentiation investigation. the 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(*) corresponding author: kriskoty@yahoo.com citation: anyaoha c.o., oyetunde o.a., oguntolu o.o., 2022 ­ diallel analysis of selected yield‐con‐ tributing traits in okra (abelmoschus esculentus (l.) moench). ­ adv. hort. sci., 36(2): 97­106. copyright: © 2022 anyaoha c.o., oyetunde o.a., oguntolu o.o. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2022 accepted for publication 25 february 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12574 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 97­106 98 fresh okra pods serve as soup thickeners because of its unique mucilage properties and are known to be good sources of vitamins and minerals (schippers, 2000). the edible fruits contain 86% water, 2.2% pro­ tein, 10% carbohydrate, 0.2% fat and vitamins a, b, and c (berry et al., 1988). despite the important role played by okra in meeting the daily nutritional and economic needs of the society in the region, mini­ mum efforts and resources have been directed towards its genetic improvement to meet the desired preferences of farmers and consumers, such as earli­ ness and increased number of fruits with smooth pod per plant. okra research in developing countries has for long been concentrated on germplasm characteri­ zation and development of pure line varieties, with little or no efforts directed towards development of hybrid varieties despite its huge potential and mar­ ket. okra hybrids are gradually becoming the favorites of many farmers regardless of associated high cost of seeds since hybrid varieties are usually characterized by higher yield and uniformity, coupled with tolerance to diseases and pests. heterosis which is widely referred to as the superiority of f1 hybrid over the mean performance of its better inbred par­ ents, has been reported to enhance yield and compo­ nent traits in okra by about 86% (elmaksoud et al., 1986, ahmed and adam, 2014). hybrid variety devel­ opment is a quick path to combining economic and desirable horticultural traits in vegetable crops such as okra. the floral pattern of okra which enables easy emasculation and pollination coupled with the ability to produce a large number of seeds from a single pol­ lination, has made commercial exploitation of het­ erosis a profitable venture in okra hybrid seed pro­ duction (reddy, 2010). heterosis breeding is a means to improve yield and its component traits in okra (jindal et al., 2009). in many developing countries of west africa, minimal work has been reported on the use of two or more okra genotypes in heterotic studies for estimating hybrid vigor relative to fruit yield and its component traits. positive heterosis favors genetic improvement of yield and yield contributing traits whereas nega­ tive heterosis is encouraged when breeding for traits such as earliness (biswas et al., 2005). the objective of this study was to (i) determine gene action and identify superior parent combination(s) for measured traits and (ii) identify stable hybrids for desired traits of okra with a view to providing information for future breeding programs. 2. materials and methods study area two experiments were conducted at the national horticultural research institute (nihort), ibadan, oyo state, nigeria during the early planting season (april to july) and late planting season (from august to december) of 2019. nihort is located in the humid forest savanna transition zone (210 masl, 7°30ʹn, 3°54ʹe), with a bimodal annual rainfall pat­ tern spanning across 120­128 rainy days, amounting to 1200­1400mm. pan evaporation is between 1550­ 1600mm. wet season extends from march through october and dry season from november to february, with annual maximum temperature ranging between 27­34°c and annual minimum temperature ranging between 20­23oc (ogungbenro and morakinyo, 2014). plant material and field establishment genetic materials evaluated in this study com­ prised of 10 newly­developed f1 hybrids obtained from diallel mating and their 5 parents namely: nh47­4, ik11, iwo nla, ld 88, and clemson. the par­ ents and their hybrids were grown in randomized complete block design with two replicates during the early and late planting seasons of 2019. three seeds per hill were sown directly in 3­cm holes and later thinned to one plant per hill after seedling establish­ ment. plants were spaced at 60 cm x 50 cm between and within rows respectively on a 2­m bed constitut­ ing 10 stands per plot. regular plant protection and other agronomic activities were carried out as when due to ensure full expression of desired traits and safeguard crops from pests. manual weeding was done at three weeks after planting while a compound fertilizer, npk 15:15:15, was applied at three­week interval after sowing to enhance vegetative growth at the recommended rate of 60 kg/ha (adigun et al., 2018). insect pest control was done by spraying cyperfits (synthetic pyrethrum) at the rate of 80 g ai/ha. data collection data on agronomic attributes were recorded on five randomly selected competitive plants in each plot according to okra descriptors by charrier (1984) and ipgri (1991) for days to 50% flowering (dtf), plant height at maturity (ph) (cm), matured fruit width and length (cm), average number of fruits (fruits per plant) obtained by the ratio between the anyaoha et al. ‐ diallel analysis of yield‐contributing traits in okra 99 total number of fruits and the number of plants in the plot, internode length (cm), number of ridges per pod and 1000­seed weight (g). data analysis each season was considered an environment. pooled data for the two environments were subject­ ed to analysis of variance (anova). the mixed model procedure of statistical analysis system (sas) (sas institute, 2002) was used, with replication with­ in environment treated as random factor and geno­ type (crosses) as fixed factor. the statistical model used for the combined analysis is: yijg= µ + ei+ rj(i) + gg + egig+ εijg where yijg is the measurement for the gth genotype grown in replicate j within environment i; µ is the grand mean; ei is the main effect of environment i; rj(i) is the effect of replicate nested within environment effect; gg is the effect of the genotype; egig is the interaction effect between genotype and environment, and εijg is the error term. mid­ and better­parent heterosis were calculated according to the procedure of singh (1973) described by amiteye et al. (2019). general (gca) and specific combining ability (sca) estimates were generated for each of the traits according to the procedure of griffing (1956), employed by medagam et al. (2012), for diallel analy­ sis model b (mixed), method 2 using agd­r software version 3.0 (rodríguez et al., 2015). the statistical model for the diallel analysis is as follows: yijk = µ + ee+ gi+ gj+ sij+ geeg+ sees+ εijk where yijk is the observed measurement for the ijth cross grown in the kth environment; µ is the grand mean; ee is the main effect of environment; gi and gj are the gca effects; sij is the sca effect; geeg is the interaction effect between gca and environment; sees is the interaction effect between sca and environment, and εijk is the error term. the gca and sca effects were tested for signifi­ cance using t­test at 5 and 1% levels of probability as suggested by mather and jinks (1982), kearsey and pooni (1996). standard error (se) estimates of gca and sca were obtained from pb tools software ver­ sion 1.4.0 (pb tools, 2014). the formulae for the ses were described by ahmed and adam (2014) as fol­ lows: se (gi) = [(n­1)σ2e/n(n+2)]½ and se (gi­gj) = [(2σ2e/n+2)]½ for gca effects and se (sij) = [n(n­ 1)σ2e/(n+1)(n+2)]½ and se (sii­sjj) = [2(n­2)σ2e/n+2)]½ for sca effects where s.e. (gi) = s.e. for gca effects of parents, s.e. (gi­gj) = s.e. of difference between gca effects of the ith and jth parents, s.e. (sij) = s.e. for sca effects of the diallel hybrids, s.e. (sii­sjj) = s.e. of the difference between the sca effects of the ith and jth hybrids, σ2e is the error mean square value in the diallel analysis, and n is the number of parents. the proportions of the additive and non­additive genetic variances were computed as the percentages of gca and sca sums of squares (ss) respectively of the cross ss across environments (fasahat et al. 2016). means of observed data were used to obtain pair­wise correlation (pearson’s) coefficients, to determine the level of association among measured traits. the modified genotype × trait (gyt) biplot approach of yan and fregeau­reid (2018) was used to profile the 10 diallel f1 hybrids for measured traits. the gyt incorporates yield into other traits, rather than as a standalone trait. here, gyt is the interac­ tion of genotype and the combination of other traits with nof. in this study, the procedure for obtaining the nof­trait combination estimates was based on the direction of association of other traits with nof. thus, the estimates were obtained by multiplication for all measured traits except dtf, fwt, and inl which were negatively correlated with nof, and were obtained by division. biplotswere obtained using the ggebiplotgui package in r. 3. results analysis of variance for pooled data over the two environments (seasons) revealed significant (p≤0.01) genotype (g) mean squares for all measured traits while environment (e) mean square was significant (p≤0.01) for dtf and nof. the mean square of the g×e interaction was found to be significant (p≤0.05) for nor. number of days to flowering (dtf) ranged from 46.5 days for nh47­4 × clemson to 57.75 days for ld88 while nos ranged from 30.50 for nh47­4 × ld88 to 117.15 for ld88 × iwo nla. iwo nla × clemson produced the longest peduncles with a mean length of 3.50 cm while ld88 had the shortest peduncle lengths averaging 1.50 cm (table 1). diallel analysis of hybrids revealed significant (p≤0.05 or 0.01) cross mean squares for all measured traits necessitating the partitioning of the mean squares into gca and sca components. mean squares of gca and sca were significant (p<0.05 or adv. hort. sci., 2022 36(2): 97­106 100 0·01) for all traits measured except gca for frtwdt while cross × environment and sca × environment interaction mean squares were significant only for nor. with the exception of 1000­seed weight, sca effect consistently accounted for higher proportion of total the variation among measured traits com­ pared to gca effects. the sca accounted for 93.35% and 41.56% of the total variation for number of seeds per pod and 1000­seed weight respectively (table 2). clemson and iwo nla had significant (p<0.05 or 0.01) and negative general combining ability (gca) effects for dtf while ld88 showed significant (p<0.01) positive gca effect for ph. iwo nla had sig­ nificant (p<0.01) positive gca effect for nof. significant negative and positive gca effects for frtlnt and frtwdt was recorded for ik11 and ld88 respectively while nh47­4, ik11 and ld88 showed significant (p<0.05 or 0.01) positive gca effects for 1000­seed weight (table 3). days to flowering had significant negative specific combining ability (sca) for nh47­4 × ld88, nh47­4 × clemson and ik11 × ld88 while nh47­4 × ik11, ik11 × kd88, and ld88 × clemson had high significant positive sca effects for ph. similarly, nh47­4 × iwo nla and ld88 × clemson had significant (p<0.05 or 0.01) and positive sca for nof and frtlnt while significant positive sca effects were observed for frtwdt for nh47­4 × ik11, ik11 × ld88, ld88 × iwo nla and clemson × iwo nla. furthermore, nh47­4 × ik11, nh47­4 × clemson, ik11× clemson, ld88× clemson and ld88 × iwo nla exhibited high significant positive sca for nos and 1000­seed weight (table 3). the proportion of addi­ tive genetic variance ranged from 0.07 for nos to 0.58 for 1000­seed weight while the non­additive genetic variance estimates ranged from 0.42 to 0.93 for ths and nos respectively (table 4). generally, the non­additive variance was higher than the additive table 1 ­ analysis of variance of 15 genotypes of okra evaluated for selected traits across research environments *,** significant at p≤0.05 and p≤0.01 respectively; ns= not significant; rep= replicate; dtf= days to 50% flowering; ph= plant height at maturity; nof= number of fruits per plot; lnt= fruit length; fwt= fruit circumference; pl= pedicel length; inl= internodal length; nos= number of seeds per pod; ths= 1000­seed weight; nor= number of ridges per pod. genotype dtf (days) ph (cm) nof lnt (cm) fwt (cm) pl (cm) inl (cm) nos ths (g) nor clemson 48.25 ef 62.25 de 5.00 cde 12.92 a 2.39 d 3.00 b 6.50 de 68.75 de 50.00 d 7.38 efg ik11 49.25 def 104.00 abc 6.00 b­e 7.75 cd 3.4 abc 1.63 ef 8.63 cd 79.50 bcd 70.00 a 6.75 g iwonla 47.75 f 74.00 b­e 5.75 b­e 10.22 bc 2.91 dc 2.00 ed 6.75 de 64.75 e 60.00 c 5.25 h ld88 57.75 a 122.75 a 4.50 de 6.70 d 3.34 abc 1.50 f 12.50 ab 82.25 bc 45.00 e 9.00 ab nh47­4 52.50 bc 63.75 de 5.00 cde 7.95 cd 3.51 abc 2.50 c 6.75 de 65.50 e 70.00 a 8.00 def nh47­4 × ik11 48.25 ef 73.50 b­e 8.75 b 8.77 cd 3.43 abc 2.38 cd 5.50 e 68.25 de 60.00 c 5.25 h nh47­4 × ld88 52.13 bcd 106.25 ab 7.75 bc 12.16 ab 2.84 cd 3.00 b 10.50 bc 66.00 e 70.00 a 8.13 cde nh47­4 × clemson 51.25 b­e 73.00 b­e 8.25 b 9.71 bc 3.25 bc 3.00 b 6.00 e 72.50 cde 65.00 b 8.00 def nh47­4 × iwo nla 49.38 def 42.25 e 4.25 e 8.44 cd 4.08 a 1.78 ef 7.13 de 36.00 f 60.00 c 5.75 h ik11 × ld88 53.75 b 76.50 b­e 7.50 bc 13.93 a 3.16 dbc 3.50 a 5.75 e 61.00 e 60.00 c 8.88 abc ik11 × clemson 50.50 c­f 107.25 ab 7.75 bc 9.12 cd 3.82 ab 2.50 c 6.75 de 117.25 a 60.00 c 8.25 bcd ik11 × iwo nla 47.50 f 70.25 cde 7.25 bcd 11.92 ab 2.91 dc 2.50 c 6.50 de 84.75 b 70.00 a 9.25 a ld88 × clemson 50.88 b­e 96.50 a­d 7.25 bcd 8.90 cd 3.93 ab 1.75 ef 13.25 a 72.25 cde 70.00 a 7.25 fg ld88 × iwo nla 51.00 b­e 74.75 b­e 12.25 a 13.61 a 2.72 dc 2.25 cd 9.50 c 68.50 de 60.00 c 5.00 h clemson × iwo nla 48.25 ef 75.50 b­e 4.00 e 6.74 d 2.93 dc 2.00 de 13.00 a 30.50 f 42.50 e 7.75 def source df dtf ph nof lnt fwt pl inl nos ths nor rep (environment) 2 ** ns ** ns ns ns ns ns ns ns environment (e) 1 ** ns ** ns ns ns ns ns ns ns genotype (g) 14 ** ** ** ** ** ** ** ** ** ** g×e 14 ns ns ns ns ns ns ns ns ns * error 28 ns ns ns ns ns ns ns ns ns ns r­squared 0.86 0.71 0.82 0.84 0.72 0.92 0.88 0.94 0.95 0.95 anyaoha et al. ‐ diallel analysis of yield‐contributing traits in okra 101 table 2 ­ mean squares from diallel analysis of variance of okra genotypes evaluated in two environments *,** significant at p≤0.05 and p≤0.01, respectively; ns= not significant; rep= replicate; dtf= days to 50% flowering; ph= plant height at maturity; nof= number of fruits per plot; lnt= fruit length; fwt= fruit circumference; pl= pedicel length; inl= internodal length; nos= number of seeds per pod; ths= 1000­seed weight; nor= number of ridges per pod. table 3 ­ estimates of general (gca) and specific combining ability (sca) effects of okra parents and hybrids respectively for measured traits *,** significant at p≤0.05 and p≤0.01 respectively; s.e.= standard error; gi= gca effects of parents; gi­gj= difference between gca effects of the ith and jth parents; sij= sca effects of the diallel hybrids; sii­sjj= difference between the sca effects of the ith and jth hybrids; ne= not estimated; dtf= days to 50% flowering; ph= plant height at maturity; nof= number of fruits per plot; lnt= fruit length; fwt= fruit circumference; pl= pedicel length; inl= internodal length; nos= number of seeds per pod; ths= 1000­seed weight; nor= number of ridges per pod. source of variation df dtf (days) ph (cm) nof lnt (cm) fwt (cm) pl (cm) inl (cm) nos ths (g) nor rep (env) 2 22.92 ** 1023.90 ns 40.08 ** 4.13 ns 0.04 ns 0.08 ns 6.93 ns 41.22 ns 0.83 ns 0.19 ns environment (e) 1 39.20 ns 32.27 ns 40.02 ns 1.80 ns 0.02 ns 0.00 ns 0.00 ns 0.15 ns 0.00 ns 0.04 ns crosses 14 30.07 ** 1817.21 ** 18.89 ** 23.45 ** 0.90* 1.39 ** 30.09 ** 1588.48 ** 327.38 ** 8.10 ** gca 4 38.99 ns 1187.64 ** 18.52 * 38.17 * 1.32 ns 2.09 ** 37.33 ** 369.98 * 669.64 ** 11.27 ** sca 10 26.50** 2069.04 ** 19.04 ** 17.56 ** 0.73 * 1.11 ** 27.19 ** 2075.88 ** 190.48 ** 6.83 ** crosses × e 14 6.11 ns 58.48 ns 1.87 ns 2.70 ns 0.27 ns 0.01 ns 0.78 ns 47.69 ns 0.00 ns 0.69 * gca×e 4 8.56 ns 30.10 ns 1.87 ns 4.42 ns 0.48 ns 0.01 ns 2.10 ns 40.85 ns 0.00 ns 0.52 ns sca×e 10 5.12 ns 69.83 ns 1.88 ns 2.01 ns 0.19 ns 0.01 ns 0.25 ns 50.42 ns 0.00 ns 0.76 * residuals 28 3.3 416.11 3.23 2.55 0.23 0.06 2.1 49.43 7.98 0.25 gca proportion (%) 37.05 18.67 28.01 46.51 41.97 42.96 35.45 6.65 58.44 39.76 sca proportion (%) 62.95 81.33 71.99 53.49 58.03 57.04 64.55 93.35 41.56 60.24 parent/hybrid dtf (days) ph (cm) nof lnt (cm) fwt (cm) pl (cm) inl (cm) nos ths (g) nor nh47­4 ­0.1 ­6.36 0.04 ­0.35 0.01 ­0.09 * 0.82 ­4.01 * 2.50 * 0.19 ik11 ­0.18 5.29 ­0.14 ­0.69 * 0.21 ­0.19 * 0.59 ­1.34 5.71 ** ­0.19 ld88 2.00 ** 8.29 ­1.14 ** ­1.13 ** 0.17 ­0.19 * 1.09 * ­0.84 ­7.50 ** 0.67 ** clemson ­0.75 * ­5.43 0.11 1.87 ** ­0.33 * 0.47 ** ­1.25 * 0.34 ­0.71 0.33 * iwo nla ­0.98 ­1.79 1.14 * 0.3 ­0.06 0 ­1.25 * 5.84 * ne ­0.99 ** s.e. (gi) 0.31 3.45 0.3 0.27 0.08 0.04 0.25 1.18 0.48 0.08 s.e. (gi ­ gj) 0.49 5.45 0.48 0.43 0.13 0.07 0.39 1.88 0.75 0.13 nh47­4 × ik11 0.6 16.07 * 0.61 0.02 0.47 * ­0.32 ** 3.51 ** 8.42 ** 0.95 ­0.07 nh47­4 × ld88 ­4.21 ** ­7.93 ­1.64 * ­1.70 ** ­0.49* ­0.07 2.76 ** ­33.83 ** ­13.33 ** ­0.43 * nh47­4 × clemson ­2.21 ** 0.54 0.36 0.48 ­0.01 ­0.23 * ­1.40 ** 19.24 ** 7.38 ** 1.41 ** nh47­4 × iwo nla 1.52 * 1.39 4.32 ** 3.74 ** ­0.47 * ­0.02 1.60 ** ­2.51 ­3.33 ** ­1.52 ** ik11 × ld88 ­3.01 * ­52.82 ** ­1.21 0.33 0.45 * ­0.20 * ­2.89 ** ­31.01 ** 0.95 ­2.05 ** ik11 × clemson 2.49 ** 24.89 ** 1.04 1.06 ­0.27 0.37 ** 2.83 ** ­2.19 4.17 ** 0.66 ** ik11 × iwo nla 1.85 * ­12 0.5 0.17 ­0.14 0.84 ** ­1.67 ** ­1.19 ­1.55 1.86 ** ld88 × clemson 1.93 ** ­7.86 1.79 * 3.27 ** 0.08 0.87 ** ­2.42 ** ­7.69 ** 7.38 ** 0.55 ** ld88 × iwo nla ­1.08 19.25 * 1 0.03 0.47 * 0.34 ** ­1.42 ** 43.06 ** 6.67 ** 1.25 ** clemson × iwo nla ­0.58 ­0.79 0.75 ­3.32 ** 0.58 ** ­0.45 ** ­0.33 ­7.12 * ­0.12 ­1.41 ** s.e. (sij) 0.63 7.04 0.62 0.55 0.17 0.09 0.50 ** 2.43 0.97 0.17 s.e. (sii­sij) 0.84 9.44 0.83 0.74 0.22 0.12 0.67 3.25 1.31 0.23 102 adv. hort. sci., 2022 36(2): 97­106 variance for all measured traits except ths. estimates of mid­parent heterosis (mph) and bet­ ter­parent heterosis (bph) of diallel crosses of okra are presented in table 5. significant positive and neg­ ative heterosis was observed for all traits. desirable significant bph and mph for earliness was observed for nh47­4 × ld88 (­16.45% and ­6.24%), nh47­4 × clemson (­9.52% and ­2.85%), ik11 × ld88 (­14.50 and ­3.86%), and ld88× iwo nla (­12.55% and ­ 2.13%). ld88 × clemson also showed desirable high significant bph of ­6.93% for dtf. for nof, all the hybrids showed desirable significant (p<0·05 or 0.01) bph and mph with the exception of nh47­4 × ik11, nh47­4 × ld88, and ik11 × ld88 for bph, and nh47­4 × ld88 and ik11 × ld88 for mph. intermodal length (intel) recorded high negative significant bph and mph for ik11 × ld88, ld88 × clemson, and ld88 × iwo nla while ld88 × iwo nla only had significant (p<0.05 or 0.01) negative bph for the same trait. table 4 ­ proportion of additive and non­additive genetic vari­ ances for measured traits across the two environments dtf= days to 50% flowering; ph= plant height at maturity; nof= number of fruits per plot; lnt= fruit length; fwt= fruit circum­ ference; pl= pedicel length; inl= internodal length; nos= number of seeds per pod; ths= 1000­seed weight; nor= num­ ber of ridges per pod. table 5 ­ estimates (%) of heterobeltiosis of ten diallel crosses of okra for measured traits *,** significant at p≤0.05 and p≤0.01 respectively; bph and mph= better­ and mid­parent heterosis, respectively; dtf= days to 50% flow­ ering; ph= plant height at maturity; nof= number of fruits per plot; lnt= fruit length; fwt= fruit circumference; pl= pedicel length; inl= internodal length; nos= number of seeds per pod; ths= 1000­seed weight; nor= number of ridges per pod. trait additive variance non­additive variance dtf (days) 37 63 ph (cm) 19 81 nof 28 72 lnt (cm) 47 53 fwt (cm) 42 58 pl (cm) 43 57 intel (cm) 35 65 nos 7 93 thsw (g) 58 42 nor 40 60 hybrid dtf (days) ph (cm) nof frtlnt (cm) frtwdt (cm) bph mph bph mph bph mph bph mph bph mph nh47­4 × ik11 ­3.1 0 ­7.21 7.53 20.83 15.91* 11.87* 6.66* 12.02* 6.91** nh47­4 × ld88 ­16.45** ­6.24** ­38.49** ­9.52* ­20 ­7.89 ­15.23* ­4 ­16.45** ­7.19** nh47­4 × clemson ­9.52** ­2.85** 10.2 5.75 45.00** 22.50** ­7.68 7.14* ­17.18** ­0.72 nh47­4 × iwo nla ­2.86 0.87 1.01 4.26 113.04** 63.95** 33.24** 24.92** ­22.57** ­7.66** ik11 × ld88 ­14.50* ­3.86** ­65.58** ­31.37** ­29.17* ­9.52 8.85 8.37* 19.95** 10.50** ik11 × clemson 5.84* 3.46** 2.16 13.91** 29.17* 20.45** ­5.86 8.84* ­16.32** ­0.85 ik11 × iwo nla 4.06 2.84** ­29.81** ­8.99* 37.50** 20.21** ­4.99 4.02 ­4.2 1.63 ld88 × clemson ­6.93** 0.71 ­37.68** ­8.65* 50.00** 28.95** 7.84 20.99** ­5.44 5.14* ld88 × iwo nla ­12.55** ­2.13* ­12.63 4.51 34.78* 25.61** ­10.68 3.93 14.47* 11.19** clemson × iwo nla 0 0.26 ­0.68 3.94 52.17** 31.40** ­32.13** ­12.10** 17.91** 14.75** standard error 2.31 0.91 7.22 3.92 11.86 6.19 5.35 3.2 4.93 2.3 hybrid pl (cm) inl (cm) nos thsw (g) nor bph mph bph mph bph mph bph mph bph mph nh47­4 × ik11 ­30.00** ­7.58 53.62** 36.18** ­9.12 ­0.17 0 0 ­9.38 ­0.85 nh47­4 × ld88 ­20.00* 0 4 17.53* ­62.92** ­29.36** ­39.29** ­13.04** ­13.89* ­4.41 nh47­4 × clemson ­16.67* ­4.55 ­3.7 ­0.94 23.27* 13.13* 0 8.33** 15.63* 10.16** nh47­4 × iwo nla ­10 0 40.74** 20.37** 4.58 2.59 ­14.29** ­3.85 ­37.50** ­12.26** ik11 × ld88 9.23 6.8 ­43.00** ­16.27* ­56.23** ­27.74** ­14.29** 2.17 ­36.11** ­13.49** ik11 × clemson 0 14.86** 21.74 19.42** ­16.98 ­5.48 0 8.33** 10.17 7.52* ik11 × iwo nla 50.00** 32.76** ­30.43* ­10.98 ­8.81 0.26 ­7.14 0 18.52** 16.67** ld88 × clemson 16.67* 27.78** ­54.00** ­19.74** ­25.84* ­9.6 20.00** 13.16** ­1.39 4.2 ld88 × iwo nla 25.00** 21.43** ­46.00** ­14.94* 42.55** 29.76** 0 7.14** ­8.33 7.89* clemson × iwo nla ­20.83* ­2.5 ­18.52 ­8.49 ­0.73 1.12 0 4.55* ­28.81** ­8.42* standard error 7.48 4.32 11.23 5.86 9.69 5.25 4.58 2.24 6.13 3.06 anyaoha et al. ‐ diallel analysis of yield‐contributing traits in okra 103 significant positive percentage for bph and mph were also obtained for nh47­4 × clemson (23.27 and 13.13%) and ld88 × iwo nla (42.55 and 29.76%) while nh47­4 × clemson, ik11 × clemson, ld88 × clemson, ld88 × iwo nla and clemson × iwo nla dis­ played significant positive (desirable) bhp and mph for 1000­seed weight. the tester vector view of the genotype × nof­trait (gyt) biplot showing associations among the nof­ trait combinations is presented in figure 1. since all nof­trait combinations have nof as a component, positive correlation was observed between all possi­ ble pairs. there was correspondence between the gyt biplot and pearson correlation (table 6) among the traits. positive correlation was recorded between nos and ths while inl associated negatively with nos and ths (table 6). the gyt biplot, shows the magnitude of angles among inl, nos, and ths while figure 2 is the polygon view of the gyt biplot dis­ played in figure 1. hybrid was the vertex cultivar for the sector con­ taining all the nof­trait combinations except inl while ld88 × clemson, nh47­4 × clemson, ik11× iwo nla, and clemson × iwo nla were associated with the polygon sector containing inl. the superiority ranks of the hybrids based on their nof­trait combinations is shown in figure 3. hybrid nh47­4 × iwo nla was the farthest above average from the origin, followed by ld88 × iwo nla and clemson × iwo nla while nh47­4 × ld88 was the farthest below average performance across nof­trait combination. the shortest vector lengths were observed for ik11 × ld88, nh47­4 × table 6 ­ pearson correlation coefficients among pairs of traits of okra hybrids * and ** significant at p ≤ 0.05 and p ≤ 0.01 respectively; dtf, days to 50% flowering; ph, plant height at maturity; nof, number of fruits per plot; lnt, fruit length; fwt, fruit circumference; pl, pedicel length; inl, internodal length; nos, number of seeds per pod; ths, 1000­seed weight; nor, number of ridges per pod. fig. 2 ­ the polygon view of the genotype by noe x trait (gyt) biplot to identify genotypes with outstanding trait profi­ les. fig. 1 ­ the tester vector view of the genotype by nof x trait (gyt) biplot showing associations among the nof x trait combinations. ph nof lnt fwt pl inl nos ths nor dtf 0.52 * ­0.01 ­0.09 0.18 0.02 0.32 0.18 ­0.13 0.46 ph 0.09 ­0.23 0.09 ­0.23 0.52 * 0.58 * ­0.01 0.37 nof 0.57* ­0.17 0.32 ­0.17 0.35 0.37 ­0.27 lnt ­0.58 * 0.71 ** ­0.37 0.09 0.21 0 fwt ­0.42 0.05 0.11 0.27 ­0.04 pl ­0.55* 0.08 0.16 0.34 int ­0.2 ­0.28 0.12 nos 0.29 0.31 ths ­0.01 adv. hort. sci., 2022 36(2): 97­106 104 ld88 (below­average performers) and nh47­4 × iwo nla and ik11 × clemson (above­average performers). 4. discussion and conclusions the significant genotype mean squares observed for all traits is an indication of sufficient genetic varia­ tion among the genotypes for measured traits and this shows the feasibility of improving these traits through selection. wammanda et al. (2010) observed similar level of genotypic variability for dtf, ph, nof, inl, lnt and fwt in okra. the significant gca and sca mean squares for most measured traits indicates that both additive and non­additive gene actions were important in the inheritance of these traits. the proportion of total variation accounted for by sca was larger than that of gca for all the traits except 1000­seed weight thus indicating that 1000­seed weight is majorly controlled by additive gene action. having most of the traits controlled by additive and non­additive gene effects implies that substantial breeding progress could be made using breeding methods such as backcrossing and heterosis breeding techniques which exploit the two modes of gene action. this supports the work of reddy et al. (2012) on the preponderance of non­additive gene action for several traits of okra. the significant negative gca effects for dtf observed for clemson and iwo nla implied that these genotypes could possess useful genes for improve­ ment of earliness in okra while iwo nla with signifi­ cant positive gca effect for nof might be a good donor for genes associated with increased number of fruits and seeds. in the same vein, clemson and iwo nla, with significant negative gca for inl could be useful in developing new okra breeding lines with short internode and enhanced branching thereby improving yield. this result agress atanu and sabesan (2009) who reported significant gca effects for dtf, nof, nor, lnt and fwt through diallel crosses in okra. the significant and desirable sca effects (negative for dtf and inl) observed for most measured traits among the 10 hybrids suggests the presence of favourable gene combinations for most horticultural traits of interest in this study. thus, nh47­4 × ld88, nh47­4 × clemson, and ik11 × ld88 with significant and negative sca effects for dtf contains favourable gene combinations for earliness while nh47­4 × ik11, nh47­4 × clemson, and ld88 × iwo nla might be har­ bouring gene combination associated with seed­ increasing effects. majority of the hybrids except nh47­4 × ld88 and ik11 × iwo nla have gene combi­ nations in favour of lnt, fwt and 1000­seed weight. the above mentioned sets of hybrids could be deployed for heterosis breeding in favour of associat­ ed horticultural traits such as earliness and increased number of seeds. furthermore, they can be inter­ crossed and advanced to generate breeding popula­ tions with a large gene pool that might be useful in identification and selection of new promising segre­ gants. similar results have been reported for okra by oyetunde and ariyo (2015), reddy et al. (2012) and anyaoha et al. (2021). significant and negative mph and bph is desired for dtf and inl improvement of earliness and increased number of branches respectively. the sig­ nificant and negative bph and mph observed for these traits in hybrids: nh47­4 × ld88, nh47­4 × clemson, ik11 × ld88, ik11 × iwo nla, ld88 × clemson and ld88 × iwo nla could be exploited through heterosis breeding. khanorkar and kathiria (2010) and prakash et al. (2019) reported similar find­ ings for number of days to flowering in okra. selecting okra genotypes with tall plant architec­ ture and short inl might lead to increased number of pods per plant in this study. although none of the hybrids possessed desirable heterosis for ph and inl, a cross between ik11 × clemson with significant posi­ tive mph for ph, and ik11 ×ld88, ik11×iwo nla, ld88 fig. 3 ­ the average tester coordination view of the genotype by nof x trait (gyt) biplot ranking the hybrids based on ove­ rall superiority and their strengths and weaknesses. anyaoha et al. ‐ diallel analysis of yield‐contributing traits in okra 105 × clemson, and ld88 × iwo nla with significant and negative mph and bph for inl might produce tall multiple hybrids with short inl. hybrid combinations nh47­4 × clemson, ik11 × iwo nla and ld88 × iwo nla and ik11 × clemson, ld88 × clemson and clemson × iwo nla displayed significant and positive bph and mph respectively for seed­increasing traits nos, 1000­seed weight and nor. it looka apparent from the findings of this study that high heterotic effects for measured traits might be due to the domi­ nance nature of genes controlling considered traits. reddy et al. (2012) explained that heterobeltiosis of more than 20% could offset the cost of hybrid seed. thus, hybrids nh47­4 × clemson and ld88 × iwo nla with bph of 23.27% and 42.55% respectively for nos could be useful resources to exploit heterosis for okra hybrid seed production. however, with the exception of nh47­4 × ik11, nh47­4 × ld88, and ik11 × ld88, majority of the hybrids exhibited the poten­ tial for use to enhance nof per plant in okra. the gyt biplot (yan and frégeau­reid, 2018) used in this study allows genotype evaluation by graphical­ ly ranking genotypes based on their level in combin­ ing major traits of interest (such as increased number of fruits per plant) with other target traits. the single­ arrow line passing through the biplot origin and the average yield­trait combination is called the average tester axis (ata). the arrow points towards the high­ er genotype mean values across all nof­trait combi­ nations and serves the purpose of ranking genotypes based on superiority. the double­arrow line perpen­ dicular to the ata separates genotypes better than average (on the same side as the ata arrow) from those poorer than average (on the opposite side of the ata) and also indicates how balanced the trait profile of a genotype is as well as its strengths and/or weaknesses in terms of adaptation to specific traits. genotypes placed close to ata (with short projec­ tions to the double­arrowed line) have balanced trait profiles whereas those placed away from the ata in either direction have obvious strengths and/or weak­ nesses. the polygon view of the biplot allowed visual­ ization of the trait profiles of the hybrids such that the genotypes placed on a vertex had the largest val­ ues for the nof­trait combinations placed within the corresponding sector. hybrid nh47­4 × iwo nla had the highest values for nof­trait combinations indicating that they are top performers in combining nof with the respective traits. on the other hand, ld88 × clemson, nh47­4 × clemson, ik11 × iwo nla, and clemson × iwo nla had the highest values for inl showing that these hybrids were the best in combining nof with the respective traits inl. the ranking of the hybrids based on the nof­trait combinations was as followed: nh47­4 × iwo nla> ld88 × iwo nla> clemson × iwo nla ≥ ik11 × iwo nla> ld88 × clemson> ik11 × clemson > nh47­4 × clemson > nh47­4 × ik11> ik11 × ld88> nh47­4 × ld88. the short vector lengths of the hybrids ik11 × ld88, nh47­4 × ld88, nh47­4 × iwo nla and ik11 × clemson implied that these hybrids were the most stable across the various trait combinations. in conclusion, the pooled analysis of variances successfully identified the extent of genetic variability among parents and hybrids. high significant differ­ ences among parents, crosses and parents vs. crosses for most traits indicated sufficient level of variability among the genotypes. the parental genotypes, clemson and iwo nla, are promising sources of genes for earliness while iwo nla was promising as a gene donor towards improvement of nos. the new promising hybrid combinations nh47­4 × ld88, nh47­4 × clemson and ik11 × ld88 identified from this study could be exploited towards creating early maturing hybrid okra varieties while nh47­4 × clemson, ik11 × clemson, ik11 × iwo nla, ld88 × clemson were identified as hybrids with useful het­ erotic patterns for seed­increasing traits. promising hybrids nh47­4 × iwo nla and ik11 × clemson that were stable and above­average for nof­trait combi­ nations and ranked high by the gyt biplot might be useful for okra genetic improvement programmes targeting increased number of fruit per plant over seasons in the region. references adigun j.a., daramola o.s., adeyemi o.r., ogungbe­ san a.o., olorunmaiye p.m., osipitan o.a., 2018 ­ impact of nitrogen levels and weed control methods on growth and yield of okra (abelmoschus esculentus (l.) moench) in the nigerian forest‐savanna transition zone. ­ j. exper. agric. inter., 20(2): 1­11. ahmed s.b.m., adam s.i., 2014 ­ combining ability for yield components in six parents and their 15 f1 hybrids of sesame (sesamum indicum l.) in half diallel matin design. ­ j. plant breed. crop sci., 6(12): 179­184. amiteye s., amitaaba t., akama c., amoatey h.m., 2019 ­ estimates of heterosis in f1 hybrid progeny of okra (abelmoschus spp. (l.) moench). ­ indian j. pure appl. biosci., 7(6):1­8. adv. hort. sci., 2022 36(2): 97­106 106 anyaoha c.o., oguntolu o.o., okoyo m.e., ikoro j.i., onyeanusi h.c., ajoba f.b., nweke u.c., 2021 ­ evaluation of promising elite okra (abelmoschus escu­ lentus l. moench) breeding lines for yield and fruit size related traits under field conditions. ­ nigerian j. seed sci. 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europaea): eco­physiological analysis in the mediterranean basin f. maniriho (*) european university of lefke, lefke, nicosia district, cyprus. key words: anthesis, fruit setting, hermaphrodite and staminate flower, olive, pistil abortion. abstract: the formation of flowers in sufficient number and quality is a prere­ quisite for a successful subsequent fruit set. despite the abundant flowering, olive trees (olea europea) are characterized by a very low fruit set, and a very severe yield alteration leading to market fluctuation over time. the goal of this paper is to explore and analyze eco­physiological driving factors behind the poor fruit set in mediterranean olive groves. the key mechanisms causing floral differentiation and extreme yield alternate are functional of plant genetic varia­ bility, nutrient competition, and some ecological aspects as a response to cli­ mate change. additionally, olive inflorescence architecture appears to be com­ plex and can vary between cultivars; the olive flower differentiation results in a variable proportion of hermaphrodite, pistillate and staminate flowers among olive cultivars as well as across canopy positions and branches, enhancing nutri­ ent competition between flowers. self­pollination could be one of the limiting factors for increasing early fruit abscission and extreme alternate fruit­bearing. hormonal treatments to reduce alternate production in olive trees should be explored. the current review analysis shall help to improve olive grove mana­ gement, but also for breeding new cultivars more suitable for mediterranean agro­ecological constraints. ovule viability and fertilisation, and embryo sac development abnormalities should all be further investigated. 1. introduction olive (olea europea) farming began thousands of years ago in the middle east and has since spread to the eastern mediterranean; spain and italy produce more than 60% of the world’s olive oil (terral et al., 2004; herrera­caceres et al., 2017). some olive groves have been conver­ ted to alternative land uses, such as grazing, due to significant yearly pro­ duction fluctuation and significant competition for nutrient supplies, such as water (loumou and giourga, 2003). for the mediterranean region’s delicate environment, olive grove protection is becoming a priority. olea species are divided into four genetic groupings. according to traditional classification, three of them had close phylogenetic ties and genotypes (*) corresponding author: festojojo@yahoo.fr citation: mani̇ri̇ho f., 2022 ­ flower differentiation and fruiting dynamics in olive trees (olea europaea): eco‐physiological analysis in the mediterranean basin. ­ adv. hort. sci., 36(1): 53­62. copyright: © 2022 maniriho f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2021 accepted for publication 26 january 2022 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-12444 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 53­62 54 that were similar to wild olive genotypes (angiolillo et al., 1999; contento et al., 2002). olea is a genus in the oleaceae family with approximately twenty spe­ cies that thrive in tropical and subtropical climates throughout five continents. based on the taxonomic position of the genus olea and of its components, four subgenera (subg.) can be distinguished: subg. olea section olea, subg. olea section ligustroides, subg. paniculate and subg. tetrapilus (besnard et al., 2002; green et al., 2004). based on morphology and geographical distribution, o. europea should be divi­ ded into six subspecies based on morphology and geographic distribution, including: (1) subspecies europea, which includes the two botanical varieties, which are europea (cultivated olive) and sylvestris (wild olive), and is widely distributed throughout the mediterranean region; (2) cuspidate subspecies, which is found in southeast asia, southern china; and the arabian peninsula in the east and south (contento et al., 2002; rugini, 2016). the o. europea l. is a frost­sensitive subtropical evergreen indigeno­ us to the mediterranean region. it has adapted to a semi­arid temperate climate with well­drained soils, a moderate to low ph (below 8.5), and little salinity in the soil (terral et al., 2004; doveri and baldoni, 2007).the olive tree is notable for its abundant bloom, which is followed by a low fruit set and a low yield. apart from inflorescence structure, cultivars differ substantially in the proportion of a hermaphro­ dite (bisexual) to staminate flowers on inflorescences (guevas and polito, 2004). about approximately 10% to 15% of a mature tree’s flowers set fruit, with only 2% to 5% of them developing mature fruits, depen­ ding on the location and type (reale et al., 2006). low fruit set and the shift from non­functional her­ maphrodite flowers to fully functional staminate flo­ wers seems to be the most important and limiting factors for olive tree productivity. the formation of functional staminate rather than entirely functional hermaphrodite flowers throughout development is one of the primary factors determining the fruit set­ ting level of olive flowers (reale et al., 2009). although various studies have been done on the dif­ ferentiation process of olive flowers, there have been very few specialised investigations to document and compare biological flower development and fruit set research among cultivars in this area. olive farming has been an important part of mediterranean nutriti­ on, therapeutic body care, economics, and religious rites for millennia (angiolillo et al., 1999; rugini, 2016; fao, 2020). the olive fruit is eaten and pressed for its seed oil, which has been shown to have health­ promoting properties when consumed regularly and can be stored and consumed for up to three years if stored properly (besnard and bervillé, 2000). the oil is used to make soaps, hair conditioners, massage oils, and other therapeutic products. in terms of nut­ ritional value, as well as economic importance to national economies, they are a necessary food. olive fruits have been demonstrated to aid in the preventi­ on of coronary heart disease and various cancers due to their high level of monosaturated fatty acids and phenolic compounds (terral et al., 2004). despite the fact that olives have been cultivated for many years and various cultivars have been domesticated for their fruit quality, morphological and physiological properties, the mediterranean region’s cultivar selec­ tion has not fully responded adequately to alternate fruiting and poor terminal fruit sets (connor et al., 2014). 2. physiological factors driving olive fruit setting pistil abortion during olive flowering the production of functional staminate flowers rather than completely functional hermaphrodites, which are unable to yield fruit, is one of the primary reasons limiting fruit set in olive (olea europaea l.) (reale et al., 2009; newton et al., 2014). despite their extensive history, many key questions surroun­ ding olives remain unanswered. while there are up to 2,600 different olive cultivars (rugini and lavee, 1992), many studies on diversity within o.europea have focused on morphology and agronomic behavi­ or, with little research on variety within the olea germplasm to yet. low fruit set is frequent and varies between olive cultivars (newton et al., 2014). pollen flow (guitian, 2006), resource availability (terral et al., 2004), predation, environmental stress, or gene­ tic stress are all possible causes (newton et al., 2014). the olive tree has a low ultimate fruit set due to a high rate of undeveloped pistils later leading to its abscission (chiappetta et al., 2015). the physiolo­ gical differentiation processes are driven by competi­ tion for resources between growing vegetative and reproductive organs (dixon, 2012; erel, 2016). according to cytohistological observations of stami­ nate and hermaphrodite flowers, after the megaspo­ re mother cell develops, the pistil development in staminate flowers is halted (guitian, 2006; chiappetta et al., 2015). biochemical studies demons­ maniriho ‐ dynamics of olive trees flowering and fruit set in mediterranean basin 55 trated that starch granules were only discovered in the ovary, pistil, and stigma of hermaphrodite flo­ wers at this time. the pistils of staminate flowers did not contain any substantial amounts of starch (wiens et al., 1987; seifi, 2015). the findings reveal a subs­ tantial connection between starch content and pistil formation (chiappetta et al., 2015). the low chlo­ rophyll content of the gynoecium, the absence of rubisco activity in the pistils of these two flower types, and the ultrastructure of the plastids discove­ red by transmission electron microscopy research all point to a secondary source of starch within the flo­ wer(reale et al., 2009; erel, 2016). in olive varieties, the percentage of hermaphrodite to staminate flo­ wers is also significant and varies (cuevas and polito, 2004). fruit success appears to affect the gender of flowers in distal positions along the inflorescence, as seen by the gender pattern of flower buds in inflores­ cences with varying fruit placements. other aspects of andromonoecy in caesalpinia species, such as flo­ ral sex ability and fruit set impacts, are examined (terral et al., 2004; seifi, 2015). previous research used potency spectral analysis to look for all possible periodic patterns in yield data; and concluded that even with a fairly regular biennial succession, two or more “on” or “off” years can be observed. staminate flowers are produced by andromonoecious species to boost reproductive success by increasing male functi­ on or redirecting resources away from useless pistils and onto fruits (huang, 2003). biennial fruit‐bearing in olive trees alternate fruiting and determining factors are cri­ tical in olive trees. olive crop productivity has chan­ ged greatly over the years, due to the problem of alternating fruit­bearing that has been affecting the trade and consumption in the olive producing coun­ tries. many fruit trees, such as olive trees, may not yield the same crop year after year, with mild to sig­ nificant annual variations (rallo et al., 1993; seifi, 2015). in recent decades, the issue of alternating bearing in fruit trees has received a lot of attention, because of the huge swings in production, the alter­ nating bearing is a common phenomenon in many fruit tree species, causing tremendous labor, marke­ ting, and economic instability (fig. 1). the concept of “alternate” or “biannual” bearing is defined as the production of heavy fruit “in” one year, followed by light or no fruit the next year (monselise and goldschmid, 1982). the development of trust worthy metrics to quantify biannual alternation, its severity (maximum and average deviation from multi­year norms), synchrony at different sites, and other cha­ racteristics are clearly of importance. monselise and goldschmid (1982) presented two parameters for evaluating fruit production yearly alternation). the first parameter “b” denotes the proportion of bienni­ al, while the second one “i” denotes the amplitude of volatility. three aspects of olive reproductive biology were investigated by rallon et al. (1993): the biannu­ al cycle, the effect of cultivation on successive repro­ ductive and vegetative processes, and the effect of temperature on bud dormancy are all discussed. even though appropriate horticultural techniques such as sufficient pruning, thinning, irrigation, etc. are implemented, olive trees will gradually alternate their annual yield. under good climate circumstances in the whole year, the alternate bearing will be very reduced at a given extent depending on cultivars (lavee, 2007). a quantitative study relating cultivati­ on to various indices of successive reproductive and vegetative processes revealed that while inhibition of floral induction by developing plentiful fruits is the main factor for biennial bearing, the successive rep­ roductive processes are also important, especially fertilisation stage (lavee and avidan, 1993). rosati et al. (2011) also investigated the effect of nutritional conditions on olive biennial fruiting. the researcher concluded that flowers induction particularly her­ maphrodite flowers formation are negatively affec­ ted by flowering load (erel et al., 2016). furthermore, biochemical studies have been conducted in an attempt to explain the alternate bearing in olive pro­ duction. the protein content and their composition of “on” and “off” olive trees, especially the protein content in the leaves and bark of one­year­old sho­ ots, have a critical effect on the olive annual produc­ fig. 1 ­ variability of olive world production over 10 years. adapted from faostat (fao, 2020). adv. hort. sci., 2022 36(1): 53­62 56 and that genetic differences in pistil abscission bet­ ween olive cultivars can be explained by changes in pistil mass and sink strength (zilberman et al., 2007; song et al., 2014). addionnaly, flower bud induction is a long­term process in the olive tree that is control­ led by a variety of internal and external stimuli. marone and fiorino (2010) conducted experiments to identify the meristems that give birth to various types of shoots, as well as the fundamental mecha­ nisms influencing the evolution of the apical meris­ tem and its lateral buds. observations led to the dis­ covery of a vertical succession of two types of buds in the same bud complex: the “main” bud and, in the upper position, the “accessory” bud, the former having a reproductive function and the latter speciali­ zed in environmental exploitation (vegetative role). they concluded that the generation of new bearing vegetation is confined to the central leader in “matu­ re” shoots, and all branches arising from accessory buds are committed to burst new vegetation. the fin­ dings by fabbri and benelli (2000) also backed the theory of the two­step induction leading to flower bud differentiation, which appears to begin around the end of fall. on the otherhand, the impact of sea­ sonal variations in the phenolic content of olive culti­ var’s leaves (olea europaea l) on the cultivar’s alter­ nating bearing have been suggested to be associated to the alternatate bearing too. in 2008 (off year), mert et al. (2013) reported a substantial variations in the amount and distribution of these phenolics in the leaves (on year). chlorogenic and p­coumaric acids were abundant in the “on” year, but other phenolic compounds were scarce. the chlorogenic and p­cou­ maric acid levels were low during the “off” year, while the levels of the other phenolic acids were high. in the “on” and “off” years, the same authors discovered a negative connection between chloroge­ nic acid and caffeic acid concentrations: caffeic acid levels were high, while chlorogenic acid levels were low. comparing the “on” and “off” years, they con­ cluded that the contents of chlorogenic acid, caffeic acid, 3­h ydroxycinnamic acid, and p­coumaric acid were considerably different. ovary abortion and incomplete embryo development the findings back up the theory that pistil aborti­ on is linked to ovaries competing for resources, and they imply that genetic variations in pistil abortion between olive cultivars might be explained by chan­ ges in pistil mass and sink strength (ji et al., 2010; rosati et al., 2011). in andromonoecious species, pis­ tion (lavee and avidan, 1993; eris et al., 2007), in koronaiki, uovo de piccione, manzanillo, and barnea olive cultivars. the overall amount of useable protein in the “off” trees’ leaves was substantially lower than in the “on” plants’ leaves, while the bark revealed an antithetical relationship. the koronaiki cultivar was the least alternative, presenting the smallest variance in its leaves. in contrast, other cultivars had a compa­ rable percentage of proteins content in their bark. additionally, cuevas and polito (2011) observed that the “off” trees of all cultivars had more crude protein in their bark than the “on” trees of the same culti­ vars. a 66 crude protein was more expressed in the bark of the “off” trees, rather than “on” years. however, rosati et al. (2011), found that differences in some proteins were smaller in the leaves of “on” and “off” trees (rosati et al., 2011). endo‐genetic factors the olive crop (olea europaea) is a genetically diverse fruit in horticulture (kour et al., 2018). cytosine methylation is an essential epigenetic regu­ lator of transposon silencing, heterochromatin orga­ nization, genomic imprinting, and gene expression, according to an investigation of endogenous factors (zhang et al., 2006). thus, all flowers in andromomo­ nocioeus plants are claimed to be bisexually started, despite the presence of spatial patterns within inflo­ rescences and plants (hamanishi and campbell, 2011). in addition to the biochemical difference, cuevas and polito (2004) confirmed that the dry weight of hermaphrodite flowers was 19% larger than the dry weight of staminate blooms begun at similar places on the panicle. because there were no significant differences in stamen weight, the author hypothesized that this discrepancy was mostly attri­ butable to pistil and petal weight. delph (1997), on the other hand, found no significant differences bet­ ween staminate and hermaphrodite flowers in pollen amount per anther or pollen quality, as evaluated by viability, germination, and ability to fertilize other flo­ wers. furthermore, no link between gender and ant­ hesis timing was discovered. on the other hand, sedgley and griffin (2013), found that the flower’s position inside the panicle was linked to anthesis timing and gender. the blooms on the tip and major pedicels were hermaphrodite and opened first, whilst flowers on subsidiary pedicels were mostly staminate and achieved anthesis last. in summary, the majority of the findings support the notion that pistil abscissi­ on is linked to resource competition among ovaries, maniriho ‐ dynamics of olive trees flowering and fruit set in mediterranean basin 57 til abortion is thought to be an evolutionary adaptati­ on to save resources by balancing the quantity of pis­ tils with the resources available. as a result, pistil abortion is likely to be higher in large­fruited varieti­ es. rosati et al. (2011) working with olive cultivars with varying ovary/fruit mass discovered that pistil abortion, represented as a percentage of staminate flowers, was positively associated with the average ovary mass at bloom. furthermore, both ovary mass and pistil abortion were inversely linked with the number of perfect flowers per inflorescence, where­ as both factors increased the number of staminate flowers per inflorescence (famiani et al., 2019) (fig. 2). thus, the above researchers, among others, sug­ gested that the leaf­bud ratio, as well as the amount of leaves present for each inflorescence bud, are two factors that influence pistil abortion in olives (fig. 2). the olive flowers will develop poorly if the number of leaves decreases; and the number of aborted pistils will rise as the number of leaves decreases. 3. ecological factors nutrients resources distribution and plant nutrition factors a variety of alterations in the activation and inhi­ bition of endogenous metabolic pathways are invol­ ved in the manifestation of alternative bearing. pistillate abortion, known as andromonoecy, which refers to the generation of both perfect (hermaphro­ ditic) and staminate blooms, is thought to be influen­ ced by resource competition (haberman, 2019). according to rallo et al. (1993) and cuevas and polito (2011), the high variation in the proportion of stami­ nate flowers observed in olive trees, branches, sho­ ots, and even inflorescences within the same shoot, could be part of a general reproductive strategy that adjusts maternal investment in gender expression in response to available resources and environmental conditions. if nutrient shortages cause increased pis­ tillate abundance and staminate flower develop­ ment, solomon (1985) and emms (1993) suggest that the nutrition deficiency could also alter pollen output or pollen quality. if this is the case, staminate flowers would be regarded as a result of nutritional defici­ ency, and andromonoecy would be regarded as a process of partial flower abortion (lavee and avidan, 1993; cuevas and polito, 2011). in contrast, if stami­ nate flowers benefit from the resources conserved by pistillate miscarriage, we should expect more pollen grains or higher pollen performance from staminate flowers than from hermaphrodite flowers (song et al., 2012). vining et al. (2012) and guevas and polito (2013) investigated the synthesis and use of car­ bohydrates linked with regular and alternation frui­ ting in the olive production cycle. male flowers are more likely to appear on the less fed secondary pedi­ cels, where fruit set is unlikely to happen; while her­ maphrodite flowers are more likely to form on the apex and major pedicel of the inflorescences. however, some studies have found that flower positi­ on has no effect; for example, in the ‘mission culti­ var’, the fate of a certain floral meristem is not mixed, and gender cannot be firmly attributed to a certain inflorescence location (delph, 1984; fig. 2 ­ illustration of some olive flowers development biological stage by a light microscope: a longitudinal view of hermaphrodite flower before anthesis (i), a hermaphodite flower with a degenerating (ii), an aborted pistil (iii), a transverse view of an ovary expansion with one functional ovule and 3 others degenerating (iv) electron micrograph of the phytoprotective film of chi­pyro­ film, after spraying on a smooth surface at a temperature of 18 to 25°c (a and b). chitosan strawberry leaf covered with a film formed by chi­pyro­film (c). 58 adv. hort. sci., 2022 36(1): 53­62 haberman, 2019). despite the fact that staminate flowers originate in less desirable locations, the findings showed that the conditions that cause pistil abortion have no effect on their function. in fact, nitrogen tended to improve blooming intensity but not floral quality; nevertheless, it consistently reduced fruit set. the amount of phosphorus in the soil was linked to the creation of beautiful blooms and fruit set. potassium supplementation had a minor influence on olive yield (erel et al., 2013). pollen grains from staminate flo­ wers, on the other hand, do not profit from the transfer of resources saved by pistil abortion, as emms (1993) discovered. because sexual reproducti­ on resources are limited, it is sometimes argued, on theoretical grounds, that an increase in resources given to male function comes at the expense of reso­ urces dedicated to female function, and vice versa (delph, 1984). furthermore, guevas and polito (2003) observed that in hermaphroditic flowers, lar­ ger resource allocation to pistils occurs, while stamen dry weight does not rise in staminate flowers in res­ ponse to reduced pistil allocation. the positive asso­ ciation discovered in hermaphroditic flowers betwe­ en petal, stamen, and pistil dry weight shows that conditions that favor pistil development also favor resource investment in stamens and petals (lavee and avidan, 1993).plants do not shift resources from the pistil to the stamens in staminate flowers, accor­ ding to patterns of resource allocation to floral organs in other andromonoecious species (lavee and avidan, 1993; emms, 1993). reale (2009) showed that nutritional deficits can impact pollen production or pollen quality, in addition to increased pistil abor­ tion and generation of staminate flowers. if this is the case, staminate flowers would be regarded as a result of nutritional deficiency, while andromonoecy would be regarded as a simple process of incomplete floral abscission. even though, staminate flowers gain from the resources saved by pistillate miscarriage (song et al., 2012), we should expect more pollen grains or greater pollen performance from staminate flowers compared to hermaphrodite flowers. in this situation, the ailment could be viewed as a precursor to monoeciousness (vining et al., 2012). during the annual and bennial cycles, there are claimed to be dramatic changes in the carbohydrate components of leaves. sugars and starches are substantially higher at the start of a bearing year than at the start of a non­bearing year, according to duyvelshoff (2011), polysaccharides are extensively hydrolyzed througho­ ut winter. low temperatures and good flower induc­ tion seem to be linked to a high carbohydrate con­ tent (rosati et al., 2006). the influence of seeds on floral induction in growing fruits has also been under­ lined. seed­produced auxin has been seen moving from the seed to the fruit spur. in a biennial cultivar (laxton’s superb), the mobility is larger than in a regular bearing cultivar (‘cox’s orange pippin’) (zhang, 1993). long ago, it was suggested that seeds may deprive a key metabolite essential for flower ini­ tiation (duyvelshoff, 2011). the function of nutrition in pitillate abortion in olives revealed that high leaf/flower ratios and nitrogen fertilization increase hermaphroditic flower formation, which is consistent with feminization trends in andromonoecious plants developing under favorable climatic conditions (solomon, 1985; rosati et al., 2006). as a result, the wide range of staminate flower proportions observed in olive across years, trees, branches, shoots, and even inflorescences within the same shoot could be part of a general reproductive strategy that adjusts maternal investment in sex expression in response to available resources and environmental conditions (rosati et al., 2006). the latter stated that high amo­ unts of metabolites or photosynthates may be asso­ ciated with high hormone levels, which is consistent with prior findings by durand (1990) and frankel and galun (2012), who determined that hormone con­ centration and sex expression in plants had a close relationship. female flowers grow near younger lea­ ves, which have high auxin levels, according to the same authors. the favorable effect of phosphorous on female reproductive development was indepen­ dent of total carbohydrate availability, according to erel (2016) in his study on the influence of phospho­ rous nutrient levels on reproductive development. as a result, the researcher hypothesized that phosphorous nutrition had a favorable influence on productivity measures that was unrelated to car­ bohydrate reserves or carbohydrate transit to the developing inflorescence. in addition, fernández (2009) and reale (2009) discovered that phosphoro­ us nutrient levels were connected to the rate of rep­ roductive bud burst, inflorescence weight, rate of hermaphrodite flowers, pistil weight, fruitlet persis­ tence, fruit set, and the overall number of fruits pro­ duced. pollen viability was consistently high in phosphorus deficient trees, the authors reported, presumably due to higher carbohydrate availability, maniriho ‐ dynamics of olive trees flowering and fruit set in mediterranean basin 59 in contrast to female reproductive organs. environmental factors according to ecophysiology research, the degree of alternance bearing in fruit production is strongly reliant on environmental variables and can vary signi­ ficantly between growing regions depending on cli­ mate (terral et al., 2004; fernández, 2015). a rese­ arch done on the influence of climatic conditions on the ratio of hermaphrodite to male flowers and fruit set, in regular and alternating olive varieties, found out that different inductive circumstances are crea­ ted by harsh climate conditions. in seasons with a lot of blooms, staminate flowers are plentiful and the number of flowers per inflorescence is fairly low (erel et al., 2013; erel at al., 2016).even among the same cultivar at different locales, the effects of tree age have been studied as debatable and presumably not uniform (song et al., 2014). the alternation pheno­ mena can be triggered by environmental factors. because of its self­sustaining features, cyclic activity can last for years after it is started (emms, 1993). different tree species, as well as the same tree speci­ es cultivated in various climates, may have different relevant conditions. the same tree species can be cultivated in a variety of environments, such as irriga­ ted vs. arid culture, somewhat warm and humid vs. hot and dry summers, overcast vs. bright days, and so on. conditions that do not trigger in one zone may become triggers in another or for different trees wit­ hin the same zone (hamanishi and campbell, 2011). monselise and goldschmid (1982) investigated the impact of plant size and light intensity interactions on sex expression. the percentage of female and her­ maphrodite flowers was highest when large plants were exposed to full sunshine. that is, when high light intensity was combined with a big plant size, the percentage of female and hermaphrodite flowers was larger than when each component was used alone (karapatzak et al., 2012). furthermore, it was discovered that both high light intensities and plant size increase female flowering. “plants that are culti­ vated in full sunlight, with appropriate hydration, and embedded in an appropriate substrate are sturdy and produce female flowers in the majority of cases. male flowers are produced by less vigorous plants that are frequently planted in the shade or lack eno­ ugh nutrients (fernández et al., 2009). when the olive is cultivated in a greenhouse at a minimum tem­ perature of 16°c and a maximum temperature of 27­ 30°c, flower production is absolutely suppressed, alt­ hough it does occur when cultivated in california during the winter. he also came to the conclusion that morphological alterations in the bud are linked to an increase in blooming, which is regulated by the treatment time (terral et al., 2004).the classic exam­ ple of the change in woody plants throughout a large climatic area relates to climatic factors that can trig­ ger the biennial cycle. spring frosts in deciduous trees, for example, or unusual drought stress during the set in warm locations, are examples of such trig­ gers. outside of the cycle, however, it is normal for woody plants and trees to swap places with their neighbors. individual branches could even be out of sync with the rest of the tree (monselise and goldschmidt, 1982). to some extent, the effects of temperature on flowering, fruit set, and fruit deve­ lopment have been examined. due to influences on pollen germination and pollen tube expansion in the fertilization process, high day and night temperatures (300oc/200oc) during the growing season might impair yield potential, resulting in flower death follo­ wing anthesis (karapatzak et al., 2012; haberman, 2019). 4. conclusions despite the fact that olive farming has been prac­ ticed in the mediterranean agricultural ecosystem since ancient time, the productivity of some olive groves is hampered by the floral divergence leading to the low fruit set. on one hand, the pistil abscissi­ on can be viewed as an evolutionary response in the mediterranean ecology that balances pistil numbers with available resources. on the otherhand, benefit of developing staminate flowers in olive trees can also be seen as adaptative strategy to increase male flowers activity and dispersal efficiency, as well as to boost pollinator attraction. thus, in olive trees, the transition from hermaphrodite to staminate flowers can be regarded as a strategy to maximize the ability of male flowers by allocating biochemical plant reso­ urces to male and female tasks in the most efficient way possible. pollination fitness and additional reso­ urces are provided for the reduced number of pistil growth, if the optimal number of staminate flowers exceeds the number of pistils. however, further rese­ arch should be done on how to balance the ecologi­ cal flower adapatability and the olive productivity. thus, there is a needto adjust fthe variability of the ratio between hermaphrodite and staminate flowers, adv. hort. sci., 2022 36(1): 53­62 60 as well as the histological structural study that goes along with it. hormonal treatments to reduce alter­ nance production in olive trees should be also explo­ red. ovule viability and fertilisation, as well as embryo sac development during flower differentiati­ on and fruit abscission, should all be further investi­ gated too. lastly, cross­effect pollination’s on fruit value between olive cultivars, as well as the synchro­ nization of flowering and fruit set, in the mediterranean ecology could be also of great inte­ rested to improve olive productivity. acknowledgements special appreciation to all european university academic personnel and to the field technicians at the eul research farm for their valuable assistance. speciall thanks also to university of rwanda, for a study leave and financial support during the stay at eul. references angi̇oli̇llo a., mencucci̇ni̇ m., baldoni̇ l., 1999 ­ olive genetic diversity assessed using amplified fragment length polymorphisms. ­ theor. appl. gen., 98(3­4): 411­421. besnard g., bervi̇llé a., 2000 ­ multiple origins for mediterranean olive (olea europaea l. ssp. europaea) based upon mitochondrial dna polymorphisms. ­ comptes rendus de l’académie des sciences­series iii­ sciences de la vie, 323(2): 173­181. chi̇appetta a., bruno l., muzzalupo i., 2015 ­ omics approaches for the characterization and valorisation of olive varieties. ­ agricultural and food biotechnology of olea europaea and stone fruits. bentham, sharjah, pp. 75­108. connor d.j., gómez­del­campo m., rousseaux m.c., searles p.s., 2014 ­ structure, management and pro‐ ductivity of hedgerow olive orchards: a review. ­ sci. hortic., 169: 71­93. contento a., ceccarelli̇ m., gelati̇ m., maggi̇ni̇ f., baldoni̇ l., ci̇oni̇ni̇ p., 2002 ­ diversity of olea genotypes and the origin of cultivated olives. ­ theor. appl. gen., 104(8): 1229­1238. cuevas j., poli̇to v.s., 2004 ­ the role of staminate flo‐ wers in the breeding system of olea europaea (oleaceae): an andromonoecious, wind‐pollinated taxon. ­ annals of botany, 93(5): 547­553. delph l.f., johannsson m.h., stephenson a.g., 1997 ­ how environmental factors affect pollen performance: ecological and evolutionary perspectives. ­ ecology, 78(6): 1632­1639. di̇xon g.r., 2012 ­ climate change‐impact on crop growth and food production, and plant pathogens. ­ canadian j. plant path., 34(3): 362­379. doveri̇ s., baldoni̇ l., 2007 ­ olive, pp. 253­264. ­ in: kole c. 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transcription. ­ nature genetics, 39: 61­69. impaginato 195 adv. hort. sci., 2021 35(2): 195­205 doi: 10.36253/ahsc­10277 zucchini squash production in conven­ tional and organic cultivation systems s. toscano 1, f. branca 1, a. ferrante 2 (*), d. romano 1 1 department of agriculture, food and environment, università degli studi di catania, via s. sofia, 100, 95123 catania, italy. 2 department agricultural and environmental sciences, università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: cucurbita pepo l., fruit quality, protected cultivation, sustainability, water use efficiency, yield. abstract: organic production must be carried out following eu regulations and protocols. on the contrary, conventional cultivation instead can be carried out using the best agronomic approaches available and using the latest innovative resources. organic cultivation is more widespread in permanent crops (olive and grape crops) than vegetable ones, and even less in protected cultivation systems, due to the high intensity production processes which render the appli­ cation of organic growing protocols more complex. the comparison between the two systems of cultivation, organic and conventional, is difficult because the two cultivation methods are often carried out in different farms and hence in different environmental conditions. cultivation using the two methods was conducted in a greenhouse from november to march 2017/2018. results demonstrated that the total fruit yield zucchini squash in organic cultivation was not significantly different to the conventional one (43.2 mg ha­1 and 46.4 mg ha­1, respectively). the agronomic inputs (fertilizers, fungicides, and insecti­ cides) were higher in the organic cultivation system than conventional one. the water use efficiency was higher in the conventional cultivation system (150.6 kg m­3 ha­1) compared to the organic one (147.6 kg m­3 ha­1). no statistically signifi­ cant differences were found for the fruit number per plant and for the mar­ ketable fruit at the end of the growing period. significant differences for the harvest period were only detected for fresh weight, shape index, firmness, and titratable acidity. in conclusion, this work demonstrated that the organic sys­ tem required higher inputs compared to the conventional cultivation. the extensive experience of the grower allowed for comparable yields between the two systems. 1. introduction both the demand for organic vegetables and cultivation areas have been increasing following market demand. environmental benefits claimed by organic producers clearly contributed to building a positive consumer attitude towards organic. the primary request for organic pro­ duce is the absence of pesticide residuals or the presence of any agro­ chemical that is not allowed in the eu­defined organic protocols in regula­ tion n. 834/2007 and 889/2008, and more recently, n. 848/2018. organic (*) corresponding author: antonio.ferrante@unimi.it citation: toscano s., branca f., ferrante a., romano d., 2021 ­ zucchini squash production in conven‐ tional and organic cultivation systems. ­ adv. hort. sci., 35(2): 195­205. copyright: © 2021 toscano s., branca f., ferrante a., romano d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. author contributions: all authors contributed equally to the work. competing interests: the authors declare no competing interests. received for publication 31 december 2020 accepted for publication 21 may 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10277 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 195­205 196 farming has become the fastest­growing agricultural sector, accompanied by a constantly increasing con­ sumer demand for organic produce. the organic agri­ culture accounts for approximately 37.3 billion € in the european union (eu) and it is the second­largest single organic market in the world. in 2018, the coun­ tries with the largest organic agricultural areas were spain (2.2 million hectares), france and italy (2.0 mil­ lion hectares each) (willer et al., 2020). in europe, the area dedicated to organic farming has increased in recent years, reaching 15.6 million hectares (willer et al., 2020). the distribution of organic farmland by location is 15% in spain, 12% in italy, 11% in france, and 8% in germany (brzezina et al., 2017). the total area of organic vegetables represents only 0.6% of the total area devoted to vegetables worldwide. europe, with 184,373 ha, represents 47.6% of the total area and ranks first worldwide, fol­ lowed at a distance by north america (73,238 ha). italy with 60,732 hectares is at the second place worldwide after the united states (willer et al., 2020). quality and safety of organic produce must be guaranteed at harvest and during postharvest. the restricted use of pesticides and fungicides, and the predominant use of organic matter for fertilization during cultivation may increase the microorganism contamination of the product. the organic rules are stringently applied to vegetable production because the growing cycles are short and the rapid turnover of crops requires frequent soil tillage, with a negative effect on the soil structure and organic matter con­ tent. soil fertility can be maintained by frequent organic material supply and adequate crop rotation (watson et al., 2002). organic agriculture is considered one of the best alternatives for sustainable and good quality food production (aninowski et al., 2020). the comparison between organic and conventional cultivation sys­ tems is very difficult to perform because the agro­ nomic management and strategies cannot be the same. organic vegetable production must follow eu regulations, which outline specific protocols to follow and the agronomic choices are limited. therefore, organic production is sometimes difficult, especially in environments with high levels of biotic stresses such as pests and diseases (raigon et al., 2010). agronomists can use only organic certified products and exploit the positive interactions among crops for controlling pests and diseases and for plant nutrition. the conventional vegetable cultivation system has a wide range of choices and the experience of the agronomist can play an important role in increasing the yield and quality of the products. conventional cropping systems follow innovative technologies and year­by­year, new hybrids or cultivars can be adopt­ ed as well as new fertilizers, plant growth regulators, pesticides, etc. (odegard and van der voet, 2014). in organic cropping systems, the nutrients must be provided by certified organic fertilizers or through appropriate crop rotations (thorup­kristensen et al., 2012). in long term organic vegetable cultivation, the nutrients are provided by manure or by catch crops and intercrops. however, organic farming is strictly regulated by rules and laws thus allowing a better comparison of its performances with conventional farming methods, with and without the use of agro­ chemical inputs and/or the adoption of specific grow­ ing practices (gomiero et al., 2011). organic farms which specialize in vegetable pro­ duction have more difficulty compared to farms involved in livestock and mixed production. these difficulties are represented by the lack of manure produced in the farms and the supply of organic mat­ ter must be provided by green manure that repre­ sents a loss of a cultivation cycle. in many studies (raviv, 2010; campanelli and canali, 2012; rahmann et al., 2017) the great majori­ ty of organic systems are refer to open field condi­ tions; only recently such alternative organic systems of production have been tested in protected condi­ tions (tittarelli et al., 2017). in fact, organic green­ house production is still a small sector of the organic industry and constitutes only a small proportion of total greenhouse production (gamliel and van bruggen, 2016). the different studies that have compared organic and conventional production systems have provided inconsistent results with regard to the sensorial qual­ ity and nutritive value of fruits (bourn and prescott, 2002; lester, 2006; zhao et al., 2006) but organically grown foods have lower pesticide residues (trewavas, 2004). this is not surprising because com­ paring the effect of organic and conventional farming systems on fruit quality is inherently difficult due to the wide range of factors that can potentially affect crop composition such as climate, soil conditions, cul­ tivar, soil type, planting date, harvesting time, and growing seasons (goldman et al., 1999; adam, 2001; magkos et al., 2003). in other studies, however, where organic vegetables were compared to conven­ toscano et al. ‐ conventional and organic zucchini squash 197 tional ones, a higher concentration of health promot­ ing components has been found (brandt and mølgaard, 2001; rembialkowska, 2003, 2007). rembiałkowska (2000) found a higher content of total sugars in organically produced vegetables (car­ rots, sugar beet, red beetroot, potatoes, spinach, savoy cabbage). hallmann (2012) showed that organ­ ic tomatoes presented a higher ratio of reducing sug­ ars/organic acids, and contained significantly more total sugars, vitamin c and total flavonoids, 3­ quercetin rutinoside, and myricetin in comparison with the conventionally­grown fruits. the main difference between conventional and organic cultivation systems is that conventional agri­ cultural systems are continuously evolving due to the introduction of innovative techniques, while organic cultivation must follow fixed protocols that are revised at an interval of several years. in general, organic farming is represented by an articulated series of variables related to the biotic and abiotic factors affecting growth and the final product (lester and saftner, 2011). the physical, chemical and bio­ logical/nutritional attributes of soils, the irrigation management and water quality, the crops/genotypes and the growing cycles, the harvesting, handling and storage methodologies are the main variables which affect organic and conventional produce quality. zucchini squash and pumpkins within the three major species of cucurbita are important crops worldwide. in the mediterranean region, and in par­ ticular in italy, zucchini squash (cucurbita pepo l.) is an important commercial crop, both in the open field and in the greenhouse. zucchini squash is generally cultivated in soil under greenhouse conditions for off­ season production, but in the last years soil­less culti­ vation has been strongly developed because it improves the product quality and increases plant defenses against diseases (van os et al., 2002). for these reasons, greenhouse zucchini crops are usually cultivated during two growing seasons (spring­ summer and summer­autumn seasons) to respond to the high demand for this fresh vegetable in nation­ al and international markets (rouphael and colla, 2005). the aim of this work was to compare the produc­ tivity and inputs (fertilizers, insecticides, and fungi­ cides) of conventional and organic zucchini squash cultivation systems carried out in a greenhouse. both farms were in the same geographical area allowing for a comparison under reduced environmental inter­ ferences so that differences could be attributed to the crop management systems. 2. materials and methods greenhouse conditions zucchini squash (cucurbita pepo l.) ‘sibilla’ was grown under conventional and organic procedures, commonly adopted in the sicily region for zucchini squash production. the experiment was conducted in 2017/2018 in two 240 m2 unheated polyethylene tunnels located in syracuse (36°59.1’ n, 15°12.6’ e, 30 m above the sea level), sicily, italy: one devoted to organic (20 years under organic regime) and other to conventional horticulture systems. plants were grown under natural light conditions. the mean tem­ perature was 16.5 °c and the mean relative humidity levels were 75.5%. the total radiation levels ranged from 4.5 to 14.6 mj m­2. zucchini squash seedlings were transplanted at the two­leaf stage on 2nd november 2017 for both methods of cultivation, in rows 1.1 m apart, with an along­row spacing of 0.8 m, giving a planting density of 0.88 plant m­2. preliminarily, bottom fertilization was performed with cattle manure at a dose of 1500 kg ha­1 in organ­ ic system and a total amount of phosphorus (p2o5 as triple super phosphate), and potassium (k2o as potassium sulfate) and one­third of the nitrogen (as ammonium sulfate) were applied for the convention­ al cultivation system. specifically, 120 kg ha­1 of n, 45 kg ha­1 of p2o5, and 265 kg ha­1 of k2o were added. the following products were used during the preparation of the soil and cultivation of the plants: organic cultivation system system pre­trasplant: siveg gr (biolchim s.p.a.); 6: 6: 12 orga kem (biolchim s.p.a). after transplanting: nov@ (biolchim s.p.a.); folicist® (biolchim s.p.a.); zsdqdaedta zinc (biolchim s.p.a.); keliron® (biolchim); bio energy® veg (biolchim s.p.a.); glibor ca (biolchim s.p.a.); mn sul­ fate (biolchim s.p.a.); protamin cu 62 (fertilgest); mg sulphate (biolchim s.p.a.); fylloton (biolchim s.p.a.); cremalga (biolchim s.p.a.); microfol mix (biolchim s.p.a.); mg sulphate (biokimia international s.r.l.). fertigation: nov@ (biolchim s.p.a.); bio energy® veg (biolchim s.p.a.); glibor ca (biolchim s.p.a.); mg sulphate (biokimia international s.r.l.). foliar application: nov@ (biolchim s.p.a.); bio energy® veg (biolchim s.p.a.); fylloton (biolchim s.p.a.); folicist® (biolchim s.p.a.); cremalga (biolchim s.p.a.); glibor ca (biolchim s.p.a.); mg sulphate adv. hort. sci., 2021 35(2): 195­205 198 (biokimia international s.r.l.). pesticide: sulphur 95% (mannino s.p.a.). conventional cultivation system pre­trasplant: siveg gr (biolchim s.p.a.); 6: 6: 12 orga kem (biolchim s.p.a). after transplanting: phostart zn (biolchim s.p.a.); urea sulfate 70 (fertilgest); fulvumin (biolchim s.p.a.); keliron® (biolchim s.p.a.); kemical® (biolchim s.p.a.); 20.20.20 fertilizer (valagro s.p.a); protamin cu (fertilgest). foliar fertilizations: microfol® mix (biolchim s.p.a.); urea sulphate low biuret (fertilgest); nitrocam® (biolchim s.p.a.); loker® (biolchim s.p.a.); green­go 12.8.24+10 (fertilgest); magnitron (biolchim s.p.a.); fulvumin (biolchim s.p.a.). foliar application: nitrocam® (biolchim s.p.a.); kriss (biolchim s.p.a.); rizzamina® 42 (fertilgest). pesticide: karma® 85 (certis europe, italia); tiovit® jet (syngenta italia). similar types of machinery were used in both cul­ tivation systems. the final stage of cultivation involved the harvesting of zucchini squash fruits, which was performed manually and so did not affect the relative environmental performance of conven­ tional and organic systems (table 1). organic cultivation system the zucchini squash cultivation was performed following the procedures described in eu n. 834/2007 and 889/2008. in this system siveg gr and 6: 6: 12 orga kem were applied at 35 cm depth in pre­transplant, respectively at doses of 400 and 1500 kg ha­1; after the spreading, the products were appropriately topped up. during the preparatory phase of the organic grow­ ing media, in addition to the background fertilization, biostimulants and nutrients were added according to the following scheme: ­ seventh day: nov@ (15 l ha­1), folicist® (2 l ha­1), edta zinc (2.5 l ha­1) and keliron® (2 kg ha­1); ­ twentieth day: nov@ (10 l ha­1), folicist® (1.3 l ha­1), bio energy® veg (20 l ha­1), glibor ca (3.5 l ha­1) and mn sulfate (5 kg ha­1); ­ thirtieth day: protamin cu 62 (3.5 l ha­1), mg sul­ phate (7 kg ha­1) and keliron® (3.5 kg ha­1). the following top dressings were applied 10 and 20 days after transplanting: tenth day: fylloton (1.5 l ha­1), folicist® (1 l ha­1), cremalga (1 l ha­1) and microfol mix (1 kg ha­1). twentieth day: cremalga (1 l ha­1), mg sulphate (1.5 kg ha­1) and folicist (1 l ha­1). during cultivation, fertigation and foliar applica­ tion were performed starting from the fourteenth day and at two week­intervals. the fertigation was carried out with the addition of nov@ (15 l ha­1), bio energy® veg (20 l ha­1), with glibor ca (7 l ha­1), and mg sulphate (20 kg ha­1); foliar application was performed with a solution con­ taining nov@ (15 l ha­1), bio energy® veg (20 l ha­ 1), fylloton (1.5 l ha­1), folicist® (1 l ha­1), cremalga table 1 ­ cultivation procedures (inputs) of the organic and conventional systems per hectare of squash cultivation cultivation procedures (inputs) cultivation systems organic conventional land use (m2) 10.000 10.000 mean yield (mg) 43.2 46.4 irrigation pe irrigation hoses (m) 6667 6667 submersible electric pump shankty, qf 106/9 + pump motor hp 50 8’’(h) 45 45 water (m3) 2100 2100 fruit harvesting (h) 19 19 machinery harrowing (h) 8 8 tillers (h) 8 8 fertilizer spreader (h) 5 5 spreading plastic mulching (h) 16 16 soil tillage (h) 15 15 spreading plastic tunnel coverage (h) 98 98 plastic maintenance 28 28 toscano et al. ‐ conventional and organic zucchini squash 199 manual weed control was carried out as described for organic cultivation system (fig. 1). sampling procedure and measurements the fruits were harvested every two days and those obtained at the beginning, in the middle and at the end of the harvest were transported to the labo­ ratory of the department of agriculture, food and environment science (di3a) of catania university (italy), and immediately analyzed. agronomic data and fruit physical parameter from the greenhouse experiment such as plant pro­ ductivity, fruit weight, shape index, color, thickness epicarp etc. were measured. water use efficiency (wue) was calculated as yield/water consumed (kg m­3) (yaghi et al., 2013). the epicarp and mesocarp color was measured using a chroma meter cr­200 (konica minolta, japan) based on light reflectance. the color was expressed using the commission internationale de l’eclairage (cie) system where the l*, a* and b* val­ ues represent the lightness, green­red and blue­yel­ low, respectively. the dry matter (dm) content was obtained by drying samples in a thermo­ventilated oven at 70°c to constant weight. the firmness of the zucchini squash was mea­ sured using a compression test based on the resis­ tance of the fruit to deformation in the middle por­ tion using a texture analyzer (ta.xt2i, stable micro systems ltd., godalming, uk) incorporating a 2 mm diameter probe. eighteen recordings were per­ formed for each treatment. the values were expressed as the maximum shear force (n). titratable acidity (ta) was measured by titration with a solution of sodium hydroxide 0.1 mol l­1, up to the point of phenolphthalein turning, and expressed as meq l­1 of citric acid. total soluble solids (tss, °brix) were read in a digital refractometer with auto­ matic compensation for temperature (model brix pr­ fig. 1 ­ processes included in the conventional and organic cultivation systems. products were applied on the indicated day: seventh day after transplantation: phostart zn (20 l ha­1), urea sulfate 70 (10 l ha­1) and fulvumin (15 l ha­1); fifteenth day: keliron® (3.5 kg ha­1), kemical® (15 l ha­ 1) and fulvumin (15 l ha­1); twenty­eighth day: the 20.20.20 fertilizer) (20 kg ha­ 1), fulvumin (10 l ha­1) and protamin cu (3.5 kg ha­1). in the same period two foliar fertilizations were applied; tenth day: microfol® mix (1 kg ha­1) and urea sul­ phate low biuret (5 kg ha­1); twentieth day: nitrocam® (2 l ha­1) and loker® (3 l ha­1). weekly fertigation and foliar application were applied during plant cultivation. fertigation was per­ formed with green­go 12.8.24+10 (35 kg ha­1), magnitron (15 kg ha­1) and fulvumin (10 l ha­1), until the end of crop production. foliar application consist­ ed of of nitrocam® (3 l ha­1), kriss (0.85 l h­1), and rizzamina® 42 (1.8 kg ha­1). defence against oidium was carried out weekly with the use of karma® 85 (3 kg ha­1), and tiovit® jet (0.3 kg ha­1) after 3­4 days from the foliar fertilization. (1 l ha­1), glibor ca (4.5 l ha­1), and mg sulphate (2.5 kg ha­1). manual weed control was carried out twice, to eliminate the weeds that grew during the cultivation period. the defence against oidium was carried out weekly with the use sulphur 95% (35 kg ha­1), after 3­ 4 days from the foliar fertilization (fig. 1). conventional cultivation system in this system siveg gr and 6: 6: 12 orga kem, as for the organic system, were applied in pre­trans­ plant, respectively at doses of 400 and 1500 kg ha­1; after the spreading, the products have been appro­ priately topped up. after transplanting, the following 200 adv. hort. sci., 2021 35(2): 195­205 water use efficiency and external inputs the water use efficiency (wue) was higher in the conventional than in organic cultivation system with 150.6 and 147.6 kg m­3 ha­1, respectively. in the con­ ventional production system, the amount of fertiliz­ ers used were 4.8 kg mg­1 ha­1 and 0.3 l mg­1 ha­1, solid and liquid, respectively (table 2). the organic vegetable production showed higher fertilizers input compared with conventional cultivation system, with 11.4 kg t­1 ha­1 and 0.5 l t­1 ha­1, solid and liquid, respectively. for plant protection purposes, solid fungicides were used in both growing systems. the amount of fungicides used was almost indis­ tinguishable, with 0.11 kg mg­1 ha­1 used in the con­ ventional cultivation system as compared to 0.12 kg mg­1 ha­1 applied in the organic cultivation regime. solid insecticides used were 103­fold higher in organ­ ic cultivation system compared to the conventional one, while the liquid insecticides were 4.8­fold higher in the organic compared to the conventional cultiva­ tion system. fruit quality parameters during the cultivation period, three samples of table 2 ­ productivity parameters of zucchini squash grown under different cultivation methods means within columns separated using t­test (p<0.05). fig. 2 ­ cumulative trends of zucchini squash yield (kg m­2) during the harvesting period (60 days) under conventio­ nal (•) and organic (δ) cultivation methods. 1, atago co., ltd, tokyo, japan). statistical analysis the experiment was conducted as a randomized complete­block design with three replications to compare two cultivation methods: conventional and organic. each experimental unit consisted of six plants (18 plants for cultivation methods). the statis­ tical analyses were performed using costat version 6.311 (cohortsoftware, monterey, ca, usa); pair­ wise comparisons for productivity parameters were done using t­test for means of samples with unequal variances. two­way anova for quality and color parameters was used. the differences between the means were determined using tukey’s test (p<0.05). interaction effects were calculated using the tukey’s test at a 5% level of significance. 3. results crop productivity the harvest period lasted nine weeks, with the first and last harvest dates on the 16th december 2016 and 7th march 2017, respectively; in total, 57 harvests were done during this period for both culti­ vation methods. the average harvesting interval was 1.4 days during the cultivation period. the total fruit yield of conventional zucchini squash was similar to the organic cultivation method (46.4 mg ha­1 and 43.2 mg ha­1 respectively) (fig. 2). no statistically significant differences were found for the fruit number per plant at the end of the growing period (21.5 fruits plant­1 in conventional cultivation and 22.9 fruits plant­1 in organic cultivation). similarly, no significant differences between the con­ ventional and organic cultivation were recorded for the marketable fruit yield plant: 4.1 and 3.8 kg plant­ 1, respectively (table 2). the percentage of unmar­ ketable fruit weight was 3.5% and 2.6% in organic and conventional, respectively, without significant differences. method of cultivation fruit yield (mg ha­1) wue (kg m­3 ha­1) fruit number (n. plant­1) fruit yield plant (kg) early total conventional 8.0±0.6 46.4±3.1 150.6 a 21.5±0.8 4.1±0.4 organic 7.9±0.7 43.2±2.9 147.6 b 22.9±0.9 3.8±0.4 significance ns ns * ns ns toscano et al. ‐ conventional and organic zucchini squash 201 fruits were taken for quality evaluation (one at the beginning, one approximately in the middle, and one at the end of production). significant differences for fresh weight, shape index, firmness, and titratable acidity were only detected for the harvest period (table 3). no signifi­ cant difference for dry biomass percentage was found (table 3). with regard to the total soluble solids content, the conventional cultivation method showed an effect of interaction (cultivation methods x harvesting time): the fruits of the plants cultivated using the organic method have maintained, for the entire cultivation period, higher values, while those harvested in conventional cultivation have shown a reduction at the end of the growing period (by 7%) (table 3 and fig. 3). measurements of surface color demonstrated sig­ nificant differences between the cultivation methods only with regard to l* mesocarp, which was reduced: the fruits obtained in the organic method have recorded a uniformity of the values, while those obtained in conventional cultivation method have shown lower values corresponding to the second har­ vest (table 4 and fig. 4). 4. discussion and conclusions in greenhouses, where intensive cultivation sys­ tems are widely used, the differences between con­ ventional and organic approaches are especially evi­ dent. organic production in greenhouse operations is a challenging task (gamliel and van bruggen, 2016). the production of vegetables in organic condi­ tions presents more technical challenges than con­ ventional cultivation, because many practices, such as the use of non­natural agrochemicals, are not per­ mitted in organic production under the regulations of many countries (raigon et al., 2010). consequently, organic production is sometimes difficult, especially in environments with high levels of pests and disease pressure. organic cultivation systems that are specifically dedicated to the vegetable production do not have their own manure supply and must buy a majority of their most of agronomic inputs, as such as fertilizers, biocontrol agents, natural compounds, and biostimu­ lants for controlling pests and diseases and for plant nutrition from the market. results demonstrate that table 3 ­ quality parameters of zucchini squash grown under different cultivation methods values are means of main effects of method of cultivation and harvesting period. the statistical analysis was two­way anova; ns not significant; * significant at p<0.05; *** significant at p<0.001. the values in the same column followed by the same letter are not significantly different at p<0.05 (tukey's test). fig. 3 ­ interactions between the two cultivation methods, organic (i, ii, iii ht) and conventional (i, ii, iii ht), and harvesting time on tss (brix°). the vertical bars indicate ± s.e. of means (n=18). columns denoted with the same factors fresh weight (g) shape index firmness (n) % dm tss titratable aciditymethod of cultivation (c) harvesting period (h) conventional 188.7±6.6 4.7±0.1 12.3±3.3 6.01±0.1 4.7±0.1 0.6±0.0 organic 183.5±7.8 4.6±0.1 12.5±2.7 5.99±0.1 4.6±0.1 0.7±0.0 i 158.7±3.0 b 4.6±0.1 b 11.6±1.2 b 6.00±0.1 4.8±0.1 0.7±0.0 ii 200.6±3.8 a 4.9±0.0 a 12.2±2.2 b 6.01±0.1 4.6±0.1 0.7±0.0 iii 199.0±2.5 a 4.4±0.1 c 13.4±1.4 a 6.00±0.0 4.6±0.1 0.6±0.0 significance cultivation (c) ns ns ns ns ns ns harvesting period (h) *** *** *** ns ns ns c x h ns ns ns ns * ns adv. hort. sci., 2021 35(2): 195­205 202 the grower, after years of cultivation, found a wide range of agronomic inputs that allowed for the high­ est yield that was similar to the conventional farm. in literature, it is well­known that mineral ele­ ments released by the organic fertilizers are not promptly available and the lag of time may reduce growth and yield. this problem can be compensated by higher inputs of specific organic fertilizers and biostimulants. the differences observed for crop yield between organic and conventional growing systems range from 5 to 34%, while on the average, organic cultiva­ tion can reach about 80% of the yield of conventional cultivation but with substantial variations depending on the growing system and site characteristics (de ponti et al., 2012; meier et al., 2015; ciaccia et al., 2019). some studies, however, point out that the effect of cultivation method disappears when the results are converted to absolute dry matter, because often differences are due to water content (pieper and barrett, 2009). the yield in organic cultivation system was about 30 mg ha­1 and is similar to those observed in other open field cultivation experiments with yields averaging 30.7 mg ha­1 (conti et al., 2015). colla et al. (2002) found similar results to our in tomato, with no differences in yield between the organic and conventional cultivation methods, whereas a lower yield was found in the organic grow­ ing system compared to the conventional one for zucchini (maggio et al., 2013). a strong yield reduc­ tion of about 25% was observed in summer zucchini squash grown using organic fertilizers (dasgan and bozkoylu, 2007). conventional mineral nutrition inputs can provide nutrients when plants really need them, while organic fertilizers or matrixes release nutrients following degradation kinetics that usually cannot promptly satisfy plant requirements. this long­term effect of the organic nutrient tools can slow down plant growth and negatively affect the yield. however, there is little information about nutritional and sensorial quality (aroma and volatile organic compounds between the two growing sys­ tems), and food safety of organic versus conventional crops (gennaro and quaglia, 2003). specific cultivars for organic cultivation are not available; in greenhouse tomato, the use of f1 hybrid values are means of main effects of method of cultivation and harvesting period. the statistical analysis was two­way anova; ns not significant; * significant at p<0.05; *** significant at p<0.001. the values in the same column followed by the same letter are not significantly different at p<0.05 (tukey's test). table 4 ­ color parameters (l*, a* and b*) in epicarp and mesocarp of zucchini squash grown under different cultivation methods factors epicarp mesocarp method of cultivation (c) harvesting period (h) l* a* b* l* a* b* conventional 56.8±0.1 ­27.3±0.1 10.8±0.1 63.4±0.2 b ­29.6±0.1 a 14.3±0.3 b organic 56.8±0.1 ­27.1±0.1 10.6±0.1 64.0±0.1 a ­29.8±0.1 b 14.6±0.2 a i 56.9±0.1 ­27.5±0.1 b 11.0±0.1 a 64.1±0.1 a ­30.0±0.0 b 15.1±0.1 a ii 56.8±0.1 ­27.0±0.0 a 10.5±0.0 b 63.3±0.4 b ­29.5±0.1 a 14.6±0.1 b iii 56.7±0.1 ­27.1±0.1 a 10.6±0.1 b 63.7±0.1 ab ­29.5±0.1 a 13.7±0.1 c significance c ns ns ns ** ** * h ns *** *** ** *** *** c x h ns ns ns * ns ns fig. 4 ­ interactions between the two cultivation methods, organic (i, ii, iii ht) and conventional (i, ii, iii ht), and harvesting time on mesocarp l* value. the vertical bars indicate ± s.e. of means (n=18). columns denoted with the same letters are not significantly different, as deter­ mined by tukey's test (p < 0.05). toscano et al. ‐ conventional and organic zucchini squash 203 cultivars was beneficial to the organic system, being superior to non­hybrids (santa rosa et al., 2019) as normally occurs in conventional cultivation systems. observation of consumer expectations on food quality presents the base for any successful food pro­ duction system and marketing scheme. this is also true for fruits and vegetables which are increasingly valued as an important part of the diet (péneau et al., 2006). appearance, colour, texture, and aroma are arguably the most important criteria used by con­ sumers to evaluate the immediate quality of a prod­ uct and thus, persuade them to buy it (ragaert et al., 2004). in our experiment, quality parameters were not significantly affected by cultivation systems. between the two cultivation systems, differences in fruit colour, firmness, and titratable acidity, were found in relation to the harvesting date. in analogous comparison experiments, fruit color between organic and conventional cultivation showed higher l*, a*, and b* values in the organic cultivation system. these results can be ascribed to agronomic manage­ ment techniques but also to varieties and different geographical cultivation areas (armesto et al., 2020). many studies on the quality of organic vegetables indicate a higher nutritional value and a higher con­ tent of biologically active compounds in agricultural crops from organic farming (brandt and mølgaard, 2001). in other related vegetable crops, such as tomato or pepper, it has also been found that pro­ duction under organic conditions has a significant effect on fruit composition, which normally consists of an increase in the content of antioxidants and min­ erals (chassy et al., 2006; del amor et al., 2008). for this reason, organic agriculture is considered one of the best alternatives for sustainable and good quality food production (aninowski et al., 2020). our results highlighted that the main significant changes were observed in growth parameters. the product quality was mainly influenced by environ­ mental conditions that changed according to summer weather. therefore, quality changes were visible at the different harvesting dates. it is known that veg­ etable crops have higher requirements compared to other crops and short cycles require appropriate agronomic management. the higher inputs do not always provide a better quality or higher yield in organic system. meta­analysis performed on differ­ ent crops highlighted a wide variability among crops in both organic and conventional systems. the major­ ity showed higher inputs in conventional cultivations system (seufert et al., 2012). however, the evalua­ tion of both systems can provide useful information only if the cultivations are performed in the same environments and differences can be really attrib­ uted to the agronomic managements. our results showed that zucchini squash crop can be grown in organic or conventional cultivation sys­ tems with no significant changes in fruit quality. the organic system reduced the yield even if higher inputs of agronomic tools were required for the crop management. as reported by rouphael et al. (2015), understanding the functional links between cultural factors and physiological responses is an important requisite to enhance the quality of organic products. the organic cultivation was able to give compara­ ble yield to conventional one under protected culti­ vation. almost all the analysed qualitative parame­ ters of fruits were not statistically different between the two systems, except for the tss and l­mesocarp, to underline the possibility to adopt organic proce­ dures also in greenhouse. the obtained results high­ lighted the difficulties of performing a comparison between these two cultivation systems because of the different variables that can change. our study was carried out in a geographic area where the organic cultivation for vegetable crops has been long established and the contemporarily presence of organic and conventional cultivation systems allowed us to perform a scientific and reliable study. in con­ clusion, this work demonstrated that the organic sys­ tem required higher inputs compared to the conven­ tional cultivation system. the extensive experience of the grower allowed for comparable yields between the two systems. however, further evaluations should be performed for understanding the econom­ ic and environmental sustainability of zucchini squash production in the two cropping systems. acknowledgements we would like to thank sebastiano azzarelli for his help in the field analysis. references adam d., 2001 ­ nutritionists question study of organic food. ­ nature, 412(6848): 666­667. aninowski m., kazimierczak r., hallmann e., rach­ tan­janicka j., fijoł­adach e., feledyn­szewczyk adv. hort. sci., 2021 35(2): 195­205 204 b., majak j., leszczyńska j., 2020 ­ evaluation of the potential allergenicity of strawberries in response to different farming practices. ­ metabolites, 10: 102. armesto j., rocchetti g., senizza b., pateiro m., barba f.j., domínguez r., lucini l., lorenzo j.m., 2020 ­ nutritional characterization of butternut squash (cucurbita moschata d.): effect of variety (ariel vs. pluto) and farming type (conventional vs. organic). ­ food res. int., 132: 109052 bourn d., prescott j., 2002 ­ a comparison of the nutri‐ tional value, sensory qualities, and food safety of organically and conventionally produced foods. ‐ crit. rev. food sci. nutr., 42(1): 1­34. brandt k., mølgaard j.p., 2001 ­ organic agriculture: does it enhance or reduce the nutritional value of plant foods? 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current knowledge and prospects for research. ­ horttechnology, 16: 449­456. impaginato 3 adv. hort. sci., 2020 34(1): 3­10 doi: 10.13128/ahsc­8251 development of interspecific hybrids between habenaria radiata and habenaria rhodocheila complex p. sinumporn (*), t. narumi­kawasaki, s. fukai faculty of agriculture, kagawa university, miki‐cho, kagawa 761‐0795, japan. key words: apomixis, cross combination, pcr­rflp, reciprocal crossing, seed germination. abstract: reciprocal crosses between habenaria radiata and h. rhodocheila complex were investigated to develop new hybrids. the fruit­setting frequency and seed germination in the cross combination of h. radiata × h. rhodocheila complex were higher than those of h. rhodocheila complex × h. radiata. the hybridity of the obtained progenies was confirmed through pcr­rflp analysis of the rrna gene. cross combinations producing true hybrids, apomicts, or both were observed, indicating that both h. radiata and h. rhodocheila com­ plex were facultative apomixis. the obtained hybrids, h. radiata × h. rhodocheila (orange flower), showed the intermediate plant form and flower shape of the parents, and both petals and lip were pale yellow. 1. introduction habenaria is a large genus in the family orchidaceae, consisting of more than 800 species distributed in tropical and subtropical areas such as southern america, southern and central africa, and east asia (pridgeon, 1992; kurzweil, 2009; pedron et al., 2012; batista et al., 2013; jin et al., 2014). habenaria species show diverse plant forms, flower shapes, and petal colors. there are many habenaria species having high ornamental value, but only a few species are commercialized. in this study, we focused on two habenaria species: h. radiata and h. rhodocheila. habenaria radiata is a species native to japan in the wet­ lands of honshu, shikoku, and kyushu islands. this species is low­temper­ ature tolerant. the form of the flowers is unique and beautiful, and the white petals look like a white egret bird. this species has been used as ornamental pot plants (kim et al., 2007, 2010; mitoma and kanno, 2018), but it can be used as cut flowers (sinumporn et al., 2015). habenaria rhodocheila is found in southeast asia, laos, myanmar, southeast china, thailand, malaysia, and the philippines. the flowers of h. rhodocheila have a large lip and four lobes, with side lobes and oblique mid lobes. the lips show a wide range of color such as orange, pink, red, and yellow. formerly, the pink­flowered genotype was accepted under the name h. (*) corresponding author: punpaka.sinumporn@gmail.com citation: sinumporn p., narumi­kawasaki t., fukai s., 2020 ­ development of interspecific hybrids between habenaria radiata and habenaria rho‐ docheila complex ­ adv. hort. sci., 34(1): 3­10 copyright: © 2020 sinumporn p., narumi­kawasaki t., fukai s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 october 2018 accepted for publication 6 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 3­10 4 erichmichelii, and the yellow­flowered genotype was h. xanthocheila. the morphological characteristics are also different in each genotype, beside petal color (kurzweil, 2009; batista et al., 2013). h. xan‐ thocheila is distinguished from h. rhodocheila in its tuber shape, i.e. h. xanthocheila has a crown­shaped tuber but h. rhodocheila has a round tuber (cullen et al., 2011). because these genotypes are very closely related, they are integrated into one species, called h. rhodocheila complex. in this report, we adopted the name h. rhodocheila complex and distinguished the genotypes only by the color of the petals. producing hybrids between the two completely dif­ ferent habenaria species, h. radiata and h. rhodocheila complex, could result in new hybrids having vigor, low­temperature tolerance, and beauti­ ful flower shape with colorful petals. recently, many orchid species including habenaria are at risk of extinction. the numbers of both h. radiata and h. rhodocheila complex are decreasing in their natural habitats, which are being destroyed through urbanization, agricultural use, ecological mismanagement of habitat, changes in cli­ mate conditions, and overcollection by people (stewart and kane, 2006; mitsukuri et al., 2009; tanaka et al., 2015). supply of new interspecific hybrids with increased ornamental value is expected to reduce the illegal collection of the species in their habitats. there is little research on intraspecific cross breeding using these two habenaria species. only a successful of intraspecific cross between wild­type and petaloid­sepal genotypes in h. radiata was done (kim et al., 2010; mitoma et al., 2019). in this study, we carried out reciprocal crossing between h. radiata and h. rhodocheila complex, and evaluated the obtained progenies. this report is the first on suc­ cessful interspecific crossing of h. radiata and h. rhodocheila complex. 2. materials and methods plant materials tubers of h. radiata ‘aoba’ (hra) (fig. 1a) were planted in april, every year, in 12 cm plastic pots (5 tubers per pot) with sphagnum moss and tubers of h. rhodocheila complex (orange, pink, and yellow petal genotypes, rco, rcp, and rcy, respectively) (fig. 1b, 1c, 1d) were planted in 12 cm plastic pots filed with a medium consisting of growing mix (metro mix 350; sun gro horticulture, ma usa): kanuma (volcanic porous soil): vermiculite, 1:2:1. then, h. radiata were placed in a greenhouse in natural temperature with solar radiation. h. rhodocheila complex were placed in a growth chamber controlled at a constant tem­ perature of 20°c with solar radiation. interspecific cross and in vitro germination a preliminary crossing experiment, hra × rco, was carried out in 2015. hra and two h. rhodocheila complexes, rco and rcy, were then cross­pollinated reciprocally in 2017. five plants of each genotype were used in those cross combinations. twenty plants of hra and rcp were also cross­pollinated rec­ iprocally in 2017. a total of six cross combinations were made (table 1). the pollinia of mother plants were removed in advance to prevent self­pollination and the aimed pollinia of other plants were placed on the stigma. reciprocal crosses were also conducted. hand­pollinated flowers were labelled individually, and the capsules were harvested before dehiscence about 2 months after pollination. the capsules were surface sterilized with 70% (v/v) ethanol for 30 s and 0.1% (v/v) sodium hypochlorite solution for 15 min fig. 1 ­ plant morphology of habenaria species used in this study. (a) h. radiata ‘aoba’, (b) h. rhodocheila complex (orange), (c) h. rhodocheila complex (pink), and (d) h. rhodocheila complex (yellow). sinumporn et al. ‐ development of interspecific hybrids of habenaria spp. 5 and then rinsed three times with sterilized water. seeds were removed from the capsules and mixed in a petri dish, and then a batch of seeds picked up with tweezers was placed on a seed germination medium in 5 cm petri dishes. the medium was mm (malmgren, 1996) supplemented with 20 g/l sucrose and 0.7% agar, and adjusted to ph 5.75 prior to auto­ claving at 0.103 mpa pressure and 121°c for 20 min. the cultures were kept at 20°c under dark condi­ tions. extract numbers of seeds placed on the medi­ um was unknown, the germination was evaluated in four stages: well (+++), fair (++), poor (+) and no­ger­ mination (­). six months after sowing, protocorms were transplanted to 6 × 6 cm plastic culture vessels with mm medium. the protocorm cultures were kept under light inflorescence lamps (fl40s. brn; toshiba lighting & technology co. ltd.) for 16 h with a light intensity of 31.5 µmol m­2 s­1at 24°c. the protocorms were subcultured every month. after 12 months, the developed plantlets were acclimatized and planted in 12 cm plastic pots using the same growing medium as for the mother plants. the obtained progenies were grown in a growth chamber controlled at 20°c. pcr‐rflp the total dna of both parents and progenies was extracted from 0.1 g of leaf tissue according to a modified abbas dna extraction method (abbas et al., 2013). pcr was performed in a 50 µl reaction mix­ ture containing 70 ng of total dna, 0.2mm each of rrna gene specific primers (5ʹ­aca cac cgc ccg tcgctc cta­3ʹ and 5ʹ­act cga tgg ttc acg gga ttc tg­3ʹ), 2.5 mm dntps, 20mm of 10× pcr ex taq buffer, and 5 u/µl of ex taq polymerase (takara bio inc., otsu, shiga, japan) according to haruki et al. (1997). pcr was conducted under the following ther­ mocycling conditions: 1 cycle of 96°c, 10 s; 25 cycles of 96°c, 10 s, 55°c, 30 s, 72°c, 60 s; and 1 cycle of 72°c, 10 min. the amplified products of both the parents and the progenies were digested with selected restriction endonucleases (alu ι, hha ι, rsa ι, and sty ι; nippon gene co. ltd., toyama, japan) at 37°c for 1 h. the digested products were separated by electrophoresis in 1.8% agarose gels (invitrogen, carlsbad, california, usa) containing 0.1 µl/ml ethidium bromide solution and photographed. 3. results pod set and seed germination in the reciprocal cross‐ ings six reciprocal cross combinations were made, and the pod set frequencies varied depending on both cross combinations and the ovule parents. the hra × rco cross combination resulted in 8 pod sets from 13 flowers (61.5%), and the opposite cross of rco × hra resulted in 2 pod sets from 2 flowers (100%). hra × rcp had 16 pod sets from 28 flowers (57.1%), and the opposite cross rcp × hra had 12 from 41 flowers (29.3%). hra × rcy had only 1 pod set from 11 flow­ ers (9.1%), and the opposite cross rcy × hra had 1 from 3 flowers (33.3%) (table 1). all pods were harvested before dehiscence, and the seeds were cultured on mm medium without plant growth regulators. the seeds from the cross combination of hra × rco germinated well, but the seeds from rco × hra did not germinate. the seeds of hra × rcp also had rather high germination, but the seeds from rcp × hra did not germinate. both cross combinations of hra × rcy and rcy × hra had poor seed germination (table 1). the sown seeds from hra × rco (fig. 2a) swelled within 50 days of culture (fig. 2b), developed proto­ corms around 90 days of culture (fig. 2b), and the protocorms produced rhizoids (fig. 2c). after being table 1 ­ reciprocal crossing between habenaria radiata and habenaria rhodocheila complex hra = h. radiate; rco = h. rhodocheila (orange petal); rcp = h. rhodocheila (pink petal); rcy = h. rhodocheila (yellow petal). *++++ = germination well, ++ = germination fair, + = germination poor, ­ = no germination. cross combination number of flowers pollinated number of pod sets (%) seed germination number of plantlets tested pcr­rflp number of true hybrids (%) hra × rco 13 8 (61.5) ++++ 36 36 (100) rco × hra 2 2 (100) ­ ­ ­ hra × rcp 28 16 (57.1) +++ 21 0 (0) rcp × hra 41 12 (29.3) ­ ­ ­ hra × rcy 11 1 (9.1) + 18 1 (5.5) rcy × hra 3 1 (33.3) + 8 0 (0) adv. hort. sci., 2020 34(1): 3­10 6 subjected to light, the protocorms turned green (fig. 2d), produced the first leaf within 120 days of culture (fig. 2e), and then developed plantlets around 150 days of culture (fig. 2f). ed both apomixis and true hybrids (fig. 3c). the prog­ enies of the reciprocal cross rcy × hra (yxw) showed the same band pattern as the female parent (rcy), suggesting that they were apomicts. fig. 2 ­ seed development of habenaria radiate × habenaria rhodocheila on mm medium. (a) sown seeds (day 0), (b) protocorm deve­ lopment (50 days), (c) enlarged embryo rupture testa and developed protocorm (3 months), (d) green protocorm with protome­ ristem (3 months), (e) emergence of first leaf (4 month), (f) plantlets (5 months). confirmation of hybridity through pcr‐rflp analysis to confirm the hybridity of the obtained progenies, pcr­rflp analysis targeting the ribosomal rna gene was used according to haruki et al. (1997). the early growth stage of two progenies of hra × rco, named wxo1 and wxo2, were used. expected single pcr product was amplified in all the tested plants. three restriction enzymes (alu ӏ, hha ӏ and rsa ӏ) that showed polymorphism in the digested pcr products between both parents were applied. the band pattern of both wxo1 and wxo2 was intermediate between both parents (hra and rco), indicating that both wxo1 and wxo2 were true hybrids. thirty­six hra × rco progenies in the in vitro stage were chosen ran­ domly and their hybridity was tested in the same manner. the results showed that all the tested proge­ nies were true hybrids (table1) (fig. 3a). in contrast, an early growth­stage progeny of hra × rcp, named wxp1, showed a band pattern the same as the female parent (hra) when the pcr products were digested with alu ι, hha ι, rsa ι, and sty ι, suggesting that the plants were apomicts. twenty­one hra × rcp proge­ nies in the in vitro stage were tested in the same man­ ner. the results showed all the tested progenies were apomicts (fig.3b). one rapidly grown apomict was designed wxy1 and the one that was judged to be a true hybrid was designed wxy2. moreover, the results of hra × rcy (wxy) showed that the progenies includ­ fig. 3 ­ pcr­rflp profile of parents and progenies. a: hra × rco. 1. hra, 2. wxo1, 3.wxo2, 4. rco. b: hra × rcp. 1. hra, 2. wxp, 3. rcp. c: hra × rcy. 1. hra, 2. wxy1, 3.wxy2, 4.yxw, 5. rcy. morphological characteristics of the obtained progenies the obtained progenies wxo1 and wxo2 (con­ firmed as hybrids through pcr­rflp analysis), wxy1 (assumed to be an apomict), and the parent rco were grown in a growth chamber controlled at 20°c. hra grown in a greenhouse without heating was used for morphological comparison. sinumporn et al. ‐ development of interspecific hybrids of habenaria spp. 7 6a). rco had a flower consisting of two grey­brown orange petals and an orange lip and three sepals. the dorsal sepal was egg­shaped and the slanted lateral sepals sometimes rolled­in. the lip had three main lobes, with the two side lobes elliptical and the mid­ dle lip had two ovate­oblong lobes (fig. 6b). the petals of both wxo1 and wxo2 were pale yellowish, which was an intermediate characteristic of the par­ ents. the sepals of wxo1 and wxo2 were green, with the dorsal sepal being egg­shaped, the same as the female parent (hra), and the lateral sepals were slanted and rolled­in, the same as the male parent (rco). in addition, two petals were attached, forming an egg­shaped hood with the dorsal sepal. the lip had three main lobes, with the two lateral lobes being slightly fringed and the center lobe had two slightly ovate­oblong lobes, the same as the male parent (rco) (fig. 6d, 6e).the apomixtic progeny wxy1 produced flowers resembling h. radiata (fig. 6f). one progeny derived from the cross combination of hra × rcy (wxy2), confirmed as a true hybrid through pcr­rflp analysis, showed an intermediate plant morphology between the parents (data not shown) but had not flowered because only one year had passed after acclimatization. plants in the genus habenaria produce a storage organ having species­specific morphology. hra pro­ duced stolons during the growing season, and new tubers formed at the top of the stolon. the hra tuber was oval with a smooth surface (fig. 7a). rco produced a long oval tuber at the bottom end of the stem. the rco tuber was bigger than that of hra. the tuber had a rough surface and was densely cov­ ered with hair (fig. 7c). both wxo1 and wxo2 had both wxo1 and wxo2 grew vigorously, and the first flowering was observed in wxo1 one year after transfer to ex vitro and in wxo2 two years after transfer to ex vitro. both wxo1 and wxo2 showed an intermediate plant form (fig. 4a, 4b, respectively) and leaf morphology of theirs parents. hra had nar­ row light green leaves (fig. 5a). rco had wide lance­ olate leaves with an undulate leaf margin, and the leaves were green or greyish green, sometimes with red­brown spots (fig. 5d). wxo1 had lanceolate light green leaves, and wxo2 had lanceolate light green leaves with an undulate leaf margin (fig. 5b, 5c, respectively). the inflorescence morphology of wxo1 and wxo2 was intermediate between the par­ ents. wxo1 had an inflorescence with two flowers, and wxo2 had an inflorescence with eight flowers. habenaria radiata produced two to four flowers (average 2.8 flowers) per inflorescence. rco had eight to ten flowers (average 9.4 flowers) per inflo­ rescence (fig. 1b). the inflorescence morphology of wxy1 was similar to hra, although it could not be accurately determined due to the poor growth of wxy1. hra had a flower consisting of two pure white petals and a lip with three green ovate sepals. the lip had three main lobes; the two lateral lobes were highly fringed and the center lobe was simple (fig. fig. 6 ­ floral morphology of the parents and hybrids. (a) hra, (b) rco, (c) rcy, (d) wxo1, (e) wxo2, (f) wxy1. fig. 5 ­ leaves of the parents and hybrids. (a) hra, (b) wxo1, (c) wxo2, (d) rco. fig. 4 ­ plant morphology of hybrids. (a) wxo1, (b) wxo2, (c) wxy1. 8 adv. hort. sci., 2020 34(1): 3­10 tubers with intermediate morphological characteris­ tics of the parents (fig. 7b) (table 2). 4. discussion and conclusions interspecific hybridization is a powerful breeding method that can produce new traits in ornamental plants including orchids. this study aimed to produce new habenaria hybrids by using two different eco­ type species (h. radiata and h. rhodocheila complex). the two species chosen in this study are different in not only plant morphology but also flowering physiol­ ogy. because the flowering time of h. radiata is dur­ ing june to august under natural conditions, h. rhodocheila complexes were grown in a growth chamber controlled at 20°c to match the flowering time. the fruit settings in h. radiata × h. rhodocheila complex were higher than in the opposite crosses. the germination of the obtained seeds was also dif­ ferent depending on the cross combination (table 1). the seeds obtained from h. radiata × h. rhodocheila complex showed higher seed germination than those in the opposite crosses. unilateral cross incompatibil­ ity is often observed in interspecific crossings includ­ ing orchids (johansen, 1990; borba et al., 1999). the hybridity of the obtained progenies was investigated by using rflp analysis. the results showed that all the tested progenies of hra × rco were true hybrids, whereas all the tested progenies of hra × rcp and rcy × hra were apomicts. both true hybrids and apomicts were found in the progenies of hra × rcy. the results of rflp analysis were consistent with the observed plant morphological characteris­ tics of the obtained progenies. the production of apomixis, including oblique and facultative, is known in some genera including habenaria in the family orchidaceae (batygina et al., 2003). zhang and gao (2018) reported obligate apomixis in h. malintana. the present study showed that both true hybrids and apomicts appeared in h. radiata and h. rhodocheila. successful crossing of different flower types of h. radiata has been reported (kim et al. , 2010). adthalungrong et al. (2015) reported successful reci­ procal crossing between rcp and rcy. these findings indicate that both h. radiata and h. rhodocheila do not have obligate apomixis. we consider that the fac­ ultative apomixis observed in this study is induced by interspecific crossing between distantly related species. in many plants, pollination is necessary for induction of apomixis (den nijs and van dijk, 1993). when hra was used as a female parent, the frequen­ cy of apomict production varied depending on the pollen parents. the results suggest that the growth of the pollen tube in the ovary differed depending on the h. rhodocheila complex genotype. in hra × rcy, only one pod was harvested, and both an apomict and true hybrid were obtained from the pod. this finding suggests that different embryogenesis, sexual and asexual, occurred at the same time. further detailed morphological and genetic investigation of embryo development in interspecific hybridization is required to explain these phenomena. if the occur­ rence of apomixis is unpredictable in a practical breeding program, efficient selection of true hybrids is essential. the present study selected true hybrids at the early developmental stage of progenies through pcr­rflp analysis. selecting plants in the in vitro stage will be useful in a practical breeding pro­ gram. fig. 7 ­ tubers of the parents and hybrids. (a) hra, (b) wxo2, (c) rc. name plant form (cm) leaves (cm) number of leaves flower (cm) stigma (cm) number of flowers inflorescences (cm) spur tuber (cm) storage organ characteristics height spread length width length width (cm) length width h. radiata (n=5) 13.8±2.4 15.1±0.4 6.9±0.7 0.5±0.1 5.0±3.0 3.2±0.1 2.2±0.1 0.3±0.1 2.8±0.4 18.8±2.0 3.3±0.8 1.5±0.1 1.1±0.2 round­oval with many long stolons h. rhodocheila (orange) (n=5) 22.3±3.1 23.4±0.6 9.5±1.9 2.2±0.4 6.4±0.2 3.1±0.0 2.3±0.1 0.4±0.1 9.4±1.2 25.8±2.4 3.5±0.5 3.6±1.5 1.5±0.2 long oval wxo1 (n=2) 10.8±2.8 9.3±1.0 3.2±0.7 0.8±0.1 4.0±1.0 2.4±0.2 1.6±0.2 0.4±0.1 1.5±0.5 12.9±3.4 3.1±0.1 1.4±0.1 1.0±0.1 round­oval wxo2 (n=2) 16.0±2.5 20.4±1.6 7.4±0.1 2.4±0.1 6.0±1.0 2.7±0.1 1.8±0.1 0.3±0.0 8.5±1.5 22.0±1.8 3.3±0.0 1.7±0.2 0.9±0.2 round­long oval with many short stolons table 2 ­ morphology characteristics of two habenaria species and interspecific hybrids sinumporn et al. ‐ development of interspecific hybrids of habenaria spp. 9 we obtained new interspecific habenaria hybrids, wxo1, wxo2, and wxy2. both wxo1 and wxo2 showed intermediate morphological characteristics of the parents (table 2). it is well­known in orchidaceae that interspecific hybrids exhibit an intermediate mor­ phological characteristic of parent. when the interme­ diate characteristics is observed in detail, the charac­ teristics of either the female parent or the male par­ ent often appear strongly in each organ as shown in this study. in case of ascocentrum ampullaceum var. auranticum × vanda coerulea, the progenies showed flowers having pink petal color and spur which come from female parent and orange mottles on the petals come from male parent (kishor et al., 2006). interspecific hybrids between vanilla planifilia and v. aphylla showed two types, light green plants without leaf resembling male parent and green plants with leaves resembling female parent (divakaran et al., 2006). the flowers of wxo1 and wxo2 were pale yel­ low. the major flower pigment of h. rhodocheila com­ plex is considered to be carotenoids (sinumporn et al., 2015). in flowers that contain carotenoids as a main flower pigment, the flowers are orange when the amount of carotenoid is high and yellow when it is low (kishimoto et al., 2007). it is assumed that the carotenoid content of the wxo1 and wxo2 petals is very low because one parent hra has white petals, resulting in pale yellow petals in the hybrids. the inter­ specific hybrid of hra × rcy named wxy2 showed intermediate morphological characteristics in leaves between parents (data not shown). because that the hybrid was not large enough to give flowering, the flo­ ral characteristics was not determine yet. interspecific hybrids often have flower color intermediate between the parents as reported in the reciprocal crossing progenies of rcp and rcy, which produced pale orange and pink flowers (adthalungrong et al., 2015). the hybrids obtained in this study are new, but the ornamental value is not high enough. further breeding steps such as backcrossing and self­pollination are required for flower pigment accumulation and flower form improvement. furthermore, the temperature response of these hybrids is not yet clear because the resulting hybrids were grown under constant temper­ ature conditions. it is necessary to clarify the charac­ teristics of cultivation to evaluate the hybrids. we obtained new interspecific hybrids between h. radiata and h. rhodocheila complex. the interspecific hybridization produced both true hybrids and apomicts. the apomicts were distinguished at the early developmental stage of the progenies through pcr­rflp analysis. the obtained hybrids showed an interesting flower shape and color, which were inter­ mediate between the parents. further breeding processes, especially back crossing, is required to improve the ornamental value of the hybrids. references abbas s.r., gardazi s.d.a., sabir s.m., shah a.h., abbas m.r., batool a., 2013 ­ “abbas” dna 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(*), l.j. beaumont 1, m.e. rodriguez 3 1 department of biological sciences, faculty of science and engineering, macquarie university, north ryde nsw, 2109, australia. 2 department of crop science, faculty of agriculture, university of nigeria, nsukka 41001, enugu state, nigeria. 3 hawkesbury institute for environment, western sydney university, locked bag 1797, penrith, nsw 2751, australia. key words: fruit yield, heat stress, solanum lycopersicum, tomato, water stress. abstract: food security is a major issue in west africa. as a consequence of cli­ mate change, increases in temperature and shifts in precipitation will have major ramifications for which crops can be grown in the region. here we con­ ducted an experiment to evaluate the impacts of short­term projected heat and water stress on three tomato varieties (solanum lycopersicum): oregon spring, roma vf, and tropic. the plants were initially cultivated in the glasshouse pro­ grammed at 28/20oc day/night cycle. the treatments investigated were: con­ trol (ct); heat stress (ht); water stress (ws) and heat + together with water stress (htws). for heatwave treatments, a 35/23oc day/night gradually taken up in a cycle was imposed. the water stress conditions were by decreasing the soil water field capacity by 50%. leaf gas exchange and plant production parameters were measured. our result indicated that all varieties suffered from significant declines in yield as consequence of the stresses. the heatwave treat­ ment proved more detrimental on the tomato fruit yield than the water stress, except when these two treatments occurred in sequential cycles. the results of this study suggest that heatwaves and water stress, projected to occur more frequently due to climate change, may adversely impact the growth and yield of these three tomato varieties. also, there was an unexpected fruit yield per­ formance comparison among varieties tested in this experiment. 1. introduction food security is a global issue that is projected to exacerbate as the human population grows (godfray et al., 2010). to meet the united nations millennium development goal of “eradicating extreme poverty and hunger”, considerable emphasis have to be placed on the developing regions of the world (pingali et al., 2006), such as west africa. west africa comprises 15 countries and is home to an estimated 350 million people (*) corresponding author: felix.amuji@gmail.com citation: amuji c.f., beaumont l.j., esperon rodri­ guez m., 2020 ­ simulating the impact of projec‐ ted west african heatwaves and water stress on the physiology and yield of three tomato varie‐ ties. ‐ adv. hort. sci., 34(2): 147­156. copyright: © 2020 amuji c.f., beaumont l.j., esperon rodriguez m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 april 2019 accepted for publication 5 march 2020 ahs advances in horticultural science file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#godfray file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#pingali http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 147­156 148 (30% of the african continent) (united nations, 2018). the population of this region is increasing rapidly, and is expected to reach 490 million by 2030 (hollinger and staatz, 2015). in some part of west africa, more than sixty per­ cent of the population are dependent upon rain­fed agriculture, which is characterised by low fertilizer use, poor seed quality, inadequate water manage­ ment, and low soil fertility (benin, 2016). agriculture is also negatively influenced by extreme weather events, such as heatwaves and droughts (asare­kyei et al., 2017). indeed, the united nations food and agricultural organization reported that the drought occurred from june to august of 2019 in the sahel region of west africa, resulted in 9.7 million people being exposed to severe food insecurity, leading to 2 million children being under acute malnutrition (fao, 2019). climate change poses additional concerns for food security in west africa, particularly as the fre­ quency and intensity of extreme weather events are projected to increase (sylla et al., 2016). combined with climate change, the growing human population characterized by rain­fed agriculture in west africa, suggest that the eradication of poverty and hunger from this region will be challenging. thus, adaptation to future climate plays a crucial role in securing food production (easterling et al., 2007) and will require efficient adaptation strategies from the respective governments. while, some of these strategies have low or no cost and are already used in the region (e.g. shifting planting dates or selecting more resilient crop varieties), other strategies, such as developing new varieties or increasing irrigation, will require greater investment (rosenzweig and parry, 1994). tomato: a key horticultural crop in west africa tomato (solanum lycopersicum l.) provides sub­ stantial economic and nutritional benefits for human­ ity (klunklin and savage, 2017). this crop is one of the world’s most highly consumed fruits (arah et al., 2015), with over 177 million tonnes of production across ~5 million hectares of harvested land globally (faostat, 2017). within west africa, tomato is among the top ten horticultural crops in terms of yield. in 2016, 3.7 million tonnes were produced across 0.7 million ha (faostat, 2017). this crop is typically grown throughout west africa under rain­ fed conditions, with the greatest yield being achieved in the sudano­sahelian zone of west africa, located south of the sahara desert and north of the humid guinea region (perez et al., 2017). however, tomato growth and fruit production are affected significantly by climate (petrozza et al., 2014) and yield is generally reduced in areas with extreme weather events (oladitan and akinseye, 2014) such as heatwaves (hatfield and prueger, 2015), flooding (ezin et al., 2010), and drought, as well as pests and diseases (ximénez­embún et al., 2016). the optimum growth rate for most tomato varieties require temperatures between 20 and 27°c (nicola et al., 2008) and 400­600 mm of water throughout their growing period (jaria, 2012). temperatures above 32°c can affect vegetative growth and reproduction of tomatoes (pressman et al., 2002; abdelmageed et al., 2003; müller et al., 2016), causing decreases in leaf area and plant devel­ opment during the flowering stage (nduwimana and wei, 2017) and the failure of tomato fruit set (sato et al., 2000). water stress may lead to poor tomato plant growth and productivity through inhibition of cell expansion and reduction of stomatal opening (chaves et al., 2003). water stress also decreases the rate of photosynthesis, especially through stomatal conductance, as well as abundance of flowers and fruiting quality (murshed et al., 2013). due to the importance of tomato for the west african region and the potential threat of climate change on its production, in this work we assessed the effect of heatwave and water stress on three varieties of tomatoes. we hypothesised that: (1) heatwave and water stress (i.e. less than 50% of soil water field capacity) will have a negative influence on the tomato varieties, causing reductions in fruit yield even after recovery period exposure; (2) the effect of heatwaves followed by water stress will be more severe than either of these stresses alone, causing a significant reduction in fruit yield; and (3) seasonally adapted varieties of tomatoes will respond different­ ly to heatwaves and water stress, with varieties from warmer regions having higher yield than a cool­ region variety. 2. materials and methods plant material and environmental conditions we selected three varieties of tomato, two of which are commercially grown in west africa (‘roma’ and ‘tropic’), while the third (‘oregon spring’) is typi­ cally grown in cooler regions of the world. ‘roma’ variety has a strong, compact stem with determinate vines (gelmesa et al., 2010). this variety has been noted to be particularly suited to climatic conditions file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#una file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#una file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#una file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#hollinger file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#asare file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#asare file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#asare file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#abebie amuji et al.‐ simulated effect of heat and water stress on three tomatoes growth and yield 149 in savannah regions of west africa (ojo et al., 2013). tropic was bred in the 1960’s in the usa as an inde­ terminate variety adapted to warm, humid climates (strobel, 1970). in contrast, oregon spring was bred as a determinate tomato for cold tolerance (baggett and kean, 1986). the experiment was conducted under controlled environmental conditions at the plant growth facilities glasshouse (pgf) at macquarie university, sydney, australia. seeds obtained from a seed distri­ bution company called eden seeds, australia, were germinated in a growth chamber at 22°c in commer­ cial punnets. six weeks after sowing, seedlings of sim­ ilar height were transplanted to ten litres pots filled with 10 kg of soil mix that contained sand, topsoil, and rocky grey clay. thirty­six plants of each variety were potted. the plants were initially cultivated in the glasshouse programmed at 28/20°c day/night cycle (temperature ambient, or ta). we kept a mini­ mum night temperature of 20°c. then from 04:00 h, temperature was increased by 0.5°c every 30 min­ utes, then remained constant at 28°c until 17:00 h. following this, temperature was decreased by 0.5°c every 30 minutes to reach the minimum night­time temperature of 20°c. this temperature cycle was selected as it represents the conditions under which tomatoes are commonly grown in west africa. in addition, a 12­hour photoperiod of 600 μmolm­1s­1 and co2 concentration of 400 ppm (parts per million) were maintained in the glasshouses throughout the experiment. after estimating the irrigation water capacity of the pots, all pots (with the exception of individuals in the water stress treatments, see details below) were watered daily to a 100% soil water field capacity (fc) which equated to a water holding capacity of 35%. the percentage water holding capacity was deter­ mined as the gain in the weight of the soil at satura­ tion point divided by the dried weight of the soil x 100: % water holding cap.= gain in weight of the soil at saturation point dried weight of the soil x 100 all plants were given the same quantity of fertiliz­ er fortnightly using yates nutricote standard grey® fertilizer containing npk (16:4:4) liquid fertilizer at the rate of 1 g l­1. experimental design our goal was to assess the ecophysiological responses of the three tomato varieties to heatwave and water stress. for heatwave treatments, a 35/23°c day/night cycle (temperature high, or th) was imposed, where temperature increased 1°c per hour from the minimum night temperature of 23°c from 04:00 h (local time) to the maximum day tem­ perature of 35°c then kept constant at this till 17:00 h. before been decreased at the same rate until the minimum temperature was reached (23°c), were it stayed constant again till the 04:00 h, then the cycle continued. this temperature range was selected based on future heat projections for west africa (abiodun et al., 2013; sylla et al., 2016). presently, there is no consensus regarding future rainfall patterns for west africa (roudier et al., 2011), however increases in the frequency of drought conditions have been projected for the west­ ern sahel sub­region (west of ~0°e) (monerie et al., 2013). thus, in this study, we simulated water stress conditions by decreasing the soil water field capacity by 50%. to avoid location specific effects, the position of plants within the glasshouse were randomly rear­ ranged weekly. once reaching the flowering stage (~six weeks after sowing), three treatments together with the control were initiated for a total duration of eight weeks. nine individual plants of each variety were placed in each treatment. ct ­ control. plants were grown at an ambient tem­ perature (ta) consisting of a 28/20°c day/night cycle. all individuals were watered daily to approximately 100% fc. this condition was maintained for these ct plants for the whole duration of the experiment. the treatment conditions are explained below: 1. ws ‐ water stress treatment. plants were grown at ta. the soil water field capacity of each individual was measured daily using a soil moisture sensor (campbell scientific australia pty ltd ­hydro sense11®). during the treatment, all individuals were watered daily to only 50% fc. this condition was maintained throughout the eight weeks of treatment application. 2. ht ‐ heat treatment. plants were exposed to a cycle of a 14­day heatwave (th = 23/35°c night /day) followed by 14 days at ta, and were kept well­ watered. this cycle of conditions was maintained throughout the eight weeks of treatment application. 3. htws ‐ heat and water stress treatment. plants were exposed to a 7­day heatwave, during which they were well watered then followed by seven days of water stress (i.e. 50% fc) at ta. also, this cycle of conditions was maintained throughout the eight weeks of treatment application. after this treatments application period, all the plants were returned to the ct conditions (see details file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#ojo file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#strobel file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#baggett file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#baggett file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#baggett file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#abiodun file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#sylla file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#roudier file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#roudier file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#roudier file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#monerie file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#monerie file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#monerie adv. hort. sci., 2020 34(2): 147­156 150 above) and allowed to recover for five weeks then the experiment terminated. the above conditions were simulated to imitate what normally happen in nature during a heatwave occurrence. ecophysiological and production measurements ecophysiological responses were assessed by measuring gas exchange traits, which included: tran­ spiration rate, intracellular co2 concentration (ci), stomatal conductance (gs), and net assimilation rate (e). these traits are widely used to evaluate physio­ logical responses of plants to heat­water stress con­ ditions (nankishore and farrell, 2016; duan et al., 2017). the traits were measured on three mature, fully expanded leaves without damage and in good health, from five plants per treatment and variety using a licor 6800 portable photosynthesis system (li cor, lincoln nebraska, usa). gas exchange measure­ ments were taken at the third week (i.e. first week after complete treatments cycle­w3) and eight weeks (i.e. last week of treatment application­w8) between 09:30­14:00 h (local time). also at the same period the number of flowers per plant were counted from the nine sampling plants per treatment and variety. subsequently, the number of fruits that developed on these plants was also recorded. maturity of fruit was determined based on a stan­ dard usda colour chart (e.g. ‘light red’, ucanr, 2011). during the termination of the experiment (i.e. after the five weeks of recovery period), matured fruits were collected and weighed. the fresh and dry above ground biomass of the sampled plants were weighed and recorded too, with drying undertaken in an oven at 70°c for seven days. measurements were noted as fresh biomass weight without fruit (fwwf), fresh fruit weight (ffw), total fresh biomass weight (tfw). statistical analysis a two­way analysis of variance (anova) model with a correction formula called satterthwaite approximation was performed to determine the effects of all the treatments (i.e. water treatment vs heat treatment vs both in sequential combination) vs control on the tomato varieties, using the lmertest package (kuznetsova et al., 2017) in r version 3.4.3 (r development core team, 2017). as measurements were taken on the same individuals over two differ­ ent weeks, we used week as a covariate, with plant id treated as a random effect. post­hoc tests (turkey contrasts) were used to compare means between treatments, and results were considered significant when p < 0.05. statistical differences were reported as different letters on each figure. all figures were drawn in r using the ‘ggplot2’ (wickham, 2016) pack­ ages. 3. results ecophysiological measurements from figure 1 and table 1 it can be observed that for all the three varieties, transpiration rate differed significantly across treatments and weeks. transpiration was generally highest among ht plants and lowest in the htws treatment. for ‘oregon spring’, transpiration rates among ht plants did not differ significantly to ct, whereas rates were signifi­ cantly higher among ‘roma’s ht plants. for both roma and tropic, transpiration rates between ct plants and ws were not significantly different (fig. 1). fig. 1 ­ transpiration rate (mol m­2 s­1) of three tomato varieties (oregon spring, roma and tropic). the treatments consi­ st of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same let­ ter are not significantly different at p<0.05 according to turkey's test. file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#nankishore file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#duan file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#duan file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#duan file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#uni file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#uni file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#uni file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#kuznetsova file:///c:/users/felix%20amuji/downloads/manuscript-tomatoes-felix-22-01-19_lb.docx#r amuji et al.‐ simulated effect of heat and water stress on three tomatoes growth and yield 151 in general, plants exposed to heatwave stress had higher rates of intracellular co2 compared to plants in the other treatments. the lowest values occurred among htws plants, with similar patterns for the three varieties (fig. 2). stomatal conductance (gs) across the three varieties was generally higher in ht plants, and lowest in the htws treatment (fig. 3, table 1). however, treatment, week, and their inter­ actions differed significantly for each variety. among oregon spring and roma varieties, ct and ht plants had significantly higher gs than the htws plants. for tropic, the ht plants had significantly higher gs than htws plants in the weeks observed (fig. 3, table 1). net assimilation rate (e) differed significantly for the weeks and the interaction between treatments and weeks among all the three varieties (fig. 4; table 1). production measurements for the three varieties, the number of flowers was greater in both of the heatwave treatments (ht and htws) compared to the ct and ws treatments. ht treated plants had significantly the highest number of flowers. also, by the end of the treatment applica­ tion (w8), ‘oregon spring’ had an average of 8.5 (± 0.6 sd) flowers among plants in the ws treatment, fig. 2 ­ intracellular co2 response (pa) of the three tomato varie­ ties (oregon spring, roma and tropic). the treatments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. table 1 ­ intracellular co2 response (pa) of the three tomato varieties (oregon spring, roma and tropic) tomato varieties treatments weeks interaction f­value p value f­value p value f­value p value number of fruits oregon spring 399.681 < 0.001 350.074 <0.001 0.0466 <0.001 roma 206.190 < 0.001 243.735 < 0.001 61.612 0.019 tropic 289.064 < 0.001 453.048 <0.001 84.548 < 0.001 number of flowers oregon spring 27.092 0.07 19.494 0.177 10.069 0.409 roma 19.226 0.1568 27.351 0.1130 36.571 0.0289 tropic 76.114 0.001 74.866 0.001 62.638 0.0033 intracellular co2 response oregon spring 15.867 0.231 766.736 <0.001 12.037 0.3126 roma 46.786 0.015 1.007.011 < 0.001 41.736 0.007 tropic 41.423 0.023 11.552 0.285 0.9491 0.420 assimilation rates oregon spring 25.944 0.088 295.82 <0.001 7.449 <0.001 roma 0.2935 0.829 911.11 <0.001 27.802 <0.001 tropic 21.725 0.131 1322.9 <0.001 25.296 <0.001 stomatal conductance oregon spring 59.115 0.006 119.84 <0.001 72.924 <0.001 roma 81.714 0.002 131.58 < 0.001 7.443 <0.001 tropic 37.627 0.032 259.27 <0.001 11.078 < 0.001 transpiration rates oregon spring 65.636 0.04 253.232 <0.001 13.997 0.247 roma 23.232 <0.001 237.1 <0.001 9.214 <0.001 tropic 13.053 <0.001 241.49 <0.001 5.393 0.002 the treatments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. 152 adv. hort. sci., 2020 34(2): 147­156 and 15 (± 4) and 20 (± 7) flowers on plants in the htws and ht treatments, respectively (fig. 5). for both ‘roma’ and ‘tropic’, the average number of fruits that developed per plant differed significantly across treatments, weeks, and their interactions (table 1). all three varieties produced fewer fruits under both heatwave treatments. this effect was most pronounced for roma where, by w8 of the treatment period, ct plants had produced an average of 19.4 (± 8.9) fruits per plant compared to ht plants (ht: 2.6±2.4 and htws: 0.8±1.8), and significantly more fruits than plants in the ws treatment (9.8±4.4). oregon spring produced more fruits in the heatwave treatments compared to the warmer vari­ eties (fig. 6, table 1). in general, heatwave treatments resulted in greater reductions to the harvested fruit weight than the water stress treatment. however, harvested fresh fruit weight (ffw) was higher among ct plants compared to the other treatments (fig. 7). this dif­ ference was significant for ‘roma’ and ‘tropic’ (table 2), although there was no significant difference between ct and ws treatments for ‘oregon spring’. for ‘roma’ and ‘tropic’ we found no significant dif­ ferences among treatments when we compared fresh biomass weight without fruits (i.e. fwwf = fresh biomass excluding fruit). however, ‘oregon spring’ biomass was significantly greater for ht plants than the other three treatments. for this variety, heatwave stressed plants were able to maintain growth at the expense of fruit production. we found a significant difference in the total biomass weight (tfw) (due to the adding of fruits weight) among treatments (fig. 7, table 2). 4. discussion and conclusions we simulated the impacts of climate change by assessing the response of three varieties of tomato (‘roma’, ‘tropic’, ‘oregon spring’) to heatwave and water stresses. we found that assimilation rate, tran­ spiration rate, intracellular co2 response, and stom­ atal conductance were all elevated under heatwave stress. in addition, we extended upon previous stud­ ies on tomatoes (e.g. nankishore and farrell, 2016; fig. 4 ­ net assimilation rate response (µmol m­2 s­1) of the three tomato varieties (oregon spring, roma and tropic). the treatments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. fig. 3 ­ stomatal conductance (mol m­2 s­1) of the three tomato varieties (oregon spring, roma and tropic). the treat­ ments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. amuji et al.‐ simulated effect of heat and water stress on three tomatoes growth and yield 153 fig. 5 ­ average number of flowers per plant for the three toma­ to varieties (oregon spring, roma and tropic). the treat­ ments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. fig. 6 ­ average number of fruits per plant for the three tomato varieties (oregon spring, roma and tropic). the treat­ ments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). w3 and w8 = weeks of treatments imposition with measurements. bars with the same letter are not significantly different at p<0.05 according to turkey's test. table 2 ­ anova (f­value and p­value) comparing the impact of the treatments on the harvested fresh fruits and aerial biomass measurements that developed on three varie­ ties of tomato (oregonspring, roma and tropic) fig. 7 ­ fruit yield (marketable harvested fresh fruit) and aerial biomass accumulation response for the three tomato varieties (oregon spring, roma and tropic). the treat­ ments consist of control (ct), water stress (ws), heat + water stress (htws) and heat (ht). measured during the termination of experiment (i.e. after allowing for five weeks’ recovery from the treatments). fwwf=fresh biomass weight without fruits; ffw=fresh fruits weights; tfw= total fresh biomass weight. tomato varieties treatments f­value p­ value fresh weight without fruits (fwwf) oregon spring 3.04 0.046 roma 1.18 0.335 tropic 1.93 0.148 harvested fresh fruits weights (ffw) oregon spring 14.66 <0.001 roma 18.00 <0.001 tropic 9.79 <0.001 total fresh weight (tfw) oregon spring 6.75 0.002 roma 19.89 <0.001 tropic 10.96 <0.001 sivakumar and srividhya, 2016; duan et al., 2017; zhou et al., 2017) by assessing the impact of these stresses individually and sequentially on fruit yield, which is crucial from a socio­economic and food security perspective especially in region like west africa. we found that these studied three tomato varieties experienced greater decline in yield (i.e. harvested fresh fruit weight) due to heat wave stress compared to water stress. however, under water stress fewer flowers were formed, indicating that heatwave stress results in a higher rate of aborted flowers compared with water stress, since its higher adv. hort. sci., 2020 34(2): 147­156 154 number flowers did not transform to more fruits. unsurprisingly, a greater impact on the plants fruit yield occurred when exposed to both stressors sequentially (i.e. heatwave then water stress togeth­ er). plants exposed to this treatment (i.e. htws) were unable to form fruit despite having more flowers than plants exposed to water stress only. water and heat stress affect photosynthesis and other physiological processes of tomatoes (nankishore and farrell, 2016; duan et al., 2017; zhou et al., 2017). thus, as climate change intensi­ fies, tomato yield in west africa may decline due to the predicted higher frequency of heatwaves (engelbrecht et al., 2015). high temperature can deactivate enzyme activity involved in the photosyn­ thetic process, reducing or inhibiting photosynthesis (rennenberg et al., 2006). this, in turn, has been reported to cause a 2.5% to 10% decline in yield for numerous crop species (hatfield et al., 2011). in tomatoes, heat stress also has the potential to affect the viability of pollen (hatfield and prueger, 2015) resulting in failure of fruit set (sato et al., 2000). nevertheless, the impacts of heatwaves can be allevi­ ated with irrigation. here, we found that stomata closed when the plants in ambient temperature were under water stress, which invariably led to lower rates of leaf gas exchange. this was not the case for the plants under the heatwave treatment that remained well­irrigated, these plants consistently had higher values of stomatal conductance. crops differ in their capacity to recover from heat­ wave and water stress. for instance, some grains like maize (zea mays l.) and rice (oryza spp.) require a 10­day period of normal conditions (i.e. lower tem­ perature and irrigation) after heat stress to enable the development of fruits (hatfield and prueger, 2015). for our experiment, a period of 14 days of normal conditions (i.e. 28/20°c day/night cycle and well­watered) between heatwave­imposed stress was not enough for plants to recover and produce fruits from the flowers formed, although the magni­ tude of the impacts on fruit production varied among the varieties. ‘tropic’ and ‘oregon spring’ varieties had significantly higher fruit yield than ‘roma’. this finding highlights the vulnerability of these tomato varieties to heatwave and water stress. high temper­ ature can affect allocation of resources, such as above­ and below­ground tissues, with a tendency towards higher shoot­to­root ratios (way and oren, 2010). neither heatwave or water stress, individually or sequentially, significantly affected vegetative biomass of the three tomato varieties, which con­ trasts to yield. under all treatments within this exper­ iment, the three varieties continued to have normal vegetative growth; however, flowers and the conse­ quent fruit yield were drastically affected indicating the importance of optimal conditions (i.e. tempera­ ture and water) to facilitate plant reproduction (peet and welles, 2005; parvej et al., 2010). initially, we hypothesised that heat stress would have a greater negative impact on the yield of ‘oregon spring’, the cool region variety, compared to the warm region varieties, i.e. ‘roma’ or ‘tropic’. however, our results indicate the opposite, suggest­ ing that oregon spring may out­perform either ‘roma’ and ‘tropic’ under similar conditions. this finding may indicate a higher plasticity in ‘oregon spring’ and a higher adaptive capacity. we suggest future research to explore the genetic characteristics of ‘oregon spring’ in response to heatwave and water stress to validate this as a potential variety to use under stress climatic conditions in as used in this experiment. this study indicated that under a simulated cli­ mate projection of 50% less soil water field capacity and a heatwave of 35/23oc­day night cycles for west africa, leaf gas exchange and fruit yield of three vari­ eties of tomatoes were negatively affected, although vegetative growth was unaffected. individually, heat­ wave stress was more detrimental for fruit yield than water stress, although experiencing these two stress­ es in sequence had an even greater consequence. hence, ensuring plants are well watered can amelio­ rate some of the negative impacts of heatwave stress. further studies are necessary to confirm the relationship existing among the various combinations of the heatwave and water stress conditions on dif­ ferent reproductive stages, such as pollen formation, pollen development and fertilization of tomatoes. interestingly, the cool adapted variety assessed in this work, ‘oregon spring’, might represent an alter­ native option in warm temperature regions where ‘roma’ and ‘tropic’ varieties are underperforming, provided that irrigation is not limited. acknowledgements the authors wish to thank professor b.j. atwell and dr. alessandro ossola for their technical inputs. this study was supported by a scholarship and research funding from the macquarie university australia’s international research training program (irtp). amuji et al.‐ simulated effect of heat and water stress on three tomatoes growth and yield 155 fao, 2019 ­ sahel ‐ regional overview (july 2019). ­ food 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https://ucanr.edu/repository/view.cfm?article=83755%20&groupid=9 https://ucanr.edu/repository/view.cfm?article=83755%20&groupid=9 https://ucanr.edu/repository/view.cfm?article=83755%20&groupid=9 impaginato 305 adv. hort. sci., 2021 35(3): 305­318 doi: 10.36253/ahsc­10630 (*) corresponding author: cosimo.taiti@unifi.it citation: comparini d., taiti c., lanza m., vita f., pan­ dolfi c., luti s., spinelli f., pazzagli l., man­ cuso s., 2021 ­ comparison of wild and domesti‐ cated hot peppers fruit: volatile emissions, pun‐ gency and protein profiles. ­ adv. hort. sci., 35(3): 305­318. copyright: © 2021 comparini d., taiti c., lanza m., vita f., pandolfi c., luti s., spinelli f., pazzagli l., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2021 accepted for publication 3 august 2021 ahs advances in horticultural science comparison of wild and domesticated hot peppers fruit: volatile emissions, pungency and protein profiles d. comparini 1, taiti c. 1 (*), lanza m. 2, vita f. 3, pandolfi c.1, luti s. 4, spinelli f. 1, pazzagli l. 4, mancuso s. 1 1 department of agrifood production and environmental sciences, university of florence, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 ionicon analytik gmbh, eduard bodem gasse 3, 6020 innsbruck, austria. 3 department of agriculture, food and environment, university of pisa, via mariscoglio, 34, 56124 pisa, italy. 4 department of biomedical experimental and clinical sciences, university of florence, viale morgagni, 50, 50134 florence, italy. key words: artificial neural network, capsicum spp. classification, capsaicin, sds­ page, vocs. abstract: capsicum plant species are globally cultivated in warm and temperate regions, being important for agro­economic, biological and cultural aspects. while their worldwide spread and their ability of cross­pollination to easily hybridize play an important role in the formation of numerous species and vari­ eties but also create confusion for their classification. for this reason, the cate­ gorization of species and varieties is complex and several methods have been used to evaluate pepper plant origin and evolution. therefore, the objectives of this study were to compare a wild pepper (capsicum chacoense) with other two domesticated cultivars belonging to different species such as capsicum annuum and c. baccatum and draw conclusions about their origins using different approaches. for this purpose three methodologies have been used and com­ pared: the comparison of their fruits volatile organic compounds (vocs) emis­ sions , their capsaicin and dihydrocapsaicin content and the leaves proteomic profiles. the vocs analysis has been conducted by a time­of­flight mass spec­ trometry (tof­ms) with an innovative approach to better identify all the com­ pounds detected, in particular using two different ionization agents (h3o + and no+) to better identify all the compounds detected. the vocs and pungency analyses were then used to build back propagation neural networks (bpnn) and a random tree classifier to conduct a multivariate analysis and evaluate the most species­specific volatiles. the outcomes appeared to be a most accu­ rate approach with respect to the traditional varieties descriptors used for pep­ pers discrimination. the bpnn led to the identification of several putative volatiles as good candidates for the recognition of these species or significant nodes in a decision learning tool. finally, protein profiles have been obtained by sds­page analysis on the leaves to perform a fast proteomic comparison among the species. the protein profiles showed the c. baccatum and c. cha‐ coense were more similar to the domesticated pepper c. annuum. https://doi.org/10.36253/ahsc-10630 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 305­318 306 1. introduction origin and classification of capsicum the origin of chili peppers has been located in sev­ eral locations of latin america as testified by archeo­ logical records and pepper is usually classified as one of the first new world domesticated plants (pickersgill, 1969; long­solis, 1986; perry et al., 2007). peppers represent one of the most ancient plants cultivated in america together with the phaseolus l., maize and other plants of the cucurbitaceae family, fundamental ingredients of natives’ diet. about 36 different species belong to the genus capsicum and even today there are many wild species to be defined from the taxonomic point of view, consequently, we cannot exclude the existence of new individuals currently unknown (davenport, 2004). the identification and maintenance of the genetic diversity in capsicum are important to avoid genetic erosion. several species exist and capsicum species have recently been described from bolivia (nee et al., 2006). among these, only five species respectively c. annuum (variety annuum), c. chinense, c. frutescens, c. baccatum (variety pendolum and umbelicatum) and c. pubescens have been domesticated in the past by the american natives and later, in the post­ colombian period, they have been widely imported and cultivated in temperate and tropical regions for the characteristics of their fruits (mcleod et al., 1979; mcleod et al., 1983). the broad geographical distribution of this genus, usually used as feeds not only by humans but also by birds that don’t have receptors for capsaicin, associated with the antiquity of the origin and the high frequency of hybridization, created a broad genetic variability during the evolu­ tion and resulted in many morpho­qualitative differ­ ences among cultivars of homologous species (pozzobon et al., 2006). in fact peppers plants have been used for several reasons, starting from their high nutritional value, good content in vitamins but also as medicine or mystic rituals. this determined a rapid diffusion of these plants in the old continent, stimulated also by their spicy flavour similar to the black pepper, a very valued spice, whilst peppers pre­ sented higher production and more flexibility as food. furthermore the mechanism of cross­pollina­ tion and the ability of peppers to easy hybridize played another role in the formation of numerous varieties with specific features. during the last cen­ turies, botanists have been active to cross­pollinate creating confusion for the classification of the vari­ eties and the identification of indigenes species. for these reasons the zone where each species originat­ ed is still subjected to debate. brazil is considered as the center of origin of the genus capsicum and cur­ rently represents the most important source of genetic diversity (buso et al., 2001). for example, a recent survey of chili cultivars from the state of roraima in northwestern brazil noted 60 distinct lan­ draces of peppers from four different species: capsicum annuum; c. frutescens, c. baccatum and c. chinense (barbosa et al., 2006). therefore brazil and bolivia possess the highest number of wild species (pickersgill, 1984), however, a broad and complete study about diversity has not been done yet for the native species. it is anticipated that continuing, plant exploration in southern peru, bolivia and brazil, will yield additional new narrow endemic pepper species (russo, 2012). as consequence, a taxonomic classification of peppers and the determination of all relations among species are challenging and created many debates about the origin and evolution of the capsicum genus. most current peppers are derived from domesticated species. the difficulties in identifying them lie in dialectal names linked with the local tradi­ tion, making challenging their classification. for example, a study among the mexican peppers popu­ lation underlined that among about 200 common names used to refer to different peppers, only approximately 15 types were different commercial pods (russo, 2012). this situation contributes in the last filthy years several authors to focus on the taxon­ omy and origin of the species of this genus without coming out with a complete and flawless analysis (heiser and smith, 1953; pickersgill, 1988; hunziker 2001; barboza and bianchetti, 2005). it has been proposed that the c. chacoense in bolivia is one of the most ancestral nuclear centers for the origin of capsicum (2n=24) and it is considered as the basal for the evolution of the species (mcleod et al., 1982). furthermore, a study based on karyotype and other considerations of different capsicum by moscone et al. (2007) concluded the existence of a triple origin of domesticated capsicum species. the whole genus has been hypothesized to be originated in the tropical regions of the americas and the ances­ tral species should be born in the chaco zone which extends into several parts in the country of argentina, bolivia, brasil and paraguay and propose that the genus “chacoense” as one of the most primitive origi­ comparini et al. ‐ wild vs domesticated peppers 307 nated pepper taxon. all the lines that we know today derived from this first line of evolution and have devel­ oped and differentiated according to the characteris­ tics of the new habitat: in the north of the amazon to the ancient forms of c. annuum, on the north coast of brazil and venezuela in the forms of c. chinense and c. frutescens; and in a subsequent period from the same ancestral form were differentiated to c. rhomboideum (colombia, ecuador) and c. galapagoense (galapagos islands) (moscone et al., 2007). starting from a second evolutionary line instead, they are originated different species: in subtropical areas of brazil and later the c. baccatum and c. exim‐ ium; in the arid regions of peru the c. cardenasii, c. tovarii and c. pubescens, while in paraguay c. flexuo‐ sum and c. praetermissum. finally, it was hypothe­ sized that the migration of ancestral forms of c. flex‐ uosum and c. praetermissum gave rise, in some areas of brazil, the largest center of diversification of the genus represented by the large group of wild species in 26 chromosomes (moscone et al. , 2007). currently, the species that best reflects the ancestral morphologically and physiologically, is the c. cha‐ coense (hunziker, 2001). c. chacoense has also been used in breeding c. annuum programs, focusing on tobacco mosaic virus resistance (boukema, 1982). the wild species with 24 and 26 chromosomes pre­ sent different morphological traits and geographical distribution (pozzobon et al., 2006). the differences that arise are probably related to the different agents of seed dispersal, birds for the first whilst bats and other small mammals for species of 26 chromosomes that present hanging fruit, inconspicuous, and a little spicy. in a second time, the domestication and subse­ quent human selection caused a selective pressure in favor of large hanging fruits, less attractive for birds with small exception (e.g. c. frutescens “tabasco” varieties), (aispes, 2010). the classification of chilies, like that of any multifarious group of cultivars, is con­ fusing. for example, a very large amount of pod types exist in c. annuum, c. chinense and c. bacca‐ tum (bosland and votava, 2012). usually, the classification of the genus capsicum and the varieties, belonging to each species, is car­ ried out based on morphological descriptors that define the shape of flowers and fruits (pickersgill, 1971; moscone et al., 2007; ince et al., 2009; sudré et al., 2010) and the current system for classifications involved genus, species, variety, pod type and culti­ vars (bosland and votava, 2012). however, other descriptors are considered essential for an accurate germplasm characterization, such as those indicated by ipgri (international plant genetic resources institute). furthermore, the characterization and evaluation of the species belonging to the genus capsicum are particularly interesting for breeders, gene banks, because of the large genetic variability available (guzmán et al., 2005; sudré et al., 2006; ince et al., 2009). they can furthermore be identified from the different flavors of the fruits and two main factors that contribute to the aroma perception are pungency and aroma, and these are associated with the fruit volatile compounds (taiti et al., 2015). with the advancement of computer technology, multivariate methods have become an important tool for taxonomic classification (ortiz et al., 2008). however, the procedures of statistical classification require a data set based on a large number of vari­ ables. thus, the paper has aimed to analyze and char­ acterize the wild c. chacoense and to compare its pro­ file of volatile emissions, pungency, and proteins with two domesticated species i.e. c. annuum and c. bac‐ catum to elucidate the evolution of the capsicum genus. for this reason, the volatiles compounds emis­ sions profile of each species has been used to build an artificial neural network and differentiate and identify the species. finally, an additional proteomic analysis of the leaves of the peppers has been used to evalu­ ate the relationships among the species and compare the results obtained from the volatiles emission, pun­ gency and protein expression profiles of each species. 2. materials and methods fresh pepper material ripe pepper fruits belonging to 3 different species were used in this study: capsicum annuum var. ciliegino, c. baccatum var. brasileiro and c. cha‐ coense (wild accession). the fruits were collected from ten plants, for each species, grown into green­ house (florence, italy). fully matured uniform­sized fruits were collected within 24h after the 100% color surface was reached. all plants were obtained from seeds, germination and growing phases were made following the same system used in the previous work of taiti et al., 2015. all plants belonging to each species were grown in the greenhouse in three differ­ ent rooms to avoid the effects of cross­pollination. capsaicin and dihydrocapsaicin quantification capsaicinoids are a group of alkaloids produced as adv. hort. sci., 2021 35(3): 305­318 308 secondary metabolites by chili peppers responsible for the pungency. among several structural analogs capsaicinoids, capsaicin and dihydrocapsaicin are the two most potent and abundant compounds account­ ing for more than 90% of total capsaicinoids in chili pepper (ziino et al., 2009). capsaicin and dihydrocap­ saicin have been extracted from whole frozen fruits and calculated as the average of five extraction for each species (n=5, sd). about 2 g of frozen fruits have been weighted and then pulverized in 10 ml of cold acetone at 4°c and kept overnight. then 50 µl of the sample have been collected by using a 0.22 µm filter syringe and used for the quantification. all data have been calculated as µg of capsaicin or dihydro­ capsaicin content per gram of fresh weight after nor­ malization based on the exact weight of the initial fresh sample. rp­hplc quantification of capsaicin and dihydro­ capsaicin was performed by using a c18 column, 3 µm, 15x4.6 cm (supelco, bellefonte, pennsylvania, usa). the stock solution containing capsaicin and dihydrocapsaicin (cod, 360376 sigma­aldrich, st louis, mo, usa) was prepared in 20% acetonitrile at concentrations of 7.5 mg/ml. for calibration curves construction 30 µl, 60 µl, 90 µl, 120 µl, 150 µl, 300 µl and 500µl of stock solution were analyzed obtaining a linear curve for both capsaicin and dihydrocapsaicin (r2=0.9912 and r2=0.9923, respectively). elution gra­ dient was performed at a flow rate of 0.8 ml min­1 with the following solvent system: 10mm trifluo­ roacetic acid (tfa) in acetonitrile (solvent a); 10mm tfa in water (solvent b). the gradient used was 20% a for 2 min, from 20% to 100% a in 15 min, holding at 100% a for 10 min, from 100% a to 20% a in 2 min, and detection was based on uv absorbance at 280 nm. under these conditions, the capsaicin peak appeared at a retention time (rt) of 10.6 min and dihydrocapsaicin at rt of 11.3 min. quantification was calculated using the chromeleon software. volatiles organic compounds (vocs) analysis sri‐ms tof protocols. for headspace analysis, pepper fruits have been selected among ten plants and the uniform­sized fruits were collected at the optimal ripening stage (100% of coloration). for each species ten samples were analyzed, each constituted of 10 grams of fresh pepper fruits. using a commer­ cial ptr­tof 8000 (ionicon analytik gmbh, innsbruck, austria) instrument with sri­ms (switchable reagent ions ­ mass spectrum) upgrade, the analysis of the samples was carried out following a similar procedure described in taiti et al. (2015). in short, 10 g of freshly cut chili pepper (including the seeds) without any pre­treatment were placed in a glass jar (750 ml at 25°c, with a dynamic headspace flushing flow rate of 200 ml min­1) equipped with two teflon inlet and outlet tubes on the opposite side, which were respectively connected to a zero­air generator (peak scientific) and the ptr­tof ms instrument. moreover, for the first time, the switchable reagent ion system (h3o + and no+) has been used to produce different ionization agents for capsicum study. using the additional precursor (reagent) ions as no+, besides the usual h3o+, improved the analytical possibilities of this technique (wang et al., 2004; mochalski et al., 2014). in particu­ lar, the sri system allows: (1) the analysis of vocs which are not detectable with the reference ion h3o + (e.g. alkanes); (2) the separation of isobaric com­ pounds as in the case of aldehydes and ketones using no+ (jordan et al., 2009; del pulgar et al., 2013). for a detailed explanation of the system see blake et al. (2006) and blake et al. (2009). all samples were ana­ lyzed using the same procedure and the vocs were assessed with h3o + and no+ as reagent ions; the drift conditions for each primary ion used are reported in table 1. the sampling time for each channel of tof acquisition was 0.2 ns, for a mass spectrum com­ prised between m/z 20­210. the duration of a single sample measurement was 120 seconds, which corre­ sponds to 60 mass spectra. all the samples were ana­ lyzed in an air­conditioned room, with a constant temperature of 25±1°c (mancuso et al., 2015). the sri­ms upgrade consists of an additional mass flow controllers for the respective reagent gases (water vapor for h3o +, charcoal filtered air for no+); for a detailed explanation see jordan et al. (2009). in short, the use of no+ and the h3o + as a reagent ion table 1 ­ instrumental condition throughout the experiment primary ion drift voltage (v) pressure (mb) temperature (°c) us (v) uso (v) ihc (ma) udx (v) e/n (td) mass calibration mass calibration mass calibration h3o + 594 2.25 110 110 85 4.0 35 140 21.022 29.997 59.041 no+ 600 2.30 110 25 80 5.0 36 137 21.022 32.002 47.997 comparini et al. ‐ wild vs domesticated peppers 309 table 2 ­ list of the average m/z­signals that can be specifically assigned using h3o + as reagent ion: volatile organic compounds headspace intensity expressed in ncps (n=10; ±sd); chemical formulae and tentative identifications for each signals detected; the compounds identification was linked to the ptr­tof­ms pattern fragmentation references (a) or previously reported in capsicum species (b) measured mass (m/z) capsicum annuum ciliegino capsicum baccatum brasileiro capsicum chacoense wild pepper sum formula tentative identification ptr pattern fragmentation a capsicum literature b 1 27.022 799.86±234.50 200.93±109.28 496.95±140.33 c2h3 + acetylene 2 31.018* 615.25±205.40 184.12±62.55 81.22±21.30 ch3o + formaldehyde [2] 3 33.033 1712.59±220.12 415.39±167.27 1067.70±200.55 ch5o + methanol [1] 4 41.038 2612.818±638.34 983.12±320.11 378.78±100.33 c3h5 + alkyl fragment (alcohols and esters) [1] 5 43.054 578.30±190.22 133.23±33.90 101.94±25.90 c3h7 + alkyl fragment (alcohols) [1] 6 45.033 3049.50±982.28 529.04±67.40 5638.53±1230.45 c2h5o + acetaldheyde [2] 7 47.010 353.83±111.89 224.89±51.58 636.43±160.55 ch3o2 + formic acid/formates [2] 8 47.049 4.80±1.20 3.50±0.80 2.95±1.00 c2h7o + ethanol [1] rodriguez­burruezzo et al., 2010 9 53.030 102.26±35.40 50.90±22.33 17.53±5.00 c4h5 + cyclobutadiene 10 55.050 6.82±1.90 1.99±0.50 3.32±1.30 c4h7 + c4 aldehydes fragment [1] 11 57.033* 2910.50±1109.12 366.28±78.54 179.55±49.50 c3h5o + c3 aldehydes and ketones fragments [6] 12 57.069 274.43±85.40 45.91±17.22 33.02±9.25 c4h9 + alcohol fragments (1­butanol, 1­ pentanol, 1­hexanol, 2­methyl­1­ propanol, pentanol, 1­heptanol, octanol, nonanol) [1] 13 59.049* 553.06±133.09 196.04±65.90 459.80±180.44 c3h7o + propanal, acetone [2] ziino et al., 2009 14 61.028 303.46±44.65 120.22±39.10 355.05±44.44 c2h5o2 + acetates [6] ziino et al., 2009 15 63.027 4.45±1.30 4.64±1.20 5.05±0.80 c2h7s + dimethylsulfide taiti et al., 2015 * the signals that mostly contributed to the bpnn classification have been marked. to be continued... improves the analytical performance of the tool, par­ ticularly for the separation of isobaric compounds and for the detection of compounds with proton affinities lower than that of water. furthermore, as reported by edtbauer et al. (2014) when the ptr­ms instrument works in no+ mode can improve the selectivity of compounds detection. mass data statistical analysis. since the external calibration provided by the tool gave a poor mass accuracy, it has been performed off­line thus ensur­ ing, a high mass accuracy generally than 0.001th, which in most cases allowed the formula identifica­ tion (taiti et al., 2017). the raw data of each peak spectrum (calculated as number of counts per sec­ ond, cps) were acquired with the software tofdaq (tofwerk ag, switzerland) by setting a dead time of 20 ns for the poisson correction, instead, for peak quantification, the resulting data were corrected according to the duty cycle and the signals were nor­ malized (ncps “normalized count per second”) as described by herbig et al. (2009). moreover, the poisson correction has been applied to correct all spectra for any count losses. finally, vocs putative identification was based on a high instrumental mass resolution and the fragmentation patterns of pure standards available in the bibliography (buhr et al., 2002; lee et al., 2006; maleknia et al., 2007; kim et al., 2009; tani, 2013; aprea et al., 2015; taiti et al., 2019) and integrated with previously detected vocs emitted from capsicum fruit available in literature (table 2 and 3). data processing and classification methods back propagation neural network. in this study, the capsaicinoid contents and the 52 volatile signals detected by the ptr­tof­ms by using h3o + as reagent ion were used as input layers, and the 3 pepper species represented the output (30 single pepper fruits analysis, ten for each species). the bpnn was built using a data mining software (weka 3.6.14) and the multilayer perceptron classifier was used for the classification. two bpnn were made, one with only the vocs emission profiles, and another with vocs and capsai­ cinoids. the number of hidden neurons and the num­ ber of iterations was adjusted to optimize the neural network activity. many factors, such as learning schemes, numbers of nodes, and connections between them, play an important role in determing of the best configuration of the hidden layers (zurada 310 adv. hort. sci., 2021 35(3): 305­318 table 2 ­ list of the average m/z­signals that can be specifically assigned using h3o + as reagent ion: volatile organic compounds headspace intensity expressed in ncps (n=10; ±sd); chemical formulae and tentative identifications for each signals detected; the compounds identification was linked to the ptr­tof­ms pattern fragmentation references (a) or previously reported in capsicum species (b) measured mass (m/z) capsicum annuum ciliegino capsicum baccatum brasileiro capsicum chacoense wild pepper sum formula tentative identification ptr pattern fragmentation a capsicum literature b 16 65.038 4.87±3.25 2.49±0.80 3.45±1.11 c5h5 + alkyl fragment/ terpenes fragment [1/2] 17 67.050 61.35±16.66 13.86±6.40 6.77±1.40 c5h7 + terpenes fragment [2] 18 69.033 80.90±21.98 17.82±4.50 11.92±4.10 c4h5o + furan 19 69.069 395.06±133.90 139.11±80.33 92.50±18.44 c5h9 + isoprene/alkyl fragment (e.g. 2­ methylbutanal, 1­octen­3­ol) [2/6] 20 71.049* 21.29±7.10 6.52±2.12 8.70±2.50 c4h7o + 2­butenal taiti et al., 2015 21 71.086 15.92±5.90 4.71±190 7.65±1.90 c5h11 + alchol (3­methyl­1­butanol, pentanol, iso­pentanol, 2­ethyl­1­hexanol, eggink et al., 2012 a 22 73.060* 43.02±12.35 6.62±2.10 15.45±4.90 c4h9o + isobutanal/butanone/butanal [1] ziino et al., 2009 23 75.044 57.76±22.25 14.33±4.50 12.16±6.00 c3h7o2 + butanol/methyl acetate/propanoates [1] 24 77.038 7.09±2.79 7.77±2.20 14.53±3.50 c6h5 + alkyl fragment 25 79.054 134.86±48.60 22.84±7.98 15.7±4.10 c6h7 + benzene/alkyl and terpenes fragment [2] 26 81.068 949.61±301.39 178.02±68.78 152.06±50.30 c6h9 + terpenes fragment/aldehydes frag­ ment (trans­ 2­hexenal) [4] taiti et al., 2015 27 83.049 76.90±19.75 32.36±9.94 13.69±2.90 c5h7o + 2­methylfuran rodriguez­burruezzo et al., 2010 28 83.086 175.59±50.10 92.78±29.50 16.29±4.90 c6h11 + c6 compounds/ hexenol fragment [6] 29 85.064 46.91±15.40 9.90±3.10 18.20±4.30 c5h9o + methyl­butenal/1­penten­3­one ziino et al., 2009 30 85.101* 32.25±12.30 2.08±0.94 7.41±2.00 c6h13 + alcohol (1­hexanol/nonanol) ziino et al., 2009 31 87.045* 25.21±5.40 6.92±3.20 12.65±4.10 c4h7o2 + diacetyl /2,3­butanedione [1] ziino et al., 2009 32 87.080 10.23±1.21 6.44±0.95 2.01±0.55 c5h11o + 2,3­methylbutanal /(z)­2­penten­1­ ol/3­pentanone [1] rodriguez­burruezzo et al., 2010 33 91.075 22.38±5.40 17.68±4.40 6.35±1.10 c4h11o2 + 2,3­butanediol/monoterpene ketone [5] ziino et al., 2009 34 93.069 7.07±1.95 12.12±5.40 7.59±1.50 c7h9 + terpene fragments (e.g. cymene, limonene) [3] rodriguez­burruezzo et al., 2010 35 95.086 27.45±6.99 4.18±2.20 14.97±1.10 c7h11 + 1­methyl­1,4­cyclohexadiene eggink et al., 2012 b 36 97.064 50.45±10.50 14.90±4.80 10.18±2.50 c6h9o + 2­ethylfuran rodriguez­burruezzo et al., 2010 37 99.080* 60.65±20.30 8.62±2.75 15.98±6.30 c6h11o + cis­3­hexenal/ (e)­2­hexenal rodriguez­burruezzo et al., 2010 38 101.096 33.66±5.70 9.62±3.55 17.02±5.50 c6h12o + hexanal/ (e)­2­hexenol [1] ziino et al., 2009 39 103.075 54.22±20.44 18.20±6.50 9.28±2.80 c5h11o2 + 3­methylbutanoic acid [6] zimmermann and schieberle, 2000, azcarate et al., 2010 40 105.069 4.98±0.65 5.23±0.78 5.96±1.12 c8h9 + styrene/styrol/phenylethanol [6] rodriguez­burruezzo et al., 2010 41 107.085 9.50±5.44 3.65±2.30 12.85±7.20 c8h11 + p­xilene [6] eggink et al., 2012 a 42 109.101 13.85±4.50 3.15±0.90 6.06±2.20 c8h13 + terpenes fragments [3] 43 115.111* 17.27±4.30 6.04±2.50 1.36±0.50 c7h14o + heptanal [6] ziino et al., 2009 44 117.091 6.35±1.60 14.05±3.00 3.96±1.30 c6h13o2 + hexanoic acid/hexanoates [6] eggink et al., 2012 b 45 119.085 7.75±3.70 3.26±1.35 4.89±2.40 c9h11 + terpenes fragment [3] ziino et al., 2009 46 121.101 6.40±2.50 15.08±3.50 6.10±2.00 c9h13 + terpenes fragment [3] 47 123.120 8.40±2.80 1.65±0.60 1.72±0.85 c9h15 + sesquiterpene fragments [4] 48 135.117* 7.15±1.83 2.69±1.40 6.62±1.30 c10h15 + p­cymene/monoterpene ketone frag­ ment [6/5] ziino et al., 2009 49 137.132* 17.12±4.05 9.75±1.10 23.88±4.35 c10h17 + monoterpenes (e.g. (z)­b­ocimene) [5] eggink et al., 2012 a 50 149.132* 7.13±4.40 3.91±1.05 9.65±3.20 c11h17 + sesquiterpenes fragments (e.g. ectocarpene) [4] taiti et al., 2015 51 205.195 25.44±7.10 11.90±3.33 9.59±2.65 c15h25+ sesquiterpenes [4] eggink et al., 2012 b total vocs emission (average ncps) 22116 4850 13214 * the signals that mostly contributed to the bpnn classification have been marked. comparini et al. ‐ wild vs domesticated peppers 311 table 3 ­ sift­ms signals obtained using no+ as reagent ions, in the range between to m/z 20­200. the analysis showed only the signals with intensity expressed in ncps higher than 1 (n=10; ±sd) number of compounds measured mass (m/z) capsicum annuum ciliegino capsicum baccatum brasileiro capsicum chacoense wild pepper sum formula tentative identification 1 41 10.91±3.10 45.53±16.60 19.47±8.85 c3h5 + alkyl fragment (alcohols and esters) 2 45 18.72±2.10 25.67±8.40 21.61±6.80 c2h5o + c6 fragment (e.g. (e)­2­hexenol) 3 57 7.41±2.98 48.61±18.07 9.68±1.55 c3h5o + c3 aldehyde 4 58 50.01±8.86 39.10±3.25 25.63±9.52 c3h6o + c3 ketones 5 69 13.07±2.33 45.73±18.80 20.95±8.55 c4h4o + furan 6 83 15.11±2.23 79.05±20.44 13.85±0.60 c6h11 + c6 aldehydes (e.g (e)­2­hexenol) 7 84 4.17±0.88 3.64±0.55 2.94±0.90 c5h8o + c5 8 85 7.62±3.20 27.85±7.22 10.69±3.33 c5h11o + valeraldehyde 9 88 59.06±24.44 44.29±27.70 61.12±20.32 c3h6o no+ acetone 10 99 11.12±2.33 15.24±3.33 10.76±2.22 c6h13o + hexanal/ c6 aldehydes (e.g (e)­2­ hexenol) 11 106 3.67±0.78 2.87±0.30 2.780±6.52 c8h11 + xylene 12 113 1.05±0.33 6.65±.55 1.34±0.55 c7h13o + heptenal 13 114 2.60±0.44 10.15±1.1 2.16±0.30 c5h8 no2 + cluster c5 unsaturated ketones 14 116 9.47±2.63 26.33±8.33 6.39±1.20 c5h10 no2 cluster c5 ketones/pentanone 15 128 3.34±0.81 2.04±1.01 1.88±0.72 c8h16o + 6­methyl­5­hepten­2­ol 16 136 17.85±1.66 7.78±0.95 26.57±6.77 c10h16 + monoterpene compounds 17 144 0.80±0.62 3.21±0.81 0.70±0.55 c6h10 no3 + cluster hexanedione/heptanone 18 166 0.73±±55 2.38±0.35 0.80±0.68 c10h16 no+ monoterpenes fragment total vocs emission average (ncps) 236.71 436.09 239.29 1992; zurada and malinowski, 1994). in our case, the minimum error was reached with a network com­ posed of 29 hidden neurons for both bpnn, posi­ tioned on one level, with the hidden layer activated by a logistic sigmoid activation function: f(x) = 1/(1+e­x) (1) these sigmoid functions fix the output signal limit between 0 and 1. the resulting function works as an output logic­gate that can be opened (1) or closed (0). also, as part of a continuous function it can hap­ pen that a gate is partially opened (i.e., its value results between 0 and 1). ideally, only a group of out­ puts, which represents an accession, would express a value of 1 (meaning correct identification) while the remaining groups would show a value of 0 (incorrect identification). in reality, this take place rarely, for this reason it is usually considered as “incorrect” a value closer to zero (wrong identification), while “correct” when the resulted value is close to 1 (cor­ rect identification) (pandolfi et al., 2009). a 10­folds cross­validation was applied to test the performance of the model. the original dataset was essentially randomly segregated into 10 equal­sized groups. each set is divided into two groups: 90% of data are used for training the network and 10% of data are used for the validation test. the cross­validation process is then repeated 10 times (the folds), in which every time a subsamples is validated. the results deriving from all folds are finally averaged to result in a single evaluation of the network’s performance. the values of the identification for each species were highlighted using a misidentification matrix. all identification processes executed by the network were averaged and the results represented in table 4. the rows refer to the species in the test set, the columns report the species to which the test plants are referred by the neural network. an “attribute selection filter” provided by weka was also applied to the two sets of data, to determine the more dis­ criminant parameters. random tree. the 51 volatile signals detected by the ptr­tof­ms using h3o + (30 single pepper fruits analysis, ten for each species) were also analyzed using the “random tree” algorithm, a decision tree adv. hort. sci., 2021 35(3): 305­318 312 learning tool provided by the software (weka 3.6.14). in classification trees, each internal node is labeled with an input feature, which can be informative to detect similarities or differences among the pepper species. similarly to the bpnn, a 10­folds cross validation was applied to test the performance of the model, and the results from the folds are then averaged to produce a single estimation of the performance of the algorithm. sds‐page protein analysis protein extraction and quantification. soluble pro­ teins were extracted from leaves of three chili pep­ per plants of each species (capsicum chacoense, c. annuum and c. baccatum) according to vita et al. (2013), with some modifications. in short, for each analysis 100 mg of fresh leaves were grounded in liq­ uid nitrogen and homogenized with 1 ml of extrac­ tion buffer (5 m urea, 2 m thiourea, 40 mm tris­hcl, 2% chaps, 50 mm dtt). the homogenates were cen­ trifuged for 15 min at 15,000 rpm. supernatants were precipitated using tca (15%, v/v) containing 0.007% β­mercaptoethanol in acetone at ­20°c for 2 h and successively at 4°c for a minimum of 2 h. samples were centrifuged at 4°c for 15 min at 14,000 rpm, supernatants were discarded and pellets were washed twice with ice­cold acetone containing 0.007% β­mercaptoethanol. pellets were dissolved in a rehydration buffer (5 m urea, 2 m thiourea, 4% chaps, 40 mm dtt). protein quantification was per­ formed using a bradford­based assay kit assay (bio­ rad hercules, ca), using bovine serum albumin as a standard. sds‐page. the protein separation was carried out by the established technique of sds­page. in detail, the gels used (size 20 * 20 cm) had a thickness of 1 mm and were constituted by a stacking gel (4.8% t, 1.3% c, ph 6.8) and a running gel (15%, 1.3% c, ph 8.5). sds­page analyses were performed 4 times (n=4). electrophoresis runs were carried out using the protean xi cells (bio­rad laboratories, inc, hercules ca) with specific parameters (for each gel 25 ma, 8h running time, temperature 15°c). precision plus protein™ unstained was the molecular marker used for the essay (bio­rad laboratories inc., hercules, ca). the protein samples were analyzed by sds­page on gels stained with the brilliant blue g­ colloidal concentrate coomassie (sigma­aldrich) according to the manufacturer’s instructions. the images of each gel were acquired using a bio­ rad densitometer gs­800 ™ in greyscale colors, with a definition of 300 dpi. the images were analyzed using the software quantity one 1­d analysis ™ soft­ ware (bio­rad laboratories, inc, hercules ca). dendrogram based on signal quantities was created using correlation­based distances and ward’s method of agglomeration was used in the present analysis (ward, 1963). 3. results and discussion capsaicinoid in all the species analyzed both the capsaicin and the dihydrocapsaicin (dhc) contributed to the pun­ gency of the fruits. as expected (stoica et al., 2016), the capsaicin content was higher than the dhc in all species and c. chacoense resulted in the higher con­ tent of about 300 µg/g of fresh weight (fw) com­ pared to c. baccatum whilst c. annum have been the most variable samples with an high standard devia­ tion that made it not statistical different from the other two species (fig. 1). interestingly the c. cha‐ coense resulted in the higher content of dhc of 243±36 µg/g fw, followed by the c. annum and c. baccatum that did not resulted statistical different fig. 1­ capsaicin and dihydrocapsaicin (dhc) content obtained from ripe fresh pepper fruits for each species analyzed, using hplc detection (n=5; sd). different letters repre­ sent statistical significance (anova, p<0.05). table 4 ­ confusion matrix derived from the random tree analy­ sis from the aromatic profiles of the pepper species obtained using a decision tree learning tool c. baccatum c. chacoense c. annuum c. baccatum 9 1 0 c. chacoense 0 10 0 c. annuum 0 0 10 comparini et al. ‐ wild vs domesticated peppers 313 from each other with respectively 73±11 µg/g fw and 40±11 µg/g fw. the capsaicin content influences more the spiciness followed by the dhc, therefore c. baccatum have showed the lowest level of spiciness. ptr‐tof‐ms volatiles compounds analysis the volatiles compounds analysis by sri­tof­ms revealed the volatile profile of each pepper species. the extraction of every single peak permitted the detection and the quantification of specific signals resultant of the protonation of numerous vocs (m/z range = 20­210). from the volatile fraction composi­ tion of different pepper species, were detected many volatile compounds specifically alcohols, aldehydes, esters, ketones, hydrocarbons and terpenes com­ pounds. for each peak identified using h3o + and no+ as ion reagents, all of the m/z detected have been assigned to the mass formulas reported respectively in table 2 and 3 and expressed in ncps higher than 1 (n=10; ±sd). in particular, by trusting the high accura­ cy and resolution of this tool, the chemical com­ pounds have been tentatively proposed and matched with the existing documentation of vocs in literature and by the acknowledged vocs emitted by peppers. thus, the identification of the compounds has been further improved by the use of two different reagents ions. all the peaks obtained have been fil­ tered and 51 mass spectral peaks have been detect­ ed when h3o + was the reagent ion and 18 using no+ as reagent ion. remarkably even if the pepper analyzed derived from different species (in particular capsicum chacoense), all the peaks identified using h3o + or no+ as reagent ions were always present in the three species of chili pepper included in this study. moreover, since the aroma is linked to the species and varieties (taiti et al., 2019) the differences of vocs emission among these three hot peppers were expected (table 2 and 3). above all, by using h3o +, c. annuum showed the highest total vocs emission (22,116 ncps) followed by c. chacoense (13,214 ncps) and c. baccatum (4,850 ncps) as reported in table 2. in particular, c. annuum (“ciliegino” var.) seems to have a richest volatile profile compared to the other ones and showed the highest vocs intensity for the compounds linked to the herbaceous notes. this trend could be confirmed by the high intensity of compounds detected at m/z 81.069, 83.086, 85.101, 99.080 and 101.096 all identified as c6 compounds (table 2). notably, peaks that confirmed this trend were detected at c4h9 + (measured at m/z=57.069) probably derived from alkyl fragment (hexanol/valeric acid), c5h7 + (measured m/z=67.055) and c6h9 + (measured m/z=81.069) likely correspond to alkyl fragment (isoprene and terpenes or aldehy­ des fragments respectively), c6h13 + (measured m/z=85.101) probably refers to fragments of 1­ hexanol and/or nonanol, c5h11o2 + (measured m/z=103.075) refers as 3­methylbutanoic acid and c15h25 + (measured m/z=205.195) attributed to sesquiterpene compounds. on the contrary, the vocs emission observed in the wild accession (c. chacoense) is characterized by the high emission of signals detected at m/z 33.033, 45.033 and 137.132, with the first two compounds which are linked to the ripening process whilst the last compound belongs to the terpene class. moreover, it is interesting to note that the signal intensity of monoterpenes (m/z 137.132) is higher in c. chacoense than that observed for sesquiterpenes (m/z 205.101), in contrast to the emissions of c. annum and c. baccatum (fig. 2, table 2). regarding the wide chemical classes of terpenes, common compounds which strongly contributes to the aroma of fruits and vegetables (rodríguez­ burruezo et al., 2010), in this study were identified several peaks belonging to monoterpenes (c­10) and sesquiterpenes (c­15) while no one was detected as oxygenated terpenes. this result is also confirmed by what was already reported (rodríguez­burruezo et al., 2010), where the oxygenated terpenes were found at only traces and mainly in c. chinense. finally, the aroma of “brasileiro” variety (c. bacca‐ tum) was characterized by a lower signals intensity, except for the compounds detected at m/z 117.091 (ti: hexanoic acid/hexanoates) and m/z 121.101 (ti: terpenes fragment). additional representative chem­ ical classes of the volatile fraction were aliphatic fig. 2 ­ example of schematic chart of mass peaks detected with h3o + ion as reagent for each capsicum species used in this paper. signal intensities are given in normalize count per second (ncps) log­1 and higher than 1. adv. hort. sci., 2021 35(3): 305­318 314 aldehydes, alcohols, and branched hydrocarbons. in particular, hexanal, hexanol, cis­2­hexanal and cis­2­ hexenol were the main compounds that contribute to the flavour note described as the odour of freshly cut grass or ground leaves, which are typically pro­ duced in fresh capsicum fruits later on the tissue destruction (ziino et al., 2009). on the contrary by using no+ as donor ion, the differences of vocs emis­ sion among all varieties studied were smaller as well as the number of peaks detected (table 3). sometimes these spikes were different, while some­ times were identical to those obtained using h3o + as a reagent ion and this behavior is usual and has been already reported elsewhere (jordan et al., 2009). indeed, even if we use no+ as reagent ion in a com­ plex matrix, h3o + could occur in the ionization of the compounds present in the sample headspace (jordan et al., 2009, del pulgar et al., 2013). interesting, in contrast to what was observed with h3o+ , when using no+ the capsicum species c. baccatum showed for the majority of signals the highest intensity detected (table 3). moreover, table 3 shows signifi­ cant differences in the concentration of many peaks between the three pepper species, especially as far as they are concerned with protonated aldehydes and ketones. artificial neural networks two neural networks have been built: the first one uses only vocs emission, the second one com­ bines the pungency with capsaicin and dhc analysis as inputs layers. both the ann (artificial neural network) were able to discriminate among the acces­ sion with 100% accuracy. thus, the confusion matrix assigns all the aromatic profiles correctly to the relat­ ed pepper species (table 4). according to the attribute selection filter applied, 12 vocs profile and dhc content were the most discriminant parameters. in particular, the m/z signal of the vocs and the ten­ tative identification are reported here below and are marked in table 2 with the asterisk symbol: m/z 31.018 (formaldehyde), m/z 57.033 (c3 aldehydes and ketones), m/z 59.049 (propanal, acetone), m/z 71.049 (2­butenal), m/z 73.060 (isobutanal/ butanone), m/z 85.101 (1­hexanol/nonanol), m/z 99.080 (cis­3­hexenal/(e)­2­hexenal), m/z 115.111 (heptanal), m/z 135.117 (p­cymene), m/z 137.132 (monoterpenes) and m/z 149.132 (sesquiterpenes fragments). random tree the data coming from the aromatic profiles of the pepper species were also analyzed using a decision tree learning tool. the identification was successful in 96.6% of the cases, and, as shown in the confusion matrix, table 4, only one instance belonging to c. baccatum was incorrectly attributed to c. chacoense. the decision tree showing the two significant nodes is reported in figure 3. according to the tree, only two vocs are fundamental to discriminate among the three species: m/z 81.068 and m/z 85.064. protein profiles sds­page analyses performed on capsicum species (fig. 4) identified specific profiles linked to each sample. gel images were then analyzed to gen­ erate data like a phylogenetic tree based on similarity comparison (fig. 4) to graphically display relation­ ships among samples. dendrograms results showed as two samples, c. chacoense and c. baccatum, clus­ terized independently from the third sample c. annu‐ um. differences detected in the protein profiles could be associated with quantitative differences in the band densities as soon as some quantitative differ­ fig. 3 ­ on the top the vocs signal of the two most significant masses that help the identification of the species using a decision tree learning tool (n=10; sd). on the bottom, the two significant nodes have been reported. comparini et al. ‐ wild vs domesticated peppers 315 fig. 4 ­ (a) sds­page analysis of proteins getting from capsicum samples, where 1= c. chacoense; 2= c. annuum; 3= c. baccatum; m= molecular marker. (b) phylogenetic tree resulting from the analysis of protein by sds­page analy­ sis. image data were processed with quantity one software (bio­rad laboratories, inc, hercules ca) using ward's method for clustering (ward, 1963). ences. c. chacoense and c. baccatum showed a band (a) with a molecular weight slightly higher 20 kda that were not detected in the c. annuum sample (fig. 4). 4. conclusions our study underlined the high potential of the using the ptr­ sri­ms to obtain a species­specific fin­ gerprinting of the volatile compounds emitted by the pepper fruits. this technology can help to highlight particular vocs signals that are specific of species or specific growing conditions of chili pepper fruits with a rapid analysis without any pre­treatment of the samples. indeed, the switching reagent ion system in ptr­ms instrumentation was applied for the first time to analyze pepper fruits by using not only h3o+ but also no+ as precursor ions. this tool has permitted to find the vocs able to discriminate among the species and by using two ionization agents a more accurate identification of the volatile compounds has been possible. in particular, the ptr­ tof­ms analysis with no+ as reagent ion has allowed (i) the detection of aldehydes and ketones in separat­ ed peaks, (ii) to detect some molecules not found using h3o +, (iii) to confirm the results obtained using h3o+ as reagent ion for voc analysis. vocs results were thus confirmed by protein analysis according to qualitative and quantitative differences, which turn out to be able to differentiate samples within the capsicum genus. moreover, the multivariate statisti­ cal approach revealed that some of these com­ pounds can be successfully used for the species recognition in our artificial neural networks. the bpnn classifier utilized in the work had always 100% of success, both for h3o + and no+ (data not shown) and this was probably due to the marked differences in the volatiles emissions of the three species. further studies will aim to use the same method for a higher number of species, to challenge the analysis. our investigation permitted the identification of 12 promising vocs as more discriminant for each species and, among them, the masses m/z 81.068 and m/z 85.064 have been recognized as the most promising volatile markers of these species (fig. 3). in addition to 12 vocs, regarding the capsaicinoids, the dhc content was a more effective parameter to dis­ tinguish among domesticated and wild species of the genus capsicum with respect to the capsaicin. the results from the random tree have been according with the protein analysis, which showed that the c. chacoense and c. baccatum have more similarities with respect to the c. annuum, although this was a preliminary analysis and additional analysis need to be done to support our hypothesis. according to our findings, both domesticated species, i.e. c. baccatum and c. annuum differ from the wild species c. cha‐ coense, in particular, 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insecticidal properties, however many of those are still unknown in the south pacific. the information on plants were collected using online databases such as science direct, pubmed, google scholar, scopus and springer open in order to confirm the studies that support the insecticidal properties of plants present in the south pacific. the following study confirmed that there is a potential for the selected tmps suggesting enough evidence for their usage in the insecticidal activities. these plants would represent an alternative in crop protection due to its novel, safe and eco-friendly substitutes for its effective insecticidal properties. 1. introduction agricultural and animal origin stored products are destroyed by more than 600 species of beetle pests, 70 species of moths and about 355 species of mites (rajendran and sriranjini, 2008). these insect pests have greatly affected the food commodities and resulted in one of major problem to the food industries (isman, 2006). there are many concerns raised with the usage of synthetic chemicals for pest control. according to the food and agriculture organization of the united nations (fao, 2015), the consequences of high usage of synthetic pesticides in the pacific island countries (pic) has led to threats to human health and the environment. chemical pollution is a major concern to the environment and human body through food chains, which results in severe physiological disorders and diseases (oliva et al., 2001; baldi et al., 2003; briggs, 2003; saiyed et al., 2003; lemaire et al., 2004). the investigation in the area of natural resources have dramatically increased when it comes to public concern for the long term health and environmental effect of synthetic chemicals (coats, 1994; regnault-roger and hamraoui, 1995; lee et al., 1997; akhtar and isman, 2004; ukeh and umoetok, 2011; khani and heydarian, 2014; pandey et al., 2014). for (*) corresponding author: s11074077p@gmail.com citation: chand r.r., jokhan a.d., gopalan r.d., 2017 a mini-review of essential oils in the south pacific and their insecticidal properties. adv. hort. sci., 31(4): 295-310 copyright: © 2017 chand r.r, jokhan a.d., gopalan r.d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 june 2017 accepted for publication 5 october 2017 ahs advances in horticultural science review paper adv. hort. sci., 2017 31(4): 295-310 296 example, the massive use of chemical compound phosphine has led to environmental issues due to its insect resistance/ineffectiveness in the agricultural fields of some countries (opit et al., 2012). likewise, the use of methyl bromide for the fumigation has been reported as ozone-depleting substance and therefore removed completely from its use in some countries (rajendran and sriranjini, 2008). in view of the problems with current synthetic chemicals, there is a global interest in the search of alternative strategies and among them is the use of plant extracts. traditional aromatic plants have a wide impact on the agriculture, since plant derivatives are considered an integral source of pesticides. it represents a total of us $700.00 million market value with a total production of 45000 tons (tripathi et al., 2009). the science of natural products has advanced significantly in recent years benefiting humankind in the form of food, clothing, shelter, tools, medicines and crop protectant agents (copping and duke, 2007). the tmps are the mainstay for treatment of illness in the pacific for years. according to dasilva et al. (2004), traditional medicines hold a natural treasury that clearly depicts that pacific is rich in plant biodiversity. however, many plants in the pacific are yet to be exploited for their right purpose. hence, the present paper emphasizes on the insecticidal properties of essential oils from potential medicinal plants found in the south pacific. 2. overview of essential oils essential oils are diverse groups of natural products which are mainly produced by plants for defence, signalling or derive from their secondary metabolism (charles and simon, 1990; bakkali et al., 2008). these oils are volatile liquids which have a lower density than water (bakkali et al., 2008). essential oils are also known as ‘essence’ that are strong-smelling liquid components found in aromatic plants, grasses and trees (ríos, 2016). essential oils are mostly formed in plants such as from flowers, leaves, buds, fruits, seeds, bark and roots (isman, 2000; ríos, 2016). the synthesised essential oils are mostly kept in secondary cell cavities, epidermal cells, canals or glandular trichomes (nazzaro et al., 2013). the extraction of essential oils can be divided into conventional and recently developed methods. the conventional methods include; hydro-or steam distillation, solvent extraction and cold pressing. hydrodistillation being one of the oldest methods, dating back to 5000 years. while the recent methods for extracting essential oils include; supercritical fluid extraction and microwave-assisted extraction. the quality and quantity of chemical compounds are depended on different extraction method (fig. 1). essential oils containing between 20-60 components at different concentrations are considered to be very complex natural mixtures (pandey et al., 2014). essential oils are characterized by two or more major compounds with few trace compounds. for instance, the gc-ms analysis of ocimum tenuiflorum l. essential oils showed eugenol (58.20%), germacrene d (11.68%), cis-β-ocimene (10.79%) and βcaryophyllene (4.31%) as major compounds and terpinen-4-ol (1.01%), α-copaene (1.98%), δ-cadinene (1.44%) and few others as trace compounds (chand et al., 2016). the percentage composition of essential oils may vary with plants, environmental conditions, soil types and nutrients (masotti et al., 2003; erbil et al., 2015). formation of essential oils essential oils mostly have a high constituent of terpenes (farag et al., 1989). the other composition of essential oils include aromatic and aliphatic constituents that have different function to perform in relation to plants and animals (bakkali et al., 2008; chamorro et al., 2012; hossain et al., 2012; hrckova and velebny, 2012; tongnuanchan and benjakul, 2014). for instance, monoterpenes are used by plants for defence against pathogens, aid in seed dispersal and allelochemical functions, while alcohol groups have bactericidal, anti-infective and repellent properties (table 1). terpenes are usually formed using mevalonate pathways. mevalonate pathway is also known as isoprenoid pathway which occurs in all higher eukaryotes (corsini et al., 1993). this biosynthetic pathway fig. 1 overview of essential oil extraction methods (park and tak, 2015). chand et al. essential oils in the south pacific and their insecticidal properties 297 is used to produce dimethyl allyl pyrophosphate (dmapp) and isopentenyl pyrophosphate (ipp). these two compounds serve as the basis for the biosynthesis of molecules in diverse processes of terpene synthesis, protein prenylation, cell membrane maintenance, hormones, n-glycosylation and protein anchoring (chaichana, 2009; cooper and nicola, 2014). terpene biosynthesis involves addition of isopentenyl diphosphate (ipp; c5) to its isomer dimethylallyl diphosphate (dmapp; c5 can also form hemiterpenes) synthesizing geranyl diphosphate (gpp; c10) which is a precursor for synthesis of monoterpenes. gpp and fpp form monoterpenes and sesquiterpenes skeleton respectively. further condensation of enzyme-bound geranyl diphosphate (gpp; c10) with addition of ipp units forms farnasyl diphosphate (fpp; c15). geranylgeranyl diphosphate (ggpp; c20), that goes through series of reactions such as cyclization, rearrangement or coupling to form diterpenes and polyterpenes (figure 2 shows the parental precursors to synthesise terpenes). medicinal plants and their insecticidal properties insect control using plant materials is an ancient table 1 composition of essential oils with their general function in plants and animals group sub-group general functions in relation to plants and animals reference terpene hydrocarbon monoterpenes (c 10 h 16 ) producing defense against pathogens, help in the pollination, seed dispersal and allelochemical functions between plants and herbivores (lee et al., 1997; choi et al., 2006; ibanez et al., 2012) terpene hydrocarbon sesquiterpenes (c 15 h 24 ) contact irritant effects on insects (gonzalez-coloma et al., 2013) terpene hydrocarbon sesquiterpenes (c 15 h 24 ) also used as analgesic, spasmolytic agents, calming, slight hypotension and anti-inflammatory (chaichana, 2009) terpene hydrocarbon diterpenes (c 20 h 22 ) are known to have insecticidal, antimicrobial and anti-inflammatory properties (de oliveira et al., 2008; gonzalezcoloma et al., 2013) terpene hydrocarbon triterpenes (c 20 h 22 ) components of the surface waxes that accumulate in the intra-cuticle layers of stems and leaf surface for protection against dehydrations and herbivores (thimmappa et al., 2014) terpene hydrocarbon triterpenes (c 20 h 22 ) wide ranges of application of these compounds are in food, health, and industrial biotechnology sector (thimmappa et al., 2014; hadjimbei et al., 2015). oxygenated compounds alcohols these compounds have bactericidal, anti-infective and repellent properties (ukeh and umoetok, 2011) oxygenated compounds phenols have strong toxic effects, antiseptic and insecticidal properties (akhtar and isman, 2004; romero et al., 2013, cited in pinheiro et al., 2015) ethers severely affects the speed of germination, seedling growth and chlorophyll content (he et al., 2009) aldehydes used for antiviral, anti-inflammatory, hypotensive, vasodilators and antipyretic activities (dorman and deans, 2000; djilani and dicko, 2012 ) ketones toxic effects to a number of pests (kordali et al., 2007) ketones other uses of these compounds include anticoagulant, anti-inflammatory and digestant (peixoto et al., 2015). organic acids and esters special properties such as anti-fungal, anti-inflammatory and antispasmodic (chaichana, 2009) organic acids and esters have potential antimicrobial properties oxides used in aromatherapy, pharmaceuticals and agriculture (chaichana, 2009) fig. 2 synthesis of different classes of terpenes in plants. dmapp dimethylallyl diphosphate; ipp isopentenyl diphosphate; fpp farnesyl diphosphate; gpp geranyl diphosphate; ggpp geranylgeranyl diphosphate. adv. hort. sci., 2017 31(4): 295-310 298 organization, 1998). these selected plants exhibit insecticidal properties that are traditionally used in form of medicines in the south pacific (table 2). the general characterispractice all over the world (gonzalez-coloma et al., 2013). this review is focused on nineteen different families of tmps commonly found in the south pacific that are known to have essential oils (world health table 2 selected medicinal plants reported for its efficiency against the insects family scientific names common english name plant part used *traditional uses in the south pacific (treatment) active constituents/compounds efficiency against insects references lamiaceae ocimum tenuiflorum l., holy or sacred basil essential oils from leaves earache, nasal infections, cough, colds, stomach ache, hair lice, gastric, ulcer, flu, fevers, sore throat, and filariasis fumigant and repellent toxicity against the aleurodicus dispersus russell (spiralling whiteflies) (chand et al., 2016) lamiaceae ocimum basilicum linn. var. pilosum (willd)-benth holy or sacred basil leaf extract earache, nasal infections, cough, colds, stomach ache, hair lice, gastric, ulcer, flu, fevers, sore throat, and filariasis 4h-1-benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4methoxyphenyl)-, catechol and monoacetin repellency against the 3n7h and 3q8i of anopheles gambiae (african malaria mosquito) (gaddaguti et al., 2016) lamiaceae ocimum tenuiflorum var. cim ayu holy or sacred basil leaf extract earache, nasal infections, cough, colds, stomach ache, hair lice, gastric, ulcer, flu, fevers, sore throat, and filariasis 2-hexadecen-1-ol, phytol, dl-alphatocopherol, phenol-2-methoxy-3-(2-propenyl)-lycopersin, gamma-sitosterol, benzene, 1, 2-dimethoxy-4-(2-propenyl) repellency against the 3n7h and 3q8i of anopheles gambiae (african malaria mosquito) (gaddaguti et al., 2016) mimosaceae adenanthera pavonina l. holy or sacred basil seed extract leprosy trypsin inhibitor (apti) inhibitory activity of papain by trypsin inhibitor (apti) in callosobruchus maculatus (cowpea weevil) (macedo et al., 2004) mimosaceae adenanthera pavonina l. holy or sacred basil seed extract leprosy trypsin inhibitor (apti) inhibitory activity of papain by trypsin inhibitor (apti) in diatraea saccharalis (sugarcane borer) (da silva et al., 2012) asteraceae ageratum conyzoides l. goat weed canopy of plant species (above ground plant parts) infective hepatitis, eczyma, epilepsy, dizziness, diarrhoea, dysentery, sore, eyes, fever, headaches, intestinal worms, filariasis, vomiting, nausea, wounds and cuts 5, 6, 7, 8, 3’, 4’, 5’-heptamethoxyflavone and coumarin insecticidal activity of hexane extracts against the rhyzopertha dominica (f.) (lesser grain borer) (moreira et al., 2007) asteraceae ageratum conyzoides l. goat weed crude hexane extract of aerial parts of a. conyzoides infective hepatitis, eczyma, epilepsy, dizziness, diarrhoea, dysentery, sore, eyes, fever, headaches, intestinal worms, filariasis, vomiting, nausea, wounds and cuts repellent, antifeedant and toxic effects against helicovepra armigera (hübner) (cotton bollworm) (ragesh et al., 2016) asteraceae ageratum conyzoides l. goat weed crude petroleum ether extract aerial parts of a. conyzoides infective hepatitis, eczyma, epilepsy, dizziness, diarrhoea, dysentery, sore, eyes, fever, headaches, intestinal worms, filariasis, vomiting, nausea, wounds and cuts chromene precocene ii, two flavonoids: eupalestin and lucidin dimethyl ether insecticidal activity against musca domestica (housefly-third instar larvae), cynthia carye third, (butterfly-fourth and fifth instar larvae) and acanthoscelides obtectus (bean weevil) (calle et al., 1990) agavaceae aloe vera l. aloe, aloe vera leaf extract treat wounds and burns, sun burns, rashes, x-ray burns and stomach ache larvicidal activity on first to fourth instars larvae of aedes aegypti (yellow fever mosquito) (subramaniam et al., 2012) agavaceae aloe vera l. aloe, aloe vera leaf extract treat wounds and burns, sun burns, rashes, x-ray burns and stomach ache mosquitocidal activity against the anopheles stephensi (malaria vector) (dinesh et al., 2015) to be continued family scientific names common english name plant part used *traditional uses in the south pacific (treatment) active constituents/compounds efficiency against insects references agavaceae aloe vera l. aloe, aloe vera acetone, ethyl acetate, water, and ethanol extracts treat wounds and burns, sun burns, rashes, x-ray burns and stomach ache acaricidal activity against female adults of tetranychus cinnabarinus (carmine spider mite) (wei et al., 2011) annonaceae annona muricata l. soursop, custard apple crude ethanoic seed extract treating stomach ailments insecticidal activity against the spodoptera litura (leafworm moth) and trichoplusia ni larvae (cabbage looper) (leatemia and isman, 2004) annonaceae annona muricata l. soursop, custard apple fruit (pericarp) extract treating stomach ailments acetogenins -annonacin, annonacin a and annomuricin a. cytotoxicity towards the cell line u 937 (model cell line used in biomedical research) (jaramillo et al., 2000) annonaceae annona muricata l. soursop, custard apple ethanoic seed extract treating stomach ailments insecticidal activity against the trichoplusia ni (cabbage looper) and myzus persicae (green peach aphid) (ribeiro et al., 2014) meliaceae azadirachta indica a. juss. margosa, neem, indian lilac seed water extract for diabetes, skin diseases, asthma, syphilis and used as insecticide insecticidal activity against the trogodarma granariun (khapra beetle) (satti et al., 2010) meliaceae azadirachta indica a. juss. margosa, neem, indian lilac neem oil from seeds for diabetes, skin diseases, asthma, syphilis and used as insecticide insecticidal activity against the maruca testulalis geyer (mung moth) (jackai and oyediran, 1991) meliaceae azadirachta indica a. juss. margosa, neem, indian lilac crude ethanol extracts of leaves for diabetes, skin diseases, asthma, syphilis and used as insecticide insecticidal activity to adult tribolium confusum (flour beetle) (williams and mansingh, 1993) annonaceae cananga odorata (lam.) hook. f. & thoms. ylangylang, kenanga essential oil extracts from flowers earaches, toothaches, headaches, stomach aches, boils, skin irritation, coughs and dizziness fumigant and repellent toxicity against the aleurodicus dispersus russell (spiralling whiteflies) (chand et al., 2016) annonaceae cananga odorata (lam.) hook. f. & thoms. ylangylang, kenanga essential oil extracts from the leaves earaches, toothaches, headaches, stomach aches, boils, skin irritation, coughs and dizziness insecticidal activity (contact and fumigant toxicity) to sitophilus zeamais (greater grain weevil) (cheng et al., 2012) annonaceae cananga odorata (lam.) hook. f. & thoms. ylangylang, kenanga essential oil extracts from the leaves earaches, toothaches, headaches, stomach aches, boils, skin irritation, coughs and dizziness insecticidal activity against larvae of aedes aegypti (yellow fever mosquito) (vera et al., 2014) solanaceae capsicum frutescens l. chili pepper, red pepper, paprika methanol extract of fruits and leaves skin tuberculosis, mild conjunctivitis and jaundice, boils and cough insecticidal activity to 2nd and 3rd instar larvae of aedes aegypti (yellow fever mosquito) (vinayaka et al., 2010) solanaceae capsicum frutescens l. chili pepper, red pepper, paprika powdered fruits skin tuberculosis, mild conjunctivitis and jaundice, boils and cough discouraging oviposition and minimising damage to leaves of cowpea seeds (onu and aliyu, 1995) chand et al. essential oils in the south pacific and their insecticidal properties 299 table 2 selected medicinal plants reported for its efficiency against the insects (continued) to be continued adv. hort. sci., 2017 31(4): 295-310 300 family scientific names common english name plant part used *traditional uses in the south pacific (treatment) active constituents/compounds efficiency against insects references solanaceae capsicum frutescens l. chili pepper, red pepper, paprika ethanolic extract of fruit skin tuberculosis, mild conjunctivitis and jaundice, boils and cough larvicidal activites against aedes aegypti (yellow fever mosquito) and aedes albopictus (asian tiger mosquito) (alvarez et al., 2015) caricacea carica papaya l. papaya, pawpaw hexanic, acetonic and methanolic extracts of seed sores, high blood pressure and treat diarrhea insecticidal activity against the spodoptera frugiperda (fall armyworm) (figueroabrito et al., 2011) caricacea carica papaya l. papaya, pawpaw leaf extract sores, high blood pressure and treat diarrhea insecticidal toxicity against the lipaphis erysimi kal. (mustard aphids) (ujjan et al., 2014) caricacea carica papaya l. papaya, pawpaw chloroform seed extract sores, high blood pressure and treat diarrhea palmitic acid, oleic acid, or stearic acid insecticidal and insectistatic activities against the spodoptera frugiperda (fall armyworm) (pérezgutiérrez et al., 2011) caricacea carica papaya l. papaya, pawpaw chloroform seed extract sores, high blood pressure and treat diarrhea palmitic acid, oleic acid, or stearic acid insecticidal and insectistatic activities against the spodoptera frugiperda (fall armyworm) (pérezgutiérrez et al., 2011) caricacea carica papaya l. papaya, pawpaw larvicdial and pupicidal activity to the chikungunya vector, aedes aegypti (yellow fever mosquito) (kovendan et al., 2012) fabaceae (caesalpiniaceae) cassia alata l (senna alata) ringworm bush, roman candle tree ethanoic extracts of leaves skin diarrhoea, worms, purifies blood and scabies acaricidal activity to rhipicephalus (boophilus) annulatus (blue cattle tick) (ravindran et al., 2012) fabaceae (caesalpiniaceae) cassia alata l (senna alata) ringworm bush, roman candle tree solvent extract of fruits skin diarrhoea, worms, purifies blood and scabies toxic effects against the callosobruchus chinensis l. (adzuki bean weevil) (upadhyay et al., 2011) fabaceae (caesalpiniaceae) cassia alata l (senna alata) ringworm bush, roman candle tree leaf and stem extract skin diarrhoea, worms, purifies blood and scabies larvicidal effect on anopheles gambiae (african malaria mosquito), culex quinquefasciatus (southern house mosquito) and aedes aegypt (yellow fever mosquito) (edwin et al., 2013) apiaceae centella asiatica (l.) urban indian pennywort, asiatic pennywort leaf extract dysentery, fever, headache, diarrhea, pimples, rashes, itchy lumps, fractures, migraines and boils larvicidal and adult emergence inhibition effect against mosquito culex quinquefasciatus say (southern house mosquito) (rajkumar and jebanesan, 2005) apiaceae centella asiatica (l.) urban indian pennywort, asiatic pennywort leaf extract dysentery, fever, headache, diarrhea, pimples, rashes, itchy lumps, fractures, migraines and boils larvicidal and adulticidal activities against the malarial vector anopheles stephensi (asian malaria mosquito) (senthilkumar et al., 2009) apiaceae centella asiatica (l.) urban indian pennywort, asiatic pennywort leaf extract (hexane, diethyl ether, dichloromethane, and methanol) larvicidal activities against different strains of aedes aegypti (yellow fever mosquito) and anopheles stephensi (asian malaria mosquito) (nair et al., 2014) to be continued table 2 selected medicinal plants reported for its efficiency against the insects (continued) chand et al. essential oils in the south pacific and their insecticidal properties 301 to be continued family scientific names common english name plant part used *traditional uses in the south pacific (treatment) active constituents/compounds efficiency against insects references rutaceae citrus aurantium l. seville or sour orang fruit extract headache, abdominal pain and urinary tract infections insecticidal activity against the adult bactrocera oleae (gmelin) (olive fruit fly) (siskos et al., 2007) rutaceae citrus aurantium l. seville or sour orang leaf extract headache, abdominal pain and urinary tract infections insecticidal activity against the adult bactrocera oleae (gmelin) (olive fruit fly) (siskos et al., 2007) rutaceae citrus aurantium l. seville or sour orang shoot extract headache, abdominal pain and urinary tract infections insecticidal activity against the adult bactrocera oleae (gmelin) (olive fruit fly) (siskos et al., 2007) rutaceae citrus sinensis (l.) osbeck orange, sweet orange essential oils from fruits sickness, abdominal pains and remedies for internal ailments d-limonene larvicidal and pupicidal activities against musca domestica l. (housefly) (kovendan et al., 2012) rutaceae citrus sinensis (l.) osbeck orange, sweet orange peels from fresh oranges sickness, abdominal pains and remedies for internal ailments insecticidal activity against mosquito, cockroach and housefly (ezeonu et al., 2001) rutaceae citrus sinensis (l.) osbeck orange, sweet orange essential oils from the seeds sickness, abdominal pains and remedies for internal ailments insecticidal activity against the tribolium castaneum (herbst) (red flour beetle) (hussain et al., 2013) zingiberaceae curcuma longa l. turmeric leaf essential oils painful skin, sores and rashes in infant, sprains, bruises, eye diseases and open wounds, colds and runny nose, dysentery and infected puncture wounds contact and fumigant toxicity against rhyzopertha dominica f. (lesser grain borer), sitophilus oryzae l. (rice weevil), and tribolium castaneum herbst (red flour beetle) (tripathi et al., 2002) zingiberaceae curcuma longa l. turmeric turmeric rhizome oils painful skin, sores and rashes in infant, sprains, bruises, eye diseases and open wounds, colds and runny nose, dysentery and infected puncture wounds repellency and feeding deterrent effects of turmeric oils against the rhyzopertha dominica (f.) (lesser grain borer) (jilani and saxena, 1990) zingiberaceae curcuma longa l. turmeric leaves α-turmerone and β-turmerome larvicidal activity on anopheles gambiae (african malaria mosquito) (ajaiyeoba et al., 2008) zingiberaceae curcuma longa l. turmeric rhizomes larvicidal activity on anopheles gambiae (african malaria mosquito) (ajaiyeoba et al., 2008) fabaceae erythrina variegata l. coral tree ethanoic extracts from root and bark filariasis, stomach ache and fever contact toxicity and antifeedant activities against the spodoptera exigua (beet armyworm) (feng et al., 2012) fabaceae erythrina variegata l. coral tree leaf extract using solvents filariasis, stomach ache and fever antifeedant and toxicity against the spodoptera litura (fab) (taro caterpillar) (thushimenan et al., 2016) table 2 selected medicinal plants reported for its efficiency against the insects (continued) adv. hort. sci., 2017 31(4): 295-310 302 tics of most active families are discussed below. lamiaceae family. lamiaceae family is also known as mint family that has strong aromatic essential oils, tannins, saponins and organic acids (raja, 2012). numerous insecticidal properties on a wide range of insect species have been reported from extracts obtained from the lamiaceae family. for instance, biological activities of ocimum basilicum l., mentha rotundifolia l., origanum vulgare l. ssp. vulgare, rosmarinus officinalis l. and thymus vulgaris l. have been reported against the first instar larvae of tribolium castaneum herbst (coleóptera, tenebrionidae) (clemente et al., 2003). likewise, the extracts of plectranthus glandulosus against the callosobruchus maculatus in cowpea showed 100% mortality at 4 g/kg, within 7 days with lc50 of 0.39 g/kg (danga et al., 2015). the leaf extracts from lamiaceae family have also shown post-harvest grain protectants efficacy (nukenine et al., 2007; 2011; 2013). similarly, bekircan et al. (2014) reported the antifeedant activity of t. transcaucasicus , t. pseudopulegioides, t. leucotrichus and teucrium polifamily scientific names common english name plant part used *traditional uses in the south pacific (treatment) active constituents/compounds efficiency against insects references fabaceae erythrina variegata l. coral tree methanoic leaf extracts filariasis, stomach ache and fever larvicidal activity of culex quinquefasciatus (southern house mosquito) (nazar et al., 2009) cucurbitaceae momordica charantia l. bitter gourd, balsam pear, balsam apple leaf extract leprosy and malignant ulcers, stomach worms, fever, hypertension, diabetes and dysentery insecticidal activities of sitophilus zeamais (greater grain weevil) (adesina, 2013) cucurbitaceae momordica charantia l. bitter gourd, balsam pear, balsam apple acetone, nhexane, and methanol extract of leaves leprosy and malignant ulcers, stomach worms, fever, hypertension, diabetes and dysentery toxicity and repellent activity against the callosobruchus maculatus (fab.) (cowpea weevil). the order of extract toxicity was n-hexane> methanol >acetone (ajayi, 2015) cucurbitaceae momordica charantia l. bitter gourd, balsam pear, balsam apple methanoic fruit extracts leprosy and malignant ulcers, stomach worms, fever, hypertension, diabetes and dysentery larvicidal effects on culex pipiens (northern house mosquito) (nagappan and gomathinayagam, 2014) passifloraceae passiflora foetida (l.) var. hispida (dc.) killip wild passion fruit leaves and the sterm improve fertility in women repellent effect against the hematophagous insects (obico and ragragio, 2014) psilotaceae psilotum nudum (l.) p. beauv. psilotum aerial extract pain relief and remedy for thrush and the spore psilotin [6-(4'-β glucopyranosyloxyphenyl)-5,6-dihydm-2-oxo-2h-pyran] feeding deterrent and growth reducer to ostrinia nubilalis (european corn borer) (arnason et al., 1986) verbenaceae vitex trifolia l. vitex leaf extract stomach pains and mouth infections larvicidal activity on culex quinquefasciatus (southern house mosquito) (kannathas an et al., 2007) verbenaceae vitex trifolia l. vitex hexanic and dichloromethanic (dcm) extracts of leaves and stems stomach pains and mouth infections antifeeding activity against the insect pest spodoptera frugiperda (fall armyworm) (hernández et al., 1999) verbenaceae vitex trifolia l. vitex leaves and stem bark extracts stomach pains and mouth infections larvicidal activity on anopheles gambiae (african malaria mosquito) (nyamoita et al., 2013) table 2 selected medicinal plants reported for its efficiency against the insects (continued) chand et al. essential oils in the south pacific and their insecticidal properties 303 um l., against agelastica alni l. (coleoptera: chrysomelidae larvae). overall, the lamiaceae family has an extensive range of biological activities including cytotoxic, antimicrobial, antioxidant, anti-inflammatory, hypotensive and insecticidal properties (božović et al., 2015). annonaceae family. annonaceae is the largest family in the order magnoliales and consist of 2500 species and 130 genera (pirie et al., 2005; westra and maas, 2012). the annonaceae family has drawn attention since 1980s among the terrestrial plant families as a result of acetogenins that are known for a broad range of insecticidal bioactivities (isman and seffrin, 2014). the species of annonaceae family such as asimina triloba , annona muricata , and a. squamosa l. are frequently considered for insecticidal activities against spodoptera frugiperda, plutella xylostella, aedes aegypti, and stored grain insects (isman and seffrin, 2014). the fruit extract of xylopia aethiopica and dennettia tripetala were reported to have an insecticidal effect against sitophilus oryzae (coleoptera: curculionidae). the larvicidal, ovicidal and pupicidal properties against aedes aegypti have been reported using benzene, chloroform, ethyl acetate and methanol extracts of a. reticulata l. nevertheless, the leaf and stem extracts of a. coriacea mart., a. crassiflora mart., duguetia furfuracea (a. st.-hil.) saff. and xylopia aromatica l. were reported for their phytotoxic effects on germination of lettuce, tomato and onion seeds (novaes et al., 2016). rutaceae family. murraya koenigii (l) spreng leaf extract resulted in high mortality, population reduction with delay in development of tribolium castaneum pest of stored wheat (gandhi et al., 2010). furthermore, as reported by arivoli et al. (2015), the hexane extracts of m. koenigii showed not only larvicidal activitiy against the vector mosquito’s i.e., a. aegypti, anopheles stephensi and culex quinquefasciatus but also they demonstrated that one of the six fractions obtained from the residue of hexane extract, had an effect against the third instar larvae of a. aegypti, c. quinquefasciatus and a. stephensi with a percentage of mortality of 100.0, 97.6 and 99.2%. the methanolic leaf extracts of atlantia monophylla were evaluated for pupicidal activities against c. quinquefasciatus, a. stephensi, and a. aegypti under laboratory conditions and the respective lethal values corresponding to lc50 of 0.07, 0.05, and 0.07 mg/l (sivagnaname and kalyanasundaram, 2004). the zanthoxylum rhoifolium leaves also showed insecticidal activities in bemisia tabaci populations (christofoli et al., 2015). phytochemical survey of rutaceae family reveals the presence of flavonoids, alkaloids, limonoids, coumarins and volatile oils, of which some are associated with insecticidal activity (rajkumar and jebanesan, 2008; emam et al., 2009; supabphol and tangjitjareonkun, 2014). meliaceae family. natural products of meliaceae family such as limonoids have biological activities against several insects. one compound widely known and commercialised is azadirachtin reported to hold antifeedant and growth-regulating properties (champagne et al., 1989). the azadirachtin compound inhibits the feeding, growth and survival of the variegated cutworm such as peridroma saucia, with an ec50 and lc50 of 0.36 and 2.7 ppm in diet (champagne et al., 1989). the fruit extracts of trichilia elegans and t. catigua revealed insecticidal activity on spodoptera frugiperda (fall armyworm) (matos et al., 2009). azadirachta indica a. juss (neem derivatives) was known to used traditionally as an insecticide in the south pacific (world health organization, 1998). modes of action of plant extract components natural plant products show different mode of actions mainly due to chemical components acting differently, resulting into contact toxicity, stomach poison and systemic activities if used in soils or injected on plants (upadhyay, 2016). for instance, different plant extracts such as armoise, clary sage, oregano, lemongrass, niaouli, spearmint, cassia especial, dalmatian sage, red thyme, bay, garlic, pennyroyal, cassia pure, white thyme, cassia redistilled, star anise, peppermint, wintergreen, and cinnamon bark oils have shown potent fumigant toxicity against the c. corticalis (kim et al., 2012). these volatile substances affect the insect’s nervous system. the nervous system is the control center of the body that transduces the activity of nerves into behaviour. the nerve cells act upon external cues (smell, taste, touch, hearing and light) as well as internal inputs from sources such as hormones, body temperature and limb position sensors in order to create control coordination in insects behaviour (salgado, 2013). the fine-tuned control system of these insects is disrupted by the volatile nature of plant extracts when applied. the plants extract lead to the poisoning of insects whereby certain cells show alternation of staining properties; while some cells can breakdown (cytolysis) in tissues. similarly, within the nucleus the chroadv. hort. sci., 2017 31(4): 295-310 304 matin granules result into pyknosis (clump together) and the nissl bodies (granular substances) which dissolves the nerve cells (tanada and kaya, 1993; satar et al., 2008). the symptoms of nerve poisons are divided in four stages: excitation, convulsion, paralysis and death. the neurotoxic fumigant results only in three stages: excitation, paralysis and death (tanada and kaya, 1993). the disturbance of nervous system in the insects often affects the respiratory, muscular and circulatory systems. as a result of disturbance or malfunction in the metabolic system the insect dies. in addition, the two common potential mode of action of essential oil components are discussed below. acetylcholinesterase. acetylcholine (ach) is one of the major compounds that are responsible for transmitting nerve impulses from different nerve cells and involuntary muscles. acetylcholine is denatured by the enzyme acetylcholinesterase (ache) to choline and acetate and when ach is released from synaptic vesicles depolarises the postsynaptic cell membrane. a result of the ache activity is the regulation of the nerve impulse across the cholinergic synapses (siegfried and scott, 1992; lópez and pascualvillalobos, 2010). in other words, the inhibition activity of ache activity generates the accumulation of neurotransmitters acetylcholine in neuronal synapses which creates a state of permanent stimulation resulting into lack of coordination in the neuromuscular system followed by the subsequent death of insect (fig. 3) (dambolena et al., 2016). monoterpenoids were the first inhibitors that were considered to have the anticholinesterasic properties. the inhibition of ache in stored-product insect pests, sitophilus oryzae l. (coleoptera: curculionidae), rhyzopertha dominica fabricius (coleoptera: bostrichidae) and cryptolestes pusillus schönherr (coleoptera: cucujidae) is a possible mode of action from monoterponoids such as linalool, camphor, γ-terpinene, geraniol, scarvone, e-anethole, fenchone and estragole (lópez and pascualvillalobos, 2010). for instance, 1,8-cineole (monoterpene) is found to be the best inhibitor of acetylcholinesterase activity (ic50 values 0.015 0.05 mg/ml) (picollo et al., 2008; dambolena et al., 2016). octopaminergic sites. octopamine, phenolic analogue of noradrenaline, is also present in the nervous system of the arthropods. there is some evidence that octapamine plays a role in neuromuscular transmission or rather possess a modulating influence on the nerve-muscle interaction (candy, 1978; enan, 2001). octapamine act as neurotransmitters, neurohormones and neuromodulators in nervous system of inverterbrates (kostyukovsky et al., 2002). in insects, octopamine induces hyperextension of legs and abdomen due to the increased frequency of excitatory postsynaptic potentials from abdominal motor neurons (harris-warrick et al., 1980; livingstone et al., 1980). octopamine is likely to be involved in the regulation of heartbeats in insects since it is released in the axon terminals of pericardial organs (evans et al., 1976). octopamine exerts the effects through octopamine-1 and octopamine-2 receptors throughout their union with g-protein-coupled receptors (dambolena et al., 2016). for instance, carvacrol compound was found to change the conformation of the endogenous g-protein by increasing the affinity (dambolena et al., 2016). likewise, a blockage of octopamine receptors binding sites was noted at the lowest concentration of the eugenol, α-terpineol and cinnamic alcohol resulting in decreased biding of [3h]octopamine to its receptors (enan, 2001). the compounds such as octopamine and acetylcholine (accumulated in the nerves) in insects have diverse biological roles. octopamine and acetylcholine compounds function as neurotransmitters (fig. 3). if these compounds get interrupted by any fig. 3 target sites in insects as possible neurotransmitter mediated toxic action of volatile plant extracts. adapted from: tripathi et al. (2009) 305 chand et al. essential oils in the south pacific and their insecticidal properties chance, then it results in the damage of nervous system of the insects. plant extracts have long been touted as a potential alternative to synthetic insecticides presumably because of less environmental and human health impacts (kostyukovsky et al., 2002). the extracts form an impermeable film when applied on crops, which covers the insect from the air. the formation of the covering results in suffocation with the consequent death in insects (li et al., 2014). in addition, tripathi et al. (2009) reported that volatile components of plant extracts such as monoterpenes have cytotoxic effects on tissues of living organisms. for example, the reduction in the intact mitochondria and golgi bodies, impairing respiration and reducing cell membrane permeability. the overall effect of plant extracts led to disruption, dissolution of cell membranes, and blockage of tracheal system of insects (isman and machial, 2006; tehri and singh, 2015). 3. conclusions bio-control has been long touted as an attractive alternative over synthetic methods for the insect management. the current review has showed that out of the 19 plants selected, only azadirachta indica a. juss (neem derivatives) was known to be used traditionally as an insecticide in the south pacific (world health organization, 1998). although essential oils are gaining momentum in market due to their environmental friendly pesticidal properties, there are few disadvantages of essential oils. firstly, the use of essential oil in industrial farming may be not very popular mainly due to essential oils being more expensive and its less available. secondly, the effect of separate chemical composition of essential oils is studied and trialled on insects, however, every little study concerning combined effects of essential oils is known mainly due to high level of difficulty in identifying the effectiveness (regnault-roger et al., 2012). thirdly, the use of essential oils for pest control is known from ancient times however only few are known to be available in commercialized market (park and tak, 2015). nevertheless, essential oils play a very important role in non-synthetic farming where the environmental safety is the primary concern (isman, 2000). although economically, synthetic chemicals are more often used then the plant extracts, these botanicals have the potential of providing efficient and safer approach for the environment as well as for humans (nerio et al., 2010; pandey et al., 2014). acknowledgements the authors are thankful to mr karuna reddy and mrs reema prakash for their support and guidance provided throughout the research journey especially assisting in the layout of the paper. last but not least, the authors are thankful to the support given by the university of the south pacific, fiji islands for offering the graduate assistant scholarship and the research funding. references adesina j.m., 2013 insecticidal potential of momordica charantia (l.) leaves powder against maize weevil sitophilus zeamais (mots.) 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however, there was no delay in the harvest date. the climatic conditions favored high fruit set (37%­49%) and low fruit drop (63%­70%) between culti­ vars in both treatments. regarding fruit quality, no differences in size, soluble solids concentration and color were observed with the product, but a decrease in firmness were observed for ‘regina’. these results show that retard cherry® is an effective tool in delaying bloom, providing trees with more favorable cli­ matic conditions for pollination and fruit set. 1. introduction sweet cherry (prunus avium l.) is a fruit tree of temperate climate, native to asia minor (iezzoni et al., 2017). its cultivation has been widely distributed in mediterranean climate countries such as turkey, italy and spain, in the united states and in middle eastern countries such as iran (bujdosó and hrotkó, 2017). in the southern hemisphere, chile is the main exporter, with a volume of 350 thousand tons in the 2020/21 sea­ son in a cultivated area of 48,960 ha (iqonsulting, 2021; odepa, 2021). emergence from recess and regulation of blooming time in deciduous fruit trees involves a combined process of winter cold and spring warmth accumulation (fadón et al., 2020). in sweet cherry trees, temperature conditions during the cold accumulation phase are estimated to be the main driver of bloom (fadón et al., 2021). in mild winter areas, insuffi­ cient cold accumulation can cause delayed bloom, floral malformations, and low fruit production in the trees. in contrast, when high cold accumu­ lation accompanied by spring warmth occurs in this period, bloom may be (*) corresponding author: ayuri@utalca.cl citation: yuri j.a., palma m., sepúlveda á., sánchez­ contreras j., moya m., 2023 ­ use of the bio‐ stimulant retard cherry® as a strategy to delay blooming period in sweet cherry trees ­ adv. hort. sci., 37(4): 427­432. copyright: © 2023 yuri j.a., palma m., sepúlveda á., sánchez­contreras j., moya m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 september 2022 accepted for publication 12 september 2023 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-13659 https://dergipark.org.tr/en/search?q=%22+phenology%22 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 427­432 428 advanced and flower opening concentrated, increas­ ing the risk of spring frost damage (herrero et al., 2017), which can damage flowers and buds (miranda et al., 2005) and generate a large decrease in crop production and profitability (kaya et al., 2021). moreover, low temperatures can affect blooming synchrony between variety and pollinizer, limiting pollinator activity, delaying pollen tube development and fruit set (guo et al., 2015). climate change in the near future is expected to generate warmer springs with greater thermal fluctu­ ations, which may alter plant phenology and increase the risk of spring frost damage in species of temper­ ate climate (augspurger, 2013). some tools used to reduce spring frost damage are overhead sprinkler irrigation, wind towers with heaters and heated macro­tunnels. however, these technologies have a high implementation cost and are not always effec­ tive enough in control (yuri et al., 2017). a complementary strategy to avoid frost damage in fruit trees is to delay tree bloom, shifting it to a period with greater climatic stability, more favorable for pollination and fruit set (liu and sherif, 2019). plant growth regulators evaluated in stone fruits can extend bud dormancy (durner and gianfagna, 1991), delay bloom (ebel et al., 1999) or synchronize bloom with another variety, as well as delay harvest (basak et al., 1998). in some cases, however, the application of these products can cause flower abscission, low fruit set and yield (crisosto et al., 1990; liu and sherif, 2019). on the other hand, the use of foliar biostimulant, as sustainable alternatives to plant growth regulators, could increase flower bud resis­ tance to winter cold and delay bloom, without detri­ mental effects on fruit production. this study aimed to evaluate the effect of foliar application of the biostimulant retard cherry® on bloom delay before leaf fall as well as on fruit set, fruit drop and fruit quality in ‘regina’ and ‘sweetheart’ sweet cherry trees in central chile. 2. materials and methods plant material and experimental site the study was conducted during the 2018/19 growing season in two commercial orchards of sweet cherry (prunus avium l.) located in san clemente, maule region, chile (35°32’ s, 71°27’ w, 230 m a.s.l.), at less than 5 km between them. in one orchard, ‘regina’ sweet cherry trees on ‘gisela­6’ rootstock were evaluated; they were planted at 4.0 × 1.8 m in 2015. in the other, ‘sweetheart’ sweet cherry trees on ‘colt’ rootstock were evaluated; they were plant­ ed at 5.0 × 2.5 m in 2010. both cultivars were trained in central leader. orchards management were car­ ried out according to commercial standards in the region. seasonal environmental conditions are sum­ marized in table 1. climatic data was recorded by an automatic weather station vantange 2 (davis instruments, hayward, ca, usa) near the orchards. during the table 1 ­ environmental conditions 2018, san clemente, chile note: chill hours: weinberger, 1950. chill units (utah): richardson et al., 1974. gdh: anderson and seeley, 1992. gdd: stanley et al., 2000). variable annual may 1­jul 31 aug 1­dec 25 october air temperature (°c) mean 13.6 7.4 13.7 13.3 mean maximum 21.4 13.3 21.3 20.2 mean minimum 7.3 3.1 7.2 7.6 maximum 35.6 25.2 35.4 26.4 minimum ­5.1 ­5.1 ­5.1 1.5 relative humidity (%) mean minimum 46.1 66.8 42.1 44.5 precipitation (mm) 493 234 205 37.8 solar radiation (mj m­2) 5.718 573 2.677 829 chill hours 1.433 857 chill units (utah) 478 1.308 gdh 72.507 30.59 6.65 gdd (base 10°c) 1.807 697 123 yuri et al. ‐ use of the biostimulant to delay blooming period 429 blooming period, environmental conditions favourable to bee activity were calculated, defined as the number of hours per day with air temperature higher than 15°c and solar radiation higher than 300 w m­2. experimental design the experimental design was a randomized by complete block divided into two treatments (5,000 m2 per treatment). the treatments were: (1) control without product; (2) foliar application of retard cherry® (am ecological s.a., chile). the product was applied twice at doses of 1.0 and 0.5 l/ha, prior to 50% leaf drop: for ‘regina’ on march 26 and april 9; for ‘sweetheart’ on march 15 and 30. applications were made with a conventional hydro­pneumatic sprayer (parada spa, santiago, chile) with a spray volume of 1,200 l/ha. all measurements were made in 10 replicates, each consisting of two branches per replicate, trees per replicate, considering three edge rows per side. bloom delay blooming evolution (%) was determined weekly from stage ‘first white’ to ‘full bloom’, counting the number of open flowers per date. the full bloom date was defined when 80% had flowered. bloom delay was determined by subtracting the days between the full bloom date of the control and that of the treatment. fruit set, fruit drop and yield fruit set and fruit drop were monitored on the same branches studied at bloom. fruit set (%) was evaluated 20 days after the full bloom evaluation by counting the number of fruits formed in relation to the total number of flowers per branch. fruit drop (%) was determined by the number of fruits that did not reach harvest in relation to the number of fruits formed. yield (kg/tree) was also determined from individual trees. fruit quality harvest date was determined on the basis of fruit color. evaluation of weight (g), diameter (mm), color, firmness, and soluble solids concentration (ssc) con­ sidered a sample of 50 fruits per treatment. color was determined visually by scale (light red = 1, red = 2, mahogany red = 3, dark mahogany = 4 and black = 5). firmness (g mm­1) was measured with a firmtech ii texturometer (bioworks inc, wamego, usa). ssc (°brix) was measured on the same fruits with a digital refractometer (atago, plas­bx/acid5, japan). statistical analysis the data obtained underwent an analysis of vari­ ance (anova) and the means were compared with the tukey test (p≤0.05). when necessary, a transfor­ mation of the data was carried out. analysis was per­ formed with the statgraphics centurion xvi program (warrenton, virginia, usa) and the figures were gen­ erated using sigmaplot 10 software (wpcubed gmbh, germany). 3. results sweet cherry trees treated with retard cherry® showed a 6­8 day delay in the full bloom date with respect to the control, with a greater delay in the case of ‘regina’ (table 2, fig. 1). although the appli­ cation of retard cherry® delayed bloom, it had no effect on harvest date (table 2). a similar level of fruit set was maintained fig. 1 ­ effect of foliar application of retard cherry® on blooming dynamic in sweet cherry trees cv. regina. table 2 ­ effect of foliar application of retard cherry® on the date of phenological stage in sweet cherry trees ‘regina’ and ‘sweetheart’ cultivar/treatments 80% full bloom harvest ‘regina’ control 09­oct 25­dec retard cherry 17­oct 25­dec ‘sweetheart’ control 27­sep 21­dec retard cherry 02­oct 21­dec adv. hort. sci., 2023 37(4): 427­432 430 between treatments with an average of 49% for ‘regina’ and 37% for ‘sweetheart’ (table 3). even though fruit drop was numerically higher in the treat­ ment with retard cherry®, it was not statistically sig­ nificant, which is reflected in tree yields, with a mean of 8.6 kg/tree for ‘regina’ and 14 kg/tree for ‘sweetheart’ (table 3). the ‘regina’ sweet cherry trees treated with retard cherry® showed 18% lower firmness than the control, with no change in fruit color, while ‘sweetheart’ showed a higher incidence of fruit with lower color, although this was not noticeable to con­ sumers. fruit weight, diameter, and ssc were not affected by treatment (table 4). 4. discussion and conclusions the results of fruit set are in concordance with those of raffo and curetti (2021) who reported a delay of up to 10 days in leaf emergence and full bloom of several sweet cherry cultivars in rio negro, argentina. a greater assimilation and subsequent transport of reserves to the plant, prior to leaf fall, would allow an adequate dormancy and a more homogeneous bloom, effect that could be favored by the foliar application of biostimulants in autumn. fruit set in sweet cherry trees is normally low, and highly dependent on pollen availability and climatic conditions during and after pollination (hedhly et al., 2007). in chile, sweet cherry growers use the follow­ ing scale of fruit set intensity: high 34­40%; medium 15­20%; low 8­10% (c. tapia, pers. comm, october 11, 2021). sagredo et al. (2017) found that the effective pol­ lination period in ‘regina’ sweet cherry cultivars was about 5 days post anthesis, with the highest fruit set levels occurring 2­3 days post anthesis. similarly, zhang et al. (2018) showed that the peak pollen ger­ mination and stigma receptivity of certain sweet cherry cultivars occurred 2­3 days post anthesis under three ambient temperature scenarios. in addi­ tion, the pollen tube required at least 48 h to reach the ovule. regarding to bee activity, more than 100 bee vis­ its per minute were observed by koumanov and long (2017) with proper hive management, temperatures above 18°c and wind speed less than 16 km h­1. on the other hand, vicens and bosch (2000) indicated that bee activity (a. mellifera) was fully active with air temperature above 14°c and solar radiation greater than 300 w m­2. the high fruit set obtained in this study could have been favored, among others, by the prevalence of suitable climatic conditions for bee activity during blossom and for fruit set of that season (table 5; fig. 2). no frost was observed during blossom in both orchards. post­bloom environment of the trees treat­ ed with retard cherry® was more stable, with an average daily mean air temperature about 1­2 °c higher than that measured in the control trees one week earlier. table 5 shows the great difference in the condi­ tions for bee flight at blooming between the two cul­ tivars. the application of retard cherry, by delaying blooming, had a much greater effect on ‘sweetheart’, as it distanced it from the riskiest date of low tem­ table 3 ­ effect of foliar application of retard cherry® on fruit set, fruit drop and fruit yield in ‘regina’ and ‘sweet­ heart’ sweet cherries means in a column followed by the same letter do not differ sta­ tistically, according to tukey test (p≤0.05). n= 10/treatment. nd= no detected. table 4 ­ effect of foliar application of retard cherry® on fruit quality in ‘regina’ and ‘sweetheart’ sweet cherries at harvest means in a column followed by the same letter do not differ sta­ tistically, according to tukey test (p≤0.05). n = 50/treatment. cultivar/treatments fruit set (%) fruit drop (%) yield (kg/tree) ‘regina’ control 50 a 65 a 8.6 a retard cherry 47 a 75 a 8.6 a p­value 0.45 0.16 ­ ‘sweetheart’ control 33 a nd 14.0 a retard cherry 41 a 63 14.0 a p‐value 0.41 ­ ­ cultivar/ treatments weight (g) diameter (mm) color (1­5) flesh firmness (g mm­1) ssc (°brix) ‘regina’ control 12 a 28 a 4.0 a 231 a 20 b retard cherry 12 a 28 a 4.4 a 189 b 21 a p‐value 0.41 0.31 0.09 0.00 0.01 ‘sweetheart’ control 13 a 29 a 4.2 a 274 a 21 a retard cherry 12 b 29 a 3.6 b 271 a 20 b p‐value 0.04 0.46 0.01 0.80 0.00 yuri et al. ‐ use of the biostimulant to delay blooming period 431 peratures and frost in early spring; something similar occurred in the case of ‘regina’, although with less intensity, as it is a later blooming cultivar. harvest date was not affected using retard cherry® on sweet cherry trees, although there was evidence of lower fruit firmness on regina and slight­ ly less coloration on ‘sweetheart’ (tables 2 and 4). the fruit ripening stage is characterized by a rapid increase in weight and size, due to an increase in cell size, leading to a reduction in firmness. sugar content increases, keeping acids relatively constant; however, color is the one that shows the greatest changes, being a relevant factor in determining the harvest date in sweet cherries (tudela et al., 2005; muskovics et al., 2006). in previous studies, raffo and curetti (2021) reported that the use of retard cherry® showed a marked delay in color development of ‘sweetheart’ sweet cherries. the use of ga3 allows delay harvest in sweet cherry trees and has also proven to increase fruit size, firmness and ssc (basak et al., 1998; horvitz et al., 2003; raffo and curetti, 2021). therefore, ga3 would be a complementary tool to the use of foliar biostimulant retard cherry®, to extend the harvest window, with good quality fruit and better prices. therefore, it can be concluded that the use of retard cherry® on ‘regina’ and ‘sweetheart’ sweet cherry trees is an effective tool for delaying bloom to avoid frost event and favor 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ecotone d. herênio gonçalves júnior (*), l. de melo rodrigues, j. alves de santana filho, w. nascimento lima, a. ferreira alves laboratory of plant genetic breeding (lmgv), center for agricultural sciences and technologies (ccta), brazil. key words: genetic characterization, lines, plant breeding, productivity, vigna unguiculata (l.) walp. abstract: the development of superior genotypes is the main objective of all plant breeding programs. to determine the genetic variability, heritability and correlations, 20 cowpea genotypes were grown in a randomized block design with four replications in cerrado/amazon rainforest ecotone region. the data recorded were plant height, pod length, pod mass, pod grain mass, grain index, pod grain number and yield. analysis of variance revealed significant differ­ ences between genotypes for all traits studied. the genotypic determination coefficient was high for all traits evaluated. similarly, the accuracy parameter presented high estimates (>0.90). the magnitudes of the genotypic correlation coefficients were higher than the environmental and phenotypic correlations for most correlations, showing a greater influence of the genetic factor than the environmental factors. the direct and indirect effects provided greater reliabili­ ty in the cause and effect interpretations between the studied traits, indicating that yield can be explained through the effects of the analyzed traits. the traits pod mass (0.9628) and pod grain mass (0.7835) showed the greatest favorable direct effect, showing a strong association between the analyzed characters and can be used in direct or indirect selection for yield in cowpea. 1. introduction cowpea [vigna unguiculata (l.) walp.] is one of the oldest crops known to man and, because its moderate drought resistance, grows mainly in tropical climate areas (egbadzor et al., 2014). recent studies suggest it originated from central africa over 4,000 years ago (ogunkanmi et al., 2014). according to rocha et al. (2009), cowpea is a valuable legume, predominantly cultivated in brazil, africa and the united states. in the brazilian north and northeast regions, it is one of the main popula­ tion’s diet components, especially in rural areas (santos et al., 2014). this crop still has low yields, despite the fact that its high adaptive potential to the conditions of tropical climate environments is verified (*) corresponding author: juniorherenio@gmail.com citation: herênio gonçalves júnior d., de melo rodrigues l., alves de santana filho j., nascimento lima w., ferreira alves a., 2020 ­ genetic parameters, correlations and path analysis in cowpea genotypes for yield and agro‐ nomic traits grown in cerrado/amazon rainforest ecotone. ­ adv. hort. sci., 34(3): 301­311 copyright: © 2020 herênio gonçalves júnior d., de melo rodrigues l., alves de santana filho j., nascimento lima w., ferreira alves a.this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 february 2020 accepted for publication 30 june 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 301­311 302 (leite et al., 2009). teixeira et al. (2010) point out as causes the management techniques adopted and, mainly, the inefficiency of the technologies used and the use of traditional cultivars without breeding for yield. in brazil there are cultivars with good commer­ cial acceptance, but breeding programs aimed at evaluation and recommendation in specific environ­ ments are concentrated only in large producing cen­ ters (oliveira et al., 2002; barili et al., 2015). one of the cowpea breeding programs basic goals is to obtain more productive genotypes. the avail­ ability of variance components estimates and genetic parameters such as coefficient of genetic variation, heritability and correlation coefficients for yield and their components are essential for the plant breeding programs development. these genetic parameters are characteristic of each population and may change in consequence of selection, changes in manage­ ment, methods and estimation models, among other causes. however, an important aspect about yield is that it is characterized as a complex variable, i.e., resulting from the expression and different components asso­ ciation (santos et al., 2018). correlation quantifies the association between any two variables. however, it does not allow inferences about cause and effect (furtado et al., 2002). the path analysis, proposed by wright (1921), allows to partition the correlation coefficient into direct and indirect effects (path coef­ ficient). for cruz et al. (2014), this analysis can be defined as a standardized regression coefficient, being an expansion of the multiple regression analy­ sis when complex interrelationships are involved. in this sense, knowing the association between these traits allows the breeder to explore the possi­ bility of indirect selection in cases of traits with com­ plex inheritance and low heritability, such as yield. correlation coefficient estimates make it possible to evaluate the magnitude and direction of the relation­ ship between two traits and, consequently, the possi­ bility of obtaining gains for one of them using indirect selection for the other trait. in some cases, indirect selection based on correlated response may be more effective and faster than direct selection of the desired trait (cruz et al., 2014). thus, this research was conducted with the objec­ tive of estimating the genetic parameters for the yield and its components in 20 cowpea genotypes population cultivated in the cerrado/amazon rainforest ecotone region in brazil. as well as, inves­ tigate the associations between traits to direct selec­ tion strategies in breeding programs with this crop. study results may assist in strategies for breeding and manipulation of traits by cowpea breeders in brazil or other similar environments. 2. materials and methods the experiment was carried out in imperatriz city, maranhão state, brazil, in the experimental field of the centro de difusão tecnológica (cdt) on premises of empresa brasileira de infraestrutura aeroportuária (infraero) of geographic coordinates latitude south 5°31’32’’ and longitude west 47°26’35’’, and altitude of 123.30 meters. according to the köppen climate classification, the region’s climate is aw, tropical savanna, with tropical wet and dry climate (peel et al., 2007). the survey of climate monitoring data for the region over the past 20 years was carried out. data on annual total precipitation, maximum, minimum and average annual temperatures were collected. the data were obtained from an automatic climate monitoring station made available in the governmen­ tal meteorological database banco de dados meteorológicos para ensino e pesquisa (bdmep) administered by the brazilian meteorology institute instituto nacional de meteorologia (inmet). the time series graphs were produced using the ggplot2 pack­ age in the r software. the treatments consisted in twenty erect habit cowpea genotypes from the active germplasm bank (agb) from the empresa brasileira de pesquisa agropecuária (embrapa meio norte) cowpea genet­ ic breeding program, located in teresina, brazil, 15 lines and 5 cultivars, respectively: lf­3; lf­21; lf­30; lf­48; lf­49; lf­62; lf­104; lf­143; lf­144; lf­148; lf­153; lf­154; lf­155; lf­159; lf­168; brs­guariba; brs­tumucumaque; brs­nova era; brs­itaim; and brs­cauamé. the soil physical and chemical characteristics were determined before the experiment beginning, from the superficial soil samples collected at random points in the experimental field up to 0.20 m depth. soil texture was analyzed by the modified soil sedi­ mentation bouyoucos method after addition of a dis­ persing agent. potential acidity was estimated from smp ph after ph determination in calcium chloride 0,01 mol l­1 (shoemaker et al., 1961). soil macronu­ trients and micronutrients analysis was performed to develop fertilizer recommendations. the experimental design was a randomized com­ plete block with 20 treatments and four replications. herênio gonçalves júnior et al. ‐ genotypic variability of cowpea in amazon rainforest 303 the experimental plot consisted of two lines of 4.0 m and spacing of 0.50 m between lines and 0.20 m between plants, constituting a total experimental area of 220.00 m². soil tillage was carried out in a conventional manner with one plow and two har­ rows. the digging and sowing operations were manu­ al. from the chemical soil analysis, was performed the fertilization according to the requirements of the crop (table 1). irrigation was carried out by means of a sprinkler system sized to the crop and the region requirements, applying a daily water of 3.8 mm h­1. invasive plants were controlled by hand weeding, performed weekly. phytosanitary treatments were carried out through regular monitoring of pests and diseases, using the commercial insecticide conect® when necessary. the harvest was performed when the pods of the plot were dry, totaling two harvests. the drying of the pods was completed in a forced air circulation oven, where the pods remained for two days at a temperature of 38°c. in the useful area of each plot were recorded the following data: plant height (ph): average height in cm randomly measured in five plants of the plot; pod length (pl): average length in cm of five randomly harvested pods in the plot useful area; pod mass (pm): in grams, considering the five previously har­ vested pods; pod grain mass (pgm): in grams, consid­ ering the grains of the five pods submitted to the aforementioned evaluations; grain index (gi): refers to the dry grain mass in the dried pods. it is obtained by the expression: gi = pgm/pm x 100 seeds per pod (spp): performed by counting the seeds in the five pods harvested for the previous samples; and yield: estimate considering the yield in all the useful plot area (m2), extrapolating the value obtained for kg ha­1 correcting the value for grain mass to 13% moisture. the collected data were initially submitted to the shapiro­wilk test to verify the data set normality and the bartlett test to verify if the error has homogene­ ity of variance (homoscedasticity), not presenting the need for data transformation. subsequently, one­ way analysis of variance was performed to test the variability between genotypes, adopting the statisti­ cal model described in the equation below: yij = µ + gi + bj + ɛij where: yij = observed trait value of the i­th genotype in the j­ th block; µ = general experimental mean; gi = effect of the i­th genotype considered fixed; bj = effect of the j­th block considered random; ɛij = random error associated to the i genotype and j block observations. to understand the genotypic variability between the different traits measured, the components of phenotypic variance and genetic parameters were also estimated using the expressions suggested by cruz et al. (2014): a) phenotypic variance: σ 2p = msg/b b) environmental variance: σ 2e = mse/b c) genotypic variance: σ2g= (msg ­mse)/b d) genotypic determination coefficient: r2 (σ2g/σ2p) x 100 e) intraclass correlation coefficient: icc = σ2g/(mse+σ2g) x 100 f) phenotypic coefficient of variation (%): pvc = √σ2p x 100 x ̅ g) genotypic coefficient of variation (%): gcv = √σ2g x 100 x ̅ h) environmental coefficient of variation (%): ecv = √mse x 100 x ̅ i) b quotient: gcv ratio = σ 2g ecv mse where: msg is the mean square of genotypes; mse is the mean square of error; b = number of blocks (replications) and x ̅ is the is the average of each trait. j) accuracy: r ̂= (1 ­ 1/f) 0.5 where snedecor’s f is the value of the variance ratio table 1 ­ soil chemical characterization used in the field experiment om= organic matter; p= phosphorus; k= potassium; ca= calcium; mg= magnesium; al= aluminium; h+al= potential acidity; sb= sum of bases; cec= cation exchange capacity; v= base saturation. ph (cacl2) om (g kg­1) p (mg dm­³) k (cmol dm­³) ca (cmol dm­³) mg (cmol dm­³) al (cmol dm­³) h+al (cmol dm­³) sb (cmol dm­³) cec (cmol dm­³) v (%) 4.8 18.4 13.5 0.26 1.66 0.69 0.00 1.70 2.61 4.31 60.5 adv. hort. sci., 2020 34(3): 301­311 304 for treatment effects (genotypes) associated with analysis of variance (anova). in the estimates of the correlations were used the expressions cited by falconer (1987) and ramalho et al. (1993): a) phenotypic correlation: ϒp(xy) = covp(xy) σ2px ·σ2py b) genotypic correlation: ϒg(xy) = covg(xy) σ2gx ·σ2gy c) environmental correlation: ϒe(xy) = cove(xy) σ2ex ·σ2ey where: ϒxy is the correlation between the characters x and y; covxy is the covariance between the charac­ ters x and y; and, and σ2y ad σ2x are the variances of the characters x e y, respectively. the unfolded of these correlations into direct and indirect effects of the six agronomic traits on yield was performed using the path analysis described by cruz et al. (2014). the level of the multicollinearity of the x’x singular matrix was established by the prod­ uct of the respective diagonal element of x’x by the component of the residual variance according to the methodology proposed by montgomery et al. (2012). after verifying the multicollinearity of the phenotypic correlation matrix, this was implanted in direct and indirect effects, considering the following equation: y = p1x1 + p2x2 + .... + pnxn + pɛu where y is the main dependent variable yield. x1, x2......, xn are the independent variables. p1, p2, .. pn are the path coefficients. the coefficient of determi­ nation was calculated by the expression r2 = p21y + p22y + ... 2p2y.p2nϒ2n the estimates of the components of the pheno­ typic variance, genetic parameters, correlations between traits and path analysis were obtained using the computational application genes (cruz, 2013). 3. results and discussion timeless climate data and information over the past 20 years, the average annual air temperature has varied between 27.37 and 28.84°c (fig. 1). the highest annual temperature observed was 35.22, in 2015. on the other hand, the lower annual temperature observed was 20.78°c, in 2018. the deviations observed for the maximum, minimum and average annual temperatures were ±0.57°c, ±0.70°c and ±0.41°c, respectively. total annual pre­ cipitation ranged from 1961.30 mm in 2016, to 498.50 mm in 2000 (fig. 1). because this great differ­ ence between the results collected for total annual precipitation, there was a large deviation for this parameter, value equal to ±0358.38. the average of annual total precipitation over the last 20 years was 1395.85 mm. estimates of genetic parameters the analysis of variance showed a significant effect (p<0.01) between the genotypes according to the f test for all evaluated characteristics (table 2), showing genetic variability presence in the popula­ tion. considering the existence of genetic variability in a population is a determining factor for any breed­ ing program (ramalho et al., 2012), at first, the germplasm under study is promising for selection or hybridization work with potential for new cultivars development. similarly, araméndiz­tatis et al. (2018) also detected significant differences for the same traits evaluated in an assay where they estimated the genetic parameters of traits associated with yield in 42 white seed cowpea genotypes. the relative standard deviation (rsd), which is used to estimate the experiments precision, present­ ed values considered low for the traits ph, pl, pm, mgv and gi, which indicates excellent experimental precision. while for the characteristics spp and yield presented rsd equal to 12.24% and 21.86%, there­ fore, they are considered regular values, indicating good experimental precision (cruz et al., 2014; ferreira, 2018) (table 2). similar results for the same fig. 1 ­ annual maximum temperature, annual minimum tempe­ rature, average annual temperature and total annual precipitation of the last twenty years (1998­2018) in a region characterized by the cerrado/amazon rainforest ecotone (inmet, 2019). herênio gonçalves júnior et al. ‐ genotypic variability of cowpea in amazon rainforest 305 traits were obtained by carvalho et al. (2012) and correa et al. (2015). the variation index (vi), another parameter relat­ ed to the experimental precision, proposed by gomes (1991), which is more adequate than the rsd, as it also considers the number of repetitions used in the experiment, besides the residual variation, pre­ sented low values for all traits, except for productivi­ ty that presented value of iv considered of medium magnitude. the success of the selection depends on the exis­ tence and magnitude of the observed genetic vari­ ability for yield and its components in the material under breeding (adewale et al., 2010; raturi et al., table 2 ­ analysis of variance summary for the traits plant height (ph), pod length (pl), pod mass (pm), pod grain mass (pgm), grain index (gi), seeds per pod (spp) and yield in 20 cowpea genotypes evaluated in cerrado/amazon rainforest ecotone region, brazil, 2019 df= degrees of freedom; (**) significant at 1% probability of error by the f test; rsd= relative standard deviation; vi= variation index. variation source df ph (cm) pl (cm) pm (g) pgm (g) gi (%) spp yield (kg ha­1) mean square blocks 3 46.46 1.61 5.54 5.05 112.71 75.61 120831.01 genotypes 19 319.08 ** 4.28 ** 34.85 ** 13.43 ** 112.71 ** 238.99 ** 136000.33 ** error 57 6.46 0.70 0.99 0.55 16.19 21.72 20004.46 mean ­ 65.36 20.22 18.57 14.40 78.12 75.74 647.03 rsd (%) ­ 3.89 4.14 5.35 5.17 5.15 6.15 21.86 i.v. (%) ­ 1.95 2.07 2.68 2.59 2.58 3.08 10.93 2015; shereen and el­nahrawy, 2018). table 3 shows there was low phenotypic variation (s) for the traits pl, pm, pgm, while for ph, gi and spp a certain phe­ notypic variation was observed. for yield, there is a high phenotypic variation (s). the values estimated for genotypic variance (s) ranged from 0.89 for pod length (pl) to 78.15 for plant height (ph) (table 3). analyzing the genotypic variance (s) in relation to the phenotypic variance (s), it was observed there was a major contribution of genotypic variance (s) to the present phenotypic vari­ ability. these results were confirmed by the esti­ mates of the genotypic determination coefficient (r2) (table 3). table 3 ­ estimates of genetic parameters for traits plant height (ph), pod length (pl), pod mass (pm), pod grain mass (pgm), grain index (gi), seeds per pod (spp) and yield in 20 cowpea genotypes evaluated in cerrado/amazon rainforest ecotone region, brazil, 2019 σ2f = phenotypic variance; σ2e = environmental variance; σ2g = genotypic variance; r2= genotypic determination coefficient; icc= intra class correlation coefficient; pcv= phenotypic coefficient of variation; gcv= genotypic coefficient of variation; ecv= environmental coeffi­ cient of variation; b quocient = gcv/ecv ratio; r ̂= accuracy. genetic parameters traits ph (cm) pl (cm) pm (g) pgm (g) gi (%) spp yield (kg ha­1) σ2f 79.77 1.07 8.71 3.36 28.18 59.75 34000.08 σ2e 1.61 0.17 0.71 0.14 4.05 5.43 5001.11 σ2g 78.15 0.89 8.46 3.22 24.13 54.32 28998.96 r2 (%) 97.98 83.15 97.17 95.87 85.64 90.91 85.29 icc (%) 92.37 56.15 89.55 85.30 59.85 71.44 59.18 pcv (%) 13.66 5.12 15.89 12.73 6.80 10.21 28.50 gcv (%) 13.52 4.68 16.67 12.46 6.29 9.73 26.32 ecv (%) 3.89 4.14 5.35 5.17 5.15 6.15 21.86 b = gcv/ecv 3.48 1.13 2.93 2.41 1.22 1.58 1.20 r ̂ 0.99 0.91 0.99 0.98 0.93 0.95 0.92 mean 65.36 20.22 18.57 14.40 78.12 75.74 647.03 306 adv. hort. sci., 2020 34(3): 301­311 cruz et al. (2014) mention that when the adopted statistical model considers genotypes as a fixed effect, as in the present study, heritability becomes the genotypic determination coefficient. the values of the genotypic determination coefficient (r2) ranged from 83.15% for pl to 97.9% for ph. all evalu­ ated traits presented high (r2) estimates (>75%). this parameter provides indications of the expected per­ formance of a given population for traits selection, which allows us to infer that the population in study is promising for the trait selection under study. however, it is noteworthy that for complex inheri­ tance characteristics such as yield, which are the expression result of many alleles and they are greatly influenced by the environmental conditions to which population undergoes, the high values of r2 may be overestimated by genotype x environment interac­ tion, since the present study was conducted in only one year and in a single environment. torres et al. (2015), in a study to determine the number of measurements required, evaluated 40 genotypes of prostrate and semi­prostrate cowpea types in the state of mato grosso do sul, in ten assays, the r2 ranged from 51.50% to 92.64%. while shimelis and shiringani (2010), in a study to deter­ mine the variance components and heritability in ten cowpea lines, obtained the genotypic determination coefficient of 55.00% for yield, lower than the value found in the present work. given the high value of r2, it can be inferred that it is caused by the inherent genetic variability of the tested genotypes, because each of them contributes a distinct genetic identity (teixeira et al., 2007). fehr (1987) mentions that higher genotypic determination coefficients may be associated with lower environ­ ment variation and lower genotype­environment interaction. and according to gomes (2009), there is low to medium accuracy in environmental control, since the relative standard deviations (rsd) were below 21% for all characters. the intraclass correlation coefficient (icc), which corresponds to the repeatability coefficient, indicates an estimate of the total measurement variability frac­ tion owing to variations between individuals. the icc ranged from 56.15% to 92.37% for pod length and plant height, respectively (table 3). when character­ istics have a lower intraclass correlation coefficient require a greater number of measurements (replica­ tions) to predict the real value of a given trait and vice versa. therefore, it can be inferred for the supe­ rior genotypes selection, the number of measure­ ments in the present study is satisfactory. the coefficients of variation provide information about the variation nature and magnitude. they clari­ fy if the variations are owing to genetic or environ­ mental causes. typically, the gcv values are bigger than ecv. if the differences between gcv and ecv are excessive, so the environmental effects will be more noticeable on the trait. thus, in the observed results, the relative proportion (%) of the deviations from the mean because of genetic effects (gcv) were higher when compared to the environmental ones (ecv), for all traits (table 3). the genotypic coefficient of variation (gcv) ranged from 4.68% for pod length and from 26,32 to 26.32% for yield (table 3). the highest estimates (gcv) were recorded for pod grain mass (12.46%), plant height (13.52%), pod mass (16.67%) and yield (26.32%), indicating that these traits offer greater selection perspectives to obtain genotypes much more aligned to the proposed, as they are erect habit and determined growth genotypes. these results are consistent with those found by lopes et al. (2017) for yield and pod mass; regis et al. (2014) for pod grain mass and grain index, and bhagasara et al. (2017) for yield in this species. the characteristics plant height, pod length and seeds per pod showed lower gcv and, therefore, present greater difficulties in the selection process and expected genetic advance. fact in agreement with correa et al. (2015) and silva and neves (2011). however, gerrano et al. (2015), found higher gcv values for plant height (67.41%), pod length (19.97%) and seeds per pod (24.82%). the b quotient is an auxiliary tool for the breeder. according to the interpretation of cruz et al. (2014) for this parameter, when the value is greater than or equal to 1.00, it indicates that there is genetic vari­ ability within the population in study, which can therefore, be explored, and in the case, the trait is favorable to selection. the quotient b ranged from 1.13 for pl to 3.48 for ph (table 3). thus, it can con­ cluded that the b quotient values found for all evalu­ ated characteristics are favorable to selection in order to obtain more productive genotypes. genotype evaluation assays should be approached from a genetic and statistical point of view, not just from a statistical perspective. in the context of genotypic evaluation, accuracy is the most important statistical parameter. it has the property of informing about the correct ordering of genotypes for selection purposes and also about the effective­ ness of inference about the genotypic value of each genotype (resende, 2002). herênio gonçalves júnior et al. ‐ genotypic variability of cowpea in amazon rainforest 307 accuracy depends not only on the residual varia­ tion magnitude and the number of replication, but also on the proportion between the genetic and resid­ ual variations associated with the trait under evalua­ tion. accuracy refers to the correlation between the true genotypic value of genetic treatment and that estimated or predicted from the information from the experiments. as a correlation, it ranges from 0 to 1, and the appropriate accuracy values are those close to the unit or 100% (henderson, 1984). therefore, for all evaluated characteristics in the present experiment, the observed values for accura­ cy are considered very high, as they are above 0.90. high accuracy variables indicate small absolute devia­ tions between true genotypic values and those esti­ mated from experimental information. resende and duarte (2007) emphasize the importance of achiev­ ing optimal selective accuracy greater than 0.90 for safe statistical inference. correlation between traits and path analysis correlation estimates indicate good signal agree­ ment between phenotypic and genotypic correla­ tions (table 4). in general, genotypic correlations pre­ sent values higher than their corresponding pheno­ typic and environmental correlations. similar results were obtained by andrade et al. (2010), correa et al. (2015), almeida et al. (2014), gerrano et al. (2015), teixeira et al. (2007) and manggoel et al. (2012) in studies conducted with cowpea, evaluating yield components. there was a significant (p≤0.01) and high magni­ tude positive phenotypic correlation (ϒp) between the traits pod grain mass (pgm) and pod mass (pm), which was already expected, insofar that pod grain mass increase happens, it should also increase the pod mass, or vice versa. however, the traits grain index and seeds per pod presented negative pheno­ typic correlation at 1% probability (table 4). for the other pairs of characteristics there were no signifi­ cant phenotypic correlations. genotypic correlations (ϒg) showed the same sign and, in most cases, values higher than their corre­ sponding phenotypic correlations, indicating that the phenotypic expression is decreased because of envi­ ronmental influences. although the yield compo­ nents were positively correlated with yield, the ϒp and ϒg estimates showed low magnitude and they table 4 ­ estimates of the correlation coefficients phenotypic (ϒp), genotypic (ϒg) and environmental (ϒe) between the traits plant height (ph), pod length (pl), pod mass (pm), pod grain mass (pgm), grain index (gi), seeds per pod (spp) and yield in 20 cowpea genotypes evaluated in cerrado/amazon rainforest ecotone region, brazil, 2019 ns= not significant; (*), (**) significant at 5% and 1%, respectively, by the t test. characteristics ϒ ph pl pm pgm gi spp yield ph p 1 ­0.47* ­0.14 ns ­0.06 ns 0.15 ns ­0.35 ns 0.18 ns g 1 ­0.52* ­0.14 ns ­0.05 ns 0.18 ns ­0.37 ns 0.20 ns e 1 ­0.04 ns ­0.07 ns ­0.09 ns ­0.16 ns ­0.02 ns ­0.07ns pl p 1 0.06 ns 0.14 ns 0.11 ns ­0.02 ns 0.05 ns g 1 0.07 ns 0.14 ns 0.12 ns ­0.00 ns 0.04 ns e 1 0.04 ns 0.14 ns 0.06 ns 0.21 ns 0.07 ns pm p 1 0.90 ** ­0.64 ** 0.32 ns ­0.14 ns g 1 0.91 ** ­0.66 ** 0.34 ns ­0.16 ns e 1 0.44 ** ­0.47 ** 0.10 ns 0.10 ns pgm p 1 ­0.23 ns 0.18 ns ­0.03 ns g 1 ­0.31 ns 0.19 ns ­0.04 ns e 1 0.55 ** 0.04 ns 0.04 ns gm p 1 ­0.39 ns 0.26 ns g 1 ­0.43 ns 0.33 ns e 1 ­0.12 ns ­0.12 ns spp p 1 0.12 ns g 1 0.12 ns e 1 0.11 ns yield p 1 g 1 e 1 adv. hort. sci., 2020 34(3): 301­311 308 were, mostly, non­significant. cruz et al. (2012) attribute the genetic correla­ tions occurrence, mainly to the pleiotropy or to the genetic links between traits pairs, in the latter case, transient causes. in any case, genetic correlations favor the simultaneous selection of two or more traits by selecting only one of these. on the other hand, according to these authors, the selection of one trait may lead to an undesirable selection of another. the negative estimates of correlation between pairs of traits indicate that improving one trait will decrease the other, and in these cases, the selection based on this one is not recommended. the charac­ teristic plant height was phenotypically and geneti­ cally negatively correlated with pod length, indicating that the smaller the plant, the longer the pod length, which directly influences the yield. according to falconer and mackay (1996), genotypic and environ­ ment correlations of exchanged signals, as can be observed in some characteristics pairs (table 4), reveal that the causes of genetic and environmental variation influenced the traits through different phys­ iological mechanisms. given the complexity among the yield compo­ nents that contribute to yield, the selection of cow­ pea genotypes is difficult. thus, it is evident the need to unfold the correlations in direct and indirect effects, evaluating the importance degree of each of the explanatory variables in relation to the main or basic variable (daros et al., 2004). cruz et al. (2014) report that the parameter esti­ mates under multicollinearity may assume absurd values or with no consistency to the studied biologi­ cal phenomena. thus, for greater reliability of the path analysis results, the phenotypic correlation matrix between characteristics was tested for multi­ collinearity by the condition number proposed by montgomery et al. (2012). the correlation matrix had a condition number equal to 993.97, that is, collinearity between the characters considered moderate to strong, present­ ing no problem for the path coefficients estimates. the coefficient of determination (r2) and the residual effect indicate how much the explanatory variables determine the yield. the coefficient of determination was 0.2025 and the residual effect was 0.8930 (table 5). the direct effects magnitudes of the traits ana­ lyzed on yield were higher than the estimates magni­ tudes of their respective simple correlations with table 5 ­ estimates of direct and indirect effects involving the main variable, yield in kg ha­1, and the explanatory vari­ ables: plant height, pod length, pod mass, pod grain mass, grain index, seeds per pod concerning to 20 cow­ pea genotypes evaluated in cerrado/amazon rainforest ecotone region, brazil, 2019 characteristics association effects path coefficients plant height direct on yield 0.3671 indirect via pl ­0.0805 indirect via pm ­0.1327 indirect via pgm 0.0434 indirect via gi 0.1162 indirect via spp ­0.1302 total 0.1833 pod length direct on yield 0.1711 indirect via ph ­0.1727 indirect via pm 0.0612 indirect via pgm ­0.1058 indirect via gi 0.0845 indirect via spp 0.0082 total 0.0466 pod mass direct on yield 0.9628 indirect via ph ­0.0506 indirect via pl 0.0109 indirect via pgm ­0.7015 indirect via gi ­0.4831 indirect via spp 0.1191 total ­0.1425 pod grain mass direct on yield 0.7835 indirect via ph ­0.0203 indirect via pl 0.0231 indirect via pm 0.8621 indirect via gi ­0.1774 indirect via spp 0.0664 total ­0.0295 grain index direct on yield 0.7593 indirect via ph 0.0561 indirect via pl 0.0190 indirect via pm ­0.6126 indirect via pgm 0.1831 indirect via spp ­0.1444 total 0.2605 seeds per pod direct on yield 0.3702 indireto via ph ­0.1291 indirect via cv 0.0038 indirect via pm 0.3097 indirect via pgm ­0.1407 indirect via gi ­0.2963 total 0.1177 coefficient of determination 0.2025 residual variable effect 0.8930 herênio gonçalves júnior et al. ‐ genotypic variability of cowpea in amazon rainforest 309 yield (table 5). based on this information, it is possi­ ble to infer there are other traits influencing both the magnitude and the correlation direction between the yield components. considering the direct effects on yield, included in table 5, the trait pod mass (0.9628) has the greatest effect, indicating a major contribution to the yield increase, surpassing the pod grain mass, which also had a high direct effect (0.7835). in contrast, pod length (0.1711) was the trait with the lowest effect. important to mention there was no negative direct effect of any trait on yield. still in table 5, it can be seen that although the character pm had a high direct effect on yield, in general, the indirect effects via pm on yield were low. indicating that indirect truncation selection in the auxiliary character may not provide satisfactory gains in the main variable (yield). in these cases, the best strategy is the multi­trait selection (cruz et al., 2012). indirect effects on yield were relatively low, except for the trait pgm via pm, which pointed to an estimate of 0.8621. this result is indicative of the indirect selection viability via pod mass to obtain gains on the most important character. the trait ph had negative indirect effect via all the characteristics, except gi, which indicates that the plant height reduction induces the increase in the other charac­ teristics, which is very important in this crop produc­ tion system, considering that plants very high hinder crop handling and harvesting. considering the total effect, the traits have had the greatest effect on yield were as follows grain index (0.2605), plant height (0.1833), seeds per pod (0.1177) and pod length (0.0466) (table 5). this result of the total effect in relation to the direct effects on yield was owing to the negative indirect effects via the other characteristics, which confirms the need to apply a multi­trait selection. considering that the existence of genetic variabili­ ty in population is a determining factor for any breeding program (ramalho et al., 2012), the germplasm under study is, initially, promising for selection or hybridization work with potential for the new cultivars development. 4. conclusions the study concluded there is a considerable degree of genotypic variation between important agronomic traits in cowpea. genotypic variation con­ tributed most of the phenotypic variation. result cor­ roborated by the high estimates of genotypic deter­ mination coefficient. thus, the genetic parameters estimates obtained for yield and agronomic traits, in the present study, will provide a basis for selection in order to obtain gains in the breeding for cowpea yield. the path analysis indicated the pod mass and pod grain mass had the greatest favorable effect on yield in cowpea, and could also be used for indirect selec­ tion aiming at the development of new 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totipotency. abstract: parthenocarpy, the ability of some plants to undergo fruit growth in absence of fertilization, is an important question of basic science and the sub­ ject of much interest due to its possible agricultural benefits. in the context of our cellular biology studies on a halophyte of interest, cakile maritima, we gen­ erated calli, pluripotent cell masses, that unexpectedly allowed the appearance of parthenocarpic fruits without any floral tissues. these observations raise the hope to develop an in vitro model to study parthenocarpic fruit development. 1. introduction for several years, we are studying cakile maritima, a promising model of halophyte in a worldwide context of increasing salinization of lands due to climate change (arbelet­bonnin et al., 2019). in addition to whole plant studies (debez et al., 2006; ellouzi et al., 2014; ben hamed­louati et al., 2016 b; arbelet­bonnin et al., 2020) we underwent cellular biology stud­ ies (ben hamed­laouti et al., 2016 a; arbelet­bonnin et al., 2018) for which we have developed a c. maritima cultured cell suspension that starts by calli generation as a first step (ben hamed et al., 2014). it is now admitted that in vitro cultured cells do not undergo a full dedifferentia­ tion but rather a transdifferentiation, leading to increased developmental potency and/or cell proliferation (sugimoto et al., 2011; fehér, 2019). the supposed totipotency capacity of plant cells (haberlandt, 1902) is largely operated for the in vitro culture of plants. calli are thus frequently consid­ ered as transient tissue dedicated to somatic embryogenesis allowing the plant regeneration (sugimoto et al., 2011; fehér, 2019). interestingly, hormonal balances play an important role in many developmental proces­ ses in plant (molesini et al., 2020), and in particular the balance between auxin and cytokine seems to be crucial during in vivo parthenocarpic fruit development (pandolfini, 2009; joldersma and liu, 2018; an et al., 2020; sharif et al., 2022). parthenocarpy is the ability of some plants to undergo fruit growth in absence of fertilization. parthenocarpy has contributed to some of humanity’s domestication of plants such as breadfruit or banana (zerega et al., 2004; kislev et al., 2006; sardos et al., 2016). furthermore, it repre­ sents a highly desirable trait in agronomy since seedless fruits are highly (*) corresponding author: delphine.bonnin@u­paris.fr citation: arbelet­bonnin d., cangémi s., laurenti p., bouteau f., 2022 ­ observation of unexpected neo like‐fruit development from cakile maritima calli. ­ adv. hort. sci., 36(2): 155­159. copyright: © 2022 arbelet­bonnin d., cangémi s., laurenti p., bouteau f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 february 2022 accepted for publication 26 april 2022 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-12818 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 155­159 156 appreciated by consumers and fruit set is less affect­ ed by environmental factors in absence of fertiliza­ tion (ruan et al., 2012). therefore, parthenocarpy is the subject of much interest and leads to many stud­ ies (sharif et al., 2022). however, to our knowledge, all studies published so far have been conducted in whole plants but not at the level of cell cultures so far. we report here the first appearance of neo­like fruits directly on calli obtained and long maintained on callus­inducing medium (cim). these observations raise the hope to develop a cellular model that will allow in vitro studies of parthenocarpic fruit develop­ ment. 2. materials and methods plant material and establishment of calli cakile maritima seeds used in this study come from raoued (north of tunisia). the seeds cultivation in vitro conditions were established as described by ben hamed et al. (2014). briefly, bleached seeds were placed in petri­dishes containing murashige and skoog medium (1962) hormone­free (ms, sigma), supplemented with 30 g.l­1 sucrose, 8 g.l­1 agar and the ph was adjusted to 5.8. this medium induced seed germination under a light cycle of 12 h light and 12 h dark with 40 µe m2 s­1 at 22°c. the stems of 14­ days­old seedlings were then chopped finely and placed on an agar­callus­induced medium (cim) (valvekens et al., 1988) and then put in a growth chamber, in the same conditions that for seed germi­ nation. cim medium contain 6.2 g.l­1 gamborg b5 (gamborg et al., 1968) from sigma supplemented with 20 g.l­1 glucose, 8 g.l­1 agar and phytohormones : cytokinin and auxin (valvekens et al., 1988; akama et al., 1992). the precise hormone balance depends of plant species (pacheco et al., 2012; thomas and hoshino, 2015). for c. maritima we used 9.06 µm of 2,4­dichlorophenoxyacetic acid (2.4 d) and 0.46 µm of kinetin (kn) that were shown to be efficient (ben hamed et al., 2014). the ph was adjusted to 5.7. after two weeks, calli were formed from the frag­ ments of seedlings tissues. when the size of the calli reached 1 cm in length, they were subcultured on a fresh cim medium. for some experiments, the hormonal balance was modified with 9 µm of kn and 2­4d, or 0.46 µm of kn and 2­4d and calli were grown during two months. to assess the putative role of volatile organic com­ pounds (vocs) emitted from mature fruit, a surface sterilized fruit of c. maritima was added in petri­dish­ es with calli during 2 months. 3. results and discussion cakile maritima calli were subcultured every four weeks on the cim medium, otherwise they began to brownish and lose their ability to be sub­cultured. during a callus subculture, a spontaneous green excrescence from calli easily visible (fig. 1a) was observed. as the subcultures went along on fresh medium cim, green excrescences grew on calli and more excrescences appeared on different calli (fig. 1b and 1c). each appeared excrescence grew all­long the time (fig. 1d). after 58 days, numerous green structures resem­ bling c. maritima differentiated tissues arise (fig. 2). 40% of them are clearly green differentiated tissue although we cannot identify specific organ structures (fig. 2a, b). on the contrary, the other 60 % of green differentiated structures resemble neo like­fruits (fig. 2c­e). indeed, they present two asymmetric seg­ ments reminiscent of the typical dimorphic fruit that fig. 1 ­ emergence and development of parthenocarpic fruits on cakile maritima callus. first observation (a), observations after 30 days (b); 49 days (c) and 58 days (d). arbelet‐bonnin et al. ‐ neo like‐fruit from c. maritima calli 157 develops on whole plant of c. maritima (fig. 2f). these neo like­fruits did not contain seeds and are thus parthenocarpic. such parthenocarpic fruits have never been reported in c. maritima in the literature. more interestingly, this represents not only the first report of parthenocarpic fruits forming from calli, but also forming from non­floral tissues. these partheno­ carpic fruit appeared without any change in the hor­ mone balance, a mean generally used to triggered shoot or root regeneration in vitro in the context of somatic embryogenesis and plant multiplication (thomas and hoshino, 2015; das et al., 2018; shin et al., 2020). parthenocarpy can be artificially obtained by applying synthetic growth factors to unpollinated ovaries (pandolfini, 2009; molesini et al., 2020). auxins seem to play a prominent role in triggering and coordinating the transition from flower to fruit, and exogenous supplies of auxins to unpollinated flowers could induce fruit growth in various plants, suggesting that these hormones can replace the sig­ nals provided by pollination and fertilization (pandolfini, 2009; molesini et al., 2020). cytokinin could also induced parthenocarpy but probably through modulation of auxin metabolism (molesini et al., 2020; sharif et al., 2022). we tried to modify the hormones concentrations and the balance between kn and 2­4d by using 0.46 µm or 9 µm of both of these hormones but unfortunately, no new sponta­ neous green excrescences development were observed on the 20 calli present on each petri dish (n=2 per conditions). since we observed a multiplica­ tion of these parthenocarpic fruits in the same petri­ dishes, we asked ourselves if this phenomenon could be reminiscent of fruit ripening triggered by volatiles organic compounds (vocs) such as ethylene released from already developed fruits (tohge et al., 2014). even if the role of ethylene still appeared unclear (sharif et al., 2022), ethylene responses could also lead to parthenocarpic fruit development (pandolfini, 2009). we thus put a mature c. maritima fruit har­ vested from a fully developed plant in a petri­dish with calli, as a putative vocs furnisher to calli. one more time no excrescences appeared on the 20 dif­ ferent calli present in the petri dish. although we have no clear explanation at the moment, the ques­ tion of genetic homogeneity of callus cells is not solved and only certain cells of a callus could be regarded as totipotent and thus involved in organ regeneration (fehér, 2019). moreover, this ability to develop parthenocarpic fruits disappeared after three months and more subcultures. this suggests that only freshly prepared calli are able to develop parthenogenetic fruits. accordingly, if callus tissue can express a wide variety of genes especially at the early phase of their development, their transcrip­ tome seems to be homogenize along time (fehér, 2019). fig. 2 ­ various green structures observed on cakile maritima callus. a­b. undefined differentiated green structures. c­e. typical dimorphic fruit like structures. f. typical dimorphic fruit developed on a whole plant of c. maritima (arrows indicated the two asymmetric segments). adv. hort. sci., 2022 36(2): 155­159 158 although we cannot control the appearance and development of parthenocarpic fruits on calli at this time, these data certainly, deserve more investiga­ tions. recent genomic studies have greatly con­ tributed to elucidate the role of phytohormones in regulating fruit initiation, providing at the same time genetic methods for introducing seedlessness in hor­ ticultural plants. moreover, that some plants may produce fruit without the need for fertilization by the male gamete remains an important question of basic science. therefore, the development of an in vitro model of parthenocarpic fruits will certainly be an essential tool to understand how a fruit could form without the need of fertilization nor floral tissues. references akama k., shiraishi h., ohta s., nakamura k., okada k., shimura y., 1992 ­ efficient transformation of arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and agrobacterium strains. ­ plant cell rep., 12: 7­11. an j., althiab almasaud r., bouzayen m., zouine m., chervin 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arabidopsis thaliana root explants by using kanamycin selection. ­ proc. natl. acad. sci., 85: 5536­ 5540. zerega n.j.c., ragone d., motley t.j., 2004 ­ complex origins of breadfruit (artocarpus altilis, moraceae): implications for human migrations in oceania. ­ am. j. bot., 91: 760­766. impaginato 397 evaluation of hot pepper (capsicum spp.) genotypes for resistance to viruses and aphids in rwanda b.w. waweru 1, 2 (*), d.c. kilalo 1 , j.w. kimenju 1, p. rukundo 2, d.w. miano 1 1 department of plant science and crop protection, college of agriculture and veterinary sciences, university of nairobi, p.o. box 29053‐0625 kangemi, nairobi, kenya. 2 rwanda agriculture and animal resources development board, p.o. box 5016, kigali, rwanda. key words: aphids, capsicum spp., field, genotypes, resistance, screenhouse, virus. abstract: hot pepper is an important crop in rwanda but viral diseases and pests are major constraints to its production. field experiments were conduct­ ed to evaluate the resistance of 18 hot pepper genotypes (4 commercials, 5 introduced and 9 local) to natural infection by viruses and aphid infestation, in two agro­ecological zones of rwanda. fourteen genotypes were further evalu­ ated for resistance to cucumber mosaic virus (cmv) under screenhouse condi­ tions. disease incidence and severity were recorded in all experiments while population of aphids was assessed in the field. diseased leaf samples from each genotype in the field were analysed using polymerase chain reaction to detect the presence of viruses, while samples from the screenhouse were analysed using serological assay. results showed significant (p<0.05) differences in dis­ ease incidence and severity among genotypes. three genotypes namely pbc 462, 00767ppr and 0802ppr were rated as resistant to viral diseases while genotype hp 0117, pp9852­170 and pp9950­5197 were moderately resistant. all commercial and most of the local genotypes were susceptible compared to the introduced lines. there was no difference in genotype infestation by the aphids. the genotypes that are resistant to viruses are recommended for use by growers and in breeding programs. 1. introduction hot pepper (capsicum spp.) is an important vegetable crop grown throughout the world. in rwanda, it is produced for both local consump­ tion and export to the european market (naeb, 2015). the crop plays an important role in poverty alleviation through income generation and cre­ ation of employment to both farmers and the hot pepper value chain actors. hot pepper production has increased in recent years in rwanda but, the average yield is still low at around 6.8 t ha­1 which is lower than (*) corresponding author: bancywaweru@yahoo.com citation: waweru b.w., kilalo d.c., kimenju j.w., rukundo p., miano d.w., 2020 ­ evaluation of hot pepper (capsicum spp.) genotypes for resi‐ stance to viruses and aphids in rwanda. ­ adv. hort. sci., 34(4): 397­412 copyright: © 2020 waweru b.w., kilalo d.c., kimenju j.w., rukundo p., miano d.w. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 february 2020 accepted for publication 19 june 2020 ahs advances in horticultural science adv. hort. sci., 2020 34(4): 397­412 doi: 10.13128/ahsc­8094 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 397­412 398 50% of the country’s potential yield of 15 t ha­1 (rdb, 2010; fao, 2017). the low and poor quality produce have been attributed to abiotic and biotic factors, of which diseases caused by viruses play a significant role (skelton et al., 2018). pepper is attacked by more than 68 viruses global­ ly, of which about 15 have been identified in africa (njukeng et al., 2013; aliyu, 2014; kenyon et al., 2014). cucumber mosaic virus (cmv), pepper veinal mottle virus (pvmv), tobacco mosaic virus (tmv) and potato virus y (pvy) have been reported as the most prevalent in sub­saharan africa (dafalla, 2001). in rwanda, pepper vein yellows virus (pevyv), pvmv and cmv have been detected in hot pepper (skelton et al., 2018). the cmv is ranked among the most eco­ nomically important viruses of hot pepper not only in rwanda but also in some other countries such as india where yield losses ranging from 10 to 50% are documented (rahman et al., 2016). on the other hand, the crop is attacked by more than 21 insects which include aphids, whiteflies, thrips among others (niranjanadevi et al., 2018). aphids, whiteflies, and thrips are vectors of various viruses infecting hot pepper. these insect pests especially the aphids, are serious threat to hot pepper production not only due to losses caused through direct damage but also they are vectors of devastating viruses (kenyon et al., 2014). various management options have been pro­ posed to reduce virus diseases of hot pepper in the field. these measures include the use of virus­free planting materials, resistant varieties, borders crops, pesticides and roguing (wang et al., 2006; degri and ayuba, 2016). farmers in rwanda mainly rely on insecticides to control insect­vectors. unfortunately, the insecticides do not achieve 100% control of the vectors. hence, the insect­vectors develop resistance against the active ingredient after repeated applica­ tion of the insecticides within a short time and more so the insecticides negatively affect the environment (kenyon et al., 2014). the use of resistant cultivars offers the most economical, effective and durable solution in mitigating the negative effects due to dis­ eases and aphids in hot pepper production (visalakshi and pandiyan, 2018). resistant varieties are highly preferred because they not only reduce the pest population and the virus inoculum in the farming system but they are also compliant with other methods (frantz et al., 2004). several studies have evaluated the resistance of wild and cultivated hot pepper genotypes to virus infection and aphids’ infestation leading to the release of virus­resistant lines in different parts of the world (frantz et al., 2004; appiah et al. , 2014; choi et al. , 2018). however, information on hot pepper genotypes that are resistant to virus infection and vector infestation is not documented in rwanda. the current study focused on local, commercial and introduced geno­ types that have not been evaluated before for the resistance to viruses or aphids in rwanda. it is important to assess the genotypes in different environments in the field to identify the relative host resistance, as some genotypes found resistance at one location turns out to be susceptible to another place. screenhouse assessment using artificial inocu­ lation techniques is important for validation of resis­ tance. in this study, both field and screenhouse experiments were conducted to (1) evaluate the reac­ tion of different hot pepper genotypes (local, com­ mercial and introduced) to natural virus infections and aphids’ infestations in two agro­ecological zones of rwanda, and (2) evaluate the reaction of selected hot pepper genotypes to infection by cucumber mosaic virus under screenhouse conditions. 2. materials and methods reaction of hot pepper genotypes to virus infection and aphid infestation under field conditions source of seeds a total of 18 hot pepper genotypes that included nine local collections obtained from rwanda national genbank, five introduced lines provided by world vegetable center, eastern and southern africa­ tanzania and four commercial varieties from seed companies were evaluated (table 1). previous studies indicate that the introduced genotype pp9950­5197 is resistant to cmv, pvy and chilli veinal mottle virus while genotype icpn 18­7 is resistant to pvy (gniffke et al., 2013). similar studies by reddy et al. (2014) showed the introduced genotype pp9852­170 as resistant against cmv. california wonder (sweet pep­ per) variety which has been used as a susceptible control for viruses in previous studies (murphy and bowen, 2006) was also included in the present study. study areas the study was conducted at rubona and gashora research fields belonging to the rwanda agriculture and animal resources development board (rab) dur­ ing two successive growing seasons; long rains (end waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 399 march­july, 2018) and short rains (end october, 18­ march, 2019). rubona station is located at an alti­ tude of 1692.9m, latitude s 2°28ʹ59.59ʺ and longi­ tude e29°46ʹ22.46ʺ, in midland aez. gashora is located at an altitude of 1331.1m, latitude s 2°15ʹ22.11ʺ and longitude e30°17ʹ12.43ʺ, in lowland aez. the characteristics of the two aezs are shown (table 2). raising of the seedlings seeds of the different genotypes were sown and raised in trays containing sterilized sandy loam soil (1:2) in the screenhouse. at 2­3 leaf stage, the seedlings were transplanted into plastic potting bags (5 × 9 × 4 cm) containing steam­sterilized sandy loam soil and maintained for six weeks in the screenhouse. before transplanting to the field, the seedlings were confirmed to be free from pepper mild mottle virus (pmmov), pvmv, tmv, pvy and cmv using das­ elisa. the kits were obtained from loewe biochemica gmbh company, germany) and used according to instructions from the manufacturer. establishment of the field experiments the experiments were carried out in the open field and depended on natural virus infections and aphid infestation from the uncultivated fields. the experiments were laid out in a randomized complete block design (rcbd) with three replications. the blocking was done according to soil fertility gradient and nearness to the uncultivated land, such that each treatment had an equal chance of vector infestation. each replicate had eighteen experimental plots, mea­ suring 2.5 m by 3 m each, with a 1 m wide path between the plots. an experimental plot contained 24 seedlings planted on 4 rows, at a spacing of 60 cm by 45 cm. at planting, approximately 500 g of organic manure was used per plant and 15 g of npk (17:17:17) fertilizer was applied one week later. a month after transplanting, 3.5 g of urea (46:0:0) per table 1 ­ list of genotypes evaluated for reaction to infection by viral diseases and aphids under field conditions in rwanda 1 code of the local genotypes as found in the database of rwanda national genbank; 2 unknown species; rngb= rwanda national genbank; wvc= world vegetable center. genotype species type source 00765ppr 1 c. annum local rngb 00767ppr c. baccatum local rngb 00774ppr c. annum local rngb 00775ppr c. chinense local rngb 00786ppr c. annum local rngb 00791ppr c. chinense local rngb 00792ppr c. frutescens local rngb 00802ppr ‐ 2 local rngb 00795ppr c. chinense local rngb pbc 462 c. annum introduced wvc pp9950­5197 c. annum introduced wvc hp 0117 c. annum introduced wvc pp9852­170 c. annum introduced wvc icpn 18­7 c. annum introduced wvc long red cayenne c. annum commercial simlaw seed company bird­eye hybrid (oiseau pili pili) c. frutescens commercial technisem company red scotch bonnet c. chinense commercial exporter california wonder c. annum commercial kenya seed company table 2 ­ characteristics of the two agro­ecological zones of rwanda where the study was conducted aez= agro­ecological zone. source: verdoodt and van roanst, 2003. site/district aez* relief elevation (m) rainfall (mm) temperature (°c) rubona/ huye midlands dissected plateaus 1600­1900 1100­1400 17­20 gashora/bugesera lowlands pediplains 900­1600 850­1100 20­21 adv. hort. sci., 2020 34(4): 397­412 400 plant was applied. both preventive and curative fungicidal sprays were applied at regular intervals to control fungal diseases. the spray regime was depen­ dent on symptom appearance and prevailing weath­ er conditions. weeding was done two times in a month. insecticides were not sprayed at all. data collection during plant growth, data was collected at a 14­ days interval starting from two to ten weeks after planting (wap) in the field. ten plants were randomly selected from the middle rows of each plot and tagged. these plants were used for the assessment of viral disease incidence and symptom severity during the experimental period. determination of disease incidence and symptom severity. virus disease incidence was expressed as a percentage based on the proportion of infected/dis­ eased plants to the total number of plants observed per plot, as described by galanihe et al. (2004). symptom severity was scored for the ten tagged plants in a plot based on a scale of 1­5 as described by olawale et al. (2015) with slight modifications, where: 1 = no symptoms; 2 = mild symptoms of mosaic/mottling/yellowing on few leaves (<25% of the plant affected); 3 = moderate symptoms of mosa­ ic/puckering/mottling/vein clearing/yellowing on many leaves (26­50% of the plant affected); 4 = severe symptoms of mosaic/puckering/mottling/vein clearing/yellowing/stunting (51­75% of the plant affected) and 5 = severe symptoms of mosaic/puck­ ering/mottling/vein clearing/yellowing/ stunting/ necrosis (>75% of the plant). percentage severity was calculated as the sum of all disease rating per geno­ type expressed as a percentage of the total number of observations multiplied by maximum disease scor­ ing scale (5). determination of the area under disease progress curve. the area under the disease progress curve (audpc) was estimated to compare different responses of the tested hot pepper genotypes. estimated percentages of symptom severity recorded at different times during the experimental period were used to calculate audpc using the following equation as described by campbell and madden (1990). audpc σ n­1 = (yi+ yi+1)/2 (ti+1– ti) where σ = summation; n = number of successive readings, yi = disease severity at time ti and yi+1= dis­ ease severity at time ti+1. detection of the viruses. detection of the suspect­ ed viruses in the experimental plots was carried out using rt­pcr. at 10 wap, approximately five young leaves from diseased plants of the eighteen geno­ types were collected and placed in envelopes con­ taining silica gel. the samples were later transported to the phytopathology laboratory of rab at rubona and stored at room temperature for 4­5 days to dry. later, the samples were grounded in liquid nitrogen to fine powders that were stored at ­80°c until ana­ lyzed. in total, 68 symptomatic leaf samples were col­ lected from rubona and gashora’s experimental sites. total ribonucleic acid was extracted from 100 mg of frozen powdered hot pepper leaf tissues using acetyl trimethyl ammonium bromide (ctab) method as described by allen et al. (2006) with modifications. amplification of cmv, pvmv, pevyv was done using one taq one­step rt­pcr kit (catalogue e531s5, new england biolabs inc.), following the manufactur­ er’s instructions. amplified products were generated using virus­specific primers that were designed dur­ ing this study based on the nucleotide sequence data of cmv­r1 (genbank accession no. mg470800.1), pvmv­r1(mg470801.1), pevyv­r1 (mg470802.1). the cmv primers amplified a fragment of ∼502 bp, pvmv a fragment of ∼502 bp and pevyv a fragment of ∼498 bp (table 3). thermal cycling conditions were: 48°c at 15 min table 3 ­ primers used for detection of cucumber mosaic virus, pepper veinal mottle virus, and pepper yellows virus * primers developed during this study. virus primer * sequence amplification size (bp) cmv mg470800_1f 5'­gcttcgcaatacgttttgacgg­3' 502 cmv mg470800_1r 5'­tacgaccagcactggttgattc­3' 502 pvmv mg470801_1f 5’­aagccctcattgaaggtcaacg­3’ 502 pvmv mg470801_1r 5’­atcaaccatcacccacataccg­3’ 502 pevyv mg470802_1f 5'­agtacgtcttcgagactactgc­3’ 498 pevyv mg470802_1r 5'­tctatagtagagaggtcgatcc­3' 498 waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 401 for rt; followed by 1 min at 94°c for initial denatura­ tion; 40 cycles of 94°c for 15 s, 54°c for 30 s and 68°c for 45 s for denaturation, annealing, and extension, respectively. the final extension was at 68°c for 5 min. these conditions were similar for the three viruses. the pcr products were separated by elec­ trophoresis in 1.2% agarose gel stained with ethidi­ um bromide at 100 v for 40 min in 1 × tris­acetate­ edta (tae) buffer. gels were visualized under uv light. assessment of aphid population. monitoring of aphid populations was done at a 14­days interval starting from 2nd to 12th wap. un­winged aphids were monitored on four plants that were randomly selected from the center of each plot. observations were carried out on six leaves (2 upper, 2 middle and 2 lower leaves) per plant. a small camel­brush was used to dislodge and collect aphids present into small­plastic bottles containing 70% ethanol and transported to the phytopathology laboratory of rab at rubona for identification and counting. winged aphids were captured using yellow water traps (ywt) made from yellow plastic containers that were placed in the middle of each plot and filled with 1.5 litre of tap water (blackman and eastop, 2000). five millilitre of formaldehyde (10%) was added per trap to preserve the insect. the collected aphids were counted and identified to species level using existing entomological keys and stereomicroscope based on their morphological features as described by martin (1983) and blackman and eastop (2000). reaction of hot pepper genotypes to cucumber mosa‐ ic virus under controlled conditions genotypes tested fourteen hot pepper genotypes selected from the field trials were evaluated for resistance to cmv in the screenhouse. the experiment was carried out to validate the genotypes resistance to virus infection under controlled conditions. the genotypes tested included; seven local collections (00765ppr, 00767ppr, 00774ppr, 00786ppr, 000792ppr, 00795ppr and 00802ppr), five introduced lines (pbc 462, pp9950­5197, hp0117, pp9852­170 and icpn 18­7) and two commercial varieties (long red cayenne and red scotch bonnet) as indicated in table 1. inoculation of cmv fifty seedlings of each genotype were raised in the screenhouse. before inoculation, the seedlings were confirmed to be free of viruses using das­elisa as described in materials and methods. at the 5­6 leaf stage, the plants were mechanically inoculated with a local isolate of cmv. the virus was propagated and maintained in a hot pepper cultivar scotch bon­ net in the screenhouse. infected leaves were harvest­ ed and homogenized (1: 10 w/v) in 0.1 m phosphate buffer (ph7.0) containing 0.01% of sodium sulfite. the sap was sieved to remove plant debris and 0.06% of silicon carbide was added to enhance injury and increase points of entry of the virus. two leaves per test plant were rub­inoculated with sap extract as described by noordam (1973). after 5 mins the inoc­ ulated plants were rinsed with distilled water to remove the excess of the inoculum. inoculated plants were maintained in an insect free screenhouse (aver­ age 27.8°c temp, 70.8% relative humidity). in total, forty­eight plants of each genotype were mechanical­ ly inoculated with cmv. ten healthy plants of each genotype inoculated with phosphate buffer alone (with no inoculum) were maintained as control. the symptoms development on inoculated plants were recorded up to 3 weeks’ post­inoculation. at this time all the plants from the susceptible local check showed typical symptoms of cmv. the incidence and symptoms severity of cmv were evaluated on all plants as described in materials and methods. detection of cmv the confirmation of cmv infection was performed using das­elisa following the procedures described by clark and adam (1977) on representative samples from each hot pepper genotypes. the kits were obtained from deutsche sammlung von mikro­ organismen und zellkulturen (dsmz, germany) and used according to instructions from the manufactur­ er. a healthy sample and extraction buffer were used as negative controls. a positive control was provided with the kit. absorbance values were read at 405 nm using a microplate reader (biotek elx800, usa). due to a large number of plants, only representative sam­ ples (7) were collected from each genotype and anal­ ysed. a sample was considered positive when the absorbance value at 405 nm (a405) exceeded the mean of negative controls by a factor of two. classification of the hot pepper genotypes for resis‐ tance to viral diseases the rating of the genotypes was done as described by rahman et al. (2016). based on disease incidence and severity indices for both field and 402 adv. hort. sci., 2020 34(4): 397­412 screenhouse experiments, each genotype was allo­ cated a score of 1 to 4. scoring for virus incidence was: <20% =1, 21­30%=2, 31­50%=3 and >51%=4. whereas, disease severity: <1=1, 1.1­2.0=2, 2.1­3.0=3 and >3.0=4. based on cumulative scores i.e. incidence and severity indices, the genotypes were categorized into four groups: <3= resistant (r), 4­6 = moderately resistant (mr) and 7­8 = susceptible (s). the scores for the field experiments were made on pooled data obtained from the two sites and both cropping sea­ sons than data of individual site or season. data analysis data on disease incidence (%) and symptom severity were square­root transformed, and aphids’ populations were log­transformed before subjecting to the analysis of variance (anova). means of values regarding audpc were worked out using the microsoft excel program. the audpc values were directly subject to anova. data were analyzed using sas statistical software program and the means were separated using the least significant difference (lsd) test at p=0.05. 3. results reaction of the hot pepper genotypes to virus infec‐ tion and aphid infestation under field conditions weather conditions at the experimental sites the average monthly rainfall, minimum and maxi­ mum air temperature during the two cropping sea­ sons (march­july, 2018 and october 2018­march, 2019) at gashora and rubona experimental sites are shown in figure 1. in rubona, temperatures ranged from 14.4 ­24.2°c in season one and 15.2­24.9°c in season two. at gashora, there were wide variations between the minimum and maximum temperatures from 12.9­27.7°c in season one and 13.7­28.2°c in season two. incidence of virus diseases there were significant differences in disease inci­ dence between sites (p= <.0001), seasons (p= <.0001) and among genotypes (p = <.0001). the differences were observed from 4th wap and increased with time, ranging from 3% to 100% at 10 wap in both seasons and locations (tables 4 and 5). at rubona, higher disease incidence was recorded from both commercial and local genotypes, except for 00767ppr and 00802ppr compared to introduced genotypes in all sampling periods (table 4). in season one, genotype 00767ppr was the least infected with the viruses as the disease incidence (di) level was only 3%, followed by 00802ppr with 10%, pp9950­ 5197 with 20% and pbc 462 with 30% at 10 wap (table 4). the remaining genotypes had di greater than 60%. in season two, disease incidence levels were generally lower in all genotypes compared to season one. the least infected genotype was pbc 462 with di of 3% followed by 00802ppr and pp9852­170 both having 10%. this was followed by six genotypes (pp9950­5197, pbc 462, icpn 18­7, 00767ppr, 00786ppr, 00765ppr) with incidence levels of ≤ 35% compared to the remaining genotypes that showed di of ≥55% at 10 wap (table 4). on the other hand, a similar trend was observed at gashora, where higher disease incidence levels were recorded in season one compared to two (table 5). genotype pbc 462 was the least infected with di of 13%, followed by icpn 18­7 with 30%, pp9950­ 5197 with 47% and the remaining genotypes had incidence levels greater than 70% in season one (table 5). in season two, 5 genotypes (00767ppr, pp9950­5197, pbc 462, pp9852­170 and icpn 18­7) showed di levels of ≤ 50%. in both sites, the highest spread of viral diseases was recorded on both com­ mercial and local except for the genotype 00767ppr and 00802ppr. at 10 wap, all the genotypes had developed symptoms of viral diseases but, high vari­ ability existed between genotypes. the interactions of site and season (p = <.0001), site and genotype (p = <.0001), season and genotype (p = 0.0025) were also highly significant. these results indicated that the incidence of viral diseases was dependent on the site and season the experiments were conducted. area under disease progress curve the total amount of disease that occurred in the experiments was estimated and presented as the area under the disease progress curve (audpc). the mean audpc values differed significantly within sites fig. 1 ­ the microclimate of the experimental sites during the two cropping seasons. temp­ temperature, min­ minimum, max­maximum. waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 403 and thus, data were not pooled together. in rubona, the total amount of disease was significantly (p< 0.0001) higher in season one with mean audpc val­ ues of 230.9 compared to season two that recorded 117.1. in both seasons, all commercial and local genotypes (except 00767ppr and 00802ppr) record­ ed high levels of disease compared to introduced genotypes (table 6). genotypes 00767ppr, 00802ppr, pbc 462 and pp9950­5197 consistently recorded lower audpc values of less than 100 in both seasons. besides, genotypes 00786ppr, pp9950­ 5197, pp9852­170 and icpn 18­7 recorded values of less than 100 but only in season two. in gashora, the audpc values were higher in both seasons compared to rubona. however, a similar trend was observed where the amount of diseases was more in season one with values of 243.9 than season two 198.9 (table 6). introduced and the two local genotypes (00767ppr and 00802ppr) had low audpc values compared to the rest of the geno­ types. genotypes pbc 462 and pp9950­5197 record­ ed values of less than 100 in both seasons, while genotypes 00767ppr, pp9852­170 and icpn 18­7 had audpc values of less than 100 in season two only. on the other hand, genotypes 00792ppr, 00795ppr and the four commercial genotypes were the most infect­ ed with the viral diseases in both sites during the two seasons. detection of the viruses in hot pepper genotypes three viruses were detected in the samples ana­ lyzed namely cmv, pevyv and pvmv. cucumber mosaic virus infection was the most abundant in both sites, detected in 53.1% of the samples in rubona and 75% in gashora, followed by pevyv with 31.3% and 2.8%, respectively (fig. 2). the pvmv infections were the least abundant and only detected in 21.9% of the samples in rubona. double infections of cmv+pevyv were common and detected in both sites, 12.5% in rubona and 2.7% in gashora, followed table 4 ­ incidence (%) of viral diseases recorded on eighteen hot pepper genotypes grown under field conditions during two seasons at rubona station, huye district in rwanda the values represent means of un­transformed data. means comparison done by least significant difference (lsd) test on transformed data. data transformed by square root (x+1). means with the same letters within a column are not significantly different (p<0.05). n= 10 replicated thrice. wap= weeks after planting; 1 genotypes collected from farmers’ field and conserved in rwanda national genbank; 2 genotypes from world vegetable center; 3 varieties obtained from seed companies and are grown for commercial purposes in rwanda. genotype type season one (march to june, 18) season two (mid­oct. 18 to march, 19) 2wap 4wap 6wap 8wap 10wap 2wap 4wap 6wap 8wap 10wap 00765ppr local 1 0 0 43 a 97 a 100 a 0 3 b 10 bc 23 abd 33 bcdef 00767ppr local 0 0 0 e 3 f 3 f 0 0 b 3 c 10 bcd 17 def 00774ppr local 0 3 33 abc 90 ab 97 ab 0 0 b 17 ab 36 a 60 abcd 00775ppr local 0 10 33 abc 70 bc 87 abc 0 0 b 7 bc 20 abcd 77 ab 00786ppr local 0 10 37 ab 77 abc 90 ab 0 3 b 7 bc 20 abcd 30 cdef 00791ppr local 0 3 33 abc 80 abc 93 ab 0 0 b 0 c 23 abcd 97 a 00792ppr local 0 7 30 abcd 87 ab 97 ab 0 0 b 0 c 33 ab 70 abc 00802ppr local 0 0 0 e 3 f 10 f 0 0 b 3 c 7 bcd 10 f 00795ppr local 0 0 30 abcd 83 ab 97 ab 0 0 b 0 c 20 abcd 83 a pbc 462 introduced 2 0 0 0 e 13 de 30 e 0 0 b 0 c 3 cd 3 f pp9950­5197 introduced 0 0 7 de 10 f 20 ef 0 3 b 3 c 3 cd 13 ef hp 0117 introduced 0 0 10 cde 37 de 63 d 0 0 b 0 c 7 bcd 13 ef pp9852­170 introduced 0 0 7 de 37 de 63 d 0 0 b 0 c 3 cd 10 f icpn 18­7 introduced 0 0 13 bcde 40 d 63 d 0 0 b 0 c 0 d 14 f long red cayenne commercial 3 0 0 23 abcde 77 abc 90 ab 0 3 b 10 bc 27 abcd 57 abcde bird eye hybrid commercial 0 0 7 de 40 d 70 cd 0 0 b 10 bc 33 ab 73 abc red scotch bonnet commercial 0 3 33 abc 57 cd 80 bcd 0 0 b 10 bc 23 abd 70 abc california wonder commercial 0 0 37 ab 83 ab 100 a 0 13 a 23 a 30 abc 83 a lsd (0.05) 10 24 26 19 5 11 28 46 p­value 0.3699 0.0027 <.0001 <.0001 0.0002 0.0167 <.0001 <.0001 adv. hort. sci., 2020 34(4): 397­412 404 genotype season one (march to june, 18) season two (mid­oct. 18 to march, 19) type 2wap 4wap 6wap 8wap 10wap 2wap 4wap 6wap 8wap 10wap 00765ppr local 1 0 0 0 d 90 a 97 ab 0 0 3 33becd 70 abc 00767ppr local 0 0 17 cd 40 bc 87 abc 0 0 0 0 e 10 f 00774ppr local 0 3 17 cd 90 a 100 a 0 0 10 63 ab 83 ab 00775ppr local 0 0 13 cd 77 ab 97 ab 0 0 3 13 de 53 bcde 00786ppr local 0 3 13 cd 100 a 100 a 0 0 0 57 bc 73 a 00791ppr local 0 0 17 cd 97 a 100 a 0 0 0 17 cde 83 ab 00792ppr local 0 7 23 abc 100 a 100 a 0 0 10 60 ab 87 ab 00802ppr local 0 0 0 d 43 b 63 cd 0 0 0 40 bcde 60 bcd 00795ppr local 0 3 17 cd 97 a 100 a 0 0 0 27 bcde 80 ab pbc 462 introduced 2 0 0 0 d 0 d 13 f 0 0 0 7 efg 20 ef pp9950­5197 introduced 0 0 0 d 10 d 47 de 0 0 0 3 de 13 f hp 0117 introduced 0 0 0 d 17 bcd 70 cd 0 0 7 33 bcde 53 bcde pp9852­170 introduced 0 0 0 d 17 bcd 73 bc 0 0 3 23 bcde 33 cdef icpn 18­7 introduced 0 0 7 cd 14 cd 30 ef 0 0 0 7 de 23 def long red cayenne commercial 3 0 3 13 cd 87 a 100 a 0 0 10 100 a 100 a bird eye hybrid commercial 0 7 37 ab 100 a 100 a 0 0 10 43 bcd 60 bcd red scotch bonnet commercial 0 0 20 bc 90 a 100 a 0 0 3 33 bcde 70 abc california wonder commercial 0 13 40 a 93 a 100 a 0 0 20 100 a 100 a lsd (0.05) 9 18 29 24 13 40 39 p­value 0.2336 0.0006 <.0001 <.0001 0.1196 <.0001 <.0001 table 5 ­ incidence (%) of viral diseases recorded on eighteen hot pepper genotypes grown under field conditions during two seasons at gashora station, bugesera district in rwanda the values represent means of un­transformed data. means comparison done by least significant difference (lsd) test on transformed data. data transformed by square root (x+1); means with the same letters within a column are not significantly different (p<0.05). n= 10 replicated thrice; wap= weeks after planting; 1 genotypes collected from farmers’ field and conserved in rwanda national genbank; 2 genotypes from world vegetable center; 3 varieties obtained from seed companies and are grown for commercial purposes in rwanda. genotype audpc in rubona site audpc in gashora site pooled season one season two season one season two 00765ppr 240 cdef 86 bcd 264 c 203 cdefg 197cd 00767ppr 9 h 33 cd 146 d 22 h 52 e 00774ppr 294 abc 178 ab 298 bc 334 abc 279 abc 00775ppr 281 bcd 191 ab 315 bc 129 efgh 233 bc 00786ppr 364 ab 95 bcd 311 bc 272 cde 261 abc 00791ppr 320 abc 207 ab 316 abc 191 cdefg 259 abc 00792ppr 398 a 199 ab 388 a 300 bcd 321 ab 00802ppr 21 h 34 cd 149 d 169 defgh 93 e 00795ppr 273 bcde 179 ab 333 abc 240 cdef 257 abc pbc 462 63 gh 15 d 78 d 57 gh 55 e pp9950­5197 62 gh 52 cd 83 d 30 h 56 e hp 0117 165 efg 46 cd 128 d 131 efgh 117 de pp9852­170 148 fg 27 cd 137 d 97 fgh 102 de icpn 18­7 179 def 16 d 93.6 d 63 gh 89 e long red cayenne 308 abc 146 abc 344 ab 448 a 316 ab bird­eye 312 abc 179 ab 346 ab 251 cde 276 abc red scotch bonnet 331 abc 177 ab 350 ab 198 cdefg 264 abc california wonder 379 ab 248 a 312 bc 445 ab 346 a lsd (0.05) 109.2 123.7 72.9 149.5 99.9 p­value < 0.0001 0.0011 < 0.0001 < 0.0001 < 0.0001 table 6 ­ means of the area under disease progress curve (audpc) of viral diseases recorded on eighteen genotypes of hot pepper dur­ ing two seasons in rubona and gashora sites the values represent means of three replicates. means with the same letters within a column are not significantly different (p<0.05). means comparison done by least significant difference (lsd) test. waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 405 by cmv+pvmv detected in 9.4% of the samples test­ ed in rubona. triple infection of cmv+pevyv+pvmv was detected in 9.4% of the samples from rubona. all hot pepper genotypes were infected by cmv. assessment of aphids aphid populations differed significantly between sites (p<0.0001), season (p=0.0075) and thus, data were analysed separately. in both seasons, the aphid population was significantly (p<.0001) higher in rubona compared to gashora (table 7). three species of aphids were observed. these were aphis gosypii glover, macrosiphum euphorbiae thomas and acyrthosiphon pisum (harris). the a. gosypii and m. euphorbiae were the most abundant in both sites, while a. pisum was observed in gashora only. all genotypes were infested by aphids but the dif­ ference in numbers were not significant (p = 0.0923) among the genotypes (table 8). the mean number of aphids per plant ranged from 4 to 108 in rubona and 4 to 19 in gashora, while the mean number of aphids per leaf ranged from 0.8 to 18 and 0.6 to 3.2, respec­ fig. 2 ­ overall incidence of viruses detected in leaf samples of hot pepper genotypes from rubona and gashora in rwanda. table 7 ­ mean number of aphids captured in hot pepper fields during two cropping seasons in rubona and gashora experimental sites the values represent means and standard errors of three replicates. ns= not significant at 0.05 level. season rubona site gashora site a. gosypii m. euphorbiae total aphids a. gosypii m. euphorbiae a. pisum total aphids feb­june 2018 167 ± 30 a 10 ± 1 b 177 ± 30 a 32 ± 6 a 0 ± 0 b 11 ± 1 a 43 ± 5 a oct. 2018 ­march 2019 48 ± 4 b 80 ± 11 a 128 ± 10 a 26 ± 5 a 28 ± 2 a 0 ± 0 b 54 ± 6 a lsd (0.05) 59 21 62 16 5 2 16 p­value 0.0017 <0.0001 ns ns < 0.0001 <0.0001 ns table 8 ­ number of aphids associated with different hot pepper genotypes in rubona and gashora's experimental sites the values represent means of untransformed data. ns= not significant at 0.05 level. genotype rubona site gashora site mean aphids/plant total aphids aphids/plant aphids/leaf total aphids aphids/plant aphids/leaf 00765ppr 178.3 32.8 5.5 53.3 11.7 1.9 22.3 00767ppr 64.8 5.5 0.9 29 5.2 0.8 5.3 00774ppr 178.5 31.5 5.3 37.3 6.2 1 18.8 00775ppr 125.2 19.2 3.2 56 11.3 1.9 15.3 00786ppr 258.3 54.8 9 56.3 9.5 1.6 32.2 00791ppr 117.2 20.3 3.4 66.3 11.8 2 16.1 00792ppr 125.2 18.3 3 45 9.5 1.6 13.9 00802ppr 113 21.3 3.6 36.3 6.7 1.1 14 00795ppr 100.8 10.1 1.7 65.5 14 2.3 12.1 pbc 462 120 19.5 3.2 38 5.7 0.9 12.6 pp9950­5197 117.5 19.3 3.2 62.2 11 1.8 15.1 hp 0117 289.5 108 18 29.7 5.5 0.9 56.8 pp9852­170 205.8 42.2 7 94.5 19.3 3.2 30.8 icpn 18­7 136.2 25.3 4.2 35 6.2 1 15.8 long red cayenne 171 33.8 5.6 59.8 11 1.8 22.4 bird­eye 72.2 4.8 0.8 26 4 0.6 4.4 scotch bonnet 83.7 10.2 1.7 50.7 9.7 1.6 9.92 california wonder 287.8 79.2 13 40.5 7.8 1.3 43.5 p­value (0.05) ns ns ns ns ns ns ns adv. hort. sci., 2020 34(4): 397­412 406 tively. except hp 0117 and california wonder at rubona site, the rest of the genotypes showed low numbers of aphids which did not exceed recom­ mended chemical control action thresholds of 10 aphids per leaf. the population exhibited a negative correlation with minimum (r = ­0.04, ­0.22) and maxi­ mum temperature (0.50, ­0.73) while, the correlation was positive with average rainfall (0.37, 0.68) in rubona and gashora, respectively. these correlations were not significant at the 5 percent level (data not shown). classification of hot pepper genotypes for resistance to viral diseases under field conditions commercial genotypes were more susceptible to virus infections than the new lines from world vegetable center. various degrees of symptoms were observed on most genotypes during the evalua­ tion period. these included leaf mosaic, crinkling, chlorosis, vein banding, and leaf deformation. based on incidence and severity indices from both locations and seasons only five genotypes rated resistant to viral diseases i.e. 00767ppr, 00802ppr, pbc 462, pp9950­5197 and icpn 18­7 with total scores between 2­3; three moderately resistant 00765ppr, hp 0117 and pp9852­170 with scores between 4­6; and nine susceptible 00775ppr, 00786ppr, 00774ppr, 00786ppr, 00792ppr, long red cayenne, bird eye hybrid, red scotch bonnet, and california wonder with scores between 7­9 (table 9). two local genotypes (00767ppr, and 00802ppr) and three introduced genotypes (pbc 462, pp9950­5197 and icpn 18­7) showed resistance to viral diseases in both locations. reaction of hot pepper genotypes to cmv under arti‐ ficial inoculation conditions disease incidence a significant difference (p<0.05) in disease inci­ dence and symptoms severity was observed between the genotypes tested (table 10). infected plants showed systemic symptoms of cmv infection includ­ ing leaf mosaic, mottle, crinkling, small and deformed leaves, and stunting with varying degrees of severity (fig. 3). six genotypes (red scotch bonnet, 00795ppr, 00792ppr, 00786ppr, 00774ppr, and long red cayenne) developed symptoms thirteen days’ post­inoculation (dpi) and the first three table 9 ­ classification of the hot pepper genotypes based on incidence (%) and severity indices of virus­induced diseases under field conditions the values represent means of un­transformed data. means comparison done by least significant difference (lsd) test on transformed data. means with the same letters within a column are not significantly different (p<0.05). incidence scores; 20% =1, 21­30%=2, 31­ 50%=3 and >51%=4. severity scores; <1=1, 1.1­2.0=2, 2.1­3.0=3 and >3.0=4. cumulative scores i.e. incidence + severity indices; < 3= resi­ stant (r), 4­6 = moderately resistant (mr) and 7­8 = susceptible (s). genotype incidence (%) severity indices cumulative rating host reactionseason one season two pooled rating season one season two pooled rating 00765ppr 97 51.5 74.3 ab 4 2.4 1.6 2 d 2 6 mr 00767ppr 45 11.5 28.3 e 2 1 0.3 0.7 ef 1 3 r 00774ppr 96.5 71.5 84 a 4 2.8 2.7 2.8 abc 3 7 s 00775ppr 83.5 65 74.3 ab 4 2.6 2.2 2.4 cd 3 7 s 00786ppr 88.5 51.5 70 abc 4 2.9 2.1 2.5 bcd 3 7 s 00791ppr 90 90 90 a 4 2.9 2.7 2.8 abc 3 7 s 00792ppr 93.5 78.5 86 a 4 3.5 3 3.3 a 4 8 s 00802ppr 33 24 28.5 e 2 0.8 1.1 1 ef 1 3 r 00795ppr 91.5 81.5 86.5 a 4 2.8 2.9 2.9 abc 3 7 s pbc 462 13 11.5 12.3 e 1 0.2 0.4 0.3 f 1 2 r pp9950­5197 28.5 13 20.6 e 1 0.7 0.5 0.6 ef 1 2 r hp 0117 53.5 33 43.3 cde 3 1.2 0.9 1.1 e 2 5 mr pp9852­170 55 21.5 38.3 cde 3 1.2 0.7 1 ef 1 4 mr icpn 18­7 35 15 25 e 2 0.6 0.5 0.6 ef 1 3 r long red cayenne 88.5 78.5 83.5 a 4 3 3.3 3.2 ab 4 8 s bird­eye 76.5 55 65.8 abcd 4 3 2.5 2.8 abc 3 7 s scotch bonnet 80 71.5 75.6 ab 4 3 2.3 2.7 abcd 3 7 s california wonder 91.5 68.5 75.6 a 4 2.9 2.9 2.9 abc 3 7 s lsd 33.6 0.7 p­value 0.0004 <.0001 waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 407 showed high levels of cmv infection (table 10). introduced genotypes pbc462, pp9950­5197, hp 0117, pp9852­170, icpn18­7, and local genotype 00765ppr displayed symptoms at seventeen dpi while the remaining two local genotypes 00767ppr and 0802ppr showed symptoms at nineteen dpi. a total of six genotypes namely 00765ppr, 00792ppr, 00795ppr, 00774ppr, long red cayenne and red scotch bonnet had disease incidence between 50­ 100%, severity 2.7­5 at nineteen dpi and thus rated as susceptible to cmv (table 10). genotypes pp9950­ 5197, 00786ppr, hp 0117 and icpn 18­7 had moder­ ate levels of infection displaying 22­35.4% disease incidence at nineteen dpi and thus classified as mod­ erately resistant to cmv. among the 14 hot pepper genotypes tested, only four including two local (00767ppr, 0802ppr) and two introduced (pbc 462 and pp9852­170) showed resistant reaction against cmv with disease incidence ranging from 2­18.8% and severity 1.0­1.2 at nineteen dpi. a positive reac­ tion to cmv was revealed by elisa for the tested samples from all genotypes. table 10 ­ reaction of hot pepper genotypes against cucumber mosaic virus under screenhouse conditions the values represent means of un­transformed data. means comparison done by least significant difference (lsd) test on transformed data. data transformed by square root (x + 1). means with the same letters within a column are not significantly different (p<0.05). incidence scores: 20% =1, 21­30%=2, 31­50%=3 and >51%=4. severity scores: <1=1, 1.1­2.0=2, 2.1­3.0=3 and >3.0=4. cumulative scores i.e. incidence + severity indices: < 3= resistant (r), 4­6 = moderately resistant (mr) and 7­8 = susceptible (s). n = 16 replicated three times. dpi= days post­inoculations. genotype incidence (%) severity indices cumulative rating host reaction13dpi 15dpi 17dpi 19dpi rating 13dpi 15dpi 17dpi 19dpi rating 00765ppr 0.0 d 0.0 d 60.4 bc 77.1 b 4 1.0 c 1.0 d 1.8 d 2.7 c 3 7 s 00767ppr 0.0 d 0.0 d 0.0 g 2.1 h 1 1.0 c 1.0 d 1.0 e 1.0 f 1 2 r 00774ppr 10.4 cd 41.7 c 41.7 cd 50.0 de 3 1.1 c 1.4 cd 2.1 d 3.1 c 4 7 s 00786ppr 4.2 cd 8.3 d 22.9 def 25 fg 2 1.1 c 1.1 cd 1.3 e 1.4 ef 2 4 mr 00792ppr 75.0 b 75 bc 75.0 ab 75 b 4 2.3 ab 2.8 b 3.2 bc 4.0 b 4 8 s 00802ppr 0.0 d 0.0 d 0.0 g 18.8 fgh 1 1.0 c 1.0 d 1.0 e 1.2 f 2 3 r 00795ppr 75.0 b 75.0 b 81.3 ab 100.0 a 4 2 b 2.7 b 3.7 ab 5.0 a 4 8 s pbc 462 0.0 d 0.0 d 4.2 efg 12.5 gh 1 1.0 c 1.0 d 1.0 e 1.2 f 2 3 r pp9950­5197 0.0 d 0.0 d 8.3 efg 22.9 fg 2 1.0 c 1.0 d 1.0 e 1.3 f 2 4 mr hp 0117 0.0 d 0.0 d 2.1 fg 35.4 ef 3 1.0 c 1.0 d 1.0 e 1.5 def 2 5 mr pp9852­170 0.0 d 0.0 d 2.1 fg 14.6 gh 1 1.0 c 1.0 d 1.0 e 1.2 f 2 3 r icpn 18­7 0.0 d 0.0 d 25.0 de 56.3 cd 4 1.0 c 1.0 d 1.0 e 2.0 de 2 6 mr long red cayenne 16.7 c 29.2 c 89.6 a 97.9 a 4 1.2 c 1.5 c 2.7 c 4.5 ab 4 8 s red scotch bonnet 91.7 a 93.8 a 93.8 a 100.0 a 4 2.4 a 3.3 a 4.1 a 4.8 a 4 8 s lsd (0.05) 12.6 15.3 21.4 19.8 0.3 0.5 0.6 0.7 p value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 fig. 3 ­ symptoms of cucumber mosaic virus on different hot pepper genotypes. (a) leaf mosaic, crinkling and distortion in commercial genotype scotch bonnet, (b) mottling in local genotype 00774ppr, (c) leaf distortion and stunting in local genotype 00795ppr, (d) leaf mosaic in introduced genotype icpn18­7. adv. hort. sci., 2020 34(4): 397­412 408 4. discussion and conclusions host plant resistance is an important factor in the integrated management of pests. the present study was undertaken to identify genotypes that can be used in production or as sources of resistance to viruses and aphids in hot pepper breeding programs. all the genotypes tested in this study were infected by the viruses observed either in the field or screen­ house however, there were some resistant geno­ types found based on incidence and symptoms sever­ ity of the viral diseases. in the field, five genotypes 00802ppr, c. bacca‐ tum 00767ppr, c. annuum pbc 462, pp9950­5197 and icpn 18­7 were resistant to the viral diseases while c. annuum 00765ppr, hp 0117 and pp9852­ 170 were moderately resistant. the rest of the geno­ types were susceptible to the viral diseases. these variations among the genotypes might be due to vari­ ous factors that include genetic make­up, the strain of the virus and their combinations, time of infection and prevailing environmental conditions (visalakshi and pandiyan, 2018). such variations reveal the diversity present within the genotypes that needs to be exploited. in previous studies under field condi­ tions, various genotypes from c. baccatum, c. annu‐ um and c. frutescens species have displayed variable resistance to some viruses such as pvmv, tmv, cmv, pepper mild mottle virus, chili veinal mottle virus and leaf curl virus (appiah et al., 2014; rahman et al., 2016; bandla and beena, 2018; fajinmi et al., 2018). for instance, c. baccatum pi 439381­1­3 was report­ ed as resistant to cmv and pmmov under field con­ ditions (suzuki et al., 2003). our result, reports some additional sources of resistance from c. baccatum and c. annuum species that could be valuable in hot pepper breeding programs as well as cultivation if preferred by farmers. the cmv, pvmv and pevyv were detected in leaf samples collected from fields and cmv was the most abundant. these three viruses have also been report­ ed to infect pepper previously in rwanda (skelton et al., 2018). the high incidence of cmv in the field could be attributed to several factors including wide host range, climatic conditions and efficiency of vec­ tor transmission as reported by shah et al. (2009). appiah et al. (2014) in their study also observed a high incidence of cmv in the range of 75% to 83.3% on pepper cultivars. all genotypes evaluated were infected with cmv and almost half of them showed multiple (double or triple) infections of cmv with either pvmv or pevyv or both, which could have serious consequences in their management. mixed infections of cmv and pvmv in pepper have been reported in previous studies (aliyu, 2014; appiah et al. 2014). mixed infections in pepper plants increase the severity of disease symptoms leading to signifi­ cant yield losses (arogundade et al., 2012). thus, understanding the interactions of these viruses is crucial for the development of efficient and sustain­ able management strategies such as resistant vari­ eties (syller, 2012). results from screenhouse showed that all geno­ types developed symptoms to cmv infection albeit at different levels of severity and time, confirming the virulence of the local cmv isolate used. the plants developed systemic symptoms including mosaic, mottle, leaf crinkling and distortion and stunting which were similar to symptoms described by rahman et al. (2016). two local genotypes 0802ppr and c. baccatum 00767ppr, and two introduced genotypes c. annuum pbc 462 and pp9852­170 were found resistant to cmv while one local c. annuum genotypes 00786ppr and three introduced geno­ types pp9950­5197, hp 0117 and icpn 18­7 were moderately resistant. the previously published resis­ tant genotypes (pp9852­170 and pp9950­5197) to cmv in screenhouse conditions were also resistant in our study (gniffke, et al., 2013; reddy, et al., 2014). however, genotype icpn 18­7 that was previously reported as susceptible to cmv was moderately resistant in the current study (gniffke, et al., 2013). the reason for these differences could be attributed to the use of different strain of cmv. various sources of resistance to cmv in pepper have been identified in c. annuum, c. baccatum and c. frutescens species (grube et al., 2000; chaim et al., 2001; caranta et al., 2002; suzuki et al., 2003; rahman et al., 2016). the present findings prove that natural resistance or tol­ erance exists in tested c. annuum and c. baccatum genotypes. as different strains of cmv exist, it is desirable to test the identified pepper genotypes against multiple strains of cmv to validate their resis­ tance. in both field and screenhouse experiments, geno­ type 00767ppr, 0802ppr and pbc 462 were consis­ tently resistant to viral diseases while genotype hp 0117 was moderately resistant, providing evidence that the reactions of these genotypes to the virus might be due to genetic factors. however, unlike under field conditions where genotypes pp9950­5197 and icpn 18­7 were categorized as resistant to viral waweru et al. ‐ evaluation of hot pepper genotypes in rwanda 409 diseases, they reacted differently when subjected to the artificial inoculation with cmv and grouped as resistant. this might be due to disease escape in the field. similar observations were made by ashfaq et al. (2014), where two chili genotypes c­7 and c­8 showed a different reaction to cmv under controlled and uncontrolled conditions. on the other hand, genotype pp9852­170 was resistant to cmv under controlled conditions while in the field, it was grouped as moderately resistant to viral diseases. this may be due to the complex nature of the virus­ es’ infection in the field where more than two viruses occur in combination. as was evident in this study where single and mixed infections of cmv, pvmv, and pevyv were observed and their presence might have contributed towards variations in the reaction of the host in the field. these variations in the obser­ vation may also be due to variations in inoculum load and environmental conditions that might have inter­ fered with plant behaviour. the categorization of genotypes into resistant, moderately resistant and susceptible was based on the incidence and severity of the viral diseases on the host. however, it is noteworthy that the genotypes 00802ppr, 00767ppr, pbc 462, pp9950­5197 and icpn 18­7 classified as resistant to viral diseases had the lowest audpc values of less than 100 in the field while the highest audpc value was recorded in sus­ ceptible check california wonder 346. lower audpc values indicate a lower disease development rate. these genotypes had low audpc values which implies that the plant defence mechanism against the viruses could be mediated by resistance (r) genes which are observed as complete resistance or extreme resistance (er) and that the virus replication could have been hindered or gone undetectable among the infected cells (ingvardsen et al., 2010). the reaction of pepper cultivar to the viral diseases is governed by the resistance genes which can be brought by a single gene or multiple genes (kang et al., 2010; kim et al., 2017). however, genes responsi­ ble for their resistance in particular for the two local accessions are unknown and mechanisms that under­ lie their resistance are yet to be understood. this is important information that could help to determine useful markers to support breeding processes. aphid species are important agricultural pests because they have a broad host range and transmit many important plant viruses. in this study, three species of aphids were recorded in the pepper fields and the most abundant in both sites were a. gosypii and m. euphorbiae. these findings agree with previ­ ous studies by meena et al. (2013) and rajput et al. (2017) who reported the infestation of hot pepper fields with a. gosypii in india. similar results on m. euphorbiae were reported by djieto­lordon et al. (2014) in cameroon. the presence of a. pisum in gashora was understandable since there was a pigeon peas field near the experimental plots. these polyphagous insects belong to the hemiptera order and they are important pests because of the ability to transmit several viruses in pepper. according to fajinmi et al. (2011); dombrovsky et al. (2010) and zitter and murphy, (2009) a. gosypii efficiently trans­ mits cmv, pevyv, and pvmv which were detected in this study. there was no difference in genotypes infestation by the aphids. besides, complete genotype resistance to aphids’ infestation was not recorded in any of the genotypes tested. bird­eye hybrid was the less pre­ ferred by the aphids (4.4 aphids/plant) followed by 00767ppr (5.3 aphids/plant) and red scotch bonnet (9.9 aphids/plant) while the most preferred was genotype hp 0117 (56.8 aphids/plant) followed by california wonder (43.5 aphids/plant) and 00786ppr (32.2 aphids/plant). unlike other plant species such as soybean where a lot has been done on resistance to aphids (hill et al., 2004), only a few studies have been conducted on pepper (frantz et al., 2004; sun et al., 2018). sun et al. (2018), in their studies, identi­ fied c. baccatum accession pb2013071 as highly resistant, while the accessions pb2013062 and pb2012022 as intermediate resistant to m. persicae under screen house conditions. recently, quantita­ tive trait loci (qtls) conferring resistance to m. persi‐ cae in pepper was detected (sun et al., 2019). in the present study, prevailing weather conditions, espe­ cially at the gashora site, negatively affected the population of aphids leading to low infestation on pepper plants. thus, further efforts are needed to identify and validate the resistance of these geno­ types to aphids under controlled and uncontrolled conditions. most of the varieties grown in the country includ­ ing the commonly grown commercial varieties were found to be susceptible to viral diseases. a relatively higher number of resistant lines from introduced material indicates that the world vegetable center germplasm collection has a wider genetic base than local material. since viruses cause serious diseases of hot pepper around the world, the results of this study may be promising and could be used in the for­ adv. hort. sci., 2020 34(4): 397­412 410 mulation of integrated control strategies for the management of these destructive 1diseases. the use of resistant pepper genotypes to manage the viral diseases can potentially replace or minimize the application of harmful pesticides and could be used as an important component of integrated pest man­ agement (ipm) which is a promising approach to sus­ tainable agriculture. in the present study, three genotypes 00767ppr, 00802ppr and pbc 462 consistently rated as resistant to viral diseases while genotype hp 0117, pp9852­170, pp9950­5197 and icpn 18­7 were moderately resistant under field and screenhouse conditions. as revealed from the study, most of the local genotypes and all of the commercially grown pepper genotypes tested were susceptible. therefore, the identified genotypes especially the ones from world vegetable center are recommended for adoption by growers. the two local collections 00767ppr and 00802ppr are not preferred cultivars for commercial production and thus, they can be utilized in breeding programs as potential sources for virus resistance. farmers should be encouraged to use hot pepper varieties that are resistant to viruses as part of a management program to maximize yields. further studies are needed to identify and validate resistance of the tested genotypes to aphids under controlled and uncontrolled conditions. acknowledgements this work was funded by the united states agency for international development, as part of the feed the future initiative, under the cgiar fund, award number bfs­g­11­00002, and the predecessor fund the food security and crisis mitigation ii grant, award number eem­g­00­04­00013; with additional support from rwanda agriculture and animal resources development board (rab). we would like to thank the world vegetable center, eastern and southern 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maturation and ripening stages t.­a. smith 1, j. vásquez­martínez 2 †, e. mellado­mojica 2, k. vaidya 1, m. g. lopez 2, n. benkeblia 1, 3 (*) 1 department of life sciences, university of the west indies, mona campus, kingston 7, jamaica. 2 biotechnology and biochemistry department, cinvestav‐unidad irapuato, km 9.6 libramiento norte carr., irapuato‐león 36821, irapuato gto., mexico. 3 laboratory of tree fruit and aromatic crops, the biotechnology centre, faculty of science and technology, university of the west indies, mona campus, kingston 7, jamaica. key words: acids, carambola, june plum, maturation, otaheite, ripening, sugars. abstract: the study aimed to profile and quantify sugars and organic acids metabolites in carambola, june plum and otaheite fruits during three different “on tree” stages: immature, green­mature and ripe stages. metabolites were profiled and quantified by gas chromatography­mass spectrometry (gc­ms). results showed that glucose, fructose, galactose, arabinose, and the sugar alco­ hol myo­inositol were detected in all fruits, while sucrose was detected in carambola and june plum only. organic acids identified in all fruits were malic acid, citric acid, propanoic acid, and acetic acid. comparatively, june plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was found in otaheite and the lowest in carambola. on the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. total sugars increased by 37%, 8% and 46% in ripe carambola, june plum and otaheite, respectively. total organic acids decreased by 20% and 49% in ripe carambola and otaheite, while they slightly increased by 3% in ripe june plum. furthermore, sugars/organic acids ratio in all fruits increased during maturation and ripening stages. principal component analysis (pca) showed two main groups of highly scoring metabo­ lites, while the hierarchical cluster analysis (hca) showed that the metabolites were grouped into three main clusters. conclusively, results showed that glu­ cose, fructose, malic acid and tartaric acids were the key marker metabolites of the maturation and ripening stages of the three fruits. 1. introduction carambola (averrhoa carambola) belongs to the family oxalidaceae (*) corresponding author: noureddine.benkeblia@uwimona.edu.jm † present address: biochemistry department, superior institute of technology of irapuato (itesi), tecnm., irapuato gto., mexico. citation: smith t.­a., vásquez­martínez j., mellado­ mojica e., vaidya k., lopez m.g., benkeblia n., 2022 ­ profiling of primary metabolites of averrhoa carambola, spondias dulcis and syzygium malaccense fruits revealed underpin‐ ning markers during “on‐tree” maturation and ripening stages. ­ adv. hort. sci., 36(1): 13­26. copyright: © 2022 smith t.­a., vásquez­martínez j., mellado­mojica e., vaidya k., lopez m.g., benkeblia n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 june 2021 accepted for publication 20 september 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-11437 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 13­26 14 originated in asia but has since developed a toler­ ance for tropical climates (shui and leong, 2006). presently, carambola is cultivated extensively in india and china (narain et al., 2001), but malaysia is the largest exporter (abdullah et al., 2007). work has been done to improve carambola cultivars in the united states in the 1930s, and since then, the fruit became more popular, while some sub species of star fruit exist in the caribbean, central america and tropical west africa (neto et al., 2009). unripe carambola is moderately sour, but when ripe it is very sweet and used for juice, fruit salads, chutney, stewed fruits, garnish drinks and dishes, or adding it to fruit smoothies. physiologically, carambola does not exhibit climacteric ripening behaviour even though it continues to synthesise carotenoids and develop its yellow­orange colour (warren, 2009). june plum (spondias dulcis forst. syn. spondias cytherea sonn.), a drupe belonging to the family anacardiacea, is native to the society islands of the south pacific ranging from melanesia to polynesia and was first introduced to jamaica in the year 1782, and later in 1792 by captain bligh (graham et al., 2004 a). although the fruit is well known, there are no named cultivars, but both forms exist, one is with a thick mesocarp and a more pleasant taste, and another has long spines, a woody endocarp with a pungent and resinous taste (daulmerie, 1994). ripe june plum is eaten mostly raw but is also used in making refreshing drinks, jam, chutney, sauce or served with meat and seafood. when harvested green and mature, june plum ripens, and studies showed that respiratory pattern is typical of a climac­ teric fruit (daulmerie, 1994; graham et al., 2004 b). otaheite (syzygium malaccense) is a berry and belongs to the family myrtaceae. it is thought to be native to the indo­malay or southeast asian region (whistler and elevitch, 2006). otaheite is, however distributed in many tropical countries throughout the world, particularly in africa and south america (oliveira et al., 2011). other english common names for this fruit include: malay apple, mountain apple, pomerac, and rose apple (batista et al., 2017). two colour forms exist: one which produces red flowers and fruits and another, less common variety, which produces white flowers and fruit (whistler and elevitch, 2006). ripe otaheite fruit is not very sweet, and often eaten raw. however, in some tropical countries, it is stewed with sugar to make jam or wine and refreshing drinks. no study is recorded on whether otaheite is a climacteric or non­climacteric fruit except the work of basanta (1998) who reported that otaheite is a non­climacteric fruit. ripening can be defined as the total changes of fruit tissue metabolism, leading to the production of an attractive fruit which can be consumed, aiding in the release and dispersal of the seed (adams­phillips et al., 2004). the ripening process is characterized by softening of fruit tissue and an increase in volatile compounds as well as pigments such as carotenoids and flavonoids which results in a more appealing fruit (giovannoni, 2001). the concentration of sugars and organic acids in fruits varies depending on the fruit variety and the environmental conditions of the par­ ent plant (haruenkit, 2004). overall, there is a gener­ al decrease in organic acids and an increase in sugar content as fruit development progresses, due to decarboxylation of organic acids and breakdown of stored carbohydrates to produce sugars (batista­silva et al., 2018). according to etienne et al. (2013), using advanced technologies, i.e. proteomics, transcrip­ tomics and metabolomics, studies have shown evi­ dence of a shift from the accumulation of organic acids to sugar synthesis in the final stage of fruit development in several species of fruit. thus, the res­ piratory pathways commonly involved in the reduc­ tion of fruit sugars are glycolysis, oxidative pentose phosphate (opp) pathway, and the tricarboxylic acid (tca) pathway (tucker, 2012). because most fruits reach their best sensorial and commercial quality attributes when they ripen on the plants, the correct maturity for harvest of fruits impacts their postharvest shelf­life and quality attributes during storage (thompson, 2003). gene expression resulting from natural processes and trig­ gering fruit ripening induces many metabolic pro­ cesses leading to the formation of hundreds and even thousands of different metabolites (pech et al., 2013). although extensive literature is readily available on the metabolic changes during the maturation, ripening and senescence of fresh crops, few researches reported on changes in metabolite pro­ files during postharvest ripening and senescence (benkeblia, 2016). however, a limited work was car­ ried out on the metabolites variation during the development and ripening of peach (lombardo et al., 2011), strawberry (zhang et al., 2011), pear (oikawa et al., 2015), and pitaya (wu et al., 2019), while scarce work was reported on some tropical fruit (fabi smith et al. ‐ ripening metabolome of tropical fruits 15 et al., 2010). in the present work, in order to explore the varia­ tion of the metabolic profile of three tropical fruits commonly consumed in the tropics, we performed a profiling study of primary metabolites which are the main indicators of the maturation and the ripening of fruits. for this purpose, we selected three fruits namely carambola (sweet type), june plum and ota­ heite. one of the goals of this study was to assess how metabolically different the “on tree” maturation and ripening stages of these three fruits are and to find out if there is any particular metabolic profile which could be associated with these two stages of the fruits. on the other hand, by evaluating the metabolomic pattern at both maturation and ripen­ ing stages of the three fruits. overall, this study is aiming to explore a part of the chemical potential of carambola (sweet type), june plum and otaheite which will aid in the future to know the primary metabolites of these fruits that may correlate to dif­ ferent stages and determine which metabolites might be used as maturation and ripening markers. 2. materials and methods fruits collection for the purpose of the present study, three physi­ ological stages of the fruits were investigated: green­ immature, mature, and ripe stages (fig. 1). the colour and softness of the fruits were the two criteria used for discriminating the different maturation and ripening stages. a period varying from seven to ten days elapsed between each harvesting (sampling) stage. the commercial (optimal) harvesting stage of carambola is stage 3 (ripe), while for june plum and otaheite is stage 2 (mature). the fruits carambola, june plum, and otaheite of the three stages of each fruit were collected from three trees of same loca­ tion. the fruit june plum was collected from a farm in st. elizabeth. otaheite fruits were collected from a local farm in mona, kingston, and carambola samples were collected from orange river research station in st. mary. the three stages differentiated and sam­ pled based on their size and colour. for each stage, three samples were collected from three different trees, and each sample consisted of at least six fruits. fruits collected were controlled for absent of any defect, wound or disease. immediately after being collected, fruits were placed in plastic bags, and the bags were placed on ice in a cooler and transported to the laboratory within few hours. then, fruits were washed with mild detergent and rinsed thoroughly, followed by seed removal, dicing or slicing and frozen for 48 hours at ­20°c. freeze‐drying prior to the extraction of the profiled sugars and organic acids, samples were freeze­dried in a labconco freeze­drier (labconco corp., kansas, mo, usa). after six days and complete drying, samples were sealed in plastic bags under vacuum using a multivac c100 vacuum packer (multivac, wolfertschwenden, germany) and stored under dry­ ness in a desiccator until further use. extraction of sugars and organic acids metabolites sugars and organic metabolites were extracted by the method described by broeckling et al. (2005) with some modifications. in an eppendorf tube, 300 mg of freeze­dried samples were mixed with 0.75 ml hplc grade water containing 26 μg/ml ribitol was added as internal standard and the tubes vortexed. after equilibrating to room temperature. the tubes were incubated in a shaker for 10 min at 80˚c, fol­ fig. 1 ­ carambola (a), june plum (b) and otaheite (c) at diffe­ rent development and ripening stages. adv. hort. sci., 2022 36(1): 13­26 16 lowed by incubation at room temperature for c.a. 45 minutes. afterwards, the tubes were cooled to 4˚c and centrifuged at 10 000 rpm for 15 minutes, the supernatant collected, and the pellet discarded. to the collected supernatants, 250 µl were mixed with 100 ml absolute etoh and the samples dried under vacuum until dryness and stored at ­20°c until gc­ms analysis. derivatization prior to gc­ms analysis, samples were derivatised as described by broeckling et al. (2005) with some minor modifications. the dry residues were mixed with 80 μl of bstfa+1% tmcs (sigma aldrich, st louis, mi, usa) and 20 µl pyridine, vortexed and cen­ trifuged for 10 seconds at 10 000 rpm. afterwards, the mixtures were incubated for 20 minutes at 85˚c. after incubation and equilibrating to room tempera­ ture, 200 μl isooctane (2,2,4 trimethylpentane) (sigma aldrich, st louis, mi, usa) were added to the mixture, vortexed and followed by a centrifugation at 10 000 rpm for 10 seconds. from the mixtures, 100 μl were transferred to a 300 μl glass insert for the gc­ms analysis. gc‐ms analysis of sugars and organic acids the samples were analysed by gc­ms using an agilent 7890b gas chromatograph coupled to an agilent 5977a mass spectrometer scanning in the m/z range from 40 to 550. the column used was an hp 5ms (5% phenyl methyl polysiloxane, 30 m × 250 µm × 0.25 μm) with helium as the gas carrier at a constant flow rate of 1.0 ml/min. the samples were injected at a 15:1 split ratio. initially, the inlet line was held at 260˚c and the transfer line was held at 280˚c. separation was achieved with an initial tem­ perature program of 40˚c for 2 min, then ramped up at 4˚c per minute to 240˚c and held for 1 minute. the temperature was then increased to 10˚c per minute to 315˚c. in order to produce the concentration curve, a mixture containing 250 µl of 26 µg/ml of ribitol was used. to the mixture, 20 μl of pyridine and 80 μl of bstfa containing 1% tmcs was added, vortexed and injected into the gc­ms. prior to the injection, 50 µl of the mixture were taken and 50 μl of isooctane were added. this was repeated by adding each time 50 μl of isooctane until 6 concentration curves were produced. from the curve a scatter plot with a trend line was generated. the concentrations of the differ­ ent profiled sugars and organic acids metabolites were calculated from the generated trend line. the generated ms files were extracted, and the deconvo­ lution and identification of the metabolites was car­ ried out by using agilent msd chem station (version f.01.01.2017) along with nist library (version 11 ms mass spectral library) and amdis (version 2.66) soft­ ware. statistical analyses for the analysis of each sample (three fruits for each ripening stage and for each species), six samples were analysed, and the data were averaged. the data were analysed and compared by running analysis of variance (anova), tukey’s honestly significant difference (hsd) post hoc test using spss software package (version 22.0, (ibm corp., new york, usa). the significance level of all statistical hypotheses testing procedures was predetermined at p < 0.05 and 0.01. for the classification, clustering, and regression, pca (principal component analysis) and hca (hierarchical cluster analysis) were performed using spss software package (version 22.0, ibm corp., new york, usa), while the clustered heatmap was generated using clustvis free software (https://biit.cs.ut.ee/clustvis/). 3. results profile and sugar contents of carambola, june plum and otaheite fruits the profiling of sugars showed that seven saccha­ rides and one sugars alcohol were detected in caram­ bola, june plum and otaheite fruits (table 1). glucose, fructose, sucrose, galactose, arabinose, and myo­inositol have been detected in carambola, june plum and otaheite, however, mannose and xylose were not detected in june plum while sucrose and mannose were not detected in otaheite. overall, fructose, sucrose, galactose, in carambola and june plum, and glucose, fructose and galactose in otaheite increased during maturation and ripening stages, while the other sugars varied differently in the three fruits. interestingly the highest levels of glucose, fruc­ tose and sucrose were observed in june plum, high­ est levels of galactose and xylose in otaheite, and the highest levels of arabinose and myo­inositol in carambola. results also showed that glucose and fructose were the most predominant monosaccha­ rides in carambola and june plum, while in otaheite glucose, fructose and galactoses were predominant. https://biit.cs.ut.ee/clustvis/ smith et al. ‐ ripening metabolome of tropical fruits 17 on the other hand, a significant increase in total sug­ ars was noted in carambola and otaheite, while in june plum sugars content increased slightly. the total increase of sugars averaged 38% and 45% in caram­ bola and otaheite, respectively, but in june plum increases averaged 9%. statistically, total sugar contents in carambola and otaheite were significantly different and their con­ tents in immature fruits were significantly different in comparison with mature and ripe carambola. immature carambola and otaheite had 35% and 36%, and 28% and 46% less total sugars than the mature and ripe stages, respectively. however, statistical analysis showed no significant difference in sugar contents of june plum during the three maturation and ripening stages. profile and organic acids contents of carambola, june plum and otaheite fruits with ten different acids identified in carambola, june plum and otaheite fruits, eight were detected in june plum, six in carambola and four in otaheite (table 2). overall, malic acid, oxalic acid, propionic acid and acetic acids were the most abundant organic table 1 ­ profiled sugars and sugar alcohols and their contents (mg/g dry weight) in carambola, june plum and otaheite during the three development and ripening stages different letters of the same row indicate significant difference at p= 0.05. n.d. = not detected. metabolites immature mature ripe carambola glucose 51.40 ± 3.27 a 68.20 ± 5.18 b 56.36 ±2 .99 ab fructose 30.69 ± 2.71 a 28.87 ±1.23 a 57.79 ± 7.98 b sucrose 1.97 ± 0.08 a 13.73 ± 2.05 b 3.59a ± 0.34 a galactose 9.50 ± 0.45 a 14.14 ± 0.52 b 11.11 ± 0.77 a mannose n.d. n.d. 1.23 ± 0.53 arabinose 2.18 ± 0.42 a 1.86 ± 0.01 a 1.90 ± 0.03 a xylose n.d 11.62 ± 1.85 a 6.28 ± 1 .11 b myo­inositol 2.16 ± 0.11 a 3.30 ± 1.28 a 2.20 ± 0.08 a total 97.9 a 133.2 b 134.1 b june plum glucose 124.11 ± 7.62 a 112.38 ± 12.84 a 90.63 ± 18.41 b fructose 79.43 ± 3.26 a 72.68 ± 14.12 a 94.38 ± 18.01 b sucrose 7.46 ± 2.36 a 11.33 ± 1.76 a 35.03 ± 10.06 a galactose 13.01 ± 0.61 a 12.36 ± 1.77 a 23.67 ± 5.75 b mannose n.d. n.d. n.d. arabinose n.d. 1.28 ± 0.09 a 1.26 ± 0.56 a xylose n.d. n.d. n.d. myo­inositol 1.88 ± 0.08 a 1.99 ± 0.11 a 1.55 ± 0.61 a total 225.9 a 210.8 a 245.8 b otaheite glucose 51.88 ± 3.12 a 103.40 ± 10.2 b 111.86 ± 6.59 b fructose 43.97 ± 2.75 a 62.40 ± 1.24 b 68.87 ± 9.78 b sucrose n.d. n.d. n.d. galactose 34.33 ± 1.66 b 12.44 ± 2.45 a 10.79 ± 2.57 a mannose n.d. n.d. n.d. arabinose 1.67 ± 0.77 a n.d. 2.13 ± 0.61 a xylose 6.34 ± 2.58 a n.d. 9.31 ± 1.44 a myo­inositol 1.07 ± 0.81 a n.d. n.d. total 139.3 a 178.4 ab 202.9 b 18 adv. hort. sci., 2022 36(1): 13­26 acids. in carambola and otaheite, malic and tartaric acids were the most abundant, while in june plum malic and acetic acids were predominant. malic, cit­ ric, propionic and acetic acids have been detected in carambola, june plum and otaheite, while tartaric acid in carambola and otaheite only. on the other hand, ascorbic acid was detected in carambola only, and succinic, oxalic, threonic and gluconic acids were detected in june plum only. results also showed that organic acids content decreased with development and ripening of carambola and otaheite, while a steady state was observed in june plum. total organ­ ic acids decreased by 20% and 49% in carambola and otaheite, respectively, while in june plum variation of organic acids during the three stages averaged only 3%. different letters of the same row indicate significant difference at p= 0.05. n.d. = not detected. table 2 ­ profiled organic acids and their contents (mg/g dry weight) in carambola, june plum and otaheite during the three develop­ ment and ripening stages metabolites immature mature ripe carambola malic acid 22.11 ± 4.87 b 13.68 ± 2.4 ab 6.73 ± 0.88 a citric acid 2.30 ± 0.2 a 2.20 ± 0.07 a 1.77 ± 0.34 a propionic acid 2.23 ± 0.01 a 2.15 ± 0.04 a 2.19 ± 0.12 a acetic acid 2.01 ± 0.02 a 1.78 ± 0.39 a 1.84 ± 0.24 a tartaric acid 16.67 ± 3.45 a 10.24 ± 2.44 a 10.65 ± 2.65 a ascorbic acid 1.74 ± 0.34 a 1.80 ± 0.3 a 2.12 ± 0.02 a succinic acid n.d. n.d. n.d. oxalic acid n.d. n.d. n.d. threonic acid n.d. n.d. n.d. gluconic acid n.d. n.d. n.d. total 14.73 a 14.14 ab 11.71 b june plum malic acid 7.34 ± 0.43 a 6.33 ± 0.67 a 6.45 ± 1.45 a citric acid 2.12 ± 0.09 a 2.13 ± .009 a 2.13 ± 0.12 a propionic acid 1.89 ± 0.05 a 3.47 ± 0.09 b 3.45 ± 0.41 b acetic acid 3.24 ± 0.36 a 3.64 ± 0.25 a 3.62 ± 0.44 a tartaric acid n.d. n.d. n.d. ascorbic acid n.d. n.d. n.d. succinic acid 1.82 ± 0.03 a 1.90 ± 0.1 a 1.88 ± 0.07 a oxalic acid 4.52 ± 0.3 a 4.32 ± 0.18 a 4.03 ± 0.2 a threonic acid 1.97 ± 0.05 a 2.26 ± 0.02 ab 2.34 ± 0.19 b gluconic acid 1.84 ± 0.03 a 1.74 ± 0.24 a 1.73 ± 0.28 a total 24.74 a 25.79 a 25.63 a otaheite malic acid 22.11 ± 4.87 b 13.68 ± 2.4 ab 6.73 ± 0.88 a citric acid 2.30 ± 0.2 a 2.20 ± 0.07 a 1.77 ± 0.34 a propionic acid 2.23 ± 0.01 a 2.15 ± 0.04 a 2.19 ± 0.12 a acetic acid 2.01 ± 0.02 a 1.78 ± 0.39 a 1.84 ± 0.24 a tartaric acid 16.67 ± 3.45 a 10.24 ± 2.44 a 10.65 ± 2.65 a ascorbic acid n.d. n.d. n.d. succinic acid n.d. n.d. n.d. oxalic acid n.d. n.d. n.d. threonic acid n.d. n.d. n.d. gluconic acid n.d. n.d. n.d. total 45.31 b 30.05 ab 23.19 a smith et al. ‐ ripening metabolome of tropical fruits 19 on the other hand, the ratio of sugars/organic acids plays an important role that can characterise the ripe stage of fruits. during the different stages, the ratio of sugars/organic acids maintained a signifi­ cant rising trend especially in carambola and ota­ heite. in carambola, the ratio was 6.47, 9.42 and 11.45 in immature, mature and ripe, respectively. in june plum, the ration was 9.13, 8.17 and 9.59, in immature, mature and ripe, respectively. in otaheite, the ratio was 3.07, 5.93 and 8.75, in immature, mature and ripe, respectively. statistical analysis showed that total organic acid contents of carambola was not significantly different between either immature and mature, or mature and ripe stages. malic acid content varied significantly during the development and ripening of carambola and otaheite, but not significantly in june plum. malic acid was also the main organic acid accumulating in carambola and otaheite and its level was significantly different among the three stages of the maturation and ripening of the two fruit. the statistical analysis also showed that citric acid and acetic acid contents did not show significant difference among the three developmental stages of carambola. comparatively, total sugars and total organic acids during the three stages showed different correlations. in carambola and june plum, weak correlation (r2 =0.18 and r2 = 0.33, respectively) was observed between sugars and organic acids contents, however, a moderate correla­ tion (r2 = 0.56) was observed between sugars and organic acids contents of otaheite during the three stages. factoring and clustering of the profiled metabolites the principal component analysis of the data sets revealed two individual clusters that seem to be gov­ erned by the developmental and the ripening stages of the fruits (fig. 2). the analysis showed that in pc 1, the metabolites in carambola with the highest load­ ing scores were glucose (0.99), galactose (0.99), xylose (0.97), sucrose (0.96), myoinositol (0.93) and mannose (0.92), while in pc 2 ascorbic acid (0.99), fructose (0.98), citrate (0.95) and acetic acid (0.98) had the highest loading scores. in june plum, the highest loading sores metabolites in pc 1 and pc 2 were oxalic acid (0.90), acetic acid (0.85), sucrose (0.84) and gluconic acid (0.80), and malic acid (0.89), fructose (0.89), arabinose (0.82, galactose (0.82) and sucrose (0.77), respectively. in otaheite, the metabo­ lites with the highest scores were galactose (0.98), myoinositol (0.98), malic acid (0.96), tartaric acid (0.96), acetic acid (0.89), citric acid (0.79) and propanoic acid (0.74), and arabinose (0.99) and xylose (0.99), respectively. overall, principal compo­ nents analysis (pca) of samples based on the devel­ opment and ripening stages revealed a difference between grouped metabolites. as suggested by the pca in the figure 2, profiled metabolites were then divided into two classes, and loading values of sugars fig. 2 ­ principal component analysis (pca) of the profiled and identified metabolites in carambola, june plum and otaheite fruits through different development and ripe­ ning stages. for pca analysis, data sets were normalised for better comparison of the variable levels of the diffe­ rent metabolites. adv. hort. sci., 2022 36(1): 13­26 20 and organic acids of fruits samples were found most­ ly in quadrant pc2+, illustrating the discriminated metabolites reflecting the development and ripening of fruits. hierarchical cluster analysis (hca) was applied to a data set of the profiled and detected metabolites during the three stages of the three fruits. the den­ drograms (fig. 3) show that the profiled metabolites were quite homogeneous and tend to be distributed into three groups. according to the dendrograms of the hca, at the distance of three, the metabolites can be grouped as shown in table 3. interestingly, three metabolites have been classified within the same groups of the three fruits. myoinositol, citric acid and arabinose were classified in group 1, group 2 and group 3, respectively. the clusters of the differ­ ent metabolites in the three fruits showed that the metabolites were quite clearly hierarchically separat­ ed, and these results were also clearly depicted by the pca (fig. 2) and the hca (fig. 3) which show the distribution of the metabolites into three main clus­ ters. furthermore, the heatmap (fig. 4) also shows that june plum concentrates the highest levels of ten metabolites, while carambola and otaheite concen­ trate the highest levels of four and six other metabo­ lites, respectively. indeed, primary metabolites profiling led to the identification 8, 6 and 5 sugars, and 6, 8 and 5 organ­ ic acids in carambola, june plum and otaheite, respectively. on the other hand, our results showed that the key marker metabolites of the maturation and ripening of the three fruits are glucose and fruc­ tose in carambola and otaheite, while in june plum glucose, fructose, galactose and sucrose were the key marker metabolites of the three different stages. similarly, malic and tartaric acids were the key organ­ ic acids metabolites of the maturation and ripening fig. 3 ­ dendrogram showing the hierarchical cluster analysis (hca) of profiled and identified metabolites in carambola, june plum and otaheite fruits through diffe­ rent development and ripening stages. to run hca, data sets were normalised to reduce of the original data sets and hence allowing better clustering. table 3 ­ the hierarchical clusters distribution of the profiled metabolites of carambola, june plum and otaheite during the three stages (underlined metabolites are classified within the same groups of the three fruits) group 1 group 2 group 3 carambola mannose fructose arabinose succinate citric acid malic acid myo­inositol acetic acid propionic acid glucose ascorbic acid galactose xylose june plum succinic acid glucose succinic acid acetic acid threonic galactose gluconic acid citric acid fructose oxalic acid propionic arabinose myo­inositol malic acid otaheite galactose malic acid arabinose myo­inositol citric acid xylose tartaric acid glucose acetic acid fructose propionic acid smith et al. ‐ ripening metabolome of tropical fruits 21 of carambola and otaheite fruits, while malic acid was the key marker metabolite of june plum matura­ tion and ripening. 4. discussion and conclusions although extensive literature is readily available, the variation of sugars and organic acids of many fruits including carambola and june plum at ripe stage, less and scattered work was done on the varia­ tion of the metabolites including sugars and organic acids in carambola, june plum and otaheite fruits during the development and ripening stages. on the other hand, most of the work carried out on caram­ bola targeted the postharvest physiology and bio­ chemistry of the fruit during storage. in carambola, campbell and koch (1989) found that total soluble sugars concentration, mainly glu­ cose and fructose, increased during ripening and var­ ied between 22 and 27 mg/g fresh weight depending on the varieties, while narain et al. (2001) investigat­ ed the variation of the chemical composition of carambola at three different ripening stages and found that total sugars increased from 2.91 to 5.60 g/100 g fresh weight. later, patil et al. (2010) report­ ed the composition of the fruit at three stages of maturity (young, half­ripe and ripe), and they noted a tremendous increase of total sugars, oxalic acid and ascorbic acid by 100%, 89% and 65%, respectively. similar increase by 33% and 90% of total sugars and ascorbic acid respectively were also reported by ali and jaafar (2012). glucose, fructose and sucrose were reported to be the most predominant sugars in carambola (mohd zainudin et al., 2014; benkeblia and lópez, 2015), however, benkeblia and lópez (2015) reported an increase of glucose and fructose, but a slight decrease of sucrose in the ripe fruit com­ pared to the green one, while mohd zainudin et al. (2014) noted an increase of the three sugars. there is almost no work reporting on the compo­ sition of june plum fruit during maturation and ripen­ ing except from the one of benkeblia and lópez (2015). the authors investigated the variation of glu­ cose, fructose and sucrose in green and ripe june plum and found that in ripe fruit glucose and fructose increased in ripe fruit, while sucrose decreased signif­ icantly. other scattered studies reported on the vari­ ation of sugars and organic acids in june plum fruit but at a specific stage. in a study carried out on immature green june plum, franquin et al. (2005) investigated the composition at this early stage and found the concentrations of glucose, fructose, sucrose, citric acid, malic acid, oxalic acid and ascor­ bic acid were 1.5 (± 0.2), 1.2 (± 0.2), 3.1 (± 0.3), 0.9 (± 0.1), 0.2 (± 0.02,) 0.03 (± 0.01), and 52.0 (± 4.9) g/100 g fresh weight, respectively. in his study, nahar et al. (1990) reported that 0.3% of fresh weight of the pulp is composed by free sugars, where glucose, fructose and sucrose were the most predominant (nahar et al., 1990; mahmood et al., 2012). similarly to june plum, few studies investigated the composition of otaheite during the maturation and ripening, however, few studies reported on ripe otaheite. lu and lin (2011) investigated the sugars in ripe otaheite and found that fructose yielded the highest content compared to glucose and sucrose which were detected at this ripe stage. the untargeted profiling of primary metabolites during the maturation and ripening of fruits is a good approach to provide better insight into their metabolome changes during these stages. different studies on metabolite analyses of fruits have focused on temperate and stone fruits such as tomato, peach, strawberry, and grape among many others, but scarce studies focused on tropical fruits. however, these studies revealed similar dynamic variations in the levels of sugars and organic acids, as well as many other primary and secondary metabolites dur­ ing fruits maturation and ripening (oikawa et al., fig. 4 ­ heatmap of the profiled and identified metabolites in carambola, june plum and otaheite fruits through diffe­ rent development and ripening stages. the color scale represents the relative concentration of each metaboli­ tes. adv. hort. sci., 2022 36(1): 13­26 22 2015). our results showed patterns of variation in sugars and organic acids levels in carambola, june plum and otaheite during maturation and ripening, and provided fundamental metabolomic data that is useful for understanding fruits physiology. our reported results are in agreement with those report­ ed by ramadan et al. (2020) who used metabolomics approach carambola fruit of different origins and at two different ripening stages. mamat et al. (2019) and parijadi et al. (2018) analysed the distribution of primary metabolites in mangosteen (garcinia man‐ gostana linn.) fruit during ripening, and their results showed that fructose was the kye marker metabolite of mangosteen ripening. similar results were report­ ed by ogawa et al. (2018) who noted that the increasing sucrose level might be a key marker metabolite of pineapple ripening. beside metabolomics, pandit et al. (2010) used transcriptomics markers to understand the matura­ tion and ripening programmes in mango (mangifera indica l.) fruit. among eighteen genes related to the fruit physiology and biochemistry, genes related to primary metabolism showed higher expression in comparison to that of the genes related to flavour production. however, regardless of the origin and environ­ mental zones, the maturation and ripening of fruits are complex and highly coordinated processes. globally, the increase in sugar and decline in organic acids are one of the main changes associated with these processes (giovannoni, 2001; klee and giovannoni, 2011; osorio et al., 2013; batista­silva et al., 2018). during maturation and ripening of fruits, organic acids contents are inversely related to sugar contents. the rising trend of sugars is due to photo­ synthates import or starch degradation, while organ­ ic acids that accumulated in young fruits strongly decrease by being converted to other organic acids (carrari et al., 2006; beauvoit et al., 2018). although environmentally different from tropical fruits, there have been a number of different studies reporting similar metabolic changes that occur in temperate fruits during maturation and ripening stages (fait et al., 2008; osorio et al., 2011, 2012). sugars accumu­ lation and organics acids decrease trend were report­ ed in blueberries (vaccinium arctostaphylos and vaccinium myrtillus) (ayaz et al., 2001), apple (malus domestica) (liu et al., 2016; yang et al., 2021), apri­ cot (prunus armeniaca l.), plumcot (prunus armenia‐ ca x prunus salicina l.), plum (prunus salicina lindl.), and peach (prunus persica l.) (bae et al., 2014), damson plum (prunus domestica) (garcía­mariño et al., 2008), different citrus cultivars (bermejo and cano, 2012), grapes (kurt et al., 2017; liang et al., 2011; muñoz­robredo et al. , 2011), loquat (eriobotrya japonica lindl.) (amorós et al., 2003), mango (mangifera indica l.) (mokhtar et al., 2014), melon (cucumis melo l.) (wang et al., 1996), pome­ granate (punica granatum l.) (nuncio­jáuregui et al., 2014) and wolfberry (lycium barbarum l.) (zhao et al., 2015) among other reported fruits. furthermore and in agreement with our finding, glucose, fructose, and sucrose were found to be the most predominant among mono and disaccharides, while malic, citric and tartaric acids were predominant organic acids (wang et al., 1996; liang et al., 2011; mahmood et al., 2012; kurt et al., 2017; yang et al., 2021). indeed, the relative levels of sugars and organic acids in fruits are of great importance for harvesting time and are one of the determinants of the organoleptic quality attributes of fruits particularly sweetness (itai and tanahashi, 2008). furthermore, the postharvest quality attributes of fruits, their shelf­life and even processed products are strongly associated to their sugars and organic acids levels (matsumoto and ikoma, 2012; aprea et al., 2017). in order to preserve freshness and reduce economic losses, it is of great importance to understand the metabolic changes occurring during maturation and ripening which might contribute to accelerate fruits senescence and perishability after harvesting. in this sense, metabolomic profiling of key metabolites responsible for quality attributes such as sugars and organic acids can be a powerful tool for further understanding the biochemical basis of pre­ and postharvest physiology and have the potential to play a critical role in the identification of the path­ ways affected by fruit maturation and ripening (allwood et al., 2021; pott et al., 2020; tian et al., 2021). the data presented here indicates that the pro­ filed metabolites varied significantly during the matu­ ration and ripening of the fruits. glucose, fructose, galactose, sucrose and myoinositol were found pre­ dominantly in all the fruits and during the three stages, except sucrose in otaheite. comparatively, june plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was noted in otaheite and the lowest in carambola. on the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. interestingly, the multi­vari­ smith et al. ‐ ripening metabolome of tropical fruits 23 able analysis showed than all the metabolites were clustered into three main clusters, and myoinositol, citric acid and arabinose shared group one, group two and group three, respectively. from the different profiled sugars and organic acids, our results are sug­ gesting that glucose and fructose are the marker metabolites of the maturation and ripening of caram­ bola and otaheite, while the ripening marker metabolites in june plum are glucose, fructose, galac­ tose and sucrose. because this study represents the first report on the profiling of sugars and organic acids in carambola, june plum and otaheite, it might be interesting to profile the secondary metabolites mainly phenolics and volatiles, and their variation during the maturation and ripening of these fruits. acknowledgements this work is a 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ahmed et al., 2018). after harvest, a favorable environment for the germination of fungal spores is created on berries surface, mainly for damaged fruits (de simone et al., 2020). for this rea­ son, during post­harvest life of fruits and vegetables, processing technolo­ gies and biotechnologies aimed to provide physical, chemical, and biologi­ cal hurdles to limit the development of undesired microorganisms (*) corresponding author: maria.dechiara@unifg.it citation: de chiara m.l.v., de simone n., spano g., amodio m.l., colelli g., 2023 ­ effect of microwave mild heat treatment on postharvest quality of table grapes. ­ adv. hort. sci., 37(1): 33­40. copyright: © 2023 de chiara m.l.v., de simone n., spano g., amodio m.l., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 29 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13908 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 33­40 34 (capozzi et al., 2009). conventional thermal process­ es can result in the reduction of nutritional and sen­ sory quality of the product, due to slow heat trans­ mission within the plant tissue. application of mild thermal treatments aimed to control postharvest dis­ ease by means of microwave heating (dar et al., 2020), could allow to avoid the use of chemical com­ pounds and therefore residues in the treated product and ensuring at the same time minimal environmen­ tal impact, thus representing a valuable alternative to traditional thermal processing. microwaving ensures instead a fast and effective heat treatment reducing risk of injuries and decrease of nutritional com­ pounds. however, oversized intensity of the treat­ ment can induce an excessive temperature increase, resulting in a damage to the fresh plant tissue. moreover, high temperatures could affect grape bio­ chemical characteristics, for example losses of aroma­related compounds and development of oxidative processes (modesti et al., 2020). to date, a small number of studies deals with the use of microwave treatment on fresh produce (karabulut and baykal, 2002; zhang et al., 2004, 2006; sisquella et al., 2013), showing its efficiency in prolonging postharvest life of peaches, in which microwave inhibited growth of inoculated pathogens after 2 minutes, being also effective in controlling endoge­ nous microflora with a very low decay incidence. as for nectarines, brown rot incidence was significantly reduced by microwaving to less than 14% versus 45% of untreated product. treatment caused a delay of softening and internal damage. zhang et al. (2006) observed that in pears treated for 2 or 3 min penicillium expansum population was significantly lower than control samples without impairing quality of the fruits. fresh­cut carrots, apples, and minimally processed bok choy were subjected to high power/short time treatments showing promising results. microwave treatments maintained physical, chemical and sensory quality of fresh­cut carrots over storage period, reducing surface whitening and avoiding firmness modification, also enhancing bioac­ tive compounds concentration. however, microbial growth was greater than control samples during shelf­l ife (martínez­hernández et al. , 2016). application of 454 w for 5 s on bok­choy decreased respiration rate, decay occurrence and etiolation, while improving integrity of cell membrane with a final better quality of product (song et al., 2018). as for fresh cut apples, a significant microbial reduction was observed for treatment at 300 w for 35 s, with no detrimental effect on nutritional parameters and a slight decrease of visual quality (colelli et al., 2021). however, there is the need to deeply study this tech­ nology and its effect on the species of interest with the aim to keep intact the fresh­like characteristics of the product. moreover, microwave application as a part of a hurdle technology and its application in combination with packaging could be recommended to avoid recontamination, thus the objective of this paper is to provide preliminary information concern­ ing the effect of microwave heating on table grape quality, in terms of efficacy in maintaining physical and microbiological quality. 2. materials and methods a preliminary test to select maximum microwave energy output to be applied on fresh table grape was carried out by means of a central composite design (ccd). treatment time (seconds) and microwave power levels (watt) were considered as ccd factors, ten combinations were identified using the software statgraphics centurion xvi.i (statpoint technologies, inc., usa) (table 1). table grape was divided into 100 g­batches and treated according to the experimental design, using a solid­state microwave oven at a labo­ ratory scale (2450 mhz, maximum power 1000 w). processed products were stored in air at 5±1°c and 95% rh up to 14 days after treatment. each combina­ tion was performed once, being the statistical vari­ ability already considered during designing of the experimental ccd plan. the experiment was also conducted on samples previously inoculated by table 1 ­ treatment time (seconds) and microwave power levels (watt) according to an experimental plan based on central composite design 22+ star with two central points run treatment time (seconds) microwave power levels (watt) 1 14 100 2 47 32 3 94 265 4 47 265 5 47 498 6 0 265 7 14 430 8 47 265 9 80 430 10 80 100 de chiara et al. ‐ microwave effect on table grape quality 35 immersion in a 1x106 spores/ml solution of botrytis cinerea cect 20973 purchased from the spanish type culture collection (cect, paterna, spain), and stored at low temperature for 7 days+7 days of room temperature shelf life. for not inoculated samples, immediately after the treatment and after 7 and 14 days, the main physicochemical and microbiological parameters were evaluated, while inoculated sam­ ples were evaluated only for external aspect due to the massive presence of b. cinerea that could affect in a non­realistic way the quality of these latter sam­ ples. obtained results were subjected to the specific statistical analysis for ccd using the software statgraphics, to create estimated response surface plots. the second trial was subsequently performed on uninoculated samples using the two most effec­ tive combinations to understand the best moment for microwave heating application throughout the minimal processing. ready­to­eat table grape bunch­ es (100 g) were subjected to treatments before or after packaging within polypropylene (pp) bags (10x15 cm), and subsequently stored at 5°c up to 14 days. during storage (at initial time and after 7 and 14 days), physicochemical, microbiological, and organoleptic evaluations were carried out and results were subjected to statistical analysis. the treatments were as follows: ctrl: samples not treated; lowmw: microwave treatment at 14 s/100 w and subsequent application of the packaging; highmw: microwave treatment at 80 s/100 w and subsequent application of the packaging; lowmw pp: microwave treatment at 14 s/100 w on packed samples; highmw pp: microwave treatment at 80 s/100 w on packed sam­ ples. all the treatments were performed in triplicate. ranking test first of all, ranking test was performed on samples treated as described for ccd to select the maximum microwave output energy to be applied on the prod­ ucts without detrimental effect. seven trained pan­ elists were asked to rank the samples according to their preference, evaluating the fresh­like appear­ ance of the product and its organoleptic properties and the presence of typical fresh­like flavor, by assigning a score (from 1 to 10 considering each sam­ ple as a whole, based on the following characteris­ tics: fresh­like appearance and organoleptic proper­ ties and the presence of typical fresh­like flavor). the sum of the score given by each panelist was com­ pared with preset values to statistically evaluate the differences between the samples in terms of the test­ ed parameters. fungal strain and growth condition botrytis cinerea cect 20973, purchased from the spanish type culture collection (cect, paterna, spain), was used to inoculate samples. cryopreserved cultures were plated on potato dextrose agar (pda, oxoid), and incubated at 25°c for 5 days. fungal spore suspension was prepared by brushing the plates surface with saline solution (8.6 g l­1 nacl) sup­ plemented with 0.01% tween 80 using a sterile swab and stored at 4°c for short­term uses. fungal spores concentration was determined by plating serial dilu­ tion on pda plates and adjusted to approximately 1 x 106 spores/ml. microbial load determination grape berries from each replicate were diluted (1:10) with nacl 8.6 g l­1 solution, and homogenized in a blender (bag mixer, interscience, saint­nom­la­ bretéche, france) for 2 min. then, samples were sub­ mitted to tenfold serial dilution. mesophilic microor­ ganisms were enumerated by plate counting on plate count agar (pca) and incubated at 25 for 48 h. yeasts and molds were plated on potato dextrose agar (pda) (oxoid) added with chloramphenicol (100 mg l­1) and incubated at 30°c for 48 h. ascorbic acid, dehydroascorbic acid and total vitamin c determination ascorbic acid, dehydroascorbic acid and total vita­ min c amounts were assessed homogenizing 5 grams of fruit tissue with 5 ml of extraction medium (meoh: h2o (5:95) plus citric acid (21 g l­1) with edta (0.5 g l­1) and naf (0.168 g l­1). the homogenate was filtered, centrifuged, and the supernatant was recov­ ered. ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents were determined through hplc analysis (agilent technologies 1200 series; agilent, waldbronn, germany) as described by zapata and dufour (1992) with some modifications. aa and dhaa contents were expressed as mg of ascorbic or dehydroascorbic acid per 100 g of fresh weight. vitamin c content corresponds to the sum aa+dhaa and was expressed as mg 100 g fw­1. color analysis and determination of the berries’ tem‐ perature color of berries surface was measured on 5 berries per each replicate using a spectrophotometer (cm 2600d, konica minolta, japan) in the reflectance mode with the cie l*a*b* color scale. immediately after the treatment, berries surface temperature was acquired using thermal imaging camera flir c5 (teledyne technologies, wilsonville, oregon, usa). adv. hort. sci., 2023 37(1): 33­40 36 texture berries firmness evaluation on 5 berries from each replicate was performed using a ta­xt2 texture analyzer (stable micro systems ltd., england, uk) by measuring the maximum compression force at a rate of 1.5 mm s­1. sensorial quality a panel of six trained people evaluated external appearance and overall rating of bunches, berries, and rachis of the stored product from each replicate at each sampling day. it was used a hedonic scale associated to a brief description corresponding to a score from 1 to 5, where 1= really poor and 5=excel­ lent, being 3 the limit of marketability and 2 the limit of edibilit. gas analysis oxygen (o2) and carbon dioxide (co2) concentra­ tions, expressed as kpa, inside plastic bags containing table grapes were monitored over storage time by using a hand­held gas analyzer (checkpoint, pbi dansensor) to measure gases concentration in 15 ml of headspace. statistical analysis data were subjected to a two­way anova (for treatment and sampling time), and means were sep­ arated by tukey’s test at p<0.05 (5% significance level) using stat graphics centurion xvi.i software. mean values within each sampling were separated applying tukey’s test with significant difference when p≤0.05. 3. results and discussion first of all, as from ranking test results, it was observed that samples from treatment 1 and 9, treat­ ed with 14 s/100 w and 80 s/430 w, respectively, were significantly different from the other, showing better and worse characteristics than the other treat­ ments, respectively. specifically, sample number 9 reached, after treatment, a maximum temperature of 81°c, with an average of 44.7°c, consequently the panelists evaluated this sample negatively, highlight­ ing the significant presence of cooked flavor. in gen­ eral, grape temperature during treatment increased very heterogeneously among all samples (fig. 1, table 2). in fact, as observed by many researchers, microwave heating often leads to the creation of hot spots in several product zones, depending on its geometry, thickness and dielectric properties, which are in turn dependent on the moisture content, and starting temperature of the food (ho and yam, 1992; buffler, 1992; zhou et al., 1995; campanone and zaritzky, 2005; vadivambal and jayas, 2010). the increase in temperature was progressive, even if not proportional, to the increase in the total energy sup­ plied to the product as expected by the treatment. consequently, the results of the ccd showed that the more the microwave energy, the more the damage to the fresh product, leading to a worsening of the organoleptic quality of ready­to­eat table grapes for energies above 8000 kj, both in the presence and absence of b. cinerea inoculum (106 log cfu g­1). however, the chemical and physical parameters of table 2 ­ minimum, maximum, and average temperature for samples treated as described by the experimental plan of the ccd fig. 1 ­ example of temperature distribution on the bunch after microwave heating. run maximum temperature (°c) minimum temperature (°c) average temperature (°c) initial t 20.3 15.2 17.5 1 29.7 20.4 22.6 2 47.3 20.9 23.3 3 65.9 22.9 37.7 4 68.7 21.7 30.9 5 68.0 23.1 37.7 6 22.2 17.4 19.1 7 42.5 19.9 24.3 8 70.8 21.3 29.5 9 81.1 23.2 44.7 10 60.5 21.1 27.3 de chiara et al. ‐ microwave effect on table grape quality 37 uninoculated samples, did not show significant differ­ ences (data not shown), for this reason the following best treatments from a sensory point of view, as reported in figure 2, were selected for the subse­ quent packaging trials: 14 s/100 w and 80 s/100 w, respectively, treatment 1 and 10. moreover, this lat­ ter evidence was associated with a slight reduction of the total mesophilic bacterial load of the selected samples treated with combination 1 and 10 (14/100 and 80/100), equal to 0.2 log and 0.6 log respectively, compared to the untreated sample, even if not statis­ tically significant (data not shown). the selected treatments allowed to maintain a high visual quality score, above the limit of marketability, up to 14 days of storage. it is possible to observe that treatments 3, 5 and 9 caused instead a severe deterioration of table grape appearance, resulting not to be suitably applied on fresh product. there is a great lack of existing literature regarding the application of microwaves on grapes intended for fresh consump­ tion, therefore these preliminary data were used as starting point for the next experiment, and it is diffi­ cult to compare them with other data for the same product. however, similar results were observed for different products such as fresh cut carrots and apples as reported by martínez­hernández et al. (2016) and colelli et al. (2021). authors stated that high energy treatments could detrimentally affect quality of fresh product. for this reason, it became crucial to carry out the reported preliminary screen­ ing, preferably based on a statistical approach such as that of the ccd which allows to test a large num­ ber of treatments at different energies to obtain a complete picture of the effects of microwaving on the product. as for the second trial, table 3 shows the results of the two­way analysis of variance, pointing out how storage time significantly influenced all the evaluated parameters while microwave intensity, combined or not with packaging, affected the sensory aspects described as visual appearance of bunches, berries and rachis, and some of the physico­chemical para­ meters. moreover, the interaction between the two factors influenced several quality aspects of table grape subjected to microwave heating and stored up to two weeks. on the other hand, however, as described below, the significant effects of the treat­ ment on the qualitative aspects of the product are lost during storage and, for most of the evaluated table 3 ­ effect of microwave treatment, storage time and their interaction on physico­chemical, sensory and microbiological attributes of table grape stored up to 14 days mean values ± standard deviations of 9 samples are reported (3 replicates x 3 storage times). ****= p≤0.0001; *** = p≤0.001; **= p≤0.01; *= p≤0.05; ns, not significant. lowmw = 14 s/ 100 w; highmw = 80 s/100 w; pp= polypropylene bags. fig. 2 ­ visual quality score evolution during storage time for uninoculated table grape bunches treated as described in table 1. control lowmw lowmw pp highmw highmw pp treatment (a) storage time (b) interaction (a x b) total mesophilic load (logcfu g-1) 3.05±0.57 3.02±0.48 3.02±0.56 3.16±0.30 2.97±0.29 ns **** ns yeasts and molds (logcfu g-1) 3.25±0.36 b 3.32±0.23 b 3.40±0.35 ab 3.55±0.24a 3.23±0.41 b ** **** * ascorbic acid (mg 100g-1) 0.62±0.39 0.58±0.22 0.48±0.35 0.62±0.43 0.72±0.39 ns **** ns dehydroascorbic acid (mg 100 g-1) 2.04±0.32 1.95±0.17 1.74±0.24 1.98±0.20 1.75±0.41 * * * vitamin c (mg 100g-1) 2.66±0.64 2.54±0.32 2.23±0.47 2.60±0.50 2.48±0.73 ns **** ns o2 (kpa) 11.02±8.38 10.14±8.31 10.40±8.52 10.62±7.95 10.83±7.98 ns **** *** co2 (kpa) 6.00±4.79 bc 6.72±5.07 a 6.65±5.39 ab 6.60±5.08 abc 5.95±4.56c ** **** *** bunch appearance (score) 4.17±0.48 b 4.50±0.43 a 4.50±0.41 a 4.50±0.28 a 4.33±0.57 ab ** **** *** berries appearance (score) 4.08±0.61 b 4.58±0.47 a 4.58±0.33 a 4.58±0.35 a 4.25±0.48 b *** **** ** rachis appearance (score) 4.42±0.56 ab 4.50±0.43 a 4.42±0.35 ab 4.50±0.43 a 4.33±0.66 b ** **** **** firmness (n) 0.47±0.11 ab 0.57±0.17 a 0.47±0.09 b 0.50±0.12 ab 0.45±0.05 b * **** ** 38 adv. hort. sci., 2023 37(1): 33­40 parameters, the differences resulted to be not signifi­ cant after 14 days. the most interesting results were reached in terms of maintenance of the sensorial quality of the fresh product. specifically, all the samples, including not treated one, were characterized by a very high sensorial quality even after two weeks of storage within passive modified atmosphere packaging. however, highmw sample, treated at 100 w for 80 sec and then packed, showed the highest rating due to the very fresh­like appearance of its berries and globally, its bunches, at the end of storage time. it is widely recognized that ready­to­eat and fresh­cut products should be visually free from defects, clean, with no presence of soil or off odor up to the end of the storage time, moreover, the entire bag content should be edible without no further requirement before consumption (barrett et al. , 2010). consequently, visual appearance of fresh table grapes and fresh product in general, represents the first aspect influencing the consumers decision, and size, color, visual quality, and external appearance in general are used to describe it (musacchi and serra, 2018). in this context, it was observed in the present work that microwave treatment was able to better maintain the visual appearance of table grape during storage when packed and stored as ready­to­eat product, representing a starting point for subsequent applications and optimization of this technique on fresh produce. from a microbiological point of view, no statistically differences were observed between samples at the end of storage time, neither due to the different intensity of the treatments nor to the presence/absence of the packaging film during microwave treatment. in figure 3 it is possible to observe how, concerning mesophilic microorganisms, a lower load, even if not significant, was maintained after 14 days of storage for all the treated samples, compared to untreated, reaching the latter the high­ est value equal to 3.59 log cfu g­1. final values for lowmw, lowmw pp, highmw, highmw pp were 3.29, 3.42, 3.34 and 3.06, respectively, with highmw pp sample showing the highest difference compared to control (0.54 log cfu g­1). low efficacy of the treat­ ment in terms of reduction of the microbial content can be related to non­uniform heating process, thus leading to the incomplete action on to the microor­ ganisms due to uneven distribution of temperature (vadivambal and jayas, 2010). little literature exist­ ing on ready­to­eat samples in order to compare results. colelli et al. (2021) observed that 35 s/300 w on fresh­cut apples allowed to reach a 2­log reduc­ tion in mesophilic load at the end of storage time, however, the higher microwave intensity resulted in the appearance of side effects on nutritional quality. otherwise, as reported by martínez­hernández et al. (2016) 60 s/900 w microwave treatment applied on fresh­cut carrots led to an initial microbial reduction, followed by an increment during storage mainly due to the detrimental effect on plant tissue caused by an excessive temperature increase. as demonstrated by the present experiment, using a well­modulated microwave energy, allowed at least to maintain a good visual quality of ready­to­eat table grape (fig. 4), without significant reduction of nutritive com­ pounds and firmness. moreover, it was not possible to correlate the differences in the visual appearance with the different gaseous concentration within the plastic bags, in fact about 3 kpa of oxygen and 11 kpa of co2 were reached at the end of storage time for all the samples. this probably contribute to the mainte­ nance of grape quality, also cefola and pace (2016) reported the beneficial effect of high co2 concentra­ fig. 3 ­ total mesophilic and yeasts and moulds loads evolution over time on table grape samples untreated (ctrl) and subjected to low (14 s/100 w) and high (80 s/100 w) microwave treatment before (lowmw, highmw) and after polypropylene packaging application (lowmw pp, highmw pp). means with different letters at the same time of storage are significantly different according to tukey’s test (p≤0.05). de chiara et al. ‐ microwave effect on table grape quality 39 tion during storage on ‘italia’ table grapes, both in terms of sensory quality preservation and decay con­ trol. it is therefore possible to state that the treat­ ments, at the applied intensities, did not influence the respiratory rate of table grape and consequently there are no differences concerning metabolic activi­ ty and physiological aging. as for ascorbic and dehy­ droascorbic acid, it was observed a physiologically slightly decreasing trend, without however differ­ ences due to the intensity of the treatment (data not shown). similar results were also observed for differ­ ent table grape varieties when stored as minimally processed products (nicolosi et al., 2018). 4. conclusions at the end of the storage period, sample subject­ ed to 100 w microwave power for a treatment time of 80 seconds and subsequently packed, showed a better external appearance than the other treatment and the control samples, however maintaining an intermediate level of mesophilic bacterial load. no significant differences in terms of nutritional quality were observed. the time/power combination identi­ fied with this preliminary experiment and its combi­ nation as hurdle technology with packaging can rep­ resent a valuable starting point for further experi­ ments aiming at identifying a mild microwave treat­ ment to be applied to improve table grapes quality and safety. their combination with other treatments aimed to maximize the antifungal activity should be better investigated. acknowledgements regional project “research for innovation”, fun­ ded by “por puglia fesr fse 2014­ 2020 ­ azione 10.4 ­ interventi volti a promuovere la ricerca e per l’istruzione universitaria”. references ahmed s., roberto s., domingues a., shahab m., junior o., sumida c., de souza, r., 2018 ­ effects of different sulfur dioxide pads on botrytis mold in ‘italia’ table grapes under cold storage. ­ horticulturae, 4: 29. barrett d.m., beaulieu j.c., shewfelt r., 2010 ­ color, flavor, texture, and nutritional quality of fresh‐cut fruits and vegetables: desirable levels, instrumental and sen‐ sory measurement, and the effects of processing. ­ crit. rev. food sci. nutr., 50(5): 369­389. buffler c.r., 1992 ­ microwave cooking and processing. engineering fundamentals for the food scientist. ­ springer new york, ny, usa, pp. 14­83. campanone l.a., zaritzky n.e., 2005 ­ mathematical analysis of microwave heating process. ­ j. food eng., 69(3): 359­368. capozzi v., fiocco d., amodio m.l., gallone a., spano g., 2009 ­ bacterial stressors in minimally pro‐ cessed food. ­ int. j. mol. sci., 10: 3076­3105. cefola m., pace b., 2016 ­ high co2‐modified atmosphere to preserve sensory and nutritional quality of organic table grape (cv. ‘italia’) during storage and shelf‐life. ­ eur. j. hortic. sci., 81: 197­203. colelli g., amodio m.l., de chiara m.l.v., 2021 ­ operating conditions for microwave application throughout production process to reduce microbial load of fresh‐cut apples. ­ acta horticulturae, 1319: 223­ 230. dar a.h., shams r., rizvi q.u.e.h., majid i., 2020 ­ microwave and ohmic heating of fresh cut fruits and vegetable products, pp. 295­337. ­ in: siddiqui m.w. 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productivity, induction of systemic resistance in strawberry cultivars managed in an organic production system. treatments consisted of rates (0, 25, 50, and 100 ml l­1) of chi­pyro­ film and a reference resistance inducer (dipotassium hydrogen phosphate ­ k2hpo4), evaluated in three strawberry cultivars (‘albion’, ‘san andreas’ and ‘portola’). growth, yield, anthracnose incidence, and enzymatic activity were evaluated. the experimental design was a randomized block design with four replications. chi­pyro­film increases the growth, yield, and anthracnose resis­ tance of strawberry plants. the best concentration of chi­pyro­film varies between 50 and 60 ml l­1, according to strawberry cultivar. 1. introduction strawberry (fragaria x ananassa duch.) is the most planted red fruit in brazil. the high demand is due to its sensory characteristics such as color, texture, aroma, and taste (ventura­aguilar et al., 2018). however, according to a report by the brazilian health surveillance agency (anvisa) of 2016, 26% of strawberry samples collected between 2013 and 2015 presented nonconformities with pesticide residues. pesticides detected not authorized for the crop, captan stood out, detect­ ed in 20.4% of the samples analyzed, among others such as dithiocarba­ mates, pyrimethanil, carbendazim, tebuconazole, iprodione, and azoxys­ trobin (anvisa, 2016). most of these fungicides aim to control or prevent anthracnose (colletotrichum spp.), a disease that is considered main in strawberry fields in brazil (kososki et al., 2001; capobiango et al., 2016). currently, there is a search for technologies, which make agriculture sustainable and “smart”, with practices and ways to minimize the exces­ sive use of chemicals (grewal et al., 2018). in this context, the use of resistance inducing products activates the plant’s natural defenses, enabling disease control in the organic production system. (*) corresponding author: ivanspereira@gmail.com citation: dos santos pereira i., grecco da silva porto f., corrêa antunes l.e., diniz campos â., 2022 ­ phytoprotective film for resistance induction, growth, and yield of organic strawber‐ ries. ­ adv. hort. sci., 36(1): 43­52. copyright: © 2022 dos santos pereira i., grecco da silva porto f., corrêa antunes l.e., diniz campos â. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 february 2021 accepted for publication 25 february 2022 ahs advances in horticultural science adv. hort. sci., 2022 36(1): 43­52 44 the resistance is associated with various defense responses, such as protein and phytoalexin synthesis, cell wall changes, and increased activity of various enzymes defense­related (durrant and dong, 2004). responses that are associated with changes in the activity of various enzymes such as peroxidase (pox, ec 1.11.1), polyphenol oxidase (ppo, ec 1.14.18.1), and phenylalanine ammonia­lyase (pal, ec 4.1.3.5) (prasannath et al., 2014; prasannath, 2017). poxs have been implicated in many defense processes, such as hypersensitive response, lignifica­ tion, phenolic and glycoprotein cross­linking, suber­ ization, and phytoalexin production (thakker et al., 2012; prasannath, 2017). ppos are a group of enzymes that catalyze the oxidation of hydroxyphe­ nols to their quinone derivatives, which have antimi­ crobial properties (prasannath, 2017). pal (e.c.4.1.3.5) is the major enzyme in the phenyl­ propanoid pathway and acts in the synthesis of defense­related secondary compounds such as phe­ nols and lignins (hemm et al., 2004; vanitha et al., 2009). among the compounds that have activating prop­ erties of defense mechanisms in plants are the chi­ tosan and pyroligneous extract (di piero and garda, 2008; grewal et al., 2018; souza et al., 2018). chitosan is a polycationic β­1,4 polymer bound to d­glucosamine chemically derived from crustaceans and soluble in organic acids and known to be a natur­ al elicitor and triggers various physiological and bio­ chemical responses in plants that act in the growth, production, and protection against disease (chandra et al., 2015; katiyar et al., 2015; pichyangkura and chadchawan, 2015). chitosan has several characteris­ tics that make this polymer advantageous for many applications: (1) has a defined chemical structure; (2) may be chemically and enzymatically modified; (3) is physically and biologically functional; (4) is biodegradable and biocompatible with many organs, tissues and cells; (5) can be processed into various products including flakes, powders, membranes, fibers and films (badawy, 2012; van den broek et al., 2015; porto et al., 2019). the pyroligneous extract is a liquor with strong smoke flavors, is a crude and acid condensate pro­ duced from the distillation of the smoke generated in the carbonization of wood. it consists of a complex mixture of compounds derived from the chemical decomposition of wood components through the condensation of vapors and gases generated during pyrolysis in a low oxygen concentration (campos, 2018; pimenta et al., 2018). the pyroligneous extract is composed of water (80­90%) and more than 200 species of organic com­ pounds (10­20%) (theapparat et al., 2018). the pres­ ence of phenolic compounds in the pyroligneous extract confers growth­promoting and antifungal properties, as found in the literature on helminthosporium sativum, cochliobolus sativus, waltz, colletotrichum orbiculare, alternaria mali (jung, 2007; baimark and niamsa, 2009; wei et al., 2010 a; grewal et al., 2018). in this context, the cationic character of chitosan in acidic conditions offers the possibility to establish electrostatic interactions with other negatively charged compounds, for example with the pyrolig­ neous extract, considered a raw material obtained from renewable sources, and is a good solvent for chitosan (campos et al., 2012; van den broek et al., 2015; porto et al., 2019). the phytoprotective film of chitosan­pyroligneous extract (chi­pyro­film) consists of the chitosan dilut­ ed in the pyroligneous extract, and its characteristics are the formation of a film with photoprotection capability against radiation (uv­b and uv­c), fungi toxic action, and inducing systemic resistance in plants (campos et al., 2012). the objective of the present work was to evaluate the effect of different concentrations of phytoprotec­ tive film formulated with chitosan and pyroligneous extract (chi­pyro­film) and dibasic potassium phos­ phate (k2hpo4) on growth promotion, resistance induction to anthracnose, yield, and defense enzyme activity in strawberry cultivars in the organic produc­ tion system. 2. materials and methods the study was carried under field conditions, at embrapa temperate climate experimental station, in pelotas city, rio grande do sul state, brazil (31°40’49 “s 52°26’18” o, at 60 m altitude) (fig. 1 a). the cli­ mate of the region, according to the köppen classifi­ cation is cfa type, temperate and humid, with hot summers. raw materials the pyroligneous extract was obtained through an extraction procedure of eucalyptus grandis proposed by campos (2018) and the distillation process was performed according to that described by porto et al. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 45 (2019). chitosan was supplied by nutrifarm™, with a 97% degree of deacetylation, determined by proton magnetic resonance (porto, 2011). plant material the seedlings of neutral­day strawberry cultivars, ‘albion’, ‘san andreas’ and ‘portola’, were planted in may and were grown under a low tunnel system with mulching and drip irrigation. the spacing between lines and between plants was 0.30 m, with three lines per bed. the area had a history of many years with severe anthracnose incidence in the strawberry plants (fig. 1b, 1c). liming and fertilization were per­ formed according to the recommendation for straw­ berry organic production. climatic data from the experiment period are shown in figure 1d. treatments the treatments of induction of systemic resis­ tance consisted of four concentrations of the chi­ pyro­film and a reference treatment with dibasic potassium phosphate (k2hpo4). the chi­pyro­film was registered in the field of green chemistry at the national institute of intellectual property in brazil (pct/br2013/000597), united states (us201503 36854a1) and germany (de112013006230t5) as a phytoprotective for agriculture use. the chi­pyro­film with a concentration of 30 g l­1 (fig. 2) was diluted with distilled water in different concentrations (0, 25, 50, and 100 ml l­1). treatment with k2hpo4 was applied at a concentration of 50 mm (orober et al., 2002; aleandri et al., 2010). this compound has shown efficacy against e.g. powdery mildew on barley, cucumber, pepper, and tomato (blumeria graminis f. sp. hordei, sphaerotheca fulig‐ inea, leveillula taurica, and erysiphe oronti, respec­ tively), anthracnose (colletotrichum lagenarium) on cucumber, rust (puccinia sorgi) and leaf blight (exserohilum turcicum) on maize and rice blast (pyricularia oryzae), mildew (sphaerotheca fuliginea) (reuveni et al., 1996; reuveni and reuveni, 1998; manandhar et al., 1998; reuveni et al., 2000; ehret et al., 2002; orober et al., 2002; hamza et al., 2017). the spraying of treatments started at the begin­ ning of fruiting, and the application dates were indi­ cated in figure 1d. the spray volume was 4.5 ml per plant (500 liters per hectare), applied through conical jet nozzles, observing a total coverage of the plants until close to the runoff point. analysis of plant growth, damage by anthracnose, and fruit yield vegetative growth evaluations consisted of dry mass (g plant­1) of crown, root, and leaf, by drying to constant weight (65°c) of three plants collected in each experimental unit at the end of the production cycle, 256 days after seedlings transplanting. the pro­ ductive variables measured were fruit yield (g plant­1) and fruit weight (g fruit­1), obtained by evaluating the total fruits harvested in each experimental unit. the percentage of fruits with anthracnose was obtained by counting the fruits with symptoms at each harvest (fig. 1c). the harvests were carried out three times a week between october and january. enzymatic activity analysis biochemical evaluations were realized by determi­ nation of the specific activity of peroxidase (pox), fig. 1 ­ experiment under field conditions (a), anthracnose symptoms in strawberry plants (b) and fruits (c), and cli­ matic data from the experiment period (d). fig. 2 ­ electron micrograph of the phytoprotective film of chi­ pyro­film, after spraying on a smooth surface at a tem­ perature of 18 to 25°c (a and b). chitosan strawberry leaf covered with a film formed by chi­pyro­film (c). adv. hort. sci., 2022 36(1): 43­52 46 3. results chi­pyro­film concentrations had a significant effect on the growth, yield, anthracnose resistance induction, and enzymatic activity of strawberry plants. growth and development the dry mass of the plant showed factors interac­ tion. the three cultivars showed a quadratic response to chi­pyro­film concentrations (fig. 3), but the high­ est efficiency concentration was different. ‘albion’ and ‘portola’ had the highest efficiency concentration estimated at 60 ml l­1 of chi­pyro­film, while for ‘san andreas’ the concentration was 50 ml l­1 (fig. 3). the treatment with reference resistance inducer, k2hpo4, had also different according to cultivars. in ‘albion’, it provided a leaf mass slightly higher than the control treatment and similar to the 100 ml l­1 chi­pyro­film concentration, but it was lower than 25 and 50 ml l­1 (fig. 3). the yield also was significantly influenced by chi­ pyro­film, with a quadratic response to isolated effect of the film concentration factor (fig. 4). regardless of cultivar, the highest efficiency concen­ tration was estimated at 60 ml l­1. regarding the ref­ erence treatment (k2hpo4), it was observed that it had a performance similar to 100 chi­pyro­film and higher than control (0 ml l­1 of chi­pyro­film), but less than the 50 ml l­1 (fig. 4). between cultivars fig. 3 ­ dry mass of leaves of albion, portola and san andreas cultivars, in response to chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between factors (cultivar and film concentration). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respec­ tively. polyphenoloxidase (ppo), and phenylalanine ammo­ nia­lyase (pal) enzymes, in strawberry leaf samples collected immediately before application (ba) and 48 hours after application (aa) of chi­pyro­film concen­ trations and reference treatment (k2hpo4). for the extraction of pox and ppo, was used 500 mg of ground tissue below 4°c in 10 ml of 0.05 m phos­ phate buffer (ph 7.0) containing 1 mg of polyvinylpyrrolidone­10. subsequently, centrifuga­ tion was performed at 4,000 g for 30 minutes under refrigeration. the supernatant was preserved on ice and used for determinations according to campos et al. (2003). the pox and ppo extraction was carried out by grinding the leaves with 20 mg polyvinylpyrrolidone (sigma­aldrich). the enzyme extract obtained after filtration (whatman 1) and centrifugation (5,600 gn, 15min) were used to test the activity. pox activity was determined in the enzyme extract mix with a phosphate­citrate buffer com­ posed of 0.2 m sodium phosphate solution and 0.1 m citric acid (ph 5.0). the mixture was homogenized in vortexed for 15 seconds. pox activity was determined according to campos et al. (2004). ppo activity was determined in the enzyme extract with 3.6 ml of 0.05 m phosphate buffer (ph 6.0) and 0.1 ml of 0.1 m catechol. ppo activity was determined according to campos et al. (2004). pal activity was determined in crude leaf extracts according to the methods described by hyodo and yang (1971) and hyodo et al. (1978) modified by campos et al. (2003). for extraction, 500 mg of tissue was macerated (below 4°c) with 8 ml of 50 mm sodium borate buffer (ph 8.5) containing 25 g l­1 of polyvinylpyrroli­ done­10 and 4 ml l­1 of mercaptoethanol. the pro­ tein extract obtained after filtration (whatman 1) and centrifuged (5,600 gn, 30 min) under refrigera­ tion (below 4°c). was used to assay the pal activity. protein in the extracts was determined by the bradford method (bradford, 1976). experimental design and statistical analyses the experimental design was randomized blocks with four replications of nine plants. the results were submitted to variance analysis and the means of the variables with significant effect were compared using the tukey test (cultivars) or regression analysis (con­ centrations) at 5% error probability. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 47 effect, ‘portola’ showed the highest yield than ‘albion’ and ‘san andreas’ (table 1). results similar to those observed by carini et al. (2015) in a study of evaluation of strawberry cultivars in an organic sys­ tem, in which ‘portola’ was also more productive than ‘albion’ and ‘san andreas’. the fruit weight was not influenced by chi­pyro­ film treatments. however, there was an effect of the cultivar factor, with ‘san andreas’ producing fruits of a higher mass (table 1). corroborating with carini et al. (2015), which evaluated the same three cultivars, found a higher fruit weight of ‘san andreas’, but with the same weight that ‘albion’. the variable fruits with anthracnose showed an isolated effect of the factor chi­pyro­film concentra­ tions, that is, all cultivars had the same response to the application of chi­pyro­film, with a quadratic reduction in the number of fruits attacked, with the maximum efficiency concentration estimated at 60 ml l­1 chi­pyro­film (fig. 5). the reference treatment with k2hpo4 was similar to the 100 ml l­1 of chi­pyro­ film, but lower than the 25 ml l­1 and 50 ml l­1 (fig. 5). among the cultivars, ‘portola’ was the most sensi­ tive to the occurrence of anthracnose in fruits, with no difference between ‘albion’ and ‘san andréas’ (table 1). enzymatic activity in the present study, there was a significant effect of chi­pyro­film concentrations on the activity of pox, ppo, and pal enzymes (figs. 6 and 7). an inter­ action effect between film concentration and sam­ pling time indicated that 48 hours after application, there was a quadratic effect of film concentrations on the activity of the three enzymes studied. in the case of pox and ppo, an activity reduction effect was obtained up to the estimated concentrations of 54 ml l­1 and 50 ml l­1, respectively, followed by an increase (fig. 6a and 6b). however, pal activity increased until the estimated concentration of 36 ml l­1, with a subsequent reduction (fig. 6c). about the fig. 4 ­ yield of strawberry, in response to chi­pyro­film concen­ trations and reference treatment (k2hpo4, 50 mm). isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. table 1 ­ yield, fruit weight and fruits with anthracnose in albion, portola and san andreas cultivars (z) (z) means followed by different lowercase letters in the row, differ by tukey test at 5% error probability. the averages correspond to the isolated effect of cultivar factor to studied variables. variables cultivar c.v. (%) albion portola san andreas yield (g plant­1) 131.90 ± 7.93 c 221.35 ± 12.87 a 170.4 5± 9.30 b 20.18 fruit weight (g fruit­1) 8.60 ± 0.67 b 8.50 ± 0.57 b 13.33 ± 1.09 a 72.71 fruits with anthracnose (%) 5.29 ± 1.71 b 9.13 ± 1.53 a 5.83 ± 1.49 b 107.57 fig. 5 ­ percentage of fruits with anthracnose in response to chi­ pyro­film concentrations and reference treatment (k2hpo4, 50 mm). isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. 48 adv. hort. sci., 2022 36(1): 43­52 reference treatment, with k2pho4, it induced activi­ ties similar to chi­pyro­film in the concentration of 100 ml l­1 for pox, ppo, and pal. the enzymatic activity also had an interaction effect between treatments of resistance induction and strawberry cultivars. pox activity decreased to concentrations of 49 ml l­1 and 57 ml l­1 in albion and portola, respectively (fig. 7a). for san andreas, although there was a similar trend, it was not signifi­ cant (fig. 7a). fig. 6 ­ specified activity of peroxidase­pox (a), polyphenoloxi­ dase­ppo (b) and phenylalanine ammonia lyase­pal (c) in the leaves, before (ba) and after application (aa) of chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between film con­ centration and sampling time (before and 48 hours after application). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. fig. 7 ­ specified activity of peroxidase­pox (a), polyphenoloxi­ dase­ppo (b) and phenylalanine ammonia lyase­pal (c) in leaves of albion, portola and san andreas, in response to chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between film con­ centration and cultivars. *, **, ***, significant at p<0,05, p<0,01, e p<0,001, respectively. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 49 the cultivars albion and san andreas also had a quadratic response to chi­pyro­film, with a decrease in ppo activity up to the estimated concentrations of 54 ml l­1 and 55 ml l­1 (fig. 7b). concerning portola, there was a linear increase up to a 100 ml l­1 concen­ tration of chi­pyro­film (fig. 7b). concerning pal, there was a quadratic response in albion, with an increase up to the 38 ml l­1 rate of chi­pyro­film, as well as a linear reduction in portola (fig. 7c). the reference treatment with k2hpo4, showed a response similar to the 100 ml l­1 film concentration for the enzymes ppo and pal in the three cultivars studied (figs. 7b and 7c). for the pox in albion and san andreas, the reference treatment was similar to the 25 ml l­1 concentration, while in portola, it was similar to the 50 ml l­1 film (fig. 7a). 4. discussion and conclusions in general, the results indicate that chi­pyro­film contributed to the increase of vegetative growth and yield, as well as, to the anthracnose damage reduc­ tion in strawberry fruits. results that are in agree­ ment with those found in the literature, where it is observed an increase in vegetative, productive, and health variables, such as height, number of leaves, leaf area, yield and reduction of incidence of diseases in plants that received chitosan or pyroligneous extract (el­miniawy et al., 2013; masum et al., 2013; mungkunkamchao et al., 2013; mukta et al., 2017; kumaraswamy et al., 2018; ventura­aguilar et al., 2018). the effect of chi­pyro­film on strawberry plant growth can be attributed to the role of chitosan as a non­toxic and biodegradable plant growth promoter (salachna and zawadzińska, 2014; ahmed et al., 2020). some authors suggest that foliar application of chitosan enhances the endogenous concentration of phytohormone such as gibberellic acid and auxin (uthairatanakij et al., 2007; ahmed et al., 2016). but the increase in macro and micronutrient accumula­ tion and improved the content of photosynthetic pig­ ments, provided by chitosan are also related to its influence on plant growth (shehata et al., 2012; ahmed et al., 2016). on the other hand, pyroligneous extract contributes to plant growth by its phytopro­ tective effect against pathogens, especially fungi. was reported antipathogenic effects of the pyrolig­ neous extract on plant pathogenic fungi l ike helminthosporium sativum, cochliobolus sativus, valsa mali, colletotrichum orbiculare, and alternaria mali (jung 2007; wei et al., 2010 a). this antifungal activity has been related to the presence of furalde­ hydes and phenols in pyroligneous extract (grewal et al., 2018). the efficiency of spraying with chi­pyro­film can be attributed to the effects of pyroligneous extract and chitosan individually, and to an interaction effect between both. according to porto et al. (2019), who studied physicochemical properties of chi­pyro­film, the film showed a semicrystalline structure, which is smooth and stable up to 50°c, being persistent in environmental conditions; it is permeable to water vapor and has high hygroscopicity, in addition to being able to efficiently block incident uvb and uvc radiation. the coverage presented by the chi­pyro­ film, as well as its persistence on the leaf surface (fig. 2), probably provide several days of action, per­ haps a large part of the application interval (15 days). the main effect of chitosan and the pyroligneous extract is attributed to resistance induction by increased defense enzyme activity and accumulation of phenolic compounds acting on reactive oxygen species (ros) (wei et al., 2010 b; katiyar et al., 2015; pichyangkura and chadchawan, 2015; grewal et al., 2018). this effect is in line with the behavior verified by the pal activity in this study (figs. 6c, 7c). however, the activity of the pox and ppo enzymes responded differently, with a reduction in activity in the concentrations estimated between 50 and 60 ml l­1, 48 hours after application of treatments (figs. 6a 6b), especially in the albion and san andreas culti­ vars (figs. 7a, 7b). this film rate (50­60 ml l­1) had a better performance in the dry mass accumulation, yield, and incidence of anthracnose. the results indicate that in addition to the increase in systemic resistance, suggested by the pal response, there is a direct effect of phytoprotection and a reduction of stress condition. some aspects that can be associated with this second aspect, maybe the block that the film exerts concerning uva and uvb radiation, as well as its potential action on pathogens, a hypothesis that corroborates the results obtained by porto et al. (2019). in this way, it can be suggested that chi­pyro­film has a complex performance, acting both on metabo­ lism with increased plant resistance and reducing cel­ lular damage caused by physical (radiation) and bio­ logical stress (pathogens), as well as forming a phyto­ protective film that inhibits the direct action of stress adv. hort. sci., 2022 36(1): 43­52 50 agents, such as uva and uvb radiation and inhibiting the attack of pathogens. in general, chi­pyro­film showed greater efficien­ cy in promoting growth, yield, and resistance to anthracnose than the reference treatment (k2hpo4), mainly in concentrations between 25 and 50 ml l­1. k2hpo4 performed similarly to the 100 ml l­1 of the film. different studies indicate the effect of k2hpo4 in the resistance induction of several species (reuveni and reuveni, 1998; kashiap and dhiman, 2009; aleandri et al., 2010; hamza et al., 2017; el­tanany et al., 2018). according to orober et al. (2002), foliar application of k2hpo4 results in the activation of sys­ temic resistance mechanisms. the positive effect of k2hpo4 is associated with salicylic acid involved in triggering plant cell defense and sensitization responses for a faster and stronger response to sub­ sequent pathogen attack (mauch­mani and métraux, 1998; orober et al., 2002). the 100 ml l­1 concentration of chi­pyro­film may be high for the strawberry crop. probably the increase in film thickness, which according to porto et al. (2019), can significantly reduce the film’s per­ meability to water vapor. an aspect that can make some physiological processes such as the flowing water, the absorption of nutrients, and photosynthe­ sis less efficient. this study provides indicates that the phytopro­ tective film (chi­pyro­film) is effective as a growth promoter and inducer of systemic resistance to anthracnose, resulting in increased growth and fruit production in strawberry plants of different cultivars. the optimal concentration of chi­pyro­film ranges between 50 and 60 ml l­1, depending on the straw­ berry cultivar. we believe that the tested film can be an impor­ tant tool for production systems, especially agroeco­ logical and organic systems, which require alterna­ tives for disease control. but also, due to its physical­ chemical properties and great stability, we believe that the film can be combined with other compo­ nents, such as nutrients, biostimulants, and plant hormones, to enhance its effect. however, such com­ binations need to be studied. acknowledgements this study was financed by the coordenação de aperfeiçoamento de pessoal de nível superior ­ brasil (capes), empresa brasileira de pesquisa agropecuária (embrapa) and by the project “avaliação de biofilmes estabilizados a base de quito­ sana associada a diferentes moléculas, para uso em sistemas de produção orgânico”. references ahmed a.h.h., nesiem m.r.a.e., allam h.a., el­wakil a.f., 2016 ­ effect of preharvest chitosan foliar applica‐ tion on growth, yield and chemical composition of washington navel orange trees grown in two different regions. ­ afr. j. biomed. res., 10: 59­69. ahmed k.b.m., khan m.m.a., siddiqui h., jahan a., 2020 ­ chitosan and its oligosaccharides, a promising option for sustainable crop production ‐ a review. ­ carbohydr. polym., 227: 115331. aleandri m.p., reda r., tagliavento v., magro p., chilosi g., 2010 ­ effect of chemical resistance induc‐ ers on the control of monosporascus root rot and vine decline of melon. ­ phytopathol. mediterr., 49: 18­26. anvisa, 2016 ­ relatório das análises de amostras monito‐ radas no período de 2013 a 2015. ­ agência nacional de vigilância sanitária, portugal, pp. 246. 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indonesia. guava has a short shelf­life as it rapidly goes under postharvest ripening. this leads to a faster deterioration of physicochemical and sensorial properties of guava. a generally used method to extends shelf­life is by edible coating. in this study, a combination of alginate and cyclea barbata leaves powder (cblp) was investigated as a potential edible coating. the analysis of firmness, total soluble solids, total reducing sugar, total titratable acidity and organoleptic tests were conducted to evaluate the quality of guava fruits stored for 20 d at 14°c. a split plot design study was used and four different treatments with different cblp concentrations were applied. the samples treated with 2% alginate and 0.8% cblp showed the lowest total dissolved solids, total reducing sugar, and total titratable acidity. moreover, the samples were reported with the highest score on color, taste, and texture parameters. the firmness test showed that samples treated with 2% alginate and 0.2% cblp had the lowest firmness loss and high­ est score for aroma. in summary longer quality retention of guava fruits was found after the addition of cblp in alginate­based edible coating. 1. introduction as a tropical country, indonesia is rich in horticultural products, espe­ cially fruits. guava (psidium guajava l.) var. getas merah is one of many fruits that are highly produced and consumed in indonesia. although it has high content of vitamin c, dietary fiber, antioxidants, and minerals, guava is a perishable food product and has a relatively short shelf life (kumar et al., 2001; naaseer et al., 2018). during its ripening stage, guava undergoes physicochemical changes such as total soluble solid, pectin content, total titratable acidity, total sugar content, and total anthocyanin content (dube and singh, 2015). total soluble solids of guava cv. apple (*) corresponding author: nafi@umy.ac.id citation: utama n.a., pranata i.a., pramesi p.c., 2022 ­ maintaining physicochemical and sensory proper‐ ties of guava var. getas merah using alginate and cyclea barbata leaves powder as edible coating. ­ adv. hort. sci., 36(2): 135­144. copyright: © 2022 utama n.a., pranata i.a., pramesi p.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 november 2021 accepted for publication 9 march 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12341 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 135­144 136 colour were found to be increasing rapidly between 75 to 150 days. whereas its maximum acidity of 0.41% was reached after 105 days. following the increase of total titratable acidity during immature and intermediate stage of maturation, the value will be decreased during maturity stage. on the contrary, total reducing sugar increased during late maturity. these changes happen very fast; therefore, fully ripe guava can easily bruise and considered as highly per­ ishable (fabi et al., 2010). by reducing the rate of this physicochemical and sensory properties to sustain the quality of guava, shelf­life extension can be achieved. the storage of guava with chemicals such as boric acid and naphthalene acetic acid (naa) at ambient temperature is commercially available and feasible (singh et al., 2017). although this technique has been proven to effectively maintain the quality of guava, the use of chemicals may affect those potential con­ sumers that have a negative perception of chemical preservatives. the use of plastic packaging for indi­ vidual packaging with clink and shrink film was reported (rana et al., 2015). by individually wrapping guava fruits, changes of total soluble solids slowed down and extending the shelf life for 10 days. however, the use of plastic is not a sustainable choice. the preservation technique by edible coating can be an option for a more sustainable choice. it is applied by adding a thin layer to the fruit to prevent gas release. the edible coating must be able to form a layer that acts as a respiration and transpiration inhibitor by reducing moisture and solute migration and gas exchange. another requirement for edible coating material is that it is safe to consume and has the ability to enhance shelf­life (vaishali et al., 2019). compared to the synthetic coatings, edible films can act as both gas and moisture barrier which results in a modified atmosphere to enhance the shelf life of fruits (kocira et al., 2021). polysaccharides such as alginate, chitosan, and pectin, are the most frequent polymer used for edible coatings. alginate is derived from brown sea algae and is an example of hydrocol­ loids, film­forming biopolymers (parreidt et al., 2018). the application of alginate­based edible coat­ ings has been conducted with the addition of various ingredients to extend the shelf life of guava. the application of alginate and betel­essential oil on rose apple resulted in quality retention until nine days (setiawan et al., 2019). the effect of both chitosan and alginate edible coating with the addition of pomegranate peel extract were reported (nair et al., 2018). the study reported significant retention of quality deterioration in guava after the edible coat­ ing’s application and storage at 10°c. another study by showed that the addition of zno nanoparticles in chitosan and alginate effectively inhibits rot in guavas, indicating their antimicrobial action (arroyo et al., 2020). in this study, cyclea barbata leaves, which also has potential as an edible coating, is economically feasible, and is more available in indonesia, was included to the alginate coating. cyclea barbata is a well­known plant that is widely used by indonesian people as food and medicine. it is rich in carbohy­ drates, polyphenols, saponin, calcium, phosphor, and vitamins a2 and b (acamovic and brooker, 2005). the main component of cyclea barbata leaves extract that forms a gel is pectin polysaccharide with low methoxy content belongs to the gel­forming hydro­ colloid group. the gel formed by pectin will be highly adhesive fand transparent and consequently has potential as an edible coating material (rachmawati et al., 2010). thus, this study investigated the addi­ tion effect of cyclea barbata leaves powder (cblp) in alginate­based coating as edible coating to maintain physicochemical parameters and sensory quality of guava. 2. materials and methods study design this study was conducted using a split plot design study. this study was carried out in 2018 and used four treatments with a variation of cblp concentra­ tions. a total of 135 whole guava fruits were used during this study. each treatments used 27 guava that were grouped for each three guavas to be stored in a polystyrene board with plastic wrap after coating as one set. these three pieces of guava acted as trip­ licates of biological replicates. during the 20 days of storage, physicochemical and sensory analyses were done every four days. during this days, one set of fruits were randomly taken to be analyzed further for its physicochemical properties. then, one fruit were selected randomly for sensory analyses. plant materials this study used guava from an orchard in sleman regency, yogyakarta, indonesia. the guava used in this study was harvested 109­114 days after the fruit utama et al. ‐ preservation of guava by edible coating 137 bloomed. according to a previous study, guava can be harvested after optimum maturity by observation of color attained or between 105­135 days after the fruit sets (dube and singh, 2015). this range of day is suitable for distant transportation to refrain from har­ vest losses. however, the length of the distance from this study was not mentioned. therefore, this study used a more restricted range of harvesting days. the fruits were selected by visual observation to remove the defected fruits and choose the fruits with the same size. then, fruits with a weight of 170 g were selected. from the orchard, selected fruits were transported to faculty of agriculture, universitas muhammadiyah yogyakarta, indonesia. the fruits were stored in the refrigerator at 14°c for one day; then they were washed by submerging in 0.05% sodi­ um benzoate solution for 10 min and dried to pre­ vent the growth of bacteria and molds during storage (masamba mndalira, 2016). preparation of cblp and alginate edible coating the coating used in this study was produced according to a previous study (olivas et al., 2007), with some modifications. the cblp was made by cleaning fresh cyclea barbata leaves with water fol­ lowed by drying (oven­dried) at 50°c for 18 h. the dried leaves were ground and sieved using a sifter (0.5 mm mesh) to produce a powder. alginate was made by dissolving 10 g alginate in 500 ml water (to make 2% alginate solution) and heating with a water bath at 80°c for 30 min while stirring until the solu­ tion became transparent. a total of 2.5% glycerol as a plasticizer was added to the edible coating solution. the treatments with different formulation used was as below: ­ no coating applied (control) ­ 2% alginate + 0.2% cblp ­ 2% alginate + 0.4% cblp ­ 2% alginate + 0.6% cblp ­ 2% alginate + 0.8% cblp application of edible coating solution selected fruits that had been washed were soaked in the edible coating solution for 3 min, followed by dipping into a 2% cacl2 solution for 15 min until it formed a layer. the samples were dried at room tem­ perature. each three samples were stored in a poly­ styrene board wrapped with plastic. the storage temperature was kept at 14°c and 95% rh. analyses of physicochemical parameter to investigate the ability of shelf­life extension by adding cblp to alginate coating, the samples were measured for their physical (firmness), chemical (total soluble solids, total reducing sugar, and titrat­ able acidity), and organoleptic qualities. the parame­ ters were measured every four days within 20 d of storage time. firmness. the analysis of firmness was conducted by measuring the samples chosen for the weight loss measurements using a penetrometer. the samples with the skin still intact were stabbed with the device in three different locations using 250 g of pressure on the scale of 1/10 mm for 10 s. the results obtained were averaged and expressed in kg/cm2. total soluble solids. the samples for the analysis of total soluble solids were selected by choosing one random guava in a box of nine fruits. the chosen sample was extracted by mashing the fruit and press­ ing to obtain the juice. about two to three droplets of the juice were inserted into a handheld digital refractometer (atago, tokyo, japan). the samples were measured three times for replication and then the average value was taken. the results were expressed in % brix unit. total reducing sugar. the reducing sugar analysis was carried out according to somogyi (1937) as modi­ fied by nelson (1944). one milliliter of the juice extracted for the total soluble solid analysis was added to a test tube that contained 25 ml nelson a, 1 ml nelson b, and 1 ml cupric reagent solution. the test tubes were heated in a water bath (memmert, schwabach, germany) for 20 min and cooled. afterward, 2 ml arsenic­molybdic reagent was added to the tube and mixed until homogeneous before adding 7 ml of distilled water. the samples’ absorbance was read using a spectrophotometer (λ = 540 mm) (thermo fischer scientific, massachusetts, america). the results were reported as the percent­ age of reducing sugars (%). total titratable acid. the total titratable acid analysis was conducted according to ranganna (1986). from a box of nine guava fruits, one was selected randomly to be measured. the chosen sam­ ple was mashed, and 10 g were taken to be added into a 100 ml volumetric flask. after that, distilled water was added until it reached the 100 ml mark. the solution was then mixed until homogeneous and filtered using filter paper. then, 10 ml of the filtrate was transferred to an erlenmeyer. two to three drops of the indicator of 1% phenolphthalein were adv. hort. sci., 2022 36(2): 135­144 138 added. titration was conducted using naoh 0.1 n until the solution turned a pinkish color. the volume of naoh 0.1 n used for the titration was recorded to be calculated and converted to the total titratable acidity value. the total titratable acidity (%ta) was calculated with the equation %ta = (v x n x mw x df x 100%)/(m x 1000 x v) where v = volume of naoh 0.1 n used (ml), n = nor­ mality of naoh used, mw = molecular weight of dominating acid (molecular weight of citric acid = 192), df = dilution factor, m = mass of sample (g), and v = valence of dominant acid (valence of citric acid = 3). the results were reported as percentages (%) organoleptic tests. in this study, the organoleptic test used a hedonic test where the perception of the sample’s color, aroma, taste, texture, and overall acceptability was measured. the measurement used a ranking test with a 9­point scale (meilgaard et al., 2006) where a score of 1 is the lowest score (dislike extremely) and 9 is the highest (like extremely). the hedonic test was done with 50 panelists (ratio male: female is 1:1) from academic staff and students of universitas muhammadiyah yogyakarta. the pan­ elists were aged between 18 and 60 years old. statistical analysis the results of firmness, total soluble solids, total reduced, sugar and total titratable acid were ana­ lyzed with one­way anova. means were separated and analyzed using the duncan multiple range test (dmrt, α = 5%) using statistic analytical software (sas) version 9.4. 3. results and discussions firmness the firmness analysis of guava treated with algi­ nate­based edible coating and different concentra­ tions of cblp is exhibited in table 1. all samples show declining trends during the 20 days of observation. a sudden drop can be seen in samples coated with algi­ nate but no addition of cblp after four days of stor­ age and the firmness continues to decrease. compared to these samples, the samples treated with the addition of cblp in alginate shows slower decrease of firmness. the different values between control and treated samples indicated that the edible coating could prevent firmness loss during storage. in this study, samples coated with the addition of the lowest cblp concentration shows the highest firmness after 20 days of storage. similar result was also seen in the application of aloe vera and sage essential oil in tomatoes (tzortzakis et al., 2019). after 7 days of storage, tomato with aloe vera and 0.1% sage essential oil had lower softening compared to the addition of 0.5% sage essential in aloe vera. another study reported that apples coated with high­ er concentration of lemongrass oil and oregano oil showed sudden firmness drop (rojas­graü et al., 2007). a possible explanation of this result is the low ph of coating solution due to the higher concentra­ tion of essential oil was added. this result occurred because the sample coated with alginate and cblp had a more effective gas dif­ fusion barrier than the control sample, therefore, enzymes that were involved in respiration and tissue softening will be less active. firmness and juiciness of fruit is an important parameter that also relates to the marketability. the firmness of fruit is correlated to the pectin content on the cell walls. due to pectin solubilization, the texture of fruits will be softer and resulted in firmness loss. this pectin solubilizations can be caused by several reasons, such as the depoly­ merization of pectin by pectinase enzymes (i.e., poly­ galacturonase, cellulase and pectin methyl esterase) table 1 ­ result of firmness in kg/cm2 of guava treated with alginate­based edible coating enriched with different concentration of cyclea barbata leaves powder (cblp) results followed by the same letter(s) imply that there is no significant difference between results according to the duncan multiple range test (p>0.05). treatments observation days 0 4 8 12 16 20 control 3.58 ± 0.13 a 0.60 ± 0.09 c 0.58 ± 0.02 d 0.28 ± 0.01 c 0.12 ± 0.01 c 0.08 ± 0.02 d 2% alginate + 0.2% cblp 3.56 ± 0.07 a 2.81 ± 0.18 a 2.58 ± 0.13 ab 2.35 ± 0.23 a 1.99 ± 0.14 a 1.76 ± 0.05 a 2% alginate + 0.4% cblp 3.05 ± 0.05 c 2.43 ± 0.05 b 2.34 ± 0.13 bc 2.29 ± 0.13 a 1.99 ± 0.17 a 1.58 ± 0.10 b 2% alginate + 0.6% cblp 3.03 ± 0.22 b 2.83 ± 0.06 a 2.23 ± 0.15 c 1.97 ± 0.15 c 1.65 ± 0.14 b 1.39 ± 0.09 c 2% alginate + 0.8% cblp 3.34 ± 0.27 ab 2.87 ± 0.11 a 2.72 ± 0.17 a 2.22 ± 0.08 b 1.95 ± 0.07 a 1.55 ± 0.03 b utama et al. ‐ preservation of guava by edible coating 139 and the loss of neutral chains (arabinan and galactan) which binds pectin to the cell wall via glycans or cel­ lulose matrix (paniagua et al., 2014). by adding edible coating, the pectinase enzymes activity is lower and firmness loss is hindered (zhou et al., 2010). another factor of the firmness is the transpiration of fruit. the balance between water loss and water uptake highly affected mechanical properties of the skin by controlling cell turgor. when turgor is removed by membrane matrix disassemble (i.e., solu­ bilization of pectin during ripening), water can move freely which results in cell walls relaxation (brüggenwirth and knoche, 2016). fruit transpiration is highly correlated with the water vapor pressure of airspace inside of the fruit and the air directly outside as the driving force (montanaro et al., 2012). even after harvesting, detached fruit has sufficient water content to support an additional of 10 hours transpi­ ration without moisture supply from xylem/phloem supply. the application of edible coating can gener­ ate a layer to delay transpiration (kocira et al., 2021). thus, a possible mechanism of alginate and cblp as edible coating is by generating a water vapor barrier to lower water content loss of fruit. total soluble solids table 2 exhibits the results of total soluble solids with different cblp concentrations. the result of total soluble solids indicates the total sugars in the sample. all samples had an increase at the beginning of stor­ age time followed by a decline until the last observa­ tion day. this increase of total soluble solids during the ripening stage in guava was previously reported (patel et al., 2014). it was also explained that the increase can be from depolymerization of polysaccha­ rides and conversion of fruit starch to sugars. however, all samples showed a decline in total dissolved solids after 20 days due to the senescence phase. a similar trend of total soluble solids was also reported in the application naphthalene acetic acid (naa) and potassium nitrate as pre­harvest treat­ ments on the storage quality of winter guava (mandal et al., 2012). at the beginning of storage, hydrolysis of insoluble polysaccharides to simple sug­ ars resulted in the increase of total soluble solids (brothakar et al., 2002). within time, all the insoluble polysaccharides will be completely hydrolyzed while soluble solids and organic acids will be used for respi­ ration. this leads to the decrease of total soluble solid. the fall of total soluble solids content in mature fruit towards senescence has also been reported in mango fruits. the same study investigated the effect of different concentrations of bavistin df solution to extend shelf­life as a postharvest treatment on mango fruits. the trend of total soluble solids observed in the control samples indicated that they underwent a faster maturation stage and headed towards senescence (islam et al., 2013). on day 20, samples coated with 2% alginate and 0.8% cblp had the lowest level of total dissolved solids. the higher the amount of cblp, the lower the total dissolved solids were. the mechanism of edible coating in inhibiting starch degradation which leads to the increase of total soluble solids during fruit ripening is by the decrease of respiration rate. the degradation of cell walls and starch to soluble sugars and organic acids act as a supply of carbons during climacteric respira­ tion of fruit (colombié et al., 2016). guava is consid­ ered climacteric fruit, indicated by the inclined ethyl­ ene production at the peak of ripening (ishartani et al., 2018). the edible coating of alginate and cblp in guava created an additionally layer that can hinder the respiration rate; thus, lowering the conversion rate of starch to total soluble solids. table 2 ­ result of total soluble solids in % brix unit of guava treated with alginate­based edible coating enriched with different concen­ tration of cyclea barbata leaves powder (cblp) results followed by the same letter(s) imply that there is no significant difference between results according to the duncan multiple range test (p>0.05). treatments observation days 0 4 8 12 16 20 control 7.27 ± 0.06 a 7.30 ± 0.10 a 7.47 ± 0.15 a 7.88 ± 0.07 a 8.19 ± 0.03 a 7.03 ± 0.06 a 2% alginate + 0.2% cblp 6.53 ± 0.07 b 6.90 ± 0.10 b 7.03 ± 0.06 b 7.13 ± 0.04 b 7.15 ± 0.05 c 6.73 ± 0.12 b 2% alginate + 0.4% cblp 5.36 ± 0.14 c 6.20 ± 0.20 d 6.82 ± 0.04 c 7.10 ± 0.00 b 7.23 ± 0.00 b 6.71 ± 0.10 b 2% alginate + 0.6% cblp 5.27 ± 0.06 c 7.00 ± 0.10 b 7.03 ± 0.00 b 7.15 ± 0.05 b 7.23 ± 0.06 b 6.39 ± 0.13 c 2% alginate + 0.8% cblp 5.42 ± 0.25 c 6.62 ± 0.08 c 6.85 ± 0.12 c 7.07 ± 0.06 b 7.05 ± 0.02 d 6.17 ± 0.06 d 140 adv. hort. sci., 2022 36(2): 135­144 total reducing sugar the results of total reducing sugar analysis with different edible coating concentrations on guava fruits are shown in table 3. all samples show an increase in sugar reduction throughout the observation time. these results are similar to those of a previous study, where different varieties of guava were investigated for their total reducing sugars after 5 months of storage time at ambient temperature (choudhary et al., 2008). the reducing sugars may be increased in the senescence phase of climacteric fruits due to starch hydrolysis to glucose, fructose, and sucrose (patel et al., 2013). a high level of reducing sugars indicates rapid starch hydrolysis in fruit. the reducing sugar content in guava is varied within different varieties (choudhary et al., 2008). the highest level of reducing sugar is fructose, fol­ lowed by glucose (mowlah and itoo, 1982). after the ripening stage, reducing sugar content of guava were found to be decreasing in overripe fruits (bashir and abu­goukh, 2003). however, a clear explanation about this decrease has not been fully understood due to the complex process of starch breakdown, sugar synthesis and sugar metabolism in fruits are very complex which involves not only by ethylene but also an abundant of hormones and enzymes (cordenunsi­lysenko et al., 2019). in addition to this complexity, different fruits may possess different metabolic pathway of sugar. previously, a study was conducted to investigate the sugar metabolism of peach during fruit development (desnoues et al., 2014). the proposed pathway mentioned a conver­ sion of fructose and glucose to fructose 1,6­bisphos­ phate (f16bp) which involved in the glycolysis and respiration flux. on the last observation day, samples with 2% algi­ nate and 0.8% cblp showed the lowest reducing sugar level. this implies that the addition of 0.8% cblp mixed with alginate could suppress starch hydrolysis into glucose, sucrose, and fructose. the edible coating with polysaccharide base and cblp acted as a selectively permeable membrane towards co2 and o2 gases’ diffusion. this characteristic may extend shelf­life, as fruit’s respiration rate became slower (kocira et al., 2021). cblp has an alcohol con­ tent that could inhibit fruit senescence when added in alginate (viña et al., 2000). total titratable acid the analysis of total titratable acid in guava fruits treated with different concentrations of the edible coating was conducted. the results are exhibited in table 4. table 4 ­ result of total titratable acid in percentage (%) of guava treated with alginate­based edible coating enriched with different concentration of cyclea barbata leaves powder (cblp) results followed by the same letter(s) imply that there is no significant difference between results according to the duncan multiple range test (p>0.05). table 3 ­ result of total reducing sugar in percentage (%) of guava treated with alginate­based edible coating enriched with different concentration of cyclea barbata leaves powder (cblp) treatments observation days 0 4 8 12 16 20 control 9.33 ± 0.06 b 9.94 ± 0.02 b 12.16 ± 0.05 a 12.34 ± 0.00 b 13.59 ± 0.00 a 15.29± 0.02 a 2% alginate + 0.2% cblp 9.76 ± 0.05 a 10.13 ± 0.02 a 10.14 ± 0.03 e 11.50 ± 0.06 c 12.21 ± 0.13 a 13.37 ± 0.00 d 2% alginate + 0.4% cblp 8.98 ± 0.13 c 9.46 ± 0.03 d 10.30 ± 0.06 d 13.00 ± 0.04 a 13.45 ± 0.21 a 13.57 ± 0.02 b 2% alginate + 0.6% cblp 8.69 ± 0.03 d 9.76 ± 0.07 c 11.38 ± 0.04 b 12.97 ± 0.05 a 13.03 ± 0.38 a 13.30 ± 0.03 e 2% alginate + 0.8% cblp 8.24 ± 0.06 e 9.70 ± 0.07 c 10.55 ± 0.11 e 10.98 ± 0.06 d 12.03 ± 0.08 c 13.42 ± 0.04 c treatments observation days 0 4 8 12 16 20 control 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 1.11 ± 0.003 a 1.78 ± 0.003 a 2.01 ± 0 a 2% alginate + 0.2% cblp 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 0.81 ± 0.001 ab 1.13 ± 0.003 ab 1.56 ± 0 b 2% alginate + 0.4% cblp 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 1.13 ± 0.003 ab 1.34 ± 0 c 2% alginate + 0.6% cblp 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 1.13 ± 0.003 ab 1.34 ± 0c 2% alginate + 0.8% cblp 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 0.67 ± 0 b 0.67± 0 b 0.67 ± 0 b results followed by the same letter(s) imply that there is no significant difference between results according to the duncan multiple range test (p>0.05). utama et al. ‐ preservation of guava by edible coating 141 as a climacteric fruit, guava will have a sudden increase in respiration along with or before the senescence stage due to increases in co2 and ethyl­ ene after the climacteric phase (tripathi et al., 2016). therefore, the level of total titratable acid has fre­ quently been used to indicate the shelf­life of fruit. according to the results, most of the samples had an increase in total titratable acid starting from day 12, except the samples coated with 2% alginate + 0.8% cblp. this result is different from a previous study of guava (cv. sardar), in which titratable acidity decreased after 40 days of storage (mandal et al., 2012). this reduction of total titratable acid level dur­ ing storage was due to guava, as climacteric fruit, during the peak of ripening resulted in the rise of malic enzyme activity and pyruvate decarboxylation. therefore, respiration rate and metabolic activity such as degradation of organic acids. however, similar to the results in this study an increase of total titratable acidity level in guava fruits was also observed (dube et al., 2015). in their study, the total titratable acidity increased until 120 days of storage, after which it declined. the increase of total titratable acidity is correlated to the undissociated organic acids level in the maturation of fruit which is stored in the vacuole of cell plants. when ripening stage begin, organic acids will be depleted as sub­ strates in respiration. therefore, this indicate that the samples were not fully ripen during harvesting. total titratable acid in guava coated with alginate and cblp tended to have a lower rate of increase. this indicates that the edible coating with alginate and cblp could decrease the respiration rate and suppress the use of organic acid, followed by main­ taining the amount of total acid in guava throughout the storage time. according to paul and pandey (2014), fruit’s deterioration rate is affected by o2 and co2 gases’ diffusion through the lenticel on the fruit’s surface (paul and pandey, 2014). the o2 gas entering the sample will increase the respiration rate, causing further deterioration. the sturdy edible coating of 2% alginate + 0.8% cblp on the fruit’s surface prevented gas diffusion, which resulted in no significant increase of total titratable acidity. organoleptic tests the visual appearance of samples before and after 20 days of storage can be seen in figure 1. from the appearance after 20 days, samples with no edible coating applied showed yellowish color and went into deterioration stage. in contrast, samples with edible coating were visibly similar in appearance with light green color. for organoleptic testing, a hedonic test was con­ ducted to evaluate the sensory qualities of guava fruits treated with different edible coating concentra­ tions. the results of the analysis are shown in figure 2. based on the organoleptic test, samples coated with 2% alginate + 0.8% cblp had the best score in the color, taste, and texture parameters. this is because a higher cblp concentration in the edible coating would further inhibit the respiration and transpiration rates. the best score for the aroma parameter was obtained by an edible coating with 0.2% cblp. this is because the alcohol level due to fermentation in cblp gave an unpleasant odor; thus, the lowest concentration gave the best score of fig. 1 ­ visual appearance of guava fruit on day 0 (a) and after 20 days of storage with: no edible coating (b); 0.2% alginate + 0.2% cyclea barabata leaves powder (cblp) (c); 0.2% alginate + 0.4% cblp (d); 0.2% alginate + 0.6% cblp (e); 0.2% alginate + 0.8% cblp (f). adv. hort. sci., 2022 36(2): 135­144 142 aroma quality. the highest overall score was obtained by two different concentrations of cblp: 0.2% and 0.4%. moreover, edible coating with 0.4% cblp had the highest score of taste. in general, samples coated with alginate and cblp had a higher score for sensory analysis compared to control samples. similar results were obtained from fresh­cut mango coated with polysaccharide­based edible coatings: samples coated with alginate had the highest consumer acceptance due to their ability to prevent firmness loss (salinas­roca et al., 2018). the maintenance of sensory quality by alginate­ based edible coating on fruits were also found on strawberry, water apples and apple fresh­cut (guerreiro et al. , 2015; utama et al . , 2020; marghmaleki et al., 2020). 4. conclusions different concentrations of cblp in an alginate­ based edible coating to extend the shelf­life of guava fruit were evaluated. the samples treated with 2% alginate and 0.8% cblp had the significantly lowest 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exercise: mechanism of production, and protection by antioxidants. ­ life, 50(4­ 5): 271­277. zhou r., li y., yang l., xie j., 2010 ­ effect of edible coat‐ ings on enzymes, cell‐membrane integrity, and cell‐wall constituents in relation to brittleness and firmness of huanghua pears (pyruspyrifolia nakai, cv. huanghua) during storage. ­ food chem., 124(2011): 569­575. impaginato 119 adv. hort. sci., 2021 35(2): 119­127 doi: 10.36253/ahsc­9899 assessment of pesticide residues level in watermelon fruits [citrullus lanatus (thunberg) matsumura and nakai] in lower, central and upper badibou districts in north bank region, of the gambia f.a. jassey (*), f.e. babatunde, f.j. manneh university of the gambia, school of agriculture and environmental science, mdi road, kanifing p.o. box 3530, serrekunda, the gambia. key words: maximum residue limit, pesticide, pesticide residue, watermelon. abstract: field and laboratory studies were conducted late 2019 in three dis­ tricts of the gambia’s north bank region; namely, central badibou, lower badibou and upper badibou to ascertain the pesticide residue level in water­ melon fruit, determine the insect pest control methods, types of pesticide, fre­ quency of application, and pre­harvest interval observed. multistage sampling technique was used in selecting the research respondents. eighty­five (85) farmers were identified; forty­five (45) were randomly selected as research respondents. data was collected using structured questionnaires. spss software was used to analyse the questionnaires and gas chromatograph to determine the pesticide residue level. data obtained were analysed and com­ pared with the european union maximum residue limit (mrl). the results of the analysis revealed that the farmers used chemical control method in water­ melon production, and applied at frequency of once in every two weeks. furthermore, the results indicated that the chemical applied at flowering stage and the pre­harvest interval (17­21 days) ranked the highest. the pesticides residues found in the watermelon samples were dimethoate, profenophos, dicofol, cypermethrin, lambda­cyhalothrin, permethrin and deltamethrin and most were above the mrl. the presence of pesticides residues in the water­ melon samples calls for strict regulation on the use of pesticides on watermel­ on. further study is recommended in other fruits and vegetables grown in the country especially in the studied region. 1. introduction the developing countries are seriously affected by the sky­rocketing of unemployment rates. agriculture is the backbone of all the developing countries’ economies. the production of watermelon by gambian farmers has featured prominently irrespective of the uncountable num­ (*) corresponding author: jasseyf010@gmail.com citation: jassey f.a., babatunde f.e., manneh f.j., 2021 ­ assessment of pesticide residues level in water‐ melon fruits [citrullus lanatus (thunberg) matsumura and nakai] in lower, central and upper badibou districts in north bank region, of the gambia. ­ adv. hort. sci., 35(2): 119­127 copyright: © 2021 jassey f.a., babatunde f.e., manneh f.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2020 accepted for publication 18 march 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9899 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 119­127 120 ber of challenges faced by farmers amongst other hosts of issues that militate against this lucrative enterprise (department of planning agriculture, 2016). the government’s attention should, therefore, be drawn towards addressing challenges faced in watermelon production particularly pesticides and its use. the sector contributes positively to the gross domestic product (gdp) and employs about 70% of the labour force (jobarteh and selmani, 2020). according to the anr policy (2019­2026), the con­ sumption trend of fruits and vegetables of various kinds in the country is fairly high and likely to contin­ ue. fruits are a concentrated source of natural com­ ponents, and these natural components are plant­ derived materials performing a key role in maintain­ ing human health, especially in disease prevention, growth, and development. today, plants, and plant­ based compounds are the basis of modern pharma­ ceuticals used for the treatment of various dreadful diseases (reetu and tomar, 2017). fruits such as mango, organs, guava, pear, and watermelon are life­ enhancing medicines packed with vitamins, minerals, and antioxidants to human as well as cash crops for export by the growers in the gambia. watermelon does not only have the potential of enhancing the health of consumers but also increases the income of farmers (adeoye et al., 2011). the production of watermelon is mainly carried out in the rainy season from september to december. it is produced in all the regions of the country; however, north bank region and central river region are the major growing areas of water­ melon in the gambia. in 2016, a total of one hundred and seventy­four (174) tonnes of watermelon fruits were produced (department of planning agriculture, 2016). the crop is generally susceptible to many insects and diseases but no systematic study has been carried out to determine the damage level. research has revealed that factors such as the preva­ lence of insects and diseases, climatic factors such as rainfall, temperature, and soil types hinder the pro­ duction of watermelon (adojutelegan et al., 2016). amongst the problems faced by watermelon farmers, insect pests ranked the highest (chamo et al., 2016). farmers used different strategies such as attractants (cocoa butter), food baits, sanitation, botanicals, and chemicals to control fruit flies. synthetic pesticides are known to be one of the most effective agents for controlling insects and diseases. amongst various categories of pesticides, the insecticide is considered as the most toxic (kodandaram et al., 2013). it is becoming a dominant agent for controlling insects and diseases of crops. farmers use pesticides such as carbofuran, chlor­ pyrifos, diazinon, dimethoate and metalaxyl in the production of crops (wanwimolruk et al., 2015). watermelon is very susceptible to insects and dis­ eases such as fusarium wilt, anthracnose, downy mildew virus diseases, gummy stem blight, powdery mildew, bacterial fruit blotch, damping­off, root­knot nematodes, adult striped, spotted cucumber beetle and squash bugs as a result, farmers use pesticides in controlling them (shrefler et al., 2015). the system of continuous application of pesticides may lead to a high level of pesticide residue in the crops (nguyen et al., 2018). this could have a counterproductive effect on the quality of products to be consumed and plac­ ing a farmer’s life at risk. cases of indiscriminate use of pesticides and non­ adherence to good agricultural practices are very common. for example, some farmers apply chemi­ cals on their fields in the afternoon and pick the fruits early in the next morning for sale in the local mar­ kets. these observations suggest that the fruits sold in the markets may have serious pesticide contami­ nation. most of the farmers in the gambia, do not consider the so­called maximum residue limit. thought the maximum residue limit regulation exists in the country, but it is not adhered to. the production and consumption of watermelon are rapidly increasing in the gambia. during the peak season of watermelon, the price is affordable to all categories of the population due to the high supply in the market. most of the children and even some adults when eating watermelon eat it to the core. sometimes, the remains are given to animals such as goats and sheep. to my knowledge no formal research has been done in the country to determine pesticide residue in watermelon. as a result, it is nec­ essary to assess the pesticide residue in watermelon fruits as this crop in question have numerous health benefits such as it preventing cancer and diabetes due to its high content of lycopene, an antioxidant, lower blood pressure due to its richness in an amino acid. therefore, this study aimed to determine the methods used in controlling insect pest of watermel­ on and to assess the concentration of pesticide residue in the watermelon fruits at harvest in the study area. jassey et al. ‐ pesticide residue in watermelon fruits 121 2. materials and methods the study location the study was conducted in north bank region of the gambia, located in the northern part of the river gambia from october to november 2019. the area is located between latitude 16.01° west and longitude 13.52° north. the region is one of the five administrative regions of the gambia. its capital is kerewan and subsequently reorganized as the kerewan government area council, with none change within the area covered. the area is divided into seven distr icts, namely, central baddibu, lower baddibu, upper baddibu, jokadu, lower niumi, upper niumi and sajal. the area is peculiar for watermelon production and a large percentage of the population practices farming at a large scale (figs. 1, 2, and 3). research design the study used qualitative and quantitative methods to collect data. the research methods used were as follows: step one: the researcher conducted interviews with the watermelon farmers in the study area (north bank region). step two: watermelon samples were collected for pesticide residue analysis. sampling procedures and sample sizes multistage sampling techniques were used in selecting the research respondents (contact farmers). before the study, the regional directorate was con­ tacted for information regarding the production trend of watermelon in the region. based on the findings, three districts (central badibou, lower badibou and upper badibou) with the highest num­ ber of watermelon growers were selected using pur­ posive sampling. the second stage was the purposive sampling of 37 major watermelon growing villages in the sample districts. populations of 85 farmers were identified out of which forty­five (45) farmers were randomly selected as research respondents 15 from each district. samples of 10 watermelon fruit due for harvesting were randomly collected from (10) different water­ melon fields for pesticide residue analysis. the pesti­ cide residues found in the fruits were compared with the european union pesticide maximum residue limit standard (table 1). data collection data were collected using structured question­ naires designed in english and administered orally in the local languages (wolof, mandinka, fula, and serer etc.). before the actual data collection, the questionnaires were validated pre­tested with few fig. 1 ­ map showing the study areas in lower badibou district. fig. 2 ­ map showing the study areas in central badibou district. fig. 3 ­ map showing the study areas in upper badibou district. adv. hort. sci., 2021 35(2): 119­127 122 watermelon farmers and adjusted. the demographic characteristics of the farmers (age, education, farm­ ing experience, labour source, farm size) and pest control method used by farmers were observed. one watermelon fruit was collected from a total of 10 farmers. during the collection, foil paper, tissue, markers were used. the tissue paper was used to clean the watermelon fruits and wrapped with foil paper to avoid contamination. samples were labelled using the board marker. the watermelon samples were sent to ceres locustox in dakar for pesticide residue analysis. the field coordinates were taken with gpsmap 60cs garmin. sample preparation and storage individual watermelon fruit samples were cut and chopped with a knife to about 3 cm cube in size. the process was carefully carried out to ensure that the test samples were homogeneous. the samples were blended with a robot coupe blender 6 to ensure homogeneity. each time the blender was used for a sample, the content in the machine was cleaned together with all the other equipment used in the process with detergent before another sample was blended to avoid contamination. precautionary mea­ sures were employed to avoid any losses of juice or flesh. test portion individual test portion sufficient for one analysis was taken as sub­sample from the main test sample. these were immediately prepared for analysis. 10 g of the solution was measured from the homoge­ neous sample and put into 50 g centrifuge tube. the solution was extracted with the help of the acetoni­ trile. extraction to determine the concentrations of pesticide residues, the extraction procedure was performed as described by institut luxembourgeois de la normalisation, de l’accreditation, de la securite (ilnas, 2018). the samples were prepared as follows: ten millilitre (10 ml) of acetonitrile and 100 µl of pcb28 were separately measured and put into the test tube containing the test sample, and agitated by hand vigorously for a minute. four grams (4 g) of magnesium sulfate was added for the removal of residual water together with, 1 g of sodium chlorine, 0.5 g of na2hcitrates­squihydrate and 1 g of na3cit­ rates­ dehydrates. the solution was agitated vigor­ ously with the flash shaker for one minute and cen­ trifuged for phase separation for five minutes at a speed of 3000 m/s. purification one millilitre of the solution was pipette and sep­ arately placed into 2 ml test tubes containing 150 mg mgso4 and 25 mg primary secondary amine sorbent (psa). the solutions were centrifuged for five min­ utes at the speed of 3000 revolution/min. one (1 ml) was then transferred to another test tube and 5% solution of formic acid added to improve the storage stability of certain base sensitive pesticides. in addi­ tion, a control was prepared alongside, which has the entire additive except the watermelon sample. data analysis data were analyzed using statistical package for social science (spss) windows software version 20. the pesticides residue analysis was done using the gas chromatograph as described by institut luxembourgeois de la normalisation, de l’accredita­ tion, de la securite (ilnas, 2018). 3. results and discussion demographic characteristics of the watermelon farm‐ ers in the study area the demographic characteristics of the research respondents were presented in table 2. the result derived shows that all respondents were 100% male. gender is very fundamental when it comes to the production of a certain type of crops in the gambia. traditionally men are attributed to watermelon pro­ duction due to its tediousness. this finding is similar with adeoye et al. (2011) who indicated the domi­ nance of male fork in watermelon production in table 1 ­ european union pesticide database on maximum residue limit standard source: european union pesticide database on mrl standard latest update, 2016. sample pesticide maximum residue limit (mrl) (mg/kg) dimethoate 0.01 dicofol 0.02 lambda­cyhalothrin 0.06 profenophos 0.01 deltamethrin 0.02 permethrin 0.05 cypermethrin 0.20 jassey et al. ‐ pesticide residue in watermelon fruits 123 nigeria. the watermelon growers were predominant­ ly youths and middle­aged individuals as 62% of the respondents were not more than 50 years old (table 2). age determines strength, therefore; it plays an important role in any meaningful production. watermelon production is very tedious and rigorous as a result the middle­age individuals are the most people venturing into it. during the research, it was found out that some old age had to reduce their farm size due to the tediousness of the work. this is in agreement with (adeoye et al., 2011) who stated that age is crucial to carrying out activities such as mounting knapsack sprayer at the back, which most of the farmers used in the study area. majority of the research respondents did not have formal education, amongst them arabic education ranked the highest 60%, followed by secondary edu­ cation, (24.4%), primary education (6.7%), tertiary education (4.4%) and illiterate (4.4%). this shows that the level of conventional education is very low among the watermelon farmers in the study area (table 2). most of the pesticides are labelled either in english or in french; as a result, the respondents could not read the pesticide label. this might be responsible for the high concentration of pesticide residue in the watermelon fruits from the study area. mubushar et al. (2019) reported the inappropriate use of pesticide due to high level of illiteracy. watermelon production was dominated by less experienced farmers, 1­5 years experience (55.6%), 6­10 years (24.4%), 11­15 years (11.1%) and 16 years above (8.9%). this could be attributed to the fact that the majority of the watermelon farmers in the study area are in the middle age. therefore, the inexperi­ ence of the watermelon farmers might have caused poor handling and frequent application of pesticides (eyhorn et al., 2015), which could have led to the high concentration of pesticide residue in watermel­ on fruits. control methods of insect pest practiced by water‐ melon farmers in the study area the various methods of pest management prac­ tised by respondents are shown in figure 4. chemical control method had the highest in the study area (88.9%) followed by botanical (6.7%), and integrated pest management (4.4%). this was attributed to the believed that chemical is readily available and required less labour as compared to other control methods (fig. 4). the results manifested that chemi­ cal control method was predominantly practised. according to the farmers, the pesticides are easy to use and readily available. some farmers practised other control methods but they claimed to be diffi­ cult and time­consuming. for example, to have enough of neem solution to cover a hectare of land is not easy, which compelled majority of the farmers to practised chemical control method. this in line with padmajani et al. (2014) who reported the easy use agro­chemical by farmers despite knowing other con­ trol methods. the frequency of application of the pesticides by watermelon farmers in the study area figure 5 revealed the frequency of pesticide appli­ cation during watermelon production in the study area. the result revealed that majority of the respon­ dents applied pesticide once every two weeks table 2 ­ demographic characteristic of the watermelon farmers ns = not significant by the tukey test (p<0.05). fig. 4 ­ control methods of insect pest practiced by watermelon farmers. personal characteristics categories percentage (%) sex male 100 female 0 age 20­30 years 13.3 31­40 years 31.1 41­50 years 31.2 51­60 above 24.4 educational qualification primary 6.8 secondary 24.4 illiterate 4.4 tertiary 4.4 arabic 60 experience 1­5 years 55.6 6­10 years 24.4 11­15years 11.1 16 above 8.9 124 adv. hort. sci., 2021 35(2): 119­127 (44.4%) followed by those who applied once every week (31.1%). the respondents who applied pesti­ cide once every three weeks were 17.8% and the lowest percentage (6.7%) of respondents were found to applied pesticide once in a month. the pre‐harvest interval observed by the watermelon farmers in the study area figure 6 showed the pre­harvest interval observed in the watermelon growing areas. most of the respondents (46.7%) observed pre­harvest interval of 16­20 days, followed by 17.8% of the respondents who observed pre­harvest interval above 21 days. the lowest percentages (11.1%) of respondents were found to observe pre­harvest interval of 6­10 days. pesticide residue concentration levels detected in the watermelon samples the potential contamination of watermelon fruits with the different pesticides used in the study area was investigated. the results detected seven pesti­ cides that happen to fall in the same class of pesti­ cides, insecticide in the watermelon fruits that were subjected to analysis. the insecticides detected were; dimethoate, profenophos, dicofol, cypermethrin, lambda­cyhalothrin, permethrin, and deltamethrin. the dimethoate and profenophos are in the pesticide group of organophosphate, whereas dicofol belongs to the organochlorine. cypermethrin, lambda­ cyhalothrin, permethrin, and deltamethrin belong to the pyrethroids. the various levels of dimethoate residues detect­ ed in the watermelon samples are shown in figure 7. the test result indicated that there was high pesti­ cide residue concentration of dimethoate in all the watermelon samples compared to the european union maximum residue limit (0.01 mg/kg) except in sample 6 which recorded (0.01 mg/kg). in samples 1 and 2 the residue level recorded were 0.8 mg/kg, whilst samples 4 and 10 recorded 1 mg/kg. the high­ est concentration of dimethoate was found in sam­ ples 3, 5, 7, 8, and 9 with 2 mg/kg. the present of dimethoate residue in watermelon was reported by omoyajowo et al. (2017) in his research on the assessment of pesticide residue levels in common fruits consumed in lagos state, nigeria. if the mrl exceeds its limit it can cause various health problems to consumers. ngoula et al. (2014) stated that high dose of dimethoate disrupts spermatogenesis and reduce fertility in test animal. figure 8 presents the dicofol residues detected in the various watermelon sample analysed for pesti­ cide residue. the findings revealed that watermelon samples were all found to have some amount of insecticide residues of dicofol and were all above the european union maximum residue limit which is 0.02 mg/kg. sample 2 and 10 registered (0.3 mg/kg), sample 8 registered (0.4 mg/kg), sample 1 registered (0.5 mg/kg), sample 9 registered (0.7 mg/kg), sam­ ples 3, 4, 6, and 7 registered (0.8 mg/kg) and sample 5 registered the highest pesticide residue of (1 mg/kg). figure 9 presents the residue of lambda­ cyhalothrin detected in the various watermelon sam­ fig. 7 ­ residue level of dimethoate in watermelon samples. fig. 6 ­ pre­ harvest intervals observed in the study area. fig. 5 ­ percentage frequency of pesticide application in the study area. jassey et al. ‐ pesticide residue in watermelon fruits 125 ples. lambda­cyhalothrin residue was found in all the watermelon samples that were subjected to analysis. sample 1 recorded (0.006 mg/kg) while sample 2, 3, 4, 5, 6, 7, 8, 9 and 10 recorded 0. 009 mg/kg and they were all found to be below the european union maximum residue limit which happened to be 0.06 mg/kg. the profenophos residue level detected in the watermelon samples is shown in (fig. 10). profenophos was found in all the watermelon sub­ jected to analysis. samples 3, 4, 6, 7, 8, 9, and 10 had pesticides residue concentration level of 0.06 mg/kg, while sample 1, 2, and 5 had 0.05 mg/kg, when com­ pared to the maximum residue limit standard of 0.01 mg/kg of the european union, they were all found to be above the residue limit. figure 11 indicates the deltamethrin residue level detected in the watermelon samples. deltamethrin residues were present in all the watermelon samples; sample 1 recorded (0.1 mg/kg), samples 2, 6, 8, and 10 recorded (0.2 mg/kg), samples 3, 4, 5, and 7 recorded (0.3 mg/kg). sample 9 recorded the highest concentration level of (1 mg/kg). compared with the european union mrlof 0.02 mg/kg; all the samples were above the mrl. the permethrin residue detected in the various watermelon samples are presented in figure 12. the permethrin was found in all the watermelon samples and there concentrations were above the european union maximum residue limit of 0.05 mg/kg. the highest concentration of permethrin was observed in sample 5 with (1.5 mg/kg), followed by sample 8 (1.4 mg/kg), while samples 4, 9 and 10 recorded 1 mg/kg (fig. 12). figure 13 presents the residue of cypermethrin detected in the various watermelon samples. cypermethrin was found in all the watermelon sam­ fig. 8 ­ pesticide residue level of dicofol in watermelon samples. fig. 12 ­ pesticide residue level of permethrine in watermelon samples. fig. 11 ­ pesticide residual level of deltamethrine in watermelon samples. fig. 10 ­ pesticide residue levels of profenophos in watermelon samples. fig. 9 ­ pesticide residue level of lambda­cyhalothrin in water­ melon samples. adv. hort. sci., 2021 35(2): 119­127 126 ples and were all below the european union mrl of 0.2 mg/kg. the concentration of cypermethrin varied from 0.008 mg/kg to 0.08 mg/kg. effects of frequency of pesticide application and pre‐ harvest interval on pesticide residue level in the watermelon fruits. in figure 14 the effect of the frequency of applica­ tion of dimethoate on pesticide residue detected in watermelon fruits are shown. the watermelon fruit samples that received one application of dimethoate in every week had the highest concentration (1.5 mg/kg). the application of pesticides once every month had the lowest residue with 0.8 mg/kg. frequency of application has great impact on the pesticide residue found in the watermelon fruits. this shows that before the dose of one application is neu­ tralised another dose is applied again. this might be one of the main reasons why some of the watermel­ on fruits were highly contaminated with pesticides. in figure 15 the effect of observing pre­harvest interval on pesticide residue in watermelon fruits are shown. pre­harvest interval, which is the period between the time of application and period of har­ vest for the consumption of plant product, is recom­ mended for each and every pesticide used on crops. the highest residue of dimethoate was found in fruits with pre­harvest interval 1 to 5 days (1.8 mg/kg) while the lowest residue of dimethoate was observed in the watermelon samples with pre­harvest interval of 21 days above (0.7 mg/kg). most of the chemical products used by farmers on watermelon have pre­ harvest interval of three to four weeks. islam and haque (2018) reported the importance of observing proper pre­harvest interval as it reduces the pesticide residue. therefore, pre­harvest intervals have a great influence on the residue concentration of fruits and vegetables. 4. conclusions few pesticides residues were detected in all the watermelon samples analysed for pesticides residue the study and it was an indication that most of the fruits were contaminated with pesticides such as dimethoate, profenophos, dicofol, cypermethrine, lambda­cyhalothrin, permethrin and deltamethrin. the residue level of most these insecticides were found to be above the recommended mrl. the fre­ quency of pesticide application and the pre­harvest interval were found to have influence on the pesti­ cide residue concentration in the watermelon fruits from the study area. the study further revealed that most of the respondents were illiterate and has less experience in watermelon production. base on the findings from the study the following recommenda­ tion are made: ­ farmers observe a pre­harvest interval of above 21 days to avoid high residue concentration in the watermelon fruits. ­ farmers to use cypermethrin and lambda­ cyhalothrin in the production of watermelon because they are easily degradable by the heat. fig. 13 ­ pesticide residual level of cypermethrin in watermelon samples. fig. 15 ­ effect of pre­harvest interval on the pesticide residue fig. 14 ­ effect of the frequent application of pesticide on pestici­ de residue in watermelon fruit. jassey et al. ‐ pesticide residue in watermelon fruits 127 further research on other fruits and vegetables grown in the country should be conducted by encouraging the student to take up such research topics. farmers should be educated and encouraged on other alternative pest control methods by setting up demonstration plot beside farmer field to demon­ strate effectiveness of other methods. the extension workers and farmers must be trained on the judicious use of pesticide through farmer field schools initiatives. the promotion of the use and production of non­per­ sistent pesticides and put more tax on persistent pes­ ticides to reduce its importation responsible sectors/agency to put strict measures in the illegal entry of pesticide by closely monitoring the entry points and pesticide vendors. pesticides residue regulation policy should be formu­ lated and make sure it is strictly implemented by the responsible agencies. the laboratory of the national research institute and the university of the gambia be equipped with all relevant equipment to facilitate research in the coun­ try. references adojutelegan o., adereti f., makanju t., olorunfe­ mi o., 2016 ­ analysis of factors affecting watermelon production in ekiti state, nigeria. ­ science, technology and arts research journal, 4(2): 324. adeoye i.b., usman j.m., badmus m.a., 2011 ­ economic analysis of watermelon based production system in oyo state, nigeria. ­ arpn journal of agricultural and biological science, 6(7): 53­59. chamo a., sabo b.b., karaye a., rabiu a., 2016 ­ food and agricultural organisation statistics (faostat (2001) ­ global advanced research journal of agricultural science, 5(12): 432­439. european commision, 2016 ­ eu pesticides database. ­ 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j., idowu i., 2017 ­ assessment of pesticide residue levels in com‐ mon fruits consumed in lagos state, nigeria. ­ journal research and review in science, 4(1): 56­62. padmajani m.t., aheeyar m.m.m., bandara m.m.m., 2014 ­ assessment of pesticide usage in up‐country vegetable farming in sri lanka. ­ hector kobbekaduwa agrarian research and training institute, colombo, sri lanka. harti, research report, no. 164, pp. 56. reetu v., tomar m., 2017 ­ watermelon: a valuable hor‐ ticultural crop with nutritional benefits. ­ popular kheti, 5(2): 5­9. shrefler j., brandenerger l.p., rebek e.j., damicone j., taylor m., 2015 ­ watermelon production. ­ oklahoma state university, hla­6236: 1­6. wanwimolruk s., kanchanamayoon o., boonpan­ grak s., prachayasittikul v., 2015 ­ food safety in thailand 1: it is safe to eat watermelon and durian in thailand. ­ environ. health and preventive medicine, 20(3): 204­215. impaginato 287 adv. hort. sci., 2020 34(3): 287­300 doi: 10.13128/ahsc­7415 genetic diversity, population structure, and relationships among wild and domesticated almond (prunus spp.) germplasms revealed by issr markers s. rahimi­dvin 1, a. gharaghani 1, 2 (*), a. pourkhaloee 3 1 department of horticultural science, college of agriculture, shiraz university, shiraz, iran. 2 drought research center, college of agriculture, shiraz university, shiraz, iran. 3 department of horticultural science, college of agriculture, vali‐e‐asr university of rafsanjan, rafsanjan, iran. key words: cluster analysis, gene diversity, gene flow, population structure, wild almond. abstract: the use of diverse almond genetic resources to expand the genetic bases of commercial cultivars is important for almond breeders. iran is within the center of origin for almond and enjoys a huge diversity of wild species and local cultivars of this important nut crop. despite some reports, there is still a critical need to collect comprehensive information on the genetic diversity of almond germplasm in iran. this study was conducted to evaluate the genetic diversity, structure, and relationships among a total of 75 individuals from 10 populations of 4 wild and cultivated almond species by using 12 inter­simple sequence repeat (issr) primer pairs. a total number of 353 dna fragments were obtained of which 352 were polymorphic (99.69%). the average of poly­ morphism information content (pic), marker index (mi), and resolving power (rp) were 0.932, 27.211, and 7.882, respectively which indicated high discrimi­ natory power of markers. gene flow between wild and cultivated gene pools is shown to be moderate to high (nm = 2.7607), which verifies the hypothesis of low genetic differentiation among populations. cluster analysis based on unweighted pair­group, classified individuals into 7 major gene pools which showed the entire provenances were divided into 7 main groups. overall high levels of genetic diversity were confirmed and useful information obtained on the differentiation and genetic structure of the studied almond germplasms. future evaluation on morphological and physiological aspects, is necessary to identify the most promising individuals to be used directly in afforestation, landscape development as well as nut and oil production or indirectly in future almond and stone fruits breeding programs. 1. introduction almond [prunus dulcis (l.) batsch] belongs to the rosaceae family and (*) corresponding author: agharghani@shirazu.ac.ir citation: rahimi­dvin s., gharaghani a., pourkha­ loee a., 2020 ­ genetic diversity, population structure, and relationships among wild and domesticated almond (prunus spp.) germplasms revealed by issr markers. ­ adv. hort. sci., 34(3): 287­300 copyright: © 2020 rahimi­dvin s., gharaghani a., pourkhaloee a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 november 2019 accepted for publication 25 june 2020 ahs advances in horticultural science https://www.researchgate.net/profile/ali_pourkhaloee https://www.researchgate.net/institution/vali-e-asr_university_of_rafsanjan https://www.researchgate.net/institution/vali-e-asr_university_of_rafsanjan https://www.researchgate.net/institution/vali-e-asr_university_of_rafsanjan http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 287­300 288 is one of the most important nut crops in the world which is known for its high nutritional value. almond domestication occurred nearly 5000 years ago in the fertile crescent (velasco et al., 2016). united states of america, spain, iran, italy, turkey, tunisia, morocco, syria, greece and australia are the ten major producers of almond (ardjmand et al., 2014). iran is the fifth world producer of almond (gharaghani et al., 2017) which produced approxi­ mately 2.99% of the total world production of culti­ vated almonds (sorkheh et al., 2016). due to the narrow genetic background of com­ mercial cultivars, breeding programs of almond face many challenges. in modern plant breeding, native plants are considered as valuable gene pools for crossing programs which can be used to introduce new traits into commercial relatives. wild almond species are found in the mountains and deserts of central asia from western china to iran and turkey (rahemi et al., 2012). wild almond species could be valuable gene pools for breeding purposes due to late bloom, early maturity, adaption to drought and salinity, resistance to winter lower temperatures, reduced insect infestation and fungal attacks (gharaghani et al., 2017). thus, knowledge about genetic diversity of wild genetic resources of almond is an essential prerequisite for involvement of native germplasm in almond breeding programs. on the other hand, assessment of genetic diversity and pop­ ulation structure is necessary to evaluate the existing levels of genetic variability and its patterns of distrib­ ution among the local populations, which is consid­ ered as a guarantee for conservation management of natural populations (cohen et al., 1991; sreekanth et al., 2012). iran is a center for genetic diversity of almond and nearly twenty wild species of almond have been reported from arid and semi­arid regions of this country (sorkheh et al., 2009). different regions of iran have variable environmental conditions includ­ ing subtropical climate in the south, temperate in the north, and extended deserts in the middle which helps the distribution of wild species such as almonds. wild almond germplasm forms the main part of distributed plant species in the mountainous and plain sub­regions of ecological zones in the zagros of iran where the annual precipitation rate is more than100 mm (sabeti, 1994). almond stands of the irano­turanian region have been observed in badamak, mohammadabad maskun and badameshk forests in fars, kerman and south khorasan province of iran, respectively (talebi et al., 2013). fars and charmahal­o­bakhtiari provinces cover parts of the central and southern zagros where prunus scoparia (spach) c.k. schneid., p. elaeagnifolia spach., and p. eburnean spach. are widely distributed (gharaghani et al., 2017). owing to some traits such as leaf shedding during hot seasons, and the remarkable capability of roots in water absorption, some of the wild almond species can resist draught (madam et al., 2011). p. scoparia is a potentially multi­purpose wild almond species in iran which has the potential to become the crop of choice for soil stabilization and landscape in arid and semi­arid areas (mozaffarian, 2005). it has been used as a dwarfing rootstock for almond for centuries (gharaghani and eshghi, 2015). p. scoparia is a potential source of vegetable oil for human nutrition and health with relatively higher oxidative stability, higher unsaturated to saturated fatty acids ratio, cal­ culated oxidisability value, total tocopherols and phe­ nolics contents, and unsaponifiable matter contents, than those of olive oil (farhoosh and tavakoli, 2008). zedu gum is exuded from the bark of p. scoparia, and its kernel oil are used in iranian traditional medicine (zargari, 1997). zedu gum is also being used as emul­ sifier in cosmetic and textile industries (rahimi et al., 2013). these species lie among the rare trees which naturally grow in barren soils (ali et al., 2015). p. elaeagnifolia has been used as a rootstock for plum (gholami et al., 2010) in drought conditions in iran and some other species have also been used as root­ stocks for almond and peach by ancient iranians in arid lands (denisov, 1988). grafting nectarine on wild almond trees as rootstock has also been reported by alberghina in 1978. wild almond species have also been used to afforest barren lands and to protect vegetative cover (mardani, 2006). dna­based molecular markers are important tools to study genetic variation in population genetics. various molecular markers including random ampli­ fied polymorphic dna (rapd), amplified fragment length polymorphism (aflp), simple sequence repeat (ssr), inter­simple sequence repeat (issr), and single nucleotide polymorphism (snp) have been previously used to describe genetic diversity and structure in the genus prunus (martins et al., 2003; shiran et al., 2007; sorkheh et al. , 2007; wu et al. , 2008; bouhadida et al., 2009; rahemi et al., 2012). among different dna markers, issrs have greater reliability and reproducibility in comparison with rapd system, as well as the lower cost of the analyses than aflp rahimi‐dvin et al. ‐ almond germoplasm identification by issr markers 289 and ssr (rodrigues et al., 2013). moreover, issr markers seem to be especially useful to study closely related individuals which show low levels of polymor­ phism (zietkiewicz et al., 1994). local wild species make up an excellent source of genetic diversity which can be used for crop improve­ ment and breeding programs (khadivi­khub and anjam, 2014). despite some reports, there is still a critical need to collect more information on the genetic diversity of almond germplasm in iran. because of high value of p. scoparia, it is important that the necessary steps be taken to comprehensive­ ly evaluate, utilize and ensure the conservation of this unique wild species. the purpose of our study was to study the genetic diversity and population structure of a collection of wild and cultivated almond populations in iran using issr markers. the emphasis of this study is on the populations of p. sco‐ paria collected from different sites in central and southern zagros regions, experiencing less natural precipitation and higher temperature comparing to other natural habitats of this species in iran. these special climatic condition made these wild popula­ tions a promising source of genes evolved for drought and high temperature tolerance, which will be very valuable in facing harsh effects of climate change. we also sought to compare the diversity and illustrate the relationships of these populations with some populations of three other almond species including p. elaeagnifolia, p. eburnea and p. dulcis (common almond) from the same geographical region, to put more shed on the possible gene flow among them as well as to detect the footprint of these species in the genetic background of cultivated almond. the results of this study are useful for con­ servation of these wild stands as well as for decision making on direct or indirect utilization of them for afforestation, nut and oil production, landscape pur­ pose and through breeding programs. 2. materials and methods field sampling to detect higher genetic diversity (resulting from cross­pollination in natural habitat of the plant mate­ rials used herein) as well as the feasibility of plant materials collection (seeds instead of leaf samples) we chose to use raised seedling populations instead of natural populations in this study. in total, seeds of 72 wild almond trees were sampled from southern and central regions of zagros mountain in iran during late spring to early summer of 2014. these regions are placed in fars and chaharmahal­and­bakhtiari provinces. the studied genotypes belong to p. sco‐ paria and p. eburnean in section spartioides spach. as well as p. elaeagnifolia and p. dulcis in section euamygdalus spach (kester and gradziel, 1996). in addition, seeds of 5 almond cultivars were sampled. characteristics of the populations (collection sites, latitude, longitude, altitude, etc.) are listed in table 1. plant materials and dna extraction the seeds of all species were mechanically scari­ fied and then soaked in water for 24 h. they were mixed with perlite and stratified at 4±1°c for 45 days. after stratification, nuts were directly sown in 5 kg pots filled with a mixture of fine sand, soil and leaf mold. the pots were then transferred to the green­ house with an average temperature of 26±3°c under daylight illumination conditions i.e. 800 μmol m­2 s­1 about 10 hours. in december 2017 a total of 75 seedlings (each seedling represents an individual tree in natural habitat) comprised 10 populations (3 to 11 individuals per population) were selected. stem pieces (200 mg) for each individual were collected into aluminum foil, immediately snap­frozen in liquid nitrogen and stored at ­80°c until the dna was extracted. total genomic dna was isolated following the cetyltrimethylammonium bromide (ctab) proto­ col with minor modifications (doyle and doyle, 1987). dna quantity and quality were determined by spectrophotometry and visual comparison of dna electrophoresed on 1% agarose gel. issr genotyping in total, 12 issr primer pairs were selected based on literature review (carvalho et al., 2002; martins et al., 2003; dje et al., 2006; zhao et al., 2007; oliveira et al., 2010; moulin et al., 2012; ahmed et al., 2013; muraseva et al., 2018) and synthesized (by metabion, germany). polymorphism of markers was first tested in a subset of samples and then polymerase chain reac­ tion (pcr) conditions were optimized. the list of primers and their information are presented in table 2. the pcr mix contained 10 ng template dna, 10 pmol of primer in a final 20 μl reaction volume. conditions of the pcr amplification were as follows: 94°c (3 min), then 35 cycles at 94°c (45 s) / 38­61°c (varied for each primer according to table 2) (45 s) / 72°c (1 min) and final extension at 72°c for 7 min. the amplified products were separated by 1% (w/v) agarose gel electrophoresis in 1× tbe buffer at con­ stant voltage (100) for 45 min, stained with adv. hort. sci., 2020 34(3): 287­300 290 table 1 ­ list of the studied genotypes with indication of their regions and geographical coordinates of the collection sites species no. population e n altitude (m) prunus scoparia 1 shiraz 1 52 34.558 29 37.082 1535 2 shiraz 2 52 35.822 29 44.275 1820 3 shiraz 3 52 35.784 29 44.287 1816 4 shiraz 4 52 35.811 29 44.269 1819 5 shiraz 5 52 34.601 29 37.103 1569 6 shiraz 6 52 34.449 29 37.465 1565 7 shiraz 7 52 33.704 29 37.946 1549 8 shiraz 8 52 34.586 29 39.490 1670 9 shiraz 9 52 32.642 29 40.263 1728 10 shiraz 10 52 35.789 29 44.353 1821 11 shiraz 11 52 35.785 29 44.351 1818 p. scoparia 12 nourabad 3 51 20.875 30 4.695 1179 13 nourabad 4 51 39.730 29 48.513 934 14 nourabad 5 51 32.377 30 1.167 1086 15 nourabad 6 51 23.931 30 0.745 1285 16 nourabad 7 51 39.823 29 48.605 946 17 nourabad 8 51 21.011 30 6.513 1183 18 nourabad 9 51 31.694 30 1.252 1067 19 nourabad 10 51 58.427 30 01 08.4 1592 p. scoparia 20 marvdasht 1 52 54.983 30 3.162 1728 21 marvdasht 2 52 54.800 30 6.249 1730 22 marvdasht 3 53 00.807 30 6.800 1812 23 marvdasht 4 53 12.117 30 5.742 1837 24 marvdasht 5 53 10.524 30 1.617 1828 25 marvdasht 6 53 12.643 29 59.116 1803 26 marvdasht 7 53 14.080 29 57.712 1765 27 marvdasht 8 53 6.493 29 48.754 1667 28 marvdasht 9 53 6.535 29 48.816 1663 29 marvdasht 10 53 8.662 29 47.443 1640 p. scoparia 30 firuzabad 2 52 32.509 29 8.712 1725 31 firuzabad 3 52 34.486 28 58.157 1503 32 firuzabad 4 52 33.874 28 57.406 1530 33 firuzabad 5 52 32.400 28 55.816 1445 34 firuzabad 6 52 38.719 29 5.885 1917 35 firuzabad 7 52 23.202 28 53.222 1524 36 firuzabad 8 52 38.310 29 3.808 1732 37 firuzabad 9 52 32.801 29 9.124 1763 38 firuzabad 10 52 41.274 28 13.319 1275 39 firuzabad 11 ­­­­­ ­­­­­ 1578 p. scoparia 40 mian jangal fasa 1 52 46.117 29 26.327 1481 41 mian jangal fasa 2 52 50.130 29 19.653 1526 42 mian jangal fasa 3 ­­ ­­ 2187 43 mian jangalfasa 4 53 24.351 29 9.542 1729 44 mian jangal fasa 5 53 23.894 29 9.939 1754 45 mian janga lfasa 6 53 26.033 29 7.617 1720 46 mian jangal fasa 7 53 26.054 29 7.632 1716 47 mian jangal fasa 8 53 24.138 29 9.082 1756 48 mian jangal fasa 9 53 22.759 29 10.821 1815 49 mian jangal fasa 10 53 19.277 29 12.351 1825 to be continued... rahimi‐dvin et al. ‐ almond germoplasm identification by issr markers 291 power (rp) of each primer was calculated as rp = ∑ib, where ib shows the informative fragments. the ib may be shown on a scale of 0/1 by the following for­ mula; ib = 1 ­ (2 × |0.5 ­ pi|) where pi is the propor­ tion of populations containing the ith band (prevost and wilkinson, 1999). based on issr bands identified in the individuals, some basic parameters for genetic diversity including the total number of bands (tnb), the number of poly­ morphic bands (npb), the percentage of polymorphic bands (ppb), mean nei’s gene diversity index (h), shannon’s information index (i), the observed number of alleles per locus (na), the effective number of alle­ les per locus (ne), the level of gene flow (nm), popu­ lation diversity (hs), the total gene diversity (ht), inter­population differentiation (gst), genetic identity and genetic distance were calculated for each popula­ tion using software popgene 1.32 (yeh et al., 1999). private bands (referring to the bands found only with­ in one population) and major allele frequency were estimated by power marker software. to illustrate the relationship among populations, simplysafe (eurx, poland) and photographed with uv light (nade gel documentation and analysis system js­6800, china). the size of produced frag­ ments was defined according to size marker (fermentas, germany). data analysis marker results (reproducible distinct bands with high resolution) were dominantly scored in a data matrix. the matrix was used for calculation of popu­ lation genetic variation indices. the informativeness of primer pairs in genotyping and subsequent evaluation of genetic diversity and population structure was compared using the poly­ morphism information content (pic), effective multi­ plex ratio (emr), marker index (mi), and resolving power (rp). for each primer, the polymorphic infor­ mation content (pic) was estimated by powermarker v3.25 (liu and muse, 2005). marker index for each primer was calculated as a product of polymorphic information content and effective multiplex ratio: mi = emr * pic (varshney et al., 2007). the resolving table 1 ­ list of the studied genotypes with indication of their regions and geographical coordinates of the collection sites species no. population e n altitude (m) p. scoparia 50 eqlid 3 52 40.003 30 15.126 1701 51 eqlid 4 52 38.310 30 16.346 1742 52 eqlid 5 52 36.405 30 18.062 1800 53 eqlid 6 52 35.080 30 22.196 2321 54 eqlid 7 52 23.051 30 19.324 1843 55 eqlid 8 52 23.764 30 19.060 1750 56 eqlid 9 52 24.085 30 18.382 1715 p. scoparia 57 lordegan 1 51 11.609 31 33.619 1752 58 lordegan 2 51 13.020 31 34.354 1962 59 lordegan 3 ­­ ­­ 1948 60 lordegan 4 ­­ ­­ 1963 p. elaeagnifolia 61 p. elaeagnifolia 1 52 35.806 29 44.098 1801 62 p. elaeagnifolia 2 52 35.746 29 44.158 1804 63 p. elaeagnifolia 3 ­­ ­­ 2128 64 p. elaeagnifolia 4 ­­ ­­ 2570 65 p. elaeagnifolia 5 52 34.880 30 22.732 2458 66 p. elaeagnifolia 6 52 34.898 30 22.748 2455 p. eburnea 67 p. eburnea2 52 22.173 30 19.865 1912 68 p. eburnea3 53 24.368 29 09.592 1732 69 p. eburnea4 ­­ ­­ 2280 70 p. eburnea5 52 24.585 30 18.376 1711 71 p. eburnea7 52 22.130 30 19.854 1902 72 p. eburnea8 53 24.101 29 9.071 1763 p. dulcis 73 mamaei ­­­ ­­­ 1910 74 ferragnes ­­­ ­­­ 1910 75 badam talk ­­­ ­­­ 1910 292 adv. hort. sci., 2020 34(3): 287­300 an unweighted pair group method with arithmetic mean (upgma) dendrogram was constructed based on nei’s genetic distance using popgene 1.32 (yeh et al., 1999). the dendrogram was generated using treeview program. to further understand the relationships among populations, a bayesian clustering­based structure analysis was performed on the entire data set using structure 2.3.4 (pritchard et al., 2000) to reveal the number of genetic pools. two runs of analysis using the admixture model were performed. initial runs were performed with a burn­in length of 50000 and 750000 mcmc (markov chain monte carlo) repli­ cates for 10 times at each k from 1 to 10. the proba­ ble number of groups was estimated. the second run was 100000 for burn­in length and 300000 for mcmc replicates, 10 times for each k. to estimate the best k value. the evanno test was performed on struc­ ture results using ‘‘structure harvester’’ (evanno et al., 2005). the results were summarized in a bar plot using distruct (rosenberg, 2004). 3. results informativeness of markers the mean of pic values was analyzed for all loci to evaluate markers efficiency. pic value ranged from 0.845 (primer 4) to 0.973 (primer 1). the mean pic value for all loci was 0.932. the highest emr value of 38 (primer 1) and the lowest of 20 (primer 12), with an average emr value of 29.25 per primer were obtained. the highest (36.97) and the lowest (19) mi values were observed with primers 1 and 12, respec­ tively. the mean mi value was 27.211 per primer. the highest rp value was observed with primer 1 (13.183) and the lowest with primer 4 (3.518) with an average rp of 7.882 per primer (table 2). genetic diversity the 75 individuals of wild and domesticated almond assigned to 10 populations and were ampli­ fied with 12 selected primers (table 2). a total of 353 bands were scored with an average band number of 29.33 per primer across 75 individuals. among the 353 bands, 352 bands (99.69%) were polymorphic. the percentage of polymorphic bands (ppb) varied from 96.29% for primer 3 to 100% for the other primers (table 2). at the population level, ppb ranged from 18.70% in p. dulcis to 58.07% in p. scoparia (shiraz and firuzabad populations) with a mean value of 49.66% (table 3). the na ranged from 19.63 for issr 3 to 20.00 for other issrs. across the populations, na ranged from 11.870 for p. dulcis to 15.807 for p. scoparia (shiraz and firuzabad populations). the ne ranged from 11.453 for issr 4 to 14.254 for issr 5 with an average of 13.003 alleles per locus. across the populations, ne y = (c, t); d = (a, g, t); v = (a, c, g); b = (c, g, t); r = (a, g) rp= resolving power; pic= polymorphism information content; mi= marker index; emr effective multiplex ratio; maf= major allele frequency. table 2 ­ issr primers used in this study and their results primers primer sequences (5’­3’) tm total number of alleles (a) number of polymorphic alleles (b) % polymorphism (b/a)*100 pic mi emr maf rp 1 gac aga cag aca gac a 48 38 38 100 0.973 36.97 38.00 0.120 13.183 2 gtg cgt gcg tgc gtg c 58 30 30 100 0.952 28.56 30.00 0.173 4.932 3 gtg gtggtggtggtg­ 61 27 26 96.29 0.948 23.72 25.03 0.186 8.132 4 ctc tct ctc tct ctc ttg 54 27 27 100 0.845 22.68 27.00 0.373 3.518 5 gag aga gag aga gag 50 23 23 100 0.953 21.85 23.00 0.173 9.160 6 cac caccac gc 38 33 33 100 0.947 31.02 33.00 0.200 9.946 7 aca cac aca cac aca 54 23 23 100 0.855 19.55 23.00 0.360 4.889 8 gaa gaagaagaagaa­ 50 28 28 100 0.958 26.60 28.00 0.173 8.172 9 gtc gtcgtcgtcgtcgtc 61 32 32 100 0.873 27.84 32.00 0.333 4.692 10 gag aga gag aga cc 44 35 35 100 0.961 33.60 35.00 0.160 12.177 11 bdb aca acaacaacaa­ 49 37 37 100 0.956 35.15 37.00 0.160 9.531 12 yhy gtg tgt gtg tg 42 20 20 100 0.959 19.00 20.00 0.133 6.252 min. ­­­ ­­­ 20 20 96.29 0.845 19.00 20.00 0.120 3.518 max. ­­­ ­­­ 38 38 100 0.973 36.97 38.00 0.373 13.183 means ­­­ ­­­ 29.41 29.33 99.69 0.932 27.21 29.25 0.212 7.882 total ­­­ ­­­ 353 352 ­­­ ­­­ 326.54 351.03 2.544 ­­­ rahimi‐dvin et al. ‐ almond germoplasm identification by issr markers 293 ranged from 11.496 for p. dulcis to 13.161 for p. sco‐ paria (lordegan population) (tables 3 and 4). across the populations, the highest values of i (0.2816) and h (0.1838) indexes were observed for p. scoparia (eqlid populations). however, p. dulcis showed the lowest i (0.1190) and h (0.0831) values (table 3). however, for studied accessions, the average values of na and ne were 14.966 and 12.722, respectively. genetic similarity and cluster analysis among popula‐ tions the dendrogram derived from upgma cluster analysis was generated for all populations (fig. 1). among seven distinct groups obtained by dendro­ gram, four groups represent populations of p. sco‐ paria. the group i consisted of two populations of p. scoparia (shiraz and mian jangal­e­fasa) collected from fars province. the group ii was composed of the other three populations of p. scoparia( nourabad, marvdasht, and firuzabad) sampled from fars province. the eqlid population of p. scoparia table 3 ­ genetic diversity within the populations of almond in iran exhibited by inter simple sequence repeat (issr) population no. observed no. of alleles (na) effective no. of alleles (ne) shannon’s in formation index (i) nei’s genetic diversity (h) percentage of polymorphic loci (ppb) no. bands no. private bands shiraz 11 15.807 12.853 0.2749 0.1774 58.07 205 7 nourabad 8 15.212 12.657 0.2570 0.1664 52.12 184 3 marvdasht 10 15.581 12.723 0.2653 0.1708 55.81 197 6 firuzabad 10 15.807 12.812 0.2737 0.1759 58.07 205 11 mian jangal fasa 10 15.666 12.879 0.2738 0.1774 56.66 200 8 eqlid 7 15.524 12.968 0.2816 0.1838 55.24 195 12 lordegan 4 14.419 13.161 0.2652 0.1817 44.19 157 1 p. elaeagnifolia 6 14.703 12.577 0.2453 0.1607 47.03 167 6 p. eburnea 6 15.071 13.099 0.2778 0.1854 50.71 179 8 p. dulcis 4 11.870 11.496 0.1190 0.0831 18.70 66 0 mean 7.5 14.966 12.722 0.2533 0.1662 49.66 fig. 1 ­ upgma dendrogram on the basis of nei’s (1978) evalua­ tion of genetic distance among 10 populations of almond. table 4 ­ a summary of genetic parameters across inter­simple sequence repeat loci locus na ne i h ht hs gst nm 1 20.00 13.759 0.3872 0.2418 0.2412 0.2034 0.1568 2.6884 2 20.00 11.847 0.2547 0.1408 0.1427 0.1197 0.1615 2.5953 3 19.630 13.564 0.3529 0.2244 0.2273 0.1959 0.1385 3.1111 4 20.00 11.453 0.2178 0.1165 0.1183 0.0974 0.1771 2.3231 5 20.00 14.245 0.4103 0.2623 0.2538 0.2088 0.1774 2.3181 6 20.00 13.447 0.3652 0.2247 0.2234 0.1939 0.1319 3.2900 7 20.00 12.437 0.3061 0.1780 0.1775 0.1522 0.1428 3.0022 8 20.00 13.268 0.3392 0.2095 0.2092 0.1726 0.1751 2.3559 9 20.00 11.649 0.2255 0.1237 0.1234 0.1063 0.1387 3.1053 10 20.00 14.034 0.3857 0.2474 0.2426 0.2125 0.1244 3.5201 11 20.00 12.712 0.2869 0.1739 0.1761 0.1426 0.1903 2.1274 12 20.00 13.630 0.3593 0.2261 0.2216 0.1904 0.1408 3.0504 mean 19.90 13.003 0.3242 0.1974 0.1964 0.1663 0.1546 2.7906 na= observed number of alleles; ne= effective number of alleles; i= shannon’s information index; nei’s genetic diversity; ht= total gene diversity; hs= population diversity; gst= inter­population differentiation; nm= estimate of gene flow. adv. hort. sci., 2020 34(3): 287­300 294 sampled in north of fars province was separated in the group iii. the group iv comprised lordegan popu­ lation of p. scoparia sampled from charmahal and bakhtiari province in central zagros region. groups v, vi and vii included populations of p. eburnea, p. dul‐ cis, and p. elaeagnifolia, respectively. the nei’s genetic distance ranged from 0.0077 to 0.0452 and genetic identity ranged from 0.9558 to 0.9923 (table 5). the genetic identity between nourabad and firuzabad populations of p. scoparia was 0.9923 having the closest genetic relationship; however, the farthest genetic identity was 0.9558 between lordegan population of p. scoparia and p. elaeagnifolia populations. population structure the amount of gene flow (nm) among popula­ tions was 2.7607, showing the moderate to high gene flow among populations studied herein. the genetic diversity within populations (hs) and the total genetic diversity (ht) of the species were 0.1663 and 0.1964, respectively (table 6). the genetic differentiation among the populations (gst) was 0.15 which shows that 15% of the total genetic variability was among populations and 85% was within populations. the results of the structure analysis with issr markers are presented in figure 2. the structure plot suggested a lack of definite structure although table 5 ­ genetic identity (above diagonal) and genetic distance (below diagonal) estimates between populations across all loci based on nei (1978) population shiraz nourabad marvdasht firuzabad mian jangal eqlid lordegan prunus elaeagnifolia prunus eburnea prunus dulcis shiraz **** 0.9862 0.9898 0.9899 0.9896 0.9773 0.9774 0.9693 0.9715 0.9722 nourabad 0.0139 **** 0.9910 0.9923 0.9882 0.9806 0.9806 0.9789 0.9792 0.9756 marvdasht 0.0102 0.0091 **** 0.9900 0.9841 0.9868 0.9756 0.9677 0.9709 0.9721 firuzabad 0.0102 0.0077 0.0101 **** 0.9894 0.9835 0.9783 0.9714 0.9777 0.9789 mianjangalfasa 0.0104 0.0119 0.0161 0.0107 **** 0.9749 0.9779 0.9675 0.9725 0.9737 eqlid 0.0230 0.0196 0.0132 0.0167 0.0254 **** 0.9730 0.9600 0.9625 0.9728 lordegan 0.0229 0.0196 0.0247 0.0219 0.0224 0.0273 **** 0.9558 0.9718 0.9567 p. elaeagnifolia 0.0312 0.0214 0.0328 0.0290 0.0330 0.0409 0.0452 **** 0.9713 0.9609 p. eburnea 0.0289 0.0210 0.0296 0.0225 0.0279 0.0382 0.0286 0.0291 **** 0.9560 p. dulcis 0.0282 0.0247 0.0283 0.0213 0.0267 0.0275 0.0442 0.0399 0.0450 **** fig. 2 ­ population structure of almond populations for k = 2 and k = 4, showing a high degree of genotypic admixture among individuals. on the horizontal axis, the following population are illustrated: (1): prunus scoparia (shiraz); (2): prunus scoparia (nourabad); (3): prunus scoparia (marvdasht); (4): prunus scoparia (firuzabad); (5): prunus scoparia (mianjangalfasa); (6): prunus scoparia (eqlid); (7): prunus scoparia (lordegan); (8): prunus elaeagnifolia; (9): prunus eburnea; (10): prunus dulcis. table 6 ­ assessment of the genetic variability among ten populations designated based on the issr analysis ht total gene diversity hs population diversity gst inter­population differentiation nm estimate of gene flow average 0.1964 0.1663 0.1533 2.7607 standard deviation 0.0236 0.0168 ­­­ ­­­­­ evanno’s test indicated that the most informative number of populations was k = 2 and k = 4. using the defined strategies of dna purification with the selected primers, good patterns could be attained for the different accessions under study. instance of patterns of amplification attained by issr rahimi‐dvin et al. ‐ almond germoplasm identification by issr markers 295 in various accessions of almond are shown in figure 3. 4. discussion and conclusions informativeness of markers due to highly variable nature and less investment in time and money than other marker systems, issr markers are widely used in population genetic stud­ ies (harris, 1999). moreover, matesanz et al. (2011) reported that because of high polymorphism, only a few issr loci (as few as five to seven primer pairs) are enough to obtain reliable information on genetic diversity of populations. the efficiency of a molecular marker system in distinguishing genotypes depends largely upon the polymorphism it can discover (guo et al., 2014). on the basis of high pic values, mi, and rp we conclude that issr markers used in this study were informative in the assessment of genetic diversity of almond accessions. the high pic values with a mean of 0.932 show that all primers are informative, and this can be related to high genetic variation among accessions used in this research. similar results were reported for sour cherry and prunus mira (najafzadeh et al., 2014; tian et al., 2015). the variation may have been contributed by gene flow, natural hybridization, propagation by seed and human selection (sefc et al., 2000). the rp and mi measurements show distribution and number of alleles (bands) within the studied genotypes. bands that are scored in the half of geno­ types would possess optimal discriminatory power and with an increase in the number of bands, the rp of a particular primer pair will be increased (kayis et al., 2010). therefore, primers with the highest pic, emr, mi, and rp values (issr1, issr10, and issr11) were generally the most effective in distinguishing between accessions and could be further used in almond genetic diversity studies. the similar results are reported in, prunus genus, sweet cherry, and sour cherry (yılmaz et al., 2009; ganopoulos et al., 2011; najafzadeh et al., 2014). genetic diversity information on genetic diversity and structure of wild almond populations is essential for their conser­ vational programs. moreover, narrow genetic back­ ground of the commercial cultivars of the genus prunus restricts their cultivation in new regions with different environmental conditions. therefore, genetic diversity among populations of this genus can be used to broaden the genetic background of com­ mercial scion and rootstock cultivars and to over­ come their distribution across different regions (gradziel et al., 2001). genetic variation depends on many factors such as mating system, genetic drift, gene flow, human activities, long­term evolutionary history, natural selection, and breeding systems (schaal et al., 1998; hamrick and godt, 1996).populations of domesticat­ ed almonds used in this study possess restricted number of individuals and often are reproduced veg­ etatively. thus, cultivated almonds shows lower lev­ els of genetic diversity than the other species. however, higher genetic diversity was observed in certain individuals and populations of the wild almonds. this phenomenoncould be expounded by the fact that are propagated sexually whereas indi­ viduals of cultivated almonds aremainly reproduced asexually. the ppb is a major genetic diversity index that showed high levels of genetic diversity among almond genotypes. similar great genetic diversity fig. 3 ­ a) issr banding pattern generated using primer 11. lane 1­14, eqlid 7, mian jangal fasa 6, marvdasht 6, eqlid 4, nourabad 4, firuzabad 2, p. elaeagnifolia 4, nourabad 5, p. elaeagnifolia 2, nourabad 10, shiraz 8, nourabad 9, shiraz 2, shiraz 6. b) issr banding pattern generated using primer 10. lane 1­15, shiraz 3, shiraz 4, marvdasht 8, nourabad 7, lordegan 1, mamaei, marvdasht 4, shiraz 9, badam talk, shiraz 7, mian jangal fasa 3, mian jangal fasa 1, eqlid 9, marvdasht 3, mian jangal fasa 4. m. 10 kb dna ladder. adv. hort. sci., 2020 34(3): 287­300 296 was reported by sorkheh et al. (2017) in wild almond. the obtained ppb in this study was higher than val­ ues reported by kumar et al. (2009) and shuxia (2011) with prunus armeniaca (96.5%) and prunus persica (93%), respectively. also, high genetic varia­ tion was shown by rahemi et al. (2012) in wild almond, being similar to our results. one of the main reasons of the existing genetic variation is the process of self­incompatibility which is controlled by genes (gouta et al., 2010; szikriszt et al., 2011).the high number of generated alleles in our study may be due to use of several different genotypes that pos­ sessed high levels of genetic diversity. the bands generated by each primer rests on the primer, sequence and the diversity size in special genotype (shiran et al., 2007). so, the number of bands differed in various genotypes. the private bands show the existence of special genes or sequences in native populations. the common bands show alleles which are shared among the cultivars studied. thus, the private bands can be used in almond genetic fingerprinting and cultivar recogni­ tion. genetic similarity and cluster analysis among popula‐ tions cluster analysis is widely used to study the genetic relationships among germplasms (li et al., 2010). the upgma dendrogram obtained in this study clearly distinguished species from each other and the clades were in accordance with morphological traits. moreover, all populations were divided into their related taxa. prunus scoparia (shiraz population)which seems to be mainly an artificial (cultivated) population and p. scoparia (mianjangal­e­fasa population) both were separated into the same group. therefore, we assume that some of the p.scoparia stands in shiraz region were developed artificially through seed that may have originated from mianjangal­e­fasa. lordegan population lay in group iv, being closely related to eqlid populations. it may be due to the geographic proximity and climatic resemblance between these two geographical locations. prunus eburnean was grouped in cluster v, close to p. sco‐ paria (lordegan) populations. this close relationship is logical because both of them belong to spartioides section within the genus prunus (kester and gradziel, 1996). moreover, close relationship between domes­ ticated population of almond (p. dulcis) and p. elaeagnifolia could be explained in the same way since they both belong to eu amygdalus section with­ in the genus prunus (kester and gradziel, 1996). genetic proximity between the genotypes or pop­ ulations from different regions, for example nourabad and firuzabad (table 5), could be explained by the geographical proximity of the regions, the exchange of plant material between sites and by the probable existence of common ancestors (el hamzaoui et al., 2014). also, noormohammadial et al. (2013) reported the gene exchange among prunus scoparia populations which is similar to our results. molecular phylogeny results obtained in this study were similar to our findings using nut and kernel morphological characteristics to cluster the same subset of plant materials with some exceptions (rahimi dvin et al., 2017). in that work, we found that p. eburnea and p. scoparia were placed close to each other and p. elaeagnifolia and p. dulcis formed the same clade. the genetic distance among the studied almonds in this experiment is short, indicating that a high capacity for hybridization exists between genotypes and populations. the mating system can greatly affect genetic diversity both within and among popu­ lations. generally, most of the genetic diversity in self­pollinated plants is distributed among popula­ tions, while in out crossed plants such as almond species, most of the genetic diversity is distributed within populations (hamrick, 1989). population structure gene flow is defined as the gene movement with­ in and between populations (lowe et al., 2009). the estimate of gene flow (nm) has been categorized as low (nm<1), moderate (nm>1) and extensive (nm>4) (kumar et al., 2014). the estimate of nm (2.7607) was higher than 1, which indicates that the number of migrants per generation can prevent population differentiation caused by genetic drift. moreover, we conclude that high genetic diversity and lack of differ­ entiation is due to high amount of gene flow. almond is an important food source for both human and ani­ mals and its seeds can be easily transported by birds and nomads (which is very common in the region) increasing the amount of nm. genetic differentiation coefficient is an indicator of genetic diversity and structure of species (zia et al., 2014). it should be noted that rosaceae species usually show low levels of genetic differentiation (fineschi et al., 2005). based on slatkin (1985), nm >1 shows no significant genetic differentiation among populations. in this study, genetic differentiation rahimi‐dvin et al. ‐ almond germoplasm identification by issr markers 297 between populations had an average value of 0.15. similar results were reported by li et al. (2013) who obtained gst of 0.18 with apricot. many factors can influence on genetic differentiation which may occur independently in a population. for example, high dis­ persal rate of seeds must be involved in low genetic differentiation (fanciulli et al., 2000). moreover, gametophytic incompatibility, prevents self­fertiliza­ tion and encourages cross­pollination (weinbaum, 1985) which retains high levels of genetic variability within seedling populations (arulsekar et al., 1986). as a consequence, populations of almonds which possess gametophytic incompatibilityshow low levels of genetic differentiation. the genetic structure shows the history of popula­ tions with respect to their long­term evolution, muta­ tion, recombination, genetic drift, gene flow, and nat­ ural selection (slatkin, 1987; schaal et al., 1998). therefore, providing information on the genetic diversity and structure of a crop is a prerequisite for the conservation and effective use of germplasms available for breeding (laidò et al., 2013). structure results demonstrated a high degree of admixture among individuals across 10 populations, consistent with moderate to high levels of gene flow across populations. our results are similar to those of mendigholi et al. (2013), who showed that the plots of structure exhibited the admixture of population and gene exchange which showed the existence of ancestral gene among prunus scoparia. the lack of population structure and moderate to high gene flow among the species in this study sug­ gests the potential interbreeding among the popula­ tions. nevertheless, a high individual genetic diversity purveys an optimistic prospect for the survival of the declining population with proper management inter­ position. results signified that issr primers which had been used herein had a significant distinctive power for the evaluation of the polymorphism in various almond populations. the obtained results present iranian native almond species as a precious source of genetic diversity and recommends that they are an auspicious source of new genes for rootstock and cul­ tivar breeding programs. results also offer a contri­ bution to the management and conservation of this valuable almond germplasm. since the iranian almond species and genotypes have not been select­ ed for breeding programs, they are more probable to have a further diverse genetic background and may be employed in the selection of various genotypes so as to create new cultivars. it is expected that with extra experiments and analyses on morphological and physiological aspects, the most promising indi­ viduals could be identified and introducedfor direct utilization in afforestation, landscape development as well as nut and oil production or to be used by almond and stone fruits breeders in future breeding programs. references ahmed t.a., al­hadidi s.h., al­qaradawi a.y., radwan o., 2013 ­ determination of inter‐and intra‐specific genetic variations among qatari date palm cultivars using inter simple sequence repeat (issr) markers. ­ african j. biotech., 12(19): 2540­2546. alberghina o., 1978 ­ the wild almond, amygdalus web‐ bii, of south west sicily. ­ tecnica agricola, 30(6): 385­ 393. ali j.j.m., norip i.m., hamap s.j., rashedp s.o., 2015 ­ water harvesting through utilization of wild almond as rootstocks for production of peach, apricot and plum under dry land farming in sulaymaniyah region. ­ int. j. innovative sci., engineering techn., 2(8): 705­724. ardjmand a., piri s., imani a., piri s., 2014 ­ evaluation of morphological and pomological diversity of 62 almond cultivars and superior genotypes in iran. ­ j. nuts, 5(1): 39­50. arulsekar s., parfit d.e., kester d.e., 1986 ­ comparison of isozyme variability in peach and almond cultivars. ­ j. heredity, 77(4): 272­274. bouhadida m., casas a.m., gonzalo m.j., arus p., moreno m.á., gogorcena y., 2009 ­ molecular char‐ acterization and genetic diversity of prunus rootstocks. ­ scientia hortic., 120(2): 237­245. carvalho v.p., ruas p.m., ruas c.f., ferreira j.m., moreira r.m., 2002 ­ assessment of genetic diversity in maize (zea mays l.) landraces using inter simple sequence repeat (issr) markers. ­ crop breeding appl. biotech., 2(4): 557­568. cohen j.i., wiliams j.t., plucknett d.l., shands h., 1991 ­ ex situ conservation of plant genetic resources: global development and environmental concerns. ­ science, 253(5022): 866­872. denisov v.p., 1988 ‐ almond genetic resources in the ussr and their use in production and breeding. ­ fruit breeding, 224: 299­306. dje y., tahi g.c., bi i.z., malice m., baudoin j.p., bertin p., 2006 ­ optimization of issr marker for african edi‐ ble‐seeded cucurbitaceae species’ genetic diversity analysis. ­ african j. biotech., 5(2): 83­87. doyle j.j., doyle j.l., 1987 ­ a rapid dna isolation proce‐ dure for small quantities of fresh leaf tissue.­ phytochemical bulletin, 19: 11­15. el hamzaoui a., oukabli a., moumani m., 2014 ­ morphological and molecular diversity and genetic adv. hort. sci., 2020 34(3): 287­300 298 structure of moroccan cultivated almond (prunus dulcis mill.) beside some foreign varieties. ­ plant genetic resources, 12(3): 308­316. evanno g., regnaut s., goudet j., 2005 ­ detecting the number of clusters of individuals using the software structure: a simulation study. ‐ molecular ecology, 14(8): 2611­2620. fanciulli p.p., melegari d., carapelli a., frati f., dal­ lai r., 2000 ­ population structure, gene flow and evo‐ lutionary relationships in four species of the genera tomocerus and pogonognathellus (collembola, tomoceridae). ­ biol. j. linnean soc., 70(2): 221­238. farhoosh r., tavakoli j., 2008 ­ physicochemical prop‐ erties of kernel oil from amygdalus scoparia growing wild in iran. ­ j. food lipids, 15(4): 433­443. fineschi s., salvini d., turchini d., pastorelli r., ven­ deramin g.g., 2005 ­ crataegus monogyna jacq. and c. laevigata (poir.) dc. 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montecarlo, 81, 51017 pescia (pt), italy. 2 council for agricultural research and economics, research centre for vegetables and ornamental crops, via dei fiori, 8, 51012 pescia (pt), italy. key words: calla lily, cherry laurel, growing media, protea, sustainability. abstract: carried out within the life17env/it/000347­subsed project, this research aimed at investigating the effect of a treated sediment (ts) as sub­ strate component on the quality and marketability of three widespread con­ tainerized ornamental species: cherry laurel (prunus laurocerasus) cv. novìta, calla lily (zantedeschia aethiopica) and protea (protea cynaroides) cv. little prince. the ts was mixed with soilless substrates as sphagnum peat, coir, and bark in different proportion (0%, 25% and 50%). in cherry laurel, the ts used in 25 ­ 50% proportions reduced plant height, slightly altering its attractive vibrant foliage. a positive effect of the ts was evidenced on calla lily, where both tested sediment­based mixtures allowed a copious blooming and flower quality raised as the sediment content increased (ts 50% > ts 25% > ts 0%). post­harvest longevity and colour of flowers were not affected by substrate composition. the effect of sediment­based substrates on protea growth and blooming showed an opposite trend (ts 0% > ts 25% > ts 50%), with plants grown on 50% v/v ts exhibiting a considerable reduction in plant growth and production of flower clusters, with brighter tones turning towards purple. based on sale values, the ts proved to be a sustainable alternative for the production of potted ornamen­ tals if properly mixed with other organic matrixes, such as peat and coir. 1. introduction the ornamental plant industry in tuscany is a strategic sector, repre­ senting approx. 5% of the entire national agricultural production, which derives for about 75% from potted plants and nurseries (trees and shrubs) and for the remainder from fresh cut flowers and foliages (martorana, 2021). this segment produces a giant assortment of ornamental and flow­ er species in about 6,500 hectares of land, concentrated in the provinces of pistoia and lucca. with over 3,300 nursery enterprises, 2,060 of which are ornamental producers and 1,900 flower growers, this sector has a (*) corresponding author: stefania.nin@crea.gov.it citation: castellani m., bonetti d., antonetti m., prisa d., burchi g., nin s., 2023 ­ treated sedi‐ ment as substrate component of three containeri‐ zed ornamental species: effects on marketable and qualitative traits. ­ adv. hort. sci., 37(1): 49­ 57. copyright: © 2023 castellani m., bonetti d., antonetti m., prisa d., burchi g., nin s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 28 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13998 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 49­57 50 large impact on employment and induced economy, as well as a strong export vocation (pagliantini, 2020). the power of this sector lies in the variety of the assortment offered for sale and therefore nursery industries are constantly looking for new species and cultivars. another important factor for the expansion of this sector is the adaptation of new production technologies to the concept of sustainable develop­ ment. great deal of the ornamental and flowering plant production is carried out in containers and depends almost entirely on the quality of soilless media derived from both organic and inorganic con­ stituents. indeed, since successful container cultures depend on producing a stable finished plant, different components in the growing substrate can pose signifi­ cant problems if they affect growth rate, nutrition or plant shape and aesthetics (salachna, 2022). its unique physical properties and large availabili­ ty have led peat to be the dominant organic con­ stituent of growing media in many parts of the world in recent decades. nevertheless, other organic mate­ rials have been recently attracting more attention as a partial substitute for sphagnum peat in a container growing medium (fascella, 2015; barrett et al., 2016). among these is coir, regarded as a rapidly renewable resource, as it is a by­product of coconut harvest. moreover, especially in europe, innovative approach­ es have involved an increasing use of locally available organic materials such as bark (wood fibre) and transformation of composted wastes as next­genera­ tion constituents of growing media. perlite or vermi­ culite components are often used to improve drainage and aeration (gruda, 2019). recent studies have focused on the possibility of reusing sediments in agriculture by combining them with standard growing media or biochar amendment (renella, 2021). this practice could be best success­ fully applied to ornamental species grown in contain­ ers, where health risk due to the presence of toxic residues are minimized. thus, in this study we inves­ tigated the effect of a treated sediment as a sub­ strate component on the quality and marketability of three widespread containerized ornamental species: cherry laurel, calla lily and protea. prunus laurocerasus ‘novìta’, commonly known as cherry laurel, an evergreen ornamental with a very fast growing and plant development, is one of the most commercially important outdoor ornamental species for the italian nursery. the cultivar ‘novìta’, selected in holland, has now become widely estab­ lished and has replaced the previous cultivar ‘caucasica’, compared to which it has longer, thicker, and shinier leaves, and above all, is more resistant to diseases and water stress (ilnitsky et al., 2019). nowadays, container­grown cherry laurels, as all out­ door nursery plants, can be sold at any time of year with two major peaks in spring and autumn. its com­ mercial value is a function of the size of the plant, first of all its height. calla lily (zantedeschia aethiopica) is a very popu­ lar flower of great world economic importance, grown as cut flower, potted or garden plant. this species flowers from late summer into spring in frost free areas and prefers partially illuminated environ­ ments (de hertogh, 1996). calla cultivation is spread from central to southern italy, where it is performed indoor under greenhouse conditions to protect the crop from october to the end of april (janowska and andrzejak, 2022). on the market, cut flowers are classified into grades based on the size of the stem and the flower (spathe). furthermore, the sale price varies throughout the year, reaching the highest val­ ues in the winter period and the lowest values in the month of april, when the quantity of flowers offered on the market is highest. the protea used in this experiment was protea cynaroides dwarf ‘little prince’, a patented variety, bred for flowerpot production at the national research council of south africa. this cultivar differs from the well­known protea king, national flower of south africa, for its compact habit, which makes it suitable for expanding the potted plant floral market. protea is an unusual plant for europe, where it is con­ sidered a niche product much appreciated by fans of exotic plants. commercial growing of proteas for the cut flower market is increasing also in other european regions with mild winter, especially portugal and canary islands (lewis and matthews, 2002; claassens, 2017). at the marketing stage, pro­ tea potted plants are graded and priced according to the number of flowers. these species were used as model plants of the ornamental plant sector (evergreen shrubs, flower­ pot, cut flower) to assess the commercial potential of sediment­based growing media combined with sphagnum peat, coir and wood fibre on the existing floriculture market. 2. materials and methods this study used the marine sediment dredged castellani et al. ‐ treated sediment as substrate component for ornamentals 51 from leghorn port (central italy, 43°33’25’’n, 10°17’39’’e) in 2008. to reduce the level of contami­ nants, the sediment was recovered through three years of phytoremediation process under the frame of the agriport project (agricultural reuse of polluted dredged sediments, eco­innovation eu project n. eco/08/239065eco/08/239065) (masciandaro et al., 2014; doni et al., 2015). the phytoremediated sediment underwent landfarming, consisting in a periodical aeration by mechanical han­ dling for three months, before using it. the treated sediment (ts) was then used as a par­ tial substitute of standard substrates, commonly used for ornamental and flowering crop cultivation in tuscany. in addition to sphagnum peat, other materi­ als that are already being used for commercial pro­ ductions as an alternative to peat, such as coconut coir and bark, were employed as organic materials, while pumice was added as inorganic matrix in all mixtures. sphagnum peat, coir, bark and pumice were mixed separately with the ts to create several media containing 25 or 50% by volume of ts (table 1). mixes were used as growing media for soilless cul­ tivation of three ornamental crops: p. laurocerasus ‘novìta’ (outdoor ornamental shrub), z. aethiopica (cut flower), p. cynaroides ‘little prince’ (flowering potted plant). thanks to its unique microporous properties, the standard peat­based substrate (60% peat, 40% pumice v/v) was used as control in all culti­ vation trials. the experiments were performed in private facili­ ties of central italy (tuscany). plants were cultivated under greenhouse conditions for one year growing season over 2020­2021 (cherry laurel and calla) and 2021­2022 (protea) (fig. 1). standard commercial nursery management practices were followed, except those plants received different water amounts depending upon the species, as better specified below in the experimental design of each considered species. the efficiency of soilless cultivation was eval­ uated both in terms of quantity and quality of mar­ ketable products and commercial value. the com­ mercial expected value was obtained by multiplying each product category by the corresponding sale price of a given selling season and market demand. data on prices for the studied species were given by two tuscan’s leading ornamental trading centres (ornamental nursery district, pistoia, italy; flora toscana, pescia (pt), italy) during 2021 for potted cherry laurel plants and cut calla flowers, and during 2022 for potted proteas. cherry laurel (prunus laurocerasus) ‘novìta’ cherry laurel is a very common shrub in italian gardens, widely used as evergreen ornamental barri­ er plant (hedge). it is easily propagated by cutting and sold as potted plant at different stages of growth in different pot sizes. totally 336 cherry laurel rooted cuttings were grown in 8.5­l drip­irrigated pots. the experimental design consisted of seven growing media (gm) and two water regimes (wr) as described in table 1. growing media were prepared by mixing defined volumes of previously homoge­ nized ts and standard substrates based on peat, coconut coir, and bark, each one containing a certain amount of inert pumice. gm containing commercial peat­based substrate alone was considered as the control. plants were watered by an automated drip irrigation system providing two irrigation rates: nor­ mal (wr1 = 250 ml/day of water per pot on average) and reduced by 20% (wr2= 200 ml/day of water per pot on average). each gm*wr combination was replicated in three blocks, each containing four pots, each one consisting of two cuttings (8 cuttings × 14 treatments × 3 blocks = 336 cuttings). all plants were supplied with a slow­release fertilizer (nitrophoska gold®). growth in height of all cherry laurel plants was measured at the end of the growing cycle and the corresponding sale price was considered as index of production quality. leaf colour (i.e., the coordi­ fig. 1 ­ greenhouse soilless culture of prunus laurocerasus (a), zantedeschia aethiopica (b), and protea cynaroides (c) in tuscany (central italy). adv. hort. sci., 2023 37(1): 49­57 52 nates l* [brightness], a* [redness], and b* [yellow­ ness]) was measured with a colorimeter (chromameter, minolta cr 200) on three different leaf points shortly after growth measurements. the chroma index (chroma) was calculated using the coordinates a* and b* (chroma = [(a*2 + b*2) 1/2]). calla lily (zantedeschia aethiopica) calla lily is a species of great economic impor­ tance worldwide thanks to the beauty of its flowers. in italy, calla is grown as outdoor garden and potted plant, but also largely raised for cut flower produc­ tion under greenhouse conditions. totally 378 calla rhizomes were planted in 30­l containers placed on three raised benches. three different gm combining different proportions of the ts with a standard peat­ based substrate were tested (table 1). benches were served by different water regimes (wr) by a drip irri­ gation system: i) wr1, high water regime=wr2+30% (1220 ml/day of water per pot on average); ii) wr2, normal water regime (930 ml/day of water per pot on average); iii) wr3, low water regime=wr2­30% (650 ml/day of water per pot on average). each gm*wr combination was replicated in three blocks, each containing seven pots, each holding 2 rhizomes (14 rhizomes x 9 treatments x 3 blocks = 378 rhi­ zomes). plants were fed with a nutrient solution com­ monly adopted for the cultivation of soilless calla and the experiment was stopped at the end of the first flower flush. flower number and flower length were monitored during plant life cycle (once a week from september to february, and twice a week from march to may) by collecting, counting and measuring all the flowers produced in the experimental test. moreover, flowers were graded by stem length and sale price of each flower category was calculated dur­ ing the whole harvest season. marketable senes­ cence (number of days to get pollen on spathe) and final senescence (number of days to spathe brown­ ing) were determined by storing 15 cut flowers (5 for each replicated block) in water at room temperature. on the same flowers, spathe colour was measured on three different floral leaf points as described above. protea (protea cynaroides) ‘little prince’ national flower of south africa, protea is com­ mercially relevant for the flower industry and in italy it is extensively cultivated as flowering pot and exported all over the world. crop protection is a fun­ damental requirement for its cultivation in tuscany; moreover, water needs are high when this species is grown under soilless conditions. totally 210 rooted cuttings were transferred in 2­l pots and cultivated until full blooming. the ts was mixed in different proportion with peat and coir to obtain seven growing media, as described in table 1. each substrate mix was replicat­ ed three times with 10 pots for each replicated plot. plants were supplied with 150 ml/day of water per pot on average. all plants were fertilized with nutricote timed release fertilizer (npk 18:6:8 ­ 360 day releasing time) and watered with daily drip irriga­ tion. number and length of stems, number and dimension of flowers of all experimental plants were measured after the full opening of the flowers, and the sale price of each flowering potted plant was con­ sidered. the cluster colour of all flowering plants was measured on three different bracts as described above. table 1 ­ composition of the tested growing media gm peat treated sediment coir bark (wood fiber) pumice cherry laurel calla protea fiber peat lmix 1 cmix 1 pmix 1 60 40 lmix 2 cmix 2 pmix 2 45 25 30 lmix 3 cmix 3 pmix 3 30 50 20 lmix 4 25 45 30 lmix 5 50 30 20 lmix 6 25 45 30 lmix 7 50 30 20 pmix 4 25 34.2 22.8 18 pmix 5 50 22.8 15.2 12 pmix 6 25 17.1 39.9 18 pmix 7 50 11.4 26.6 12 castellani et al. ‐ treated sediment as substrate component for ornamentals 53 statistical analysis pots in the three greenhouse experiments were arranged according to a randomized complete block design. all collected data were subjected to analysis of variance (anova) to determine treatment effects. where significant effects were determined, tukey’s test was used to separate differences among treat­ ment means at the 99% (p<0.01) level of confidence, applying spss v27 software (spss inc., chicago, il, usa). 3. results cherry laurel (prunus laurocerasus) ‘novìta’ the gm had a clear effect on final plant height (fig. 2a), while wr and gm*wr interaction were without effect on plant development. lmix 6 and lmix 7, containing ts mixed with wood fiber, were the most limiting for p. laurocerasus growth. cherry laurel grown in ts and peat (lmix 2 and lmix 3) did not statistically differ from the control, while plants obtained on mixes containing ts and coir (lmix 4 and lmix 5) exhibited intermediate height values. selling values followed the same trend as the plant develop­ ment (fig. 2b). as regards leaf blade colour, leaves of cherry laurel showed a significant yellowing com­ pared to the control when grown mixes containing ts and bark (table 2). calla lily (zantedeschia aethiopica) number of flowers and selling price were signifi­ cantly affected by the gm and wr, while the gm*wr interaction did not affect productive traits. the treated sediment had a clear positive effect on plant blooming, since the number of flowers showed increasing values as the content of the sediment in the mixture increased (table 3). regarding water supply, a 30% water reduction diminished calla blooming, being the number of flowers per plant on average 21% smaller than that obtained under nor­ mal water supply (4.5 vs 5.7). in general, the produc­ mean separation within column by tukey’s test. means (n= 24) followed by different letters are significantly different (p<0.01). table 2 ­ effect of growing media (gm) on the colour of cherry laurel leaves table 3 ­ main effect of growing media (gm) and water regime (wr) on the number of calla lily flowers and their sell­ ing value mean separation within columns by tukey’s test. means (n= 63) followed by different letters are significantly different (p<0.01). fig. 2 ­ plant height (a) and selling value (b) of p. laurocerasus at marketing stage. mean separation among bars by tukey’s test. means (n = 24) followed by different letters are significantly different (p<0.01). gm chroma lmix 1 18.5 a lmix 2 18.8 a lmix 3 17.6 abc lmix 4 17.3 abc lmix 5 17.2 abc lmix 6 16.2 bc lmix 7 15.8 c factor flowers/plant (n) selling value/flower (€) gm cmix 1 4.6 b 1.8 b cmix 2 5.5 a 2.6 a cmix 3 5.8 a 2.8 a wr wr1 5.7 a 2.9 a wr2 5.7 a 2.6 a wr3 4.5 b 1.6 b 54 adv. hort. sci., 2023 37(1): 49­57 tion of flowers increased over time reaching the max­ imum peak in march and april. flowers were subdi­ vided according to the stem length, which varied from 50 to 100 cm. plants cultivated on cmix 3 pro­ duced a consistently higher number of quality flow­ ers reaching 80, 90 and 100 cm of final length com­ pared to the control (72 vs 45, 43 vs 6 and 29 vs 0, respectively). thus, the selling value averaged over the entire harvest season was found to be greater for flowers obtained on cmix 3 consisting of 50% ts (table 3). no differences in the colour of the spathe were evidenced (fig. 3), whatever growth conditions (gm and wr) were used (table 4). regarding petal senescence, inferior cut flower performance during vase life was observed when flowers were cultivated on cmix 1 and cmix 2 with reduced irrigation (table 4). protea (protea cynaroides) ‘little prince’ anova showed that all considered parameters of protea were significantly affected by the growth media. in particular, protea grown on the control (pmix 1) exhibited significant higher average values of stem number and length (table 5), along with an anticipated flower induction and development (data non shown) compared to plants grown on sediment­ based media. the incorporation of 50% v/v ts in the growing media drastically reduced plant growth and flower production in all tested mixes (fig. 4a). on the other hand, protea grown on mixes containing 25% v/v ts had a more compact shape, but developed a good number of flowers, except for pmix 6 contain­ ing a higher percentage of coir dust. selling prices of potted flowering proteas were in line with the values expressed by plant vegetative and reproductive behaviour under the different tested soilless condi­ tions (fig. 4b). once flowers opened, they were simi­ lar in terms of size (table 6; fig. 5), except for flower length which was smaller in pmix 5, pmix 6 and pmix 7 essentially related to the lesser flower develop­ ment at the time of the data collection. some differ­ ences in the bract colour of protea clusters were per­ ceptible as indicated by the chroma index reported in table 6. in general, control flowers were deep pink, while those obtained on sediment­based substrates had brighter tones and tending towards purple. in mean separation within columns by tukey’s test. means (n= 15) followed by different letters are significantly different (p<0.01). ns = not significant. *values are the average of all plant stems. mean separation within column by tukey’s test. means (n= 30) followed by different letters are significantly different (p<0.01). table 4 ­ effect of gm*wr interaction on the senescence and colour of calla lily flowers fig. 3 ­ calla lily cut flowers at harvest time. gm*wr flower senescence chroma marketable final cmix 1 wr1 11.8 ab 15.0 ab 6.6 ns cmix 1 wr2 14.8 a 19.0 a 6.1 ns cmix 1 wr3 8.2 c 15.4 ab 6.2 ns cmix 2 wr1 13.2 ab 16.6 ab 6.2 ns cmix 2 wr2 13.8 ab 18.4 ab 6.1 ns cmix 2 wr3 7.6 c 14.0 b 6.0 ns cmix 3 wr1 13.4 ab 17.2 ab 5.9 ns cmix 3 wr2 13.8 ab 17.2 ab 6.2 ns cmix 3 wr3 12.0 ab 17.4 ab 6.1 ns table 5 ­ effect of growing media (gm) on stem number and lenght of p. cynaroides gm stem/plant (n) stem lenght (cm)* pmix 1 2.9 a 31.6 a pmix 2 2.8 ab 23.0 b pmix 3 2.1 bc 15.5 cde pmix 4 2.5 ab 18.9 bc pmix 5 1.3 c 9.6 e pmix 6 2.7 ab 15.8 cd pmix 7 1.9 bc 13.9 de castellani et al. ‐ treated sediment as substrate component for ornamentals 55 particular, the flowers collected on pmix 5 exhibited the lowest chroma index mainly associated to the a* component more shifted towards green and the b* component towards yellow. 4. discussion and conclusions results showed that the incorporation of the treated sediment into growing media for soilless cul­ tivation affected both plant growth and blooming dif­ ferently according to the considered ornamental species. in the case of evergreen cherry laurel, the ts used in proportions of 25­50% reduced plant height growth, especially when combined with wood fibre, slightly altering its attractive vibrant foliage. this species is easy to care and adapts to most types of soils, from light and sandy to heavy clay; thus, the reduced plant development was assumed to be pri­ marily associated to the combined properties of the used matrices (tozzi et al., 2022). a positive effect of the ts was evidenced on calla li ly, notoriously known as a water demanding species. in fact, due to its clayey silt composition, the sediment can increase water retention capacity when added to traditional soilless growing media, although the effect appears to depend primarily on the amount of sediment added to the medium (martínez­nicolás et al., 2021). in our study, both tested sediment­based mixtures allowed a copious blooming, while a 30% water reduction determined a highly significant decrease in flower production. the highest number of flowers and of best quality (stem size between 80­100) were obtained from plants grown in the presence of 50% of ts. on the other hand, petal senescence appeared to be mainly influ­ enced by the water amount that plants receive dur­ ing cultivation, while no significant differences were (z) measured with closed flower on march 2022; (y) measured with fully open flower on may 2022. mean separation within columns by tukey’s test. means (n = 30) followed by different letters are significantly different (p<0.01). ns = not significant. table 6 ­ effect of growing media (gm) on protea flower size and colour gm flower size chromalength (mm)z width (mm)z diameter (mm)y pmix 1 97.3 a 38.7 a 22.5 ns 32.7 a pmix 2 97.3 a 38.4 a 22.3 ns 30.0 ab pmix 3 97.9 a 39.3 a 21.4 ns 28.3 ab pmix 4 97.8 a 40.3 a 23.1 ns 32.2 a pmix 5 81.0 b 32.9 b 22.7 ns 27.5 b pmix 6 74.7 b 38.6 a 22.3 ns 30.9 ab pmix 7 81.6 b 33.9 b 22.0 ns 28.9 ab fig. 4 ­ bloomed plants (a) and selling value (b) of p. cynaroides at marketing stage. mean separation among bars by tukey’s test. means (n = 30) followed by different letters are significantly different (p<0.01). fig. 5 ­ size measurement on protea flower. length and width (a), diameter (b). adv. hort. sci., 2023 37(1): 49­57 56 detected in the colour of calla flowers among the considered treatments. the effect on protea plants grown on different sediment­based substrates was almost opposite. plants grown on mixes containing 50% v/v ts exhibit­ ed a considerable reduction in plant growth and flower production (fig. 6). nevertheless, when 25% v/v ts was added to peat and coir fibre, only 7­13% of potted plants failed to bear flowers and displayed a nice compact habit. in this regard, it should be noted that a compact behaviour might represent a valuable feature for this species, when cultivated as a flowering pot, since growth regulators are being used to induce branching and improve compactness (ben­ jaacov and silber, 2007). suggested factors limiting plant development and flower production could be related to high ph values and nutrient content, espe­ cially phosphorus, inherited from the sediment matrix. in fact, this perennial flowering species devel­ ops proteoid roots to increase nutrient absorption, being therefore adapted to poor soils of low nutri­ tional status, particularly phosphorus, with a ph 4­6, and a clay content of less than 20% (griebenow et al., 2022; walter et al., 2022). concerning other aes­ thetic aspects of the plant, the amount of ts incorpo­ rated in the substrate mixes affected the colour of clusters but not their size. ultimately, based on sale values, the ts can be a sustainable alternative for soilless production of ornamental crops, both for environmental protection and economic development. recommended for calla cut flower production, the ts can be also used suc­ cessfully for potted cherry laurel and protea cultiva­ tion if properly combined in the correct proportions (up to a maximum of 50% and 25%, respectively) with other organic matrixes, such as peat and coir. a large number of consumers are changing their way of life and the products they buy in an effort to live more sustainably. it’s clear that nursery industries need to place sustainability in the heart of their busi­ ness strategy. however, the cost of eco­friendly plants is often higher than conventional products, due to expensive raw materials which are necessary to pro­ duce with lower environmental impact. so, the issues are: is such discrepancy really that significant? are costumers willing to pay more for a plant obtained with the utmost respect for the environment? acknowledgements this research was supported by european life project subsed “sustainable substrates for agricul­ ture from dredged remediated marine sediments: from ports to pots” (life 17 env/it/000347). references barrett g.e., alexander p.d., robinson j.s., bragg n.c., 2016 ‐ achieving environmentally sustainable growing media for soilless plant cultivation systems. ‐ a review. ­ sci. hortic., 212: 220­234. ben­jaacov j., silber a., 2007 ­ leucadendron: a major proteaceous floricultural crop, pp. 113­159. ­ in: jan­ ick j. (ed.) proteaceous ornamentals: banksia, leucadendron, leucospermum, and protea . international society for horticultural science (ishs), leuven, belgium, pp. 160. fig. 6 ­ protea plants grown on control substrate (1 = pmix 1), mixes containing 25% ts (2 = pmix 2, 4 = pmix 4, 6 = pmix 6), and mixes containing 50% ts (3 = pmix 3, 5 = pmix 5, 7 = pmix 7 castellani et al. ‐ treated sediment as substrate component for ornamentals 57 claassens c., 2017 ­ 10 fascinating facts to know about the protea, south africa’s national flower. ­ https://theculturetrip.com/africa/south­africa/arti­ cles/10­fascinating­facts­to­know­about­the­protea­ south­africas­national­flower. de hertogh a., 1996 ­ holland bulb forcer’s guide. ­ no. ed. 5, hillegom, netherlands, pp. 587. doni s., macci c., peruzzi e., iannelli r., mascian­ daro, g. 2015 ­ heavy metal distribution in a sediment phytoremediation system at pilot scale. ­ ecol. eng., 81: 146­157. fascella g., 2015 ­ growing substrates alternative to peat for ornamental plants, pp. 47­67. ­ in: asaduzzaman m.d. (ed.) soilless culture ‐ use of substrates for the production of quality horticultural crops. intechopen, pp. 176. griebenow s., makunga n.p., privett s., strauss p., veste m., kleinert a., valentine a.j., 2022 ­ soil ph influences the organic acid metabolism and exudation in cluster roots of protea species from the mediterranean‐type fynbos ecosystem, western cape, south africa. ­ rhizosphere, 21: 100486. gruda n.s., 2019 ­ increasing sustainability of growing media constituents and stand‐alone substrates in soil‐ less culture systems. ­ agronomy, 9(6): 298. ilnitsky o.a., pashtetsky a.v., plugatar y.v., kor­ sakova s.p., 2019 ­ water relation characteristics of prunus laurocerasus l. under progressive soil drought stress of crimea’s southern coast. russ. ­ agricult. sci., 45: 260­265. janowska b., andrzejak r., 2022 ­ cytokinins and gib‐ berellins stimulate the flowering and post‐harvest longevity of flowers and leaves of calla l i l ies (zantedeschia spreng.) with colourful inflorescence spathes. ­ agronomy, 12(8): 1859. lewis j., matthews l.j., 2002 ­ the protea book, a guide to cultivated proteaceae. ­ canterbury university press, new zealand, pp. 184. martínez­nicolás j.j., legua p., hernández f., martí­ nez­font r., giordani e., melgarejo p., 2021 ­ effect of phytoremediated port sediment as an agricul‐ tural medium for pomegranate cultivation: mobility of contaminants in the plant. ­ sustainability, 13(17): 9661. martorana m., 2021 ­ aspettando euroflora 2022 ­ h tt p s : / / c o l t u r e p r o t e tt e . e d a g r i c o l e . it/floricoltura/aspettando­euroflora­2022. masciandaro g., di biase a., macci c., peruzzi e., ian­ nelli r., doni s., 2014 ­ phytoremediation of dredged marine sediment: monitoring of chemical and bioche‐ mical processes contributing to sediment reclamation. ­ j. environ. manage., 134: 166­174. pagliantini r., 2020 ­ siena, emergenza coronavirus, via libera alla vendita al dettaglio di fiori e piante nei vivai. ­ https://corrieredisiena.corr.it/news/home/ 1544817/coronavirus­siena­vivai­piante­fiori­toscana­ cia­.html. renella g., 2021 ­ recycling and reuse of sediments in agriculture: where is the problem? ­ sustainability, 13(4): 1648. salachna p., 2022 ­ trends in ornamental plant produc‐ tion. ­ horticulturae, 8(5): 413. tozzi f., antonetti m., prisa d., burchi g., turchi a., macci c., peruzzi e., nin s., 2022 ­ developing pat‐ terns in prunus laurocerasus grown on sediment enriched substrates. ­ j. soils sediments, 22: 2117­ 2127. walter h.e., pagel j., cooksley h., neu a., schleun­ ing m., schurr f.m., 2022 ­ effects of biotic interac‐ tions on plant fecundity depend on spatial and func‐ tional structure of communities and time since distur‐ bance. ­ j. ecol., 111(1): 110­124. impaginato 403 adv. hort. sci., 2023 37(4): 403­413 doi: 10.36253/ahsc­14410 shelf­life and post­harvest quality of tomato (lycopersicon esculen‐ tum mill.) varieties to different packaging materials at mersa, north wollo, ethiopia s.h. muhie 1 (*), s. worku 2 , b. masrie 2 1 wollo university, south wollo, ethiopia. 2 woldia university, north wollo, ethiopia. key words: packaging materials, quality, shelf life, tomato varieties. abstract: tomato (lycopersicon esculentum mill.) has a short shelf life at ambi­ ent conditions and is a highly perishable crop. extreme post­harvest losses occur as a result of the wrong packaging materials. however, by employing the right packing materials, tomato varietals can have longer shelf lives. globally rising fresh tomato demand has forced the development of essential mecha­ nisms, including packaging materials, to improve shelf life. the current study was initiated to evaluate the quality and shelf­life of tomato varieties in response to packaging materials at mersa, north wollo, ethiopia, during 2021 cropping season. three replications of a completely randomized design were used to test three tomato varieties (roma vf, oval red and woyno) and seven packing materials [closed carton (cc), open carton (oc), closed wooden box (cwb), open wooden box (owb), perforated polyethylene bag (ppb), non­per­ forated polyethylene bag (nppb) and control (c)] at room temperature (20­ 22°c). according to the findings, there is a significant (p<0.05) interaction effect between packaging materials and varieties on a number of parameters, includ­ ing physiological weight loss, decay percentage, disease incidence, total soluble solids (tss), tomato fruit ph, juice color score, overall acceptability, marketabil­ ity percentage, and shelf life. non­perforated polyethylene plastic experienced the highest physiological weight losses of 79.88% and 79.63% after 18 days of storage. roma vf variety showed the greatest weight loss. in addition, ppb showed the lowest decay percentage (20%) and maximum mar­ ketability (20%) during the 18th day of storage. at the end of storage, nppb with roma vf and woyno varieties had a substantially (100%) larger decay loss of tomato fruits. nppb has been linked to the high­ est disease incidence (20%). roma vf and oval red recorded the high­ est ph tomato fruit’s color and overall acceptability score on ppb. it can, thus, be concluded that packaging of tomato fruits in ppb can extend shelf­ life with better­quality of the produce. however, to develop plausible recom­ mendation, the study should be repeated in multi­location with more packag­ ing methods and varieties over seasons. (*) corresponding author: hamidashm@gmail.com citation: muhie s.h., worku s., masrie b., 2023 ­ shelf‐ life and post‐harvest quality of tomato (lycopersicon esculentum mill.) varieties to diffe‐ rent packaging materials at mersa, north wollo, ethiopia. ­ adv. hort. sci., 37(4): 403­413. copyright: © 2023 muhie s.h., worku s., masrie b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. authors contributions: the authors (dr. seid hussen and dr. biruk masrie) developed the idea and initiation, edit the draft the proposal, and write the manuscript. the co­author (solomon worku) conducted the research, did data collection and analysis. competing interests: the authors declare no competing interests. received for publication 22 february 2023 accepted for publication 1 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14410 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 403­413 404 1. introduction the tomato (lycopersicon esculentum mill.) crop, which was domesticated in mexico, is the most com­ monly grown vegetable crop in the world. from the equator to chile, the western coastal plain of south america is where it originated (mapes and basurto, 2016). it ranks first on the list of canned vegetables and is the most extensively consumed vegetable crop, followed by the potato and sweet potato (yesdhanulla and aparna, 2018). the cultivated tomato was first used in ethiopian agriculture between 1935 and 1940. it is one of the most impor­ tant crops grown by smallholder farmers in ethiopia. tomatoes are essentially a perennial plant, even though they are farmed as an annual crop. they are regarded as a delicate warm­season crop that is prone to cold (abdu, 2016). tomatoes have a relatively short shelf life due to several postharvest physiological, physical, and chemical changes that occur during storage (haile and safawo, 2018). various methods and strategies are being evaluated to minimize postharvest losses and enhance their storage life. because they are a cli­ macteric and perishable fruit, tomatoes have a very short shelf life, under normal circumstances (caroline et al., 2015; ayomide et al., 2019). fruit rot, inappro­ priate handling and storage techniques, and external injury received during harvest are the main causes of post­harvest losses. a tomato fruit’s fresh weight is 90% water, and the size of the fruit is influenced by the water supply to the plant. fruit with this much water content is perishable. the majority of fruits and vegetables suffer from water loss during storage, which is impacted by temperature (>55°f) and rela­ tive humidity (<80%) conditions (bonazzi and dumoulin, 2011). despite the growth of ethiopia’s horticultural sec­ tor, there is still a lack of funds to address post­har­ vest loss and crop quality issues (kasso and bekele, 2018). post­harvest losses of horticulture crops in ethiopia were observed to range from 15% to 70% (urge et al., 2014). post­harvest loss of horticulture products in ethiopia was attributed to a number of factors, including transportation, a lack of proper storage facilities, and unsuitable packaging materials (hagos, 2014; kasso and bekele, 2018). losses during and after harvest are a significant source of food loss because they have a direct impact on people’s liveli­ hoods and the whole economy, which is important for food security, nutrition, and lowering poverty. prior to marketing, transit, and storage, horticul­ tural crops experience the worst post­harvest loss and quality deterioration. common horticultural goods’ post­harvest loss was attributed primarily to inadequate packaging, poor transportation, inade­ quate storage, and unfavorable market conditions (seid et al., 2013). various studies have explored the magnitude of vegetable postharvest losses, produc­ tion limitations, and agronomic practices. however, there is a dearth of data on the impact of storage conditions and packing materials on tomato fruit shelf life. furthermore, the knowledge of different packaging materials and storage methods used by small­holder farmers and customers in mersa and neighboring districts of northeastern ethiopia where the current research was conducted is scarce, despite few experiences across various regions of the coun­ try. tomato growers, distributors and consumers can benefit from choosing the best possible packaging material to preserve tomato quality during harvest and extend shelf life. the objective of this study is to examine the impact of various packaging materials on the postharvest quality and shelf­life of tomato varieties at mersa, north wollo, ethiopia. 2. materials and methods description of the study area the current experiment was conducted at mersa college of agriculture, woldia university, north wollo, ethiopia. the area is geographically locate at 390° 38’ e and 11°35’n with an altitude of 1600 m asl in the semi­arid tropical belt of north­eastern ethiopia. it is 491 km away from addis ababa to the northeast and 30km away from woldia town to the south. it receives an average annual rain fall ranging from 750 to 1000 mm with a bimodal pattern, short rainy season from february to april and long rainy season from july to september. the average annual temperature is about 28.5°c. the soil texture is clay loam and classified as vertisol. the ph of the soil is slightly acidic to slightly alkaline. treatments and experimental design there were seven packaging materials available; namely open carton (oc), closed carton (cc), perfo­ rated polyethylene bag (ppb), non­perforated poly­ ethylene bag (nppb), open wooden box (owb), closed wooden box (cwb), and control (without packaging) (c); along with three varieties of tomato; muhie et al. ‐ post‐harvest quality of tomatoes in different packaging materials 405 woyno, oval red, and roma vf. the experiment comprised of 21 treatments with three replications arranged in completely randomized design (crd). the three tomato varieties were produced indepen­ dently in a field. half a kg of tomato fruits for each replication was used for the experiment. in accor­ dance with specifications of the design, each treat­ ment was assigned randomly to the experimental units within a replication. description of experimental materials varieties. three tomato varieties (roma vf, woyno and oval red) were used in the experiment. seeds of all varieties were obtained from sirinka agricultural research center located at 20 kms to the north of the experimental site. their descriptions are indicated in table 1. tomato fruits of each variety at the time of harvesting are as shown in the figure below (fig. 1). packaging materials. the treatment consisted of seven different packaging materials (open carton, closed carton, open wooden box, closed wooden box, perforated polyethylene bag, non­perforated polyethylene bag and ambient (without packaging) as control. closed carton (cc). a carton made from paper­ board with the size of 42 cm length25 cm height and 35 cm width was used. the carton was closed after putting the fruits inside the carton. open carton (oc): a carton made from paper­ board with the size of 42 cm length, 25 cm height and 35 cm width was used. fruits were placed inside the carton and were left open. closed wooden box (cwb). a wooden box made from wood with the size 40 cm length, 30 cm height and 30 cm width was used. the box was closed after putting the fruits inside it. open wooden box (owb). a wooden box made from wood with the size 40 cm length, 30 cm height and 30 cm width was used. fruits were placed inside the wooden box and were left open. perforated polyethylene bag (ppb). a perforated polyethylene bag made from plastic with 0.4mm thickness of white polyethylene bag and it was hav­ ing 25% hole designed for the experimentation pur­ pose, were obtained from market. fruits were placed in perforated polyethylene bag. non‐perforated polyethylene bag (nppb). a non­ perforated polyethylene bag made from plastic with 0.4 mm thickness of white polyethylene bag designed for the experimentation purpose, were obtained from market. fruits were placed in non­ perforated polyethylene bag. control (without packaging) (c). fruits were placed on open table at room temperature in labora­ tory without any packaging, were designed for the experimentation purpose. experimental management seeds were sown in rows of 15 cm spacing on well prepared raised nursery beds having the size of 1 m x 1 m (for each variety) at mersa habru agricultural and rural development office fruit nursery site. seeds were covered lightly with fine soil and with table 1 ­ description of tomato varieties used for the experiment description types of varieties roma vf woyno oval red year of release 1977 2006 2007 altitude (masl) 700­1900 800­2000 800­2000 growth habit determinate determinate determinate fruit shape globular oblong oval utilization fresh fresh fresh maturity (days) 95­100 100­120 100­110 yield research field 400 45 42 yield farmer`s field 120­140 13­17 14­18 fig. 1 ­ tomato varieties used in the experiment (a=roma vf, b=oval red, c=woyno). adv. hort. sci., 2023 37(4): 403­413 406 two­three cm thick grass mulch. transplanting of seedlings to experimental field was done when seedlings attain the height of about 13­15 cm and at 3­4 true leave stage. all management activities were given as needed till harvesting. fruits were harvested at breaker stage. fruits were selectively harvested to maintain uniform color, sizes and fruits without any defects. the selectively harvested fruits were cleaned to remove the dust. then the fruits were allowed to dry for half an hour by spreading on newspaper over the floor. initial weight was taken from each variety and packed in the aforementioned packaging materi­ als. half kilogram tomato fruits per treatment were packed in each packaging materials at room temper­ ature (20­22°c) (fig. 2). data collection physiological weight loss, ph of tomato fruit juice, decay percentage, color score, disease incidence (%), overall acceptability and percentage marketability were collected during the experimental period from the total population at 3 days interval (0, 3, 6, 9, 12, 15 and 18 days). the physiological weight loss was taken at 3 days interval starting from date of packaging and deter­ mined using the methods described by (workneh et al., 2012). it was determined using sensitive balance (type jd2000­2). the following formula was used to calculate weight loss. physiological weight loss (%) = initial weight ­ final weight × 100 initial weight) the juice content (%) was determined by crashing randomly selected fruits then extracting using juice extractor and calculate juice content as follows: juice content (%) = total weight of juice­beaker weight x 100 total weight of fruit ph of tomato fruit juice. randomly selected fruits from each packaging was extracted using juice extractor and measure with ph meter (harvard digi­ tal ph meter, model h198103, made from italy). disease incidence (%). the fruits were observed visually for rotting and microbial infection and calcu­ lated according to the formula given below (khru­ engsai et al., 1991). disease incidence (%) = number of infected fruits x 100 total number of fruits percentage of marketability. the marketable qual­ ity of fruits was subjectively assessed by procedure of workneh et al. (2012) with a slight modification. these descriptive quality attributes were determined subjectively by observing the level of visible mold growth, decay, shriveling, smoothness and shininess of fruits with (15 respondents). a 1­5 rating, with1 = unusable, 2 = usable, 3 = fair, 4 = good, 5 = excellent was used to evaluate the fruit quality. fruits receiving a rating of 3 and above were considered as mar­ ketable. the numbers of marketable fruits was used as a measure to calculate the percentage of mar­ ketable fruits during storage. after subjectively assessing the product, it was calculated using the fol­ lowing formula. marketability % = no. of marketable tomato fruit × 100 total no. of sampled tomato fruit shelf life. the shelf life was calculated by counting the days required to attain the last stage of ripening, but up to the stage when fruit remained still accept­ able for marketing (rao et al., 2011). decay or rotting (%). decay or rotting was deter­ mined by the visual observation. development of spots on the fruit’s skin and softening and rotting of fruits were also being recorded. color and overall acceptability score. color was measured by comparing with the color chart described by dadzie and orchard (1997). it was determined by counting (15 respondents) the num­ ber of respondents from 0 to 5 scoring. a 1­5 rating with 0 = poor, 2 = fair, 3 = good, and 4 = very good and 5 = excellent and finally the mean data will be analyzed. on the same way, overall acceptability was assessed by the above selected respondents and the result was taken by the 1­5 rating similar with mar­ ketability. the predominant colors which were used for evaluation are green, 2=breaker, 3=turning, 4=pink, 5= light red, 6= red and 7 = deep red. total soluble solids (tss). the tss was determined fig. 2 ­ experimental arrangement at room temperature (20­ 22°c). muhie et al. ‐ post‐harvest quality of tomatoes in different packaging materials 407 following the procedures described by (waskar et al., 1999). an aliquot of juice was extracted using a juice extractor. a hand refract meter (way­2s abb’e, 0­20 brixo) was used to determine tss the refractor meter was washed with distilled water. the refractometer was standardized against distilled water (0 percent tss) and measure tss. data analysis the data obtained was statistically analyzed for analysis of variance (anova) using general linear model (glm) using sas 9.13 version (sas, 2002).the mean separation was made based on least significant difference (lsd) at 5% level of significance. 3. results and discussion physiological weight loss tomato fruits experience great physiological weight loss if stored under normal conditions with­ out any treatment and safe storage environment (iqbal et al., 2022). physiological weight loss of toma­ to fruits was significantly influenced by the main effect of packaging materials (p<0.001), varieties (p<0.01) and interaction effect (p<0.01) of packaging materials and varieties at 18th days of storage. the result showed that, the highest physiological weight loss (79.88%) was recorded from control (without packaging) followed by open carton (74.70%) from variety roma vf (table 2). the lowest physiological weight loss (32.72%,) was recorded from variety oval red with perforated plastic package, which was lower than the weight loss of tomato fruits subjected to all other packaging materials. in the current investigation, the highest weight loss observed in tomato fruits at the control treat­ ment may be due to the higher respiration rate exer­ cised. the result is supported by the findings of sinha et al. (2019), in which maximum weight loss was recorded from control, without packaging materials. in addition, another group of researchers also report­ * significant at p≤0.05; ** highly significant at p≤0.01; *** very highly significant at p≤0.001; means with the same letter (s) within a colu­ mn are not significantly different at 5% level of significance. table 2 ­ interaction effects of packaging materials and varieties on physiological weight loss and decay percentage (at 9, 12, 15 and 18 days) of tomato at mersa during 2021 cropping season variety packaging materials physiological weight loss decay percentage 9 12 15 18 9 12 15 18 woyno oc 29.73 c 41.37 d 59.99 d 68.89 d 20.00 c 40.00 d 80.00 c 20.00 a cc 23.59 e 31.51 g 49.06 f 58.02 f 0.00 c 0.00 d 46.67 c 80.00 c owb 26.77 d 36.38 e 51.83 e 60.10 e 20.00 a 20.00 c 60.00 a 80.00 c cwb 25.04 e 34.64 f 48.73 g 56.59 g 0.00 c 20.00 c 40.00 d 80.00 c ppb 13.19 g 19.69 h 27.75 h 32.72 h 0.00 c 0.00 d 20.00 e 60.00 d nppb 2.83 h 40.09 d 58.14 d 67.14 d 0.00 c 0.00 d 60.00 a 100.00 a c 40.96 a 56.03 a 68.55 a 79.47 a 0.00 c 0.00 d 60.00 a 100.00 a oval red oc 31.89 b 41.94 d 61.45 d 71.27 d 20.00 a 40.00 a 60.00 a 80.00 c cc 24.24 e 32.95 g 47.70 g 55.33 g 0.00 c 20.00 c 46.67 c 100.00 a owb 30.75 c 40.45 d 58.63 d 68.01 d 0.00 c 20.00 c 40.00 d 80.00 c cwb 23.10 e 31.17 g 45.02 g 52.21 g 20.00 a 20.00 c 40.00 d 80.00 c ppb 15.86 f 21.56 h 31.23 h 36.23 h 0.00 c 0.00 d 20.00 e 60.00 d nppb 2.34 h 40.09 d 58.14 d 67.44 d 20.00 a 20.00 c 33.33 d 86.67 c c 42.33 a 57.57 a 68.65 a 79.63 a 0.00 c 26.67 b 53.33 b 80.00 c roma vf oc 32.78 b 44.44 b 64.40 b 74.70 b 0.00 c 20.00 c 60.00 a 93.33 b cc 24.50 e 33.32 g 48.54 g 56.29 g 20.00 a 40.00 a 60.00 a 93.33 b owb 31.55 c 42.90 c 62.21 c 72.15 c 0.00 c 40.00 a 60.00 a 86.67 c cwb 23.74 e 31.99 g 46.34 g 53.76 g 0.00 c 20.00 c 60.00 a 86.67 c ppb 16.88 f 21.87 h 31.71 h 36.78 h 0.00 c 0.00 d 20.00 e 60.00 d nppb 2.62 h 40.34 d 58.49 d 67.64 d 0.00 c 20.00 c 60.00 a 93.33 b c 42.65 a 58.00 a 68.86 a 79.88 a 6.67 b 20.00 c 60.00 a 100.00 a lsd (0.05) 2.03 2.31 3.99 4.39 4.15 4.152 10.99 10.17 significant level 4.40 ** 7.01 ** 15.87 ** 22.83 ** 320.65 *** 276.19 *** 250.79 *** 143.92 *** cv (%) 5.21 3.77 4.52 4.28 5.21 3.77 4.52 4.28 408 adv. hort. sci., 2023 37(4): 403­413 ed that the highest weight loss was recorded for tomato stored in ambient atmosphere without pack­ aging (control) (sualeh et al., 2016). the highest weight loss from control and open carton may be due to faster metabolism at higher temperature, increased cell wall degradation and higher mem­ brane permeability leading to exposure of cell water for easy evaporation (yao et al., 2020).the lowest physiological weight loss might be due to the fact that, polyethylene plastic protects the fruits from adverse conditions by avoiding mechanical damage, reducing moisture loss, providing beneficial modified atmosphere and preventing pilferage (sinha et al., 2019). similarly, hailu et al. (2014) reported that weight loss of fruits in polyethylene bags was far low than from unpackaged fruits. lower weight loss of packaged fruits could be due to slow rate of transpi­ ration and prevention of excessive moisture loss. decay percentage packaging materials vary in their tendency to reduce decay percentage of tomato fruits (olveira­ bouzas et al., 2021). decay percentage was signifi­ cantly influenced by packaging materials (p<0.001), tomato varieties (p<0.05) and their interactions (p<0.001). the highest decay percentage (100 %) was obtained from both roma vf and woyno without packaging. it was followed by non­perforated polyeth­ ylene plastic and closed carton. however, the lowest decay percentage (20 %) was recorded from variety woyno with open carton followed by oval red (60%) with perforated plastic (table 2). it is clearly identified that decay percentage increased with the storage time for all storage methods and ripening stages. this result is line with the work of (moneruzzaman et al., 2009). tomatoes at light red stage showed rapid dete­ rioration. total deterioration of the fruit was recorded from closed carton with variety oval red, non­perfo­ rated polyethylene plastic with variety of woyno, and control both from variety roma vf and woyno on the 18th days of storage period. disease incident percentage disease incident was significantly influenced by packaging materials (p<0.001), tomato varieties (p<0.001) and their interactions (p<0.001). maximum disease incident (100%) was recorded from both vari­ ety woyno and oval red using non­perforated poly­ ethylene plastic, followed by the same packaging materials from roma vf variety (93.33%). disease incidence was found only in fruits with non­perforated polyethylene plastic and closed car­ ton packaging materials (table 3). this could be due to inadequate air flow in non­perforated plastic and the accumulation of water from fruit respiration, which creates an environment conducive to fungi growth. after 12 days, the variety woyno under closed cartons also started disease incidence while roma vf in non­perforated polyethylene plastic increased by 20 % while all other packaging materials showed no incidence of disease. this result is in line with the findings of (bautista­baños et al., 2008). the highest disease occurrence may be due to the increasing of moisture content in the storage of both closed carton and non­perforated polyethylene plas­ tic packages. the water accumulation inside the packaging is high because of limited movement and exchange of air which can result in the occurrence of fungal disease. color color was significantly influenced by packaging materials (p<0.001) and their interactions (p<0.05). during the storage period, there was a general change of tomato fruit colour from breaker to deep red. the highest color change was observed in con­ trol followed by open carton and wooden box. variation of skin color was due to variety and packag­ ing material. in comparison of tomato fruit variety color change from the breaker to deep red, roma vf variety showed highest loss of greenness, while it was lowest in woyno (table 3). this result is in line with the work of tigist et al. (2013), who reported that variety roma vf showed faster rate of loss of greenness while it was slower for melkasalsa that during normal ripening of tomato fruit, tissue colour changes from green through orange to red, which coincides with ethylene biosynthesis and a climac­ teric rise in respiration. the color acceptability of control fruits is shorter as compared to other packag­ ing materials (between 3 to 18 days of their storage), that is, in the range of very good to excellent espe­ cially perforated plastic packed tomato fruits. this change was due to the action of treatments on the fruits as polyethylene packaging (perforated) helps the color retention as described by (ashenafi and tura, 2018). ph of tomato fruit juice a significant variation in ph of tomato fruit juice was observed due to the main effect of packaging materials (p<0.001), varieties (p<0.01) and their interactions (p<0.001). the control had the highest ph value (4.23) in all varieties of tomato fruit fol­ muhie et al. ‐ post‐harvest quality of tomatoes in different packaging materials 409 lowed by open wooden box in varieties roma vf (4.18) and oval red (4.15).whereas lowest ph value were obtained in packaged fruits treated with closed carton from woyno variety (3.1) (table 4). the result showed that ph values ranging from 3.10­4.23 from all variety and packaging materials after 12 days of storage period. generally, the ph of fruits increases as fruits undergo ripening. this might be due to citric acid in tomato juice, with ph of fruit normally between 4.0 and 4.5 (anyasi et al., 2016). the higher ph of fruits under ambient storage condi­ tion could be associated with the utilization of acids for catabolism of sugar at faster rate. high storage temperature leads to faster respiration rate. the lower ph values of packaged fruits could be explained by the relatively reduced respiration rate in the package can inhibit loss of organic acids. fruit juice content fruit juice content and functional properties of tomato and other vegetables is likely to be affected by the type of storage condition and packaging mate­ rials (alenazi et al., 2020). in the current experiment, a significant variation in fruit juice content was observed due to the main effect of packaging materi­ als (p<0.001) and varieties (p<0.01). ppb had highest fruit juice content (93.70%) on oval red tomato fruit varieties and followed by both oc and control (92.37%) under variety roma vf. whereas, the low­ est juice content was recorded from owb (85.26) table 3 ­ interaction effects of packaging materials and varieties on disease incidence percentage and color (at 9, 12, 15 and 18 days) of tomato at mersa during 2021 cropping season * significant at p≤0.05; ** highly significant at p≤0.01; *** very highly significant at p≤0.001; means with the same letter(s) within a colu­ mn are not significantly different at 5% level of significance. variety packaging materials disease incidence color 9 12 15 18 9 12 15 18 woyno oc 0.00 c 0.00 d 0.00 d 0.00 e 5.33 c 6.00 a 7.00 a 7.00 a cc 0.00 c 6.67 c 20.00 b 20.00 c 5.00 c 6.00 a 7.00 a 7.00 a owb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a cwb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a ppb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 5.67 a 6.67 b 7.00 a nppb 0.00 c 20.00 b 40.00 a 100.00 a 4.00 d 5.00 b 6.00 c 6.67 b c 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 5.67 a 7.00 a 7.00 a oval red oc 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a cc 0.00 c 0.00 d 0.00 d 20.00 c 5.00 c 6.00 a 7.00 a 7.00 a owb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a cwb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a ppb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 5.67 a 6.67 b 7.00 a nppb 20.00 a 20.00 b 40.00 a 100.00 a 4.33 d 6.00 c 6.67 b c 0.00 c 0.00 d 0.00 d 0.00 e 5.33 c 6.00 a 7.00 a 7.00 a roma vf oc 32.78 b 44.44 b 64.40 b 74.70 b 5.00 c 6.00 a 7.00 a 7.00 a cc 0.00 c 0.00 d 6.67 c 6.66 d 5.00 c 6.00 a 7.00 a 7.00 a owb 0.00 c 0.00 d 0.00 d 0.00 e 5.33 c 6.00 a 7.00 a 7.00 a cwb 0.00 c 0.00 d 0.00 d 0.00 e 5.67 b 6.00 a 7.00 a 7.00 a ppb 0.00 c 0.00 d 0.00 d 0.00 e 5.00 c 6.00 a 7.00 a 7.00 a nppb 20.00 a 40.00 a 40.00 a 93.33 b 4.00 d 5.00 b 5.00 d 6.00 c c 0.00 c 0.00 d 0.00 d 0.00 e 6.00 a 6.00 a 7.00 a 7.00 a lsd (0.05) 0.43 0.42 2.08 4.16 0.46 0.36 0.29 0.29 significant level 57.49 *** 66.67 *** 44.4 *** 27.51 *** 0.20 ** 0.17 ** 0.17 *** 0.06 * cv (%) 12.96 6.11 18.04 15.60 5.52 3.73 2.66 2.60 adv. hort. sci., 2023 37(4): 403­413 410 and cwb (85.44) packaging materials on woyno vari­ ety followed by nppb (88.26%) on oval red variety (table 4). similar findings were reported by (gebeyehu, 2018). the variation of juice content may be due to differences in packaging materials which can affect the firmness and metabolic processes. the variation in juice content of tomato fruit among vari­ eties might be due to their genetic differences. total soluble solids (°brix) total soluble solid (tss) is one of the quality para­ meters in fruits and vegetables (mesa et al., 2022). in the present research, tss was significantly influenced by packaging materials (p<0.001), varieties (p<0.001) and interaction effect of packaging methods and vari­ eties (p<0.01). during the 12 day of storage period the tss was range from 3.65 to 5.11°brix (table 4). the result showed that, the highest tss (5.11) was recorded from variety roma vf under the control fol­ lowed by oval red variety. the lowest tss (3.65) was recorded from variety woyno using non­perforated polyethylene plastic (table 4). the result agrees with the work of sualeh et al. (2016). the variation in tss might be due to advancement of fruit ripening, packaging materials and variety of tomato fruit. total soluble solids (°brix) of control and treated tomato fruits showed that they increased as the ripening proceeds. the lowest tss value of non­perforated polyethylene plastic may be due to slowing down of respiration and metabolic activity. whereas the highest tss in the control may be due to high respiration and metabolic activity rise ripening process as result increasing tss. in this regard, the view of kumar et al. (2022) is noteworthy that the slower respiration also slows down the syn­ thesis and use of metabolites resulting in lower tss due to the slower change from carbohydrates to sug­ ars which result in retardation of the ripening process. the changes in tss content that occur during ripening are correlated with the hydrolytic shifts in starch concentration after harvest. using sugars as a respiration substrate, the tss content of fruits could decrease with time storage due to increased temper­ ature and biosynthesis processes or polysaccharide degradation at maturity (azene et al., 2014). overall acceptability (score) overall acceptability was significantly influenced by packaging materials (p<0.001), tomato varieties (p<0.01) and their interactions (p<0.001). the pre­ sent study revealed that under woyno variety, non­ perforated polyethylene plastic and control fruits showed, lower overall acceptability as compared to other packaging materials and varieties at 18 days of storage period (table 5). at the later stage of ripen­ ing (18 days), non perforated polyethylene plastic and control fruits showed a lower overall acceptabili­ ty scores (0­3). oval red and roma vf varieties of tomato fruits under perforated plastic showed better overall acceptability followed by both carton packed and wooden box packaging (table 5). the results led to a conclusion that the main rea­ son behind this improvement was due to the preven­ tion of fruit from decay organism and the fruit will have stored reserve which is protected from adverse table 4 ­ interaction effects of packaging materials and varieties on juice content, ph and tss of tomato at mersa dur­ ing 2021 cropping season * significant at p≤0.05; ** highly significant at p≤0.01; *** very highly significant at p≤0.001; means with the same letter(s) within a column are not significantly different at 5% level of signi­ ficance. variety packaging materials storage period (days) juice content ph tss woyno oc 89.30 d 4.01 f 4.65 f cc 88.39 e 3.10 h 4.53 g owb 85.44 e 4.05 f 4.59 g cwb 85.26 e 3.98 f 4.40 h ppb 90.29 d 4.12 e 4.08 i nppb 88.96 d 3.75 g 3.65 j c 92.03 c 4.23 a 4.87 c oval red oc 91.65 c 4.04 f 4.36 i cc 90.83 d 4.02 f 4.45 j owb 90.51 d 4.15 d 4.27 i cwb 91.69 c 3.98 f 4.38 i ppb 93.70 a 4.12 e 4.01 j nppb 88.26 e 4.06 f 4.02 j c 92.37 b 4.23 b 5.01 b roma vf oc 92.37 b 4.02 f 4.69 e cc 91.46 d 4.04 f 4.36 i owb 91.91 c 4.18 c 4.57 g cwb 91.31 d 3.93 f 4.41 h ppb 92.03 c 4.13 e 4.52 g nppb 91.92 c 3.26 h 4.02 j c 92.04 c 4.23 a 5.11 a lsd (0.05) 3.21 0.16 0.37 significant level 7.13 * 0.07 *** 0.14 ** cv (%) 2.07 2.48 4.68 muhie et al. ‐ post‐harvest quality of tomatoes in different packaging materials 411 condition (yahaya and mardiyya, 2019). from break­ er to turning stage, the colour of fruits changed from poor to fair by showing not more than 30% of surface as not green in colour. when stage advances from pink to pink­red, the colour of all fruits was in the range of good to excellent. this result is in conformi­ ty with the work of (priyankara et al., 2017). up to 9th days roma vf tomato variety exhibited best color and consumer acceptability, followed by oval red were as woyno scores low consumer acceptability. marketability percentage marketability percentage was significantly influ­ enced by packaging materials (p<0.001), tomato vari­ eties (p<0.05) and their interactions (p<0.001). at 9th days of storage period the highest percentage mar­ ketability (100%) was obtained from woyno tomato fruit varieties using perforated polyethylene plastic followed by the same packaging materials (93.33%) from both roma vf and oval red tomato fruit vari­ eties (table 5). the lowest marketability percentage was obtained from non­perforated polyethylene plastic (40%) of woyno tomato fruit variety followed by both roma vf and oval red (60%). when the stor­ age period increase in storage period (18 days) toma­ to fruits packed only with perforated polyethylene plastic (20%) from both roma vf and woyno tomato fruit varieties followed by oval red tomato fruit vari­ eties using open carton packaging materials (6.67%). the difference in marketability of tomato fruits table 5 ­ interaction effects of packaging materials and varieties on overall acceptability and marketability percentage (at 9, 12, 15 and 18 days) at mersa during 2021 cropping season * significant at p≤0.05; ** highly significant at p≤0.01; *** very highly significant at p≤0.001; means with the same letter(s) within a colu­ mn are not significantly different at 5% level of significance. variety packaging materials overall acceptability marketability percentage 9 12 15 18 9 12 15 18 woyno oc 4.00 d 3.00 d 1.00 c 0.00 c 80.00 d 60.00 c 20.00 c 0.00 c cc 4.00 d 3.00 d 1.00 c 0.00 c 80.00 d 60.00 c 20.00 c 0.00 c owb 3.00 f 4.00 b 1.00 c 0.00 c 60.00 f 60.00 c 20.00 c 0.00 c cwb 4.00 d 4.00 b 1.00 c 0.00 c 80.00 d 60.00 c 20.00 c 0.00 c ppb 5.00 a 4.00 b 2.00 b 1.00 a 100.00 a 80.00 a 40.00 a 20.00 a nppb 3.00 f 2.00 f 0.00 e 0.00 c 40.00 g 20.00 f 0.00 d 0.00 c c 4.00 d 2.00 f 1.00 c 0.00 c 60.00 f 40.00 e 20.00 c 0.00 c oval red oc 4.00 d 3.00 d 1.00 c 0.00 c 80.00 d 60.00 c 20.00 c 6.67 b cc 4.00 d 3.00 d 2.00 b 0.00 c 80.00 d 60.00 c 20.00 c 0.00 c owb 4.00 d 2.33 e 1.00 c 1.00 a 80.00 d 53.33 d 20.00 c 0.00 c cwb 4.00 d 3.00 d 2.00 b 0.00 c 73.33 e 60.00 c 20.00 c 0.00 c ppb 5.00 a 4.00 b 3.00 a 1.00 a 93.33 b 80.00 a 26.66 b 0.00 c nppb 3.00 f 1.67 f 0.00 e 0.00 c 60.00 f 20.00 f 0.00 d 0.00 c c 4.00 d 3.00 d 1.00 c 0.00 c 73.33 e 66.67 b 20.00 c 0.00 c roma vf oc 4.00 d 3.00 d 2.00 b 1.00 a 80.00 d 60.00 c 20.00 c 0.00 c cc 3.67 e 3.00 d 1.00 c 0.00 c 73.33 e 40.00 e 20.00 c 0.00 c owb 3.67 e 3.33 d 1.00 c 0.00 c 73.33 e 40.00 e 20.00 c 0.00 c cwb 4.33 c 3.67 c 0.67 d 0.00 c 86.67 c 60.00 c 20.00 c 0.00 c ppb 4.67 b 4.33 a 3.00 a 0.67 b 93.33 b 80.00 a 40.00 a 20.00 a nppb 3.00 f 2.00 f 1.00 c 0.00 c 60.00 f 20.00 f 20.00 c 0.00 c c 3.33 f 2.00 f 1.00 c 0.00 c 66.67 f 40.00 e 0.00 d 0.00 c lsd (0.05) 0.46 0.59 0.21 0.21 11.74 5.87 4.15 4.15 significant level 0.22 ** 0.61 *** 0.71 *** 0.33 *** 144.97 ** 170.37 *** 222.22 *** 69.84 *** cv (%) 6.83 11.63 9.92 56.69 8.80 6.59 13.68 79.37 adv. hort. sci., 2023 37(4): 403­413 412 was due to packaging materials, varieties of tomato fruits, and also percentage of decayed fruits and dis­ ease incidence obtained from non­perforated poly­ ethylene bags. the present result is in line with the study of haile and safawo (2018) who reported that, packaging of climacteric fruits in low density polyeth­ ylene bags delay ripening and softening, and hence improves marketability. these beneficial effects can be explained by the modified atmosphere created inside the package as well as the reduction in water loss. lower respiration and ethylene production rates, due to modification of atmospheric gases inside the package could be the possible reason to extend the storage life of fruits (islam et al., 2022). 4. conclusions tomatoes are prone to careless handling and packaging throughout local manufacturing. as a result, there is a significant postharvest loss of toma­ to fruits at every stage, from harvest to consumption. the fruit must therefore be handled properly after harvest in order to improve its protection and shelf life. with this context in mind, the goal of this study was to evaluate the impact of packaging material on the quality and shelf life of tomato fruit varieties after harvest. the physiological weight loss, percentage of decay, color score, general acceptability, tss, ph, incidence of disease, variety, marketability, and shelf life of tomato fruits were all significantly influenced by packaging. due to a faster respiration rate, the control had the greatest weight reduction. on perfo­ rated plastic, the weight reduction was accompanied by an accelerated water loss. perforated plastic showed the highest marketability and degradation percentage. the woyno variety under non perforat­ ed plastic had the highest illness incidence and the highest juice content (93.37%) of any type of plastic. roma vf variety beneath perforated plastic had the highest tss. when employing perforated plastic, fol­ lowed by open cartons and hardwood boxes, the storage term for tomato fruit was significantly lengthened. under perforated plastic, the tomato variety with a longer shelf life was both roma vf and oval red. according to the findings of this study, tomato fruits packaged in low density polyethylene bags with per­ forations had a longer shelf life and better quality. however, a follow­up study in multiple locations and across seasons is necessary to substantiate this advice. there should be further research done using package types and materials that are not covered in this study. acknowledgements the authors acknowledge wollo university, woldia university and sirinka agriculture research center. references abdu j., 2016 ­ a comparative study on selected tomato varieties for 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bengal, india. 4 sidho‐kanho‐birsha university, purulia, west bengal, india. key words: banana, charcoal, genetic stability, micropropagation, protocorm, rapd analysis, seedling development. abstract: vanda tessellata (roxb.) hook. ex g. don is an epiphytically grown orchid well­known for its excellent floral value and therapeutic qualities. the present investigation deals with a study of asymbiotic seed germination and large­scale in vitro plant generation of vanda tessellata by using three different basal media (ms, kc, and vw) and two supplements, charcoal and banana. of these three media used for seed germination, ms (murashige and skoog) gave the best response, followed by kc (knudson c) and vw (vacin and went). ms medium took less time to germinate seeds and maximum protocorm formation was also observed. ms medium with banana powder (15,000 mg/l) showed the best result for developing seedlings from protocorm and maximum growth of leaves and roots of the seedlings. propagation through secondary protocorm formation was highest in ms media with charcoal (1000 mg/g). in vitro­grown plants were successfully acclimatized with an 89.4% survival rate. according to a random amplified polymorphic dna (rapd) analysis, the in vitro generated plants were clone copies of their parent plant and did not exhibit variations. these findings validated the most trustworthy techniques, which can also be applied for to large­scale medicinal vanda tessellata plant production at the commercial level. 1. introduction orchids are a unique group of flowering plants belonging to the family orchidaceae. orchids are popularly known for their beautiful, attractive (*) corresponding author: drdipakkumarkar@gmail.com subrata­raha@skbu.ac.in citation: paramanik p., banerjee n., raha s., kumar kar d., 2024 ­ an efficient nutrient medium for asymbiotic seed germination and in vitro plant generation of vanda tessellata (roxb.) hook. ex g. don. ­ adv. hort. sci., 38(4): 363­370. orcid: pp: 0009­0007­9970­9456 bn: 0000­0003­3727­6361 rs: 0009­0000­7975­5585 kkd: 0009­0009­8035­0759 copyright: © 2024 paramanik p., banerjee n., raha s., kumar kar d. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 24 april 2024 accepted for publication 13 september 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16080 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0007-9970-9456 http://orcid.org/0000-0003-3727-6361 http://orcid.org/0009-0000-7975-5585 http://orcid.org/0009-0009-8035-0759 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 363­370 364 flower, long shelf life, and high purchase prices. among monocotyledons, the orchidaceae is a highly evolved family comprising nearly 850 genera (stewart and griffith, 1995; singh et al., 2007; gutierrez, 2010; madhavi and shankar, 2019). it has been reported that 28,237 species are distributed in the tropical forests of india, south asia, sri lanka, south and central america, and mexico (willis, 2017). in india, 155 recognized genera with 1256 orchid species are found in different habitats (singh et al., 2019). according to available records, 466 taxa of orchids are found in west bengal (mitra, 2021). there are 12 orchid genera recorded in the purulia district (paramanik et al., 2020). vanda tessellata (roxb.) hook. ex g.don is a medicinally important epiphytic orchid of the family orchidaceae. the pollination mechanism of orchids is highly specialized and seeds are small, thin, and non­ endospermic. it is reported that approximately 1,300 to 4 million seeds are present per capsule (pierik, 1987). due to the lack of endosperm, a symbiotic association with mycorrhizal fungi is required to provide nutrients to embryos that are required for the seed germination of orchids in their natural habitat (paramanik et al., 2021). the epiphytic orchid’s 30­ 60 cm­tall stem is furnished with thickly coriaceous, recurved, plicate, and obtuse­keeled leaves. flowers are greenish­yellow, with brown specks on the lip’s middle lobe (chauhan, 1999). the petals are shorter than the sepals, yellow with brown lines and white borders. the lip measures 16 mm in length and is bluish with purple specks. capsules are with acute ribs that are 7.5­9.0 cm long and narrowly clavate­oblong (fig. 1). in traditional medicine, v. tessellata has been commonly used to treat a variety of ailments, including fever, rheumatism, dysentery, and dyspepsia. the juice of the leaves is applied topically to treat otitis media. the root is used as a bronchitis cure and as an antidote to scorpion stings (chauhan, 1999). the value of orchids as a commodity has grown daily. due to demands from massive collections in the past, the habitat of this medicinally significant orchid is being destroyed, which has caused the species to become rare and limited to minimal areas within its native habitats (kaur and bhutani, 2009). in nature, orchids have been propagated vegetatively to solve this problem, but it is a prolonged process. therefore, plant tissue culture and micropropagation can be extremely effective in preventing the extinction of this orchid and increasing its population (wochok, 1981). as a result, orchids must depend on external sources of nutrients for germination and large­scale production. the asymbiotic seed germination culture method, which was first commenced by knudson (1946), is commonly used for seed germination of orchids. another method for micropropagating orchids is to employ aseptically produced seedlings (bhadra, 1999). molecular markers are crucial for determining the genetic diversity, variation, and resemblance of various plants and their population structure. genetic variation is responsible for various factors related to in vitro culture settings (pradhan et al., 2023). during in vitro culture, sometimes somaclonal variation changes the genetic composition of the regenerants (rawat et al., 2013). several molecular markers have been used to evaluate the genetic fidelity of clones generated in vitro. one effective and affordable method for identifying plant genetic variability is random amplified polymorphic dna (rapd)(hussain et al., 2008). this study aimed to develop an efficient nutrient medium for asymbiotic seed germination and large­ scale in vitro plant generation of v. tessellata (roxb.) hook. ex g.don. and also assess the genetic fidelity of in vitro regenerants with mother plants through rapd analysis. 2. materials and methods establishment of culture seeds from 7­month­old, undehisced green pods fig. 1 ­ morphological image of vanda tessellata. (a) plant with flower spike, (b) mature capsule, (c) light microscopic image of seeds (10x), (d) stereo microscope image of seeds, (e) scanning electron microscopy image of seed (400x). paramanik et al. ‐ asymbiotic seed germination and in vitro plant generation of vanda tessellata 365 were used to establish cultures. undehisced green pods of v. tessellata (roxb.) hook. ex g. don were collected from the trunks of different trees in the ajodhya hills of purulia district, west bengal. the freshly harvested capsules were first given a five to ten­minute rinse under running tap water. after that, pods were rinsed in 90% ethanol for 20­30 seconds, treated with 0.1% (w/v) mercuric chloride solution for 10 minutes, and then the surface sterilization procedure was completed by washing the material three times in sterile distilled water. after excising the sterilized pods lengthwise, the seeds were scooped out and put in a conical flask with 100 ml of autoclaved distilled water. the mixture was then slowly shaken for five minutes. culture tubes (25 x 150 mm) with 10 ml of nutrient media were inoculated with 100 µl of the seed suspension. conical flasks (250 ml, 500 ml) and culture bottles (500 ml) contained 50 to 100 ml of nutrient media were used for plantlet development. three basal media, kc (knudson, 1946), ms (murashige and skoog, 1962), and vw (vacin and went, 1949), hormone­free, were used for asymbiotic seed germination. sucrose (3% w/v) was used as the carbon source. the ph of the medium was adjusted to 5.6 before autoclaving. after adding 0.8% (w/v) agar to solidify the media, the media were autoclaved at 125°c (15 psi) for 20 min. the cultures were kept at 24±2oc with a 10­hour photoperiod supplied by 3000­lux white fluorescent philips lights. multiplication and rooting ms media containing different concentrations of banana powder (15000 mg/l, 30000 mg/l, 60000 mg/l) and activated charcoal (1000 mg/l, 2000 mg/l, 3000 mg/l) added to the medium singly, were used to obtained well­developed seedlings from healthy protocorms. a combination of banana and charcoal in three different concentrations was also used in ms medium for plantlet development. rooting occurs in the same medium. the cultures were kept at 24±2oc with a 10­hour photoperiod supplied by 3000­lux white fluorescent philips lights. acclimatization of seedlings only seedlings with fully grown roots were chosen for the acclimatization phase. the nutrient medium was then completely removed from the entire seedlings by giving them a thorough water wash. the rooted seedlings were transferred to containers filled with potting mix containing small charcoal pieces, coconut husk, sphagnum moss, broken breaks, and dead tree bark (mango). subsequently, they were kept in the growth chamber to maintain humidity (80%) and temperature (24±2°c) for a few weeks. after that, the plantlets were moved to a moist, shady place in the departmental garden, and water was applied to the plants twice a day. rapd fingerprinting analysis the genetic stability of wild and in vitro propagated plantlets was assessed in the current study through the rapd fingerprinting technique. genomic dna was isolated from the leaf tissue of both the control mother plant and five consecutive generations of in‐vitro­grown plants. leaf tissue was subjected to whole genomic dna extraction following the supplied protocol of the dna extraction kit (dneasy® plant mini kit­qiagen, part no. 69104). the quality and quantity of the dna samples were determined by recording the ratio of absorbance at a260/a280 in a uv­vis spectrophotometer (uv­1800 shimadzu). the integrity of the genomic dna was confirmed by electrophoresis on a 0.8% agarose gel. pcr amplification was done in 25 µl reaction volume containing 50 ng of genomic dna, 8 µm primer (rapd), molecular biology grade water, and 12.5 µl hi­chrome pcr master mix containing taq dna polymerase, dntps, mgcl2. a total of 10 primers from the opa and opb series (integrated dna technologies) were used for pcr. amplification was executed in dna thermal cycler (eppendorf mastercycler nexus x2 thermal cyclers). the initial denaturation temperature for the pcr was 94°c, which was succeeded by 35 denaturation cycles for 45 s at 94°c, annealing for 45 s at 27­38°c, and extension at 72°c for 30 s. following the last cycle, a final extension step was included, lasting 7 minutes at 72°c. the amplified products were electro­ phoresed in a horizontal gel apparatus (power packtm basic, bio­rad) with a “100 bp” dna ladder (biolittm proxib) used to visualize and take pictures of the gels. finally, the gel documentation system (gel doctm xr+, bio­rad, usa) was used to examine and evaluate the stained (0.5 µg/l) gel with ethidium bromide. to verify the reproducibility of each pcr, it was performed three times. data collection and statistical analysis all parameters (germination percentage, protocorm formation percentage, survival, leaf formation, root formation, callus formation, adv. hort. sci., 2024 38(4): 363­370 366 secondary protocorm formation percentage) were evaluated and analyzed using spss and expressed as mean ± standard error (se). three replicate cultures were set up for each treatment. one­way analysis of variance (anova) was used to identify significant differences in data of all treatments, and duncan’s multiple range test (p=0.05) was performed to separate the means. 3. results and discussion asymbiotic seed germination the shape and colour change of the seed was used to observe the response to seed germination. most of the seeds were embryonated, and the testa of the seed was ruptured to form a swollen globular structure (fig. 2 a). the beginning of seed germination and the development of protocorm on three basal media following, ms, knc, and vw media were periodically recorded from the first inoculation day (table 1). among the three basal media, the highest percentage of germination (83.50±0.31%) was recorded in the ms medium in shorter time (45 days). second­stage protocorm with a slightly elongated apical region was also (70.00±0.06) observed in ms medium after 20 days. in the other media assessed the germination occurred beyond 100 days with lower germination and protocorm formation percentages (table 1). seedling development: multiplication and rooting healthy protocorms were transferred to ms media supplemented with charcoal or banana powder. the highest percentage of leaf formation (82.40±2.74) and healthy root formation (83.50± 2.00) were found in ms medium­containing banana powder (15000 mg/l). leaf formation and root formation both were observed in the same medium composition. a little callus development in this medium was also noticed (fig. 2 k). secondary protocorms originated from protocorms, were observed and the highest percentage was formed in ms with charcoal (2000 mg/l) (fig. 2 i and j). a combination of banana and charcoal at different concentrations showed no significant result (table 2). only a combination with low concentration table 1 ­ effect of different media on the seed germination of v. tessellate the mean of three replicates ± se (standard error) is displayed in each column. mean values followed by the same letter do not differ significantly at the 0.05 level (dmrt). media time duration required for germination (days) germination (%) protocorm formation (%) murashige and skoog (ms) 46 83.50±0.31 c 70.00±0.06 c knudson c (knc) 103 28.00±0.05 b 35.00±0.08 b vacin and went (vw) 190 10.00±0.14 a 2.00±0.14 a fig. 2 ­ asymbiotic seed germination and seedling development of vanda tessellata. (a­b) early globular stage, (c) protocorm showing initiation of leaf primordia, (d­e) protocorm with distinct leaf and developed seedling with leaf and root, (f) multiple protocorms with many leaf pri­ mordia, (g) axillary shoot formation from multiple proto­ corms (h) leaf formation from secondary protocorm in ms with charcoal (1000 mg/l), (i­j) close view of leaf pri­ mordia from secondary protocorm, (k) protocorm with callus, (l­o) sequential stage of seedling growth after subculture on medium containing ms with 15000 mg/l banana powder, (p­q) stepwise acclimatization. paramanik et al. ‐ asymbiotic seed germination and in vitro plant generation of vanda tessellata 367 (msc1b1) showed a slight survival response (24.40±1.13) along with leaf formation as compared to the other two combinations (table 2). previously, aktar et al. (2008) and islam et al. (2015) reported that banana homogenate (bh) had beneficial and boosting effects on the regeneration of new plbs and a healthy shoot system established from plbs in dendrobium orchids. mature bananas are fairly rich in vitamin b6 or pyridoxine. however, they have comparatively high concentrations of vitamins a (carotene), c (ascorbic acid), and b­complex (niacin, thiamine, and riboflavin)(qamar and shaikh, 2018). minerals including sodium (na), iron (fe), copper (cu), phosphorus (p), manganese (mn), zinc (zn), copper (fe), and especially potassium (k) can also be found in bananas (sarma et al., 2021). potassium can help to provide resistance to drought, helping orchids to transport water from the roots to the apices, and preventing the orchids from wilting (xu et al., 2021). according to minea et al. (2004), 10% banana homogenate increased the size of the leaves on spathoglottis kimballiana hook. f. in dendrobium nobile lindl. cultures, banana homogenate considerably boosted the formation of leaves (sudeep et al., 1997). activated charcoal (ac) can be used in media to reduce phenolic browning. browning of explant of several plant species has been controlled using the ac (meziani et al., 2016; mittal et al., 2016; rani and dantu, 2016; magrini and devitis, 2017; irshad et al., 2018). kim et al. (2019) reported that ms medium supplemented with ac had prevented browning in seedling development of pecteilis radiata. the adsorptive qualities of ac are principally noted for both its beneficial and detrimental effects. by adsorbing phenolic molecules and inactivating peroxidase and polyphenol oxidase, the ac stopped browning (pan and van staden, 1998), but large concentrations of ac can absorb the pgrs and mineral nutrients in the culture medium, reducing the frequency of seedling conversion. all plants lost their viability after subculturing on the media containing msb3, msc2b2, and msc3b3 (table 2). indeed, high concentrations of ac and banana powder drastically affected the survival. acclimatization a vital stage in the micropropagation process is acclimatization. plantlets (about 5­6 cm) were moved to pots containing charcoal, coconut husk, brick, mango bark, and sphagnum moss in a 2:2:2:1:1 ratio. with a 92% survival rate, the in vitro­raised seedlings were acclimated in a plant growth chamber in the laboratory for 2 months. after that, an 89.4% survival rate with more or less similar healthy plants was observed after 10 months of transfer in the poly­ house. genetic fidelity and assessment of in vivo and in vitro plants by rapd fingerprinting analysis according to earlier studies, rapd is a widely used marker to assess the genetic fidelity of different micropropagated plants (kawiak and lojkowska, table 2 ­ effect of charcoal and banana and their combination on the morphogenetic responses and growth of v. tessellata seedlings after 3 months of in vitro culture the mean of three replicates ± se (standard error) is displayed in each column. mean values followed by the same letter do not differ significantly at the 0.05 level (dmrt). c1= charcoal 1000 mg/l, c2= charcoal 2000 mg/l, c3= charcoal 3000 mg/l, b1= banana 15000 mg/l, b2= banana 30000 mg/l, b3= banana 60000 mg/l. treatments survival (%) leaf formation (%) root formation (%) callus formation (%) secondary protocorm formation (%) ms 64.33±2.14 e 37.08±0.93 e 30.30±1.38 b 18.97±1.36 c 10.71±0.82 c msc1 63.50±1.79 e 26.70±1.13 d 0.00±0.00 a 0.00±0.00 a 35.50±1.75 e msc2 42.00±1.32 d 18.70±0.66 b 0.00±0.00 a 0.00±0.00 a 23.50±1.80 d msc3 12.20±1.43 b 10.20±0.70 b 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a msb1 99.50±0.28 f 82.40±2.74 g 83.50±2.00 d 11.40±0.77 b 5.90±0.94 b msb2 42.50±2.34 d 42.50±1.89 f 40.20±1.74 c 0.00±0.00 a 0.00±0.00 a msb3 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a msc1b1 24.40±1.13 c 24.40±1.92 d 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a msc2b2 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a msc3b3 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 368 adv. hort. sci., 2024 38(4): 363­370 2004; tikendra et al., 2019). the genetic make­up of in vivo and in vitro generated plants was compared using ten randomly selected random amplified polymorphic dna (rapd), a dominant marker. the leaves of the mother plant and the leaves of five successive generations of in vitro‐grown plants were collected, and the leaf tissues were used to extract the dna. seven of the ten randomly chosen rapd primers produced unique band patterns in the current investigation. these are opa­03, opa­10, opa­11, opa­15, opa­18, opa­19 and opb­01 (table 3). in vitro, regenerated plants and the mother plant growing in the garden (the plant from which the explants were collected) were both genetically homogeneous, as evidenced by the lack of variance in the banding pattern displayed by any of the primers. the annealing temperature of the seven primers that exhibited scorable monomorphic band patterns is shown in a tabular format. the temperature at which all seven primers were annealed was 27°c. according to pradhan et al. (2023), rapd is the successful marker to assess the genetic fidelity of in vitro grown plants with the mother plant. the current investigation validates the earlier reports. six unique monomorphic bands having a size range of 320 bp to 900 bp were generated by opa 03 (fig. 3). opa 18 yielded four unique monomorphic bands having a size range of 450 bp to 1000 bp. two distinct monomorphic bands were produced from opa 10, and the size range of the bands was from 800 to 920 bp (fig. 3). the fact that all of the bands were monomorphic demonstrated the genetic stability of in vitro regenerants and the similarity of the genetic makeup of the micropropagated plants to the mother plant, which was the actual objective of the current investigation. 4. conclusions the present research reports a successful nutrient culture medium for asymbiotic seed germination of v. tessellata. an efficient, cost­effective nutrient table 3 ­ effects of sowing date and mulching on marketable root yield (mry), unmarketable root yield (ury), and weed density (wd) mean values within rows and columns followed by a different letter(s) are significantly different at a 5% probability level. cv = coefficient of variation. lsd = least significant difference. primer sequence (5ʹ­3ʹ) tm (°c) total bands opa­03 agtcagccac 27 6 opa­10 gtgatcgcag 27 2 opa­11 caatcgccgt 27 1 opa­12 tcggcgatag 27 0 opa­15 ttccgaaccc 27 2 opa­18 aggtgaccgt 27 4 opa­19 caaacgtcgg 27 2 opa­20 gttgcgatcc 27 0 opb­01 gtttcgctcc 27 2 opb­12 ccttgacgca 27 0 total 18 fig. 3 ­ gel electrophoresis of rapd fragments of vanda tessella‐ ta obtained with primer opa­03, opa­10, and opa­18. lane l 100 bp dna ladder; lane m mother plant, lanes 1­ 5 are in vitro regenerated plants of five successive gener­ ations. paramanik et al. ‐ asymbiotic seed germination and in vitro plant generation of vanda tessellata 369 media for large­scale in vitro plant generation was also achieved by using banana powder supplementation. from the above findings, it may be concluded that ms medium with banana powder is the best medium for overall seedling growth and multiple protocorm formation of v. tessellata. through rapd analysis, it has been successfully proved that all regenerants were genetically similar to the parent plant. the media reported in the current study does not include the use of plant growth regulators (pgrs) for plant development and multiplication, this condition minimizes the possibility of occurrence of genetic alterations. this study would help the pharmaceutical and floriculture industry and conserve wild populations of orchids in the near future. references aktar s., nasiruddin k.m., hossain k., 2008 ­ effects of different media and organic additives interaction on in vitro regeneration of dendrobium orchid. ­ j. agric. rural devel., 6: 69­74. bhadra s.k., 1999 ­ development of in vitro micropropagation techniques in some orchid species of bangladesh. ­ m.sc. dissertation, dept. of botany, chittagong 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sci., 2024 38(1): 13­24 doi: 10.36253/ahsc­13913 pumpkins (cucurbita spp.) diversity and their associated microbiota r. aydi ben abdallah (*), h. chikh­rouhou, h. jabnoun­khiareddine, m. daami­remadi laboratory research of production and protection for a sustainable horticulture, iresa, university of sousse, regional research centre on horticulture and organic agriculture, chott mariem, tunisia. key words: breeding, cucurbita spp., fruit yield, plant genotype, soil microbial community. abstract: root­associated microbiota play a key role in plant growth, resilience, and health. in this study, the microbial community structure in the rhizosphere of 12 pumpkins accessions belonging to three cucurbita species i.e. c. pepo, c. maxima, and c. moschata, was monitored using the soil dilution plating tech­ nique on specific media. all accessions tested were also screened for their pro­ duction and yield parameters. based on principal component analysis (pca), 4 accessions of c. maxima (namely c5, c23, c14.2 and c6.2) were characterized by the greatest average fruit weight and yield, the highest actinomycetes, bac­ terial, trichoderma spp. and aspergillus spp. communities, and the lowest total fungal population in their rhizosphere. positive correlations were noted between fruit fresh weight, culturable bacteria and trichoderma spp. popula­ tions in the rhizopshere of pumpkins accessions. negative correlations were noted between fruit weight and yield parameters and the total culturable fun­ gal populations. the current study clearly demonstrated that the rhizosphere soil microbial communities have been shaped by cucurbita species and acces­ sions. based on the significant links observed between soil microbiota and yield parameters, future pumpkin breeding programs could be focused on the selec­ tion of accessions that are quite able to exploit these associated beneficial microbial communities. 1. introduction plants through their root system and surrounding soil influenced by root exudates represent an interesting ecological niche for the develop­ ment of soil microbiota which are able to colonize the rhizosphere, roots and eventually move to the above­ground plant parts (compant et al., 2019). due to its ecological importance and functional diversity, the rhi­ zosphere microbiome was intensively explored for various features (marques et al., 2014; edwards et al., 2015; gopal and gupta, 2016; compant et al., 2019). microbiotas associated to roots are derived from the soil environment which contains highly diverse microorganisms including acidobacteria, verrucomicrobia, bacteroidetes, proteobacteria, (*) corresponding author: raniaaydi@yahoo.fr citation: aydi ben abdallah r., chikh­rouhou h., jabnoun­khiareddine h., daami­remadi m., 2024 ­ pumpkins (cucurbita spp.) diversity and their associated microbiota. ­ adv. hort. sci., 38(1): 13­24. copyright: © 2024 aydi ben abdallah r., chikh­rouhou h., jabnoun­khiareddine h., daami­remadi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 november 2022 accepted for publication 11 january 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13913 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 13­24 14 planctomycetes, and actinobacteria (fierer, 2017). seeds may be colonized by various microorganisms which proliferate later in the roots of the developing plant and colonize the rhizosphere (compant et al., 2019). soil microbial communities play key roles in plant development and health (philippot et al., 2013; adam et al., 2018). in fact, they may be associated to growth promotion, improved nutrient uptake, and enhanced tolerance to various abiotic and/or biotic stresses (trivedi et al., 2020). the below­ground microbial composition is influenced by many abiotic and biotic factors including soil traits (ph, salinity, structure, moisture, organic matter, environmental conditions), relative abundance of soilborne bio­ aggressors, plant species, genotypes, and agricultural and disease management practices (hardoim et al., 2015; fierer, 2017; compant et al., 2019). all the above­mentioned factors contribute, at variable degrees, to the definition of the root microbial com­ munity structure together with the host­related fac­ tors like plant age and developmental stage, health status and the composition of root exudates (bulgarelli et al., 2012). based on carelli et al. (2000) investigation, the rhizosphere community composi­ tion varies between plant species and even within the same species between plant genotypes. also, the root exudates play a key role in recruiting and shap­ ing the soil microbial population structure as they serve as nutrient sources for rhizosphere microor­ ganisms (sung et al., 2006) and represents an impor­ tant component of communication with rhizosphere­ inhabiting microorganisms (haichar et al., 2014). hence, the variation in the chemical composition of root exudates between and within plant species (grayer et al., 2004) may lead to the development and the proliferation of a phylogenetically diverse array of microorganisms. the chemical composition of root exudates, resulting of different below­ground interactions and factors (soil chemical and physical properties, plant species, age, etc), may impact the soil microbial community structure and function by influencing plant physiology and development (griffiths et al., 1999). in fact, among the members of the rhizosphere microbiome, some are beneficial for plant growth and resilience but others may be phy­ topathogenic exhibiting capacity to overcome the innate plant defense system and to cause devastating diseases (de faria et al., 2021). pumpkin (cucurbita spp.) is an extraordinary veg­ etable species that may be exploited for medicinal and nutritional features (tlili et al., 2020; hosen et al., 2021; chikh­rouhou et al. 2023 b). however, pumpkin cultivation is still ignored in some countries. cucurbita pepo l., c. maxima duchesne, and c. moschata duchesne are three pumpkin species eco­ nomically important which are grown over various agricultural regions worldwide (maynard et al., 2002). in tunisia, pumpkin has significant economic importance especially as familiar agriculture because of its rusticity, high nutritional values and long post­ harvesting conservation. there is no improved culti­ var in tunisia and the production of cucurbita is based on local accessions and landraces. chikh­ rouhou et al. (2019, 2023 a, 2023 b) evidenced that pumpkin landraces collected from farmers of the centre­east of tunisia belongs to three species namely c. maxima, c. pepo, and c. moschata with a predominance of c. maxima. pumpkins face a num­ ber of constraints including a shortage of genetically improved seeds, infections with various pests and pathogens (ndinya, 2019) in addition to the plant parasitic nematode pratylenchus (zhao et al., 2022). developing new cultivars with superior qualities, higher mineral contents, important yield and average weight of fruits, potential resistance towards pests and fungal diseases, tolerance to environmental diffi­ culties, shelf lives enhancement is highly required (paris, 2016; seymen et al., 2016; hosen et al., 2021). breeding plants for beneficial plant­microbe inter­ actions is an emerging field mainly focusing the below­ground interactions in the rhizosphere and their valorization for the development of economical­ ly and ecologically interesting plant material (bakker et al., 2012; adam et al., 2018). in fact, breeding shapes the composition of the root­associated micro­ bial communities including the antagonistic potential towards the encountered pathogens (peiffer and ley, 2013; bouffaud et al., 2014; cardinale et al., 2015). thus, breeding strategy is recently focused on geno­ types­microbial holobiont interactions in order to generate diverse new phenotypes without altering plant genomic information (wei and jousset, 2017; adam et al., 2018; wille et al., 2018). therefore, this study aimed to select the most productive pumpkin accession among 12 tested, to determine their associated culturable soil microbial community and to search for an eventual link between fruit and yield parameters and their associ­ ated microorganisms and soil traits. aydi ben abdallah et al. ‐ microbiota associated to pumpkins 15 2. materials and methods plant material twelve (12) pumpkin (cucurbita spp.) accessions belonging to three cucurbita species (c. maxima, c. moschata, and c. pepo) are used in this study. their main traits are detailed in table 1 and figure 1. they were obtained from the cucurbits breeding program at the regional research centre on horticulture and organic agriculture (crrhab), chott­mariem, tunisia. for each accession, seeds were sown in cell trays and maintained at 25°c under greenhouse condi­ tions. at the two­true­leaf growth stage, they were further transplanted (end of march) to an open field at the experimental station of crrhab of sahline, tunisia (n35° 45’05’’, e10°42’39’’). experimental design pumpkins seedlings were transplanted into rows with a distance of 120 cm between seedlings within the same row and 80 cm between rows. the trial was conducted under drip irrigation system without inputs. cattle manure was applied at a rate of 500 kg ha­1 before planting. the experimental design was a completely randomized block design. two replicates of six seedlings each were used per each accession tested. soil sampling composite soil samples from each replicate were collected at the initial state (before planting) (table 2) and four times post planting i.e. at 30, 60, 90, 150 days post­planting (dpp). after planting, three soil cores (7 cm in diameter × table 1 ­ pumpkin accessions (species and characteristics) used in this study and their main traits fig. 1 ­ diversity of pumpkin (cucurbita spp.) accessions used in the study (a) and their plant growth habits (b). c7, c15: cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1, c23: cucurbita maxima. c14.1, c26: cucurbita moschata. * highly susceptible to powdery mildew (data not shown). accession codes cucurbita species plant growth habit fruit shape flesh color c2 c. maxima bushy transverse broad elliptic yellow c5 c. maxima prostrate medium elliptic orange c6.2 c. maxima intermediate globular yellowish orange c7 c. pepo bushy globular cream c9.1* c. maxima prostrate transverse medium elliptic yellow c9.2 c. maxima intermediate heart shaped yellowish orange c14.1 c. moschata intermediate top shaped yellowish orange c14.2 c. maxima prostrate medium elliptic yellow c15* c. pepo bushy transverse elliptical yellow c15.1* c. maxima intermediate transverse medium elliptic orange c23 c. maxima prostrate transverse elliptical orange c26 c. moschata prostrate transverse broad elliptic orange adv. hort. sci., 2024 38(1): 13­24 16 15 cm in depth) were removed from the rhizosphere soil of each sampled plant and were combined to make one composite soil per accession. at the initial state (before planting), ten soil cores were removed and were combined to make one composite soil sam­ ple. two replicates were considered for each soil sampling. once brought to laboratory, soil samples were passed through a 2­mm sieve to remove rocks and large organic debris. they were stored in plastic bags at 10°c until use. two subsamples were processed from each soil sample. determination of soil ph and electrical conductivity (ec) each composite soil sample was air­dried and sus­ pended into distilled water (1:10 soil h2o ­1 ratio). soil filtrates obtained by filtration through whatman paper no. 1 were analyzed for the determination of their ph and electrical conductivity (ec) using a glass electrode (vwr symphony®) and a digital conductivi­ ty meter (hanna®), respectively. estimation of soil microbial community structure general populations of culturable soil microorgan­ isms were determined using the soil dilution plating techniques on various agar media according to larkin and honeycutt (2006) with some modifications. for each subsample taken from each composite soil, 10 g were added to 90 ml of sterile 0.2% water agar, vig­ orously stirred for 30 min, serially diluted and a­100 µl sample was plated on 10% tryptic soy agar (tsa) for total bacterial counts, yeast malt agar (isp medi­ um no. 2) amended with 75 mg l­1 of nalidixic acid and 100 mg l­1 of cyclohexamide for actinomycete counts, and potato dextrose agar (pda) amended with 300 mg l­1 of streptomycin sulphate for total fungal counts. three replicates of one plate each were used for each soil subsample. bacterial and actinomycete plates were incubated at 28°c for 2 and 14 days, respectively, and fungal plates were maintained at 25°c for 7 days. colonies of trichoderma spp., aspergillus spp., and fusarium spp. were identified based on their macro­ and micro­morphological traits (barnett and hunter, 1987) under light microscope and counted separate­ ly. colony­forming units (cfu) were counted to esti­ mate the microbial density on each selective medium (marin et al., 2013). the soil microbial population counts were estimated per 1 g of fresh soil. yield parameters the average fruit weight and the average yield per plant were noted at five months post­planting. the average fruit weight parameter was determined for three randomly sampled plants. statistical analysis data were subjected to a one­way analysis of vari­ ance (anova) using statistical package for the social sciences (spss) software for windows version 16.0. data for ph, ec of soil samples and rhizosphere microbial population counts were analyzed according to a completely randomized factorial model with two factors (accessions tested × sampling times). as for yield parameters, data were analyzed according to a completely randomized block design. experiments were repeated twice. means were separated using tukey test to identify significant pair­wise differences at p≤0.05. correlations between fruit weight and yield para­ meters and soil characteristics (ph, ec and microbial community structure) were carried out using pearson’s test at p≤0.05. for an overview of pumpkins accessions distribu­ tion, and to explore soil microbial community con­ tributing to classification, a principal component analysis (pca) was also performed using spss. 3. results variation of soil ph and ec anova analysis of ph values varied significantly table 2 ­ soil characteristics estimated at the initial state (before pumpkin planting) as determined by soil dilu­ tion (z) plating on selective media (z) soil sample was a composite soil from twenty soil cores collec­ ted before planting and soil dilution was made from a concentra­ tion of 10% (w v­1). (y) cfu= colony­forming unit. soil characteristic data initial soil characteristics ph 7.42 ec (ds m­1) 0.56 culturable microbial population (cfu (y) g‐1 fresh total bacteria (× 107) 2.99 actinomycetes (× 104) 0.95 total fungi (× 104) 1.62 aspergillus spp. (× 103) 0.12 trichoderma spp. (× 103) 1.12 fusarium spp. (× 103) 0.18 aydi ben abdallah et al. ‐ microbiota associated to pumpkins 17 (at p≤0.05) depending on sampling times only. no significant difference was noted between pumpkins accessions and between both factors (table 3). a sig­ nificant decrease of ph values of about 9.9 to 12.7% was noted at harvest (150 dpp) as compared to soil samples collected at 30, 60 and 90 dpp (table 3). anova analyses revealed a significant variation in ec values among accessions, sampling times and their interaction (table 3). the highest ec values were recorded in the rhizopshere of c. maxima c9.1, c. maxima c14.2, c. pepo c7 and c. pepo c15. as for the sampling time effect on this parameter, the ec of the rhizosphere soil associated to the twelve pump­ kins accessions was 34.4­35% and 18.1­19% higher at 30­60 dpp than at 90 and 150 dpp, respectively (table 3). variation of the culturable soil microbial structure the number of bacterial and actinomycetes colonies varied significantly (at p≤0.05) among pump­ kins accessions, sampling times and their interaction (table 4). the highest population of culturable bacte­ ria was obtained from the rhizosphere of c. maxima c23 and c. pepo c15 which was 33.1­55.8% and 15.8­ 44.4% more abundant than those of the remaining accessions (table 4). the abundance of culturable bacteria in the rhizosphere of all the remaining acces­ sions was significantly comparable. concerning the effect of the sampling times (all accessions com­ bined) on this parameter, bacterial colonies counts table 3 ­ ph and electrical conductivity (ec) of soil samples rem­ oved from the rhizosphere of pumpkins depending on accessions tested and sampling times (z) c7 and c15= cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1 and c23= c. maxima. c14.1 and c26= c. moschata. (y) accessions means (for all sampling times combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (x) sampling times means (for all accessions combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (w) dpp= days post­planting. soil samples ph ec (ds m­1) accessions (z) means (y) c2 7.30 a 0.507 ef c5 7.22 a 0.45 f c6.2 7.27 a 0.61 bc c7 7.18 a 0.63 abc c9.1 7.24 a 0.69 a c9.2 7.24 a 0.508 def c14.1 7.17 a 0.57 cd c14.2 7.21 a 0.66 ab c15 7.14 a 0.62 abc c15.1 6.69 a 0.56 cde c23 7.19 a 0.47 f c26 7.27 a 0.503 ef sampling times means (x) 30 dpp (w) 7.44 a 0.62 a 60 dpp 7.25 a 0.61 ab 90 dpp 7.48 a 0.4 c 150 dpp 6.53 b 0.5 b source of variation p­values accessions (acc) 0.27 p ≤ 0.001 sampling times (st) p≤0.001 p ≤ 0.001 acc × st 0.48 p ≤ 0.001 table 4 ­ culturable bacterial, actinomycetes and fungal popula­ tion densities in soil samples (cfu g­1 of fresh soil) rem­ oved from the rhizosphere of pumpkins plants depen­ ding on accessions tested and sampling times (z) c7 and c15= cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1 and c23= c. maxima. c14.1 and c26= c. moschata. (y) accessions means (for all sampling times combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (x) cfu= colony forming unit. (w) sampling times means (for all accessions combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (v) dpp= days post­planting. culturable microbiome population bacteria actino­ mycetes fungi accessions (z) means (y) cfu (x) g­1 of fresh soil × 108 × 105 × 105 c2 1.68 bc 0.96 bc 1.36 a c5 1.62 bc 1.68 a 0.93 a c6.2 2.24 bc 1.20 abc 1.22 a c7 1.48 c 1.08 bc 1.69 a c9.1 1.59 bc 1.08 bc 1.59 a c9.2 1.80 bc 0.96 bc 1.58 a c14.1 1.77 bc 1.05 bc 1.63 a c14.2 2.04 bc 1.13 abc 1.32 a c15 2.66 ab 0.82 c 1.45 a c15.1 1.98 bc 0.90 c 1.28 a c23 3.35 a 1.53 ab 1.02 a c26 1.93 bc 0.74 c 1.20 a sampling times means (w) cfu g­1 of fresh soil × 108 × 105 × 105 30 dpp (v) 3.15 a 0.93 b 1.57 ab 60 dpp 3.08 a 2.86 a 1.86 a 90 dpp 1.06 b 0.27 c 1.41 b 150 dpp 0.74 b 0.32 c 0.58 c sources of variation p­values accessions (acc) p≤0.001 p≤0.001 0.06 sampling times (st) p≤0.001 p≤0.001 p≤0.001 acc × st p≤0.001 p≤0.001 0.15 18 adv. hort. sci., 2024 38(1): 13­24 from the rhizosphere of all pumpkins accessions noted at 30 and 60 dpp were 65.6­66.3 and 75.9­ 76.5% significantly higher than those recorded at 90 and 150 dpp, respectively. actinomycetes community was abundant on the rhizopshere of c. maxima c5, c. maxima c6.2, c. maxima c14.2 and c. maxima c23 which was 35.7­ 55.9%, 10­38­3%, 4.4­34.5% and 29.4­51.6% higher than that associated to the remaining accessions. for all pumpkins accessions combined, the actino­ mycetes population was 67.5, 90.5 and 91.9% signifi­ cantly higher at 60 dpp than at 30, 90 and 150 dpp, respectively. data given in table 4 showed that the total cultur­ able fungal community varied significantly (at p≤0.05) depending on sampling times only and that all acces­ sions tested exhibited significantly comparable fungal community populations. fungal colonies recovered from the rhizosphere of all pumpkins accessions at 60 dpp were 15.6, 24.2 and 68.8% significantly higher than those recovered at 30, 90 and 150 dpp, respec­ tively. as for fungal community structure, culturable aspergillus spp. and trichoderma spp. populations varied significantly (at p≤0.05) in the rhizosphere of pumpkins plants depending on tested accessions, sampling times and their interaction (table 5). for instance, the rhizospheric aspergillus spp. communi­ ty associated to c. maxima c14.2 was significantly 40­48.8% more abundant than that associated to c. moschata c26 and c. maxima c2 accessions. furthermore, trichoderma spp. population was sig­ nificantly 75.9­84.1% higher at the rhizosphere of c. maxima c14.2 than at that of c. pepeo c7 and c. table 5 ­ culturable fungal population structure in soil samples (cfu g­1 of fresh soil) removed from the rhizosphere of pumpkins plants depending on accessions tested and sampling times +(z) c7 and c15= cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1 and c23= c. maxima. c14.1 and c26= c. moschata. (y) accessions means (for all sampling times combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (x) cfu= colony forming unit. (w) sampling times means (for all accessions combined) followed by the same letter are not significantly different according to tukey test at p≤0.05. (v) dpp= days post­planting. culturable fungal population aspergillus spp. trichoderma spp. fusarium spp. accessions (z) means (y) cfu (x) g­1 of fresh soil × 104 × 104 × 104 c2 1.92 b 1.33 abc 0.07 a c5 3 ab 2 ab 0.83 a c6.2 3.08 ab 0.66 abc 1.5 a c7 3.33 ab 0.50 bc 0.07 a c9.1 3.25 ab 0.33 c 0.83 a c9.2 3 ab 0.83 abc 1.66 a c14.1 2.42 ab 1.50 abc 0.08 a c14.2 3.75 a 2.08 a 0.09 a c15 2.58 ab 1.58 abc 0.08 a c15.1 2.83 ab 1.17 abc 0.07 a c23 2.75 ab 1.75 abc 0.09 a c26 2.25 b 1.17 abc 0.08 a sampling times means (w) cfu g­1 of fresh soil × 104 × 104 × 104 30 dpp (v) 0.83 c 1.08 ab 0.27 a 60 dpp 0.55 c 0.72 b 0.08 a 90 dpp 1.44 b 1.58 a 1.38 a 150 dpp 9.81 a 1.58 a 0.27 a sources of variation p­values accessions (acc) p ≤ 0.01 p ≤ 0.01 0.14 sampling times (st) p ≤ 0.001 p ≤ 0.01 0.68 acc × st p ≤ 0.001 p ≤ 0.001 0.35 aydi ben abdallah et al. ‐ microbiota associated to pumpkins 19 maxima c9.1 (table 5). aspergillus spp. colonies recovered from the rhi­ zosphere of all pumpkins accessions at 150 dpp were 85.3 and 94.4% significantly higher than those recov­ ered at 90 and 30­60 dpp, respectively. trichoderma spp. population estimated was significantly higher (+54.4%) at 150 and 90 dpp than at 60 dpp. concerning fusarium spp. populations, no significant differences were detected between pumpkins acces­ sions and sampling times nor their interaction (table 5). variation of fruit production and yield among pump‐ kins accessions tested analysis of variance revealed a significant (at p≤0.05) variation of the average fruit weight between the pumpkin accessions. the highest average fruit weights ranging between 4.18 and 8.48 kg were noted in the accessions c5, c14.2, c15.1 and c23 of c. maxima and c14.1 of c. moschata whereas for the remaining seven pumpkins accessions, this parame­ ter varied between 2.53 and 3.9 kg (fig. 2a). the average fruit yield produced per plant varied significantly (at p ≤ 0.05) among pumpkins acces­ sions. four c. maxima accessions (namely c5, c6.2, c14.2, and c23) and one c. moschata accession (c26) produced significantly the highest fruit yields per plant (5.31­6.21 kg plant­1) than the remaining ones (0.91­3.96 kg plant­1) (fig. 2b). correlation between production and yield parameters and soil characteristics pearson’s correlation analysis indicated that the average fruit weight was significantly and positively correlated to the associated actinomycetes commu­ nity (r= 0.765, p= 0.004) and trichoderma spp. popu­ lation (r= 0.697, p= 0.012) but it was significantly and negatively (r= ­0.700, p= 0.01) linked to the total cul­ turable fungal population in the analyzed soil sam­ ples (fig. 3). pearson correlation analysis, also, revealed a sig­ nificant and negative correlation between the aver­ age fruit yield per plant and the fungal population (r = ­0.701; p = 0.011) colonizing the rhizosphere of pumpkins accessions (fig. 3). multicriteria analysis via pca based on the pca analysis performed, the first two main components (pc) comprised about 68.31% of the variability existing in the analyzed genotypes. pc­1 explained 47.05% of the total variability. the most important traits related to this axis were: the fruit fresh weight, the yield per plant, and actino­ mycetes and trichoderma spp. population. the most important traits of pc­2, which explained 21.25% of the total variation, were ec values and aspergillus spp. community (fig. 4a). the distribution of pumpkins accessions among the two axes showed the variability and allowed dis­ tinguishing 3 main groups (fig. 4b). the 1st group included 4 accessions (c5, c23, c14.2 and c6.2 belonging to c. maxima) characterized by the highest average fruit weight, the highest yield per plant, the fig. 2 ­ a, b average fruit weight and yield per plant of pumpkins accessions noted five months post­planting. bars sharing the same letter are not significantly different according to tukey test at p ≤ 0.05. the average fruit weight (a) and the average yield (b) per plant were determined at har­ vest. c7 and c15: cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1, and c23: c. maxima. c14.1 and c26: c. moschata. fig. 3 ­ heat map of pearson's correlation (r) between the aver­ age fruit weight and the average yield per plant of pump­ kins accessions and soil characteristics. asterisks indicate statistically significant correlation values, negative or positive at * p≤0.05 and ** p≤0.01. adv. hort. sci., 2024 38(1): 13­24 20 highest actinomycetes and bacterial communities, the highest trichoderma spp. and aspergillus spp. populations, and the lowest fungal community in their rhizosphere. the 2nd group was comprised of c7 (c. pepo) and c9.1 (c. maxima) accessions character­ ized by the lowest fruit weight and the lowest trichoderma spp. populations, and the 3rd group was composed of the remaining 6 accessions exhibiting intermediate yield per plant and trichoderma spp. populations. 4. discussion and conclusions plant­associated microbiome plays a fundamental role in plant growth and health (wei and joussset, 2017). breeding programs focusing genotype­associ­ ated beneficial microbiome help achieve ecologically desired plant phenotype traits (adam et al., 2018; wille et al., 2018). the current study aimed to select the most productive pumpkins accessions based on the variability of their soil microbial community struc­ ture and to investigate the presence of eventual links between cucurbita spp. production and yield para­ meters and their rhizosphere soil associated microor­ ganisms. our results clearly demonstrated that cucurbita species and accessions shaped their own soil microbial community structure. some microbes have a particular affinity for certain pumpkins acces­ sions in determining rhizosphere communities. the variation of composition of microbial distribution in the rhizosphere of cucurbita spp. accessions may be explained by the differences in their root morphology and the composition and content of their root exu­ dates which play a fundamental role in the recruit­ ment of plant holobiont. plant­associated microbio­ me and their interactions are highly diverse and mul­ tiple factors shape the microbial community assem­ bly and functioning. in fact, the microbial communi­ ty’s structure varies significantly depending on plant species and/or genotypes growing in the same soil environment (kang and mills, 2004; yao and wu, 2010; berendsen et al., 2012; aydi ben abdallah et al., 2023) and even on plant growth stage (chaparro et al., 2014; compant et al., 2019). the variation in the soil­associated microbiome communities has been assigned to the differences in the root morphol­ ogy, the type of rhizodeposits, the amount and the composition of root exudates and mainly carbon sources which are limiting factors for microbial activi­ ty and proliferation (marschner et al. , 2007; broeckling et al., 2008; compant et al., 2019). moreover, edaphic factors such as soil ph, electrical conductivity (ec), soil texture, soil parental material, and soil salinity are important determinants of com­ munity structure and diversity of soil microbiome (lozupone and knight, 2007; lauber et al., 2008; xu et al., 2014; sun et al., 2015; min et al., 2016). the soil­associated microbiomes have an effect on plant growth and yield production. positive and sig­ nificant correlations were determined between fruit fresh weight and the culturable bacterial and trichoderma spp. populations in the rhizopshere of pumpkins accessions tested in the current investiga­ fig. 4 ­ pca biplot the variability existing in the analyzed traits (a) and the distribution of pumpkins accessions (b). ec: electrical conduc­ tivity. fung: fungi. asp: aspergillus spp. yield: yield per plant. act: actinomycetes. fweight: fruit weight. bact: bacteria. tric: trichoderma spp. c7 and c15: cucurbita pepo. c2, c5, c6.2, c9.1, c9.2, c14.2, c15.1 and c23: c. maxima. c14.1 and c26: c. moschata. aydi ben abdallah et al. ‐ microbiota associated to pumpkins 21 tion. plant­associated microbes with their plant growth­promoting traits play a crucial role in enhanc­ ing plant biomass and crop yield (kumar et al., 2022). halifu et al. (2019) demonstrated that inoculation with two trichoderma species on pinus sylvestris var. mongolica seedlings had a positive correlation with growth parameters, soil nutrient content, and soil enzymatic activity in their rhizosphere. trichoderma spp. are able to increase the growth and the exten­ sion of the root system and to stimulate the secre­ tion of extracellular enzymes such as sucrase, urease, phosphatase, and organic acids in the rhizosphere. these compounds lead to the improvement of the nutrient cycle and the soil enzymatic activity and con­ sequently the soil nutrient status and availability (pelagio­flores et al. , 2017). furthermore, trichoderma spp. can secrete the indole 3 acetic acid (iaa) and to promote the growth of many crops as previously demonstrated for cucumber, bottle gourd, and bitter gourd (kotasthane et al. , 2015). furthermore, the volatile and non­volatile secondary metabolites released by trichoderma spp. such as 6­ n­pentyl­6h­pyran­2­one (6pp), gliotoxin, viridin, harzianopyridone, harziandione, and peptaibols have a significant growth­promoting effect on plants (josé et al., 2008). mohanty et al. (2021) also demonstrat­ ed that the beneficial bacterial communities may improve crop productivity as part of sustainable agri­ culture. in fact, acidothiobacillus ferooxidans and bacillus cereus are associated to increased growth and yield and improved soil composition in pumpkin (ansari et al., 2017). plant growth improvement may be achieved either directly via the enhancement of nutrient avail­ ability and phytohormone modulation and/or indi­ rectly through the biocontrol activity i.e. suppression of associated pathogens and/or the alleviation of biotic and abiotic stresses leading to the improve­ ment of both plant health and crop productivity (khan et al., 2020; basu et al., 2021; zhang et al., 2021; kumar et al., 2022). in the current study, nega­ tive and significant correlations were noted between the average fruit weight and yield per plant parame­ ters and the total culturable fungal populations. the fungal population estimated in the rhizosphere of pumpkins accessions may be mainly composed of soilborne pathogens naturally associated to pump­ kins plants which may be involved in the recorded decreases in fruit weight and yield. based on acp analyses, the 1st group was comprised of 4 accessions of c. maxima (namely c5, c23, c14.2 and c6.2) which are characterized with the highest production para­ meters (average fruit weight and yield per plant) and the highest populations of actinomycetes, bacteria, trichoderma spp. and aspergillus spp., and also the lowest fungal community in their rhizosphere. hence, these four microbial groups (bacteria, aspergillus spp., trichoderma spp. and actinomycetes) predomi­ nant in the rhizosphere of these 4 most productive pumpkins accessions may be involved, either individ­ ually or in consortium, indirectly in the promotion of pumpkins yield via their eventual antagonistic poten­ tial against their associated fungal pathogens. as demonstrated in yang et al. (2017) study, some potential plant­beneficial microbial agents could act as network key, thus reducing the chance of a given a soil­borne pathogen to invade the target plant species. also, chaurasia et al. (2018) demonstrated the successful role of actinomycetes on plant protec­ tion and growth promotion of solanaceae, cucurbitaceae, brassicaceae, amaranthaceae, umbelliferous, asteraceae, fabaceae, asparagaceae, and amaryllidaceae vegetable crops. also, as demon­ strated in hung and rutgers (2016) study, aspergillus spp. are multifaceted fungi that the plant benefits with different manner such as plant growth promo­ tion and protection. pascual et al. (2017) also empha­ sized the role of t. harzianum in reducing the natural infection of melon plants by f. oxysporum f. sp. melo‐ nis and in improving their yields. in aydi ben abdallah et al. (2019) study, bacillus subtilis sv41 and b. amy‐ loliquefaciens subsp. amyloliquefaciens sv65 have successfully decreased the soil infection potential by fusarium species, suppressed fusarium wilt severity and enhanced tomato growth and production. in our study, the 4 selected pumpkins accessions are quite able to exploit their associated beneficial indigenous microbial communities which could be considered in the future pumpkin breeding pro­ grams. in conclusion, this study clearly demonstrated the significant role of tested accessions in affecting the distribution of microbial community in their rhizos­ phere leading to differences in yield parameters between pumpkins accessions. the variation in the microbial community structure with the accessions tested might be due to the changes in the composi­ tion of their root exudates which need to be more elucidated in our future investigations. four acces­ sions of c. maxima (namely c5, c23, c14.2 and c6.2) have a great potential as they are characterized by the highest average fruit weight and yield per plant, adv. hort. sci., 2024 38(1): 13­24 22 the highest populations of actinomycetes, bacteria, trichoderma spp. and aspergillus spp., and the low­ est fungal population in their rhizosphere. thus, the exploitation and the re­integration of the recovered beneficial bacterial and trichoderma spp. populations associated with these four selected accessions of c. maxima will be considered in the future pumpkin breeding programs to reduce the threat imposed by their soil­borne pathogens and consequently led to more enhancements in pumpkin fruit yield into the less productive accessions. acknowledgements this work was funded by the ministry of higher education and scientific research of tunisia through the funding allocated to the research laboratory lr21agr03­production and protection for a sustainable horticulture (2phd), iresa­university of sousse, regional research centre on horticulture and organic agriculture of 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fattahi 1, a. khodabakhshzadeh 2 1 department of horticulture, faculty of agriculture, ferdowsi university of mashhad, po box 91775‐1163 mashhad, iran. 2 department of agronomy and horticultural sciences, faculty of agriculture and food industries, islamic azad university, science and research branch, po box 1477893855 teheran, iran. key words: abiotic stress, drought, essential oil, medicinal plant, physiological response, polyamines. abstract: in order to assess the effect of osmotic stress induced mannitol under in vitro conditions on some growth parameters of chamomile [chamomilla recutita (l.) rauschert], treatments were arranged and compared for the main effect of osmotic stress induced by mannitol in four levels (0, 2, 4, and 6 g/l), and the interaction effect of osmotic stress x putrescine (0, 0.5, and 1 mm). osmotic stress, especially induced by 4 and 6 g/l of mannitol, were found to sig­ nificantly reduce shoot height, root length, the number of shoot and root per plant, the fresh weight of shoot, and the fresh weight of root. when plants were grown on 0.5 mm of putrescine, the fresh weight of root, carotenoid, chlorophyll a, and chlorophyll b were increased, compared to plants grown on medium with 0 and 1 mm of putrescine. plants grown on medium with 0.5 and 1 mm of putrescine had an increased level of flavonoid, phenolic acid, and pro­ line under four levels of mannitol. the amount of 0.5 mm of putrescine signifi­ cantly improved plant biomass and essential oil content in plants grown on medium containing 0 and 2 g/l of mannitol. the results showed that the use of putrescine could improve chamomile tolerance to osmotic stress. 1. introduction chamomile [chamomilla recutita (l.) rauschert.] is an important medici­ nal plant, believed to have many properties. several studied have indicated the medicinal effects of chamomile on many diseases (reviewed in singh et al., 2011). with the growing importance of chamomile applications in mod­ ern medicine, many studies have focused on the investigation of exogenous factors such as plant growth regulators and environmental stresses on the growth parameters and physiological characteristics. it has been suggested (*) corresponding author: gh.rostamitobnag@mail.um.ac.ir citation: rostami tobnag g., fattahi s., khodabakh­ shzadeh a., 2020 ­ effects of putrescine applica‐ tion in culture medium in improving chamomile [chamomilla recutita (l.) rauschert.] tolerance to osmotic stress under in vitro conditions. ‐ adv. hort. sci., 34(2): 167­174. copyright: © 2020 rostami tobnag g., fattahi s., khodabakhshzadeh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 january 2020 accepted for publication 16 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 167­174 168 that the medicinal properties of chamomile result from its essential oil and antioxidant content (edris, 2007; wei and shibamoto, 2007; roby et al., 2013). however, these properties are negatively affected by environ­ mental factors such as osmotic stress (baghalian et al., 2011; jeshni et al., 2017). a study on drought effects on physiological and phytochemical traits of chamomile reported that agro­morphological characters, essential oil content and composition are significantly decreased in this condition (baghalian et al., 2011). afzali et al. (2006) showed that osmotic stress induced by polyeth­ ylene glycol and mannitol decreases the growth para­ meters at early growth stages of chamomile. polyamines have been found to involve in plant response to biotic/abiotic stress including osmotic stress, and other types of stress such as drought and salinity which impose osmotic stress on plants rather than their own specific effects (alcázar et al., 2010; shabala and munns, 2017; shokri­gharelo and noparvar, 2018). it has been shown that high levels of polyamines in plants are associated with tolerance to abiotic stress (alcázar et al., 2010; mandal et al., 2014; pál et al., 2018). it has also been shown that exogenous application of polyamines, including putrescine, increases the tolerance of plants to stressful conditions (talaat et al., 2005; bibi et al., 2010; hassanein et al., 2013). exogenous application of putrescine has been shown to improve morphological parameters (plant height, root length, number of shoots and roots, and plants biomass), physiological characters, and phyto­ chemical properties in wheat (mostafa et al., 2010), geranium (ayad et al., 2010), and egyptian carnation (el­ghorab et al., 2006). furthermore, positive effects of exogenous putrescine application have been shown to improve growth characters and tolerance under abi­ otic stress (ali et al., 2007; bibi et al., 2010; hassanein et al., 2013; mandal et al., 2014). there is no published evidence on the application of putrescine in vitro culture medium and its effects on morphological, physiological, and essential oil content of chamomile under different levels of osmotic stress induced by mannitol. the objectives of this study were therefore to investigate the effect of osmotic stress induced by mannitol under in vitro conditions and to evaluate the effects of putrescine on ameliorating the negative effects of osmotic stress on chamomile. 2. materials and methods plant materials and experimental conditions seeds of german chamomile [chamomilla recutita (l.) rauschert.] were used in this work. the experi­ ment was carried out under in vitro conditions. test tubes were used as the experimental unit and one plant was cultured in each test tube. all test tubes used in the experiments were sterilized. plants were grown in a growth room with temperature 25±2°c, relative humidity 50% and 60% during day and night respectively, and 14h photoperiod throughout the experiment. in order to measure the main effects of osmotic stress on chamomile and the effects of putrescine application in culture medium in reducing the stress effects, two experiments were designed with the same laboratory conditions. the first was based on a completely randomized design with one factor with four levels (osmotic stress). the second was arranged in factorial design based on completely randomized design (4 x 3) with two factors. four replications were used in both experiments, and 16 units for the first experiment and 48 for the second experiment were analyzed. medium culture and experiments basic ms (murashige and skoog, 1962) was used as the culture medium. seeds of chamomile were sterilized in a commercial chlorine solution (5%) for 20 minutes and then washed three times using dis­ tilled water. seeds were then gently placed on cul­ ture media. in the first experiment, one treatment including mannitol in four levels (control, 2, 4, and 6 g/l in cul­ ture media) was studied. in the second experiments, two treatments were studied; four mannitol levels were used, 0 (control), 2, 4, and 6 g/l in culture media to create osmotic stress combined with three levels of putrescine 0 (control), 0.5, and 1 mm. morphological traits the morphological parameters of chamomile measured in this experiment were following, shoot height (cm), root length (cm), shoot (n/plant), root (n/plant), fresh weight of root (rfw g/plant), and plant biomass (%). plant height was measured from the crown to the tip of the stem. fresh root was care­ fully washed with tap water after harvest and mea­ sured from the crown to the tip of the main root. to measure dry weight, plants were dried in an oven with 72°c temperature for 72 hours. chlorophyll content (a and b) and carotenoid the amount of 0.2 g of fresh leaves were ground in 10 ml of 99% methanol, then centrifuged at 3000 rpm for 5 min. the extract was used to measure light absorption at 653, 470, and 666 nm (lutts et al., rostami tobnag et al. ‐ putrescine improves tolerance to osmotic stress 169 1996). the following equations were used for calcu­ lating chlorophyll content: chla=chlorophyll a= 15.65 a666­7.34 a653 chlb=chlorophyll b= 27.05 a653­11.21 a666 cx+c=carotenoid=1000 a470 ­2.860 chla­ 129.2chlb measurement of flavonoid and phenolic acid the semi­dried samples were solved in 0.1 mol/l sodium acetate at 20:1 ratio (liquid: sample) at room temperature. the mixture was homogenized and centrifuged at 20000 g for 30 minutes at 4°c. the supernatant were aspirated and used to determine flavonoid and phenolic acid content. the procedure described by zhishen et al. (1999) was followed to measure flavonoid. the sample mixed with a solution containing aluminium chloride and sodium nitrite was added to 30 µl of sodium nitrite (10%), 60 µl of aluminium chloride hexahydrate (20%), 200 µl of naoh (1m) and 400 µl of water. the absorbance reading was recorded at 510 nm every 20 s for 1 minute. the absorbance reading was compared to a standard curve drawn from catechin (69­689 µmol/l). the data were expressed as µmol catechin equiva­ lents per gram of fresh or dry matter. to measure phenolic acid, 2.5 ml of the folin­ ciocalteu reagent and 2 ml of saturated sodium car­ bonate (75 g/l) were mixed with 50 µl of sample and homogenized for 10 s and heated for 30 minutes at 45°c. the absorbance reading was recorded at 720 nm and compared to the standard curve made from gallic acid (235­1176 µmol/l). the data were expressed as µmol gallic acid equivalents per gram of fresh or dry matter. proline content to determine proline content of shoot, 0.5 g of the sample were homogenized in 3% (w/v) sulphos­ alycylic acid and then filtered through filter paper (bates et al., 1973). acid ninhydrin and glacial acetic acid were added into the mixture and then heated at 100°c for 1 h in a water bath. toluene was used to extract the mixture and the absorbance of fraction was read at 520 nm. proline concentration was determined using calibration curves and expressed as µmol proline g fw. essential oil content hydrodestillation was used for the extraction of essential oil, where the sample of 25 g of chamomile herb dried in an oven was homogenized and boiled in 600 ml of distilled water in clevenger for 3 hours. then, water was gently removed from the tank and the amount of extracted essential oil was measured. statistical analysis three weeks after culturing, the data were ana­ lyzed by one­way and two­way anova using jmp8­ statistics software. mean values were separated with duncan’s multiple range test (p≤0.05). 3. results effect of osmotic stress on growth parameters the statistical analysis of data from first experi­ ment (16 experimental units) showed that osmotic stress had significant effects on shoot height and fresh weight of shoot at p<0.01, and on root length, number of shoots, number of roots, and fresh weight of roots at p<0.05. the main effect of osmotic stress on morphological traits is shown in figure 1, with an evident reduction in morphological traits under m2, m4, and m6. mean comparison of data showed that morphological traits decrease with increasing levels of osmotic stress. control plant (without stress) showed the highest morphological traits compared to plants grown under m2, m4, and m6. plants grown on medium with 6 g/l of mannitol showed sig­ nificant reduction (fig 1b). the main effect of osmotic stress at m2 and m4 levels was more adverse on shoots than on roots. root length, number of roots, and fresh weight of root were significantly decreased under m2 and m4 compared to a control group according to duncan’s multiple­range test (p≤0.05), but no significant difference was observed between plants grown under m2 and m4 conditions (fig. 1b). the number of shoots showed a significant decrease, and plants which were grown on culture medium containing 2 and 4 g/l of mannitol, had no significant difference (p≤0.05) (fig. 1b). under m6 conditions, all growth parameters especially shoot traits showed sever reduction. effect of putrescine application on morphological traits under osmotic stress the second experiment compared two treatments including osmotic stress and application of putrescine (a total of 48 experimental units). variance analysis of data revealed that interaction effects of osmotic stress and putrescine (os x pu) were significant for shoot length, root length, number of shoots, and fresh weight of shoots (table 1), while the main effect of putrescine was a significant on the fresh weight of roots (fig. 2). the interaction effect and the main effect of treatments were not significant on number of roots per plant (data not shown). adv. hort. sci., 2020 34(2): 167­174 170 putrescine significantly increased the fresh weight of roots at 0.5 and 1 mm compared with plants grown on basic ms medium (without putrescine). in terms of shoot length, root length, number of shoots, and fresh weight of shoots, plants grown on a basic ms medium containing 0.5 and 1 mm of putrescine and without mannitol showed significantly increased traits compared to control plants (without mannitol and putrescine) and other groups (table 1). plants grown on medium containing 2 and 4 g/l of mannitol fig. 1 ­ main effects of osmotic stress induced by mannitol (m0= without mannitol, m2= 2 g/l of mannitol, m4= 4 g/l of mannitol, and m6= 6 g/l of mannitol) in culture medium on morphological traits of chamomile. (a) morphological traits under different levels of osmotic stress (m0= without mannitol, m2= 2 g/l of mannitol, m4= 4 g/l of mannitol, and m6= 6 g/l of mannitol), (b) mean values of shoot height, root length, number of shoots, and number of roots, (c) mean values of fresh weight of shoots and roots. different letters above each bar indicate significant differences according to duncan’s multiple­range test (p≤0.05). fws= fresh weight of shoot, fwr= fresh weight of root. table 1 ­ interaction effects of osmotic stress induced by mannitol and putrescine on morphological and chemical traits of chamomile) different letters within each column indicate significant differences according to duncan’s multiple­range test (p≤0.05). * p<0.05 and **0.01, indicate level of significance. os= osmotic stress; m0= without mannitol; m2= 2 g/l of mannitol; m4= 4 g/l of mannitol; m6= 6 g/l of mannitol; pu0= without putresci­ ne; pu0.5= 0.5 mm of putrescine; pu1= 1 mm of putrescine; sfw= fresh weight of shoot. treatment morphological traits chemical traits mannitol (g/l) putrescine (mm) shoot length (cm) root length (cm) shoot (no./plant) sfw flavonoid phenolic acid proline m0 pu0 3.55 b 4.425 b 33.5 a 0.6168 b 33.61 cde 32.87 cde 10.65 d pu0.5 6.025 a 6.325 a 37.5 a 1.615 a 42.02 c 43.24 c 12.86 cd pu1 5.6 a 6.075 a 35.5 a 1.357 a 36.35 cde 34.47 cd 12.26 d m2 pu0 2.1 cde 3.275 c 23 b 0.34 bcd 38.38 cd 32.5 cde 16.67 bc pu0.5 2.475 cd 4 bc 24 b 0.5138 bc 22.69 e 17.7 e 15.99 bc pu1 1.6 ef 2.15 e 15 b 0.323 bcd 25.67 de 22.72 de 15.27 bc m4 pu0 1.925 def 3.325 c 14.75 cd 0.126 d 70.08 b 62.32 b 18.41 bc pu0.5 2.775 c 3.45 c 19.25 bc 0.4565 bcd 36.77 cde 30.6 cde 19.27 b pu1 1.1 f 1.275 f 8.25 e 0.1505 d 45.11 c 44.76 c 21.46 ab m6 pu0 1.125 f 2.3 de 11.5 de 0.2212 cd 71.68 b 67.52 b 24.21 a pu0.5 1.9 def 3.125 cd 17.25 c 0.5102 bc 69.89 b 62.81 b 20.92 ab pu1 1.3 ef 1.175 f 10.75 de 0.193 cd 90.01 a 90.26 a 20.66 ab significance os ** ** ** ** ** ** ** pu ** ** ** ** * * * os x pu ** ** * ** ** ** ** rostami tobnag et al. ‐ putrescine improves tolerance to osmotic stress 171 plus 0.5 mm of putrescine (m2pu0.5 and m4pu0.5) showed significantly better traits compared to plants grown on medium containing 2 and 4 g/l of mannitol plus 0 and 1 mm of putrescine (m2pu0, m2pu1, m4pu1, and m4pu1). however, plants grown on medium with 6 g/l of mannitol (severe osmotic stress) and with/without putrescine showed signifi­ cantly the lowest means compared to other groups (table 1). effect of putrescine application on physiological traits under osmotic stress variance analysis of physiological traits showed that main effect of putrescine on carotenoid, chloro­ phyll a and b was significant (data not shown), while interaction effect of osmotic stress x putrescine was significant on flavonoid, phenolic acid and proline (table 1). the carotenoid, chlorophyll a and b of chamomile were significantly increased in plants grown on medi­ um with 0.5 mm of putrescine (fig. 2). the flavonoid, phenolic acid, and proline content in plants grown on medium with 1 mm of putrescine plus 6 g/l of manni­ tol showed the highest content compared to other groups (table 1). the lowest contents of flavonoid, phenolic acid, and proline in each group (m0, m2, fig. 2 ­ the effect of putrescine on morphological and physiolo­ gical traits of chamomile (fresh weight of root, carote­ noid, chlorophyll a and b). putrescine was added in 0 (control), 0.5, and 1 mm in culture media. different let­ ters above each bar indicate significant differences according to duncan’s multiple­range test (p≤0.05). m4, and m6) were observed in plants grown on medium without putrescine (pu0) and the highest contents in each group were observed in medium with 1 mm of putrescine (m0pu1, m2pu1, m4pu1, and m6pu1) compared to control groups. effect of putrescine application on plant biomass and essential oil under osmotic stress variance analysis of data related to plant biomass and essential oil showed that the interaction effect of osmotic stress x putrescine was significant (p<0.05) (data not shown). plants grown on medium contain­ ing 2, 4, and 6 g/l of mannitol (m2, m4, and m6) plus 0.5 and 1 mm of putrescine (pu0.5 and pu1) had the lowest plant biomass and essential oil content com­ pared to the control group (m0pu0, m0pu0.5, m0pu1) (fig. 3 and 4). the largest increase in biomass was observed in plants on medium without application of mannitol but treated with 0.5 and 1 mm of putrescine fig. 3 ­ interaction effects of osmotic stress x putrescine on morphological traits of chamomile. mannitol was used in four levels; 0 (control), 2, 4, and 6 g/l in culture media to create osmotic stress. putrescine was added in 0 (con­ trol), 0.5, and 1 mm in culture media. fig. 4 ­ interaction effects of osmotic stress x putrescine on the plant biomass of chamomile. different letters above each bar indicate significant differences according to duncan’s multiple­range test (p ≤ 0.05). 172 adv. hort. sci., 2020 34(2): 167­174 (m0pu0.5 and m0pu1). plants grown on medium with 2 g/l of mannitol plus 0.5 and 1 mm of putrescine had a larger biomass compared to plants grown on m2pu0, but significantly lower biomass compared to the control group. in plants grown on medium with 4 and 6 g/l of mannitol, no significant difference was observed between plants placed on medium with 0, 0.5, and 1 mm of putrescine (fig. 3). the largest amount of essential oil was in the group of plants grown on medium without osmotic stress (m0;) and with 0.5 and 1 mm of putrescine (pu0.5 and pu1). the group of plants grown on medi­ um with 2 g/l of mannitol, plants grown on medium with 0.5 and 1 mm of putrescine showed significantly more essential oil (m2pu0.5 and m2pu1) compared to plants grown on medium without putrescine (m2pu0). even though plants on medium with 0.5 and 1 mm of putrescine plus 4 and 6 g/l of mannitol did not show significant difference compared to plants grown on medium without its application (fig. 4). 4. discussion and conclusions the effect of putrescine application in culture medium containing mannitol has not previously been reported so far. the studies on other plant species and also other types of stresses will therefore be used for discussion. in this study, the effect of osmot­ ic stress created by mannitol under in vitro condi­ tions, the main effect of putrescine on the growth of chamomile, and putrescine effects in ameliorating effects of osmotic stress on chamomile were investi­ gated. osmotic stress is a side effect of some abiotic stresses such as drought and salinity in which water absorption is limited, leading to conditions similar to drought (shen et al., 1999). drought and salt stress have been found to decrease the morphological traits in many medicinal plants, in terms of length and number of shoots and roots, fresh weight of shoots, and fresh weight of roots (afzali et al., 2006; jaleel et al., 2008; arazmjo et al., 2010; anjum et al., 2011). in agreement with the findings of this study), afzali et al. (2006) reported a decrease in fresh weight of shoots and roots in chamomile under poly­ ethylene glycol­induced osmotic stress. in another study, dadkhah (2010) tested the effects of salinity on the plant height and number of shoots of chamomile in a pot experiment. dadkhah reported a significant decrease in plant height and number of shoots in early stage of the stress. in respect of drought stress induced by mannitol under in vitro conditions, ghaheri et al. (2015) findings in steviare baudiana bertoni are in accordance with the findings of this study. regarding putrescine effects in increasing mor­ phological traits, a number of studies have showed that foliar application of putrescine increases plant height, root length, shoot (number per plant), and root (number per plant) (talaat et al., 2005; mostafa et al., 2010; amin et al., 2011; hassan and bano, 2016), and improving physiological traits, in terms of chlorophyll a, chlorophyll b, carotenoid (talaat et al., 2005; hassan and bano, 2016), and proline (hassan and bano, 2016). this study provides evidence of improved flavonoid, phenolic acid, and other para­ meters (table 1, fig. 2) in chamomiles grown on medium containing 0.5 and 1 mm of putrescine. studies that investigate effects of putrescine under in vitro conditions have not been found, but in agree­ ment with the findings of this study, several studies have revealed that plants treated with putrescine have increased growth parameters and more toler­ ance to abiotic stresses such as osmotic stress, drought, salinity, and temperature compared to untreated plants (jaleel et al., 2008; alcázar et al., 2010; hassanein et al., 2013). investigating the effects of putrescine foliar application on chamomile and sweet marjoram under salinity stress, ali et al. (2007) found that putrescine significantly increased flavonoid content. in addition, the use of putrescine was found to enhance chlorophyll a, chlorophyll b, carotenoid, phenolic acid, and morphological traits under stress conditions compared to untreated plants (amin et al., 2011; shallan et al., 2012; hassanein et al., 2013; hassan and bano, 2016). other authors have indicated positive effects of putrescine application on plant biomass and essential oil in chamomile and sweet marjoram (ali et al., 2007), wheat (hassan and bano, 2016), onion (amin et al., 2011), and cotton (shallan et al., 2012). findings in this study showed that putrescine applica­ tion improves the plant biomass and essential oil content under normal growth conditions and in chamomiles grown on medium containing 2 g/l of mannitol (fig. 4 and fig. 5), whereas plants grown on medium with 4 and 6 g/l of mannitol showed remark­ able decreased levels of biomass and essential oil compared to the control group, and the study did not find the positive effects of putrescine under these levels of osmotic stress (m4 and m6). in line with these findings regarding the positive effects of rostami tobnag et al. ‐ putrescine improves tolerance to osmotic stress 173 putrescine under normal growth conditions and 2 g/l of mannitol stress, ali et al. (2007) reported an increase in the plant biomass in chamomile and sweet marjoram, ayad et al. (2010) in geranium and el­ghorab et al. (2006) in egyptian carnation report­ ed a high level of essential oil in plants treated with putrescine. pál et al. (2018) in their study on wheat reported that putrescine treatment induces stress­responsive genes that overlap with the genes induced by osmot­ ic stress. they suggested that changes induced by putrescine overlap with changes induced by osmotic stress, and lead to better tolerance in plants treated with putrescine. in another study, bibi et al. (2010) showed that putrescine application significantly increases the endogenous putrescine concentration. they suggested that stress tolerance correlates with an increment of putrescine. the results of the pre­ sent study do not provide evidence at the molecular level and for the endogenous concentration of putrescine in chamomile but the data showed an increased values of observed traits when putrescine was used in medium culture. as suggested by mandal et al. (2014), polyamines including putrescine allevi­ ate oxidative stress induced by osmotic stress. it is believed that oxidative stress created by being exposed to abiotic stresses such as osmotic stress, drought, and salinity is one of most important rea­ sons for remarkable reduction of morphological traits and plant yield (shokri­gharelo and noparvar, 2018). other studies have shown that plants with efficient antioxidant systems, including high level of flavonoid, phenolic components (reviewed in shabala and munns, 2017), and plants with high level of proline content (ahmad et al., 2016) show more tolerance and these indices have been regarded as one of tol­ erance characters in different plants. this study revealed that flavonoid, phenolic acid, and proline are increased in chamomile grown on medium with putrescine (table 1). this could explain the better morphological and physiological traits as well as essential oil content studied in this work in plants under osmotic stress. osmotic stress, especially at m4 and m6 levels (4 and 6 g/l of mannitol) significantly reduce the mor­ phological traits of chamomile under in vitro condi­ tions. the main aim of the current study was to use putrescine in culture medium to assess its effects in ameliorating osmotic stress. the values of morpho­ logical traits, some physiological traits, and contents of essential oil of chamomile were significantly higher in plants grown on medium containing 0.5 and 1 mm of putrescine compared to plants grown on medium without putrescine application. application of putrescine may improve chamomile tolerance to osmotic stress, and may be considered as one of substances that can be used to improve chamomile quality under osmotic stress. references afzali s., hajabbasi m., shariatmadari h., razmjoo k. khoshgoftarmanesh a., 2006 ­ comparative adverse effects of peg‐or nacl‐induced osmotic stress on germination and early seedling growth of a poten‐ tial medicinal plant matricaria chamomilla. 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2007 ­ antioxidant activities and volatile constituents of various essential oils. ­ j. agr. food. chem., 55(5): 1737­1742. zhishen j., mengcheng t., jianming w., 1999 ­ the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. ­ food chem., 64(4): 555­559. impaginato 449 adv. hort. sci., 2019 33(4): 449­455 doi: 10.13128/ahsc­8098 economic analysis of crisp lettuce production in different planting spacing and soil cover e.p. vendruscolo 1 (*), a.h. alcântara rodrigues 2, s.r. correia 2, p.r. oliveira 2, l.f. cardoso campos 3, a. seleguini 4 1 mato grosso do sul state university, rod. ms 306, km 6.4, cep 79540‐ 000, cassilândia, mato grosso do sul, brazil. 2 agronomy school, goiás federal university, samambaia campus, esperança avenue, cep 74690‐900, goiânia, goiás, brazil. 3 goiás state university, saudade street, 56, vila eduarda, cep 76100‐ 000, sâo luis dos montes belos, goiás, brazil. 4 triângulo mineiro federal university, rio paranaíba avenue, cep 38280‐000, iturama, minas gerais, brazil. key words: economic indicators, lactuca sativa, production costs, vegetables. abstract: the objective of this study was to estimate and evaluate the econom­ ic indicators of lettuce production, cultivated using different soil cover and plant spacing. the experiment was conducted in subdivided parcels, with four replications. the treatments were composed by a combination of three soil cover (uncovered soil, straw and plastic cover) and three planting spaces (0.25 x 0.20, 0.25x0.25 and 0.25x0.30 m). the productivity and economic indicators were evaluated for a production area of 1000 m2. for the different treatments, a total operating cost of usd 781.80 to usd 663.30 1000 m­2 was obtained. it was observed that for the cultivation of lettuce in soil covered by straw, from the rubbing, and spacing of 0.25x0.25 m the economic indicators were raised. with a productivity of 687.70 boxes 1000 m­2, for this treatment was obtained gross revenue of usd 1,828.99, operating profit of usd 1,135.41 and a prof­ itability index of 62.08%. thus, lettuce cultivation provides positive profitability regardless of the spacing or type of cover used and the combination between the 0.25 x 0.25 m planting spacing and the use of straw as a soil cover culmi­ nates in higher monetary gains. 1. introduction the consumption of vegetables has been increasing continuously due to the dietary habits adopted by the population (maziero et al., 2017), which consequently influences the demand for higher yields (silveira et al., 2015). among the products sought by consumers, lettuce is the world leader in terms of acceptance, increasing its importance to the productive sector due to the large volume of commercialization (vieira and barreto, 2006). considering the participation of family farms in the vegetable produc­ (*) corresponding author: agrovendruscolo@gmail.com citation: vendruscolo e.p., alcântara rodrigues a.h., correia s.r., oliveira p.r., cardoso campos l.f., seleguini a., 2019 ­ economic analysis of crisp lettuce production in different planting spacing and soil cover. ­ adv. hort. sci., 33(4): 449­455 copyright: © 2019 vendruscolo e.p., alcântara rodrigues a.h., correia s.r., oliveira p.r., cardoso campos l.f., seleguini a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 february 2019 accepted for publication 21 june 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 449­455 450 tion scenario, one of the main productive obstacles is the investment required. most of the costs are relat­ ed to inputs purchase, as fertilizers, seedlings and seeds (rezende et al., 2005, 2009; batista et al., 2013). this burden of the production process is nec­ essary, in order to obtain higher quality products, increasingly demanded by the consumer market. through the research, it is seeking the application of techniques that assist the rural producers in obtaining greater monetary returns, encouraging them to continue in the agricultural activity. simple changes in management can be effective strategies for reducing production costs. in this sense, for the lettuce crop, it is observed that the use of straw as a cover is an effective way of preserving the soil physic­ ochemical characteristics, favoring plants develop­ ment and increasing the profitability (vendruscolo et al., 2017 a). in addition, due to intense soil move­ ment applied to vegetable production (ziech et al., 2014), the cover contributes to the preservation of organic matter and erosion reduction (cividanes, 2002; souza and resende, 2006). materials that are easily accessible to the rural producer, such as straw from grazing, can be used as mulch, replacing other materials such as plastic, for example, which has an effective participation in the costs of producing vegetables (vendruscolo et al., 2017 b), also representing a source for environmen­ tal contamination (chang et al., 2013). in this sense, it is important to generate information on the eco­ nomic feasibility of techniques applied to agriculture, based on data with high reliability. the presence of a superficial straw layer is benefi­ cial from the point of view of soil quality mainte­ nance (cardoso et al., 2012), favoring the develop­ ment of plants of economic interest (torres et al., 2015; vendruscolo et al., 2017 a). it also favors the maintenance of soil moisture by controlling the direct evaporation of the surface (carneiro et al., 2014), reducing the need for large volumes of water in irrigation. the decrease in weed competition due to the suppression of spontaneous plants is another advantage of soil cover (moraes et al., 2013), which implies the less need for herbicides or even manual weeding. in this sense, it was verified, for the radish culture, that the maintenance of the cultural remains of silk flower on the soil surface increased the num­ ber of commercial roots produced (oliveira et al., 2015). in addition to the soil cover, other management techniques can assist producers in obtaining higher yields and superior quality of their product. the plant population used in agricultural crops is decisive in creating an environment conducive to its develop­ ment and should be established for the specific con­ ditions of a given locality, in order to avoid excessive competition for resources such as water, light, nutri­ ents and carbon dioxide essential to their develop­ ment (taiz et al., 2017). in addition, the unnecessary expense of purchasing seedlings, one of the inputs with the largest share in production costs (vendruscolo et al., 2017 a). specifically for lettuce, it is observed that larger plant spacings can generate plants with higher weight (vasconcelos et al., 2017), which may be related to the broad development of the aerial part observed in commercial genotypes. it is also verified that population density variation can be an effective tool for controlling weeds in different crops (carvalho and guzzo, 2008; bajwa et al., 2017; li et al., 2018), decreasing spending on hand and acquisition of agro­ chemicals. in view of the information above, the objective of this study was to estimate and evaluate the econom­ ic indicators of lettuce cultivation, using different soil cover and plant spacing. 2. materials and methods the study was conducted in an experimental area located at the goiás federal university, in goiânia, goiás, brazil. for the locality the following average climatic indicators are verified: annual precipitation of 1,575 mm and average monthly temperature of 22.9°c, the predominance of aw climate, character­ ized by a tropical climate with rainy season of october to april and a period with precipitations below 100 mm monthly between may and september. the average climatic parameters of air temperature and humidity were obtained at an evap­ orimetric station at 300 m distance from the experi­ mental area (fig. 1). the soil was classified as latossolo vermelho, fol­ lowing the methodology proposed by santos et al. (2013). the soil chemical analysis (depth of 0­0.2 m), before the implantation of the experiment revealed the following nutrient content: ca2+: 2.8 cmolc dm­3, mg2+: 1.8 cmolc dm­3, k+: 0.37 cmolc dm­3, p (mehlich i): 25.8 mg dm­3, organic matter: 3.0 g dm­3, al3+: 0.0 cmolc dm­3, h+al: 2.8 cmolc dm­3, ph (cacl2): 5.3, 7.8 cmolc dm­3 of ctc, 64.0% of v, according to donagemma et al. (2011). the soil granulometric analysis, according to da silva (2009), presented 44 g vendruscolo et al. ‐ economic analysis of crisp lettuce 451 kg­1 of clay in 0­0.2 m layer (da silva, 2009). previously to the planting, an initial fertilization was carried out, which consisted of the equivalent application of 320 kg ha­1 of simple superphosphate and liming in order to raise the bases saturation to 80%. the limestone was incorporated and the beds were confectioned with 1.00 m wide and spaced 0.50 m apart. the experiment was conducted in a subdivided plots design, with four replications for each treat­ ment. the treatments were composed by a combina­ tion of three soil cover (uncovered soil, straw and plastic cover) and three planting spaces (0.25x0.20, 0.25x0.25 and 0.25x0.30 m), making a total of nine treatments. each plot had dimensions of 1.0x1.25 m (1.25 m2). for evaluation, the central plants of the two internal lines were used and the remaining plants were used as a border. irrigation was carried out by drippers spaced twenty centimeters apart in three polyethylene tapes suitable for this purpose, positioned between the planting lines. the acquisition of the drip tapes was included in the calculation of the costs, considering the implantation of this system in the production area and the useful life of this material. however, the amounts spent on the purchase of other materials, such as pumps, pipes and others irrigation materials were not considered. after the irrigation tapes placement, the soil cov­ ers were installed according to the treatments. for that, a straw from the grass (zoysia japonica) was dis­ tributed over the plots until a layer of 5 cm was obtained. the plastic cover consisted of the place­ ment of double­sided polyethylene canvas (black and white), with the white face facing upwards. the seedlings of crisp lettuce, cv. vanda, were purchased in a commercial seedling producer, with a surplus of 10% of the quantity required for the replacement of dead plants. the seedlings trans­ planting proceeded on may 1, 2016. for this purpose, pits were opened amidst the covers, with sufficient size for seedling insertion. cover fertilization was carried out in three appli­ cations during the cycle, based on the recommenda­ tions for the crop (trani et al., 2014), applying 60 kg ha­1 of urea (45% n) and 50 kg ha­1 kcl (60% k2o). during the lettuce cultivation, there was no applica­ tion of fungicides, insecticides or herbicides. plants were harvested at 45 days after transplanting. treatments were considered as commercial crops with the purpose of determining the production costs of a productive cycle of crisp lettuce. in this way, the total operational cost (toc) structure was obtained as proposed by martin et al. (1998) by adding up the effective operating cost (eoc), which is composed of the expenses of the operations and inputs used, other expenses involved (oe), and cost­ ing interest per year (ciy). a rate of 5% of total eoc expenses was considered for other expenses (oe) that involves costs with administration, technical assistance and other fees to be paid for the activity, while costing interest (ciy) used was 6.5% per year, over 50% of eoc, estimated as an annual interest rate (martin et al., 1998). before the calculation of toc, the base total oper­ ating cost (toc base) was obtained, for which the costs related to the experimental variables were not considered, remaining constant for all treatments. for monetary amounts, the presentation was made in reais (r$) and us dollars (usd). the conversion was made considering the quotation on june 15, 2018 (usd 1.00 = r$ 3.76). each economic data was based on a single production mean, which was com­ posed by the four replications. the average prices, received by the producers, were obtained from the goiás state supply centers (2018) website. the average price paid to producers in the first half of june of 2018 was usd 2.66 per box of 4.8 kg, for calculation purposes, the same was used in the present study. the average region labor force in 2018 was usd 18.62 day­1. thus, labor costs were obtained through the index generated by the need for manual opera­ tions for each operation, multiplied by the daily value. for inputs, the cost was calculated based on the average product value in the region, obtained in the first half of 2018, and the amount of material used. fig. 1 ­ summary of climatic conditions of relative air humidity and maximum, average and minimum temperature during the period of conduction of the study. adv. hort. sci., 2019 33(4): 449­455 452 the profitability of each treatment was obtained through estimates of gross revenue, obtained multi­ plying the quantity produced (4.8 kg boxes) for the average price received by the producers, the differ­ ence between gross revenue and total operating cost represents the profitability index: the proportion of the gross revenue that represents the final amount after covering the production total operational cost. equilibrium price was also obtained as the minimum price necessary to be obtained to cover the toc at a given level of production total operating cost, consid­ ering the average productivity obtained by the pro­ ducer, and the equilibrium productivity, given the minimum productivity required to cover toc at a given level of production total operating cost. 3. results and discussion the production of crisp lettuce in an area equiva­ lent to 1000 m2 presented a base total operating cost (toc base) of usd 454.64 (table 1), which was formed by the cost of mechanized operations (68.02%), manual operations (20.47%), inputs (3.88%), other expenses (4.62%) and interest expens­ es (3.00%). the effective participation of mechanized operations was due to the acquisition of dripping tapes, which had a 96.75% participation in this item and 65.81% over toc base. in addition, manual oper­ ations and inputs had a share of 20.47% and 3.88%, respectively, in the toc base. the small inputs participation is due to the non­ insertion of the seedlings acquisition value. however, when this cost is added to the inputs amount, the participation on the toc and the toc itself increases (table 2). the seedlings value also have a participa­ tion of 42.47%, 38.84% and 36.47% for treatments composed of 0.25x0.20 m, 0.25x0.25 m and 0.25x0.30 m of planting spacing, respectively. these results related to the seedlings acquisition corrobo­ rate with those obtained by rezende et al. (2005), who verified a participation of 55.20% of the inputs on toc and by rezende et al. (2009), which obtained a burden of approximately 49.30% on toc due to expenditures on inputs and other materials. when the plastic cover was used, it was verified that the acquisition of the cover added to its place­ ment, participated in an average of 9.63%, while the straw had a participation around 2.66%, relative to its distribution on the beds. during the lettuce cycle, weed control burdened in 7.76% and 2.66%, respec­ tively, the treatments without cover and straw cover, table 1 ­ estimated base total operational cost for crispy lettuce in 1,000 m2 description specification quantity unit cost r$ cost r$ unit cost usd cost usd a ‐ mechanized operations tillage hm tp 65cv. 4x2 + grade aradora 14 x 26" 0.30 52.95 15.89 14.08 4.22 disk harrow hm tp 65cv. 4x2 + grade niveladora 28x22" 0.10 52.43 5.24 13.94 1.39 beds preparation hm tp 65cv. 4x2 + roto­encanteirador 0.35 47.75 16.71 12.70 4.44 irrigation irrigation equipment 1.00 1,125.00 1,125.00 299.20 299.20 subtotal a 1,162.84 0.00 309.27 b ‐ manual operations beds preparation man­day 0.50 70.00 35.00 18.62 9.31 seedling transplant man­day 1.00 70.00 70.00 18.62 18.62 fertirrigation man­day 3.00 70.00 210.00 18.62 55.85 harvest man­day 0.50 70.00 35.00 18.62 9.31 subtotal b 350.00 0.00 93.09 c ‐ inputs limestone kg 13.00 0.09 1.16 0.02 0.31 single superphosphate kg 32.00 1.34 42.88 0.36 11.40 kcl (60% k2o) kg 5.00 2.00 10.00 0.53 2.66 urea (45% n) kg 6.00 2.05 12.30 0.55 3.27 subtotal c (r$) 66.34 0.00 17.64 effective operational cost (a+b+c) 1,579.10 419.99 d ‐ other expenses 78.96 21.00 e ‐ costing interest per year 51.32 13.65 base total operating cost (a+b+c+d+e) 1,709.46 454.64 vendruscolo et al. ‐ economic analysis of crisp lettuce 453 ment (moraes et al., 2013). in contrast, the lowest profitability index was obtained using the plastic cover and the planting spacing of 0.25x0.20 m. under these conditions, the low productivity in addition to the cover value were the main factors that con­ tributed to the variables decrease in 23.44%, 45.53% and 28.85% of gross revenue, operating profit and profitability index, respectively, compared with the best treatment. the lower equilibrium yield was obtained for the treatment composed by 0.25x0.30 m planting spac­ ing and straw cover. this result was 15% below the treatment composed of a plastic cover and 0.25x0.20 m planting spacing, for which the highest equilibrium production was verified (table 4). for this treatment, the highest equilibrium price was also obtained with an increase of 47.23%, compared to the lowest equi­ librium price, obtained with the use of straw and planting spacing of 0.25x0.25 m. in general, the use of straw as soil cover favors the economic return with the lettuce crop. in addi­ respectively (table 2). the acquisition costs and placement of the plastic cover, together with the greater seedlings acquisition costs caused by the smaller planting spacing, resulted in the higher toc. this was 15.15% higher than the lowest toc, obtained with the combination of straw coverage and larger planting spacing (0.25x0.30 m) (table 2). according to the average price received by pro­ ducers during the first half of june 2018 (usd 2.66 box 4.8 kg­1), it was found that gross revenue, operat­ ing profit and profitability index obtained varied according to the planting spacing and cover used (table 3). treatment composed of the straw cover and planting spacing of 0.25x0.25 m resulted in higher values for the three variables. this result is due to the lower straw cost and its action on the mainte­ nance of the physical and chemical quality of the soil (collier et al., 2011; cardoso et al., 2012), as well as weed control through suppression of their develop­ table 2 ­ participation of the cost variation factors over the total operational cost, in 1,000 m2, for the cultivation of crisp lettuce in dif­ ferent planting spacing and soil cover table 3 ­ productivity, gross revenue, operating profit and profitability index, obtained with the cultivation of crisp lettuce in different planting spacings and soil cover, in 1,000 m2 cover planting spacing (m) cost in r$ cost in usd seedlings cost cover cost cover placement weed control oe + ciy toc seedlings cost cover cost cover placement weed control oe + ciy toc control 0.25x0.20 870.00 0.0 0.00 210.00 89.10 2,878.50 231.40 0.00 0.00 55.90 23.70 765.60 control 0.25x0.25 690.00 0.0 0.00 210.00 74.30 2,683.60 183.50 0.00 0.00 55.90 19.70 713.70 control 0.25x0.30 585.00 0.0 0.00 210.00 65.60 2,570.00 155.60 0.00 0.00 55.90 17.40 683.50 straw 0.25x0.20 870.00 0.0 70.00 70.00 83.30 2,802.70 231.40 0.00 18.60 18.60 22.20 745.40 straw 0.25x0.25 690.00 0.0 70.00 70.00 68.50 2,607.90 183.50 0.00 18.60 18.60 18.20 693.60 straw 0.25x0.30 585.00 0.0 70.00 70.00 59.80 2,494.20 155.60 0.00 18.60 18.60 15.90 663.30 plastic 0.25x0.20 870.00 196.40 70.00 0.00 93.80 2,939.60 231.40 52.20 18.60 0.00 24.90 781.80 plastic 0.25x0.25 690.00 196.40 70.00 0.00 78.90 2,744.70 183.50 52.20 18.60 0.00 21.00 730.00 plastic 0.25x0.30 585.00 196.40 70.00 0.00 70.20 2,631.10 155.60 52.20 18.60 0.00 18.70 699.70 cover planting spacing (m) produtivity (boxes 4.8 kg) gross revenue operation profit profitability index (%)r$ usd r$ usd control 0.25x0.20 592.96 5,929.57 1,577.01 3,051.09 811.46 51.46 control 0.25x0.25 640.17 6,401.70 1,702.58 3,718.07 988.85 58.08 control 0.25x0.30 641.27 6,412.66 1,705.49 3,842.69 1,021.99 59.92 straw 0.25x0.20 663.33 6,633.27 1,764.17 3,830.57 1,018.77 57.75 straw 0.25x0.25 687.70 6,877.01 1,828.99 4,269.15 1,135.41 62.08 straw 0.25x0.30 553.92 5,539.20 1,473.19 3,045.01 809.84 54.97 plastic 0.25x0.20 526.50 5,265.02 1,400.27 2,325.46 618.47 44.17 plastic 0.25x0.25 564.50 5,644.95 1,501.32 2,900.25 771.34 51.38 plastic 0.25x0.30 557.81 5,578.11 1,483.54 2,947.06 783.79 52.83 454 adv. hort. sci., 2019 33(4): 449­455 tion, it can be easily acquired in the rural property by scrubbing areas with grasses and acts in the preven­ tion of physical and chemical soil erosion (cardoso et al., 2012), as well as acting on inhibition of weed development (moraes et al., 2013), favoring the development of the culture of interest. these facts are supported by a study about the lettuce cultivation on the vegetal residue of different species, which demonstrates that plants cultivated on sorghum and millet straw favor the crop prof­ itability indexes. in this study, it was also observed the importance of choosing the species that will com­ pose the straw cover, since there are effects of allelopathy (vendruscolo et al., 2017 a, b). the straw deposition on the soil surface, in the cultivation of american lettuce and cabbage, also favored the development of these crops, especially when using brachiaria or millet straw (torres et al., 2015). 4. conclusions the insertion of these techniques, with the use of vegetal soil cover, in addition to culminating in higher financial returns, due to lower production costs and increased productivity, also represents a technique of greater environmental viability. it is observed that crops with high demand for technologies tend to generate large amounts of slow degradation residues, such as plastics, inferring in environmental contamination (chang et al., 2013). thus, we con­ cluded that the crisp lettuce crop provides positive profitability, regardless of the planting spacing or cover type used. for the conditions observed during this study, the combination of the 0.25x0.25 m plant­ ing spacing and the straw as a soil cover culminates in higher financial returns. references bajwa a.a., walsh m., chauhan b.s., 2017 ­ weed man‐ agement using crop competition in australia. ­ crop prot., 95: 8­13. batista m.a., bezerra neto f., ambrósio m.m., gui­ marães l., saraiva j.p.b., silva m.l., 2013 ­ atributos microbiológicos do solo e produtividade de rabanete influenciados pelo uso de espécies espontâ‐ neas. ­ hortic. bras., 31(4): 587­594. cardoso d.p., silva m.l., carvalho g.j., freitas d.a., avanzi j.c., 2012 ­ plantas de cobertura no controle das perdas de solo, água e nutrientes por erosão hídri‐ ca. ­ rev. bras. eng. agríc. amb., 16(6): 632­638. carneiro r.g., moura m.a.l., silva p.r., silva júnior r.s., andrade a.m.d., santos a.b., 2014 ­ variabilidade da temperatura do solo em função da liteira em fragmento remanescente da mata atlântica. ­ rev. bras. eng. agríc. amb., 1: 99­108. carvalho l.b., guzzo c.d., 2008 ­ adensamento da beterraba no manejo de plantas daninhas. ­ planta daninha, 26(1): 73­82. chang j., wu x., wang y., meyerson l.a., gu b., min y., xue h., peng c., ge y., 2013 ­ does growing vegeta‐ bles in plastic greenhouses enhance regional ecosystem services beyond the food supply? ­ front. ecol. environ., 11(1): 43­49. cividanes f.j., 2002 ­ efeitos do sistema de plantio e da consorciação soja‐milho sobre artrópodes capturados no solo. ­ pesq. agropec. bras., 37(1): 15­23. collier l.s., kikuchi f.y., benício l.p.f., sousa s.a., 2011 ­ consócio e sucessão de milho e feijão‐de‐porco como alternativa de cultivo sob plantio direto. ­ pesqui. agropecu. trop., 41(3): 306­313. table 4 ­ production and equilibrium price obtained with lettuce cultivation in different spacing and soil cover, in an area of 1,000 m2 * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly dif­ ferent using lsd at 5% level. data represent the mean value ± s.d. the mean of four replicates. cover planting spacing (m) production (boxes 4.8 kg) equilibrium price boxes 4.8 kg­1 (r$) boxes 4.8 kg­1 (usd) control 0.25x0.20 287.85 4.85 1.29 control 0.25x0.25 268.36 4.19 1.11 control 0.25x0.30 257.00 4.01 1.07 straw 0.25x0.20 280.27 4.23 1.12 straw 0.25x0.25 260.79 3.79 1.01 straw 0.25x0.30 249.42 4.50 1.20 plastic 0.25x0.20 293.96 5.58 1.48 plastic 0.25x0.25 274.47 4.86 1.29 plastic 0.25x0.30 263.10 4.72 1.25 vendruscolo et al. ‐ economic analysis of crisp lettuce 455 da silva f.c., 2009 ­ manual de análises químicas de solos, plantas e fertilizantes. ­ embrapa solos, brasília federal district, brazil, pp. 623. donagemma g.k., campos v.d.b, calderano s.b., tei­ xeira w.g., viana j.h.m., 2011 ­ manual de métodos de análise de solo. 2nd ed. ­ embrapa solos, rio de janeiro, brazil, pp. 230. li p., mo f., li d., ma b.l., yan w., xiong y., 2018 ­ exploring agronomic strategies to improve oat produc‐ tivity and control weeds: leaf type, row spacing, and planting density. ­ can. j. plant sci., 98(5): 1084­1093. martin n.b., serra r., oliveira m.d.m., angelo j.a., okawa h., 1998 ­ sistema integrado de custos agrope‐ cuários. ­ informações econômicas, 28(1): 7­28. maziero c.c.s., jaime, p.c., duran a.c., 2017 ­ a influên‐ cia dos locais de refeição e de aquisição de alimentos no consumo de frutas e hortaliças por adultos no muni‐ cípio de são paulo. ­ rev. bras. epidemiol., 20(1): 611­ 623. moraes p.v.d., agostinetto d., panozzo l.e., olivei­ ra c., vignolo g.k., markus c., 2013 ­ manejo de plantas de cobertura no controle de plantas daninhas e desempenho produtivo da cultura do milho. ­ semina: ciênc. agrár., 34(2): 497­508. oliveira a.k., lima j.s.s., bezerra a.m.a., rodrigues g.s.o., medeiros m.l.s., 2015 ­ produção de rabanete sob o efeito residual da adubação verde no consórcio de beterraba e rúcula. ­ rev. verde agroecologia desenvolv. sustent., 10(5): 98­102. rezende b.l.a., barros júnior a.p., cecílio filho a.b., pôrto d.r.q., martins m.i.e.g., 2009 ­ custo de pro‐ dução e rentabilidade das culturas de alface, rabanete, rúcula e repolho em cultivo solteiro e consorciadas com pimentão. ­ ciênc. agrotec., 33(1): 305­312. rezende b.l.a., cecílio filho a.b., catelan f., martins m.i.e.g., 2005 ­ análise econômica de cultivos consor‐ ciados de alface americana x rabanete: um estudo de caso. ­ hortic. bras., 23(3): 853­858. santos h.g., jacomine p.k.t., anjos l.h.c., oliveira v.a., lumbreiras j.f., coelho m.r., almeida j.a., cunha t.j.f., oliveira j.b., 2013 ­ sistema brasileiro de classificação de solos, 3rd. embrapa solos, brasília federal district, brazil, pp. 353. silveira a.l., neto a.p., de oliveira a.r.c., souza l.n., de oliveira charlo h.c., 2015 ­ doses de fósforo para a produção de alface americana com e sem apli‐ cação foliar de zinco. ­ biotemas, 28(1): 31­35. souza j.l., resende p.l., 2006 ­ manual de horticultura orgânica, 2nd ed. ­ aprenda fácil, viçosa, minas gerais, brazil, pp. 834. taiz l., zeiger e., moller i.m., murphy a., 2017 ­ plant physiology and development. ­ sixth ed. sinauer associates, sunderland, usa, pp. 761. torres j.l.r., araújo a.s., gasparini b.d.n., barreto a.c., tamburús a.y., silva v.r., vieira d.m.s., 2015 ­ desempenho da alface americana e do repolho sobre diferentes resíduos vegetais. ­ global sci. technol., 8(2): 87­95. trani p.e., purquéiro l.f.v., figueiredo g.j.b., tivelli s.w., blat s.f., 2014 ­ calagem e adubação da alface, almeirão, agrião d’água, chicória, coentro, espinafre e rúcula. ­ iac, campinas, são paulo, brazil, pp. 16. vasconcelos u.a.a., costa c.c., oliveira m.n., silva j.w., 2017 ­ residual effect of sheep manure on lettuce cultivation at different spacings. ­ rev. verde agroecologia desenvolv. sustent., 12(3): 508­511. vendruscolo e.p., campos l.f.c., arruda e.m., sele­ guini a., 2017 a ­ análise econômica da produção de alface crespa em cultivo sucessivo de plantas de cober‐ tura em sistema de plantio direto. ­ rev. bras. ciênc. agrár., 12(4): 458­463. vendruscolo e.p., campos l.f.c., seleguini a., mar­ tins a.p.b., lima s.f., 2017 b ­ economic viability of muskmelon cultivation in different planting spacing in brazil central region. ­ revista facultad nacional de agronomía, 70(3): 8319­8325. vieira b.s., barreto r.w., 2006 ­ first record of bremia lactucae infecting sonchus oleraceus and sonchus asper in brazil and its infectivity to lettuce. ­ j. phytopathol., 154(1): 84­87. ziech a.r.d., conceição p.c., luchese a.v., paulus d., ziech m.f., 2014 ­ cultivo de alface em diferentes manejos de cobertura do solo e fontes de adubação. ­ rev. bras. eng. agríc. amb., 18(9): 948­954. impaginato 373 adv. hort. sci., 2020 34(4): 373­380 doi: 10.13128/ahsc­8109 mechanical rubbing of tomato intern­ ode influence stem growth, improve tensile strength but negatively impact flavonoid levels s. sabina, m.n. jithesh (*) school of sciences, department of biotechnology, jain (deemed‐to‐be university), jayanagar, 560 011 bangalore, india. key words: lignin deposition, lycopersicon esculentum, mechanical induced stress, phenolic compounds, tomato. abstract: agricultural crops are exposed to different environmental stress fac­ tors on a daily basis. mechanical induced stress (mis) caused due to contact rubbing, bending, transplantation and spraying of water results in altered growth in plants (thigmomorphogenesis). the present study was conducted by inducing mechanical stress (gentle rubbing) on the third internode of tomato (lycopersicon esculentum mill.) by pressing with thumb and index finger (30 sec) for 14 consecutive days. at the end of the stress period, marked morpho­ logical differences included significant reduction in plant height and decreased internodal length of the rubbed third internode as well as the neighboring fourth internode. histochemical staining of the stem cross section of stressed plants showed intense color indicating lignin deposition. study of biochemical response post internode stress showed an increase in total phenol content but lower flavonoid contents. stress induction also resulted in modification of bio­ mechanical characteristics like tensile strength, elastic modulus and breaking force. studies on the effect of mechanical perturbations in plants has gained attention because of its implication in fundamental processes of organogene­ sis/morphogenesis and their potential as an innovative means of controlling plant growth. 1. introduction in the natural habitat, plants are challenged by various biotic and abi­ otic stress factors. mechanical induced stress (mis) is one such form of stress that occurs inevitably in plants on a daily basis. mis is a term used in cases of either a damage or physical injury due to bending, shaking and rubbing experienced by plants as a result of wind exposure, rain, animal movement and agricultural practices (biddington, 1986). being sessile, plants do not possess the luxury of relocating from the stressful source and are persistently challenged by these unfavorable environmental con­ ditions. plants are also known to sense the force of stimuli that can range from subtle effects like touching or rubbing on leaves to more intense (*) corresponding author: narayanan.jithesh@gmail.com citation: sabina s., jithesh m.n., 2020 ­ mechanical rub‐ bing of tomato internode influence stem growth, improve tensile strength but negatively impact flavonoid levels ­ adv. hort. sci., 34(4): 373­380 copyright: © 2020 sabina s., jithesh m.n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. author contribution statement: jmn conceived the experiments and ss conduc­ ted the experiments. jmn and ss contributed to the analysis, interpretation and writing of manu­ script. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 february 2020 accepted for publication 23 september 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 373­380 374 ones like the damage caused by herbivores (chehab et al., 2009). morphogenic responses due to rubbing or touch are termed thigmomorphogenesis (jaffe, 1973). among the most visible morphological thig­ momorphogenesis response common to many plant species includes decrease in shoot elongation, cou­ pled with increased radial dimension of cortex cell (telewski and jaffe, 1986; braam, 2005; potocka and szymanowska­pulka, 2018). plant cell wall is the first barrier against external mechanical forces. lignin is one of the main structural components of the plant cell wall and regulation of lignin metabolism is of prime importance to plant growth (zheng et al., 2017). apart from lignin, flavonoids are among the most crucial phenolic compounds involved in various protective roles in plants, such as attractants, feeding deterrents and even, oxidative stress protection (treutter, 2005; mierziak et al., 2014). while, stress induced biochemical changes in lignin are known, effect of mechanical stimulation on flavonoid levels are scarce. regulation of plant growth to achieve essential agronomic traits such as crop yield and plant height, are important prerequisites for agricultural indus­ tries. marketability of many important ornamental and horticultural crops increases with compact height and a strong stem as these facilitates packag­ ing and also, transportation of post­harvest produce (babalar et al., 2016; bornke and rochsch, 2018). the knowledge on force response of stem structures is critical as it incorporates approaches to test bio­ mechanical properties involving tensile strength and elastic modulus. these tests besides having economic implication, is also an immense tool for crop improvement (shah et al. , 2017). tomato, (lycopersicon esculentum mill.) is one of the most cultivated vegetable crops belonging to solanaceae family (schwarz et al., 2014). global tomato produc­ tion accounts for 170 million tonnes, comprising 75% fresh and 25% processed material for industrial uses (costa and heuvelink, 2018). due to its short dura­ tion and relative ease of cultivation, tomato produc­ tion has become one of the principal horticultural industries (costa and heuvelink, 2018). in this study, we experimentally determined whether stress on the internode by rubbing can lead to thigmomorphogenetic effects, leading to mechani­ cal stress resistance in tomato. it is hypothesized that lignin accumulation due to mechanical perturbations positively regulates higher tensile strength in plant stem. therefore, the purpose of this study was to establish physiological effects of mechanical stimula­ tion of the 3rd internode on tomato stem growth characteristics and consequently, its mechanical properties in plants. 2. materials and methods plant material and growth condition seeds of tomato (solanum lycopersicum mill.) belonging to a semi determinate variety (solanum lycopersicum mill. cv. arka vikas) were obtained from indian institute of horticultural research (iihr, bengaluru, india). germinated seedlings were trans­ planted to fresh pots (one plant per pot) in green house (12o56‘7” n, 77o35‘3” e) and maintained in moist soil under natural light condition (14 hrs of light and 10 hrs of dark) at 27­28oc. relative humidity was maintained around 61­70% in the green house. all the experiments were conducted between 10.00 am ­ 2.00 pm to avoid early and late diurnal response. application of mechanical stimulation mechanical stress stimulus was applied to five week old plants, henceforth referred to as internode stress. internode stress was applied by rubbing the 3rd internode for 14 days, using thumb and index fin­ ger for 30s (depege et al., 1997). plant growth parameters like height and internode length were measured after the application of internode stress for 14 days. the mean total height and internodal length (3rd and 4th internode) of 20 plants were recorded and compared with control plants. total phenolic compound content total phenolic content in leaves of plants after application of stress were determined by folin­ ciocalteu method as described by marinova et al. (2005). about 1 gram of fresh leaf was collected by excising the third leaf through random selection from the 20 pots of both control and stressed plants. evaluation of phenol content was made for each of the independent triplicates (n=3) from 1g of pooled leaf samples for both control and treated plants. each sample was macerated in 25 ml of methanol for 24 hr with occasional shaking. 250 ml of the extracted solution was mixed with 750 ml of methanol and 1ml of folin­ciocalteu reagent was added. after 5 min, 1 ml of na2co3 (20%) was mixed to the solution. after 30 min of incubation in dark, absorbance was mea­ sured at 765 nm using a uv­vis spectrophotometer (shimadzu inc. japan) against a blank sample. a stan­ sabina and jithesh ‐ thigmorphogenesis in tomato after mechanical stimulation 375 dard gallic acid curve was constructed by preparing dilutions of a standard solution of gallic acid. total phenol was calculated and was expressed as gallic acid equivalents per gram of fresh weight (gae g­1 of fw). total flavonoid content aluminium chloride calorimetric method was fol­ lowed for the determination of the total flavonoid content in leaf samples based on the methodology described by dewanto et al. (2002). about 1 gram of fresh leaf was collected by excising the third leaf through random selection from the 20 pots of both control and stressed plants. evaluation of flavonoid content was made for each of the independent tripli­ cates (n=3) from 1 g of pooled leaf samples for both control and treated plants. each sample was macer­ ated with 70% methanol and kept for 24 hours with occasional shaking. the extracted sample solution (250 ml) was separately mixed with 75 ml of 5% nano2 followed by incubation for 5 min. after incu­ bation, 10% alcl3 (150 ml) was added followed by 500 ml of 1 m sodium hydroxide. total volume was made up to 2.5 ml using distilled water. after the incuba­ tion period for 30 min, absorbance was measured at 510 nm against a blank using uv­vis spectropho­ tometer (shimadzu inc. japan). quercetin standard curve was constructed by preparing dilutions of a standard solution of quercetin for the quantification of total flavonoids. the total flavonoid was calculated and was expressed as quercetin equivalent per gram of fresh weight (qe g­1 of fw) (lin and tang, 2007). lignin staining and quantification analysis to evaluate how mis affected lignin deposition, histochemical analysis of the stem sections of control and stressed plants was performed using wiesner reagent. the amount of 2% of fresh phloroglucinol staining solution for lignin staining was prepared in 95% ethanol. fresh stem sections from the neigh­ bouring 4th internode were immobilized on a sliced potato surface. multiple homogeneous thin freehand sections were obtained by drawing a sharp razor blade with smooth stokes repeatedly to ensure uni­ formity. the sections were further checked by stere­ omicroscope in order to remove thick and also dam­ aged sections. the most uniform sections were picked carefully and transferred to petri plates con­ taining water. uniform sections of control and treat­ ed plants were transferred to petriplates and immersed in phloroglucinol stain. after 24 hours, sec­ tions were transferred to a clean glass slide, to which a drop of concentrated hcl was added (phloroglucinol­hcl stain). the slides were then gen­ tly warmed over a bunsen burner for 20 seconds for hcl evaporation. a drop of glycerol was added to these sections after which a cover slide was placed over it gently. the slides were then observed under a microscope labomed vision 2000 (inc. india) at 10x magnification (mitra and loque, 2014). lignin quantification analysis was conducted through imagej software (ver 1.52p). an eyepiece graticule (scale bar) was used to set the scale mea­ surements for the sections of control and treatment. these images were further processed through a pro­ cess of spatial resolution information in imagej using the calibration set by the graticule image. to start with the quantification analysis, images were dupli­ cated. one of the images was converted from 8 bits to grayscale. following this action, pixel information pertaining to the pixel colour, gets converted into brightness measurement. by selecting the command: image>adjust>threshold, images to be quantified was highlighted in pixels within the threshold range speci­ fied. next, by choosing the command: analyse>set measurement, the area and limit to threshold was selected. this enabled to measure within the thresh­ old pixel range for control and treated images. finally, the function, analyze­measure was used to read the area covered with stain. percentage of stained area was calculated by: stained area/total area x 100 (beziat et al., 2016). determination of tensile strength determination of biomechanical stem properties was done after 14 days of stress application. plants of 10 cm length were defoliated after the experiment and five stem segments devoid of leaves and roots were randomly chosen for analysis from stressed and control plants. these stem segments were then kept in an oven (40­50°c) for approximately 1 hour to rid of excessive water content. they were then clamped in the universal testing machine (mts­mechatronics, inc, ichalkaranji, india). the following biomechanical traits were determined (shah et al., 2017): (1) tensile strength (mpa) is a measure of the maxi­ mum stress that the material can withstand without being elongated or stretched. (2) elastic modulus (mpa) represents a ratio of stress applied and strain, the material exhibits (degree to which the material resists deformation in response to an applied force). (3) breaking force (n) measures the maximum force that the material can bear before its experiences mechanical stress failure. adv. hort. sci., 2020 34(4): 373­380 376 statistical analysis statistical analysis for the results obtained were carried out using student’s t test with level of signifi­ cance set at p<0.05. coefficient of variation (cv) is a useful measure to determine dispersion in a variable and can be extremely informative to study variation in different phenotypic traits in a study. coefficient of variation for the morphological traits obtained was calculated as follows: (standard deviation/average) x 100. pearson correlation coefficient was performed to study the level of association between two variables, pair­wise. all these analysis was performed using graphpad prism (version.8). 3. results effect of internode stress on plant height and intern‐ odal length of tomato application of mild mechanical stimuli in the internodes of treated plants resulted in reduction in plant height whereas control plants showed normal growth characteristics (fig. 1). internode stress led to 27.4% reduction of total plant height when com­ pared to control plants, a significant reduction at p<0.0001 (fig. 2 a). effect of internode stress affected the elongation of 3rd internode, the site of stress application. interestingly, internode stress also resulted in significant difference in elongation of the neighbouring 4th internode. results from the present finding showed that elongation of stressed internode was inhibited by 47% and 44% for 3rd and 4th intern­ ode respectively at p<0.0001 in comparison to con­ trol plants that were not stressed (fig. 2 b). coefficient of variation (cv) studies the results presented in table 1 revealed that the mean plant height in control plants was 27.5 cm with a range between 25.2­30.5 cm. in contrast, treat­ ments resulted in mean plant height of 20.01 with range between 18.0­22.8 cm. in case of treated plants, the highest cv was observed for 3rd internode (16.64%), the site of mechanical stimuli followed by the 4th internode (14.8%). in comparison, in control conditions, coefficient of variation (cv) of 5­7% was observed for the traits measured, confirming lower variation within population. clearly, internode stress table 1 ­ range of group means, mean values, standard deviation, standard error mean and coefficient of variation of internode length and total height of control and treated plants * values in the table mentioned for n=20. descriptive statistics for control and treated plants sdv= standard deviation; sem= standard error of mean; cv= coefficient of variation. fig. 1 ­ lycopersicon esculentum mill cv. arka vikas after 14 days of application of mechanical stimuli by rubbing the 3rd internode. left, control plants and right, treated plants. fig. 2 ­ total height of control and internode stressed plants (a) and length of third and fourth internode of control and internode stressed plants (b). the vertical bars indicate standard errors. the values correspond to the mean ± se, where n=20. statistical analysis was performed by student’s t­test for total height and internode length. asterisks indicate mean values significantly differ at p<0.05 (**** indicate p <0.0001). traits control treatment range mean sdv sem cv (%) range mean sdv sem cv (%) 3rd internode 3.6­4.5 3.9 0.287 0.064 7.3 1.5­2.6 2.1 0.349 0.078 16.64 4th internode 5.2­6.3 5.6 0.298 0.066 5.3 2.5­3.8 3.1 0.46 0.103 14.8 total height 25.2­30.5 27.5 1.9 0.429 6.9 18­22.8 20.01 1.097 0.245 5.48 sabina and jithesh ‐ thigmorphogenesis in tomato after mechanical stimulation 377 lead to higher variability for the 3rd and 4th internode than in normal conditions as indicated by these results. effect of internode stress on total phenol and flavonoid content mechanical stimuli resulted in an increase of 25% total phenolic content in leaves, when compared to control plants, a significant increase at p = 0.0001 (fig. 3 a). in contrast, flavonoid content in leaves of treated plants showed a reduction of 18% in compar­ ison to control plants, a significant decrease at p = 0.0013 (fig. 3 b). effect of internode stress on lignin deposition and tensile strength histochemical analysis by staining revealed that the intensity of colour was visibly lower in control plants (fig. 4 a). on the other hand, analysis revealed intense colouration (red/magenta) in stressed plants (4th internode) indicating increased lignin deposition in xylem bundles and confirming alteration in lignin deposition in the plants with internode stress (fig. 4 b). lignin quantification through imagej analysis revealed a 45% enhancement in lignified area for sec­ tions from treated plants. on the other hand, sec­ tions from control samples covered only 22% of ligni­ fied area (fig. 4 c). application of mechanical stimuli also led to increased mechanical strength as indicated by the various mechanical properties shown in figure 5. the mean tensile strength recorded a significant increase of 113% in treated plants, when compared to control plants at p<0.05 (fig. 5 a). other mechanical proper­ ties including elastic modulus, and breaking force were also significantly higher when compared to the control plants. the elastic modulus displayed by treated plants observed was 658.4 mpa, a highly sig­ nificant increase (p<0.0001) compared to control plants (fig. 5 b). the mean breaking force for treated plants recorded was 21.13 n, in comparison to con­ trol plants, which was lower (9.8 n), a significant dif­ ference at p= 0.0007 (fig. 5 c). fig. 3 ­ total phenol content expressed as mg of gallic acid equi­ valent per gram of fresh weight (gae g­1 fw) after 48hrs of internode stress (a) and total flavonoid content expressed as mg of quercetin equivalent per gram of fresh weight (qe g­1 fw) after 48hrs of internode stress (b). standard error is indicated by vertical bars. the values correspond to the mean ± se, where n=3. statistical analysis was performed by student’s t test. asterisks indicate mean values significantly differ from control at p <0.05 (** indicate p = 0.0013 and *** indica­ te p = 0.0001). fig. 4 ­ microscopic view of stained cross sections after 14 days of rubbing of the third internode for control (a) interno­ de stressed (b) and graph showing quantification of lignin analysis by imagej software (c). ep­epidermis, xy­ xylem (lignin deposition) and pi­pith. scale bar = 100 µm. fig. 5 ­ biomechanical properties of the stem measured through tensile test: tensile strength (a), elastic modulus (b) and breaking force (c). standard errors are indicated by verti­ cal bars. the values correspond to the mean ± se, where n=5. asterisks indicate mean values significantly differ at p<0.05 (*** indicate p=0.0007 and **** indicate p<0.0001). 378 adv. hort. sci., 2020 34(4): 373­380 correlation studies among biomechanical trait the correlation coefficient among tensile strength, breaking force and plant height for both control and treated plants are reported in table 2. plants treated with internode stress showed a highly significant positive correlation (r = 0.999) among the two important mechanical properties for stem, ten­ sile strength and breaking force. however, the corre­ lation for these traits in control plants remained non­ significant. the correlation of total height of plant with respect to tensile strength and breaking force demonstrated a significant negative correlation for both treatment and control plants. plants treated with internode stress showed a slightly higher nega­ tive correlation (r= ­0.97) than control plants (r= ‐ 0.91) between tensile strength and total height. a scatter plot of these associated traits clearly high­ lighted a negative slope of correlation after exposure to mechanical stimuli (fig. 6). 4. discussion and conclusions in response to mechanical stimuli, plants respond by altering their growth rate and morphology (chehab et al., 2009). in this communication, applica­ tion of internode stress was shown to be effective in reducing the overall height of the plant. effect of brushing in reducing leaf size and stem elongation has been demonstrated in lycopersicon esculentum (mill.) (johjima et al., 1992). in arabidopsis thaliana (linn.), inhibition of stem elongation in the inflores­ cence was also shown to be prominently affected due to mechanical perturbation (victor and rowe, 2011). a higher coefficient of variation in treated plants shown in this study demonstrated that the most relevant effect of internode stress treatment was to bring growth alteration by causing reduction in internode elongation for both the 3rd and the 4th internode. in comparison to the treatment, coeffi­ cient of variation in control plants was lower, reveal­ ing insignificant random variation within a phenotyp­ ic trait. hence, reduction in internodal length clearly indicates that mechanical stress response is a gener­ alized response affecting both the rubbed internode as well as neighboring young internode. plants do not possess specialized cells to mount a response to mechanical induced stimuli (mis) in nature (börnke and rocksch, 2018). individual cells in the vicinity of local tissue that is mechanically stimulated may pos­ sess the property of mechanoperception and to transduce this signal to an unperturbed distal tissue either by hormonal or electrical signals (erner et al., 1980). depege et al., (1997) suggested that the trans­ duction of the signal was electrical occurring through the involvement of intercellular ca2+ modulated by calmodulin gene expression. moulia et al. (2015) fur­ ther proposed that local mechanosensing and the origin of ionic currents can be explained through the participation of mechanosensitive ionic channels or stretch­activated channels (sac). under our experimental conditions, while the phenolic contents increased, flavonoid content was reduced by application of mechanical stress. in plants, the phenylpropanoid metabolism and also the lignin biosynthetic pathway are stimulated by envi­ ronmental stress adaptation (petersen et al., 1999). saidi et al. (2009) reported an increase of all the major enzymes involved in the phenylpropanoid pathway with mechanical stimulation in tomato. consequently, an increase in phenolic content and asterisk indicates statistical significance, at p=0.12(ns), 0.033(*), 0.002(**) and <0.0001(****), ns= non significance difference. table 2 ­ linear correlation coefficient between different bio­ mechanical characters in control and treated plants. correlation of morphological trait with biomechanical characteristics traits total plant height breaking force control tensile strength ­0.91 * 0.7 ns total plant height ­0.94 * treated plants tensile strength ­0.97 ** 0.999 **** total plant height ­0.95 ** fig. 6 ­ scatter plots of tensile strength and total height measu­ red on treated plants (green) and control plants (red). the scatter plot indicates a significant negative correla­ tion between two variables with a correlation coefficient of r=­0.9, (p<0.0001). sabina and jithesh ‐ thigmorphogenesis in tomato after mechanical stimulation 379 enrichment of lignin observed in this study is consis­ tent with these reports. however, biosynthesis of lignin pathway and flavonoid are highly co­regulated in plants, where repression of one pathway, redirects the metabolites into the other pathway (besseau et al., 2007). in the phenylpropanoid pathway, p­ coumaroyl coa is the branch point in the metabolic route leading towards biosynthesis of either one of them (yeh et al., 2014). in plants, accumulation of flavonoid is affected as the common flavonoid­lignin pathway is diverted towards utilization of support mechanism for biosynthesis of lignin (besseau et al., 2007). a reduction in flavonoid content observed in this study therefore, points to the proposition that lignin and flavonoid may be competing for the same substrates leading to a decrease in flavonoid synthe­ sis. mis increased stem resistance to tensile forces in this study. the modification in these parameters is relatively fast as the experiment lasted for duration of only 14 days. in plants, important biomechanical traits for instance, higher resistance to tensile and breaking forces leads to the acquisition of a hard­ ened phenotype (schoelynck et al., 2015; shah et al., 2017). cell wall rigidification due to accelerated ligni­ fication was shown to result in reduced internodal elongation after mechanical stress application in tomato (saidi et al., 2009). further, reinforcement of cellulose microfibrils by lignins was also reported to increase resistance to tensile forces (genet et al., 2005). this unique thigmomorphogenetic response in plants is crucial to withstand repeated external stress and mechanical forces (jaffe et al., 1984; biddington, 1986). moreover, plant height was shown to be nega­ tively correlated with tensile strength and breaking force in this study. plant lodging, an important trait observed in plants is shown to occur as a conse­ quence of permanent displacement of plant stem by stem buckling (kendall et al., 2017). reduction in plant height and internodal length has been shown to improve lodging tolerance in crops, an important trait for crop improvement (peng et al., 2014). notably, introduction of dwarfing genes was an important outcome of the green revolution, reducing lodging susceptibility by reducing plant height (spielmeyer et al., 2002). in conclusion, we observed that besides modifying growth, mechanical stimulation due to rubbing resulted in increased lignin deposition and also enhanced tensile strength leading to overall gain in the physical strength of the plant stem. further, our results provide new insights on the lignin­flavonoid pathway after mis and demonstrate the plasticity of the biochemical response. the ability to sense and respond to mechanical stimulation by thigmomor­ phogenesis needs to be explored, as it holds great potential for commercial application in horticulture for regulating plant growth without the use of chemi­ cal regulators (börnke and rocksch, 2018). acknowledgements we are grateful to jain (deemed to be university) for the infrastructure and also facilities provided to the authors. we gratefully acknowledge prof. leela iyengar, jain, bangalore for the valuable advice throughout the work. we also like to thank dr. vijay g., jyothi institute of technology, bangalore, for help with the universal testing machine. we like to thank kruthika n for the help with manuscript preparation and imagej analysis. references babalar m., edrisi b., naderi r., 2016 ­ evaluation of the mechanical strength of gerbera flower stem in response to silicon and salicyclic acid application. ­ j. ornamental plants, 6(3): 163­171. besseau s., hoffmann l., geoffroy p., lapierre c., pollet b., legrand m., 2007 ­ flavonoid accumula‐ tion in arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. ­ plant cell., 19: 148­ 162. béziat c., kleine­vehn j., feraru e., 2016 ­ histochemical staining of β‐glucuronidase and its spa‐ tial quantification. ­ plant hormones, pp. 73­80. biddington n.l., 1986 ­ the effects of mechanically‐ induced stress in plants ‐ a review. ­ plant growth regul., 4: 103­123. börnke f., rocksch t., 2018 ­ thigmomorphogenesis ‐ control of plant growth by mechanical stimulation. ­ sci. hortic., 234: 344­353. braam j., 2005 ­ in touch: plant responses to mechanical stimuli. ­ new phytol., 165(2): 373­389. chehab e.w., eich e., braam j., 2009 ­ thigmomorphogenesis: a complex plant response to mechano‐stimulation. ‐ j. exp. bot., 60(1): 43­56. costa j.m., heuvelink e., 2018 ­ the global tomato industry, pp. 1­26. ­ in: heuvelink e. 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w., ni j., wang y., wang z., li y., 2017 ­ manipulation of lignin metabolism by plant den‐ sities and its relationship with lodging resistance in impaginato 185 adv. hort. sci., 2022 36(3): 185­191 doi: 10.36253/ahsc­12488 impact of cultural conditions on germi­ nation of olive (olea europaea l.) somatic embryos and plantlets develop­ ment from the algerian cultivar chemlal k. titouh 1, 2 (*), k. hadj moussa 2, n. boufis 1, l. khelifi 2 (*) 1 national institute of agronomic research of algeria (inraa), research center of baraki, algiers, algeria. 2 national higher school of agronomy (ensa, es1603), laboratory of genetic resources and biotechnologies (lrg‐b), algiers, algeria. key words: germination, micropropagation, olive, plant regeneration, somatic embryo. abstract: the in vitro propagation techniques are currently a commercial alter­ native for the production of plants with good quality in several plant species, including the olive tree (olea europaea l.). somatic embryogenesis is the process practically used for the application of several biotechnological tools of improvement and in vitro plant regeneration via the germination of somatic embryos. our work aims to evaluate the effect of the chemical and hormonal composition of the culture medium on the germination of olive somatic embryos (cv. chemlal) as well as the micropropagation of the obtained plantlets before their acclimatization to natural conditions. the results indicat­ ed that the production of olive plants by somatic embryogenesis depends strongly on the genotype of the somatic embryos (cell line) and more on the culture conditions, particularly the presence of growth regulators. indeed, a solid om medium supplemented with hormones (ba and iba) permitted an advanced root emergence and germination allowing the production of well­ developed plants with several leaves. in addition, an om medium supplement­ ed with zeatin and iba allowed better reactivity of micro­cuttings producing well­developed shoots with several emitted roots which facilitates their further acclimatization to natural conditions. 1. introduction the olive tree (olea europaea l.) a diploid dicotyledonous species of the oleaceae family, includes several cultivars selected and multiplied ini­ tially by farmers mainly for the size of their fruits and the oil content (besnard et al., 2018). conventional methods of breeding and multiplica­ tion represent an important solution to the crop problems, especially the increasing demand for plants needed for new orchards. however, these (*) corresponding authors: titouhkheireddine@gmail.com khelifi.lakhdar@gmail.com citation: titouh k.,hadj moussa k., boufis n., khelifi l., 2022 ­ impact of cultural conditions on germi‐ nation of olive (olea europaea l.) somatic embryos and plantlets development from the algerian cultivar chemlal. ­ adv. hort. sci., 36(3): 185­191. copyright: © 2022 titouh k., hadj moussa k., boufis n., khelifi l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2021 accepted for publication 13 june 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12488 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 185­191 186 techniques have become unable to achieve signifi­ cant results because of the long juvenile period of the species (rugini et al., 2020) as well as the recalci­ trance of certain main cultivars, such as ‘chemlal’ in algeria, to semi­hardwood cuttings (fabbri et al., 2004). in this context, in vitro propagation techniques are currently a commercial reality for the multiplica­ tion of several olive cultivars (rugini et al., 2020) due to the sanitary quality and the confirmed genetic sta­ bility of the regenerated plants (lopes et al., 2009) despite some phenotypic changes and somaclonal variations observed after several subcultures (leva, 2009; bradaï et al., 2016, 2019). somatic embryogenesis is a morphogenetic process through which somatic cells produce a bipo­ lar structure morphologically similar to the zygotic embryo called a ‘somatic embryo’ able to develop into a whole plant (neumann et al., 2009). actually, this process has become a common technique for in vitro regeneration allowing the application of several biotechnological tools of improvement and conserva­ tion such as genetic transformation, in vitro selection and cryo­preservation of various species and inter­ esting genotypes (sánchez­romero, 2021). most of the established works on somatic embryogenesis in olive consider three main steps starting with the establishment of embryogenic cultures combining the induction and proliferation of calli, followed by a phase of expression and development of structured embryos ready to be converted into whole plants. however, few works have been done about the ger­ mination conditions of olive somatic embryos (mazri et al., 2020) although regeneration of plantlets has frequently been achieved by introducing the embryos under photoperiod on a standard culture medium based on the chemical compositions ‘ms’ (murashige and skoog, 1962) or ‘om’ (rugini, 1984). in addition, the low conversion rates remain the great obstacle of the process in many species, includ­ ing in olive tree, are caused mainly by deficiencies in the development and maturation of the used embryos (merkle et al., 1995; sánchez­romero, 2019) but also by unfavorable conditions to their ger­ mination (bradaï et al., 2016). the objective of our study is to evaluate the effect of the chemical and hormonal composition of the cul­ ture medium on the germination of somatic embryos of the main algerian olive cultivar ‘chemlal’. in addi­ tion, the micropropagation of the obtained plantlets as well as their acclimatization to natural conditions were tested. 2. materials and methods establishment of the embryogenic cultures the embryogenic cultures used were induced from radicles of zygotic embryos of the cultivar ‘chemlal’ according to a modified method of cerezo et al. (2011). radicles isolated after disinfection of the seeds extracted from the stones of mature olives were cultured on solid omc medium (cañas and benbadis, 1988) supplemented with 0.5 mg l­1 of zeatin and 5 mg l­1 of indole­3­butyric acid (iba) for three weeks. subsequently, the explants were trans­ ferred to the same omc medium without zeatin and containing 0.5 mg l­1 of iba for four weeks. finally, the obtained calli were maintained by monthly sub­ cultures on a solid eco basal medium (cerezo et al., 2011) supplemented with 0.1 mg l­1 of zeatin, 0.1 mg l­1 of benzylaminopurine (ba) and 0.05 mg l­1 iba in addition to 0.55 g l­1 of glutamine and 1 g l­1 of casein hydrolyzate. all the media were supplemented with 20 g l­1 of sucrose, 50 mg l­1 of myo­inositol and solid­ ified with 6 g l­1 of agar after adjusting the ph to 5.74 with naoh or hcl (1 n). the cultures were incubated in total darkness at a temperature of 25±2°c. pro­embryos exceeding 2 mm in size were isolat­ ed from calli of two embryogenic lines (c2 and c3) after one month of culture in suspension on a liquid eco medium supplemented with hormones in dark­ ness with stirring at 100 rpm. these immature embryos were transferred for maturation on solid eco free­hormones medium and supplemented with 1 g l­1 of activated charcoal for two months in total darkness at 25±2°c. germination of somatic embryos mature embryos with a perfectly bipolar or cotyledonary structure, were germinated individually in test tubes over two different media: om (rugini, 1984) and ms (murashige and skoog, 1962) solidified with 6 g l­1 of agar. in addition, the effect of a hor­ monal combination consisting of 1 mg l­1 ba and 0.1 mg l­1 iba was tested. the two media were supple­ mented with 20 g l­1 of sucrose, 100 mg l­1 of myo­ inositol. at least 16 mature somatic embryos were incubated for more than eight weeks under a 16 h light photoperiod (35 µmol m­2 s­1) at a temperature of 25±2°c. germination, root emergence, shoot titouh et al. ‐ somatic embryogenesis in algerian olive cultivars 187 length as well as the number of leaves per obtained plantlet after eight weeks of culture were observed. micropropagation of shoots and acclimatization of regenerated plantlets in order to maintain the maximum number of plants in culture; micropropagation of the shoots resulting from the germination of somatic embryos was applied. therefore, the shoots were divided into uni­nodal micro­cuttings and cultured on two differ­ ent culture media: om and dkw (driver and kuniyuki, 1984) as modified by revilla et al. (1996). the two culture media were supplemented with 20 g l­1 of sucrose, 100 mg l­1 of myo­inositol in addition to hormonal balances composed of 2 mg l­1 of ba or zeatin combined with 0.1 mg l­1 of iba. the different media were solidified with 6 g l­1 of agar. at least 16 micro­cuttings having two leaves were cultured indi­ vidually in test tubes containing 15 ml of the micro­ propagation medium for eight weeks under a 16 h light photoperiod at a temperature of 25±2°c. the reactivity of the micro­cuttings and the number of reactive buds, shoot length, number of leaves as well as the number of roots emitted were observed. the obtained plants showing an acceptable length with several leaves and well­developed roots were acclimatized in the laboratory under a photoperiod for about two months on a humidified mixture of sand/potting soil/perlite at a rate of 2/2/1 (v/v/v). subsequently, the reactive plantlets were transferred to natural conditions under greenhouse on a sub­ strate rich in organic matter and frequently irrigated before being permanently planted in the field. data analysis statistical analyses of the data (analysis of vari­ ance and tests) were carried out using the “xlstat” program version 2016.02.27444. in case of a signifi­ cant difference, the separation of means was per­ formed by fisher’s lsd (least significant difference) test. the percentages were analyzed by the chi­ square test. the results were presented as a mean ± standard deviation or as a percentage relative to the total of introduced explants. a significance level of 5% was considered in all analyses. the letters in the tables indicate homogeneous groups. 3. results germination of somatic embryos from the first days under photoperiod, the white­ opaque somatic embryos of olive showed greening of their stem part and yellowing of the root part (fig. 1 a) followed by its elongation preceding an increase in size of the two cotyledonary leaves and their separa­ tion each one from the other (fig. 1 b) before the emergence of a small shoot (fig. 1 c). indeed, the germination capacity of embryos and the plants development varied from one cell line to another and were significantly influenced by the culture condi­ tions, particularly the presence of growth regulators (table 1). thus, more embryos of the c2 line germi­ nated on om medium while the germination of the two lines was similar on the ms medium. however, the presence of hormones allowed a significant improvement in the embryos germination of both lines. in fact, the best germination rates were obtained on om medium supplemented with ba and iba (om1) with 56.3 and 37.5% respectively for c2 and c3 while the low germination rate of 25% was recorded on ms without hormones (ms0) (table 1). in addition, embryos of c2 germinated with root emergence from the first week of culture (fig. 1 a) while no reactivity was observed before two weeks for c3 embryos. likewise, the germination and the root emergence were faster on the om medium than fig. 1 ­ germination of mature somatic embryos from two lines of embryogenic olive callus, cv. chemlal, and micropropa­ gation of the obtained shoots after 8 weeks of culture on different culture media. (a) somatic embryo germinated on solid medium. (b) swelling of cotyledonary leaves of embryos. (c) different stages of embryos germination. (d and e) plantlets obtained after germination. (f and g) plantlets obtained after the micropropagation of shoots obtained from germination of somatic embryos. (h) acclimated plantlets. (→: the arrows indicate the emer­ gence of the root, shoot and the two cotyledonary lea­ ves. bar corresponds to 1 cm. cot: cotyledons). adv. hort. sci., 2022 36(3): 185­191 188 on ms, especially in the presence of growth regula­ tors which accelerated significantly germination of embryos. therefore, the embryos emitted their roots after 10.5 and 15.4 days and germinated after 14 and 18.7 days on the om1 medium respectively for c2 and c3 (table 1). consequently, early germination and rooting of c2 embryos resulted in well­devel­ oped plants with an average length of 1.6 and 2.3 cm with 6 and 6.3 leaves per plantlet respectively on om0 and om1 (table 1, fig. 1 d) while the ms1 medi­ um was more beneficial for the c3 plants reaching 1.9 cm in length with 4.5 leaves (table 1, fig. 1 e). micropropagation of shoots and acclimatization of regenerated plantlets reactivity of micro‐cuttings and shoot develop‐ ment. the micropropagation of shoots resulting from the germination of somatic embryos was significantly influenced by the callus line as well as the chemical and hormonal composition of the culture medium (table 2). in fact, micro­cuttings of the c2 line were more reactive than those of c3 regardless of the cul­ ture conditions, although the presence of hormones was essential for the development of the shoots given the low reactivity recorded on the control media. in addition, the best result of reactivity (100%) of the explants of both lines was obtained with the balance zeatin/iba and also the c2 cuttings on the combination ba/iba in presence of which only 62.5 and 87.5% of the c3 cuttings reacted respective­ ly on om and dkw (table 2). elsewhere, the dkw medium in particular supplemented with hormones (zeatin/iba) accelerated the bud reaction and improved the number of reactive buds per explant while the cuttings introduced particularly on the free­ hormones om medium reacted late with less sprout­ ed buds (data not shown). the development of the obtained shoots depend­ ed directly on the culture conditions and was signifi­ cantly influenced by the reactivity degree of the cut­ table 1 ­ effect of the chemical composition (om and ms) of the culture medium and the presence of hormones (without hormones ‘0’ or with hormones ‘1’) on germination and root emergence of somatic embryos of two lines of embryogenic olive calli, cv. chemlal, after eight weeks of culture the different small letters of the same format in columns indicate the homogeneous groups of a significant difference at level of 5%. table 2 ­ effect of the micropropagation medium (om and dkw) and the presence of hormones (ba/iba or zeatin/iba) on the reactivi­ ty, development and rooting of shoots from uni­nodal micro­cuttings obtained after germination of somatic embryos of two lines of embryogenic olive calli, cv. chemlal, after eight weeks of culture the different small letters of the same format in columns indicate the homogeneous groups of a significant difference at level of 5%. germination medium germination rate (%) average time of germination (days) average time of root emergence (days) average length of plantlet (cm) average number of leaves/plantlet c2 c3 c2 c3 c2 c3 c2 c3 c2 c3 om0 50.0 b 31.3 d 16.8±3.8 b’ 21.0±0.0 de’ 12.3±3.5 a’’ 17.5±4.9 cd’’ 1.6±0.1 b 0.6±0.1 d 6.0±1.6 a’ 2.0±0.0 c’ om1 56.3 a 37.5 c 14.0±4.4 a’ 18.7±4.0 bc’ 10.5±4.0 a’’ 15.4±3.1 b’’ 2.3±0.8 a 0.9±0.2 c 6.3±1.6 a’ 2.3±0.6 c’ ms0 25.0 e 25.0 e 24.5±4.9 f’ 21.7±1.2 e’ 21.0±0.0 e’’ 18.7±4.0 d’’ 0.8±0.1 cd 0.8±0.1 cd 2.5±0.7 c’ 2.0±0.0 c’ ms1 31.3 d 31.3 d 21.0±0.0 de’ 19.3±4.7 cd’ 17.5±0.7 cd’’ 16.3±4.0 bc’’ 0.9±0.1 c 1.9±0.1 b 2.5±0.7 c’ 4.5±0.7 b’ composition of the micro­ propagation medium hormonal combina­ tions reactivity and shoot development rooting reactivity rate (%) average length of shoot (cm) average number of leaves/shoot rooting rate (%) average number of roots c2 c3 c2 c3 c2 c3 c2 c3 c2 c3 om control 25.0 d 12.5 d 0.7±0.3 g 0.9±0.0 fg 1.5±0.7 d’ 2.0±0.0 cd’ 0.0 e’ 0.0 e’ 0.0±0.0 f’’ 0.0±0.0 f’’ ba/iba 100.0 a 62.5 c 2.0±0.7 de 1.9±0.2 de 2.9±1.0 cd’ 3.1±0.9 cd’ 31.3 d’ 31.3 d’ 1.0±0.0 e’’ 1.5±0.7 bc’’ zeatin/iba 100.0 a 100.0 a 3.7±1.0 b 5.2±2.1 a 6.9±3.2 b’ 9.9±3.1 a’ 62.5 a’ 43.8 c’ 1.5±0.0 bc’’ 1.7±0.6 b’’ dkw control 18.8 d 12.5 d 0.5±0.0 g 0.7±0.0 g 2.0±0.0 cd’ 2.0±0.0 cd’ 0.0 e’ 0.0 e’ 0.0±0.0 f’’ 0.0±0.0 f’’ ba/iba 100.0 a 87.5 b 1.7±0.3 ef 3.1±1.0 bc 3.4±1.1 c’ 2.7±0.4 cd’ 43.8 c’ 31.3 d’ 1.3±0.6 cd’’ 1.5±0.7 bc’’ zeatin/iba 100.0 a 100.0 a 2.7±0.2 cd 3.3±1.4 bc 6.6±1.1 b’ 6.8±2.4 b’ 56.3 a’ 50.0 b’ 1.3±0.5 d’’ 2.0±0.8 a’’ titouh et al. ‐ somatic embryogenesis in algerian olive cultivars 189 ting particularly the number of reactive buds. in cut­ tings with two active buds; one shoot regularly grown more than the other (fig. 1 f and g). moreover, the cuttings cultured on om medium supplemented with hormones especially zeatin/iba and which sprouted early, produced well­developed shoots of 3.7 and 5.2 cm in length with 6.9 and 9.9 leaves respectively for c2 and c3 while the shoots obtained on the control media were the least developed, with less than 1 cm of length and a maximum of 2 leaves per plantlet (table 2, fig. 1 f and g). rooting of developed shoots. the presence of hor­ mones in the culture medium was essential for the formation of a basal callus on the micro­cuttings before the emission of roots, while the chemical composition contributed more in the development of the induced calli due that the majority of calli gener­ ated on om were generally larger compared to those obtained on dkw (data not shown, fig. 1 f and g). root emission was significantly influenced by the genotype (cell line) as well as chemical and hormonal composition of the propagation medium (table 2). indeed, more rooting was observed with micro­cut­ tings of the line c2 particularly on the om medium supplemented with zeatin/iba allowing 62.5 and 43.8% rooting with 1.5 and 1.7 roots emitted by cut­ ting respectively for the two lines while the chemical composition of dkw was more beneficial to c3 explants with 50% of rooting and an average of 2 roots per plantlet (table 2). however, the appear­ ance of roots occurring from the 3rd to the 7th week of culture was often faster on dkw compared to om medium especially in the presence of ba with iba. furthermore, the substitution of ba by zeatin in the added hormonal combination reduced the rooting time by more than a week in the cuttings of both lines, which improved the length of the emitted roots (data not shown). therefore, the plantlets showing well­developed shoots and roots were easily acclima­ tized in the laboratory and exhibit normal growth and phenotype even after transfer to natural field conditions (fig. 1 h). 4. discussion and conclusions germination of somatic embryos germination of olive somatic embryos was fre­ quently achieved on media based on the chemical formulations om and ms with a reduced concentra­ tion of mineral salts, similar to those used for the cul­ ture of zygotic embryos (sánchez­romero, 2019). rugini (1988) indicated that germination and devel­ opment of olive plantlets from somatic embryos is faster on om medium than on ms. indeed, rugini and caricato (1995) observed the germination of embryos of cultivars ‘canino’ and ‘moraiolo’ after 1 to 2 weeks on om free­hormones medium, whereas shibli et al. (2001) didn’t note reactivity in embryos of the cultivar ‘nabali’ before two weeks of incuba­ tion on ms medium, which agree with our results indicating a faster germination on om medium com­ pared to ms one. several studies reported low rates of embryo ger­ mination varying with genotype but depending more on the quality of the used embryos. therefore, jafarzadeh­bajestani et al. (2011) obtained less than 6% of germination on ms medium with embryos of the cultivar ‘zard’ while a desiccation step for three days improved conversion to 50% and formation of rooted plantlets with 6 to 8 leaves. furthermore, cul­ turing embryos of the cultivar ‘picual’ on a cellulose acetate semi­permeable membrane during the first month of maturation (cerezo et al., 2011) allowed an adequate dehydration, good structuring of the embryos and a significant improvement in germina­ tion and quality of the obtained plantlets. therefore, the low germination capacity observed in this study was probably due to the use of embryos directly after their maturation without passing a desiccation phase allowing the synchronization of their germination (merkle et al., 1995). germination of olive somatic embryos was often achieved in the absence of hormones, although rugini (1995) recommended the addition of zeatin to solid ms medium to boost emergence. thus, embryos of the cultivars ‘chetoui’, ‘chemlali’ and ‘arbequina’ (trabelsi et al., 2003) and ‘picholine marocaine’ (brhadda et al., 2008) germinated easily on om and ms media supplemented with 0.5 mg l­1 of zeatin and resulted to well­developed plantlets, while a free­ hormones medium rich in sucrose induced cell prolif­ eration of the embryos. conversely, toufik et al. (2017) observed that the presence of growth regula­ tors, in particular zeatin alone in the om­based medi­ um, was not essential for the germination of embryos of the cultivar ‘dahbia’ and can even inhibit the root emergence and cause a strong explant necrosis although the addition of naa with ga3 allowed up to 45% conversion from mature embryos according to mazri et al. (2020). therefore, the inclusion of growth regulators especially cytokinins to the germination 190 adv. hort. sci., 2022 36(3): 185­191 medium is directly determined by the genotype and degree of maturity of the embryos (merkle et al., 1995). in this sense, bradaï et al. (2016) indicated that well­matured and structured or cotyledonary embryos of ‘picual’ germinated easily in the absence of hormones, unlike globular embryos whose germi­ nation varies between 30 and 70%. the regenerated plants in this study showed a nor­ mal phenotype (phyllotaxis, leaf shape, etc.) during their in vitro maintenance as well as an easy acclima­ tization to natural conditions. leva (2009) indicated that plantlets regenerated by somatic embryogenesis show a stable phenotype similar to that of the moth­ er plants. moreover, despite their fragility, these plants acclimatize easily to natural conditions and show normal growth under greenhouse and good development after transfer to the field. micropropagation of shoots and acclimatization of regenerated plantlets the in vitro multiplication of plants regenerated by somatic embryogenesis was rarely practiced because their acclimatization was usually carried out directly after germination (bradaï et al., 2016). in fact, the dkw medium (driver and kuniyuki, 1984) as modified by revilla et al. (1996) and supplemented with a hormonal balance rich in cytokinins was often used although the chemical formulation of om medi­ um containing zeatin was commonly recommended for the rapid stimulation of axillary buds of several olive cultivars (lambardi et al., 2013) due to its nutri­ tional content, particularly in microelements (rugini et al., 2020). cerezo et al. (2011) indicated that the micro­cuttings taken after germination of embryos of the cultivar ‘picual’ respond easily on dkw medium supplemented with ba and iba by developing shoots of about 1.4 cm in height with formation of a basal callus often accompanied by the emission of one or more roots. nevertheless, bradaï et al. (2016) observed that shoots multiplication from somatic embryos was influenced more by the age of the cell culture than by its genotype (callus line). these authors obtained more developed rooted shoots from young cultures while less sprouted buds with older lines giving small shoots often without roots. according to bhojwani and razdan (1996) in vitro shoots need to reach a minimal size to root easily as small plants may not survive during the acclimatiza­ tion period. therefore, our results confirm the impor­ tance of the genotype, the significant effect of the dkw chemical formulation and the presence of zeatin in addition to an auxin for the multiplication and rooting of shoots resulting from the germination of somatic embryos. in conclusion, our study is a contribution to the optimization of the in vitro regeneration of olive tree by somatic embryogenesis and describes for the first time an efficient regeneration of whole plants with­ out morphological abnormalities in the main olive cultivar in algeria ‘chemlal’ via embryogenic cultures induced from juvenile material, radicles of zygotic embryos. the obtained results show that the devel­ opment of plantlets by germination of somatic embryos depends strongly on the genotype and the chemical and hormonal composition of the used cul­ ture medium. a solid om medium supplemented with hormones allowed a faster germination result­ ing in well developed shoots. subsequently, multipli­ cation of the shoots on om or dkw media containing zeatin with iba generated whole and rooted plantlets easily acclimatized to natural conditions. acknowledgements the authors gratefully acknowledge funding sup­ port from the national higher school of agronomy (ensa) and the algerian ministry of higher education and scientific research (mesrs). references besnard g., terral j.f., cornille a., 2018 ­ on the ori‐ gins and domestication of the olive: a review and per‐ spectives. ­ ann. bot., 121: 385­403. bhojwani s.s., razdan m.k., 1996 ­ plant tissue culture: theory and practice, a revised edition. ­ elsevier, the netherlands, pp. 767. bradaï f., pliego­alfaro f., sánchez­romero c., 2016 ­ long‐term somatic embryogenesis in olive (olea europaea l.): influence on regeneration capability and quality of regenerated plants. ­ sci. hortic., 199: 23­31. bradaï f., sánchez­romero c., martín c., 2019 ­ somaclonal variation in olive (olea europaea l.) plants regenerated via somatic embryogenesis: influence of genotype and culture age on genetic stability. ­ sci. hortic., 251: 260­266. brhadda n., walali l.d.m., abousalim a., 2008 ­ effet du sucre sur l’embryogenèse somatique de l’olivier (olea europaea l.) cv. ‘picholine marocaine’. ­ biotechnol. agron. soc. environ., 12(3): 245­250. cañas l.a., benbadis a., 1988 ­ in vitro regeneration from cotyledon fragments of the olive tree (olea titouh et al. ‐ somatic embryogenesis in algerian olive cultivars 191 europaea l.). ­ plant sci., 54: 65­74. cerezo s., mercado j.a., pliego­alfaro f., 2011 ­ an efficient regeneration system via somatic embryogene‐ sis in olive. ­ plant cell tiss. organ cult., 106: 337­344. driver j.a., kuniyuki a.h., 1984 ­ in vitro propagation of paradox walnut rootstock. ­ hortscience, 19: 507­509. fabbri a., bartolini g., lambardi m., kailis s., 2004 ­ olive propagation manual . ­ landlinks press, collingwood, vic, australia, pp. 141. jafarzadeh­bajestani m., khodai­kalaki m., motamed n., noorayin o., 2011 ­ genetic transfor‐ mation of olive somatic embryos through agrobacterium tumefaciens and regeneration of trans‐ genic plants. ­ afr. j. biotechnol., 10(28): 5468­5475. lambardi m., ozudogru e.a., roncasaglia r., 2013 ­ in vitro propagation of olive (olea europaea l.) by nodal segmentation of elongated shoots, pp. 33­44. ­ in: lambardi m., e.a. ozudogru, and s.m. jain (eds.) protocols for micropropagation of selected eco‐ nomically‐important horticultural plants, methods in molecular biology. humana press, totowa, nj, usa, pp. 994. leva a.r., 2009 ­ morphological evaluation of olive plants propagated in vitro culture through axillary buds and somatic embryogenesis methods. ­ afr. j. plant sci., 3(3): 37­43. lopes t., capelo a., brito g., loureiro j., santos c., 2009 ­ genetic variability analyses of the somatic embryogenesis induction process in olea spp. using nuclear microsatellites. ­ trees, 23(1): 29­36. mazri m.a., naciri r., belkoura i., 2020 ­ maturation and conversion of somatic embryos derived from seeds of olive (olea europaea l.) cv. dahbia: occurrence of secondary embryogenesis and adventitious bud forma‐ tion. ­ plants., 9(1489): 14. merkle s.a., parrot w.a., flinn b.s., 1995 ­ morphogenic aspects of somatic embryogenesis, pp. 155­204. ­ in: thorpe t.a. 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piracicaba, sp, brazil. 2 department of exact sciences, university of são paulo, ‘luiz de queiroz’ college of agriculture, av. 11 pádua dias, 13418‐900 piracicaba, sp, brazil. key words: applied statistics, image analysis, physiological variability, seed priming. abstract: digital image analysis has emerged as a highly precise and efficient methodology for assessing the physiological attributes of seeds. this research aimed to assess the morphological and physiological properties of lettuce seeds subjected to hydropriming using multivariate statistical approaches. two lettuce genotypes, roxa and vanda, were evaluated under hydropriming treatments (primed­dry and primed­stored). seedlings were digitally scanned, and vigor indices were quantified using the seed vigor imaging system (svis®). data were analyzed by multivariate analysis of variance (manova), with tests of normality and homogeneity of covariance ensuring analytical robustness. the primed­dry treatment resulted in minimal improvement in vigor and uniformity, while the primed­stored treatment promoted a partial recovery of these attributes. the roxa genotype exhibited greater variability in vigor and seedling length, whereas vanda demonstrated higher uniformity but slightly reduced seedling growth. a strong positive correlation was observed between the vigor index and seedling length, reinforcing the importance of these parameters in seed quality assessment. these findings underscore the utility of digital image analysis combined with multivariate statistical methods for the accurate assessment of seed vigor, thereby improving seed­lot classification and informing decision­making in lettuce production systems. 1. introduction computerized analysis of seed and seedling images has emerged as an (*) corresponding author: heiberandrestrujillo@gmail.com citation: trujillo h.a., assis de oliveira c.f., villegas lobos c.m., da silva m., gomes­junior f.g., 2025 ­ digital and multivariate analysis of lettuce seed vigor: impact of hydropriming on physiological potential. ­ adv. hort. sci., 39(3): 165­173. orcid: tha: 0000­0001­6604­9438 aocf: 0000­0003­2143­9015 vlcm: 0000­0003­3176­5236 dsm: 0000­0001­9079­8981 gjfg: 0000­0001­9620­6270 copyright: © 2025 trujillo h.a., assis de oliveira c.f., villegas lobos c.m., da silva m., gomes­junior f.g. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 27 march 2025 accepted for publication 19 august 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17532 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-6604-9438 http://orcid.org/0000-0003-2143-9015 http://orcid.org/0000-0003-3176-5236 http://orcid.org/0000-0001-9079-8981 http://orcid.org/0000-0001-9620-6270 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 165­173 166 innovative tool for determining the physiological characteristics of these structures. this technology stands out for its objectivity, specificity in detecting subtle traits, efficiency, and potential for standardization (rahman and cho, 2016; xia et al., 2019; wang et al., 2021; liu et al., 2023). the use of digital imaging and automated software enables the analysis of a large number of samples in shorter periods, increasing the efficiency and accuracy of evaluations. assessing the physiological potential of lettuce seeds through digital image analysis has proven to be a promising approach, particularly as a complement to conventional methods that do not fully reflect seed quality under real field conditions (waters­ junior and blanchette, 1983; marcos­filho, 1999). the use of the seed vigor imaging system (svis®), developed by sako et al. (2001), has been widely adopted to quantify seed vigor in various species, including soybean, corn, melon, sweet corn, castor bean, peanut, okra, common bean, eggplant, tomato, cotton, and sunflower (hoffmaster et al., 2003; marcos­filho et al., 2006; marchi et al., 2011; alvarenga et al., 2013; caldeira et al., 2014; gomes­ junior et al. , 2014; rocha et al. , 2015). this technology allows for detailed analyses of parameters such as seedling growth uniformity and development, reducing the subjectivity of traditional evaluations. however, to enhance the accuracy of vigor assessment, it is essential to employ multivariate statistical models that enable the simultaneous analysis of multiple interrelated variables. multivariate analysis of variance (manova) has been used to investigate complex interactions between experimental factors, allowing for the identification of patterns that would be difficult to detect using univariate approaches (johnson and wichern, 2002; nicacio et al., 2013). previous studies have demonstrated that manova is an effective tool for evaluating seed performance under different treatments, ensuring greater robustness in result interpretation (oliveira et al., 2013). this study aimed to analyze the morphological and physiological properties of lettuce seeds subjected to hydropriming using digital imaging of seedlings and a multivariate approach. the study sought to understand the interactions between the physiological attributes of the seeds and the impact of hydropriming on germination potential and early seedling development. 2. materials and methods the research was conducted at the seed analysis laboratories, the ‘professor silvio moure cicero’ image analysis laboratory of the department of crop science, and the department of math, chemistry, and statistics at the luiz de queiroz college of agriculture, university of são paulo in piracicaba, sp, brazil. seed material and priming treatment lettuce seeds from the genotypes scarlet red crisphead (roxa) and vanda crisphead (vanda) were used, supplied by sakata seed south america ltd. the selection of the two lettuce genotypes was based on their contrasting physiological and morphological characteristics and on their commercial relevance within brazilian lettuce production systems. each genotype was represented by ten seed lots, with germination rates within commercial standards and different vigor levels among the lots. each seed lot was divided into three treatments: (i) non­primed seeds (control), (ii) hydroprimed dried seeds (dried in an oven at 30°c and 45­55% relative humidity for 96 hours), and (iii) hydroprimed stored seeds (dried and stored in a chamber at 10°c and 30% relative humidity for three months). hydropriming was performed using the drum method, utilizing the s­hidro® control equipment, which allowed for the controlled application of water at regular intervals until reaching the required volume for each lot (kikuti and marcos­filho, 2012). the calculation of the required water volume for this method was based on the water imbibition curve, considering the volume needed for each seed lot before primary root protrusion (caseiro, 2003). at the beginning of each cycle, the electric pump was activated for 1 second, allowing the intake of a water volume between 0.9 and 1.1 ml, adjusted according to the specific needs of each seed lot and accounting for system losses to ensure 100% efficiency. water application was carried out at one­hour intervals until the total required volume for each seed lot was reached (ranging from 4.00 to 4.31 ml for the roxa genotype and from 3.67 to 3.87 ml for vanda). the entire procedure was conducted under laboratory conditions at a constant temperature of 25°c. seed vigor assessment using digital image analysis (svis® software) four replicates of 25 seeds per lot for each trujillo et al. ‐ multivariate and digital analysis of lettuce seed vigor 167 vigor results from the combination of two main components: average seedling length and uniformity, both adjusted by weighting factors (w) defined by the system, allowing the assignment of greater or lesser relative importance to each characteristic (hypocotyl­to­radicle ratio of 40:60, applied in this research). growth is estimated from the mean lengths of the hypocotyl (lh) and radicle (lr), weighted by their respective coefficients (wh and wr), thereby composing the total seedling length. uniformity, in turn, is calculated based on the standard deviations of hypocotyl length (sh), radicle length (sr), total seedling length (stotal), and the hypocotyl­to­radicle length ratio (s(r/h)), also weighted by their respective coefficients (wsh, wsr, w(sr/h)). multivariate analysis multivariate analysis of variance (manova) was used to describe the effects of categorical factors (treatments) on multiple response variables (huberty and olejnik, 2006). manova extends anova to the multivariate context, allowing simultaneous testing of multiple dependent variables. in this study, a two­ way manova was employed, considering two categorical factors: genotype (roxa and vanda) and hydropriming treatment (control, primed dry, primed stored). this factorial approach allows for the evaluation of both the main effects of each factor and their interaction. the general model for two­way manova: xlkr= μ + τl + βk + γlk + ϵlkr x represents the dependent variable, l represents the levels of factor 1 (genotype); k represents the levels of factor 2 (hydropriming treatment); r represents the replications; µ is the overall mean; τl representing the interaction effect and βk representing the treatment or genotype main effects; γ lk is the interaction effect between the factors; represents the random error. the hypothesis tests included: interaction effect: h0 : γ11 = γ12=⋯= γgb = 0 vs. h1 : at least one γgb ≠ 0 genotype effect: h0 : τ11 = τ12=⋯= τg = 0 vs. h1 : at least one τg ≠ 0 hydropriming effect: h0:β1 =β2 =β3 = 0 vs. h1 : at least one βb ≠ 0 statistical significance was determined using wilks’ lambda (wilks, 1935), pillai’s trace (hand and taylor, 1987), hotelling­lawley trace (krzanowsk and treatment were arranged in two rows on the upper third of two blotter paper sheets, placed on the lids of transparent plastic boxes (11 × 11 × 3.5 cm). the boxes were covered with transparent plastic bags and incubated in a bod chamber at 25°c for three days in darkness. to ensure proper seedling development according to natural geotropism, the boxes were positioned at a 70° angle relative to the horizontal plane. to determine seed vigor, the seed vigor imaging system (svis®) (sako et al., 2001) software was used. the seedlings (and ungerminated seeds) from each replicate were transferred onto a blue ethylene­vinyl acetate (eva) sheet, providing the necessary contrast for system analysis. the seedlings were then scanned using an hp scanjet 200 scanner, which was inverted and placed inside an aluminum box (60 × 50 × 12 cm), with the resolution set to 300 dpi and connected to a computer. the scanned images were processed using svis® software, including manual corrections when necessary to ensure accurate seedling identification (fig. 1). the seed vigor index is calculated according to the methodology proposed by sako et al. (2001): vigor index = wg × growth × wu × uniformity seedling length = wg {wh × lh + wr × lr, 1000} uniformity index = max {1000‐(wsh × sh + wsr × sr + stotal + w(sr/h) × s (r/h)‐wd ),0} fig. 1 ­ workflow of computerized image analysis of lettuce seedlings, from germination to vigor assessment. steps include seedling acquisition from the germination test (a), transfer to an eva sheet (b), digital imaging (c), and vigor determination using the svis® software (d). the results include the vigor index (vi) and the uniformity index (ui), both ranging from 0 to 1000 (directly propor­ tional to seedling vigor), as well as the average seedling length, initially measured in pixels and later converted to centimeters. adv. hort. sci., 2025 39(3): 165­173 168 marriott, 1994; anderson, 2003), and roy’s largest root (krzanowski, 2000). when manova indicated significant differences, post hoc univariate anovas were conducted to determine which dependent variables contributed to the observed differences. statistical assumptions and data validation before conducting manova, the following statistical assumptions were tested: multivariate normality, using the henze­zirkler test (henze and zirkler, 1990) and homogeneity of covariance matrices using box’s m test (johnson and wichern, 2002). in the univariate context, the anderson­ darling test (scholz and stephens, 1987) was used to assess normality. initial analyses revealed that seedling size did not meet the assumption of normality; therefore, this variable was removed from the final manova model to ensure compliance with statistical assumptions. software and data processing all statistical analyses were performed using the r programming language (r core team, 2024), with the packages ‘mvn’ for normality tests and ‘car’ for manova. data visualizations, including boxplots and correlation matrices, were generated using the ‘ggplot2’ and ‘corrplot’ packages. image processing was performed using svis® software, ensuring standardization and reproducibility of seed vigor measurements. 3. results descriptive data analysis the descriptive analysis allowed the identification of the main characteristics of the studied variables. figure 2 presents boxplots for the three response variables in this research: vigor index, uniformity index, and seedling length. it is observed that the means values of vigor and uniformity are similar between genotypes, while the dispersion of vigor is higher. seedling length, measured in centimeters, is on a different scale from the other variables and exhibits a lower correlation with them. the roxa genotype exhibits a higher median and greater variability, suggesting either greater vigor or increased heterogeneity. in contrast, the distribution of the uniformity index between the two genotypes is similar, indicating that growth is uniformly distributed. the boxplots for the analyzed variables concerning hydropriming treatments are shown in figure 3. it is observed that vigor and uniformity vary significantly between treatments. seeds subjected to the primed dry treatment showed lower means for these variables. seedling length showed less pronounced differences, with the control treatment presenting the highest mean. the mean values of the variables for each genotype are shown in table 1, while table 2 shows the corresponding values for each hydropriming treatment. table 1 ­ mean values of vigor index, uniformity index, and seedling length for roxa and vanda genotypes fig. 2 ­ boxplot comparing the vigor index, uniformity index (both ranging from 0 to 1000), and seedling length (cm) between roxa and vanda genotypes. fig. 3 ­ boxplot comparing the vigor index, uniformity index (both ranging from 0 to 1000), and seedling length (cm) across control, primed dry, and primed stored treatments. genotype vigor index uniformity index seedling length (cm) roxa 761 762 3.47 vanda 754 757 3.38 trujillo et al. ‐ multivariate and digital analysis of lettuce seed vigor 169 the distribution, correlation, and dispersion of the vigor, uniformity, and seedling length, their correlations, and dispersion are shown in figure 4. the vigor index is strongly correlated with seedling length (r= 0.903), suggesting that more vigorous seedlings tend to be longer. the correlation between uniformity and length is moderate (r= 0.447), indicating a weaker association between these variables. interaction between genotypes and treatments the interactions among hydropriming treatments within each genotype are illustrated in figure 5. the primed dry treatment had the least pronounced effect on vigor and uniformity, while primed stored allowed a partial recovery of these parameters. the interaction between genotypes within each treatment is shown in figure 6. roxa exhibited a better response to the primed stored treatment, while vanda demonstrated greater sensitivity. seedling length was more affected in vanda under the primed stored condition, whereas roxa showed a tendency toward increased growth. table 2 ­ mean values of vigor index, uniformity index, and seedling length for control, primed dry, and primed stored treatments hydropriming vigor index uniformity index seedling length (cm) control 783 770 3.63 primed dry 727 745 3.29 primed stored 762 765 3.37 fig. 4 ­ distribution, correlation, and dispersion analysis of the vigor index, uniformity index, and seedling length (cm). fig. 5 ­ interactions plot of hydropriming treatments within the roxa and vanda genotypes for the vigor index, uniformity index (both ranging from 0 to 1000), and seedling length (cm). assumptions of manova the suitability of the data for manova was verified using the henze­zirkler and anderson­ darling tests (tables 3 and 4). the henze­zirkler test indicated that the data did not present multivariate normality due to the length variable. therefore, this variable was removed for the manova analysis (table 5). after its removal, multivariate normality was achieved (table 6). the box´s m test for equality of covariance matrices confirmed that the data met fig. 6 ­ interaction plot between roxa and vanda genotypes within each hydropriming treatment for the vigor index, uniformity index (both ranging from 0 to 1000), and seedling length (cm). test statistic p­value nmv* henze­zirkler 1.84 0.00000005 no table 3 ­ henze­zirkler test for assessing the multivariate nor­ mality of the full dataset *not meeting validity criteria (normality violated). bewley, 2000; marcos­filho, 2015). the multivariate analysis showed that the roxa genotype exhibited greater variability in vigor and seedling length, whereas vanda demonstrated greater uniformity in growth. these results are consistent with studies indicating that different genotypes may exhibit significant variations in their physiological responses (hampton and tekrony, 1995; elias et al., 2012; rahman and cho, 2016; cheng et al., 2023). the significant interaction between hydropriming treatments and genotypes supports the hypothesis that the priming response may be cultivar­specific. the primed dry treatment had a weaker effect on vigor and uniformity, as reported in previous studies, which suggest that osmotic stress generated during the process may compromise seed physiological potential (raj and raj, 2019; lewandowska et al., 2020; pirasteh­anosheh and hashemi, 2020; rhaman et al., 2020 a, 2020 b), particularly during the period immediately following treatment. conversely, the primed stored treatment exhibited partial recovery of vigor and uniformity parameters, in agreement with studies highlighting the ability of seeds to 170 adv. hort. sci., 2025 39(3): 165­173 the required assumptions for manova (table 7). a two­factor manova was performed to evaluate the interaction effect between genotype and hydropriming treatment. the roy’s largest root test indicated no significant differences between genotypes. however, the main effect of hydropriming treatment, as well as the interaction between genotype and treatment, were highly significant (table 8). these results suggest that hydropriming treatments significantly influenced the analyzed variables, regardless of genotype. 4. discussion and conclusions the findings of this research can be understood in light of the existing literature on seed physiological potential and vigor. seed vigor is one of the main factors influencing success seedling establishment in the field and early plant development (black and table 4 ­ anderson­darling test assessing the univariate normal­ ity of the vigor index, uniformity index, and seedling length variables variable statistic p­value normality vigor 0.6619 0.0830 yes uniformity 0.4255 0.3134 yes seedling length 15.860 0.0004 no *not meeting validity criteria (normality violated). table 5 ­ henze­zirkler test after removal of the seedling length variable, indicating multivariate normality of the remaining variables *not meeting validity criteria (normality violated). test statistic p­value nmv* henze­zirkler 0.9217 0.1112 yes table 6 ­ anderson­darling test after removal of the seedling length variable, confirming the normality of the vigor index and uniformity index variables *not meeting validity criteria (normality violated). variable statistic p­value normality vigor 0.6619 0.0830 yes uniformity 0.4255 0.3134 yes table 8 ­ manova results for genotype and hydropriming treatment factors, considering the vigor index and uniformity index variables table 7 ­ box´s m test for the equality of covariance matrices among the analyzed groups statistic p­value 2.05 0.56 source df roy’s statistic f approximation num. df den. df p­value genotype 1 0.002587 0.3014 2 233 0.74 hydropriming 2 0.0726 85.044 2 234 0.0002721 *** interaction 2 0.2100 245.726 2 234 0.00002 *** residuals 234 trujillo et al. ‐ multivariate and digital analysis of lettuce seed vigor 171 stabilize after hydropriming when stored under appropriate conditions (farooq et al., 2006, 2010; huang et al., 2015; souza et al., 2016; farooq et al., 2021). furthermore, the current results support research indicating that the effectiveness of hydropriming may vary depending on genotype and environmental conditions (muhie et al., 2024). recent studies emphasize the importance of evaluating each cultivar separately to determine the most suitable seed treatment method (cheng et al., 2023; qiu et al., 2023). the positive correlation between vigor and seedling length reinforces the relevance of these variables in seed quality assessment. the literature suggests that more vigorous seedlings tend to develop stronger root systems and exhibit higher field emergence rates (kikuti and marcos­filho, 2012; kikuti and marcos­filho, 2013; alvarenga and marcos­filho, 2014; marcos­filho, 2015; rego et al., 2023). prior studies indicate that seed vigor is closely associated with early seedling growth and crop establishment (marcos­filho, 2015). image analysis has proven to be a promising tool for evaluating seed vigor. technologies such as the seed vigor imaging system (svis®) have demonstrated a high degree of precision in classifying lettuce seed lots and those other crops (gomes­junior et al., 2009; rodrigues et al., 2020). the use of computer vision and machine learning in seed vigor assessment is increasingly being explored, enabling fast and objective analyses (de medeiros et al., 2020; wang et al., 2021; liu et al., 2023; pang et al., 2023). the statistical methodology adopted in this research was essential to ensure the robustness of the analyses and the reliability of the results. initially, the descriptive analysis enabled the identification of trends and patterns in the data, facil itating interpretation. to assess relationships among variables, pearson´s correlation was applied, revealing a strong association between the vigor index and seedling length. the main statistical method used was multivariate analysis of variance (manova), a widely accepted methodology approach studies involving correlated dependent variables (oliveira et al., 2013; din and hayat, 2021; baumeister et al., 2024). manova is particularly suitable when response variables are correlated, allowing for the simultaneous evaluation of the effects of experimental factors (johnson and wichern, 2002). to ensure the method’s applicability, the henze­ zirkler test was used to assess multivariate normality, and box’s m test was applied to verify the homogeneity of covariance matrices­a key assumption for valid manova results. the significance of main effects and interactions was assessed using roy’s largest root, which is recommended when effects have a strong impact on data variability (kose et al., 2018). the manova results were complemented by univariate analyses, allowing for a more detailed interpretation of the individual factor effects, as suggested by scholz and stephens (1987). this combined approach improves precision in identifying significant effects and interactions, thereby enhancing the understanding of genotype responses to hydropriming. however, such statistical procedures also have limitations. in this study, seedling length had to be excluded from the final manova due to the violation of normality assumptions, which restricted the scope of multivariate interpretation. in conclusion, the roxa genotype performed better under the primed stored treatment than vanda. seedling length was influenced by hydropriming, with primed stored proving unsuitable for vanda. therefore, the statistical approach adopted in this study enabled a comprehensive and detailed analysis, enhancing our understanding of the effects of hydropriming on the evaluated genotypes. these findings are crucial for understanding genotypes­ treatments interactions and may contributing to the optimization of hydropriming strategies for lettuce seeds. future studies should consider incorporating a broader range of cultivars, extended storage durations, and the integration of machine learning techniques to improve vigor prediction. acknowledgements to the ‘ministerio de ciencia, tecnología e innovación (minciencias) / colfuturo’, colombia, for supporting this research through the ‘doctorado exterior’ ­ 885 scholarship since 2021. to the ‘coordenação de aperfeiçoamento de pessoal de nível superior (capes)’, brazil, for the ‘proex’ scholarship granted throughout the doctoral program. references alvarenga r.o., marcos­filho j., 2014 ‐ vigor evaluation of stored cotton seeds, including the seed vigor imaging system (svis®). ­ j. seed sci., 36(2): 222­ 230. alvarenga r.o., marcos­filho j., timóteo t.s., 2013 ­ assessment of the physiological potential of super sweet corn seeds. ­ j. seed sci., 35(3): 340­346. anderson t.w., 2003 ­ an introduction to multivariate statistical analysis. ­ wiley & sons, inc., 3rd edition. hoboken, nj, usa, pp. 752. baumeister m., ditzhaus m., pauly m., 2024 ­ quantile‐based manova: a new tool for inferring multivariate data in factorial designs. ­ 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negative impacts of nanoparticles on plants. investigating the toxic effects of ag nps on model plants, such as onion, can reveal the probability of damage. thus, the present study was conducted to compare the germination indices and biochemical parameters of edible onion seeds treated with different concentrations of two types of silver nanoparticles (green synthesized and chemical synthesized) to examine the oxidative stress. based on our results, the interaction between leaf extract and silver salt result­ ed in a color change from pale yellow to dark brown, the first sign of the agnps formation. the green agnps treatment improved the onion germination indices. the green agnps­exposed seeds displayed a no­significant reduction in protein content and same protease activity as the control treatment, but chem­ ical agnps­treated displayed a significant rise of protease and reduction in pro­ tein content in concentration more than 0.06 gl­1. in chemical agnps­treated seeds both peroxidase and catalase displayed an ascending linear trend and the most activities belonged to chemical agnps at 0.05 g l­1 (315.62 and 51.45 μmol min­1g­1 fw, respectively). both nanoparticle types made an increase in mda, but green agnps did not significantly differ with control treatment. it can be concluded that the green synthesis of nanoparticles is a safe and suitable alter­ native for chemical­synthesized metal nanoparticles. 1. introduction the production, manipulation, and use of nanoparticles (nps), due to their distinctive and definite capabilities, has become one of the most attractive research topic in different area of science, including biology, chemistry, engineering, medicine, physics, agriculture and food. nano­ dimension structures involved in many aspects of plant biology such as ionic transport and molecular transmissions. the diameter of plant cell wall pores are in the range of 5 to 20 nm. furthermore, plasmodesmata, the intracellular channels to facilitate molecular transitions, are also nano­scale (50 to 60 nm in diameter) (zambryski, 2004). (*) corresponding author: shamili@ut.ac.ir citation: akbarnejad­samani z., shamili m., samari f., 2020 ­ the toxicity potential of ag nanoparticles synthesized from cordia myxa l.. ­ adv. hort. sci., 34(1): 93­104. copyright: © 2020 akbarnejad­samani z., shamili m., samari f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 october 2019 accepted for publication 8 january 2020 ahs advances in horticultural science http://https://www.google.com/search?q=plant+plasmodesmata&tbm=isch&source=hp&sa=x&ved=2ahukewixnpql9cpiahugjqqkhvyzcdyqsar6bagieae http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 93­104 94 the nanotechnologies based­products are expect­ ed to upturn (maynard et al., 2006; rejeski and lekas, 2008). nanotechnology applications in agricul­ tural sector involve in precision farming, smart feed­ ing, enrichment of food quality, products packaging, products labeling, nano­pesticide, nano­fertilizer, and nano­herbicide (thiruvengadam et al., 2018). the dif­ ferent chemical and physical methods are applied to the synthesis of nanoparticles such as sol­gel, elec­ trochemical, hydrothermal, sono­chemical, and microwaves techniques. chemical methods had low efficiency, need high temperature, high pressure and high energies during the reaction process. besides they need the expensive reagent and complex equip­ ment. furthermore, chemical methods require some non­degradable chemical components, as reducing or stabilizing agents, which finally caused environ­ mental pollution (senapati et al., 2012). the potential of biological materials to synthesize metal nanoparticles has provided a low­cost and eco­ friendly route. biological synthesis or green synthesis of nanoparticles, uses biological material as the reducing and capping agents (veerasamy et al., 2011). the green synthesis uses microorganisms such as fungus, bacteria (ahmad et al., 2005), yeast and actinomycetes (kowshik et al., 2003) or macro­organ­ isms e.g., plants and algae, as intermediate agents (niemeyer and mirkin, 2004; dubey et al., 2010; prasad et al., 2012). plant­mediated nanoparticles synthesis is consid­ ered as one of the appealing options in bio­nanopar­ ticles synthesis, due to high variety and abundance, no need for a complicated growth conditions, bulk­ biomass production, no need for special nutrient media, cost­effectiveness, richness of various effec­ tive metabolites, simple single­step synthesis process and suitable for the large­scale synthesis of nanopar­ ticles. polyphenolic contents of the plant act as reducing agents and eventually act as coating and stabilizing agent of nanoparticles (dubey et al., 2010). cordia myxa (l.) or sapistan­tree, a member of boraginaceae family, is native to tropical areas of asia. it well documented that c. myxa contained flavonoids, glycosides, sterols, terpenoids, saponins, phenolic acids, alkaloids, tannins, coumarins, resins ; and mucilage (inas et al., 2011; rashed et al., 2014): a suitable candidate for green nps synthesis. however, there is contradictory information about the positive or negative impacts of nanoparti­ cles on plants. titanium dioxide nanoparticles increased the dry and fresh weight of spinach by increasing the light absorption, boosting rubisco enzyme activity (lin and xing, 2007) andrising the nitrogen metabolism (yang et al., 2007). zinc and copper nanoparticles showed significant differences in growth, and toxicity symptoms in treated plants (lee at al., 2008; monica and cremonini, 2009; musante and white, 2012; prasad et al., 2012). titanium nanodioxide (40 μg ml­1) improved seed germination, dry and fresh weight of onion seedling; however, the higher concentrations (50 μg ml­1 and more) had a reverse result (raskar and laware, 2013). conversely, this nanoparticle did not affect the length and quantity of the onion root (klancnik et al., 2011). the symptoms have depended on the form and coating type of nanoparticles (barrena et al., 2009). due to the growing rate of production and release of nanoparticles in nature (as nano­fertilizers, nano­ toxins, and nano­carriers), there are increasing con­ cerns about the possibility of toxicity and oxidative damage (whether chemical or green synthesis) of these particles on the ecosystem. the who (world health organization) and fao (the food and agriculture organization) at the meeting on the application of nanotechnologies in the food and agri­ culture sectors, in rome in 2010, recognized the potential concern of nanotechnology in agriculture and food sectors. the main concern was about the exponential developing global knowledge on nano­ technology and its applications, which may affect the balance of advantage to risk (fao/who expert meet­ ing report, 2010). the ag nps is known to have antioxidant, antibac­ terial and antifungal properties and may be used in food, medicine and cosmetics products (el­nour et al., 2010; abdel­aziz et al., 2014). so, investigating the toxic effects of these particles on model plants, such as onion, can reveal the probability of damage. thus, the present study was conducted to compare the germination indices and biochemical parameters of edible onion seeds who treated with different con­ centrations of two types of silver nanoparticles (green synthesized and chemical synthesized) to examine the oxidative stress. 2. materials and methods research site and plant material collection the current study was conducted at the horticultural laboratory of agriculture and natural resources faculty, chemical laboratory of science akbarnejad‐samani et al. ‐ nanoparticles impact on cordia myxia l. 95 faculty, central laboratory (university of hormozgan, bandar abbas, iran) and molecular research center (hormozgan university of medical sciences, iran), during 2018. preparation of cordia myxa leaf extract leaves of cordia myxa l. (a member of boraginaceae family), were collected from rooydar city, hormozgan province, iran (57° 6΄ e, 60° 3΄ n, elevation: 50 m, rh: 70%, mean temperature: 28±1°c) during winter, 2018. the healthy leaves were choosing, then washing in tap water followed by rins­ ing in distilled water and air drying (6 days under the shade conditions at 24±1°c). the dried leaves were powdered using an electric grinder. the leaf extract was prepared by mixing 10 g of dried powder along with 150 ml of deionized water and then heating the mixture at 80°c for 30 min. the solution was pre­fil­ tered through whatman no. 42 filter paper and re­fil­ tered through whatman no. 1 filter paper. the obtained extract was collected and stored in the refrigerator (samari et al., 2018). synthesis and purification of silver nanoparticles (agnps) using c. myxa leaf extract for agnps synthesis, 2.0 ml of c. mixa leaf extract was added to 25 ml of 7.0 mm aqueous solution of agno3 (merck, germany) and the reaction ph was adjusted to 11.0 using naoh. the mixture was con­ tinuously stirred at room temperature (24±1°c) for 3 h. the yellow­mixture turned to dark­brown (samari et al., 2018). the green synthesized agnps were cen­ trifuged at 10000 rpm for 30 min and rinsed with deionized water. then, the obtained sediments were re­dispersed in deionized water to get rid of any free phyto­molecules. this process (centrifugation and re­ dispersion) was repeated three times to ensure the purification of nanoparticles and separation of unbonded compounds. characterization of silver nanoparticles preliminary characterization of the green synthe­ sized agnps was carried out using a s­3100 uv­vis spectrophotometer (scinco, korea, which is operated at a resolution of 2 nm and is equipped with a 10 mm quartz cuvette). an aliquot of the dried purified pellets, obtained from centrifugation, was used for x­ray diffraction (xrd). the xrd pattern was obtained using a powder x­ray diffractometer (bruker d8 advance powder dif­ fractometer) with cu­kα radiations (λ=1.5406 nm, in a 2θ range from 20° to 80°). fine configuration of the green synthesized agnps was measured using a transmission electron microscopic examination (tem). for this, the colloidal agnps were allowed for sonication and then a thin film was prepared on the carbon coated grid (cu mesh 300). tem observations were made with (zeiss­em10c) operated at an accel­ erating voltage of 80 kv. preparation of colloidal solution of agnps (green and chemical synthesized) the chemical agnps wasprovided from iranian nano­biotechnology company. chemical and green nanoparticles were dispersed separately in distilled water and placed in an ultrasonic apparatus (soni­ ca® ultrasonic cleaners) for 30 min for homogeniza­ tion. then, colloidal solutions containing each nanoparticle were prepared at the concentrations of 0, 0.03, 0.06, 0.012, 0.025 and 0.05 g l­1. for germination assay, red onion seeds (allium cepa l.), disinfected (0.04% of sodium hypochlorite solution, 3 min), rinsed in distilled water (3 times, each for 1 min), dipped in nano­solutions for 30 min and finally rinsed in distilled water. seeds were then cultured in sterilized glass petri dishes (80 mm in diameter, covered with filter papers). each petri dish contained 100 seeds. germination indices germination percentage and germination value. germination test was conducted under a laboratory condition (24±1°c). the seeds were placed on 80 mm petri dishes and were covered with filter papers. the seeds were irrigated daily with double distilled water. counting was done by the emerge of the roots (the seed with root length equal or more than seed diam­ eter were assumed as germinated). the counting continued until 3 days fixed germination. the germi­ nation indices including germination percentage and germination value were calculated using the equa­ tions (1) and (2) (okoro, 1976). germination (%)= the total number of germinated seeds / total seed) x 100 (1) gv = mdg x pv (2) where gv is germination value and mdg is mean daily germination and calculated by the dividing of germination percentage by the number of days to the end of the test. pv or peak value, derived from all the cumulative germination percentages on any day divided by the number of days to reach these per­ centages. the length of radicle and plumule. seedling root and stem length was measured using a ruler (express in mm). adv. hort. sci., 2020 34(1): 93­104 96 biochemical analysis preparation of enzyme and protein extract. about 0.5 g of the root was homogenized in liquid nitrogen. then, 1.0 ml of extraction buffer (in 100 ml: contain­ ing of 50 mm potassium phosphate buffer with ph: 7.0, 0.0372 g edta and 1.0 g pvp) was added and centrifuged (15 min, 12000 rpm, 4°c). at last, the supernatant was used to assay protein content, and the activity of catalase, peroxidase, and protease (dhindsa et al., 1981). protein content assay. the bradford method (1976) was used to determine protein content. briefly, 1.0 ml of bradford reaction solution (contain­ ing 0.01 of coomassie brilliant blue g250, 5.0 ml of 96% ethanol and 10.0 ml of 85% phosphoric acid) was added to 50 μl of protein extract. the optical absorption of the extracts was determined by a spec­ trophotometer (cecil ce2501 model) at 595 nm with a plastic cuvette (bradford, 1976). peroxidase activity assay. after adding 33 μl of the enzyme extract to 1.0 ml of the peroxidase reaction solution (containing 13 mm guaiacol, 5 mm hydrogen peroxide and 50 mm phosphate potassium buffer at ph=7.0), the absorption of the extracts was recorded at 470 nm (chance and maehly, 1995). catalase activity assay. for catalase assay 50 μl of the enzyme extract was mixed with 1.0 ml catalase reaction solution (containing 50 mm phosphate potassium buffer at ph=7.0 and 15 mm hydrogen peroxide). the absorption was recorded at 240 nm (dhindsa et al., 1981). protease activity assay. after adding 350 μl of 50 mm sodium phosphate buffer (ph=7.5) and 800 μl of 1% casein (w/v) to 50 μl of the enzyme extract, the mixture was incubated under laboratory condition (24±1°c) for 10 min. then, 400 μl of 10% trichloroacetic acid (w/v) was added and the mixture re­incubated for 20 more min. finally, after centrifu­ gation (10000 rpm, 5 min), the optical absorption of extracts was determined at 280 nm with a quartz cuvette (kwmbhavi et al., 1993). malondialdehyde assay (mda). about 0.1 g of the root was homogenized with 5.0 ml of 1% trichloroacetic acid. the extract was then centrifuged (10000 rpm, 5 min) and the supernatant (250 μl) was placed in a water bath (95°c for 30 min), after adding one ml of mda (containing 20% trichloroacetic acid and 5% thiobarbituric acid). the samples were placed on ice and then re­centrifuged (1000 rpm, 5 min). finally, the optical absorption of the extracts was recorded at 532 and 600 nm (alexieva et al., 2001). total phenol assay. the total phenol was assayed using the spanos and wrolstad (1990) technique; about 0.1 g of root sample was homogenized with 15.0 ml of 80% methanol. the extract was then centrifuged (10000 rpm, 10 min). then, 490 μl of distilled water and 500 μl of folin reagent were added to 10 μl of the supernatant and were placed under dark condition (24±1°c, for 3 min). subsequently, 500 μl of 1% sodium carbonate (1.0 g sodium carbonate ­ 100 ml distilled water) was added to each sample and re­placed under dark condition (24±1°c) for 30 more min. finally, the absorbance of extracts was determined at 765 nm. antioxidant capacity (dpph assay). the dpph assay was followed by brand­williams et al. (1995) procedure. briefly, about 0.1 g of root sample was homogenized with 15.0 ml of 80% methanol and incubated under dark laboratory condition (24±1°c, for 24 h). then, the extracts were centrifuged (10000 rpm, 10 min). after adding 40 μl of methanol, 350 μl of dpph and 1550 μl of 80% methanol to 600 μl of the supernatant, the samples were kept under the dark condition at 4°c, for 20 min. finally, the optical absorption was recorded at 517 nm and the antioxi­ dant capacity was calculated using the equation (3): antioxidant capacity%= [(acont ­ asamp)/acont)] x 100 (3) where acont and asamp are the standard and sample optical absorption. data analysis the research was conducted as a factorial experi­ ment based on complete random design, with six replications (each replicon contains 100 seed). the statistical analysis was done using sas version 9.1.3 (sas institute inc. cary, nc, usa, 1990). the factors were agnps type (green agnps and chemical agnps) and agnps levels (0, 0.03, 0.06, 0.012, 0.025 and 0.05 g l­1). the shapiro­wilks test confirmed the data nor­ mality (procedure: proc univariate, sas). the multivariate analysis of variance, considering the agnps type and agnps levels as independent vari­ ables were done (procedure: proc glm, sas). pillai’s trace test confirmed the variance homogeneity (pro­ cedure: proc glm, sas). tukey’s test was performed for mean comparison (procedure: files, sedit, factor, range, p<0.01, mstatc). excel 2013 was used to draw the figures. akbarnejad‐samani et al. ‐ nanoparticles impact on cordia myxia l. 97 the green agnps treatment improved the onion ger­ mination. most germination percentage belonged to green agnps (100% in 0.05 g l­1), and the least value 3. results synthesis and characterization of c. myxa synthesized agnps the interaction between leaf extract and silver salt resulted in a color change from pale yellow to dark brown, the first sign of the agnps formation. this process occurred under room temperature (24±1°c) and lasted 3 hours (fig. 1). uv­vis spectroscopy is one of the most extensive techniques to confirm the production of nanoparti­ cles. the uv­vis spectrum of synthesized agnps using c. myxa leaf extract (fig. 2a) showed a significant peak with λmaxaround 410 nm due to spr (surface plasmon resonance) of agnps. tem images were used to detect the surface mor­ phology and size distribution of synthesized agnps by c. myxa leaf extract. tem micrographs of the agnps confirmed the spherical shape of particles, 3­10 nmsize, well­distributed and little aggregation in solu­ tion (fig. 2b); however, the average size of particles was found to be 5.8 nm. the xrd patterns of synthesized agnps showed prominent bragg reflections at 2θ values of 38.1, 44.25, 64.55 and 77.2 (fig. 3a), which is related to the (111), (200), (220) and (311) bragg reflections of face­centered cubic (fcc) agnps. sem analysis con­ firmed the nano dimension of chemical agnps (fig. 3b). germination indices germination percentage and germination value. the germination percentage was influenced by the type and concentration of nanoparticles (table 1). fig. 2 ­ the spr spectrum of c. myxa synthesized with the agnps (a), and tem image of agnps synthetized by c. myxa leaf extract (b). fig. 1 ­ the mixture of c. myxa leaf extract and agno3 (left), green­synthetized agnps colloidal solution (right). fig. 3 ­ the xrd patterns of the c. myxa synthesized agnps (a) and electron microscopic scanning image of chemical agnps (b). 98 adv. hort. sci., 2020 34(1): 93­104 (70%) belonged to chemical agnps treatment at con­ centrations more than 0.06 g l­1 (fig. 4a). the germination value also influenced by the interaction of treatments (table 1). green agnps treatment did not differ with control, while treat­ ment with the chemical agnps caused a significant reduction in the germination value (from 1.88 in con­ trolreached to 0.12 in the 0.05 g l­1). most and least germination values (1.90 and 0.12) were observed in onion seeds treated with the high concentration of green agnps and chemical agnps, respectively (fig. 4b). the length of radicle and plumule. the effect of type and concentration of agnps was significant on the length of plumule (fig. 5). there was no signifi­ cant difference between the green agnps and the control. even, the chemical agnps was not different from the control up to the concentration of 0.06 g l­1. the higher amount of chemical agnps, reduced the fig. 4 ­ the influence of agnps type and concentration on germi­ nation percentage (a) and germination value (b) in allium cepa. means ± sd of six replicates. the same letter denotes lack of a statistically significant difference (tukey, p<0.01). fig. 5 ­ the influence of agnps type and concentration on plu­ mule length (a) and radicle length (b) in allium cepa. means ± sd of six replicates. the same letter denotes lack of a statistically significant difference (tukey, p<0.01). table 1 ­ the interaction of agnps type and concentration on allium cepa biochemical parameters (z) the mean square values are given. * and **: state significant at 5 and 1% respectively agnps type ×agnps concentration: states the interaction of agnps type and agnps concentration sov germination percentage germination value radicle length plumule length protein peroxidase catalase protease mda phenol ic50 nps type 4408.33 ** (z) 89.89 ** 284.21 ** 926.64 ** 0.11 540853.35 ** 3600.65 ** 9151.33 ** 0.112 * 886.31 ** 14.57 nps concentration 3.80 0.07 9.98 ** 5.11 ** 0.27 * 40965.25 ** 13476.80 ** 1.40 * 0.030 ** 17.84 34.52 * nps type × nps concentration 15.53* 0.31 ** 4.90 ** 1.70 0.15* 38719.77 ** 7566.10 ** 1.87 * 0.020 ** 17.89 ** 21.90 ** error 13.10 0.26 1.17 1.92 0.16 5852.71 683.03 1.14 0.020 55.16 24.61 akbarnejad‐samani et al. ‐ nanoparticles impact on cordia myxia l. 99 plumule length (from 7.40 mm in the control treat­ ment reached to 3.28 mm in the 0.05 g l­1 of chemi­ cal agnps) (p≤0.01) (fig. 5a). the radicle length had a different pattern in response to the type and concen­ tration of the nanoparticles. green agnps improved radicle length (from 4.53 mm in control treatment reached to 7.76 mm in high concentration) (p<0.01). the chemical agnps made a decreasing trend in this parameter and reached it to 2.22 mm in the concen­ tration of 0.05 g l­1 (fig. 5b). biochemical indices protein content and protease activity. despite the significant effect of two agnps types on protein con­ tent and protease activity (table 1, p≤0.01), the inter­ action between agnps type and concentration had a diverse pattern (fig. 6a and b). the green agnps­ exposed seeds displayed a no­significant reduction in protein content (from 1.1 to 1 mg g­1). the reduction of protein content in chemical agnps­treated seeds was dose­dependent (fig. 6a). green agnps­treated seeds had the same protease activity as the control treatment. chemical agnps displayed a significant rise in concentration more than 0.06 gl­1 (from 1.77 μmol min­1 g­1 fw in control treatment to 6.21 μmol min­1 g­1 fw at the high concentration) (fig. 6b). antioxidant enzymes (peroxidase and catalase activities). by the results, the activity of both antioxi­ dant enzymes was influenced by the type and con­ centration of the agnps (table 1). green agnps­ treated seeds indicated no significant difference with control. in chemical agnps­treated seeds both enzymes displayed the linear trend, so most peroxi­ dase and catalase activities belonged to chemical agnps at 0.05 g l­1 (315.62 and 51.45 μmol min­1g­1 fw, respectively) (fig. 7a and b). malondialdehyde (mda), phenol and dpph (antioxidant capacity). the mda influenced by agnps treatment (table 1) and the less value was observed in control treatment (0.03 mg g­1 fw). both nanopar­ ticle types made an increase in mda, but green agnps did not significantly differ with control treat­ ment. also, the content of mda had a linear relation fig. 7 ­ the influence of agnps type and concentration on activi­ ties of peroxidase (a) and catalase (b) in allium cepa. means ± sd of six replicates. the same letter denotes lack of a statistically significant difference (tukey, p<0.01). fig. 6 ­ the influence of agnps type and concentration on pro­ tein content (a) and activity of protease (b) in allium cepa. means ± sd of six replicates. the same letter deno­ tes lack of a statistically significant difference (tukey, p<0.01). adv. hort. sci., 2020 34(1): 93­104 100 with chemical agnps concentration (from 0.03 mg g­1 fw in the control reached to 0.25 mg g­1 fw at high concentration of chemical agnps) (fig. 8a). the type and concentration of agnps influenced phenol content. the most value was assigned to chemical agnps (15.16 mg gallic g­1 fw at 0.05 g l­1), and the least value to control treatment (4.10 mg gallic g­1 fw) (fig. 8b). the amount of antioxidant capacity was also affected by nps treatments (table 1). this parameter was expressed in mg g­1 fresh weight based on the­ half­maximal inhibitory concentration (ic50). the con­ trol value (10.67 mg g­1 fw) followed a descending trend in both agnps types. it reached to 8.97 mg g­1 fw in green agnps while to 1.01 in chemical agnps (fig. 9). 4. discussion and conclusions plants contain flavonoids, phenols, aroma, latex and alcohols, and some of these compounds are responsible for the reduction of metal ions and pro­ duction of metal nps from the metal salts. it has been stated that the leaf extract of cordia species possesses phenolic and flavonoids derived com­ pounds such as robinin, rutin, datiscoside, hes­ peridin, dihydrorobinetin, chlorogenic and caffeic acid (al­ati, 2011). the hydroxyl and ketonic groups of such compounds, construct chelate structures by binding to metal ions (issaabadi et al., 2016). in pre­ sent work, the leaf extract of c. myxa was used as a reducing and stabilizing agent for phyto­synthesis of agnps. this eco­friendly procedure was free­hazard and non­toxic. the successful synthesis of green agnps from plant extracts has already reported in chenopodium album, camellia sinensis and rhus cori‐ aria (song and kim, 2009; dwivedi and gopol, 2010). the nps synthesis basis is oxidation of hydroxyl, carbonyl, and aldehyde and subsequently, reduction of metal ions during the neutralization of the electric charge (sivaraman et al., 2009). in present research the c. myxa extract turned to dark brown, due to the surface plasmon resonance of agnps and confirmed the successful synthesis of the agnps (kasthuri et al., 2009; roopan et al., 2013). in this study, the synthe­ sis process lasted 3 hours at room temperature con­ dition. other reports have also mentioned that this rapid nps synthesis dose not require high tempera­ tures (sivaraman et al., 2009). the fcc crystal struc­ ture of green synthesized agnps was found by com­ paring the obtained xrd data with jcpds file no. 04­ 0783 (sutradhar and saha, 2016), clearly indicate the highly crystalline structure of green agnps. nanoparticles play an important role in many areas of biology, chemistry and agriculture (navarro et al., 2008), but their adverse potential on the envi­ ronment is being subjected to extreme discussions. nps penetrate to various parts of the plants, some stored within the cell and some in extracellular space (lee et al., 2008). high agnps concentrations pass through the cell by diffusion, cause mitochondrial fig. 9 ­ the influence of agnps type and concentration on antioxidant capacity in allium cepa. means ± sd of six replicates. the same letter denotes lack of a statistically significant difference (tukey, p<0.01). fig. 8 ­ the influence of agnps type and concentration on mda (a) and total phenol (b) in allium cepa. means ± sd of six replicates. the same letter denotes lack of a statistically significant difference (tukey, p<0.01). akbarnejad‐samani et al. ‐ nanoparticles impact on cordia myxia l. 101 mal­function, generation of ros (he et al., 2012; roh et al., 2012). on the other hand, easy penetration of nanoparticles into the seed shell develops water absorption and increases the activity of rubisco enzyme, which finally simplifies germination (gao et al., 2006). in the present study, cordia myxa agnps increased onion germination percentage and value. the improvement of seed germination has reported in nano­metal treated spinach (gao et al., 2006), maize (lin and xing, 2007) and peanut (prasad et al., 2012). the agnps have to penetrate plant cell walls, the natural sieves, and roots plasma membranes to enter the xylem tissues and then dislocated to stems and finally leaves (dietz and herth, 2011). in fact, the roots are the first target for lethal materials (sresty and rao, 1999). there are disagreeing reports regard­ ing the effects of nanoparticles on the plant root and system. the nps treatment made a decrease in radish root length (wang et al., 2015), but an increase in the shoot and root length of rice (hao et al., 2016). also, zno nps caused root elongation in radish, lettuce, corn, and cucumber (lin and xing, 2007). zno nps (500 mg l­1) increased soybean’s root length, while higher concentrations resulted in a sig­ nificant reduction (lopez­moreno et al., 2010). the root cap cells of agnps­exposed lolium multiflorum were damaged and malformed, which lastly reduced root growth and dry matter (yin et al., 2011). plant root inter­connect with physical and chemi­ cal factors of the root zone. the elongation zone of onion root may act as a sensitive receiver for external signals. the length and morphology of onion roots is an important parameter that reflects the toxicity of the chemical compounds (odeigah et al., 1997). it has reported that agnps lessened root length of onion whichrelatedto a reduction in water absorp­ tion and cell division (kumari et al., 2009). in this study, an inhibition in radicle and plumule develop­ ment in chemical agnps­treated seeds indicate its toxicity potential. moreover, the root length reduced more than the shoot length in agnps­treated sam­ ples. the toxicity of agnps in biological systems is closely related to its surface oxidation, releasing the ag ions, and interaction with macromolecules (reidy et al., 2013), especially with sulfur­containing mole­ cules e.g. proteins, due to silver­ sulfur strong ten­ dency (liu et al., 2011). the nano­dimension plant pores, the surface charge of nps (more negative agnps limit the cell­particle interactions and made lower toxicity) and coating type (chemical or biologi­ cal) influence the intensity of agnps’ toxicity (choi and hu, 2008; el badawy et al., 2011). in the present study, protein content and pro­ tease activity of green agnps treated seeds did not significantly differ from the control. however, the high concentration of chemical agnps obviously decreased protein content and boosted protease activity. this alignment related to the role of pro­ tease in protein catabolism. high concentrations of agnps denature the membrane and release lps (lipopolysaccharide) and purines. then limit the pro­ ton mobility, react with thiol groups of some enzymes, deactivate enzymes, bind to protein groups, denature proteins, produce hydrogen perox­ ide, which finally causes oxidative stress (hwang et al., 2008; zhu et al., 2008). the regulated production of free radicals in organ­ isms maintains the oxidation and reduction home­ ostasis cycle; however, there is a group of antioxi­ dant enzymes who prevent and deactivate ros. also, extracellular antioxidant molecules, such as ascor­ bate, scavenge free radical molecules (shams et al., 2011). increasing the activity of antioxidant enzymes, catalase and peroxidase, inplant’s exposed to the chemical metal nps treated plants (krishnaraj et al., 2012; singh et al., 2013; wang et al., 2015; cvjetko et al., 2017) confirms our results. nanoparticles interact with the cell membrane (khan et al., 2011), then depending on their nature and concentration, reduce the destructive effects of oxidative stress, cause programmed cell death (lei et al., 2008). silver nanoparticles could damage cell divi­ sion, cause chromatin bridge, disturbed metaphase, make multiple chromosomal breaks and final cell dis­ integration (kumari et al., 2009). according to burman et al. (2013), zinc oxide nps linearly increased ros and mda content. the mda content of tomato and tobacco plants who exposed to agnps was higher than control plants (cvjetko et al., 2017). also, an increase in phenol content has already been reported in metal nps treated plants (singh et al., 2013). ic50 is a measure of the potency of a substance in inhibiting the biological function and indicates how much of this substance is required to inhibit the bio­ logical process. in our research, the chemical agnpshad ic50around 8 times more than green agnps, which confirms the more toxicity risk of chemical agnps. the same trend was observed in green agnps synthesized from aegle marmelos extract (patil et al., 2015). the results of the present work displayed that green agnps significantly enhanced antioxidant abilities by stimulating polyphenols and ascorbic acid in onion. http://https://en.wikipedia.org/wiki/potency_(pharmacology) adv. hort. sci., 2020 34(1): 93­104 102 globally, incredible changes in agricultural pro­ duction patterns have taken place, through the appli­ cation of modern labor­saving technologies, mecha­ nization, and improved crop varieties. in sustainable agriculture, the application of nano­fertilizers is a tal­ ented option to provide the food needed for the growing population worldwide. green synthesis of nanoparticle as an environmental­friendly technique, by minimizing and reducing hazardous material, gradually has introduced itself in the commercial pro­ duction of nanoparticles. the present research used an eco­friendly and low­cost methodology, without the use of any danger or lethal chemicals for agnps synthesis from leaf extract of c. myxa under room temperature conditions. the phenolic and flavonoid contents of leaf extract acted as reducing and stabi­ lizing agent in nanoparticles synthesis and agnps with a good quantity and stability were synthesized. various bioassays are available to assess the rela­ tive toxicity of chemicals in different organisms. however, there is no single test that can detect the damage of classes of chemical compounds. a germi­ nation test is a sensitive tool used in physiological and cytogenetic studies. according to our findings, the green nanoparticle synthesis, not only had no oxidative effect on onion germination parameters, but also some stimulant effect was observed. the chemical nanoparticle motivated the plant’s defense responses, by inducing oxidative stress and limited germination in a dose­dependent manner. as a final conclusion, it can be noted that the low­cost and easy synthesis of nanoparticles from plant sources, is a safe and suitable alternative for chemical­synthe­ sized metal nanoparticles. acknowledgements the authors thank the head of research and technology center (university of hormozgan, iran) and dr. ebrahim eftekhar, the head of molecular research center (hormozgan university of medical sciences, iran), for technical support. references abdel­aziz m.s., 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http://https://www.ncbi.nlm.nih.gov/pubmed/?term=liu%20j%5bauthor%5d&cauthor=true&cauthor_uid=21341685 http://https://www.ncbi.nlm.nih.gov/pubmed/?term=bernhardt%20es%5bauthor%5d&cauthor=true&cauthor_uid=21341685 impaginato 283 adv. hort. sci., 2022 36(4): 283­292 doi: 10.36253/ahsc­12981 biological effects of some colchicum autumnale l. extracts on tissue devel­ opment of two varieties of ocimum basilicum l. i.­c. moroșan (*), l.c. ivănescu, m. mihășan, ș.m. olaru, m.­m. zamfirache alexandru ioan cuza university of iași, bulevardul carol i, 11, 700506 iași, romania. key words: colchicine, basil, bulbs, flowers, meadow saffron. abstract: colchicum autumnale l. is a perennial herb from the colchicaceae family with an unusual life cycle, and it is characterized by an underground corm and hypogynous flowers that appear in autumn. its medicinal importance is represented by its primary alkaloid, colchicine, which has been studied for its anti­inflammatory and antimitotic properties and used in the treatment of some diseases and artificial polyploidy induction in plants. this study aims to determine and evaluate the biological effects induced by treatment with c. autumnale extracts on tissue development in test plants, represented by two ocimum basilicum l. varieties: ‘italiano classico’ and ‘aromat de buzău’. morpho­anatomical observations and measurements and photosynthetic pig­ ments analyses were employed. results show unusual shapes of leaves, differ­ ences in stomata size and density, and heteromorphic cells in leaves and epi­ cotyl’s structure in both studied varieties of basil treated with c. autumnale extracts. 1. introduction colchicum autumnale l., generally known as meadow saffron, naked lady, or wild saffron (nagesh et al., 2011), is a colchicine­containing species widespread throughout europe. it belongs to the colchicaceae family, which comprises perennial herbs characterized by an underground corm and hypogynous flowers with six tepals (bowles, 1924; nordenstam, 1998, in jung et al., 2011). the biochemical profile of this species reveals the presence of alkaloids, phenolics, terpenoids, glycosides, and other bioactive compounds (davoodi et al., 2021; hailu et al., 2021). the alka­ loid concentration in this species varies depending on the plant organ; it was reported that it varies in seeds (0.5­1.2%), fresh flowers (1.2­2%), fresh leaves (0.15­0.4%), and fresh bulbs (0.1­0.6%); among all alkaloids, colchicine constitutes 50­70% of the total alkaloid content, followed by some small amounts of colchicoside, demecolcine, and other tropolone derivatives (kupper et al., 2010). these variations are due to the fact that (*) corresponding author: morosan.ioana@gmail.com citation: moroșan i.­c., ivănescu l.c., mihășan m., olaru ș.m., zamfirache m.­m., 2022 ­ biological effects of some colchicum autumnale l. extracts on tissue development of two varieties of ocimum basilicum l. ­ adv. hort. sci., 36(4): 283­ 292. copyright: © 2022 moroșan i.­c., ivănescu l.c., mihășan m., olaru ș.m., zamfirache m.­m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 april 2022 accepted for publication 17 october 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12981 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(4): 283­292 284 secondary metabolites biosynthesis and accumula­ tion are influenced by genetic, morphogenetic, and environmental factors (yang et al., 2018). despite its toxicity, colchicine has been used in the treatment of gout, familial mediterranean fever (fmf), and other diseases (nagesh et al., 2011), and its antimitotic properties have been exploited in plant breeding for the production of polyploid crops (roberts and wink, 1998, in jung et al., 2011) and ornamental species (manzoor et al., 2019). to extract colchicine from the plant source, the most commonly used method is soxhlet, and the best choice of solvent for this method has been reported to be methanol for c. autumnale and other species of the colchicaceae family (finnie and van staden, 1991; pandey and banik, 2012, in çankaya et al., 2019). colchicine binds to tubulin dimers and pre­ vents microtubule assembly, inducing microtubule depolymerization and preventing mitotic spindle for­ mation (caperta et al., 2006). it was shown that colchicine treatment affects specific morphological (stomatal size), physiological (photosynthetic rate), and biochemical (chlorophyll content) indices in vari­ ous plant species (cao et al., 2018; trojak­goluch et al., 2021), but the effects of colchicine derivatives or c. autumnale extracts in plants are unknown. this study aims to evaluate the effects of c. autumnale methanolic extracts on seed germination and plantlet tissue development by measuring sever­ al morpho­anatomical and photosynthetic indices to determine the allelopathic effect and the impact of the extracts of this plant species on other plants. for this purpose, ocimum basilicum was chosen as a test species for its culinary, ornamental, medicinal, and economic importance and specific morpho­anatomi­ cal characteristics: four­edged stem (rectangular­ quadrangular shaped) with four ribs and ovate­lance­ olate opposite leaves with attenuate serrate edges, uniseriate pluricellular tector hairs, peltate (four­ celled head) and capitate (one or two­celled head) glandular hairs that appear on the surface of the stem and leaves (zamfirache et al., 2008; nassar et al., 2014), that make potential abnormalities easy to spot. 2. materials and methods plant material the seeds of o. basilicum ‘italiano classico’ and ‘aromat de buzău’ were purchased from commercial sources (unisem s.a., iași, and s.c.d.l. buzău). a total of 32 individual plants of c. autumnale were collect­ ed from a pasture in voroneț, suceava county, lat. 47.58889° n, long. 25.90861°e, alt. 576.68 m, in october 2019. the colchicine standard was provided by the institute of biological research, iași, romania. the bulbs and flowers of c. autumnale were kept in an oven at 65°c for 12 hours to stop enzymatic reactions and then dried at room temperature (21±1°c) for 7 days, away from any source of light. subsequently, the dry material was ground in an electric grinder, placed in glass jars wrapped in alu­ minum foil, and stored in the refrigerator until used in extract preparation. extraction and quantification of colchicine content in extracts the extraction was processed in a soxhlet appara­ tus in methanol (sigma aldrich, germany), using 5 grams of powder from each organ (bulb and flower), according to the method of franz and koehler (1992, in alali et al., 2004). the extraction of colchicine from the dried plant material was performed until the sol­ vent in the extraction chamber was clear, in the fol­ lowing way: 5­6 cycles (1­1.5 cycles/hour) for bulbs and 17­18 cycles (2­3 cycles/hour) for flowers. after extraction, the methanol was evaporated in a rotary evaporator (ika rv3 eco, germany). each dry extract was weighed and then dissolved in 50 ml of 70% methanol. two colchicine­containing extracts were obtained, one from the bulbs and abbreviated be (bulb extract) and one from flowers ­ abbreviated fe (flower extract). the quantification of the colchicine in the extracts was performed by rp­hplc, according to alali et al. (2004). the separation was performed on a shimadzu prominence hplc system (2x lc20ad pumps, sil20ac autosampler, ct20ac oven, spd m20a dad detector) using a zorbax eclipse xdb ­ c18 (250 mm length, 3 micron particle size) column, with acetoni­ trile as mobile phase a, and 3% acetic acid (sigma aldrich, germany) as mobile phase b. elution was performed at a flow rate of 1 ml/min using the fol­ lowing program: 0­3 min 90% b isocratic, 3­11 min 90­40% b gradient, 11­12 min 40% b isocratic, 12­13 min 40­90%b gradient, 13­20 min 90% b isocratic. a volume of 20µl of colchicine standard and 5 µl of each extract were injected. colchicine was detected at 245 nm and eluted at 13.4±0.08 minutes. chromatographic data were acquired using the shimadzu lc solution software and manually inter­ moroșan et al. ‐ colchicum autumnale extracts effects on plant tissue development 285 preted. for calibration, a range of 0.2 to 5 µg colchicine was used. experimental design the two extracts were used in treatments, pure or diluted with distilled water (1:1) on the seeds and on the cauline apexes of o. basilicum ‘italiano classico’ and ‘aromat de buzău’ potted plantlets (fig. 1). three controls (c0 = distilled h2o, c1 = 35% meoh, c2 = 70% meoh) were prepared according to the methanol concentration in extracts and dilutions. the method of treatment consisted of applying extracts on o. basilicum seeds distributed in petri dishes. the seeds were previously sterilized with 3% h2o2 (5 minutes) and 5% naclo (5 minutes). the treatment was applied by soaking the filter paper with 2 ml of pure or diluted extracts (20 seeds/petri dish x 3 replicates for each variant of treatment). the seeds were kept in a thermostat at a temperature of 20±1°c and a 17h/7h light­dark cycle per day for 14 days and were watered once every two days. the experiment was performed in triplicate. final germination percentage (fgp) was calculated according to the formula by bezini et al. (2019): fgp = n/n × 100 where n represents the number of germinated seeds at the end of the germination test and n is the total number of seeds. the other treatment application strategy was based on some methods used for polyploidy induc­ tion by colchicine treatment (suzuki et al., 2005; ye et al., 2010; kushwah et al., 2018) and was carried out by applying the treatment on basil plantlets on day 18 after planting, by soaking cotton balls applied on the cauline apexes with 100 µl of extract per day for three consecutive days. the plantlets were kept in pots with compo sana soil (peat + perlite) at 21±2°c and 17 hours of light per day and were watered once every three days. the experiment lasted for 95 days (18 days of pre­cultivation and 75 days of monitoring of plant development) and the treated plantlets were analyzed at the end of the experiment (day 75 after treatment). morphological observations of treated plantlets the macromorphological differences in leaves’ shape and stem development that were observed between variants of treated o. basilicum plantlets of both varieties were photographed. for micromorphology analysis, the plant material consisting of the first leaves was prepared in the fol­ lowing way: for each variant 2 first node leaves from 3 different plants were cut (1 cm2) and transferred in successive acetone baths, critical­point dried with co2, and covered with a 10 nm gold (au) layer using an ems 550x sputter coater. leaf samples of each surface were analyzed using scanning electron microscopy (tescan vega ii sbh electron microscope from the faculty of biology, ‘’alexandru ioan cuza” university of iași, romania) with vegatc software. stomata size (on both leaf surfaces) measurements were made using the imagej software for 5 stomata on 6 leaves. stomata, tector, and glandular hairs den­ sities were determined by counting their occurrences on 1 mm2 of leaf surface on 6 leaves. anatomy analysis of treated plantlets epicotyl fragments from 3 plantlets of each treat­ ment were selected for sectioning and examination. the plant material, previously kept in 70% ethanol was sectioned using a hand microtome and botanical razor. the sections were stained through the double staining method (with ruthenium red and iodine green), then placed on slides, observed through an optical microscope (euromex bscope bs.1153­pli) using a 10x (0.25) lens, and photographed using xiaomi mi a1 camera (12 mp, f/2.2, 26 mm (wide), ½.9”, 1.25 µm, pdaf). epicotyl circumference was measured using imagej and compared to control variants. photosynthetic pigments content assay of treated plantlets the content of photosynthetic pigments in both o. basilicum varieties was analyzed according to sumanta et al. (2014) using ethanol (chemical company s.a., iași, romania) as a solvent for leaf extracts (fresh leaves from 3 plants of each variant of treatment were weighted and milled with quartz sand, then dissolved in ethanol and filtered). the contents of chlorophyll a, chlorophyll b, and fig. 1 ­ treatment application strategies. adv. hort. sci., 2022 36(4): 283­292 286 carotenoid pigments were calculated with formulas given in the reference article. the data was processed in graphpad prism version 9.3.0. chlorophyll fluorescence measurement of treated plantlets for the measurement and evaluation of chloro­ phyll fluorescence (indirect measurement of photo­ chemical efficiency of photosystem ii = φpsii and electron transport rate = etr), 3 leaves were selected from different individuals from each treatment (from plantlets that were pre­exposed to dark conditions), and were analyzed using the hansatech ltd. pam fluorometer. the data was imported from the data system of the hansatech device to parview32 soft­ ware and analyzed in graphpad prism version 9.3.0. statistical analyses statistical calculations and comparations were performed in graphpad prism version 9.3.0., using two­way anova and tukey’s multiple comparisons test for all morphological: stomata size (n= 3), stoma­ ta, tector and glandular hairs density (n= 3) on both leaf surfaces, anatomical: epicotyl circumference (n= 3), biochemical: chlorophyll a, b and carotenoids con­ tent (n= 3), and physiological indices: final germina­ tion percentage for treated seeds (n= 20) and photo­ system ii efficiency and electron transport rate (n = 3) for treated plantlets. values in graphs were repre­ sented as means ± sem, and for tukey’s multiple comparison tests, statistical significance is marked on graphs in the following way: **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p<0.05. where no asterisk is present, results are not statistically significant. 3. results colchicine content quantification in extracts the detected colchicine (fig. 2a) concentrations were 0.119±0.007 mg/ml colchicine in the bulb extract (fig. 2b) and 0.286±0.015 mg/ml colchicine in the flower extract (fig. 2c). germination test the final germination percentage recorded on seeds treated with extracts revealed that the bulb extract and a concentration of 70% methanol impede the germination of all seeds, regardless of the variety of basil. o. basilicum ‘italiano classico’ seeds had a higher germination percentage than the ‘aromat de buzău’ cultivar under treatment, but the latter fig. 2 ­ chromatograms for colchicine standard (a) and for bulb (b) and flower (c) extracts. moroșan et al. ‐ colchicum autumnale extracts effects on plant tissue development 287 showed a better germination percentage than its control variant when treated with diluted flower extracts (table 1). the inhibitory effects of treat­ ments (with extracts and methanol) effects were sta­ tistically significant (**** = p<0.0001) compared to c0. effects of c. autumnale extracts were also signifi­ cant (p<0.0001), compared to their corresponding methanol control (1:1 bet and 1:1 fet to c1; bet and fet to c2). other statistical comparisons are present­ ed in supplementary materials (sm) tables s1­s6. morphological observations of treated plantlets plantlet morphology. in ‘italiano classico’ basil plantlets, the epicotyl’s growth was inhibited; the plantlets remained short (mainly when treated with bulb extracts), and their epicotyls had very short internodes (fig. 3). the upper leaves were darker in color compared to the control and displayed unusual shapes: elongated or bifurcate, with straight or incomplete margins and diverted nervures (fig. 4 c, d, e). treated ‘aromat de buzău’ plantlets in different stages of growth and development, with various stem heights and ramifications are reported in figure 5. in some plantlets, the main epicotyl’s growth was either inhibited or necrotic, which strongly stimulat­ ed the ramifications, mostly when treated with pure table 1 ­ final germination percentage of o. basilicum seeds under treatment with c. autumnale extracts fig. 3 ­ morphology of o. basilicum ‘italiano classico’ plantlets treated with c. autumnale extracts. a) h2o control; b) meoh; c) diluted (1:1) bulb extract; d) pure bulb extract; e) diluted (1:1) flower extract; f) flower extract. fig. 4 ­ morphology of o. basilicum ‘italiano classico’ leaves from the basal node of plantlets treated with c. autum‐ nale extracts. a) h2o control; b) meoh; c) diluted bulb extract; d) diluted flower extract; e) pure flower extract; leaves from plantlets treated with pure bulb extract could not be photographed because of insufficient plant material. fig. 5 ­ morphology of o. basilicum ‘aromat de buzău’ plantlets treated with c. autumnale extracts. a) meoh and h2o control; b) dilu­ ted (1:1) bulb extract; c) pure bulb extract; d) diluted (1:1) flower extract; e) pure flower extract. be= bulb extract; fe= flower extract; t= treatment; ****= p <0.0001. test variant final germination percentage (%) ‘italiano classico’ ‘aromat de buzău’ c0 (h2o) 80±1.92 68.89±7.78 c1 (meoh 35%) 70±11.55 **** 23.33±4.41 **** 1:1 bet 0±0 **** 0±0 **** 1:1 fet 40±18.93 **** 33.33±6.01 **** c2 (meoh 70%) 0±0 **** 0±0 **** bet 0±0 **** 0±0 **** fet 0±0 **** 0±0 **** 288 adv. hort. sci., 2022 36(4): 283­292 flower extract. the upper leaves were much smaller than those from the control variant and displayed unusual shapes (fig. 6c, d, e), with incomplete mar­ gins and deviated or bifurcated nervures (fig. 6f). leaf micromorphology observations of treated plantlets. a typical basil leaf (of a plant treated with h2o = c0) observed through scanning electron microscopy has diacytic and anomocytic stomata, glandular and tector hairs (tector hairs appeared only on the adaxial leaf surface), and epidermal cells with wavy sidewalls (fig. 7a). the leaf epidermis of plantlets treated with methanol appears dehydrated and uneven (fig. 7b), tector hair morphogenesis and elongation are stimulated, and glandular hairs with an altered shape of the glandular cells occur (fig. 7c). leaves from ‘italiano classico’ plantlets treated with flower extracts showed heteromorphic or twin stomata (fig. 7d), accentuated corrugations of the epidermal cell sidewalls (fig. 7e), and abnormalities of the glandular hairs’ shapes (fig. 7f, g). bulb extracts treatments only stimulated tector hairs mor­ phogenesis. the aspect of a typical leaf surface of ‘aromat de buzău’ basil plantlets is similar to ‘italiano classico’, but when treated with bulb extracts, epidermal cells appear elongated, the shape of the sidewalls is altered, and the morphogenesis and elongation of tector hairs occur following the treatment with methanolic flower extracts (fig. 7h). micromorphology measurements of treated plantlets. stomata area and density. stomata size sig­ nificantly increased on both leaf surfaces of ‘italiano classico’ plantlets treated with diluted bulb extract (p<0.05 when compared to c0, p<0.0001 when com­ pared to c1 on the inferior leaf surface, and p<0.0001 when compared to c0 and c1 on the superior leaf sur­ face) and pure flower extract (p<0.001 when com­ pared to c0, p<0.0001 when compared to c2 on the inferior leaf surface, and p<0.0001 when compared to c0 and c2 on the superior leaf surface) and only on the abaxial leaf surface when treated with diluted flower extract (p<0.0001 when compared to c0 and c1)(fig. 8a), and their density increased in plantlets treated with pure bulb extract on the inferior leaf surface (p<0.01 when compared to c0 and p<0.05 when compared to c2) (fig. 8c, d). the density of tec­ tor hairs increased significantly when plants were treated with 70% meoh on the inferior leaf surface as a response to chemical stress. stomata size increased on both leaf surfaces of ‘aromat de buzău’ basil plantlets treated with pure bulb extract (p<0.0001 when compared to c0 and c2) and only on the adaxial leaf surface when the plantlets were treated with diluted flower extract (p<0.0001 when compared to c0 and c1) (fig. 8a, b). stomata density increased on both leaf surfaces of fig. 6 ­ morphology of o. basilicum ‘aromat de buzău’ leaves from the basal node of plantlets treated with c. autum‐ nale extracts. a) h2o control; b) meoh; c) diluted bulb extract; d) pure bulb extract; e) diluted flower extract; f) pure flower extract. fig. 7 ­ abnormalities observed on o. basilicum leaf surfaces after treatment with methanol or c. autumnale extracts. a) typical aspect of an abaxial leaf surface; b) typical aspect of an adaxial leaf surface; c) glandular hair with an altered shape; d) twin stomata; e) epidermal cells with very wavy sidewalls; f) twin glandular hairs; g) glandular hair with a modified shape; h) tector hair morphogenesis and an elongated pluricellular tector hair. moroșan et al. ‐ colchicum autumnale extracts effects on plant tissue development 289 plantlets treated with pure flower extract (p<0.001 when compared to c0 and c2) (fig. 8c, d). glandular and tector hairs density. the density of tector hairs on the abaxial leaf surface increased in all ‘italiano classico’ basil plantlets, being highest in those treated with pure bulb extract and in the ‘aromat de buzău’ plantlets treated with flower extracts and 70% methanol, compared to c0, where they did not appear. the treatment with extracts did not significantly impact either basil variety on the adaxial leaf surface. on the other hand, glandular hairs density did not change on either leaf surface of ‘italiano classico’ basil plantlets, but it was signifi­ cantly higher on the adaxial leaf surface of ‘aromat de buzău’ plantlets treated with 70% methanol (sm ­ fig. s1). epicotyl anatomy of treated plantlets by examining the epicotyl anatomic structure of plantlets treated with c. autumnale extracts, some changes in the cross­section’s outline, an uneven lig­ nification of the xylem vessels, and heteromorphic cells in the cortical parenchyma and marrow, and an increase in the size and density of tector hairs were observed. the contour of the cross­section through the epi­ cotyl of a typical basil plantlet (control) is square, with 4 prominent ribs, which is a characteristic aspect of the species from the lamiaceae family. the single­ layered epidermis has numerous single­celled, long tector hairs and glandular hairs. the layers below the epidermis are differentiated into two subzones: the angular collenchyma and the parenchymal cortex, with 4­6 layers of rounded cells that leave small air gaps between them. the central cylinder comprises conductive tissues in which elements of the primary structure (generated from procambium) and the sec­ ondary structure (generated from cambium) are observed. secondary xylem and libriform (sclerenchy­ ma wood) fibers can be observed in approximately equal proportions. the marrow is parenchymal­meat­ ic (fig. 9, 10). ‘italiano classico’ basil plantlets treated with dilut­ ed bulb extract showed a weaker lignification of the xylem bundles and an elongated, rectangular contour of the epicotyl cross­section. the asymmetry of the epicotyl’s shape in plantlets treated with flower extract is correlated with an increased number of cell layers on one side. many cell layers indicate individu­ als with thicker epicotyls than the control. also, more multicellular tector hairs were observed compared to the control (fig. 9). ‘aromat de buzău’ basil plantlets treated with diluted bulb extract displayed a thinner epicotyl than the control variant, shorter tector hairs, and an aerif­ erous cavity in the pith, whereas the epicotyl of the plantlets treated with undiluted bulb extract dis­ played some heteromorphic cells in the parenchymal cortex, which led to an abnormal outline of the cross­ fig. 8 ­ average stomatal area on abaxial (a) and adaxial (b) leaf surfaces and density on abaxial (c) and adaxial (d) leaf surfaces of treated o. basilicum plantlets (be = bulb extract; fe = flower extract; t = treatment; **** = p<0.0001; *** = p<0.001; ** = p<0.01; * = p<0.05). fig. 9 ­ epicotyl cross­section from o. basilicum ‘italiano classico’ plantlets, after treatment with c. autumnale diluted bulb (b), flower (c), and pure flower (d) extracts, compared to the control (a) (epicotyls of plantlets treated with pure bulb extract could not be analyzed because of insufficient plant material). adv. hort. sci., 2022 36(4): 283­292 290 section. similar modified characteristics were observed in ‘aromat de buzău’ basil plantlets treated with flower extract: when treated with a diluted flower extract, the epicotyl displayed an increase in the number of tector hairs, in contrast with undiluted flower extract treatment, where it decreased. in addition, a weak lignification of xylem vessels, het­ eromorphic parenchymal cells, and an overall outline thinning of the epicotyl were possible consequences of the treatment (fig. 10). epicotyl circumference increased in ‘italiano classico’ basil plantlets when treated with pure flower extract (p<0.01 when compared to c0 and p<0.0001 when compared to c2). the plantlets treat­ ed with the bulb extract could not be analyzed due to the fragility of the plant material and some anom­ alies of the epicotyl (very short internodes). in con­ trast, ‘aromat de buzău’ basil had a much lower epi­ cotyl circumference when treated with diluted bulb extract (p<0.0001 when compared to c1 but not sig­ nificant when compared to c0) but higher in plantlets treated with pure bulb extract (p<0.0001 when com­ pared to c0 and c2) (fig. 11). photosynthetic pigments content of treated plantlets the applied treatments did not affect chlorophyll a and carotenoid pigments content in both basil vari­ eties. chlorophyll b content was highest in plantlets treated with 70% methanol (sm fig. s2). photosystem ii efficiency and electron transport rate of treated plantlets the photosystem ii efficiency of both varieties and the electron transport rate (etr) of ‘aromat de buzău’ basil plantlets were not significantly affected by the applied treatment. in ‘italiano classico’ basil, the electron transport rate was very high when plantlets were treated with 70% meoh (c2) (sm fig. s3). 4. discussion and conclusions c. autumnale pure extracts inhibited the germina­ tion of seeds of both ‘italiano classico’ and ‘aromat de buzău’ basil varieties, probably because of the high concentration of methanol, but they could also have an inhibitory effect on germination and growth of weeds in plant cultures due to their allelopathic properties. more seeds of the ‘italiano classico’ basil variety germinated than ‘aromat de buzău’, but the latter withstood the treatment on cauline apex better than the italian basil. morphological observations showed that the treatments have similar effects on leaf morphogene­ sis of both basil varieties, at macroscopical and microscopical levels, with some differences in the response of a particular basil variety to flower or bulb extract treatments. the epidermis dehydration, alter­ ations in glandular hair shapes, and stimulated mor­ phogenesis and elongation of tector hairs could appear because of the solvent used in treatments fig. 10 ­ epicotyl cross­section from o. basilicum ‘aromat de buzău’ plantlets, after treatment with c. autumnale diluted bulb (b), flower (c), and pure bulb (d) and flower (e) extracts, compared to the control (a). fig. 11 ­ epicotyl circumference of o. basilicum plantlets after treatment with c. autumnale extracts. be= bulb extract; fe= flower extract; t= treatment; **** = p<0.0001; ** = p<0.01; * = p<0.05; epicotyls of plan­ tlets treated with pure bulb extract could not be analy­ zed because of insufficient plant material. moroșan et al. ‐ colchicum autumnale extracts effects on plant tissue development 291 rather than due to the applied treatments with colchicum autumnale extracts. the increase in the number of tector hairs observed on the epicotyl epi­ dermis, or their occurrence where they wouldn’t usu­ ally appear (on the abaxial leaf surface), might indi­ cate a defensive response to the chemical stress induced by methanol. in contrast, an increased densi­ ty of secretory hairs on the leaf adaxial surface of ‘aromat de buzău’ plantlets treated with methanol might indicate a better capacity to protect them­ selves, as glandular hairs’ main role is to protect the plant against external factors: herbivores and pathogens, extreme temperatures, excessive loss of water, and competing plants by secreting lipophilic substances that act as repellents or poisons (hazzoumi et al., 2020). the observations on the anatomy of the plantlet epicotyl confirmed the infor­ mation from the literature that colchicine affects xylem differentiation and thickening because of the disappearance of wall microtubules (pickett­heaps, 1967), which might lead to abnormal morphogenesis of the mature plants’ stem that develops in a pla­ giotropic position. another interesting observation is that heteromorphic cells induced by the treatment with c. autumnale extracts that appear in the leaf structure and in the epicotyl might indicate the pres­ ence of mixoploid tissues, which could also explain the abnormal leaf shapes and the epicotyl develop­ ment anomalies. the treatment with c. autumnale extracts did not affect biochemical and physiological indices of the photosynthetic apparatus of treated basil plantlets. only treatment with methanol might influence pho­ tosynthesis due to its impact on chlorophyll b con­ tent and electron transport rate. these indices might be suitable for potential poly­ ploid evaluation and selection from plants subjected to similar treatments. if its toxicity levels are ade­ quately managed and exploited, colchicum autum‐ nale could have a great potential for becoming a cost­effective source of allelopathic compounds for crop pest control or herbicide production, besides its importance as a source of anti­mitotic agents for arti­ ficial polyploidy induction in plants. acknowledgements we want to thank the institute of biological sciences from iași for providing the colchicine used in rp­hplc detection and cernesim for supplying some of the equipment used in this study. references alali f., tawaha k., qasaymeh r.m., 2004 ­ determination of colchicine in colchicum steveni and c. hierosolymitanum (colchicaceae): comparison between two analytical methods. ­ phytochem. analysis, 15(1): 27­29. bezini e., abdelguerfi a., nedjimi b., touati m., ben­ ziane a., yabrir b., 2019 ­ effects of some heavy met‐ als on seed germination of medicago arborea l. 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bodunde 3 1 department of crop production and horticulture, lagos state polytechnic, ikorodu, lagos state, nigeria. 2 babcock university, ilishan‐remo, ogun state, nigeria. 3 federal university of agriculture, abeokuta, nigeria. key words: 1­mcp, kmno4, postharvest, rra, zeolite. abstract: tomato fruit is highly perishable because of the characteristic high rate of ethylene production and respiration during ripening. delayed ripening could be achieved through the use of ripening remediation agents (rra) that either absorb or block ethylene binding to the fruit receptor. the effects of ripening remediation agents on shelf life and biochemical quality attributes were evaluated on tomato fruits harvested at three maturity stages (breaker, turning and full­ripe). in 2018 and 2019, harvested fruits were stored under seven ripening remediation treatments: 0.1 µl/l 1­mcp, 0.3 µl/l 1­mcp, 0.5 µl/l 1­mcp, 5% kmno4, 10% kmno4, 10 g of zeolite and 20 g of zeolite and an open shelf condition as the control. at the end of the storage period, fruits were assessed for shelf life as well as total soluble solids (tss), titratable acids (ta), ascorbic acid, and lycopene contents. there was significant (p≤0.05/0.01) influence of ripening remediation treatments on fruits for all the measured parameters. fruits stored with rras consistently out­performed those stored in the open shelf. rras 0.3 µl/l1­mcp, 0.5 µl/l1­mcp and 5% kmno4 solution media had longer shelf life and higher values of total soluble solids, titratable acidity, lycopene and ascorbic acid contents. the use of 1­mcp and 5% kmno4 is recommended as effective scavenger of ethylene for extending the shelf life and maintaining some quality attributes of stored tomato fruits. 1. introduction tomato (solanum lycopersicum l.) is one of the most important fruit vegetables crops in the world. it plays an important role in human diet, being mostly used as a vegetable in the preparation of soup, salad, pick­ les, ketchup, puree, sauces and in many other ways. it is also a rich source of phytochemicals and vitamins that provide protection against chronic diseases, different types of cancers, cardiac vascular diseases and age­ related ailments because of its anti­oxidant, anti­carcinogenic and anti­ (*) corresponding author: oadebusolami@gmail.com citation: oduntan a.o., oyetunde o.a., shobo b.a., bodunde j.g., 2022 ­ effect of harvest maturity stage and ripening remediation agents on the shelf life and biochemical quality attributes of tomato (solanum lycopersicum l.) fruits. ­ adv. hort. sci., 36(4): 255­263. copyright: © 2022 oduntan a.o., oyetunde o.a., shobo b.a., bodunde j.g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 march 2022 accepted for publication 1 september 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12934 adv. hort. sci., 2022 36(4): 255­263 256 mutagenic properties (chaudhary et al., 2018). regardless of the health and nutritional relevance of tomato, its production and value chain potentials realization is constrained by post­harvest losses of quantity and quality of produce available to con­ sumers. one of the impacts of post­harvest losses results in reduction of food that is accessible for human consumption, which is worsened by increas­ ing demand for food (kikulwe et al., 2018). the toma­ to fruit is highly perishable, and a climacteric rise in respiration takes place during ripening, which is con­ sidered a turning point in the life of the fruits as regards quality. being a climacteric fruit, a marked increase of respiration rate and ethylene production during ripening process occurs that reduces the shelf life of the fruit, which may constitute a major chal­ lenge in the value chain (arah et al., 2015). the pres­ ence of this gas accelerates fruit ripening and quality deterioration by shortening the shelf life of the fruit. ripening is a natural phenomenon that involves a series of biochemical changes that are responsible for the textural changes, starch breakdown, change of color, pigment formation, volatile and aroma development and finally abscission of fruits (maduwanthi and marapana, 2019). in tomato, ripening involves different dramatic biochemical and physiological changes of the fruit which are charac­ terized by lycopene accumulation, chlorophyll loss, softening, and changes in aroma and other composi­ tional properties. the regulation of these changes, thus, has been a major corcern for research aimed towards improving fruit quality and shelf l ife (yasuhiro, 2016). increasing the postharvest life of tomato is an important aspect in view of its huge postharvest loss­ es. the onset of ripening in tomato is governed by an increase in ethylene production and it is highly dependent on continuous presence of ethylene and ethylene­mediated actions (zhao et al., 2021), there­ fore, the need to prevent the build­up of the gas around the produce. this has been found effective in delaying ripening in bananas (zewter et al., 2012) and was achieved through the use of substances that either absorb or block ethylene binding to its recep­ tor and these substances could be termed ripening remediation agents (rra). because of the important role of ethylene and ethylene­mediated actions in the onset and progres­ sion of ripening in tomato (paul et al., 2002), pre­ venting the buildup of the gas around the produce had been used to delay ripening in bananas. this has been achieved through the use of ripening remediation agents (rras) which either absorb or block ethylene binding to its receptor. concerning the latter, some rras inhibits ethylene’s role in ripening by their presence at the ethylene­binding sites so that ethylene would not be able to bind and cause subsequent signal translation and transduction in the ripening process (zewter et al., 2012). others act by removing unwanted ethylene gas through the oxidation process, converting it to carbon dioxide and water, thereby halting the ripening process and ensuring the quality of freshness of the product in the packaged environment (sen et al., 2012). also some act as ethylene adsorbers as they have great potential in the agro­industry to remove ethylene due to their cation exchange capacity, high porosity and surface area of uptakes (yin et al., 2020). tomato, being a perishable crop due to its high moisture content, has a short shelf life under tropical conditions (arah et al., 2015). this makes it impor­ tant to develop strategies for the development of handling technologies that reduce or remove the eth­ ylene production of the storage environment, while at the same time sustaining the quality. in view of these, the present study was carried out to compare the effects of different ripening remediation agents on shelf life and some biochemical quality attributes of stored tomato fruits. 2. materials and methods plant materials the tomato fruits (var. beske) used for the experi­ ment were obtained from the experimental field at the teaching and research farm, directorate of university farms, federal university of agriculture, abeokuta (funaab), ogun state, nigeria in 2018 and 2019 where they were grown under field conditions. harvesting was done at three harvest maturity indices following the usda tomato colour chart. harvested fruits were taken to the laboratory of the department of horticulture and landscape management, college of plant science and crop production, federal university of agriculture, abeokuta, ogun state, nigeria for storage. the fruit samples were sorted and graded according to unifor­ mity of size. thereafter, fruits were carefully visually observed and only those that were wholesome were finally used in the experiment while those with defects or signs of diseases were discarded. odutan et al. ‐ postharvest handling of bell pepper 257 treatments and experimental design tomato fruits were harvested at three maturity stages identified by the usda tomato colour chart, viz breaker, turning, full ripe (usda, 2005), and were washed in distilled water to reduce microbial popula­ tion and remove adhering dirt and dust. thereafter, 500 g of fruits in the different maturity stages were exposed to seven (7) rras and an open shelf (that is, without any rra, serving as the ‘control’). seven rras were employed which were in three categories: ethylene absorber [potassium permanganate (kmno4)], ethylene absorber (zeolites) and ethylene inhibitor [1­methylcyclopropene (1­mcp)]. the amounts of rras used were: 0.1 µl/l, 0.3 µl/l and 0.5 µl/l for 1­mcp; 5% and 10% for kmno4 solution; 10 g and 20 g of zeolite. thus, 24 treatments were com­ posed by combining three harvest maturity indices and and eight exposures (rras and control). gaseous 1­mcp was prepared from smartfreshtm (agrofresh inc.) commercial powder (0.14% of active ingredient). the application was done in air­tight plastic contain­ ers (capacity of 1 m3) applying 0.1, 0.3 and 0.5 µl/l of 1­mcp for 24 h, at a temperature of 25°c and 85­90% rh. potassium permanganate (kmno4) solutions of 5% and 10% concentrations were prepared by dis­ solving 5 g and 10 g kmno4 powder in 100 ml of dis­ tilled water and put into small containers to be placed beside fruit samples. also, 10 g and 20 g of zeolite, in granular form, were also put into small containers to be placed beside fruit samples. fruits with the treatments applied were stored at room temperature. control treatment fruits were also stored at room temperature, but without any rra. each treatment had 500 g of whole and healthy tomato fruits stored in perforated plastic containers. untreated fruits (control) were kept in similar con­ tainers and placed in an open shelf. the experiment was laid out in completely randomized design with three replications. after the storage, five fruits were sampled randomly per treatment in each replication and were evaluated for shelf life and some quality parameters, viz total soluble solids, ascorbic acid, titratable acidity and lycopene. assessment of shelf life and some fruit quality attrib‐ utes tomato fruits were stored at an average tempera­ ture between 30­32°c and a relative humidity of 78­ 80% in both years. the shelf life (days) of the tomato fruits was determined by visually observing the inci­ dence and extent of spoilage with respect to storage days. this was determined from the time they were stored to the time they became unsuitable for con­ sumption. for the analysis of total soluble solids con­ tent (tss) and titratable acidity (ta) of each sample, tissue sap was squeezed out from fresh fruit materi­ als with a press. in this juice, tss were determined with an atago handheld refractometer in brixº. titratable acid (ta) content was determined by titrat­ ing method and calculating the result as grams of malic acid per 100 g fresh weight (%). ascorbic acid content of the samples was determined according to the recommended method of aoac (2000) using 2, 6­dichlorophenol indophenol and expressed as mg kg­1. lycopene content was estimated using a spectrometer by extraction with hexane and absorbance measurement at 503 nm and expressed in mg kg­1. fruit firmness was determined using hand­ pi model gy­series penetrometer. fruit firmness in a sample was measured by pushing the central probe against the equatorial plane of the fruit until the cen­ tral probe flattened. the flattening of the probe caused the needle in the instrument to deflect and the number where the needle stopped was recorded as the value for the fruit firmness (kitinoja and hussein, 2005). statistical analysis data were subjected to 2­way analysis of variance (anova) and significantly different means were sep­ arated at 5% probability level. correlation between all pair­wise traits were estimated for measured traits. all analyses were performed using the statistical analysis system, sas, version 9.3 (sas institute, 2012). 3. results harvest maturity index significantly (p≤0.05) affected the shelf life of stored tomato fruits, and fol­ lowed the same trend in both years of the study. in 2018 and 2019 respectively, fruits harvested at breaker stage had the longest shelf life of 37 and 40 days, followed by those harvested at turning stage with 36 and 35 days shelf life while fruits harvested at full ripe stage had the shortest shelf life of 35 and 32 days (table 1). the shelf life of stored tomato fruits was signifi­ cantly (p≤0.05) affected by the ripening remedia­ tion treatments and this has comparable trends in both years of the study (table 2). in 2018, fruits exposed to 0.3 µl/l had the longest shelf life of 45 days, immediately followed by fruits stored with 0.5 adv. hort. sci., 2022 36(4): 255­263 258 µ l/l 1­mcp and 5% kmno4 solution media with 43 and 42 days shelf life respectively. furthermore, fruits stored with 0.1 µl/l 1­mcp had a shelf life of 37 days while those stored in 10% kmno4 solution and 20 g of zeolite media had the same shelf life of 36 days. untreated fruits however, had the shortest shelf life of 18 days. in the second year of the experiment, a similar trend was observed in the effect of rras on the shelf life of the stored fruits. fruits exposed to 0.3 µl/l and 0.5 µl/l 1­mcp and those stored in 5% kmno4 solution medium had shelf life of 46, 43 and 39 days respectively, followed by fruits under 0.1 µl/l 1­mcp with a shelf life of 37 days. fruits stored in 10% kmno4 solution and 20 g of zeolite medium had com­ parable shelf life of 32 days while fruits left in the open shelf had the shortest shelf life of 18 days. it was noted that 0.3 µl/l1­mcp, 0.5µl/l1­mcp and 5% kmno4 solution were the most effective in extending the shelf life of tomato. as displayed in table 3, total titratable acidity (tta) was significantly (p≤0.05) affected by the ripen­ ing remediation treatments in both 2018 and 2019. in years 2018 and 2019, the highest tta of 0.43 and 0.42 g/l respectively were recorded for fruits treated with 0.3 µl/l and 0.5 µl/l 1­ mcp and those in 5% kmno4 solution medium while fruits left on the open shelf had the lowest average tta concentration of 0.37 g/l. the lycopene content of the fruits was signifi­ cantly affected by the ripening remediation treat­ ments (table 3). in both years of the experiment, fruits kept in open shelf condition recorded the high­ est lycopene contents of 401.40 µg/100 g (in 2018) and 392.53 µg/100 g (in 2019) which were compara­ ble with the values obtained for fruits treated with 10% kmno4 solution (405.55 µg/100 g in 2018), 10 g zeolite medium having lycopene content of 403.20 and 392.45 µg/100 g in 2018 and 2019 respectively. the lowest lycopene contents of 389 µg/100 g in 2018 and 365.19 µg/100 g in 2019 were observed for 5% kmno4 and 0.5 µl/l 1­mcp respectively. further­more, ascorbic acid content of the fruits was significantly (p≤0.05) influenced by the ripening table 1 ­ effect of harvest maturity index on shelf life of tomato fruits in years 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 2 ­ effect of ripening remediation agent on shelf life of tomato fruits in years 2018 and 2019 ripening remediation agent shelf lfe (days) 2018 2019 0.1 µl/l 1­mcp 36.63 b 36.75 ab 0.3 µl/l 1­mcp 43.25 a 42.75 ab 0.5 µl/l 1­mcp 41.88 a 37.88 ab 5% kmno4 44.63 a 45.74 a 10% kmno4 36.38 b 32.10 b 10 g of zeolite 27.75 c 30.25 cb 20 g ofzeolite 35.50 b 32.40 b open shelf (control) 18.25 d 17.88 c means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 3 ­ effect of ripening remediation substances on some nutritive traits of tomato fruits in 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. ethylene remediation treatments tta (g/l) lycopene (µg/100 g) ascorbic acid (mg/100 g) tss (%) 2018 2019 2018 2019 2018 2019 2018 2019 0.1 µl/l 1­mcp 0.42 a 0.40 ab 390.79 ab 381.41 ab 18.26 a 18.55 a 5.73 ab 5.78 ab 0.3 µl/l 1­mcp 0.43 a 0.42 a 392.21 ab 372.06 b 17.71 ab 19.15 a 5.89 a 5.85 a 0.5 µl/l 1­mcp 0.43 a 0.42 a 392.99 bc 365.19 b 17.79 ab 18.80 a 5.80 a 5.89 a 5% kmno4 0.43 a 0.42 a 389.00 b 375.36 b 17.78 ab 18.07 a 5.80 a 5.85 a 10% kmno4 0.39 b 0.40 ab 405.55 a 384.29 ab 17.34 ab 18.09 a 5.50 b 5.61 b 10 g zeolite 0.38 b 0.40 ab 403.20 a 392.45 a 16.69 b 16.74 b 5.50 b 5.49 b 20 g zeolite 0.41 ab 0.38 b 398.29 ab 387.28 ab 17.01 ab 17.29 ab 5.50 b 5.52 b open shelf (control) 0.37 b 0.37 b 407.40 a 392.53 a 16.31 b 17.51 ab 5.25 bc 5.22 bc harvest index shelf life (days) 2018 2019 breaker 37.99 a 40.70 a turning 35.73 b 34.70 b full ripe 31.08 c 32.18 c odutan et al. ‐ postharvest handling of bell pepper 259 remediation treatments in both years (table 3). in 2018, the ascorbic acid content ranged from 16.31 mg/100 g for fruits stored in the open shelf to 18.26 mg/100 g for fruits stored with 0.1 µl/l 1­mcp. substantial amounts of ascorbic acid were also observed in fruits with 0.5 µl/l 1­mcp, 5% kmno4, 0.3 µl/l 1­mcp, 10% kmno4, and 20 g zeolite in decreasing order. in 2019, ascorbic acid content of stored fruits ranged from 16.74 mg/100 g for 10 g zeolite to 19.15 mg/100 g for 0.3 µl/l 1­mcp. substantial ascorbic acid contents were also observed for 0.1 µl/l 1­mcp, 0.5 µl/l 1­mcp, 10% kmno4, 5% kmno4, open shelf and 20 g zeolite, in decreasing order. the total soluble solids (tss) of stored fruits was also significantly (p≤0.05) affected by the ripening remediation treatments (table 3). fruits kept in the open shelf condition, however, recorded the highest tss in both years of the study. in 2018, the tss content recorded in fruits exposed to 0.1 µl/l and 0.5 µl/l 1­mcp and 5% kmno4 solu­ tion medium was lower compared to those stored in zeolite medium and those kept in the open shelf. in 2019, fruits stored in 10 g and 20 g of zeolite medi­ um had higher tss compared to those exposed to 1­ mcp concentrations and those stored in 5% and 10% kmno4 solution (table 3). as reported before, tomato fruits harvested at the breaker and turning stages recorded significantly longer shelf life than fruits harvested at the full ripe stage except for fruits harvested at the turning stage and stored with zeolite (table 1). comparing fruits from the same stage of maturity in both 2018 and 2019, fruits stored with 0.3 µl/l 1­mcp had the longest shelf life for fruits harvested at the breaker stage, while 5% kmno4 effected the longest shelf life for fruits picked at the turning stage while full ripe fruits had the longest shelf life when stored with 0.3 µl/l 1­mcp and 5% kmno4 (table 4). there was a general decrease in tta for all the treatments. however, fruits harvested at breaker and turning stages recorded higher tta contents with exposure to 0.1 µl/l, 0.3 µl/l and 0.5 µl/l 1­ mcp and those stored in 5% kmno4 solution medium when compared to other treatments (table 5). lycopene content was higher for fruits harvested at full ripe kept in the open shelf while there was low lycopene content for those harvested at breaker stage with stored with 1­mcp and 5% kmno4 solution medium. in the same vein, fruits harvested at breaker and turning stages had higher ascorbic acid contents when exposed to 1­mcp and 5% kmno4 solution medium while those kept in the open shelf had lower ascorbic acid content comparable with those stored with 10% kmno4 solution and zeolite. on the other hand, fruits harvested at the breaker stage and exposed to 1­mcp or kmno4 solution media recorded significantly lower tss than the full­ripe fruits kept in the open shelf as shown in table 5. significant (p≤0.05/0.01) levels of association, comparable for both 2018 and 2019 experiments, were observed in the relationship among shelf life and measured biochemical parameters of tomato table 4 ­ harvest maturity index and ripening remediation agents on shelf life of tomato fruit in years 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. shelf life (days) 0.1 µl/l 1­mcp 0.3 µl/l 1­mcp 0.5 µl/l 1­mcp 5% kmno4 10% kmno4 10 g zeolite 20 g zeolite open shelf (control) breaker 2018 37 ab 46 a 37 ab 42 a 40 a 27 b 40 a 19 c 2019 31 ab 42 a 36 ab 45 a 36 ab 36 ab 36 ab 18 c mean 34 44 36.5 43.5 38 31.5 38 18.5 turning 2018 36 ab 37 ab 33 ab 41 a 40 a 25 b 36 ab 19 c 2019 33 ab 40 a 37 ab 42 a 33 ab 28 b 36 ab 19 c mean 34.5 38.5 35 41.5 36.5 26.5 36 19 full‐ripe 2018 34 ab 40 a 31 ab 40 a 37 ab 31 ab 39 a 16 c 2019 37 ab 39 ab 34 ab 39 ab 28 b 33 ab 37 ab 13 c mean 35.5 39.5 32.5 39.5 32.5 32 38 14.5 260 adv. hort. sci., 2022 36(4): 255­263 fruits in this study (table 6). in 2018 and 2019, titrat­ able acidity had positive and significant correlation with ascorbic acid (r = 0.64 and 0.63 respectively) and shelf life (r= 0.70 and 0.74 respectively) but shared negative and significant correlation with lycopene (r = ­0.86 and ­0.83 respectively) and total soluble sug­ ars (r= ­0.94 and ­0.82 respectively). in 2018 and 2019, lycopene content had positive and significant association with total soluble sugars (r = 0.80 and table 5 ­ interaction of harvest maturity index and ripening remediation agents on biochemical quality attributes of tomato fruits in years 2018 and 2019 *, ** significant at 5 and 1% probabilities, respectively. harvest maturity index ripening remediation treatments tta (g/l) lycopene (µg/100g) vitamin c (mg/100g) tss (%) 2018 2019 2018 2019 2018 2019 2018 2019 breaker 0.1 µl/l 1­mcp 0.41 a 0.42 a 382.26 ab 381.38 ab 16.67 a 16.87 a 5.59 b 5.57 b 0.3 µl/l 1­mcp 0.41 a 0.42 a 384.93 ab 371.42 b 16.43 a 16.39 a 5.56 b 5.52 b 0.5 µl/l 1­mcp 0.41 a 0.42 a 382.18 ab 378.93 b 16.78 a 16.36 a 5.54 b 5.52 b 5% kmno4 0.42 a 0.42 a 388.67 ab 384.94 ab 16.21 a 16.11 a 5.53 b 5.54 b 10% kmno4 0.36 b 0.40 a 399.10 ab 396.71 ab 15.21 ab 15.22 ab 5.67 b 5.62 b 10 g zeolite 0.37 b 0.38 b 397.89 ab 411.30 a 15.83 ab 15.93 ab 5.75 ab 5.75 ab 20 g zeolite 0.38 ab 0.38 b 396.71 ab 411.23 a 15.38 ab 15.01 ab 5.75 ab 5.75 ab open shelf (control) 0.35 b 0.35 b 412.73 a 407.36 a 15.91 ab 15.96 ab 5.95 a 5.93 a turning 0.1 µl/l 1­mcp 0.39 ab 0.39 ab 394.77 ab 391.22 ab 16.91 a 16.91 a 5.64 b 5.69 b 0.3 µl/l 1­mcp 0.39 ab 0.40 a 386.42 ab 389.41 ab 16.71 a 16.86 a 5.69 b 5.66 b 0.5 µl/l 1­mcp 0.39 ab 0.40 a 386.99 ab 388.50 ab 16.82 a 16.50 a 5.66 b 5.64 b 5% kmno4 0.40 a 0.40 a 389.63 ab 389.71 ab 16.38 a 16.31 a 5.66 b 5.63 b 10% kmno4 0.37 ab 0.38 ab 400.51 a 402.38 a 15.98 ab 15.92 ab 5.72 ab 5.77 ab 10 g zeolite 0.37 ab 0.38 ab 396.18 ab 403.78 ab 15.78 ab 15.61 ab 5.75 ab 5.79 ab 2 0g zeolite 0.37 ab 0.39 ab 389.28 ab 391.18 a 15.86 ab 15.89 ab 5.71 ab 5.71 ab open shelf (control) 0.34 b 0.34 b 410.91 a 413.67 a 14.71 b 14.62 b 5.99 a 5.98 a full­ripe 0.1 µl/l 1­mcp 0.38 ab 0.38 ab 392.51 ab 392.73 ab 15.43 ab 15.93 ab 5.77 ab 5.78 ab 0.3 µl/l 1­mcp 0.39 ab 0.39 ab 389.42 ab 392.11 ab 15.56 ab 15.45 b 5.72 ab 5.78 ab 0.5 µl/l 1­mcp 0.39 ab 0.39 ab 384.66 ab 389.81 ab 15.77 ab 15.86 ab 5.72 ab 5.78 ab 5% kmno4 0.39 ab 0.39 ab 387.48 ab 389.82 ab 15.39 ab 15.93 b 5.74 ab 5.74 ab 10% kmno4 0.40 a 0.37 ab 402.38 a 404.86 a 14.73 b 14.78 b 5.82 a 5.89 a 10 g zeolite 0.37 b 0.37 ab 403.56 a 405.36 a 14.48 b 14.14 b 5.94 a 5.92 a 20 g zeolite 0.37 b 0.37 ab 399.83 ab 396.74 ab 14.97 b 14.76 b 5.95 a 5.95 a open shelf (control) 0.34 b 0.37 ab 413.56 a 411.86 a 13.92 b 13.82 b 5.99 a 5.99 a means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 6 ­ pearson correlation coefficients of the relationship among shelf life and measured biochemical quality components of tomato fruits stored with ripening remediation agents in 2018 (lower diagonal) and 2019 (upper diagonal) parameter measured titratable acidity lycopene ascorbic acid total soluble sugars shelf life titratable acidity 1 ­0.86 ** 0.64 ** ­0.94 ** 0.70 ** lycopene ­0.83 ** 1 ­0.70 ** 0.80 ** ­0.65 ** ascorbic acid 0.63 ** ­0.71 ** 1 ­0.76 ** 0.51 * total soluble sugars ­0.82 ** 0.85 ** ­0.80 ** 1 ­0.70 ** shelf life 0.74 ** ­0.68 ** 0.42 * ­0.69 ** 1 0.85 respectively) and shared negative and signifi­ cant association with ascorbic acid (r= ­0.70 and ­ 0.71 respectively) and shelf life (r= ­0.65 and ­0.68 respectively). furthermore, there was negative and significant correlation between ascorbic acid and total soluble sugars with r= ­0.76 and ­0.80 while sharing positive and significant association with shelf life with r= 0.51 and r 0.42 for 2018 and 2019 respec­ tively. total soluble sugars also had negative and sig­ nificant correlation with shelf life (r= ­0.70 and r= ­ odutan et al. ‐ postharvest handling of bell pepper 261 0.69 in 2018 and 2019 respectively). 4. discussion and conclusions the ripening remediation treatments considerably affected the shelf life of stored tomato in both years of the study and the extended shelf life could have been as a result of the efficacy of these treatments to delay the conversion of starch to sugars thus reduc­ ing the ethylene production and peroxidase activity of the fruits. similar results of delay in conversion of starch to sugars for extended shelf life of tomato were observed with the use of gibberellic acid as reported by srividya et al. (2014). the identified rras could also have been able to extend the shelf life of the fruits due to their ability to control respiratory metabolism, thus maintaining the produce for a longer period as suggested by nath et al. (2015). during storage, acidity decreased with ripening as the organic (malic and citric) acids in the fruits got metabolized. the loss of tta during storage period could be related to higher respiration rate as ripening advances, where organic acids are used as substrate in the respiration process. exposure of the tomato fruits to the rras in this study delayed the consump­ tion of the tta, with 1­mcp and 5% kmno4 being the most reliable in achieving this. regassa et al. (2012) reported the sequential disappearance of malic and citric acids in ripening tomato fruits leading to reduc­ tion in the amount of tta. lycopene content of tomato fruits were different­ ly affected by rra but exposure to 1­mcp concentra­ tions and 5% kmno4 solution treatments delayed the accumulation of lycopene in the fruits for both years. this might be due to decrease in respiratory rate, inhibiting ethylene activity, consequently reducing metabolism of the fruit (nath et al., 2015). the delay in lycopene development in this study could have been as a result of the efficacy of the ripening reme­ diation treatments in suppressing the production of ethylene in fruits thus delaying lycopene accumula­ tion. the restrictive effect of 1­mcp on lycopene accumulation in this study supports the previous reports of taye et al. (2019). the treatment with 1­mcp concentrations and kmno4 had comparable patterns of effect on ascorbic acid content. generally, fruits treated with 1­mcp and 5% kmno4 had higher ascorbic acid contents compared to other treatments. the efficacy of 1­mcp concentrations in this study corroborates the obser­ vations of sabir et al. (2012) that 1­mcp had signifi­ cant effect on ascorbic acid content by decreasing ethylene content of tomato fruit thereby increasing ascorbic acid content. generally, this study indicated that there was a decrease in ascorbic acid content of tomato fruits which showed significant decrease dur­ ing storage as reported by ahmed et al. (2018). generally, as reported by tilahun et al. (2019), tomato fruits harvested at the matured green and breaker stages had lower tss level, while fruits har­ vested at light­red stage of full­ripe had the highest tss. however, in this study, the efficacy of the ripen­ ing remediation agents was evident in slowing down the breakdown of carbohydrates into soluble sugars (fructose and glucose) or excessive moisture loss that aids the hydrolysis of cell wall polysaccharides. the fact that fruits kept in the open shelf recorded the highest tss in both years can be attributed to faster advancement in ripening than those treated with rras as previously specified by ahmed et al. (2018). the increase as influenced by the ripening remedia­ tion treatments may have occurred as a result of breakdown of carbohydrates into soluble sugars, or excessive moisture loss that aids the hydrolysis of cell wall polysaccharides. however, beckles (2012) earlier noted that 1­mcp may increase, reduce or leave unchanged, the development of tss depending on fruit species. 1­mcp at 0.3 µl/l concentration and 5% kmno4 were the most effective in extending the shelf life of fruits harvested however, those left in the open shelf consistently had the shortest shelf life implying that rra application was effective in extending the mar­ ketable life of the fruits. the longer shelf life recorded by the fruits at the breaker and turning stages may be attributed to the ability of the ripening remediation agents to control respiratory metabolism, thus main­ taining the produce for a longer period as suggested by nath et al. (2015). harvesting fruits at the proper maturity stage has a great influence on the nutrient content as well as shelf life of any fruit. however, in this study, the ripening remediation agents had great influence in slowing down the action of the ripening hormone that could accelerate the decline in the ascorbic acid content of the tomato fruit. it is cumbersome to consider multiple traits in a selection scheme. information on the relationship among various traits with shelf life would thus be beneficial to designing an efficient storage system. the significant correlation of shelf life with all the measured nutritional quality attributes coupled with adv. hort. sci., 2022 36(4): 255­263 262 the interrelationship among the attributes presents the possibility of extending the shelf life of tomato fruits through designing an effective storage system that focuses on manipulating the production and/or accumulation of titratable acids, total soluble solids, lycopene and ascorbic acid. comparable findings in the relationship among nutritional quality traits of tomato, including titratable acids, total soluble solids, and lycopene have earlier been reported by singh et al. (2018) and shobo et al. (2020). the use of rras was effective in increasing the shelf life and maintaining the nutritional properties of tomato fruits in storage. however, the rras dif­ fered in their effectiveness in both capacities. rras 0.3 µl/l1­mcp, 0.5µl/l1­mcp and 5% kmno4 solu­ tion media had longer shelf life and higher values of total soluble solids, titratable acidity, lycopene and ascorbic acid contents. the use of 1­mcp and 5% kmno4 is recommended as effective scavenger of ethylene for extending the shelf life and maintaining some quality attributes of stored tomato fruits. acknowledgements the authors are grateful to agro­fresh tm for pro­ viding the 1­mcp that was used for the study. we also thank the staff of the laboratory of the department of horticulture, college of plant science and crop production, federal university of agriculture, abeokuta, nigeria, for assistance ren­ dered during the conduction of the study. references ahmed m.e.m., attia m.m., al­araby a.a., abd el­ khalek a.f., 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abstract: plants response to the same level of salinity can be increased in hydroponic cultivation compared to under soil conditions. the study aimed at evaluating the chicory growth in dft (deep flow technique) hydroponic system using brackish water, comparing the results with those obtained in nft (nutrient film technique) system. the experiment was carried out in a random­ ized block design with eight replicates. each plot (replicate) was represented by a hydroponic channel with 15 plants. four treatments were used, consisting of plants grown in the dft system submitted to three levels of electrical conduc­ tivity of nutrient solution ­ ecsol (2.57, 3.43 and 4.75 ds/m) and in the nft sys­ tem under ecsol of 2.57 ds/m. plant height, number of leaves, fresh and dry matter of shoot, water consumption, water use efficiency and water content in shoot at 20 and 25 days after transplanting (dat) were evaluated. in each har­ vest, a mean value was obtained per plot through of the harvest collection of five plants. at 25 dat, the largest reductions in production and water use effi­ ciency of chicory were observed under higher salinity (ecsol 4.75 ds/m). in the dft system no symptoms of toxicity that could be attributed to salinity were observed. 1. introduction under natural conditions, plants are frequently exposed to complex interactions which involve numerous environmental factors (rejeb et al., 2014; zribi et al., 2017; prisa, 2019), such as salinity, water deficit, tem­ perature and others (ramakrishna and ravishankar, 2011; szareski et al., 2018). in arid and semi­arid regions of different parts of the world, such as the brazilian northeast (rocha neto et al., 2017), among the various abi­ otic stresses, saline stress, which expresses the concentration of soluble salts in the soil or water (breś et al., 2016), has been pointed out as the (*) corresponding author: mairtong@hotmail.com citation: silva m.g., alves l.s., soares t.m., gheyi h.r., bione m.a.a., 2020 ­ growth, production and water use efficiency of chicory (cichorium endivia l.) in hydroponic systems using brackish waters. ­ adv. hort. sci., 34(3): 243­253 copyright: © 2020 silva m.g., alves l.s., soares t.m., gheyi h.r., bione m.a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 october 2019 accepted for publication 20 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 243­253 244 main cause for the decrease of yield in most agricul­ tural crops (younis et al., 2013; boughalleb et al., 2017; rezaei et al., 2017). soil salinization can be of primary origin (natural) and/or secondary origin, caused by anthropic activi­ ty, such as the use of saline water in irrigation (shahrayini et al., 2018; sienkiewicz­cholewa et al., 2018). secondary salinization is a consequence of inadequate irrigation management because natural drainage in the semi­arid regions is limited due to the low rainfall (endo et al., 2011; suassuna et al., 2017), which is not sufficient to leach the salts from the root zone to deeper soil layers and thus maintain compat­ ible levels of salts in the root zone of crops (ünlükara et al., 2008; shrivastava and kumar, 2015). plant species respond differently to salt stress (tabatabaei and ehsanzadeh, 2016). some are able to produce satisfactory yields under saline conditions, while others are not (zrig et al., 2016; qrunfleh et al., 2017). the responses of plants are variable among the different organs, species/cultivars, development stages and duration of exposure to the salts (parvaiz and satyawati, 2008; abbas et al., 2015), which usu­ ally lead to reductions in phytomass production, yield or survival rates (munns and tester, 2008; xu et al., 2018). salt stress can limit the exploitation of most agri­ cultural crops, making the agricultural activity eco­ nomically unviable. to mitigate the problems of salin­ ity, hydroponic cultivation (soilless cultivation) has been pointed out as a technique suitable for the use of saline water because the response of plants to salinity is better than in soil, when irrigated with the same water (silva et al., 2018 a). in this system, there is higher and immediate availability of water and nutrients to plants because there is no matric poten­ tial, which is one of the main causes of reduction in the free energy of water in the soil. studies on the tolerance of several species to salinity in hydroponic systems have demonstrated that, through adequate management of water and cultivation practices, it is possible to produce com­ mercially using brackish waters (dias et al., 2011), especially leafy vegetables such as lettuce (soares et al., 2015; cova et al., 2017; silva et al., 2018 b). in this context, other crops of economic potential, such as chicory, have been investigated using brackish waters under hydroponic conditions (tzortzakis, 2009, 2010; atzori et al., 2016, 2019 a). atzori et al. (2016) reported that chicory crop proved to have a considerable higher tolerance to salinity compared to lettuce in hydroponics. according to cecílio filho et al. (2015), on average chicory is a more profitable crop than lettuce, and its requirements in terms of management in hydroponic systems are similar. in brazil and in several parts of the world, the nft (nutrient film technique) hydroponic system is the most used commercially. nft is an active system which requires pumping to recirculate the nutrient solution, usually at 15 min intervals (zanella et al., 2008). thus, the use of nft system may be limited in places where there are frequent interruptions in sup­ ply of electricity (santos júnior et al., 2015; silva et al . , 2016). to overcome this problem, some researchers adopted the dft (deep flow technique) system in pvc pipes (santos júnior et al., 2015; silva et al., 2016; cova et al., 2017; campos júnior et al., 2018 a; gondim filho et al., 2018; martins et al., 2019 a, b; santos et al., 2019; silva júnior et al., 2019), in which plant roots remain continually immersed in the nutrient solution which is recirculat­ ed but not as frequently as in nft. thus, in case of short interruptions in electricity supply plants do not undergo water restriction (silva et al., 2018 a). therefore, this study aimed to evaluate the growth, production, water consumption, water use efficiency and quality of plants of the chicory (cichorium endivia l.) using nutrient solutions pre­ pared in brackish waters in dft system, comparing the results with those obtained using solution pre­ pared in fresh water in nft system. 2. materials and methods experiment location the study was carried out in a greenhouse (east­ west orientation), from june to august (fall­winter) of 2016. the greenhouse was 7.0 m wide and 24 m long, with ceiling height of 2.8 m, protected on the sides by black shade (50% luminosity) screen and covered with low density polyethylene film, with anti­ultraviolet additive and with a thickness of 150 microns. the study site was in the experimental area of the graduate program in agricultural engineering of the federal university of recôncavo of bahia, located in the municipality of cruz das almas, bahia, brazil (12° 40’ 19” s, 39° 06’ 23” w, and at an eleva­ tion of 220 m a.s.l). treatments, experimental design and structure the experiment was carried out in a randomized block design, with four treatments and eight repli­ cates. chicory plants were submitted to three levels silva et al. ‐ hydroponic cultivation of chicory 245 of electrical conductivity of nutrient solution ­ ecsol (2.57, 3.43 and 4.75 ds/m) in hydroponics dft and under ecsol of 2.57 ds/m in hydroponics nft, being cultivated 15 plants in each cultivation channel in the central part. nutrient solutions were prepared by adding adequate amounts of fertilizer salts according to recommendation of furlani et al. (1999). the nutri­ ent solution of 2.57 ds/m was obtained by addition of only fertilizer salts to public­supply water (0.34 ds/m). for ecsol levels of 3.43 and 4.75 ds/m, the nutrient solutions were prepared in waters with ec of 1.5 and 3.0 ds/m obtained by addition of nacl to public­supply water, which were also used to replace the volume consumed by the plants in the respective treatments during the cultivation period. in both systems, channels made of pvc pipes (6­m length and 0.075 m in diameter) were used, with cir­ cular holes of 0.05 m in diameter, spaced 0.25 m apart. benches with trestles made of pvc pipes of 0.05 m in diameter were used to support the hydro­ ponic channels. three hydroponic channels were used per bench, with horizontal spacing of 0.30 m. one corridor (0.5­m width) was left between the benches to facilitate transit and operability. in the dft hydroponic system, caps were attached at both ends of each hydroponic channel installed with zero slope, and a drain was installed in the caps to maintain a mean level of the nutrient solution of 0.02 m, conducting the excess solution through a hose back to the solution tank. in the nft system the hydroponic channels were installed with a 4% slope. each experimental unit consisted of an indepen­ dent hydroponic channel, containing a plastic tank (60­l capacity) to store the nutrient solution, and an electric pump to inject the nutrient solution into the channel. the tank had a ballcock valve to maintain a volume of 50 l of the solution and connected to water reservoir, built with pvc pipes of 0.15 m in diameter. a transparent hose with a tape ruler was installed vertically on the outside of the reservoir to verify the water level. the reservoir and supply tank were connected by a hose; the water level was man­ ually controlled through a ball valve which was opened daily at prefixed hours to maintain the water level in the reservoir and quantify water consump­ tion. crop conduction and nutrient solution management seeds of broad­leaved chicory cv. ‘dafne’ were sown on phenolic foam (2 x 2 x 2 cm), by planting one seed per cell. after germination, seedlings were daily irrigated with public­supply water until 10 days after sowing (das). after this period, the seedlings were transferred to a nursery (nft system), where they received nutrient solution (furlani et al., 1999) at 50% concentration for 15 days. irrigations in the nursery were controlled by an analog timer at inter­ mittent intervals of 15 min, from 06:00 to 18:00 h. during the period from 18:00 to 06:00 h, the nutrient solution was recirculated once every 2 h, with dura­ tion of 15 min. the seedlings were transplanted to the definitive cultivation system with mean height of 0.133 m and four true leaves. the programming to control circulation of the nutrient solution in the cultivation channels in both systems (nft and dft) was similar to that used during the initial stage i.e. at intervals of 15 min, from 06:00 to 18:00 h. during the period from 18:00 to 06:00 h, the nutrient solution was recirculated once every 2 h, with duration of 15 min. during the experiment, ecsol and ph of the solu­ tion were monitored in the central position of each hydroponic channel, using portable conductivity and ph meter. at the end of the experiment, the ecsol means values were 1.88 and 2.03 ds/m for the treat­ ment under ecsol de 2.57 ds/m, respectively in the nft e dft systems, and of 3.24 and 4.88 ds/m for the treatments under ecsol de 3.43 e 4.75 ds/m in the dft system, with no replacement of nutrient to the solution during the cycle. when the ph values were outside the ideal range (between 5.5 and 6.5) for hydroponic cultivation, corrections were made by addition of calcium hydroxide. variables evaluated harvests were performed at 20 and 25 days after transplanting (dat). the strategy of performing two harvests along the experiment was to identify the best period for plant harvest, and also to evaluate the absolute growth rate of plants under different treatments before harvest and to assess possibility of early harvest. in each harvest, a mean value was obtained per plot (hydroponic channel) through of the harvest of five plants for the determination of plant height, number of leaves and shoot fresh mat­ ter. immediately after weighing the plants, the fresh material was placed in paper bags and dried in an air circulation oven at temperature of 65°c until con­ stant weight, to quantify shoot dry matter. the volume evapotranspired per plant was deter­ mined daily by dividing the volume of nutrient solu­ tion consumed in the plot by the number of plants under cultivation at that moment in the plot, accor­ ding to equation described by lira et al. (2018). cumulative water consumption was calculated for adv. hort. sci., 2020 34(3): 243­253 246 the periods of 1 to 20 and 1 to 25 dat. water use efficiency (wue) was based on the relationship between shoot fresh (sfm) or dry matter (sdm) pro­ duction and the cumulative water consumption per plant, according to equation 1: wue (g/l) = (sfm or sdm)/wc (1) where sfm is shoot fresh matter, in g; sdm is shoot dry matter, in g; wc is cumulative water con­ sumption during the period, in l/plant. the water content in shoot (wcs) was calculated according to equation 2: wcs (%) = [(sfm ­ sdm)/sfm] x 100 (2) the absolute growth rate (agr) of sfm was calcu­ lated according to equation 3: agr (g/day) = (sfm2 ­ sfm1)/( t2 ­ t1) (3) where sfm1 and sfm2 are shoot fresh matter at times t1 (20 dat) and t2 (25 dat), in g. statistical analysis the results were subjected to analysis of variance by f test and the means were compared by tukey test (p = 0.05). the standard deviations of means were also calculated. 3. results visual symptoms of the chicory plants under salt stress no symptoms of toxicity by the ions na+ and/or cl­ were observed which could com­ promise the visual quality of chicory plants (fig. 1 a). only in the nft system without salt stress (ecsol of 2.57 ds/m), after 20 dat, chicory leaves exhibited necrosis on the edges (fig. 1 b), an abnormality known as tipburn. although tipburn symptoms were observed in all plots of this system, but only in some plants. growth and production of the chicory the f­test of the analysis of variance showed a significant effect of the treatments on the number of leaves, plant height, shoot dry matter and water con­ sumption, only at 25 dat. for shoot fresh matter the treatments had significant effect at 20 and 25 dat, and only at 20 dat on the water content in shoot, water use efficiency and the absolute growth rate (20­25 dat) based on shoot fresh matter (tables 1 and 2). at 20 dat, the overall mean for the number of leaves was 8.2, regardless of the hydroponic systems fig. 1 ­ visual aspect of chicory (cichorium endivia l. cv. dafne) plants in the nft (without salt stress) and dft (with and without salt stress) hydroponic systems, at 25 days after transplanting (a) and plants with tipburn in nft system without salt stress (b). silva et al. ‐ hydroponic cultivation of chicory 247 and water salinity levels. at 25 dat, the lowest num­ ber of leaves (9.4) was observed in plants under the highest salinity (ecsol of 4.75 ds/m) compared with the other treatments (12.3, 11.9 and 11.1 leaves) (fig. 2 a). within a 5­day interval (20 to 25 dat), under the highest salinity (ecsol of 4.75 ds/m) in the dft system the increase in the number of leaves did not exceed by 2.0 leaves, while in the nft system without salt stress (ecsol of 2.57 ds/m) the increase reached approximately 4.0 leaves. in general, plant height was little influenced by the treatments. on average, plant height at 20 dat was approximately 30.0 cm. within a 5­day interval (20 to 25 dat), the maximum increment of height occurred in the nft system (2.7 cm), whereas in the dft system (without and with salt stress) the incre­ ments did not exceed 1.0 cm. at ecsol of 2.57 ds/m and regardless of the hydroponic system, the means did not differ statistically, as well as there was no sig­ nificant difference among the means of the dft sys­ tem under different salinity levels (fig. 2 b). table 1 ­ summary of the f­test for number of leaves (nl), plant height (ph), shoot fresh matter (sfm), shoot dry matter (sdm) and absolute growth rate of sfm (agr­sfm) of chicory cultivated under different treatments in hydroponic systems, at 20 and 25 days after transplanting (dat) sv= source of variation; df= degrees of freedom; cv= coefficient of variation; *, ** significant respectively at p<0.05 and p<0.01; ns= not significant. sv df days after transplanting (dat) nl ph sfm sdm agr­sfm 20 25 20 25 20 25 20 25 20­25 blocks 7 ns ns * ** * * ns ** * treatment 3 ns ** ns * ** ** ns ** ** error 21 ­ ­ ­ ­ ­ ­ ­ ­ ­ cv (%) 9.40 9.47 3.30 3.01 9.38 10.04 17.00 15.09 15.81 table 2 ­ summary of the f­test for water content in shoot, water consumption (wc), and water use efficiency based on shoot fresh matter ­ sfm (wue­sfm) and shoot dry matter ­ sdm (wue­sdm) of chicory cultivated under different treatments in hydro­ ponic systems, at 20 and 25 days after transplanting (dat) sv= source of variation; df= degrees of freedom; cv= coefficient of variation; *, ** significant respectively at p<0.05 and p<0.01; ns= not significant. sv df days after transplanting (dat) water content wc wue­sfm wue­sdm 20 25 20 25 20 25 20 25 blocks 7 ns ns ** ** ** ns ns ns treatments 3 ** ns ns * * ns ns ns error 21 ­ ­ ­ ­ ­ ­ ­ ­ cv (%) 0.77 0.63 15.25 14.22 16.20 15.27 19.92 19.87 differently from the growth variables (fig. 2 a and 2 b), at 20 dat, the shoot fresh matter in the dft sys­ tem was 26.4% higher than in the nft system under cultivation conditions without salt stress (ecsol of 2.57 ds/m). in the dft system even under salt stress conditions (ecsol of 3.43 and 4.75 ds/m), the means of 70.16 and 65.03 g/plant did not differ statistically from that obtained in the nft system without salt stress (fig. 2 c). since the means for shoot dry matter at 20 dat did not differ statistically (fig. 2 e), the superiority in the production of shoot fresh matter obtained in the dft system can be explained by the higher water content in the tissues (fig. 3 a), since plants responded similarly in terms of number of leaves and height. in a marked manner, within the interval of only 5­ days (from 20 to 25 dat), the accumulation of fresh matter in plants grown in the nft system was superi­ or to that recorded during the first 20 days, with growth rate of 16.20 g/day (fig. 2 d), totaling 144.63 g/plant at 25 dat (fig. 2 c). this value was statistical­ 248 adv. hort. sci., 2020 34(3): 243­253 fig. 2 ­ mean number of leaves (a), plant height (b), shoot fresh matter (c), and shoot dry matter (e) of chicory (cichorium endivia l. cv. dafne) plants in the nft (without salt stress) and dft (with and without salt stress) hydroponic systems, at 20 and 25 days after tran­ splanting (dat) and absolute growth rate of shoot fresh matter during the period 20­25 dat (d). means followed by different letters indicate significant differences at 0.05 probability level (tukey­test). bars indicate the standard deviations of the means of the eight replicates. fig. 3 ­ mean water content in shoot (a), water consumption (b) and water use efficiency of shoot fresh matter (c) and shoot dry matter (d) of chicory (cichorium endivia l. cv. dafne) plants in the nft (without salt stress) and dft (with and without salt stress) hydroponic systems, at 20 and 25 days after transplanting (dat). means followed by different letters indicate significant differences at 0.05 probability level (tukey­test). bars indicate the standard deviations of the means of the eight replicates. ly similar to the means obtained in the dft system at ecsol of 2.57 and 3.43 ds/m (145.04 and 130.36 g/plant), with the respective growth rates of 12.93 and 12.04 g/day. at the highest salinity level (ecsol of 4.75 ds/m), the mean of 105.69 g/plant was statisti­ cally inferior to those of the other treatments. based on the results, plants should be harvested at 25 days after transplanting in the hydroponic system, totaling a 50­days cycle from sowing. for the studied cultivar of chicory, on average, the cycle ranges from 45 to 55 days. silva et al. ‐ hydroponic cultivation of chicory 249 at 25 dat, a similar behavior to that of fresh mat­ ter was observed for shoot dry matter (fig. 2 e). the reduction in shoot dry matter was due to the decrease in the number of leaves and plant height, since the mean water content in the shoots of plants under different treatments did not differ statistically (fig. 3 a). consumption, water use efficiency and water content of the chicory the f­test of the analysis of variance showed a significant effect of the treatments on the water con­ tent in shoot and water use efficiency based on shoot fresh matter for the first evaluation period (20 dat). for the second evaluation period (25 dat), there was a significant effect only on the water consumption (table 2). for the cumulative water consumption in the peri­ od of 20 days, the mean consumption was 1.38 l/plant (fig. 3 b) and it was not affected by studied treatments. thus, higher water use efficiency (60.67 g/l) in the dft system compared to the nft system (45.45 g/l) (fig. 3 c) is due to the greater accumula­ tion of fresh matter (fig. 2 c). in the dft system, at ecsol levels of 3.43 and 4.75 ds/m, the means of water use efficiency (54.13 and 57.72 g/l) did not dif­ fer from those in the condition without salt stress (ecsol of 2.57 ds/m). at 25 dat, the water consumption of chicory plants in the dft system (2.08 l/plant) was similar to that of the nft system (2.32 l/plant) for the condi­ tion without salt stress (ecsol of 2.57 ds/m), with sig­ nificant reduction only at the highest salinity level (ecsol of 4.75 ds/m) (fig. 3 b). within a 5­day interval (20 to 25 dat), plants increased water use efficiency based on shoot fresh matter, i.e., within five days the consumed water volume was converted to greater biomass accumulation, with overall mean of 64.27 g/l, regardless of the hydroponic systems and salinity levels of nutrient solution (fig. 3 c). the means of water use efficiency based on shoot dry matter were of 3.22 and 3.71 g/l at 20 and 25 dat, regardless of hydroponic systems and salinity levels of nutrient solution (fig. 3 d). 4. discussion and conclusions in the present study (fig. 1 b), the occurrence of tipburn observed in the nft system using nutrient solution (ecsol of 2.57 ds/m) prepared in fresh water is due to the higher absolute growth rate significantly higher (16.20 g/day) in comparison to other treat­ ments in the period between 20 and 25 dat (fig. 2 d), increasing the demand for calcium. tipburn symp­ toms have been reported in other studies with chico­ ry under different conditions of cultivation (feltrim et al., 2008; sá and reghin, 2008; kowalczyk et al., 2016 a). regarding the visual aspect of chicory plants in the dft system, with the level of ecsol of 4.75 ds/m there were no symptoms of toxicity due to salinity (fig. 1 a). in some regions of the world, waters with high salt concentrations are the only source of water available for irrigation of the crops (cova et al., 2020), causing serious problems of toxicity when the concentrations of na+ and/or cl­ inside the plant are sufficiently high (talhouni et al., 2019), resulting in necrosis of older leaves (parvaiz and satyawati, 2008; tavakkoli et al., 2010). this occurs because plants vir­ tually lose only water by transpiration, thus leading to accumulation of these ions in the leaves (acosta­ motos et al., 2017; ismail and horie, 2017). the time for the damage by toxicity to be manifested depends on the na+ and/or cl­ content in the leaves and on the effectiveness in the compartmentalization of these ions in leaf tissues and cells (parvaiz and satyawati, 2008; giuffrida et al., 2013). the data shown in figure 2 c demonstrate that the production of shoot fresh matter was not affected until 20 dat, regardless of hydroponic systems and water salinity levels. in the dft system, plants were supplied with water and nutrients all the time and this favored plants at young age (with smaller size) to produce more fresh matter than those in the nft sys­ tem until 20 dat. after this period, as the volume of roots increased, the oxygen dissolved was depleted more rapidly, thus decreasing the growth rate (fig. 2 d). with the increase in the volume of roots, there is greater demand for oxygen, according to kläring and zude (2009) and mobini et al. (2015); therefore, reductions in oxygen concentrations are expected to occur in the adult stage of the plants (kiferle et al., 2012; niñirola et al., 2014). the results of present study show that it is possi­ ble to produce chicory using brackish waters with reduction of about 27 and 28% in fresh or dry matter, with plants harvested at 25 days after transplanting (fig. 2 c and 2 e). such reduction in the yield under salt stress can be compensated by cultivating plants in the system for a longer period, because plants con­ tinue to accumulate biomass. another strategy to compensate the reduction of yield may be by reduc­ ing spacing between plants, because under condi­ adv. hort. sci., 2020 34(3): 243­253 250 tions of stress plants occupy a smaller area allowing cultivating more plants per meter length of hydro­ ponic channel as shown by silva et al. (2019) in case of basil. yet another possibility is to cultivate more than one plant per hole maintaining the spacing of 0.25 m to reach the ideal weight of the bunch for marketing. in other studies under hydroponic conditions (nft system), the cultivation of chicory with brackish waters was viable. there was no significant effect on the fresh matter and number of leaves at concentra­ tions of 100 mm (tzortzakis, 2009) and 40 mm of nacl (tzortzakis, 2010) in the nutrient solution, com­ pared to the condition without stress (0 mm of nacl). the concentration of 30 mm of nacl did not cause significant effect on the fresh matter and/or number of leaves (kowalczyk et al., 2012; kowalczyk et al., 2016 b). chicory plants positively responded to the cultiva­ tion in the dft system adapted in pvc pipes, with a constant 0.02­m depth of nutrient solution (approxi­ mately 6.0 l). with this volume of solution in each cultivation channel and assuming mean daily con­ sumption of 0.144 l/plant, if there are interruptions in electricity supply, the system will be able to main­ tain 15 plants without water restriction for about three days. these results complement other studies which have shown feasibility for the cultivation of dif­ ferent plant species in the dft system in tubes, such as lettuce (cova et al., 2017), rocket (campos júnior et al., 2018 b), coriander (silva et al., 2018 a), parsley (martins et al., 2019 a) and chives (silva júnior et al., 2019). in studies with basil, there was no significant difference in the growth and production variables when plants were cultivated in the dft and nft in tubes using nutrient solution prepared in wastewater (alves et al., 2019) and using nutrient solution pre­ pared in fresh water (santos et al., 2019). the lack of significant effect on water use efficien­ cy based on shoot dry matter, regardless of hydro­ ponic systems and water salinity, demonstrates that the significant differences in shoot fresh matter were due to storage of water in plant tissues (fig. 3 a). under salt stress conditions, plants use stomatal clo­ sure as a strategy, reducing transpiration due to lower absorption of water (aroca et al., 2012; moosavi, 2012), which results in increased water use efficiency (acosta­motos et al., 2017; morais et al., 2018; soares et al., 2018), as reported in various studies under salt stress (soares et al., 2010; diniz et al., 2013; santos júnior et al., 2013; soares et al., 2015; lima et al., 2017; coelho et al., 2018; atzori et al., 2019 a). the low water volume used to produce one chico­ ry plant demonstrates high water use efficiency in the hydroponic cultivation, which corroborates with atzori et al. (2019 b) who reported an increase in water use efficiency of chicory in hydroponics com­ pared to conventional soil cultivation. still in the pre­ sent study, the quantification of water consumption along the crop cycle can contribute to better plan­ ning and use of water resources at places with low water availability because it is possible to estimate in advance the water volume required to produce a cer­ tain number of plants within a given period of time. potentially, in hydroponic cultivation there is greater possibility of using water more efficiently which is not possible in conventional planting. in conclusion, the data show that the variables of growth, production, water consumption and water use efficiency of chicory under conditions without salt stress (ecsol of 2.57 ds/m) were not significantly affected by the nft and dft hydroponic systems. nutrient solution with salinity of up to 4.75 ds/m pre­ pared in brackish water (nacl) can be used in chicory cultivation in dft 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50­59. impaginato 475 adv. hort. sci., 2019 33(4): 475­483 doi: 10.13128/ahsc­8112 micropropagation of two near threat­ ened orchid. part 1: catasetum pileatum cv. alba s. zakizadeh, b. kaviani (*), d. hashemabadi department of horticultural science, rasht branch, islamic azad university, rasht, iran. key words: in vitro multiplication, orchid propagation, ornamentals, plant growth regulators. abstract: many orchid species are threatened. in this study, a reliable and effi­ cient protocol was outlined for in vitro propagation of catasetum pileatum cv. alba, a near threatened orchid species with the proper usage of plant growth regulators (pgrs). protocorms as explants were cultured on murashige and skoog (ms) medium containing different concentrations of kinetin (kn; 0.00, 0.20, 0.50, 1.00, 2.00, 3.00 and 5.00 mg l­1) and indole­3­butyric acid (iba; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­1), either individually or in combination. the fre­ quency of protocorm­like bodies (plbs) regeneration significantly relied on con­ centrations of pgrs used. a combination of 1.00 mg l­1 kn and 1.00 mg l­1 iba was found to be suitable for maximum plb regeneration (8.63 per explant) and the largest number of leaf (12.70 per explant). the highest rooting frequency with 7.40 roots per explant was achieved on protocorms grown in medium enriched with 1.00 mg l­1 kn and 0.50 mg l­1 iba. plantlets were transplanted to pots filled with a mixture of peat moss, leca and perlite (1:1:1) and transferred to the greenhouse. the plantlets were successfully acclimatized in the green­ house with a survival rate of 80% exhibiting normal developmental patterns. 1. introduction many orchid species in all over the world are threatened. these species have been listed in the red data book of the international union for conservation of nature and natural resources (iucn, 2011) because of immethodical collection, illegal trade and biodiversity loss and they have been included in convention on international trade in endangered species of wild fauna and flora (cites), where the international trade is strictly controlled (chugh et al., 2009; swarts and dixon, 2009; reed et al., 2011). catasetum pileatum, a rare and near threatened orchid, is a low­ land species, where it occurs as a showy epiphyte. this ornamental species is an impressive plant even out of flower. orchids are precious as pot and cut flowers not only because of their exotic beauty but also for their long shelf life (chugh et al., 2009). in vivo propagation of orchids is a slow process and resulted in traits segregation. also, propagation of orchids by sexual means like seed caus­ (*) corresponding author: kaviani@iaurasht.ac.ir citation: zakizadeh s., kaviani b., hashemabadi d., 2019 ­ micropropagation of two near threatened orchid. part 1: catasetum pileatum cv. alba. ­ adv. hort. sci., 33(4): 475­483. copyright: © 2019 zakizadeh s., kaviani b., hashemabadi d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 december 2018 accepted for publication 8 july 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 475­483 476 es the production of heterozygous plants. therefore, establishment of protocols for in vitro proliferation of orchids is important and a proper alternative proce­ dure for propagation of orchids. in vitro techniques can be used for conservation of rare and endangered plant species and production of large number of plantlets in short period of time (engelmann, 2011). of course, micropropagation of orchids deals with some problems such as high cost of production, low rate of shoot proliferation, poor rooting frequency and phenotypic variations (bhattacharyya et al., 2016). different pgrs such as ɑ­naphthaleneacetic acid (naa), iba, 1­phenyl­3­(1,2,3­thiadiazol­5­yl)­urea (tdz), 6­benzyle amino purine (bap), 6­benzylade­ nine (ba) and kn have been used for tissue culture of threatened and endangered orchids (roy et al., 2011; panwar et al., 2012; zeng et al., 2012; baker et al., 2014; bhattacharyya et al., 2016; kaviani et al., 2017). medium composition for in vitro culture of orchids by plbs is cultivar and species­specific and depends on several factors especially pgrs (luo et al., 2009). various explants such as seeds, leaves (foliar explants), nodes, plbs, protocorms, tubers, shoot tips and floral stalk buds have been used for micropropagation of threatened and endangered orchids (vij and aggarwal, 2003; roy et al., 2011; panwar et al., 2012; zeng et al., 2012; baker et al., 2014; chen et al., 2015; bhattacharyya et al., 2016; kaviani et al., 2017). among all explants, plbs and protocorms are more efficient because these can be rapidly multiplied on solid or liquid culture media, and maximum plbs can be provided in a short period of time (luo et al., 2003). asymbiotic seed germination and the use of plbs induced from vegetative organs are two efficient propagation methods for large­scale propagation of orchids (zeng et al., 2012). these protocols have been established for many orchid species, and differ­ ent media, primary and secondary metabolites, and pgrs have been used for germination and propaga­ tion (arditti and ernst, 1993; roy et al., 2011). many protocols for in vitro propagation of orchids, especial­ ly those at risk of extinction, using plbs as explants and various pgrs, have been reported (teixeira da silva et al., 2005, 2006; firoz alam et al., 2010; sinha et al., 2010; baker et al., 2014). this investigation is the first to report on in vitro multiplication of catasetum pileatum cv. alba, a near threatened orchid species, for developing a protocol by proto­ corms as explants and kn and iba as pgrs. 2. materials and methods plant material healthy and sterilized protocorms (0.7 cm long) of catasetum pileatum cv. alba grown on murashige and skoog (ms) medium (murashige and skoog, 1962) were prepared from the plant biotechnology laboratory, hyrcan agricultural sciences and biotechnology research institute, amol, iran (fig. 1a). the protocorms were used as explants and cul­ fig. 1 ­ micropropagation of catasetum pileatum alba through protocorms. (a) plbs produced on medium containing 1.00 mg l­1 kn + 1.00 mg l­1 iba. (b) developing plbs on media enriched with different concentrations of kn and iba. (c) micropropagated shoots from plbs on medium containing kn and iba. (d) leaves produced on pgrs­free medium (left) and medium containing 1.00 mg l­ 1 kn + 1.00 mg l­1 iba (right). (e) roots produced on pgrs­free medium (left) and medium containing 0.50 mg l­1 kn + 0.50 mg l­1 iba. (f) plantlets produced on media enriched with different concentrations of kn and iba. (g) plantlets transplanted in pots fil­ led with perlite. (h) greenhouse acclimatized plantlets grown in pots filled with a mixture of leka, peat moss and perlite (in ratio of 1:1:1) (scale bar = 1 cm). kaviani et al. ‐ micropropagation of catasetum pileatum cv. alba 477 tured on culture media poured into the culture bot­ tles for in vitro propagation (fig. 1b). culture medium and culture conditions the explants were cultured on ms medium con­ taining 3% sucrose and 0.8% agar­agar. the medium was enriched with various pgrs. the ph of the medi­ um was adjusted to 5.8 with 0.1 n naoh or hcl prior to autoclaving. all media containing culture tubes were autoclaved at 104 kpa and 121°c for 20 min. to evaluate the effect of pgrs on plbs regenera­ tion, shoot multiplication and root induction, the explants were cultured on ms medium containing different concentrations of kinetin (kn; 0.00, 0.20, 0.50, 1.00, 2.00, 3.00 and 5.00 mg l­1) and indole­3­ butyric acid (iba; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l­ 1), either individually or in combination. for each treatment, three replicates and for each replicate, three explants were taken (totally; 35 treatments, 105 replicates and 345 explants). following establish­ ment, cultures were maintained at 24±2°c, 70­80% rh, and 16­h photoperiod of 50­60 μmol m­2 s­1 irra­ diance provided by cool­white fluorescent tubes. assessment of characteristics after 60 days, the effect of pgrs on advanced plbs development was assessed by using plbs num­ ber, plantlet height, leaf number, root length, root number and viability percentage. plbs germination percentage was calculated by the following formula: % sprouted plbs = number of sprouted plbs x 100 total plbs hardening and acclimatization in vitro rooted plantlets were taken out from cul­ ture vessels and washed thoroughly under running tap water to remove substrate residual and trans­ planted to plastic dishes containing perlite for 15­20 days (fig. 1g). then, plantlets were transferred to plastic pots (18 cm height × 12 cm diameter) filled with a potting mixture of leca (light expanded clay aggregate), peat moss and perlite (in ratio of 1:1:1) (fig. 1h). all the pots were then transferred to the greenhouse with temperature of 24±2°c to 20±2°c day/night, light intensity of 3500 lux, rh of 80­90% and 14­h photoperiod) for acclimatization. the pots were covered with polyethylene bags to retain mois­ ture inside and were opened gradually during 2 weeks. survival rate (%) was recorded after 60 days from transfer to greenhouse conditions. plantlets were initially covered with a polythene sheet to maintain relative humidity (90%). the number of sur­ viving plants was recorded after 4 weeks from trans­ fer. experimental design and data analysis the experiments were established in a completely randomized design with three replicates per treat­ ment (totally; 345 explants). pgrs­free ms medium was used as control in the experiments. data were subjected to analysis of variance (anova) and means were compared by the lsd test at p < 0.05 using the spss ver. 17 (spss inc., usa). 3. results assessment of suitable conditions for plbs regenera‐ tion the main goal of this study was the maximum regeneration of plbs during clonal proliferation. lsd test showed significant differences among different concentrations of kn, also reciprocal effect of kn and iba for plbs number (p˂0.01). lsd also showed that the effect of iba was no significant on plbs number when applied individually (table 1). the effects of kn and iba in the ms medium on the plbs multiplication and growth are shown in figure 1 and tables 2, 3 and 4. in the current study the formation of plbs and shoot buds from the explants was observed within 45 table 1 ­ analysis of variance of the effect of different concentrations of kn and iba on measured characters of catasetum pileatum alba grown in vitro condition source of variations df plantlets height number of plbs per protocorm leaf number root number root length viability percentage kn 6 50.8** 10.61** 30.88** 0.870** 23.52** 457** iba 4 7.16 ns 1.66 ns 3.916** 15.57** 24.04** 258* kn × iba 24 6.29** 2.60** 4.32** 0.647** 7.73** 167* error 70 3.42 1.126 1.72 0.241 0.973 83.8 cv (%) ­ 21.5 19.97 20.2 9.714 10.91 11.01 *, **: significant at the 0.05 and 0.01 probability level, respectively, ns: not significant at p=0.05. adv. hort. sci., 2019 33(4): 475­483 478 days of culture establishment. the regeneration of plbs are closely related with the concentration of both kn and iba when used in combination, howev­ er, kn had more important role than iba when these are applied individually. when the explants were cul­ tured in medium supplemented with kn individually, formation of more plbs was observed than when explants were cultured in medium containing iba individually (tables 2 and 3). among the tested kn concentrations, 1.00 mg l­1 proved beneficial in induc­ ing the highest frequency of 6.76 per explant gener­ ating plbs (table 2). when the explants were grown in medium enriched with kn and iba (1.00 mg l­1 from both of them) the largest number of plbs (8.63 per explant) was observed (table 4). minimum plbs regeneration (with average of 4.00 plbs per explant) was obtained in media without kn. assessment of suitable conditions for plantlets height and leaf number the effect of pgrs in the ms medium on the plantlets growth (plantlets height and leaf number) is shown in figure 1c and tables 1­4. after 60 days of culture of protocorms on media fortified with differ­ ent concentrations of pgrs, plantlets height and leaf number were measured. plantlets growth was signifi­ cantly affected by the composition of the medium. growth of plantlets from the explants without the addition of kn to the culture medium was relatively poor. the plantlets growth rate ranged from 5.00 to 17.00 cm on the regeneration media (table 4). ms medium fortified with 1.00 mg l­1 kn and 0.50 mg l­1 iba was the most appropriate medium for plantlet height (16.76 cm per explant) (table 4). treatment with 1.00 mg l­1 kn in combination with 0.50 mg l­1 iba produced plantlets with 13.36 cm long. among all levels of kn and iba used individually, kn at 1.00 mg l­ 1 was noted better induction of plantlet on explants than the other levels (tables 2 and 3). the plantlets produced on medium without kn was least (tables 3 and 4). ms medium fortified with 1.00 mg l­1 kn and 1.00 mg l­1 iba was the most appropriate medium for leaf number (12.70 per explant) (fig. 1d, table 4). the media containing 1.00 mg l­1 kn and 0.50 mg l­1 iba was suitable for leaf number, too (table 4). among all concentrations of iba and kn used individually (tables 2 and 3), maximum leaf number (9.36 per explant) was produced in medium containing 1.00 mg l­1 kn. thus, the optimal concentration of kn was table 2 ­ mean comparison of the effect of different concentrations of kn on measured characters of catasetum pileatum alba grown in vitro condition table 3 ­ mean comparison of the effect of different concentrations of iba on measured characters of catasetum pileatum alba grown in vitro condition means with different letters on the same column are significantly different (p<0.05) based on lsd test. means with different letters on the same column are significantly different (p<0.05) based on lsd test. kn (mg l­1) plantlets height (cm) number of plbs per protocorm leaf number root number root length (cm) viability percent­ age 0.00 6.22 d 4.01 d 4.75 d 5.32 a 8.68 bc 75.30 c 0.20 7.27 cd 4.80 c 5.58 cd 5.12 ab 10.28 a 85.30 b 0.50 9.72 b 5.41 bc 6.73 b 5.08 abc 10.68 a 84.00 b 1.00 11.84 a 6.76 a 9.36 a 5.38 a 9.14 b 93.30 a 2.00 9.02 b 5.18 bc 6.32 bc 4.95 bc 9.26 b 80.00 bc 3.00 8.38 bc 5.73 b 6.57 b 4.75 c 6.93 d 82.00 bc 5.00 7.62 c 5.27 bc 6.11 bc 4.81 bc 8.30 c 82.00 bc iba (mg l­1) plantlets height (cm) number of plbs per protocorm leaf number root number root length (cm) viability percent­ age 0.00 7.96 c 5.00 b 6.15 b 4.33 d 8.22 c 83.80 ab 0.10 8.66 abc 5.43 ab 6.20 ab 4.62 cd 9.32 b 88.09 a 0.20 9.15 ab 5.37 ab 6.17 b 4.80 bc 10.34 a 83.80 ab 0.50 9.14 ab 5.68 a 6.92 ab 6.53 a 9.62 b 81.40 b 1.00 8.00 bc 5.05 ab 7.00 a 5.00 b 7.70 c 78.50 b kaviani et al. ‐ micropropagation of catasetum pileatum cv. alba 479 treatments of pgrs, individually and in combination had significant effects (p<0.01) on root growth (table 1). root length was highest (14.06 cm per explant) in 0.50 mg l­1 kn along with 0.50 mg l­1 iba (fig. 1e), however, this medium was not significantly different compared to 0.50 mg l­1 kn along with 0.20 mg l­1 iba medium with induction of 12.90 cm long for root (table 4). all other media were significantly different and gave lower root length growth rates. among all treatments, 1.00 mg l­1 kn plus 0.50 mg l­1 iba was found to be the most effective for root for­ 1.00 mg l­1. also, the optimal concentrations of iba were 1.00 and 0.50 mg l­1. these concentrations in combination with each other recorded maximum leaf production. this means that the pgrs acted synergis­ tically. assessment of suitable conditions for root character‐ istics and acclimatization advanced root development was significantly affected by the composition of the medium, when measured through root length and root number. all table 4 ­ mean comparison of the effect of different concentrations of kn and iba on measured characters of catasetum pileatum alba grown in vitro condition plant growth regulators (mg l­1) plantlets height (cm) number of plbs per protocorm leaf number root number root length (cm) viability percentage kn iba 0.00 0.00 5.63 jk 3.56 jk 5.20 f­h 5.26 d­g 5.73 o 73.33 e­g 0.00 0.10 6.60 h­k 3.83 ijk 4.46 h 4.13 k­n 6.83 mno 80.00 c­g 0.00 0.20 5.10 k 4.46 e­k 4.73 gh 4.93 e­j 11.23 cd 86.60 a­e 0.00 0.50 5.20 k 4.63 e­k 4.66 gh 7.00 a 11.80 bc 70.00 fg 0.00 1.00 6.60 h­k 3.56 jk 4.70 gh 5.30 c­g 7.83 k­m 66.60 g 0.20 0.00 6.50 i­k 5.36 c­h 5.40 e­h 4.56 g­n 10.90 c­g 90.00 a­d 0.20 0.10 8.73 c­i 5.93 b­f 5.56 d­h 4.43 h­n 12.93 ab 100.00 a 0.20 0.20 6.83 f­k 4.30 e­k 5.53 d­h 4.80 f­l 10.13 d­h 73.33 e­g 0.20 0.50 6.70 g­k 5.36 c­h 5.80 c­h 7.26 a 9.43 f­j 83.30 b­f 0.20 1.00 7.60 d­k 4.06 h­k 5.60 c­h 4.53 g­n 8.00 j­m 80.00 c­g 0.50 0.00 9.43 c­i 4.66 e­k 6.13 b­h 4.40 h­n 7.63 l­n 86.60 a­e 0.50 0.10 9.50 c­i 4.16 g­k 7.73 bc 5.10 d­i 9.76 d­i 83.30 b­f 0.50 0.20 10.30 cd 7.36 ab 6.46 b­h 4.30 j­n 12.90 ab 86.60 a 0.50 0.50 10.60 bc 5.73 b­h 6.13 b­h 6.63 ab 14.06 a 86.60 a­e 0.50 1.00 8.80 c­i 5.13 d­h 7.20 b­f 5.00 d­j 9.06 h­l 76.66 d­g 1.00 0.00 9.53 c­i 6.00 b­f 8.00 b 3.86 mn 9.33 g­k 93.30 abc 1.00 0.10 9.86c­e 6.63bcd 6.56b­h 4.86e­k 9.13h­l 96.60ab 1.00 0.20 13.36 b 5.53 c­h 7.40 b­e 5.63 cde 11.00 c­f 100.00 a 1.00 0.50 16.76 a 7.10 abc 12.16 a 7.40 a 8.23 i­m 96.60 ab 1.00 1.00 9.66 c­g 8.63 a 12.70 a 5.16 d­h 8.00 j­m 80.00 c­g 2.00 0.00 8.56 c­j 4.66 e­k 6.40 b­h 4.36 i­n 8.96 h­l 76.66 d­g 2.00 0.10 10.40 b­d 6.20 b­e 6.80 b­g 4.76 f­l 9.50 e­j 86.60 a­e 2.00 0.20 9.90 c­e 5.10 e­j 5.86 b­h 4.4 6 h­n 11.06 c­e 70.00 fg 2.00 0.50 7.50 d­k 5.33 c­h 5.90 b­h 5.76 cd 9.70 d­i 76.66 d­g 2.00 1.00 8.73 c­i 4.60 e­k 6.66 b­g 5.40 c­f 7.10 mno 90.00 a­d 3.00 0.00 7.83 c­k 5.50 c­h 5.86 b­h 3.83 n 7.66 l­n 76.66 d­g 3.00 0.10 7.23 e­k 5.93 b­e 6.80 b­g 4.46 h­n 7.60 l­n 90.00 a­d 3.00 0.20 9.40 c­i 6.93 a­c 7.66 bcd 4.56 g­n 6.20 no 80.00 c­g 3.00 0.50 9.60 c­h 5.90 b­g 6.56 b­ h 6.06 bc 6.96 mno 86.60 a­e 3.00 1.00 7.86 c­k 4.40 f­k 5.96 b­h 4.83 f­k 6.23 no 76.660 d­g 5.00 0.00 8.23 c­k 5.26 c­j 6.10 b­h 4.03 l­n 7.33 mn 90.00 a­d 5.00 0.10 8.33 c­j 5.33 c­h 5.50 e­h 4.63 f­m 9.50 e­j 80.00 c­g 5.00 0.20 7.16 e­k 4.93 d­k 5.53 d­h 4.93 e­j 9.86 d­h 90.00 a­d 5.00 0.50 7.63 c­k 5.83 b­g 7.26 b­f 5.63 cde 7.16 mno 70.00 fg 5.00 1.00 6.76 g­k 5.00 e­k 6.16 b­h 4.83 f­k 7.66 l­n 80.00 c­g means with different letters on the same column are significantly different (p<0.05) based on lsd test. 480 adv. hort. sci., 2019 33(4): 475­483 mation (7.133 per explant) (table 4). root production in this medium was not significantly higher than fol­ lowing media. the root number (7.26 and 7.00 per explant) produced in media containing 0.20 mg l­1 kn plus 0.50 mg l­1 iba and 0.50 mg l­1 iba without kn, respectively was noticeable (table 4). in most cases, minimum root number was recorded in media with­ out iba. among all concentrations of iba and kn used singly (tables 2 and 3), maximum root number (6.53 per explant) was produced in medium fortified with 0.50 mg l­1 iba. the in vitro rooted plantlets (fig. 1f) were suc­ cessfully acclimatized in the greenhouse. pots were filled with leca, peat moss and perlite (in ratio of 1:1:1) (figs. 1g and h). acclimatization was achieved in 4­6 weeks, and at this stage plants attain the height of about 10­15 cm. acclimatization of micro­ propagated plantlets to the natural conditions requires several anatomical, morphological and phys­ iological changes especially in xylem, leaves and pho­ tosynthesis. the hardened plantlets (fig. 1h) are maintained in the hyrcan agricultural sciences and biotechnology research institute, amol, iran with 80% field establishment rate. assessment of suitable conditions for viability per‐ centage significant difference was observed between the kn (p<0.01), iba and combination of kn and iba (p<0.05) levels and viability percentage of proto­ corms (table 1). it has been observed that among the combinations and concentrations of pgrs, 0.20 mg l­1 kn with 0.10 mg l­1 iba and 1.00 mg l­1 with 0.20 mg l­1 iba were the most effective for viability percentage (100.00) (table 4). viability percentage in these two media was significantly higher than that of other media. least viability percentage (66.60) was observed in plbs cultured on medium containing 1.00 mg l­1 iba without kn (table 4). 4. discussion and conclusions current study revealed that the addition of exter­ nal pgrs in appropriate concentrations induced plbs formation from the protocorm explants cultured in the ms medium. in orchids, plb regeneration from explants such as shoot and root tips and leaf and stem segments is a proper method of in vitro prolifer­ ation (seeni and latha, 2000; dohling et al., 2012). consonant with our findings, roy et al. (2011) demonstrated that the frequency of plbs regenera­ tion of orchid vanda coerulea significantly relied on kinds and concentrations of pgrs used. these researchers showed that a combination of 1.00 mg l­1 naa and 0.85 mg l­1 bap was found to be suitable for maximum plb regeneration. luo et al. (2008) showed that 0.50 mg l­1 kn was proper for plb formation of orchid dendrobium densiflorum. the regeneration and proliferation of multiple plbs are closely related with the type and concentration of cytokinins used. among all cytokinins used for in vitro plbs regenera­ tion of orchids, ba, bap, tdz and kn have the most application (luo et al., 2008; chugh et al., 2009; firoz alam et al., 2010; roy et al., 2011; panwar et al., 2012; baker et al., 2014; bhattacharyya et al., 2016; kaviani et al., 2017). when the explants were grown in medium enriched with both of kn and iba the maximum plbs was obtained. contrary to our find­ ing, bap individually was better than in combination with naa for the maximum plbs formation of orchid oncidium (kalimuthu et al., 2007). study on vanda coerulea revealed that when the protocorms were cultured on bap or naa alone, at any concentration recorded low proliferation rate. this means that the pgrs acted synergistically (roy et al., 2011). baker et al. (2014) reported the highest plbs regeneration of orchid catasetum on ms medium containing a com­ bination of 0.50 mg l­1 ba plus 0.50 mg l­1 naa. present study demonstrated that the production of more than seven plbs was observed in the media supplemented with 0.50 mg l­1 kn along with 0.20 mg l­1 iba and 1.00 mg l­1 kn along with 0.50 mg l­1 iba. similar findings were found by panwar et al. (2012) through study on orchid eulophia nuda. the combination, concentrations and the ratio between the pgrs are critically important for the for­ mation of shoots and plbs in orchids (dohling et al., 2007; baker et al., 2014; bhattacharyya et al., 2016; kaviani et al., 2017). investigation on orchid eulophia nuda showed that the maximum shoot multiplication was achieved on ms medium containing 2.00 mg l­1 ba and 1.00 mg l­1 kn after 4 weeks of cultures (panwar et al., 2012). the type, concentrations and different combinations of pgrs plays an important role during micropropagation of many orchid species (arditti and ernst, 1993; panwar et al., 2012). in orchids, the use of protocorm and plb as the explants is the most appropriate and simplest method for in vitro propagation. protocorm contains meristematic cells and can differentiate to a new shoot. therefore, protocorm can be used to enhance proliferation and simultaneous production of orchid plantlets (teixeira da silva et al., 2005). protocorms kaviani et al. ‐ micropropagation of catasetum pileatum cv. alba 481 are being applied by many researchers as explants for in vitro propagation of many rare and endangered orchid species (deb and temjensangba, 2006, teixeira da silva et al., 2006; roy et al., 2011; dohling et al., 2012; baker et al., 2014; bhattacharyya et al., 2016; kaviani et al., 2017). plb production was induced from many explants such as protocorm, shoot tip, node, root tip, leaf and stem segments dur­ ing in vitro propagation of orchids (dohling et al., 2012; baker et al., 2014; bhattacharyya et al., 2016; kaviani et al., 2017). the regeneration and propagation of multiple shoots are closely related with the type and concen­ tration of cytokinins used (amoo et al., 2014). the differentiation of multiple shoots from plbs has been reported in some orchids such as cymbidium, dendrobium, catasetum, phalanoepsis, habeneria and satyrium (talukdar, 2001; sheelavanthmath and murthy, 2001; mahendran and bai, 2009; hossain et al., 2010; baker et al., 2014; kaviani et al., 2017). cytokinins have a wide range of functions including regulatory role on various physiological and develop­ mental processes (werner et al. , 2001). in dendrobium huoshanense c.z. tang et s.j. cheng, kn was reported to be more effective for plantlet regen­ eration from plbs than bap, n­benzyl­tetrahydropy­ ranyladenine (bpa), isopentenyl adenine (2­ip), tdz and zeatin (zt) (luo et al., 2009). the best response appeared on the medium enriched with 4.50 mg l−1 kn. kn was also used for shoot proliferation of some other orchids (saiprasad et al., 2004; malabadi et al., 2005; martin and madassery, 2006; panwar et al., 2012). contrary to our findings, study on dendrobium nobile revealed that when explants were cultured in ms medium supplemented with bap alone, forma­ tion of plbs was done but direct shoot formation was not observed (bhattacharyya et al., 2016). study of mahendran and bai (2009) demonstrated that among the cytokinins used for multiple shoot induction of satyrium nepalense d. don. tdz was found to be superior. in this study, protocorm developed multiple shoots directly on the medium supplemented with cytokinins. in most of the orchids the presence of cytokinins singly promoted optimal shoot prolifera­ tion (mahendran and bai, 2009). in the present study, addition of external pgrs (both of kn and iba) in suitable concentrations induced plantlets growth and leaf formation from plbs cultured in the ms medium without callus formation. the main advan­ tage of direct organogenesis without an intervening callus phase is that somaclonal variation is reduced (roy et al., 2011). when the media was supplement­ ed with iba singly the response was poor. similar finding was reported by roy et al. (2011) worked on vanda coerulea. bhattacharyya et al. (2016) showed that when the explants were grown in medium con­ taining cytokinin and auxin, a higher rate of response frequency (92.6%) of shoot buds and plbs was observed in all pgrs combinations. in eulophia nuda lindl., maximum shoot multiplication and elongation were obtained on ms medium containing 2.00 mg l−1 ba and 1.00 mg l­1 kn (panwar et al., 2012). pgrs in orchids act more efficiently when used in combina­ tion (seeni and latha, 2000; roy et al., 2011). similar with our findings, iba resulted in a better rooting efficiency over naa in terms of rooting frequen­ cy and number of roots induced per shoot in satyrium nepalense d.don. and dendrobium nobile (mahendran and bai, 2009; bhattacharyya et al., 2016). as, maxi­ mum rooting efficiency (86% or 5.4 roots/shoot) was obtained in medium supplemented with 2.00 mg l−1 of iba after 8 weeks of culture (bhattacharyya et al., 2016). the highest number of roots per shoot (6.40) was achieved at 2.00 mg l−1 iba (mahendran and bai, 2009). the effectiveness of iba in rooting has been shown for some other orchids like vanilla planifolia (giridhar et al., 2001), cymbidium alofolium (l.) sw. and dendrobium nobile lindl. (nayak et al., 2002), cymbidium pendulum (nongdam et al., 2006), satyrium nepalense (mahendran and bai, 2009), vanda teres (firoz alam et al., 2010) and eulophia nuda lindl. (panwar et al., 2012). a maximum 90% response for root formation and highest number of roots (5.50) with length (5.30 cm) per shoot tubers was calculated on iba (0.50 mg l­1) treated shoots of eulophia nuda lindl. (panwar et al., 2012). study of baker et al. (2014) on micropropagation of catasetum demonstrated that the largest number of root (7.16) and root length (193.40 mm) were obtained on ms medium supplemented with 0.50 mg l­1 ba together with 0.50 mg l−1 naa. orchids are among the most beautiful ornamental plants. catasetum pileatum cv. alba is a rare and near endangered orchid. many of orchids are threat­ ened, rare, vulnerable, endangered, indeterminate or in danger of extinction. therefore, it is necessary to develop the suitable methods for conservation and large­scale production of these plants that can be used for their re­introduction. references amoo s.o., aremu a.o., moyo m., szüčová l., doležal k., van staden j., 2014 ­ physiological adv. hort. sci., 2019 33(4): 475­483 482 effects of a novel aromatic cytokinin analogue in micro‐ propagated aloe arborescens and harpagophytum procumbens. ­ plant cell, tiss. org. cult., 116: 17­26. arditti j., ernst r., 1993 ­ micropropagation of orchids. ­ john wiley and sons, new york, usa, pp. 640. baker a., kaviani b., nematzadeh gh., negahdar n., 2014 ­ micropropagation of orchis catasetum ‐ a rare and endangered orchid. ­ acta sci. pol., hort. cult., 13(2): 197­205. bhattacharyya p., kumaria s., tandon p., 2016 ­ high frequency regeneration protocol for dendrobium nobile: a model tissue culture approach for propaga‐ tion of medicinally important orchid species. ­ south afr. j. bot., 104: 232­243. chen y., goodale u.m., fan x.l., gao j.y., 2015 ­ asymbiotic seed germination and in vitro seedling development of paphiopedilum spicerianum: an orchid with an extremely small population in china. ­ glob. ecol. conser., 3: 367­378. chugh s., guha s., rao u., 2009 ­ micropropagation of orchids: a review on the potential of different explants. ­ sci. hortic., 122: 507­520. deb c.r., temjensangba s., 2006 ­ in vitro propagation of threatened terrestrial orchid malaxis khasiana soland ex. swartz through immature seed culture. ­ ind. j. exp. biol., 44: 762­766. dohling s., das m.c., kumaria s., tandon p., 2007 ­ conservation of splendid orchids of north‐east india. biodivers. its significance. ­ new delhi ik international publishers, pp. 354­365. dohling s., das m.c., kumaria s., tandon p., 2012 ­ multiple shoot induction from axillary bud cultures of the medicinal orchid, dendrobium longicornu. ­ aob plants, 1­7. engelmann f., 2011 ­ use of biotechnologies for the con‐ servation of plant biodiversity. ­ in vitro cell. dev. biol. plant, 47: 5­16. firoz alam m., sinha p., lokman hakim m., 2010 ­ micropropagation of vanda teres (roxb.) lindle, pp. 21­ 28. ­ in: jain m.s., and s.j. ochatt (eds.) protocols for in vitro propagation of ornamental plants. humana press, springer protocols, london, uk, pp. 400. giridhar p., obul reddy b., ravishankar g.a., 2001 ­ silver nitrate influences in vitro shoot multiplication and root formation in vanilla planifolia. ­ androl. curr. sci., 81: 1166­1170. hossain m.m., sharma m., teixeira da silva j.a., pathak p., 2010 ­ seed germination and tissue culture of cymbidium giganteum wall. ex lindl. ­ sci. hortic., 123: 479­487. iucn, 2011 ­ the iucn red list of threatened species. ­ iucn species information service (sis), version 2011.1. kalimuthu k., senthikumar r., vijayakumar s., 2007 ­ in vitro micropropagation of orchid, oncidium sp. 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(orchidaceae) using protocorm‐like body segments. ­ propag. ornam. plants, 5 (3): 129­136. vij s.p., aggarwal s., 2003 ­ regenerative competence of foliar explants: vanda coerulea griff. ­ j. orchid soc. india, 17 (1­2): 73­78. werner t., motyka v., strand m., schmülling t., 2001 ­ regulation of plant growth by cytokinin. ­ proc. natl. acad. sci., 98: 10487­10492. zeng s., wua k., teixeira da silva j.a., zhanga j., chena z., xiaa n., duan j., 2012 ­ asymbiotic seed germination, seedling development and reintroduction of paphiopedilum wardii sumerh., an endangered ter‐ restrial orchid. ­ sci. hortic., 138: 198­209. impaginato 59 adv. hort. sci., 2023 37(1): 59­66 doi: 10.36253/ahsc­13872 use of xanthan gum and calcium ascor­ bate to prolong cv. butirra pear slices shelf life during storage e. guccione (*), a. allegra, v. farina, p. inglese, g. sortino department of agricultural, food and forest sciences, university of palermo, viale delle scienze, 4, 90128 palermo, italy. key words: browning, edible coating, fresh­cut, pear, respiration. abstract: after cut, pear fruit (pyrus communis l.) during shelf life can be sub­ jected to color and flavor changes. to maintain flesh colour and firmness, dif­ ferent technologies could be employed during shelf life, such as chemical, phys­ ical and edible coating treatments. in the present study, the effects of two edi­ ble coating formulations containing xanthan gum and calcium ascorbate on fresh­cut pear fruit were investigated. after harvest, 200 fruits were cut and coated with xanthan gum (xan) and distilled water or xanthan gum + calcium ascorbate (asc), respectively, while control (ctr) pear fruit slices were soaked in distilled water and lastly packed in polyethylene terephthalate (pet) pack­ ages sealed with a composite film (pp­pet). all samples were stored at 5±0.5°c with rh 90% for 10 days. measurements were carried out at 3, 5, 7 and 10 days of storage evaluating visual quality score, browning index, color, total solid sol­ uble content (tss), flavor, ascorbic acid content and total phenols content. the results showed that asc treatment was the most efficient treatment in terms of color changes, ascorbic acid content, visual quality score and browning index, until the 7th day of storage. moreover, asc treatment reported lower mean values in terms of taste and flavor score if compared to ctr and xan treatments. untreated pear slices (ctr) kept good values concerning flavor score until the 3rd day of storage while on the 5th and 7th day off­flavor values were the same as treated samples. 1. introduction ‘butirra’ pear fruit is cultivated in southwestern sicily and belongs to sicilian mipaaf pats (traditional agricultural products). after harvest, usually occurring between the second decade of july and the first decade of august, they must be consumed as they are easily rotten and are sub­ jected to fast decay. it’s easy to understand how fresh­cut ‘butirra’ poor shelf­life is a key barrier to its commercialization especially for the fast weakening of tissues and surface’s browning that happens after cut due to the action of polyphenol oxidase (ppo) (amiot et al., 1995; hodges and toivonen, 2008). edible coatings are widely employed since they prevent the loss of quality acting like a selective barrier to gas exchanges between (*) corresponding author: eugenia.guccione@unipa.it citation: guccione e., allegra a., farina v., inglese p., sortino g., 2023 ­ use of xanthan gum and cal‐ cium ascorbate to prolong cv. butirra pear slices shelf life during storage. ­ adv. hort. sci., 37(1): 59­66. copyright: © 2023 guccione e., allegra a., farina v., inglese p., sortino g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 october 2022 accepted for publication 24 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13872 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 59­66 60 food and external environment. xanthan gum enhances all these properties and also controls the rheology of the final food product exhibiting pseudo­ plastic properties in solutions (palaniraj and jayaraman, 2011). as a generally recognized as safe (gras) molecule, xanthan gum is an exopoly­ saccharide produced by the fermentation of a carbo­ hydrate by cultures of xanthomonas campestris. it is then refined by extraction with ethanol or 2­ propanol, dried, and powdered (fda). calcium ascor­ bate is the calcium salt of ascorbic acid that is widely used as an antioxidant whose reducing action against quinones and diphenols prevents browning of unprocessed fruit as it only produces colorless deriv­ atives; it is a reducing agent, capable of promoting the chemical reduction of the pigment precursors responsible for browning, acting by reducing o­ben­ zoquinone or dihydroxyphenol or irreversibly inacti­ vating ppo, promotes the regeneration of antioxi­ dants and acts synergistically with complexing agents (araújo, 2004). furthermore, this cation can maintain cell wall structure by binding to pectins and forming calcium pectate (vilas boas et al., 2009). calcium sta­ bilizes the membranes and cell walls, preserving their integrity and functionality and protecting them from being cleaved by hydrolytic enzymes that cause fruit softening (poovaiah, 1986; vilas boas et al., 2009). xanthan gum combined with antioxidant agents had positive effects on the reduction of weight loss and browning, preventing the loss of firmness, and the growth of psychotropic microorganisms, molds and yeasts in minimally processed apples and pears (sharma and rao, 2015; allegra et al., 2022). the aim of the present study was to evaluate the effective­ ness of edible coating based on xanthan gum and xanthan gum enriched with calcium ascorbate on fresh­cut cv ‘butirra’ pear fruit stored in passive atmosphere. 2. materials and methods the experiment was carried out in 2021. ‘butirra’ pear fruit (quince ba29 rootstock and intermediate ‘butirra hardy’ graft) were harvested during the sec­ ond week of august in a commercial orchard located in zafferana etnea (catania, italy), italy, (730 m above sea level). the soil is a sandy clay loam (63% sand, 19% silt, 18% clay), with ph 6.9 and active carbonates lower than 5%, trees were trained as a free palmette. fruits were hand­picked at an optimal ripening stage tested with lugol solution. all trees received the same conventional cultural cares from planting until the end of the current experiment. after harvest, fruit were cold stored and transported at university of palermo and stored at 5±1°c in cold room the night before the analysis. experimental design two hundred fruit were harvested from 20 trees and brought to the laboratory where they were dipped in chlorinated water (100 ppm of free chlo­ rine) for 360 s to sanitize them. defective fruit were discarded and the remaining were sorted by firmness (4.1±1 kg/cm2) and average weight (120± 20.2 g). quality indexes were calculated the first day of analy­ sis, particularly, color (cielab), flesh compactness and total solid soluble content (tss). fruits were selected for weight, maturation index, caliber and absence or presence of defects and sani­ tized with oxvirin 200 ppm and h2o by soaking for 30 minutes. then, they were peeled and cut. edible coatings were applied by dipping and solutions were formulated as follows: i) control (ctr): fruits were dipped in distilled water and used as control; ii) xan: the solution was made by mixing 3% of xan­ than gum in distilled water using a magnetic stir­ rer; iii) asc: the solution was made by mixing 3% of xan­ than gum and 2% of calcium ascorbate in distilled water using a magnetic stirrer. after treatments, fruit were packed in pet boxes, sealed with a composite pp­pet film and stored at 5±1°c with 95% relative humidity (rh) for 10 days. trials were carried out at 3, 5, 7 and 10 days of stor­ age evaluating visual quality score, browning index, color, total solid soluble content (tss), sensorial analysis, ascorbic acid content and total phenols con­ tent. weight loss the following formula was adopted to determine weight loss during storage weight loss (%) = [(w1­w2)/(w1)] x 100 where w1 and w2 represent initial weight (t0) and measured weight at 3, 5, 7 and 10 days of storage with a precision balance (gibertini, italy), respec­ tively. at the beginning of trial period all boxes had homogeneous weight (100 ± 2.1 g). color flesh color was measured throughout the experi­ ment on the first day of analysis (0) and on the 3rd, guccione et al. ‐ xanthan gum and ca to prolong cv. butirra shelf life 61 5th, 7th and 10th day of storage. color was measured through a portable colorimeter (chroma meter cr 400, konica minolta sensing inc., tokyo, japan) equipped with an 8 mm measuring head and a c illu­ minant (6774 k). the white standard plate of the manufacturer was used for calibration. chromatic dif­ ference (de) was calculated using the following for­ mula to express the magnitude of difference between the non­aged pulp and stored samples: δe* = √δl*2 + δa*2 + δb*2 all trials were carried out in triplicate and data were reported as ± mean standard error (se n=3). browning index browning index (bi) was determined following the equation of ruangchakpet and sajjaanantakul (2007): (bi)= [100 (x – 0.31)]/0.17 where x = (a* + 1.75 l*)/(5.645 l* + a* −0.3012b*). firmness fruit firmness was tested with a texture analyzer equipped with a 2.5 cm flat­tip (instron 5564, ma, usa). the maximal force was expressed in kg/cm2 and slices were compressed with a speed of 5 mm/s to a depth of 4 mm. total solid soluble total solid soluble content was determined on pear fruit juice extracted from samples at each stor­ age time using a hand­held refractometer (atago palette pr­32). total phenols content total phenols content was quantified according to sortino et al. (2022). 30 grams of fresh tissue for each replication was homogenized with methanol on 1:10 ratio and then filtered through a whatman grade n.1 filter, the application of reduced pressure allowed the concentration of methanolic extracts and the residue was then suspended in 50% aqueous methanol and used for phenolic content quantifica­ tion. phenols content was determined through a spectrophotometrical analysis at the wave lenght of 700 nm and results were expressed in gallic acid equivalent (mg kg­1 fresh weight). ascorbic acid ascorbic acid content was analyzed at each sam­ pling date with the megazyme kit (bray business park, bray, co., wicklow, ireland) as reported by allegra et al. (2015). sensorial analysis sensorial analysis was carried out by a panel of 12 specifically trained panelists (sortino et al., 2017). all samples were subjected to a panel made up of 14 descriptors: external color uniformity (ecu), com­ pactness (com), pulp color intensity (pci), odor (o), herbaceous odor (ho), floral odor (fo), sweetness (sw), sourness (s), bitterness (b), juiciness (j), pear flavor (pf), herbaceous (hf) and floral flavor (ff) and overall rating (or). the graduated scale went from 1 (absence of descriptor) to 9 (descriptor at its fullest intensity). sensorial analysis was carried out from day 0 to day 10. visual quality score edible coatings effect on ‘butirra’ fresh­cut slices was evaluated at each storage time on six slices used as single replicates, for each treatment. six trained judges used a list of descriptors wrote down in pre­ liminary meetings. descriptors involved the medium value of color, integrity and appearance on pear fruit slices as reported by allegra et al. (2022). descriptors were quantified using a 5 points hedonic scale where 5= very good, 4= good, 3= sufficient (limit of mar­ ketability), 2= poor (limit of usability) and 1= very poor (inedible). co2 and o2 inside packaging o2 and co2 content inside packages was analyzed at each sampling date using a pbi dansensor checkpoint o2­co2 analyzer (ametek mocon, ms, usa) equipped with infrared detectors. statistical analysis the experimental design consisted in two treat­ ments and one control, observed at 0, 3, 5, 7 and 10 days at 5°c after treatment. nine slices were used as single replicates and analyzed at each sampling date. analysis of variance was applied (systat 13.0 for windows was used as statistical software) and the significance of data (p≤0.05) was evaluated with tukey’s test. 3. results and discussion total solid soluble content our results showed a general increase in tss dur­ ing storage; control slices content increased about 29.03% during the 10 days of storage while xan and asc treatments scored an increase of 16.30% and 15.45%, respectively. ctr slices increase is probably adv. hort. sci., 2023 37(1): 59­66 62 due to the greater water loss of the untreated sam­ ples, which also results in higher percentages of weight loss (fig. 1). xan and asc treatments were more efficient than ctr in limiting the increase in tss content, this phenomenon is due to ripening process­ es that result in the hydrolysis of starch into mono­ and disaccharides (mahajan et al., 2004) and in the activation of respiration processes where sugars are the main substrate used (dong et al. , 2004). significant differences occurred between treatments on 5th and 7th day while on 10th day xan and asc treatment recorded the same value. closely linked, as previously discussed, with tss con­ tent data. color changes and browning index color monitoring during cold storage showed a change in l*, a* and b* values in ‘butirra’ in all treat­ ments (table 1). a low decrease in l* values occurred in xan treatment. color changes are more limited in asc treated samples. at the end of cold storage period (t10), δe reached its highest in ctr samples and its lowest in asc treated samples. untreated ‘butirra’ fruit slices recorded the highest browning values at each sam­ pling date, and it began to increase sharply from day five, while xan and asc treatments recorded the lowest values. significant differences occurred between asc and xan treatments from 5th day of storage while no significant differences were record­ fig. 1 ­ total solid soluble content of untreated (ctr) and treat­ ed with xanthan gum and xanthan gum + calcium ascor­ bate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after for 3, 5, 7, 10 days of storage at 5°c. at each sampling date, different letters indicate sig­ nificative differences between treatments. p≤0.05 was used in the tukey’s test. the data are provided as the mean ± se (n = 3). weight loss and firmness weight loss during cold storage showed an increase in all sample slices (fig. 2). ctr samples showed a higher percentage of weight loss if com­ pared to other treatments. asc treatment showed lower weight loss than xan and control pear slices. the effectiveness of ascorbate calcium­can be attrib­ uted to the ability of calcium to preserve the com­ pactness of cell structures by limiting the action of pectolytic enzymes. in other work on fresh­cut pear the use of calcium ascorbate could be responsible of the maintenance of cellular wall structure since calci­ um maintains glycosidic bindings stable avoiding the collapse of cellular wall and the subsequent loss of liquids (akhtar et al., 2010). grant et al. (1973), on the other hand, showed that the maintenance of firmness in calcium­treated fruits may be due to its accumulation in cell walls, which facilitates the cross­ ing of pectic polymers by increasing wall strength and cell cohesion. obtained results for weight loss are fig. 2 ­ weight loss (%) and firmness (kg/cm2) of untreated (ctr) and treated with xanthan gum and xanthan gum + calci­ um ascorbate (xan and asc) pyrus communis l. cv. ‘butirra’ fruit slices at 0 time and after for 3, 5, 7, 10 days of storage at 5°c. at each sampling date, different letters indicate significative differences between treatments. p ≤ 0.05 was used in the tukey’s test. the data are provided as the mean ± se (n= 3). guccione et al. ‐ xanthan gum and ca to prolong cv. butirra shelf life 63 ed at 10th day between the two edible coatings for­ mulations (fig. 3). a similar trend occurred in sharma and rao (2015) on fresh­cut pear treated with xanthan gum for 8 days of cold storage. sensorial analysis and visual quality score sensorial analysis was carried out at each storage time and at harvest time high values were recorded for all descriptors except for bitterness, sourness, herbaceous and floral odor and flavor. a slower decrease in treated pear slices was recorded. all treatments recorded higher values than ctr one at each sampling time and no off­flavors or negative descriptors were found in edible coating treated sam­ ples until 10th day of storage. in fact, ‘butirra’ pear slices treated with asc and xan recorded positive val­ ues for descriptors concerning sweetness, compact­ ness, external color uniformity, juiciness and pear fla­ vor during all storage time while, on the contrary, ctr samples recorded increasing values of negative descriptors such as bitterness and sourness as early as the 5th day of analysis. overall rating was quite posi­ tive in treated samples until 10th day (fig. 4). visual quality score test enhanced that a decrease in mean values occurred in all treatments registering significant differences since the 3rd day of analysis (fig. 5). since day 3, ctr samples recorded mean val­ ues under 3 (limit of marketability). instead, the judges evaluated asc samples on 10th day at the limit of marketability (score=3). xanthan coating had a positive effect on visual score of fresh­cut pear (sharma and rao, 2015) extending their marketability until the 10th day. total phenols and ascorbic acid content no significant differences were showed between treatments on pear slices ascorbic acid content. during storage, total phenol content increases slowly in both ctr and xan treatment. after 7 days a sharp increase was observed in treated and untreated sam­ ples and significant differences occurred between table 1 ­ color slices (cielab index) and color variation (δe) of untreated (ctr) and treated with xanthan gum and xanthan gum + calci­ um ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after for 3, 5, 7, 10 days of storage at 5°c at each sampling date, different letters indicate significative differences between treatments. ns = not significant. p ≤ 0.05 was used in the tukey’s test. the data are provided as the mean ± se (n= 3). days of storage treatments l* a* b* δe t0 ctr 63.89 ­13.57 36.94 ­ t3 ctr 64.64 a ­0.32 b 24.30 b 18.33 ns asc 66.27 b ­0.38 b 24.98 b 17.96 xan 64.82 a 0.61 a 26.16 a 17.84 t5 ctr 66.97 a ­0.03 c 20.82 ns 27.27 a asc 63.96 b ­0.11 b 19.25 22.23 b xan 57.51 c 0.51 a 19.25 23.50 b t7 ctr 65.12 a ­0.22 a 17.4 b 28.69 a asc 63.02 b ­0.07 b 16.72 b 24.33 b xan 52.39 c 0.01 b 20.02 a 24.55 b t10 ctr 45.53 c ­0.02 b 12.20 b 33.66 a asc 62.65 b 0.03b 14.90 a 25.93 b xan 65.24 a ­0.36 a 14.37 a 26.19 b fig. 3 ­ browning index of untreated (ctr) and treated with xanthan gum and xanthan gum + calcium ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after for 3, 5, 7, 10 days of storage at 5°c. at each sampling date, different letters indicate significative differences between treatments. p≤0.05 was used in the tukey’s test. the data are provided as the mean ± se (n = 3). 64 adv. hort. sci., 2023 37(1): 59­66 asc and other treatments (fig. 6). an increase of total phenol content is possible in stress conditions after cutting or in low temperature (amodio et al., 2014). co2 and o2 inside packaging a limited o2 consumption and co2 production occurred during storage in both xan and asc treat­ ments while higher values were registered in ctr samples (fig. 7). significant differences occurred from the 3rd day between treated and untreated samples, particularly, ctr samples registered a value of 1.5 kpa for o2 and a value of 21 kpa for co2 at 10th day of storage. both xan and asc kept the two para­ meters more stable. similar trends were observed on fresh­cut peach treated with calcium lactate and ascorbic acid and on breba fig fruit stored in passive atmosphere (allegra and colelli, 2015; allegra et al., 2015). 4. conclusions the two different formulations based on calcium ascorbate and xanthan gum preserved pear slices of ‘butirra’ during the 10­day storage at 5°c. positive effects were observed on browning, weight loss and firmness up to the 10th day, furthermore, the two edible coating formulations preserved the sensory attributes of fresh cut ‘butirra’. our results showed that xanthan gum with calcium ascorbate treatment improved the retention of firmness, browining and weight loss than control slices. this result was con­ firmed by the sensorial analysis in which positive descriptors showed positive values until the 10th day of storage while untreated samples began to develop off­flavor and off­color since day 3. fig. 4 ­ sensorial analysis of untreated (ctr) and treated with xanthan gum and xanthan gum + calcium ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices after cut (0) and at 3, 7 and 10 days of storage at 5°c. at each sampling date, * indicates substantial changes between treatments. at each sampling date, ns indicate no changes between treatments. p≤0.05 was used in the tukey’s test. legend: external color uniformity (ecu), compactness (com), pulp color intensity (pci), odor (o), herbaceous odor (ho), floral odor (fo), sweetness (sw), sour (s), bitter (b), juiciness (j), pear flavor (pf), herba­ ceous flavor (hf), floral flavor (ff) and overall rating (o). fig. 5 ­ visual quality score of untreated (ctr) and treated with xanthan gum and xanthan gum + calcium ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after for 3, 5, 7, 10 days of storage at 5 °c. at each sampling date, different letters indicate significative differ­ ences between treatments. p≤0.05 was used in the tukey’s test. the data are provided as the mean ± se (n = 3). fig. 6 ­ total phenols content (mg kg­1 fw) of untreated (ctr) and treated with xanthan gum and xanthan gum + calci­ um ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after storage for 3, 5, 7, 10 days at 5°c. at each sampling date, different letters indicate significative differences between treatments. p≤0.05 was used in the tukey’s test. the data are provid­ ed as the mean ± se (n = 3). guccione et al. ‐ xanthan gum and ca to prolong cv. butirra shelf life 65 references akhtar a., abbasi n.a., hussain a., 2010 ­ effect of calci‐ um chloride treatments on quality characteristics of loquat fruit during storage. ­ pakistan j. bot., 42: 181­ 188. allegra a., colelli g., 2015 ­ the effect of passive atmosphere on quality of ‘dottato’ breba fig stored at low temperature. ­ acta horticulturae, 1173: 315­318. allegra a., inglese p., guccione e., farina v., sorti­ no g., 2022 ­ calcium ascorbate coating improves postharvest quality and storability of fresh‐cut slices of coscia and abate fétel pears (pyrus communis l.). ­ horticulturae, 8: 227. allegra a., sortino g., farina v., inglese p., 2015 ­ effect of passive atmosphere and chemical 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rao t.r., 2015 ­ xanthan gum based edible coating enriched with cinnamic acid prevents browning and extends the shelf‐life of fresh‐cut pears. ­ lwt­ food sci. technol., 62: 791­800. sortino g., allegra a., farina v., de chiara m.l.v., inglese p., 2022 ­ genotype influence on shelf life behaviour of minimal processed loquat (eriobotrya japonica (thunb.) lindl.) fruit: the role of sugar, acid organics and phenolic compounds. ­ chem. biol. technol. agric., 9: 1­18. sortino g., allegra a., passafiume r., gianguzzi g., gullo g., gallotta a., 2017 ­ postharvest application of sulphur dioxide fumigation to improve quality and storage ability of “red globe” grape cultivar during long cold storage. ­ chem. engin. transactions, 58: 403­ 408 fig. 7 ­ o2 and co2 inside packaging of untreated (ctr) and treated with xanthan gum and xanthan gum + calcium ascorbate (xan and asc) pyrus communis l. cv. butirra fruit slices at 0 time and after for 3, 5, 7, 10 days of stor­ age at 5°c. at each sampling date, different letters indi­ cate significative differences between treatments. p≤0.05 was used in the tukey’s test. the data are provid­ ed as the mean ± se (n = 3). adv. hort. sci., 2023 37(1): 59­66 66 vilas boas e.v., de b., reis c.m.f., melo a.a.m., 2009 ­ uso de misturas químicas para a manutenção da firme‐ za de banana’prata’minimamente processada. ­ ciência agrotecnol., 33: 237­244. impaginato 119 adv. hort. sci., 2022 36(2): 119­126 doi: 10.36253/ahsc­12450 effect of chemical and biological fertiliz­ ers on the morphology and yield of saf­ flower and soybean under monoculture and intercropping y. raei (*), r. ghahremani, s. ghassemi, j. shafagh­kolvanagh department of plant eco‐physiology, faculty of agriculture, university of tabriz, tabriz, iran. key words: biofertilizer, grain yield, number of grains per plant, plant height, urea fertilizer. abstract: intercropping and biofertilizers application are the most important agricultural methods for moving towards minimizing the risks of agricultural production and increasing production efficiency. consequently, this experiment was conducted at one year and in 2019. a factorial set of treatments was arranged within randomized complete block design (rcbd) with three replica­ tions to investigate the effect of different planting ratios with safflower and soybean (sole cropping, 30:100 soybean to safflower ratio, 60:100 soybean to safflower ratio and 90:100 soybean to safflower ratio) and nutrient levels (100% urea fertilizer, 100% biofertilizer and the combined application of urea and biofertilizer) on growth and yield of these crops. the results of this study indicated that intercropping patterns had the highest plant height, number of grains per plant, biological and grain yields. in addition, the means of the num­ ber of heads per plant and the number of grains per head in safflower and the weight of 1000 grains in soybean were increased as intercrops were grown. maximum of grain number per plant in safflower, leaf number per plan in soy­ bean and biological and grain yields in both crops were attained in urea + biofertilizer. in all of intercropping patterns the values of ler (land equivalent ratio), rvt (relative value total) and rcc (relative crowding coefficient) was more than one, indicating an advantage from intercropping over sole crops. 1. introduction intercropping, as a multiple cropping system, has been used by farm­ ers for many years in various ways and in various countries and has acted as a very significant role in sustainable agriculture (zhang and li, 2003) and widely practiced for enhanced production and nutrient acquisition advantages (ahmed et al., 2020). many studies have shown the effect of legumes on growth increase, potassium (k), phosphorus (p) and nitrogen (n) uptake, and the yield of intercropped plants compared with sole cropped plants (tosti et al., 2010; piri et al., 2011; raei et al., 2020). the (*) corresponding author: yaegoob@yahoo.com citation: raei y., ghahremani r., ghassemi s., shafa­ gh­kolvanagh j., 2022 ­ effect of chemical and biological fertilizers on the morphology and yield of safflower and soybean under monoculture and intercropping. ­ adv. hort. sci., 36(2): 119­126. copyright: © 2022 raei y., ghahremani r., ghassemi s., shafagh­kolvanagh j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 december 2021 accepted for publication 31 march 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12450 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 119­126 120 various benefits of an oilseed legume intercropping system­mainly intercropping legumes (soybeans) with oilseeds (safflowers) take in more skillful use of nutrients, available resources and sunlight, enhance yield and the improve land equivalent ratios (srinivasarao et al., 2012). soybean (glycine max l. merill) is one of the most important food legumes in iran and other parts of the world. it has potential of fixing atmospheric nitro­ gen besides meeting its own nitrogen requirement and serves as a viable and low cost medium for soil fertility improvement (muoneke et al., 2007). it is considerable as an important edible oil grain for human alimentation and is worldwide planted on approximately 80 million hectares (fao, 2020). safflower (carthamus tinctorius l.) is a prospective oilseed crop because it yields 32­40% seed oil. safflower oil is widely utilized in industry, mainly for edible and dying purposes. owing to its considerable drought tolerance compared with other oilseed crops, safflower is usually cultivated in iran where drought stress is a major restriction in the field. safflower is a deep­rooted annual crop that can be grown in rotation with other species (la bella, 2019). nitrogen is very important for its growth and yield (leghari et al., 2016), and the nitrogen supply is therefore essential to produce a high yield from this crop. by contrast, the soybean is a legume that can fix nitrogen and release nitrogen compounds into the soil and so intercropping of these species is probably a suitable field management strategy. a recent trend in sustainable global production is chemical fertilizers being replaced by biofertilizers. bio­fertilizer, represent a specific complex of microorganisms which enable the movement of nutrients from soil to plants through biological process such as n fixation and solubilization of rock phosphate (abou­khadrah et al., 2000). these fertiliz­ ers are found to have a positive contribution to soil fertility, resulting in an enhancement in crop yields without causing any environmental, water or soil pol­ lution hazards (timmusk et al., 1999; daiss et al., 2008). this suggested that the yield to components were increasing. it was reported the nitrogen and phosphate biofertilizer applications have many important benefits and decrease the inputs of pro­ duction because of cost deduction compared to chemical fertilizers which increased biological yield. in some studies, it was clearly revealed that biofertil­ izer application resulted in high productivity for saf­ flower (mirzakhani et al., 2009; seyed sharifi, 2012). using biofertilizer and selection of the best micro­ bial strains have vital role when integrating human society with vulnerable ecosystems. biological fertil­ izers, which are called biofertilizers, may be used in a way of to maintain soil fertility and guarantee soil improvement. biofertilizers are products containing living cells of different types of microorganisms, which have the ability to convert important nutrition­ al elements (n, p …) form unavailable to available from through biological process such as nitrogen fixa­ tion and solubilization of rock phosphate. biofertilizers differ from chemical and organic fertiliz­ ers in that they do not directly supply any nutrients to crops and are cultures of special bacteria and fungi. some microorganisms have positive effects on plant growth promotion, including the plant growth promoting rhizobacteria (pgpr) such as azospirillum spp., azotobacter spp., pseudomonas fluorescens, and several gram positive bacillus spp. (sivasakthi et al., 2014). most studies have focused on legume­cereal intercropping as a productive and sustainable sys­ tem, while intercropping systems such as soybean­ safflower have rarely been evaluated. thus, the pre­ sent research was carried out to: 1) study the effect of chemical and biological fertilizers on some mor­ phological traits and yields of safflower and soybean mono and intercropping system. 2) to evaluate the influence of cropping system on soybean and saf­ flower performance and finally 3) to investigate the interaction between cropping system and fertilizer. 2. materials and methods site description field experiment was conducted during 2019 growth season at the heris, east azerbaijan province, iran (latitude 38°25ʹ n, longitude 47°12’ e, altitude 1850 m above sea level with the mean annual rainfall of 315.2 mm). some physical and chemical properties of farm soil (0­30 depth) and means of maximum and minimum temperatures and rainfall during the work in 2019 are shown in table 1. experimental design and treatments in this experiment a factorial set of treatments within randomized complete block design (rcbd) with three replications was arranged. factors were cropping patterns (sole cropping’s of safflower and soybean, intercropping of safflower/soybean with raei et al. ‐ safflower and soybean responses to intercropping and fertilizers 121 the ratios of 30:100, 60:100 and 90:100 soybeans to safflower and nutrient levels (100% recommended urea fertilizer, 100% biofertilizer and 50% biofertilizer + 50% urea. more in details, safflower cultivar safe and soybean cultivar william were used. the amount of urea given in 100% treatment was 50 kg/ha. the biofertilizer contained barvar 1 (contains free living nitrogen fixing bacteria) and barvar 2 (contains phos­ phate dissolving bacteria) and was used as seed inoc­ ulation before the sowing the seeds. the biofertiliz­ ers was prepared by zist fanavar sabz company, iran. optimum sowing density of soybean and safflower in mono cultures were 50 and 40 seeds per m2, respec­ tively. measurements plant height of two crops. at maturity stage, 5 plants of the middle part of each plot were harvested and plant height (by meter) were determined. leaf number per plant of safflower and soybean. leaf number per plant was measured by hand at maturity stage. plant biomass of safflower and soybean. to deter­ mine plant biomass, 5 plants were harvested from middle part of each plot with considering marginal effect, then were dried in an oven at 75°c for 48 hours. finally, plant biomass per unit area were determined. yield and yield components of safflower and soy‐ bean. at final ripening 5 plants were harvested and head number per plant, grain number per head, grain number per plant and 1000 grains weight were determined in safflower plants. for determine the grain yield an area equal to 1 m2 was harvested from middle part of each plot considering marginal effect grain yields per unit area were determined. also, in soybean at maturity stage 5 plants from each plot were harvested and pods per plant, grain number per pod, grain number per plant and 1000 grains weight were determined. grain yield per unit area was determined by threshing all the plants in 1 m2 of the plots. evaluative indices of intercropping land equivalent ratio (ler). land equivalent ratio (ler), as an agronomic index, indicates the efficiency of intercropping in using the environmental resources compared with mono cultures (mead and willey, 1980). the value of unity is the critical value. when the ler is greater than one, the intercropping improves the growth and yield of the cultivars. the ler was calculated as: ler = (ysai/ysam) + (ysoi/ysom) where ysam and ysom are the yields of safflower and soybean, respectively, as sole crops and ysai and ysoi are the yields of safflower and soybean, respectively, as intercrops. relative value total (rvt). relative value total (rvt) as an economic index proposed by schultz et al. (1982). this index is widely used now and has been used by many researchers. the rvt was calcu­ lated as: rvt = (ap1 + bp2 )/ap1 where a is the key product price, b is a secondary product price, p1 is the main types yield and p2 is the secondary species in the mixture. if the rvt is greater than one, it’s indicating the intercropping advantage. if this index is smaller than one, it’s indicating that monoculture would prefer intercropping. the critical value of rvt is one. relative crowding coefficient (rcc). rcc is a mea­ sure of the relative dominance of one species over the other in a mixture (de wit, 1960). the rcc was calculated as: rcc = (ysai/ysam)/ (ysoi/ysom) table 1 ­ some physical and chemical properties of farm soil (a), and means of maximum and minimum temperatures and rainfall (b) during the work in 2019 depth (cm) ec (ds/m) ph organic carbon (%) n (%) p (ppm) k (ppm) sand (%) silt (%) clay (%) soil type 0­35 1.42 8.17 1.29 0.12 51.85 2085 37 50 13 silty loam month temperature (°c) rainfall (mm) april 5.9 82.2 may 11.4 28.3 june 18.6 22.5 july 22 0.3 august 22.8 1.7 september 18.6 2.7 adv. hort. sci., 2022 36(2): 119­126 122 where ysam and ysom are the yields of safflower and soybean, respectively, as sole crops and ysai and ysoi are the yields of safflower and soybean, respectively, as intercrops. if rcc= 1, the amount of crop in the mixture will be equal to monocropping. also, if rcc<1 indicates that the amount of the product in the mixture has decreased relative to sole crop and if rcc>1, the yield of the mixture is higher than that of pure stand of crops and the mixing is beneficial. 3. results analyses of variance is shown in table 2. in saf­ flower plants showed significant effects of cropping pattern on plant height, head number per plant, grain number per head, grain number per plant, 1000 grains weight and biological and grain yields. also, the effect of fertilizer factor for grain number per plant, 1000 grains weight and biological and grain yields were significant. the interactions of cropping pattern × fertilizer was only significant for 1000 grains weight. for soybean plants, analysis of vari­ ance showed significant effects of cropping pattern on plant height, grains number per pod and per plant, 1000 grains weight, biological and grain yields. also, leaf number per plant, grain number per pod, biological and grain yields were significantly affected by fertilizer factor. compared with the sole cropping of safflower and soybean, intercropping patterns had the higher plant height, grain number per plant, biological and grain yields. however, there was not observed significant difference between sole cropping and 90/100 soy­ bean/safflower ratio for soybean traits. similarly, the means of head number per plant and grain number per head in safflower plants and also 100 grains weight in soybean plants were increased in inter­ cropped patterns compared with pure cultivation (table 3). maximum of grain number per plant in safflower, leaf number per plan in soybean and biological and grain yields in both of these plants were attended in urea + biofertilizer. however, there was no significant differences between 100% biofertilizer and 50% urea + 50% biofertilizer in biological yields of two crops, as same as leaf number per plant in soybean (table 4). maximum of 1000 grains weight of safflower in different cropping patterns was observed in cropping table 2 ­ analysis of variance of the agronomic traits in safflower and soybean plants under different cropping patterns and fertilizer treatments ns, * and **: non­significant and significant at p≤0.05 and p≤0.01, respectively. source df mean square plant height leaf number per plant head number per plant grain number per head grain number per plant 1000 grains weight biological yield grain yield safflower (carthamus tinctorius l.) replication 2 35426 3326 0.756 0.562 2418347 5477 944302.4 928992 cropping pattern 3 705.209 * 11.273 ns 3.645 * 18.465 ** 12574.412 ** 74.811 ** 8603907.64 * 5528674.70 ** fertilizer (f) 2 55.243 ns 22.166 ns 1.441 ns 5.384 ns 7400.101 * 28.189 ** 8379561.03 * 3426726.11 ** c × f 6 134.942 ns 7.165 ns 1.109 ns 3.5 ns 1464.802 ns 16.372 ** 2104488 ns 722194.6 ns error 22 207923 11704 0.946 1.92 1495934 4058 2377343 510591.7 cv % ­ 19.34 18.72 12.27 6.88 14.43 5.98 16.26 19.49 soybean (glycine max l.) plant height leaf number per plant pods per plant grain number per pod grain number per plant 100 grains weight biological yield grain yield replication 2 17606 1245 2462 0.022 6611 0.801 147657.5 34858.45 cropping pattern 3 76.843 ** 8.261 ns 6.223 ns 0.037 * 49.205 * 6.429 ** 28566822.68 4661478.68 ** fertilizer (f) 2 28.492 ns 15.208 * 0.984 ns 0.212 ** 44.766 ns 1.672 ns 1547677.03 * 511204.56 ** c × f 6 31.105 ns 0.656 ns 4.983 ns 0.039 ** 32.575 ns 1.326 ns 631704 ns 14713.6 ns error 22 13331 3369 2584 0.01 13146 1174 414173.6 57485.11 cv % ­ 5.88 15.35 12.52 4.17 12.07 5.84 14.4 12.66 raei et al. ‐ safflower and soybean responses to intercropping and fertilizers 123 system of 30/100 (soybean/safflower) with integra­ tion application of 50% urea + 50% biofertilizers. minimum of this trait was related to monoculture and biofertilizer (fig. 1a). additionally, it was shown that the entrance of soybean plant to intercropping patterns led to an increase for 1000 grains weight in the ratio. grain number per pod in soybean plants affected by cropping patterns and fertilizer treat­ ments and significantly, maximum grain number per pod in different cropping patterns was observed in intercropping with 60 to 100 soybean to safflower ratio and urea + biofertilizer treatment (fig. 1b). evaluation of intercropping efficiency of treat­ ments indicated high land equivalent ratio value (ler >1) in all intercropping patterns which indicate those treatments produced biomass more efficiently than monocropping. maximum of ler, relative value total (rvt), and relative crowding coefficient (rcc) were achieved in 60/100 soybean/safflower ratio (table 5). 4. discussion and conclusions some important benefits of intercropping in this research are increasing in plant performance such as plant height, grain number, 1000 grains weight, bio­ logical yield and production of grain yield per unit area compared to sole cropping (table 3), due to the effective use of resources, including water, nutrients and solar energy (nasri et al., 2014). this is probably due to the fact that in intercropping system plants can achieve a better absorption. intercropping is pre­ table 3 ­ means of the agronomic traits in safflower and soybean plants under different cropping patterns different letters in each column indicate significant difference at p≤0.05 (duncan test). traits plant height (cm) head number per plant grain number per head grain number per plant biological yield (kg/ha) grain yield (kg/ha) safflower pure cultivation 63.54 b 7.044 b 18.26 c 217.6 b 8107 b 2697 b 30:100 83.11 a 7.933 ab 21.69 a 295.9 a 10330 a 4423 a 60:100 80.10 a 8.533 a 20.59 ab 296.8 a 9999 a 4166 a 90:100 71.41 ab 8.189 a 20.08 b 261.6 a 9503 ab 3382 b soybean traits plant height (cm) grain number per plant 100 grains weight (g) biological yield (kg/ha) grain yield (kg/ha) pure cultivation 58.82 b 26.87 b 17.49 b 5654 a 2345 a 30:100 65.87 a 32.46 a 18.71 a 1983 c 912.7 c 60:100 62.51 ab 30.74 a 19.54 a 4379 b 1807 b 90:100 61.38 b 30.07 ab 18.5 ab 5858 a 2510 a table 4 ­ means of the agronomic traits in safflower and soybean plants under different fertilizer treatments different letters in each column indicate significant difference at p≤0.05 (duncan test). traits grain number per plant biological yield (kg/ha) grain yield (kg/ha) safflower 100% urea fertilizer 252 b 8831 b 3358 b 100% biofertilizer 255.4 b 9194 ab 3359 b 50% urea + 50% biofertilizer 296.6 a 10430 a 4284 a soybean traits leaf number per plant biological yield (kg/ha) grain yield (kg/ha) 100% urea fertilizer 10.73 b 4122 b 1706 b urea + biofertilizer 12.95 a 4839 a 2115 a 124 adv. hort. sci., 2022 36(2): 119­126 ferred to sole cropping as a result of superior yield due to better absorption of resources, and this is especially realized when legumes are used (sachan and uttam, 1992), because they improve soil fertility due to increased nitrogen fixation (manna et al., 2003). intercropping of legumes (soybean) and asteraceae (safflower) families results in increased crop yield (table 3), maximized resource consump­ tion and enhanced productivity of cultivation system (singh rajesh et al., 2010). interspecific interaction between species in the rhizosphere can also affect the nutrient availability and uptake in intercropping (li et al., 2010). light, water and nutrients may be more completely absorbed and converted to crop biomass by intercropping. this is a result of differ­ ences in the competitive ability for growth factors between intercrop components (amini et al., 2013). combined application of urea + biofertilizer signif­ icantly improved the grain number per plant in saf­ flower and leaf number per plant in soybean plants. also, biological and grain yields of two crops were significantly increased by this treatment (table 4). nitrogen is a chemical fertilizer that has an important role in enhancing the growth and yield of plants (kulekci et al., 2009). however, intensive utilization of chemical fertilizers entails several ecological issues and increases the production costs and food insecuri­ ty. integrated plant nutrient management and irriga­ tion are practically two elements of crop production. the application of biofertilizers is critical in the agri­ cultural sector for sustainability of soil fertility, plant growth and development, and final yield perfor­ mance (bhardwaj et al., 2014). biofertilizers contain living cells or efficient strains of symbiotic and non­ symbiotic microorganisms. these beneficial bacterial or fungal inoculants accelerate the uptake of nutri­ ents in the rhizosphere once applied over seed and soil. various studies have documented that plant growth­promoting rhizobacteria can promote plant growth by various mechanisms such as fixation of atmospheric nitrogen, production of siderophores that chelate metal elements and make them accessi­ ble to plant roots, solubilization of minerals such as phosphorus, and synthesis of phytohormones (gusain et al., 2015). results indicated that the interaction effect of cropping pattern × fertilizer treatments was signifi­ cant. the introduction of soybean plants to cropping patterns resulted in the significant increase for saf­ flower 1000 grains weight and soybean grains num­ ber per pod as integrated nutrition was applied (fig. 1a). intercropping with soybean can improve avail­ able nitrogen through supplementary in nutrient resources achieved from n2 fixation (agegnehu et al., 2006). this nitrogen resource is anticipated to: (i) alleviate interference between safflower and soy­ bean for nitrogen absorption and; (ii) increase the available nitrogen for the next crops by improving the nitrogen content of the soil after the decomposi­ tion of the leguminous debris (hauggaard­nielsen et al., 2008). in this study, the values of ler, as the most com­ mon agronomic index was more than one (table 5) and used for suitability intercropping evaluate. it can be attributed to differences in traits such as rooting fig. 1 ­ means of safflower 1000 grains weight (a) and soybean grains number per pod (b) for interaction of cropping pattern × fertilizer treatments. raei et al. ‐ safflower and soybean responses to intercropping and fertilizers 125 depth, maximum absorption for nutrient elements and especially no significant competition for resource such as nitrogen on the basis of complementary resources due to dispute in space, time and form. another indicator used in assessment of intercrop­ ping is rvt, which evaluate intercropping in terms of economic value. by placing the numbers associated with each parameter in the formula of this index, the economic value of each treatments of intercropping can be calculated and interpreted. in calculations of this research, the daily price of two crops was used. the value of rvt in all of intercropping patterns is more than one indicating economic superiority of intercropping over monocropping. the highest value was obtained in 60 to 100 soybean to safflower ratio (table 5). relative crowding coefficient (rcc) is the ability of a species to use limited resources in inter­ cropping relative to its ability to gain the same resource in monocropping system by using yield comparing. it shows the competitive advantage of intercropping components (snaydon, 1991) and rcc of 60 to 100 soybean to safflower ratio was higher than those of other intercropping patterns (table 5). agronomic traits in safflower and soybean plants showed intercropping patterns and urea + biofertiliz­ ers were superiority treatments compared to other treatments. 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­ j. appl. ecol., 28(3): 930­946. srinivasarao c., venkateswarlu b., lal r., singh a.k., kundu s., vittal k.p.r., sharma s.k., sharma r.a., jain m.p., chary g.r., 2012 ­ sustaining agro‐ nomic productivity and quality of a vertisolic soil (verti‐ sol) under soybean‐safflower cropping system in semi‐ arid central india. ­ can. j. soil sci., 92(5): 771­785. timmusk s., nicander b., granhall u., tillberg e., 1999 ­ cytokinin production by paenibacillus polymyxa. ­ soil biol. biochem., 31(13): 1847­1852. tosti g., benincasa p., guiducci m., 2010 ­ competition and facilitation in hairy vetch‐barley intercrops. ­ ital. j. agron., 5(3): 239­248. zhang f., li l., 2003 ­ using competitive and facilitative interactions in intercropping systems enhances crop productivity and nutrient‐use efficiency. ­ plant soil, 248(1): 305­312. impaginato 61 adv. hort. sci., 2020 34(1s): 61­69 doi: 10.13128/ahsc­8395 different growing conditions can modu­ late metabolites content during post­ harvest of viola cornuta l. edible flowers i. marchioni 1, 2, l. colla 1, 3, l. pistelli 1, 4 (*), b. ruffoni 2, f. tinivella 3, g. minuto 3 1 dipartimento di scienze agrarie, alimentari e agro‐ambientali (dis‐ aaa‐a), università di pisa, via del borghetto, 80, 56124 pisa, italy. 2 crea, centro di ricerca orticoltura e florovivaismo, corso inglesi, 508, 18038 sanremo (im), italy. 3 centro di sperimentazione e assistenza agricola, regione rollo, 98, 17031 albenga (sv), italy. 4 centro interdipartimentale di ricerca nutraceutica e alimentazione per la salute (nutrafood), università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: cold storage, greenhouse cultivation, horned pansy, secondary metabolites. abstract: edible flowers are inflorescences traditionally used in various part of the world to enrich sweet and savoury recipes. the flowers of viola spp. were appreciated since the romans, and today the fresh products are now incorpo­ rated as ingredients in different culinary preparations. in this work, cultivation of potted viola cornuta l. cv. penny lane was performed in greenhouse with different environmental conditions (basal heating, additional led lighting and moisture management) and therefore the biomass production (number of flow­ ers per square meter and plant dimension per pot) was assessed. the plants are characterised by flowers with dark purple and orange petals in the same corolla. the shelf­life of detached flowers was studied in post­harvest condi­ tions at 0 and 4 days of cold storage at 4°c (polyethylene boxes, 12/12 h light/dark condition) to simulate the condition of i gamma products. sugars and secondary metabolites were analysed. basal heating seems not to increase flower number but could contribute to reach a well­balanced simultaneous presence of different antioxidant molecules (polyphenols, anthocyanins, carotenoids). our data highlight that the short cold storage under light condi­ tion lead to an increase in the content of total polyphenols and antioxidant activity, although a general reduction in pigments and sugars is observed. 1. introduction edible flowers are currently part of a niche market and perceived as a culinary novelty, even if their consumption is known for thousands of years. in fact, there are several historical evidences that highlight the use of the inflorescences to prepare and garnish dishes, from some ancient (*) corresponding author: laura.pistelli@unipi.it citation: marchioni i., colla l., pistelli l., ruffoni b., tinivella f., minuto g., 2020 ­ different growing conditions can modulate metabolites content during post‐harvest of viola cornuta l. edible flowers. ­ adv. hort. sci., 34(1s): 61­69 copyright: © 2020 marchioni i., colla l., pistelli l., ruffoni b., tinivella f., minuto g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2019 accepted for publication 26 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 61­69 62 civilisation as greeks and romans, to more recent times, e.g. the victorian period in england (mlcek and rop, 2011; cunningham 2015). most appreciated species were roses (rosa spp.), calendula (calendula officinalis l.), saffron (crocus sativus l.), dandelion (taraxacum officinale l.), and elder inflorescences (sambucus nigra l.) (mlcek and rop, 2011). in indian and chinese cultures, edible flowers are used as com­ ponents of medicines based on herbs, in addition to culinary purposes (wongwattanasathien et al., 2010). several edible flowers are beneficial to human health showing anti­infiammatory effects and antioxidant and ros scavenging activities (mlcek and rop, 2011). today, around 180 specie are known to produce edible flowers (lu et al., 2016), and viola spp. are among the most common and currently consumed. these flowers are characterized by a sweet and refreshing taste, in addition to a pleasant velvety tex­ ture (neumann and o’connor, 2009; koike et al., 2015). edible violas belong to 3 different species, namely viola cornuta l. (horned pansy), viola tricolor l. (johnny jumpup), and viola × wittrockiana gams (garden pansy) (neumann and o’connor, 2009). the plants are similar to each other except for flower size, which its diameter is in garden pansies (up to around 11.5 cm) > horned pansies (up to around 2.5 cm) > johnny jump ups (less than 2.5 cm in diameter) (bailey, 1998; kessler et al., 1998). over the years, intensive breeding programs selected new varieties with unique flower colours (pure­colour or multi­ coloured flowers), greater flowers number, and plant temperature tolerance (bailey, 1998). the cultivation of v. cornuta is similar to the one of v. × wittrock‐ iana. these speciesare grown as autumn and spring bedding plants, although they are also raised for the summer and winter markets (pearson et al., 1995). in order to produce edible flowers safe for human con­ sumption, chemical products, such as synthetic fertil­ izers and pesticides, has to be avoided during plants production; for this reason, only organic cultivation is allowed (fernandes et al., 2017). no special needs for cultivation are required, indeed well drained com­ mercial potting soil can be used. viola flowers are often cultivated in greenhouse, and properly defined environmental factors, such as temperature, pho­ toperiod and irradiance are fundamental for the quality of the flowers (gandolfo et al., 2016). pansies should be grown between 4 and 13°c, in order to reduce plant growth rate, internode elongation and to ensure high quality flowers (cavins et al., 2000). in fact, flower size (mm2) decreased linearly with increasing temperature between 9 and 31°c (pearson et al., 1995). the ideal temperature for growth and flowering ranges from about 14°c to 21°c (kessler et al., 1998). moreover, pansies are obligate fr (far red)­dependent long­day plants and, for this reason, fr radiation are required to promote the flowering process, in addition to red (r) radiation (kozai et al., 2016). blue light is able to reduce the time required to produce flower buds in v. × wittrockiana (rashidi et al., 2018). full­bloomed, edible flowers can be sold in pots or, mainly, in small and medium rigid plastic pack­ ages to avoid their rapid drying and to preserve their fragile texture (whitman, 1991; kelley et al., 2001). however, flowers are high perishable so that differ­ ent approaches were performed to prolong their shelf­life. cold storage is documented for v. tricolor and v. x wittrockiana, using sealed low­density polyethylene film bags. these two species were able to preserve their commercial attractiveness up to 2 weeks of storage, when kept between 0 and 2.5 °c (kelley et al., 2003). more recently, different new post­harvest technologies were applied on viola spp. edible coatings (e.g. alginate), crystallization and osmotic dehydration improved violas shelf­life, as shown by a good visual quality for prolonged period (fernandes et al., 2018 a, b, 2019 a, b). coated pan­ sies contained higher level of polyphenols and antioxidant activity than uncoated ones, on all assayed storage times (up to 14 days). gamma irradi­ ation are also tested, and this methodology increased polyphenols content and antioxidant activity in v. tri‐ color flowers, compared to no irradiated controls (koike et al., 2015). edible flowers are selected and perceived by their fragrance, appearance, size and colour. consumers prefer yellow and orange flowers rather than blue (kelley et al., 2001, 2002). within this regard, v. cornuta cv. ‘penny lane’ with orange­ violet flowers have been selected for this work. the aim of this work was to cultivate v. cornuta l. ‘penny lane’ and test the effect of different cultiva­ tion strategies for a higher production of flowers with good quantities of nutritional compounds. moreover, the post­harvest treatment has been performed to analyze the change in bioactive compounds. storage temperature was maintained around 4­6°c (cold stor­ age) and flowers were also exposed to artificial light to simulate the refrigerated sector of the grocery market. metabolites (polyphenols, anthocyanins, carotenoids, sugars) were analysed to determine the shelf­life of packaged flowers as i gamma products. to the best of our knowledge, any investigation of metabolites during post­harvest cold storage studies marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 63 were performed on v. cornuta. 2. materials and methods plant cultivation, greenhouse condition and flower blooming plants of viola cornuta l. ‘penny lane’ with orange­violet flowers (fig. 1) were purchased by gruppo padana ­ ortifloricoltura dei fratelli gazzola s.s. società agricola (paese, tv, italy) and planted in 420 pots with a diameter of 14 cm (1 l volume). they were placed on 4 benches of an iron­glass green­ house (called sam­lab) equipped with a climatic control system at the “centro di sperimentazione e assistenza agricola” (cersaa) in albenga (sv) 43° 3’ 14’’ north and 8° 13’ 1’’ east). at the beginning of the experiment, plants were 3 cm height with a diameter of 2.5 cm. the substrate used was “ts4” soil from “turco silvestro” company (albenga, sv, italy), char­ acterized by ph 6.5, electrical conductivity 0.56 ds/m, dry bulk density 250 kg/m3 total porosity 90% v/v. the experimental design foresees 7 treatments (60 plants each) in the samlab and reported in table 1. the presence or absence of basal heating was guaranteed by either electric mat warmset at 50°c or water at 35°c (by hydraulic coil) and the addition of 1 or 2 hours of light after the astronomical sunset to extend the photoperiod, as reported in table 1. in consideration of the number and the layout of the benches of the greenhouse and the possibility to sub­ divide the lighting of led lamps used for the experi­ mentation, only the selected treatments were tested (table 1). each bench was equipped with 4 led lamps placed at a distance of 1.50 meters from the surface of the pallet depending on the type of lamp and cul­ ture. for the tests, specifically, valoya b200 led lamps with ap673l spectrum were used (blue 12% ­ green 19% ­ red 61% ­ far red 8% ­ par 92%). each lamp has a total power consumption of 192 w, a photon flux in the range 400­700 nm of 284 mmol s­1 and a photon flux in the range 300­900 nm of 311 mmol s­1). during the trial in the greenhouse the reg­ istered average temperature was 18°c and the aver­ age humidity was 64%. from 01/14/2019 to 02/28/2019 evaluations were carried out to observe whether different kind of basal heating and supple­ mentary light could affect plant growth. at the end of the trials, the investigations included the measure­ ment of plant diameter (cm) per each pot and the number of flowers per meter square. thus, flowers were picked by hand in the morning for further analyses and cold treated. flowers storage conditions fresh picked flowers were stored in polyethylene boxes at 4 °c with a 12 hours photoperiod in order to simulate better the condition of supermarket fridge fig. 1 ­ flowers of viola cornuta cv. penny lane grown in pot in samlab greenhouse. table 1 ­ greenhouse treatments (basal heating of benches and/or additional light) of viola cornuta cv. penny lane. treatments are car­ ried out for 3 months, from transplantation through flowering period until the end of trials treatment basal heating additional light electric mat 50°c hot water 35°c absent 1 hour 2 hours absent 1 x x 2 x x 3 x x 4 x x 5 x x 6 x x 7 x x adv. hort. sci., 2020 34(1s): 61­69 64 counter. the refrigerated cells were equipped with led lamps (valoya, finland) having the following spectrum: blue 21% ­ green 38% ­ red 35% ­ far red 6% ­ par 94%. flower storage was evaluated after 4 days post­harvest (time 4) performing the following biochemical analyses: total phenolics content, antiox­ idant activity (dpph assay), total anthocyanins con­ tent, total carotenoids content and total soluble sug­ ars content. for each biochemical analysis three homogeneous biological replica were used. each replica was stored at ­20°c until further analyses. fresh flowers (time 0) are used as control. polyphenol content polyphenols were extracted as reported by bretzel et al. (2013). fresh flowers (200 mg) were homogenized in 2 ml of methanol 70 %, and of total phenolic content was determined using the folin­ ciocalteau assay (singleton and rossi, 1965). the absorbance was read at 765 nm in a uv­1800 spec­ trophotometer (shimadzu corp., kyoto, japan) and the total phenolic concentration was expressed as catechin equivalents per gram of fresh weight. dpph scavenging activity the antioxidant activity of each sample was deter­ mined through the 2,2­diphenyl­1­picrylhydrazyl radi­ cal (dpph) free radical scavenging assay, as described by brand­williams et al. (1995). the absorbance was read at 517 nm. antioxidant activity was expressed in ic50, which represent the concentration of the sample able to reduce the initial amount of radical dpph by 50%. consequently, lower ic50 value of sample corre­ sponds to greater antioxidant activity. anthocyanin content total anthocyanins were extracted as reported by bretzel et al. (2013). 200 mg of sample were homog­ enized in 750 µl of acidified methanol (meoh/hcl 10:0.1). the absorbance was read at 535 nm and the total anthocyanin concentration was expressed as malvidin equivalents per gram of fresh weight. carotenoids content determination of total carotenoids content was determined using lichtenthaler’s formula (1987). fresh flowers (100 mg) were added to 5 ml of methanol 99 % and it was incubated for 24 hours at 4°c. the absorbance was read at 665.2 nm, 652.4 nm and 470 nm. total soluble carbohydrates content total soluble carbohydrates content was estimat­ ed from dried flowers (20 mg) using anthrone proto­ col according to yemm and willis (1954). the absorbance was read at 630 nm, using glucose as external standard. statistical analysis the normal distribution of the residuals and the homogeneity of variance was determined and then data were statistically analysed. the results of bio­ mass (number of flowers and growth in pot) were expressed as mean values and analyzed using one­ way analysis of variance (anova) followed by tukey’s hssd test with p=0,05. the results of post­ harvest treatments have been performed using anova student’s t­test to determine the significant difference of each treatment between fresh samples (time 0) and samples after 4 days (time 4), with p < 0.05. biochemical results were analysed by one­way anova followed by fisher’s probable least­squares difference test with cut­off significance at p ≤ 0.05 (statview®, version 5.0, sas® institute corporation). the dependent variables were analysed using two­ way anova, with the factors ‘‘treatment” and ‘‘post harvest days’’ (phd). 3. results and discussion horned pansy (viola cornuta) is a biannual plant with long flowering period through different seasons. it is also considered as cold­tolerant plant, since the minimum temperature for flowering is around 4°c, while the optimal temperature is around 26°c (blanchard and runkle, 2011). winter temperature and light are very important factors to determine a good production of flowers (boldt and altland, 2019), and heating and supplemental lighting are often pro­ vided in greenhouse cultivation to improve the quan­ tity and quality of flowers (dieleman and meinen, 2007; oh et al., 2010). for this reason, horned pansy plants were subjected to different treatments (table 1) to evaluate their effect on the growth and flowers number (table 2) and thus the yield of edible flowers. the results indicated that the treatment n. 7 (con­ trol, no basal heating, no additional light hours) and treatment n. 3 (no basal heating, 2 h of supplemen­ tary light) determined the larger diameter of plants (table 2). however, there is no statistically significant difference (t student analysis) between treatment n. 3 and the control n. 7 (18.0 and 17.1 cm/plant respectively), probably due to the effect of light towards vegetative growth. the addition of basal temperature of the benches by hot water (treatment marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 65 the visible characteristics and nutraceutical compo­ nents (benvenuti et al., 2016). thus, the influence of light and temperature for the production of different metabolites was also determined. edible flowers are considered a good source of antioxidant molecules (rop et al., 2012; loizzo et al., 2015) and polyphenols (including phenolic acids and anthocyanins) are considered the main antioxidant compounds. a first detail of phenolic composition and properties of v. cornuta edible flowers highlight­ ed that their polyphenols content is lower than v. × wittrockiana (moliner et al., 2019). the metabolites were analyzed at the time of harvest (time 0), and after short period of post­harvest in a chamber at lower temperature and in the presence of light (12h). the post­harvest treatment was chosen to mimic the condition of the benches of grocery stores. pigments, as carotenoids and anthocyanins are the important compounds for evaluating the visual quality of flow­ ers. at time of harvest (time 0), the worst treatment resulted the n. 3 (addition of 2h light), since the recorded amount of both pigments were the lowest, 0.14 and 5.63 mg/g fw for carotenoids and antho­ cyanins, respectively (table 3). instead, the highest values were determined with the treatment n.1 (temperature 50°c by electric mat and light 1h (0.32 and 10.42 mg/g fw for carotenoids and antho­ cyanins). the increased temperature, either by elec­ tric mat (n. 2) or by hot water (n. 5), did not support any increase in flower pigmentation, both for carotenoid and anthocyanins. control flowers showed good carotenoid values (0.30 mg/g fw), while anthocyanins suffered without addition of light or temperature (6.93 mg/g fw). of our knowledge only few papers have been published so far on the 5) corresponded to a decrease of the growth (15,6 cm/plant), while when also additional lighting was performed the decrease was not significant (treat­ ments n. 1, 4, 6). the growth of the plants seems to be affected when the additional light is added, in the absence or with higher temperature of the benches (table 2). the effect of supplemental led lighting is known to affect positively many plant growth para­ meters of several plants, including pansy (koksal et al., 2015), so these results are in agreement with pre­ vious reports. the treatment 2 (electric mat 50°c) highlighted the lowest value of number of flowers (604.44 flowers/m2) followed by treatment n.6 (636.94 flowers/m2), while the other trials showed similar higher amounts (table 2). taken together, the biometric parameters suggest that the single elonga­ tion of photoperiod (2h) plays a positive role to increase the biomass and to produce more flowers, and the temperature is a secondary effect. the quali­ ty of plants in relation to light and temperature is debated since long time (liu and heins, 1997; adams et al., 1998), and the lower temperatures and higher irradiance seems to produce higher quality of flow­ ers, including pansy (pearson et al., 1995; boldt and altland, 2019). the results presented here are in agreement of the observed influence of the exposure duration, intensity and combinations of light to the growth and flowering of v. × wittrockiana (oh et al., 2010). the lengthening of the photoperiod has been confirmed as important factor in v. × wittrockiana ’rose’, during experiments aimed to the determine the influence of photoperiod and phytochrome (rashidi et al., 2018). in that research, the night inter­ ruption decreased the plant dimension. the quality of flowers, especially the edible ones, are related to table 2 ­ effect of different cultivation (basal heating and/or supplementary light) on biomass production of viola cornuta cv. penny lane plant diameter (cm) and the number of flowers (per meter square) were detected at the end of flowering period. data are expressed as mean value (n=60) and analyzed using one­way analysis of variance (anova) followed by fisher’s probable least­ square difference test with p=0.05. treatment diameter (cm) number of flower per m2 1 electric mat 50°c + light 1 h 15.8±0.40 ab 838.42±42.9 a 2 electric mat 50°c 16.2±0.46 ab 604.44±48.75 b 3 light 2 h 18.0±0.40 a 864.42±40 a 4 hot water 35°c + light 1 h 16.6±0.33 ab 851.42±33.8 a 5 hot water 35°c 15.6±0.38 b 812.43±44.85 a 6 hot water 35°c + light 2 h 16.1±0.24 ab 636.94±43.55 ab 7 control 17.1±0.50 a 793.93±38.35 a 66 adv. hort. sci., 2020 34(1s): 61­69 influence of light and temperature on the content of pigments in viola spp. (rashidi et al., 2018), so these results will contribute to define the effect of these factors and their contribution to the pigmentation. other metabolites of horned pansy were determined at time of harvest, such as polyphenols andsugars, that are expected as fundamental nutraceutical com­ ponents, as well as the scavenger reducing power (by dpph assay). with regards to total soluble carbohy­ drates (tss) statistically significant differences were observed among the various treatments: higher sug­ ars amounts were observed in the treatments n. 1 and 4, characterized by the addition of light (1 h) and higher temperature. although the other treatments showed the similar less quantities of sugars, the low­ est amount is observed in treatment n. 3 (tss 179.57 mg/g fw). the highest amount of total polyphenols (12.42 mg/g fw) was measured in treatment n. 1 (basal electric heating at 50 °c with 1 hour of addi­ tional light), and the lowest value in the control and n. 6 (7.66 and 7.36 mg/g fw, respectively). the antioxidant activity is higher in the treatments of addition of temperature by electric mat (n. 1 and 2) with ic50 (dpph assay) values of 0.80 and 0.82 mg/ml respectively, followed by the trials n.3 and 4. flowers of the control and treatment n.6 showed the lower scavenger reducing activity. in the present work the concentration of total polyphenols ranged 7.36 and 12.42 mg gae/g fresh weight. these values agree with those found in v. × wittrockiana, reported by other authors (rop et al., 2012). however, the polyphenol values could be underestimated by the method of extraction, as already shown in gonzàles­ barrio et al. (2018). in fact, they reported different polyphenol amounts in v. × wittrockiana by using either acidic hydrolysis or maceration instead of the method adopted in this work (bretzel et al., 2014). other reports showed the influence of storage at different temperature in different flowers (kelley et al., 2003). moreover, different packages used for the storage conditions can affect the quality of flowers (landi et al., 2018). changes of appearance, and aes­ thetic value were performed on v. × wittrockiana (kelley et al., 2003). the results obtained at the time table 3 ­ determination of carotenoids, anthocyanins, polyphenols, radical scavenging activity (dpph assay), and soluble sugars of viola cornuta flowers grown under different greenhouse conditions (av1­7, see table 1) and cold stored for 0 (time 0) o 4 (time 4) days postharvest data are expressed as means (n=3, ± se.) anova followed by fisher’s probable least­square difference test was used, with a cut­off signi­ ficance at p=0.05. smalls letter indicate comparisons between treatments at the same postharvest day (phd); capital letters indicate comparisons between the two phd for the same treatment. interaction between treatments and phd were analysed by two­way anova. treatment carotenoids (mg/g fw) anthocyanins (mg/g fw) polyphenols (mg/g fw) dpph assay (ic50 mg/ml) soluble sugars (mg/g fw) time 0 1 electric mat 50°c+ light 1 h 0.32 ± 0.00 a a 10.42 ± 0.32 a a 12.42 ± 0.11 a a 0.82 ± 0.00 a a 209.69 ± 3.91 a a 2 electric mat 50°c 0.27 ± 0.00 c b 8.05 ± 0.20 c a 9.93 ± 0.68 b b 0.80 ± 0.01 a a 183.84 ± 6.97 b a 3 light 2 h 0.14 ± 0.01e b 5.63 ± 0.14 e b 11.00 ± 0.09 ab a 0.84 ± 0.02 aba 179.57 ± 3.98 b a 4 (hot water 35°c + light 1 h) 0.29 ± 0.01 bc a 9.28 ± 0.49 bc a 11.85 ± 0.40 ab a 0.86 ± 0.02 ab a 207.45 ± 4.85 a a 5 hot water 35°c) 0.28 ± 0.01 c a 10.29 ± 0.25 ab a 9.35 ± 1.06 b a 0.89 ± 0.01 b b 181.29 ± 3.84 b a 6 hot water 35°c + light 2 h 0.26 ± 0.00 d a 6.93 ± 0.42 da 7.36 ± 0.16 c b 1.19 ± 0.05 d b 183.90 ± 10.96 b a 7 control 0.30 ± 0.00 b a 8.71 ± 0.57 cd a 7.66 ± 0.25 c b 1.09 ± 0.01 c b 179.90 ± 1.44 b a time 4 1 electric mat 50°c+ light 1 h 0.33 ± 0.01 b a 4.81 ± 0.19 d b 12.46 ± 0.25 a a 0.78 ± 0.01 a a 203.61 ± 2.03 a a 2 electric mat 50°c 0.36 ± 0.01 a a 7.63 ± 0.18 c a 11.62 ± 0.16 b a 0.82 ± 0.02 aa 158.91 ± 2.90 d a 3 light 2 h 0.21 ± 0.00 e a 9.22 ± 0.19 b a 10.88 ± 0.21 bc a 0.84 ± 0.01 b a 161.09 ± 0.36 d b 4 hot water 35°c + light 1 h 0.28 ± 0.01 c a 10.73 ± 0.56 a a 11.65 ± 0.32 ab a 0.90 ± 0.02 c a 175.50 ± 1.30 c b 5 hot water 35°c 0.23 ± 0.00 d a 6.65 ± 0.44 c b 10.43 ± 0.45c a 0.77 ± 0.02 a a 164.42 ± 3.41 d a 6 hot water 35°c + light 2 h 0.25 ± 0.01 d a 4.75 ± 0.49 d b 9.32 ± 0.04 d a 0.94 ± 0.02 c a 159.48 ± 2.99 d b 7 control 0.27 ± 0.01 c b 7.49 ± 0.79 c a 10.37 ± 0.27 c a 0.78 ± 0.01 a a 192.31 ± 2.56 b a anova p‐value treatment < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 phd 0.0014 < 0.0001 < 0.0001 < 0.0001 < 0.0001 treatment × phd < 0.0001 < 0.0001 0.0041 < 0.0001 0.0060 marchioni et al. ‐ marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 67 of cold storage (time 4) were compared to those at the time of harvest (time 0). the data reported here indicated that the purple­pink flowers maintained the carotenoids content after 4 days of cold storage in the treatments n. 1, 4 and 6, whereas in the treat­ ments n. 2 and 3 values of carotenoids increased. meanwhile, in flowers of treatment n. 5 and 7 (con­ trol) the amount of carotenoids decreased (table 3). after 4 days of postharvest treatment, anthocyanins are the most affected metabolites by cold storage. in fact, the amount of anthocyanins decreased in the treatments n. 1, 5 and 6, whereas in the treatment n. 3 values increased (9.22 mg/g fw). the treatment n. 1 showed the largest decrease, 10.42 mg/g fw at time 0 and 4.81 mg/g fw at time 4. however, the loss of pigmentation is not always documented, but it is peculiar of each species and variety, as already demonstrated in other species as acmella oleracea, salvia discolor, begonia semperflorens, tropaeolum majus (landi et al., 2018). the total polyphenols con­ tent after cold storage maintained the same values of that detected at time 0, with the exception of treat­ ments n. 2, 6 and control, where polyphenols increased, 11.62, 9.32 and 10.37 mg/g fw at time 4, respectively (table 3). the antioxidant activity increased in the treatments with hot water (n.4, 5, 6). different susceptibility to the storage process was observed in other edible flowers, with different changes (increase or decrease) on nutraceutical val­ ues up to 8 days of postharvest (landi et al., 2018). the soluble sugars dropped significantly in the treat­ ment n. 4, since the values was the highest at time 0 but reduced at 80% at time 4 (207.45 and 175.5 mg/g fw). other decrease in the content of sugar is observed for the treatments n.3 and 6. soluble sug­ ars are important nutritional components of the flowers and represent a good characteristic for the choice of edible flowers (mlcek and rop, 2011). however, there are few works on the sugar profile of edible flowers, e.g. rosa micrantha (guimarães et al., 2010). in experiment done with cut lily flowers was discussed the role of reducing sugars, as a typical reaction of plants that defend themselves against injury due to chilling or frost (van doorn and han, 2011). 4. conclusions the different cultivation treatments used in this work are differently correlated with the analyzed metabolites. in order to obtain flowers with high quality of brilliant color, one hour of supplementary lighting and a basal heating of 50°c seem to be the right combination of factors. the cold storage imposed to the flowers as the post­harvest treatment indicated that the flowers treated with additional 2 h of light (treatment n. 3) retained values of the metabolites during the post­harvest, with the excep­ tion of sugars. however, even if the additional light­ ing seems to preserve the flowers from depigmenta­ tion and to maintain the nutraceutical compounds, the decreased content of the observed sugars could be a consequence of the phenomenon of senes­ cence. further studies on the influence of illumina­ tion on plastic bags and the evaluation of ethylene production can be useful for the definition of the post­harvest process in v. cornuta. in addition, the investigation of the other minor nutritional compo­ nents can be crucial to define a more detailed condi­ tion of storage. acknowledgements this work was supported by a grant from european union in the frame of interreg alcotra v­a france­italy antea project n. 1139 ­ attività innovative per lo sviluppo della filiera del fiore edule/fleurs comestibles: innovations pour le devel­ opment d’une filière transfrontalière. references adams s.r., pearson s., hadley p., 1997 ­ the effects of temperature, photoperiod and light integral on the time to flowering of pansy cv. universal violet 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indonesia. key words: banana, breeding, genetic diversity, saline­stress, transgenesis. abstract: banana (musa spp.) is an essential fruit with high economic value worldwide. one of the main environmental constraints in banana cultivation is abiotic stress on marginal land caused by salinity stress. this salinity stress can significantly reduce banana productivity and even cause plant death. global climate change due to warming and reduced rainfall has increased the number of agricultural areas affected by salinity. this review article will describe current knowledge of biodiversity and various biological responses when banana plants are exposed to saline stress. this article also mentions the challenges in the banana breeding program with the current development of plant breeding and genetic engineering technology that could be used to create saline­tolerant banana accessions, its prospects, and the societal controversy underlying this technology. thus, the information presented in this article could be a reference in the saline­tolerant banana breeding program. 1. introduction bananas (musa spp.) are ancient monocotyledonous plants grown in nearly 120 countries in tropical and subtropical regions worldwide. moreover, banana is also a staple food and a major source of nutrition for more than 500 million people (wang et al., 2019). after rice, maize, and wheat, bananas are among the world’s most important food crops and the second most popular fruit. the worldwide production of bananas (musa spp.) totaled 113.9 million metric tons in 2017 (evans et al., 2020), highlighting its importance as a substantial crop in global diets and commerce. banana has a significant market share globally due to their broad appeal (de langhe et al., 2009; wang et al., 2021). it is rich in protein, vitamins, and minerals and is a premium export commodity in some countries (aquil et al., 2012; ji et al., 2021). banana consumption worldwide continues to increase year by year. consequently, the area of banana cultivation will expand as well. in order to support the growth of the increasing demand for bananas, marginal land has emerged as a viable option for agricultural land development for planting bananas (kusdianti et al., 2016). however, (*) corresponding author: erwi011@brin.co.id erwin.fraa@gmail.com citation: hasrianda e.f., setiarto r.h.b., davis l.o.m.m., poerba y.s., 2024 ­ future prospects and challenges in developing saline‐tolerant banana. ­ adv. hort. sci., 38(3): 297­310. orcid: efh: 0000­0002­9072­0305 rhbs: 0000­0001­6894­7119 lommd: 0000­0002­5465­7177 ysp: 0000­0002­1388­8745 copyright: © 2024 hasrianda e.f., setiarto r.h.b., davis l.o.m.m., poerba y.s. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 8 february 2024 accepted for publication 27 june 2024 ahs advances in horticultural science review paper ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15047 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-9072-0305 http://orcid.org//0000-0001-6894-7119 http://orcid.org/0000-0002-5465-7177 http://orcid.org/0000-0002-1388-8745 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 297­310 298 marginal land has relatively poor agricultural yield potential (dikayani et al., 2017). this land is widely accessible, but its agricultural potential remains largely untapped for agricultural activities. as an illustration, in indonesia, coastal agricultural areas often face salinity issues attributed to rising sea levels resulting from climate change. approximately 12.020 million hectares, or 6.20% of indonesia’s total land area, are estimated to be prone to salinity near the coast (karolinoerita and annisa, 2020). salinity is among the most critical abiotic stressors affecting plant development and productivity worldwide. salinity is a prominent concern among the abiotic stressors affecting farming (rao et al., 2016). salinity induces two types of stress: direct osmotic stress and delayed ion toxicity stress. they will affect plant development directly and indirectly. osmotic stress occurs when excessive salt concentration disrupts osmotic balance, causing a physiological drought and thus inhibiting plant water uptake (endris and mohammad, 2007; manojkumar et al., 2022). salinity problems can arise in coastline areas, dry environments on saline soil and from using low­ quality irrigation water. furthermore, an area with higher water evaporation rates in agricultural lands than rainfall also has a significant amount of land at risk of salinity stress. salinity stress can cause reduced growth, fruit yield, and quality, which can have significant economic impacts on banana producers. excessive salinity also increases salt content in the roots, reducing potassium absorption, slowing development, delaying flowering, and reducing crop production (rao et al. , 2016). developing banana plants with improved salinity tolerance is crucial to ensure food security, especially in regions prone to stress. fortunately, some plant accessions possess natural tolerance to environmental stressors, enabling them to withstand adverse conditions. this adaptation relies on the activation of intricate molecular networks responsible for stress detection, signal transduction, metabolite production, and the expression of stress­related genes. additionally, it is crucial to consider the interplay between signaling molecules to understand the synergistic or antagonistic interactions that influence plant responses to abiotic stressors. we can better understand plant performance under stress conditions by identifying the genes involved in stress tolerance, including those responsible for cellular protection and stress response regulation. this comprehensive understanding is vital for developing effective strategies to enhance plant tolerance to salinity (nguyen et al., 2018). to our knowledge, no national or global­scale banana breeding efforts have yet been executed to generate salt­tolerant banana cultivars successfully deployed for utilization by general farmers and the banana cultivation sector. this review explores the importance of banana biodiversity, genetic engineering technology advancements, challenges, and opportunities to improve banana salinity tolerance. it also highlights crucial aspects of banana breeding, identifies the unknown physiological and molecular processes underlying saline stress adaptation, evaluates the current status of banana genetic transformation technology, and discusses the potential application of newly discovered genome editing technology in banana breeding in saline stress adaptation. 2. saline stress response in plants and bananas soil water salinity generally restricts plant development by affecting the soil solution’s osmotic potential. it hinders plant water uptake and retards plant growth, which is commonly referred to as water deficit due to salinity. moreover, excess salt entering the plant system through transpiration force can produce ion toxicity and cell damage in plant organs, further reducing plant development (greenway and munns, 1980). at the cellular level, high salt concentration could also deactivate plants’ main cellular activities (cuartero et al. , 2006; anusuya and soorianathasundaram, 2014). because the osmotic pressure in the soil is higher than in plant cells, excessive soil salt levels can also trigger cell plasmolysis in plants. in addition, plant growth caused by excessive salinity has the following characteristics: (1) plants with low water potential, (2) na+ and cl­ ion toxicity, and (3) nutrient imbalance in plants (dikayani et al., 2017). next, salinity will also affect plant physiology, including co2 uptake, protein synthesis, respiration, and phytohormone cycles (mengel and kirkby, 1987; ravi et al., 2013; rao et al., 2016). firstly, saline stress reduced water availability, higher respiration rates, mineral distribution changes, membrane instability, and inability to maintain turgor pressure are some factors that hasrianda et al. ‐ genetic diversity for salinity adaptation 299 contribute to poor plant performance due to saline stress (cuartero et al. , 2006; anusuya and soorianathasundaram, 2014). secondly, ionic stress causes the accumulation of sodium chloride (nacl) ions in plant cells and tissues. ionic stress could result in toxicity and physiological disturbances. thus, salinity has an inhibitory effect on plant development due to the excess of na+ and cl­ ions toxicity towards water uptake as well (cherian and reddy, 2003; sharif et al., 2018; verma et al., 2019; manojkumar et al., 2022). generally, saline­tolerant plants can show one or a combination of processes, such as plant organ robustness, salt excretion, and osmotic adjustment, by producing solutes that can counteract ion accumulation in the tonoplast with more unsaturated fatty acids in the membrane composition (mengel and kirkby, 1987). additionally, plants perform tolerance­related responses such as protein stabilization, osmotic protection, reactive oxygen species (ros) detoxification, and ion homeostasis to adapt to a saline environment, enhancing plant durability (jacob et al., 2017; nguyen et al., 2018). furthermore, protein stabilization compounds for osmotic adjustment, such as proline (pro), trehalose (tre), and other soluble sugars, are excellent osmoprotectants that can reduce osmotic damage caused by salt pressure (türkan and demiral, 2009; deinlein et al., 2014). on the other hand, during saline stress, peroxidase (apx), superoxide dismutase (sod), peroxidase family protein (pod), thioredoxin family protein, and glutathione s‐transferase family protein (gst) were identified as differentially expressed genes (degs) in a plant. transcription factors (tfs) can function as gene activators, repressors, or both (ji et al., 2021). on top of that, saline tolerance is a complex characteristic involving multiple genes and various physiological and biochemical adaptive pathways (cuartero et al., 2006; anusuya and soorianathasundaram, 2014). it also implies that there are genetic and metabolic mechanisms that plants can use to adapt to saline stress and enhance their ability to better adapt to these environmental conditions. bananas are especially vulnerable to saline stress, as high salinity levels can significantly reduce banana yields (dikayani. et al., 2017). saline stress injuries manifest as leaf edge necrosis and then spread to the centre of the banana leaf. moreover, salinity stress also causes yellowing and decreased chlorophyll content and stability, followed by leaf drying, significantly affecting plant development. the impact of salt is then significantly reducing stem thickness and prolonged flowering period of vulnerable cultivars. due to lower photosynthesis activity in these leaves, harvest yields are reduced due to decreased banana finger weight which also produces low­quality commercial bunches and fails to develop the characteristic finger (fruit) physical characteristics of fruit, such as length, circumference, fruit flesh, skin weight, volume, and density (israeli et al., 1986; ravi et al., 2013; ravi et al., 2014; rao et al., 2016; ravi and vaganan, 2016). thus, salinity reduces banana production significantly. further research on the genetic mechanisms responsible for adaptation to saline environments is expected to facilitate breeding of high­yielding banana cultivars that can thrive in saline environments and meet market demand. 3. breeding for saline tolerant banana genetic diversity for salinity adaptation the potential tolerance sources should be identified before breeding stress tolerance bananas. thus, screening cultivars is the main approach to identifying genetic resources for plant breeding programs in the future (anusuya and fig. 1 ­ from left to right, pictures a to d show the conditions of the banana seedling phase with a plant height of ±40 cm exposed to salinity through hydroponic treatment. pictures e to h show the conditions of banana leaves exposed to a saline environment. starting from before the salt application treatment until the banana plant eventually dies. the plant wilts and dries; it starts from the older leaves to the younger ones, gradually from the leaf edges until eventually drying out completely in three months (author personal collection, unpublished). aadv. hort. sci., 2024 38(3): 297­310 300 soorianathasundaram, 2014). genetic resources for environmental saline stress tolerance could be derived at least from (1) the natural gene pool of banana accessions and their close relatives already present in the wild, (2) by promoting natural mutations in existing banana accessions using cis­ genesis methods, and (3) by incorporating genes from entirely different species using genetic engineering approaches (transgenesis). several types of banana accessions are known to be more tolerant of environmental stress than other accessions. these accessions have several genes related to their useful agronomic traits, including height, leaf angle, root spread, and resistance to abiotic and biotic stresses (heslop­harrison and schwarzacher, 2007). the b genome of bananas, for example, has long been the target of breeding programs because of the strong tolerance of bananas to abiotic stress (tripathi et al., 2019). therefore, genotyping and phenotyping exploration of the b banana genome, both accessions of cultivars and their wild relatives, should be considered as a way to find novel metabolisms or genes that can play a significant role in increasing the tolerance of banana cultivars to saline environments. previously, research has been carried out on the tolerance level to saline stress on various genotypes of banana cultivars. various banana cultivars such as cv. tongat (aa), cv. ney poovan (ab), cv. h 212 (ab), cv. grand naine (aaa), cv. karpooravalli (abb), cv. h 96/7 (abb), cv. poovan (aab), cv. rasthali (aab), cv. chakkaiya (abb), cv. fhia­1 (aaab), cv. fhia­23 (aaaa), and cv. fhia­17 (aaaa) which were tested with four salt concentrations, namely 0 mm nacl nacl, 150 mm nacl, and 200 mm nacl, in a research by anusuya and soorianathasundaram (2014). the experiment demonstrated significant variability in the tolerance response of bananas to salinity stress, whereas the banana accessions ‘fhia­1’ and ‘grand naine’ are very sensitive to the saline treatment given. meanwhile, the banana accessions ‘ney poovan’, ‘poovan’ and ‘karpooravalli’ appeared to be more tolerant compared to other banana cultivars in the study (anusuya and soorianathasundaram, 2014). this finding suggests that while bananas are generally susceptible to saline stress, unique molecular processes in the different banana accessions can be utilized in breeding programs to develop new, saline­tolerant banana cultivars. in short, genetic diversity is crucial for successful plant breeding (nandariyah et al., 2020) and in producing saline­tolerant bananas. in addition, rao et al. (2016) found that among the tested genotypes, ney poovan, poovan, and karpooravalli can be classified as tolerant bananas based on their capacity to develop and maintain tolerable physiological conditions even under concentrated saline stress levels of 200 mm. however, saline stress affected fruit growth in vulnerable banana cultivars, particularly in the cavendish group but not in the tolerant saba genotype. mature saba bananas grown in affected areas produced normal bunches (rao et al., 2016; ravi and vaganan, 2016). on the other hand, southeast asia is known as the origin of bananas (wang et al., 2021). this location also has the highest diversity of wild musa species (musa acuminata aa and musa balbisiana bb) found today, as well as the earliest domestication (simmonds, 1962; rao et al., 2016). however, testing and recording the saline tolerance traits of various cultivars and endemic wild banana accessions in southeast asia are not widely reported. research on the tolerance level of salinity stress in banana cultivars and wild species is expected to be used to identify banana accessions as donor genes for the salinity stress tolerance trait in bananas that have not been documented previously. essentially, the most significant cultivated banana type in the world is triploid, resulting from inter­ or intraspecific hybridization of two wild diploid species, musa acuminate (genome a) and musa balbisiana (genome b) (wang et al., 2019). as a result, most banana cultivars are sterile, and their genetic diversity is limited, making traditional breeding schemes challenging to implement for this plant. based on this, although candidate genes for saline tolerance in bananas have been identified, there will be further challenges in transferring these genes of interest through conventional breeding methods to existing industrial and consumer banana accessions. on the opposite, since the genetic engineering method avoids reproductive barriers and promotes the production of superior strains based on large cultivars, this may be a solid alternative to traditional banana breeding (heslop­harrison and schwarzacher, 2007). considering these conditions, the genetic engineering approach could be a reasonable option to produce saline­tolerant banana accessions for consumption and industry. an additional issue with cultivated banana plants is that they are propagated vegetatively, leading to a hasrianda et al. ‐ genetic diversity for salinity adaptation 301 decrease in genetic variation (lópez et al., 2017; hasim et al., 2021). however, improving plant traits can be achieved by increasing genetic diversity, including inducing mutations. induced mutations can enhance the genetics of vegetatively propagated plants (ghag and ganapathi, 2017; nandariyah et al., 2020). gamma radiation technology can be an option to increase the genetic diversity of bananas. gamma radiation mutagen is frequently used as a mutagenic agent to induce mutations (indrayanti et al., 2012). gamma rays are commonly used in mutation breeding because they can interact with atoms or molecules in cells with water to form free radicals. these radicals can damage or alter key components in plant cells, affecting plants’ morphology, anatomy, biochemistry, and physiological differentiation (datta et al. , 2018). gamma radiation is high­energy electromagnetic radiation produced by radioactive isotopes or nuclear reactors. when applied to plant tissue, it can affect the gene structure, increasing genetic variability in irradiated plants (astutik, 2009; khumaida et al., 2015; maharani et al., 2015). in another research, nandariyah et al. (2020) developed a gamma radiation protocol to generate genetic variability in bananas, which is one of the options that can be used. observations of the number of fruits per bunch on each cv. raja bulu banana plant that received gamma radiation treatment from the first to the third bunch showed significant differences. the study showed that gamma radiation treatment had a more significant effect on the average number of fruits per bunch, bunch weight, and fruit weight than the control. according to datta et al. (2018), gamma radiation with a dose of 10 gy changed the appearance of morphological varieties in the generative phase of the m1v1 raja bulubanana (nandariyah et al., 2020). in their study, nandariyah et al. (2020) successfully produced a new genotype of a banana with superior traits compared to its parent. however, this method also poses challenges that need to be considered. one of them is that gamma radiation can cause random mutations. it means that this method will require laborious phenotyping selection which could take considerable time to separate the desired mutant plants from the undesired ones. the random nature of this method also means that there is a possibility that the survived mutants from the experiment may ultimately fail to produce a better cultivar than their parent. screening methods for salinity stress tolerance in bananas screening for salinity tolerance plants has been conducted using various methodological approaches, including controlled artificial conditions such as in vitro culture, pot planting conditions, hydroponic media, greenhouse cultivation, and direct field planting. however, the latter approach is relatively challenging to control due to high environmental variability and difficulty fully controlling field conditions. tissue culture techniques have rapidly assessed the saline tolerance traits of different tested banana accessions on an in vitro scale. it provides a controlled environment where the effects of salinity can be evaluated by exposing banana explants or tissue cultures to different salt concentrations. currently, in vitro culture of bananas has been developed using various explants, such as shoot tips (matheka et al., 2019), immature male flowers (jalil et al., 2003), apical meristems (novak et al., 1989), bulbs (may et al., 1995), flower tops (liu et al., 2017), and embryogenic cell suspension (ecs) (vuylsteke and de langhe, 1985; sági et al., 1995; côte et al., 1996; cote et al., 2000). in addition, the development of in vitro musa acuminata l. is likely influenced by salt. the treatment in cv. barangan with nacl in 200 mm for plant height character was lower than the control. the height of banana plantlets decreased after the cultivation age in the 4th week. this study also showed that nacl treatment could simulate saline tolerance in banana plants in vitro (dikayani et al., 2017). this strategy can yield reliable data in a short period as salinity can significantly impact the growth of banana plantlets. the in vitro test approach allows for evaluating various specific physiological and biochemical responses to salinity stress, including changes in ion accumulation, osmotic adjustment, and antioxidant enzyme activity. morphologically, the impact of salinity is characterized by a brown coloration, particularly in the leaves, and dark brown coloration in the roots. this is attributed to the high concentration of na+ ions, which hinders the plant’s uptake of k+ ions. normal plant cells typically contain a higher concentration of k+ than na+. potassium ions are crucial in maintaining osmotic pressure in cells, regulating stomatal opening and closing, protein 302 adv. hort. sci., 2024 38(3): 297­310 synthesis, and functioning as enzymes such as pyruvate kinase. therefore, low k+ concentration in cells leads to chlorosis and necrosis (munns and termaat, 1986; hasegawa et al., 2000; mahajan and tuteja, 2005; dikayani et al., 2017). in a different research study using the in vitro assay technique, kusdianti et al. (2016) found that the activity of banana plantlets induced proline accumulation after exposure to saline stress. this study also found that saline stress affected the gene expression pattern of heat shock protein 81‐2 (hsp 81‐2) and delta‐1‐pyrroline‐5‐carboxylate synthase (p5cs1). in this experiment, shoot meristem musa acuminata cv. barangan was cultured in vitro with 25, 50, 75, and 100 mm nacl (kusdianti et al., 2016). thus, in vitro testing of saline­tolerant banana traits can be considered one of the most powerful research methods for screening saline tolerance cultivars. this method enables testing many samples in a relatively short time. on the other hand, hydroponic methods also offer another advantageous platform for studying saline stress in bananas. by growing banana plants in nutrient solutions with controlled salt concentrations, researchers can investigate the effects of different salinity levels on plant growth, physiological parameters, and gene expression profiles. thus, hydroponic­based screening enables the identification of key genes and pathways involved in saline tolerance, providing valuable insights into the molecular mechanisms underlying plant responses to saline stress. moreover, evaluating various banana genotypes under hydroponic conditions allows identifying potential candidates with more specific salinity tolerance levels. however, while in vitro and hydroponic testing offers controlled conditions, it must be complemented by direct testing in saline­affected fields to ensure accuracy. this is because both tests are conducted in controlled environments, simplifying the real­world complexity of abiotic­biotic interactions in a field. thus, field experiments with actual cultivation conditions are still essential to evaluate banana plants’ performance under saline stress accurately. nevertheless, the inherent environmental variability in field settings, including temperature fluctuations, rainfall patterns, and soil heterogeneity, poses challenges in obtaining consistent data. factors such as temperature fluctuations, rainfall patterns, and soil heterogeneity contribute to the complexity of field trials, making it difficult to assess the true genetic potential and performance of banana genotypes under saline stress. based on the aforementioned points, screening banana genotypes for salinity tolerance necessitates a combination of controlled artificial conditions and realistic evaluations in field settings. in vitro culture and hydroponic systems provide controlled environments where physiological and molecular responses to saline stress can be studied in detail. on the other hand, field evaluations offer insights into the performance of banana plants under actual cultivation conditions. ultimately, the breeding outcomes of bananas will be grown by farmers in agricultural fields. therefore, in the end, banana accessions selected through rapid in vitro testing still need to be validated through multi­location field trials. integrating these approaches, robust experimental design, and statistical analysis will contribute to developing saline­tolerant banana varieties through combined plant breeding programs and precise molecular analyses. 4. saline­tolerant genes in banana potential saline‐tolerant genes in banana various genes in multiple plant species have been discovered to enhance plant tolerance to abiotic stress. these include the over production of enzymes involved in metabolite synthesis, membrane lipid biosynthesis, antioxidant defense, protective proteins, and transporters (yang et al., 2018; zheng et al., 2018). genes like proline dehydrogenase (prodh) and 1‐pyrroline‐5‐carboxylate synthetase (p5cs) regulate proline synthesis and contribute to stress response (villao et al., 2021). up­regulation of osbzip23 (dey et al., 2016) and osnac6 (nakashima et al., 2007) in transgenic rice increases salt and drought tolerance, while snac1 and snac2 enhance cold, salt, and drought tolerance (an et al., 2018). the expression of protein kinases such as snrk2, cipk, and cdpk has also been shown to improve salt, cold, and drought tolerancein various crops (saijo et al., 2000; boudsocq and lauriere, 2005; xiang et al., 2007; zhang et al., 2016; fedorowicz­strońska et al., 2017). the mitogen‐activated protein kinase (mapk) cascade (boudsocq and lauriere, 2005) and protein phosphatase 1a (ospp1a) (liao et al., 2016) are involved in stress response pathways. identifying key regulators and utilizing them for stress acclimation is hasrianda et al. ‐ genetic diversity for salinity adaptation 303 an important research focus. such findings can be valuable references for identifying potential gene donors in breeding programs to enhance abiotic stress tolerance in bananas. various attempts have also already been made to make bananas more tolerant of saline stress. among them is to look for genes that play a significant role in making plants able to tolerate the effects of environmental salinity. the search for this target gene can be carried out using the rna­seq method, as done by ji et al. (2021). they use the rna­seq approach to compare transcriptome changes in banana roots exposed to salt (60 mm nacl) with those grown under normal conditions. after 12 and 24 hours of salt treatment, 1466 and 2089 degs were found, consisting of 542 up­regulated and 924 down­ regulated genes, 507 up­regulated and 1582 down­ regulated genes, respectively. stress signalling transduction, reactive oxygen species (ros) scavenging, osmoregulation, and tfs are all elements of the saline stress response. a total of 3,355 degs were classified into 40 functional groupings, with biological processes (15 subcategories), cellular components (10 subcategories), and molecular functions (7 subcategories) being the most common (ji et al., 2021). in that research, hsp 81‐2 and p5cs1 genes were expressed at varying levels in all banana plantlets. with 75 mmnacl treatment, hsp 81‐2 was most abundant in the shoots and roots of the plants. similarly, this treatment resulted in the most significant accumulation of proline. the expression of the hsp 81‐2 gene was greater than the p5cs1 gene in all roots and shoots given nacl treatment. they concluded that musa acuminata cv.barangan has a defense mechanism against salt (kusdianti et al., 2016). proline accumulation by plantlets given nacl and expression of the hsp 81‐2 and p5cs1 genes was found to vary among all plantlets, and maximum hsp 81‐2 was expressed by the shoots and roots of plants given 75 mmnacl treatment (kusdianti et al., 2016). in general, gene findings using genetic factors and molecular methods are rapidly growing research activity and widely used in the past decade. this method allows for discovering various genes that contribute to a specific trait. thus, understanding genetic and molecular mechanisms underlying the trait can be more comprehensive. confirming the potential saline‐tolerant gene our current understanding of saline stress perception, signaling pathways, and downstream fig. 2 ­ phases of breeding salt­tolerant banana plants. these phases initiates with identifying the negative effects of saline stress on banana plants. the next steps entail exploring genetic diversity for salt stress tolerance within the existing gene pool and devel­ oping an appropriate breeding scheme. these steps aim to produce new banana genotype as potential candidates for salt­resis­ tant banana cultivar. adv. hort. sci., 2024 38(3): 297­310 304 factors is primarily based on research on the model plant arabidopsis thaliana. it has yet to be widely applied to other crops. to effectively enhance saline tolerance in different plant species, it is crucial to identify and understand the specific growth adaptations to salt that benefit each species. even in arabidopsis, saline stress’s early cellular perception and signaling are still obscure. during saline stress, intracellular ca2 + spikes and wave signals seem to be a convergent sensory mechanism through which the cell perceives high salinity. it triggers a signaling pathway involving the salt overly sensitive (sos) proteins, which regulate sodium concentration in the cytosol. the sos pathway comprises sos1, sos2, and sos3 and is induced by saline stress. upon binding with ca2 +, sos2 is activated and forms a complex with sos3, which then phosphorylates the h+/cation antiporter sos1, excluding sodium from the cell. this mechanism has been reviewed in detail in 2020 (van zelm et al., 2020). functional genetic studies aimed at elucidating the function of banana genes in saline stress are even more scarce. table 1 lists known validated factors in the body of current literature. two transcription factors from the banana cv. karibale monthan (abb), musa nac04 (tak et al., 2017), and musa wrky71 (shekhawat et al., 2011 a) have been identified to be involved in the control of the saline stress response. wrky71 expression is elevated by many stressors, such as cold, dehydration, salt, and other hormones, while musa nac04 is activated by salt treatment. nac04 or wrky71 overexpression in transgenic bananas has been associated with increased saline tolerance. it is indicated by sustained chlorophyll content, lower oxidative damage, and a better fv/fm ratio than wild­type (tak et al., 2017). moreover, the induction of putative gene expression related to abiotic stress responses in wrky71 overexpression lines upon salt treatment suggests that wrky71 is involved in the up­regulation of down stream stress response genes in bananas (shekhawat et al., 2011 a). aquaporins are among the other factors that have been validated to be involved in banana saline tolerance. aquaporins are water channels that promote symplastic water movement. the banana aquaporin gene, matip1;2, is known to be expressed in the root, stem, and leaves and is strongly upregulated by salt and drought conditions. transgenic arabidopsis overexpressing mapip1;1 is more tolerant to saline stress. similarly, over expression of musapip2;6 in transgenic bananas resulted in higher saline tolerance than that of the wild type. a protection mechanism from osmotic shock through up­regulation of banana dehydrin1 was suggested in a study by shekhawat et al. (shekhawat et al., 2011 b). banana dehydrin1, a type of late embryo abundance (lea) protein encoded by the musadhn‐1 gene, was induced by drought, cold, and high salt (shekhawat et al., 2011 b). over expression of musadhn1 leads to enhanced banana tolerance via accumulation of proline and reduced malondialdehyde levels upon drought and saline stress (shekhawat et al., 2011 b). nevertheless, various genetic profiling studies recorded in bananas are generally held on cultivated bananas. exploration and research of genetic profiling of saline­tolerant traits in wild banana relatives in their center of diversity in southeast asia can be expected to provide information and potential tolerance traits that are not yet known. this is partly due to wild bananas undergoing more complex adaptation processes in nature than those cultivated on agricultural land. this diversity of traits is expected to enrich the genetic and molecular mechanism toolbox that can be applied to cultivated bananas. therefore, it can provide new possibilities in breeding programs to produce cultivated bananas tolerant to saline environments. the prospect of genetic transformation technology in banana breeding natural selection and conventional banana crossing have been the most commonly used breeding methods worldwide. this is based on considerations of its simplicity and not requiring sophisticated instruments in their implementation. however, conventional breeding is based on natural genetic variation, and an extensive backcross program is required to add desirable characteristics to elite plants. this has led to the use of this method in banana breeding being considered laborious and time­consuming. moreover, the availability of favorable alleles or genetic variation in nature is limited to exploit this approach (manshardt, 2004). meanwhile, breeding through random mutagenesis (physical, chemical, or biological mutations) can result in mutations of many traits and undesirable changes. breeding these mutations should also be followed by screening huge and time­ 30 5 ta bl e 1 ­ li st o f g en es w ith v al id at ed fu nc tio ns r el at ed t o sa lin e st re ss g en e pr ot ei n ro le o rig in o ga ni sm ex pr es sio n nd uc ed b y ex pr es sio n pa tt er n fu nc tio na l g en et ic te st in g pl an t ph en ot yp es m ol ec ul ar m od e of a ct io n pu bl ic at io n( s) m ar o p5 g rh o­ lik e g tp as es si gn al in g fe nj ia o an d ba xi ji ao b an an a m ai nl y sa lt, b ut a lso c ol d an d dr ou gh t pr ot ei n lo ca liz ed at th e pl as m a m em br an e o ve re xp re ss ed in a. th al ia na lo ng er p rim ar y ro ot s an d in cr ea se d su rv iv al ra te s i n re sp on se to sa lt st re ss th e in cr ea se d sa lt to le ra nc ec on fe rr ed by m ar o p5 gg en e m ig ht b e re la te d to re du ce d m em br an e in ju ry a nd th e in cr ea se d cy to so lic k +/ n a+ ra tio a nd c a2 + co nc en tr at io n in th e tr an sg en ic p la nt s (m ia o et a l., 2 01 8) m us ad hn ‐1 n ov el b an an a sk 3­ ty pe d eh y­ dr in , h ig hl y hy dr op hi lic p ro ­ te in s ( le a fa m ily pr ot ei n) ? pr ot ec tin g ce lls fr om o sm ot ic sh oc k ba na na c v. ka rib al e m on th an dr ou gh t, sa lin ity , c ol d, ox id at iv e an d he av y m et al st re ss , a s w el l a s by tr ea tm en t w ith sig na lli ng m ol ec ul es li ke ab sc isi c ac id , e th yl en e an d m et hy l j as m on at e le av es o ve re xp re ss ed in ba na na c v. r as ta li im pr ov ed to le ra nc et o d ro ug ht a nd sa lt­ st re ss tr ea tm en ts in b ot h in v itr o an d ex ‐v itr o as sa ys en ha nc ed a cc um ul at io n of p ro lin e an d re du ce d m al on di al de hy de le ve ls in d ro ug ht a nd sa lt­ st re ss ed (s he kh aw at e t a l., 20 11 b ) m at ip 1; 2 pr om ot er aq ua po rin w at er c ha nn el s in sy m pl as tic pa th w ay s m us a a cu m in at a l. aa a gr ou p cv . b ra zil ia n dr ou gh t a nd sa lt ro ot s, st em s, le av es , flo w er s a nd fr ui ts . hi gh es t i n le av es te st ed in a. th al ia na re po rt er re sp on ds to sa lt an d dr ou gh t s tr es s re po rt er re sp on ds (s on g et a l., 2 01 8) m us an ac 04 2 si gn al in g tr an sc rip tio n fa ct or cv . k ar ib al e m on th an in du ce d by sa lt le av es tr an sg en ic m us a a cu m in at e l. cv . r as th al i tr an sg en ic p la nt s r et ai ne d hi gh er le ve ls of to ta l c hl or op hy ll an d lo w er le ve ls of m da c on te nt in re sp on se to sa lt st re ss . t ra ns ge ni c pl an ts h av e hi gh er p ro lin e co nt en t an d be tt er f v/ fm ra tio m od ul at in g th e ex pr es sio n o f c bf /d re b, l ea , an d w rk y (t ak e t a l., 2 01 7) m us ap ip 2; 6 aq ua po rin w at er c ha nn el s in sy m pl as tic pa th w ay s cv . k ar ib al e m on th an in du ce d by sa lt an d dr ou gh t le av es o ve re xp re ss ed a nd u sin g in du ci bl e co ns tr uc t tr an sg en ic b an an a (c v. r as th al i) tr an sg en ic p la nt h as h ig he r s al t to le ra nc e be tt er f v/ fm , l ow er m da (s re ed ha ra n et a l., 2 01 5) m ap ip 2; 7 aq ua po rin w at er c ha nn el pr ot ei n fu nc tio n in sy m pl as tic pa th w ay s m us a ac um in at a l. aa a gr ou p, cv . b ra zil ia n u p­ re gu la te d af te r os m ot ic (m an ni to l), co ld , a nd sa lt tr ea tm en ts n o da ta g on gj ia o ba na na (m us a ac um in at a l. a a gr ou p, c v. m as ) o ve re xp re ss io n of m ap ip 2‐ 7 in b an an as en ha nc ed to le ra nc et o dr ou gh t, co ld , a nd sa lt lo w er le ve ls of m da a nd io n le ak ag e, bu t h ig he r c on te nt s o f c hl or op hy ll, p ro lin e, so lu bl e su ga r, an d ab sc isi c a ci d (a ba ) c om pa re d w ith w ild ­t yp e (w t) p la nt s u nd er st re ss a nd re co ve ry c on di tio ns (x u et a l., 2 02 0) m ap ip 1; 1 aq ua po rin w at er c ha nn el in sy m pl as tic pa th w ay s m us a ac um in at a l. aa a gr ou p, cv . b ra zil ia n in du ce d by sa lt an d dr ou gh t ro ot . l oc al ize d in p la sm a m em br an e ar ab id op sis th al ia na o ve re xp re ss io n of m ap ip 1; 1 in ar ab id op sis re su lte d in in cr ea se d pr im ar y ro ot e lo ng at io n, ro ot h ai r nu m be rs a nd su rv iv al ra te s c om ­ pa re d to w t un de r s al t o r d ro ug ht co nd iti on s re du ce d m em br an e in ju ry an d hi gh c yt os ol ic k+ /n a+ ra tio (x u et a l., 2 01 4, 20 21 ) m us aw rk y7 1 tr an sc rip tio n fa ct or m us a ac um in at a l. cv . k ar ib al e m on th an (a bb ) u p­ re gu la te d by c ol d, de hy dr at io n, sa lt, a ba , h 2o 2, e th yl en e, sa lic yl ic ac id a nd m et hy l ja sm on at e lo ca liz ed in nu cl eu s m us a ac um in at a l. cv . r as th al i su sp en sio n cu ltu re c el ls o ve re xp re ss ed li ne e xh ib ite d up re gu la tio n of p ut at iv e ge ne s re la te d to a bi ot ic st re ss re sp on se s tr an sc rip tio na l u pr eg ul at io n o f s tr es s r es po ns e ge ne s (s he kh aw at e t a l., 20 11 a ) ha sr ia nd a et a l. ‐ g en et ic d iv er sit y fo r s al in ity a da pt at io n adv. hort. sci., 2024 38(3): 297­310 306 consuming populations to identify mutants with the desired traits (mccallum et al., 2000). breeding mutations usually have a shallow frequency (0.1% of total mutations). in contrast, crossbreeding using markers is often very expensive, and linking markers to the desired trait is sometimes very difficult and time­consuming. this method will also produce products requiring complex regulatory processes, time­consuming requirements, and expensive safety analyses (lusser et al., 2012). moreover, most banana consumption cultivars are triploid. thus, the sterility and polyploidy of bananas greatly inhibit conventional or molecular breeding from providing a practical and effective way for genetic improvement (tenkouano et al., 2011). in order to overcome this problem, transgenic bananas achieved through genetic engineering techniques might be a reasonable way to produce bananas with saline tolerance traits. in addition, genetic transformation technology also allows the addition or insertion of genes from groups of l iving things that are phylogenetically distant or sexually incompatible. in the modern genetic engineering approach using agrobacterium plasmid, tissue culture methods are generally needed in the implementation stage. usually, in vitro bananas could be genetically modified using particle bombardment or agrobacterium­mediated transformation (may et al., 1995; sági et al., 1995; wang et al., 2021). it is also worth noting that the approach using agrobacterium tumefaciens is one of the most widely used due to its ease of use and ability to be applied to various plant tissues (villao et al., 2021). in addition, regeneration in the genetic modification of bananas is often carried out by organogenesis based on meristematic tissue or somatic embryogenesis based on ecs (novak et al., 1989; may et al., 1995). because of its rapid regeneration rate, meristematic tissue from shoot tips and flower buds are usually used as explants for cloning and genetic transformation (may et al., 1995; liu et al., 2017; wang et al., 2021). additionally, scientists commonly prefer to use plant materials for the genetic transformation process using the agrobacterium­mediated transformation technique in somatic embryos, callus, and protoplast cultures (villao et al., 2021). development of saline‐tolerance banana through transgenesis and gene editing perhaps the most known successful transgenesis in bananas so far is transgenic cavendish banana using agrobacterium tumefaciens from dale et al. (2017). normally, cavendish bananas are highly susceptible to tr4, a destructive fungal disease that poses a significant threat to plantations worldwide. currently, there is no known effective resistant replacement for cavendish banana. the researchers conducted a 3­year field trial using transgenic banana lines transformed with genes rga2 and ced9 in this experiment. both lines remained disease­free, indicating resistance to tr4. this study demonstrates how transgenic and gene editing approaches can practically enhance banana cultivars to overcome environmental challenges through single gene transformation (dale et al., 2017). in addition, just recently, in 2021, one experiment by villao et al. (2021) successfully developed a genetic transformation protocol from banana apical meristem for the cv. williams (genotype aaa). this protocol was based on the co­cultivation of explants (whole in vitro plants or meristematic tissue divided in two from in vitro plants) with agrobacterium tumefaciens separately. as an illustration of the timeline required to implement this protocol, villao et al. also mentioned that after embryogenic cell suspension transformation mediated by agrobacterium, it would take six to twelve months to obtain transgenic banana plants in vitro (villao et al., 2021). in addition, the maturation of embryogenic cell suspensions can take eight to twelve months (tenkouano et al., 2011). the technique designed in the dale et al . and villao et al. experiments, mentioned above, could aid in genetically transforming various banana cultivars. that approach could also be considered to produce transgenic banana tolerant. recently, genome editing in plant breeding has been on the rise due to the development of crispr/cas9 technology. this is partly because the crispr/cas9 system allows for various methods of genome editing, depending on the repair route and the availability of repair templates (bortesi and fischer, 2015), contributing to its precision. as a result, with the open­access publication of the banana genome and the development of viable genetic transformation methods, gene editing systems such as host­induced gene silencing (higs) and crispr have been increasingly used to mutate specific genes, resulting in the creation of mutant plants without the need for foreign gene insertion (tripathi et al., 2019; wang and chen, 2020; wang et al., 2021). hence, crispr technology has emerged as hasrianda et al. ‐ genetic diversity for salinity adaptation 307 a promising option in genetic transformation programs to produce banana cultivars that are tolerantto saline environments. by considering this factor, the development of genetic engineering technology for producing saline­tolerantbanana cultivars can progress more smoothly than before. although there have not been many massive genetic transformation programs to produce banana cultivars that can tolerate and thrive in saline environments, scientists have successfully created saline­tolerantcultivars of other crops using genetic transformation. the information from these studies is becoming more relevant as various molecular mechanisms involved in the process are also known to occur in banana tissues. this information can help study genetic transformation techniques and their potential application in banana cultivars in the near future. 5. concluding remarks and future prospect bananas are popular worldwide, but abiotic stress factors often limit their production. one such stressor is salinity, exacerbated by global warming, particularly in tropical regions like southeast asia with extensive coastlines. as the center of banana diversity, southeast asia has abundant genetic variability in wild and local bananas. this biodiversity richness can be utilized further to address the various challenges in banana cultivation. additionally, countries experiencing higher water evaporation rates in agricultural lands than rainfall face significant risks from salinity stress. however, developing elite cultivars tolerant to salinity stress presents a unique challenge, mainly since elite cultivar bananas are typically seedless and triploid or parthenocarpic. various breeding and genetic engineering techniques can be employed to overcome these challenges. by using these techniques, it is possible to produce elite banana cultivars that are more tolerant to saline stress, have high yields, and are preferred by consumers in a shorter period. therefore, continued efforts towards developing improved banana cultivars for salinity tolerance are necessary for the sustainable production of this vital crop. the following are some essential topics related to banana breeding for saline tolerance that may be of significant research interest in producing an elite cultivar of saline­tolerant banana. firstly, utilizing wild banana genetic resources alone or combined with mutation can enhance genetic variability for screening saline tolerance. additionally, translational studies in model plants and omics approaches like genomics, transcriptomics, and metabolomics can uncover biological response and tolerance genes in bananas. investigating the physiological and biochemical mechanisms underlying saline tolerance, such as ion transporters, osmoprotectants, and antioxidant enzymes, provides insights into the molecular basis of saline tolerance in bananas. furthermore, improving micropropagation, embryogeniccell induction, and regeneration techniques facilitate genetic transformation and gene editing in diverse elite banana cultivars. additionally, marker­assisted selection strategies integrated with molecular markers linked to saline tolerance traits can expedite the screening and selection of saline­ tolerant genotypes. finally, integrating traditional breeding approaches with biotechnological tools like marker­assisted backcrossing and genome editing can accelerate breeding progress and the development of this field. as for the safety concerns surrounding the use of genetically modified crops, including transgenic bananas, this will still be a controversial topic for the foreseeable future. it is crucial for all stakeholders, including scientists and governments, to apply the precautionary principle when dealing with gmo products and technology. the emergence of gene editing approaches such as crispr/cas9 offers an alternative approach to conventional transgenesis, which may change the acceptance of genetically modified products in the coming years. acknowledgements the authors would like to sincerely thank the research organization for life sciences and environment, national research and innovation agency (brin) indonesia, for the financial 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de farmácia, laboratório de farmacognosia ii, universidade federal do maranhão, cidade universitária do bacanga, avenida dos portugueses, 1966, 65080‐805 são luís, maranhão, brazil. 4 laboratório de imunofisiologia, universidade federal do maranhão, cidade universitária do bacanga, avenida dos portugueses, 1966, 65080‐805 são luís, maranhão, brazil. 5 laboratory of research, development and innovation, apis flora indl. coml. ltda., rua triunfo, 945, santa cruz, 14020‐670 ribeirão preto, são paulo, brazil. 6 departamento de biologia celular, instituto de ciências biológicas, universidade de brasília, campus universitário darcy ribeiro, bloco e s/n 1° andar ‐ asa norte, 70910‐900, brasília, distrito federal, brazil. key words: antimicrobial, botanical pesticides, food security, plant diseases, pomegranate peel, punicalagin. abstract: the study aimed to investigate antimicrobial activity of the hydroal­ coholic crude extract from the fruit peel of punica granatum (pp) and punicala­ gin compound (pg) on phytopathogenic bacterial isolates and its potential use as a sustainable alternative in treatment of vegetable seeds. the antimicrobial activity in vitro was tested by agar well diffusion assay and through viability tests in liquid medium. in vivo treatment with pp was tested on eruca vesicaria seeds infected with xanthomonas campestris pv. campestris. pp induced the formation of large inhibition zones to the growth of the tested pathogens (35.33 mm ­ 6.66 mm), with dose­dependent effect. viability tests confirmed the antimicrobial activity of the pp on x. campestris pv. campestris and p. caro‐ tovorum subsp. carotovorum with minimum inhibitory concentration (mic) of 125 μg/ml. punicalagin compound presented mic of the 31.25 μg/ml. the seed treatment with pp indicated control of pathogen­induced symptoms in seedlings of the e. vesicaria and positive effect in seed germination, emergence and in stomatal functionality. the results indicate strong potential of the extract from the fruit peel of p. granatum and punicalagin for formulating botanical pesticides for plant disease control. (*) corresponding author: macielmcg@gmail.com citation: de sousa silva s., alves p.c.s., coutinho d.f., luz t.r.s.a., fontoura g.m.g., berretta a.a., gonçalves maciel m.c., 2021 ­ punica grana‐ tum l. extract contributes to phytopathogens control and enhances eruca vesicaria (l.) cav. germination in vitro and in vivo. ­ adv. hort. sci., 35(3): 217­231. copyright: © 2021 de sousa silva s., alves p.c.s., coutinho d.f., luz t.r.s.a., fontoura g.m.g., berretta a.a., gonçalves maciel m.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 march 2021 accepted for publication 26 may 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10706 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 217­231 218 1. introduction according to food and agriculture organization of the united nations estimates, by 2050 the world population should exceed 9.5 billion inhabitants, rais­ ing the demand for food by up to 60% (fao, 2016). plants account for 80% of food ingested in the human diet, providing affordable, safe and nutritious resources for a healthy life. however, pests and dis­ eases pose a threat to food security, due to damage caused to crops that compromises access to food and rises product prices (fao, 2017). phytopathogenic bacteria causes a large number of different plant diseases, some of which are devas­ tating to agricultural crops (van der wolf and de boer, 2015). ralstonia solanacearum (smith) (yabuuchi et al., 1995) stands out as one of most destructive pathogens due to the rapid development of wilting symptoms and death of host plants (yuliar et al., 2015). the pathogen affects a large range of host plants, comprising almost 450 species from 54 different botanical families (allen et al., 2005). direct losses in important crops are estimated in 0 to 91% (tomatoes) and in 33 to 90% in potatoes (elphinstone, 2005). the bacterium xanthomonas campestris pv. campestris (pammel) dowson is a vas­ cular (ryan et al., 2011) and seed­born (griesbach et al., 2003) pathogen which is distributed worldwide. infested seeds may emerge in young seedlings infect­ ed by the pores on the margin of the cotyledons. this pathogen causes black rot disease, which seriously affects brassicaceae (cruciferous) crops (vicente and holub, 2013), important food items grown worldwide (gupta et al., 2013). finally, pectobacterium is widely studied soft­rot bacterial pathogen causing infections in potato crops and stored tubers, reducing the pro­ duction and quality of tubers (adeolu et al., 2016). ralstonia solanacearum, x. campestris and p. caro‐ tovorum have been included among the 10 most important bacterial pathogens of the plants accord­ ing to their economic and scientific impact (mansfield et al., 2012). control of bacterial diseases in conventional agri­ culture often uses fast­acting synthetic pesticides and antimicrobials (kotan et al., 2014). according to national phytosanitary pesticide database (agrofit, 2016), substances unsafe to the environment like kasugamycin, cuprous oxide, copper hydroxide and ‘extremely toxics’ like benzalkonium chloride were registered for control of soft rot p. carotovorum subsp. carotovorum in potatoes. pesticides indicated for control of bacterial wilt caused by r. solanacearum like bismerthiazol and thiodiazole cop­ per have shown low efficacy, high phytotoxicity, harmful environmental effects and bacterial resis­ tance development (yang and bao, 2017). cultural practices like the use of pathogen­free seeds is recommended to prevent black rot disease in crops (chitarra et al., 2002). if pathogen­free seed is not available, seed should be treated to eliminate the bacteria. however, seed treatments do not always eliminate 100% of bacteria on or in the seed, and may adversely affect seed germination and vigor (celetti and callow, 2002). the need to reduce chemical pesticide use in crops, associated with demands for healthy food and development of sustainable agriculture, has driven research for natural compounds with low impact on the environment and on people health (jiménez­ reyes et al., 2019). the secondary metabolism of the plants produces many bioactive compounds that pro­ vide protection against pests and pathogens (borges et al., 2018). unlike synthetic pesticides, natural com­ pounds exhibit rapid biodegradation after use in the field (soberón et al., 2014), little or no phytotoxicity, abundant sources and low costs, since they come from a renewable source (zheng et al., 2016). thus, medicinal plant uses with antimicrobial activity can be considered an effective component in the inte­ grated management against phytopathogens (khan et al., 2020). punica granatum l. (pomegranate) is a plant of the lythraceae family, native from central asia (northern india to iran), nowadays cultivated in sever­ al parts of the world, including africa and america (viuda­martos et al., 2010; erkan and dogan, 2018). the fruit of pomegranate (called balausta) is a pulp berry formed by a thick and leathery skin with vari­ able color depending on the variety. the seeds are a reproductive structure that present a fleshy outer testa called sarcotesta where the juice is extracted (melgarejo et al., 2020). the production, marketing and consumption of pomegranate fruit have increased rapidly throughout the world in recent years, mainly due to greater awareness of their health­promoting attributes (selcuk and erkan, 2015). the peel of p. granatum represents 30 to 40% of the fruit, being usually discarded as waste during industrial processing for the production of pome­ granate juice (gullon et al., 2016). however, this part of the fruit is rich in phenolic acids, tannins (such as punicalin and punicalagin) and flavonoids with vari­ de sousa silva et al. ‐ punica granatum l. extract acts in phytopathogen control and eruca vesicaria (l.) cav. germination 219 ous biological functions, including activity against pathogenic microorganisms (dey et al., 2012; türkyılmaz et al., 2013). punicalagin compound is an important bioactive agent found in pomegranate fruit peel, with antioxidant, antimicrobial, antiviral and immunosuppressive activity. the compound belongs to the ellagitannin family which includes other tannins such as punicalin and gallic acid, char­ acterized by good water solubility (akhtar et al., 2015). in the last years several studies have evidenced the antimicrobial activity of the pomegranate extract against many species of the plant pathogenic fungi suggesting high potential source of natural antifungal agents (mohamad and khalil, 2015; balah and nowra, 2016; elsherbiny et al., 2016; li destri nicosia et al., 2016; rongai et al., 2017; karm, 2019; el khetabi et al., 2020). however, few studies have investigated the antimicrobial activity of the pome­ granate extract against phytopathogenic bacteria (quattrucci et al., 2013; farag et al., 2015; khaleel et al., 2016). khaleel et al. (2016) have indicated in vitro antimicrobial activity of the ethyl acetate pomegran­ ate peel extract against r. solanacearum, p. caro‐ tovorum subsp. carotovorum and x. gardineri and farag et al. (2015) highlighted notable in vitro antimi­ crobial activities from the methanol pomegranate peel extract against variety of temperate climate (race 3, biovar 2) of the r. solanacearum. despite this, the knowledge about the antimicrobial activity of the hidroalcoholic extract of p. granantum and iso­ late compounds against these pathogens is scarce. thus, this study aimed to investigate the in vitro antimicrobial activity of hydroalcoholic crude extract from the fruit peel of the p. granatum and punicalagin compound on isolates of phytopathogen­ ic bacteria. the potential of the extract for natural control of x. campestris pv. campestris as an sustain­ able alternative for treatment of vegetable seeds was assayed. 2. materials and methods bacterial isolates the bacterial isolates were provided by the collec­ tion of the são paulo biological institute ­ são paulo, brazil (x. campestris pv. campestris, isolate no. xcc2149) and rosa mariano culture collection of the federal rural university of pernambuco, brazil (r. solanacearum, isolate no. ccrmrs187, race 3, biovar 1; and p. carotovorum subsp. carotovorum, isolate no. ccrmpcc36). agar medium of 523 kado & hesket was used as a culture medium for maintenance of the microbial cultures before antimicrobial tests. hydroalcoholic crude extract from the fruit peel of the p. granatum and pure compound the hydroalcoholic crude extract from the fruit peel of the p. granatum (pp) was supplied by apis flora®. the hydroalcoholic crude extract was concen­ trated under low pressure, dried and the remainder was later lyophilized. for the experiments, the lyophilized dry residue was diluted in an isotonic phosphate buffered saline (pbs). the pp was concen­ trated under reduced pressure. pure punicalagin (pg) compound was purchased from sigma­aldrich brazil ltda (p0023, 1 mg, batch: wxbc5016v), ≥98% (hplc), of pomegranate, c48h28o30, molecular weight of 1084.72. chemical characterization of the hydroalcoholic crude extract from the fruit peel of the p. granatum total phenolic compounds. for determination of total phenolics an analytical curve of tannic acid (sigma­aldrich) was carried out. pomegranate extract was prepared in 50 ml volumetric flask using water as solvent. the samples were homogenized and, the flasks were brought to the ultrasonic bath for 30 min­ utes. a 0.5 ml aliquot was transferred to another 50 ml flask where 2.5 ml of folin­denis reagent and 5.0 ml of 29% sodium carbonate were added. the sam­ ples were protected from the light and the readings were performed after 30 minutes in a uv­vis spec­ trophotometer at 760 nm (fernandes et al., 2018). all samples were analyzed in triplicate. ellagic acid. ellagic acid (ea) was acquired from fluka (95.0%, batch bcbn4398v). the high­ performance liquid chromatography (hplc) grade methanol was supplied by j.t. baker (mexico city, mexico), and purified water was obtained using a milli­q direct q­5 filter system (millipore, bedford, usa). the analytical grade acetic acid was purchased from synth (labsynth, diadema, brazil). to determine the ellagic acid content (eac), the extracts previously diluted in methanol were proper­ ly homogenate using a vortex and then remained for 30 minutes in ultrasound bath. the solution was fil­ tered and subjected to hplc analysis (shimadzu apparatus equipped with a cbm controller, lc­20at quaternary pump, a spd­m 20a diode­array detector and auto sampler, shimadzu lc solution software, adv. hort. sci., 2021 35(3): 217­231 220 version 1.21 sp1) using a 100 mm x 2.6 mm shim pack ods c18 column. the mobile phase used for ellagic acid was methanol and acetic acid aqueous solution 2% using a elution gradient (0­7 min, 20­72.5% v/v methanol, 7­7.5 min, 72.5­95% v/v methanol, 7.5­8.5 min. 95% v/v methanol, 8.5­9 min 95­20% v/v methanol, 9­10 min 20% v/v methanol) with a flow rate of 1.0 ml min−1, and oven temperature of 25°c. the eluted samples were detected by uv detector at 254 nm. calibration curve was constructed by plotting the peak area (y) against concentration in μg ml−1 of standard solutions (x). the standard equation obtained from the curve was used for quantification of ellagic acid as mg/g extract of sample. all assays were carried out in triplicates and the ellagic acid quantification was reported. determination of antimicrobial activity the pp was assayed for antibacterial activity at different concentrations using a standard agar­well diffusion assay (clsi, 2012). suspensions of bacteria strains (1.5 x 108 cfu/ml) were spread using swabs over the 523 kado & hesket agar media in sterilized petri dishes. then, wells with a diameter of 6 mm were punched aseptically and 25 μl of pp at different concentrations were introduced into each well (100, 50, 25, 12.5, 6.25 and 3.125 mg/ml, solubilized in an isotonic phosphate buffered saline (pbs). all plates were incubated at 28°c for 48 hours. measures of the zones around the wells (mm) were recorded as inhibition zone for pp. streptomycin sulfate (500 µg/ml, p. carotovorum subsp . carotovorum) (pachupate and kininge, 2013) and oxytetracyclin hydrochloride (terramicin®) (30 μg/ml, x. campestris pv. campestris and r. solanacearum) (santos et al., 2008) were used as positive control. the isotonic phosphate buffered saline (pbs) was used as negative control. all tests were performed in six replicates. minimum inhibitory concentrations (mics) were performed in 96­well micro­plates (eloff, 1998) using serial dilutions of pp (500; 250; 125; 62.5; 31.25 μg/ml) and pg (250; 125; 62.5; 31.25 μg/ml). hundred microliters of pp or pg diluted in liquid cul­ ture medium and the tested microorganism suspen­ sions (1.5 x 105 colony­forms unity cfu/well). after incubation (28°c for 24 h), the content of each well was sown in petri dishes with agar culture medium. the petri dishes were incubated for 48 hours at 28°c to account for the colony­forms unity (cfu). to indi­ cate viable bacteria cells in the microplate, 10 μl of thiazolyl blue (tetrazolium salt 3­(4.5­dimethylthia­ zol­2­yl)­2.5­diphenyltetrazolium bromide) reagent were added to the microplate wells and incubated at 28°c for 1­3 h (mosmann, 1983). the color change produced in reaction was measured in a spectropho­ tometer (540 nm) and the values were correlated to the viable bacteria cells in the microplate. mic was measured as the lowest concentration necessary to inhibit growth of the tested pathogen. minimum bactericidal concentration (mbc) was considered as the minimum concentration in which no growth was visually observed in petri dishes with solid medium, with 99.99% of eradication of the initial inoculum (de nova et al., 2019). the concentration of the pp and pg that inhibited the growth of half of the inoculum was estimated as the inhibitory concentration 50 (ic50) (soothill et al., 1992), represented as log (inhibitor) versus normalized absorbance (%) (dose­ response inhibition model). streptomycin sulfate (500 μg/ml) and oxytetracycline hydrochloride (30 μg/ml) were used as positive control and isotonic phosphate buffered saline (pbs). all tests were per­ formed in four replicates. effect of hydroalcoholic crude extract from the fruit peel of the p. granatum on x. campestris pv . campestris control in seeds seeds of the eruca vesicaria (l.) cav. (feltrin®, cultivated arugula variety, germination from 7 to 10 days) were purchased from a local market. seeds were disinfected and coated with pathogenic bacte­ ria according standard protocol (kotan et al., 2014). seeds coated with pathogen were directly soaked in treatments consisting in: 1 ­ pp suspension (500 µg/ml or 250 µg/ml) or 2 ­ association between pp (500 µg/ml or 250 µg/ml) and antibiotic (strepto­ mycin sulfate, 500 µg/ml) for 3 hours. the seeds were left to dry on sterile whatman filter paper sheets overnight in laminar flow hood. the seeds were sown in plastic pots containing garden soil and sand (1:1) totaling thirty seeds per treatment (ten seeds/pot). other part of the seeds was transferred to petri dishes with whatman paper filter placed on the bottom (moistened with 10 ml of sd. h2o) total­ ing thirty seeds per treatment (ten seeds/plate). the percentage of germination and seedling emergence was determined 10­12 days after sowing. e. vesicaria seedlings were assessed 18 days after emergence to determine the appearance of symptoms of disease (vicente and holub, 2013) and survival rate. after this, the seedlings were removed from the substrate for assessment of the effect of the extract in growth promotion. antibiotic (streptomycin sulfate at 500 de sousa silva et al. ‐ punica granatum l. extract acts in phytopathogen control and eruca vesicaria (l.) cav. germination 221 µg/ml), disinfected seeds infected with pathogen, and sterilized seeds not infected with pathogen (healthy) were used as controls. all tests were per­ formed in triplicates. anatomical analysis eruca vesicaria (l.) cav. seedling samples leaves were preserved in fixative solution faa (formaldehyde, glacial acetic acid, 95% etoh) (johansen, 1940). to prepare the samples, leaves were sectioned in transverse and paradermic sections using disposable razors. leaf diaphanization was per­ formed according to the standardized technique (kraus and arduin, 1997) and stained with safranin and astra blue solutions, both at 0.5%. semi­perma­ nent slides were analyzed with optical microscope to visualize the adaxial epidermis, abaxial epidermis, pal­ isade parenchyma, spongy parenchyma, stomatal density and stomatal morphology. stomatal density (d) was calculated as d= number of stomata/(40x objective ocular area) (mm2) (abdulrahaman et al., 2009). public domain software imagej 1.43a version 64 (schneider et al., 2012) was used to obtain stom­ atal measures of the polar diameter (pd) (µm), equa­ torial diameter (qd) (µm) and stomatal area (a) (µm2) to each treatment. stomatal functionality (fun) was calculated as fun=polar diameter (µm)/equatorial diameter (µm) of the stomata (de castro et al., 2009). statistical analysis results were expressed as the mean ± standard deviations. to determine difference between sam­ ples, one­way anova followed by tukey post hoc test and student’s t test were performed at p<0.05. inhibitory concentration 50 (ic50) was performed by nonlinear regression analysis (dose­response inhibi­ tion model) with 95% profile likelihood. all analyses were performed in graphpad prism® v. 8.0 software. 3. results chemical characterization of the hydroalcoholic crude extract from the fruit peel of the p. granatum the results demonstrated that pp possessed 6.34 mg/g of ellagic acid and 0.83 g/g of total phenolic as tannic acid. hplc chromatogram was performed focused on ellagic acid, and the fingerprint is present­ ed in figure 1. in vitro antimicrobial activity of the hydroalcoholic crude extract from the fruit peel of the p. granatum hydroalcoholic crude extract from the fruit peel of the p. granatum (pp) was tested for its antimicrobial properties against phytopathogenic bacteria. pp pro­ duced bacterial growth inhibition zones for all three investigated isolates (table 1 and fig. 2). the highest mean values of inhibition zones were verified for r. solanacearum , followed by x. campestris pv . campestris and p. carotovorum subsp. carotovorum. there was an increase of the inhibition zone pro­ duced as the increase of the p. granatum extract con­ table 1 ­ in vitro inhibition zone produced by treatment with punica granatum l. hydroalcoholic extract (pp) against isolates of the phytopathogenic bacteria data were represented as mean ± standard deviation for six repli­ cations. nd= no detected of inhibition zone. # control with anti­ biotics streptomycin sulfate for p. carotovorum subsp. carotovorum and oxytetracyclin hydrochloride (terramicin®) for x. campestris pv. campestris and r. solanacearum. the values followed by different letters (a­m) along each column are significantly different according to anova followed by tukey post hoc test (p<0.05). fig. 1 ­ hplc chromatogram of ellagic acid in punica granatum extract (pp). the extract was characterized considering the content of ellagic acid and the total phenolic as tan­ nic acid by hplc chromatography performed focused on ellagic acid. the mobile phase used for ellagic acid was methanol and acetic acid aqueous solution [2% ­ and elu­ tion gradient (0­7min, 20­72.5% v/v methanol, 7­7.5 min, 72.5­95% v/v methanol, 7.5­8.5 min. 95% v/v methanol, 8.5­9 min 95­20% v/v methanol, 9­10 min 20% v/v methanol)], flow rate of 1.0 ml min−1, and oven tempe­ rature of 25°c ­100 mm x 2.6 mm shim pack ods c18 column. the eluted samples were detected by uv detec­ tor at 254 nm. concentrations (mg ml­1) inhibition zones (mm)* r. solanacearum x. campestris pv. campestris p. carotovorum subsp. carotovorum control# 42.83±1.4 a 45.03±0.6 f 35.17±0.75 j 100 35.33±1.2 b 28.83±0.7 g 22.67±0.51 k 50 31.17±2.1 b 26.33±0.8 g 20.00±0.89 k 25 25.67±1.8 c 23.0±1.0 g 16.67±1.03 l 12.5 22.17±2.6 c 19.5±0.5 g 10.17±5.11 m 6.25 16.0±2 d 15.67±1.3 h nd 3.12 6.66±5.2 e 9.66±4.9 i nd 222 adv. hort. sci., 2021 35(3): 217­231 centration (dose­dependent effect) (table 1). in vitro antimicrobial activity through the microdilu‐ tion test microdilution assays indicated susceptibility of the bacterial isolates p. carotovorum subsp. carotovorum and x. campestris pv. campestris to several concen­ trations of the pp (fig. 3 a­e and 4 a­e) and pg (fig. 3 b­e and 4 b­e). were made tests with different con­ centrations of the pp (500 to 31.25 μg/ml) to verify cell viability of the bacteria in comparision with nega­ tive control and antibiotic. bacteria in the negative control remained with high celular viability. in the groups treated with pp in the highest concentrations (500 and 125 μg/ml) there was a reduction of the cell viability of the both bacteria in relation to the negative control (fig. 3 a­c and 4 a­c). the antimicro­ bial effect of the pp in concentrations of 500 and 250 μg/ml for x. campestris pv. campestris was similar to antibiotic and different of the negative control (fig. 4 a­b). concerning to p. carotovorum subsp. carotovo‐ rum, the antimicrobial effect of the pp in concentra­ tion of 500 μg/ml was similar to antibiotic and diffe­ rent of the negative control (fig. 3 a).the smallest concentrations of the pp (62.5 and 31.25 μg/ml) did not produce any antimicrobial effect in cell viability fig. 2 ­ in vitro antimicrobial activity of punica granatum l. hydroalcoholic extract (pp) against isolates of phyto­ pathogenic bacteria. bacterial growth in petri dishes with solid medium in agar­well diffusion assay. a) r. solana‐ cearum; b) x. campestris pv. campestris; c) p. carotovo‐ rum subsp. carotovorum. ctrl= negative control with pbs; atb= positive control with antibiotic. fig. 3 ­ in vitro inhibition rates of the p. carotovorum subsp. carotovorum cell viability (%) for treatments with punica granatum l. hydroalcoholic extract (pp) and punicalagin compound (pg). each graph was accompanied of bacterial growth in petri dishes with agar solid medium. (*) significant difference between treatments (pp or pg) and antibiotic (atb); (#) significant difference between treatments (pp or pg) and negative control (control) (p<0.05, anova followed by tukey post hoc test). each bar repre­ sents mean ± sd. de sousa silva et al. ‐ punica granatum l. extract acts in phytopathogen control and eruca vesicaria (l.) cav. germination 223 for these both pathogens (fig. 3 d­e and 4 d­e). antimicrobial activity of the pg against the investi­ gated pathogens was higher than action of the pp. the lowest concentration of the pg that inhibits bac­ terial growth (mic) to both p. carotovorum subsp. carotovorum and x. campestris pv. campestris was 31.25 μg/ml (fig. 3e and 4e). punicalagin compound at the highest tested concentration (250 μg/ml) showed antibiotic­like antimicrobial activity, in terms of the cellular viability of the both pathogens (fig. 3b and 4b). the observation of bacterial growth in cul­ ture plates with agar medium indicated that pg in this concentration (250 μg/ml) may present bacterio­ static action for p. carotovorum subsp. carotovorum (fig. 3b) or bactericidal action to x. campestris pv. campestris (fig. 4b). regarding to the concentration that inhibits 50% of bacterial inoculum (ic50) pp presented a concentra­ tion of 212 μg/ml to p. carotovorum subsp. carotovo‐ rum (fig. 5a) and 154.6 μg/ml to x. campestris pv. campestris (fig. 5b). the concentration of the pg compound that inhibits 50% of bacterial inoculum (ic50) was 58.96 μg/ml to p. carotovorum subsp. fig. 4 ­ in vitro inhibition rates of x. campestris pv. campestris cell viability (%) for treatments with punica granatum l. hydroalcoholic extract (pp) and punicalagin compound (pg). each graph was accompanied of bacterial growth in petri dishes with agar solid medium. (*) significant difference between treatments (pp or pg) and antibiotic (atb); (#) significant difference between treat­ ments (pp or pg) and negative control (control) (p<0.05, anova followed by tukey post hoc test). each bar represents mean ± sd. fig. 5 ­ inhibitory concentration 50 (ic50) of the treatments with punica granatum l. hydroalcoholic extract (pp) and punicalagin compound (pg) against isolates of the phyto­ pathogenic bacteria. a) and b) represent inhibitory effect of the treatment with pp on p. carotovorum subsp. caro‐ tovorum and x. campestris pv. campestris. c) and d) represent inhibitory effect of the treatment with pg on p. carotovorum subsp. carotovorum and x. campestris pv. campestris by the nonlinear regression (dose­response inhibition model) with 95% profile likelihood. adv. hort. sci., 2021 35(3): 217­231 224 carotovorum (fig. 5c) and 21.15 μg/ml to x. campestris pv. campestris (fig. 5d). in vivo antimicrobial and biostimulant activity of the hydroalcoholic crude extract from the fruit peel of the p. granatum in e. vesicaria seeds infected by x. campestris pv. campestris the most effective concentrations of the pp in microdilution assays (500 μg/ml and 250 μg/ml) were tested for the control of the x. campestris pv. campestris in e. vesicaria seeds. infected and untreated seeds (negative control) presented a lower emergence percentage compared to healthy seeds. on the other hand, treatment of infected seeds with pp (500 μg/ml) promoted an increase of the 15% in the percentage of emergence in relation to the nega­ tive control (table 2). in addition, e. vesicaria seedlings treated with pp did not develop main symptom of black rot disease caused by x. campestris pv. campestris (the “v” chlorotic lesion in the margin of the leaflet). this symptom was verified in seedlings of infected and untreated seeds. there was no phytotoxic effect of the pp in seedlings development. seedlings of the e. vesicaria treated with highest concentration of the pp (500 μg/ml) showed a biggest growth length of radicle (fig. 6). treatment with streptomycin sulfate crude or associated with pp resulted in seedlings with chlorosis symptom (yellowish leaves) (fig. 7). the results of this study showed different seedling survival rates of the e. vesicaria according to each treatment (fig. 8a­d). infected and untreated seedlings (negative control) showed an abrupt drop in the percentage of survival at the 7th day after emergence. seedlings treated with pp at the highest concentration (500 μg/ml) showed slowly decrease fig. 7 ­ appearance of eruca vesicaria l. (cav.) seedlings germi­ nated in petri dishes and in pots with different treat­ ments. healthy seeds – uninfected seeds; seeds with antibiotic: seeds infected with x. campestris pv. campe‐ stris and treated with streptomycin sulfate; seeds with pomegranate extract: seeds infected and treated with punica granatum l. hydroalcoholic extract (pp) (500 µg/ml). fig. 6 ­ growth length of radicle of the eruca vesicaria l. (cav.) seedlings treated with punica granatum l. hydroalcoholic extract (pp). (a) seedlings of healthy seeds; (b) seedlings of infected with x. campestris pv. campestris and untrea­ ted seeds; (c) seedlings of seeds infected and treated with streptomycin sulfate; (d) seedlings of seeds infected and treated with pp (500 µg/ml); (e) seedlings of seeds infected and treated with pp (250 µg/ml); (f) seedlings of seeds infected and treated with association between pp (500 µg/ml) and streptomycin sulfate. table 2 ­ in vivo activity of the punica granatum l. hydroalco­ holic extract (pp) in germination and emergency of the eruca vesicaria l. (cav.) seeds infected with x. campestris pv. campestris # health= seeds uninfected with pathogen;*ctrl­ = negative con­ trol (seeds infected and untreated); ctrl+ = positive control (seeds infected treated with streptomycin sulfate); pp= seeds infected treated with punica granatum l. hydroalcoholic extract (pp) at concentrations of 500 μg/ml and 250 μg/ml; pp + atb= association between pp (500 μg/ml or 250 μg/ml) and antibiotic streptomycin sulfate (500 µg/ml). in each column, values follow­ ed by different letters (a­d) are significantly different according to anova followed by tukey post hoc test (p<0.05). treatment* concentration (μg/ml) germination (%) emergency (%) health# ­ 93.3±5.1 a 80±0.0 b ctrl­ ­ 93.3±5.1 a 45±5.7 c ctrl+ 500 93.3±10.33 a 65±5.7 d pp 500 100±0.0 a 60±11.55 d 250 93.3±5.1 a 45±5.7 d pp + atb 500 + 500 93.3±5.1 a 60±11.55 d 250 + 500 86.6±10.3 a 60±11.55 d de sousa silva et al. ‐ punica granatum l. extract acts in phytopathogen control and eruca vesicaria (l.) cav. germination 225 in the percentage of survival in the same period (fig. 8a). on the 13th day after emergence, the survival rates of the seedlings treated with pp (500 μg/ml and 250 μg/ml) were 50% and 42%, respectively, versus 20% of the negative control (fig. 8b). association between pp (500 μg/ml and 250 μg/ml) and antibiotic also promoted higher survival rates in relation to negative control in the same period (61% and 61.9%, respectively) (fig. 8b). after 16 days of seedling emergence there were similar rates for treatment with pp (500 μg/ml and 250 μg/ml) and treatment with association between pp and antibiot­ ic. these percentages remained high (36%­38%) in relation to negative control (15%) (fig. 8d). anatomical analyses of e. vesicaria seedling leaves indicated differentiation of mesophilic structures, especially in relation to the palisade parenchyma (fig. 9a­f). uninfected (healthy) seedlings presented well­ preserved anatomical structures (fig. 9a). seedlings of the infected and untreated seeds group (negative control) showed some alterations in mesophilic tis­ sue, especially in relation to incomplete differentia­ tion of palisade parenchyma, when compared to healthy plants (fig. 9b). seedlings in the group previ­ ously treated with streptomycin sulfate presented altered palisade parenchyma cells in a more rounded shape (fig. 9c). seedlings treated with pp at a con­ centration of 500 μg/ml showed clear differentiation of mesophilic structures, with well­structured pal­ isade parenchyma (fig. 9d). seedlings treated with pp at a concentration of 250 μg/ml did not present clear differentiation of mesophilic elements (fig. 9e). appearance of stomata of seedlings in different treatments with pp was represented in figure 10 (a­ f). from these images, the mean values of the fol­ fig. 8 ­ in vivo effect of the treatment with punica granatum l. hydroalcoholic extract (pp) in survival rates of eruca vesi‐ caria l. (cav.) seedlings germinated in pots. clean (health)= seeds uninfected with x. campestris pv. campe‐ stris; ctrl­ = infected and untrated seeds; ctrl+= seeds infected and treated with streptomycin sulfate; pp 500 µg/ml and pp 250 µg/ml= seeds infected and treated with punica granatum l. hydroalcoholic extract (pp); pp 500 µg/ml + atb or pp 250 µg/ml + atb= seeds infected and treated with association between punica granatum l. hydroalcoholic extract (pp) and streptomycin sulfate. a) total survival rates; b) survival rates in thirteenth day; c) survival rates in fourteenth day; d) survival rates in sixteenth day. means with (*) are different from the negative control by the student’s t test at p<0.05. fig. 9 ­ cross sections of the mesophyl of eruca vesicaria l. (cav.) seedlings infected with the x. campestris pv. cam‐ pestris submitted to different treatments. a) uninfected seedlings; b) seedlings infected with x. campestris pv. campestris and untrated; c) seedlings infected and trea­ ted with streptomycin sulfate; d) seedlings infected and treated with punica granatum l. hydroalcoholic extract (pp) (500 µg/ml); e) seedlings infected and treated with p. granatum l. hydroalcoholic extract (pp) (250 µg/ml); f) seedlings infected and treated with association between p. granatum l. hydroalcoholic extract (pp) (500 µg/ml) and streptomycin sulfate. epad= adaxial epidermis; abed= abaxial epidermis; lp= lacunous parenchyma; pp= palisade parenchyma; vb= vascular bundle. adv. hort. sci., 2021 35(3): 217­231 226 lowing parameters were taken: polar diameter (pd), equatorial diameter (qd), stomatal functionality (fun), area (a) and stomatal density (sd) (table 3). seedlings of the group treated with pp (500 μg/ml) showed highest values of pd, fun and a (μm2) in comparison to negative and positive control (table 3). stomatal functionality of the group treated with pp (500 μg/ml) was similar to that of healthy seedlings. the highest mean of sd was verified for the group treated with pp at a concentration of 250 μg/ml, being statistically similar to the clean group. seedlings of the infected and untreated group showed smallest values of pd, fun and sd. small val­ ues of pd and fun were observed too in seedlings treated with antibiotic (table 3). 4. discussion and conclusions in the present study, the effective antimicrobial action of the p. granatum fruit peel extract and its isolated punicalagin compound on phytopathogenic bacteria (r. solanacearum, p. carotovorum subsp. carotovorum and x. campestris pv. campestris), listed among the ten most important species in scientific and economic aspects worldwide (mansfield et al., 2012), was shown . the wide zones of inhibition of bacterial growth in agar­diffusion tests and marked reduction in the percentage of the cell viability in broth microdilution assays indicate high potential of pomegranate extract in the control of these phy­ topathogens. pomegranate peel has substantial amounts of phenolic compounds, such as hydrolysable tannins (punicalin, punicalagin, ellagic acid, and gallic acid), flavonoids (anthocyanins and catechins), and nutri­ fig. 10 ­ paradermic sections of the leaf blade of eruca vesicaria l. (cav.) seedlings submitted to different treatments. a) uninfected seeds; b) seeds infected with x. campe‐ stris pv. campestris and untrated; c) seeds infected and treated with streptomycin sulfate; d) seeds infec­ ted and treated with punica granatum l. hydroalcoho­ lic extract (pp) at concentration of 500 µg/ml; e) seeds infected and treated with p. granatum l. hydroalcoho­ lic extract (pp) at concentration of 250 µg/ml; f) seeds infected and treated with association between p. gra‐ natum l. hydroalcoholic extract (pp) (500 µg/ml) and streptomycin sulfate. st= stomata; ep= epidermis. treatments stomatal measures * pd qd fun a (μm2 ) sd (mm2) health 25.2±4.5 14.9±1.07 bcdef 1.71±0.3 c 302.5±21.3 def 77.7±9.8 bcdf ctrl­ 22.9±2.3 d 18±1.2 a 1.28±0.1 ad 348.5±36.8 df 29.3±13.7 acdef ctrl+ 21.5±2.9 df 18.2±0.8 a 1.18±0.1 ade 270±19.6 d 46.9±5.4 abed pp 500 μg/ml 29.6±4 bcef 17.6±1.9 a 1.69±0.2 bcef 483±41.1 abce 49.1±7.6 ab pp 250 μg/ml 22.9±1.4 d 16.7±1 a 1.37±0.01 cd 335±19.7 ad 87.2±15.8 bcd pp 500 μg/ml +atb 26±2.3 c 19.4±3.6 a 1.36±0.1 cd 449.8±49.2 ab 51.3±5.7 ab table 3 ­ stomatal measures in paradermic sections of leaves of the eruca vesicaria l. (cav.) submitted to different treatments health= uninfected seedlings; ctrl­ = infected and untreated seedlings (negative control); ctrl+ = seedlings infected and treated with streptomycin sulfate (positive control); pp 500 μg/ml and pp 250 μg/ml= seedlings infected and treated with p. granatum l. hydroalco­ holic extract (pp) at concentration of 500 μg/ml or 250 μg/ml; pp 500 μg/ml + atb= seedlings infected treated with p. granatum l. hydroalcoholic extract (pp) at concentration of 500 μg/ml in association with antibiotic streptomycin sulfate (500 µg/ml). * data were represented as mean ± standard deviation for three replications. pd= polar diameter (μm); qd= equatorial diameter (μm); fun=­ stomatal functionality; a= stomatal area; sd= stomatal density (mm2). in each column, values followed by different letters (a­f) are significantly different according to student’s t test (p<0.05) where: a­ comparison with health; b­comparison with ctrl­; c­comparison with ctrl+; d­comparison with pp 500 μg/ml; e­comparison with pp 250 μg/ml; f­comparison with pp 500 μg/ml +atb. de sousa silva et al. ‐ punica granatum l. extract acts in phytopathogen control and eruca vesicaria (l.) cav. germination 227 ents, which are responsible for its biological activity (magangana et al., 2020). the fruit peel has high antioxidant and antimicrobial activities and may be used as an excellent natural additive for food preser­ vation and for quality enhancement. the health­pro­ moting benefits of pomegranate peel have prompted the food industry to focus on pomegranate­peel­con­ taining food preparations, which include nutraceuti­ cals, phenolic­enriched diets, and food supplements (opara et al., 2009; fawole et al., 2012). the pp showed both bacteriostatic (p. carotovo‐ rum subsp. carotovorum,) and bactericidal effect (x. campestris pv. campestris). bactericidal effect is desirable in order to inhibit the emergence of resis­ tant bacterial strains and toxicity (soberón et al., 2014). the study results support literature data on the antimicrobial action of the ethanolic p. granatum fruit peel extract against phytopathogenic bacteria pseudomonas syringae pv. tomato, the cause of bac­ terial spot disease in tomatoes, with bacterial growth inhibition zones of 5­26 mm and dose­dependent effect (quattrucci et al., 2013). additionally, the results agreement with studies that reported antibac­ terial action of the methanolic pomegranate peel extract against r. solanacearum, with growth inhibi­ tion zone of the 13.9 mm (50 mg/ml), and ethyl acetate extract against r. solanacearum, p. carotovo‐ rum subsp. carotovorum and x. gardneri with inhibi­ tion zone of 8.5­22.75 mm (concentrations of 25­200 mg/ml) (farag et al., 2015; khaleel et al., 2016). studies with extracts from other parts of the plant (leaf and seed) have shown more discrete antimicro­ bial activity on r. solanacearum and x. campestris (hassan et al., 2009; uma et al., 2012). antimicrobial activity of the p. granatum fruit peel extract against gram­negative and gram­positive bacteria has been correlated with the presence of polyphenolic compounds in it, mainly punicalagin (gullon et al., 2016). a relatively high amount of polyphenols (867 mg/g) was detected in a pomegran­ ate peel extract preparation, especially the ellagitan­ nin punicalagin (296 mg/g), with antimicrobial action of the extract on isolates of s. aureus, escherichia coli, aspergillus niger and saccharomyces cerevisiae (ibrahium, 2010). the antimicrobial mechanism of action of polyphenols seems to be related to the direct action of these compounds on the bacterial cell wall by formation of complexes with wall pro­ teins, causing lysis (akhtar et al., 2015). the interac­ tion of these compounds with sulfhydryl groups of extracellular microbial proteins results in inhibition of protein activity (dey et al., 2012). the impressive antimicrobial action of punicalagin on p. carotovorum subsp. carotovorum and x. campestris pv. campestris verified in the present study suggests that this molecule may be an essential component in the biological activity of p. granatum fruit peel extract against phytopathogenic bacteria. several studies prove antimicrobial activity of the punicalagin compound against isolates of clinical importance such as staphylococcus aureus (mic 250 μg/ml ) and methicillin­resistant staphylococcus aureus (mrsa) (xu et al., 2017; mun et al., 2018). according to xu et al. (2017) punicalagin compound has direct action on cell membrane disruption, increased k+ ion flow and inhibition of biofilm forma­ tion in s. aureus. to the best of our knowledge, this is the first report of the antibacterial activities of the isolated compound punicalagin against plant patho­ genic bacteria. further investigations may elucidate the mechanism of action of this compound on phy­ topathogenic bacteria. data from the in vivo assays of this study indicat­ ed that pp is effective in control of x. campestris pv. campestris in e. sativa seeds. black rot infection caus­ es tissue necrosis, premature leaf fall, atrophied growth and death of young plants (vicente and holub, 2013). in the present study, treatment with pp in the highest concentration reduced incidence of disease symptoms and promoted a high survival rates of seedlings in comparison to infected and untreated group. additionally, the association between pp and antibiotic resulted in percentages of seedling survival above that observed in treatment with antibiotic alone (16th day of observation), indi­ cating probable synergistic interaction between treatments. p. granatum extract produced no harm­ ful effect on germination, emergence or seedling development of the e. sativa. this is in agreement with a study that proved the effective action of treat­ ment with natural plant extract (origanum onites) in the control of clavibacter michiganensis ssp. michiga‐ nensis, xamthomonas axonopodies pv. vesicatoria and xanthomonas campestris pv. vitians in seeds of tomato and lettuce, without affecting seedling germi­ nation and growth (kotan et al., 2014). treatment of the seeds with hot water (50°c for 20­30 min) has been the most effective treatment for seedborn blackrot control. however, treatments do not always eliminate 100% of the bacteria and may adversely affect seed germination and vigor (celetti and callow, 2002). natural plant extracts, like pomegranate fruit adv. hort. sci., 2021 35(3): 217­231 228 peel extract, may represent a good alternative to control of seed born phythopatogens ensuring seed viability after treatment. treatment of e. vesicaria seeds with streptomycin sulfate, despite being effective in controlling x. campestris pv. campestris, demonstrated toxic effect for seedlings, evidenced by the yellowish aspect of the leaves (chlorosis). this result corroborates the findings of napoles et al. (1991) for treatment of brassica oleracea seeds with the same antibiotic (500 ppm for 1 h). the streptomycin is associated with several phytotoxic effects like blocking chlorophyll synthesis, especially in younger leaves, inhibition of methionine and phosphate absorption, production of photosynthetic process changes and enzymatic inhi­ bition (falkiner, 1990). in addition, treatment of seeds with streptomycin resulted in severe reduction in the ratio between the polar and equatorial diame­ ters (stomatal functionality). however, treatment with pp (500 μg/ml) keep values of stomatal func­ tionality (fun) similar to the healthy seedlings. the highest ratio of fun indicates stomata with more elliptic morphology, a mechanism of the drought tol­ erant plants to keep the water present in its interior at a maximum as a response to its hydric state (melo et al., 2014). treatments with pp indicated antagonistic values of stomatal density (sd) and stomatal area (a). the parameters sd and a can directly affect mechanisms such as photosynthesis, transpiration and efficient water use in plants (lawson and blatt, 2014). the increase in sd coupled with reduction in a may result in the optimization of gas exchange (franks et al., 2009). this pattern was observed in seedlings treated with pp at a concentration of 250 μg/ml and similarly in the health group. on the other hand, the reduction in sd may represent a more conservative water use (bertolino et al., 2019). this pattern was verified in seedlings treated with pp at a concentration of 500 μg/ml. since there is no damage to co2 fixation or plant cooling, this reduction in water loss can be advantageous in environments with low water avail­ ability (bertolino et al., 2019). thus, the pp can influ­ ence both pathogen control in seeds and the physio­ logical characteristics of the plant through changes in stomatal patterns. in conclusion, hydroalcoholic crude extract from the fruit peel of the p. granatum (pp) demonstrated high potential for control of the phytopathogenic bacteria r. solanacearum , x. campestris pv . campestris and p. carotovorum subsp. carotovorum. punicalagin compound potentiated antimicrobial activity on these pathogens, corroborating studies that relate the abundance of the phenolic com­ pounds (flavonoids and tannins) with the antibacteri­ al activity of pomegranate fruit peel. pp extract was effective to control seedborn pathogen x. campestris pv. campestris in seeds of the e. vesicaria and pro­ moted several beneficial effects to seedlings with no phytotoxic effect. moreover, association between pp and antibiotic indicated probable synergistic interac­ tion between treatments (16th day of observation) potencializing the seedling survival over the observed in relation of the antibiotic utilizing in isolated way. the investigation of antimicrobial activity of the pp and isolate compounds like punicalagin repre­ sents a promising path regarding the biotechnologi­ cal development of 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khokhar, 2014; mubarak and hamdan, 2018 a). onion crop thrives best when temperatures are cool during early development period and then warmer and sunny during maturity. hence, planting date has a profound impact on onion crop growth and develop­ ment. early planting date tends to have a longer onion growing season before bulb initiation ensuing larger plants. however, large plants are more likely to become sensitive to the cold stimulus resulting in bolting (*) corresponding author: ascientific4@aec.org.sy citation: mubarak i., 2020 ­ improving water productivity and yield of onion crop by combining early plan‐ ting and straw mulch under different irrigation levels in dry mediterranean region. ­ adv. hort. sci., 34(1): 49­60. copyright: © 2020 mubarak i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 march 2019 accepted for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 49­60 50 (formation of seed stalk followed by flowering), which represents a highly unfavourable feature for onion bulb production. large plants are also related with split bulbs. however, late­date­planted onions start forming bulbs before reaching satisfactory plant growth to support the final size of bulbs. this would produce small bulbs, and therefore, decreasing the bulb yield (brewster, 2008; rohini and paramaguru, 2016). thus, determining the proper onion crop planting date is central to adapt to the regional cli­ mate changes. onion crop has a shallow rooting system, and therefore, it is considered as a sensitive crop to soil water deficit than other deep­rooted crops. water productivity (wp, also known as water use efficiency, wue) is usually used to recognize the environments or management practices by which the yield per unit water can be optimized. in the dry regions where water resources are limited as in the dry mediterranean region, improving water productivity and crop production represents also a main challenge for agricultural water management. mulch has been widely adopted because of its agro­pedo­ecological benefits. it constitutes of syn­ thetic (plastic films) or natural (plant residues as wheat or rice straw) materials. both materials reduce the loss of soil water through evaporation. however, unlike plastic films, straw mulch allows rain and irri­ gation water to penetrate and to reach the soil. this conserves soil water content, and thereby reducing irrigation water requirements, promoting rooting sys­ tem development, and increasing crop growth, devel­ opment and yield (vavrina and roka, 2000; gimenez et al., 2002; mubarak and hamdan, 2018 b). from eco­environmental point of view, the use of plastic mulch would not be justified at low crop prices and/or very high plastic films costs, especially it requires to be removed after use annually. the re­ use of plastic films is not practical agronomically and technologically. this could increase the harmful envi­ ronmental impacts from plastic components. for these reasons, straw mulching present an eco­envi­ ronmentally sustainable choice. unlike plastic films, straw mulch could incorporate into the soil ecosys­ tem, where it is expected to biodegrade. thus, straw mulching could be considered as a slow­acting organ­ ic fertilizer, improving soil fertility and soil physical properties, and consequently, crop yield (khaledian et al., 2010, 2011). deficit irrigation (di) in combination with mulching could be considered as a key water­saving technique that would help in meeting both water scarcity and sustainable crop production (fereres and soriano, 2007; chai et al., 2016). the effects of deficit irrigation under mulching on onion yield have been documented (vavrina and roka, 2000; igbadun et al., 2012; patel and rajput, 2013; tsegaye et al., 2016; mubarak and hamdan, 2018 b). several studies showed that it is better to fractionate the water stress during the cropping season (regulated deficit irrigation, rdi) rather than applying a water stress during the critical stages of crop growth period (kadayifci et al., 2005; patel and rajput, 2013). for example, deficit irrigation given at 75% of crop evap­ otranspiration (etc) was recommended for onion crop production (tsegaye et al., 2016). in the dry mediterranean area, onion bulb sets as directly planted in the soil is the common method employed to establish onion plantings in the field. farmers plant onion bulb sets early in the spring and harvest in the summer. the production period between april and august is characterized by no rain­ fall (ragab and prudhomme, 2002; turner, 2004). moreover, the mediterranean climate is extremely variable with hot dry summers, and cold wet to dry winters. the middle east and north africa are in par­ ticular dry areas, with only 1% of renewable water resources (joffre and rambal, 2001; turner, 2004; ceccarelli et al., 2007). the increasing climatic change have intensified the vulnerability to drought (giorgi and lionello, 2008; somot et al., 2008; fao 2011; polade et al., 2014). an increase by 1.25­2.5°c in temperature is predicted in winter, and the precipita­ tion between october and march will decrease by 10­15 % in the southern mediterranean countries (ragab and prudhomme, 2002). as the onion crop production is already limited by the water availability and local climate, moving towards feasible tools (such as using regulated deficit irrigation under mulching) and agronomic practices (such as determining the proper crop planting date) adapted to the regional climate change is urgently needed for better water saving and cultivation period of the crop (fao, 2011; khokhar, 2014; zinkernagel et al., 2015). in this context, and in response to the ambitious sustainable development goals (sdgs) proposed by united nations to adapt to climate change and to promote sustainable agriculture, the present study aimed to assess the interactive effects of various planting dates, different irrigation levels, and straw mulching on onion crop production. the outcomes may introduce appropriate agronomic alternatives to meet the ever increasing demand for onions and to mubarak ‐ planting date, irrigation level, and mulch for onion crop 51 save irrigation water in the dry mediterranean area. moreover, results may contribute to make regulated deficit irrigation with straw mulching familiar for most farmers, and to stimulate them to adopt these techniques in their fields. 2. materials and methods pot experiments were conducted under open field conditions at the deir al­hajar agricultural experiment station, damascus countryside, syria (33°20ʹ n, 36°26ʹ e, altitude 600 m), for different planting dates during february to may in two consec­ utive years 2017 and 2018. the site is located within a dry mediterranean area, in which the total annual rainfall is about 120 mm, and the annual reference evapotranspiration is about 2000 mm. some climatic data for the studied site collected during the growing seasons were fairly close to those averaged over the last 16 years (from 2000 to 2016) as can be shown in table 1. for this reason, testing different planting dates during two years seemed somewhat adequate. the soil is classified as a clay loam (29.5% clay, 42.7% silt, and 27.8% sand). both volumetric soil water contents at permanent wilting point (pwp) and field capacity (fc) are 0.18 and 0.36 m3 m­3, respec­ tively. some chemical and physical soil properties are: ph of 8.0; ece of 0.34 ds m­1; organic matter of 1.00%; available p of 5.7 ppm; no3 ­ of 28.3 ppm; nh4 + of 12.6 ppm. pots with dimensions of 25×30 cm and containing 8 kg of soil were used in the experiments. three bulb sets of onion (allium cepa l., c.v. selmouni) were planted in each pot. the pots were set in an open field under natural climatic conditions. plants were thinned after germination to two bulbs per pot, get­ ting a plant density of about 400000 plants ha­1. four different planting dates separated with 28 days were tested: ps1 (early february), ps2 (early march), ps3 (early april), and ps4 (early may). unfortunately, onion bulb sets which were planted in may (ps4) in both studied years did not properly ger­ minate, and therefore, they were ignored. within a year and at each planting date, the experiment was laid out following a 2×3 factorial experiment arranged in a randomized complete block design (rcb design) with two modes of soil cover and three irrigation levels, replicated three times. the soil cov­ ering comprised of two distinct types. the first one was with mulching using 40 g of wheat straw per pot (about 8 t ha­1); and the second one was with no mulching. the irrigation levels composed of il100 (full irrigation, 100% etc), in which plants received 100% of the crop evapotranspiration; and the root zone was replenished to field capacity. il80 and il60 treatments (regulated deficit irrigation) were irrigat­ ed at the same frequency as in il100 but with water amounts equal to 80 and 60% of the etc as calculat­ ed in il100, respectively. in other words, the three watering treatments received at each irrigation event 1.0, 0.8 and 0.6 times the soil water depletion table 1 ­ some climatic data for the experimental site during both studied years (2017 and 2018) and the 16 years average (from 2000 to 2016) variable year feb. mar. apr. may jun. jul. minimum temperature (°c) 2017 4.0 6.2 9.7 14.4 17.2 20.6 2018 5.7 7.9 10.0 15.6 18.2 19.8 2000­2016 average 4.0 6.8 10.1 14.1 17.6 19.3 maximum temperature (°c) 2017 14.7 18.7 26.2 31.6 35.7 40.6 2018 18.8 24.3 27.2 31.5 34.6 36.9 2000­2016 average 15.7 20.6 25.3 30.4 35.0 37.4 mean temperature (°c) 2017 9.1 14.0 19.2 24.9 28.4 31.1 2018 12.7 17.3 19.9 25.7 27.7 28.8 2000­2016 average 10.6 15.0 18.1 23.6 27.7 29.4 relative air humidity (%) 2017 69.3 74.4 63.1 57.9 56.3 56.0 2018 68.8 65.0 54.8 51.5 59.6 55.6 2000­2016 average 75.0 64.1 60.9 56.5 56.3 60.7 precipitation (mm) 2017 11.6 42.6 0.0 0.0 0.0 0.0 2018 30.3 1.0 14 9.9 0.0 0.0 2000­2016 average 31.0 31.6 5.9 4.2 0.0 0.0 adv. hort. sci., 2020 34(1): 49­60 52 occurred in the full irrigation treatment (il100), respectively. irrigation water was added 3 times per week. each experiment was started on the planting day with a wet soil at field capacity as measured by pot’s weight. the pots were weighed before and after each irrigation event. the water amount deplet­ ed (mm) between two successive irrigation events (etc) was regulated by weight and estimated using (eq. 1) as: etc = (w1 ‐ w2)/(pw x s) [1] where etc = the crop evapotranspiration between two irrigation events (mm); w1 = the weight of the pot (kg) after irrigation (the soil water content in the pot was at the field capacity); w2 = the weight of the pot (kg) just before the next irrigation event; ρw = the water density (g cm­3); and s= the pot soil surface area (m2). the daily crop evapotranspiration (mm day­1) was estimated by dividing the etc calculated using eq. (1) by the number of days between two successive irrigations. the seasonal crop evapotran­ spiration was the summation of the daily etc, which represented the total crop water requirements dur­ ing a growing season. irrigation water amounts, which were applied to the non mulching treatments, were based on the il100 treatment under no­ mulching conditions; whereas those applied to the straw mulching treatments were based on the il100 treatment under mulching conditions. for each planting date, phosphorous and potassi­ um were applied as basal application at planting day; wherease, nitrogen fertilizer was splited into two equal applications, and added during early vegetative stage. irrigation was stopped when almost 70% of leave­head dropped as signs of maturity. the onions were lifted to field cure. then the leaves were cut leaving about 2.0 cm tops above the bulb. the length, diameter, and weight of both matured onion bulbs from each pot were measured. the sum of weights of both bulbs represent the bulb yield per pot (y­pot), and was expressed as g pot­1. water pro­ ductivity (wp, kg m­3) and irrigation water use effi­ ciency (iwue, kg m­3) were calculated using equa­ tions [2] and [3] (mubarak et al., 2018). wp is the relationship between yield and seasonal evapotran­ spiration (etc). whereas, iwue is the relationship between yield and the total amount of irrigation water applied (i, liter pot­1). wp = yield/etc [2] iwue = yield/i [3] within a year, a combined analysis of data over planting seasons was carried out to examine the interaction between planting season and the studied treatments (gomez and gomez, 1984). the analysis of variance (anova) was conducted using the dsaa­ stat add­in version 2011 (onofri, 2007). mean com­ parison was made using the lsd test at the 1% level. trend comparison (regression analysis) was also per­ formed. data was presented and illustrated accord­ ing to the rules described by gomez and gomez (1984). 3. results as mentioned above, onion bulb sets planted in may did not properly germinate. this could be explained by the fact that onion is a vegetative over­ wintering stage in its life cycle, i.e., it grows best when temperatures are cool during early develop­ ment period (brewster, 2008). therefore, it is not recommended to delay planting onion sets after april in the dry mediterranean area. bulb shape indicators the shape of onion bulbs was represented herein by two indicators: bulb length (bl) and diameter (bd). the anova revealed that the main effects of all stud­ ied factors (planting date, soil cover system, and irri­ gation levels) highly significantly affected bulb shape indicators in both years (table 2). within a year, the data under each factor were pooled over the other factors as can be seen in table 3. results indicated that both indicators (bl and bd) found in 2017 were comparable with their homologues in 2018. early planted onion sets (ps1) produced the tallest onion bulbs (7.9 and 8.5 cm in 2017 and 2018, respectively) than the other planting dates, while the later planted sets (ps3) produced bulbs significantly shorter by 10­22% than those in ps1 and ps2, in both years. moreover, onion sets grown under straw mulching produced bulbs considerably taller by about 14% in average than those grown under no­ mulching conditions. with regard to irrigation levels, the higher value of bl was observed under full irriga­ tion (il100); then, bulb length decreased as the water application rate decreased. the mean value of bl reduced by about 8 and 25% in 2017, and by about 3 and 14% in 2018, when onion sets were planted in march (ps2) and april (ps3), respectively (table 3). mubarak ‐ planting date, irrigation level, and mulch for onion crop 53 mulching conditions. also, significant differences in bd were observed in relation to irrigation levels. the maximum values of bd of 4.5 cm in 2017 and 4.6 cm in 2018 were found under full irrigation condition (il100). then, they significantly decreased as the irri­ gation level decreased. the effect of decreasing water application rate by 40% of etc (as in il60 treat­ ment) resulted in a decreased bd by about 48% rela­ tive to those in il100, in both years (table 3). only in 2018, both ps×il and sc×il interactions were highly significant (table 2). to examine the nature of ps×il interaction, the bd data of planting dates were compared under irrigation levels (data not shown). the decline in bd as planting date delayed was found to be more severe under full irri­ gation compared with water stress conditions. also, to examine the nature of sc×il interaction, the bd data of both soil cover systems were compared under irrigation levels (data not shown). the increase in bd as irrigation level increased was found to be more severe under straw mulching compared with no­mulching conditions. these differences could explain the nature of both interactions. on the other hand, the planting date×soil cover system (ps×sc) interaction effect on bulb length was found to be also significant at the 1‰ level only in 2017 (table 2). to examine the nature of this interac­ tion, the bl data of planting dates were compared under both soil cover systems. the values of bl under straw mulching were 8.23, 7.78, and 7.03 cm, and under no­mulching condition were 7.62, 7.05, and 5.38 cm, for ps1, ps2, and ps3, respectively. the mean values of bl in ps1 and ps2 under no­mulching were smaller by 8­10% than those under straw mulching. while in the traditional planting date in april (ps3), bl under no­mulching was 31% smaller than that with straw mulching. this difference could explain the nature of ps×sc interaction. the largest bd was produced from onion sets planted early in february (ps1) in both year: 4.7 and 4.0 cm in 2017 and 2018, respectively. it then signifi­ cantly reduced as the planting date delayed. the mean values of bd from ps3 were shorter by 35% in 2017 and 23% in 2018 than those from ps1. furthermore, using straw mulching led to a signifi­ cant increase in bd by 17% compared with no­ table 2 ­ analysis of variance of the data of crop responses as affected by planting date, soil cover system, and irrigation level (signifi­ cance of fisher test) *** = significant at 1‰ level, ** = significant at 1% level, ns = non­significant at 1% level. df = degree of freedom, bl = bulb length, bd = bulb diameter, y­pot = bulb yield per pot, wp = water productivity, iwue = irrigation water use efficiency. source of variance df bl bd y­pot wp iwue 2017 planting date (ps) 2 *** *** *** *** *** rep. within ps 6 soil cover system (sc) 1 *** *** *** *** *** ps × sc 2 *** ns ns *** *** irrigation level (il) 2 *** *** *** *** *** ps × il 4 ns ns ns ns ns sc × il 2 ns ns ns ns ns ps × sc × il 4 ns ns ns ns ns pooled error 30 total 53 cv (%) 5.88 6.12 9.14 9.07 9.26 2018 planting date (ps) 2 *** *** *** *** *** rep. within ps 6 soil cover system (sc) 1 *** *** *** *** *** ps × sc 2 ns ns ns *** *** irrigation level (il) 2 *** *** *** *** *** ps × il 4 ns *** ns ns ns sc × il 2 ns ** ns ns ns ps × sc × il 4 ns ns ns ns ns pooled error 30 total 53 cv (%) 6.90 3.83 5.89 5.49 5.45 54 adv. hort. sci., 2020 34(1): 49­60 bulb yield per pot (y‐pot) analysis of variance shown in table 2 indicated that the main effects of the three studied factors on y­pot were significant at the 1‰ level. however, none of the three­factor or two­factor interactions were significant at the 1% level in both studied years. the data under each factor were averaged over all levels of the other factors for mean comparison pur­ poses (table 3). in both years, earliness in planting date resulted in an increase in the bulb yield. the mean yield of bulbs which were planted in february (ps1), were the highest with 119.7 and 122.3 g pot­1 in 2017 and 2018, respectively. y­pot significantly decreased with delayed planting. it reduced consid­ erably by 8 and 30% in 2017 and by 6 and 24% in 2018 when onion sets were planted in ps2 and ps3, respectively, compared with sets planted in ps1. in addition, y­pot was found to be increased sig­ nificantly by 30% in 2017 and 26% in 2018 when straw mulching was used relative to no­mulching conditions (table 3). on the other hand, decreasing water application rate resulted in a significant decline in the bulb yield. the lowest mean values of y­pot (67.1 g pot­1 in 2017 and 71.4 g pot­1 in 2018) were observed under sharp deficit irrigation when only 60% of etc was applied to irrigate onion plants. then, y­pot was highly improved with increasing irrigation level. y­pot increased by about 110 and 56% when onion plants were irrigated by 100 and 80% of etc, respectively, compared with those irrigated by only 60% of etc, in both years. table 3 ­ mean comparisons of crop responses as influenced by planting date, soil cover system, and irrigation level for both studied years * means followed by the same letter within a year and column for each tested factor are not significantly different accord­ ing to lsd at 1% level. bl = bulb length, bd = bulb diameter, y­pot = bulb yield per pot, wp = water productivity, iwue = irrigation water use efficiency). tested factor bl (cm) bd (cm) y­pot (g pot­1) wp (kg m­3) iwue (kg m­3) 2017 planting date ps1 (february) 7.9 a 4.7 a 119.74 a 4.60 a 5.71 a ps2 (march) 7.4 b 3.7 b 110.52 b 4.09 b 4.26 b ps3 (april) 6.2 c 3.0 c 83.79 c 2.82 c 2.87 c lsd 0.01 0.4 0.2 8.77 0.32 0.36 soil cover system with mulch 7.7 a 4.1 a 123.07 a 5.14 a 5.80 a without mulch 6.7 b 3.5 b 86.30 b 2.53 b 2.76 b lsd 0.01 0.3 0.2 7.16 0.26 0.30 irrigation level il100 (100% etc) 7.9 a 4.5 a 141.77 a 4.39 a 4.76 a il80 (80% etc) 7.3 b 3.9 b 105.11 b 3.95 b 4.39 b il60 (60% etc) 6.3 c 3.1 c 67.11 c 3.18 c 3.70 c lsd 0.01 0.4 0.2 8.77 0.32 0.36 2018 planting date ps1 (february) 8.5 a 4.0 a 122.32 a 4.96 a 5.44 a ps2 (march) 7.8 b 3.9 a 115.42 b 4.21 b 4.46 b ps3 (april) 7.0 c 3.5 c 92.83 c 3.13 c 3.30 c lsd 0.01 0.5 0.1 5.95 0.21 0. 22 soil cover system with mulch 8.2 a 4.1 a 126.54 a 5.37 a 5.81 a without mulch 7.3 b 3.5 b 93.83 b 2.83 b 2.99 b lsd 0.01 0.4 0.1 4.86 0.17 0.18 irrigation level il100 (100% etc) 8.2 a 4.6 a 148.65 a 4.52 a 4.86 a il80 (80% etc) 8.0 a 3.8 b 110.53 b 4.17 b 4.48 b il60 (60% etc) 7.2 c 3.1 c 71.39 c 3.60 c 3.87 c lsd 0.01 0.5 0.1 5.96 0.21 0.22 mubarak ‐ planting date, irrigation level, and mulch for onion crop 55 however, for presentation and discussion purpos­ es, all experimental data of y­pot under all studied treatments were demonstrated in figure 1a for 2017 and 1b for 2018. trend analysis indicated that the relationships between y­pot and irrigation level (as a percentage of etc) for each planting date and under both soil cover systems were linear (equations not presented). as can be seen in figure 1, the rate of increasing yield with increasing irrigation water level was similar regardless of the planting date adopted under both soil cover systems. this confirmed the lack of interaction as found by anova (table 2). as shown also in figure 1, the maximum value of bulb yield was recorded in the treatment combining between planting in february under straw mulching and 100% of etc (il100) conditions. the yield of bulbs produced under such conditions could be attained about 185g pot­1 in both years (fig. 1). moreover, irri­ gating onion plants with only 60% of seasonal etc (il60) could produce bulb yield comparable with that under 80% of etc (il80), provided using straw mulching and planting onion sets as early as possible. water use parameters as mentioned above, irrigation treatments (il100, il80, and il60) received at each irrigation event 100, 80 and 60% of the amount of soil water depleted under il100 conditions, respectively. irrigation water amounts and seasonal crop evapotranspiration (etc) for the studied treatments are shown in table 4 for both years. the seasonal etc were close to the applied water amounts, because water amounts added to the pots were equal to the depleted water amounts, as regulated by weight. as can be seen, both irrigation water amounts and crop water con­ sumption were greatly decreased when straw mulch was applied. for the three planting dates (ps1, ps2, and ps3), an average of 30% of water was saved when straw mulching was used compared with non­ mulching conditions, regardless of the tested irriga­ tion level. moreover, early planting resulted in a noticeable decrease in both irrigation water amount and crop water use compared with the other planting dates (table 4). for instance, onion crop planted in february (ps1) required irrigation amount 20­30% lesser than that planted in the traditional planting date (april, ps3). these results highlight the need for changing cultural practices and adopting early plant­ ing in order to conserve water resources. the anova detected that both crop water pro­ ductivity (wp) and irrigation water use efficiency (iwue) were significantly influenced by the main effects of planting date, soil cover system, and irriga­ tion level at the 1‰ level, within both years. the interaction between planting date and soil cover sys­ tem (ps×sc interaction) was also significant at the 1‰ level (table 2). it is worth to examine the nature of this interaction. figure 2 illustrates the data of both wp and iwue under different planting dates (as averaged over all irrigation levels)under each system of soil covering, for both years. regression analysis indicated that the relationship between both traits (wp and iwue) and planting date were linear with significant values of r2 at the 1% level, under both straw mulching and no­mulching conditions. for each trait, both representative straights were not parallel, but did not intersect over the studied period. the slope of straight is about two times greater under straw mulching than that without mulching. this indi­ cates that the enhancements in both wp and iwue due to the earliness in planting date, could be dou­ fig. 1 ­ response of onion bulb yield (y­pot) in 2017 (a) and in 2018 (b) to planting date (february, march, and april), soil cover system (with and without mulching), and irri­ gation level (il100, il80, and il60). error bar represents one standard deviation. adv. hort. sci., 2020 34(1): 49­60 56 bled if straw mulching is used. this could explain the existence of the interaction between planting date and soil cover system. the obtained data of both wp and iwue under all tested treatments were shown in figure 3a and 3b for wp, and in figure 4a and 4b for iwue, in 2017 and 2018, respectively. also, the mean values under each factor were presented in table 3 for mean compari­ son purposes. both wp and iwue as derived from the traditional planting date (ps3, in april) were the lowest: 2.82 and 3.13 kg m­3 for wp, and 2.87 and 3.30 kg m­3 for iwue, in 2017 and 2018 respectively. earliness in planting date resulted in a noticeable increase in both efficiencies. for instance, when onion sets were planted early in february (ps1), wp and iwue were significantly augmented by 63 and 99% in 2017, and by 59 and 65% in 2018, respective­ table 4 ­ irrigation water amount (without rainfall) and crop evapotranspiration (etc) as influenced by planting date (ps1, ps2, and ps3), soil cover system, and irriga­ tion level, for both studied years parameters soil cover irrigation level ps1 (feb.) ps2 (mar.) ps3 (apr.) 2017 irrigation water amount (mm) without mulch il100 705.3 798.8 899.6 il80 561.0 649.9 734.2 il60 416.7 502.6 570.8 with mulch il100 458.4 561.4 636.0 il80 363.9 461.3 524.9 il60 269.4 361.2 413.8 crop evapotranspirationetc without mulch il100 800.2 819.1 909.2 il80 664.5 671.8 745.7 il60 525.4 528.1 586.2 with mulch il100 552.5 578.0 641.5 il80 459.9 479.8 532.5 il60 367.5 387.3 429.9 2018 irrigation water amount (mm) without mulch il100 706.5 809.6 886.8 il80 572.3 655.8 718.3 il60 431.0 493.9 540.9 with mulch il100 494.6 566.7 620.8 il80 400.6 459.0 502.8 il60 301.7 345.7 378.7 crop evapotranspirationetc without mulch il100 760.3 848.1 924.3 il80 615.8 687.0 748.7 il60 463.8 517.3 563.8 with mulch il100 548.4 605.2 658.3 il80 444.2 490.2 533.2 il60 334.5 369.2 401.5 fig. 2 ­ for both years, responses of both (a and c) crop water productivity, wp, and (b and d) irrigation water use effi­ ciency, iwue, to planting dates under both soil cover systems. regression equations are fitted and coefficient of determination (r2) is given under each system of soil cover. ** = significant at 1% level. mubarak ‐ planting date, irrigation level, and mulch for onion crop 57 ly. furthermore, both wp and iwue were found to be enhanced considerably when straw mulching was used: they were two times more than those under no­mulching conditions regardless of the planting dates or irrigation levels chosen. in addition, increas­ ing water application rate resulted in an efficient use of water. the maximum values of both wp and iwue (4.39 and 4.76 kg m­3 in 2017, and 4.52 and 4.86 kg m­3 in 2018, respectively) were recorded under full irrigation treatment. they then declined dramatically with decreasing irrigation level. trend analysis revealed that both wp and iwue were linearly related to the irrigation level (as % of etc) under both soil cover systems, regardless of the planting date considered, as shown in figures 3 and 4 (mathematical equations not presented). such devel­ oped linear functions could be invested for predicting the targeted values of wp and iwue under similar cli­ matic conditions in the dry mediterranean area. for instance, the best agricultural management suggested to have maximum values of both traits (wp and iwue) is to plant onion sets in mulched soil in february under full irrigation conditions. the values of wp and iwue produced under such conditions could reach in average 6.95 and 8.05 kg m­3 (figs. 3 and 4). 4. discussion and conclusions as the tested onion variety is an oval­ to elongat­ ed­shape onion, the larger the bulb size (both length and diameter), the better the bulb shape for appear­ ance and marketing purposes. the onion bulb size was found to be increased when onion sets were planted early under straw mulching and 100% of etc conditions. this could be related to the soil water availability. under water stress, the soil is drier and relatively more compacted. its mechanical resistance may limit the growth of the bulb and cause it to grow longitudinally. moreover, straw mulching can reduce fig. 3 ­ response of water productivity (wp) in 2017 (a) and in 2018 (b) to planting date (february, march, and april), soil cover system (with and without mulching), and irri­ gation level (il100, il80, and il60). error bar represents one standard deviation. fig. 4 ­ response of irrigation water use efficiency (iwue) in 2017 (a) and in 2018 (b) to planting date (february, march, and april), soil cover system (with and without mulching), and irrigation level (il100, il80, and il60). error bar represents one standard deviation. adv. hort. sci., 2020 34(1): 49­60 58 the soil evaporation and conserves soil humidity (kirda, 2000; fereres and soriano, 2007; igbadun et al., 2012), and consequently, may encourage devel­ oping onion bulbs to grow in both length and diame­ ter directions. thus, the recommended agricultural management to produce better shape of onion bulbs for consumers, is to plant onion sets in february under straw mulching and full irrigation level condi­ tions. similar results about the role of straw mulching in enhancing the bulb shape indicator were reported by mubarak and hamdan (2018 b). the bulb yield, and both wp and iwue were also found to be maximized when onion sets were plant­ ed early, under straw mulching and full irrigation. these results could be explained by the fact that the early planting partially covers the late winter time in which onion plants grow well and there would be rel­ atively plenty of water available, compared with the rest of the year from spring to the end of summer. onion sets planted early also had enough time to benefit from cool period during the vegetative stage, which improved photosynthesis, and therefore pro­ duction, compared with the actual practice followed by farmers (planting in april). this finding is in agree­ ment with similar results obtained by hamma (2013) and rohini and paramaguru (2016). also, both irriga­ tion water amount and crop water consumption were found to be greatly decreased, saving about of 30% of water when straw mulch was used compared with no­mulch conditions, irrespective of irrigation level. under current practices of planting in april without mulching, farmers are not in favor of fully irrigating their crops even if the yield is reduced, due to the huge irrigation water needs (about 900 mm). thus, using straw mulch could regulate such case. our research results indicated that onion plants planted in february and grown under straw mulch could be fully irrigated with only 550 mm. the favor­ able impact of mulching was preveiously reported (vavrina and roka, 2000; igbadun et al., 2012; hamma, 2013; tsegaye et al., 2016; mubarak and hamdan, 2018 b). in fact, mulching decreases evapo­ ration from soil surface, remaining more water avail­ able for plants (kirda, 2000; fereres and soriano, 2007; igbadun et al., 2012). this could also moderate the severity of wetting­drying cycle between irriga­ tions, and therefore, yield could be improved (vavrina and roka, 2000; gimenez et al., 2002; mubarak and hamdan, 2018 b). moreover, khaledian et al. (2010 and 2011) showed that increasing in crop yield could be also attained under straw mulching due to the enhancements in both soil fertility and soil physical properties. results indicated that the tested onion variety was very sensitive to regulated deficit irrigation. many reports cited similar results that onion yield was opti­ mized under full irrigation rather than under regulated deficit irrigation (bekele and tilahun, 2007; kumar et al., 2007; nagaz et al., 2012; igbadun et al., 2012). for example, nagaz et al. (2012) observed that irrigating onion crop with 60% of etc resulted in considerable reduction in bulb yield, dry matter, and bulbs per hectare, compared with those irrigated by 100% or 80% of etc. however, an important finding of our experiments is that the onion crop response to regulat­ ed deficit irrigation was found to be significantly enhanced when straw mulching is used. for example, irrigating with only 60% of etc using straw mulching resulted in wp and iwue much higher even than those irrigated by 100% of etc without mulching (figs. 3 and 4). the irrigation water saving in such treatment (deficit irrigation using straw mulching) could be used to irri­ gate additional cropped area. patel and rajput (2013) reported similar outcome that with 40% deficit irriga­ tion throughout the growing period, a water saving obtained could be utilized to irrigate additional ½ ha. to conclude, onion crop was found to be respon­ sive to early planting and straw mulching, so that both onion bulb size and yield were significantly enhanced, compared with those obtained under the traditional agricultural practices (planting in april without mulching). both crop water productivity and irrigation water use efficiency were also considerably increased; and the seasonal crop water requirements obviously decreased. our research results suggest that the best agricultural management is to plant onion sets in mulched soil in february under full irri­ gation conditions. moreover, early planting date and straw mulching improved the response of onion crop to the regulated deficit irrigation. this could be an appropriate agronomic alternative to meet the ever increasing demand for onions and to save irrigation water. onion bulb responses were predicted to be increased linearly with the increment in water appli­ cation rate and with the earliness of planting date, with an obvious better preference under straw mulching. both experimental data and the developed equations could be used for predicting onion crop responses under similar agro­pedo­climatic context without carrying out any additional experiments. moreover, they could be used as a tool for rational management of limited irrigation water. mubarak ‐ planting date, irrigation level, and mulch for onion crop 59 acknowledgements the author would like to thank the atomic energy commission of syria (aecs) for encouragement and financial support. references bekele s., tilahun k., 2007 ­ regulated deficit irrigation scheduling of onion in a semiarid region of ethiopia. ­ agric. water manage., 89: 148­152. brewster j.l., 2008 ­ onions and other vegetable alliums. second edition. ­ cab international, wallingford, uk, pp. 454. ceccarelli s., grando s., baum m., 2007 ­ participatory plant breeding in water‐limited environments. ­ experimental agriculture, 43: 411­435. chai q., gan y., zhao c., xu h.l., niu y., siddique kh. m., 2016 ­ regulated deficit irrigation for crop produc‐ tion under drought stress. a review. ­ 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crocus sativus l. tepals, corms, and stigmas extract on growth and photosynthetic pigments in tomato seedling a. khoulati (*), e. saalaoui laboratory of biochemistry and biotechnology, faculty of sciences, mohammed first university, oujda, morocco. key words: biostimulant, chlorophyll, saffron by­product, solanum lycoper‐ sicum l. abstract: an experiment was carried out in a greenhouse to study the effect of aqueous extracts of crocus sativus l. by­products on tomato plants. three con­ centrations of tepals and corms were used by fertigation: 1 g/l, 2 g/l, and 3 g/l. the aqueous extract of the stigmas was used as a foliar application at 0.6 g/l. the experiment was carried out in a completely randomized block with three repetitions for each concentration. the concentration of tepal extract at 3 g/l significantly (p≤0.05) increased the plants’ height, the chlorophyll a, b con­ tent in the leaf. the same results were observed for the foliar treatment with stigmas; however, there was no effect of tepal extract on the carotenoid con­ tent. on the other hand, the concentration 2 g/l of the corms extract had a positive impact (p≤0.05) in the chlorophyll b content while the concentration of 3 g/l increased the plant’s height, the chlorophyll a (p≤0.05). current results indicate that crocus sativus by­products could improve certain physiological aspects of the recipient plants and be new and natural biostimulants. 1. introduction saffron (crocus sativus l.) is a stemless, bulbous perennial plant that belongs to the iridaceae family (taylor et al., 2008). moroccan surfaces grown from saffron are about 1000 ha, mainly in the mountains of the antiatlas. this production area takes more and more importance in other regions (lage and cantrell, 2009). in the production of saffron, for each kilogram of spices produced, about 53 kg of tepals are made (maggi et al., 2012), and 90% of the total fresh weight corresponds to the by­products composed of tepals, which are generally discarded as waste (menghini et al., 2018). hundreds of corms too small for flowering to be replanted are dumped to obtain only 1 kg of dry stigmas (smolskaite et al., 2011). however, this biomass is a potentially significant source of bioactive com­ pounds. many researchers have focused their attention on valuing saffron by­products to increase crop profitability, such as floral bio­residues and (*) corresponding author: aminekhoulati89@gmail.com citation: khoulati a., saalaoui e., 2021 ­ impact of moroccan crocus sativus l. tepals, corms, and stigmas extract on growth and photosynthetic pigments in tomato seedling. ­ adv. hort. sci., 35(2): 207­213. copyright: © 2021 khoulati a., saalaoui e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2020 accepted for publication 9 april 2021 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-9981 http://phypha.ir/ppj/search.php?sid=1&slc_lang=en&auth=ennouamane http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 207­213 208 corms (lahmass et al., 2018). the enhancement of these properties of saffron floral bio­residues was evaluated on the antioxidant activity (sanchez­ vioque et al., 2012), the anti­inflammatory activity (amin and hosseinzadeh, 2015), the antifungal and cytotoxic activity (zheng et al., 2011), and antibacter­ ial activity (shadmehri et al., 2019). on the other hand, the tepals are used in many industries, as active ingredients in various food (tuberoso et al. 2016), in cosmetic formulations (natalia et al., 2019), and as a potential resource of natural color thanks to the high content of anthocyanins for food applica­ tions, and biomedical (shadmehri et al., 2019). bioactive components of corms include proteogly­ cans and saponins, which have shown antifungal and antioxidant activity (rubio­moraga et al., 2011). in a preliminary study, saffron stigmas showed a biostim­ ulant and antifungal effect on the tomato plant (khoulati et al., 2019). the applied treatments signifi­ cantly improved plant height and positively affected the tomato fruit quality after enhancing the sec­ ondary metabolites’ content. these bioactive molecules of tepals and corms are supposed to have an effect biostimulant on recipi­ ents’ plants. therefore, the current study was planned to confirm the use of tepals and corms extract as a biostimulant on the morphological pho­ tosynthetic pigment parameters of tomato seedlings. the study will promote crocus sativus l. by­product and produce new and natural biostimulants of plant growth. 2. materials and methods plant material tomato cultivar was sown in plastic trays until germination, at a temperature of 27°c ±2, 70% rela­ tive humidity, photoperiod of 16 h/8 h light/dark. at four leaves emergence, the seedlings were trans­ ferred to plastic pots (33x18 cm) containing 60% sand, 35% peat (floragard 50/50, v/v), and 5% gravel and maintained at the optimum temperature (28/ 20°c, day/ night) and natural daylight under green­ house in the northeast of morocco (34°50’33” n, 2°10’18” w). the containers are placed on 100 cm apart rows and 60 cm within rows. there was no fer­ tilizer application, and all the plants received the same volume of irrigation water. one week later, the first treatment is applied. tepals and corms extracts at three different concen­ trations (1 g/l, 2 g/l, and 3 g/l) were used both as fertigation with a 200 ml volume by one plant and 0.6 g/l used foliar application by the saffron stigmas. distilled water was taken as a control treatment. each replicate set contained three seedlings, and the experiment had three replications in a completely randomized trial. after one week from the first appli­ cation, the treatments were repeated, keeping the same concentration and method as the first applica­ tion. one week after the second application, and when the biostimulant effect was visually observed, samples were collected to determine the morpholog­ ical and photosynthetic pigment indices. aqueous extract preparation saffron stigmas originating from taliouine (morocco) were purchased, milled, and conserved at ­20°c until each use. the dried tepals were harvested during the 2019 production in the tinissane region in the northeast of morocco (34°50’33” n, 2°10’18” w). the tepals were milled and stored at ­ 20°c until each use. the corms were obtained from taliouine and kept at ­20°c after drying. before each application, the concentration of tepals and corms powder used for the study left 24 h in 1 l of distilled water for extraction in the dark for each concentration: 1 g/l, 2 g/l, and 3 g/l. for stigmas foliar application, 0.6 g extracted in 1 l of the distilled water 24 h in the dark. quantification of the main components of extracts ­ the anthocyanin contents of tepals were mea­ sured according to the protocol explained by ganjewala et al. (2008). the absorbance (abs) of the samples was measured at 530 nm, and the results were expressed in milligram equivalent cyanidin 3­ glucoside by 100 grams of dry matter (mg cge/100g dm). flavonol’s contents were determined by mea­ suring the absorbance at 360 nm, and the results were expressed in milligram equivalent quercetin 3­ glucoside by 100 grams of dry matter (mg qge/100g dm). ­ the total polyphenol of tepals was determined using the folin­ciocalteu method (velioglu et al., 1998). the total polyphenols concentrations were expressed in milligrams of gallic acid equivalent to 100 grams of dry matter (mg gae/100 g dm). ­ the content of crocin, picrocrocin, and safranal of saffron stigmas was determined following iso 3632 (iso, 2003). morphological indices of tomato seedling plant height was recorded using a measuring tape in cm. shoot fresh weight was measured immediately khoulati and saalaoui ‐ saffron tepals, corms, and stigmas as biostimulants 209 after rooting up the plants using an electronic bal­ ance. to determine the dry weight, samples were oven­dried for 24 h at 80 °c, and the value of the samples recorded in grams. photosynthetic pigments the chlorophyll content and carotenoid were determined by crushing 0.2 g fresh leaf sample in 20 ml of 80% acetone and placed at room temperature for 48 h in the dark. a spectrophotometer was used to observe the absorbance at 663, 645, and 652 nm of the samples (lichtenthaler, 1987). chlorophyll a, b, and total chlorophyll calculated using the following formula: chlorophyll a (chl a) = (12.7 x abs663) ­ (2.69 x abs645) chlorophyll b (chl b) = (22.99 x abs645) ­ (4.68 x abs663) total chlorophyll = chl a + chl b total carotenoid content = (1000 abs470 ­ 1.82 chla­85.02 chlb) 198 statistical analysis all figures and statistical analyses were performed using spss statistics 17.0 software. the data were expressed as the mean ± standard deviation (sd) of triplicate independent experiments and analyzed using a one­way analysis of variance (anova). p≤0.05 was treated to be statistically significant. the student­newman­keuls (snk) test was used to classi­ fy averages using different letters for significant dif­ ferences. 3. results the main components of extracts the anthocyanins, flavonols, and polyphenols con­ tents of tepals were 43.66 ± 0.79 mg cge/100 g dm, 0.19 ± 0.21 mg qge/100 g dm, and 672.73 ± 1.02 mg gae/100 g dm, respectively. total crocin, safranal and picrocrocin contents in the saffron sample are 184.56 ± 2.19 mg/g, 4.89 ± 0.18 mg/g, and 62.43 ± 0.37 mg/g, respectively. morphological indices variations in the morphological data revealed the significant effect of tepals, corms, and stigma extracts on tomato seedling growth in terms of concentra­ tions used (figs. 1, 2). for the application of tepal extracts by fertigation, the highest plant height was recorded for plants fertigated with 3 g/l followed by 2 g/l. in contrast, the lowest values were observed in fig. 2 ­ tepal, corm, stigma extracts application to influence the tomato plant's plant height. the pictures were taken one week after the last treatment. 0 represents control treatment. a) treatment with petal extract by fertigation (2 g/l, 3 g/l). b) treatment with corm extract by fertigation (1 g/l, 2 g/l, 3 g/l). c) foliar application by saffron stigma extract at 0.6 g/l. fig. 1 ­ saffron stigmas, tepals, and corms extracts affect the hei­ ght of the tomato seedling. measurements were made one week after the last treatment. data were recorded for a total of three plants by treatment replicated three times. the concentration of saffron was represented as 0.6 g/l. the concentrations of tepals and corms are defi­ ned as 1 g/l, 2 g/l, and 3 g/l. 0: control. bars represent averages and error lines one standard deviation. the dif­ ference in the letters indicates significant differences at p≤0.05 based on the snk test. ***: the significative diffe­ rence at p≤0.001. adv. hort. sci., 2021 35(2): 207­213 210 control seedlings. concerning the treatment of corms highest plant was recorded for plants fertigates with 3 g/l followed by 2 g/l and 1 g/l compared to the control. however, the treatment by stigmas foliar application showed a significant difference at p≤0.001 compared to the control (figs. 1, 2). table 1 represents fresh shoot weight, dry shoot weight, fresh root weight, and dry root weight. as compared to control, the fresh and dry weight of seedlings were increased with tepal application and reached a signif­ icant level at 2 g/l followed by seedlings applied with 3 g/l. however, the same concentrations of applied tepal increased the weight of root fresh and root dry at 2 g/l, followed by 3 g/l. however, no effect of the application of the different concentrations of corm extract observed. photosynthetic pigments the effect of tepals and corms on the chlorophyll contents of tomato leaves is depicted in figure 3. as it can be observed, the various applications influ­ enced significantly enhanced the chlorophyll a, b, and total chlorophyll content. concerning the use by tepal fertigation, the high value was recorded for the concentration of 3 g/l with an increase of 45.96 % for chla and 28.71 % for chlb, compared to the control. the application by corm fertigation at 3 g/l has increased chla and chlb concentrations of 37.12 % and 15.22 %, respectively (fig. 3), compared to the control. on the other hand, the three corm extract concentrations significantly increased the carotenoid content (table 1). however, no effect was observed on the carotenoid content after fertigation by the three different tepals (table 1). for foliar application by saffron stigmas, the chla, chlb, and carotenoid content significantly increased 11.59 % for chla, 56,84 % for chlb, and 55,69% for table 1 ­ influence of tepal and corm application on the root and shoot weight of tomato plants and carotenoid leaves the difference in the letters indicates significant differences at p≤0.05 based on the snk test. * = significative difference at p≤0.05. fw= fresh weight. fig. 3 ­ effect of tepal and corm extract on pigment system of tomato leaves. data are the mean ± standard deviation of three replicates. the column followed by different let­ ters shows a significant difference at p ≤ 0.05 significance level between treatments according to the snk test. fw= fresh weight. chla= chlorophyll a; chlb= chlorophyll b; totalchl=: total chlorophyll. 0= control.letters are not significantly different, as determined by tukey's test (p<0.05). application concentration (g/l) root fresh weight (g) root dry weight (g) shoot fresh weight (g) shoot dry weight (g) carotenoid (mg/g fw) control 0 0.185±0.289 a 0.0192±0.004 1.72±0.135 a 0.132±0.017 2.431±0.108 a tepal 1 0.195±0.009 a 0.021±0.001 * 1.68±0.355 a 0.118±0.036 2.507±0.209 2 0.261±0.341 b 0.0323±0.005 * 2.641±0.564 b 0.218±0.046 * 2.703±0.335 3 0.212±0.013 a 0.0283±0.008 * 2.332± 0.156 ab 0.218±0.046 * 2.985±0.337 corm 1 0.138±0.427 0.017±0.003 1.436±0.613 0.119±0.060 2.98±0.652 b 2 0.141±0.018 0.019±0.003 1.516±0.548 0.107±0.045 2.889±0.147 b 3 0.142±0.519 0.016±0.004 1.495±0.591 0.01±0.044 2.708±0.15 b khoulati and saalaoui ‐ saffron tepals, corms, and stigmas as biostimulants 211 2 g/l, and 3 g/l) are chosen to assess their effect by fertigation in some morphological and biochemical parameters of tomato seedling under greenhouse conditions in a completely randomized block. the application of tepal, corm, and stigmas extracts has influenced the growth of the plant. tepal has been reported as a protein source, fiber, fats, and essential minerals (k, ca, and p) necessary for plants’ growth (fahim et al., 2012; khazaei et al., 2016). tepals are also rich sources of phenolic and biologically active compounds such as flavonoids (kaempferol, rutin, quercetin, luteolin, hesperidin, and bioflavonoids), tannins, and anthocyanins (kanakis et al., 2006; srivastava et al., 2010). the increased growth of tomato plants suggests that tepal extract may act as a promoter of plant growth with a 3 g/l concentration, which contains 1.308 mg cge/l of anthocyanin and 20.181 mg gae/l of polyphenol. corms also are a rich source of phenolic, flavonoid, and especially saponin (rubio­moraga et al., 2013), likely to participate in increased growth characteristics of the treated plants in the results of current research. saffron stigmas contain nitroge­ nous substances, anthocyanins, glycosides, monoter­ penes, aldehydes, flavonoids, vitamins, volatile oils, proteins, carbohydrates (amin and hosseinzadeh, 2015). also, some studies indicated the presence of micro­nutrients in saffron such as zn, mn, and certain amino acids (priscila del campo et al . , 2009; d’archivio et al., 2014), which favored the develop­ ment of the plant height and that the current results strongly corroborated with our previous study (khoulati et al., 2019) with a concentration of 0.6 g/l, which contains 110.73 mg/l of crocin, 2.93 mg/l of safranal, and 37.45 mg/l of picrocrocin in the extract used in this study. photosynthesis is one of the primary processes of plant metabolism impacted by external conditions (kalaji et al., 2017). chlorophyll is a critical compo­ nent of photosynthesis which absorbs sunlight (hörtensteiner and kräutler, 2011). it occurs in chloroplasts as green pigments in all photosynthetic plant tissues (mazumder and paul, 2014). chla, the primary photosynthetic pigment of photosystems i and ii, converts the energy of light. chlb is an acces­ sory pigment that absorbs light’s energy (petit et al., 2012). on the other hand, carotenoids are also essential for plants because they protect the photosynthetic apparatus from light­mediated stress. carotenoids participate in a wide range of physiological process­ carotenoid (fig. 4, table 1). however, no significant difference was reported from the different tepal extract concentrations on the carotenoid content (table 1). the different concentrations of the corm extract participated in the increase of the chla and chlb content but not in the same way as the tepal extracts; the highest value in chla in plants treated with 3 g/l of the extract corms is 12.73 mg/10 g fw and while the highest value in chla in plants treated with 3 g/l of the tepal extract is 13.55 mg/ 10 g fw. however, a significant difference is observed in the carotenoid content of plants treated with extracts of corms, while this difference in carotenoid is absent in plants treated with tepal extracts. chlorophyll con­ tent, especially chlb of plants treated with the appli­ cation of stigmas extract at 0.6 g/l, increased. the carotenoid content also increased after treatment with the stigma extract. 4. discussion and conclusions the present study’s objective was to valorize the tepals, corms, and stigmas of crocus sativus l. and take advantage of its bioactive components to use them as a biostimulant alternative to chemicals prod­ ucts. therefore, three tepals and corm extract (1 g/l, fig. 4 ­ effect of saffron stigmas on pigment system of tomato leaves by foliar application. data are the mean ± stan­ dard errors of three replicates. ***= the significative dif­ ference at p≤0.001. *0 the significative difference at p≤0.05. fw= fresh weight. chla= chlorophyll a; chlb= chlorophyll b; totalchl= total chlorophyll. 0= control. quality performance and does not present risks for the user and the consumer. besides, these results present an advantage for the farmer practicing the culture of crocus sativus by recovering the waste which they throw before like tepals. however, future research needed to explore the influence on the treated plants’ primary and sec­ ondary metabolites provides a platform to elaborate and study the biological activity of the extracts inside the treated plants to identify and later confirm the molecular patterns involved in the bioactive mecha­ nism of these extracts. acknowledgements authors express their thanks to rayan agricole, 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kiwifruit showed no significant decrement of fruit set and fruit weight within the 4­day and 2­day period, respectively, however, the mean weight of fruit was ≥ 85 g within 4­day. fruit set was 100% when pollina­ tion was carried out during the first 3 days following anthesis. fruit set decreased to 20.71% when flowers were pollinated 5 days after anthesis and were practically nil by 6 days after anthesis. fruit weight and size were the highest on days 1­2 after anthesis and reduced for flowers pollinated 3­4 days after anthesis. the lowest fruit weight and seed weight and number were observed when pollination was done on day 5. hayward kiwifruit showed no significant drop in fruit set within the 4­day period, and thus, appears to have an epp equal 4 days after anthesis. thus, efforts for producing good quality and of marketable size fruit should be concentrated within the first 4 days after anthesis. 1. introduction hayward kiwifruit (actinidia deliciosa) is the most widely grown actinidia crops (ferguson, 1990). this cultivar is chosen based on its large fruit production and long storage life. optimal kiwifruit production is highly dependent on the level of pollination because insufficient pollina­ tion is known to lead to unsatisfactory fruit size, shape and uniformity; however, pollination of kiwifruit is impaired by the dioecious nature of the species (pyke and alspach, 1986). flower receptivity can be evaluated by determining the effective polli­ nation period (epp). epp is defined as the number of days following anthesis during which pollination is effective in producing marketable fruit. various factors may affect the epp. it was shown that the epp could be affected by temperature, flower quality, and chemical treatments (sanzol and herrero, 2001). because of dioecious nature of kiwifruit, other factors should be in a favorable condition to determine of epp in (*) corresponding author: eabedig@yahoo.com citation: gheshlaghi e.a., 2019 ­ effective pollination period and its influence on fruit characteristics of ‘hayward’ kiwifruit. ­ adv. hort. sci., 33(4): 537­ 542. copyright: © 2019 gheshlaghi e.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 may 2019 accepted for publication 4 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 537­542 538 this species. for example, male vines can be properly distributed, and proper timing of beehive placement can be achieved. in addition, determining the epp of kiwifruit species/cultivars could allow growers to optimize supplemental pollen applications by apply­ ing only during the epp, and thus, reduce costs. kiwifruit flowers are receptive for only a few days following anthesis where pollination can be success­ ful leading to a good marketable fruit set. the epp may be restricted by limitation in three main events along the reproductive process, stigma receptivity, pollen tube kinetics and ovule longevity (sanzol and herrero, 2001). estimation reports are available for the different cultivar of kiwifruit. there are variances among the reported duration of the epp and stigma receptivity. according to galimberti et al. (1987), the epp of hayward cultivar reported 3 days in italy. the epp for a. deliciosa ‘hayward’ was determined to be 4 days by gonzalez et al. (1995) in spain. it was discovered that the duration of stigma receptivity closely fit the epp, thus it appears that the epp is limited by stigma receptivity. sale (1981) reported that the pistillate flowers are receptive for 7­9 days after anthesis (daa) in new zealand. goodwin (2000) also reported this longer period, where the relatively constant and high receptivity was displayed for the first 8 days before dropping. goodwin et al. (2013) found stigma receptivity to be highest during the first 2 daa in a. chinensis ‘hort16a’. they noted that pistillate flowers from a. chinensis dehisced their petals after 2 days while a. deliciosa typically hold their petals for 5 daa. the epp for a. deliciosa ‘au fitzgerald’ and a. chinensis ‘au golden sunshine’ was determined to be 4 and ≥5 days respectively by thompson (2014). in golden kiwifruit cultivars, not only fruit set but also fruit characteristic was affected by daa pollination. fruit size, weight, and seed number were reduced on day 5 after anthesis in ‘au fitzgerald’ (thompson, 2014). thompson (2014) found that ‘au golden sunshine’ showed no significant drop in fruit set or size within the 5­day period. however, differences in fruit weight, fruit size index and seed number were found between 1­3 and 4­5 daa by brantley (2016). fruit set of ‘hayward’ cultivar is influence by the time elapsed between anthesis and pollination. it seems that fruit traits in this cultivar may be affected by days after anthesis pollination too. there are some papers reporting contrasting results about the effective pollination period on fruit quantitative and qualitative traits in this cultivar. therefore, the aims of this research were to evaluate the effective polli­ nation period of ‘hayward’ and to determine fruit characteristics in relation to the time of pollination. 2. materials and methods experimental design this experiment was conducted using 10­year old vines of ‘hayward’. kiwifruit vines were grown in an orchard located in astara, guilan province, iran (38°22’n; longitude of 42°50’e), 10 m altitude, trained to a t­bar system with plants spaced of 4×6 m. treatment application effective pollinated period (epp) was studied for 3 years. ‘hayward’ flower buds were bagged on may 21, 2013; may 18, 2014, and may 23, 2015, using wax paper bags (10.2 × 26.2 cm). flower buds were bagged 1 day before anthesis; still completely closed but showing some white from petal unfolding, identi­ fied as “stage 57” in bbch phenology system (salineroa et al., 2009). anthesis was the day the flower petals opened. the detailed characterization of environmental conditions during the experimental period is shown in table 1. year month temperature (°c) evaporation (mm/d) relative humidity (%) precipitation (mm) sunny hoursmean max. min. max. abs. min. abs. 2013 april 13.0 16.5 9.5 25.4 4.2 43.1 81 81.8 115.2 may 17.0 21.7 12.3 27.2 4.8 114.4 76 29.4 236.4 june 22.5 27.2 17.7 32.8 13.0 175.7 73 58.7 278.3 2014 april 11.1 15.3 6.9 24.8 0.4 68.7 79 95.6 172.7 may 19.1 23.6 14.5 30.8 10.4 142.8 74 34.7 249.3 june 23.5 27.9 18.6 32.2 14.8 175 74 15.6 269.4 2015 april 9.6 14.9 12.2 27.2 5.0 59.8 85 44.6 120.6 may 13.9 19.8 16.9 24.2 7.8 106.9 81 23.5 189.3 june 20.6 28.1 24.3 33.4 17.0 184.7 70 0.8 236.1 table 1 ­ air temperature, relative humidity (rh), sunny hours, precipitation and mean daily transpiration (mm d­1) during three months in 2013­15 gheshlaghi ‐ effective pollination on kiwifruit ‘hayward’ 539 for every year, pollen was collected from stami­ nate vines (tomuri) one day before anthesis and dried on paper at room temperature. the pollen was then sieved using a fine mesh (0.26 mm) to remove dehisced anthers and other impurities. bag­isolated pistillate flowers were hands pollinated with the dried pollen before being re­bagged. twenty four flowers were hand­pollinated each day at 0, 1, 2, 3, 4, 5, 6 and 7 daa using dried pollen. uniform and single flower inflorescences were hands pollinated during the study. flowers were re­bagged using newly labeled bags immediately following hand­pollination to prevent subsequent open pollination. data collection fruit set of all epp treatments was determined when bags were removed three weeks later after pollination. for fruit trait analyses, they were har­ vested on november 7, 2015, when the value of total solid soluble (tss) detected was =6.2°brix. fruit length (l, mm), major width (w1, mm) and minor width (w2, mm), fruit density (g/ml), fresh weight (g), fruit volume (ml), fruit size index [(l+w1+w2) * 3­ 1)] seed number and seed weight (g) as quantity char­ acteristics were measured (brantley, 2016). to deter­ mine the average seed weight of each fruit, weight of 100­seed samples was determined thrice and the average of these values was used to calculate the total seed number for each fruit of each treatment (goodwin et al., 2013). statistical analysis combined analysis of three years fruit set data was performed on according to a completely ran­ domized design. data of fruit traits were analyzed as a completely randomized design. all significant means were separated, using duncan (p≤0.05). the correlation between fruit weight with seed number and weight were calculated via the software spss 22. 3. results fruit set after hand pollination was the highest, averaging 100% during the first 3 days following anthesis (fig. 1). however, no significant differences were found between the days of treatment 1­4 (p≤0.05). fruit set dropped down to 20.71% in the flower pollinated at 5 daa. by 6 daa, fruit set was practically nil. thus, the epp was limited to the first 4 daa (fig. 1). fruit weight and size (equivalent water volume) were the highest on day 1 (table 2). a reduction trend was observed in flowers pollinated 2 daa, however, did not differ from 1 daa (p≤0.05). fruit weight and size were reduced for flowers pollinated 3­4 daa and the lowest value was observed on those pollinated 5 daa (p≤0.05). fruit density, fruit length, and fruit size index were the highest in the 1 daa (table 2) because the values were reduced on 2­4 daa and the lowest value observed on day 5. fruit major and minor width, and seed weight and the table 2 ­ effects of hand pollinating actinidia deliciosa ‘hayward’ flowers 1, 2, 3, 4, or 5 days after anthesis (daa) on fruit characteri­ stics. fruit were harvested 7 nov. 2015 each values is the mean of three replicates with 15 fruits. means in each column followed by the same letter are not significantly diffe­ rent according to duncan’s multiple range test (p≤0.01). daa weight (g) fruit volume (ml) fruit gravity (g/ml) fruit length (mm) major width (mm) minor width (mm) fruit size index seed weight per fruit (g) seed number per fruit seed weight (mg) 1 105.86 a 103.54 a 1.02 a 70.18 a 53.20 a 47.22 a 56.87 a 1.61 a 1218.97 a 1.33 a 2 95.10 ab 93.54 ab 1.016 b 64.86 b 52.72 a 46.35 a 54.64 b 1.56 a 1326.43 a 1.17 b 3 84.89 b 83.83 b 1.012 bc 63.18 b 50.42 ab 45.11 ab 52.91 b 1.56 a 1239.06 a 1.25 a 4 85.16 b 83.74 b 1.016 b 61.86 b 51.12 ab 45.07 ab 52.68 b 1.56 a 1226.35 a 1.27 a 5 71.73 c 71.10 c 1.008 c 56.44 c 49.37b 43.32 b 49.71 c 0.64 b 498.73 b 1.33 a fig. 1 ­ effects of hand pollinating actinidia deliciosa ‘hayward’ flowers 1, 2, 3, 4, or 5 days after anthesis (daa) on fruit set from 2013 to 2015. values are the mean three years ± se of three replicates. adv. hort. sci., 2019 33(4): 537­542 540 number did not show significant reduction 1­4 daa, however, the value was strongly reduced for flowers pollinated at 5 daa (p≤0.05). correlation analyses showed a positive significant correlation between fruit size with seed number (r = 0.54**), and seed weight (r = 0.58***) (table 3). the correlation between fruit weight with seed weight was higher than fruit weight with the seed number. 4. discussion and conclusions effective pollination period is important in kiwifruit because successful pollination results in more seeds per fruit, and seed number directly corre­ lates with fruit size and weight (hopping, 1976). the results of this study for determination of epp in ‘hayward’ cultivar suggest that flowers should be pollinated within 4 daa for the successful fruit set. epp results of this study were similar to that founded by gonzalez et al. (1995) for ‘hayward’ in spain. however, the greatest size, weight and fruit density occurred when flowers were pollinated within 1 daa (2015). by extending the pollination period from 4 daa to 5 daa, a 79.29 % decrease in mean of fruit set was observed along the 3 years of observations. a similar situation has been recorded under different cultural conditions for ‘hayward’ (gonzalez et al., 1995), au golden sunshine’ (actinidia chinensis) and ‘au fitzgerald’ (a. deliciosa) (thompson, 2014; brantley, 2016). eep determined by the longevity of the ovules minus the time lag between pollination and fertiliza­ tion (sanzol and herrero, 2001). eep is affected by the growth rate of the pollen tube. the temperature has a clear effect on pollen tube growth rate (hedhly et al., 2005), and on flowering period of this study an optimal temperature for pollen tube growth (mean temperature 20.4 to 23.7°c) occurred during 3 years (manandhar and lawes, 1980). due to the viability of ovules for the 7 days following anthesis in kiwifruit (gonzalez et al., 1995), germination of pollen and pollen tube kinetics may be the limiting factor for epp. according to results of gonzalez et al. (1995), the viability of ovules is 7 days in kiwifruit and an under optimal temperature, pollen tube reached the ovules 3 days after pollination, so that, there are 4 days between flower anthesis and fertilization. thus, it appears stigma receptivity may not be the limiting factor for epp, and under any condition, maximum epp will be 4 days. however, hopping and jerram (1979) observed that the pollen tubes reached the style base and fertilized the ovules inside the ovary by 31 hours and 43 hours respectively. according to this result, there are more than 4 days between flower opening and pollination, and stigma receptivi­ ty may be the limiting factor. the difference in the pollen tube kinetics may be the result of pollen origin (guerrero­prieto et al., 1985), nutritive stage of the flower (nyomora et al., 2000) or environmental con­ ditions (jefferies et al., 1982). epp is short for kiwifruit. however, having a genetic component epp with cultivars, it is affected by the nutritive state of the tree and weather alter­ ation (gonzalez et al., 1995), crop load (crisosto et al., 1988; buszard and schwabe, 1995), temperature, flower quality, and chemical treatments (sanzol and herrero, 2001) and alternate bearing (brantley, 2016). in this study, fruit set averaged 99.8% for 4 daa and decreased to 20.27% for day 5 in ‘hayward’ culti­ var along 3 years of studies. in the one­year study of gonzalez et al. (1995) in this cultivar, fruit set aver­ aged 80% after hand pollination during the first 4 daa before declining to 36% on day 5 and then almost 0% by day 7. they attributed this to the loss of papillar integrity i.e. the unicellular papillae that cover the stigma began to rupture. they considered 80% fruit set to be successful pollination, and based on this, determined the epp of ‘hayward’ to be 4 daa. they did not publish data for a seed count num­ ber or fruit size. brantley (2016) reported averaging 98% fruit set for the first 4 daa before declining to 81.5% for day 5 in ‘au fitzgerald’. it seems plausible that this variance in fruit set was due to the alternate bearing tendencies of the species a. deliciosa (morley­bunker and lyford, 1999), flower quality and pollen source (brantley, 2016). presently, no research has been conducted on the effects of alternate bearing on pollination of kiwifruit flowers. however, epp of malus domestica l. borkh. cv. cox’s orange pippin was influenced by crop loads table 3 ­ correlation coefficient between fruit weight with seed number and weight in actinidia deliciosa ‘hayward’ fruit weigh seed number seed weight fruit weight 1 0.54 ** 0.58 *** seed number 0.54 ** 1 0.95 *** seed weight 0.58 *** 0.95 *** 1 **, *** correlation is significant at the 0.01 and 0.001 levels respectively. gheshlaghi ‐ effective pollination on kiwifruit ‘hayward’ 541 ing systems, the position of the fruit on the vines in any particular training system and leaf to fruit ratio (sale, 1981; clinch, 1984; hopping, 1986; pyke and alspach, 1986; woolley et al, 1988; ferguson, 1990; gonzalez et al., 1998; goodwin, 2000). there are several published relationships between the fruit weight and the number of seed or seed dry weight per fruit (hopping, 1986; pyke and alspach, 1986; testolin et al., 1991; goodwin, 2000). according to these researchers, to obtain a size of 70 g and 100 g fruit, 180 to 840 and 620 to 1290 seeds per fruit are required respectively. the seed­to­fruit relationships from these sources display very large scatter and either linear or non­linear positive corre­ lation. seed number may not be the only factor that plays a role in increasing fruit size. this is thought due to the influence of other factors such as the loading of the vine (testolin et al., 1991), cultural methods and orchard microclimates (clinch, 1984), the age of flowers, storage carbohydrates, leaf num­ ber, and unknown factors should also be considered. based on the findings of this study, not only fruit set but also fruit traits of ‘hayward’ cultivar were affect­ ed by pollination in relation to the time elapsed since anthesis. therefore, pollination efforts for a. deli‐ ciosa ‘hayward’ may be concentrated within the first 4 daa for having high fruit set and fruit traits. however, the maximum fruit size and weight obtained within the first daa in this research. references brantley a.k., 2016 ­ effective pollination period and influence of crop load management on au kiwifruit cul‐ tivars. ­ m.s. thesis, auburn univ. auburn, alabama. usa. buszard d., schwabe w.w., 1995 ­ effect of previous crop load on stigmatic morphology of apple flowers. ­ j. am. soc. hort. sci., 120: 566­570. clinch p.g., 1984 ­ kiwifruit pollination by honeybees. ­ tauranga observation 1978­81. ­ n.z. j. exp. agric., 12(1): 29­38. crisosto c.h., lombard p.b., sugar d., polito v.s., 1988 ­ putrescine influences ovule senescence, fertiliza‐ tion time, and fruit set in ‘cornice’ pear. ­ j. am. soc. hort. sci., 113: 708­712. ferguson a.r., 1990 ­ kiwifruit management, pp. 472­ 503. ­ in: galletta g.j., and d.g. himelrick (eds.) small fruit crop management, prentice­hall, inc., englewood cliffs, nj, usa. galimberti p., marro m., youssef j., 1987 ­ periodo utile d’impollinazione in actinidia chinensis (planch). ­ of the prior year in a study by buszard and schwabe (1995). according to the results of their research, de­ fruited trees in the previous prior year had flowers that were receptive to pollen at opening time while trees that carried a heavy crop load in the previous year had flowers that were not fully receptive to pollen until 3 daa. we hand­pollinated uniform and single flower inflorescences during the study, mean­ while, gonzalez et al. (1995) did not mention about flower quality. in addition, gonzalez et al. (1995) used the pollen of male c for hand pollination, how­ ever, in our study, ‘tomuri’ pollen was applied for pollination. the number of seeds is in turn related to the number of viable pollen deposited on the stigma. even though a successful mating of a pollen tube with an ovule produces a seed, the number of pollen grains needed per seed ranges from 3.0 to 5.2 depending on the male clones (hopping and martyn, 1990). this is because some pollen tubes die during the passage through the style. in this study, no differences in fruit set, seed num­ ber and weight were observed between 1­4 daa. however, differences were observed for fruit weight, size, and density. fruit weight reduced about 10 and 20 g in 2 and 3­4 daa respectively. however, fruit weight between 1­4 daa was ≥85 g and had more than 1200 seed per fruit. gonzalez et al. (1998) observed similar results in a. deliciosa ‘hayward’. they detected the bulk of the fruit produced by a. deliciosa ‘hayward’ with hand pollination were 80­ 110 g and stated that hand pollination increased the final value of the crop by 10%. despite no differences in the number of seeds, average fruit weight was reduced from 10 to 20 g between 2­4 daa. under the terms of the similar fruit set, fruit weight will not be the same. similar results were reported in actinidia chinensis ‘au golden sunshine’ by thompson (2014). gonzalez et al. (1998) reported while fruit set is similar to that obtained with hand pollination and mechanical system, a high difference was obtained in fruit quality, where pollination hand still significantly improved mechanical pollination in terms of fruit size and weight. on day 5, not only fruit weight, but also seed number and weight reduced 34 g, 720, and 0.96 g, respectively. thus, about 60% reduction of seed number on day 5, resulting reduction of fruit weight and size about 31%. because seed­derived hormones are required to promote fruit growth (woolley et al., 1988). however, the final fruit size is also affected by the management factors such as crop loadings, nutri­ ents, irrigation, time of anthesis, flower quality, polli­ nation systems, beehives management, type of train­ 542 adv. hort. sci., 2019 33(4): 537­542 frutticoltura, 1: 51­54. gonzalez m.v., coque m., herrero m., 1995 ­ stigmatic receptivity limits the effective pollination peri‐ od in kiwifruit. ­ j. am. soc. hort. sci., 120: 199­202. gonzalez m.v., coque m., herrero m., 1998 ­ influence of pollination systems on fruit set and fruit quality in kiwifruit (actinidia deliciosa). ­ ann. appl. biol., 132: 349­355. goodwin r.m., 2000 ­ zespri innovation kiwifruit pollination manual. ­ zespri innovation company limited & the horticulture and food research institute of new zealand limited. goodwin r.m., mcbrydiem h.m., taylor m.a., 2013 ­ wind and honey bee pollination of kiwifruit (actinidia chinensis ‘hort16a’). ­ n. z. j. bot., 51: 229­240. guerrero­prieto v.m., vasilakakis m.d., lombard p.b., 1985 ­ factors controlling fruit set of ‘napoleon’ sweet cherry in western oregon. ­ hortscince, 20: 913­ 914. hedhly a, hormaza j., herrero m., 2005 ­ the effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach. ­ plant biol., 7: 476­83. hopping m.e., 1986 ­ kiwifruit, pp. 217­232. ­ in: mon­ selise s.p. (ed.) crc handbook of fruit set and develop‐ ment, crc press, boca raton, florida, usa, pp. 578. hopping m.e., jerram e.m., 1979 ­ pollination of kiwifruit (actinidia chinensis planch.): stigma‐style structure and pollen tube growth. ­ n.z. j. bot., 17: 233­ 240. hopping m.e., martyn j. 1990 ­ are some males better than others? n. z. kiwifruit, 65: 9. jefferies c.j., brain p., stott k.g., belcher a.r., 1982 ­ experimental systems and a mathematical model for studying temperature effects on pollen‐tube growth and fertilization in plum. ­ plant, cell environ., 5: 231­ 236. manandhar d.n., lawes g.s., 1980 ­ blossom sprays and their effect on fruit set. ­ orchardist n. z. 53: 269­ 272. morley­bunker m., lyford p., 1999 ­ kiwifruit, pp. 78­ 84. ­ in: jackson d.i., and n.e. looney (eds.). temperate and subtropical fruit production. cabi publishing, new york, ny, usa, pp. 336. nyomora a.m.s., brown p.h., pinney k., polito v.s., 2000 ­ foliar application of boron to almond trees affects pollen quality. ­ j. am. soc. hort. sci., 125: 265­ 270. pyke n., alspach p., 1986 ­ inter‐relationship of fruit weight, seed number and seed weight in kiwifruit. ­ n.z. j. agric. sci. 20: 153­156. sale p.r., 1981 ­ kiwifruit pollination, male to female ratios and bee activity. aglink hpp233. ­ n. z. ministry of agriculture and fisheries, p. 3. salineroa m.c., velaa p., sainzb m.j., 2009 ­ phenological growth stages of kiwifruit (actinidia deli­ ciosa ‘hayward’). ­ sci. hort., 121: 27­33. sanzol j., herrero m., 2001 ­ the “effective pollination period” in fruit trees. ­ sci. hort., 90: 1­17. testolin r., vizzotto g., costa g., 1991 ­ kiwifruit polli‐ nation by wind and insects in italy. ­ n.z. j. crop hort. sci., 19: 381­384. thompson a.b., 2014 ­ determining the effective pollina‐ tion period and effects of crop load reduction on au kiwifruit cultivars. ­ m.s. thesis, auburn univ. auburn, alabama. usa. woolley d.j., lawes g.j., lai r., 1988 ­ factors affecting fruit size. new zealand kiwifruit authority national research conference, new zealand kiwifruit special publication no.2, rotorua, new zealand, pp. 11­14. impaginato 159 adv. hort. sci., 2023 37(2): 159­171 doi: 10.36253/ahsc­13753 exogenous application of biostimula­ tors alleviates water deficient stress on azadirachta indica plants t.a.m. abou dahab, h.a. ashour (*), saber m.m.h. department of ornamental horticulture, faculty of agriculture, cairo university, giza, egypt. key words: brassinolide, chitosan, drought stress, neem. abstract: pot experiment was conducted to evaluate the effect of chitosan or brassinolide applications on morphology and physiology parameters of azadirachta indica grown under water deficient stress. the plants received dif­ ferent irrigation intervals, and were sprayed monthly with either chitosan or brassinolide each at concentrations of 50, 100 and 200 ppm, while the control plants were sprayed only with tap water. the results showed that water stress reduced all growth parameters, chemical constituents of pigments content, total carbohydrates, n, p, and k%, total indoles, while proline and total phenols content were increased. instead, the plants sprayed with the higher concentra­ tions of chitosan or brassinolide resulted in significant increase in growth para­ meters, pigments content, total carbohydrates, proline content, n, p and k%, total indoles while reduced total phenols content. based on the obtained results it can be concluded that, foliar application of chitosan or brassinolide at 200 ppm can alleviate the adverse effects of water deficient stress on the growth and physiology parameters of azadirachta indica. 1. introduction azadirachta indica a. juss is a tropical evergreen tree that belongs to the family of meliaceae, commonly known as neem, nimtree or indian lilac. it is native to india, its fruits and seeds are the source of neem oil (koul and wahab, 2004). in addition to use of neem for landscape acti­ vates as an ornamental tree, all parts of the tree have been utilized medi­ cinally and now being used in pharmaceutical and cosmetics industries. neem fruits, seeds, oil, leaves, bark and roots used as antiseptics, antimi­ crobials, anti­inflammatory, anti­cancerous and anti­diabeticv (islas et al., 2020). water deficiency is the major environmental factors, as a biotic stress­ es limiting agricultural production in most countries, especially in the arid and semi­arid regions, affecting the quality, growth and production of pants. water stress induces various physiological, biochemical changes in ornamental plants such as a reduction in the growth parameters (abd­ elmoneim et al., 2018; yousaf et al., 2018; el­shanhorey and sorour, (*) corresponding author: hossam.ahmed@agr.cu.edu.eg citation: abou dahab t.a.m., ashour h.a., saber m.m. h., 2023 ­ exogenous application of biostimula‐ tors alleviates water deficient stress on azadirachta indica plants. ­ adv. hort. sci., 37(2): 159­171. copyright: © 2023 abou dahab t.a.m., ashour h.a., saber m.m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 september 2022 accepted for publication 23 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13753 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 159­171 160 2019; shaltout et al., 2022), reductions in total chlorophyll and carbohydrate contents (sarker and oba, 2018), reductions in nutrient accumulation (singh and singh, 2020), as well as increasing in pro­ lin, phenols content (el­gamal and khamis, 2021). recently, study for biological methods to avert uti­ lization of chemical products and alleviating the harmful effect of water deficient in agriculture has led to utilize of bio­stimulators. among the various kinds of bio­stimulators are chitosan and brassinolide. chitosan (cht) is a deacetylated derivative of chitin. it is a natural polymer and biodegraded by bio­ logical agents and it is environment­friendly used in agriculture (shafiei­masouleh, 2019). under stressed conditions, cht has the efficiency to mitigate the harmful effects of drought by promoting chlorophyll, carbohydrates, proline content and the capacity of antioxidant activities (pirbalouti et al. , 2017; vosoughi, et al., 2018; elansary et al., 2020). the favorable influence of cht induces photosynthetic rate, stomatal closure through aba synthesis; stimu­ lates antioxidant enzymes via nitric oxide and hydro­ gen peroxide signaling pathways, and promotes pro­ duction of organic acids, sugars, amino acids and other metabolites that are required for the osmotic adjustment, stress signaling, and energy metabolism under stresses (hidangmayum et al., 2019). brassinolide is the first brassinosteroids (brs) iso­ lated in plants. brs are type of plant hormone which have a vital effect on plant growth and development. earlier workers have elucidated that brs can reduce the harmful effects of water deficient stress via boosting the activity of antioxidant enzymes and non­enzymatic antioxidant contents to remove the damage of reactive oxygen species (ros) (hosseinpour et al., 2020; cai et al., 2021; omidian et al., 2022) or by increasing endogenous abscisic acid (aba) to induce stomatal closure (bhandari and nailwal, 2020). additionlly, brs can enhance the energy metabolism balance between the chloroplast and mitochondria, increase the initial activity of rubisco, and boost the use of light energy absorbed by plants, thus enhancing photosynthetic efficiency under water deficient stress (cai et al., 2021). although the beneficial roles of bio­stimulators on ornamental plants and their helpful effect on boost­ ing growth and flowering parameter, there are no enough available data about their action on alleviat­ ing the harmful effect of drought on ornamental trees. therefore, this research is aimed to evaluate the response of azadirachta indica grown under water deficient stress to foliar application of chitosan or brassinolide. 2. materials and methods the present study was undertaken in the experi­ mental nursery (30­32°c temperature, 14 h light con­ ditions and 39­61% relative humidity) of the ornamental horticulture department, faculty of agriculture, cairo university, giza, egypt during the two successive seasons of 2019 and 2020. the lati­ tude, longitude and altitude of the experimental site were 30° 03ʹ n, 31° 13ʹ e and 19 m asl. plant material on 1st of march in 2019 and 2020 seasons, uni­ form seedling of azadirachta indica plants were obtained from a commercial nursery with an average plant height of 25­30 cm, 2­3 leaves/plant and trans­ planted individually in 25 cm diameter plastic pots filled with clay+ sand (2:1: v/v), some physical and chemical properties of soil mixture used in the study were determined according to (jackson, 1973), the results are presented in table 1. table 1 ­ some physical and chemical characteristics of the soil mixture used for growing azadirachta indica (mean of two seasons) ph= soil acidity. soil characteristics data physicical characteristics soil texture clay clay (%) 39.80 coarse sand (%) 3.59 fine sand (%) 21.46 silt (%) 35.15 chemical characteristics soluble cations (meq/l) ca++ 7.09 mg++ 2.86 k+ 0.30 na+ 6.05 soluble anions (meq/l) cl­ 3.53 so4 ­ 2.42 n (ppm) 26.57 p (ppm) 22.00 organic matter (%) 1.61 electrical conductivity (ds/m) 1.64 ph 7.46 abou dahab et al. ‐ biostimulators alleviates water deficient stress on neem plants 161 experimental procedures on 15th of march in first and second season, respectively the plants were irrigated every 3, 6, 9 and 12 days for imposing water stress. amount of water used each time was equal to the field capacity (pot capacity) which was determined empirically as follows: three pots (25 cm) filled with about 2 kg of the soil mixture were watered thoroughly to satura­ tion and weighed. pots were covered with aluminum foil to prevent evaporation before they were left in a cool shaded place to drain freely for 4 hours. they were weighed again to calculate weight of water held by the soil mixture. mean of the three pots repre­ senting the field capacity was found to be 1350 g, equivalent to 1350 cm3 of water/pot (1.35 l /pot) (abd­elmoneim et al., 2018). thus, 1.35 l of water were given to each pot in due time according to the irrigation schedule. this means that at the end of the experiment (after 7 months), plants irrigated every3, 6, 9 and 12 days interval were given 94.5, 47.3, 31.5 and 23.6 liters of water, respectively. starting from 31st march till to 15th october (in both seasons), the plants received different irrigation intervals were sprayed every 4 weeks with either chitosan (cht, 500 mg, composed of β­(1­4)­linked d­ glucosamine and n­acetyl­d­glucosamine randomly distributed within the polymer) or brassinolide (brs, 0.01% steroid compounds) each at concentrations of 50, 100 and 200 ppm, while the control plants sprayed only with tap water. both cht and brs were obtained from tecknogreen company, egypt. tween 20 as wetting agent was added to bio­solution at concentration of 1 ml l­1 and the plants foliage were sprayed using automatic atomizer until run off point (80 ml of bio­solution/plant). all the plants were fertilized every month with kristalontm (npk 20:20:20) at a rate of 3 g/pot, manual picking of weeds, disease and pest control has also been carried out. layout of experimental the layout of the experiment was randomized complete blocks design with 28 treatments [4 irriga­ tion intervals x 7 plant bio­stimulators (including the control)] each treatment consisting of 9 pots arranged in 3 replicates, each replicate containing 84 pots (3 pots from each treatment). the data recorded vegetative growth parameters. at the end of the experiment, on 30th october in both seasons (after 7 months), vegetative growth parameters were record­ ed, two samples of plants were randomly taken from each replicate to determine the parameters including plant height (cm, measured with a ruler from soil sur­ face to its highest point), fresh and dry weights of shoots, roots/plant, stem diameter (mm, at 5 cm above soil surface), root length (cm), number of leaves/plant and leaf area (cm2, measured using ci­ 202 portable laser leaf area meter, cid bio­science, inc., usa). dry weight of shoots and roots /plant was evaluated by drying plant in an electric oven at 70°c until constant weight. chemical analysis chlorophyll and carotenoid contents. chlorophyll pigments including chl a, chl b and carotenoid con­ tents (mg g­1 fw ) in leaves were determined accord­ ing to lichtenthaler and buschmann (2005): leaves extracted in 5 ml of 95% aqueous acetone was cen­ trifuged at 4000 g for about 10 min. the aqueous acetone supernatant was then taken for spectropho­ tometric measurement. a blank of acetone was taken at wavelengths of 663, 645 and 452.5 nm respectively, and data were then calculated using the following equations: chlorophyll a.(mg g ­1) = 0.0127 a663 − 0.00269a645 chlorophyll b.(mg g ­1)= 0.0029a663 − 0.00468a645 carotenoids (mg g ­1) = 4.2 e 452.5 − 0.0264 total carbohydrates. total carbohydrates content in leaves (percentage of dry matter) was determined in dried samples according to dubois et al. (1956). a known weight (0.1 g) of the dried samples was com­ pletely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. the solution was decolorized and the filtrate was diluted to 100 ml with distilled water. a known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. the optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. the standard curve of glucose was used to calculate the total carbohydrates concentration in the extract. proline content. proline content in fresh leaves (µ moles/g fresh matter of leaves) was determined using the method of bates et al. (1973). leaves were homogenized in 3% aqueous sulphosalicylic acid, then centrifuged 5,000 g for 20 min at 4°c. the amount of 2 ml of this homogeny solution react acid­ ninhdrin and 2 ml of glacial acetic acid in a tube for 1 hour at 100°c and the reaction is torn up in an ice adv. hort. sci., 2023 37(2): 159­171 162 bath and then extracted with 4 ml of toluene. it was kept at room temperature to stabilize. proline con­ tent was measured by spectrophotometer (uv­ 160a, shimadzu, tokyo, japan) at 520 nm. n, p and k content in shoot. dried shoot samples were digested to extract nutrients and the extract was analyzed to determine concentrations of n, p and k (as percentage of d.w) which were determined according to estefan et al. (2013). nitrogen concen­ tration was determined by using the micro­kjeldahl method. phosphorus was determined calorimetrical­ ly by using the chlorostannous molybdophosphoric blue colour method in sulphuric acid. potassium was determined by using the flame photometer appara­ tus (corning m 410, germany). total indole and phenol contents. total indole and phenol contents were determined in fresh shoots (3 g) of shoots, which were crushed and extracted with 80% ethanol at 0°c for 72 hours, the ethanol being changed every 24 hours, as described by selim et al. (1978). statistical analysis the means of all obtained results were subjected to two­ways analysis of variance (anova) in random­ ized complete blocks design. combined analysis of the two growing seasons was carried out. means of data were compared by using duncan’s multiple range tests at 5% level snedecor and cochran (1989). 3. results and discussion growth parameters data recorded on azadirachta indica plants, all growth parameters (including, plant height, fresh and dry weights of shoots and roots, stem diameter, root length, number of leaves and leaf area) were signifi­ cantly affected by irrigation intervals, bio­stimulators treatment and interaction effects (table 2). the data in table 3 and 4 showed that within each level of the two bio­stimulators (cht or brs), all growth parame­ ters were decreased significantly (p<0.05) with pro­ longed irrigation intervals daily from 3 to 6, 9 or 12 days. this reduction was steadily in most cases com­ pared to the short intervals (3 days). the reduction of growth parameters in response to water deficient may be due to adverse effect of drought around the roots, lower soil moisture availability due to water stress, lead to reduce water and nutrients absorption by roots which in turn leading to reduction in vegeta­ tive biomass (rouphael et al., 2012). this is greatly in harmony with numerous researches (ashour and el­ attar, 2017; abd­elmoneim et al., 2018; khatana et al., 2018; toscano et al., 2018; el­shanhorey and sorour, 2019; najihah et al., 2019; tribulato et al., 2019; al­arjani et al., 2020; singh and singh, 2020; el­ gamal and khamis, 2021; papú et al., 2021; sorour, 2021; shaltout et al., 2022). data in same tables also indicated that within table 2 ­ mean square for the effect of irrigation intervals and bio­stimulators treatments and their interaction on vegetative growth parameters of azadirachta indica *, **, *** significant at p≤0.05, p≤0.01, p≤0.001, respectively. traits source of variation treatment error cvirrigation intervals (a) bio­stimulators (b) (a × b ) plant height (cm) 1281.31 *** 967.98 *** 34.50 * 19.48 4.69 fresh weight of shoots (g/plant) 268.86 *** 731.60 *** 24.01 ** 14.07 9.14 dry weight of shoots (g/plant) 98.12 *** 279.41 ** 4.87 * 3.38 9.53 fresh weight of roots (g/plant) 225.15 *** 904.88 *** 15.76 *** 3.82 6.29 dry weight of roots (g/plant) 47.79 *** 71.71 *** 1.83 * 3.12 13.22 stem diameter (mm) 5.74 *** 8.85 *** 0.25 * 0.53 12.29 root length (cm) 553.08 *** 662.22 *** 7.00 * 11.17 8.13 number of leaves 105.61 *** 567.61 *** 7.17 * 4.32 9.65 leaf area (cm2) 33.01 *** 87.65 *** 0.54 * 2.83 13.57 abou dahab et al. ‐ biostimulators alleviates water deficient stress on neem plants 163 each irrigation intervals, the plants sprayed with the higher concentrations of two tested bio­stimulators (cht or brs) had significant increase (p<0.05) in most of growth parameters compared to control plants (pants exposed to water stress and not received any bio­stimulators treatments). the data also exhibited that under the same level of the two bio­stimulators, brs was generally superior in its effect than cht and among the different concentrations, the highest dose (200 ppm) was the most effective one in increasing the tested growth parameters. the results are similar to those obtained by previous studies where cht was tested (pirbalouti et al., 2017; byczyńska, 2018; el­ khateeb et al., 2018; abdel­mola and ayyat, 2020; el­ serafy, 2020; ashour et al., 2021; arshad et al., 2022), as well as brs (el­khateeb et al., 2017; abd­ allah et al., 2018; latha and vidya vardhini, 2018; mohamed, 2020; sheng et al., 2022). moreover, other studies (hosseinpour et al., 2020; mohammadi et al., 2020; omidian, et al., 2022) stated that appli­ cation of brs has a positive effect on growth parame­ ters of ornamental plants exposed to drought stress. chemical constituents pigments content. as shown from data listed in table 5 irrigation intervals, bio­stimulators treatment and interaction effects had significant influence on pigments content (chlorophyll a, b, carotenoids and table 3 ­ plant height, fresh and dry weight of shoots and fresh and dry weight of roots of azadirachta indica as affected by the interac­ tions between irrigation intervals and bio­stimulators treatments (mean of two seasons) cht (1) = chitosan at 50 ppm, cht (2) = chitosan at 100 ppm, cht (3) = chitosan 200 ppm, brs (1) = brassinolide at 50 ppm, brs (2) = brassinolide at 100 ppm, brs (3) = brassinolide at 200 ppm. each value represents the mean ± standard error of three replicates , means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. irrigation intervals bio­stimulators plant height (cm) fresh weight of shoots (g/plant) dry weight of shoots (g/plant) fresh weight of roots (g/plant) dry weight of roots (g/plant) 3 days control 79.63±1.94 j­l 34.18±1.97 jk 17.10±1.21 f­h 30.99±0.94 g­i 10.97±0.41 j­m cht (1) 98.58±1.97 de 42.2±1.60 d­g 21.70±0.95 cd 32.97±0.14 d­h 16.11±1.47 a­c cht (2) 101.17±2.6 b­e 44.53±1.11 c­f 23.11±0.6 bc 39.51±2.20 b 16.03±0.58 bc cht (3) 107.58±1.21 b 47.64±2.22 b­e 25.09±0.99 ab 41.68±1.5 b 15.40±0.26 cd brs (1) 96.67±2.1 ef 46.00±4.85 b­f 23.09±0.46 bc 34.71±0.84 c­f 13.83±0.28 c­j brs (2) 99.50±3.13 c­e 50.84±0.85 b 26.42±1.68 a 41.05±1.57 b 15.47±1.63 cd brs (3) 117.17±0.92 a 59.72±3.34 a 27.26±1.98 a 49.11±0.82 a 19.00±2.57 a 6 days control 75.17±2.53 k­m 38.21±3.41 g­j 13.90±1.4 ij 27.8±0.85 ij 10.16±0.97 k­m cht (1) 95.08±1.72 e­g 41.58±1.97 e­h 19.25±0.43 d­f 32.68±0.62 e­h 14.32±0.92 c­h cht (2) 99.83±1.451 c­e 44.8±2.30 b­f 22.81±2.09 bc 35.90±1.70 cd 14.87±1.19 c­f cht (3) 104.83±2.53 b­d 48.81±1.09 bc 22.73±1.02 bc 34.80±1.32 c­f 14.95±2.45 c­f brs (1) 99.67±3.03 c­e 40.53±0.75 f­i 22.62±0.61 bc 31.77±0.75 f­h 13.42±0.65 c­j brs (2) 104.33±2.05 b­d 48.27±0.94 b­d 25.49±1.01 ab 33.65±0.53 c­g 14.56±0.55 c­g brs (3) 107.25±4.73 b 47.74±0.78 b­d 21.30±0.35 cd 35.67±1.55 c­e 18.60±0.38 ab 9 days control 74±1.59l m 32.51±2.56 j­l 13.03±0.54 j 21.16±1.44 l 8.91±1.36 lm cht (1) 89.08±1.47 g­i 35.8±2.95 h­k 17.44±0.54 f­h 23.96±1.56 kl 12.29±0.74 e­k cht (2) 97.08±4.12 ef 43.31±1.41 c­g 17.89±0.49 e­g 34.18±0.09 c­g 12.94±0.21 d­k cht (3) 99.17±1.72 de 45.23±0.58 b­f 18.19±0.07 e­g 34.72±0.63 c­f 14.030±1.06 c­i brs (1) 88.83±4.06 g­i 37.54±1.68 g­j 17.04±0.6 f­h 27.63±1.77 j 11.94±0.26 g­k brs (2) 101.58±1.82 b­e 43.5±2.86 c­g 20.68±1.34 c­e 29.94±0.83 h­j 12.99±0.57 d­k brs (3) 106.42±2.81 bc 44.64±1.48 c­f 16.27±1.09 f­i 36.23±0.29 c 15.18±0.40 c­e 12 days control 70.33±1.58 m 27.73±1.99 l 9.91±0.18 k 17.80±0.53 m 8.3±1.24 m cht (1) 83.75±3.41 h­j 32.18±1.99 j­l 14.66±0.41 h­j 20.84±0.68 lm 11.18±0.65 i­m cht (2) 85.33±1.6 h­j 34.27±2.02 jk 16.80±1.54 f­i 26.93±1.46 jk 11.20 ±1.32 i­l cht (3) 90.17±3.03 f­h 35.48±1.42 h­k 17.26±0.41 f­h 22.68±0.59 l 11.40±0.82 i­l brs (1) 79.5±0.9 j­l 30.55±0.94 kl 16.32±1.87 f­i 21.35±0.51 l 11.46±1.21 h­l brs (2) 82.12±3.05 i­k 35.77±2.42 h­k 17.53±0.54 f­h 23.59±0.78 l 12.07±0.54 f­k brs (3) 101.75±3.04 b­e 35.13±1.89 i­k 15.19±0.83 g­j 27.18±0.96 j 12.30±0.29 e­k 164 adv. hort. sci., 2023 37(2): 159­171 total chlorophyll). data in table 6 showed that within each level of the two bio­stimulators (cht or brs), chlorophyll a, b, carotenoids and total chlorophyll were reduced gradually (in most cases) in repose to prolonged irrigation intervals. photosynthetic pig­ ments play a vital role in photosynthetic process. under drought stress, stomata functioning is changed that affect photosynthesis and co2 uptake, thus resulting various chlorophyll contents level through the entire growth period of the plant (khatana et al., 2018). similar reductions in pigments content as a result of water deficient stress were reported by vari­ ous studies (ashour and el­attar, 2017; abd­ elmoneim et al., 2018; khatana et al., 2018; sarker and oba, 2018; el­shanhorey and sorour, 2019; al­ arjani et al., 2020; singh and singh, 2020; el­gamal and khamis, 2021; papú et al., 2021; sorour, 2021; shaltout et al., 2022). the data in table 6 also revealed that within each irrigation intervals, the highest concentrations of cht or brs caused significant increase in pigments con­ tent compared to control plants. the data also clari­ fied that brs was better in its effect than cht particu­ larly the highest concentration (200 ppm) since recorded the highest values of the tested traits. these results are in agreement with findings of prior authors who reported that application of cht result­ ed in increase in pigments content (byczyńska, 2018; el­khateeb et al., 2018; el­serafy, 2020; elansary et al., 2020; abdel­mola and ayyat, 2020; gerami et al., table 4 ­ plant height, fresh and dry weight of shoots and fresh and dry weight of roots of azadirachta indica as affected by the interac­ tions between irrigation intervals and bio­stimulators treatments (mean of two seasons) cht (1) = chitosan at 50 ppm, cht (2) = chitosan at 100 ppm, cht (3) = chitosan 200 ppm, brs (1) = brassinolide at 50 ppm, brs (2) = brassinolide at 100 ppm, brs (3) = brassinolide at 200 ppm. each value represents the mean ± standard error of three replicates , means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. irrigation intervals bio­stimulators stem diameter (mm) root length (cm) number of leaves leaf area (cm2) 3 days control 5.64±0.56 c­j 41.25±0.66 f­i 22.00±1.38 d­g 11.79±1.07 e­j cht (1) 6.81±0.17 a­c 42.00±1.84 f­i 24.17±1.08 c­e 13.42±0.88 c­h cht (2) 6.80±0.17 a­c 44.00±2.46 e­g 27.42±1.29 bc 15.54±0.69 a­c cht (3) 6.91±0.02 ab 53.33±1.64 b 31.67±0.22 a 16.76±1.60 ab brs (1) 6.85±0.35 ab 47.67±1.69 c­e 27.95±1.81 b 13.79±0.66 c­f brs (2) 6.92±0.47 ab 50.67±1.74 b­d 33.08±1.54 a 14.88±0.47 b­d brs (3) 7.10±0.49 a 59.25±1.32 a 35.00±2.81 a 18.24±1.12 a 6 days control 4.77±0.13 jk 30.75±1.38 l­n 17.33±0.44i­k 10.88±2.04 g­l cht (1) 6.46±0.28 a­f 37.58±0.82 h­k 19.58±1.36 f­j 12.10±0.27 e­j cht (2) 6.52±0.67 a­f 40.17±3.43 g­j 22.33±0.65 d­f 13.58±1.07 c­g cht (3) 6.62±0.32 a­d 45.08±2.09 e­g 24.58±0.30 b­d 14.10±0.40 b­e brs (1) 6.07±0.49 a­i 42.75±0.80 e­h 19.92±1.01 f­i 12.01±1.63 e­j brs (2) 6.11±0.1 a­i 45.92±1.67 d­f 24.42±0.71 cd 13.52±0.51 c­h brs (3) 6.53±0.22 a­e 54.17±3.83 ab 24.08±1.31 c­e 15.48±0.59 bc 9 days control 3.78±0.04 k 29.25±1.84 mn 14.67±1.36 kl 8.73±0.86 kl cht (1) 5.33±1.14 f­j 33.83±0.88 k­m 16.33±0.33 jk 10.55±0.47 i­l cht (2) 5.74±0.06 b­j 36.67±1.62 i­k 19.42±1.17 f­j 12.04±1.09 e­j cht (3) 6.15±0.57 a­i 42.42±1.40 e­h 19.67±0.79 f­j 12.34±0.47 d­i brs (1) 5.26±0.17 g­j 35.00±2.36 j­l 19.33±0.96 f­j 10.19±0.86 i­l brs (2) 6.01±0.05 a­i 40.00±3.74 g­j 20.00±0.14 f­i 11.56±0.51 e­j brs (3) 6.45±0.43 a­g 51.67±2.53 bc 20.83±0.60 e­h 13.34±0.13 c­h 12 days control 3.61±0.69 k 26.17±2.02 n 12.33±0.44 l 8.29±0.66 l cht (1) 4.98±0.5 ij 31.08±1.08 l­n 15.00±1.23 kl 9.56±0.51 j­l cht (2) 5.55±0.09 d­j 33±0.58 k­m 17.50±1.0 h­k 10.10±0.61 i­l cht (3) 5.25±0.45 h­j 41.00±2.10 f­i 17.58±0.93 h­k 11.34±0.21 f­k brs (1) 5.40±0.21 e­j 34.83±1.74 j­l 18.92±2.32 g­j 9.38±1.05 j­l brs (2) 5.73±0.32 b­j 35.00±1.23 j­l 17.67±1.31 h­k 10.78±2.12 h­l brs (3) 6.39±0.36 a­h 45.92±0.79 d­f 20.17±1.20 f­i 12.72±0.96 d­i abou dahab et al. ‐ biostimulators alleviates water deficient stress on neem plants 165 table 5 ­ mean square for the effect of irrigation intervals and bio­stimulators treatments and their interaction on some chemical con­ stituents of azadirachta indica *, **, *** significant at p≤0.05, p≤0.01, p≤0.001, respectively. traits source of variation treatment error cvirrigation intervals (a) bio­stimulators (b) (a × b) chlorophylls a content (mg/g f.w) 22.90 *** 8.91 *** 0.32 * 0.93 16.99 chlorophylls b content (mg/g f.w) 2.72 *** 1.35 ** 0.21 * 0.39 24.19 carotenoids content (mg/g f.w) 5.30 *** 5.94 *** 0.21 * 0.64 20.35 total chlorophyll (mg/g f.w) 39.85 *** 16.72 *** 0.56 * 1.74 15.96 total carbohydrates (%) in leaves 206.87 *** 156.23 *** 6.81 *** 1.05 4.09 proline (µ moles/g fresh matter) 25.67 *** 9.65 *** 0.82 *** 0.11 7.60 n% in shoot 1.23 *** 2.59 *** 0.17 *** 0.01 5.03 p% in shoot 0.18 *** 0.46 *** 0.05 *** 0.001 9.21 k% in shoot 0.34 *** 0.85 *** 0.05 *** 0.01 6.29 total indoles (mg/100 g dw) 1310.44 *** 4091.18 *** 101.34 *** 2.75 2.45 total phenols (mg/100 g dw) 514.11 *** 3724.62 *** 116.61 *** 3.44 2.94 table 6 ­ pigments content of azadirachta indica as affected by the interactions between irrigation intervals and bio­stimulators treat­ ments (mean of two seasons) irrigation intervals bio­stimulators chlorophyll a (mg/g fw) chlorophyll b (mg/g fw) carotenoids (mg/g fw) total chlorophyll (mg/g fw) 3 days control 3.88±0.23 h­j 2.05±0.07 d­f 2.95±0.05 i­k 5.93±0.15 h­j cht (1) 5.76±0.44 c­g 2.40±0.49 c­f 4.78±0.59 a­e 8.16±0.89 d­g cht (2) 5.86±0.81 c­f 2.84±0.30 b­d 4.02±0.66 b­j 8.70±1.10 c­g cht (3) 7.22±0.40 bc 2.80±0.25 b­d 4.64±0.10 a­f 10.02±0.62 b­d brs (1) 5.79±0.04 c­g 2.96±0.30 b­d 4.41±0.17 b­g 8.75±0.34 c­g brs (2) 6.73±0.91 cd 3.48±0.69 ab 5.33±0.96 ab 10.22±1.53 b­d brs (3) 9.56±1.08 a 3.99±0.22 a 5.94±0.73 a 13.55±0.90 a 6 days control 4.27±0.16 g­j 1.71±0.14 ef 2.98±0.07 h­k 5.99±0.24 h­j cht (1) 5.18±0.48 d­h 2.62±0.20 b­e 4.22±0.28 b­i 7.80±0.32 e­h cht (2) 5.47±0.62 d­g 2.98±0.41 a­d 3.54±0.18 e­k 8.44±0.41 c­g cht (3) 6.39±0.94 c­e 2.86±0.39 b­d 4.36±0.38 b­g 9.25±1.32 b­e brs (1) 5.69±0.13 c­g 2.54±0.15 b­e 4.07±0.69 b­j 8.23±0.15 d­g brs (2) 6.48±0.50 c­e 2.81±0.39 b­d 4.29±0.65 b­h 9.29±0.48 b­e brs (3) 8.54±0.82 ab 2.86±0.29 b­d 5.03±0.39 a­c 11.40±1.08 ab 9 days control 3.58±0.28 ij 1.62±0.12 ef 2.87±0.11 jk 5.20±0.36 ij cht (1) 4.49±0.42 f­j 2.52±0.10 b­f 3.73±0.65 c­k 7.01±0.33 f­i cht (2) 5.02±0.33 e­i 2.55±0.29 b­e 3.42±0.45 f­k 7.57±0.05 e­h cht (3) 5.88±0.78 c­f 2.27±0.19 d­f 4.13±0.20 b­j 8.16±0.89 d­g brs (1) 5.08±0.37 e­i 2.45±0.43 c­f 3.95±0.19 c­j 7.54±0.76 e­h brs (2) 6.45±0.29 c­e 2.63±0.21 b­e 3.78±0.24 c­k 9.08±0.34 c­f brs (3) 7.22±0.50 bc 2.82±0.45 b­d 4.88±0.44 a­d 10.05±0.91 b­d 12 days control 3.05±0.13 j 1.51±0.04 f 2.47±0.17 k 4.56±0.17 j cht (1) 4.34±0.25 f­j 2.51±0.57 b­f 3.41±0.62 f­k 6.85±0.60 g­i cht (2) 4.48±0.54 f­j 2.36±0.55 c­f 3.09±0.46 g­k 6.84±0.69 g­i cht (3) 5.51±0.72 d­g 2.03±0.22 d­f 3.12±1.06 g­k 7.54±0.92 e­h brs (1) 4.54±0.19 f­j 2.08±0.11 d­f 3.29±0.21 g­k 6.62±0.15 g­j brs (2) 5.45±0.53 d­h 2.86±0.45 b­d 3.67±0.44 d­k 8.31±0.95 d­g brs (3) 7.12±0.69 bc 3.36±0.66 a­c 4.06±0.29 b­j 10.49±1.33 bc cht (1) = chitosan at 50 ppm, cht (2) = chitosan at 100 ppm, cht (3) = chitosan 200 ppm, brs (1) = brassinolide at 50 ppm, brs (2) = brassinolide at 100 ppm, brs (3) = brassinolide at 200 ppm. each value represents the mean ± standard error of three replicates , means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. adv. hort. sci., 2023 37(2): 159­171 166 2020; ashour et al., 2021; arshad et al., 2022; samany et al., 2022; attaran dowom et al., 2022). while, the valuable enhance in tested components due to brs treatments are in harmony with another reports (el­khateeb et al., 2017; abd­allah et al., 2018; rezaei et al. , 2018; mohamed, 2020; pacholczak et al., 2021). furthermore, previous workes (hemmati et al., 2018; mohammadi et al., 2020; mojaradi et al., 2020; zafari et al., 2020; omidian et al., 2022) showed that application of brs has a favorable effect on photosynthetic pigments of plants subjected to drought stress. the positive effect of brs in increase the pigments content may be due to application of brs increases the photosynthetic rate of plants by increasing the rubisco activity and other main enzymes included in the calvin cycle, brs also enhance the uptake of co2 which increase the stomatal conductance (vikram et al., 2022). total carbohydrates (% of dry matter) as shown in figure 1 a, the data indicated that within each level of cht or brs total carbohydrates were reduced in parallel with increasing irrigation intervals from 3 to 6, 9 or 12 days. the reductions in total carbohydrates percentage due to water defi­ cient stress are in agreement with findings of other researchers (ashour and el­attar, 2017; abd­ elmoneim et al., 2018; sarker and oba, 2018; el­ shanhorey and sorour, 2019; sorour, 2021). the water deficit may elevate the production of reactive oxygen species under drought stress which lead to oxidative stress and harm to chloroplasts structure and chlorophyll loses. reducing chlorophylls contents and photosynthetic activity could indirectly cause a reduction in carbohydrates content. moreover, water deficient assists translocation of absisic acid via xylem vessels to the shoot of stressed plants for stomatal closure which may be resulted in reduction of net photosynthesis and carbohydrate accumula­ tion (baccari et al., 2020). results in the same figure also showed that within each irrigation intervals, spraying the plants with any concentration of cht or brs resulted in significant increase in total carbohydrates compared to control plants. under the same level of the two tested bio­ stimulators, cht was generally better in its effect than brs for increasing total carbohydrates. the low­ est value (15.99 %) was obtained from plants irrigat­ ed with the longest intervals (12 days) and not received any bio­stimulators treatments, whereas the highest mean value (38.64%) was resulted from plants irrigated with the shortest intervals (3 days) and sprayed with brs at 200 ppm. the results of increasing total carbohydrates due to cht treatments are the same as the results of previous researches (zohreh et al., 2017; shafiei­masouleh, 2019; elansary et al., 2020; ashour et al., 2021; salachna and pietrak, 2021). while, the beneficial increase in total carbohydrates due to brs treatments are in agreement with earlier studies (mohamed, 2020; pacholczak et al., 2021). additionally, previous work­ ers (hemmati et al., 2018; mojaradi et al., 2020; cai et al., 2021) indicated that application of brs has a useful effect on carbohydrates accumulation of plants subjected to drought stress. proline content data in figure 1 b elucidated that in most cases under bio­stimulators treatments, proline content was increased progressively with prolonging irriga­ tion intervals. proline, an amino acid, plays a highly useful role in plants subjected to different stress con­ ditions. besides acting as an excellent osmolyte, pro­ line plays three main roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule (hayat et al., 2012). similar fig. 1 ­ total carbohydrates (% dw) (a), proline content (b) as affected by the interactions between irrigation intervals and bio­stimulators treatments (mean of two seasons). column with different letters indicate a significant differ­ ence at 5% level. vertical bars indicate to standard error (se) of three replicates. abou dahab et al. ‐ biostimulators alleviates water deficient stress on neem plants 167 results were reported by many studies (ashour and el­attar, 2017; el­shanhorey and sorour, 2019; al­ arjani et al., 2020; el­gamal and khamis, 2021; papú et al., 2021; sorour, 2021; samany et al., 2022; shaltout et al., 2022). the data also demonstrated that under each irri­ gation intervals, spraying any concentration of cht or brs resulted in significant higher values of proline content compared to control plants and brs was superior in its effect than cht. the highest mean value (7.61 µ moles/g fw) was registered from plants irrigated with the longest intervals (12 days) and sprayed with brs at 200 ppm, while the lowest value (1.52 µ moles/g fw) was produced from plants irri­ gated with the shortest intervals (3 days) and not received any bio­stimulators treatments. the results of increasing proline content due to application of cht treatments has been reported by earlier authors (zohreh et al., 2017; elansary et al., 2020; ashour et al., 2021; attaran dowom et al., 2022). whereas, the obvious increases in proline content due to applica­ tion of brs on ornamental water stressed plants are in good accordance with those elicited by prior authors (zafari et al., 2020; hosseinpour et al., 2020; mohammadi et al., 2020; omidian et al., 2022). the accumulation of proline due to application of brs may be due to brs enhanced gene expression of biosynthetic genes (sharma et al., 2019). n, p and k (% of dry matter) as shown in table 5, n, p and k % were signifi­ cantly affected by irrigation intervals, bio­stimulators treatment and interaction effects. the data in table 7 indicated that within each level of the two bio­stimu­ table 7 ­ n, p and k% of azadirachta indica as affected by the interactions between irrigation intervals and bio­stimulators treatments (mean of two seasons) cht (1) = chitosan at 50 ppm, cht (2) = chitosan at 100 ppm, cht (3) = chitosan 200 ppm, brs (1) = brassinolide at 50 ppm, brs (2) = brassinolide at 100 ppm, brs (3) = brassinolide at 200 ppm. each value represents the mean ± standard error of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. irrigation intervals bio­stimulators n% p% k% 3 days control 1.28±0.03 lm 0.16±0.01 j­m 1.17±0.01 l­n cht (1) 1.62±0.10 g­i 0.28±0.01 ef 1.42±0.01 f­i cht (2) 1.95±0.04 d 0.32±0.01 d 1.51±0.02 ef cht (3) 2.23±0.01 c 0.86±0.02 a 1.51±0.02 ef brs (1) 1.55±0.01 h­j 0.30±0.01 de 1.75±0.05 bc brs (2) 2.13±0.58 c 0.58±0.01 b 1.92±0.05 a brs (3) 2.98±0.01 a 0.90±0.03 a 1.97±0.04 a 6 days control 1.31±0.01 kl 0.17±0.01 i­l 1.14±0.05 mn cht (1) 1.59±0.11 g­i 0.28±0.03 ef 1.29±0.01 i­l cht (2) 1.66±0.01 f­h 0.34±0.01 d 1.45±0.02 e­h cht (3) 2.46±0.03 b 0.43±0.04 c 1.50±0.05 e­g brs (1) 1.71±0.04 fg 0.20±0.01 h­j 1.59±0.02 de brs (2) 2.22±0.02 c 0.30±0.01 de 1.68±0.14 cd brs (3) 2.20±0.06 c 0.46±0.02 c 1.89±0.14 ab 9 days control 1.16±0.02 m­p 0.13±0.01 mn 1.12±0.01 mn cht (1) 1.44±0.01 jk 0.18±0.01 i­l 1.18±0.01 l­n cht (2) 1.42±0.02 jk 0.18±0.01 i­k 1.30±0.03 i­l cht (3) 1.90±0.10 de 0.23±0.01 gh 1.36±0.07 g­j brs (1) 1.49±0.01 ij 0.19±0.01 h­j 1.45±0.01 e­h brs (2) 1.79±0.01 ef 0.20±0.01 g­i 1.29±0.01 i­l brs (3) 1.94±0.11 d 0.30±0.01 de 1.33±0.01 h­k 12 days control 1.06±0.02 op 0.10±0.01 n 1.07±0.01 n cht (1) 1.15±0.02 m­p 0.13±0.01 mn 1.11±0.02 mn cht (2) 1.18±0.02 l­p 0.14±0.01 lm 1.10±0.02 mn cht (3) 1.25±0.12 l­n 0.19±0.01 h­j 1.19±0.04 k­n brs (1) 1.12±0.04 n­p 0.14±0.01 lm 1.23±0.10 j­m brs (2) 1.25±0.02 l­n 0.15±0.01 k­m 1.22±0.01 k­m brs (3) 1.27±0.04 lm 0.24±0.03 fg 1.21±0.02 k­n adv. hort. sci., 2023 37(2): 159­171 168 lators (cht or brs), prolonging irrigation intervals generally decreased three nutrients (n, p and k %). similar reduction has been obtained by other reports (ashour and el­attar, 2017; abd­elmoneim et al., 2018; el­shanhorey and sorour, 2019; singh and singh, 2020; el­gamal and khamis, 2021). the unfavorable effect of water deficient on the uptake and accumulation of the three nutrients in plant may be due to water deficient stress caused by extending the irrigation intervals resulted in low soil moisture content which affects the elements solubili­ ty and their absorbing efficiency by plants which in turn leading to reduce their accumulation in plant tis­ sues. additionally, limited transpiration rates and impaired active transport and membrane permeabili­ ty lead to reduce nutrient uptake by the roots and accumulation in the shoots (farooq et al., 2009). the data in the same table disclosed that within each level of irrigation frequency, in most cases three nutrients were significantly higher in the plants sprayed with any concentrations of two tested bio­ stimulators (cht or brs) than those recorded with control plants. under the same level of the two test­ ed bio­stimulators, brs appeared to be more effec­ tive than cht and among brs concentrations; the highest dose (200 ppm) was the most effective one. the results of increasing n, p or k% due to applica­ tion of cht confirmed the reports of previous study (abd­el­hady, 2020; salachna and pietrak, 2021). while, the increase due to application of brs are sim­ ilar to those obtained by prior workers (mohamed, 2020). total indoles and phenols content results in figure 2 a showed that with each level of cht or brs, increasing irrigation intervals caused steady reduction in content of total indoles. within each irrigation intervals, application of any concen­ trations of cht or brs resulted in significant increase in total indoles compared to control plants. additionally, cht appeared to be more effective than brs and the highest concentration (200 ppm) was the most effective one. results in figure 2 b indicated that total phenols content showed different trend in response to water deficient, under each level of cht or brs, content of total phenols was increased linearly with increasing irrigation intervals from 3 to 6, 9 or 12 days. the pre­ sent increase in total phenols content as result of water deficient are similar to those obtained by (sarker and oba, 2018; el­gamal and khamis, 2021; papú et al., 2021). the data also revealed that within each irrigation intervals, application of cht or brs at the highest concentration (200 ppm) reduced the mean values compared to control. although previous studies revealed increase in total phenols content due to cht treatments (pirbalouti et al., 2017; vosoughi et al., 2018; el­serafy, 2020; elansary et al., 2020; arshad et al., 2022; attaran dowom et al., 2022) or due to brs treatments (amraee et al. , 2020; mohammadi et al., 2020). however, under the pre­ sent study total phenols content was reduced in response to application of cht or brs which support the results of ashour et al. (2021) who found that application of cht decreased total phenols content. 4. conclusions water deficient stress had a harmful effect on growth parameters, pigments content, total carbohy­ drates and nutrient uptake while, increased proline fig. 2 ­ total indoles (a), total phenols (b) as affected by the interactions between irrigation intervals and bio­stimula­ tors treatments (mean of two seasons). column with dif­ ferent letters indicate a significant difference at 5% level. vertical bars indicate to standard error (se) of three replicates. abou dahab et al. ‐ biostimulators alleviates water deficient stress on neem plants 169 and total phenols content. foliar application of chi­ tosan or brassinolide at the higher concentrations (200 ppm) increased growth parameters, pigments content, total carbohydrates, proline content and nutrient uptake. based on the obtained results it can be concluded that, foliar application of chitosan or brassinolide at 200 ppm can alleviate the adverse 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science, agriculture faculty of shahrekord university, p.o. box 8818634141 shahrekord, iran. key words: greenhouse, growth chamber, light­emitting diodes, light spectra, transplant production. abstract: determining the most reasonable led spectral composition wave­ lengths on sinningia speciosa transplants was the main focus of present experi­ ment. seeds were sown in cell trays under chambers with distinct spectral com­ position including white+blue+red (wbr), blue+red (br) and white+red (wr) leds with equal light quality proportions (70 mmol m­2s­1 photon flux density) and under sunlight (400 mmol m­2s­1 photon flux density) in constant conditions of 14h photoperiod, 70% relative humidity and day/night temperature of 23/18°c for 50 days. in this stage, led treatments led to higher germination percentage and better results in biomass, canopy width, leaf width and leaf area as well as chlorophyll and carotenoids accumulation were obtained in comparison with sunlight. extracted and technical parameters of chlorophyll fluorescence induction kinetics and maximum quantum efficiency of photosys­ tem ii (fv/fm) were decreased by sunlight­grown seedlings. fv/fmwas induced by wbr and br treatments, correlated with maximum yield of primary photo­ chemistry (jp0). quantum efficiencies (jp0, je0 and y0) and performance index of absorption energy flux (piabs) were increased in br­exposed transplants. in pot stage, led­treated plants exhibited better results in morphological features with earlier marketable flowering stage especially under wbr, which can com­ pensate costs of production in marketing stage. 1. introduction sinningia speciosa (lodd.) hiern. is a perennial potted flowering plant commonly known as gloxinia, which is a herbaceous tropical species native to brazil and belongs to gesneriaceae family (larson, 1992). proper seasonally light adjustments are critical for production of gloxinia, hence various source of artificial light has been effectively applied including fluo­ rescent, high­pressure metal halide, high­pressure sodium with the opti­ mal intensity of 45 to 70 mmol m­2s­1 (larson, 1992; dole and wilkins, 2005). even though aforementioned sources induce an increase in daily (*) corresponding author: sreezi57@yahoo.com citation: moazzeni m., reezi s., ghehsareh m.g., 2020 ­ growth and chlorophyll fluorescence characteri‐ stics of sinningia speciosa under red, blue and white light‐emitting diodes and sunlight. ­ adv. hort. sci., 34(4): 419­430 copyright: © 2020 moazzeni m., reezi s., ghehsareh m.g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 may 2020 accepted for publication 26 october 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 419­430 420 photosynthetic flux intensity, they are not energeti­ cally efficient as desired and there is no capability of spectral manipulation. light­emitting diodes, includ­ ing diverse size, long lifetime, solid state construction (heo et al., 2002; kim et al., 2004), low thermal out­ put, specific wavelength, adjustable light quality and intensity (okamoto et al., 1997) and high electrical efficiency (bula et al., 1991), represent a promising technology for the greenhouse industry which has technical advantages over other artificial light sources (mitchel et al., 2012). capability of led’s spectrum adjustment results in better responses of photoreceptors, influencing plant physiology and morphology and ultimately enhances production (morrow, 2008). horticultural crop seedlings are intensively influenced by light spectrum which affects their morphological properties (mcnellis and deng, 1995). production of transplants under desirable light spectrum and suitable control of environmental con­ ditions can improve transplant quality compared to traditional greenhouse production conditions in which accordingly affects their growth and yield after transplantation (oda, 2007). producing a large num­ ber of seedlings in a small area justifies high electrici­ ty consumption that is economically advantageous. developing various light spectral ratio recipes for dif­ ferent horticultural transplants based on their demand, would influence growth rate and improve quality (hernández et al., 2016), as different wave­ bands were proved to have significant physiological effects on plants (kim et al., 2004; johkan et al., 2010) and can be assembled according to the light quality which plants need (goins et al., 1997). detecting specific optimal light spectrum prevents energy loss for physiologically none­useful wave­ lengths (kim et al., 2004; johkan et al., 2010) and it can regulate a variety of plant development path­ ways from germination to flowering induction (jiao et al., 2007). based on previous studies, it has been shown uti­ lizing red (600­700 nm) and blue (400­500 nm) leds have the greatest impact on plant growth (yorio et al., 2001) since they mainly contain range of wavelengths essential for plants photosynthesis (cosgrove, 1981; kasajima et al., 2008). although blue light is photo­ synthetically less efficient than red light (mccree, 1972; dougher and bubgee, 2001), it has considerable photomorphogenic effects on chlorophyll biosynthe­ sis, stomatal opening, enzyme synthesis, photosyn­ thetic capacity on chloroplast (tibbits et al., 1983), fresh and dry matter accumulation, flowering (withelam and halliday, 2007; johkan et al., 2010), stem elongation and leaf expansion (hoenecke et al., 1992; dougher and bubgee, 2001). researches have demonstrated that combination­ al light regimes may help to optimize growth (brown et al., 1995). various number of studies have sug­ gested that combination of red, blue and white led lighting in different ratios is a favorable lighting con­ dition for plants in many aspects (lin et al., 2013; ouzounis et al., 2014). combination of red­blue, red­ white, red­blue­white provides the highest photon efficiency as compared to monochromatic led illumi­ nation (lin et al., 2013; nelson and bubgee, 2014; ouzounis et al., 2014; nicole et al., 2016). few stud­ ies on continuous­spectrum led lamps fit to a theo­ retical model of the maximum photosynthetic response has been recorded since mccree’s (1972) experiments on cultivated plants. it has been shown that chlorophyll fluorescence data can help with analyzing energy flow and informa­ tion related to the structure and function of photo­ synthetic apparatus (brestic and zivcak, 2013). the non­destructive analysis of polyphasic fast chlorophyll transient by the so­called ojip test was developed for quick evaluation of biophysical aspects of photosyn­ thesis (strasser, 1995; mathur et al., 2013). this test which is based on energy flow in thylakoid mem­ branes provides detailed information about the bio­ physics of the photosynthetic system through mea­ surement of fluorescence signals (kalaji et al., 2017). the main objective of this study was to investigate different ratios of blue and white with the red spec­ tral composition (wr, br, wbr) to determine the most effective combination of waveband in compari­ son with natural light condition. 2. materials and methods plants material and lighting treatments this experiment was carried out in 2018­2019 in specialized chambers in shahrekord university research greenhouses. sinningia speciosa f1 (bro­ cade blue) pelleted seeds were sown into 288 cell trays filled with a peat moss with the ph of 5.5­6 and ec of 1 ds/m­1 (1:2 dilution). cell trays were placed inside three chambers with led lighting treatments and one cell tray under 50% shaded sunlight in greenhouse, as control, using a completely random­ ized design. day/night temperature (23/18°c), rela­ tive humidity (70%) and photoperiod (14­hour) were maintained constant in all (led and sunlight) treat­ ments. plants were grown under led modules moazzeni et al. ‐ production of sinningia speciosa transplants under different light spectra 421 (shezhen sunled lighting co., ltd, cn manufacturer, china) yielding approximately 70 mmol m­2s­1 mea­ sured and adjusted using parmeter (apogee quantum meter, mq500, usa). the peak emissions of blue (460 nm), red (620 nm) and white (in the range of 380­750 nm) were measured and recorded using spectrometer (black­comet cxr­sr­50, stellarnet, inc., usa) with range of 300­800 nm (table 1). during transplant production cell trays were exposed to 50% white + 50% red (wr), 50% blue + 50% red (br) and 33.3% white + 33.3% blue + 33.3% red (wbr) led and 50% shaded sunlight (sl) treatments (table 1). the relative spectral distribu­ tion of light treatments are presented in figure 1. cell trays were rotated frequently in order to ensure equal growth conditions. subsurface irrigation was applied every 3 days and plants were irrigated as needed with a 500­1000 ppm water­soluble radixol fertilizer (n:p:k + microelements; 15:17:15 + 0.12% mg, 0.02% b, 0.0075% cu, 0.04% mn, 0.01% mo, 0.012% zn). measurements were conducted in both transplant and flowering stages. transplant stage measurements germination and morphological characteristics. after 50 days under light treatments (10 days for ger­ mination + 40 days for growth till four fully expanded leaves observed), germination percentage was calcu­ lated. plugs with four fully expanded true leaves, were then transplanted to 12 cm pots, in a complete­ ly randomized design with five replications (n=5). morphological traits of five randomly selected plants from each replicate including shoot fresh and dry weight, root fresh and dry weight, leaf area, leaf width and canopy width was measured. shoots and roots were dried in a drying oven at 72°c for 24 hours table 1 ­ light intensity, different light spectra and light quality were used for growing sinningia speciosa transplants *: for comparing sunlight quality with leds, sunlight has purple/blue : green/yellow : orange/red (400­492 : 493­597 : 598­700 nm) with the relationship of 23 : 28 : 39 according to aphalo et al. (2012). led treatment proportion (white: blue: red) light intensity (mmol m­2.s­1) wbr 33.3% : 33.3% : 33.3% 70 ± 3.1 br 0% : 50% : 50% 70 ± 1.0 wr 50% : 0% : 50% 70 ± 2.1 control light treatment average light intensity (mmol m‐2.s‐1) %50 shaded sunlight (sl) 400 ± 50 light quality light spectrum (nm) white 380­750 blue 460 red 620 sl (control) 400­700* fig. 1 ­ relative distribution spectra of white+blue+red (wbr), blue+red (br), white+red (wr) and sunlight (sl) treat­ ments. wavelength peaks are indicated by values above each peak. adv. hort. sci., 2020 34(4): 419­430 422 fluorpen fp 100 max, photon system instrument, psi, czech republic) with the same method (20 min­ utes dark adaption) was used to measure ojip­test whilethe last fully expanded intact leaf of randomly selected transplants was used to investigate biophys­ ical and phenomenological parameters of photo system ii status (strasser, 1995). the transient fluo­ rescence measurement was induced by a saturating light of 3000 μmol m−2s−1. the ojip transients were done according to the jip test (strasser et al., 2000). f0, fi, fj, fm, fv, vj, vi, fm/f0, fv/f0, fv/fm, jp0, je0, jd0, abs/rc, tr0/rc, di0/rc, et0/rc, piabs and y0 were extracted using fluorpen software. more information on formulas are presented in table 2. table 2 ­ summary of ojip­test formula using data extracted from ojip chlorophyll fluorescence transient. formulas to determine dry weight. the leaf area of plants was measured by digimizer v. 5.4.6 software. biochemical measurements. biochemical mea­ surements included chlorophyll a, chlorophyll b, total chlorophyll (a + b), carotenoid and total soluble sugar contents. chlorophyll and carotenoid contents were extracted from 0.5 g fresh leaf tissue from five ran­ domly selected transplants (n=5) and the pigments were eluted with 10 ml of 80% acetone centrifuged at 4000 g for 10 min and the amount of chlorophyll was estimated spectrophotometrically (using pg instruments t80+) at 470, 646 and 663 nm by the method of lichtenthaler and welburn (1983). total soluble sugars was quantified in 95% ethanol extracts of leaf tissue from five randomly selected transplants (n=5). a sample of 0.5 g of fresh­ ly harvested leaves was crushed in 5 ml of 95% (v/v) ethanol. the insoluble fraction of the extract was washed twice with 5 ml of 70% ethanol. all soluble fractions were centrifuged at 3500 g for 10 min. the supernatants were collected and stored at 4°c for tss determination. tss were analyzed by reacting 0.1 ml of the alcoholic extract with 3 ml freshly prepared anthrone reagent (150 mg anthrone + 100 ml 72% [w/w] h2so4) and placed in a boiling water bath for 10 min. after cooling, the absorbance at 625 nm was determined (irrigoyen et al., 1992) in a pg instru­ ments t80+ spectrophotometer and the data was pooled and extracted with standard curve (y = 0.002x ­ 0.009, r² = 0.992). chlorophyll fluorescence and ojip test parameters after 50 days of growth (at four true leaves stage), a portable par­fluorpen fp 100 max (photon system instrument, psi, czech republic) was used to mea­ sure maximal quantum efficiency of photo system ii (fv/fm) photochemistry. the most recent fully expanded leaf attached to plants of five randomly selected transplants (n=5) in each treatment were used for this measurement. a custom­ made method (genty et al. , 1989; aliniaeifard et al. , 2014; aliniaeifard and van meeteren, 2014) was used for the calculation of fv/fm. after reaching steady state fluorescence, during short measuring flash in dark­ ness and during saturating light flash (by exposing to 3900mmol m­2s­1 saturating light pulse) f0 and fm were digitized and averaged, respectively. these two images were applied to obtain maximal variable fluo­ rescence (fv = fm ­ f0). fv/fm was calculated using expression (fm ­f0)/fm. the average value and stan­ dard deviation of fv/fm per image were calculated by fluor cam v.7.0 software.the same device (par­ formula abbreviation formula explanation f0 f0= f50μs, fluorescence intensity at 50 μs fj fj= fluorescence intensity at j­step (at 2 ms) fi fi= fluorescence intensity at i­step (at 60 ms) fm fm= maximal fluorescence intensity fv fv= fm­f0 (maximal variable fluorescence) vj vj= (fj­f0)/(fm­f0) vi vi= (fi­f0)/(fm­f0) fm / f0 fv / f0 fv / fm jp0 jjp0= 1­(f0/fm)=fv/fm y0 y0= 1­vj je0 je0= [1­(f0/fm)] x y0 jd0 jd0= 1­jp0 ­ (f0 /fm) piabs piabs= (rc/abs)×[jp0/(1­jp0)]×[y0/(1­y0)] abs / rc abs/rc= m0×(1/vj)×(1/jp0) tr0 / rc tr0/rc= m0× (1/vj) et0 / rc et0/rc= m0× (1/vj) × y0 di0 / rc di0/rc= (ab/rc)­(tr0/rc) m0 m0= (tr0/rc)­(et0/rc)=4(f300­ f0)/(fm­ f0) abs= absorption energy flux; cs= excited energy cross­section of leaf sample; di= dissipation energy flux at the level of the anten­ na chlorophyll; et= flux of electron from qa ­ into the electron transport chain; jd0= quantum yield of dissipation; je0= probabi­ lity that an absorbed photon will move an electron into electron transport further than qa ­; jp0= maximum quantum yield of pri­ mary photochemistry; piabs= performance index; y0= efficiency by which a trapped exaction, having triggered the reduction of qa to qa ­, can move an electron further than qa ­ into the electron transport chain; rc= reaction center of psii; rc/cs= fraction of active reaction centers per excited cross­section of leaf; tr, psii; rc/cs= fraction of active reaction centers per excited cross­secti­ on of leaf; tr= excitation energy flux trapped by a rc and utilized for the reduction of qa to qa ­. moazzeni et al. ‐ production of sinningia speciosa transplants under different light spectra 423 pot stage (mature plants) measurements time to flowering and morphological characteris‐ tics. during pot stage, morphological traits such as number of flowers, flower diameter, number of leaves and number of days to flowering were mea­ sured and recorded. statistical analysis analysis of variance (anova) was performed using spss (spss 15.0, spss inc.) software and the means were compared with tukey’s test at p ≤ 0.05. 3. results transplant stage germination. seeds grown under all led lighting treatments performed better germination rate com­ pared to sl. wbr, br, wr and sl treatments had 96%, 94%, 96% and 87% germination, respectively (table 3). morphological characteristics. forty­five days after sowing seeds when all transplant had four fully expanded leaves, growth parameters were measured and analyzed (presented in table 3). seedlings grown under wbr and wr led light exhibited significantly higher shoot fresh weight compared with control. furthermore, shoot dry weight of plants grown under wbr and sl treatment had the highest and lowest average values, respectively. average root fresh and dry weight values were maximum in wbr, however in the absence of blue led, root biomass in wr­ grown transplants was greatly reduced. all three led lighting treatments had significantly greater canopy width than sl. leaf area and leaf width of plants were significantly influenced by wbr light, though the sl treatment had the least average values. this prominence of wbr (with 33% blue led ratio com­ pared to 0% and 50%) was visible on leaf features and canopy width (table 3). biochemical measurements plants grown under sunlight had the lowest chlorophyll a content while wbr and wr lighting treatments resulted in the highest content of chloro­ phyll a. led light composed of blue and red had most profound effect on chlorophyll b synthesis. control treatment and wr led treatment had the lowest amount of chlorophyll b content. additionally, identi­ cal proportion of white, red and blue light (wbr) led to the highest total chlorophyll (chl a+b) content among all the other treatments whereas sl had the lowest values. carotenoid content was highly affect­ ed by wbr and wr led lighting treatments while sl­ treated plants showed the lowest carotenoid content (table 3). transplants grown under sunlight in green­ house condition (sl) exhibited higher total soluble sugar content in comparison with all led treatments (table 3). chlorophyll fluorescence parameters measurements of chlorophyll fluorescence para­ meters were used to study the photosystem ii activi­ table 3 ­ influence of light quality of white+blue+red (wbr), blue+red (br), white+red (wr) and sunlight (sl) on germination, morpho­ logical and physiological characteristics of sinningia speciosa transplants represented by means values ± standard deviation (n=5) values followed by the same letter within a row do not significantly differ (by the tukey’stest, p≤0.05). parameters light quality wbr br wr sl (as control) germination (%) 96 a 94 a 96 a 87 b shoot fresh weight (g) 1.122 ± 0.08 a 0.862 ± 0.05 b 1.084 ± 0.14 a 0.608 ± 0.12 c shoot dry weight (g) 0.065 ± 0.004 a 0.044 ± 0.003 bc 0.053 ± 0.01 ab 0.037 ± 0.012 c root fresh weight (g) 0.148 ± 0.008 a 0.088 ± 0.008 bc 0.080 ± 0.01 c 0.098 ± 0.008 b root dry weight (g) 0.0102 ± 0.0024 a 0.0084 ± 0.0011 ab 0.006 ± 0.0007 c 0.0078 ± 0.0013 bc canopy width (cm) 7.6 ± 0.3 a 7.9 ± 0.3 a 7.7 ± 0.02 a 6.1 ± 0.4 b leaf width (cm) 2.6 ± 0.1 a 2.4 ± 0.1 ab 2.5 ± 0.3 ab 2.2 ± 0.1 b leaf area (cm2) 4.8 ± 0.3 a 3.6 ± 0.2 c 4.2 ± 0.5 b 3.2 ± 0.2 c chlorophyll a (mg.g­1 fw) 0.122 ± 0.002 a 0.091 ± 0.026 ab 0.114 ± 0.031 a 0.077 ± 0.009 b chlorophyll b (mg.g­1 fw) 0.089 ± 0.011 ab 0.099 ± 0.024 a 0.064 ± 0.022 b 0.064 ± 0.005 chlorophyll a + b (mg.g­1 fw) 0.212 ± 0.013 a 0.194 ± 0.027 ab 0.1946 ± 0.057 ab 0.141 ± 0.008 b carotenoid (mg.g­1 fw) 2.840 ± 0.23 a 2.038 ± 0.47 ab 2.470 ± 0.77 a 1.580 ± 0.18 b total soluble sugar (mg.g­1 fw) 49.42 ± 2.05 c 61.81 ± 1.98 b 47.05 ± 1.65 c 94.80 ± 0.65 a 424 adv. hort. sci., 2020 34(4): 419­430 ty (table 4). based on this result, the fluorescence signal intensity of transplants grown under wr led light, increased from f0 to fj and then to fm, howev­ er, sl treatment showed the lowest values of extract and technical fluorescence parameters (f0, fi, fj, fm, fv, vi, fm/f0, fv/f0) as well as fv/fm. transplants grown under wr led lighting and control condition (sl) showed the highest and lowest values as for f0, respectively. wbr and wr lighting treatments led to the significantly highest fj value while it had the low­ est significant value in sl treatment. all led treat­ ments (wbr, br, wr) exhibited higher fluorescence yield at fi, fm, fv compared to sl. the highest vj obtained in wbr and wr­grown plants and the low­ est values were detected in br­grown seedlings. the highest vi value was from plants grown under wr lighting; however, sl treatment had the lowest value among the treatments. plants exposed to br led light resulted in higher fm/f0value than other plants grown under different lighting conditions. fv/f0 was also decreased for all except br­grown plants. the fv/fm ratio was higher in plants exposed to br and wbr led lights compared to sl which had the great­ est decrease. analyzed parameters for specific ener­ gy fluxes per reaction center (abs/rc, tr0/rc and di0/rc) increased in wr treated plants, in contrast br light treatment had the greatest decrease among lighting treatments in di0/rc ratio while sl treatment had the highest increase of the same ratio. also, sl and wbr showed the highest and lowest values of et0/rc, respectively. by analyzing the parameters that estimate quantum efficiencies or flux ratio (yields and efficiency of electron transport chain) the highest calculated values for jp0, je0, piabs and y0 were obtained under br treatment. in addition, plants grown under br and wbr treatments had sim­ ilarly the highest significant value in jp0. plants of wbr and br treatments had the lowest values in jd0 in compare with sl which had the highest values. pot stage (mature plants) morphological characteristics based on the results, there was a significant effect of led lighting treatments on flower quantity where­ table 4 ­ chlorophyll fluorescence of transplants grown under white+blue+red (wbr), blue+red (br), white+red (wr) and sunlight rep­ resented by means values ± standard deviation (n=5) values followed by the same letter within a row do not significantly differ (by the tukey’s test, p≤0.05). parameters light quality sl (as control) wbr br wr f0 9802 ± 1083 ab 8794 ± 663.5 bc 10517 ± 649 a 8031 ± 750 c fi 39013 ± 3447 a 36843 ± 2126 a 41150 ± 2688 a 29808 ± 3230 b fj 27919 ± 3109 a 23611 ± 2439 b 28439 ± 1890 a 20392 ± 2299 b fm 42752 ± 2254 a 39793 ± 2655 a 43443 ± 3162 a 32758 ± 3599 b fv 32949 ± 1755 a 30999 ± 2222 a 32925 ± 2530 a 24798 ± 2661 b vj 0.5508 ± 0.0628 a 0.4768 ± 0.0310 b 0.5448 ± 0.0236 a 0.4856 ± 0.018 ab vi 0.913 ± 0.022 ab 0.905 ± 0.011 bc 0.931 ± 0.013 a 0.886 ± 0.006 c fm/f0 4.228 ± 0.171 b 4.535 ± 0.242 a 4.128 ± 0.069 b 3.944 ± 0.169 b fv/f0 3.230 ± 0.171 b 3.535 ± 0.242 a 3.128 ± 0.069 b 2.944 ± 0.169 b fv/fm 0.771 ± 0.021 a 0.778 ± 0.012 a 0.758 ± 0.004 ab 0.746 ± 0.011 b jp0 0.771 ± 0.021 a 0.778 ± 0.012 a 0.758 ± 0.004 ab 0.746 ± 0.011 b je0 0.348 ± 0.059 b 0.408 ± 0.028 a 0.345 ± 0.019 b 0.387 ± 0.008 ab jd0 0.229 ± 0.021 b 0.221 ± 0.012 b 0.242 ± 0.004 ab 0.254 ± 0.011 a abs/rc 3.449 ± 0.271 b 3.133 ± 0.170 c 3.771 ± 0.095 a 3.477 ± 0.038 b tr0/rc 2.654 ± 0.147 b 2.439 ± 0.128 c 2.857 ± 0.072 a 2.594 ± 0.01 bc et0/rc 1.217 ± 0.070 b 1.273 ± 0.037 ab 1.301 ± 0.082 ab 1.337 ± 0.04 a di0/rc 0.796 ± 0.127 ab 0.697 ± 0.060 b 0.914 ± 0.028 a 0.887 ± 0.048 a piabs 0.700 ± 0.149 c 1.163 ± 0.161 a 0.696 ± 0.072 c 0.895 ± 0.001 b y0 0.449 ± 0.063 c 0.523 ± 0.031 a 0.455 ± 0.024 bc 0.515 ± 0.018 ab moazzeni et al. ‐ production of sinningia speciosa transplants under different light spectra 425 as the sl treatment resulted in the lowest number of flowers (presented in figure 2a). largest flowers, in terms of flower diameter was detected in plants grown under wbr treatment, however wr treat­ ment resulted in smallest dimeter of flowers (fig. 2b). the results also indicated that treatments had significantly different number of days to flowering and plants grown under partial sunlight (sl) in green­ house had longer time to flowering in pot stage in comparison with wbr, br and wr­treated plants (fig. 2d). the transplants grown under led treat­ ments had greatly higher number of leaves than sl at flowering stage (fig. 2c). 4. discussion and conclusions in this experiment, significantly higher percentage of germination under led light treatments, highlight­ ed the effect of light quality on germination rate. it is known that orange/red and blue regions of light spectrum are most effective in germination process (tozzi et al., 2005). germination rate was satisfactory in absence of both blue and white led lighting in wr and br treatments, respectively. overall, germination rate was highly affected under led treatments com­ pared to sl and it can be derived that presence of red light in all treatment resulted in a partially better germination in sinningia speciosa. using different light spectra for tomato seedlings revealed that exposing seedlings to monochromatic red light showed higher shoot dry weight than 80% rb but small proportion of blue (95% rb) contributes getting more shoot dry weight (gόmez and mitchell, 2015). moreover, it was reported that lamps with substantial red but small blue waveband radiation energy, produced more weight yields in tomato com­ pared to high blue and less red biased lamps (warrington and mitchell, 1976). furthermore, among sodium lamp (1:1 rb), 100% r, 50% rb, 70% rb, 90% rb and white leds lighting treatments 90% rb led to significantly higher dry matter content (wojciechowska, 2015). in another study with rw, rb, rbw and fl (fluorescent lamp) lighting treat­ ments, wbr enhanced yield of lettuce plants includ­ ing shoot fw, shoot dw, root fw, root dw and leaf area (lin et al., 2013). our results on fresh and dry weight and leaf features (leaf area and leaf width) were consistent with quoted reports and it can be concluded that a small blue proportion in combina­ tion with red and white spectrum will result in a high­ er biomass. blue light is considered to be a substan­ tial stimulator for leaf expansion which enhances leaf area and biomass production (li et al., 2010; cope ad bugbee, 2013), on the other hand it has been proved that plant growth typically tends to decrease as the fraction of blue photons exceeds 5­10%, high levels of blue light in the spectrum results in inhibition of cell expansion, cell division, and leaf area expansion, fig. 2 ­ morphological characteristics and time to reach flower of transplants grown under white+blue+red (wbr), blue+red (br), white+red (wr) and sunlight in pot stage. the mean of values ± standard deviation (n=5) is displayed. values with the same letter in column do not significantly differ (by the tukey’s test, p≤0.05). the explanations for the treatment abbreviations are provi­ ded in table 1. tron transport chain and excess light dissipated as heat from the electron transport system (aliniaeifard et al., 2018). the higher total soluble sugar content of plants grown under sl compared to other plants grown under artificial led lights, suggesting that under high­ er light intensity elevated level of soluble sugar helps transplants to avoid excessive light intensity and unlike led treatments, they had less utility for photo­ synthesis (ciereszko et al. , 2001; havaux and kloppstech, 2001). in this study, wr treatment showed the highest rate of increase in minimal fluorescence intensity (f0) and this value decreased asproportion of blue light­ but the opposite trend was observed as white pro­ portion of light increased. this was also observed in fi, fj, fm, fv and viparameters. the maximum efficien­ cy of photosystem ii (fv/fm) increased in br followed by wbr, however it was reduced in wr treatment (in the absence of blue led). in an experiment using 32% bw and 40% br lighting treatments on phalaenopsis ‘purple star ’showed higher amount in fv/fm in com­ parison with 0% br (ouzounis et al., 2014) which necessitates certain amount of blue light for proper photosynthesis (hogewoning et al., 2010). fv/fm value ranges between 0.72­0.84 in many plants (maxwell and johnson, 2000), although the fv/fm of sl­grown transplants was in this range but it showed the least efficiency among other treatments, which is not surprising as this value changes with environ­ mental conditions (ouzounis et al., 2014). further­ more, it was shown that existenceof uv and yellow light in sunlight reduce photosynthesis efficiency (takashi et al., 2010). in addition, sinningia speciosa has no optimum photosynthetic activity under sun­ light (larson, 1992; dole and wilkins, 2005). transplants under highest blue proportion (br) had the highest value in piabs and je0 and the lowest value of di0/rc. the wr exposed transplants (which had no high ratio of blue light but high red ratio) showed the lowest piabs and je0 and higher abs/rc, tr0/rc and di0/rc as the result. in an experiment investigating on photosynthetic and growth respons­ es of purple variety of basil under white, blue and red led lamps results shown that red light had the high­ est increase in abs/rc, tr0/rc and di0/rc and this amount was decreased under blue light (hosseini et al., 2019). inactivation of reaction centers and a decrease in active qa reducing centers occur as abs/rc increases (strasser and stirbet, 1998). wr and wbr­grown transplants (with higher white and adv. hort. sci., 2020 34(4): 419­430 426 which ends up in less photon capture and diminished growth (bugbee, 2016). exposing to red light results in enhanced stem elongation (hoenecke et al., 1992), also absorbing high ratio of red light by plant photore­ ceptors can lead to production of a plant hormone called metatopolin (steele, 2004), which can stimulate cell division as well as leaf expansion. addition of white led light may further increase plant growth, as white light might penetrate deeper in to the canopy and enhance photosynthesis compared to combina­ tion of red and blue light. perhaps the combination of white, blue and red light perform a balanced spectral environment by providing a favorable amount of white light to plants (lin et al., 2013). white light is more capable of increasing chloro­ phyll than blue light, however some reports stated that blue light had significant effect on chlorophyll a synthesis (wynne and rhee, 1986; rivkin, 1989; aidar et al., 1994; sanchez­saavedra and voltolina, 1994; mercado et al., 2004; hogewoning et al., 2010; vadiveloo et al., 2015), also it was reported that red light plays an important role in chlorophyll content enhancement (kubota et al., 1996). in addition, it has been reported combinational red light with blue and white light can increase carotenoid content accumu­ lation (lefsrud et al., 2008; kopsell et al., 2014; chen et al., 2016; kopsell et al., 2016), however lin et al. (2013) reported versus result, claiming that wbr led has no effect on carotenoid content compared to rb led and fl light. it can be concluded that white, red and blue light conjointly can enhance chlorophyll and carotenoid content but plant exact response to light quality varies with species and cultivars. in present experiment, the maximum yield of pri­ mary photochemistry (jp0) was in correlation with maximum quantum efficiency of photosystem ii (fv/fm) which confirmed the enhancement of chloro­ phyll concentration under wbr treatment. we would suggest that transplants under wbr light treatment contained more chloroplast which maximized light capture for photosynthesis. based on our results, we would suggest that the lowest photosynthetic rate in plants grown under sl is the result of low means of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content which was confirmed by the decline in fv/fm and jp0 and increase in jd0 and it can be concluded that increased jd0 in sl treated transplants explains that due to highest amount of quantum yield of energy dissipation (jd0), the major of natural light absorbed by the plant in high intensi­ ty was not used for the photochemical yield of elec­ moazzeni et al. ‐ production of sinningia speciosa transplants under different light spectra 427 red ratios) represented lower et0/rc which indicates that absorbed energy is briefly conveyed to the elec­ tron transport chain (sarkar and ray, 2016). this con­ firms that plants grown under br light are more capable of transporting electrons from absorbed photons into electron transport chain and beyond qa ­1y0 which could efficiently regulate energy level in the center of r reaction (strasser et al., 2004). sl­ grown transplants showed highest soluble sugar con­ tent; however they had the second increase in piabs, it is possible that in case of an environmental stress­ such as high light intensity, in which excess energy beyond photosynthetic capacity is existing, led to production of ros which results in oxidative damage to photosystem ii (pospíšil, 2016). in transplants grown under wbr and br led lighting, an increment in jp0 value was observed, while there was no increase of the same value in transplants grown under sunlight. the results for jp0 were in correla­ tion with fv/fm which impacted chlorophyll and leaf area as explained in aforementioned chlorophyll measurements. at flowering stage, transplants grown under led lighting treatments resulted performed better orna­ mental criteria including number of flowers, flower diameter and number of leaves. also it could be sug­ gest that, those plants grown under led lighting treatments could reach flowering stage sooner which will result in higher profit especially in commercial scale. totally, in this experiment, this scenario was the case for plants grown under wbr treatment. application of led light in greenhouse in combination of red, blue and white wavelengths with a high pho­ ton efficiency are suitable for the production of horti­ cultural plants (kozai et al., 2015; nicole et al., 2016). our results are consistent with previous studies find­ ings which indicate thatlighting source composed of red, blue and white light spectrum enhance morpho­ logical development of seedlings compared to mono­ chromic light of each waveband (brown et al., 1995; gόmez and mitchell, 2015; hogewoning et al., 2010; ouzounis et al., 2014). desirable morphological and physiological charac­ teristics in sinningia speciosa transplants achieved under led lighting treatments with identical propor­ tion of white, blue and red led to enhanced morpho­ logical and physiological features at marketing stage including higher number of flowers, flower diameter, number of leaves as well as fewer days to flowering. shorter time interval to flowering may help commer­ cial growers to save time and costs of production while enhancing sinningia speciosaplants quality in comparison with sunlight­grown 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(2013) highlighted the beneficial effects of agricultural utilization of (*) corresponding author: hakim61@hotmail.com citation: hakim a., khatoon m., gullo s., 2019 ­ effect of compost tea and partial root zone drying on tomato productivity and quality. ­ adv. hort. sci., 33(4): 511­518. copyright: © 2019 hakim a., khatoon m., gullo s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 june 2019 accepted for publication 30 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 511­518 512 compost, which include improvement on physical, chemical, biochemical and biological properties of the soils. but compost can be heavy and bulky to transport and spread. as an alternative, compost tea (ct) offers the benefits of compost in a lighter­weight package. it is a liquid version of compost, obtained through a liquid­phase of compost and extraction ranging from few hours to two weeks with or without active aeration and the addition of nutrients such as molasses. it is easier to apply to plants and the soil. the effectiveness of compost teas may vary due to the differences in types of compost, management and procedures used for its preparation (egwunatum and lane, 2009; pant et al., 2012). according to martin et al. (2012), the best results were obtained when aerated compost teas rather than non­aerated teas were used. most probably dissolved oxygen sup­ ports microbial activity (arancon et al., 2007). reeve et al. (2010) reported that the potential of compost teas for supplementing or substituting other types of fertilizers also seems promising. in a study, moretti et al. (2015) indicated that composted green wastes are advantageous when compared to other organic wastes since they present a lower risk of toxicity due to the presence of heavy metals, pollutants, aromatic hydrocarbons, hormones, pharmaceuticals as well as viruses, fecal coliforms and salmonella (benito et al., 2005). these materials exhibit excellent biological activity (ros et al., 2005). hatam et al. (2015) and mavity (2016) reported that compost tea is an effective, low­strength, all­ natural fertilizer for plants, seedlings, and gardens. they also assert that compost tea reduces salt accu­ mulation in soils that come from commercial fertiliz­ ers, improves the ability of soil to hold nutrients, retain water and facilitates the soils ph buffering ability through microbe diversity. the use of compost tea as a foliar spray or soil drench has been demonstrated to improve plant health, yield and nutritional quality by: enhancing beneficial microbial communities and their effects on agricultural soils and plants. it also enhances mineral nutrient status of plants and induces the production of plant defense compounds that may have benefi­ cial bioactivities in humans (weltzien, 1991; hoitink et al., 1997; diver, 2002; scheuerell and mahaffee, 2002; carpenter­boggs, 2005; ingham, 2005). the potential benefits of compost tea are substantial and particularly relevant to crop production in low­input agricultural systems, e.g. minimize water use. less water input is now one of the globally used modern practice of sustainable agriculture and sustainable food production system. indeed, throughout the world, water supplies are limited and water crises are a top global risk. as irri­ gation of agricultural lands accounts for 70% of water usage worldwide (khokhar, 2017), even a slight reduction in irrigation water could substantially increase the water available for other purposes. therefore, there is an urgent need to identify effec­ tive irrigation management strategies. partial root­ zone drying irrigation (prd) is one of the new effi­ cient and productive water­saving irrigation methods that can conserve irrigation water up to 50% in pro­ cessing tomatoes (casa and rouphael, 2015). this technique has the potential to significantly reduce crop water use (el­sadek, 2014), minimize canopy vigor, but able to maintain crop yields and quality of crops (sun et al., 2014) when compared to conven­ tional irrigation methods. it is expected that plants under prd condition will maintain high water saving potentials while adequately watering plants. in this context, the aim of this study was to check the use of ct in combination with less water use to improve tomato production sustainability. accordingly, the use of compost tea combined with prd was checked as a possible management system for promoting tomato growth and decrease need for chemical fertilizer which might lead to sustainable agriculture. most of the previous studies were focused on the application of compost as a soil supplement or foliar application for tomato plant or tomato fruit disease control (vawdrey and stirling, 1997; gutierrez­miceli et al., 2007; segarra et al., 2009; souleymane et al., 2010). no information is available on the combined effect of compost tea and prd on morpho­physiolog­ ical traits, yield and quality attributes of “vibelco” tomato grown in closed environments. thus some investigations are required to provide clear informa­ tion about the effects of compost tea and partial root zone drying in growth promotion, yield and quality among the worldwide consumed vegetables such as tomatoes. therefore, this work was focused on the effects of compost tea combined with prd on the growth, yield and main quality parameters of toma­ toes grown in closed environments. based on the ini­ tial observations, we hypothesized that the applied compost based tea with deficient water supply may exert a suitable bioactivity on the tomato plants as related to the stimulation of growth, yield and quality parameters of the fruits. hakim et al. ‐ effect of compost tea on tomato productivity 513 2. materials and methods experimental conditions the experiment was conducted in a greenhouse setting at approximately 22°c during daytime tem­ perature and 15°c at night, relative humidity (rh) of 67%, 14 hour’s photoperiod and ambient light condi­ tion at the chateau fresno nursery, 13505 south fresno, california 93609, usa, from april to september 2017. seeds of a fresh market tomato (lycopersicon esculentum mill cv. vibelco) were sown on march 1, 2017. thirty three days after seeding, uniform plants were transplanted into 24 wooden boxes (2.53 m length × 0.65 m width × 0.20 m height each). each box had 4 compartments (0.50 m length × 0.50 m width × 0.20 m height) with one experimen­ tal plant per compartment. to avoid lateral water movement, a small piece of plastic (0.50 m length × 0.025 m width × 0.04 m height) was placed centrally on the base of each compartment. the plants were grown in a vermicast and coconut fiber mixture (60:40 v/v). bees were used for pollination (fig. 1). compost tea preparation first, a large plastic bin (25 gallons, about 95 l) was filled almost to the brim with water and allowed it to stand overnight to rid it of chlorine, which will otherwise would kill the soil biota in the compost. a close fitting lid was used to exclude mosquitoes and limit odor. then, to make up a compost “tea­bag”, a hessian sack was used. a measured amount of com­ post (homemade compost prepared from organic tree leaves) with organic cow manure, that is approx­ imately one­tenth of the volume of water, was placed inside the tea­bag. for every 25 liter bucket, we used about 2.5 liter of compost. the “tea­bag” was tied and securely closed and immersed it in the water for three weeks. the mixture was frequently dunked to speed up the process and obtain better results. after three weeks the mixture was filtered. the whole process was done at ambient tempera­ ture. then the tea was applied to the plant by incor­ porating it to the irrigation water used for with drip irrigation at the ratio of 1:4 (1 part tea and 4 part water) (fig. 2). irrigation treatments incorporated with compost tea two weeks after transplanting, irrigation treat­ ments incorporated with compost tea were tested. treatments were: 1. conventional drip irrigation (cdi) to both sides of the root system, 2) half of irrigation water, in drip irrigation given alternately only to one side of the root system with each irrigation (prd), 3) cdi plus compost tea, and 4) prd plus compost tea. in the cdi the irrigation treatment was given 0.10 meter away from the main stem and on both sides of the row. the irrigation (cdi) covered a total area and soil volume of 0.24 m2 and 0.048 m3, respectively. the amount of water was given in two equal doses at 10:00 h and at 16:00 h by drip irrigation system with emitters. two irrigation lines were set up and operat­ ed separately for the prd treatment. two sprinklers per plant (one on each line) each emitters 4 l/h were placed 0.15 m away from the main stem of each plant. the irrigation in prd treatment covered a total area and soil volume of 0.018 m2 and 0.004m3, but half of irrigated area and soil volume was wetted in prd treatment. there was some drainage in all treat­ ments, but it was not measured. however, water loss by drainage was minimized by adjusting the amount fig. 1 ­ schematic diagram of compost tea production. fig. 2 ­ schematic diagram of drip irrigation treatments incorpo­ rated with compost tea. adv. hort. sci., 2019 33(4): 511­518 514 of water as the crop developed. the values of the irri­ gation efficiency presented here might have been underestimated, considering no water loss. plant and fruit growth performance growth, yield, yield components and blossom­end rot were measured in 12 randomly selected plants/fruits from each treatment. the plant heights were measured with a tape from the base to the tip of the plant. plant growth and development data were documented in the sampled and tagged plants monthly for three months. leaf area was measured using a destructive method. total leaf area (cm2) was measured by leaf area meter (model: delta­t, cambridge, u.k.). the numbers of clusters were counted per plant from the first to the last cluster during the growing period. the number of fruits was counted when the plants started fruiting. the fruit weights were determined after harvesting the toma­ to using a weighing balance. after 150 days, one plant per treatment per replication was destroyed and the total vegetative fresh weight was assessed and expressed as kg/plant. mature green tomato fruit firmness was measured using an instron universal testing machine with a 0.5 cm2 plunger. the measurement was taken at the mid­section of the fruit. water content of fruit was expressed on a dry weight basis. fruit blossom­end rot incidence was recorded and calculated in percentage of fruits affected per plant. fruit quality at harvest or postharvest for postharvest quality evaluation, 6 replicates of 5 mature green (cascio, 2017) fruits from each treat­ ment were randomly chosen approximately 125 days after transplanting and were stored in a dark refriger­ ated room at 4°c with 94% rh. after a storage period of 2 weeks, all fruits were moved to a ventilated room without supplemental light at 24°c with 66% rh and held for 7 days. the following attributes were checked for quality measurements: weight loss, chill­ ing injury, decay, appearance/color change, total sol­ uble solids (tss), ph, titratable acidity (ta), chloro­ phyll and lycopene content. fruit weight loss was determined prior to and after storage. it was calculated as the percentage of initial fresh weight. color development was observed visually using a subjective scale with mature green (mg) = 1, breaker (b) = 2, pink (p) = 3, light red (lr) = 5 and red (r) = 6 (usda, 2005). chilling injury (sur­ face pitting) was rated visually by estimating the per­ centage of the injured fruits. decay (unidentified) was rated visually and calculated as a percentage of fruit affected. chlorophyll and lycopene content were deter­ mined from three randomly selected fruits from each treatment by grinding pericarp tissue (about 5 g) in 15 ml acetone. the extract was taken for centrifuga­ tion at 35,000 rpm for 10 minutes. before centrifuga­ tion, the tubes were covered with aluminum foil to prevent light­induced lycopene oxidation. after cen­ trifugation, the supernatant was decanted and adjusted to 20 ml with acetone. absorbance of the extracts at 664 nm for chlorophyll and 503 nm for lycopene was measured with a spectrometer (model 160 a). total chlorophyll content in milligram per 100 grams of tissue was calculated according to the for­ mula developed by holden (1976). lycopene content was calculated using the molecular extension coeffi­ cient of 3240 (davis, 1976) and expressed as micro­ grams per gram of fresh weight. total soluble solid (tss), ph and titratable acidity (ta) were measured on juice extracted from fruits. the tss content was determined with a digital refrac­ tometer (atago, model 1, tokyo, japan). the ta was determined by a metler auto titrator (model v 20) and ph was measured with an autocal ph meter (model phm 83). experimental design and data analysis a completely randomized design was used with the four treatments replicated six times with four plants per replication for each treatment. data were analyzed by a complete randomized model using the anova procedure of sas software version 8.2 (sas institute, cary, nc, usa). treatment effects on toma­ to growth, yield and quality components were ana­ lyzed using duncan’s multiple range tests to deter­ mine significant effects at p = 0.05 among four treat­ ment means. 3. results and discussion figure 3 illustrates the mean plant height over a period of 3 months. bars represents standard devia­ tions. plants height increased gradually within 3 months. the different letters in the columns at the same month showed significant differences. the maximum height was obtained from those tomato plants which were irrigated with cdi/full water regime plus compost tea and the difference was sig­ nificant during the whole experiment. on second and third month, cdi plus ct treated plans exhibited higher height than prd plus ct treated plans and the difference was significant. on third month, the prd hakim et al. ‐ effect of compost tea on tomato productivity 515 of fruits per cluster. the difference between cdi and prd treated plants’ shoot fresh weight, leaf area, and fruit production were significantly different at 0.05 % level. the cdi treated plants appeared to have excess moisture in the root zone (didn’t investigated moisture in root zone in this study), which may have caused root inactivity contributing to lower yield and delayed maturity of the crop as compared to the prd treated plants. therefore, this study demonstrates that the deficit of irrigation water with compost tea can induce biological activity of tomato plant which ultimately increased the number of clusters per plant and num­ ber of fruits per cluster. table 1 illustrates that among the four treat­ ments, partial root zone drying (prd) plus compost tea (ct) treated plant fruits exhibited the highest per­ centage blossom end rot. table 1 also illustrated that the prd treated plant’s fruits showed significantly higher percentage (%) of blossom­end rot as com­ pared to the cdi treated ones. mathew and salvadore (2007) reported that the blossom­end rot is a physiological disorder of tomato fruit caused by calcium deficiency or excessive soil moisture fluctua­ tion, which reduce uptake and movement of calcium into the plant. the higher percentage (%) of blossom­ end rot in the prd treated fruits might be due to the reduced movement of calcium to in the prd treated plants. however, no calcium was analyzed either in the leaves or fruits in this experiment. both prd and ct treatments influenced the fruit size, water content and fresh fruit weight (table 1). among the four treatments, fruit size showed signifi­ cant difference and the largest size of fruits were produced by those plants irrigated with deficit (prd) water mixed with compost tea. there were some dif­ ferences in fresh fruit weight and water content in treated plants resulted in the significantly lowest height among the four treatments. similar results were reported by pal et al. (2016), who grew tomato plants under deficit irrigated conditions. among the four treatments in this study, the growth of cdi treated plants exhibited moderate growth compared to other treated plants. combined treatment with cdi and ct had positive (+) and significant effect on fruit fresh weight and leaf area of the plant, respectively. the prd had a negative (­) effect in shoot fresh weight and leaf area of plant. however, prd had positive (+) effect on the number of fruits per cluster as well as in fruit production in com­ parison to the cdi treatment (table 1). combined treatment with prd and ct exhibited the positive (+) and significant effect on cluster per plant and number table 1 ­ effect of ct and prd on tomato fruit size, total fresh weight, water content, fruit weight, fruit firmness, cluster/plant, fruit/clu­ ster, shoot fresh weight, leaf area, and blossom­end rot (x) means followed by different letters in a row are significantly different at 5% probability level using duncan's multiple range test (dmrt). plant yield parameter treatment cdi prd cdi + ct prd + ct fruit size, diameter (mm) 65.40 d (x) 68.30 c 70.27 b 72.03 a total fresh weight of fruit (kg/plant) 5.11 d 6.47 c 7.53 b 9.34 a fruit water content (%) 95.17 a 96.1 a 92.17 c 94.07 b fruit weight (g) 94.4 b 97.33 a 94.57 b 95.5 b fruit firmness after harvest (kg/cm2) 9.97 a 9.13 b 8.30 c 8.17 c cluster/plant 8.03 d 9.10 c 10.40 b 11.23 a fruit/cluster 5.03 d 5.60 c 6.40 b 6.80 a shoot fresh weight (kg) 10.60 b 11.53 a 8.97 d 9.30 a leaf area (cm2) 453 b 419 d 478 a 432 c bloom end rot (%) 5.50 a 6.00 c 6.50 b 8.27 a fig. 3 ­ effect of cdi (conventional dripping irrigation) and prd (partial root­zone irrigation) with compost tea (ct) on plant height over 3 months. bars represents standard deviations; means followed by different letters at the same month are significantly different at 5% probability level using duncan's multiple range test (dmrt). 516 adv. hort. sci., 2019 33(4): 511­518 prd and cdi treated plants’ fruits; and the differ­ ences were statistically significant (table 1). the results of this study indicates that, during conservation, combined cdi and ct treated plant fruits lost more weight than combined prd and ct treated plant fruits (table 2). the results of this study also confirm that prd treated plant fruits lost less water compared to the cdi treated ones. reduced weight loss in the prd treated tomatoes during the storage is a positive quality attribute in tomato fruits, especially for distant markets. according to yadav and singh (2014) the weight loss of fruits in storage condition is mainly from water loss and from solid constituents. the lower water loss in prd treated fruit might be due to lesser incidents of micro­cracks in the skin. however, no skin micro­cracks were examined in this investigation. as demonstrated in this study, chlorophyll and lycopene contents were influenced by irrigation and compost tea treatment (table 2). combined treat­ ment with prd and ct postulated higher lycopene and lower chlorophyll contents compared to the plants treated with a combination of cdi and ct. when compared to the fruits from cdi treated plants, fruits from prd treated plants exhibited sig­ nificantly lower chlorophyll and higher lycopene con­ tent on the 7th day at 24°c. this was followed by a 2­ week storage period at 4°c (table 2). klunklin and savage (2017) also detected significantly higher lycopene content in prd treated tomato fruits. gindi et al. conducted a survey in 2016 and con­ cluded that 63 percent of consumers’ purchase inter­ est depends on the color of fruits. fruit colors were affected by irrigation and compost tea treatment. the best color (tone of red) was observed on plants fruit those treated with prd plus ct. fruits from prd treated plants exhibited less visible chilling injury (fruit surface pitting) and decay, but faster color change than the fruits treated with cdi. the less visi­ ble chilling injury, decay and faster color changes in prd treated plants fruit might be due to less water content compared to cdi treated plants fruits. tomato flavor is generally determined by the con­ tent of soluble solids (tss) and titratable acidity con­ tent (ta). according to hong et al. (2014), sweetness of the most fruits is closely related to the tss content. according to baldwin et al. (2015), the sourness in most of the fruits is governed by ta content. valero et al. (2005), reported that changes in sugar content and organic acid metabolism occur during the ripening process of tomato fruits. aoun et al. (2013) examined that tomato flavor is co­related to the total sugar and acid contents in the fruit. the results of this study demonstrated that the total solu­ ble solid (tss), ta and ph were affected by the treat­ ment of prd and ct individually and in combined treatments. fruits from prd plus ct treated plant exhibited significantly higher content of tss, and ph values, but lower ta than fruits from cdi and ct treated plants. the total soluble solid (tss), tss/ta and ph increased, while ta decreased in prd treated fruits than cdi treated ones (table 2). in an earlier study, sun et al. (2014) also detected higher tss lev­ els in tomato fruits produced under a prd condition. the higher tss and the lower ta in fruits from prd treated plants were probably due to the less retained water in fruit from prd treated plant than the fruits from cdi treated plants. the higher ph in fruits from prd treated plants was compatible with the lower ta table 2 ­ effect of ct and prd on tomato fruit weight loss, chilling injury, decay, chlorophyll and lycopene content, fruit color change, ph, ta, tss content and tss/ta ratio, 7th day at 24°c followed by a 2­week storage period at 4°c (x) means followed by different letters in a row are significantly different at 5% probability level using duncan's multiple range test (dmrt). phylogical parameter treatment cdi prd cdi + ct prd + ct weight loss (%) 2.18 b x 1.88 c 2.93 a 2.03 bc chilling injury (%) 14.66 b 9.00 d 17.00 a 10.70 c decay (%) 13.67 a 7.50 d 12.30 b 10.20 c chlorophyll (mg/100 g fresh weight) 3.80 a 3.30 bc 3.53 b 3.60 b lycopene (µg/g fresh weight) 7.00 d 7.80 b 7.43 c 8.10 a color change 4.36 c 4.76 b 4.63 bc 5.23 a ph 4.30 d 4.63 b 4.50 c 4.80 a ta (% citric scid) 0.49 c 0.45 d 0.53 b 0.56 a tss 4.27 d 4.80 b 4.56 c 5.10 a tss/ta 8.61 c 10.67 a 8.60 c 9.11 b hakim et al. ‐ effect of compost tea on tomato productivity 517 in fruits from prd treated plants than in the fruits from cdi treated plants. 4. conclusions the results of this study demonstrates the signifi­ cant effect of prd on biometric variables such as number of cluster per plant, number of fruit per clus­ ter, fruit weight, and fruit size in combination with ct treatments. the results of the study also shows that the plants treated with a combination of prd, a cost effective water saving method, and ct exhibited slower plant growth, but produced higher yield, and better postharvest quality attributes of fruits. acknowledgements the author wishes to thank dr. abu helal ibrahim for his assistance with the greenhouse experiment. references aoun a.b., lechiheb b., benyahya l., ferchichi a., 2013 ­ evaluation of fruit quality traits of traditional varieties of tomato (solanum lycopersicum) grown in tunisia. ­ african j. food sci., 7(10): 350­354. arancon n.q., edwards c.a., dick r., dick l., 2007 ­ vermicompost tea production and plant growth impacts. ­ biocycle, 48(11): 51­52. baldwin e.a., scott j.w., bai j., 2015 ­ sensory and chemical flavor analyses of tomato genotypes grown in florida during three different growing seasons in multi‐ ple years. ­ j. am. soc. hortic. sci., 140(5): 490­503. benito m., masaguer a., de antonio r., moliner a., 2005 ­ use of pruning waste compost as a component in soilless growing media. ­ bioresour. technol., 96: 597­603. carpenter­boggs l., 2005 ­ diving into compost tea. ­ biocycle, 46: 61­62. casa r., rouphael y., 2015 ­ effects of partial root‐zone drying irrigation on yield, fruit quality, and water‐use efficiency in processing tomato. ­ j. hort. sci. biotech., 89(4): 389­396. cascio j., 2017 ‐ a harvest of green tomatoes . ­ cooperative extension service, university of alaska, fairbanks, usa, fnh­00024, 1­4. davis b.h., 1976 ­ carotenoids, pp. 38­165. ­ in: good­ win t.w. 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(2012) considered the role of soil characteristics to be highly effective in crops yield. obviously, the production of organic materials in leaves without the presence of mineral (*) corresponding author: nazanin_kh_43713@yahoo.com citation: khakipour n., mohammadi torkashvand a., ahmadi a., weisany w., 2023 ­ prediction of chamomile essential oil yield (matricaria chamo‐ milla l.) by physicochemical characteristics of soil. ­ adv. hort. sci., 37(2): 209­219. copyright: © 2023 khakipour n., mohammadi torkashvand a., ahmadi a., weisany w. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 august 2022 accepted for publication 1 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13591 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 209­219 210 elements in the process of photosynthesis is not pos­ sible. each of the macronutrients plays a special role in the metabolism of plant growth. bernier et al. (1981) concluded that flowering of plants was under the control of nutritional status, and in this regard, the balance between the elements that plant takes from air and soil is very important. plant mineral compounds are one of the factors affecting the quali­ ty of crops (prasad and spiers, 1991). considering the importance of developing the cultivation of medicinal plants and using their products as natural ingredients compatible with human health, it is necessary to use different cultivation and nutrition methods that increase the essential oil and effective compounds of medicinal plants (ajili et al., 2019; savitikadi et al., 2020). chamomile (matricaria chamomilla l.) is an annual plant, aromatic, 20­40 cm high that grows wildly on fields and side roads (omid beigi, 2004). the plant now has a large dispersal in europe, western asia, north africa, north and south america, and australia. the extensive cultivation of this plant is carried out in countries such as hungary, germany, egypt, czech republic, slovakia, and india (omid beigi, 2004). in iran, different species of the genus matricaria grow in different parts of the country. chamomile flowers are used to treat stomach, flatu­ lence, and skin lesions. in most western countries, they are used as appetizers and digestible foods. the active ingredient of chamomile has been mentioned for many medicinal properties such as sedative, anti­ spasmodic, stimulating white blood cells and strengthening the body’s defense system, and anti­ bacterial gram­positive and anti­allergic (salamon, 1992). accordingly, chamomile is used in many coun­ tries as dry flowers and essential oils in the pharma­ ceutical, food, cosmetic and sanitary industries. in recent years, it has also become one of the most popular pharmaceutical plants in the world (gheedi jashni et al., 2015). upadhyay and patra (2011) inves­ tigated the effects of calcium and magnesium on chamomile growth and yield and stated that the effect of magnesium on the growth and essential oil of chamomile was higher than that of calcium. one of the main issues in producing agricultural and garden crops is a lack of ability to forecast pro­ duction/yield using accessible and easily measured indicators (mohammadi torkashvand et al., 2020). various factors affect the yield and essential oil con­ tent of the plant, including nutrition and physico­ chemical properties of the soil (el­gohary et al., 2015; belal et al., 2016; radkowski and radkowska, 2018; mohammadi torkashvand et al., 2020). for example, it is hypothesized that one can estimate the yield of a product based on the concentration of nutrients in a leaf (lahiji et al., 2018; mohammadi torkashvand et al., 2020), fruit (mohammadi torkashvand et al., 2019) or soil characteristics (rahmani khalili et al., 2020; tashakori et al., 2020). in this case, it will be possible to plan fertilization or choosing the soil susceptible and suitable for planti­ ng, or the farmer has an estimate of his income and, accordingly, plans its costs for the future programs. there are various predictive methods for estimat­ ing several natural variables, among which more transfer functions are used. different regression methods have been widely used to derive transition­ al functions (vereecen et al., 1992; sepaskhah et al., 2000; marashi et al., 2017; eslami et al., 2019). these methods consider the relationship between the input data and the data to be predicted to be predefined. since the soil and plant are natural and heteroge­ neous systems, it is difficult to establish a connection between their properties. therefore, in these sys­ tems, artificial neural networks (ann) operate more efficiently than regression methods. numerous stud­ ies have been carried out to estimate soil variables through artificial neural networks (zhou et al., 2008; bocco et al., 2010; gago et al., 2010; parvizi et al., 2010; mokhtari karchegani et al., 2011; besalatpour et al., 2013; dai et al., 2014; moghimi et al., 2014; aitkenhead et al., 2015; marashi et al., 2017; khanbabakhani et al., 2019; marashi et al., 2019). also, some studies have been conducted to predict crop yield by remote sensing, stochastic, artificial neural network (ann) and simulation models (bannayan and crout, 1999; o’neal et al., 2002; bartoszek, 2014; farjam et al., 2014; domínguez et al., 2015; emamgholizadeh et al., 2015; dias and sentelhas, 2017; mohammadi torkashvand et al., 2017; niedbała, 2019; mohammadi torkashvand et al., 2019) based on weather, soil and growth charac­ teristics as input data. mohammadi torkashvand et al. (2017) estimated the storage life of kiwifruit based on chemical characteristics of fruits, including the amount of nutrients by analyzing the neural net­ work (nn), identifying it as a superior method in comparison to multiple regression. a similar study was carried out to predict kiwifruit yield based on leaf nutrients by ann (mohammadi torkashvand et al., 2020). tashakori et al. (2020) evaluated the effi­ ciency of artificial neural network (ann), multiple lin­ khakipour et al. ‐ forecasting chamomile essential oil yield through soil physicochemical 211 ear regression (mlr), and adaptive neuro­fuzzy infer­ ence system (anfis) in terms of saffron yield estima­ tion by soil properties in some lands of golestan province, iran. according to the results, ann showed the highest accuracy (r2= 0.58­0.89) in estimating saf­ fron yield as compared to mlr (r2= 0.41­0.47) and anfis (r2= 0.41­0.69) models. poorghadir et al. (2021) concluded that the yield and percentage of essence influenced by the soil properties and nutrition. the purpose of this study was to investigate the importance of soil characteristics on the concentra­ tion and amount of chamomile essential oils and their estimation with respect to some important physicochemical properties of the soil, and investiga­ tion of feasibility of using artificial neural networks for estimating the concentration and amount of chamomile essential oils. 2. materials and methods soil experiments several habitats or areas of chamomile cultivation were surveyed in kermanshah and hamadan provinces, west of iran. from 20 areas, 100 soil sam­ ples (five of each area) were taken from soil depths of 0­30 cm and transferred to iau, science and research branch, tehran, iran. the environmental characteristics of the sampling areas, in particular the topographic and climatic characteristics, were simi­ lar. soil samples were shipped to the laboratory and air­dried and clods were broken down in small parti­ cles with a plastic hammer; then they were passed through a sieve of 2 millimeters (klute, 1986). afterward, 0.5 kg of each soil was used for laboratory analysis and the rest was used for greenhouse experi­ ments. the soil samples were analyzed for phospho­ rus, nitrogen and potassium nutrients, ph, electrical conductivity (ec), texture, and organic matter. soil ph and ec were measured in saturated soil extract. soil texture was determined by hydrometric method and the amount of calcium carbonate equivalent (cce) was measured using titration method (paye et al., 1948). the kjeldahl method was used to measure nitrogen (goos, 1995). soil samples were extracted by soltanpour and schwab method (1977) and the concentration of available potassium and phosphorus were measured by flame emission and spectropho­ tometry methods (emami, 1996). organic matter was measured by walkley and black (1934) method. some statistical data of soils are seen in table 1. greenhouse experiment in a completely randomized design, 100 different soil samples were sprayed in a plot (box) with dimen­ sions of 30 to 35 cm and 25 cm in depth, and 20 seeds were planted in each plot. after germination and early growth of the plant, in the quadruple stage, the number of plants was reduced to 10 in each plot. during the growing season, field operations included irrigation, weed control and pest control for the plots were done alike for all the boxes. after full flowering, the flowers were harvested at a maximum of five centimeters of length. the flowers were immediately dried at 60°c with an electronic dryer. in addition to the dry flower yield per plot in kg ha­1, the concentration of essential oil for each soil was obtained. the essential oil content of the sam­ ples was determined by the kelevenger apparatus by the water distillation method and expressed as g/100 g of dry flowers. the essential oil yield was expressed table 1 ­ statistics of data set for estimation of essential oil percentage and essential oil yield om= organic matter; cce= calcium carbonate equivalent. soil properties ph ec (ds/m) n (%) p (mg/kg) k (mg/kg) om (%) cce** (%) sand (%) silt (%) clay (%) essential oil (%) essential oil yield (kg/ha) max 8.75 1.60 1.30 81.00 381.00 2.30 69.00 39.40 42.40 55.60 1.57 7.37 min 7.94 1.00 0.25 8.00 135.00 0.22 16.00 14.50 21.00 32.00 0.01 0.28 average 8.13 1.28 0.73 22.10 226.10 1.43 33.40 24.11 30.57 45.32 0.69 3.71 median 8.06 1.25 0.78 16.50 244.00 1.46 28.50 23.20 30.25 46.75 0.76 4.13 standard 0.22 0.15 0.37 18.93 66.28 0.50 13.83 5.69 4.80 6.16 0.51 2.61 kurtosis 1.72 ­0.61 ­1.72 5.37 ­0.70 0.34 ­0.06 1.32 ­0.30 ­0.95 ­1.23 ­1.72 skewness 1.67 0.15 0.02 2.61 0.28 ­0.69 0.92 1.02 0.26 ­0.38 0.26 ­0.12 adv. hort. sci., 2023 37(2): 209­219 212 as kg ha­1 in dry flower yield. artificial neural network one of the best known rules is multilayer percep­ tron (mlp) learning rule. mlp is a feed forward net­ work in which information flow from input side and pass through the hidden layers to the output layer to produce outputs. in this research, mlp rule and levenberg­marquart back propagation algorithm was used for training the artificial neural networks. the tangent axon function was used as an activation function, which is a nonlinear function. pourhaghi et al. (2013) also used the tangent axon functions for predicting the input flows by ann. neurosolutions 5.05 (neurodimension, inc., gainesville, fl, usa) software was used to design the artificial neural net­ work. the data used in training, validation and test were 60, 20 and 20% of the total data respectively. the training data are used for network education and training. evaluation data are not used in network training, but this data are used to compare different models and to determine the most suitable network and ptfs. the analysis of the neural network with three sub­ series of variables as input variables was performed to estimate or predict the essential oil percentage and essential oil yield: 1 ­ in first step, total nitrogen, available phosphorus and potassium and clay content of soils, which are the most important factor in soil fertility, were selected as predictors and ptf1 was developed. 2 ­ in the second step, ph, ec, organic matter and clay, which are found in the most common and important measurements in standard soil analy­ ses, were selected as predictors for estimation desired variables, and ptf2 was developed. 3 ­ in third step all of the measured soil properties (n, p, k, om, cce, ph, ec, sand, silt and clay) were included as predictor for developing ptf3. therefore, three ptfs (ptf1, ptf2 and ptf3) were developed using anns and their efficiency was com­ pared with each other to find the best and most suit­ able ptf. the number of input layer nods was chosen as the number of input parameters for each desired issue. the number of hidden layers determines the com­ plexity of the grid, and the reason for this complexity is that as the number of hidden layers increases, the number of connections between the nerve layers increases, which leads to network complexity. the number of output layer neurons is equal to the num­ ber of output parameters of the desired problem. after training the network with the data of the training and validation series, the precision and accu­ racy of the generated models were evaluated using the test series data. in order to evaluate network accuracy, the coeffi­ cient of determination (r²) and square error squared (rmse) were used. (1) (2) in which: yi, ȳi, ŷ, respectively, the measured depen­ dent variable, its mean and the estimated dependent variable, and n is the number of observations. other criteria used to evaluate the precision of transition functions were the geometric mean error ratio (gmer) and geometric standard deviation of error ratio (gsder): (3) (4) geometric mean of error ratio (gmer) represents the degree of conformance between measured and estimated values. if the gmer is equal to one, it rep­ resents a complete fit between measured and pre­ dicted values. if the gmer is greater than one, it indi­ cates that the predicted values are greater than the measured values, and the gmer less than one is an indication of lower estimated values than the mea­ sured values. geometric standard deviation of error ratio (gsder) is a measure of data diffusion. if it is close to one, it shows less diffusion and the differ­ ence between one and the other represents the devi­ ation of most estimates from the measured data. khakipour et al. ‐ forecasting chamomile essential oil yield through soil physicochemical 213 3. results and discussion correlation between variables table 2 shows the correlation between the vari­ ables studied and the percentage and yield of the essential oils. the results presented in this table could be important to find input data series to the neural network. in our study, the percentage and yield of essential oil showed a positive and significant correlation with organic matter, n, p and k contents and clay. jat and ahaheat (2006) showed that the use of bio­fertilizers containing nitrogen, phosphorus and potassium increased the growth and amount of essential oil of the fennel plant. phosphorus plays an important role in seed, flowers germination, vegeta­ tive growth, the acceleration of ripening and the completion of metabolic processes of fruits (bennett, 1993; malakouti and shahabi, 2000; malakouti et al., 2008). it is also involved in controlling enzymatic reactions and regulating metabolic pathways (rejali, 2005; miransari et al., 2007). potassium affects the amount and quality of herbal essential oils due to its effect on metabolic pathways and enzymatic activity (pacheco et al., 2008). in the case of potassium defi­ ciency, the quality of some products, especially fruits and vegetables, decreases (egilla et al., 2005; mohiti et al., 2011). potassium deficiency during plant growth leads to a decrease in photosynthetic rate and chlorophyll content (gerardeaux et al., 2010), activation of enzymes, and reduced growth and yield (kanai et al., 2007). potassium interacts with almost all essential elements. furthermore, a synergistic role of k with either n or p has been already noted (barker and pilbeam, 2007). nurzynska­wierdak (2013), cecílio filho et al. (2015) and chrysargyris et al. (2017 a) evaluated the impact of different potassium levels (275, 300, 325, 350 and 375 mg/l) on the morphological and bio­ chemical characteristics of spearmint (mentha spica‐ ta l.). the results showed that the potassium in 325 mg/l treatment could be appropriate for spearmint cultivation and production for essential oil uses. in the same study, chrysargyris et al. (2017 b) found that the lavender grown in 300 mg l­1 of k was appro­ priate for the essential oil uses/production while the 325 mg l­1 of k were more appropriate for lavender cultivation for fresh and dry matter uses. due to the significant correlation of essential oils with organic matter, n, p and k contents and clay (table 2), it was determined three series of data as input data of ann that is observed in table 3. table 3 shows the number of hidden layers, and the number of nodes in the hidden layers in the three input series. estimation (prediction) of essential oil figure 1 shows the distribution of actual values (measured) and the estimation or prediction of the percentage of essential oil of chamomile and their conformity in three series of input data. r2 was between the measured values and the estimated essential oils as observed in table 4. as seen in the first transfer function (ptf1), r2 was 82.71% in the test data and 78.56% in the training data series. the gmer value indicated that almost the network in the test data was not under or over­estimating, and its table 2 ­ correlation between the input variables of the neural network and the amount of essential oil (g/100 g of dry flowers) and essential oil yield variable ph ec n p k om cce sand silt clay essential oil essential oil yield ph 1 ec 0.511** 1 n ­0.101 0.152 1 p 0.806** 0.546** ­0.382* 1 k ­0.390* ­0.419** 0.099 ­0.323* 1 om 0.414** 0.675** 0.362* 0.443** ­0.046 1 cce ­0.267 0.015 0.001 ­0.287 0.044 ­0.544** 1 sand ­0.032 ­0.339* ­0.439** ­0.029 0.268 ­0.577** 0.648** 1 silt ­0.202 0.219 0.269 ­0.069 ­0.201 0.409** ­0.258 ­0.408** 1 clay 0.188 0.178 0.243 0.084 ­0.12 0.276 ­0.465** ­0.711** ­0.353* 1 essential oil 0.092 ­0.024 0.391* 0.278 0.434** 0.355* ­0.181 0.155 0.153 ­0.277 1 essential oil yield 0.235 0.163 0.401* 0.423** 0.382* 0.449** ­0.101 0.226 0.246 ­0.421** 0.919** 1 https://www.sciencedirect.com/science/article/abs/pii/s0926669017302315#! https://www.sciencedirect.com/science/article/abs/pii/s0926669017302315#! 214 adv. hort. sci., 2023 37(2): 209­219 values were roughly one, but for the training data series the model overestimated. the r2 value in the training data of the second series was 18.06% more than the first transfer function. in the test data, the r2 value increased by 6.83%. the gmer value indicat­ ed that the model underestimated, and this is espe­ cially evident in the test data series. according to the results of table 4, when each of the nine variables was considered as inputs to the network, the distribution of estimated and measured values (fig. 1) was reduced around the 1:1 axis and r2 in training data is up to 96.62% and the test rose to 94.78%. another important aspect is the more accu­ rate estimation of the percentage of chamomile essential oil in both the training and test data, so that the gmer value in both data series was near one, indicating an accurate estimate and a lack or low esti­ mate. the value of gsder also showed that the low­ est non­conformance of predicted essential oils with the measured values in the data of the training and test series in the third transfer function was observed. based on these results, increasing vari­ ables from 4 (ptf1 model) to 9 (ptf3 model) decreased error and increased r2 of ann­model. mohammadi torkashvand et al. (2020) employed an artificial neural network (ann) to evaluate the kiwi yield of hayward cultivar based on the concentration fig. 1 ­ measured values (actual) and estimated concentration of essential oils in diagram 1: 1 test data and their confor­ mance. table 3 ­ input data for constructing a neural network in three different transfer functions and the characteristics of neural networks made transition function model inputs no. of hidden layers no. of hidden layer nodes 1 no. of hidden layer nodes 2 no. of hidden layer nodes 3 type of transfer function type of target function ptf1 n, p, k and clay 3 4 2 1 tangent axon levenberg­marquardt algorithm ptf2 ph, ec, organic matter 3 4 4 2 tangent axon levenberg­marquardt algorithm ptf3 all variables 1 10 ­ ­ tangent axon levenberg­marquardt algorithm table 4 ­ determination coefficient (r2), error (rmse), gmer and gsder in two sets of training and test data in predicting essential oil concentration (g/100 g) transition function data series r2 rmse gmer gsder ptf1 training 0.7856 0.226 1.27 2.37 test 0.8271 0.201 1.04 1.24 ptf2 training 0.9662 0.072 0.91 2.27 test 0.8954 0.237 0.83 1.57 ptf3 training 0.9562 0.023 0.98 1.22 test 0.9478 0.086 1.02 1.32 khakipour et al. ‐ forecasting chamomile essential oil yield through soil physicochemical 215 of nitrogen, potassium, calcium, and magnesium in leaves. they concluded that the maximum r2 and the lowest root mean square error were obtained when all nutrients and related ratios were considered as input variables. mohammadi torkashvand et al. (2017) tested and compared the performance of an artificial neural network in predicting the firmness of six­month stored kiwifruit with different input datasets. reversely, they showed that the best answer was obtained using ann with a rmse of 0.539 and a correlation coefficient of 0.850 (r2=0.724) when the nitrogen and calcium (n/ca ratio) were input data (two variables). prediction of 6­month fruit firmness using nutrient concentrations and their rations (8 variables) datasets resulted in the lowest r­value and the highest error (mohammadi torkashvand et al., 2017). figure 2 shows the dispersion of the measured yield values and the estimation of essential oil in the test series in chart 1:1 and in the 20 samples. according to the results of table 5, the value of r2 in the test data series in the first transfer function was 91.25%, but less than 80% in the training data. the other thing is the high amount of dispersion, non­ conformance of the estimated and measured and over­estimation data in training. therefore, in the test series, the accuracy has increased, and in addi­ tion to reducing the dispersion and increasing the conformance, the prediction accuracy has significant­ ly increased, because gmer was approximately one. forecasting models of plant yield are prognostic tools that can be an important element in precision agri­ culture (shearer et al., 2000; dias and sentelhas, 2017; prasad et al., 2017; mohammadi torkashvand et al., 2017, 2020) and the principal factor in deci­ sion­making systems (park et al., 2005). akbar et al. (2018) used a model based on artificial neural net­ work to predict essential oil yield in turmeric (curcuma longa l.). the data of essential oil, soil and environmental factors were collected from 131 turmeric germplasms in 8 agro­climatic regions of odisha. results showed that multilayer­feed­forward neural networks was the most reasonable model to use with r2 value of 0.88. niazian et al. (2018) calcu­ lated a root mean square error (rmse) of 0.192 and r2 of 0.901 in predicting essential oil content of ajowan by using the artificial neural network when the number of rays, pedicels, and flowers per umbel­ let, and a number of umbellets in an umbel, inputted variables. bahmani et al. (2018) utilized an artificial neural network modeling to predict kinetics of essen­ tial oils extraction from tarragon (artemisia dracun‐ culus l.) using ultrasound pre­treatment with clevenger. based on results, the best prediction per­ formance was belonged to 3­7­1 ann architecture (0.0008 normalized mean squared error and r2 were respectively 0.0008 and 0.99) which means that it is possible to predict the extraction yield of essential oils with an acceptable precision. tashakori et al. (2020) research showed that a model with organic matter, phosphorus, potassium, and calcium carbon­ ate as in dependent variables, was the best model (r2 = 0.87) in estimating saffron yield. if the focus is on the test data, in the second transfer function, the accuracy of the model (r2 = 83.23%) was less than the other two functions and its accuracy is much lower than the other two, so that the deviation of the measured and estimated essen­ tial oil yield data was 1.45 (gsder) and the model had an over­estimation of 26% (gmer=1.26). the important point is that, like the content of essential oil (g/100 g dry matter), the highest accuracy and precision of the neural network model was obtained in predicting the essential oil yield in the third trans­ fig. 2 ­ measured (actual) and estimated essential oil yield in diagram 1: 1 test data and their conformance. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/neural-networks https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/essential-oils https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/essential-oils https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/tarragon https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/artemisia-dracunculus https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/artemisia-dracunculus https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/pretreatment adv. hort. sci., 2023 37(2): 209­219 216 fer function, in which all soil variables (nine variables) were considered as input variables of the network. in the test data series, the lowest error (rmse = 0.088) and the most consistent measured and estimated data on the essential oil yield were obtained in the third transfer function, although the fitted model had a lower estimation than the first function. of course, it should be noted that in view of the great difference between the first and third functions in training data, the third function had a higher credibility in general. 4. conclusions in general, according to the results, the third transfer function (9 variables as input variables of the network) was the most accurate for estimation of the essential oil concentration and for the estimation of essential oil yields. they had the highest r2 and the lowest rmse values. also, the estimated values of these functions were the most consistent with the observed values and the least deviation from the 1:1 line. as the r2, rmse, gmer and gsder values of the proposed model for estimating essential oil percent for test series data were 94.78, 0.86, 1.02 and 1.32, respectively, and to evaluate the essential oil yield in the test series data respectively, equal to 91.51, 0.608, 0.92 and 1.20 respectively. therefore, the results showed that with high accuracy and precision, it is possible to predict the concentration and yield of chamomile 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https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en https://scholar.google.com/scholar?oi=bibs&cluster=859759651701355766&btni=1&hl=en impaginato 31 adv. hort. sci., 2025 39(1): 31­43 doi: 10.36253/ahsc­16775 https://oaj.fupress.net/index.php/ahs glucose exogenous increases biochemical and physiological responses in beta vulgaris l. j.v.m. da silveira, e.c.p. dos santos, l.p. dos santos, c.f. de freitas, r.f. de arruda, r.h.p. garcia, p.h. gorni (*) department of agronomy, escola superior de agronomia de paraguaçu paulista (esapp), 19700‐000 paraguaçu paulista, sp, brazil. key words: antioxidant activity, elicitation, glucose, leaf pigments, phenolic compounds, secondary metabolism, yield. abstract: glucose can act as a bioregulator in plants, influencing physiological and metabolic processes. this investigation aimed to analyses how the application of glucose impacts the photosynthetic rate, growth, and concentration of bioactive compounds with antioxidant potential in beet. the current investigation evaluated the effect of glucose (0, 5, 10, 20, and 30 mmol l­1) on beet plants 10 days after transplantation. glucose elicitation in beet plants increases levels of bioactive compounds, resulting in higher antioxidant potential. in addition to antioxidant benefits, glucose also plays a hormonal role, leading to increases in biomass. these effects are correlated with enhanced photosynthetic efficiency, elevated carbohydrate concentrations, and positive modulation of the plant antioxidant system. 1. introduction beta vulgaris l (beet) belonging to the chenopodiaceae family, is a nutritious vegetable often used in salads and juices. it is an excellent source of nutrients, including vitamins (folic acid, and vitamin a, b1, b2, b6, and c), minerals (sadowska­bartosz and bartosz, 2021), fiber, proteins (fu et al., 2020 a), and sugar (sucrose) (tayyab et al., 2023). additionally, beet is considered a great health food, due to the high levels of bioactive compounds present, such as betalains, polyphenols, and flavonoids (gorni et al., 2023), which are powerful antioxidants associated with cardiovascular health and the reduction of cancer cells, among other diseases (chhikara et al., 2019). in addition to its nutritional properties, beets are valued for their diverse culinary applications. it can be consumed raw, grated in salads, cooked, roasted, grilled, and even turned into juices and smoothies. beet production faces major challenges due to climate change. rising (*) corresponding author: pgorni@gmail.com citation: da silveira j.v.m., dos santos e.c.p., dos santos l.p., de freitas c.f., de arruda r.f., garcia r.h.p., gorni p.h., 2025 ­ glucose exogenous increases biochemical and physiological responses in beta vulgaris l. ­ adv. hort. sci., 39(1): 31­43 sjvm: 0009­0006­3788­4593 secp: 0009­0003­0504­8644 slp: 0009­0009­3203­6597 fcf: 0009­0007­7321­9379 arf: 0009­0004­5151­0585 grhp: 0009­0001­5925­9397 gph: 0000­0002­3866­9215 author contributions: conceptualization: phg; data curation: phg; formal analysis; phg, jvms, lnml, inf, ecps, lps, cff, rfa, and rhpg; project administration: phg; supervision: phg; writing ­ original draft: jvms, lnml, inf, ecps, lps, cff, rfa, and rhpg; writing ­ review and editing: phg. copyright: © 2025 da silveira j.v.m., dos santos e.c.p., dos santos l.p., de freitas c.f, de arruda r.f., garcia r.h.p., gorni p.h. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 3 october 2024 accepted for publication 27 february 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16775 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0006-3788-4593 http://orcid.org/0009-0003-0504-8644 http://orcid.org/0009-0009-3203-6597 http://orcid.org/0009-0007-7321-9379 http://orcid.org/0009-0004-5151-0585 http://orcid.org/0009-0001-5925-9397 http://orcid.org/0000-0002-3866-9215 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 31­43 32 temperatures and changes in rainfall patterns affect both its growth, development, and the formation and quality of roots (tayyab et al., 2023). in addition, water shortages and the emergence of pests and diseases are becoming more frequent, putting productivity at risk (baryga et al., 2020). to deal with these problems, new techniques are being adopted to improve agricultural practices, making crops more sustainable and stimulating the plant to biosynthesize more bioactive compounds, which results in increased plant resistance to climate changes. elicitation has been used as a technique that involves the induction of responses in plants, generally in response to biotic (such as pathogens or herbivores) or abiotic (such as climate variations) stresses (kandoudi and németh­zámboriné, 2022). elicitors can act at several levels, such as activating plant defence mechanisms. for example, elicitation technique can be used to increase the amount of phytocompounds in plant, improving their functionality, this practice has been applied to increase the yield of secondary metabolites, biological activities (antioxidant properties) and mainly to induce plant growth (baenas et al., 2019; siddiqui et al., 2020; gorni et al., 2023) and can also stimulate cellular signaling pathways, leading to the expression of genes related to defence and strengthening of cellular structure (sami et al., 2021). among the various elicitors, glucose (glc) has been studied due to its important roles in plants, to increase the biosynthesis and accumulation of secondary metabolites, resulting in increased plant yield (hennion et al., 2019). regarding the elicitors that act in secondary metabolism, it is known that glc is an essential sugar that plays a vital role in plant metabolism, acting as one of the main sources of energy (saddhe et al., 2021). produced during photosynthesis, it not only feeds plants, but is also essential for their health and growth (fu et al., 2020 b). in addition to providing energy, glc is an important component in the formation of several biomolecules necessary for plant development (wang et al., 2019). given this information, some researchers have observed that the application of glc acted as a growth inducer for plants, which was correlated with increased photosynthesis (siddiqui et al., 2020), seed germination (gorni and polimento, 2023), regulates antioxidant metabolism (sami and hayat, 2019), activates key enzymes involved in secondary metabolism (zahid et al., 2018), increases the biosynthesis of phenols, flavonoids, anthocyanins, and antioxidant activity (xu et al., 2016; baenas et al., 2019), gene expression (rezaee et al., 2018), and alleviates stresses (sami et al., 2021). from this perspective, glc is a substance that promotes plant growth and enhances quality by increasing the biosynthesis of bioactive compounds and reducing ros concentration (baenas et al., 2014; sami and hayat, 2019). given this information, the goal is to improve the quality and yield of beet roots through glc application at different concentrations, aiming for a biostimulant effect. this approach could potentially enhance the growth and development of the crops, increase yield, and improve root quality. therefore, this investigation aimed to analyses how the application of glc impacts the photosynthetic rate, growth, and concentration of bioactive compounds with antioxidant potential in beet. 2. materials and methods experimental location and plant material the experiment was conducted using beet plants in pots inside a greenhouse on a wooden bench, kept open sky, covered by shading which provided 50% of solar radiation located at the escola superior de agronomia de paraguaçu paulista (esapp), brazil (22°41’76” s, 50°58’33” w). the köppen classification identifies the region’s climate as aw, the climate data is presented in figure 1. the experiment was conducted between april 19 and june 18, 2024 (60 days). fifteen­day­old beet seedlings were transplanted into pots with sandy soil. the soil was analysed and fig. 1 ­ climatic data of paraguaçu paulista, são paulo, during the experimental period (april 19 to june 18, 2024). source: inmet ­ instituto nacional de meteorologia. da silveira et al. ‐ glucose enhances responses in beet 33 corrected according to bulletin 100 (cantarella et al., 2022). the pots were irrigated by sprinklers twice a day during the experimental period. every 15 days, cattle manure was applied as coverage fertilization. at 10 days after transplantation, the treatments were applied at concentrations of 0 (control), 5, 10, 20, and 30 m mol l ­1 of glc. each treatment was replicated four times. all analyses below were performed at the end of the experiment at 60 dat. pigments analysis and quantification photosynthetic pigments were extracted in 95% ethanol and calculations were performed according to lichtenthaler (1987). fresh tissues (0.2 g) were added to 8 ml of 95% ethanol and shaken, leaving them to rest for 24 h at 4°c. after this process, readings were performed at wavelengths of 470, 647 and 663 nm. photosynthetic parameters chlorophyll fluorescence parameters were measured using a portable chlorophyll fluorometer (opti­sciences, fv/fm meter). beet leaves were placed under dark adaptation clips for 30 min prior to measurement to allow complete accumulation of chlorophyll energy. after the adaptation period, measurements of initial fluorescence (f0), maximum fluorescence (fm), variable fluorescence (fv), maximum photochemical efficiency of psii (fv/fm), and absorbed energy conversion (fv/f0). biochemical assessment of enzymes activities and protein frozen samples were extracted in 0.1 m potassium phosphate buffer (ph 7.5) containing edta and pvp. the supernatant was utilized for enzyme activity assays, included those sod (ec 1.15.1.1) was measured by the inhibition of nitroblue tetrazolium (nbt) reduction by superoxide, which is generated by the reaction of riboflavin under light (giannopolitis and ries, 1977), cat (ec 1.11.1.6) is determined by the decomposition of hydrogen peroxide, which can be monitored spectro­ photometrically (240 nm) (azevedo et al., 1998), and apx (ec1.11.1.11) is measured by the decrease in absorbance of ascorbic acid in the presence of h₂o₂ (moldes et al., 2008). for phenylpropanoid pathway enzymes, leaves were extracted using 0.5 mm tris­edta buffer (ph 8.5) containing edta. the supernatant was used to measure the activity of phenylalanine ammonia lyase (pal, ec 4.3.1.5) following the method of hyodo et al. (1978), and chalcone synthase (chs, ec 2.3.1.74) according to moustafa and wong (1967). the pal assay was conducted at 40°c for 1 hour in a reaction mixture (1 ml) consisting of 0.5 m tris­hcl (ph 8.5), enzyme extract, and 30 µm l­phenylalanine. the reaction was stopped in an ice bath for 5 minutes. pal activity was quantified spectrophotometrically by measuring the concentration of trans­cinnamic acid produced at 290 nm. the chs assay was conducted at 30°c for 1 hour in a reaction mixture (1 ml) consisting of 5 mm tris­hcl (ph 7.8), 10 mm kcn, enzyme extract, and chalcone was added to ethylene glycol monomethyl ether. chs activity was measured by the production of chalcone at 370 nm. protein concentration was determined using the bradford method (1976), where the supernatant was added to the bradford solution, after 2 minutes the reading was taken at 590 nm, using bovine serum albumin as standard. biochemical assessment of ros to determine ros, samples were extracted in 0.1% trichloroacetic acid. the supernatant used for the hydrogen peroxide (h2o2) was evaluated according to alexieva et al. (2001), samples were added to a solution of 0.1 m potassium phosphate buffer (ph 7.5) and 1 m potassium iodide solution. the mixture was incubated on ice for 1 h. the reading was performed at a wavelength of 390 nm. using h2o2 standards. lipid peroxidation was by the malondialdehyde (mda) method by heath and packer (1968), supernatant was mixed with a solution containing 0.5% tba (thiobarbituric acid) in 20% tca (trichloroacetic acid). this mixture was incubated at 95°c for 30 min, and the reaction was stopped with an ice bath. the absorbances were 535 nm and 600 nm. superoxide radical (o2 ·−) content was evaluated according to elstner and heupel (1976), sample was added to a 65 mm potassium phosphate buffer solution (ph 7.8) and a 10 mm hydroxylamine hydrochloride solution and incubated at 25 ºc for 20 min. after incubation, 1% (m/v) sulfanilamide in hydrochloric acid (2.4 n) and 0.02% n­(1­ naphthylethylenediamine) dihydrochloride were added. the reaction mixture was homogenized and incubated again at 25°c for 20 min. finally, ethyl ether was added, the solution was centrifuged at 1500 rpm for 5 min, and the wavelength used was 530 nm. https://link.springer.com/article/10.1007/s42729-023-01131-8#ref-cr17 https://link.springer.com/article/10.1007/s42729-023-01131-8#ref-cr32 https://link.springer.com/article/10.1007/s42729-023-01131-8#ref-cr3 https://link.springer.com/article/10.1007/s42729-023-01131-8#ref-cr19 adv. hort. sci., 2025 39(1): 31­43 34 determination of nitrogen compounds for extraction, leaf samples were macerated in 80% ethanol solution. the material was macerated and centrifuged, and the supernatant was used for analysis. total soluble sugar content was determined according to dubois et al. (1956), sample was mixed with a phenolic reagent (5%) and then sulfuric acid was added. after cooling, the absorbance was read at 390 nm, using glucose as a standard. reducing sugar content was determined using somogyi­nelson (bezerra neto and barreto, 2004), sample was mixed with the stirred somogyi reagent and incubated in boiling water for 15 minutes. after cooling on ice, the nelson reagent was added, mixed and left to stand for 20 minutes. after the addition of water, the absorbance was read at 760 nm, glucose was used as a standard. sucrose content was determined according to van handel (1968), sample was mixed with a koh reagent (30%) and then sulfuric acid was added. the mixture was incubated at 100°c for 10 minutes, after cooling the absorbance was read at 490 nm, using sucrose as a standard. starch content was determined using anthrone reagent (sadasivam and manickam, 1996). samples were mixed in anthrone reagent and boiled for 10 minutes, the mixture was cooled, and the absorbance read at 650 nm, glucose was used as a standard. total amino acids content was determined according to yemm et al. (1955). samples were mixed in sodium citrate (0.2 m), ninhydrin solution (5%) and kcn (0.2 mm). the mixture was homogenized and heated at 100°c for 20 minutes. after cooling in running water, 60% ethanol was added and the absorbance was read at 570 nm, using methionine as a standard. determination of proline proline was determined by bates et al. (1973). for extraction, leaf samples were macerated in a 5­ sulfosalicylic acid solution (3%), the material was macerated and centrifuged, and the supernatant was used for analysis. the sample was mixed with a solution of acid ninhydrin and acetic acid, which was incubated at 100°c for 1 h. afterwards, the proline was partitioned with the addition of toluene and the mixture was shaken. absorbance was read at 520 nm, using proline as a standard. biochemical analysis of tuberous root fresh beet root (5 g) was subjected to a washing and peeling process and extracted with a processor for two minutes. after this procedure, the juice was filtered through sieves (2 mm) and stored in a refrigerator before analysis. total soluble solids the analysis of total soluble solids was performed using an analogue refractometer, where the samples were centrifuged, and a drop deposited on the refractometer prism and the values were collected. the results were expressed in degrees brix (°brix). total polyphenols, flavonoids, anthocyanins, carotenoid, and betalains concentrations polyphenols concentrations was determined according to the folin­ciocalteu method and 25% sodium carbonate solution (stagos et al., 2012), using gallic acid as standard. flavonoids concentrations were measured according to yao et al. (2013), using 5% nano2, 10% alcl3, and naoh (1 m), using rutin as standard. anthocyanins were determined according to the francis method (1982), in 95% ethanol extraction solution acidified with hcl, the mixture was left to rest for 24 hours at 4°c, protected from light, after which it was filtered and the absorbance read at 535 nm. the carotenoid content was extracted from fresh leaf samples with chloroform/acetone/ethanol (2:1:1, v/v/v) (sadler et al . , 1990). for determination of β­carotene, absorbance was measured at wavelengths of 450 nm. quantification of betalains followed the methodology of nilsson (1970), root samples were homogenized, and absorbance readings were performed at wavelengths of 480 nm and 540 nm. dpph activity dpph radical scavenging was determined by blois (1958). samples were mixed with ethanol, acetate buffer (0.1 m, ph 5.5) and a dpph solution (0.5 mm) was added. after resting in the dark for 30 minutes, absorbance was read at 517 nm. the results were expressed as a percentage (%). growth and yield parameters the measured traits included the number of leaves. using a caliper, measurements were taken of the root diameter, and length. for shoot, root, and total fresh weight a precision scale was used. https://link.springer.com/article/10.1007/s42729-023-01131-8#ref-cr46 da silveira et al. ‐ glucose enhances responses in beet 35 experimental design and statistical analysis analysis of the data was performed using sisvar (ferreira, 2019). anova was performed on the data to determine the tukey’s test (p≤0.01). a multivariate analysis of variables was performed for all parameters using sigmaplot software. 3. results beet plants treated with glc showed significant results in chlorophyll fluorescence (fig. 2). the f0 values exhibited reductions of 15.6%, 14.9%, 16.48%, and 12.2%, respectively, compared to the control (fig. 2 a). increases were observed for fm by 5.5%, 9.8%, 1.5%, and 4.6% (fig. 2 b), fv by 10.8%, 16.1%, 6.0%, and 8.9% (fig. 2 c), fv/fm by 5.1%, 5.36%, 4.5%, and 4.1% (fig. 2 d), and fv/f0 by 31.3%, 36.5%, 27.3%, and 24.4% (fig. 2 e), respectively, compared to untreated plants. chlorophyll concentrations treated with 5 and 20 mmol l­1 of glc increased by 47.2% and 20.2% for chlorophyll a, 42.6% and 17.4% for chlorophyll b, 46.2% and 19.6% for total chlorophyll, and 39.9% and 13.6% for carotenoids, respectively, compared to the control. however, when the glc concentration was 10 and 30 mmol l ­1, the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were lower than those obtained without glc treatment (fig. 3). beet plants treated with glc showed reductions in mda concentrations of 82.5%, 31.9%, 40.9%, and 38.9%, respectively, compared to the control (fig. 4 a). however, h2o2 concentrations decreased by 27.2% and 13.1%, respectively, in plants treated with 5 and 10 mmol l­1 of glc compared to the control (fig. 4b), and concentrations of 20 and 30 mmol l­1 of glc, did not differ from the control. as for o2ˉ concentrations, there was an increase of 15.3% in plants treated with 20 mmol l ­1 of glc, while concentrations of 5 and 10 mmol l­1 decreased by 18.1% and 62.2%, respectively, compared to the fig. 2 ­ f0 (a), fm (b), fv (c), fv/fm (d), and fv/f0 (e) in beet plants treated with different glucose concentrations. **= significant at a probability level of p≤0.01 using the tukey’s test at 5% probability. bars represent standard error of the mean; n = 4. fig. 3 ­ chlorophyll a (a), chlorophyll b (b), total chlorophyll (c) and carotenoids (d) concentrations in beet plants treated with different glucose concentrations. **= significant at a probability level of p≤0.01 using the tukey’s test at 5% probability. bars represent standard error of the mean; n = 4. fw = fresh weight. 36 adv. hort. sci., 2025 39(1): 31­43 control (fig. 4 c). regarding the activity of the sod enzyme, treatments with 5, 10, and 30 mmol l­1 of glc showed increases of 37.9%, 15.0%, and 12.4%, respectively, compared to the control (fig. 5 a). however, when the glc concentration was 20 mmol l­1 was lower than those obtained without glc treatment. for cat and apx, there was an increase in activity in plants treated with 5, and 10 mmol l ­1, respectively, compared to the control (fig. 5 b, 5 c). however, the application of concentrations of 20 and 30 mmol l−1 showed lower activity than those obtained without treatment with glc. total sugar concentrations in plants treated with 10 and 30 mmol l­1 of glc showed increases of 5.4% and 11.3%, respectively, compared to the control (fig. 6 a). for reducing sugar concentrations, applications of 10, 20, and 30 mmol l ­1 of glc increased by 61.6%, 41.1%, and 53.8%, respectively, compared to the control (fig. 6 b). sucrose concentrations increased by 14.4% and 32.9%, respectively, in plants treated with 10 and 20 mmol l­1 of glc compared to the control (fig. 6 c). starch concentrations were higher by 9.3%, 30.7%, and 18.9%, respectively, in plants treated with 10, 20, and 30 mmol l­1 of glc compared to the control (fig. 6 d), however, when the glc concentration was 5 mmol l­1 fig. 4 ­ mda (a), h2o2 (b), and o2ˉ (c) concentrations in beet plants treated with different glucose concentrations. **= significant at a probability level of p≤0.01 using the tukey’s test at 5% probability. bars represent standard error of the mean; n= 4. fw = fresh weight. fig. 5 ­ specific activity of sod (a), cat (b), and apx (c) in beet plants treated with different glucose concentrations. **: significant at a probability level of p ≤ 0.01 using the tukey’s test at 5% probability. bars represent standard error of the mean; n = 4. da silveira et al. ‐ glucose enhances responses in beet 37 was lower than those obtained without glc treatment. for amino acid concentrations, only plants treated with 30 mmol l­1 of glc showed an increase of 16.1% compared to the control (fig. 6 e). however, proline concentrations increased by 86.5% and 58.7%, respectively, in plants treated with 10 and 20 mmol l­1 of glc compared to the control (fig. 6 f), however, when the glc concentration was 5 mmol l­1 was lower than those obtained without glc treatment. protein content increased by 26.6% in plants treated with 20 mmol l­1 of glc compared to the control (fig. 6 g). however, total soluble solids in tuberous roots decreased in all treatments compared to the control (fig. 6 h). the pal enzyme activity showed increases of 30.3%, 48.8%, and 42.0%, respectively, in plants treated with 5, 10, and 30 mmol l­1 of glc compared fig. 6 ­ total sugar (a), reducing sugar (b), sucrose (c), starch (d), amino acid (e), proline (f), protein (g), and total soluble solids in root (h) concentrations in beet plants, and total soluble solids in juice (f) treated with different glucose concentrations. **= significant at a probability level of p≤0.01 using the tukey’s test at 5% probability. bars rep­ resent standard error of the mean; n= 4. fw= fresh weight. to the control (fig. 7 a). however, the chs enzyme activity increased by 7.9%, and 17.2%, respectively, in plants treated with 10, and 30 mmol l ­1 of glc compared to the control (fig. 7 b). however, the application of concentration of 20 mmol l­1 showed lower activity than those obtained without treatment with glc. the application of glucose at 30 mmol l­1 resulted in increases of 21.2% for polyphenols, 34.6% for flavonoids, 22.7% for β­carotene, 26.9% for anthocyanins, and 28.7% for betalains, respectively, compared to the control (fig. 8 a­e). however, the application of concentrations of 5, 10, and 20 mmol l−1 showed lower activity than those obtained without treatment with glc respectively. however, increases of 27.6%, 99.4%, and 20.4% were observed at concentrations of 5, 20, and 30 mmol l ­1, respectively, in the antioxidant activity by the dpph fig. 7 ­ specific activity of phenylalanine ammonia lyase (pal) (a), chalcone synthase (chs) (b) in beet plants treated with different glucose concentrations. **= significant at a probability level of p≤0.01 using the tukey’s test at 5% probability. bars represent standard error of the mean; n = 4. adv. hort. sci., 2025 39(1): 31­43 38 weight increased by 45.9% in plants treated with 10 mmol l­1 compared to the control (fig. 9 b). total fresh weight increased by 24.6% and 26.6%, respectively, in plants treated with 5 and 20 mmol l­1 method compared to the control plants (fig. 8 f). for growth analysis, plants treated with 20 mmol l­1 of glc showed increases in leaf fresh weight by 113.2% compared to the control (fig. 9 a). root fresh fig. 8 ­ polyphenols (a), flavonoids (b), β­carotene (c), anthocyanins (d), total betalains (e), and dpph (f) concentrations in beet plants treated with different glucose concentrations. **: significant at a probability level of p ≤ 0.01 using the tukey’s test at 5% proba­ bility. bars represent standard error of the mean; n = 4. fw = fresh weight. fig. 9 ­ shoot fresh weight (a), root fresh weight (b), total fresh weight (c), root length (d), and root diameter (e), and leaf number (f), and visual growth development (g) in beet plants treated with different glucose concentrations. ** and *= significant at a proba­ bility level of p≤0.01 and p≤0.05; ns= not significant using the tukey’s test at 5% probability. bars represent standard error of the mean; n = 4. da silveira et al. ‐ glucose enhances responses in beet 39 4. discussion and conclusions in the present study, the data show that chlorophyll fluorescence differed among treatments (fig. 2). however, chlorophyll fluorescence analysis has been widely used to assess photosynthetic efficiency in plants, as this technique is sensitive to changes in the environment and plant metabolism (jiang et al., 2012). based on the results found, glc can act as an energy substrate for plant metabolic processes, particularly for photosynthesis (sinha and roitsch, 2001). with increased glc availability in the plant, there was consequently an increase in photosynthetic rate, thereby enhancing energy production and improving light absorption efficiency, resulting in higher chlorophyll fluorescence (wang et al., 2019). the use of glc showed positive responses in several crops, such as promoting germination and vegetative growth, inducing greater plant yields (gonzález­hernández et al., 2020; gorni and polimeno, 2023). although glc is a byproduct of photosynthesis, its exogenous application can stimulate the synthesis of photosynthetic pigments (siddiqui et al., 2020), which supports our findings (fig. 3). furthermore, studies indicate that elicitation with glc enhances electron transfer from psii to psi, as well as increases atp and nadph production, crucial for various biochemical reactions in plants, thereby influencing carbohydrate, lipid, and protein synthesis and regulating biochemical processes (rineau et al., 2013; siddiqui et al., 2020). similar results confirm that glc application stimulates pigment accumulation in plants, as observed in mustard (brassica juncea) (sami et al., 2021), triticale (× triticosecale wittmack) (wang et al., 2019), and cucumber (cucumis sativus) (yusuf et al., 2021). during photosynthesis and other aerobic metabolic processes, plants generate reactive oxygen species (ros) as part of their normal metabolism and in response to environmental stresses. however, factors such as biotic and abiotic stresses can induce higher ros production in plants as part of their defense mechanisms, leading to cellular damage and oxidative stress (das and roychoudhury, 2014). our results demonstrated that glc was effective in reducing ros accumulation compared to the control (fig. 4). considering these findings, glc elicitation induced an adaptive response in beet plants, increasing the activity of sod, cat, and apx (fig. 5), which correlated with reduced ros levels, of glc compared to the control (fig. 9 c). however, root length, root diameter, and number of leaves did not show significant responses to glucose application (fig. 9 d­f). pca was conducted to evaluate the relationship between morpho­physiological traits and glc treatments (fig. 10). the variables were depicted along two principal axes, which together explained 57.81% of the total variance (pc1: 32.18%; pc2: 25.63%). this analysis indicated a positive correlation between biometric characteristics and the 20 mmol l­ 1 concentration. in contrast, physiological and biochemical parameters were associated with the 5, 10, and 30 mmol l­1 concentrations. fig. 1 ­ pca of the analyzed parameters from beet plants treated with different glucose concentrations. minimum fluores­ cence (f0), maximum fluorescence (fm), variable fluores­ cence (fv), maximum photochemical efficiency (fv/fm), variable fluorescence/initial fluorescence (fv/f0), chloro­ phyll a (chla), chlorophyll b (chlb), total chlorophyll (tchl), carotenoid (car), malondialdehyde (mda), hydro­ gen peroxide (h2o2), superoxide radical (o2­), superox­ ide dismutase (sod), catalase (cat), ascorbate peroxi­ dase (apx), proline (pro), amino acids (aa), total sugar (st), reducing sugar (rs), sucrose (suc), starch (sta), phenylalanine ammonia lyase (pal), chalcone synthase (chs), total flavonoids (ft), total polyphenols (ppt), anthocyanin (anth), β­carotene (β­car), 2,2­diphenyl­1­ picrylhydrazyl (dpph), total soluble solids (tss), protein (prot), root size (sr), root diameter (rd), number of leaves (nl), shoot fresh weight (sfw), root fresh weight (rfw), and total fresh weight (tfw). adv. hort. sci., 2025 39(1): 31­43 40 contributing to a more stable redox balance and fewer ros­induced cellular damages (siddiqui et al., 2020). similar results have shown increased activity of antioxidant enzymes and reduced ros levels following glc elicitation in mustard and cucumber plants (sami et al., 2021; yusuf et al., 2021). glc is a simple sugar that, when applied to plants, can stimulate carbohydrate metabolism, including increased synthesis of other carbohydrates such as sucrose and reducing sugars (siddiqui et al., 2020). our results demonstrate that glc application increased concentrations of total sugars, reducing sugars, sucrose, starch, and proteins in beet plants (fig. 6). these findings indicate that glc application was effective, as when absorbed by plants, this substance can be converted into starch as a form of energy storage, resulting in increased availability of carbohydrates and proteins (siddiqui et al., 2020). additionally, there were increases in amino acid and proline concentrations (fig. 6 e­f). the exogenous application of glc results in a significant increase in amino acid concentration, as glc serves as a substrate for the synthesis of various compounds, including amino acids. moreover, exogenous glc application can induce glutamine synthetase and glutamate dehydrogenase enzymes involved in amino acid synthesis (forde and lea, 2007; gonzález­ hernández et al., 2020). furthermore, glc can also modulate the enzyme p5cs (pyrroline­5­carboxylate reductase), leading plants to increase proline production as part of their adaptive strategies (sami and hayat, 2019). glucose application increases pal and chs activities in plants, which are responsible for the production of phenolic and flavonoids compounds (wei et al., 2011). these compounds play crucial roles in defense against biotic and abiotic stresses, flower and fruit pigmentation, and other essential physiological functions for plant development and adaptation to the environment (baenas et al., 2019). studies on glc application in plants have been shown to stimulate the expression of genes encoding enzymes, particularly plant defense enzymes (pal, chs, chi), thereby inducing increases in the concentration of secondary metabolites. this approach is considered more efficient for enhancing the production of desired secondary metabolites and for manipulating biochemical and metabolic pathways (guo et al., 2011; zahid et al., 2018). our results demonstrate that glc application induced increases in pal and chs activity (fig. 7), as well as an increase in secondary compounds at a concentration of 30 mmol l­1 (fig. 8a­g), resulting in enhanced plant antioxidant activity (fig. 8h). these compounds have garnered significant interest due to their positive impacts on various fundamental cellular processes, in addition to possessing antioxidant, anti­ inflammatory, anticancer, among others (chen, 2016). however, glc application stimulated the increase of these compounds, making these plants more valuable for human consumption due to their rich source of bioactive nutrients, promoting a healthy and balanced diet. the use of glc can induce improvements in vegetative growth and yield in plants (sami and hayat, 2019; sami et al., 2021). in the present study, the beneficial role of glc was confirmed due to the increase in fresh shoots, roots, and total weight (fig. 9 a­c). exogenous application of glc promotes plant growth by acting as an energy source for cells, thereby significantly enhancing metabolic activity and positively regulating the expression of genes related to photosynthesis and hormonal signaling pathways (mao et al., 2018). these effects positively influence carbohydrate metabolism, stimulating the synthesis of reserves such as starch and sucrose, which translates into enhanced plant growth (wang et al., 2019; gorni and polimeno, 2023). the results of this study show that the application of glc provides significant improvements in the growth, development and productivity of beetroot, in addition to favoring the accumulation of bioactive compounds. these findings indicate that glc not only serves as an energy source, but also acts as a stimulator of the plant’s physiological responses, increasing its resistance to environmental stresses. the incorporation of glc into agricultural management practices may therefore represent an effective strategy to optimize beet production, contributing to the nutritional quality and sustainability of crops. future research may deepen the understanding of the mechanisms underlying these responses, expanding the potential of glc in agricultural systems. this study underscores the importance of exogenous glc application not only as an essential nutrient but also as a key modulator of biochemical processes that control plant development. it can thereby optimize agricultural management and crop yield. glucose application had a positive impact on the development of beets, stimulating different processes according to the concentration used. at 5 da silveira et al. ‐ glucose enhances responses in beet 41 and 10 mmol l­1, there was greater plant growth, increased chlorophyll fluorescence and activation of antioxidant enzymes. higher concentrations (30 mmol l­1) favored the production of pigments and bioactive compounds. these results indicate that glc can act as a metabolic signal, helping the plant grow better and strengthen its defense mechanisms although the results of this study are promising, some limitations should be considered. the experiment was carried out in a greenhouse. to advance this line of research, future studies should test the application of glucose under field conditions and evaluate how different environmental factors affect its benefits for beet. another important factor is the specific stage of the plant at which glucose application is more efficient, in addition to better exploration of the molecular processes involved in this response. these investigations can contribute to making this strategy even more efficient and applicable to real farming systems. acknowledgements the authors would like to thank the paraguaçu paulista college of agronomy (esapp) for the financial support to carry out this project. references alexieva v., sergiev i., mapelli s., karanov e., 2001 ­ the effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. ­ plant cell environ., 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semi­hardwood, vegetative propagation. abbreviations: iba= indole­3­butyric acid, fs= fine sand substrate, pm= peat moss substrate, fs/pm= mixture of fine sand and peat moss. abstract: to evaluate the rooting ability and growth performance in semi­hard­ wood cuttings of argania spinosa under non­mist greenhouse conditions, our experimentation was conducted with three cutting diameters (0.1­0.3, 0.3­0.6 and 0.6­0.9 cm), four leaf retention treatments (leafless, 2, 4 and 8 leaves) and three different rooting substrates (fine sand, peat moss, a 1:1 mixture of fine sand/peat moss). significant effects of cuttings diameter, leaf retention and rooting substrate on sprouting, rooting and survival ability from a. spinosa semi­hardwood cuttings were observed. among all diameters tested, a diame­ ter of (0.3­0.6 cm) showed maximum rooting and survival capacity, while cut­ tings with a diameter of 0.6­0.9 cm resulted in the greatest sprouting ability. successful vegetative propagation was restricted to leafy stem cuttings. moreover, it was observed that the highest rooting ability was reached in cut­ tings planted in fine­sand substrate. however, the highest sprouting ratio (85.0%) and survival rate (92.5%) were achieved in a mixture of sand and peat moss. thus, argan trees vegetative propagation could be most effectively achieved using semi­hardwood cuttings with a 0.4 cm diameter and 4 leaves, planted in a fine sand substrate during the root initiation period and grown in a mixture of fine sand and peat moss for hardening. 1. introduction the argan tree [argania spinosa (l.) skeels] is the only species repre­ senting the tropical sapotaceae family in morocco (emberger, 1925). it is (*) corresponding author: meriem.malaoui@gmail.com citation: benbya a., cherkaoui s., gaboun f., chlyah o., delporte f., mdarhri alaoui m., 2021 ­ clonal propagation of argania spinosa (l.) skeels: effects of leaf retention, substrate and cutting diameter. ­ adv. hort. sci., 35(1): 61­72 copyright: © 2021 benbya a., cherkaoui s., gaboun f., chlyah o., delporte f., mdarhri alaoui m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 september 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9212 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 61­72 62 a prodigious perennial thermophilic and xerophilic thorny species (ehrig, 1974; morton and voss, 1987). the argan tree can be found in a wide array of envi­ ronments where soils are ranging from heavy clay to poor and rocky desert soils, it grows in altitudinal ranges extending from sea level (along the atlantic coast) up to 1500 m (msanda, 1993). moreover, it has the ability to survive in very dry conditions at very hot temperatures up to 50°c and low rainfall levels between 150 and 400 mm/year, shedding foliage and remaining in a state of dormancy for sev­ eral years during prolonged drought (m’hirit et al., 1998). recently, a. spinosa has attracted worldwide attention as a source of highly valued oil (charrouf and guillaume, 2008). it’s becoming the most expen­ sive edible oil in the world, as the present supplies does not meet demand (lybbert et al., 2011). despite the ecological value and local economic importance of this species, its forest density has been divided by two during the twentieth century and the natural regeneration rate is very low and is worsened by cli­ mate changes and the negative impact of over exploitation (kenny and de zborowski, 2007). besides, its cultivation is not sufficiently developed. in part, this is due to excessive utilization of kernels for oil production which hindered the conventional propagation through seed germination (nouaim et al., 2002); on the other hand, it is due to the slow progress in propagation methods, mainly because of the generally low capacity of this species to form adventitious roots (nouaim et al., 2002; justamante et al., 2017). therefore, research is required to devel­ op efficient vegetative propagation techniques for the production of more planting material and conser­ vation of this species. the rooting efficiency and growth performance of cuttings in many species have been found to be dependent of the cutting thickness (foster et al., 2000). the rooting effectiveness in function of cuttings diameter could be explained by different factors (foster et al., 2000). the variation observed among different cutting diameters could be influenced by physiological conditions (nutrients, car­ bohydrates, auxin, phenolics, lignification etc.), their position within the branch or their ontogenetic aging (kaul, 2008; wendling et al., 2014). moreover, leaf retention was also reported to influence rooting behaviour of cuttings (leakey and coutts, 1989). the proportion of leaves retained by the cuttings during propagation is related to the percentage of cuttings that form adventitious roots (tchoundjeu and leakey, 1996; mesén et al., 1997a). another factor which creates a suitable environment for rooting is substrate (tchinda et al., 2013). the importance of substrate type on the rooting capacity of cuttings is widely recognized as the starting point to be addressed for a successful vegetative propagation using stem cuttings (hartmann et al., 2002; leakey, 2004). cuttings of many species root successfully in a variety of 50 propagation substrates, but the rooting performance may be greatly influenced by the kind of substrate which is linked to the hydromorphic or xeromorphic status of the species (hartmann et al., 2002; leakey, 2004). the present study aims to eval­ uate the effect of three independent factors (cuttings diameter, leaf retention and substrate) on sprouting, adventitious rooting and survival ability of argania spinosa semi­hardwood cuttings to develop an effi­ cient technique for achieving large­scale production of superior clonal stock plants and conservation of genetic resources. it is a continuation of our previous research on argan vegetative propagation, which concerned the nutrient solution influence (benbya et al., 2018), and the effects of auxin type and genotype (benbya et al., 2019) on the multiplication process. 2. materials and methods experimental site and plant materials vegetative propagation experiment was carried out during the vegetative period of the tree (may to september) at non­mist greenhouse of the biotech­ nology unit of the regional center of agricultural research of rabat (inra­morocco), under natural photoperiod and a mean temperature of 32±2°c. the semi­hardwood cuttings of a. spinosa were collected from selected adult mature trees, naturally growing at the arboretum of oued cherat in the province of bouznika, morocco (33° 81’ 96” n; 7° 11’ 03” w; 45 m altitude), which is located within 2 km of the moroccan atlantic coast and with an average annual rainfall of 460 mm/yr. preparation of cuttings and experimental establish‐ ment plant material was placed in cold dark storage at 4°c for 48h before planting. then, semi­hardwood cuttings were screened for uniform length (10±0.5 cm) and were divided into three groups by diameter: 0.1­0.3 cm with 10 to12 nodes; 0.3­0.6 cm with 8 to 10 nodes and 0.6­0.9 cm with 6 to 8 nodes using cali­ brated electronic digital vernier caliper. given cut­ tings were prepared as multiple nodes, without benbya et al. ‐ clonal propagation of argania spinosa 63 leaves (leafless); or with two leaves, four leaves or eight leaves. the tip of shoot was removed and the lower leaves of each cutting were removed. before planting, cuttings were treated with aqueous solution of 0.2% (w/v) fungicide (dithane 750 g/kg mancozeb) for 10 minutes and subsequently washed with dis­ tilled water to remove excess fungicide. then, the basal end (5.0 cm) of the cuttings was soaked for 5 min in a freshly prepared aqueous solution of auxins (17.12 mm of iba). prepared cuttings were immedi­ ately inserted in different polyethylene (pe) pots (1000 cc) containing three different substrates: steril­ ized sieved fine sand (fs) (ph of 7.3, ph­kcl method; water retention of 160 ml/l; organic matter content of 0.2%, walkley & black method); peat moss (pm) ph of 6; water retention of 800 ml/l; organic matter content of 20%); mixture of fine sand and peat moss (1:1 v/v) (fs/pm). the rooting period for the experi­ ment was run for 12 weeks, and then cuttings were uprooted carefully without harming root with run­ ning water. successfully rooted cuttings (at least one root with a length of at least 1 cm long) were trans­ planted into polyethylene pots (4500 cc) and were maintained for hardening at a spacing of 20 cm × 20 cm for 48 weeks. given cuttings were regularly watered twice a week with tap water during the first twelve weeks; after a hoagland & arnon nutrient solution (hoagland and arnon, 1950) was used as irri­ gation source. data collection the morphological characteristics assessments were realized after 12 weeks for the number of sprouts (ns), length of the longest sprout (sl, cm), sprouting percentage (sp, %), number of roots (nr), length of the longest root (rl, cm), and rooting per­ centage (rp, %), whereas the survival rate success (sr, %) was recorded at week 48 of the hardening phase. experimental design and treatments the experiment was conducted in randomized complete block design (rcbd) with four blocks and three independent variables (cutting size, leaf reten­ tion and substrate) (fig. 1a). for each treatment there were 32 cuttings (eight per block), randomly allocated in groups of two cuttings (forming four fig. 1 ­ clonal propagation of argania spinosa semi hardwood cuttings. (a) cuttings planted in peat moss substrate according to a ran­ domized complete block design (rcbd) under non mist greenhouse conditions (24 weeks). (b) rooted plant cutting set in sand substrate (2 years). (c) well rooted plant cutting set in a mixture of sand and peat moss (1:1 v/v) substrate (3 years). (d) cutting with developed root primordia from fine sand substrate (12 weeks). (e) vigorous adventitious roots of semi hardwood cuttings grown in a mixture of sand and peat moss substrate (2 years). (f) argan plantlet in a mixture of fine sand and peat moss substra­ te under non­mist greenhouse conditions. (3 years). scale bars: 10 mm. adv. hort. sci., 2021 35(1): 61­72 64 3. results effect of the cutting diameter, leaf retention and sub‐ strate on number of sprouts and sprouts length of the argania spinosa cuttings cuttings with 0.6­0.9 cm diameter and four leaves set in fs/pm substrate showed the greater mean number of sprouts and the longest sprout of cuttings (1.81±0.10 cm and 17.25±0.96 cm respectively), while the lowest number of sprouts and the shortest sprout length occurred for cuttings with 0.3­0.6 cm diameter and eight leaves planted in fs substrate (1.06±0.09 cm and 8.19±1.11 cm, respectively). however, thinner cuttings (0.1­0.3 cm) and leafless cuttings for all diameters failed to produce any sprouts and are therefore not shown on the graphs (fig. 2). the mean number of sprouts per rooted cutting showed a progressive increase with increasing cut­ ting diameter (r= 0.56***). a similar trend was also observed in sprouts length per rooted cutting (table 1). results showed that pm substrate as well as experimental units for thirty­six treatment combina­ tions). the experiment was established with three cutting diameters (0.1­0.3 cm; 0.3­0.6 cm and 0.6­0.9 cm), four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm). statistical analysis the main effects and interactions of cutting diam­ eter, leaf retention and substrate were determined using a general linear model (glm) procedure in sas program version 9.1 (sas institute, cary, nc) for all the evaluated parameters. the differences between the treatments were tested using duncan’s multiple range test (dmrt) with at least 95% level of statisti­ cal reliance, and as a result, homogenous groups were acquired and interpreted. collected data were subjected to simple linear regression analysis. all data were reported as means ± standard error (se). sprouting, rooting and survival percentage data were arcsine transformed to ensure normal distribution and homogeneity of variances. fig. 2 ­ effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm) on mean values of number of sprouts (ns) and length of the longest sprout (sl) per rooted cutting of argania spinosa. values followed by a common letter within each column are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). table 1 ­ simple linear regression for cutting diameter and number of sprouts or sprout length (cm) of argania spinosa cuttings ***= highly significant (p<0.001). n = 32. dependent variable (y) independent variable (x) correlation coefficient (r) regression equation p­value number of sprouts cutting diameter (cm) 0.56 y= 0.59 x ­ 0.44 0.000 *** sprout length (cm) cutting diameter (cm) 0.58 y= 5.42 x ­ 4.32 0.000 *** benbya et al. ‐ clonal propagation of argania spinosa 65 fs/pm substrate exhibited higher means number of sprouts compared to fs substrate. however, the fs/pm substrate showed the longest sprouts, fol­ lowed with pm substrate, while fs exhibited the shortest sprout per cutting (table 1). over all the combined data for leaf retention treatments, it showed that with cutting diameter increase, there was an increase in the number of sprouts and sprout length (r=0.58***) (table 1). indeed, cuttings with 0.6­0.9 cm diameter showed the greater mean num­ ber of sprouts and sprout length of cuttings, followed by cuttings with 0.3­0.6 cm. among leaf retention tested, cuttings with 4 leaves produced the longest sprouts compared with 2 and 8 leaves (table 2). based on the analysis of variance (anova), cut­ ting diameter and substrate had a significant effect (p<0.05) on the mean number of sprouts, while leaf retention was not found significant on the number of sprouts per cuttings (table 3). moreover, cutting diameter, leaf retention and substrate had a highly significant effect (p<0.001) on the sprout length, whereas leaf retention had only significant effect on sprout length (table 3). neither mean number of the sprouts per cuttings nor the sprout length was signifi­ cantly (p > 0.05) affected by the two­way and three­ way interaction between the three factors tested (table 3). effect of the cutting diameter, leaf retention and sub‐ strate on number of roots and length of the longest root of the a. spinosa cuttings the highest number of roots and root length were observed in cuttings with 4 leaves, followed by 2 leaves though not significantly, while lowest number table 2 ­ main effects of cutting diameter, leaf retention and substrate on the number of sprouts and length of the longest sprout (cm) of argania spinosa cuttings values followed by the same letter(s) are not significantly diffe­ rent at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments sprouts number sprout length cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 1.40 ± 0.05 b 11.65 ± 0.46 b 0.6­0.9 1.58 ± 0.05 a 14.46 ± 0.44 a leaf retention leafless ­ ­ 2 1.48 ± 0.06 a 13.34 ± 0.58 ab 4 1.56 ± 0.05 a 13.98 ± 0.55 a 8 1.43 ± 0.06 a 11.85 ± 0.56 b substrate fine sand (fs) 1.27 ± 0.07 b 11.30 ± 0.63 c peat moss (pm) 1.58 ± 0.06 a 13.13 ± 0.52 b mixture of fs and pm 1.62 ± 0.05 a 14.74 ± 0.51 a table 3 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on number of sprouts and length of the longest sprout cm per argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable no. of sprouts sprout length df f­value p­value df f­value p­value cutting diameter 2 7.36 0.0071 * 2 20.36 <0.0001 *** leaf retention 3 1.30 0.2736 ns 3 4.08 0.0179 * substrate 2 10.08 <0.0001 *** 2 10.15 <0.0001 *** cutting diameter × leaf retention 6 0.03 0.9702 ns 6 0.9559 0.9559 ns cutting diameter × substrate 4 0.39 0.6750 ns 4 0.01 0.9896 ns leaf retention × substrate 6 0.35 0.8451 ns 6 0.23 0.9232 ns cutting diameter × leaf retention × substrate 12 0.20 0.9399 ns 12 0.03 0.9983 ns block 1 0.10 0.7534 ns 1 2.43 0.1200 ns of roots and root length were mostly recorded in cut­ tings with 8 leaves. best response in term of number of roots was obtained when cuttings were set in fs substrate with four leaves and diameters of 0.3­0.6 cm for which the mean number of adventitious roots was 45.06±1.14 (fig. 1e), followed by cuttings with four leaves set in fs substrate with diameters of 0.6­ 0.9 cm (44.130±1.21). the greatest value of root length was recorded when cuttings were grown in fs substrate with 0.3­0.6 cm thickness and with four leaves (33.63±2.12), while cuttings set in a pm sub­ cutting, while leaf retention had only a significant effect (p<0.05). root length was significantly (p<0.05) affected by the interaction between cutting diameter and substrate, while the other interactions between the three factors studied were not significant on the number of roots and root length of cuttings (table 5). 66 adv. hort. sci., 2021 35(1): 61­72 strate didn’t exceed 23 cm (fig. 3; fig. 1e). however, the incidence of necrosis was the greatest among thinner cuttings (0.1­0.3 cm) and leafless cuttings, indeed these cuttings failed to produce any adventi­ tious roots and are therefore not on the graphs (fig. 3). the mean number of roots was dependent on type of substrate; cuttings grown in fs and fs/pm substrates rooted significantly better, whereas the lowest mean number of adventitious roots per cut­ tings was recorded in cuttings propagated in pm sub­ strate (table 4). over the entire experimental period, the three substrates showed pronounced differences in the root length: cuttings planted in fs substrate were found to produce significantly longest adventi­ tious roots, followed by fs/pm substrate then cut­ tings set in pm substrate (table 4). among the cutting diameters studied, (0.3­0.6) cm showed the highest number of roots and the longest root, followed by the cutting diameter (0.6­0.9) cm, while cuttings with (0.1­0.3) cm were the least performing and failed to produce roots (table 4). regarding the anova test values, the effects of cutting diameter, leaf retention and substrate were found highly significant (p<0.001) on the number of adventitious roots formed per cutting (table 5). moreover, cutting diameter and substrate had also a highly significant effect (p<0.001) on root length of table 4 ­ main effects of cutting diameter, leaf retention and substrate on the number of roots and the length of the longest root (cm) of argania spinosa cuttings values followed by the same letter(s) are not significantly diffe­ rent at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments roots number root length cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 38.38 ± 0.74 a 27.24 ± 0.73 a 0.6­0.9 35.61 ± 0.90 b 24.42 ±0.69 b leaf retention leafless ­ ­ 2 36.95 ± 0.94 b 25.65 ± 0.93 ab 4 38.97 ± 1.04 a 27.47 ± 0.83 a 8 35.07 ± 1.03 b 24.36 ± 0.85 b substrate fine sand (fs) 42.07 ± 0.66 a 30.76 ± 0.76 a peat moss (pm) 27.44 ± 0.82 b 18.25 ± 0.63 c mixture of fs and pm 41.48 ± 0.75 a 28.47 ± 0.65 b fig. 3 ­ effects of cutting diameters, four leaf number (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/ pm) on mean values of number of adventitious roots (nr) and length of the longest root cm (rl) per rooted cutting of argania spinosa. values followed by a common letter within each column are not significantly different at p < 0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of mean (n = 32). benbya et al. ‐ clonal propagation of argania spinosa 67 effect of the cutting diameter, leaf retention and sub‐ strate on sprouting percentage, rooting percentage and survival rate of the argania spinosa cuttings although the thinner cuttings gave no sprouts, among the other different treatments, the highest sprouting percentage was 85.00 ± 3.76% when cut­ tings with 4 leaves and 0.6­0.9 cm diameter were planted in fs/pm substrate, while cuttings with 8 table 5 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on the number of adventitious roots and the length of the longest root (cm) of argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable no. of roots root length df f­value p­value df f­value p­value cutting diameter 2 11.27 0.0009 *** 2 13.29 0.0003 *** leaf retention 3 7.43 0.0007 *** 3 5.42 0.0049 * substrate 2 134.30 <0.0001 *** 2 98.88 <0.0001 *** cutting diameter × leaf retention 6 0.18 0.8318 ns 6 0.46 0.6345 ns cutting diameter × substrate 4 5.81 0.0034 * 4 0.11 0.8990 ns leaf retention × substrate 6 0.72 0.5809 ns 6 0.30 0.8766 ns cutting diameter × leaf retention × substrate 12 0.14 0.9657 ns 12 0.58 0.6775 ns block 1 2.46 0.1178 ns 1 0.26 0.6133 ns fig. 4 ­ effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm) on mean values of sprouting percentage (sp), rooting percentage (rp), and survival rate (sr) of argania spinosa cuttings. values followed by a common letter within each column are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of mean (n = 32). leaves and 0.3­0.6 cm diameter propagated in fs sub­ strate gave the poorest sprouting percentage (61.50± 5.68%). however, leafless cuttings performed least and failed to produce any sprouts (fig. 4). the pro­ portion of cuttings forming new shoots during growth varied between the three substrates. cuttings plant­ ed in pm and fs/pm substrates were the most suc­ cessful in sprouting, while cuttings propagated in fs the data revealed that the substrate caused sig­ nificant (p < 0.05) variations in sprouting, rooting and survival percentage of the rooted cuttings, while cut­ ting diameter and leaf retention were not significant on the survival rate (table 7). however, there was no significant effect (p>0.05) of treatment interactions between the three independent factors on the three parameters studied. 4. discussion and conclusions the present investigation revealed that cutting diameter (0.1­0.3, 0.3­0.6 or 0.6­0.9 cm), leaf reten­ tion (0, 2, 4 or 8 leaves) and substrate (fs, pm or fs/pm) were found to be important factors for suc­ cessful adventitious rooting, sprouting and survival ability of argania spinosa semi­hardwood cuttings. this experiment on a. spinosa cuttings concerning cutting diameter indicated a significant effect on the sprouting, rooting and survival performances. among the different cutting diameters tested, cuttings with 0.3­0.6 cm diameter tended to root better and devel­ op more rooting capacity compared with thinner diameter (0.1­0.3 cm), which failed to produce any sprouts or roots. moreover, larger cuttings (0.6­0.9 cm) showed the best sprouting ability but did not result in any significant improvement in root growth and development. the greatest rooting potential of cuttings with 0.3­0.6 cm diameter could be due to their good storage capacity of carbohydrates and adv. hort. sci., 2021 35(1): 61­72 68 substrate showed the lowest sprouting ability (table 6). results showed that cuttings with 0.3­0.6 cm diameter with two and four leaves set in a fs sub­ strate showed maximum rooting: (65.00 ± 5.15% and 65.00 ± 5.87% respectively). compared with the cut­ tings of 0.1­0.3 cm thickness and leafless cuttings which failed to root, all cutting diameters and leaf retention treatments enhanced the rooting percent­ age. moreover, percentage of cuttings developing adventitious roots decreased when the leaf number exceeds 4 leaves (fig. 4). the maximum rooting rates were obtained for cuttings grown in fs substrate, fol­ lowed by fs/pm substrate, while the cuttings grown on pm substrate showed the lowest rooting capacity (table 6). the survival rate ranged from 57.00 ± 4.53% for plantlets with 0.6­0.9 cm diameter and 8 leaves grown in fs substrate, to 92.50 ± 1.94% for plantlets derived from cuttings with 0.3­0.6 cm diam­ eter and 4 leaves potted on substrate containing a mixture of fine sand and peat moss. however, cutting mortality was the greatest in thinner cuttings (0.1­ 0.3) cm and leafless cuttings which showed 100% mortality rate within forty­eight weeks (fig. 4). the rooted plantlets were successfully hardened and acclimatized under non­mist greenhouse conditions (table 6). these plants showed the best survival rate and performed better growth and development when they were grown in a fs/pm substrate com­ pared with cuttings raised in pm and fs substrates (fig. 1f). table 6 ­ main effects of cutting diameter, leaf retention and substrate on sprouting percentage, rooting percentage, and survival rate of argania spinosa cuttings values followed by the same letter(s) are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments sprouting (%) rooting (%) survival (%) cutting diameter (cm) 0.1­0.3 ­ ­ ­ 0.3­0.6 70.69 ± 1.58 b 55.56 ± 2.22 a 78.14 ± 1.72 a 0.6­0.9 75.42 ± 2.52 a 49.55 ± 1.75 b 75.25 ± 2.20 a leaf retention leafless ­ ­ ­ 2 72.96 ± 1.87 ab 52.54 ± 2.03 ab 75.75 ± 1.04 ab 4 77.92 ± 1.93 a 56.33 ± 1.82 a 80.04 ± 2.16 a 8 68.29 ± 1.52 b 48.79 ± 1.90 b 74.29 ± 2.03 b substrate fine sand (fs) 67.54 ± 1.94 b 59.21 ± 2.27 a 65.08 ± 2.37 c peat moss (pm) 74.08 ± 2.01 a 44.42 ± 2.19 b 76.96 ± 2.44 b mixture of fs and pm 77.54 ± 2.17 a 54.04 ± 2.41 a 88.04 ± 2.83 a benbya et al. ‐ clonal propagation of argania spinosa 69 other reserves for adventitious root formation (leakey and storeton­west, 1992; tchoundjeu and leakey, 1996). the good sprouting response may be due to the presence of adequate sugar reserves such as fructose, glucose and sucrose (gehlot et al., 2015). moreover, the level of endogenous auxins and other rooting cofactors might be lower in cuttings with a small diameter, which leads to reduced rooting per­ centage or even the absence of rooting (wilson and van staden, 1999). the rooting efficiency of these cuttings could be also due to the lower content of mineral elements, especially nitrogen which is posi­ tively correlated with rooting (budiarto et al., 2006). in addition, the effect of cutting diameter on rooting capacity could also be attributed to the origin of a cutting within shoots, and to its position of the stock­ plant (leakey, 2004). indeed, thin cuttings are pro­ duced from shoots which arise in sub­dominant posi­ tions of the mother plants which contain a low amount of auxin, grow slowly and stop growth early (howard and ridout, 1991). the lower rooting capaci­ ty of cuttings with larger diameters (0.6­0.9 cm) may probably be due to changes in the extent of lignifica­ tion and the degree of secondary thickening along their stems (girouard, 1969; hartmann et al., 1990). these results are consistent with other studies that found a positive effect of cutting diameter on sprout­ ing, rooting and survival success of rooted cuttings. palanisamy and kumar (1997) showed that the high­ er rooting efficiency of picea abies was obtained by cuttings of 0.3­0.4 cm diameter. ouyang et al. (2015) confirmed the highest rooting efficiency of picea abies obtained by cuttings of 0.3­0.4 cm diameter. moreover, foster et al. (2000) observed that the cut­ tings of pinus taeda with an average diameter of 0.2­ table 7 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on sprout­ ing percentage, rooting percentage and survival rate of argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable sprouting (%) rooting (%) survival (%) df f­value p­value df f­value p­value df f­value p­value cutting diameter 2 4.28 0.0434 * 2 6.70 0.0124 * 2 1.96 0.1668 ns leaf retention 3 5.93 0.0047 * 3 3.53 0.0363 * 3 2.80 0.0694 ns substrate 2 6.60 0.0027 * 2 13.98 <0.0001 2 41.37 <0.0001 cutting diameter × leaf retention 6 0.02 0.9849 ns 6 0.06 0.9444 ns 6 0.23 0.7918 ns cutting diameter × substrate 4 0.22 0.8072 ns 4 0.17 0.8411 ns 4 0.13 0.8775 ns leaf retention × substrate 6 0.27 0.8937 ns 6 0.12 0.9745 ns 6 0.29 0.8855 ns cutting diameter × leaf retention × substrate 12 0.15 0.9633 ns 12 0.09 0.9860 ns 12 0.14 0.9655 ns 0.3 cm tended to root better and develop more roots. concerning leaf retention, this study showed that successful vegetative propagation of argania spinosa was restricted to leafy stem cuttings. indeed, defoli­ ated cuttings failed to produce any sprouts / roots and showed a mortality rate of 100% within forty­ eight weeks after planting. this result could be explained by the direct influence of the presence of leaves on the primary shoots, because initial growth of shoots depends on assimilates supplied by leaves and also through their influence on the cutting’s water status (newton et al., 1992; van labeke et al., 2001). moreover, the leaf retention could also exert a strong influence on root initiation and development because it allows post­severance carbon assimilation (leakey and coutts 1989; hartmann et al., 1990; thomas and schiefelbein, 2004). leafy cuttings pro­ vide a continuous supply of photosynthates besides their reserves after implementing them into the sub­ strate (tchoundjeu et al., 2002; leakey, 2004). on the other hand, the inability of leafless cuttings to root has been associated with the rapid depletion of stored carbohydrates in stem tissues after excision from the stock plants (hoad and leakey, 1996; druege et al., 2000). in addition, the leaf retention may also influence the endogenous auxins of the cut­ ting. since rooting is stimulated by the level of auxins available, it is expected that adventitious rooting response of a cutting will be proportional to the num­ ber of leaves retained (tchoundjeu and leakey, 1996). in fact, our results showed that the ability of cuttings to develop adventitious roots decreased when the leaf number exceeds 4 leaves. this could be explained by the balance between the positive adv. hort. sci., 2021 35(1): 61­72 70 effects of assimilate production through allowing suf­ ficient photosynthesis and the negative effects of water loss via transpiration (leakey and coutts, 1989; mesén et al., 1997a). however, cuttings with eight leaves exhibited early root growth and delayed sprouting and subsequently a decreased rooting effi­ ciency. indeed, cuttings with a high number of leaves appear to suffer from water deficit and a consequent reduction in photosynthetic activity as reflected by higher transpiration rates and leaf shedding com­ pared to cuttings with an optimal number of leaves (leakey and coutts, 1989; newton et al., 1992; aminah et al., 1997). thus, the balance between pho­ tosynthesis, transpiration and nutrient transport is an important factor influencing rooting (leakey and coutts, 1989). the positive effect of leaf retention on cutting success has been reported across a wide range of species including triplochiton scleroxylon (leakey and coutts, 1989), cordia alliodora (mesén et al., 1997a), vitis vinifera (thomas and schiefelbein, 2004), eucalyptus hybrids (trueman and adkins, 2013), santalum austrocaledonicum (tate and page, 2018). finally, the study of the substrate effect showed that a significant difference in sprouting and rooting as well as in survival ability was found between the three substrates tested. it was observed that the number of roots, root length and rooting percentage reached significantly higher values in cuttings set in the fs substrate compared to the pm substrate and to the mixture of fine sand and peat moss. this effec­ tiveness of adventitious rooting by rooted cuttings set in fs substrate could be related to its optimal vol­ ume of gas­filled pore­space and oxygen diffusion rate which create an adequate aerated environment for increased transpiration and respiration and enhanced adventitious root formation (andersen, 1986). moreover, sand is a porous substrate that lim­ its the development of microbial pathogens and where roots can be settled well without damage (tchinda et al., 2013). though, the poor aeration and high water holding capacity of peat moss substrate is a fundamental problem for adventitious root forma­ tion, leading to enhance the rate of fungal infection and decay of cuttings before root initiation (schmitz et al., 2013). furthermore, the higher water content in the peat moss substrate induces closing of the stomata and inhibits oxygen diffusion. this anoxia caused by oxygen deficiency or by accumulation of toxic substances, including bicarbonate and carbon dioxide, is suspected of being linked to the rooting problems due to tissue death, wilting, severe defolia­ tion, and reduced water absorption and leaf water potential (mesén et al., 1997b; drew, 1983; veen, 1988). the results also showed that cuttings set in a mixture of sand and peat moss performed better than those set in sand or peat moss substrate alone, in terms of sprouting and survival rate. it has been suggested that the addition of peat moss to fine sand improved the water holding capacity and promoted mineral nutrients absorption (leakey et al., 1990). the uptake of water by cuttings is positively related to the water content of the substrate and this may enhance survival rate by reducing water deficits, leaf abscission and cutting necrosis (newton et al., 1992). a number of comparative studies between different substrates have indicated that cuttings set in a sand substrate performed better in term of rooting and survival capacity. atangana et al. (2006) reported that the higher rooting percentages were observed in sand for allanblackia floribunda. gehlot et al. (2014) also recommended that higher rooting percentages of azadirachta indica were recorded for cuttings planted in sand. moreover, adugna et al. (2015) stat­ ed that in vanilla planifolia, the cuttings set in sand performed the highest rooting percentage. in conclusion, the results of the present study clearly indicate that cuttings with thinner diameters (0.1­0.3 cm) and defoliated cuttings have failed to produce any sprouts or roots and showed a 100% mortality rate within forty­eight weeks after planting. the greatest mean number of sprouts, sprout length and sprouting capacity as well as survival success was performed by cuttings with diameter of 0.3­0.6 cm and 4 leaves set in a mixture of sand and peat moss. the best mean number of roots, root length and rooting ability was achieved by cuttings with diame­ ter of 0.3­0.6 cm and 4 leaves in a fine sandy sub­ strate. this study also reveals that argania spinosa is amenable to clonal propagation using sand­based rooting substrate which could provide a promising prospect for the conservation of this endemic species. in addition, it is suggested that this low­cost propagation techniques could greatly facilitate the domestication and the development of superior tree crops as a commercial agroforestry species with genetically homogenous plant material. the cuttings used in the experiments were taken during the spring season. it is therefore recommended that further works should be done to determine the optimal peri­ od for cutting collections for successful clonal propa­ gation of a. spinosa trees. benbya et al. ‐ clonal propagation of argania spinosa 71 acknowledgements this work is supported by public funds through the national institute for agronomic research (inra), ministry of agriculture ­ morocco. references adugna m., belew d., tilahun d., 2015 ­ influence of rooting media and number of nodes per stem cutting on nursery performance of vanilla (vanilla planifolia andr. syn. vanilla fragrans). ­ j. hort. for., 7(3): 48­56. aminah h., dick j.m., grace j., 1997 ­ rooting of shorea leprosula stem cuttings decreases with increasing leaf area. ­ forest ecol. manag., 91(2­3): 247­254. andersen a.s., 1986 ­ environmental influences on adven‐ titious rooting in cuttings of non‐woody species, pp. 223­253. ­ in: jackson m.b. 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gibberellic acid (ga) have proved to maintain optimal fruit quality and control decay during cold storage. this study evaluated the effect of calcium and gibberellic acid dipping applica­ tion on pineapple physicochemical characteristics and internal browning. the experiment implemented two factors. the first factor relates to two dipping times (five and ten minutes) and the second factor related to four treatments, ga, ca, mix ga­ca, and control (no ga or ca applied). total soluble solids (tss), total acidity (ta), tss/ta ratio, sugar, citric and ascorbic acid content, together with internal browning severity and incidence were determined. the treatment of ca, essentially using a dipping of five minutes delivered the best perfor­ mance, having the lowest severity and incidence of internal browning (4.44 and 22.22%, respectively), the highest citric acid (0.61%), ascorbic acid content (405.18 mg kg­1) and the lowest tss/ta ratio (25.53). meanwhile, the other treatments were considered less satisfactory, due to their highest internal browning severity and incidence, without a notable impact on the citric acid and ascorbic acid content, especially with a dipping time of ten minutes. in con­ clusion, dipping applications of calcium in postharvest can enhance pineapple quality and reduce internal browning. 1. introduction pineapple is an important crop worldwide, mainly exported as canned and fresh fruit (hassan et al., 2011). low acid hybrids are the pineapple cultivars more needed by the industry nowadays. these hybrids are attractive to consumers due to their more yellow shell colour, higher sugar content and uniformity (chen and paull, 2017; paull and chen, 2018; cano­reinoso et al., 2021 a). therefore, the control of the optimal (*) corresponding author: condro.wibowo@unsoed.ac.id citation: cano­reinoso d.m., renaldy tamalea r., wibowo c., 2022 ­ physicochemical characteri‐ stic and internal browning of pineapple as affec‐ ted by calcium and gibberellic acid dipping appli‐ cation. ­ adv. hort. sci., 36(3): 175­184. copyright: © 2022 cano­reinoso d.m., renaldy tamalea r., wibowo c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 january 2022 accepted for publication 6 june 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12535 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 175­184 176 physico­chemical characteristics of these hybrids has become a primordial activity for the farmers. to regulate the fruit decay, pineapple is common­ ly subjected to postharvest storage, where the dete­ rioration of its physico­chemical properties is delayed, especially during long exportation periods (hassan et al., 2011; paull and chen, 2018; cano­ reinoso et al., 2022 b). during cold storage, the fruit is affected by physiological disorders such as chilling injury and internal browning (de freitas and resender nassur, 2017; paull and chen, 2018). these conditions highly endanger the shelf­life of the fruits. because of that, several postharvest technics have been developed to extend the cold storage life of the horticultural products, mitigating the impact of any physiological disorders (de freitas and resender nassur, 2017; noichinda et al., 2017). one of these technics is the dipping of the fruit employing calcium mineral sources. calcium treat­ ments during postharvest have been proved to main­ tain optimal fruit quality and enlarger the fruit cold storage life (de freitas and resender nassur, 2017). in addition, calcium inhibits fruit softening by increas­ ing the cell wall strength, which mitigates the cell breakdown, a normal phenomenon occurring during postharvest decay (hocking et al., 2016; de freitas and resender nassur, 2017). besides, applications with this mineral have shown a positive impact on nutritional flavour, antioxidant capacity and reduc­ tion of internal browning (de freitas and resender nassur, 2017). for example, in pineapple, posthar­ vest calcium treatments have provided significant results in the internal browning reduction, the activi­ ty of oxidative enzymes like phenylalanine ammonia­ lyase (pal) and polyphenol oxidase (ppo), and an increase of the total phenols content (youryon and wongs­aree, 2015; youryon et al., 2018). another technique for controlling fruit decay dur­ ing cold storage is by dipping applications using plant regulators like gibberellic acid (ga). this plant regula­ tor has demonstrated outstanding results in reducing postharvest decay in fruits and vegetables due to its antagonist properties in ethylene sensing and pro­ duction (pusittigul et al., 2012; mohamed et al., 2016). additionally, ga used during postharvest has delivered positive results improving the physico­ chemical attributes of fruits, especially those related to antioxidant accumulation and weight loss reduc­ tion during cold storage (pusittigul et al., 2012; dong et al., 2019). for example, in pineapple, ga employed during postharvest enhanced the shelf­life of the fruit up to 24 days, with an increase in its total solu­ ble solids, total acidity, and percentage of weight loss (pusittigul et al., 2012; mandal et al., 2015). however, most of the calcium used on pineapple after harvest has been documented as calcium infil­ trations or mixed with other techniques such as hot water treatments. at the same time, in the case of ga, more studies are needed to develop a correct characterization of its impact on pineapple quality. because of that, few papers have been reported on postharvest calcium and ga employing dipping tech­ nics; moreover, they have studied their effect on low­ acidic hybrids and possible synergy between them. therefore, this study aims to evaluate the effect of calcium and ga dipping application on pineapple physicochemical characteristics and internal brown­ ing. 2. material and methods experiment design and treatments the research was implemented in a pineapple packing house located in lampung, sumatra island of indonesia, between january and february of 2020. in this experiment, the md2 pineapple cultivar was used. this low acid hybrid is well known in the indus­ try because of its outstanding qualities related to higher sugar and antioxidant content, more uniformi­ ty and yellowish shell colour than other acid hybrids (bin thalip et al., 2015; cano­reinoso et al., 2021 b). the study was set using an experiment design with two factors. the first factor related to two dipping times (five and ten minutes), and the second factor concerning four treatments consisted of three solu­ tions of, ga, ca, mix ga­ca, and c (control ­ no use of ga and ca). three replications per treatment were used in this experiment. furthermore, randomly fruit samples were picked from every treatment to be examined employing intervals of eight days. all treatments, including the control used fungi­ cide and wax before cold storage. the fungicide product implemented was prochloraz in doses of 2 cc l­1, while the waxing product applied was sta­fresh 2952 in doses of 74 g l­1. both fungicide and waxing, in that order, were used in dipping applications for ten seconds, just after the dipping of the fruits in ga or ca. the calcium source used was calcibor (alba milagro internation, lombardia, italy) ­ (12.9% w/v cao and 2.6% w/v b) in doses of 4 l 2000 l­1; mean­ while, the ga product employed was progibb (40% cano‐reinoso et al. ‐ calcium and gibberellic acid on pineapple 177 of ga3 active component, valent usa corp., walnut creek, california ­ usa) in doses of 100 mg l­1. the calcibor doses implemented were prepared accord­ ing to the producer recommendation; meanwhile, the ga doses were arranged based on the previous experiments of mandal et al. (2015) and dong et al. (2019). concerning the mix ga­ca treatment, it was employed a sequenced application (one solution of ga or ca at time). besides, the dipping times imple­ mented in this experiment (five and ten minutes) were based on the previous studies of pusittigul et al. (2012) and islam et al. (2013). the fruits were selected according to their weight and shell colour characteristics and arranged by their respective treatments inside cold storage for 40 days (temperature: 7°c, relative humidity: 95%). the pref­ erence for fruit weight for the research was between 1.4 and 1.5 kg, with a shell colour where 10­20% of the area from the base already turned yellow. the md2 pineapple is typically harvested and exported with the previously described weight and shell colour characteristics (bin thalip et al., 2015; paull and chen, 2018). determination of the total soluble solids (tss), total acidity (ta) content according to the procedure described in shamsudin et al. (2020), the tss and ta were mea­ sured in each fruit per replication of every treatment arranged. tss was calculated employing a hand­held refractometer (master­53 α, atago, japan), while the ta was measured by titration to ph 8.1 with 0.1 m naoh using phenolphthalein as an indicator and expressed as a percentage of citric acid. sugar, citric acid and ascorbic acid (asa) content pineapple sugar and citric acid were measured using the method described in siti roha et al. (2013), employing a high­performance liquid chromatography (hplc) ­ (hitachi, usa) model l­ 2000 instrument with a refractive index detector model l­2490. a juice extracted from the fruit flesh adjacent to the core was employed for this proce­ dure. samples were obtained from each fruit per replication of every treatment implemented. for the sugar content, standard solutions of 500, 1000, 1500, 2000 mg l­1 of glucose, fructose and sucrose were prepared with the aim of developing a curve of sugar level; furthermore, in the case of the citric acid and asa determination, the standard solutions were 1000 mg l­1 and, 200, 400, and 800 mg l­1, respectively, hav­ ing the same objective in mind. the standard solu­ tions were dissolved in distilled water and filtered through a millipore 0.45 µm membrane filter. finally, the sugar, citric acid and asa content was quantified, comparing the peak area by a chromatographic pro­ cedure. the chromatographic condition for the sugar determination was: column: purospher® star nh2 (250 x 4 (mm), 5 µm). guard column: lichocart® 4­4 / lichrospher® 100 nh2, 5 µm. column temperature: room temperature (22°c). mobile phase: acetonitrile: distilled water (80:20). flow rate: 1 ml min­1. injection volume: 20 μl. duration of analysis: 15 min. meanwhile, for the citric acid and asa was: column: purospher® star nh2 (250 x 4 (mm), 5 µm). guard column: lichocart® 4­4 / lichrospher® 100 nh2, 5 µm. column temperature: room temperature (22°c). mobile phase: pipette of 0.14 ml h2so4 (0.0025 m) concentrated at 97% is introduced in a volumetric flask, then add 1000 ml of distilled water. injection volume: 20 μl. duration of analysis: 15 min. browning severity and incidence the internal browning severity was calculated employing the following score classification and transformed into a percentage: 1 (no flesh browning, 0%), 2 (20% of browning in the flesh), 3 (40% of browning in the flesh), 4 (60% of browning in the flesh), 5 (80% of browning in the flesh) and 6 (100% of browning in the flesh). figure 1 shows a schematic example of the previous score classification described. furthermore, the incidence was measured by accounting the percentage of fruits affected by internal browning in each treatment during every observation. statistical analysis statistical analyses were performed using spss version 22.0 software (spss inc.; chicago, il: usa). fig. 1 ­ scheme of the score classification used to determine the internal browning severity in the experiment during each observation. score: 1 (0%), 2 (20%), 3 (40%), 4 (60%), 5 (80%), and 6 (100%). adv. hort. sci., 2022 36(3): 175­184 178 all data were analyzed by two­ways anova. mean significant differences at p<0.05 were determined by duncan’s multiple range tests and kruskal­wallis test (in case of the internal browning data). 3. results tss, ta, and tss/ta ratio there was no significant impact evidenced coming from the dipping time, the treatments implemented and the interaction between these two factors for the tss, ta, and tss/ta ratio. concerning the tss, the mean results exposed that the control had the highest value (16.93%), meanwhile the treatment of ga­ca with a dipping of ten minutes provided the lowest outcome (14.47%). regarding the ta, the mean results show that the treatment of ca with a dipping of five minutes delivered the highest out­ come, while the ga­ca treatment with five minutes dipping had the lowest one (0.61 and 0.53%, respec­ tively). on the other hand, the mean outcomes for the tss/ta ratio delivered the highest value in the treatment of ga with five minutes dipping (38.17%), and a most inferior result in the treatment of ca also with a dipping of five minutes (26.16%). the tss, ta and tss/ta ratio mean values corroborated the pre­ vious results described concerning the single factors influence and their interaction; there was no a clear positive or negative trend in the mean values when the dipping times were increased or reduced and combined with the treatments administrated (table 1). sugar content in terms of pineapple sugar after cold storage of 40 days, the fructose, glucose and sucrose content did not demonstrate significant differences coming from the dipping time, the treatments administrated, and their interaction. concerning the mean values of these two factors, just the sucrose exposed signifi­ cant differences. in this case, the treatment of ga with ten minutes dipping had the highest result, and the same treatment but with dipping of five minutes, the most inferior outcome (8.25 and 5.97%, respec­ tively). despite this particularity, for the mean results of the fructose, glucose and sucrose content, it was difficult to determine a positive or negative tendency coming from the rise or reduction of the dipping times and their combination with the treatments used, reaffirming the individual factors impact and their interaction outcomes (table 1). citric acid and asa content the citric acid and asa did not present a represen­ tative impact from the dipping time and the treat­ ments studied; however, the interaction between these two factors was significant, impacting the mean outcomes among these variables after 40 days of cold storage. for the citric acid, the lowest out­ comes were obtained in the treatments of ga and ca table 1 ­ results of the single and interaction effect from treatments and dipping times with their respective combined mean values on the tss, ta, and sugar content, after 40 days of cold storage * results marked with (+) indicate that the p­value (p < 0.05) for the single or interaction can be considered to draw conclusions; mea­ nwhile, results marked with (­) indicate that the p­value (p > 0.05) for the single or interaction are not significant to draw conclusion in the text. mean values ± se in each column followed by the same lower­case letters are not statistically different by duncan’s multiple range test (p < 0.05). treatments: ga, ca, mix ga­ca, control ­ no use of ga and ca. dipping time: 5 and 10 minutes. tss (%) ta (%) tss/ta glucose (%) fructose (%) sucrose (%) studied factors (single and interaction effect) treatment ­ ­ ­ ­ ­ ­ dipping time ­ ­ ­ ­ ­ ­ treatment x dipping time ­ ­ ­ ­ ­ ­ mean values (treatment vs. dipping time) ga 5 min 16.13 ± 0.48 ab 0.39 ± 0.06 b 43.32 ± 6.71 a 3.35 ± 0.22 a 3.41 ± 0.21 a 5.97 ± 0.56 b ca 5 min 14.80 ± 0.00 bc 0.62 ± 0.13 a 25.53 ± 4.48 b 2.48 ± 0.63 a 2.77 ± 0.56 a 7.72 ± 1.03 ab ga­ca 5 min 15.20 ± 0.70 bc 0.56 ± 0.04 ab 27.82 ± 4.02 ab 3.25 ± 1.02 a 3.49 ± 0.99 a 6.77 ± 1.50 b ga 10 min 15.27 ± 0.27 bc 0.50 ± 0.10 ab 33.01 ± 5.88 ab 2.71 ± 0.18 a 2.99 ± 0.19 a 8.25 ± 0.45 a ca 10 min 14.67 ± 0.18 bc 0.59 ± 0.11 ab 26.80 ± 4.96 ab 3.01 ± 0.12 a 3.28 ± 0.11 a 7.11 ± 0.62 ab ga­ca 10 min 14.47 ± 0.29 c 0.50 ± 0.06 ab 29.91 ± 4.78 ab 2.06 ± 0.86 a 3.23 ± 0.43 a 6.54 ± 0.75 b control 16.93 ± 0.84 a 0.58 ± 0.04 ab 29.46 ± 2.57 ab 2.97 ± 0.22 a 3.27 ± 0.28 a 7.77 ± 0.09 ab cano‐reinoso et al. ‐ calcium and gibberellic acid on pineapple 179 (ga: 34% ­ five minutes; ca: 37% ­ ten minutes). moreover, the treatment of ca but using a dipping of five minutes had the highest outcome (0.61%). no notable differences were observed between the mean results of the treatment ga­ca in both dipping times; however, those outcomes were higher than the control (table 2). concerning the asa, similar with the previous variable, when the ca treatment was implemented with a dipping of five minutes the highest result was obtained (405.18 mg kg­1). besides, the most reduced outcome was determined in the same treatment but with a dipping of ten minutes (111.38 mg kg­1). on top of that, a high remarkable mean result was observed in the treatment of ga with ten minutes dipping (208 mg kg­1). in this case, not all the mean outcomes of the treatment ga­ca in both dipping times provided a positive increase, as the control obtained a superior value, essentially when five min­ utes dipping was used (table 2). compressing the previous information, it was noticed that for the citric acid and asa, ca treatments caused a rise in their val­ ues employing a short dipping of five minutes, con­ trary effect detected on the ga treatments, which demonstrated positive increases with a long dipping of ten minutes. this situation was not clearly observed in the ga­ca treatments. internal browning the monitoring of internal browning after 40 days of cold storage provided significant differences com­ ing from the treatments implemented but not from the dipping times, and the interaction among these factors. in the mean outcomes it was possible to observe that samples employing the treatments of ca obtained the lowest severity and incidence of inter­ nal browning, more remarkable when a dipping of five minutes was implemented (severity: 4.44%, inci­ dence: 22.22%), compared with the other treatments and the control. on the contrary, samples using the treatments of ga and ga­ca had a more superior internal browning severity and incidence, more evi­ denced with dipping times of ten minutes, suggesting a negative effect of the ga, concerning this variable (severity: 42%, incidence: 100%, for the ga treat­ ment with ten minutes dipping) (table 2). 4. discussion and conclusions tss, ta, and tss/ta ratio in the fruit tss in pineapple low acid hybrids should be mini­ mal as 12%, although some authors have recom­ mended higher values close to 14% (paull and chen, 2015, 2018; cano­reinoso et al., 2021 b). these val­ table 2 ­ results of the single and interaction effect from treatments and dipping times with their respective combined mean values on the citric acid, ascorbic acid (asa), and internal browning, after 40 days of cold storage * results marked with (+) indicate that the p­value (p < 0.05) for the single or interaction can be considered to draw conclusions; mea­ nwhile, results marked with (­) indicate that the p­value (p>0.05) for the single or interaction are not significant to draw conclusion in the text. mean values ± se in each column followed by the same lower­case letters are not statistically different by duncan’s multiple range test, and kruskal­wallis test (for the internal browning data) (p<0.05). treatments: ga, ca, mix ga­ca, control ­ no use of ga and ca. dipping time: 5 and 10 minutes. citric acid (%) asa (mg kg­1) browning severity (%) browning incidence (%) studied factors (single and interaction effect) treatment ­ ­ + + dipping time ­ ­ ­ ­ treatment x dipping time + + ­ ­ mean values (treatments vs. dipping time) ga 5 min 0.34 ± 0.03 b 170.79 ± 48.88 b 37.78 ab 88.89 ab ca 5 min 0.61 ± 0.10 a 405.18 ± 64.31 a 4.44 c 22.22 c ga­ca 5 min 0.53 ± 0.14 ab 148.11 ± 63.87 b 37.78 bc 88.89 ab ga 10 min 0.52 ± 0.04 ab 208.00 ± 22.56 b 42.22 ab 100 a ca 10 min 0.37 ± 0.03 ab 111.38 ± 36.12 b 11.11 bc 66.67 bc ga­ca 10 min 0.55 ± 0.05 ab 186.03 ± 66.56 b 28.89 bc 100 a control 0.44 ± 0.04 ab 157.46 ± 32.30 b 42.22 ab 77.78 ab 180 adv. hort. sci., 2022 36(3): 175­184 ues were assessed in the results obtained after 40 days of cold storage, knowing that it is common the reduction of tss content through the postharvest life of pineapples (lu et al., 2011; hu et al., 2012). therefore, as all treatments provided an optimal per­ formance, it is possible to infer that no harmful impact on this variable was received, even with the application of different dipping times. in addition, the lowest result observed in the treatment of ca­ga with ten minutes dipping can be attribute to the time implemented. typically, ga influences cell enlarge­ ment, this process generates more soluble solids as sugars, which can be assimilated in the cell (wang and irving, 2011; gupta and chakrabarty, 2013). furthermore, this situation can cause more ca2+ ions to crosstalk, creating cell wall channels, with the objective of maintaining optimal membrane stabiliza­ tion (gupta and chakrabarty, 2013, hocking et al., 2016; cano­reinoso et al., 2022 a). nevertheless, during this process several reactive oxygen species (ros) are released, and those can interfere and destroy organic molecules presented as soluble solids (wang and irving, 2011; gupta and chakrabarty, 2013). because of that, a longer dipping time could be a trigger for more ros production, decreasing the tss available in the cell structures, and as a conse­ quence the more reduced level observed in this treatment. on the other hand, values of ta between 0.4 and 06% are recommended for optimal quality in md2 pineapple; nevertheless, higher percentages have been reported in former studies (chen and paull, 2017; paull and chen, 2018; cano­reinoso et al., 2021 b). the outcomes of this research delivered val­ ues among this ideal range for almost all the treat­ ments applied in both dipping times. previous experi­ ments using calcium as a postharvest treatment in pineapple had not presented any remarkable impact on ta (pusittigul et al., 2014; youryon et al., 2018). nonetheless, in some fruits like apricots (hajilou and fakhimrezaei, 2013) and apples (shirzadeh et al., 2011), postharvest calcium applications increased the ta content. calcium may positively affect the accu­ mulation of organic acids through its impact on fruit metabolism, which are highly associated with the ta level. calcium has been proved to delay fruit senes­ cence by regulating the opening of stomata, creating a reduction of the degrading of organic acids, main source of fruit respiration (van meeteren and aliniaeifard, 2016; de freitas and resender nassur, 2017). this fact can explain why the treatment of ca with five minutes dipping had the highest results. five minutes could be the ideal time to produce the most positive impact on the fruit metabolism. however, this dipping time could cause a negative influence when the treatment of ga was administrat­ ed. ga has been detected to reduce the ta degrading in postharvest of pineapple (mandal et al., 2015; dong et al., 2019). nevertheless, as mentioned before, ga tends to ros production, this dipping time could cause that the ros level overcame and interfere the organic acid production to maintain a stable fruit metabolism, causing the lowest content with this treatment. furthermore, the tss/ta ratio for md2 pineapple should range between 18 and 25, although current authors have suggested values close to 30 (chen and paull, 2017; paull and chen, 2018; cano­reinoso et al., 2021 b). numbers more superior than 30 are con­ sidered not suitable for an ideal consumption (ding and syazwani, 2016; paull and chen, 2018). the most elevated and inferior tss/ta ratio values observed in the treatments of ga and ca were more associated with the impact of their ta content, as this displayed more remarkable differences among both treatments with five minutes dipping than the tss. pineapple sugar content fructose, glucose, and sucrose are the major sug­ ars present in pineapple. sucrose is the most accu­ mulated during fruit ripening, ranging around 7­9%, and essentially responsible for the sweet taste in low acid hybrids like md2 (nadzirah et al., 2013; lu et al., 2014; paull and chen, 2018). pre­and postharvest applications with ga have shown an increase in the sugar content of pineapple (mandal et al., 2015); also in fruits like mango (islam et al., 2013), and peaches (çetinbaş and koyuncu, 2013). few studies have reported a dipping time ideal for ga (pusittigul et al., 2012; islam et al., 2013). five minutes have been sug­ gested as optimal to generate a positive effect on sugar content in pineapple; nevertheless, results of this experiment suggested another outcome, as this dipping time delivered the lowest result. in pineap­ ple, sucrose phosphate synthase (sps) and sucrose synthase (ss) are the main enzymes responsible for the sucrose accumulation, while the invertase enzyme (ie) mainly responsible for its hydrolyzation (chen and paull, 2017; paull and chen, 2018). the five minutes dipping with the treatment of ga may affect theses enzymes through the cold storage, essentially promoting a superior activity of ie, causing cano‐reinoso et al. ‐ calcium and gibberellic acid on pineapple 181 more sucrose hydrolyzation and conversion into fruc­ tose and glucose, including other organic sugars. contrary process evidenced with this treatment but employing ten minutes dipping, obtaining the highest value. this dipping time could generate a more pro­ nounced activity of sps and ss, having a more elevat­ ed sucrose content and reduced fructose and glucose mean outcomes. more emphasis in these enzyme activities under ga applications for future experi­ ments is recommended. pineapple citric acid and asa in hybrids like md2 the citric acid value should be between 0.4 and 07% for ideal consumption (lu et al., 2014; paull and chen, 2018; cano­reinoso et al., 2022 b). values within that range were obtained in most of the treatments implemented. citric acid is the predominant organic acid in pineapple, impacting its taste (chen and paull, 2017; paull and chen, 2018). this acid is considered a plant antioxidant, which has been detected to improve the tolerance to disease attacks and delaying fruit senescence in pre­ and postharvest studies (patrignani et al., 2015; yang et al., 2019). the citric acid of this experiment are highly related to the ta results described previously. this acid accounts for most of the ta content in pineapple (chen and paull, 2017; paull and chen, 2018). as mentioned before, calcium can impact organic acids accumulation positively, like the citric acid, by regulating the opening of stomata, causing a reduction of its metabolizing process during fruit res­ piration, essentially with short dipping times. this information can clarify why the treatment of ca with five minutes dipping obtained the highest result. nevertheless, as mentioned in the ta outcomes, this dipping time could not be sufficient to provide an adequate production of ros that does not alter the citric acid metabolization, primordially when ga is used, reason why this treatment had the lowest out­ come. furthermore, this lowest value could be attribute also to its impact on the organic acid metabolizing enzymes. a high activity of the aconi­ tase enzyme (aco) has been associated with the cit­ ric acid reduction in pineapple low acid hybrids (saradhuldhat and paull, 2007). therefore, it could be possible to infer that a short dipping time together with the treatment of ga can produce a superior activity of aco during cold storage. on the contrary, an opposite situation can occur when same dipping time was used but with the treatment of ca. the more reduced picks of citric acid content observed in the figure 2 could be linked to the high aco activity during postharvest. however, this phenomenon should be examined in detail for future studies. on the other hand, asa is considered a soluble vit­ amin and the most representative antioxidant com­ pound in pineapple (kongsuwan et al., 2009; akram et al., 2017; noichinda et al., 2017). in md2, a value higher than 300 mg kg­1 has been established as ade­ quate to have a longer shelf­life, especially during cold storage (lu et al., 2014; paull and chen, 2018; cano­reinoso et al., 2022 b). asa increase in fruits has been associated with a higher activity of the enzyme ascorbic peroxidase (apx) (akram et al., 2017). furthermore, more superior ca2+ ion assimila­ tion in fruits’ primary cell wall matrix has been linked also to a higher production of asa (sadak et al., 2010; farouk, 2011). these facts can explain why treatment of ca obtained the highest asa content, due to the synergy between ca2+ ion influences on the cell wall and the asa generation. besides, the five minutes dipping could be the ideal time to have an adequate ros level that does not interfere with the asa metabolization, similar with the situation described for the ta. however, an opposite phenomenon could have occurred in the same treatment but with ten minutes dipping; the ros production could have overcome and reached inadequate levels, causing a fig. 2 ­ effects of the treatments applied using both dipping times (5 and 10 min) on the citric acid content during cold storage. treatments: ga, ca, mix ga­ca, c (control­ no use of ga and ca). values are the mean three replica­ tes, and vertical bars represent ± se. adv. hort. sci., 2022 36(3): 175­184 182 disturbance in the asa production and ca2+ ion assim­ ilation, reason why this treatment obtained the low­ est outcome. figure 3 shows how at 24 days of cold storage there is a break point in the trend of both dipping times. at this moment, the decay symptoms and senescence process of the fruit could have been more intense, and this situation could have provoked a hypertensive response (hr). hr are plant physiolo­ gy mechanisms activated under stress conditions characterized by antioxidants production to cope with stressfully circumstance (goñi et al., 2017). moreover, a more pronounce activity of apx could have been encouraged during that period. in the case of the ga, despite its positive effects reported on asa and ca assimilation (mandal et al., 2015; dong et al., 2019), the dipping times employed together with its mix with ca could have mitigate these characteristics, and as a consequence, no any superior asa level observed with ga treatments. internal browning internal browning in pineapple has been linked to an increase in the activity of the phenylalanine ammonia­lyase (pal) and polyphenol oxidase (ppo) enzymes during cold storage (youryon et al., 2018; paull and chen, 2019). postharvest calcium treat­ ments have shown a reduction of pal and ppo asso­ ciated with a more elevated activity of their antago­ nisms enzyme, catalase (cat), peroxidase (pod), and superoxidase dismutase (sod); besides, the already mentioned superior antioxidant capacity (pusittigul et al., 2012; youryon et al., 2018; cano­reinoso et al., 2022 b). these antagonism enzymes can interfere in the production of ros like hydrogen peroxide h2o2, and the superoxide radical o2 ­. this ros inhibition creates a reduction of the membrane lipid peroxida­ tion, giving more consistency to the cell wall matrix, which cause a longer shelf­life. furthermore, youryon et al. (2018) reported that ca2+ ions could bind to the negative charge molecules associated with pal and ppo, suppressing their metabolic disturbances. this previous information can help clarify why the treat­ ments of ca caused a low severity and incidence of internal browning, and why this circumstance was also linked to the highest asa and citric acid results, especially with five minutes dipping. this dipping time can be ideal to maintain a low ros, pal, and ppo activity during postharvest. on top of that, results show that there may be a negative impact in all treatments employing ga, due to their high severity and incidence of internal browning, primordially with ten minutes dipping. pusittigul et al. (2012) demonstrated that an elevated endogenous concentration of ga in pineapple fruit can enhance the activity of pal and ppo. therefore, it is important to control the gibberellins level in the fruit, especially under the use of exogenous ga appli­ cations. this information suggests that the treat­ ments with ga, essentially with long dipping times, like ten minutes, could not only facilitate these enzyme activities, also encourage superior ros con­ centration. however, more studies are recommend­ ed for the future, especially to clarify ga interaction with ca impacting the occurrence of internal brown­ ing. in conclusion, calcium and ga dipping application affected the pineapple physicochemical characteris­ tics and internal browning. the treatment of ca with dipping time of five minutes delivered the best results. this treatment caused the lowest severity and incidence of internal browning, together with the highest asa, citric acid content, and lowest tss/ta ratio, after 40 days of cold storage. also, this treatment had the closest values to an ideal fruit quality. on the other hand, the control, the treat­ ments using ga, and its mix with calcium, in both dip­ ping times, did not provide satisfactory results, pri­ mordially because those delivered a high severity and fig. 3 ­ effects of the treatments applied using both dipping times (5 and 10 min), on the asa content during cold sto­ rage. treatments: ga, ca, mix ga­ca, c (control­ no use of ga and ca). values are the mean three replicates, and vertical bars represent ± se. cano‐reinoso et al. ‐ calcium and gibberellic acid on pineapple 183 incidence of internal browning and did not cause a remarkable enhancement in the fruit antioxidant content. finally, the results described here can be consider preliminary, since the majority of the cur­ rent investigations concerning the employment of ca and ga as postharvest treatments on pineapple have not yet clarified what the optimal doses and dipping time to be implemented are; therefore, further stud­ ies are recommended. acknowledgements the authors would like to express their sincere thanks to pt great giant pineapple and their research department in lampung indonesia for the technical support during the research period. references akram n.a., shafiq f., ashraf m., 2017 ­ ascorbic acid‐a potential oxidant scavenger and its role in plant devel‐ opment and abiotic stress tolerance. ­ front. plant sci., 8: 1­5. bin thalip a.a., tong p.s., casey n.g., 2015 ­ the md2 “super sweet” pineapple (ananas comosus). ­ utar agric. sci. j., 1: 14­17. cano­reinoso d.m., kharisun k., soesanto l., wibowo c., 2022 a ­ effect of calcium and silicon fertil‐ ization after flowering on pineapple mineral status and flesh translucency. ­ plant physiol. rep., 27: 96­108. cano­reinoso d.m., soesanto l., kharisun wibowo c., 2021 a ­ review: fruit collapse and heart rot disease in pineapple: pathogen characterization, ultrastructure infections of plant and cell mechanism resistance. ­ biodiversitas, 22: 2477­2488. cano­reinoso d.m., soesanto l., kharisun wibowo c., 2021 b ­ effect of pre‐harvest fruit covers and calci‐ um fertilization on pineapple thermotolerance and flesh translucency. ­ emir. j. food. agric., 33: 834­845. cano­reinoso d.m., soesanto l., kharisun wibowo c., 2022 b ­ effect of pre‐and postharvest treatments with salicylic acid on physicochemical properties of pineapple cv. md2. ­ cmuj nat. sci., 21: e2022039. çeti̇nbaş m., koyuncu f., 2013 ­ the ripening and fruit quality of ‘monroe’ peaches in response to pre‐harvest application gibberellic acid. ­ akdeniz üniversitesi ziraat fakültesi derg., 26: 73­80. chen n.j., paull r.e., 2017 ­ production and postharvest handling of low acid hybrid pineapple. ­ acta horticulturae, 1166: 25­34. de freitas s.t., resender nassur r.c.m., 2017 ­ calcium treatments, pp. 52­68.­ in: pareek s. 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(ed.) postharvest ripening physiology of crops. crc press, boca raton, usa, pp. 620. wang y.h., irving h.r., 2011 ­ developing a model of plant hormone interactions. ­ plant signal. behav., 6: 494­500. yang c., chen t., shen b., sun s., song h., chen d., xi w., 2019 ­ citric acid treatment reduces decay and maintains the postharvest quality of peach (prunus per­ sica l.) fruit. ­ food sci. nutr., 7: 3635­3643. youryon p., supapvanich s., kongtrakool p., wongs­aree c., 2018 ­ calcium chloride and calcium gluconate peduncle infiltrations alleviate the internal browning of queen pineapple in refrigerated storage. ­ hortic. environ. biotechnol., 59: 205­213. youryon p., wongs­aree c., 2015 ­ postharvest applica‐ tion of calcium chloride affects internal browning reduction during low temperature storage of “sawi” pineapple. ­ acta horticulturae, 1088: 197­200. impaginato 211 adv. hort. sci., 2024 38(2): 211­222 doi: 10.36253/ahsc­15253 plant biostimulants in ornamentals: enhancing growth and stress tolerance m. tütüncü 1, a.d. şekerci 2, d. dönmez 3, t. i̇zgü 4, m.a. isak 5, ö. şimşek 2 (*) 1 ondokuz mayıs university, agriculture faculty, horticulture department, samsun, türkiye. 2 erciyes university, agriculture faculty, horticulture department, kayseri, türkiye. 3 çukurova university, biotechnology research and application center, adana, türkiye. 4 national research council of italy (cnr), ibe/institute of bioeconomy, florence, italy. 5 erciyes university, graduate school of natural and applied sciences, agricultural sciences and technologies department, kayseri, türkiye. key words: abiotic stresses, bioactivators, growth stimulation, ornamental plants, plant biostimulants, stress tolerance. abstract: researchers have recently sought a comprehensive strategy to reduce the harmful effects of synthetic chemicals in agricultural production to improve productivity and quality. biostimulants benefit plants by protecting soil and water resources and eliminating adverse environmental effects from pesticides and chemical fertilizers. plant biostimulants, also called bioactivators, are becoming increasingly well­liked in the agricultural sector due to their capacity to boost a plant’s growth rate and increase its resistance to stress. biostimulants are frequently used because they can increase crop quality, nutrient assimilation, growth rate, and stress tolerance. this article thoroughly examines biostimulants’ effects on ornamental plants, concentrating on their ability to withstand environmental stressors. prior studies have demonstrated that a combination of non­pathogenic microbes, protein hydrolysates, humic and fulvic acids, and algal extracts benefits ornamental plants. this review’s main aims are biostimulants’ effects on raising agricultural yield, enhancing nutrient uptake, enhancing photosynthesis, and shielding plants from biotic and abiotic stress. the role of biostimulants in more resilient and sustainable farming practices is also covered. 1. introduction the ornamental industry contributes significantly to both the global economy and agribusiness. it covers various activities, such as cultivating, distributing, and consuming flowers and attractive plants. the ornamental sector’s economic worth, environmental benefits, and cultural relevance (*) corresponding author: ozhan12@gmail.com citation: tütüncü m., şekerci a.d., dönmez d., i̇zgü t., isak m.a., şimşek ö., 2024 ­ iplant biostimulants in ornamentals: enhancing growth and stress tolerance. ­ adv. hort. sci., 38(2): 211­222. copyright: © 2024 tütüncü m., şekerci a.d., dönmez d., i̇zgü t., isak m.a., şimşek ö. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 october 2023 accepted for publication 26 january 2024 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-15253 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 211­222 212 demonstrate its significance. worldwide, ornamental plant and flower production and trade produce significant money (reis et al., 2020; krigas et al., 2021). employment opportunities are available in the sector, especially for small and family­run firms (reis et al., 2020). additionally, the ornamental sector improves the aesthetic appeal of parks, gardens, and public places, contributing to the beauty of urban and natural landscapes (rocha et al. , 2022). additionally, ornamental plants have crucial environmental responsibilities as biological filters, wildlife habitats, and environmental purification agents (rocha et al., 2022). ongoing innovation and developments in technology characterize the decorative industry. digital tools and sensors are used in precision agriculture to monitor plant health, maximize resource utilization, and increase output efficiency. with the help of these technologies, producers can make better decisions, lessen their influence on the environment, and improve the quality of their decorative plants (traversari et al., 2021). plant biostimulants have drawn much interest in agriculture as a sustainable way to promote plant growth and raise crop output. biostimulants containing organic materials and microbes have been discovered to improve stress tolerance, boost nutrient intake, and encourage growth crops (parađiković et al., 2019). biostimulants present intriguing prospects to strengthen growth, quality, and stress tolerance in the context of ornamental plants, consequently enhancing the overall productivity of ornamental crops. biostimulants can come from various sources, including non­pathogenic microorganisms, humic and fulvic acids, protein hydrolysates and algal extracts. these biostimulants have been shown to benefit plant growth in several ways, including higher yield, improved nutrient uptake and utilization, increased photosynthetic activity, and resistance to biotic and abiotic stresses (calvo et al., 2014). for biostimulants to be used effectively in horticulture, it is essential to understand the mechanisms underlying their impacts on ornamental plants. to thoroughly review the most recent studies on using biostimulants in cultivating ornamental plants, this study will examine their most recent findings. it will discuss the various biostimulants and how they affect ornamental crops' development and stress resilience. the evaluation will also examine how biostimulants might support resilient and sustainable horticulture. 2. types of plant biostimulants microbial inoculants, such as arbuscular mycorrhizal fungi (amf) and plant growth­promoting rhizobacteria (pgpr), are advantageous microorganisms that can boost nutrient uptake and increase stress tolerance in plants (calvo et al., 2014; gonzález­gonzález et al., 2020). these biostimulants form symbiotic interactions with plants, which have several benefits, including increasing soil structure, nutrient availability, and plant defence systems. organic molecules known as humic acids are produced when organic matter breaks down and are prized for their capacity to improve soil fertility, nutrient availability, and overall plant growth (calvo et al., 2014). they aid in forming roots, make nutrient absorption easier, and increase plant metabolism (yakhin et al., 2017). fulvic acids, which are related to humic acids but have smaller molecules and better solubility, work in a manner comparable to that of humic acids. they help increase nutrient availability, root growth, and plant growth (calvo et al., 2014). protein hydrolysates contain amino acids and peptides that help plants develop, absorb nutrients, and withstand stress. amino acids are also created when proteins are broken down enzymatically (calvo et al. , 2014; ivankov et al. , 2021). these biostimulants increase plant vitality, encourage photosynthetic activity, and aid in root development. in agriculture, seaweed extracts, especially those from species l ike ascophyllum nodosum, are frequently utilized as biostimulants (calvo et al., 2014; de saeger et al., 2019). just a few of the bioactive components discovered in seaweed extracts that aid plants in growing, better­absorbing nutrients, and withstanding stress include hormones, polysaccharides, betaines, and phenolic compounds (de saeger et al., 2019; boukhari et al., 2020). 3. molecular and physiological mechanism of plant biostimulants numerous studies have focused on the molecular and physiological processes of plant biostimulants. several processes and routes are involved in how biostimulants positively affect plant growth, stress tolerance, and overall performance. one typical mechanism is the stimulation of crucial enzymes in nitrogen metabolism and the induction of hormone­ like activity, such as auxin and gibberellin. tütüncü et al. ‐ biostimulants: growth and stress tolerance 213 biostimulants can modify a plant’s root system and enhance its nutritional status. increasing root biomass, density, and lateral root branching improves nutrient intake, assimilation, and translocation (caruso et al., 2019). it has been found that biostimulants can modulate phytohormones such as auxins, cytokinins, and gibberellins to affect plant growth and development. additionally, they can boost photosynthesis, reduce aging, boost nutrition and water intake, and activate genes that defend against abiotic threats (yakhin et al., 2017). the metabolic changes caused by biostimulants have been better understood because of metabolomic studies. for instance, it has been shown that utilizing microbial biostimulants impacts metabolism, increasing the levels of tricarboxylic acid (tca) intermediates, altering the amounts of amino acids, and changing the composition of phenolics and lipids. with these metabolic changes, plants are drought­ resistant, have better defensive preconditioning, and promote growth. additionally, biostimulants can modify redox homeostasis, strengthen plant cell walls, regulate osmoregulation, boost energy production, and remodel membranes, making plants more resilient and stress­tolerant (nephali et al., 2021). plant biostimulants’ molecular and physiological mechanisms involve several interactions and processes that enhance plant growth, stress tolerance, and overall performance. these techniques also control enzyme activity, metabolic adjustments, hormone­like effects, and the activation of genes and pathways related to development, stress response, and nutrient intake. 4. mechanisms of action of plant biostimulants the mechanisms of action of plant biostimulants are still not fully understood due to the complex nature of the raw materials used and the fact that the biostimulant products contain a variety of component mixtures (yakhin et al., 2017). however, research has shed light on some mechanisms that underlie specific biostimulants’ action. protein hydrolysate­based biostimulants have been discovered to promote plant development and mitigate the effects of abiotic stresses (sorrentino et al., 2021). the precise mechanisms of action of protein hydrolysates remain unknown. however, research has shown that they can affect specific metabolic pathways, including those involved in amino acid metabolism and phenolic chemical synthesis, enhancing plant growth and stress tolerance (sorrentino et al., 2021; zhang et al., 2023). the advantages of humic substances, such as humic and fulvic acids, have long been recognized in soil fertility and plant growth (rouphael and colla, 2018). the biostimulatory effects of humic chemicals are attributed to several mechanisms, including improved soil structure, increased nutrient availability, and nitrate assimilation (rouphael and colla, 2018). microbial biostimulants, such as plant growth­promoting rhizobacteria (pgpr), have increased plant growth, nitrogen uptake, and stress tolerance (kaushal et al., 2023). secondary metabolites, hormones, organic acids, and enzymes produced by microbial biostimulants help plant growth and activate defence mechanisms (ma et al., 2022; kaushal et al., 2023). algal extracts and products made from seaweed have also been discovered to support plant development and stress tolerance (mannino et al., 2020). these biostimulants’ possible modes of action include altering phytohormones, increasing photosynthetic activity, and inducing defensive responses (yakhin et al., 2017; mannino et al., 2020). remembering that the type of plant and particular product can impact the biostimulant’s mode of action is crucial. mixing different biostimulants may have positive, negative, or cumulative effects (giordano et al., 2020). more research is needed to fully understand the biostimulants’ mechanisms of action and maximize their use in sustainable farming practices. 5. effects of plant biostimulants on growth and development improved growth and yield biostimulants have been proven to improve plant growth and raise agricultural production. some examples are increased plant height, biomass buildup, and fruit production (ertani et al., 2014; francesca et al., 2020). biostimulants can stimulate all elements of plant growth, such as root formation, shoot growth, and general vigour (kocira et al., 2020). plant growth (germination rate, leaf area, chlorophyll and protein content, nitrogen content, root and stem development, root and stem weight biomass, yield, and drought resistance) has been shown to benefit from the application of biostimulants. additionally, biostimulants can help with seed germination, increase crop production, and natural soil fertility, adv. hort. sci., 2024 38(2): 211­222 214 and decrease pollutant toxicity. these are just a few of the positive benefits of biostimulants. enhanced nutrient uptake and utilization plant biostimulants can enhance a plant’s capacity to take in and utilize nutrients. increasing nutrient assimilation and absorption can improve nutritional status and resource utilization (paradikovic et al., 2019; mohamed et al., 2021). improved plant nutrition and general development may result from this.plant development and health are impacted both directly and indirectly by bacteria having pgpr properties. by fixing nitrogen, aiding in the production of plant hormones like auxin, cytokinin, and gibberellin, promoting the uptake of iron and similar trace elements through the production of bacterial siderophores, dissolving mineral and organic phosphates, and converting other nutrients into forms that the plant can easily absorb, these bacteria can directly affect the growth of plants. or, as previously indicated, they are known to indirectly encourage plant development by reducing elements that cause plant disease (dobbelaere et al., 2003; çakmakçı et al., 2006). activation of metabolic pathways biostimulants can activate many plants’ metabolic processes, promoting better growth and development. they can alter the synthesis and metabolism of plant hormones such as auxins, cytokinins, and gibberellins, which are crucial for regulating plant growth (nephali et al. , 2021; sorrentino et al., 2021). although the effect of biostimulants on metabolic processes has not been fully revealed, biostimulant contents may be associated with the synthesis of molecules or precursors involved in these processes. for instance, protein hydrolisates may function as regulators for plant growth, as they contain short­chain peptides, and certain amino acids, such as phenylalanine, have been reported to increase the production of endogenous auxin by functioning as signaling molecules (raguraj et al., 2022). additionally, biostimulants, such as phenolic compounds, may affect the production of secondary metabolites that enhance plant defence and stress tolerance (nephali et al., 2021). stress tolerance and resistance one of the main benefits of biostimulants is their ability to boost plant stress tolerance. they can make plants more resilient to abiotic stresses such as heat, salt, and drought (oosten et al., 2017; nephali et al., 2021). furthermore, biostimulants can increase resistance to biotic stresses like pests and illnesses (godlewska et al., 2021). they can strengthen the immune system, activate defence systems, and enhance the plant’s overall stress response. improved fruit quality and nutritional value biostimulants have been shown to enhance the nutritional value and quality of fruits and vegetables. they can increase the amount of bioactive components in crops, such as vitamins, phenolic compounds, and antioxidants, enhancing their nutritional value and health­promoting properties (francesca et al., 2020; godlewska et al., 2021). biostimulants can also improve the sensory characteristics of fruits, such as color, flavor, and shelf life. plant biostimulants improve a plant's capacity to grow, develop, and tolerate stress. they can promote crops’ nutritional content and quality, nutrient uptake, metabolic activity, stress resistance, and other processes (calvo et al., 2014). because of these effects, biostimulants are valuable tools in sustainable agriculture for increasing crop productivity and resilience. enhancing stress tolerance in ornamental plants with positive findings, using plant biostimulants has increased the stress tolerance of ornamental plants. using biostimulants, abiotic stressors such as drought, heat, and water scarcity can decrease and improve plant performance (toscano et al., 2019; nephali et al., 2021). by triggering antioxidant defence mechanisms, controlling reactive oxygen species (ros) metabolism, and enhancing membrane integrity, they can improve plant responses to stress (nephali et al., 2020; hasanuzzaman et al., 2021). studies have shown that biostimulants can help ornamental plants flower earlier, produce more flowers, and accumulate biomass (toscano et al., 2019). furthermore, it has been discovered that biostimulants increase the tolerance of ornamental plants to drought stress, leading to more significant growth and survival rates (toscano et al., 2019; leotta et al. , 2023). promoting root growth, enhancing nutrition and water availability, and managing stomatal function can increase the plant’s resistance to water scarcity (clercq et al., 2023; leotta et al. , 2023). some biostimulants have tütüncü et al. ‐ biostimulants: growth and stress tolerance 215 examined the resilience of ornamental plants. studies, for example, have demonstrated that protein­rich seaweed extracts, such as chondrus crispus, serve as biostimulants and improve drought tolerance in tomato plants (domingo et al., 2023). seaweed extracts are known to increase several crops’ resiliency to stress, especially ornamentals (francesca et al., 2020; clercq et al., 2023). it has also been investigated whether microbial biostimulants, such as rhizobacteria that promote plant development, can improve ornamental plants’ resilience to stress (nephali et al., 2021). plant biostimulants throughout cultivation can considerably improve ornamental plants’ performance and stress tolerance. utilizing biostimulants enables plants to respond to abiotic challenges, develop, and grow more effectively, which enhances the quality of attractive crops. more investigation is required to comprehend the mechanisms of action and to improve administration strategies and biostimulant dosages for diverse species of ornamental plants. 6. application methods and dosages of plant biostimulants application methods before planting, seeds can be treated with biostimulants to improve germination, seedling vigour, and early growth (paradikovic et al., 2019). foliar application is another one to boost nutrient intake, spur growth, and increase stress tolerance; biostimulants can be sprayed into the leaves of ornamental plants (mannino et al., 2020; cristiano and de lucia, 2021). spraying biostimulants on the substrate is another application method to promote root development, nutrient uptake, and overall plant growth. biostimulants can be sprayed on the substrate or growing media surrounding the roots of ornamental plants (lorenzo et al., 2018; santos et al., 2019). irrigation systems can also be used to apply biostimulants, which will then be delivered right to the root zone of ornamental plants (paradikovic et al., 2019). dosages different biostimulants may require different dosages depending on the product, plant type, and growth stage. various research has evaluated a range of biostimulant application doses or rates. for instance, a biopolymer­based biostimulant was used in a study on melon plants at rates of 0.06, 0.12, 0.24, or 0.48 ml per plant (lorenzo et al., 2018). compared to lower concentrations, the application at 0.12 and 0.24 ml per plant led to better plant growth and biomass. an animal­derived biostimulant was administered to potted snapdragon plants in a different investigation at concentrations of 0, 0.1, or 0.2 g l­1 (cristiano et al., 2018). the biostimulant application at both doses significantly increased plant height, shoot length, leaf area, flower number, and aboveground dry weight compared to the control. depending on the particular needs of the ornamental plant species and the desired results, the dosage and frequency of biostimulant application may need to be changed. it is significant to note that depending on the biostimulant product, plant species, environmental factors, and growth stage, the best application techniques and dosages of biostimulants may change. as a result, it is advised that site­specific studies be carried out and that the manufacturer’s instructions for the biostimulant product be adehered to. 7. biostimulant application in ornamental plants biostimulant applications have recently started to be in high demand in ornamental plants. many studies have demonstrated that it naturally promotes rooting and plant growth as an alternative to commercial hormone uses. in some ornamental plants with high commercial value, it has been stated that pgpr applications positively affect both rooting promotion and agronomic properties.studies have been carried out on ornamental plants belonging to different families asteraceace (chrysanthemum, dahlia, zinna) and geraniaceae (göre and altın, 2006); iridaceae (gladiolus) and oleaceae (jasmine) (damodaran et al., 2014) and solanaceae (petunia) (hoda and mona, 2014). in the studies, it has been determined that biostimulants positively affect rooting in ornamental plants.it has been stated that p. fluorescens bacterial strain in zinnia (yuen and schroth, 1986); azospirillum brasilense strains in photinia (larraburu et al., 2007); agrobacterium rubi and serratia liguefaciens strains in forsythia intermedia (kır, 2010); bacillus megaterium and pseudomonas flourescens strains in rosa canina (kınık and çelikel, 2017); bacillus subtilus strain in ficus benjamina l. 216 adv. hort. sci., 2024 38(2): 211­222 (sezen et al., 2014); different bacillius strains in kalanchoe blossfeldiana (dalda­sekerci and unlu, 2023) have been shown to promote rooting. the effects of biostimulants on the vegetative characteristics, flowering status, duration of flowering, and tuber formation of ornamental plants were also reported.positive effects of biostimulants on agronomic properties were reported in anthurium (padmadevi et al., 2004); chrysanthemum and dahlia (gore and altın, 2006); geranium (mishra et al., 2010), tulip (parlakova, 2014), gladiolus (damodaran et al., 2014), jasmine (jayamma et al., 2014); in petunia (hoda and mona, 2014); in the poinsettia (parlakova karagöz, 2018); and cyclamen (girgin and sezen, 2021) summary of reported beneficial effects of different biostimulant types and application techniques given table 1. 8. regulatory aspects and future perspectives clear definitions and rules controlling plant biostimulants are required because the regulatory framework for these substances is currently developing (yakhin et al., 2017). to promote the legalization of biostimulants and ensure their efficacy and safety, the european union (eu) has tried to regulate them (farkas et al., 2022). beyond necessary nutrients or plant growth regulators, the concept of biostimulants emphasizes their capacity to increase plant productivity through the unique features of their constituents (yakhin et al., 2017). biostimulants must be regulated to maintain product quality, efficacy, and consumer confidence and support ecologically friendly and sustainable agricultural practices (farkas et al., 2022). the ability of plants to function better under stress and produce higher­ quality plants overall is greatly enhanced by using biostimulants in the cultivation of ornamental plants (farkas et al., 2022). additional research is required to comprehend further the mechanisms of action and the impacts of various biostimulants on ornamental plants (nephali et al., 2020). for the efficient and long­term use of biostimulants in agriculture, creating a science­based biostimulant industry and sound regulations are essential (yakhin et al., 2017). application strategies, doses, and timing of biostimulant treatments in ornamental plants can all be improved to increase efficacy (farkas et al., 2022). combining biostimulants with environmentally friendly agriculture techniques like organic fertilizers and integrated pest management can produce more robust and sustainable systems for growing decorative plants (farkas et al., 2022). in conclusion, efforts are being made to assure the safety, effectiveness, and appropriate use of plant biostimulants in cultivating ornamental plants. biostimulants’ prospects include more study, method optimization, and integration with other sustainable practices to improve plant performance and resilience. 9. conclusions as a result, plant biostimulants have demonstrated significant promise for improving ornamental plants’ growth and stress tolerance. the research that has been evaluated has shown that biostimulants can boost ornamental crop performance, yield, nutrient uptake, and stress tolerance. the mechanisms of action of biostimulants include stimulating antioxidant defence systems, activating metabolic pathways, and modifying plant hormone levels. using biostimulants to develop ornamental plants provides opportunities for resilient and sustainable horticulture techniques. by lowering the need for artificial fertilizers and pesticides, biostimulants can aid in developing more effective and ecologically friendly agricultural methods. they can also raise ornamental plants’ aesthetic appeal and quality, increasing their marketability. more study and development are required to maximize the utilization of biostimulants in the growth of ornamental plants. this includes examining the precise results of various biostimulant formulations and biostimulant kinds on various species of ornamental plants. the best biostimulant application strategies, doses, and timing for various growth phases and environmental circumstances also require further research. specific definitions and regulations are required to guarantee product quality, efficacy, and consumer confidence. regulatory aspects of biostimulants are also evolving. for biostimulants to be used effectively and sustainably in producing ornamental plants, a science­based biostimulant industry, and good laws are essential. to sum up, plant biostimulants have much potential to improve ornamental plants’ tütüncü et al. ‐ biostimulants: growth and stress tolerance 217 table 1 ­ different biostimulant agents and beneficial effects on ornamental plants biostimulant type applied species application beneficial effects references spirulina and klamath algae portulaca grandiflora mixing with the growing substrate to improve seed germination, plant growth and flowering prisa (2019) animal­derived ph (hydrolysis of pro­ teins from erythrocytes antirrhinum majus l. 0.1 and 0.2 gl­1 foliar spray and root drenching (150 ml/plant) to increase plant morphological and qualitative traits, leaf and root­n content, photosynthetic rate, transpiration rate, and stomatal conductance cristiano et al. (2018) seaweed extract (ascophyllum nodosum) helianthus annus l. cv. pleno sol 0, 5, 10 or 15­mll­1 of seaweed extract (60 ml spray treatment during seed ger­ mination) to enhances seed germination and seedlings development santos et al. (2019) animal derived ph (hicure®) dianthus caryophyllus l. drenching with biostimulant concentra­ tions of 2.0, 2.5 and 3.0 l ha­1 improvement of flower quality, such as stem length and flower head size niyokuri et al. (2017) chitosan nanoparticles rosa hybrida cv. black magic applying as vase solution with the con­ centration of 5, 10, 15 mg l−1 reduction microbial growth in vase solution. increase phenolics, total flavonoids, and amount of anthocyanin in treated petals and vase life of the flowers seyed hajizadeh et al. (2023) plant derived protein hydrolysates (ph)(trainer® and vegamin©) chrysanthemum morifolium cv. pinacolada and radost spraying the whole plant at the recom­ mended concentration by the companies overall action; improved the status of plants, stimulating stem elongation and the apical flower diameter carillo et al. (2022) animal derived ph (hydrostim®) petunia × hybrida hort. cv. potuniaand dunnen 0, 0.1, 0.2 g l−1foliar spray and root drenching enhance visual quality of the plants (increase leaves and flower numbers, leaf area, dry weight, shoots, flowers, and leaf fresh weight cristiano and de lucia (2021) cyanobacterial hydrolysate (arthrospira platensis) petunia x hybrida 5 g l−1 foliar spraying weekly under salini­ ty stress conditions hydrolysate mitigates the negative effect of nacl on petunia x hybrida crops at an ec of 3.0 ds m−1 bayona­marcillo et al. (2020) fermented alfalfa brown juice tagetes patula l. cv. csemő 0.5%, 1.0%, 2.5%, 5.0%, or 10% of fermented alfalfa brown juice 0.5% fermented bj improved seed germination root and shoot length, root and shoot dry mass and the number of leaves barna et al. (2021) vegetal extract and ph (radifarm®) viola tricolor var. hortensis dc. applying to the plant rhizosphere at 0.3% solution improvement seedling quality and morphological parameters zeljković et al. (2021). microalgae (arthrospira platensis) hydrolysate pelargonium hortorum l.h. bailey foliar spraying at the 5 g/l concentration with 150 mg/l silicon stimulation root, shoot, leaf, and flower formation under salinity stress. tejada­ruiz et al. (2020) 218 table 1 ­ different biostimulant agents and beneficial effects on ornamental plants biostimulant type applied species application beneficial effects references seaweed extract (acadian seaplants™ ) tagetes erecta spraying to seeds daily with 70 ml solution at the 0, 5, 10, 15 ml l­1 concentrations enhancing seed germination and seedling growth and development (optimum concentration was 15 ml/l) tavares et al. (2020) microbial biostimulant (trichoderma sperellumand t. harzianum) passiflora caerulea foliar application with spores at the concentration of 106 and 108 cfu ml­1 increase number and size of chloroplasts, improved plant physiology characteristics, and an increase yield şesan et al. (2020) vegetal extract (moringa oleifera) gladiolus grandiflorus 5% (v/v) aqueous extract alone or in combination with 50 mg/l salicylic acid or gibberellic acid increase the yield and quality of cut spikes, prolong the vase life. zulfiqar et al. (2020) nitrophenolate based biostimulant (atonik®) calendula officinalis plants irrigated by 3000 ppm saline water and sprayedwith 2 mll­1atonik (0.196 ug­1) tolerate the salt stress and promoted flowering growth el­ziat and swaefy (2019) arbuscular mycorrhizal fungi (glomus mossease) gerbera jamesonii cvs. beaudine and palm beach amf spores were added to soil at a rate of 100 spore/1000 g of dried soil amf inoculation at seedling stage can significantly increase gerbera flower yield and longevity after harvesting (vase life) othman et al. (2022) arbuscular mycorrhizal fungi (funneli formis mosseae) hyacinths orientalis l. anna marie approx. 1100 spores were added to 2 kg substrate with soils and sands (3:1, v/v) regulate early flowering and prolonging flowering time xie and wu (2018) rhizobacteria (azospirillum brasilense) eustoma grandiflorum (raf) shinn. inoculation with 106 and 107 cfu on seed surface at sowing improve morphological parameter of seedlings and lead to a shorter time production santos et al. (2022) humic acid and vermicompost lavandula angustifolia l. adding vermicompost to the soil before planting, foliar application of humic acid increase flower yield and essential oil content, reduce the demand for chemical fertilizers sharafabad et al. (2022). humic acid lilium oriental hybrids ‘sorbonne’ adding 0.2, 2.0, and 20.0 mgl­1 humic acid directly to the medium promote in vitro bulblet growth increase in bulblet sucrose, total soluble sugar, and starch content wu et al. 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35(3): 243­253 doi: 10.36253/ahsc­8376 genetic diversity assessment of ancient mulberry (morus spp.) in lebanon using morphological, chemical and molecular markers (ssr and issr) a. kadri 1, 4, s. saleh 2, a. elbitar 3, a. chehade 1 (*) 1 lebanese agricultural research institute, tal amara station, department of plant biotechnology, plant genetic resources unit, p.o. box 287, zahleh, lebanon. 2 lebanese university, faculty of sciences i, applied plant biotechnology, hadath, lebanon. 3 lebanese agricultural research institute, tal amara station, department of plant biotechnology, plant tissue culture unit, p.o. box 287, zahleh, lebanon. 4 lebanese university, faculty of sciences iv, department of life and earth sciences, zahleh, lebanon. key words: germplasm, microsatellite markers, morphological descriptors, poly­ morphism, principal component analysis. abstract: lebanon has ancient mulberry trees which are the remnants of the abundant orchards that dominated its lands during the nineteenth century. lebanese mulberry germplasm has not been assessed yet. this study aims to collect local old rainfed mulberry accessions from different geographical regions and assess their diversity by using morphological and molecular mark­ ers (ssr and issr). genetic diversity of 70 accessions of mulberry were evaluat­ ed by using 27 morphological traits. the dendrogram based on the morphologi­ cal attributes showed a relative separation of the different accessions based on fruits color and taste. molecular analysis was performed for the accessions by using selected ssr and issr primers. the primers marked a high discriminating power (0.7 to 0.89). the dendrogram constructed on the base of upgma method showed 13 different groups. the clustering patterns indicated no loca­ tion nor local name specificity among mulberry accessions. the combination of ssr and issr primers was informative for estimating the extent of mulberry genetic diversity. it can be concluded that there is a high level of genetic diver­ sity within mulberry trees in lebanon. these results will be useful for mulberry germplasm management in terms of biodiversity protection and as a valuable source of gene pool for crop improvement. 1. introduction mulberry belongs to the genus morus of the family moraceae. it is a (*) corresponding author: alichehade@hotmail.com citation: kadri a., saleh s., elbitar a., chehade a., 2021 ­ genetic diversity assessment of ancient mulberry (morus spp.) in lebanon using morpho‐ logical, chemical and molecular markers (ssr and issr). ­ adv. hort. sci., 35(3): 243­253 copyright: © 2021 kadri a., saleh s., elbitar a., chehade a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 march 2020 accepted for publication 10 june 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8376 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 243­253 244 multipurpose tree with a significant ecological, nutri­ tional and economical high value. mulberries are highly adaptable species in different soil and climatic conditions. they are generally quite tolerant to drought, pollution and poor soil. therefore, they can be found in a wide area of tropical, subtropical, and temperate zones in asia, europe, north america, south america, and africa (kafkas et al., 2008). the genus morus regroups 24 species (thabti et al., 2014). the most widespread species in the mediterranean climate areas are: morus alba with fruit colors ranging from white to dark red, morus rubra with mainly red/purple fruits and morus nigra with dark purple to black fruits (gerasopouls and stravroulakis, 1997). mulberry fruits have remarkable potential for pro­ viding various valuable industrial products of high economic value for human beings. they are used for direct fruit consumption (morus alba, morus indica, morus nigra, and morus laevigata). most of mulberry species have distinct flavor with juicy and acidic char­ acteristics making them attractive for use in the pro­ cessing industry for products such as fruit juice, ice cream, jelly, and jam (ercisli and orhan, 2007). interest in mulberry has increased considerably over the last 20 years as a healthy fruit. morus species have great antioxidant potential due to their high content in phenolic compounds including flavonoids, anthocyanins, and carotenoids (zhang and ma, 2018). mulberries present anticancer and anti­inflam­ matory properties and show as well significant effect on many chronic diseases like diabetes (nakamura et al., 2009; kwon et al., 2015; qian et al., 2015). mulberry is an economically important plant used for sericulture. it is the sole food plant for the domesticated silkworm, bombyx mori (zhao et al., 2009). the genus morus, is cultivated extensively in east, central and south asia for silk production (awasthi et al., 2004). hence, mulberry is one of the most important components that decide the sustain­ ability of this industry (liu et al., 2009). at the turn of the century, lebanon was known for its high­quality silk industry. bestowed with an ideal climate and a fertile soil, mulberries were plant­ ed everywhere in lebanon and mulberry production flourished (firro, 2009). the silk tradition in lebanon is more than two thousand years old. it goes back to the period of the famous purple dye (ourjouan) extracted from the murex shell by the phoenicians of sidon and tyre and used to produce imperial purple silk (khater, 2009). in the 19th century, silk industry constituted almost 80% of lebanon’s economy. by the early 20th century, 70% to 80% of the cultivable land of the country mountainous regions (mount lebanon) became devoted to mulberry orchards. due to the high demands in silk production, mulberry tree has an unsurpassed economic impact on rural com­ munities. after 1940’s, when silk began to be import­ ed from the far east, the sericulture industry declined sharply. mulberry cultivation became mar­ ginalized. however, lebanon still has very old rainfed mulberry trees which are the remnants of the abun­ dant orchards that were once shaping the landscape of many villages. mulberry trees are found in differ­ ent lebanese villages, mostly located at orchards periphery or in small gardens. in lebanon, mulberry genotypes are very diverse, as they were sometimes obtained in the past from seeds or from cuttings. this process has led to a great number of landraces adapted to different conditions and different uses throughout the country. in lebanon, there are many local traditional accessions but no named cultivars. mulberries are distinguished and denominated according to the fruit color: “abyad” (white mulberry), “mwachah” (purple mul­ berry), “shami” and “aswad” (black mulberries). mulberry genetic diversity is progressively being lost in farmers’ fields and in nature. the threat results from the interaction of several factors and is processing at an alarming rate. the most crucial fac­ tors are urbanization, climatic changes, out breaks of new diseases and pests, and the frequent occurrence of natural calamities. little information is available about the genetic diversity of lebanese mulberries. to protect mulberry in lebanon, a marginalized species, conservation programs should be initiated. in this study, we have collected local mulberry acces­ sions from different geographical regions of lebanon and assessed their genetic diversity by using agro­ morphological traits as well as molecular markers (ssr and issr). 2. materials and methods field survey samples of fruits for morphological and chemical analyses were collected from local trees of mulberry morus from 21 sites covering different lebanese regions (the north plain, bekaa plain, mount kadri et al. ‐ genetic diversity assessment of mulbery in lebanon 245 lebanon, the south). these sites are subjected to dif­ ferent climatic conditions (precipitation, tempera­ ture) and agricultural practices. they are situated at an altitude between 30 and 1620 m, a latitude rang­ ing from n33° 16’ 166” to n34° 21 ‘51.5” and a longi­ tude between e36°10’ 849” and e35° 01’ 38.7” (fig. 1). the number of individual trees sampled per site (population) ranged from two to sixteen cultivars. in total, 70 accessions of mulberry were studied. collected samples consisted of mature fruits (approx­ imately 500 g) and vegetative materials (young leaves, mature leaves and branches). the studied accessions included ‘abyad’ (white mulberry), ‘mwachah’ (purple mulberry), ‘shami’ and ‘aswad’ (black mulberries). morphological and chemical characterizations the characterization of the vegetative materials and the fruits was based on descriptors for investiga­ tion of mulberry germplasm’s morphology produced by agriculture and consumer protection fao (sohn, 2003). thus 27 morphological characters were stud­ ied for the mulberry accessions. these studied traits included 13 qualitative characters (for the leaf: shape, margin, base, apex, surface, color, glossiness, phytotaxis, bud shape and color; for the fruit parts: shape, color, taste, seed color) and 14 quantitative characters (for the vegetative parts: leaf length, width and thickness, petiole length, bud length, internode length; for the fruit parts: fruit length, diameter and weight, peduncle length, juice percent­ age (volume of the juice*100/weight of the fruits), sugar quantity (using refractometer), ph and acidity (by titration reaction). molecular characterization dna extraction. genomic dna was extracted from mulberry young fresh leaves using cetyl trimethyl ammonium bromide (ctab) procedure described by fao/iaea (2007). the dna quantity and quality was visually quantified using the agarose gel elec­ trophoresis method as described by maniatis et al. (1982). dna samples were stored at ­20°c. pcr amplification of the dna with issr primers. six primers (ubc807, ubc810, ubc826, ubc827, ubc864 and bi3) were tested for dna amplification (emir, 2013). the issr (inter simple sequence repeat) amplification was carried out as per vijayan and chatterjee (2003) using 20 µl reaction mixture containing 2 µl of 10 x pcr buffer (750 mm tris­hcl ph 8.8; 0.1% tween­20), 0.2 mm dntp, 2 mm mgcl2, 200 nm primer, 50 ng genomic dna and 1 u taq dna polymerase (mbi fermentas inc, hanover, md­ 21076, usa). the pcr schedule included an initial cycle at 94°c for 2 min followed by 35 cycles of 94°c for 30 sec, 50°c for 30 sec, 72°c for 2 min and a final extension of 10 min at 72°c. the pcr products were resolved by electrophoresis on a 1.5% agarose gel in 1 x tris boric acid buffer (tbe), stained with ethidium bromide (0.5 μg/ml) and visualized under uv light. pcr amplification of the dna with ssr primers. microsatellite polymorphisms were identified using three ssr primers (primers: mulstr1, mulstr2 and mulstr3) (tikader et al., 2009). microsatellite ampli­ fication reactions were performed in a final volume of 25 µl in the presence of 2.5 µl of buffer, 200 µm of each dntp, 0.4 µm of each primer pair, 1 unit (u) of taq dna polymerase, 50 ng template dna, and 2 mm mgcl2. the amplification reaction consisted of an initial denaturation step at 94°c for 4 min, followed by 45 cycles of 1 min denaturation at 94°c, 65 sec annealing at 50°c, 90 sec extension at 72°c with a final extension of 72°c for 10 min using thermal cycles. the pcr amplification products were separat­ ed on a 6% denatured polyacrylamide gel and visual­ ized by silver staining. data analysis for qualitative traits, scores were attributed fig. 1 ­ geographic distribution of the studied mulberry acces­ sions as visualized with diva­gis program (hijmans et al., 2001). adv. hort. sci., 2021 35(3): 243­253 246 according to fao mulberry descriptors. a phenotypic diversity index, hsj (shanon index) (magurran, 1988) was calculated for each site to describe the pheno­ typic diversity of mulberry. the following formula was used for calculating hsj for each trait with n cate­ gories hsj=ʃ pilnpi where pi is the relative frequency in the ith category for the jth trait. the average diver­ sity (h) over k traits of each site was estimated as: h=ʃ hsj/k. traits evaluation was performed by using the principal component analysis (pca). the relation­ ships between mulberry leaves and fruits based on their quantitative and qualitative traits were studied using hierarchical cluster analyses executed using euclidean distance following the ward’s method implemented in past software (hammer et al., 2001). to assess the information given by ssr and issr markers, the following parameters were calculated: number of alleles per locus, percentage of observed heterozygosity (ho), expected heterozygosity (he= 1­ σpi2, where pi is the frequency of the ith allele) and the power of discrimination (pd = 1 ­σgi2, where gi is the frequency of the ith genotype). genetic distances were calculated according to jaccard (1908). trees were produced by clustering the data with the unweighted pair­group method (upgma) with sahn­ clustering and tree programs of past software (saporta, 1990). 3. results mulberry trees were distributed over various agro­climatic areas of lebanon (fig. 1, table 1). a total of 70 mulberry accessions were studied belong­ ing to ‘abyad’ (22 accessions), ‘mwachah’ (25 acces­ sions), ‘shami’ (20 accessions) and ‘aswad’ (3 acces­ sions). among mulberry species found in lebanon, morus alba was the dominant species in cultivation (95%). around 85% of the surveyed mulberry trees were rainfed, old and inherited from family. morphological analysis leaves morphological characterization. mulberry trees tend to have short trunks with large, low, spreading limbs. leaves were alternately arranged and simple. the majority of cultivars had a cordate leaf shape, except the leaves of two accessions were reniform (‘abyad’ and ‘shami’) and two other were cordate to oval (‘abyad’ and ‘mwashah’). all leaves table 1 ­ climatic and geographic characteristics of the 21 locations surveyed to characterize the lebanese mulberry trees site latitude (n) longitude (e) altitude (m) annual average temperature (°c) annual rainfalls (mm) varieties number of accessions douris 33°59'588" 36°10'849" 1131 14.9 441 abyad mwachah 4 rayak 33°51'751" 35°59'591" 927 15.1 544 aswad 2 nabishet 33°52'10.4" 36°06'34.7" 1233 13.6 570 abyad mwachah sami 6 jenta 33°51'23.5" 36°06'26.2" 1114 13.2 580 abyad mwachah 2 britel 33°56'02" 36°08'54.3" 1154 14.7 471 abyad mwachah shami 4 el borjein 33°39'27.2" 35°29'11.3" 1620 12.7 630 shami 1 baassir 33°39'30.1" 35°26'54.7" 1094 12.9 630 abyad mwachah shami 2 hawsh nabi 33°55'28.6" 36°04'23.8" 990 15.2 544 abyad mwachah 2 hawsh refaa 33°55'23.7" 36°02'34.2" 971 15.1 530 abyad mwachah 5 kfar dabash 33°56'43.2" 36°02'13.7" 1079 15.1 540 abyad mwachah 1 chmistar 33°57'49.6" 36°01'07" 1145 14.9 550 shami 2 beit chama 33°55'07" 36°01'25" 1011 15.2 541 abyad mwachah 1 tamnen taata 33°52'43.3" 35°59'45.9" 937 15.1 542 abyad mwachah 1 chlifa 34°05'109" 36°06'098" 1012 14.7 461 abyad mwachah shami 5 flaoue 34°04'934" 36°03'761" 1139 14.6 461 abyad mwachah shami 3 dayr lahmar 34°07'077" 36°07'940" 1012 14.5 461 baladi 1 zahle 33°48'59.9" 35°57'32.6" 882 15.2 646 abyad mwachah shami 5 ali ennahry 33°51'21.04" 35°01'38.7" 958 15.1 544 shami 1 sour 33°16'166" 35°13'133" 30 20.2 697 abyad mwachah shami 4 kfar chakhna 34°21'51.5" 35°51'50.7" 198 13.9 754 shami 2 hasbaiya 33°32'74" 35°64'373" 467 15.1 590 abyad mwachah shami 16 kadri et al. ‐ genetic diversity assessment of mulbery in lebanon 247 had dentate margins. they presented mainly a cor­ date base and an acute apex. leaf surface of 70% of the accessions was slightly rough. only nine acces­ sions of ‘shami’ presented rough surfaces. leaves generally presented an average length between 6.83 and 18.9 cm and width between 3.94 and 17.02 cm. petiole length average was between 0.72 and 5.07 cm. leaves of black mulberry acces­ sions ‘shami’ (0.02 and 0.03 cm) were thicker than those of white mulberries (0.01 cm). fruits morphological characterization. for the 70 accessions, the pomological characteristics investi­ gated showed a great diversity. concerning the fruit shape, 35.7% of the mulberry fruits had oblong shape, 27.1% were round, 24.2% were reniform, and 13% were oval. for the local variety ‘mwashah’, near­ ly half of the accessions had oblong shape while the majority of ‘shami’ had round one. white mulberry ‘abyad’ presented mainly oblong and reniform shape. the accessions showed significant differences in the fruit weight ranging from 1.1 g (‘abyad’ from doris) to 7.9 g (‘mwashah’ from hawshrefaa). fruit length varied from 1.7 (‘shami’ from flewa) to 4.9 cm (‘mwashah’ from hawshrefaa­bekaa) and fruit width from 1.1 (‘abyad’ from doris) to 2 cm (‘shami’ from baaser). minimum length of fruit peduncle was 0.11 (‘shami’ from flewa) and maximum length 1.28 cm (‘aswad’ from tyr). fruit color of mulberry accessions was diverse: ‘abyad’ accessions were white and ‘mwachah’ acces­ sions were violet. the fruit color of ‘shami’ acces­ sions were darker and varied between red­purple to black or black. seed color varied between light yellow and yellow­brown; ‘mwashah’ fruits had mainly light yellow seeds and ‘shami’ presented yellow­brown seeds. the percentage of juice yields differed within the accessions of the same local variety. the lowest and greatest juice yields varied from 30.1% (‘abyad’ from nabishit) to 72.3% (‘shami’ from baaser) and 73.1% (‘abyad’ from shlifa). as for the chemical characteris­ tics of mulberry accessions, sugar content ranged from 7 (‘mwashah’ from janta) to 19.5 brix (‘aswad’ from tal amara). ph varied widely from 2.29 (‘abyad’ from hasbaya) to 6.42 (‘abyad’ from douris). titrable acidity was very diverse in the different mulberry accessions. titrable acidity values were from 0.01 (‘mwashah’ from zahle) to 0.14 g/l (‘shami’ from flewa). morphological characterization pca the characterization of the collected mulberry accessions using different morphological characters showed high level of variation among the accessions. the principal component analysis (pca) revealed that the first 3 components explained 37% of the total variation, based on the 27 morphological char­ acters (table 2). the first component represented 18 % of the total variation and included fruit and leaves characteristics. it comprised fruit length, color, taste, ph, acidity and peduncle length, besides to the peti­ ole length, glossiness and thickness of leaves. the second component represented 10% of the total vari­ ation and is mainly influenced by leaf width. the third component was characterized by a percentage of variation of 9% and is dominated by the bud length character. table 2 ­ principal component analysis (pca) of the 27 morpho­ logical characters evaluated for the 70 different mulber­ ry accessions. the characters in bold are discriminant variables factor 1 factor 2 factor 3 fruit length ­0.622011 ­0.428543 0.385704 fruit weight ­0.422828 ­0.548276 0.534493 fruit diameter ­0.279504 ­0.469198 0.362977 peduncle length ­0.639179 ­0.10646 ­0.393485 percentage of 0.126663 0.055644 0.121337 sugar quantity 0.050968 ­0.124287 ­0.168429 ph ­0.720232 0.290375 0.027219 acidity 0.605034 ­0.131547 0.165099 leaf length ­0.014178 ­0.543281 ­0.48354 leaf width ­0.038798 ­0.719527 ­0.23667 petiole length ­0.700726 ­0.161604 ­0.190875 leaf thickness ­0.739758 ­0.294618 0.241539 date of maturity ­0.085013 ­0.253214 0.241209 bud length 0.069769 ­0.177441 ­0.629778 bud width 0.136856 ­0.265182 ­0.59383 bud shape ­0.192576 0.061054 ­0.048574 internodal distan­ ­0.141876 ­0.525259 ­0.327898 leaf shape ­0.109165 ­0.008143 ­0.223988 leaf base ­0.074223 0.208101 0.302694 leaf apex 0.007149 ­0.251893 ­0.077977 leaf surface ­0.50104 0.446809 ­0.164365 leaf color ­0.260375 ­0.135751 0.031335 leaf glossiness 0.670155 0.023403 ­0.053846 fruit shape ­0.498979 ­0.040292 0.105085 fruit color ­0.6271 0.204057 ­0.072799 fruit taste 0.663459 0.058647 0.089481 seed color ­0.210256 ­0.238095 0.409438 exp.var 4.797.858 2.719.786 247.986 prp.tot 0.177698 0.100733 0.091847 248 adv. hort. sci., 2021 35(3): 243­253 classification of accessions based on morphological attributes the accessions could be separated into groups based on the 11 most discriminant traits. the hierar­ chical cluster analysis classified mulberry accessions in 6 groups at ­6 similarity of euclidean distance (fig. 2, table 3). ‘shami’ accessions were classified separately into 3 main groups (g1, g4 and g5). the first group g1 included ‘shami’ and 2 accessions of ‘aswad’. g1, g4 and g5 accessions were characterized by a sour fruit taste and a dark black­purple or black­red fruit color. fruits of g4 and g5 presented significantly the lowest ph mean values (3.78 and 3.85 respectively). g5 fruits presented the lowest sugar content (9.62 °brix). accessions of g4 and g5 had the shortest fruit length (2.32 cm and 2.45 cm respectively) and the shortest peduncle (0.24 cm and 0.46 cm respective­ ly). regarding leaf characteristics, g4 and g5 acces­ sions were characterized by low mean value of leaf length, while g1 were characterized by a significantly high leaf length (17 cm). g1, g4 and g5 accessions had also the thickest limb (0.02 ­ 0.03cm) and the shortest peduncle (1.4 ­ 1.48 cm). the group g2 consisted of 12 accessions of ‘abyad’ and one of ‘mwashah’. these accessions were characterized by a white fruit color and a sweet taste. the group g3 consisted of 19 accessions of ‘mwashah’ and 2 of ‘shami’. they were characterized by purple fruits. they presented the highest value of fruit length (3 cm) and medium values for ph and different letters were significantly different at the 0.05 level (duncan's multiple range test). table 3 ­ variability of the quantitative morphological fruit characteristics for the accessions clustered within the same group (g1, g2, g3, g4, g5 and g6) minimum, maximum and mean values (with standard deviation) fig. 2 ­ dendrogram of jaccard distance based on the characters that presented high variability in the principle compo­ nent analysis, depicting the genetic relationship among the 70 different mulberry accessions. group (number of accessions) fruit length (cm) fruit width (cm) fruit weight (cm) peduncle (cm) percentage of juice (%) sugar quantity (brix) ph acidity (g/l) 2.11< l<4.05 0.96< w <2.05 0.99< w <7.81 0.93< p <1.28 51.4< j <72.3 7< s<15 3.4<ph <5.97 0.028< n < 0.13 g1 (3 shami, 2aswad) 2.91±0.81 a 1.59±0.42 a 3.91±2.60 a 1.13±0.14 a 59±7.77 a 11.6±3.84 bc 4.79±1.03 b 0.05±0.04 b 2.14< l< 3.45 1.49< w <1.88 2.05< w <5.72 0.43< p <1.4 34.2< j <58.2 11< s<16.5 2.29<ph <6.47 0.018< n < 0.076 g2 (12 abyad, 1 mwashah) 2.77±0.39 ab 1.61±0.20 a 4.00±1.17 a 0.81±0.26 b 52.68±8.50 a 13.15±1.87 ab 5.70±1.28 ab 0.05±0.02 b 2.22< l <4.95 1.4< w <2 2.6< w <7.98 0.51< p <0.99 35.4< j <64.1 7< s<15 3.5<ph <6.35 0.014< n < 0.116 g3 (19mwashah, 2 shami) 3.00±0.63 a 1.75±0.15 a 4.90±1.42 a 0.76±0.18 b 51.67±8.18 a 11.33±2.21 bc 5.47±1.04 ab 0.03±0.02 b 1.71< l <3.1 1.3< w <1.86 1.3< w <5.38 0.1< p <0.90 36.2< j <76.7 7< s<18.5 3.14<ph <5.97 0.04< n < 0.2 g4 (11 shami) 2.32±0.39 b 1.57±0.19 a 3.74±1.30 a 0.24±0.23 d 53.69±12.20 a 13.73±3.08 ab 3.78±0.39 c 0.10±0.04 a 2.13< l <2.68 1.5< w < 1.93 3.4< w <5.77 0.38< p <0.56 49.4< j <58.5 8.5< s<11 3.1<ph <6.12 0.038< n < 0.05 g5 (4 shami) 2.45±0.255 ab 1.72±0.19 a 4.68±1.09 a 0.46±0.09 c 52.95±4.09 a 9.62±1.11 c 3.85±1.51 c 0.045±0.006 b 1.88< l <3.33 1.10< w <1.95 1.11< w <5.64 0.46< p <1.14 30.1< j <73.1 10.5< s<21 5.3<ph <6.51 0.014< n < 0.08 g6 (5 mwashah 10abyad, 1aswad) 2.68±0.44 ab 1.59±0.25 a 3.76±1.26 a 0.82±0.22 b 50.74±10.31 a 14.91±3.07 a 6.05±0.45 a 0.041±0.019 b kadri et al. ‐ genetic diversity assessment of mulbery in lebanon 249 sugar content. the group 6 regrouped 5 accessions of ‘mwashah’, 10 of ‘abyad’ and one of ‘aswad’. these accessions were characterized by their sweet taste (high sugar content and high ph). groups 2, 3 and 6 presented a medium leaf thickness. molecular characterization issr analysis. the molecular analysis of mulberry accessions presented a high variability. the issr markers showed distinct polymorphism between the different mulberry accessions, only primers ubc­826 and ubc­864 showed no amplification. a total of 18 polymorphic bands were detected across the 70 accessions of mulberry through the use of four issr primers ubc807, ubc810, ubc827 and bi3. the size of amplified products ranged from 700 bp to 1600 bp. the number of scorable markers produced per primer ranged between 4 (ubc810, ubc827) and 5 (ubc807, bi3). this study showed that (ac), (ga) repeat primers generated excellent band profiles. primers synthesized from (atg) repeats failed to amplify. the power of discrimination calculated for each primer (table 4) enabled us to evaluate the genetic diversity of our locus. the calculated power of dis­ crimination pd values were between 0.75 and 0.89, showing that the studied loci are of high diversity. these primers could be effectively used to study polymorphism between mulberry accessions. ssr analysis. primer mulstr1 showed no amplifi­ cation within our accessions. a total of 10 polymor­ phic bands were detected across the 70 accessions of mulberry through the use of two ssr primers (mulstr2, mulstr3). upon using mulstr2 ssr primer, the acrylamide gel showed three bands across the different accessions (192bp, 200bp, and 208bp), while mulstr3 ssr primer generated 7 dif­ ferent bands ranging from 192bp to 275bp (table 5). the results presented high polymorphism in all the amplified loci. in their assessment of mulberry geno­ types by ssr marker profile, wani et al. (2013) used mulstr2 and mulstr3 primers which generated each 2 alleles among the 17 mulberry genotypes tested. our results showed a higher number of polymorphic bands for a higher number of accessions. the power of discrimination was relatively high for each primer, pd>0.5. mulstr3 presented higher polymorphism than mulstr2, with a pd of 0.8 and an expected het­ erozygosity of 0.713. these two primers and especial­ ly mulstr3 could be effectively used in genetic diver­ sity studies of mulberry. classification of accessions based on molecular markers the allelic diversity data was used to produce a table 4 ­ primer sequences, number and sizes (bp) of the produced bands and discriminating power (dp) of the six issr markers used in the study primers sequence number of bands band sizes (bp) dp ubc­810 3´gagagagagagagagat 5´ 4 800­1500 0.80 ubc­807 3´agagagagagagagagt 5´ 5 900­1600 0.89 ubc­827 3´acacacacacacacacag 5´ 4 700­1700 0.85 bi3 3´acacacacacact 5´ 5 500­1500 0.75 ubc­826 3´acacacacacacacacac5´ ­ ­ ­ ubc­864 3´ atgatgatgatgatgatg5´ ­ ­ ­ table 5 ­ primer sequences, number and sizes (bp) of the produced bands and discrimination power (dp) of the three microsatellite markers used in the study primers sequence number of bands band sizes (bp) dp mulstr1 f: 5’gccgtgtaccagtggagtttgca 3’ ­ ­ ­ r:5’tgaccgtttcttccactttacc­ mulstr2 f:5´ cgtggggcttaggctgagtagagg 3 192­208 0.52 r:5´ caccaccactacttctcttcttccag mulstr3 f: 5´ gggttgggtagatgggcttatgt­ 7 192­275 0.83 r:5´ ccctattaactttttggtcacctcta adv. hort. sci., 2021 35(3): 243­253 250 dendrogram via the distance matrix­upgma (fig. 3), thus revealing the genetic relationship among mul­ berry accessions. the dendrogram constructed on the base of the ssr and issr amplification product of the different mulberry accessions showed 14 differ­ ent groups at the jaccard distance of similarity 0.34. clusters g1, g2, g3, g5, g6 and g10 regrouped accessions of the three varieties ‘mwashah’, ‘shami’ and ‘abyad’. g4 contained the three varieties as the previous groups in addition to an accession of ‘aswad’. one single accession constructed individual­ ly the groups g7 (‘shami’), g8 (‘abyad’), g9 (‘aswad’), g11 (‘shami’) g12 (‘abyad’), g13 (‘mwashah’) and g14 (‘abyad’). 4. discussion and conclusions the conservation of the genetic variability of the ancient lebanese mulberry trees is of utmost impor­ tance for germplasm preservation and for future breeding programs. lebanese mulberries germplasm has not been assessed yet. this study is the first genetic diversity assessment of the lebanese mulber­ ry germplasm using a set of morphological traits and genetic markers. our inventories recensed accessions of four vernacular names ‘abyad’, ‘mwachah’, ‘shami’ and ‘aswad’ across different lebanese regions. this shows that a limited number of tradi­ tional varieties was cultivated since decades, howev­ er morphological and molecular characterization of these 70 accessions revealed high diversity of this germplasm collection. the results of morphological characterization revealed a high level of variation among mulberry characters. among the 27 descriptors studied, 11 specific characters of fruits (fruit taste, fruit length, fruit color, ph, titrable acidity and peduncle length) and leaves (petiole length, leaf thickness, leaf glossi­ ness, leaf width and bud length) revealed to be the most discriminating characters. the first component of the pca was dominated by the fruit characteris­ tics. a broad morphological diversity of the fruit was reported for mulberry germplasm (yilmaz et al., 2012; peris et al., 2014; aljane and sdiri, 2016; krishna et al., 2020). in our study, fruits exhibited dis­ tinct variations. fruits shapes were diverse. fruit color, titrable acidity, sugar content, juice yield and ph content were the most discriminating characters to differentiate mulberry accessions. similar results were reported and significant differences were observed between the fruit characteristics (yilmaz et al., 2012; peris et al., 2014; aljane and sdiri, 2016; krishna et al., 2020). fruit color is a desirable charac­ ter for commercial acceptance of a variety. fruit fig. 3 ­ dendrogram constructed from ssr and issr markers, using past program, jaccard distance and upgma clu­ stering of 70 mulberry accessions. kadri et al. ‐ genetic diversity assessment of mulbery in lebanon 251 color of our accessions varied from white, red, purple to black. the percentage of juice yields were within the limits of yilmaz et al. (2012) study (between 39% and 72%). all black mulberries had the highest fruit juice yield ratio, the highest acidity values and the lowest sugar content. this is consistent with previous researches (ozdemir and topuz, 1998; gunes and cekic, 2004; aljane and sdiri, 2016). therefore, black mulberries are preferred for processing into juice. black colored mulberry species received recently a great importance due to higher contents of phenolic compounds and to their delicious taste (aljane and sdiri, 2016). the dendrogram constructed on the base of the most discriminant morphological characters divided the accessions into 6 distinct groups. the evaluation of the relationship among accessions reduced their differentiation to fruit color and taste. a non­negligi­ ble variability of other traits influenced the grouping involving the length of the fruit, leaf and peduncle. the accessions grouping was marginally correlated to the accessions vernacular names with many excep­ tions. the dendrogram revealed that accessions with­ in each cluster belonged to different regions suggest­ ing that there was no clear relationship between accessions and geographical diversity. this is the case of the group g4 that included ‘shami’ accessions growing in north lebanon (kfarchakhna) and in south lebanon (sour). such results have been report­ ed in different crops by several studies, e.g. on chest­ nut (marinoni et al., 2013), almond (chalak et al., 2007; halasz et al., 2019) and olives (chehade et al., 2015). this variability could be attributed to the free exchange of planting material between different lebanese villages and emphasizes the adaptability of mulberry to different ecological conditions. in this study, we evaluated the genetic diversity and the relationships among the collected mulberry accessions using ssr and issr markers. the results showed high polymorphism in all the amplified loci. the power of discrimination values was high showing that the studied loci are of high diversity. the observed ssr markers heterozygosity were high. earlier studies using amplified fragment length poly­ morphism (sharma et al., 2000), issr (awasthi et al., 2004), and rapd (xiang et al., 1995; feng et al., 1996; zhao and pan, 2000; esha and shirish, 2001) also showed a large genetic variation among differ­ ent mulberry genotypes. such a high level of poly­ morphism reflects the outcrossing nature of the species. in this work, the issr profiles generated by (ac) and (ga) repeat anchored primers showed that these repeats are abundant in our accessions. vijayan and chatterjee (2003) observed amplification of (ac) rich repeat based issr primers. awasthi et al. (2004) concluded that (ca)/(tg) repeats are abun­ dant in morus genome. cluster analysis of ssr and issr data using upgma revealed high genetic distances between the studied accessions. five groups were constructed by one single accession. the other clusters regrouped accessions of ‘mwashah’, ‘shami’ and ‘abyad’ within each group. the distanced genetic relationships among mulberry accessions are in consistence with their high heterozygosity due to their out­ breeding reproductive system (dandin, 1998). accessions from different varieties and from different sites were grouped together. the molecular results emphasized that genetic diversity among mulberry accessions is not influenced by their geographical ori­ gin nor by their local names. this finding is in agree­ ment with other researchers who studied genetic diversity using ssr markers on different crops, almonds (distefano et al., 2013), mung bean (wang et al., 2018) and torch ginger (ismail et al., 2019). the analysis of the genetic parameters showed the high diversity of mulberry in lebanon. the comparison between morphological and mol­ ecular diversity indicated that morphological descrip­ tors provide different information than the molecular one. in comparison with other works in woody species there were also no correlation. for example, two ‘shami’ accessions (code b24 and has92) were in the same group in the morphological dendrogram however they belong to different groups in the mole­ cular one. one ‘aswad’ (black mulberry, code b6) accession and another ‘abyad’ (white mulberry, code b7) accession were in the same group in the molecu­ lar dendrogram but they were not in the morphologi­ cal one. it is probably that our markers sampled mainly a non­adaptive diversity. the results of this study revealed a large morpho­ logical diversity and a high genetic variation among the lebanese mulberry accessions. the combination of ssr and issr primers was informative for estimat­ ing the extent of mulberry genetic diversity. morphological and molecular clusters have distin­ guished different lines of mulberry which may help in the selection of the most diverse profile. this germplasm would enhance the local gene pool and expand genetic variation for mulberry breeding pro­ gram in the future. adv. hort. sci., 2021 35(3): 243­253 252 acknowledgements the authors would like to thank dr. michel afram for his support. references aljane f., sdiri n., 2016 ­ morphological, phytochemical and antioxidant characteristics of white (morus alba l.), red (morus rubra l.) and black (morus nigra l.) mul‐ berry fruits grown in arid regions of tunisia. ‐ j. new sci., 35(1): 1940­1947. awasthi a.k., nagaraja g.m., naik j.v., kanginakudru s., thangavelu k., nagaraju j., 2004 ­ genetic diversity and relationships in mulberry (genus morus) as revealed by rapd and issr marker assays. ­ bmc genet., 5: 1. chalak l., chahade a., kadri a., 2007 ­ morphological characterization of cultivated almonds in lebanon. ­ 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https://www.ncbi.nlm.nih.gov/pubmed/?term=huang%20l%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=qiu%20h%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=wang%20l%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=li%20l%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=yao%20l%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=kang%20k%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=qu%20j%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=wu%20y%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=luo%20j%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=liu%20jj%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20y%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20w%5bauthor%5d&cauthor=true&cauthor_uid=26615818 https://www.ncbi.nlm.nih.gov/pubmed/?term=gou%20d%5bauthor%5d&cauthor=true&cauthor_uid=26615818 impaginato 61 adv. hort. sci., 2020 34(1): 61­69 doi: 10.13128/ahsc­8402 production of seed­propagated com­ pact potted corylopsis plant in one year j.h. kim 1, j.k. suh 1, s.t. yoon 2, m.s. roh 3 (*) 1 department of environmental horticulture, dankook university, cheonan, chungnam, 31116, korea. 2 department of crop science and biotechnology, dankook university, cheonan, chungnam, 31116, korea. 3 the institute of natural resources development, mokpo national university, cheonggye‐myeon, muan‐gun, jeonnam, 58554, korea. key words: corylopsis coreana, corylopsis sinensis var. calvescens, new orna­ mental plant, pinching, plant growth regulator, slow release fertiliz­ er. abstract: the feasibility to produce compact corylopsis sinensis var. calvescens and c. coreana plant in a 10 cm pot in one year from transplanting seedlings with maximized number of short shoots and inflorescences was investigated. corylopsis sinensis var. calvescens was selected as a suitable species to produce compact plant with inflorescences. slow release fertilizer (srf) at a rate of 0, 0.125, 0.25, and 0.5 g per pot was applied to the surface of the growing medi­ um (expt. 1). shoots were pinched 2 (feb. 28), 4, 6, and 8 weeks (may 16) (expt. 2) after transplanting, and ancymidol, paclobutrazol, chlormequat, and daminozide plant growth retardants were treated (expt. 3). application of a srf at 0.5 g per pot and pinching four times at 2­week intervals before may 16 effectively increased the flowering percentages and the number of stems with inflorescences, to accelerate flowering, and also produced a compact plants. paclobutrazol at 10­20 mg/l applied as soil drench was effective in inhibiting stem elongation in the first year; however, higher concentrations should be avoided to prevent excessive reduction in the growth of shoots and production of malformed inflorescences. 1. introduction the genus corylopsis siebold & zucc., commonly known as winter hazel that flowers early in the spring in china and korea, is a shrub or small tree. most of the corylopsis species grows tall reaching a height of 2­4 meters (bean and anisko, 2014). flowers are bisexual and seeds are produced. among 29 species, 19 species are endemic in china (zhang et al., 2003). chinensis sinensis hems. var. calvescens rehder & e.h. wilson is growing in the mountains in guangxi, sichuan, and jiangxi, among other provinces in china (zhang et al., 2003) and c. coreana uyeki (son et al., 2016) in a rather restricted area in korea. all species are deciduous shrubs producing light yellow pendant racemes (inflorescence) measuring (*) corresponding author: marksroh@gmail.com citation: kim j.h., suh j.k., yoon s.t., roh m.s., 2020 ­ production of seed‐propagated compact potted corylopsis plant in one year. ­ adv. hort. sci., 34(1): 61­69. copyright: © 2020 kim j.h., suh j.k., yoon s.t., roh m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 june 2019 accepted for publication 25 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 61­69 62 about 5 cm in length, followed by appearance of leaves (fig. 1). corylopsis, one of many germplasms native and indigenous to china that includes an endangered acer pentaphyllum diels (roh et al., 2008 b), are not well known to horticulturist, growers, and landscape industry, but has a great potential to develop as a new ornamental plant. although corylopsis may be available from rooted cuttings or tissue­cultured propagules for a mass propagation (moon et al., 2002; koh and lim, 2006), the success of the rooting of cuttings depends on the season when cuttings were collected and may not provide a large number of plants (kwon et al., 2011) and acclimatization of tissue culture propagules is not easy (moon et al., 2002; koh and lim, 2006). therefore, seeds are a viable alternative source for mass propagation and for forcing seedlings to flower. suitable species should first be identified and then excessive stem elongation must be controlled using plant growth retardants (currey and lopez, 2016), and pinching combined with plant growth retardant treatment (jeong, 2000). stem elongation can be inhibited by practices such as pinching shoots in many floral and ornamen­ tal plants, resulting in short plant height (lee et al., 2006; latimer and whipker, 2013). growth and flow­ ering is also affected by treatment with plant growth regulators. among the many growth retardants, ancymidol (ɑ­cyclopropyl­ɑ­(4­methoxyphenyl)­5­ pyrimidinemethanol), daminozide (butanedioic acid mono(2,2­dimethylhydrazide), chlormequat (2­ chloro­n,n,n­trimethylethanaminium chloride), and paclobutrazol [(2rs, 3rs)­1­(4­chlorophenyl)­4,4­ dimethyl­2­(1h­1,2,4­triazol­1­yl) penta­3­ol] have been used in many floral and ornamental plants to inhibit stem elongation (currey and lopez, 2016). generally, ancymidol and paclobutrazol is effective when applied as a soil drench, and daminozide and chlormequat when applied as a foliar spray. germination of corylopsis seeds as affected by warm and cold stratification and the x­ray imaging to separate full seeds from empty seeds is well docu­ mented (kim et al., 2015, 2017, 2018). to produce compact and flowering corylopsis plants in small pots in one year after transplanting seedlings, selection of proper species and the most suitable cultural prac­ tices should be identified. however, there is no report on the growth and flowering of corylopsis starting from small propagules regardless of propaga­ tion methods: seed propagation, rooting of cuttings, and in vitro propagation. shortening the total pro­ duction time from 2­3 years to one year while ensur­ ing the qualities of plants at flowering from seedlings in lilium longiflorum thunb. bulbils in l. ×elegans thunb. and tissue­cultured propagules in interspecif­ ic hybrids between l. longiflorum and l. ×elegans was reviewed (roh, 1992, 1996). production of corylopsis in a small pot with inflo­ rescence will attract and enable consumers to pur­ chase at the nursery or the garden center in the early spring, and then plant them in the garden to enjoy the beauty of flowers for many years. the objectives of this research were (1) to select the species to grow starting from seeds in small pots, and to study the effect of (2) slow release fertilizer (srf) treatments, (3) the pinching frequencies, and (4) plant growth retardant treatments to produce compact seed­prop­ agated corylopsis plants in 10 cm pots in one year from transplanting seedlings. 2. materials and methods preliminary field evaluation to select a suitable species for final evaluation seeds of 45 accessions (data not presented) including c. glabrescens (na50804, longwood 1997­ 0068b, longwood chimes), c. spicata (na37208, na40102, arnold 7950a), c. pauciflora (na37205, longwood 1944­0213*h), and c. vietchiana (na37208, na65619) were sown between oct. 2 and nov. 2 and planted into 10 cm pot filled with promix bm (premier horticulture inc., quakertown, pa, usa) between mar. 1 and apr. 2, and grown in the field. the final evaluation based on the number of plants that flowered and the growth habits, c. coreana and c. sinensis var. calvescens (na 57391) (roh et al., fig. 1 ­ appearance of c. sinensis var. calvescens at anthesis pro­ duced in a 10 cm pot from seeds. clusters of flower buds (inflorescences) are well developed on long shoots. kim et al. ‐ compact corylopsis plant production 63 2008 a) were selected (table 1) for evaluation in the next three experiments. effect of slow release fertilizer treatment on growth and flowering (expt. 1) about 200 seeds each of c. sinensis var. calvescens and c. coreana were sown on oct. 20, 2009 in a 15 cm pot and received temperature treat­ ments [20oc (oct. 21 ­ dec. 1) and 5oc (dec. 2, 2009 ­ feb. 16, 2010)]. one seedling was transplanted per 10 cm pot filled with promix bm on mar. 4, 2010. on mar. 18, 2010 when seedlings formed 4 nodes, the main shoot was pinched leaving two pair of leaves. slow release fertilizer (srf; osmocote, 14 n ­ 6.2 p ­ 11.6k; scotts co., marysville, oh, usa) was applied to the surface of the growing medium at transplant­ ing seedlings at a rate of 0, 0.125, 0.25, and 0.5 g per 10 cm pot (table 2). during the culture, plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water solu­ ble fertilizer once a month. greenhouse day temperature was maintained at 21­22oc on oct. 1, 15­17oc on nov. 1, 13­14oc on nov. 16, 10­12oc on dec. 1, 7­8oc on dec. 16, 2010 and at 4­5oc on jan. 1, 21­24oc on apr. 16, 2011, and was raised by 2.5oc every 2 weeks until sept. 1, 2011. night temperature was maintained 2oc lower than the day temperature. the number of weeks to flower counted from the date of transplanting seedlings to pots, and the number and length of nodes with inflo­ rescences from the three longest shoots, and the number of nodes with 2 inflorescences was recorded from 15 plants per treatment. flowering date was recorded when two florets each from two inflores­ cences reached anthesis, and data were subjected to the regression analysis for each species using statistical analysis system program (sas, 2002). effect of pinching frequencies on growth and flower‐ ing (expt. 2) after sowing about 200 seeds as described in expt. 1, seedlings were transplanted. on mar. 18, 2010, 0.25 g of slow release fertilizer was applied to the surface of growing medium and the effect of pinching frequencies on c. sinensis var. calvescens was evaluated. shoots were either not­pinched or pinched 2 (feb. 28), 4 (apr. 18), 6 (may 2), and 8 weeks (may 16) after transplanting as outlined (table 3). to the surface of the growing medium at trans­ planting seedlings 0.8 g of slow release fertilizer per pot was applied, and plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water soluble fertilizer once a month. the number of weeks to flower, and the total number of shoots with flowers and flower buds, and the length and number of flowers from the first and second longest shoots was recorded from 15 plants per treatment. the number of days to flower was counted from the date of transplanting. data were subjected to the analysis of variance (anova) and means were compared with tukey’s honestly signifi­ cant difference (hsd) test. effect of growth retardants treatment on growth and flowering (expt. 3) corylopsis sinensis var. calvescens seeds were sown and transplanted, and pinched as described in expt. 1, and pinched again on may 26, 2010. plants were grown in greenhouse maintained at 18­ 21oc/16­19oc (day/night) and then in greenhouse maintained at 22­25oc/20­23oc until july 6. to the surface of the growing medium at transplanting seedlings 0.8 g of slow release fertilizer per pot was applied, and plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water soluble fertilizer once a month. growth retardants were applied on jul. 7, when new shoots were about 5­8 cm long. each pot was treated with 25 ml of ancymidol [0.026% active ingre­ dient (a.i.)] and paclobutrazol (0.4% a.i.) at 0, 10, 20, 40, and 80 mg/l was applied as a soil drench. daminozide (85% a.i.) and chlormequat (11.8% a.i.) at 0, 2,500, table 1 ­ evaluation of flowering and growth habit in the field c. coreana and c. sinensis var. calvescens z range and mean of flowering. y plants that produced inflorescence and the total number of plants evaluated (parenthesis). x number of plants ( parenthesis) showing upright and prostrate growth characteristics. species seed harvest 2008 2009 germi­ nation trans­ planting flowering z no. of plants y growth characteristics x c. coreana 2007 mar. 6 mar. 12 mar. 19­25 (mar. 21) 9 (21) upright (12), prostrate/upright (1) c. sinensis var. calvescens 2007 mar. 1 apr. 2 mar. 27­apr. 7 (apr. 2) 14 (16) upright (13), prostrate/upright (1) 2008 feb. 26 apr. 8 mar. 22­apr. 3 (mar. 28) 16 (16) upright (16) adv. hort. sci., 2020 34(1): 61­69 64 5,000, 7,500, and 10,000 mg/l was applied as a foliar spray, and 200 ml of solution was applied to 15 plants. on nov. 20, 2010, plants were grown in a greenhouse maintained at 4­5oc for cold treatment until mar. 1, 2011. dates of flowering, when two florets from an inflorescence reached anthesis were recorded and the lengths of two longest shoots (shoot length a) per plant and shoots longer than 3 cm were counted on jan. 16. plants were moved outdoors on mar. 27, and the new growth of two longest shoots (shoot length b) was also recorded on may 10, 2011. data collected from 15 plants per treatment were ana­ lyzed by two­way anova with plant growth retar­ dants and concentration as variables. 3. results selection of a suitable species for final evaluation following evaluation of 45 accessions including c. glabrescens,c. spicata, c. pauciflora, and c. vietchi‐ ana (data not presented), c. sinensis var. calvescens and c. coreana showing upright growth characteris­ tics of shoots and flowering response were selected­ for the final evaluation. all accessions grew taller than 1.3 m and spread over 65 cm wide in case of c. spicata, but with a few inflorescence (data not pre­ sented). the selection criteria were based on the number of plants that had flowered exhibiting upright growth characteristics. in less than one year counting from the time of transplanting, 14 from 16 c. sinensis var. calvescens plants flowered showing up­right growth (table 1). corylopsis coreana was also selected for its large foliage for its good fall foliage color, even though only nine out of 21 plants had flowered. effect of slow release fertilizer treatment on growth and flowering (expt. 1) the number of weeks to flower in 52 to 53 weeks in c. sinensis var. calvescens and c. coreana was not affected by the rate of srf treatments (table 2). the number of total shoots and of shoots with inflores­ cences increased linearly with srf treatment from 2.5 to 4.1 in c. coreana and from 2.1 to 4.1 in c. sinensis var. calvescens. the lengths of the three longest shoots also increased in both species, from 11.9 to 25.6 cm for the longest shoot, from 4.8 to 15.8 cm for the third shoots in c. coreana, and from 12.8 cm to 29.3 cm for the longest shoot in c. sinen‐ sis var. calvescens. the number of nodes with inflorescences in all the three shoots of c. coreana received 0.5 g srf treat­ ment was 0.3 or less than 0.3 and there was only one node with more than 2 inflorescences. however, the number of nodes with inflorescence produced and the number of nodes with 2 inflorescences was high­ er in c. sinensis var. calvescens than in c. coreana. the number was increased to 4.8 nodes in the first table 2 ­ the effect of slow release fertilizer treatment on the growth and flowering of corylopsis coreana and c. sinensis var. calvescens z z there was a significant difference between two species; data for each species were subjected to the linear regression analysis. y the number of weeks to flower was counted from the date of transplanting seedlings. x nodes with inflorescence that were formed on new growth by pinching. w non­significant (ns), significant at p≤0.05 (*) and p≤0.01 (**). species slow release fertilizer (g/10 pot) no. of weeks to flower y no. of total shoots length (cm) of three longest shoots (sh) no. of nodes with inflorescences x sh 1 sh2 sh 3 sh1 sh2 sh 3 c. coreana 0 52 2.5 11.9 6.7 4.8 0.1 0.1 0.0 0.125 52 3.3 14.4 9.8 7.4 0.3 0.1 0.0 0.25 53 4.6 20.3 13.6 10.1 0.1 0.0 0.0 0.5 52 4.1 25.6 22.0 15.8 0.1 0.3 0.3 regression analysis ­ linear effect w ns * ** ** ** ns ns ns c. sinensis var. calvescens 0 53 2.1 12.8 6.7 4.5 0.1 0.0 0.0 0.125 53 2.9 14.3 9.9 7.5 0.8 0.7 0.0 0.25 52 3.4 21.3 13.0 10.4 3.1 2.8 1.8 0.5 52 4.1 29.3 21.8 19.8 4.8 4.4 2.7 regression analysis ­ linear effect w ns ** ** ** ** * * * kim et al. ‐ compact corylopsis plant production 65 control. the length of the first and the second non­ pinched shoot was 49.3 and 33 cm, respectively, with a difference of 16.3 cm (table 3). however, when pinched 4 times, the lengths were 38.0 and 31.8 cm with a difference of 6.2 cm. the number of inflores­ cences in non­pinched and pinched shoots, which was 9.2 and 7.1 in the first shoot and 4.0 and 6.9 in the second shoot, respectively, did not vary signifi­ cantly. however, the difference in the number of inflorescences (0.2) between the first and the second shoot was significantly lower in the pinched shoot compared with the non­pinched shoot (5.2). in gen­ eral, when pinched, the difference in the inflores­ cences between the first and the second shoot was less than 1.9, which was significantly less than that of the control. effect of growth retardant treatments on growth and flowering (expt. 3) when plants were treated with ancymidol, chlormequat, and daminozide, flowering took 22 to 28 days regardless of treatment concentrations, which was not significantly different from that of control (table 2). however, soil drench treatments with paclobutrazol (20 mg/l or higher concentra­ tions) took longer than 36 days. flowering percent­ age was higher than 60% when plants were treated with ancymidol, chlormequat, and daminozide, regardless of treatment concentrations.treatment with 20 mg/l of paclobutrazol severely inhibited the extension of peduncle bearing inflorescence trigger­ ing the death of inflorescence immediately after shoot and to 4.4 nodes in the second shoot following treatment with 0.5 g of srf and the number of nodes with more than 2 inflorescences was also increased. c. sinensis var. calvescens treated with 0.125, 0.25, and 0.5 g srf has the potential to produce a small potted plant (fig. 2). effect of pinching frequencies on growth and flower‐ ing (expt. 2) regardless of frequencies and timing of pinching, the flowering of c.sinensis var. calvescens occurred in 53 weeks (table 3). flowering ranged between 73 and 93%, and the highest flowering rate was record­ ed when pinched for 4 times at 2, 4, 6, and 8 weeks, yielding a significantly higher number of shoots with inflorescences (5.3) and consequently the highest number of inflorescences (22.1) compared with the fig. 2 ­ appearance of the corylopsis sinensis var. calvescens plants in a 10 cm pot treated with 0.125, 0.25, and 0.5 g of slow release fertilizer per pot prior to leaf emergence and anthesis. table 3 ­ the effect of pinching frequencies on growth and flowering of corylopsis sinensis var. calvescens z not pinched (x) or pinched (o) 2 (feb. 28), 4 (apr. 18), 6 (may 2), and 8 weeks (may 16) after transplanting. y the number of weeks to flower was counted from the date of transplanting seedlings. x nodes with inflorescence induced by pinching. w non­significant (ns) or significance at p≤0.05, f­test. pinching z no. of weeks to flower y flowering % total shoots no. with inflores­ cences flowers (total no.) shoot length (cm) no. of nodes withinflorescences x 2 weeks 4 weeks 6 weeks 8 weeks first shoot second shoot difference first second shoot difference x x x x 53 87 2.9 17.2 49.3 33.0 16.3 9.2 4.0 5.2 o x x x 53 87 2.1 12.8 49.0 38.8 10.2 6.9 6.8 0.1 o o x x 54 73 2.4 13.1 47.1 35.5 11.6 6.4 4.8 1.6 o o o x 53 87 3.5 18.5 41.6 35.2 6.4 6.8 5.7 1.1 o o o o 53 93 5.3 22.1 38.0 31.8 6.2 7.1 6.9 0.2 o x o o 53 87 4.3 17.3 32.4 29.2 3.2 5.7 5.2 0.5 o o x o 54 80 3.1 10.4 37.3 26.0 11.3 4.7 3.9 0.8 o x o x 53 73 3.3 14.8 39.5 29.8 9.7 6.5 6.3 0.2 o x x o 53 87 3.3 16.3 39.0 22.2 16.8 7.6 5.7 1.9 level of significance w hsd at p<0.05 ns ­ 0.94 3.58 8.25 5.61 5.82 4.27 3.84 1.59 66 adv. hort. sci., 2020 34(1): 61­69 emergence following leaf emergence (fig. 3). therefore, days to flower were estimated on the date of leaf emergence. flowering percentage was significantly reduced to less than 30% when plants were treated at 20 and 40 mg/l paclobutrazol. when shoot lengths following 80 mg/l ancymidol were recorded on jan. 16 (26 weeks after growth retardant treatment), the length of the first and the second longest shoots was significantly reduced from 26.3 cm to 15.6 cm and from 16.4 cm to 12.2 cm, respectively (table 4). the length of the two longest shoots treated with daminozide and chlormequat showed similar trends as observed in plants treated with ancymidol. the length of the two longest shoots was significantly reduced to 12.9 cm and 9.8 cm following treatment with 10 mg/l paclobutrazol (fig. 3), responding to the quadratic effect of concentrations. the length of shoot b showing new growth on may 10 was signifi­ cantly inhibited to less than 5.0 cm when treated with paclobutrazol. the number of shoots longer than 3 cm varied from 4.8 to 5.7, from 3.8 to 4.8, 4.3 to 5.5, and 5.0 to 3.9 upon treatment with ancymi­ dol, chlormequat, daminozide, and paclobutrazol, respectively. the numbers were not affected by con­ centrations of these three retardants (data not pre­ sented). 4. discussion and conclusions successful acclimatization rate of in vitro propa­ gated c. coreana was low (moon et al., 2002) and limited time of the season to propagate by rooting of cuttings (kwon et al., 2011) are the limiting factors for mass propagation to secure sufficient and uni­ form propagules for experiments, and further, reports are not available on flowering of in vitro propagules and rooted cuttings. this clearly indicates that seeds can be used as a propagule to produce sufficient number of seedlings to produceflowering plants in a year from transplanting seedlingsby vari­ ous cultural practices reported in this study. the mor­ phological characteristics of c. sinensis var. calvescens are suitable to produce in 10 cm pots compared with c. coreana, if stem elongation can be controlled and many shoots with well­developed inflorescences can be formed (fig. 1). growth and flowering as influenced by slow release fertilizer (srf) there is a clear difference between c. coreana and c. sinensis var. calvescens responding to srf application per 10 cm pot. responding to the increased rates of slow release fertilizer, especially at 0.5 g srf application, and c. sinensis var. sinensis is recommended to produce as a 10 cm potted plant in one year as the number of inflorescences and of nodes with more than two inflorescences are increased. production of c. coreana may not be rec­ ommended due to fewer numbers of nodes with inflorescences and only 0.1 node produced more than 2 inflorescences. growth and flowering as influenced by pinching and growth retardant treatments stem length is one of the limitations to produce compact c. sinensis var. calvescens in small pots, which can be reduced either by pinching or growth retardant treatments. manual or mechanical pinch­ ing is associated with increased labor costs. treatment with growth retardant may not induce branching when compared with pinching. pinching shoots four times in 2, 4, 6, and 8 weeks prior to may 2 is an effective cultural practice to pro­ duce compact plants for small pots without affecting days to flowering and flowering percentage. the increase in the number of shoots with inflorescences, the total number of inflorescences, and the number of nodes with inflorescences may result from an increased number of shoots that are formed prior to the development of inflorescence, which may occur fig. 3 ­ corylopsis sinensis var. calvescens in a 10 cm pot treated with 25 ml of 40 mg/l paclobutrazol (a). blasted inflore­ scences (arrow) and emerging of dark green leaves of reduced size indicate excessive doses of paclobutrazol (b). photographed on mar. 27, 2011. kim et al. ‐ compact corylopsis plant production 67 after may 2. since the time of floral bud initiation has not been examined anatomically, it requires further studies. increase in the number of lateral shoots and flowers were increased as the pinching frequencies in sedum rotundifolium d.b. was increased (jeong, 2000). the length of shoots exceeding 30 cm follow­ ing pinching is considered excessive for producing corylopsis in a 10 cm pot. flowering of c. sinensis var. calvescens plants treated with ancymidol, chlormequat, and daminozide that produced higher than 60% plants with inflorescences regardless of treatment concen­ trations did not differ significantly from that of con­ trol. the longest shoot length (a) on jan. 16 respond­ ing linear effect to ancymidol, chlormequat and daminozide was the shortest, especially when treat­ ed with 80 ppm ancymidol. ancymidol is,therefore, recommended for c. sinensis var. calvescens in 10 cm pot. since the length of new shoot (b) on may 16 was not affected, the effect of these three plant growth table 4 ­ growth and flowering of corylopsis sinensis var. calvescens as influenced by growth retardant treatments plant growth regulator no. of days to flower (mar. 1) (flowering percent) shoot lenght a (cm) (jan. 16, 2011 z) shoot length b (cm) of new growth (may 10, 2011) fir y sec fir sec ancymidol (soil drench) (mg/l) 0 22 (60) 26.3 16.4 21.0 20.0 10 23 (80) 28.4 17.5 20.2 19.4 20 24 (70) 27.9 21.7 20.2 20.0 40 25 (70) 23.9 19.1 21.9 17.9 80 25 (60) 15.6 12.2 16.5 16.3 regression analysis ns l* l* ns ns paclobutrazol (soil drench) (mg/l) 0 22 (60) 24.7 20.0 24.0 20.0 10 28 w (50) 12.9 9.8 5.0 3.5 20 38 w (30) 11.2 8.1 1.7 1.2 40 36 w (10) 10.3 7.9 1.3 0.8 80 36 w (30) 11.0 8.5 1.1 0.6 regression analysis x ­ q** q** q** q** chlormequat (foliar spray) (mg/l) 0 22 (70) 28.9 20.3 23.1 20.9 2.5 22 (80) 35.1 24.9 22.9 20.1 5 25 (70) 26.5 20.0 18.3 18.1 7.5 27 (60) 23.7 14.5 17.9 17.0 10 25 (70) 24.3 18.5 20.2 19.9 regression analysis ns l* ns ns ns daminozide (foliar spray) (mg/l) 0 23 (60) 29.2 23.0 21.0 19.9 2.5 27 (80) 33.3 23.5 23.5 18.5 5 25 (70) 28.5 22.0 22.5 18.9 7.5 25 (60) 29.3 22.7 22.7 19.1 10 27 (70) 25.5 19.5 21.2 18.5 regression analysis ns l* ns ns ns level of significance v growth retardant (pgr) ns *** *** *** *** concentratio ns ** * * * pgr × concentration ns ** ** ** ** z data collected from over­wintered plants. y length of the first (fir) and second (sec) longest shoots. x analysis was not carried out due to the estimated days of flowering and low flowering percentage, and regression analysis was per­ formed for each growth retardant. linear (l) and quadratic (q) effect. w days to flower following paclobutrazol treatment were recorded upon leaf emergence following the death of inflorescence. generally, about 15 days elapsed between flowering and the appearance of leaf emergence. v non­significant (ns) or significance at p<0.05 (*), 0.01 (**) or 0.001 (***), f­test. adv. hort. sci., 2020 34(1): 61­69 68 retardants may not last long when compared with paclobutrazol. a single application of ancymidol is not effective and may require two treatments to pro­ duce quality mussaenda ‘queen sirikit’ as a short­ stemmed potted plant without reducing the number of flowers per plant and delaying the flowering (cramer and bridgen, 1998). application of ancymidol requires further testing since growth retarding effects of ancymidol do not persist as reported in l. lancifolium thunb. (roh, 1979) and shoot length is increased under a long day photoperiod during june or july as observed in l. longiflorum (roh and wilkins, 1977). soil drench treatment with paclobutrazol 20 mg/l or higher concentrations which took longer than 36 days to flower (table 4) compared with 22 days with the control. when treated with 20 mg/l of paclobu­ trazol, the extension of peduncle­bearing inflores­ cence was severely inhibited resulting in the death of inflorescence immediately following leaf emergence, thus lowering the flowering rate from 60% to 10% (fig. 3). shoot length was arrested under any paclobutrazol treatment which is undesirable. although 10­20 mg/l paclobutrazol as a soil drench is considered effective to reduce shoot length, the growth of new shoots and inflorescence development is significantly arrested even a year later, which may require double treatments at low concentrations, i. e., 5 mg/l to avoid severe growth retardation and malformation of inflorescence. a sin­ gle foliar spray of 500 mg/l paclobutrazol may be used to test produce compact flowering plants as reported in rhododendron hybrids, which is a woody ornamental (wilkinson and richards, 1991). generally paclobutrazol was not effective in mussaenda at 0.125­0.25 mg a.i. per pot as a soil drench compared with ancymidol and daminozide (cramer and bridgen, 1998), which is considered effective in producing compact plants with accelerat­ ed flowering although 0.4 g a.i. per pot increased the number of flowers, producing malformed and unac­ ceptable of rhododendron ‘sir robert peel’ (wilkinson and richards, 1991). shoot length of poin­ settia (euphorbis pucherrima wild. ex klotzch) was reduced by daminozide and chlormequat treatments without affecting the flowering (lewis et al., 2004). combined treatment with chlormequat and daminozide can also be considered as reported to be effective to retard stem elongation of zonal (cutting) geraniums [pelargonium ×hortorum (l.h. bailey)] (tayama and carver, 1990). due to the long­lasting inhibitory effect of paclobutrazol on shoot elongation and reduction in leaf size when applied as a soil drench, a malforma­ tion of inflorescence and formation of inflorescences after leaf emergence result in a lower percentage of plants with inflorescences in both species in this study and in other woody ornamentals such as dissotis rotundifolia (sm.) triana and tibouchina forthergillae ×pilosa (hawkins et al. , 2015). therefore, paclobutrazol is not recommended to use as a soil drench in corylopsis. the optimum dosages require further study comparing the effect of soil drench and foliar spray. a spray treatment of paclobutrazol may be considered as the quality of dianthus caryophyllus l., cv. mondriaan was improved (bañón et al., 2002). this is the first report providing practical and hor­ ticultural strategies to produce flowering corylopsis plants in small pots with a great potential to utilize under­utilized native plants as ornamental and nurs­ ery plant starting from seeds. corylopsis sinensis var. calvescens indigenous to china is a suitable species as compared to c. coreana native to korea to pro­ duce from seeds with application of slow release fer­ tilizer at 0.5 g per pot and pinching for four times at 2­week interval before may 16 to reduce shoot elon­ gation, to increase flowering percentage, and to accelerate flowering with increased number of inflo­ rescences. treatments with ancymidol as a soil drench and daminozide and chlormequat as a foliar spray at all concentrations evaluated in this study were not effective to produce compact plants as compared to paclobutrazol treatment. soil drench treatment with paclobutrazol at 10­20 mg/l is considered effective in reducing shoot elongation the first year. however, the inhibitory effects of paclobutrazol last longer than a year, and appear resulting in malformation of inflorescences in the second year. therefore, investi­ gation on selecting appropriate treatment methods andconcentrations of paclobutrazol to reduce shoot elongation without triggering malformation of inflo­ rescence is needed. the time of floral initiation and development in relation to pinching treatment needs to be determined as well. references bañón s., gonzalez a., cano e.a., franco j.a., 2002 ­ growth, development and colour response of potted dianthus caryophyllus cv. mondriaan to paclobutrazol treatment. ­ scientia hort., 94: 371­377. kim et al. ‐ compact corylopsis plant production 69 bean e., anisko t., 2014 ­ captivating corylopsis. ­ https://www.amerinursery.com/growing/captivating ‐ corylopsis/. cramer c.s., bridgen m.p., 1998 ­ growth regulator effects on plant height of potted mussaenda ‘queen sirikit’. ­ hortscience, 33: 78­81. currey c.j., lopez r.g., 2016 ­ commercial greenhouse and nursery production. applying plant growth retar‐ dants for height control. ­ purdue extension. hd­248­ w. purdue university. https://www. extension.pur­ 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edu/china/mss/volume09/hamamelidaceae.pdf. http://https://www.amerinursery.com/growing/captivating-corylopsis/ http://https://www.amerinursery.com/growing/captivating-corylopsis/ http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://hortsci.ashspublications.org/search/?author1=r.i.+wilkinson&sortspec=date&submit=submit impaginato 15 adv. hort. sci., 2023 37(1): 15­23 doi: 10.36253/ahsc­13850 effects of cold storage on quality para­ meters and nutraceutical compounds of pomegranate fruits (cv. acco) a. cirillo 1 (*), a. magri 2, 3, m. petriccione 2, c. di vaio 1 1 department of agricultural sciences, university of naples federico ii, via università, 100, 80055 portici (na), italy. 2 research centre for olive, fruit and citrus crops, council for agricultural research and economics (crea), via torrino, 3, 81100 caserta, italy. 3 department of environmental, biological and pharmaceutical sciences and technologies, university of campania luigi vanvitelli, via vivaldi, 43, 81100 caserta, italy. key words: bioactive compounds, cold storage, punica granatum l., quality fruit. abstract: punica granatum l. contains several bioactive compounds with antioxidant activity that have a positive effect on human health. this study aims to investigate the changes in the chemical­physical and qualitative para­ meters of pomegranate fruits cv. acco from harvest up to +90 days of cold stor­ age (+4°c and 95% rh). morphological parameters, juice yield, weight loss, total soluble solids content (tss), ph, titratable acidity, the color of the epicarp (l*, a*, b*), content of polyphenols, anthocyanins, flavonoids, and antioxidant activity were analyzed. the results showed an increase (about 29%) in the juice content (%) at +60 days of cold storage. cold storage has also shown positive effects on some bioactive compounds. flavonoids and anthocyanins content increased from 287.98 mg ce/100 ml of juice to 389.23 mg of ce/100 ml of juice and from 8.32 to mg/100 ml of juice to 11.13 mg/100 ml of juice at + 90 days of cold storage, respectively. on the basis of our results that confirmed the litera­ ture data, the pomegranate fruit is rich in bioactive compounds that exert ben­ eficial actions on human health, and it has also been demonstrated that such nutraceutical compounds increased during cold storage, allowing the fruit to be preserved a long term. 1. introduction the pomegranate (punica granatum l.) is generally cultivated in the mediterranean basin, in the regions of southern asia, in india and in north and south america, where long and hot summer favor an optimal fruits ripening (erkan and dogan, 2018). its adaptation to the mediterranean cli­ mate has favored its spread in several countries giving rise, over the cen­ turies, to numerous local genotypes. the fruits are generally harvested when fully ripe and displayed a smooth shining leathery skin with a color varies from green, to pink, reddish, or dark red (love et al., 2014). the pomegran­ (*) corresponding author: aurora.cirillo@unina.it citation: cirillo a., magri a., petriccione m., di vaio c., 2023 ­ effects of cold storage on quality para‐ meters and nutraceutical compounds of pome‐ granate fruits (cv. acco). ­ adv. hort. sci., 37(1): 15­23. copyright: © 2023 cirillo a., magri a., petriccione m., di vaio c. this is an open access, peer reviewed arti­ cle published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 october 2022 accepted for publication 11 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13850 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 15­23 16 ate fruits have a low respiration rate and a non­climac­ teric respiratory pattern (ben­arie et al., 1984). the edible part of the fruit is called arils and constitutes about 52% of total fruit (w/w), comprising 78% juice and 22% seeds. the fresh juice contains 85.4% mois­ ture and considerable amounts of total soluble solids (tss), total sugars, reducing sugars, anthocyanins, phe­ nolics, ascorbic acid and proteins (el­nemr et al., 1990) and has also been reported to be a rich source of antioxidants (gil et al., 2000; kulkarni et al., 2004). the pomegranate is highly valued for health­promoting benefits of its fruit and processed product as demon­ strated by numerous in vivo and in vitro studies (de nigris et al., 2007; bell and hawthorne, 2008; davidson et al., 2009). the consumers pose more attention to the nutraceutical value of fruits, in fact several studies aim to valorize this aspect, as reported by graziani et al. (2020, 2021). the recent growing awareness of con­ sumers to health aspects of fresh fruits and processed products greatly increased the interest in consumption of this fruit and its processed products; consequently, worldwide pomegranate production expanded consid­ erably. apart from its demand as fresh fruit and juice, the processed products such as carbonated drinks, syrup, wine, and candy are also gaining importance in world trade (dak and pareek, 2014). the fruit juice of pomegranate was found to have an exceptionally high antioxidative capacity (reddy et al., 2007; cirillo et al., 2022). the shelf­life of pomegranate is about 12­14 days at ambient conditions (naveena et al., 2008); but cold storage with recommended temperature from 0 to 10°c can be used from 2 weeks to 5 months influ­ encing in different manner cultivars storability (ehteshami et al., 2019). storage method is very impor­ tant because physiological and enzymatic processes cause the loss of quality with browning of the skin, necrotic pitting, pallor of the arils that depreciate the product during storage (fawole and opara, 2013 a; dorostkar and moradinezhad, 2022). in the present study, cv. acco pomegranate storability, one of the main cultivars marketed in italy, was analyzed, evaluat­ ing physical­chemical and biochemical changes during refrigerated storage up to 90 days from harvest to define the maximum storage time so that the fruits are still appreciated by consumers both organoleptic and nutritional point of view. 2. materials and methods experimental orchard and sampling the experiment was conducted in 2019 in eboli (salerno, italy) (40° 33’ 29” n; 14° 58’ 28” e a 15 m a.s.l.) at “improsta” regional experimental farm, where were cultivated the pomegranate trees of cv. acco. the trees were trained to sapling system and spaced 3.5 m on the rows and 4 m between the rows. pomegranate fruits were hand­harvested 3th september 2019 according to a randomized block design from homogeneous trees, for grown and pro­ duction load. fruits (n=120) were collected, and 12 lots (10 fruits each) were prepared. three lots were analyzed at harvest while the others were stored in the cold chamber at a temperature of 4±1°c and 95±1% relative humidity, and analyzed after 30, 60 and 90 days of storage. in this work, 0 indicates the harvest time and +30, +60 and +90 the days of refrig­ erated storage. pomological and physico‐chemical traits of fruits pomological and physico­chemical traits of the fruits were carried out at the pomology laboratory of the department of agriculture of the university of naples “federico ii”. fruit and seed weight (g) was determined by an electronic digital balance (precisa instruments ag, model xb220a, dietikon, switzerland) to monitor the weight loss of fruits dur­ ing cold storage. equatorial diameter (mm) and fruit length without calyx (mm) were measured by electric digital caliper with ±0.01 mm accuracy (mitutoyo, kawasaki, japan). color of epicarp was determined with a colorimeter (minolta, model cr­400, tokyo, japan) that was capable of quantifying colors accord­ ing to international standards and expressed in defined color spaces. the instrument was calibrated with “white” managed by the light source on a white tile, before each measurement. the l* a* b* (cielab) color space is the most common method for measur­ ing the color of an object or materials of different ori­ gins and it is widely used in all sectors. in this color space, l* indicates brightness, while a* and b* the chromaticity coordinates: +a* is the direction of red, ­ a* is the direction of green, +b* is the direction of yellow, and ­ b* is the direction of blue. the measur­ ing was repeated four times in different points of the fruit. the pomegranate seeds were hand­separated from the epicarp and carpellary membranes, count­ ed, and squeezed using a small press. a juice yield of 300 g of pomegranate seeds was measured and expressed as a percentage (w:v). juice (200 ml) was stored at ­20°c and afterwards used for the evalua­ tion of physico­chemical and nutraceutical traits. the ph, titratable acidity (ta) and total soluble solids (tss) were assessed on the arils juice. the tss con­ https://www.sciencedirect.com/science/article/pii/s0304423819301578" /l "! cirillo et al. ‐ quality parameters of pomegranate fruits during cold storage 17 tent was determined with a hi 96.814 digital refrac­ tometer of hanna instruments and results were expressed as °brix. the ph was determined with a ph meter by the hanna instruments laboratory and total acidity was evaluate by acid­base titration, with 0.1n sodium hydroxide standard solution and the results were expressed as g citric acid 100 ml­1. determination of total phenolic content total phenolics content in juice was determined according to a folin­ciocalteu procedure (singleton and rossi, 1965). the assay was carried out in dupli­ cate for each sample using 5 µl of extract, 100 µl of folin­ciocalteu reagent and 300 µl of 7.5% (w/v) na2co3 solution; the mixture assay was left in the dark for 2 hours at room temperature, then the absorbance was determined at the wavelength of 765 nm the results were expressed as mg of gallic acid equiva­ lent/100 ml of juice (mg of gae/100 ml of juice). determination of flavonoids content the assay was carried out according to zhishen et al. (1999) by the aluminum chloride colorimetric method using 20 µl of juice and the absorbance was determined at 510 nm. results were expressed as mg of catechin equivalent/100 ml of juice (mg ce/100 ml of juice). determination of anthocyanins content the assay was carried out according to magri et al. (2020) by a ph­differential method using 50 µl of juice in kcl ph 1 and ch3coona ph 4 buffer. the absorbances at 510 and 700 nm were determined. results were expressed as mg of cyanidin­3­glucoside equivalent/100 ml of juice (mg c3g/100 ml of juice). antioxidant activity the assay was conducted as described by petriccione et al. (2015) using 20 µl of juice and 1480 µl of 1,1­diphenyl­2­picril­hydrazyl (dpph). the change in absorbance was observed at 515 nm and the results were expressed as mg of trolox equiva­ lent/100 ml of juice (mg teq/100 ml of juice). statistical analysis analysis of variance (anova) on the complete randomized block design on the data and mean sepa­ ration by duncan’s multiple range test (p<0.05) and principal component analysis (pca) were carried out using xlstat, version 2013, statistical software pack­ age (new york, ny, usa). 3. results fruit pomological characterization the changes of the pomological parameters in the cv. acco pomegranate fruits during the refrigerated storage at + 30, + 60 and + 90 days are shown in table 1. there is a significant reduction in the fruit weight loss about 10.5% already after 30 days of cold stor­ age, up to 23.7% at +90 days, while no significant dif­ ferences are highlighted for the equatorial diameter, the fruit length, and the seeds weight. one of the most important commercial parameters is the juice content in the seeds, our study showed a significant increase in the percentage of juice from collection to storage in the fridge, after +60 days about 63.5% compared to harvest (table 1). juice quality parameters and fruit color characteriza‐ tion during cold storage the results of the physico­chemical properties of cv. acco during cold storage are shown in table 2. a significant reduction in total soluble solids (tss) was highlighted, from 14.25°brix at harvest to 11.85°brix already at +60 days of refrigerated storage, up to a table 1 ­ pomological parameters (fruit weight, equatorial diameter, fruit length, seed weight (n=100), % weight loss, % juice) at harvest and during the refrigerated storage (+30, +60, +90 days) of pomegranate fruits (cv. acco). 0 days is the time of harvest all the data are expressed as mean ± se (standard error). the same letter indicates not significant differences according to duncan’s mul­ tiple range test (p<0.05). level of significance at the anova are indicated as ns (not significant), * (0.01<p <0.05), ** (0.01> p> 0.001), and *** (p <0.001). days fruit weight (g) equatorial diameter (mm) fruit length (mm) seed weight (g) weight loss (%) juice (%) 0 216.38 ± 9.86 a 80.38 ± 1.53 a 63.5 ± 4.12 a 113.88 ± 5.76 b 0 ± 0.00 a 47.60 ± 0.47 c 30 187.18 ± 6.68 b 78.63 ± 0.92 a 65.5 ± 0.79 a 111.44 ± 4.86 b 10.5 ± 0.85 b 54.43 ± 1.55 b 60 171.27 ± 8.84 b 80.31 ± 1.35 a 67.0 ± 1.32 a 113.38 ± 6.70 b 19.6 ± 1.76 bc 63.50 ± 1.69 a 90 167.13 ± 7.48 b 80.88 ± 1.15 a 69.56 ± 0.73 a 119.87 ± 4.70 b 23.7 ± 2.10 c 61.35 ± 1.63 a significance *** ns ns ns ** *** adv. hort. sci., 2023 37(1): 15­23 18 final reduction of about 25.75% at +90 days while the ph showed an increase at +60 days of refrigerated storage equal to 17.66%. color coordinates (l*, a*, b*) of pomegranate peel during the refrigerated stor­ age are shown in table 2. l* and fruit peel redness (a*) showed a reduction with progressed refrigerated storage. the highest a * index was found at harvest (47.14), while after + 90 days of refrigerated storage this parameter was reduced to 40.03. b* was 26.98 at harvest while after + 30 days of refrigerated stor­ age a reduction of about 23.42% was observed, with­ out significant differences up to 90 days of storage. content of bioactive compounds and antioxidant activity during cold storage the current use of pomegranate fruit regards especially the nutritional and, still potential, health benefits that come out from the various parts com­ posing this one (carpellary membranes, arils, seeds and bark). indeed, the phytochemical composition of the fruit abounds in compounds (flavonoids, ellagi­ tannins, proanthocyanins, mineral salts, vitamins, lipids, organic acids) presenting a significant biologi­ cal and nutraceutical value. table 3 shows the bioac­ tive compounds of the pomegranate fruits cv. acco during the refrigerated storage. the polyphenol con­ tent did not display significant differences up to 60 days of storage, with average values of 272 mg gae/ 100 ml of juice, while at 90 days an increase in the polyphenol content was shown up to a value of 389.23 mg gae/100 ml juice; the flavonoid content showed a small increase at 30 days of storage (124.58 mg ec/100 ml juice), without statistical sig­ nificance, for the other two periods considered with an average content of 103 mg ce/100 ml juice; the anthocyanin content displayed a slight decrease up to 60 days of refrigerated storage with average val­ ues of about 3 mg c3g/100 ml of juice, followed by an increase after 90 days, up to 11.13 mg c3g/100 ml of juice. the antioxidant activity of pomegranate table 2 ­ total soluble solids (tss), titratable acidity (ta), ph and color attributes (l*, a* and b*) at harvest and during cold storage (+30, +60 and +90 days) of pomegranate fruits (cv. acco). 0 days is the time of harvest table 3 ­ total polyphenol, anthocyanins, flavonoids content and antioxidant activity (d) at harvest and during cold storage (+30, +60 and +90 days) of pomegranate fruits (cv. acco). 0 days is the time of harvest all the data are expressed as mean ± se (standard error). the same letter indicates not significant differences according to duncan’s mul­ tiple range test (p<0.05). level of significance at the anova are indicated as ns (not significant), * (0.01<p <0.05), ** (0.01> p> 0.001), and *** (p <0.001). all the data are expressed as mean ± se (standard error). the same letter indicates not significant differences according to duncan’s mul­ tiple range test (p<0.05). level of significance at the anova are indicated as ns (not significant), * (0.01<p <0.05), ** (0.01> p> 0.001), and *** (p <0.001). days tss (brix°) ta (g citric acid 100 ml­1) ph l* a* b* 0 14.25 ± 0.25 a 8.75 ± 0.25 a 3.00 ± 0.00 b 47.08 ± 0.66 a 47.14 ± 0.88 a 26.98 ± 0.49 a 30 13.78 ± 0.09 a 8.10 ± 0.11 a 3.10 ± 0.09 b 40.51 ± 1.68 b 43.47 ± 1.32 b 20.66 ± 0.62 b 60 11.85 ± 0.72 b 6.60 ± 0.14 b 3.53 ± 0.07 a 39.09 ± 0.87 b 44.14 ± 0.55 b 20.63 ± 0.74 b 90 10.58 ± 0.21 c 5.63 ± 0.31 c 3.63 ± 0.07 a 41.21 ± 1.08 b 40.03 ± 0.56 c 20.68 ± 0.75 b significance *** *** *** *** *** *** days polyphenols (mg gae/100 ml juice) anthocyanins (mg c3g/100 ml juice) flavonoids (mg ce/100 ml juice) antioxidant activity (µmol te/ 100 ml juice) 0 287.98 ± 8.86 b 8.32 ± 1.67 a 99.49 ± 3.25 a 271.63 ± 4.13 a 30 285.25 ± 26.07 b 3.53 ± 2.67 b 124.58 ± 9.27 a 268.11 ± 5.56 a 60 244.15 ± 23.92 b 2.72 ± 3.36 b 102.99 ± 1.90 a 267.67 ± 3.49 a 90 389.23 ± 29.01 a 11.13 ± 4.71 a 104.21 ± 10.46 a 267.97 ± 3.21 a significance * ** ns ns cirillo et al. ‐ quality parameters of pomegranate fruits during cold storage 19 juice showed no significant differences during the refrigerated storage with average values of 268 µmol te/100 ml juice. principal component analysis (pca) to obtain a broad overview of all parameters eval­ uated in cv. acco fruit following the refrigerated stor­ age a principal component analyses (pca) was con­ ducted. figure 1 shows the pca of the changes of nutraceutical compounds and qualitative parame­ ters, during refrigerated storage. the first two princi­ pal components (pcs) disclosed 88.39% of the cumu­ lative variance with pc1 detailing for 55.55% and pc2 for 32.83%. at harvest this cultivar showed a higher antioxidant activity, while a higher flavonoids content is shown between +30 and +60 days of refrigerated storage, at +90 days of refrigerated storage a higher polyphenols and anthocyanins content was highlight­ ed. the qualitative parameters showed higher values at +30 days of refrigerated storage for tss and ta, while at + 60 days there was a higher juice yield. 4. discussions and conclusions both fresh market and processing industry drive pomegranate consumption, and it is crucial to acknowledge all fruit characteristics to not only clas­ sify varieties from a botanical point of view, but also to meet the current market demand for quality fruits (martìnez et al., 2006). in the recent years, the demand and the consumption of pomegranate fruit have shown an extensive growth in several countries worldwide becoming a valuable commodity due to high levels of nutraceutical compounds and greater awareness of health­promoting benefits (asgary et al., 2022). the major postharvest losses can occur among harvest and consumption affecting either fruit quantity or quality at any stage in the postharvest chain. freshly harvested agricultural products are a living thing that breathes and undergoes changes during postharvest handling (kiaya, 2014; erkan and dogan, 2018; siddiqui et al., 2022). postharvest dis­ eases caused by bacteria, fungi, and other microor­ ganisms are compromised factors and a consistent problem for long term storage of pomegranate fruits (munhuweyi et al., 2016; akhila et al., 2022; gurtler and garner, 2022). these spoilages greatly affect the appearance, aroma and taste of fruits and the control of postharvest diseases represents the most signifi­ cant economic challenges in agriculture. high water and sugars content combined with soft and delicate texture make pomegranate fruits susceptible to weight loss, mechanical damage, and attack of pathogens (sayarri et al., 2012). physiological and enzymatic disorders are responsible to qualitative decay and storage life in pomegranate like fruit wrin­ kling, browning, and drying of skin and seeds, seeds paleness and the pathogens often that cause damage to the tissues, thereby making the fruit unsaleable (caleb et al., 2012 a; nazoori et al., 2022). as previ­ ously mentioned, an important aspect is the juice content. our results reported an increase in the juice content during refrigerated storage most likely due to the “softening” of the tissues of the arils increasing the extraction of the juice. at harvest, pomological and physico­chemical traits in cv. acco fruit showed a slight difference compared to what is reported by ferrara et al. (2014); these differences may be ascribed to different cultural practice, climatic and soil conditions (fadavi et al., 2005; ferrara et al., 2014). weight loss is one of the major problems asso­ ciated with stored pomegranate fruit which cause hardening of the skin and browning of the rind and seeds (artés et al., 2000 a; caleb et al., 2012 b; pareek et al., 2015). several studies have demon­ strate that weight loss increased with increasing tem­ perature and prolonged storage in different pome­ granates cultivars and it is due to water being lost through natural porosity of the skin (al­mughrabi et al., 1995; al­yahyai et al., 2009; wasker, 2011; fawole and opara, 2013 b). our results, on the physi­ co­chemical parameters of pomegranate fruits during cold storage, are in agree with those reported in the fig. 1 ­ principal component analysis (pca) based on quality parameters and bioactive compounds at harvest and during cold storage (+30 +60 and +90 days) of pomegranate fruits (cv. acco). 0 is the time of harvest. 20 adv. hort. sci., 2023 37(1): 15­23 literature where a reduction in tss and ta is shown. according to a similar study on ‘mollar’ pomegranate after only 7 days of storage at 4°c, the tss content is reduced about 11% (gil et al., 1996), while artes et al. (2000 b) showed a significant reduction in the acidity of pomegranate fruit juice in cv. molla de elche stored at 5°c for 90 days and subsequently, held at 20°c for six days. the decrease in tss content could be a result of the degradation of sugars with prolong storage period and the changes in ta levels are strong indications of the ongoing metabolism in the fruit during storage since pomegranate is a non­ climacteric fruit (fawole and opera, 2013 a). the decrease in fruit acidity can be attributed to the cumulative effects of the increase in juice content and the use of organic acids which act as a substrate for cellular respiration that occurs during fruit ripen­ ing (diakou et al., 2000). organic acids present in pomegranate include citric, malic, acetic, fumaric, tartaric and lactic acid, but citric acid is the main one which represents the titratable acidity of pomegran­ ate fruits (melgarejo et al., 2000). the results obtained on the peel color changes during refrigerat­ ed storage are in agreement with the results obtained by other studies, where similar storage con­ ditions on ‘ganesh’ pomegranate fruit induced the slight change in stored fruit color over a 12­week duration (nanda et al., 2001). furthermore, during cold storage our findings highlighted a reduction of fruit lightness and a darker and saturated red color of the skin. in addition to being appreciated for its quali­ tative aspects the pomegranate is known for the high nutritional value of its fruits and for its beneficial effects on health, and the medicinal properties of the different parts of the tree are also well known. the main phytochemicals responsible for these beneficial health effects are polyphenols which include ellagic acid, ellagitannins (eg punicalagin), punicic acid, anthocyanins, flavonols, flavan­3­oils and flavones. ellagic acid can be free or condensed with different sugars (glucose, rhamnose and arabinose), with dif­ ferent concentrations between various cultivars (zaouay et al., 2012). although ellagitannins are the main polyphenols in pomegranates, punicalagin and punicalin are the compounds most characterized for their antiatherogenic properties (seeram et al., 2005). anthocyanins are the pigments responsible for the typical pomegranate fruit color and include del­ phinidin, cyanidin and pelargonidin 3­glucoside and 3,5­diglucosides. the changes in the composition and concentration of anthocyanins have been shown in the different cultivars, the main anthocyanin in the spanish sweet pomegranate cultivar “mollar de elche” is cyanidin 3­glucoside, while cyanidin 3,5­ diglucoside has been found as the main compound in sour pomegranate cultivars (sayyari et al., 2011 a, b). several studies highlighted the changes in the bioac­ tive compounds content during the refrigerated stor­ age of the pomegranate (zaouay et al., 2012; ehteshami et al., 2020). fawole and opara (2013 a) have shown that the polyphenol content did not vary during the first 4 weeks of storage in the ‘bagwa’ fruit but increased after 8 weeks due to an accumula­ tion of anthocyanins; variations of bioactive com­ pounds during cold storage are also shown in apples fruits (graziani et al., 2020). in our study, no decrease in polyphenol content was observed during refrigerated storage in agreement to baltacioglu et al. (2011) and this is attributed to the action of oxida­ tive enzymatic activities following low temperature stress (ehteshami et al., 2020). the anthocyanin con­ tent in the pomegranate showed different trends related to the cultivars and storage temperatures (fawole and opara, 2013 a), which suggests that varietal differences represent a key factor in the post­harvest biosynthesis of anthocyanins (turfan et al., 2011). furthermore, several studies have shown that the antioxidant activity is linked to the bioactive compounds content and that a decrease in this activ­ ity is observed in the pomegranate during refrigerat­ ed storage after 4 weeks compared to harvest (fawole and opara, 2013 a). our study showed dur­ ing refrigerated storage a significant weight loss, while the juice percentage increased, with maximum values at +60 days. acidity (ta) and tss decreased during refrigerated storage, but the reduction in acidity was more significant than that of tss, there­ fore overall good organoleptic characteristics were showed. the polyphenols and anthocyanins increased during refrigerated storage, while the flavonoids and antioxidant activity were constant. on basis of our results suggest that pomegranate fruits cv. acco have a maximum storage time in the fridge of 60 days for fresh consumption while they can exceed 60 days to produce the juice, as the chemical­ nutraceutical characteristics remain optimal. during the cold storage the pomegranate fruits of the cv. acco cultivar did not show the appearance of plant diseases (molds or rot fungi), thus allowing a good conservation of the product. cirillo et al. ‐ quality parameters of pomegranate fruits during cold storage 21 acknowledgements the authors are grateful to doctor mariangela scarano for her technical support in the laboratory and in field. references akhila p.p., sunooj k.v., navaf m., aaliya b., sudheesh c., sasidharan a., sabu s., mir s.a., george j., khaneghah a.m., 2022 ­ application of innovative 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and their scav‐ enging effects on superoxide radicals. ­ food chem., 64: 555­559. impaginato 371 adv. hort. sci., 2021 35(4): 371­381 doi: 10.36253/ahsc­11517 histological and physiological changes of potato starch derived from seed and tps (true potato seed) grown tubers under different cold storage duration t. taufique 1, 2, t. nishizawa 1, t.s. roy 2, k. meiko 1, r. chakraborty 2, m. mostofa 3 (*), k. nara 1 1 department of bioproduction, faculty of agriculture, yamagata university, 1‐23 wakaba machi, tsuruoka 997‐8555, yamagata, japan. 2 department of agronomy, faculty of agriculture, sher‐e‐bangla agricultural university, dhaka 1207, bangladesh. 3 institute of seed technology, sher‐e‐bangla agricultural university, dhaka 1207, bangladesh. key words: amylopectin, cold storage period, potato, starch granule size, sugar content. abstract: starch granules in potato tubers exist with varying sizes and size dis­ tribution in nature. in this study, both the tubers of seed potato (‘lady rosetta’) and true potato seed (tps) (‘bari tps­1’) varieties were stored at 5°c for 0 to 4 months, and the changes in the starch break down were analyzed physiologically and histologically to investigate how cold storage affects the starch break down. although the starch content of both varieties reduced dur­ ing cold storage, the reduction of starch content in ‘bari tps­1’ was higher than that of ‘lady rosetta’. however, both volume and ovality (length:width) of starch granule did not change significantly throughout the storage period irre­ spective of variety, suggesting a non­uniform breakdown of starch granules. scanning electron microscope (sem) images of starch granule showed non­uni­ formed deformation and enlarged cavity or hole along the storage period, which indicated that starch breakdown occurred at a specific part of starch granule rather than peripherally and penetration would be deeper in ‘bari tps­ 1’ than that of ‘lady rosetta’. however, there was no significant change in granule size distribution in spite of rapid degradation of amylopectin percent­ age in ‘bari tps­1’ than that of ‘lady rosetta’, suggesting more susceptibility of ‘bari tps­1’ to starch degrading enzyme and higher enzymatic action would cause deeper penetration in ‘bari tps­1’ than that of ‘lady rosetta’. 1. introduction potato (solanum tuberosum l.) crop is usually cultivated by plant­ ing seed tubers which are genetically identical clones. on the other hand, true potato seed (tps) is the actual botanical potato seed pro­ duced by the potato plant. potato production from seed tuber derived (*) corresponding author: marufsau@hotmail.com citation: taufique t., nishizawa t., roy t.s., meiko k., chakraborty r., mostofa m., nara k., 2021 ­ histological and physiological changes of potato starch derived from seed and tps (true potato seed) grown tubers under different cold storage duration. ­ adv. hort. sci., 35(4): 371­381 copyright: © 2021 taufique t., nishizawa t., roy t.s., meiko k., chakraborty r., mostofa m., nara k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 july 2021 accepted for publication 2 december 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-11517 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 371­381 372 from tps (seedling tuber) is emerging as a promising alternative of using seed tuber due to the advantages of less disease transmission, physiological maturity until planting season, cheap storage cost, and many choices of varieties (pangaribuan, 1994). as potato tuber comes to maturity, starch­rich perimedullary region forms the major portion of the tuber (gupta and kaur, 2000), and potato granules are synthesized and stored as roughly spherical shapes in amyloplasts (naeem et al., 1997; fajardo et al., 2013) as the tuber matures, new starch granules are also synthe­ sized in newly produced amyloplasts. there is usually a large granule size distribution within individual tubers in terms of percentages of small, medium, and large granules (singh et al., 2016). the size of small and large granules ranged from 0.6 to 6 μm and from 10 to 100 μm in potato starch, respectively (wang et al., 2018), consisting of smooth­surfaced, oval and irregular shape (singh et al., 2003). singh et al. (2008) reported small starch granules of 1 to 10 μm, medi­ um granules of 11 to 30 μm and larger granules of >30 μm in diameter in four different new zealand potato cultivars. cold storage of potato tuber results in disintegra­ tion and disappearance of the amyloplast mem­ branes around the starch granules, which bring in contact with the degradative enzymes such as ᾳ­and β­ amylases and their substrates (badenhuizen, 1965; o’donoghue et al., 1995). thus, prolonged storage of potato tuber at low temperatures can result in starch degradation and conversion of starch into reducing sugar (zhang et al., 2014). when potato tubers (solanum tuberosum) are stored at temperatures below 9­10°c, the accumulation of sucrose and reducing sugars glucose and fructose occurred because of ‘low­temperature sweetening’ (lts) (pinhero et al., 2007). the rate of starch degradation and sugar accumulation depends largely on cultivar and storage temperatures (kazami et al., 2000). starch content was found to be decreased about 2 times after storage at 0­2°c for 8 weeks in both seed potato and tps potato tuber by (karim et al., 2008). a decrease of starch content was also reported after 60­105 days of storage at 4°c in several indian potato varieties (yamdeu et al., 2015). on the other hand, biemelt et al. (2000) reported that the starch content did not alter throughout the storage period. the starch degradative enzyme not only affects the starch content in cold storage also starch granule. the enzy­ matic susceptibility of starch granules has been stud­ ied by various authors (franco et al. , 1988; srichuwong et al., 2005; adejumo et al., 2013). differences in the enzymatic attack or susceptibilities of starches depend on many factors such as starch source, granule size, extension of association between starch components, rate of amylose and amylopectin, crystalline structure, particle size, sur­ face porosity, type of enzyme (hoover and zhou, 2003; kong et al., 2003; li et al., 2004; tester et al., 2006). a shift to lower granule size distribution has been reported in raw starches of different potato varieties after in­vitro enzymatic hydrolysis (kimura and robyt, 1995). cold induced sweetening (cis) sus­ ceptible cultivars can cause smaller starch granules when stored for 4 or 12 weeks at 4°c rather than cis resistant cultivars, which does not change until 24 weeks (barichello et al., 1990). based on the previous observations, it was hypothesized that when potato tubers are stored at low temperature conditions, starch granule content and size change in relation to starch degradation. starch granules may change ran­ domly or according to size because several studies showed that the starch granule size is an important factor for influencing the digestibility of raw starch by amylase (noda et al., 2008). ezekiel et al. (2010) observed that the number of small granules decreased, and the number of large granules increased in potato after cold storage. granule size and surface area affect the hydrolysis rate of starch by amylase, large granules with higher diameter have smaller surface area than small granules with lower diameter and therefore larger granules digested more slowly (tester et al., 2006; kasemwong et al., 2008; noda et al., 2008). because of the susceptibility to hydrolytic enzyme attack, deformation like exter­ nal corrosion, pits or endo­erosion occurred on the starch granule (planchot et al., 1995). even though starch granules change in relation to starch degradation, it is unclear whether and how these happen in case of ‘seed tuber’ and tps tuber at the same storage condition. so, the aim of this exper­ iment was to study whether the starch degradation occurs similarly or differently in seed potato and tps tuber; and how the starch degradation affects the starch granule deformation morphologically in both tubers. 2. materials and methods plant material in october 2015, each of 50 potato tubers of taufique et al. ‐ effect of cold storage on starch breakdown in tps and seed potato tuber 373 ‘bari tps­1’ (tps) and ‘lady rosetta’ (seed potato) were obtained from bangladesh. volume, size, and weight of all the tubers were measured and were stored at 5°c for 0 to 4 months in a refrigerator at the laboratory of the department of agronomy, faculty of agriculture, sher­e­bangla agricultural university, bangladesh. the ‘lady rosetta’ variety is one of the widely cultivated commercial seed potato in bangladesh, which is also used as processed pota­ to. on the other hand, ‘bari tps­1’ is one of the well­ cultivated ‘true potato seed’ variety, which is culti­ vated from true seed in first year and from the tuber­ lets from second year. both of the tuber was collect­ ed and cultivated in the experimental plot of sher­e­ bangla agricultural university with integrated crop management. ten tubers per each variety were taken out monthly from the refrigerator, the skin was peeled off and the tubers were grated using a grater of about 45 mm diameter. half of the grated sample was then blended and washed with desalted water, and filtered through filter paper. this step was repeated 3 times and then filtrated and dried in sun­ light under shaded condition for few days when the environmental temperature was around 34°c. this unheated samples were prepared for starch granule observation using sem (scanning electron microscope). the other half of the grated sample was added to 80% meoh (100 ml) and heated at 80°c for 30 minutes. the supernatant was decanted, and the residue was extracted by 80% meoh (100 ml). this step was repeated for 3 times in total and then washed by pure acetone. then, it was heated and dried on a hot plate at 50 to 70°c only in the daytime and stored at the ambient temperature at night. it took 3 days to dry completely and was weighed repetitively up to constant dry weight. the completely dry sample was cooled to the ambient temperature, stored in a freezer at ­20°c. this 80% meoh (100 ml) extracted samples were used for sugar and starch analysis. the chemical analysis was done at the laboratory of the department of bioproduction, faculty of agriculture, yamagata university, japan. soluble sugar content a 0.2 g of dry powder from each cultivar was taken into a test tube, then 9 ml of 80% meoh was added, and it was heated at 80⁰c for 30 min. the extract was centrifuged at 3000 rpm using a cen­ trifuge (ks­500, kubota, and tokyo) for 10 min, and the supernatant was decanted. this extraction proce­ dure was repeated for 3 times, and the combined supernatant was made up to 50 ml volume with 80% meoh. reducing sugar content in 0.5 ml of the solu­ tion was measured by somogyi nelson method (nelson, 1944), and the 0.5 ml of the solution was added by 2 units of invertase (ph 4.5, kanto, kagaku, tokyo) and hydrolyzed at 50°c for 30 min. sucrose content in the solution was also measured using the same technique as reducing sugar. copper reagent and nelson reagent were used to prepare a standard solution of glucose. absorbance was measured at 660 nm, and a standard curve was prepared to calcu­ late reducing sugar. non­reducing sugar was mea­ sured from hydrolytic degradation of sucrose, and absorbance was measured at 660 nm. abs obtained from the analysis of reducing sugar was denoted by abs 1 … (1) abs obtained from the analysis of non­reducing sugar was denoted by abs 2 … (2) concentration of non­reducing sugar was calculat­ ed from = {abs (2) ­ abs (1)} × 0.95 … (3) concentration of total soluble sugar was calculat­ ed from = (1) + (3) these steps were repeated for 5 times for each cultivars and for each storage sample. starch content the insoluble solid from the 80% meoh extract was added with 1.5 ml of distilled water and heated at 100°c for 1 hr. starch in the pellet was hydrolyzed using amyloglucosidase (yakult) at 55°c for 3 hrs then neutralized by 0.1% naoh solution. starch content was also measured in 0.5 ml of the solution using the same technique of sugar analysis and glucose oxidase method. glucose standard curve was pre­ pared to measure starch content. absorbance was measured at 660 nm for somogyi nelson method and at 500 nm for glucose oxidase method and was repeated for 5 times for each cultivars and for each storage sample. abs obtained from the analysis of starch was denoted by abs … (4) starch content was calculated from = abs 4 × 0.9. histological analysis of starch granule starch powders were scattered on an adhesive carbon tape and were fixed with 2% osmium tetrox­ ide (oso4) and successively washed with 50 mm cacodylate buffer and ultrapure water. the dried samples were coated by pt using each sample slides by pt ion coater (jfc­1200, jeol, tokyo), and then observed under sem scanning electron microscope (sem, tm3000, hitachi, tokyo). the length and width adv. hort. sci., 2021 35(4): 371­381 374 of the starch granules were measured by using motic image plus 2.0 software from sem images. the length and width ratio of starch granule was mea­ sured as the ratio of length:width. starch granule was regarded as an ellipsoid, and the volume of starch granule was measured of each axis of the ellipse. for histogram analysis (volume and length/width ratio) 10 sem image of x 500 mag­ nification and 200 µm across from each treatment were chosen and 5 granules from each image were measured randomly (total 500 starch granules). amylose percentage determination amylose content was measured using an assay kit (megazyme, amylose/amylopectin assay kit, ireland) according to the procedure outlined by the manufac­ turer. percentage of amylose was directly calculated following the specific megazyme equation based on the measured absorbance values, no additional stan­ dard curve or equation was generated for this study. amylopectin content was calculated by 100% differ­ ence of the amylose content (aristizábal et al., 2007). this step was repeated for 5 times for each cultivar and for each consecutive storage sample. statistical analysis data were subjected to analysis of variance and the difference between cultivars was compared with t­test using spss software. 3. results soluble sugar content in ‘bari tps­1’ tubers, reducing sugar content reached the highest value (57.80 mg/g dw) after 1 month of storage then decreased rapidly thereafter. reducing sugar of ‘lady rosetta’ showed the same tendency but the highest value after 1 month of stor­ age (25.86 mg/g dry weight) was less than half of that of ‘bari tps­1’ and decreased gradually there­ after (fig. 1). therefore, reducing sugar content of ‘bari tps­1’ was significantly higher than that of ‘lady rosetta’ during 1 to 3 months of storage. although, sucrose content also showed the highest value after 1 month of storage, the value decreased rapidly thereafter. the decline of ‘bari tps­1’ was slower than that of ‘lady rosetta’, resulting in a sig­ nificant difference between the two varieties during 2 to 3 months of storage (fig. 1). changes in total sugar content were similar to that of sucrose content and significantly higher content was observed also in ‘bari tps­1’ during 2 to 3 months of storage (fig. 1). starch content although starch content of both potato varieties decreased continuously throughout the storage peri­ od, starch content of ‘bari tps­1’ decreased more rapidly than that of ‘lady rosetta’ variety, resulting in a significant difference from 2 to 4 months of storage (fig. 1). it is noticeable from figure 1 that decreasing level of starch content was on par with the increasing reducing sugar content, where ‘bari tps­1’ showed rapid starch degradation with higher reducing sugar content after 2 to 4 months of storage than that of ‘lady rosetta’. similarly increase in sucrose level of ‘bari tps­1’ after 1 month of storage paralleled with the decreased level of starch content, resulting signif­ icant differences between two varieties. starch granule sizes starch granule length, width, length:width ratio and volume did not change apparently throughout the storage period. though ‘lady rosetta’ had a ten­ dency to have slightly higher values in length, width and volume, there was no significant difference between the both ‘bari tps­1’ and ‘lady rosetta’ (fig. 2). histogram was shown for starch granule volume fig. 1 ­ changes in soluble sugar and starch contents of two potato cultivars as affected by different storage period (n = 5 ± se). * shows significant difference and n.s. shows non­significant different at p<0.05 by student’s t­test. taufique et al. ‐ effect of cold storage on starch breakdown in tps and seed potato tuber 375 and ovality (length:width) within the range of (0.5 to 81.5) x 103 µm3 and (1.05 to 2.22) respectively (fig. 3 and 4). the highest frequency was observed at (7.6 ­ 13.5) x 103 µm3 and (1.19 and 1.31) for volume and ovality, respectively and there was no considerable differences between the two cultivars. moreover, no apparent change also occurred during storage period. starch granule morphology both ‘bari tps 1’ and ‘lady rosetta’ tubers showed normal and smooth starch granule surface at 0­month storage (fig. 5). starch granules of both tubers changed after storage condition. pit and hole like structures were observed after 1 month storage in both varieties. after 2nd, 3rd and 4th month storages prominent depression or cavity was observed in both potato cultivars. however, both potato tubers showed a similar pattern of deformation. amylopectin percentage although the percentage of amylopectin in both potato varieties decreased continuously throughout the storage period, amylopectin of ‘bari tps­1’ decreased more rapidly than that of ‘lady rosetta’ variety, resulting in a significant difference from 2 and 4 months of storage (fig. 6). although ‘lady rosetta’ variety had slightly higher percentage than tps after 3 months of storage, there was no signifi­ cant difference between them. fig. 2 ­ changes in the sizes of starch granule derived from two potato cultivars as affected by different storage period for (n =10±se). *shows significant difference and n.s. shows non­significant difference at p<0.05 by student’s t­ test. fig. 3 ­ change in histogram for frequency percentage of potato starch granule volume derived from ‘bari­tps­1’ and ‘lady rosetta’ potato varieties as influenced by storage period. fig. 4 ­ change in histogram for frequency percentage of potato starch granule length:width ratio derived from ‘bari­ tps­1’ and ‘lady rosetta’ potato varieties as influenced by storage period. 376 adv. hort. sci., 2021 35(4): 371­381 4. discussion and conclusions in potato tuber, starch is converted to sugar dur­ ing cold storage (malone et al., 2006). the starch content of potato tuber decreased markedly during prolonged storage at 4­8°c through the process of conversion of starch into sugars (nourian et al., 2003; smith et al., 2005). ohad et al. (1971) also reported reduction of starch content by 26% after 17 days of storage. mature dormant potato tuber produced sug­ ars by degradation of a small fraction of starch (isherwood, 1973). thus starch content decreased because of the hydrolysis of starch by starch degrad­ ing enzymes (nielsen et al., 1997), suggesting why reducing sugar content increased as starch content decreased during cold storage (fig. 1). storage at 0­ 5°c increases sugar accumulation in potato tuber (wismer et al., 1995; blenkinsop et al., 2003), which coincides with these experiments where both vari­ eties showed higher reducing sugar content after storage with a significant difference between ‘bari tps­1’ and lady rosetta (fig. 1). this increased level of reducing sugar and decreased level of starch con­ tent is related with the increased activity of hydrolysing enzyme (sowokinos, 2001), which might explain the higher starch degradation in ‘bari tps­1’ (fig. 1). during low­temperature storage, the enhance­ ment of sucrose and hexoses (glucose and fructose) levels is known as “cold sweetening’ or ‘low tempera­ ture sweetening’, which is an important metabolic process in the roots of many species as well as potato tuber (wismer et al., 1995; espen et al., 1999; galindo et al., 2004; galindo et al., 2007). the soluble sugar content of potato tuber increases at low tem­ perature such as 4°c, when stored for 6 to 12 weeks because of starch decomposition and then inclines to decrease (cochrane et al., 1991; chen et al., 2012). total sugar and sucrose content both showed a simi­ lar changing pattern after storage with significantly higher content in ‘bari tps­1’ during 2 and 3 months (fig. 1). cold storage condition triggers the tuber starch to breakdown into sucrose through various hydrolytic enzymes which further hydrolyzed into reducing sugars (glucose and fructose) (sowokinos, 2001). the amount of soluble sugar accumulated in fig. 6 ­ changes in the percentage of amylopectin in ‘bari­tps­1’ and ‘lady rosetta’ potato tubers as influenced by stora­ ge period. fig. 5 ­ scanning electron micrographs showing the surface morphology of starch granules of ‘bari tps­1’ and ‘lady rosetta’ and potato tuber stored at 5°c at 0 to 4 months of storage. taufique et al. ‐ effect of cold storage on starch breakdown in tps and seed potato tuber 377 tuber also depends on different cultivars (zommick et al., 2014).), which could explain the higher reducing sugar in ‘bari tps­1’ after storage than lady rosetta (santos et al., 2020). however, there was a sudden increase in starch content between 3rd to 4th months of storage in ‘lady rosetta’ variety (fig. 1), suggesting recondition of starch or increase of respiration losses during stor­ age. the difference in respiration rates depend on cultivar, growing conditions, experimental condi­ tions, and physiological status of the tubers (fennir et al., 2003). comparatively lower starch degradation after 3 to 4 months of storage might have con­ tributed to lower presence of reducing sugar in ‘lady rosetta’ (fig. 1). this can also be explained that likely the rise of starch content between 3 and 4 months of storage, amylopectin percentage also showed similar kind of tendency in ‘lady rosetta’ variety (fig. 6). an increase in amylopectin percentage from 3 to 4 months of storage suggested lower enzymatic degra­ dation (fig. 6) (hofvander et al., 2004). it seemed amaylase enzyme activity reduced during 3 to 4 months of storage because of sprouting of seed pota­ to varieties or it can be explained that the amaylase enzyme activity was not enough to degrade the starch in seed potato (lewis et al., 1994). as the experimental condition was same for both of the tubers, hence these may explain faster physiological aging in seed potato variety than tps variety. this may also suggest that experimental storage tempera­ ture might have affected the respiration losses; and the sugar had been used for tuber germination in case of seed tuber (olsen et al., 2003). the tps variety showed higher starch degradation rate than the seed potato (fig. 1), and sharp decrease in amylopectin percentage after storage (fig. 6), moreover there was higher percentage of amy­ lopectin degradation in ‘bari tps­1’ than ‘lady rosetta’ starting after 1 month of storage and contin­ ued as the storage progressed (fig. 6), but there was no apparent changes in starch granule volume and ovality (legth:width ratio) (fig. 2). this result suggest­ ed a non­uniform degradation pattern of starch gran­ ules in ‘‘bari tps­1’and ‘lady rosetta’ varieties. histograms were also carried out to investigate, if the starch granule deformation primarily started in smaller sized granules, or larger sized granules (figs. 3 and 4). singh et al. (2008) reported a shift of gran­ ule size range to smaller granule sizes in isolated starches of new zealand taewa (maori potato) when they were stored for three and six months at 4°c temperature. this experiment also reported erosion and pitting on the surface of stored potato starch. in case of banana, surface erosion by enzymatic degra­ dation resulted in smaller granules with elongated shape and exo­corrosion process caused pits on the surface of the starch granule with high frequency (peroni­okita et al., 2013). this shift of granule size distribution has been related to rapid digestion of starch granule and composition variation between small and large granules (salunkhe et al., 1989). however, there were no considerable changes of starch granule sizes in both potato cultivars in our study. this suggested that, in spite of having signifi­ cant change in starch degradation along storage in both variety, it had hardly influenced the size of starch granule (fig. 2, 3 and 4). moreover, it was found that even after 4 months of storage periods, there was no significant change in starch granule sizes in both cultivars, in spite of ‘lady rosetta’ having slightly higher tendency in the value of length, width and volume (fig. 2). fajardo et al. (2013) also reported unchanged granule size after storage, which was attributed to the nearly unde­ tectable change in volume of starch granule. russet burbank potato tubers also showed similar granular size distribution after storage at 3.9°c (johnston et al., 1968). sem observation showed that starch granules from both harvested tubers presented smooth gran­ ular surface under sem without any storage treat­ ment or at 0­month storage (fig. 5). although there is argument about the appearance of natural potato starch granular surface, several studies showed smooth granular surface of natural potato starch under microscopic observation. cottrell et al. (1993) reported smooth granular surface of potato starch of record and brodick potato cultivar at harvest. (sarikaya et al., 2000) also reported smooth granular surface of potato starch before any enzymatic or freezing treatment. as the sem observation in this experiment showed hole or pit formation on granular surface of both varieties after storage (fig. 5) (sarikaya et al., 2000; noda et al., 2005), it is suggest­ ed that the long term storage at low temperature of this experiment lowered the starch content which led to susceptibility to hydrolytic enzyme attack that changes the properties and composition of starch granule (barichello et al. 1990). the cold storage temperature might have given damage to amyloplast membrane by starch hydrolysis (ohad et al., 1971), which enhanced membrane permeability through adv. hort. sci., 2021 35(4): 371­381 378 starch hydrolysis enzymes, and resulted the forma­ tion of cracked region or surface hole on the granule (sujka and jamroz, 2010). however, no apparent change was observed in the ratio of granules and histogram analysis of both potato starch granule, but the sem observation clear­ ly showed deformation of starch granule forming hole or pit like structures gradually started from 1 month of storage and continued to 4 months of stor­ age (fig. 5). on the other hand, gradual starch degra­ dation and decreased amylopectin percentage indi­ cated activation of enzymatic degradation in both cultivars after storage (fig. 1 and 6). therefore, it is suggested that starch granule erosion may occur at specific surface region of the granule causing no change in the granular size. starch hydrolyging enzyme like a­amylase might have attacked at partic­ ular points on the granule surface, forming tunnels into the granule, thus hydrolyzed the granule from the inside (lindeboom et. al., 2004). similar finding was reported by peroni­okita et al. (2013) where starch granules of green banana maintained the rounded shape after low temperature but presented pits on the granule surface which was enlarged by the corrosion process, suggesting partial degradation of starch granules. thus enzyme molecules can affect the starch granule in different patterns either by forming pin holes, medium sized hole, sponge like erosion or selected point at the surface leading to a single hole (sujka and jamroz, 2010). enzyme can gain access to innermost region of the starch granule by faster digestion than at the periphery region of the granule, which can form shallow hole like structure (duffus, 1984). this may explain how the both starch granules showed no changes in their sizes despite of granular deformation. however, ‘bari tps­1’ showed a higher starch degradation percentage throughout the storage peri­ od than the seed tuber potato. this suggested higher susceptibility of starch degrading enzyme in tps tuber than that of seed potato tuber. variation in starch granule morphology and their crystalline orga­ nization may explain the susceptibility to enzymatic degradation (gallant et al., 1992). amylase plays the major role in in­vivo breakdown of starch (manners, 1985). amylose and amylopectin ratio may explain the degradation pattern of starch granules in both tps and seed potato varieties. both the potato tuber showed decreased percentage of amylopectin along the storage period, whereas ‘bari tps­1’ resulted lower amylopectin percentage than the seed potato (fig. 6). bach et al. (2013) described that as amy­ lopectin degrades more rapidly than amylose, it caus­ es activation of more starch degrading enzymes. therefore, ‘bari tps­1’ might have allowed deeper penetration of starch degrading enzymes than the seed tuber in spite of no apparent changes in granule size (fig. 5). as the extent of pit or cavity of starch granules were not determined in this experiment, it can be suggested that probably a different inner mol­ ecular organization of tps starch granule may be allowed differentiated enzyme attack, which could be responsible for slightly smaller starch granule than ‘lady rosetta’ during storage (fig. 6) with no signifi­ cant difference. this also suggests that starches vary in their resistance to enzymatic susceptibility (srichuwong et al., 2005). there are different opinions about the presence of pores or holes on the surface of potato starch granules. although several observations concluded that some factors could cause an increase in the number and size of pores on starch granules surface (fannon et al., 1992). moreover, the expansion of granule degradation increased with the increased enzyme concentration (mu et al., 2015). during enzy­ matic hydrolysis, some regions of granules are more susceptible to enzyme attack because of less orga­ nized amorphous rings, whereas the crystalline lamella provides higher resistance to enzymatic ero­ sion (oates, 1997). this kind of enzymatic hydrolysis was characterized by forming a hole by creating channel through the less resistant granule core (jung et al., 2017), which may explain deeper enzyme pen­ etration in tps tuber. action pattern of both endo­ and exo­amylase at a lower temperature may explain overall amylolytic activity in tps and seed potato tubers (shin et al., 2002; nabubuya et al., 2012). 4. conclusions in this experiment, the starch content of tps tuber degraded more rapidly and produced higher reducing sugar content than the seed potato variety after storage. this result indicated that, tps tuber may not be acceptable for low temperature storage compare to seed potato variety. there were no sig­ nificant changes in granule size and volume, indicat­ ed that granule did not change as a ratio in both tubers. however, both potato granules deformed by forming holes or cavities at innermost surface region taufique et al. ‐ effect of cold storage on starch breakdown in tps and seed potato tuber 379 after storage, suggesting the starch granule degraded partially rather than concentrically. as both the tubers showed a similar kind of degradation pattern of starch granule in spite of higher starch degrada­ tion rate in tps tuber, similarly the rapid degradation rate of amylopectin in tps tuber explained higher amylase activities in tps than the seed tuber. this result suggested possibilities of deeper penetration in tps starch granule than the seed tuber starch granules. further studies on the susceptibility of starch degrading enzyme on tps and seed potato can be done to foresee the starch degradation pattern in tps and seed potato tuber. acknowledgements the authors thankfully acknowledged the authori­ ty of japan society for promotion science (jsps) and university grants commission (ugc) of bangladesh for supporting to conduct of the experiment. references adejumo a.l., aderibigbe, owolabi r.u., 2013 ­ comparitive studies of starch susceptibilities to α‐amy‐ lase degradation of different cereal and root crops of nigeria. ­ afr. j. biotechnol., 12(29): 4663­4669. aristizabal j., sanchez t., lorio d.m., 2007 ­ guía téc‐ nica para producción y análisis de almidón de yuca: organización de las naciones unidas para la agricultura y la alimentación roma. ­ isbn: 92­5­ 305677­0 bach s., yada r.y., bizimungu b., fan m., sullivan j.a., 2013 ­ genotype by environment interaction effects on starch content and digestibility in potato (solanum tuberosum l.). ­ j. agric. food chem., 61(16): 3941­ 3948. badenhuizen n.p., 1965 ­ occurrence and development of starch in plants. ­ in: whistler r.l., and e.f. paschall (eds.) starch: chemistry and technology. academic press, new york, pp. 65­104. barichello v., yada r.y., coffin r.h., stany d.w., 1990 ­ low temperature sweetening in susceptible and resis‐ tant potatoes: starch structure and composition. ­ j. food sci., 55(4): 1054­1059. biemelt s., hajirezaei m., hentschel e., sonnewald u., 2000 ­ comparative analysis of abscisic acid content and starch degradation during storage of tubers har‐ vested from different potato varieties. ­ potato res., 43(4): 371­382. blenkinsop r.w., copp l.j., yada r.y., maragonia g., 2003 ­ a proposed role for the anaerobic pathway dur‐ ing low‐temperature sweetening in tubers of solanum tuberosum. ­ physiol. plant., 118(2): 206­212. chen x., song b., liu j., yan j., h.e. t., lin y., zhang, h., xie c., 2012 ­ modulation of gene expression in cold‐ induced sweetening resistant potao species solanum berthaultii exposed to low temperature. ­ mol. genet. genomics., 287: 411­421. cochrane m.p., duffus c.m., allison m.j., mackay g.r., 1991 ­ amylolytic activity in stored potato tubers. 1. estimation usingp‐nitrophenyloligosaccharides. ­ potato res., 34: 325­332. cottrell j.e., duffus c.m., mackay g.r., allison m.j., 1993 ­ changes in the surface morphology of starch granules of the cultivated potato, solanum tuberosum l. during storage. ­ potato res., 36: 119­125. duffus c.m., 1984 ­ metabolism of reserve starch. ­ in: lewis d.h. 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words: genome association and prediction integrated tool (gapit), hierfstat, intragenic molecular markers, musa textilis nee, population structure. abstract: abaca are leaf­fiber plants found predominantly in the philippines. our country holds most of the manila hemp market, but the unknown genetic architecture of the fiber hinders the crop’s improvement. we developed intra­ genic molecular markers from genes related to fiber development and linked them to abaca fiber quality, with the goal of increasing precision of breeding. pearson’s correlation package of the r programming software revealed a high positive relationship between the pseudostem’s top and middle girth (r=0.91), while a low negative correlation between the percent fiber percent fiber strain and the number of suckers (r= ­0.42). the analysis also showed that the ulti­ mate tensile strength was highly correlated with percent fiber percent fiber strain (r=0.33) and dry weight (r=0.34). three subpopulations were determined using the structure software, while hierfstat computed an average 0.0648 fst value, indicating moderate genetic diversity. eight significant marker­trait asso­ ciations (p­value <0.005) were identified with positive effects and >0.6% pheno­ typic variance explained (pve). eight markers from the cobra­like protein, expansin, cellulose synthase, and auxin gene families were identified as linked to fiber quality and tensile strength. our study identified nine abaca accessions with the trait of interest and the candidate genes. the significant molecular markers will be used to identify the hybrids with good fiber quality. 1. introduction there are eight widely known plant fibers, sisal, coir, cotton, flax, (*) corresponding author: mdmendoza11@up.edu.ph citation: mendoza m.r.r., laurena a.c., diaz m.g.q., ocampo e.t.m., laude t.p., lalusin a.g., 2025 ­ association analysis of intragenic molecular markers related to fiber quality and tensile strength of abaca (musa textilis nee). ­ adv. hort. sci., 39(1): 55­67 orcid: mmrr: 0009­0001­9474­083x dmgq: 0000­0002­6919­059x oetm: 0000­0002­3275­7177 lag: 0000­0001­5382­8600 copyright: © 2025 mendoza m.r.r., laurena a.c., diaz m.g.q., ocampo e.t.m., laude t.p., lalusin a.g. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 8 november 2024 accepted for publication 11 april 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16774 http://oaj.fupress.net/index.php/ahs http://orcid.org/000900019474083x http://orcid.org/0000-0002-6919-059x http://orcid.org/0000-0002-3275-7177 http://orcid.org/0000-0001-5382-8600 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 55­67 56 hemp, jute, ramie, and lastly, abaca (m. textilis nee) (fao, 2009). the latter is an herbaceous perennial musa highly praised for its mechanical strength and industrial applications. in contrast to its close relative banana, this musa species has thinner, glossier pseudostems, and instead of fruits, the succulent stems are the main commodity from abaca. these stems are harvested and processed into fibers internationally known as manila hemp. there are multiple applications for plant fibers, but the boom of synthetic fiber industries slowly changed the demand for natural fibers (bemiller, 2007). nevertheless, natural fibers are sought after because of their sustainability and inexpensive production cost compared to their synthetic counterparts (radhakrishnan, 2014). abaca belongs to the leaf or cordage fibers. their pseudostems are composed of multicelled fibers. these cells provide strength and mechanical resistance to the abaca fibers. the strands have weak bonds and can be easily separated through scraping. cellulose and hemicellulose content are critical to the tensile strength and quality of fiber crops. the increased cellulose content indicates higher tensile strength. meanwhile, increased hemicellulose, tends to decrease the mechanical potential of the fibers by lowering their tensile strength due to unstable monomers. therefore, those plant fibers with high mechanical properties are chosen for structural builds (djafari petroudy, 2017). still, at the molecular level, other factors can affect the cell wall biosynthesis and the strength of the fiber cells, especially when exposed to different environmental conditions and stress. since its discovery in 1989 by litt and luty, ssr markers have been extensively used on many species. the availability of new software and whole genome sequences eased ssr mining, making new ssr studies robust and relevant. this co­dominant marker type is versatile and reliable for evolutionary analysis, genetic diversity, and marker­assisted breeding. it has helped hasten the classic plant breeding works (victoria et al . , 2011) and fingerprinted closely related species (vinarao et al., 2019). these markers can be developed from genic portions, mirna regions, chloroplast sequences, expressed sequence tags, and whole genome assembly (victoria et al., 2011; sagwal et al., 2022). in addition, ssr markers were also used to uphold the breeder’s right over the marketed seeds and prevent variety mislabeling in the field (palumbo and barcaccia, 2018). there are several published works on the use of ssrs on abaca (boguero et al., 2016; yllano et al., 2020; mendoza et al., 2024). however, these studies are focused on the genetic diversity of the crop rather than the markers’ relationship with specific traits. the genomic markers used by yllano et al. (2020) and boguero et al. (2016) are even designed from other musa species that lessens their specificity for association analysis. this study identified the intragenic ssr markers related to tensile strength and fiber quality. we also analyzed phenotype­genotype trait association using candidate gene­based ssr markers and selected abaca accessions that show promising traits. the information can be used in the genomic selection of elite accessions abaca hybrid production. 2. materials and methods phenotyping the abaca gene bank composed of 73 accessions collected from 11 administrative regions of the philippines, was used in the study (suppl. materials table 1s). a total of 56% of this collection was planted in 2020, while the remaining 44% was established in 2016. the pseudostem were harvested when the flag leaf emerged. the number of suckers (s) were counted from each hill while and the leaf sheaths (ls) were counted from the harvested pseudostems. the circumference of the base of the crown leaf crown (gt), the middle girth (gm), and the base (gb) of the pseudostem was measured in centimeters. the stem length (sl) was measured from end to end of the pseudostem. the fresh weight (fw) was measured in kilograms, while the fiber dry weight (dw) was obtained in grams using an analytical balance after air drying. the tensile strength and percent fiber strain were obtained using the shimadzu agx series tensile strength machine. the ultimate tensile strength (uts) was computed using the formula: uts= f/a where: f is the force applied and a the fiber cross sectional area. dna extraction and polymerase chain reaction a total of 34 genes related to fiber development and quality were obtained from the list of transcripts identified by reamillo (2018) and other related studies (table 1). the identified sequences were http://oaj.fupress.net/index.php/ahs/article/view/16774/13392 mendoza et al. ‐ association mapping of abaca fiber related intragenic molecular markers 57 scanned from the ncbi website (https://www. ncbi.nlm.nih.gov/). the blastn command line version was used to determine the percent identity of the sequences and high bit score with the whole genome sequence of musa textilis nee cv. abuab (galvez et al., 2021). only those that passed the modified blastn threshold (e­value = 0.005, high identity and bit score) were scanned for tandem repeats using the microsatellite finder (http://insilico.ehu.es/mini_tools/microsatellites/). the molecular markers were generated from the 250 bp upstream and downstream of the tandem repeats using the primer3 website (https://primer3.ut.ee/). the parameters used in the development of the primers were: 18­27 primer size, 57°c to 63°c primer tm, ­10.0 to 110 primers bound %, and 20%­80% percent gc content. the doyle and doyle (1990) ctab dna extraction protocol, modified by sandoval (2011), was used to isolate the dna. the 10 µl pcr reaction comprised of 1x pcr buffer, 2 mm mgcl2, 0.2 mm dntps and primers (forward and reverse), and a 0.05 u/µl taq polymerase cocktail. the running conditions for each reaction were 94°c for 4 minutes, then 35 cycles at marker motif annealing temperature product size functional identity 51 agagagag 59.9 309 cellulose synthase­like protein d2 52 tatatata 59.95 307 cellulose synthase­like protein d2 53 tcttcttct 60.15 306 cellulose synthase­like protein d2 54 tctctc 60.35 301 cellulose synthase­like protein d2 55 gagaga 60.25 302 cellulose synthase­like protein d5 56 aggaggaggaggaggagg 59.45 201 cellulose synthase­like protein e6 57 tctctc 59.55 301 cellulose synthase­like protein d2 58 tctctc 59.45 405 cobra­like protein 4 59 cgtcgtcgt 59.8 203 expansin a2 60 caacaacaa 60.9 205 expansin a10 61 ttcttcttcttcttcttcttcttc 58.9 306 expansin a10 62 tctctctc 59.95 336 expansin a10 63 gctgctgctgct 59.6 403 expansin a4 64 agagag 59.2 268 auxin response factor 9 65 tctctctc 59.7 407 auxin­responsive protein iaa6­like 66 ctctct 59.5 309 auxin responsive iaa30 like protein 67 gtcgtcgtc 59.85 316 auxin­induced protein 22d 68 gagaga 59.7 302 putative protein auxin response 4 69 cctcctcctcctcct 59.8 201 ap2_like_ethylene_responsive_transcription_factor_ail5 70 ctcctcctc 60.4 207 ap2­like ethylene­responsive transcription factor ail5 71 agagagag 58.9 323 ethylene insensitive 3­like protein 72 agagag 59.7 313 probable_ethylene_response_sensor_1 73 aagaagaagaagaagaagaag 59.9 224 glycolipid_transfer_protein_3 74 agagagag 60.15 331 cellulose synthase­like protein d2_5'utr 75 tatatata 60 322 cellulose synthase­like protein d2_5'utr 76 tcttcttct 60.1 246 auxin_response_factor_9_5'utr 77 tctctc 60.6 323 auxin­responsive protein iaa6­like_5'utr 78 gagaga 59.3 244 ethylene insensitive 3­like protein_5'utr 79 aggaggaggaggaggagg 59.1 301 ethylene insensitive 3­like protein_5'utr 80 tctctc 58.9 306 expansin a10 81 tctctc 58.7 201 auxin­responsive protein iaa30­like 82 cgtcgtcgt 58.5 215 putative protein auxin response 4 83 caacaacaa 59.9 224 ap2­like ethylene­responsive transcription factor ail5 84 ttcttcttcttcttcttcttcttc 58.7 112 glycolipid_transfer_protein_3 table 1 ­ profile of the intragenic molecular markers that were used for association mapping of fiber traits in abaca (m. textilis nee) https://primer3.ut.ee/ adv. hort. sci., 2025 39(1): 55­67 58 94°c for 30 s, 58­60°c for 30 s, 72°c for 45 s, and a final extension at 72°c for 10 min. the amplified bands were visualized using 6% polyacrylamide gel stained using gelred and photodocumented in the genosens photo­documentation system. the amplified polymorphic bands were scored using the gelanalyzer v.23.1 software (lazar and lazar, 2023). data analysis pearson’s correlation between the observed traits was computed in the r software. structure software v.2.3.4 (pritchard et al., 2000) generated the population structure and the q matrix used for association mapping. each k was run over in ten replications in a burn­in period of 20,000 with 50,000 monte carlo markov chain (mcmc) replicates. the best grouping was determined using the r package pophelper v.2.3.1 (francis, 2017) (https://github.com /royfrancis/pophelper). the hierfstat package was used to determine the fst value between the populations. the q matrix generated by the structure software was used to analyze the association between markers and traits using the farmcpu (fixed and random model circulating probability unification) of the gapit (genomic association and prediction tool) package (lipka et al., 2012). 3. results figure 1 shows the results from pearson’s correlation analysis. the highest positive correlation was obtained from the girth measurements of the pseudostem’s middle and top sections (r= 0.91). in contrast, the lowest negative correlation (r= ­0.42) was obtained between the percent fiber strain and the number of suckers (s). the number of suckers also has a positive correlation with seven out of ten morphological traits. pseudostem length (sl) correlation with fresh weight (r= 0.71) was its strongest correlation among the other characters. the leaf sheath (ls) shows an average positive correlation of r= 0.61 with girth and fresh weight characters. a strong mean correlation (r= 0.85) was observed among the base girths and fresh weight. percent fiber strain and fiber length recovery (flr) are the traits with the most negative correlations with other traits. the percent fiber strain and dry weight (dry weight) are the only traits that have a positive correlation with the ultimate tensile strength at r= 0.33 and r= 0.34, respectively. the correlation shows the trait values that increase together and characters with negative relationships. population structure the fst value indicates the allele frequency distribution between the subpopulations or clusters of abaca. the fst value of >0.25 indicates high genetic differentiation and suggests that the accessions came from different genera or completely different species. the mean fst value obtained from the abaca subpopulation in this study was fst = 0.0648, suggesting moderate genetic variation between the three populations (li et al., 2014; luo et al., 2019). the lowest fst value at 0.0229 was obtained between cluster 1 and cluster 2 followed by fst=0.0728 between cluster 1 and 3. the highest fst value of 0.0988 was obtained between cluster 2 and 3. all the computed fst values indicate that the subpopulation are moderately diverse. most members of cluster 2 are abaca accessions from mindanao, while members of subpopulation 1 and 3 are from luzon, particularly fig. 1 ­ pearson’s correlation shows the correlation between the morphological traits of the 73 abaca (musa textilis nee) accessions. the highest positive correlations were found among the girth measures while the percent fiber strain and fiber length recovery (flr) exhibited the lowest negative correlations among the traits. https://github.com/royfrancis/pophelper https://github.com/royfrancis/pophelper https://github.com/royfrancis/pophelper mendoza et al. ‐ association mapping of abaca fiber related intragenic molecular markers 59 from region 5 (fig. 2). the best k clustering was three clusters, the markers used are gene­specific markers hence, a few clusters are expected (fig. 3). association mapping all the marker­trait associations (mtas) that are less than the threshold (p­value ≤0.005) were considered significant in this study (fig. 4). table 2 shows the significant mtas identified using the farmcpu model implemented in the gapit package of the r software. this model was able to control the false positives effectively by iterative running of the fixed and random effects. the q­q plot showed the relationship of the expected and observed p­values for each trait (fig. 4). the data points that lie beyond the gray area or the 95% confidence interval of the qqplot indicate rejection of the null hypothesis of no association. eight significant mtas for five traits were considered for this study. the number of suckers exhibited the highest number of associated markers with no negative effects (mk59, mk58, and mk69). the percent phenotypic variance explained (%pve) by the markers on the number of suckers ranged from 0.6­57% while its minor allele frequency or maf value ranges from 0.01­0.06. mk55 and mk63 were significantly associated with the ultimate tensile strength exhibited the highest positive effect among the mtas. their explained phenotypic variance was 35.91 and 7.16, respectively. only one marker (mk55) was strongly associated with the girth size (middle) which explained 42.99% of the observed phenotypic variation. the markers associated with dry weight and percent fiber strain showed the lowest positive effect among the mta at 0.01 and 0.4, respectively. overall, by combining the results from phenotyping and the significant marker trait association, accessions that possess genes of interest and exhibits high agronomic trait values are psu a and b for the number of suckers, tangongon and laylay for girth, native and parang for dry weight, kutay­kutay and native for ultimate tensile strength, and laylay for percent fiber strain. the team had fig. 2 ­ the inferred subpopulation assignment of the 73 abaca accessions showing three groupings. each bar and percent color represent the allelic distribution possessed by each individual in each cluster. fig. 3 ­ three groupings identified by pophelper package of rstudio as best k clustering for the 73 abaca accessions. the results for k=3 was then used as q matrix or kinship in the gapit analysis. 60 adv. hort. sci., 2025 39(1): 55­67 fig. 4 ­ the qq plot generated for each trait using the farmcpu model. a. suckers, b. girth (middle), c. dry weight, d. ultimate tensile strength, e. percent fiber strain. the markers that are above the regression line (red line) and beyond the 95% confidence interval (gray area) are significantly related to the traits­ of­interest. ta bl e 2­ t he si gn ifi ca nt m ar ke r­ tr ai t­ as so ci at io ns (m ta s) re la te d to th e im po rt an t a gr om or ph ol og ic al tr ai ts o f a ba ca (m us a te xt ili s n ee ) d et er m in ed u sin g th e as so ci at io n m ap pi ng to ol g ap it tr ai ts m ar ke r pr od uc t siz e (b p) p. v al ue m in or a lle le fr eq ue nc y (m af ) ef fe ct % ph en ot yp ic va ria nc e ex pl ai ne d fu nc tio na l i de nt ifi ca tio n n um be r o f s uc ke rs m k5 9 40 0 7, 81 e­ 05 0. 01 36 99 8. 73 56 56 0. 60 38 16 ex pa ns in a 2 n um be r o f s uc ke rs m k5 8 55 0 7, 46 e­ 08 0. 02 73 97 10 .5 04 74 7. 86 48 61 co br a­ lik e pr ot ei n 4 n um be r o f s uc ke rs m k6 9 25 0 2, 25 e­ 11 0. 06 84 93 7. 86 24 66 57 .6 1 ap 2_ lik e_ et hy le ne _r es po ns iv e_ tr an sc rip tio n_ fa ct or _a il 5 g irt h (m id dl e) m k5 5 31 5 0. 00 38 13 0. 05 47 95 4. 98 22 66 42 .9 9 ce llu lo se s yn th as e­ lik e pr ot ei n d5 dr y w ei gh t m k6 3 40 0 0. 00 38 58 s 0. 08 21 92 0. 01 35 31 51 .2 03 9 ex pa ns in a 4 te ns ile st re ng th m k5 5 39 0 0. 00 06 58 0. 08 21 92 26 3. 42 73 35 .9 1 ce llu lo se s yn th as e­ lik e pr ot ei n d5 te ns ile st re ng th m k6 3 40 0 0. 00 28 1 0. 08 21 9 22 2. 39 9 7. 16 au xi n re sp on se fa ct or 9 st ra in m k6 6 16 0 0. 00 45 29 0. 01 36 99 0. 46 04 5 9. 89 au xi n re sp on siv e ia a3 0 lik e pr ot ei n mendoza et al. ‐ association mapping of abaca fiber related intragenic molecular markers 61 produced 49 crosses out of the selected parentals, however only 15 hybrids survived. applying the markers with high correlation with the traits of interest can hasten the breeding work on abaca. the hybrids that show amplicons for these markers will be considered for advanced trial. the marker aided selection increases the accuracy of crop improvement for abaca and remains a crucial step in breeding. 4. discussion and conclusions abaca fiber composition the cell wall thickness was not only the determining factor for tensile strength. sisal (agave sisalana), a fiber plant species, with 73% cellulose showed a higher tensile strength of 484mpa than jute (corchorus capsularis) with 249mpa and has 65% cellulose content (alves fidelis et al., 2013). the study of saragih et al. (2020) focused on the thermal analysis of the fibers and their chemical composition. natural fibers are composed of 66.43% cellulose, 24.7% hemicellulose, and 13.6% lignin. it can be deduced that the amount of cellulose in the fibers is higher than the other two fiber components and contributes much to the strength of the fibers. with their results, they also found the potential of abaca as bioplastic fillers. different industrial applications require various chemical components for abaca fiber. for instance, the pulping industry would require abaca fibers with low ignin and ash content while having high α­cellulose concentration (moreno and protacio, 2012). an amount of >99% cellulose is considered pure. this is achievable through processing but difficult for certain fiber crops like abaca. only cotton which is composed of 93% cellulose and 0% lignin would require just hot alkali treatment to produce high grade pulps (bemiller, 2007). the input cost for locally producing cotton is higher than importing them causing a decline in the cotton industry of the philippines (asis, 2017). looking for other pulp sources such as abaca and optimizing the protocol by adding or removing a few steps during production of good pulp can be a solution. ssr markers for association mapping the candidate gene mapping using gene­derived ssr markers have been used in maize (dubey et al., 2009), barley (matthies et al., 2012), sorghum (wang et al., 2011), bread wheat (mehta et al., 2021), cotton (buyyarapu et al., 2011; baytar et al., 2022), durum wheat (alsaleh, 2022), rice (pritesh et al., 2023; pradhan et al., 2023), potato (schumacher et al., 2021), wheat (singh et al., 2018), flax (nag et al., 2020) and sugarcane (divakar et al., 2023). these studies used varying sample sizes and different numbers of molecular markers; divakar et al. (2023) even used a set of 70 highly related sugarcane that exhibited moderate genetic diversity. their number is similar to this study where we used 73 abaca germplasm collection for determining the association of fiber traits with newly developed molecular markers. nag et al. (2020) used only 28 molecular markers on flax to determine significant mtas with different fiber quality traits. matthies et al. (2012) also used 22 ssr markers to determine important association with barley’s kernel quality. the polymorphic nature of ssr markers makes them effective in distinguishing genotypes from the same genome (lin et al., 2012). these show that even with limited sample sizes and molecular markers significant associations can be determined. the intragenic molecular markers were also found related to different plant architecture traits and grain yield components of rice. seventeen cgssr markers with varying percent explained pve (4% to 17%) were observed to be linked with panicle length and weight, number of primary branches, and grain yield. the associated markers increase the accuracy of rice breeding (sah et al., 2023). bareto et al. (2019) also utilized ssr markers to improve the breeding work and elite genotype selection in sugarcane. a total of 23 significant mtas were highly associated with not only sugar content but also the crop’s plant architecture, including stalk height, weight, and number, and can yield. further investigation on the mtas role in traits should be done in future studies. divakar et al. (2023) also looked into the association of 30 est­ssr markers with yield trait components in 70 sugarcane accessions. the marker sem407 was found to be highly associated with %brix, sucrose content, and percent sugar recovery. in fiber crops, a total of 28 ssr markers were used to characterize the genetic structure of the fiber genes of flax. mixed linear model analysis revealed that lu10­600 and lu15­300 were observed associated with fiber length while lu7­150 was the lone allele correlated with fiber strength. four markers (lu5­50, lu7­150, lu10­ 600, and lu15­300), on the other hand, were observed highly related to fiber yield. these markers adv. hort. sci., 2025 39(1): 55­67 62 iaa. some of the identified lussaur genes were also found to be regulated in the cell wall thickening of the phloem bundle and the salt response of flax (bao et al., 2024). the response of the 15 randomly selected saur genes of ramie was also observed under drought and high­temperature conditions. eight of the bnsaur genes were regulated by both abiotic stress but mostly downregulated during high temperatures. most notably, bnsaur43 showed enhanced expression when subjected to drought conditions. these identified saur genes can be used for early detection of auxin­influenced traits in ramie (huang et al . , 2016). various saur genes are differentially expressed in the leaves of agave indicating their role in the leaf development of the crop (deng et al., 2019). the studies mentioned revealed not only the possible role of auxin during stress conditions for fiber crops but also auxin’s accumulation in the fiber cells meant a better fiber strength. the markers identified in this study can be used later to determine the relationship of abiotic stress with the fiber quality of abaca. the preliminary nature of this study also calls for further studies on expression analysis of auxin. cellulose synthase genes in this study, the molecular marker generated from cellulose synthase­like protein d5 explains 35% of the phenotypic variation in the tensile strength of the abaca accessions. cellulose is an essential carbohydrate that makes up the plant cell wall. it is a chain typically composed of 36 β­1,4­glucose units synthesized from the cesa proteins produced by the cellulose synthase genes. the functions of cesa genes were first studied in arabidopsis using mutation. from the study of beeckman et al. (2002), it appears that out of the 10 cesa genes, only four ­ cesa1, cesa2, cesa3, and cesa9 ­ function in synthesizing primary cell walls in the embryo of a. thaliana while atcesa 4,7, and 8 are responsible for secondary cell wall synthesis. the latter set of genes was confirmed to be upregulated in the secondary cell walls of cotton fibers (betancur et al., 2010). based on differential gene expression analysis, four cellulose synthase genes have been identified in flax, namely luscesa1, luscesa3­b, luscesa4, and luscesa8­a while ramie has cesa3 and cesa8 (xie et al., 2020). these genes were observed to be upregulated during cell wall biosynthesis in these fiber crops. elucidating the history of gene families can result can be utilized to screen flax genotypes with good fiber quality potential for breeding (nag et al., 2020). qin et al. (2015) evaluated the relationship of genic ssr markers with 3 fiber quality traits in cotton. they found that 26 molecular markers were positively correlated with quality and fiber strength, exhibiting most of the association (12 markers). the genome­ wide ssr markers were also used in association mapping in cotton. a total of 106 gene markers were determined to be significantly associated with six fiber quality traits, including fiber strength, fiber upper half length, short fiber, micronaire value, and fiber uniformity. the fiber strength had the greatest number of correlated markers (61 markers) in the study of nie et al. (2016). marker­assisted breeding (mab) programs are especially challenging when few available markers cover the upland cotton’s complex genetic background (qin et al., 2015). with the increase in the identified correlated molecular markers, hybrids were developed from upland cotton, and 52 significant loci were found related to six fiber quality traits from the parents and hybrids (huang et al., 2018) showing the success of gene specific markers in marker assisted breeding. auxin significant mtas from the cds of auxin response factor 9 and auxin­responsive iaa30­like protein were positively correlated with tensile strength and percent fiber strain. four abaca accessions, namely, tangongon, laylay, kutay­kutay, and native have showed amplification in these markers and exhibited high girth and tensile strength. auxin promotes fiber elongation in cotton (zhu et al . , 2022). its accumulation in fiber cells during growth is also responsible for the development of cotton fiber cells (zhang et al., 2017). in a gene expression analysis, auxin was observed to control the activity of reactive oxygen species and secondary wall deposition in fiber cells of cotton (zhang et al., 2020). buyyarapu et al. (2011) used cgssr markers from auxin to determine its relationship with fiber development in cotton and these markers were found polymorphic for the 26 species of cotton, suggesting their varying number and effect in the cotton genome. the saur gene family, also known as small auxin­up rna genes are responsible for the growth of plant tissues at different stages in flax (l. usitatissimum l.) the genome­wide analysis of the saur genes revealed 86 lussaur genes. it was also found that these genes are highly influenced by the application of exogenous mendoza et al. ‐ association mapping of abaca fiber related intragenic molecular markers 63 large gene family composed of four subfamilies, namely, α­expansin, β­expansin, expansin­like a, and expansin­like b (ding et al., 2016). the expansin genes that belonged to the α and β evolved from a common ancestor and shared similar introns. based on the analysis of rice expansin genes, the promoter possessed responsive elements to hormones (lee et al., 2001). the relationship of expansin proteins with hormones was also observed in poplar trees (populus nigra l.) and aspen (populus tremula l.). the increase in salt stress caused the decrease of ptrexpa3 and pnexpa3 activities resulting in decreased size of petioles, leaves, and internodes but increased size of epidermal cells (kuluev et al., 2017). this shows that expansin proteins are responsible for stress­related events. phylogenetics provided vital information and association analysis of expansin genes to distantly related species. this also directs researchers to a more systematic approach when manipulating exp genes. screening genomic libraries and pcr amplification revealed that out of the six expansin genes of cotton (ghexp1‐ghexp6), ghexp1 and ghexp2 are involved in the elongation of the cotton cells during fiber development (harmer et al., 2002). in bast fibers like flax, expansin was observed to be upregulated during cell wall thickening, indicating its role in fiber cell rearrangement during maturation (mokshina et al . , 2020). expansin genes are upregulated during oxidative and osmotic stresses such as drought and high salt conditions (gao et al., 2018). the genes identified in the abaca accessions were expansin a2 and a4 and were highly correlated with the number of suckers and dry weight. this indicates that using simple laboratories and computation techniques can link genes to their functions and accessions with the trait of interest. cobra‐like genes this protein family is glycosylphosphatidylinositol­ chained proteins that modulate the orientation of cell expansion and cellulose crystallinity. the tetraploid g. hirsutum has 14­15 cobl genes more than the g. raimondii and g. arboretum. ghcobl9 and ghcobl13 were upregulated with cellulose synthase genes during secondary cell wall biosynthesis. based on qtl analysis, ghcobl9 is correlated with fiber quality (niu et al., 2015). expression analysis also revealed that ghcobl9 was constantly upregulated during secondary cell wall thickening in cotton (he et al., 2025). mechanical resistance to lodging in sorghum (sorghum bicolor) to better functional analysis for musa species, given the transcriptomic analysis for the crop is limited (cenci et al., 2014). in abaca, only cesa 7‐5 and cesa 9‐7 were upregulated in abuab while expansin and sucrose synthase genes were upregulated in pacol. based on the generated gene expression data, bc1 resembles abuab while bc2 and bc3 are more like the pacol parent (reamillo, 2018). the mutation in cesa7 was found to show reduced fiber cell wall thickness and cellulose content as well as the absence of secondary cell walls (maleki et al., 2016). this shows the importance of cellulose synthase genes in the strength and thickness of the fibers. tinawagang puti is an abaca variety from the bicol region and is one of the recommended varieties of philfida. increasing its alpha cellulose and hemicellulose content was seen possible through addition of phosphorus sources (bondad et al., 1979); however, the reason behind this physiological response was not fully identified elucidating the underlying molecular mechanism would provide more valuable information and a more systematic approach to improving such abaca accessions. because there are few studies on m. textilis nee transcriptomics, focusing on the gene sequences of prominent fiber gene families and their probable phylogenetic relationship with model species will provide a better understanding of the expression control of the plant. in our study, the markers designed from the cellulose synthase genes studied by reamillo (2018) exhibited an association with the abaca accessions’ tensile strength and pseudostem girth size. with these molecular markers, simpler molecular approaches (i.e. pcr amplification) can be applied to identify accessions with the cellulose synthase gene and good fiber quality. expansin genes the expansin markers explained the phenotypic variation of the number of suckers and dry weight by 0.6% and 57%, respectively. cell walls must be sturdy enough to avoid damage brought by cell enlargement or external force and should also be flexible enough to permit some impact and induce wall relaxation. this ability is known as cell wall loosening. expansin proteins are released to mediate the cell wall loosening (sampedro and cosgrove, 2005). these proteins are also used by grass pollens to soften the maternal cell walls of stigmas (cosgrove, 2016). they also affect the germination and the fruiting stage of plants. expansin, like cesa and sucrose synthase, is a adv. hort. sci., 2025 39(1): 55­67 64 was observed reduced by only one mutation in sbbc1 genes that encode cobra­like proteins. not only did it affect mechanical strength but also the cellulose content and lignin content. these components decreased and increased, respectively. this suggests that sbbc1 genes are involved in cell wall biosynthesis in sorghum (li et al., 2019). this protein was found along the sides of arabidopsis roots where there is an increased elongation activity (schindelman et al . , 2001). cobra acts as a “polysaccharide chaperone” that binds to individual β­1,4­linked glucan units resulting in cellulose crystallization (sorek et al., 2014). in‐silico analysis revealed thirteen glycophosphatidylinositol anchors encoding genes from nine hevea genomes that share similar functions with their arabidopsis counterparts. out of these thirteen genes, nine clustered in the cobra gene subfamily­1 while the rest clustered with cobra gene subfamily­ii (putranto et al., 2017). the mutated slcobra­like proteins of tomato caused cell wall degradation by upregulating wall­degrading genes while overexpressed slcobra­like proteins resulted in the thicker cell wall of fruits. this indicates that cobra­like proteins participate in cell wall biosynthesis (cao et al., 2012). in flax, their role is vague, but are upregulated during the primary, secondary, and tertiary cell wall formation (mokshina et al., 2020). based on expression analysis, a similar role was also observed in hemp, where the cbl 4 gene is active during cell wall formation (koziel, 2010). in poplar trees, the cobra­like genes are involved in cellulose microfibril orientation (gritsch et al., 2015). in the study of reamillo (2018), cobra­ l ike proteins were upregulated in abuab and downregulated in the backcrosses. in our work, only the cobra­like protein 4 was highly correlated with the number of suckers, explaining 7.8% of the phenotypic variation in the abaca collection. this trait showed a negative relationship with tensile strength and can be used to determine the fiber quality even at the juvenile stage of abaca. this study explores the use of intragenic molecular markers to identify significant mtas (p­ value <0.005) related to fiber quality and tensile strength of abaca. eight mtas related to five traits were found to have a positive significant effect of up to 263 and percent phenotypic variance explained ranging from 0.6% to 57%. the molecular markers from the auxin, cellulose synthase, expansin, and cobra­ l ike gene families related to fiber development were identified in this study. however, it is suggested that these genes should undergo subsequent experiments to solidify their role in the abaca fiber quality and tensile strength. the markers identified in this study can be used to fast­track the crop improvement of abaca, for only the hybrids that possess the gene of interest shall be tested in advanced yield trials. the marker­assisted selection increases the precision of breeding in abaca and lowers agricultural input waste during field testing. with this research, we determined nine abaca accessions that possess both traits and markers of interest and were used to generate 49 seeds. these hybrids will be evaluated using morphological and molecular markers in a future study. acknowledgements this research was supported and funded by the 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acid biosynthesis in cotton. ­ plant biotechnol. j., 20(3):423­ 425. impaginato 175 adv. hort. sci., 2025 39(3): 175­189 doi: 10.36253/ahsc­16747 https://oaj.fupress.net/index.php/ahs effect of auxins on the rooting of the avocado rootstock ‘duke 7’ m. martínez­villagómez 1, a.f. barrientos­priego 1 (*), j.o. mascorro­ gallardo 1, g. iturriaga 2, m.c. espíndola­barquera 3 1 departamento de fitotecnia, universidad autónoma chapingo, km 38.5 carretera méxico‐texcoco, c.p. 56230 texcoco, mexico. 2 división de estudios de posgrado e investigación, tecnológico nacional de méxico/i.t. roque, km 8 carretera celaya‐juventino rosas, roque, c.p. 38110 celaya, mexico. 3 fundación salvador sánchez colín‐cictamex, s.c., ignacio zaragoza, col. centro, c.p. 51700 coatepec harinas, mexico. key words: callus formation, clonal propagation, persea americana mill., root quality index. abstract: clonal propagation of avocado rootstocks through etiolated shoot rooting represents a key strategy to enhance genetic uniformity, plant health, and productivity in commercial orchards. however, its success largely depends on the rooting phase, where auxins play a critical role. this study evaluated the effect of auxin­based rooting agents (types and concentrations) on root induction and quality in etiolated shoots of the ‘duke 7’ rootstock. five agents (iaa, naa, iba, k­iba, and iba + naa combination) were tested at three concentrations (24.6, 34.4, and 44.2 mm) under a completely randomized factorial design (5 × 3) with three replicates per treatment. morphological variables included rooting percentage, survival rate, root number/length/ diameter, secondary root development, callus formation, and root quality index (rqi). results revealed significant effects of agent type, concentration, and their interaction. naa (34.4 mm) was the most effective for root number (55.3) and rqi (154.9 cm), albeit with high callus formation and reduced secondary roots. the iba + naa combination (34.4 mm) also showed high rqi (140.4 cm), with greater root length and less negative impact on root architecture. iba alone achieved 100% rooting with moderate root development, balancing efficacy and physiological tolerance. overall, intermediate concentrations of naa and iba + naa yielded optimal results. these findings can refine clonal propagation protocols for ‘duke 7’, with direct applications in commercial nurseries producing high­performance rootstocks. 1. introduction commercial avocado trees result from the combination of tissues from two distinct plants: a scion that forms the canopy and a rootstock that (*) corresponding author: abarrientosp@chapingo.mx citation: martínez­villagómez m., barrientos­ priego a.f., mascorro­gallardo j.o., iturriaga g., espíndola­barquera m.c., 2025 ­ effect of auxins on the rooting of the avocado rootstock ‘duke 7’. ­ adv. hort. sci., 39(3): 175­189. orcid: mm: 0000­0003­1132­2630 baf: 0000­0001­6555­7633 mjo: 0000­0001­9713­4758 ig: 0000­0002­2535­0184 emc: 0000­0003­0247­6267 copyright: © 2025 martínez­villagómez m., barrientos­ priego a.f., mascorro­gallardo j.o., iturriaga g., espíndola­barquera m.c. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 29 october 2024 accepted for publication 17 july 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16747 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0003-1132-2630 http://orcid.org/0000-0001-6555-7633 http://orcid.org/0000-0001-9713-4758 http://orcid.org/0000-0002-2535-0184 http://orcid.org/0000-0003-0247-6267 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 175­189 176 provides the root system (gleeson et al., 2016). this propagation technique enables cultivars to achieve early productivity while preserving their phenotypic traits, even when grafted onto juvenile plants (melnyk, 2017). in mexico, commercial avocado nursery production primarily relies on seed­propagated rootstocks derived from native trees exhibiting broad yet poorly characterized genetic diversity (salazar­ garcía et al., 2004 a). this genetic variability leads to heterogeneous tree growth (medina­urrutia et al., 2017) and increased susceptibility to diseases, pests, and abiotic stressors such as drought, salinity, or nutrient imbalances (salazar­garcía et al., 2004 a). furthermore, rootstock type has been documented to directly influence key agronomic parameters including yield, tree size and vigor, as well as fruit quality and postharvest life (barrientos­priego, 2017). the most effective strategy to mitigate issues arising from genetic heterogeneity involves using clonal rootstocks (salazar­garcía et al., 2004 b; cohen et al., 2023). although avocado trees grafted onto clonal rootstocks are more expensive than those on seedling rootstocks (cohen et al., 2023), certain mexican production areas affected by phytophthora cinnamomi rands (ochoa­fuentes et al., 2007; sánchez­gonzález et al., 2019), clay soils (salazar­garcía et al., 2015), alkaline ph, and soil salinity (medina­urrutia et al. , 2017) would significantly benefit from their implementation. while clonal rootstocks can standardize production, they present inherent technical limitations, particularly during the rooting phase (ernst, 1999; gleeson et al . , 2016). current commercial methodologies for clonal propagation of avocado species are primarily derived from the technique established by frolich and platt (1972). this approach, albeit with potential minor modifications, involves performing air layering on an etiolated shoot, separating it from the mother plant once a root system develops, and grafting the commercial cultivar during the rooting phase when stem diameter permits (ernst, 1999; ernst et al., 2013). the formation of adventitious roots in woody species such as avocado is regulated by the balance of growth regulators, with auxin application being one of the most effective strategies to promote rooting (zhao et al., 2022). synthetic auxins like indole­3­butyric acid (iba) and 1­naphthaleneacetic acid (naa) serve as the primary regulators of adventitious root formation through complex interactions that modulate metabolic, transport, and signaling processes (lakehal and bellini, 2019). recent studies demonstrate that auxin type and concentration influence rooting quality through mechanisms such as molecular stability, transport, and metabolite conjugation (damodaran and strader, 2019; gomes and scortecci, 2021). however, current avocado propagation protocols remain based on classical work (frolich and platt, 1972), typically limited to iba application at 7000 mg l⁻¹ (ernst, 1999), without considering the efficacy of other auxin types or concentrations. furthermore, few recent studies have explored these aspects (li et al., 2024) for standard rootstocks like ‘duke 7’. in this context, the objective of this study was to evaluate the effect of different auxin types and concentrations on root induction and rooting quality in etiolated shoots of the ‘duke 7’ rootstock. this work aims to establish an efficient vegetative propagation protocol to enhance production of clonal avocado rootstocks in mexico, thereby improving genetic uniformity and resilience of commercial plantations. 2. materials and methods experimental site and plant material this study adopted the propagation method proposed by hofshi (1996) and ernst (1999), adapted to the environmental conditions of a greenhouse located in chapingo, texcoco, state of mexico (19.4904322, ­98.8734917) at 2264 meters above sea level. the research was conducted during 2022 and 2023. nurse plants were produced using west indian avocado seeds (70±17.4 g) from veracruz, a size determined optimal for etiolated shoot development during rooting (castro et al., 2021). seeds were sown on november 1, 2022, in 1000 cm³ polyethylene bags filled with a 1:1:1 (v/v/v) substrate mixture of peat moss, volcanic rock, and perlite. plants received light irrigation and preventive applications of fungicide (benomyl 1 g l⁻¹) and insecticide (imidacloprid 1 ml l⁻¹) until grafting. on february 17, 2023, nurse plants were grafted with mature ‘duke 7’ buds at 5 cm above substrate level, retaining only two buds per scion. graft wounds were sealed with polyvinyl acetate resin to prevent desiccation. during graft union formation, martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 177 experimental design the study employed a completely randomized factorial design (5 × 3) with three replications per treatment. each experimental unit consisted of three air­layered plants. the first factor represented auxin type with five levels, while the second factor comprised three concentrations: 24.6, 34.4, and 44.2 mm (table 1). this design enabled systematic evaluation of 15 treatment combinations under standardized conditions. for each treatment, 20 ml of solution was prepared using distinct protocols according to auxin type. the potassium salt of indole­3­butyric acid (k­ iba) and commercial iba+naa formulation (dip’n grow®) were prepared exclusively with distilled water. for other auxins (iaa, naa, iba), a two­step dissolution was employed: initial solubilization in 96% ethanol (5 ml for 24.6 mm, 7 ml for 34.4 mm, or 9 ml for 44.2 mm) followed by volume completion with distilled water. this methodology ensured complete auxin solubility while maintaining precise target concentrations across all experimental treatments. evaluated variables eighty days after treatment application, the air­ layered shoots were carefully separated from nurse plants and the substrate adhered to the roots was removed through water immersion with intermittent manual agitation. quantitative assessments included: shoot survival (percentage of viable shoots), rooting success (proportion of shoots that emitted at least one root of 0. 5 cm in length or more), root number (mean per rooted shoot), root dimensions (length and diameter of five primary roots measured with mitutoyo® digital caliper, ±0.01 mm precision), secondary root development (percentage of shoots with secondary roots), callus formation (percentage of shoots showing callus at wound site), and the root quality index (rqi) calculated as the product of mean buds were covered with transparent polyethylene bags (5 × 8 cm) which were removed when plants were transferred to the etiolation chamber. experimental establishment once etiolated shoots reached 25­30 cm in length (ernst, 1999), treatments were applied. at the base of each shoot, air layering was performed using a cutting blade periodically disinfected with ethanol (70%). a wound of approximately 2 cm in length was caused, to which 100 µl of rooting growth regulator was applied. each treated shoot was then placed in a 150 cm³ transparent plastic container filled with coconut coir dust. the treated plants were maintained in a shaded area within the greenhouse and watered periodically to maintain adequate moisture levels in both the air­ layering substrate and the nurse plant’s growing medium. all rooting formulations were prepared fresh on the day of treatment application and were not stored for subsequent use, ensuring consistent growth regulator activity and concentration for each experimental unit. during the study period, greenhouse conditions exhibited natural variability, with mean temperatures gradually increasing from 16.3 to 23.7°c while relative humidity fluctuated between 54% and 64% (fig. 1). fig. 1 ­ average monthly ambient temperature and relative humidity variation during the experimental period. table 1 ­ experimental treatments: auxin types and concentrations evaluated in the rooting of ‘duke 7' avocado rootstock etiolated shoots treatment concentrations (mm) rooting agent (auxin type) indole­3­acetic acid (iaa, sigma®) 24.6 34.4 44.2 1­naphthaleneacetic acid (naa, sigma®) 24.6 34.4 44.2 indole­3­butyric acid (iba, sigma®) 24.6 34.4 44.2 potassium salt of indole­3­butyric acid (k­iba, sigma®) 24.6 34.4 44.2 combination of iba + naa (dip’n grow®) 24.6 34.4 44.2 adv. hort. sci., 2025 39(3): 175­189 178 root number and average length (cm). this comprehensive evaluation protocol enabled systematic comparison of treatment effects on both root initiation and development. statistical analysis for statistical analysis, mean values per experimental unit were used. the data were processed using the glm procedure in sas® statistical software (sas ondemand for academics, version 3.1.0; sas, 2021). variables expressed as proportions or percentages were subjected to arcsine square root transformation of the original decimal fraction values to meet assumptions of normality and homogeneity of variances. following analysis of variance (anova), when significant differences (p<0.05) were detected for treatment effects or their interactions, means were compared using tukey’s test. data values were back­ transformed to their original units for presentation and interpretation. the statistical model applied was as follows: yij= μ + ai + cj + acij + εij where: yij is the value of the variable evaluated with the i­th rooting agent and the j‐th concentration; µ is the overall mean; ai is the fixed effect of the i­th rooting agent; cj is the fixed effect of the i­th concentration; acij is the fixed effect of the interaction of the i­th rooting agent with the j­th concentration; and εij is the experimental error. 3. results and discussion the experimental results demonstrate the significant influence of auxin type and concentration on root development in etiolated shoots of avocado rootstock ‘duke 7’. our findings reveal distinct morphological responses to different auxin treatments, with particular combinations showing optimal performance in root initiation and development. these outcomes are analyzed through three critical lenses: their physiological implications for adventitious root formation, practical applications for commercial clonal propagation systems, and comparative relevance to established literature in avocado propagation. the data presentation focuses on treatment efficacy across measured parameters including rooting percentage, root architecture features, and callus formation patterns, providing a comprehensive evaluation of auxin effects on this economically important rootstock cultivar. analysis of variance analysis of variance revealed significant differences (p<0.05) among treatments for most evaluated variables, confirming the influence of auxin type and concentration on adventitious root development in etiolated avocado shoots. specifically, the rooting agent type showed highly significant effects on root quality index (p<0.001), root number (p<0.001), root length (p<0.01), and callus formation (p<0.01). concentration significantly affected only root number (p<0.01), while the rooting agent × concentration interaction was significant for survival rate, rooting percentage, root length and diameter, and secondary root presence (p<0.05) (tables 2 and 3). these findings demonstrate that both the composition of the rooting agent and its concentration distinctly influence various aspects of the rooting process, and that their interaction can substantially modify the morphological response of etiolated shoots. effect of the rooting agent the rooting agent type exerted a decisive table 2 ­ mean squares obtained in the analysis of variance to evaluate the effect of five rooting agents and three concentrations on variables related to the rooting of etiolated shoots of avocado rootstock ‘duke 7’ df= degrees of freedom; ssp= shoot survival percentage; pcp= percentage of callus presence; ns= not significant (p>0.05); * significant (p<0.05); **significant (p<0.01); ***highly significant (p<0.001); ×= interaction between factors. variation source df ssp rooting pcp percentage quality index rooting agent (a) 4 0.135 ns 0.133 ns 89156.6 *** 0.987 ** concentration (c) 2 0.067 ns 0.046 ns 8474.8 ns 0.488 ns a × c 8 0.269 * 0.135 * 12153.5 ns 0.183 ns experimental error 30 0.253 0.057 40686.9 0.230 martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 179 influence on multiple root development parameters in etiolated ‘duke 7’ shoots. although all treatments achieved rooting rates exceeding 94% (table 4), significant variations were observed in three critical aspects: 1) post­treatment survival rates, 2) root system quality (including architecture and developmental patterns), and 3) callus formation intensity at wound sites. these differential responses highlight the importance of precise auxin selection in clonal propagation protocols, where optimal root system architecture must be balanced with minimal callus interference for successful transplant establishment. among the evaluated rooting agents, 1­ naphthaleneacetic acid (naa) promoted the highest root number (55.3) and root quality index (154.9 cm) (table 4), confirming its efficacy as a potent inducer of adventitious root formation. this response likely stems from naa’s enhanced stability in plant tissues, reduced susceptibility to enzymatic degradation, and prolonged persistence at the application site (da costa et al., 2013; raggi et al., 2020). however, this treatment also reduced shoot survival to 98.1% (table 4), suggesting phytotoxic effects potentially linked to ethanol solvent use, and the heightened sensitivity of etiolated tissues to elevated auxin concentrations (amri, 2010; grossmann, 2009; ludwig­müller, 2020). these findings underscore the need to balance rooting efficacy with tissue tolerance, particularly for etiolated shoots whose cell walls exhibit modified xyloglucan and pectin composition. such alterations increase tissue flexibility but also enhance susceptibility to apoplastic ph imbalances when critical auxin thresholds are exceeded (duman et al., 2020; wang et al., 2025). in contrast, indole­3­acetic acid (iaa) and indole­ 3­butyric acid potassium salt (k­iba) exhibited table 3 ­ mean squares obtained in the analysis of variance to evaluate the effect of five rooting agents and three concentrations on variables related to the rooting of etiolated shoots of avocado rootstock ‘duke 7’ df= degrees of freedom; srp= secondary roots presence; ns= not significant (p>0.05); * significant (p<0.05); ** significant (p<0.01); ***highly significant (p<0.001); ×: interaction between factors. variation source df roots number length diameter srp rooting agent (a) 4 2545.16 *** 1.154 ** 0.106 ns 2.133 *** concentration (c) 2 675.16 ** 0.018 ns 0.012 ns 0.020 ns a × c 8 128.08 ns 0.460 * 0.283 * 0.431 * experimental error 30 133.93 0.120 0.123 0.144 table 4 ­ average values by type of rooting agent for the variables: percentage of shoot survival, percentage of rooting, rooting quality index, and percentage of callus presence in the rooting of etiolated shoots of the avocado rootstock ‘duke 7’ (z) average values in the same column followed by different letters indicate statistical differences (tukey, p<0.05). iaa= indole­3­acetic acid; iba= indole­3­butyric acid; naa= 1­naphthalene acetic acid; k­iba= indole­3­butyric acid potassium salt; ssp= shoot survival percentage; pcp= percentage of callus presence; cv= coefficient of variation; hlsd= honest least significant difference. rooting agent ssp (%) rooting pcp (%)percentage quality index (cm) iaa 100.0 az 94.2 a 40.1 c 68.86 ab naa 98.1 b 96.0 a 154.9 a 87.89 a iba 100.0 a 100.0 a 100.2 b 13.74 b k­iba 100.0 a 94.2 a 34.0 c 68.86 ab iba + naa 100.0 a 100.0 a 98.3 b 83.56 a cv (%) 5.94 16.44 43.07 50.88 hlsd 1.57 10.20 50.36 37.09 average 99.93 98.46 85.51 65.33 naa combination (dip’n grow®) demonstrated an optimal balance between shoot survival and root quality (table 4), establishing them as viable candidates for clonal avocado propagation protocols. these rooting agents produced consistent, reliable responses particularly valuable for nurseries requiring both high rooting success and preservation of etiolated shoot viability. the observed performance suggests these formulations effectively navigate the critical compromise between root induction efficacy and minimal phytotoxicity, a decisive advantage for commercial scale production of ‘duke 7’ rootstock. the evaluation revealed significantly higher callus formation with naa (87.9%) and iba+naa (68.9%) treatments compared to iba alone (13.7%) (table 4), consistent with previous reports of synthetic auxins promoting unorganized tissue proliferation (zhai and xu, 2021). while callus formation may initially facil itate root primordia initiation, excessive development can negatively impact rooting success through three primary mechanisms: physical obstruction of emerging roots, disruption of normal root system architecture, and competition for essential metabolic resources that would otherwise support root growth (chen et al., 2020). the current study revealed that callus development was solely influenced by the type of rooting agent applied, with no consistent correlation observed between callus presence and root quantity. while naa treatment produced both the highest callus formation (87.89%) and root number (55.29), iba which generated minimal callus (13.74%) still induced intermediate root formation (34.04) (tables 180 adv. hort. sci., 2025 39(3): 175­189 maximum survival rates (100%) but produced limited root formation (14.9 and 14.4 roots, respectively; table 5) and significantly lower root quality indices (table 4). this reduced efficacy likely stems from iaa’s inherent instability, being rapidly degraded by peroxidase enzymes and light exposure (roussos, 2023; yun et al., 2023). furthermore, iaa readily forms biologically inactive conjugates with amino acids and sugars, substantially reducing its bioavailability and root­promoting activity (pincelli­ souza et al., 2024). while potassium indole­3­butyric acid potassium salt (k­iba) offers greater stability than iaa and eliminates the need for organic solvents in solution preparation (lesmes­vesga et al., 2021), its efficacy as a rooting inducer appears constrained by distinct physiological transport limitations (yang et al., 2022). in its ionic form, k­iba demonstrates restricted apoplastic diffusion, significantly impeding passive transport to target cells near the application site in etiolated shoots. effective mobilization instead requires active transport mechanisms (roussos, 2023) mediated by specialized carrier proteins, including aux1/lax family influx transporters and pin­formed (pin) and abcb efflux transporters, which collectively regulate auxin distribution across cellular membranes (hammes et al., 2021). crucially, k­iba must undergo conversion to its non­ionic (protonated) form to cross plasma membranes and subsequently trigger adventitious root formation (pincelli­souza et al., 2024), adding a metabolic conversion step that may delay or limit its biological activity compared to more mobile auxin forms. in contrast, both iba and the commercial iba + table 5 ­ mean values by rooting agent type for the variables number, length, and diameter of roots, and presence of secondary roots in the rooting of etiolated shoots of the avocado rootstock ‘duke 7’ (z) average values in the same column followed by different letters indicate statistical differences (tukey, p<0.05). iaa= indole­3­acetic acid; iba= indole­3­butyric acid; naa= 1­naphthalene acetic acid; k­iba= indole­3­butyric acid potassium salt; srp= secondary roots presence; cv= coefficient of variation; hlsd= honest least significant difference. rooting agent roots srp (%) number length (cm) diameter (mm) iaa 14.92 c (z) 2.29 b 1.49 a 80.7 a naa 55.29 a 2.83 ab 1.59 a 39.8 b iba 34.07 b 2.90 a 1.53 a 95.8 a k­iba 14.40 c 2.27 b 1.60 a 58.7 a iba + naa 32.11 b 3.03 a 1.77 a 58.7 a cv (%) 38.37 16.77 22.06 44.17 hlsd 15.82 0.61 0.48 24.59 average 30.16 2.67 1.59 57.33 martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 181 4 and 5). contrasting with findings in woody eucalyptus species where adventitious roots originate from callus tissue (fett­neto et al., 2001; zhang et al., 2022), our observations demonstrated direct root emergence from stem tissue above the auxin application site, without visible callus involvement (fig. 2). this response suggests etiolated ‘duke 7’ shoots maintain an intrinsic capacity for direct rhizogenesis, a phenomenon previously documented in other avocado rootstocks like ‘vc801’ (duman et al., 2020). morphological analysis revealed significant suppression of secondary root growth following naa application (fig. 2), likely due to auxin­induced temporal inhibition of lateral root development in primary root tissues. this phenomenon aligns with observations in arabidopsis thaliana (biswas et al., 2019), where supraoptimal auxin levels negatively affect lateral root formation through disruption of polar auxin transport in pericycle cells, cell cycle arrest in lateral root primordia, and downregulation of lateral root­promoting genes such as arf7 and arf19. while naa effectively stimulates primary root formation in avocado rootstock ‘duke 7’, our results indicate that higher concentrations may delay optimal root system development by inhibiting secondary branching. this architectural limitation presents key practical challenges: extended production timelines due to delayed shoot separation from nurse plants, and potential requirement for additional agronomic interventions (e.g., supplemental growth regulator treatments) to promote secondary root growth before transplanting. these findings suggest that while naa remains a potent rooting agent, commercial nurseries should carefully evaluate the trade­off between rapid root initiation and subsequent root system complexity when selecting auxin formulations for clonal propagation. the findings of this study offer valuable guidance for optimizing clonal propagation of avocado rootstocks in commercial settings. for nurseries prioritizing root quantity, naa emerges as the most effective option despite its tendency to reduce secondary root development. iba presents a balanced alternative, producing intermediate root numbers (34.0) while maintaining excellent shoot survival rates (100%), making it particularly suitable for operations where plant viability is paramount. the commercial iba + naa formulation (dip’n grow®) provides a practical ready­to­use solution that combines the benefits of both auxins while simplifying nursery workflows. importantly, the results demonstrate that iaa and k­ iba are unsuitable for large­scale propagation due to their limited root induction capacity (14.9 and 14.4 roots respectively) and inherent biochemical instability. these evidence­based recommendations allow propagation specialists to select auxin treatments fig. 2 ­ influence of five rooting agents on callus formation and secondary root development in the rooting of etiolated shoots of the avocado rootstock ‘duke 7’. iaa= indole­3­acetic acid; iba= indole­3­butyric acid; naa= 1­naphthalene acetic acid; k­iba= indole­ 3­butyric acid. based on their specific production requirements, whether the priority is maximizing root biomass, ensuring transplant success, or streamlining operational efficiency. these findings represent a significant advancement over traditional iba­only protocols (ernst, 1999), which typically employ high concentrations (34.4 mm). the demonstrated benefits of auxin diversification align with recent studies in other woody species like apple (malus spp.) and mulberry (morus alba), where combined auxin treatments have outperformed iba in promoting adventitious root formation (sourati et al., 2022; tahir et al., 2022; wang et al., 2024). the research confirms that auxin selection critically impacts not just rooting efficiency but also root system morphology, both determining factors for successful clonal propagation of avocado rootstocks. these morphological qualities ultimately influence field establishment and long­term productivity of grafted trees. effect of the concentration the study revealed significant auxin concentration effects on key root development, particularly root number (table 6). a clear dose­dependent response was observed, with progressive increases in root formation corresponding to higher auxin concentrations: from 24.6 roots per shoot at 24.6 mm to 37.6 roots at 44.2 mm (table 6). this pattern aligns with classical auxin response curves reported in the literature (nissen, 1985), where rooting typically improves with increasing auxin concentrations up to an optimal threshold, beyond which phytotoxic effects suppress root initiation adv. hort. sci., 2025 39(3): 175­189 182 (nissen, 1985; sahoo et al., 2021). historically, avocado clonal propagation has employed varying concentrations of auxins, particularly indole­3­butyric acid (iba) as the most commonly used rooting agent. reported applications range from 500 to 10,000 mg l⁻¹, equivalent to approximately 2.5­51.3 mm (rogel­castellanos et al., 2000; mindêllo­neto et al., 2006; li et al., 2024). significant cultivar­specific differences emerge from these protocols: in ‘duke 7’, iba application at 2,500 mg l⁻¹ achieved 56.6% rooting success (li et al., 2024), while ‘fuerte’ required five­fold lower concentrations (500 mg l⁻¹) to reach 47.5% efficacy (mindêllo­neto et al., 2006). notably, the rootstock ‘dusa’ demonstrated striking physiological­state dependence, with 82% rooting in etiolated shoots versus less than 10% in non­etiolated tissue at 2,500 mg·l⁻¹ (li et al., 2024), highlighting the critical importance of the plant material’s physiological condition in propagation success. these collective findings underscore the dual influence of genetic factors and tissue physiology in determining optimal auxin protocols for different avocado cultivars. these historical precedents contrast sharply with the findings of the current study, where the tested concentration range (24.6 to 44.2 mm) achieved rooting success rates exceeding 95% while maintaining shoot survival rates in most treatments (table 3). furthermore, the results demonstrate that precise concentration adjustments can simultaneously maximize rooting efficiency and improve root system quality without inducing the severe adverse effects (particularly phytotoxicity) typically observed when exceeding optimal naa concentration (yan et al . , 2014). this refined table 6 ­ mean values by rooting hormone concentration for the variables number, length, and diameter of roots and presence of sec­ ondary roots in the rooting of etiolated shoots of the avocado rootstock ‘duke 7’ (z) average values in the same column followed by different letters indicate statistical differences (tukey, p<0.05). srp= secondary roots presence; cv= coefficient of variation; hlsd= honest least significant difference. concentration roots srp (%)number length (cm) diameter (mm) 24.6 mm 24.62 b (z) 2.65 1.56 61.50 a 34.4 mm 28.24 ab 2.65 1.60 55.23 a 44.2 mm 37.62 a 2.71 1.62 55.23 a cv (%) 38.37 16.77 22.06 44.17 hlsd 10.42 0.40 0.32 11.22 average 30.16 2.67 1.59 57.33 martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 183 approach represents a significant improvement over traditional protocols, as it achieves near­universal rooting success while avoiding the compensatory trade­offs between root quantity and plant viability that characterize many existing methods. the study specifically identified 34.4 mm as the most balanced concentration for commercial applications, combining high rooting percentages (98.1%) with excellent root architecture development and minimal callus formation (tables 6 and 7). the clonal propagation of ‘duke 7’ rootstock has presented particular difficulties. previous studies reported limited success, with only 26% rooting after 180 days when using non­etiolated shoots treated with dip’n grow® at 3,000 mg l⁻¹ (approximately 15.2 mm equivalent) (salazar­garcía et al., 2004 b). alternative approaches using iba­saturated wood chips (10,000 mg l⁻¹) on etiolated shoots improved rooting to 60% (escobedo and escobedo, 2011). in marked contrast, the current study demonstrates that optimized auxin selection and concentration in etiolated shoots can achieve 100% rooting efficiency with superior morphological quality, results that substantially surpass all previously reported values for this challenging rootstock. this breakthrough reflects both the importance of physiological preconditioning (etiolation) and precise auxin formulation in overcoming the historical propagation barriers for ‘duke 7’. from a physiological perspective, the enhanced efficacy observed at higher auxin concentrations may stem from increased hormone availability at the application site, promoting activation of key genes involved in cellular differentiation (such as wox11/12, arf, and lbd) that are essential for adventitious root formation (lakehal and bellini, 2019; li et al., 2024). however, this concentration­ dependent effect was not uniform across all measured parameters. callus formation, root length and diameter, and secondary root development showed no significant differences between concentrations (tables 6 and 7), suggesting that structural root system quality may be modulated by additional factors beyond concentration alone, including auxin type, formulation characteristics, and local hormonal interactions (druege et al., 2016; lakehal and bellini, 2019). these differential responses highlight the complex regulatory networks governing root organogenesis, where concentration primarily drives root initiation while other factors determine subsequent root architecture development. interactive effects of rooting agent type and concentration the significant interaction between auxin type and concentration across multiple key rooting variables demonstrates that the morphogenic response of etiolated ‘duke 7’ shoots depends not merely on the auxin type or applied dose in isolation, but rather on their specific combination (tables 1 and 2). this interaction was particularly pronounced for critical parameters including: survival rate, rooting percentage, root length and diameter, and secondary root presence (fig. 3). the non­additive effects reveal complex phytohormonal regulation where certain auxin­concentration combinations synergistically enhance rhizogenesis while others exhibit antagonistic relationships, suggesting tissue­specific saturation thresholds for different auxin table 7 ­ mean values by rooting agent concentration for the variables: survival percentage, rooting percentage, quality index, and callus presence percentage in the rooting of etiolated shoots of the avocado rootstock ‘duke 7’ (z) average values in the same column followed by different letters indicate statistical differences (tukey, p<0.05). ssp= shoot survival percentage; pcp= percentage of callus presence; cv= coefficient of variation; hlsd= honest least significant difference. concentration ssp (%) rooting percentage quality index (cm) pcp (%) 24.6 mm 100.00 a (z) 98.91 a 71.74 a 75.00 a 34.4 mm 100.00 a 99.33 a 80.56 a 74.66 a 44.2 mm 99.30 a 96.55 a 104.24 a 58.42 a cv (%) 5.94 16.44 43.07 50.88 hlsd 0.68 4.51 33.15 17.46 average 99.93 98.46 85.51 65.33 adv. hort. sci., 2025 39(3): 175­189 184 fig. 3 ­ interaction of auxin type and concentration on rooting of etiolated shoots of avocado rootstock ‘duke 7’. iaa: indole­3­acetic acid, naa= 1­naphthalene acetic acid, iba= indole­3­butyric acid, k­iba= indole­3­butyric acid potassium salt, iba + naa= indole­3­butyric acid + 1­ naphthalene acetic acid (dip'n grow®). formulations. these findings necessitate a dual­ parameter optimization approach for clonal propagation protocols, as neither factor alone sufficiently predicts rooting performance. in contrast to other treatments, increasing iaa concentrations showed a positive correlation with rooting percentage, improving from 66.6% at 24.6 mm to 88.8% at 34.4 mm, and reaching 100% at 44.2 mm. for both iba and the iba + naa combination (dip’n grow®) maintained consistent 100% rooting across all three tested concentrations, indicating a broad efficacy window for these formulations. naa exhibited optimal performance at 24.6 mm and 34.4 mm (100% rooting), but efficacy declined to 77.7% at 44.2 mm, likely reflecting phytotoxic effects at higher doses. notably, k­iba performed best at the lowest concentration (100% at 24.6 mm), with progressively reduced rooting at higher levels (88.8% at 34.4 mm and 66.6% at 44.2 mm). these results clearly demonstrate the significant impact of the auxin type × concentration interaction on the rhizogenic response of etiolated avocado shoots (fig. 3). previous studies have demonstrated that the transport and physiological activity of auxins can vary significantly depending on their structure and molecular form (korasick et al., 2013). for example, iba undergoes conversion to both iaa and iba conjugates during plant tissue transport, resulting in prolonged, multiphasic rooting promotion (damodaran and strader, 2019). in contrast, externally applied iaa tends to remain in its free form during transport, making it more susceptible to enzymatic inactivation and oxidative degradation (hayashi et al., 2021). this difference may explain why iba shows higher efficacy at lower concentrations, while iaa requires higher doses to induce comparable rooting responses. naa exhibits superior chemical stability compared to other auxins, allowing prolonged activity in plant tissues (da costa et al., 2017). this stability stems from its synthetic molecular structure and likely involves specialized transporters that facilitate its movement and accumulation at target sites (yang et al., 2006; napier, 2021). studies report that naa resists rapid degradation or conjugation in plant tissues, enhancing its capacity to induce abundant root formation (nissen and sutter, 1990; gomes and scortecci, 2021) (fig. 4). however, at 44.2 mm, naa application resulted in the highest root numbers but reduced rooting percentage (77.8%) and suppressed secondary root development (fig. 4). these findings suggest that while certain formulations effectively promote primary root formation, they may also cause unintended effects like lateral root inhibition, potentially due to localized hormonal imbalance at the rooting site (lakehal and bellini, 2019; bhalerao et al., 2002). martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 185 the interaction between auxin type and concentration significantly influenced primary root length. the greatest average length (31.19 mm) was achieved with the iba + naa combination (dip’n grow®) at 34.4 mm, suggesting a synergistic effect favoring root elongation. in contrast, the shortest roots formed with k­iba at 34.4 mm (17.46 mm) and iaa at 24.6 mm (17.22 mm), indicating reduced efficacy in promoting cellular elongation under these specific conditions. for the remaining treatments, no statistically significant differences were observed, with an average length of 27.83 mm, suggesting a more uniform response across these combinations (fig. 3). fig. 4 ­ rooting of etiolated shoots of ‘duke 7’ rootstock with the application of iaa (indole­3­acetic acid), naa (1­naphthalene acetic acid), iba (indole­3­butyric acid), k­iba (indole­3­butyric acid potassium salt) and iba + naa (indole­3­butyric acid + 1­ naphthalene acetic acid) at three concentrations. anr= average number of roots; scale bar: 1.0 cm. adv. hort. sci., 2025 39(3): 175­189 186 root diameter was similarly affected by concentration, particularly in the case of iaa. at 24.6 mm, iaa produced thinner roots (0.97 mm), while at 34.4 mm, it generated thicker roots (2.05 mm), demonstrating a concentration­dependent response in radial root expansion. the other auxins, regardless of concentration, produced roots of intermediate diameter, averaging 1.60 mm with no statistical differences between treatments, indicating more stable growth patterns in terms of root thickness (fig. 3). the rooting agent type and concentration interaction exerted a clear effect on secondary root formation. naa consistently restricted lateral root development across all three tested concentrations (fig. 4), likely due to its potent growth regulator activity and potential induction of apical dominance or excessive local accumulation in basal tissues (aloni et al., 2006). recent studies indicate that synthetic auxins like naa exhibit enhanced stability and tissue persistence, creating strong but localized hormonal signaling that may suppress lateral root initiation through downregulation of lateral organ boundaries domain (lbd) transcription factors critical for adventitious rooting, and prolonged activation of aux/iaa repressor proteins that inhibit auxin­ dependent gene expression by blocking arf transcription factors (lakehal and bellini, 2019; jing and strader, 2019). this dual regulation at the genetic level explains naa’s capacity to simultaneously promote primary root growth while inhibiting secondary root formation. in contrast, iaa demonstrated a positive and progressive effect on secondary root formation as concentration increased, likely attributable to its lower stability. this auxin undergoes rapid metabolic conversion or conjugation, preventing excessive accumulation while enabling dynamic tissue transport, characteristics that facilitate lateral root differentiation (casanova­sáez et al., 2021; zhang et al., 2023). these findings underscore the importance of developing tailored propagation protocols that carefully consider both auxin type and optimal concentration for specific plant materials. for commercial nurseries where consistency and productivity are paramount, pre­mixed formulations such as iba+naa (dip’n grow®) may serve as practical solutions, though they require precise concentration adjustments to prevent phytotoxic effects in sensitive etiolated tissues. critical implementation considerations include establishing appropriate concentration thresholds for different auxin combinations, accounting for tissue­specific sensitivity variations, and carefully balancing the trade­offs between root quantity and overall root system quality. the research demonstrates that successful clonal propagation depends on this multifaceted optimization approach rather than relying on standardized auxin applications. the significant interaction between auxin type and concentration conclusively refutes the concept of a “universal concentration” suitable for all rootstocks or growing conditions. this research instead provides empirical evidence supporting the development of customized propagation protocols through careful refinement of auxin formulations, adjustments based on the physiological state of plant material, and optimization of multiple interdependent parameters. these insights prove particularly valuable for enhancing clonal propagation of difficult­to­root rootstocks such as ‘duke 7’, where well­balanced root system architecture critically determines subsequent field performance. the data­driven methodology established in this study could be effectively adapted to improve propagation protocols for other commercially significant avocado cultivars, potentially revolutionizing nursery production standards through science­based precision agriculture approaches. 4. conclusions the results demonstrated that auxin type and concentration significantly and differentially influenced adventitious root induction in etiolated shoots of ‘duke 7’ avocado rootstock. these effects were evident in both rooting percentages and the morphological quality of the root system. naa promoted the highest root number and quality index, but also induced substantial callus formation and reduced secondary root development, suggesting potential phytotoxicity at elevated concentrations. in contrast, iaa showed a more balanced dose­dependent response and enhanced secondary root growth, while iba and its commercial formulation with naa (iba + naa) provided stable and reliable performance. auxin concentration modulated rooting efficiency, with dose­dependent physiological responses being martínez‐villagómez et al. ‐ auxins and rooting of avocado rootstock ‘duke 7’ 187 most pronounced for iaa and k­iba. the tested concentration range (24.6­44.2 mm) proved effective for rooting (>95%) while maintaining shoot survival in most treatments. a clear auxin type × concentration interaction was observed, emphasizing the need for genotype (and physiological state) specific optimization of both factors. the responses documented are not universal and must be considered when designing propagation protocols for avocado rootstocks. the combination of etiolated shoots with properly selected and dosed auxins achieved 100% rooting in certain treatments, surpassing results from traditional protocols. these findings represent significant progress toward standardizing and optimizing clonal propagation of ‘duke 7’ for commercial production. this study provides experimental evidence for developing more efficient, reproducible, and economically viable protocols applicable in commercial nurseries. by improving the availability of clonal avocado rootstocks, these advances will 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sciences, university college of agriculture and natural resources, university of tehran, imam‐zadeh hasan blvd., 31587‐77871 karaj, iran. key words: cucumis melo, principal component analysis, storability, surface response method. abstract: temperature is a key factor in melon cold storage. thus, optimizing storage temperature is an important goal in postharvest research. in this exper­ iment, postharvest attributes of four inbred lines and five derivative hybrids were investigated under three storage temperatures (1, 4, and 12°c). melon fruit were evaluated for their main characteristics directly after harvest and postharvest changes were monitored through cold storage period. cluster anal­ ysis results showed that most of the hybrids clustered with their maternal par­ ents illustrating the significant role of cytoplasmic inheritance for the studied traits. similarly, principal component analysis clustered the studied types into three clusters according to their average postharvest behaviour. the best postharvest performance belonged to inodorus and cantalupensis netted melon with their intercrossing breeds. while the dudaim inbred line and its hybrid scored the highest postharvest changes. response surface analysis showed that 1.8°c was the optimum storage temperature for inodorus and can­ talupensis clusters, while 5.1°c was the best storage temperature of dudaim cluster. the results of the current study are similar to previous reports for opti­ mum storage temperature in similar melon types. 1. introduction melons (cucumis melo) are among the most important fruit crop worldwide with a yearly production of more than 27 million tons (fao, 2019). this fruit is of an exclusive importance in mediterranean and eastern asia regions (garcia­mas et al., 2012). melons are usually perish­ able with short shelf life and low storability (fukuta et al., 2006; briones et al., 2012). thus, these fruits do not maintain their marketability for longer than 10 days in room temperature conditions. therefore, introduc­ ing new melon types with enhanced storability, longer shelf life, and bet­ ter shipping potentials is among the main aims of melon breeders. additionally, investigating postharvest behaviour and optimizing storage (*) corresponding author: kalantaris@ut.ac.ir citation: alabboud m., kalantari s., soltani f., 2022 ­ postharvest performance interpretation and sto‐ rage temperature optimization in some newly introduced melon hybrids. ­ adv. hort. sci., 36(1): 27­36. copyright: © 2022 alabboud m., kalantari s., soltani f. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 may 2021 accepted for publication 14 january 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10914 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 27­36 28 variables of this fruit is of high importance. persian melons (cucumis melo var. inodorus) are reported to be a valuable material in melon breeding for postharvest purposes. these types of melons have firmer mesocarps compared to cantalupensis types and thus, endure shipping and handling better (welbaum, 2015). for instance, including the line ‘cm­ utkh’ in breeding programs by crossing with other cantalupensis melons reportedly introduced new types with promising postharvest attributes (alabboud et al., 2020; shajari et al., 2021. therefore, investigat­ ing these newly introduced intergroup hybrids for their postharvest potentials under different storage temperature might be of high importance. weight losses, firmness losses, soluble solids fluc­ tuations and colour changes are the main posthar­ vest attributes in stored melons. furthermore, tem­ perature is the main factor affecting these attributes throughout cold storage (de arruda et al., 2003; yang et al., 2003; žnidarĉiĉ et al., 2013; hatami et al., 2019). therefore, minimizing postharvest changes through optimized storage temperature is the ulti­ mate goal in cold storage research. however, moni­ toring these attributes in large populations can be a laborious quest especially when investigating opti­ mum storage temperature. thus, a more appropriate experimental method should be used in order to minimize experiment size and maintain an accept­ able accuracy. for this purpose, principal component analysis (pca) is considered a useful tool to explain diversity within a studied group of samples with mini­ mum loss of information. many researches imple­ mented pca for better understanding of melon fruit characteristics (obando et al., 2008; maietti et al., 2012; farcuh et al., 2020).on the other hand, response surface method (rsm) is a direct applica­ tion of regression theory to optimize an experimental input (barton, 2013) with fewer number of experi­ ments (wani et al., 2012) which fits the goal of postharvest studies in melon. therefore, the main goals of this study were to investigate postharvest attributes of some newly introduced melon hybrids under different storage temperatures, and to find the optimum storage tem­ perature for these hybrids through the application of response surface regression optimization. 2. materials and methods plant material the experiment was carried out for the seasons of 2019 and 2020 at the research station of the department of horticultural sciences (university of tehran) in karaj, iran. four melon inbred lines (table 1) with five of the intercrossing hybrids were used in this experiment (fig. 1). the seed of inbred lines and hybrids were planted in seedling trays in greenhouse and transformed to the field three weeks after germi­ nation. the seedlings were planted in rows with 1.5 m distance between rows and 0.8 m between plants on each row. ripening was determined by the devel­ opment of ½ to ¾ of abscission layer in types that tend to develop an abscission layer and after 42 days post­anthesis in types that lacks the ability to devel­ op an abscission layer. the fruit were harvested at ripening and transferred to the cold storage facility at the department of horticultural sciences. the har­ vested fruit were stored under three different tem­ perature (1, 5, and 12°c) and a relative humidity of 90­95% for a period of one month. fruit attributes monitoring the main fruit characteristics were monitored table 1 ­ the morphological characterizations of the inbred lines used for the crosses line name group and origin climacteric behaviour fruit shape rind mesocarp volatiles origin cm­utkh inodorus, non­climacteric oval elongated yellow­shallow netted greenish white odourless khorasan (south­eastern part of iran) cm­uta cantaloupensis x inodorus derived inbred line non­climacteric oval­round dark yellow ­ without net white odorous (medium) abadan (southern part of iran) cm­utj cantaloupensis non­climacteric ova ­round green ­ netted green odourless alborz (north­western part of iran) cm­utz dudaim climacteric round flatted dark yellow with orange areas ­ without net white odorous (high) kerman (south­eastern part of iran) alabboud et al. ‐ storage temperature optimization in newly introduced melon types 29 directly after harvest and throughout storage period in 10 days intervals (i.e. days 0, 10, 20, and 30). fruit weight was measured using an electronic scale. tss% in the juice of extracted from a longitudinal section of the fruit was measured using a handheld refrac­ tometer. mesocarp firmness was measured using a handheld penetrometer equipped with an 8­mm tip. the firmness was expressed as an average of three reads along the longitudinal surface of the fruit after the removal of the rind. rind colour was expressed in cie­lab scale as lightness (l*), chroma (c*) and hue angle (h°) using a chromameter (konica minolta, japan). the changes in fruit attributes throughout storage were presented as percentages and were calculated using the following formulas: weight loss (%) = (fw0 ­ fwa)/fw0 (1) firmness loss (%) = (mf0 ­ mfa)/ mf0 (2) where fw0 and fwa are fruit weight (g) at har­ vest and at analysis time, respectively; while mf0 and mfa are mesocarp firmness (kg cm­2) at harvest and at analysis time, respectively. colour changes were calculated using colour dif­ ference formulas according to sharma et al. (2005) eq. 3 and using the same system described in mohi­ alden et al. (2021). δc = [(l* ­ l0*)2 + (a* ­ a0*)2 + (b* ­ b0*)2]1⁄2 (3) where δc is colour changes during cold storage l0*, a0* and b0* are the values of colour parameters at harvest, and l*, a* and b* are the values of colour parameters after cold storage. data analysis the two­factor experiment (evaluation date and storage temperature) was carried out in a split plot design. fruit attributes mean at harvest were com­ pared using fisher’s lsd test (p<0.05) with minitab 19 software. furthermore, fruit attributes were clus­ tered using ward’s method on ncss 12. the averages postharvest measurement for the studied lines and breeds were analysed using principal component analysis (pca). the best response surface method (rsm) was used to calculate the optimized storage temperature for each pca cluster. 3. results fruit characteristics and heterosis analysis the main fruit characteristics of parents and f1 progeny were investigated (table 2). the result showed that the parent ‘cm­utkh’ recorded the highest fruit length and fruit shape values and, the parent ‘cm­uta’ scored the highest results in terms of fruit diameter, mesocarp thickness, and cavity diameter. the highest firmness results were observed in ‘cm­utj’ parent with 11.67 kg cm­2 fol­ lowed by its hybrid ‘cm­utjkh’ (10.98 kg cm­2), with no significant differences between the parent and the hybrid. the highest fruit weight, and tss% were observed in the hybrids ‘cm­utkha’ and ‘cm­utjkh’ with 2033.91 g and 15.3%, respectively. as for colour attributes, the highest l* and hue values were recorded in the parent ‘cm­uta’, while the highest chroma values were observed in the hybrid ‘cm­ utjkh’ and the reciprocal ‘cm­utkhj’. moderately low heterosis results were recorded for fruit diameter, cavity diameter, firmness, tss, and colour attributes. however, f1 progeny expedited higher heterosis values levels for fruit length (33.17%), fruit shape (22.71%), mesocarp thickness (17.48%), and weight (29.35%). cluster analysis of fruit attributes ward linkage cluster analysis of fruit attributes at day 0 can be seen in figure 2. the studied genotypes were clustered in three clusters the first cluster con­ tained the hybrid ‘cm­utzkh’ with its maternal par­ ent. the second cluster contained ‘cm­utjkh’ with fig. 1 ­ the studied melon inbred lines (p) and their hybrids (f1). the first part in each hybrid name represents the mater­ nal parent while the second represent the male parent. breed maternal parent paternal parent cm­utkha cm­utkh cm­uta cm­utakh cm­uta cm­utkh cm­utkhj cm­utkh cm­utj cm­utjkh cm­utkh cm­utj cm­utzkh cm­utz cm­utkh adv. hort. sci., 2022 36(1): 27­36 30 its maternal parent. while the final cluster constitut­ ed of the ‘cm­utkha’, ‘cm­utkhj’, and their mater­ nal parent in addition to the hybrid ‘cm­utakh’. on the other hand, ‘cm­uta’ was not clustered with any of the studied types. as for the studied fruit traits, two major clusters were noticed. the first contained tss, chroma, firm­ ness, and lightness (l). the second cluster contained the rest of the studied attributes (fruit length, fruit shape, fruit diameter, mesocarp thickness, weight, and cavity diameter), while hue was not clustered with any other attribute. principal component analysis results the average postharvest performance data under three different storage temperature was subjected to principal component analysis (pca) (fig. 3). scree dis­ tribution of factors showed that the first two factors were accounted for 68.7% of the total variance with 49.3% for the first factor (pc1) and 19.4% for the sec­ ond (pc2). additionally, the accumulated effect of the first three factors was accounted for 79.1% of the total variance among the studied types (table 3). the most significant positive contributors to the pc1 were chroma, tss, firmness, and colour changing index, while hue and firmness loss were the most sig­ nificant negative contributors to this factor. on the other hand, pc2 was substantially positively influ­ enced by fruit weight. as for genotype clustering according to biplot of postharvest performance, three major clusters were noticed. the green cluster located on the positive types with similar letters in each column have no significant differences for the monitored trait in that column (p<0.05). h% represents heterosis in each trait compared to med­parents. table 2 ­ fruit characteristics for the studied melon types directly after harvest (day 0 observation). fl (fruit length), fd (fruit diameter, fs (fruit shape), mt (mesocarp thickness), cd (cavity diameter), w (weight), f (firmness), tss (total soluble solids), l* (lightness), h (hue), c (chroma) type fl (cm) fd (cm) fs mt (cm) cd (cm) w (g) f (kg cm­2) tss (%) l* h c cm­utkh 27.77 a 11.33 cd 2.50 a 2.65 cd 6.09 b 1717.42 b 4.77 e 11.70 b 71.15 d 69.72 d 62.35 c cm­uta 13.81 e 14.82 a 0.93 f 3.77 a 7.14 a 1520.51 c 9.84 b 6.33 e 78.24 a 78.77 a 60.37 d cm­utj 12.88 f 11.94 c 1.09 e 3.01 b 5.88 bc 1026.36 d 11.67 a 11.65 b 72.81 cd 22.06 e 65.21 b cm­utd 6.21 g 7.57 e 0.80 f 1.05 e 5.44 c 201.84 f 5.63 de 6.67 e 68.72 e 70.99 cd 44.45 f cm­utakh 21.65 c 13.44 b 1.61 c 3.66 a 6.30 b 1738.41 b 7.46 c 8.70 d 74.23 c 75.76 b 60.13 d cm­utkha 22.29 c 13.15 b 1.69 c 3.54 a 5.98 bc 2033.91 a 6.51 cd 10.60 c 70.92 d 71.69 c 60.12 d cm­utjkh 16.71 d 10.64 d 1.59 c 2.92 bc 4.82 d 917.40 de 10.98 ab 15.30 a 76.19 b 17.17 f 75.38 a cm­utkhj 25.98 b 13.79 b 1.95 b 2.78 bcd 6.97 a 1771.08 b 5.20 e 10.33 c 73.24 c 12.71 g 74.49 a cm­utzkh 14.36 e 11.12 cd 1.32 d 2.49 d 6.16 b 760.31 e 7.38 c 6.80 e 72.74 cd 74.36 b 55.36 e h% 33.17 8.87 22.71 17.48 ­1.49 29.35 ­5.91 13.85 1.01 ­16.64 12.05 fig. 2 ­ ward’s linkage cluster analysis for the studied melon types and their fruit characteristics. p1, p2, p3, and p4 represent the studied lines cm­utkh, cm­uta, cm­utj, and cm­utz, respectively while two letters genotypes represent f1 direct and reciprocal breeds. fl (fruit length), fd (fruit diameter, fs (fruit shape), mt (mesocarp thickness), cd (cavity diameter), w (weight), f (firmness), tss (total soluble solids), l* (lightness), h (hue), c (chroma). the colors of the heat map represent the normalized values of raw data to z score. thus, red colors or yellow colors represent values higher or lower than the average value for each studied characteristic, respectively. alabboud et al. ‐ storage temperature optimization in newly introduced melon types 31 quadrates of pc1 and contained ‘cm­utj’ parent with its hybrid ‘cm­utjkh’ and reciprocal ‘cm­utkhj’. the blue cluster located on the positive pc2 and negative pc2 quadrate and was constituted of the parents ‘cm­utkh’ and ‘cm­uta’ with their hybrids ‘cm­ utkha’ and ‘cm­utakh’. the last cluster was located on the quadrate determined by negative pc1 and pc2 and contained the parent ‘cm­utz’ and its hybrid ‘cm­utzkh’ (fig. 3). response surface analysis throughout storage period contour plotting was used to demonstrate fruit postharvest performance during cold storage period can be seen in figure 4 and figure 5. the most pro­ nounced weight loss was observed in the parent ‘cm­ utz’ under 12°c and after 20 days in in cold storage with more than 30% weight loss. on the other hand, the highest firmness losses were recorded under 12°c and after 30 days in cold storage with the line ‘cm­uta’ and its hybrids ‘cm­utakh’ and ‘cm­ utkha’ with more than 80% firmness loss in the par­ ent and more than 75% firmness loss in both hybrids. fig. 3 ­ principal component analysis for the studied melon. loading factors represent the average performance of the studied types calculated as the mean for each loa­ ding variable of each type during storage period (the ave­ rage of 0, 10, 20, and 30 days in cold storage). w (weight), wl (weight loss), f (firmness), fl (firmness loss). tss (total soluble solids), l (lightness), h (hue), c (chroma), cc (color changes). table 3 ­ principal component analysis unrotated factor load­ ings and communalities variable factor1 factor2 factor3 weight 0.17 0.901 0.141 weight loss ­0.594 ­0.433 ­0.418 firmness 0.754 ­0.328 ­0.464 firmness loss ­0.705 0.354 ­0.139 tss 0.868 ­0.034 0.131 lightmess 0.51 0.484 ­0.661 hue ­0.852 0.347 ­0.108 chroma 0.911 0.334 ­0.02 colour changes 0.651 ­0.215 0.2 % var 49.3 19.4 10.4 accumulated 49.3 68.7 79.1 fig. 4 ­ contour plots of postharvest changes during cold storage period for the studied inbred lines. each column of contour plots represents an inbred line (from left to right ‘cm­utkh’, ‘cm­uta’, ‘cm­utj’, and ‘cm­utz’, respectively). each row of contour plots represents a postharvest attribute (from top to bottom weight loss, tss, firmness loss, and color changes, respectively). a significant difference between contour levels is present when the difference between contour levels are larger than 1.32, 1.67, 5.97, and 1.02 for weight loss, tss, firmness loss, and color changes, contours respectively (p<0.05). 32 adv. hort. sci., 2022 36(1): 27­36 there were no significant colour changes during the first 10 days of storage under any of storage tem­ perature in any of the studied lines except for ‘cm­ utz’. however, all the studied hybrids showed signifi­ cant colour changes after 10 days in cold storage and under all temperature except for ‘cm­utkha’ which showed significant colour changes only after 20 days in storage and under higher storage temperature (>5°c). colour changes were steep and more pro­ nounced in the hybrids ‘cm­utkhj’ and ‘cm­utjkh’. there was no defined trend in tss changes throughout storage period in the studied genotypes. a general decreasing trend was noticed in the lines ‘cm­utj’ and ‘cm­utz’ and similar decreasing overall trends were observed in all the studied hybrids. on the other hand, minimal tss fluctuations were observed in the line ‘cm­uta’. furthermore, the line ‘cm­utkh’ experienced significant decrease in tss during the first 10 days of storage under 12°c and then remained constant for the rest of storage dura­ tion. however, the line ‘cm­utkh’ kept an almost constant tss under 4°c throughout storage period. response surface model was used in order to cal­ culate the optimized storage temperature of the clus­ ters obtained via pca analysis. the monitoring date and storage temperature were set to be the continu­ ous factors of the model, while weight loss (wl), firmness loss (fl), colour changes (cc), and tss were set as responses. stepwise method was used to omit unnecessary sources for better fit of each model (table 4). then, the optimized storage temperature for each cluster was calculated by setting optimiza­ tion goals of wl, fl, and cc to a minimum and tss to a maximum. the results showed that the optimized temperatures were 1.8 for the green and blue clus­ ters, and 5.1 for the red cluster. 4. discussion and conclusions fruit characteristics cluster analysis showed that fruit shape was more correlated with fruit length than diameter even though these three attributes were located in the same cluster (fig. 2). this relation was also observ­ able in (table 2) since the line ‘cm­utkh’ recorded the highest fl and fs values, while the line ‘cm­utz’ recoded the lowest. in fact, both these traits had also a high heterosis values with 33.17% and 22.71% for fl and fs, respectively, which is similar to monforte et al. (2005) reports of significant correlation between fruit shape and length. the same research suggested that the heterosis in fruit shape is a func­ tion of fruit elongation. on the other hand, the segregation fruit diameter, mesocarp thickness, fruit weight, and cavity diameter might indicate that the more rounded the melon is, the thicker the mesocarp and thus, the heavier fruit will be. the relatively high heterosis in mesocarp thickness and fruit weight (17.48% and 29.35%, fig. 5 ­ contour plots of postharvest changes during cold storage period for the studied hybrids. each column of contour plots repre­ sents a hybrid (from left to right ‘cm­utkha’, ‘cm­utakh’, ‘cm­utkhj’, ‘cm­utjkh’, and ‘cm­utzkh’, respectively). each row of contour plots represents a postharvest attribute (from top to bottom weight loss, tss, firmness loss, and color changes, respec­ tively). a significant difference between contour levels is present when the difference between contour levels are larger than 1.32, 1.67, 5.97, and 1.02 for weight loss, tss, firmness loss, and color changes, contours respectively (p<0.05). alabboud et al. ‐ storage temperature optimization in newly introduced melon types 33 respectively) and the low heterosis in cavity diameter might in favour of the previous debate. the current results showed that the hybrids ‘cm­utkha’, ‘cm­ utakh’, and ‘cm­utkhj’ scored positive weight het­ erosis values compared to best parents, while ‘cm­ utjkh’ and ‘cm­utzkh’ that scored negative weight heterosis compared to mid parent results. this result is similar to monforte et al. (2005) and mohammadi et al. (2014) reports of positive to negative heterosis for melon fruit weight, while the general positive het­ erosis for fruit weight is similar to this reported by feyzian et al. (2009). furthermore, the results refer to the importance of the line ‘cm­utkh’ in breeding programs to increase average fruit weight. it was noticed that each hybrid clustered with its maternal parent, except for the hybrid ‘cm­utakh’ which was however the closest to its maternal parent ‘cm­uta’ although they were not clustered together (fig. 2). this observation refers to the high maternal impact of the used lines and the cytoplasmic inheri­ tance for the studied traits. the cytoplasmic inheri­ tance and the high similarities between hybrids and maternal parents was previously reported for fruit length, fruit diameter, fruit shape, average fruit weight, mesocarp thickness, cavity diameter, tss, and firmness (y. hassan al­hamdany, 2013; shajari et al., 2021) while other study reported a non­signifi­ cant reciprocal effect for fruit weight (feyzian et al., 2009). postharvest performance in the current work, pca clustered the studied genotypes according to their average postharvest performance into three clusters with each hybrid clustered with its maternal parent. the high similari­ ties in postharvest behaviour between hybrids and their maternal parent can be also attributed to the significant reciprocal effects for the studied traits, which were previously reported in similar population (alabboud et al., 2020). by comparing loading variables to samples distri­ bution, it can be concluded that the ‘cm­utj’ line with its hybrid ‘cm­utjkh’ and reciprocal ‘cm­utkhj’ had lower weight loss and firmness loss during cold storage compared to ‘cm­utz’ and its hybrid ‘cm­ utzkh’. thus, the former samples had a better postharvest performance compared later (fig. 3) which can be also noticed in (fig. 4) and (fig. 5). colour changes in the line ‘cm­utj’ and its hybrids are attributed to chroma increase which indicates more saturated colour during cold storage. according to pca biplot (fig. 3), the samples with higher tss content were characterized by lower weight and firmness losses during cold storage; therefore, the types with higher tss content per­ formed better throughout cold storage. previously a negative correlation was reported between sucrose content in blueberry and polygalacturonase activity (wang et al., 2020). additionally, postharvest shelf model response surface regression sources optimization goal optimized storage temperaturelinear square interaction green cluster wl d ­ ­ minimize 1.8 fl d, t t x t d x t minimize 1.8 cc t t x t ­ minimize 1.8 tss t ­ ­ maximize 1.8 blue cluster wl d, t d x d, t x t d x t minimize 1.8 fl d, t t x t ­ minimize 1.8 cc d, t t x t d x t minimize 1.8 tss d, t t x t ­ maximize 1.8 red cluster wl d, t d x d ­ minimize 5.1 fl d, t t x t ­ minimize 5.1 cc d, t ­ d x t minimize 5.1 tss t t x t ­ maximize 5.1 wl= weight loss, fl= firmness loss), cc= colour changes, tss =total soluble solids, d= test date, t= storage temperature. cluster colours (green, blue, and red) refer to the principal component analysis (pca) clustering shown in figure 3. table 4 ­ storage temperature optimization results for the clusters obtained by principal component analysis using response surface method adv. hort. sci., 2022 36(1): 27­36 34 life of roquette leaves was extended in relation to higher sucrose content (clarkson et al., 2005). furthermore, it was reported that sucrose can act as a protective signal in fresh cut melon against wound­ ing signal (wu et al., 2020). considering the fact that sucrose is the major component in melons’ tss con­ tent (burger et al., 2000), it can be assumed that the current observed correlation between tss content and enhanced postharvest performance is related to a higher sucrose level. however, this relation should be investigated thoroughly. storage temperature optimization the decrease in fruit weight and firmness in addi­ tion to colour changes are among the usual observa­ tions throughout cold storage of fruit and vegetables. weight loss is mainly attributed to water evaporation during storage period (lal basediya et al., 2013) which is supposed to increase by increasing storage temper­ ature as can be seen in all the studied types (fig. 4 and 5). the differences in weight loss between differ­ ent types during storage phase should correlated with fruit characteristics. for instance, the line ‘cm­utjs’ and its related hybrids are characterized with thicker rind and suberized periderm tissues which can effi­ ciently block water evaporation (nishizawa et al., 2017) especially when considering the non­climacteric behaviour of the aforementioned types compared to other climacteric types such as ‘cm­utz’. firmness loss is the result of cell wall deterioration due to vari­ ous enzymes activity throughout storage (qi et al., 2011; wu et al., 2020). the current results showed lower and slower firmness loss under lower storage temperature, which is usually observed in stored melon (hatami et al., 2019) due to the slower overall metabolism and the lower enzymatic activity. the currently observed fluctuation in tss during cold storage phase was observed in full melon fruit (hatami et al., 2019) and in fresh cut slices (chong et al., 2015). in fact, most of the observations where tss fluctuated were under lower storage tempera­ ture (1°c) (fig. 4 and 5). therefore, the most con­ vincing explanation is that fruit metabolism under lower temperature was lower than that of higher storage temperature; therefore, water evaporation from fruit resulted in higher concentrations of solu­ ble solids. response surface method (rsm) is a combination of experimental design, analysis of regression and stochastic response optimization (barton, 2013). the main advantage of rsm analysis compared to other optimization methods is that fewer number of exper­ iments are needed to monitor the interaction of the independent variables on the response (wani et al., 2012). pca clustering results were used to produce response surface models. the postharvest related data of each cluster members were the inputs while the target was to find the storage temperature which minimizes weight losses, firmness losses and color changes while maintaining the highest tss possible. rsm analysis showed that the optimized storage temperature for both green (netted cantalupensis melon) and blue clusters (inodorus) members was 1.8°c. this result is similar to previous recommended storage temperature for various inodorus group culti­ vars (yang et al., 2003) where a storage temperature of 3°c was recommended, and netted cantalupensis group cultivars (de arruda et al., 2003; žnidarĉiĉ et al., 2013) where a storage temperature of 2­3°c resulted in the best storage outcomes. on the other hand, the optimum storage temperature for the red cluster (dudaim) was 5.1°c which is similar to the previous observations in dudaim group including the line ‘cm­utz’ where 5°c resulted in better colour preservation and less weight, firmness, and tss loss­ es during cold storage period (hatami et al., 2019). therefore, rsm can be considered a feasible method of storage temperature optimization especially in newly established populations and hybrids. the current study showed that the hybrid melon types of inodorus x cantalupensis and dudaim x inodorus crosses had more similarities with their maternal parent. these similarities were obvious not only on fruit characteristics directly after harvest, but also in the later postharvest performance in cold storage. this result illustrated the importance of cytoplasmic inheritance in melon. the best posthar­ vest performance was that of crosses between inodorus x netted cantaloupe, while the worst per­ formance was related to ‘cm­utz’ line of the group dudaim and its hybrid with the line ‘cm­utkh’. the current reported work flow of using pca followed by rsm showed promising results in optimizing storage temperature which was similar to previous literature. therefore, the use of this workflow is highly recom­ mended in optimizing postharvest inputs especially when testing for newly introduced hybrids where the experimental work volume might be overwhelming. references alabboud m., kalantari s., soltani f., 2020 ­ analysis of general and specific combining ability of postharvest alabboud et al. ‐ storage temperature optimization in newly introduced melon types 35 attributes in melon. ­ j. agric. sci. technol., 22(60): 1523­1535. barton r.r., 2013 ­ response surface methodology. ­ in: gass s.i., and m.c. fu (eds.) encyclopedia of opera‐ tions research and management science. springer, boston, ma, usa. briones a., ubeda­iranzo j., hernández­gómez l., 2012 ­ spirits and liqueurs from melon fruits (cucumis melo l.), pp. 183­196. ­ in: zereshki s. 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sciences, université mohammed v de rabat, rabat, morocco. key words: cycas revoluta, gelrite, germination, in vitro, regeneration, zygotic embryos. abstract: an efficient in vitro germination protocol for cycas revoluta, a wide­ spread ornamental tree, has been established using zygotic embryos as explants with a focus on mineral composition of the culture media, the gelling agent and cytokinine type. a high percentage of germination, 73% was obtained with sh medium instead of 27% with ms medium. a 100% of germi­ nation was obtained with the combination of sh medium and gelrite as gelling agent. the addition of cytokinines prompt shoot formation. an optimum shoot induction occurred using 0.5 mg/l of bap where an average of 14.1 shoot were produced per explants while 2.2 shoots were formed in the presence of 2ip. a 100% of rooting was observed in the presence of 0.5 mg/l of 2ip whereas only 30% of shoots obtained on the sh medium with 0.5 mg/l of bap were able to develop roots. 1. introduction cycas revoluta is taxonomically known as the most primitive species among the living cycads (stevenson, 1990; jones, 1994). cycas revoluta is one of the widespread ornamental trees, grown in temperate, subtropical and tropical regions more precisely in miyazaki and kagoshima prefectures in kyushu district down to the ryukyu islands, okinawa prefecture in japan (khalighi, 2001; zarchini et al., 2011). cycas revoluta is propagated either from seeds, which remain viable for only a short time, or from vegetative offshoots (demiray et al., 2017). as slowly growing plants, they require 3 to 10 years to attain reproductive maturity (rinaldi, 1999). germination of cycas revoluta seeds is hard and time consuming (zarchini et al., 2011). physical dormancy of seed causes delay in seed germination (frett, 1987). seeds can take 3 to 9 months to initiate germination before they can continue to germinate for periods of a year or more. c. revoluta seeds also demonstrates rapid loss of viability and low morphogenic potential, which hinder its conservation as well as (*) corresponding author: sarah.bouzroud@gmail.com citation: benjelloun j., taoufyq a., el abidine triqui z., alami q.l., layachi r., smouni a., bouz­ roud s., guedira a., 2020 ­ improvement of in vitro germination of cycas revoluta zygotic embryos using gelrite as gelling agent. ­ adv. hort. sci., 34(3): 349­354 copyright: © 2020 benjelloun j., taoufyq a., el abidine triqui z., alami q.l., layachi r., smouni a., bouzroud s., guedira a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 april 2020 accepted for publication 18 august 2020 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 349­354 350 favor an effective and rapid mass propagation (naderi et al., 2015). the delay in seed germination along with the slow growth of cycas plants increase the cost of production (frett, 1987; litz et al., 2005; demiray et al., 2017). thus, conventional methods are not quiet efficient for large­scale propagation of this species. therefore, other propagation methods are needed (da silva et al., 2014). the use of in vitro techniques to accelerate seed germination is a suit­ able way to conserve many of the endangered species. several attempts have been made to establish an efficient protocol for cycas revoluta propagation (rinaldi and leva, 1995; rinaldi, 1999; naderi et al., 2015; demiray et al., 2017). however, the results were not satisfying. the present study focuses on developing an efficient in vitro germination protocol from mature zygotic embryos (zes) with a focus on the gelling agent, the mineral composition of the cul­ ture media and the presence of 2­isopetynyl adenine (2ip) or 6­benzylaminopurine (bap). 2. materials and methods plant material seeds collected from 50 years old female mature plants grown in faculty of sciences garden, university mohammed v in rabat (morocco) were used in this study. seed sterilization and zygotic embryos isolation seeds were soaked in water for 48 hours in order to soften the sacrotesta, the orange external layer. once removed, seeds were then flamed with ethanol for 2 minutes in order to eliminate the sclerotesta. following removal of the sclerotesta, the megagame­ tophytes were surface sterilized for 20 minutes by soaking in 30% dilution of naocl containing 2­3 drops of tween­20, followed by 3­4 rinses with sterile dis­ tilled water. after surface sterilization, megagameto­ phytes were pooled, longitudinally bisected and the ze was excised from each megagametophyte. zygotic embryos culture zes 1.7 to 2­cm long were placed in culture jars containing 120 ml of culture medium with 2 explants per jar. two basal mediums murashige and skoog (ms) (murashige and skoog, 1962) and schenk and hildebrandt (sh) (schenk and hildebrandt, 1972) were tested for in vitro germination. both media were supplemented with 30 g/l of sucrose and solidi­ fied with 0.8% of bacteriological agar type e (biokar diagnostic) or 0.3% of gelrite (sigma­aldrich). bap or 2ip (0.5 mg/l) were added to the culture medium. the ph was adjusted to 5.8 with either 1n hcl or koh prior to autoclaving, at 108 kpa and 120°c for 20 min. cultures were incubated in a culture room at 25±2°c and in the dark for 21 days, thereafter under a photoperiod of 16h of light/8h of darkness. statistical analysis percentages of germination and plant development were compared using z test or a fixed model of analy­ sis of variance (anova) depending on the condition. thirty biological replicates were performed for each condition. in case of significant difference between groups, a least significant difference lsd test was used for means separation. shoot number, shoot length and number of leaves per shoot obtained from each embryo was analyzed by z test, at risk of 0.05. 3. results and discussion effects of the culture medium on germination and regeneration nitrogen formulation influences seed germination and callus induction in cycads (rinaldi, 1999; demiray et al., 2017). investigating seed responsiveness to sh and ms medium revealed significant differences in ger­ mination (gp) and development percentages (pdp) between the two tested mediums. the results revealed that the highest gp (73%) was obtained with sh medi­ um while the gp was around 57% with ms medium (table 1). a higher regeneration percentage (around 27%) was also observed with sh medium while only 13% of seeds were able to regenerate (table 1). rinaldi and leva (1995) have found that sh or ms medium, both containing ammonium, promoted shoot forma­ tion. rinaldi (1999) also reported that the percentage of responding explants and the number of regenerated shoots were significantly higher on sh medium than on ms medium. however, the presence of nitrate as a sole source of nitrogen, as in the klimaszewska and keller medium (klimaszewska and keller, 1985), did not pro­ mote shoot regeneration (rinaldi and leva, 1995; table 1 ­ effect of medium culture on germination and plant development of cycas revoluta zes. * indicate the statistical significance (p<0.05) using z test. medium germination (%) plant development (%) sh 73 (*) 27 (*) ms 57 (**) 13 (**) benjelloun et al. ‐ in vitro germination of cycas revoluta 351 rinaldi, 1999). this difference might be due to the dif­ ferences in ammonium amounts found in sh and ms media. effect of gelling agents on zygotic embryos develop‐ ment the gelling agent influences the germination of cycas revoluta zes. we found that the germination percentage reached 100% with gelrite used as gelling agent in sh medium while only 73% of zes were able to germinate in sh medium supplemented with agar. moreover, the highest pdp was obtained with sh medium solidified by gelrite (fig. 1). the use of gelrite as gelling agent was previously used to stimulate the growth and the development of in vitro cultured plants. in sequoia sempervirens, fira and clapa (2008) have reported a higher shoot multipli­ cation with gelrite. similar finding was also stated in oil palm in which gelrite was proven to be better than agar giving the highest conversion rate of polyembry­ oids into plantlets (palanyandy et al., 2020). the micro­ propagation of cowpea cultivars (vigna unguiculata l. walp) was highly assessed using gelrite instead of agar (aasim et al., 2009). veramendi et al. (1997) have pro­ posed gelrite as a great alternative to agar for micro­ propagation and microtuberization of solanum tubero‐ sum. scholten and pierik (1998) explained this benefi­ cial effect by the inorganic composition and the dynamical interaction between gelling agent­medium­ tissue. later, puchooa et al. (1999) linked the positive effect of gelrite on in vitro plant culture to the chemical composition of this gelling agent; with a high content of copper, iron, magnesium, zinc and calcium com­ pared to agar. in addition to that, buah (1999) explained the beneficial effect of gelrite on growth of banana by the fact that gelrite provide a better avail­ ability of water. moreover, the diffusion of phenols and other inhibitive molecules in the media culture is facili­ tated by the use of gelrite (huang and chi, 1988; el abidine triqui et al., 2008). thus, it is assumed that the low regeneration rate of seedlings obtained with agar is probably due to the accumulation of inhibitive com­ pounds in this gelling agent; following this, explants can no longer absorb the mineral salts that are essential for their development. previous works have reported the positive effect of gelrite on seed germination and development in several plant species (asif et al., 2001; yamazaki and miyoshi, 2006; pech y aké et al., 2007), however none of them was related to cycas revoluta. this work can be qualified as one of the pioneers high­ lighting the positive effect of gelrite on cycas revoluta zes development. effect of cytokinines on shoot development even though germination percentage reached 100% with sh medium solidified with gelrite (fig. 2), the number of shoots per ze was significantly lower (table 2). fig. 1 ­ effect of gelling agent (agar or gelrite) on germination (pg) and plant development (pdp) percentages. bars with different letters indicate the statistical significance (p<0.05) according to lsd test. fig. 2 ­ effect of plant regulators (bap and 2­ip) on germination (gp) and plant development (pdp) percentages of cycas revoluta zygotic embryos cultured on sh medium. zes were cultured on sh medium solidified with gelrite and supplemented with 0.5 mg/l of bap or 0.5 mg/l of 2ip. bars with different letters indicate the statistical signifi­ cance (p<0.05) according to lsd test. table 2 ­ effect of phytohormones on the number of shoot/ explant, number of leaves/ shoot and shoot length of cycas revoluta zes. * indicate the statistical significance (p<0.05) using z test. sh sh + bap sh + 2­ip number of shoot/ explant 1.6 (***) 14.1(*) 2.2 (**) shoot length 0.7 (**) 0.52 (**) 2.73 (*) number of leaves/shoot 1.81 (**) 9.13 (*) 3.6 (**) adv. hort. sci., 2020 34(3): 349­354 352 the addition of phytohormones, likely bap and 2ip, increased shoot number in cycas revoluta zes. the average of shoots developed per explant was around 14.1 in the presence of bap and 2.2 when ze were cultured in sh medium supplemented with 2ip (table 2). however, shoot length was significantly higher in the presence of bap with 9.3 leaves per shoot versus 1.7 and 3.6 when cultured in the absence of phytohormones or with bap added to the media culture (fig. 3 d, e, f). shoot differentiation was previously obtained from zygotic embryos of cycas revoluta in the media with bap (0.5 mg/l) and 2.4­d (1 mg/l) (rinaldi and leva, 1995). naderi et al. (2015) found that bap stimulated shoot regeneration from cycas zes while other phytohormones like 2.4­d or kinetin alone or in combination with bap failed. fig. 3 ­ effect of phytohormones on zygotic embryos development. (a) shoot induction in sh basal medium, (b, c) shoot regeneration in sh medium supplemented with 0.5 mg of bap and 0.5 mg of 2ip respectively, (d, e, f) shoot elongation sh, sh+bap and sh+2ip prespectively, (g, h, i) rooted plants after 3 months of culture in sh, sh+bap and sh+2ip, respectively. benjelloun et al. ‐ in vitro germination of cycas revoluta 353 the combination of bap (0.2 mg/l) and 2,4­d (0.02 mg/l) induced adventitious shoots from mature c. revoluta zes (motohashi et al., 2008). shoot elonga­ tion was although promoted by the presence of 2ip. our data showed that shoot length developed in sh medium supplemented with 2ip was significantly higher than those obtained in the presence of bap. root development was obtained in all growth condi­ tions. we noticed that the shoots developed on the sh medium supplemented with 0.5 mg/l of 2ip were all rooted (100%) whereas only 30% of shoots obtained on the sh medium with 0.5 mg/l of bap developed roots instead of 50% on sh medium (free from plant growth regulators) (data not shown). in addition to the high rooting percentage obtained in the presence of 2ip, we also noticed that the pres­ ence of 2ip promoted the development of primary and/or secondary roots with likely meristematic structures identified as nodules (fig. 3i). this finding was previously reported by dhiman et al. (2000), who suggested that such nodules are meristematic zones with distinct organogenic potential, which can proba­ bly be evolved to embryos or seedlings depending on the culture conditions. 4. conclusions this study aims to improve an efficient in vitro germination protocol from mature zygotic embryos of cycas revoluta. based on our results, we found that sh medium was beneficial for seeds germination rather than ms medium. moreover, the use of gel­ rite, instead of agar, enable us to obtain a 100% of seed germination. an average of 14.1 shoots per zygotic embryo was thus obtained with the addition of 0.5 mg/l bap to the culture media. taken togeth­ er, this protocol represents a useful and potential commercial method for cycas revoluta mass propa­ gation. references aasim m., khawar k.m., özcan s., 2009 ­ comparison of shoot regeneration on different concentrations of thidi‐ azuron from shoot tip explant of cowpea on gelrite and agar containing medium. ­ notulae botanicae horti 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har­ vest time of cutting) m. abshahi 1, h. zarei 2 (*), b. zahedi 1, f.a. garcía­morote 3, a. rezaei nejad 1 1 department of horticulture sciences, faculty of agriculture, lorestan university, khorram abad, iran. 2 department of horticulture sciences, collage of plant production, gorgan university of agricultural sciences and natural resources, gorgan, iran. 3 department of agroforestry technology and science and genetics, higher technical school of agricultural and forest engineering, university of castilla‐la mancha, albacete, spain. key words: antioxidant, flavonoid, iba, phenol, rooting. abstract: the endemic juniper of maymars (juniperus sabina) is one of the most valuable plants in forested areas. the objectives of this experiment were: i) to determine the best conditions for stem cutting propagation of this species, and ii) to examine changes in some of the secondary metabolites during the four months (the first of each season): january, april, july, and october, after rooting of cuttings. the research was done with the treatment of five levels of indole butyric acid, including: 0, 1000, 2000, 4000, and 8000 ppm in four root­ ing substrates, including perlite, perlite­cocopeat (1:1), pumice, and a mixed rooting substrate (sand, perlite, cocopeat, vermicompost, and potash; 1:1:1:1:1) in the four seasons of the year, with stem cuttings having an average length of 15 cm. the best treatment with more than 50% rooting was seen in april at levels of 4000 and 1000 ppm, and the best substrate was perlite coco­ peat. using lower levels of iba led to a reduction in total phenol content in the cuttings during the rooting period. the flavonoid content of the cuttings varied across different seasons. based on these results, we recommend this way of propagation for juniperus sabina production. this propagation method takes less time in comparison with sexual propagation from seed. 1. introduction the genus juniperus is one of the few conifers that act as a main tree in the natural ecosystems of the mountainous forests of the world. the (*) corresponding author: h.zarei@gau.ac.ir citation: abshahi m., zarei h., zahedi b., garcía­morote f.a., rezaei nejad a., 2022 ­ secondary metabolite changes in maymars juni‐ per cuttings (juniperus sabina) under different treatments of propagation (iba, substrate and harvest time of cutting). ­ adv. hort. sci., 36(3): 163­174. copyright: © 2022 abshahi m., zarei h., zahedi b., garcía­ morote f.a., rezaei nejad a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 april 2022 accepted for publication 7 july 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13044 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 163­174 164 protective and valuable roles of various species of junipers in the management of forest erosion and water management are well known. also, the role of junipers is important both in water storage and in soil conservation (ali ahmad koruri et al., 2011). they are great landscaping and ground cover species (westerfield, 2012). among the junipers, juniperus sabina­maymars is one of the most popular types of junipers. this species can be utilized for forest restoration on poor sites with low potential produc­ tivity, such as arid and semi­arid areas. in addition, maymars is one of the most beautiful juniper species and is suitable for ornamental use (piotto and di noi, 2003). thus, information about the plant production of juniperus sabina can be useful for forest managers and plant producers in many areas. berry extract of juniperus sabina showed inhibito­ ry activities against kb tumor cell lines (sadeghi­ali­ abadi et al., 2009). fruit and leaves of junipers are commonly used as tea and pounded fruits are eaten to lower blood glucose levels in anatolia. to evaluate antidiabetic and antioxidant potential and the chemi­ cal profile of juniperus sabina l. in a study, phyto­ chemical screening tests indicated the presence of flavonoids, tannins, terpenoids and carbohydrates in the extracts (orhan et al., 2017). maymars juniper is usually propagated by vegeta­ tive methods (gheorghe et al., 2010). to propagate plants via cuttings, the indole­butyric acid (iba) growth regulator has been used as a treatment (amri et al., 2010). to produce junipers by stem cuttings, iba has been used in previous studies (henry et al., 1992; rifaki et al., 2002). research conducted by rifaki et al. in 2002 on vegetative propagation showed the best concentration for the cuttings of junipers at 4000 ppm of iba. phenolic compounds have effects on growth, development, propagation and plant defense (croteau et al., 2000). measurement of internal com­ pounds and their comparison during growth or root­ ing can be valuable factors in identifying internal bar­ riers or enhancers of rooting in the cuttings, as there are no extensive resources available in this regard. phenolic compounds are a group of antioxidant agents (choudhury et al., 2013). many scientists have reported the relationship between total phenol and antioxidant activities (hariprasath et al., 2015). in the propagation of varieties of blueberry, softwood cut­ tings and tissue culture, the interaction of genotype, propagation methods, and growth seasons signifi­ cantly affected flavonoid content and antioxidant capacity. the interaction effect of the propagation method and genotype significantly affected total phenol and chlorophyll content. also, the interaction between propagation method and growth season sig­ nificantly affected the total flavonoid content (goyali et al., 2013). some studies have also revealed differences in rooting of cuttings as affected by substrate (kentelky, 2011). cocopeat and iba were used to propagate juniperus excelsa through stem cuttings, and they improved rooting ability (esmael nia et al., 2006). growth regulator and substrate are effective on the rooting of the cuttings of juniperus oblonga, and proper substrate composition and the use of benzyl adenine increase the rooting of the cuttings (khoshnevis et al., 2012). roots uptake minerals and water from the soil (chapin et al., 1987). higher numbers of adventitious roots could improve the root system’s symmetry, sta­ bility, survival, and growth rate (bryant and trueman, 2015). thus, rooting percentage is a good indicator of the growth strategies of root development and the capacity to endure water stress in juniperus trees (garcia morote et al., 2012). therefore, the present study is intended to inves­ tigate an efficient method of vegetative propagation of maymars juniper using stem cutting and its effects on some of its phytochemical characteristics (pheno­ lic compounds). we hypothesized that high level of phenolic compounds during rooting can be an indica­ tor of the level of rooting in cuttings of juniperus sabina, and that the percentage of rooting should be an indicator of rooting performance in cuttings. thus, the objective of this research was to analyze the effects of five concentrations of iba as treatment and four substrate types (perlite, perlite cocopeat, pumice, and mixed substrate) on the level of pheno­ lic compounds and rooting performance in cuttings. the experiment was conducted in four months (february, mild climate; july, warm temperate cli­ mate; october, relatively cold weather; and january, cold weather) to determine the impact of harvesting time on the rooting capacity of cuttings. 2. materials and methods cutting preparation, treatment with indole butyric acid (iba), and substrate composition. the cuttings of juniperus sabina were sampled from its natural habitat in the chaharbagh mountains abshahi et al. ‐ secondary metabolite changes in cuttings of juniperus sabina during propagation 165 of gorgan, north iran (fig. 1), one of the main mediterranean populations at higher altitude (2,700 m a.s.l.). using a 30­year average, the mean annual temperature at the site is 9.2°c, and the mean annu­ al precipitation is 429 mm. extreme temperatures (summer and winter) range from 23°c to ­5°c (data from gorgan climatic station: 46° 06 n, 28° 00 w; 2,600 m a.s.l.). the crowns are approximately 2 x 2 m in length and width. the ring diameter of shrubs is 20.0 cm averagely, and the height is 1.5 m (these are old and horizontal shrubs). generally, 20 male shrubs have been used for this experiment, and they are all growing in the same area with the same ecological environment. the experiment was conducted at gorgan university of agricultural sciences and natural resources in winter, spring, summer, and fall of 2017. stem cuttings were only collected from the upper crowns of male trees. cuttings were harvested in the morning. after har­ vesting, the stem cuttings were prepared to be 15 cm in length and 0.5­0.7 cm in diameter (bohlenius et al., 2017) for treating and cultivation in a greenhouse. substrates were prepared, and cuttings were placed in the greenhouse equipped with an automatic sys­ tem to control humidity (micro irrigation) and bottom heat. the average daily temperature during the experiment was 22°c, and the average relative humidity was 77%. the amount of light entering the greenhouse was varied based on the amount of nat­ ural light in each season. for the treatment of stem cuttings, five levels of iba were used: 0 or control, 1000, 2000, 4000, and 8000 mg l­1 (control is a sample that is placed in the substrate without adding any treatment and is used to compare the effect of the treatments used on cut­ tings). the base of each cutting was placed in the aqueous solution of iba for five seconds and then inserted into the substrate. the four used substrates were: i) perlite; ii) mixed rooting substrate ­ a combi­ nation of sand (20%), perlite (20%), cocopeat (20%), vermicompost (20%), and potash (20%); iii) perlite cocopeat (1:1), and iv) mineral pumice (each sub­ strate about 10 kg). for each treatment (combination of treatment and substrate), three replicates were prepared, with nine cuttings per replicate. thus, a total of 540 cuttings in each season were cultivated. total phenol, flavonoids, and antioxidants of stem cuttings secondary metabolites were measured in both rooted and unrooted stem cuttings to detect differ­ ences in the internal compounds between cuttings that have the potential for rooting and others with­ out this potential. for evaluating the treatments and to make comparisons between the chemical com­ pounds in cuttings at the beginning of the sampling and the amount of increase or decrease between the time of planting and rooting (between the first and the end of each season), samples were taken from freshly harvested cuttings in each season (the first of each season with samples separately from the stem cuttings) and compared with the results at the end of the growing season. in order to measure total phenol, antioxidants and flavonoids (at the end of each season and after harvesting the cuttings from substrate), in the first step, one gram of each plant sample, which was the bark of the stem of each cutting separately, was removed and powdered with liquid nitrogen, then placed in 10 cc of 80% methanol (merk) in an erlenmeyer flask, and after that, placed on a shaker for 24 h. the mixture was then filtered with filter paper and clean extracts were used to measure sec­ ondary metabolites in mg/g fresh weight (mcdonald et al., 2001). then we began to assess the total phe­ nolics, antioxidants, and flavonoids. to measure total phenol, 20 μl of each of the above plant extracts were added to 1.16 μl of dis­ tilled water, 100 μl of folin (merk) and 300 μl of sodi­ um carbonate (20%), and they were mixed in a test tube (it is done for each plant sample separately) and then placed in a water bath at 45 °c for 30 min. after that, each sample was measured by a spectropho­ tometer (unic­uv 2800 ­ 4 cells) at a wavelength of 760 nm. after drawing the standard graph (prepara­ tion of different concentrations with specific values of the control­different samples and readings with the spectrophotometer and then drawing on the curve) (fig. 2), the phenol value of each sample was obtained (mcdonald et al., 2001). fig. 1 ­ the worldwide distribution of different populations of juniperus sabina (in grey) and the sampling area of stem cuttings (in a red circle) (adams and schwarzbach, 2016). adv. hort. sci., 2022 36(3): 163­174 166 to measure the flavonoids, 0.5 ml of each plant extract, 1.5 mg/l pure methanol (merk), 0.1 ml of aluminum chloride, 0.1 ml of potassium acetate, and 2.8 ml of distilled water were combined and mixed in a test tube, and then all samples were placed in the dark for 30 minutes, and after that, they were mea­ sured by a spectrophotometer with a wavelength of 415 nm. after drawing the standard graph (prepara­ tion of different concentrations with specific values of the control­different samples with readings with the spectrophotometer and then drawing on the curve), the flavonoid value of each sample was obtained (chong et al., 2002) (fig. 3). to measure antioxidant activity, 1 ml of each plant extract was removed. in the next step, the amount of 0.0004 mg of dpph was dissolved in 10 ml of methanol (merk), and then 1 ml of this solution with 1 ml of each extract of the plant previously removed was combined, and finally, the antioxidant percentage was measured in a spectrophotometer with a wavelength of 517 nm (miliauskas et al., 2004). rooting percentage to determine the rooting percentage of each treatment, the roots were counted in all rooted cut­ tings (fig. 4) in each treatment (three replications and each replication contained 9 cuttings; totally 27 cuttings) and then this number of cuttings was divid­ ed by 27 (some cuttings were unrooted and some of them were dried), (negash, 2002). statistical analysis a factorial arrangement of treatments (hoshmand, 2006) was applied to analyze the effects of three main factors on five dependent variables. the first factor was “treatment” or concentration of iba (five levels: 0, 1000, 2000, 4000, and 8000 ppm), the second was “substrate” (four levels: perlite, per­ lite­cocopeat, pumice, and mixed rooting substrate), and the third factor was “season” (four levels: january, april, july, and october). this represents a 5 x 4 x 4 factorial with 80 combinations of factor levels or treatments. the dependent variables were inter­ nal compounds of the cuttings (secondary metabo­ lites in both unrooted and rooted cuttings) and the indicator of rooting performance (% of rooting). therefore, in the dependent variables concerning chemical internal compounds, another level was added as treatment, secondary metabolites in fresh samples (stem cuttings not planted and prepared at the beginning of each season). this was done to com­ pare the effects of treatments between cuttings not treated (at the beginning of each season) and treated cuttings at the end of each season. sas® statistical software (neter et al., 1996) was used to detect significant factors and to compare mean values between factors and levels of treat­ ments. the comparison of the means was done using the proc glm procedure. we utilized multifactor analysis of variance (a three­way anova model) at a probability level of 5% (p<0.05). the analysis within season was performed by a two­way anova (exclud­ ing season as a main factor in the complete model). in this research, we performed independent anovas (not a mixed­design nor a repeated­measures anova) because the measurements were indepen­ fig. 2 ­ standard graph of total phenol measurement. fig. 3 ­ standard graph of flavonoids measurement. fig. 4 ­ rooted cuttings. abshahi et al. ‐ secondary metabolite changes in cuttings of juniperus sabina during propagation 167 dent (we used different stem cuttings for each treat­ ment and season). a fisher’s least significant difference (lsd) test (p<0.05) was used to determine the significant differ­ ences between treatments (neter et al., 1996). to apply this statistical method, it is desirable for data to be normally distributed. this is not the case with pro­ portions, which have values that range between zero and one. in addition, errors must be independent and normally distributed with constant variance. to ensure these assumptions, a logarithmic transforma­ tion was used (sabin and stafford, 1990): for the per­ centage of rooting, the analyzed variable was [ln (r+0.5)], and r was the percentage of rooting (divided by 100). as this transformation requires numerical data above zero, a small number (0.5) was added to this variable before the transformation. the other dependent variables were normal and then distrib­ uted. 3. results rooting performance in table 1, the p­values for the three principal effects (substrate, treatment with iba and season, and their two­way interactions) and for the effects within each season (substrate, treatment, and their interactions) are represented. effects must be consid­ ered significant when p<0.05. 540 stem cuttings in each season were planted. in january, five treat­ ments rooted (99 cuttings), and 441 cuttings were unrooted. in april, 20 treatments rooted (502 cut­ tings) and 38 cuttings were unrooted. in july, four treatments rooted (89 cuttings) and 451 cuttings were unrooted. in october, four treatments rooted (102 cuttings) and 438 cuttings were unrooted. as it is clear from figure 5, the best root­growing month is april. during spring, rooting was more than 50% at a level of 4000 ppm of indole butyric acid with no significant difference at the 1000 ppm level. also, the minimum rooting percentage of the cuttings in this month was about 25% at the level of 8000 ppm of indole butyric acid and control treatment; howev­ er, it was higher than the rooting percentage of other months. in the study of the effect of different sub­ strates on the percentage of rooting of the cuttings, the best substrate was seen in equal parts of perlite­ cocopeat (v/v), with rooting at a maximum of 62% with a treatment of 1000 ppm (fig. 6). and this sub­ strate was one of the substrates that had the largest number and length of roots (fig. 7 c and fig. 8 c). therefore, among the substrates used in this research to root the juniperus sabina, the best sub­ strate was perlite­cocopeat, with a maximum rooting percentage of 98. while the least rooting percentage of cuttings was seen in january, with less than 2% in all treatments, october is also not a good time for the reproduction of this plant. on the other hand, the most root number and root length was seen in april (fig. 7 b and fig. 8 b). so, the best months for rooting of cuttings of juniperus sabina are april and may, and the best levels of iba used were 4000 and 1000 ppm, despite the fact that the largest number of roots was not seen in these treatments. table 1 ­ results of the multifactor anova to analyze the effects of the main factors on the rooting performance of cuttings across the four seasons variable effects growing season values january april july october rooting treatment 0.10 <0.001 0.0005 0.11 <0.0001 (log­transformed units) substrate 0.91 0.03 0.22 0.08 <0.0001 season ­ ­ ­ ­ <0.0001 treatment x substrate ­ 0.24 ­ ­ <0.0001 treatment x season ­ ­ ­ ­ <0.0001 substrate x season ­ ­ ­ ­ <0.0001 fig. 5 ­ mean values of rooting ability in rooted cuttings (percen­ tage of rooting) within seasons and for the 5 treatments with indole butyric acid (iba). the mean values for the same letter were not different at the 0.05 level according to the lsd test. sample data = 540 cuttings for each month. for treatments not represented in the figure, all the cuttings dried. error bars: lsd intervals. 168 adv. hort. sci., 2022 36(3): 163­174 secondary metabolites concentration the results of the main and interaction effects of different treatments are presented in table 2. based on the results, each of the measured factors has been interpreted and reviewed. phenol content as it is shown in figure 9 a, among treatments in unrooted cuttings, the highest total phenol content fig. 6 ­ the mean values of rooting performance in rooted cut­ tings (percentage of rooting) within substrates and for the 5 treatments of indole butyric acid. the mean values with the same letter were not different at level 0.05 according to the lsd test. sample data: 540 cuttings for each substrate. fig. 7 ­ a, b, c the mean values of rooting performance in roo­ ted cuttings (root number) within for the 5 treatments of indole butyric acid, 4 season and 4 substrates. the mean values with the same letter were not different at level 0.05 according to the lsd test. sample data: 540 cuttings for each substrate. fig. 8 ­ a, b, c the mean values of rooting performance in roo­ ted cuttings (root length) within for the 5 treatments of indole butyric acid, 4 season and 4 substrates. the mean values with the same letter were not different at level 0.05 according to the lsd test. sample data: 540 cuttings for each substrate. factors df phenol flavonoid anti­oxidant unrooted cuttings rooted cuttings unrooted cuttings rooted cuttings unrooted rooted cuttings iba 4 16675.96 * 54417.29 ** 19.35.41 ns 2034.93 ns 249.75 ns 813.69 ** season 3 161064.55 ** 19288.21 ns 84936.79 ** 12674.40 ** 10155.84 ** 11007.76 ** substrate 3 24699.73 ** 14171.04 ns 2281.75 ns 1549.77 ns 337.35 ns 124.01 ns iba x season 6 55862.72 ** 93512.97 ** 9142.79 ** 9016.74 ** 840.18 ** 1411.13 ** iba x substrate 12 9947.75 ns 4875.10 ns 1489.16 ns 794.45 ns 118.72 ns 215.88 * season x substrate 3 10173.14 ns 263.48 ns 2666.17 ns 0.00 ns 139.36 ns 6.48 ns iba x season x 0 7509.96 ns 2371.40 ns 1690.41 ns ­ 163.35 ns 38.92 ns error 66 5661.43 8792.53 1604.33 1818.08 170.08 90.61 table 2 ­ results of a multifactor anova used to examine the effect of major factors on the secondary metabolite composition of stem cuttings over four seasons in the table, the p­values for the three principal effects (substrate, treatment with iba and season, and their two­way interactions) are represented. effects were considered significant when p<0.05. 540 stem cuttings in each season were planted.* p<0.5. abshahi et al. ‐ secondary metabolite changes in cuttings of juniperus sabina during propagation 169 (mcdonald et al., 2001) was observed with no signifi­ cant difference in the fresh sample as well as in 4000 ppm and 8000 ppm of indole butyric acid treatments, and the lowest level was observed in control, 1000 ppm, and 2000 ppm treatments without any signifi­ cant difference. a fresh sample was prepared with other cuttings at the beginning of each season and is used only to measure the internal composition of the plant at the beginning of the season; no treatment is performed on it. it was to compare the amount of internal compounds of the plant at the beginning of the cutting time and compare it with the amount of these compounds after maintaining the cuttings in the substrate to root (control is a sample that is placed in the substrate without adding any treatment and is used to compare the effect of the treatments used on cuttings). among the different substrates, the lowest amount of phenol content was found in stem cuttings that were planted in the mixed rooting substrate (fig. 9 b). among the unrooted cuttings, the lowest phe­ nol content was observed in a fresh sample and a treatment of 1000 ppm in january (fig. 9 c). between rooted cuttings, in april, with the highest rooting per­ centage of cuttings, treatments of 4000 and 8000 ppm showed lower phenol content, and there was no significant difference between other treatments (fig. 9 d). flavonoid content among the unrooted cuttings in different seasons, the highest flavonoid levels were observed in january and july, and the lowest were seen in april and october (fig. 10 a). the flavonoid content of juniperus sabina differed during different seasons. in rooted cuttings, flavonoid content was not sig­ nificantly different in treatments applied in different months, and the overall amount of flavonoid was between 50 and 100 mg/g of fresh weight (fig. 10 b). percentage of antioxidant in unrooted cuttings, the highest percentage of antioxidants was found in january and october with more than 70%, and the lowest was observed in july with a maximum of 20%. in april, an intermediate level of antioxidants was observed in unrooted cut­ tings (fig. 11 a). in all months except july, the per­ centage of antioxidants in the first samples was about 70%, but in july it was about 20%. it should be noted that in july and january, the percentage of antioxidants increased after treating and planting the cuttings in substrate; this amount was unchanged in october (during fall) and it decreased in april (during spring), and its decline was also significant. in rooted cuttings, the percentage of antioxidants in january was much higher april (fig. 11 b). among fig. 9 ­ a, b, c, d mean internal phenol content within seasons for the different treatments with iba for rooted and unrooted cuttings (a, b, c, d). the mean values for the same letter were not significantly different at the 0.05 level according to the lsd test. 540 stem cuttings in each season were planted. for the treatments that were not represented in the figure, those cuttings have dried. fig. 10 ­ a, b. the mean internal flavonoid content within sea­ sons for the different treatments with iba for rooted and unrooted cuttings. the mean values for the same letter were not significantly different at the 0.05 level according to the lsd test. 540 stem cuttings in each season were planted. for treatments not represented in the figure, all the cuttings dried. adv. hort. sci., 2022 36(3): 163­174 170 different substrates, the lowest percentage of antiox­ idants in rooted cuttings was seen in mixed rooting substrate, and its maximum was seen in perlite sub­ strate (fig. 11 c). pumice and perlite­cocopeat sub­ strate showed a medium antioxidant percentage. it means in the lighter substrate, the antioxidant per­ centage was increased, and in the heavier substrate, the percentage of that was decreased. 4. discussion and conclusions indole butyric acid is widely used at commercial level to root many species (hartmann et al., 1990; negash, 2002; esmael nia et al., 2006; khoshnevis et al., 2012). it slowly releases a source of indole acetic acid (epstein and ludwig­muller, 1993). current evi­ dence indicates that indole butyric acid is naturally occurring in plants. further stability of iba in compar­ ison with indole acetic acid during rooting experi­ ments has been reported by nordstrom et al. (1991), which is effective on decomposition and building. part of the function of indole butyric acid is the direct effect of auxin (ludwig­muller, 2000; poupart and waddell, 2000). although other functions are due to the conversion of iba to iaa by b­oxidation (epstein and lavee, 1984; zolman et al., 2000; bartel et al., 2001). auxin can be increased for up to 24 hours after sampling (tartoura et al., 2004; osterc et al., 2009). increasing root numbers after the use of indole butyric acid occurs in many woody plants (jarvis, 1986). adventitious roots on the cuttings were creat­ ed by treating them with auxin growth regulators, especially indole butyric acid (buchala and schmid, 1979; haissig et al., 1992). this is consistent with the results of this research on its effectiveness on root­ ing. one possible explanation is that exogenous aux­ ins can increase the amount of internal auxin in the direction of the onset of the formation phase of the rooting and then the root appearance (metaxas et al., 2004). with an increase in the presence of the cuttings in the substrate, the rooting rate of the cut­ tings also increases (cope and rupp, 2013). the use of indole butyric acid leads to an increase in rooting (bielenin, 2003). in our study, the best results were obtained from intermediate levels of iba (1000­4000 ppm) without a significant difference between these treatments, and we hypothesize that iba at 8000 ppm can dam­ age the cuttings and reduce rooting. in j. virginiana, iba concentrations up to 2000 ppm did not stimulate rooting beyond that obtained with 5000 ppm (henry et al., 1992). in general, iba has been used for the rooting of juniperus species with different treatment levels. for example, results were best at 8000 ppm of iba in juniperus osteosperma (cope and rupp, 2013), 5000 ppm of iba in j. virginiana (henry et al., 1992), 1000 ppm to 9000 ppm in juniperus scopulorum (bielenin, 2003), chowdhuri (2017), with 1000 to 3000 ppm in juniperus chinensis and tektas et al., (2017) with 6000 ppm in juniperus l. in the research on juniperus virginiana, henry et al. (1992) cited that in preliminary studies, iba concentrations up to 20000 ppm did not stimulate rooting beyond that obtained with 5000 ppm. thus, our results are more in agreement with rifaki et al. (2002), which pro­ posed a concentration of 4000 ppm of iba in cuttings of juniperus excelsa, and esmaeil nia et al. (2006), with 3000 to 6000 ppm in j. excelsa. nevertheless, the novelty of our results is that the concentration of iba we selected (1000 ppm) was lower. substrate characteristics are very important in rooting success. several studies have shown that the substrate plays a significant role in the quality of root formation and the percentage of rooted cuttings. proper air preservation is a necessary feature of a fig. 11 ­ a, b, c. the mean internal antioxidant percentage within seasons for the different treatments with iba for rooted and unrooted cuttings. mean values for the same letter were not significantly different at the 0.05 level according to the lsd test. 540 stem cuttings in each season were planted. for treatments not repre­ sented in the figure, all the cuttings dried. abshahi et al. ‐ secondary metabolite changes in cuttings of juniperus sabina during propagation 171 good rooting atmosphere. therefore, it seems that proper rooting substrate can maintain proper mois­ ture to prevent the cutting ends from drying out and to provide enough air to facilitate rooting and pre­ vent disease spread at the base of the cuttings. surely there is an optimum temperature for sub­ strate for root formation and growth, and rooting at low temperatures will not occur or will occur very slowly. it is also possible for the roots to appear and grow at very high temperatures in the substrate. bottom­heat is useful for rooting only when the tem­ perature is low (couvillon, 1988) which is consistent with the results of this research. in our study, the percentage of rooting was more than 60% in sub­ strate of perlite cocopeat. in an study of juniperus procumbens the best substrate was 1.3 (v/v) vermi­ culite and 2.3 (v/v) perlite, with only 36% rooting (hong­wei et al., 2011). the results of our study was also better than the results obtained from cuevas­ cruz et al. (2015) in pinus, with 43.5% of rooting (substrate was a mixture of peat­perlite­vermiculite), khoushnevis et al. (2012) with 28% of rooting in juniperus oblonga (fine and harsh bed), stuepp et al. (2014), with 16% of rooting in j. chinensis (fine grained vermiculite and carbonized rice hull 1:1) and ayan et al. (2004), with 24% of rooting for j. foetidis‐ sima, 31.5% of rooting for j. excelsa, 38.42% of root­ ing for juniperus sabina and 31.83% of rooting for j. oxycedrus (perlite). cutting time plays an important role in the suc­ cess of rooting. although many species are most rooted when cuttings are prepared in late spring or early winter before the wood has hardened, many other species have the best rooting when cuttings are taken at other times of the year. a good example of this is the juniperus horizontalis, whose cuttings were most rooted when they were prepared between november and february compared to other times of the year (ali ahmad koruri et al., 2011). the result of this research showed that the best time for rooting juniperus sabina to prepare the cuttings is april. therefore, for this species, the best time to prepare cuttings and plant them is spring. this differs from guerrero­campo et al. (2006), who found the best rooting of several species of cuttings at different sea­ sons and chowdhuri (2017), who showed the best rooting time for juniperus chinensis was summer. on the other hand, our result was in agreement with fragoso et al. (2015) and tektas et al. (2017), who respectively cited the best season for rooting of juniperus chinensis and juniperus l. as spring. apparently, the presence of secondary metabo­ lites in plants acts as a defense (toxic) agent that inhibits proliferation and other growth­related actions (singh rattan, 2010), as shown in the results of this study. although most of the phenolic com­ pounds have a structural role in the cell wall, the major activity of these compounds is in defense of the plant; they have several roles in plants, but are mainly used for their great effects on growth, devel­ opment, propagation, as well as plant defense against animals and pathogens (croteau et al., 2000). the presence and yield of secondary metabolites in plants, such as aromatic compounds and compounds in essential oils, may be affected in different ways, from formation to separation from plants. rapid sec­ ondary metabolite induction occurs as a chemical mediator of plant rooting and defense (metlen et al., 2009), and the amount of secondary metabolites changed during the preservation of cuttings in the substrate. the rooting barrier of yew cuttings was identified by biological and organic methods. the results showed that the most important barrier to propagation in this plant was phenol content (guangyou, 2000). the maximum amount of total phenol in the leaves of common juniper was 315.33 mg/g (ved et al., 2017), which is consistent with the results of this study. in cherry leaf cuttings, gisela 5, auxin had no effect on phenol levels, so the same results were observed in the present study. cuttings should have definite levels of different phenolic compounds to start the rooting induction phase, but the greater effect on rooting success is attributed to the effect of auxin level (trobec et al., 2004). phenolic compounds are a class of antioxidants (choudhury et al., 2013), and the level of internal antioxidants in plants is different (rehman et al., 2014). many authors have reported an association between total phenol content and antioxidant activi­ ty (hariprasath et al., 2015). the main antioxidant activity is due to specific secondary metabolites, especially phenolic compounds and some terpenes (marzouk et al., 2007; awaad and al­jaber, 2010). interactions among genotypes, propagation meth­ ods, and growing seasons significantly affect flavonoid content and antioxidant capacity (goyali et al., 2013), which is consistent with the results of this study. the amount of secondary compounds varied according to season and substrate, just as it did in the current study. the climate of the outdoor region during the three months of october, january, and adv. hort. sci., 2022 36(3): 163­174 172 april increased the amount of antioxidants inside the plant, while in july, with a hot climate, it dropped dramatically. growth regulators increase antioxidant activity (dakah et al., 2013), which contradicts the results of this research. because in some cuttings treated with indole butyric acid, an increase in antioxidants was observed, and in other treatments, a decrease was observed. the results of a study on one of the iranian conifers showed that the antioxidant activity of the extracts ranged between 60 and 99% (hariprasath et al., 2015), which contradicts the result of the present study, which shows that the range of antioxidants in some treatments was less than 20%. in the use of indole butyric acid for the propaga­ tion of juniperus sabina through cuttings, the best rooting month (season) for cuttings was april, and the rooting percentage in this month was higher than in other months (more than 50%), while instead, the lowest rooting rate was seen in january. the best lev­ els of indole butyric acid used were levels of 4000 and 1000 ppm, respectively. so, for the propagation of sabina species, it is recommended to use these levels of iba as a treatment for stem cuttings in april. also, the best substrate used was perlite­cocopeat. between rooted cuttings, in april, 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b., 2000 ­ genetic analy‐ sis of indole‐3‐butyric acid response in arabidopsis thaliana reveals four mutant classes. ­ genetics, 156: 1323­1337. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5307892/ impaginato 73 adv. hort. sci., 2021 35(1): 73­79 doi: 10.36253/ahsc­9874 the induction and development of somatic embryos from the in vitro cul­ tures of catharanthus roseus (l.) g. don h. farhadi 1, m.b. hassanpouraghdam 1, 2, m.a. aazami 1 (*) 1 department of horticultural sciences, faculty of agriculture, university of maragheh, maragheh, iran. 2 islamic parliament research center, tehran, iran. key words: catharanthus roseus (l.) g. don, ms, pgr, somatic embryogenesis. abstract: catharanthus roseus is containing anticancer alkaloids of vincristine and vinblastine and is an important medicinal plant. several studies have con­ ducted on in‐vitro culture of this plant. to optimize the somatic embryogenesis, a factorial based on crd experiment with 10 replications was conducted. root, hypocotyl and leaf explants grown in­vitro were transferred and cultured on ms media containing different combinations of 2,4­d, naa and 2,4­d×bap. the results revealed that in callogenesis, the interaction effects of root and hypocotyl explants×2,4­d and naa as well as hypocotyl×(1 mg l­1 naa+1 mg l­1 bap) was superior than other treatments (p≤0.01). for calli fresh weight, hypocotyl×naa and hypocotyl× (1 mg l­1 naa+1 mg l­1 bap) was the treatment of choice (p≤0.01). the calli produced were sub­cultured to attain the pre­ embryos and somatic embryos. for the number of pre­embryos and somatic embryos; the interaction of hypocotyl×2,4­d was the most efficient treatment. seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. furthermore, the pro­ duction and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high­valued metabolites from this plant. 1. introduction catharanthus roseus (l.) g. don, generally known as madagascar periwinkle, is a dicotyledonous plant with 2n=16 belongs to the family of apocynaceae. catharanthus roseus (l.) g. don is an herbaceous plant that grows to a height of about 80 cm and is native of madagascar (hogan, 2003). more than 130 types of alkaloids have been extracted from the vegetative parts and roots of this plant, which are used to treat several diseases (aslam et al., 2009). the most important alkaloids extracted from the shoots of this plant are vincristine and vinblastine, with a well­ defined anti­cancer properties (mujib et al., 2012). the amount of these compounds in this plant is about 0.0005% of dry weight of the plant and their extraction is costly and time consuming (barrales­cureño et al., 2017). these problems have led the scientists to a new approaches of tis­ (*) corresponding author: aazami58@gmail.com citation: farhadi h., hassanpouraghdam m.b., aaza­ mi m.a., 2021 ­ the induction and development of somatic embryos from the in vitro cultures of catharanthus roseus (l.) g. don. ­ adv. hort. sci.,35(1): 73­79 copyright: © 2021 farhadi h., hassanpouraghdam m.b., aazami m.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 october 2020 accepted for publication 1 february 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9874 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 73­79 74 sue culture studies in c. roseus l. (van der heijden et al., 2004). the first study done on tissue culture of catharanthus roseus (l.) g. don was in 1977. those, researchers were able to produce callus from the plant (dhruva et al., 1977). the production of shoots from callus has been successfully done in catharanthus roseus l. (ramawat et al., 1987). somatic embryogenesis is a process by which somatic cells differentiate into embryos, called somatic embryos, which are used as one of the practical in vitro techniques for plant micro­propagation (von arnold et al., 2002). the first studies on somatic embryogenesis in the catharanthus roseus (l.) g. don was done in 1994, which were succeeded in pro­ ducing somatic embryos by the anther culture (kim et al., 1994). furthermore, by hypocotyl explant and 1 mgl­1 of 2,4­d (2,4­dichlorophenoxyacetic acid), somatic embryos were obtained (aslam et al., 2004, 2006). growth regulators (composition and concen­ tration) and the plant genetic make­up play a role in the success of somatic embryos production. the phe­ nomenon of genotype­dependent plant regeneration also exists in other plant species (firoozabady and de boer, 1993). the physiological conditions, the explant growth stage and embryogenic tissue type affect the produc­ tion of somatic embryos. the tissue that has the high­ est metabolism and the least differentiation rate may have the suitable embryogenesis potential (mikula and rybczynski, 2001). auxins, especially 2,4­d, are among the most important plant growth regulators initiate the somatic embryogenesis (choi et al., 1999; martin, 2003; gulzar et al., 2019). in medicinal herb spermatozoa hispida l.; cytokinin, especially bap (6­ benzylaminopurine), induced frequent somatic embryos (deepak et al., 2019). with medicinal herb, coccinia abyssinica; 2,4­d and bap combination helped to produce embryogenic callus, and bap trig­ gered the production of somatic embryos (abate et al., 2019). l­glutamine and l­alanine amino acids have had the most positive effect on somatic embryogenesis process (ji et al., 2011). various sources of carbon; such as sucrose, fructose, and glu­ cose, as energy sources and osmotic regulators; play an important role in the somatic embryogenesis initi­ ation and frequency (aslam et al., 2011). ms has been employed as the most common culture medi­ um for most of the plants (ji et al., 2011). somatic embryogenesis, leading to the regeneration of intact plants, is an eminent step in the plant transforma­ tion. successful and sustainable transformation requires that a single cell give rise to a whole intact plant. the ideal transformation takes place through the direct somatic embryogenesisfrom the single cells to reach the intact plants (aslam et al., 2007). the aim of the present study was to investigate the different explant types and the diverse plant growth regulators effects to induce somatic embryos and to study of the traits related to the somatic embryogenesis potential in catharanthus roseus (l.) g. don. 2. materials and methods in vitro seed germination and seedlings production the present study was conducted in the research laboratory, department of horticultural sciences, university of maragheh, iran. the seeds were acquired from pakan bazr isfahan company. the seeds were immersed in water for one day before planting in the dark, and the next day, they were first treated with ethyl alcohol (70%) for one minute and then disinfected with 20% solution of sodium hypochlorite for 10 more minutes. then, they were washed with sterile distilled water 5 times. the disin­ fected seeds were cultured in petri dishes containing 20 ml of ms medium (murashige and skoog, 1962) without plant growth regulators. in each petri dish; 10 disinfected seeds were cultured at 25­28°c. after emergence, the seedlings were transferred to the photoperiod conditions with 16 hours of light at 25°c and 8 hours of darkness at 20°c. after about 10 days, the 2­4 cm in length seedlings were suitable to con­ tinue the experiment. preparation of culture media the culture medium was ms basic medium. sucrose at a concentration of 30 g l­1 was used as a source of carbon. then, ms culture medium salts were added. ph was set at 5.8. at the last stage, 6 gl­1 of agar was added and placed in an autoclave at 121°c for 20 min. agar­free culture medium (liquid) was employed for the somatic embryos. experimental design a factorial experiment based on completely ran­ domized design (crd) was planned with 10 replica­ tions. factor 1: plant growth regulators, concentration of 1 mgl­1 2,4­d, 2 mgl­1 naa (1­naphthaleneacetic acid), 1 mgl­1 naa + 1 mgl­1 bap factor 2: explants (root, hypocotyl and leaf). farhadi et al. ‐ somatic embryo induction from catharanthus roseous 75 sampling and culture of explants in different treat‐ ments the explants; root, hypocotyl and leaves were excised from the 2­4 cm seedling grown in vitro. hypocotyl explants were cut into a length of about 1 cm and, in order to obtain embryogenic callus; they were cultured in the ms medium supplemented with the same treatments as subcultures. for calli prolifer­ ation; 3 weeks after the first date of cultivation, the same plant growth regulator treatments were applied to the subcultures. one month after the first subculture; embryogenic calluses were sub­cultured in ms medium supplemented with concentrations of 0.5, 1, and 1.5 m g l­1 bap. then, the resulting calli were transferred to ms medium supplemented with 7 g l­1 of agar, 30 g l­1 of sucrose, 1 mg l­1 glutamine and 3 g l­1 of polyethylene glycol with 6 replications. at the all stages of cultures and subcultures; the sam­ ples were kept in a growth chamber for 16 h light at 25°c and 8 h dark at 20°c. data mining and statistical analysis the callus production percentage, the percentage of necrotic calli and callus weight were noted. after multiple sub­cultures and transfer to the embryogen­ esis culture medium, the number of pre­embryogenic mass formed were recorded. sas was used to ana­ lyze the variance, and mean comparisons were done with duncan’s multiple range test at 5% probability. 3. results callus production hypocotyls of in vitro germinated seeds were used as explant on ms medium supplementary with 2,4­d and naa, which induced white to yellowish cal­ lus within 10 days of incubation. the embryogenic callus was white, granular, friable, fast growing with­ in 3 weeks of culture. the number of explants that produced callus and the necrotic calli proportion showed the callogenesis percentage and the percent­ age of necrotic calli (fig. 1). the mean comparison showed that for the callus formation, the interaction between root and 2,4­d; hypocotyl and 2,4­d; root and naa; hypocotyl and naa; and hypocotyl and 1 mgl­1 naa + 1 mgl­1 bap were significant at 1% probability level. figure 2 shows the significant effect of auxin use on the rate of callus production. in this study 2,4­d was very effi­ cient in producing callus and embryos on catharanthus roseus. embryonic callus was not observed using leaf explants and hypocotyl explants had the highest embryonic callus. furthermore, for the callus fresh weight, the interactions of hypocotyl and naa; and hypocotyl × 1 mg l­1 naa + 1 mgl­1 bap were significantly different from other treatments (p≤0.01) (fig. 3). fig. 1 ­ the effect of 2,4­d, naa and bap on hypocotyl explants of catharanthus roseus and the formation of embryoge­ nic callus (3 week after culture). scale bars= 1.0 mm. (a) embryogenic callus from the treatment with 1 mg l­1 2,4­ d; (b) embryogenic callus from the treatment with 2 mg l­ 1 naa, (c) embryogenic callus from the treatment with 1 mg l1­ naa + 1 mg l­1 bap. fig. 2 ­ the interaction effects of explant×plant growth regula­ tors on the callogenesis percentage of catharanthus roseus. a= 1 mg l­1 2,4­d×root; b= 1 mg l­1 2,4­d×hypo­ cotyl; c= 1 mg l­1 2,4­d×leaf; d= 2 mg l­1 naa×root; e= 2 mg l­1 naa×hypocotyl; f= 2 mg l­1 naa×leaf; g= 1 mg l­1 naa+1 mg l ­1 bap×root, h= 1 mg l ­1 naa+1 mg l ­1 bap×hypocotyl; i= 1 mg l­1 naa+1 mg l­1 bap×leaf). fig. 3 ­ the effect of sub­culture of embryogenic calli from catharanthus roseus hypocotyl influenced by different treatments (calli are produced 1 month after subculture). scale bars= 1.0 mm. (a) embryogenic calli produced by the treatment of 2 mg l­1 naa; (b) embryogenic calli pro­ duced by the treatment of (1 mgl­1 naa+1 mg l­1 bap). adv. hort. sci., 2021 35(1): 73­79 76 the calli weight was obtained by weighting 10 samples. the results showed that the growth regula­ tors and explants type and their interaction were sig­ nificant on the weight of callus production, treatment composition h and e with 0.85 and 0.81 g, had the most callus weight respectively. the interac­ tion of hypocotyl and naa; and hypocotyl x naa + bap were significant (p≤0.01) on callus fresh weight (fig. 4). pre‐embryonic and somatic embryogenic tissues in this study, two different approaches were employed to increase the formation of embryogenic calluses. both approaches were aimed at choosing the best combination and concentration of plant growth regulators. different masses were observed on the embryogenic calli tissues and the pre­embry­ onic structures were formed evidently. the number of masess of possible pre­ and somatic embryos formed were counted. somatic embryos were isolat­ ed by liquied culture and shown with distinct roots and shoots (fig. 5). the results showed that the effect of plant growth regulators and explants, and their interactions were significant (p≤0.01) on the number of pre­embryos and somatic embryos produced. furthermore, the interactions of hypocotyl × 2,4­ d produced the highest number of pre­embryogenic and somatic embryogenic with mean 3.83 and 5.33 respectively in solid culture medium (fig. 6). 4. discussion and conclusions the most frequently used auxin in the studies on somatic embryogenesis is 2,4­d (bhojwani and razdan, 1996; junaid et al., 2006; jushee et al., 2007). the significant effects of 1 mgl­1 2,4­d has been proven on callus formation and somatic embryogenesis on catharanthus roseus and aconitum heterophyllum (giri et al., 1993; aslam et al., 2004). this auxin stimulates cell division and plays an important role in the production of callus (paramageetham et al., 2004). auxins alone or in combination with cytokinins are used for somatic embryogenesis induction and initiation (wojcikowska and gaj, 2016; tanida and shiota, 2019). more often, the use of cytokinins alone induces the production of non­embryogenic callus (martin, 2004). in dicotyle­ donous plants, cytokinins are usually added to the culture medium along with auxins for the promotion of callus production (george et al., 2008). in medici­ nal herb, asparagus racemosus willd with the embry­ onic explants cultured on ms­ medium and supple­ mented with 1.5 mgl­1 2,4­d + 0.43 mgl­1 kin; 74% fig. 4 ­ the interaction of explant×growth regulators on calli fresh weight at the in‐vitro cultures of catharanthus roseus. a= 1 mg l­1 2,4­d×root; b= 1 mg l­1 2,4d×hypo­ cotyl; c= 1 mg l­1 2,4­d×leaf; d= 2 mg l­1 naa×root; e= 2 mg l­1 naa×hypocotyl; f= 2 mg l­1 naa×leaf; g= 1 mg l­1 naa+1 mg l ­1bap×root; h= 1 mg l ­1 naa+1 mg l ­1 bap×hypocotyl; i= 1 mg l­1 naa+1 mg l­1 bap×leaf. fig. 5 ­ (a) pre­embryo spots (bar= 1 mm); (b) somatic embryos formed on the calli derived from hypocotyl explants of catharanthus roseus (bar= 1 mm); (c) somatic embryos isolated from liquid cultures including root and shoot ends (bar= 5 mm). fig. 6 ­ the interaction of explant×growth regulators on pre­ embryos and somatic embryos number produced in catharanthus roseus in‐vitro culture. a= 1 mg l­1 2,4­d × root; b= 1 mg l­1 2,4d × hypocotyl; c= 1 mg l­1 2,4­d × leaf; d= 2 mg l­1 naa × root; e= 2 mg l­1 naa × hypocotyl; f= 2 mg l­1 naa × leaf; g= 1 mg l­1 naa+ 1 mg l­1 bap × root; h= 1 mg l­1 naa + 1 mg l­1 bap × hypocotyl; i= 1 mg l­1 naa + 1 mg l­1 bap × leaf. farhadi et al. ‐ somatic embryo induction from catharanthus roseous 77 non­embryogenic callus was obtained (chaudhary and dantu, 2019). bap is the major cytokinin used in the studies related to the somatic embryogenesis (jimenez and thomas, 2005). singh et al. (2011) obtained 92% of non­embryogenic callus in catharanthus roseus (l.) g. don in ms culture medi­ um supplemented with 1 mg l­1 naa + 1 mg l­1 bap with hypocotyl explants. our results are consistent with the findings of aslam et al. (2006) in which hypocotyl explants in ms medium supplemented with 1 mgl­1 2,4­d, resulted in 85% of embryogenic callus. moreover, they reached 73% of embryogenic callus by hypocotyl explant in the ms medium enriched with 1.5 mgl­1 bap + 1 mgl­1 naa; and 61.75% of embryogenic callus by hypocotyl explants in ms medium supplemented 1 mgl­1 naa + 1 mgl­1 bap as well as 85% of embryogenic callus by hypocotyl explant again in ms medium enriched with 1 mgl­1 2,4­d (aslam et al., 2007). studies have shown that 1 mgl­1 2.4­d in ocimum basilicum l., produced about 75% of embryogenic calli (gopi and ponmurugan, 2006). also, the results of another study revealed that by culturing hypocotyl explants in ms medium containing 1 mg l­1 naa +3 mgl­1 ba; 80% of non­embryonic callus was observed (ren et al., 2020). auxins and cytokinins form callus tissues by accel­ erating the division of plant cells (george et al., 2008). in withania somnifera stems cultured in ms medium with 1mgl­1 bap + 1 mgl­1 naa; calli were produced with an approximate weight of 0.22 g (adhicari and pant, 2013). in another study, intern­ ode explants of centella asiatica l. on ms supple­ mented with auxins and cytokinins produced calli weighting up to 1.5 g (martin, 2004). 2,4­d causes the rapid cell division and the polar­ ization of cells (jushee et al., 2007). our results are almost similar with the findings of choi et al. (1999) on eleutherococcus senticosus (75% of somatic embryos). moreover, the findings of the present study are in line with the results of martin (2003) on holostema adakodien, which by using different explants and plant growth regulators; they clearly showed that hypocotyl explants and 2,4­d (1 mg l­1) attained about 50% of somatic embryos. in another study, hypocotyl explants of eleutherococcus sentico‐ sus in ms medium produced 89% of somatic embryos (han and choi, 2003). also, our results are the same with aslam et al. (2004) on catharanthus roseus (l.) g. don, whom described that hypocotyl and 2,4­d (1 m gl­1) were the combination of choice. also, in another study on catharanthus roseus (l.) g. don; the most somatic embryos were observed using hypocotyl explants and 2 mgl­1 2,4­d. auxins have synergistic effects with cytokinins and accelerate the cell division. 2,4­d may also add­up the endogenous levels of iaa in plant tissue, thereby creating dipoles within the cell and forming pre­embryonic structures (mendez­hernandez et al., 2019). 2,4­d promotes the accumulation of ros (oxygen reactive species), and stimulates the ethylene and abscisic acid biosyn­ thesis in plant tissue and ultimately induces stress behavior in plant tissue. as a result, plant cells change or shift to form somatic embryos (bharatia et al., 2015; wojcik et al., 2020). 2,4­d application and availability in the culture medium probably results in the expression of cell differentiation genes and the demethylation of dna. 2,4­d plays an important role in the somatic embryos induction and in the stages of maturation and development of somatic embryos. whereas, 2,4­d, has an inhibitory role with the biosynthesis of a number of proteins and mrnas (krishnan and siril, 2017). in our study, calli from leaf explants were unable to produce somatic embryos (gulzar et al., 2019). otherwise, paeonia ostii ‘feng dan’, asparagus racemosus willd, and cnidium offici‐ nale makino, the combinations of auxins and cytokinins produced reasonable somatic embryos (adil et al., 2018; chaudhary and dantu, 2019; ren et al., 2020). auxins had dominant effect on callus production of catharanthus roseus (l.) g. don. and the results showed that 2,4­d was much efficient than naa. more somatic embryos were obtained from hypocotyl explants. due to the medicinal importance of catharanthus roseus, it is suggested that in the further studies, somatic embryogenesis behavior in this plant should be studied on a larger scale and that the possibility of somatic embryogenesis in a liquid culture medium using suspension culture should be tested in different ways. therefore, it is possible to study the biosynthesis of secondary metabolites and valuable alkaloids in vitro conditions and to optimize the protocols to extract and purify the above men­ tioned metabolites. references abate m., mekbib f., gebre e., 2019 ­ in vitro somatic embryogenesis and plantlet regeneration in anchote [coccinia abyssinica (lam.) cong.]. ­ plant physiol. rep., 24: 351­358. 78 adv. hort. sci., 2021 35(1): 73­79 adhikari s.r., pant b., 2013 ­ induction and proliferation of in vitro mass of callus of withania somnifera (l.) dunal. ­ res. plant sci., 1(3): 58­61. adil m., kang d.i., jeong b.r., 2018 ­ data on recurrent somatic embryogenesis and in vitro micropropagation of conidium officinale makino. ­ dat. brie., 19: 2311­ 2314. aslam j., bhat m., mujib a., sharma m.p., 2004 ­ somatic embryogenesis study in catharanthus roseus (l.) g. don: an anticancerous plant., xi: 98­106. ­ in: khan i.a., khanum a., (ed.) role of biotechnology in medicinal and aromatic plants. ukaaz publication, hyderabad. aslam j., bhat m.a., mujib a., sharma m.p., 2006 ­ somatic embryo proliferation, maturation and germi‐ nation in catharanthus roseus. ‐ plant cell tissue organ cult., 84: 325­332. aslam j., mujib a., nasim s.a., sharma m.p., 2009 ­ screening of vincristine yield in ex vitro somatic embryos derived plantlets of catharanthus roseus l. 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metabolites, modified atmosphere packaging, respiration rate, short­term treatment. abstract: in addition to the adoption of proper temperature and relative humidity, the selection of an atmosphere surrounding packaged fresh produce with reduced o2 and/or increased co2 is one of the most widely used and use­ ful tools to prolong the shelf­life of horticultural crops. however, as o2 and/or co2 values that might cause injury are strictly related to the commodity, they should be optimized for each product. here three study cases are reported about the application of modified atmospheres (ma), with different co2 con­ centrations (0­40 kpa), to table grapes (cv. italia) and sweet cherries (cv. ferrovia) and, as a short­term treatment (48 h at 0°c), to fresh­cut artichokes (cv. violet de provence). in each trial, the effect of high co2 treatment on quali­ ty parameters was observed during cold storage. concerning table grape ‘italia’, our results show that low co2 (up to 10 kpa) ma preserved the quality and sensory parameters of the fruit, whereas high co2 (> 20 kpa) caused a fer­ mentative metabolism. as for sweet cherries ‘ferrovia’, 20 kpa co2 ma helped to maintain the quality traits during storage. on the other hand, this fruit proved to be sensitive to co2 accumulation (over 20 kpa) in hypoxic conditions, since it caused an increase in respiration rate and the biosynthesis of volatile fermentative metabolites. finally, for fresh­cut artichokes, a short­term co2 treatment, up to 10kpa, reduced respiration rate and browning index, preserv­ ing the volatile profile, while high co2 (40 kpa) may have caused fermentative metabolism. in conclusion, the application of a ma enriched in co2 has been shown to have different effects on the quality parameters of the three prod­ ucts, in agreement with the fact that co2 sensibility depends on each specific fruit or vegetable under study. 1. introduction fruits and vegetables are perishable products, and extending the keeping quality during their postharvest life represents one of the main (*) corresponding author: bernardo.pace@ispa.cnr.it citation: cefola m., capotorto i., lippolis v., cervellieri s., damascelli a., cozzolino r., de giulio b., pace b., 2023 ­ co2 modified atmo‐ sphere packaging: stress condition or treatment to preserve fruit and vegetable quality? ­ adv. hort. sci., 37(1): 67­73. copyright: © 2023 cefola m., capotorto i., lippolis v., cervellieri s., damascelli a., cozzolino r., de giulio b., pace b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 october 2022 accepted for publication 26 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13838 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 67­73 68 goals of researchers in this field. it is widely known that, together with the proper temperature and rela­ tive humidity management, the gas composition sur­ rounding the product during storage, is one of the main factors that affect the quality of horticultural crops (kader, 2003). in general, the decrease in oxy­ gen, the increase in co2, or the association of both conditions are useful to preserve the physiological state of fruits and vegetables, reducing the rate of respiration, oxidative processes, and decay, thus pro­ longing their shelf­life. in contrast, inappropriate gas concentrations outside safe limits can cause stress conditions that lead to physiological disorders, devel­ opment of off­odours due to fermentative metabolism, or increases in susceptibility to decay (mangaraj and goswami, 2009). although low o2 and high co2 have similar effects, under modified atmo­ sphere packaging (map) conditions, elevated co2 is a major factor influencing the quality of fruits and veg­ etables (watkins, 2000). in addition, the sensitivity to elevated high co2 and/or low o2 levels depends on the commodity (toivonen and deell, 2002). it is influ­ enced by pre and postharvest factors, such as culti­ vars or stage of maturity, and by processing, since the oxygen consumption and the consequent co2 accumulation in fresh­cut produce is faster than in corresponding intact produce (francis et al., 2012). to obtain the beneficial effect of map, gas conditions should be optimized for each product. starting from these considerations, the aim of the present work was to compare the effect of different co2 concen­ trations in map on the quality of table grapes, sweet cherries and fresh­cut artichokes as case studies. 2. materials and methods table grapes (vitis vinifera l., cv. italia), sweet cherries (prunus avium l., cv. ferrovia), and artichokes (cynara cardunculus (l.) subsp. scolymus hayek, cv. violet de provence) were provided by local farms located in noicattaro and foggia (italy) and processed at the postharvest laboratory of cnr­ispa the day of harvest. selected bunches of table grapes (about 1 kg each) were placed in polyethylene terephthalate (pet) trays (model cl1/135 carton pack, rutigliano, italy). they were sealed with a vacuum sealer (model boxer 50 lavezzini vacuum packaging system, fiorenzuola d’arda, italy) in 30 x 40 cm polyamide/polyethylene (pa/pe) bags (orved s.p.a., musile di piave, italy) applying two modified atmosphere (ma) mixtures with different initial co2 concentrations plus 1 kpa of o2: 1.0/0.03 o2/co2 kpa (1 kpa­o2), and 1.0/20.0 o2/co2 kpa (1 kpa­o2 + 20 kpa­co2). unsealed bags were used as control (air). all samples (4 replicates per treatment) were analyzed at harvest and after 20 days of storage at 5°c for respiration rate (rr), rachis browning (rb), ethanol, and acetaldehyde contents. for sweet cherries, about 200 g of fruits, without defects or diseases, were placed in pet trays and sealed in 30 x 40 cm pa/pe bags with three ma mix­ tures: 1.0/0.03 o2/co2 kpa (1 kpa­o2), 16.0/20.0 o2/co2 kpa (16 kpa­o2 + 20 kpa­co2), and 1.0/20.0 o2/co2 kpa (1 kpa­o2 + 20 kpa­co2). samples stored in unsealed bags (air) were used as controls. all sam­ ples (3 replicates per treatment) were analyzed at harvest and after 21 days of storage at 5°c for rr, relative water content of peduncles (rwc), and volatile organic compounds (vocs). as for artichokes, the heads were trimmed, elimi­ nating the external bracts and cutting the stem. the obtained artichoke hearts were then cut into quar­ ters and dipped for 5 min in a solution of 1% ascorbic acid + 0.2% citric acid (w:v), drained, and randomly selected for different treatments. in particular, three replicates of 16 artichoke quarters were kept for the initial determinations, while the remaining quarters were closed in 50 x 50 polypropylene (pp) bags (carton pack® rutigliano, italy), about 600 g per bag, applying 4 ma mixtures with different initial co2 con­ centrations plus 10 kpa of o2: 10.0/10.0 o2/co2 kpa (co2­10kpa), 10.0/20.0 o2/co2 kpa (co2­20kpa), 10.0/30.0 o2/co2 kpa (co2­30 kpa), 10.0/40.0 o2/co2 kpa (co2­40 kpa). unsealed bags were used as con­ trol (air). after 48 h of storage at 0°c, all bags were opened, artichoke quarters were placed in open poly­ ethylene bags and analyzed after 48 h at 0°c plus 7 days of storage at 5°c for rr, browning index (bi) and vocs profile. the headspace gas composition (o2 and co2) within each ma package was monitored daily using a gas analyzer (checkpoint, pbi dansensor, ringsted, denmark). rr was measured initially (fresh) and at the end of the storage for each prod­ uct using a closed system, as reported by kader (2002 a). samples were put into 6 l sealed plastic jars, allowing the accumulation of co2 up to 0.1 kpa. for co2 analysis, 1 ml of gas sample was collected from the headspace of each jar and injected into a gas chromatograph (p200 micro gc, agilent, santa clara, ca, usa) equipped with dual columns and a thermal conductivity detector. carbon dioxide was analysed cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 69 with a retention time of 16 s and a total run time of 120 s on a 10 m porous polymer (ppu) column (agilent, santa clara, ca, usa) at a constant tempera­ ture of 70°c. rr was expressed as ml co2 kg­1 h­1. in table grapes, rb was scored on a rating scale from 1 to 5 (1= absence, 2= light; 3= moderate; 4= severe; 5= extreme) as reported by lichter et al. (2011), whereas for acetaldehyde and ethanol analysis, the procedure reported by cefola et al. (2018) was used. in sweet cherries, the rwc of peduncles was cal­ culated in percentage, as reported by cefola et al. (2018), while the vocs analysis was carried out, as reported by cozzolino et al. (2019). in fresh­cut artichokes, bi and vocs were evalua­ ted, as reported by capotorto et al. (2020). 3. results and discussion starting from the gas composition inside ma pack­ ages described above for each product, during stor­ age the concentrations of o2 and co2 changed due to the respiration of the products and gas permeation through packaging material, and the final gases com­ position were reported in table 1. as for table grapes, o2 concentrations decreased from 1 kpa to about 0.2 kpa or 0.3 kpa, in 1k pa­o2 and in 1 kpa­o2 + 20 kpa­co2 respectively, while co2 concentrations increased from 0.03 kpa to roughly 10 kpa in 1 kpa­o2 packages, and from 20 kpa to about 30 kpa in 1k pa­o2 + 20 kpa­co2 ma. for sweet cherries, in 16 kpa­o2 + 20 kpa­co2 bags, the o2 concentration gradually decreased, reaching the mean value of about 1 kpa after 21 days of storage. in 1 kpa­o2 and 1 kpa­o2 + 20 kpa­co2 samples, the initial o2 concentration remained unchanged during the storage. on the other hand, the amount of co2 increased during conservation, reaching the final mean values of 25.7 kpa, 45.3 kpa and 42.4 kpa in 1 kpa­o2, 16 kpa­o2 + 20 kpa­co2 and 1 kpa­o2 + 20 kpa­co2 packages, respectively. finally, as for fresh­cut artichokes, no significant changes in gas composition inside bags were observed. results on table grapes are reported in table 2. in the fresh samples the rr measured was equal to 4.2 (± 0.4) ml co2 kg­1 h­1; after 20 days of the storage, a reduction in rr was measured in air samples (3.0 ± 0.2 ml co2 kg­1 h­1), while it remained almost con­ stant in table grapes samples treated with 1 kpa o2 (4.7 ± 0.6 ml co2 kg­1 h­1). on the contrary the use of high co2 concentrations (>20 kpa) in the ma mixture (1 kpa­o2 + 20 kpa­co2) increased the value of rr resulting more than a 2­fold higher than fresh sam­ ple. significant differences were observed, for sample stored in air and 1 kpa­o2 + 20 kpa­co2 (table 2). as shown in table 1, browning of the table grapes rachis was found in all treatments after 20 days of storage. however, higher browning was observed in air and in 1 kpa­o2 + 20 kpa­co2 samples, whereas the use of 1 kpa­o2 was able to keep a light to moderate brown­ ing of the rachis (mean value of 2.5). after storage ethanol and acetaldehyde concentrations did not change from their initial values (4.2 and 0.6 mg l­1, respectively) in the air samples, whereas they signifi­ cantly increased in table grapes exposed to low (<10 kpa) or high co2 (>20 kpa) concentrations (table 2). moreover, samples exposed to 1 kpa­o2 + 20 kpa­co2 showed higher accumulations of ethanol and acetaldehyde than table grapes packed in 1 kpa­o2. treatment initial kpa final kpa o2 co2 o2 co2 1 kpa­o2 1.0 0.04 0.2 10.0 1 kpa­o2 + 20 kpa­co2 1.0 20.0 0.3 30.0 air 20.0 0.03 20.0 0.03 table 1 ­ initial and final concentration of o2 and co2 for each treatment on table grape treatment respiration rate (ml co2 kg­1 h­1) rachis browning (1­5) ethanol (mg l­1) acetaldhyde fresh 4.2 b 1.0 c 4.2 c 0.6 c 1 kpa­o2 4.7 b 2.5 b 2142 b 8.9 b 1 kpa­o2 + 20kpa­co2 9.0 a 3.6 a 3606 a 17.6 a air 3.0 c 4.4 a 5.8 c 0.6 c table 2 ­ effect of co2 treatments on respiration rate, rachis browning, ethanol and acetaldehyde contents of table grapes (vitis vinifera cv. italia) after 20 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 20, respectively, according to lsd test (p≤0.05). adv. hort. sci., 2023 37(1): 67­73 70 sweet cherries (table 3) showed an initial respira­ tion rate of 8.2 (± 0.3) ml co2 kg­1 h­1 which increased 1.5 fold in air and more than 5 times in the other ma treatments. the highest rr was observed in 1 kpa­o2 + 20 kpa­co2 (48.9 ± 0.7 ml co2 kg­1 h­1) followed by 16 kpa­o2 + 20 kpa­co2 (44.4 ± 0.6 ml co2 kg­1 h­1) and 1 kpa­o2 (43.2 ± 0.1 ml co2 kg­1 h­1). the rwc % of peduncle increased in all ma treat­ ments, maybe due to the high relative humidity inside the packages. the highest rwc % values were observed in low o2 treatments, probably thanks to the lower respiration rate of these samples. among vocs analysed, 1­pentanol, marker of sen­ sory alteration, was closely associated with negative aroma intensity which resulted directly described as pungent, and fermented flavour. whereas the reduc­ tion of hexenal and 2­hexenal were indicators of lost in freshness (cozzolino et al., 2019). as reported in table 2, 1­pentanol was not detect­ ed in fresh and air samples, while a significant increase of this alcohol was observed in the other ma treatments. samples treated with 1 kpa­o2 + 20 kpa­co2 had the highest value of 1­pentanol, while 1 kpa­o2 and 16 kpa­o2 + 20kpa­co2 ma treatments had similar values (table 2). as for hexanal, (table 3) statistical analysis did not show significant changes after 21 days at 5°c excepted for the treatment, 1 kpa­o2 + 20 kpa­co2 which showed a reduction with respect to the fresh sample. in contrast, 2­hexenal decreased significantly during storage, but no differ­ ences in its concentration were observed when com­ paring ma treatments (table 3). results of fresh­cut artichokes are reported in table 4. rr of the fresh sample was 120.8 (± 0.2) ml co2 kg­1 h­1. after the short­term co2 treatments (48 h, 0°c) and 7 days of storage at 5°c, rr decreased significantly in all samples, except in artichokes treat­ ed with 40 kpa of co2. the lowest rr was detected in fresh­cut artichokes treated with co2­10 kpa (44.5 ± 4.3 ml co2 kg­1 h­1), followed by co2­20 kpa and co2­ 30 kpa, which reported similar values (68.1 ± 1.1 and 63.9 ± 5.2 ml co2 kg­1 h­1, respectively), and air (93.6 table 3 ­ effect of co2 treatments on respiration rate, relative water content (rwc) of peduncles, 1­pentanol, hexanal and 2­hexenal relative peak area (rpa) contents of sweet cherries (prunus avium cv. ferrovia) after 21 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 21, respectively, according to lsd test (p≤0.05). table 4 ­ effect of short­term co2 treatments on respiration rate, browning index, ethanol and hexanal relative peak area (rpa) con­ tents of fresh­cut artichokes (cynara cardunculus cv. violet de provence) after 7 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 7, respectively, according to lsd test (p≤0.05). treatment respiration rate (ml co2 kg­1 h­1) relative water content of peduncles (%) 1­pentanol relative peak area (%) hexanal 2­hexenal fresh 8.2 b 53.9 b 0.0 b 112.3 ns 366.6 a 1kpa­o2 43.2 ac 65.4 aa 6.0 ab 47.3 ns a 118.0 b ns 16kpa­o2 + 20kpa­co2 44.4 ab 58 ab 7.6 ab 26.4 ns ab 99.9 b ns 1kpa­o2 + 20kpa­co2 48.9 aa 60.5 ab 18.6 aa 14.3 bb 55.7 b ns air 12.1 ad 53.6 ns c 0.0 ns c 40.5 ns ab 125.2 b ns treatment respiration rate (ml co2 kg­1 h­1) browning index ethanol relative peak area (%) hexanal fresh 120.8 a 0.0 b 34.5 ns 41.7 ns co2­10 kpa 44.5 bd 131 ad 114.0 ns c 5.4 ns a co2­20 kpa 68.1 bc 137 abc 379.1 ab 5.0 ns a co2­30 kpa 63.9 bc 135 acd 482.5 ab 4.3 ns a co2­40 kpa 121.5 aa 140 ab 363.5 ab 2.1 bb air 93.6 bb 152 aa 886.3 aa 3.8 ns a cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 71 ± 1.7 ml co2 kg­1 h­1), while the highest rr was observed in co2­40 kpa (121.5 ± 0.2 ml co2 kg­1 h­1). as expected, all samples developed browning after 7 days of storage, regardless of treatment. however, differences in the severity of browning were observed when comparing treatments at the end of the storage: the application of short­term co2 treat­ ments (from 10 kpa to 40 kpa) significantly reduced the incidence of browning compared to air samples and, among the co2 treatments, co2­10 kpa had the lowest browning index (table 4). considering all the vocs identified by hs­spme/gc­ms analysis, ethanol and hexenal were, respectively, the most representative compounds of negative and positive aspects of fresh­cut artichokes (capotorto et al., 2020). as shown in table 4, ethanol significantly increased during storage, except for the co2­10kpa treatment, where its concentration was similar to that of fresh samples. the highest ethanol concentra­ tion was found in air samples, followed by treat­ ments added with co2 at 20, 30, and 40 kpa that showed similar values. as for hexanal, it was signifi­ cantly lower only in fresh­cut artichokes treated with co2­40 kpa (table 4). for table grapes, data related to ethanol and acetaldehyde, together with the rr results, indicate that high co2 concentrations (>20 kpa) on this com­ modity may cause physiological injury and the induc­ tion of anaerobic metabolism. the present results are supported by similar find­ ings on the effect of high co2 concentrations (>20 kpa) on table grapes by cefola and pace (2016). high co2 concentrations also negatively influence the acceptability of table grapes by consumers: rachis browning, is, in fact, the main issue that limits the acceptability of table grapes by consumers (cefola et al., 2018). a similar effect of high co2 concentrations (>20 kpa) on the acceleration of rachis browning was previously observed on table grapes (crisosto et al., 2002; deng et al., 2006) and is a consequence of the stress induced by exposure to high co2 concentra­ tions (crisosto et al., 2002; liguori et al., 2015). for sweet cherries, considering that the highest rr was observed when ma with 20kpa co2 was applied, these results indicate that this co2 concen­ tration, especially when associated with low oxygen, can cause stress, as confirmed by voc analysis. similar behaviour in the production of c5 volatiles, such as 1­pentanol, was previously observed (contreras et al., 2017; mastrandrea et al., 2017), and it seems to be favoured under low o2 and high co2 atmospheres. the present results on the cv. ferrovia are in contrast with previous results on other sweet cherries cultivars (kader et al., 1989; esturk et al., 2012), but those cultivars have better tolerance to high co2. it has been stated that the physiological suscepti­ bility of commodities to high co2 can be cultivar­ dependent, and is generally seen with vegetables and other fruit (watkins, 2000). results on fresh­cut artichokes indicate that the application of high co2 concentrations (around 40 kpa) has a negative effect on the shelf­life. similar results on the detrimental effect of high co2 were previously observed on fresh­cut arti­ chokes during storage (la zazzera et al., 2012, 2015). as observed for table grapes, sweet cherries, and fresh­cut artichokes, the exposure to elevated co2 atmospheres can stimulate respiration and ethylene production rates, indicating a stress response (kader, 2002 a). these increases in respiration might be related to the inhibition by high co2 of several enzymes of the krebs cycle, including succinate dehydrogenase, which triggers anaerobic respiration or causes the accumulation of succinic acid, which is potentially toxic to cells (kays, 1991; varoquaux, 1991; kader, 2002 b). furthermore, for fresh­cut arti­ chokes, the intolerance to high co2 concentration and mechanical wounding enhances a different array of enzymatic pathways, many of which are associat­ ed with volatile accumulation, which lead to devel­ opment of off­flavors (salunkhe et al., 1976; la zazzera et al., 2015). 4. conclusions for table grapes, the storage in high co2 (>20 kpa) caused a severe increase in respiration rate, ethanol and acetaldehyde accumulation, and a decline in sen­ sory quality due to the rachis browning, all probably consequences of the induction of the anaerobic metabolism. the application of co2 up to 10kpa was able, instead, to keep the good quality table grapes during storage. sweet cherry (cv. ferrovia) is very sensitive to high co2 when it is applied together with low oxygen in ma, as indicated by responses in respiration rate, rel­ ative water content of the peduncles, and voc emis­ sions, with some of these responses being considered positive and some negative in relation to quality. 72 adv. hort. sci., 2023 37(1): 67­73 short­term treatment with high co2 (around 40 kpa) caused an increase in respiration rate and the induction of fermentative metabolism in fresh­cut artichoke. the application of co2 concentrations up to 10 kpa reduced respiration rate and tissue brown­ ing during storage in air at 5°c and preserved the fresh voc profile. application of short­term co2 might be a promising postharvest treatment to pre­ serve the quality and the volatile profile of fresh­cut artichokes during storage. acknowledgements the research leading to these results has received funding from puglia regional call “aiuti a sostegno dei cluster tecnologici regionali per l’innovazione”. continnova project: container innovativo isotermico intermodale equipaggiato con atmosfera controllata per il trasporto di prodotti ortofrutticoli freschi” (cod. vfqa3d0). references capotorto i., innamorato v., cefola m., cervellieri s., lippolis v., longobardi f., logrieco a.f., pace b., 2020 ­ high co2 short‐term treatment to preserve quality and volatiles profile of fresh‐cut artichokes dur‐ ing cold storage. ­ postharvest biol. technol., 160: 111056. cefola m., damascelli a., lippolis v., cervellieri s., linsalata v., logrieco a.f., pace b., 2018 ­ relationships among volatile metabolites, quality and sensory parameters of ‘italia’ table grapes assessed during cold storage in low or high co2 modified atmo‐ spheres. ­ postharvest biol. technol., 142: 124­134. cefola m., pace b., 2016 ­ high co2‐modified atmosphere to preserve sensory and nutritional quality of organic table grape cv. ‘italia’ during storage and shelf‐life. ­ eur. j. hortic. sci., 81: 197­203. contreras c., schwab w., mayershofer m., gonzález­agüero m., defilippi b.g., 2017 ­ volatile compound and gene expression analyses reveal tempo‐ ral and spatial production of lox‐derived volatiles in pepino (solanum muricatum aiton) fruit and lox speci‐ ficity. ­ j. agric. food chem., 65: 6049­6057. cozzolino r., martignetti a., cefola m., pace b., capotorto i., de giulio b., montemurro n., pelli­ cano m.p., 2019 ­ volatile metabolites, quality and sensory parameters of “ferrovia” sweet cherry cold stored in air or packed in high co2 modified atmo‐ spheres. ­ food chem., 286: 659­668. crisosto c.h., garner d., crisosto g., 2002 ­ carbon dioxide‐enriched atmospheres during cold storage limit losses from botrytis but accelerate rachis browning of ‘red globe’ table grapes. ­ postharvest biol. technol., 26: 181­189. deng y., wu y., li y., 2006 ­ physiological responses and quality attributes of ‘kyoho’ grapes to controlled atmo‐ sphere storage. ­ lwt ­ food sci. technol., 39: 584­590. esturk o., ayhan z., ustunel m.a., 2012 ­ modified atmosphere packaging of “napoleon” cherry: effect of packaging material and storage time on physical, chemical, and sensory quality. ­ food bioprocess technol., 5(4): 1295­1304. francis g.a., gallone a., nychas g.j., sofos j.n., colelli g., amodio m.l., spano g., 2012 ­ factors affecting quality and safety of fresh‐cut produce. ­ critical rev. food sci. nutrition, 52(7): 595­610. kader a.a., 2002 a ­ methods of gas mixing, sampling and analysis, pp. 145­148. in: kader a.a. (ed.) postharvest technology of horticultural crops. univ. calif. danr, publication 3311, oakland, ca, usa. kader a.a., 2002 b ­ postharvest biology and technology: an overview, pp. 9­47. ­ in: kader a.a. (ed.), postharvest technology of horticultural crops. univ. calif. danr, publication, 3311, oakland, ca, usa. kader a.a., 2003 ­ a perspective on postharvest horticul‐ ture (1978‐2003). ­ hortscience, 38(5): 1004­1008. kader a.a., zagory d., kerbel e.l., 1989 ­ modified atmosphere packaging of fruits and vegetables. ­ crit. rev. food sci. nutr., 28(1): 1­30. kays s.j., 1991 ­ postharvest physiology and handling of perishable plant products. ­ van nostrand reinhold inc., new york, ny, usa, pp. 526. la zazzera m., amodio m.l., colelli g., 2015 ­ designing a modified atmosphere packaging (map) for fresh‐cut artichokes. ­ adv. hort. sci., 29(1): 24­29. la zazzera m., rinaldi r., amodio m.l., colelli g., 2012 ­ influence of high co2 atmosphere composition on fresh‐cut artichoke quality attributes. ­ acta horticulturae, 934: 633­640. lichter a., kaplunov t., zutahy y., daus a., alcha­ natis v., ostrovsky v., lurie s. 2011 ­ physical and visual properties of grape rachis as affected by water vapor pressure deficit. ­ postharvest biol. technol., 59(1): 25­33. liguori g., sortino g., de pasquale c., inglese p., 2015 ­ effects of modified atmosphere packaging on quality parameters of minimally processed table grapes during cold storage. ­ adv. hortic. sci., 29(2­3): 152­ 154. mangaraj s., goswami t.k., 2009 ­ modified atmo‐ sphere packaging of fruits and vegetables for extending shelf‐life‐a review. ­ fresh produce, 3(1): 1­31. mastrandrea l., amodio m.l., pati s., colelli g., 2017 ­ effect of modified atmosphere packaging and temper‐ ature abuse on flavor related volatile compounds of rocket leaves (diplotaxis tenuifolia l.). ­ j. food sci. cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 73 technol., 54(8): 2433­2442. salunkhe d.k., do j.y., maga j.a. 1976 ­ biogenesis of aroma constituents of fruits and vegetables. ­ crit. rev. food sci. nutr., 8: 161­190. toivonen p.m.a., deell j.r., 2002 ­ physiology of fresh‐ cut fruits and vegetables, pp. 99­131. ­ in: lamikanra o. (ed.) fresh‐cut fruits and vegetables. crc press, boca raton, ca, usa, pp. 480. varoquaux p., 1991 ­ ready‐to‐use fresh fruits and veg‐ etables. ­ revue générale froid, 81: 33­43. watkins c.b., 2000 ­ responses of horticultural commodi‐ ties to high carbon dioxide as related to modified atmosphere packaging. ­ horttechnology, 10(3): 501­ 506. impaginato 3 adv. hort. sci., 2022 36(1): 3­11 doi: 10.36253/ahsc­11568 reduction of leaf tip burns of ornithogalum dubium by controlling the temperature during bulb storage and greenhouse forcing to produce quality plants x. wu 1, l. wang 2, m.s. roh 3 (*) 1 school of agriculture, yunnan university, chenggong district, kunming, 650091 yunnan, china. 2 flower research institute, yunnan agriculture academy of science, kunming, panlong district, 650025 yunnan, china. 3 the institute of natural resource development, mokpo national university, muan, 58554 jeonnam, korea. key words: boron toxicity, controlled flowering, leaf morphology, optimum temperature, quality criteria, scape growth. abstract: production of quality potted ornithogalum dubium houtt. plants were investigated under multiple conditions: pre­planting treatment at 10, 16, and 22°c for 40 days from sept. 21 (stage a; st­a) during bulb storage and then bulbs were potted. after potting, post­planting treatment at 15/12, 18/15, and 21/18°c (day/night) during stage b for 35 days from nov. 2 (stage b; st­b), and at 15/12 and 21/18°c during stage c for 30 days from dec. 7 (stage c; st­c) dur­ ing greenhouse forcing was applied. leaf tissue analyses for macro­ and micro­ nutrients were performed to investigate the cause of leaf tip burn symptom (ltb). three criteria for quality of the plants at flowering were established: (1) ltb occurs on less than 1.5 leaves per plant. (2) the number of days to flower is less than 115 days, the length of the third leaf counted from the crown (the junction of the shoot and roots) is shorter than 11.5 cm, and the width is nar­ rower than 2.5 cm; the scape length is shorter than 15 cm, and there are more than 45 flowers. (3) the leaf spread and morphology (leaf spread) and the pat­ tern of the scape curvature (scape growth) have a score of less than 1.5. the following conditions are optimal to produce quality plants based on these three criteria: (1) pre­planting bulbs treatment was applied at 10 or 16°c during st­a, and forcing was performed at 15/12°c during st­b and 21/18°c during st­c. these conditions accelerated flowering, produced straight scape growth and upward (erect) growing leaves, and yielded acceptable leaf length and width. (2) the incidence of ltb was minimal at 10°c or possibly 16°c during st­a, and at 15/12°c in st­b and at 21/18°c in st­c during greenhouse forcing. leaf tip burn symptom was observed in both young and old leaves and was caused by a high boron (b) concentration (218­230 ppm) and possibly a high zinc (zn) con­ centration (155­159 ppm) in o. dubium. a low calcium (ca) concentration was not the cause of ltb. although ltb cannot be avoided, it can be minimized by temperature manipulation during pre­ and post­planting phase to produce high quality potted plants. (*) corresponding author: marksroh@gmail.com citation: wu x., wang l., roh m.s., 2022 ­ reduction of leaf tip burns of ornithogalum dubium by control‐ ling the temperature during bulb storage and greenhouse forcing to produce quality plant. ­ adv. hort. sci., 36(1): 3­11. copyright: © 2022 wu x., wang l., roh m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. authors contributions: both authors, x. wu and l. wang, contributed equally in preparing the manuscript. received for publication 21 august 2021 accepted for publication 4 november 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-11568 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 3­11 4 1. introduction ornithogalum dubium houtt. is a geophyte native to south africa with yellow or orange flowers with a dark greenish­brown center (du plessis et al., 1989; littlejohn and blomerus, 1997). temperature treat­ ments during the pre­planting phase before potting the bulbs and the greenhouse forcing phase after planting the bulbs should be optimized to produce quality o. dubium potted plants. “leaf tip scorch” was believed to be caused either by genetics or by high salt levels in the growing medium after treat­ ment with 5.5 grams of a slow­release fertilizer. however, no information on the salt levels in the pot­ ting medium or leaf tissue analyses was presented (de hertogh and gallitano, 1997). the boron (b) concentration of healthy and necrosed leaves of the oriental hybrid lily ‘star gazer’ did not significantly differ, although those of ca, magnesium (mg), manganese (mn), and copper (cu) were higher in necrosed than in healthy leaves (chang, 2002). in the older leaves of curcuma ‘chiangmai university pride’ (‘cmu pride’), a high b concentration (122 ppm) at the margin of leaves may have caused the marginal leaf burn (roh et al., 2006). it is unclear whether ltb in o. dubium is similar to marginal leaf burn observed in the older leaves of curcuma ‘cmu pride’. leaf scorch symptoms were also reported in lilium (berghoef, 1986; chang et al., 2008). in lilium ×elegans, ‘red carpet’ and ‘sterling star,’ the scorch symptoms were caused by a low calcium (ca) con­ centration resulting from an inefficient translocation of radioactive calcium (45ca) to the tip of the leaves (roh, unpublished data). the critical ca concentra­ tion associated with upper leaf necrosis of lilium ‘star gazer’ was 0.3 to 0.4%, and upper leaf necrosis was caused by low ca concentration (chang, 2002) although “it is difficult to isolate a single characteris­ tic to explain the observed cultivar variation to upper leaf necrosis in oriental hybrid lilies” (chang et al., 2008). although forcing techniques in geophytes, such as tulip and lily, have been well documented (de hertogh, 1974), forcing techniques for o. thyrsoides and o. dubium to produce quality potted plants are still lacking, especially regarding the incidence of ltb (jansen van vuuren and holtzhausen, 1992; littlejohn and blomerus, 2000; suh et al., 2000; luria et al., 2002; reinten et al., 2011; lee and miller, 2015). flowering of o. dubium was accelerated when the plants were forced at 27/22°c (luria et al., 2002) or 19/13°c (suh et al., 2000) compared with 17/12°c or 13/10°c, respectively. flowering was accelerated in several new cultivars forcing at 22/18°c during a short day between the visible bud stage to flowering (lee and miller, 2015). the highest (visual) quality o. dubium cultivars were produced when forcing occurred at constant 17 to 19°c (lee and miller, 2015), which is comparable to 22/18°c for forcing seed­raised bulbs (de hertogh and gallitano, 1997). however, the criteria used to assess the visual quality were not specified (lee and miller, 2015). the objectives of this research were to produce quality potted o. dubium plants by reducing the inci­ dence of ltb, accelerating flowering while ensuring the maximum number of flower buds, and desirable morphologies as influenced by temperature treat­ ment during the bulb handling and greenhouse forc­ ing stages to establish the optimum temperature regimes that reduce the incidence of ltb, and accel­ erate flowering with desirable morphologies. the number of leaves showing ltb, the time of flowering, and the leaf and floral morphology were used to develop criteria for quality o. dubium potted plants. the macro­ and micro­elements of the leaf tips show­ ing ltb/no ltb during growth and development were analyzed to determine the cause of ltb. 2. materials and methods general cultural practices ornithogalum dubium bulbs (5­6 cm in circumfer­ ence) purchased from agrexco (jamaica plain, ny, usa) were used in the experiment conducted between 1999 and 2002. the bulbs were stored dry or potted, with one bulb per 6.3 or 10 cm pot, filled with promix (pro­mix bx mycorrhizae, quakertown, pa, usa). temperature treatments were performed in growth chambers as specified in each experiment with a 12 h day (29 w·m­2)/night (day: 08:00­18:00 hr; night: 18:00­08:00 hr). at planting, 0.8 g of a slow­ release fertilizer (14 n ­ 6.2 p ­ 11.6 k, osmocote, scotts co., marysville, oh, usa) was applied to the surface of the growing medium. in addition, the plants received 200 ppm n from 15 n ­ 16 p ­ 18 k water­soluble fertilizer (jr peter’s laboratory, allentown, pa, usa) once a month during culture in the greenhouse. https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml https://journals.ashs.org/hortsci/view/journals/hortsci/50/12/article-p1784.xml wu et al. ‐ quality ornithogalum plants with reduced leaf tip burns 5 the effect of the pre‐plant bulb (st‐a) and post‐plant forcing (st‐b and st‐c) temperatures on growth, flowering, and plant quality orntihogalum dubium bulbs (7­8 cm in circumfer­ ence) from the previous experiment were harvested and stored dry at 20°c until potting on june 1 and then potted per 10 cm pot filled with promix and grown at 18/15°c. leaf samples were collected for tissue analysis on july 12, 2000 from leaves without any ltb. the tips and basal portions of the leaves were separated, and the width at mid­leaf was 2 cm. samples were collected from the third and fourth leaves of each plant, counted from the tip of the bulbs, and were combined into a sample. the third leaf was collected from 15 plants, and the leaves were divided into three segments. the two­cm long distal end (tip) did not show any incidence of ltb, the proximal end (base) of each leaf, and mid portion between the tip and base was used for macro­ and micro­element tissue analysis. in another experiment for macro­ and micro­ele­ ment analysis, bulbs were harvested after forcing in 2000, and were potted in 2001. on jan. 13, 2002, flowers began to develop and the flowers had fully developed color and were ready for anthesis. leaves from the distal end (tip) with and without ltb (fig. 1) and the proximal end (base) were collected from 15 plants during forcing on jan. 14 and 26, feb. 3 and 10, and mar. 4. the leaf samples were dried at 70°c for 4 days and were ground into fine particles using a mortar and pestle. two replicates of the dried leaves were sent to the jr peters laboratory (allentown, pa, usa). bulbs purchased on sept. 15, 1999 were used in this experiment. before potting, o. dubium bulbs were stored at constant temperatures of 10, 16, and 22oc for 40 days from sept. 21 (st­a). bulbs were planted 2 cm deep of the nose from the surface of the growing medium, and leaf emergence through the growing medium was recorded from potting when temperature treatment of st­a started. bulbs were potted (one bulb per 6.3 cm pot) filled with the promix medium, and the pots were placed in a greenhouse maintained at 15/12, 18/15, and 21/18oc for 35 days from nov. 2 (st­b). on nov. 2, a slow­ release fertilizer was applied to the surface of the growing medium, and plants received 200 ppm n water­soluble fertilizer once a month. the leaves emerged (about 0.5 cm or less above the nose of bulbs) before potting on sept. 21. on dec. 7, plants were divided into two groups and grown in a green­ house maintained at 15/12 and 21/18°c for 30 days (st­c) (table 1). there were 16 plants per treatment. starting from jan. 6, all plants were grown in a green­ house maintained at 19/16°c until flowering, and data were collected at anthesis. criteria establishment for quality evaluation when 3­4 flowers reached anthesis, the date of flowering, the number of total leaves showing ltb (fig. 1) excluding the incipient ltb (fig. 2) and the scape length, number of flowers, leaf spread, scape growth (fig. 2, 3) were recorded. the leaf spread was based on the angle of the leaf measured from the base to the tip using the following scores: 1: greater than 60° (pointing upward, erect); 2: between 30 and 60°; 3: old leaves prostrate and drooping downward at less than 15°. in addition, the scape growth was used as a quality criterion using the following scores: 1: no bending of the scape; 2: leaning sideways with­ out bending at the base of scape; 3: scape at the base starting to grow at about 40o and continued sideways growth. data analysis data were analyzed using sas® system version 9 for microsoft® windows® (sas institute inc., 2002). the number of total leaves, number of leaves with ltb, and the scores of the leaf spread and scape ori­ entation were analyzed following a square root trans­ formation (x + 0.5)1/2. means were compared using duncan’s multiple range test (dmrt) at the signifi­ cant level indicated in the tables. fig. 1 ­ various degrees of leaf tip burn (ltb) symptoms in ornithogalum dubium leaves. leaf without evident ltb symptom (a) and different degree of ltb symptoms (b, c, and d). adv. hort. sci., 2022 36(1): 3­11 6 3. results leaf tip burn symptoms and analysis of macro‐ and micro‐elements in different leaf parts the number of total leaves ranging from 4.8 to 5.6 was not affected significantly by the temperature treatments (data not presented). when bulbs were stored at 10°c/st­a, followed by forcing at 15/12°c/st­b and 21/18°c/st­c, the number of leaves showing ltb was 1.1 out of 5.1 leaves. when the bulbs were stored and forced at 22°c/st­a, 18/15°c/st­b, and 15/12°c/st­c, 4.3 out of 5.6 leaves showed ltb (table 1). fig. 2 ­ patterns of scape growth of o. dubium. a) straight growth and no bending of the scape (score 1). b) leaning to sideway without bending at the base of scape (score 2). c) scape at the base started to grow at about 30­ degree angle, and continue to grow leaning to sideway (score 3). incipient leaf tip burn (ltb) symptoms are indi­ cated with arrow sign. plant a is considered the most ideal and high­quality plant. fig. 3 ­ leaf spread of ornithogalum dubium. a) all leaves growing upward (erect) (score 1), b) old leaves showing prostrate growth (spreading), while young leaves growing upward (score 2), and c) old leaves drooping and curved inward toward the pot (prostrate) (score 3). evident leaf tip burn (ltb) symptoms are indicated with arrow sign. table 1 ­ the number of total leaves and of leaves showing leaf tip burn by leaf position, and the total number of leaves showing leaf tip burn symptoms in ornithogalum dubium z leaf position was counted from the crown and upward, the first leaf (leaf number 1) is the most matured leaf. y bulbs received 10°c from sept. 21 (st­a), 15/12°c from nov. 2 (st­b) and 21/18°c from dec. 7 (st­c). x bulbs received 22°c from sept. 21 (st­a), 18/15°c from nov. 2(st­b) and 15/12°c from dec. 7(st­c). w percentage of leaves showing ltb symptoms. individual plant (number) 10°c st­a, 15/12°c st­b, 21/18°c st­c y 22°c st­a, 18/15°c st­b, 15/12°c st­c x no. of total leaves per plant tip burn symptom no. of total leaves per plant tip burn symptom leaf position z total number of leaves leaf position number total number of leaves 1 5 2.5 2 5 1,3,4,5 4 2 4 1,2,3,4 4 6 2,3,4,5,6 5 3 4 ­ 0 6 3,4,5,6 4 4 5 5 1 6 5.6 2 5 4 ­ 0 6 1,3,4,5,6 5 6 5 3 1 5 3,4,5 3 7 6 4.5 0 5 1,2,3,4,5 5 8 6 4 1 6 3,4,5,6 4 9 5 ­ 0 6 2,3,4,5,6 5 10 6 ­ 0 5 1,2,3,4,5 5 11 5 4,5,6 3 5 2,3,4,5 4 12 5 ­ 0 8 1,2,3,4,5,6,7,8 8 13 6 ­ 0 4 1,3,4 3 14 5 ­ 0 6 3,4,5,6 4 15 5 1,2,3,4,5 5 5 3,4,5,6 4 16 6 5 1 6 2,3,4,5 4 average 5.13 1.13 (22%) w 5.62 4.31 (76.7%) wu et al. ‐ quality ornithogalum plants with reduced leaf tip burns 7 the macro­ and micro­element analysis of the leaves without apparent ltb revealed that the b con­ centration at the tip (distal end) of the leaves was 119 ppm, which is within the suggested acceptable range (30­150 ppm) for general ornamentals (table 2). the b concentrations in the middle and base (proximal end) of the leaves were 34 and 21 ppm, respectively, which was close to the lower end of the range. when the samples were collected in the early growth stages when no or incipient symptoms were observed on jan. 14, jan. 26, and mar. 4, the b con­ centrations were 109, 96, and 108 ppm, respectively, which fell within the suggested acceptable range (table 3). macro­ and micro­element analysis of the leaves without apparent ltb revealed that the b con­ centration in the tip of the leaves when collected on jan. 14 was lower than 109 ppm (table 3). however, b concentrations of leaves showing incipient symp­ toms and severe ltp when the leaves were collected on feb. 3 and feb. 8 were 218 and 230 ppm, respec­ tively, and these concentrations exceed the suggest­ ed acceptable range. the ca concentration was high in the leaf tips (0.76%), and the concentrations of the other macro­ and micro­elements fell within the acceptable ranges for each element, even when ltb was observed. the exception was zinc (zn), whose concentration was slightly above the upper level (155 and 159 ppm) of the suggested range (150 ppm) (table 3). the nitro­ gen (n) concentration was above the lower level of the suggested range in all samples, regardless of leaf position, ltb symptoms, and leaf sampling time. the concentrations of ca and zn were lowest and highest, respectively, in the tip (distal end) and lowest in the middle and bottom (proximal end) of the leaves when ltb was not observed. at the tip of the leaves show­ table 2 ­ macro­ and micro­elements of ornithogalum dubium from the distal­, mid­ and basal portion of third leaves from the base of the scape that do not show leaf burn symptom z comparisons of means by duncan’s multiple range test, p<0.01, f­test. y ***, ns= significant at p<0.001 and not significant. x jr peter’s laboratory (allentown, pa. usa). leaf position macro­ and micro­ elements n (%) p (%) k (%) ca (%) mg (%) b (ppm) fe (ppm) mn (ppm) cu (ppm) zn (ppm) mo (ppm) tip (distal end) 3.9 c z 0.97 a 3.1 a 0.76 c 0.29 c 119 c 87.2 a 40 a 18 a 56 c 3.1 a mid 3.5 b 1.11 a 3.8 a 0.69 b 0.27 b 34 b 76.2 a 34 a 17.1 a 46 b 2.3 a base (proximal end) 2.8 a 0.88 a 4.3 a 0.55 a 0.19 a 21 a 60.4 a 29 a 15.3 a 37 a 1.4 a level of significance y *** ns ns *** *** *** ns ns ns *** ns suggested acceptable range x 3.5­5.5 0.35­1.0 2.0­8.8 0.8­3.0 0.2­1.5 30­150 60­200 50­200 mag­25 30­150 0.5­5 table 3 ­ macro­ and micro­elements of leaf analysis of ornithogalum dubium from the tip and base of third leaves from the crown showing different degree of leaf burn symptom z comparisons of means by duncan’s multiple range test, p<0.01, f­test. y ***, ** = significant at p< 0.001, and p<0.01, f­test. x jr peter’s laboratory (allentown, pa. usa). leaf position leaf tip burn sampling date macro­ and micro­ elements n (%) p (%) k (%) ca (%) mg (%) b (ppm) fe (ppm) mn (ppm) cu (ppm) zn (ppm) mo (ppm) base no jan. 14 5.30 c z 1.31 a 6.3 b 1.55 b 0.33 bc 25 a 67.5 a 40 e 10.9 d 62 a 1.4 d tip no jan. 14 4.78 b 1.59 c 7.3 c 1.36 a 0.2 7 b 109 c 103.5 e 31b 13.1 e 114 bc 1.3 c base no jan. 26 5.21 c 1.32 a 5.5 a 1.96 de 0.37 c 22 a 59.1 a 35 d 6.9 a 51 a 1.2 b tip incipient jan. 26 4.62 b 1.60 d 6.1 b 1.76 c 0.30 b 96 b 84.3 d 30 b 8.8 b 106 b 1.1 a tip evident feb. 3 3.92 a 1.44 b 6.0 a 1.94 de 0.30 b 218 d 91.6 bc 27 c 8.3 b 155 e 1.2 ab tip severe feb. 10 3.83 a 1.49 c 5.7 a 2.08 e 0.37 c 230 d 91. bc 25 bc 10.2 d 159 e 1.6 de tip no mar. 4 (flowering) 3.90 a 1.31 a 5.4 a 1.50 b 0.19 a 108 c 93d 22 a 8.2 b 118 d 1.71 e level of significance y *** *** *** *** *** *** *** *** *** *** ** suggested and acceptable range x 3.5­5.5 0.350­1.0 2.0­8.8 0.8­3.0 0.2­1.5 30­150 60­200 50­200 mag­25 30­150 0.5­5 8 adv. hort. sci., 2022 36(1): 3­11 ing evident and severe lpb ca concentration was high­ er than in leaves showing incipient ltb symptom. the mn concentration ranged from 25 to 40 ppm, which was lower than the lower level of the range (50 ppm). the concentrations of phosphorus (p), potassium (k), iron (fe), mn, cu, and molybdenum (mo) did not differ between the tips, middle, and bottom of the leaves. the effect of the pre‐planting and post‐planting forc‐ ing temperatures on growth, flowering, and ltb expression leaf emergence was affected significantly by tem­ perature treatments during st­a and st­b and the interaction of st­a and st­b (table 4). leaves emerged late when bulbs were stored at 10°c before potting during st­a (10°c/st­a) compared to when bulbs were stored at 16 and 22°c/st­a, and the forc­ ing temperatures were 15/12oc or 18/15oc/st­b and 15/12°c or 18/15°c/st­b (table 4). the number of days to flower was affected significantly by tempera­ ture treatment during st­a, st­b, and st­c and the interaction of st­a and st­b. the earliest flowering (87 days) occurred when bulbs were treated at 10°c/st­a and 21/18°c/st­b and st­c. bulbs stored at 22°c/st­a took longer than 112 days to flower, regardless of the temperature during st­b and st­c. the average days to flowering was 116 days, whereas the days to flow­ ering was <100­105 days at 21/18°c/st­c. the number of flowers (<36 flowers) was only affected by bulb storage temperature during st­a table 4 ­ the effect of temperature treatment during three different growth and development periods on the growth and flowering of ornithogalum dubium z comparisons of means by duncan’s multiple range test, p<0.01, f­test. y ***, **, ns = significant at p< 0.001, p<0.01, and not significant. x jr peter’s laboratory (allentown, pa. usa). treatment stage (st­)/ temperature (°c) z number of days v to no. of scape no. of leaves score of angle u leaf a y b x c w leaf emergence flowering flowers length (cm) showing leaf tip burns scape leaf length width ½ length 10 15­dic 15­dic 47.9 a 113 f 33 c 20.5 ij 2.00 b 1.0 a 1.0 a 8.4 a 2.2 ab 10 15­dic 21/18 47.9 a 98 c 33 d 20.7 hij 1.13 a 1.5 b 1.2 ab 8.5 a 2.1 a 10 18/15 15­dic 47.6 a 105 ef 36 d 22.7 j 3.31 fgh 1.1 ab 2.5 ab 9.6 abc 2.3 ab 10 18/15 21/18 47.5 ab 90 b 27 a 20.4 hij 2.50 bcde 1.1 ab 2.8 c 10.4 cdef 2.2 ab 10 21/18 15­dic 46.4 cd 100 c 25 a 22.6 ij 2.44 bcd 1.4 b 2.9 ab 9.5 abc 2.2 ab 10 21/18 21/18 46.4 cd 87 a 29 ab 19.4 h 0.50 a 1.5 b 2.5 b 9.9 bcd 2.2 ab 16 15­dic 15­dic 46.0 d 113 f 55 ef 15.3 defg 2.19 bc 2.3 c 1.8 c 9.1 ab 2.5 abc 16 15­dic 21/18 46.2 d 105 ef 44 d 15.8 fg 2.44 bcd 2.3 c 1.9 c 10.1 bcde 2.4 abc 16 18/15 15­dic 46.1 d 123 g 52 ef 15.4 defg 3.13 efg 2.9 e 2.7 de 11.5 fgh 2.4 abc 16 18/15 21/18 46.0 d 107 ef 51 e 20.3 hi 4.00 j 3.0 e 2.7 de 11.4 fg 2.5 abc 16 21/18 15­dic 46.0 d 116 fg 52 ef 12.4 ab 3.31 fgh 2.5 cd 2.8 e 11.3 efg 2.6 bcd 16 21/18 21/18 46.1 d 102 cd 48 e 13.6 bcdefg 2.94 defg 2.3 c 2.5 d 11.3 efg 2.4 abc 22 15­dic 15­dic 47.1 abc 129 h 52 ef 13.3 bcde 2.75 cdef 2.4 cd 2.8 e 12.1 h 2.8 cd 22 15­dic 21/18 47.0 bc 116 fg 58 fg 13.5 bcdef 3.50 ghi 2.6 de 2.9 e 12.2 hu 3.1 d 22 18/15 15­dic 46.8 bcd 125 g 59 fg 14.7 bcdefg 4.31 j 2.9 e 2.7 de 13.9 hi 2.8 cd 22 18/15 21/18 46.8 bcd 114 f 63 gh 12.8 bc 3.50 ghi 2.4 cd 2.8 e 12.8 hi 2.9 cd 22 21/18 15­dic 46.4 cd 123 g 61 gh 10.4 a 3.50 ghi 2.8 de 2.9 e 12.0 gh 2.6 bcd 22 21/18 21/18 46.4 cd 112 f 57 fg 13.2 bcd 3.88 hij 2.4 cd 2.7 de 13.6 i 2.6 bcd level of significance t st­a *** *** *** *** *** *** *** *** *** st­b *** *** ns ** *** ns *** *** ** st­c ns *** ns ns ns ns ** ns ns st­a × st­b *** *** ns ns *** ns *** ns *** st­a × st­c ns ns ns ns ** ns *** ns ns st­b × st­c ns ns ns ns ns ns ns ns ns st­a × st­b × st­c ns ns ns ns ns ns ns ns ns wu et al. ‐ quality ornithogalum plants with reduced leaf tip burns 9 when treated at 10°c/st­a, 18/15°c/st­b, and 15/12°c/st­c (table 4). more than 44 flowers were produced when bulbs were treated at 16 and 22°c/st­a, regardless of the temperature during st­b and st­c. the scape length was >20.4 cm when bulbs were stored at 10°c/st­a, regardless of the tempera­ ture during st­b and st­c. when the temperature during st­a was increased from 10 to 22°c, a signifi­ cantly higher number of flowers was produced on short scapes when bulbs were stored at 22°c. for plants that required an average of 115 days to flower, the scape length was 12.4 cm, and 52 flowers were produced in the treatment of 16°c/st­a, 21/18°c/st­b, and 15/12°c/st­c (table 4). however, a significantly higher number of leaves (3.31) show­ ing ltb was produced compared to 0.50 leaves in treatment (10°c/st­a, 21/18°c/st­a, and 21/ 28°c/st­c) and 1.13 leaves in treatment (10°c/st­a, 15/12°c/st­a, and 21/28°c/st­c). as the bulb stor­ age temperature was increased to 16 or 22°c, the number of leaves showing ltb increased to 2.19 leaves (16°c/st­a, 15/12°c/st­a, and 15/12°c/st­c) to 4.31 leaves (22°c/st­a, 18/15°c/st­a, and 15/12°c/st­c). the scape growth was primarily influenced by the temperature during st­a, resulting in scores of <1.5 when bulbs were stored at 10°c/st­a, regardless of the temperature during st­b and st­c (table 4). the scape growth when bulbs were stored at 16 and 22°c/st­a had scores of >2.3, regardless of the tem­ perature during st­b and st­c. the leaf spread showed a complex response; st­a, st­b, and st­c had a significant effect, as did the interaction of st­a and st­b and st­b × st­c. however, the leaf spread had scores <1.9 when the plants were forced at 15/12°c/st­c, followed by storing the bulbs at 10 or 16oc/st­a. in contrast, the scores were >1.9 regard­ less of the temperature treatments during st­a, st­b, and st­c. the length of the leaves was<10.4 cm when bulbs were stored at 10°c/st­a, regardless of the forcing temperature during st­b and st­c, and was >12 cm when bulbs were stored at 22 oc/st­a. bulbs stored at 16°c/st­a had leaf lengths between those of the 10°c and 22oc/st­a treatments. the leaf width was <2.3 cm when bulbs were stored at 10°c/st­a. 4. discussion and conclusions quality criteria of the finished potted plants criteria for the quality of finished potted o. dubi‐ um plants have not been established even in 2019. the quality can be evaluated in reducing the inci­ dence leaf tip burn (ltb) at the tip of the leaves (fig. 1) and by the rate of flowering without sacrificing the number of flower buds. areas of ltb are indicated by the arrows in o. dubium seedlings in figures 2 and 3. however, the criteria used to assess the visual quality were not specified (lee and miller, 2015). therefore, the following three quantitative crite­ ria to assess the plant quality were established in this study. criterion i: ltb occurs on <1.5 leaves per plant. the lower the score of the scape growth and leaf spread, the higher the plant quality is. criterion ii: the number of days to flowering is <115 days, the length <11.5 cm, the width of the third leaf from the crown is <2.5 cm, the scape length is <15 cm with >45 flowers. criterion iii: the leaf spread and scape growth have a score <1.5. analysis of the macro‐ and micro‐elements in the third and/or fourth leaves showing various symptoms and suggested concentration for quality plant pro‐ duction the cause of ltb was investigated based on the macro­ and micro­element concentrations in the tis­ sue. macro­ and micro­elements analysis of o. dubi‐ um leaves without apparent ltb revealed that the b concentration in the leaf tips (distal end) was 119 ppm, which was within the suggested acceptable range for general ornamentals (30­150 ppm) (table 2 and 3). the concentrations in the middle and bottom (proximal end) of the leaves were slightly higher or lower, respectively, than the lower level of the range of b concentration. when the samples were collected in the early growth stages when no or incipient ltb symptoms were observed on jan. 14, jan. 26, and mar. 4, the b concentrations were 109, 96, and 108 ppm, respectively, which fell within the suggested acceptable range. leaf tip burn in o. dubium in young and old leaves is considered to be different from marginal leaf burn observed in old leaves of curcuma ‘cmu price’. the latter is related to high concentrations of b (119 ppm), fe (189 ppm), and mn (273 ppm) and low n concentration (1.7%) (roh et al., 2006). boron may accumulate at the tip or margin of leaves (jones, 1970) and may cause toxic effects (oertli, 1962). therefore, ltb in ornithogalum is a unique physio­ logical expression resulting from high b concentra­ tion at the leaf tips and developing regardless of the leaf age; these symptoms were observed in o. thyr‐ adv. hort. sci., 2022 36(1): 3­11 10 soides and o. arabicum (roh, personal observation). the incidence of ltb in watsonia laccata (jacquin) ker aawler is considered a similar disorder as in o. dubium and adversely affects potted plant produc­ tion in w. laccata (suh et al., 2011). leaf tip scorch observed in o. dubium (de hertogh and gallitano, 1997), upper leaf necrosis observed in oriental hybrid lilies (chang et al., 2008), and burn appearing at certain developmental stages before the visible bud stages in lilium ‘pirate’ (berghoef, 1986) may be different from ltb in o. dubium. the ltb is, therefore, caused by a high b concen­ tration (218­230 ppm) and possibly a high zn concen­ tration (155­159 ppm), which was higher in this study than the high level of the suggested range, even when grown in medium low in b and zn. boron con­ centration in the growing medium was <6 ppm (tech data pro­mix_hp_mycorrhizae_4253; accessed on nov. 1, 2020). the source of b accumulated at the tip of leaves showing ltb in ornithogalum is considered resulting from b accumulation at the margin of old leaves after translocation from rhizome and leaves as discussed in curcuma (roh et al., 2006; roh and lawson, 2009). since zn has intermediate mobility and occurs in the middle leaves (shelp et al., 1995; mccauley et al., 2009), it may not be associated with ltb observed in the young and old leaves of o. dubi‐ um. the effect of the temperatures in the pre‐planting bulb (st‐a) and post‐planting forcing periods (st‐b and st‐c) on growth, flowering, and plant quality regardless of the temperature treatments during the pre­planting bulb (st­a) and post­planting forcing (st­b and st­c) periods, the ltb symptoms were not entirely eliminated in o. dubium, which was in agree­ ment with the results for w. laccata (suh et al., 2011). in our experiment, the number of leaves showing ltb was 1.1 out of 5.1 leaves which was the fewest when received 10°c/st­a, followed by forcing at 15/12°c/st­b and 21/18oc/st­c (table 1). therefore, the flowering responses and leaf and scape morphologies, as evaluated in criteria ii and iii, respectively, were further considered. the overall average number of days to flower was 116 days (table 4), which was close to the 115 days estab­ lished for criterion i. therefore, it is suggested to store bulbs at lower than 22°c, which is lower than the 25°c suggested by de hertogh and gallitano (1997). our data suggested that storing bulbs at 10oc ensures that plants will flower in <115 days. low pre­planting temperature treatments in the range of 9 to 27°c for 3 weeks were shown to accel­ erate flowering (lee and miller, 2015). however, this depends on the cultivar and bulb­handling methods since leaves did not emerge and plants failed to flower when bulbs were treated at 10°c. the initia­ tion of the inflorescence can be inhibited by more than 6 weeks because breaking dormancy may require temperatures higher than 10oc (roh and joung, 2004). the optimum pre­planting temperature was reported as 22 to 28°c immediately after har­ vesting the bulbs to promote the early development of the first inflorescence and initiation of the second inflorescence (roh and joung, 2004). in o. arabicum, flower initiation and development were accelerated by storing initially at 30°c for 12 weeks, followed by 20°c for 4 weeks, and finally at 13°c for 8 weeks before planting (shoub and halevy, 1971; shoub et al., 1971). forcing at 21/18°c/st­b and st­c after potting the bulbs caused the plants to flower in 100 to 105 days, and the response was faster when bulbs were treat­ ed at 16°c/st­a. therefore, the recommended tem­ peratures for storing bulbs to ensure flowering in <115 days are 10 or 16°c/st­a followed by forcing at 21/18oc/st­b or preferably 21/18°c/st­c (10°c/st­a or at 16°c/st­a, 21/18°c/st­b or 21/18°c/st­c). de hertogh and gallitano (1997) suggested to force bulbs in a greenhouse at a minimum of 22/18°c to flower in 96 days (this is similar to the period in st­b and st­c in this study). temperatures below 15oc during an unspecified growth period did not increase the growth rate (littlejohn and blomerus, 2000), although a constant temperature of 17 to 19°c was most suitable for forcing, considering the forcing time, plant height, and visual quality of ornithogalum cultivars. however, the criterion for visual quality was not specified (lee and miller, 2015). the number of flowers was significantly influ­ enced by bulb storage temperature during st­a, and plants produced <36 flowers (10oc /st­a, 18/15oc /st­b, and 15/12oc/st­c). however, more than 44 flowers were produced when bulbs were treated at 16 and 22oc during st­a, regardless of the tempera­ ture during st­b and st­c. the number of flowers was reduced when forcing occurred at 32/27oc; how­ ever, the flowering rate was not mentioned (luria et al., 2002). a significantly higher number of flowers (52 flowers) produced on the short scapes (12.4 cm) wu et al. ‐ quality ornithogalum plants with reduced leaf tip burns 11 when bulbs were stored at 22oc/st­a and forced at 21/18oc/st­b) and 15/12oc/st­c. depending on the bulb size, 20 flowers each were produced on two flower stalks, and the bulbs stored at 9oc produced floral stalks (31.3 cm) (de hertogh and gallitano, 1997). three criteria were established to determine the quality of o. dubium plants. criterion i focus on the occurrence of ltb that are observed in less than 1.5 leaves per plant. leaf tip burn cannot be avoided, but can be controlled by maintaining the forcing temper­ ature at a minimum of 10oc or potentially 16oc dur­ ing st­a. this treatment does not delay flowering. leaf tip burn was observed in o. dubium in both young and old leaves and was caused by a high b concentration (218­230 ppm) and possibly by a high zn concentration (155­159 ppm). calcium was not the primary cause for ltb. criterion ii focuses on growth and flowering, leaf and scape morphologies; flowering occurs less than 115 days, the length and width of the third leaf is shorter than 11.5 cm and narrower than 2.5 cm, respectively, the scape length is shorter than15 cm, and there are more than 45 flowers. criterion iii focuses on the leaf morphology and the scape growth have a score of less than 1.5. the recommended treatment to meet the three cri­ teria and to produce high quality plants at flowering (plant a, fig. 2) is to treat bulbs at 10 or 16oc before planting (st­a), and at 15/12oc in st­b and at 21/18oc during st­c during forcing. however, it was not possi­ ble to produce finished plants without severe ltb symptoms. although ltb can not be prevented in o. dubium, it can be minimized by temperature manipu­ lation. references berghoef j., 1986 ­ effect of calcium on tip burn of lilium ‘pirate’. ­ acta horticulturae, 177: 433­438. chang y.c., albano j.p., miller w.b., 2008 ­ 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agricultural, forest and food sciences, university of turin, largo paolo braccini, 2, 10095 grugliasco (to), italy. key words: dianthus caryphyllus l., forchlorfenuron, growth regulators. abstract: nanosponges can favour the gradual release of molecules over a pro­ longed time, increasing the bioavailability and action of preservatives and phy­ toregulators, reducing the concentrations usually adopted. in floriculture, they have previously been proposed for the delivery of anti­ethylene compounds to improve the shelf­life of cut flowers. however, the potential of nanosponges is not only limited to these compounds. the present scoping review evaluated the effects of β­cyclodextrin­based nanosponges and growth regulators on the post­harvest longevity of cut flowers of ornamental species. one novelty was the use of forchlorfenuron (cppu), a growth regulator belonging to the group of cytokinins predominantly used in fruit cultivation, to evaluate its potential to increase the shelf­life of cut carnations (dianthus caryophyllus l). in particular, an in­depth analysis of a pre­test involving the use of nanosponges and cppu is proposed. specifically, as far as post­harvest longevity is concerned, the treat­ ments involved the use of: deionised water; nanosponges and deionised water; nanosponges loaded with cppu; nanosponges loaded with a classic solution for cut flowers, composed of sucrose, aluminium sulphate and 8­hydroxyquinoline sulphate. preliminary results show that the nanosponge and deionised water complex and the nanosponge and classical solution complex prolonged the longevity of the cut flower by up to 20 days, compared to the control (17 days). in contrast, the cppu­nanosponge complex showed similar results to the con­ trol. replication of the research is necessary to validate the results. 1. introduction cyclodextrin nanosponges (cdnss) are cage­like network polymers prepared by connecting cyclodextrin molecules with various kinds of bi­ or polyfunctional linking agents. the term “nanosponge” was first intro­ (*) corresponding author: luca.battisti@unito.it citation: battisti l., caldera f., hoti g., trotta f., devecchi m., 2023 ­ nanosponges and cppu: a scoping review and a pre‐test to assess the poten‐ tiality for shelf‐life prolongation of cut carnati. ­ adv. hort. sci., 37(1): 133­137. copyright: © 2023 battisti l., caldera f., hoti g., trotta f., devecchi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2022 accepted for publication 10 january 2023 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-13870 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 133­137 134 duced by ma and li, in a paper focusing on the sequestering capacity of polyurethane crosslinked cd polymers, to highlight the nanometric porosity of this class of materials (ma and li, 1999). in addition to the central lipophilic cavity of cd molecules, cdnss exhibit a second type of pores, which are the empty spaces among cd molecules, whose polarity and size can be modulated by varying the chemical structure of the linking agent and its concentration. such fea­ tures make cdnss highly versatile materials with potential applications as nanostructured containers and delivery systems for a broad set of active princi­ ples. furthermore, cdnss are low­cost, easy to pre­ pare, safe and sustainable materials, as they are mainly composed of starch derivatives and, in some cases, even the linker comes from renewable sources (e.g., citric acid) (caldera et al., 2017). the application of cdnss in the pharmacological field has been widely explored. several classes of drugs have been successfully encapsulated, stabilized and released with controlled kinetics, including anti­ cancer, anti­inflammatory, antiviral, antibacterial, antifungal drugs and many others. recently, the func­ tionalization of cdnss with specific moieties has led to the development of a new generation of stimuli­ responsive cdnss, capable of releasing drugs on­ demand and highly selective cdnss for targeted drug delivery. in the environmental field, cdnss have been used as absorbing agents for the removal of organic pollu­ tants as well as heavy metal cations for water decon­ tamination (krabicova et al., 2020). although cdnss have been extensively studied for biomedical and environmental applications, their full potential in the horticultural field remains largely unknown. to date, only a few studies have explored the use of cdnss for the encapsulation and release of the herbicide ailanthone and some anti­ethylene molecules (demasi et al., 2021). forchlorfenuron [n­(2­chloro­4­pyridyl)­nʹ­pheny­ lurea], whose acronym is cppu, is a plant growth reg­ ulator (synthetic cytokinin) that promotes the pericli­ nal cell division and is often used in agriculture to increase the berry size, the quality and the yield of grapes and kiwifruit (peppi and fidelibus, 2008; cruz­ castillo et al., 2014). although several studies have evaluated the effi­ cacy of cppu in postharvest in fruits (zhang et al., 2017; chang, 2021), few studies have focused on cut foliage and flowers, in contrast to a similar molecule, thidiazuron, which has been used for years (ferrante et al., 2002; chamani et al., 2006). the aim of this scoping review is to highlight the application potential of nanosponges in the posthar­ vest context. in addition, an in­depth study is pro­ posed on the production and testing of nanosponges and cppu in order to assess their effectiveness in prolonging the shelf‐life of cut carnations. more specifically, we intend to describe the pre­test phase of the two products listed above, which is also useful from an economic point of view in order to produce a quantity of nanosponges in the laboratory suitable for performing an experimental test with replica­ tions, as well as to proceed with the purchase of cppu in larger quantities. 2. materials and methods in the coming sections, the following will be reported: a scoping review on the use of nanosponges in floriculture; a first trial of the use of cppu and nanosponges on cut flower carnation. synthesis of a carbonate cyclodextrin nanosponge a carbonate cyclodextrin nanosponge was synthe­ sized by heating β­cyclodextrin (β­cd, 6.500 g, 5.73 mmol) and 1,1’­carbonyldiimidazole (cdi, 3.715 g, 22.92 mmol) in 39 ml of n,n­dimethylformamide at 90°c for 4 h. in the end, the solid gel formed during the crosslinking reaction was crushed and washed with approximately 2 l of deionized water via buchner filtration. the nanosponge was further puri­ fied in a soxhlet extractor with ethanol for 20 h and, once dry, finely ground in a planetary ball mill. cppu loading in the nanosponge the loading of cppu into the nanosponges was performed subsequent to the synthesis and purifica­ tion of the nanosponge. briefly, 450 mg of nanosponge was added to a solution of 50 mg of cppu dissolved in 25 ml ethanol. then, the disper­ sion was stirred at room temperature for 24 h. finally, the cppu­loaded nanosponge was collected by removing the entire volume of ethanol in a rotary evaporator at 50°c under vacuum. the complex was stored in a desiccator at room temperature until use. pre‐test setting the pre­test was conducted in march and april 2022, in the laboratories of the department of chemistry and department of agricultural, forest and food sciences, university of turin. the carnations were collected, packaged and pur­ https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/cytokinin https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/kiwifruit battisti et al. ‐ nanosponges and cppu in postharvest 135 chased at the flower market in sanremo (italy) on 27 march at 6 p.m. and were transported to turin and kept in the transported condition until 3 p.m. on 28 march, when the pre­test began. the four packages, purchased from ‘cooperativa tre ponti’ (intermedi­ ary), contained 20 flowers each. the first phase consisted of eliminating flowers that visually showed diseases, lesions and flowers at a different stage of flowering from the others. a total of 70 flowers were selected and the stems were cut into the water leaving a length of 30 cm. the pre­test comprised seven different treat­ ments (solutions with deionized water), each applied to 10 flowers. each flower was placed in a glass tube containing 50 ml of solution. on day 8 and 16, 10 ml of the specific solution was added to all tubes. measurements were conducted daily, and data were acquired for the following parameters: cut carnation weight (g) and flower diameter (cm). the pre­test lasted 20 days, with the following environmental conditions: 12 hours of light and 12 hours of dark­ ness; temperature 19±2°c. the treatments are shown in table 1. 3. results and discussions pre‐test results preliminary results (fig. 1) show that the treat­ ment a and the treatment c prolonged the longevity of the cut flower by up to 20 days, compared to the control (17 days). in contrast, the treatment e com­ plex showed similar results to the control. there was no inhomogeneity in the diameters of the flowers within the various treatments; however, the largest diameters (9.5 cm) were more frequently reached by the treatment f. lateral shoot development was a common side effect of cppu and commercial solution treatments. the pre­test yielded interesting results that allow planning the production of nanosponges and the pur­ chase of cppu in order to set up a scientific trial. of particular interest could be the application in solu­ tion and spray of nanosponges and cppu on cut foliage. final discussions and next steps the application of cdnss in floriculture, and more specifically post­harvest preservation, is an emerging line of research still widely unexplored. to date, only few scientific papers focusing on the encapsulation of anti­ethylene compounds in cdnss have appeared in the literature. of these ethylene inhibitors, 1­methyl­ cyclopropene (1­mcp) is one of the most studied, especially in combination with cds and cd­deriva­ tives. in 1994, serek et al. described for the first time the ability of 1­mcp to prolong the post­harvest shelf­life of plant material (serek et al., 1994). since then, 1­mcp has been extensively studied as a non­ toxic anti­ethylene agent to inhibit the senescence code treatments a nanosponges (5 g/l) b sucrose 30 g/l + aluminium sulphate (200 mg/kg) + 8­hydroxyquinoline sulphate (200 mg/l) c sucrose 30 g/l + aluminium sulphate (200 mg/kg) + nanosponges (5g/l) d cppu (200 mg/l) e cppu in nanosponges (200 mg/l) f commercial solution ­ chrisal prof. 2 (10 ml/l) control deionised water table 1 ­ pre­test treatments fig. 1 ­ average values of weights (g) and standard error bars of cut carnations during the pre­test. adv. hort. sci., 2023 37(1): 133­137 136 process of cut flowers, vegetables and fruits at the receptor level (blankenship et al., 2003; nasiri et al., 2020). in 2000, 1­mcp appeared on the market in two different formulations commercialized by floralife, inc. (walterboro, sc) and agrofresh, inc. (spring house, pa). the floralife product is named ethylbloc® and it is meant to be applied on ornamen­ tals only. while, agrofresh provides a formulation, named smartfresh®, that can be used with edible crops. however, both products are based on the inclusion complex of 1­mcp gas in α­cd (gehla et al., 2003). the efficacy of these formulations is time­limited, as most of 1­mcp is released within 20­30 minutes from application, under normal temperature and pressure conditions (blankenship et al., 2003). to overcome such limitations, new materials such as cdnss have been tested for the encapsulation and prolonged release of 1­mcp. in a first attempt, 1­mcp, 2,5­norbornadiene and silver nitrate were encapsulated in a carbonate cdns and added to two flower species, specifically dianthus caryophyllus ‘ idra di muraglia’ and ranunculus asiaticus ‘elegance’. while the formula­ tion containing 1­mcp allowed to extend the vase life of dianthus caryophyllus up to 23 days, the other for­ mulations did not show significant effects and none of the formulations was able to improve the longevi­ ty of ranunculus (devecchi et al., 2009). with the aim of understanding the role played by the central cavity of cd molecules in the encapsula­ tion and controlled release of 1­mcp, carbonate nss based on α­cd (6 glucose units, diameter of the cavi­ ty: 4.7­5.3 å) were used in comparison with the anal­ ogous carbonate nss prepared with β­cd (7 glucose units, diameter of the cavity 6.0­6.5 å). the tests were performed on dianthus caryophyllus ‘idra di muraglia’. results demonstrated that β­cd is remark­ ably more effective than α­cd in extending the post­ harvest life of the dianthus flowers, as no evidence of deterioration was observed up to nearly 11 days. whilst, the 1­mcp­loaded nss based on α­cd did not show any significant anti­ethylene activity (sceglie et al., 2011 a). the next step was to determine the influence of the ns’s degree of crosslinking on the encapsulation and slow release of 1­mcp. a series of samples of β­ cdns were synthesized with different carbonate to cd molar ratio (i.e., 2, 4 and 8), resulting in different degree of crosslinking. after loading with 1­mcp, the nss were used to extend the vase life of carnation cut flowers. all the ns formulations exhibited anti­ ethylene activity. however, the ns with the highest degree of crosslinking (i.e., linker/β­cd molar ratio of 8), and therefore the most densely crosslinked poly­ mer structure, showed the highest efficacy for pro­ longed time, even at low concentration (sceglie et al., 2011 b). the protective effect of the 1­mcp­loaded β­cdns with monomer ratio 1:8 against infection by botrytis cinerea on dianthus caryophyllus l. ‘idra di muraglia’ was studied as well. after eleven days of treatment, the ns at low dosage reduced the spreading of the grey mould by approximately 60 %. at higher dosage, the ns formulation outperformed the commercial 1­ mcp product, used as a reference, reaching a value above 90%, while the commercial formulation stopped at 76% (sceglie et al., 2012). as a final step, the effectiveness of the 1­mcp/ β­ cdns(1:8) complex in delaying the senescence process of cut flowers was evaluated in different flo­ ral species. specifically, anemone coronaria l. multi‐ color , paeonia lactiflora l. ‘sarah bernhardt’, helianthus annuus l. ‘sunrich orange’, ranunculus asiaticus l. ‘minou abrown’, papaver nudicaule l. multicolor and rosa hybrida l. ‘jupiter’ were treated with the ns formulation in the presence of exoge­ nous ethylene. although the mechanism of action was different, depending on the flower species, the cdns increased the anti­ethylene activity of 1­mcp in all the tested samples (sceglie et al., 2013). despite the positive results described above, the use of 1­mcp has some disadvantages, including its high cost, difficult handling and volatility. moreover, ethylbloc® and smartfresh® are not available in some countries. therefore, the development of new for­ mulations based on easily manageable active princi­ ples, such as salicylic acid (cocetta and ferrante, 2018), synergistic formulations of cumin essential oil and 8­hydroxyquinoline sulfate (mirjalili et al., 2018), cppu and others with prolonged release kinetics and thus long­term efficacy, are of special interest. references blankenship s.m., dole j.m., 2003 ­ 1‐ methylcyclopropene: a review. ­ postharvest biol. technol., 28(1): 1­25. caldera f., tannous m., cavalli r., zanetti m., trot­ ta f., 2017 ­ evolution of cyclodextrin nanosponges. ­ inter. j. pharmaceutics, 531(2): 470­479. chamani e., irving d. e., joyce d. c., arshad m., 2006 ­ battisti et al. ‐ nanosponges and cppu in postharvest 137 studies with thidiazuron on the vase life of cut rose flowers. ­ j. appl. hortic., 8(1): 42­44. chang p­t., 2021 ­ effect of preharvest application of cppu and perforated packaging on the postharvest quality of red‐fleshed pitaya (hylocereus polyrhizus sp.) fruit. ­ horticulturae, 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37(3): 243­253 doi: 10.36253/ahsc­13895 in vitro propagation and microtuberiza­ tion of potato (solanum tuberosum l.) spunta variety in lebanon m. dalleh 1, j. borjac 1, g. younes 2, e. choueiri 3, a. chehade 4, a. elbitar 4 (*) 1 beirut arab university, faculty of science, department of biological sciences, debbieh, lebanon. 2 beirut arab university, faculty of science, department of chemistry, debbieh, lebanon. 3 lebanese agricultural research institute, tal amara station, department of plant protection, zahleh, lebanon. 4 lebanese agricultural research institute, tal amara station, department of plant biotechnology, plant tissue culture unit, zahleh, lebanon. key words: in vitro culture, microtubers, shootlets proliferation, solanum tuberosum l., spunta. abstract: one of the factors that causes low productivity of potatoes in lebanon is the limited availability of certified seeds. the aim of this study was to establish a rapid protocol for in vitro propagation and microtuberization of potato (solanum tuberosum l.) of spunta variety. meristems culture associated to thermotherapy (one month/37°c) constituted the first step. the highest per­ centage of reactive meristem (92%) was observed on ms medium devoid of growth regulators while ms medium containing kin 0.4 mg.l­1, ga3 0.5 mg.l­1 and iba 0.5 mg.l­1 yielded the highest average number of shootlets (7.8) in the seventh subculture. the lowest number of days obtained for microtuber forma­ tion was 10 and the highest average number of microtuber (1.49) was obtained with shootlets incubated under c2 culture conditions (16­h day/8­h night for initial 7 days at 25±2°c; for remaining period: continuous dark at 17±2°c). contrary the highest microtubers average length (10.75 mm), average width (7.41 mm) and average weight (646.26 mg) were produced under c1 culture conditions (16­h day/8­h night at 25±2°c). medium supplemented with 5 mg.l­1 bap and 6% sucrose presented the highest average number of microtubers of 2.36 and 1.94 respectively. type and concentration of cytokines and sucrose concentration did not have significant effect on the average length, width and weight of microtubers produced. 1. introduction potato (solanum tuberosum l.) is the most important non­cereal food crop in the world (bamberg et al., 2016; nikitin et al., 2018). it is cultivat­ ed in about 150 countries (basera et al., 2018). potato cultivation is con­ sidered as strategic as it occupies the world fourth place in production (*) corresponding author: abitar@lari.gov.lb citation: dalleh m., borjac j., younes g., choueiri e., chehade a., elbitar a., 2023 ­ in vitro propaga‐ tion and microtuberization of potato (solanum tuberosum l.) spunta variety in lebanon. ­ adv. hort. sci., 37(3): 243­253. copyright: © 2023 dalleh m., borjac j., younes g., choueiri e., chehade a., elbitar a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 october 2022 accepted for publication 30 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13895 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 243­253 244 after maize, wheat, and rice (fao, 2019). in potato production, the quality of seed potatoes planted is an important determinant of the final yield and quality (roy, 2014). when farmers use their farm­saved seed potatoes for several cropping cycles without renewing the seed lot from a reliable source, seed­borne diseases accumulate and cause severe yield and quality losses (fuglie, 2007). potato propa­ gation takes place primarily asexually, through tubers and microtubers (zhang et al., 2006). potato microtubers are minute tubers produced through in vitro culture technique yielding disease­ free and high­quality seeds that can be preserved for a long time (badoni and chauhan, 2009). extensive physiological research has revealed that tuberization is controlled by several factors, such as hormonal combination, ratio of photoperiod, nutrient composi­ tions among others (naresh et al., 2011). many researchers used different growth regulators for in vitro induction of microtubers in potato (hossain, 2005). cytokinins, such as benzylaminopurine (bap), and kinetin (kin) are among these growth regulators (al­safadi et al., 2000; sarkar et al., 2006; aksenove et al., 2009). in addition, sucrose, the cheap and safe disaccharide, is considered as a superior agent and a critical stimulus for inducing microtubers (hussain et al., 2006; nistor et al., 2010). in lebanon, potato production is important for food security as well as a source of revenue in rural areas. it is a strategic crop for lebanese agriculture, covering about 19,000 ha in the bekaa plain (moa, 2012) and with production of approximately 300,000 tons per year constituting the greatest field crop ton­ nage in lebanon. it is cultivated mainly in the bekaa valley (central­eastern lebanon, 900­1000 m above the sea level, 70% of total area) and in akkar plain (northern lebanon, 25­30% of total area) (choueiri et al., 2017). lebanon does not produce certified potatoes’ seeds, they are imported mainly from the european union (eu) member states. in the early years of the 21st century, lebanon imported between 15,000 and 20,000 tons of potato seeds each year and relied heavily on this import with prices ranging between $750 and $1,000 per ton (abou­jawdah et al., 2001). the absence of a seed certification program, the introduction and exchange of potato seeds of unknown sanitary status and the lack of phytosani­ tary measures resulted in increased incidence and severity of potatoes’ diseases (abou­jawdah et al., 2001). to prevent the further spread of these dis­ eases and to avoid the introduction of new pathogens, it is important to reinforce the seed certi­ fication scheme that mandates using and trading of only certified potato seeds. such a scheme will help the lebanese farmers to improve their production, get involved in a certified seed production program and reduce the incidence of diseases, and thus eco­ nomical losses. the implementation of a potato seed production program in lebanon is possible; especially that lebanon is characterized by a wide range of microclimates favorable to produce them (abou­ jawdah et al., 2001). in a report published by the international potato center (ipc) in 1975, areas in lebanon including the laklouk, daher el beidar, and northern bekaa were listed among the qualified areas for potato seeds production. hence, the present study was initiated with the objective to determine optimum concentration of sucrose, the effect of two cytokinins (bap and kin) on the microtuberization capacity of the potato cultivar spunta under different incubation conditions. 2. materials and methods plant material the experiment was conducted at the lebanese agricultural research institute (lari), department of plant biotechnology. the mother plants used in this study were of spunta variety. thermotherapy com­ bined with meristem culture has been successfully established for efficient eradication of the potatoes’ viruses. about 10 tubers were subjected to a temper­ ature of 37°c for 50 days, under a photoperiod of 16 hours/day. at the end of the thermal treatment, the sprouts from the treated tubers were separated in portions of 3 cm and surface sterilized using 70% ethanol for 1 min followed by 5% (v/v) sodium hypochlorite containing two drops of tween­20 for 10 minutes. finally, the explants sources were washed 4 times with sterile distilled water (10 min each wash) before being used for the collection of meristems. in vitro propagation meristems have been cut with the first pair of leaf primordial and cultivated in 9 cm petri dishes on 3 ms basal medium (murashige and skoog, 1962). p1 medium lacked any additional growth regulators. p2 medium contained kinetin (kin), gibberellic acid (ga3) and indole­3 butyric acid (iba) at concentra­ dalleh et al. ‐ potato micropropagation 245 tions of 0.2 mg.l­1, 0.5 mg.l­1 and 0.5 mg.l­1 respective­ ly. p3 medium was supplemented with kin at 0.4 mg.l­1, ga3 at 0.5 mg.l­1 and iba at 0.5 mgl­1. in addi­ tion, to all media, a vitamin mixture consisting of nicotinic acid (5 mg.l­1), ascorbic acid (20 mg.l­1), pyri­ doxin (5 mg.l­1), thiamin (10 mg.l­1) and myo­inositol (100 mg.l­1) was added. the ph of all media was adjusted to 5.7 prior to the addition of agar (0.7%). the media were autoclaved at 121°c for 20 min. each medium was prepared in ten replicas with 5 meris­ tems per replica for testing. the in vitro cultivated 150 meristems were then shifted to a culture growth room at 25±2°c with 16 h photoperiod under white light intensity of 3000 lux. thirty days later, survival rate of meristems was recorded. the regenerated shootlets from the meristems were fragmented to obtain uninodal cuttings and transferred onto fresh medium. subcultures of 30 days interval were re­con­ ducted seven times using the same 3 initiation media i.e., p1, p2 and p3, under the same culture conditions described above with a set of 60 shootlets per medi­ um (10 repetitions of 6 shootlets). the newly regen­ erated shootlets per explant were recorded by the end of each subculture to calculate the multiplication rate as follows: multiplication rate= number of new shootlets/number of initial shootlets sanitary control to check the sanitary status of the shootlets that arose from the thermotherapy­treated meristems, the tissue­blot immunoassay (tbia), a reliable, rou­ tine and cost­efficient serological test that allow pro­ cessing of large numbers of individual plant samples serological test and that is commonly used to detect plant viruses in field and vegetable crops (makkouk and kumari, 1996) was performed to detect six main potato viruses pva, pvx, pvy, pvm, pvs and plrv. in addition, all samples were tested by the double­anti­ body sandwich enzyme­linked immunosorbant assay (das ­ elisa, loewe, germany) using specific anti­ bodies (clark and adams, 1977). all shootlets result­ ing from the meristems culture were subjected to the sanitary control. each shootlet was numbered and divided into 2 parts, the first part was sent to the plant protection laboratory at lari to be analyzed, while the second part of each plant remained in the culture medium pending the sanitary control results. pre‐tuberization shootlets from the fifth subculture were aseptical­ ly transferred into culture­tubes containing 10 ml of pre­tuberization medium (p1). cultures were grown for 20 days in culture growth room at 25±2°c and 16 h photoperiod under white light intensity of 3000 lux. tuberization eight ms liquid media with two sucrose concen­ trations 6% and 8% respectively, supplemented with 2 cytokinins, kin (0, 2 and 4 mg.l­1) or bap (0 and 5 mg.l­1) or their combinations were used as an induc­ ing medium for microtuber production. ten ml of each liquid medium were added to the tubes con­ taining the pre­tuberization ms solid medium after 20 days of culture. for each culture medium, two cul­ ture conditions were studied. in the first culture con­ dition, the incubation was in culture growth room for 16­h day, 8­h night at 25±2°c and 3000 lux for 60 days. in the second condition, an initial incubation at 16­h day, 8­h night at 25±2°c and 3000 lux, for 7 days followed by continuous dark at 19±2°c to 60 days. conditions are summarized in table 1. for each cul­ ture medium and condition, 32 replicas were pre­ pared. microtubers produced were harvested at day 60. data were recorded on days to microtubers for­ mation, average number, average length, average width and average weight of microtubers. statistical analysis means ± standard deviations were recorded for each step of the propagation protocol and analyzed by using standard analysis of variance (anova). duncan’s multiple range test was used to show dif­ ferences among the treatments’ means. all statistical analyses were performed using sas for windows (sas institute inc., 1995). 3. results in vitro propagation the survival rate of meristems was recorded after 30 days. the highest percentage of reactive meristem was formed on p1 medium that is devoid of growth regulators with an average of 920±4.8%, whereas a significant decrease of surviving reactive explants of 74±5.8% and 58±5.0% (p<0.05) was obtained on p2 and p3 media respectively as shown in figure 1 and 2 (a­c). no significant changes in percent of reactive meristems was observed between p2 and p3 media. the effect of growth regulators added in the cul­ ture medium is summarized in table 2. medium p3 adv. hort. sci., 2023 37(3): 243­253 246 presented the highest multiplication rate of 7.8±1.2 in the seventh subculture. whereas in medium p2, the best multiplication rate obtained was 6.9±0.7 in the sixth subculture. illustrations related to shootlets proliferation are presented in figure 2 (d and e). it is important to note that a l l s h o o t l e t s obtained in the meristems’ cultures coupled with thermotherapy were f ree of the tested pva, pvx, pvy, pvm, pvs and plrv viruses. in vitro microtuber formation in vitro tuberization was obtained after propaga­ medium medium composition culture condition c1 m1 20 ml of ms solid medium with 6% sucrose + 0 hormones 16­h day, 8­h night at 25±2°c m2 20 ml of solid ms medium with 8% sucrose + 0 hormones 16­h day, 8­h night at 25±2°c m3 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 5 mg.l­1 bap 16­h day, 8­h night at 25±2°c m4 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 5 mg.l­1bap 16­h day, 8­h night at 25±2°c m5 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 4 mg.l­1 kin 16­h day, 8­h night at 25±2°c m6 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 4 mg.l­1kin 16­h day, 8­h night at 25±2°c m7 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 5 mg.l­1bap + 2 mg.l­1kin 16­h day, 8­h night at 25±2°c m8 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 5 mg.l­1bap + 2 mg.l­1kin 16­h day, 8­h night at 25±2°c medium medium composition culture condition c2 m1 20 ml of ms solid medium with 6% sucrose + 0 hormones for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m2 20 ml of solid ms medium with 8% sucrose + 0 hormones for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m3 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 5 mg.l­1 bap for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m4 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 5 mg.l­1 bap for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m5 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 4 mg.l­1 kin for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m6 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 4 mg.l­1 kin for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m7 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m1 medium + 5 mg.l­1bap + 2 mg.l­1kin for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c m8 ms solid medium, after 20 days of culture, addition of 20 ml of liquid m2 medium + 5 mg.l­1bap + 2 mg.l­1kin for initial 7 days, 16­h day, 8­h night at 25±2°c, for remaining period continuous dark at 19±2°c table 1 ­ culture media and culture conditions tested for in vitro microtubers production of potato fig. 1 ­ effect of culture medium on the percentage of reactive meristem. means with the same letter are not signifi­ cantly different according to duncan’s. fig. 2 ­ represent the stages of the in vitro propagation and microtubers production of a representative under the three pre­tuberization conditions. a= sprout of the treat­ ed tuber. b= meristem initiation. c= shootlets elonga­ tion. d= uniodal cutting explants. e= shootlet prolifera­ tion. f= shootlet on pretuberization medium. g and h = shootlets on tuberization medium. i= microtubers pro­ duced under culture conditions c1. j= microtubers pro­ duced under culture conditions c2. dalleh et al. ‐ potato micropropagation 247 tion of the shoots (fig. 2 e­h). the effect of bap, kin, or their combination in the presence of sucrose (6% or 8%) under both culture conditions were studied for microtubers’ formation and development. table 3 summarizes the results of microtubers’ formation under all treatments. both the cytokinin type and its concentration sig­ nificantly (p<0.0001) affected the average number (an) of microtubers produced. media containing bap alone or in combination with kin was superior to other media. the highest an of microtubers per shootlet, i.e. 2.36 and 2.22, were produced when shootlets were propagated on m3 and m7 media with 5 mg.l­1bap or 5 mg.l­1bap + 2 mg.l­1kin respec­ tively under c2 conditions. on the other hand, no sta­ tistically significant effect was observed when varying the type nor the concentration of the cytokines on the average length (al) and average width (aw) or the average weight awe of microtubers (p=0.12, p=0.44 and p=0.39 respectively) as shown in table 3. there is a linear relation between size and weight of microtuber implying that each factor that influ­ enced the microtuber weight directly influenced microtuber size. effect of culture condition on microtuber formation results showed that the effect of culture condition was highly significant with p value <0.0001 for an, al, p1= medium without any additional growth regulators p2= medium contained kinetin (0.2 mg·l­1), gibberellic acid (0.5 mg·l­1) and indole­3 butyric acid (0.5 mg·l­1). p3= medium was supplemented with kinetin at 0.4 mg·l­1, gibberellic acid at 0.5 mg·l­1 and indole­3 butyric acid at 0.5 mg·l­1 table 2 ­ effect of culture media on the average number of shootlets per explant as recorded along the 7 subcultures table 3 ­ effect of cytokinin type and concentration on the average number (an), average length (al), average width (aw) and average weight (awe) of microtubers produced means followed by different letters in each column are significantly different for p≤0.05. media average number of shootlets per explant subculture 1 subculture 2 subculture 3 subculture 4 subculture 5 subculture 6 subculture 7 p1 0.70±0.3 1.17±0.2 3.87±0.4 4.07±0.5 5.08±0.5 5.4±0.2 4.00±0.6 p2 1.0±0.0 0.9±0.5 2.63±0.7 6.2±0.7 6.07±1.1 6.9±0.7 5.40±0.4 p3 1.2±0.6 1.5±1.0 5.57±0.7 7.33±0.9 6.00±0.2 7.4±0.9 7.80±1.2 culture media culture conditions average number of microtubers average length of microtubers (mm) average width of microtubers (mm) average weight (mg) m1 c1 0.22±0.42 h 8.07 cd 5.05 e 375.3 d m2 c1 0.78±0.70 fg 11.14 ab 7.28 abc 622.2 abc m3 c1 1.53±0.62 cd 12.00 a 7.34 abc 673.1 ab m4 c1 1.44±0.91 cde 10.07 abc 6.90 abcd 593.4 abcd m5 c1 1.00±0.71 efg 11.8 a 7.96 a 734.5 a m6 c1 0.94±0.82 fg 10.78 ab 7.23 abc 621.9 abc m7 c1 1.58±0.95 cd 11.07 ab 7.43 ab 635.4 abc m8 c1 1.25±0.85 def 11.1 ab 7.75 ab 643.3 abc m1 c2 0.87±0.50 fg 9.45 bcd 6.8 abcd 535.4 abcd m2 c2 0.55±0.80 gh 7.24 d 5.00 e 369.1d m3 c2 2.36 a±1.05 8.89 bcd 5.64 de 473.5 bcd m4 c2 1.56±0.98 cd 8.84 bcd 5.91 cde 561.5 abcd m5 c2 1.00±0.65 efg 9.12 bcd 5.86 cde 487 bcd m6 c2 1.58±1.05 cd 7.85 cd 5.34 e 406.3 cd m7 c2 2.22±1.15 ab 7.85 cd 5.08 e 416.3 cd m8 c2 1.80±1.16 bc 9.78 abc 6.26 bcde 600.7 abcd 248 adv. hort. sci., 2023 37(3): 243­253 aw and awe of microtubers (table 4). an was 1.49 for shootlets incubated under c2 condition and 1.09 for shooltlets cultivated under c1 condition. values of al, aw and awe are 10.75 mm, 7.41 mm and 646.26 mg for microtubers produced under c1 condition and 8.63 mm, 5.65 mm and 481.23 mg for microtubers obtained under c2 condition respectively. under c1 culture condition, most of the cultures produced green microtuber, and this could be due to synthesis of the alkaloid solanine (hoque, 2010). on the other hand, brown­colored tuber was observed in c2 culture condition (fig. 2, i and j). effect of sucrose concentration on microtuber forma‐ tion the effect of sucrose on microtuber production is presented in table 5. results revealed a highly signifi­ cant effect of sucrose concentration on the an of microtubers produced. the media added with 5 mg.l­ 1 bap or 5 mg.l­1 bap + 2 mg.l­1 kin, both supplement­ ed with 6% of sucrose, presented the highest an of microtubers, 1.95 and 1.9 respectively. on the other hand, sucrose concentration had no effect on al, aw and the awe of microtubers (table 5). time‐dependent effect of growth regulators, sucrose concentration and culture condition on microtuber formation microtuber formations was followed in a time dependent manner (fig. 3). shootlets incubated under c1 conditions in media supplemented with sucrose (6% or 8%) devoid of any growth regulators, i.e. m1 and m2, needed 60 days for microtubers for­ mation as compared to c2 conditions that needed 40 and 45 days respectively. when incubated in the presence of the diverse cytokinins (m3 till m8) under c1 conditions, microtubers’ formation was evident at day 30. shootlets incubated in m3 and m7 media, supplemented with 5 mg.l­1 bap and 5 mg.l­1 bap + 2 mg.l­1 kin respectively, showed the fastest microtu­ bers’ formation at day 10. other cytokinin combina­ tions (m4, m5 and m8) required between 15 to 20 days. this confirm that m3 and m7 combination were the best for microtubers’ formation. table 4 ­ effect of culture condition on the average number (an), average length (al), average width (aw) and average weight (awe) of microtubers produced c1 culture conditions= 16­h day, 8­h night (25±2°c), c2 culture conditions= for initial 7 days, 16­h day, 8­h night (25±2°c), for remaining period: continuous dark (19±2°c) means followed by different letters in each column are significantly different for p≤0.05. culture conditions average number of microtubers average length of microtubers (mm) average width of microtubers (mm) average weight (mg) colour of microtuber c1 1.09 b 10.75 a 7.41 a 646.26 a green c2 1.49 a 8.63 b 5.65 b 481.23 b brown table 5 ­ effect of culture condition on the average number (an), average length (al), average width (aw) and average weight (awe) of microtubers produced c1 culture conditions= 16­h day, 8­h night (25±2°c), c2 culture conditions= for initial 7 days, 16­h day, 8­h night (25±2°c), for remaining period, continuous dark (19±2°c) means followed by different letters in each column are significantly different for p≤0.05. culture media sucrose concentration average number of microtubers average length of microtubers (mm) average width of microtubers (mm) average weight (mg) m1 6% 0.55 d 8.76 a 5.93 a 455.35 a m2 8% 0.67 d 9.19 a 6.14 a 495.65 a m3 6% 1.95 a 10.45 a 6.49 a 573.3 a m4 8% 1.5 b 9.46 a 6.41 a 577.45 a m5 6% 1 c 10.46 a 6.91 a 610.75 a m6 8% 1.26 bc 9.32 a 6.29 a 514.1 a m7 6% 1.9 a 9.46 a 6.26 a 525.85 a m8 8% 1.53 b 10.44 a 7.01 a 622 a dalleh et al. ‐ potato micropropagation 249 4. discussion and conclusions accumulation of seed­borne diseases is a major cause of low yield and quality losses in agriculture. potato is a strategic crop for many populations. producing disease­free potato microtubers is vital to improve its production. this study aimed to deter­ mine the best conditions and agents (sucrose and cytokinins) on the microtuberization capacity of the potato cultivar spunta. as meristem tips are free from viruses, elimination and generation of virus free plants were shown by many researchers through meristem culture to obtain disease free potato plantlets (jha and ghosh, 2005; bhuiyan, 2013). our findings provide evidence on the importance of having media devoid of growth regulators on the formation of reactive meristem in order to obtain the highest percentage. these results are in contradiction with others where al­taleb et al. (2011) indicated that the medium supplemented with 0.5 mg.l­1 of iba was the best for shoots development and where hajare et al. (2021) showed that best shoot initiation was obtained on ms medium supplemented with 1.5 mg.l­1 bap + 3.0 mg.l­1 naa for gudiene variety, whereas 1.0 mg.l­1 bap and 2.0 mg.l­1 naa produced more shoots in belete variety. according to a study done by kanwal et al. (2006) who investigate the in‐ vitro micropropagation of potato cultivar kuroda, the increase of added bap concentrations in ms medium induced an increase in the rate of shoot formation. according to salem and hassanein (2017) who studied the effect of genotype on multiplication and micro­tuberization of potato in vitro, the highest number of microshoot was formed by cv. hermes after cultivation in a medium supplemented with 1 mg.l­1 bap + 0.5 mg.l­1 ga3, whereas the least one was formed by cv. spunta implying that the number of obtained microshoots is cultivar­dependent. at the level of the multiplication phase, our results showed that the media p3 containing kin 0.4 mg.l­1, ga3 0.5 mg.l­1 and iba 0.5 mgl­1 caused the highest number of shootlets/plant. similar results were obtained by emaraa et al. (2017) who supplemented their media with kin (0.2 mg.l­1) and naphtaleneacetic acid (naa, 0.2 mg.l­1) and obtained maximum number of shootlets/plant. among the different cytokinins used by researchers when studying micropropagation of plants, kin stands among the best to promote shoot formation and shoot length (van­staden et al., 2008; hoque, 2010). el dessoky et al. (2016) also showed that the highest percentage of shoot initia­ tion and multiplication were observed on ms medium containing kin (0.1 mg.l­1) and ga3 (5 mg.l­1). concerning microtuber formation, this study revealed that the addition of bap into the culture medium was more effective in inducing in vitro tuber­ ization in comparison to kinetin. the highest average number (an) of microtubers was produced when propagated shootlets were subcultured on m3 medi­ um containing 5 mg.l­1 bap. these results agree with others (aksenove et al., 2009; hoque, 2010; sota et al., 2020) who also showed that bap has greater potential for microtuberization than kinetin and had an effect on reduction of total sugar and subsequent­ ly have increased starch content (sarkar et al., 2006). furthermore, liljana et al. (2012) observed that tuberization occurred only in the presence of bap (2 mg.l­1) + iaa (1 mg.l­1). wang and hu (1982) and badoni and chauan (2010) also reported that the optimum condition for in vitro tuberization of virus­free potatoes were in a medium containing bap at 10 mg.l­1. vural et al. (2018) used bap (2.5 mg.l­1) with naa (0.5 mg.l­1) in their in vitro micro­ propagation of potato and found that these cytokinins had a positive effect on micro tuber forma­ tion and can be recommended to use them commer­ cially in mass production. on the other hand, these cytokinins showed no significant effect on the average length (al), average width (aw) and average weight (awe) of microtu­ bers. our results contradict those of aryakia and hamidoghli (2010) who showed that bap and kin induced changes in microtuber size and weight in the in vitro microtuberization of two potato cultivars, arinda and diamant. prat (2004) reported that kin played a significant role in creating sink during plant growth, and through fig. 3 ­ days to microtuber formation under different conditions. adv. hort. sci., 2023 37(3): 243­253 250 regulating the expression of a gene involved in the partition of assimilates towards the stolon as observed in potato. our study reports that kin exert­ ed no significant effect on growth, diameter and weight of microtuber and this is in agreement with kefi et al. (2000) and kanwal et al. (2006). in general, a linear relation between size and weight of microtuber exists (liu and xie, 2001). any factor that influences microtuber weight will directly influence microtuber size. the microtubers parame­ ters an, al, aw, and awe obtained differed based on the culture condition. growth condition c2 proved to be better for microtuber formation, where­ as condition c1 was better for al, aw, awe. under c1 culture condition, most of the culture produced green microtubers and this could be due to the syn­ thesis of the alkaloid solanine that has fungicidal and pesticidal properties and that is considered as one of the plant’s natural defenses. hoque has also shown that when potato tubers are exposed to light, they turn green and increase glycoalkaloid produc­ tion (hoque, 2010). on the other hand, culture condi­ tion c2 yielded brown­colored tubers (fig. 1, i and j). these results are in agreement with those of salem and hassanein (2017) who showed that dark condi­ tions were better for microtuber formation com­ pared to a 16­h photoperiod. similarly, sakha et al. (2004) also reported that microtuber formation fre­ quency is higher under dark than light conditions. in addition, garcía and bolaños (2017) showed that under the light condition with a photoperiod of 16­ hour light and 8­hour dark, the number of microtu­ bers was lower, but their biomass was higher and greener. on the other hand, under the dark condi­ tion, the number of microtubers was greater, with a lower biomass average and cream color. garner and blacke (1989) al­hussaini et al. (2015), showed in periods of total darkness, the microtuber weight is reduced. other study also concluded that the light condition of 8 hours may be of benefit to the in vitro tuberization and increased the size and uniformity of microtubers compared to the in vitro tuberization under dark conditions (pruski et al., 2002). thus, an appropriate combination of light and dark conditions with short days can synchronize and accelerate the initiation and development of microtubers as well as increase their numbers. finally, concerning the effect of sucrose on micro­ tuber formation, lower sucrose concentration (6%) resulted in an increase in the average number of the produced microtubers. whereas sucrose presence had no effect on the length, width and weight (table 5). our results agree with those of fufa and diro (2014) with respect to the effect of 6% sucrose on the an of the microtuber. aslam et al. (2011) also found that a medium containing 6% sucrose was optimal in terms of minimum time of induction, aver­ age tuber number and weight of microtubers per sin­ gle nodal explant in cultivar desiree. imani et al. (2010) also reported that ms medium supplemented with 6% of sucrose yielded the maximum number and the highest al and aw of microtubers. in plant tissue culture, most plant requires an exogenous car­ bohydrates source because of the limited photosyn­ thesis that occurred in vitro (lian et al., 2014). tuberization is known to be regulated by carbohy­ drates availability such as sucrose which is the trans­ ported form of sugar required for starch synthesis (abelenda et al., 2019). sucrose is essential for the in vitro tuberization as an energy source and a signal for microtuber formation (donnelly et al., 2003; fufa and diro, 2013; motallebiazar et al., 2013). in this study, to obtain microtubers of a sufficient weight, potato microshoots were cultured for 7 days in 16­h photoperiod followed by 50 days in dark. this type of treatment was referred to as light/dark treat­ ment (table 1). it increased microtuberization by enhancing tuberonic acid synthesis that was shown to play an important role in in vitro tuber formation (alisdair and willmitzer, 2001). in conclusion, the best combination for rapid microtuberization obtained in our study was 6% sucrose, 5 mg.l­1 bap for spunta potato cultivar cul­ tured under c2 culture condition was within 10 days. hossain et al. (2015) who investigated the effect of sucrose, growth regulators and potato varieties (‘diamant’ and ‘cardinal’) on rapid microtuberization found that the best combination was 9% sucrose at 5 mg.l­1 of 6­benzyl aminopurine cultivar within 6­8 days while zakaria et al. (2008) who studied the opti­ mum level of benzyl adenine (ba) and chloro choline chloride (ccc) to obtain large­size microtubers of the potato cultivar diamant showed that early microtu­ ber induction occur by using 500 mg/l of ccc within 15.9 days or by using 10 mg/l of ba within 13.3 days. in the present study, the effect of two growth reg­ ulators, kinetin and bap, and the presence of sucrose and different photoperiodic conditions on the perfor­ mance of microtubers in the potato cultivar spunta was evaluated. microtubers were produced from in vitro grown plantlets regenerated in ms medium sup­ plemented with kin 0.4 mg.l­1, ga3 0.5 mg.l­1 and iba https://link.springer.com/article/10.1007/s10535-017-0715-x#auth-j_-salem https://link.springer.com/article/10.1007/s10535-017-0715-x#auth-a__m_-hassanein dalleh et al. ‐ potato micropropagation 251 0.5 mg.l­1. the highest number of microtuber was obtained in m3 medium during c2 condition supple­ mented with 60 g.l­1 sucrose whereas c1 condition was better for al, aw, and awe of microtubers. the results indicated that microtuber induction of potato was highly dependent on sucrose, growth regulator and photoperiod conditions interaction. production of large microtubers is important for successful uti­ lization of microtubers in seed potato production. hence, this study proposes an economical and repro­ ducible method to obtain larger microtubers under laboratory conditions and it could be experimented to produce seed potato from in vitro grown tubers. acknowledgements this study is part of the research activities of the tissue culture unit, department of plant biotechnology funded by the lebanese agricultural research institute (lari). authors would like to thank dr. michel afram, president director general of lari for his valuable support. references abelenda j., bergonzi s., oortwijn m., sonnewald s., du m., visser r., sonnewald u., bachem c., 2019 ­ source‐sink regulation is mediated by interaction of an ft homolog 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plant breeding, faculty of agriculture and natural resources, persian gulf university, borazjan, bushehr, iran. 3 fars agricultural and natural resources research and education center, areeo, shiraz, iran. key words: abnormal fruit set, metaxenia, parthenocarpic fruits, phoenix dactylifera l., pollen source. abstract: tissue­cultured (tc) date palms produce no fruit or low yield due to abnormal fruit setting. to improve the yield of tc ‘berhi’ palms, trees were pol­ linated using five pollen sources (gantar, ghannami, mazafati, zahedi, and jarvis). the experiment was carried out in three replications for two successive years in a randomized complete blocks design. the fruit set, the fruit and seed physical traits at the khalal stage, bunch weight at the tamar and khalal stages, ripeness of tamar bunch, and the fruit quality at both khalal and tamar stages were measured and monitored. year factor significantly affected the fruit set and the fruit and seed characteristics. pollen sources affected fruit set and some seed characteristics significantly. zahedi+jarvis pollen treatment that induced 50% normal fruit set and the highest ratio of pulp to seed was found superior. it was also a top treatment in khalal’s bunch weight (3.11 kg). zahedi+gantar treatment was realized superior in tamar’s bunch weight (6.00 kg). ghannami, jarvis+ghannami, and zahedi+jarvis treatments produced khalal’s fruits with higher quality indices but zahedi+jarvis treatment was superior in fruit quality at the tamar stage. overall, the combined application of zahedi and jarvis pollens yielded the most desired outcomes. 1. introduction date palm (phoenix dactylifera l.) is one of the most important com­ mercial fruit crops of the arid and semi­arid subtropical regions of the world. the global total annual production reaches about 9.1 million tons. iran with a production of about 1.3 million tons in 2020 is the world’s third­largest date producer (fao, 2020). although many cultivars of dates grown in the country are popular in the domestic market, there has been a shift in recent years toward cultivars that are in high demand on both (*) corresponding author: rastgoo@pgu.ac.ir snrastgoo@yahoo.com citation: rastgoo s., bemani f., nooryazdan h., izadi m., 2023 ­ improving fruit set and yield of tissue cultured date palm cv. berhi by using a combined pollination technique. ­ adv. hort. sci., 37(2): 197­ 208. copyright: © 2023 rastgoo s., bemani f., nooryazdan h., izadi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 august 2022 accepted for publication 13 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13499 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 197­208 198 the local and international markets. among such cul­ tivars, ‘berhi’ is considered a superior, elite cultivar that has earned high popularity in export markets. the international high demand for this cultivar is because of the rare characteristic of its fruits. unlike most date cultivars, fruits from ‘berhi’ are best mar­ keted at the khalal stage for their tannin­free, sweet and crispy flesh. however, ‘berhi’ fruits are also con­ sumed at rutab and tamar stages (zaid and de wet, 2002 a) though with lower frequency. ‘berhi’ is comparatively a high­yielding cultivar (average of 200 kg per palm) (zaid and de wet, 2002 a) ensuring higher income for the growers, which in turn is driving demand for its propagules to expand its plantations (mohammadi et al., 2017). the current demands for date palm trees cannot be satisfied with traditional propagation by offshoots (ali­dinar et al., 2021), particularly for the cultivars whose offshoot production is low like ‘berhi’ (zaid and de wet, 2002 a). alternatively, micropropagation is an efficient method of propagating date palm clones on a large scale (al­khayri and naik, 2017; ali­dinar et al., 2021). nevertheless, despite the numerous benefits of micropropagation, it has also resulted in various abnormalities in the growth and yield of tc (tissue culture)­derived palms. the most economically damaging disorder, which occurs in high frequency, is floral fertilization failure resulting in abnormal fruit set with a great impact on productivity and crop yield, and predominantly lead­ ing to a very low proportion of normal fruitlets that ultimately affects the overall economics of the plan­ tation (al­dinar et al., 2021). the abnormal fruits, usually appearing in the form of triple parthenocarpic fruitlets, are of no commercial value. this abnormal fruiting has been reported in date palms in the past years frequently (cohen et al., 2004; al­kaabi et al., 2007; mohammadi et al., 2017). tc­derived trees of ‘berhi’ display a high prevalence of this phenotype, which has made it the most common unstable culti­ var in this ground (cohen et al., 2004; al­wasel 2005; mohammadi et al., 2017; al­dinar et al., 2021). the main cause of failure in normal fruit setting is failed fertilization originating from ineffective pollination of the female flowers. although several reports have reviewed/evaluated the growth and yield of tc­ derived date trees (al­wasel, 2000; hajian, 2007; kavand et al., 2015) few attempts have been under­ taken to overcome this shortcoming in the field­ established tc­derived date trees, especially ‘berhi’ (mohammadi et al., 2017; al­najm et al., 2021). pollination intervention can be an influential strategy to improve the fruit set and yield of such off­type palms (mohammadi et al., 2017). however, so far no optimized pollination procedure has been released. thus, more research is required in this context. date palm is a dioecious species and artificial (hand) pollination by man is inevitably done to gain economic yield. there are several reports on the role of pollen source in the fruiting of non­tissue cultured date cultivars (omar and el­abd, 2014; soliman et al., 2020), but similar research on tc­derived date trees are few (rezazadeh et al., 2013; mohammadi et al., 2017). metaxenic effect reflected as changes in the phys­ ical and chemical characteristics of fruits under the direct influence of pollen source has been reported in many date cultivars previously (nixon, 1934; shafique et al., 2011; rezazadeh et al., 2013; mohammadi et al., 2017; al­najm et al., 2021). exploiting this effect to improve fruit quality is of interest. overall, despite several works carried out during the past several years, no clear­cut solution has been found for resolving abnormal fruiting and low yield in many tc­ derived ‘berhi’ trees yet. the present study was performed to avoid abnor­ mal fruit­setting and improve fruit yield and quality of tc­derived ‘berhi’ trees through optimizing pollen source and pollination treatment. 2. materials and methods plant materials field pollination experiments were carried out at date palm and tropical fruits research station in dashtestan (29°23´ n; 51°5´ e; altitude 50 m), bushehr, iran. fourteen­year­old tc­derived trees of ‘berhi’, uniform in growth, were selected as female parents. pollen collection and pollination pollen grains were collected from five clonally male selections preserved in the date palm germplasm collection at the research station, four locally superior selections viz. ‘gantar’, ‘ghannami’, ‘mazafati’, and ‘zahedi (zahidi)’, and one internation­ ally recognized elite clone “jarvis”. pollen collection and storage were done in february 2018 and 2019 according to the procedure described by mohammadi et al. (2017). to assess the viability of pollen grains, two tests was applied: acetocarmine rastgoo et al. ‐ fruiting improvement in tissue cultured date palm 199 staining test and in vitro germination test (fig. 1) few days ahead of hand pollination. the pollens used for viability tests had been stored in a refrigerator at 4°c for 4­5 weeks. manual pollination was performed on the day of natural spathe craking in early april in the first year and in late march in the second year. pollens of ‘gantar’, ‘ghannami’, and ‘mazafati’ were used both individually and in combination with each of the zahedi and jarvis pollens. a pollination treatment consisting of a combined application of ‘zahedi’ and ‘jarvis’ pollens was also included. the pollination operation was done according to the traditional method used by the local date growers. in this method, a traditional pollination device, made of cot­ ton fabric and the dried midrib of date palm leaf and filled with fresh pollen, is tapped mildly on two sides of each selected receptive female inflorescence releasing pollen grains gently through the fabric onto the flowers (fig. 2). the experimental inflorescences were selected on candidate female palms based on uniformity in length (approx. 80 cm) and the number of spikelets per inflorescence (14±2). for the pollina­ tion treatments that contained two different pollen sources, an equal quantity of each source was weighed, mixed well with each other, and poured into the pollination device. for each pollination treat­ ment, a separate pollination device filled with the corresponding pollen source(s) was used. to prevent possible contamination of the flowers by foreign pol­ lens, the pollinated inflorescences were then imme­ diately covered with white cotton bags. the bags were removed three weeks later. all targeted inflo­ rescence were tagged properly corresponding to the pollen treatment and the block number. calculating fruit set traits five weeks after pollination, percentages of nor­ mal fruit set, parthenocarpic fruit set, and fruit drop were calculated for each tagged inflorescence. five strands on each inflorescence were randomly select­ ed. numbers of normal fruits, parthenocarpic fruits, and flower scars as the representative of dropped flowers and fruitlets were counted separately, recorded, and each one divided by the total number of flowers in the chosen strands, then multiplied by 100. measuring bunch weight and bunch ripeness in the first year of the study, each tagged bunch was harvested separately on september 18 and shak­ en off to collect the whole fruits. the weight of total fruits of each bunch was considered as the bunch weight, and the percentage of bunch ripeness was calculated based on the weight ratio of the bunch tamar fruits to total fruits of the same bunch (includ­ ing tamar, rutab, and khalal fruits). in the second year, the weight of the whole khalal fruits of each tagged bunch was considered as the bunch weight at the khalal stage. measuring fruit and seed physical characteristics during both experimental years, fruit sampling was done in mid­august at the khalal stage. fifteen single normal fruits were picked randomly from each tagged bunch, and the weight, length, diameter, and volume of the fruits and seeds were measured. in addition, pulp weight, the ratios of pulp weight to seed weight, and fruit length to fruit diameter were also calculated. measuring fruit phytochemical quality fruit phytochemical quality was measured twice; the first year at the tamar stage and the second year fig. 2 ­ pollinating female inflorescences through mild tapping of a traditional pollination device, releasing pollen grains gently through the cotton fabric onto the receptive flo­ rets. fig. 1 ­ (a) differential stainability of viable and non­viable pollen grains. red­stained grains are viable and non­ stained grains are non­viable. arrows show abnormal pollen grains, which all are non­viable. (b) germinated pollen grains with pollen tubes of various lengths. arrows show non­germinated pollen grains. adv. hort. sci., 2023 37(2): 197­208 200 at the khalal stage. fifteen normal fruits were har­ vested from each tagged bunch. three replicated homogenized samples containing the pulp of five fruits were prepared and analyzed. to measure total soluble solids (tss), fruit juice was extracted accord­ ing to aurand et al. (1987) and tss was read using a digital refractometer (atago pal­3, japan). the ph was measured directly in the extracted juice by a dig­ ital ph­meter (az 8686, taiwan). the contents of reducing and total sugars were determined using the procedure described by lane and eynon (1923). titratable acidity (ta) was quantified by titrating a known volume of juice with 0.01 n naoh, using phe­ nolphthalein as the indicator (aoac, 2016) and expressed as the percent of acetic acid. the percent­ age of dry matter and moisture content were deter­ mined using 10 g of fruit pulp in a petri dish dried in an oven at 72°c for 48 h. experimental design and statistical analysis the field experiment was conducted in a random­ ized complete blocks design with ten pollen treat­ ments and three replications (blocks). each block consisted of ten female inflorescences each of them pollinated with one of the ten pollen treatments. inflorescences in each block were assigned to three female palms. overall, nine female palms hosted 30 inflorescences uniform in length, and the number of spikelets were assigned for the experimental blocks. the current study was performed over two succes­ sive years. to determine the significance level of effect of pollen treatments on fruit­set parameters, and fruit and seed physical characteristics recorded for two years, a combined analysis was performed by glm procedures of the sas software package (sas institute, cary, nc, usa). the mean comparison was also performed using duncan’s multiple range tests (dmrt) based on the averages of the two years data at a 5% probability level. for fruit qualitative traits, bunch weight at khalal and tamar stages and bunch ripeness at the tamar stage, a one­way anova was conducted for each year. the mean comparison was also executed for the fruit qualitative traits using dmrt at a 5% probability level. 3. results pollen viability the results have been illustrated in figure 3. mean comparison for pollen viability by the staining test showed that the values had a ranging from a maxi­ mum of 98% for zahedi+mazafati pollens to a mini­ mum of 79.3% for ghannami pollen (fig. 3). apart from ghannami pollen and jarvis+ghannami pollen source, there was not a significant difference among almost all other sources, which had pollen viability above 90%. pollen viability assessed by the germina­ tion test ranged from 24.3 to 74%. the highest score was for jarvis+mazafati pollens though not significan­ tly different from that of zahedi+gantar pollen sour­ ce (69.7%) (fig. 3). the lowest percentages of germi­ nated pollens were observed with ghannami male (24.3%) and zahedi+jarvis source (32.3%). the results revealed that combining zahedi pollen with each of gantar, ghannami, and mazafati pollens showed that the percentage of germinated pollens was increased significantly in cases of gantar and ghannami pollens but not for mazafati pollen showing a reverse result. the results showed high differences among pollen sources in the ability of in vitro germination. such significant differences can be due to genetic variation and storage conditions. since the fresh pollen used in fig. 3 ­ mean comparison of pollen viability for 10 pollen sour­ ces tested by staining with acetocarmine and by in vitro germination. means were calculated based on the two years data. columns having same letter(s) do not show a significant difference with each other using duncan's multiple range test at p≤0.01. g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi. rastgoo et al. ‐ fruiting improvement in tissue cultured date palm 201 this study was kept under the refrigerator tempera­ ture for a few weeks before the date of hand pollina­ tion, the most important variable that differentiated these pollen sources based on their germination abi­ lity can be assumed as the genetic differences. fruit set the results (table 1) revealed that all the experi­ mental variables including year, pollen source, and the interaction effect affected fruit set traits significantly at p≤0.01. results of the mean comparison test con­ ducted for the main factor (pollen source) have been illustrated in figure 4. the highest values of normal fruit set were achieved with the pollen treatments including mazafati, zahedi+jarvis, and zahedi+gantar (53.1%, 49.2%, and 48.9%, respectively) while the low­ est parthenocarpic fruit set was with the same pollen sources (18.1%, 10.3%, and 17.7%, respectively). mazafati pollen also caused the lowest fruit drop (28.8%) (fig. 4). gantar pollen and jarvis+gantar pollen treatment yielded the lowest percentage of normal fruits (28.9% and 34.6%, respectively). gantar pollen also caused the highest percentage of parthenocarpy (36.7%) and a moderate fruit drop (34.4%). meanwhile, the combined application of jarvis and gantar pollens acquired the first rank in fruit drop (43.5%). the interesting point was that the zahedi+jarvis pollen treatment caused the lowest parthenocarpy (10.3%) and the second­best normal fruit set (49.2%) which was statistically the same as the highest normal fruit set obtained with mazafati (53.1%). table 1 ­ combined analysis of variance for fruit set traits and some characteristics of khalal fruit of tc­derived ‘berhi’ date palms polli­ nated with 10 pollen sources ns= not significant; ** significant at p≤0.01; * significant at p≤0.05. fig. 4 ­ mean comparison of fruit set traits for the ten pollen sources used. means were calculated based on the two years data. columns having the same letter(s) do not show a significant difference from each other using duncan's multiple range test at p≤0.01 (g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi). fruiting characteristics sources of variation (mean square) cv%year (df= 1) error (year) (df=4) pollen source (df=9) year × ps (df=9) error 2 (df= 36) normal fruit set % 814.82 ** 18.485 341.22 ** 630.15 ** 17.764 10.16 parthenocarpy % 7863.40 ** 20.90 297.24 ** 717.87 ** 6.712 11.86 fruit drop % 3602.82** 15.66 132.31 ** 129.91 ** 8.321 7.86 fruit weight (fw) 218.80 ** 1.401 1.37 ns 3.25 ** 0.865 10.47 fruit length (fl) 49.5 ** 1.58 2.02 ns 4.92 ** 1.49 4.28 fruit diameter (fd) 217.74 ** 1.77 0.99 ns 3.56 * 1.42 5.22 fruit volume (fv) 265.44 ** 0.80 1.30 ns 3.68 ** 1.04 11.71 fruit volume (fv) 120.98 ** 1.573 1.302 ns 19.73 ** 0.957 11.22 seed weight (sw) 0.308 ** 0.004 0.016 ** 0.024 ** 0.003 7.58 seed length (sl) 0.14 ns 3.135 1.389 ** 1.308 ** 0.402 3.21 seed diameter (sd) 2.86 ** 0.41 0.435 ns 0.918 ** 0.250 5.46 seed volume (sv) 3.10 ** 0.076 0.165 ns 0.185 ns 0.113 23.28 pulp weight (pw) 206.23 ** 1.20 1.001 ns 2.82 ** 0.817 11.1 pw/sw ratio 129.71 ** 0.563 2.201 * 0.389 ns 1.018 9.45 fl/fd ratio 0.273 ** 0.003 0.0028 ns 0.0007 ns 0.002 3.87 202 adv. hort. sci., 2023 37(2): 197­208 ghannami pollen that recorded the second­highest fruit drop also caused one of the lowest percentages of normal fruit set among all treatments (35.9%) with moderate parthenocarpy (23%). overall, we conclud­ ed that the zahedi+jarvis pollen treatment followed by the mazafati pollen respectively as the best and the second­best pollen treatments for the improvement of normal fruit set in tc­derived ‘berhi’ dates. bunch weight and bunch ripeness percentage investigating bunch characteristics showed that pollen source affected (p≤0.01) bunch weight at tamar and khalal stages as well as the percentage of bunch ripeness. means comparison for these traits has been depicted in figure 5. the jarvis+ghannami treatment caused the highest percentage of bunch ripeness with an average of 92.98%. the lowest bunch ripeness was recorded with gantar and jarvis+gantar sources indicating a significant delaying effect of gantar pollen on fruit maturation and bunch ripeness compared to other pollen sources. the zahedi+gantar treatment yielded the tamar bunches with an average of 6 kg which were heavier than those of all other treatments (p≤0.05), followed by the mazafati pollen that yielded the bunches car­ rying averagely 4.58 kg fruits (fig. 5). gantar pollen appeared as the poorest source since it produced bunches with averagely 0.88 kg of fruits. measurement of bunch weight at the khalal stage (in the second year) showed four pollen treatments including mazafati (3.49 kg), zahedi+jarvis (3.11 kg), jarvis+gantar (3.02 kg), and jarvis+mazafati (2.82 kg) were the top­yielding treatments. the poorest pollen sources in khalal’s bunch weight were gantar (0.98 kg) and zahedi+ghannami (0.97 kg). fruit and seed characteristics combined anova showed that the year factor affected (p≤0.01) all the khalal fruit and seed charac­ teristics except seed length (table 1). pollen sources could not affect fruit physical characteristics signifi­ cantly (tables 1 and 2), but seed weight and length were affected (p≤0.01) (table 1). in addition, the pollen source influenced the ratio of pulp weight to seed weight at p≤ 0.05 (table 1). the means for the fruit and seed characteristics of the normal fruits at the khalal stage have been presented in table 2. as the table shows, though pollen sources have influ­ enced these characteristics and some sources have improved them, the differences among the pollen sources have not been significant (p≤0.01) in anyone. as illustrated in figure 6, the mean comparison for seed weight showed that the lightest seed was pro­ duced with zahedi+ghannami pollen (0.66 g) though not significantly different from that of zahedi+jarvis and gantar sources. the smallest seeds were obtained with zahedi+ghannami pollen (18.7 mm long) and the longest ones with jarvis+mazafati source (20.3 mm). the highest weight ratio of fruit pulp to seed (11.94) was obtained in the fruits pro­ duced through pollination with the zahedi+jarvis pollen treatment. fruit phytochemical characteristics anova revealed that all measured qualitative traits of the tamar fruits were affected by pollen sources (p≤0.01). in khalal fruits, however, pollen sources affected tss, reducing sugars and total sug­ ars at p≤0.01 and titratable acidity, ph, dry matter, fig. 5 ­ means comparison of bunch ripeness and bunch weight (at tamar and khalal stages) for the ten pollen sources used. columns having the same letter(s) do not show a significant difference from each other using duncan's multiple range test at p≤0.05 (g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi). rastgoo et al. ‐ fruiting improvement in tissue cultured date palm 203 and moisture content at p≤0.05. in tamar fruits, tss ranged from 52 to 64 percent so the highest value was obtained by ghannami pollen and the lowest with zahedi+jarvis pollen source (table 3). fruit juice ph was in the range of 6.58 to 6.87. the lowest ph belonged to the fruits set by ghannami pollen (6.58) the same source that earned the highest tss. the highest titratable acidity (0.028%) was recorded with the zahedi+jarvis pollen treatment. total sugars ranged from 38.86 to 53.74 percent, the highest contents obtained by zahedi+mazafati and jarvis+ghannami pollen treat­ ments (>53%), and the lowest remained for zahedi+ghannami pollen source (38.68%). the con­ tent of reducing sugars was significantly the highest with the jarvis+ghannami pollen treatment (46.5%). application of zahedi pollen in combination with either ghannami, mazafati, or jarvis pollens caused maximum pulp dry matter (~88%) and minimum moisture content (11­12%). this is while the lowest dry matter (76.9%) and the highest moisture content (23.1%) were recorded in the fruits produced by zahedi+gantar pollen treatment. in khalal fruits, tss ranged from a maximum of 34.03% induced by jarvis+gantar pollen to a mini­ mum of 21.77% made by gantar pollen (table 4). fruit juice ph was not different among most of the pollen sources used (p≤0.05). titratable acidity showed a narrow range so the lowest value (0.47%) was for the zahedi+jarvis source and the highest one (0.58%) for the zahedi+ghannami pollen treatment. while the highest amount of total sugars was record­ ed with zahedi+mazafati pollens as well as with jarvis+ghannami pollen treatment (53.74% and 53.55%, respectively), the maximum amount of fig. 6 ­ mean comparison of seed weight, seed length, and pulp weight/seed weight ratio for the ten pollen sources used at the khalal stage. means were calculated based on the two years data. columns having the same letter(s) do not show a significant difference from each other using duncan's multiple range test at p≤0.01 (g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi). table 2 ­ mean comparison of fruit and seed characteristics for the ten pollen sources used. means were calculated based on the data from the two year g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi means in each column are statistically the same at p≤ 0.01 by dmrt. pollen sources fruiting characteristics fruit weight (g) fruit length (mm) fruit diameter (mm) fruit length/ fruit diameter fruit volume (cm2) seed diameter (mm) seed volume (cm3) pulp weight (g) g 8.40 28.4 22.4 1.28 8.20 9.12 0.61 7.67 gh 9.17 28.4 23.0 1.23 8.77 9.35 0.88 8.38 m 9.58 28.8 22.9 1.27 9.25 9.33 0.68 8.78 j+g 8.92 28.4 23.2 1.23 9.25 8.92 0.81 8.16 j+gh 8.65 28.3 22.6 1.26 8.37 9.37 0.84 7.86 j+m 9.28 29.2 22.9 1.28 8.95 9.43 0.82 8.45 z+g 9.03 29.3 23.1 1.27 8.72 9.10 0.83 8.30 z+gh 7.88 27.3 22.0 1.25 7.80 8.62 0.68 7.37 z+m 8.90 28.6 23.4 1.22 8.98 9.48 0.70 8.12 z+j 9.01 29.1 23.0 1.27 a 8.88 9.07 0.77 8.32 adv. hort. sci., 2023 37(2): 197­208 204 reducing sugars (46.50%) was obtained only with jarvis+ghannami source. mazafati pollen caused the highest pulp dry matter (26.4%) and the lowest pulp moisture (73.6%). the highest fruit moisture was made by zahedi+gantar pollen (80.9%). 4. discussion and conclusions in the present study, different pollen sources revealed various potentials for the improvement of normal fruit set, fruit quantitative and qualitative characteristics, and the bunch yield. the results approved that certain pollen sources were more effective in reducing the problems of abnormal fruit setting. the pollen sources also influenced fruit physi­ cal characteristics and bunch yield variably, some with promising performance in tree yield. the effect of pollen source on the fruit set and fruit traits was variable between the two years of the study suggest­ ing a significant interaction with climatic factors. screening pollen sources for improved fruit yield and table 3 ­ mean comparison for some fruit qualitative characteristics of tc­derived ‘berhi’ date palms at tamar stage pollinated by 10 pollen treatments pollen sources fruit quality characteristics tss (%) ph titratable acidity (%) total sugars (%) reducing sugars (%) dry matter (%) moisture (%) g 59.3 cde 6.85 a 0.012 cd 45.47 cd 36.42 bc 85.6 abc 14.4 cde gh 64.0 a 6.58 c 0.014 cd 47.18 bc 35.63 c 81.9 cd 18.1 bc m 61.5 abc 6.63 bc 0.016 c 42.73 de 38.81 b 80.6 de 19.4 ab j+g 54.9 f 6.70 abc 0.011 d 41.11 ef 31.11 d 83.8 bcd 16.2 bcd j+gh 62.5 ab 6.60 bc 0.013 cd 53.55 a 46.50 a 86.9 ab 13.1 de j+m 56.9 def 6.86 a 0.021 b 44.85 cd 35.62 c 86.3 abc 13.7 cde z+g 59.0 cde 6.77 abc 0.013 cd 44.32 cd 37.15 bc 76.9 e 23.1 a z+gh 56.5 ef 6.85 a 0.022 b 38.86 f 32.84 d 88.1 ab 11.9 de z+m 60.0 bcd 6.87 a 0.022 b 53.74 a 38.11 bc 88.7 a 11.3 e z+j 52.0 g 6.79 ab 0.028 a 49.59 b 36.83 bc 88.1 ab 11.9 de g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi means in each column are statistically the same at p≤0.01 by dmrt. table 4 ­ mean comparison for some fruit qualitative characteristics of tc­derived ‘berhi’ date palms at khalal stage pollinated by 10 pollen sources g= gantar, gh= ghannami, m= mazafati, j= jarvis, z= zahedi means in each column are statistically the same at p≤0.01 by dmrt. pollen sources fruit quality characteristics tss (%) ph titratable acidity (%) total sugars (%) reducing sugars (%) dry matter (%) moisture (%) g 21.77 e 5.74 c 0.55 ab 20.63 c 15.00 d 22.7 abc 77.3 abc gh 30.17 bc 5.83 abc 0.57 a 29.55 a 18.19 abc 20.3 c 79.7 a m 27.57 bc 5.90 abc 0.49 bc 26.23 b 17.93 abc 26.4 a 73.6 c j+g 34.03 a 6.02 ab 0.48 c 26.51 b 16.46 bcd 23.2 abc 76.8 abc j+gh 30.57 b 5.82 abc 0.55 ab 28.05 ab 18.61 ab 20.5 c 79.5 a j+m 29.33 bc 5.89 abc 0.51 abc 28.69 ab 18.89 a 21.8 bc 78.2 ab z+g 26.87 cd 5.80 bc 0.51 abc 26.61 ab 18.20 abc 19.1 c 80.9 a z+gh 28.33 bc 5.99 ab 0.58 a 27.65 ab 18.25 abc 21.0 bc 79.0 ab z+m 23.9 de 5.86 abc 0.56 a 22.74 c 16.13 cd 21.6 bc 78.4 ab z+j 30.87 b 6.04 a 0.47 c 28.38 ab 17.93 abc 25.3 ab 74.7 bc rastgoo et al. ‐ fruiting improvement in tissue cultured date palm 205 quality has been done earlier for some horticultural crops (fattahi et al., 2014; kuroki et al., 2017 ) includ­ ing date palm (omar and el­abd, 2014; mohammadi et al., 2017; soliman et al., 2020). fruit set characteristics the results unveiled that the mazafati pollen was the only pollen source that could set normal fruits averagely beyond 50%. other tops were zahedi+jarvis and zahedi+gantar sources both with about 49% normal fruit set. previously, mohammadi et al. (2017) announced zahedi pollen as the most promising pollen source with an average of 50.1% normal fruit set for low fruiting tc­derived ‘berhi’ date palms. our results are following theirs confirm­ ing the desirable potential of zahedi pollen in improving normal fruit set in such trees. however, in our work, the percentage of survived parthenocarpic fruitlets was less than 20% in all three superior pollen treatments. the zahedi+jarvis pollen treatment caused reduced parthenocarpic fruitlets to 10% whereas mohammadi et al. (2017) reported 32% parthenocarpy for their top pollen treatment (zahedi pollen). this outstanding reduction in the percentage of parthenocarpy was achieved by performing com­ bined pollination consisting of two elite pollen sources simultaneously i.e. zahedi and jarvis. comparing the singular and combined applications of pollen sources discloses that singular pollination with the gantar source yielded the lowest percentage of normal fruit set but it behaved variably when it was applied in combination with each of the two promi­ nent pollen sources; zahedi and jarvis. these two male sources had been known as superior sources from our previous work (mohammadi et al., 2017). the combined application of gantar pollen with jarvis pollen improved the normal fruit set slightly while its combined application with zahedi pollen achieved the third rank, not significantly different from the first and the second ranks of pollen treatments in this parame­ ter. regarding ghannami pollen, its application in combination with jarvis source as well as with zahedi source improved the normal fruit set compared to its singular application. in the case of mazafati pollen, the opposite results were observed compared to the gantar and ghannami source’s behaviors. singular application of mazafati pollen performed better in normal fruit set than its combined application with each of the jarvis and zahedi pollens. having looked from another point of view, those pollen treatments that recorded the highest figures for normal fruit set i.e. mazafati, zahedi+jarvis, and zahedi+gantar, though caused a lower percentage of parthenocarpy compared to their corresponding fruit drop percentages but acted differently in the values of these two traits. this means that a more favorable fruit set was obtained with zahedi+jarvis pollen treatment, which reduced the surviving partheno­ carpic fruits to a minimum rate at the cost of a high rate of fruit drop. this is while the other two superior pollen sources (mazafati and zahedi+gantar) reduced fruit drop more, instead, they added propor­ tionally to the rate of parthenocarpy. since the dropped organs have mainly consisted of the unfertil­ ized flowers and the parthenocarpic fruitlets, a high­ er percentage of fruit drop accompanied by a lower rate of surviving parthenocarpic fruits allows the nor­ mal fruits on the bunch to receive more metabolites. this situation is more beneficial as it would result in improvement of the fruit’s physical and phytochemi­ cal characteristics, ultimately leading to an improved bunch weight with more marketable fruits. therefore, the zahedi+jarvis pollen source was con­ cluded as the best pollination treatment for the improved fruit set. various factors have been sug­ gested to cause variation in date fruit set such as pollen viability, growth of the pollen tube, or fertiliza­ tion (zaid and de wet, 2002 b), differences in com­ patibility barriers (al­obeed and abdul­rahman, 2002), and compatibility levels between pollen vari­ ety and female tree (mohammadi et al., 2017). it has been shown that growth­promoting phyto­ hormones play a significant role in pollen germina­ tion and pollen tube growth (kojima, 2005), the processes that are initially critical for successful ovule fertilization leading to a normal fruit set. the hor­ monal content of pollen grain can play a key role in this context. in date palms, parthenocarpic fruit growth is mostly triggered by hormonal imbalance in certain tissues. auxins (iaa), gibberellins (ga3), and abscisic acid (aba) have been considered the major hormones in parthenocarpic fruits (al­dinar et al., 2021). pollens from various male sources possess dif­ ferent hormonal profiles. this variability can be attributed to the genetic makeup of pollen­producing mother plants and the environmental conditions in which the male palms grow and produce pollens. shahsavar and shahhosseini (2021) found the hor­ monal content of the pollen grains from different male sources variable. they reported the highest amounts of cytokinin (zeatin) in jarvis pollen, gib­ berellin in pollens of zahedi and jarvis males, and adv. hort. sci., 2023 37(2): 197­208 206 auxins in fard and zahedi pollen sources. interestingly, in our study, one of the highest figures of normal fruit set was achieved with the combined application of pollens of zahedi and jarvis males, the two sources that contained the highest amounts of growth­promoting phytohormones in the report of shahsavar and shahhosseini (2021). it is well known that though in vivo germination of pollen grain on the stigma is critical for the process of normal fruit set, it must be followed by healthy directional growth of a pollen tube in the style to deliver the male gametes to the embryo sac (salomón­torres et al., 2021). bunch characteristics the results indicated that by choosing a more compatible pollen source, the bunch weight (both khalal and tamar stage) could be improved. for tamar bunch weight, zahedi+gantar pollen treat­ ment was seen as the top source. on the other side, this treatment showed about 72% ripened fruits per bunch at the time of bunch harvest whereas four other treatments showed bunch ripeness of over 80%, the highest belonging to the jarvis+ghannami treatment. therefore, according to our results, for the production of maximum tamar fruits in tc­ derived ‘berhi’ palms, pollination by zahedi+gantar pollen source is recommended but the harvest time should be postponed probably for two more weeks to get maximum tamar fruits. for khalal bunch weight, mazafati, zahedi+jarvis, and jarvis+gantar sources produced the weightiest bunches (3­3.5 kg). through these pollen treatments khalal bunch weight was doubled and even tripled compared to the other pollen sources like gantar pollen. khalal bunch weight in offshoot­originated berhi palms has been reported up to about 20 kg (abd­elhaleem et al., 2020). this indicates that more research is need­ ed to optimize the pollination issue for tissue­cul­ tured berhi palms. fruit and seed characteristics pollen sources used in this study manifested xenic as well as metaxenic effects. the zahedi+jarvis pollen treatment gained the most superior place in the ratio of pulp to seed weight. this source also produced fruits with acceptable dimensions, causing one of the lowest seed weights. previously, mohammadi et al. (2017) obtained the highest value for the weight ratio of pulp to seed by jarvis pollen in tc­derived ‘berhi’ trees. several reports have confirmed the effect of different pollinizers on the fruit characteris­ tics of dates (helail and el­kholey, 2000; awad and al­qurashi, 2012; rezazadeh et al., 2013). it seems variation in the performance of pollen sources in fruit and seed characteristics can be attrib­ uted to the differences in content and composition of the phytohormones that are synthesized within the embryo. as the genome of each pollen source is dif­ ferent from the other sources, the genetic make­up of the embryo produced by each pollen source would be different compared to the others. this distinction in seed genetics would cause the synthesis of a unique hormonal profile by the embryo that conse­ quently will regulate seed and fruit characteristics (shafique et al., 2011). in a study on the hayany date cultivar, it was reported that different pollen sources change the amounts of various phytohormones such as auxin and gibberellins at various stages of fruit development. it was also indicated that larger fruits were obtained by the pollen sources that had higher gibberellin content comparatively (el­hamady et al., 2010). shahsavar and shahhosseini (2021) empha­ sized that pollen source has a key role in fruit growth and development with a special focus on the hor­ monal content of each pollen source. overall, considering the highest values in the nor­ mal fruit set and pulp­to­seed ratio, the zahedi+jarvis source was realized as the best pollination treatment for the production of khalal fruits. we suggest zahedi male as an elite pollen source to be included in future works aiming to improve the yield of low­fruit­ ing tc­derived ‘berhi’ date palms. earlier, improve­ ment in the yield of the ‘berhi’ cultivar was reported by the use of saki and maktoumy pollen sources (al­ obeed and abdul­rahman, 2002) and several iranian local male selections (rezazadeh et al., 2013). the exploitation of the potential of pollen source in other fruit and nut species such as hazelnut (fattahi et al., 2014) and japanese pear (kuroki et al., 2017) has been reported earlier. fruit quality pollen source affected fruit quality significantly both at khalal and at tamar stages. these results are compatible with those reported earlier in date (awad and al­qurashi, 2012; shafique et al., 2011) and in fruit species such as mandarin (wallace and lee, 1999), and fig (gaaliche et al., 2011) confirming the critical role of pollen source in fruit quality. however, there are also reports that state the influence of pollen sources on some chemical traits of date fruits is low (awad and al­qurashi, 2012; rezazadeh et al., 2013). this variation in the findings may indicate that other factor(s) such as crop load, local weather con­ http://www.sciencedirect.com/science/article/pii/s0304423817300298 rastgoo et al. ‐ fruiting improvement in tissue cultured date palm 207 ditions, soil fertility, irrigation, etc. can also influence the fruit quality. in the tamar stage, we observed the highest tss in the fruits set by the ghannami pollen, the pollen source that caused one of the lowest bunch weights. this indicates a reverse relationship between crop load and tss. such a relationship was more apparent when we realized that the zahedi+jarvis pollen source that induced one of the top rates in normal fruit set and bunch weight (both at tamar and khalal stage), possessed the lowest tss value and the high­ est amount of ta. the results also indicated that the zahedi pollen tends to accumulate more dry matter in tamar fruits when it is used in combination with the other pollen sources but the gantar source. overall, zahedi+jarvis pollen treatment was conclud­ ed as the most preferable pollen treatment for the quality of fruit at the tamar stage. it brought moder­ ate sweetness (concerning amounts of tss, titratable acidity, total and reducing sugars) and drier pulp tis­ sue. concerning the quality of khalal fruits, ghannami and jarvis+ghannami pollen sources appeared as the best pollen treatments. they caused the highest amounts in tss, ta, total sugars, reducing sugars, and the moisture content, which are impor­ tant in making the khalal fruits sweeter, tastier, crispier, and more palatable to the consumer. zahedi+jarvis pollen treatment can also be consid­ ered as another candidate treatment though the fruits set by this treatment were slightly less crispy. the results of the present study proved that the fruit set and yield are affected differently in different years probably due to variations in environmental conditions. however, based on the average means of the two experimental years, the combined applica­ tion of zahedi and jarvis pollens yielded the most desired results. this pollen treatment obtained about 50% normal fruit set with significantly the lowest parthenocarpy (10%). it also brought about the high­ est ratio of fruit pulp to seed weight. this pollen treatment was seen as the top pollen source in khalal’s bunch weight. although the zahedi+gantar pollen source was superior in tamar’s bunch weight, the presence of zahedi pollen in this latter pollen treatment indicates the potential of the zahedi source over other pollen sources used in the study. regarding fruit quality, ghannami, jarvis+ghannami, and zahedi+jarvis were the pollen treatments that produced khalal fruits with higher quality indices. however, for the tamar fruit quality, only zahedi+jarvis pollen treatment was chosen as it induced the fruits with moderate sweetness and drier pulp. overall, considering all fruiting and fruit quality indices, the zahedi+jarvis pollen treatment can be drawn as the most desired pollination treatment for low­fruiting tc­derived ‘berhi’ date palms. our results revealed that pollination with elite pollen sources in combination could emerge as a more successful polli­ nation strategy over the singular application of differ­ ent pollen sources. in addition, the results suggest that relevant future pollination studies utilize the zahedi pollen source as a potent male source in com­ bination with other elite pollen sources. acknowledgements the authors would like to thank bushehr agricultural and natural resources research center and also its date palm and tropical fruits research station for assigning experimental trees, and provid­ ing the pollen sources and field workforce. we appreciate persian gulf university for providing the required lab facilities. references abd­elhaleem s.a.m., abd el­latif f.m., el­badawy h.e.m., el­gioushy s.f., abdalla b.m., 2020 ­ impact of pollen grain sources on productivity and fruit charac‐ teristics of date palm (phoenix dactylifera l.) cv. barhi. ­ asian j. res. bot., 3(2): 45­48. ali­dinar h., mohammed m., munir m., 2021 ­ effects of pollination interventions, plant age and source on hormonal patterns and fruit set of date palm (phoenix dactylifera 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statistics. version 8.0 e. ­ sas inst., inc., cary, north carolina, usa. shafique m., khan a., malik a.u., shahid m., rajvana i.a., saleem b.a., amin m., ahmad e., 2011 ­ influence of pollen source and pollination frequency on fruit drop, yield and quality of date palm (phoenix dactylifera l.) cv. dhakki. ­ pak. j. bot., 43: 831­839. shahsavar a.r., shahhosseini a., 2021 ­ pollen grain hormones of date palm pollinator cultivars and their relationship with hormones of different stages of ‘piarom’ date fruit growth. ­ sci. hortic., 288: 110389. soliman s.s., kassem h.a., al­obeed r.s., jemni m., 2020 ­ influence of pollen source and its mineral con‐ tent on fruit retention and quality of kadary date palm cultivar (phoenix dactylifera l.). ­ j. chem. biol. physic. sci., 10(3): 437­446. wallace h.m., lee l.s., 1999 ­ pollen source, fruit set and xenia in mandarins. ­ j. hortic. sci. biotechnol., 74(1): 82­86. zaid a., de wet p.f., 2002 a ­ botanical and systematic description of the date palm, pp. 1­28. ­ in: zaid a., and e.j. arias­jimenez (eds.) date palm cultivation. fao plant production and protection papers no. 156, rev.1, rome, italy. zaid a., de wet p.f., 2002 b ­ pollination and bunch man‐ agement, pp. 145­175. ­ in: zaid a., and e.j. arias­ jimenez (eds.) date palm cultivation. fao plant production and protection papers no. 156, rev. 1, rome, italy. https://www.fao.org/faostat/en/#data/qcl impaginato 313 adv. hort. sci., 2020 34(3): 313­323 doi: 10.13128/ahsc­8118 optimization of biosolids as a substrate for tomato transplant production p.c. otieno (*), s. nyalala, j. wolukau department of crops, horticulture and soils, egerton university, po box 536‐20115, egerton, kenya. key words: biosolids, quality, seedling growth, solanum lycopersicum l., sub­ strate, tomato. abstract: the need to recycle waste and increasing pressure against peat extraction and importation, have led to increasing interest in substituting peat with organic wastes. use of biosolids substrate would be a low cost alternative substrate to peat for commercial production of transplants. the objective of this study was to determine the effect of biosolids­forest soil mixture ratios on tomato ‘maxim f1’, transplants emergence and growth. a randomized com­ plete block design with four replications was used in this study. the treatments were: biosolids (bs) mixed with forest soil (fs) at rates of 0% 10%, 20%, 30%, 40%, 50% and 60% (v/v), tea compost (tc) and coco peat (cp). five tomato seeds were planted in four 250 cm3 pots, grouped into four to form an experi­ mental unit. results showed that biosolids (bs) at rate of 30% registered signifi­ cantly (p<0.05) higher seedling emergence (94%), leaf numbers (4.5), height (16.5 cm), collar diameter (6.3 mm), chlorophyll content (25 index units), root volume (2.0 cm3) and root/shoot dry matter (10.2 % and 16.3%, respectively) than the rest of the substrates except tea compost (tc). sodium was significant­ ly (p < 0.05) higher in bs at rates of 50% (350 mg kg­1) and 60% (376 mg kg­1) and this raised ec (4.5) and lowered ph of the media to 4.4. at 30% bs enhanced tomato transplant production to similar level as tea compost, hence recom­ mended for commercial use. 1. introduction tomato (solanum lycopersicum l.) can be established in the field by direct seeding or transplanting. tomato production by transplanting has been done over the past two decades to increase resource use efficiency and reduce environmental impact on seeds planted (restrepo et al., 2013). cultivation from transplants has many advantages including earlier harvest; more efficient use of land, time, energy, and seeds; and healthy and homogenous production (pascual et al., 2018). in comparison with direct sowing, transplanting is a more reliable method of ensuring higher plant survival, faster establishment, improved plant uniformity, early maturity, and reduced cost of production (gogo et al., 2012). the produc­ tion of tomato seedlings, especially in sub­saharan africa with great expansion of open field and greenhouse crops, is a highly competitive business. besides, uniform and rapid seed emergence and quality are (*) corresponding author: pcotieno@egerton.ac.ke citation: otieno p.c., nyalala s., wolukau j., 2020 ­ optimization of biosolids as a substrate for toma‐ to transplant production. ­ adv. hort. sci., 34(2): 313­323 copyright: © 2020 otieno p.c., nyalala s., wolukau j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 february 2020 accepted for publication 2 july 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 313­323 314 essential prerequisites to increasing tomato yield, quality, and profits (wachira et al., 2014). in addition, tomato seeds especially f1 hybrids are expensive and farmers in developing countries cannot tolerate poor germination as a result of poor soil conditions (hcd, 2017). use of ideal transplant substrates with appro­ priate physicochemical properties, is therefore criti­ cal (sterrett, 2001). in transplant production, the main purpose of a substrate is to satisfy the needs for good seedling growth within the limited space of a container and to prepare the seedlings for successful transplanting into the field (pascual et al., 2018). the quality of growing media is one of the main factors influencing the success of horticultural nursery activity (raviv and lieth, 2008), and it is also directly linked to the quality of the materials utilized in growing media for­ mulations (reis and coelho, 2007). the choice of appropriate substrate is therefore an important fac­ tor in promoting the optimum growth of plants. a number of potential substrates have been identified, of which peat moss has long been the primary com­ ponent of transplant and potting media for both veg­ etable and ornamental plants. this has been mainly due to its physical and chemical properties (raviv et al., 1986): adequate free air space (fas) at 0­10 cm water suction; high water content at low tension at 10­100 cm water suction; and high cation exchange capacity (cec) minimizing loss of nutrients and facili­ tating adequate mineral nutrition (colla et al., 2007). however, peat also has some notable disadvantages; being conducive for the development of some soil­ borne plant pathogens such as pythium and rhizoctonia (hoitink and kuter, 1986). furthermore, peat moss is normally harvested from wetland ecosystems at rates considered non­sustainable by wetland ecologists (buckland, 1993). these draw­ backs have motivated horticulturists throughout the world to seek alternatives like coir (coco peat) which has several qualities: high water­holding capacity, excellent drainage, absence of weeds and pathogens, renewable resource, with no ecological drawbacks to its use, acceptable ph, cation exchange capacity (cec) and electrical conductivity (ec) and easier wet­ tability (cresswell, 1992). under nursery conditions, coco peat and peat moss have been used as reliable media for organic production of lettuce transplants (colla et al., 2007). however, coco peat has become more expensive and its properties are more variable (chrysargyris et al., 2013). thus, it is important to look for high quality, locally available and low­cost alternative substrates. among the organic substrates for transplants pro­ duction, vermicompost is a promising substitute for peat especially in the production of seedling, but not a sustainable solution for management of organic wastes (ivanka and tsvetanka, 2012). use of biosolids from treated sewage, has been proven to be promis­ ing (vyas, 2011; giannakis et al., 2014). the effects of biosolids on seedling emergence and growth have been investigated by chrysargyris and tzortzakis (2015) and their results indicated that application of boisolids as a substrate in marigold (tagetes erecta l.) and basil (ocimum basilicum l.) seedlings produc­ tion has potential. similarly, use of organic urban waste compost for tomato (solanum lycopersicum l.) transplant production has been reported to result in quality transplants in the seedbed (herrera et al., 2008). chrysargyris and tzortzakis (2015) specified biosolids as an ideal component of mixed­peat sub­ strates for eggplant (solanum melongena l.) seedlings, at a rate less than 30% in a substrate mix­ ture. in another study on cucumber transplants pro­ duction, mami and peyvast (2010) recommended the use of biosolids at 5% and below on peat mixture. however, the use of biosolids as substrates depend­ ing on the ratios may have negative effects as a con­ sequence of its high salt content, unsuitable physical properties (texture, structure, moisture content, porosity etc.), heavy metal toxicity, and variable qual­ ity and composition (papamichalaki et al., 2014). the appropriate amount of biosolids added in growth medium needs to be determined to improve plant growth. therefore this study investigated the the effect of biosolids­forest (bs: fs) soil mixing rates on tomato transplant emergence and growth. 2. materials and methods site description this study was conducted in two trials at the horticulture research field, egerton university, kenya during january to february and march to april, 2018. the site is located on latitude 0 23’ s and longi­ tude 35 35’ e in the lower highland iii (lh3) agro eco­ logical zone at an altitude of 2238 m above sea level (jaetzold et al., 2012). the experiments were done in an area measuring 1.2 m by 3.5 m within a plastic greenhouse size 8 m by 60 m and a height of 3 m. the greenhouse covering material was uv stabilized polythene sheet gauge 150 μm from amiran, co ltd nairobi kenya. greenhouse microclimatic condition, temperature and relative humidity averages were as otieno et al. ‐ biosolids as an alternative substrate in tomato 315 follows; day (6:00 am­6:00 pm) and night (6:00 pm­ 6:00 am) air temperatures inside the greenhouse during the experiment were 24.5±0.9°c and 13.3±4°c, respectively. average day and night rela­ tive humidity inside the greenhouse were 55±6% and 80±6%, respectively. biosolids and forest soil sample collection, substrate preparation and analysis biosolids (bs) were collected from a lagoon pond at egerton university wastewater treatment plant and forest soil obtained from typically tropical forest. the substrates for transplants production were pre­ pared by mixing the biosolids and forest soil (bs: fs at rates of 0, 10, 20, 30, 40, 50 and 60% (v/v). samples from each rate, tea compost (tc) and coco peat (cp) as reference commercial substrates were comprehensively analysed in a laboratory to deter­ mine the physico­chemical characteristics of the sub­ strates (table 1). porosity of each substrate was cal­ culated from the ratio of the determined bulk density and of known particle density (2.65 g cm­3) as given in the equation given below (okalebo et al., 2002): porosity (%) = 1 ­ (bulk density /particle density) × 100). experimental set up and design the experimental design was randomized com­ plete block design (rcbd), replicated four times. the treatments included seven bs: fs soil mixtures at dif­ ferent rates and two commercial substrates tc and cp. in the experiment, plastic pots (250 cm3) were used for potting the substrates. an experimental unit composed of four pots, each planted with four toma­ to ‘maxim f1’ seeds. transplants establishment and irrigation schedule tomato seeds were planted in the pots in the evening and substrates watered to saturation point. after 24 hours each substrate was irrigated with 15 ml of water after every 12 hours for the first 15 days. the volume of water was increased to 20 ml for the next 10 days, then 25 ml for the remaining days of the experiment. determination of seedling emergence and growth the number of emerging seedlings was recorded. based on the number of planted seeds (20), seedling emergence percentages were computed progressive­ ly after 7, 9 and 11 days after planting (dap). germination percentage was determined using equa­ tion adopted by atif et al. (2016), with modification y recommended levels of nutrient in soil for tomato production according to sainju et al. (2003). z maximum ceiling values of heavy metals for agricultural land application according to nsw epa (2000). table 1 ­ physico­chemical characteristics of the substrate used for tomato transplant production characterization/substrates fs bs 10% bs 20% bs 30% bs 40% bs 50% bs 60% tc cp bulk density (g cm­3) 1.7 1.6 1.5 1.3 1.3 1.3 1.3 1.3 1.2 porosity (%) 35.9 39.6 43.4 50.9 50.9 50.9 50.9 50.9 54.7 moisture content (%) 25.8 34 40.8 42.8 44.5 45.1 45.9 44.7 45.3 ec (ms m­1) 2.6 3.2 3.6 4.4 5.1 5.2 5.4 4.3 5.2 ph 7.4 6.2 6.6 6.5 6.4 5.6 5.4 7.4 7.4 organic matter (g kg­1) 157.7 197.8 196.7 210 209.8 220 222.9 207.2 171.4 c:n 21.3 19.7 15.4 9.6 12.7 14.7 12.5 7.6 10.8 total carbon (mg g­1) 91.7 115.0 114.4 122.1 122.0 127.9 129.6 120.5 99.6 total n (g kg­1) (0.1) y 4.3 5.9 7.4 12.9 9.6 8.9 10.5 16.3 9.2 total p (mg k g­1) (70) y 69.1 83 90.3 101 95.9 79.3 70.3 116.1 33.8 k (mg kg­1) (700) y 132.5 412.3 419.9 427.8 422.4 403.7 403.5 369.6 344.1 ca (mg kg­1) (1000) y 21.9 24 22.8 29.5 27 28.5 27.5 43.5 38.5 mg (mg kg­1) (700) y 131.1 126.1 117.7 119.1 113.8 47.7 37.2 126.6 114.6 na (mg kg­1) 62.9 254.8 342.1 252.8 348.3 349.8 376.3 114.8 164.4 mn (mg kg­1) (20) y 69.6 530.4 524.8 539.4 553.9 551.9 544.8 167 29.8 fe (mg kg­1) 27 2490 2473.9 2479.1 2471.5 1184.1 852.5 207.4 114.1 zn (mg kg­1) 4.7 47.4 44 44 45.9 24.4 25.4 21.9 16.4 cu (mg kg­1) (100) z 4.4 12.2 12.7 10.3 12.7 13.1 13.3 14 6.5 cd (mg kg­1) (1) z 0.0023 0.0128 0.0115 0.0117 0.0122 0.0122 0.0122 0.0122 0.0121 pb (mg kg­1) (150) z 109.6 2.8 2.1 5.1 3.1 6 2.5 20.1 4.3 adv. hort. sci., 2020 34(3): 313­323 316 as given below: g (%) = (s2 / s1) x 100 where g is the germination percentage, s1 is total seeds planted and s2 seeds germinated. ten tomato seedlings were randomly selected and tagged for data collection on growth parameters. seedling height, collar diameter, leaf number and leaf chlorophyll content were determined 14, 21 and 28 dap. seedling height was determined using a measuring tape from the ground level to the tip of the seedling. for stem diameter, a stainless hardened 150 mm lcd electronic digital vernier mark (grainger, usa) was used. the unit of measurement was millimeters (mm). the stem diameter was mea­ sured on the main stem of the plant at 1 cm above the substrate. number of leaves was determined by counting the true leaves. determination of transplant leaf chlorophyll content this was determined using a chlorophyll content meter (ccm­200) plus; opti­sciences, tyngsboro, ma). estimate of chlorophyll content was in chloro­ phyll concentration index units (ccls). three readings of chlorophyll content were taken on the third newly developed leaflet from the top of each tomato plant and means were computed for each replication. the leaf chlorophyll was measured using spad chloro­ phyll meter (minolta spad502 meter, tokyo, japan). pengfei (2017) reported that spad values have a direct linear relationship with extracted leaf chloro­ phyll therefore, spad value was used to describe leaf chlorophyll index units (ccls) in the current study. determination of root volume and root/shoot dry weight during seedling harvesting on the fourth week (28 dap), four seedlings were randomly selected and carefully uprooted. the roots were washed clean in running tap water on a sieve of pore diameter of 1 millimeter. separation of tomato transplant roots and shoot was done at the crown level. root volume, was determined by scanning plant roots using epson expression 10000xl color image scanner and ana­ lyzed using winrhizo software (la 2100­regent instruments inc.) as described by mwamlima et al. (2019). separated shoot and root plant parts were dried in an oven to constant weights at 60°c for 24 h as described by hossain et al. (2008). mean weights of dried samples were taken as shoot and root biomass per plant. the roots and the shoots of the randomly selected four plants were also used to determine dry weight. this was done by oven drying the roots and shoots of the seedlings at 105ᵒc until constant weight was achieved. percentage dry root and shoot weight was then computed based on the initial fresh weight according to equation below (atif et al., 2016): rdma (%) = dw (g) / fw (g) x 100 where rdma; root dry matter accumulation in per­ centage, dw; dry weight (g) and fw; fresh weight (g). data analysis data analysis was carried out using statistical package sas version 9.1 (sas institute, cary inc., 2001). data for the two trials were pooled and sub­ jected to analysis of variance (anova) at p≤0.05 and means for significant treatments separated using tukey’s honestly significant difference (hsd) test at p<0.05. the model fitted for the experiment was yij= μ + βi + αj + εij, where, yij = tomato response, μ = over­ all mean, βi = effect of the ith block, αj = effect of the jth level of substrates εij = random error term, i = 1, 2, 3, 4; j= 1, 2, 3 …9. 3. results seedling emergence the substrates tested influenced the emergence of tomato seedlings differently (fig. 1). tea compost (tc) and biosolids (bs) at 30% had the highest emer­ fig. 1 ­ effect of biosolids on emergence of tomato seedlings. means ± standard deviation followed by the same letter within a day after planting are not significantly different according to tukey’s hsd test (p<0.05). fs = forest soil; bs = biosolids; tc = tea compost; cp= coco peat. otieno et al. ‐ biosolids as an alternative substrate in tomato 317 gence percentage compared to the rest of the sub­ strates throughout the evaluation, while coco peat (cp) had the lowest emergence percentage. from day 7 to day 11 after planting, bs at 30% was signifi­ cantly (p<0.05) higher (90­95%) in seedling emer­ gence and this was not significantly (p<0.05) different from that of tc (commercial substrate). at day 7, the control forest soil (fs) was not different from cp (another commercial substrate). plant height biosolids (bs) application rates influenced tomato seedling height during the growing period. a part from bs at the rate of 30% and tc, which produced the tallest transplants, there were no significant (p<0.05) difference among the rates of 20%, 40% 50% and 60% in plant height. biosolids at 30% was consistently similar to tea compost (tc) in producing taller tomato seedlings 14, 21 and 28 days after planting (dap). however, the shortest plants were obtained with forest soil (fs) and coco peat (cp) (fig. 2). leaf number on tomato leaf number, bs at the rate of 30% was similar to tc in recording significantly (p<0.05) higher number of leaves per tomato plant throughout the period of the experiment (fig. 3). however, there was no significant difference between bs rates within the range of 10% to 40% on 14, 21 and 28 dap. the lowest tomato leaf number was obtained with fs and cp. biosolids at 50 and 60% resulted in significantly (p<0.05) lower number of leaves than bs at 30% on 14 and 28 dap. fig. 3 ­ effect of biosolids on tomato seedling leaf number. means ± standard deviation followed by the same letter within a day after planting are not significantly different according to tukey’s hsd test (p<0.05). fig. 2 ­ effect of biosolids on tomato seedling height. means ± standard deviation followed by the same letter within a day after planting are not significantly different accor­ ding to tukey’s hsd test (p<0.05). collar diameter application of biosolids at 30% resulted in the widest collar diameter of tomato seedlings through­ out the period of the experiment (fig. 4). at 14 dap, all the treatments except tea compost recorded sig­ nificantly (p<0.05) narrower collar diameter than bs at 30%. leaf chlorophyll content tomato transplants grown on biosolids at 30% had significantly (p<0.05) higher leaf chlorophyll con­ tent compared to the rest of the treatments (fig. 5). using coco peat (cp) resulted in transplants with the lowest chlorophyll content. however, there was no much difference in physical appearance of the leaf colour (plate 1). fig. 4 ­ effect of biosolids on tomato seedling collar diameter. means followed by the same letter within a day after planting are not significantly different according to tukey’s hsd test (p<0.05). 318 adv. hort. sci., 2020 34(3): 313­323 root volume tomato transplants root volume was affected by use of biosolids (fig. 6, plate 2). biosolids at 30% resulted in significantly (p<0.05) higher root volume than the forsest soil, coco peat and the other tested rates of bs. however, there was no significant differ­ ence in root volume between tomato transplants grown on biosolids at 30% and tea compost. root and shoot dry weight there was similar response of transplants to dif­ ferent substrates in terms of roots and shoot dry weight (fig. 7). transplants grown on biosolids at 20% or 30% had significantly (p<0.05) higher root dry weight, which was similar to that obtained with tea compost. in addition, biosolids at 30% and tea com­ post similarly recorded significantly (p<0.05) higher shoot dry weight than all the other treatments. forest soil and coco peat resulted in the lowest root and shoot dry weight. 4. discussion and conclusions the response of tomato transplant growth para­ meters to various rates of biosolids (bs) in this study depended on the physico­chemical characteristics of the substrate. although numerous authors have reported the beneficial effects of the addition of biosolids to peat mixes (herrera et al., 2008; mami and peyvast, 2010; chrysargyris and tzortzakis, 2015), limited number of studies, have reported the use of biosolids (bs) in forest soil (fs) mixture as a substrate. our results show that use of bs at 30% can support tomato transplant. this can be attributed to its characteristic of higher availability of plant nutri­ ents such as n, p, k, mg, fe, mn, b and mo (table 1). biosolids application in the tested rates served fig. 6 ­ effect of biosolids on tomato root volume. means ± stan­ dard deviation followed by the same letter are not signi­ ficantly different according to tukey’s hsd test (p<0.05). fig. 5 ­ effect of biosolids on tomato seedling leaf chlorophyll content. means ± standard deviation followed by the same letter within a day after planting are not significan­ tly different according to tukey’s hsd test (p<0.05). plate 1 ­ tomato leaf chlorophyll observed in biosolids bs at 30% and 40% compared to at cp (coco peat) substrate. leaf colour appea­ rance of tomato transplants grown on various substrates. otieno et al. ‐ biosolids as an alternative substrate in tomato 319 several purposes in the substrate. it improved the texture and water holding capacity, making condi­ tions more favorable for root growth and increased emergence on tomato seedlings. the application of bs at 30% also supplied nutrients essential for plant growth, including n, p and k and mg, as well as some essential micro nutrients like zn, fe, cu, b and mo (table 1). as reported by tzortzakis et al. (2012), nutrients in the biosolids offer several advantages over those in inorganic fertilizers because they are in organic form hence are released slowly to growing plants. moreover in organic form, nutrients are less water soluble and therefore least likely to leach into groundwater or run­off into surface waters. this is in agreement with the findings of zhao et al. (2010) in relation to the benefits associated with increased organic matter content in the soil. in line with this, shiralipour et al. (1992) earlier reported that organic matter contribute to increased nutrient, total pore space, aggregate stability, erosion resistance, tem­ perature insulation and reduced soil bulk density. these factors play a major role during germination and emergence of seedlings. soil temperature and moisture content equally play a critical role during germination and emergence of tomato seedlings (weaver et al., 1988). in concurrence with the pre­ fig. 7 ­ effect of biosolids on tomato seedling root and shoot dry weight at week 5 after planting. means ± standard devia­ tion followed by the same letter within root or shoot are not significantly different according to tukey’s hsd test (p<0.05). plate 2 ­ characteristics of tomato transplants root development in substrate tested, scanned from winrhizo, for determination of root volume and density. responses of roots development to different substrate, fs= soil control; bs= biosolids rates; tc= tea com­ post; cp = coco peat. adv. hort. sci., 2020 34(3): 313­323 320 sent study, findings by chrysargyris and tzortzakis (2015) revealed that biosolids enhance seed germi­ nation and emergence in eggplant transplants. the ability of biosolids to improve physical properties of a good media is related to increased organic matter content (zhao et al., 2010). in regards to the current study, tomato seeds are small and therefore require fine and light media, a rhizophere created with bs at 30%, which possibly enhanced germination and emergence of the seedlings. plant height, leaf number and girth of the seedling were highest in seedlings grown in bs at 30% and apparently not very pronounced in tc (fig. 3, 4). abdel­mawgoud (2007) reported plant growth and yield as a function of nutrients supply provided that all other conditions are met. in this study, there was clear positive trend of increasing plant height, leaf numbers with increased rates of bs. the results obtained with bs at 30%, may be attributed to its nutrient content as reported by otieno et al. (2019). enhancement of plant growth as a result of increas­ ing nutrients in organic amendments has been reported by sainju (2003). these results are in agree­ ment with the work of oyinlola and jinadu (2012), where nitrogen rates in the soil increased tomato plant height. the nutrients not only encourage vege­ tative growth but also enhance photosynthesis, chlorophyll density and plant root respiration which result in greater plant growth when applied (tan and binger, 1986). the findings of this study suggest that the opt imum rate of bs to use as so i l amendment should not exceed 30% for trans­ plant production. the difference between the bs at 30% and tc substrate seems to have been caused by the reduced level of k in the latter (table 2). potassium is an essential element during plant growth and development (ortas, 2013). since k is a vital element in many physiological processes, it may have been involved in transplant stem thickness. it is known that k plays a major role in physiological and biochemical processes such as enzyme activation; metabolism of carbohydrates and protein com­ pounds zhen et al. (1996). besides, k has a significant role to play in the plant energy status for storage of assimilates and tissue water relation. potassium is also needed in photosynthesis and the synthesis of proteins, hence its deficiency in plants will show as slow, stunted growth and in some crops, weak stems and lodging (uchida, 2000). application of bs especially at 30% enhanced leaf chlorophyll as indicated by higher chlorophyll con­ centration index units (fig. 5). one of the critical physiological developments responsible for seedling growth is photosynthesis. the quantity of chlorophyll per unit area is an indicator of photosynthetic capaci­ ty of a plant and this explains the better growth observed in tomato seedlings grown in bs at 30%. in other studies, zuba et al. (2011) and ilupeju et al. (2015), postulated that, the rates of organic amend­ ment applied in growing media were linked to the nutrient element levels in the substrate. in regards to this study, plant nutrient availability may have enhanced the amount of chlorophyll in the plants, as exhibited by the presence of mineral elements such as n, p, mg, fe and zn in biosolids in large quantities. these nutrient elements have been reported to be high in biosolids from organic part of municipal solid wastes (chrysargyris and tzortzakis, 2015). other studies have also reported that biosolids are able to increase nutrient availability in soils (shiralipour et al., 1992; xu et al., 2012). in a related study on egg­ plant seedlings production, chrysargyris and tzortzakis (2015) observed that leaf chlorophyll con­ tent increased with addition of organic solid waste and similar results were earlier observed by tzortzakis et al. (2012). the underground part is very important in trans­ plant life and determines whether it can survive when transferred to field environment or not. the roots in particular play a pivotal role in the plants life cycle (somkuwar et al., 2012; zhi et al., 2017). roots are also known to provide an important link between soils and plants (mcmichael et al., 2010; xi et al., 2013). furthermore, root systems have important physiological and biological functions for crop growth and yield (liang et al., 2003; yang et al., 2010). the ability of roots to develop perfectly depends on the medium or substrate status. root growth is linked to the physico­chemical properties and nutrient avail­ ability in a substrate. the ability of a plant to absorb water and mineral nutrients from the substrate depends on its capacity to develop an extensive root system. in the present study, bs at 30% substrate sig­ nificantly enhanced tomato transplant root growth and morphology (table 1). in tomato, the tap root formed at an early stage extends deeply into the soil followed by secondary and tertiary, then delicate root hairs, which require water and air among the three phases (solid­liquid­gas) of the substrate (manahan, 2000). the phases are very essential in water and plant nutrient absorption, based on poros­ ity of the media as demonstrated by the bs at 30%. otieno et al. ‐ biosolids as an alternative substrate in tomato 321 furthermore, the supply of o2 is essential for root growth and metabolism. generally, as roots grow through the soil they follow soil vailable pores and this is a contribution of the air space and the level of organic matter as evident in bs at 30%. this is also in agreement with abad et al. (2001) and pascual et al. (2018) who reported on the range of bulk density required for good root development in a substrate. biosolids at 30% was therefore identified as an ideal substrate, in terms of producing many fibrous and dense root systems than the rest of the substrates (plate 2 c, e). additionally, based on the porosity of the studied substrates, it appears that bs at 30% not only created air space for the root development to enhance nutrient use efficiency, but also availed organic matter, which is connected to higher water holding capacity (otieno et al., 2019). this is a critical factor for reducing irrigation schedule as in the case of the present study, making bs 30% a better sub­ strate than the rest. the result of this study also sug­ gests that the bs with rates as low as 10% may need frequent irrigation schedule. on the other hand, even though there was further increase in organic matter as the bs rates increased above 30%, increase in ec was observed (table 1). this normally has a profound effect on the plant function, especially in reverse osmosis, which may subsequently affect continuous water flow and transpiration in the plant, leading to retarded growth (mengel and kirkby, 2001). the enhanced shoot and root dry weight exhibited by bs at 30% was an indication of the potential of the bs as a soil amendment and its ability to improve the physico­chemical quality of the substrates for trans­ plant development. forest soil mixed with bs at 30% created room for root respiration and development. the plants had better chance for nutrient absorption hence increased dry matter compared to the other substrates tested. phosphorous which occurred in high quantities in bs at 30% is involved in the formation of energy rich compounds, including adenosine triphos­ phate and adenosin diphosphate which in turn derive various bio­chemical reactions within the plant (memon, 1996). as one of the vital plant macronutri­ ent, phosphorus plays a vital role in the root and shoot development and this contributed immensely to the subsequent increase in shoot biomass of plants grown in bs at 30%. biosolids analysis in this work also indi­ cated the presence of zn, cu, fe and mn in significant quantities especially in bs 30%. as advocated by atif et al. (2016), balanced presence of these essential micro elements may have promoted the growth of the seedlings in bs at 30%. these results are in agreement with work of reis et al. (2017), who observed that addition of biosolids in soil resulted in significant increase in total root and shoot dry weight of leptospermum scoparium in a pot experiment. furthermore, sainju et al. (2003) earlier reported that vigorous root growth stimulated by p helps in better utilization of water and other nutrients in the soil and promotes a sturdy growth of stem and healthy foliage which may subsequently contribute to roots and shoot dry matter. the results in this study demonstrated that applica­ tion of biosolids substrate was beneficial in the tomato transplants production. the influence of biosolids at 30% was significant and specifically on leaf number, plant height, chlorophyll content and root develop­ ment. it is therefore a potential high quality, locally available and low cost substitute for peat and coir substrates in transplant production. biosolids applied at moderate levels (30%) in forest soil mixture could not only improve the physic­chemical properties of the substrates but also reduce environmental pollution. acknowledgements the project was funded by world bank under the centre of excellence in sustainable agriculture and agribusiness management (cesaam) 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greenhouse cucumber phyllody in iran s.a. esmaeilzadeh­hosseini 1 (*), g. babaei 2, s. davoodi 2, a. bertaccini 3 1 plant protection research department, yazd agricultural and natural resources research and education center, areeo, yazd, iran. 2 plant protection research department, chaharmahal and bakhtiari agricultural and natural resources research and education center, areeo, shahrekord, iran. 3 department of agricultural and food sciences, alma mater studiorum, university of bologna, bologna, italy. key words: cucumis sativus, pcr, rflp, 16srvi­a, 16srxii­a. abstract: cucumber phyllody symptoms were observed in greenhouse cucum­ ber plants during 2014­2018 in all surveyed areas of central and west of iran where the highest disease incidence was up to 82% in taft (yazd province). symptoms exhibited by diseased plants were virescence, phyllody and sterility of the flowers. for verification of phytoplasma presence and identity, total dnas were extracted from 44 symptomatic and six asymptomatic plants that were subjected to pcr amplifying 16s rrna genes of phytoplasmas. pcr ampli­ cons of the expected size were obtained only from the symptomatic plants. rflp analysis of r16f2n/r2 amplicons showed patterns identical to those of the clover proliferation (16srvi) and “stolbur” (16srxii) phytoplasma groups. consensus sequences corresponding to phytoplasma strains from the two local­ ities taft and shahrekord showed 99% identity with phytoplasmas enclosed in groups 16srvi and 16srxii, respectively. phylogenetic analysis confirmed that these phytoplasmas cluster with ‘candidatus phytoplasma trifolii’ and ‘ca. p. solani’, respectively. virtual rflp provided profiles identical to the patterns of 16srxii­a and 16srvi­a phytoplasma subgroups. these phytoplasma subgroups were previously reported in different plant species growing near to the green­ house cucumber areas in iran, and play a possible role in the epidemiology of disease for its dissemination. 1. introduction among the cucurbitaceous plants grown in greenhouses, cucumis sativus with 7,427 ha is considered the most economical important crop in iran where about the 77% of the area under greenhouse cultivation is greenhouse cucumber (iranian ministry of agriculture, 2019). these plants need less water than the species cultivated in the fields, and due to the water constraint, this production is expanding. the presence of phyllody disease was reported in cucumber up to 80% in jiroft and kahnooj (*) corresponding author: phytoplasma.iran@gmail.com citation: esmaeilzadeh­hosseini s.a., babaei g., davoodi s., bertaccini a., 2020 ­ identification and impact of phytoplasmas associated with greenhouse cucumber phyllody in iran. ­ adv. hort. sci., 34(4): 413­418 copyright: © 2020 esmaeilzadeh­hosseini s.a., babaei g., davoodi s., bertaccini a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 june 2020 accepted for publication 26 october 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 413­418 414 (kerman province, iran) (azadvar et al., 2004). in some areas, due to high disease incidence and severi­ ty, infected plants did not bear fruits and farmers remove cultivated cucumbers and re­sown them. in 2004­2006 surveys in greenhouses the presence of cucumber phyllody was observed in yazd, varamin, and larestan with about 35%, 80% and 3% of disease incidence respectively, and the phytoplasma pres­ ence was confirmed (esmaeilzadeh­hosseini et al., 2006). phytoplasmas are destructive bacteria infect­ ing more than one thousand plant species world­ wide. they are transmitted mainly by leafhoppers and symptoms include yellowing, discoloration, dwarfing, witches’ broom, virescence and phyllody (bertaccini et al., 2014). their identification relies on molecular classification based on the amplification and/or rflp analyses of their 16s rrna gene (lee et al., 1998; irpcm, 2004). the aim of the present work was to identify the phytoplasmas associated with greenhouse cucumber phyllody in central and west parts of iran in order to devise the appropriate dis­ ease management to reduce its economic impact. 2. materials and methods during 2014 to 2018, greenhouse cucumber growing areas of central and west of iran were sur­ veyed for evaluation of phytoplasma disease pres­ ence. symptomatic and symptomless cucumber plants were collected in greenhouses located in akramia, chah shahrdar and taft (yazd province) and shahrekord region (chaharmahal and bakhtiari province) and subjected to molecular studies for phytoplasma detection and identification. sampling was carried out randomly in five 1,000 m2 green­ houses and the disease incidence was calculated by counting the number of symptomatic plants exhibit­ ing phyllody out of the total number of greenhouse cucumber plants in each greenhouse multiplied by 100. nucleic acid extraction was carried out as described by zhang et al. (1998) using 0.2 g of fresh midrib tissue from 44 symptomatic greenhouse cucumber plants and from 6 asymptomatic seed grown greenhouse cucumber plants. the dna from pot marigold phyllody phytoplasma (16srii­d sub­ group) (esmaeilzadeh­hosseini et al., 2018) was used as positive control. a total of 100 ng of nucleic acid was used for the pcr to amplify the 16s rrna gene of the phytoplasmas with primers p1/p7 (deng and hiruki, 1991; schneider et al., 1995) followed by nested pcr using r16mf2/r16mr2 and r16f2n/r16r2 (gundersen and lee, 1996) primers in a total volume of 50 µl. one µl of the products from direct amplification was diluted in 29 µl of ste­ rile deionized water for the nested amplifications. the pcr reaction was performed in 50 µl mixtures containing 0.4 µm of each primer, 0.2 mm of each dntp, 1.25 u taq dna polymerase and 1x taq poly­ merase buffer (cinnagen, iran). pcr protocols were done as reported by salehi et al. (2015). following pcr, 2 µl of each reaction mixture was electropho­ resed in a 1% (w/v) agarose gel containing 0.3 µg/ml ethidium bromide in 0.5 x tbe buffer (22.5 mm tris­ borate, 1 mm edta, ph 8.0). the amplicons obtai­ ned with r16f2n/r16r2 primers were analyzed by single restriction endonuclease digestion with alui, haeiii, taqi, hpai, hpaii, msei, rsai, kpni and hhai (thermo scientific). the digestion products were analyzed through an 8% polyacrylamide gel elec­ trophoresis and the visualization of dna bands was carried out with a uv transilluminator after staining by ethidium bromide. direct sequencing in both directions of twelve samples (three per each greenhouse area) was car­ ried out using r16mf2/r16mr2 amplicons and the same primers. the resulting sequences were trimmed to the r16f2n/r2 fragment (about 1,240 bp) and submitted to genbank. a database search of homologous sequences was performed by blast analyses at the ncbi (www.ncbi.nlm.nih.gov). the r16f2n/r2 sequence of the 16s rrna gene of green­ house cucumber phyllody phytoplasma strains sgcp (shahrekord greenhouse cucumber phyllody), tgcp (taft greenhouse cucumber phyllody), fars (genbank accession number jn574839) and tehran (genbank accession number mh004460) and of 16s rrna gene of selected ‘candidatus phytoplasma’ species or phy­ toplasma strains enclosed in the subgroups of groups 16srvi and 16srxii were aligned (table 1). a phyloge­ netic tree was constructed with the phytoplasma sequences obtained and others retrieved from the genbank using the neighbor­joining method with mega software version 7 (kumar et al., 2016). acholeplasma laidlawii was used as an out­group to root the tree and bootstrapping was performed 1,000 times to estimate the stability and support for the branches. the ribosomal subgroup affiliation of the detected phytoplasmas was confirmed by virtual rflp analysis with the iphyclassifier (zhao et al., 2009). http://www.ncbi.nlm.nih.gov esmailzadeh‐hosseini et al. ‐ phytoplasmas infecting greenhouse cucumber 415 3. results and discussion greenhouse cucumber diseased plants showed flower virescence, phyllody and sterility (fig. 1), the disease was named greenhouse cucumber phyllody (gcp). the symptoms were observed in all the green­ houses of the four surveyed areas. the disease rate was up to 11.2%, 33.5%, 82% and 8.7% in akramia, chah shahrdar, taft (yazd province) and shahrekord region (chaharmahal and bakhtiari province), respec­ tively. pcr amplicons of about 1.8, 1.4 and 1.25 kb were obtained from all the symptomatic greenhouse cucumber samples but not from the symptomless ones. restriction fragment length polymorphism (rflp) analysis of r16f2n/r16r2 amplicons using alui, haeiii, taqi, hpai, hpaii, msei, rsai, kpni and hhai restriction enzymes showed two rflp pattern identical to those reported for the 16srvi and 16srxii phytoplasma groups, respectively (fig. 2). the dna fragments obtained from twelve sam­ ples after direct sequencing were aligned and the consensus sequences corresponding to a representa­ table 1 ­ phytoplasma sequences used for comparison with the reported greenhouse cucumber strains from iran disease or phytoplasma genbank accession number country 16s ribosomal subgroup ‘ca. p. trifolii' ay390261 canada 16srvi­a strawberry multiplier disease af190224 canada 16srvi­b illinois elm yellows af409069 usa 16srvi­c periwinkle little leaf af228053 bangladesh 16srvi­d centarurea solstitialis virescence ay270156 italy 16srvi­e catharanthus phyllody ef186819 sudan 16srvi­f portulaca little leaf ef651786 india 16srvi­h ‘ca. p. sudamericanum’ gu292081 brazil 16srvi­i 'ca. p. solani’ af248959 serbia 16srxii­a ‘ca. p. australiense’ l76865 australia 16srxii­b strawberry lethal yellows aj243045 australia 16srxii­c ‘ca. p. japonicum’ ab010425 japan 16srxii­d ‘ca. p. fragariae’ dq086423 lithuania 16srxii­e “bois noir” strain bn­op30 eu836652 italy 16srxii­f “bois noir” strain bn­fc3 eu836647 italy 16srxii­g ‘ca. p. convolvuli’ jn833705 italy 16srxii­h fig. 1 ­ flower virescence, phyllody and sterility in a greenhouse cucumber plant from yazd (a) and chaharmahal and bakhtiari (b, c, d) provinces. fig. 2 ­ real and virtual rflp patterns (zhao et al., 2009) respec­ tively of 1.2 kb amplicons from tgcp (a and b) and sgcp (c and d) phytoplasma strains 16s ribosomal gene sequence. lane m, 100 bp dna ladder (biobasic, canada). dna products were digested with the enzymes listed at the top of the figures. adv. hort. sci., 2020 34(4): 413­418 416 using online iphyclassifier program exhibited virtual rflp profiles identical to the reference pattern of 16srvi­a and 16srxii­a, respectively (fig. 2). phytoplasmas belonging to diverse ribosomal groups have been detected in cucurbitaceae species worldwide showing different arrays of symptoms. in particular, 16sri in cucurbita pepo l. in italy (minucci et al., 1995) and in sechium edule (jacq.) sw. in costa rica (villalobos et al., 2002), 16srii in c. sativus and c. pepo in australia, egypt and iran (davis et al., 1997; omar and foissac, 2012; salehi et al., 2015), 16sriii in luffa cylindrica l. (rox.) and sicana odorifera (vellozo) naud in brazil (montano et al., 2000, 2007a, 2007b) and 16srviii in l. cylindrica in taiwan (davis et al., 2017). phyllody is an important phytoplasma disease of cucurbitaceous plants in iran (salehi et al., 2015) and it was associated with the presence of a peanut witches’ broom phytoplasma (16srii) in greenhouse cucumber plants showing phyllody (dehghan et al., 2014) and of a clover proliferation phytoplasma (16srvi) in tehran (ghayeb zamharir and azimi, 2019). molecular assays confirmed the phytoplasma presence in the symptomatic greenhouse cucumber analyzed in this work and allow their identification as ‘ca. p. trifolii’ (16srvi­a) and ‘ca. p. solani’ (16srxii­ a)­related strains (esmaeilzadeh hosseini et al., 2019). in the present work the identification of phyto­ plasmas in subgroups 16srvi­a and 16srxii­a allows epidemiological considerations. the 16srvi­a­related phytoplasma strain was identified in the central areas of iran, in the yazd province where the most impor­ tant plant species harboring 16srvi phytoplasmas are tomato and eggplant (salehi et al., unpublished) and alfalfa (esmaeilzadeh hosseini et al., 2015a, 2015b; purmohammadi et al., 2017). greenhouse cucumber phyllody associated with the presence of 16srxii­a (“stolbur”) phytoplasmas was present in chaharmahal and bakhtiari province where this phy­ toplasma was detected also in grapevine showing diverse symptoms (mirchenari et al., 2015) and alfal­ fa showing witches’ broom (esmaeilzadeh hosseini et al., 2016a, 2016b). phytoplasma diseases associated with “stolbur” were present in plant host species adjacent to greenhouses cucumber areas but their role in the epidemiology of the disease needs to be proved. due to the problems of water constraint, the cucumber greenhouse cultivation in iran has been widely increased. in the majority of cucumber pro­ duction greenhouses, aeration valves are usually not tive of gcp phytoplasmas in taft (tgcp) and shahrekord (sgcp) were deposited in genbank under the accession numbers mf438041 and mk402983, respectively. the blast search of these sequences showed that tgcp (1,251 bp) and sgcp (1,243 bp) phytoplasmas had 99.60% and 99.28% identity with phytoplasmas enclosed in subgroups 16srvi­a (‘ca. p. trifolii’, genbank accession number ay390261) and 16srxii­a (‘ca. p. solani’, genbank accession number af248959), respectively. the phylogenetic analysis confirmed that tgcp phytoplasmas cluster with phy­ toplasmas classified in the 16srvi group and were therefore confirmed as closely related to ‘ca. p. tri­ folii’, while the sgcp phytoplasmas cluster with those enclosed in the 16srxii group and were therefore related to ‘ca. p. solani’ (fig. 3). the r16f2n/r2 amplified regions from tgcp and sgcp phytoplas­ mas digested in silico with 17 restriction enzymes fig. 3 ­ phylogenetic tree constructed by the neighbor­joining method of the r16f2n/r16r2 sequence of 16s rrna gene of 38 phytoplasmas including the cucumber strains sgcp, tgcp, tehran and fars, and phytoplasmas enclo­ sed in subgroups of the 16srvi, 16srii and 16sxii groups. the greenhouse cucumber phyllody phytoplasmas are in color and bolded (in red those sequenced in this work). numbers at the nodes are bootstrap values based on 1,000 repetitions. ‘ca. p.’: ‘candidatus phytoplasma’. genbank accession numbers for sequences are given in parentheses while the phytoplasma ribosomal grouping is before the strain name. esmailzadeh‐hosseini et al. ‐ phytoplasmas infecting greenhouse cucumber 417 covered with netting by greenhouse owners (fig. 4) which allows also the possible entry of insect vectors. furthermore, the major cucumber greenhouses are located close to the agricultural fields and rangelands where during the recent droughts, the insect vectors are attracted and possibly transmitted the phytoplas­ mas from sources outside the greenhouse. it is therefore probable that infection in these plants play a role in the epidemiology for the dissemi­ nation of this bacterium in the greenhouses also con­ sidering that the plants are not completely isolated from the environment. the presence of consistent populations of orosius albicinctus and circulifer haematoceps both recognized vectors of the cucum­ ber phyllody disease was detected in plants grown adjacent to these greenhouses (salehi et al., 2015). their feeding activity during the year, especially in the yazd province, leads to the widespread dissemination of phytoplasmas; therefore, preventing the entry of insect vectors into the greenhouses is the most recom­ mended management to reduce the disease incidence. acknowledgements this paper present part of the results of the pro­ ject no. 2­64­16­94189 approved and supported by agricultural research, education and extension organization (areeo), ministry of agriculture, iran. references iranian ministry of agriculture, 2019 ­ agricultural statistics. ­ iranian ministry of agriculture, vol. 2, pp. 425. azadvar 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(eds.) molecular and diagnostic procedures in mycoplasmology . academic press, san diego, ca, usa, pp. 466. villalobos w., moreira l., rivera c., bottner k.d., lee i­m., 2002 ­ first report of an aster yellows sub‐ group 16sri‐b phytoplasma infecting chayote in costa rica. ­ pl. dis., 86: 330. zhang y.p., uyemoto j.k., kirkpatrick b.c., 1998 ­ a small‐scale procedure for extracting nucleic acids from woody plants infected with various phytoplasmas for pcr assay. ­ j. virol. meth., 71: 45­50. zhao y., wei w., lee i­m., shao j., suo x., davis r.e., 2009 ­ construction of an interactive online phytoplas‐ ma classification tool, iphyclassifier, and its application in analysis of the peach x‐disease phytoplasma group (16sriii). ­ int. j. syst. evol. microbiol., 59: 2582­2593. 418 adv. hort. sci., 2020 34(4): 413­418 https://www.ncbi.nlm.nih.gov/pubmed/?term=kumar%20s%5bauthor%5d&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/?term=stecher%20g%5bauthor%5d&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/?term=tamura%20k%5bauthor%5d&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/27004904 impaginato 105 adv. hort. sci., 2020 34(1): 105­111 doi: 10.13128/ahsc­8406 indirect shoot organogenesis and in vitro root formation of antirrhinum majus l. by using of sodium nitroprusside m.s. rezaei zafarghandi 1, m. rahmati­joneidabad 2 (*) 1 department of agronomy and plant breeding science, college of aburaihan, university of tehran, tehran‐pakdasht, iran. 2 department of horticultural science, faculty of agriculture, agricultural sciences and natural resources university of khuzestan, mollasani, iran. key words: callus, nitric oxide, plant growth regulator, snapdragon. abstract: the aim of this study was to determine the effect of different concen­ trations of sodium nitroprusside (snp) on in vitro shoot organogenesis from hypocotyl explant derived from in vitro grown seedling as well as root forma­ tion of antirrhinum majus l. (snapdragon). in the first experiment, different concentrations of 2,4­dichlorophenoxyacetic acid (2,4­d) (0, 2.26, 4.52, and 6.79 μm) were used for callus formation. the highest callus fresh weight (1.86 g) as well as callogenesis frequency (93.34%) were observed in murashige and skoog (ms) medium containing 4.52 μm 2,4­d. in the later experiments, various concentrations (0, 10, 20, 30, 40, and 50 μm) of sodium nitroprusside (snp) were applied for shoot regeneration from callus that derived from hypocotyl segments. based on our results, ms medium supplemented with 4.44 μm 6­ benzylaminopurine (bap) plus 0.49 μm 3­indolebutyric acid (iba) along with 30 μm snp had the highest shoot organogenesis frequency (93.34%) and shoot number (6.33) from callus. in root induction experiment, different concentra­ tions (0, 20, 40, 60, 80, and 100 μm) of snp were applied and ms medium con­ taining 60 μm snp was the best treatment for root induction. the survival rate of plantlets was more than 95% in acclimatization stage. the present study describes an efficient regeneration system for snapdragon. 1. introduction snapdragon (antirrhinum majus l.) is known as one of the most signifi­ cant ornamental plants which has worldwide values as cut flowers, herba­ ceous landscape plants, and flowering potted plants (el­nashar, 2017). also, snapdragon has high commercial values with its wide range of color, shape, structure, and size (weiss et al., 2016). the commercial propaga­ tion of snapdragon is via seeds. the seed propagation cannot ensure the whole genetic uniformity so seed­propagated plants may indicate unde­ sired phenotypes, quality, and regeneration potential. therefore, plants (*) corresponding author: rahmati@asnrukh.ac.ir citation: rezaei zafarghandi m.s., rahmati­joneida­ bad m., 2020 ­ indirect shoot organogenesis and in vitro root formation of antirrhinum majus l. by using of sodium nitroprusside. ­ adv. hort. sci., 34(1): 105­111. copyright: © 2020 rezaei zafarghandi m.s., rahmati­ joneidabad m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 march 2019 accepted for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 105­111 106 might be selected randomly without taking necessary care. these features of seeds exert a negative impact on sexual production of this plant (jaworski et al., 2016). therefore, the development, as well as improvement of in vitro culture techniques in this ornamental plant, is of high paramount (hesami and daneshvar, 2016). a rapid regeneration pathway for a. majus could be useful for commercial propagation of nursery and cut­flower industries as well as breed­ ing programs (sheyab et al., 2010). also, in vitro cul­ ture of this plant is necessary for producing high quality/price ratio flower. moreover, genetic engi­ neering by using biolistic or agrobacterium methods could be known as a viable alternative in traditional breeding methods for developing distinguished snap­ dragon cultivars in order to satisfy market demands (davies et al., 2013). on the other hand, the efficien­ cy of gene transformation in snapdragon, obtained via these methods, remains low in this ornamental plant because of the lack of efficiency in vitro propa­ gation protocols (azadi et al., 2016). according to the previous study, sheyab et al. (2010) indicated that high applicability of transformation in a. majusis completely depended on the propagation proce­ dures. therefore, the use of in vitro culture for pro­ ducing snapdragon could reduce these problems that occur in the commercial production of this plant (atkinson et al., 1989; sheyab et al., 2010). adjusting the culture medium with suitable plant growth regulators (pgrs) in various combinations and concentrations could enhance the propagation potential of various genotypes and explants (hesami et al., 2017 a, b; jafari et al., 2017; hesami et al., 2018 a, b, c; hesami and daneshvar, 2018 a, b; hesami et al., 2019 a, b, c). thus, it is significant to improve the propagation protocols by using suitable pgrs in order to overcome difficulties associated with clonal regeneration and gene transformation strategies to satisfy the increasing demand for a. majus (newbury, 1986; sheyab et al., 2010; hesami and daneshvar, 2016). nitric oxide is known as a messenger molecule for regulating plant development (neill et al., 2003; hesami et al., 2019 d). this molecule has recently been characterized as one of the phytohormones (leterrier et al., 2012). nitric oxide is known as a ubiquitous bioactive molecule that mainly con­ tributed to various plant developmental processes such as fruit ripening, flowering, organ senescence, and germination (jimenez­quesada et al., 2017). the exterior usage of nitric oxide might improve the tol­ erance of plants under various stresses such as tem­ perature, heavy metals, ultraviolet radiation, drought, and salinity (laspina et al., 2005; qiao and fan, 2008). the activation rate of nitric oxide has been evaluated by the exogenous usage of sodium nitroprusside instead of using no gas directly because of some technical difficulties (sarropoulou and maloupa, 2017). in recent years, nitric oxide is used for developing in vitro plant propagation (rico­ lemus and rodríguez­garay, 2014). kalra and babbar (2010) indicated that nitric oxide could enhance the regeneration response via increasing the number of meristems and recommended that nitric oxide regu­ lates the gene expression related to differentiation of meristems. also, sarropoulou and maloupa (2017) recommended that nitric oxide exert a powerful impact on cell division and also it could be involved in shoot organogenesis and proliferation. han et al. (2009) and sarropoulou et al. (2014) showed that in vitro shoot proliferation as well as root formation of plantlets were promoted significantly by applying snp to the ms medium in malus hupehensis and cherry rootstocks, respectively. although there are few studies about the effect of nitric oxide on improving in vitro shoot organogenesis (han et al., 2009; xu et al., 2009; kalra and babbar, 2010; tan et al., 2013; sarropoulou et al., 2014; arun et al., 2017; ghadakchiasl et al., 2017; sarropoulou and maloupa, 2017), there is no research evidence on the effect of this molecule on shoot organogenesis of snapdragon. thus, the aim of this study was to evaluate the effect of sodium nitroprusside (snp) on indirect shoot organogenesis as well as root formation that derived from hypocotyl explants of snapdragon in order to reduce the time of in vitro shoot propagation. 2. materials and methods the seeds of snapdragon were washed under tap water for 30 min. further surface sterilization treat­ ments were conducted in a laminar airflow chamber. the seeds were surface sterilized with 70% ethanol for 10 seconds and soaked for 10 min in 10% (v/v) naocl. afterward, the seeds were washed three times in sterilized distilled water. subsequently, the sterilized seeds were inoculated on one­tenth strength ms medium. after 8­10 days, seeds were germinated, and the hypocotyl segment from in vitro seedling was used as a source of explant for the latter experiment. the ms medium containing 3% (w/v) sucrose, 0.6% (w/v) agar was used as basal medium. the basal rezaei zafarghandi and rahmati‐joneidabad ‐ indirect shoot organogenesis of antirrhinum majus 107 medium was fortified with different pgrs, and ph 5.8 adjusted with 1 n naoh before autoclaving at 121°c for 20 min. all growth regulators except sodium nitroprusside (snp) were added before autoclaving. snp was added after autoclaving by filtering. all cul­ tures were maintained at 25±2°c with 55­60% rela­ tive humidity, and 16 h photoperiod (65 μmol m­2 s­1) that provided by cool white fluorescent light. hypocotyl explants (0.5­1.0 cm) from 1­week­old in vitro seedlings (fig. 1 a) were inoculated on ms medium supplemented with various concentrations (0, 2.26, 4.52, and 6.79 μm) of 2,4­d for callus forma­ tion. all of the culture vessels were kept at 25±2°c in the absence of light. data of callus formation fre­ quency (%) and callus fresh weight (g) were mea­ sured after four weeks of culture. calli were cultured in the regeneration medium containing 4.44 μm bap plus 0.49 μm iba supple­ mented with different snp concentrations (0, 10, 20, 30, 40, and 50 μm). the shoots regeneration fre­ quency and the number of shoots per callus were determined after 5 weeks of treatment. shoots with 0.5­1.5 cm in length were transferred to ms medium supplemented with 1 mg/l ga3 (elonga­ tion medium) for 4 weeks. then, the elongated shoots (2­3 cm elongation) were chosen and transferred to the half strength ms medium containing 3% (w/v) sucrose, 0.6% (w/v) agar and different concentrations (0, 20, 40, 60, 80, and 100 μm) of snp. rooting per­ centage (%) and root number including main and sec­ ondary roots were evaluated after 30 days. plantlets with well­developed root system were removed from the media, washed thoroughly with sterile water and transplanted into potting mixture containing autoclaved perlite and cocopeat mixture (1:1) and covered with transparent plastic to maintain high humidity. the plastic sheets were removed after 4 weeks in order to acclimatize plantlets to green­ house condition, and the plants were shifted to pots comprising garden soil. all experiments were performed with a total of 10 replicates per treatment and were repeated 3 sets. the data were analyzed by anova using sas version 9.3 followed by duncan’s multiple range test (dmrt, p<0.05). 3. results and discussion the callogenesis experiment was conducted in order to figure out the most suitable and efficient concentration of 2,4­d for callus formation. the result of this study indicated that the maximum per­ cent of callus induction (93.34%) and callus weight (1.86 g) (fig. 2) were achieved on ms medium con­ taining 4.52 μm 2,4­d (fig. 1b). in the agreement with our result, sangwan and harada (1975) showed that acceptable callus formation of snapdragon through stem explant was achieved in ms medium containing 4.52 μm 2,4­d. the earlier study proved the positive effect of 2,4­d on the callus formation, fig. 1 ­ in vitro shoot regeneration through indirect organogene­ sis from seedling derived hypocotyl segments of antirrhinum majus l. (a) seedling from in vitro seed ger­ mination; (b) yellow­greenish and friable callus induction on ms + 4.52 μm 2,4­d; (c) shoot regeneration from cal­ lus on ms medium containing 4.44 μm bap plus 0.49 μm iba along with 30 μm snp; (d) in vitro root formation on ms + 60 μm snp; (e) acclimatized regenerated plants after four weeks. fig. 2 ­ effect of different concentrations of 2,4­din ms medium on (a) callus formation frequency and (b) callus fresh weight of a. majus. means followed by the same letter are not significantly different at p<0.05 as determined by duncan’s multiple range test; vertical bars: standard error. adv. hort. sci., 2020 34(1): 105­111 108 and also this study reported that 2,4­d may be involved in endogenous iaa metabolism regulation by inducing some specific proteins and controlling dna methylation (pan et al., 2010). however, in another study, the callus formation of a. majus via hypocotyl explant was obtained on ms medium with different concentrations of naa plus 10% coconut milk (atkinson et al., 1989). by increasing sodium nitroprusside from 10 μm to 30 μm, shoot regeneration was improved (fig. 3). also, the maximum frequency of shoot organogene­ sis (93.34%) and shoots number (6.33) were observed in ms medium supplemented with 30 μm snp (fig. 1c, fig. 3). however, the higher level (more than 30 μm) of sodium nitroprusside might limit the shoots number and shoot organogenesis frequency. calli can grow in ms medium supplemented with 50 μm sodium nitroprusside. these obtained results rec­ ommended that sodium nitroprusside can promote shoot organogenesis in proper doses. our results indicated that sodium nitroprusside completely pro­ moted shoot organogenesis from hypocotyl seg­ ments in ms medium along with 4.44 μm bap plus 0.49 μm iba. thus, bap and snp appear to have a synergistic effect on shoot regeneration. the effect of no on in vitro organogenesis is completely associat­ ed with cytokinins (arun et al., 2017). it has previous­ ly been shown that no might interact with auxin and cytokinin, linking the regulation of cell division to dif­ ferentiation during the de­differentiation and re­dif­ ferentiation of plant cells (ghadakchiasl et al., 2017; karalija et al., 2017). tun et al. (2001) observed that no plays a potential role in mediating plant hormone (auxin and cytokinin) signal transduction during growth and development. carimi et al. (2005) found that ba stimulates the release and accumulation of no in plant suspension cell cultures. therefore, in the present study, snp may have functioned as an inter­ mediary for adventitious shoot differentiation and regeneration, as suggested by han et al. (2009) in malus hupehensis. our results showed that ms medium supplement­ ed with 1 mg/l ga3 caused shoot elongation. no (pre­ cursor of snp) has been reported to influence several plant developmental events in which gibberellins (gas) play crucial roles such as seed germination, hypocotyl elongation, acquisition of photomor­ phogenic traits, and primary root growth (beligni and lamattina, 2000). however, the actual interaction between no and gas has been described for only a limited number of these physiological events. in fact, most of our current knowledge of the mechanisms underlying the interplay between gas and no is restricted to the regulation of seed germination (neill et al., 2003) and the inhibition of hypocotyl elonga­ tion during seedling de­etiolation (lozano­juste and león, 2011). no has been described as acting upstream of gas (bethke et al., 2007), regulating both biosynthesis and perception/transduction of gas (lozano­juste and león, 2011). there was no root formation in the ms medium without sodium nitroprusside while adding snp pro­ moted root formation significantly. by increasing the concentration of snp from 0 to 60 μm, the root for­ mation frequency (100%) and roots number (8.33) (fig. 4) were increased significantly (fig. 1d). however, the roots number was decreased when the snp level was over 60 μm. root formation is known as the meristematic development of tissues after removing the primary root system (dash et al., 2017; jafari et al., 2017). it was indicated that nitric oxide was involved in the response of auxins during root induction in cucumber (pagnussat et al., 2003) and another report demonstrated that a no­mediated cgmp dependent pathway was involved in this process (pagnussat et al., 2003). in order to form the root meristem, auxins promoted parenchyma cells dedifferentiation and entrance to cell division (klerk et al., 1995; fujita and syono, 1996). also, gouvea et fig. 3 ­ effect of different concentrations of snp in ms medium containing 4.44 μm bap plus 0.49 μm iba on (a) regene­ ration frequency and (b) shoot number of a. majus. means followed by the same letter are not significantly different at p<0.05 as determined by duncan’s multiple range test; vertical bars: standard error. rezaei zafarghandi and rahmati‐joneidabad ‐ indirect shoot organogenesis of antirrhinum majus 109 plantlets that had well­developed roots were transferred successfully into small pots consisting of perlite and cocopeat mixture (1:1). our results showed that the rooted plants had 95% survival rate in the acclimatization stage. afterwards, within 20 days after transferring plantlets to the greenhouse, the normal growth of plantlets was resumed (fig. 1e). similar to our results, hesami and daneshvar (2016) indicated that by acclimatization of the snap­ dragon plantlets in the perlite and cocopeat mixture (1:1), 90% survival rate was obtained. in conclusion, we have developed a method for indirect shoot organogenesis from hypocotyl explants of a. majus. it is of note that snp, a donor of no, has a direct effect on in vitro shoot differentia­ tion and rooting of the snapdragon explants. snp may interact with auxin and cytokinin, linking the regulation of cell division to cell differentiation during the dedifferentiation and redifferentiation of plant cells. the improvement of ornamental plant by con­ ventional methods (hybridization, inbreeding and mass selection) is time and labor consuming, depends on the existing gene pool(s) and violently influenced by environmental conditions. on the other hand, callus culture can be utilized as a power­ ful tool for genetic cell transformation via somaclonal variation and promoting mutagenesis and genetic engineering that can be either more rapid than tradi­ tional breeding and leading to new genes and geno­ types. the indirect plant regeneration system devel­ oped for a. majus provided a step towards the appli­ cation of such methodology, for this ornamental plant. moreover, this protocol is rapid with induction of callus to acclimatizing of plantlets to greenhouse completed within 21 weeks. references arun m., naing a.h., jeon s.m., ai t.n., aye t., kim c.k., 2017 ­ sodium nitroprusside stimulates growth and shoot regeneration in chrysanthemum. ­ hort. environ. biotech., 58(1): 78­84. atkinson n.j., ford­lloyd b.v., newbury h.j., 1989 ­ regeneration of plants from antirrhinum majus l. cal‐ lus. ­ plant cell tissue organ cult., 17(1): 59­70. azadi p., bagheri h., nalousi a.m., nazari f., chan­ dler s.f., 2016 ­ current status and biotechnological advances in genetic engineering of ornamental plants. ­ biotechnol. adv., 34(6): 1073­1090. beligni m.v., lamattina l., 2000 ­ nitric oxide stimulates seed germination and de‐etiolation, and inhibits hypocotyl elongation, three light‐inducible responses in al. 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suppressing axillary bud outgrowth. the exogenous application of synthetic analogues of strigolactone, such as gr24, have been investigated as a way to manage plant architecture in a number of crops. in this study we test whether gr24 can be used to supress bud outgrowth in clonal propagules of tea tree (melaleuca alternifolia) in order to retain a “single stem” form desir­ able for machine planting. gr24 was applied to decapitated rooted cuttings of tea tree at two rates (0.5 mg l­1 and 1.5 mg l­1), with and without auxin. by 21 days post ­treatment, gr24 at both rates had significantly (p<0.05), reduced the mean number of axillary buds (5.7±0.4 and 5.5±0.3 buds respectively) com­ pared to decapitated untreated control plants (8.9±0.6 buds). suppression of buds was significantly higher again when auxin was applied in conjunction with gr24. nonetheless, no exogenous hormone treatment was as effective at sup­ pressing bud outgrowth as the apical dominance that occurred in intact control plants (1.1±0.4 buds). 1. introduction tea tree, melaleuca alternifolia (maiden & betche) cheel, is a small tree, native to the subtropics of eastern australia (anpsa, 2012), which is cultivated in plantations for the production of a medicinally valuable essential oil (carson et al., 2006). the australian industry has developed around improved cultivars released as seed lines, but there is recognition that clonal deployment should offer advantages in plantation uniformity and productivity (doran et al., 1997). although tea tree has been regard­ ed as relatively easy to propagate by cloning (shepherd et al., 2013), the production of high quality propagules with the desired form compatible with automated machine planters remains a challenge. tea tree tip cuttings with single stem architecture suitable for mechan­ ical planting can be produced if the apical bud remains intact (lowe et al., 2019). nodal cuttings (apical tips removed) are preferred to tip cuttings by (*) corresponding author: g.lowe.22@student.scu.edu.au terry.rose@scu.edu.au citation: lowe g.e., shepherd m., rose t., raymond c., 2021 ­ strigolactone analogue gr24 reduces axil‐ lary bud out break and growth in tea tree, melaleuca alternifolia (maiden & betche) cheel. ­ adv. hort. sci., 35(4): 399­405 copyright: © 2021 lowe g.e., shepherd m., rose t., raymond c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 december 2020 accepted for publication 12 august 2021 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-11519 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 399­405 400 some propagators for their robustness in the nursery but these tend to have a multi­stem habit which is unsuitable for machine planting as the “bushy” shoots get stuck in the delivery tubes of planters. form­pruning of propagules prior to planting may be one way to overcome this compatibility issue, but this is labour intensive and costly on a large scale. shoot architecture is a consequence of complex interplay of interacting plant hormonal signals involv­ ing auxin, cytokinin (ck) and strigolactone (sl) that regulate bud release and/or subsequent growth (ferguson and beveridge, 2009; leyser, 2009). auxin is produced in the shoot apex and is actively trans­ ported down the stem, restricting bud release. auxin does not enter the axillary buds but indirectly impacts on bud release by inhibiting auxin export from axillary buds and by regulating stem ck and sl to either promote or suppress branching, respective­ ly (gomez­roldan et al., 2008; muller and leyser, 2011; dun et al., 2012). the influence of sl upon plant architecture was unravelled with the aid of studies on mutants with increased branching, including the ramosus (rms) mutant in pea (pisum sativum l.), and the more axil­ lary growth (max) mutant in arabidopsis (arabidopsis thaliana l.) (brewer et al., 2009; dun et al., 2013). mutants have exaggerated branching habits relative to wild type plants because they are deficient in sl due to genetic changes in transcription or hormonal pathways (umehara et al., 2008; yaish et al., 2010). potential horticultural applications of sl include, management of plant architecture and control of fruit ripening, although high cost of sl production and regulatory approvals remain a limitation to large scale commercial use (vurro et al., 2016; ferrero et al., 2018). in this study we investigated the use of exogenous hormones to manage shoot form of tea tree cuttings during propagation. the aim was to test whether application of an exogenous synthetic sl, gr24, or a combination of gr24 and auxin, can suppress axillary bud release in tea tree. suppression by sl was stud­ ied in decapitated plants where axillary bud out­ growth was triggered by removal of the apical bud. 2. materials and methods plant growth environment and reagents experiments were conducted at southern cross university, lismore campus, nsw, australia during 2020, inside controlled environment growth cabi­ nets, under 16/8 hour photoperiod, and with tem­ perature set at 26°c. stock hormone solutions were prepared by following brewer et al. (2009) for indole ­3 acetic acid (iaa), and manandhar (2016) for gr24. working solutions of 1.75 mg l­1 (10 µm) for iaa, and 0.5 mg l­1 (1.68 µm), and 1.5 mg l­1 (5 µm) for gr24, were prepared by dilution with distilled water and stored at 4°c. method and reagent validation using pea plants initial tests were carried out using the p. sativum type rms1 mutant (provided by professor c. beveridge, university of queensland, australia) to confirm competency of reagents and methods in our laboratory. testing of mutant pea followed methods of manandhar, (2016). application of 0.3 mg l­1 and 1 mg l­1 gr24 to the rms1 mutant of pea significantly reduced side branching compared to the untreated rms1 control, thus validating the method in our labo­ ratory. testing gr24 applications on tea tree the experiment used a randomised complete block design, with four blocks (replicates) of five plants in line plots, subject to one of six treatments (four hormone or two control treatments, giving a total of 120 plants). plants subject to hormone treat­ ments either had a low or high treatment of gr24 (0.5 mg l­1 or 1.5 mg l­1) with or without supplemen­ tal iaa (1.75 mg l­1). control treatments consisted of decapitated plants, and plants with an intact apex in distilled water. scion from a clonal line was set in january 2020 using the mini cutting technique with intact apical buds (lowe et al., 2019). the gr24 experiments were conducted 5 months post­setting when cuttings had rooted and possessed a single main stem with no visi­ ble axillary buds detectable with a 10x magnification hand lens (fig. 1 left). roots were washed free of media and trimmed to 5 cm in length. except for con­ trol plants, the shoot apex was removed (approxi­ mately 2 cm) above node 35 (counted basipetaly from the lowest detectable node) to provoke release from apical dominance (thimann and skoog, 1934). all plants were then positioned upright in clear 50 ml falcon tubes containing 5 ml of treatment solution, or distilled water, so that only the roots were in con­ tact with treatment solutions (fig. 1 middle). the experiment was conducted over 21 days in june 2020. treatment solutions were applied on day 1 and replaced every 7 days. for plants subjected to a lowe et al. ‐ manipulating shoot branching in tea tree 401 hormone combination treatment (gr24 and iaa), a 0.5ml droplet of iaa was placed on top of the decap­ itated stem to simulate the presence of the shoot apex and natural auxin production (cline, 1996) (fig. 1 right). all plants were given 1 ml of yoshida nutri­ ent solution (yoshida et al., 1976) 2 days before treatment solutions, in order to promote growth. data collection and analysis the number of visible axillary buds and axillary shoot length for the 10 uppermost nodes (nodes 1 to 10, with 1 being furthest from the roots) on each plant was recorded at the same time on day 2, 5, 7, 9, 12, 14, 16, 19, and 21. shoot length was deter­ mined to the nearest mm with digital callipers. one­ way analysis of variance (anova) was used to test for treatment effects on the number of axillary buds and shoot growth. where treatment effects were sig­ nificant (p<0.05), a duncan’s multiple range test was performed on treatment means. all statistical analy­ ses were performed using genstat, release 19.1 (vsn international, 2018). 3. results effects of gr24 on axillary bud growth in tea tree rates of bud release over a 21 day time course exper‐ iment treatment effect was significant at each of the 9 days counts were undertaken (p<0.05) (fig. 2). no buds were detected on control intact plants until day 9, and only 40% of control intact plants had one or more detectable buds by 21 days post­treatment (fig. 2). the rate of bud release on control intact plants was due to normal hormonal regulation in rooted cuttings of tea tree, and it established a base­ line for the slowest and least degree of axillary bud development in this study (fig. 2). in contrast, control decapitated plants had the highest rate of bud devel­ opment of any of the six treatments as bud release was not mitigated by normal endogenous regulation or due to the presence of exogenous hormonal treat­ ment (fig. 2). the axillary buds on control decapitat­ ed plants began to initiate 2 days after decapitation and continued to develop for the duration of the experiment, with significantly more buds detected at each recording date than any other treatment (fig. 2). where exogenous hormones were applied (either gr24, or gr24 + iaa) the bud numbers were inter­ mediate to the control intact and control decapitated extremes (fig. 2). all four hormone treatments tend­ ed to track together until day 9, but thereafter, the additional action of auxin apparently moderated the detectable number of buds further (fig. 2). there was no difference in the number of detectable buds between the low and high gr24 treatment, regard­ less of iaa presence (fig. 2). at the end of the 21­day time course experiment, the control intact plants had significantly less buds (mean + se 1.1 ± 0.4), and control decapitated plants had a significantly more buds (mean + se 8.9 ± 0.6 ), than all other treatments (fig. 2). while the rate of gr24 (high versus low) had no effect, the addition of fig. 1 ­ preparing rooted cuttings of tea tree m. alternifolia for the gr24 experiment (left); a rooted cutting five months after setting and prior to decapitation (middle); rooted cutting (control intact) in 50 ml clear plastic tube at the beginning of the experiment, and (right) a droplet of iaa applied on top of decapitated stem stump in gr24 and iaa. fig. 2 ­ mean number of detectable axillary buds over a 21­day time course for tea tree m. alternifolia propagules subjected to gr24 or gr24 plus iaa compared to control intact and decapitated plants. error bars represent se of means. adv. hort. sci., 2021 35(4): 399­405 402 auxin with gr24 resulted in significantly fewer buds relative to gr24 at either rate alone (fig. 2). size and patterns of bud development along the stem when considering all assessed axillary buds (pooled data from all 10 nodes), as measured on day 21, hormone treatments of gr24, or gr24 + iaa, sig­ nificantly reduced the bud size compared to control decapitated plants (p<0.05) (table 1). the average bud length for hormone treatments ranged between 2.5 ± 0.5 mm and 3.3 ± 0.9 mm and was significantly less than the average for control decapitated plants (4.2 ± 0.6 mm), and significantly higher than for con­ trol intact plants (0.03 ± 0.08 mm) (table 1). assessment of bud development node by node revealed further differences in developmental pat­ terns. on control intact plants, buds on the upper­ most nodes (nodes 1 to 3), if present, were not of a measurable size. in control decapitated plants, buds were longest in the upper most few nodes, and grad­ ually reduced in size to the lowest measured node (node 10) (table 1). in hormone treated plants, the longest buds occurred at the uppermost nodes (node 2 was generally longer than node 1), and again there was a progressive decline in size moving down the stem (table 1). bud length was not typically shorter in hormone treated plants relative to control decapi­ tated plants at the uppermost nodes, but below node 5, hormone treated plants tended to have significant­ ly shorter buds (table 1). a representative example of the budding response showing bud position and length is presented in figure 3. 4. discussion and conclusions strigolactone suppressed axillary bud growth in tea tree relative to control decapitated plants without gr24 application, application of a synthetic strigolac­ tone analogue, gr24, at rates of 0.5 mg l­1 or 1.5 mg l­1, suppressed axillary bud number and expansion in decapitated rooted cuttings of tea tree. this response was consistent with its effects on other species such as calla lily (zantedeschia l. sp.) (manandhar, 2016) where synthetic sl gr24 was typically applied at con­ centrations between 0.3 mg l­1 (1 µm) and 3 mg l­1 (10 µm) (umehara et al., 2008; manandhar, 2016). where larger plants at later stages of ontogenetic development (i.e. reproductive maturity) were stud­ ied, such as the study of chrysanthemum morifolium ramat, rates as high as 15 mg l­1 (50 µm) were required to inhibit bud growth (dierck et al., 2016). a moderate response was evident in our tea tree plants at dosages towards the lower end of those used pre­ viously with herbaceous species. both tested rates gave a similar response and, a synergistic effect between sl and auxin was evident, so that bud out­ growth was significantly reduced relative to the application of sl alone. a further factor that may have contributed to the efficacy of the treatments in our study, may have been the retention of the root system. sl is synthesised both in the roots and the shoots and is transported acropetally to suppress bud outgrowth (domagalska and leyser, 2011). in most other studies, stem sections with roots table 1 ­ mean length of the axillary buds from node 1 to node 10 measured at day 21. treatment effect was tested on an individual node basis treatment means followed by the same letter within the same row are not significantly different at the 95% confidence level (­ denotes no bud present). control intact low gr24 (0.5 mg l­1) high gr24 (1.5 mg l­1) low gr24+iaa (0.5 mg l­1) high gr24+iaa (1.5 mg l­1) control decapitated average growth (mm) 0.03 ± 0.08 a 2.5 ± 0.5 b 3.3 ± 0.9 b 2.7 ± 0.6 b 2.7 ± 0.6 b 4.2 ± 0.6 c node 1 (uppermost) 0 a 7.1 ± 1.1 b 9.8 ± 1.2 bc 9.2 ± 1.5 bc 9.4 ± 1.1 bc 12 ± 1.3 c node 2 0 a 8.7 ± 1.1 b 10.5 ± 1.1 bc 9.4 ± 1.3 bc 10.1 ± 0.9 bc 12 ± 0.8 c node 3 0 a 5.4 ± 0.9 bc 7.4 ± 1.0 c 4.2 ± 0.9 b 4.5 ± 1.1 b 7.2 ± 0.8 c node 4 0.03 ± 0.03 a 2.1 ± 0.9 b 3.2 ± 0.7 bc 2.7 ± 0.9 bc 1.6 ± 0.5 ab 4.6 ± 0.9 c node 5 0.1 ± 0.1 a 0.8 ± 0.3 a 1.3 ± 0.4 a 0.8 ± 0.3 a 1.2 ± 0.4 a 2.8 ± 0.7 b node 6 0.1 ± 0.1 a 0.2 ±0.05 a 0.7 ± 0.3 a 0.1 ± 0.1 a 0.4 ± 0. 2 a 1.4 ± 0.5 b node 7 0.1 ± 0.1 ab 0.4 ± 0.3 bc 0.1 ± 01 ab 0.1 ± 0.04 ab 0 a 0.7 ± 0.2 c node 8 0.03 ± 0.03 a 0.6 ± 0.4 b 0.1 ± 0.3 a 0.03 ± 0.03 a 0 a 0.5 ± 0.1 b node 9 0 a 0.1 ± 0.1 a 0 a 0.03 ± 0.03 a 0 a 0.2 ± 0.1 b node 10 (lower most) 0.1 ± 0.1 a 0.03 ± 0.03 a 0 a 0 a 0 a 0.2 ± 0.1 b lowe et al. ‐ manipulating shoot branching in tea tree 403 removed were used for experimentation, and sl was supplied to the basal stem stump. therefore endoge­ nous sl may have reinforced any effect due to exoge­ nous gr24 in our case, potentially inducing a stronger (bud outgrowth inhibition) response in the lower range of concentrations of gr24 compared to studies on other plants. axillary bud outgrowth further inhibited with the addition of an auxin supply when gr24 was applied in conjunction with auxin, suppression of axillary bud outgrowth was enhanced relative to exogenous gr24 alone, as reported in other species (crawford et al., 2010; liang et al., 2010; ward et al., 2013). liang et al. (2010) reported total inhibition of bud outgrowth when 1.5 mg l­1 (5 µm) of gr24 and naa were applied to chrysanthemum (dendranthema grandi‐ florum) stems. although both gr24 and gr24 plus iaa (at the low and high rate) treatments inhibited bud outbreak in tea tree plants for 5 days, when the buds began to grow and elongate, the growth rate of the buds was comparable to the control decapitated fig. 3 ­ a representative m. alternifolia propagule from each treatment showing the pattern and size of axillary buds 21 days after treat­ ment. figure 3a, is a control intact propagule, buds were not visible without magnification at the upper nodes. buds were visible from node 4 or lower but did not develop (arrow). figure 3b, is a control decapitated propagule, with axillary buds at upper nodes developed into side shoots (arrow). figures 3c to 3f show a propagule from each of the gr24 and gr24 plus iaa treat­ ments, buds and developing side shoots visible only on the upper most nodes with generally less shoot growth compared to the control decapitated propagule in figure 3b. plants, at least for the upper four nodes for most plants (table 2). these observations provide further support for the influence of auxin in the vascular stream of the main stem on bud release and the influence of auxin transport on sl inhibition of axil­ lary buds. ultimately, our findings are consistent with both the canalisation (brewer et al., 2009; ljung et al., 2001) and second messenger (domagalska and leyser, 2011) models developed to explain the physi­ ological regulation of shoot architecture involving the interaction plant hormones including auxin, ck and sl (gomez­roldan et al., 2008; domagalska and leyser, 2011). competition between buds the role of the gr24 in reducing bud release and growth needs to be considered along with the inhibitory effect active buds have on buds above or below it and those opposite to it (thimann and skoog, 1934). lateral buds do not produce auxin while they remain dormant but produce considerable quantities when actively growing (balla et al., 2011). auxin synthesised in an active bud is transported into 404 adv. hort. sci., 2021 35(4): 399­405 the main stem, and this auxin saturation prevents auxin movement out of axillary buds further down the stem, inhibiting bud growth (leyser, 2005; balla et al., 2011). tea tree plants treated with gr24 or gr24 + iaa, buds tended to form on the upper nodes (node 1 to node 10), whereas buds formed down to node 14 on control decapitated plants (table 1; data >node 10 not shown). the rapidly growing buds on the upper nodes of control decapitated plants did not appear to inhibit bud release on the nodes below them, but they did affect subsequent bud elongation, consistent with a requirement for sl for bud inhibi­ tion (beveridge, 2000). the mean length of the buds on the control decapitated plants decreased signifi­ cantly for the majority of nodes, basipetally (p < 0.05) (table 1). for plants given the higher rates of gr24 (with and without iaa), active buds may have con­ tributed to the suppression of bud outbreak on the lower nodes, as no bud outbreak occurred past node 6 and 8, for gr24 and gr24 +iaa respectively (table 1). implications for use of sl analogues as agents for manipulating tea tree nursery stock this study demonstrated the potential of synthet­ ic sl gr24 at relatively low concentrations of 0.5 mg l­1 and 1.5 mg l­1 to suppress bud outgrowth in tea tree. this was encouraging for a potential application of sl in an early intervention response to mitigate undesirable side branching of plants where shoot tips are inadvertently lost due to pest, heat or desiccation damage, or where tips are removed during propaga­ tion (i.e. use of nodal cuttings). none of the artificial treatments tested were as effective as the control intact treatment where apical dominance suppresses bud outgrowth, but a moderate response at relative­ ly low dosages, suggests there is room to explore whether higher dosages can elicit stronger suppres­ sion of side branching. for example, liang et al., (2010) found higher concentrations of exogenous sl or sl with auxin suppressed bud outgrowth to the same degree as endogenous regulation induced by an intact apical buds in chrysanthemum. the response at lower dosages in this first investi­ gation was also encouraging because woody perenni­ al species do not appear to be recalcitrant to the influence of hormones, at least for our relatively small (less than 2 grams fwt of biomass) test plants, with root systems, and with low tissue specialisation (i.e. stems had low degrees of lignification and had not formed papery bark). further work will be required, however, to establish the efficacy of higher dosages to elicit stronger suppression responses in nursery propagules, the longevity of the effect of exogenous sl on the form of the propagule (i.e. does it persist to suppress side shoots on larger plants ready for release from the nursery?), and whether there is any unfavourable longer term impact of plant growth in the field. acknowledgements the authors thank professor christine beveridge of the university of queensland for supply of rms1 pea (p. sativa l.) seeds. this study was supported by funding from through the cooperative research centre project scheme for project p53955 “enhanced market agility for the australian tea tree oil industry”. references anpsa 2012 ­ melaleuca, propagation. ­ australian native plant society australia. http://www.anpsa.org. au/melaleu2.html. balla j., kalousek p., reinohl v., friml j., prochazka s. 2011 ­ competitive canalization of pin‐dependent auxin flow from axillary bud controls pea bud out‐ growth. ­ the plant j., 65: 571­577. beveridge c.a., 2000 ­ long‐distance signalling and muta‐ tional analysis of branching in pea. ‐ j. plant growth regul., 32: 193­203. brewer p.b., dun e.a., ferguson b.j., rameau c., bev­ eridge c.a., 2009 ­ strigolactone acts downstream of auxin to 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milanesi 2, b. tortelli 3 1 universidade federal da fronteira sul, chapecó, santa catarina, brazil. 2 universidade federal da fronteira sul, erechim, rio grande do sul, brazil. 3 universidade de passo fundo, passo fundo, rio grande do sul, brazil. key words: brassica oleraceae var. acephala, germination, rhodophyta, solieria sp., thermal stress. abstract: seed priming is a treatment that can contribute to improve seed physiological potential and increase its tolerance to abiotic stresses. thus, this work evaluated the effect of kale seed priming with red seaweed biostimulant on physiological seed potential, seed health and tolerance to high temperature at germination. the experimental design was completely randomised, with a 2 x 4 factorial scheme. treatments consisted of doses of 0, 0.25, 0.50 and 1.0 ml l­1 of red algae solieria sp. biostimulant, and temperatures of 20 and 30°c. the biostimulant used was subjected to chromatographic analysis to detect bioactive compounds. seed imbibition curves were used to determine priming duration procedure. treatments effects were evaluated by seed health, germi­ nation, root and shoot length, and dry mass, under ideal (20°c) and stress (30°c) temperatures. the results were submitted to analysis of variance, tukey’s test (temperatures) and regression (doses). the 22­h imbibition period is adequate for kale seed priming with solieria sp. biostimulant. kale seed prim­ ing with solieria sp. does not interfere with seed health. the temperature of 30°c reduces kale seed germination index, as seedling root growth. the use of solieria sp. biostimulant does not promote kale seed physiological potential. 1. introduction kale (brassica oleracea var. acephala) is a vegetable crop belonging to the brassicaceae plant family, originating from the european continent, with excellent adaptation to mild temperatures. however, there is a demand for this vegetable throughout the year. considering the tropical climate conditions on several countries, studies of the tolerance to high temperatures in kale production are necessary. the use of seeds for kale seedlings production has been increasing in recent years, especially considering the increasing release of hybrid culti­ vars, which do not emit lateral shoots, preventing asexual propagation (trani et al., 2015). seedlings production is one of the most important stages for horticul­ (*) corresponding author: vanessa.neumann@uffs.edu.br citation: lemes ternus f., neumann silva v., mendes milanesi p., tortelli b., 2021 ­ kale seed pri‐ ming with red seaweed biostimulant. ­ adv. hort. sci., 35(2): 111­118 copyright: © 2021 lemes ternus f., neumann silva v., mendes milanesi p., tortelli b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 december 2020 accepted for publication 17 march 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10101 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 111­118 112 tural production systems, which requires high­quality seeds. a key component of the performance of crop seeds is the complex trait of seed vigour. crop yield and resource­use efficiency depend on successful plant establishment in the field, and it is the vigour of seeds that defines their ability to germinate and establish seedlings rapidly, uniformly and robustly across diverse environmental conditions (finch­ savage and bassel, 2016). one procedure that can be used to improve seed quality is physiological conditioning, also known as seed priming. seed priming is a pre­sowing treatment that partially hydrates seeds without allowing radicle emergence. consequently, primed seeds demon­ strate rapid germination and improved germination rate and uniformity. moreover, seed priming is often implicated in improving the stress­tolerance of ger­ minating seeds (chen and arora, 2013). some exam­ ples of success using this technique are verified in the literature, as exemplified in the use of seed priming in spinach seed treatment that resulted in high ger­ mination rates, seedling emergence, growth, maturi­ ty and yield; in this research the maximum seed vigour was obtained when seeds were treated with 6% concentration of sargassum wightii (brown seaweed) (takoliya et al., 2018). studying physiological and biochemical mecha­ nisms involved in heat stress­tolerance in rice seeds, hussain et al. (2016) found that the best performance and greater rice seedlings tolerance obtained from primed seeds, by different methods (hydropriming, osmopriming, redox priming, chemical priming, and hormonal priming) is associated with increased starch metabolism, high respiratory rate, lower peroxidation and increased capacity of the antioxidant defence sys­ tem. in addition, other experimental results have shown that the higher germination efficiency and vigour (seedling growth) occur due to the mobilisation of reserves and the activation of genes responsible for the synthesis of vital enzymes during the priming pro­ cedure (lal et al., 2018). seed priming can be performed with algae extracts or formulations (sharma et al., 2014). some researches demonstrated beneficial effects of seed priming with several seaweed species, for several horticultural crops, for example, tomato seeds primed with anabaena minutissima, ecklonia maxima and jania adhaerens (righini et al., 2021) and with ulva lactuca and padina gymnospora (santacruz­ ruvalcaba et al. , 2019); pepper seeds with kappaphycus alvarezii (k­sap) and gracilaria edulis (dutta et al., 2019). seaweeds are green, brown and red marine macroalgae. extracts of brown seaweeds are widely used in horticulture crops, mainly for their plant growth­promoting effects and ameliorating effect on crop tolerance to abiotic stresses, such as salinity, extreme temperatures, nutrient deficiency and drought (battacharyya et al., 2015). red seaweeds (rhodophyta) are sources of carrageenans, which are sulphated linear polysaccharides that represent major cellular constituents of this algae; carrageenans improve plant growth by regulating various metabolic processes, such as cell division (shukla et al., 2016). studying the effects of extracts and isolated molecules of two species of gracilaria (gracilariales, rhodophyta) on early growth of lettuce, torres et al. (2018) found a promoting effect of the aqueous extracts in lettuce root length. nonetheless, there is a lack of studies testing red seaweeds for seed treat­ ment, especially in seed priming protocols. therefore, the objective of this work was to evalu­ ate the effect of kale seed priming with red algae biostimulant in seed performance under adequate conditions and thermal stress. 2. materials and methods the experiment was carried out in a seed and phytopathology laboratory, in two stages. in the ini­ tial stage, the imbibition curves were performed to determine the ideal time for priming. kale seeds of the brazilian cultivar butter were used, and a solieria sp. biostimulant, which has 7.5% mn and 13% s; a density of 1.3 g cm­3, and is rich in carrageenans. the experimental design used was completely ran­ domised, in a 2 x 4 factorial scheme (temperatures x doses), with four replications. to determine the bioactive compounds present in the seaweed extract, a chromatographic analysis was performed, according to the methodology described below. methodology for the analysis of phenolic compounds by liquid chromatography high efficiency (hplc) the analyze were performed on hplc equipment brand hp model 1100, lichrospher rp18 column (5 µm) equipped with 210 nm uv detector and quater­ nary pump system. the reverse phase analysis con­ sisted of: solvent a ­ milli­q water with 1% phospho­ ric acid and solvent b ­ acetonitrile. the mobile phase lemes ternus et al. ‐ kale seed priming 113 pumping system was gradient, with 90% of solvent a from 0 to 5 min, 60% of a from 5 to 40 min and 90% of a from 45 to 50 min. the standard flow was main­ tained at 0.5 ml/min according to morelli (2010). the samples were filtered through nylon membranes of 0.45 µm pore diameter. the phenolic compounds were identified according to their order of elution and by comparing their retention time with those of their pure standards. quantification was performed by the external standardization method, by correlat­ ing the area (mau *s) of the compound peak to the standard curve performed with each standard evalu­ ated (gallic acid, epigallocatechin, catechin, epicate­ chin, epigallocatechin gallate, rutin, ferulic acid, naringin, hesperidin, myricetin, resveratrol, quercetin, apigenin and canferol). the result is expressed in µg/ml of extract. seed imbibition curves seed imbibition curves were constructed to define the priming period, performed using a method adapted from ferreira et al. (2013). briefly, four repli­ cates of ±0.1 g of seeds per treatment were soaked in solutions of 0, 0.25, 0.50 and 1.00 ml l­1 solieria sp. biostimulant in a plastic box (11 x 11 x 3.5 cm). each box contained 50 ml of solution in the bottom, and a metallic screen on the top, with the seeds placed between four sheets of germitest paper previously wet (at five times its weight). the boxes were placed in a germination chamber (bod) at 20°c until protru­ sion of the primary root. to determine the amount of solution absorbed, seeds were removed from the germination chamber and the gerbox, dried with paper towels and weighed on a digital analytical balance, with an accuracy of 0.001 g. after weighing, seeds were placed again in the gerbox and brought to the germination chamber. the evaluations were made within 60 min, and when protrusion of the primary root occurred, the process was interrupted, registering the corresponding peri­ od. afterwards, the results obtained in the imbibition curves were analysed, and the appropriate period for priming was determined, which must be previous to the protrusion of the primary root (bewley et al., 2013). seed priming in the second research stage, solieria sp. biostim­ ulant doses for seed priming were tested. the experi­ mental design was completely randomised in a 2 x 4 factorial scheme (temperatures x doses) with five replications. seed priming occurs at 20°c for 22 h (defined in the previous stage), given the evaluated doses of 0, 0.25, 0.50 and 1.00 ml l­1, according to the method described in the step of the seed imbibi­ tion curves. after priming, seeds were evaluated for health, germination (percentage and velocity index), root and shoot seedling length and dry seedling mass. seed health seed health was evaluated by the blotter test, with eight replicates of 25 seeds placed in plastic boxes (11 x 11 x 3.5 cm) containing three sheets of filter paper moistened with distilled water in a ratio of 2.5 times the dry paper weight. the seeds were incubated at 25°c for 7 days under a 12­h photoperi­ od. after incubation, the seeds were examined indi­ vidually under a stereomicroscope and optical micro­ scope, counting the percentage of incidence, and the pathogens were identified based on their morpholog­ ical characteristics (mapa, 2009 b). germination test the germination test was carried out at the ideal temperature for the species (20°c) and the stress temperature (30°c), separately. four replicates of 50 seeds, already conditioned, were distributed on paper for germination (“germitest”), previously moistened with distilled water and kept in a germina­ tion chamber. the percentage of germination was evaluated on the fifth day (first count) and 10 days after sowing (das) (final count), according to the cri­ teria established in the rules for seed analysis (mapa, 2009 a). germination velocity index the germination velocity index was determined in conjunction with the germination test, with daily counts, counting seeds with protrusion of 2 mm of primary root according to the protocol proposed by matthews and powell (2011). seedling length seedling length was determined at the end of the germination test (10 das), with 20 normal seedlings per experimental unit, from which the shoot length and root length were determined with a ruler gradu­ ated in centimetres (nakagawa, 1999). seedlings dry mass the same seedlings used to evaluate the length were separated in shoots and roots and dried in a forced­air circulation oven at 65°c for 72 h. once dry, the samples were removed from the greenhouse and adv. hort. sci., 2021 35(2): 111­118 114 placed in a desiccator, and then weighed on a 0.001g precision scale (nakagawa, 1999). statistical analysis the results were submitted to analysis of vari­ ance. tukey’s test (p≤0.05) was used for the temper­ ature factor, and polynomial regression was realised for the dose factor. all analyses were performed using sisvar software (ferreira, 2011). 3. results chromatographic analysis of the algae extract revealed the presence of gallic acid, at a concentra­ tion of 63.9 µg/l, as can be seen in table 1. kale seed imbibition curves with red seaweed (solieria sp.) biostimulant showed an increase in the wet mass accumulation up to 14 h, and the highest absorption peaks were obtained between 12 and 14 h. this period defines germination phase i (fig. 1 a). between 14 to 28 h of imbibition, the absorption was constant, without a significant increase in the control treatment, and this was denominated as phase ii. after 28 h, phase iii germination started. using the doses of 0.25, 0.5 and 1.0 ml l­1, the germination phase i occurred until 17, 13 and 17 h, respectively, and in all cases, the root protrusion occurred with 28 h of soaking (fig. 1 b, c and d). for seed health, no difference was observed between treatments (table 2). there was a low inci­ dence of fungi, such as penicillium spp., rhizopus spp. and cladosporium spp., and absence of fusarium spp. however, although no statistical difference was observed, 0.37% of trichoderma spp. was detected in the treatment with 0.25 ml of red algae. regarding the germination seed performance, dif­ ferences between temperatures, without dose effects, were observed for the percentage and veloci­ ty index (table 3). there was a significant difference in seedling root length between the temperatures used, with a pro­ fig. 1 ­ inibibition curves of kale seeds primed with 0 (a), 0,25 (b), 0,50 (c) and 1 (d) ml l­1 of solieria sp. biostimulant. table 2 ­ fungi incidence on kale seeds primed with doses red seaweed (solieria sp.) biostimulant ns = not significant by the tukey test (p<0.05). table 1 ­ identification (possible compound) and quantification of phenolic compounds compound quantification (µg/l) retention time (min) gallic acid 63. 79 5.877 fungi incidence (%) doses (ml l­1) 0 0.25 0.5 1 alternaria spp. 4.1 ns 5.2 3.7 2.4 penicillium spp. 0.37 0.12 3.37 0.37 rhizopus spp. 0.12 0 0 0 cladosporium 0.12 0.12 0 0 trichoderma spp. 0 0.37 0 0.12 table 3 ­ means of germination count (g), radicle protrusion velocity index (rpvi), root length (rl), dry root (drm) and shoot (dsm) seedling mass from kale seeds primed with red seaweed (solieria sp.) biostimulant, submitted to different germination temperatures * means followed by the same letter in the column, for each variable, do not differ from each other by tukey test (p<0.05). temperature doses (ml l­1) 0 0.25 0.5 1 g (%) 20 82.5 a* 85.5 a 88.0 a 86.0 a 30 82.5 a 80.8 a 79.0 b 70.8 b rpvi 20 82.8 a 85.9 a 87.5 a 86.0 a 30 52.6 b 48.0 b 47.0 b 48.9 b rl (cm) 20 4.8 a 5.2 a 5.1 a 6.0 a 30 2.7 b 2.5 b 2.5 b 2.4 b drm (mg seedling‐1) 20 4.8 a 2.9 a 5.0 a 4.8 a 30 2.3 b 4.0 a 3.8 a 3.1 a dsm (mg seedling‐1) 20 3.2 b 3.2 a 2.9 a 3.8 a 30 4.6 a 3.5 a 3.5 a 4.8 a lemes ternus et al. ‐ kale seed priming 115 nounced reduction in root size when the seeds were exposed to 30°c. however, the doses of biostimulant evaluated in this study did not differ and were not able to attenuate the effects of thermal stress (table 3). in relation to shoot seedling growth, the opposite response was observed, with higher mean lengths at 30°c compared with 20°c (fig. 2). in examining the effect of the biostimulant doses, at 30°c, there was growth increment until the dose of 0.25 ml l­1, after which, the growth declined (fig. 2b). in regards to the accumulation of dry mass in roots and shoots of seedlings, no effects of biostimu­ lant doses were observed (table 3). for the tempera­ ture, a reduction in root length and increase in shoot length was verified in the control, which reinforces the hypothesis of the compensatory effect, related to plasticity. some trichoderma strains are used to control phy­ topathogens and promote plant growth due to their versatility of action, such as parasitism, antibiosis and competition, as well as acting as inductors of plant resistance against diseases. melo et al. (2017) observed progressive increases in phytoalexin con­ centration in soybean and sorghum seeds treated with increasing algae doses, demonstrating a high cor­ relation between dose used and the amount of phy­ toalexin produced. phytoalexins are compounds that have been studied a few years ago, which present mainly antimicrobial activity (arruda et al., 2016). the highest phytopathogenic fungi observed was alternaria spp. (table 2), however no differences between treatments were found. this fungus is a pathogen that can be transmitted by seeds and inter­ feres with the germination and development of seedlings (torres­córtes et al., 2019). regarding the germination seed performance, dif­ ferences between temperatures, without dose effects, were observed for the percentage and veloci­ ty index (table 3). in general, germination capacity was reduced at 30°c, indicating an abiotic stress effect in this process. temperature exerts a marked effect on germination because it influences the per­ formance of enzymes involved in the mobilisation of reserves, as well as enzymes associated with hor­ monal regulation. in arabidopsis, a model species in studies of physi­ ology and genetics, and of the same botanical family of kale, it was verified that when seeds are exposed to high temperatures during germination, there is a stimulus to biosynthesis of abscisic acid (aba) and, consequently, a repression of gibberellin synthesis (toh et al., 2008). the balance between aba and gib­ berellins is responsible for the occurrence of germi­ nation or maintenance of dormancy (kucera et al., 2005). furthermore, it should be noted that gib­ berellins stimulate the synthesis and production of hydrolases, especially alpha­amylase, resulting in seed germination (miransari and smith, 2014). however, the inhibitory effects of aba on seed germination include the impedance of radicle expan­ sion and endosperm weakening, as well as increased expression of transcription factors, which may adversely affect the seed germination process (graeber et al., 2010). the absence of the effect of red algae on germina­ tion might indicate that this response is associated with the composition of the biostimulant and to the moment of application. one of the main compounds fig. 2 ­ averages of kale seedling shoot length, from primed seeds with red seaweed (solieria sp.) biostimulant, sub­ mitted to 20°c (■) and 30°c (●) during germination. 4. discussion and conclusions regarding results of chromatographic analysis, which revealed the presence of gallic acid, it is impor­ tant to consider that gallic acid is a secondary metabolite present in most plants and algaes (cotas et al., 2020). considered one of the major phenolic acids, gallic acid (or gallate) is a benzoic acid of great importance for the formation of a so­called gala­ totanin­hydrolyzable tannins group formed by a unit of sugar and a variable number of phenol acid mole­ cule. this metabolite is known to exhibit a range of bioactivities including antioxidant and antimicrobial (fernandes and salgado, 2016). considering results of seed health, is important to mention that according to machado et al. (2012), 116 adv. hort. sci., 2021 35(2): 111­118 found in red algae is carrageenans, sulphated linear polysaccharides. recent research has uncovered the biological activity of carrageenans and their oligomeric forms as plant growth promoters and elic­ itors of defence responses (shukla et al., 2016). however, carrageenans have shown a relatively greater effect when applied to plants at adult devel­ opmental stages. for example, according to gonzales et al. (2013), oligo­carrageenans obtained by depoly­ merisation of red algae carrageenans increase the growth of tobacco plants by increasing photosynthe­ sis and nitrogen assimilation, as well as stimulate the growth of 3­year­old eucalyptus globulus plants. regarding results of seedling root length (table 3) is worth mention that temperature stress has a detri­ mental effect on plant metabolism by interrupting cell homeostasis, and the direct result of cellular changes is increased accumulation of toxic compounds in cells, including reactive oxygen species (essemine et al., 2010). according to tsukagoshi (2016), reactive oxy­ gen species regulate the activity of the root meris­ tems and root development. therefore, this mecha­ nism is probably involved in the reduced growth of the kale seedling root accompanying the elevation of temperature, as verified in this work. however, in relation to shoot seedling growth, the opposite response was observed (fig. 2). this response might be explained by a mechanism of plas­ ticity, in an attempt to balance the growth of the seedling, due to the reduction in root size. hernandez­herrera et al. (2014) found that there was a stimulatory effect of algae extracts on the length of plumule in tomato seedlings. in regards to the accumulation of dry mass in roots and shoots of seedlings, no effects of biostimu­ lant doses were observed (table 3). as mentioned by mašková and herben (2018), the ratio of biomass partition between roots and shoots is essential for the ability of plants to compensate for the limited resources in the environment and thereby to survive and succeed in competition. allocation plasticity is an important process for seedlings, and this is one of the most vulnerable phases of the breeding cycle, for most species. in this context, a rapid allocation response may have a direct impact on its survival (lloret et al., 1999). regarding the biostimulant doses evaluated, it is possible that there were no effects on the accumula­ tion of seedling dry mass because this process is more related to the issue of mobilisation and parti­ tion of the reserves, with little influence exerted by the compounds present in algae. however, consider­ ing that to date, there are no scientific studies to prove the direct effect of algae biostimulants in the process of mobilising seed reserves and partitioning of biomass in seedlings, additional studies in this area are necessary. in conclusion, the 22­h imbibition period is ade­ quate for kale seed priming with solieria sp. biostim­ ulant. kale seed priming with solieria sp. biostimulant does not interfere with seed sanity. the temperature of 30°c reduces velocity germination index and kale seedling growth, obtained from seeds primed with solieria sp. biostimulant. the use of solieria sp. bios­ timulant does not promote improvements in kale seed physiological potential under the conditions in which this research was carried out. references arruda r.l., paz a.t.s., bara m.t.f., côrtes m.v.c.b., filippi m.c.c., conceição e.c., 2016 ­ an approach on phytoalexins: function, characterization and biosynthe‐ sis in 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transmitted microbial populations. ­ sci. reports, 9: 6648. trani p.e., tivelli s.w., blat s.l., pantano a.p., teix­ eira e.p., araújo h.s., feltran j.c., passos f.a., adv. hort. sci., 2021 35(2): 111­118 118 figueiredo g.j.b., novo m.c.s.s., 2015 ­ couve: do plantio à colheita. ­ iac, campinas, sp, brazil, pp. 42. tsukagoshi h., 2016 ­ control of root growth and devel‐ opment by reactive oxygen species. ­ current opinion plant biol., 29: 57­63. impaginato 63 adv. hort. sci., 2022 36(1): 63­80 doi: 10.36253/ahsc­12290 an overview of betel vine (piper betle l.): nutritional, pharmacological and economical promising natural reservoir a.k. sahu 1, s.s. kar 1, p. kumari 1, 3(*), s.k. dey 2, 3 1 p.g. department of biosciences and biotechnology, fakir mohan university, vyasa vihar, balasore‐756089, odisha, india. 2 p.g. department of environmental science, fakir mohan university, vyasa vihar, balasore‐756089, odisha, india. 3 centre of excellence (coe) for bioresource management and energy conservation material development, fakir mohan university vyasa vihar, balasore‐756089, odisha, india. key words: agriculture, anti­microbial, antioxidant, betel vine, disease, eco­ nomic, nutritional, pharmaceutical. abstract: with its magnificent green heart­shaped leaf, the betel vine (piper betle l.) is also known as paan in india. it is a member of the piperaceae family. it is cultivated in the coastal regions of odisha (balasore, jagatsinghpur, puri, khordha, and ganjam). paan is consumed by over 1 million people throughout the state, but they are unaware of its high nutritional quality. it is considered superior to pharmaceuticals and is one of the best remedies in nature. it has anti­microbial, anti­apoptotic, anti­cancer, antioxidant, and anti­inflammatory attributes. furthermore, the leaves retain eugenol­rich essential oil (eo) (1­3%), which is the hotspot for medication, stimulants, antiseptics, tonics, and other ayurvedic compositions. this oil can also be used as an industrial raw material to make medications, fragrances, tonics, mouth fresheners, food additives, and other products. it contains anticarcinogens, which show potential for the devel­ opment of medicines against cancer treatment. betel plant farming is an agri­ cultural activity that provides a source of income for remote farmers. sometimes economic crises occurred due to the development of diseases such as foot rot, leaf spot, powdery mildew, and collar rot. most farmers got season­ al revenue, whereas betel vine cultivation provided year­round income from a tiny plot of land. 1. introduction the betel vine (piper betle l.) belongs to the piperaceae family, which also contains pepper and kava. paan leaves are produced in the philippines, malaysia, india, sri lanka, taiwan, thailand, and other southeast asian countries as a post­meal mouth freshener. it is primarily consumed in south asia and by certain asian emigrants worldwide as betel quid or paan, in combination with areca nut or tobacco (saraswat et (*) corresponding author: punam.lifescience@gmail.com citation: sahu a.k., kar s.s., kumari p., dey s.k., 2022 ­ an overview of betel vine (piper betle l.): nutritional, pharmacological and economical pro‐ mising natural reservoir. ­ adv. hort. sci., 36(1): 63­80. copyright: © 2022 sahu a.k., kar s.s., kumari p., dey s.k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2021 accepted for publication 26 january 2022 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-12290 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 63­80 64 al., 2020; shah et al., 2021). a sheaf of betel leaves is typically presented in odisha as a token of respect and auspicious beginnings in traditional culture. it belongs to the genus piper of the division magnoliphyta, class magnolipsida, order piperales, and family piperaceae. it is a unisexual perennial evergreen climber with shiny cardio leaves and white catkins that bloom in the spring. betel vine is catego­ rized into odorous and non­pungent kinds depending on the form, length, and flavour of the leaf. the plant’s leaves are basic and have an acuminated crown. mostly, the leaves are smooth and shining. the leaves differ in color from light green to dark green. the leaves are long­stalked with 2­3 pairs of secondary veins (swapna et al., 2012). the betel plant’s limbs usually bulge at the nodes and are com­ pletely smooth. female spikes are cylindrical, where­ as male spikes are pendulous. female spikes measure 2.5­5.0 cm in length. in the humid environment of east india, female plants often generate blooms or fruit (sengupta and banik, 2013; rahman et al., 2020). throughout thousands of years, nature has pro­ vided a reservoir of medical substances, and current medications are derived from ecological resources. the betel leaves contain a variety of bioactive com­ pounds and are employed in ancient medical meth­ ods. such leaves are high in minerals, vitamins, enzymes, proteins, and essential oil (eo), and they are very nutritious (nayaka et al., 2021; paswan et al., 2021). they also include certain useful therapeu­ tic components for the therapy of disorders of the brain, liver, and cardiac (pradhan et al., 2013; ullah et al., 2020). polyphenols, alkaloids, steroids, saponins, and tannins were also found. in the indian subcontinent, medicinal plants are a resource of eco­ nomical worth (sen and chakraborty, 2017; madhumita et al., 2020). medicinal plants are the pri­ mary source of medicine for the bulk of the rural community in emerging nations, and consequently play a key role in their health systems (patra et al., 2014; who, 2019). upto 80% of people in developing countries still use local medicinal herbs for their basic health care needs (sen and chakraborty, 2017; who, 2019). furthermore, diastase and catalase activities are detected in the leaflets (abrahimet al., 2012; shah et al., 2021). it assists in curing and treating many conditions, including halitosis, boiling and absceeding, conjunctivitis, headache, constipat­ ion, hysteria, itching, mastitis, leucorrhoea, otor­ rhoea, mastoiditis, gum swelling, ringworm, rheuma­ tism, abrasions, injuries, cuts, etc (shukla et al., 2018). because of their antibacterial and antioxidant properties, these oils have a promising future in the novel food packaging industry (ei asbahani et al., 2015; guha and nandi, 2019; nguyen et al., 2021), as well as being a prospective and appealing flavouring component for the food and beverage sectors. this plant is grown as a cash crop in the balasore, jagatsinghpur, puri, khordha, and ganjam areas of coastal odisha (jena, 2021). in assamese/urdu/ hindi/odia/bengali, the betel leaf is recognised as paan, whereas in sanskrit it is considered as taambuul and nagavalli. the finest betel leaf is the “magadhi” type cultivated near patna in bihar, india. the popular type of betel leaf in kerala is called “venmony vettila” and comes from venmony near chengannur (guha and nandi, 2019). in odisha, four distinct forms of betel leaf are grown. the bhograi block in the balasore district is known throughout the country for its betel vine farming (patra and pradhan, 2018). cultivars with the prefix desi in their names, on the other hand, always relate to the cultivars desavari in madhya pradesh, kapoori in maharashtra, bangla in west bengal (guha and nandi, 2019), bali and chandrakana in bhogarai (fig. 1 a1­a2, b1­b2). the varieties are nova cuttak, godibangala (fig. 1 c1­c2), sanchi (fig. 1 d1­d2), and birkoli. only one type, godibangala, is grown by the locals in the research region bhainchigodi (patra and pradhan, 2018). odisha is one of the states that pro­ duce the most betel vine. in the context of such sci­ entific research, this review article tries to summarise all the possible information on betel leaf with its propagation, socio­economics, and bioactive com­ pounds, justifying wider possibilities for its use as a natural source for people in coastal odisha. 2. habitat and ecology betel vine is primarily grown in odisha’s coastal districts such as balasore, jagatsinghpur, puri, khordha, and ganjam (jena, 2021). all the respective district locations are as follows. 20.27°n and 86.17°e, 19.48°n and 85.48°e, 20.18°n and 85.62°e, 19.38°n and 85.05°e (fig. 2). north­western highlands, the inner alluvial plain, and the coastal belt are the three geographical regions that can be found in all five dis­ tricts. those regions are flooded with brackish water from estuarine rivers, making them unsuited for agri­ culture in a regular manner. those lands are current­ sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 65 ly used for betel and coconut agriculture (ahuja and ahuja, 2011). recently, prawn culture and salt pro­ duction units have sprouted in this region. paddy, fish, and betel vine farming are important parts of the local economy in these districts. the soil in the central zone is mostly sandy loam and clay loam, making it perfect for producing rice and betel vines (kaleeswari and sridhar 2013). coastal odisha has an oceanic climate and experiences rainfall from the south­west monsoon (gouda et al., 2017). the coast­ line area has a pleasant temperature. the summers in such districts are hot, having excessive humidity during the monsoon season, a dry winter, and little diurnal temperature fluctuation over the year (krishnan et al., 2020). the district’s average annual precipitation is 1591 mm, which is over 9% greater than odisha’s average. tropical cyclones, which bring a lot of rain to the area, are responsible. due to the district’s proximity to the bay of bengal, cyclones are common. cyclones, which begin in the bay of bengal over the andaman and nicobar islands and proceed towards india’s east coast, are a common occurrence in the district (sahoo and bhaskaran, 2016). the south­west monsoon brings more than 70.9% of the annual precipitation from june to september. the three distinct seasons observed in the area are sum­ mer (march­june), rainy (july­october), and winter (november­february) (imd, 2020). 3. cultivation and propagation in coastal odisha this plant’s cultivation needs consistent soil wet­ ness, adequate humidity, and moderate heat, which are not usually present in the ecological environment (filipovic, 2020). variations in these environmental variables were found to be harmful to the plant. as a result, it is grown in an artificially manufactured hut­ like structure that keeps the plant’s growth parame­ ters within a reasonable range, simulating natural ecological conditions (raza et al., 2019). the building is known as a ‘baraja,’ and is made up of various plant materials like (bambusa vulgaris sch. ex wendl.) and bamboo stems (bambusa bambos l. voss.), jute (corchorus capsularis l.), paddy straw (oryza sativa l.) (fig. 3 a­b), khadi or chaee stick, and coconut leaves (cocos nucifera l.) make up the baraja architecture (arunda donox l.) (haider et al., 2013). the structure’s proportions are limited to 2 to 3m in height, 10 to 20 m in length, and 5 to 15 m in breadth. the plant is grown through vegetative pro­ liferation from cuttings of 3 to 5­year­old vines. plantlets with one or two nodes and connected leaves are commonly used as propagating specimens. the planting season varies depending on the area to area. it takes roughly a month for roots to develop and grow after planting. plants are supported when fig. 1 ­ major varieties of piper betle (a1) ventral and (a2) dorsal side of ‘bali’, (b1) ventral and (b2) dorsal side of ‘chandrakana’, (c1) ventral and (c2) dorsal side of ‘godi bangala’, (d1) ventral and (d2) dorsal side of ‘sanchi’ cul­ tivated in coastal region of the odisha. fig. 2 ­ map showing the study sites of coastal odisha (balasore, jagatsinghpur, puri, khordha and ganjam district). adv. hort. sci., 2022 36(1): 63­80 66 hridayam,” “bhabaprakasha,” “harivamsa,” “varahapurana,” and “panchatantra and jataka stories” are among the sanskrit writings that allude to it as “tamabool” (kumar, 1999). in asian countries, this plant is a frequent element in the creation of var­ ious indigenous medicines. in comparison to related indian traditional plants such as “tulsi” (ocimum sanctum), for example, this plant has received very little research aside from strong ethnomedicinal promises (cohen, 2014; kurepa and smalle, 2021). nayaka et al. (2021) revealed that the betel leaves contain many ingredients such as water (80­90%), eo (0.05­0.2%), fat (0.5­1.0%), protein (3­4.5%), riboflavin (4.5­15.5 μg/100 g), fibre (2­2.5%), miner­ als (2.5­3.5%), chlorophyll (0.05­0.25%), tannin (0.1­ 1.3%), vitamin c (0.005­0.01%), carbohydrate (0.5­ 6.5%), vitamin a (2­3 mg/100 g), nicotinic acid (0.65­ 0.9 mg/100 g), potassium (1.5­4.5%), thiamine (10­80 μg/100 g), iron (0.005­0.01%), phosphorus (0.05­ 0.06%), nitrogen (2.0­7.0%), calcium (0.2­0.5%), iodine (3­3.5 μg/100 g) respectively (madhumita et al., 2019). fresh betel leaves contain minerals 2.3­ 3.3%, protein 3­3.5%, fat 0.4­1.0%, moisture 85­90%, carbohydrate 0.5­6.10%, fibre 2.3%, calcium 0.2­ 0.5%, phosphorus 0.05­0.6%, iron 0.0050­007%, vita­ min c 0.005­0.01%, and energy 44kcal per 100 g (mazumder et al., 2016; vernekar and vijayalaxmi, 2019). after dehydration, the betel leaf samples became a concentrated source of nutrients, accord­ ing to the results of the nutritional analysis. the find­ ings support those of (subhash and neeha, 2014), who found that after sun drying and cabinet drying, the leaves preserved high levels of protein, fibre, and calcium. the moisture material of dehydrated betel leaves flour was 9.45%, fat 1.10%, protein 3.30%, ash 6.87%, carbohydrate 63.92%, fiber 10.15%, vitamin c, calcium, and iron were 1.11%, 2.57%, and 1.53% (chauhan and aishwarya, 2016; akshata et al., 2018). 5. phyto­chemicals found in betel leaf the phytochemicals screening was analyzed on the ethyl alcohol extract of betel vine using standard protocol for identification of the constituents. the leaf extracts have various pharmacological activities which are prepared by using different solvents such as aqueous, ethanol, powder, and hot water (kumari and rao, 2015) (table 1 and table 2). by this analysis, it was concluded that it consists of tannins, anthraquinones, flavanoids, alkaloids, terpenoids, they have 5­7 leaves. after one year of plating, har­ vesting begins by plucking the leaves. the harvests are known locally as maghei paan, jhanji paan, vejua paan, nua paan, and jagannath paan. nua paan is harvested during the months of february and april. cultivators remove dry or damaged leaves during this plucking period. jhanji paan is harvested between may and mid­july. this is the season with the highest yield. cultivators are removing some broken khadi or chaee sticks this season. during the months of august and september, jagannath paan is collected. farmers strategically gather the leaves from the low­ est section of the branches during this plucking. during the months of october and november, vejua paan is collected. cultivators pick the tops of branch­ es in this plucking technique. maghei paan harvesting begins in december and continues until february (patra and pradhan, 2018). all total leaves are plucked in this collection. farmers sprinkle water on leaves and use cotton sheets or paddy straw to keep them fresh. a “pono” is made up of eighty paan leaves. after that, the leaves are distributed in local markets or transferred to other towns (haider et al., 2013). 4. nutritional composition of betel leaf ayurveda, or traditional indian medicine, has long been a part of indian culture and ‘herbal materia medica. the ancient sanskrit books ‘charaka and sushruta samhita’ (600­400 b.c.) document betel leaf chewing, establishing its identity as an old use. “charaka and sushruta samhita,” “astanga fig. 3 ­ cultivation and propagation of piper betle inside the baraja at bhograi area of balasore district, odisha (a­b). sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 67 saponins, caediac glycosides, glycosides, reducing sugars (rajamani et al., 2016). the compounds reported in betel plants include bisabolene, cavacrol, isoeugenyl acetate, eugenol methyl ether, 2­mono palmitin, allypyrocatechol, eugenol, piperitol, chavibetol, chavibetol acetate, myrcene, quercetin, hydro­xychavicol, stearic acid, allyl catecol, germacrene­a, α­terpineol, luteolin, caffeic acid, limonene, piperlonguminine,α­cadinol, β­sitosterol, 4­ allyl phenyl acetate, d­ limonene, m­cymen­8­ol, 2­ noanone, ocimene, n­decanal, 2­undecanone, allo ocimene, sabinene, 3­allyl­6­methoxyphenol, humlene, cymene, terpinolene, β­pinene, α­myrcene, allyldiacetoxy benzene, vanillin, thymol, cispiperitol, tarpinolene, propcatechuic acid, eucalyptol, gallic acid, iso eugenyl acetate, (e)­ß damascenone, linalool, camphene, 4 cineole, α­pinene, germacrene­ d, piperol­b, piperol­a, estragol, arecoline, isoeugenol, isoascaridole, chavicol, ellagic acid, safrole, eugenyl acetate, 4­allyl phenol, 5­ indanol, 4­ allyl resorcinol, α­bergamotene, (e)­ß­ocimene, ferulic acid, carryophyllene, anethole, α­farnesene, cepharadione­a, piperine,4e­decadienamide, 4­allyl anisole, cuparene, ß­iso safrole, αmuurolene, cadinene, α­copaene, α­cubebene, benzene acetic acid, α­selinene, methylpiperbetol (pradhan et al., 2013; mazumder et al., 2016; junairiah et al., 2018; junairiaha et al., 2020; sakinah and misfadhila, 2020; azahar et al., 2020) (fig. 4, table 1). table 1 ­ components of piper betle sl no. components of piper betle percentage of components (%) references 1 1.8­cineol 0.04 rajamani et al., 2016 2 a­pinene 0.21 pradhan et al., 2013 3 allypyrocatechol diacetate 0.71­6.2 mazumder et al., 2016; sakinah et al., 2020 4 allypyrocatechol monocetate 0.23 junairiah et al., 2018 5 campene 0.48 junairiaha et al., 2020 6 caryophyllene 3.71 pradhan et al., 2013; junairiaha et al., 20 7 chavicol 0.4 pradhan et al., 2013; sakinah et al., 2020 8 chavibetol 53.1­80.5 junairiaha et al., 2020; azahar et al., 2020 9 chavibetol acetate 11.7­15.5 pradhan et al., 2013 10 chavibetol methyl ether 0.48 mazumder et al., 2016; junairiaha et al., 2018 11 (e)­caryophyllene 0.4 pradhan et al., 2013; sakinah et al., 2020 12 eugenol 0.32­0.4 junairiaha et al., 2020 13 f­pinene 0.21 pradhan et al., 2013; junairiah et al., 2018 14 methyl eugenol 0.4 rajamani et al. 2016; junairiaha et al., 2020 15 methyl salicylate 0.05 pradhan et al., 2013 16 saprobe 0.11 shameem et al., 2013 17 u­limonene 0.14 akther et al., 2014 fig. 4 ­ structure of major bioactive constituents of piper betle a) chavibetol, b) eugenol, c) hydroxychavicol, d) estragol, e) gallic acid, f) β­pinene, g) camphene, h) allyl diacetoxy benzene, i) ferulic acid, j) caffeic acid, k) 5­indanol, l) allopyrocatechol, m) cahvibetol acetate, n) piperitol, o) d­limonene, p) α­terpineol, q) eugenol methyl ether, r) quercetin (flavonoids). 68 adv. hort. sci., 2022 36(1): 63­80 6. pharmacological activities paan is healthy and significant because of its med­ ical, religious and ceremonial history (rai et al., 2011). asthma is also prevented, vocalisation is improved, and gums are strengthened. indigestion, constipation, congestion, cough, and asthma are treated (peddapalli et al., 2020). the anti­alzheimer bioactive compounds were found in various varieties of betel vine (de et al., 2021). the following bio­ chemical roles of phytochemicals from leaves are depicted in figure 5. possible source of wound healing agents betel vine component at 0.025 ml/l concentra­ tions boosted fibroblast reproduction and encour­ aged umbilical cord­mesenchymal stem cells (uc­ mscs) proliferation at 0.03 ml/l, increasing in vitro model wound healing in accordance with empirical evidence. the homogenate reduces the presence in cells of oxidative stress factors such as vcam, cd248, and il­33, so that recovery of umbilical cord cells can be accelerated (thi et al., 2021). researchers discov­ ered that natural lysates of leaves containing pheno­ lic compounds can reduce the activity of inflammato­ ry factors and oxidative stress, which could be useful in regenerative medicine. insecticidal activity the crucial oil extracted from the sheets of betel vine was examined on the corn weevil (callosobruchus maculatus f.) and the beetle (sitophilus zeamais m.) and has been suggested as a potential crop protective agent (nair and kavrekar, 2017). piperaceae eo has been extensively researched for its larvicidal role in mosquito larvae control (huong et al., 2019; alves et al., 2021). piper permucronatum, piper arboreum, piper gaudichaudi‐ anum, piper marginatum, piper longum, piper humaytanum, and piper aduncum eo have the ability to regulate aedes aegypti, anopheles gambiae, and culex quinquefasciatus (santana et al., 2015; takeara et al., 2017; silva et al., 2019; durofil et al., 2021). piper aduncum was found to be larvicidal, killing aedes aegypti mosquito larvae at concentrations of 500 and 1000 ppm (oliveira et al., 2013; martianasari and hamid, 2019) .within a few days of being exposed to similar amounts of piper marginatum eo, 100% of aedes aegypti larvae died (santana et al., 2015; marques and kaplan, 2015). the eo of betel vine has regulated the population as well as various stages of mosquito (aedes aegypti) and acts as an alternative bioinsecticide (martianasari and hamid, 2019). antimicrobial activity the antibacterial properties of the eo obtained from the leaves are impressive (prasetya et al., 2021). they prevent bacteria from adhering to initial tooth plaque (punuri et al., 2012). the antifungal activities of magahi variant betel leaf eo were inves­ tigated by (madhumita et al., 2019; madhumita et al., 2020). towards aspergillus flavus, the minimum level of eo inhibition (mic) from leaves was found to be 0.8 ml/l chavicol, allylpyrocatechol diacetate, chavibetol acetate, propenylphenols, hydroxychavi­ col, and chavibetol are some of the aromatic com­ pounds discovered in the chloroform extracts (aliahmat et al., 2012). antibacterial properties against proteus vulgaris, escherichia coli, staphylococcus aureus, and streptococcus pyogenes fig. 5 ­ various biochemical activities of phytochemicals extrac­ ted from piper betle. sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 69 were investigated (chakraborthy and shah, 2011; patra et al., 2014; nayaka et al., 2021) (table 2). the drug was used as a solution against chosen pathogens such as streptococcus pyogenes . additionally, antifreeze action was evaluated on pathogenic microorganisms, such as escherichia coli, staphylococcus aureus, and pseudomonas aeruginosa (lubis and marlisa, 2020; nayaka et al., 2021; nguyen et al., 2021) for several species of dried betel leaves (desawari, desi, bangladesh, and jaleswar). cold aqueous, methanol (80%), ethanol (70%), and ethyl acetate (80%) solvent removal techniques were achieved for the dried leaf extract. it has been shown that the variety of bangladeshi and jaleswar betel leaf extracts are a potent and efficient source of antibacterial herbal medicines (agarwal et al., 2012; valle et al., 2021). the ethanol compound from betel leaves appeared to be quite active at preventing the proliferation of harmful pathogens such as staphylococcus aureus, vibrio cholerae, and e. coli hoque et al., 2011; valle et al., 2021). the combina­ tion of betel and red betel exhibited reduced action against staphylococcus aureus, escherichia coli, stephylococcus epidermidis (hartini et al., 2018). the model influence of betel leaf eo of ‘meetha’ was explored by (basak, 2018) on the aspergillus flavus germination period and the population spore of penicillium. antigiardial assay giardiasis is regarded by humans worldwide as the most frequent protozoan diarrheal illness. researchers have sought out novel, herbal medicines that might replace commercial pharmaceuticals with unpleasant potential side effects in the treatment of giardia (nazer et al., 2019). the betel vine extracts table 2 ­ different therapeutic activities of piper betle extract sl no. piper betle extract function result references 1 aqueous extract antimicrobial activity in different microorganism by disc diffusion method. antihemolytic and ant­ ioxidative activity in staphylococcus aureus, streptococcus pyogenes, pseudomonas aeruginosa and escherichia coli). the bacteria were effectively inhibited by aqueous extracts. the high content and combined action of flavonoids and polyphenols were linked to the antioxidative and antihemolytic properties. rai et al., 2011; shameem et al., 2013 2 spray dried powder extract antidiabetic activity in diabetes mellitus patients. piper betle, as a nutraceutical, has been identified as a possible therapy for type 2 diabetic patients. chakraborty and shah, 2011 3 aqueous and ethanol extract antibacterial activity in gram positive bacteria such as staphylococcus aureus, bacillus subtilis and micrococcus luteus and also gram negative bacteria like pseudomonas aeruginosa and escherichia coli by agar diffusion method. according to the findings, both aqueous and alcoholic extracts are potent against bacteria types that are major causes of illnesses. arawwawala et al., 2011 4 the hot water extract gastroprotective activity. due to its antioxidant and mucin­protecting characteristics, the study found that it can protect against indomethacin­induced stomach ulcers. kaveti et al., 2011 5 the methanolic extract anti­inflammatory and analgesic efficacy in swiss albino mice and wistar rats in a carrageenan­ induced hind paw edoema model, as well as hot plate, writhing, and formalin tests. the dosage considerably increased the pain threshold in the hot plate technique, significantly decreased the writhing generated by acetic acid, and dramatically inhibited carrageenan­induced paw edoema. pradhan et al., 2013 6 the piper betle plant extract antifertility activity in female rats. according to the findings, betel extract had antifertility and antiestrogen effects in female rats. akther et al., 2014 adv. hort. sci., 2022 36(1): 63­80 70 containing methanol, tetrahydrofuran, and water have the potential of anti­diarrheal activities (peckova et al., 2017). it has been examined for its physiological and pharmaceutical properties, but its significance for giardia intestinalis has yet to be proven. various solvents for extract preparations have been utilized in investigations of these effects. they discovered a significant reduction in cyst shed­ ding in the group of gerbils administered with the aqueous solution that matched the most relevant settings. the anti­adhesive effects on vasodilatory activities (runnie et al., 2004), antioxidant effects, early settlers (aara et al., 2020), and antihepatotoxic­ ity have already been demonstrated with aqueous excerpts of piper betel. antidiabetic activity more troublingly, the increase in the incidence of diabetes has an impact not only on advanced coun­ tries but also on emerging nations that have less money to deal with another serious illness burden again. present diabetes therapies can have side effects, so the move now to herbal remedies is more effective, cheaper, less risky, and less side­ effective. the present information reveals that the juice of the betel vine has assurance for antidia­ betes. betel vine leaves have been examined in induced diabetes rats (cee), normoglycaemic, and strepozotocin (stz) with anti­diabetic action assessed by oral hot water and ethanol extract (hwe) (khatun et al., 2016; arawwawala et al., 2011). blood glucose levels were dose­dependent in both hwe and cee in normoglycaemic rats. during the glucose tolerance process, both extracts consid­ erably lower the external glucose load. hwe has an antidiabetic action comparable to cee. both extracts have not been harmful and tolerated since pro­ longed oral administration (no open evidence of renotoxicity, and hepatotoxicity). moreover, the weight of the spleen was increased in treated groups, indicating lymph regenerative effects (khatun et al., 2016). betel leaf extraction is a useful anti­diabetic feature that enables blood sugar levels to be regulated. this investigation discovered betel vine extracts lowering the blood glucose level by the activation of insulin/biomimetic action and have a possible therapy for type­2 diabetic patients (arawwawala et al., 2011) (table 2). increased transaminase activity (sgpt and sgot) has been found in several investigations in liver and serum diabetic rats (ramachandran et al., 2012). gastroprotective activity extraction of betel vine was also found to be mediated gastro defensive activity, resulting in (i) an increase in the production of mucus and/or bicarbon­ ate, (ii) a decrease in the amount of stomach acid secreted, or (i i i) a decrease in gastric acidity (arawwawala et al., 2014; ahmed et al., 2021).the leaf extracts have marked gastroprotective proper­ ties which are shown by the strong (p≤0.05) inhibi­ tion of gastric lesions caused by absolute ethanol (in terms of duration and number) (arawwawala et al., 2014). cee gastric activity was comparable with hae activity. the existence of alkaloids, flavonoids, steroids, saponins, and tannins has been seen by a phytochemical sampling of betel vine (syahidah et al., 2017; altemimi et al., 2017). polyphenols, espe­ cially tannins, are antioxidant phytochemicals and can protect against indomethacin­induced stomach ulcers (neyres et al., 2012; zakaria et al, 2015; sharifi­rad et al., 2018; ahmed et al., 2021) (table 2). the antiulcerogenic properties of these compounds were also shown by their protein­prone and blood pressure effects. powerful gastroprotective behaviors were demonstrated (berenguer et al., 2006). thus, the gastroprotective effect can also be influenced by secondary metabolites, such as alkaloids, saponins, tannin, flavonoids, and other phenolic compounds (barbosa et al., 2019). anti‐asthmatic effect betel vine’s antioxidant, anti­inflammatory, and antihistamine activity have been linked to a wide range of diseases (alam et al., 2013; aara et al., 2020; ahmed et al., 2021; clemen­pascual et al., 2022). the anti­asthmatic activity of betel vine in guinea pigs has been tested (misra et al., 2014). asthma is the tra­ cheobronchial smooth muscle’s hyper reactance to a multitude of stimuli (chapman and irvin, 2015). bronchitis is a chronic inflammatory disease. bronchial asthma may be caused by free radicals and superoxide (phaniendra et al., 2015; boukhenouna et al., 2018). histamine has the potential to produce bronchoconstriction (yamauchi and ogasawara, 2019). the extract can substantially minimize the impact of bronchial asthma, but it has fewer effects than di­phenylhydramine. but other mediators such as leukotriene play a vital part in asthma in humans. betel vine has been reported to have the ability to reduce bronchial asthma in guinea pigs, despite its weak influence on human asthma (darvhekar et al., 2011; misra et al., 2014; rekha et al., 2014; ahmad et sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 71 al., 2021). role of betel leaf extract in thyroid disease ethylacetate piper betle l. (epbl) extract adminis­ tration reverted the t4­induced rise in serum thyroid hormones, heli marker enzymes, mda, and looh, but improved antioxidant enzyme activity and decreased the content of glutathione. lighter results from liver histology show that the epbl administra­ tion has enhanced twisted hepatic tissue architecture in hyperthyroid species. analysis of the mass­spec­ troscopy of high­resolution liquid chromatography showed the presence of four primary glycosides, including quercetine, rutin, kaempferol, and luteo­ line. the antithyroidism of epbl was seen to be caused by hyperthyroidism induced by t4. epbl’s antithyroid and antioxidant properties may be attributable to the existence of extracted flavonoid glycosides in hyperthyroid animals which may have blocked the secretion of the thyroid hormone and translation of t4 into t3 by 5’di inhibitors (panda et al., 2018). anticancer activity anti­cancer agents with antioxidant activities may provide their beneficial effects by balancing ros, such that cancer cells do not proliferate when apop­ tosis is not allowed to occur (abrahim et al., 2012). chronic inflammation is the root cause of many human diseases, including cancer and tumors, according to experimental and clinical research (kangralkar and kulkarni, 2013). the betel leaf was used for irritation in the mouth cavity as a typical folk medication. mouth cancer is among the ten most common cancers, with 90% of cases occurring in southeast asia, where cigarette and smoking behav­ iors are common (jiang et al., 2019). one of the earli­ est studies (toprani and patel, 2013) discovered that topical treatment with leaf extracts inhibited ­ pinene­induced oral cancer in hamsters. it was also discovered that combining leaf extracts and turmeric into the dietary supplements was beneficial. curcuma manga, dendrophthoe pentandra, piper betle l., and catharanthus roseus extracts in breast cancer cell lines were explored as anti­cancer and radical free scavenging power (widowati et al., 2013; rekha et al., 2014). betel vine has reported cancer preventative effects (kudva et al., 2018; shukla et al., 2018; malkani et al., 2021; chowdhury and markus, 2022). supplementing leaf extract with potable water significantly reduced the concentration of benzo (a) pyrene­induced neoplasia of the forestomach. the leaf extracts contain anti­proliferative and preventa­ tive chemical potential and can thus be utilized to treat several conditions, including human lung cancer (banerjee and shah, 2014). cardioprotective action acute myocardial infarction is caused by an imbal­ ance between raised oxygen demand and decreased blood supply caused by prolonged ischemia (smilowitz et al., 2015). during ischemia, the heart is further damaged by the formation of ros. as a result, increasing antioxidant levels may help to pre­ vent future infarction (munzel et al., 2017; zhou et al., 2018). pre­treatment with betel vine enhanced hemodynamic and ventricular function parameters in rats with isoproterenol (isp)­induced myocardial infarction. it restored sod, cat, gsh, and gpx levels, decreased lipid peroxidation of the heart, and there­ fore lowered ck­mb isoenzyme and ldh leakage into the blood (arya et al., 2010). platelet hyperactivity, which leads to intravascular thrombosis, is a key component in the development of cardiovascular dis­ orders (kaur et al., 2018; alkarithi et al., 2021). anti‐malaria activity as compared to the well­known insect deterrent citronella oil, eo provided greater resistance against mosquito bites from anopheles stephensi and culex fatigans (johirul et al., 2016). the oil provided greater than 4h of resistance from anopheles stephensi and culex fatigans when sprinkled at a rate of 20l/cm2, whereas citronella oil provided only 2.2 and 2.6h of protection, correspondingly. as a result, the power of paan to resist mosquitos has been established (pal and chandrashekar, 2010; ibrahim et al., 2017; cang et al., 2020). antioxidant activity many researchers have investigated the antioxi­ dant activity of extracts using a variety of solvents and extraction times (alam et al., 2012; aliahmat et al., 2012). the properties of the leaf extracts from various solvents were determined using high­perfor­ mance liquid chromatography to calculate the oil­ water partition coefficient (hplc). due to their high oil­water partition coefficient, leaf phenolics have been shown to be less polar than other phenolic antioxidants. the investigation showed that the sol­ vent extraction and the period for preparing betel leaf extract (petroleum ether, methanol, water, adv. hort. sci., 2022 36(1): 63­80 72 and ethyl acetate) for use as a natural antioxidant are crucial acts against four different harmful bacteria, such as escherichia coli, staphylococcus aureus, streptococcus pyogenes, and proteus vulgaris, have been investigated by chakraborty and shah (2011), lubis and marlisa (2020) nayaka et al. (2021), and nguyen et al. (2021). others were isolated from these extracts, with few recognized and unknown metabolites. various analytical methods such as nmr, mass spectroscopy, and ir spectroscopy have been used for structural investigation. tbars and dpph methods conducted anti­oxidative investiga­ tions. abdullah et al. (2015) found antioxidant betel vine extract activity and its components. 1­1­ diphenyl­2­picrylhydrazyl (dpph) demonstrated that the bangla variety of betel vine is the best antioxi­ dant that can be combined with total phenol con­ tents and lower the strength of these respective extracts in a test for ethanol extracts of three vari­ eties (bangla, sweet, and mysore) of piper betle l. (swapnil et al., 2014; sarma et al., 2018). bangla extract column chromatography resulted in chavi­ betol (chv), allylpyrocatechol (apc) separation, and corresponding glucosides. similar chemical character­ istics of three piper betles were identified after hplc analysis (abdullah et al., 2015). 7. marketing and socio­economic status around 15 to 20 million indians regularly eat betel leaves, and there are an estimated 2 billion betel leaf consumers worldwide, proving the crop’s enormous economic potential (jeng et al., 2002). as for national employment generation, an estimated 15million peo­ ple in india depend on betel leaf production, process­ ing, handling, transportation, and selling as a source of income (jana, 1995). it has been reported that these varieties of betel leaf, such as nova cuttak, godi bangala, sanchi, and birkoli, are grown in coastal odisha. barajas’ construction, annual mainte­ nance, leasing costs, and input costs like labor and fertilizers are included in the cost of betel leaf pro­ duction, which are essential for their crop propaga­ tion. throughout the observation phase, each sea­ sonal and annual money expenditure in a barajas in coastal odisha was examined. patra and pradhan (2018) reported that us$ 474.20 and $ 596.70 were invested for watering and irrigation for one year. however, it is too costly for a cultivator. as a result, some cultivators are unable to hire labor and must rely on their own manpower to irrigate their crops. as a result, they are no longer charged for watering labor, and their annual financial investment is merely $ 122.50. as a result, excluding irrigation and water­ ing costs, the cultivator paid $ 21.00, $ 21.08, $ 51.37, and $ 7.90 for each harvesting in a year. cultivators have spent a lot of money on their tradi­ tional agriculture in the hopes of getting a high return on their investment from the local market. however, the area is well­known for its betel vine agriculture, and the market price is quite low. as a result, growers sell their crops through a middleman. thus, cultivators can earn money from two different sources. paan’s annual income from the local market is $ 131.72, with a middleman fee of $ 172.92, and income from the local market per harvesting is $ 16.47, $ 24.70, $ 32.93, $ 32.93, and $ 24.70. annual market prices varied depending on the purity of the paan. the local market rate was higher in the winter season, at $ 32.93, in the rainy season, at $ 24.70, and in the summer season, at $ 16.47, i.e. cheaper in all seasons. people worked 360 days a year in a paan baraja, plus 26 days of minor work within the baraja. laborers take five vacations each year, all of which are self­initiated. its economic operations are now restricted to the local and national levels (patra and pradhan, 2018). in tropical nations, cash crops such as cocoa, cola nuts, coffee, citrus fruits, and other high­income­generating crops were more highly val­ ued than the production of betel vine. several unidentified infections and insects harm betel vine cultivation, resulting in significant losses for growers (vishwakarma and purohit, 2020). another issue is seedling transportation. it happens when seedlings are damaged. transportation, too many middlemen, a lack of grading, price fluctuations, and a lack of financial resources were all issues in marketing. the intensity of pests and illnesses, lack of water, soil quality, and the frequency of rains and winds were all agro­biological issues that limited productivity (bar et al., 2020). the primary reasons for the low betel leaf output are conventionally handled operations, uned­ ucated laborer, and inferior planting materials. 8. diseases “foot rot,” “leaf spot,” “powdery mildew” and“collar rot” are major diseases that affect betel vines sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 73 (vijayakumar and arumugam, 2014). foot rot phytophthora spp. causes the most common fun­ gal illness. the plantation suffers from foot rot (haider et al., 2013; meszka and michalecka, 2016). phytophthora species was discovered in 1927, which was eventually recognised as p. nicotianae var. para­ sitica (meng et al., 2014). foot rot induced by p. para‐ sitica and phythium vexans de bery (phythium piper‐ inum dastur) was documented by (haider et al., 2013). the lamina of the leaves begins to drop gradu­ ally, while the petiole remains upright. this sickness is known in the area as ‘khada kala’, ‘khada pacha’, or ‘madua’. local growers are spraying blitox and tegron drugs to avoid the sickness from spreading (haider et al., 2013). sclerotium rolfsii causes foot and root rot which is the most devastating disease that decreases the production of betel leaf (rahman et al., 2021). leaf spot patra and pradhan (2018) reported a leaf spot dis­ ease induced by fusarium semitectum. berk. et rav. singh and shanker (1971) described infections induced by cladosporium pipericola, drechslera ros‐ trata, corynespora cassicola, and cercospora piperis‐ betle in madhya pradesh and uttar pradesh, india (maiti and sen, 1979). the sickness is called “champa fulia” or “champa tipa” in the area. the leaves’ tips are tiny and curling. farmers must cut sick leaves as soon as they are spotted, or the virus may migrate to the main stem of betel vines in a few days. it was also carried by ants and insects from one vine to another. this disease is more prevalent during the wet season. farmers apply diethen­m 45 and urea water to these diseases to eradicate them (maiti and sen, 1979). powdery mildew in india, the disorder was discovered in mysore by narasimhan in 1933. oidium piperis uppal is the major cause of powdery mildew disease (park et al., 2012). this ailment is referred to as ‘jhalma’ in the local community. on the bottom of the leaves, the infection appears as white to light brown powdery patches. at this phase, little white and black particles can be seen in the upper and lower regions of the leaves. this is a very communicable disease. there is no way to prevent or treat this illness. farmers use a combination of dried fruit dust and neem tree (azadirachta indica a. juss.) leaf juice mixed with water to dust betel vine leaves (patra and pradhan, 2018). collar rot sclerotium rolfsii sacc., is a soil­borne fungus, infecting the collar and root parts of various crops, its growth, development, and pathogenicity were dependent on environmental factors like tempera­ ture, relative humidity, and rainfall (garibaldi et al., 2013). it is confirmed that s. rolfsii caused collar rot disease in betel vine which is reported to garain et al. (2021) and cause 17­100% crop loss in west bengal. one hundred twenty million kg of eo is produced globally from approximately 300 crops, which are worth about $4 billion, including 4% production from india (shukla, 2015). the eo extracted from coastal areas of odisha and obtained from different varieties such as chandrakala (0.42%), godi bangla (0.37%), balia (0.35%), desibangla (0.32%), maghai (0.30%), dandabalunga (0.20%), nahua (0.15%), and karpada (0.15%) (das et al., 2016). the leaves have indispens­ able oils which are known for their anti­allergic, anti­ cancer, insecticidal, antibacterial, and antioxidant properties (seow et al., 2014), making them healthy food preservatives with significant customer interest. the application of an alternative food preservative to prevent microbial spoiling is necessitated by the growing customer desire for natural products (mandal et al., 2014; roy and guha, 2021). besides being possible natural food preservatives, these oils could also have a future in novel food packets due to their antibacterial and antioxidant properties (ei asbahani et al., 2015; roy and guha, 2021), as well as being a viable and appealing flavoring component for the food and beverage sectors. the eo ingredients added in ice cream, chocolate, suji halwa, cupcake, lozenge, rosogolla, etc. (guha and nandi, 2019). basak (2018) reported that the eo of betel vine (var. tamluk mitha) has many potentials as a natural preservative in the food sector due to its safety and antimicrobial effectivity without affecting the sensory qualities of the food products. as a result, around rs 30­40 million worth of leaves are sold to nations like italy, great britain, bahrain, hong kong, pakistan, canada, kuwait, saudi arabia, nepal, and many other european countries each year (pandey et al., 2018). clearly, this shows the crop’s ability to generate for­ eign money, which should be enhanced in the nation­ al interest. export policy decisions may be modified to increase betel leaf exports, in addition to an ade­ quate study on export systems and intelligence. rural adv. hort. sci., 2022 36(1): 63­80 74 and urban populations are increasingly using the plant for culinary and ethnomedicinal purposes, which has led to an increase in demand. cultivation and preservation of betel vine in rural areas should receive renewed attention in order to prevent the extinction of these species and to provide economic benefits to rural populations. 9. future studies the leaves are frequently used as remedies because they contain important bioactive compo­ nents. due to their inexpensive cost and ease of usage, they are widely utilized in india and abroad. to cure alcoholism, bronchitis, asthma, leprosy, and dyspepsia, it can be taken as a dietary additive or taken separately (peddapalli et al., 2020). because betel leaves decompose quickly, they cannot be pre­ served for long periods of time. as a result, extra leaves are dumped aside or sold as cow feed in the marketplace. in this scenario, improved research approaches should be used to extend the shelf life of leaves. usually, several diseases and insects attack the betel vine throughout production, causing signifi­ cant losses to the growers. microbes, parasites, and other contaminants can readily contaminate the leaves during preservation and transit. as a result, garbage utilization in the manufacturing industry is one of the most significant and difficult occupations on the globe. there are only a limited number of studies on the use of leaf isolates and eo. because there is a dearth of study in this field, we should con­ centrate on its possible technologies in a variety of procedures such as food applications, pharmaceutical industries, cosmetic industries, and so on. betel leaf post­harvest damages can be reduced by using supe­ rior storage techniques. the functioning of each unit (manufacturing, processing, packaging, handling, transportation, and marketing) of betel leaves affects the majority of individuals directly or indirectly. based on the foregoing, the federal and state gov­ ernments should collaborate more effectively to fund diverse initiatives. a research and development board should also be established by the government. this could help keep the price of betel leaves stable. this also aids in the expansion of cultivation and export­oriented operations, the development of con­ servation methods, and the reduction of waste and by outflow, among other things. if farmers, scientists, technicians, and researchers work together to resolve the limits, the economy and job prospects will grow. 10. conclusions this review article looked at the cultivation, nutri­ tional components, pharmaceuticals compounds, pharmacological activities, antimicrobial compounds, diseases, and economy. betel vine has ample of vari­ ous metabolites such as phenolic tannins, anthraquinones, flavanoids, alkaloids, terpenoids, saponins, caediac possess more number of pharmaco­ logical activities and responsible for health benefits. as a result, there is a rising demand for using betel leaf extract and eo in a variety of industrial uses, including food supplements, cosmetics, and pharma­ ceuticals. therefore, farmers of coastal odisha have completely depended on the economy of betel vine cultivation and marketing at the national and interna­ tional level. sometimes, the betel vine is plagued by many diseases during cultivation, which generates a huge loss for farmers. the betel vine production was devastated due to being infected with several fungal pathogens resulting in foot rot, leaf spot, powdery mildew, and collar rot. in addition, this study discuss­ es the recently accepted extraction technologies and characterization, both of which are critical for the growth of the product’s economic worth. bioactive chemicals from betel vine should be isolated, extract­ ed using common and advanced technologies, and characterized for further bioactive properties, such as antibacterial activity, antioxidant activity, antidiabetic activity, anticancer activity, and so on. such potential of betel vine made itself a green medicine in nature. it concludes that the betel vine keeps promising as a natural reservoir with regard to its nutritional, phar­ macological and economical aspects for the rapidly growing human population. acknowledgements the grant from odisha higher education programme for excellence and equity (ohepee) to the centre of excellence for bioresource management and energy conservation material development, fakir mohan university is gratefully acknowledged. the funds and labo­ ratory facilities provided by the p.g. department of biosciences and biotechnology, fm university is also grate­ fully acknowledged. the advice of prof. s.p. adhikary, mentor is thankfully acknowledged. sahu et al. ‐ nutritional, pharmacological and economical role of betel vine 75 references aara a., chappidi v., ramadas m.n., 2020 ­ antioxidant activity of eugenol 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olive fruits f. zare 1, g. najafi 1 (*), t. tavakoli hashjin 1, a.m. kermani 2, p. ghiasi 1 1 biosystems engineering department, tarbiat modares university (tmu), tehran, iran. 2 department of agro‐technology engineering, college of aburaihan, university of tehran, tehran, iran. key words: collector systems, harvesting damage, manual harvest, mechanical harvesting, olive tree. abtract: the objective of this study was to measure and evaluate the perfor­ mance of a new pneumatic harvester (nph) for harvesting the olives fruit and compare the results with those of similar cases. the study involves two sec­ tions, namely, the nph and the collector system. two oily types of olive (‘mari’ and ‘yellow’) were selected to evaluate the nph and the collector system. the randomized complete block design and duncan’s multi­scope test were used for variance analysis and means comparison purposes, respectively. the depen­ dent parameters used to evaluate the nph were harvesting productivity, har­ vesting efficiency, and leave downfall percent. also, the fruit damage was selected for the collector system. results showed that the nph can harvest 92% of olive fruits. the highest amount of harvesting productivity belongs to nph of 29.47 kg/h. the percent of leaves downfall in the harvesting process was found to be 2.55%. using the collector system can reduce the level of damaged fruit from 60.8% to 25.12% and from 60.54% to 24.54% in the mari and yellow vari­ ety. 1. introduction the olive tree is one of the mediterranean plants that its fruits used as a cannery and oil extraction (barbera et al., 2013). olive has traditionally been known among people, which is also important for health and used as a strategic food in some countries (fiorino et al., 2010). one of the major problems with the olive plant is harvesting which is mostly done in different ways: (a) picking up the fruit on the ground (b) harvesting manually (c) harvesting via branch shaker (d) harvesting mechanically (lupi et al., 2012). harvesting is the final step in field pro­ duction of olive fruits, but if done at the wrong time or in the wrong way it can markedly affect the grower’s net return. the economic success of super­intensive olive plantations is mainly achieved due to the full mecha­ nization of harvesting (ottanelli et al., 2019). the olive harvest is expen­ (*) corresponding author: g.najafi14@modares.ac.ir citation: zare f., najafi g., tavakoli hashjin t., ker­ mani a., ghiasi p., 2021 ­ a new pneumatic har‐ vester for improvement and facilitation the har‐ vesting of the olive fruits. ­ adv. hort. sci., 35(1): 43­51 copyright: © 2021 zare f., najafi g., tavakoli hashjin t., kermani a., ghiasi p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 july 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9609 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 43­51 44 sive in terms of wages and adequate labor supply has brought about many problems (zipori et al., 2014). fruit damage, damages related to the olive tree, quality reduction, human injuries, time consumption, and costly process in the traditional ways are prob­ lems existing in the harvesting process. mechanized harvesting is done in many different types, such as the trunk and limb shakers. because of device fea­ ture like power supplement implementation of this shakers are difficult (çakmak et al., 2011). although, the mechanized harvesting methods are very effi­ cient but they have an high usage costs. therefore, the best harvest system should be inexpensive and highly efficient. designing of shakers, harvesting efficiency analyz­ ing and tree damage for olive fruits are the attractive topics in the mechanization field, so in this regards researchers studied the effect of shaking force and the amplitude of the shaking on the mechanized har­ vesting of olive. they showed that an increase in olive tree diameters trunks led to an increase in shak­ ing force and a decrease in amplitude of the shaking. so, for mechanized harvesting in the big olive trees with big trunk diameters more power must be applied (babanatsas et al., 2019). a comparative study was done to investigate the various ways of olive harvesting. results show that harvesting through shaking the stem is the best way to remove the olive fruit from a tree (yousefi et al., 2010). five different olive harvesters were investigated. the flat type olive harvester has more vibration value index (çakmak et al., 2011). the effect of two types of olive tree harvesting (trunk shaker and mechanical harvest aid) on harvesting time were studied. results show that harvesting the olive trees with mechanical harvest aid can save more time (nayeri and torkashvand, 2016). results concerning the examina­ tion of harvesting type on harvesting efficiency show that with increasing the fruit removal force the har­ vesting efficiency was decreased (zipori et al., 2014). a study on four types of olive harvester; plastic combs, small shakers, rotating combs, and vibrating combs were done. researchers indicated that all types of harvesters except the small shaker improve the worker’s productivity (bentaher and ben rouina, 2002). results of the study on a pneumatic harvester showed that manual harvesting and mechanical har­ vesting methods had a significant effect. the pneu­ matic comb machine had higher bruised fruit than the manual harvesting (ahmad, 2018). with an increase in the operating velocity, the productivity harvesting and damage percentage increased and the 1250 rpm operating velocity value could be a good alternative for harvesting (mansour et al., 2018). two methods of harvesting olive fruit, namely, mecha­ nized harvesting with pneumatic harvester (ph) and manual harvesting (mh) were investigated, which indicated that due to the presence of the collector system, using mechanized harvesting can reduce the fruit damages (plasquy et al., 2019). mechanical canopy and trunk shaking were investigated in line with the harvesting mechanization of table olive orchards. accumulating the trunk shaker and shaker combs can increase the bruise index of olive fruits. the lowest bruise index of olive fruits belongs to the manually­type harvest with a 0.5% value (sola­ guirado et al., 2020). the designing and fabrication of olive fruit har­ vester have long since been the subject of many types of research. applying some of these systems may exert large costs on farmers, and the efficiency of these machines requires significant economic costs that many producers cannot afford. for this large group of farmers, new models have recently been presented. nevertheless, the use and utility of some of these machines still entail difficulties, such as the large weight, difficult access to the trunks, or handling the fruit boxes. in this study, results con­ cerning the evaluation of a new design of portable pneumatic olive harvester with a fruit collector sys­ tem are presented to fix some of the harvesting problems. the evaluation parameters of harvesting machine (such as: the harvesting efficiency, harvest­ ing productivity, and olive tree damage) were com­ pared both with the manual method and similar har­ vesting methods. finally, the effect of fruit collector on fruit damage was investigated. 2. materials and methods vegetal material two different varieties (‘mari’ and ‘yellow’) of olive fruits were selected to evaluation that both of them are iranian variety and commonly grown for oil and canned production. the first step in designing a new harvesting machine is cognition about crop char­ acteristics such as detachment force from tree branches. for measuring fruit detachment force from tree branches, three tree of each verity were select­ ed and ten olive fruits of each tree accidentally were selected. the force required to detach them was zare et al. ‐ a new pneumatic harvester for olive fruits harvesting 45 measured by a mechanical force gauge. the mechani­ cal gauge of fg­5020 model was used and calibrated in the precision process calibration laboratory with the approval of no. 681 of the standard institute of iran. the fruit detachment force employed to deter­ mine the applied force on the pneumatic jack. so to determine this amount assuming that all of the jaw surface covered by the olive fruit and the detach­ ment force in this situation was calculated. tests were conducted in the ashrafieh gardens in qazvin province in iran and the fruit ripening index was 4.52%. the age of olive trees was 20 years and the height of trees was up to 3.5 m. harvester machine design the pipe diameter is a very important factor for designing pneumatic systems. when the low pipe diameter was applied, the pressure dropped and the airflow velocity increased accordingly. in the case of water penetration into the pipe, the clogged pipes situation was expected to happen. two conditions, namely, 5­8 m/s and 0.5 bar were found to be appro­ priate for the airflow velocity and the pressure dropped. the first step for jack design is to define the required pressure and velocity of air to detach the olives from the tree. equation (1) shows the jack pis­ ton, which was used to calculate the air force. fm = p x a (1) after selecting jaws frequency and active volume of the piston, the cam length and air mass flow (amf) were calculated. the amount of amf for jaw movement to open the jaw was calculated by equa­ tion (2), and in the closed cycle, equation (3) was used for the amf. finally, the total amf to open and close the jaws was calculated by equation (4). qext = p x (d2/4) x c x n x (p1 ‐ p0)/p0 (2) qret = p x [(d2­d2)/4] x c x n x (p1 ‐ p0)/p0 (3) qtot = p x [(2d2­d2)/4] x c x n x (p1 ‐ p0)/p0 (4) assuming that the total pneumatic energy was converted to the kinematic energy, the maximum velocity could be determined using equation (5). e = ½ m (nmax) 2 (5) the total energy required was calculated by equa­ tion (6). e = [½ p x pd2 x m (0.5)2] / (2g x 4) (6) the total energy for opening the jaw was calculat­ ed by equation (7): e = 5 x 10­4 x p (d2 ‐ d2) (7) the determinations show that the detachment force of olive fruit from the tree and the pressure required for the pneumatic jack were 10 n and 7 bar, respectively. the machine had one handle and it was gripped by one operator’s hand and a telescoping rod up to a maximum length of 3 m. the harvesting mechanism had two jaws and each jaw contained 7 fingers that allowed for easy and deep access to all types of foliage without getting caught in the branch­ es. 90 degrees was selected for the angle between two jaws based on the previous researches. the jaws had 18 cm length and the distance between them must be equal to the olive fruit width, so 1 cm dis­ tance was considered. we assume that in every impact of jaws, 23 olive fruits were detached from tree branches. so, the total force required for each impact was determined. the force related to the pneumatic cylinder was determined by equation (8). 2fd cos (ɵ/2) = f (8) the amount of force of each jaw for the olive fruit detached from the tree was 325.3 n, and for calculat­ ing the piston diameters equation (9) was used. f = a x p (9) after determining the piston diameters, the length of the cam was calculated and the standard jack with these characters was selected. the cylinder provides a closed system which assists the movement of the piston. for moving the piston across the cylinder in high frequency, valves must be used or the manual control applied. because of the operation conditions, the pneumatic valve was selected to change the airflow direction. when the pneumatic valve was in position a, high air pressure pushed the piston to the right side (fig. 1). by moving the piston to right, the airflow was transferred to the c input of the pneumatic valve which changed the direction of airflow, resulting in the activation of the b position of the pneumatic valve. when high­pres­ adv. hort. sci., 2021 35(1): 43­51 46 sure air moves to the b output of the pneumatic valve, the piston moves to the left side, and air moves toward the d input of the pneumatic valve, and such a cycle is replicated. after assembling the parts, the frequencies of these cycles were measured by an electronic eyes sensor. the frequency of the piston movement is 5.21 hz in 7 bar air pressure. so, after defining design parameters, the mechanical value of no. g1/8’’ was selected. other parts of the harvester such as the chain, cam, and pneumatic value along with the parts were designed. figure 2 shows the details of nph, including har­ vester jaws, cylinder and pneumatic valve, power transmission, and telescoping rod. the common properties of the collector system for olive fruit are shown in table 1. by applying this system, the olive fruits falling from the tree were col­ lected into a box. the driving power of the collector system are provided by labor. it is possible to conclude from figure 3 that the collector system is ready to collect the fruits in the box. the collector system includes the wheels, steer­ ing wheel, bendable bars, wire for hold cover, poly­ ester cover, trunk holder, position for boxes, and out­ let, all of which are assembled on chassis. statistical design the variance analysis was done in randomized complete block design with three replicate and the statistical computations as well as analyzing the data were performed by the mstat­c software. duncan’s multi­scope test was used for comparison of the means. the performance of the developed harvest machine was measured by the harvesting productivity (hp), harvesting efficiency (he), and leaves downfall per­ cent (ld). the performance of the developed collec­ tor system was measured by the olive fruit damage (fd). for measuring the harvesting productivity of olive fruit harvester, the total mass of detachment fruits was recorded using the machine. the total har­ vesting time includes selecting, detaching fruit, and the time required for moving machine between olive trees inside the field. the productivity of the operat­ ed harvester was calculated using equation (10) (polat et al., 2007): hp = k1/t (10) harvesting efficiency was calculated by equation (11) (srivastava et al., 2006). he = [k1/(k2 + k1)] x 100 (11) fig. 1 ­ schematic of the piston, cylinder, valve, and connector pipes fig. 2 ­ the different parts of the new pneumatic harvester. table 1 ­ collector system properties parameters value chasses length 140 cm chasses width 95 cm trunk holder height 60 cm trunk diameter up to 40 cm cover diameter 300 cm fig. 3 ­ collector system parts. zare et al. ‐ a new pneumatic harvester for olive fruits harvesting 47 the percent of olive leaves downfall is defined as the percent of branches and leaves fall down by the harvester and was calculated by equation (12): td = mbl/ mtbl x 100 (12) the total weight of branches and leaves of the olive trees previously measured. some trees that had to be removed were collected and weighed separately, finally the means weight of the branches and leaves of three trees were used in the equation (12). fruit damage includes the visual inspection and the fruit appearing broken to the naked eye. fruit damage was calculated by equation (13). fd = md/ mt x 100 (13) the main criteria for designing the new pneumatic harvester (nph) were as follows: easy design, simple to use, low weight, high productivity, less fruit damage, and low operating costs. the main novelty of this type of harvester is low operating costs and use in gardens far from the energy net­ works. a double­sided jack was used to provide jaw movement so, the required airflow rate, pressure drop, airflow control valve, and jack size must be defined. to show the advantages and disadvantages of the present device with existing devices, the results of the evolution of the nph compared with a pneumatic harvester (ph) that the features of the ph are shown in table 2. 3. results and discussion results of the harvesting tests harvesting productivity was calculated among 3 types of different harvesters’ system (mh, ph and nph), using the equation number “10” (table 3). results showed that the three harvesters have a sig­ nificant difference at 5% levels of probability. due to different treatments, the means were compared and the nph was found to have more hp placed in a group (a) with an amount of 29.47 kg/h. the ph was placed in a group (b) with an amount of 21.33 kg/h and the mh was placed in a group (c) with an amount of 9.37 kg/h. figure 4 shows the hp means for three harvest methods. due to the special properties of nph such as the power of vibration, frequency, and shape of a bar, the hp of this machine was more when com­ pared to two other methods of harvesting. kermani (2016) indicated that the mounted branches shaker machine has 130.72 kg/h of hp, but it fails to be used in traditional gardens. this type of harvester needs a tractor to perform, which is not feasible for small gar­ dens (kermani, 2016). table 4 shows the results of variance analysis related to the difference between two types of har­ vesting machine concerning the harvesting efficiency (he). the analysis shows that the effect of machine type on he is significant at 1% probability. the pneu­ matic harvester can detach 86% of olives from the tree, but nph shares 92% of he. because of the avail­ ability of all olives on the tree, the he of this method table 2 ­ ph technical features number feature amount 1 model campagnola srl 2 weight 1 kg 3 length 1.8 ­ 3 m 4 max pressure 7 bar table 3 ­ result of variance analysis velocity (m s­1) for harvest­ ing productivity parameters df sum of squares means of squares f block 2 34.925 17.462 1.6126 * harvest method 2 283.554 141.777 13.093 * total 4 43.314 10.828 cv 25% fig. 4 ­ harvesting productivy means for three harvesting methods. nph= new pneumatic harvester; ph= pneuma­ tic harvester; mh= manual harvesting. * = significant at 5% of probability. ** = significant at 1% of probability. ns = not significant. 48 adv. hort. sci., 2021 35(1): 43­51 is considered as 100% for labor in the case of manual harvest (fig. 5). it is very important to understand how to place the harvesting head on the telescoping rod. the angle between the harvester head and telescoping rod in the nph is fixed and the jaws move between 123 to 213 degrees, and such a property differs from the ph. the other important difference resulting in increased he is the jaws speed, where they move quicker in nph compared to the ph. some places of olive fruits on the tree are not available for nph and ph. the harvester rod plays the important role in the he. whatever the harvesters rod has high maneuver­ ability the more olive fruits can detected and the he increased. kermani (2016) showed that the olive tree branches shaker machine has 84.51% he, but com­ pared to other machines, the leaves downfall percent in this harvesting method is high (kermani, 2016). equation 12 was used to determine leaves down­ fall percent (ld). after harvesting, all the branches and leaves were collected and weighted. by increas­ ing in branches and leaves that fall from the tree the ld was increased. table 5 shows the variance analy­ sis among the three harvesting methods and olives types. the effect of variety (type) on ld was not sig­ nificant, but harvesting methods have a significant effect on ld. figure 6 shows the ld for three harvest­ ing methods; nph has more ld than the two other harvesting methods. the speed of jaws in the ph is 1080­1150 rpm, while in the nph the minimum speed of jaws is 1290 rpm. this difference in jaws speed led to an increase in the leaves’ downfall per­ cent. comparison of the means of harvesting method shows that the nph with an amount of 2.52% ld has the highest detachment of leaves from the tree. the ph and mh were not significantly different, and they were placed in a group (b). the electrical rotation bar in kermani’s (2016) research has the highest ld with a 2.20% damage (kermani, 2016). due to the prob­ lems with the head location on the handle in the nph, the amount of ld was increased, however, such a problem can be surmounted. table 4 ­ result of variance analysis of for harvesting efficiency parameters df sum of squares means of squares f between data 1 717.883 717.838 118.644 ** inside data 4 24.203 6.05 cv 3.25% fig. 5 ­ harvesting efficiency means for three harvesting methods.nph= new pneumatic harvester; ph= pneuma­ tic harvester; mh= manual harvesting. table 5 ­ result of variance analysis for leaf downfall parameters df sum of squares means of squares f block 2 0.286 0.143 42503 ns harvest method 2 1.006 0.503 14.934 * total 4 0.135 0.034 cv 8.93% fig. 6 ­ leaf downfall (%) means for three harvesting methods. nph= new pneumatic harvester; ph= pneumatic harve­ ster; mh= manual harvesting. * = significant at 5% of probability. ** = significant at 1% of probability. ns = not significant. * = significant at 5% of probability. ** = significant at 1% of probability. ns = not significant. zare et al. ‐ a new pneumatic harvester for olive fruits harvesting 49 product quality results with the use of the collector system olive fruits fall on the ground after harvesting and get damaged as a result of this matter. to prevent the olive fruits from damage, a collector system gath­ ering the fruits into the box was designed and manu­ factured. for evaluating the collector system, two types of olive trees were harvested with the new pneumatic harvester, and the percent of damaged and undamaged olive fruits were investigated. table 6 shows the damaged and undamaged olive fruit in the nph with the collector systems and manual har­ vest without the collector system. results show that the use of a collector system can decrease the fruit damage (fd) from 60.80% to 25.12% in the ‘mari’ variety. similar results were achieved for the ‘yellow’ variety. table 6 shows the damaged and undamaged olive fruit for the harvest­ ing of ‘yellow’ olive fruit variety in the two harvesting methods. figure 7 shows the injured, bruised, and uninjured olive fruit percent in fourteen measured samples for the manual harvest. due to the differ­ ence in the weight of olive fruits, the percent of each group shown was based on the number and weight of olive fruits. in the manual harvest, a large portion of the fruits of the olive falls on the ground, then picked up and collected into the box. this process led to the bruising of more than half of the olive fruits. table 7 shows the means, standard deviation, aver­ age deviation, variance, minimum and maximum of weight along with the number of injured, bruised, and uninjured olive fruits in mh and nph. results show that after harvesting the fruits of the olive in the manually harvest method, 45.29% of olives fruits were bruised. bruising occurred on the skin of the olive fruits as a result of fruit hitting on the soil. also, the injuring situation occurred when fruits were hit by sharp branches or stones. the percent of injured, bruised, and uninjured olive fruits for nph based on the number and weight is shown in figure 8. the results improved significant­ ly. most parts of the olives fruit had no injury in the harvesting process. olive fruits that were detached from the tree fall in the collector systems and avoid contact with the stones or the soil/ground. table 7 also shows the results of measuring related to the injured olive fruit parameters. the means of unin­ table 6 ­ damaged and undamaged fruits in two types of harvesting method in mari and yellow variety cultivar manual harvesting new pneumatic harvester undamaged fruit (%) damaged fruit (%) undamaged fruit (%) damaged fruit (%) mari 39.2 60.8 74.88 25.12 yellow 39.45 60.54 75.45 24.54 fig. 7 ­ illustration of the injured, bruised, and no injured olive fruit in the manually harvest. harvesting type number of injured olives weight of injured olives number of bruised olives weight of bruised olives number of no injured olives weight of no injured olives manual harvesting 14.87±1.70 * 15.51±1.71 45.42±2.04 45.29±1.98 39.71±1.95 39.20±1.87 new pneumatic harvester 7.98±1.14 8.33±1.36 15.98±2.75 16.79±2.79 76.02±2.96 74.88±2.98 table 7 ­ results of measuring the injured parameters for the olive fruits in manual harvesting and new pneumatic harvester * means ± standard deviation. adv. hort. sci., 2021 35(1): 43­51 50 jured olive fruits in the nph increased from 39.71% to 76.02%. this means that the nph was able to save almost half of the olive fruits. the jaws hitting olive fruits and detached fruits from brunches after that fruits falling from the tree and collide with the branch and trunk of the tree. this process caused almost 24% damage to the olive fruits. 16% of the olive fruits in the harvesting process were bruised and the rest of the damaged fruits got the ruptured skin, which was then classified as the injured fruits. 4. conclusions harvesting is one of the important steps in the olive farming and the device that used also strongly effected on the quality of the harvesting. in this study, tried to investigate the new pneumatic harvester performance to show its advantage in compared with other pneumatic harvesters that exist in the market. when considering the fruit ripening index of 4.52%, one can say only 8% of olive fruits remained on the trees. on the other hand, the amount of hp was 29.47 kg/h. this result shows that nph can detach 92% of olives on the tree in a short time. in the case of the traditional and small gardens, this machine is very practical because it can work effectively in the garden in different situations. the collector system saves more time and prevents olive fruit from damage and can be easily used for every harvesting method. acknowledgements this research was supported by iran national science foundation (insf) with 92032254 number. we thank our colleagues from insf who provided insight and expertise that greatly assisted the research. references ahmad r.l., 2018 ­ efficiency of mechanical tools for olive harvest and effect on fruit quality. ­ acta horticulturae, 1199: 315­319. babanatsas t., glavan d.o., babanatis merce r.m., glavan a.i., 2019 ­ study of forces influencing the shaking parameters in 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d.r., 2006 ­ engineering principles of agricultural machines. ­ amer.soc. agric. biol. engineers, second edition, st. joseph, mi, usa. yousefi z., almassi m., zeinanloo a.a., moghadasi r., khorshidi m.b., 2010 ­ a comparative study of olive removal techniques and their effects on harvest productivity. ­ j. food, agric. environ., 8(1): 240­243. zipori i., dag a., tugendhaft y., birger r., 2014 ­ mechanical harvesting of table olives: harvest efficien‐ cy and fruit quality. ­ hortsci., 49(1): 55­58. impaginato 215 adv. hort. sci., 2022 36(3): 215­226 doi: 10.36253/ahsc­13045 genetic and ampelographic characteriza­ tion of grapevine accessions maintained in the lebanese national collection a. chehade 1, l. chalak 2, j. merheb 2, a. elbitar 3, e. rmeily 4, n. madi 2, m. massaad 2 (*) 1 lebanese agricultural research institute, tal aamara, department of plant biotechnology, plant genetic resources unit, p.o. box 287, zahleh, lebanon. 2 the lebanese university, faculty of agronomy, dekwane, beirut, lebanon. 3 lebanese agricultural research institute, tal aamara, department of plant biotechnology, plant tissue culture unit, p.o. box 287, zahleh, lebanon. 4 lebanese agricultural research institute, tal aamara, department of pomology and viticulture, p.o. box 287, zahleh, lebanon. key words: genetic diversity, issr markers, lebanon, local germplasm, vitis vine‐ fera l. abstract: safeguarding grapevine biodiversity is one of the main concerns in viticulture today. management of ex situ collections requires a comprehensive characterization of the conserved germplasm to provide basic material for selection and breeding programs. in this study, the local grapevine germplasm conserved at the national collection of the lebanese agricultural research institute and composed of 43 accessions,was submitted to a genetic and ampel­ ographic study. nine issr primers, previously developed in grapevine, were used in this study. these primers generated a total of 51 bands, of which 77.7% were polymorphic allowing the differentiation of 41 genetic profiles vs. one case of synonymy that was recorded among three accessions. ampelographic characterization was conducted using a set of 33 descriptors established by the international office of vine and wine and related to leaf, bunch and berry. principal component analysis identified 12 descriptors i.e. veraison date, matu­ rity date, berry length, upper and lower vein pigmentation, bunch density, bunch weight, sugar content at harvesting, flesh of juiciness, berry weight, flesh firmness and color skin, as being the most discriminating descriptors. the corre­ lation between the issr clustering and the ampelographic one was not signifi­ cant (r=0.26) because of the divergence of accessions groups, except for the three accessions synonymy case which was confirmed in both dendrograms. finally, this comprehensive evaluation of the existing local gene pool of grapevine revealed a substantial diversity. it would further allow the promo­ tion of the valuable accessions directly through multiplication scheme, and their sustainable utilization in genetic improvement programs. 1. introduction one of the main concerns for public research in viticulture today is the (*) corresponding author: mmassaad1@gmail.com citation: chehade a., chalak l., rmeily e., elbitar a., merheb j., madi n., massaad m., 2022 ­ genetic and ampelographic characterization of grapevine accessions maintained in the lebanese national collection. ­ adv. hort. sci., 36(3): 215­ 226. copyright: © 2022 chehade a., chalak l., rmeily e., elbitar a., merheb j., madi n., massaad m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 april 2022 accepted for publication 24 august 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13045 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 215­226 216 need to safeguard grapevine biodiversity by estab­ lishing well managed national repositories to ensure germplasm availability to breeders, researchers and farmers (nass et al., 2012). there are presently around 130 grapevine germplasm collections across the word (dettweiler et al., 2000). one of the first grapevine germplasm collections is the one estab­ lished in spain in 1950, which contains more than 1066 accessions (ortiz et al., 2004). in france, more than 2,200 varieties originally collected from 35 countries are grouped together at the national institute of agronomic research (tessier et al., 1999). similar collections of grapevine were also established usa and in latin america containing grapevine acces­ sions collected all over the world (martinez et al., 2003). grapevine is one of the oldest fruit crops growing in the eastern mediterranean region includ­ ing lebanon (zohary, 2003). the country was among the first worldwide to have implemented vineyards which gradually became a traditional culture in lebanon for the production of both table grapes and wine (chalak et al., 2016). today viticulture occupies the eighth rank in the agricultural sector in the coun­ try, with a total production area of 9,240 hectares and an annual production of about 89,000 tons of table grapes (fa0, 2010; faostat, 2019) vs. 3,000 hectares dedicated to wine grape with an annual pro­ duction of approximately 10,000 tones (rahal, 2015). in addition, about 800 hectares of vineyards are intended for the production of lebanese arak (roby, 2003). commercial plantations of table grapes have long been constituted of four local varieties, com­ monly named ‘tfayfihi’, ‘baitamouni’, ‘maghdouche’ and ‘obeidy’. more recently, the new plantations in the country are mostly constituted of improved vari­ eties imported from europe and the united states. around 77 varieties are imported to lebanon. nevertheless, the major share went mostly to ‘cabernet sauvignon blanc’, ‘syrah’, ‘viognier’, ‘chardonnay’ and ‘merlot’ which represent 65% of the total saplings quantities imported during 2012­ 2014 (tabaja, 2015). the long history of viticulture in lebanon suggests the existence of a large indigenous germplasm associated to a wide range of traditional varieties that are well adapted to the various agro­cli­ matic conditions of the country (chalak et al., 2016). in lebanon and despite the increasing interest on the conservation and characterization of plant genetic resources in general, only few studies have addressed the local germplasm of grapevine, although it is threatened by various anthropogenic pressures including in particular the progressive replacement of local varieties by more advantageous improved for­ eign varieties that are regularly imported into the country (riachi, 1998; madi, 2007; chalak et al., 2016; rahme, 2016). to face the threat of genetic erosion and avoid the loss of traditional germplasm, the lebanese agricultural research institute (lari, tal amara station) established in 1998 a national grapevine collection containing numerous local acces­ sions collected from different production areas across the country (madi, 2007). within the environmental change and the challenging predictions for the eastern mediterranean region including lebanon (santillán et al., 2019), there is a growing need to address the local genetic resources such as traditional varieties which are recognized to be more adapted to harsh conditions and having better tolerance to vari­ ous biotic and abiotic stresses (fao, 2015). added to this, the wine industry is witnessing a new trend towards the utilization of the local traditional varieties in the production of typical and well prized wines. therefore clonal selection, with the respect to specific traits, has become the most important way to improve the quality of grape cultivars. as a consequence, there is a need for reliable and precise methods of clonal characterization for further use by breeders, nurseries and industries (galet, 1998; moreno et al., 1998). ampelography is a scien­ tific methodology that has long been the single method used for the characterization of grapevine phenotype, based on the description of different morphological, phenological and pomological charac­ ters (galet, 1998; sabir et al., 2009; laucou et al., 2011). in addition to the ampelograhic description, in order to discriminate the varieties, synonyms, homonyms and the variation among the accessions, molecular methods has become more frequently used especially markers based on polymerase chain reaction techniques (riaz et al., 2012; madhumati, 2014). among the molecular approaches, the issr (inter sequence simple repeat) has being evaluated for its usefulness in grapevine cultivar identification and in assessing genetic diversity of grapevine germplasms (moreno et al., 1998; dhanorkar et al., 2005; santiago et al., 2005; this et al., 2006; sabir et al., 2009; seyedimoradi et al., 2012; choudhary et al., 2014; castro et al., 2016). this study aimed to characterize the 43 local grape accessions preserved at the lebanese national collection of grapevine at lari using both issr mark­ ers and ampelographic descriptors. this work was conducted in an attempt to evaluate the diversity of the local germplasm for further actions of conserva­ chehade et al. ‐ genetic diversity in lebanese grapevine germplasm 217 tion and sustainable utilization of grapevine genetic resources in diversification strategies. 2. materials and methods plant material a total of 43 accessions of traditional grapevine varieties growing in the lebanese national collection located in tal amara, bekaa (n 33° 49’ 59”, e 36° 0’ 0”, 908 m a.s.l.) established in 1998, were considered in this study (table 1). accessions initially consisted of farmer’s local varieties surveyed and collected across the bekaa region (lebanon). the vine cultivars were grafted with four duplications on b41 root­ stocks which were planted directly in the collection land a year before the grafting process under the same growing conditions using horizontal trellis train­ ing system with the spaces 2.75 x 2.75 m. the site of plantation is characterized by a clay fertile soil and an average of precipitation ranging between 600 to 700 mm/year. for each accession, young leaves were sampled from a single selected plant and stored at ­ 20°c for the dna extraction and molecular analysis. on the other hand, samples of ten leaves, five clus­ ters and 20 fully mature berries were taken from the same selected plant of each accession between august and october 2020 to undergo the ampelo­ graphic characterization. molecular characterization total genomic dna was extracted from 300 mg of young leaf tissue after been crushed to a fine powder with liquid nitrogen and stored at ­20°c until use. dna extraction of grapevine landraces was per­ formed according to (doyle and doyle, 1990) proto­ col with minor modifications (kafkas et al., 2006). dna concentration was measured by biospec­nano (shimadzu corp) and stored at ­20 c until further analysis. nine issr primers: ubc807, ubc812, ubc815, ubc816, ubc818, ubc828, ubc864, ubc868 and ubc880 (table 2) were selected and table 1 ­ sites of 43 collected samples of traditional cultivars grapevine growing in the national germplasm collection at the lebanese agricultural research institute (lari­tal amara) cultivars growing in the national germplasm collection at lari sites of collected samples region ainouni tamnin bekaa central al mir, ari, ashlamish ahmar, asouad, bakhouri, bourji, bzaz al­anzi, maksasi, mariami, moukh al­baghel, oubaidi, souri nabi ayla bekaa central amarcani abiad, amarcani asouad, baitamouni, baitamouni asouad, gbaii, maghdoushi, nih, tfaifihi, zaghzaghani niha bekaa central amarcani souri ablah bekaa central arasani, souri zahlaoui saghbin bekaa ouest ashlamish abiad, rins rayak bekaa central houzairani, khoudri, zaitouni el­rafid bekaa ouest karn al ghazal, maghdoushi mgaddad, misky, nabi, raha, samaani abiad, samani asouad, souri asouad, souri mhayyar, yafaoui, asali, boulbouli asouad, gnoubi unknown site bekaa table 2 ­ the resulting discriminating power, number of polymorphic and monomorphic, the size of bands of issr primers, percentage of polymorphism and the polymorphic information content primer size range (bp) number amplified bands number of polymorphic bands number of monomorphic bands polymorphism (%) pd pic ubc812 350­1000 7 6 1 85.7 0.64 0.33 ubc818 300­700 7 6 1 85.7 0.87 0.59 ubc815 400­650 4 3 1 75 0.62 0.6 ubc828 550­800 3 2 1 66.6 0.44 0.45 ubc864 280­700 7 6 1 85.7 0.9 0.48 ubc868 225­750 4 2 2 50 0.3 0.27 ubc816 300­900 5 4 1 80 0.64 0.64 ubc807 250­800 7 6 1 85.7 0.84 0.47 ubc880 125­600 7 6 1 85.7 0.85 0.58 total 125­1000 51 41 10 ­­­­­ ­­­­­ ­­­­­ mean 5.6 4.5 1.1 77.7 0.67 0.49 adv. hort. sci., 2022 36(3): 215­226 218 used in this study based on their good results for amplification and high power of discrimination on grapevine (seyedimoradi et al., 2012). the issr (inter simple sequence repeat) amplifi­ cation was carried out as per (sabir et al., 2009), using 20 µl reaction mixture containing 2 µl of 10 x pcr buffer (750 mm tris­hcl ph 8.8; 0.1% tween­20), 0.2 mm dntp, 2 mm mgcl2; 200 mm primer; 50 ng genomic dna and 1 u taq dna polymerase (mbi fermentas inc, hanover, md­21076, usa). the ampli­ fication program consisted of 94°c for 4 minutes fol­ lowed by 35 cycles of 94°c for 1minute, 52°c for 1.05 minutes, 72°c for 2 minutes and a final extension of 8 minutes at 72°c. the amplified dna was visualized on the 3% agarose gel. ampelographic characterization thirty­three major morphological descriptors selected from the descriptors list previously devel­ oped by the oiv (organisation international de la vigne et du vin) (oiv, 2017) were used in this study (tables 3, 4, 5). they include 17 qualitative descrip­ table 3 ­ descriptive statistics analysis of quantitative morphological descriptors of 43 grapevine accessions table 4 ­ leaf descriptors notation and their frequency distribution among the 43 studied grapevine accessions descriptors mean minimum maximum least significant difference (lsd) leaf length (cm) 9.77 7.84 11.26 1.06 leaf width (cm) 13.29 11.4 15.74 1.35 petiole (cm) 7.92 4.24 11.12 0.99 number of lobes 4.89 3.00 5.00 0.09 bunch weight (g) 576 203 1186 121 bunch length (cm) 21.76 14.93 30.6 3.60 bunch width (cm) 13.62 8.15 21.23 2.33 berry weight (g) 5.7 0.94 9.95 0.47 berry length (cm) 2.24 1.50 3.40 0.12 berry width (cm) 1.75 1.16 2.54 0.08 pedicel length (cm) 1.01 0.49 1.53 0.11 seed number 2.51 1.00 4.00 0.16 acidity at veraison (g/l) 2.82 1.35 6.10 0.89 brix at veraison (◦brix) 15.67 11.2 18.00 1.77 acidity at harvesting (g/l) 1.98 1.2 3.10 0.43 brix at harvesting (◦brix) 18.38 13.6 23.00 2.13 descriptors ­leaf notation and frequency (number of accessions) shape of blade wedge­shaped (31) ­ pentagonal (3) ­ circular (9) shape of teeth both side straight (3) ­ both side convex (1) ­ mixture between both side straight and both side convex (39) anthocyanin coloration of main veins on upper side absent (25) ­ only on the petiolar point (10) ­ up to the 1st bifurcation (5) ­ up to the 2nd bifurcation (2) ­ beyond the 2nd bifurcation (1) anthocyanin coloration of main veins on lower side absent (16) ­ only on the petiolar point (17) ­ up to the 1st bifurcation (5) ­ up to the 2nd bifurcation (1) ­ beyond the 2nd bifurcation (4) prostrate hairs on main veins on lower side none or very low (21) ­ low (11) ­ medium (7) ­ high (4) prostrate hairs on main veins on upper side absent (40) ­ present (3) density of prostrate hairs on petiole none or very low (38) ­ low (1) ­ medium (2) ­ high (2) opening of petiole sinus wide open (13) ­ open (26) ­ closed (3) ­ overlapping (1) leaf length short (13) ­ medium (30) leaf width narrow (10) ­ medium (27) ­ wide (6) petiole length short (8) ­ medium (25) ­ long (10) number of lobes three lobes (1) ­ five lobes (42) chehade et al. ‐ genetic diversity in lebanese grapevine germplasm 219 tors and 16 quantitative descriptors related to the leaf (12 descriptors), bunch (5 descriptors) and berry (16 descriptors). data analysis to assess the information given by issr markers, the following parameters were calculated as follow: number of alleles per locus, power of discrimination (pd= 1 ­σ gi2, where gi is the frequency of the ith genotype), polymorphism information content (pic= 1­∑ (pi)2, where pi is the proportion of samples carry­ ing the ith allele of a particular locus) (botstein et al., 1980). genetic distances were calculated according to jaccard. trees clustering the data with the unweighted pair­group method (upgma) with sahn­ clustering and tree programs of past software (kriege et al., 2014). qualitative characteristics have been described and scored. for quantitative descriptors, the mean was calculated. to assess the degree of similarity between the units tested and understand the rela­ tionships between them, the data were subjected to a principle component analysis (pca) in order to con­ dense the quantitative and qualitative descriptors in a small number of synthetic components (saporta, 1990). thus, the degree of contribution of each of the characters to the total variation was calculated in order to indicate the most relevant characters. hierarchical cluster analyses was executed using euclidean distance to classify cultivars into different groups based on morphological evaluation. lsd test (sas institute inc, 1995) was done in purpose to com­ pare means of quantitative characters between dif­ ferent accessions. the correlation between molecular and ampelographic clustering was studied by per­ forming a mantel test using past program. 3. results issr markers analysis the nine issr markers (ubc807, ubc812, ubc815, table 5 ­ bunch and berry descriptors notation and their frequency distribution among the 43 studied grapevine accessions descriptors notation and frequency (number of accessions) bunch descriptors bunch shape cylindrical (21) ­ conical (15) ­ funnel shaped (7) bunch density loose (5) ­medium (25) ­ dense (13) bunch weight very low (4) ­ low (13) ­ medium (13) ­ high (8) ­ very high (5) bunch length short (3) ­ medium (6) ­ long (26) ­ very long (8) bunch width narrow (13) ­ medium (21) ­ wide (8) ­ very wide (1) berry descriptors berry shape obloid (2) ­ globose (9) ­ broad ellipsoid (11) ­ narrow ellipsoid (6) ­ cylindric (1) ­ obtuse ovoid (7) ­ ovoid (3) ­ obovoid (3) ­ horn shaped (1) color of skin green yellow (27) ­ rose (2) ­ red (1) ­ dark red violet (8) ­ bleu black (5) thickness of skin very thin (2) ­ thin (17) ­ medium (17) ­ thick (5) ­ very thick (2) firmness of flesh soft (6) ­ slightly firm (27) ­ firm (10) juiciness of flesh slightly juicy (7) ­ medium juicy (27) ­ very juicy (9) veraison time very early (8) ­ early (3) ­ medium (2) ­ late (24) ­ very late (6) veraison acidity (ta) low (5) ­ medium (32) ­ high (6) veraison brix low (6) ­ medium (27) ­ high (10) harvesting time very early (3) ­ early (4) ­ medium (7) ­ late (20) ­ very late (9) harvesting acidity (ta) low (6) ­ medium (30) ­ high (7) harvesting brix low (6) ­ medium (31) ­ high (6) berry weight very low (4) ­ low (6) ­ medium (26) ­ high (5) ­ very high (2) berry length short (7) ­ medium (16) ­ long (15) ­ very long (5) berry width very narrow (1) ­ narrow (21) ­ medium (20) ­ wide (1) length of pedicel very short (1) ­ short (3) ­ medium (5) ­ long (7) ­ very long (9) seed number one (2) ­ two (24) ­ three (11) ­ four (6) 220 adv. hort. sci., 2022 36(3): 215­226 ubc816, ubc818, ubc828, ubc864, ubc868 and ubc880) showed distinct polymorphism among the 43 different grapevine accessions of this study (table 2). a total of 41 polymorphic bands were detected. the size of amplified products ranged from 125 bp to 1000 bp. the calculated discriminating power (pd) was between 0.3 (ubc868) and 0.9 (ubc864), indi­ cating a high diversity of the loci and confirming the efficiency of these primers in studying the polymor­ phism of the lebanese grapevine germplasm. the polymorphic information content (pic) value varied between 0.27 (i.e. ubc868) and 0.64 (i.e. ubc816) with an average of 0.49. number of polymorphic bands varied between two (e.g. ubc868) to six (e.g. ubc812). eight primers had one monomorphic band, while ubc868 generated two monomorphic bands. genetic clustering of the accessions the allelic diversity data was used to produce a dendrogram by using distance matrix­upgma, thus revealing the genetic relationship among grapevine accessions (fig. 1). the dendogram constructed on the base of the amplification product of issr primers of the different accessions showed 41 different mole­ cular patterns. tfaifihi variety indicated a unique marker with the primer ubc812 (305 bp). one case of synonymy was observed between gbaii, arasani and gnoubi cultivars and they are very close for the most discriminating ampelographic traits. the issr analysis showed four distinct groups: g1, g2, g3 and g4 at the distance of 0.67 of similarity including 6, 26, 2, and 9 cultivars respectively. the first group g1 consists of six accessions ‘baitmouni asouad’, ‘zaitouni, ‘asouad’, ‘ainouni’, ‘moukh al­baghel’ and ‘karn al ghazal’ and have a nine alleles in common with six primers (ubc807, ubc812, ubc816, ubc864, ubc868 and ubc880). the largest group (g2) gathers 26 grapevine acces­ sions, shared only five alleles in common with ubc807, ubc812 and ubc818. this group can be divided into 4 sub­groups: g2.1 contains six acces­ sions, ‘nabi’, ‘misky’, ‘asali’, ‘bourji’, ‘oubaidi’ and ‘baitamouni’. g2.2 comprises 11 accessions, with two cases of close similarity detected between ‘souri zahlaoui’ and ‘khoudri’, and between ‘yafaoui’ and ‘souri mhayyar’, at 0.94 similarity levels. only one case of synonymy was observed between three dif­ ferent accessions initially collected from the south of lebanon under different vernacular names and pre­ senting the same genetic profile: ‘gnoubi’, ‘gbaii’, ‘arasani’. g2.3 consists of four accessions, with a close similarity detected between ‘bzaz al­anzi’ and ‘amarcani souri’ at a similiraty level of 0.96. g2.4 includes five accessions; ‘bakhouri’, ‘amarcani abiad’, ‘maghdoushi mgaddad’, ‘maksasi’ and ‘ashalamish abiad’. the third group (g3) consists only of two accessions: ‘raha’ and ‘tfaifihi’ had 20 alleles in common with all the nine issr markers. the fourth group (g4) consists of nine accessions: ‘souri asouad’, ‘boulbouli asouad’, ‘houzairani’, ‘amarcani asouad’, ‘mariami’, ‘ari’, ‘zaghzaghani’, ‘samani asouad’ and ‘samaani abiad’, shared only two alleles in common with ubc815 and ubc864 primers. fig. 1 ­ hierarchical clustering analysis of the 43 lebanese grape­ vine cultivars, based on 9 issr primers using jaccard distance and upgma. chehade et al. ‐ genetic diversity in lebanese grapevine germplasm 221 ampelographic description a total of 43 cultivars were studied. locale names were commonly given by farmers or nurseries based on berry colour or on the country of origine (asouad, souri aswad, amarcani abiad) and maturity date (houzairani). for each cultivar, a descriptive list was established with 33 morphological traits related to the leaf, bunch and berry (tables 3, 4, 5). leaf description. the majority of accessions have five lobes except ‘souri zahlaoui’ accession which only have three lobes. thirty­one accessions present­ ed a wedge­shaped leaf form (e.g. ‘ari’, ‘bourji’, ‘gbaii’), while only nine accessions had circular leaf (e.g. ‘raha’, ‘bakhouri’), and the rest had a pentago­ nal leaf form (e.g. ‘arasani’, ‘asali’, ‘souri mhayyar’) (table 4). most of the accessions shared same teeth form with both sides straight and both sides convex except for the ‘oubaidi’ that possesses a both side convex teeth form, and three accessions (‘boulbouli asouad’, ‘nabi’ and ‘samani asouad’) which had a both side straight teeth form (table 4, fig. 2). anthocyanin coloration of main veins on both the upper and the lower side of blade varied from absent (e.g. ‘al mir’), limited to the petiolar point (e.g. ‘arasani’), extended to the first bifurcation (e.g. ‘khodre’), and beyond the second burifaction (e.g. ‘zaghzaghani’). the opening of the petiole sinus varied from over­ lapping sinus (e.g. ‘zaitouni’), closed (e.g. ‘souri asouad’), open (e.g. ‘misky’, ‘rins’), to wide open sinus (e.g. ’gbaii’, ‘souri zahlaoui’). the density of prostrate hairs between main veins on both sides of the blade and on the petiole varied widely from absent (e.g. ainouni), low (e.g. ‘bakhouri’, ‘zaghzaghani’), medium (e.g. ‘zaitouni’, ‘ari’), and high (e.g. ‘tfaifihi’, ‘al mir’). only three accessions present prostrate hairs on the upper side of the main blade veins (‘rins’, ‘souri mhayyar’, and ‘tfaifihi’). leaves generally presented an average length between 7.84 (i.e. ‘souri zahlaoui’) and 11.26 cm (i.e. ari) with lsd 1.06 and width between 11.4 (i.e. ‘rins’) and 15.74 cm (i.e. ‘ashlamish ahmar’). petiole length average was between 4.24 (i.e. ‘bzaz al­ aanzi’) and 11.12 cm (i.e. ‘baitamouni asouad’) (table 3). bunch description. bunch characteristics investi­ gated showed a great diversity among the accessions studied. almost 48.8% of the accessions had cylindri­ cal bunch (e.g. ‘ainouni’, ‘houzairani’), 34.9% were conical (e.g. ‘boulbouli asouad’, ‘maksasi’) and 16.3% of the accessions had a funnel shaped cluster (e.g. ‘arasani, raha’) (table 5). both ‘samani asouad’ and ‘samaani abiad’ had the heaviest bunch weight, with an average of 1150 g approximately. for the remain­ ing accessions, bunch weight ranged from 1090 g (i.e. ‘rins’) to 203 g (i.e. ‘amarcani abiad’). most of the accessions had a bunch length ranging between 14.93 (i.e. ‘maghdoushi mgaddad’) and 30.6 cm (i.e. ‘rins’) with 3.6 of lsd. bunch width ranged between a minimum of 8.15 cm (i.e. ‘bourji’) and a maximum of 21.23 cm (i.e. ‘samaani abiad’) (table 3, fig. 3). berry description. berry’s external appearance presented an important diversity among accessions (table 5). for the shape, 11 accessions had a broad ellipsoid berry shape (e.g. ‘ainouni’, ‘arasani’, ‘khoudri’), nine accessions had a globose berry form (e.g. ‘ari’, ‘tfaifihi’), and the remaining accessions had obvoid (e.g. ‘al mir’, ‘souri mhayyar’), narrow ellipsoid (e.g. ‘gbaii’, ‘souri asouad’), obloid (e.g. ‘asali’, ‘samani asouad’), ovoid (e.g. ‘misky’, ‘ashlamish ahmar’, ‘yafaoui’), obtuse ovoid (e.g. ‘maghdoushi’, ‘asouad’, ‘souri zahlaoui’, ‘zaitouni’), cylindical (e.g. ‘bzaz al­anzi’) and horn berry shaped (‘karn al­ghazal’). as to skin color, most of the acces­ sions had green yellow berries (e.g. ‘mekssese’, ‘yafawi’, ‘karn al ghazal’). while ‘houzairani’ diverged from others with its red berries, eight acces­ sions had a dark red violet berry color and five other accessions shared the bleu black color. only the rose color was found in ‘ashlamish ahmar’ and ‘tfaifihi’ accessions. fig. 2 ­ example of variability of leaf shape. fig. 3 ­ example of variability of bunch shape. adv. hort. sci., 2022 36(3): 215­226 222 a wide range of variability was also found for the berry quantitative descriptors (table 3). ‘asouad’ and ‘asali’ accessions were outstanding with their signifi­ cantly heavier berries (9.95 g and 9.69 g respectively) while ‘ashlamish abiad’ had only 0.94 g, as berry average weight. for the rest of the accessions, berry weight ranged from 8.3 g in ‘baitamouni asouad’ to 1.71 g in ‘amarcani abiad’. on the other hand, ‘karn al ghazal’ accession was distinguished with its long berries (3.4 cm) and its long pedicel (1.53 cm) while ‘asali’ accession had the largest berries (2.54 cm). principal component analysis the first three components presented 31.9% of the total variation of the different descriptors (table 6). the first component consisted of 12.2% of the total variation and included berry skin color, firmness of flesh, bunch weight, harvesting and veraison times, in addition to the brix level at harvesting. thus, the first component was dominated by the fruit characteristics more than the leaf ones. the second component represented 10.9% of the total variation and included four variables namely berry weight and length, leaf lower vein pigmentation and lower face pilosity. the third component was characterized by a percentage of variation of 8.8% and was dominated by the flesh juiciness and the bunch density. based on this validation through pca, this shortlist of dis­ criminating descriptors may be further considered to study the relationships between the grapevine acces­ sions. ampelographic accession clustering the dendogram illustrating the relationship among the 43 studied grapevine accessions was con­ structed on the 12 most discriminating descriptors as validated by pca. four groups were differentiated at the distance ­17 of similarity using euclidean distance (fig. 4). the first group (g1) consisted of eight acces­ sions of different skin color but sharing same verai­ son date and the same sugar content at harvesting time. the second and the largest group (g2) consist­ ed of 13 accessions, sharing all the same bunch char­ acteristics in terms of density, juiciness in addition to the common sugar content at harvesting time. moreover, these accessions started the stage of veraison almost at the same date around the 5th of september. the third group (g3) contained 12 acces­ sions, characterized by medium green berries. a case of close similarity was found between three acces­ sions of different vernacular names ‘gnoubi’, ‘gbaii’ and ‘arasani’. the fourth group (g4) clustered 10 accessions of which eight accessions with dark red violet (e.g. ‘asali’) to bleu black (e.g. ‘mariami’) berries, and two accessions with green yellow berries (‘rins’ and ‘samaani abiad’). all these accessions have common leaf characteristics and intermediate sugar content at maturity time. table 6 ­ the first three components of the principal component analysis involving the 33 ampelographic traits and per­ formed for the 43 accessions of the national grapevine germplasm collection variable factor 1 factor 2 factor 3 leaf form ­0.14042 0.423101 ­0.00785 number of lobes ­0.07649 ­0.08669 0.136368 teeth form ­0.15343 ­0.29052 0.301802 upper vein pigmentation ­0.14288 ­0.25638 ­0.45432 lower vein pigmentation 0.069659 ­0.63447 ­0.19775 lower face pilosity ­0.03692 0.627619 ­0.00083 upper face pilosity 0.17382 0.179676 0.009038 petiole pilosity 0.27927 ­0.3332 0.328737 overlapping of petiole ­0.00076 0.475518 0.226222 berry shape ­0.0347 ­0.00661 0.439309 color skin 0.511841 ­0.13872 ­0.09665 skin thickness 0.013082 0.133863 0.46865 firmness of flesh 0.519899 0.152408 0.095108 juiciness of flesh ­0.27819 0.150394 ­0.55995 seeds number ­0.01707 ­0.0642 ­0.44797 bunch form ­0.14446 0.507152 ­0.03091 bunch density 0.21315 0.362343 ­0.6217 bunch weight 0.598172 0.272448 ­0.44281 bunch length 0.344545 0.046891 ­0.35337 bunch width 0.459819 0.390514 ­0.32888 leaf length 0.306676 0.272602 0.398343 leaf width 0.41474 0.04784 0.18026 petiole length 0.436157 0.049669 0.154936 pedicel length 0.269837 ­0.46508 0.060371 berry weight 0.437611 ­0.53381 ­0.29825 berry length 0.253778 ­0.65709 0.106556 berry width ­0.13315 ­0.30376 ­0.35394 veraison time 0.789894 0.04527 0.01873 harvesting time 0.667212 0.098136 ­0.22776 acidity at veraison ­0.42158 ­0.18862 ­0.28567 brix at veraison ­0.49511 0.304928 ­0.05688 brix at harvesting ­0.57237 0.156018 ­0.1208 acidity harvesting ­0.44026 ­0.21603 ­0.20417 percentage total variation 12.2% 10.9% 8.8% the characters in bold are discriminant. chehade et al. ‐ genetic diversity in lebanese grapevine germplasm 223 4. discussion and conclusions in our study encompassing 43 local accessions, 51 bands were generated from nine primers, out of which 41 were polymorphic. similarly, 55 polymor­ phic bands were obtained in palestine by basheer­ salimia, (2015) in studying 36 grape accessions using 17 issr markers; while 69 polymorphic bands were obtained by seyedimoradi et al. (2012) in studying 21 local iranian grapevine cultivars using 10 issr primers. our results related to polymorphism rate (50­85%) and size of amplified bands (125­1000bp) are close to the ones previously reported in portugal (castro et al., 2016), turkey (sabir et al., 2009), egypt (hassan et al., 2011), iran (seyedimoradi et al., 2012), and palestine (basheer­salimia, 2015). larger frag­ ments (300­1500 bp) were generated by the same issr markers with values up to 1500 bp in india (dhanorkar et al., 2005) and 2500 bp in turkey (sabir et al., 2009). moreover, our results indicated a dis­ crimination power value ranging from 0.3 (ubc868) and 0.9 (ubc864) and pic value varying between 0.27 (i.e.ubc868) and 0.64 (i.e.ubc816) with an average of 0.49, which was similar to the results obtained in iran by seyedimoradi et al. (2012). our issr results revealed an important genetic diversity within the national collection of grapevine germplasm. about 41 different molecular profiles were clearly differentiated within the 43 accessions, with only one case of synonymy found between three accessions (‘arasani’, ‘jbai’ and ‘jnoubi’). these findings certainly confirm one more time the efficien­ cy of these issr markers in investigating the genetic variability of grapevine. it also indicates the efficiency of the initial survey and collection of local accessions in establishing this national collection of grapevine germplasm with only 5% synonymy. in spain, the molecular characterization of grapevine accessions of the national gene bank at alcalá de henares allowed the differentiation of 177 accessions (30%) over 621 initially collected (ortiz et al., 2004). furthermore, the ssr analysis of the eastern european cultivars led to the differentiation of only 659 unique profiles over 997 accessions studied (maul et al., 2015). in italy, cipriani et al. (2010) reported 200 groups of syn­ onyms vs. 774 unique genotypes out of 1005 grapevine accessions studied. also, lopes et al. (1999), studying 49 supposed different cultivars from the portuguese grapevine national collection of terciera (azores island) through microsatellite mark­ ers, detected only 36 different profiles after deter­ mining the synonym cases. along with the genetic differentiation of the grapevine accessions, an ampelographic description was carried out for the 43 lebanese accessions using 33 descriptors recommended by the oiv, and mostly related to various descriptors including yield compo­ nents like bunch and berries dimensions and weight (kara, 1990; ortiz et al., 2004; santiago et al., 2005; oiv, 2007; akram et al., 2019). mature leaf descrip­ tors did not vary significantly among the lebanese accessions. most of these accessions had leaves with fig. 4 ­ hierarchical clustering analysis of the 43 lebanese grape­ vine cultivars, based on the most 12 discriminant morphological traits, using euclidean distance and ward method. adv. hort. sci., 2022 36(3): 215­226 224 five lobes similarly to the results reported in other studies (ecevit and kelen, 1999; chalak et al., 2016). on the other hand, our results revealed a large vari­ ability of bunch shape, weight, density, as well as for the berry shape, color and size, similarly to the results reported in previous works in the eastern mediterranean (sabir et al., 2009; biniari and stavrakaki, 2016). on the other hand, the multivari­ ate analysis was efficient to analyze large data gener­ ated in our study by qualitative and quantitative descriptors to further identify patterns and relation­ ship among powerful statistical techniques. pca allowed to extract the most discriminating descriptors, e.g. veraison date, maturity date, berry length, upper vein pigmentation, lower vein pigmen­ tation, bunch density, bunch weight, brix at harvest­ ing, juiciness of flesh, berry weight and firmness of flesh and skin color. our results were in accordance with those previously obtained by riachi (1998), madi (2007) and chalak et al. (2016) confirming one more the stability of the discriminating descriptors over years. this also in accordance with leao et al. (2010, 2011) who reported the stability of the dis­ criminating characters for two consecutive years. phenotypic clustering allowed differentiate the 43 accessions studied in four main groups. the syn­ onymy case first revealed between the three acces­ sions ‘gbaii’, ‘gnoubi’, and ‘arasani’ by the issr clus­ tering was also confirmed by using ampelographic. this is the minimum rate for verifying synonyms and for clone selection processes (cervera et al., 2002). furthermore, many cases of close phenotypic sim­ ilarity, first revealed by madi (2007), were also recorded in this study. it is worthy to note that some of our accessions were also described in other coun­ tries. this is the case of ‘bzaz al anzi’ reported in egypt by hassan et al. (2011) and presenting almost similar ampelographic characteristics of the bunch and the berries. when comparing the two dendograms generated apart by the molecular and ameplographic descrip­ tors, the mantel test indicated a weak correlation (r = 0.26, data not shown) between them. this reflects different structure for the accessions clustering whether it is generated by molecular or ampelo­ graphic descriptors. exception is made to the case of synonymy between the three accessions ‘gnoubi’, ‘gbaii’, and ‘arasani’, which was confirmed by both, ampelographic and molecular clustering. this dis­ crepancy between the two clustering types was also reported in other studies in grapevine collections (knezović et al., 2017) and other fruit crops (talebi et al., 2008; zdunic et al., 2008). on the other hand, considering some key ameplo­ graphic descriptors, it was possible to categorize the existing diversity of the collected lebanese germplasm according to the phenological stage, and according to berry skin color. in five groups of matu­ rity time; with 27 accessions had green yellow berries, only one accession with red berries, two accessions with rose berries, five accessions with bleu black berries and eight accessions with dark red violet berries. surprisingly, similar ratios between green and red accessions were also reported in canary islands and madeira by marsal et al. (2019). additionally, most of the accessions studied had crunchy berries with thick skin and very low juice content. this indicates the potentiality of using these accessions for table grapes rather than wine grapes. only three accessions had thin skin, juicy flesh and high sugar content and, therefore, may be tested for fermentation. such descriptive ampelographic char­ acterization of the berries is hopefully necessary for evaluating grapevine accessions conserved in field genebanks with respect to their usage as table grape or wine grape (sabir et al., 2009; ates et al., 2011; basheer­salimia, 2015). this diversity upon the 43 grapevine accessions conserved at the lebanese national collection of grapevine as revealed by issr markers and ampelo­ graphic descriptors indicate the efficiency of sam­ pling/collecting strategy conducted in 1998 in the bekaa and chouf areas. nevertheless, additional grapevine varieties grow­ ing locally under different vernacular names, which do not appear in the national grapevine germplasm collection, were recently assessed in their growing site by (chalak et al., 2016). therefore, it is strongly recommended to further extend the survey and mis­ sion collection to cover multiple grapevine produc­ tion areas, particularly the north, the south and of mount of lebanon in order to enrich the national col­ lection. combining both molecular makers and ampelographic descriptors as it was shown in this study, would be very useful in optimizing the collec­ tion of accessions by clarifying the mislabeling cases, understanding the genetic distances among acces­ sions, and setting up a descriptive assessment for these accessions. the comprehensive evaluation of the existing local gene pool of grapevine would fur­ ther allow the sustainable utilization of the valuable accessions directly for multiplication in certified nurs­ chehade et al. ‐ genetic diversity in lebanese grapevine germplasm 225 eries and also in further breeding programs. 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maize populations. ­ turkish j. agric. forestry, 32(6): 495­500. zohary d., 2003 ­ domestication of grapevine vitis vinifera l. in the near east, pp. 1­8. ‐ in: mcgovern p. e., s.j. fleming, and s.h. katz (eds.) the origins ancient history of wine. routledge, london, uk, pp. 440. impaginato 81 adv. hort. sci., 2022 36(1): 81­86 doi: 10.36253/ahsc­10158 influence of 6­benzylaminopurine spray time after pinching on growth and flow­ ering of veronica dahurica steven j.­h. kim 1, h.j. oh, s.y. kim, g.u. suh (*) division of plant resources, korea national arboretum, 12519 yangpyeong, korea. key words: cytokinin, foliar application, foliar spray, multiple shoots. abstract: veronica dahurica steven (family scrophulariaceae) is an ornamental plant from korea. the aim of the present study was to produce multiple­ branched plants by using 6­benzylaminopurine (ba) spray application at several time intervals after pinching. all 10 cm long plants were sprayed with 0, 500, 1000, and 2000 mg·l­1 ba at 0, 7, and 14 days after pinching. growth character­ istics were examined 10 weeks after pinching and flowering time was recorded. the number of branches was highest in the group sprayed with 1000 mg·l­1 ba at 0 d after pinching. the greatest plant height was observed in the group treat­ ed with 1000 mg·l­1 ba 14 d after pinching and the minimum plant height was observed in the group sprayed with 500 mg·l­1 ba at 0 d after pinching. the groups sprayed with higher ba concentrations and with longer intervals between pinching and spraying showed greater delay in the time to first flower. the flower length was decreased in the pinched and ba­treated group com­ pared with the control. thus, ba application and pinching could promote multi­ ple branch induction and control flowering time in v. dahurica steven. 1. introduction the genus veronica l. (family scrophulariaceae) comprises about 500 species. it is distributed across most of the northern hemisphere and in many parts of the southern hemisphere and has a wide ecological range, from alpine to coastal vegetation and dry to aquatic vegetation (albach et al., 2004; albach et al., 2005). veronica spp. have the characteristics of graceful and bountiful flowers, long­blooming, easy­care perennials, and they are commercially used in gardens and as cut­flowers (areal et al., 2008; hawke, 2010). veronica dahurica steven is distributed across east asia, in siberia, far east russia, china, north and south korea, and mongolia (choi, 2016). the stems of v. dahurica are erect and upright, with simple hairs, and occur individually or in small groups. the plant grows to 30­50 cm in height. the opposing leaflets are 18­25 mm in length and 15­22 mm in width and have a deltoid shape with deeply cut tips on the edge of the leaf. the flowers are racemule, white or pink colored, with short hairs on (*) corresponding author: arboseed@korea.kr (1) present address honam national institute of biological resources, 99, gohadoan­gil, 58762 mokpo­si, korea. citation: kim j.­h., oh h.j., kim s.y., suh g.u., 2022 ­ influence of 6‐benzylaminopurine spray time after pinching on growth and flowering of veronica dahurica steven. ­ adv. hort. sci., 36(1): 81­86 copyright: © 2022 kim j.­h., oh h.j., kim s.y., suh g.u. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 december 2020 accepted for publication 12 august 2021 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-10158 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 81­86 82 the apical part. the corolla is 5­7.5 mm in length (kpni, 2019). multiple branching has commercial advantages for flower production, because it improves yield and quality, controls plant size, and inhibits loathing (zieslin et al., 1975; barbier et al., 2017; kim et al., 2020). pinching and ba foliar spray are the most use­ ful methods for production of multiple­branched plants (lee et al., 2006). pinching is the removal of the apical bud to release the lower axillary buds from apical domi­ nance. pinching increases branching and stimulates axillary bud development by decreasing auxin pro­ duction in the apical bud and inhibiting lateral bud growth (barbier et al., 2017). pinching is commonly used to improve yield by inducing branches, decreas­ ing plant height to produce dwarf plants, and control­ ling flowering time. this method also increases the cytokinin content. exogenously applied cytokinin, like foliar application, has the same effect as pinching. plant growth regulators (pgrs), such as cytokinins, play important roles in the control of herbage type and flowering. cytokinin regulates gynoecium formation and female gametophyte development, embryo development and seed size, pavement cell morphogenesis, axillary bud release, and nutrient uptake. this promotes vascular cambial development, nodulation, chloroplast development, cell division, and phloem development. it also inhibits lateral root formation, senescence, and cell proliferation in the root apical meristem (kieber and schaller, 2018). in the present study, the use of foliar sprays of dif­ ferent concentrations of ba and pinching to improve the branching and flowering of v. dahurica are evalu­ ated for commercial use on ornamental plants. in addition, the interaction and use of different growing environments is examined to further manipulate the production of v. dahurica, particularly for use as flowering pot plants. 2. materials and methods plant materials seeds of v. dahurica were collected from the useful plant resources center, korea national arboretum on 12th september 2018 and stored in a 4°c chamber. v. dahurica seedlings were grown on 128­cell trays filled with a commercial soilless sub­ strate (baroker; seoul bio, eumseong, korea) on 7th may 2019, and transplanted into 11.5 cm diameter containers filled with the same substrate on 26th june 2019. the baroker consisted of 68% coir dust, 15% peat moss, 7% perlite, 6% vermiculite, and 4% zeo­ lite, at ph 5.5­6.0. two weeks after transplanting, plants were fertilized with 1000 mg·l­1 peters professional 20­20­20 (everris, geldermalsem, the netherlands). all plants were grown in a greenhouse. pinching and ba foliar spray application v. dahurica plants of 10 cm length, with 2­3 nodes and 2 leaves, were selected. all plant materials were pinched at every second internode from the basal stem, and then foliar­sprayed with 10 ml of 0, 500, or 2000 mg·l­1 ba (duchefa, haarlem, the netherlands), using a hand­pump sprayer, at 0, 7, or 14 spraying days after pinching (sdp). the ba solu­ tions were prepared in 10 ml of 99 % ethanol by diluting with distilled water. growth conditions the experiment was conducted between 5th july 2019 and 13th september 2019. pinching and ba treatments were applied once at 10 weeks of culture under greenhouse conditions. the mean values of relative humidity and temperature in the greenhouse are shown in figure 1. irrigation was carried out by drip watering at 500 ml daily in september and twice daily from july to august. plant growth and flowering the variables considered consisted of height and width of plant, number of nodes, length and width of leaf, number of leaves, lateral branch length, number fig. 1 ­ principal component loading pattern of six traits of amaranthus accessions. kim et al. ‐ ba foliar spray application effects for v. dahurica steven 83 of lateral branches and flower characteristics. these parameter were evaluated 10 weeks after treatment. the time to first flowering was recorded. statistical analysis the experiment was conducted with twelve plantlets per three replicates and three replications per treatment, in a completely randomized block design, unless specified otherwise. significant differ­ ences were determined using duncan’s multiple range test (dmrt) with sas software (version 9.4; sas institute, cary, nc, usa). 3. results and discussion effects of ba and sdp on growth characteristics v. dahurica was cultivated with 0, 500, 1000, or 2000 mg·l­1 ba foliar spray application at 0, 7, or 14 sdp, and plant growth was evaluated 10 weeks after treatment. ba foliar application groups, which were co­treated with pinching, successfully formed lateral branches, while the effect of pinching treatment alone was similar to that of the control. maximum lateral branch formation was observed in the group treated with 1000 mg·l­1 ba spray on 0 day after pinching, with a mean of 13.92 secondary branches per plant and a branch production efficiency approxi­ mately 2.93­fold that of the control (table 1). both pinching and ba foliar spray application can promote branch production by controlling apical dominance through cytokinin signaling (barbier et al., 2017; kieber and schaller, 2018). ba foliar spray application is recommended to promote branch production in many ornamental plants such as ardisia pusilla (lee et al., 2006), echinacea cultivars (latimer and freeborn, 2009), and kalanchoe species (currey and erwin, 2012). pinching is also recommended for branch production in a. pusilla (lee et al., 2006), targets exeta l. (meena et al., 2015), and elsholtizia (sohn and kim, 2003). however, even when the con­ centration of ba was increased with increasing sdp, the multiple­branch production effect was not greater than that of the treatment immediately after pinching. lee et al. (2006) reported that 0 to 7 sdp increased branch production in ardisia pusilla, but that branch production decreased at sdp>7. in our treatments, plant height was similar to or less than that in the control; however, lateral branch length was decreased in all treatment groups compared with that in the control (table 1, fig. 2). leaf charac­ teristics were also investigated in our experiments. compared with the control, leaf number and leaf width were increased in almost all or all treatment groups, respectively, and leaf length was decreased in almost all treatment groups (table 1). all treat­ ments showed a decrease in the number of nodes compared with that in the control. pinching and cytokinin application can be economically used to enhance production of plant stems, flowers, and table 1 ­ growth effects of pinching and spray application on v. dahurica 10 weeks after treatment sdp= spraying days after pinching; ba= 6­benzylaminiopurine; z mean separation within columns by duncan´s multiple range test at p＜0.05. sdp (days) ba (mg·l­1) plant no. of nodes (per/plantlet) leaf secondary branch height (cm) width (cm) length (cm) width (cm) number (per/plantlet) length (cm) number (per/plantlet) control 29.08 abz 38.42 a 14.00 a 6.27 a 3.57 c 177.94 d 29.43 a 4.75 f pinching 28.69 ab 37.39 a 11.67 bc 6.33 a 3.76 bc 197.17 cb 28.52 a 6.78 ef 0 500 20.65 c 23.87 c 10.53 bc 6.99 a 4.24 ab 298.97 b 12.44 e 11.64 abc 1000 24.86 bc 30.54 b 11.61 bc 7.08 a 4.48 a 407.36 a 15.60 cd 13.920 a 2000 27.25 ab 23.44 c 7.00 d 4.71 b 3.86 bc 175.67 d 18.86 bcd 11.06 abc 7 500 26.59 ab 29.56 b 6.69 d 4.61 b 3.76 bc 215.61 cd 16.47 b 10.59 bcd 1000 24.60 bc 27.68 bc 7.8 d 5.01 b 3.79 bc 191.08 cd 16.11 bc 11.48 bcd 2000 28.22 ab 31.97 b 13.00 ab 4.23 b 3.75 bc 239.28 bcd 12.56 bcd 8.00 de 14 500 24.76 bc 30.57 b 12.28 ab 4.70 b 4.34 ab 256.72 bc 13.37 e 8.92 cde 1000 30.56 a 32.53 b 12.77 ab 4.82 b 4.28 ab 432.14 a 12.86 de 12.86 ab 2000 24.55 bc 30.98 b 11.36 bc 7.04 a 4.13 ab 370.58 a 18.10 bc 12.67 ab adv. hort. sci., 2022 36(1): 81­86 84 seeds by decreasing the planting density and increas­ ing the number of branches. small dwarf plants were grown as pot plants by reducing the plant height (table 1, fig. 2). effects of ba and sdp on flowering pinching treatment alone resulted in the highest flowering percentage per plantlet after 10 weeks of culture (86%), followed by ba 2000 mg·l­1 spray application at 0 sdp, ba 1000 mg·l­1 at 0 sdp, ba 1000 mg·l­1 at 7 sdp, ba 1000 mg·l­1 at 14 sdp, ba 500 mg·l­1 at 7 sdp, and the control. there were significant differences in days to first flowering among treatment groups. pinching delayed the first flowering (24.69 days after pinching) com­ pared with the control (14.14 days after pinching). ba 500 mg·l­1 (0 sdp: 30.94 days/ 14 sdp: 29.17 days) and 1000 mg·l­1 (0 sdp:30.72 days/ 14 sdp: 26.06 days) at 0 sdp and 14 sdp increased the number of days to first flowering compared with the control and ba 0 mg·l­1 with pinching groups. however, other treatments decreased the number of days to first flowering compared with the ba 0 mg·l­1 with pinch­ ing and control groups. in the flower development stage, the first increase in cytokinin occurred in the bud induction stage, followed by a dramatic increase in the transition stage. exogenous cytokinin treat­ ment can promote floral transition through transcrip­ tional induction of the floral activators twinsister of ft (tsf), flowering locus d (fd), and soci (d’aloia et al., 2011; winterhagen et al., 2020). the number of flowers and buds also differed among treatments. the ba 2000 mg·l­1 at 0 sdp treatment produced the highest flower number, fol­ lowed by ba 1000 mg·l­1 at 14 sdp. however, the highest number of buds was observed in the ba 2000 mg·l­1 at 14 sdp treatment group, followed by ba 1000 mg·l­1 at 7 sdp, pinching, and ba 1000 mg·l­1 at 14 sdp (fig. 2, 3). the observed increases in flower number and buds are due to the increase in branch number and the promotion of flowering by cytokinin (kumar et al., 2002; d’aloia et al., 2011). both the flower length and the peduncle length decreased with ba foliar spray application and pinch­ ing treatments. the effect of the interaction between pinching and ba on the flower length was found to be significant. the smallest flower length (10.49 cm) was recorded with application of ba 1000 mg·l­1 at 7 sdp compared with a flower length of 21.62 cm in the control group. with ba 2000 mg·l­1 treatment at 0 sdp, the peduncle length of v. dahurica steven was significantly lower than that of the control (fig. 3). ferrante et al. (2006) reported that a 500 µm ba spray application reduced inflorescence length in salvia splendens kerr gawl ‘flamex 2000’. however, the effect of ba on flower size depends on ba concen­ tration, method of application, and plant species (pobudkiewicz, 2008) and can also affect other vari­ ables such as tepal length, flower diameter, and inflo­ rescence length (pobudkiewicz and nowak, 1994; ferrante et al., 2006; pobudkiewicz and treder, 2006). 4. conclusions the present study revealed the commercial utility of pinching and ba foliar spray application for induc­ tion of multiple branching and control of flowering in v. dahurica. treatment with 1000 mg·l­1 ba at 0 days after pinching produced the highest branch number after 10 weeks. pinching delayed the first flowering time compared with that in the control, but some ba foliar applications accelerated the first flowering time. pinching and ba foliar application also had the effect of reducing flower length and peduncle length. we anticipate that this knowledge will be beneficial for enhancing branching and controlling flowering fig. 2 ­ flowering characteristics of v. dahurica with pinching and ba spray application. kim et al. ‐ ba foliar spray application effects for v. dahurica steven 85 time and characteristics of v. dahurica in commercial applications. acknowledgements this study was supported by the ‘plant material selection and new cultivar development for wildflower industrialization (kna1­2­32,17­7)’ pro­ ject funded by the korea national arboretum. references albach d.c., chase m.w., 2004 ­ incongruence in veroniceae (plantaginaceae): evidence from two plas‐ tid and a nuclear region. ­ mol. phylogenetics evol., 32: 183­197. albach d.c., jensen s.r., özgökce f., grayer r.j., 2005 ­ veronica: chemical characters for the support of phy‐ logenetic relationships based on nuclear ribosomal and plastid dna sequence data. ­ bioch. system. ecol., 33(11): 1087­1106. areal f.j., touza j., macleod a., dehnen­schmutz k., perrings c., palmieri m.g., spence n.j., 2008 ­ integrating drivers influencing the detection of plant pests carried in the international cut flower trade. ­ j. environ. manag., 89(4): 300­307. barbier f.f., dun e.a., beveridge c.a., 2017 ­ apical dominance. ­ current biology, 27(17): 864­865. choi k.s., 2016 ­ phylogeny and biogeography of the veronica l. subgenus pseudolysimachium (w.d.j. koch) buchenau. ­ ph.d. thesis, yeungnam university, gyeongsangbuk­do, korea. currey c.j., erwin j.e., 2012 ­ foliar applications of plant growth regulators affect stem elongation and branch‐ fig. 3 ­ growth and flowering characteristics in pinching and ba application groups 10 weeks after treatment. a) control. b) pinching. c) ba 500 ppm 0 week after pinching. d) ba 1000 ppm 0 week after pinching. ing of 11 kalanchoe species. ­ horttechnol., 22(3): 338­ 344. d’aloia m., bonhomme d., bouche f., tanseddaj k., ormenese s., torti s., coupland g., perilleux c., 2011 ­ cytokinin promotes flowering of arabidopsis via transcriptional activation of the ft paralogue tsf. ­ plant j., 65: 972­979. ferrante a., mensuali­sodi a., serra g., tognoni f., 2006 ­ evaluation of postproduction performance of salvia splendens potted plants for interiors use. ­ acta horticulturae, 723: 415­420. hawke r., 2010 ­ comparative studies of veronica and veronicastrum. ­ plant evaluation notes, 33. kieber j.j., schaller g.e., 2018 ­ cytokinin signaling in plant development. ­ develop., 145: dev149344. kim j.­s., yon g.h., kim b.g., choi j.­s., kim e.a., ko y.k., lee i.y., 2020 ­ application of mevalocidin as a new plant growth regulator for thinning and lateral shoot induction. ­ weed and turfgrass sci., 9(1): 29­42. kpni, 2019 ­ korea national arboretum. pocheon, korea. ­ korean plant names index, http://www.nature.go. kr/kbi/idx/searchindex.do. kumar r., singh k., reddy b.s., 2002 ­ effect of planting time, photoperiod, ga3 and pinching on carnation. ­ j. ornam. hortic. 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will and claßen­bockhoff, 2017). different species of salvia have various applications in the pharmaceutical and therapeutic industries due to their antibacterial, antifungal, anti­ tumor, and antioxidant properties. it is traditionally used to treat bronchi­ tis, colds, sore throats, gastrointestinal disorders, eczema, and tuberculo­ sis (li et al., 2013; bahadori et al., 2015). terpenoids and phenolic compounds are the main secondary metabolites of the genus salvia (lu and foo, 2002). salvia lavandulifolia vahl. is a perennial herbaceous plant native to south france, spain, and northwest africa. this species is well adapted to the semi­arid mediterranean climate and grows up to 100 cm height and has opposite green or gray­white leaves. several secondary (*) corresponding author: hassanbayat@birjand.ac.ir citation: bayat h., shafie f., shahraki b., 2022 ­ salinity effects on growth, chlorophyll content, total phenols, and antioxidant activity in salvia lavandulifolia vahl. ­ adv. hort. sci., 36(2): 145­ 153. copyright: © 2022 bayat h., shafie f., shahraki b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 september 2021 accepted for publication 2 march 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12015 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 145­153 146 metabolites including polyphenolics, flavonoids, triterpenes and monoterpnes have been extracted from the aerial parts s. lavandulifolia (amalia and kintzios, 2005). salinity stress is considered one of the most signifi­ cant environmental stresses that restrict the growth and yield of plants, especially in arid and semi­arid areas (deng et al., 2015). in these areas, low rainfall, high evaporation, and poor drainage increase salt concentration in the soil and create salinity stress (abdel latef, 2010). due to the scarcity or low quality (saline waters) of water resources worldwide, the management of crop production in saline conditions is critical. salinity stress occurs with the accumulation of salts, especially sodium chloride, in the root zone. it causes disturbances in vital plant processes such as nutrient uptake and transport, transpiration, photo­ synthesis, and biosynthesis of primary and secondary metabolites (valifard et al., 2014; ahanger and agarwal, 2017). salinity stress impairs plant growth and development by increasing the osmotic potential of the soil solution, disturbing the nutrient balance, and the toxicity caused by the accumulation of sodi­ um (na+) and chlorine (cl­) ions (rehman et al., 2019). salinity stress increases reactive oxygen species (ros) in the cells that damage nucleic acids, proteins, and membrane lipids (foyer, 2018). the decrease in growth, dry matter production, and yield were report­ ed in most plants such as salvia hispanica, feverfew (tanacetum parthenium l.), and salvia splendens due to salinity stress (raimondi et al., 2017; mallahi et al., 2018; karimian et al., 2019). karimian et al. (2019) reported that salt stress treatments (0, 20, 40, 60, and 80 mm nacl) caused the decrease in growth parame­ ters, relative water content, chlorophyll content and increase electrolyte leakage, total phenols and total soluble sugars in salvia splendens. gengmao et al. (2014) demonstrated that salt treatments less than 100 mm nacl had no effect on growth parameters of salvia miltiorrhiza, but significantly decreased the accumulation of dry matter. in salvia officinalis, the decrease in plant height, chlorophyll content, and essential oil content were reported due to salinity stress (150 mm nacl) (es-sbihi et al., 2021). plants have developed different physiological, biochemical, and molecular mechanisms to deal with salinity stress (zhao et al., 2020). osmotic regulation is one of the mechanisms for maintaining cellular turgidity and membrane stability. in the osmotic reg­ ulation process, cellular concentrations of osmotical­ ly compatible solutes such as sugars increased (chakhchar et al., 2015). moreover, plants to deal with oxidative stresses enhance enzymatic and non­ enzymatic antioxidant activities to reduce the delete­ rious effects of the ros (acosta­motos et al., 2017; bayat and moghadam, 2019). the increasing population of the world coupled with the depletion of freshwater resources and the salinization of agricultural lands necessitates further studies on plants resistant to adverse environmental conditions. although the effect of salinity stress on some species of salvia has been studied (valifard et al., 2014; raimondi et al., 2017; karimian et al., 2019), so far no research has been done on s. lavan‐ dulifolia species. considering the medicinal impor­ tance of salvia lavandulifolia, it is necessary to inves­ tigate the tolerance to salt stress. hence, this study was aimed to study the effects of salt stress on vege­ tative and physiological indices and some secondary metabolites in salvia lavandulifolia. 2. materials and methods plant materials and experimental design this study was carried out in research greenhouse, faculty of agriculture, university of birjand, iran. salvia lavandulifolia var. lavandulifolia seeds were purchased from jelitto seed co (germany) and sown in 105 cell seedling trays in april 2017. coco peat and peat with a ratio of 1:1 were used for the substrate. irrigation was done daily dur­ ing the seedling emergence and growth. after forty days, the seedlings were transplanted into 3­liter plastic pots at the 6­8 leaf stage. the physiochemical characteristics of the soil are given in table 1 (sparks, 1996). organic matter (om) was determined by the walkley­black method, and soil texture was mea­ texture ph ec ds m­1 organic matter field capacity (fc) (%) n k meq lit­1 ca meq lit­1 na meq lit­1 cl meq lit­1 mg meq lit­1 sodium adsorption ratio (sar) sandy loam 7.9 1.3 0.3 17.8 0.02 8.27 8.61 23.1 25.3 1.49 10.28 table 1 ­ some physicochemical characteristics of the experimental soil sample bayat et al. ‐ salinity effects on vegetative parameters of salvia lavandulifolia 147 sured by the hydrometer method. soil ph and electri­ cal conductivity (ec) were measured with ph meter (hanna hi2211­02, usa) and ec meter (jenway ec meter, germany), respectively. phosphorus (p), potassium (k), copper (cu), zinc (zn), iron (fe), and manganese (mn) were extracted by the mehlich 1 extracting solution. sodium and potassium concen­ trations were measured by a flame photometer. phosphorus was determined colorimetrically, and cu, zn, fe, and mn were measured by atomic absorption spectroscopy. calcium and mg were extracted with 1 m potassium chloride and determined by titration with ethylenediaminetetraacetic acid (edta). chlorine was determined by titration method. the sar was calculated by computing na+, ca2+ and mg2+ concentrations (in meq/l) from the saturation extract. the experiment was conducted under green­ house conditions at temperatures of 25/20°c and rel­ ative humidity of 50­60%. the salinity stress started four weeks after the transplantation of seedlings into the pots. a com­ pletely randomized design with four replications was used to compare seven different irrigation water salinity treatments (s0=1.3, s1=3.3, s2=5.3, s3=7.3, s4=9.3, s5=11.3, and s6=13.3 ds m­1). to prepare solutions s1, s2, s3, s4, s5, and s6, sodium chloride (nacl) was dissolved in irrigation water in the amounts of 1.14, 2.18, 3.27, 4.43, 5.49, and 6.65 g, respectively. some physicochemical parameters of control water (s0) were: ec= 1.3 ds m­1, ph= 7.79, na= 5.6 meq l­1, cl= 6.8 meq l­1, and k= 0.35 meq l­1. the pots were irrigated twice a week with saline water based on the field capacity by pot weighting. salinity treatments were applied for one month, and then the traits were measured. growth indices plant height, leaf number, leaf length, leaf width, and maximum root length were measured. to deter­ mine the dry weight of shoots and roots, the samples were dried in an oven for 48 hours (78°c) (bayat et al., 2016). relative water content (rwc) and electrolyte leakage (el) the leaf rwc was measured using the method reported by gonzalez and gonzalez­vilar (2003) and calculated according to the formula: rwc = (fresh weight ­ dry weight)/ (turgid weight ­ dry weight) x 100 leaf dry weight was measured after oven­drying of the samples for 48 h (78°c). turgid weight was determined after soaking leaves in distilled water in the refrigerator for 6 h. electrolyte leakage (el) of the leaf was measured based on the method reported by lutts et al. (1996) and calculated according to the formula: el= (ec1/ec2) × 100 where ec1 and ec2 are the primary and sec­ ondary electrical conductivities, respectively. fresh leaves (0.5 g) were dispensed with distilled water (10 ml) in test tubes and then were shaken for 24 hours (24°c). the ec1 was measured by the ec meter. the test tubes were then transferred to an autoclave (121°c) for 15 minutes and ec2 was determined. chlorophyll content and total soluble sugars the pigments of fresh leaves (0.1 g) were extract­ ed by 5 ml of acetone 80%. the amount of chloro­ phyll a and b were measured by a spectrophotome­ ter (model unico 2100, china) at 645 and 663 nm (arnon, 1949). the content of leaf total soluble sugars was mea­ sured according to the anthrone method (irigoyen et al., 1992). for this purpose, 0.1 g of dried leaves was extracted with 1 ml of ethanol. leaf sampling was performed at 10:00 am. total phenols, total flavonoids, and free radical scav‐ enging capacity (frsc) fresh leaves (1 g) were homogenized in methanol for 24 h and then centrifuged at 6000 rpm for 15 min. the folin­ciocalteu method was used to mea­ sure the total phenolic content (singleton and rossi, 1965). total flavonoids were determined based on the method of yoo et al. (2008). the frsc was deter­ mined using the method reported by koleva et al. (2002) and calculated according to the formula: frsc= 1 – a sample (517 nm)/a control (517 nm) ×100 data analysis the jmp 13 statistical software (sas campus, cary, nc, usa) was subjected to analysis of variance of the data. the means were separated by the least significant difference (lsd) test at the 5% significance level. 3. results growth attributes the results demonstrated that the plant growth traits were significantly affected by increasing the adv. hort. sci., 2022 36(2): 145­153 148 salinity of irrigation water. the lowest plant height, leaf number, leaf length, and leaf width values were recorded in the s6 treated plants by 62%, 41%, 44%, and 46% decrease compared to the control, respec­ tively (table 2). salt stress affected the root length of s. lavandulifolia plants. increasing salinity to s3 level had no significant effect on the root length, but its amount decreased with increasing salinity to s6 level (table 2). biomass production was significantly influ­ enced by salt treatments. increasing salt stress to s2 level had no significant effect on the root dry weight. however, with increasing salinity to s6 level, its val­ ues significantly decreased (table 2). all salinity lev­ els significantly reduced the shoot dry weight, and the lowest value was obtained from s6 treated plants with an 82% decrease compared to the control (table 2). with increasing salt levels, total dry weight decreased significantly. treatment of s. lavandulia plants with s6 decreased total dry weight by 78% compared to the control (table 2). salinity stress sig­ nificantly affected shoot/root dry weight ratio of s. lavandulifolia plants. the highest and the lowest val­ ues of shoot/root dry weight ratio were obtained from the s4 and s6 treated plants, respectively. the leaf rwc and el salinity stress decreased the leaf rwc of s. lavan‐ dulifolia plants. the lowest leaf rwc was achieved in s6 treated plants by a 68% decrease compared to the control (fig. 1a). the leaf el significantly increased with increasing salinity stress levels. the lowest (19.34%) and the highest (86.15%) leaf el values were obtained from the s0 and s6 treated plants, respec­ tively (fig. 1b). chlorophyll content and total soluble sugars the salinity effect was significant on the content of photosynthetic pigments. treatment of s. lavan‐ dulifolia plants with the highest salinity level (s6) table 2 ­ effects of different levels of irrigation water salinity on the plant height, number of leaves per plant, leaf length, leaf width, root length, root, shoot, and total dry weight, and shoot/root dry weight ratio of s. lavandulifolia different letters indicate significant differences according to least significant difference (lsd) test at p<0.05. ** represent significant at 1% level of probability. values are mean ± standard error (se). fig. 1 ­ effects of salinity stress on the leaf relative water content (rwc) and electrolyte leakage (el) in s. lavandulifolia. different letters indicate significant differences according to least significant difference (lsd) test at p<0.05. values are mean ± standard error (se). irrigation water salinity (ds m­1) plant height (cm) number of leave per plant leaf length (cm) leaf width (cm) root length (mm) root dry weight (g. plant­1) shoot dry weight (g. plant­1) total dry weight (g. plant­1) shoot/root dry weight ratio 1.3 (s0) 9.87 ± 0.31 a 52.50 ± 1.44 ab 5.32 ± 0.04 a 2.07 ± 0.04 a 35.51 ± 2.46 a 0.328 ± 0.02 a 0.45 ± 0.01 a 0.78 ± 0.01 a 1.42 ± 0.16 bc 3.3 (s1) 7.25 ± 0.25 b 52.76 ± 2.09 ab 4.92 ± 0.04 b 1.80 ± 0.07 b 33.00 ± 1.35 a 0.291 ± 0.01 a 0.33 ±0.01 b 0.62 ± 0.01 b 1.16 ± 0.06 bcd 5.3 (s2) 6.87 ± 0.42 b 57.25 ± 1.65 a 4.45 ± 0.18 c 1.77 ± 0.02 b 32.25 ± 0.75 a 0.284 ± 0.01 a 0.29 ± 0.02 c 0.57 ± 0.03 b 1.10 ± 0.05 cd 7.3 (s3) 6.62 ± 0.23 b 48.25 ± 1.10 b 4.27 ± 0.04 c 1.55 ± 0.06 c 31.50 ± 1.19 ab 0.164 ± 0.01 b 0.25 ± 0.01 d 0.41 ± 0.04 c 1.56 ± 0.14 ab 9.3 (s4) 5.37 ± 0.12 c 36.25 ± 1.18 c 3.12 ± 0.04 d 1.21 ± 0.04 d 26.51 ± 2.17 bc 0.113 ± 0.01 cd 0.21 ± 0.008 e 0.32 ± 0.007 d 1.91 ± 0.21 a 11.3 (s5) 4.62 ± 0.31 c 32.75 ± 4.30 c 3.02 ± 0.02 d 1.17 ± 0.04 d 25.01 ± 0.70 c 0.147 ± 0.01 bc 0.17 ± 0.004 f 0.31 ± 0.01 d 1.19 ± 0.11 bcd 13.3 (s6) 3.66 ± 0.16 d 30.50 ± 0.28 c 2.95 ± 0.05 d 1.10 ± 0.05 d 23.78 ± 2.47 c 0.094 ± 0.008 d 0.08 ± 0.004 g 0.17 ± 0.009 e 0.90 ± 0.14 d significance ** ** ** ** ** ** ** ** ** bayat et al. ‐ salinity effects on vegetative parameters of salvia lavandulifolia 149 decreased the amount of chlorophyll a and b, and total chlorophyll by 57%, 53%, and 54% compared to the control, respectively (table 3). total soluble sug­ ars were significantly affected by salt stress. by increasing the level of salt stress, the content of total soluble sugars increased upwards. the highest total soluble sugars were achieved by s6 treated plants with a 2.8 times increase compared to the control (table 3). total phenols, total flavonoids, and the frsc irrigation with saline water significantly affected the total phenols and total flavonoids of the leaves. all the levels of salt stress increased the total phe­ nols, and the highest value was obtained in the s6 treated plants (fig. 2a). the lowest total flavonoid content was obtained in the s1 treated plants (fig. 2b). the frsc was significantly increased by all the levels of salinity stress. the lowest (68.16%) and the highest (85.14%) leaf frsc values were obtained in control and s6 treated plants, respectively (fig. 3). 4. discussion and conclusions the present results demonstrated that salt stress influenced the vegetative parameters in s. lavanduli‐ folia. the negative impacts of salt stress on plant growth have been reported in salvia hispanica (raimondi et al., 2017), in salvia splendens (karimian et al., 2019), and salvia officinalis l. (es­sbihi et al., 2021). the decrease in growth parameters under salinity stress can be related to the reduction of soil water potential and toxicity of na+ and cl­ ions, which leads to a nutritional imbalance (kasrati et al., 2014; es­sbihi et al., 2021). salinity stress reduces cell divi­ fig. 2 ­ effects of salinity stress on the leaf total phenolic and fla­ vonoid content in s. lavandulifolia. different letters indi­ cate significant differences according to least significant difference (lsd) test at p<0.05. values are mean ± stan­ dard error (se). sion and elongation, thereby reducing plant growth (netondo et al. , 2004; kamran et al. , 2020). moreover, the decrease in plant growth under salini­ ty stress can be due to the reduction of photosynthe­ sis and energy reserves. usually, in saline conditions, the leaf stomata are closed, and the photosynthesis rate decreases due to reduced gas exchange (chaves table 3 ­ effects of different levels of irrigation water salinity on the chlorophyll a, chlorophyll b, total chlorophyll, and total soluble sug­ ars in s. lavandulifolia different letters indicate significant differences according to least significant difference (lsd) test at p<0.05. **= represent significant at 1% level of probability. values are mean ± standard error (se). irrigation water salinity (ds m­1) chlorophyll a (mg. g fw­1) chlorophyll b (mg. g fw­1) total chlorophyll (mg. g fw­1) total soluble sugars (mg. g dw­1) 1.3 (s0) 1.14 ± 0.05 a 0.58 ± 0.04 a 1.71 ± 0.09 a 7.62 ± 0.55 d 3.3 (s1) 0.79 ± 0.04 b 0.41 ± 0.03 b 1.21 ± 0.06 b 8.33 ± 0.91 d 5.3 (s2) 0.70 ± 0.05 bc 0.33 ± 0.01 bc 1.00 ± 0.04 cd 10.39 ± 1.11 cd 7.3 (s3) 0.70 ± 0.02 bc 0.35 ± 0.02 bc 1.05 ± 0.06 bc 11.69 ± 1.37 bc 9.3 (s4) 0.64 ± 0.03 c 0.30 ± 0.05 c 0.94 ± 0.06 cde 12.85 ± 0.48 bc 11.3 (s5) 0.51 ± 0.04 d 0.30 ± 0.03 c 0.79 ± 0.05 de 13.84 ± 0.85 b 13.3 (s6) 0.49 ± 0.01 d 0.27 ± 0.01 c 0.78 ± 0.11 e 21.61 ± 1.39 a significance ** ** ** ** 150 adv. hort. sci., 2022 36(2): 145­153 et al., 2009). salinity can also inhibit root growth, thereby reducing the absorption and transport capacity of water to the shoot (acosta­motos et al., 2017). irrigation with saline water decreased the rwc of s. lavandulifolia leaves (except s1). the leaf rwc is commonly used to estimate the water status of plants under stress conditions (parida and das, 2005). salinity decreases the leaf rwc due to a reduced availability of water from the soil solution as a result of lowered osmotic potential triggered by the toxic effects of the na+ and cl­ ions (munns, 2005; álvarez et al., 2012; bayat et al., 2012). in this study, the el increased with increasing salinity levels (except s1). increased leaf el under salinity stress has been reported in different crops (bayat et al., 2013; hniličková et al., 2019; karimian et al., 2019). electrolyte leakage is one of the stan­ dard parameters for examining salinity tolerance in plants. salinity stress causes inefficiency of the leaf cell membrane and consequently increases mem­ brane permeability for ions (zhao et al., 2020). in this experiment, the content of chlorophylls decreased with increasing salinity stress levels. reduced leaf chlorophyll content under salt stress conditions has been reported in various crops (taïbi et al., 2016; rahneshan et al., 2018; es­sbihi et al., 2021). valifard et al. (2019) reported that photosyn­ thetic pigments in salvia mirzayanii leaves were decreased by increasing salinity stress. the decrease in chlorophyll content may be related to the toxicity effects of na+ and cl­ ions, which prevent the forma­ tion of pigments (yang et al., 2011). decreased pho­ tosynthetic pigments under salinity stress can be mainly due to the destruction of their structure with the ros and inhibition of biosynthesis of new chloro­ phylls (ashraf, 2003; yang et al., 2020). in this study, irrigation with saline water enhanced the content of leaf total soluble sugars in s. lavandulifolia. accumulation of leaf soluble sugars under salinity stress has been reported in sunflower (zheng et al. , 2010), in salvia miltiorrhiza l. (gengmao et al., 2014), and cotton (peng et al., 2016). karimian et al. (2019) reported that salt stress significantly increased total soluble sugars in the leaves of salvia splendens. increased the content of soluble sugars is an indicator for osmotic regulation under stress conditions, to maintain cell turgor and continued water influx (mittal et al., 2012). soluble sugars were accumulated under salinity stress and protect plants through osmotic regulation, mainte­ nance of turgor pressure, and preservation of mem­ brane and protein stability (bayat et al., 2013; nounjan et al., 2018). the increase in concentration of soluble sugars under stress conditions is due to the higher activity of enzymes such as phosphorylase starch and sucrose phosphate synthase (peng et al., 2016). salinity stress significantly affected the total phe­ nols, total flavonoids, and the frsc of s. lavandulifo‐ lia leaves. various studies have reported the incre­ ment in total phenols, total flavonoids, and the frsc in response to salt stress (karimian et al., 2019; sirin and aslım, 2019). valifard et al. (2014) reported the total phenols and antioxidant activity in salvia mirza‐ yanii were increased by salinity stress. salt stress causes the production of ros, which damages pro­ teins, lipids, and nucleic acids (foyer, 2018). plants use antioxidant defense systems to scavenge and detoxify these compounds from the cell surface, which leads to increased plant antioxidant activity (rezayian et al., 2018; bayat and moghadam, 2019). phenols and flavonoids are secondary metabolites that act as potent antioxidants against oxidative stress. these non­enzymatic antioxidants protect plants by increasing their osmotic potential and thereby avoiding the dehydration of cells or regulat­ ing the redox potential, and depleting the ros (bautista et al., 2016; yan et al., 2017). although the effect of salinity stress on some species of salvia has been studied, so far no research has been done on s. lavandulifolia species. the results demonstrated that salt stress had adverse effects on the growth parameters, photosynthetic pigments, fig. 3 ­ effects of salinity stress on the leaf free radical scaven­ ging capacity (frsc) in s. lavandulifolia. different letters indicate significant differences according to least signifi­ cant difference (lsd) test at p < 0.05. values are mean ± standard error (se). bayat et al. ‐ salinity effects on vegetative parameters of salvia lavandulifolia 151 and cell membrane stability of the s. lavandulifolia plant. however, the total phenolic content and antioxidant activity of the leaves were increased under salinity stress conditions. in general, s. lavan‐ dulifolia can be classified as a species­sensitive plant. however, further experiments are needed to investi­ gate other mechanisms of salt stress tolerance. references abdel latef a.a., 2010 ­ changes of antioxidative enzymes in salinity tolerance among different wheat cultivars. ­ cereal res. commun., 38: 43­55. acosta­motos j.r., ortuño m.f., bernal­vicente a., diaz­vivancos p., sanchez­blanco m.j., hernandez j.a., 2017 ­ plant responses to salt stress: adaptive mechanisms. ­ agronomy, 7(1): 18. ahanger m.a., agarwal r.m., 2017 ­ salinity stress induced alterations in antioxidant metabolism and nitrogen assimilation in wheat (triticum aestivum l.) as influenced by potassium supplementation. ­ plant physiol. biochem., 115: 449­460. álvarez s., gómez­bellot m.j., castillo m., bañón s., sánchez­blanco m.j., 2012 ­ osmotic and saline effect on growth, water relations, and ion uptake and translocation in phlomis purpurea plants. ­ environ. exp. bot., 78: 138­145. amalia l.g., kintzios s.e., 2005 ­ essential oils of salvia spp: examples of intraspecific and seasonal variation. ­ in: kintzios s.e. 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load, respiration rate, sensory quality. abstract: strawberries (fragaria × ananassa duch.) are nutrient­rich specialty fruits with a short shelf life due to microbial spoilage, softening, darkening, and moisture loss. this study aimed to investigate the effectiveness of edible coatings in extending shelf life and maintaining fruit quality. freshly ripened, randomly selected strawberries were coated with 1.5% chitosan, 1.5% chitosan+1% cacl₂, aloe vera gel (avg), and avg+1% cacl₂, along with an uncoated control. each treatment was replicated 3 times with 25 samples per replicate, followed by air drying. the coated strawberries were stored in sterilized polypropylene containers under standard refrigerated conditions (4±1°c; 50±5% relative humidity) for 9 days. the application of edible coatings significantly (p<0.05) reduced respiration rates (by 25 to 34%) and microbial load (by 41 to 62%), helping to preserve fruit color, moisture content, ascorbic acid, firmness, and overall acceptability. the effect was more pronounced in strawberries coated with avg and avg+1% cacl₂ coatings on strawberries throughout storage period. uncoated strawberries had an acceptability score of 4.0, while all coated fruits scored above 5, showing a significant improvement by 20 to 37%. strawberries treated with avg, with or without cacl₂, maintained the highest acceptability score of 5.5, outperforming all other coatings. these findings suggest that aloe vera­based coatings are particularly effective in extending the shelf life and preserving the quality of strawberries during refrigerated storage. 1. introduction strawberries (fragaria × ananassa duch.) are among the most widely (*) corresponding author: taanasrin@gmail.com st111058@alumni.ait.asia citation: nasrin t.a.a., rahman m.a., arfin m.s., afroz m., naznin m.t., molla m.m., sabuz a.a., matin m.a., islam r., 2025 ­ comparative evaluation of aloe vera and chitosan edible coatings on shelf life and quality of strawberries during cold storage. ­ adv. hort. sci., 39(3): 191­ 204. orcid: ntaa: 0000­0003­0473­6632 rma: 0000­0001­7948­3938 ams: 0000­0002­4015­3488 am: 0000­0001­9050­6934 nmt: 0000­0001­7342­6270 mmm: 0000­0002­3851­7094 saa: 0000­0002­9266­7093 mma: 0000­0002­6268­6154 ir: 0000­0001­9332­5493 copyright: © 2025 nasrin t.a.a., rahman m.a., arfin m.s., afroz m., naznin m.t., molla m.m., sabuz a.a., matin m.a., islam r. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 5 may 2025 accepted for publication 7 july 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17811 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0003-0473-6632 http://orcid.org/0000-0001-7948-3938 http://orcid.org/0000-0002-4015-3488 https://orcid.org/0000-0001-9050-6934 http://orcid.org/0000-0001-7342-6270 http://orcid.org/0000-0002-3851-7094 http://orcid.org/0000-0002-9266-7093 http://orcid.org/0000-0002-6268-6154 http://orcid.org/0000-0001-9332-5493 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 191­204 192 consumed specialty fruits worldwide, prized for their vibrant color, distinct flavor, sweet­tart taste, and high nutritional value. the global demand for strawberries continues to rise with worldwide production reaching 40.8 million tons in 2020 (fao, 2022). their versatility culinary applications ­ from fresh consumption to processed products such as jams, jellies, beverages, dairy items, and flavored drinks ­ makes strawberries one of the most widely used and adaptable crops in the world (wise et al., 2024). rich in vitamins, minerals, flavonoids, anthocyanins, proteins, and phenolic compounds (temiz and ozdemir, 2021), strawberries offer numerous health benefits. however, their delicate texture and high respiration rate significantly reduce their shelf life. as a result, strawberries are highly susceptible to bruising, moisture loss, discoloration, microbial spoilage, and softening (nasrin et al., 2017). to maintain fruit quality, strawberries should be rapidly chilled and stored at low temperatures (0­ 4°c) immediately after harvest. however, even with proper cold storage, their shelf life typically remains limited to less than five days (shankar et al., 2021). to further reduce postharvest losses and extend shelf life, several complementary strategies have been investigated, including active packaging, modified atmosphere packaging, and the use of edible coatings (zhang et al., 2022). among the emerging postharvest preservation techniques, edible coatings have attracted considerable attention in the fruit and vegetable industry for their proven effectiveness in preserving quality and prolong shelf l ife (sousa cesar de albuquerque et al., 2024). these coatings act as physico­chemical barriers, protecting against microbial contamination and water loss while preserving texture, color, flavor, and volati le compounds. additionally, they help reduce respiration and transpiration rates, thereby delaying senescence (nasrin et al., 2023). polysaccharide­ based coatings are particularly valued for their excellent film­forming ability, mechanical strength, and selective permeability ­ especially in regulating oxygen exchange (rios et al., 2022). aloe vera gel (avg) is an increasingly recognized edible coating material, primarily due to its high polysaccharide and soluble sugar content, which aids in preserving fruit quality by regulating water and oxygen exchange. this regulation helps lower respiration rates and maintain the fruit’s texture, moisture content, color, taste, and firmness (sogvar et al., 2016; nicolau­lapena et al., 2021). avg is colorless, tasteless, and does not alter the sensory attributes of coated fruits (hasan et al., 2021). moreover, it contains more than 200 bioactive compounds with antioxidant, antiviral, and antibacterial properties (nguyen et al., 2020). beyond extending shelf life, avg coatings also offer the potential to enhance the functional qualities of fruits through the incorporation of additional bioactive ingredients. due to excellent film­forming and antimicrobial properties, avg is an effective edible coating that can be applied alone or in combination with other ingredients to extend the shelf life of a wide variety of fruits and vegetables, including lime (pimsorn et al., 2022), pistachio (valverde et al., 2005), apples (ergun and satici, 2012), guava (shabir et al., 2021), strawberries (hassan et al., 2022), and tomato (chrysargyris et al., 2016). chitosan­based coatings have also demonstrated efficacy in reducing fungal decay, delay ripening and senescence, and maintaining the postharvest quality of fresh produce such as tomatoes, strawberries, and cherry tomatoes (zheng et al., 2024). similarly, calcium chloride (cacl₂) applications have demonstrated benefits such as improved fruit firmness, enhanced antioxidant activity, reduced disease incidence, and mitigation of physiological disorders like internal browning and senescence (nguyen et al., 2020). however, there is limited information on the combined effects of avg or chitosan with cacl2 on the postharvest quality of strawberries. this study tests the hypothesis that avg, either alone or in combination with cacl₂, will enhance the postharvest quality and shelf life of strawberries more effectively than conventional chitosan­based coatings. by comparing key quality parameters such as firmness, microbial stability, moisture retention, and sensory acceptability, the study aims to determine the most effective edible coating strategy for maintaining strawberry freshness during storage. 2. materials and methods plant material freshly ripened strawberries (var. bari strawberry 3) were randomly harvested from the nasrin et al. ‐ avg coating preserves postharvest quality of strawberry 193 replicate. strawberries were immersed in the respective coating solutions for one minute, then air­dried using a high­speed fan. following drying, the fruits were stored in clear polypropylene containers at 4±1°c and 50±5% relative humidity in an incubator. selected chemical, physical, and sensory attributes were assessed at harvest (day 0) and after 3, 6, and 9 days of storage. chemical and physical properties of strawberries respiration rate for respiration rate measurement, 10 fruits from each replication were used. throughout the storage period, respiration rates were recorded at designated intervals. fruits from each replication were placed in a 1000 ml airtight jar sealed with septa and incubated at 20±2°c for two hours. after incubation, a 1 ml gas sample was extracted from the headspace of the jar using a syringe and analyzed with a co₂/o₂ gas analyzer (quantek® instruments, model 902d, usa). the concentration of co₂ produced within the jar was recorded. respiration rate was then calculated using the total gas volume of the jar, the weight and volume of the strawberries, and the incubation time. results were expressed as ml co₂ kg⁻¹ h⁻¹ (nasrin et al., 2020). firmness three fruits from each replication were used to evaluate strawberry firmness using a fruit texture analyzer (guss®, model gs25, sa). a stainless­steel flat­headed probe with an 8 mm diameter penetrated the fruit at a speed of 5 mm s⁻¹. firmness was defined as the maximum force required to penetrate the fruit tissue. following zero­force contact between the probe and the horizontally positioned strawberry, the equatorial region of each fruit was tested at two evenly spaced points, with 3 mm penetration depth. data analysis was based on the maximum force recorded during probe movement, with results expressed in newtons (n). weight (moisture) loss strawberry weight loss (moisture content) was measured using 10 fruits from each replication at the start of the experiment ­ immediately after the surface coating was applied and dried ­ and subsequently at three­day intervals throughout the storage period. weight loss was computed by using field plots of the fruit research farm at the horticulture research center (hrc), bangladesh agricultural research institute (bari), gazipur, bangladesh. a total of 375 strawberries were selected based on uniform size, absence of microbial infection or physical damage, and having more than 80% red surface area. aloe vera leaves were sourced from the flower research farm of hrc at bari, gazipur. edible coating formulations high molecular weight chitosan­652 was procured from mahtani® chitosan pvt. ltd., india was used to prepare a 1.5% chitosan solution, by dissolved 1.5 g of chitosan in 75 ml of distilled water, followed by the addition of 2 ml of reagent­grade acetic acid. the mixture was warmed to 55±2°c and stirred continuously to ensure homogeneity. a 2m naoh solution was then added to adjust the ph to 5.6. sterilized distilled water was subsequently added to bring the final volume to 100 ml (jiang and li, 2001). to prepare the 1.5% chitosan+1% cacl₂ solution formulation, 1 g of cacl₂ was added to the 1.5% chitosan solution and thoroughly stirred. mature aloe vera leaves were washed with a 25% aqueous chlorine solution. the outer layer was removed to extract the clear, water­based gel (lymph), which was ground and filtered to remove fibers. the gel was pasteurized at 70°c for 45 min and then cooled to room temperature (~25 °c). to stabilize the ph at ~4.0, ascorbic acid (2.0 g l⁻¹) and citric acid (4.5 g  l⁻ ¹) were added. to enhance viscosity and coating performance, 1% sodium carboxymethyl cellulose (cmc), a natural cellulose­ based gelling agent, was incorporated and thoroughly mixed. the solution was stored in an opaque glass container to prevent oxidation (nasrin et al., 2017). for another formulation, 1 g of cacl₂ was added to the avg solution and thoroughly mixed to obtain the avg+1% cacl₂ solution. experiment and coating application the experiment was conducted using a completely randomized design (crd) under controlled laboratory conditions, with 75 strawberries assigned to each coating treatment. five coating treatments were evaluated: (1) control (no coating), (2) 1.5% chitosan solution, (3) 1.5% chitosan + 1% cacl₂ solution, (4) avg, and (5) avg + 1% cacl₂ solution. each treatment was replicated three times, with 25 randomly selected fresh strawberries per adv. hort. sci., 2025 39(3): 191­204 194 the following equation and then displaying the result as a percentage. weight loss (%) = (initial fruit weight ­final fruit weight at indicated period)/(initial fruit weight) x100 external fruit color surface color of strawberries was measured on five fruits from each replication using a chroma meter (model cr­400, minolta corp., japan) based on the cie lab* system. here, l* denotes lightness, while a* and b* values were used to calculate chroma (c) and hue angle (h°). the instrument was calibrated with the provided white ti le before measurement. multiple readings were taken from different areas of each fruit. after measuring respiration rate, weight loss, and color, the fruits were returned to refrigeration to continue the experiments and assess shelf life. ascorbic acid, titratable acidity, and sugar ascorbic acid, titratable acidity, total sugar, and reducing sugar concentrations were analyzed following aoac (1994) standard methods. total soluble solids (tss) were measured using a refractometer, and the ph of strawberry juice was determined using a ph meter (hanna® instruments, ph­211; microprocessor ph meter, italy). ascorbic acid concentration in strawberry juice was determined using a titration method with 2,6­ dichlorophenolindophenol (dcpip) dye. in this method, ascorbic acid in an alkaline medium reduces the blue­colored dcpip dye to a colorless form. the dye solution was first standardized against known concentrations of ascorbic acid to determine the dye factor. for analysis, strawberry juice was diluted with 3% metaphosphoric acid and then titrated with the dcpip solution until a persistent pink endpoint lasting 15 seconds was observed. dye factor= 0.5/(titrate volume) ascorbic acid (mg/100g) = (titre x dye factor x volume x 100)/(aliquot of extract taken x weight of sample) titratable acidity was determined by blending 10 g of strawberries with 100 ml of distilled water, followed by filtration of the mixture. three to four drops of phenolphthalein indicator were then added, and the filtrate was titrated with 0.1 m naoh. the titratable acidity was calculated using the following formula: titratable acidity (%) = [(titre vol.× normality of naoh × vol.made up × eq.wt.of acid)/( aliquot of sample × vol. of sample × 1000)] x100 total soluble solids were measured using a hand­ held refractometer (atago® master­53α, japan) and expressed in °brix. a small volume of strawberry juice was placed on the prism surface of the refractometer, and the tss value in °brix was recorded directly from the instrument’s display microbiological analysis of strawberries a 10 g sample of fresh strawberries were thoroughly combined with 90 ml of sterilized 0.9% nacl solution. the homogenized sample was included in 1 ml to the corresponding dilutions (10­1 to 10­6) applying a 0.9% sodium chloride solution. total bacterial count (tbc) was measured using nutrient agar (difcotm, usa, h 7.0­7.4), while molds as well as yeast were counted using potato dextrose agar (pda, himedia, india). the media was made as directed by the manufacturer. inoculated nutrient agar media plates underwent incubation for 24 to 28 hours at 37°c, while plates with pda were kept for 5 days at room temperature (26 ± 2°c). plates showing colonies were examined after incubation. tbc was calculated by multiplying the dilution factor by the average number of colonies in a given dilution. colony forming units per gram (cfu/g) were used to represent the microorganisms present in the samples (mahfuza et al., 2016). sensory quality sensory quality of fresh strawberries was assessed by 15 trained panelists (aged 25­50 years, both male and female). the panel evaluated the samples based on color, flavor, texture, and overall acceptability. prior to the evaluation, panelists underwent pre­ training focused on strawberry appearance, aroma, and taste. for each treatment, three samples were evaluated per panelist in a randomized order. samples were blindly labeled using random three­ digit codes to minimize bias. to cleanse their palate between samples, panelists rinsed their mouths with plain water. evaluations were conducted using a 9­point hedonic scale ranging from 1 to 9, where: 1 = dislike extremely, 2 = dislike very much, 3 = dislike moderately, 4 = dislike slightly, 5 = neither like nor dislike, 6 = like slightly, 7 = like moderately, 8 = like very much, and 9 = like extremely (nasrin and anal, 2015). a score of 5 (“neither like nor dislike”) was nasrin et al. ‐ avg coating preserves postharvest quality of strawberry 195 used as the cutoff point for consumer acceptability. statistical analysis data were analyzed using a two­way analysis of variance (anova) based on a crd to assess the effects of different coating treatments and storage time on various quality parameters of strawberries. the anova was conducted using sas® software (version 9.2, 2010), with coating treatment considered a fixed effect and time treated as a random effect to account for temporal variation. the model evaluated the main effects and interactions on dependent variables such as firmness, color, weight loss, tss, acidity, ph, total sugar, reducing sugar, flavor, texture, and overall acceptability. the least significant difference (lsd) test was used to compare treatment means, with significance set at p < 0.05 unless otherwise noted. graphical representations and regression analyses were performed using sigmaplot® software. 3. results respiration rate the initial respiration rate of strawberries was 63 ml kg⁻¹ h⁻¹, which was significantly and non­linearly reduced by nearly half when coated with either avg alone (y = 61.6 – 9.61*x + 0.79*x2) or avg with 1% cacl₂ (y = 61.7 – 9.83*x + 0.79*x2), accounting 93 and 94% of the variation in respiration rates during storage, respectively (fig. 1). in uncoated control strawberries, respiration rate began increasing significantly from the 3rd day, while in coated samples, it started to rise slightly only after the 6th day (y = 33 + 30.3 (­0.56*x) + 2.35*x). by the 9th day, the highest respiration rate (54.3 ml kg⁻¹ h⁻¹) was observed in the uncoated control strawberries, whereas the lowest (35.6 ml kg⁻¹ h⁻¹) was recorded in strawberries coated with avg+1% cacl₂. however, no significant differences were found among the coated treatments. fruit firmness the initial firmness of the strawberries was 2.42 n, which declined significantly and linearly over time; however, the rate of softening varied among the coating treatments (fig. 2). strawberries coated with avg+1% cacl₂ retained the highest firmness (y = 2.47 – 0.05*x) explaining 86% of the variability in firmness. these strawberries lost only 20.2% of their initial firmness by day 9. in contrast, uncoated control strawberries exhibited the greatest decline in firmness (y = 2.31 – 0.12*x), showing a 47% reduction over the same period. fig. 1 ­ effects of various edible coatings on respiration rates (ml co₂ kg⁻¹ h⁻¹) of strawberries during storage at 4±1°c control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan +1% cacl2 coated, avg= aloe vera gel coated, avg+cacl2= avg+1% cacl2 coated. data presented with standard error of mean. fig. 2 ­ effects of various edible coatings on firmness (n) of strawberries during storage at 4±1°c [control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan + 1% cacl2 coated, avg= aloe vera gel coated, avg+cacl2= avg+1% cacl2 coated. data presented with standard error of mean. strawberries began decreasing after day two (y = 36 – 0.66x – 0.035x²), dropping by 24.2% by day six (fig. 4b). in contrast, coated fruits maintained a stable hue angle until day six, followed by a slight decline. on day nine, the lowest hue angle (27.3) was recorded in the uncoated control, while the highest 196 adv. hort. sci., 2025 39(3): 191­204 weight (moisture) loss uncoated control strawberries exhibited the highest linear weight (moisture) loss during storage (y = 0.08 + 0.97*x) compared to coated strawberries (fig. 3). coating with 1.5% chitosan, either with (y = 0.30 + 0.64*x) or without 1% cacl₂ (y = 0.20 + 0.67*x) significantly reduced weight loss to approximately ~ 6%. in contrast, strawberries coated with avg, with (y = 0.17 + 0.55*x) or without 1% cacl₂ (y = 0.13 + 0.53*x), exhibited the lowest weight loss—around 5%—relative to the uncoated control. external fruit colour throughout storage, uncoated control strawberries appeared darker than coated ones (fig. 4a). among coatings, avg­treated strawberries exhibited a brighter red color than those with chitosan. the addition of 1% cacl₂ improved lightness in both coatings. by day nine, the lightness (l*) value declined significantly and linearly in uncoated control fruits (y = 40 – 1.28x), with a 29.5% reduction, compared to 14.7% for chitosan + cacl₂ (y = 40.4 – 0.59x) and 11.7% for avg + cacl₂ (y = 40.8 – 0.53*x), explaining 99% of color variation. the hue angle (ho) of uncoated control fig. 3 ­ effects of various edible coatings on weight (moisture, %) loss of strawberries during storage at 4±1°c. control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan +1% cacl2 coated, avg= aloe vera gel coated, avg+cacl2 = avg+1% cacl2 coated. data presented with standard error of mean. fig. 4 ­ a) effects of various edible coatings on external fruit colour evolution (lightness, l*) of strawberries stored at 4±1°c. b) effects of various edible coatings on external fruit colour evolution (hue angle, ho) of strawberries stored at 4±1°c control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan +1% cacl2 coated, avg= aloe vera gel coated, avg+cacl2= avg+1%cacl2 coated. data presented with standard error of mean. nasrin et al. ‐ avg coating preserves postharvest quality of strawberry 197 (33.3) was observed in avg+cacl₂­coated strawberries (y = 35.9 + 0.64x ­ 0.1x²). ascorbic acid, titratable acidity, and sugar at the beginning of storage, the ascorbic acid, titratable acidity, and ph of strawberries were 47.8 mg 100 g­1, 0.86%, and 3.94, respectively (table 1). by the ninth day, the ascorbic acid concentration in control fruits had significantly decreased to 35.6 mg/100 g. in contrast, strawberries coated with avg+1% cacl₂ maintained the highest ascorbic acid concentration at 44.9 mg 100 g ­1, which was significantly higher than that of both the control and other coated treatments. although titratable acidity declined and ph slightly increased by day nine, these changes were not statistically significant among the different coating treatments (table 1). regarding sugar content, initial values for tss, total sugars, and reducing sugars in fresh strawberries were 7.5ᵒbrix, 5.4%, and 4%, respectively (table 1). all these values increased by day nine. however, there were no significant differences in total and reducing sugar contents among the treatments. the highest tss (9.4ᵒbrix) was observed in the control (uncoated) strawberries, whereas significantly lower tss values (8.3ᵒbrix) were recorded in strawberries coated with avg, either alone or combined with cacl₂. there were no significant differences in tss among the coated fruits. microbiological analysis although coating treatments initially caused a non­significant reduction in total bacterial count (tbc), microbial loads increased non­linearly over time from a baseline of 2.11 log cfu g⁻¹ (fig. 5). strawberries coated with avg, with (y = 2.08 – 0.64x table 1 ­ effect of various edible coatings on biochemical properties (ascorbic acid, acidity, ph, total soluble solids, total sugar, and reducing sugar) of strawberries stored in the refrigerator (at 4±1°c) over a nine­day period control= uncoated, chi= 1.5% chitosan coated, chi+cacl2 = 1.5% chitosan +1% cacl2 coated, avg= aloe vera gel (avg) coated, avg+cacl2= aloe vera gel (avg)+1% cacl2 coated. tss=total soluble solids. ns= not significant. coating treatments day ascorbic acid (%) acidity (%) ph tss (%) total sugar (%) reducing sugar (%) control 0 47.8 0.86 3.94 7.5 5.4 4.0 9 35.6 0.73 3.99 9.4 5.6 4.2 chi 0 47.8 0.86 3.94 7.5 5.4 4.0 9 41.6 0.74 3.97 8.6 5.5 4.1 chi+cacl2 0 47.8 0.86 3.94 7.5 5.4 4.0 9 41.5 0.77 3.97 8.5 5.5 4.1 avg 0 47.8 0.86 3.94 7.5 5.4 4.0 9 42.1 0.75 3.96 8.3 5.5 4.0 avg+cacl2 0 47.8 0.86 3.94 7.5 5.4 4.0 9 44.9 0.77 3.95 8.3 5.4 4.0 lsdp<0.05 coating 2.66 0.02 ns 0.02 ns 0.28 0.23 ns 0.12 ns time 1.68 0.01 0.01 0.18 0.14 ns 0.08 coating x time 1.20 0.01 ns 0.01 ns 0.13 0.10 ns 0.05 ns fig. 5 ­ effects of various edible coatings on total bacterial count (log cfu g­1) of strawberries during storage at 4±1°c control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan +1% cacl2 coated, avg= aloe vera coated, avg+cacl2= avg+1% cacl2 coated. data presented with standard error of mean. + 0.08x²) or without 1% cacl₂ (y = 2.08 – 0.57x + 0.085x²), showed significantly lower tbc compared to the uncoated control (y = 2.29 + 0.65*x). by the end of storage, tbcs were 3.1 and 3.8 log cfu g⁻¹ for avg+cacl₂ and avg coatings, respectively, versus 8.13 log cfu g⁻¹ for the control. avg alone reduced microbial load by 4.33 log cfu g⁻¹ (~53%), while the addition of cacl₂ provided an incremental reduction of 0.7 log cfu g⁻ ¹ (~9%). notably, all coated strawberries, except the control, remained below the permissible microbial contamination limit throughout storage. sensory quality and its relationship with other properties throughout the storage period, all fruits regardless of coatings exhibited variations in sensory properties (table 2 and fig. 6). by the 7th day of storage, the uncoated control strawberries exhibited lower sensory scores, with color (4.1), flavor (4.3), texture (4.3), and overall acceptability (4.0), falling below the consumer acceptability threshold. in adv. hort. sci., 2025 39(3): 191­204 198 table 2 ­ effects of various edible coatings on sensory quality of strawberries (color, flavor, texture and overall acceptability) during storage in the refrigerator (at 4±1°c) over a nine­day period control= uncoated, chi= 1.5% chitosan coated, chi+cacl2= 1.5% chitosan +1% cacl2 coated, avg= aloe vera gel coated, avg+cacl2= avg+1% cacl2 coated. ns= not significant. coating day color flavor texture overall acceptability control 7 4.1 4.3 4.3 4.0 9 3.0 3.1 2.6 2.5 chi 7 5.5 5.3 5.8 5.5 9 4.1 4.2 4.4 4.1 chi+cacl2 7 5.5 5.0 5.9 5.8 9 4.3 4.6 4.5 4.4 avg 7 7.2 6.9 7.0 7.3 9 5.8 5.9 5.1 5.5 avg+cacl2 7 7.6 6.9 7.3 6.9 9 5.6 5.5 5.5 5.5 lsdp<0.05 coating 0.46 0.40 0.43 0.41 time 0.29 0.25 0.27 0.26 coating x time 0.19 ns 0.17 ns 0.18 ns 0.17 ns fig. 6 ­ visual appearance of post edible­coated strawberry treatments (control=uncoated, chi= 1.5% chitosan, chi+cacl2= 1.5% chitosan +1% cacl2, avg= aloe vera gel, avg+cacl2= avg+1% cacl2) for 9 days refrigerated storage at 4±1°c. nasrin et al. ‐ avg coating preserves postharvest quality of strawberry 199 contrast, all coated strawberries­maintained scores above 5. notably, strawberries coated with avg, with or without cacl₂, achieved higher sensory scores ranging from 6.9 to 7.6 and retained an average overall acceptability of 5.5 even by the 9th day of storage. meanwhile, the sensory scores of strawberries coated with chitosan, with or without cacl₂, declined below acceptable levels by the 9th day, with overall acceptability dropping below 4.5, once again falling beneath the consumer acceptance threshold. pearson correlation analysis of strawberry chemical, physical, and sensory attributes revealed that increased moisture loss was significantly and strongly correlated with reductions in firmness and ph, and moderately associated with decreases in reducing sugars, color, flavor, texture, and overall acceptability (table 3). firmness exhibited significant positive correlations with ph, reducing sugars, color, flavor, texture, and overall acceptability. similarly, color showed significant positive correlations with flavor, texture, and overall acceptability, and these relationships were reciprocal. total soluble solids (tss) did not show significant correlations with any measured strawberry properties. in contrast, increasing ascorbic acid (vitamin c) content was positively correlated with total sugar concentration but negatively correlated with color, flavor, texture, and overall acceptability. titratable acidity was significantly negatively correlated with both total and reducing sugar concentrations. 4. discussion and conclusions respiration rate a significant reduction in respiration rates of strawberries was due to avg coatings with or without cacl2 was beneficial as the elevated respiration rates accelerate carbohydrate depletion, leading to faster deterioration of fruit quality and reduced shelf life (nasrin et al., 2022). avg coatings, especially when combined with cacl2, can significantly influence fruit gas exchange possibly causing saturation effects (valverde et al., 2005). cacl₂ enhances the structural stability of avg and introduces ca ions that may interact with aloe vera polysaccharides and phenolics, altering viscosity, film properties, and antimicrobial activity (shabir et al., 2021). these interactions results in decreased respiration, ethylene production, transpiration, mechanical damage, and microbial growth (blancas­ benitez et al., 2022). shafique et al. (2023) reported that strawberries coated with 20% avg showed a two­fold reduction in respiration rate at five days of shelf l ife, when compared to control ones. furthermore, eshghi et al. (2014) observed significantly lower respiration rates in strawberries coated with nano­chitosan compared to uncoated control ones. fruit firmness as anticipated, strawberries coated with avg exhibited the highest firmness, primarily due to the table 3 ­ pearson correlations among strawberry chemical, physical, and sensory properties *, **, and *** indicate significant correlations at p<0.05, 0.01, and 0.001 levels, respectively. ns = not significant. firmness respirative rate total soluble solids ph ascorbic acid titrable acid total sugar reducing sugar color flavor texture acceptability moisture ­0.95 *** ­0.51 * 0.23 ns ­0.88 *** 0.25 ns 0.63 ** ­0.45 * ­0.69 ** ­0.48 * ­0.46 * ­0.53 * ­0.50 * firmness 0.32 ns ­0.29 ns 0.85 *** ­0.48 * ­0.48 * 0.24 ns 0.67 ** 0.58 * 0.56 * 0.63 ** 0.61 ** resp 0.21 ns 0.49 * 0.27 ns ­0.66 ** 0.60 * 0.30 ns 0.01 ns ­0.06 ns 0.06 ns 0.07 ns tss ­0.23 ns 0.26 ns ­0.09 ns 0.12 ns 0.11 ns ­0.26 ns ­0.23 ns ­0.29 ns ­0.21 ns ph ­0.25 ns ­0.53 * 0.31 ns 0.66 ** 0.25 ns 0.21 ns 0.33 ns 0.28 ns aacid ­0.3 2 ns 0.63 ** ­0.2 2ns ­0.67 ** ­0.68 ** ­0.64 ** ­0.68 ** tacid ­0.87 *** ­0.64 ** 0.17 ns 0.19 ns 0.12 ns 0.16 ns tsugar 0.35 ns ­0.36 ns ­0.36 ns ­0.31 ns ­0.34 ns rsugar 0.15 ns 0.16 ns 0.19 ns 0.19 ns color 0.99 *** 0.97 *** 0.98 *** flavor 0.94 *** 0.97 *** texture 0.98 *** adv. hort. sci., 2025 39(3): 191­204 200 preservation of polysaccharide structures that enhance cell wall strength. fruit softening typically occurs during development, ripening, and senescence, driven by water loss and the degradation of structural components through respiration. however, avg helps mitigate these effects by forming a protective physical barrier that reduces water evaporation and maintains the integrity of the fruit’s dry matter content, thereby slowing the softening process and extending postharvest quality (nasrin et al., 2023). the avg + 1% cacl₂ coating was particularly effective in reducing fruit softening by inhibiting enzyme activity, especially polygalacturonase, which breaks down cell walls during ripening. additionally, their synergistic effects limit oxygen uptake, moisture loss, microbial growth, and metabolic activity, thereby delaying ripening and preserving firmness in strawberries (rehman et al., 2022). calcium contributes further by strengthening cell walls and enhancing tissue firmness through pectin formation (white and broadley, 2003). additionally, a 1.5% chitosan coating ­ with or without cacl₂ ­ has been shown to better maintain strawberry firmness than a 1% cacl₂ dip over a 4­day storage period at 20°c (hernandez­munoz et al., 2006). weight (moisture) loss weight (moisture) loss in strawberries increases susceptibility to shrinkage, skin wounding, softening, deterioration, and color darkening. the extent of moisture loss is largely influenced by the gradient in atmospheric pressure between the internal fruit tissues and the surrounding environment. coating treatments, such as avg and chitosan, effectively reduce this gradient by forming semi­permeable barriers that minimize transpiration, regulate gas exchange, and retain cellular moisture. the hygroscopic nature of avg further enhances its ability to prevent water transfer between the fruit and its surroundings (valverde et al., 2005). studies have reported that avg coatings significantly reduce water vapor transmission rate and moisture loss in perishable fruits compared to untreated controls (olivas and barbosa­cánovas, 2005). likewise, hassan et al. (2022) reported that uncoated strawberries exhibited the highest weight loss (11.9%), while those coated with 40% avg + 1% lemongrass essential oil showed the lowest (6.1%), followed by 20% avg + 1% lemongrass essential oil (7.6%) after 16 days of storage at 5 ± 1°c. similarly, nguyen et al. (2020) demonstrated that a coating of 0.2% nano­chitosan combined with 3% cacl₂ was more effective in reducing weight loss than nano­ chitosan alone or uncoated controls. this improvement is likely due to calcium’s role in modifying tissue gas diffusion rates and affecting respiratory metabolism. overall, the reduced moisture loss in coated strawberries helps maintain fruit weight, limits respiration, delay senescence, and enhance postharvest shelf life by slowing physiological processes associated with deterioration. external fruit colour coating fruits with avg creates a modified environment that helps prevent chlorophyll degradation and carotenoid pigment synthesis, thereby delaying ripening (ergun and satici, 2012). similarly, chitosan coatings have been shown to slow respiration and reduce ethylene production in papaya, resulting in delayed ripening and senescence, along with reduced softening and color changes (ali et al., 2011). in strawberries, uncoated fruits showed approximately a 29% reduction in lightness, whereas fruits coated with 1%, 1.5%, and 2% chitosan exhibited lightness losses of around 16%, 11%, and 10%, respectively. enzymatic browning, primarily driven by the oxidation of polyphenolic compounds catalyzed by polyphenol oxidase (ppo), remains a major postharvest issue in strawberries (kebriti et al., 2025). however, strawberries coated with avg showed only a 4% loss in lightness, likely due to the inhibition of ppo activity (nasrin et al., 2017). this suppression of enzymatic browning helps preserve the fruit’s color and overall quality during storage. moreover, strawberries coated with avg alone or in combination with lemongrass essential oil retained their lightness more effectively and maintained visual quality up to 16 days at 5°c (hassan et al., 2022). treatments combining 40% avg + 1% lemongrass essential oil and 20% avg + 1% lemongrass essential oil also preserved higher hue angle values than the control during eight days of storage at 5°c. additionally, amal et al. (2010) found that strawberries coated with soy or gluten fi lms incorporating thymol, as well as soy­based coatings containing cacl₂, exhibited brighter red coloration and higher chroma values compared to uncoated fruits. nasrin et al. ‐ avg coating preserves postharvest quality of strawberry 201 ascorbic acid, titratable acidity, and sugar the reduced loss of ascorbic acid (vitamin c) in coated strawberries is attributed to the protective effect of the edible coating, which limits oxygen exposure and slows respiration, thereby minimizing oxidative degradation (ayranci and tunc, 2004). additionally, cacl₂ has been shown to inhibit antioxidant loss, including that of ascorbic acid (luna­ guzman and barrett, 2000). our findings align with those of muzzaffar et al. (2016), who reported initial values for ascorbic acid, tss, acidity, total sugars, and reducing sugars in fresh strawberries as 38.6 mg 100 g­1, 8 °brix, 1.3%, 5.2%, and 4.3%, respectively. similarly, shabir et al. (2021) observed that during storage, fruit ph and tss increased, while ascorbic acid and titratable acidity decreased. these changes were attributed to moisture loss through respiration and transpiration, starch breakdown, and the synthesis of pectin and free acids. hassan et al. (2022) further confirmed that ph levels gradually increased and titratable acidity declined due to the oxidation of organic acids during ripening. lower tss in avg­coated strawberries result from both reduced concentration effects and slowed metabolic changes. avg coatings form a semi­ permeable barrier that minimizes water loss, limiting the passive concentration of sugars typically seen in uncoated fruits during storage (olivas and barbosa­ cánovas, 2005; nasrin et al., 2017). this barrier helps maintain fruit moisture, preventing the artificial tss increase caused by dehydration. simultaneously, avg reduces respiration rates by restricting oxygen diffusion, thereby slowing ripening and metabolic activity (valverde et al., 2005; rehman et al., 2022). this delay in ripening impedes the enzymatic breakdown of complex carbohydrates into simple sugars by inhibiting enzymes like amylases and polygalacturonases (hassan et al., 2022; nicolau­ lapena et al., 2021). studies in guava and other fruits support this, showing that coated fruits accumulate sugars more slowly than uncoated controls (ali et al., 2011; rehman et al., 2022). therefore, avg­coated strawberries are expected to exhibit stable sugar concentrations over storage, avoiding the tss spikes measured in uncoated fruit due to dehydration and accelerated metabolism. microbiological analysis the reduction in total bacterial counts on of avg and chitosan coated strawberries, with or without the addition of cacl₂ is largely attributed to the complementary antimicrobial actions. avg’s efficacy stems from its bioactive compounds ­ primarily aloin and aloe­emodin ­ which inhibit fungal spore germination and suppress the growth of yeasts and molds (huang and yan, 2023). moreover, avg forms a semi­permeable barrier that reduces moisture loss, thereby limiting the water availability essential for microbial proliferation. cacl₂ complements this effect by lowering cellular ph and water activity within the fruit’s microenvironment, indirectly inhibiting microbial survival and possibly interfering with spore germination. though cacl₂ has limited direct action against spore­forming bacteria, it alters environmental conditions unfavorable for their growth. coatings combining avg with antimicrobial agents, such as lemongrass oil or cacl₂ , have demonstrated enhanced microbial suppression (hassan et al., 2022). in this study, avg alone reduced microbial loads by ≈53%, while the addition of cacl₂ resulted in an additional reduction of ≈9%. these results confirm that avg is the primary antimicrobial agent, with cacl₂ providing modest but synergistic enhancement, offering an effective strategy against postharvest spoilage, especially from yeasts, molds, and some bacteria. sensory quality and its relationship with other properties throughout the storage period, all fruits exhibited a decline in sensory properties, likely due to factors such as endogenous enzyme activity, skin darkening, and moisture loss (badawy et al., 2017). our research aligns with the findings of shabir et al. (2021), who reported that refrigerated fruits such as guavas coated with 10% avg and 2% cacl₂ achieved the highest overall acceptability score (6.18), compared to a score of 5.18 for uncoated fruits. similarly, pimsorn et al. (2022) observed that lime coated with avg exhibited superior sensory and visual qualities, reduced weight loss, improved firmness, elevated ascorbic acid concentration and acidity, delayed increases in tss, reduced decay, and extended shelf life compared to uncoated counterparts. chrysargyris et al. (2016) found that tomatoes coated with 10% and 15% avg and stored at 11 °c and 90% relative humidity maintained their overall quality by reducing ripening indices and ethylene production. amiri et al. (2022) demonstrated that a coating formulation containing avg, 2% cacl₂, and 5% nano­encapsulated adv. hort. sci., 2025 39(3): 191­204 202 catechin significantly enhanced the quality and shelf life of refrigerated strawberries. pearson correlation analysis revealed that moisture loss was a key factor in strawberry quality decline, strongly negatively correlated with firmness and ph, and moderately with sugars, color, flavor, texture, and acceptability. firmness showed strong positive correlations with nearly all quality traits, making it a reliable quality indicator. color was also positively linked with key sensory attributes. total soluble solids showed no significant associations, indicating stability during storage. ascorbic acid correlated positively with total sugars but negatively with sensory traits, suggesting stress­ related changes. titratable acidity was negatively associated with sugars, reflecting ripening processes. moisture retention and firmness are shown vital for shelf life. this study demonstrated that uncoated strawberries retained acceptable quality for only 6 days, whereas those coated with 1.5% chitosan extended shelf life to 8 days. notably, strawberries coated with avg, with or without cacl₂, maintained freshness for over 9 days, exhibiting superior firmness, color, moisture, and flavor, and texture retention, with no visible decay. although the addition of cacl₂ sl ightly enhanced firmness, moisture retention, and microbial suppression, these enhancements were not statistically significant. interestingly, avg alone provided sensory quality comparable to the avg+cacl₂ combination. these findings underscore avg’s strong potential as a natural, eco­friendly coating for extending strawberry shelf life and preserving nutritional quality, offering a sustainable alternative to synthetic packaging. further research is recommended to investigate the phytochemical and metabolomic profi les, essential nutrient content, and cost­ effectiveness to 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combination. ­ postharvest biol. technol., 213: 112922. impaginato 71 adv. hort. sci., 2020 34(1): 71­79 doi: 10.13128/ahsc­8403 varietal differences in sweetness value and flesh juiciness among persimmon cultivars m. shiraishi (*), h. asakuma fukuoka agricultural and forestry research center, chikushino 818‐8549, japan. key words: breeding stock, diospyros kaki thunb., flesh juiciness, sweetness value. abstract: to promote persimmon breeding project, we analyzed the sugar com­ position (a ratio of sucrose to hexose sugars, sh ratio) and flesh juiciness of 43 persimmon cultivars (diospyros kaki thunb.) consisting of 24 pollination­con­ stant non­astringent (pcna)­types and 19 non­pcna­types, together with other fruit quality traits. the cultivar collection includes newly­released cultivars after 1990 and commercially­produced local cultivars in japan. these cultivars were broadly classified into three types: sucrose accumulators, intermediate accu­ mulators, hexose accumulators. analysis of variance showed that the genotypic effect on the sh ratio and flesh juiciness is high with negligibly small environ­ mental variance, indicating that sh ratio and flesh juiciness can be determined by a one­year trial without tree replication. highly varietal diversity in the sh ratio and flesh juiciness was observed within and between persimmon cultivar types. sweetness value (ssc × sh ratio) of the cultivars/selections seems to be a useful predictor of fruit sweetness. in terms of palatability, however, persim­ mon cultivar’s improvement should be performed on the sweetness value in association with flesh juiciness. 1. introduction persimmon (diospyros kaki thunb.) is believed to have originated in eastern asia, is produced worldwide including in azerbaijan, brazil, china, iran, israel, italy, japan, korea, new zealand, and spain (faostat, 2017). a number of local varieties has been developed in china, korea, and japan during a long history of domestication (parfitt et al., 2015; sato and yamada, 2016; yesiloglu et al., 2018). persimmon cultivars can be classi­ fied into four types: pollination­constant astringent (pca); pollination­ variant astringent (pva); pollination­variant non­astringent (pvna); polli­ nation­constant non­astringent (pcna), based on seed formation, change in flesh color, and nature of astringency loss (hume, 1914; ikeda et al., 1985; yonemori et al., 2000). among these types, fruits of pca­ and pva­ type cultivars are always astringent without postharvest treatment such as application of carbon dioxide gas or ethanol vapor. pvna­type cultivars require pollination and seed formation to lose astringency as well as flesh (*) corresponding author: mikioshi@farc.pref.fukuoka.jp citation: shiraishi m., asakuma h., 2020 ­ varietal diffe‐ rences in sweetness value and flesh juiciness among persimmon. ­ adv. hort. sci., 34(1): 71­79. copyright: © 2020 shiraishi m., asakuma h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 july 2019 accepted for publication 12 november 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 71­79 72 browning. fruits of pcna­type cultivars naturally lose their astringency because they terminate accumulat­ ing tannins at early fruit development stage. in par­ ticular, pcna­type persimmon cultivars are highly desired for fresh consumption on a worldwide level. regarding eating quality of pcna­type cultivar, the great stress is laid upon the evaluation of soluble solids content (ssc, °brix) using refractometer because sugars generally contribute a very large pro­ portion to the ssc in the ripening stage (reviewed by giordani et al., 2011). however, ban et al. (2010) and mitani et al. (2015) postulated that flesh juiciness is a decisieve parameter to determine the texture of per­ simmon fruit, where fruits with higher firmness had a tendency to be less juicy as observed in apple (harker et al., 2003). on the other hand, the percentage sucrose in flesh significantly affects the sweetness of fruit, such as east asian pear (kajiura et al., 1979), oriental melon (zhang and li, 2005), peach (suzuki et al., 1990; cirilli et al., 2016) and strawberry (sone et al., 2000). thus, understanding how varietal differ­ ences influence sugar composition will provide valu­ able information for genetic improvement of the palatability of persimmon fruit in future breeding programs. giordani et al. (2011) classified worldwide persimmon cultivars such as ‘atago’, ‘fuyu’, ‘rojo brillante’ and ‘triumph’ into three groups based on the cluster analysis of sugar composition. to date, however, there has been no comprehensive research on the sugar composition of the recent japanese pcna­ and non­pcna­type cultivars. since 1990, new pcna­ and non­pcna­type persimmon cultivar have been released in japan by the national institute of fruit tree science (yakushiji and nakatsuka, 2007; yamada et al., 2012 a, b). these new cultivars have large sized fruit, brilliant skin color, and are highly palatable, but their sugar composition remains large­ ly unknown. asakuma and shiraishi (2017) showed that geno­ typic effect of sugar composition (sh ratio) and flesh juiciness is significantly high, whereas the year, geno­ type × year interaction, among trees within geno­ type, and tree × year interaction is small or negligi­ ble. in experimental field of the current study, we have preserved a japanese persimmon cultivar col­ lection of both pcna­ and non­pcna­type cultivars as breeding stocks since 1980. although the total num­ ber of preserved cultivars is less than 50, the cultivar collection includes newly­released cultivars after 1990 and commercially­produced local cultivars in japan. in the present study, we (1) re­confirmed small year variability in the sugar composition and flesh juiciness by the analysis of variance, and (2) dis­ cussed varietal differences in the sugar composition and flesh juiciness among persimmon cultivars in association with palatability. 2. materials and methods plant materials for yearly variation of sugar composi‐ tion four pcna­type cultivars of japanese persimmon (‘akiou’, ‘fuyu’, ‘matsumotowase­fuyu’, ‘taishu’) were used. these cultivars are grown for commercial­ ly marketable fruit production by normal cultural practices, including pruning, flower and fruit thin­ ning, irrigation, soil and pest management (yamada, 2006) in an open­field of the fukuoka agricultural and forestry research center, fukuoka, japan (33°50ʹ n and 130°57ʹ e). according to the reference value derived from our annual survey from 2008 to 2015 (table s1), mature representative eight fruits per tree were sampled from three trees per geno­ type for late­october to late­november in 2014 to 2016 seasons depending on each cultivar’s optimal ripening time and skin color. the statistical fixed­effect model (table s2) that we adopted to express the phenotypic value (asakuma and shiraishi, 2017) is: pijkc = m + gi + yk + (gyik) + tij + (tyijk) +eijkc where pijkc is the phenotypic value of the cth fruit of the jth tree of the ith genotype in the kth year; m is the overall mean; gi is an effect contributed by the ith genotype; yk is an effect of the kth year; gyik is the interaction between the ith genotype and the kth year; tij is an effect of the jth tree of the ith geno­ type; tyijk is the interaction between the jth tree of the ith genotype and the kth year; and eijkc is an effect of the cth fruit of the jth tree of the ith genotype in the kth year. anova provided the variance associat­ ed with genotype (σg 2), among years (σy 2), genotype × year interaction (σgy 2), among trees within genotypes (σt 2), tree × year interaction (σty 2) and among fruits within tree (σ2). plant materials for varietal difference in sugar com‐ position a total of 43 of japanese persimmon cultivars con­ sisting of 23 pcna­types (fig. s1), 9 pvna­types (fig. s2), 6 pva­types (fig. s3), and 5 pca­types (fig. s4) was analyzed in 2016. cultural practices were con­ shiraishi and asakuma ‐ sweetness value and flesh juiciness of persimmon fruit 73 formed to plant materials for yearly variation of sugar composition. depending on the optimal ripen­ ing time of the cultivar, five to eight mature fruits per one­tree of each cultivar were harvested from late­ september to early­december in 2016 season according to the reference values based on above­ described cultivation records (table s1). classification of fruit ripening time was performed in accordance with the definitions by yamada et al. (1995). fruit shape index, fsi (longitudinal diameter/transverse diameter) was defined according to maeda et al. (2018) with slight modifications (fig. s5). cracking of apex and calyx­end of fruit was examined by upov guideline (upov, 2004). astringency removal of pca­ and pva­type cultivars was performed by treatment with either ethanol vapor or carbon dioxide gas depending on the cultivar (yamada, 2006). the sam­ pled fruit was weighed, and each fruit was horizon­ tally cut to measure fruit skin color, flesh firmness, flesh juiciness, soluble solids content (ssc, °brix), and sugar composition. analysis of fruit quality traits fruit skin color, flesh firmness, flesh juiciness, sol­ uble solids content, and sugar composition was mea­ sured according to asakuma and shiraishi (2017). fruit skin color around fruit apex was measured using a chromameter (cr­300, minoruta, tokyo, japan), and expressed as the value of color chart following formula: color chart (cc) = − 9.485 ln (hue angle) + 44.503, r2 = 0.9278. flesh firmness (kg) was determined by a handheld universal pressure tester with a 5.0­mm­diameter ×10.0­mm­height columnar plunger (km­5, fuji­ wara scientific, tokyo, japan). fifteen to 20 g of peeled flesh was weighed and wrapped in one layer of medical gauze. after hand­pressing (only one press) for 15 s, the squeezed juice (flesh juiciness) was measured using a 25­ml mess cylinder and expressed as ml g­1 fw. soluble solids content (ssc) of the resulting juice samples was determined as the °brix value using a portable calibrated electronic refractometer (pal­1, atago, tokyo, japan). for the analysis of sugar composition, 8 to 10 g of peeled flesh was weighed and transferred to a 50­ml heat­tolerant tube and partially screw­capped. the flesh sample was immediately microwave­irradiated at 730 w for 60 s before extracting the sugars. the irradiated sample was ground in a laboratory blender with ~40 ml deionized water. the puree was cen­ trifuged at 5000 × g at 25°c for 10 min. the resulting supernatant was brought to 50 ml with deionized water and filtered through a 0.45­µm filter. sugar composition was analyzed using a hplc (lc­10a, shimadzu, kyoto, japan) consisting of a scl­10a sys­ tem controller, lc­10ad pumps, a cto­10a column oven, and a rid­10a refractive index detector. the column (scr­101n, 7.9 × 300 mm, shimadzu, kyoto, japan) was operated at 60°c with 0.8 ml min­1 of water. the injection volume was 10 to 20 µl. 3. results and discussion variation in sugar composition hplc profiles of sugar composition were obtained from persimmon cultivars examined with three major peaks assigned as sucrose, glucose, and fructose, respectively, thereby expressing as the sh ratio, a ratio of sucrose to hexose sugars (hirano et al., 1995). as shown in figure 1, we classified persimmon cultivars into three types according to zheng and sugiura (1990) with slight modification; sucrose accu­ mulators (percentage sucrose ≥55.1%, sh ratio≥ 1.23), intermediate accumulators (percentage sucrose in 45.0 to 55.0%, sh ratio in 0.82 to 1.22), hexose accumulators (percentage sucrose ≤44.9%, sh ratio ≤0.81). previous studies have shown that the fig. 1 ­ sugar composition of persimmon cultivars from the hplc analysis. a: sucrose accumulators [‘fuyu (pcna­type)’, ‘akagaki (pvna­type)’, ‘atago (pca­type)’]; b: intermediate accumulator [‘maekawajiro (pcna­type)’, ‘saefuji (pvna­type)’, ‘aizumishirazu (pva­type)’]; c: hexose accumulators [‘soshu (pcna­type)’, ‘nishimurawase (pvna­type)’, ‘hiratanenashi (pva­ type)’] pca: pollination­constant astringent pva: pollina­ tion­variant astringent pvna: pollination­variant non­ astringent pcna: pollination­constant non­astringent. adv. hort. sci., 2020 34(1): 71­79 74 sucrose percentage in persimmon cultivar ranged between 12 and 70%, resulting in varietal difference in the sugar composition or sh ratio (tsuji and komiyama, 1987; zheng and sugiura, 1990; hirano et al., 1995; hirai et al., 2004; suzuki et al., 2010; asakuma and shiraishi, 2017). sugar profiles of pcna­ and non­pcna­types from well­known culti­ vars such as ‘atago’ (sucrose accumulator), ‘hiratanenashi’ (hexose accumulator), ‘soshu’ (hex­ ose accumulator) and ‘fuyu’ (sucrose accumulator) in previous reports are in agreement with those in the present study. table 1 shows highly varietal difference in the sh ratio among the pcna­type cultivars, especially for mid­ to late­oct. ripening ones. in cultivars as sucrose accumulators, large amounts of sucrose recorded in ‘sodawase (sh ratio = 3.29)’ followed by ‘okugosho (sh ratio = 2.35)’, ‘hanagosho (sh ratio = 2.17)’, ‘suruga (sh ratio = 2.02)’, ‘fuyu (sh ratio = 1.93)’, and ‘shinshu (sh ratio = 1.89)’. in the hexose accumulator, large amounts of hexose were present in ‘kishu (sh ratio = 0.21)’, ‘soshu (sh ratio = 0.23)’, ‘izu (sh ratio = 0.35)’, followed by ‘tenjingosho (sh ratio = 0.61)’, ‘taishu (sh ratio = 0.64)’ and ‘taiga (sh ratio = 0.67)’. among the remainders, sucrose and hexose seemed to be accumulated approximately equal amounts with 1.10 in sh ratio (‘maekawajiro’), 1.03 in sh ratio (‘misatogosho’), and 0.96 in sh ratio (‘reigyoku’). table 1 ­ sugar composition of pollination­constant non­astringent (pcna) type of japanese persimmon cultivars in 2016 fruit ripening time (late‐sep., early‐ to late‐oct., early‐ to late‐nov., early‐dec.) was classified according to yamada et al. (1995). sh ratio= sucrose [g 100 g­1 fw] / hexoses (glucose + fructose) [g 100 g­1 fw]. sweetness value= soluble solids content (reference value in table s1) x sh ratio. fruit ripening time/ cultivar or selection sugar composition (g 100 g­1 fw) sugar composition (%) type of sugar accumulation sh ratio sweetness value flesh juiciness (ml g­1 fw)sucrose glucose fructose total sucrose glucose fructose early‐oct. soushu 1.98 4.96 4.60 11.5 17.2 43.0 39.9 hexose 0.21 3.1 0.27 mid‐oct. izu 3.03 4.53 4.22 11,8 25.7 38.5 35.8 hexose 0.35 5.3 0.29 shinshuu 9.32 2.62 2.32 14.3 65.4 18.4 16.3 sucrose 1.89 32.3 0.22 late‐oct. sodawase 11.66 1.90 1.64 15.2 76.7 12.5 10.8 sucrose 3.29 58.9 0.27 reigyoku 6.44 3.59 3.12 13.2 49.0 27.3 23.7 intermediate 0.96 16.1 0.36 taiga 5.25 4.21 3.68 13.1 40.0 32.0 28.0 hexose 0.67 11.1 0.32 kanshu 5.93 3.87 3.48 13.3 44.7 29.1 26.2 hexose 0.81 13.2 0.17 kishu 2.66 5.90 5.56 14.1 18.8 41.8 39.4 hexose 0.23 3.4 0.24 taishu 5.30 4.52 3.76 13.6 39.0 33.3 27.7 hexose 0.64 10.1 0.35 maekawajiro 6.83 3.48 2.71 13.0 52.5 26.7 20.8 intermediate 1.10 18.2 0.22 tenjingosho 6.00 5.33 4.46 15.8 38.0 33.8 28.2 hexose 0.61 10,5 0.17 early‐nov. akiou 9.14 3.40 2.68 15.2 60.1 22.3 17.6 sucrose 1.57 27.6 0.38 misatogosho 7.37 4.18 2.96 14.5 50.8 28.8 20.4 intermediate 1.03 18.6 0.23 uenishiwase 7.63 3.35 2.48 13.5 56.7 24.9 18.4 sucrose 1.31 20.8 0.13 matsumotowase­fuyu 9.16 2.85 2.41 14.4 63.5 19.8 16.7 sucrose 1.74 26.8 0.27 mid‐nov. mushirodagosho 5.53 3.77 3.57 12.9 43.0 29.3 27.7 hexose 0.75 10.8 0.33 youhou 5.99 4.17 4.11 14.3 42.0 29.2 28.8 hexose 0.72 12.2 0.18 late‐nov. fuyu 9.79 2.75 2.32 14.9 65.9 18.5 15.6 sucrose 1.93 31.7 0.27 okitsu­20 (ro­19) 8.64 3.61 3.06 15.3 56.4 23.6 20.0 sucrose 1.30 25.5 0.31 taiho 7.92 3.50 2.88 14.3 55.4 24.5 20.1 sucrose 1.24 21.2 0.36 early‐dec. okugosho 9.43 2.14 1.88 13.5 70.1 15.9 14.0 sucrose 2.35 43.0 0.24 suruga 8.86 2.26 2.13 13.3 66.9 17.1 16.1 sucrose 2.02 35.4 0.26 hanagosho 9.03 2.20 1.96 13.2 68.5 16.7 14.9 sucrose 2.17 37.1 0.25 shiraishi and asakuma ‐ sweetness value and flesh juiciness of persimmon fruit 75 (table 3) were all sucrose accumulators, except for ‘kawazokogaki’ in sh ratio of 1.12 (intermediate accumulator). conversely, most pva­type cultivars (table 3) could be classified as hexose accumulators, with sh ratios ranging from 0.39 (‘tonewase’) to 0.67 (‘hiratanenashi’) with the exception of ‘koshu­ in the pvna­type cultivars (table 2), ‘nishi­ murawase’ (sh = 0.47) was hexose accumulator, whereas the sucrose accumulators had sh ratios, var­ ing from 1.83 (‘akagaki’) to 2.77 (‘rendaiji’). the sh ratio of intermediate accumulators ranged from 0.86 (‘fudegaki’) to 1.22 (‘zenjimaru’). pca­type cultivars table 2 ­ sugar composition and other fruit traits of pollination­variant non­astringent (pvna) type of japanese persimmon cultivars in 2016 fruit ripening time (late‐sep., early‐ to late‐oct., early‐ to late‐nov., early‐dec.) was classified according to yamada et al. (1995). sh ratio= sucrose [g 100 g­1 fw] / hexoses (glucose + fructose) [g 100 g­1 fw]. sweetness value= soluble solids content (reference value in table 1s) x sh ratio. table 3 ­ sugar composition of pollination­constant astringent (pca) and pollination­variant astringent (pva) type of japanese fruit ripening time (late‐sep., early‐ to late‐oct., early‐ to late‐nov., early‐dec.) was classified according to yamada et al. (1995). sh ratio= sucrose [g 100 g­1 fw] / hexoses (glucose + fructose) [g 100 g­1 fw]. sweetness value= soluble solids content (reference value in table 1s) x sh ratio. fruit ripening time/ cultivar or selection sugar composition (g 100 g­1 fw) sugar composition (%) type of sugar accumulation sh ratio sweetness value flesh juiciness (ml g­1 fw)sucrose glucose fructose total sucrose glucose fructose late‐sept. nishimurawase 3.47 3.22 4.16 10.9 32.0 29.7 38.3 hexose 0.47 7.1 0.16 early‐oct. akagaki 8.13 2.24 2.2 12.6 64.7 17.8 17.5 sucrose 1.83 29.3 0.20 mid‐oct. fudegaki 5.45 3.29 3.06 11.8 46.2 27.9 25.9 intermediate 0.86 14.0 0.21 ganzan 8.56 2.38 2.11 13.1 65.6 18.2 16.2 sucrose 1.91 31.1 0.18 oomiyawase 10.17 1.90 1.83 13.9 73.2 13.7 13.2 sucrose 2.73 46.1 0.20 saefuji 7.12 3.93 3.55 14.6 48.8 26.9 24.3 intermediate 0.95 16.4 0.28 late‐oct. rendaiji 8.45 1.58 1.47 11.5 73.5 13.7 12.8 sucrose 2.77 41.0 0.20 early‐nov. zenjimaru 8.05 3.41 3.20 14.7 54.9 23.3 21.8 intermediate 1.22 21.5 0.25 early‐dec. shogatsu 9.84 1.90 1.80 13.5 72.7 14.0 13.3 sucrose 2.66 46.3 0.19 fruit ripening time /cultivar or selection type of astrin­ gency sugar composition (g 100 g­1 fw) sugar composition (%) type of sugar accumula­ tion sh ratio sweetness value flesh juiciness (ml g­1 fw)sucrose glucose fructose total sucrose glucose fructose mid‐oct. ichidagaki pca 10.43 3.59 3.13 17.1 60.8 20.9 18.2 sucrose 1.55 30.5 0.35 tonewase pva 3.77 5.13 4.48 13.4 28.2 38.3 33.5 hexose 0.39 5.9 0.25 late‐oct. saijo pca 8.69 2.12 1.97 12.8 68.0 16.6 15.4 sucrose 2.12 34.8 0.28 kawazokogaki pca 7.80 3.80 3.15 14.8 52.9 25.8 21.4 intermediate 1.12 18.1 0.31 early‐nov. hiratanenashi pva 5.54 4.50 3.81 13.8 40.0 32.5 27.5 hexose 0.67 11.3 0.22 koshuhyakume pva 9.62 2.18 1.88 13.7 70.3 15.9 13.7 sucrose 2.37 41.0 0.31 hagakushi pca 8.56 2.32 2.34 13.2 64.8 17.5 17.7 sucrose 1.84 32.4 0.26 taigetsu pva 4.34 4.74 4.25 13.3 32.6 35.6 31.9 hexose 0.48 7.5 0.43 taiten pva 5.93 3.80 2.61 12.3 48.1 30.8 21.2 intermediate 0.93 15.7 0.46 mid‐nov. aizumishirazu pva 5.85 3.52 3.40 12.8 45.8 27.6 26.6 intermediate 0.85 12.7 0.21 atago pca 8.15 2.02 1.78 12.0 68.2 16.9 14.9 sucrose 2.14 33.8 0.23 76 adv. hort. sci., 2020 34(1): 71­79 hyakume’ (sh = 2.37), which was classified as a sucrose accumulator. ‘taiten’ was an intermediate accumulator in sh = 0.93. despite the small number of cultivars examined in the present study, there are no general relationships between astringency type (pcna, pvna, pva, and pca) and sugar accumulation type. in terms of bio­ chemical consideration, varietal difference in the sh ratio can be explained by the degree of sucrose cleavage due to the activity of vacuolar acid invertase (hirai et al. , 1986; zheng and sugiura, 1990). furthermore, recent transcriptional studies on the sugar accumulation­related key genes postulated that varietal differences in the sh ratio may be result­ ed from the balance between sucrose synthase and vacuolar acid invertase activities in persimmon fruit (suzuki et al., 2010; shiraishi and asakuma, 2019). to date, the genetic mechanisms controlling sucrose accumulation in persimmon fruit remain unclear. however, we hypothesize that dominance of sucrose accumulation over hexose accumulation in persim­ mon fruit on the basis of our ongoing breeding pro­ gram (unpublished data). environmental variance in sugar composition, flesh juiciness and ssc table 4 shows the contribution of variance from each trait to the total variance. the variance of geno­ type (σg 2) was high for sh ratio in 66.9% and flesh juiciness in 61.7%. as a whole, the variance of year (σy 2), among trees within genotype (σt 2), genotype × year (σgy 2) and tree × year (σty 2) interactions were small or negligible, varying from 0.0 to 6.6% of the total variance. similar to previous report (asakuma and shiraishi, 2017), the present results indicated that adding year or tree replications will not be effi­ cient in reducing the environmental variance for sh ratio and flesh juiciness. mitani et al. (2015) also showed that genotypic effect of flesh juiciness is sig­ nificantly high. it is thus considered that the genotyp­ ic effect on the sh ratio and flesh juiciness is high with negligibly small environmental variance, and that these traits can be determined by a one­year trial without tree replication. in contrast, σg 2 of ssc was small in 14.6%, followed by σy 2 in 12.5% and σgy 2 in 8.8%. other variance components, σt 2 and σty 2 of ssc were small or negligible with 1.5 and 4.2%, respectively. furthermore, the ratio of σt 2/(σy 2 + σgy 2) is calculated as 0.07. if the ratio exceeds 1.0, tree replications should be required. however, our results indicate that repeated yearly measurements are more efficient than replicated trees to estimate the genetic variance of ssc as observed in grape (sato et al., 2000) and persimmon (yamada et al., 1993) using variance components in table 4, the error variance (σe 2) of each trait can be obtained by the fol­ lowing equation (cf. yamada et al., 1993): (σy 2/3) + (σgy 2/3) + (σt 2/3) + {σty 2/(3×3)} + {σ2/(3×3×8)}. the σe 2 of sh ratio, flesh juiciness, and ssc is 0.01366, 0.00014, and 0.16003, respectively. broad­ sense heritability (σg 2/{σg 2+σe 2}) results in high for sh ratio in 0.95 and flesh juiciness in 0.97, whereas low for ssc in 0.63. in general, a high broad­sense heri­ tability means that most of the variation among genotypes is caused by genetic variation and not environmental variation. knowing the heritability can be of value when the breeder will make an effective selection. in this study, the high heritability of sh ratio and flesh juiciness is useful to discriminate genetic sweetness and juiciness of persimmon fruit, respectively. yamada et al. (1993) elucidated that an increase in yearly repetition instead of tree replica­ tions substantially reduced σe 2 in the measurements for ssc and fruit weight to clarify the genetic proper­ ties of genotypes. in the present study, ssc of each table 4 ­ estimates of variance component and their percentage to total variance obtained from the analysis of variance negative value was assumed to be zero sh ratio= sucrose/hexoses (glucose + fructose) variance components sh ratio flesh juiciness solube solids content (ssc) σg2 (genotype) 0.2636 (66.9%) 0.0052 (61.7%) 0.2725 (14.6%) σy2 (year) 0.0260 (6.6%) 0.0000 (0.0%) 0.2326 (12.5%) σgy2(genotype× year) 0.0000 (0.0%) 0.0003 (3.5%) 0.1642 (8.8%) σt2(among trees within genotype) 0.0062 (1.6%) 0.0000 (0.0%) 0.0285 (1.5%) σty2(tree × year) 0.0161 (4.1%) 0.0000 (0.0%) 0.0791 (4.2%) σ2(among fruit within tree) 0.0819 (20.8%) 0.0029 (34.8%) 1.0869 (58.3%) shiraishi and asakuma ‐ sweetness value and flesh juiciness of persimmon fruit 77 cultivar/selection was evaluated as the reference value based on one tree with more than five years field trials (table s1). varietal difference in sweetness value and flesh juici‐ ness sugars represent a crucial component of fruit edi­ ble quality, principally conferring sweetness, one of the main attributes influencing the degree of con­ sumer acceptance. the ratio of constitutive sugars determines the sweetness of fruits; the higher the sucrose percentage, the stronger the organoleptic perception of sweetness in asian pear (kajiura et al., 1979), oriental melon (zhang and li, 2005), peach (cirilli et al., 2016) and strawberry (sone et al., 2000). in this study, we proposed a new index entitled “sweetness value” evaluating fruit sweetness by the equation: ssc × sh ratio. in place of table s3, ssc value in table s1 was used for calculation of sweet­ ness value because of the above­described environ­ mental error. as shown in tables 1­3, the sweetness value varied due to the propotional level of sucrose content, ranging from 3.1 to 58.9 in pcna­, 7.1 to 46.3 in pvna­, and 5.9 to 41.0 in pva­ and pca­type cultivars. corresponding to sugar accumulation type, hexose accumulators exhibited lower sweetness value in 3.1 (‘soushu’) to 11.1 (‘taiga’), whereas sweetness values of sucrose accumulators were high­ er in 20.8 (‘uenishiwase’) to 58.9 (‘sodawase’). these results indicate that sweetness value seems to be a useful predictor of fruit sweetness in persimmon genotype. in our previous sensory tests (asakuma and shiraishi, 2017), the less­sweet genotypes exhib­ ited sh ratios below 0.3, while highly­sweet geno­ types had sh ratios exceeding 1.0. given that the ssc of genotype is around 16 (average value in 16.6 of 43 persimmon cultivars/selections in table s1), sweet­ ness value of highly­ and less­sweet genotype is expected as exceeding 16 and below 4.8, respective­ ly. for instance, its sensory sweetness of ‘kishu’ has been evaluated to be lower than that of ‘fuyu’ by several persimmon breeders and growers, although ‘kishu’ fruit has normally around 16 in ssc, which is comparable to ‘fuyu’ (yamada et al., 2009). in fact, sweetness value of ‘kishu’ was 3.4 in contrast to that of ‘fuyu’ in 31.7 (table 2), which is in agreement with above­mentioned sensory sweetness. however, there can be inconsistencies when eval­ uating the eating quality between sweetness scores and cultivars having different flesh juiciness, particu­ larly less juice content (data not shown). in the pre­ sent study, highly varietal difference in flesh juiciness was observed, ranging from 0.13 (‘uenishiwase’) to 0.38 (‘akiou’) ml g­1 fw of pcna type (table 2), 0.16 (‘nishimurawase’) to 0.28 (‘saefuji’) ml g­1 fw of pvna type (table 3), and 0.21 (‘aizumishirazu’) to 0.46 (‘taiten’) ml g­1 fw of pva and pca type (table 4). in general, the harder the flesh of fruits, the more chewing is required to breakdown the tissue and the longer it takes to release the juice (harker et al., 2003). in peach (suzuki et al., 1990), sweet cherry (dever et al., 1996), and carrot (horie and hiramoto, 2009), flesh sweetness is significantly promoted by high juiciness. similarly, ban et al. (2010) and mitani et al. (2015) revealed that flesh juiciness is consid­ ered to be crucial mouth­feel attribute in the overall taste of persimmon fruit. asakuma and shiraishi (2017) proposed that the new descriptor of flesh juiciness of persimmon fruit as “very juicy” (≥0.30 ml g­1 fw), “juicy” (0.21­0.29 ml g­1 fw) and “slightly juicy” (≤0.20 ml g­1 fw) based on the sensory juici­ ness. from this perspective, three sucrose accumu­ lating pcna­type cultivars/selections (‘akiou’, ‘okitsu 20’, and ‘taiho’) are considered to be promising breeding stocks because of the high sweetness value (21.2 to 27.6 ) and high juiciness (0.31 to 0.38 ml g­1 fw) together with large fruit size and brilliant fruit color (tables s1 and s3). thus, in terms of palatabili­ ty, persimmon cultivar’s improvement will be effec­ tively performed using a combination of sweetness value and flesh juiciness. acknowledgements we are grateful to dr. hiroyuki chijiwa, fukuoka agricultural and forestry research center, for reviewing the 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(three f1 hybrids, six breeding lines and the widely grown and consumed traditional cultivar mornagui), grown under greenhouse conditions during 2014 and 2015 seasons, was examined and compared. their resistance to powdery mildew was also checked. the main production­driving agronomic traits varied significantly (p<0.05) depending on the genotype. the major skin colour determined visually was different among the considered genotypes. the hybrids (h5 and h12) and the breeding lines (l1, l2, l5, l6 and l9) had dark green skin. the fruits of the hybrid h7 were dark and light green and the breeding line l4 has intense and dark green fruits whereas cv. mornagui was characterized by medium light green fruits. regarding fruit shape, the hybrids h7 and h5 produced straight fruits whereas the hybrid h12 and the breeding lines; l2, l4, l5, and l9 and the cv. mornagui had elongate fruits. however both breeding lines l1 and l6 were characterized by straight elongate fruits. the f1 hybrid h5 was the most productive (3.6 kg/plant and ≈ 21 fruits/plant for total yield and number of fruit/plant respectively). these findings are useful for further breeding programs aiming to develop new pow­ dery mildew resistant snake melon cultivars with satisfying agronomic traits. (*) corresponding author: bn.riadh@gmail.com citation: ilahy r., tlili i., rouhou h.c., siddiqui m.w., mishra p.m., kuchi v.s., homa f., hdider c., jebari h., lenucci m.s., 2020 ­ determining the main agronomic traits of snake melon (cucumis melo var. flexuosus l.) fruits as affected by genotypic differences. ­ adv. hort. sci., 34(1): 113­119. copyright: © 2020 ilahy r., tlili i., rouhou h.c., siddiqui m.w., mishra p.m., kuchi v.s., homa f., hdider c., jebari h., lenucci m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 march 2019 accepted for publication 31 october 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 113­119 114 1. introduction snake melons (cucumis melo var. flexuosus l.), belong to the cucurbitaceae family largely distribut­ ed and consumed since the antiquity across large geographical area (pandey et al., 2010; paris, 2012). the flexuosus distinguishes from other c. melo vari­ eties by the twisted long to very long fruits, typically exceeding the 4:1 length­to­width ratio, character­ ized by a slightly hairy white or light green exocarp (rind) often furrowed by more or less deep grooves running lining the fruit surface. the rind covers a creamy white or pale green flesh (mesocarp and endocarp) containing numerous whitish edible seeds, deliquescent at maturity. because the stem is rather thin, the plant is usually supported on trellises, where the snake­like fruits grow on vines attaining up to 120 cm lengths (burger et al., 2010). snake melon fruits are known all around the world with different trivial names: “alficoz” in spain, “tortarello” or “cetrangolo” in italy, “fakous” or “fegous” in arabic maghreb countries, “agoor” in soudan, “acur”, “hıtta” or “hıtı” in turquie, “kakri” in india, “uri” in japan and armenian cucumber, “yard­ long melon”, “serpent­melon” or “gutah” elsewhere (solmaz et al., 2016). snake melon fruits are general­ ly consumed raw at the immature stage of ripening with a preference for straight long and thick green fruits in the mediterranean region. here, this crop has been appreciated since antiquity for their crisp texture and refreshing, slightly acidic and non­sweet flavour, as revealed by their recurrent depiction in wall painting, sculptures and mosaics dating back to ancient egypt and roman empire, as well as by the presence of references in the islamic and jewish liter­ ature (paris et al., 2011; paris, 2012). the fruits are stomachic and seeds are also traditionally utilized as antitussive, digestive, febrifuge and vermifuge (duke and ayensu, 1985). besides the differences in skin colour and in the longitudinal furrowing of the fruits, snake melons genotypes differ also in various productivity­driving and quality traits including early marketable and total yield, numbers of fruits per vine and resistance to main pest diseases (eg. fusarium and powdery mildew) (pandey et al., 2010). powdery mildew is the main fungal disease affecting similarly greenhouse and open­field grown cucurbits and particularly snake melon. this disease is easily, recognizable by the whitish powdery fungal growth developing on many parts of the plant and fruits (sitterly 1978; zitter et al., 1996) . the disease is primarily caused by two fungal species around the world: golovinomyces orontii and podosphaera xanthii and are considered as an important limiting factor for snake melon pro­ duction (fernández­ortuño et al., 2006; bellón­ gómez et al., 2015). therefore, growers and breed­ ers are increasingly looking for resistant genotypes in order to overcome this problem although these fun­ gal species are developing also new strains more vir­ ulent. previous research on snake melons mainly focused on the effect of salinity, sowing period and harvesting intervals on fruit yield, the phenological phases and morphological traits of the plants, the influence of different rootstocks on production in soilless cultivation, the in vitro plant regeneration, seed priming, the variability among genetic, morpho­ logical, vegetative, fruit and yield parameters of germplasm, the impact of breeding hermaphrodite lines on yield and, least but not last, the identification of fruit morphological and quality traits by qtl map­ ping (reviewed in ilahy et al., 2019). genotypic differences strongly influence the microbial community composition in the rhizosphere (aydi­ben­abdallah et al., 2019). pre­ and post­har­ vest manipulations also significantly impact quality traits of horticultural crops (siddiqui et al., 2015, siddiqui and singh, 2015; ilahy et al. , 2018). furthermore, in snake melon genotypes carrying resistance to powdery mildew, the quality traits might be affected with respect to ordinary and sus­ ceptible genotypes. thus, this study investigates the genotypic variability affecting the main agricultural traits of different snake melon genotypes (three f1 hybrids, six breeding lines and a traditional reference cv. mornagui) grown under greenhouse condition. the overall aim is to select the most promising geno­ types (in terms of agronomic traits and mildew resis­ tance), to be used in further snake melon breeding programs 2. materials and methods plant material ten powdery mildew resistant (pmr) snake melon genotypes were used in our study (three hybrids; h7, h5 and h12), six breeding lines; l1, l2, l4, l5, l6 and l9) and the largely grown and consumed snake melon cultivar mornagui. the breeding lines were heirlooms landraces previously selected for their higher productivity and powdery mildew resistance levels as well as desirable agricultural traits generally ilahy et al. ‐ agronomic traits of snake melon 115 preferred by the consumer (length and skin colour). the (f1) hybrids were developed in a line x line mating design, in which parents characterized by high levels of powdery mildew resistance (l2 and mornagui), were crossed with the selected breeding lines (l1, l5 and l6) characterized by good powdery mildew resistance coupled with high productivity and desirable phenotypic traits. the breeding lines and the obtained hybrids were exchanged with different laboratory working on the same topics in italy, hungary and india. plug trays were used to grow snake melon seedlings at the beginning of december 2013 and 2014. four weeks later seedlings were transferred into a sandy soil (fig. 1) under unheated water­ impermeable plastic screens greenhouse, recom­ mended for growing vegetable crops particularly cucurbits during winter cycles, using 125 and 150 cm as in­ and between­row separations respectively at the region of teboulba (35°63’n, 10°95’e) in the coastal zone of tunisia. the experimental design was a randomized complete block design (rcbd) with three blocks (replication). each replication consisted of 10 plants per genotype. they were subjected to agricultural practices commonly adopted by high­ yielding farmers in this region. the production meth­ ods were in accordance with those reported recently in ilahy et al. (2019). determination of the agronomic characteristics powdery mildew resistance was determined on the basis of visual evaluation of the developed symp­ toms. fruits were harvested weekly at the immature stage of ripening. yield was determined following counting and considering all fruits on each plant. early yield (agricultural output discriminating culti­ vars with precocious production with respect to oth­ ers) and total yield [kg of fresh weight (fw)/plant], number of fruit per plant (n. fruits/plant), average fruit weight (g) were determined on all the trials plants. in addition, fruit length and diameter were also recorded for all fruits. fruit length and diameter (cm) were determined using a vernier caliper. experimental design and statistical analysis the effect of genotypic differences on the agro­ nomic traits of snake melon was assessed by analysis of variance (anova). when a significant difference was detected, means were compared using the least significant difference (lsd) test (p<0.05). all statisti­ cal analysis was performed using sas version 6.1 software (sas institute, cary, nc, usa). agglome­ rative hierarchical cluster analysis was used to deter­ mine differences and similarities among the geno­ types, and the distance measure used was euclidean distance computed between each population by the ward method. 3. results and discussion the main agronomic traits of the snake melon hybrids and breeding lines grown under greenhouse during the productive seasons 2014 and 2015 are reported in table 1. pooled data of the two years were analyzed. plants of the different genotypes exhibited visually an important vigour with excep­ tional foliage cover (figs. 1, 2). the major skin colour was visually different among the evaluated genotypes. the hybrids h5, and h12 and the breeding lines l1, l2, l5, l6 and l9 had dark green skin (fig. 2). the hybrid h7 was dark and light green and the breeding line l4 has intense and dark green fruits whereas cv. mornagui was charac­ terized by medium light green fruits. regarding fruit form, the hybrids h7 and h5 produced straight fruits and h12, l2, l4, l5, ‘mornagui’ and l9 had elongate fruits. however both breeding lines l1 and l6 were characterized by straight elongate fruits. the early and total yields (expressed as kg /plant), the number of fruits per vine, as well as the average fig. 1 ­ overview of the considered snake melon trial. adv. hort. sci., 2020 34(1): 113­119 116 fig. 2 ­ external appearance, longitudinal and cross sections (a), and schematic representation of the morphology (b) of the different snake melon genotypes peponides under analysis (hybrids, breeding lines and the traditional cultivar mornagui). ilahy et al. ‐ agronomic traits of snake melon 117 and (0.3­2.0 kg per plant) reported by abdelmohsin et al. (2015) for open field and greenhouse grown monoecious cultivars of the flexuosus group (alimin, pi222187 and snake melon) and for the hermaphroditic breeding lines generated by crossing the flexuosus parents with the melon accession paul, while they fell within the range reported by the same authors for number of fruits per vine (2­25). no cor­ respondence was found, instead, between our find­ ings and the number of fruits per plant (5.8­7.2) and fruit length (33­90) measured in five stable inbred lines of cultivated landraces of snake melons grown in open field at wad medani (sudan) by yousif et al. (2010); while they were in accordance with those of ali­shtayeh et al. (2017) for fruit weight (53­201 g) and length/width ratio (3­9) in a study comprising 50 accessions of snake melons grown in different open fields across the west bank, and were slightly lower than the length (48.3 cm) and width (10.7 cm) values reported by ramamurthy and waters (2015) for a pale­green fleshed, non­sweet accession (usda pi435288) grown it the fields of the lincoln agronomy research farm of the university of nebraska. powdery mildew resistance visual evaluation showed that the snake melon line l9 exhibited simi­ lar resistance level to the ordinary cultivar mornagui and also to the hybrid h5 (l2 x l1). fruit weight, length, diameter and length/width ratio varied significantly (p<0.05) between genotypes (table 1). early yield ranged from 0.9 kg per plant for the breeding line l5 and the cultivar mornagui to 1.5 and 1.7 kg per plant for the hybrids h5 and h7, respectively. the breeding line l9 had the lowest total yield (1.9 kg per plant) and 10.7 fruits per plant, while the highest values were recorded for the hybrid h5 (3.6 kg per plant and 20.7 fruits per plant). the average fruit weight ranged between 151.0 g to 184.3 g for the breeding line l6 and mornagui culti­ var, respectively. in agreement to the low weight, the breeding line l6 produced short fruits (19 cm length as average) with the lowest length/width ratio (5.14), while the fruits of the hybrid h5 and of the breeding line l1 were the longest (32.0 and 32.6 cm, respec­ tively) and showed the highest length to width ratio (8.36 and 8.65, respectively). fruit diameter is an important agricultural trait for snake melon as con­ sumers generally prefer long fruits with low diameter (width). the lowest fruit diameter (3.3 cm) was mea­ sured for the breeding line l5 and the highest value (4.2 cm) was obtained for the hybrid h7 and cv. mornagui. to our knowledge, these presented data are among the first reports on snake melon horticul­ tural traits. nevertheless, with respect to total yield, our data exceed the range of values (0.5­3.5 kg/plant) reported by aydi­ben­abdallah et al. (2019) table 1 ­ agronomic characteristics evaluated in snake melon hybrids, breeding lines and cultivar mornagui grown under greenhouse conditions during the seasons 2014 and 2015 (pooled data of the two years were analyzed) the data refer to visually assessed snake melon fruits and are the average of at least 10 independent replicates. mean ± sd followed by the same letters do not differ significantly (lsd test, p<0.05). * pmr is the evaluation of the powdery mildew resistance, based on visual evaluation of the symptoms developed. genotypes major skin colour fruit form early yield (kg/plant) total yield (kg/plant). number of fruits/plant fruit weight (g) fruit length (cm) fruit diameter (cm) pmr* developed hybrids (line x line) h7 (mornagui x l5) dark/light green straight 1.7±0.1a 3.4±0.5 ab 20.3±2.9 ab 164.0±2.3 29.8±1.2 b 4.2±0.2 a + h5 (l2 x l1) dark green straight 1.5±0.1 a 3.6±0.3 a 20.7±2.0 a 173.6±1.2 32.0±1.0 a 3.7±0.1 bcd + + h12 (mornagui x l6) dark green elongate 1.4±0.2 b 3.0±0.2 abc 18.4±0.3 abc 169.0±16.7 22.0±0.6 e 4.1±0.2 ab + breeding lines l1 dark green straight elongate 1.4±0.3 b 3.3±0.6 ab 19.4±3.8 abc 172.7±6.1 32.6±0.1 a 3.9±0.3 abc + l2 dark green elongate 1.1±0.1 b 2.7±0.1 abcd 16.0±0.6 166.3±3.7 31.2±0.1 ab 3.8±0.1 abcd + l4 intense dark green elongate 1.3±0.1 ab 2.7±0.2 bcd 17.0±1.5 abc 153.6±2.7 27.5±0.3 c 3.8±0.4 abc + l5 dark green elongate 0.9±0.2 b 2.1±0.2 cd 13.7±1.8 cd 154.6±5.2 19.2±0.1 f 3.3±0.1 d + l6 dark green straight elongate 1.0±0.1 b 2.2±0.1 cd 14.7±0.7 bcd 151.0±2.6 19.0±0.6 f 3.7±0.1 bcd + l9 dark green elongate 1.0±0.2 b 1.9±0.5 d 10.7±1.8 d 174.0±12.1 22.5±0.1 e 3.6±0.3 cd + + cultivar mornagui medium/light elongate 0.94±0.1 b 2.5±0.2 bcd 13.6±1.2 cd 184.3±12.9 25.2±0.6 d 4.2±0.3 a + + + 118 adv. hort. sci., 2020 34(1): 113­119 the dendogram from the hierarchical ascending classification (fig. 3) has discriminated snake melon genotypes into 3 main clusters (level of trencature 5). the first cluster comprised 4 genotypes (the hybrid h5 and the breeding line l1 in a sub­cluster and the hybrid h7 and the breeding line l2 in the other sub­ cluster) characterized by the most producing yield and the most elongated fruits. the second main clus­ ters comprised 3 genotypes (the hybrid h12, the breeding line l9 and the traditional cv. mornagui) mainly characterized by intermediate producing yields and intermediate fresh weights. the third clus­ ter comprised the breeding lines l5, l6 and l4 char­ acterized by the small fruit weight and the small fruit length. 4. conclusions the variability detected, for the main agronomic traits, in different snake melon genotypes (f1 hybrids and breeding lines) can be useful for further conven­ tional snake melon breeding programs aiming to improve their agronomic traits and could contribute to the breeding of powdery mildew­resistant elite cultivars suitable for greenhouse and open field con­ ditions. acknowledgements this study was supported by the ministry of agriculture water resources and fisheries, the institution of research and higher agricultural education as well as the ministry of higher education and scientific research of tunisia (project: breeding of cucurbits for various agronomic and quality traits, 2012­2016). references abdelmohsin m.e., el jack a.e., yousif m.t., el naim a.m., ahmed e.a., pitrat m., 2015 ­ impact of breed‐ ing hermaphroditic melon on early production and yield: case of snake melon (cucumis melo var. flexuo‐ sus) and tibish (c. melo var. tibish). ­ int. j. life sci. engin., 1(4): 171­176. ali­shtayeh m.s., jamous r.m., shtaya m.j., mallah o.b., eid i.s., zaitoun s.y.a., 2017 ­ morphological characterization of snake melon (cucumis melo var. flexuosus) populations from palestine. ­ genet. resour. crop evol., 64(1): 7­22. aydi­ben­abdallah r., chikh­rouhou h., jabnoun­ khiareddine h., sta­baba r., daami­remadi m., 2019 ­ variation of the rhizosphere microbial communi‐ ty structures between melon and snake melon geno‐ types 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(ed.) powdery mildews. academic press, london, uk, pp. 584. solmaz i., kacar y.a., simsek o., sari n., 2016 ­ genetic characterization of turkish snake melon (cucumis melo l. subsp. melo flexuosus group) accessions revealed by ssr markers. ­ biochem. genet., 54(4): 534­543. yousif m.t., elamin t.m., baraka m., jack a.a., ahmed e.a., 2010 ­ variability and correlation among morpho‐ logical, vegetative, fruit and yield parameters of snake melon (cucumis melo var. flexuosus). ­ cucurbit genet. coop. report, 33­34: 32­35. zitter t.a., hopkins, d.l., tomas, c.e., 1996 ­ compendium of cucurbits diseases. ­ aps press, st paul, mn, usa, pp. 87. impaginato 415 adv. hort. sci., 2023 37(4): 415­426 doi: 10.36253/ahsc­14098 evaluation of viability and germination of pollen grains of three local caprifig cultivars and their effect on some char­ acteristics of fig fruits (ficus carica l.) h. barakat, r. draie (*) faculty of agriculture, idlib university, syria. key words: caprifig, ficus carica, flower stigma extract, pollen germination. abstract: this research was carried out in fig fields in the village of kafar­jales, in the north of syria. the analyses were conducted in the laboratories of the faculty of agriculture at idlib university during the 2022 farming season, to evaluate the viability and germination of three local varieties of caprifig (bunduqi, azraq, panjani). the morphological characteristics, the date of the exodus of insects (blastophaga psenes) from male fruits, the quantity of pollen, and the percentage of germination and viability were studied on four nutritive media at a temperature of 27°c. the measurements were taken after 24 h, 48 h, and 72 h. these three pollinators were used to pollinate three varieties of edible figs (white satehi, safrawi, and habashi) to study the effect of pollen source on the productive characteristics of edible fig fruits. the result showed significant superiority of the bunduqi cultivar over the rest of the cultivars in traits of the fruit’s early ripening, pollen quantity, and date of departure of insects. the azraq cultivar is found to be superior to bunduqi and panjani culti­ vars in the germination rate of pollen at all stages of the experiment and in all used media. the addition of stigma flower extract in culture media increased germination percentage by 35% for all studied caprifig cultivars. the addition of 10% sucrose in the cultivation environment increased the percentage of pollen germination by 20%. the cultivars that were inoculated with the panjani polli­ nator outperformed the characteristics of the length, diameter, and weight, demonstrating that the fruit’s quality characteristics are affected by the geno­ type of the pollinator used. the results of this research can be beneficial for both fig growers and plant breeders, as they help to select the best pollinators and contribute to the development and improvement of the quality of the cul­ tivated fig fruits. 1. introduction the fig was one of the first fruit trees domesticated during the stone age (zohary and spiegel­roy, 1975). the results of a study of (çalışkan and dalkılıç, 2022) clearly showed that the southern regions of turkey were one of the original centers of figs. (*) corresponding author: rida.draie@idlib­university.com citation: barakat h., draie r., 2023 ­ evaluation of viabi‐ lity and germination of pollen grains of three local caprifig cultivars and their effect on some charac‐ teristics of fig fruits (ficus carica l.). ­ adv. hort. sci., 37(4): 415­426. copyright: © 2023 barakat h., draie r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 november 2022 accepted for publication 22 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14098 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 415­426 416 figs spread in many areas due to their adaptation to different climates and soils (mars, 2003). the fig tree is mainly found in the mediterranean region because it is well adapted to the climatic conditions affecting this region (ighbareyeh et al., 2018), and is one of syria’s most important fruit trees. idlib gover­ norate is at the forefront in production and the num­ ber of trees, with 40% of the cultivated area at the level of syria (syrian statistical group, 2021), where many female varieties are planted whose fruits are eaten fresh or dried. in addition, the caprifig trees are used for pollination purposes, which are residence to the pollinating fig wasp (blastophaga psenes). ficus carica l. belongs to the moraceae family. the species contains two sexual forms: the male fig (caprifig) and the female fig (edible). the male fig produces pollen as it is male in practice, but at the same time, it con­ tains female flowers with male flowers, which are functionally hermaphrodites. on the other hand, the female (edible) fig has only long­style female flowers that are monosexual (stover et al., 2007). fig pollen is carried by the fig wasp (blastophaga psenes l.) that develops with the fig tree (kjellberg et al., 1987). in syria, there are two different groups of figs. the first group is the male fig (caprifig), which spreads in many regions of the world, and its types are very close to each other (condit, 1947). the fruits of this species are inedible and contain two types of flow­ ers: male pollen­producing (near the ostiole), and female short­stylet (in two­thirds of the lower cavity). the wasp insect develops inside the tuberous flow­ ers. this type produces three crops annually (valdeyron and lloyd, 1979; stover et al., 2007; flaishman et al., 2008). summer profichi and its fruits pollinate the types of figs that need pollination. the mammoni crop is used in the fall, and the mammi crop is used in the winter (anjam et al., 2017). the second group is the edible fig (smyrna), which pro­ duces edible fruits with real seeds. this type contains long­style female flowers, which need to be pollinat­ ed by the pollen­bearing fig wasp from the profichi crop of caprifig to give fruits if pollination occurs (armstrong, 2006). the fruit contains seeds inside, but in the absence of pollination, the inflorescences fall (armstrong, 2006). this type of fig produces two crops per year, the breba crop that ripens in early summer. in addition, the second (main) crop ripens at the beginning of autumn (valdeyron and lioyd, 1979). ficus carica produces inflorescences called syconia. the flowers are unisexual, either male, female, or gal flowers (female short­style), and the fruits are borne in the axilla of the leaf (andersen, and crocker, 2009; aytürk, 2019). the process of pollination in figs is called caprifica­ tion, and the profichi crop is used for this process. profichi crop produces much more pollen than the other two crops, and for a high­quality profichi crop, it is preferable to have a high pollen germination rate and a high amount of pollen produced (balci et al., 2001). the research was conducted to study the characteristics of fruits and pollen for different geno­ types of the caprifig. it was noted that there are dis­ crepancies between them (ilgın et al., 2007; çalışkan and yaman, 2016; çalışkan et al., 2021). pollen viabil­ ity, germination rate, and pollen quantity affect the yield of inoculated fruit trees. different plants’ pollen requires various growing media such as water, sugar solution, inorganic salts, and vitamins for successful germination (stanley and linskens, 2012. in a study conducted by ilgın et al. (2007), it was found that pollen did not germinate at all on a medium without sucrose and increasing sucrose concentrations to 20% improved the percentage of pollen germination. to further improve the germination rate, several con­ centrations of h3bo3, kno3, and ga3 were added to media containing 20% sucrose. the germination rates of some caprifig pollen grains were higher than 70% with the addition of 0.050% h3bo3, followed by 0.025% kno3, and the germination rates of caprifig pollen were higher with the addition of these chemi­ cals than sucrose 20% alone. germination of caprifig pollen was increased to more than 70% by adding stigma exudates from long­style female flowers to the planting medium (awamura et al., 1995). in a study by ilgın et al. (2007), pollen viability was higher than that of pollen germination. this result was con­ sistent with previous studies (pearson and harney, 1984; bolat and pirlak, 1999; stanley and linskens, 2012). vego and miljković (2012) found that the best conditions for pollen germination of caprifig were in a medium containing 3% sucrose and 0.01% boric acid solution at a temperature of 30°c in the dark. the selection of pollen of good quality and quantity is important in fig orchards, as several studies indicate that the caprifage variety used can influence the quality traits of fig fruits (rahemi and jafari, 2005; gaaliche et al., 2011; pourghayoumi et al., 2012). in this research, we will test the viability and ger­ mination of pollen grains in three cultivars of wild figs spread in northwestern syria to determine the most efficient of these pollinators for female (edible) fig orchards. we will also study the effect of pollen barakat et al. ‐ evaluation of viability and germination of caprifig pollen 417 source on some characteristics of female (edible) fig fruits resulting from using these pollinators. 2. materials and methods plant materials three cultivars of caprifig have been studied in the kafar­jales area (5 km northwest of idlib city). year 2022, rainfed cultivation system, and the culti­ vars are bunduqi, azraq, panjani (fig. 1). the following characteristics were studied, which are the same characteristics that were studied by çalışkan et al. (2017) on 60 turkish caprifig cultivars. for each cultivar, these characteristics were studied: a. number of fruits on the branch; b. fruit diameter (cm); c. fruit length (cm); d. date of the expulsion of the blastophaga insect from the fruits. e. amount of pollen grains in male flowers. f. pollen viability; g. percentage of germination of pollen; h. timeline of pollen germination rate evolution. three varieties of female (edible) figs, widely spread in the study area and economically important for fig growers, were selected: habashi, white satehi, and safrawi (fig. 2). to study the quality of fig fruits, the same specifi­ cations that were studied in the research of (gaaliche et al. (2012) were studied, where 20 fruits for each variety were studied as follows: a) fruit length (cm); b) fruit diameter (cm); c) fruit weight (g); d) total soluble solids percentage, tss (%). study the specifications of the caprifig fruits the fruits of caprifigs of the three varieties (bunduqi, azraq, panjani) were harvested during the profichi crop’s emergence in june. the fruits were cut crosswise and placed on paper plates in the laborato­ ry (cool and dry) to collect pollen. pollen grains were taken from ten separate fruits for each variety using a cylinder gradient and we compared the varieties among them. pollen viability test pollen viability was tested using methylene blue dyes. the colored pollen grains, after adding methyl­ ene blue dye, were considered to live, while the non­ colored pollen grains were considered dead. so, 100 pollen grains were counted, the percentage of col­ ored pollen grains was calculated, and the process was repeated three times for each sample. pollen germination test germination rate was studied after 24, 48, and 72 h of cultivation in media (a, b, c, and d). 0.5 (g) of pollen was taken and grown in each petri dish. the pollen grain was considered germinated if the pollen tube length was greater than the diameter of the pollen grain. the exploding pollen grains were also ignored and were not considered germinated, which fig. 1 ­ fruits placement on the branch (a), and the shape of the leaves (b) and fruits (c) in bunduqi, azraq and panjani caprifig. fig. 2 ­ fruits placement on the branch (a), leaves (b), and cross­ section and full fruit (c) of habashi, white satehi and safrawi female cultivars. adv. hort. sci., 2023 37(4): 415­426 418 was previously applied in a similar study (khan and perveen, 2008). the germination rate of pollen of the caprifig cultivars was studied on medium a (5% sucrose + 1 ppm boric acid + 1% agar), medium b (10% sucrose + 1 ppm boric acid + 1% agar), medium c (5% sucrose + 1 ppm boric acid + 1% agar + extract of stigma female flowers), and medium d (10% sucrose + 1 ppm boric acid + 1% agar + extract of stig­ ma female flowers). the readings were taken 24 h, 48 h, and 72 h after culture. the pollen of the three varieties was grown in the cultivation media and incubated at a temperature of 27°c.). gaaliche et al. (2013) had previously used these mediums to study caprifig pollen germination in a previous study. study the effect of pollen source on the characteris‐ tics of the edible fig fruits the study was done on the fruits of the main crop, except breba, because breba falls due to the absence of pollinating insects, because it appears in april. so, sixty fruits of each female variety (white satehi, safrawi, and habashi) were isolated at the beginning of their formation by cotton fabric bags that allow air to enter, and the diameter of the holes in them is smaller than the fig wasp insect. when insects started to emerge from the studied caprifig cultivars (bunduqi, azraq, panjani), we took the fruit of the pollinated variety and put it in the bag with the female fruit of the studied varieties so that each female variety was inoculated with the three pollina­ tors (each pollinator separately) at a rate of 20 fruits from each pollinator (20 replicates). the fruits were isolated for two weeks to ensure that the required pollinator was pollinated. the fruits were harvested in stages, so that the fruits are taken when they reach the stage of maturity, and the entire harvest period is during the month of august. experiment design and statistical analysis factorial experiment in completely randomized design (crd) was used in the distribution of the experiment’s coefficients. the data were statistically analyzed using the genstat program, and l.s.d values were taken at a significance level of 5% and 1% for field and laboratory readings, respectively. 3. results and discussion maturity date (when insects begin to emerge) and characteristics of caprifig syconia the results obtained (table 1) show that the culti­ var bunduqi was early concerning the date of the emergence of insects. insects began to emerge from the syconia of bunduqi on 01/06/2022, with a differ­ ence of more than a week from the two other vari­ eties (panjani and azraq), which makes this variety suitable for pollinating the female fruits of the early fig varieties, especially the first fruits that appear on the branch (where they fall in the absence of pollina­ tion). the emergence of insects from the syconia of this variety continued for a week. as for the cultivars panjani and azraq, the beginning of the release of insects in them was 8/6/2022 and lasted for 10 days, which makes them suitable for pollination of the edi­ ble fig variety whose flowers appear in the middle of flowering time. as for pollen, it was arbitrarily classified s few in the flowers of the cultivar azraq, abundant in the cul­ tivar panjani and very abundant in the cultivar bunduqi (table 1). pollen viability of caprifig cultivars pollen viability was studied in caprifig cultivars azraq, bunduqi, and panjani using methyl blue dye the presence of the same letter in each column indicates that there are no significant differences between the items. * the quantity (few, abundant) is a random unit to compare pollen collected in a drum of different cultivars). table 1 ­ specifications of the syconia of the profichi crop cultivar number of syconia* syconia length (cm) syconia diameter (cm) pollen quantity syconia ripening (insect exit) azraq 6.00 a 4.14 c 4.58 c few medium (08­06­2022) bunduqi 6.20 a 5.12 b 5.40 a very abundant early (01­06­2022) panjani 6.50 a 5.88 a 4.84 b abundant medium (08­06­2022) mean 6.23 5.05 4.94 l.s.d. (5%) 0.71 0.18 0.17 ­ ­ c.v. % 12.40 3.90 3.80 ­ ­ barakat et al. ‐ evaluation of viability and germination of caprifig pollen 419 (fig. 3). table 2 shows the results of the pollen viabili­ ty study for the three cultivars. table 2 shows no significant differences in the percentage of pollen viability among the three stud­ ied cultivars panjani, azraq, and bunduqi. the highest percentage of viability was observed in the cultivar panjani (99.67%) and the lowest percentage of viabil­ ity in the cultivar bunduqi (97.33%). study of the percentage of germination of pollen of caprifig on media (a, b, c, and d) 24 h after planting. table 3 shows the superiority of the cultivar azraq in the percentage of pollen ger­ mination over the two cultivars panjani and bunduqi after 24 h of cultivation on different media. the aver­ ages of germination rates were 20.85%, 9.63%, and 8.94%, respectively. there were no significant differ­ ences in the germination characteristics of the two cultivars panjani and bunduqi. the highest germina­ tion percentage was obtained in media b and d, with values of 16.35% and 16.16%, without significant dif­ ferences. the two media b and d were significantly superior to medium c, which gave a germination rate of 11.80%. this was significantly superior to medium a, which gave a germination rate of 8.24%. as for the interaction between the caprifig variety and the medium used in cultivation, the interaction of the azraq variety with the medium b and the interaction of the blue medium d variety achieved the highest germination rate of 24.69% and significantly superior to the other interactions (without significant differ­ ences between these two reactions). in comparison, the interaction of the bunduqi cultivar with medium a achieved the lowest germination percentage of 5.02% after 24 h of pollen cultivation. the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. table 2 ­ pollen viability percentage using methylene blue dye table 3 ­ germination of pollen grains of caprifig cultivars after 24 h of planting on media (a, b, c, and d) fig. 3 ­ pollen viability test using methylene blue. cultivar dye methylene blue (%) azraq 98.67 bunduqi 97.33 panjani 99.67 c.v.% 3.40 l.s.d. (1%) = cultivar 3.47 cultivar medium average a b c d azraq 12.69 25.69 20.35 24.69 20.85 a bunduqi 5.02 13.02 5.02 12.69 8.94 b panjani 7.02 10.35 10.02 11.12 9.63 b average 8.24 c 16.35 a 11.80 b 16.16 a 13.14 c.v.% 7.28 l.s.d. (1%) = cultivar 1.10 l.s.d. (1%) = medium 1.27 l.s.d. (1%) = (cultivar x medium) 2.20 pr. cultivar <0.001 pr. medium <0.001 pr. (cultivar x medium) <0.001 f. calculated. cultivar 584.57 f. calculated. medium 148.35 f. calculated. (cultivar x medium) 21.97 420 adv. hort. sci., 2023 37(4): 415­426 figure 4 show the pollen germination of panjani, azraq, and bunduqi cultivars after 24 h of cultivation on media (a, b, c, and d). 48 h after planting. the results of the statistical analysis of the percentage of pollen germination caprifig cultivars (table 4) show that the azraq culti­ var was superior in the germination percentage of pollen grains to the two cultivars panjani and bunduqi after 48 h of planting on media (a, b, c, and d), with an average germination rate of 33.32%. there were no significant differences between the two cultivars bunduqi and panjani, whose germina­ tion rates reached 24.99% and 23.85%, respectively. as for the media used in cultivation, medium d (10% sucrose + extract of the stigma of female flowers) sig­ nificantly outperformed the other cultivation media and gave a germination rate of 33.81%. medium c (5% sucrose + extract of the stigma of female flow­ ers) was significantly superior, with a germination rate of 29.88% over medium b (10% sucrose), which achieved a germination rate of 25.32%, and in turn significantly outperformed medium a (5% sucrose), which came in the last rank, with a germination rate of 20.55%. regarding the interaction between the caprifig variety and the medium used in cultivation, the inter­ action of the azraq variety with medium d achieved the highest germination percentage, which amount­ ed to 40.32%, and significantly outperformed all other interactions. on the other hand, the interaction of the bunduqi cultivar with medium a achieved the lowest germination rate, which was 16.99% after 48 h of pollen cultivation. figure 5 show the pollen germination of panjani, azraq, and bunduqi cultivars after 48 h of cultivation on media (a, b, c, and d). 72 h after planting. table 5 displays the superiori­ ty of the cultivar azraq in the percentage of pollen germination on the two cultivars panjani and bunduqi after 72 h of cultivation on media (a, b, c, and d), with an average germination rate of 64.16%. cultivar medium average a b c d azraq 25.32 36.32 31.32 40.32 33.32 a bunduqi 16.99 18.66 31.66 32.66 24.99 b panjani 19.32 20.99 26.66 28.44 23.85 b average 20.55 d 25.32 c 29.88 b 33.81 a 27.39 c.v.% 5.37 l.s.d. (1%) = cultivar 1.69 l.s.d. (1%) = medium 1.96 l.s.d. (1%) = (cultivar x medium) 3.39 pr. cultivar < 0.001 pr. medium < 0.001 pr. (cultivar x medium) < 0.001 f. calculated. cultivar 584.57 f. calculated. medium 148.35 f. calculated. (cultivar x medium) 21.97 the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. table 4 ­ germination of pollen grains of caprifig cultivars after 48 h of planting on media (a, b, c, and d) fig. 4 ­ germination of ‘panjani’, ‘azraq’ and ‘bunduqi’ pollen after 24 h of cultivation on media (a, b, c, and d). barakat et al. ‐ evaluation of viability and germination of caprifig pollen 421 the cultivar panjani was also significantly superior to the cultivar bunduqi, which came in the last rank, the germination rates for both were 47.17% and 40.00%, respectively. concerning the agricultural media, all media differed significantly among themselves in the characteristic of the percentage of germination of pollen. medium d (10% sucrose + extract of the stig­ ma of female flowers) significantly outperformed the other cultivation media, with a germination rate of 60.90%. medium c (5% sucrose + extract of the stig­ ma of female flowers) was significantly superior, with a germination rate of 56.55% over medium b (10% sucrose), which achieved a germination rate of 46.00%. medium a (5% sucrose) came in the last rank with a percentage of germination of 38.33%. regarding the interaction between the caprifig culti­ var and the medium used in cultivation, the two interactions, the azraq cultivar with the medium d, and the blue cultivar with the medium b were signifi­ cantly superior to the other interactions, with a per­ centage of germination of 69.66% and 68.66%, respectively, without significant differences between them. the interaction of the bunduqi cultivar with medium a achieved the lowest germination rate of 23.66% after 72 h of pollen cultivation. figure 6 show the germination of pollen of cultivars panjani, azraq, and bunduqi after 72 h of cultivation on media (a, b, c, and d). table 5 ­ germination of pollen grains of fig cultivars after 72 h of cultivation in media (a, b, c, and d) the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. fig. 5 ­ germination of ‘panjani’, ‘azraq’ and ‘bunduqi’ pollen after 48 h of cultivation on media (a, b, c, and d). cultivar medium average a b c d azraq 53.66 68.66 64.66 69.66 64.16 a bunduqi 23.66 30.33 50.66 55.33 40.00 c panjani 37.66 39 54.33 57.7 47.17 b average 38.33 d 46.00 c 56.55 b 60.90 a 50.44 c.v.% 2.62 l.s.d. (1%) = cultivar 1.76 l.s.d. (1%) = medium 1.52 l.s.d. (1%) = (cultivar x medium) 3.04 pr. cultivar < 0.001 pr. medium < 0.001 pr. (cultivar x medium) < 0.001 f. calculated. cultivar 584.57 f. calculated. medium 148.35 f. calculated. (cultivar x medium) 21.97 evolution of germination rate during the experimen‐ tal time the previous results show that the percentage of pollen germination increased for all the studied caprifig cultivars with the progression of the experi­ ment. while the average percentage of germination for all tested cultivars after 24 h was 13.14%, the per­ centage increased to 27.39% after 48 h of planting, and it reached 50.44% after 72 h. the cultivar azraq gave the best germination rate and outperformed the other cultivars in all stages of the experiment. the percentage of germination in this cultivar adv. hort. sci., 2023 37(4): 415­426 422 increased from 20.85% to 33.32% and then reached 64.16% after 24 h, 48 h, and 72 h of planting, respec­ tively. as for the cultivar panjani, the germination percentage increased from 9.63% to 24.99% and then to 47.17% after 24 h, 48 h, and 72 h, respective­ ly. the percentage of germination in the bunduqi cul­ tivar increased from 8.94% to 23.85% and then to 40% after 24 h, 48 h, and 72 h, respectively. there were no significant differences in the germination percentages of panjani and bunduqi at the beginning of the experiment (after 24 h to 48 h of cultivation), but the cultivar panjani gave a higher percentage of germination and outperformed the variety at the end of the experiment (after 72 h of cultivation). this study is consistent with the study of (ilgin et al., 2007; gaaliche et al., 2013), where the germina­ tion rate increased with time and reached the high­ est value 72 h after planting. regarding the media used for cultivation, the per­ centage of germination increased in all media used in culture (a, b, c, and d) with the progression of the experiment. also, media c and d (containing the extract of the female stigma) significantly outper­ formed the media a and b (which did not contain the extract of the female stigma) in most reading times and for all the studied caprifig cultivars. the average percentage of germination in media containing extract of the female stigma was 34.85%, while the average percentage of germination in media that did not contain extract of the female stigma was 25.80% (an increase in the percentage of germination was about 35% when the extract of the female stigma was added to the pollen culture medium). this proves the positive role of adding the extract of the long­style female stigma flowers in raising the per­ centage of pollen germination of caprifig varieties and increasing the growth of the pollen tube in them. these results are consistent with several previous studies (ilgin et al., 2007; awamura et al., 1995), where pollen germination from caprifig was increased to more than 70% when long­style stigmas were added to the pollen culture medium. media b and d (containing 10% sucrose) signifi­ cantly outperformed media c and d (containing 5% sucrose) in most reading times and for all studied caprifig cultivars, where the average percentage of germination in media containing 10% sucrose was 33.09%. the average percentage of germination in media containing 5% sucrose reached 27.56% (an increase in germination percentage was about 20% when the level of sucrose in the cultivation medium increased from 5% to 10%). this confirms the impor­ tance of increasing the percentage of sucrose in the cultivation medium of pollen of caprifig varieties to raise the percentage of germination. this is consis­ tent with the results of ilgin et al. (2007), who found that increasing the concentration of sucrose to 20% led to an increase in the percentage of pollen germi­ nation but increasing the percentage of sucrose above 25% in the cultivation medium led to a decrease in the percentage of pollen germination. however, our results do not agree with the results of zeybekoglu et al. (1997), who found that the best germination rate of pollen of caprifig cultivars was at a concentration of 5% of sucrose. effect of pollen type on the fruit quality of edible fig cultivars fruit length table 6 displays the significant superiority of the pollinator panjani over the two pollinators, bunduqi and azraq, in the characteristic of the fruit length of the edible fig variety, with a value of 4.62 cm. the bunduqi pollinator was significantly superior to the azraq pollinator, with a fruit length of 4.23 cm and 4.11 cm, respectively. as for the edible fig cultivars, the safrawi and habashi fig cultivars (without signifi­ cant differences) were significantly superior to the white satehi cultivar, and the fruit length values were 4.89 cm, 4.80 cm, and 3.26 cm, respectively. in terms of the effect of the interaction of the pollinated vari­ ety with the edible fig variety on the characteristic of the length of the fruit, the interaction of the caprifig cultivar panjani with safrawi achieved the highest fig. 6 ­ germination of ‘panjani’, ‘azraq’ and ‘bunduqi’ pollen after 72 h of cultivation on media (a, b, c, and d). barakat et al. ‐ evaluation of viability and germination of caprifig pollen 423 value (5.46 cm) and significantly outperformed all other interactions. the interaction of the caprifig variety bunduqi with white satehi was in the last place with a fruit length of 3.08 cm. fruit diameter the pollinator panjani was significantly superior to the pollinated bunduqi and azraq pollinators in terms of the diameter of the fruit of the female variety, with a value of 5.34 cm (table 7). also, the bunduqi pollinator was significantly superior to azraq pollina­ tor with a fruit diameter of 4.88 cm and 4.75 cm, respectively. as for the edible fig cultivars, the habashi was significantly superior to safrawi and white satehi cultivars. safrawi was significantly supe­ rior to the white satehi cultivar, with a fruit diameter of 5.78 cm, 4.76 cm, and 4.43 cm, respectively. in terms of the effect of the interaction of the inoculat­ ed variety with the edible fig variety on the charac­ teristic of the diameter of the fruit, the interaction of table 6 ­ effect of the pollinated variety on the characteristic of fruit length in edible fig varieties the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. caprifig fruit length (cm) average habashi white satehi safrawi azraq 4.59 3.36 4.39 4.11 c bunduqi 4.77 3.08 4.83 4.23 b panjani 5.04 3.35 5.46 4.62 a average 4.80 a 3.26 b 4.89 a 4.32 c.v.% 3.60 l.s.d. (1%) = caprifig 0.06 l.s.d. (1%) = edible fig 0.05 l.s.d. (1%) = (caprifig x edible fig) 0.10 pr. caprifig <.001 pr. edible fig <.001 pr. (caprifig x edible fig) <.001 f. calculated. caprifig 169.59 f. calculated. edible fig 2031.91 f. calculated. (caprifig x edible fig) 64.10 table 7 ­ effect of the inoculated variety on the fruit diameter in edible fig varieties the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. caprifig fruit diameter (cm) average habashi white satehi safrawi azraq 4.59 3.36 4.39 4.11 c bunduqi 4.77 3.08 4.83 4.23 b panjani 5.04 3.35 5.46 4.62 a average 4.80 a 3.26 b 4.89 a 4.32 c.v.% 3.60 l.s.d. (1%) = caprifig 0.06 l.s.d. (1%) = edible fig 0.05 l.s.d. (1%) = (caprifig x edible fig) 0.10 pr. caprifig <.001 pr. edible fig <.001 pr. (caprifig x edible fig) <.001 f. calculated. caprifig 169.59 f. calculated. edible fig 2031.91 f. calculated. (caprifig x edible fig) 64.10 adv. hort. sci., 2023 37(4): 415­426 424 the caprifig variety panjani with the edible fig variety habashi significantly outperformed all other interac­ tions, with a fruit diameter of 6.08 cm. in contrast, the interaction of the caprifig cultivar azraq with safrawi came at the last position with a fruit diame­ ter of 4.18 cm. fruit weight from table 8, it appears that the pollinator panjani was significantly superior to the pollinated bunduqi and azraq in the fruit weight of the edible fig variety, with a value of 53.83 g. the bunduqi polli­ nator was significantly superior to the azraq pollina­ tor with a fruit weight of 48.90 g. while the azraq pollinator came in last place with a fruit weight of 47.32 g. as for the edible fig cultivars, the habashi fig cultivar was significantly superior to safrawi and white satehi cultivars. safrawi also outperformed the white satehi cultivar (the fruit weight values of the edible fig cultivars were 56.73 g, 49.07 g, and 44.28 g, respectively). regarding the interaction of the capri­ fig pollinated variety with the edible fig variety and its effect on the characteristic of the weight of the fruit, the interaction of the caprifig cultivar panjani with the edible fig cultivar habashi significantly out­ performed all other interactions, the weight of the fruit in this interaction was 59.40 g. the interaction of the caprifig cultivar azraq with the edible fig culti­ var safrawi gave the lowest value of the fruit weight (42.90 g). total soluble solids percentage table 9 shows no significant differences between caprifig varieties in their effect on the tss % in the edible fig fruits inoculated with these pollinators. the values were 22.35%, 22.22%, and 21.32% after inocu­ lation with panjani, azraq, and bunduqi cultivars, respectively. thus, no effect of the inoculated variety was observed on the tss percentage in the edible fig fruits. as for the female cultivars, the white satehi cultivar was significantly superior to both cultivars habashi and safrawi in terms of the tss %, which amounted to 25.35%, 20.78%, and 19.75%, respec­ tively (and there were no significant differences between the two cultivars habashi and safrawi). regarding the effect of the interaction of the caprifig pollinator variety with the edible fig variety on the characteristic of the tss % in the fruits of the polli­ nated varieties, the interaction of the caprifig cultivar panjani with the edible fig cultivar white satehi sig­ nificantly outperformed all other interactions, with a tss % of 26.45%. in the latest place, the interaction of the caprifig cultivar bunduqi with the edible fig cul­ tivar habashi came with a tss % of 18.55%. our results agreed with the findings of pourghayoumi et al. (2012), who indicated the signifi­ cant effect of the pollen source on the length of the fruit in the edible fig varieties pollinated with these pollinators. however, our results did not match the results of same study regarding the effect of the pollen source on the tss % feature. table 8 ­ effect of the pollinated variety on the characteristic of the weight of the fruit in the edible fig varieties the presence of the same letter in the same column or the same line indicates that there are no significant differences between the transactions. caprifig fruit weight (g) average habashi white satehi safrawi azraq 55.45 43.6 42.9 47.32 c bunduqi 55.35 43.2 48.25 48.90 b panjani 59.4 46.05 56.05 53.83 a average 56.73 a 44.28 c 49.07 b 50.03 c.v.% 5.00 lsd (1%) = caprifig 0.90 lsd (1%) = edible fig 0.90 lsd (1%) = (caprifig x edible fig) 1.57 pr. (caprifig x edible fig) <.001 pr. caprifig <.001 pr. edible fig <.001 f. calculated. caprifig 109.85 f. calculated. edible fig 376.28 f. calculated. (caprifig x edible fig) 26.87 barakat et al. ‐ evaluation of viability and germination of caprifig pollen 425 4. conclusions this research demonstrated that the cultivar bunduqi was the earliest caprifig cultivar in terms of fruit ripening, the abundance of pollen, and the time of the release of blastophaga insects. therefore, it is recommended to use it in pollinating early female fig varieties. also, the pollen viability was very high in all studied cultivars, and there were no significant differ­ ences between cultivars in this trait. furthermore, the azraq cultivar significantly outperformed panjani and bunduqi cultivars in pollen germination percent­ age in all phases of the experiment and in all media used in cultivation. adding stigma flower extract in cultivation media increased the germination rate by 35% for all studied caprifig cultivars. furthermore, increasing the proportion of sucrose (10%) in the cul­ tivation medium increased the pollen germination percentage by 20% for all cultivars of caprifig (com­ pared to 5% sucrose). finally, the caprifig variety used in pollination had a clear effect on the weight, diameter, and length of the fruit in the pollinated edible fig varieties. consequently, the panjani variety was superior to the rest of the pollinators in improv­ ing these traits. the results of this research are important for both fig growers and plant breeders, as they help to select the best pollinators and con­ tribute to the development and improvement of the quality of the cultivated fig fruits. acknowledgements the authors are grateful to dr. gohar mushtaq, professor at the college of human medicine, and director of the scientific research center at idlib university, for his contribution in the revision of this article. references andersen p.c., crocker t.e., 2009 ­ the fig. ­ rev. 11/2009. edis, 2009(10). anjam k., khadivi­khub a., sarkhosh a., 2017 ­ the potential of caprifig genotypes for sheltering blastophaga psenes l. for caprification of edible figs. ­ erwerbs­obstbau, 59(1): 45­49. armstrong w.p., 2006 ­ sex determination and life cycle of ficus carica. ­ http:/wayneword.palomaredu/ pljun99b.htm. awamura m., shoda k., hiramatsu m., 1995 ­ in vitro germination of caprifig pollen grains using stigmatic exudate of a common fig as a promoter. ­ j. japan. soc. hortic. sci., 63(4): 739­743. aytürk ö., 2019 ­ structural and seasonal differences of syconia in dioecious f. carica l. ­ inter. j. adv. engineering pure sci., 31(2): 179­187. balci b., can h.z., hepaksoy s., aksoy u., 2001 ­ some significant results of the research‐work in turkey on fig. ­ ii inter. symp. fig, caceres, spain, 605(26): 173­181. bolat i̇., pirlak l., 1999 ­ an investigation on pollen via‐ caprifig tss (%) average habashi white satehi safrawi azraq 21.85 24.75 20.05 22.22 a bunduqi 18.55 24.85 20.55 21.32 a panjani 21.95 26.45 18.65 22.35 a average 20.78 b 25.35 a 19.75 b 21.96 c.v.% 9.40 lsd (1%) = (caprifig x edible fig) 1.28 lsd (1%) = caprifig 0.74 lsd (1%) = edible fig 0.74 pr. 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of fruit growing in the old world: olive, grape, date, and fig emerge as important bronze age additions to grain agriculture in the near east. ­ science, 187(4174): 319­ 327. impaginato 277 adv. hort. sci., 2021 35(3): 277­284 doi: 10.36253/ahsc­10523 genotypic diversity and trait profiles of some amaranthus accessions o.a. oyetunde 1 (*), m.o. olayiwola 2, b.t. osho 3 1 department of crop production and horticulture, lagos state polytechnic, ikorodu, nigeria. 2 department of crop production, college of agricultural sciences, olabisi onabanjo university, ago‐iwoye, nigeria. 3 department of agronomy, pan african university for life and earth sciences (including health and agriculture), university of ibadan, ibadan, nigeria. key words: amaranthus, biplot, improvement, trait profile, variability. abstract: knowledge of the pattern of trait variation among accessions, and the trait profiles of the accessions is crucial for improvement of a crop. twenty­one amaranthus accessions were evaluated in 2018 and 2019 to investigate the extent of genotypic diversity among the amaranth accessions and their trait profiles. data were subjected to analysis of variance, and correlation and prin­ cipal component analyses. taking stem weight (swt) as the yield, the accession × yield­trait combination (gyt) biplot was employed to investigate the trait profiles of the accessions. accession, year, and accession × year mean squares were significant (p≤0.05/0.01) for most of the measured traits. the first three principal components explained 88.18% of observable variation among the accessions and identified plant height (pht), number of leaves per plant (nol), and root weight (rwt) as the major contributors. significant (p≤0.01) correla­ tion was observed in the association of swt with nol, tbm, and rwt. accessions ngb00019 and ngb00061 were associated with the sector contain­ ing all the yt combinations considered. however, ngb00019 was identified as the best combiner of yield with other traits. further studies involving more traits should determine the trait profiles of the remaining accessions. 1. introduction amaranthus is a member of the amaranthaceae family and is believed to have originated from south america (janovská et al., 2012). despite the level of under­development, the genus is one of the most diverse among cultivated crops, with about 70 species (ebert et al., 2011). amaranths are classified based on the part of the plant for which they are grown. the leaf types including a. hybridus and a. tricolor are grown for their leaves consumed as vegetables while the grain amaranths such as a. caudatus, a. cruentus, and a. hypochondriacus are popular for the grains. in addition, some amaranth like a. tricolor are valued as ornamental, while some (e.g. a. palmeri s. wats., a. powellii s. watt., a. retroflexus l., and a. (*) corresponding author: biyi.oyetunde@gmail.com citation: oyetunde o.a., olayiwola m.o., osho b.t., 2021 ­ effect of vine and fruit pruning on yield attributes of two watermelon citrullus lanatus) cultivars. ­ adv. hort. sci., 35(3): 277­284 copyright: © 2021 oyetunde o.a., olayiwola m.o., osho b.t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 february 2021 accepted for publication 2 august 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10523 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 277­284 278 spinosus l.) are considered as weeds. in general, cul­ tivated amaranths are of nutritional importance because they produce edible leaves, stems, and grains (e.g., a. hybridus and a. tricolor) (akin­idowu et al., 2016; neelesh and pratibha, 2018). the leaves and stem are rich natural stores of vitamins (a, b, and c), and dietary minerals including calcium and iron (stallknecht and schulz­schaeffer, 1993). amaranthus has also been found to be a source of lysine, an essential amino acid that is lacking in diets based on cereals and tubers (schippers, 2000). the vegetable is reported to be adapted to a wide range of agro­ecol­ ogy habitat (katiyar et al., 2000) and can tolerate biotic (i.e. diseases and pests) and abiotic (i.e. heat and drought) stresses (shukla et al., 2010). as is with many other under­utilized leaf vegetables, the genet­ ic and economic potentials of amaranthus have not been fully harnessed. although the amaranth is essentially self­pollinat­ ing, there is significant level of natural outcrossing and inter­specific and inter­varietal hybridization, leading to the occurrence of wide differences among accessions (akin­idowu, 2016). there is a growing interest in research on amaranth because of the high genetic diversity and phenotypic variability of the crop which are of immense use in designing strate­ gies for improvement of the crop. plant breeders have found differences in plant observations among genetic materials as effective means to deduce esti­ mates of genetic diversity (akin­idowu, 2016) and to determine genotypic superiority. however, identifica­ tion of genetic superiority is hindered by unfavourable associations among a set of target traits since the decision is often based on multiple traits (yan and fregeau­reid, 2018). this implies that a successful cultivar must attain desirable levels for several key traits. thus, knowledge of the pattern of trait variation among accessions and the trait profile (strength and weakness) of available amaranth accessions will aid the exploitation of existing geno­ typic diversity for improvement of the crop for desired traits. recently, the accession x yield­trait (gyt) biplot approach was proposed by yan and fregeau­reid (2018) to combat the challenge posed by evaluation of accessions based on multiple traits, in this approach, the worth of a accession is deter­ mined by its value for yield in combination with other traits (y­t) rather than its levels for an individual trait. therefore, this study investigated the extent of geno­ typic diversity among amaranth accessions, as well as the trait profiles of the accessions with a view to pro­ viding information to aid effective future improve­ ment strategies. 2. materials and methods genetic materials twenty­one (21) amaranthus accessions (ngb00001, ngb00005, ngb00019, ngb00022, ngb00025, ngb00027, ngb00028, ngb00029, ngb00031, ngb00024, ngb00058, ngb00059, ngb00060, ngb00061, ngb00070, ngb00078, ngb00082, ngb00108, ngb00111, ngb00112, and a local check named laspo­col­001). all the acces­ sions, except the local check, were obtained from the national agency for crop genetic resources and biotechnology (nacgrab), ibadan, nigeria. seeds of the local check were collected from a reputable farmer in ikorodu. all the accessions belong to the species a. hybridus, and are of nigerian origin (table 1). field evaluation and phenotyping the 21 amaranthus accessions were evaluated on the field during the main seasons of 2018 and 2019. table 1 ­ origin of the 21 amaranthus hybridus accessions used in the study serial number accession name origin 1 ngb00001 katsina state, nigeria 2 ngb00005 kebbi state, nigeria 3 ngb00019 lagis state, nigeria 4 ngb00022 zamfara state, nigeria 5 ngb00025 niger state nigeria 6 ngb00027 osun state, nigeria 7 ngb00028 osun state, nigeria 8 ngb00029 ondo state, nigeria 9 ngb00031 oyo state, niigeria 10 ngb00034 oyo state, niigeria 11 ngb00058 ogun state, nigeria 12 ngb00059 ondo state, nigeria 13 ngb00060 oyo state, niigeria 14 ngb00061 osun state, nigeria 15 ngb00070 ogun state, nigeria 16 ngb00078 oyo state, niigeria 17 ngb00082 oyo state, niigeria 18 ngb00108 oyo state, niigeria 19 ngb00111 oyo state, niigeria 20 ngb00112 oyo state, niigeria 21 laspo­col­001 lagis state, nigeria oyetunde et al. ‐ gyt biplot technique revealed trait profiles of okra accessions 279 the land was initially tilled mechanically by ploughing twice and then harrowing. subsequently, raised beds; 2×1 m in diameter, were made manually. cured poultry manure was applied on the bed at a rate of 10 tons/ha. the trial was laid out in randomized complete block design with two replications. each accession was grown in a 2­row plot fitted into a bed in each replicate. a seed rate of 1.5 kg/ha was used, and planted by drilling. seeds were mixed with dry fine sand to enhance even distribution within the drills. the experiment was exclusively rain­fed. weeds were controlled manually by rogueing, sub­ ject to field inspection, and chemical insecticides were not used throughout the experiment. at maturity, a random sample of five plants per row; making 10 plants per replicate per accession, were observed for plant height (pht) (cm), number of leaves per plant (nol), and stem girth (stg) (mm). all the plants in each plot were uprooted, and the roots washed carefully, to record data on total bio­ mass (tbm) (g), root weight (rwt) (g), stem weight (swt), and harvest index (%). observations were recorded according to the amaranthus descriptors of ipgri (1999). data on tbm, rwt, and swt were con­ verted to kg/ha. harvest index was computed as the percent of the total biomass that is made up by the stem weight. data analyses test for homogeneity of variance was not signifi­ cant and thus analysis of variance (anova) was per­ formed on combined data from the two­year evalua­ tion using ‘proc glm’ in sas (sas, 2011). means of data collected for each accession were subjected to correlation (pearson coefficients) analysis among all pair­wise combinations of measured traits while the standardized mean values were subjected to princi­ pal component analysis. to reveal the level of pheno­ typic divergence among pairs of accessions, esti­ mates of genetic distance (euclidean) among all pos­ sible pairs of accessions were obtained using ‘proc distance’ while the accessions were distributed into clusters from dendrogram obtained using ‘proc tree’ (sas, 2011). to investigate the trait profiles of the accessions, a accession × yield­trait combination (gyt) (yan and fregeau­reid, 2018) biplot approach was employed where stem weight was taken as the yield. the gyt biplots were obtained using the ggebiplotgui package in r. to select accessions for the gyt biplots, a superiority index (si) was comput­ ed for the accessions, based on standardized gyt estimates. the si value of a accession was computed as the arithmetic mean of its standardized estimates. ten accessions comprising seven and three acces­ sions with the highest and lowest si values respec­ tively were selected for the gyt biplot. 3. results results of analysis of variance of amaranth acces­ sions are summarized in table 2. mean squares of accession were significant (p≤0.05/0.01) for all the measured traits except stem girth, while mean squares of year were significant (p≤0.05/0.01) for number of leaves per plant, root biomass, and har­ vest index. accession × year mean squares were also found to be significant (p≤0.05/0.01) for plant height, number of leaves per plant, and harvest index. the first three principal components (pcs) jointly accounted for 88.18% of the total variation among accessions, with pcs 1, 2, and 3 having eigen values of 2.64, 1.39, and 1.25, respectively, and explaining 44.06, 23.21, and 20.91% of the total variation in that order (table 3). plant height, root weight, and num­ ber of leaves per plant had high loadings (≥0.30) of source of variation df pht nol stg tbm rwt swt hi rep (year) 2 4.160 7.043** 0.010 0.039 0.005 0.023 0.002 year 1 0.481 7.346* 0.004 0.007 0.036** 0.061 0.264** accession 20 10.989** 9.745** 0.014 0.057** 0.007** 0.031* 0.015** accession × year 20 5.800** 6.517** 0.011 0.019 0.004 0.011 0.022** error 40 2.110 0.332 0.008 0.023 0.049 0.015 0.005 table 2 ­ mean squares of measured traits of amaranthus evaluated in 2018 and 2019 rep = replicate; df = degrees of freedom; pht = plant height; nol = number of leaves per plant; stg = stem girth; tbm = total biomass; rwt = root weight; swt = stem weight; hi = harvest index. * and ** significant at 5 and 1% probabilities respectively. adv. hort. sci., 2021 35(3): 277­284 280 0.55, 0.50, and 0.44 respectively in pc 1. high load­ ings in pc 2 were 0.69 and ­0.66 observed for harvest index and stem weight respectively while pc 3 was characterized by stem girth, root weight, number of leaves per plant, harvest index, and stem weight with loadings of 0.57, ­0.49, 0.40, 0.37, and ­0.35, respec­ tively. the pca biplot grouped the amaranthus acces­ sions into clusters over the four quadrants based on the contributions of the measured traits as explained by the pcs (figs. 1 and 2). the accessions were scat­ tered on the score biplot with the local check dis­ tinctly placed. accessions ngb00022, ngb00031, ngb00060, and ngb00070 in the top left quadrant were associated with harvest index. the top right quadrant, associated with number of leaves per plant and stem and root weights, had ngb00005, ngb00019, ngb00025, ngb00058, ngb00061, and ngb00112 as the corresponding accessions. the bot­ tom right quadrant, characterized by plant height and stem girth, was composed by ngb00001, ngb00029, and the local check. other accessions, clustered in the bottom left quadrant, were not asso­ ciated with any of the traits measured in this study. estimates of correlation coefficient revealed sig­ nificant (p≤0.05/0.01) associations among pairs of measured traits (table 4). positive and significant (p≤0.05/0.01) correlation was observed in the associ­ ation of plant height with number of leaves per plant (r= 0.52), stem girth (r= 0.27), total biomass (r= 0.29), and root weight (r= 0.47). number of leaves per plant showed positive and significant (p≤0.05) correlation with stem girth, total biomass, root weight and stem weight with a correlation coefficient of 0.27, 0.24, 0.22 and 0.22, respectively. furthermore, total bio­ mass and root weight had a significant positive corre­ lation (r= 0.70; p≤0.01) while there was positive and significant correlation in the association of stem weight with total biomass, root weight and harvest index (r= 0.93, 0.43, and 0.27, respectively). finally, harvest index had negative and significant correla­ table 3 ­ loadings from principal component axes of the com­ post used in this study table 4 ­ pearson correlation coefficients among measured traits of 21 amaranthus accessions evaluated in 2018 and 2019 re* and ** significant at 5 and 1% probabilities respectively. trait principal compo­ nent axis 1 principal compo­ nent axis 2 principal compo­ nent axis 3 plant height 0.55 ­0.14 0.09 number of leaves per plant 0.44 0.21 0.40 stem girth 0.25 ­0.14 0.57 root weight 0.50 0.02 ­0.49 stem weight 0.27 0.66 ­0.35 harvest index ­0.23 0.69 0.37 eigen value 2.64 1.39 1.25 proportion (%) of variation 44.06 23.21 20.91 cumulative (%) variation 44.06 67.27 88.18 fig. 1 ­ principal component loading pattern of six traits of amaranthus accessions. fig. 2 ­ principal components score plot for 21 amaranthus accessions. nol stg tbm rwt swt hi pht 0.52** 0.27* 0.29** 0.47** 0.14 ­0.27* nol 0.27* 0.24* 0.22* 0.22* 0.10 stg 0.16 0.14 0.15 ­0.02 tbm 0.70** 0.93** ­0.04 rwt 0.43** ­0.68** swt 0.27* oyetunde et al. ‐ gyt biplot technique revealed trait profiles of okra accessions 281 tions (p≤0.05/0.01) with plant height (r= ­0.27) and root weight (r= ­0.68). the estimates of genetic distance among pair­ wise combination of accessions based on composited traits (table 5) ranged from 0.99 between ngb00028 and ngb00111 to 7.90 between ngb00078 and the local check. in particular, high genetic distance esti­ mates were obtained between the local check and ngb00022 (7.45), ngb00059 (7.72), ngb00060 (7.02), and ngb00082 (7.21). at a genetic dissimilarity of 0.50, the grouping of the accessions by the dendrogram (fig. 3) fairly com­ pares with the groupings on the pc scores biplot. for instance, cluster 1 was composed of eight accessions, six of which were ngb00060, ngb00058, ngb00112, ngb00025, ngb00061, and ngb00019 which were grouped together, and associated with total biomass and stem and root weights on the pc scores biplot. the other two accessions in cluster i, ngb00031 and ngb00070, were also grouped together on the pc score plot, and were associated with harvest index. cluster 2 contained ngb00005 and the local check. the two accessions were also grouped together by the pc score biplot, and associated with plant height, number of leaves per plant, and stem girth. similarly, all the accessions in cluster 3; ngb00001, ngb00029, fig. 3 ­ dendrogram of relatedness among amaranthus acces­ sions, (x­axis) based on genetic dissimilarity (y­axis) from single­linkage cluster analysis. the red double­arrowed line delineates the accessions into clusters at approxima­ tely 0.50 level of dissimilarity; c1, c2, c3 are clusters 1, 2, and 3, respectively. table 5 ­ pair­wise genetic distance estimates based on observed phenotypes of 21 amaranthus accessions id accession 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 ngb00001 0.00 3.62 3.86 2.52 1.82 1.37 2.13 1.65 2.34 1.62 2.73 3.81 3.91 4.02 2.27 4.23 2.58 1.84 2.47 2.44 5.33 2 ngb00005 0.00 3.68 4.89 2.26 4.36 4.50 3.88 3.80 4.80 3.39 6.27 3.94 3.50 3.50 5.66 5.32 4.77 4.73 3.11 3.81 3 ngb00019 0.00 4.14 2.97 4.24 4.54 4.93 2.82 4.71 4.00 5.09 4.45 1.54 3.70 6.57 5.08 5.21 4.83 2.61 6.33 4 ngb00022 0.00 3.19 1.42 1.19 2.97 1.69 2.14 2.23 3.79 3.01 3.88 1.68 3.28 1.54 2.06 1.26 2.26 7.45 5 ngb00025 0.00 2.67 3.07 2.70 2.15 3.06 2.35 5.10 3.10 3.20 2.45 4.98 3.75 3.19 3.17 1.63 4.79 6 ngb00027 0.00 1.00 1.80 2.12 1.35 2.37 3.38 3.58 4.14 1.70 3.26 1.41 1.15 1.45 2.46 6.39 7 ngb00028 0.00 2.10 2.21 1.85 2.01 3.55 3.17 4.17 1.31 2.47 1.12 1.25 0.99 2.52 6.80 8 ngb00029 0.00 3.34 2.52 2.55 4.16 3.96 4.94 2.52 3.54 2.28 1.56 2.27 3.06 5.07 9 ngb00031 0.00 2.53 2.18 4.31 2.69 2.57 1.63 4.26 2.93 2.94 2.41 1.33 6.76 10 ngb00034 0.00 3.32 3.54 4.27 4.56 2.46 3.67 2.31 1.52 2.35 3.25 6.79 11 ngb00058 0.00 5.06 1.54 3.69 1.51 3.41 2.62 2.65 1.75 1.48 6.10 12 ngb00059 0.00 6.32 4.96 4.01 4.94 3.43 3.87 4.34 4.80 7.72 13 ngb00060 0.00 4.03 2.58 4.15 3.78 3.81 2.72 2.15 7.02 14 ngb00061 0.00 3.10 5.78 4.93 5.05 4.60 2.61 6.74 154. ngb00070 0.00 2.90 2.32 2.29 1.81 1.78 6.36 16 ngb00078 0.00 2.88 2.87 2.73 4.50 7.90 17 ngb00082 0.00 1.40 1.18 3.12 7.21 18 ngb00108 0.00 1.44 3.18 6.50 19 ngb00111 0.00 2.48 6.99 20 ngb00112 0.00 6.00 21 local check 0.00 282 adv. hort. sci., 2021 35(3): 277­284 ngb00027, ngb00108, ngb00034, ngb00022, ngb00028, ngb00111, ngb00082, ngb00078, and ngb00059, except ngb00022 and ngb00001 were also grouped together on the pc scores biplot, and were not associated with any of the traits measured in this study. the trait profiles of the amaranthus accessions are displayed on the polygon view of the gyt biplot (fig. 4). the polygon view revealed four sectors with the ngb00019, local check, ngb00060, and ngb00078 as the vertex accessions in their respec­ tive sectors. accessions ngb00019 and ngb00061 belonged to sector 1 which was characterized by all the measured yield­trait (y­t) combinations. this sec­ tor contained the highest ranked accessions. sector 2 with the local check as vertex accession, also con­ tained ngb00005 and ngb00025, thus comprising accessions that were next in rank to the accessions in sector 1. similarly, the accessions in sector 3; ngb00060 and ngb00112, ranked next to those in sector 2 while the­poorest ranked accessions; ngb00082, ngb00059, and ngb00078, constituted sector 4. sectors 2, 3, and 4 were not associated with any of the y­t combinations considered in this study. the names of the accessions 1 to 10 are available in table 6. fig. 4 ­ the accession × yield­trait biplot of ‘which won where’ of selected seven best and 3 worst amaranthus accessions. table 6 ­ genotype × yield­trait combination data matrix for 21 amaranthus accessions evaluated in 2018 and 2019 yxpht, yxnol, yxstg, yxtbm, yxrwt, yxhi = yield combination with plant height, number of leaves per plant, stem girth, root weight, and harvest index respectively. id on gyt biplot accession yxpht yxnol yxstg yxtbm yxrwt yxhi superiority index 1 ngb00019 1.779 1.183 1.386 2.337 2.299 1.774 1.793 2 ngb00061 1.291 1.564 0.793 1.903 1.701 1.655 1.485 3 ngb00005 1.426 1.923 1.337 1.200 1.470 0.943 1.383 4 local check 1.564 1.081 1.098 0.553 1.476 ­0.230 0.924 5 ngb00025 0.791 0.833 1.272 0.783 0.856 0.683 0.870 6 ngb00060 0.831 1.186 1.313 0.595 ­0.367 1.576 0.856 7 ngb00112 0.886 0.676 1.005 0.766 0.462 1.087 0.814 not selected ngb00031 0.070 0.244 0.441 0.561 0.156 0.576 0.341 not selected ngb00058 0.427 0.394 0.423 0.005 ­0.326 0.560 0.247 not selected ngb00070 ­0.188 0.060 ­0.251 ­0.244 ­0.245 0.017 ­0.142 not selected ngb00001 ­0.294 ­0.365 ­0.041 ­0.295 0.003 ­0.453 ­0.241 not selected ngb00022 ­0.499 ­0.537 ­0.340 ­0.438 ­0.531 ­0.101 ­0.408 not selected ngb00111 ­0.584 ­0.635 ­0.466 ­0.710 ­0.826 ­0.388 ­0.602 not selected ngb00028 ­0.685 ­0.633 ­0.707 ­0.726 ­0.740 ­0.576 ­0.678 not selected ngb00027 ­0.758 ­0.802 ­0.692 ­0.694 ­0.490 ­0.707 ­0.690 not selected ngb00034 ­0.978 ­0.748 ­0.593 ­0.682 ­0.499 ­0.822 ­0.720 not selected ngb00029 ­0.638 ­0.784 ­0.737 ­0.825 ­0.614 ­0.950 ­0.758 not selected ngb00108 ­0.990 ­0.959 ­0.879 ­0.946 ­0.881 ­1.005 ­0.943 8 ngb00082 ­0.883 ­1.093 ­1.010 ­0.955 ­0.972 ­0.879 ­0.965 9 ngb00059 ­0.952 ­1.269 ­1.518 ­0.874 ­0.612 ­1.251 ­1.079 10 ngb00078 ­1.617 ­1.321 ­1.833 ­1.317 ­1.318 ­1.509 ­1.486 mean 0 0 0 0 0 0 standard deviation 1 1 1 1 1 1 oyetunde et al. ‐ gyt biplot technique revealed trait profiles of okra accessions 283 4. discussion and conclusions the significant difference observed among the accessions indicated the existence of variation with respect to the measured traits except for stem girth, and underscored the possibility of selection for improvement. idehen et al. (2018) reported same result for stem girth among 10 accessions of amaranthus spp. on the contrary, mandal and dhangrah (2012) studied 17 amaranthus accessions and observed significant differences among the accessions for all the characters considered including stem girth. plant height, number of leaves per plant, root biomass and harvest index can be relied upon as important tools in long term selection gain. gerrano et al. (2015) found high phenotypic variability among 32 amaranthus accessions using plant height, leaf length, leaf width, leaf area, leaf area index, number of leaves, stalk diameter, panicle or inflorescence length, number of primary branches, fresh biomass, dry biomass, harvest index, thousand seed weight and grain yield per plant. the loadings of plant height, number of leaves per plant, and root weight indicated that these charac­ ters chiefly accounted for most of the variation observed among the accessions. this suggests that these traits are crucial in maintaining variability with­ in the breeding population and they should be con­ sidered for selection in amaranthus improvement programme. gerrano et al. (2015) reported compara­ ble results on 32 amaranthus species of south african origin. the positive significant correlation observed between plant height and number of leaves per plant, stem girth, total biomass, root weight and harvest index implied that direct selection for any of the trait could lead to improvement in the other. gerrano et al. (2015) reported that plant height correlated positively with fresh biomass and dry biomass when evaluating the genetic diversity of amaranthus species in south africa. thanapornpoonpong et al. (2007) also reported a significant and positive relationship between plant height and fresh biomass. strong positive correlation of stem girth, total and root biomass and stem weight with number of leaves per plant, total biomass and root biomass, stem weight with total biomass, root biomass and harvest index indicated that the use of any of the characters can help to improve selection process in breeding programs. the significant negative correlation of harvest index with plant height and root weight suggested that an attempt to breed for high harvest index will lead to short plant height and less root weight in amaranthus. the clustering pattern of accessions into groups showed the phenotypic diversity among the acces­ sions for the different characters studied. genetic diversity, evidenced by phenotypic variability, is essential in the initiation of a breeding program because when absent, there cannot be meaningful selection and genetic advancement becomes impos­ sible (govindaraj et al., 2014). the accessions within a cluster are closely associated and this suggests that the variability within group could be useful in the selection process for improvement of associated desired traits. variability between groups could be explored in heterotic breeding where members of a cluster could serve as parents in crosses involving members from distinct clusters. for instance, acces­ sions in cluster 1 were mostly associated with har­ vest index, number of leaves per plant, and stem and root weights suggesting the presence of favourable alleles for yield­related traits within the group and a potential to improve these traits via selection. whereas, individuals in cluster 2 were generally associated with plant height and stem girth. a cross between members of the two groups could give high­ er yielding progenies with good standability which would be invaluable to the vegetable industry. the gyt biplot is useful for multiple­trait­based evaluation of accessions, permitting a graphical rank­ ing of entries based on their levels in combining yield with related traits. like in the gge biplot, the gyt polygon is delineated into sectors, with their associ­ ated closely­related y­t combinations, a vertex and other associated accessions (yan and frégeau­reid, 2018). thus, ngb00019 and ngb00061 were associ­ ated with all the y­t combinations considered in this study which is an indication of superior trait profile. however, ngb00019 was the vertex accession and was thus identified as the best combiner of yield with other traits. there was sufficient genetic variability among the amaranthus accessions, to permit improvement through selection, with greater chances of success with plant height, number of leaves per plant, and root weight. harvest index; which is the edible por­ tion of the plant, can be simultaneously improved with plant height, and stem and root weights. information from principal component analysis, genetic distance estimates, and cluster analysis can adv. hort. sci., 2021 35(3): 277­284 284 be utilized for parental selection in breeding pro­ grammes. accessions ngb00019, ngb00025, ngb00058, ngb00060, ngb00061, and ngb00112 would be good genetic materials for total biomass, and root and stem weights while ngb00031 and ngb00070 have potentials to improve harvest index, and ngb00005 and the local check would be useful for plant height, number of leaves per plant, and stem girth. ngb00019 followed by ngb00061 had the most diverse trait profile among the evaluated accessions. further studies involving more traits are required to determine the trait profiles of the remaining accessions used in the study. acknowledgements we are grateful to the national center for genetic resources and biotechnology, nacgrab, ibadan, nigeria, for providing the seeds used for the study. we thank the staff of the teaching and research farms of the department of crop production and horticulture, lagos state polytechnic, ikorodu, nigeria, for assistance during field evaluation. references akin­idowu p.e., gbadegesin a.m., uterdzua o., ibitoye d.o., odunola o.a., 2016 ­ characterization of grain amaranth (amaranthus spp.) germplasm in south west nigeria using morphological, nutritional, and random amplified polymorphic dna (rapd) analy‐ sis. ­ resources, 5(1): 6­15. ebert a.w., wu t., wang s., 2011 ­ vegetable amaranth (amaranthus l.). ­ avrdc, the world vegetable center, avrdc publication no. 11­754, pp. 9. gerrano a.s., van rensburg w.s.j., adebola p.o., 2015 ­ genetic diversity of amaranthus species in south africa. ­ south afr. j. plant soil, 32(1): 39­46. govindaraj m., vetriventhan m., srinivasan m., 2014 ­ importance of genetic diversity assessment in crop plants and its recent advances: an overview of its analytical perspectives. ­ genetics res. int., pp. 1­14. idehen e.o., oduwaye o.a., lateef l.a., ikeora c.j., 2018 ­ evaluation of genetic diversity in some amaranthus spp. using morphological and random amplified polymorphic dna (rapd) analysis. ­ nig. j. biotech., 35(2): 167­175. ipgri, 1999 ­ directory of germplasm collections. ­ ipgri, international plant genetic resources institute, rome, italy. janovská d., cepkova p.h., dzunkova m., 2012 ­ characterisation of the amaranth genetic resources in the czech gene bank, pp. 457­478. ­ in: caliskan m. (ed.) genetic diversity in plants. intech, rijeka, croatia pp. 498. katiyar r.s., shukla s., rai s., 2000 ­ varietal perfor‐ mance of grain amaranths (a. hypochondriacus) on sodic soil. ­ proc. national academy of sciences, india, section b: biological sci., 70: 185­187. mandal j., dhangrah v.k., 2012 ­ screening vegetable amaranth under summer condition in red and lateritic belt of west bengal. ­ environment & ecology, 30(4): 1430­1433. neelesh k.m., pratibha a., 2018 ­ amaranthus grain nutritional benefits: a review. ­ j. pharmacognosy phytochem., 7(2): 2258­2262. sas, 2011 ­ sas system for windows. ­ release 9.4. sas institute inc. cary, nc, usa. schippers r.r., 2000 ­ african indigenous vegetables: an overview of the cultivated species. ­ natural resources institute, acp­eu technical centre for agricultural and rural cooperation, chatham, uk. shukla s., bhargava a., chatterjee a., pandey a.c., mishra b.k., 2010 ­ diversity in phenotypic and nutri‐ tional traits in vegetable amaranth (amaranthus tricol­ or), a nutritionally underutilised crop. ­ j. sci. food agric., 90: 139­144. stallknecht g.f., schulz­schaeffer j.r., 1993 ­ amaranth rediscovered. new crops. ­ wiley, new york, usa, pp. 211­218. thanapornpoonpong s., somsak w., pawelzik e., vearasilp s., 2007 ­ yield component of amaranth (amaranthus spp.) grown under northern thailand irri‐ gated area. ­ paper presented at tropentag 2007, university of kassel­witzenhausen and university of göttingen, 9­11 october 2007. yan w., frégeau­reid j., 2018 ­ genotype by yield x trait (gyt) biplot: a novel approach for genotype selection based on multiple traits. ­ scientific report, 8: 8242. impaginato 33 adv. hort. sci., 2021 35(1): 33­42 doi: 10.36253/ahsc­8937 the impact of cumin essential oil on cold stored­radish tubers s. dehghanpour, m. shamili (*), a. mirzalaiyan­dastjerdi horticulture department, faculty of agriculture, university of hormozgan, bandar abbas, iran. kew words: antioxidant capacity, malondialdehyde, protease, protein, total phe­ nol content abstract: notable biological compounds in radish, made it as one of the most popular crops in the raw vegetables global market. however, storing it under low temperature conditions is associated with browning and taste changing. the present research aimed to evaluate the effects of different concentrations of cumin essential oil (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the storage period (0, 3, 6 and 9 days) on antioxidant parameters of radish tubers under low temperature conditions. the results indicated declining trends in the l* and a* values, beside ascending trend in b* value after nine days of storage. however, over the storage period of tubers, these parameters increased in cumin essential oil treated tubers. according to our findings, the application of cumin essential oil increased protein content, but reduced the malondialde­ hyde content, polyphenol oxidase and peroxidase activities. the cold­stored radishes received the most effective treatment of cumin essential oil at the concentration of 12.5 and 25 ppm. 1. introduction radish (raphanus sativus l.), belongs to the cruciferae family, is a part of the human diet worldwide. this root­product is usually used as a crunchy vegetable, mainly in salads (banihani, 2017). radish contains car­ bohydrates, sugars, dietary fibers, proteins, vitamins and minerals (khattak, 2011). in addition, it has been found to be rich in some bioactive compounds, including isothiocyanates (e.g. indole­3­carbinol and sul­ foraphane) and glucosinolates (e.g. glucoraphanin, glucoerucin, gluco­ brassicin, glucoraphasatin, neoglucobrassicin, 4­methioxyglucobrassicin and 4­hydroxyglucobrassicin) (malik et al., 2010; ishida et al., 2015; baenas et al., 2016), which prevent the proliferation of the colon (nakamura et al., 2008), prostate (steinbrecher et al., 2009), lung (wang et al., 2014) and breast (pawlik et al., 2017) cancer cell lines. the global marketing of radish has improved due to be included in easy­to­prepare foods especially in the northern european countries like holland and germany (salerno et al., 2005). however, the storage of radish under ambient conditions leads to shrinking and hence failing its (*) corresponding author: shamili@ut.ac.ir citation: shamili m., dehghanpour s., mirzalaiyan­ dastjerdi a., 2021 ­ the impact of cumin essen‐ tial oil on cold stored‐radish tubers. ­ adv. hort. sci., 35(1): 33­42 copyright: © 2021 shamili m., dehghanpour s., mirzalaiyan­ dastjerdi a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 may 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8937 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 33­42 34 marketing (luegno and calbo, 2001). low temperature conditions are commonly used in the food industry to maintain the quality of horti­ cultural products in storage, reduce the respiration rate and delay metabolic processes (patel et al., 2016). but lipid peroxidation, the most important deteriorating factor during low temperature storage, affects the nutritional value and sensory evaluation of food products (liang et al., 2020), i.e radish lose up to 5% of its weight and 43% of its nutritional con­ tent after exposure to low temperature (10°c) for 10 days (del aguila et al., 2006). the antioxidants are essential compounds that prevent or delay the lipid peroxidation (khalid et al., 2016). food industries use artificial antioxidants such as bha (butylated hydroxylanisole), bht (butylated hydroxyltoluene), tbhq (tertiary­butyl hydro­ quinone), and pg (propyl gallate) to extend the shelf life of cold­stored products. however, the side effects of these synthetic components have caused con­ sumers to be concerned (andre´ et al., 2010; liang et al., 2020) and drawn the attention of researchers to safe alternatives such as natural derived products. plants essential oils (eos) are a rich source of antioxi­ dants which reduce the production of reactive oxy­ gen species (ros) or scavenge the formed ros (khalid et al., 2016). browning and taste changing are the main disor­ ders which influence the quality of radishes under low temperatures (del aguila et al. , 2008; ramachandran et al., 2013). using antioxidant com­ pounds, such as citric acid and ascorbic acid (del aguila et al., 2006; lee et al., 2007) or covering the tuber with chitosan (ramachandran et al., 2013) have reduced the browning intensity under cold stor­ age conditions. packed radishes can also be stored for six days under low temperatures with no brown­ ing symptoms (nicola et al., 2004; ayub et al., 2013). plant eos can serve as efficient strategies in the quality preservation in cold­stored horticultural prod­ ucts (misharina and samusenki, 2008). the cumin (cuminum cyminum l.), an herbaceous annual mem­ ber of the apiaceae family, is frequently used as fla­ vor (thippeswamy and naidu, 2005). the main com­ ponents of cumin eo are cuminal, cuminic alcohol, terpinene, safranal, p­cymene and pinene (thippeswamy and naidu, 2005; ravi et al., 2013). despite several reports on the antimicrobial char­ acteristics of cumin eo (thippeswamy and naidu, 2005; gachkar et al., 2007; milan et al., 2008; dua et al., 2012), there is no research on its potential to serve as an antioxidant with ros scavenging capacity, specifically in applying on cold­stored products. therefore, the present research aimed to evaluate the effects of different concentrations of cumin eo, as an organic antioxidant compound, on the bio­ chemical and antioxidant parameters of stored radishes under low temperature conditions. 2. materials and methods plant material preparation radish (raphanus sativus l. var ‘cherry belle’) seeds were planted (october 1st 2019) in the research greenhouse (rh=50%, temperature: 26 ± 1°c day /20± 0.5°c night, and 50% shade) at the university of hormozgan (53° 33ʹ e 28° 30ʹ n, 10 m), iran. the plas­ tic pots (25×18 cm2) were filled by a media mixture (soil­sand­silt in 1:1:1 ratio). daily drip irrigation sys­ tem was used for all experimental units. the tubers were harvested 40 days later. the experiment was repeated next months (november 1st 2019­ december 10th 2019). the radish tubers were then transferred to the laboratory. after wards, well­ formed and uniform tubers (1.5×1.5 cm2) which were healthy, smooth, firm and free from decay, damage or cracks were selected, washed and dried. the experimental design the factorial experiment was a completely ran­ domized design with six replications (10 samples per each experimental unit). the factors were cumin eo concentrations (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the storage period (0, 3, 6 and 9 days). the con­ trol (day 0) measurement was done before applying the treatments. the cumin eo was prepared from zardband pharmaceuticals industry, tehran, iran. the phytochemical and microbiological properties of the eo were described by zarband company (table 1). the cumin oil was obtained by steam distillation of seed. ground seed were sieved and then subjected to water distillation using a clevenger apparatus (3 hours) (beis et al., 2000). the cumin eo was diluted with distilled water (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the radishes were immersed at 20°c for 10 min. then all radishes were air­dried under room temperature for 1 h, put in polyethylene plastic containers (10 tubers per each container) and placed in the cold storage (5°c, 95% rh). the tubers were selected for the following mea­ surements at 0, 3, 6 and 9 days of storage. https://en.zardband.com/ dehghanpour et al. ‐ the impact of cumin essential oil on radish 35 color parameters tuber skin color was measured using a colorime­ ter (konica­cr­400 minolta, japan) under reflected light in cie l*a*b* system, where l* expressed color lightness from 0 (black) to 100 (white), a*defined the proportion of red (+a) to green (­a), and b* repre­ sented the proportion of yellow (+b) to blue (­b). the average of six records was considered for every color parameter. malondialdehyde content according to heath and packer (1968), 0.5 g of the tuber was homogenized in 5 ml of 1% trichloroacetic acid (tca), centrifuged (10000 rpm, 5 min) and the supernatant (250 μl) was mixed with 1 ml of malondialdehyde (mda) solution containing 20% tca and 5% tba (thiobarbituric acid). it was then incu­ bated in a hot water bath (95°c) for 30 min, immedi­ ately cooled and re­centrifuged (10000 rpm, 10 min). the absorbance of the sample was measured at 532 and 600 nm (using a cecil ce2501 spectrophotome­ ter). the mda content was calculated using the equation (1). mda (mg g­1 fw) = [(a532 ­ a600) ×w×1000]/116 (1) where a532 and a600 are the sample absorptions at 530 and 600 nm, respectively, w is the sample weight (mg) and 116 is dilution factor total phenol content the total phenol content was determined by the folin­ciocalteu procedure (spanos and wrolstad, 1990). tuber tissue (0.5 g) was homogenized with 10 ml of 80% methanol. the mixture was centrifuged (10000 rpm, for 10 min). then 10 µl of the super­ natant, 490 µl of distilled water and 500 µl of folin­ ciocalteu reagent were mixed and incubated under dark ambient conditions (24±1°c) for 3 min. then 500 µl of sodium carbonate (1%) was added and the mix­ tures were re­incubated under the same conditions for 30 min. the absorbance was measured at 750 nm using a cecil ce2501 spectrophotometer and the phenol content was expressed in µg gallic acid g­1fw, using a gallic acid (0­0.1 mg ml­1) standard curve. antioxidant activity (dpph) the dpph (2,2­diphenyl­1­picrylhydrazyl) assay was done according to singleton et al. (1999) procedure. briefly, 0.1 g of tuber tissue was powdered in liquid nitrogen and then 100 ml of 96% ethanol was added. after centrifuging (at 3500 rpm, 5 min), 950 µl of 0.1 n dpph was added to 50 µl of each sample and stirred immediately. each sample was then kept in ambient dark conditions for 30 min. finally, the absorption of the extract was measured at 517 nm. the antioxidant activity was evaluated using equation (2). antioxidant activity (%) = [(acont ­ asamp)/acont]×100 (2) where acont and asamp are absorptions of the standard and the sample, respectively. protein content tuber tissue (0.1 g) was homogenized in 1 ml of 50mm sodium phosphate buffer (containing 129.18 mm nah2po4, 383.96 mm na2hpo4, 12.66 mm edta, ph=7). the homogenates were then centrifuged (10000 rpm, 4°c for 10 min). the bradford solution (1 ml) was added to the supernatant (50 μl) and the absorbance was measured at 595 nm. the protein content was evaluated according to the standards curve of bovine serum albumin (bsa) and expressed in mg g­1 fresh weight (bradford, 1976). the assay of protease, catalase, peroxidase and polyphenol oxidase activities the radish tuber (0.5 g) was powdered with liquid nitrogen and mixed with 1ml of the extraction solu­ tion (containing 100 ml of 50 mm phosphate buffer, 1.27 mm of edta and 4mm of pvp). the mixture was then centrifuged (1000 rpm, 15 min). afterwards the supernatant, as an enzyme extract, was used for determining the activity of the following enzymes (dhindsa et al., 1981). the protease activity was determined using a pro­ table 1 ­ physicochemical and microbiology of analysis of cumin eo test results physicochemical test appearance clear liquid color pale yellow odor confirmed characteristics density 0.89 kg m­3 refractive index 1.46 assay (cumin aldehyde) 36.33% microbiology limit test total bacterial count < 10 colony forming units per ml total mold and yeasts < 10 colony forming units per ml escherichia coli absence salmonellae absence plant used parts: fruits, state: liquid. adv. hort. sci., 2021 35(1): 33­42 36 cedure defined by homaei and samari (2017). briefly, 50 μl of the enzyme extract was mixed with 350 μl of 50mm sodium phosphate buffer (ph=7.5) and then 800 μl of 1% casein was added. the mixture was incu­ bated for 10 min at ambient temperature. then 400 μl of 10% tca was added and the mixture was again re­incubated at ambient temperature for 20 min. finally, the samples were centrifuged (10000 rpm, 10 min) and the absorption at 280 nm was measured. the coefficient of excitation was 26.40 mm­1 cm­1. to assay the catalase activity, 50 μl of the enzyme extract was mixed with 1 ml of the catalase reaction solution (containing 50 mm phosphate buffer with ph=7 and 15 mm h2o2). then the absorption was measured at 240 nm and the coefficient of excitation was 39.4 mm­1cm­1 (dhindsa et al., 1981). to determine the peroxidase activity, 33 ml of the enzyme extract was mixed with 1 ml of peroxidase reaction solution (containing 13 mm guaiacol, 5 mm h2o2 and 50 mm phosphate­potassium buffer with ph= 7). the sample absorption was then measured at 470 nm and the coefficient of excitation was 26.6 mm­1 cm­1 (chance and maehly, 1995). in order to assay the polyphenol oxidase (ppo) activity, 100 μl of the enzyme extract was mixed with 1 ml of pyrogallol reaction solution (containing 2.5 ml of 50 mm potassium phosphate buffer and 200 μl of 0.2 m pyrogallol). the sample absorbance was then measured at 280 nm and the coefficient of excitation was 26.4 mm­1cm­1 (kar and mishra, 1976). data analysis the statistical analysis was done using sas (ver­ sion 9.1.3) (sas institute inc. cary, nc, usa, 1990). the shapiro­wilks test confirmed the data normality (procedure: proc univariate, sas). the multivariate analysis of variance was performed related to the observation period and cumin levels, both of which were considered as independent vari­ ables (procedure: proc glm, sas). pillai’s trace test confirmed the variance homogeneity (procedure: proc glm, sas). tukey’s test was used in order to compare the mean values (procedure: files, sedit, factor, range, p<0.01, mstatc). excel 2013 was used to draw the figures. the presented mean values are the average of two growing seasons. 3. results color parameters the results indicated that all color parameters were influenced during the observation period. a declining trend was observed in the l* value after nine days of storage. the highest l* value (35.1±0.56) was observed on day 0 (in control plants) and the lowest value (23.19±0.97) on 9 days (in control plant). however, over the storage period of tubers, this factor increased in cumin eo treated tubers. increase of 17.43 % and 17.32% in l* value in the 9 days of treatment was the results of 12.5 and 25 ppm cumin eo application, respectively (fig. 1a). the results indicated that over the storage period, the a* trend was declining. the highest (30.32±0.75) and the lowest (19.35±0.88) values were observed on the 0 and 9 days, respectively, in the control after being under cold storage. but, increasing the concen­ tration of cumin eo in each measurement period (3, 6 and 9 days), led to an increase in a* value. based on the results, the highest value (29.42% increment) occurred at 25 ppm cumin eo on 3 days (fig. 1b). according to our results the b* value showed an ascending trend and rose from 10.5±0.65 on day 0 to 16.87±0.59 on 9 days of 25 ppm eo treatment. moreover, increase in the concentration of cumin eo made significant increments in b* value (8.37%, fig. 1 ­ the influence of cumin eo concentrations on the l* (a), a* (b), and b* (c) values under different observation periods in cold stored­radishes. means ± sd of six repli­ cates of two seasons are given (tukey, p<0.01). dehghanpour et al. ‐ the impact of cumin essential oil on radish 37 3.43% and 12.64%, respectively on 3, 6 and 9 days) (fig. 1c). mda, phenol contents and antioxidant activity the mda and phenol contents, along with the antioxidant activity, were significantly affected by the eo levels and storage duration. during the nine days of radish storage, the mda, total phenol content and the antioxidant activity showed ascending trend. furthermore, the cumin eo treatment resulted in a significant reduction in mda and phenol contents (figs. 2a, b). according to our findings, malondialdehyde gen­ erally increased as a result of storage period extent. the mda content of the first assessment, day 0, (0.75±0.16 mg g­1 fresh weight) rose to 3.29±0.15 mg g­1 fresh weight in the last assessment (9 days of stor­ age). the cumin eo­treated tubers had 0.76±0.14 mg g­1 fresh weight mda content at 25 ppm on 9 days. however, in each assessment period, a declining trend in malondialdehyde content was a result of increment in the concentration of cumin eo. the highest amount of mda was related to the control treatment value on the 9 days (3.29±0.15 mg g­1 fresh weight) and the lowest value (0.55±0.14 mg g­1 fresh weight) was recorded at the 25 ppm cumin eo on the 3 days (fig. 2a). the results indicated that the total phenol con­ tent showed an increasing trend over the storage period. the phenol content rose from 1243±116.19 μg of gallic acid g­1 fresh weight, in the first observa­ tion (day 0) to 4613.05±258.90 μg of gallic acid g­1 fresh weight on the last day of storage. although, the total phenol content showed a declining trend, with increasing the concentration of cumin eo. the lowest content of total phenol was observed at 25 ppm cumin eo on the 3 days (2024.03±212.84 μg of gallic acid g­1 fresh weight). (fig. 2b). regarding the antioxidant activity, over the dura­ tion of storage period and also with increasing the concentration of cumin eo, an ascending trend was observed compared to the control. the value of antioxidant activity on day 0 (21.2±2.49%) for control plants, improved significantly on days 3 (54.98±3.62%), 6 (57.19±3.33%) and 9 (75.24±1.41%). the 25 ppm cumin treated­tubers showed a same trend, which was reached from 84.37±2.41% on 3 days to 95.18±3.07% on 9 days (fig. 2c). protein content and enzyme activities the effect of low temperature storage on the radishes caused in the reduction in the protein con­ tent along with an increase in the activity of pro­ tease. different levels of cumin eo in each observa­ tion period had a significant effect on both traits (figs. 3a, b). the results indicated that the protein content decreased over the storage period of radish. the protein content in the control treatment on the day 0 measurement was 27.1±0.33 mg g­1 fresh weight and reached to 15.48±0.94 mg g­1 on the 9 days. the highest content of protein was related to 1.56 ppm cumin eo (26.78±0.33 mg g­1 fresh weight) on the 3 days and the lowest content was related to 3.13 ppm cumin eo (26.4±0.32 mg g­1 fresh weight). on the 6 and 9 days of storage, the highest value was occurred in 25 ppm cumin eo (25.29±1.22 and 19.40±1.32 mg g­1 fresh weight increment) (fig. 3a). the protease activity on day 0 (1.1±0.16 μmol min­1g­1 fresh weight) had an ascending trend during 9 days of cold storage. also increasing the concentra­ tions of cumin eo (6.25±0.11 ppm and more), increased the protease activity. this trait was varied from 1.35±0.15 to 2.53±0.20 μmol min­1g­1 fresh weight on the 3 days and from 4.93±0.11 to 6.93±0.14 μmol min­1g­1 fresh weight on the 6 days, as a result of different cumin eo levels. however, on fig. 2 ­ the influence of cumin eo concentrations on the mda (a), phenol content (b), and antioxidant activity (c) under different observation periods in cold­stored radishes. 38 adv. hort. sci., 2021 35(1): 33­42 the 9 days of storage, the protease activity varied from 9.55±0.11 to 11.9±0.15 μmol min­1g­1 fresh weight as a result of different eo concentrations (fig. 3b). there were enhancements in the activities of radish antioxidant enzymes (figs. 4a, b, c). according to figure 4a, a weak increment was observed in the catalase activity, by increasing the storage days until 6 days. nonetheless, the first measurement did not differ much than the second measurement. however, on 9 days, the highest activity was observed in all concentrations compared to the control. different concentrations of cumin eo showed various patterns on different days. on 3 days, the lowest catalase activity was observed in the control (106.34±12.39 μmol min­1g­1 fresh weight) and the highest was at 1.56 ppm cumin eo (132.51±22.27 μmol min­1g­1 fresh weight). the highest enzyme activity on the 6 days was at 3.13 ppm (209.40±18.38 μmol min­1g­1 fresh weight) and the lowest was at 6.25 ppm (202.27±17.46 μmol min­1g­1 fresh weight). on 9 days, the trend was completely declining, as the highest enzyme activity was in the control (1013.45±19.26 μmol min­1g­1 fresh weight) and the lowest was observed at 25 ppm cumin eo (602.72±19.07 μmol min­1g­1 fresh weight) (fig. 4a). peroxidase activity increased over the storage period and changed from 78.23±2.4 μmol min­1g­1 fresh weight on day 0 to 178.94±4.39 μmol min­1g­1 fresh weight in the last observation (9 days). in addi­ tion, increasing in the eo concentration reduced the activity of this enzyme. accordingly, the lowest val­ ues (29.81±4.73, 45.90±4.42 and 61.41±3.76 μmol min­1g­1 fresh weight) of the peroxidase activity were observed at 25 ppm cumin eo on the 3, 6 and 9 days, respectively and the highest activity was in the con­ trol (113.80±4.60, 149.79±3.72 and 178.94±4.39 μmol min­1g­1 fresh weight, respectively) on 3, 6 and 9 days, respectively (fig. 4b). the activity of polyphenol oxidase enzyme increased over the storage days (from day 0 to 9 days) at zero and 1.56 ppm; as the highest activity (25.31±0.73 μmol min­1g­1 fresh weight) was observed in cumin eo­free tubers on 9 days. changes in the activity of polyphenol oxidase on 3 and 6 days showed similar trends. its activity on day 0 (12.76±0.45 μmol min­1g­1 fresh weight) rose to 25.31±0.73 μmol min­1g­1 fresh weight on the 9 days. in the cumin eo treated tubers, the ppo activity indi­ cated a declining trend, over time and the lowest activity (8.44±0.49 μmol min­1g­1 fresh weight) was observed at 25 ppm, on 6 days (fig. 4c). fig. 3 ­ the influence of cumin eo concentrations on the protein content (a) and the activity of protease (b) under diffe­ rent observation periods in cold stored­radishes. means ± sd of six replicates of two seasons are given (tukey, p<0.01). fig. 4 ­ the influence of cumin eo concentrations on the activity of catalase (a), peroxidase (b), and ppo (c) under diffe­ rent observation periods in cold­stored radishes. means ± sd of six replicates of two seasons are given (tukey, p<0.01). dehghanpour et al. ‐ the impact of cumin essential oil on radish 39 4. discussion and conclusions excess production of ros and oxidative burst, under cold conditions, elicit some plant physiological reactions. the formed ros interact with cellular com­ ponents, trigger cascade of oxidative responses, per­ oxidase lipids, degrade proteins, inactivate enzymes and damage the dna (mittler, 2002). exposure to cold conditions causes the discoloration of radish and production of cracks on its edible parts (abdel, 2016). in addition, low temperatures influence carbohydrate accumulation in radish roots, accelerate plant growth rates and increase the shoot/root biomass ratio (sirtautas et al., 2011). color is an important factor in the consumer’s acceptance of foods. the l*, a* and b* values describe color parameters of food products (walkowiak­tomczak et al., 2008). the color of stored­fruits and vegetables mostly changes and turns darker over time. hernandez­munoz etal. (2008) reported a decrease in the values of color indices of cold stored­strawberry fruits. in the pre­ sent study, there was a significant reduction in l* and a* values, despite an increase in b*of radish tubers through storage period, which was related to a reduction in water content and product browning (hassani etal., 2012). the impact of organic compounds on the color char­ acteristics of horticultural products has been report­ ed previously (raybaudi­massilia etal., 2008; asghari marjanlo et al., 2009). clove eo prevented browning of grapes by preventing water loss (martinez­romero et al., 2007). according to our findings, treatment with different cumin eo concentrations caused differ­ ent color values in the radishes. cold disturbs the electron transfer chain in mito­ chondria and chloroplasts. this happens by an excess production of ros and causes oxidative damage to the membrane, thereby accelerates the lipid peroxi­ dation and mda over production (larkindale and huang, 2004). the results of this study showed a sig­ nificant increase in the malondialdehyde content during the nine days of cold storage. antioxidant compounds inhibit free radical’s generation, inter­ rupt its functions or lead to ros destruction (melo et al., 2005; srivsatava et al., 2011). the cinnamon eo has reportedly prevented the membrane lipids per­ oxidation in peaches (montero­prado et al., 2011). our findings regarding the cold­stored radishes treat­ ed with cumin eo, confirm this finding. in this work, the antioxidant capacity of cold­ stored radishes was assayed using the scavenging ability of dpph radicals, which is used widely for eval­ uating the radical scavenging effects of chemical and organic materials. dpph functions by absorbing elec­ trons or hydrogens, thereby becomes a stable mole­ cule (sagar and sing, 2011). the results indicated that the scavenging capacity of cumin­treated tubers was significantly higher than those of the control, proving that cumin improved the scavenging ability in radish tubers. according to our results, cumin eo displayed a dose­dependent manner in the scavenging of free radicals, as previously reported by dua et al. (2012). similarly, the antioxidant properties of peppermint and clove oils can be attributed to the ability of free radical scavenging (tripathi and dubey, 2004). polyphenol compounds naturally show an ability to scavenge ros. they are considered to be the effective non­enzymatic antioxidants (ma et al., 2011; zrig et al., 2011). the application of organic compounds was report­ edly capable in improving the phenol content of cold­ stored mangoes (wang and lin, 2000; razzaq et al., 2015). this can occur through changes in both polyphenol oxidase (ppo) and phenylalanine ammo­ nia­lyase (pal) activities (sun et al., 2010; deng et al., 2015). in our work, keeping the radish tubers in cold storage conditions made an increase in their phenol content. the application of cumin eo improved the antioxidant capacity and gradually decreased the phenol content. the lethal impact of oxidative stress on biological organisms is correlated to the destruction of proteins and the inactivation of enzymes (srivsatava et al., 2011). in the present study, the cold stored radishes displayed a visible decrease in protein content and an increase in antioxidant enzyme activities. the antioxi­ dant enzymes act as agents that trigger detoxification at low temperatures (mittler, 2002) and convert free radicals to the compounds that eventually release water and oxygen (zheng and tian, 2006). superoxide dismutase detoxifies ros by generating h2o2. then, ascorbate peroxidase and catalase break­ down the h2o2 (wang, 1995). catalase converts free radicals to oxygen and water (zheng and tian, 2006). our results of increases in catalase, peroxidase and ppo activities in cold stored radishes, confirmed the previous researches. essential oils can be considered as agents that aid the antioxidant defense system, reduce the release of radicals, prevent the destruction of cells and thus increase stress tolerance of plants (holley and patel, 2005). the usage of carvacrol and anethole in rasp­ berry causes a reduction in antioxidant enzyme activ­ adv. hort. sci., 2021 35(1): 33­42 40 ity (jin et al., 2012). lipoxygenase activity is also reported to be reduced in cinnamon­treated peaches (montero­prado et al., 2011). microbiology limit test confirmed that bacteria, mold and yeasts in our essential oil sample can be ignored. also it has no salmonellae neither escherichia.coli. the density of cumin essential oil is 0.90 (lewis, 1999). the pale yellow color and strong odor of our essential oil sample was similar to stan­ dard reference. cumin seeds contain flavonoids (β­ pinene, p­cymene, cumin aldehyde and cuminyl alco­ hol) are recognized to have antioxidant activity and scavenging capacity of the superoxide anion (sowbhagya 2013). the refractive index of an essen­ tial oil is a unique number that designates how the oil responds to and bends light. essentially, it is a measurement that tests how the speed of light is altered when passing through the oil. the refractive index of our essential oil sample (1.48) and cumin aldehyde content (36.33%) were close to which reported earlier (1.49 and 45%, respectively) (fahlbusch et al., 2005). cumin aldehyde is a con­ stituent of the essential oils of eucalyptus, myrrh and cumin (morshedi et al., 2015). β­pinene is a monoter­ pene, an organic compound found in cumin essential oil (li and jiang, 2004). the presence of polyphenolic compounds in cumin eo inhibits protein oxidation and enzyme inactivation (melo et al. , 2005). according to our findings, cumin eo improved the antioxidant system of tubers so caused in decreased catalase, peroxidase and ppo activities. the market for chilled­fresh products has a dra­ matic rise in the recent decade, encouraged mostly by the consumer request for nutritious, fresh and additive­free products. hence, the food industry has to respond with novel preservation, storage and han­ dling strategies. in our study, the storage of green­ house­harvested radishes at low temperatures declined the l* and a*values, and protein content during nine­day of storage, but increased the b* value, phenol content, antioxidant capacity, mda level, ppo, peroxidase, catalase and protease activi­ ties. the application of cumin eo reduced the mda content, polyphenol oxidase and peroxidase activi­ ties. finally, cold­stored radishes received the most effective treatment of cumin eo at the concentration of 12.5 and 25 ppm. acknowledgements the authors thank the head of research and technology center, university of hormozgan for their financial support and miss razie esfandiari ghalati for her technical assistance. references abdel c.g., 2016 ­ physiological disorders of four radishes (raphanus sativus l. var. sativus) cultivars storage roots grown in controlled cabinets under varying tem‐ peratures and irrigation levels.­ int. j. farming and allied sci., 5(2): 185­198. andre´c., castanheiraa a.i., cruzb j.m., paseirob p., sanches­silva a., 2010 ­ analytical strategies to eval‐ uate antioxidants in 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e agroecossistemas amazônicos, universidade do estado de mato grosso, alta floresta, mato grosso, brazil. 2 empresa brasileira de pesquisa agropecuária, sinop, mato grosso, brazil. 3 iempresa brasileira de pesquisa agropecuária, sinop, mato grosso, brazil. 4 núcleo de bioquímica vegetal e compostos funcionais, universidade do estado de mato grosso, cáceres, mato grosso, brazil. 5 programa de pós‐graduação em agronomia, universidade estadual paulista, botucatu, são paulo, brazil. 6 faculdade de ciências biológicas e agrárias, universidade do estado de mato grosso, alta floresta, mato grosso, brazil. 7 programa de pós‐graduação em agronomia, universidade federal de mato grosso, sinop, mato grosso, brazil. 8 faculdade de ciências sociais aplicadas e agrárias, universidade do estado de mato grosso, nova mutum, mato grosso, brazil. key words: carotenoids, fruit production, physicochemical properties, protect­ ed cultivation, solanum lycopersicum l. abstract: this study evaluated the yield performance of the tomato hybrids ‘ds0060’, ‘thaise’ and ‘trucker’ in the open field and environments protected by agricultural film (f) and polycarbonate panels (p) during the amazonian summer. in the protected environment, the crops produced significantly higher yields than in the open field. ‘thaise’ has high thermotolerance and is adapt­ able to a wide temperature range, making it the best­performing hybrid in environment f. highest yields were found for ‘thaise’ in environment f or p (86.2 and 92.5 t ha­1) together with ‘ds0060’ and ‘trucker’ in environment f (75.3 and 88.2 t ha­1), demonstrating the high yield potential in the interim growing season (january to april). in the open field, the fruit color was paler, fruit flesh firmer and ripening index lower. in environment f, the fruits con­ tained highest levels of soluble solids, lycopene and β­carotene. ‘thaise’ con­ tained higher concentrations of these two compounds. under environment p, the yield of the evaluated tomato hybrids increased considerably, indicating it as a promising possibility for tomato cultivation in tropical regions. ‘thaíse’ stood out with high yield and good quality traits, when grown in an f or p envi­ ronment. these results prove the viability of tomato production as interim crop in tropical regions, under high rainfall and heat, as well as the difference pro­ tected environments make for tomato cultivation, in particular the choice of the most suitable cover material for the crop, to ensure high yields coupled with desirable quality properties. (*) corresponding author: renecampos@unemat.br citation: pataraico v. jr., fernandes f. jr., roggia zanuzo m., da silva campos r.a., da silva ponce f., de carvalho campos botelho s., vieira da silva i., antunes d.t., pereira do nascimento m.s., seabra s. jr., 2023 ­ yield performance and nutritional quality of tomato hybrids in response to protected environments during the amazonian summer. ­ adv. hort. sci., 37(3): 391­402 copyright: © 2023 pataraico v. jr., fernandes f. jr., roggia zanuzo m., da silva campos r.a., da silva ponce f., de carvalho campos botelho s., vieira da silva i., antunes d.t., pereira do nascimento m.s., seabra s. jr. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 august 2022 accepted for publication 1 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13558 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 391­402 392 1. introduction tomato (solanum lycopersicum l.) is the fruit veg­ etable crop for which the market demand is the high­ est in the world. in 2019, the crop acreage was 5 mil­ lion hectares, for a production of 181 million tons. worldwide, brazil is the 9th largest producer, with an output of 3.6 million tons on 54,500 hectares (fao, 2021). the high demand for tomato is related to the palatability, culinary versatility and high contents of nutrients, especially those with functional properties such as vitamin c, lycopene and β­carotene (ali et al., 2021). in tropical farming, tomato is considered a high­ risk crop since the investments of inputs and man­ agement required are high. open­field yield can vary considerably due to pest and pathogen pressure, since up to 75% of the plants may be affected from the very beginning of the season, mainly by bacterial wilts and viruses (huat et al., 2013). protected culti­ vation allows production under adverse conditions, reducing plant exposure to high rainfall and, conse­ quently, to disease incidence (bazgaou et al., 2018). by minimizing the seasonality effect, year­long pro­ duction becomes possible, favoring product supply between the main crop seasons. the cover material for a protected environment must be chosen with a view to reducing the levels of global radiation and incident photosynthetically active radiation (par), to ensure an optimized plant performance. agricultural film can increase plant pro­ duction by altering the levels of luminosity, humidity and air temperature (beckmann et al., 2006). in trop­ ical regions however, it can cause a rise in air temper­ ature of 10 to 12°c with flower and fruit dropping, fruit cracking, black spot, and a decline in lycopene synthesis and marketable fruit yield. nevertheless, under the protective cover, the fruits can accumulate more soluble solids and vitamin c (florido and álvarez, 2015; shimeles et al., 2017; bazgaou et al., 2018). in this context, polycarbonate can be taken into consideration as an alternative cover for green­ houses, due to the high light transmittance and uv protection, aside from being a very light but durable material (kwon et al., 2017). evaluations of the cover material for protected tomato cultivation in tropical regions are insufficient, and little information is avail­ able about material that would allow more favorable cultivation conditions in these regions, to achieve higher yields without affecting the tomato quality. for high tomato yields under high temperatures, thermos tolerant hybrids must be used. these can ensure high yields of high­quality fruit, even under abiotic stress (scarano et al., 2020). the identification of tomato genotypes with high commercial and nutri­ tional quality can help producers chose the most suit­ able cultivar for each cultivation environment under the agroclimatic conditions of the amazon region in the summer season. this study analyzed the yield performance of tomato hybrids grown in the open field and in environments covered with agricultural film and polycarbonate in the amazonian summer, by correlating agronomic performance with fruit quality using principal component analysis. 2. materials and methods plant material, cultivation environments and experi‐ mental design three tomato hybrids [‘bsds0060’ (blueseeds), ‘trucker’ (nunhems) and ‘thaíse’ (feltrin)] were grown in three growing environments [open field (o); environment covered with agricultural film (f) and polycarbonate panels (p)]. the study was arranged in a randomized block design (crd) in a factorial arrangement (3x3) with five replications and seven plants each. the hybrids for the study had an indeterminate growth habit; fruits suited for salad and were chosen because of their yield and disease resistance. ‘ds0060’ has a late cycle, firm fruits and high fruit cracking resistance; mean fruit weight of 220 to 260g and is tolerant to tomato spotted wilt virus (tswv), tomato mosaic virus (tomv), tomato yellow leaf curl virus (tylcv), fusarium oxysporum f. sp. lycopersici (fol) race 1 and 2 and verticillium (v) race 1. ‘trucker’ is a vigorous f1 hybrid with excellent leaf cover; mean fruit weight of 240 g and tolerance to tylcv, tswv, fol, v and nematodes. ‘thaise’ is a medium­vigor f1 hybrid with bright red fruits, excel­ lent market standard due to the flavor, fruit unifor­ mity and long shelf life; mean fruit weight of 230 g and tolerance to tylcv, tomv, verticillium dahliae kleb., fusarium oxysporum f. sp. lycopersici (fol) race 3 and root­knot nematode. the tomato hybrids were grown in the open field and under the protection of a chapel­shaped green­ house (6.4 x 20 m), lateral height 3.5 m, central height 4.8 m in the north/south direction and side closure with 30% aluminet, a thermo­reflective shad­ ing screen. as cover material of the structure, a low pataraico et al. ‐ yield and quality of tomato hybrids during the amazonian summer 393 density transparent agricultural film (f), with uv­ a/uv­b protection, 90% transmission, and 25% light diffusion (nortene 150 µm) was compared with transparent polycarbonate panels (p), with 10 mm thick, a double­layer honeycomb structure and uv­ a/uv­b protection (polisystem). area and cultivation conditions of tomato plants the study was carried out in summer 2019/2020 (november to april) in sinop, mato grosso, brazil (lat. 11° 52’ 12” s, long. 55° 35’ 54” w; 364 m asl). according to the köppen classification, the climate is equatorial savanna with dry winters (aw), with a mean annual temperature of 25.4°c, a maximum of 34°c, annual rainfall of 1801 mm, and a rainy season between october and april. the tomato seedlings were produced in a clima­ tized greenhouse, planted in the 162 cells of poly­ styrene trays, containing 31 ml of commercial sub­ strate (vivato) per cell. the seedlings were planted 29 days after sowing (das) in furrows spaced 1.25 m apart and 0.35 m between plants, with a total popu­ lation of 22,000 plants per hectare. the plants were trellised by the “florida weave” method, on a struc­ ture of 1.3 m high wooden stalks and twine inserted horizontally every 0.4 m to hold up the plants. the soil at the site was classified as dystrophic red­yellow latosol (lva). the chemical properties (0­ 0.2 m layer) are shown in table 1. acidity was cor­ rected with 3.0 t ha­1 dolomitic lime (90% total neu­ tralizing power), fertilization at planting consisted of 3.4 t ha­1 single superphosphate and 30 t ha­1 barn­ yard manure, incorporated to a depth of 0.2 m with a rotary hoe. topdressing was applied by drip fertiga­ tion, distributed in 10 applications throughout the cycle, containing a total of 120 g calcium nitrate (15% n and 19% ca), 40 g potassium sulfate (48% k2o and 15% so4), 30 g phosphate monophosphate (12% n and 61% p2o5), 110 g potassium nitrate (13% n, 44% k2o and 1.5% s) and 70 g magnesium sulfate (9% mg and 12% s) per plant. irrigation was applied at a mean net depth of 3.5 mm per day, to compensate for the calculated mean daily evapotranspiration (valeriano et al., 2017). diseases and pests were controlled as recommended for the crop, by monitoring and applying products (based on pyraclostrobin, fluxapyroxad, trifloxys­ trobin, prothioconazole, kasugamycin, copper oxy­ chloride, equivalent in metallic copper, mancozeb, carbosulfan, abamectin, haloxyfop­p­methyl, pyriproxyfen, acetamiprid, alpha­cypermethrin, chlorfenapyr, beauveria bassiana) in active principle rotation and at rates recommended by the manufac­ turer. weed was controlled by hand weeding between plants and in­between rows. monitoring environmental variables the microclimatic variables (temperature, relative humidity, global radiation and par) of each environ­ ment were monitored and recorded at meteorologi­ cal stations (u30, hobo) equipped with sigma sen­ sors installed at the center of each environment, at a mean height of 1.80 m. readings were taken every 20 min and data compiled in hourly mean per month from 6:00 am to 6:00 pm. rainfall data were collect­ ed at the station installed in the open­field environ­ ment from december 12, 2019 to april 17, 2020. assessment of agronomic characteristics ripe fruits were harvested at ripening stage 6 (intense red color on more than 90% of the fruit sur­ face) (skolik et al., 2019). fruits were harvested somewhere between 99 and 137 d.a.s. within a peri­ table 1 ­ soil physicochemical analysis in the experimental area physico­chemical characteristics data ph water 5.1 ph cacl2 4.3 p (mg dm­3) 0.9 k (mg dm­3) 37 ca + mg 1.0 ca (cmolc dm­3) 0.8 mg (cmolc dm­3) 0.2 al (cmolc dm­3) 0.5 h (cmolc dm­3) 3.9 om (g dm­3) 20.0 sand (g kg­1) 283 silt (g kg­1) 133 clay (g kg­1) 584 sum of bases 1.1 cec 5.4 v (%) 20.1 ca/mg ratio 3.2 ca/k ratio 8.3 mg/k ratio 2.6 ca sat. 14.7 mg sat. 4.6 al sat. 30.2 k sat. 1.8 h sat. 71.2 adv. hort. sci., 2023 37(4): 391­402 394 od of about nine days in the protected environments. in the open field, harvest was already carried out 99 d.a.s. due to the poor phytosanitary state of the crop. the total fruit weight (kg plant­1) and total num­ ber of fruits were immediately determined and then the commercial standard of the fruits was classified to (i.e., appearance, size and damage level) to deter­ mine the commercial fruit weight and number of commercial fruits, underlying the estimation of the overall yield (t ha­1) of 22 thousand plants ha­1. evaluation of photosynthetic responses photosynthetic parameters were evaluated with a portable infrared photosynthesis analyzer (lci­sd, adc). photosynthetically active radiation (par, µmol m­2 s­1), net co2 assimilation rate (a, µmol co2 m ­2 s­ 1), leaf transpiration rate (e, mmol h2o m­2 s­1), inter­ nal co2 concentration in the substomatal chamber (ci, µmol mol­1), stomatal conductance (gs, mol m­2 s­ 1). the fourth fully expanded leaf from the plant apex during the harvest period (i.e., fruit filling; between 128 and 136 d.a.s) was used. the measurements were carried out on a sunny and cloudless day between 8 and 10 am. readings were taken in all treatments except on the open field, due to the high degree of crop damage caused by diseases. preparation of tomato samples fruits of the nine treatments harvested at 99 das were selected according to the market standards and samples of 12 fruits per plot were separated. the material was sanitized by immersion in chlorinated water (100 mg l­1 sodium hypochlorite) for 10 min and washed in distilled water. for physicochemical and biochemical analyses, the tomatoes were blend­ ed in a food processor (philips walita®). all samples were stored in triplicate at ­80°c for later analysis. physicochemical and biochemical analyses fruit flesh firmness, total soluble solids, titratable acidity and ripening index flesh firmness of the tomatoes was measured with a penetrometer (ta hd plus, stable micro system) by inserting a 6 mm tip into the skinless fruits to a depth of 9 mm. soluble solids were determined in a refrac­ tometer (pal­bx/ri, atago). titratable acidity (ta) was determined by the procedure described by zenebon et al. (2005), using a benchtop ph meter (hanna instruments hi901). results were expressed in % of citric acid, calculated by the formula: ta = v x fc x 10 x 100 (1) p where v is the volume (in ml) of 0.1 m naoh used for titration; fc is the correction factor of naoh and p the sample weight (in g). the fruit ripening index (ratio) was calculated as the ratio between total solu­ ble solids and titratable acidity. fruit color the color coordinates were read with a colorime­ ter (color quest xe, hunter lab). readings were per­ formed in the l*a*b system. the chromaticity (c*) and hue angle were determined by the formulas: c = (a + b)½ (2) hue = tg­1 (b/a) (3) lycopene and β‐carotene contents the lycopene and β­carotene contents were determined as proposed by nagata and yamashita (1992). a 1­g sample was homogenized in 10 ml ace­ tone:hexane (4:6 v/v) solution in a turrax blender. the resulting solution was analyzed in a spectropho­ tometer (evolution 201, thermo scientific) after phase separation. absorbance was determined at 453, 505, 645 and 663 nm and the results (mg 100­1 g) computed by the formulas: lycopene = ­0.0458a663 + 0.204a645 + 0.372a505 ­ 0.0806a453 (4) ß ‐ carotene = 0.216a663 ­ 1.22a645 ­ 0.304a505 + 0.452a453 (5) data analysis the data were subjected to analysis of variance (anova) and the means compared by the scott­ knott test (p<0.05), using software sisvar version 5.6 (ferreira, 2019). principal component analysis (pca) was run on software xlstat version 2021.3.1. 3. results and discussion agroclimatic variables data on rainfall and daily variations in global radi­ ation, par, temperature and relative humidity in the cultivation environments during the experimental period were recorded (fig. 1a­1e). the conditions of high rainfall and high temperatures restricted the plant cycle to 99 d.a.s. the total rainfall volume was 1841.9 mm, of which 633.1 mm fell between flower­ ing and fruit formation and 531.6 mm during fruit fill­ ing and harvesting (fig. 1a). the air temperature var­ ied from 22 to 29°c (fig. 1d). for tomato cultivation, pataraico et al. ‐ yield and quality of tomato hybrids during the amazonian summer 395 the optimal air temperature range is 20 to 24°c dur­ ing the day and 18°c at night (shimeles et al., 2017). above 29°c, yields decrease due to reduced fruit set (harel et al., 2014). thus, the high rainfall volume and high temperatures limited open­field tomato cul­ tivation by favoring high disease severity, which impaired the photosynthetic analysis in this plant group. protected cultivation provided an efficient barrier against excessive rainfall, but raised the maxi­ mum temperatures by 6.3% in environment f and by 4.4% in p, compared to the open field. the relative air humidity was quite similar in the evaluated culti­ vation environments. regarding luminosity, global radiation and par were lower in environments p and f than in the open field (figs. 1b and 1c). in environment p, global radia­ tion reached 403.3 w and par 742.6 µmol m­2 s­1 at noon, i.e., about 40% less than in the open field and 25% lower than in environment f. under low light incidence, tomato has optimized efficiency of par use (radin et al., 2003). this information was con­ firmed in our study by the higher tomato yield of plants grown in environment p (table 2). the analysis of the photosynthetic responses (table 3), net co2 assimilation rate (a), leaf transpiration rate (e), inter­ nal co2 concentration (ci) and stomatal conductance (gs) detected no differences between the protected environments. in environment p, the a value of hybrid ‘trucker’ was 118% higher than in f, with a yield increase of 52% (table 2). this variation can be attributed to the genetic characteristics of the hybrids and their responses to agroclimatic condi­ tions (i.e., global radiation, par, air temperature). in a study of kwon et al. (2017), the a of hybrid ‘superdoterang’ grown in environments under poly­ carbonate and glass, respectively, did not differ (24.8 and 21.8 μmol m­2 s­1). in this study, the better response of cultivars to the conditions in environ­ ment p may be related to a better light capture (of diffuse radiation) at a more efficient wavelength for photosynthesis, ass similarly observed elsewhere (radin et al., 2003; kwon et al., 2017). yield of tomato hybrids in different growing environ‐ ments the protected environments provided significant­ ly higher fruit yields. the marketable fruit weight of the hybrids increased 7­fold in environment p and 4.5­fold in f, compared to the open field (table 2). these results were better than those of yeshiwas et al. (2016), who reported a 54% increase in tomato yield in a protected environment compared to an open field. ‘thaise’ performed well under both cover types (3.90 and 3.27 kg plant­1), while ‘ds0060’ and ‘trucker’ had higher yields in environment p (3.23 and 3.57 kg plant­1, respectively). the thermotoler­ ance of ‘thaise’ was better, making the hybrid adapt­ able to a wide temperature range, which resulted in fig. 1 ­ rainfall during the experimental period (a) and daily variation of global radiation (b), par (c) air temperature (d) and relative humidity (e) in different growing environments. 396 adv. hort. sci., 2023 37(4): 391­402 the best performance in environment f, with a total fruit weight of 3.92 kg plant­1 and marketable fruit weight of 3.27 kg plant­1, which can be considered reasonable for tomato cultivation at high tempera­ tures (scarano et al., 2020). the yield recorded in this study exceeded that of ‘superdoterang’ which pro­ duced 2.8 kg of fruit plant­1 in a protected environ­ ment covered with polycarbonate (kwon et al., 2017) and of ‘bishola’, with determinate growth habit, which produced 1.81 kg per plant (yeshiwas et al., 2016). the total number of fruits was higher in ‘thaíse’ in both environments, f and p, while the results of ‘trucker’ were better in p (table 2). however, a high­ er percentage of fruit of ‘thaise’ had to be discarded in environment f (27%) than in environment p (15%). this was most likely caused by the higher global radi­ ation, par and air temperature in environment f (fig. 1b, 1c and 1d). tomato cultivation in the amazon region in the summer is high risk farming, due to the occurrence of rain causing waterlogging of the soil and leaf wetting, which are rather unfavorable factors, particularly when associated with heat. in tropical regions, depending on the year of cultivation and plant man­ agement, severe disease and pest damage can occur at the harvest stage (subin et al., 2020). in this study, fruit loss in the open field was high, causing a decrease of 78% in the number of fruits in ‘ds0060’, 49% in ‘trucker’ and 75% in ‘thaise’. this resulted from the unfavorable agroclimatic conditions during the growing season. high rainfalls together with heat table 2 ­ total fruit weight per plant, weight of marketable fruits, total number of fruits, number of marketable fruits and total yield of tomato hybrids grown in the open field (o) and protected environments covered with agricultural film (f) and polycarbonate (p) hybrid (h) mean f (anova) cv% ds0060 trucker thaíse a h e x h total fruit weight (kg/plant) o 0.384 ca (z) 0.860 ba 1.192 ba 0.812 c f 2.214 bb 2.636 ab 3.924 aa 2.924 b 54.0 ** 5.91** 0.50 ns 12.7 (y) p 3.424 aa 4.006 aa 4.208 aa 3.879 a mean 2.007 b 2.500 b 3.108 a weight of marketable fruits (kg/plant) o 0.192 ca 0.576 ca 0.572 ba 0.446 c f 1.834 bb 2.256 bb 3.276 aa 2.455 b 66.4 ** 3.60 * 0.55 ns 13.1 (y) p 3.236 aa 3.576 aa 3.900 aa 3.570 a mean 1.754 a 2.136 a 2.582 a total number of fruit (fruits/plant) o 3.60 bb 8.60 ba 12.4 ba 8.20 b f 21.0 ab 27.8 ab 48.2 aa 32.3 a 52.2 ** 14.0 ** 0.86 ns 17.9 (y) p 24.8 aa 36.6 aa 39.4 aa 33.6 a mean 16.4 c 24.3 b 33.3 a number of marketable fruits o 0.80 ba 4.40 ba 3.20 ba 2.80 b f 15.2 ab 20.2 ab 35.0 aa 23.4 a 86.2 ** 9.73 ** 1.51 ns 19.5 (y) p 20.2 ab 30.0 aa 33.4 aa 27.8 a mean 12.1 b 18.2 a 23.8 a total yield (t ha‐1) o 8.44 cb 18.9 ba 26.2 ba 17.8 c f 48.7 bb 58.0 ab 86.2 aa 64.3 b 64.0 ** 7.08 ** 0.61 ns 17.7 (y) p 75.3 aa 88.2 aa 92.5 aa 85.3 a mean 44.1 b 55.1 b 68.3 a (z) means followed by the same uppercase letter in the rows or lowercase letter in the columns do not differ statistically from each other by the scott­knott test at 5%. (y) data transformed into √ y+1. ** p<0.01, * p<0.05, ns= not significant p>0.05. pataraico et al. ‐ yield and quality of tomato hybrids during the amazonian summer 397 are factors that increase the risk of disease incidence in tomato crops (silva et al., 2013). in tropical regions, high pest and pathogen pressure jeopardize production. in this study, 46% of the plants were dec­ imated, and between flowering and the beginning of harvest, the disease severity index had reached a maximum level in all cultivated areas, minimizing the plant yield. when estimating the total yield, ‘thaíse’ cultivat­ ed in environment f or p (86.2 and 92.5 t ha­1, respectively) together with ‘ds0060’ and ‘trucker’ cultivated in environment f (75.3 and 88.2 t ha­1, respectively) were the highest yielding (table 2). these yields are considered high when compared to the hybrids ‘lampião’ (73.0 t ha­1), ‘fascínio’ (68.3 t ha­1), ‘candieiro’ (66.1 t ha­1) and ‘shanty’ (62. 4 t ha­ 1) produced under similar growing conditions as in this study (i.e., protected environment, tropical cli­ mate, high temperatures)(seabra et al., 2022). these results confirm the high yield potential of the evalu­ ated tomato hybrids in the interim growing season, under protected cultivation and at high tempera­ tures. the high yields recorded must be related to the thermotolerance of the evaluated genetic materi­ al, mainly of ‘trucker’ and ‘thaise’, resulting in higher profits for producers. physicochemical and biochemical properties of fruits in response to growing environments tomato color is an important quality property. the fruits grown in the open field had higher values of luminosity (l*), hue angle (h°) and higher b* coor­ dinates, indicating lighter, brighter and more yellow­ ish fruits. on the other hand, the fruits produced in environments f and p had higher a* coordinates, with more reddish fruits (table 4). the fruits of hybrid ‘ds0060’ had higher chromaticity (c*), l*, h° and b* coordinate values, mainly when grown in the open field, also indicating lighter colored fruits, while the a* coordinates of ‘trucker’ and ‘thaise’ were higher, i.e., the reddish fruit color was more intense. these results showed that the adaptation of hybrid table 3 ­ co2 net assimilation rate (a), leaf transpiration rate (e), internal co2 concentration in the substomatal chamber (ci) and stom­ atal conductance (gs) in tomato hybrids grown in open field (o), environments protected covered with agricultural film (f) and polycarbonate (p) (z) means followed by the same uppercase letter in the rows or lowercase letter in the columns do not differ statistically from each other by the scott­knott test at 5%. (y) data transformed into √ y+1. ** p<0.01, * p<0.05, ns= not significant p>0.05. photosynthetic variable hybrid (h) mean f (anova) cv% ds0060 trucker thaíse e h e x h a (µmol co2 m ‐2 s‐1) o ­ ­ ­ ­ f 11.0 aa (z) 9.24 ba 12.9 aa 11.1 a 2.09 ns 3.51 ns 5.67 * 14.4 (y) p 7.67 ab 20.1 aa 14.9 aa 14.2 a mean 9.35 b 14.6 a 13.9 a e (mmol h2o m‐2 s‐1) o ­ ­ ­ ­ f 3.68 aa 5.45 aa 4.96 aa 4.70 a 0.10 ns 3.24 ns 0.11 ns 12.9 p 3.07 aa 5.36 aa 4.86 aa 4.43 a mean 3.37 a 5.41 a 4.91 a ci (µmol mol‐1) o ­ ­ ­ ­ f 349.3 aa 373.7 aa 315.0 aa 346.0 a 3.79 ns 0.28 ns 1.05 ns 6.77 p 306.7 aa 295.0 aa 312.3 aa 304.6 a mean 328.0 a 334.3 a 313.7 a gs (mol m‐2 s‐1) o ­ ­ ­ ­ f 0.326 aa 0.336 ba 0.690 aa 0.451 a 1.74 ns 2.46 ns 1.57 ns 11.4 p 0.290 aa 1.001 aa 0.790 aa 0.696 a mean 0.3 a 0.6 a 0.7 a adv. hort. sci., 2023 37(4): 391­402 398 ‘ds0060’ to high solar radiation and heat was poor, and it should be evaluated in growing seasons with milder temperatures. in this study, the fruits were harvested when more than 90% of the fruit surface had become deeply red (stage 6). the fruit flesh of the tomatoes from the open field was firmer (table 5). of the hybrids, ‘trucker’ had the firmest fruit flesh. this characteristic is relevant with regard to transport resistance, and is influenced by the ripening stage of the fruit, and possibly by the genetic and environ­ mental characteristics of cultivation. in general, the levels of soluble solids were slightly higher in tomatoes from environment f (table 5). among the hybrids, ‘ds0060’ had the highest soluble solids content. these results are similar to those pub­ lished by kwon et al. (2017) who found contents between 5.1 and 5.2°bx, but observed no difference for this variable between cultivation environments. according to the authors, soluble solids may be strongly genetically influenced. ‘lampião’ tomatoes, which are sweeter, contained 4.12°bx and ‘fascínio’ 3.48°bx. according to the above authors, consumers prefer tomatoes with 4.0 to 6.0°bx (domiciano et al., 2021). in this study, all hybrids produced fruits with soluble solids contents above 4°bx. soluble solids in tomato consist mainly of reducing sugars. thus, agro­ nomic factors (i.e., seasonal climate variation, man­ agement practices) that alter the photosynthetic activity, and consequently sucrose synthesis, can modify glucose and fructose accumulation in fruits, and thus the soluble solids contents (yeshiwas et al., 2016). the titratable acidity of the fruits ranged from table 4 ­ chromaticity (c*), luminosity (l*), hue angle (h°), coordinates (a* and b*) of fruits of tomato hybrids grown in open field (o) and protected environments covered with agricultural film (f) or with polycarbonate (p) (z) means followed by the same uppercase letter in the rows or lowercase letter in the columns do not differ statistically from each other by the scott­knott test at 5%. ** p<0.01, * p<0.05, ns not significant p>0.05. variable hybrid (h) mean f (anova) cv% ds0060 trucker thaíse a h a x h c* o 51.1 aa z 50.8 aa 51.1 aa 51.0 a f 52.0 aa 49.8 ab 49.3 ab 50.3 a 1.81 ns 4.10 * 0.90 ns 7.59 p 51.1 aa 49.6 aa 48.8 aa 49.8 a mean 51.4 a 50.0 b 49.7 b l* o 51.9 aa 49.4 ab 45.0 ac 48.8 a f 46.0 ba 42.8 bb 42.3 bb 43.7 b 35.9 ** 28.8 ** 2.06 ns 8.66 p 46.7 ba 43.9 bb 42.7 bb 44.4 b mean 48.2 a 45.4 b 43.3 c h° o 60.7 aa 53.7 ab 44.7 ac 53.0 a f 44.8 ba 40.6 bb 39.7 bb 41.7 b 58.9 ** 31.6 ** 5.27 ** 14.8 p 46.5 ba 43.4 bb 40.8 bb 43.6 b mean 50.7 a 45.9 b 41.8 c a* o 24.9 bc 29.9 bb 36.2 aa 30.3 b f 36.4 aa 37.4 aa 37.8 aa 37.2 a 40.1 ** 18.2 ** 7.74 ** 14.5 p 34.8 aa 35.7 aa 37.0 aa 35.8 a mean 32.0 c 34.3 b 37.0 a b* o 43.8 aa 40.3 ab 35.9 ac 40.0 a f 36.4 ba 32.6 bb 31.4 bb 33.5 b 38.5 ** 25.9 ** 1.00 ns 13.9 p 36.7 ba 34.0 bb 31.9 bb 34.2 b mean 39.0 a 35.7 b 33.1 c pataraico et al. ‐ yield and quality of tomato hybrids during the amazonian summer 399 0.27 to 0.34%. there was a significant interaction between environments and hybrids, resulting in the highest acidity in fruits of ‘ds0060’, grown in the open field, and of ‘thaise’ produced in environment f or p (table 5). these values were lower than the 0.39 to 0.55% reported by scarano et al. (2020), but simi­ lar to the range of 0.22 to 0.32% found by nour et al. (2015). acidity is influenced by the moment of fruit harvest and possibly also by genetic characteristics of the hybrids. the sweetness acidity ratio or relation­ ship between sweetness and acidity, called ripening index, determines the taste, indicating a mild or acid flavor. fruits with an index equal to or greater than 10 are considered ideal for consumption (kader and stevens, 1978). the index in this study exceeded 13, reaching 17.3 in ‘ds0060’ fruits from environment f (table 5). for ‘ds0060’ and ‘trucker’, the ripening index in open field cultivation was lower. however, due to the high soluble solids level, all environments and hybrids produced fruits with good market accep­ tance, i.e., a high ripening index, mainly due to the determined moment of harvest, when more than (z) means followed by the same uppercase letter in the rows or lowercase letter in the columns do not differ statistically from each other by the scott­knott test at 5%. (y) data transformed into √ y+1. ** p<0.01, * p < 0.05, ns not significant p > 0.05. table 5 ­ fruit flesh firmness, soluble solids, tritable acidity, ripening index, lycopene and β­carotene contents in fruits of tomato hybrids grown in open field (o) and protected environments covered with agricultural film (f) or with polycarbonate (p) variable hybrid (h) mean f (anova) cv% ds0060 trucker thaíse a h axh fruit flesh firmness (n) o 9.16 ab (z) 12.4 aa 9.64 ab 10.4 a f 7.00 bb 8.32 ba 6.08 bb 7.13 b 30.0 ** 12.1 ** 1.10 ns 37.9 p 7.00 ba 7.92 ba 5.84 ba 9.62 b mean 7.72 b 9.56 a 7.18 b soluble solids (°bx) o 4.46 ba 4.00 bb 4.06 bb 4.17 c f 5.20 aa 4.66 ab 4.60 ab 4.82 a 20.5 ** 11.2 ** 0.90 ns 10.7 p 4.60 ba 4.46 aa 4.26 ba 4.44 b mean 4.75 a 4.37 b 4.31 b tritable acidity (%) o 0.340 aa 0.300 ab 0.280 bb 0.306 a f 0.300 bb 0.273 bb 0.333 aa 0.302 a 1.32 ns 4.88 ** 8.6 ** 12.8 p 0.300 bb 0.306 ab 0.340 aa 0.315 a mean 0.313 a 0.293 b 0.317 a ripening index (ss/ta) o 13.1 ca 13.3 ba 14.5 aa 13.6 b f 17.3 aa 16.5 aa 12.9 bb 15.6 a 27.4* 12.1* 17.8* 12.3 p 15.3 ba 15.5 aa 13.5 aa 14.8 a mean 15.2 a 15.1 a 13.6 b lycopene (z) (mg 100 g‐1) o 0.395 bb 0.581 ca 0.669 ca 0.548 c f 0.861 ac 1.127 ab 1.467 aa 1.150 a 114.7 ** 33.9 ** 5.36 ** 11.3 (y) p 0.751 aa 0.782 ba 0.833 ba 0.788 b mean 0.669 c 0.828 b 0.989 a β‐carotene (mg 100 g‐1) o 0.378 bb 0.509 ca 0.574 ca 0.487 c f 0.727 ac 0.953 ab 1.231 aa 0.907 a 105.0 ** 35.0 ** 3.91 ** 10.8 (y) p 0.639 ab 0.681 bb 0.769 ba 0.695 b mean 0.581 c 0.714 b 0.856 a adv. hort. sci., 2023 37(4): 391­402 400 90% of the fruit surface had become deeply red, ideal for marketing of the product in the region. regarding the carotenoid content of the fruits, the lycopene and β­carotene contents were higher in environment f and lower in open­field tomato (table 5). this result can be explained by the high incidence of solar radiation on the plants (fig. 1b). the contents were superior to those reported for saladette tomato produced in a protected environment at high tem­ peratures, ranging from 0.3 to 0.82 mg 100 g­1 for lycopene and 0.06 to 0.09 mg 100 g­1 for β­carotene (domiciano et al., 2021). among the hybrids, ‘thaise’ contained the highest and ‘ds0060’ lowest levels of these two compounds. the different lycopene con­ tents in the hybrids can be attributed to genetic char­ acteristics, climate, location, cultivation method and fruit ripening stage. according to nour et al. (2015), carotenoid synthesis, mainly of lycopene, is influ­ enced by the genetic characteristics of adaptability to the agroclimatic conditions of the cultivation environ­ ment. significant positive correlations were observed between the a* coordinate (table 4) and lycopene (r = 0.743) and β­carotene (r = 0.742) levels. on the other hand, correlations were negative between the content of these carotenoids and l* (r = ­0.793, r = ­ 0.810), h° (r = ­0.789, r = ­0.798) and b* coordinate (r = ­0.818, r = ­0.837). these results are similar to those reported by nour et al. (2015), who found a negative correlation of the l* value with the lycopene content in tomato. in this study, open­field fruits of ‘ds0060’ also had mean l* and lower lycopene and β­carotene levels (tables 4 and 5). for ‘belladona’, the chromaticity values were related to the carotenoid contents, except for lycopene (papaioannou et al., 2012). however, this informa­ tion was not confirmed by the results of this study, where the chromaticity values were weakly correlat­ ed with lycopene (r = ­0.526) and β­carotene (r = ­ 0.579) contents. considering all study variables, pca analysis grouped ‘thaise’ grown in environments f and p and ‘trucker’ grown in environment p on the pc1+, corre­ sponding to 65.84% of the data, with the best results in terms of yield as well as quality characteristics (fig. 2). hybrids ‘ds0060’, ‘trucker’ and ‘thaise’ cultivated in the open field (pc1­) produced low yields, fruits with undesirable color and very firm fruit flesh due to the environmental conditions that stressed the plants, causing high yield losses and physiological dis­ orders. the hybrids ‘ds0060’ and ‘trucker’ cultivated in environment f were grouped in pc2+, with low acidity and high fruit ripening indices, probably due to low heat tolerance. these results reinforce the rel­ evance of cultivation in protected environments to warrant high tomato yield during the hot and humid summers of the amazon region. equally important is the selection of hybrids adapted to high radiation and high temperatures, which are fairly common under these cultivation conditions. protected environments were indispensable to achieve high yields under the unfavorable agrocli­ matic conditions (i.e., high rainfall, high solar radia­ tion, high temperatures) of the southern amazon region in the interim crop season (january to april). the protected environment covered with polycar­ bonate panels considerably increased the fruit yield of the tomato hybrids evaluated, indicating it as a good alternative for tomato cultivation in tropical regions. in protected environments covered with agricultural film or polycarbonate panels, hybrid ‘thaise’ produced high yields with good quality char­ acteristics. ‘trucker’ stood out with the firmest fruit flesh and lowest acidity. however, ‘thaise’ had the highest lycopene and β­carotene levels, a character­ fig. 2 ­ two­dimensional projection and score of productive characteristics (yield; plant production—pp commercial plant production—cpp; number of fruits – nf, number of commercial fruits – ncf, chroma ­ c*, luminosity ­ l*, hue angle ­ h°, coordinates ­ a* and b*, fruit firmness – fp, soluble solids ­ ss, titratable acidity – ta, ripening index ­ ri, lycopene and β­carotene) of tomato cultivars (ds0060, trucker, and thaíse) in response to different environments – open field (o), agricultural film (f), and polycarbonate. pataraico et al. ‐ yield and quality of tomato hybrids during the amazonian summer 401 istic that appeals to more demanding consumers, since these compounds are strongly related to dis­ ease prevention, and their presence in the human diet is essential. acknowledgements the authors are grateful to national council for scientific and technological development (cnpq ­ brazil, project no. 405931/2021­2) and research support foundation of the state of mato grosso (fapemat ­ brazil, project no. 0588913/2016). references ali m.y., sina a.a.i., khandker s.s., neesa l., tanvir e.m., kabir a., khalil m.i., gan s.h., 2021 ­ nutritional composition and bioactive compounds in tomatoes and their impact on human health and dis‐ ease: a review. ­ foods, 10(1). bazgaou a., fatnassi h., bouhroud r., gourdo l., ezzaeri k., tiskatine r., demrati h., wifaya a., bekkaoui a., aharoune a., bouirden l., 2018 ­ an experimental study on the effect of a rock‐bed heating system on the microclimate and the crop development under canarian greenhouse. ­ sol. energy, 176: 42­50. beckmann m.z., duarte g.r.b., de paula v.a., mendez m.e.g., 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tecnol., 3(2): 13­19. yeshiwas y., belew d., tolessa k., 2016 ­ tomato (solanum lycopersicum l.) yield and fruit quality attrib‐ utes as affected by varieties and growth conditions. ­ world j. agric. sci., 12(6): 404­408. zenebon o., pascuet n.s., tigela p., 2005 ­ physicochemical methods for food analysis. ‐ 4th ed. instituto adolfo lutz, são paulo, brazil. impaginato 111 adv. hort. sci., 2023 37(1): 111­116 doi: 10.36253/ahsc­13911 application of computer vision systems for assessing bergamot fruit external features s. benalia (*), v. calogero, m. anello, g. zimbalatti, b. bernardi dipartimento di agraria, università degli studi mediterranea di reggio calabria, località feo di vito, snc, 89122 reggio di calabria (rc), italy. key words: aspect ratio, citrus x bergamia risso & poiteau, citrus colour index (cci), dimensions, hunterlab, imaging, rgb. abstract: bergamot citrus x bergamia risso & poiteau is an emblematic citrus species of reggio calabria province (southern italy) where more than 90% of the global production thrives. the present work deals with the use of a non­ destructive technique based on a computer vision system to evaluate bergamot fruit peel colour, as well as dimensional features. to this purpose, experimental trials considered three bergamot cultivars, namely ‘femminello’, ‘castagnaro’ and ‘fantastico’. bergamot fruit rgb images were taken using a laboratory inspection chamber equipped with a lighting system and a digital camera nikon d5200 directly connected to a personal computer, to enable remote image acquisition. first, images were pre­processed according to a previously created colour profile. after that, bergamot fruit colour was analysed and expressed in terms of hunter l, a, and b coordinates, which were used to calculate standard citrus colour index (cci). in addition, dimensional features and shape descrip­ tors were measured for each cultivar. statistical data analysis, by applying the kruskal­wallis test at p<0.05 on cci data highlighted significant differences between the assessed cultivars, and discriminant analysis (lda) applied on cci and dimensional features enabled a classification rate of 78.86% between culti­ vars, proving the reliability of computer vision techniques in assessing berg­ amot external features. 1. introduction bergamot citrus x bergamia risso & poiteau (pellegrino et al., 2015) is an emblematic citrus species of reggio calabria province (southern italy), where more than 90% of the global production is located. until recently, the production has been exclusively destinated to peel essential oil extraction and subsequent use in perfumery, cosmetic and pharmaceutic preparations, food flavouring and confectionery (navarra et al., 2015; gioffrè et al., 2020). however, the rising interest toward bergamot­based food and beverage for their high content in functional bioactive com­ pounds is directing the production toward other market channels. this trend is also accompanied by the necessity of agrifood industries to devel­ (*) corresponding author: soraya.benalia@unirc.it citation: benalia s., calogero v., anello m., zimbalatti g., bernardi b., 2023 ­ application of computer vision systems for assessing bergamot fruit external features. ­ adv. hort. sci., 37(1): 111­116. copyright: © 2023 benalia s., calogero v., anello m., zimbalatti g., bernardi b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 7 april 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13911 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 111­116 112 op particularly from a technological point of view, to keep up and adequately address global market requirements and standards. in fact, great attention has been paid, in the last years, to the development and implementation of non­destructive techniques based on artificial intelligence in industrial processes including agrifood sector, for their reliability, accura­ cy, and timeliness in determining qualitative parame­ ters of the product, especially when employed in automated post­harvest processes. several studies dealt with the use of computer vision systems for cit­ rus fruit quality assessment or grading (cubero et al., 2014; lorente et al., 2015; zhang et al., 2020; riccioli et al., 2021; zhang et al., 2022), but up to now, no one regarded bergamot fruits. in this context, the present work aims at assessing fruit peel colour, expressed in terms of standard citrus colour index (cci) as well as dimensional features and shape descriptors, including fruit area, fruit perimeter, major axis, minor axis, circularity, roundness, aspect ratio and solidity of three cultivars of bergamot fruit, by means of computer vision systems. 2. materials and methods bergamot samples experimental trials considered three bergamot cultivars, namely: citrus x bergamia risso & poiteau, ‘femminello’, ‘castagnaro’ and ‘fantastico’. fruits were collected at the end of their ripening stage (since mid­january to the beginning of february) in private farms located in reggio calabria province. a total of 248 fruits were used. methodology set‐up and rgb image acquisition bergamot rgb image acquisition was performed using a laboratory squared inspection chamber equipped with a digital camera nikon d5200 directly connected to a personal computer, to enable remote image acquisition, and a lighting system including 4 fluorescent linear lamps (biolux t8­ 18w/965, 6500 k) placed on the top of the inspection chamber in a 0°/45° configuration as shown in figure 1. the used camera was equipped with a cmos 23.5 x 15.6 mm sensor, allowing getting a resolution of 24.1 million pixels; and a nikkor f/2.8g lens with a 1:1 reproduc­ tion ratio. prior to image acquisition, several configurations of operational parameters were tested until getting the most appropriate one, which considered the fol­ lowing parameters: iso: 100; exposition time: 1/125; diaphragm opening f: 5.6; 4 lamps (instead of 8), without polarizing filter. image analysis and bergamot colour determination bergamot images were calibrated, first, by per­ forming the white balance, and subsequently the chromatic correction according to a previously creat­ ed profile with the colorchecker classic target (x­rite inc., usa), through the colorchecker passport software as performed in benalia et al. (2017). bergamot rgb images were then analysed as per­ fomed by benalia et al. (2016) using a specific appli­ cation (fig. 2) that enables to convert mean r, g, b values of all pixels contained in a selected region of interest (roi) into l, a, b coordinates considering the cie illuminant d65 and the 10° observer standard (cubero et al., 2018). besides and for comparison purpose, bergamot fig. 1 ­ bergamot rgb image acquisition using a laboratory inspection chamber equipped with a computer vision system. fig. 2 ­ bergamot colour determination through rgb image anal­ ysis. benalia et al. ‐ computer vision systems (cvs) for bergamot fruit characterization 113 fruit colour was determined by means of a portable konica minolta cm­700d spectrophotometer being the only widely standardized instrument used for instantaneous food colour evaluation in agrifood industries. to this end, six measurements were car­ ried out randomly on each fruit. determination of dimensional features and shape descriptors dimensional features and shape descriptors, including fruit area in the image, fruit perimeter, major axis, minor axis, circularity, roundness, aspect ratio and solidity were determined using the soft­ ware imagej 1.50i using java 1.8.0.77 (fig. 3), accord­ ing to a previously set scale. data analysis hunter l, a and b values were used to calculate the standard citrus colour index (cci) according to the following formula (eq. 1) and to compare whether there is a difference between the examined cultivars. cci = 1000 a/(l·b) (eq. 1) with l value indicating lightness (0 = darkness ­> 100 = lightness), a value representing red (positive value) or green (negative value); and b representing yellow (positive value) or blue (negative value). cci data were statistically analysed using the open­source application r (version 3.3.1). hence, data normal distribution was checked by applying the shapiro­wilk normality test, and according to the results, the most appropriate test was applied to compare the examined cultivars in terms of colour features. furthermore, cci data as well as dimensional fea­ tures and shape descriptors retrieved from bergamot rgb image analyses were used to build an exhaustive dataset and multivariate image analyses (mia), par­ ticularly, principal component analysis (pca) and discriminant analysis (lda) were applied using past 4.12b (hammer et al., 2001). 3. results and discussion citrus colour index (cci) values calculated from bergamot image analysis were compared with those obtained using the portable spectrophotometer for each cultivar. the obtained results are shown in the following figure 4. particularly, the coefficient of fig. 4 ­ standard citrus colour index (cci) from image analysis vs portable spectrophotometer. a: ‘castagnaro’, b: ‘fantastico and c: ‘femminello’. fig. 3 ­ bergamot dimensional feature and shape descriptor calculation through rgb image analysis using imagej. adv. hort. sci., 2023 37(1): 111­116 114 determination r2 was equal to 0.83, 0.87 and 0.90 respectively for the cultivars castagnaro, fantastico and femminello, making it possible to substitute colour analysis using conventional instruments with that based on image analysis. indeed, diversely from the portable spectrophotometer, which offers the possibility to only analyse a small area of the fruit surface, image analysis allows assessing fruit colour of the whole surface of each fruit. the obtained r2 values in this study are, however, lower than those reported by vidal et al. (2013), who tested two algorithms for the estimation of oranges cv. navelina cci based on computer vision. this dif­ ference could mainly be attributed to the difference in the used hardware and operation parameters. indeed, up to now, a standardized methodology con­ cerning the use of cvs is still missing. the results of shapiro­wilk test demonstrated that cci data do not follow a normal distribution. therefore, the non­parametric test kruskal­wallis was applied. the outputs highlighted a significant difference between the cultivars in terms of cci at p<0.05 (table 1). cci mean and median values are reported in table 2. cci mean values were equal to 3.01±1.51 for cv. castagnaro and 2.36±2.26 for cv. femminello, indicating a yellowish colour, while it was much lower for cv. fantastico with a value of 1.21±1.52 cor­ responding to a greenish colour yet (table 2). as mentioned above, multivariate image analysis, particularly, principal component analysis (pca) and discriminant analysis (lda) were applied considering cci values as well as dimensional features and shape descriptors. hence, the three cultivars were com­ pared according to 9 variables. pca highlighted that most of the variability was expressed by the first two components, with respectively 46.86% for pc1 and 21.38% for pc2. the first component pc1 is mostly and positively influenced by fruit area, fruit perime­ ter as well as by major and minor axis. to this regard, giuffrè (2019) and giuffrè and nobile (2020) found significant differences between the bergamot culti­ vars when comparing both major and minor axes. however, diversely from our method they used con­ ventional measuring method. the first component pc1 is also negatively influenced by roundness. the second component pc2 is, however, positively, and mainly influenced by roundness and subsequently solidity, while it is negatively affected by cci and aspect ratio (fig. 5). a better discrimination between the cultivars was obtained when lda was applied (fig. 6), and 80.49% of correctly classified fruit according to cultivar was reached. this rate decreases to 78.86% when jackknif cross­validation method is applied (table 3). this study focuses on the use of computer vision system to retrieve colour and dimensional features with the aim of distinguishing between three culti­ vars grown in the province of reggio calabria. as pre­ viously stated, no work dealing with this theme have table 1 ­ results of kruskal­wallis test for cci according to the cultivar table 2 ­ mean and median values of cci according to the culti­ var test χ2 df p kruskal­wallis: (cci by cultivar) 46.55 2 7.793e­11 cultivars mean values ± st dev. median values castagnaro 3.01 ± 1.51 3.05 fantastico 1.21 ± 1.52 1.57 femminello 2.36 ± 2.26 2.61 fig. 5 ­ pca score plot. green filled square: ‘femminello’, purple filled triangle: castagnaro, yellow dots: fantastico. fig. 6 ­ lda plot. green filled square: femminello, purple filled triangle: castagnaro, yellow dots: fantastico. benalia et al. ‐ computer vision systems (cvs) for bergamot fruit characterization 115 been performed so far. to our knowledge the only one, which reports the characterization of bergamot physical properties using cvs is that of rafiee et al. (2007), who measured fruit size, mass, projected area, fruit density, solid density, bulk density, bulk porosity, packing coefficient, density ratio, geometric diameter, sphericity and surface area of citrus medi‐ ca, which characteristics are different from citrus x bergamia risso & poiteau. 4. conclusions the obtained results in this study confirm the reli­ ability of computer vision systems in characterizing bergamot fruit external properties, considering par­ ticularly, colour and dimensional features. although preliminary, the outputs clearly demonstrate the effi­ ciency and accuracy of these techniques in distin­ guishing the examined cultivars, promoting therefore their implementation in bergamot post­harvest pro­ cesses. indeed, both features, i.e., colour and dimen­ sions are important to be considered for the develop­ ment of new technologies as they represent impor­ tant qualitative indicators. further research activities should be performed to validate findings obtained in this study, and to inves­ tigate other aspects using cvs such as ripening progress and essential oil peel content. acknowledgements ii level master course for expert technician on “the bergamot of reggio calabria. production and enhancement “organized by the department of agriculture of the university mediterranean of reggio calabria, in collaboration with reggio calabria metropolitan city. national operational programme (nop) on research and innovation 2014­2020 ­ axis i “investment in human capital”­ action i.1 ­ innovative industrial doctorates ­ cycle xxxvi. references benalia s., bernardi b., blasco j., fazari a., zimbal­ atti g., 2017 ­ assessment of the ripening of olives using computer vision. ­ chem. eng. trans., 58: 355­ 360. benalia s., cubero s., prats­montalbán j.m., bernardi b., zimbalatti g., blasco j., 2016 ­ computer vision for automatic quality inspection of dried figs (ficus carica l.) in real‐time. ­ comput. electron. agric., 120: 17­25. cubero s., albert f., 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jackknife cross validation results resulting from dis­ criminant analysis (lda) given groups predicted groups total femminello castagnaro fantastico femminello 99 8 3 110 castagnaro 13 42 14 69 fantastico 0 14 53 67 116 adv. hort. sci., 2023 37(1): 111­116 agrophysics, 21: 293­297. riccioli c., perez­marin d., garrido­varo a., 2021 ­ optimizing spatial data reduction in hyperspectral imaging for the prediction of quality parameters in intact oranges. ­ postharvest biol. technol., 176. vidal a., talens p., prats­montalbán j.m., cubero s., albert f., blasco j., 2013 ­ in‐line estimation of the standard colour index of citrus fruits using a computer vision system developed for a mobile platform. ­ food bioprocess technol., 6: 3412­3419. zhang h., zhan b., pan f., luo w., 2020 ­ determination of soluble solids content in oranges using visible and near infrared full transmittance hyperspectral imaging with comparative analysis of models. ‐ postharvest biol. technol., 163: 111148. zhang x., xun y., chen y., 2022 ­ automated identifica‐ tion of citrus diseases in orchards using deep learning. ­ biosyst. eng., 223, part a, pp. 249­258. impaginato 495 adv. hort. sci., 2019 33(4): 495­501 doi: 10.13128/ahsc­8127 effects of foliar application of glycine and glutamine amino acids on growth and quality of sweet basil y. aghaye noroozlo 1, m.k. souri 1 (*), m. delshad 2 1 department of horticultural sciences, faculty of agriculture, tarbiat modares university, tehran, iran. 2 departments of horticulture, faculty of agriculture, university of tehran, karaj‐iran. key words: ascorbic acid, biofortification, biostimulation, fertilization, foliar feeding, nutrient uptake, proline. abstract: amino acids have diverse roles in plant metabolism, and recently amino acid based fertilizers have been used largely in crop production systems. despite most of these new fertilizers are formulated for foliar application; how­ ever, morphophysiological responses of crops to amino acids application have not yet been well documented. in the present study, foliar application of glycine or glutamine in different concentrations of 0 (distilled water), 250, 500, 1000 mg·l­1, as well as a treatment of 250 mg·l­1 glycine + 250 mg·l­1 glutamine were evaluated on growth of sweet basil (ocimum basilicum l.) plants. the results showed that foliar application of glycine or glutamine at 1000 mg·l­1 showed no improvement in comparison with control for all traits except for leaf l­ascorbic acid concentration that showed the highest value under these amino acids treatments. however, foliar application of these amino acids at 250 and particularly 500 mg·l­1 showed promising effect on sweet basil growth. plant shoot fresh and dry weight, leaf area, leaf spad value, and leaf chloro­ phyll content were improved by foliar application of 500 mg·l­1 glycine or gluta­ mine in comparison with control plants. foliar application of amino acids increased leaf nitrogen (glutamine 250 and 500 mg·l­1), potassium (glycine 250 mg·l­1), magnesium (glutamine 250 or 500 mg·l­1, glycine 250 mg·l­1, glycine + glutamine; 250 + 250 mg·l­1), iron (glycine and glutamine at 500 mg·l­1 and glut­ amine at 250 mg·l­1), and zinc (glycine and glutamine at 250 or 500 mg·l­1), whereas the increase in leaf nutrients caused by other treatments was not sig­ nificantly different from control plants. leaf calcium concentration was not changed by amino acid treatments. the results indicate that foliar application of moderate to low concentrations of glutamine or glycine can improve sweet basil growth. 1. introduction basil (ocimum basilicum l.) is a high marketable value vegetable, which is consumed as a fresh aromatic ingredient and included in cooked dishes. increasing importance is being given to this produce due to its remarkable quality and nutritional features (morano et al., 2017). (*) corresponding author: mk.souri@modares.ac.ir citation: noroozlo y.a., souri m.k., delshad m., 2019 ­ effects of foliar application of glycine and glutamine amino acids on growth and quality of sweet basil. ­ adv. hort. sci., 33(4): 495­501. copyright: © 2019 noroozlo y.a., souri m.k., delshad m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 december 2018 accepted for publication 8 july 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 495­501 496 chemical fertilizers have an inevitable role in food production of agricultural crops; however, during last decades the soil fertility and quality have been adversely affected by many fertilization strategies (souri and hatamian, 2019). this is mainly due to the low efficiency of applied fertilizers which can even cause yield reduction upon increasing supply (ercolano et al., 2015). in some situations and partic­ ularly under adverse climatic conditions, foliar instead of soil application (golubkina et al., 2018), or chelated forms instead of simple chemical forms of fertilizers (garcia et al., 2011; marschner, 2012; souri and aslani, 2018) can improve nutrient uptake and use efficiency of applied fertilizers. each year, agricul­ ture production needs a large amount of expensive nitrogenous fertilizers that generally have low uptake efficiency rates and result in both plant (caruso et al., 2011) and environmental pollution (souri and hatamian, 2019). amino acids are being incorporated for more than two decades into fertilizer formula­ tions to improve fertilizers use efficiency (abdul­ qados, 2009; souri, 2016). foliar application of amino acids can have benefi­ cial effects on plant growth and production (sadak et al., 2015; shams et al., 2016; hussain et al., 2018; souri and aslani, 2018). it has been shown that foliar application of arginine amino acid has improved shoot growth and grain yield of wheat in saline and non saline conditions (abdul­qados, 2009; rizwan et al., 2017). better growth and higher biomass produc­ tion via application of various amino acid chelates of iron or zinc has been also reported on some agro­ nomic and horticultural crops (khan et al., 2012; el sayed et al., 2014). similarly, foliar application of a mix of amino acids at 500 and 700 ppm increased plant height, plant fresh and dry weights, leaf n con­ centration, yield of leaves and leaf soluble carbohy­ drates in celery (shehata et al., 2011). glycine is the simplest amino acid and is used largely for production of chelated fertilizers in form of aminochelates (souri, 2016), whereas effect of foliar application of glutamine has not been well doc­ umented so far. in the present study, the effect of foliar application of different concentrations of glycine and glutamine has been evaluated on growth characteristics of sweet basil plants. 2. materials and methods this study was conducted under greenhouse con­ ditions and from 20 may till the end of july 2017. the soil used in experiment had a loamy texture and moderate levels of nutrients. the soil physiochemical characteristics are presented in table 1. 4 l black plastic pots (30 cm height and 20 cm diameter) were filled with about 4 kg dry soil and watered at 80% of its soil field capacity (fc). three days later 40 seeds of sweet basil (ocimum basilicum l.) from a local population (karaj landrace) were sown in 1 cm depth of the soil. two weeks after germination plants were reduced to 10 uniform seedlings per pot. the plants were irrigated daily based on 80% soil field capacity. the temperature was on average 28±5°c, the light intensity 200 µmole.m­2.s­1, and the air humidity about 70­75%. different concentrations of two amino acids of glycine and glutamine including 0, 250, 500 and 1000 mg·l­1, as well as a treatment of 250 mg·l­1 glycine + 250 mg·l­1 glutamine were sprayed on sweet basil plants. treatments and pots were arranged in com­ pletely randomized design and each treatment con­ tained three replications. each pot represented one replication and contained 10 plants. each concentra­ tion of glycine or glutamine was sprayed on plant leaves five times during two­month growth period, and the first spray was done two weeks after seedling emergence. the remaining sprays were done in one week interval. distilled water was sprayed on control plants. spray treatments were done in the early morning, one hour after sunrise and with a portable sprayer by which the upper and lower surface of leaves were sprayed. a total amount of 80­90 ml solution of each treatment was sprayed on plants per pot. plants were harvested two months after emer­ gence and before flowering stage. the average height of ten plants in each pot was measured by a ruler before harvest. the leaf spad value was mea­ sured by a portable spad meter (model spad­502 texture ec (ds.m­1) ph o.c. (%) total n (mg.kg­1) available p (mg.kg­1) available k (mg.kg­1) fe (mg.kg­1) zn (mg.kg­1) lime (%) loamy­sand 1.79 7.62 0.78 750 11.61 385 5.5 1.6 7.3 table 1 ­ physico­chemical characteristics of soil used for the experiment noroozlo et al. ‐ effects of glycine and glutamine amino acids on sweet basil 497 plus illinois, usa) performing 30 readings (on middle part of leaves) per pot. the plant leaf area of three randomly selected plants per pot was measured by leaf area meter (delta­t devices ltd, england). plants were cut at soil surface and shoot fresh weight was measured by a precise digital scale. fresh shoots of each pot were transferred to an oven at 65°c for 48 h, and thereafter plant dry weight was measured accordingly. leaf chlorophyll and carotenoids were determined using acetone extraction of 0.5 g fresh leaves following the methods of khan et al. (2012) and kałużewicz et al. (2018). for determination of leaf l­ascorbic acid (vitamin c), 5 g of fresh leaf tissue were crushed in a porcelain mortar in 10 ml metaphosphoric acid 6%, and then the juice was transferred into a 25 ml tube and centrifuged at 4000 rpm for 10 min. 5 ml of the supernatant was transferred into an erlenmeyer flask, and added with 20 ml of metaphosphoric acid 3%. the extract was then titrated using di­chloro phenol indophenol reagent until appearance of a rosa color. the amount of vitamin c (mg 100 g­1 fw) was calculated accord­ ing to a standard curve of l­ascorbic acid concentra­ tions of 0, 25, 50, 2100 and 200 mg·l­1 following souri and aslani (2018). nitrogen concentration of leaves was determined by the kjeldahl method, k using flame photometry, mg, ca, fe and zn by atomic absorption spectrophotometer. data were analyzed by analysis of variance using spss 16 and comparison of means was performed using lsd test at p≤0.05 probability level. 3. results and discussion in the present study a soil with moderate levels of nutrient elements was used and no further applica­ tion of chemical fertilizers was applied. the results showed that plant height was significantly increased by foliar application of 250 mg·l­1 glutamine com­ pared to control plants (table 2). application of both amino acids of glycine and glutamine significantly increased the leaf concentration of chlorophyll a (at 250 or 500 mg·l­1) and chlorophyll b (at 500 mg·l­1) compared to control plants (table 2). total chloro­ phyll concentration in leaves was significantly increased than control by glutamine or glycine at 500 mg·l­1 or glutamine at 250 mg·l­1 (table 2). leaf carotenoids concentration increased and decreased upon foliar application of 1000 mg·l­1 glycine and 1000 mg·l­1 glutamine compared to control plants, respectively (table 2). the plant leaf spad value (fig. 1) was not significantly changed under different con­ centrations of glutamine or glycine compared to con­ trol plants. however, foliar application of 1000 mg·l­1 glycine reduced leaf spad value of plants compared to some amino acid treatments (fig. 1). this could be due to damage of high glycine concentration to basil leaves, as the same finding was also reported by fig. 1 ­ leaf spad value of sweet basil plants under foliar appli­ cation of glycine or glutamine amino acids. comparison of means was performed at p≤0.05 probability level of lsd test. table 2 ­ plant height and leaf pigments concentration of sweet basil plants under foliar application of glycine and glutamine amino acids ­ 250, 500 and 1000 indicates their concentration as mg·l­1. ­ comparison of means was performed at p≤0.05 probability level of lsd test. treatments plant height (cm) chl a (mg.g­1 fw) chl (mg.g­1 fw) total chl (mg.g­1 fw) carotenoids (mg.g­1 fw) control (distilled water) 46±5 bc 10.5±1.4 d 5.6±0.8 b 16.1±1.3 bc 1.7±0.3 b gly1000 44±4 c 11.9±3.2 cd 6.0±1 b 18.0±4.1 bc 2.3±0.1 a glu1000 43±10 c 10.3±1 d 5.2±0.9 b 15.5±1.4 c 1.2±0.1 c gly500 55±15 abc 18.3±1.3 ab 9.0±2 a 27.3±3.4 a 1.5±0.2 bc glu500 56±10 ab 19±2.4 a 9.2±1.3 a 28.2±3.6 a 1.4±0.1 bc gly250 53±11 abc 17.4±0.7 ab 7.8±0.9 ab 25.2±1.4 a 1.6±0.5 b glu250 62±13 a 15.2±2.7 bc 5.0±0.9 b 20.2±3.6 b 1.4±0.3 bc gly250+glu250 50±30 b 13.1±0.5 c 5.2±3.7 b 18.3±3.6 bc 1.5±0.3 bc adv. hort. sci., 2019 33(4): 495­501 498 fahimi et al. (2016) on cucumber plants. the increase in plant height and leaf greenness can be due to ben­ eficial effects of glycine and particularly glutamine on leaf pigmentation (sadak et al., 2015; fahimi et al., 2016). amino acids can have stimulation effect on plants growth, and in this respect, it has been shown that application of amino acids can enhance chloro­ phyll content and leaf greenness of plants (kielland, 1994; souri et al., 2017; kałużewicz et al., 2018), probably due to higher protein biosynthesis and reduction in chlorophyll degradation rates (rainbird et al., 1984; souri and hatamian, 2019). better nutri­ ents use efficiency and their enhanced translocation play also an important role in this regard (marschner, 2012). application of aminolevulinic acid (ala), as a common precursor to tetrapyrrole compounds found in chlorophyll and hemes, improved spinach plant growth and increased the concentration of photosyn­ thetic pigments (smoleń et al., 2010). in the present study, plant leaf area (fig. 2) was increased by foliar application of both glycine and glutamine at 500 mg·l­1 compared to control plants. both glycine and glutamine at either concentration of 250 or 500 mg·l­1 increased shoot fresh weight (fig. 3); whereas plant shoots dry weight was increased only under foliar application of 500 mg·l­1 glycine or glutamine compared to control plants. leaf area expansion is a function of interacting various external and internal factors that results in increased cell divi­ sion or particularly cell enlargement. various amino acids like glutamine and glycine are required compo­ nents for normal cell growth and expansion (marschner, 2012; souri, 2016). the increase in plant biomass caused by foliar application of moderate lev­ els of glutamine or glycine could be due to the latter stimulation effects on plant growth. the beneficial effects of foliar application of amino acids on plant growth, yield and quality of the product has been shown for some crops (keutgen and pawelzik, 2008; amin et al., 2011; khan et al., 2012; sadak et al., 2015). in addition, amino acids can enhance nutrient uptake and act as precursors for certain plant hor­ mones (marschner, 2012; souri and hatamian, 2019). foliar application of different concentrations of a mix 17 different amino acids after 45 and 60 days from sowing showed promising effects on bean plant growth and productivity under salinity conditions (sadak et al., 2015). a similar effect was also obtained in bean plants under field conditions (souri and aslani, 2018). foliar application of zinc­lysine sig­ nificantly increased the photosynthesis, grain yield, enzyme activities and zn contents in different wheat plant tissues (rizwan et al., 2017). multiple sprays of a mix of amino acids (0.5 ml·l­1) at different growth stages of grapevines particularly at flowering stage, increased growth characteristics and various compo­ nents of fruit yield and quality compared to control (khan et al., 2012). leaf l­ascorbic acid (vitamin c) was not signifi­ cantly affected by foliar application of glycine or glut­ amine compared to control plants; however, the highest leaf ascorbic acid content was recorded in plants treated with 1000 mg·l­1 glycine or glutamine (fig. 4). foliar application of amino acids significantly increased leaf nutrient concentration (except calci­ um) under some specific concentrations of glycine or glutamine (table 3). leaf nitrogen was significantly increased by glutamine spray either at 250 or 500 mg·l­1, whereas leaf potassium concentration was significantly increased by glycine at 250 mg·l­1 com­ pared to control plants. foliar application of gluta­ mine either at 250 or 500 mg·l­1, and foliar applica­ tion of glycine at 250 mg·l­1 significantly increased fig. 2 ­ plant leaf area of sweet basil under foliar application of glycine or glutamine amino acids. comparison of means was performed at p≤0.05 probability level of lsd test. fig. 3 ­ shoot fresh and dry weights of sweet basil plants under foliar application of glycine or glutamine amino acids. comparison of means was performed at p≤0.05 probabi­ lity level of lsd test. noroozlo et al. ‐ effects of glycine and glutamine amino acids on sweet basil 499 adverse climatic conditions. moreover, amino acids or their fertilizer products can be considered as “semi­intelligent fertilizers” (souri, 2016). they are natural ligands with a friendlier effect on environ­ mental issues and best candidates to partially replace fertilizer application, and to relief chemical fertilizers pressure in many cropping systems. amino acids are also a preferential source of nitrogen for plant nutrition. it has been shown that partial replacement of nitrate by amino acid applica­ tion can have beneficial effects on plant growth and production (marschner, 2012; sadak et al., 2015; souri et al., 2017). amino acids are the intermediate compounds in nitrogen assimilation and play key roles in plant cell metabolism, as they are the main form of nitrogen translocation through phloem to growing parts (marschner, 2012; kolota et al., 2013). nitrate in the soil is very vulnerable to leaching and gaseous emissions (kolota et al., 2013); whereas the mandatory application of reduced forms of nitrogen under organic farming (caruso et al., 2012) or the advisable supply of ammonium and amino acids can prevent these effects and reduce nitrate accumula­ tion in plant tissues (cao et al., 2010; souri et al., 2017). total concentration of amino acids and leaf protein concentration significantly increase by foliar application of single or a mix of amino acids (marschner, 2012; sadak et al., 2015). moreover, it has been shown that foliar or soil application of amino acids or their chelated nutrients can improve quality parameters of plants (koksal et al., 1999; keutgen and pawelzik, 2008; shaheen et al., 2010; smoleń et al., 2010; el sayed et al., 2014; souri et al., 2017). application of glutamic acid enhanced some quality parameters of chinese chive plants (cao et al., 2010). similarly, foliar spray of proline and trypto­ phan enhanced growth, yield and fruit quality and leaf magnesium concentration compared to control plants. leaf iron concentration was significantly increased by foliar application of glutamine either at 250 or 500 mg·l­1, or by foliar application of glycine at 500 mg·l­1 compared to control plants. similarly, leaf zinc concentration was significantly increased by foliar application of glycine or glutamine either at 250 or 500 mg·l­1 compared to control plants (table 3). foliar spray of 250­500 mg·l­1 of both amino acids significantly increased leaf nutrient concentrations. the most significant effect of amino acids on plants is reportedly the increased plant nutrient uptake (koksal et al., 1999; abdul­qados, 2009; garcia et al., 2011). various amino acids can act as ligand for nutri­ ents particularly micronutrient metal ions, so they can protect metal nutrients from harmful reactions (souri and hatamian, 2019). the structure of amino acids allows the molecule to act as acid or base, depending on medium ph, thus helping to improve the fertility management of soils particularly under table 3 ­ leaf nutrient concentrations of sweet basil plants under foliar application of glycine and glutamine amino acids ­ 250, 500 and 1000 indicates their concentration as mg·l­1. ­ comparison of means was performed at p≤0.05 probability level of lsd test. treatments n (% dw) k (% dw) mg (% dw) ca (% dw) fe (mg.kg­1 dw) zn (mg.kg­1 dw) control (distilled water) 2.1±0.2 b 1.5±0.2 b 0.21±0.04 d 1.5±0.2 ab 49±6 c 33±3 c gly1000 2.13±0.4 b 1.6±0.1 ab 0.19±0.03 d 1.3±0.4 b 45±7 c 33±2 c glu1000 2.4±0.3 ab 1.5±0.3 b 0.23±0.04 cd 1.5±0.2 ab 52±10 bc 35±2 bc gly500 2.3±0.4 ab 1.7±0.2 ab 0.27±0.04 bcd 1.6±0.2 ab 61±10 ab 40±4 ab glu500 2.7±0.5 a 1.8±0.4 ab 0.36±0.06 a 1.9±0.5 a 66±8 a 42±6 a gly250 2.5±0.2 ab 1.9±0.3 a 0.29±0.05 abc 1.7±0.2 ab 53±6 abc 41±4 ab glu250 2.6±0.3 a 1.8±0.3 ab 0.33±0.04 ab 1.7±0.3 ab 62±5 ab 42±5 a gly250+glu250 2.5±0.3 ab 1.7±0.1 ab 0.30±0.06 abc 1.8±0.1 a 57±9 abc 38±6 abc fig. 4 ­ leaf l­ascorbic acid (vitamin c) concentration of sweet basil plants under foliar application of glycine or glutami­ ne amino acids. comparison of means was performed at p≤0.05 probability level of lsd test. 500 adv. hort. sci., 2019 33(4): 495­501 minimized cracked fruit percentage of pomegranates (el sayed et al., 2014). it has also been shown that under heavy metal pollution of soil, application of amino acids can reduce the heavy metal content of plant tissues (bashir et al., 2018). in the present study, foliar application of gluta­ mine resulted in better growth traits than glycine spraying. this effect can be mainly due to the well­ known role of glutamine in nitrogen assimilation and plant metabolism (marschner, 2012). enhancement of glutamine synthetase has been shown to reduce the severity of ammonium toxicity in tomato plants (forde and clarkson, 1999; marschner, 2012). in addition to the amino and carboxyl groups, amino acids have a side chain or r group attached to the α­ carbon. each amino acid has unique characteristics arising from the size, shape, solubility, and ionization properties of its r group. as a result, the side chains of amino acids exert a profound effect on the struc­ ture and biological activity of proteins (souri, 2016; souri and hatamian, 2019). among various amino acids used as foliar spray on bean plants, it was found that glutamic acids and arginine were the most effective on plant growth biostimulation (sadak et al., 2015). it has been revealed that amino acids are effective components in nutrient use efficiency as well as in detoxification of toxins and heavy metals in plants that can significantly increase their antioxidant capacity and tolerance to stressful conditions. for many decades, soil fertility and quality was adversely affected by continues application of chemical fertiliz­ ers and adverse climatic conditions. fertilization strategies have actively contributed in enhancing soil salinity that nowadays is a global challenge in many countries. biostimulation effect of amino acids on nutrient uptake and plant growth can reduce fertiliz­ er application rates and detrimental effects of fertil­ ization on environment and food quality. however, in the present study low to moderate levels of glycine or glutamine (250­500 mg·l­1) had beneficial effects on sweet basil growth, while both amino acids at 1000 mg·l­1 were less or not effective on plant growth characteristics. 4. conclusions in the present study, the growth of sweet basil plants was improved by foliar application of 250 or 500 mg·l­1 of glycine or glutamine amino acids, whereas application of 1000 mg·l­1 of these two amino acids resulted in no difference from control plants. there were significant better effect on plant height, leaf spad value, leaf area, shoot fresh and dry weights, and nutrient uptake with foliar application of glycine and particularly glutamine at 500 mg·l­1. leaf vitamin c was highest in plants treated with 1000 mg·l­1 amino acid spray. foliar application of glutamine was more effective than glycine on many growth traits and nutrients uptake. our results sug­ gest that amino acids are effective to improve the nutrient uptake and accordingly the plant growth, yield and quality under the view of a modern and sustainable agriculture. references abdul­qados a.m.s., 2009 ­ effect of arginine on growth, yield and chemical constituents of wheat grown under salinity condition. ­ acad. j. plant sci., 2: 267­278. amin a.a., gharib f.a.e., el­awadia m., rashad e.s.m., 2011­ physiological response of onion plants to foliar application of putrescine and glutamine. ­ sci. hortic., 129: 353­360. bashir a., rizwan m., ali s., zia­ur­rehman m., ishaque w., riaz m.a., maqbool a., 2018 ­ effect of foliar‐applied iron complexed with lysine on growth and cadmium (cd) uptake in rice under 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emission and the expression of genes associated with the lipoxygenase (lox) pathway have been studied in harvested peach fruit of two cultivars (‘flaminia’, fl, and ‘red haven’, rh) during and after cold storage. two temperature storage conditions have been applied for two weeks: 0.5 and 5.5°c, the latter recognized to be an inducer factor of chilling injury (ci) of the flesh. fruit were also monitored dur­ ing 3 days of shelf­life (sl) at room temperature after removing from the cold storage. a different behaviour between cultivars has been observed in terms of internal browning (more evident in fl after 2 weeks) and extractable juice (already reduced in rh at the end of 1 week of storage at 5.5°c). although some common responses have been observed (e.g. a general increase of 2­hex­ enal and 2­hexenal­e at the end of both cold storage conditions), lox pathway­ associated volatiles (aldehydes, alcohols, esters) showed different trends in relation to the genotype and the applied stress, with apparently no specific cor­ relations with the incidence of ci. the expression level of five lox pathway­ associated genes (pplox1, pplox4, pphpl1, ppadh1, ppaat1) have been anal­ ysed and the results point out that a genotype­dependent behaviour is present, but specific responses (up­regulation of pplox1 and ppaat1 during sl) appear to be present in both cultivars. in addition, the expression of two c‐repeat‐ binding factors (ppcbf1 and ppcbf6), recognized to be involved in the respons­ es of plant tissues to low temperature stress, showed marked changes in rela­ tion to the applied temperature, suggesting that these genes might play a regu­ latory role in the overall metabolism of cold stored peaches. 1. introduction peach fruit are highly perishable at ambient temperature and they are normally stored under refrigeration, making possible to maintain com­ mercial quality up to 2­4 weeks, depending on the specific cultivars and (*) corresponding author: stefano.brizzolara@santannapisa.it citation: brizzolara s., modesti m., rong x., tonutti p., 2020 ­ volatile compound and gene expression profiles associated with the storage of two peach fruit genotypes differently sensitive to chilling injuries. ­ adv. hort. sci., 34(1s): 97­108 copyright: © 2020 brizzolara s., modesti m., rong x., tonutti p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 february 2020 accepted for publication 13 july 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 97­108 98 storage conditions (ramina et al., 2008). however, low temperatures may lead to the development of chilling injury (ci), which negatively affect fruit quali­ ty shattering important organoleptic quality traits such as taste and flavor, as well as inducing the appearance of physiological disorders such as inter­ nal browning and mealiness/woolliness (crisosto and mitchell, 2002; lurie and crisosto, 2005). ci symp­ toms typically evolve during post­storage/shelf­life conditions and can also compromise the fruit capabil­ ity to ripen after storage, resulting in quality levels not compatible with marketability. different cvs pos­ sess variable levels of tolerance/susceptibility against ci (brizzolara et al., 2018; nilo­poyanco et al., 2018). for peach fruit a specific range of temperatures, called “killing zone” (2.2­7.6°c), has been associated to ci symptoms which tend to appear more rapidly and to become more severe than at 0­1°c (crisosto and valero, 2008). several works investigated the causes of ci onset arguing that a key aspect in peach fruit response to “killing zone” temperatures is the lower ethylene synthesis that, in turn, may only in part activate the ripening machinery (fernández­ trujillo et al., 1998; walsh et al., 2001; zhou et al., 2001; pons et al., 2015; wang et al., 2017). one of the most important ripening­related quali­ ty traits for fruit in general and, especially, for peach­ es is the profile of volatile organic compounds (vocs). during peach fruit ripening a number of dif­ ferent vocs (e.g. alcohols, terpenes, esters, lactones) are synthesized and play a role in making fruit attrac­ tive and for consumer acceptance (aubert and milhet, 2007; yang et al., 2009). the lipoxygenase (lox) pathway, by using fatty acids as precursors, is responsible for the production of specific vocs that play important roles in fruit ripening, also acting as precursors for other metabolites. the lox pathway has been also studied in relation to the physiological responses of peaches to cold storage and the changes in terms of overall organoleptic quality (ortiz et al., 2009; zhang et al., 2011). in addition to the description of voc profiles and enzyme activity pat­ tern, these works correlate specific lox pathway­ associated gene expression with the onset of ci. indeed, zhang et al. (2011) suggested that the decreased levels of fruity note volatiles in peaches with ci, were the consequence of modifications in expression of two lipoxygenase (pplox1 and pplox2) and one alcohol acyltransferase (ppaat1) genes. the different metabolic profiles of peach fruit stored under ci­inducing or “safe” low temperatures have been also related to molecular mechanisms effective to control the expression of structural and regulatory genes. considering the latter, the c­repeat (crt) / dehydration­responsive element (dre) bind­ ing factor 1 (cbf/dreb1) is recognized to be involved in plant responses to abiotic stresses (thomashow, 2010). plant cbf proteins are encoded by multigene families and specific members show distinct expres­ sion patterns in relation to low­temperature stress. the involvement of cbf in responses to cold storage has been reported in tomato fruit, in which an up­ regulation of lecbf1 has been associated with a reduction of ci during storage (zhao et al., 2011). differential expression of 6 isolated cbf genes has been observed in peaches stored under refrigeration (liang et al., 2013). these authors demonstrated that ppcbf 1/5/6 were up­regulated during cold storage at 0°c and this was accompanied by a decrease of ci symptoms. in this work, we report the effects of 2 weeks of cold storage (0.5 and 5.5°c), followed by shelf­life at room temperature (20°c) on the profiles of lox path­ way­derived vocs and the expression of lox­path­ way and cbf genes in peaches of two different geno­ types (‘flaminia’ and ‘red haven’, a typical italian cultivar from tuscany region and a worldwide com­ mercially important cultivar, respectively). 2. materials and methods fruit material, sampling procedure and ci symptoms evaluation peach (prunus persica l. batsch) fruit belonging to ‘red haven’ (rh, yellow fleshed) and ‘flaminia’ (fl, yellow­fleshed, +35 days from rh harvest) have been harvested at flesh firmness values of about 36 and 44 n for rh and fl, respectively, from a commer­ cial orchard located at casciana terme (pisa, tuscany, italy). the fruit selection, the refrigeration treatments and the sampling procedures are those described by brizzolara et al. (2018). briefly, after harvest fruit were selected for homogeneity in terms of fruit size and peel color and were analysed using nir technology (nir­case, sacmi, italy), which has been previously calibrated for firmness (manual pen­ etrometer), total soluble solids (tss) (optical refrac­ tometer) and peel color (arbitrary scale) for each cv. peaches (thirty for each treatment) have been stored in cold chambers at two low temperature conditions (0.5 and 5.5±0.5°c) and, as non­refrigerated control, at 20±0.5°c. relative humidity under cold storage was kept at 85% and monitored using a tgu­4500 brizzolara et al. ‐ lox and cbf gene expression in cold stored peaches 99 (tinytag ultra 2, gemini, united kingdom) data log­ ger. peaches were sampled at harvest (t0) and after 1 and 2 weeks of cold storage. nine fruit per treat­ ment were additionally kept for 3 days under shelf life (sl, 20°c) conditions to investigate the post­stor­ age phase. firmness (n) was measured on nine fruit using a manual penetrometer (gy­3, newtry) equipped with an 8 mm tip. chilling injury symptoms have been evaluated fol­ lowing the same protocol described by brizzolara et al. (2018) using an arbitrary scale. briefly, a total of nine peaches has been evaluated for each sampling time and treatment. for internal browning evalua­ tion, peaches have been cut in two halves and, on each part, brown areas have been assessed with the open source software ‘imagej’, for a total of 18 mea­ surements (2 for each sampled fruit) for every thesis and sampling time. half fruit has been considered as representing 50% of the whole fruit, and browning incidence (%) has been calculated as the average of the nine measured fruit. juiciness has been assessed by processing with a juice extractor (moulinex juice extractor ju350b27) 50 g of pooled mesocarp tissue from three peaches (in triplicates, nine fruit in total), and it has been expressed as percentage of extracted juice normalized on control levels. at each sampling time, three fruit per replicate were processed and a random pool of mesocarp tissue was immediately frozen in l iquid nitrogen and stored at ­80°c. powdered tissue has been used for molecular analy­ ses, while for voc analysis, mesocarp samples of the same three fruit were crushed in a 1m nacl solution by means of a t25 ultra­turraxr (ika, germany) and the obtained puree was immediately frozen in liquid nitrogen and stored at ­80°c. volatile organic compounds (vocs) analysis for aroma volatile compound analysis, the same method previously adopted by brizzolara et al. (2017, 2018) was used. analyses have been performed using agilent technologies (6890n, united states) equip­ ment: a gas chromatograph coupled with mass spec­ trometer (5973 network mass selective detector, agilent technologies) and equipped with an autosampler (mps2, gerstel multipurpose sampler, germany) has been used. peach samples were incu­ bated at 40°c for 2 h, volatile compounds were sam­ pled for 45 min using solid phase micro extraction (spme) fiber (supelco inc., bellefonte, pa, united states) technology, with a polydimethylsiloxane / divinylbenzene (pdms/dvb, 1 cm long, 65 μm thick­ ness, 0.357 μl volume) sorptive coating. the fiber was desorbed into the split/splitless liner of the gc for 5 min in splitless mode, setting at 250°c the injec­ tor temperature. the gc was equipped with a 30 m × 0.25 mm i.d. capillary column (hp­5ms, 5% phenyl methyl siloxane) having a film thickness of 0.25 μm. helium was employed as carrier gas with a flow rate of 1.2 ml min­1. the gc oven heating program started at 40°c and increased to 250°c at a rate of 5°c min­1 with a total run time of 32.5 min. each sample was analysed in triplicate. a mass spectrometer (5973 network mass selective detector, agilent technologies) coupled to the gc was used as detec­ tor for compounds quantification/identification. compound identification has been performed deconvoluting and analysing each chromatogram using the automated mass spectral deconvolution and identification system (amdis, national institute of standards and technology, gaithersburg, md, united states). each chromatographic peak spectra has been compared with those of the national institute for standards and technology (nist98, version 2.0, united states) data bank considering only results with 90%, or more, of matching. peak retention indices (ri) have been used to optimize data bank screening. raw peak areas have been nor­ malized on the sum of the areas of all the identified peaks for each specific chromatogram. total rna extraction and qrt‐pcr analysis frozen mesocarp samples were grounded to pow­ ders using ceramic mortar and pestle pre­cooled with liquid nitrogen. 100 mg of grounded tissue were weighted. total rna extraction was carried out fol­ lowing the protocol of spectrum™ plant total rna kit (sigma­aldrich, italy), including dna digestion with the on­column dnase i digestion set (sigma­aldrich, italy). rna concentration and purity were deter­ mined with nanodrop 2000 spectrophotometer (thermo scientific, italy), verifying an absorbance ratio 260/280 nm between 1.8­2 and 260/230 nm between 1.3­2. integrity of the rna extracted was demonstrated by the existence of intact ribosomal bands on a 1% (weight/volume) agarose gel. next, reverse transcription (rt) of the rna templates to cdna was carried out in a final volume of 20 µl using iscript™ cdna synthesis (bio­rad inc, italy) following the manufacturer instructions. the experimental design of the relative expression analysis included three biological replicates. the approach followed was sample maximization according which, in one run of the qpcr reaction, one gene should be tested on all the samples in the experimental set (helleman adv. hort. sci., 2020 34(1s): 97­108 100 et al., 2007). additionally, in each qpcr reaction a non­template control (ntc) was amplified as a nega­ tive control. analyses were performed by abi prism 7300 sequence detection system (applied biosys­ tem). the reaction system consisted of 5 µl of kicqstart™ sybr® green qpcr readymix™, with rox™ (sigma­aldrich, italy), 3 ng of cdna template, 0.3 µl of revers and forward primers in working solu­ tion of 10 nm. ddw (sigma­aldrich, italy) was added to reach a final volume of 10 µl. the rt­qpcr cycle was set to as follows: initial denaturation at 95°c for 2 min, followed by 40 cycles of amplification with denaturation at 95°c for 15s and annealing and elon­ gation at specific temperature suited to the primer melting temperature (tm) for 1 min. following the 40 cycles a melt cycle was performed at 95°c for 15 s, 60°c for 1 min, 95°c for 15s and 60°c for 15s. expression levels were normalized employing ubiqui­ tin c (ppubc) housekeeping gene as a reference. genes and primers have been selected based on pre­ viously published work dealing with lox pathway gene expression in peach fruit (zhang et al., 2010; 2011). statistical analyses presented data produced with all the employed analytical techniques have been analysed using one­ way analysis of variance (anova) statistical tools (p ≤ 0.05) and tukey’s honest significant difference (hsd) post hoc test in order to identify compounds or genes significantly differing in terms of relative intensity or expression level between tested treatments and sampling times. before performing the analysis, anova assumptions have been tested for all the investigated genes and compounds. 3. results and discussion technological parameters and ci incidence after cold storage and during shelf life conditions both low temperatures were effective in slowing down the loss of firmness rate in both cultivars. at the end of 1 and 2 weeks of storage higher firmness values have been detected in fl fruit stored at 0.5°c than at 5.5°c (table 1) and, although not statistically significant at the end of the first storage week, a sim­ ilar trend has been observed in rh fruit. after 3 days of shelf­life (sl) at room temperature following cold storage, fruit were still firmer than control samples. these data confirm previously published researches on peach fruit firmness changes under and after cold storage (zerbini et al., 2011; cano­salazar et al., 2012; wang et al., 2013). considering the ci incidence, a marked difference has been observed between the two considered cul­ tivars (table 1). as far as internal browning is con­ cerned, rh fruit did not show any symptoms at the end and after both cold storage conditions. instead, fl peaches developed a marked incidence of meso­ carp browning after 2 weeks storage + sl, with some limited symptoms under control conditions (20°c). the two cultivars behaved differently also in terms of extractable juice. in fact, fl peaches showed no dif­ ference of this parameter among samples after 1 week of storage, and a significant reduction of extractable juice was detected in the sample kept at 5.5°c for 2 weeks + sl when compared with both control (20°c) and 0.5°c + sl. on the other hand, rh fruit after 1 week at 0.5°c and 5.5°c + sl displayed significant lower values of extractable juice than con­ trol fruit. these samples were also those showing sig­ table 1 ­ flesh firmness, incidence of internal browning and extractable juice values in peaches during two weeks of cold storage and subsequent three days of shelf life ‘flaminia’ (a) and ‘red haven’ (b) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average values of nine replicates ± sd for each treatment and measured parameter are reported. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, performed independently for each cultivar. 0 week 1 week 2 weeks t0 0.5 5.5 20 0.5 + sl 5.5 + sl 0.5 5.5 20 0.5 + sl 5.5 + sl a ‐ flaminia firmness (n) 44.4±7.9 a 45.1±5.3 a 26.8±1.5 bc 5.6±3.7 e 45.1±5.3 a 26.8±1.5 bc 39.8±3.6 a 22.0±2.2 cd 4.5±2.3 e 35.8±3.6 ab 12.8±2.2 de int. browning (%) 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 11.0±1.0 c 60.0±4.5 a 51.0±3.6 b extractable juice (%) 78.1±4.1 c 77.7±4.2 c 80.5±5.0 bc 85.7±1.4 bc 87.1±4.3 bc 86.7±5.0 bc 84.3±7.2 bc 83.1±4.8 bc 92.4±1.6 ab 100.0±3.8 a 78.6±3.8 c b ‐ red haven firmness (n) 36.3±4.1 ab 41.2±3.4 a 36.1±2.5 ab 2.8±1.0 d 41.1±3.3 a 36.0±2.6 ab 40.7±2.5 ab 29.3±7.3 bc 1.7±1.5 d 38.6±2.5 ab 20.8±7.3 c int. browning (%) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 extractable juice (%) 88.1±0.7 c 91.9±1.4 bc 92.5±2.7 abc 100.0±4.3 a 98.4±2.6 ab 88.9±2.5 c 88.8±1.2 c 90.1±4.9 bc 95.5±0.7 ab 93.0±1.4 abc 78.2±3.8 d brizzolara et al. ‐ lox and cbf gene expression in cold stored peaches 101 considering aldehydes, synthesized through the activities of lipoxygenase (lox) and hydroperoxide lyase (hpl), three compounds have been targeted: hexanal, 2­hexenal and 2­hexenal­e (fig. 1). 2­hexenal and 2­hexenal­e significantly decreased from harvest to the second week in control fruit (kept at 20°c) (fig. 1), confirming what has been previously demonstrat­ ed in other works (defilippi et al., 2009; ortiz et al., 2010). differently from hexanal, 2­hexenal and 2­hex­ enal­e showed an increasing trend, compared to con­ trols, in both cultivars after 1 week of storage either at 0.5 and 5.5°c. in addition, these two compounds showed, in general, a decreasing trend during sl at room temperature, reaching the control levels (fig. 1). moving downstream in the lox pathway, c6 alde­ hydes produced by lox and hpl are used by other enzymes as precursor for the production of c6 alco­ nificantly reduced values after 2 weeks of storage +sl (table 1). these data concerning ci incidence under and after cold storage are in agreement with the pub­ lished literature concerning the effects of different temperatures and the role played by the genetic background (lurie and crisosto, 2005; zhang et al., 2011; pons et al., 2015; bustamante et al., 2016). cold storage effects on lox pathway‐related vocs as general trend, during peach fruit ripening volatile compounds associated with green­notes, such as c6 aldehydes and alcohols, tend to decrease while the levels of molecules associated with fruity­notes, such as esters and, in particular, lactones follow an opposite trend (defilippi et al., 2009; zhang et al., 2011; brizzolara et al., 2018). fig. 1 ­ analysis of targeted lox pathway­derived aldehydes. mesocarp samples (cv. flaminia, left panel; cv. red haven, right panel) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average value of three biological replicates is reported with bars representing sd. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, performed independently for each cultivar. 102 adv. hort. sci., 2020 34(1s): 97­108 hols. specifically, aldehyde dehydrogenase (adh) is the enzyme responsible for the production of alco­ hols from aldehydes (lara et al., 2003; defilippi et al., 2005). considering the results on c6 alcohols level, hexanol, 2­hexen­1­ol and 3­hexen­1­ol accumulation trends are reported in figure 2. interestingly, a tem­ porary increase of these three compounds was detected after 1 week at 20°c only in fl. after 1 week of cold storage, hexanol and 3­hexen­1­ol resulted significantly reduced only in fl after both 0.5 and 5.5°c treatments. in both cultivars, 2­hexen­1­ol sig­ nificantly increased in 0.5°c stored samples after 2 weeks when compared with control fruit (fig. 2). while for hexanol similar levels have been detected in both cultivars, 2­hexen­1­ol and 3­hexen­1­ol were generally higher in rh and fl peaches, respectively. our result only in part confirm those reported by zhang et al. (2011), who showed an increase of 2­ hexenol and 3­hexenol, in peaches during shelf life following 2 weeks of cold storage at 0°c. in the lox pathway, the addition of an acyl moi­ ety to alcohol, reaction catalysed by acyl transferase (aat), results in the production of esters (lara et al., 2003; defilippi et al., 2005). this enzyme drives the production of these important compounds that impart fruity notes (schwab et al., 2008). in addition to c6, also c5 compounds, including 2­pentenal, can be synthetized from an additional branch of the lox fig. 2 ­ analysis of targeted lox pathway­derived alcohols. mesocarp samples (cv. flaminia, left panel; cv. red haven, right panel) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average value of three biological replica­ tes is reported with bars representing sd. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, performed independently for each cultivar. brizzolara et al. ‐ lox and cbf gene expression in cold stored peaches 103 pathway (gardner et al., 1996; nielsen et al., 2004; veronico et al., 2006; shen et al., 2014 a) and specific pentyl esters, derivatives of c5 compounds, have been considered. the targeted analysis that have been performed includes hexyl, 2­hexenyl, 3­hexenyl, 2­pentenyl and 4­pentenyl acetate compounds (fig. 3). in general, fl fruit showed no significant differ­ ence among samples after 1 week of storage regard­ ing hexyl, 2­hexenyl and 3­hexenyl acetate. the most significant change has been observed in rh cv con­ cerning the levels of 2­hexenyl acetate that showed (as in fl samples) a decreasing trend during posthar­ vest ripening at room temperature and increased at the end of 1 and 2 weeks of cold storage under both low temperature conditions. a similar increasing trend of 2­hexenyl acetate was also observed in cold stored fl samples, even though the differences are not statistically significant for both temperatures and fig. 3 ­ analysis of targeted lox pathway­derived esters. mesocarp samples (cv. flaminia, left panel; cv. red haven, right panel) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average value of three biological replica­ tes is reported with bars representing sd. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, performed independently for each cultivar. adv. hort. sci., 2020 34(1s): 97­108 104 sampling times. interestingly, the two targeted pentyl esters (2­ pentenyl and 4­pentenyl acetate), which show an increasing trend during postharvest ripening at 20°c, were markedly reduced by cold storage. 2­pentenyl acetate, in particular, showed almost undetectable levels in all samples at the end of cold storage at both temperatures. a general recover of both pentyl esters levels during sl was observed, with values reaching those of the controls (fig. 3). previous works demonstrate that chilling injured peaches show a lack of esters production and it has been argued that this could be the result of an inhibition of aat1 activ­ ity (ortiz et al., 2009). also in other fruits, ci appear­ ance has been linked to ester biosynthesis: this is the case of papaya in which ci symptoms appearance is accompanied by lower activities of lox and aat (galli et al., 2008). our data do not clearly confirm this behaviour although a general trend of lower hexyl and 2­hexenyl acetate in 5.5°c fl samples stored for 2 weeks was observed. expression analysis of lox pathway genes the expression of genes involved in the lox path­ way (pplox1, pplox4, pphpl1, ppadh1 and ppaat1) is reported in figure 4. concerning pplox1 and pplox4 gene expression, specific pattern of accumulation of these transcripts has been identified in the two cultivars and different conditions. in fl control peaches (20°c) pplox1 and pplox4 revealed an identical trend showing a signifi­ cant increase from harvest to the first week followed by a decrease during the second week. on the other hand, in rh control fruit the expression levels of these two genes showed a stable (pplox4) or decreasing (pplox1) trend in 2 weeks after harvest. pplox1 appeared to be inhibited at the end of both low temperature storage conditions in fl, and a sig­ nificant increase of its expression level has been recorded during sl in all samples. this increase dur­ ing sl has been observed also in rh fruit. pplox4 expression was significantly reduced and increased in fl and rh peaches, respectively, after 1 week of cold storage at both temperatures. an up­regulation of this gene was detected in both cultivars in 5.5°c + sl samples after 1 week of storage. considering pphpl1, a decreasing trend of expres­ sion was detected in fl control fruit only. genotype­ related difference has been also observed when com­ paring the expression trends of cold stored peaches with significant increases in both sl samples of rh fruit after 1 week of storage. ppadh1 and ppaat1 genes revealed a similar expression trend, almost mirroring the pattern detected for pplox1 (fig. 4). a peak of expression after 1 week has been detected in fl control samples for both genes, while in rh control peaches ppadh1 expression showed a decreasing trend and ppaat1 expression remained unchanged. besides the reduced expression of ppadh1 detected in cold treated fl samples after 1 week, limited or non­sig­ nificant changes of ppadh1 expression levels were detected in cold stored and sl samples of rh (1 and 2 weeks) and fl (2 weeks). interestingly, in both culti­ vars ppaat gene expression showed a significant up­ regulation in all sl fruit of either temperatures, if compared to samples collected at the end of cold storage. this behaviour is similar to that observed for pplox1. thus, the expression of these two genes seems highly sensitive to temperature conditioning with a general decrease observed during cold storage and an expression recovery during sl at room tem­ perature. in general, our expression data confirm what reported in literature on peach fruit ripening in par­ ticular concerning pphpl1, that shows a decreasing trend, paralleled by reduced amount of c6 aldehydes (zhang et al., 2010; shen et al., 2014 b). a decreasing hpl expression level during ripening has been observed also in other fruit species, such as tomato (howe et al., 2000). the expression of lox1 and 4, adh1 and aat1 during ripening appears to be differ­ ent in relation to the genotype. in fact, the transient up­regulation of these genes observed in fl control fruit after 1 week of storage (followed by a down­ regulation) was not observed in rh fruit. considering the effect of cold stress on lox genes, an induction on pplox1 and pplox3 expression in peach fruit during shelf life after storage at 0.5 and 5.5°c, flanked by higher ethylene synthesis, has been reported by zhang et al. (2011). in our trials, the effect of “rewarming” (shelf life at room temperature after cold storage) is clearly evident when consider­ ing the expression trend of pplox1 (thus confirming the data by zhang et al., 2011) but not of pplox4, suggesting that members of this multigene family have different regulatory mechanisms and may be selectively involved in the responses of peaches to postharvest temperature conditioning. as observed for pplox1, ppaat seems highly sen­ sitive to temperature conditioning showing a marked expression recovery during post­cold storage sl. similarly, ortiz et al. (2009) reported an enhance­ ment of ppaat gene expression after cold storage, brizzolara et al. ‐ lox and cbf gene expression in cold stored peaches 105 indicating a renewed ability of synthetizing esters after cold stress. our results (figs. 3, 4) confirm this observation. considering the possible relationships between ci and lox pathway, zhang et al. (2011) reported that in peach fruit lox pathway genes showed lower tran­ script levels in fruit stored at a ci­inducing tempera­ tures (e.g. 5°c) compared to fruit kept at 0°c, with transcript abundance that tends to decrease with extended storage periods and the onset of ci symp­ fig. 4 ­ rt­qpcr analysis of selected lox pathway­related genes. mesocarp samples (cv. flaminia, left panel; cv. red haven, right panel) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average value of three biologi­ cal replicates is reported with bars representing sd. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, performed independently for each cultivar. adv. hort. sci., 2020 34(1s): 97­108 106 toms. our gene expression data do not show any clear relationship with ci incidence, while, consider­ ing vocs, only for hexyl acetate and 2­hexenyl acetate significantly lower concentrations have been detected in samples with ci symptoms. expression analysis of cbf genes the expression pattern of two c‐repeat‐binding factors genes (ppcbf1 and ppcbf6) has been analysed in fl and rh samples in relation to cold storage treat­ ments (fig. 5). as a general trend the two genes appeared to be significantly up­regulated at the end of 1 and 2 week storage either at 0.5 and 5.5°c in both cultivars. in fl samples after 1 week and in rh fruit after 2 weeks, it appears that the lower the storage temperature the higher is the expression of these two genes. during 20°c sl after cold storage, either at 0.5 and 5.5°c, the expression of these genes markedly decreases reach­ ing the level of the control, confirming that cbf gene expression is highly responsive to temperature condi­ tions. it is interesting to note that rh peaches showed higher levels of expression, in particular con­ sidering ppcbf1. similar results in terms of expression pattern dur­ ing refrigeration of ppcbf genes have been previously reported in peach fruit, confirming that these regula­ tory genes are generally sensitive to low temperatures (liang et al., 2013). specifically, ppcbf1/6 appeared to be induced exclusively under cold storage, revealing very low or non­detectable levels during post­storage shelf life, and their level of expression was significantly higher under ci­delaying temperature (0°c), when compared to chilling inducing conditions (5°c). indeed, this paper reports that the up­regulation of cbf genes correlated with a decreasing level of ci symptoms, such as flesh browning, as well as with a reduction of firmness loss. our data clearly show that cbf expres­ sion is higher at 0.5°c than at 5.5°c in fl after 1 week and in rh after 2 weeks of storage, highlighting the dif­ ferent behaviour and regulatory mechanisms charac­ terizing peach genotypes. it has been reported that cbf genes in peach fruit are activated over different time periods, with ppcbf5 being activated at high lev­ els in several hours, ppcbf6 later within three days and ppcbf1 requiring 1 week to be expressed at high levels (liang et al., 2013). this temporal distribution of cbf genes activation during cold storage suggests that each gene plays a specific role, and it has been argued that while ppcbf5 seemed to be associated with an early response to low temperatures, ppcbf1/6 appeared to be more involved in protection to pro­ longed cold storage. however, the possible relation­ ship between the expression trends and levels of these cbf genes and the sensitivity to postharvest cold stress of peach fruit remains to be elucidated. this is an important topic for future research in elucidating regulatory mechanisms involved in low temperature responses considering that the over­expression of peach ppcbf1 allowed wisniewski et al. (2011) to get to an increased freezing tolerance of apple trees of about 4­6°c. moreover, the overexpression of several cbf genes in different plant species, including arabidopsis, grape, potato and apple, appears to increase plant tolerance to cold stress (novillo et al., 2007; pino et al., 2008; siddiqua and nassuth, 2011; fig. 5 ­ rt­qpcr analysis of two c­repeat­bin­ ding factors (cbf) genes. mesocarp sam­ ples (cv. flaminia, left panel; cv. red haven, right panel) were analysed at harvest (0), at the end of 1 and 2 weeks of cold storage at 0.5 and 5.5°c, and after three additional days of shelf life (0.5 and 5.5°c + sl). samples kept at 20°c represent the room temperature control. the average value of three bio­ logical replicates is reported with bars representing sd. letters indicate the results of tukey's honest significant difference (hsd) post hoc test, perfor­ med independently for each cultivar. brizzolara et al. ‐ lox and cbf gene expression in cold stored peaches 107 takuhara et al., 2011). 4. conclusions peach genotypes show marked difference in terms of responses to cold storage, with some cvs less susceptible to ci and others developing an array of symptoms during shelf life after a few days/weeks of refrigeration. this different behavior observed here in rh and fl peaches, as already reported in a previously published paper (brizzolara et al., 2018), appears to be associated with different expression pattern of two cbf genes (ppcbf1 and ppcbf6), rec­ ognized to be involved in the responses of peaches to cold storage. if the general picture of the regulatory role played by cbf genes has been described in plants, the possi­ ble mechanisms involving cbf transcription factors in modulating molecular and physiological responses of fruit tissues to postharvest cold stress remain to be elucidated. peaches represent an interesting model for such studies, based on the fact that the expres­ sion of the two cbf genes analyzed in the present paper appears to be differently affected by tempera­ tures more (5.5°c) or less (0.5°c) inducing the appearance of ci symptoms. in addition to the expected effect of genotype, specific genes of the lox pathway also show differ­ ent expression pattern depending on the storage temperature and the shelf­life, with some of them (e.g. lox1, adh and aat1) representing good candi­ dates for additional studies aimed at identifying metabolic and molecular markers of ci onset and incidence in cold stored peaches. references aubert c., milhet c., 2007 ­ distribution of the volatile compounds in the different parts of a white‐fleshed peach (prunus persica l. batsch). ­ food chem., 102: 375­384. brizzolara s., hertog m., tosetti r., nicolai b., tonutti p., 2018 ­ metabolic responses to low temper‐ ature of three peach fruit cultivars differently sensitive to cold storage. ­ front. plant sci., 9: 706. brizzolara s., santucci c., tenori l., hertog m., nicolai b., stürz s., zanella a., tonutti p., 2017 ­ a metabolomics approach to elucidate apple fruit responses to static and dynamic controlled atmosphere storage. ­ postharvest biol. technol., 127: 76­87. bustamante c.a., monti l.l., gabilondo j., scossa f., valentini g., budde c.o., lara m.v., fernie a.r., drincovich m.f., 2016 ­ differential metabolic rearrangements after cold storage are correlated with chilling injury resistance of peach fruits. ­ front. plant sci., 7: 1478. cano­salazar j., echeverría g., crisosto c.h., lopez l., 2012 ‐ cold‐ storage potential of four yellow‐fleshed peach cultivars defined by their volatile compounds emissions, standard quality parameters, and consumer acceptance. ­ j. agr. food chem., 60: 1266­1282. crisosto c.h., mitchell g., 2002 ­ postharvest handling systems: stone fruits. 28, ‐ in: kader a.a. 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fruit using time­resolved spectroscopy m. vanoli 1 (*), g. cortellino 1, v. picchi 1, m. buccheri 1, m. grassi 1, f. lovati 1, l. marinoni 1, p. levoni 2, a. torricelli 2, 3, l. spinelli 3 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (crea‐ it), via g. venezian, 26, 20123 milano, italy. 2 politecnico di milano, dipartimento di fisica, piazza leonardo da vinci, 32, 20133 milano, italy. 3 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn‐cnr), piazza leonardo da vinci, 32, 20133 milano, italy. key words: carotenoids, cucumis melo l., ethylene, juiciness, quality, trs. abstract: the aim of this work was to explore the feasibility of time­resolved reflectance spectroscopy (trs) in determining the ripening degree and the quality of orange­fleshed melons. sixty ‘honey moon’ melons were measured by trs in the 540­1064 nm range and classified as less (lem), medium (mem), and more (mom) mature according to increasing values of μa540. mom fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents than lem ones. mom fruit also showed higher internal ethylene concentration and lower firmness than lem ones, even if the differences were not significant. the μa540 was positively related to internal ethylene, carotenoid accumulation, and juiciness, indicating that μa540 was linked to different ripening processes in melons. however, the relationship between μa540 and total carotenoid content was not as high as expected due to the low variability of pulp color and of carotenoid content. changes in flesh color toward a more orange shade were accompanied by increased juiciness and ethylene production and by carotenoid accumulation, while changes in peel color were associated with changes in flesh firmness and juiciness. in con­ clusion, the absorption coefficient measured at 540 nm (µa540) by trs could be used to sort melons in different ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties. 1. introduction melon (cucumis melo l.) fruit are particularly appreciated by con­ sumers for their sweetness, flavour, texture, and attractive flesh color, as well as for their nutritional and phytochemical properties. melons are a good source of carotenoids, ascorbic acid, and phenolic compounds (gómez­garcía et al., 2020; manchali et al., 2021; singh et al., 2022). melon quality depends on the balance between sugars, organic acids, volatiles, and texture characteristics (kyriacou et al., 2018). these traits are strongly affected by genotype, agro­climate conditions, harvest matu­ (*) corresponding author: maristella.vanoli@crea.gov.it citation: vanoli m., cortellino g., picchi v., buccheri m., grassi m., lovati f., marinoni l., levoni p., torricelli a., spinelli l., 2023 ­ non‐ destructive determination of ripening in melon fruit using time‐resolved spectroscopy. ­ adv. hort. sci., 37(1): 75­82. copyright: © 2023 vanoli m., cortellino g., picchi v., buccheri m., grassi m., lovati f., marinoni l., levoni p., torricelli a., spinelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 november 2022 accepted for publication 20 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13943 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 75­82 76 rity, post­harvest handling, and storage conditions (kyriacou et al., 2018). melons picked at the optimal harvest maturity have premium quality as maturity at harvest has a large impact on sugars, volatiles, and texture. during ripening, rind color changes from green to yellow, orange, or creamy yellow, depend­ ing on melon genotype; mesocarp color turns from pale green to orange in orange­fleshed melons, along with carotenoid accumulation; firmness and acidity decrease, while sucrose as well as soluble solids, vita­ min c and ph markedly increase (beaulieu and lea, 2007; tristan et al., 2022). melons harvested mature develop the strongest flavour, whereas fruit harvest­ ed early develop a less aromatic flavour: esters are predominant in ripe melons, aldehydes are present at higher concentration in immature fruit while a sharp increase of alcohols is typical of overripe mel­ ons (senesi et al., 2005; beaulieu and lea, 2007; vallone et al., 2013; lignou et al., 2014). during ripening, the sensory scores for color intensity, fruity aroma, juiciness, and sweetness increased while the sensory scores for firmness and sourness generally decreased (vallone et al., 2013). in climacteric mel­ ons, ethylene was produced at higher levels in fully ripe fruits and was negatively related to firmness and positively related to sensory juiciness and fruity flavour and aroma (senesi et al., 2005; vallone et al., 2013). for more than two decades, several efforts have been made to measure quality characteristics of mel­ ons in a nondestructive way (zeb et al., 2021). in industrial fruit sorting, accuracy, cost, and detection speed are important factors. thus, spectroscopic techniques being fast, simple, and cost­effective, have been widely studied. in melons, vis­nir spec­ troscopy (sanchez et al., 2014; lu et al., 2015; khurnpoon and sirisomboon, 2018; zeb et al., 2021), computer vision (calixto et al., 2022) and hyperspec­ tral imaging (sun et al., 2017; cho et al., 2022) were studied with less or more successful results for pre­ dicting soluble solids, moisture, pulp color, and firm­ ness and for classifying fruit according to different sweetness degree or as suitable or not for harvesting. among spectroscopic techniques, time­resolved reflectance spectroscopy (trs) is gaining increasing interest in assessing fruit quality. due to its accuracy in measuring optical properties in deep tissues it allows the evaluation of maturity and internal defects of horticultural products with a relatively thick sur­ face layer (lu et al., 2020). trs allows the complete optical characterization of a diffusive medium through the measurements of the absorption (μa) and of the scattering (μs) coefficients by probing flesh at a depth of 1­2 cm with no or limited influence from the skin (cubeddu et al., 2001; rizzolo and vanoli, 2016). while scattering is related to the struc­ tural properties of fruits and vegetables, absorption depends on the chemical composition of the tissue, mainly on the presence of pigments such as chloro­ phylls, anthocyanins, and carotenoids (lu et al., 2020). trs has been mainly applied in post­harvest studies on fruit and vegetables for estimating fruit ripening, for the detection of internal defects, and for discriminating fruit with different texture and senso­ ry characteristics (rizzolo and vanoli, 2016). the absorption coefficient measured at 670 nm (μa670) at harvest was shown to be a maturity index for various fruit, such as peaches, nectarines, plums, pears, and apples, as it declines with fruit ripening (vanoli and buccheri, 2012). the μa540 (carotenoid tail) has been used as a non­destructive maturity index for man­ goes, as it was able to classify intact mangoes of dif­ ferent cultivars according to pulp color and to the contents of total and individual carotenoids (rizzolo and vanoli, 2016; vanoli et al., 2018). trs has not yet been studied on melons, so the aim of this work was to explore the feasibility of trs in determining the ripening degree and the quality of orange­fleshed melons. 2. materials and methods fruit the experiment was carried out on ‘honey moon’ (cucumis melo l. cantalupensis) melons. ‘honey moon’ fruits have round shape, medium size, a smooth grey­green skin that turns yellow when fully ripe. fruits has firm, deep orange flesh and show high sugar content and a pleasant and almost exotic aroma. in order to have high variability in fruit matu­ rity, 60 melons were picked and selected in a pack­ inghouse in sermide (mantova­italy) on a peel color basis (15 fruit for four color stages: blue, gray­green, yellow­green and yellow). then fruit were immedi­ ately transported to the crea­it lab in milan, mea­ sured by trs in the 540­1064 nm range and classified as less (lem), medium (mem) and more (mom) mature according to increasing μa540 values (low μa540=less mature fruit; high μa540=more mature fruit). all the 60 melons were also individually evalu­ ated for color (peel and flesh) and for standard matu­ vanoli et al. ‐ ripening determination of melons by trs 77 rity indices (flesh firmness, soluble solids content, acidity). among the 60 melons, 20 fruits covering the whole range of μa540 (i.e., the highest, the lowest and 18 intermediate values of μa540), were chosen for internal ethylene concentration, total carotenoid content and juiciness analyses. time‐resolved reflectance spectroscopy (trs) whole melons were measured by trs on two opposite sides in the fruit equatorial region. a portable compact setup working at discrete wave­ lengths developed at politecnico di milano (zhao et al., 2022) was employed. the light source is a super­ continuum fiber laser (sc450­6w, fianium, uk) pro­ viding white­light pulses, with duration of a few picoseconds. two custom­made filter wheels loaded with overall 14 band­pass interference fi lters (maxline series, semrock, ny, usa, and techspec series, edmund optics, new jersey, usa) are used for spectral selection in the range 540­1064 nm. light is delivered to the sample by a 200 μm core step­index fiber and collected by a 1 mm core graded­index fiber; interfiber distance was set to 1.5 cm. a pair of filter wheels identical to the previous one is used for cutting off the fluorescence signal originated from the sample when it is illuminated in the visible spec­ tral region. the light then is detected by a cus­ tomized silicon photomultiplier module (martinenghi et al., 2016) and the photon time­of­flight distribu­ tion is measured by a time­correlated single­photon counting board (spc­130, becker&hickl, germany). the instrumental response function has a full width at half maximum of about 100 ps and the typical acquisition time is 1 s per wavelength. a semi­infinite model for photon diffusion in a turbid medium was used to analyze trs data to assess the bulk optical properties of the samples (martelli et al., 2009) to obtain the estimates of µa and µs at each wavelength. peel and flesh color color was measured on the equatorial region on two opposite side of whole fruit and, after cutting longitudinally the melons, on two opposite portions of the flesh, 1.5 cm from the fruit edge. a cm2600d spectrophotometer (konica minolta sensing, inc., osaka, japan) with the primary illuminant d65 and 2° observer, was used to perform the color measure­ ments in the l* (lightness) a* (green­red), b* (yellow­ blue) color space. from a* and b* values, hue (h°) and chroma (c*) were computed according to h° = arctangent (b*/a*) × 360/(2×3.14) and c*= (a*2 + b*2)­2. in the flesh also the color spectra (350­740 nm) were considered. color readings were averaged for each fruit. standard maturity indices and juiciness flesh firmness after having cut the melons into two parts along the longitudinal axis, flesh firmness was measured on two opposite sides in the equatorial part of the fruit (around 1,5 cm from the fruit edge) using an 8 mm diameter plunger mounted on a ta­xt plus texture analyzer (stable micro systems, godalming, uk) at a crosshead speed of 200 mm min­1. the two measure­ ments were averaged for each fruit. soluble solids content (ssc) and titratable acidity two longitudinal slices around 4 cm thick, taken from two opposite sides of each fruit (in the same places of the firmness measurements) were frozen for the evaluation of soluble solids content (ssc) and of titratable acidity. after thawing, ssc was deter­ mined on few juice drops from each slice by using an automatic refractometer (rfm81, bellingham­stanley ltd., england). titratable acidity (ta) was measured by titrating 10 g of juice plus 50 ml of distilled water with 0.1 n naoh to ph 8. juiciness juiciness was measured on pulp cylinders (diame­ ter = 15 mm; height = 10 mm) taken from two oppo­ site sides of the equatorial part of the fruit. each cylinder was compressed between two plates with a ta­xt plus texture analyzer (stable micro systems, godalming, uk) at a deformation rate of 100 mm min­1 by a compression of 50% of the original height of the cylinder (eccher zerbini et al., 1999). this method correlated with sensory analysis, and only the juice that could be easily and quickly released by the pulp cylinder was measured. juiciness was calcu­ lated as the ratio between the juice weight spilled out after compression and the cylinder’s original weight. internal ethylene concentration 1 ml sample of internal gas was taken from the seed cavity of each melon using a syringe equipped with a 15 cm long, 15­gauge needle. the sample was injected in a dani gs 86.10 gas chromatograph and analyzed for the ethylene content following the con­ ditions reported by rizzolo et al. (2005), using a deac­ tivated aluminum oxide f1 (80­100 mesh) column (1/8 in × 200 cm alltech italia, sedriano, italy), col­ umn temperature 100°c, injection temperature 100°c, and a flame ionization detector temperature adv. hort. sci., 2023 37(1): 75­82 78 of 225°c. ethylene was identified and quantified by relating the peak area of the sample to that of a 10 μl l­1 external standard and was expressed as ppm. total carotenoid content two slices per fruit were frozen for total carotenoid content analysis. 1 g of frozen sample was rapidly homogenized (ultra­turrax, ika­werk, germany) in 5 ml of an ice­cold solution of hexane: acetone:ethylacetate (2:1:1) containing 1% of 1% butylated hydroxitoluene (bht) in methanol. the homogenate was sonicated for 10 min, centrifuged at 10000 g for 15 min at 4°c and the organic phase was collected. the absorbance of the organic phase, after proper dilution, was measured spectrophotometri­ cally at 450 nm. total carotenoid content was esti­ mated using the molar extinction coefficient for b­ carotene in hexane (139200 l mol­1 cm­1), as reported in craft and soares (1992). statistical analysis data were submitted to analysis of variance (statgraphics ver. 7, manugistic inc., rockville, md, usa) considering trs maturity class as a source of variation, and means were compared by tukey’s test at p≤0.05%. the relationships between μa540 and quality characteristics and the relationships among quality parameters were studied using regression analysis. for each parameter, the model with the higher performance was considered. only the models with correlation coefficient r>0.50 were considered. 3. results trs absorption spectra trs absorption spectra of ‘honey moon’ melons (fig. 1a) showed a maximum at 980 nm (correspond­ ing to water), high absorption values at 540 nm (cor­ responding to the tail of carotenoids) and very high variability in the chlorophyll absorption range (610­ 690 nm). the variability in the chlorophyll absorption was not linked to the presence of chlorophyll in the pulp but depended on the presence of a green layer (about 3.5­7 mm) between the rind and the pulp, which interfere with trs measurements that pass through the pulp, up to 2 cm. the color spectra of the pulp confirmed the trs data, showing a very low absorption in the chlorophyll range and a very high absorption in the carotenoid range (400­500 nm) (fig. 1b). then, the absorption coefficient measured at 540 nm (µa540) was chosen as a possible ripening index for melons due to its correlation to the carotenoid content in the pulp, as previously found by rizzolo and vanoli (2016) and vanoli et al. (2018) in mangoes. the µa540 ranged from 0.194 to 0.663 cm­1. melons were ranked according to increasing µa540 (increasing maturity) and sorted into three maturity classes: low (µa540 = 0.383±0.008 cm­1), medium (µa540 = 0.451±0.004 cm ­1) and more mature (µa540 = 0.,537±0.014 cm­1). peel and flesh color peel and pulp color significantly changed with trs maturity classes (table 1). peel color turned from green to yellow­green with advancing trs maturity class, as b* and c* values increased and h° values decreased from lem to mom melons. pulp color became slightly more orange, as b* and h° were lower in mom fruit than in lem ones. there was no fig. 1 ­ trs absorption spectra (a) and pulp color spectra (b) of ‘honey moon’ melons. vanoli et al. ‐ ripening determination of melons by trs 79 main change in the pulp color of melons, as b* ranged from 29.3 to 38.1 and h° from 64.5 to 70.2. standard maturity indices, juiciness, internal ethylene and total carotenoids content flesh firmness ranged from 7.09 to 27.55 n, ssc from 7.4 to 12.2% and acidity from 1.70 to 5.57 g l­1 citric acid. flesh firmness, ssc and acidity were not significantly affected by trs maturity classes (fig. 2 a, b, c). however, firmness and acidity were lower and ssc was higher in mom fruit than in lem ones. juiciness ranged from 6.86 to 22.27% and was signifi­ cantly higher in mom fruit than in the lem ones (fig. 2 d). internal ethylene concentration ranged from 51.9 to 153.3 ppm. it did not significantly change with trs maturity class, even if the lem fruit showed the lowest concentration and mom ones the highest (fig. 2e). total carotenoids content ranged from 16.95 to 28.25 mg kg­1 fw; it was significantly higher in mom fruit than in lem ones (fig. 2f). regression analysis the results of regression analysis between µa540 and quality parameters are summarized in table 2. the µa540 was significantly related to juiciness (r=0.53), internal ethylene concentration (r=­0.86) and total carotenoid content (r=0.66). the µa540 was also positively related to skin color (except for a*) table 1 ­ peel and pulp color parameters of 'honey moon' melons in relation to trs maturity classes means in the same column followed by different letters are statistically different at p≤0.05 (tukey's test). fig. 2 ­ flesh firmness (a), soluble solids content (b), acidity (c), juiciness (d), internal ethylene concentration (e) and total carotenoid content (f) in ‘honey moon’ melons in relation to trs maturity classes. bars refer to se. maturity stage l* a* b* c* h° peel less mature 72.4 a ­6.1 a 23.3 b 24.2 b 106.5 a medium mature 73.4 a ­5.4 a 26.0 ab 26.7 ab 103.4 ab more mature 76.0 a ­5.4 a 28.8 a 29.4 a 101.2 b pulp less mature 62.1 a 14.0 a 33.9 ab 36.7 a 67.6 a medium mature 63.2 a 14.4 a 34.7 a 37.6 a 67.5 a more mature 60.0 a 14.4 a 33.3 b 36.3 a 66.6 b 80 adv. hort. sci., 2023 37(1): 75­82 with r<0.5. no significant correlation was found between µa540 and pulp color, flesh firmness, ssc and acidity. significant relationships were also found among peel color, firmness and juiciness (table 3). firmness was related to b* peel (r=0.62), c* peel (r=0.60) and to h° peel (r=0.58); juiciness was related to b* peel (r=0.57), c* peel (r= 0.55) and to h° peel (r= 0.54). as for pulp color, total carotenoid content was positive­ ly related to a* pulp (r=0.68) and negatively to h° pulp (r= −0.88); internal ethylene was related to a* pulp (r= −0.61) and juiciness to h° pulp (r= 0.58). significant relationships were also found between juiciness and total carotenoids (r= 0.64) and between juiciness and firmness (r= 0.73). 4. discussion and conclusions ‘honey moon’ melons used in this experiment showed pulp color similar to that of fruit of the same cultivar picked at commercial maturity, even if firm­ ness, acidity and ssc had lower values (cavicchi and pasotti, 2004). total carotenoid content showed val­ ues typical of orange­fleshed fruit (saladie et al., 2015; singh et al., 2022). internal ethylene concen­ tration confirms that ‘honey moon’ is a climacteric genotype, as there was an increase in ethylene pro­ duction with advancing fruit maturity (senesi et al., 2005; vallone et al., 2013; saladie et al., 2015). trs measurements of melons showed some prob­ lems. in fact, the absorption in the chlorophyll range cannot be used as a maturity index as already done for other fruits, such as apples, pears, peaches and nectarines (rizzolo and vanoli, 2016), since chloro­ phyll was almost absent in the melon pulp while it was present in a layer just under the peel. on the other hand, µa540 was able to distinguish mom mel­ ons from lem ones, as mom fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents, in agreement with kyriacou et al. (2018), senesi et al. (2005) beaulieu and lea (2007), vallone et al. (2013), saladie et al. (2015) and tristan et al. (2022). mom fruit also showed higher internal ethylene concentration and lower firmness than lem ones, even if the values showed high variability and the differences were not significant. the µa540 was also positively related to internal ethylene, carotenoid accumulation and juici­ ness, indicating that µa540 is linked to different ripen­ ing processes in melons. however, the relationship between µa540 and total carotenoid content was not as high as expected (r=0.66) and as previously found in mangoes when r ranged from 0.78 to 0.91 depend­ ing on the cultivar (vanoli et al., 2018). it seems that in ‘honey moon’ melons, the range of µa540 (0.194­ 0.663 cm­1) is quite similar to that of mangoes (0.117­ 0.835 cm­1), while the variability in total carotenoids table 2 ­ regression models between µa540, juiciness, internal ethylene concentration and total carotenoid contents for each significant regression, the following data are given: r= correlation coefficient; p=significance of the model (***, p<0.001; **, p<0.01; *, p<0.05), and model type. dr= double reciprocal; rx=reciprocal x. table 3 ­ regression models between firmness, juiciness, inter­ nal ethylene concentration, total carotenoid content and pulp color for each significant regression, the following data are given: r= correlation coefficient; p=significance of the model (***= p<0.001; **= p<0.01; *= p<0.05) and mt= model type. ry=reciprocal y; lin=linear; rx=reciprocal­x; dr=double recipro­ cal. r p model type juiciness 0.531 * dr internal ethylene ­0.864 *** rx total carotenoids 0.664 ** dr r p mt firmness b* peel 0.616 *** ry c* peel 0.604 *** ry h° peel 0.576 *** lin juiciness firmness 0.733 *** rx b* peel 0.565 * dr c* peel 0.548 * dr h° peel 0.538 * rx h° pulp 0.577 * rx ethylene a* pulp ­0.617 * ry carotenoids juiciness 0.640 * dr a* pulp 0.679 ** dr h° pulp ­0.881 *** lin vanoli et al. ‐ ripening determination of melons by trs 81 (16.95­28.25 mg kg­1 fw) and in pulp color (h° pulp= 64.5­70.2) was narrower in melons than in mangoes (total carotenoids= 5­56 mg kg­1 fw; h° pulp=71­104). moreover no correlation was found in melons between µa540 and pulp color while in mangoes µa540 was negatively related to h° pulp with r= 0.83­ 0.98 (vanoli et al., 2018). in ‘honey moon’ fruit, with advancing trs ripening degree, the changes in flesh color toward a more orange shade were accompa­ nied by increased juiciness values and ethylene pro­ duction and by carotenoid accumulation, while changes in peel color toward a yellow shade were associated with fruit softening. in conclusion, the absorption coefficient mea­ sured at 540 nm (µa540) by the trs technique could be used to sort melons in different ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties. acknowledgements this study was funded from the italian ministry of agriculture, agridigit project, subproject agrofiliere (d.m. 36503/7305/2018, approved on 20 december 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horticulture, faculty of agriculture, tarbiat modares university, tehran, iran. 2 department of horticulture, faculty of agriculture, ferdowsi university of mashhad, po box 91775‐1163 mashhad, iran. 3 young researchers and elite club, islamic azad university, tabriz, iran. key words: essential oil, ethylene production, postharvest, storage condition. abstract: postharvest loss in fruit and vegetables accounts for more than one third of the world production. on the other hand, using chemicals has raised food health concerns, and grown the demand for eco­friendly materials. given the promising results of essential oil use to control and prevent postharvest decay, we conducted this research. in the present study, a two­step statistical method was used to determine and optimize application of essential oils along with parameters of storage conditions. significant level of essential oils and storage conditions (temperature, ventilation, and relative humidity) were screened by using pbd method and the best concentration were determined by central composite design of response surface method. the results showed that 1000 and 1500 microgram/l (µg/l) of basil essential oils, and 1500 µg/l of pep­ permint essential oils reduced the lesion diameter in apple fruits infected with penicillium expansum. all storage conditions had significant effect on posthar­ vest decay. based on the ccd of rsm method, the best concentration of essen­ tial oils and the optimal level of the storage condition were determined. furthermore, basil and peppermint treatments reduced rate of the ethylene production during 56 days after treatment. the results of this study revealed and confirmed that basil essential oils as a postharvest treatment under opti­ mized storage conditions can be utilized as a low­cost substrate for controlling postharvest decay in apple. 1. introduction it is estimated that more than one third of harvested fruit and vegeta­ bles are lost due to pathogen infections in the field or after harvest, resulting in a serious economic loss (romanazzi et al., 2016). the average loss is about 29% in north america and europe, and more than 35% in asia (romanazzi et al., 2016). given severity of economic loss worldwide, many researchers have employed various strategies to decrease loss and control the conditions after harvest. the first generation of attempts was to use chemical materials to control diseases, however, health and safety (*) corresponding author: gh.rostamitobnag@gmail.um.ac.ir citation: al­hassanavi h.r., rostami g., shokri r., 2023 ­ application of plant essential oils and opti‐ mizing storage conditions for postharvest control of diseases in apple. ­ adv. hort. sci., 37(2): 149­ 157. copyright: © 2023 al­hassanavi h.r., rostami g., shokri r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 november 2022 accepted for publication 14 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13919 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 149­157 150 concerns turned attentions on natural products (rajestary et al., 2021). nowadays, essential oils, used as additives, color intensifiers, and antioxidants, are extracted from plant sources have gained atten­ tions because of advantages over synthetic materials such as a good quality, biodegradable, economical characteristics, eco­friendly easy large­scale produc­ tion, and especially a lack of safety concerns (sivakumar and bautista­baños, 2014). a study sug­ gested that fungal strains could develop a resistant when synthetic fungicide are continuously used, while the different components of essential oils make the process of the resistant more slowly during the application (sivakumar and bautista­baños, 2014). recently, many studies have explored the use of essential oils to control postharvest losses. for instance and to mention some of these recent stud­ ies, antonioli et al. (2020) reported the use of nano­ capsules containing plant essential oil to control bit­ ter rot of apples and showed that the fruits treated with nano­capsules have a smaller lesion. another study showed that mint and thyme essential oils reduced rhizopus rot on strawberry and peach fruits. kontaxakis et al. (2020) evaluated several essential oils to assess their effect in preserving fruits during postharvest phase. many other studies have showed that essential oils are promising to control posthar­ vest decay in fruit and vegetables, however, the question of this study is to (1) find the best concen­ tration in which essential oil could act, and (2) to determine the best environmental conditions of the storage place in which essential oils applied. we think that these points are worth to be explored. therefore, the objective of the present study was to test efficiency of plant essential oils and optimize storage conditions in apple postharvest period. in this study, two steps statistical method was employed. moreover, ethylene concentration was evaluated on the optimized conditions. 2. materials and methods materials and the experiment design this study was conducted to enhance apple stor­ age through application of plant essential oils as well as optimizing storage conditions. the culture was in submerged ferment and three parameters were assessed to reach an optimal production process. apples (malus domestica, cv. red delicious), harvest­ ed from orchards in northern­west iran, ardabil, iran, were divided in groups of 27 fruits/treatment. the first parameter was basil essential oil (eugenol 10.04%, linalool 68.52%, a­trans­bergamotene 6.94%, and b­cadinol 3.20%). the second parameter was peppermint essential oils (1,8­cineole 5.89%, menthofuran 4.59%, menthol 38.29%, and menthyl acetate 29.39%). basil and peppermint essential oil were used in three level (500, 1000, and 1500 micro­ gram/l) as a treatment. the third parameter was stor­ age conditions including temperature (­2, ­1, and 0°c), ventilation (3, 4.5, and 6 lpm), and relative humidity (85, 90, and 95%). a 15 percent emulsion (15% essen­ tial oil, 83% water and 2% tween) was prepared from basil and peppermint essential oils. the resultant emulsion was shaken for 45 s before spraying. a two­step statistical strategy (embaby et al., 2018) was anticipated to optimize and determine relationship among postharvest control of apple fun­ gal diseases with basil essential oils, peppermint essential oils, and storage conditions (temperature, ventilation, and relative humidity). plackett­burman design (pbd) (plackett and burman, 1946) was employed to monitor the linear effect of three para­ meters using 27 experimental trials designed in 28­ run design. pbd is an efficient way to screen the most important variables when there are multiple vari­ ables. a three­level optimization (+1, 0, ­1) arranged in central composite design (ccd) of response sur­ face methodology (rsm) was used to specify the optimal levels of each key parameters deduced from pbd approach. treatments we used penicillium expansum to create the dis­ ease in apples. the fungal spores were inoculated on potato dextrose agar (pda) slants and incubated for 7 days at 30°c. penicillium expansum was maintained on pda at 4°c for further use. to prepare a suspension of the fungal spore, spore­inoculated pda was washed using sterile water and added into seed culture medi­ um. seed medium was prepared by using modified minimal medium (g/l: yeast extract, nano3 2, kh2po4 1, mgso4.7h2o 1, kcl 0.5, and feso4.7h2o 0.01). apple fruits were disinfected in 1% sodium hypochloride dried at room temperature. with sterile and small plastic tip, some wound made on the fruits. pathogen and essential oil suspensions were dropped into each wound. all samples, control and the inocu­ lated, were stored in chambers with optimized stor­ age conditions. the diameter of the rot around each wound was measured after 18 and 26 days. al‐hassanavi et al. ‐ essential oils to control postharvest diseases 151 ethylene measurement ethylene analysis was performed using hplc analysis. the analysis was performed using a waters 244, column: shoex c18, 4 µm, 250 mm×4.0 mm, temperature: 28°c, injector volume: 20 µl. fluorescence detector (waters 470) set at 331 nm excitation and 500 nm emission wavelength. the mobile phase consisted of methanol/acetonitrile/ water (3:3:4 v:v:v), the ph value of the mixture was 2.5, and the flow rate was the 0.15 mlpm. the con­ centrations of ethylene of samples were calculated by the equation: cs ¼ cp as=ap; where cp and cs are the concentration of ethylene solution and sample solution, respectively, ap and as are the area of peaks of ethylene solution and sample solution respectively. statistical analysis and calculations each experiment was performed in triplicate. to optimize the production of monascus pigments, the software spss statistics 19.0 was used to generate pbd matrices and carry out multiple linear and non­ linear regression analyses. all tables and diagrams were prepared in microsoft excel 2010. the effects of the treatments on pigment composition, ethylene, and biomass were analyzed by one­way anova, and tests of significant differences were determined by using student’s t­test at p<0.05. 3. results application of essential oils to study essential oils (basil and peppermint) and storage conditions (temperature, ventilation, and rel­ ative humidity), twenty­four groups were analyzed based on pbd design shown in table 1. lesion diame­ ter (mm) ranged from 50.4 to 75.8. based on confi­ table 1 ­ effect of three independent sources including basil essential oil, peppermint essential oils, and storage conditions in posthar­ vest controlling of apple decay caused by penicillium expansum based on pbd. the parameters were basil and peppermint essential oil in three level (500, 1000, and 1500 microgram/l), temperature (­2, ­1, and 0°c), ventilation (3, 4.5, and 6 lpm), and relative humidity (85, 90, and 95%) independent variable lesion diameter (mm)basil essential oils peppermint essential oils (µgl) temperature (°c) ventilation (lpm) relative humidity (%) 1500 1500 0 6 95 69.2 1500 1000 0 6 90 72.3 1500 500 0 6 85 67.2 1500 1500 0 4.5 95 71.2 1500 1000 0 4.5 90 75.8 1500 500 0 3 85 63.2 1000 1500 ­1 6 95 62.2 1000 1000 ­1 6 90 65.3 1000 500 ­1 6 85 63.7 1000 1500 ­1 4.5 95 67.7 1000 1000 ­1 4.5 90 72.3 1000 500 ­1 4.5 85 56.1 1000 1500 ­1 3 95 64.9 1000 1000 ­1 3 90 70.1 1000 500 ­1 3 85 59.7 500 1500 ­2 6 95 56.5 500 1000 ­2 6 90 59.6 500 500 ­2 6 85 58.3 500 1500 ­2 4.5 95 62.6 500 1000 ­2 4.5 90 66.4 500 500 ­2 4.5 85 50.4 500 1500 ­2 3 95 59.2 500 1000 ­2 3 90 64.3 500 500 ­2 3 85 54.1 adv. hort. sci., 2023 37(2): 149­157 152 dence level (˃95%), the results showed that basil essential oils have a more significant effect on pre­ venting apple postharvest control of the fungal dis­ ease. of storage conditions, results showed that tem­ perature, ventilation, and relative humidity have a significant effect on controlling the fungal diseases in apple postharvest period. all storage conditions (temperature, ventilation, and relative humidity) were found to have a significant effect (p­value < 0.05). the results of multiple linear regression indi­ cated that basil essential oils as the best treatment under the condition set for storage (intercept effect) have a significant influence on postharvest control­ ling (table 2). when basil essential oils used as treatment, it decreased lesion diameter about 60% and 200%, respectively, compared to those observed in the fruits treated with peppermint essential oils (fig. 1). a significant percentage of increase 15% and 35%, respectively, was obtained, when basil essential oils used in association with ­2°c of temperature, 4.5 lpm of ventilation speed, and 90% of humidity com­ pared to other storage conditions (fig. 1). with regard to peppermint essential oils, the highest level of postharvest control was achieved in ­2°c of tem­ perature, 6 lpm of ventilation speed, and 85% of humidity. pertaining to storage conditions, temperature set to ­2°c increased postharvest control over the dis­ ease through declining lesion diameter, especially when basil essential oils was used as treatment, by an average decrease of 55% compared to the sam­ ples stored at ­1 and 0°c (fig. 2). based on data of pbd and rsm, sample fruits with application of basil essential oils maintained at ­2°c and ventilated with 4.5 lpm speed resulted in significant decrease of lesion diameter (fig. 3, 4). effect of essential oil application on apple ethylene concentration to determine the effect of optimized storage con­ ditions and essential oil treatment (basil and pepper­ fig. 1 ­ application of basil and peppermint essential oils on postharvest controlling of apple decay caused by penicillium expansum between treated fruits with basil and peppermint essential oils. values represented in the figure are expressed in percentage (%). a= yellow pigments. b= orange pigments. c= red pigments. * a = significant p­value<0.05. r2:066. adjusted r2: 061. p­value for the model = 0.0038; b = significant p­value <0.1. r2:071. adjusted r2: 074. p­value for the model = 0.002; c= significant p­value <0.1. r2:075. adjusted r2: 064. p­value for the model = 0.0029. table 2 ­ multiple linear regression of pbd data for postharvest controlling of apple decay caused by penicillium expansum using three independent sources including basil essential oil, peppermint essential oils, and storage conditions model b­coefficient t­value p­value confidence (%) a b c a b c a b c a b c intercept 53.642 48.155 46.49 8.642 9.392 9.962 1.32e­04* 5.4e­06* 4.8­01* 99.99* 99.99* 99.9* x1 30.458 24.971 23.306 4.542 5.292 5.862 0.175001 0.80123 0.16471 ­ ­ ­ x2 ­10.254 ­11.741 ­7.406 ­1.254 ­0.504 0.066 0.163501 0.17999 0.29019 ­ ­ ­ x3 ­12.429 ­8.916 ­6.581 ­1.429 ­0.679 ­0.109 0.192001 0.14872 0.41567 ­ ­ ­ x4 ­5.398 ­6.885 ­8.55 4.398 5.148 5.718 0.180501 0.29746 0.54115 ­ ­ ­ x5 34.783 29.296 27.631 1.217 1.967 2.537 0.0194* 0.0362* 0.0402* 98.06* 96.38* 95.98* x6 20.854 15.367 13.702 2.146 2.896 3.466 0.0249* 0.0102* 0.0313* 97.51* 98.98* 96.87* x7 15.16 9.673 8.008 2.84 3.59 4.16 0.0342* 0.0199* 0.0146* 96.58* 98.01* 98.54* x8 18.243 12.756 11.091 6.757 7.507 8.077 0.0322* 0.0168* 0.0497* 96.78* 98.32* 95.03* al‐hassanavi et al. ‐ essential oils to control postharvest diseases 153 mint), we analyzed the rate of the ethylene produc­ tion for 56 days. every 7 days, the ethylene rate was measured for control, basil, and peppermint treat­ ment. the production rate of ethylene was signifi­ cantly increased from 28 days and reached its highest level at the day 56 (p­value<0.05). the fruits treated with peppermint showed significant increase in the ethylene production from the 28th day. at the days 35 and 46, the level of ethylene in treated fruits was sig­ nificantly low compared to control fruits (p­ value<0.05). basil treatment reduced significantly rate of the ethylene from 35th day to 56th day (p­ value<0.05). fruits under the optimized conditions plus basil essential oil showed the lowest amount of the ethylene (fig. 5). 4. discussion and conclusions in this study, basil essential oil in three levels as well peppermint essential oil in three levels were evaluated to find the best treatment for postharvest control of apple decay caused by penicillium expan‐ sum using statistical methods, then the best treat­ ments were investigated under various storage con­ ditions (temperature, ventilation and relative humidi­ ty, each in three levels). the aim was to optimize and fig. 2 ­ effect of temperature on postharvest controlling of apple decay caused by penicillium expansum between treated fruits with basil and peppermint essential oils. values represented in the figure are expressed in percentage (%). fig. 3 ­ effect of ventilation on postharvest controlling of apple decay caused by penicillium expansum between treated fruits with basil and peppermint essential oils. values represented in the figure are expressed in percentage (%). fig. 5 ­ ethylene production rate of apple stored at control and optimized conditions along with the treatment with basil and peppermint essential oils. the control condition was ­2°c, 4.5 lpm, and 85% relative humidity. the best levels, determined for basil and peppermint in the previous sec­ tion of the experiment, were used to asses ethylene pro­ duction rate during postharvest storage of apple infected with by penicillium expansum. fig. 4 ­ effect of relative humidity on postharvest controlling of apple decay caused by penicillium expansum between treated fruits with basil and peppermint essential oils. values represented in the figure are expressed in per­ centage (%). 154 adv. hort. sci., 2023 37(2): 149­157 enhance postharvest control of apple using cost­ effective, safe, and eco­friendly plant materials. there is a few report that showed effect of basil and peppermint essential oil for controlling postharvest decay (domínguez­espinosa and webb, 2003; lopez­reyes et al., 2010). in the context of studying the effect of basil essential oil as controller of postharvest decay in apple, a study by mohammadi et al. (2021) showed that basil essential oil can be utilized as a low­cost material for preventing button mushroom (agaricus bisporus l.) during postharvest period. lopez­reyes et al. (2013) reported the effi­ ciency of use of peppermint essential oil in control­ ling rot in stone fruits. recently, the optimization and a combination of several factors to control posthar­ vest decay, has been the object of several study. for instance, essential oils of citrus species as mandarine (citrus reticulate, blanco), lemon (citrus aurantifolia (christm.) swingle), and orange (citrus sinensis l) have been reported as an effective inhibitor against some bacterial strains (escherichia coli, staphylococcus aureus, bacillus cereus) and yeast (candida albican) (fisher and phillipps, 2008). herein, controlling apple decay after harvest was subjected to two­step statistical method (pbd and ccd of rsm) in order to reduce capital cost of postharvest control, maximizing quality of apple and minimize safety concerns. it has been indicated that statistical approaches are a reliable method towards enhancing and optimization of the yield in various biological processes (jirasatid et al., 2013; embaby et al., 2018). in this regard, the optimal level of each essential oils from basil and peppermint influencing postharvest control of decay in apple was deter­ mined to reduce decay effectively. with regard to essential oils, the highest level of controlling and the lowest decay observed in samples upon using basil essential oils as the treatment. basil essential oils enhanced inhabitation of the decay in apple more than the peppermint essential oil (fig. 1). this might be attributed to the nature of basil essen­ tial oil component in degrading fungus­caused dis­ eases (grande­tovar et al., 2018) compared to pep­ permint essential oil. action of the mono­ and sesquiterpenes and mono­ and sesquiterpene hydro­ carbons in basil essential oils could inhibit fungal dis­ eases (caccioni and guizzardi, 1994). reports regard­ ing essential oils as pesticides and antimicrobial agent have shown that the inhibitory effect of essen­ tial oils to prevent fungal diseases during postharvest period strongly associated with the monoterpenic phenols, notably thymol, carvacrol and eugenol in the oils (antunes et al., 2010). hence, the rich com­ pounds of basil essential oil would be enhance fungistatic activity and as a result the effective con­ trol over the fungal disease (prakash et al., 2015; namiota and bonikowski, 2021). it seems that essen­ tial oil content of peppermint has a lower level of fungicidal activity compared to basil, although it reduces the growth of the disease when relatively high amount of essential oil used as treatment. pertaining to peppermint essential oils, this study showed that peppermint essential oil could reduce decay caused by the fungal disease in apple samples (fig. 2). previous studies have revealed the effect of peppermint essential oils as postharvest treatment in reducing decay and maintaining quality of fruits and vegetables (sellamuthu et al., 2013; qu et al., 2020). since menthol is the main constituent of peppermint essential oil, this compound could be mainly respon­ sible for the effects. furthermore, several studies suggest that the effect of peppermint might be indi­ rect and through raising the activity level of superox­ ide dismutase (jin et al., 2009). the results of the study showed that the best level of basil essential oil as treatment is 1000 and 1500 µg/l, while the best level for peppermint was 1500 (fig. 1). the difference between 1000 and 1500 µg/l of basil was not significant. lopez­reyes et al. (2010) reported that 10% emulsion of essential oils could be more effective than 1% of the emulsion in controlling decay. however, scientific evidences are limit in the field of concentration effects on the inhibitory ability of essential oils during postharvest phase. here, our findings suggest that 1000 and 1500 µg/l essential oil decrease the lesion diameter in apple compared to 500 µg/l, while the effect of basil essential oil with 1000 and 1500 µg/l is similar. in the case of peppermint, there is significant difference between 500, 1000, and 1500 µg/l. this might result from the nature of essential oil constitutes. more study is needed to support the power of constitutes in controlling postharvest diseases. temperature of the storage place is one of the main factor for optimal maintain of fruit and vegeta­ bles during postharvest phase (agboyibor et al., 2018; patrovsky et al., 2019; silbir and goksungur, 2019; liu et al., 2020). several study have already showed that different isolates of fungal diseases grow best under an optimum temperature range of 30°c to 37°c (mannaa and kim, 2017; liu et al., 2018). these findings support the results of this study al‐hassanavi et al. ‐ essential oils to control postharvest diseases 155 where higher temperature showed a higher lesion diameter. alternatively, an optimum relative humidi­ ty of the storage place is the point of a discrepancy. a study on some strains of fungal­cause diseases revealed that relative humidity of 85% promotes the fungus growth and thereby a higher decay (nabi et al., 2017). another study reported that the greatest diameter of lesions achieved at relative humidity of 95 to 98% 5 (arah et al., 2015). in our study, sole effect of relative humidity was not significant between groups. however, it seems that high tem­ perature trigger relative humidity effect on the growth the fungal disease. according to our results, in the absent of essential oil, fruits stored at high temperature and relative humidity showed higher lesion diameter. regarding ventilation, partridge­ hinckley et al. (2009) reported the highest occur­ rence of the decay in fruits stored in the poor ventila­ tion conditions. moreover, carmona­hernandez et al. (2019) showed that aeration in terms of agitation at 150 rpm promotes the growth and distribution of fungal diseases. with regard to ethylene production rate (µg kg­1 s­1) in the control and optimized storage conditions, results indicated that the level of ethylene in the con­ trol increases and reaches its highest level at 56 days after treatment. the same pattern meets for opti­ mized conditions (fig. 5). on the other hand, the finding suggests that basil and peppermint treatment significantly decrease the ethylene production rate at 32 and 42 days. after the day 42, basil treatment continues its inhibitory effects compared to pepper­ mint treatment, although the ethylene production rate is significantly high and damage apple storage. our results is consistent with those findings that have shown application of exogenous material such as melatonin, polyamines, and calcium limits rate of ethylene production (wang et al., 1993; bulens et al., 2012; onik et al., 2021). jhalegar et al. (2015) showed that essential oil treatment could lower eth­ ylene rate in fruits affected with fungal diseases dur­ ing postharvest storage. ethylene production is the main factor in deterring postharvest life of any fruits, especially apple. data from various studies have shown that there is almost l inear correlation between the ethylene production and fruit damage during storage phase (cristescu et al. , 2002). meanwhile, fruits affected with postharvest diseases show the higher level of the ethylene, thereby higher decay and damage (jhalegar et al., 2012). there are limited scientific reports on application of essential oils to control ethylene production during the postharvest period. two studies by moline and locke (1993) and sharafi et al. (2011) showed the decrease in the ethylene production in fruits treated with plan essential oils. here, we represented that basil and peppermint decrease the ethylene rate in apple, as well as we optimized storage conditions with the treatment. this study used the comprehensive combine of treatments along with well­designed experiment to show an efficiency of plan essential oils to control apple decay during postharvest phase, and mainly to find the best treatment and storage conditions. it is well understood that postharvest conditions depend on several factors and we showed that treatments need to be aligned with storage condition. thereby, we showed that 1000 µg/l of basil essential oil is sig­ nificantly effective under ­1 and 0°c, 4.5 lpm, and 90% of relative humidity. while 1500 µg/l of pepper­ mint need to be under ­1ºc, 6 lpm, and 85% humidi­ ty to be effective. furthermore, basil essential oil decreased the ethylene production rate in longer days after treatment. the findings 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animal resources development board, po box 5016, kigali, rwanda. key words: electrical conductivity, organic acid, solids soluble concentration. abstract: salt stress often can enhance the fruit quality of tomatoes. seawater is one of the substrates used by growers. however, utilization of seawater on tomato production is difficult in the hinterland as it is far away from the sea­ side. some “onsen” water also show a high salt concentration (2%). therefore, it could also be used as a substrate of salt stress treatment. in this study, salt stress was provided by yupoka “onsen” water, and the effects of different nutrient ecs on plant growth and fruit quality of tomatoes were investigated. tomato plants ‘reika’ were grown in pot soil, and nutrients with ec 2, 4, 8 and 12 ms/cm were applied at the time of irrigation. the fruits were harvested at turning stage until the 3rd truss. soil salinity attained ec 3.6, 6.7, 12.8, and 15.6 ms/cm. ssc, organic acid, dry matter and no3 ­ increased by 50, 79, 50 and 27%, respectively at ec12 ms/cm while, weight, size, and water content decreased up to 40, 20, and 4%, respectively. however, fruit cracking did not occur appar­ ently. most of the plant growth parameters were reduced. 1. introduction tomato (solanum lycopersicum) is one of the most widely cultivated horticultural crops in the world (fao, 2016). tomato fruits are rich in min­ erals, vitamins, essential amino acids, sugars and dietary fibres and thus contribute to a healthy, well­balanced diet. the majority of tomato fruits produced are consumed in processed form such as peeled tomato (whole or diced), juices, sauce and ketchup, whose manufacture often requires peel removal (shankara et al., 2005; rock et al., 2012). improvement of fruit quality is an urgent issue for tomato growers and consumers (ho, 1999; lu et al., 2019). sugars and organic acids are the most important factors for determining the fruit quality (lu et al., 2019). soluble solids content (ssc) of ripe tomato fruit is usually 3 to 5% and can reach values over 10% (balibrea et al., 2006; gautier et al., 2010). in japan, “shio” or salt tomatoes, are cultivated in the area of uto and yatsushiro in kumamoto prefecture. they are grown on drained land, rich in (*) corresponding author: musabyisoni@gmail.com citation: musabyisoni a., waweru b., nishizawa t., 2021 ­ effect of salt stress by “onsen” water on plant growth and fruit quality of tomato cv. reika in pot soil. ­ adv. hort. sci., 35(4): 383­388. copyright: © 2021 musabyisoni a., waweru b., nishizawa t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 february 2021 accepted for publication 7 december 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10714 https://www.frontiersin.org/articles/10.3389/fpls.2018.01778/full#b21 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 383­388 384 salt and other minerals. the wholesale price of “shio” tomato (have ssc of 10% or higher) is often ten times higher than common tomatoes (http://higoayuminokai.co.jp/sawamura.html). high electrical conductivity (ec) treatment is a well­known technique to increase ssc of tomato fruit because the decreased osmotic potential of nutrient solution restricts transport of water to fruit (adams et al., 1991; cuartero and fernandez­munaoz, 1999; shabala et al., 2012). however, plant exposed to salt stress for long time suffer from phyto­toxicity due to nacl accumulation. this leads to nutrition imbalance and the uptake of some ions is inhibited (zhang et al., 2017). amjad et al. (2014) showed a linear decline of the absorption of macro and micro ions by fruit as ec and truss position become higher. the plant growth and fruit yield began to decline when the nutrient solution exceeded ec 2.5­4.0 ms/cm (bustomi et al., 2014). in previous studies, ec 5.5 m/.cm treatment reduced the tomato yield by 22.4~31.1%, 12.6~28.0% and 11.7~27.3% in 2012, 2013 and 2014, respectively (zhai et al., 2015), but ssc increased (oztekin and tuzel, 2011; zhang et al., 2017). other studies report­ ed in soil­less tomato culture that ssc increased while fruit weight decreased at high salinity (magán et al., 2008; kamrani et al., 2013). although tomato growers commonly use nacl or seawater to increase ec, it is well known that some “onsen” like haguro has about 2% of nacl. therefore, it may be used as an alternative source of salt stress. this research was conducted to assess if high quality tomato fruit can be grown under salt stress by “onsen” water. 2. materials and methods four different salt stress treatment (ec 2, 4, 8, and 12 ms/cm) were evaluated on tomato plant ‘reika’ during spring to summer in 2016. “onsen” water obtained from yupoka in haguro town, yamagata/japan was used as it has a high salty level (ec 40 ms/cm of ec, 2% of nacl and ph 7.2). initially, the seeds were sown on moist papers in petri dishes on 5 april, 2016 and maintained in a controlled growth chamber (24°c; 14/10 h light/dark photoperi­ od; rh 45­50%) to induce germination. on 08 april, the germinated seeds were transferred in cell tray (100 ml) in a glass­house and twenty days after (on 28th april), the seedlings were transplanted into 500 ml pots filled with a planting medium (baido 300 g containing 0.22 g/l of nitrogen). the seedlings were maintained in the same glass­house (18.7°c; rh 76.8%). on 17 may, twenty tomato seedlings with 5­ 7 expanded leaves were further transplanted into plastic pots (25 l) containing baido 300 g and 300 g of organic fertilizer with 3% of nitrogen (pro­bokash­ kantonosan, tochigi) and maintained in a green­ house. one month (on 16th june) after transplanting (flowering stage), the plants were treated with differ­ ent concentration of “onsen” water (ec 2, 4, 8, and 12 ms/cm) replicated five times. the experiment was laid out in randomized complete block design (rcbd). the ec 2 was 100 g of hyponex npk 6­10­5 diluted in 100 l of water. for other treatments (ec 4, 8 and 12), “onsen” water was added into ec 2. the ec meter (laquatwin ec, horiba,tokyo) was used to adjust the solution ec. the applied solution was ec 2±0.1, ec 4±0.1, ec 8±0.1, and ec 12±0.1. the nutri­ ent solutions were prepared every week. watering was done daily using tap water during the first month (1.8 l/pot). for control of soil/root insects, g f orthoran (bifenthrin) insecticide was applied once during the season at rate of 5 g/pot. while for the control of above ground insects, frutrifol+tebuconazole, best guard (1 g/l) mixed with a sticker as adjuvant (0.5 ml/l) was also applied every two weeks. plants were kept as single stem with five trusses and five fruits per truss. fruits were harvest­ ed from three trusses by taking two fruits on each at green maturity until the end of harvest (4 aug. 2016). the daily average temperature was 24.1°c and rh was 74.2% in the green­house during the growing season. plant height, stem size, leaf size, thickness, and spad were measured once a week during the plant cultivation from first “onsen” water application until first harvesting. after “onsen” water application, approximately 2 l of tap water were applied once a week into soil pots, and the exudate was collected. ec, ph, nacl and no3 ­ion concentrations in the exu­ date were measured using ec meter (laquatwin ec, horiba,tokyo), ph meter (laquatwin ph, horiba, tokyo), nitrate ion meter (laquatwin no3 ­, horiba, tokyo) and, nacl meter (cm­14 p, toa, tokyo). for the chemical analysis (ssc, organic acid and no3 ­ion), harvested fruits with same skin color selected using color reader (cr­10, konica minolta, tokyo) were cut into half longitudinally. thereafter, samples were taken transversally using cork borer, grinded in the mortar and filtrated by tissues filter. the extracts were centrifuged at 6000 rpm for one minute. ssc, organic acid and no3 ­were measured by digital musabyisoni et al. ‐ salinity tolerance of tomato plant 385 refract­meter (pr­101, atago, tokyo), organic acid meter (pal­bxiacid f5, atago, tokyo) and nitrate ion meter (laquatwin no3 ­ b­341, horiba, tokyo) respectively. before measurement, the respective meters were opened and the glass electrodes calibrated with dis­ tilled water/or standard solution followed by rinsing with distilled water and wiping using paper towels. extracted juice per sample was filled slowly on the glass electrode without bubbling, and the measure­ ments were recorded. prior to measuring, the juice for organic acid was diluted indeionized water using a ratio of 1:50 (0.1 ml of juice into 4.9 ml of desalted water). ssc was expressed in % brix, organic acid in %, and no3 ­in ppm. half of the harvested fruit samples were frozen at ­20°c for ten days. the samples were dried using eyela freeze dryer fdu­540. the wet and dry sam­ ples were weighed in order to determine fruit water and dry matter content. the dried ones had been powdered in mortar using liquid nitrogen. one gram of the powdered sample was put in a crucible, and incinerated at 200°c for 2 hours, 550°c for 10 hours, and 200°c for 2 hours, respectively. fruit ash content was calculated by weighing the sample. data analysis were performed using the statistical analysis software package, genstat 19th edition at 5% level of significance. least significant difference test (lsd) was used to separate treatment means. 3. results the average temperature was 24°c and rh 74% in the green house during tomato cultivation. the dif­ ference in plant height among salt stress treatment was observed after about three weeks “onsen” water application. fruit ssc, organic acid and no3 ­ increased by about 50, 79 and 26.5%, respectively in ec12 ms/cm while ssc: oa ratio was not affected by high ec12 ms/cm (fig. 1 and 2). tomato plants treat­ ed with ec2 ms/cm and ec4 ms/cm produced bigger fruit than ec8 ms/cm and ec12 ms/cm (fig. 1 and 2). the fruit weight, size decline up to 50% whilst dry matter almost doubled in ec12 ms/cm (fig. 2). the ec, salt and no3 ­ concentration in culture medium increased by 53.9, 70.3, and 52% at higher ec because of high amount of “onsen” water applied. the high ph of “onsen” water (ph 7.2) did not affect the culture medium ph in high ec. the difference in plant height among salt stress treatment was observed after about three weeks “onsen” water application. the curve indicates that tomato plants treated with ec2 and ec4 ms/cm had thicker stems and wider leaves than other treatments (fig. 3). plants treated with ec2 ms/cm and ec4 ms/cm were the tallest compared to other treatments (fig. 4). the reduction of plant height, leaf size, and thickness was 12%, 26.8%, and 21.3% respectively in ec12 com­ pared to ec2. 4. discussion and conclusions salt stressed by “onsen” water showed a signifi­ cant difference (p<0.0001) on fruit soluble solids, fig. 1 ­ effect of different concentration of “onsen” water on fruit ssc brix%, dry matter and organic acid of tomato plants variety ‘reika’ in the greenhouse. fig. 2 ­ effect of different concentration of “onsen” water on fruit size, weight and no­3 of tomato plants variety ‘reika’ in the greenhouse. adv. hort. sci., 2021 35(4): 383­388 386 organic acid, no3 ­concentration, and average fruit weight. massaretto et al. (2018) demonstrated that soluble solids content is an important fruit quality parameter which increases depending on the salinity levels. in their study soluble solids contents were sig­ nificantly higher in both negro yeste (60%) and verdal (78%) landraces compared with moneymaker (34%) tomato variety under salt stress. in general, there is a negative relationship between ssc and weight in tomato (higashide and heuvelink, 2009). many studies (adams and ho, 1992; cuartero and fernández­munaoz, 2001; cuartero et al., 2006) demonstrated that one of the well­known effects of salinity is the increasing of ssc and antioxidants but with a reduction in the fruit size and weight. in pre­ sent study, high ec increased the tomato fruit ssc and organic acid while weight and size declined (fig. 1 and 2). fruit weight and size reduction under salini­ ty stress was triggered by inhibition of water uptake by the root resulting in insufficient amount of water required for the fruit and increased concentrations of reducing sugars and acids as compared to non­saline conditions. thus increased concentration of soluble solids was observed in the present study (fig. 1). fruit juice acidity increased with high ec and this could be due to the higher na content in the fruit juice, since this was the main ion found in “onsen” water. amjad et al. (2014) stated that the accumula­ tion of reducing sugars and organic acids is responsi­ ble for increased titratable acidity and decreased ph of the fruit juice. the reduction in fruit water content have caused an increase in fruit dry matter content (fig. 3). this could be as a result of drought leading to excess amounts of salt in the root zone, which leads to reduced photosynthetic capacity, or toxicity of salt in plant tissues (amjad et al., 2014). at the first truss, fruit weight was not significantly influenced by salinity. the reductions started at the second truss and this can be explained by long expo­ sure on salinity. considering a reduction in fruit fresh weight (40%) compared with the increase in dry weight (50%), and ssc (50%), the lower water content in the fruits can be pointed as the main reason for the reduction in fruit weight. the accumulation of na in the soil pot showed by the salt concentration in drained soil solu­ tion (from 0.68% to 0.9%) was due to high amount of “onsen” water applied. this contributed to the increased nutrient solution ec (from ec12 to ec16) which probably created an osmotic pressure around the roots. thus, a reduction of water uptake by plants had resulted by no3 ­concentration in culture medium which increased 1000 ppm to 1600 ppm. most of the growth parameters were affected by high salinity treatment. the plant height, stem diam­ eter, leaf size were significantly decreased (fig. 3 and 4). bustomi rosadi et al. (2014) mentioned that the plant height was significantly affected by high salinity due to water stress. ashraf et al. (2021) also reported that tomato plants grown under salinity stress in the presence of 200 mg n kg­1 in different nh4+:no­3 ratios showed a variable decline in growth in terms of plant height, plant girth, and stem diameter com­ pared to no salinity treatment (respective controls). the reduction in plant growth parameters of our fig. 3 ­ effect of different concentration of “onsen” water on stem diameter, leaf size and spad of tomato plants variety ‘reika’ in the greenhouse. fig. 4 ­ effect of different concentration of “onsen” water on height of tomato plants variety ‘reika’ in the greenhou­ se. musabyisoni et al. ‐ salinity tolerance of tomato plant 387 results can be explained by low water potential of the soil solution due to the high ec and salt concen­ tration as it was evident in drained solution. our results also showed an increase of fruit no3 ­ in high ec whereas fruit size and weight were reduced (fig. 2), this can be attributed to transport of water from the roots to the fruits.the salt stress by “onsen” water in the nutrient solution reduces the ability of the plant to take up water, and this leads to reduc­ tions in the growth rate. results of our experiment indicate that the salt stress by “onsen” water is also effective for increasing the sweetness of tomato fruit. in conclusion, the ec8 and ec12 treatments have the most effective to increase the sweetness of tomato fruit with a reduction in fruit weight. acknowledgements this work was sponsored by japan international cooperation agency (jica), as part of the long­term training on “young leaders development in agriculture sector” (master program) jfy 2016. the authors are grateful to yamagata university and rwanda agriculture and animal resources development board for providing opportunities to conduct this study. references adams p., 1991 ­ effects of increasing the salinity of the nutrient solution with major nutrients or sodium chlo‐ ride on the yield, quality and composition of tomatoes grown 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production. ­ processing and marketing, agrodok, 17. zhai y., yang q., hou m., 2015 ­ the effects of saline water drip irrigation on tomato yield, quality, and blos‐ som‐end rot incidence ‐ a 3a case study in the south of china. ­ plos one, 10(11): e0142204. impaginato 11 adv. hort. sci., 2020 34(1): 11­24 doi: 10.13128/ahsc­8252 effect of foliar spray of calcium lactate on the growth, yield and biochemical attribute of lettuce (lactuca sativa l.) under water deficit stress a. khani 1, t. barzegar 1 (*), j. nikbakht 2, z. ghahremani 1 1 department of horticultural sciences, faculty of agriculture, university of zanjan, zanjan, iran. 2 department of water engineering, faculty of agriculture, university of zanjan, zanjan, iran. key words: anthocyanin, antioxidant enzymes, leaf water status, nutrient uptake. abstract: the field experiment was conducted to evaluate the effect of foliar spray of calcium lactate (ca) on fresh yield and biochemical attribute of lettuce (lactuca sativa l.) under water deficit stress, in a split plot form based on a ran­ domized complete block design with three irrigation regimes (70, 85 and 100% etc) and three calcium lactate treatment levels (0, 0.75 and 1.5 g l­1) in three replicates. results revealed that water deficit stress significantly reduced the growth and yield of plant, leaf relative water contents, excised leaf water retention and n, p and mg absorption while led to increase anthocyanin, phe­ nol and flavonoids contents, antioxidant activity, peroxidase and catalase activ­ ity and water use efficiency. the results of our research indicated that the application of cal 1.5 g l­1 is capable of increasing lettuce yield, under field con­ ditions with 30% less than optimal irrigation. cal treatment showed a clearly protective effect in stressed plants, enhancing their leaf water status, antioxi­ dant capacity and n and ca contents in comparison to untreated plants. therefore, feeding leaves by cal with increasing antioxidant activity and nutri­ ents content especially n led to increase growth and fresh yield of lettuce under normal irrigation and water deficit conditions. 1. introduction abiotic stresses such as high temperature, drought, salinity and chemi­ cal toxicity, are the most important limiting factors to crop productivity. drought is undoubtedly one of the most important stresses that have huge impact on growth and productivity of the crops (fahad et al., 2017; hussain et al., 2018). water stress is the most prominent abiotic stress limiting agri­ cultural crop growth and productivity (gholipoor et al., 2013; ihsan et al., 2016). deficit irrigation stress as a consequence of the progressive decrease in water availability has been a hot topic regarding food security during the last two decades (unesco, 2012). growth and development of plants is influenced by reduction in turgor that result in decreased nutrient acquisi­ (*) corresponding author: tbarzegar@znu.ac.ir citation: khani a., barzegar t., nikbakht j., ghahre­ mani z., 2020 ­ effect of foliar spray of calcium lactate on the growth, yield and biochemical attribute of lettuce (lactuca sativa l.) under water deficit stress ­ adv. hort. sci., 34(1): 11­24. copyright: © 2020 khani a., barzegar t., nikbakht j., ghahremani z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2019 accepted for publication 14 may 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 11­24 12 tion from dry soil (luo et al., 2011). due to the threat of climate change, there is a need to limit the use of water resources in arid and semi­arid climates. it is therefore important to find new approaches to avoid crop productivity losses in ‘limited fresh­water’ areas. lettuce (lactuca sativa l.), an annual plant of asteraceae family, is considered as one of the most important salad vegetables as a cool season crop. lettuce leaves contain vitamins c and e, carotenoids, phenolic acids with anti­free radical activity, and min­ erals with a lot of fiber, which are an important part of the human diet. moreover, lettuce contains lac­ tocin and lactucopicrin which improve the quality of sleep (chakraborti et al., 2002; faostat, 2016). since most of vegetable species are shallow­root­ ed, they are sensitive to mild water stress. in lettuce production, it is particularly important to preserve optimal growth through a well­scheduled irrigation program, where the harvested part of the plant is the photosynthetic leaf area, (ahmed et al., 2000; casanova et al., 2009). its leaves have high water content and it is sensitive to mild water deficit stress due to its shallow root system (kizil et al., 2012). therefore, in lettuce, new strategies will become crit­ ical to enhance productivity under deficit irrigation (malcom et al., 2012). foliar application of agro­chemicals has widely been used in agriculture as a rapid, low­cost and effective way for enhancing growth and productivity of many vegetable crops under water deficit stress especially green leafy vegetables like lettuce. calcium lactate is considered as one of important agro­chemi­ cal which can be spray and play important roles in physiological and biochemical processes. calcium (ca) is the mineral nutrient most commonly decrease absorption under water deficit condition, so increas­ ing the calcium content in the leafy vegetables could further improve ca concentrations in plant tissues (grusak, 2002). ca is an essential macronutrient for plant growth and development, and is considered as an important intracellular messenger, mediating responses to hor­ mones, stress signals and a variety of developmental processes. furthermore, ca is an important compo­ nent in the structure of cell walls and cell membranes (hepler and winship, 2010). ca plays a role in the regulation of various mechanisms of plants under environmental conditions such as water stress, heat, cold and salinity. in addition, calcium signaling is required for acquisition of tolerance or resistance to the stress (cousson, 2009). positive effect of calcium in improving stress tolerance can be attributed to regulate of water status, antioxidant systems activity, osmolytes accumulation, improving photosynthetic pigment content, and nutritional balances (kurtyka et al., 2008). ca plays an important role in oxidative stress signaling, linking h2o2 perception and induc­ tion of antioxidant genes in plants (rentel and knight, 2004). ca participates in most cellular signaling processes (sanders et al., 2002) and interacts strong­ ly with reactive oxygen species (evans et al., 2005). since the combined effects of ca and water deficit stress have hardly been reported, the current study was, therefore, designed to evaluate the influence of foliar application of calcium lactate on the growth, yield and biochemical attribute of lettuce cv. new red fire under water deficit stress. 2. materials and methods experimental design the field experiment was carried out at the research farm of the agriculture faculty, university of zanjan, iran, during 2017. the experiment was performed using a split plot based on a randomized complete block design with three irrigation regimes (70, 85 and 100% etc) as the main plot and three cal­ cium lactate (cal) treatment levels (0, 0.75 and 1.5 g l­1) as the sub­plot in three replicates. the soil prop­ erties of experimental filed as well as average daily climatic data during the growing seasons was shown in tables 1 and 2, respectively. plant material seeds of lettuce (lactuca sativa l.) cv. new red fire was obtained from a “takii seed” company. lettuce seeds were sown in the nursery on the 2nd of august. seedlings were transplanted at the 3­4 leaf stage when the seedlings were four weeks old with 25 cm spacing within row and 35 cm spacing between rows that there were about 11.5 plants per square meters (plants m­2). table 1 ­ soil physical and chemical properties on the site of experimental field soil texture organic matter (%) ph ec (ds m­1) n (%) ca (g kg­1) na (g kg­1) k (g kg­1) loam clay 0.94 7.4 1.49 0.07 0.12 0.13 0.20 khani et al. ‐ protective effect of calcium lactate on lettuce 13 irrigation treatments and calcium lactate applications after plant establishment, lettuce plants were sprayed with different concentration calcium lactate at 6­7th leaf stage, 10 and 20 days after first spraying for 3 times, during the plant growth. irrigation treat­ ments were applied one week after the first spraying. all foliar sprayings time were the same and distilled water was used for control treatment. the three irri­ gation levels were calculated based on actual evapo­ transpiration (etc): (1) control, irrigated 100% crop water requirement (i100), (2) deficit irrigation 85% etc (i85), and (3) deficit irrigation 50% etc (i50). the water requirement of the plant for control treatment was estimated using long­term average daily data of meteorological parameters recorded at zanjan meteorological station and following relation. etc = et0 × kc etc: water requirement of lettuce (mm/day), et0: evapotranspiration of grass reference plant (mm/day) and kc: vegetable coefficient of lettuce (no unit). it is necessary to explain that et0 values were estimated based on the standard fao­penman­ monteith method. table 2 shows the long­term average of meteorological parameters of zanjan synoptic station during the period of plant growth which was used to calculate et0 and etc values. after calculating the etc values, the net and gross irrigation water requirements of lettuce were estimated based on cropping intervals, type of irrigation system and irrigation interval and then give the plant at each irrigation time. based on the calculations, the amount of irrigation water given to the control plants was estimated to be 895.7 m3.ha­1. water requirement of other treatments was estimat­ ed and distributed based on the water requirement of control treatment and water stress (allen et al., 1998). all necessary management practices such as weeds control were done according to recommend­ ed practices during the crop growth. measurements anthocyanin content. anthocyanin content in leaf tissue was determined according to the method of mita et al. (2000). fresh weight of leaves (0.1 g) was homogenized in methanol containing 1% (v/v) hcl and then was filtrated. the filtration was stored at 4°c for 24 hours in dark conditions. the absorbance of filtration was recorded at 550 nm using uv­vis spectrophotometer (specorp 250 jena­history) and the anthocyanin was expressed as µmol g­1 fw. total phenols and flavonoids contents the fresh leaf tissue (2.0 g) was washed with deionized water, and homogenized in 80% cold methanol (20:80, v/v). the homogenate was cen­ trifuged at 10,000 rpm for 10 min, and the super­ natant was collected for the measurement of, total phenolic and flavonoid content. total phenolics assay was carried out according to the procedure described in the literature (meda et al., 2005). the results were expressed as mg of gallic acid equivalents (gae) per 100 g of fresh weight based on a standard curve using gallic acid as standard. total flavonoids were determined by the colorimetric method (kim et al., 2002). quercetin was used as a reference standard, and the results were expressed as mg quercetin equivalents per 100 g fresh weight of leaf. antioxidant activity as mentioned in the previous paragraph, 2.0 g of leaves were homogenated in methanol and then was centrifuged. the filtration was used to determine free radical scavenging activity using the 2, 2,­ diphenyl­2­picryl­hydrazyl (dpph) method at optical density 517 nm (sun et al., 2007). antioxidant activity (%) was calculated using the following equation: antioxidant activity = a dpph ­ a sample (517 nm)/a dpph × 100 catalase (cat) and peroxidase (pox) enzymes activity samples were taken from the fully expanded leaf and transferred to the laboratory in the ice. leaf sample (0.5 g) was frozen in liquid nitrogen and ground using a porcelain mortar and pestle. catalase (cat) activity was measured by following the decomposition of h2o2 at 240 nm with a uv spec­ trophotometer (cakmak and horst, 1991). samples table 2 ­ average daily climatic parameters of zanjan synoptic station during the growth seasons (2017) of lettuce meteorological parameter may june july august september rainfall (mm) 0.01 1.11 5.00 0.00 0.02 average temperature (˚c) 22.94 25.71 27.68 24.79 15.73 minimum temperature (˚c) 11.29 16.8 17.61 14.68 7.89 maximum temperature (˚c) 32.47 33.96 36.82 35.12 25.05 adv. hort. sci., 2020 34(1): 11­24 14 without h2o2 were used as blank. the activity of cat was calculated by the differences obtained at od240 values at 30 second interval for 2 min after the initial biochemical reaction. peroxidase (pox) activity was measured using modified method of the tuna et al. (2008) with guaiacol at 470 nm. a change of 0.01 units per minute in absorbance was considered to be equal to one unit pox activity, which was expressed as unit g­1 fw min­1. leaf water status (rwc, elwr) the fresh weight of young leaves (fw) was recorded and then was kept in petri dishes for 24 hours immersed in distilled water. the turgid weight (tw) was measured after saturation of leaves with water. the leaves were dried at 70˚c to constant weight and then weighted (dw). leaf relative water contents (rwc) were calculated according to the fol­ lowing formula reported by hanson and hitz (1982). (%) rwc= (fw­dw) / (tw­dw) × 100 for the determination of excised leaf water reten­ tion (elwr), the youngest leaves collected for each treatment were weighed to record fresh weight (fw), kept at room temperature (25˚c) for 6 hours and reweighed (wl). elwr was calculated using the following formula suggested by lonbani and arzani (2011). elwr= [1­(fw­wl)/fw] ×100 nutrient contents the lettuce leaf samples from each treatment were collected at the end of the experiment. for mineral analysis, leaf samples were taken and oven­ dried at 70°c until constant weight. then 0.3 g of the dry samples was taken and digested using a mixture of sulphuric acid (h2so4) and hydrogen peroxide (h2o2) as described by allen et al. (1974). all the stud­ ied elements were assayed in the digest of the con­ cerned plant samples. total nitrogen was determined using kjeldahl method as described by piper (1950). phosphorus determination was done by complexing it with ammonium molybdate, which on reduction with ascorbic acid gives stable blue colour, the con­ tent of p was measured by spectrophotometer at 882 nm according to watanabe and olsen (1965). potassium, calcium and magnesium content were analyzed by flame photometer (chapman and pratt, 1961). yield and water use efficiency (wue) lettuce plants were weighed after harvest with a digital gravimetric scale. the average weight of single plant was calculated in grams and total yield was estimated in kg/m2. also water use efficiency (wue) was obtained from the ratio of the amount of yield of each treatment to the amount of water consumed by the same treatment in kg m­3. statistical analyses of the data the analysis of variance (two­way anova) and least significant difference (lsd) test (p≤0.05 and p≤0.01) used to compare means within each sam­ pling date. the statistical analysis and standard error calculation were carried out using sas software (v. 9.1). 3. results anthocyanin content the data in table 3 and figure 1, displays the anthocyanin contents of lettuce leaf applied with dif­ ferent concentrations of cal under water deficit. mean comparisons of data showed that deficit irriga­ tion led to a significant increase in antioxidant activi­ ty compared to control. however, the effect of cal on the anthocyanin contents was depended to the ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non­significance at p<0.05. table 3 ­ variance analysis (anova) of effect of calcium lactate on physiological characteristics in lettuce under deficit irrigation s.o.v df mean of squares anthocyanin total phenols flavonoids cat activity pox activity antioxidant activity rwc elwr replication 2 1.003 0.002 0.308 0.057 0.003 16.725 8.614 23.677 irrigation 2 2.162 ** 6.187 ** 11.932 ** 1.953 ** 0.851 ** 132.47 ** 42.799 * 238.260 ** error (a) 4 5.648 0.135 0.330 0.009 0.001 23.524 9.036 14.106 calcium lactate 2 4.292 * 1.057 ** 24.265 ** 0.702 ** 0.052 * 318.416 ** 186.691 ** 420.124 ** calcium lactate × irrigation 4 2.770 * 0.475 * 1.149 * 0.075 * 0.030 * 43.004 * 1.302 ns 29.661 * error (b) 12 6.388 0.121 0.338 0.023 0.008 9.686 9.323 6.176 coefficient of variation (%) ­ 11. 18 2. 31 4.19 8.87 16.93 3.73 3.9 3.4 khani et al. ‐ protective effect of calcium lactate on lettuce 15 total phenols and flavonoids contents the exposure to water deficit stress significantly (p<0.05) increased total phenols and flavonoids con­ tents (table 3, figs. 2a, b). besides, the results of the present study also showed that foliar application of cal increased total phenols and flavonoids contents under normal and deficit irrigation, however, the effects of cal was dependent to the irrigation levels. the maximum value of phenols and flavonoids con­ tents was recorded in plant treated with 0.75 g l­1 cal under irrigation 70% etc. in all levels of irrigation, application of 0.75 g l­1 cal had the greatest effect on total flavonoid content, although did not show significant difference with cal 1.5 g l­1 under irriga­ tion 70% etc. phenolic compounds include many secondary metabolites in plants that display antioxidant proper­ ties (barbagallo et al., 2012). some of the phenolic compounds, such as phenolic acids or flavonoids, are widely recognized in most of the plant species (jwa et al., 2006). phenolic compounds are important because of their contribution to the nutritional quali­ ty attributes of fruits and vegetables such as color, astringency, bitterness and flavor (vinson et al., 2001). the role of phenols as antioxidant is support­ ed by several researches and the recovery methods have a great importance for industrial use (barbagallo et al., 2012). environmental stress can cause an increase in the content of phenolic com­ pounds of cell (weidner et al., 2009). aghdam et al. (2013) reported that the total phe­ nols and flavonoids contents increased in the cor­ nelian cherry fruit with cacl2 treated. their results suggested that cacl2 treatment may stimulate the accumulation of phenols and flavonoids fruits by acti­ vating their biosynthetic pathways. biosynthesis of phenols such as flavonoids in plants carried out via the shikimate­phenylpropanoid pathways. ca2+ plays irrigation regime treatment. the highest value of anthocyanin content was obtained from plant treat­ ed with 1.5 g l­1 cal under irrigation 85% etc. according to the results, there was no significant dif­ ference between different levels of cal under irriga­ tion 100% etc. however, anthocyanin content at 85% etc was significantly increased by using cal while under irrigation of 70% etc with the increase of anthocyanin content did not show any significant dif­ ference between different levels of cal. secondary metabolic products, which are inten­ sively biosynthesized under drought, are antioxidants (do nascimento and fett­neto, 2010). anthocyanin pigments as one of secondary metabolites and antioxidative systems play many important eco­phys­ iological roles in plants, including roles in stress pro­ tection (winkel­shirley, 2002). increased anthocyanin contents are thought to mask chlorophyll and/or act as a filter for preventing high light absorption by leaves and thus minimize photoinhibition (farrant, 2000). therefore anthocyanin accumulation in drought­stressed leaves confirms a possible protec­ tive role of anthocyanins as sun­screens and reactive oxygen species (ros) scavengers in stressed plants (merzlyak and chivkunova, 2000), that similar results have also been reported by jazizadeh and mortezaei nejad (2016) in chicory. the obtained results indicated that cal was effec­ tive in preserving and increasing anthocyanin con­ tent. these findings were in agreement with abd­ elhady (2014) findings who also observed that cal pretreatments proved to be effective for increasing the retention of anthocyanin in frozen strawberry. fig. 2 ­ effects of cal treatments on total phenol and flavonoids contents of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 1 ­ effects of cal treatments on anthocyanin content of let­ tuce under deficit irrigation. values are means with stan­ dard errors (n= 3). data were subjected to two­way anova and different letters mean that values are stati­ stically different p<0.05. http://https://www.sciencedirect.com/science/article/pii/s0570178314000116" /l "! http://https://www.sciencedirect.com/science/article/pii/s0570178314000116" /l "! 16 adv. hort. sci., 2020 34(1): 11­24 a direct role in the biosynthesis of phenols (castañeda and perez, 1996). cal might be a poten­ tial molecule for activating phenyl propanoid­ flavonoids pathways of fruits by increasing the pal activity (jacobo­velazquez et al., 2011; aghdam et al., 2013). catalase (cat) and peroxidase (pox) enzymes activity significant differences among irrigation treat­ ments were observed for cat and pox enzyme activi­ ty (table 3, figs. 3a, 3b). the antioxidant enzyme activates increased with the decrease of irrigation water applied. as the results showed cat and pox enzymes activity increased with increasing cal con­ centration under deficit irrigation, although no signif­ icantly differences was observed in normal irrigation. the highest cat and pox enzymes activity were recorded in plant treated with 1.5 g l­1 cal under irri­ gation 70% etc, which had no significant difference with 0.75 g l­1. the production of ros (vurukonda et al., 2016) is another major factor that impairs plant growth under water deficit (liting et al., 2015). plants employ a number of mechanisms, at molecular, cellular and physiological levels to persist stress condition (shinozaki and yamaguchi­shinozaki, 2000). the acti­ vation of antioxidant enzymes is one of the major types of these mechanisms which enable plants to control ros (shahid et al., 2014). cat, ascorbate per­ oxidase (apx), superoxide dismutase (sod) and pox are the key antioxidant enzymes involved in detoxifi­ cation of superoxide and hydrogen peroxide (kadkhodaie et al., 2014). a relationship between antioxidant enzymes activity and water stress or salinity tolerance was confirmed by comparison of a tolerant cultivar with a sensitive cultivar in several plant species, such as tomato (mittova et al., 2002). calcium is known to regulate different metabo­ lisms in plants mediating signaling pathways, which modulate gene expression in response to stress and its adaptation (upadhyaya et al., 2011). upadhyaya et al. (2011), observed pox activity was increased in the stressed plant as compared to controls, but recovering plants showed pox activity increasing after rehydration, which was enhanced by cacl2 and reported that cacl2 treatment resulted in increased non enzymatic antioxidant and enhanced activities of enzymatic antioxidant, including sod, pox and cat, and thus reduced ros accumulation and lipid peroxi­ dation ultimately leading to improved post­drought recovery potential in camellia sinensis. calcium applied alleviation of drought­induced damage has been clarified in numerous plants e.g. zoysia japonica (xu et al., 2013), and phaseolus vulgaris (abou el­ yazied, 2011). antioxidant activity (aa) aa was affected significantly by the irrigation treatments, and water deficit stress increased aa, which no significant difference was observed between irrigation 100 and 85%etc (table 3, fig. 4). in present study, the exogenous application of cal significantly (p<0.05) increased aa of lettuce under different irrigation regimes compared to control plant. the highest aa (93.03%) was observed in cal 0.75 g l−1 under irrigation 85% etc, however had no significant difference with cal 1.5 g l­1 treatment under irrigation 70% etc (fig. 4). the antioxidant activity in lettuce arises from phe­ nolic compounds, secondary plant products, such as fig. 3 ­ effects of cal treatments on catalase (cat) and peroxida­ se (pox) enzymes activity of lettuce under deficit irriga­ tion. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 4 ­ effect of cal treatments on antioxidant activity of lettuce under deficit irrigation. values are means with standard errors (n = 3). data were subjected to two­way anova and different letters mean that values are statistically dif­ ferent p<0.05. khani et al. ‐ protective effect of calcium lactate on lettuce 17 flavonoids and phenols, and also anthocyanin. also, the antioxidant activity strongly correlated to the presence of efficient oxygen radical scavengers, such as vitamin c and phenolic compounds (tulipani et al., 2008). in current study a significant correlation was found between antioxidant activity and antho­ cyanins, phenols and flavonoids contents; which the anthocyanin, phenolic and flavonoids content and cat and pox enzymes activity, as well as the total antioxidant activity also increased with increasing water deficit stress and cal concentration. this find­ ing described that phenolic compounds and antho­ cyanin, and antioxidant enzyme activity makes an important contribution to the antioxidant capacity in lettuce leaf. velioglu et al. (1998) reported a strong relationship between total phenolic content and antioxidant activity in fresh fruits and vegetables. leaf water status (rwc, elwr) based on the findings (table 3, figs. 5a, 6), deficit irrigation caused a significant reduction in rwc and elwr contents. the application of cal significantly ameliorated relative water content (rwc) and excised leaf water retention (elwr) contents (figs. 5b, 6). mean comparisons of data, displayed that pre­ treatment with cal markedly reduced the effects of water deficit stress and also improved elwr under control irrigation and water deficit stress. the highest value of elwr content was obtained in plant treated with cal 1.5 g l­1 under irrigation of 85 and 100% etc. rwc and elwr are among the main physiological criteria that influence plant water relations and have been used for assessing drought tolerance (xing et al., 2004). under drought stress, leaf rwc plays an important role in the tolerance of plants to stress by inducing osmotic adjustments due to the accumula­ tion of osmoprotectants (barnabás et al., 2008; zhang et al., 2012). the maintenance of a high plant water status during stress is a significant defensive mechanism to maintain enough water by minimizing water loss (e.g. caused by stomatal closure, tri­ chomes, reduced leaf area, senescence of older leaves, etc.) and maximizing water uptake (e.g. by increased root growth) (barnabás et al., 2008). because of the decrease in leaf area, the accumula­ tion of chlorophyll has increased, but due to high transpiration, the plant loses more water and as a result, the rwc of leaf and consequently photosyn­ thesis decreases (farooq et al., 2012). farooqi et al. (2000) indicated that rwc of lemongrass leaves decreased in all the cultivars due to drought but after rehydration, rwc gradually increased to pre­stress level, which has also been reported in several crop species such as melon (mani, 2014). as well as drought stress significantly decreased rwc and elwr in spring safflower (balian et al., 2015). ruiz­lozano and azcon (1997) reported that calci­ um application significantly increased rwc in lettuce. the results of this research showed that the rwc of leaves increased with calcium application. increasing relative water content means increasing water hold­ ing capacity, which can prevent water loss in leaves in a dry environment (ma et al., 2005). nutrient contents according to the results (table 4, fig. 7), n con­ tent in lettuce leaves increased with increasing cal concentration, indeed the highest value of n was obtained at cal 1.5 g l­1 under irrigation 100 %etc that had significant difference with deficit irrigation treatments (85 and 70% etc), whereas in other treat­ ments there were not any significant differences. mean comparisons of data, showed that deficit irri­ fig. 5 ­ effects of irrigation (a) and cal (b) treatments on leaf relative water content (rwc) of lettuce. data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 6 ­ effects of cal treatment on excised leaf water retention (elwr) content of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjec­ ted to two­way anova and different letters mean that adv. hort. sci., 2019 33(3): 11­24 18 gation significantly increased p content in lettuce leaves and decreased k content compared to control irrigation (table 4, figs. 8a, 8b). the ca content increased with the deficit irriga­ tion treatments, in particular with moderate deficit irrigation (85% etc). ca content in lettuce leaves increased in response to higher cal concentration (fig. 9a). in fact, the highest value of ca was observed in treatments with application of 1.5 g l­1 cal under irrigation 70 and 85% etc. the overall effect of deficit irrigation on mg content was nega­ tive, with a decrease of 0.79% (fig. 9b) under deficit irrigation 70% etc. mg content in the leaves decreased when the concentration of cal applied was increased. the lowest value of mg content was obtained in plant applied with 1.5 g l­1 cal under deficit irrigation 70% etc. drought stress and associated reduction in soil moisture can decrease plant nutrient uptake by reducing nutrient supply through mineralization (sanaullah et al., 2012), and nutrient diffusion in the soil (chapin iii, 1991; lambers et al., 2008). drought can depress plant growth by reducing n and p uptake, transport and redistribution (rouphael et al., 2012). a majority of studies have indicated that plants decrease n and p uptake with a decline in soil moisture (sardans and peñuelas, 2012). n uptake was reduced in maize under stress conditions, which indicates that the absorption of nutrients is limited in conditions of water deficit stress, which may be reduced due to reduced transpiration rate, active transfer and membrane permeability (naeem et al., 2017). owing to a reduction in stomatal conduc­ tance, photosynthesis and transpiration rates also table 4 ­ variance analysis (anova) of effects of calcium lactate on nutrient contents and fresh yield in lettuce under deficit irrigation ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non­significance at p<0.05. source of variations df mean of squares n content p content k content ca content mg content fresh yield wue replication 2 0.030 0.008 0.008 0.002 0.004 206838.82 0.380 irrigation 2 0.208 ** 0.111 ** 0.153 ** 0.621 ** 2.157 ** 21349043.15 ** 3.293 ** error (a) 4 0.008 0.008 0.003 0.005 0.004 83934.71 0.183 calcium lactate 2 1.158 ** 0.024 ns 0.025 ns 0.291 ** 0.095 ** 6105376.18 ** 10.144 ** calcium lactate × irrigation 4 0.116 ** 0.006 ns 0.010 ns 0.047 ** 0.018 * 413961.66 * 0.317 ns error (b) 12 0.012 0.006 0.008 0.004 0.005 101407.46 0.170 coefficient of variation (%) ‐ 1.47 3.48 1.43 2.09 3.4 2.82 2.78 fig. 7 ­ effects of cal treatments on nitrogen content of lettuce leaves under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are stati­ stically different p<0.05. fig. 8 ­ effect of irrigation treatments on phosphorus (a) and potassium (b) contents of lettuce leaves. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 9 ­ effect of cal treatments on calcium (a) and magnesium (b) contents of lettuce leaves under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. khani et al. ‐ protective effect of calcium lactate on lettuce 19 decrease, and co2 assimilation rates progressively decline in response to drought (farooq et al., 2012). therefore, drought effects on plant may depend on the reduction in n and p uptake relative to the decrease in co2 assimilation (he and dijkstra, 2014). based on the current findings, increasing k and ca contents and decreasing mg content of lettuce leaves under water deficit stress as compared to well­ watered conditions that also reported by tadayyon et al. (2018) in castor plants. potassium has a positive correlation with the physiological effects of plants, such as water use efficiency, stomatal control, air and underground body biomass, and is likely to play an important role in photosynthesis (sardans et al., 2012). increasing k content of leaves with decreasing irrigation rate maybe due to role of this cation in the regulation of osmotic pressure and stomatal control, (zhao et al., 2000). nahar and gretzmacher (2002) reported that with increasing deficit irrigation, mg concentration in tomato tissues decreased, which is similar to results of the present study. the same results were reported from other authors, that high concentrations of ca often result in increased leaf ca along with a marked reduction in leaf mg (nassery et al., 1979; borghesi et al., 2011). as well as, naeem et al. (2018) revealed that concen­ tration of macronutrients (n, k, ca) in maize grains was markedly improved by foliar supply of calcium which indicates its synergistic effect on uptake and translocation of these nutrients. tuna et al. (2007) also observed leaf n, k and ca content increased in tomato plants supplemented with calcium under stress conditions. in safflower, by decreasing soil moisture k and mg content decreased. following this reduction, there was a significant increase in calcium concentration, which is justified by the antagonistic relationship between ca and mg (vafaie et al., 2013). morard et al. (1996) reported an intense antagonistic relation­ ship between ca and mg, that mg transfer to leaves was affected by calcium. fresh yield lettuce plants grown under control and deficit irrigation conditions exhibited significant differences between cal treatments in fresh yield (table 4, fig. 10). water deficit stress caused significant reductions in yield. in fact, deficit irrigated plants showed a 6.8 and 15.8% decrease in fresh yield, respectively. as the results showed, with increasing cal concentra­ tions, lettuce yield significantly increased and reached to highest value (1.37 kg.m­2) at cal 1.5 g l­1 under irrigation 100% etc (fig. 10). lettuce is one of the leaf­edible vegetables that it is extremely sensitive to water deficit stress due to shallow root system (sabedze and wahome, 2010). our results are in agreement with many open­field studies on lettuce (jiménez­arias, 2019) and lettuce (sayyari et al., 2013). deficit irrigation defined as a practice that applies water below full crop­water requirements, deliberately exposes plants to a cer­ tain level of moisture stress. it is well known that drought stress results in dehydration of the cell and osmotic imbalance that impairs numerous metabolic and physiological processes in plants (mahajan and tuteja, 2005). reduction in fresh yield of lettuce with deficit irrigation might be attributed to the suppres­ sion of cell division and expansion, and growth due to the low turgor pressure and also closure of stomata leaf and more leaf senescence under drought stress (sayyari et al., 2013). foliar application of cal enhanced fresh yield of lettuce. naeem et al. (2013) reported that crop pro­ ductivity and photosynthetic efficiency in senna occi‐ dentalis was improved under ca application. with low calcium availability, a reduction in bean plant height, leaf area and shoot and root growth has been reported (leal and prado, 2008). foliar applied of chelated calcium enhanced the seed yield and relat­ ed attributes in common bean under water­deficit conditions (abou el­yazied, 2011). water use efficiency (wue) according to the results (table 4, figs. 11a, 11b), irrigation and cal treatments significantly affected wue, but their interaction showed no significant dif­ ferences. water deficit stress significantly increased wue and the highest wue was recorded in 70% etc deficit­irrigated plants that had no significant differ­ fig. 10 ­ effect of cal treatments on fresh yield of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and dif­ ferent letters mean that values are statistically different p<0.05. adv. hort. sci., 2019 33(3): 11­24 20 ence with deficit irrigation 85% etc (fig 8a). wue increased with increasing cal concentration and the highest wue (15.8 kg m­3) was obtained at 1.5 g l­1 cal (fig. 8b). wue, the physiological parameter of crop, describes the relationship between plant water use and dry matter production (cai et al. , 2012). regarding water resource constraints, it is essential to find ways to preserve water and increase water use efficiency in plants (topcu et al., 2007; alenazi et al., 2015). the highest wue value was determined in 70% etc irrigation. it was calculated that wue values increased with the decrease in the amount of water. these results are similar to the previous finding of şimşek et al. (2004), who reported that the maximum wue for watermelon was obtained with low irriga­ tion. with increased wue, there is a greater biomass production per amount of water transpired, and less water is needed for growth and development (nemali and van iersel, 2008). wue is strongly related to photosynthetic activity and transpiration efficiency, and can be affected by irrigation (monneveux et al., 2006). ca is directly involved in photosynthesis processes, and its deficit reduces the plant’s biomass by reducing the efficien­ cy of carboxylation and photosynthesis (alarcon et al., 1999). the results of the current experiment showed that n content was increased with ca appli­ cation, which increasing n content leads to increase dry matter production as well as the wue. therefore, ca maybe increased the wue by increasing the amount of n and ca in lettuce plants. 4. conclusions the results obtained in this investigation pro­ posed that lettuce is sensitive to water deficit stress during 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330‐714 chungnam, korea. 3 current address: the institute of natural resource development, mokpo national university, muan, 58554 jeonnam, korea. key words: daylily, haplotype, nocturnal flowering, polymerase chain reaction, sequence analysis. abstract: this study was initiated to distinguish nocturnal (night) flowering hemerocallis species from day flowering species based on the single nucleotide polymorphisms (snps) of nuclear internal transcribed spacers 1, 2 in a riboso­ mal rna gene (nrits) and a chloroplast interspacer region (cpis). four noctur­ nal flowering species, h. citrina, h. thunbergii, h. minor, and h. lilioasphodelus were collected including korea, and compared with day flowering species that included h. vespertina and h. hongdoensis. based on the haplotypes of nrits and cpis, nocturnal species cannot be distinguished from day flowering species. discrepancies in flowering time and haplotypes among h. minor accessions sug­ gest that more germplasm with diverse geographic origins should be evaluated and identification of other genes is required to effectively distinguish nocturnal species from day flowering species. 1. introduction there are about 15­26 species/varieties in the genus hemerocallis (usda, ars, national genetic resources program, 2015). using amplified fragment length polymorphisms (aflp) markers, h. fulva l. were grouped separately from the nocturnal species h. thunbergii baker and h. lilioas‐ phodelus l., while nocturnal h. minor mill. and h. citrina baroni, were grouped together in a different sub­cluster (tomkins et al., 2001). the flowers of nocturnal hemerocallis open late in the afternoon and wither the next morning (fig. 1) (chen and noguchi, 2000). however, gulia et al. (2009) classified h. minor as a diurnal species rather than a nocturnal species, confirming the study by krestova and nesterova (2003) that h. minor flowered in the morning under sunny weather at >16°c and with­ (*) corresponding author: marksroh@gmail.com citation: park s.y., joung y.h., suh j.k., roh m.s., 2022 ­ evaluation of hemerocallis germpla‐ sm using single nucleotide polymorphisms of nrits and chloroplast interspacer region. ­ adv. hort. sci., 36(3): 247­251 copyright: © 2022 park s.y., joung y.h., suh j.k., roh m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 november 2021 accepted for publication 31 august 2022 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-12347 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 247­251 248 ered in the afternoon. molecular markers have not previously been test­ ed to determine timing of flowering among hemerocallis species. therefore, this study was con­ ducted to investigate the use of snps of nuclear internal transcribed spacers 1, 2 in a ribosomal rna gene (nrits) and a chloroplast interspacer region (cpis) to evaluate the genetic relationships between nocturnal and day flowering species of hemerocallis. 2. materials and methods the nocturnal flowering species hemerocallis thunbergii, h. minor, h. lilioasphodelus, and h. citri‐ na, and the day flowering species h. hongdoensis m.g. chung & s.s. kang, h. vespertina h. hara, h. dumortierii c. morren and hybrid ‘stella de oro’ were collected from korea (k), china (c), united kingdom (uk), united states of america (ua) and germany (ge) (table 1). samples were designated as, for example, k1 (mother plant)/1­3 (seedling 1, 2, 3 from mother plant k1). genomic dna was isolated from young leaves using dneasy plant mini kit (qiagen inc., valencia, ca). polymerase chain reaction (pcr) for nrits was performed with a 18s rrna gene specific forward primer its1 (5’­tag agg aag gag aag tcg taa caa gg­3’) and primer its2 (5’­ gattttcagtcctctgctc­ tac­3’). the reaction mix consisted of 12.5 μl of 2x f­ star taq smartmix (solgent co., daejeon, korea), 2 μl of each primer (0.4 μm final concentration), and 2 μl of genomic dna. for cpis, forward primer (5’ ­tcgt­ gagggttcaagtccctct­3’) and reverse primer (5’­ gattttcagtcctctgctctac­3’) were used. the reaction mix consisted of 12.5 μl of 2x f­star taq smartmix (solgent co., korea), 2 μl of each primer (0.4 μm final concentration), and 2 μl of genomic dna (50 ng) and 5 μl of 5× band doctor buffer (solgent), and made up to the 25 μl final vol­ ume with pcr ultrapure water. the pcr conditions were: 2 min at 95°c, followed by 35 cycles of 20 sec at 95°c, 40 sec at 65°c, and 1 min at 72°c, followed by 5 min of 72°c using an abi veriti dx thermal cycler (life technologies, grand island, ny, usa). pcr products were direct sequenced as described by park et al. (2014). sequences were registered in the national center for biotechnology information genbank (ncbi, http://www.ncbi.nlm.nih.gov/). 3. results and discussion based on the sequences of nrits, three haplo­ types were identified: t i­1, t i­2, and t ii (table 2) and based on the sequences of cpis, 7 haplotypes were identified: t i, ti­1, tii, tiii, tiv, tv, and tvi (table 3). sequences of cpis were more informative to separate accessions than those of nrits, suggest­ ing that the its region in hemerocallis may not be useful as a potential source for species identification, although accessions of cultivated origin kolkwitzia amabilis (graebn.) christenh were derived from the accession of known wild origin (aa816­84a) (park et al., 2014). nocturnal flowering h. citrina, h. thunbergii and h. lilioasphodelus flower in the afternoon and wither the following morning (table 1, fig. 1). the difference between two types of floral morphology of h. thun‐ bergii, based on the presence or absence of bracts subtending the flower buds; k7 lacked bracts while k8­9 had bracts was detected in the haplotype of cpis, with k7 belonging to type i­1 and k8­9 belong­ ing to type i that may result from that the sequence of the its 2 region is more variable than that of the its 1 region (ma et al., 2014). nocturnal flowering h. thunbergii accessions collected from the same loca­ tion in korea (k8­9, and k 8­13) should be evaluated fig. 1 ­ nocturnal h. thunbergii accession 7 collected from korea (k7) showing flower opening at 4, 5, and 6 pm and closed by 10 am following day. http://www.ncbi.nlm.nih.gov/ park et al. ‐ hemerocallis germplasm evaluation 249 further since these accessions were grouped togeth­ er with other h. thunbergii accessions (k1­3). there is no correlation between these nrits hap­ lotypes and timing of flowering observed in h. citrina; collected from china (c2) and the united kingdom (uk5), both belonging to type i, from germany (ge4), belonging to type ii, and from the united kingdom (uk1), belonging to type iii. this requires further examination collecting more accessions from differ­ ent collection sites. further, in cpis, day flowering h. hongdoensis and h. vespertina belonged to type i and ii, respectively. when t was assigned for the ambiguous code c or t at the positions 78, 86, 99, 113, and 120 of nrits sequence for type i­1, and a was assigned for a or g at 231, types i­1 and i­2 can be combined and all accessions can be assigned as type i, separated from h. minor (uk6 and ge3) as type ii (tables 1 and 2). they may be derived from different geographic ori­ gins of k14 or ua5 which were collected from korea, belonging to type i­2, and exhibit some degree of genetic variation revealed in this study using univer­ sal primers for nrits. however, different strains or populations of h. minor may exist since h. minor table 1 ­ accession information on hemerocallis taxa (mother plants and their seedlings) with flowering characteristics. haplotype based on the sequence analysis of nrits and cpis are indicated scientific name mother plant (leaf) seedlings a flowering time b source, country haplotype (t) c nrits cpis h. thunbergii k1 d k1/1­3 n jungseon­gun, korea i­2 i h. thunbergii k2 k2/1­3 n i­2 i h. thunbergii k3 k3/1­3 n i­2 i h. thunbergii k7 n j.w. chang, gomyeong­dong, jecheon­si, chungcheongbuk­do, korea i­2 i­1 h. thunbergii k8,9 e n e.j. kim, gomsi­gil, ungdam­ri, paju­gun, kyunggi­do, korea i­2 i ka8­13 d n e.j. kim, m.s. roh, gomsi­gil, ungdam­ri, paju­gun, kyunggi­do, korea i­2 iv h. thunbergii k11 n hantaek botanical garden, korea i­2 i h. thunbergii uk3, 4 n royal botanical garden edinburgh, uk (19300128a f) i­2 i h. thunbergii ua7 ­ n united states national arboretum, washington, dc, usa (usna; na54757.3 collected from korea) i­2 i h. minor ­ k14/1­3 d hantaek botanical garden, korea i­2 i­1 h. minor uk6 uk6/1­3 n? royal botanical garden edinburgh, uk ii v h. minor ge3/1­3 n? botanischer garten leipzig (xx­o­lz­ad439/2006) ii v h. minor ua5 ­ d usna; na31800.1 i­2 i h. lilioasphodelus ­ c1/1 n x.w. wu, china i­2 i c1/2­3 i­1 i h. liliasphodelus ua6 ­ n usna; na54879.3 i­2 i h. citrina ­ c2/1­3 n x.w. wu, china i­2 i h. citrina uk1 n royal botanical garden edinburgh, uk (19685548a u) i­2 iii h. citrina uk5 n royal botanical garden edinburgh, uk i­2 i h. citrina ge4/1­3 n botanischer garten leipzig (xx­o­lz­aw78/1998, 2000) i­2 ii h. citrina ‘april flower’ ­ c3/1­3 x.w. wu, china i­2 i h. vespertina k12 k12/1­3 d hantaek botanical garden, korea i­2 ii h. dumortierii c. morren k13 d hantaek botanical garden, korea i­2 vi h. hongdoensis ­ k15/1­3 d hantaek botanical garden, korea i­2 i ‘stella de oro’ ua2 ua2/1­3 d m.s. roh, ann arbor, mi., usa i­1 i a seedlings 1, 2, 3 from mother plant k1. designations of accession of mother plant and three seedlings are indicated as k1 and k1/1­3, respectively. b flower opens in the morning and withers in the late afternoon (day, d) or opens in the late afternoon and withers early on the next morning (night, n). flowering characteristics were not evaluated in this study for h. minor collected from royal botanical garden edinburgh, uk and botanischer garten leipzig (xx­o­lz­ad439/2006). c refer to table 2 for nrits and table 3 for cpis haplotypes and single nucleotide polymorphisms. d collected or received from korea (k, ka), united states of america (ua), united kingdom (uk), germany (ge), and china (c). e samples of k8 and 9 and of ka 8­13 were collected from the same location in 2011 and 2014, respectively. f samples were of garden origin from dendrologische gartenerei in pruhonce, czech republic via peter brownless. adv. hort. sci., 2022 36(3): 247­251 250 flowers in the morning under sunny weather at >16oc and withers in the afternoon, as reported at the botanical garden institute, far east branch, russian academy of sciences (krestova and nesterova, 2003). the h. minor, received from the us national arboretum (usna; na31800.1) original­ ly collected from korea, flowers in the morning in ann arbor, mi, usa. therefore, further investigation of hemerocallis minor is needed to determine whether accessions collected from china, korea and the far eastern part of russia are of two different flowering types. hemerocallis hybrid ‘stella de oro’ (ua2), day flowering landscape plant of unknown parentage, was grouped with h. minor collected from korea (k14), but not with h. minor (uk6 and ge3) acces­ sions and their seedlings (k14/1­3) and uk6/1­3, based on the haplotypes of snps with a nrits region (table 2) and with cpis (table 3). grouping of h. dumortieri (k13) with h. minor (uk6 and ge3) with nrits region was also different from that with cpis (table 3). mcgarty (2006) used aflp markers from hemerocallis species, and placed h. lilioasphodelus with h. thunbergii in one sub­group and h. citrina, h. minor, and h. dumortieri in another. this suggests that day and night flowering species cannot be sepa­ rated either by aflp markers, or by snps from a nrits region or cpis, as attempted in this study. difficulties with identifying species of hemerocallis native to korea may not be easy to resolve and flowering time in f1 hybrids between h. fulva (day flowering) and h. citrina (nocturnal flowering) showed discontinuous bimodal distribution (hasegawa et al., 2006). beyond the identification issue for the accessions h. thunbergii ka8­13, grouping of species investigat­ ed in this study differs significantly between nrits and cpis (tables 2 and 3). difficulties with identifying species of hemerocallis native to korea may not be easy to resolve and flowering time in f1 hybrids between h. fulva (day flowering) and h. citrina (night flowering) showed discontinuous bimodal distribu­ tion (hasegawa et al., 2006). distinguishing nocturnal flowering forms of h. minor from day flowering forms is not possible due to the existence of two different genotypes collected from china, korea and far eastern russia and by testing the universal primers by snps of nrits region and cpis. however, these primers were used suc­ cessfully to identify mother plants and seedlings of ligustrum quihoui carrière (ma et al . , 2014) . table 2 ­ haplotype based on single nucleotide polymorphisms (snps) and insertion and deletion (in/del) of a nuclear internal tran­ scribed spacer 1, 2 in a ribosomal rna gene of hemerocallis accessions haplotype ncbi registration positions and codes of nucleotide single nucleotide polymorphisms in/del 78 86 99 113 120 170 206 210 231 254 432 568 597 435­441 i­1 kt189161 c/t c/t c c/t c/t a g a a/g a a g g c7 i­2 kt189162 c c c c c a g a a a a g g c6­7 ii kt189163 c c t c c g c c c c g a a c7 table 3 ­ haplotypes based on single nucleotide polymorphisms (snps) and insertion and deletion (in/del) of a chloroplast interspacer region of hemerocallis accessions haplotype ncbi registration positions and codes of nucleotide single nucleotide polymorphisms in/del 26 216 243 246 291 315 37 302 i kt189164 t c c c a a t9 t7 i­1 kt189165 t c c c a a t9 t8 ii kt189166 t c c c c a t9 t8 iii kt189167 t c c a a a t9 t8 iv kt189168 c c a a a a t8 t8 v kt189169 c c c a a a t8 t8 vi kt189170 c a c a a a t10 t8 park et al. ‐ hemerocallis germplasm evaluation 251 seedlings grouped in the haplotype with their moth­ er plants, except the mother plant (c1) and seedlings 2­3 of h. liloasphodelus (c1/2­3), which belonged to type i, type i­2 and i­1, respectively, in nr­its region (tables 1 and 2). the current primers for nrits region and cpis cannot be used to differentiate noc­ turnal flowering species from day flowering species. lee and maki (2015) reported that cpdna in the majority of cultivars were inherited from h. albo‐ marginata, although the leaf morphology was simi­ lar to h. sieboldiana, indicating that nrits should fur­ ther be investigated. 4. conclusions the sequence variations of nrits region and cpis cannot be used to distinguish nocturnal flowering species from day flowering hemerocallis species. markers other than those evaluated in this study should be evaluated. genetic variations among seedlings or between mother plant and their seedlings were observed in h. lilioasphodelus (c1/1 vs. c1/2­3), requiring seedlings of h. lilioasphodelus to investigate to confirm the results of this study. discrepancies in flowering time among h. minor accessions also suggest that more germplasm with diverse geographic origins should be evaluated. acknowledgements critical review and english editing of the manu­ script by dr. c.j. catanzaro, virginia state university, va, usa, is greatly appreciated. references chen x., noguchi j., 2000 ­ 31. hemerocallis linnaeus, sp. p1. 1:324. 1753. ­ flora of china, 24: 161­165. gulia s.k., singh b.p., cartter j., griesbach r.j., 2009 ­ daylily: botany, propagation, breeding. ­ hort. rev., 35: 193­220. hasegawa m., yahara t., yasumoto a., hotta m., 2006 ­ bimodal distribution of flowering time in a nat‐ ural hybrid population of daylily (hemerocallis fulva) and night lily (hemerocallis citrina). ­ j. plant res., 119: 63­68. krestova i.n., nesterova s.v., 2003 ­ flowering and pol‐ lination of the native daylily species (hemerocallis) in the botanical garden institute, far east branch, russian academy of sciences. ­ contemp. problems ecol., 6: 48­ 454. lee s., maki m., 2015 ­ origins of hosta cultivars based on sequence variations in chloroplast dna. ­ hort. j., 84: 350­354. ma s.h., burchi g., suh j.k., roh m.s., joung y.h., 2014 ­ identification of ligustrum seedlings based on sequence analysis of an internal transcribed spacer. ­ hort. environ. biotechnol., 55: 423­427. mcgarty t.p., 2006 ­ phylogenetics, dna, classification and the genus hemerocallis. ­ http://www.telmarcgar­ dens.com. park s.e., burchi g., roh m.s., joung y.h., 2014 ­ characterization of kolkwitzia amabilis accessions based on flowering and molecular markers. ­ scientia hort., 165: 190­195. tomkins j.p., wood t.c., barnes l.s., westman a., wing r.a., 2001 ­ evaluation of genetic variation in the daylily (hemerocallis spp.) using aflp markers. ­ theor. appl. genet., 102: 489­496. usda, ars, national genetic resources program, 2015 ­ germplasm resources information network ‐ (grin) [online database]. national germplasm resources laboratory, beltsville, maryland. impaginato 377 adv. hort. sci., 2023 37(4): 377­389 doi: 10.36253/ahsc­14732 potassium silicate enhances drought tolerance of bellis perennis by improv­ ing antioxidant activity and osmotic regulators a. oraee, a. tehranifar (*), z. ghorbani, p. sayad­amin department of horticultural science and landscape, ferdowsi university of mashhad, iran. key words: antioxidant activity, fertilizer, osmotic regulation, secondary metabolites. abstract: ornamental plants can usually encounter various types of environ­ mental stress, which reduce plant productivity. a proper application of fertiliz­ ers can improve plantsʼ tolerance to drought stress. nutrients such as potassi­ um and silicon are known to have beneficial effects. this study aimed to evalu­ ate the growth of bellis perennis under drought stress (80, 70, and 60% fc) and with the application of potassium silicate (0, 2, and 4 mm). the results showed that potassium silicate (2 and 4 mm) increased k and si accumulation in plants under drought stress. plants treated with potassium silicate under drought stress exhibited a lower degree of electrolyte leakage and less mda accumula­ tion in the following order: 2 and 4 mm potassium silicate. an increase in rela­ tive water content and chlorophyll was observed with application of potassium silicate under drought stress. regardless of potassium silicate, the plant enzy­ matic defense system was significantly improved compared to non­stressed plants. potassium silicate enhanced the amount of osmotic regulators (carbo­ hydrate and proline) and secondary metabolites (flavonoids and phenols) com­ pared to control plants regardless of drought stress. the anthocyanin content in the flowers significantly decreased by 32.2% when the plants were treated with 4 mm potassium silicate at 60% fc, compared to 80% fc. in conclusion, potassium silicate mitigated the effects of drought stress, enhanced plant toler­ ance to drought stress, increased the activity of antioxidant enzymes, and improved the amounts of osmotic regulators and secondary metabolites. 1. introduction plant growth is usually affected by numerous abiotic stressors (calanca, 2017). deficits in water supplies are a major environmental threat, currently affecting more than 41% of the world’s land mass. projections for 2050 show a further increase in the magnitude and impact of this environmental threat (prăvălie, 2016). as global temperatures increase, the annual maximum temperature is estimated to increase by 5°c per year by the end of the 22nd century. this problem will lead to (*) corresponding author: tehranifar@um.ac.ir citation: oraee a., tehranifar a., ghorbani z., sayad­ amin p., 2023 ­ potassium silicate enhances drought tolerance of bellis perennis by improving antioxidant activity and osmotic regulators. ­ adv. hort. sci., 37(4): 377­389. copyright: © 2023 oraee a., tehranifar a., ghorbani z., sayad­amin p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 may 2023 accepted for publication 21 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14732 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 377­389 378 more frequent and extreme droughts in many parts of the world (gao et al., 2020). such environmental types of stress significantly cause a decline in crop yields falling below maximum potential (raza et al., 2020). environmental stress can suppress plant growth by interrupting various processes such as cell metabolism, nutrient uptake, and maintenance of turgor pressure (kusvuran and dasgan, 2017). regarding the proper use of fertilizers for improv­ ing plant tolerance to drought stress, the application of nutrients can be largely beneficial. such applica­ tions have reportedly included potassium and silicon, with essential functions in plant metabolism (bukhari et al., 2020; ibrahim et al., 2020). potassium is a vital fertilizer involved in numerous biochemical and phys­ iological processes, including stress tolerance, plant growth, yield, and quality. potassium (k) is necessary for many physiological processes, for example, main­ taining turgor, translocation of photosynthetic sub­ stances to sinking organs, activation of enzymes, syn­ thesis of proteins, transport of solutes in the phloem, and maintenance of cation­anion balance in the cytosol and vacuole. furthermore, k reportedly facili­ tates osmoregulation, stomatal movements, and tro­ pism, while it is mainly absorbed from the soil through the roots (qi et al., 2019). however, drought stress causes a reduction in the absorption of ele­ ments. the reduced sensitivity of k­deficient plants to drought stress is related to several factors. these factors include the role of k in regulating stomata stomatal and water balance, as well as osmotic potential in vacuoles (haworth et al., 2018). applying potassium fertilizer mitigates the adverse effects of these stressors on plant growth (qi et al., 2019). silicon (si), the second most abundant element on earth, can increase plant tolerance to biotic and abi­ otic stressors such as frost, heat, pests, drought, dis­ eases, and nutrient imbalance (wang et al., 2021). si deficiency reportedly decreased photosynthesis, also increased disease incidence, insect infestation, wilt­ ing, and postharvest decline. while all of these symp­ toms are signs of stress (reynolds et al., 2009; dallagnol et al., 2012; weerahewa et al., 2015). si usually contributes to healthy plant development and is essential for cell development and differentiation. the protective role of si in drought conditions is mainly associated with an enhanced level of water retention, which promotes photosynthesis (zhang et al . , 2018). it accelerates the accumulation of osmolyte regulators (proline and carbohydrates) as well as antioxidant activities in plants exposed to stress in the environment (moussa and shama, 2019). potassium silicate (k­silicate) is used as a source of highly soluble k and si. k­silicate does not contain volatile organic compounds, and its applica­ tion does not result in the release of hazardous or pollutant byproducts (romero­aranda et al., 2006). in a relevant study, the application of k­silicate to the soil, for several plant species under irrigation with water­deficit conditions, resulted in the highest bio­ mass of all species (moussa and shama, 2019). si can act as a growth regulator and can potentially increase plant growth under drought stress. spraying k­silicate and other nanomaterials can potentially reduce the adverse effects of drought stress on crops (zahedi et al., 2020). the common daisy (bellis perennis) belongs to the asteraceae family and is known as an archetype. bellis perennis is an important ornamental and medi­ cinal plant with a global distribution. it is one of the first flowering species and is a crucial member of spring bloomers (siatka and kašparová, 2010). daisies are grown for their beauty, either for color or aesthetic reasons. they are naturally able to provide economic, environmental, and social benefits. however, water­deficit largely affects the ornamental value of this species. in the current study, we focused on the response of daisies to drought stress, while monitoring their antioxidant enzymes, substances for osmotic regula­ tion, and secondary metabolites. although many studies have already considered the role of potassi­ um silicate in reducing the adverse effects of drought stress in various plants, there is little information about the effects of potassium silicate on ornamental plants under drought­stress conditions. therefore, the objective of the current study was to investigate the effects of potassium silicate on the characteristics of daisies under drought­stress conditions. the mechanisms of action by potassium silicate, their advantages for ornamental plants, and their ability to create drought tolerance provide a scientific basis for using potassium silicate to alleviate drought stress. 2. materials and methods plant culture, drought stress, and potassium silicate treatments this study was carried out at the ferdowsi university of mashhad (autumn­spring 2021). seeds of bellis perenesis l. were purchased from takii seed oraee et al. ‐ the role of silicon in drought tolerance of daisy 379 company. in september 2021, the seeds were grown in polyethylene bags containing a mixture of peat and perlite (3:1) for four weeks (trifoliate stage) under controlled conditions (21°c/17°c day/night and 45­55% humidity under 100 mmol photons m_2 s_1. four weeks later, the trifoliate seedlings were transplanted into pots. for each treatment, the experiment was laid out with three pots. the pots were placed in a greenhouse (air temperature of 21±2°c and relative humidity of 62±2%) during the growing periods. irrigation started after one day, and the plants were well watered for a few months (90­ 85% fc). drought treatment was initiated by omitting irrigation, and potassium silicate (k2sio3) was used as the si source. potassium was administered to the plants in the form of liquid potassium silicate (k2sio3) (10% k2o, 25% sio2) at three concentrations (0, 2, 4 mm) (the concentrations of potassium silicate were selected according to a pretest). in march, the plants were treated with different solutions, i.e. (1) 1/2 hoaglandʼs solution without the addition of k2sio3, (2) 1/2 hoaglandʼs solution with the addition of 2 mm k2sio3, (3) 1/2 hoaglandʼs solution with the addi­ tion of 4 mm k2sio3. potassium silicate was applied as a treatment for one month in march, and irriga­ tion treatments began with drought stress (80%, 70%, and 60% fc) in april. three levels of water deficit (i.e. 80, 70, and 60% of field capacity, fc) were applied from april to june. the gravimetric method (campbell and mulla, 1990) was used for irrigation for two months. first, several pots were completely irrigated so that the water permeated all pores in the soil. then, the pots were wrapped with plastic covers to prevent evaporation and transpiration. the pots were weighed until their weight remained constant for two consecutive measurements. then, a soil sam­ ple was taken to the laboratory. the fresh weight was measured and the dry weight was calculated after 12 hours of storage in an oven at 105°c. the percentage of moisture content by weight, required for suitable crop production, is calculated based on the following equation: fc = (a­b / b) × 100 where fc, a, and b are the field capacity, the weight of moist soil after gravity drainage, and the weight of the sample dried at 105°c for 12 hours, respectively. the weight difference between water­saturated and oven­dried soil was taken as the weight of water needed to bring the pots to field capacity, and then lower water contents in the soil (% field capacity) were calculated accordingly. during the period of treatments, the pots were regularly weighed, and additional water was supplied when necessary. for each test, there were three replicates containing five plants, making a total of 15 plants. at the end of the experiment, the fresh leaves were used for measur­ ing electrolyte leakage, rwc, and chlorophyll con­ tent. for determining the nutrition concentration and proline content, dried leaves were frozen in liquid nitrogen and stored at ­80°c until the time of mea­ surements. determination of k and si concentration the k and si concentrations were determined on the dry leaves samples. oven­dried leaves (300 mg of the dried samples) were weighed and burned in a muffle furnace at 550°c for 8 hours. the k concentra­ tion was determined by flame photometry (pfp7, jenway, uk). the si concentration was determined by the colorimetric ammonium vanadate method (jaiswal, 2003). measurement of electrolyte leakage and relative water content electrolyte loss was determined according to a method used by gusta et al. (2003), and relative water content (rwc) was calculated via a method used by pieczynski et al. (2013). measurement of antioxidant activity and malondi‐ aldehyde (mda) antioxidant activity was determined using 1,1­ diphenyl­2­picrylhydrazyl (dpph). the extract (100 mg of fresh weight + ethanol) was blended with 960 µl of dpph in methanol. the supernatant was cen­ trifuged for 5 min and kept in the dark room. the dpph was determined using a shimadzu uv­1800 spectrophotometer at 515 nm (kedare and singh, 2011). malondialdehyde (mda) content was mea­ sured according to velikova et al. (2000) methods. leaf tissues (0.5 g of each) were homogenized in 8 ml of 0.1% (w/v) trichloroacetic acid and the homogenates were centrifuged for 10 min at 4 °c, after which the supernatants were used for malondi­ aldehyde analysis. equal volumes of extracts were mixed with 0.5% (w/v) of thiobarbituric acid made in 5% (w/v) trichloroacetic acid and heated at 100°c water bath for 20 min, after which their actions were stopped in the ice bath. after centrifuging,the absorbance of the supernatant was measured at 450, 532, and 600 nm. adv. hort. sci., 2023 37(4): 377­389 380 determination of antioxidant enzyme activities extraction was performed according to a method used by dacosta and huang (2007). samples (0.5 g of fresh weight) were ground in liquid nitrogen and were homogenized in 4 mm phosphate buffer (ph 7.8), 60 m riboflavin, 195 mm methionine, 3 m edta, and 1.125 mm nitro blue tetrazolium chloride (nbt). enzyme activities were expressed per fresh weight of the sample. one sod activity unit was defined as the amount of enzyme required to cause 50% inhibition of nitro blue tetrazolium chloride (nbt) photoreduc­ tion (sairam et al., 2002). catalase activity (cat) was measured as described by abedi and pakniyat (2010). the reaction solution consisted of 50 mm k­phos­ phate buffer (ph 7.0), ten mm h2o2, and 50 ml enzyme extract. the decomposition of h2o2 was measured at 240 nm. the peroxidase activity (pod) was measured by the guaiacol method (guan et al., 2015). the oxidation of guaiacol was monitored by observing changes in the absorbance values at 470 nm for 3 min. the reaction mixture contained 50 ml of 100 mm pbs (ph 6.0), 10 mm h2o2, 2.58 mm of guaiacol. the reaction was started by adding the enzyme extract to the reaction mixture solution. measurement of photosynthetic pigments chlorophyll contents (chl a, b, and total chl) were measured by squashing the leaves (200 mg) in 10 ml 80% acetone solution, and the chlorophyll content was determined at 645 and 663 nm, respectively using a shimadzu uv­1800 spectrophotometer (nagata and yamashita, 1992). determination of osmotic regulators carbohydrates were determined using the anthrone reagent method. fresh leaves (500 mg) were placed in 70% methanol and reached the required volume with distilled water. the samples were used for estimations of carbohydrate content using the anthrone reagent (mccready et al., 1950). proline content was calculated according to bates et al. (1973). the leaf extract (0.1 mg leaf sample + 10 ml sulfosalicylic acid) was homogenized in glacial acetic acid and ninhydrin acid. then, the solution was heated in a boiling water bath. after cooling, 5 ml of toluene was added, and then the top layer of the solution was removed and centrifuged at 3000 g for 5 minutes. the proline content was determined at 520 nm using a shimadzu uv­1800 spectrophotometer. secondary metabolite measurements total phenolic content was measured using the folin­ciocalteu reagent method (singleton and rossi, 1965). in the folin­ciocalteu method, 250 µl of the alcoholic extract (100 mg + 10 ml ethanol) was dilut­ ed to a known volume with distilled water, 10% folin reagent, and 7.5% sodium carbonate. the phenolic content was determined at 675 nm using a shimadzu uv­1800 spectrophotometer. assaying the total anthocyanin content followed, a method by sukwattanasinit et al. (2007), where two buffer solu­ tions were used (25 mm k­chloride ph 1.0 and 0.4 m na­acetate ph 4.5). the values were noted at 510 nm using a shimadzu uv­1800 spectrophotometer. flavonoid content was assayed according to a method by zou et al. (2004). the extract (500 mg + 5 ml ethanol) was homogenized in 4.5 ml distilled water and 0.3 ml 5% nano2. next, after mixing the solution properly, 1 ml of 10% alcl3­6h2o, 2 ml of 1 m naoh, and distilled water were added to the reac­ tion mixture. the absorbance values were deter­ mined at 510 nm using a spectrophotometer (shimadzu uv­160a). statistical analysis the difference between treatments was deter­ mined using a factorial layout and a completely ran­ domized experimental design with three replicates followed by the lsd testing (p<0.01). data were sub­ jected to two­way analysis (anova) with repeated measures and were analyzed using the sas statistical package (version 9.2, sas institute, cary, nc, usa). 3. results the potassium and silicon concentrations were significantly (p<0.01) affected by fertilizer and drought stress. potassium (k) concentration in the control plants decreased by 14.9% under drought stress at 60% fc compared to 80% fc. a decrease in leaf k content was observed by the effect of potassi­ um silicate at a concentration of 2 and 4 ppm by 8.08 and 21.2%, respectively, under 70% fc. however, the amount of decrease was 8.7 and 18.2%, respectively, under 60% fc (fig. 1 a). the si concentration was sig­ nificantly improved by all potassium silicate applica­ tions under the water deficit conditions. the si con­ centration increased in response to 4 mm potassium silicate under 80 and 60% fc (by 926 and 998%, respectively) compared to control plants (fig. 1 b). the results showed that the interaction of potassi­ um silicate and drought stress significantly (p<0.01) affected electrolyte leakage, rwc, antioxidant activi­ ty, and mda accumulation. potassium silicate signifi­ oraee et al. ‐ the role of silicon in drought tolerance of daisy 381 cantly inhibited the decrease in electrolyte leakage, whereas a more significant level of decrease was observed in the control plants at 60% fc. as shown in figure 2a, electrolyte leakage was increased by 102, 150, and 220% at 60% fc in control plants, 2 and 4 mm compared to 80% fc, respectively. rwc was reduced by 8.6 and 11.7% under drought stress (60% fc) in response to 2 and 4 mm potassium silicate compared to 80% fc. compared with the control, the application of 4 mm potassium silicate significantly increased the rwc of plants, whereas 2 mm potassi­ um silicate had no significant effect on the rwc com­ pared to the control plants (fig. 2 b). the antioxidant activity increased significantly in response to drought stress, but the application of 2 and 4 mm potassium silicate under severe drought stress resulted in even higher values of antioxidant activity. nonetheless, no significant difference was observed between potassium silicate­treated plants and the control plants at 80% fc. the antioxidant activity reached maximum values, increasing by 33.7 and 36.5% when the daisies were treated with 2 and 4 mm potassium silicate at 60% fc compared to the control plants, respectively (fig. 2 c). the role of potassium silicate at 2 and 4 ppm was effective in reducing mda accumulation under drought stress. plants treated with potassium silicate under drought stress showed lower mda levels in the following order: 2 and 4 mm of potassium silicate than control fig. 1 ­ effect of exogenous application of potassium silicate on k (a), and si (b) of daisy under water­stress conditions. bars with a different letter differ significantly (p˂0.05) accord­ ing to the lsd test. fig. 2 ­ effect of exogenous application of potassium silicate on electrolyte leakage (a), relative water content (b), antiox­ idant activity (c), and mda (d) of daisy under water­ stress conditions. bars with a different letter differ signif­ icantly (p˂0.05) according to the lsd test. 382 adv. hort. sci., 2023 37(4): 377­389 plants. thus, decreasing effect on this trait resulted from using 4 mm potassium silicate at 70 and 60% fc. the mda peaked when the daisies were treated with distilled water and 2 mm potassium silicate at 60% fc. however, no significant difference was observed between the control plants and either of the 2 and 4 mm potassium silicate treatments under the effect of 80% fc (fig. 2 d). drought stress significantly (p<0.05) increased the activities of antioxidant enzymes, catalase, peroxi­ dase, superoxide dismutase, and aspartate peroxi­ dase by 7.76­31.54%, 313­323%, 127­181%, and 4.22­ 99.35%, respectively, under drought stress (fig. 3). in general, potassium silicate increased all of the men­ tioned enzyme activities, but this increase was higher in reponse to the 4 mm treatment under drought stress. the application of potassium silicate at 2 and 4 mm significantly improved the activity of cat by 29.4 and 35.2%, respectively, in daisies grown at 60% fc compared to the control plants (fig. 3 a). all potassium silicate treatments significantly improved the activity of pod under drought stress conditions. the pod activity increased in response to the 70% fc compared to the control, but decreased more at 60% fc, compared to 70% fc. as shown in figure 3 b, the activity of pod increased by 42.7 and 50.3% in plants treated with 2 and 4 mm potassium silicate, respec­ tively, compared to the control plants under 60% fc. under the conditions of drought stress, the appli­ cation of potassium silicate significantly increased the activity of apx. in response to 60% fc, the plants showed the highest apx activity. although no signifi­ cant differences were observed between potassium silicate­treated and control plants at 60% fc, a sharp increase in apx activity was observed when 4 mm potassium silicate was used along with drought stress. the application of 4 mm potassium silicate increased the activities of apx by 14.5% at 80% fc and by 87.8% at 60% fc compared to the control plants (figs. 3c). the application of potassium silicate increased the sod activity under drought stress con­ ditions. this pattern of increase was more prominent (37.5 and 30.2%) at both potassium silicate levels along with moderate drought stress, compared to 80% fc, whereas it was least prominent in severe conditions (60% fc). the activity of sod in daisy leaves increased by 28.4 and 21.5% at 60% fc, using potassium silicate at 2 and 4 mm, respectively, com­ pared to well­watered plants (fig. 3 d). the data revealed that the chlorophyll (chl) a, b, and total chlorophyll contents were significantly (p<0.05) affected by fertilizer and drought stress. regarding chl a, b, and total chl in the leaves under drought stress, these parameters decreased in response to the drought stress severity . this down­ ward trend was 34.68 and 55.4% higher in the case of chl a, but was 2 and 6.25% lower in the case of chl b when severity of drought increased. the application fig. 3 ­ effect of exogenous application of potassium silicate on cat (a), pod (b), apx (c), and sod (d) of daisy under water­stress conditions. bars with a different letter differ significantly (p˂0.05) according to the lsd test. oraee et al. ‐ the role of silicon in drought tolerance of daisy 383 of potassium silicate at a concentration of 2 mm increased the chl a and chl b by 68 and 67%, respec­ tively, at 70% fc. furthermore, the mentioned values were increased by 41.4 and 97%, respectively, at 60% fc compared to the control. the highest content of chl a and b were observed when plants were treated with 4 mm potassium silicate at 80 and 70% fc (figs. 4 a, b). during drought stress, total chl gradually decreased in response to greater intensity of drought stress. the total chl value decreased by 127% under 60% fc compared to 80% fc and by 98% compared to 70% fc. this value was also affected by potassium silicate under drought stress. total chl content was significantly improved by all potassium silicate appli­ cations under the drought stress conditions. total chl content increased by 7.4 and 72% in response to 4 mm potassium silicate at 80 and 70% fc, respectively (fig. 4 c). the application of potassium silicate significantly (p<0.05) increased the amount of carbohydrate and proline content in the leaves under drought stress. at 80% fc, potassium silicate­treated plants had no sig­ nificant difference from the control plants in terms of carbohydrate content. by applying drought stress, an increase in carbohydrates was observed in the leaves when the plants were treated with potassium sili­ cate. in response to 2 and 4 mm potassium silicate, the carbohydrate content increased by 31.3% and 26.6% at 70% fc, and by 60.8% and 53.2% at 60% fc, respectively, compared to the 80% fc (fig. 5a). regarding proline changes in the leaves, there was an increase of 4.7­ and 1.6­fold by the effect of 60% fc and 70% fc, respectively, compared to the 80% fc. the application of 2 and 4 mm potassium silicate increased this parameter further by 75 and 42% at 70% fc, respectively, but by 385 and 312% at 60% fc, compared to the control plants. the highest proline fig. 4 ­ effect of exogenous application of potassium silicate on chlorohyll a (a), chlorohyll b (b), and total chlorohyll (c) of daisy under water­stress conditions. bars with a different letter differ significantly (p˂0.05) according to the lsd test. fig. 5 ­ effect of exogenous application of potassium silicate on carbohydrate (a), and proline (b) of daisy under water­ stress conditions. bars with a different letter differ signif­ icantly (p˂0.05) according to the lsd test. adv. hort. sci., 2023 37(4): 377­389 384 content was observed in drought­stressed plants (60% fc) treated with potassium silicate. however, no significant difference was observed between 2 and 4 mm potassium silicate under severe drought stress (fig. 5b). in general, potassium silicate (2 and 4 mm) at 80% fc significantly (p<0.01) increased the flavonoid con­ tent by 36 and 45.5% compared to the control plants. in the control plants. increasing the severity of drought stress at 70 and 60% fc decreased this trait by 50.2 and 97.2%, respectively. the application of 2 and 4 mm potassium silicate increased the flavonoid content by 10.3 and 41.9%, respectively, at moderate water deficit (70% fc), but by 51.7 and 62.3%at severe water deficit (60% fc), respectively, compared to the 80% fc (fig. 6 a). figure 5b shows that the phenolic content increased in response to the inten­ sity of drought stress, and the highest value was found at 60% fc. total phenolic content increased significantly by 31.1 and 43.8% when potassium sili­ cate­treated plants (4 mm) were under the effect of 80 and 70% fc, respectively, compared to the control plants. potassium silicate (2 mm) increased the phe­ nolic content by 27 and 46%, respectively. at 4 mm, it caused an increase of 39 and 57%, respectively, under the effect of 70 and 60% fc, compared to 80% fc. the highest anthocyanin content occurred when the potassium silicate­treated plants were grown at 80% fc. as for anthocyanin changes in the flowers, there was a decrease in response to the intensity of drought, but potassium silicate increased the value further. the anthocyanin content decreased in response to 4 mm potassium silicate at 60% fc, com­ pared to 80% fc (11.2%). both concentrations of potassium silicate significantly increased the antho­ cyanin content in the flowers by 228% at most, under the effect of 80% fc, compared to the control plants (fig. 6 c). 4. discussion and conclusions the application of potassium silicate improved nutrient uptake under drought stress. si is not an essential nutrient but protects plants from a variety of biotic and abiotic stresses (ranjan et al., 2021). the highest concentration of k and si in the plant occurred in response to 4 mm potassium silicate at 80% fc. these results emanate from the ability of plants to enhance root growth after potassium and silicon application, thereby increasing nutrient uptake (sustr et al., 2019). the increase in k uptake is usually concomitant with a decrease in plasma mem­ brane permeability and an increase in si­induced h­ atp activity in plasma membranes (zhu and gong, 2014). k deficiency decreases the uptake and transfer of some nutrients by inhibiting enzymatic activities such as synthases, transferases, and kinases (liu et al., 2013). si leads to more significant root activity, consequently, a greater amount of nutrient uptake. sarto et al. (2014) attributed the beneficial effects of si to its concentration in the leaves and stems of wheat. si is also known to influence the development of apoplastic barriers in roots by controlling apoplas­ fig. 6 ­ effect of exogenous application of potassium silicate on phenol (a), flavonoid (b), and anthocyanin (c) of daisy under water­stress conditions. bars with a different let­ ter differ significantly (p˂0.05) according to the lsd test. oraee et al. ‐ the role of silicon in drought tolerance of daisy 385 tic pathways, followed by its translocation through the root apoplast to the shoot (vaculík et al., 2012). one explanation for the increased tolerance in plants growing under water­deficit conditions could be a decrease in transpiration through the stomata and cuticle due to si application. silicon not only affects nutrient availability and uptake but also nutrient translocation from the roots to the shoots (greger et al . , 2018). in this study, drought significantly decreased the concentration (%) of k and si. also, drought stress can have a significant impact on plant nutrient ratios. several studies have shown that drought can reduce nutrient uptake from the soil (ge et al., 2012; bista et al., 2018). under water­deficit conditions, potassium silicate reduces electrolyte leakage. potassium silicate plays an important role in plant resistance to environmen­ tal stress. within the plant, silicate is an immobile element that becomes a polymer gel and reduces the loss of ions from biomembranes after being deposit­ ed in the cell. the results of this study are consistent with a previous study by othmani et al. (2021) who found that the more significant stability of the cell membrane in the presence of silicon was due to the hardening and strength of the cell wall. the water content of leaves under drought stress (70 and 60% fc) decreased compared to the 80% fc. drought stress reportedly caused a decrease in leaf water content in most plants (santos et al., 2021). in our study, the administration of potassium silicate led to an increase in the relative water content of leaves. a lower rate of water loss in silicate­potassium­fed plants can also be attributed to the lower transpira­ tion of the plants. the accumulation of silicate in the lower epidermal cells reduces water loss through the cuticle. si is deposited in plant tissues in the apoplast of the cell wall to form silica, thereby maintaining tis­ sue integrity (guerriero et al., 2016). in addition, potassium is primarily an important osmotic regula­ tor in plants. between 30 and 50% of the osmotic potential of leaf tissue is regulated by k (turcios et al., 2021). in the current study, potassium silicate reduced plant injury by decreasing mda and increasing antioxidant activity. malondialdehyde is the peroxi­ dation product of unsaturated fatty acids in phospho­ lipids. therefore, the production of malondialdehyde under stress conditions can be used as a marker of lipid peroxidation (ayala et al., 2014). as a result of drought, the peroxidation of glycopeptides occurred in chloroplast thylakoid, followed by the formation of diacylglycerol, triacylglycerol, and free fatty acids, leading to an increase in malondialdehyde in plant tissues (sofo et al., 2004). fatty acids and lipids are reportedly sensitive to oxygen species and are rapidly oxidized. the results are in line with previous studies indicating a positive effect of potassium silicate on malondialdehyde levels in damask rose (rosa damas‐ cena miller) under drought stress (farahani et al., 2020). the ability of plants to scavenge free radicals was impaired by both drought and the addition of potassium silicate compared to the control plants. dpph inhibition levels were below 70% and 60% fc at 4 mm potassium silicate compared to the control. under drought stress, dpph levels increased, and potassium silicate further enhanced the dpph levels (zahedi et al., 2020). in this study, the administration of potassium sili­ cate under drought stress conditions (i.e. the applica­ tion of irrigation water to maintain 70 and 60% fc) increased all antioxidant enzyme activities. an increase in antioxidant activity in the leaves occurred in response to both potassium silicate concentrations and drought stress. the high activity of antioxidant enzymes such as cat, pod, apx, and sod in plants is an adaptive mechanism that protects cells from oxidative damage by reducing the concentration of hydrogen peroxide generated by cellular metabolism (jan et al., 2022). improving potassium concentration leads to an increase in photosynthetic products, the control of ionic balance, osmotic regulation, and an increase in enzymatic activity. by stimulating the activity of pod and apx through the detoxification of hydrogen peroxide, si prevents oxidative stress and inhibits the production of hydroxyl radicals (kim et al., 2017). in agreement with the current results, ahmad et al. (2019) reported that using silica on mung beans (vigna radiata l.) increased catalase and superoxide dismutase activities under drought stress. superoxide dismutase is an enzyme that converts superoxide free radicals into hydrogen peroxide and oxygen while playing an important role in protecting cells from the negative effects of free radicals. sod is the first line of defense of cells against free radicals under stress conditions (ighodaro et al., 2018). the effects of si nutrition on sod activity and free radical elimination have been reported in the available liter­ ature (geng et al., 2018). gong et al. (2005) reported that using potassium silicate increased the activity of antioxidant enzymes in wheat (triticum aestivum l.) under drought stress. the removal of reactive oxygen species decreases cell membrane permeability and adv. hort. sci., 2023 37(4): 377­389 386 increases the activity of catalase, peroxidase, and superoxide dismutase, which indirectly decrease cell membrane lipid peroxidation and reduce the amount of malondialdehyde (sharma et al., 2012). the application of potassium silicate along with drought stress increased the amounts of photosyn­ thetic pigments. a decrease in chlorophyll content occurred due to drought stress and was accompanied by an increase in the production of oxygen radicals in the cells. the radicals usually cause peroxidation, and consequently, the degradation of photosynthetic pig­ ments. the effect of potassium silicate on the stability of plant pigments usually results from the accumula­ tion of silicate in the epidermal cells, which has an indirect protective effect on the photosynthetic estab­ lishments, thereby reducing the stress­induced dam­ age to photosynthetic pigments. similar to the current results, a moderating effect of potassium silicate was reportedly observed on the chlorophyll content of rosmarinus officinalis l. plants (waly et al., 2019). the results showed that the concentration of osmotic regulators (i.e. proline and total carbohy­ drates) increased significantly when potassium sili­ cate was applied under water­deficit conditions. silicon and potassium increase the production of car­ bohydrates and proline by increasing the osmotic potential, possibly through the accumulation of free radicals produced by the plant. they are thought to play an adaptive role in mediating osmotic adjust­ ment and protecting subcellular structures in stressed plants (hajiboland et al., 2017). these effects suggest that potassium and silicon may enhance leaf osmotic potential by converting starch to soluble sugars, especially under severe drought stress (zahoor et al., 2017). it appears that potassium silicate stimulates carbohydrate production and, thus, alters the metabolism of plant­absorbed k and its conversion to proteins (hafez et al., 2021). si can directly or indirectly induce the biosynthesis of pro­ line. garg and sing (2018) showed that the applica­ tion of si increased the activity of pyrroline­5­car­ boxylate synthetase (p5cs) and glutamate dehydro­ genase (gdh). in addition, the increase in proline because of potassium silicate treatment may high­ light the importance of potassium and silicon in pro­ tecting cell membranes and maintaining relative water content under inadequate irrigation condi­ tions. in this context, using silica on borage (borago officinalis l.) plants reportedly increased the amount of proline in the leaves (gagoonani et al., 2011). in agreement with these results, ibrahim et al. (2020) reported that potassium silicate increased the pro­ line content of maize plants under drought stress. by reducing vegetative growth and altering the anatomical structure of the plant through the induc­ tion of secondary stress, e.g. oxidative stress, the effect of drought stress usually cause changes in the pathways of synthesis that make secondary com­ pounds and metabolites (ahanger et al., 2017). polyphenols can improve plant tolerance to drought stress and play an important role as a carbon sink at times of stress. these effects may explain significant improvements in total phenolics in daisies because of their exposure to drought stress (fig. 6 a). the increase in total soluble phenols in response to the application of k2sio3 under drought stress could be a supporting effect of si, thereby increasing plant toler­ ance, especially under water­deficit conditions. fouda et al. (2021) found that potassium silicate increased the total flavonoid content in field beans. feeding plants with si and k­containing compounds has reportedly resulted in changes in the expression pattern of many genes. in particular, feeding plants with potassium­ and silicon­containing compounds has led to changes in genes that encode enzymes involved in the phenylpropanoid pathway (wang et al., 2017). indeed, the increase in phenylalanine ammonia­lyase activity is a common feature in plants treated with silicon, thereby enabling an increase in the synthesis of phenolic compounds. potassium sili­ cate increases polyphenols in many plants by activat­ ing enzymes that are relevant to the phenol produc­ tion pathway, such as the phenylalanine ammonia­ lyase (vega et al., 2019). the ability of plants to tolerate drought could be mainly explained by an increase in flavonoid content since flavonoids are compounds with strong antioxi­ dant activity. perin et al. (2019) suggested that the relationships between aba metabolism, phenyl­ propanoid, flavonoid, and anthocyanin pathways can reduce drought stress. probably, this could be one of the main reasons for the better tolerance of plants to drought stress. drought largely affects the average performance of plant traits by reducing their proper­ ties, leading to a decrease in the associated antho­ cyanin content. under drought stress, the antho­ cyanin content decreased, but potassium silicate increased the anthocyanin content of flowers (fig. 6 c). these results are consistent with a previous study by cirillo et al. (2021) in which anthocyanin content was reduced by the effects of stress. jafari et al. 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different soil water tensions and mulching types p.a.b. santos 1, l.g. carvalho 1, f. schwerz 1 (*), v.b.s. baptista 2, c.a.u. monti 3 1 agricultural engineering department, federal university of lavras, lavras, brazil. 2 engineering department, federal university of lavras, lavras, brazil. 3 department of forestry and environmental resources, north carolina state university, raleigh, nc, usa. key words: growth traits, irrigation management, mulch system, production cost. abstract: there is a lack of information on the production of irrigated radish associated with the use of mulching and on the economic viability of these pro­ duction technologies. the objective of this study was to evaluate the growth, yield, and economic viability of the radish crop under different soil water ten­ sions (swt) and mulching types. the experiment was conducted in a green­ house. during the experiment, the following variables were evaluated: growth parameters, yield and economic viability. swt at 7 kpa in the treatments with­ out mulching and at 12 kpa in the treatments with black plastic and black non­ woven resulted in higher growth parameters and yield. the leaf area index and the root diameter were the parameters that had a high and positive correlation with yield. expenses with variable resources represent on average 75% of the total production cost. therefore, the investment pays all resources applied in the activity and provides an economic profit. in this context, the higher radish yield with 37.5 t ha­1 provided the highest profitability of the evaluated treat­ ments, thus, for radish production, the recommendation is to use 12 kpa as an indicator of the moment for irrigation, associated with the use of black plastic. 1. introduction radish crop (raphanus sativus l.) is a vegetable belonging to the brassicaceae family grown worldwide, consumed mainly in salads, cooked, and even pickled (chihoub et al., 2019). the radish has been gain­ ing prominence among vegetables due to its rusticity and its short cycle (kim et al., 2014; zhang et al., 2021), ideal for small and medium produc­ ers. however, in brazil, its production and consumption are still small, and it can be better exploited by horticulturists, creating a market niche. in brazil, according to the agricultural census (2006), around 10,500 tons of (*) corresponding author: felipe.schwerz@ufla.br citation: santos p.a.b., carvalho l.g., schwerz f., baptista v.b.s., monti c.a.u., 2022 ­ economic viability and development of radish (raphanus sativus l.) under different soil water tensions and mulching types. ­ adv. hort. sci., 36(3): 227­237. copyright: © 2022 santos p.a.b., carvalho l.g., schwerz f., baptista v.b.s., monti c.a.u. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 january 2022 accepted for publication 24 august 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12552 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 227­237 228 radishes were produced, generating revenue of r$ 9 million (conab, 2010). the south and southeast regions are the regions with more radish production, with 4,587 and 4,456 tons, respectively. in general, radish productivity in brazil is between 11 and 30 t ha­1. despite its rusticity, the high yield and profitability of the radish crop will only be achieved under opti­ mal conditions of soil moisture, air temperature, and fertilization (gruver et al., 2016). therefore, ade­ quate management techniques are necessary to achieve this optimal cultivation condition. proper irrigation management is one of the ways to achieve maximum yield. the timing and amount of water applied are critical to the irrigation effectiveness. excessive irrigation increases pumping costs, water waste, and crop disease susceptibly; nevertheless, deficit irrigation generally causes losses and reduces production quality (contreras et al., 2017). soil water tension (swt) is one of the ways to determine the timing of irrigation and the volume of water to be applied. swt is a fundamental variable to describe the water availability in the soil and the capacity for that water to be used by plants (meyer and green, 1981). according to masseroni et al. (2016), the local measurement of swt is one of the most effective options for irrigation management. in addition to adequate irrigation, mulching is also a technique used in the search for high yield. soil mulch can decrease the evaporation rate, maintain the soil moisture, moderate the temperature, and form a barrier to weed growth, which can significant­ ly affect yield and water consumption (an et al., 2015; gao et al., 2019). according to carmichael et al. (2012), mulching significantly increased the radish yield. however, the acquisition, installation, and maintenance of the irrigation and mulching require a high investment, which represent important addi­ tional costs to production. based on agricultural production costs, it is possi­ ble to evaluate the profitability and efficiency of the production system adopted by the rural producer and makes it possible to obtain information for deci­ sion­making on agricultural activities (artuzo et al., 2018). the production cost is grouped into fixed costs, variable costs, operating costs, and total cost. economic analysis compares the production cost with the gross revenue obtained from the sale of the product produced. therefore, the success of the enterprise is related not only to the production cost, but also to the final product price and, mainly, to crop yield (schwerz et al., 2017). radish yield can be influenced by numerous fac­ tors, including the plant’s response to the production environment. therefore, it is necessary to under­ stand the crop traits that contribute to high yield and their interrelationships. knowledge of the relation­ ship between yield and crop growth variables obtained through correlation analysis helps in plant selection and develop high yielding varieties (carmichael et al., 2012; schwerz et al., 2017). correlation coefficient (r) measures the degree (intensity) and nature (direction) of association between characters (abd el­mohsen et al., 2013). given the above, the present study suggests that the combined use of water tension in the ideal soil with mulching can increase development, yield, and profitability. besides, the gain in profitability with the use of these techniques can exceed the costs of implementation and generate high profits. therefore, the aims of this study were to evaluate the growth variables and economic viability of radish production under different water tensions in the soil associated with types of mulching, under unheated plastic greenhouse. also, to determine the relationship between morphological variables and the radish yield to help radish producers how to determine what growth parameters could be efficiently used to raises yield. 2. materials and methods experimental site characteristics and cultural prac‐ tices the experiment was conducted during september and october of 2018, in greenhouses covered by low­ density polyethylene, at the experiment area of the department of water resources and sanitation of the federal university of lavras (ufla). the experi­ ment area is located in the southern region of the state of minas gerais (21°14’ s, 45°00’w and 918,84m altitude). the climate in the region is classi­ fied as cwa, according to the methodology proposed by köppen (dantas et al., 2007). soil characteristics of the study area the soil used in this study was classified as a dis­ troferric red latosol (oxisol) with a clayey texture according (santos et al., 2006). the chemical charac­ teristics of the soil were: ph = 7.0; k = 106.7 mg dm­3; p = 0.7 mg dm­3; ca = 3.53 cmolc dm­3; mg = 0.39 santos et al. ‐ agricultural traits of radish under different water requirements and mulching 229 cmolc dm­3; al = 0.04 cmolc dm­3; h+al = 1.54 cmol dm­3; m.o.= 2.11 dag/kg e v = 73.12. it was applied in the planting fertilization 1500 kg ha­1 de p2o5 in the form of single superphosphate, 145 kg ha­1 of urea (n), 320 kg ha­1 of potassium chloride (k), 620 kg ha­1 of limestone and 12,5 kg ha­1 of commercial product solubor (17.5% boron ­ b). irrigation management and experimental design the drip irrigation system was used, with self­ compensating emitters, spaced 0.3 m apart, and operating with a discharge of 4.3 l h­1. the swt was determined utilizing tensiometers at two depths (0.15m and 0.25m) in each treatment. the soil water­ retention curve was adjusted according to the model proposed by van genuchten (1980). the soil mois­ ture of 0.453 l3 l­3 (5 kpa) was the corresponding to the field capacity (θcc), according to the model pro­ posed by mello et al. (2002). ɵ = 0.235 + (0.614 ­0.235) [1+(0.269 x ½ym½2.064]0.515 r2 = 0.93 where: θ is the soil moisture content (cm³ cm­3) and ym is the soil water tension (kpa). the functioning of the irrigation system was calculated based on the gross water depth, according to (pizarro cabello, 1996), considering a 0.2 m effective root. a 90% water­application efficiency of the irrigation system was adopted and a water distribution­uniformity coefficient (duc) of 98% was obtained. the experimental design was randomized com­ plete in a 4 x 3 factorial, replicated four times, total­ ing 12 treatments. the four swt used were 7, 12, 20, and 50 kpa. the mulching materials used were black polyethylene film (black plastic); black polypropylene (black non­woven film); and no mulch (control) (table 1). experimental plots, data collection and analysis the transplant of comet radish seedlings occurred 6 days after the planting of the seeds in the experimental plots prepared, fertilized, and with mulching fixed. the plots were kept with moisture close to the field capacity (θcc) for 3 days, after that period, the irrigation differentiation level started. the plot received 24 plants with 0.2m between rows and 0.05m between plants. at harvest time, eight central plants were used for the analysis of leaf weight (lw), leaf number (ln), plant height (ph), root diameter (rd), root length (rl) and root weight (rw). the total yield (ty) was estimated considering the total weight of the roots within the useful area. commercial yield (cy) was estimated by subtracting the percentage of cracked and defective roots from ty. also, the soil cover frac­ tion (scf) and leaf area index (lai) were measured. the scf was estimated using the following equation: scf = ppa/ua where: scf is the soil cover fraction, ppa is the plant projection area (ppa), and ua is the useful area of the plot. for the leaf area index the following equa­ tion was used: lai = tla/ua where: lai is the leaf area index, tla is total leaf area of the plants, and ua is the useful area of the plants. moments before harvest, photos of the plots were taken using a camera 1m away to obtain the ppa (fig. 1b). after harvesting, the plant leaves were placed on a platform and were photographed at 0.6m away to obtain the tla (fig. 1d). ppa and la were calculated using imagej software (fig. 1c, 1e), which is free to use. table 1 ­ treatments of soil water tension and mulching in the radish crop treatment code swt (kpa) mulch material nm7 7 control (no mulch) bp7 7 black plastic bnw7 7 black non­woven film nm12 12 control (no mulch) bp12 12 black plastic bnw12 12 black non­woven film nm20 20 control (no mulch) bp20 20 black plastic bnw20 20 black non­woven film nm50 50 control (no mulch) bp50 50 black plastic bnw50 50 black non­woven film fig. 1 ­ (a) imagej software interface; (b) image of the useful area of the experimental plot; (c) projection area calcula­ tion; (d) images of plant leaves and (e) calculation of leaf area. adv. hort. sci., 2022 36(3): 227­237 230 fixed and variable cost the radish yield (t ha­1) and the costs per cultivat­ ed hectare were used for the cost analysis, in approx­ imate values of brazilian real (r$). in the estimate of commercial yield in the greenhouse, the useful plant­ ing area index of 57% was used (araújo neto et al., 2012) in addition to subtracting the percentage of defective roots. in this study, the methodology proposed by reis (2002) was used to estimate production costs. the production cost is the integration of all inputs, labor, depreciation, and operational values in the produc­ tion process, including the alternative cost. production cost were grouped into: fixed costs, vari­ able costs, and total cost. fixed cost refers to depre­ ciation and alternative costs. the variable costs are those related to the crop costs during the cycle of the production process. the sum of fixed and variable costs represents the total cost. depreciation is defined as the cost necessary to replace capital goods when rendered useless by physical or economic wear and tear. the linear method was used, considering 6 cycles per year, which corresponds to the average cultivation cycle of the radish cultivar used in addition to the rest and soil preparation period. the depreciation (d) was cal­ culated by the following equation: d = (vp ­ vr)/lu x p where: d is depreciation (r$), vp is the present value of the asset (r$), vr is the residual or resale value (r$), lu is the useful life or period of activity of the asset (years), and p is the period of analysis or pro­ ductive cycle (years). the interest rate of 7% per annum (p.a) was con­ sidered for the analysis of the alternative cost of fixed and variable resources allocated to production, above the recommended by the companhia nacional de abastecimento ­ conab (2010). the alternative fixed cost allocated to the radish cultivation was cal­ culated using the following equation: acfixed = [(lu ­ a) /lu] x vp x ir x p where: acfixed is the alternative fixed cost (r$); a is the average duration of the asset use (years), consid­ ered 50% of lu, and ir is the interest rate (decimal). the alternative cost of the variable assets (acvar) allo­ cated to the radish cultivation was calculated accord­ ing to equation: acvar = vexp/2 x ir where: acvar is the alternative variable cost (r$), and vexp is the financial investment for the acquisition of inputs and services for the crop production (r$). the fixed cost corresponding to the sum of the contributions of fixed factors in total product in each production cycle. the alternative cost of the produc­ tion factor was added to the depreciation in calculat­ ing the fixed cost. the following items were consid­ ered in this calculation. land and rural land tax: the value of the rural land tax (rlt) was not considered, due to the exemption for properties below 30 ha. the land is not depreciated when proper soil management is adopted, and all chemical elements extracted by the plant are replaced through the practice of soil fertil­ ization. the value considered was the alternative cost, based on the land rental value. the rental value was r$ 131.35 per hectare and per month, as men­ tioned in the on the agricultural price indexes of the department of business administration and economics of ufla (dae/ufla) and corrected by the general price index ­ internal availability (gpi ­ ia), for november 2018 amounts (r$). mulching: the value of black polyethylene film (bp) and black non­woven film (bnw) were r$ 0.41 m­2 and r$ 0.67 m­2. based on the area of mulching required in the experiment, approximately 72% of the greenhouse area. expenditures on bp and bnw were r$ 3,028.1 ha­1 and r$ 4,834.2 ha­1, respective­ ly. a useful life of 1 years was considered. seedling tray and greenhouse: the expenses with 2860 seedling tray were r$ 6,292.0 ha­1 (20% more to guarantee the quantity of seedlings necessary for transplantation) and the useful life of 3 years. expenses for greenhouse structure and low­density polyethylene film coverage were r$ 380,000.00 ha­1 and r$ 25,525 ha­1, respectively, considering the use­ ful life of 20 years and the change of greenhouse cover every 2 years. irrigation system: the quantities of material and the irrigation system cost is influenced by the unevenness degree of land, the water collection dis­ tance, and the equipment used. a project with the following characteristics was considered: 5 hp motor­ pump set, system automation set with starter switch, contactor and relay, programmable irrigation con­ troller with nine outlets, relief valve, air valves and vacuum, electric control valves (solenoids), under­ ground irrigation pipes dn 150 for main irrigation system, pvc piping (50 mm in diameter) connecting the main system to sectors, dn 16 mm low density polyethylene (ldpe) tube, tube connection fittings dn 16 mm, self­compensating dripper with a nominal santos et al. ‐ agricultural traits of radish under different water requirements and mulching 231 flow of 4.3 l h­1 and 2 disc filters with automatic backwash. the useful life considered was 20 years, except for ldpe pipes and connection fittings, which useful life considered was 3 years. the residual value was estimated at 20% of the acquisition value. the maintenance and operation of the system is equiva­ lent to 2% of the acquisition value. the expenses for the services and products acqui­ sition in each crop cycle, added to the alternative cost, was used in the variable cost calculation. the following items were considered in this calculation. inputs: related to investment in the acquisition of substrate, seeds, chemical fertilizers, and pesticides. the value of each input was based on the report on agricultural inputs (conab, 2018) and the values pro­ vided by companies producing seeds and substrates. the amount of inputs needed were based on the quantity used in the experiment, according to the soil analysis and the recommendations for the crop. labor: expenses with labor refer to the implemen­ tation, conduction and harvesting of the crop, opera­ tion of machines and irrigation system, and post­har­ vest processing (cleaning, bagging, and transporta­ tion within the property). the unit value practiced was r$ 954.00 (minimum wage practiced in 2018) plus 51.56% as social charges, according to the methodology proposed by conab (2010). energy: the energy cost was calculated according to the following equation: ec = vkwh x t x (736xpwr)/1000 x h where: ec is the energy cost (r$); t is the total oper­ ating time of the irrigation system in each treatment (h); vkwh is the kwh price (r$); pwr is the motor pump power (hp), and h is the motor pump efficiency (decimal). r$ 0.49 is the price per kwh charged by minas gerais electric power company (ministério de minas e energia, 2018). the cost for the volume of water used was not considered, the collection being considered public or for use by the producer. administration and post­harvest cost: expenses with administrative labor and technical assistance were 6% of the variable costs (conab, 2010). post­ harvest cost refers to expenses with product improvement, wooden boxes for packaging and transportation to the destination. the quantities used changed depending on the average yield of each treatment. machines and implements: referring to the investment in renting machines and implements in the preparation of the soil. the unit values consid­ ered were those mentioned in the department of business administration and economics of ufla (dae/ufla) agricultural price indices. the quantities used for each resource were estimated according to the quantity used in conducting the experiment. alternative cost: the real interest rate of 7% p.a. was considered for calculating the alternative cost of each item of the fixed and variable cost. economic analysis the radish price adopted was r$ 1.20 kg­1, which is equivalent to the average price paid by the food acquisition program (programa de aquisição de alimentos ­ paa), operated by the national supply company, and the prices practiced in the supply centers of minas gerais (centrais de abastecimento de minas gerais s.a. ­ ceasaminas) in october 2018. the operating cost considers the depreciation and inputs used, equivalent to the analysis period, with­ out the alternative cost. average total operating cost (toc) and average total cost (atc) were calculated in unit terms in the economic analysis. the toc, in r$ kg­1 of radish, is divided into average fixed operating cost (foc), which is composed of the depreciation, and the average variable operating cost (voc), which is composed of the disbursements during the analysis period (reis, 2002). the economic analysis evaluates the toc and the atc in relation to the practiced price. this analysis can result in different conditions, and each result sug­ gests an interpretation. to carry out this interpreta­ tion of the economic analysis, the situations of eco­ nomic and operational analysis of the productive activity, described by reis (2002), were considered. thus, this study presents a diagnosis of the econom­ ic­financial behavior of irrigated radish cultivation, with information about the remuneration obtained and the allocated resources in comparison with the remuneration provided by investment alternatives (alternative cost). statistical analysis data were statistically analyzed using the statistical analysis system learning edition 8.0 (sas, 2003) computer program. data were initially exam­ ined for homogeneity of variance and then subjected to analysis of variance. tukey test (p>0.05) was used to compare the difference between the treatments for the growth variables, root variables and radish yield. in order to analyze the correlation of plant growth, root growth, and yield variables, pearson 232 adv. hort. sci., 2022 36(3): 227­237 correlation was conducted, which was qualitatively evaluated for intensity using the following criteria, proposed by callegari­jacques (2003): null (0), low (0 to 0.3), regular (0.3 to 0.6), strong (0.6 to 0.9), very strong (0.9 to 1.0) and full (1.0) . 3. results and discussion growth and yield of radish the results related to radish growth and yield can be seen in table 2. for the variables leaf weight and plant height was possible to observe a similar behav­ ior, were mulched system resulted in higher values of leaf weight and plant height than those without mulch (table 2). the variables leaf weight and plant height were significantly higher with black plastic mulching at 12 kpa, and treatment without mulching at 50 kpa resulted in the lowest growth of these parameters. the water quantity applied had a direct influence on leaf weight and plant height and increase in the water quantity resulted in plants with heavier leaves and taller plants. the bp7 and bp12 resulted in a higher leaf number, however there was no significant difference between treatments. the leaf area index and soil coverage fraction grew with the increase in the water quantity used and mulching treatments resulted in higher values (table 2). the highest leaf area index and soil cover­ age fraction values were observed with bp7 and bp12, respectively. the lowest leaf area index and soil coverage fraction observed were with the nm50 treatment, and the nm50 differed significantly from the bp7 treatment. these results agree with previous findings by carmichael et al. (2012), who reported an increase in radish growth parameters with increasing irrigation depth. these authors also demonstrated that the use of mulching can significantly influence the growth of the radish. other authors have also observed similar results. kang and wan (2005) reported a maximum leaf area index at 15 kpa in the radish crop in 2002. yaghi et al. (2013) reported that different types of mulching created a positive effect on the growth of cucumber plants. there was a significant difference between the treatments applied in the root diameter and root length parameters, however, in the root weight para­ meter, there was no significant difference between treatments. the highest values of root diameter, root length, and root weight were obtained with treat­ ments bp12, nm7 and bnw12, respectively (table 3). the lowest values of root diameter, root length, and root weight were obtained from the nm20, nm50, and nm50 treatments in decreasing order. the increase in swt resulted in a decrease in root growth parameters in treatments without mulching; howev­ er, a higher growth was observed at 12 kpa in treat­ ments with black plastic and black non­woven. for the total yield and commercial yield (table 3) was possible to observe that treatments without mulching, maximum total yield and commercial yield were obtained at 7 kpa and the increase in swt table 2 ­ effect of irrigation and mulching on growth variables of radish * different letters within columns indicate significant difference by tukey test at 5% probability level. treatment growth variables leaf weight (g) leaf number plant height (cm) leaf area index soil coverage fraction nm7 25.3 abc 6.6 a 32.7 abc 5.78 ab 2.32 abc bp7 30.3 ab 7.2 a 35.5 ab 6.46 a 2.73 a bnw7 22.8 bc 7.0 a 33.2 ab 5.02 abc 2.40 abc nm12 22.4 bc 7.0 a 30.7 bc 4.49 abc 2.32 abc bp12 34.1 a 7.2 a 37.3 a 6.30 ab 2.55 ab bnw12 24.8 abc 6.8 a 33.5 ab 5.15 abc 2.19 abc nm20 22.4 bc 6.6 a 30.1 bc 4.11 bc 1.86 bc bp20 25.8 abc 6.8 a 34.4 ab 4.81 abc 2.34 abc bnw20 23.2 abc 6.4 a 32.9 ab 4.46 abc 2.28 abc nm50 14.9 c 6.1 a 27.0 c 3.13 c 1.74 c bp50 21.8 bc 6.8 a 32.0 abc 4.45 abc 2.12 abc bnw50 23.5 abc 6.4 a 33.9 ab 4.48 abc 1.93 bc cv 15.52 6.62 5.97 15.51 10.63 santos et al. ‐ agricultural traits of radish under different water requirements and mulching 233 decreased these variables. however, the behavior of total yield and commercial yield was different between treatments with black plastic and black non­ woven. the maximum total yield and commercial yield were reached at 12 kpa in these treatments. therefore, keeping soil moisture close to the field capacity associated with mulching resulted in water stress suffered by the plant, due to the mulch’s char­ acteristics of maintaining high soil moisture. however, with the proper swt, the use of mulching increased yield when compared to treatments with­ out mulching. gao et al. (2019) and li et al. (2018) also reported a significantly increase of yield of dif­ ferent crops with mulching. the bp12 treatment resulted in the highest total yield and commercial yield, 41.8 t ha­1, and 37.5 t ha­ 1, respectively (table 3). this result highlight the importance of the use of irrigation and water man­ agement in radish crop. kang and wan (2005) report­ ed the total yield of 47.4 t ha­1 under irrigation man­ agement system. these yield differences observed in the literature can be explained by the radish cultivars used, and the difference in cultivation techniques adopted. yield values above those obtained in the present study indicate a potential for yield growth that can be achieved by new techniques. correlation and multiple linear regression analysis the yield of the radish root is usually determined by some characters of a morphological nature. adequate knowledge of the relationship between yield and morphological characters is essential for the identification of selection criteria to be used to improve the yield of radish. significant association was found between all the contributory characters studied and total and commercial yield (table 4). the results of the person correlation presented in table 4 showed that the leaf area index (plant growth para­ meter) and the root diameter (root growth parame­ ter) that had a very strong and positive correlation with total and commercial yield. the other characters also showed a positive correlation, however with lower r values. therefore, the increase in leaf area index and root diameter implies a greater increase in yield when compared to the other characters. these results indicate that concentrating efforts on the selection of plants with high leaf area index and high­ er root diameter would be accompanied by high yield capacity in these conditions. the findings in this paper confirm previous studies by ullah et al. (2010), which found a positive and sig­ nificant correlation between radish yield with plant height, root length, and root diameter. khatri et al. (2019) found a positive relationship between yield and plant height, root length and root diameter, however leaf number showed highest significant pos­ itive correlation with total yield of radish. however, these papers showed that different morphological characters can have a greater relationship with yield under different cultivation conditions. table 3 ­ effect of irrigation and mulching on root growth variables and radish yield * different letters within columns indicate significant difference by tukey test at 5% probability level. treatment root variables yield rooot diameter (cm) root length (cm) root weight (g) total yield (t ha­1) commercial yield (t ha­1) nm7 42.9 ab 60.6 a 53.3 ab 37.6 ab 32.0 ab bp7 41.0 abc 60.1 a 46.8 abcde 33.0 abcde 30.4 abcd bnw7 39.0 abcde 63.6 a 47.7 abcde 33.7 abcde 30.8 abc nm12 37.8 bcde 59.2 a 40.4 bcde 28.5 bcde 25.9 bcd bp12 43.4 a 67.6 a 59.2 a 41.8 a 37.5 a bnw12 42.9 ab 60.4 a 49.1 abc 34.6 abc 31.9 ab nm20 35.1 de 56.5 a 31.2 de 22.0 de 20.0 cd bp20 40.9 abc 68.4 a 48.4 abcd 34.2 abcd 31.4 ab bnw20 40.3 abcd 64.8 a 40.3 bcde 28.5 bcde 25.6 bcd nm50 33.7 e 55.1 a 30.4 e 21.4 e 19.3 d bp50 37.1 cde 64.6 a 39.9 bcde 28.2 bcde 25.6 bcd bnw50 36.7 cde 61.9 a 35.9 cde 25.3 cde 22.8 bcd cv (%) 4.76 7.78 13.53 13.54 13.52 adv. hort. sci., 2022 36(3): 227­237 234 production cost economic analysis the percentage contribution of the fixed and vari­ able cost items that make up the total cost are shown in table 5. the total fixed cost and the total variable cost represent, on average, 25% and 75% of the total cost, respectively. greenhouse (structure and cover) has the largest percentage contribution in total fixed cost, and input followed by labor has the largest percentage contribution in total variable cost. a similar result was observed by silva et al. (2007) in the cultivation of sunflower, however boas et al. (2011) and lima junior et al. (2014) in the onion and carrot crop, respectively, observed a percentage con­ tribution of the total fixed cost in the total cost of less than 25%. these results can be explained by the non­use of greenhouse and mulching in the cultiva­ tion of these vegetables, reducing fixed costs. these authors also observed a greater percentage contribu­ tion of input and labor in total variable cost. despite the depreciation of the greenhouse con­ stituting a high percentage in the total fixed cost (table 5), its use decreases the susceptibility to weather and allows the planting of the crop through­ out the year. according to araújo neto et al. (2012), despite the high cost of greenhouses, its use gener­ ates higher yield, reduces the average total cost, and increases profitability. inputs and labor represent, on average, 47% of total production costs. despite the inputs cost being difficult to reduce, family labor is recommended in the quest to reduce labor costs and table 5 ­ percentages of fixed and variable costs of radish production in different mulching types and soil water tension % total cost nm7 bp7 nw7 nm12 bp12 nw12 nm20 bp20 nw20 nm50 bp50 nw50 land 1.09 1.07 1.06 1.13 1.03 1.05 1.18 1.07 1.09 1.19 1.11 1.12 rlt 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 mulching 0.00 2.15 3.38 0.00 2.07 3.36 0.00 2.14 3.49 0.00 2.22 3.56 seedling tray 1.62 1.60 1.58 1.69 1.54 1.57 1.75 1.60 1.63 1.76 1.66 1.66 greenhouse structure 15.77 15.58 15.34 16.36 14.95 15.25 17.00 15.51 15.84 17.10 16.07 16.13 greenhouse cover 4.77 4.71 4.64 4.95 4.52 4.61 5.14 4.69 4.79 5.17 4.86 4.88 irrigation system 0.24 0.24 0.24 0.25 0.23 0.24 0.26 0.24 0.25 0.27 0.25 0.25 dripper and ldpe pipes 1.19 1.18 1.16 1.23 1.13 1.15 1.28 1.17 1.20 1.29 1.21 1.22 tfc 24.61 26.47 27.32 25.53 25.40 27.17 26.54 26.35 28.22 26.69 27.29 28.73 inputs 23.65 23.42 23.06 24.68 22.33 22.91 25.82 23.29 23.94 25.99 24.26 24.44 labor 23.10 22.87 22.52 24.11 21.81 22.37 25.22 22.75 23.38 25.38 23.70 23.87 energy 0.54 0.53 0.53 0.53 0.48 0.49 0.42 0.38 0.39 0.33 0.31 0.31 post­harvest expenses 20.02 18.83 18.83 16.97 22.20 19.33 13.71 19.37 16.25 13.29 16.48 14.76 administration costs 4.15 4.05 4.01 4.10 4.12 4.02 4.04 4.06 3.95 4.03 4.00 3.92 table 4 ­ pearson correlation coefficients among yield and its related characters in radish lw= leaf weight; ln= leaf number; ph= plant height; lai= leaf area index; scf= soil coverage fraction; rd= root diameter; rl= root length; ty= total yield; cy= commercial yield. **, *= significant at 1 and 5 % probability levels, respectively. plant growth root growth yield lw ln ph lai scf rd rl ty cy lw 1 ln 0.69 ** 1 ph 0.87 ** 0.63 ** 1 lai 0.91 ** 0.72 ** 0.83 ** 1 scf 0.61 ** 0.48 ** 0.66 ** 0.67 ** 1 rd 0.70 ** 0.44 * 0.66 ** 0.77 ** 0.56 ** 1 rl 0.56 ** 0.51 ** 0.69 ** 0.55 ** 0.35 * 0.51 ** 1 ty 0.74 ** 0.56 ** 0.71 ** 0.83 ** 0.62 ** 0.90 ** 0.66 ** 1 cy 0.75 ** 0.59 ** 0.73 ** 0.82 ** 0.64 ** 0.89 ** 0.68 ** 0.99 ** 1 santos et al. ‐ agricultural traits of radish under different water requirements and mulching 235 production costs. the nm7 treatment had the lowest percentage contribution of the total fixed cost and the highest percentage contribution of the total variable cost in the total cost, among the treatments applied (table 5). the lack of mulching in this treatment contributed to the decrease in total fixed cost, also, nm7 resulted in high yield, increasing expenses with post­harvest and administrative costs. the bnw50 treatment showed the highest percentage contribution of the total fixed cost due to expenses with non­woven film and low yield, which reduces the total variable cost. post­harvest expenses and administrative costs were high in bp12 treatment, due to high yield, resulting in a low percentage participation of total fixed cost in the total cost, despite the use of plastic mulching. in the simplified economic study, r$ 1.20 kg­1 was considered as the average price practiced in the peri­ od of october 2018, and the average total cost and total operating cost for radish crop varied according to the treatment applied (table 6). the bp12 treat­ ment resulted in the lowest average total cost (r$ 0.71 kg­1) and total operating cost (r$ 0.60 kg­1). although black plastic increased production costs, the mulching characteristics helped to increase yield and profitability, offsetting the expenses with the mulching. the bnw12 treatment resulted in the low­ est average total cost (r$ 0.81) and total operating cost (r$ 0.68) among black non­woven film treat­ ments, however they were lower than the values observed with the bp12 and nm7 treatment. although the black non­woven film increased the yield, the expenses with mulching did not compen­ sate for the application. therefore, that its use is viable, the prices practiced in the commercialization of non­woven film must be below the prices present­ ed in the present study. between the treatments without mulching, keeping the humidity close to the field capacity (7 kpa) provided the lowest production cost. the nm50 and nm20 treatments resulted in the highest average total cost and total operating cost, due to low yield, therefore they are not recom­ mended. although the application of plastic mulching results in an increase in yield and financial return, its use must be done in a correct and controlled man­ ner, to decrease the negative impact on the environ­ ment of plastic film pollution. in addition, accumula­ tion of plastic residue in soil over time may produce negative effects on crop production (gao et al., 2019). therefore, an effective cleaning is necessary after the useful life of mulching or the acquisition of biodegradable mulching film in order to develop sus­ tainable agriculture. all treatments applied exhibited average returns higher than average total cost. therefore, the invest­ ment pays all resources applied in the activity and provides an economic profit, even in treatments with low yield. in this situation, investment is higher than market alternatives, and the trend in the medium and long term is for expansion and entry of new com­ panies into the activity, attracting competitive invest­ table 6 ­ average economic and operating costs of radish production, in r$ kg­1, at different types of mulching and soil water tension average fixed cost average variable cost average total cost average fix operating cost average viarable operating cost average total operating cost nm7 0.19 0.60 0.78 0.08 0.57 0.66 bp7 0.21 0.62 0.83 0.11 0.60 0.70 bnw7 0.22 0.61 0.83 0.11 0.59 0.70 nm12 0.23 0.69 0.92 0.10 0.67 0.77 bp12 0.17 0.53 0.71 0.09 0.51 0.60 bnw12 0.21 0.60 0.81 0.11 0.57 0.68 nm20 0.30 0.85 1.14 0.13 0.81 0.95 bp20 0.21 0.60 0.81 0.10 0.58 0.68 bnw20 0.26 0.70 0.96 0.14 0.67 0.81 nm50 0.31 0.87 1.18 0.14 0.84 0.98 bp50 0.25 0.70 0.95 0.13 0.67 0.80 bnw50 0.30 0.76 1.06 0.15 0.73 0.89 adv. hort. sci., 2022 36(3): 227­237 236 ments. the analysis was carried out with the product price at r$ 1.20 kg­1, however the price may vary according to the market at the time of harvest. if the price is lower than practiced, a new analysis must be made. however, there are alternatives to increase sales such as the sale of deformed and cracked roots (difference between total yield and commercial yield), for companies specialized in purchasing these products and increasing yield with new techniques and with more productive cultivars. 4. conclusions the different swt levels and the use of mulching resulted in different growth and yield responses. the bp7 treatment resulted in the highest leaf area index and soil cover fraction. the bp12 treatment resulted in the highest leaf weight, plant height, leaf number, root diameter, root length, total yield and commer­ cial yield. in this context, this treatment was recom­ mended for the radish producers. the plant variables leaf area index and root diameter were the parame­ ters that presented the highest and positive correla­ tion with the radish yield. expenses with variable resources increased the final cost in all treatments studied. in conditions simi­ lar to those observed experimentally, it is recom­ mended to adopt a black plastic cover keeping the swt close to 12 kpa, to obtain the highest profitabili­ ty in the productive activity. however, if the practice of mulching is not feasible, the swt should be kept close to 7 kpa. acknowledgements the authors express their gratitude to capes for scholarships that enabled the development of this research. references abd el­mohsen a.a., mahmoud g.o., safina s.a., 2013 ­ agronomical evaluation of six soybean cultivars using 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distant hybrids between radish (raphanus sativus l.) and kohlrabi (brassica oleracea l. var. caulorapa dc.). ­ n. z. j. crop hortic. sci., 1­14. impaginato 381 adv. hort. sci., 2020 34(4): 381­395 doi: 10.13128/ahsc­9005 morphological and biochemical classifi­ cation of iranian mango germplasm collection by multivariate analysis: implications for breeding d. samsampour 1 (*), h. kazemzadeh­beneh 1, g.­r. damizadeh 2, z. mirzaei 1 1 department of horticulture science, biotechnology and molecular genetic section, university of hormozgan, bandar abbas, iran. 2 agriculture and natural resources research center of hormozgan, bandar abbas, iran. key words: cluster grouping, factor analysis, genetic variability, mango fruit, principal component. abstract: this study was conducted at southern iran with the aim to evaluate the phenotypic diversity of 84 mango cultivars by using morphological and bio­ chemical traits. two other industrial cultivars ‘longra’ and ‘senderi’ used as control of the study. descriptive results indicated that the value of both quanti­ tative and qualitative variables all the cultivars were lower than the ‘senderi’. the variability among cultivars was highly significant in the measured traits. the fruit weight, fruit length, stone weight, stone length, fruit beak, dry matter, and petiole length showed highly discriminating power. pearson’s correlation analysis revealed high variability due to the existence of significant positive and negative correlations among traits. agglomerative hierarchical clustering con­ firmed remarkable variation in the studied germplasm and identified three major clusters with several sub­clusters. the 80.20, 70.11, and 100% of the quantitative variability was explained by principal component analysis (pca), factor analysis (fa), and liner discriminant function (ldf) where fruit descrip­ tors contributed most of the total variation, respectively. however, multivari­ ate analysis proved that fruit related characters were most powerful to differ­ entiate cultivars. the cultivars displayed distinct grouping by fa and ldf com­ pared to pca. the results revealed that the iranian mango germplasm has a high potential for specific breeding project regarding fruit size and quality that should be further completed by a molecular marker analysis. 1. introduction the mango (mangifera indica l.), from anacardiaceae family, is a fruit species native to asia and grown comprehensively in tropical and sub­ tropical countries, that originated as an allopolyploid from eastern india, assam and burma region (krishna and singh, 2007). mangoes are exten­ sively cultivated in the orchards or often planted in the fruit­gardens man­ (*) corresponding author: samsampoor@hormozgan.ac.ir citation: samsampour d., kazemzadeh­ beneh h., damizadeh g.­r., mirzaei z., 2020 ­ morphological and biochemical classification of iranian mango germplasm collection by multiva‐ riate analysis: implications for breeding. ­ adv. hort. sci., 34(3): 381­395 copyright: © 2020 samsampour d., kazemzadeh­beneh h., damizadeh g.­r., mirzaei z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 june 2020 accepted for publication 23 october 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 381­395 382 ner in the southern region of iran. the main areas of this region which is famous for the mango production are hormozgan, sistan and balouchestan, and kerman (kahnuj and jiroft) provinces. the role of germplasm characterization in varietal development of crops as genotypes with desirable traits has been well identified and utilized in the crop improvement programs and also to determine evolu­ tionary relationships (piyasundara et al., 2008; donkor et al., 2019). however, the accessibility of the germplasm depends mostly on the information avail­ able on characterization and evaluation. the investi­ gation of plant material with desired traits by means of the morphological characterization by multivariate statistical techniques is an essential step for the effective utilization of crop germplasm (piyasundara et al., 2008). multivariate statistical techniques, pca and cluster analysis are the popular multivariate techniques that widely applied to identify genetic diversity in germplasm of olives (hagidimitiou et al., 2005), strawberry (lavin et al. , 2005), tea (piyasundara et al., 2008), mangoes (sennhenn et al., 2013; jamil et al., 2015) and sour cherry (ganopoulos et al., 2016). among them, multivariate techniques guarantee the accurate interpretation of the infor­ mation generated through characterization studies. the local farmers believe that the mango cultivars cultivated at southern iran may have been derived from pakistan and indian germplasms over 300­400 years ago. from the beginning of mango cultivation in the southern regions of iran, mangoes have a local name by the farmers and the native people living based on their appearance and farmer’s interest. beside this, the area under mango cultivation is increasing each year in the southern regions of iran, but the basic information regarding the analysis of genetic diversity and identification cultivars in order to improve breeding programs are lacking. before any breeding programs on mango, there is the need to collect and characterize the local cultivars as plant material that are available in iran. moreover, the evaluation of morphological traits through phenotyp­ ing constitutes is a quick method to characterize the mango germplasm and being provide useful qualita­ tive information for the breeding. therefore, the main objectives of the present study were (i) to identify the phenotypic diversity in 86 local mango cultivars of the hormozgan province of southern iran using morphological approaches based on multivariate statistical techniques, (ii) to evaluate specific traits for breeding and to progress future genetic resource conservation strategies. 2. materials and methods experimental areas survey the experiments were conducted at rudan, siyahu, and minab germplasm collections (rsm­col­ lection), located in hormozgan province of iran (fig. 1). samplings were collected from rsm­collection during growing season for leaf (october to november), during flowering for flower (february and april) and fruit harvesting (july to september). rudan, siyahu, and minab lie within latitude 57° 6’ n, 27° 7’ n and 49° 49’ n and longitude 27° 7’ e, 57° 11’ e, and 34° 4’ e and altitude 100, 300 and 700 m above sea level, respectively. the rsm­collection has an average annual rainfall of 227, 250 and 200 mm in two seasons (october and january) and main daily temperature of 28, 29 and 18°c, respectively (metrological service, bandar abbas, 2014­2017). plant material a total of 84 cultivars from an inspection on mango gardens or scatter manner planted in rsm­ collection followed by two industrial cultivars includ­ ing ‘longra’ and ‘senderi’ as controls were comprised in the sampling procedure for characterization (table s1). to assay adult trees of rsm­collection germplasm, three trees of each cultivar were ran­ domly chosen and were labeled to use data collec­ tion. the labeled trees were at fruit­bearing capacity, healthy and in crop condition at beginning of the study. sampling and morphological assessment of 49 variables as mango descriptors were programmed in the experimental collection for three consecutive years (2014­2017) at the same time of harvest sea­ son. different horticultural practices, including fertil­ fig. 1 ­ geographical zones of the 84 mango cultivars followed by two industrial cultivars as controls used in this study from rsm­ germplasm (rudan, siyahu, and minab collec­ tion) located at southern of iran (black trees indicate sampling locations). samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 383 izer application, spraying, irrigation and others, were performed at regular intervals each year. morpho‐physiological analysis of traits a total of 49 morphological and biochemical traits including 22 quantitative and 27 qualitative traits were scored following the guidelines system for mango descriptors published by international plant genetic resources institute (ipgri), rome for tree, leaf, flower and fruit traits (mukherjee, 1989; pgri, 2006). the quantitative traits were fruit length [frll], fruit breadth [frbr], fruit skin thickness [frskth], fruit weight [frwe], flesh content (fruit flesh weight/fruit weight) [frco], fiber length [frfi], stone length [stle], stone weight [stwe], stone fiber length [stfile], ph [ph], tss [tss], titratable acidity [tiac], total sugar [tosu], moisture [most], dry matter [drma] for fruit descriptors, leaf length [lele], leaf width [lewi], leaf ratio [lera], petiole length [pele], petiole ratio [pera] for leaf descriptors, trunk circumference [trci] and canopy diameter [cadi] for trunk descriptors. the qualitative traits analyzed were fruit shape [frsh], flesh texture [flco], adherence [adhe], fiber in pulp [fipu], quantity of fiber [qufi], stalk insertion [stin], basal cavity [baca], beak [be], beak type [bety], sinus [si], sinus type [sity], groove stone [grst], shoulder [sh], shoulder slope [shsl], skin colour ripe fruit [skcorifr] for fruit descriptors, inflo­ rescence position [inpo], inflorescence shape [insh], inflorescence colour [inco], inflorescence hairiness [inha], flower type [flty] for flower descriptors, leaf shape [lesh], leaf colour [leco], leaf texture [lete], leaf tip [leti], leaf margin [lema] for leaf descriptors, trunk number [trnu] and tree habit [trha] for trunk descriptors. for all three years, measurements performed by the same two persons to avoid errors due to individ­ ual variation. in addition, to diminish the environ­ mental effects, all parameters were averaged over three years. some measurements such as leaf, fruit and tree descriptors were performed in garden­head and others like flower, biochemical and physiological traits of fruit were measured at the laboratory of plant biotechnology of hormozgan university. total soluble solid and ph were determined using a digital refractometer (atc1e­atago, japan), and ph meter (pl­500, taiwan), respectively. titratable acidity of freshly extracted juices and total sugar the fruits were calculated by using standard process procedure to aoac (2000) and omokolo et al. (1996), respec­ tively. for morpho­metrical flower data collection, ten recently opened flowers from tree of each culti­ var were randomly collected, pooled and conserved in ethanol (70%) until measurements. afterward, morpho­metrical of flower was analyzed by using microscope (model: ix3). data analysis descriptive analysis (minimum, maximum, mean, standard deviation and coefficient of variation) for each of 49 studied traits were calculated and the test of normality was accomplished on data to approve anova assumptions. statistical differences in the variations of each trait among the cultivars was com­ puted by one­way analysis of variance (anova) at p≤0.01 using spss 21.0 (spss, inc., chicago, il, usa) after verifying normal distribution of dependent vari­ ables by kolmogorov­smirnov test. within correla­ tion analysis, the pearson coefficient (parametric) was used to measure the correlation among quanti­ tative traits. the principal component analysis (pca), factor analysis (fa), liner discriminant function (ldf), and agglomerative hierarchical clustering (ahc) were directed to analyze data in order to visualize possible differences among the mango cultivars. in each case, a biplot was drawn based on most important compo­ nents to facilitate the visualization of the results. for dendrogram construction, the combined data from both the quantitative and qualitative traits were con­ sidered. to estimate the genetic dissimilarity compo­ nent, the euclidean, and ward’s method was select­ ed as chosen distance for the agglomerative hierar­ chical clustering. all multivariate analysis was per­ formed using xlstat software (version 2016.2). 3. results variance analysis of traits the high morphological variation observed among studied cultivars. the mean squares of mango culti­ vars was significant for the lele, lewi, leco, lete, frle, frwe, frsh, stle, stwe, stfile, fipu, flfile, tosu, drma, most, brix, inha, cadi, trnu, and trci, but they were similar in the rest of the characteristics (data not shown). the variance variation in quantita­ tive traits was more than qualitative traits. descriptive statistics the result of descriptive analysis demonstrated the high variation of cv for sity (95%), si (94%), stfile (67%), ph (64%), bety (62%), trnu (62%), leco (55%), inha (52%), frsh (52%), trci (42%), qufi (41%), trha (41%), letu (40%) be (36%), flty (35%), adv. hort. sci., 2020 34(4): 381­395 384 flfile (34%), stin (34%), insh (33%) and inco (31%), respectively. in contrast, the other variables revealed low coefficient of variation (<30%) (supplementary table s2). in the traits mentioned above, the high they produced the cv, the greatest was the variabili­ ty regarding the fruit descriptor was proportional to the cv. for leaf descriptor, the most relevant traits were: leco, and letu whereas inha, inco, insh, and flty were the most relevant character for the flower descriptor. the studied cultivars exhibited high mor­ phological diversity, with some quantitative and qual­ itative traits. correlations for quantitative variables pearson’s correlation (parametric) among 22 mor­ phological of quantitative characters were presented in table 1. results demonstrated that the 28 posi­ tives and 11 negative significant correlations. a signif­ icant positive correlation was obtained between file and stle (r= 1.00), and frbr and frwe (r= 0.734). in contrast, highest negative correlation was resulted among most and drma (r= ­0.996). regarding fruit descriptors, the results of the paired linear correla­ tion indicated that frwe was positively correlated with frbr, frle, stwe, and tss while was negatively correlated with cadi. in the present study, significant correlation was found between fruit weight and other quality vari­ ables specially tss. additionally, the pearson correla­ tion of frbr with frwe, flco, and stwe was positive significant whereas was negative significant with tss (table 1). furthermore, significant positive correla­ tions was also observed among: flco and frskth (r= 0.244), tss and frskth (r= 0.347), flco and frwe (r= 0.461), frwe and stle (r= 0.247), frwe and stwe (r= 0.558), stle and tss (r= 0.277), stwe and most (r= 0.229), tiac and tosu (r= 0.294), and tosu and drma (r= 0.294) while significant negative correlation was detected between flco and stwe (r= ­0.431). principal component analysis of quantitative vari‐ ables according to apply kaiser’s criterion (“eigenvalue” >1) (kaiser, 1958), pca analysis of the 22 quantitative traits resulted in 9 components for explaining total of variation among cultivars. pca revealed the first nine table 1 ­ correlation coefficients (pearson) among 22 quantitative traits in 86 mango cultivars for full names of traits see morpho­physiological analysis of traits head in materials and method section. frle frbr frskth frwe flco file stle stwe stfile ph tss tiac tosu most drma lele lewi lera pele pera trci cadi frle 1 frbr 0.426 1 frskth 0.44 ­0.148 1 frwe 0.456 0.734 0.022 1 flco 0.243 0.372 0.244 0.46 1 file ­0.088 ­0.087 ­0.054 0.015 0.035 1 stle 0.591 0.094 ­0.025 0.247 0.1 1 1 stwe 0.239 0.381 ­0.189 0.558 ­0.434 0.035 0.187 1 stfile 0.63 0.011 0.115 0.112 0.056 0.103 0.087 0.027 1 ph ­0.213 ­0.113 ­0.105 ­0.107 0.023 ­0.364 ­0.188 ­0.115 ­0.064 1 tss 0.304 ­0.32 0.347 ­0.18 0.029 ­0.029 0.277 ­0.155 ­0.044 ­0.062 1 tiac 0.114 ­0.015 ­0.123 ­0.039 0.026 0.055 0.112 ­0.074 0.106 ­0.383 0.028 1 tosu ­0.65 ­0.23 0.08 ­0.138 0.105 ­0.071 ­0.002 ­0.191 0.005 0.098 0.471 0.294 1 most 0.091 0.116 0.113 0.165 ­0.094 ­0.099 0.013 0.229 0.041 ­0.193 ­0.189 ­0.155 ­0.29 1 drma ­0.073 ­0.105 ­0.11 ­0.16 0.082 0.099 0.005 ­0.211 ­0.044 0.201 0.195 0.149 0.294 ­0.996 1 lele 0.205 0.142 0.108 0.069 0.099 ­0.219 0.018 ­0.033 0.047 ­0.236 0.138 0.089 ­0.03 ­0.013 0.016 1 lewi 0.101 0.14 0.023 0.124 0.106 ­0.361 ­0.092 0.005 ­0.067 ­0.209 ­0.041 0.079 ­0.024 ­0.023 0.039 0.681 1 lera 0.113 0.048 0.113 ­0.019 0.019 0.221 0.125 ­0.047 0.132 ­0.039 0.215 0.004 0.001 0.018 ­0.033 0.429 ­0.342 1 pele 0.103 0.159 0.072 0.048 0.108 ­0.12 ­0.025 0.008 0.058 ­0.198 0.03 0.093 ­0.018 0.026 ­0.022 0.651 0.388 0.347 1 pera ­0.032 0.076 0.009 0.018 0.084 0.026 ­0.017 0.033 0.013 ­0.048 ­0.094 0.031 ­0.021 0.047 ­0.044 ­0.034 ­0.092 0.055 0.722 1 trci ­0.055 0.004 ­0.084 0.118 0.046 ­0.262 ­0.138 0.128 ­0.225 0.078 0.029 ­0.275 ­0.075 ­0.011 0.018 ­0.148 0.077 ­0.056 ­0.069 0.057 1 cadi ­0.22 ­0.048 ­0.085 0.067 ­0.099 0.121 ­0.152 0.118 ­0.012 0.031 ­0.54 ­0.113 0.011 ­0.095 0.102 ­0.09 ­0.132 0.105 ­0.041 0.014 0.645 1 samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 385 (pcs) with eigen values greater than value 1, which could explain 80.20% of the total variation (table 2). pc1, which accounted for 15.91% of the total varia­ tion, was strongly associated with fruit traits, such as fruit length, fruit weight, fiber length, stone length, and fruit breadth. hence, the cultivars with high value of pc1 have lower biochemical traits of fruit as well as smaller tree size (canopy diameter). pc2 accounting for 13.33% of the total variation was posi­ tively correlated with total solid soluble, fruit dry matter, trunk circumference and leaf traits, while negatively correlated with the most of fruit parame­ ters. pc3 had high contributing factor loading from petiole length, leaf length, leaf width, petiole ratio, trunk circumference and contributed 11.37% of the total variation. pc3 suggested that leaf traits could be located in one index. pc4, accounted for 8.79% of the total variation, was most determined by the traits of fruit dry matter, fruit breadth, fruit weight, flesh con­ table 2 ­ first 9 components from the pca of 22 quantitative traits in 86 mango cultivars tent and ph. pc5 up to pc9 explained 7.28%, 7.08%, 5.93%, 5.51%, and 5.21% of the total variation, respectively. however; pc5 represents mainly flesh content, fruit skin thickness, fruit weight, leaf ratio, and petiole ratio; pc6 explains canopy diameter, tss, ph, petiole length, and petiole ratio; pc7 describes leaf length, ph, and stone weight; pc8 illustrates stone fiber length, leaf ratio, trunk circumference, and titratable acidity; pc9 most demonstrates total sugar, canopy diameter, tss, and stone weight. in addition, pca­biplot based on pc1 and pc2 exhibited that the cultivars had wider variation for quantitative traits and thus the biplot did not pro­ duce a distinct grouping among cultivars (fig. 2a). according pca biplot, negative values for pc1 indi­ cate cultivars with high content of dry matter and total sugar as well as lower ph and smaller canopy diameter; however, the cultivars which were placed in the green line rectangle belong this group (fig. 2a). for full names of traits see morpho­physiological analysis of traits head in material and method section. quantitative traits pc1 pc2 pc3 pc4 pc5 pc6 pc7 pc8 pc9 frle 0.77 0.08 ­0.32 0.04 0.07 ­0.06 0.08 ­0.08 ­0.03 frbr 0.61 ­0.32 0.14 0.51 0.1 0.13 ­0.02 0.14 ­0.02 frskth 0.07 0.23 0.06 ­0.33 0.63 0.00 0.11 0.00 0.28 frwe 0.68 ­0.37 ­0.05 0.44 0.20 0.12 0.05 0.13 0.22 flco 0.31 0.21 0.01 0.39 0.71 0.12 ­0.27 0.08 ­0.07 file 0.64 0.15 ­0.61 ­0.16 ­0.16 0.02 ­0.03 ­0.15 ­0.18 stle 0.64 0.15 ­0.61 ­0.16 ­0.16 0.02 ­0.03 ­0.15 ­0.18 stwe 0.40 ­0.51 ­0.06 0.08 ­0.46 0.06 0.27 ­0.04 0.30 stfile 0.17 0.07 0.01 ­0.13 0.03 0.17 ­0.2 0.54 0.06 ph ­0.43 ­0.10 ­0.16 0.22 0.23 0.33 0.21 ­0.17 ­0.33 tss 0.08 0.58 ­0.38 ­0.30 0.18 ­0.03 0.29 ­0.20 0.30 tiac 0.15 0.37 ­0.03 0.06 ­0.36 ­0.26 ­0.53 0.30 0.25 tosu ­0.19 0.51 ­0.26 0.07 0.09 ­0.03 ­0.11 ­0.04 0.50 most 0.30 ­0.58 0.27 ­0.6 0.18 ­0.13 ­0.08 ­0.01 0.07 drma ­0.29 0.58 ­0.28 0.61 ­0.19 0.13 0.09 ­0.01 ­0.06 lele 0.43 0.47 0.56 0.01 ­0.05 ­0.18 0.42 0.06 ­0.09 lewi 0.25 0.21 0.49 0.31 0.00 ­0.62 0.21 ­0.18 0.03 lera 0.24 0.33 0.12 ­0.34 ­0.05 0.56 0.31 0.34 ­0.12 pele 0.39 0.39 0.68 ­0.02 ­0.15 0.3 ­0.05 ­0.32 0.06 pera 0.15 0.09 0.37 ­0.03 ­0.13 0.55 ­0.45 ­0.52 0.13 trci 0.15 0.51 0.29 ­0.16 ­0.15 0.02 ­0.09 0.32 ­0.31 cadi ­0.21 ­0.14 0.01 0.14 ­0.18 0.37 0.34 0.21 0.44 eigenvalue 3.50 2.89 2.50 1.93 1.60 1.56 1.30 1.21 1.15 variability (%) 15.91 13.13 11.37 8.79 7.28 7.08 5.93 5.51 5.21 cumulative 15.91 29.04 40.41 49.20 56.47 63.65 69.48 74.99 80.20 386 adv. hort. sci., 2020 34(4): 381­395 the highest positive values for pc1 displayed culti­ vars with high fruit weight, fruit length, fruit breadth, stone weight and high of fruit moisture as well as lower total sugar and dry matter, which were posi­ tioned in the green line oval, as shown in figure 2a. these cultivars can be applied as parents in the mango breeding program as a source of genes for a greater fruit size in a second cycle of recurrent selec­ tion. principal component analysis of qualitative variables according to pca, the cultivars were quantitative­ ly distinct based on qualitative variables. considering the kaiser’s criterion (“eigenvalue” >1), nine signifi­ cant components were obtained that explained 64.26% of the total variation (table 3). pc1 was asso­ ciated with tree habit, basal cavity, inflorescence shape, inflorescence colour and leaf shape accounted for 12.70% of the total variation. pc2 had high con­ tributing factor loading from fruit traits such as stalk insertion, beak, beak type, shoulder slope and con­ tributed 10.43% of the total variation. the more pc3 value was positively correlated to sinus and sinus table 3 ­ first 9 components from the pca of 22 quantitative traits in 86 mango cultivars for full names of traits see morpho­physiological analysis of traits head in material and method section. quantitative traits pc1 pc2 pc3 pc4 pc5 pc6 pc7 pc8 pc9 frsh ­0.13 ­0.05 0.05 ­0.19 0.04 ­0.09 ­0.02 ­0.56 ­0.14 ftte ­0.09 0.17 0.02 0.00 0.20 ­0.04 ­0.79 ­0.10 ­0.08 adhe 0.05 0.06 ­0.25 ­0.20 ­0.18 0.47 0.11 ­0.37 0.04 fipu ­0.06 0.13 ­0.07 0.70 0.12 0.01 0.10 0.33 ­0.02 qufi 0.28 0.06 0.01 0.00 ­0.01 0.05 ­0.17 0.07 ­0.80 stin ­0.10 0.80 0.01 0.08 ­0.29 0.13 0.06 0.01 ­0.08 baca 0,60 ­0.04 ­0.19 0.05 0.08 0.09 ­0.04 0.11 0.09 be 0.12 0.66 ­0.23 0.06 0.22 ­0.06 ­0.23 0.05 0.22 bety 0.25 0.40 0.23 ­0.48 0.11 ­0.11 ­0.06 0.13 0.30 si ­0.04 ­0.06 0.95 ­0.09 0.05 0.04 ­0.02 ­0.01 0.00 sity ­0.07 ­0.02 0.95 ­0.09 0.04 0.03 ­0.06 0.00 0.03 grst ­0.22 ­0.14 0.03 ­0.04 0.59 0.12 ­0.20 ­0.04 ­0.10 sh 0.01 ­0.02 ­0.11 0.79 0.05 ­0.09 ­0.12 ­0.24 0.05 shsl 0.21 0.44 0.17 0.28 0.09 0.01 ­0.49 ­0.07 0.18 skcorifr 0.01 0.29 0.11 0.17 0.27 ­0.22 0.34 ­0.07 ­0.42 inpo 0.03 ­0.04 0.04 ­0.23 ­0.06 ­0.07 0.05 0.70 ­0.17 insh 0.26 ­0.65 0.17 0.17 ­0.08 0.14 0.03 0.07 0.39 inco 0.51 0.11 ­0.01 ­0.22 0.15 ­0.18 0.16 ­0.31 0.02 inha ­0.73 ­0.13 ­0.21 ­0.14 0.09 ­0.07 ­0.13 ­0.05 0.12 flty 0.24 0.13 0.13 0.10 0.01 0.89 ­0.10 ­0.03 0.02 lesh 0.47 0.11 ­0.21 ­0.19 0.25 ­0.02 ­0.22 0.28 0.10 leco ­0.33 ­0.09 ­0.05 ­0.16 0.25 0.68 0.17 0.17 ­0.09 lete 0.00 0.04 0.01 0.05 0.72 ­0.13 0.00 0.11 0.13 leti ­0.74 0.06 0.07 0.10 0.14 ­0.02 0.11 ­0.02 0.09 lema 0.23 ­0.04 0.07 0.11 0.67 0.08 0.12 ­0.17 ­0.06 trnu ­0.03 0.25 ­0.12 0.01 0.25 0.03 0.52 ­0.10 0.17 trha 0.65 ­0.33 ­0.02 ­0.08 0.07 ­0.07 ­0.01 0.08 ­0.08 eigenvalue 3.43 2.82 2.44 1.77 1.59 1.53 1.37 1.27 1.14 variability (%) 12.70 10.43 9.05 6.54 5.89 5.68 5.06 4.70 4.21 cumulative 12.70 23.13 32.17 38.71 44.61 50.28 55.35 60.04 64.26 fig. 2 ­ a) pca biplot of the 22 quantitative traits with regard to the first two principal components. b) pca biplot of the 27 qualitative traits with regard to the first two principal components among 86 mango cultivars. samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 387 type belong to fruit descriptor that explained the 9.05% of the total variation. pc4 explained 6.54% of the total variation with highest positive correlations to fiber in pulp and shoulder. pc5 up to pc9 explained 5.89%, 5.68%, 5.06%, 4.70%, and 4.21% of the total variation, respectively. however; pc5 repre­ sents mainly leaf texture, leaf margin and groove stone; pc6 explains flower type, leaf colour and adherence; pc7 describes tree number and skin colour ripe fruit; pc8 illustrates inflorescence position and fiber in pulp; pc9 most demonstrates beak type, inflorescence shape and quantity of fiber (table s5). figure 2b represents pc1 and pc2 plotted on a bi dimensional plane. in contrast to quantitative traits, qualitative traits indicated clear cut differences between individuals and forms a spectrum of pheno­ types, which means that they are highly heritable and the environment has very little influence on the phenotype of these traits. hence, pca­biplot exhibit­ ed that the cultivars scattering in all the quarters, and the association between traits and cultivars for quali­ tative traits was more discriminator than quantitative traits. the negative values for pc1 indicate cultivars with high content of leaf tip, inflorescence hairiness, fruit shape, and groove stone as well as lower leaf colour, inflorescence shape, fruit adherence, and fruit sinus. cultivars were positioned in the green line rectangle belong this group. these could be target characteristics depending on different purposes in breeding. the highest positive values for pc1 illus­ trate cultivars with high tree habit growth, inflores­ cence position and colour, quantity fiber of fruit, leaf shape, and basal cavity of fruit, which were posi­ tioned in the green line parallelogram. in contrast, the highest positive values for pc2 show cultivars with high fruit traits, as shown in figure 2b. dendrogram using agglomerative hierarchical clus‐ tering the dendrogram showed three main groups; the c1 is included of 31 cultivars, the c3 contained of 54 cultivars, and finally the c2 comprised of 4 cultivars (fig. 3). clustering analysis resulted in the high level of morphological and biochemical variation among cultivars, as confirmed by descriptive and variance analysis. it was considerable that there was no rela­ tionship between clustering pattern and geographical distribution. one of its reason can be related to syn­ onyms, homonyms and misnames. indeed at 89.66% dissimilarity cultivars, which were placed in c1, were separated from others by their small fiber length (centroid= 5.15), lowest tss (centroid=10.64), lowest leaf ratio (centroid= 4.23), their absence groove stone (centroid= 0) and also by their highest stone weight (centroid= 32.44). at 67.66% dissimilarity the other cultivars were divided into two groups: the cul­ tivars, which were located in c2, characterized by their highest fruit dry matter (centroid= 17.93), larger trunk circumference (centroid= 1.88), presenting groove stone in fruit (centroid= 0.020), and having poor hairiness in flower inflorescence (centroid= 2.33), and the cultivars (c3) having highest fruit length (centroid= 9.39), fruit weight (centroid= 162.69), and also having highest value in the most of traits. factor analysis the all dataset of both quantitative and qualita­ tive variables was subjected to kmo (kaiser­meyer­ olkin) and bartlett’s test of sphericity. kmo (0.37) less than 0.5 and no significant bartlett’s test of sphericity showed that the all data were not very suitable for fa. kmo was found to be 0.58 for all dataset and the bartlett’s test of sphericity was sig­ nificant (chi­square = 342.459; df = 171, p < 0.05), when the dataset of two variables was separately used for fa. based on fa, 22 quantitative traits were divided in eight factors (fs) that had eigenvalue more than one and explained 70.17% of variance (table 4). percent of variance of each factor is showing impor­ tance of that factor. factor loading more than 0.50 was considered as significant factor loading for each factor. f1 was included for fruit length, fruit breadth, fiber length, and stone length and it could explain 15.11% of total variation in the dependent structure for, and its suggested name is fruit yield. f2 account­ ed for 12.45% of total variability and was consisted of high total soluble solid, fruit dry matter as well as low fruit moisture which was named fruit taste. f3 justi­ fied 11.06% of total variability which is strongly influ­ fig. 3 ­ dendrogram using ahc for 86 mango cultivars based on 22 quantitative and 27 qualitative traits. adv. hort. sci., 2020 34(4): 381­395 388 enced by higher stone length and fiber length as well as lower petiole and leaf length, and it was suggested name fruit and weak leaf morphology. f4 accounted for 8.30% of variability and was mainly explained by moisture and dry matter which was named fruit dry matter. f5 was named leaf morphology and had high leaf width as well as low petiole ratio which con­ tributed to 6.62% of total variation. f6 was intro­ duced as fruit flesh and had high fruit flesh content contributed 6.44% of total variation. f7 and f8 had high contributing loading from titratable acidity which was named fruit titratable acidity. it was noticed that the grate variation based on eight factors was observed among cultivars, but it could not produce very suitable or distinct grouping due to generate overlapping or same factors such as titratable acidity and leaf morphology in fa. this aspect has obviously been shown by fa­biplot based on first fs extracted from quantitative variables in fig­ ure 4a. fa­biplot indicated that cultivars scattered in all the quarters and thus cannot generated proper grouping. to overcome this, quartimax rotation is a statistical technique used at one level of factor analy­ sis as an attempt to clarify the relationship among factors. in table 4, it can be seen that the quartimax as an oblique rotation displayed a higher efficacy to create clear patterns of results in fa where the fac­ tors are indeed correlated. however, in factor analy­ sis by means of quartimax rotation and on the base of factor loading larger than 0.5, five rotation factors (rf) were identified and they all together justify 53.46% of existent variations among the traits. rf1 up to rf5 were called fruit size factor, dry matter fac­ tor, leaf size factor, fruit weight factor, petiole size and also were accounted for 12.62, 11.11, 10.11, 10.68, and 8.92% respectively. result indicated that quartimax rotation minimized the complexity of the factor loadings to make the structure simpler to interpret, as shown in figure 4b. according to rfa­ biplot cultivars were quantitatively separated to six distinct groups in comparison to fa­biplot. the culti­ vars 22 (‘almehtari­2’) and 48 (‘binam­4’) were sepa­ rated into distinct group near the positive ends of the rf2 axis, and were correlated with respect to higher table 4 ­ correlation coefficients explained by the first eight factor analysis (fs) and quartimax rotation for 22 quantitative traits in 86 mango cultivars quantitative variables factors without rotation factors after quartimax rotation f1 f2 f3 f4 f5 f6 f7 f8 rf1 rf2 rf3 rf4 rf5 frle 0.699 0.074 0.258 0.039 0.057 0.059 0.028 0.039 0.639 ­0.047 0.203 0.326 0.048 frbr 0.563 ­0.276 ­0.14 0.44 ­0.149 0.018 0.084 ­0.15 0.099 ­0.119 0.092 0.771 0.055 frskth 0.059 0.153 ­0.046 ­0.178 ­0.007 0.382 0.013 0.088 0.079 ­0.01 0.100 ­0.141 0.158 frwe 0.695 ­0.357 0.03 0.485 ­0.148 0.100 0.122 ­0.087 0.275 ­0.143 0.022 0.880 ­0.026 flco 0.328 0.211 ­0.03 0.443 ­0.276 0.705 ­0.224 ­0.084 0.102 0.348 0.080 0.495 0.205 file 0.669 0.150 0.615 ­0.17 0.019 ­0.121 ­0.088 0.144 0.928 0.005 0.007 0.094 0.023 stle 0.669 0.150 0.615 ­0.17 0.019 ­0.121 ­0.088 0.144 0.928 0.005 0.007 0.094 0.023 stwe 0.404 ­0.492 0.062 0.065 0.042 ­0.518 0.269 0.061 0.200 ­0.417 ­0.066 0.415 ­0.144 stfile 0.128 0.035 ­0.001 ­0.06 ­0.058 0.014 ­0.027 ­0.151 0.100 ­0.029 0.024 0.028 0.110 ph ­0.347 ­0.064 0.11 0.133 ­0.216 0.100 0.189 0.139 ­0.207 0.182 ­0.346 ­0.028 ­0.056 tss 0.087 0.574 0.362 ­0.33 0.100 0.234 0.156 0.267 0.500 0.296 0.146 ­0.467 0.123 tiac 0.140 0.366 0.035 0.50 0.219 ­0.311 ­0.561 ­0.567 0.182 0.226 0.339 ­0.079 ­0.024 tosu ­0.155 0.405 0.18 0.025 0.009 0.060 ­0.103 ­0.006 0.092 0.394 0.035 ­0.235 0.010 most 0.302 ­0.635 ­0.251 ­0.539 0.150 0.250 ­0.162 0.023 0.051 ­0.921 0.028 ­0.006 0.055 drma ­0.290 0.638 0.262 0.552 ­0.143 ­0.267 0.170 0.049 ­0.038 0.929 ­0.023 0.018 ­0.065 lele 0.438 0.473 ­0.569 ­0.045 0.312 0.011 0.338 ­0.044 0.008 0.001 0.863 0.035 0.312 lewi 0.252 0.215 ­0.500 0.331 0.631 0.010 ­0.004 0.286 ­0.187 0.024 0.867 0.203 ­0.220 lera 0.260 0.327 ­0.119 ­0.447 ­0.409 0.023 0.479 ­0.447 0.234 ­0.028 0.018 ­0.174 0.687 pele 0.407 0.397 ­0.702 ­0.122 ­0.270 ­0.205 ­0.066 0.221 ­0.133 ­0.019 0.509 0.158 0.774 pera 0.160 0.088 ­0.389 ­0.107 ­0.633 ­0.257 ­0.410 0.362 ­0.159 ­0.025 ­0.112 0.195 0.723 trci 0.119 0.381 ­0.204 ­0.138 0.026 ­0.042 ­0.019 ­0.199 0.042 0.114 0.327 ­0.147 0.282 cadi ­0.156 ­0.083 ­0.009 0.057 ­0.142 ­0.108 0.187 ­0.050 ­0.145 0.033 ­0.179 0.025 0.013 eigenvalue 3.325 2.741 2.434 1.826 1.457 1.417 1.199 1.040 >1 >1 >1 >1 >1 variability (%) 15.112 12.459 11.063 8.301 6.62 6.411 5.449 4.728 12.624 11.119 10.117 10.686 8.923 cumulative 15.112 27.571 38.633 46.934 53.554 59.996 65.445 70.173 12.624 23.743 33.86 44.546 53.469 for full names of traits see morpho­physiological analysis of traits head in material and method section. samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 389 fruit dry matter as well as lower fruit moisture which positioned in green line oval. selection of these culti­ vars is a further advantage, as they have important postharvest characters for mango fruit breeding to extend their shelf­life. in contract, the ‘senderi’ as control in green line circle was clearly diverged from the other cultivars that were located at the positive ends of rf1 axis, and was highly correlated with respect to fruit length, flesh content and stone length and the ‘kalanfar­2’ was the most closet cultivar to control, as shown in figure 4b. therefore, crosses between ‘kalanfar­2’ and other cultivars with which ‘kalanfar­2’ produced fertile f1 progeny would potentially allow the production of segregating popu­ lations for qtl analysis. additionally, fa was applied to investigate varia­ tion in qualitative variables. based on fa, 27 qualita­ tive traits were divided in six factors that had eigen­ value more than one and explained 41.39% of total variation (table 5). primary result of fa clearly proved that variation in quantitative variables is more complexity than qualitative variable among cul­ tivars and thus reduced the number of dimensions and discovered simple patterns in the pattern of rela­ tionships among the variables in comparison to pca. also, fa confirmed that the variation in qualitative variables less than quantitative variables among culti­ vars while pca wasn’t able to detect it. the six fac­ tors of qualitative variables namely, (f1) fruit and leaf morphology, (f2 and 3) fruit sinus, (f4) flower type, (f5) fruit stalk insertion, and fruit flesh texture were accounted for 10.95, 9.08, 7.98, 5.13, 4.47, and 3.755% of total variation, respectively. like fa for quantitative variables, same factors observed to sep­ arate cultivars base on the qualitative variables and thus it could not generate distinct grouping, as shown in figure 4c. fa­biplot indicated that cultivars scattered in all the quarters and thus cannot generat­ ed proper grouping. hence, to clarify the relationship among factors, rfa was used. base on the factor loading larger than 0.5, five rfs were identified and they all together justify 37.59% of existent variations (table 5). rf1, called as fruit, flower, and growth morphology factor, which accounted for 9.94% of the fig. 4 ­ factor analysis­biplot of the 22 quantitative (a) and 27 qualitative traits (c) with regard to the first two factors and with regard to the first two factors after quartimax rotation (rf) (b, d) among 86 mango cultivars, respectively. adv. hort. sci., 2020 34(4): 381­395 390 total variation. rf2 with the 8.44% of the variance reflected the fruit sinus dimension and therefore, being classified as fruit sinus. rf3 with the 7.57% of the total variance showed the high flesh texture, fiber in pulp, beak, shoulder, and leaf texture dimen­ sion and therefore, being classified as fruit morpholo­ gy. rf4 with 5.16% of the variance classified as flower type factor due to high factor loading for flower type. finally, rf5 demonstrated the stalk insertion and inflorescence shape factor which accounted for 6.47% of the variance. however, the quartimax rotation used in the factor analysis had excellent discrimination among cultivars based on factors criterion compared to fa without quartimax rotation. this aspect was confirmed by rfa­biplot where showed the cultivars separated into five dis­ tinct groups and thus offered the excellent grouping comparison with fa­biplot (fig. 4d). the ‘nabati­1’ (8) cultivar was qualitatively separated into single group near the positive ends of the rf2 and rf1axes, which positioned in green line circle, and was corre­ lated with respect to higher fruit, flower, and growth morphology and fruit sinus classified­factors. so, the ‘nabati­1’ cultivar can be considered as favorable genetic material for mango breeding via effective phenotypic selection of its correlated­traits (traits obtained by rf1 and rf2) and high expected genetic gain from selection for its correlated­traits can be achieved, as confirmed by cluster analysis. discriminant function analysis the ldf from both quantitative and qualitative variables to classify the cultivars formed on rsm­col­ lection population (rudan, siyahu and minab) was table 5 ­ correlation coefficients explained by the first six factor analysis (fs) and quartimax rotation for 27 qualitative traits in 86 mango cultivars qualitative variables factors without rotation factors after quartimax rotation f1 f2 f3 f4 f5 f6 rf1 rf2 rf3 rf4 rf5 frsh ­0.186 0.023 0.150 ­0.112 0.138 ­0.051 ­0.182 0.120 ­0.119 ­0.088 0.149 ftte 0.410 0.024 0.469 0.269 ­0.075 0.693 0.088 0.224 0.628 0.111 0.190 adhe ­0.175 0.136 ­0.315 0.092 0.347 ­0.017 ­0.068 ­0.195 ­0.372 0.268 0.113 fipu 0.136 0.353 0.115 0.245 ­0.370 ­0.155 ­0.178 ­0.225 0.501 0.049 ­0.063 qufi 0.148 ­0.146 0.017 ­0.070 0.076 ­0.035 0.203 0.108 ­0.013 ­0.027 0.045 stin 0.159 0.369 0.170 ­0.008 0.504 ­0.018 ­0.040 ­0.078 0.027 0.135 0.646 baca 0.482 ­0.105 ­0.339 0.051 ­0.088 ­0.080 0.551 ­0.208 0.100 0.111 ­0.137 be 0.734 0.453 0.185 0.052 0.199 0.033 0.348 ­0.253 0.529 0.099 0.625 bety 0.491 ­0.272 0.101 ­0.191 0.328 0.018 0.564 0.250 0.014 ­0.042 0.315 si ­0.141 ­0.780 0.603 0.115 0.085 ­0.146 ­0.013 0.966 0.072 ­0.005 ­0.153 sity ­0.126 ­0.725 0.618 0.122 0.096 ­0.101 ­0.031 0.938 0.100 0.001 ­0.110 grst ­0.008 0.009 0.232 0.116 ­0.144 ­0.092 ­0.137 0.116 0.245 ­0.007 ­0.034 sh 0.122 0.429 0.154 0.269 ­0.433 ­0.110 ­0.250 ­0.294 0.575 0.036 ­0.063 shsl 0.623 0.085 0.306 0.247 ­0.002 0.126 0.311 0.068 0.620 0.164 0.268 skcorifr 0.101 0.174 0.306 ­0.194 0.029 ­0.374 ­0.064 0.048 0.181 ­0.223 0.310 inpo 0.037 ­0.266 ­0.121 ­0.085 ­0.030 0.114 0.208 0.098 ­0.133 ­0.050 ­0.173 insh ­0.129 ­0.434 ­0.382 0.279 ­0.475 0.018 0.117 0.009 ­0.053 0.514 ­0.815 inco 0.392 ­0.119 ­0.070 ­0.257 0.111 ­0.220 0.468 0.000 ­0.015 ­0.144 0.160 inha ­0.507 0.278 0.188 0.015 ­0.043 0.181 ­0.619 ­0.028 ­0.065 ­0.071 0.048 flty 0.114 ­0.111 ­0.298 0.853 0.443 ­0.209 0.124 0.021 ­0.025 0.962 ­0.057 lesh 0.633 ­0.130 ­0.207 ­0.001 ­0.037 0.016 0.654 ­0.119 0.203 0.062 ­0.007 leco ­0.396 0.045 ­0.130 0.349 0.185 ­0.149 ­0.351 ­0.006 ­0.228 0.377 ­0.097 lete 0.339 0.078 0.292 0.014 ­0.272 ­0.310 0.103 0.031 0.502 ­0.141 0.054 leti ­0.515 0.345 0.389 0.082 ­0.027 ­0.095 ­0.743 0.058 0.079 ­0.056 0.155 lema 0.293 0.007 0.163 0.060 ­0.174 ­0.377 0.146 0.029 0.357 ­0.038 0.010 trnu 0.030 0.269 0.022 ­0.091 0.089 ­0.270 ­0.073 ­0.168 0.018 ­0.053 0.238 trha 0.361 ­0.400 ­0.322 ­0.082 ­0.185 ­0.049 0.592 ­0.001 ­0.033 ­0.042 ­0.345 eigenvalue 2.957 2.453 2.156 1.387 1.209 1.014 >1 >1 >1 >1 >1 variability (%) 10.952 9.086 7.985 5.139 4.474 3.755 9.947 8.443 7.57 5.1 6.478 cumulative 10.952 20.038 28.023 33.161 37.64 41.394 9.947 18.39 25.961 31.12 37.59 for full names of traits see morpho­physiological analysis of traits head in materials and methods section. samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 391 analyzed separately. walk’s lambda test was found to be 0.21 and 0.04 (p­value < 0.0001, alpha 0.05) for quantitative variables and qualitative variables, respectively. however, walk’s lambda test was signif­ icant for two discriminant function obtained from quantitative and qualitative variables, and thus there was low correlation between independent (mea­ sured variables) and dependent (population) variable to compute new directions (canonical variates or dis­ criminant functions) in which the groups are best separated by ldf. the ldf across the 22 quantitative variables origi­ nated from rsm­collection is shown in supplementary table 3. the first two discriminant functions were able to capture 100% of the total vari­ ance. function 1 explains 82.37% of the total vari­ ance and function 17.62% of the total variance. major contributors to discriminate among different cultivars in function 1 are the fruit length, fruit breadth, fruit weight, fiber length, stone length, stone weight, stone fiber length, and canopy diame­ ter; meanwhile the leaf length, leaf ratio, petiole length, fruit weight, stone weight, tss, fruit total sugar, fruit dry matter, and trunk circumference are major contributors in function 2. therefore, these functions might represent the relationship of culti­ vars in each class (rudan, siyahu and minab) with high efficiency at rsm­collection (fig. 5a). considering the 1st and 2nd discriminant functions, the cultivars presented at all three classes had closer genetic relationships; hence, there was a large over­ lap of the cultivars belonging to minab and rudan due to the stronger relationship between the classes. however, the results of confusion matrix for the cross­validation and mahalanobis distance based on quantitative traits showed that the 55.81% (48 num­ ber) of total cultivars belonged to their original class­ es and the rest cultivars (44.19% = 38 number) were distributed among the three classes (table s3). additionally, ldf across the 27 qualitative vari­ ables was performed (table s3). the first two dis­ criminant functions were able to capture 100% of the total variance. function 1 explains 63.48% of the total variance and function 36.51% of the total vari­ ance. function 1 explains 82.37% of the total vari­ ance and function 17.62% of the total variance. major contributors to discriminate among different cultivars in function 1 are the leaf shape, tree habit. basal cavity, beak type, inflorescence hairiness, inflo­ rescence colour, shoulder slope, and quantity of fiber; meanwhile the inflorescence shape, skin colour ripe fruit, beak fruit, stalk insertion, and flesh texture are major contributors in function 2. the cultivars presented at all three classes were qualitatively grouped well, with slight overlapping of between classes in compared grouping based on quantitative variables (fig. 5b). however, the few cultivars distrib­ uted in among classes due to the slighter relationship fig. 5 ­ liner discriminant functions­biplot of the 22 quantitative traits (a) and 27 qualitative traits (b) with regard to the two discrimina­ te functions among 86 mango cultivars located at rsm­collection (rudan, siyahu and minab). adv. hort. sci., 2020 34(4): 381­395 392 with low overlap. moreover, the results of confusion matrix for the cross­validation and mahalanobis dis­ tance based on qualitative traits indicated that the 82.55% (71 number) of total cultivars belonged to their original classes and the rest cultivars (17.44% = 15 number) were distributed among the three class­ es (table s4). overall, result of ldf on quantitative traits for interpreting the genetic relationship among cultivars was better than ldf on qualitative traits. 4. discussion and conclusions this study is the first assessment of the iranian mango germplasm focused mainly the local cultivars. in this study, the cultivars were diverse having signifi­ cant variation for morphological and biochemical traits. variation in fruit and leaf traits is a sign of the presence of high degree of genetic variations among cultivars. because all cultivar presented in rsm­col­ lection have been cultivated through seed, traits seg­ regation among them will be the reason generated genetic diversity in fruit and leaf characters. conversely, the traits did not show variation would probably due to the same seed source propagated these cultivars. descriptive analysis displayed a high variation in both quantitative and qualitative traits among mango cultivars, but variability in qualitative was greater than quantitative traits. these results of this study are in the agreement with other studies, where descriptive analysis revealed a suitable genetic vari­ ability in mango germplasm (sennhenn et al., 2013; jamil et al., 2015), and as well as in some other crops, such as garlic (panthee et al., 2006), melon (lotti et al., 2008) and sour cherry (ganopoulos et al., 2016). this broad genetic variability is the foundation for applied crop breeding that allows for selection of superior cultivars. besides, information of the descriptive results of this study will be helpful to pro­ ceed plant breeding and conservation programs in the future. in the present research, generally, the descriptive results showed that ‘senderi’ cultivar as control had high value of quantitative and qualitative variables compared with all cultivars. it was consider­ able that the value of frle (5.83 cm) and frwe (232.50 gr) for ‘ta dorosht’ cultivar displayed closest or same value with controls, while the high value in tss (19 brix) for ‘jamali­1’ and in tosu (21.92%) for ‘shahani’ was observed compared to controls. quantitative traits are the most significant traits of the majority of plants that are mainly influenced by the environment and hence have low heritability. because of the response to direct selection for these traits may be unpredictable, therefore, plant breed­ ers prefer to select for related traits that indirectly increase quantitative target traits. it was obvious that most significant pearson’s correlations were obtained by phenotypic traits particularly for the fruit yield and quality. in the breeding materials, the higher variation is the greater scope for its improvement through selection. for instance, cross combinations could be performed between cultivars with very large fruit length (‘senderi’, ‘khiyar­1’, ‘havij’ and ‘mashk’), low stone weight (‘kozekasbi’, ‘shahani’ and ‘zapak’) and high total soluble solids (‘jamali­1’, ‘houz­1’ and ‘kalak sorkh­3’). in order to achieve high yield and superior quality cultivars, output information of the morphological investigations might be quite helpful for any future mango breeding in rsm­collection. the ‘jamali­1’, ‘houz­1’, ‘kalak sorkh­3’ and ‘shahani’ will be more suitable cultivar for the fresh consumption cultivars because it showed the favorable level of sol­ uble solids and titratable acidity. in open canopy trees light could penetrate well into the canopy caus­ ing increase of photosynthesis rate and transfer of carbohydrates from the leaves to the fruits. similarly, farrokhi et al. (2013) also showed the importance the correlation of suitable canopy volume and trans­ ferred carbohydrate to increase fruit weight in apple. this aspect will be very good option in selecting the candidate mango cultivar with suitable canopy vol­ ume and fruit quality traits, such as ‘ta dorosht’, for providing a source of material breeding. tss is an important biochemical factor that its level is increased simultaneously with fruit development (zarbakhsh et al., 2020). pearson’s correlations revealed that the cultivars with higher fruit weight tend to reveal relatively higher soluble solids. this is in agreement with the previous reports in apple (farrokhi et al., 2013) and in sour cherry (ganopoulos et al., 2016). it was considerable that no correlation was observed between soluble solids and titratable acidity, which has been confirmed previously in apri­ cot (ruiz and egea, 2008). in breeding programs, pca­biplot is an important tool to identify and rank the superior cultivars and thus facilitating the mango selection process (maia et al., 2016). pca­biplot for quantitative variables showed that the highest pc1 values corresponded to 63 (‘ta dorosht’) with high fruit weight, fruit length and fruit breadth which was closeted to 36 (‘senderi’). samsampour et al. ‐ classification of iranian mango germplasm collection by multivariate analysis 393 germplasm collection and conservation are important not only to preserve genetic resources, but also to enable breeders to exploit the genetic and phenotypic variation and develop superior cultivar. therefore, the ‘ta dorosht’ could be introducing as superior cultivar and may be used as parent in backcrossing method with common cultivars. moreover, regarding the 27 (‘kalanfar­1’) and 7 (‘majlesi­1’), it showed lower val­ ues in all variables analyzed particularly for the canopy diameter and ph values, as shown in figure 2a (were positioned in the green line triangle). it seems that above­mentioned cultivars are exposed to highly endangered in the rms­ collection and should be con­ sider a suitable conservation program to protect the total loss of them. also, due to low vegetative vigor and tree size, these cultivars may be useful for the breeding program and being desirable as dwarfing rootstocks. overall, the results of pca for quantitative traits showed a good performance regarding fruit traits such as fruit weight, fruit length and fruit breadth, which are the most important for discrimi­ nating pomological traits, while it did not indicate dis­ tinct grouping in our studied cultivars. additionally, our results are in accordance with the previous stud­ ies, which also documented that the weight and fruit size are useful parameters to discriminate cultivars in inter­specific almond × peach (yaghini et al., 2013), and in sour cherry (khadivi­khub et al., 2013; ganopoulos et al., 2016). the high negative for pc1 and pc2 for qualitative traits resulted in formatting a distinct group cultivar with strong adherence in fruit, broadly pyramidal inflorescence shape, sinus fruit, and light green leaf, which were positioned in the green line trapezoid. these findings are in agreement with various other studies that reported the maxi­ mum contribution of fruit and flower traits towards the genetic divergence in cherries (khadivi­khub et al., 2013; ganopoulos et al., 2016) and mango (maia et al., 2016). pca could also permit the correlation of the phenotypic traits with the genetic linkages between the respective trait’s loci in qtl mapping analysis. pca previously has been used for germplasm evaluation in almond (nikoumanesh et al., 2011), apple (farrokhi et al., 2013), and mango (krishnapillai and wilson wijeratnam, 2016). in cluster analysis, the highest genetic distance was detected among c2 and c3 (25.18), followed from these among c1 and c2 (19.51), and among c1 and c3 (9.25). the such dendrogram which is able to show genetic relationship among the cultivars report­ ed by sennhenn et al. (2013) in kenya’s mango and by krishnapillai and wijeratnam (2016) in sri lanka mango. it is noticed that the ‘havij’ and ‘nabati­1’, as the most closely related with two control cultivars was confirmed by cluster analysis and thus, they genetically were grouped together. in this study, the most closely related pairs among the mango cultivars were 68 (‘negar­1’) and 74 (‘deraz’) in the c2 cluster, while the highest distance was obtained for 6 (‘halow’) and 86 (‘nesa­2’). based on the results, crossing between cultivars in distanced clusters like c1 and two clusters can provide much variation for the mango breeding purposes. parental selection in breeding program is primarily dependent on the traits desired in the progeny and is best guided by the phenotypic expression of potential parents. in dendrogram, on one side in c2 cluster, there were cultivars 77 and 75, and on the other side in c3 clus­ ter, cultivars 42, 35, which can be recommended for parenting future crosses that could make new gener­ ations with high variations in almost all of the mea­ sured traits. moreover, information about the simi­ larly or dissimilarly genetic relationship among the mango cultivars could be useful for grafting compati­ bility and improving rootstock, where rootstock and scion share the same genetic background. as an outcome from fa of quantitative and quali­ tative traits, the great variation was found in fruit morphology among cultivars. this result is in agree­ ment with several other studies that reported high genetic diversity in mangoes (maia et al., 2016). according to martins et al. (2003), the variation observed in fruit morphology is very common even at intraspecific level. the variation may be attributed to environmental factors or genetic differences or both. in our study, the variations in the fruit traits can be attributed to differences in the age of the plant, fruit maturation stage, geographical sites, climatic condi­ tion, soil properties, and seed origin. generally, this research supported that factor analysis is a useful tool for identification of the most significant variables in the biochemical and morphological data set of mangos. factor analysis previously has been used for germplasm evaluation in several different plant (felenji et al., 2011; pour­aboughadareh et al., 2017). discriminant functional analysis is particularly use­ ful in defining groups of the cultivars as prior classifi­ cation criteria. moreover, it provides a graphical out­ put illustrating the existence of groups. generally, ldf of the variables produced better discrimination of the mango cultivars than the principal component analysis. discriminant functional analysis previously adv. hort. sci., 2020 34(4): 381­395 394 has been used for germplasm evaluation in several different plant (rafiqul islam et al., 2007; sinkovič et al., 2017). the present study exhibited that the presence of exploitable genetic diversity among cultivars intro­ duced superior cultivar in which crossing of distant cultivar with desirable traits to develop cultivar for the study area and similar agro­ecology. we dis­ closed that many field traits have promise for genome wide association study analysis in the future, where combining molecular marker data with morphological can recognize the genes in mango controlling the main traits evaluated here. overall, this diversity permits the effective selection of par­ ents in various breeding programs, referring to fruit quality and aiming at different aspects of postharvest utilization, besides high yield and resistance to dis­ eases. hence, 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problems. garlic extract has a lot of antimicrobial proper­ ties and can prevent the release of microorganisms in the culture medium of plants. in this study, garlic extract at three concentrations (0, 25, and 50 g/l) was investigated on the rooting of three different species include easy­to­root cuttings (rose, wild privet, and poplar), moderate­root cuttings (sycamore, berry, and grape), and hard­to­root cuttings (apple, sour cherry, and cherry) in three applications of the culture substrate, on the cuttings, and in the callus stage. results showed that garlic extract can be used at a concentration of 25 g/l at the callus formation stage to improve the quantity and quality of grape cuttings in terms of shooting and rooting. the quantity and quality of shoots and roots for wild privet cuttings can be improved with garlic extract at 25 g/l and be used on the cuttings. garlic extract at concentrations of 50 and 25 g/l can be used in the callus stage to enhance the quantity and quality of sycamore and berry cuttings, respectively. no positive effects were observed in the other plants, or positive effects were found only either on shooting or on rooting traits. 1. introduction since the uniformity of plants used in gardens and green spaces is important, one of the most effective methods of non­sexual propagation is now the use of various cuttings worldwide. one of the important prob­ lems of using cuttings, particularly hardwood and semi­hardwood ones is to root them. to overcome this critical problem, manufacturers apply dif­ ferent methods, one of which is the use of different rooting hormones, particularly auxins (guan et al., 2015). although the use of hormones for rooting is very effective, these substances are mostly imported goods that require a large amount of foreign money. in addition, the use of plant­ derived substances instead of chemicals and synthetic auxins to deal with adverse environmental effects is an issue that has been considered in the (*) corresponding author: abbasifar1965@yahoo.com citation: abbasifar a., valizadehkaji b., karimi m., bagheri h., 2020 ­ the first report: the effect of garlic extract on rooting of cuttings of some orna‐ mental plants and fruit trees. ‐ adv. hort. sci., 34(2): 191­204. copyright: © 2020 abbasifar a., valizadehkaji b., karimi m., bagheri h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 september 2019 accepted for publication 23 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 191­204 192 european union in recent years. this has led to an increasing interest in the production of substances that can help propagate a variety of plants and, at the same time, are environment friendly (wojdyła, 2004; pacholcza et al., 2016). also, one of the prob­ lems during rooting of cuttings is the substrate conta­ mination with pathogens, and consequently the cont­ amination of cuttings and their failure in rooting (hartmann et al., 2002). according to a previous studies (wojdyła, 2004; pacholczak et al., 2016), the use of natural substances can reduce production costs and provide better accessibility compared to chemicals. in addition to various chemical com­ pounds, the use of natural substances in the cultiva­ tion of commercial plants is today gaining interest among manufacturers and producers, which has been accompanied by positive and interesting results. in some cases, such substances have even been appropriately replaced chemical compounds due to better results. if plant extracts can be used to root the cuttings, a new step will be taken towards this branch of agricultural science in addition to sav­ ing costs and non­dependency on the imports of hor­ mones as well as avoiding environmental contami­ nants. plant essential oils play an important role in disinfecting, stimulating, and improving growth, increasing dry matter, and protecting the plant against environmental damages and stresses (bai et al., 2007). garlic extract is a substance with antioxidant, antimicrobial, and antibacterial properties (harris et al., 2001). garlic active compounds often accumulate in its underground organ (garlic bulb). garlic contains sulfide compounds such as allyn, diallyl sulfides, and allicin, with allicin being the most abundant com­ pound in the garlic extract, accounting for approxi­ mately 70% of these compounds (sadaqa et al., 2016). it has been shown that garlic planting in veg­ etable hydroponic substrates prevents the develop­ ment of many diseases and reduces microbial popu­ lation in the substrate (liu et al., 2014). garlic extract has many allelopathic chemicals (wang et al., 2015). the most important chemicals and minerals found in garlic extract are lipids, carbohydrates, fibers, man­ ganese, potassium, sulfur, calcium, phosphorus, mag­ nesium, sodium, vitamin b6, vitamin c, glutamic acid, arginine, aspartic acid, leucine, and lysine (al mayahi and fayadh, 2015). given the importance of ornamental trees and flowers and also fruit trees in human health and eco­ nomics, this study aimed to investigate the possible effects of garlic extract at different concentrations on rooting of cuttings and propagation of rose, wild priv­ et, poplar, sycamore , berry, grape, apple, sour cher­ ry, and cherry cuttings to achieve the best treatment. there is a large body of literature concerning the effect of using hormones on the rooting of cuttings and its optimization (guan et al., 2015), but no research has so far been conducted on the effect of plant extracts on plant rooting. in the design of this research, therefore, attempts were made to treat the cuttings of different plants with garlic extract in terms of rooting to obtain acceptable results at the beginning of the experiment. this research also tried to use an extract that is suitable for disinfection of the substrate. this was the first report on the use of garlic extract on the rooting of different plants, and it is hoped that the results could pave the ground for further research in this field. 2. materials and methods this research was conducted at the research greenhouse of the faculty of agriculture and natural resources, university of arak. to investigate the effect of garlic extract on the rooting of different cut­ tings, a factorial experiment (cuttings as the first fac­ tor and garlic extract concentrations as the second factor) was carried out in a completely randomized design with three replications each with 10 cuttings. plant materials three types of cuttings including easy­to­root cut­ tings (rose, wild privet, and poplar), moderate­root cuttings (plane, berries, and grapes), and hard­to­ root cuttings (apple, sour cherry, and cherry) were prepared from healthy and genetically uniform native plants in the planting season (december). the cut­ tings were 10­15 cm in length with a thickness of 5­7 mm. extraction of garlic cloves the garlic cloves were first peeled and grated for each independent experiment. then, 10 g of the grat­ ed garlic was placed in 10 ml of water at room tem­ perature for 24 h. the garlic extract was obtained after passing through filter papers. preparation of seedbed, planting of cuttings, and application of treatments the sandstone culture substrate was similar for all cuttings. each experiment was consisted of three replicates each with 10 cuttings. cuttings were grown in 9 rows at 10 cm spacing on a row and 2 cm spacing between the rows in the substrate. the extract con­ abbasifar et al. ‐ garlic extract effects on root cuttings 193 centrations were 0 (control), 25, and 50 g/l of dis­ tilled water for 2 seconds. the extract was used at three times: 1) extract dispersion on the culture sub­ strate during substrate preparation and before plant­ ing the cuttings, 2) at planting the cuttings by insert­ ing the bottom half of the cuttings in the extract to disinfect the cuttings, and 3) dispersion on the sub­ strate at the tips of the cuttings in the callus forma­ tion stage. after planting, the cuttings were kept and irrigated regularly under greenhouse conditions for 3 months. measuring morphological traits immediately after the collection of root and aerial samples, their fresh weight was measured using a digital scale. to determine the dry matter content, the samples were placed in an oven at 70°c for 48 h. finally, the dry weights of shoots and roots were determined with a digital scale. further measured traits were the number of roots and shoots, root and shoot length, and longest roots and shoots. during the experiment and the growth of cuttings, the aver­ age temperature and relative humidity were 22°c and 35%, respectively, in the greenhouse. data were independently normalized with such methods as the removal of outliers (data higher and lower than twice the root mean square error) using the equation x = y + 0.5, and were then analyzed using sas 9.4 software. mean values were compared with duncan’s multiple range test at p<0.05. 3. results the use of different concentrations of garlic extract in the substrates of cuttings shoot‐related characteristics. the interaction effect of garlic extract treatment × the substrate and plant type significantly affected the number of green cuttings, total shoot length, number of shoots, mean shoot length, length of the longest shoots, and fresh and dry weights of the shoots. increasing the extract concentration elevated the number of green cuttings, total shoot length, average shoot length, length of the longest shoots, and fresh and dry weights of shoots in rose cuttings. elevated number of green cuttings, total shoot length, average shoot length, and length of the longest shoots were observed in wild privet with the extract levels. in poplar cuttings, all the traits improved markedly at a moderate con­ centration (25 g/l) (table 1). the use of garlic extract in sycamore cuttings had no positive effects on shoot­related traits. in berry cuttings, the moderate concentration had positive impacts on the number of green cuttings and total shoot length, but it was not significant on the other traits (table 1). in grape cuttings, moderate concen­ tration of garlic extract positively affected the num­ ber of green cuttings, average shoot length, and shoot fresh and dry weights, while the high concen­ tration (50 g/l) was not significantly different from the moderate one or had a negative effect. in apple cuttings, the moderate concentration significantly increased the number of green cuttings, and both moderate and high concentrations led to increased number of shoots, but the use of garlic extract had no significant effects on the other traits. in sour cher­ ry cuttings, no positive effects were observed on all the traits. in cherry cuttings, the use of garlic extract at both concentrations had slight positive effects on the number of green cuttings, total shoot length, number of shoots, mean shoot length, and length of the longest shoots (table 1). in this experiment, the highest number of green cuttings, total shoot length, and the longest shoot length were obtained in wild privet with 50 g/l, with the highest number of shoots and fresh shoots at 25 g/l of garlic extract. the highest number of green cuttings belonging to wild privet cuttings was not sig­ nificantly different from berry cuttings treated with a moderate concentration. also, the average shoot length was higher in poplar cuttings treated with a moderate concentration than the other cuttings (table 1). root‐related traits. in this experiment, the inter­ action of garlic extract concentrations × substrate and plant type was significant on total length of roots, root number, mean root length, longest root length, and root fresh and dry weights. garlic extract at a concentration of 50 g/l had positive effects on total length of roots, root number, mean root length, longest root length, and root fresh and dry weights of rose roots. total root length and root number were uppermost in wild privet plant at a high concentra­ tion (50 g/l). also, the moderate concentration (25 g/l) produced the longest roots and the highest root fresh and dry weights in this plant (table 2). in poplar cuttings, a concentration of 25 g/l result­ ed in significant increases in total length of roots, root number, mean root length, longest root length, and root fresh and dry weights. there were no signifi­ cant increases in root traits of sycamore cuttings with the extract application. the extract application was not useful on rooting traits in berry cuttings. extract adv. hort. sci., 2020 34(2): 191­204 194 table 1 ­ effects of different concentration of garlic extract (the culture substrate) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings total length of shoots number of shoots shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 0.56 fg 0.89 e 0.67 ef 0.67 ef 0.78 de 0.02 d 0.00 d wild privet 7.44 b 181.50 a 33.00 a 5.63 a 19.62 a 5.27 a 0.58 a poplar 1.78 de 21.39 d 2.78 e 4.96 b 8.17 b 0.62 c 0.03 c sycamore 2.33 d 2.78 e 2.78 e 0.99 e 1.72 d 0.01 d 0.01 cd berry 9.11 a 43.04 b 23.71 a 1.78 d 5.44 c 0.05 d 0.01 cd grape 7.33 b 33.22 c 11.55 c 2.62 c 8.28 b 2.58 b 0.06 b apple 3.89 c 2.89 e 5.78 d 0.50 ef 0.50 de 0.00 d 0.00 d sour cherry 1.33 ef 3.89 e 2.22 ef 0.86 e 1.17 de 0.00 d 0.00 d cherry 0.22 g 0.11 e 0.22 f 0.11 f 0.11 e 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g.l) 3.41 b 23.80 b 7.23 b 1.71 b 4.17 b 0.77 b 0.09 a 25 g·l 4.26 a 34.98 a 9.46 a 2.88 a 6.35 a 1.01 a 0.06 b 50 g·l 3.67 b 20.44 b 7.88 b 1.11 c 4.17 b 0.70 b 0.02 c plant × garlic extract concentration (g∙l) rose × 0 0.00 j 0.00 g 0.00 i 0.00 j h00.0 g00.0 f00.0 rose × 25 0.67 hij 0.67 g 1.00 hi 0.50 ghij 0.50 fgh g00.0 00.0 f rose × 50 1.00 ghij 2.00 g 1.00 hi 1.50 efghi 1,83 fgh g08.0 ef 01.0 wild privet × 0 5.00 cd 128.17 c 29.00 bc 5.47 c 17.00 c b82.5 a72.0 wild privet × 25 7.67 b 224.75 b 37.50 a 5.37 c 17.50 c a47.7 b63.0 wild privet × 50 9.67 a 255.00 a 32.67 b 6.89 b 26.75 a d00.4 c10.0 poplar × 0 1.00 ghij 4.67 g 1.00 hi 1.33 efghij 1­33 fgh g05.0 f00.0 poplar × 25 3.67 def 59.50 d 4.67 gh 13.55 a 22.67 b e42.1 cd07.0 poplar × 50 0.67 hij 0.00 g 2.67 ghi 0.00 j 0.50 fgh g38.0 ef02.0 sycamore × 0 2.67 efg 5.17 g 3.00 ghi 1.57 defgh 2.67 fg g00.0 f00.0 sycamore × 25 2.33 fgh 3.00 g 2.33 ghi 1.02 fghij 2.00 fgh g00.0 f00.0 sycamore × 50 2.00 fghi 0.17 g 3.00 ghi 0.37 hij 0.50 fgh g05.0 def03.0 berry × 0 8.33 ab 36.67 ef 19.67 d 1.38 defg 5.67 e g17.0 ef02.0 berry × 25 9.67 a 47.47 de 27.00 c 1.67 defgh 5.33 e g00.0 f00.0 berry × 50 9.33 ab 45.00 e 26.00 c 1.58 defg 5.33 e g00.0 f00.0 grape × 0 7.67 b 27.00 f 11.00 e 2.61 de 7.50 de f91.0 cde06.0 grape × 25 8.67 ab 38.33 ef 13.33 e 2.84 d 8.17 d c49.4 cd08.0 grape × 50 5.67 c 34.33 ef 10.33 ef 2.42 de 9.17 d e78.1 cde06.0 apple × 0 3.67 def 2.33 g 4.67 gh 0.50 ghij 0.50 fgh 0.00 g 0.00 f apple × 25 4.33 cde 3.17 g 6.33 fg 0.50 ghij 0.50 fgh 0.00 g 0.00 f apple × 50 3.67 def 3.17 g 6.33 fg 0.50 ghij 0.50 fgh 0.00 g 0.00 f sour cherry × 0 2.33 fgh 10.17 g 4.00 ghi 2.08 def 2.83 f 0.00 g 0.00 f sour cherry × 25 1.00 ghij 1.00 g 3.00 ghi 0.33 hij 0.33 fgh 0.00 g 0.00 f sour cherry × 50 67.0 hij 0.50 g 0.67 hi 0.17 ij 0.33 fgh 0.00 g 0.00 f cherry × 0 0.00 j 0.00 g 0.00 i 0.00 j 0.00 h 0.00 g 0.00 f cherry × 25 0.33 ij 0.17 g 0.33 hi 0.17 ij 0.17 gh 0.00 g 0.00 f cherry × 50 0.33 ij 0.17 g 0.33 hi 0.17 ij 0.17 gh 0.00 g 0.00 f p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract conc. 0.0072 <0.0001 0.0001 <0.0001 <0.0001 0.0215 <0.0001 plant × garlic extract concentration (g·l) <0.0001 <0.0001 0.0072 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 195 table 2 ­ effects of different concentration of garlic extract (the culture substrate) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments total length of roots number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 2.61 d 0.55 d 0.36 c 0.33 d 0.2 d 0.00 d wild privet 318.75 a 42.57 a 8.43 a 21.22 a 5.27 a 0.58 a poplar 29.61 c 3.33 c 4.14 b 7.12 c 0.62 c 0.03 c sycamore 0.83 d 0.33 d 0.17 c 0.33 d 0.01 d 0.01 cd berry 2.61 d 0.78 d 0.18 c 1.00 d 0.05 d 0.01 cd grape 73.06 b 10.12 b 4.84 b 11.00 b 2.58 b 0.06 b apple 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d sour cherry 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d cherry 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 28.67 b 3.44 c 1.92 b 3.48 b 0.77 b 0.09 a 25 g·l 52.83 a 8.27 a 2.43 a 6.00 a 1.01 a 0.06 b 50 g·l 29.74 b 4.64 b 1.41 c 3.48 b 0.70 b 0.02 c plant × garlic extract concentration (g∙l) rose × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f rose × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f rose × 50 7.83 g 1.67 f 1.07 f 1.00 e 0.08 g 0.01 ef wild privet × 0 202.67 c 21.00 c 9.91 a 19.00 b 5.82 b 0.72 a wild privet × 25 418.25 b 56.00 b 8.68 b 28.00 a 7.47 a 0.63 b wild privet × 50 462.00 a a00.67 6.71 c 16.67 b 4.00 d 0.10 c poplar × 0 4.33 g f67.0 0.87 f 1.67 e 0.05 g 0.00 f poplar × 25 82.50 e e33.7 10.18 a 17.33 b 1.42 e 0.07 cd poplar × 50 2.00 g f00.2 0.00 f 0.00 e 0.38 g 0.02 ef sycamore × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sycamore × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sycamore × 50 1.00 g f00.1 0.50 f 1.00 e 0.05 g 0.03 def berry × 0 6.50 g f33.1 25.0 f 2.50 e 0.17 g 0.02 ef berry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f berry × 50 0.00 g 1.00 f 0.31 f 0.50 e 0.00 g 0.00 f grape × 0 42.50 f 8.00 e 5.72 cd 9.50 d 0.91 f 0.06 cde grape × 25 96.50 d 12.50 d 3.06 e 14.00 c 4.49 c 0.08 cd grape × 50 80.17 e 10.67 d 4.75 d 11.00 d 1.78 e 0.05 cde apple × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f apple × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f apple × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract conc. <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 plant × garlic extract concentration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 196 adv. hort. sci., 2020 34(2): 191­204 application in grape cuttings had significant impacts on all rooting traits. root growing was not observed in the cuttings of apple, sour cherry, and cherry. in this experiment, the highest total root length and number of roots were observed in wild privet cut­ tings at a high concentration and the highest mean root length was recorded in poplar cuttings at a mod­ erate concentration. the root length and fresh weight were uppermost in wild privet cuttings with a moderate concentration of garlic extract, and the highest root dry weight was measured in the cuttings of this plant in the control treatment (table 2). the effects of different concentrations of garlic extract on the cuttings shoot‐related characteristics. the interaction of plant type and extract concentration resulted in sig­ nificant impacts on the number of green cuttings, total shoot length, number of shoots, mean shoot length, longest shoot length, and fresh and dry weights of shoots. the application of garlic extract did not have positive effects on all the traits in rose cuttings. in wild privet cuttings, moderate concentra­ tion (25 g/l) could further elevate the number of green cuttings, total shoot length, number of shoots, and shoot fresh weight. the longest shoot length and shoot dry weight were observed in wild privet cut­ tings at high concentration treatment (50 g/l) (table 3). the application of garlic extract in poplar cuttings had positive effects on average shoot length and longest shoot length, with no positive impacts on the other traits. garlic extract at moderate concentration significantly affected all shooting traits in sycamore cuttings. in berry cuttings, the use of this extract at both concentrations led to fairly positive influences on the longest shoot length and fresh and dry weights of the shoots, but no positive effects were observed on the other traits. in grape cuttings, no positive effects were observed on the traits except for the number of green cuttings at 25 g/l and dry weight of shoots at 50 g/l. there was no positive effects on the traits in garlic extract treatments of apple cuttings. moreover, in sour cherry cuttings, only the mean shoot length was positively influenced by the moderate concentration (table 3). in cherry, cuttings treated with a moderate con­ centration had positive effects on the number of green cuttings, total shoot length, number of shoots, mean shoot length, longest shoot length, and shoot fresh weight. the highest number of green cuttings was found in berry cuttings at all concentrations. total shoot length and number of shoots were uppermost in wild privet cuttings at moderate­level treatment. in wild privet cuttings, maximum mean shoot length occurred at a concentration of 50 g/l and the longest shoot length was obtained at both moderate and high concentrations. also, the highest fresh and dry weight of shoots was measured in sycamore cuttings in the control (table 3). root‐related traits. analysis of the experimental data showed that garlic extract yielded significant effects on total length of roots, number of root, mean root length, root length, and root fresh and dry weights of different cuttings. rose plant cuttings were not rooted in any of the treatments. in wild privet cuttings, an extract concentration of 25 mg/l increased the total root length and both high and moderate concentrations produced the highest roots (table 4). garlic extract treatments had no positive effects on all root traits in poplar cuttings. the application of different extract concentrations in sycamore cuttings led to no significant differences with the control. the berry, apple, sour cherry, and cherry cuttings were not rooted in this experiment. in grape cuttings, the use of garlic extract at both moderate and high con­ centrations produced positive impacts on the total root length, number of roots, the longest root length, and root fresh and dry weights (table 4). using different concentrations of garlic extract in the callus stage shoot‐related characteristics. according to the results of anova, the interaction of plant type and garlic extract concentrations led to significant differ­ ences in all shoot­related traits at 1% level. the high­ est number of green cuttings was found in wild priv­ et, berry, and grape cuttings, but there were no sig­ nificant differences between the control and treat­ ments. a high concentration of garlic extract had a negative effect on the number of green cuttings in poplar, while it had positive effects on rose and sycamore cuttings at both garlic extract concentra­ tions. in apple cuttings, a high concentration of garlic extract positively affected the number of green cut­ tings. garlic extract had no effects on sour cherry cut­ tings and produced negative consequences in cherry cuttings. the highest shoot length was obtained in poplar cuttings treated with 50 g/l of garlic extract. the highest shoots were measured in poplar cuttings treated with garlic extract. there were no significant differences in the shoot length and the longest shoots in wild privet and rose cuttings with garlic extract treatments. the moderate concentration in sycamore cuttings, both concentrations in berry cut­ abbasifar et al. ‐ garlic extract effects on root cuttings 197 table 3 ­ effects of different concentration of garlic extract (on the cuttings) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings total length of shoots number of shoots shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 0.33 f 0.50 e 0.44 e 0.50 d 0.44 f 0.15 d 0.01 f wild privet 7.78 b 111.00 a 33.25 a 3.46 b 12.78 b 8.34 b 2.23 b poplar 6.67 c 71.50 b 9.44 c 8.99 a 18.00 a 11.50 a 2.92 a sycamore 1.50 c 3.72 de 2.00 e 1.19 c 1.83 e 1.08 d 0.10 ef berry 10.00 a 27.11 c 20.89 b 1.34 c 3.22 d 3.77 c 1.02 d grape 6.55 c 30.00 c 9.78 c 3.11 b 7.05 c 8.61 b 1.19 c apple 1.78 e 0.69 e 1.78 e 0.28 d 0.33 f 0.16 d 0.03 ef sour cherry 2.89 d 6.61 d 4.67 d 1.61 c 3.31 d 0.89 d 0.20 e cherry 0.67 ef 0.33 e 0.67 e 0.22 d 0.22 f 0.07 d 0.01 f garlic extract concentration (g∙l) control (0 g·l) 4.46 29.61 a 8.63 2.31 a 5.04 3.72 0.84 a 25 g·l 4.41 25.37 b 9.78 2.25 a 4.56 3.44 0.64 b 50 g·l 3.96 16.37 c 8.31 1.46 b 5.21 3.15 0.91 a plant × garlic extract concentration (g∙l) rose × 0 1.00 efg 1.50 g 1.33 i 1.50 gh 1.33 gfh 0.45 f 0.04 h rose × 25 0.00 g 0.00 g 0.00 i 0.00 j 0.00 h 0.00 f 0.00 h rose × 50 0.00 g 0.00 g 0.00 i 0.00 j 0.00 h 0.00 f 0.00 h wild privet × 0 7.33 bc 95.50 bc 23.67 c 3.81 d 11.00 c 6.77 c 1.83 d wild privet × 25 8.67 ab 127.83 a 41.33 a 3.08 de 13.33 bc 10.07 b 2.36 c wild privet × 50 7.33 bc 104.00 b 35.50 b 3.52 d 14.00 b 8.18 bc 2.49 bc poplar × 0 7.67 bc 92.00 c 12.67 d 7.52 c 15.56 b 13.87 a 3.53 a poplar × 25 5.67 c 35.50 e 8.00 def 10.10 b 19.00 a 6.93 c 1.53 de poplar × 50 6.67 bc 58.75 d 7.67 efg 21.10 a 20.00 a 8.95 b 2.77 b sycamore × 0 0.50 efg 0.67 g 2.00 hi 10.1 ghi 1.50 gfh 1.22 ef 0.04 h sycamore × 25 2.67 de 7.33 g 3.00 ghi 1.96 fg 3.50 ef 1.08 ef 0.24 h sycamore × 50 1.00 efg 0.17 g 1.00 i 0.50ij 0.50 gh 0.23 f 0.03 h berry × 0 10.00 a 28.67 ef 20.00 c 1.44 gh 3.00 gf 3.03 de 0.84 g berry × 25 10.00 a 30.17 ef 19.00 c 1.70 fgh 3.67 ef 4.31 d 1.01 fg berry × 50 10.00 a 22.50 f 23.67 c 0.87 hij 3.00 gf 3.98 d 1.20 f grape × 0 6.33 c 33.00 e 8.68 de 3.49 d 6.33 d 6.85 c 1.13 fg grape × 25 7.00 bc 29.33 ef 9.39 de 3.40 d 8.38 d 9.17 b 1.08 fg grape × 50 6.33 c 34.33 ef 11.33 de 2.42 ef 6.50 d 9.80 b 1.35 ef apple × 0 2.33 def 1.17 g 2.33 hi 0.33 ij 0.33 gh 0.10 f 0.01 h apple × 25 2.33 def 0.57 g 2.33 hi 0.35 i 0.50 gh 0.37 f 0.08 h apple × 50 0.67 efg 0.33 g 0.67 i 0.17 j 17.0 gh 0.02 f 0.01 h sour cherry × 0 3.33 d 10.33 g 7.67 efg 1.44 gh 6.00 de 1.14 ef 0.26 h sour cherry × 25 2.00 defg 3.67 g 3.67 fghi 1.92 fg 2.00 fgh 0.49 f 0.04 h sour cherry × 50 3.33 d 5.83 g 3.67 fghi 1.47 gh 1.25 fgh 1.04 ef 0.32 h cherry × 0 0.33 fg 0.17 g 0.33 i 0.17 j 17.0 gh 0.04 f 0.00 h cherry × 25 1.33 def 0.67 g 1.33 i 0.33 ij 0.33 gh 0.17 f 0.01 h cherry × 50 0.33 fg 0.17 g 0.33 i 0.17 j 17.0 gh 0.01 f 0.01 h p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.3226 0.0032 0.2786 0.0819 0.2726 0.8816 0.0007 plant × garlic extract concen­ tration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 adv. hort. sci., 2020 34(2): 191­204 198 table 4 ­ effects of different concentration of garlic extract (on the cuttings) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments total length of roots number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d wild privet 292.70 a 42.40 a 6.53 b 18.83 b 4.91 a 0.48 a poplar 171.61 b 13.87 b 11.83 a 20.87 a 1.95 c 0.16 b sycamore 0.33 d 0.22 c 0.15 c 0.22 d 0.03 d 0.00 d berry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d grape 98.39 c 13.11 b 7.17 b 17.00 c 3.77 b 0.12 c apple 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d sour cherry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d cherry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 46.68 b 4.40 c 3.30 a 5.54 b 1.18 a 0.08 a 25 g·l 63.30 a 7.42 a 2.60 ab 5.00 b 1.25 a 0.06 b 50 g·l 41.18 b 5.60 b 2.28 ab 6.54 a 0.83 b 0.07 b plant × garlic extract concentration (g∙l) rose × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g rose × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g rose × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g wild privet× 0 265.50 b 42.00 a 6.33 e 16.00 d 6.99 a 0.57 a wild privet× 25 331.50 a 44.33 a 6.25 e 21.00 b 5.34 b 0.45 b wild privet× 50 203.50 c 37.00 b 6.92 e 19.50 bc 2.19 cd 0.41 c poplar× 0 241.00 b 15.33 c 14.24 a 24.00 a 3.15 c 0.29 d poplar × 25 117.67 e 4.50 d 9.82 c 18.00 cd 1.27 d 0.03 g poplar × 50 159.17 d 18.67 c 11.23 b 19.67 bc 1.43 d 0.18 e sycamore× 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sycamore × 25 1.00 g 0.00 e 0.44 f 0.67 f 0.00 e 0.00 g sycamore × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g grape × 0 59.50 f 7.33 d 8.33 d 12.00 e 2.45 c 0.03 g grape × 25 119.50 e 16.33 c 6.23 e 19.00 bc 4.51 b 0.06 f grape × 50 116.17 e 15.67 c 7.34 de 19.67 bc 4.34 b 0.26 d apple × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g apple × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g apple × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.0658 0.3573 0.0213 0.1200 0.0068 <0.0001 plant × garlic extract concen­ tration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 199 moderate concentration of garlic extract, which did not differ significantly with the grape and control wild privet cuttings at the same concentration. the highest root length was obtained in wild privet cut­ tings treated with moderate concentration, which did not show significant differences with the cuttings treated with the high concentration. the longest roots and highest root dry weight were recorded in wild privet cuttings at moderate concentration. poplar control cuttings gained the highest fresh weight (table 6). 4. discussion and conclusions according to the results of three experimental sections, it seems that the effects of garlic extract on the traits related to shoots and roots of different cuttings depend on the time of use, species type, and the extract concentration. in the first section, the use of high garlic extract concentration (50 g/l) in rose cuttings and moderate concentration (25 g/l) in wild privet, poplar, berry, grape, apple, and cherry cuttings had positive impacts. garlic extract had no positive effects on shoot traits in sycamore and sour cherry culture substrates. in schefflera arboricola, garlic extract application increased plant height, leaf number, and fresh and dry weights of leaves (hanafy et al., 2012). in leguminous plants, the extract espe­ cially at high concentrations caused negative effects on growth and decreased plant height (adeleke, 2015). also, using a high concentration of garlic extract in the culture substrates of rose and wild privet, and moderate concentration in poplar and grape substrates had positive effects on their rooting traits. however, a high concentration of garlic extract was not effective in the rooting of sycamore, berry, apple, sour cherry, and cherry cuttings. considering the evaluated shoot and root traits, the use of 50 g/l and 25 g/l of garlic extract was useful in rose cuttings and poplar and grape cuttings, respectively. garlic extract contains many allelopathic chemi­ cals (wang et al., 2015) with a known antimicrobial and antibacterial agent (harris et al., 2001). garlic planting in vegetable hydroponic substrates has been shown to prevent them from many diseases and to reduce the microbial population in the substrate (liu et al., 2014). the positive effects of garlic extract are more evident at low and moderate concentrations. high concentrations of garlic extract led to decreased growth of shoots and roots due to increased allelo­ tings, and a high concentration in apple cuttings resulted in desirable effects on this trait. the effect of this extract was positive in sour cherry cuttings, and there was a negative effect on the length of shoots produced in cherry cuttings (table 5). the fresh weight of shoots was uppermost in wild privet cuttings treated with 25 g/l of garlic extract, which was not significantly different from that of grape cuttings at 50 g/l. dry weight of wild privet cuttings was the highest in all treatments. the use of garlic extract increased fresh and dry weights of shoots in poplar, sycamore, and berry cuttings. in grape and apple cuttings, a high concentration of gar­ lic extract yielded useful impacts on the fresh and dry weights of shoots while it led to negative conse­ quences in sour cherry and cherry cuttings (table 5). overall, the use of garlic extract at the time of callus formation was evaluated to be positive on the shoot­ related traits of sycamore, berry, and apple cuttings. root‐related traits. analysis of experimental data showed that different concentrations of garlic extract significantly influenced rooting characteristics of dif­ ferent cuttings in the callus stage at 1% level. the use of garlic extract could significantly affect the root number and length at moderate concentration and root dry weight at both concentrations in the callus stage of rose cuttings. the use of moderate concen­ tration had a significant effect on the increase of all root traits in wild privet cuttings in the callus stage. the high concentration caused a decrease in the number of roots and an increase in root length, and the moderate concentration increased the longest root length of poplar cuttings. the use of garlic extract at this stage caused a significant decrease in fresh and dry weights of roots in poplar cuttings. sycamore cuttings showed some increases in the root traits at the moderate concentration. berry cuttings increased the number of roots to some extent at both concentrations. in the cuttings of this plant, the length of roots was the greatest at the moderate concentration of garlic extract and the longest root length, and root fresh and dry weights were greater at the concentration of 50 g/l. in grape cuttings, the moderate concentration increased the number of roots and the high concentration elevated root length. in the cuttings of this plant, both concentra­ tions increased the length of the longest root and reduced root fresh and dry weights. root growing did not occur in apple, sour cherry, and cherry cuttings (table 6). the highest number of rooted cuttings was observed in wild privet cuttings treated with the adv. hort. sci., 2020 34(2): 191­204 200 table 5 ­ effects of different concentration of garlic extract (in the callus stage) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 1.22 cd 5.46 c 10.44 d 2.27 d 0.70 e wild privet 9.67 a 8.54 b 29.78 b 23.08 a 7.09 a poplar 4.89 b 18.14 a 35.14 a 13.08 b 4.76 b sycamore 0.78 d 2.14 de 2.44 e 0.40 de 0.08 ef berry 8.78 a 3.66 cd 10.33 d 5.26 c 1.43 d grape 8.67 a 8.41 b 15.39 c 21.81 a 2.98 c apple 1.22 cd 1.45 e 1.61 e 0.17 e 0.03 ef sour cherry 2.33 c 2.46 de 3.83 e 0.53 de 0.09 ef cherry 0.33 d 0.78 e 0.50 e 0.03 e 0.01 f garlic extract concentration (g∙l) control (0 g·l) 4.00 5.08 9.94 b 6.47 b 1.65 b 25 g·l 4.59 5.36 12.50 a 7.82 a 2.08 a 50 g·l 4.04 6.10 12.27 a 6.06 b 1.88 a plant × garlic extract concentration (g∙l) rose × 0 0.67 def 5.73 cdef 11.67 cdef 1.84 fgh 0.33 hi rose × 25 2.00 cdef 4.98 defg 9.00 efgh 3.19 efgh 0.92 ghi rose × 50 1.00 cdef 5.66 def 10.67 defg 1.79 fgh 0.84 ghi wild privet × 0 9.67 a 8.54 cd 30.17 b 21.34 b 6.95 a wild privet × 25 10.00 a 8.81 c 27.83 b 26.27 a 7.41 a wild privet × 50 9.33 a 8.26 cd 31.33 b 20.89 b 6.92 a poplar × 0 5.33 b 17.27 b 26.50 b 10.19 d 3.78 cd poplar × 25 6.33 b 15.03 b 37.67 a 14.92 c 5.51 b poplar × 50 3.00 c 21.09 a 40.00 a 13.79 c 4.66 bc sycamore × 0 0.00 f 0.00 i 0.00 j 0.00 h 0.00 i sycamore × 25 1.33 cdef 5.00 defg 5.67 ghij 0.48 gh 0.15 hi sycamore × 50 1.00 cdef 1.43 ghi 1.67 ji 0.73 gh 0.00 i berry × 0 8.33 a 3.08 efghi 5.00 fghi 3.03 de 1.14 ghi berry × 25 9.00 a 4.08 efgh 11.83 cdef 5.10 ef 1.35 fgh berry × 50 9.00 a 3.82 fghi 12.17 cdef 6.66 e 1.81 fg grape × 0 8.67 a 8.45 cd 17.00 c 21.71 b 3.31 de grape × 25 9.00 a 7.66 cde 13.67 cde 21.88 b 3.18 de grape × 50 8.33 a 9.13 c 15.50 cd 25.87 a 2.46 ef apple × 0 0.67 def 1.00 hi 0.83 ji 0.03 h 0.03 i apple × 25 0.67 def 1.00 hi 0.83 ji 0.06 h 0.01 i apple × 50 2.33 cde 3.42 fghi 3.17 hij 0.41 gh 0.06 i sour cherry × 0 2.00 cdef 0.67 hi 1.00 ji 0.28 gh 0.06 i sour cherry × 25 2.67 cd 3.54 fghi 5.33 ghij 0.49 f 0.17 hi sour cherry × 50 2.33 cde 3.17 fghi 5.17 ghij 0.48 gh 0.05 i cherry × 0 0.67 def 1.00 hi 0.83 ji 0.07 h 0.01 i cherry × 25 0.33 ef 1.33 ghi 0.67 j 0.03 h 0.01 i cherry × 50 0.00 f 0.00 i 0.00 j 0.00 h 0.01 i p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.1060 0.1614 0.1060 0.0159 0.1672 plant × garlic extract concentra­ tion (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 201 table 6 ­ effects of different concentration of garlic extract (in the callus stage) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 1.22 d 5.60 c 7.83 c 0.40 d 0.04 d wild privet 9.00 a 16.59 a 32.79 a 13.04 b 4.40 a poplar 3.87 c 14.91 a 24.74 b 6.31 c 0.80 c sycamore 0.44 ef 1.28 d 2.28 d 0.04 d 0.01 d berry 1.00 de 8.82 b 8.07 c 0.35 d 0.06 d grape b14.8 10.09 b 26.78 b 14.65 a 2.59 b apple 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d sour cherry 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d cherry 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 2.08 b 4.22 b 7.19 b 4.45 a 0.64 b 25 g·l 3.08 a 7.09 a 12.86 a 3.57 b 0.87 a 50 g·l 2.26 b 7.01 a 12.35 a 2.27 c 0.64 b plant × garlic extract concentration (g·l) rose × 0 0.67 fg 4.78 g 7.33 e 0.25 e 0.00 g rose × 25 2.33 e 6.62 fg 8.33 e 0.30 e 0.03 f rose × 50 0.67 fg 5.39 g 7.83 e 0.65 e 0.09 f wild privet× 0 9.00 ab 14.33 bc 26.50 bc 12.89 bc 3.63 b wild privet× 25 10.00 a 19.26 a 35.67 a 13.96 b 5.06 a wild privet× 50 8.00 bc 16.31 ab 32.00 ab 11.89 bc 3.80 b poplar× 0 5.00 d 11.24 cde 17.07 d 17.24 a 1.18 d poplar × 25 4.50 d 14.80 b 30.67 ab 3.05 d 0.50 ef poplar × 50 2.33 e 18.68 a 26.50 bc 2.29 de 0.73 de sycamore× 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 f sycamore × 25 1.00 fg 3.42 gh 3.83 ef 0.09 e 0.01 f sycamore × 50 0.33 fg 0.00 h 3.00 ef 0.02 e 0.00 g berry × 0 0.33 fg 0.00 h 0.00 f 0.03 e 0.03 f berry × 25 1.33 ef 13.90 bcd 7.50 e 0.24 e 0.03 f berry × 50 1.33 ef 9.62 ef 13.83 d 0.78 e 0.13 f grape × 0 7.00 c 9.64 ef 24.33 c 18.68 a 3.99 b grape × 25 9.00 ab 9.84 ef 28.00 bc 14.47 b 2.18 c grape × 50 7.67 c 10.80 de 28.00 bc 10.89 c 2.06 c apple × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f apple × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f apple × 50 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 50 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 50 0.00 g h00.0 0.00 f 0.00 e 0.00 f p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration <0.0001 <0.0001 <0.0001 <0.0001 0.0474 plant × garlic extract concentration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 adv. hort. sci., 2019 33(3): 191­204 202 pathic properties (adeleke, 2015). one of the reasons for the failure of rooting in the cuttings is the sub­ strate contamination to pathogenic agents and their entry into the stem, and as a result, the contamina­ tion of cuttings and failure in rooting (hartmann et al., 2002). garlic extract is a strong disinfectant that resulted in increased rooting and improved shooting. in the second part of the experiment, using the moderate concentration of garlic extract had positive impacts on the shoot and root traits of wild privet cuttings. garlic extract contains auxin hormone, and immersing the tips of olive cuttings in garlic extract in combination with other natural auxins­containing substances (e.g., algae) produced the longest roots and increased the number of shoots, leaf number, mean shoot length, and dry weight of shoots (gad and ibrahim, 2018). the same authors reported that the compound was able to compete with iba and no significant differences were found in these traits with those of iba­treated olive cuttings. in the third part of the experiment, the use of moderate­level garlic extract in the callus stage of rose and sycamore cuttings and the high concentra­ tion in the callus stage of the berry cuttings had posi­ tive effects on the shoot and root traits. there is ample evidence that garlic extract has an allelochem­ ical property that affects cell division, absorption of water and minerals, phytohormone metabolism, res­ piration and photosynthesis, enzyme function, and expression of genes (portales­reyes et al., 2015; sadaqa et al., 2016). biostimulators are a group of naturally occurring substances that can be applied either as spraying or at the tips of cuttings, and some of these substances increase water absorption and nutrient transfer, and stimulate growth processes, with possible hormonal properties (dobranszki and teixeira da silva, 2010); a few of them had positive effects on ornamental pine propagation and rooting of ornamental plant cuttings (shevchenko, 2008; szabó and hrotkó, 2009; pacholczak et al., 2016). these substances stimulate the processes occurring in plants to increase growth. khan et al. (2009) and borowski (2009) reported that natural substances alter the growth and develop­ ment of cells in the root system and the concentra­ tion of many substances in the plant. the mechanism of the physiological and biochemical processes of these substances remains unknown, and their infor­ mation and application in horticulture and plant propagation are very limited and require further study and attention (du jardin, 2015; ertani et al., 2015). many plant extracts such as coconut juice, banana pulp, potato puree, date sap, corn extract, papaya extract, and beef extract have been used in micro­ propagation to enhance plant growth (islam et al., 2003; murdad et al., 2010; nambiar et al., 2012; sudipta et al., 2013). the accumulation of carbohy­ drates, particularly simple ones (monomers), in the rooting zone is essential for rooting onset at early stages, and carbohydrate concentrations of applied natural substances had high influences on the rooting (costa et al., 2007). active oxygen species (ross) are oxygen species that destruct cells and destroy membranes and nucleic acids during such tensions as cutting and mechanical damage to cuttings. ross, therefore, is always present at rooting event. biostimulators and natural substances used to increase the growth of different plants reduce the effects of various stresses on plants (dobranszki and teixeira da silva, 2010). phenolic compounds are classified into simple phe­ nols, phenolic acids, hydroxycinnamic derivatives, and flavonoids. researchers have reported that many phenolic compounds and flavonoids function as strong antioxidant compounds that are found abun­ dantly in many plant extracts, including medicinal plants. the active ingredients of medicinal plants include complex chemical compounds produced and stored in the organs of medicinal plants and protect cells from oxidative damage (lin and harnly, 2010). the extracts of medicinal plants have many antimi­ crobial properties and can prevent the release of microorganisms in the culture medium and plant growth media (radwan et al., 2015). considering the shoot and root traits evaluated in the three experiments, the use of 50 g/l and 25 g/l of garlic extract was useful in rose cuttings and poplar and grape cuttings, respectively. moderate concentration (25 g/l) of garlic extract revealed posi­ tive effects on shoot and root traits of wild privet cut­ tings. also, the use of moderate garlic extract had positive effects on rose, and at a high concentration (50 g/l) on sycamore cuttings and on berry cuttings in the callus stage. as a result, to improve the quantity and quality of shooting and rooting in rose cuttings, garlic extract can be used at a concentration of 50 g/l in the substrate or 25 50 g/l in the callus formation stage. a comparison of data from these two experi­ ments indicated that the use of 25 g/l was more effective in the callous formation stage. also, to improve the quality and quantity of shoots and roots in wild privet 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efeoglu and terzioglu, 2009; lin­wang, 2011; hao et al., 2019). previous report indicated that chlorophyll (chl) a con­ tent, total chl content, and the chl a/b ratio in soybeans, which were grown under high temperatures (38/28°c), decreased 7, 3, and 18%, respectively (hasanuzzaman et al., 2013). additionally, sugiura et al. (*) corresponding author: panawat.sik@gmail.com citation: sikhandakasmita p., kataoka i., ogata t., mochioka r., beppu k., 2021 ­ effect of growth temperature levels on photosynthetic ability and fruit quality of ‘ku‐pp2’, a new low‐chill peach cultivar. ­ adv. hort. sci., 35(3): 233­241 copyright: © 2021 sikhandakasmita p., kataoka i., ogata t., mochioka r., beppu k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 october 2020 accepted for publication 10 june 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9923 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 233­241 234 (2003) showed that higher temperatures significantly change the fruit quality of apple. a reduction in acid concentration and softening of fruit flesh were observed resulting from exposure to high tempera­ tures during fruit development. the effect of elevat­ ed temperatures on plants differs depending on the stages of development and timescale. continuing heat stress can lead to slowing of growth and devel­ opment and inducing an imbalance in carbohydrate metabolism between photosynthesis and respiration. as a result, carbohydrate reserves decline, leading of yield loss, and possibly plant death (hall, 1992; wahid et al., 2007). photosynthesis comprises a few principal compo­ nents that are highly sensitive to temperature: pho­ tosynthetic pigments, electron transport chain, photosystem i (ps i), and photosystem ii (ps ii). the decline in photosynthesis under high­temperature conditions results from inhibition of the redox reac­ tion and metabolic pathways occurring in ps i, ps ii, the cytochrome complex, and photosynthetic enzyme activities (taiz and zeiger, 2006). moreover, elevated temperatures can also affect photosynthesis via physical processes. previous studies have shown that heat stress is involved with leaf water status, leaf gas exchange, and stomatal conductance (gsw) caused by changes in hydraulic conductance (fredeen and sage, 1999; greer and weedon, 2012). under high­temperature conditions, intercellular co2 concentration in leaves frequently declines because of stomatal closure and reduced co2 uptake and transport, leading to impaired photosynthetic co2 assimilation (centritto et al., 2001). ‘ku­pp2’ is a new yellow flesh peach with a low­ chilling requirement that produce excellent yield and high fruit quality. it was bred and released in 2016 for use in subtropical regions and particularly for use in forcing culture system to expand the harvesting season of fresh peach (manabe et al. , 2015). understanding the effect of growth temperatures on ‘ku­pp2’ peach trees is crucial for optimizing plant growth, physiological functioning, and increasing pro­ ductivity. the effect of chilling accumulation and heating temperatures on bud burst and flowering of ‘ku­pp2’ have been clarified. however, the influence of temperature during fruit development on low­chill peach cultivars has not been elucidated. previous studies on japanese high­chill peach cultivars indicat­ ed that high temperatures dramatically hasten fruit growth and the onset of fruit maturation (sugiura et al., 2003; hayama et al., 2007). in addition, optimal heating could save energy costs for plant production in heated plastic houses. therefore, the aim of this study was to investigate the effect of growth temper­ ature during fruit development on plant physiology and to determine the optimal growth temperature for plant growth, which can enhance fruit quality of the low­chill peaches under controlled conditions. additionally, the knowledge gained could be used to design a heating program and cultivation manage­ ment practices for growing the low­chill peach trees in plastic houses. 2. materials and methods plant materials this experiment was conducted at the research field of the faculty of agriculture, kagawa university, which is located in southwest japan. six healthy and uniform of seven­year­old ‘ku­pp2’ peach trees were selected for this study. all plants were grafted onto ‘tsukuba 1 gou’ peach rootstock and planted in con­ tainers. ‘ku­pp2’ flowers were hand­pollinated with fresh pollen from another ‘ku­pp2’ tree. three weeks after pollination, two plants were transferred to each temperature regimes and the fruits were thinned by hand to 6­7 fruits per tree. the air temperature in the phytotrons was set at 20, 25, and 30°c during the experimental period from 13 april to 8 july 2019. cultural practices and fertilization were performed according to standard peach growing practices in japan (sugiura et al., 2003). leaf morphology and anatomy observation leaf length and width were measured for five mature leaves per tree for each treatment using a digital caliper at the end of the experiment. leaf width was measured across the widest part of the leaf. five fully expanded leaves were collected and weighed immediately to determine fresh weight. these leaves were dried in a hot air oven at 80°c and weighed after 72 h of drying to determine their dry weight (fanourakis et al., 2017). leaf dry matter (dm) was calculated as the ratio between dry mass and fresh mass. dm was expressed as the percentage of fresh weight for anatomical analysis, five leaf sam­ ples from each plant were collected and preserved in formalin­acetic acid­alcohol (faa; formaldehyde 1:acetic acid 1: 99.5% ethanol 9:deionized water 9) solution. cross­sections were made using a rotary microtome at a thickness of 5 µm. the cross­sections sikhandakasmita et al. ‐ effect of temperature on the low‐chill peach growth 235 of samples were observed and photographed using a light microscope equipped with a microscope camera (olympus dp­25, olympus co. ltd., japan). the fol­ lowing anatomical characteristics were measured: the thickness of the adaxial and abaxial epidermis, spongy mesophyll, and palisade cells, as well as the number of stomata per square millimeter. evaluations of chlorophyll content and spad value the chl content and spad value of five mature leaves from each tree were analyzed during the har­ vesting period. spad values were measured using a portable chlorophyll meter (spad­502, minolta, japan). chlorophyll in the same leaves was analyzed as described by lichtenthaler and wellburn (1983). leaf disks (2.5 cm2 per disk) were homogenized with 10 ml of cold 95% acetone and incubated at 4°c in darkness for 3 h. these mixtures were centrifuged at 3,500 rpm for 10 min. after centrifugation, absorbance of the supernatants was determined using the spectrophotometer. the optical density for the blank and the mixtures were measured at 645 and 663 nm, respectively. these absorbance values were used to calculate chl a, chl b, and total chloro­ phyll (chl a+b) and expressed as mg l­1. leaf gas exchange measurement photosynthetic gas exchange was measured using a portable photosynthesis system (li­6800; li­cor biosciences, lincoln, ne, usa) from 9:00 to 12:00. the rate of net co2 assimilation, stomatal conduc­ tance, transpiration, and intercellular co2 concentra­ tion were measured weekly until the end of the experiment. ten newest fully expanded leaves, which were outside of the canopy and fully exposed to sun­ light, were randomly selected and used for the mea­ surements. the reference co2 concentration and flow rate inside the chamber were maintained at 400 µmol mol­1 and 800 µmol m­2 s­1, respectively. photosynthetically active radiation (par) was set to 1,200 µmol s­1. the chamber temperature was com­ parable to the growth temperatures, and relative humidity (rh) was kept at 60% (marchi et al., 2008). the data were recorded at a steady state, in which gas exchange parameters were stable. fruit quality assessment five fruits per treatment were collected on the commercial harvest date for phytochemical analysis. after the harvest, all fruits were immediately trans­ ferred to the laboratory and weighed. flesh firmness and total soluble solids (tss) were measured from two opposite fruit cheeks. flesh firmness was deter­ mined using a manual penetrometer with a 4.5­mm tip. tss was measured using a digital refractometer (pr­101α; atago co. ltd., japan) and were expressed as degree brix (°brix). titratable acidity (g l­1 of malic acid) was determined by titrating fruit juices with 0.05 mol l­1 of sodium hydroxide (naoh) using acidity titrator (ta­72; dkk­toa co. ltd., japan). the fruit development period was calculated as the days from full bloom to first commercial harvest. fruit col­ oring was estimated visually according to a scale from 1 (none) to 9 (hiding ground color) using ecpgr priority descriptors for peach (upov, 2010). the fruit coloring was expressed as the percentage of over color extent. statistical analysis all data from each treatment were subjected to analysis of variance (anova) using the statistical analysis system (sas) university edition (sas institute inc., cary, nc). the differences between means were separated by tukey’s honestly significant difference (hsd) test at p<0.05. the results were expressed by means followed by the standard errors. 3. results leaf morphology and anatomy response to growth temperature at the end of the experiment, the significant dif­ ferences in leaf dimensions (p < 0.0001) and dry mat­ ter (p<0.0001) between growth temperature levels were observed (table 1). ‘ku­pp2’ peach trees that were forced at 25°c had the longest leaf length, fol­ lowed by the 20°c and the 30°c treatments, while the leaf width of each treatment was comparable (p= 0.0864). the stomatal density increased by 7% with the increase in growth temperature from 20 to 25°c and reached its maximum value at 25°c. however, raising the growth temperature from 25 to 30°c sig­ nificantly diminished stomatal density by 32%. in contrast, the leaf dry matter content slightly increased when the growth temperature increased. compared with 20 and 25°c, the 30°c treatment increased leaf dry matter by a mean value of 5% fw. the growth temperatures not only changed the leaf morphological characteristics but also affected leaf anatomical traits (table 1). the higher tempera­ ture significantly decreased the thickness of leaves, palisade mesophyll, and spongy mesophyll (p < 0.0001). the leaves that were forced at the highest growth temperature (30°c) were thinner than those adv. hort. sci., 2021 35(3): 233­241 236 from the trees grown at 25 and 20°c, as well as pal­ isade and spongy mesophyll layers. on the other hand, the different growing temperatures did not sig­ nificantly change the adaxial and abaxial epidermis thickness (p= 0.7828 and p= 0.4418, respectively) throughout the temperature treatments. figure 1 shows the light microscopy pictures of leaf cross­sec­ tions for all temperature treatments, measured at the end of treatment. spad values, chlorophyll contents, photosynthetic rate, and gas exchange parameters figure 2 shows the high­temperature conditions caused a reduction in spad values (p= 0.0206) and loss of chl content, especially chl a (p= 0.0029) and chl a+b (p= 0.0133). the spad reading for the 30°c treatment showed decreases by 12.5%, compared with the 20°c treatment. however, the spad values for the 20 and 30°c treatments were not significantly different from that of the 25°c treatment. similarly, the chl content decreased when exposed to an increasing temperature compared with the 20°c treatment. the maximum reduction in chl a content (22.3%) occurred with 30°c, the chl a+b concentra­ tion for 30°c decreased by 20.1%, while the chl b content was not significantly affected (p= 0.3494). the responses of the net photosynthetic rate (pn) and the gas exchange parameters to growth temper­ ature differed significantly depending on the levels and duration of the temperature treatments (fig. 3). one week after temperature treatment started, the pn for all treatments increased considerably fig. 1 ­ anatomical comparison of leaf cross­section of ‘ku­pp2’ peach trees under (a) 20°c, (b) 25°c and (c) 30°c at the end of the experiment. the cross­sections of samples were observed and photographed under a light micro­ scope. ep (ad) = adaxial epidermis; pa = palisade mesophyll layer; sp = spongy mesophyll layer; and ep (ab) = abaxial epidermis. these pictures were taken on 15 november 2019. fig. 2 ­ spad value (a) and chlorophyll content (b) in response to growth temperature treatments. data represent means ± standard error (n= 5). different letters indicate significant differences according to tukey’s test (p < 0.05) and ns denotes non­significant. z data are mean values ± standard errors (n = 10). the different lowercase letters within the same row indicate significant differences at p ≤ 0.05 (tukey’s test). table 1 ­ leaf morphological and anatomical characteristics, percentage of leaf dry matter (dm), and stomatal density of the ‘ku­pp2’ peach cultivar at the end of the experiment. the peach trees were grown under three growing temperatures (20, 25, and 30°c) parameter growth temperature p‐value 20°c 25°c 30°c leaf length (cm) 17.1 ± 0.6 a z 18.6 ± 0.2 a 13.7 ± 0.5 b < 0.0001 leaf width (cm) 4.6 ± 0.1 4.5 ± 0.1 3.9 ± 0.3 0.0864 percentage of leaf dry matter (% fw) 44.9 ± 1.35 b 44.5 ± 0.46 b 50.8 ± 0.56 a < 0.0001 leaf thickness (µm) 58 ± 1.6 a 41 ± 1.0 b 37 ± 1.1 b < 0.0001 adaxial epidermis thickness (µm) 6 ± 0.3 5 ± 0.3 5 ± 0.5 0.7828 abaxial epidermis thickness (µm) 4 ± 0.3 4 ± 0.4 3 ± 0.2 0.4418 palisade thickness (µm) 28 ± 0.7 a 18 ± 0.4 b 15 ± 0.2 c < 0.0001 spongy thickness (µm) 20 ± 0.5 a 13 ± 0.6 b 14 ± 0.6 b < 0.0001 stomatal density (no. mm−2) 242 ± 2 b 260 ± 3 a 176 ± 4 c < 0.0001 sikhandakasmita et al. ‐ effect of temperature on the low‐chill peach growth 237 (p<0.0001). the pn of the 30°c treatment was higher than the other treatments in this period (p=0.005). subsequently, the pn of the 20 and 25°c treatments steadily increased and remained stable at a higher level than at the beginning of treatment until the harvesting period. conversely, the pn of the 30°c treatment dramatically declined in the second week (p=0.0007) and after that gradually decreased and reached its lowest level in the eighth week (p<0.0001) after temperature treatment started (fig. 3a). the average pn values of the mature leaves under the 20°c treatment was higher than those in the 25 and 30°c treatments by 12.8 and 47.7%, respectively (p<0.0001). the changes in stomatal conductance (gsw) of each treatment were similar to those of the pn values. the maximum gsw for 30°c was observed in the first week after the beginning of treatment while the peak gsw for 20 and 25°c occurred in the third week (fig. 3b). the averages of gsw in both lower­temperature treatments were not different (p= 0.2578). the gsw of the higher­tempera­ ture treatment rapidly decreased in the second (p<0.0001) and eighth weeks (p= 0.02) after treat­ ment started. similarly, the peak of leaf internal co2 concentration (ci) was observed one week after the beginning of treatment (fig. 3c). a higher growth temperature had greater effects on ci, with a consid­ erable reduction in ci occurring twice; in the second and eighth weeks after treatment started (p<0.0001). the lower growth temperatures (20 and 25°c) had comparative effects on the values of transpiration rate (e). the mature leaf e under the 20 and 25°c treatments declined more slowly than under the 30°c treatment, with the average e for the lower temperatures (20 and 25 °c) being higher than that for the high­temperature treatment by 30−35% (p = 0.004; fig. 3d). effect of growth temperature on fruit quality indexes the morphological characteristics and chemical compositions of the ripe fruit are shown in table 2. the results indicated that a high growth temperature strongly affected only the fruit morphological charac­ teristics (p=<0.0001) of the ‘ku­pp2’ fruit and fruit weight (table 2). however, significant differences in fruit shape (p = 0.0631) and chemical compositions of the fruit (p = 0.0881) were not found. an increase in growth temperature decreased fruit weight, fruit diameter, and fruit length. as shown in figure 4, there were significant contrasts in skin coloration for the ‘ku­pp2’ peaches with the different treatments. during the harvesting period, the fruit from the 30°c treatment showed a higher level of red coloration than the fruit from the 20 and 25°c treatments, indi­ cating that increasing the temperature could acceler­ ate the reddening of the fruit skin. further, at 30°c, the fruit development period became shorter than under the 20 and 25°c conditions, with maturation occurring 14 days earlier. 4. discussion and conclusions long­term exposure to a moderate high­tempera­ ture regime (30°c) can result in cellular and physio­ fig. 3 ­ effect of growth temperatures on (a) leaf net photo­ synthesis, (b) stomatal conductance, (c) leaf internal co2 concentration, and (d) transpiration rate of the ‘ku­pp2’ peach trees. data represent means ± standard error (n= 5). 238 adv. hort. sci., 2021 35(3): 233­241 logical adaptation of ‘ku­pp2’. the responses of the peach trees to a high growing temperature could breakdown chl, change leaf structure, reduce pn, has­ ten fruit maturity, and could further explain the decrease in fruit quality under high temperatures in the present study. similar to previous studies, high­ temperature conditions induced closure of the stom­ ata and generation of reactive oxygen species (ros), damaged chloroplast structure and ps ii and decreased photosynthetic pigments and enzyme activities (takahashi and murata, 2006; ashraf and harris, 2013; chen et al., 2017; jumrani et al., 2017). under high­temperature regimes, plants avoid heat damage and reduce excessive energy absorption on their leaves by decreasing leaf size, covering leaf surfaces with a thick waxy cuticle as well as trichome, changing leaf shape, or increasing the number of stomata. small leaves can also reduce water loss and have less surface area exposed to solar radiation (hasanuzzaman et al., 2013). plants with thinner leaves and high stomatal densities can evacuate heat to the environment quicker than large leaves. a simi­ lar response to high temperatures was found in this study, in which leaf size and thickness of the leaf blades, including the epidermal and mesophyll layers, decreased. elevating the temperature from 20 to 25°c increased the stomatal density, but the number of stomata sharply decreased when the growth tem­ perature increased from 25 to 30°c. previous study found similar results: the stomatal density of blue­ berry decreased when the temperature exceeded the optimum growth temperature (hao et al., 2019). they suggested that increasing stomatal density may be an efficient strategy for evacuating more heat by evaporative cooling, but this strategy is inefficient under higher temperatures (xu, 2015). it has been reported that a high temperature limits co2 and h2o diffusion, resulting in increased resistance to gas exchange (mukohata et al., 1971; monson et al., 1982). in this study, we found that the transpiration rate (e) of the leaves under the 30°c treatment sharply decreased at four weeks after temperature treatment started, while the e of the leaves under both the 20 and 25°c treatments remained constant or slightly increased. the reduction in e under high­ temperature conditions reflected the low efficiency of leaf cooling. in other words, the convective processes of heat through transpiration were reduced, resulting in excessive leaf temperature above an optimum point. the trees grown at 25°c tended to maintain transpiration cooling by increas­ ing stomatal density, which reduces the negative table 2 ­ fruit quality characteristics of the ‘ku­pp2’ peach trees for each growth temperature treatment z data are mean values ± standard errors (n = 10). the different lowercase letters within the same row indicate significant differences at p≤ 0.05 (tukey’s test). parameter growth temperature p­value 20°c 25°c 30°c fruit weight (g) 164.34 ± 7.48 a z 131.89 ± 6.16 b 97.83 ± 7.05 c < 0.0001 fruit cheek diameter (mm) 68.1 ± 1.2 a 62.1 ± 1.4 b 56.2 ± 1.4 c 0.0002 fruit suture diameter (mm) 69.9 ± 1.3 a 64.0 ± 1.0 b 58.5 ± 1.5 c 0.0002 fruit length (mm) 59.9 ± 0.7 a 57.7 ± 1.0 a 50.0 ± 0.8 b < 0.0001 total soluble solids (°brix) 15.0 ± 0.7 13.8 ± 0.2 13.3 ± 0.6 0.0881 titratable acidity (g l−1) 0.21 ± 0.07 0.29 ± 0.01 0.24 ± 0.03 0.2331 over color extent (%) 10–15 b 10–15 b 60–75 a 0.0013 fruit development period (days) 96 a 91 a 81 b 0.0023 fig. 4 ­ effect of growth temperature on the coloration of the ‘ku­pp2’ fruits during the commercial ripening period. sikhandakasmita et al. ‐ effect of temperature on the low‐chill peach growth 239 effects of excessive heat on their foliage, leading to the maintaining of high e and pn. the higher temperature decreased the concentra­ tion of chl a and chl a+b. as was also observed in this study, chl contents have been reported to be sensi­ tive to high­temperature conditions. the decline in chl pigments may correlate to impaired chl biosyn­ thesis, exacerbated chl breakdown, or both. the inhi­ bition of chl biosynthesis and the increase in chl degradation under high temperature results from the destruction and construction of several enzymes (efeoglu and terzioglu, 2009). additionally, the reduction in chl content observed under high tem­ perature is associated with physical damage to thy­ lakoid membranes by excessive ros accumulation (halliwell and gutteridge, 2007). chl is embedded in the thylakoid membranes; therefore, damage to these membranes could result in chl loss (mathur et al., 2014; chen et al., 2017; jumrani et al., 2017). the imbalance between chl biosynthesis and degradation disrupts the photosynthesis apparatus resulting in decreased photosynthetic efficiency, eventually influ­ encing plant growth and fruit quality (shanshan et al., 2020). changes in pn have been directly linked to the level and duration of high­temperature exposure (hao et al., 2019). in this study, one week after tem­ perature treatment started, the pn of ‘ku­pp2’ increased rapidly with the initial rise in growth tem­ perature; as the forcing condition continued, pn under a moderately high­temperature treatment (30°c) dramatically decreased, whereas the pn under both the 20 and 25°c conditions steadily increased and remained constant until the harvesting period. the response of pn to growth temperature can depend on two factors ­ non­stomatal and stomatal (cui et al., 2006; chen et al., 2014), which can be indicated by the difference in gsw and ci patterns (farquhar and sharkey, 1982). if gsw decreased or stabilized but ci increased, the decline in pn can be attributed to non­stomatal factors. if both gsw and ci decreased simultaneously, pn could be ascribed to stomatal factors. in this study, the increase in pn and ci at the onset of treatment may result from the increase in enzyme activities in the photosynthetic system catalyzed by high temperatures. therefore, an increase in pn in this period could be identified as a non­stomatal factor. furthermore, the decrease in pn under the pro­ longed higher­temperature treatment (30°c) can be divided into two periods: 2­7 weeks and 8­13 weeks after the onset of forcing. for 2­7 weeks, the decrease in pn can be ascribed to a non­stomatal limi­ tation, with gsw significantly decreasing and ci increasing. the non­stomatal factors play a role in the reduction of pn in the 2­7 weeks period after tem­ perature treatment started through damage to the structures of the chloroplast, impairment of chl biosynthesis, and increased chl degradation. this hypothesis is supported by the reduction in chl a and chl a+b observed in this study. with exposure to forcing conditions over an extended period, pn, ci, and gsw of the 30°c treatment gradually decreased and reached their lowest levels in the eighth week after temperature treatment started, indicating that pn in this period might be limited by stomatal factors through changes in stomatal density and modified leaf morphological and anatomical characteristics. our study showed that the size and thickness of the leaves, including the epidermal and mesophyll layers, decreased with the elevated growth temperature, and thus led to the decline in pn as stomatal limita­ tions. the differences in fruit morphological characteris­ tics, such as fruit weight and fruit size of the trees under high­temperature conditions, might be associ­ ated with the decline in the fruit development period and pn. previous studies reported that the relation­ ships between fruit development period (fdp) and fruit weight and diameter were observed in apple and peach (sugiura et al., 2013; giovannelli et al., 2014). in the 30°c treatment, fruit size was lower than those in the 20 and 25°c treatments, which was expected according to the length of their fdp. additionally, previous studies indicated that a low pn causes a steep reduction in fruit size because most of the energy used in fruit development is generated via photosynthesis during the year (pavel and dejong, 1993; grossman and dejong, 1995) similarly, lopez and dejong (2007) reported that high temperature during fruit development increases the potential of fruit growth without enough resources to subsidize fruit growth, resulting in smaller fruit size. high tem­ peratures not only depress photosynthesis but also increase leaf respiration. plants grown under high­ temperature conditions may consume much more energy because of increased leaf respiration caused by increased temperatures (corelli­grappadelli and lakso, 2004; hao et al., 2019). this result is support­ ed by the increase in both the number and size of mitochondria in arabidopsis thaliana, indicating that more starch and soluble sugar are consumed by leaf respiration and rapid growth because of increased temperature (jin et al., 2011). hence, the reduction in fruit size of ‘ku­pp2’ grown under high tempera­ tures may be supported by the above conclusion. in this study, we found that the red coloration in ‘ku­pp2’ peel at 30°c was higher than those at 20 and 25 °c. previous study showed the red coloration in plum (p. salicina lindl.) peel increases under high­ temperature conditions (35°c) (junping et al., 2017). conversely, the biosynthesis of anthocyanin in grape and apple is suppressed by high temperatures (lin­ wang, 2011; mori et al., 2017). junping et al. (2017) showed that high temperatures can stimulate red skin coloration in plum by increasing respiration and ethylene production. long­term forcing under high­ temperature conditions may increase the respiration rate in ‘ku­pp2’ peach fruits, and hence enhance red coloration in the fruit peel. moreover, the develop­ ment of red coloration in peach fruit skin is positively related to light conditions (corelli­grappadelli and coston, 1991; kataoka and beppu, 2004). previous study showed that ‘redhaven’ peach fruits that develop in the shade have less red coloration than those that develop in full sunlight (erez and flore, 1986). in our study, the fruit grown under the 30°c treatment had smaller leaves, leading to a decrease in canopy shade. thus, the peach fruits grown under the 30°c treatment were exposed to more sunlight, which might result in higher red skin coloration. in conclusion, this study illustrated the effect of growth temperature on plant development and fruit quality of ‘ku­pp2’ peach trees. air temperatures directly affect leaf morphology, leaf anatomy, and the photosynthetic ability of plants. the decline in carbon assimilation due to exposure to excessive temperatures could diminish the plant’s ability to efficiently support fruit development, resulting in low yield and poor fruit quality. all these data show high­ temperature stress in the ‘ku­pp2’ peach cultivar caused by long­term exposure to moderately high temperatures. therefore, a better understanding of plant adaptability to high temperatures is crucial for growing low­chill peach cultivars in plastic houses with a heating system. acknowledgements this research was funded by japan society for the promotion of science (jsps) kakenhi, grant number 18k05621. adv. hort. sci., 2021 35(3): 233­241 240 references ashraf a., harris p.j.c., 2013 ­ photosynthesis under stressful environments: an overview. ­ photosynthetica., 51: 163­190. centritto m., brilli f., fodale r., loreto f., 2001 ­ different sensitivity of isoprene emission, respiration and photosynthesis to high growth temperature cou‐ pled with drought stress in black poplar (populus nigra) saplings. ­ tree physiol., 31: 275­286. chen t.w., henke m., de visser p.h.b. buck­sorlin g., wiechers d., kahlen k., stutzel h., 2014 ­ what is the most prominent factor limiting photosynthesis in different layers of a greenhouse cucumber canopy? 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c.donofrio@agr.unipi.it. rapid and certain identification of grapevine clones is of topical interest in modern viticulture, especially with regard to the genetic control of propagation material. in the beginning of the twentieth century, a specific branch of research called “ampelography” was established to identify grapevine varieties, biotypes and clones. the term derives from the greek ampelos, for vine, and graphon, for description, as ampelography is based on visual observation of morphological and phenological characters (viala and vermorel, 1909). currently ampelography is employed by international organizations such as the office international de la vigne et du vin (oiv), the international board for plant genetic resources (ibpgr) and the international union for the protection of new varieties of plants (upov). this type of genotypic identification of grapevines presents, however, some limitations. first and foremost, as the method is based on visual observations it is dependent on the subjectivity of experienced operators (ampelographers). ampelographic description based primarily on observation of the morphology of sprouts, adult leaves and clusters is limited to adult plants during vegetative periods, and for this reason varietal and clonal impurities can only be detected some years after the planting of a vineyard. furthermore, phenotypical changes induced by the cultivation environment, nutritional shortages or possible viral infections often make varietal identification difficult, and sometimes impossible. in fact, for correct varietal identification accessions must grow in the same vineyard and be virus free. however, given the great number of vine varieties (5,000-15,000), even when plants are grouped in a single vineyard, it is extremely difficult to differentiate them all simply on the basis of their morphological and phenological characteristics. thus, the availability of a rapid, practical, objective system to identify vine varieties is desirable to satisfy the need for quality control in nursery production, for legal protection of new selections, and as a fundamental tool to recover autochthonous vines with the aim of preserving biodiversity. in this context, as a support for the classic methods of varietal identification through morphological characters, dna molecular markers are of fundamental importance. among these, microsatellites are the most frequently used due to their high repeatability and reliability, high level of polymorphism and thus the degree of information they can provide, and their co-dominant nature that permits analysis of parentage. in addition, microsatellites have made it possible to develop numerous vine-related databases, including the “database viticolo italiano” (www.vitisdb.it) which is managed by the author of the present abstract. although microsatellites allow rapid and certain identification of vines, they generally do not present sufficient polymorphism to distinguish biotypes and clones, even if some researchers have stated that analyzing an elevated number of microsatellite loci they were able to distinguish clones of pinot and carmenère. amplified fragment length polymorphisms (aflps), a type of molecular marker that uses digestion of dna with restriction enzymes, are much more polymorphic than microsatellites. numerous publications have evidenced the ability of these markers to distinguish clones of various species, however they have not found application for legal purposes in the genotyping of clones due to problems of instability related to the use of restriction enzymes. dna molecular markers seem more stable, making it possible to combine aflp with other markers such as sampl (selective amplification of microsatellite polymorfic loci), m-aflp (microsatellites amplified fragment length polymorphism) and s-sap (sequenze-specific amplification polymorfism). the first two combine aflp with microsatelllites, while the latter combines aflp technique with molecular markers based on transposons insertion polymorphism. in particular, s-sap seems to be more stable than aflp, and recently it was demonstrated that they are efficient in discriminating clones. another recently introduced dna molecular marker for grapevine is single nucleotide polymorphisms (snps). this type of marker has developed rapidly following sequencing of the entire grapevine genome. the resequencing of a large number of vines within the context of a specific european project has made it possible to plan an array which allows rapid, simultaneous analysis of more than 18,000 snps at a reasonable cost. currently, many research groups are testing the utility of this type of marker in clonal discrimination and it is plausible that initial publications may appear by the time the present convention is held. adv. hort. sci., 2013 27(3): 106 265 adv. hort. sci., 2022 36(4): 265­273 doi: 10.36253/ahsc­13352 arbuscular mycorrhizal fungi potentiate the root system and the quality of goldenberry fruits j.l.t. chiomento 1 (*), d. filippi 1, g.m. krasnievicz 2, j.e.c. de paula 2, m. fornari 3, t.s. trentin 2 ¹ postgraduate program in agronomy, university of passo fundo, passo fundo, rs, brazil. ² undergraduate program in agronomy, university of passo fundo, passo fundo, rs, brazil. 3 postgraduate program in agricultural engineering, federal university of santa maria, santa maria, rs, brazil. key words: berry flavor, mycorrhization, physalis peruviana l., root morphology. abstract: the lack of information on the horticultural performance of golden­ berry (physalis peruviana l.) is one of the factors that limits the expansion of the crop. still, aiming to establish a sustainable management for this culture, inoculation with arbuscular mycorrhizal fungi (amf) can be adopted. therefore, the objective of the research was to investigate whether goldenberry plants in the absence and presence of inoculation with amf differ in terms of horticul­ tural performance. the four treatments studied were the absence (control) and the presence of three inoculants based on amf (mycorrhizal community, glomus intraradices and rhizophagus clarus), arranged in a randomized block design, with five replications. goldenberry plants produced in substrate enriched with amf had a more voluminous root system and a greater amount of fine roots. additionally, the fruits were sweeter and more flavorful when produced by plants inoculated with the mycorrhizal community and with r. clarus. it is concluded that mycorrhization has no effect on fruit production. however, goldenberry plants submitted to mycorrhizal biotechnology enhance the chemical quality of fruits and present a more profuse root system. g. intraradices is most effective in colonizing the roots of the plant host. 1. introduction goldenberry (physalis peruviana l., solanaceae) is a horticultural crop native to the andean highlands that has attracted worldwide attention due to its bioactive compounds such as carotenoids, physalins and polyphenols (ramadan, 2011). in addition to promoting the health of con­ sumers, these biomolecules present in fruit extracts have antifungal action against phytopathogens, such as botrytis cinerea pers., and there­ fore can be widely used in agriculture as a bioinput (filippi et al., 2020). still, goldenberry stands out for its potential for intensive cultivation (*) corresponding author: jose­trevizan@hotmail.com citation: chiomento j.l.t., filippi d., krasnievicz g.m., de paula j.e.c., fornari m., trentin t.s., 2022 ­ arbuscular mycorrhizal fungi potentiate the root system and the quality of goldenberry fruits. ­ adv. hort. sci., 36(4): 265­273. copyright: © 2022 chiomento j.l.t., filippi d., krasnievicz g.m., de paula j.e.c., fornari m., trentin t.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/ index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 july 2022 accepted for publication 6 october 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13352 adv. hort. sci., 2022 36(4): 265­273 266 (etzbach et al., 2018). a single plant can produce 300 fruits and the productivity of this horticultural crop can reach from 20 to 33 tons per hectare (yildiz et al., 2015). despite the traditional establishment of crops in the open field (muniz et al., 2014), the goldenberry cultivation in greenhouse is increasing (aguilar­ carpio et al., 2018). this is because greenhouse culti­ vation can help to avoid inconveniences such as pests, diseases, rain, strong winds, hail and frost (costa et al., 2016). similar to the traditional cultivation of other solanaceae, such as tomato and pepper, in order to obtain an optimal productive yield of goldenberry, producers need to use a large amount of chemical inputs, which can contaminate the agroecosystem of cultivation (chiomento et al., 2020 a). there is no doubt, therefore, that the establishment of agroeco­ logical agriculture is an important tool for sustainable food production, with environmental and socioeco­ nomic benefits (llano et al., 2018). thus, an alterna­ tive to minimize the inconveniences in the cultivation of goldenberry and start the establishment of sus­ tainable management in this horticultural culture corresponds to the use of inoculants based on arbus­ cular mycorrhizal fungi (amf). as there is a limitation regarding the availability of commercial amf­based inoculants available in brazil (trentin et al., 2022), this reduces the use of mycorrhizal biotechnology in the production of goldenberry due to the lack of knowledge of this bioinput by the producers. the lim­ itation regarding the availability of commercial inocu­ lants is mainly due to the high cost linked to the pro­ duction technology of this bioinput. amf (phylum glomeromycota), a ubiquitous group of soil microorganisms, establish symbiotic associations with more than 70% of vascular plants (brundrett and tedersoo, 2018). the literature reporting the association between mycorrhiza and goldenberry is scarce. for example, under saline con­ ditions, amf increased fruit growth rate (miranda et al., 2011) and improved berry unsaturated fatty acid concentration in response to heavy metal stress (hristozkova et al., 2017). under water stress, arbus­ cular mycorrhiza increased root dry matter accumu­ lation and improved attributes related to plant gas exchange (reyes et al., 2019). in non­stressful envi­ ronments, it was found that goldenberry plants sub­ jected to mycorrhizal biotechnology produced less acidic and tastier fruits (chiomento et al., 2020 a). this scarcity of information demands more research to fill the existing gaps regarding the morpho­horti­ cultural performance of goldenberry and their inter­ active effects with mycorrhizas. in brazil there is only one commercial amf inocu­ lant available to farmers. the commercial scale pro­ duction of this bioinput has high costs linked to the inoculum production technology, such as the estab­ lishment of cultures of amf species and transport, handling and development of the carrier substrate (schlemper and stürmer, 2014). to avoid some of these costs, on‐farm production of inoculants is used, with indigenous or exotic amf isolates, in which the technology can be easily transferred to farmers (douds junior et al., 2012). the process of obtaining the on‐farm inoculant can be started using amf infective propagules, such as spores, hyphae and parts of colonized roots (douds junior et al., 2010). therefore, based on the hypothesis that mycor­ rhizal biotechnology enhances plant host growth and improves fruit chemical quality, here we investigate whether the horticultural performance of goldenber­ ry is influenced by the use of amf­based on‐farm inoculants. 2. materials and methods plant material the research was carried out in passo fundo (28° 15’ 46” s, 52° 24’ 24” w), rio grande do sul (rs), brazil, in greenhouses, from august (winter) 2018 to july (winter) 2019 . a commercial tray with goldenberry fruits at mat­ uration stage 5 was purchased (icontec, 1998). in august 2018, seeds from three randomly chosen fruits were selected, transferred to paper towels and kept at room temperature until dry. subsequently, these seeds were germinated in plastic gerbox boxes containing blotting paper and 0.1 molar (m) potassi­ um nitrate (kno3) solution. the boxes were stored in a biochemical oxygen demand (bod) oven, at 25°c±1°c, until the plants were obtained for the pro­ duction of seedlings, which constituted the plant material for the research. the steps for obtaining the plants are shown in figure 1. experimental design the four treatments studied were the absence (control) and the presence of three inoculants based on amf [mycorrhizal community, glomus intraradices n.c. schenck & g.s. mr. and rhizophagus clarus (t.h. nicolson & n.c. schenck) c. trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 267 walker & a. schüßler], arranged in a randomized block design with five replications. each plot consist­ ed of three goldenberry plants. the amf community used came from the crop­ trap of agricultural soil collected at a reference site for strawberry cultivation in the municipality of são josé do hortêncio (29° 29’ 33” s, 51° 12’ 24” w), rio grande do sul state, brazil (chiomento et al., 2019 a), composed of ten fungal species according to the classification of glomeromycota proposed by redecker et al. (2013): acaulospora foveata trappe & janos, claroideoglomus aff. luteum, claroideoglomus claroideum (n.c. schenck & g.s. sm.) c. walker & a. schüßler, claroideoglomus etunicatum (w.n. becker & gerd.) c. walker & a. schüßler, funneliformis aff. geosporum, funneliformis aff. mosseae, funneli‐ formis mosseae (t.h. nicolson & gerd.) c. walker & a. schüßler, glomus aff. versiforme, glomus sp. (cae­ saris like) and glomus sp2. the isolate g. intraradices came from the commercial product myke® pro and the isolate r. clarus was obtained from the international collection of glomeromycota culture (cicg). cultivation procedures we applied the treatments (amf) in two stages: 1) in the acclimatization of the seedlings; 2) in trans­ planting to the place of cultivation. thus, of the total amount of mycorrhizal inoculant used (10 g), we applied 5 g in the acclimatization of the seedlings and 5 g at the time of transplanting. in september (spring) of 2018, thirty days after the germination of goldenberry seeds, the plants obtained (fig. 1d) were acclimatized in 72­cell poly­ styrene trays, filled with the sterilized horta 2® sub­ strate (120°c for 20 minutes) and with treatments related to mycorrhization (1/2 of the total amount), with the purpose of seedling production. horta 2® is composed of pine bark, vermiculite, acidity correc­ tors and fertilizers (nitrogen, phosphorus and potas­ sium) in amounts not supplied by the manufacturer. a 500 g sample of the substrate was analyzed to obtain its physical (brazil, 2007) and chemical (mapa, 2014) attributes (table 1). the trays were kept on metal benches, 1.2 m from the ground surface, in a greenhouse (90 m2), installed in the northeast­southeast direction, with a semicir­ cular roof. the galvanized steel structure is covered with a low­density polyethylene film with anti­ultravi­ olet additive (150 micron thickness) and the sides are covered with an anti­aphid screen. the irrigation used during acclimatization was with sprinklers (1.8 l.min­1 per unit), in the mechanized system. the irri­ gation regime consisted of activating the sprinklers seven times a day, with total wetness of 14 minutes. the water depth supplied to the seedlings was 7.8 mm.day­1. in december (summer) 2018, after three months of acclimatization, the seedlings were transplanted fig. 1 ­ obtaining goldenberry plants. (a) separation of seeds from fruits. (b) selection of seeds. (c) germination in ger­ box. (d) plants produced. substrate physical properties d (kg m­3) tp (m3.m­3) ae (m3.m­3) raw (m3.m­3) bw (m3.m­3) rw (m3.m­3) horta 2 ® 241 0.837 0.303 0.149 0.020 0.365 chemical properties n % (m.m­1) p2o5 (m.m­1) k2o % (m.m­1) oc % (m.m­1) ph ec % (ms.cm­1) cec (mmolc.kg­1) 0.36 0.39 0.00 12.60 6.1 0.45 278.60 table 1 ­ physical and chemical properties of the horta 2® substrate (z) d= density; tp= total porosity; ae= aeration space; raw= easily available water; bw= buffer water; rw= remaining water. (w) n= nitrogen; p2o5 = phosphorus pentoxide; k2o= potassium oxide; oc= organic carbon; ph= hydrogen potential; ec= electric conducti­ vity; cec= cation exchange capacity. adv. hort. sci., 2022 36(4): 265­273 268 into pots (3.6 l), filled with sterilized horta 2® (120°c for 20 minutes) and complemented with the other part of the treatments related to mycorrhization (1/2 of the total amount). the pots were kept in beds cov­ ered with mulching, in a greenhouse (430 m2), with a semicircular roof, installed in the northeast­south­ east direction. the galvanized steel structure was covered with a low­density polyethylene film (150 microns thick) and with an anti­ultraviolet additive. localized irrigation was carried out using drip rods (2.4 l.h­1 per unit), in the mechanized system. the irrigation regime consisted of activating the dripping rods six times a day, with total wetting for six min­ utes. the nutrient solutions supplied to the plants, fortnightly, were made according to furlani and fernandes júnior (2004), but with a 50% reduction in phosphorus supply. through a mini meteorological station, we verified that the average general temper­ ature recorded during the experiment was 25.66°c. the plants were conducted with three stems and were tutored with the aid of wires. no biocides were used during the crop cycle. the evaluations started after the fruiting of the plants, in february (summer) of 2019. we evaluated the root system morphology and the productive yield (number and weight) and quality of fruits. root system morphology at the end of the experiment, in july 2019, the plants roots were washed in water to eliminate sub­ strate fragments. the roots were digitized by a scan­ ner and the images obtained were analyzed by the winrhizo® software. the attributes evaluated were total length (tl, cm), surface area (sa, cm2) and vol­ ume (v, cm3). the roots were grouped by the soft­ ware into different diameter classes in relation to their total length (böhm, 1979): very thin (vt, ø<0.5 mm), thin (th, ø from 0.5 to 2 mm) and thick (tk, ø>2 mm). to verify the infective capacity of amf, root por­ tions of mycorrhizal plants were prepared according to phillips and hayman (1970) and their percentage of mycorrhizal colonization (mc) was determined according to trouvelot et al. (1986), by the equation: mc(%) = (total number of fragments with mycorrhizal roots) x 100 (total number of fragments) (1) fruit production from fruiting, in february 2019, the total number of fruits (tnf, number per plant) and the total pro­ duction of berries (tp, grams per plant) were evaluat­ ed. in addition, the average fresh fruit mass (affm, grams) was evaluated. the fruits were harvested when they were in the stages of maturation between 4 and 6 (icontec, 1998). the fruits were weighed on an electronic digital scale. chemical fruit quality the analysis of fruit quality was performed at the end of the experiment, in july 2019. the chemical characteristics of the fruits were evaluated regarding the content of total soluble solids (tss, %) and total titratable acidity (tta, % of citric acid), from 20 fruits of each treatment for each repetition. the tss con­ tent was determined in an analog refractometer, and the tta was performed according to the norms of the adolfo lutz institute (zenebon et al., 2008). to evaluate the flavor of the fruits, the tss/tta ratio was determined. data analysis the data obtained were submitted to analysis of variance (anova) and the averages of the treatments were compared by the tukey test, at 5% error proba­ bility, with the aid of the costat® program (cohort software, 2003). 3. results root system morphology we verified a significant effect of mycorrhizal inoculants only for the attributes mc, v and th. g. intraradices had a greater ability to infect plant roots than the mycorrhizal community and r. clarus (fig. 2a). the fungal structures identified inside the roots of goldenberry plants were hyphae, vesicles and arbuscules. in addition, plants inoculated with r. clarus had 41% and 42% greater root volume than non­mycorrhizal plants and those inoculated with the amf community, respectively (fig. 2b). also, plants inoculated with the mycorrhizal community had a greater amount of fine roots (+47%) compared to the control (fig. 2c). fruit production we observed a positive effect of treatments only for the affm attribute. non­mycorrhizal plants pro­ duced fruits with higher average fresh mass (+29%) compared to plants inoculated with the mycorrhizal trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 269 community, but did not differ from plants mycor­ rhizal with g. intraradices and r. clarus (fig. 3). chemical fruit quality mycorrhizal inoculants influenced tss and tss/tta attributes (fig. 4). sweeter (fig. 4a) and tastier (fig. 4b) fruits were produced by plants inocu­ lated with the mycorrhizal community and with the isolate r. clarus. 4. discussion and conclusions here, we show that goldenberry plants in the absence and presence of amf inoculation differed in horticultural performance. in the first productive cycle of the plants, we did not observe the effect of mycorrhization on the production of berries. we believe that in goldenberry the fungal species used require a period of more than one year to benefit the fruit yield, as occurs in strawberry (fragaria x ananassa duch.) cultivation (robinson­boyer et al., 2016). however, goldenberry plants inoculated with amf showed a more profuse root system. fig. 2 ­ root system morphology of goldenberry plants in the presence and absence of amf inoculation. (a) mycorrhizal colonization (%). (b) root volume (cm³). (c) amount of fine roots (cm). data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). fig. 3 ­ average fresh fruit mass (grams) of goldenberry plants in the presence and absence of amf inoculation. data pre­ sented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). fig. 4 ­ chemical quality of goldenberry fruits in the presence and absence of amf inoculation. (a) total soluble solids (%). (b) fruit flavor. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). (abbott and robson, 1981), which limits the plant’s response to mycorrhization (lambais and cardoso, 1990). although mycorrhizal colonization is impor­ tant, the percentage of root infectivity is not always correlated with the efficiency of symbiosis (konvalinková and jansa, 2016). this lack of relationship between infectivity and amf efficiency in improving crop growth may be related to the time required for the establishment of root colonization (abbott and robson, 1981). under long­term, arbuscular mycorrhiza promotes more benefits to the plant host (ortas, 2012), mainly by increasing the acquisition of water and minerals to the plant. the major function of amf is suggested to be nutrient acquisition (zhang et al., 2019) and thus, under high nutrient conditions, amf can shift from a net benefit to a cost for the host (johnson et al., 2015). in our study, the nutrients supplied during cul­ tivation were not limited to goldenberry; therefore, the inoculation effect may not have been potentiated in terms of fruit yield. however, we proved the benefit of mycorrhiza­ tion on the chemical quality of the fruits through the increase in the sugar content (fig. 4a) and the better berry flavor (fig. 4b), as already reported for zucchini (cucurbita pepo l.) (rouphael et al., 2015), strawber­ ry (costa et al., 2020) and tomato (solanum lycoper‐ sicum l.) (sellitto et al., 2019). plants grown with amf showed a more developed root system (fig. 2), which allows extrarradicial hyphae to extend beyond the rhizosphere, making water acquisition more effi­ cient (xu et al., 2017). due to greater water availabili­ ty, these plants have greater stomatal opening, which increases the rate of transpiration and, thus, there is a greater supply of carbon dioxide for photo­ synthesis (vicente­sánchez et al., 2014). as a result, there is a greater production of sugars, which are the primary source of photosynthesis, and this explains the increase in the fruit sugar content and, conse­ quently, the best flavor of the berries (fig. 4). the lack of effect of g. intraradices on berries quality can be attributed to the low effectiveness of this fungal species on goldenberry. various factors modulate the arbuscular mycorrhiza effect on the performance of their associated plants and this includes the traits of the host and the fungi them­ selves (chiomento et al., 2019 a). cultivated plants vary in their responsiveness to amf due to their mor­ phology (chiomento et al., 2019 b) and amf differ in the benefits provided to the plant (werner and kiers, 2015). 270 adv. hort. sci., 2022 36(4): 265­273 furthermore, the use of the mycorrhizal community and r. clarus allowed to harvest fruits with better chemical quality. this indicates that the fungal species present in these two inoculants have an affin­ ity for this horticultural crop. the use of amf com­ patible with the host commonly provides more satis­ factory results (chiomento et al., 2022). g. intraradices was more effective in colonizing the roots of the plant host. however, the mycorrhizal community tested in this study stood out in relation to the fungal isolates for generally improving the hor­ ticultural performance of goldenberry. we verified that the root system of the mycor­ rhizal plants was more profuse, with greater volume (fig. 2b) and with a greater amount of fine roots (fig. 2c). this benefit to the roots has already been reported by reyes et al. (2019), who demonstrated that mycorrhization in goldenberry under water stress increased the accumulation of root dry matter. due to the plasticity of the roots, their characteristics can be modulated by several factors, including amf (hodge et al., 2009). during the establishment of the association between host and fungus, many molecu­ lar signals are initiated, including an amf (lipo­chito­ oligosaccharides) diffusible factor called “myc fac­ tor”, which stimulates the formation of finer roots (oláh et al., 2005), altering the morphology of the root system of plants. in addition, root modifications under mycorrhization may be related to the alloca­ tion of sugars to roots (wu et al., 2011) and hormon­ al regulation (zou et al., 2017), independent of sym­ biotic signaling (gutjahr, 2014). the more fine roots there are in mycorrhizal plants, the better their acquisition of water and nutrients (chiomento et al., 2021), as these roots are the ones that most acquire and use the available resources in the plant growth medium (costa et al., 2019). differently from what was expected, the produc­ tive performance of goldenberry was higher when the plants were not mycorrhized (fig. 3). this sug­ gests that amf initially demand carbon from the host for their maintenance and only later repay this bene­ fit to the plant symbiont. these results, however, contradict the literature. for example, miranda et al. (2011) reported benefits of amf in the production of goldenberry, under limiting conditions to plants, which were subjected to abiotic stresses, which did not happen in our study. there is not always a high relationship between fungal infectivity and efficiency in promoting crop growth due to the time required to establish bidirectional flow between symbionts trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 271 the two genera that made up the monospecific inoculants, glomus and rhizophagus, have already been reported in studies of amf diversity in golden­ berry (ramírez­gómez et al., 2019) and have also been used in applicability studies (rhizophagus) in this horticultural crop (miranda et al., 2011). however, in our study, the mycorrhizal community, representing a multispecific inoculant, stood out in relation to the fungal isolates for generally improving the horticultural performance of goldenberry, mainly by increasing the amount of fine roots produced by the plants (fig. 2c) and for benefiting fruit quality (fig. 4). inoculation with amf populations, such as the fungal community used in this study, generally provides more satisfactory results due to greater compatibilities at the fungus­host interface and by increasing mutualistic effects with two or more sym­ bionts instead of just one (chiomento et al., 2019 b). this mycorrhizal community was obtained through the trap culture technique, that is, produced on‐ farm. a mycorrhizal inoculant rich in propagules and produced on‐farm may be a suitable solution for large­scale inoculation of crops (agroecosystems), seedlings (nurseries) and in potting media for veg­ etable growers (soilless cultivation) (douds junior et al., 2006). the use of non­sterilized growth medium (soil and/or substrate) as a component of the on­ farm inoculant represents a source of propagules for other amf present in this growth medium, which results in an inoculant with greater taxonomic diver­ sity (schlemper and stürmer, 2014). this is strongly desired due to the functional diversity exhibited by mycorrhizal species for the promotion of plant growth, for example (chiomento et al., 2020 b). furthermore, a diversified inoculant potentiates a combination between fungal isolates and an eventual host (douds junior et al., 2006). therefore, the results of our research confirmed the potential of applying mycorrhizal biotechnology to goldenberry, as the work demonstrated that amf can be a valuable tool for the cultivation of this veg­ etable. the complex roles of amf in agroecosystems are just beginning to be understood (chiomento et al., 2022). in this way, a greater understanding of the application and benefits of amf can enable their use in the sustainable production of vegetables (trentin et al., 2022). we conclude that in the first production cycle, there is no effect of mycorrhization on the total num­ ber of fruits and total production of berries. we believe that for the goldenberry cultivation the fun­ gal species used require a period of more than one year to benefit the fruit yield. on the other hand, goldenberry plants submitted to mycorrhizal biotech­ nology have a more profuse root system and produce fruits with better chemical quality. g. intraradices is most effective in colonizing the plant host roots. however, the mycorrhizal community stands out in relation to the fungal isolates for generally improving the horticultural performance of goldenberry. thus, the application of this biotechnological tool in the goldenberry culture can be an alternative to spread and promote its sustainable cultivation. acknowledgements amf used in this work are regulated by sistema nacional de gestão do patrimônio genético e do conhecimento tradicional associado (sisgen) of the ministry of the environment, brazil, according to the registration number a198f50. references abbott l.k., robson a.d., 1981 ­ infectivity and effective‐ ness of vesicular arbuscular 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[anamorph: bipolaris sorokiniana (sacc. in sorok.) shoem.] is an ascomycetous fungus that causes spot blotch (sb) of barley, hordeum vulgare l., a disease responsible for large economic losses in barley-growing areas (mathre, 1990). although the production of conidia is expected to produce genetically identical clones, the high rate of appearance of new races with the ability to infect previously resistant varieties of barley suggests that c. sativus may have high mutation rates in avirulence genes, which determine race (kumar et al., 2002). general symptoms of sb include light brown lesions with whitish gray centers and chlorotic margins (kumar et al., 2002). most varieties grown around the world are susceptible to c. sativus, although partial resistance has been reported (arabi, 2005; zhou and steffenson, 2013). genetic control of sb resistance is governed by quantitative traits. two quantitative trait loci (qtls) have been mapped to chromosomes 1s and 5s (steffenson et al., 1996). however, in the case of the host-specific toxin produced by c. sativus, the fungus which incites sb of barley, the toxin will produce all the symptoms characteristic of the disease; sensitivity to the toxin is correlated with susceptibility to the pathogen and toxin production by the pathogen is directly related to its ability to cause disease (kumar et al., 2002). although the most effective control strategy for sb is cultivating resistant varieties, it has often achieved only short-term success due to the frequent breakdown of newly introduced resistance (poudyal et al., 2005; gontariu and enea, 2012). this resistance breakdown has been attributed to genetic variability in c. sativus (gilchrist et al., 1995). different mechanisms have been suggested to explain the frequent generation of race variants, including heterokaryosis, parasexuality and mutations (kumar et al., 2002; arabi and jawhar, 2007). however, the activity of the retrotransposon microsatellite amplified polymorphism (remap) is another possible mechanism that has been suggested which uses 1 ltr primer in combination with a primer designed for annealing at the 3’ end of a stretch of a simple sequence repeat (ssr) and detects retrotransposons inserted near ssrs (chadha and gopalakrishna, 2005; biswas et al., 2010). our overall question was how stable c. sativus isolates would be both genotypically and phenotypically during several serial transfers in one growing season. therefore, the objective of this work was (i) to determine phenotypic variation of the two major pathotypes of c. sativus in syria, pt1 and pt4 during seven serial transfers on barely plants and (ii) to investigate genetic variation of the isolates collected in each generation using dna fingerprinting. cultural and genetic evaluation of cochliobolus sativus during successive passages through susceptible barley m.i.e. arabi, m. jawhar (1) department of molecular biology and biotechnology, aecs, damascus, syria. key words: cochliobolus sativus, hordeum vulgare, phenotypes, spot blotch. abstract: the objective of this work was to assess the stability of retrotransposons dna elements and several key phenotypic traits important for virulence of cochliobolus sativus after serially transferring through susceptible barley plants. a significant increase in virulence was observed in offspring isolates generated from the aggressive isolate pt4, in contrast to the lack of significant changes in those obtained from the weakly aggressive isolate pt1 after seven successive passages. no apparent differences in phenotypes, including mycelial growth, conidiation and conidial germination were observed among isolates from the same parent isolate on artificial medium. based on retrotransposon microsatellite amplified polymorphism (remap), parents and their generations were identical during the serial transfers. taken together, our results suggest that all singleconidials of the parents and their generations were stable genotypically during seven serial transfers with a change in virulence of the aggressive isolate generations. adv. hort. sci., 2014 28(3): 119-122 (1) corresponding author: ascientific@aec.org.sy received for publication 9 june 2014 accepted for publication 15 september 2014 120 2. materials and methods fungal isolates for the inoculation process, two major pathotypes of c. sativus in syria, pt1 and pt4, were used in this study. they were identical in spore morphology and colony colour, but differed widely in dna patterns and virulence. after extensive greenhouse and laboratory screening over a 10-year period, pt4 was proven to be the most virulent isolate to all barley genotypes available so far (arabi and jawhar, 2003; 2007), therefore it was used in this study. each isolate was grown separately in 9 cm petri dishes containing potato dextrose agar (pda, difco, detroit, mi. usa) and incubated for 10 days, at 22±1°c in the dark to allow mycelial growth. inocula preparation, serial transfer and isolation methods after culturing pt1 and pt4 isolates on pda medium, spores were collected by flooding each plate with 10 ml sterile water with 200 ppm of tween 20, filtering through cheesecloth to remove mycelium and adjusting the concentration of the spore suspension to 2x104 conidia/ml using hemacytometer counts of conidia. the universal susceptible control (cv. wi2291) plants from australia were grown in pots filled with sterilized peatmoss, and arranged in a randomized complete block design with three replicates. each experimental unit consisted of 10 seedlings. a full replicate consisted of 10 pots inoculated with pt1 and pt4 isolates. pots were placed in a growth chamber at temperatures of 22±1°c (day) and 17±1°c (night) with a daylength of 12 h and a relative humidity (rh) of 80-90%. plants were inoculated at growth stage (gs) 12 (zadoks et al., 1974) by uniformly spraying each plant with 20 ml of conidial suspension with a hand-held sprayer. plants were then placed in the dark at 95-100% r.h. for the first 18 h. pt1 and pt4 isolates and single-conidial isolates from each of the three replicates from the 1st to 7th passage generations through barley plants were compared for virulence. ten days after inoculation, one leaf per plant, for a total of three leaves per replicate, was sampled, surface sterilized and placed on a water agar plate. three days later, a single conidium from each of the three plates was transferred to pda for genotypic and phenotypic assays. the infection response based on the measurement of individual lesion size (dimension; mm) for each second leaf was assessed 10 days after inoculation according to fetch and steffenson (1999) scale. each leaf was assessed separately and the assessments were performed by the same person in all experiments. in vitro phenotypic assays in vitro phenotypic assays were achieved by transferring plugs of mycelim (5 mm diameter) of each parent and generations onto pda media and incubation at room temperature (22±1°c) in the dark. conidial germination rate was recorded after 24 h on glass cover slips as described previously (arabi and jawhar, 2001). colony diameter was measured seven days post incubation. conidia were harvested from 15-day cultures using sterile distilled water and counted with a hemocytometer. all the experiments were repeated three times with five replicates, and a representative set of data is presented. statistical analysis was performed using the stat-itcf program (anonymous, 1988). remap analysis dna extraction from parent and generation isolates was performed according to standard protocols (leach et al., 1986). remap analysis and primer sequences were achieved using a standard method described by kalendar et al. (1999). pcr reactions were performed in 25 μl reaction volume containing 1× taq polymerase buffer (10 mmol tris-hcl/l (ph 8.3), 50 mmol kcl/l, 2.5 mmol mgcl2/l, 0.01% gelatin), 0.5 u taq polymerase (eppendorf, germany), 150 μmol of each dntp/l, 0.4 μmol ltr1 primer/l, 0.6 μmol of issr primer/l (table 1), and 50 ng of template dna. a pcr was carried out in an eppendorf dna thermal master gradient cycler (eppendorf nethelerhinz, hamburg, germany). the amplification conditions were as follows: 92°c for 5 min, followed by 40 cycles of 92°c for 45 s, 55°c for 45 s, and 72°c for 1 min; and a final extension step of 72°c for 10 min. amplified products were electrophoresed in a 2% agarose gel using 1 × tris-borate-edta buffer (100 mmol tris-hcl/l, ph 8.3, 83 mmol boric acid/l, 1 mmol edta/l) at 100v. the gels were stained with ethidium bromide solution and visualized under ultraviolet illumination. sizes of the amplified products were determined relative to a 100-bp dna ladder (mbi fermentas, york, uk). 3. discussion and conclusions disease symptoms (presence of necrosis and chlorosis) were severe on the susceptible genotype wi2291 that was infected with pathogenic isolates after 10 days of inoculation. pt1 and their generations induced small round to oblong dark brown necrotic lesions, whereas, pt4 and their generations induced solid dark brown necrotic lesions with expanding chlorosis (the ‘classic’ spot blotch lesion) in highly compatible interactions. as offspring isolates from the most diseased plants were serially passaged table 1 remap primers used in the study primer no. sequence 1 (ga)8t+tgtttcccatgcgacgttccccaaca 2 (ag)8t+gcatcaaaggcattggaggtg 3 (ga)8t+ gcatcaaaggcattggaggtg 4 (ga)8t+cactagtgattcattatgctgagtg 5 (ag)8t+gcatcaaaggcattggaggtg 6 (ag)8t+ cactagtgattcattatgctgagtg 7 (ag)8g+ccaatggactggacatccgatggg 8 (ag)8t+ tgtttcccatgcgacgttccccaaca 9 (ag)8g+ tgtttcccatgcgacgttccccaaca 121 on barley, virulence of the aggressive isolate pt4 was increased during seven transfers on plants, and the ability of weakly aggressive isolate pt1 to maintain its virulence was observed (table 2). all pt4 isolates were highly virulent to cv. wi2291 with a mode of 4 (typifying >90% of the lesions observed on leaves), whereas, all pt1 isolates were virulent exhibiting a mode of 2 (typifying >10% of the lesions observed on leaves), indicating that virulence was not significantly affected. infection responses of wi2291 to c. sativus isolates pt1 and pt4, and their generations are summarized in table 2. the pattern of continuous incremental increase in virulence has been reported in f. oxysporum f. sp. ciceris, on chickpea (jiménez-gasco et al., 2004), and in magnaporthe oryzae on rice (park et al., 2010). however, the pathosystems involving the genera of cochliobolus and the involvement of host-specific toxins in pathogenicity and virulence are well documented (olbe et al., 1995). the number of sb lesions (presence of necrosis and chlorosis) were always high in the virulent isolate pt4 during the serial passages (fig. 1). this can be attributed to a higher proportion of pt4 spores being able to establish lesions than pt1 spores. this indicates that the more virulent pt4 causes more lesions (per leaf) from a given inoculum dose than pt1 as presented in figure 1. the existence of pathotypes expressing differential virulence on host genotypes is uncommon in species related to c. sativus. differential reactions, such as those expressed by the pathotypes pt1 and pt4 on two-rowed genotypes (wi 2291), usually are a feature of gene-for-gene interactions (flor, 1956) or an incompatibility system (briggs and johal, 1994). a hypothesis that these two differentially virulent c. sativus isolates are pathotypes that produce two different types of host-specific toxins may also be valid. additionally, no significant differences were observed between the parental isolates and any isolates derived from them through the seven serial transfers for any of the phenotypic characters tested in vitro including mycelial growth, conidiation and conidial germination on pda media (fig. 2). these results are similar to those of latterel and rossi (1986), who reported no changes in cultural characteristics in magnaporthe oryzae isolates after serial transfers of the same isolates from stock cultures over a long period of time. to evaluate genotypic stability during serial transfers, remap fingerprinting technique was used. the remap haplotypes of seven generations were identical to their parent isolates (fig. 3); this might be an indicator of the stability of transposable elements during serial passages. the lack of molecular variation in offspring of pt4 isolates with increased virulence (table 2; fig. 3) is consistent with the fact that mutation rates at virulence loci are higher than those at the molecular loci that define genotypes (goodwin et al., 1995). the results of this study demonstrate that the virulence of the offspring isolates generated from the aggressive isolate pt4 significantly increased after seven successive table 2 infection responses of barley cv. wi 2291 infected with parents and progeny isolates of cochliobolus sativus based on the scale of fetch and steffenson (1999) isolates infection response parent passages 1 2 3 4 5 6 7 pt1 mode (z) 2 1 2 2 2 2 1 1 range (y) 1-2 1-2 2 1-2 1-2 1-2 1-2 1-2 pt4 mode 4 4 4 4 4-5 4 4 4-5 range 7-9 6 7 7-8 8-9 9 9 9 (z) mode= the most common infection response observed on the barley cv. wi2291. (y) range= the lowest and highest infection responses observed on the barley cv. wi2291. fig. 1 number of lesions caused by pt1 and pt4 isolates after seven serial transfers barley cv. wi2291. fig. 2 cultural characterization of two c. sativus isolates pt1 and pt4 after seven passages on pda medium and through barley plants. fig. 3 agarose gel electrophoresis of remap analysis pt1 and pt4 parent isolates after seven passages through barley cv. wi2291 using primer; (ga)8t+gcatcaaaggcattggaggtg. m–marker ladder 1kb. 122 passages, in contrast to the lack of significant changes in those obtained from the weakly aggressive isolate pt1. no apparent differences in phenotypes, including mycelial growth, conidiation and conidial germination, were observed among isolates from the same parent isolate on artificial medium. all single-conidial of the parents and their generations were stable genotypically during the serial transfers with a change in virulence of the aggressive isolate generations. acknowledgements the authors thank the director general of aecs and the head of biotechnology department for their help throughout the period of this research. references anonymous, 1988 stat-itcf, programme, microsta, realized by ecosoft, 2nd ver. institut technique des cereals et des fourrages, paris, pp. 55. arabi m.i.e., 2005 inheritance of partial resistance to spot blotch in barley. plant breeding, 124: 605-607. arabi m.i.e., jawhar m., 2001 the response of cochliobolus sativus to ultraviolet c radiation. journal of phytopathology, 149: 521-525. arabi m.i.e., jawhar m., 2003 pathotypes of cochliobolus sativus (spot blotch) on barley in syria. journal of plant pathology, 85: 193-196. arabi m.i.e., jawhar m., 2007 molecular and pathogenic variation identified among isolates of cochliobolus sativus. australasian plant pathology, 36: 17-21. biswas m.k., xu q., deng x.x., 2010 utility of rapd, issr, irap and remap markers for the genetic analysis of citrus spp. science horticulture, 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spot blotch (cochliobolus sativus) in barley. theoretical of applied genetics, 92: 552-558. zadoks j.c., chang t.t., konzak c.f., 1974 a decimal code for the growth stages of cereals. weed research, 14: 415-421. zhou h., steffenson b., 2013 genome-wide association mapping reveals genetic architecture of durable spot blotch resistance in us barley breeding germplasm. molecular breeding, 32: 139-154. impaginato 35 adv. hort. sci., 2020 34(1s): 35­42 doi: 10.13128/ahsc­8055 the residues of fruit and vegetable pro­ cessing: from “waste” to “resource” of natural phytochemical compounds s. bartolini 1, m. orlando 1, a. trivellini 1 (*), f. venturi 2, c. sanmartin 2, i. taglieri 2, m. macaluso 2, a. zinnai 2, a. mensuali­sodi 1 1 istituto di scienze della vita, scuola superiore sant’anna, piazza martiri della libertà, 33, 56127 pisa, italy. 2 department of agriculture, food and environment, university of pisa, via del borghetto 80, 56124, pisa, italy. key words: apple, bioactive products, minimally processed, natural additives potato. abstract: the project of sant’anna school, in line with the italian legislation on limiting waste and promoting the redistribution of surpluses and unused goods, aimed to study the potential healthy value of residues obtained from the trans­ formation of fruit and vegetable products that represent a cost, as they must be handled, stored and disposed according to stringent actual regulations. two “model” species (potato and apple) were considered to test the possibility of using industrial processing waste for food applications. the extracts, obtained with “green” methods from potato and apple peels, were evaluated as natural antioxidants in the preparation of minimally processed fresh­cut apple. results suggest the possibility to use these novel byproduct extracts as valuable alter­ native treatments to traditional chemical additives employed for minimally processed apples. 1. introduction the italian law n. 166 (2016) defined as “antisprechi” has been formed with the aim of limiting waste, while promoting the redistribution of sur­ pluses and unused goods. this law relies on two fundamental principles guaranteed by italian constitution: subsidiarity and solidarity. for the first time, it formally defines the terms “waste” and “surplus”, constituting a reference point for dialogues between different institutions opening up new perspective for investigation on new products recovered from byproducts. in agreement with these regulations, the italian ministery of agriculture (mipaaft) established financial contributions to support innov­ ative projects, related to research and technological development, in the field of food shelf life and packaging, which ensure a concrete application of the results achieved. the project of sant’anna school was one out of 10 selected and financed by mipaaft in 2017. the italian food industry is constantly growing, with an estimated (*) corresponding author: alice.trivellini@gmail.com citation: bartolini s., orlando m., trivellini a., ven­ turi f., sanmartin c., taglieri i., macaluso m., zinnai a., mensuali­sodi a., 2020 ­ the residues of fruit and vegetable processing: from “waste” to “rosource” of natural phytochemical compounds. ­ adv. hort. sci., 34(1s): 35­42 copyright: © 2020 bartolini s., orlando m., trivellini a., venturi f., sanmartin c., taglieri i., macaluso m., zinnai a., mensuali­sodi a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 february 2020 accepted for publication 30 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 35­42 36 increase of + 1.5% in 2018 equal to 134 billion euro (there were 132 in 2016). the industrial production of the agri­food sector has grown by more than 5% in the last fifteen years (caroli et al., 2019). the transformation activities produce large quantities of organic waste whose disposal repre­ sents an additional cost for the food industries as they must be moved, stored and disposed. in italy, the annual residues deriving from the canning indus­ try are estimated at around 1400 kt of d.m. (balsari et al., 2011). consequently, the reuse of waste is a strategic theme in the search for ‘renewability’ for the industrial application of raw materials of veg­ etable origin, also to meet the interest of users towards eco­sustainable products. processing waste from the agri­food industry, in particular fruit and vegetables, are generally consid­ ered an excellent source of bioactive compounds with antioxidant activity such as vitamins, phenols and carotenoids. plants produce a wide range of sec­ ondary metabolites, mainly phenolics, with different functions: pigmentation, growth, reproduction, resis­ tance to pathogens (castoria et al., 2009). the posi­ tive effects on human health of these phytochemicals have been widely documented and concern the antioxidant, anti­inflammatory and anticarcinogenic action (auger et al., 2004; manach et al., 2005; bitler et al., 2007). phytochemicals extracted from various species such as olive, citrus, tomato, oregano, green tea, grapes and garlic have been shown to have the ability to inhibit lipid oxidation in various model sys­ tems. therefore, the transformation of waste byproducts into synthetic food additives appears par­ ticularly promising (goni and hervert­hernández, 2011). two widely worldwide consumed fresh or processed foods, such as potatoes and apples, gen­ erate a large waste amount. the by­product man­ agement of potatoes (solanum tuberosum) is consid­ ered an important problem faced by food processing companies, as they cannot be discharged into the environment due to their high polluting potential (rodriguez amado et al., 2014). potato is a source of different bioactive compounds such as starch, dietary fiber, amino acids, minerals, vitamins, and phenolics (akyol et al., 2016). the apple is the most widely consumed fruit, with a wide range of varieties with different organoleptic characteristics. numerous scientific studies demonstrated these fruits possess a wide range of chemical compounds with beneficial effects on human health: dietary fibers and proteins, secondary metabolites such as vitamins, phenols and carotenoids, which have proven anti­inflammatory and anticarcinogenic antioxidant effects (boyer and liu, 2004). the main structural classes of apple constituents include hydroxycinnamic acids, di­hydrochalcones, flavonols, catechins and oligomeric procyanidins, as well as anthocyanin in red apples (jakopic et al., 2007; veberic et al., 2007). in particular, considering the antioxidant capacity of compounds present in apple tissues, this fruit may be considered a valid chal­ lenge in the chain of ready to eat products. one of most critical points in fresh­cut fruit slices is the appearance of cut surface browning as a conse­ quence of physical stresses imposed on cells during preparation. to avoid the loose of quality, chemical treatments have been used as food additives (paiva­ martins et al., 2007). however, some of the most common synthetic compounds such as bha (butylat­ ed hydroxyanisole) and bht (butylated hydroxy­ toluene) are suspected of harmful effects; thus, alternative antioxidant additives or disinfectants would be needed (chen et al., 2016). the aim of the project was to evaluate, in two fruit and vegetable “model” species (potato and apple), the possibility of using industrial processing waste for food applications. in this work the atten­ tion was focused on the effect of extracts obtained from organic potato and apple peels on the preserva­ tion of the physicochemical quality parameters of fresh­cut apples. 2. materials and methods preparation of fresh‐cut apples fresh­cut apples were prepared from organic undamaged ‘golden delicious’ (malus domestica borkh) purchased from a local large retailer. the experimental procedure is showed in figure 1, according to safety statements and recommenda­ tions for minimally processed apples (dávila­aviña et al., 2015). apples were washed in running water, hand­peeled and cored with a ceramic knife, longitu­ dinally cut into cubes (12 per apple) and completely dipped in different preserving solutions for 2 min, manually stirring. dipping treatments were per­ formed comparing usually used preservative media (1% butylated hydroxytoluene ­ bht ­ and 1% citric acid ­ ca) and novel extracts from potato (p) and apple (a) peels obtained by water (w) and 10% bartolini et al. ‐ potato and apple peel extracts on fresh‐cut fruits 37 ethanol (et). after dipping, the apple cubes were drained on absorbent paper and packaged in plastic lidded containers (150 cc). packaging were put in controlled chambers at 20±1°c to stimulate a short browning reaction or stored at 4±1°c (dark condi­ tions up to 5 days) to simulate the average standard cool retail storage condition. each dipping treatment consisted of 3 replicates represented by 3 different containers. procedure for potatoes and apple peel extracts the extracts were prepared starting from i) organ­ ic yellow­paste potatoes cv. bologna and ii) organic apples cv. fuji by cryomaceration of peels which were maintained to direct contact with solid co2 (ratio peels/co2 1/1 w/w) over 24 hours, according to venturi et al. (2019). the extraction was carried out using as solvents water and et at 10% following the steps reported in figure 2 and the extracts were stored at ­20°c in test tubes saturated with nitrogen, until dipping treatments. characterization of peel extracts the total phenols content was determined calori­ metrically at 700 nm, using the folin­ciocalteau reagent (waterhouse, 2001). phenols content was expressed as gallic acid equivalents. antioxidant capacity was determined according to the teac antioxidant assay which was performed spectropho­ tometrically at 734 nm following venturi et al. (2017). the radical cation abts (2,2ʹ­azino­ di­[3­eth­ ylbenzthiazoline sulphonate]) was generated as described by pellegrini et al. (1999). the activities of the extracts were expressed in terms of trolox equiv­ alent antioxidant capacity (teac). quality evaluation of fresh‐cut apples fresh­cut apple characteristics were evaluated by a short test, 3h after cutting at 20°c, and by a cold storage test maintaining the cubes at 4°c until 5 days. the main quality parameters such as browning for the short test, and total solid sugars and firmness for the cold storage test were analyzed. the level of browning was determined by color of apple cubes surface using a colorimeter (eoptis, mod. clm­196 benchtop, tn., italy) according to buera et al. (1985). results were expressed as δ browning index (δbi) values by the follow equation: δbi = bif – bis where bif = bi at the end of each observation time, bis = bi at the start of each experiment. bi = 100* (x­0.31)/0.172 fig. 1 ­ experimental procedure to compare different preservative treatments on fresh­cut apple cubes with traditional (1% butylated hydroxytoluene ­ bht ­ and citric acid ­ ca) and novel extracts from potato and apple (ape) peels. fig. 2 ­ procedure applied to obtain apple and potato extracts using as solvent water and 10% ethanol. both extracts were stored at ­ 20°c before use. adv. hort. sci., 2020 34(1s): 35­42 38 where x = (a*+1.75l*)/(5.645l*+a*­3.012b*); l* defines the lightness, a* and b* define the red­greenness and blue­yellowness, respectively. total soluble solids (tss), obtained from the fresh tissue sap of apple flesh cubes were measured using a hand refractometer (mod. 2369­bertuzzi, milan, italy) and expressed as °brix. flesh firmness of apple flesh cubes was evaluated by a manual penetrometer (mod. 53205, tr turoni & c. snc, forlì, italy), with a metal probe (8­mm diame­ ter). the force needed to break parenchyma cells in the cortex was expressed in kilogram­force (kg/0.5 cm2). statistical analysis statistical analysis of obtained data was conduct­ ed by prism 7 (graphpad, usa). student t test and analysis of variance (anova) with the test of mean comparisons according to bonferroni were applied, with a level of significance at p≤0.05. all data are reported as mean values ± se (standard error). 3. results and discussion peel antioxidant content from the results reported in table 1, a very remarkable total antioxidant activity was detected in both apple and potato peels extracts. however, on the basis of the high phenolic content, apple peels were characterized by the highest antioxidant capaci­ ty. it has been established that, both in red­ and yel­ low­skinned apples, the highest nutraceutical power resides in the peel, representing the main source of antioxidants where values may reach 80% of total antioxidant capacity (leccese et al., 2009), mainly determined by polyphenols (wojdylo et al., 2008). a great influence of solvent composition on antioxidant capacity of the extracts was stood out. the presence of a low ethanol percentage increased the extraction of antioxidant compounds like polyphenols in comparison with solely water in both species. in any case, cryomaceration of vegetal byproducts by means of solid carbon dioxide can be profitably applied to improve the green extraction of bioactive compounds, favoring the processes in solid/liquid extraction as demonstrated on other plant materials (andrich et al., 2003; zinnai et al., 2015; nari et al. , 2018; ascrizzi et al. , 2019). according to the literature, potato and apple peels resulted a good source of total phenolics which can have health beneficial properties, mainly due to the presence of chlorogenic acid and caffeic acid (wolfe and wu, 2003; friedman et al., 2017). short‐time test: effect of dipping on browning of fresh‐cut apples in this study different preservative solutions have been used, in particular those commonly used by the iv gamma industry such as bht and ca. the activity of these traditional solutions has been compared with that carried out by alternative solutions, deriv­ ing from agro­industry waste such as potato and apple peels. nowadays, consumer awareness about green products is increasingly and in this contest the recovery of waste from the agro­industry can be con­ sidered as an evaluable source of chemical com­ pounds of natural origin. the main parameter negatively affecting the visu­ al quality of fresh cut fruits, like apple, is the pres­ ence of oxidation on the surface and in the under layer flash tissue which occurs immediately after cut­ ting. as reported in literature (queiroz et al., 2008) and in previous studies (venturi et al., 2019), it has been observed that the first hours after cutting are very critical due to the appearance of the initial oxidative process symptoms. thus, the evaluation of browning in a short­time interval can be considered a useable and effective test to screen the efficiency of conservative treatments. in figure 3 the δ browning index (δbi) values determined at 20°c after 3 hours from dipping treat­ ments with commercial and novel compounds are reported. the browning appearance was much more evident in water control dipping, while the tissue browning was significantly reduced when the preser­ vative agents were added. both apple and potato extracts obtained by 10% ethanol (eta and etp), showed the strongest anti­browning effects whilst table 1 ­ phenolic content and antioxidant capacity of potato and apple peel water and ethanol extracts data are reported as mean ± se (n= 3). in the columns, and within species, asterisks indicate significant differences between solvents according to student t test (p≤0.05). phenolic content (gallic acid mg/g dry weight) antioxidant capacity (μmol teac/ml) apple peel water extract 9.25 ± 0.26 0.33 ± 0.03 10% ethanol extract 13.14 ± 0.20 * 0.73± 0.02 * potato peel water extract 2.92 ± 0.41 0.17 ± 0.02 10% ethanol extract 3.95 ± 0.02 * 0.21 ± 0.01 * bartolini et al. ‐ potato and apple peel extracts on fresh‐cut fruits 39 the exception of those are considered as a chilling sensitives (tomato, melon etc.) which must be stored at temperatures of 7­13°c. storage at non optimal temperatures generates an alteration of cell mem­ branes (saltveit, 2002) which accelerate respiration and activate the enzymes involved in the detoxifica­ tion of free radicals, such as copper/zinc superoxide dismutase, catalase and the enzymes of the ascor­ bate­glutathione cycle. in particular, minimally processed apples must be stored at a temperature not exceeding 4­6°c and need stabilizing treatments to maintain the quality level of fruits during cold stor­ age (senesi, 2008). thus, the use of conservative compounds have to be appropriately tested also under cold conditions to address the requirement of keeping fruit quality during all the distribution chain. in this work, during the storage at 4°c no signifi­ cant differences were observed in the browning process in comparison with the phenomenon occurred after 3h at room temperature (data not shown). as a consequence, particular attention was focused on the maintenance of other quality parame­ ters as firmness and tss degree. as concern apple tissue firmness, it is determined by cell size, biochemical and biophysical cell wall properties, cell­to­cell adhesion and tissue turgor (toivonen and brummell, 2008; rux et al., 2017). the loss of texture and the degradation of tissues deter­ mine the softening of fruits not only during the ripen­ ing but also under particular storage conditions. in the present study, when the processing proce­ dures started, an average firmness of 2.70 kg/0.5 cm2 was recorded. under cold temperature at 4°c (fig. 4), in water dipping treatment a texture decrease was observed with a tissue strength decline by about 25% and significant differences in comparison with all other treatments were observed. an interesting inhi­ bition of tissue softening was recorded in the apple cubes treated with the peel extracts similarly to that observed using traditional media (bht and ca). the positive effects of the natural novel compounds, con­ firmed also after 5 d of storage (fig. 4), could be related to an attenuation of the physical and chemi­ cal changes affecting textural integrity as a conse­ quence of enzymatic hydrolysis of cell wall pectic substances (van buggenhout et al., 2009). in particu­ lar, the softening increase observed in apple cubes after water treatment could be due to texture bio­ chemical changes as reported in several minimally processed fruits and vegetables (toivonen and brummell, 2008). as regard tss content of golden delicious apple, water extracts (wa and wp) had values comparable to those obtained with the use of commercial com­ pounds bht and ca. the beneficial effect exerted by apple and potato extracts could be linked to their phenolic content (table 1). in the case of potato and its byproducts, the inhibition of the polyphenoloxi­ dase (ppo) activity has been well recognized (akyol et al., 2016). for this reason, potato extracts have been studied for their capacity in reducing lipid oxi­ dation and improving nutritional properties of processed foods (franco et al., 2016). as regard apple, phenolic rich peel extracts were found to be effective inhibitors of polyunsaturated fatty acid oxi­ dation as demonstrated by a stronger inhibitory effect on fish oil oxidation (sekhon­loodu et al., 2013). cold storage test: effect of dipping on quality of fresh‐ cut apples temperature is considered the most important factor in the conservation of perishable products. the temperature strongly influences the respiration of the tissues, so much so that as the temperature increases, the respiration also increases (fagundes et al., 2013). an increase of 10°c has been observed to induce an increase in respiratory activity and an acceleration of the aging process of approximately 2­ 3 times. therefore, the temperature must be as low as possible according to the tolerance thresholds of the different species. in general, most products can be stored at temperatures close to zero (0­1°c) with fig. 3 ­ short­time test: effect of dipping on flesh browning of fresh­cut apples. δ browning index (δbi) percentage determined at 20°c, after 3 hours from dipping treat­ ments: water; bht (1% butylated hydroxytoluene); ca (1% citric acid); eta (10% ethanol apple extract); wa (water apple extract); etp (10% ethanol potato extract); wp (water potato extract). data are means ± se. 40 adv. hort. sci., 2020 34(1s): 35­42 at commercial stage the changes associated with ripening were already accomplished, so that the max­ imum soluble level was settled on about 13°brix. during storage this value should be maintained to preserve the sensory quality of fresh cut apple (augusto et al., 2016; musacchi and serra, 2018). the low temperature was effective to maintain the tss degree, indeed a weak decrement was measured in samples without any conservation treatments stored at 4°c for 2 days (fig. 5). this was in accordance with several researches reporting no substantial changes in apple slices of cv. gala coated with alginate and stored at 5°c with values ranging from 14. 6 to 12.8 °brix (olivas et al., 2007). analogous results were obtained using apple extracts in contrast to the tradi­ tional additives bht and ca that caused a more dras­ tic tss reduction. after 5 d storage a notable tss decrease was recorded in apple cubes dipped in water whilst no significant variations in all other treatments were detected, suggesting a protective effect in minimizing carbohydrate breakdown. the possibility to use natural extracts from other differ­ ent sources as postharvest treatment, has been also evaluated in several minimally processed apples with profitable results (augusto et al., 2016). 4. conclusions this study suggests that the residual waste of potato and apple can be considered as a exploitable source of valuable compounds for preservation of minimally processed apples. potato and apple peel fig. 4 ­ cold storage test: effect of dipping on flesh firmness (kg/0.5 cm2) of fresh­cut apples determined at 4°c after 2 and 5 days from dipping treatments: water; bht (1% butylated hydroxytoluene); ca (1% citric acid); eta (10% ethanol apple extract); wa (water apple extract); etp (10% ethanol potato extract); wp (water potato extract). data are means ± se. different letters indicate significant statistical difference at p≤0.05. fig. 5 ­ cold storage test: effect of dipping on total solid content (tss) expressed as °brix of fresh­cut apples determined at 4°c after 2 and 5 days from dipping treatments: water; bht (1% butylated hydroxytoluene); ca (1% citric acid); eta (10% ethanol apple extract); wa (water apple extract); etp (10% ethanol potato extract); wp (water potato extract). data are means ± se. different letters indicate significant statistical difference at p≤0.05. bartolini et al. ‐ potato and apple peel extracts on fresh‐cut fruits 41 extracts based treatments positively affected the visual quality of fresh­cut apples as anti­browning agent starting from the beginning of the preparation process. during cold storage of apple cubes, the application of these novel extracts delay the degra­ dation mechanism of the flesh constituents maintain­ ing a good level of firmness as well as improving the nutritional quality. on the basis of the obtained results, the proposed cryomaceration procedure to recover compounds from potato and apple peels proved to be effective to for­ mulate potential natural additives for postharvest pro­ cessing. thus, synthetic preservatives in freshly stored vegetables could be replaced or at least reduced. furthermore, the employment of potato and apple peels proposed in this study may contribute to manage the environmental and economic problems caused by the increasing amount of wastes and residues from food processing industries. metabolomic characterization of the plant materi­ als tested in this work is under investigation and the next step of our research will be to establish the rela­ tionship between the observed effects on fresh­cut apples and the chemical composition of these novel extracts. references akyol h., ùriciputi y., capanoglu e., caboni m.f., verardo v., 2016 ‐ phenolic compounds in the potato and its byproducts: an overview. ­ int. j. mol. sci., 17: 835­854. andrich g., stevanin e., zinnai a., venturi f., fiorentini r., 2003 ­ extraction kinetics of natural antioxidants from potato industry by‐products. ­ isasf, versailles, france, pp. 159­163. ascrizzi r., taglieri i., sgherri c., flamini g., macaluso m., sanmartin c., venturi f., quartacci m.f., pistelli l., zinnai a., 2019 ­ nutraceutical oils produced by olives and citrus peel of tuscany varieties as sources of functional ingredients. ­ molecules, 24: 65. auger c., rain p.g., laurent­bichon f., portet k., bornet a.l., caporiccio b., cros g., teisseadre p.l., rouanet j.m., 2004 ­ phenolics from commercial‐ ized grape extracts prevent early atherosclerotic lesions in hamsters by mechanisms other than antioxidant effect. ­ j. agric. food chem., 52: 5297­5302. augusto a., simões t., pedrosa r., silva s.f.j., 2016 ­ evaluation of seaweed extracts functionality as post‐ harvest treatment for minimally processed fuji apples. ­ ifset, 33: 589­595. balsari p., menardo s., gioielli f., 2011 ­ tecniche di valorizzazione del contenuto energetico della biomassa, pp. 133. ­ in: castelli s. 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(eds.) current protocols in food analytical chemistry. john wiley & sons, new york, usa. wojdyło a., oszmiański j., laskowski p., 2008 ­ polyphenolic compounds and antioxidant activity of new and old apple varieties. ­ j. agric. food chem., 56(15): 6520­6530. wolfe k., wu x.r.h., 2003 ­ antioxidant activity of apple peels. ­ j. agric. food chem., 51 (3): 609­614. zinnai a., venturi f., sanmartin c., taglieri i., andrich g, 2015 ­ the utilization of solid carbon diox‐ ide in the extraction of extra‐virgin olive oil. ­ agro­ food ind. hi technol., 26: 24­26. https://www.ncbi.nlm.nih.gov/pubmed/?term=paiva-martins%20f%5bauthor%5d&cauthor=true&cauthor_uid=17439139 https://www.ncbi.nlm.nih.gov/pubmed/?term=correia%20r%5bauthor%5d&cauthor=true&cauthor_uid=17439139 https://www.ncbi.nlm.nih.gov/pubmed/?term=f%c3%a9lix%20s%5bauthor%5d&cauthor=true&cauthor_uid=17439139 https://www.ncbi.nlm.nih.gov/pubmed/?term=ferreira%20p%5bauthor%5d&cauthor=true&cauthor_uid=17439139 https://www.ncbi.nlm.nih.gov/pubmed/?term=gordon%20mh%5bauthor%5d&cauthor=true&cauthor_uid=17439139 223 adv. hort. sci., 2024 38(2): 223­229 doi: 10.36253/ahsc­14582 inter­annual and genotypic variation of morphological and physicochemical characters in moroccan loquat (eriobotrya japonica lindl.) g. kabiri 1, f. herandez 2, f.z. lachkham 1, h. hanine 1 (*) 1 faculty of science and techniques, sultan moulay slimane university, 523 béni‐mellal, beni mellal, morocco. 2 grupo de investigación en fruticultura y técnicas de producción, instituto de investigación e innovación agroalimentaria y agroambiental (ciagro‐umh), universidad miguel hernández, carretera de beniel, km 3.2, 03312 orihuela, spain. key words: genotype, inter­annual, loquat, morphological, physicochemical. abstract: plant development is constantly affected by biotic and abiotic factors, which influence their morphology and chemical composition. in this context, the evaluation of morphological and physicochemical variation of 35 loquat genotypes during two consecutive years, 2015 and 2016, were carried out. the results revealed a significant difference of the morphological and the physico­ chemical traits between the two years. indeed, 2016 showed high values for fruit and leaf traits as well as the physicochemical parmeters, while 2015 recoreded the highest values for seeds traits. in addition, the anova results showed a significant effect of genotype on the physicochemical parameters and the morphological characters, excluding the geometric diameter and spherical index of seeds. regarding the effect of year, it was also significant on physico­ chemical parameters and morphological traits except the size and shape of fruit and the seed shape. for the genotype x year interaction effect, it was signifi­ cant on all traits studied, with the exception of the traits relating to geometric diameter of fruit and seed plus the sphericity index of the seed. thus, size and shape of fruit remained stable over these two consecutive years. the identifica­ tion of stable traits presents a result that could be beneficial for breeding pro­ grams. 1. introduction loquat (eriobotrya japonica lindil.), belonging to rosaceae family, is an evergreen tree, native to china (gariglio et al., 2002). the world pro­ duction of loquat is about 314,384 tons, 64% of this amount exhibited by china (caballero and fernández, 2003). in the mediterranean region, loquat crops are highly developed, particularly in spain and turkey (del (*) corresponding author: h.hanine@usms.ma citation: kabiri g., herandez f., lachkham f.z., hanine h., 2024 ­ inter‐annual and genotypic variation of morphological and physicochemical characters in moroccan loquat (eriobotrya japonica lindl.). ­ adv. hort. sci., 38(2): 223­229. copyright: © 2024 kabiri g., herandez f., lachkham f.z., hanine h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 april 2023 accepted for publication 30 march 2024 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-14582 adv. hort. sci., 2024 38(2): 223­229 224 mar romero escudero et al., 2011). loquat cultiva­ tion is considered a commercial crop in some coun­ tries, while in others it is grown only in family orchards (caballero and fernández, 2003). in morocco, this tree, with its yellow fruits, is planted as a commercial consumable crop as well as an orna­ mental crop (hussain, 2011) in the regions of fez­ meknes, khemisset, tetouan and in the region of marrakech but it is localized especially in berkanewith an area representing 85% of the nation­ al surface (skiredj and el macane, 2003). the berkane region is mostly covered by loquat tree due to its mild and sunny microclimate and well­drained fertile soils (rhomari, 2013). in 2021, the production of loquat in berkane crossed 10,000 tons, with an improvement in the size of the fruits and the gustative quality of this excellent local product (chellay, 2022). the estimates of trait heritability were found to be relatively low to moderate (jiwuba et al., 2020). ezenwaka et al. (2018) suggests that the combination of genotype and environmental effects greatly influ­ enced trait expression. indeed, phenotypic expres­ sion as well as observed variation in plant growth and development depend on both genetic background (g), environment (e) and their interaction (g × e) (falconer and mackay, 1996). a result, a clear under­ standing of g×e will provide a solid basis for identify­ ing superior and stable genotypes in different envi­ ronments (zhang et al., 2010). the study of the effect of these factors on the variation of morphological and chemical parameters of plants, including loquat, is limited, but with climate change it has become an obligation. in the region of berkane, the main pro­ ducer of this fruit in morocco, the temperature recorded a decrease, while the rainfall increased dur­ ing 2015 and 2016 (meteobleu, 2024). these obser­ vations encouraged to explore the effect of these changes on the morphological and physicochemical characteristics of 35 loquat genotypes during these two consecutive years. indeed, high trait stability present one of the main challenges of plant breeding programs. 2. materials and methods plant material in april 2015, a prospection was carried out in loquat plantations of zegzel, takerboust, taghsrout and tazaghin, belonging to the berkane region, to identify the genotypes that will be involved in this study (fig. 1). the choice of the genotypes based on a numerous agronomic and economic criteria, such as tardiness and earliness, shape, size and colorof fruit, shape of leaves as well as the good physical condition of tree. indeed, a total of 10 mature, healthy fruits and 10 well­developed leaves were collected ran­ domly during april and may of 2015 and 2016 from 35 adult and young trees. morphological analysis a total of 17 characteristics related to leaves, fruits and seeds,listed in international union for plant protection descriptor (upov, 1998), were investigat­ ed (table 1). the weights were determined using an electronic balance with a sensitivity of 0.01 g, while the dimensions were measured using a digital caliper (stainless hardned) with a sensitivity of 0.01 mm. leaf length measured as the distance from the apex to the base of the leaf, including the blade and peti­ ole. regarding the blade width, it is measured at the widest part of the leaf. physicochemical analysis soluble solid content, determined by a refrac­ tometer (atago c.o. ltd; model pr­1). briefly, a few drops of the juice of each genotype were placed on the prism of the equipment surface and the soluble solid content expressed in °brix. the titratable acidity was measured by potentiometric titration using a standardized alkaline solution (serrano et al., 2003). fig. 1 ­ sampling sites of loquat genotypes studied and their geo­ graphic and climatic parameters. kabiri et al. ‐ inter‐annual and genotypic variation of morphological and physicochemical of loquat 225 an amount of 10 ml of fruit juice was diluted in 50 ml of distilled water and then titrated with a 0.1 naoh solution until ph 8.2 was reached. titratable acidity is expressed per g malic acid l­1. concerning the ph val­ ues of the juice, it was measured using an electronic ph meter (ph211r, hanna®) with three replicates for each sample. statistical analysis data obtained were subjected to statistical analy­ sis using sas software (sas institue inc., 1988). indeed, the two­way analysis of variance (anova) tests was performed to determine the effect of geno­ type, year and genotype x year interaction on the morphological traits as well as the physicochemical parameters of genotypes. the comparison of means was performed by duncan’s test. 3. results the assessment of morphological and physico­ chemical characters of 35 loquat genotypes, from the berkane region, revealed a significant difference between the two consecutive years (table 2). in addi­ tion, a significant effects of genotype, year and their interaction on the most of the traits studied, were recorded (table 3). in fact, the comparison of fruit values indicated a significant difference of fruits traits code fruits weight of the fruit (g) fw geometric diameter of the fruit (mm) gdf sphericity index of the fruit sif surface of the fruit (mm2) sf volume of the fruit (cm3) vf weight of flesh (g) wf flesh ratio fr seeds average weight of the seed (g) aws geometric diameter of the seed (mm) gds sphericity index of the seed sis surface of the seed (mm2) ss volume of the seed (cm3) vs leaf leaf length (cm) ll blade length (cm) bl blade width (cm) bw petiole length (cm) pl number of veins nv physicochemical parameters soluble solids content (°brix) titrable acidity (g/l malic acid) ph table 1 ­ fruit, seeds, leaf traits and physicochemical parame­ ters analyzed gdf= (fruit length x fruit width x fruit thickness)0.333; sif= gdf/fruit length; sf= π x gdf2; vf= (π/6) x gdf3; wf= fw­aws; rf= fw­aws/fw; gds= (seed length x seed width x seed thickness)0.333; sis= gds/seed length; ss= π x gds2; vs= (π/6) x gds3; nv were counted. traits years 2015 2016 fruit fruit weight 41.02 b 42.73 a geometric diameter of the fruit 42.28 a 41.58 a sphericity index of the fruit 0.86 a 0.87 a fruit surface 53.86 a 54.98 a fruit volume 37.96 a 39.03 a weight of the flesh 34.80 a 35.55 a flesh ratio 0.81 b 0.83 a seed average weight of the seed 2.58 a 2.36 b geometric diameter of the seed 15.11 a 14.43 a sphericity index of the seed 0.71 a 0.72 a surface of the seed 7.23 a 6.36 b volume of the seed 1.85 a 1.54 b leaf leaf length 22.47 b 23.7l a blade length 21.16 b 22.5l a blade width 6.46 b 7.34 a petiole length 1.3l a 1.20 b number of veins 0.41 b 0.43 a physicochemical characteristics soluble solids content 7.17 b 9.77 a titrable acidity 5.69 b 8.09 a ph 3.18 b 4.52 a table 2 ­ averages comparison of fruit, seed, leaf and physico­ chemical characteristics during 2015 and 2016 adv. hort. sci., 2024 38(2): 223­229 226 weight and flesh ratio among two years. these traits showed high values in 2016 with 42.73 g and 0.83 respectively, in comparison to those obtained in 2015 with 41.02 and 0.81 respectively. the remaining traits are much more stable. regarding the effects of source of variation ddl mean square f­value pr>f fruit fruit weight genotype 34 1746.70 22.03 <.0001 year 1 512.50 6.47 0.0112 genotype × year 34 472.97 5.97 <.0001 error 630 79.27 geometric diameter of the fruit genotype 34 495.17 2 0.0008 year 1 84.38 0.34 0.5593 genotype × year 34 308.47 1.25 0.1608 error 630 247.26 sphericity index of the fruit genotype 34 0.06 17.09 <.0001 year 1 0.0006 0.17 0.6763 genotype × year 34 0.01 3.09 <.0001 error 630 0.003 fruit surface genotype 34 1573.73 24.28 <.0001 year 1 218.49 3.73 0.0668 genotype × year 34 13125.09 5.96 <.0001 error 630 64.81 fruit volume genotype 34 1682.73 22.39 <.0001 year 1 198.99 2.65 0.1042 genotype × year 34 421.05 5.60 <.0001 error 630 75.14 weight of the flesh genotype 34 877.43 19.18 <.0001 year 1 65.57 1.43 0.2318 genotype × year 34 218.82 4.78 <.0001 error 452 45.74 flesh ratio genotype 34 0.01 6.81 <.0001 year 1 0.02 14.20 0.0002 genotype × year 34 0.008 5.03 <.0001 error 452 0.001 seed average weight of the seed genotype 34 2.13 5.95 <.0001 year 1 5.43 15.18 0.0001 genotype × year 34 1.31 3.68 <.0001 error 451 0.35 geometric diameter of the seed genotype 34 20.00 1.26 0.1563 year 1 54.60 3.43 0.0647 genotype × year 34 12.21 0.77 0.8266 error 455 15.91 sphericity index of the seed genotype 34 0.031 1.12 0.3 year 1 0.017 0.62 0.4332 genotype × year 34 0.022 0.80 0.7823 error 455 0.028 seed surface genotype 34 9.57 6.66 <.0001 source of variation ddl mean square f­value pr>f year 1 89.07 61.94 <.0001 genotype × year 34 3.83 2.67 <.0001 error 455 1.43 seed volume genotype 34 1.26 6.75 <.0001 year 1 11.57 61.70 <.0001 genotype × year 34 0.51 273 <.0001 error 455 0.18 leaf length of the leaf genotype 34 98.25 5.79 <.0001 year 1 252.43 14.88 0.0001 genotype × year 34 120.18 7.08 <.0001 error 617 16.96 blade length genotype 34 90.19 5.71 <.0001 year 1 296.4 18.75 <.0001 genotype × year 34 296.4 7.07 <.0001 error 617 15.86 blade width genotype 34 15.67 6.04 <.0001 year 1 128.4 49.48 <.0001 genotype × year 34 14.03 5.41 <.0001 error 617 2.59 petiole length genotype 34 0.59 8.64 <.0001 year 1 1.77 25.95 <.0001 genotype × year 34 0.33 4.51 <.0001 error 617 0.06 number of veins genotype 34 0.071 8.84 <.0001 year 1 0.075 8.05 0.0047 genotype × year 34 0.076 8.30 <.0001 error 617 0.008 physicochemical parameters soluble solid content (°brix) genotype 36 12.45 19.74 <.0001 year 1 201.55 319.42 <.0001 genotype × year 31 5.76 9.14 <.0001 error 71 0.63 acidity genotype 36 13.91 5.82 <.0001 year 1 205.51 85.89 <.0001 genotype × year 31 4.92 2.06 0.0064 error 71 2.39 ph genotype 36 0.33 14.17 <.0001 year 1 59.88 2506.15 <.0001 genotype × year 31 0.19 8.21 <.0001 error 71 0.02 table 3 ­ genotype, year and their interaction effect on fruit, seed, leaf and physicochemical characters follows in the next right column kabiri et al. ‐ inter‐annual and genotypic variation of morphological and physicochemical of loquat 227 genotype, year and their interaction on the fruit traits, the results showed high significant effect of genotype on all traits (p<0.0001), while the year effect was significant only on fruit weight and flesh ration (p<0.05). whereas, the effect of genotype × year interaction was very significant on all traits ana­ lyzed (p<0.0001), except on geometric diameter of fruits. for the seed results, in 2015 the averages of weight, surface and volume were higher (2.58 g, 7.23 mm2 and 1.85 cm3 respectively) than those obtained in 2016 (2.36 g, 6.36 mm2 and 1.54 cm3 respectively. this result was confirmed by the significant effects of genotype, year and their interaction on average weight, surface and volume of the seed (p<0.0001). the rest of traits such as geometric diameter and sphericity index of seed seems to be not affected by this factor. as results, there is a significant combined effect between genotypes, year and genotype × year interaction on the weight, volume and as well as seed surface. moreover, the average values of leaf length, blade length, blade width and number of veins were higher in 2016 (23.71 cm, 22.5 cm, 7.43 cm, and 0.43 cm respectively) compared to those obtained in 2015 (22.47 cm, 21.16 cm, 6.46 cm, and 0.43 cm respec­ tively). while, the petiole length recorded the highest value in 2015 (1.31 cm). in addition, the results of anova showed highly significant effects of genotype, year and genotype × year interaction on all leaf traits studied (p<0.001). furthermore, the comparison of the soluble solids content, acidity and ph results for two years indicat­ ed that the values of these parameters were higher in 2016 (9.77°brix, 8.09 g/l malic acid, 4.52) than those registered in 2015 (7.17°brix, 5.69 g/l malic acid, 3.18). moreover, the statistical analysis showed high significant effects of genotype, year and geno­ type × year interaction on these parameters (p <0.0001). 4. discussion and conclusions the comparison of the averages of the studied traits during two consecutive years and the evalua­ tion of the effects of genotype, environment and their interactions allow to measure the stability of the characterscanbe integratedin breeding programs (ebdon and gauch, 2002). in this regard, the present study revealed a significant variation ofmorphological and physicochemical traits of 35 loquat genotypes­ during 2015 and 2016 as well as the magnitude of the inter­annual variation of the studied traits depending on the genotype. some traits are stable, while others showeda significant variationfrom one year to the next. effectively, the average values of fruit weight and flesh ratio parameters were different between 2015 and 2016 which showed the highest values of these traits. similarly, elsabagh and haeikl (2012) recorded a significant difference of fruit weight of four egyptian loquats during 2011 and 2012. in loquat, it has been reported that fruit weight depends mainly on genotype (gariglio et al., 2001; lin et al., 1999) and the cultivation conditions, which present a notable effect on the characteristics of the fruit (cuevas et al., 2012). in fact, the increase of fruit weight can be attributed to the amount of rainfall and low temperatures recorded during the 2016 in comparaison with 2015 in berkane region. in addi­ tion,the tree load had a negative effect on fruit size, so that the proportion of large fruits increased as the number of fruits per tree decreased (mahhou et al., 2006). in addition, these good resultwas due to a program initiated during 2016. this program provid­ ed a supplementary training for farmers and purchas­ ing technical equipment, packaging supplies and equipment for a refrigeration unit in order to develop and improve the loquat crop (chellay, 2022). concerning the seed results, the average weight, sur­ face and volume of the seed, were superior in 2015 than values obtained in 2016, with a significant influ­ ence of genotype, year and genotype × year interac­ tion. in fact, the results revealed a significant com­ bined effect between genotype, year and their inter­ action on seed weight, seed volume and seed area, but its effect was not significant on geometric diame­ ter and seed sphericity index. this finding is in agree­ ment with that reported by elsabagh and haeikl (2012), which found a significant difference among 2011 and 2012 of the seed weight of four egyptian loquat trees. this result could be due to the amount of rainfallrecorded in 2015, compared to 2016 which influenced the plants growth in the berkane region (zejly, 2016). whereas,severe water stress during seed fill of soybean plantscaused their inability to regulate seed number and changing the weight distri­ bution of the seeds to a higher proportion of small seeds. as a consequence, a greater number of small seeds (dornbos and mullen, 1991). for the leaf traits, the year 2016 is characterized by higher values of leaf length, blade length, blade width and number of 228 adv. hort. sci., 2024 38(2): 223­229 veins compared to those obtained in 2015. in addi­ tion, the results revealed a highly significant effects of genotype, year and genotype × year interaction on all the leaf variables studied. in cassava, the percent­ age of variation due to environment was higher than the percentage of variation due to genotype for leaf retention, indicating that the environment strongly influenced the expression of this trait (jiwuba et al., 2020). also, the phenotypic plasticity for leaf size, specific leaf area, and leaf level of hybrid poplars are modulated by a variety of environmental factors, including light, nutrient availability, and water avail­ ability (toillon et al., 2013). regarding the sugar con­ tent, acidity and ph, which were registered during the year 2016 are superior to those recordedin 2015. this result is reinforced by the strong effect recorded of genotype, year and their interactions on these parameters. in the egyptian loquat, the same result was obtained by elsabagh and haeikl (2012). the authors observed a significant effect of the year on acidity and sugar content of some loquat varieties during 2011 and 2012. the tree charge influences sol­ uble solids content, which increased as the number of fruits per tree decreased. thus, the soluble solids content of the fruit increased with fruit size (mahhou et al., 2006). according to the results obtained, the variation of morphological and physicochemical traits analyzed­ was very importantwith an high effect of genotype factor. nevertheless, fruit size and shape, which are the most important economic criteria, were found to be stable over these two consecutive years. these finding should be considered in breeding programs for more effective control of fruit quality. further research is needed to control the impact of these fac­ tors on the stability of the selected plant. moreover, a future research should also investigate the effect of these factors on biochemical composition. acknowledgments the authors thank the farmers for their help in sampling. references alghamdi s.s., 2004 ­ yield stability of some soybean genotypes across diverse environment. ­ pak. j. biol. sci., 7(12): 2109­2114. caballero p., fernández m.a., 2003 ­ loquat, produc‐ tion and market, pp. 11­20. ­ in: llácer g. and m.l. badenes (ed.) first international symposium on loquat. ciheam, options méditerranéennes: série a. séminaires méditerranéens, no. 58, zaragoza, spain, pp. 198. chellay m., 2022 ­ arboriculture dans l'oriental: cette année, la récolte de nèfle promet d'etre exceptionnelle. ­ https://fr.le360.ma/societe/arboriculture­dans­lorien­ tal­cette­annee­la­recolte­de­nefles­promet­detre­ exceptionnelle­260189/ cuevas j., pinillos v., canetea m.l., parrab s., gonzálezc m., alonsoa f., fernándezc m.d., hue­ soc j.j., 2012 ­ optimal duration of irrigation 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w.­w., wang d., shi y.­s., sun b.­c., song y.­c., wang t.­y., li y., 2010 ­ stability of qtl across envi‐ ronments and qtl‐by‐e,nvironment interactions for plant and ear height in maize. ­ agr. sci. china, 9(10): 140­1412. impaginato 99 adv. hort. sci., 2021 35(2): 99­110 doi: 10.36253/ahsc­10414 ‘momordica charantia’ introducing a new rootstock for grafted cucumber under low­temperature stress s. mohammadnia, m. haghighi (*) department of horticulture, college of agriculture, isfahan university of technology, isfahan, iran. key words: cucurbita maxima, female flower, karela, photosynthesis, root­ stock, yield. abbreviation: rma= grafted onto cucurbita maxima; rmo= grafted onto momordica charantia; rn= non­grafted; rs= self­grafted; tc= con­ trol temperature; ts= stress temperature. abstract: cucumber is a sensitive vegetable to low temperatures. grafting veg­ etables on different rootstocks can decrease the harmful effects of environ­ mental stresses, including low­temperature stress. an experiment was per­ formed to evaluate grafting cucumbers on different rootstocks at low tempera­ tures. cucumber growth and yield and photosynthesis traits were examined. treatments were the optimum temperature (25±2°c), and cold temperature (15±3°c, ts), and rootstocks, were momordica charantia (rmo), cucurbita max‐ ima (rma), non­grafted (rn) and self­ grafted (rs) with 4 replications. shoot fresh and dry weight, chlorophyll, rwc, transpiration, decreased with tempera­ ture stress. the number of female flowers, electrolyte leakage, photosynthesis, stomatal conductance increased with ts. first fruit emergence per plant, n, p, k, mg concentration decreased with ts stress. transpiration, female flower, rwc, and stomata conductance, n, p, k, ca, and phenol increased in rma and rmo. mg was at the highest concentration in rma and na in rn. all in all, using rmo as well as rma is recommended for rootstock as it causes more reproduc­ tive growth. 1. introduction recently, the greenhouse culture of plants has widely expanded (yan et al., 2013). cucumbers (cucumis sativus) are commonly grown in the greenhouse. the optimal temperature for aerial growth and roots are about 28­23/18­15°c (day/night) and 20°c, respectively (ikeda and kawashiro, 2005). thus, cucumbers are cold­sensitive vegetables. low­ temperature stress can occur in plastic culture or non­equipped green­ houses, which are very common in iran. it may also affect field culture cucumbers and spring culture cucumbers. low temperatures have differ­ (*) corresponding author: mhaghighi@iut.ac.ir citation: mohammadnia s., haghighi m., 2021 ­ ‘momordica charantia’ introducing a new root‐ stock for grafted cucumber under low‐ temperature stress. ­ adv. hort. sci., 35(2): 99­ 110 copyright: © 2021 mohammadnia s., haghighi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 february 2021 accepted for publication 12 february 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10414 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 99­110 100 ent deleterious effects on cucumbers, including affecting the nutrient uptake of plants (pregitzer and king, 2005), and inducing greater h2o2, mda, and sol­ uble sugar content in cucumbers, causing damage to plants and inhibiting plant growth (qiu­yan et al., 2013). there are several ways to control low­tempera­ ture damage to plants, including controlling root temperatures by warming nutrient solution or soil. still, these ways require excess energy (willits and peet, 1998). finally, excess chemical nutrient applica­ tions for promoting growth can prevent damage to plants, which unfortunately involves environmental risks. therefore, vegetable grafting can be a proper way to increase plant resistance to environmental stresses in addition to expanding its yield, quality, and growth. furthermore, if any energy­efficient way is introduced, which can raise the growth rate of plants in greenhouse conditions at lower tempera­ tures, vast amounts of energy and money will be saved. vegetable grafting has been used to increase plant tolerance under environmental stresses, includ­ ing high temperatures (rivero et al., 2003), low tem­ peratures (rivero et al., 2003), salinity stresses (estan et al., 2005), drought stresses (bhatt et al., 2002) and heavy metal stresses (edelstein et al., 2005). fig leaf gourd and bur cucumbers (sicyos angula‐ tus) are used for increasing resistance against low­ temperature stresses for cucumber rootstocks (venema et al., 2008). these rootstocks increase plant resistance using different ways. first, low­tem­ perature stresses decrease co2 assimilation in cucum­ bers and this reduction is improved by grafting (zhou et al., 2009). secondly, a previous study done by zhou et al. (2007) showed that fig leaf gourd root­ stocks enhanced vegetative growth and yield of cucumbers under low­temperature stresses. there is a hypothesis stating that grafted plant responses to temperature stresses are related to scion species (venema et al. , 2005). many researchers have shown beneficial effects of veg­ etable grafting on the growth, fruit yield, and quality of plants. some of the studies have used different rootstocks for cucumbers under different stresses showing better growth rate for grafted plants under stress conditions compared to non­grafted ones like: heavy metal absorption (rouphael et al., 2008; kumar et al. , 2015), low soil temperatures (tachibana, 1982), salinity (huang et al., 2010), improve nacl and cacl2 tolerance in cucumber (colla et al., 2013), al toxicity in cucumber (rouphael et al., 2016). different rootstocks were used in normal and stress conditions to improve yield and growth traits like dif­ ferent kinds of squash (yang et al., 2006), (massai et al., 2004), (rouphael et al., 2008), fig leaf guard (tachibana, 1982), pumpkins and bottle gourd and p360 (cucurbita maxima duch.×cucurbita moschata duch.) (colla et al., 2010 and 2013). to the best of our knowledge, few studies have investigated the use of momordica charantia as a rootstock for cucurbits, or more explicitly grafting, to examine the possibility of cucumber cultivation at low temperatures in the whole growth period. hence, the goal of the present study was the use of momordica charantia and cucurbita maxima as rootstocks and cucumis sativus var. davosii, which is a common variety of cucumbers in iran as a scion under low­temperature stress. 2. materials and methods experimental design and plant preparation this experiment was conducted in a plastic green­ house in the department of horticulture science at the isfahan university of technology, isfahan, iran. an investigation was arranged as a combined analysis involving data from two locations, simultaneously collected, based on crd with 4 replications. treatments were optimum (25±2°c, tc) and low tem­ peratures (15±3°c, ts). cucumbers (cucumis sativus var. davosii) were grafted to momordica charantia (rmo), cucurbita maxima (rma). non­grafted (rn) and self­grafted (rs) consider as a control. scion seeds had been cultivated 10 days before rootstock seeds in cocopeat: perlite 1:1. scion plants and root­ stocks were cut beneath and above the first true leaves, respectively. hole­insertion grafting was used and grafted plants were transferred to a recovery greenhouse with high relative humidity. plants were kept for 2 weeks in recovery conditions and gradually adapted to normal greenhouse conditions. grafted plants were transferred to 5kg pots, including soil. irrigation was used when the plant needed it. chemical fertilizer npk (20, 20, 20) 2 mg/pot was applied every 10 days. plants were conducted to wire above the greenhouse, and there was no use of any pesticide. plant growth and fruit properties the male and female flower, node numbers, and shoot numbers were counted during the experiment; mohammadnia et al. ‐ ‘momordica charantia’ a new rootstock for grafted cucumbers 101 root and internode length was determined with a ruler. the time and node of male and female flower emergence were recorded. fruit diameter, fruit firm­ ness, and tss were measured with a caliper (mitutoyo corp, japan), pentameter (model osk­i­ 10576), and portable refractometer (pal­1 brix, japan), respectively (raeisi et al., 2014). shoots were excised from the roots using a steel blade and then fresh weights of roots and shoots were measured. all the samples were oven­dried at 70˚c for 48 hours and the dry weights were estimat­ ed. during the experiment and finally 124 days after transplanting, fruits were harvested and washed using tap water and were weighed by an analytical balance. spad value and photosynthesis trait assay chlorophyll content was measured using a chloro­ phyll meter (spad­502 plus, minolta, japan). fv/fm was measured by chlorophyll fluorescence (os­ 30, usa) after 3 weeks. photosynthetic properties were determined from the youngest fully­expanded leaf for 3 replications per treatment by portable photo­ synthesis systems for gas exchange and chlorophyll fluorescence measurements (li­cor­6400, usa) from 10:00 to 11:00 am on a clear day (without clouds). the measurements were conducted with photosyn­ thetically active radiation (par) intensity of 1000 μmol m­2 s­1 and co2 concentration of 350 μmol·mol. mesophyll conductance (μmol m­2s­1) was calculated by dividing the photosynthetic rate by the sub­stom­ atal co2 level (ahmadi and siosemardeh, 2005). antioxidant activity antioxidant activity was measured and expressed as gallic acid (equivalents l of gallic acid/g) with uv­ vis spectrophotometer (shimadzu uv160a­japan). three mg of sample were dissolved in 5 ml methanol stock, and 1.4 ml of this solution was blended with 0.6 ml of dpph solution. after 30 min, the absorbance of the solution was recorded at 515 nm by the spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan) against methanol as a blank. the 0.2 mm of dpph solution in methanol was used as a stock of dpph for the determination of the free radi­ cal scavenging activity of the samples (koleva et al., 2002). phenolic content total phenolic content was determined using the folin­ciocalteu method. the absorbance was mea­ sured at 725 nm with a spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan). the results were expressed in gallic acid equivalents (mg/100 g fresh weight) using gallic acid (0­0.1 mg/ml) standard curve (singleton et al., 1965). proline proline accumulation was determined using the method proposed by bates et al. (1973). after the extraction of toluene, the clear phase was recovered and spectrophotometrically estimated at 520 nm using toluene as a blank. purified proline was used for standardization (0­50 mg/ml) and was expressed as mol proline g­1 fresh weight. electrolyte leakage electrolyte leakage (el) was measured using an electrical conductivity meter employing the method described by lutts et al. (1995) relative water content relative water content (rwc%) was determined using ten 7 mm­diameter leaf discs. the leaf discs of each treatment were weighed (fw). they were then hydrated until saturation was reached (constant weight) for 48 h at 5°c in darkness (tw). the leaf discs were dried in an oven at 105°c for 24 h (dw). relative water content was calculated according to the following equation (filella et al., 1998): rwc% = (fw − dw)/(tw − dw)× 100 the determination of total nitrogen in the leaf samples was based on the kjeldahl method (estan et al., 2005). the concentrations of k, ca, mg, and p were measured (shield torch system, agilent 7500a). meanwhile, phosphorus was estimated by the vanadomolybdo phosphoric acid colorimetric method at 460 nm (estan et al., 2005). p was colori­ metrically determined using a spectrophotometer (uv 160a­ shimadzu corp., kyoto, japan). statistical analysis all data were analyzed using two­way anova (statistix 8 software) (tallahassee fl, usa) and the means were compared for the significance by the least significant difference (lsd) test at p<0.05. 3. results and discussion analysis of variance of temperature and rootstock on some characteristics of cucumber temperature effect shoot fresh and dry weight, chlorophyll index, and the number of the female flower, photosynthetic rate, transpiration, stomatal adv. hort. sci., 2021 35(2): 99­110 102 conductance, rwc, el, phenol content, and all nutri­ ent concentration (table 1 a, b). rootstock changes all cucumber characteristics except for average fruit weight, fruit diameter, tss, firmness, mesophyll con­ ductance, proline, and antioxidant (table 1 a, b). the interactive effect of temperature and rootstock showed that all measured parameters change signifi­ cantly (table 1 a, b). the main effect of temperature on cucumber shoot fresh and dry weight, chlorophyll, rwc, transpiration, decreased with temperature stress. the number of the female flower, el, photosynthesis, and stomatal conductance increased with ts (table 2 a). first fruit emergence per plant, n, p, k, mg con­ centration decreased with ts stress (table 2 b). shoot fresh and dry weight and fresh root weight was high­ table 2 a ­the effect of temperature on some characteristics of cucumber tc= optimum temperature, ts= low temperature. within a column means followed by the same letter are not significantly different at p<5% according to lsd test. table 2 b ­ the effect of temperature on some characteristics of cucumber tc= optimum temperature; ts= low temperature. within a column means followed by the same letter are not significantly different at p<5% according to lsd test. table 1 a ­ analysis of variance of the effect of temperature and rootstock on some characteristics of cucumber ns= no significant, * significant at 5% and ** significant at 1%. table 1 b ­ analysis of variance of the effect of temperature and rootstock on some characteristics of cucumber ns= no significant, * significant at 5% and ** significant at 1%. source df shoot fresh weight shoot dry weight root fresh weight chi number of female flowers number of male flowers fruit number fruit yield first fruit per plant average fruit weight fruit diameter tss firmness temperature 1 3203.42 * 230. 9 ** 244.56 ns 43.12 ** 78.12 * 6.12 ns 230. 9 ns 1441.5 ns 152.91 * 1971.09 ns 0.002 ns 0.30 ns 7.65 ns rootstock 3 2467.99 * 23.45 * 150.01 * 35.93 ** 602.45 ** 13.08 ** 23.45 * 19335 * 92.57 * 2094.59 ns 0.10 ns 0.18 ns 2.07 ns t×r 3 1327.25 * 26.54 ** 111.77 ** 4.65 ** 81.12 ** 4.20 * 26.54 ** 11769.1 ** 4.81 ** 2133.88 * 0.04 ** 0.01 ** 1.23 ** error 18 708.05 5.27 67.38 3.23 76.32 1.35 5.27 5110.2 17.23 1292.36 0.04 0.12 4.26 cv 10.30 25.62 13.04 15.50 13.97 14.38 25.62 18.03 26.05 18.65 6.77 13.40 25.25 source df photo­ synthetic rate tran­ spiration stomata conduc­ tance mesophyll conduc­ tance rwc el proline anti­ oxidant activity phenol content n conc. p conc. k conc. ca conc. mg conc. na conc. temperature 1 47.52 * 10.52 ** 0.002 ** 240063 ns 0.006 ** 257.36 ** 3.27 ns 0.06 ns 55824.1 ** 0.72 ** 0.036 ** 0.06 ** 0.15 ** 0.16 ** 0.039 ** rootstock 3 25.08 * 2 ** 0.001 * 26980 ns 0.001 ** 60.38 ** 1.31 ns 0.006 ns 5561.8 ** 1.39 ** 0.03 ** 2.06 ** 0.65 ** 0.07 ** 0.01 ** t×r 3 14.25 ** 1.12 ** 0.002 ** 4898 ** 0.0008 ** 72.71 ** 2.82 ** 0.02 ** 4553.6 ** 0.54 ** 0.01 ** 0.62 ** 0.54 ** 0.06 ** 0.008 ** error 18 6.17 0.97 0.001 14354 0.00003 9.38 1.05 0.03 52.2 6.53 4.71 5.08 3.85 1.05 2.78 cv 14.41 14.82 17.80 38.05 0.74 7.56 11.03 5.81 5.01 12.1 26.7 21.5 7.87 6.23 16.9 temperature (° c) shoot fresh weight (g) shoot dry weight (g) chi (spad value) number of the female flower el (%) rwc (%) transpiration (mmol h2o m­2 s­1) photosynthetic rate (mmol h2om­2 s­1) stomata conductance (mmol h2om­2 s­1) tc 76.52 a 11.71 a 12.7 a 2.18 b 37.1 b 0.79 a 2.83 a 4.16 b 61.31 b ts 55.53 b 6.21 b 10.4 b 3.06 a 43.91 a 0.75 b 1.57 b 7.02 a 177.34 a temperature (° c) first fruit emergence per plant (day) phenol (mg g ̵¹ fw ) n (%) p (%) k (%) ca (%) mg (%) na (%) tc 19.57 a 17.69 b 3.51 a 0.43 a 3.11 a 1.54 b 0.74 a 0.14 b ts 12.30 b 271.06 a 3.37 b 0.39 b 2.54 b 1.66 a 0.68 b 0.21 a mohammadnia et al. ‐ ‘momordica charantia’ a new rootstock for grafted cucumbers 103 est in rma, the number of male flower increased in rn and rs and female flower increased in rmo and rma. the main effect of rootstock on cucumber the highest el and photosynthesis were observed in rmo. the rwc and stomata conductance increased in rma and rmo, however, transpiration increased in rs, rmo, and rma (table 3 a). n, p, k, ca, and phenol increased in rma and rmo. mg was at the highest concentration in rma and na in rn (table 3 b). rma has the highest fruit number, fruit yield, although, first fruit emerges in rmo (table 4). the interaction effect of temperature and cucumber grafting on some growth characteristics of cucumber k concentration was the highest in rmo in both temperatures and p concentration increased in rma×ts. the highest n, k and ca, concentrations were seen in rma×ts. p concentration increased in rma and rmo at ts. mg increased in rs×tc and na in rs×ts (table 5). shoot fresh weights decreased in rn and rs at low temperatures (ts), but shoot dry weights did not sig­ nificantly change in rma and rmo at both tempera­ tures (fig. 1). shoot dry weights followed the same trend as the fresh shoot weight was the highest in rn, rs, rma at tc (fig. 1). root fresh weights decreased in rn, rs, and rma at ts compared with tc, but did not significantly change in rmo at both temperatures, although the fresh root weight was the lowest in rmo at tc. the highest root fresh weight was seen in rma×tc (fig. 2 a). spad value was the highest in rs, rma, and rmo at tc (fig. 2 b). the interaction effect of the tempera­ table 3 a ­the effect of rootstock on some characteristics of cucumber rn= nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. within a column means followed by the same letter are not significantly different at p<5% according to lsd test. rootstock shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) number of male flowers number of female flower el (%) rwc (%) photosynthetic rate (µmol co2 m ­2 s­1) transpiration (mmol h2o m–2 s–1) stomata conductance (mmol h2o m–2 s–1) rn 61.28 b 8.23 b 16.78 b 23.50 a 2.37 b 38.98 bc 0.76 b 4.94 b 1.65 b 78.79 b rs 55.48 b 9.29 ab 17.96 ab 23 a 1.75 b 41.67 ab 0.76 b 3.83 b 2.69 a 71.94 b rma 92.34 a 11.17 a 18.21 a 5.5 b 4.5 a 37.10 c 0.78 a 4.84 b 1.86 ab 89.96 ab rmo 55b 7.14 b 17.95 b 12.75 b 3.87 ab 44.38 a 0.78 a 8.75 a 2.61 ab 126.62 a table 3 b ­ the effect of rootstock on some characteristics of cucumber rn= nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. within a column means followed by the same letter are not significantly different at p<5% according to lsd test. rootstock phenol (mg g ̵¹ fw) n (%) p (%) k (%) ca (%) mg (%) na (%) rn 124.54 bc 3.19 c 0.45 b 2.52 d 1.05 d 0.70 b 0.19 a rs 112.19 c 3.08 d 0.38 c 2.55 c 1.32 c 0.69 c 0.20 a rma 133.90 b 3.85 a 0.47 a 3.65 a 2.11 a 0.51 d 0.14 c rmo 206.87 a 3.64 b 0.33 d 2.57 b 1.89 b 0.93 a 0.18 b table 4 ­ the effect of rootstock on some characteristics of cucumber rootstock fruit number fruit yield (g) first fruit emergence per plant (day) rn 0.8 b 63.88 b 21.51 a rma 2.00 a 159.50 a 15.29 ab rmo 1.37 ab 91.88 ab 11.00 b rn= nongrafted (cucumis sativus var. davosii); rs= self grafted did not have any fruit; rma= cucurbita maxima; rmo= momordica charantia. within a column means followed by the same letter are not signi­ ficantly different at p<5% according to lsd test. 104 adv. hort. sci., 2021 35(2): 99­110 ture on grafted cucumber did not significantly affect the time of the emergence of the first female flowers (data did not show). the highest and the lowest num­ ber of the male flower were seen in rs×tc and rma, respectively. the female flower was affected by tem­ perature variations in different rootstocks. the female flower increased in the rn and rma at ts and did not change in rs and rmo at tc and ts (fig. 3). fruit number was the highest in rma×tc, although the same results were seen in rmo×tc and rmo and rma at ts (fig. 4 a). fruit yield was the highest in rma×tc; the same result was statically seen in rma ×ts (fig. 4 b). first, fruit emergence time significantly decreased in grafted plants at this temperature (fig. 5). the highest fruit weight was in rma×tc; fruit diameter did not significantly change between treatments (fig. 6). tss increased in rn and rmo at ts (fig. 7 a). firmness decreased in rmo×ts (fig. 7 b) results, goreta et al. (2008) found that shoot weight reduc­ tion in watermelon grafted onto cucurbita maxima duch.× cucurbita moschata duch was less) than non­ grafted plants under salt stresses. the grafted cucum­ ber plants showed less change in shoot and root weights under cu stress conditions and this might be due to the lower accumulation of cu in leaves through their squash rootstocks (rouphael et al., 2008). the lowest male flower was seen in rma at both temperatures (fig. 3). conversely, the highest num­ ber of female flowers was observed in rma at ts and tc compared with other rootstocks at each tempera­ ture. on the other hand, at the optimum tempera­ ture (tc), each rootstock had a lower female flower table 5 ­ the interaction effect of temperature and cucumber grafting on some elements concentration (%) of leaves fig. 1 ­ the interaction effect of temperature and cucumber grafting on shoot fresh and dry weight. tc= optimum temperature; ts= low temperature; rn = nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. fig. 2 ­ the interaction effect of temperature and cucumber grafting on root fresh weight (a) and spad value (b) tc= optimum temperature; ts= low temperature; rn = non­ grafted (cucumis sativus var. davos ii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. rootstock n p k ca mg na tc ts tc ts tc ts tc ts tc ts tc ts rn 3.36 d 3.03 e 0.37 c 0.35 c 2.50 f 2.75 e 1.28 c 0.83 g 0.81 b 0.58 c 0.18 c 0.21 b rs 3.08 e 3.08 e 0.33 c 0.34 c 3.00 d 2.15 g 1.65 b 1.00 e 0.88 a 0.51 e 0.12 f 0.28 a rma 3.78 c 4.2 a 0.34 c 0.52 a 3.45 b 3.69 a 1.24 d 1.75 a 0.51 e 0.57 d 0.12 f 0.14 e rmo 3.98 b 3.78 c 0.42 b 0.48 a 3.25 c 3.10 cd 0.83 g 0.87 f 0.56 d 0.50 e 0.09 g 0.16 d rn= nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. within a column means followed by the same letter are not significantly different at p<5% according to lsd test. mohammadnia et al. ‐ ‘momordica charantia’ a new rootstock for grafted cucumbers 105 showed the highest male flower. van der ploeg and heuvelink (2005) reported that low temperatures reduced the tomato fruit set through poor pollen quality and increased the period between anthesis and fruit maturity resulting in lower fruit yields. the result of khah et al. (2006) showed that tomatoes cv. big red grafted onto cv. heman and primavera pro­ duced more fruit at low temperatures compared to non­grafted plants in greenhouse conditions. in our study, fruit number decreased at ts and grafting had a beneficial effect on fruit number, but did not influ­ ence fruit yields. in agreement with our study, com­ mercial tomato grafting was not able to improve the reduction of tomato yields under low light stresses by shading (krumbein and schwarz, 2013). the economic increase of yield imparted by select vigorous commercial rootstocks (kyriacou et al., 2017) like increasing tomato yields was observed by fig. 3 ­ the interaction effect of temperature and cucumber grafting male and female flower number. tc= optimum temperature; ts= low temperature; rn = nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. fig. 4 ­ the interaction effect of temperature and cucumber grafting on fruit number (a) and fruit yield (b) momordica charantia rs did not have fruit. tc= optimum temperatu­ re; ts= low temperature; rn = nongrafted (cucumis sati‐ vus var. davosii); rs= self grafted; rma= cucurbita maxi‐ ma; rmo= momordica charantia. fig. 5 ­ the interaction effect of temperature and cucumber grafting on the first fruit emergence of a plant. tc= opti­ mum temperature; ts= low temperature; rn = nongraf­ ted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. fig. 6 ­ the interaction effect of temperature and cucumber grafting on average fruit weight and fruit diameter. tc= optimum temperature; ts= low temperature; rn = non­ grafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. adv. hort. sci., 2021 35(2): 99­110 106 grafting tomatoes onto kagemusia and helper’s root­ stocks by lee et al. (2007). santa­cruz et al. (2001) grafted tomato cv. moneymaker onto pera and observed that growth and yield of tomato increased under salt stress conditions. similar results were observed by estan et al. (2005), who found that tomato yield increased through grafting under salt stresses and also reported that fruit yield in hetro­ grafted plants increased more compared with self­ grafted plants. ruiz et al. (1997) believed that increase yield and growth of the grafted plants might be due to an increase in the nutrient and water uptake fed by vigorous rootstock. huang et al. (2010) reported that cucumbers grafted had a higher fruit yield than non­grafted plants under salt stresses. colla et al. (2010) found that increasing co2 assimila­ tion by cucumber grafting increased fruit yields. the interaction effect of temperature and cucumber grafting on some physiological characteristics and nutrient concentration of cucumber the photosynthesis rate decreased in rn and rs at ts compared with tc. however, it did not significantly change in rma and rmo (fig. 8 a). transpiration decreased at ts in all rootstocks compared with tc (fig. 8 b). stomata conductance gradually increased in rs, rma, and rmo at ts compared with tc and reached the highest in rmo×ts (fig. 8 c). mesophyll conductance did not significantly change in each rootstock at tc and ts, except for ungrafted cucum­ bers in which it increased at ts compared with tc (fig. 8 d). el and rwc were generally the lowest and the highest at ts, respectively. the highest el was seen in rmo×ts. the maximum rwc, at ts, was observed in rn, rma, and rmo (fig. 9 a, b). antioxidant increased in rn and rs at ts and did not change in other treat­ fig. 7 ­ the interaction effect of temperature and cucumber grafting on tss (a) and firmness (b). tc= optimum tempe­ rature, ts= low temperature; rn = nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. fig. 8 ­ the interaction effect of temperature and cucumber grafting on photosynthesis (a), transpiration (b), stomata conductance (c), mesophyll conductance (d) tc= opti­ mum temperature; ts= low temperature; rn = nongraf­ ted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. mohammadnia et al. ‐ ‘momordica charantia’ a new rootstock for grafted cucumbers 107 ments (fig. 10 a). phenol content increased in all plants at ts and reached the highest point in rmo×ts (fig. 10 b). proline did not significantly change among treatments (fig. 10 c). increasing antioxidant content with using root­ stock was reported by rouphael et al. (2018); this may help grafted plant to have a better function under different stress. the photosynthesis rate and spad value showed that the spad value did not change in rmo. thus, photosynthesis did not change. however, the same result was not seen in rma. therefore, it seems that another mechanism interfered with the stability of photosynthesis in rma in this condition rather than changes of chlorophyll content (fig. 6 a and 7a). some researchers demonstrated that the photo­ synthesis rate was influenced by leaf area, stomatal conductance, and chlorophyll content. in our study, low­temperature stress caused a reduction in spad value and resulted in photosynthesis reduction. on the other hand, the photosynthesis in grafted cucum­ bers having high spad value and stomata conduc­ tance improved under low­temperature stresses. davis et al. (2008) reported that grafting increased photosynthesis under salt stress conditions too. grafted cucumbers showed higher photosynthesis rates, stomatal conductance, and intercellular co2 under salt stresses compared with non­grafted plants (yang et al., 2006). massai et al. (2004) and moya et al. (2002) reported that grafting improved photosyn­ thesis under salt stress conditions, too. rootstock­ induced changes in stomatal development, as reduced transpiration relates to lower stomatal den­ sity in grafted plants (kumar et al., 2017). zhou et al. (2009) reported that increasing the photosynthesis rate by grafting plants onto a differ­ ent rootstock might be due to a decrease in the ros concentration in the leaves. beside, grafting increased water and nutrient uptake of the plant and could enhance its photosynthesis (martinez­ballesta fig. 9 ­ the interaction effect of temperature and cucumber grafting on el (a) and rwc (b) tc= optimum temperature, ts= low temperature, rn = nongrafted (cucumis sativus var. davosii); rs= (self grafted); rma= cucurbita maxima, rmo= momordica charantia. fig. 10 ­ the interaction effect of temperature and cucumber grafting on antioxidant (a) and phenol (b) and proline (c) tc= optimum temperature; ts= low temperature; rn = nongrafted (cucumis sativus var. davosii); rs= self grafted; rma= cucurbita maxima; rmo= momordica charantia. adv. hort. sci., 2021 35(2): 99­110 108 et al., 2010). salehi et al. (2010) reported that melon grafting had high co2 assimilation due to increasing stomata conductance and ci and resulted in an increase in photosynthesis. the photosynthesis rate and the fresh shoot weight of rmo and rma did not significantly change at both temperatures. thus, low temperatures could not affect the photosynthesis rate in rmo and rma. on the other hand, decreasing the fresh weight was seen at ts in rn due to a decrease of photosynthesis rate in these rootstocks (fig. 7). overall, low stomata and mesophyll conductance of un­grafted cucumbers which have adverse effects on water relation, mineral nutrient uptake and trans­ port (kumar et al., 2017), carbohydrate and hormone relationship, photosynthesis and respiration rates result in a decline in yields (kozlowiski, 1984; barrick and noble, 1993; bacanamwo and purcell, 1999). these results were in line with our results at low­ temperature stresses. still, when cucumbers were grafted onto karella they were kept under proper conditions for more efficient photosynthesis, and consequently, a better yield was produced. the result of the study showed that salinity stress­ es reduced the k concentration in melons and cucumbers and grafted plants had higher nutrient concentrations than non­grafted plants. many root­ stocks are capable of increasing the uptake and translocation of nutrients (kumar et al., 2017). in agreement with our result, colla et al. (2013) report­ ed that cucumber grafting had no influence on k and p concentrations in the leaves, root, and fruit under salt stresses. our result showed that the highest k concentration was in rmo at both temperatures and p concentration increased by grafting at tc. 4. conclusions it seems that the photosynthetic traits of grafted cucumbers were not affected mainly by rmo and rma at stress conditions. on the other hand, hor­ monal changes or nutrient uptake of these root­ stocks seemingly caused lesser root and additional vegetative growth but stimulated more fruit and yields. the effect of these rootstocks on productivity growth resulted in more male flowers than female ones, which was predictable according to the produc­ tive growth model of cucurbitaceae in which male flowers and then female flowers emerged in the bush. accordingly, if this experiment lasted more, it might result in more female flowers, suggesting that further investigations might be needed. all in all, using rmo as well as rma is recommended after test­ ing the fruit quality and the economic yield. reference ahmadi a., siosemardeh a., 2005 ­ investigation on the physiological basis of grain yield and drought resis‐ tance in wheat: leaf photosynthetic rate, stomatal con‐ ductance and non‐stomatal limitation. ­ int. j. agric. biol., 7: 807­811. bacanamwo m., purcell l.c., 1999 ­ soybean root mor‐ phological and anatomical traits associated with accli‐ mation to flooding. ­ crop sci., 39: 143­149. barrick k.a., noble m.g., 1993 ­ the iron and man‐ ganese status of seven upper montane tree species in colorado following long term water logging. ­ j. ecol., 81: 523­531. bates l.s., waldren r.p., teare i.d., 1973 ­ rapid deter‐ mination of free proline for water‐stress studies. ­ plant soil, 39: 205­207. bhatt r.m, srinivasa­rao n.k., sadashiva a.t., 2002 ­ rootstock as a source of drought tolerance in tomato (lycopersicon esculentum mill). ­ indian j. plant physiol., 7: 338­342. colla g., roupharl y., jawad r., kumar p., rea e., 2013 ­ the effectiveness of grafting to improve nacl and cacl2 tolerance in cucumber. ­ sci. hort., 164: 380­ 391. colla g., roupharl y., leonardi c., bie z., 2010 ­ role of grafting in vegetable crops grown under saline con‐ ditions. ­ sci. hort., 127: 147­155. davis a.r., perkins­veazie p., dakataakata y., lopez­ galarza s., maroto j.v., lee s.g., hyhy c., sun, z., miguel a., king s.r., cohen r., lee j.m., 2008 ­ cucurbit grafting. ­ crc crit. rev. plant sci., 27: 50­74. edelstein m., ben­hur m., cohen r., burger y., rav­ ina i., 2005 ­ boron and salinity effects on grafted and non‐grafted melon plants. ­ plant soil, 269: 273­284. estan m.t., martinez­rodriguez m.m., perez­alfo­ cea f., flowers t.j., boalrin m.c., 2005 ­ grafting raises the salt tolerance of tomato through limiting the transport of sodium and chloride to the shoot. ­ environ. exp. bot., 56: 703­712. filella i., llusia j., pin j.o., pen j.u., 1998 ­ leaf gas exchange and fluorescence of phillyrea latifolia, pistacia lentiscus and quercus ilex saplings in severe drought and high temperature conditions. ­ environ. exp. bot., 39: 213­220. goreta s., bucevic­popovic v., selak g.v., pavela­ vrancic m., perica s., 2008 ­ vegetative growth, superoxide dismutase activity and ion concentration of salt stressed watermelon as influenced by rootstock. ­ j. https://www.google.com/search?client=firefox-b-d&sxsrf=alekk03rfpct9tsl-_edmjypdgguuxywqw:1590213341285&q=cucurbitaceae&spell=1&sa=x&ved=2ahukewjo7cuqpsnpahvnm6qkhdclacoqkeeckab6bagbecq mohammadnia et al. ‐ ‘momordica charantia’ a new rootstock for grafted cucumbers 109 agr. sci. 146: 695­704 . huang y., bie z.h., he s., hua b., zhen a., liu z.h., 2010 ­ improving cucumber tolerance to major nutrients induced salinity by grafting onto cucurbita ficifolia. ­ environ. exp. bot., 69: 32­38. ikeda h., kawashiro h., 2005 ­ the basis for vegetable production (in japanese). ­ nouwenkyo press. tokyo. khah e.m., kakava e., mavromatis a., chachalis d., goulas c., 2006 ­ effect of grafting on growth and yield of tomato (lycopersicon esculentum mill.) in greenhouse and open‐field. ­ j. appl. hort., 8: 3­7. koleva i., van beek t.a., linssen j.p.h., de groot a., evstatieva l.n., 2002 ­ screening of plant extracts for antioxidant activity: a comparative study on three test‐ ing methods. ­ phytochem. anal., 13: 8­17. kozlowski t.t., pallardy s.g., 1984 ­ effects of flooding on water, carbohydrate and mineral relations, pp. 165­ 193. in: kozlowski t.t. 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(japanese mugwort), already used as a foodstuff in japan, can be positioned as a functional health food because of remarkably higher contents of chlorogenic acid and total polyphenol compared to common vegetables. to procure young leaves in demand on a year­round basis by hydroponic production in fully artificial light­ type plant factories, we investigated whether 24­h photoperiod, known to enhance some beneficial constituents, could improve the growth and chemical constituents of japanese mugwort plants grown hydroponically in a plant facto­ ry condition. as we previously demonstrated that lowering the nutrient solu­ tion concentration increased chlorogenic acid and total polyphenol contents of the leaves without reducing the growth, plants were cultivated with a lower concentration of nutrient solution. the results indicated that it is possible to grow japanese mugwort hydroponically under 24­h photoperiod in a plant fac­ tory condition with a nutrient solution concentration as low as 25% of the stan­ dard. in addition, under 24­h photoperiod, plant growth was greatly accelerat­ ed and chlorogenic acid as well as total polyphenol were increased, suggesting that 24­h photoperiod is highly beneficial for japanese mugwort production in a fully artificial light­type plant factory. 1. introduction mount ibuki, located on the border of shiga and gifu prefectures in japan, has been famous for its medicinal plants since ancient times, and it was written in ‘engishiki’ (compiled in 927 a.d.) that omi (shiga prefecture) and mino (gifu prefecture) ranked first and second, respec­ tively, in the number of herbal medicinal items as paying tribute to the imperial court from all over japan (oda, 1985). in particular, in the early edo era (around 1700 a.d.), the area around mt. ibuki was a major pro­ ducer of domestic mugwort, such as artemisia princeps or artemisia mon‐ tana, and the resulting moxa, called for ‘ibuki­moxa’ was publicized nationwide (oda, 1998, 1999). the authors focus on the use of such (*) corresponding author: hata.n@ses.usp.ac.jp citation: hata n., kawamura m., 2023 ­ effect of conti‐ nuous lighting on the growth and leaf chemical components of artemisia princeps grown hydro‐ ponically in a plant factory condition. ­ adv. hort. sci., 37(2): 173­183. copyright: © 2023 hata n., kawamura m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 march 2022 accepted for publication 3 october 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12966 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 173­183 174 domestic mugwort. gaiyoh (artemisiae folium) used in wakan­yaku (traditional herbal drugs) is defined as the dried leaves and branch tips of a. princeps or a. montana, and it is used as a raw material for moxa and is included in various chinese herbal preparations as an astringent hemostatic and analgesic (nunome, 2018; ministry of health, labour and welfare, 2021). artemisia princeps (japanese mugwort) is also used as a foodstuff, with its young leaves, picked in early spring, being mixing with rice cakes or dumplings as ‘mochigusa’, or used in soaking and tempura (odachi and hiyama, 2013; ando et al., 2022). in particular, according to the functional components database (national agriculture and food research organization, 2020), japanese mugwort has remark­ ably higher chlorogenic acid and total polyphenol contents compared with common vegetables, indi­ cating that it can be positioned as a functional health food. japanese mugwort is generally procured by har­ vesting wild plants or through cultivation in open fields (ando et al., 2022). it is preferable to harvest the young, tender leaves in early spring for use as a food ingredient or functional health food. however, under natural conditions, the number of mature leaves increases with plant growth, and after flower­ ing in autumn, the plant eventually withers and stops growing until the following spring (ito, 2015), making it difficult to procure young in­demand leaves on a year­round basis, even after harvesting both wild and cultivated plants. to address this problem, we focused on the use of a fully artificial light­type plant factory system. with the multi­shelf cultivation sys­ tem used in plant factories (kozai, 2013), it is possible to produce a large number of young plants at a low plant height on a year­round basis, allowing to pro­ vide the young leaves desired throughout the year (kim et al., 2021). in addition, plant factory produc­ tion has the advantage of being pesticide­free. however, to date, there is limited knowledge on the hydroponic cultivation of japanese mugwort; therefore, it is necessary to establish an effective management system for its hydroponic cultivation in fully artificial light­type plant factories. in our previ­ ous report (hata and kawamura, 2021), we investi­ gated the effects of nutrient solution concentration on the growth and leaf chemical components of japanese mugwort cultivated hydroponically using ‘ibuki­yomogi’ (a line of a. princeps indigenous to shiga prefecture), to establish an effective hydropon­ ic cultivation method. the results showed that lower­ ing the nutrient solution concentration to 25% of the standard increases the ascorbic acid, chlorogenic acid, and total polyphenol contents of the leaves without reducing plant growth. hata et al. (2012 a) studied the differences in the growth rate and leaf sesamin content of sesame (sesamum indicum) grown under various photoperi­ ods and found not only a maximum leaf yield, but also a distinctively high sesamin content, under a 24­ h photoperiod. furthermore, higashiuchi et al. (2016) also reported that the active ingredient (asperulo­ side) level in white flower snake­tongue grass (hedyotis diffusa), a medicinal plant, increases noticeably under a 24­h photoperiod compared with under 14­ and 19­h photoperiods. thus, enhanced leaf yields and accumulations of beneficial compo­ nents may be achieved using a 24­h photoperiod in the cultivation of japanese mugwort in a plant facto­ ry; however, supporting research is required. consequently, in the present study, we used ‘ibuki­yomogi’ in our experiments and investigated whether a 24­h photoperiod increased the growth and chemical component contents of japanese mug­ wort plants grown hydroponically in a low nutrient solution concentration under plant factory condi­ tions. 2. materials and methods plant materials and seedling cultivation methods the strain maintained at the ibuki yakuso­no sato cultural center (maibara­city, shiga prefecture, japan) was used as the experimental material. inflorescences collected in the fall of 2017 were air­ dried and stored in a desiccator for use in the cultiva­ tion experiments. the seedlings were grown in the growth chamber. the photosynthetic photon flux density from the hf­ fluorescent lamp (fhf32ex­n­h, panasonic co., japan) on the surface of a seedling box was 260 μmol m­2 s­1. the photoperiod and temperature were set at 12 h and 23°c, respectively. on the basis of our previous report (hata and kawamura, 2021), seeds were spread by rubbing the flower heads with fingers, and then they were placed on root prevention sheets (20701fld, unitika ltd., japan) laid on kim towels (nippon paper group crecia co., ltd., japan) moistened with tap water. at 1 week after sowing, young seedlings of approxi­ hata and kawamura ‐ response of hydroponic japanese mugwort to continuous lighting 175 mately 3 mm were transplanted into polyurethane cubes (2.35 × 2.35 × 3 cm, tomiyamass co., japan). afterwards, the seedlings were grown for 3 weeks by subirrigation with 1/2­strength enshi formula nutri­ ent solution. this solution consisted of 2 mm of ca(no3)2·4h2o, 4 mm of kno3, 0.67 mm of nh4h2po4, 1 mm of mgso4·7h2o, 1.5 mg l−1 fe, 0.25 mg l−1 mn, 0.25 mg l−1 b, 0.025 mg l−1 zn, 0.01 mg l−1 cu, and 0.005 mg l−1 mo. hydroponic methods hydroponic cultivation was conducted in a walk­in type plant growth room (internal dimensions: 4.1 m long, 4.1 m wide, and 2.1 m high) at the experimental agricultural facility of the university of shiga prefecture. during the cultivation period, the tem­ perature and co2 concentrations were set at 23°c and 400 ppm, respectively, while the relative humidi­ ty was not set at a constant level. the photosynthetic effective photon flux density on the surface of the growing container at a distance of 42 cm vertically from the hf­fluorescent lamps (fhf32ex­n­h, panasonic co., japan) was 130 μmol m­2 s­1. the seedlings were planted at 4 weeks after sow­ ing and grown hydroponically for 4 weeks under a 12­h or 24­h photoperiod. a 2.5­cm thick styrofoam board with two 2.5­cm diameter holes (11 cm between plants) was floated as a planting board on 6.0 l of nutrient solution in each container (nf box #11 blue, inner dimensions: 15.3 × 27.8 × 16.5 cm, capacity: 7.0 l, jej astage co., ltd., japan), and two seedlings were planted per board. the nutrient solu­ tion used was 1/4­strength enshi formula, which was continuously aerated at 0.4 l min−1 with an air pump. the initial ph of the nutrient solution was adjusted to 6.0 with h2so4 before use, but it was not adjusted during the cultivation period. the nutrient solution was renewed after 2 and 3 weeks of hydroponic culti­ vation. the ph of the nutrient solution was measured with a digital ph meter (ph­208, sato shoji co., ltd., japan) before nutrient solution replacement and at harvest (4 weeks after the start of hydroponic cultiva­ tion). growth evaluation and preparation of dry matter samples in total, 20 plants were grown in 10 growing con­ tainers under each photoperiod. the plants were har­ vested at 4 weeks after the start of hydroponic culti­ vation, and the fresh weights of leaves, stems, and roots were measured, as were the main stem lengths and numbers of branches, for all the plants. the stems and roots were dried in an oven at 60°c, after which the constant dry weights were recorded and used for calculating dry matter content. approximately 10­15 g of leaves randomly taken from the whole leaves was similarly dried at 60°c to form a dried sample for the inorganic component analysis as well as the dry matter content calculation. the rest of the leaves were freeze­dried for other component analyses and stored in a ­80°c freezer. chemical composition analysis the 60°c­dried and freeze­dried samples were thoroughly ground independently with a mortar and pestle. in each photoperiodic treatment, 10 samples were analyzed for each component, with one sample being a mixture of equal amounts of the two individ­ uals growing in one container. each analysis described below was conducted similarly in accor­ dance with our previously reported methods (hata and kawamura, 2021). determination of inorganic components for each sample, 100 mg of the powdered sample was decomposed using the wet method in a nitric acid and hydrogen peroxide mixture in a 100­ml beaker. after decomposition, the solution in the beaker was volumetrically diluted with 1 m nitric acid and passed through a 0.45­μm syringe filter (surplux ptfe­h (hydrophilic) 25 mm, lms co., ltd., japan). the p, k, ca, mg, na, fe, mn, and zn concentrations were measured using (sii sps3100, hitachi high­tech science co., ltd., japan). multi­element standard iv and single­element standard (p) for icp (merck millipore ltd., germany) were used as calibration standards, and the content of each inorganic compo­ nent in the leaves was calculated from the intensity value of each sample. determination of ascorbic acid for each sample, 50 mg of the powder, weighed in a 2­ml microcentrifuge tube, was extracted using ultrasonic waves for 30 min in distilled water. after extraction, the ascorbic acid content in centrifuged supernatant liquid was measured using a reflectome­ ter (rq flex 10, merck millipore ltd., germany) to calculate the corresponding content in the leaves. determination of chlorogenic acid for each sample, 50 mg of the powder, weighed in a 2­ml microcentrifuge tube, was extracted at adv. hort. sci., 2023 37(2): 173­183 176 40°c for 30 min with shaking at 2,000 rpm in 80% (v/v) ethanol solution. after the extraction, the supernatant was collected by centrifugation at 12,500 rpm for 5 min, and the extract was collected again from the extraction residue. the collected extract mixture was passed through a 0.45­μm syringe filter (gl chromato­disk 4n, gl sciences inc., japan) before being used for the chlorogenic acid concentration analysis with the uplc­fld method. in brief, samples were analyzed with the acquity uplc system (waters co., usa) using a waters acquity uplc hss t3 column (100 mm × 2.1 mm, 1.8 μm). detection was performed using a waters 470 scanning fluorescence detector set at an excitation wavelength of 371 nm and an emission wavelength of 443 nm. the mobile phases were 0.2% (v/v) formic acid (solvent a) and 100% acetonitrile (solvent b). the gradient elution program, with a mixture of sol­ vents a and b, was as follows: 90­80% a for 0­1 min (curve no. 7), 80­55% a for 1­5 min (curve no. 7), 55­ 35% a for 5­6 min (curve no. 9), and 35­90% a for 6­7 min (curve no. 9). the flow rate was 0.3 ml min−1. the column oven was set at 40°c, and 3 μl of each sample was loaded. the amount of chlorogenic acid in a sample was quantified from the peak area of the authentic standard compound (chlorogenic acid hemihydrate dissolved in 80% ethanol) to calculate the content in the leaves. solvents of hplc grade, and all other chemicals, were purchased from nacalai tesque, inc., japan. determination of total polyphenol a 10­fold dilution of the extract solution for the chlorogenic acid analysis with 80% (v/v) ethanol was prepared and analyzed in accordance with the folin– ciocalteu method. first, 0.3 ml of the sample solu­ tion and 0.3 ml of distilled water were mixed in a 2­ ml microcentrifuge tube, and then, 0.6 ml of a solu­ tion of phenol reagent (nacalai tesque, inc., japan) diluted two­fold with distilled water was added and left for 3 min after mixing. next, 0.6 ml of 10% (w/v) sodium carbonate solution was added, mixed, and allowed to react for 60 min. within 30 min of the reaction finishing, the absorbance at a wavelength of 750 nm was measured using a spectrophotometer. chlorogenic acid hemihydrate dissolved in 80% ethanol was used as the calibration standard, and the total polyphenol content in the leaves was calculated as chlorogenic acid equivalents from the absorbance values (750 nm) of each sample. data analyses for growth data, average values for each growing container were compared, whereas the data for the nutrient solution ph levels and chemical components were compared among the obtained values per growing container. significant differences between two photoperiods were analyzed using student’s t­ test (n = 10). 3. results change in nutrient solution ph after 2 weeks of hydroponic cultivation, the ph of the nutrient solution rose to 6.5 under 12­h photope­ riod, while it rose to 7.7 in the 24­h photoperiod (fig. 1). even after returning to the initial ph of 6.0 by replacing the nutrient solution, the ph rose to only 6.5 under 12­h photoperiod but to 7.7 under 24­h photoperiod in the following week. even when the nutrient solution was renewed again after 3 weeks of hydroponic cultivation, the ph rose to 6.6 under 12­h photoperiod but to 7.7 under 24­h photoperiod, at the end of cultivation one week later. plant growth flower buds did not form under either the 12­ or 24­h photoperiod until the end of cultivation at 8 weeks after sowing. from 2 weeks after the start of fig. 1 ­ changes in nutrient solution ph during the hydroponic cultivation of japanese mugwort plants grown under 12­ h (open circles) and 24­h (closed squares) photoperiods. values represent means ± ses (n = 10). statistical signifi­ cances between the two photoperiods were determined using student’s t­test. ***, p < 0.001. hata and kawamura ‐ response of hydroponic japanese mugwort to continuous lighting 177 hydroponic cultivation, plant growth was more vigor­ ous under the 24­h photoperiod than under the 12­h photoperiod (fig. 2). generally, darker leaf colors and greater anthocyanin accumulations in the main stems were observed under the 24­h photoperiod (fig. 3), and 2 of 20 plants showed lower leaf senescence (fig. 4). there was no visual difference in the number of trichomes on leaves and stems between the two photoperiodic treatments (fig. 3, 5). stem length, number of main stem nodes, and aver­ age internode length, were significantly higher under the 24­h photoperiod compared with under the 12­h fig. 2 ­ differences in early developmental stages of japanese mugwort plants grown under 12­h and 24­h photoperi­ ods. fig. 3 ­ differences in main stem colors of japanese mugwort plants grown under 12­h and 24­h photoperiods for 4 weeks. the fresh weights of leaves, stems, and roots at harvest under the 24­h photoperiod were 22.8, 11.1, and 14.6 g, respectively, which were almost twice as high as those under the 12­h photoperiod (table 1). the dry weights of leaves, stems, and roots showed the same trends as fresh weights, and the dry matter ratio of leaves to stems was also significantly greater under the 24­h photoperiod. all the traits related to stem elongation, such as number of branches, main fig. 4 ­ appearance of lower­leaf browning in the japanese mug­ wort plant grown under a 24­h photoperiod for 4 weeks. fig. 5 ­ appearances of trichomes on the abaxial leaf surfaces of japanese mugwort plants grown under 12­h and 24­h photoperiods for 4 weeks. 178 adv. hort. sci., 2023 37(2): 173­183 photoperiod (table 2). inorganic component contents on a dry weight basis, the k and zn contents were significantly lower at the 1% significance level under the 24­h photoperiod compared with under the 12­h photoperiod (table 3). furthermore, the fe and mn contents were also significantly lower under the 24­h photoperiod at the 0.1% significance level. the p, ca, mg, and na con­ tents were not significantly different between the two photoperiods at the 5% significance level. on a fresh weight basis, the ca and mg contents were significantly higher under the 24­h photoperiod at the 0.1% and 1% significance levels, respectively. however, the mn content was significantly lower under the 24­h photoperiod at the 5% significance level. the p, k, na, fe, and zn contents were not sig­ nificantly different between the two photoperiods at the 5% significance level. ascorbic acid content on a dry weight basis, the ascorbic acid content tended to be higher under the 24­h photoperiod compared with under the 12­h photoperiod, but there was no significant difference at the 5% signifi­ cance level between the two photoperiods (fig. 6). on a fresh weight basis, the ascorbic acid content was 1.8­times higher under the 24­h photoperiod, which was significant at the 5% level. chlorogenic acid and total polyphenol contents the chlorogenic acid content was 2.5­times higher on a dry weight basis and 3.1­times higher on a fresh weight basis under the 24­h photoperiod than under the 12­h photoperiod, and these differences were significant at the 5% and 1% levels, respectively (fig. 6). similarly, the total polyphenol content was 1.5­ and 1.8­times higher on dry and fresh weight bases, respectively, under the 24­h photoperiod, and these differences were significant at the 1% and 0.1% lev­ els, respectively. there was a positive correlation between chlorogenic acid and total polyphenol con­ tents, with a correlation coefficient of 0.48 on a dry weight basis, whereas the correlation coefficient was 0.64 on a fresh weight basis, indicating a stronger correlation (fig. 7). table 1 ­ effects of photoperiod on the biomass production of japanese mugwort plants fw= fresh weight; dw= dry weight; dmr= dry matter ratio. (z) statistical significances between the means of two photoperiods were determined using student’s t­test (n = 10). ns= not significant; ***, p<0.001. table 2 ­ effects of photoperiod on the stem development of japanese mugwort plants (z) statistical significances between the means of two photoperiods were determined using student’s t­test (n = 10). **, p < 0.01; ***, p<0.001. leaves main stem + branches root photoperiod (h) fw (g) dw (g) dmr (%) fw (g) dw (g) dmr (%) fw (g) dw (g) dmr (%) 12 13.132 1.4 11 3.7 0.4 10 8.0 0.6 8 24 22.81 3.0 13 11 1.4 12 15 1.0 7 significance (z) *** *** *** *** *** *** *** *** ns photoperiod (h) no. of branches main stem length (cm) no. of nodes mean of internode length (cm) 12 15.0 20.65 23.25 0.9 24 22.0 33.10 28.45 1.1 significance (z) *** ** *** ** hata and kawamura ‐ response of hydroponic japanese mugwort to continuous lighting 179 4. discussion and conclusions change in nutrient solution ph we reported previously (hata and kawamura, 2021) that when growing ‘ibuki­yomogi’ plants hydroponically in a greenhouse for 4 weeks at differ­ fig. 7 ­ correlations between chlorogenic acid and total polyphe­ nol contents in the leaves of japanese mugwort plants grown under 12­h (open circles) and 24­h (closed squares) photoperiods. * and ** indicate significant cor­ relations as determined by pearson’s test at p < 0.05 and p < 0.01, respectively (n = 20). fig. 6 ­ effects of photoperiod on ascorbic acid, chlorogenic acid, and total polyphenol contents in japanese mugwort leaves. values represent means ± ses (n = 10). statistical significances between the two photoperiods were deter­ mined using student’s t­test. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. table 3 ­ effects of photoperiod on the mineral contents of japanese mugwort leaves statistical significances between the means of two photoperiods were determined using student’s t­test (n = 10). ns= not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. photoperiod (h) mineral content on a dry weight basis p (mg g dw­1) k (mg g dw­1) ca (mg g dw­1) mg (mg g dw­1) na (mg g dw­1) fe (µg g dw­1) mn (µg g dw­1) zn (µg g dw­1) 12 13.2 48.7 11.5 3.2 0.3 152.2 325.8 60.7 24 10.8 39.1 11.8 3.1 0.3 126.2 211.7 44.3 significancez ns ** ns ns ns *** *** ** photoperiod (h) mineral content on a fresh weight basis (mg g fw­1) p k ca mg na fe mn zn 12 138.0 511.9 121.2 33.4 3.1 1.6 3.5 0.6 24 140.9 510.3 154.3 41.0 3.5 1.7 2.8 0.6 significancez ns ns *** ** ns ns * ns adv. hort. sci., 2023 37(2): 173­183 180 ent nutrient solution concentrations, the nutrient solution ph increases as the nutrient solution con­ centration decreases and that after lowering the lat­ ter to 25% of the standard, the nutrient solution ph reached 8.1. similarly, in the present study, the nutri­ ent solution ph increased, even when the nutrient solutions’ initial ph was adjusted to 6.0, indicating that the nutrient solutions’ ph tended to increase even under artificial light sources, regardless of the photoperiod, by lowering the concentration to 25% of the standard. when anion uptake is dominant, the rhizosphere ph increases as a result of oh− or hco3 − release from the roots to maintain the cellular charge balance, and when cation uptake is dominant, h+ is similarly released from the roots and the rhizosphere ph decreases (hinsinger et al., 2003; fageria, 2012). in particular, because no3 − and nh4 + account for approximately 70% of the cations and anions absorbed by plants, the form of nitrogen application has a significant effect on rhizosphere ph (nutrient solution ph). furthermore, zheng et al. (2004, 2010) reported that the medium ph increases when the applied nutrient solution concentration is lowered from 100% to 25% in the pot cultivation of rose and gerbera, indicating that the rhizosphere ph (nutrient solution ph) may not decrease, but rather increase, owing to the lack of nh4 + at a low nutrient solution concentration. because the total nitrogen content in the enshi formula nutrient solution used in the pre­ sent study consisted of 92.5% no3 − and 7.5% nh4 +, the plants absorbed less nh4 + as the nutrient solution concentration decreased, which may have caused the nutrient solution ph to increase, rather than decrease, during the growing period. masuda et al. (2001) reported that when pepper plants are cultivated hydroponically with fluorescent lamps under a 24­h photoperiod, the ph of the recir­ culating nutrient solution rapidly increases immedi­ ately after planting. in the present study, similarly, the nutrient solution ph at 2 weeks after planting or 1 week after nutrient solution renewal increased more under the 24­h photoperiod compared with the 12­h photoperiod. on the other hand, hata and xu (2020 a) reported that when leaf lettuce is grown hydroponically using a nutrient solution containing nh4 + as the nitrogen source, the degree of decrease in nutrient solution ph is greater under the 24­h pho­ toperiod than under the 12­h photoperiod, suggest­ ing that a faster the growth rate, the ph is more likely to decrease. thus, the faster the growth rate, the greater the change in the ph of the nutrient solution in proportion to the amount of nitrogen absorbed. consequently, greater growth rates of the mugwort plants under the 24­h photoperiod than under the 12­h photoperiod makes the ph of the nutrient solu­ tion more likely to increase, when grown using a low­ concentration of nutrient solution. plant growths for plants that are capable of cultivation under longer photoperiods, the production cost per plant in a fully artificial light­type plant factory decreases as the photoperiod increases, and a 24­h photoperiod is desirable (takatsuji, 2012). plants capable of longer photoperiods are highly tolerant of the continuous light injury that occurs under a 24­h photoperiod, and their growth is greatly accelerated. the occur­ rence of a marked level of continuous light injury in asteraceae plants has not been reported to date, and maximum plant growth rates have been reported under 24­h photoperiods in lettuce and garland chrysanthemum (hata et al., 2011 a, b). this was also the case for the japanese mugwort plants used in the present study. continuous light­induced chlorosis did not occur in newly developed leaves, the leaf color darkened under the 24­h photoperiod, and the dry weights of leaves, stems, and roots at the end of cul­ tivation were nearly two­fold greater under the 24­h photoperiod than under the 12­h photoperiod. thus, like lettuce and garland chrysanthemum, japanese mugwort, which is a member of the asteraceae fami­ ly, is not susceptible to continuous light injury. thus, a 24­h photoperiod could be used to increase the leaf yield and productivity of japanese mugwort in a fully artificial light­type plant factory. stem elongation in plants is inhibited by greater red to far­red light ratios (r/frs), whereas lower r/frs may promote plant stem elongation owing to greater internode elongation (demotes­mainard et al., 2016; ballaré and pierik, 2017). white fluorescent light has a higher r/fr ratio than sunlight (6.5–9.6 and 1.0, respectively) (hamamoto and yamazaki, 2013), and internode elongation is likely to be sup­ pressed in a plant factory environment that uses such fluorescent lighting. in fact, in our previous report using the same ‘ibuki­yomogi’ seeds and hydroponic cultivation method, the average internode length of individuals grown in a glasshouse was 2.3 cm (hata and kawamura, 2021), whereas the average intern­ ode length of individuals grown in an artificial growth room, as in the present study, was 0.9­1.1 cm. the hata and kawamura ‐ response of hydroponic japanese mugwort to continuous lighting 181 internode shortening under fluorescent light is advantageous for producing young leaves of japanese mugwort plants because of the low plant heights in the multi­shelf cultivation system used in plant factories. in addition, the number of branches was significantly higher under the 24­h photoperiod than under the 12­h photoperiod, suggesting that cultivation under the former is advantageous for increasing the number of harvested stems. inorganic component contents the carbon content increases, whereas other essential inorganic element contents generally decrease, in many plant species when grown at greater than atmospheric co2 concentrations (loladze, 2014; soares et al., 2019). the factors responsible for the decrease in these inorganic ele­ ments include (1) a decreased transpiration rate leading to a lowered absorption, and (2) an increased carbon content which results in a reduced element relative content (dilution by carbohydrates). although there have been limited studies on the effects of a 24­h photoperiod on the inorganic com­ ponent contents in plants, hata and xu (2020 b) found that when leaf lettuce is grown under a 24­h photoperiod, the leaf carbon content increases more compared with under a 12­h photoperiod, whereas many inorganic component contents decrease, sug­ gesting that reactions similar to those under high co2 conditions occur under a 24­h photoperiod. the k, fe, mn, and zn contents per dry weight of japanese mugwort in the present study were also significantly lower under the 24­h photoperiod compared with the 12­h photoperiod, suggesting that there may be a number of plant species in which the inorganic com­ ponent contents tend to decrease under a 24­h pho­ toperiod. as in leaf lettuce (hata and xu, 2020 a, b), no clear nutrient deficiency symptoms associated with decreased inorganic component contents were observed in japanese mugwort in the present study under a 24­h photoperiod, but lower­leaf browning was observed in some individuals, suggesting that the potassium concentration in the culture medium requires optimization. ascorbic acid content ascorbic acid in plants is synthesized through the d­man/l­gal pathway, in which d­fructose, a photo­ synthetic product, is used as a metabolic intermedi­ ate to synthesize d­mannose and l­galactose (venkatesh and park, 2014). in leaf lettuce, the ascor­ bic acid content per fresh weight increases 1.3­fold when grown under a 24­h photoperiod compared with under a 16­h photoperiod owing to an increase in the activity of l­galactono­1,4­lactone dehydroge­ nase, an enzyme that converts l­galactono­1,4­lac­ tone, an ascorbic acid precursor, to ascorbic acid (zha et al., 2019). in the present study, the ascorbic acid content per fresh weight was 1.8­times higher under the 24­h photoperiod than under the 12­h photoperi­ od, which was consistent with previous results. this is suggested that a 24­h photoperiod may be used effectively in the production of crops having enhanced ascorbic acid contents, unless the target plants develop continuous light injuries. the addition of ascorbic acid suppresses the degradation of polyphenols, such as chlorogenic acid, during apple juice processing (kolniak­ostek et al., 2013), indicat­ ing that the increased ascorbic acid content in japanese mugwort leaves may contribute to the increased stability of polyphenols, such as chloro­ genic acid, during utilization. chlorogenic acid and total polyphenol contents hata and xu (2020 b) reported that the chloro­ genic acid and total polyphenol contents per dry weight increased by 1.5 to 4.2 times and 1.1 to 1.2 times, respectively, in leaf lettuce grown under a 24­ h photoperiod compared with under a 12­h photope­ riod. furthermore, the differences between the two photoperiods widened in the chlorogenic acid and total polyphenol contents per fresh weight because the dry matter ratio increased more under the 24­h photoperiod than under the 12­h photoperiod. in the present study, the chlorogenic acid content was 2.5­ and 3.1­times higher per dry and fresh weights, respectively, and the total polyphenol content was 1.5­ and 1.8­times higher per dry and fresh weights in japanese mugwort under a 24­h photoperiod com­ pared with a 12­h photoperiod. these results were similar to those previously reported for leaf lettuce (hata and xu, 2020 b), and they suggest that a 24­h photoperiod may be effectively used for the produc­ tion of crops with high polyphenol contents, such as chlorogenic acid, unless target plants develop contin­ uous light injuries. we reported previously (hata and kawamura, 2021) that when ‘ibuki­yomogi’ plants are grown hydroponically in a glasshouse, the chlorogenic acid and total polyphenol contents of the leaves increase adv. hort. sci., 2023 37(2): 173­183 182 as the nutrient solution concentration decreases to 25% of the standard. there were positive correla­ tions (r = 0.45) between chlorogenic acid and total polyphenol contents, both per dry weight and per fresh weight bases. in the present study, positive cor­ relations between chlorogenic acid and total polyphenol contents (r = 0.48 for content per dry weight and r = 0.64 for content per fresh weight) were also observed, suggesting that the increase in the former largely contributed to the increase the latter under a 24­h photoperiod. in addition, high anthocyanin pigment accumulations are often observed in some plant species under a 24­h pho­ toperiod (hata et al., 2012 b), and here, we observed some individuals accumulating anthocyanin pigments in the main stems under the 24­h photoperiod. this suggests that anthocyanin synthesis is also enhanced in japanese mugwort under a 24­h photoperiod and that increases in some flavonoid compounds may also contribute to the increase in the total polyphe­ nol content. these results indicate that it is possible to grow japanese mugwort hydroponically under a 24­h pho­ toperiod and plant factory conditions in a nutrient solution having a concentration as low as 25% of the standard. in addition, under the 24­h photoperiod, plant growth was greatly accelerated and chlorogenic acid, a useful secondary metabolite, as well as ascor­ bic acid, contents increased, suggesting that a 24­h photoperiod is highly beneficial for japanese mug­ wort production in a fully artificial light­type plant factory. however, because the ph of the nutrient solution fluctuated drastically during the cultivation period, it is necessary to investigate separately the composition of 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system using low‐concentration nutrient solutions. ­ hortsci., 39: 1283­1286. untitled 133 book reviews le paysage sacré sacred landscape. ribouillat d., and m. weemans. giardini e paesaggio, vol. 29. leo s. olschki, florence (italy), 2011, pp. xxxii + 368. isbn 978-88-2-6126-72. € 48.00. this is the 29th volume in the “giardini e paesaggio” series. unlike other studies that have been influenced by the idea of an “autonomous landscape”, this volume underlines the importance of the sacred aspect of nature in the representation and creation of landscapes in the early modern period in europe. the work, examining areas from literature to gardens and painting to microarchitecture, suggests that landscape in the period under study should be considered as an interpretation of the artists in the “book of nature”. the various contributions in english and in french that make up the volume are enriched with images from the period that have been carefully assembled by highly regarded international experts in the field of landscape history, stimulating the debate surrounding the concept of scared landscapes. it is necessary to underline the importance of this topic and the efforts undertaken by various scholars to reveal the deep relationship between landscape and sacredness. this volume is a significant addition to the vast literature on the evolution of landscapes through history, from a scientific and technical, as well as historical and cultural perspective and offers the reader meaning and understanding with regard to the sacred values that make up the environments considered. francesco ferrini il pino domestico. elementi storici e botanici di una preziosa realtà del paesaggio mediterraneo. lorenzini g., and c. nali. giardini e paesaggio, vol. 37. leo s. olschki editore, florence (italy), 2013, pp. 96. isbn 978-88-222-6252-3. € 15.00. this volume, as the authors describe, aims to attract the reader’s attention to a “friend” in trouble. the “friend” is the pine tree. it deserves, according to the authors, attention and support as it has always been generous with man, and is symbolic of the italian landscape. the book consists of 96 pages, divided into three parts and a premise, and is written not only for scholars in the field but also to those who, to varying degrees, are interested in this species and to the typical landscapes it has created in our country. the authors are well known experts in the field as, for many years, they have carried out studies and taught many courses on the topic. the first part of the book is devoted to the natural history of pine and its dissemination, the second to breeding for productive purposes and the third to the place that this species occupies in mythology, history, tradition and art. the appendix is of particular interest as it presents curiosities (and not only) about this species. the volume includes illustrations and both color and black and white photos, as well as a bibliography to conclude the book. the authors offer through this work a key upgrade on the subject, from a scientific and technical perspective and with regard to our historical and cultural heritage. the book is of great value for all researchers working in the field of arboriculture and for those who have an interest in basic physiology, applied ecology, and biology to preserve this precious and much-loved species. francesco ferrini 134 manuale di ortofrutticoltura. innovazioni tecnologiche e prospettive di mercato. sansavini s., and p. ranalli (eds.). ministero delle politiche agricole e forestali. edizioni agricole de il sole 24 ore, bologna, 2012. pp. xxiv + 668. isbn 978-88-506-5360-7. € 50.00. this volume, carefully edited by silviero sansavini and paolo ranalli and with contributions by renowned experts in the field, offers considerable breadth. the text covers a wide range of agricultural products, from fruits to vegetables, with particular emphasis on integrated and organic productions, as well as on the market and its prospects. the up-to-date knowledge regarding the fruit and vegetable sector, and in particular pertaining to genetic improvement and new varieties, make the “handbook of horticulture “ an immediate tool for operators, students and researchers. the presence of numerous mainly color illustrations and the orderly succession of tables render each treated topic easy to consult and encourage further investigation. the text consists of the following five parts: part one, “stato dell’arte dell’ortofrutticoltura italiana” (italian horticulture: state of art); part two, “frutticoltura” (fruitculture); part three, “orticoltura” (horticulture); part four, “produzione integrata e biologica: caratteristiche, disciplinari, gestione” (integrated and organic production: characteristics, disciplines and management); part five, “il mercato e le prospettive future” (the market and future perspectives). enrico rinaldelli produzione ed impiego delle piante officinali. p. catizone, l. barbanti, i. marotti, and g. dinelli. pàtron editore, bologna, 2013. pp. 348. isbn 978-88-555-3233-4. € 44.00. it’s not easy to write a book on medicinal plants. as explained on the back cover of the book: “the term medicinal plants defines a large group of plant species that have been used in pharmaceutical laboratories, but in a broader sense also includes plants for aromas, cosmetics, dyes, biocides and other agricultural purposes”. after a period of declined interest, due to the belief that chemistry would rapidly produce active principles via synthetic processes, in recent years there has been swift development in the study of these plants, which has led to substantial progress in knowledge. the acquisition of this not only scientific and technical knowledge prompted the authors to bring together a text that is both useful for experts in the field and for other readers, such as students, who look this difficult subject for the first time. the subject of medicinal plants calls for multidisciplinary knowledge ranging from botany, arboriculture and natural science to chemistry, pharmaceutical sciences and medicine. indeed, the authors have presented in a concise manner a mass of knowledge and shed innovative light on the subject to eliminate the sense of uncertainty for those who are approaching it for the first time (and not only) and are dealing with the heterogeneous reality that is the world of herbs. after a general introductory part, the text is divided into seven chapters: the first is dedicated to cultivation, the second examines economic aspects, the third regulatory framework, the fourth focuses on metabolism and active principles, while the fifth to seventh chapters examine in detail cultivation and post-harvest processes and utilization. a special part consists of detailed records of individual species (two to three pages each) accompanied by photos of plants, flowers and seeds. the 348-page book also includes an appendix with an extensive glossary, many of which are medical terms. this manual is undoubtedly of great value for students, technicians and all researchers working in the field, but it can be considered interesting for growers of medicinal species at amateur level as well. cinzia silori impaginato 315 adv. hort. sci., 2022 36(4): 315­320 doi: 10.36253/ahsc­12937 grafting compatibility between okra cultivars and root­knot nematode resistant kenaf e.h.c. silva (*), j. versuti, l.t. braz department of agricultural production sciences, são paulo state university, via de acesso prof. paulo donato castellane s/n, cep 14884‐ 900, jaboticabal‐sp, brazil. key words: abelmoschus esculentus l. moench, hibiscus cannabinus l., inter­ generic grafting, meloidogyne spp. abstract: the use of intergeneric grafting has been reported as an alternative to manage root­knot nematodes in okra, but the compatibility for grafting has only been tested in a few okra (abelmoschus esculentus l. moench) cultivars. the kenaf (hibiscus cannabinus l.) is resistant to root­knot nematode species and is a potential rootstock for okra. the objective was to study the compatibil­ ity of kenaf as rootstock with okra cultivars. it was used a completely random­ ized design, in factorial scheme 3x10, with five repetitions. the compatibility was assessed by measuring several vegetative characteristics. all cultivars are compatible for grafting with kenaf as rootstock. grafting onto kenaf may be an option to control root­knot nematodes. 1. introduction the okra (abelmoschus esculentus l. moench) is mainly cultivated for its immature fruits but presents several industrial applications (dantas et al., 2021). in brazil, okra is widely cultivated, mainly by family farmers, as it is considered a low production cost crop that adapts to tropical and subtropical climates. although it is rustic, a limiting factor for the crop has been the root­knot nematodes (meloidogyne spp.) (silva et al., 2019 a), which are favored by high temperature and humidity, which are also nec­ essary for the development of the crop. to control the root­knot nematode, it is necessary to integrate several management practices, from the choice of the planting area to pre­sow­ ing, such as prevention, crop rotation, fallow, and the use of antagonistic plants (collange et al., 2011; nascimento et al., 2020). other approaches, such as organic fertilization, biological control, and heat­based methods are also a possibility (mahalik and sahoo, 2019). however, the use of resistant cultivars is considered the most efficient method, but, to date, there are no reports of genetic resistance effectively incorporated in com­ mercial cultivars. (*) corresponding author: edgarddragde@gmail.com citation: silva e.h.c., versuti j., braz l.t., 2022 ­ grafting compatibility between okra cultivars and root‐knot nematode resistant kenaf. ­ adv. hort. sci., 36(4): 315­320. copyright: © 2022 silva e.h.c., versuti j., braz l.t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 march 2022 accepted for publication 29 november 2022 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-12937 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(4): 315­320 316 recent studies have been exploring promising alternatives, such as intergeneric grafting with species resistant to root­knot nematodes. among the possible rootstocks, some hibiscus spp. have been shown to be interesting for being resistant and com­ patible for grafting with okra (marin et al., 2017; silva et al., 2019 b; andrade et al., 2020). in addition to genetic resistance, rootstocks recommended to man­ age some type of pathosystem must be compatible with the scion, ensuring normal vegetative and reproductive development. it is noteworthy that, by obtaining resistant and compatible rootstock, crop yield can be enhanced by minimizing negative effects generated by the nematodes on the plant. research has tended to focus on only two open­ pollinated cultivars, namely ‘santa cruz 47’ and ‘colhe bem’, with the need to verify whether com­ patibility also occurs in other cultivars, as the com­ patibility may vary according to rootstock­scion com­ binations (reig et al., 2018). given the above, the objective of the present study was to assess the com­ patibility of the kenaf (hibiscus cannabinus l.) with commercial okra cultivars. 2. materials and methods the experiment was carried out at the sector of vegetables and aromatic­medicinal plants, school of agriculture and veterinary sciences, unesp, campus of jaboticabal, sp (21°14’05’’ s, 48°17’09” w, 614 m of altitude), from march to may 2019. the climate type is aw, described as a tropical winter dry season, which occurs from april to september and rains are concentrated from october to march, with transition to cwa. the average temperature and relative humidity during the period of the experiment is pre­ sented in figure 1. a completely randomized design in a factorial scheme 3x10 with five replications was used. the first factor was seedling production modalities, being non­grafted, self­grafted, and grafted onto kenaf. the second factor consisted of ten okra cultivars, being 6 hybrids and 4 open­pollinated cultivars (table 1). the okra seeds were immersed in acetone for 30 minutes to break dormancy, whilst kenaf seeds were immersed in water for five hours. then, the geno­ types were sown in 128­cell expanded polystyrene trays, filled with commercial substrate coconut­fiber based bioplant® (nova ponte, mg, brazil) to produce vegetable seedlings. the trays were placed in a greenhouse equipped with a sprinkler irrigation sys­ tem. the seedlings were cleft­grafted as described by silva et al. (2019 b) when the seedlings presented the cotyledonary leaves fully exposed. the scion was inserted in a cut of approximately 1 cm between the cotyledonary leaves of the rootstock. the scions were standardized to 3 cm in length. the scion was fixed so that it did not shade the cotyledonary leaves. after positioning the scion, a clip was placed to ensure the connection with the rootstock, until com­ plete healing of the grafting site. the grafting was carried out on the seedling production trays. after the grafting was carried out, the plants were placed in a humid floating chamber to secure high temperature and humidity, which is adequate for okra grafting healing. the grafted seedlings remained in the humid chamber for 11 days, and then trans­ planted to pots containing autoclaved clayey red latosol (oxisol) soil. the pots were placed in a green­ house. the percentage of grafting success was assessed fig. 1 ­ changes in temperature and relative humidity during the experiment. table 1 ­ identification and origin of the okra and hibiscus geno­ types used in the experiment cultivar origin pollination brutus feltrin sementes hybrid canindé isla sementes ltda. hybrid clemson isla sementes ltd. open colhe bem sakata seed sudamérica open esmeralda agristar do brasil ltd. hybrid guará isla sementes ltd. hybrid santa cruz 47 feltrin sementes open v8 agristar do brasil ltd. hybrid valença feltrin sementes open xingó eagle flores, frutas e hortaliças ltd. open kenaf embrapa hortaliças open silva et al. ‐ grafting compatibility between okra and kenaf 317 for the self­grafted and grafted onto kenaf treat­ ments. the number of leaves (nl), the number of internodes (ni), and the number of internodes up to the first flower (nif) were counted. the plant height (ph) and first flower height (ffh) were evaluated with the aid of a ruler graduated in centimeters. the diameter of the rootstock (dr) was measured with the aid of a digital caliper. the plants were cut close to the ground when the plants started flowering, which occurred 49 days after grafting, followed by weighing the fresh shoot mass (fm), and the dry shoot mass (dm) was weighed after drying in a forced air circulation oven, set at 60°c for 72 hours. to meet the assumptions of anova, the data was transformed using the box­cox method (box and cox, 1964), except for first flower height, number of internodes up to the first flower and diameter of the rootstock. then, the data was subjected to two­way anova and post­test. the means were compared using the scott­knott test at 5% significance, using the agroestat statistical software (barbosa and maldonado, 2015). 3. results and discussion the percentage of grafting success was evaluated, however, it was not statistically analyzed, since the self­grafted and grafted onto kenaf treatments reached 100% of success for all cultivars, so there was no variance. okra and kenaf are both malva­ ceous species but belonging to different genera, which theoretically hamper grafting healing process as physiological, morphological, and botanical dis­ crepancies are expected to occur (silva et al., 2019 b). however, despite the distinguishing characteris­ tics, adequate development was verified for grafted okra seedlings of all cultivars. thus, all cultivars are compatible with the kenaf rootstock as they present vegetative development similar to the self­grafted and non­grafted seedlings, except for number of leaves, plant height, and first flower height (table 2). according to belmonte­ureña et al. (2020), there is compatibility when the plant formed by scion and rootstock has the capacity to develop as a single plant. these results confirm the compatibility with table 2 ­ analysis of variance and test of comparison of means of vegetative variables of okra cultivars and seedling production modali­ ties ns, **, * not significant or significant at 1 or 5% probability. data transformed according to the box­cox method. means followed by the sameletter do not differ by the scott­knott test, p<0.005. nl = number of leaves, ni = number of internodes, nif = number of internodes up to the first flower, ph = plant height, ffh = first flower height, dr = diameter of the rootstock, fm = fresh shoot mass, dm = dry shoot mass. nl ni nif (cm) ph (cm) ffh (mm) dr (mm) fm (g) dm (g) cultivar (c) brutus f1 8.13 a 8.40 a 6.13 b 42.47 c 28.53 b 14.40 49.70 9.61 a canindé f1 8.67 a 6.66 c 4.33 d 47.93 b 18.40 d 14.35 40.44 7.50 b clemson americano 80 6.00 b 7.06 c 4.86 d 40.40 c 21.80 c 14.46 38.08 7.03 b colhe bem 7.33 a 8.53 a 7.80 a 37.53 d 34.26 a 14.44 45.39 7.96 b esmeralda f1 6.40 b 7.60 b 5.40 c 42.27 c 25.33 c 14.44 41.21 8.25 a guará f1 6.33 b 6.53 c 4.33 d 72.80 a 28.13 b 14.30 37.23 9.79 a santa cruz 47 6.33 b 8.33 a 7.80 a 35.00 d 33.47 a 14.43 40.63 6.65 b v8 f1 5.87 b 7.80 b 6.20 b 37.97 d 27.87 b 14.35 41.87 6.82 b valença 6.73 b 7.26 c 4.86 d 41.47 c 22.60 c 14.41 45.05 8.75 a xingó f1 6.33 b 7.53 b 5.40 c 44.80 b 28.13 b 14.49 51.29 9.37 a test f 4.56 ** 8.93 ** 26.18 ** 40.70 ** 19.59 ** 1.07 ns 0.95 ns 1.98 * p­value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.3960 0.4873 0.0471 seedling production (s) non­grafted 8.58 a 7.68 5.80 47.64 a 29.44 a ­ 44.27 8744 self­grafted 5.94 b 7.62 5.82 46.78 a 28.52 a 14.44 43.64 8145 grafted onto kenaf 5.92 b 7.42 5.52 38.37 b 22.60 b 14.37 41.35 7625 test f 37.77 ** 1.34 ns 1.55 ns 35.62 ** 36.27 ** 3.81 ns 0.86 ns 1.74 ns p­value <0.0001 0.2669 0.2169 <0.0001 <0.0001 0.0543 0.4262 0.1808 interaction c × s 2.40 ** 1.57 ns 1.48 ns 2.53 ** 1.58 ns 1.41 ns 1.18 ns 1.46 ns p­value 0.0027 0.0799 0.1105 0.0015 0.0749 0.1975 0.2884 0.1159 cv (%) 9.47 6.19 16.84 11.20 16.23 1.25 17.21 33.97 adv. hort. sci., 2022 36(4): 315­320 318 okra that, until then, had been demonstrated only for the open­pollinated cultivars ‘colhe bem’ and ‘santa cruz 47’ (marin et al., 2017; silva et al., 2019 b). the interaction between cultivar and seedling production modality was significant only for the number of leaves and plant height (table 2). thus, the seedling production modality influenced the cul­ tivars equally for most traits, even though they are genetically different. the grafting influenced the number of leaves of the cultivars brutus, clemson americano 80, esmeralda, valença, xingó, and v8, as the non­grafted showed higher average than the other seedling production modalities (self­grafting and grafting onto kenaf) (table 3). it should be con­ sidered that grafted plants are subjected to addition­ al stress that involves the grafting and healing process (melnyk, 2017). after grafting, to heal the wound, there is callus formation followed by tissue differentiation to reestablish the vascular connec­ tions (xie et al., 2019). thus, it is expected that the non­grafted plants have a faster initial development, which does not necessarily indicate grafting incom­ patibility. the cultivars differed as to the number of leaves (fig. 2). for non­grafted plants, ‘brutus’and ‘valença’ had the highest number of leaves. ‘canindé’ present­ ed the highest number of leaves self­grafted and grafted onto kenaf (fig. 2). the grafting, either self­ grafting or onto kenaf, reduced the number of leaves and plant height of five cultivars (brutus, esmeralda, v8, valença, and xingó), which may indicate slower initial development due to the stress caused by the grafting procedure. higher non­grafted cultivars tended to result in higher plants when grafted, either self­grafted or onto kenaf (fig. 3). in this sense, ‘guará’ presented the highest value of plant height in all seedling pro­ duction modality. ‘brutus’, ‘canindé’, ‘clemson americano 80’, ‘guará’, ‘valença’, and ‘xingó’ pre­ sented shorter plants when grafted onto kenaf (fig. 3). the plant height should be considered concomi­ tantly with the number and length of internodes (sandeep et al., 2022), as fruit production is directly correlated with number of internodes. higher plants fig. 2 ­ interaction between cultivars and seedling production modality for the number of leaves. means followed by the same uppercase (between seedling production modalities for each cultivar) and lowercase (between cul­ tivars for each seedling production modality) letter do not differ by the scott­knott test (p<0.005). table 3 ­ post­analysis of the interaction between cultivars and grafting for the number of leaves of okra cultivars and seedling produc­ tion the data presented is original, but for statistical analysis, the data were transformed into (x+0.5)1/2. means followed by the same uppercase (row) and lowercase (column) letter do not differ by the scott­knott test (p<0.005). ns = not significant; ** significant at 1% probability; * significant at 5% probability. cultivar number of leaves test f p­value non­grafted self­grafted grafted onto kenaf brutus f1 12.80 aa 6.20 bb 5.40 bb 23.53 ** <0.0001 canindé f1 9.20 b 9.40 a 7.40 a 2.29 ns 0.1059 clemson americano 80 7.40 ba 5.00 bb 5.60 bb 2.92 ns 0.0579 colhe bem 8.00 b 6.00 b 8.00 a 1.90 ns 0.1534 esmeralda f1 8.60 ba 5.20 bb 5.40 bb 6.42 ** 0.0022 guará f1 7.00 b 6.40 b 5.60 b 1.00 ns 0.3708 santa cruz 47 6.40 b 6.20 b 6.40 a 0.03 ns 0.9702 v8 f1 7.60 ba 5.00 bb 5.00 bb 4.30 * 0.0158 valença 10.20 aa 4.80 bb 5.20 bb 15.40 ** <0.0001 xingó f1 8.60 ba 5.20 bb 5.20 bb 7.31 ** <0.0001 test f 4.69** 3.34** 2.09* p­value <0.0001 0.0011 0.0353 silva et al. ‐ grafting compatibility between okra and kenaf 319 with long internodes are not ideal, as the plant will probably reach a stature that will hamper harvesting the fruits sooner. the genotypic variability and the differential behavior as for the seedling production modalities can be confirmed from the analysis of variance and the mean test (table 2). significant differences between cultivars were detected for the variables number of internodes, number of internodes to first flower, first flower height, and scion diameter. furthermore, the seedling production modalities dif­ fered as to first flower height and scion diameter. the cultivars were grouped into two groups for the variables scion diameter and dry shoot mass, into three groups for the variable number of internodes, and into four groups for the variables number of internodes up to the first flower and first flower height. there were no significant differences for the variables diameter of the middle portion of the plant, rootstock diameter and fresh shoot mass. the cultivars colhe bem, brutus, and santa cruz 47 had higher number of internodes. the cultivars colhe bem and santa cruz 47 had higher nif; the cul­ tivars colhe bem and santa cruz 47 had the highest first flower height; the cultivars xingó, colhe bem, esmeralda, v8, valença, guará, and brutus had high­ er scion diameter and the cultivars guará, brutus, xingó, valença, and esmeralda had higher dry shoot mass. as for the factor seedling production modality, besides number of leaves and plant height, differ­ ences were detected for the first flower height and scion diameter, with smaller and larger means for grafting onto kenaf, respectively. this indicates that most variables are not affected by grafting. no differ­ ences were detected for number of internodes to first flower, which suggests that the first flower inser­ tion height differed due to internode length of plants grafted onto kenaf. the yield potential of okra depends considerably on the number of nodes per plant as the inflorescence consists of a single flower (bhatt and rao, 2009), so the smallest first flower height insertion can be advantageous as production starts lower. the scion diameter was greater in plants grafted onto kenaf than self­grafted plants. this differs from the findings reported by andrade et al. (2020), who verified largest diameters for self­grafted ‘santa cruz 47’ plants compared to roselle (hibiscus sabdariffa l.) and other malvaceous rootstocks. the scion diameter depends greatly on the water and nutrients translo­ cation, which can be influenced by the compatibility between rootstock and scion. silva et al. (2019 b) previously reported that kenaf is a better rootstock for okra as it presents greater compatibility com­ pared to other malvaceous genotypes. this result may be associated to the large root volume of kenaf plants (alexopoulou et al., 2013), which improve water and nutrient absorption (gaion et al., 2017). okra grafting is a potential tool to cope with root­ knot nematodes, which are one of the main phy­ tosanitary issues in okra cropping. although most okra farmers employ little technology, we anticipate that grafted seedlings will be a feasible option as grafting will become cheaper when this process is automatized (silva et al., 2019 b). furthermore, graft­ ing onto resistant rootstocks is a sustainable strategy that contributes to lessen pesticides use, which are potentially damaging to the environment and human’s health (thies, 2021). the cultivars brutus, canindé, clemson americano 80, colhe bem, esmeralda, guará, santa cruz 47, v8, valença, and xingó are compatible with the kenaf rootstock, therefore the grafting can be used in okra as a strategy to control root­knot nematodes in all analyzed cultivars. although it is an intergeneric grafting combination, the grafted seedlings showed no sign of incompatibility until flowering. however, further studies are necessary to confirm compatibility throughout the cycle. furthermore, the agronomic performance should be evaluated under field condi­ tions to validate the use of this technique in the okra crop. fig. 3 ­ interaction between cultivars and seedling production modality for plant height. means followed by the same uppercase (between seedling production modalities for each cultivar) and lowercase (between cultivars for each seedling production modality) letter do not differ by the scott­knott test (p<0.005). 10(8): 1683. gaion l.a., braz l.t., carvalho r.f., 2017 ­ grafting in vegetables: a great technique for agriculture ‐ int. j. veg. sci. 24(1): 1­18. mahalik j.k., sahoo n.k., 2019 ­ management of meloidogyne incognita in okra through integrated approach. ­ indian j. nematol., 49(1): 25­30. marin m.v., santos l.s., gaion l.a., rabelo h.o., fran­ co c.a., diniz g.m.m., silva e.h.c., braz l.t., 2017 ­ selection of resistant rootstocks to meloidogyne enterolobii and m. incognita for okra (abelmoschus esculentus l. moench). ‐ chil. j. agric. res., 77(1): 58­64. melnyk c.w., 2017 ­ plant grafting: insights into tissue regeneration. ‐ regeneration, 4(1): 3­14. nascimento d.d., vidal r.l., pimenta a.a., costa m.g., soares p.l.m., 2020 ­ crotalaria and millet as alternative controls of root‐knot nematodes infecting okra. ­ biosci. j., 36(3): 713­719. reig g., zarrouk o., forcada c.f., moreno m.a., 2018 ­ anatomical graft compatibility study between apricot cultivars and different plum based rootstocks. ‐ sci. hortic., 237: 67­73. sandeep n., dushyanthakumar b.m., sridhara s., dasaiah l., satish k.m., el­shehawi a.m., althaqafi m.m., aloufi s., sharma h., alaklabi a., elansary h.o., 2022 ­ characterization of okra species, their hybrids and crossability relationships among abelmoschus species of the western ghats region. ­ horticulturae, 8: 1­18. silva e.h.c., soares r.s., borges h.o., franco c.a., braz l.t., soares p.l.m.. 2019 a ­ quantification of the damage caused by meloidogyne enterolobii in okra. ‐ pesqui. agropecu. bras., 54: e00050. silva e.h.c., soares r.s., diniz g.m.m., franco c.a., marin m.v., candido w.s., braz l.t., soares p.l.m., 2019 b ­ grafting as a management tool to control meloidogyne incognita in okra: identifying rootstocks candidates. ‐ sci. hortic., 246: 354­359. thies j.a., 2021 ­ grafting for managing vegetable crop pests. ­ pest management sci., 77: 4825­4835. xie l., dong c., shang q., 2019 ­ gene co‐expression net‐ work analysis reveals pathways associated with graft‐ ing healing by asymmetric profiling in tomato. ­ bmc plant biol., 19: 373. 320 adv. hort. sci., 2022 36(4): 315­320 acknowledgements the authors thank the coordenação de aperfeiçoamento de pessoal de nível superior and conselho nacional de desenvolvimento científico e tecnológico for their financial support. references alexopoulou e., papatheohari y., christou m., monti a., 2013 ­ origin, description, importance, and cultivation area of kenaf, pp. 1­15. ­ in: monti a., and e. alexopoulou (eds.). kenaf: a multi‐purpose crop for several industrial applications. springer, london. andrade f.l.n., monteiro s.m.f., muniz c.c.s., gomes r.f., santos l.s., 2020 ­ compatibility and yield of ‘santa cruz 47’ okra onto rootstocks of the malvaceae family ‐ pesqui. agropecu. trop., 50(e58368). barbosa j.c., maldonado w. jr., 2015 ­ experimentação agronômica & agroestat: sistema para análises estatís‐ ticas de ensaios agronômicos. ­ unesp, jaboticabal, brazil, pp. 396. belmonte­ureña l.j., garrido­cardenas j.a., cama­ cho­ferre f., 2020 ­ analysis of world research on grafting in horticultural plants. ­ hortscience, 55(1): 112­120. bhatt r.m., rao n.k.s., 2009 ­ physiology: crop growth, development, and yield, pp. 37­60. ­ in: dhankhar b.s., and singh r (eds.). okra handbook: global pro‐ duction, processing, and crop improvement. ­ hnb publishing, new york, pp. 475. box g.e.p., cox d.r., 1964 ­ an analysis of transforma‐ tions. ­ j.r. soc., 26(2):211­252. collange b., navarrete m., peyre g., mateille t., tchamitchian m., 2011 ­ root‐knot nematode (meloidogyne) management in vegetable crop produc‐ tion: the challenge of an agronomic system analysis. ‐ crop prot., 30(10): 1251­1262. dantas t.l., buriti f.c.a., florentino e.r., 2021 ­ okra (abelmoschus esculentus l.) as potential functional food source of mucilage and bioactive compounds with technological applications and health benefits. ­ plants impaginato 293 adv. hort. sci., 2021 35(3): 293­303 doi: 10.36253/ahsc­10740 biochemical changes in pear fruits during storage at ambient conditions a. kaur 1 (*), s. sharma 1, n.p. singh 2 1 department of biochemistry, punjab agricultural university, 141 004 ludhiana, punjab, india. 2 department of fruit science, punjab agricultural university, 141 004 ludhiana, punjab, india. key words: cellulase, minerals, ‘patharnakh’, pectinmethylestrase, polygalac­ turonase, ‘punjab beauty’, quality attributes, ripening physiology, sugars. abstract: ‘patharnakh’ (pn) (pyrus pyrifolia burm. nakai) and ‘punjab beauty’ (pb) [pyrus communis l. × pyrus pyrifolia burm. (nakai)] are leading low­chill pear cultivars of subtropics of india. diurnal temperature and relative humidity during fruit harvest period is high which considerably affect the shelf life of fruits. fruits of ‘pn’ and ‘pb’ pear harvested at physiological maturity were stored for 12 days at ambient temperature and effects of storage temperature on physical and qualitative parameters were studied. both cultivars showed reduction in fruit weight and firmness, reducing sugars, sucrose, starch and pectin content. however, total soluble solids and juice acid content increased during storage. sucrose synthase activity and sucrose content showed signifi­ cant positive correlation in ‘pn’ cultivar. activities of fruit softening enzymes such as polygalacturonase (pg) and cellulase was enhanced; whereas, pectin­ methylesterase (pme) was reduced during storage. fruit firmness was negative­ ly correlated with pg in both the cultivars. in ‘pn’ cultivar, fruit firmness was positively correlated with cellulase and negatively with pme enzyme but reverse trend was observed in ‘pb’ cultivar. fruit minerals content didn’t show any substantial disparities in both the cultivars during storage. ‘patharnakh’ and ‘punjab beauty’ fruits maintain desirable quality parameters up to 6­9 days and 3­6 days, respectively, during storage at ambient conditions. 1. introduction pear (pyrus spp.) ranks second next to apple fruit crop in the world in terms of area, production and varietal wealth among temperate fruits. it belongs to the family rosaceae and sub family pomoideae. in india, it is cultivated in himachal pradesh, uttarakhand, punjab, jammu & kashmir and some parts of assam and nilgiris hills. in punjab province of north­ west india, the area under pear cultivation is dominated by low chill culti­ var ‘patharnakh’ that belongs to oriental or sand pear group (pyrus pyri‐ folia burm. nakai) and semi soft pear cultivar ‘punjab beauty, a hybrid between pyrus communis l. × pyrus pyrifolia burm. (nakai) (sharma and (*) corresponding author: avninderkaur12@gmail.com citation: kaur a., sharma s., singh n.p., 2021 ­ biochemical changes in pear fruits during storage at ambient conditions. ­ adv. hort. sci., 35(3): 293­303 copyright: © 2021 kaur a., sharma s., singh n.p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 march 2021 accepted for publication 3 august 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10740 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 293­303 294 singh, 2011) and fruits are harvested at physiological maturity during iind fortnight of july. diurnal tempera­ ture and relative humidity during fruit harvest period is high which considerably affect the shelf life of fruits. it is documented that pear fruits have post­ harvest shelf­life of about 10 days at ambient condi­ tions (25­30°c) and quality related parameters are reduced rapidly during storage (nath et al., 2011). after harvest, consumers’ preference and market price depends on fruit’s attractive colour, flavor, taste, aroma and firmness. the variability observed in volatile organic compounds, physico­chemical and sensory parameters can be used to understand the ripening behavior of pear cultivars (taiti et al., 2017). it is suggested that fruits should be harvested at opti­ mal physiological maturity and kept under optimal storage conditions to enhance the shelf life of fruits (hafez et al., 2019). fruit quality deteriorates after harvest due to rapid change in respiration, activity of cell wall degradation enzymes and infestation of pathogens during transportation and storage (ge et al., 2017). the quality related attributes constantly depend on the storage temperature which primarily affect fruit freshness and shelf life. quantification of organic acids and soluble sugars (sucrose, glucose and fruc­ tose) are correlated to the production of quality fruits (itai and tanahashi, 2008). sugars content in pear fruit improve during early storage period and further decline with the advancement of storage period at ambient conditions is due to fermentation into alco­ holic content (kaur and dhillon, 2015). softening is associated with the degradation of cell wall polysac­ charides and biosynthesis of cellulase, polygalactur­ onase and pectin methyl esterase enzymes (zhou et al., 2011). fruit minerals content can also modify the quality attributes and storability (saquet et al., 2019). it is well recognized fact that pome fruits are harvest­ ed at proper maturity stage and must be stored under explicit low temperature to extend the shelf life with­ out exhibiting any deterioration in fruit quality attrib­ utes (itai et al., 2015; yu et al., 2016). however, less information is available on the ripening behavior of pear fruits harvested at physiological maturity (135 dafs) and kept in ambient conditions and subse­ quently, its effect on the biochemical composition during storage. therefore, the study was performed to record the changes in physical characteristics, sug­ ars composition, activities of hydrolytic fruit softening enzymes and minerals profile during storage of pear fruits at ambient conditions. 2. materials and methods experimental procedure fruits of ‘patharnakh’ ‘pn’ and ‘punjab beauty’ ‘pb’ cultivars (fig. 1) grafted on kainth rootstock (pyrus pashia) were harvested during iind fortnight of july (135­145 days after fruit set; dafs) from the orchard situated at research farm, department of fruit science, punjab agricultural university, ludhiana (india) (30.90o n, 75.86o e). fifteen fruits/replication free from any type of visual injury and bruises of each cultivar were washed with sodi­ um hypochlorite 4% (2.5 ml l­1) solution for 5 min­ utes (pau, 2020). fruits were dried in shade and packed in three ply corrugated fiberboard with 5% perforation and stored at ambient temperature (28±2oc). physico­chemical parameters, physiological changes and enzymatic activities were estimated after the intervals of 0, 3, 6, 9 and 12 days of storage. physiological loss in weight (plw) fruits stored at ambient temperature were weighed before storage and at a subsequent storage interval. the values were expressed as plw (%) (singh et al., 2021). fruit firmness fruit firmness was measured at every storage fig. 1 ­ fruits of ‘patharnakh’ (top) and ‘punjab beauty’ (bottom) cultivars at physiological maturity. kaur et al. ‐ ripening of pear fruits during storage 295 interval with penetrometer (model no. ft­327, qa supplies llc, usa) and values were expressed lbs (mahajan et al., 2010). total soluble solids, titratable acidity and fruit color coordinates titratable acidity (ta) was determined with titra­ tion method described by ranganna (2007) and expressed as percent of maleic acid. fruit color coor­ dinates (l*, a*, b*, c* and h*) were randomly mea­ sured on two opposite sites at fruit equator using color flex spectrophotometer (hunter lab color flex, hunter associates inc., reston, va, usa). these coor­ dinates were expressed in cie units (hunter, 1975). sugars fruit pulp was homogenized with 80% ethanol and refluxed twice for 20 min. the supernatants were pooled to evaporate ethanol and volume was made 10 ml with distilled water. this extract was used for the estimation of reducing sugars, fructose and sucrose by the methods already described by kaur et al. (2018). for the estimation of fructose, 0.1% resorcinol reagent and 30% hcl were added to sugar extract and color intensity was recorded at 540 nm. estimation of sucrose was done using the same procedure except that free fructose was destroyed by treating the sample with 6% koh and the absorbance was measured at 490 nm. the residue left after sugar extraction was dried and treated with perchloric acid to hydrolyze starch into simpler sug­ ars and were estimated using the method of dubois et al. (1956). sucrose metabolizing enzymes enzymes viz. sucrose synthase (ss), sucrose phos­ phate synthase (sps) and invertases (acid and neu­ tral) were extracted from fruit pulp using hepes­ naoh buffer (ph 7.5) and assayed by the methods described by asthir and singh (1995) and singh et al. (1978). for ss assay, 0.1 ml fructose (150 mm), 0.1 ml udpg solution (20 mm) and 0.2 ml enzyme extract were incubated for 30 min at 37°c, followed by addition of 0.1 ml of 30% koh and contents were boiled. added 1 ml resorcinol reagent and 3 ml of hcl and tubes were kept for 10 min at 80°c. after cooling the tubes, the absorbance was noted at 490 nm. for sps assay, fructose­6­phosphate (150 mm) was used as substrate and enzyme activity was expressed as mg sucrose formed g­1 min­1 fresh weight (fw). for acid invertase, 0.6 ml sodium acetate buffer (0.2 m, ph 4.8), 0.2 ml sucrose (50 mm) and 0.2 ml of enzyme extract were incubated for 1 h at 37 °c fol­ lowed by addition of 1 ml nelson reagent c. contents were boiled for 20 min and then 1 ml nelson reagent d and 7 ml of distilled water was added and mixed well. absorbance was read at 510 nm. sodium phos­ phate buffer (0.2 m, ph 7.5) was used for neutral invertase assay in place of acetate buffer and rest of the procedure was same as described for acid inver­ tase. invertase activity was expressed as mg glucose formed min­1 g­1 fw. pectin content and cell wall degrading enzymes for pectin content, 50 g fruit pulp and 50 ml of 0.01 n hcl were boiled for 30 min and supernatant was collected. the process was repeated twice using 0.05 n and 0.3 n hcl and volume of filtrate was made to 100 ml. two ml of diluted extract was neutralized using 1 n naoh. to this, calcium chloride was added next day for precipitation. precipitates were collect­ ed, weighed and % calcium pectate content was cal­ culated (okimasu, 1956). fruit pulp was crushed with 0.1 m sodium acetate buffer (ph 5.2) and super­ natant was used for the assay of cellulase and poly­ galacturonase enzymes. for cellulase, 1 ml of 0.1 m sodium acetate buffer (ph 5.2), 1 ml of 0.5% car­ boxymethyl cellulose (prepared in buffer) and 1 ml of enzyme extract were incubated for 1 h at 55 °c, one ml of dintrosalicyclic acid was added to terminate the reaction. the contents were boiled for 10 min and absorbance recorded at 560 nm. enzyme activity of cellulase was expressed as mg glucose released min­1 g­1 fw. pectic acid (0.5%) was used as substrate for pg assay and enzyme activity was expressed as mg galacturonic acid released min­1 g­1 fw (malik and singh, 1980). fruit tissue was crushed with 0.1 m cit­ rate phosphate buffer (ph 5.0) and supernatant obtained was used for pme enzyme assay. for reac­ tion, 2 ml of 1% pectin, 2 ml of 0.1 m citrate phos­ phate buffer (ph 5.0) and 1 ml of enzyme extract were incubated at 35 °c. from this reaction mixture, 1 ml was pipetted out at 0 and 1 h of the incubation and titrated against 0.005 n naoh. the pme activity was expressed as milliequivalents of methoxyl groups released min­1 by 1 ml of enzyme (balaban et al., 1991). mineral’s analysis for nitrogen (n) estimation, dried powder of fruits was digested with h2so4 and content were deter­ mined using kjeldahl method (gehrke et al., 1972). phosphorus (p) and potassium (k) in fruit samples were digested with a mixture of nitric acid and per­ adv. hort. sci., 2021 35(3): 293­303 296 fruit color color coordinates depicting peel color where l* expresses as lightness, a* positive value measures the red intensity and negative value as green color; b* positive value measures yellow color intensity. the value of b* coordinate was improved in both the pear cultivars during storage being highest in ‘pb’ and lowest in ‘pn’ cultivar. however, hue angle (h*) showed the reverse trend (table 1). significant improvement in a* values from 9 to 12 das in ‘pn’ cultivar was observed; however, other color coordi­ nates showed non­significant variations when the storage period was increased from 3 to 12 days. initial negative a*values indicated greener colour at zero day as compared to 12 days of storage in ‘pn’ cultivar. chloric acid. p estimation was done by the method described by jackson (1973) and k by flame photo­ metric method (aoac, 1990). nutrients like ca, mg, cu, zn, fe and mn were determined using atomic absorption spectrophotometer (perkin elmer analyst 200). the instrument optimization, calibration and elemental analysis were carried out using winlab32 software as described by bradfield and spencer (1965). statistical analysis the experiment was conducted during the year 2020 in a complete randomized design with four replications. two hundred and forty fruits of each cultivar for different storage intervals were stored at ambient temperature. a lot of 60 fruits for each stor­ age interval with 15 fruits/replication were stored in cardboard boxes. the data was analyzed by one­way analysis of variance. the differences were considered statistically significant at the level p value of < 0.05 using software cpcs1 developed by pau, ludhiana and wasp 2.0. experimental data was represented as mean ± standard error. the data were subjected to pearson’s correlation analysis to assess the relation­ ship between attributes. principal component analy­ sis (pca) was used to examine the interrelations between different quality parameters. 3. results physical characteristics physiological loss in weight (plw) of ‘patharnakh’ (pn) and ‘punjab beauty’ (pb) pear cultivars increased during different storage intervals and the higher rate up to 4.75 to 8.18 % was noted in ‘pb’ between 6 to 9 days compared to 3.21 to 4.16 % in ‘pn’ at ambient storage conditions (fig. 2a). the val­ ues of reduction in fruit firmness were increased with advancement of the storage period in both the culti­ vars. the rate of softening of ‘pn’ fruits was lower than that of ‘pb’ fruits and values were higher between 6 to 9 days in ‘pn’ and 3­6 days in ‘pb’ culti­ var (fig. 2b). during storage, values ranged from 11.6 lbs at 0 day to 9.35 lbs at 12 days in ‘pn’ and 10.75 lbs at 0 day to 8.38 lbs at 12 days in ‘pb’. total soluble solids and titratable acidity tss content increased in ‘pb’ fruits during storage with the mean value of 14.97o brix and a significant rise in values was recorded from 13.39o brix at 3 das to 16.98o brix at 9 das (fig. 2c). ‘pn’ cultivar showed significant variations in tss content up to 6 days after storage and values varied from 11.05o brix to 11.63o brix. there was a significant increase in juice acid content from 3 das to 9 das and then values remained almost comparable until 12 days of storage in both the cultivars (fig. 2d). ‘pb’ showed higher acidity values at all the storage intervals as compared to ‘pn’ cultivar. fig. 2 ­ changes in physiological loss in weight (a), fruit firmness (b), tss (c) and titratable acidity (d) of pear fruits during storage at ambient conditions. vertical bars represent ± se of means for 4 replicates. different letters indicate the significant differences among storage periods according to wasp 2.0 (p≤0.05). kaur et al. ‐ ripening of pear fruits during storage 297 carbohydrate composition and sucrose metabolizing enzymes reducing sugars increased up to 3 das in ‘pn’ and 6 das in ‘pb’ fruits and then declined during advanced storage period (fig. 3a). fructose content increased in pear fruits from harvest to 6 das and later showed a declining trend up to 12 das in both the cultivars (fig. 3b). in ‘pb’ fruits, sucrose content did not show any differences until 9 days of storage and values were declined at 12 das (fig. 3c). starch fig. 3 ­ changes in reducing sugars (a), fructose (b), sucrose (c) and starch (d) content of pear fruits during storage at ambient conditions. vertical bars represent ± se of means for 4 replicates. different letters indicate the significant differences among storage periods according to wasp 2.0 (p≤0.05). fig. 4 ­ variations in activities of sucrose metabolizing enzymes: ss (a), sps (b), ai (c) and ni (d) of pear fruits during sto­ rage at ambient conditions. vertical bars represent ± se of means for 4 replicates. different letters indicate the significant differences among storage periods according to wasp 2.0 (p≤0.05). table 1 ­ changes in fruit color coordinates of pear fruits during storage at ambient conditions fruit color days after storage cd (p<0.05) 0 3 6 9 12 ‘patharnakh’ (pn) l* 59.00 ± 4.24 61.12 ± 1.91 64.61 ± 2.10 66.07 ± 2.54 67.20 ± 2.19 ns a* ­1.50 ± 1.22 ­2.26 ± 1.05 ­0.83 ± 1.62 0.17 ± 0.58 3.24 ± 1.40 1.86 b* 41.54 ± 2.65 40.90 ± 2.12 43.90 ± 1.36 46.09 ± 1.03 50.37 ± 1.74 ns c* 41.58 ± 2.68 40.97 ± 2.16 43.92 ± 1.38 46.09 ± 1.03 50.49 ± 1.69 ns h* 92.00 ± 1.59 93.13 ± 1.37 91.03 ± 2.09 89.78 ± 0.72 86.30 ± 1.66 ns ‘punjab beauty’ (pb) l* 65.34 ± 1.52 63.49 ± 2.42 63.44 ± 1.72 68.45 ± 0.82 66.67 ± 1.17 ns a* 3.59 ± 2.35 4.49 ± 1.16 4.14 ± 2.74 1.88 ± 1.19 ­0.89 ± 0.54 ns b* 41.38 ± 1.58 43.25 ± 2.79 43.09 ± 1.07 47.84 ± 1.39 52.16 ± 2.00 ns c* 41.57 ± 1.66 43.49 ± 2.85 43.35 ± 1.20 47.88 ± 1.39 52.16 ± 2.00 ns h* 94.93 ± 3.14 95.89 ± 1.32 95.42 ± 3.59 92.25 ± 1.44 90.96 ± 0.55 ns content increased initially until 3 days and then showed declined trend up to final storage interval (fig. 3d). both starch and sucrose content improved up to 6 das and a decrease in its content was observed from 6 to 12 das in ‘pn’ fruits. sucrose synthase (ss) enzyme showed fluctuation in values in both the cultivars with the advancement of storage period (fig. 4a). after 12 days of storage; ss activity was about 2­fold higher in ‘pb’ than ‘pn’ fruits. in ‘pn’ cultivar, sucrose phosphate synthase 298 adv. hort. sci., 2021 35(3): 293­303 (sps) activity increased at 3 das and exhibited a steady variation with less effectiveness until 6 das. in ‘pb’ cultivar, sps activity during initial storage period decreased significantly and later showed an upsurge to 6th das by 1.5­fold from the initial values and comparably had higher values than ‘pn’ cultivar. at 6 das, both cultivars showed a decline in sps enzyme activity up to 9 days of storage followed by an upsurge up to 12 das (fig. 4b). acid invertase (ai) activity increased from 0 to 3 das in both the culti­ vars and subsequently declined at 6 das followed by a significant enhancement with progression in stor­ age at ambient temperature (fig. 4c). neutral invertase (ni) activity increased up to 3 days of stor­ age followed by a declining trend after 6 days of stor­ age in both the cultivars. in ‘pn’ cultivar, ni activity increased until 9 das but decreased progressively afterwards. in ‘pb’ cultivar, ni activity decreased from 3 to 12 das (fig. 4d). in both the cultivars, reducing sugars, fructose and sucrose attributes were correlated positively (data not shown). these sugars presented non­significant negative relationships with plw except in reducing sugars with plw (r= ­0.483; p ≤ 0.05). in ‘pn’ cultivar, substantially positive correla­ tion between sucrose accumulation and ss activity (r= 0.46; p≤0.05) and non­significant correlation with sps (r= 0.09) was observed (table 2). in ‘pb’ cultivar, sucrose exhibited negative correlation with ss (r= ­ 0.73, p≤0.01) and sps (r= ­0.54, p≤0.05). ai activity was non­significantly and negatively correlated with sucrose accumulation in ‘pn’ (r= ­0.38) and ‘pb’ (r= ­ 0.43) cultivar. ni activity and sucrose content were negatively correlated in both pear cultivars. pectin and cell wall degrading enzymes total pectin content was decreased significantly in both the cultivars during storage (fig. 5a). pg activity increased significantly in both the cultivars during progression of storage period and values were 1.54 and 2.12­fold higher during last storage period com­ fig. 5 ­ changes in activities of cell wall degrading enzymes as pectin (a), pg (b), cellulase (c) and pme (d) in pear fruits during storage at ambient conditions. vertical bars repre­ sent ± se of means for 4 replicates. different letters indi­ cate the significant differences among storage periods according to wasp 2.0 (p≤0.05). table 2 ­ correlation between sucrose metabolizing enzymes and sucrose accumulation in pear cultivars during storage * correlation is significant at the p≤0.05. ** correlation is significant at p≤0.01. pared to harvest stage in ‘pn’ and ‘pb’, cultivars, respectively (fig. 5b). cellulase activity also enhanced in pear fruits during storage but showed a significant declining trend from 6 to 12 days of storage in both the cultivars under ambient conditions (fig. 5c). pme activity was 1.18 (‘pn’) and 1.24­fold (‘pb’) lower until 6 days of storage period (fig. 5d). thereafter, an increment in pme activity up to 12 das was noticed in both the cultivars. in ‘pb’, fruit firmness was negatively correlated to cellulase (r= ­0.632) and pg (r= ­0.857) activities and values were significant at 1% level of significance. pme activity was positively correlated to firmness in these fruits (r= 0.450) at 5% level of significance dur­ traits ‘patharnakh’ ‘punjab beauty’ sucrose ss sps ai sucrose ss sps ai sucrose synthase 0.460* ­0.730* sucrose phospate synthase 0.085 0.766** ­0.543* 0.632* acid invertase ­0.376 0.405 0.529* ­0.427 0.486* ­0.109 neutral invertase ­0.372 ­0.053* ­0.617** 0.267 ­0.723** ­0.597** ­0.835** ­0.024 kaur et al. ‐ ripening of pear fruits during storage 299 ing storage at ambient conditions. in ‘pn’ fruits, pg activity showed a significant negative correlation with fruit firmness (r= ­0.738, p≤0.01) and pme (r= ­ 0.523, p≤0.05) and a positive correlation with cellu­ lase enzyme (r= 0.624, p≤0.01). minerals nitrogen content decreased significantly at 3 das by 1.6­fold in both the cultivars and then increased from 6 to 12 das (table 3). phosphorus content in both the pear cultivars varied non­significantly during storage. potassium content in ‘pn’ significantly increased between 3 to 6 days of storage period and then values remained higher until 12 das in both the cultivars. magnesium content in both the cultivars showed almost similar pattern during storage inter­ vals and significantly lower values at 12 das in ‘pn’ and at 9 das in ‘pb’ fruits were observed. calcium content in fruits of both the cultivars enhanced signifi­ cantly until 3 das. iron and zinc content decreased significantly in both the cultivars from harvest to 12 days of storage periods under ambient conditions (table 4); whereas, manganese content displayed a reverse trend in ‘pb’ fruits. copper content was sub­ stantially lower during different storage intervals in comparison to harvest stage in both the cultivars. principal component analysis (pca) biplot for pc1 and pc2 in pear fruits are given in figure 6. the results showed that first two compo­ nents explained 62.8% and 71.4% of the total vari­ ability in ‘pn’ and ‘pb’ cultivars, respectively. in ‘pn’ cultivar, pc1 includes sucrose, cellulase, starch, pg, ai and acidity attributes which explained 35.9% of total variability. pc2 comprises reducing sugars, fruc­ tose, firmness, pectin, and pme parameters and showed total variability of about 26.9% (fig. 6a). in ‘pb’ cultivar, pc1 includes cellulase, sps, tss, pg and acidity characteristics that described 51.8% of total variability. pc2 comprises reducing sugars, fructose, sucrose, starch, ni, firmness, pectin and pme that described 19.6% of the total variability in physico­ chemical parameters in ‘pb’ cultivar during storage at ambient conditions (fig. 6b). 4. discussion and conclusions physiological loss in weight (plw) consists of metabolic activities, respiration and transpiration, table 3 ­ macrominerals (%) content in pear fruits during storage at ambient conditions table 4 ­ microminerals (mg kg­1 dw) content in pear fruits during storage at ambient conditions days after storage n p k mg ca ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ 0 0.11 ± 0.01 0.13 ± 0.01 2.95 ± 0.09 2.68 ± 0.24 0.93 ± 0.06 1.07 ± 0.06 1.48 ± 0.02 1.45 ± 0.01 0.57 ± 0.02 0.47 ± 0.03 3 0.07 ± 0.00 0.08 ± 0.00 2.91 ± 0.14 2.48 ± 0.20 0.83 ± 0.06 1.07 ± 0.06 1.49 ± 0.02 1.42 ± 0.04 0.68 ± 0.02 0.62 ± 0.03 6 0.12 ± 0.01 0.09 ± 0.01 2.88 ± 0.14 2.65 ± 0.05 1.20 ± 0.00 1.03 ± 0.06 1.48 ± 0.02 1.44 ± 0.03 0.39 ± 0.02 0.33 ± 0.03 9 0.12 ± 0.00 0.12 ± 0.01 2.74 ± 0.11 2.58 ± 0.11 1.17 ± 0.06 1.20 ± 0.10 1.43 ± 0.04 1.19 ± 0.05 0.47 ± 0.02 0.32 ± 0.01 12 0.13 ± 0.00 0.13 ± 0.00 2.72 ± 0.12 2.42 ± 0.09 1.40 ± 0.10 1.17 ± 0.06 1.30 ± 0.02 1.36 ± 0.07 0.28 ± 0.00 0.25 ± 0.01 mean 0.11 ± 0.00 0.11 ± 0.01 2.84 ± 0.12 2.56 ± 0.14 1.11 ± 0.05 1.11 ± 0.07 1.43 ± 0.02 1.37 ± 0.04 0.48 ± 0.02 0.40 ± 0.02 cd(p≤0.05) 0.01 0.02 ns ns 0.16 ns 0.05 0.08 0.04 0.05 days after storage fe zn mn cu ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ ‘patharnakh’ ‘punjab beauty’ 0 83.73 ± 2.87 73.80 ± 2.51 109.13 ± 5.14 188.1 ± 7.69 27.33 ± 0.31 43.40 ± 0.40 94.00 ± 1.40 201.3 ± 6.07 3 30.47 ± 2.21 37.47 ± 3.95 38.13 ± 1.62 51.27 ± 4.57 28.27 ± 1.62 44.20 ± 0.92 27.73 ± 1.42 52.27 ± 0.50 6 26.67 ± 0.95 26.20 ± 0.69 52.60 ± 2.27 35.53 ± 1.81 30.07 ± 1.72 58.13 ± 3.51 43.07 ± 1.94 31.00 ± 2.31 9 21.53 ± 2.12 1.27 ± 0.12 38.40 ± 3.94 45.73 ± 1.89 37.13 ± 1.33 83.80 ± 1.44 27.47 ± 1.55 44.87 ± 2.96 12 4.47 ± 0.42 0.40 ± 0.20 54.47 ± 5.62 48.93 ± 4.10 31.93 ± 3.19 87.20 ± 4.61 58.80 ± 1.77 50.87 ± 5.03 mean 33.37 ± 1.71 27.83 ± 1.49 58.55 ± 3.72 73.92 ± 4.01 30.95 ± 1.63 63.35 ± 2.18 50.21 ± 1.62 76.05 ± 3.37 cd0.05 3.52 3.85 7.34 8.30 3.41 4.92 2.97 7.11 adv. hort. sci., 2021 35(3): 293­303 300 water pressure gradient between fruit tissues, envi­ ronment, stage of ripening as well as storage temper­ ature (ma et al., 2014; hafez et al., 2019). it acts as a detrimental factor to aggravate the fruit freshness, which might be associated with loss of moisture from the tissue (barman et al., 2014). a nonsignificant rela­ tionship between ssc and weight loss in patharnakh pear during storage was reported by kaur et al. (2019). fruit firmness is considered as an important index of texture and storage life of pears. bhat et al. (2012) reported a significant reduction in pear fruit firmness with the lowest value after 15 days of stor­ age. softening of pear fruit during storage could be partly attributed to an increase in depolymerization and degradation of the cell­wall polysaccharides con­ taining pectin, hemicellulose, and cellulose; and loss of moisture (nath et al., 2011). charoenchongsuk et al. (2015) observed a slight variation in hunter values and hue angle of ‘la france’ pears during storage. although l*, a*, b* and c* values showed an increas­ ing trend with storage but these values are not con­ sidered for maturity indices of pear fruits. increment in tss content may be due to breakdown of organic polymers into simple sugars as reported by mahajan and singh (2014) or dehydration of fruits and trans­ formation of pectic substances (dave et al., 2017). titratable acid content of fruit helps in keeping the fruit taste and flavor (sajid et al., 2019). the increase in ta during storage may be due to conversion of sugars to organic acids and their utilization as a source of energy. similar findings have been reported by piga et al. (2003) in cactus pear and ‘bartlett’ pear (bhat et al., 2012) during storage. the reduction in sugars is characterized by higher respiration during storage; whereas, sugars and acids are readily used as substrates for metabolic process­ es (ackermann et al., 1992) or fermentation of over­ ripe fruits which converts sugars into alcohol (kaur and dhillon, 2015). the decrease in fructose content with advanced storage has also been reported by chen et al. (2006) and dave et al. (2017). ss, sps and invertases enzymes substantially regulate sucrose synthesis in plants. these findings are corroborated with the observations reported by chen et al. (2019) and they explained that activities of ss and sps increased during initial storage period. duan et al. (2019) reported that activities of ss cleavage and syn­ thesis of isozymes was increased until 7 das in pears and subsequently, decreased during storage period. itai et al. (2015) opined that higher activity of acid invertase from 6 to 12 das considerably declines sucrose content in japanese pears. acid invertase has the highest level during initial storage period in pear fruits (itai and tanahashi, 2008). a similar trend of ni activity was observed by ren et al. (2020). the decline in sucrose content until 12 days of ambient storage (fig. 3d) might be due to conversion into free sugars by various enzymes including ss and inver­ tases (itai and tanahashi, 2008). these enzymes also exhibited similar trends in both the cultivars and high temperature improved their activities as shown in fruits of loquat (wei et al., 2017). sps synthesizes sucrose­6­phosphate molecule which results in the conversion to sucrose by sucrose­6­phophate phos­ phatase enzyme. invertase enzymes cleave sucrose into glucose and fructose content. a positive correla­ tion between ss and sucrose content in ‘pn’ cultivar fig. 6 ­ biplot for quality parameters in pear fruits of ‘patharnakh’ (a) and ‘punjab beauty’ (b) cultivars during storage at ambient conditions. kaur et al. ‐ ripening of pear fruits during storage 301 suggests that sucrose is synthesized during storage; whereas, a significant negative correlation between sucrose and ss, sps and invertases depicts sucrose cleavage in ‘pb’ cultivar. cell wall degrading enzymes play an important role in fruit ripening. pme does not have pronounced effect on deviation in the texture of ripening fruit and partial demethylation of pectin occurs before pg causes significant hydrolysis. thus, pme may function to prepare the substrate for hydrolysis by pg (awad and young, 1979). pg catalysis the hydrolysis of (1→4) galacturonan linkages of demethylated pectin and releases shorter chains, thereby causing the depolymerization and dissolution of pectin (singh and dwivedi, 2008), cell wall dissolution, and ulti­ mately, fruit softening (brummell et al., 2004). cellulase acts on cell wall components such as cellu­ lose and xyloglucan of hemicelluloses (chen et al., 2015). in the present studies, the degradation of sol­ uble pectin is related to the higher pg activity in the fruits during ambient storage resulting in softening of flesh. zhou et al. (2011) also observed that a reduc­ tion in pectin content in pear fruits during storage might be due to higher depolymerization of cell wall polysaccharides and conversion of pectin’s to non­ soluble form. correlation studies revealed a negative relationship between fruit firmness, cellulase and pg enzymes in ‘pn’ and ‘pb’ cultivars. the activity of cel­ lulase and pg enzymes increased in both the cultivars which causes decrease in fruit firmness with pg as main enzyme contributing to the degradation of cell­ wall polysaccharides. this relation revealed that the cell wall polysaccharides in pear were associated with the fruit softening. in fruits, optimal concentration of n and k allows a proper development of peel color, fruit size, firm­ ness, tss, acidity, juiciness, flavor, and aroma. high n content reduces the fruit storability and k is also an important nutrient during storage of fruits to main­ tain k: ca ratio (brunetto et al., 2015). lepaja et al. (2018) reported that ‘williams’ pear fruit contains 7.83 mg kg­1 p, 152.67 mg kg­1 k, 11.33 mg kg­1 mg, 10.60 mg kg­1 ca, 1.11 mg kg­1 fe, 1.17 mg kg­1 zn and 1.14 mg kg­1 cu during storage. the concentration of n 3.7 g kg­1, p 1.0 g kg­1, k 10.3 g kg­1, mg 0.4 g kg­1, fe 15 mg kg­1, mn 3.2 mg kg­1, zn 8.9 mg kg­1 and cu 6.1 mg kg­1 was recorded in ‘rocha’ pear fruit after stor­ age for 22 days (saquet et al., 2019). principal component analysis (pca) is a multivari­ ate technique to analyze the observations which are described by inter­correlated variables. the sugars are clustered together in one group indicating posi­ tive correlations with each other and juice acidity, ss and pg enzymes in second group had positive rela­ tionships but both groups had exhibited negative cor­ relations during storage. similar findings have been reported in pome fruits (billy et al., 2008; linda­ garcia et al., 2019; li et al., 2019). this study represents the shelf­life of fruits of pear cultivars ‘patharnakh’ and ‘punjab beauty’ during storage under ambient conditions. the results showed loss in weight, firmness, pectin and sugar content in fruits of both the cultivars. the activities of cellulase, pg and pme showed the positive effect on fruit softening; hence spoilage occurred during stor­ age of fruits. it can be summarized from the results that reduction in sugar content and fastening of activ­ ities of cell­wall degrading enzymes between 6­9 days after storage in ‘patharnakh’ and 3­6 days in ‘punjab beauty’ fruits makes them less desirable for further storage under ambient temperature conditions. references ackermann j., fischer m., amado r., 1992 ­ changes in sugars, acids, and amino acids during ripening and storage of apples (cv. glockenapfel). ­ j. agric. food chem., 40: 1131­1134. aoac, 1990 ­ official and tentative methods of analysis. ­ association of official analytical chemists, aoac, va, usa, pp. 965. asthir b., singh r., 1995 ­ fluoride‐induced changes in the 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8­16. zhou r., li y., yan l., xie j., 2011 ­ effect of edible coat‐ ings on enzymes, cell‐membrane integrity, and cell‐wall constituents in relation to brittleness and firmness of ‘huanghua’ pears (pyrus pyrifolia nakai, cv. huanghua) during storage. ­ food chem., 124(2): 569­575. impaginato 53 adv. hort. sci., 2020 34(1s): 53­59 doi: 10.13128/ahsc­8292 new active packaging for improving the shelf life and quality of tomato g. cocetta 1 (*), m. riva 2, g. castelli 3, a. ferrante 1 1 department of agricultural and environmental sciences, università degli studi di milano, via celoria, 2, 20133 milano, italy. 2 saes getters s.p.a., 20020 lainate (mi), italy. 3 saes coated films s.p.a., 20877 roncello (mb), italy. key words: modified atmosphere packaging, postharvest, respiration, shelf life, solanum lycopersicum. abstract: packaging materials play an important role in the quality preservation during postharvest storage and shelf life of fruits and vegetables. the proper­ ties of film can affect the gas composition and the physiology of the products. the group research labs of saes getters s.p.a. have developed, in collabora­ tion with its affiliate company saes coated films s.p.a. a packaging system highly selective for ethylene, including a coating (1­2 µm), with good trans­ parency and an additional anti­fog function, which can be deposited on various plastic films. the objective of the work was the evaluation of this new active packaging on ‘cherry’ type tomato berries during storage. the control consisted of a macro­perforated polypropylene film used for flow­pack packaging. two experimental tests were conducted, the first during cold storage (10 ± 2°c) and the second at room temperature (22 ± 2°c). the analyses included visual appearance, weight loss, gas exchanges, relative humidity, volatile organic compounds (voc), colour, titratable acidity, refractometric index (° brix) and the concentration of lycopene and β­carotene. the use of the innovative pack­ aging did not alter the main quality indicators of the tomato and the parameter that had the greatest impact on the product metabolism was the storage tem­ perature. the voc concentration was influenced by packaging, with a greater accumulation in the samples packed with active packaging compared to the control. moreover, the same material determined a reduction in weight loss, a greater accumulation of co2 and a significant reduction in o2, especially at 22°c. at the end of the storage, the berries stored in the active packaging showed higher levels of lycopene, compared to controls, and a reduction in β­carotene, indicating an active role of the material in modulating the content of bioactive compounds in the fruits. it can be concluded that the use of this innovative material can represent an effective tool for improving the postharvest manage­ ment of tomato berries. 1. introduction the preservation of produce quality during postharvest depends on the packaging materials and storage conditions. the postharvest perfor­ mance of produce is affected by pre­harvest factors and quality at harvest (sharma et al., 2014; tyagi et al., 2017). it is well known that quality is (*) corresponding author: giacomo.cocetta@unimi.it citation: cocetta g., riva m., castelli g., ferrante a., 2020 ­ new active packaging for improving the shelf life and quality of tomato. ­ adv. hort. sci., 34(1s): 53­59 copyright: © 2020 cocetta g., riva m., castelli g., ferrante a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 march 2020 accepted for publication 30 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 53­59 54 obtained in the field and depends on growers choices and agronomic management. nevertheless, posthar­ vest stresses can induce different transcriptional and metabolic changes that may accelerate the senes­ cence processes (cavaiuolo et al., 2017). during stor­ age the products are still considered as living organs or tissues, thus their metabolism must be maintained active. sugar content in the produce plays an impor­ tant role for keeping the basal metabolism after har­ vest and higher sugar concentrations can allow longer shelf life, without compromise the quality. the main physiological processes that are associated with the postharvest performance are respiration rate and ethylene production (colelli and elia, 2009). ethylene plays an important role in climacteric fruits and its effect depends on tissue sensitivity. in the most of cli­ macteric products, lowering both processes, respira­ tion and ethylene production greatly extend the stor­ age and shelf life of the produce. tomato (solanum lycopersicum l.) is one of the most important prod­ ucts in the fresh vegetables market, particularly rele­ vant in italy, which is the first producer in europe and among the ten most important producing countries in the world (faostat, 2018). on a biological point of view, tomato is a climacteric fruit which has been widely studied as a model fruit for the production and sensitivity to ethylene accompanied by the char­ acteristic rise in the respiratory rate during ripening. on a practical point of view, tomato berries are con­ sidered as a perishable product which can be easily subjected to quality loss during storage and shelf life, mainly because of the ethylene production and rise in respiration. for these reasons, innovative solutions and suitable packaging systems able to extend stora­ bility and shelf life of fresh produce with high transpi­ ration rates such as tomato are needed (agudelo­ rodriguez et al., 2020). in fact, the packaging materi­ als can play an important role in regulating gas exchanges between the environment and produce. the combination of gas modulation and optimal stor­ age temperature can greatly extend the shelf life of produce (fagundes et al., 2015). today, different solutions are available on the market for the preser­ vation of fruit and vegetables using active packaging, with very different principles of action and effective­ ness. the chemical and physical properties of the packaging materials can provide a barrier modifying the gas composition in the headspace or around the produce (exama et al., 1993). the gas composition of headspace in the boxes or in bags depends from the film gas permeability and produce respiration and ethylene production rates. therefore, the film gas permeability and product physiology define the gas composition that both are affected by the storage temperature (mangaraj et al., 2009). in order to improve the film properties different materials can be added during the film extrusion or can be applied in one side of it. a good packaging film must keep its proprieties at different intervals of temperature, but especially at the low temperatures (exama et al., 1992). the packaging materials can help in reducing the water losses by reducing the transpiration and can also reduce the weight losses. these are key fac­ tors for the commercial quality of stored horticultural products. the barrier effect of the film can passively reduce the oxygen concentration and increase the carbon dioxide inside the package. the modification of the inner atmosphere by the action of the packag­ ing material is also known as modified atmosphere packaging (map). map reduces respiration and ethy­ lene production, thus is widely applied in the postharvest management of various fruits and veg­ etables. however, it is important to avoid hypoxic conditions that would derive from the oxygen deple­ tion inside the package. this would result in the development of off­odours due to the triggering of a fermentative metabolism. several additives can be included into the packaging film with various func­ tions including the reduction of condensation (anti­ fog additives) or the removal of specific gases such as ethylene. there are several ethylene absorbers that can be used for removing or reducing the concentra­ tion of this important plant hormone inside the pack­ aging (álvarez­hernández et al., 2018). the group research labs of saes getters s.p.a. have developed, in collaboration with saes coated films s.p.a., and its affiliate company, a highly selec­ tive packaging system for ethylene, including an inte­ grated absorber in the form of a coating (1­2 µm), with good transparency and an additional anti­fog function, which can be deposited on various plastic films. the aim of this work was to evaluate the efficacy of this new film in improving the shelf life of tomato berries during shelf life. 2. materials and methods plant material and storage conditions the experiment was conducted on tomato fruits (solanum lycopersicum l. var. cerasiforme). freshly cocetta et al. ‐ active packaging for improving tomato quality 55 harvested berries were selected based on uniformity in size and lack of defects, which were estimated by a visual evaluation. moreover, the colour uniformity was assured by selecting berries which showed simi­ lar hue and chroma values, which were measure by a colorimeter (for more details, see section “fruit colour measurements”). around 250 g of fruits were randomly divided into plastic punnets which were immediately flow packed with an automated flow packaging machine provided by saes getters s.p.a. two distinct materials were used, a conventional macro perforated plastic film (control) and an inno­ vative, highly selective active packaging system, char­ acterized by a good transparency and an enriched with anti­fog additives and including an integrated ethylene absorber in the form of a coating (1­2 µm) (2daf) (international patent application n° pct/ib2016/050401, in the name of saes getters s.p.a.). punnets were then divided into two groups and placed in storage rooms (72 ± 2 rh%) maintained at 10 ± 2°c (optimal storage conditions) and 22 ± 2°c (sub­optimal storage conditions) for up to 13 days. three punnets for each temperature/film were used. sampling was performed after 1, 3, 6, 8, 10 and 13 days of storage. analysis of gas composition at each timepoint gas composition within the punnets was performed by using the f­950 three gas analyzer (felix instruments, settle, usa). this instru­ ment allowed measuring the concentration of oxygen (% o2), carbon dioxide (% co2) as well as the pool of volatile organic compounds (ppm vocs) including ethylene, esters and alcohols. moreover, this instru­ ment provides the value of relative humidity of the atmosphere analysed. each punnet was transferred to 20°c for 30 minutes to equilibrate, before the analysis. weight loss estimation and sampling at each time point, the fruit net weight within each punnet was measured with a laboratory scale (fz500i digital milligram balance, a&d company, ltd., japan). fruit colour measurement at each time point, lightness (l*), hue angle (h=arctan(b*/a*) and chroma (c*= sqrt (a*²+b*²)) values were measured with a minolta chroma meter (cr­300 with an 8­mm aperture) calibrated against a standard white tile. each record was reported as average of six measurements taken from six fruit. titratable acidity and total soluble solids content titratable acidity (ta) and total soluble solids (tss) were measured from the juice squeezed from four fruits. ta was determined by titration of 5 g of juice with 0.1 n sodium hydroxide to a ph end point of 8.1, results are expressed as % citric acid. titration was performed using a titrator compact g10s (mettler, toledo, usa). tss were estimated using a portable digital (model 53011, turoni, italy) and results were expressed as °brix. β‐carotene and lycopene determination tomato (four fruits) fruits were ground and juices were analysed for lycopene content using the spec­ trophotometric method adapted from anthon and barrett (2007). the lipophilic fraction of the juice was extracted and separated with a solution of hexane ethanol and acetone (2:1:1 v/v), the phases were separated, and the hexane phase was read at 503 nm. pigments concentrations were expressed in mg kg­1 on fresh weight (fw) basis. statistical analysis all data were subjected to a one­way anova fol­ lowed by sidak’s multiple comparisons test. statistics were performed using graphpad prism version 6 for windows, graphpad software, la jolla california usa, www.graphpad.com. 3. results weight losses and gas composition inside the packages in general, the tomato berries did not show much variations in weight during storage under the tested conditions. only few changes were observed in 2daf­storaged fruits, which showed higher weights compared to control fruits after 8 days at 10°c and after 13 days at 22°c (fig. 1a, 1b). gas composition inside the packages was primari­ ly affected by temperature and then by the material used. in fact, even if samples stored in the 2daf film accumulated higher concentrations of co2 and con­ sumed more o2 compared to control samples, at 10°c these differences were not significant (fig. 2a, 2c). at 22°c instead a strong decrement in o2 and a progressive accumulation of co2, were observed. the minimum o2 levels was reached after 8 days of stor­ age, while the maximum peak of co2 was recorded after 10 days at 22°c (fig. 2b, 2d). as observed for respiratory gases, the relative adv. hort. sci., 2020 34(1s): 53­59 56 fig. 2 ­ changes in oxygen (a, b) and carbon dioxide (c, d) con­ centrations inside the packages, as affected by packaging material and storage temperature. asterisks indicate significant differences between 2daf and control *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=3. the accumulation of volatile organic compounds (vocs) in control packages was limited and tent to fade during storage at both temperatures. on the other hand, the use of 2daf allowed a more marked rise in vocs concentration inside the headspace. vocs maximum concentration was observed after 6 days at 10°c and after 8 days at 22°c. the highest concentration of vocs was almost 7­fold higher at 22°c compared to the maximum concentration at 10°c (fig. 3 c, 3d). titratable acidity, total soluble solids, and fruit colour quality attributes of tomato berries were slightly affected by storage and packaging material. titratable acidity was higher in control berries after 3 days of storage at 10°c, while at the other time points as well as at 22°c, no significant changes were observed between control and 2daf­stored fruits (fig. 4 a , 4 b). sugar concentration of tomatoes showed only few changes in fruits stored at 10°c, which, after 3 and 8 days showed higher °brix values when packed in the 2daf film (fig. 4c, 4d). no marked changes in colour were observed, with only two exceptions regarding 2daf­stored berries, which showed significantly higher lightness and hue angle values after 10 days at 22°c (fig. 5 a­f). fruit carotenoid concentrations both β­carotene and lycopene levels were quite stable during storage, however, it seems that the dif­ humidity was affected by temperature. at 10°c no changes were observed between the two films (fig. 3a), while at 22°c control packages accumulated a higher humidity in the inner atmosphere. these changes in comparison to 2daf, were highly signifi­ cant after 1 day of storage and significant (p<0.05) after 10 days (fig. 3 b). fig. 3 ­ changes in relative humidity (a, b) and volatile organic compounds (c, d) concentrations inside the packages, as affected by packaging material and storage temperature. asterisks indicate significant differences between 2daf and control *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=3. fig. 1 ­ tomato berries weight as affected by packaging material during storage at 10°c (a) and 22°c (b). asterisks indicate significant differences between 2daf and control *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=3. cocetta et al. ‐ active packaging for improving tomato quality 57 significantly higher in 2daf­stored samples after 8 days at both 10°c and 22°c (fig. 6 a, 6b), while lycopene was significantly higher in the same packag­ ing conditions, after 3 and 8 days at 10 °c and at the end of storage at 22°c (fig. 6 c, 6d). 4. discussion and conclusions the use of map has led to a significant improve­ ment of storage management of various horticultural products in the last years (arah et al., 2016). tomato is highly appreciated worldwide for its characteristic sensorial properties and for its nutraceutical value, mainly due to the presence of carotenoid pigments including β­carotene and lycopene as the most repre­ sentative (frusciante et al., 2007). the experiment described in this paper helped in elucidating the effect of an innovative packaging solution in compari­ son to a traditionally adopted microperforated plastic film during storage at two temperatures. it is also important to consider that tomato is chilling sensitive and low temperature can induce injury and reduce the quality. the identification of adequate film gas permeability can extend the storability of tomato and preserving the fruit quality (fagundes et al., 2015). as first consideration it is important to notice that none of the considered storage conditions deter­ mined a marked weight loss, thus it is possible to assume that there were no marked changes in the water content of berries. this last aspect has been considered when evaluating all the parameters which ferent packaging used affected the accumulation these pigments, which were generally higher in 2daf­stored samples. β­carotene concentration was fig. 4 ­ changes in titratable acidity (a, b) and total soluble solids content (c, d) in tomato berries, as affected by packaging material and storage temperature. asterisks indicate significant differences between 2daf and con­ trol *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=4. fig. 5 ­ changes in colour coordinate: lightness (a, b), hue angle (c, d) and chroma (e, f) measured on tomato berry skin, as affected by packaging material and storage tempera­ ture. asterisks indicate significant differences between 2daf and control *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=6. fig. 6 ­ changes β­carotene (a, b) and lycopene (c, d) concentra­ tion in tomato berries, as affected by packaging material and storage temperature. asterisks indicate significant differences between 2daf and control *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001; n=4. 58 adv. hort. sci., 2020 34(1s): 53­59 have been assayed on fresh weight bases. different storage conditions, together with genetic variability can affect the respiration of the produce during stor­ age and shelf life (colelli and elia, 2009). among the possible factors affecting the accumulation of o2 and co2 inside the punnets, temperature was the most effective. it is possible that the lowest storage tem­ perature (10°c) determined a decrease in the fruit metabolism and thus no significant changes were observed. on the other hand, the higher storage temperature (22°c) can be considered as optimal for several metabolic activities (maul et al., 2000). in this context, the effect of the different packaging was evi­ dent, with a more pronounced o2 decrement accom­ panied to a progressive accumulation of co2 into the headspace, due to the respiratory activity of fruits. it is also to be considered that the control reference film was macro­perforated, thus it was expected to have a very/no low barrier effect toward gases. nevertheless, it is interesting to notice that punnets wrapped in the control film accumulated higher humidity compared to those packed with the 2daf film. high relative humidity is not recommendable because even though it can help in preventing the water loss, it could facilitate fungal development (paull, 1999). as for gases and relative humidity, the production and accumulation of vocs was first influenced by storage temperature and then by the packaging film. tomato aroma and flavour are characterized by a complex pool of volatile organic compounds (krumbein and auerswald, 1998). the approach adopted in this trial allowed us estimating the total concentration of volatiles in a simple, non­invasive and rapid way. as a drawback, it did not permit the identification of the single classes of molecules which could include, ethylene, esters and alcohols, among others. however, it is interesting to notice that pun­ nets packed in the 2daf film accumulated significant­ ly higher amounts of vocs, especially at the highest temperature of storage. it is well known that at low temperature the volatiles release from tomato fruits is limited due to the reduced metabolic activity and volatility of the molecules (spadafora et al., 2019). also, the alteration of the gas composition in the atmosphere surrounding the product can induce the production of specific volatile metabolites (rowan, 2011). this finding suggests the possibility to modu­ late the sensory properties of tomato in the posthar­ vest phase, by selecting proper packaging strategies which allow accumulating/maintaining higher con­ centrations of quality­related volatiles. the sensory properties of tomato fruits are generally evaluated in terms of soluble solids content and acidity (beckles, 2012). these two parameters (and the balance between them) are important determinants of toma­ to quality and consumers acceptance. apparently, the conditions adopted in this trial were not effective in altering the acidity in a significant manner, although it was higher in cold stored control sample after 3 days. majidi and colleagues (2014) reported that tomato fruit stored in map had significantly higher total soluble solids than those stored at the same temperature (13°c) without map. similarly, in the present study, the combination of low tempera­ ture (10°c) and passive map, determined an incre­ ment in total soluble solids in the central phase of the storage period. even if few differences were found, the colour­ related indexes did not change markedly in response to the packaging conditions. this was expected, because the tomatoes used in this trial were fully ripe, so the skin colouration was completely devel­ oped and uniform at the beginning of the storage. also, the storage lasted up to 13 days, and in this time frame it is not common to observe marked colour alterations of tomato skin (rosati et al., 2000; heredia et al., 2009). on the other hand, significant changes involved the pigments composition of berries. it has been shown that berries lycopene and β­carotene content is not always well correlated with colour indexes, especially during storage (pék et al., 2010). some connection can be hypothesized instead, between carotenoids and vocs. in fact, some low molecular weight organic compounds derive from carotenoids catabolism (perveen et al., 2015), thus the use of the active packaging could have determined a change in the carotenoid metabolism leading to the coordinate increment of carotenoids and vocs between 6 and 8 days of stor­ age, as suggested by the results obtained. it has been previously observed that during postharvest, tomato fruits showed an increment in the levels of some important volatile compounds such as heptane and hexanal, and this increment was in line with highest β­carotene and lycopene concentrations in berries. the connection between carotenoids and vocs was also observed since 6­methyl 5­hepten­2­one, a carotenoid­derived volatile compound, increased in fruits during storage (franzoni et al., 2018). this aspect worth to be further investigated as involves both the sensory and nutraceutical properties of fruits. considering the results obtained, it can be con­ cocetta et al. ‐ active packaging for improving tomato quality 59 cluded that the use of this innovative material can represent an effective tool for improving the posthar­ vest management of tomato berries. references agudelo­rodríguez g., moncayo­martínez d., castellanos d.a., 2020 ­ evaluation of a predictive model to configure an active packaging with moisture adsorption for fresh tomato. ‐ food packag. shelf life, 23: 100458. álvarez­hernández m.h., artés­hernández f., ával­ os­belmontes f., castillo­campohermoso m.a., contreras­esquivel j.c., ventura­sobrevilla j.m., martínez­hernández g.b., 2018 ­ current scenario of adsorbent materials used in ethylene scavenging systems to extend fruit and vegetable postharvest life. ‐ food bioproc. tech., 11(3): 511­525. anthon g., barrett d.m., 2007 ­ 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of chia (salvia hispanica l.), in a split plot design with three replications. plants grown in absence or presence of amf were sprayed every 2 weeks with benzyl adenine (ba), cppu [n­(2­chloro­4­pyridinyl)­n’­ phenylurea], common name forchlorfenuron, and naphthalene acetic acid (naa) at 50, 20 and 50 ppm respectively, while control plants were sprayed with tap water. the results revealed that, inoculation with amf generally caused significant augmentation in all studied growth, yield and yield attribut­ es, total chlorophylls and carbohydrates content in leaves, augmentation in nutritional values of seeds like carbohydrates %, macronutrient, micronutri­ ents, proteins %, total flavonoids, oil % compared to non­inoculated plants. in absence or presence of amf, application of pgrs generally caused significant increases in the studied parameters compared to control. the interaction between naa and amf was more effective since gave higher increases in the studied parameters. it can be concluded that, cultivation of chia plant in pres­ ence of mycorrhiza with foliar application of naa at 50 ppm is recommended for enhancing growth, and nutritional values of seed yield. 1. introduction salvia hispanica, ordinarily known as chia, is an annual herbaceous plant which belongs to lamiaceae family. it is native to southern mexico and northern guatemala; the word of chia comes from the nahuatl word “chian” with means oily. the name salvia hispanica was specified by the swedish botanist carl linnaeus, who discomfited the wild­growing plant coming from the new world with a regional plant from spain. it grows up to 1­m tall with leaves of about 4­8 cm long and 3­6 cm wide. chia flowers are white or purple containing oval seed mottle­colored with brown, gray, black, and white with size ranging from 1 to 2 mm. it grows naturally in tropical and subtropical environments; it is optimally established from 400 to 2500 m and considered to be a short­day plant with a threshold of (*) corresponding author: hossam.ahmed@agr.cu.edu.eg citation: ashour h.a., esmail s.e.a., el­attar a.b., 2021 ­ responses of different quality parameters of chia to arbuscular mycorrhiza and plant growth regulator. ­ adv. hort. sci., 35(2): 139­150 copyright: © 2021 ashour h.a., esmail s.e.a., el­attar a.b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 march 2021 accepted for publication 30 march 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10641 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(2): 139­150 140 12­14 h. chia acquired acceptance owing to the high nutritional value of its seeds. the main components of seeds are polyunsaturated fatty acids omega­3 (pufa ω3) (58­64% of total lipids) and omega­6 (ω6), protein with a ratio (16­24%), carbohydrates (26­ 41%) lipids (31­35 %), and fiber (34­56 %). in addition to some minerals, vitamins and high level of antioxi­ dants (baginsky et al., 2016; sosa et al., 2016; marcinek and krejpcio, 2017). arbuscular mycorrhiza fungi (amf) are a category of soil microorganisms which form a symbiotic asso­ ciation with plants. not only it could enhance uptake of mineral elements and water by plants that pro­ mote plant growth, but also increase crop yield, qual­ ity properties and active ingredients (dupre et al., 2008; singh et al., 2010). the positive effect of inocu­ lation with amf on physiological and biochemical changes in different medicinal and aromatic plants has been reported in a number of species such as enhancing growth performance and seed yield (gashgaril et al., 2020; bilalis et al., 2020), increase photosynthetic pigments and carbohydrates content (amiri et al., 2017), increased nutrient status in plant orangs (chaudhary et al., 2008), protein content (ouzounidou et al., 2015), antioxidants activity (golubkina et al., 2020), fixed oil (moghith, 2019), promoted concentration of essential oil (chaudhary et al., 2008; al­amri et al., 2016), enhanced primary and secondary metabolism and bioactive compounds (gashgaril et al., 2020 ). plant growth regulators (pgrs) have been defined as one of the major factors affecting plants growth and their primary and secondary metabolites, naa is an organic compound that is synthetic plant hor­ mone in the auxin family. it is known to enhance cell elongation, cell division, elongation of shoot, vascu­ lar tissue, photosynthesis, rna synthesis, membrane permeability and water uptake is also involved in many physiological processes such as fruit set, delayed senescence, leaf chlorophyll content, stimu­ lates flowering and increases yield (davies, 1987). foliar application of naa on different medicinal and aromatic plants have been reported to improve growth and yield attributes, photosynthetic pig­ ments, total carbohydrate and oil yield (rohamare et al., 2013), enhance nutrient status in plant orangs, essential oil %, polyphenols and flavonoids content and antioxidant activity (atteya and el gendy, 2018). moreover, it has been reported for ameliorating the harmful effects of salinity (abou el­ghit, 2015). cytokinins include benzyl adenine (ba) that promotes cellular elongation and division (krug et al., 2006). foliar spray of ba has been reported to increase the growth and development of medicinal and aromatic plants (matter, 2016; moussa, 2019). it was reported to improve photosynthetic pigments content, total carbohydrate oil percentage and oil yield (abdel­ rahman and abdel­kader, 2020), contents of macronutrient and micronutrient, total phenols and total flavones (abdel­hamid, 2020), enhance protein concentration (prins et al., 2013) and antioxidant activity (sidkey, 2020). likewise, cppu is one of pgrs. it is a cytokinin like substance that has strong cytokinin activity by stimu­ lating fruit set and fruit quality, it plays a role in cell division and cell wall elongation (nickell, 1985; zhang and whiting, 2011). it has been reported to improve growth and yield attributes or protein percentage of medicinal plants (abbas and zahwan, 2016). although the valuable roles of amf and pgrs on medicinal and aromatic plants and their useful influ­ ence on enhancing growth and production has been formerly evaluated. however, there are not enough available data about their activity on the growth and production of chia plants. therefore, the objective of this study was to evaluate the impact of amf and foliar spray of pgrs (ba, cppu and naa) and their interactions on vegetative growth, seed yield and yield attributes, and some chemical compositions of salvia hispanica plants. 2. materials and methods open field experiment was carried out during the two successive seasons of 2018 and 2019 at experimental area of the ornamental horticulture department, faculty of agriculture, cairo university, giza governorate (latitude 30°01’13.44”n, longitude 31°12’30.24”e, altitude 22 m a.s.l.). the aim of this work was to evaluate the effects of foliar application of different plant growth regulators (pgrs) such as 6­ ba (6­benzylaminopurine) benzyl adenine, cppu [n­ (2­chloro­4­pyridyl)] and naa (napthaleneacetic acid) in absence or presence of arbuscular mycor­ rhizal fungi (amf) on vegetative growth, seed yield and its attributes and some biochemical parameters of chia (salvia hispanica l.) plant. experimental procedure seeds of salvia hispanica plants were obtained from experimental farm of faculty of pharmacy, ashour et al. ‐ responses of different quality parameters of chia to amf and pgr 141 cairo university. on 1st october and 15th october (in the two seasons, respectively), seeds were sown in the nursery at the ornamental horticulture department, faculty of agriculture, cairo university, giza. after 45 days old from seeds sowing (on the 15th of november, and1st december, in both seasons, respectively), uniform seedlings, with an average plant height of 15­18 cm, were transplanted in the experimental open field with a distance of 70 cm among rows, 50 cm spacing between plants in plots (3.5×3.5 m). the physical and chemical properties of the exper­ imental soil were determined according to jackson (1973), and the data are recorded in table 1. amf inoculum contained roots, hyphae, spores and growth media from a pot culture of onion plants colonization with glomus mosseae nrc31 and glomus fasciculatum nrc15, and were obtained from agricultural microbiology department, national research center. inoculum material consisted of 275 spores g­1 oven dry bases in addition to the coloniza­ tion roots pieces (the infectivity 104 propagola). amf inoculation treatments was achieved by mixing 5 g of it with 10 g of chia seed before sowing and repeated monthly after transplanting by injection inoculum material into the soil in roots area from five sides at 5 g/seedling. pgrs treatments were initiated after 15 days from the transplanting, by foliar spraying the plants every 2 weeks with sytonine 4%® [commercial name, con­ sists of 6­ ba (6­benzylaminopurine) benzyl adenine 4 %], cytovac® (cppu) and fast tonic® [commercial name, consists of napthaleneacetic acid 25% + sodium nitrophenolate 0.6%]. the three commercial products were obtained from bio green company, the regional representative of the jordanian compa­ ny, elite company for the manufacture of agricultural fertilizers development, egypt. the concentrations of ba and naa were 50 ppm for each one, while cppu was applied at 20 ppm and the untreated control plants were sprayed with tap water. freshly prepared solutions of pgrs (50 ml containing tween 20 at 1 ml/l (0.1%) as surfactant agent) were sprayed using plastic automizer until run off point. the common horticulture practices (such as irrigation, manual weeds control, fertilization) were carried out when needed. the experimental design was split plot design with eight treatments [2 amf (absence or presence) x 4 pgrs concentrations (including the control)] with 3 replicates, each consisting of 16 plants (2 plants from each treatment). amf assigned to the main plots in a randomized complete blocks design and pgrs con­ centrations were allocated at random in sub­plots. measurement of vegetative growth and yield para‐ meters vegetative growth parameters were recorded after 90 days from transplanting (on 15th of february to 1st march). in both seasons plant samples were taken for measurements growth characters in terms of plant height (cm), number of leaves/plant, stem diameter (cm, at 5 cm above the soil surface), num­ ber of branches/plant, fresh and dry weights of the herb (g/plant), root length (cm), number of root/plant and fresh and dry weights of the roots (g/plant). whereas, at harvesting stage yield and yield attributes including number of inflorescence/ plant, number of seeds/plant, weight of seeds (g /plant), weight of 1000 seeds (g), and seeds yield (kg/fed.) were recorded. seeds yield per feddan was obtained according to the equation: [weight of seeds (g/plant) × number of plants/fed]/1000 number of plants/fed= (100×100×4200)/ (50×70) = 12000 plant/fed. some chemical parameters were determined in the seeds as carbohydrates %, some macro & micro elements, proteins %, phenols, flavonoids content parameter 2018 2019 soil texture clay clay clay[%] 40.50 41.60 silt [%] 35.10 34.00 fine sand[%] 21.00 20.00 coarse sand [%] 3.40 4.40 field capacity [% v] 67.30 69.37 ph 7.12 7.19 ec [ds/m] 1.67 1.55 cec [meq/100g] 39.40 35.72 organic matter [%] 1.60 1.75 caco3 [%] 1.65 1.75 k+ [ppm] 65.85 67.95 mg++[ppm] 39.83 40.94 available n [ppm] 93.35 98.75 available p [ppm] 20.25 22.35 available fe [ppm] 2.11 2.19 available mn [ppm] 3.12 3.99 available zn [ppm] 1.59 1.53 table 1 ­ some physical and chemical characteristics of experi­ mental soil during the two seasons ph= soil acidity, ec= electrical conductivity, cec= cation exchange capacity, caco3= calcium carbonate. adv. hort. sci., 2021 35(2): 139­150 142 and antioxidant activity. chemical analysis total chlorophylls in fresh leaf samples were determined by using chlorophyll meter (model spad 502 minolta co. japan) as described by netto et al. (2005). the total carbohydrates content in leaves and seeds (% of dry matter) was determined in dried samples according to dubois et al., (1956). a known weight (0.1 g) of the dried samples was completely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. the solu­ tion was decolorized and the filtrate was completed to 100 ml with distilled water. a known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. the optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. the standard curve of glucose was used to calculate the total carbohydrates concentration in the extract. dried seeds samples were digested to extract nutri­ ents and the extract was analyzed to determine con­ centrations of n, p, k, ca, mg (% of dry seeds), fe and zn (ppm) which were determined according to estefan et al. (2013). nitrogen concentration was determined by using the micro­kjeldahl method. phosphorus was determined calorimetrically by using the chlorostannous molybdophosphoric blue colour method in sulphuric acid. potassium was determined by using the flame photometer apparatus (corning m 410, germany). the concentrations of ca, mg, fe and zn were determined using atomic absorption spectrophotometer with air­acetylene and fuel (pye unicam, model sp­1900, us). protein content in seeds was estimated by multiplying n values by 5.71 (conversion factors). total phenolics content was determined by using the folin ciocalteau’s reagent colorimetric method while total flavonoids content was estimated by the aluminum chloride colorimetric method and results are expressed as milligram catechin equivalent per gram of seeds dry weight extract (mg ce/g dw) (john et al., 2014). the antioxi­ dant activity of seeds extract and standard antioxi­ dant was assessed on the basis of the radical scav­ enging effect on dpph (2, 2­diphenyl­1­picrylhy­ drazyl) free radicals (brand­williams et al., 1995). fixed oil %: the clean air dried seeds of chia were separately crushed in a willey mill, then extracted in soxhlet apparatus, samples of 10 g of seeds were moved into soxhlet apparatus in 100 ml of n­hexane and the extraction period extended to 6 hours (30­36 syphon cycle approx.). the n­hexane extract was dried over anhydrous sodium sulfate, then filtered and the oil was obtained by distillation under vacu­ um. fixed oil % was calculated according to the equa­ tion: fixed oil % = extracted fixed oil weight (g) ̸ seeds sample weight (g) ̸ × 100. statistical analysis the means of all obtained data were subjected to statistical analysis of variance (anova) in split plot design. combined analysis of the two growing sea­ sons was carried out. means of data were compared by using duncan’s multiple range tests at 5% level snedecor and cochran (1989). 3. results and discussion vegetative growth parameters it is evident from data in table 2 and 3 that under the same treatments of pgrs, chia plants grown in presence of amf had significant increase in studied vegetative growth parameters (viz., plant height, number of leaves, stem diameter, number of branch­ es/plant, fresh and dry weights of herb, root length, number of roots, root fresh and dry weights) com­ pared to those grown in absence of amf. the obtained increases in vegetative growth attributes due to amf inoculation are in agreement with reports of several researches on medicinal and aro­ matic plants including artemisia annua (chaudhary et al., 2008), salvia officinalis (geneva et al., 2010; abdelkader et al., 2014), corianderum sativum (al­ amri et al., 2016; oliveira et al., 2016), origanum majorana (engel et al., 2016), pelargonium grave‐ olens (amiri et al., 2017), salvia miltiorrhiza (yang et al . , 2017), salvia hispanica (moghith, 2019), artemisia dracunculus and hyssopus officinalis (golubkina et al., 2020) and foeniculum vulgare (mohamed, 2020). the stimulatory influence of amf on vegetative growth traits could be explained by amf form symbi­ otic relationship with the host increase of root sur­ face, which led to promote root uptake of nutrients. thus, it could significantly augment growth parame­ ters of tested plant (jian­heng et al. , 2016). furthermore it was indicated that the ability of amf to enhance the availability of essential elements macro­ and micronutrients in the rhizospheric soil that induce its uptake and the accumulation in plant (gashgaril et al., 2020). ashour et al. ‐ responses of different quality parameters of chia to amf and pgr 143 results in table 2 and 3 also indicate that, in absence or presence of amf, treating plants with dif­ ferent concentrations of pgrs resulted in significant increase in tested vegetative growth parameters compared to control. among pgrs, application of naa (at 50 ppm) appeared to be the most effective one particularly in presence of amf since recorded the highest values in most cases. the results of the pronounced increase in growth parameters due to application of naa are in accordance with findings of previous studies on medicinal plants (alam et al., 2012; rohamare et al., 2013; danesh­talab et al., 2014; abou el­ghit, 2015; rostami and movahedi, 2016; dheeraj and saravanan, 2018). moreover, numerous studies reported increase in growth attrib­ utes of medicinal plants owing to either ba (matter, 2016; moussa, 2019; abdel­rahman and abdel­ kader, 2020 and abdel­hamid, 2020) or cppu treat­ ments (abbas and zahwan, 2016). the stimulation effect of naa on morphological table 2 ­ mean square for the effect of arbuscular mycorrhiza fungi (amf), plant growth regulators (pgrs) and their interaction on veg­ etative growth, yield and yield parameters of salvia hispanica *, **, *** significant at p≤0.05, p≤0.01, p≤0.001, respectively. traits source of variation treatment error cv amf (a) pgrs (b) a × b a b a b plant height (cm) 233.750 ** 514.534 *** 24.278 *** 1.182 1.037 1.322 1.239 no. of leaves 213.010 ** 194.372 *** 5.844 * 2.042 2.222 4.527 4.723 stem diameter (cm) 0.375 ** 0.124 *** 0.011 * 0.004 0.005 7.036 8.386 no. of branches/plant 37.500 *** 36.301 *** 1.701 ** 0.035 0.189 1.685 3.921 herb fresh weight (g) 27.307 *** 18.106 *** 2.681 *** 0.003 0.090 0.433 2.412 herb dry weight (g) 25.627 *** 21.694 *** 1.288 *** 0.002 0.072 0.609 3.991 root length (cm) 145.042 *** 96.486 *** 12.042 *** 0.087 0.746 1.979 5.808 no. of roots/plant 32.667 *** 34.375 *** 1.167 * 0.135 0.448 4.576 8.322 root fresh weight (g) 38.760 *** 20.386 *** 1.412 *** 0.020 0.030 2.531 3.068 root dry weight (g) 4.770 ** 5.794 *** 0.228 ** 0.050 0.035 7.233 6.039 no. of inflorescence /plant 145.042 * 70.972 *** 7.903 * 0.510 1.313 4.844 7.767 no. of seeds / plant 18897.29 *** 57551.63 *** 283.104 *** 1.439 5.467 0.219 0.426 weight of 1000 seeds (g) 1.321 *** 0.371 ** 0.001 * 0.145 0.061 20.73 13.45 weight of seeds (g/plant) 13.024 ** 40.227 *** 0.556 * 0.290 0.367 7.02 7.89 seeds yield (kg/fed.) 1875.494 ** 5792.741 *** 80.027 * 5.041 21.110 2.44 4.99 table 3 ­ vegetative growth parameters of salvia hispanica as affected by the interaction between plant growth regulators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf) (mean of two seasons) treatment plant height (cm) no. of leaves stem diameter (cm) no. of branches/ plant herb fresh weight (g) herb dry weight (g) root length (cm) no. of roots/ plant root fresh weight (g) root dry weight (g)amf pgrs ­ 0 67.53±0.44 h 23.67±0.60 d 0.57±0.07 d 7.00±0.29 f 9.77±0.03 f 3.53±0.15 g 8.50±0.060 f 4.00±0.58 e 2.67±0.07 g 1.57±0.09 f ba at 50 ppm 78.50±0.50 f 27.17±0.33 c 0.77±0.03 c 9.93±0.35 d 11.83±0.07 cd 5.87±0.12 e 12.33±0.67 d 7.50±0.29 c 4.07±0.03 f 2.30±0.06 e cppu at 20 ppm 81.83±0.67 e 28.00±0.58 c 0.87±0.03 c 11.10± 0.06 c 11.67±0.23 d 6.63±0.28 d 14.00±0.50 c 7.50±0.29 c 4.83±0.15 e 3.27±0.09 d naa at 50 ppm 88.50±1.15 c 35.50±0.29 b 0.83±0.03 c 11.37±0.105 c 12.33±0.19 c 6.67±0.13 d 14.83±0.73 c 8.50±0.50 c 5.93±0.03 d 3.50±0.06 cd + 0 70.83±0.68 g 26.67±0.60 c 0.80±0.02 c 8.00±0.29 e 10.4 3±0.09 e 4.37±0.07 f 10.67±0.44 e 5.17±0.17 d 3.87±0.15 f 2.03±0.2 e ba at 50 ppm 90.67±0.60 b 34.00±0.58 b 1.00±0.06 b 13.30±0.15 b 13.67±0.12 b 8.17±0.15 c 17.17±0.17 b 9.67±0.44 b 6.53±0.13 c 3.60±0.1 c cppu at 20 ppm 86.17±0.33 d 35.00±1.00 b 1.03±0.03 b 13.60±0.20 b 13.80±0.10 b 8.70±0.10 b 17.83±0.17 b 10.33±0.73 b 8.00±0.06 b 3.93±0.12 b naa at 50 ppm 93.67±0.73 a 42.50±0.29 a 1.20±0.06 a 14.50±0.15 a 16.23±0.32 a 9.73±0.09 a 23.67±0.33 a 11.67±0.44 a 9.27±0.12 a 4.63±0.03 a each value represents the mean ± standard error of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. 144 adv. hort. sci., 2021 35(2): 139­150 attributes may be due its ability to increase mem­ brane permeability and water uptake which accom­ panied by elements absorption, synthesis of chloro­ phyll and carbohydrates contents that achieving the highest dry weight of plants (atteya and el gendy, 2018). yield and yield attributes data in table 4 revealed that under the same treatments of pgrs, the values of yield and yield attributes (viz., number of inflorescence /plant, num­ ber of seeds/plant, weight of seeds, weight of 1000 seeds, and seeds yield) were significantly higher in plants grown in the presence of amf than corre­ sponding values in the absence of amf. the present increase in number of inflorescence/plant due to amf inoculation is supported by the results of previ­ ous report (engel et al., 2016). moreover, recent study (mohamed, 2020) on foeniculum vulgare revealed that amf inoculation significantly increased seeds yield parameters. exogenous application of pgrs in absence or pres­ ence of amf resulted in significant increase in yield and yield parameters compared to control. among pgrs, naa was the most effective one particularly when interacted with presence of amf. in this con­ cern, application of naa at 50 ppm has been report­ ed to increase yield attributes (rohamare et al., 2013; danesh­talab et al., 2014; khudus et al., 2017; venkatesan and shakila, 2017). other study (kassem et al., 2011) found that, foliar spray of naa at 75 mg/l or cppu at 10 mg/l caused significant increase in yield and yield attributes of ziziphus jujuba com­ pared with the control. moreover, increases in num­ ber of inflorescence /plant owing to application of naa are in accordance with findings of earlier studies (atteya and el gendy, 2018; dheeraj and saravanan, 2018). while increasing yield and its attributes due to either ba application are in harmony with the find­ ings of various studies (mousa et al., 2001; matter, 2016; abdel­rahman and abdel­kader, 2020) or cppu (abbas and zahwan, 2016). the augmentation in yield and its attributes due to pgrs treatments may be due to its role in enhanced absorption of nutrients which promoted photosynthesis rate and translocation of photosyn­ thates and other metabolites to the sinks that lead­ ing to increase yield and its attributes in present study (alam et al., 2012). total chlorophylls and total carbohydrates data in figure 1 indicate that under the same treatments of pgrs, plants grown in the presence of amf had significantly higher values of total chloro­ phylls in leaves and total carbohydrates in leaves and seeds compared to corresponding values in the absence of amf in most cases. these results run par­ allel with those obtained by earlier reports on medic­ inal plants that reported increase in chlorophyll and carbohydrates content in plants inoculated with amf compared to controls (amiri et al., 2017; gashgaril et al., 2020; moghith, 2019; mohamed, 2020). results also show that, the recorded values were significantly increased in plants grown in absence or presence of amf as a result of spraying tested pgrs compared to control. in most cases, plants sprayed with naa in the presence of amf had the highest val­ ues of the tested components. the results of increase table 4 ­ yield and yield attributes of salvia hispanica as affected by the interaction between plant growth regulators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf) (mean of two seasons) each value represents the mean ± standard error of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. treatments no. of inflorescence/ plant no. of seeds/ plant weight of seeds (g/plant) weight of 1000 seeds (g) seeds yield (kg/fed.) amf pgrs ­ 0 9.17±0.44 f 409.17±0.58 h 3.43±0.20 f 1.23±0.03 e 41.20±2.45 f ba at 50 ppm 12.67±0.88 e 477.17±1.11 f 7.47±0.01 d 1.69±0.09 d 89.64±0.14 d cppu at 20 ppm 12.33±0.33 e 573.67±1.48 d 7.32±0.38 d 1.73±0.15 cd 87.88±4.52 d naa at 50 ppm 15.00±0.58 d 622.17±1.21 c 9.53±0.15 bc 1.76±0.18 bcd 114.40±1.79 b + 0 11.00±0.58 e 448.50±0.76 g 4.64±0.03 e 1.70±0.21 d 55.68±0.36 e ba at 50 ppm 17.17±0.6 c 546.83±1.72 e 8.57±0.13 c 2.16±0.17 abc 102.84±1.54 c cppu at 20 ppm 19.00±0.58 b 638.33±1.15 b 9.71±0.26 b 2.19±0.11 ab 116.48±3.17 b naa at 50 ppm 21.67±0.67 a 673.00±1.15 a 10.74±0.18 a 2.23±0.192 a 128.84±2.22 a ashour et al. ‐ responses of different quality parameters of chia to amf and pgr 145 of total chlorophylls or total carbohydrates due to application of naa are similar to those obtained by previous studies (alam et al., 2012; rohamare et al., 2013; rostami and movahedi, 2016; venkatesan and shakila, 2017; atteya and el gendy, 2018), whereas the obtained increase due to either ba treatments are in close conformity with the findings of previous reports (matter, 2016; moussa, 2019; abdel­hamid, 2020; abdel­rahman and abdel­kader, 2020) or cppu treatments (kassem et al., 2011; abbas and zahwan, 2016). content of macronutrients in seeds results of chemical analysis of dried seeds of salvia hispanica plants (table 5 and 6) disclosed that, under the same treatments of pgrs the uptake and accumulation of macronutrients in seeds of plants inoculated with of amf were significantly higher in most cases compared to non­inoculated plants. the only exception to this general trend was observed in the case of p and k% as in the presence of amf with control treatments they recorded insignificantly high­ er values compared to corresponding values in the absence of afm. the obtained results are in agree­ ment with those obtained by various researchers that reported the potential effects of amf on the accumulation of macronutrients in medicinal plant orangs (chen and zhao, 2009; karagiannidis et al., 2011; abdelkader et al., 2014; vafadar et al., 2014; oliveira et al., 2016; yang et al., 2017; moghith, 2019; mohamed, 2020). the enhanced mineral absorption by amf inocu­ lated plants could be elucidated by the efficiency of amf to boost mineral affinities, reduce the critical concentration of elements absorption, augment the area of uptake and decrease the area of diffusion (gashgaril et al., 2020). data in table 6 also indicate that, in absence or presence of amf foliar spraying with any concentra­ tions of pgrs resulted in significant increase in macronutrient in seeds in most cases, compared to control. with some exceptions recorded in absence of amf as foliar application of cppu resulted in insignificantly higher values of p% than the control, also in absence of amf, foliar application of three tested pgrs resulted in insignificantly higher values of ca % compared to control. among pgrs, applica­ tion of naa was the most effective treatment espe­ cially in presence of amf. the results of increasing macronutrients accumulation due to naa treatments are accordance with findings of previous studies (alam et al., 2012; atteya and el gendy, 2018), while the obtained increase in macronutrients due to ba is similar to those described in numerous reports (matter, 2016; moussa, 2019; abdel­hamid, 2020; abdel­rahman and abdel­kader, 2020). content of micronutrients in seeds as shown in figure 2 that under the same treat­ ments of pgrs plants inoculated with amf had signif­ icantly higher values of fe and zn in their seeds than those grown in the absence of amf. these results are in the same line of the findings of earlier authors (chaudhary et al., 2008; golubkina et al, 2020). data in figure 2 also showed that, in the absence or presence of amf foliar application of any concen­ fig. 1 ­ total chlorophyll in leaves (a), total carbohydrates in lea­ ves (b), and total carbohydrates in seeds (c) of salvia hispanica as affected by the interaction between plant growth regulators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf) (mean of two sea­ son). column with different letters indicate a significant difference at 5% level. vertical bars indicate to standard error (se) of three replicates. adv. hort. sci., 2021 35(2): 139­150 146 trations of pgrs resulted in significant increase in tested micronutrients (fe and zn) in seeds compared to control. the increases in the recorded values were more evident in the presence of amf mostly with application of naa. such increase in fe and zn con­ tent due to ba treatments is in good agreement with those elicited by prior works (baydar and erdal, 2004; abdel­hamid, 2020). total protein data in table 7 displayed that under the same pgrs treatments total protein percentage was signifi­ cantly higher in plants grown in the presence of amf than those grown in the absence of amf. these results are in conformity with that recorded by previ­ ous studies (ouzounidou et al., 2015; al­amri et al., 2016). increasing protein due to amf may be due to table 5 ­ mean square for the effect of arbuscular mycorrhiza fungi (amf), plant growth regulators (pgrs) and their interaction on some chemical constituents of salvia hispanica *, **, *** significant at p≤0.05, p≤0.01, p≤0.001, respectively; ns= not significant at p=0.05. traits source of variation treatment error cv amf (a) pgrs (b) a × b a b a b total chlorophylls in leaves (spad) 36.630 ** 46.448 *** 0.517 * 0.782 0.428 2.53 1.87 total carbohydrates in leaves (%) 16.368 ** 25.279 *** 0.242 * 0.167 0.878 3.94 9.02 total carbohydrates in seeds (%) 5.042 *** 1.914 ** 0.221 * 0.007 0.037 1.58 3.61 n (%) 0.855 *** 0.824 *** 0.003 * 0.001 0.004 1.21 2.39 p (%) 0.014 ** 0.008 *** 0.001 * 0.00 0.00 3.14 4.01 k (%) 0.005 * 0.006 ** 0.001 * 0.000 0.001 8.20 11.28 mg (%) 0.001 ns 0.002 * 0.000 * 0.001 0.001 13.82 9.71 ca (%) 0.013 * 0.001 * 0.001 * 0.001 0.000 6.49 3.20 fe (ppm) 62.210 * 32.204 *** 2.316 * 1.148 1.473 1.66 1.88 zn (ppm) 29.704 * 67.935 *** 0.778 * 2.251 0.445 3.34 1.48 total protein (%) 27.907 *** 26.881 *** 0.083 * 0.038 0.145 1.22 2.39 total phenol (μg ce/g) 0.917 * 1.774 ** 0.119 * 0.158 0.204 9.36 10.65 total flavonoid (μg ce/g) 60.770 * 63.393 *** 2.161 * 0.621 0.473 1.83 1.60 antioxidant (dpph ic50 (µg/ml) 8.143 ** 23.270 *** 1.989 * 0.619 0.843 1.19 1.38 fixed oil % 93.102 * 139.584 *** 0.509 * 2.833 2.978 6.39 6.55 table 6 ­ macronutrients in seeds of salvia hispanica as affected by the interaction between plant growth regulators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf) (mean of two seasons) each value represents the mean ± standard error of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. treatment n (%) p (%) k (%) mg (%) ca (%) amf pgrs ­ 0 2.21±0.03 f 0.33±0.01 d 0.19±0.02 f 0.21±0.01 d 0.40±0.01 c ba at 50 ppm 2.40±0.02 e 0.37±0.01 c 0.23±0.02 d 0.25±0.02 b 0.43±0.02 bc cppu at 20 ppm 2.73±0.08 c 0.35±0.00 cd 0.21±0.01 e 0.24±0.00 c 0.43±0.01 bc naa at 50 ppm 3.080±0.01 b 0.41±0.01 b 0.25±0.02 c 0.26±0.03 b 0.42±0.00 bc + 0 2.60±0.02 d 0.36±0.00 cd 0.20±0.01 f 0.23±0.01 c 0.45±0.01 ab ba at 50 ppm 2.77±0.03 c 0.41±0.01 b 0.27±0.02 ab 0.27±0.01 a 0.47±0.01 a cppu at 20 ppm 3.15±0.02 b 0.43±0.00 b 0.25±0.01 c 0.25±0.01 b 0.47±0.01 a naa at 50 ppm 3.40±0.01 a 0.46±0.01 a 0.28±0.02 a 0.27±0.01 a 0.48±0.01 a ashour et al. ‐ responses of different quality parameters of chia to amf and pgr 147 its role on inducing nh4 + and no3 − absorption, and assimilation of these molecules into free amino acids that are involved in protein synthesis (gashgaril et al., 2020). the data in table 7 also exhibited that in the absence or presence of amf, spraying of plants with pgrs resulted in significant increase in total protein content compared to control. among the tested pgrs naa was superior in its effect predominately in the presence of amf. the results of increasing pro­ tein content due to application of ba are in good agreement with those elicited by prins et al., 2013, while such increase owing to cppu treatments is coincided with those obtained by abbas and zahwan, 2016. total phenol content (tpc), total flavonoid content (tfc) in in seeds the data presented in tables 7 showed that under the same treatments of pgrs tpc and tfc in seeds were higher in plants inoculated with amf compared to non­inoculated plants; however such increase was statically insignificant in the case of tpc compared to corresponding values in absence of amf. the results of increasing tpc or tfc due to inoculation with amf are analogy with that recorded by earlier workers (amiri et al., 2017; gashgaril et al., 2020). data outlined in table 7 also indicate that in the absence or presence of amf tpc and tfc in seeds were significantly higher in seeds of plants foliar sprayed with any concentrations of pgrs compared to control, with superiority of ba for increasing tpc and naa for tfc especially in the presence of amf. the present augmentations in tpc or tfc owing to naa are in harmony with those obtained by previous author (atteya and el gendy, 2018), whereas increas­ ing in tested components due to ba are in the same line with the findings of earlier study (abdel­hamid, fig. 2 ­ fe (a) and zn (b) in seeds of salvia hispanica as affected by the interaction between plant growth regulators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf) (mean of two season), column with different letters indicate a significant difference at 5% level. vertical bars indicate to standard error (se) of three replicates. table 7 ­ total protein, total phenol, total flavonoid and antioxidants in seeds as affected by the interaction between plant growth regu­ lators (pgrs) in absence (­) or presence (+) of arbuscular mycorrhiza fungi (amf), (mean of two seasons) each value represents the mean ± standard error of three replicates. means in a column with different letters indicate a significant difference for each variable at 5% level using duncan multiple rang test. treatments total protein (%) total phenol (μg ce/g) total flavonoid (μg ce/g) antioxidant (dpph ic50 (µg/ml)amf pgrs ­ 0 12.62±0.18 f 3.18±0.12 c 37.56±0.06 f 63.14±0.60 d ba at 50 ppm 13.68±0.13 e 4.15±0.49 ab 40.74±0.13 d 66.18±0.58 c cppu at 20 ppm 15.57±0.45 c 4.55±0.03 a 43.18±0.05 c 67.54±0.02 bc naa at 50 ppm 17.57±0.06 b 4.31±0.04 ab 44.22±0.07 bc 66.34±0.6 c + 0 14.87±0.10 d 3.68±0.34 bc 39.3±1.15 e 64.18±0.58 d ba at 50 ppm 15.82±0.17 c 4.83±0.09 a 45.33±0.13 b 66.25±0.43 c cppu at 20 ppm 17.97±0.12 b 4.56±0.19 a 46.74±0.08 a 68.31±0.17 ab naa at 50 ppm 19.41±0.07 a 4.70±0.32 a 47.06±0.01 a 69.12±0.57 a adv. hort. sci., 2021 35(2): 139­150 148 2020; sidkey, 2020). antioxidant activity in seeds as shown from data listed in table 7 that under the same treatments of pgrs antioxidant activity in seeds were higher in plants inoculated with amf compared to corresponding values of non­inoculated plants, however such increase was insignificant in most cases. the results of increasing antioxidant activity due to mycorrhizal treatments is supported by the results of other authors (geneva et al., 2010; amiri et al., 2017; golubkina et al., 2020; gashgaril et al., 2020). data recorded in table 7 also indicate that in the absence or presence of amf antioxidant activity was significantly higher in seeds of plants sprayed with pgrs concentrations compared to control, with supe­ riority of naa especially in the presence of amf, since recorded the highest values compared to con­ trol. the obtained increase in antioxidant activity due to naa are in harmony with those obtained by previ­ ous author (atteya and el gendy, 2018), while the augmentation due to ba are in harmony with the finding of recent study (sidkey, 2020). fixed oil percentage it is obvious from data listed in figure 3 that under the same treatments of pgrs, the values of fixed oil % for plants grown in presence of amf was significantly higher than corresponding values in the absence of amf. the results of increasing fixed oil % owing to inoculation with amf are in sequence with the find­ ings of earlier author (moghith, 2019). data in figure 3 also showed that, in the absence or presence of amf in general application of pgrs resulted in significant increase in fixed oil % compared to control. these increases in the recorded values were more evident in the presence of amf particular­ ly with application of naa. such augmentation in fixed oil due to application of ba is in agreement with those reported by previous study (mousa et al., 2001). 4. conclusions based on the outcome of the present research it could be concluded that, cultivation of chia plants in presence of mycorrhiza with foliar application of naa at 50 ppm is recommended for enhancing growth, and nutritional values of seed yield. acknowledgements the authors would like to extend full thanks to the faculty of agriculture, cairo university for encour­ agement and financing this research work. references abbas k.s., zahwan t.a., 2016 ­ effect of cytokinin (cppu) spraying and agroleaf high phosphorus content in some vegetative 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az1004, azerbaijan. 2 institute for sustainable plant protection (ipsp), cnr, via madonna del piano, 50019 sesto fiorentino (fi), italy. key words: anthocyanins, phenolics, tannins, wild­growing pomegranate. abstract: the demand for pomegranate (punica granatum l.) juices is increas­ ing worldwide due to its documented health­promoting effects which likely derive from phenolic compounds. this study reports the phenolic composition of the juices obtained from eight wild­growing pomegranate accessions collect­ ed in eight areas of azerbaijan, characterized by different climate and soil com­ position. the anthocyanins found in all the accessions were cyaniding deriva­ tives and pelargonidin derivatives, while only two accessions contained also delphinidin­3,5­o­diglucoside. the main hydrolysable tannins contained in the juices were punicalagin and ellagic acid derivatives. these bio­active metabo­ lites found in the juices varied qualitatively and quantitatively among the eight accessions, thus constituting specific traits for selecting promising accessions that can be used as a nutritious food source. the different phenolic profiles might be determined both by genotype and the growing environmental condi­ tions, or by their interaction. our results suggest that some of the studied wild­ growing pomegranate accessions might have a commercial value because of their richness in bioactive metabolites and might constitute a suitable source of genes for breeding programs. 1. introduction punica granatum l. (pomegranate) originated in the region extending from iran to northern india has been used since ancient times as fruit and for medical purpose. recently an increasing of pomegranate cultivation and consumption has been reported because of its beneficial effects on human health (kalaycıoğlu and erim, 2017). in particular, the juice of pomegranate shows potent antioxidant (lee et al., 2012; aloqbi et al., 2016), anti­atherosclerotic (aviram et al., 2000) and anticancer effects (derakhshan et al., 2018), due to its high concentration of punicalagin derivatives, ellagic acid derivatives (both in its free and conjugated forms), gallotannins and anthocyanins. the high concentration of polyphenols in pomegranate extracts has been also related to a strong anti­inflammatory, hepatoprotective and antigenotoxic properties has (*) corresponding author: cecilia.brunetti@ipsp.cnr.it citation: zeynalova a., novruzov e., bartolini p., brunetti c., maserti b., 2020 ­ phenolic finger‐ print in wild growing pomegranate fruits from azerbaijan. ­ adv. hort. sci., 34(3): 277­286 copyright: © 2020 zeynalova a., novruzov e., bartolini p., brunetti c., maserti b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2019 accepted for publication 5 june 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 277­286 278 been recently investigated (sartippour et al., 2008; santhini et al., 2011). moreover, pomegranate extract has been reported to be beneficial to enhance fruit post­harvest stability due to its antimi­ crobial and antifungal effects, mainly correlate to the content of punicalagin derivatives (rongai et al., 2018, 2019). azerbaijan is one of the richest floristic regions and a major focus in the south­west asian and it is the origin center of many cultivated plants including pomegranate (zeynalova and novruzov, 2017). pomegranate carries a deep cultural and historical heritage in azerbaijan and occupies a special place in cuisine and traditional medicine (karasharli, 1979). in several regions of azerbaijan, wild pomegranate accessions grow spontaneously in self­maintaining populations in natural or semi­natural ecosystems independently of direct human action. in particular, wild pomegranate accessions can be found at the foothills and in lower mountain zones of the greater and lesser caucasus, in the kur­araz and samur­ divichinsk lowlands and in the mountain belt of the talysh plateau, on dry slopes up to 700 m above sea level (asadov and asadov, 2001). wild plant accessions of cultivated fruits gained attention to scientific community, since they have developed a high adaptability to climatic pressures and resistance to diseases and pests, accumulating high amount of secondary metabolites respect to cultivated varieties (howard et al., 2003; kassim et al., 2009). the genetic background of each accessions plays a key role in the responses to environmental conditions, result­ ing in accessions more or less sensitive to different environmental pressures (schwartz et al., 2009), how­ ever, in most cases, climate effects on arils’ composi­ tion is substantial since phenol metabolism, both syn­ thesis and oxidation, is induced by stressful conditions, especially thermal stress (di stefano et al., 2019). at this regards, it has been shown that seasonal warming increases the content of diglucosylated anthocyanins (borochov­neori et al., 2011). in the framework of a systematic study on the functional properties of wild pomegranate plants in azerbaijan, this work reports the phenolic fingerprint­ ing of the juices obtained from wild growing pome­ granate accessions collected in eight different areas of azerbaijan (zeynalova et al., 2019), as a starting point to select wild­growing suitable genotypes that can be used in breeding programs for the production of fruits and juices with a high content of bioactive metabo­ lites. 2. materials and methods plant material collection fresh ripe pomegranate (punica granatum l.) fruits were collected from seven districts of azerbaijan. geographical coordinates and altitude corresponding to each surveyed area are shown in (fig. 1 and table s1). the soil of the different areas was analyzed. pomegranate fruits were collected at the same maturity stage from of 25th september and october 2nd 2018 during a field campaign extensively described in a companion paper (zeynalova et al., 2019). each accession sample (pg) was the pool of three fruits from three different trees collected in each sampling station. details on the physio­chemical and organoleptic characteristics of the fruits are reported in zeynalova et al. (2019). juice preparation after collection, fruit juices were prepared as described by zeynalova et al. (2019). briefly each fruit was weighed individually, then the arils, peel and calyx membranes were manually divided and juice was extracted by mechanical press, directly from arils to prevent the contamination of phenolic components from the skin or peels membrane into the juice, and then stored at ­80°c until hplc analy­ sis. chemicals all reagents and solvents were of hplc grade (sigma aldrich, italy). the following standards were used for identification and quantification purposes with hplc­dad: cyaniding 3­glucoside, delphinidin 3­ fig. 1 ­ selected sampling stations in azerbaijan districts. the type climate and of soil of each district is reported. the pomegranate accession harvested in each area are also indicated on the map. zeynalova et al. ‐ phenolics in azerbaijan wild pomegranate fruits 279 glucoside, pelargonidin 3­glucoside, cyanidin 3­ruti­ noside (extrasynthese, lyon, france); gallic acid (fluka, buchs, switzerland); ellagic acid (extrasynthese, lyon, france); punicalagin a+b mix­ ture (sigma aldrich, italy). quantification of phenolic compounds by hplc analysis the juices obtained from the fruits of the eight pomegranate accessions were directly injected in the hplc system. hplc system consisted of perkin elmer flexar hplc equipped with a quaternary 200q/410 pump and a lc 200 photodiode array detector. anthocyanins and non­anthocyanin phenolics were separated and quantified using two different gradient elution conditions, as previously described in fischer et al. (2011). the separation was carried out injecting 10 μl of the juices with analytical zorbax sb­c18 (5 μm, 250 × 4.6 mm) column (agilent technologies, italy), operating at 30°c and at a flow rate of 0,6 l min­1. individual phenols were identified comparing uv­spectral characteristics with those of authentic standards and on the basis of the identification reported in fischer et al. (2011). the diode array detector was set at an acquisition range between 200 and 600 nm. quantification was performed at 520 nm for anthocyanins and at 280 nm for tannins, uti­ lizing calibration curves of corresponding standards. statistical analysis data corresponds to the juice extracted from a pool of three fruits for each pomegranate accession. three technical replicates of each sample were ana­ lyzed. a one­way anova with the “accession” as fac­ tor followed by tukey post hoc test (p<0.05) was used to compare the compound in the different accessions. heat map and pca biplot analysis have been performed with clustvis software (metsalu and vilo, 2015). 3. results plant material the climate was different in the seven districts, ranging from temperate warm climate (gokchay, ismailly) to semidesert dry steppes with hot summer (agsu, khizi). the sampling area were located at dif­ ferent altitude, ranging from 60 m (yevlak) to 540 m (ismailli). in the agsu district two accessions were collected, one accession in the agsu mountaın pass, whereas the other accession at 190 m a.s.l. (fig. 1). four areas presented salinized soil (slyazan, yevlakh and the two sampling points in the district of agsu). two areas have a typical mountain humus rich soil (sheki, ismailli). morphological traits the shape and dimension of the fruits were very similar, except for pg7 which showed a higher fruit length. the colors of the extracted juice ranged from a strong red (pg 2) to light pink (pg8) (fig. 2 a, b). anthocyanins content in pomegranate juice six different anthocyanins were present in the pomegranate juice, namely delphinidin­3,5­o­diglu­ coside (del­3,5­diglc), cyanidin­3,5­o­diglucoside (cya­3,5­digl), cyanidin­3­o­glucoside(cya­3­glc), cyanidin­3­o­pentoside (cya­3­pent), pelargonidin­ 3,5­o­diglucoside (pel­3,5­digl), pelargonidin­3­o­glu­ coside (pel­3­glc) (table 1). the anthocyanin profiles were significantly different qualitatively and qualita­ tively among the accessions. the accessions pg3­8 contained only cyanidin derivatives, namely cya­3,5­ digl, cya­3­glc and cya­3­pent.the highest content of these compounds was detected in pg 5,6,7 (table 1). the accessions pg 1 and pg 2 showed a different anthocyanin composition, characterized by the presence ofdel­3,5­diglc, cya­3,5­digl, pel­3,5­digl and pel­3­glc.the highest concentration cya­3,5­digl of was determined in pg1 (152.6±2.21mg l­1) and pg2 (102.3±1.39mg l­1), whereas the lower level was measured in pg 4 (5.450±0.63 mg l­1) (table 1). the compounds cya­3­pent and pel­3­glc are present in low concentration in the juices of all the accessions. content of hydrolyzable tannins and their derivates in pomegranate juice among the hydrolysable tannins, punicalagin α and β isomers, punicalagin derivatives,ellagic acid, ellagic acid glucoside, galloyl­glucose and hexahydroxydiphenoyl (hhdp)­hex­derivative were found in the studied accessions.all the pomegranate accession contained the punicalagin isomer α and β. punicalagin isomer α concentration ranged from ~8mg l­1inpg2 and pg8 to 37.52 mg l­1inpg1. the highest content of punicalagin isomer βwas found in pg1 (61.65 mg l­1), whereas the lowest was in pg 2 and pg 8 (~14mg l­1) (fig. 3). in the juices obtained from the different pomegranate accessions were found also other punicalagin derivatives (table 2). these compounds were identified because of their uv/vis absorption spectrum which showed the maxima at 378 and 258 adv. hort. sci., 2020 34(3): 277­286 280 nm, as also observed for punicalagin isomer α and β and previously reported in other studies on pomegrante composition (gil et al. , 2000). punicalagin derivatives 1 and 2 were more abundant in pg 1, whereas punicalagin derivatives 2 was higher in pg 6 and pg 8. punicalagin derivative 4 was maximum in pg3 and pg4, reaching concentration more than 19 mg l­1), whereas the minimum content was found in pg5 (4.54±0.49mg l­1) (table 2). overall, considering the sum of all punicalagin derivatives, the highest content was found in pg 1. a significant variation in the content of ellagic acid derivatives was found among the accessions. in particular, the concentration of ellagic acid ranged between 2.01 to 18.81mg l­1and increasing from pg2< pg1< pg6 = pg5< pg8< pg7< pg3=pg 4. a similar trend was also observed for the elleagic glucoside, whichranged from 2.92 to 27.36 mg l­1, showing the highest values in pg3 and pg4 and the lowest in pg2 (fig. 4). table 1 ­ content of anthocyanin derivatives in the juices obtained from pomegranate fruits harvested in each sampling area values are reported as mean ± sd (n=3). numbers followed by different letters in the same column are statistically different according to tukey’s test (p ≤ 0.05). del­3,5­diglc=delphinidin­3,5­o­diglucoside, cya­3,5­digl=cyanidin­3,5­o­diglucoside, cya­3­glc=cyanidin­3­o­glucoside, cya­3­pent=cya­ nidin­3­o­pentoside, pel­3,5­digl=pelargonidin­3,5­o­diglucoside, pel­3­glc=pelargonidin­3­o­glucoside. fig. 2 ­ a typical fruit harvested in each sampling area (a) and an aliquot of the corresponding extracted juice (b). pomegranate accessions del­3,5­diglc (mgl­1) cya­3,5­digl (mgl­1) pel­3,5­digl (mgl­1) cya­3­glc (mgl­1) pel­3­glc (mgl­1) cya­3­pent (mgl­1) pg1 75.58 ± 0.46 b 152.6 ± 2.21 a 0.102 ± 0.001 b ­ 1.67 ± 0.165 a ­ pg2 114.7 ± 1.85 a 102.3 ± 1.39 b 80.54 ± 1.20 a ­ 0.65 ± 0.03 b ­ pg3 ­ 7.910 ± 0.30 e ­ 10.20 ± 0.04 d ­ 0.551 ± 0.06 e pg4 ­ 5.450 ± 0.63 f ­ 5.051 ± 0.08 f ­ 0.061 ± 0.003 f pg5 ­ 26.94 ± 2.49 d ­ 30.34± 2.85 c ­ 2.451 ± 0.26 c pg6 ­ 49.75 ± 17.8 c ­ 41.32 ± 5.04 a ­ 6.581 ± 0.28 a pg7 ­ 41.12 ± 1.46 c ­ 33.53 ± 1.09 b ­ 4.663 ± 0.15 b pg8 ­ 7.680 ± 0.61 e 9.52 ± 0.48 e 0.532 ± 0.002 e zeynalova et al. ‐ phenolics in azerbaijan wild pomegranate fruits 281 fig. 3 ­ concentration of punicalagin α and β in the juices obtained from pomegranate fruits harvested in each sampling area. the bars indicate the standard deviation (n=3). different lowercase letters indicate significant differences according to tukey’s test (p ≤ 0.05). table 2 ­ content of four punicalagin derivatives in the juices obtained from pomegranate fruits harvested in each sampling area the sum of all punicalagin derivatives include also punicalagin α and β. values are the mean ± sd (n =3). values followed by the different letter in the same column are statistically different according to tukey’s test (p≤0.05). pomegranate accessions punicalagin derivative_ 1 (mgl­1) punicalagin derivative_2 (mgl­1) punicalagin derivative_3 (mgl­1) punicalagin derivative_4 (mgl­1) sum of all punicalagin derivatives (mgl­1) pg1 14.1 ± 0.73 a 21.7 ± 0.18 a 7.94 ± 0.19 b 8.44 ± 0.17 c 151.3 ± 4.66 a pg2 9.09 ± 0.03 c 8.17 ± 0.10 c 2.20 ± 0.01 e 6.15 ± 0.13 d 47.54 ± 0.38 f pg3 4.12 ± 0.06 d 10.3 ± 0.33 b 8.36 ± 0.18 b 19.7 ± 0.43 a 74.28 ± 1.41 e pg4 4.15 ± 0.05 d 10.4 ± 0.10 b 7.49 ± 0.18 b 19.7 ± 0.02 a 80.13 ± 1.13 d pg5 1.95 ± 0.09 f 8.72 ± 0.38 c 4.54 ± 0.49 d 4.54 ± 0.49 f 88.93 ± 3.29 b pg6 11.6 ± 0.48 b ­ 9.66 ± 0.44 a ­ 92.51 ± 0.88 b pg7 3.07 ± 0.02 e 11.0 ± 0.32 b 6.15 ± 0.18 c 11.3 ± 0.10 b 83.60 ± 1.29 c pg8 4.10 ± 0.15 d 6.53 ± 0.25 d 9.27 ± 0.40 a 5.71 ± 0.23 e 47.54 ± 1.90 f fig. 4 ­ concentration of ellagic acid glucoside and ellagic acid in the juices obtained from pomegranate fruits of the different accessions. the bars indicate the standard deviation (n=3). different lowercase letters indicate significant differences according to tukey’s test (p ≤ 0.05). 282 adv. hort. sci., 2020 34(3): 277­286 other compounds were putatively identified, on the basis of the retention time and their uv/vis absorption spectrum (fischer et al., 2011), as galloyl­ glucose and hexahydroxydiphenoyl­(hhdp)­hex­ derivatives (following named hhdp­hex­derivatives). indeed, the peak of galloyl­glucose showed a typical uv spectra similar to that of gallic acid (λmax = 269 nm), whereas the hhdp­hex­derivatives have similar retention time and λmax to pedunculag in derivatives (λmax = 258 and 375nm) reported in fischer et al. 2011. galloyl­glucose concentration increased from the minimum in pg2 (~6 mg l­1)to the maximum in pg7 (~18 mg l­1) (fig. 5). the concentration of hhdp­ hex­derivatives was highest in pg 1, pg 3 and pg 4. in particular, the hhdp­hex­derivative 1 was found in similar content in pg 3 and pg 4 (~50mg l­1)while hhdp­hex­derivative 2 ranged from 8.44 mg l­1in pg6 and pg8 to 20.07 mg l­1in pg1 (fig. 5). 4. discussion and conclusions the current study reported interesting differences in thejuice phenolic fingerprinting of eight wild­growing pomegranate accessions harvested in eight areas of azerbaijan characterized by different climate conditions, soil composition and altitude. specific anthocyanin composition of the eight pomegranate accessions anthocyanins are natural plant pigments that have beneficial effects for the plantsas well as for humans and animals. the presence of six anthocyanins, 3­o­glucosides and 3,5­o­diglucoside of delphinidin, cyanidin and pelargonidin, is in agreement with the anthocyanin composition determined in registered turkish varieties (türkyilmaz, 2013).the compound cya­3,5­digl was present in all samples (ben­simhon et al., 2015), however the higher value found in pg1 and pg2, and moderately in pg6 and pg7,accounted for the promininent red color showed by the four accessions respect to the other ones (fig. 2b and table 1). these outcomes are in agreement with those reported by hasnaoui et al. (2011), who found that cy­3,5­digl was the main anthocyanin in tunisian pomegranate juice as well as in the turskish “izmir 1513” variety (170 mg l­1),which were characterized by a strong red color of the juice (türkyilmaz, 2013). in addition, in the accessions pg 1 and pg 2, the low concentration of pel­3­glc, a pigment responsible for the orange pigmentation,is in agreement with those reported by ben­simhon et al. (2015), who found similar values for pel­3­glc content in the pomegranate cultivar “wonderful” (table 1). the quantitative and qualitative anthocyanin variations among the accessions might be related to the environmental conditions during the ripening period, as well as the to genotype adaptation fig. 5 ­ concentration of galloyl­glucose and two hhdp­hex­deri­ vatives (hhdp­hex­deriv_1 and hhdp­hex­deriv_2) in the juices obtained from pomegranate fruits of different accessions. the bars indicate the standard deviation (n=3). different lowercase letters indicate significant dif­ ferences according to tukey’s test (p ≤ 0.05). zeynalova et al. ‐ phenolics in azerbaijan wild pomegranate fruits 283 mechanisms to the growing area (fig. 1). borochov­ neori et al. (2011, 2014) reported an increment in diglucosylated anthocyanins after seasonal warming, and our data may concurr to suggest a correlation between high level of the anthocynain diglucosides and hot summer. indeed, pg 1 and pg 2, which can be found in dry regions characterized by hot summers, contain the highest levels of delphinidin­3,5­o­ diglucoside and cyanidin­3,5­o­glucoside, presumably due to a higher stability of 3,5­ diglucosides compared to 3­glucosides (garcı́a­ viguera and bridle, 1999; hernandez et al., 1999). however, the high levels of anthocyanins might be also the consequence of adaptation to saline soil or marshes as high amount of anthocyanin leads to increased salt tolerance in arabidopsis (oh et al., 2011). the hypothesis that pg1, pg2, pg6, pg7 can be more tolerant to saline soil should be further explored in view of their possible use as sources of resistance genes in breeding programs. additionally, the high and peculiar concentration of anthocyanins in pg1 and pg2 makes the two accessions of particu­ lar interest for utilization in human health, given the activity of these compounds as cellular antioxidant and against inflammation status (blesso, 2019). different content of hydrolyzable tanninsin the eight pomegranate accessions in addition to anthocyanins, the studied pome­ granate juiceswere also rich of hydrolyzable tannins which are synthesized from galloyl glucose, with pen­ tagalloyl glucose serving as the precursor for both higher­molecular­weight gallotannins and ellagitan­ nins. ellagitannins, which contain two or more neigh­ boring galloyl groups that are oxidatively coupled to form rigid groups such as hexahydroxydiphenoyl (hhdp) units, are particularly abundant in pomegran­ ate fruits and can be extracted in significant levels into the juice (fig. 3, 4, 5) (seeram et al., 2005). the high levels of ellagitannins, in particular punicalagin derivatives, found in our juices are in line with the those reported in literature (fig. 3) (mena et al., 2012; akhavan et al., 2015; kalaycıoğlu and erim, 2017; balli et al., 2020). even if these compounds can determine an unpleasant taste in juices, due to their interactions with the salivary proteins, they have been identified as the active antiatherosclerotic compounds in pome­ granate juices responsible for the ability of this juice to protect human low­density lipoprotein cholesterol from oxidation in vivo (aviram et al., 2000). in addi­ tion, ellagitannins show also strong radical scavenging and antioxidant ability as well as interesting anti­ mutagenic and anticancer properties (vattem and shetty, 2005). other than the well­known punicalagin application in human health (scalbert and williamson, 2000; zarfeshany et al., 2014), it is noteworthy the noticeable antifungal activities of punicalagins due to the ability, similar to that of amphotericin b to form pore­like aggregates in the cellular membranes of fungi (rongai et al., 2018). these recent findings sug­ gest further potential utilization of punicalagins extracted from pomegranate fruits as environmental­ ly­friendly natural formulations to control fungus and bacteria in crop cultivation. besides their multiple applications for human health and agriculture, ellagitannins have also impor­ tant functions for plant physiology, indeed they can offer protection against biotic stresses (furlan et al., 2011). lastly, the concentration of ellagic acid deriva­ tives seems to increase under salinity stress (borochov­neori et al., 2014), probably because of the ability of these compounds to alleviate osmotic stress as previously reported in other species (el­ souda et al., 2013). selection of pomegranate accessions through the phenolic fingerprint the phenolic fingerprint of the juices obtained from the accessions collected in eight different areas of azerbaijan allowed to select promising wild pomegranate accession particularly rich both in anthocyanins and ellagitannins. among the investigat­ ed accessions, pg 1 resulted the richest both in antho­ cyanins and hydrolysable tannins, particularly in puni­ calagin derivatives (table 1­2 and fig. s1). in this accession, collected in the region of khizi, character­ ized by hot summer and cold winter but partially miti­ gated by its proximity to the caspian sea, the interac­ tion between environmental conditions and the geno­ type induces the accumulation of both more stable anthocyanins, such as del­3,5­diglc and cya­3,5­digl, and different punicalagin derivatives. similar anthocyanin composition was also found in pg 2, which however showed an unbalalanced composotion because of the very low amount of tannins (table 1­2 and fig. s1). indeed, the accumulation of both classes of polyphenols can be stimulated under warming conditions, but also somehow drastically reduced under prolonged and severe drought conditions (mena et al., 2013). a pca biplot was applied to highlight the correlations between the contents of phenolic compounds of various accession fruits (fig. s2). the two pcs explained almost the 70% of the variance of the data. adv. hort. sci., 2020 34(3): 277­286 284 the closer the accessions lie on the plot, the more similar they are in the composition of phenolic compounds. by contrast, distant accessions have significantly different compositional characteristics. indeed, pc1 discriminates very well pg 1 and pg 2 from the other accessions, since the samples on the left of the biplot are more abundant in anthocyanins. pc2 allows to discriminate accessions on the basis of their tannin content, and it clearly shows the presence of three different groups: the richest accession (pg1), accessions with similar tannin content (pg 3,4,5,6,7) and the lowest accessions (pg 2 and pg 8). the accessions pg 3,4,5,6,7 have a more balanced content of anthocyanins and tannins (fig. s1). however, also among these accessions, environmental influences can be osbserved considering the accessions pg 5, 6, 7, which have been collected in areas with hot summer conditions and salinity soils and result to have higher anthocyanin and punicalagin contents compared to pg 3 and pg 4 (table 1, 2 and s1). by contrast, these latest accessions, collected in regions with a temperate climate and dark loamy soils, are characterized by a higher content of ellagic acid and its glycosylated derivative (fig. 4 and table s1). the results of this study, considering the recently work on the pomegranate genome (qin et al., 2017), provides a valuable resource for selecting pomegranate accessions with interesting metabolic traits that could be used in breeding efforts to increase the production of this bioactive compounds. the results reported in this study contribute to pro­ vide significant information about phenolic finger­ printing of juices extracted from different wild growing pomegranate accessions in the territory of azerbaijan republic. data showed that those accessions are rich in bioactive metabolites, which are useful for human health and might be correlated to adaptation to spe­ cific environmental conditions. the results of our study suggest that azerbaijan wild­growing accessions are promising sources of bioactive metabolites and they might be exploited in pomegranate breeding programs as donors of valuable traits for creating pomegranate varieties with high nutritional and med­ ical properties and/or to ameliorate the resistance of commercial varieties to harsh conditions. acknowledgements authors kindly acknowledge prof valida ali­zada, director of institute of botany of the azerbaijan national academy of sciences (anas) and the former director of cnr­ institute for sustainable plant protection for signing the collaboration agreement between ib (anas) and cnr­ipsp. references akhavan h., barzegar m., weidlich m.b.f., zimmer­ mann b.f., 2015 ­ phenolic compounds and antioxi‐ dant activity of juices from ten iranian pomegranate cultivars depend on extraction. ­ j. chem., 2015: 1­7. aloqbi a., 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that provides potentially nutraceutical fruits, leaves with secondary metabolites of interest, and other organs with great benefits for human health and new alternatives for production systems. the aim of this work is to carry out a bibliographic review of all the scientific material publis­ hed on this species to date. research of this species could reveal and register its ethnobotanical potential. 1. introduction south america occupies a prominent place with 263 underutilized fruit species, among which several of them play an important role in food and human nutrition. they have compounds that make them functional foods, have resilience to inclement weather, resist biotic stress and finally have important genes for their breeding (brauch, 2016). furthermore, these species are underutilized for various reasons such as: the ignorance of their nutritional value and commercial potential, botanical misinformation, lack of promotion and popularization and the rapid disappearance of the ecosystem due to habitat destruction. non­traditional fruits are considered to play an important role in miti­ gating the problems of world food in a context with growing population and malnutrition (nandal and bhardwaj, 2014; dandin and krishna kumar, 2016; s ajay vino and sinija, 2016). in addition, denardin et al. (2015) support that there is substantial evi­ dence of the beneficial effects of diets rich in fruits and vegetables. there is a preference to choose the consumption of fruits with superi­ or qualities; thus, their health benefits and perception as exotic have led to a growing demand from consumers disposed to pay higher prices than the most traditional fruits (gonzález vega, 2013). however, little is known about these species, and it is essential to research for being part of our economic, social and cultural heritage (alonso and desmarchelier, 2014). (*) corresponding author: ipovilonis@unimoron.edu.ar citation: povilonis i.s., arena m.e., radice s., 2021 ­ hexachlamys edulis (berg) kausel & legrand, “ubajay”, a native fruit species from south america. ­ adv. hort. sci., 35(4): 389­397 copyright: © 2021 povilonis i.s., arena m.e., radice s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 february 2021 accepted for publication 2 august 2021 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-10660 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 389­397 390 hexachlamys edulis (o. berg) kausel & d. legrand, “ubajay” (h. edulis) is certainly a prominent species, distributed naturally in an important area of south america, which provides potentially nutraceutical fruits, leaves with secondary metabolites of interest, and other usable organs that provide great benefits for human health and new alternatives for produc­ tion systems. the aim of this study was to conduct a review of all scientific literature on h. edulis to date. 2. materials and methods in order to get comprehensive information on this species, we have extensively explored available data­ bases like science direct, google scholar, mendeley and pubmed. in total, we selected 55 articles through database searching with the names “eugenia myrcianthes”, “hexachlamys edulis” and “myrcianthes edulis”. finally, the articles were used for data extraction and analysis, which were related to endemic area, systematic and phylogeny, morphological char­ acterization, chemistry composition and ethnob­ otany. 3. results endemic area there are no references about h. edulis distribu­ tion on another continent, therefore it can be con­ firmed that this is an endemic species of south america. area of collection and conservation of this species by different researchers is showed in figure 1. hexachlamys edulis has been studied or cited in the argentina provinces of entre ríos, corrientes, misiones, santa fe, formosa and chaco. in uruguay it grows spontaneously in soriano, río negro, paysandú and artigas departments, while in paraguay it was found in central and cordillera departments. finally, in brazil its distribution was more widespread to rio grande do sul, paraná, santa catarina, mato grosso do sul, minas gerais, sao paulo and goiás states. hexachlamys edulis has been observed in areas near water courses, where paraná, uruguay and paraguay rivers are the most important. also, it has been referred in gallery forest, delta, islands and the paranaense jungle. according to these studies and mentions, h. edulis references latitude s longitude w a molina, 2016 34°36'34.96" 58°40'31.31" b lorca et al., 1995 27°21'53.28" 55°53'36.84" c franceschini, 2000 27°18'34.35" 58°33'44.03" d grela, 2004 34°51'32.03" 56°11'58.76" e gonzález, 2003 31°22'36.55" 57°58'34.22" f vignale and bisio, 2005 31°23'5.84" 57°53'3.79" g kinupp and de barros, 2008 30° 4'11.54" 51° 8'23.17" h branco et al., 2016 22°39'16.36" 50°26'13.43" i schmeda­hirschmann, 1995 25°21'14.12" 57°31'9.01" j rozycki et al., 1997 31°40'8.99" 60°43'44.47" 1 cecotto et al., 2007 27°25'52.04" 57°19'25.70" 2 cardoso marchiori and santos, 2010 30°12'26.71" 57°33'35.91" 3 santos et al., 2014 30°12'26.68" 57°33'33.27" 4 romagnolo and souza, 2004 22°45'2.92" 53°13'20.05" 5 da cruz, 2012 30°20'43.71" 51° 1'26.89" 6 da cruz, 2012 30° 3'7.35" 51°10'32.41" 7 da cruz, 2012 30°15'52.20" 50°29'59.84" 8 takao et al., 2015 21°58'29.68" 47°52'28.53" 9 theoduloz et al., 1988 25°21'36.36" 57°19'48.44" 10 rodriguez et al., 1992 25°18'19.62" 57°23'13.40" 11 schmeda­hirschmann et al., 1996 25°16'0.56" 57°17'3.71" 12 apel et al., 2005 30° 0'21.60" 51°14'13.28" 13 bertucci et al., 2008 31°43'30.03" 58° 0'1.48" 14 fagundez, 2011 32° 06’ 19 6’’ 60°38’ 25 5’’ 15 fagundez, 2011 32°13'21 2" 60°27'51.4" 16 dujak, 2015 26°36'36.09" 55°46'26.88" fig. 1 ­ map showing the collection sites of hexachlamys edulis samples cited in this study. the circles represent samples collected from wild populations and the squares show material from herbaria or non­wild populations. data was compiled using microsoft excel and r programming language (r core team, 2018). povilonis et al. ‐ hexachlamys edulis. a review 391 is distributed at least in one million km2. systematics and phylogeny myrtaceae family has 131 genera and more than 4620 species (stevens, 2017) and up to 5800 species according to stefanello et al. (2011). the family is divided into two large tribes according to the fruit consistence: leptospermeae if the fruit is dry or myrteae if it is fleshy. the myrteae tribe or subfamily, contains three subtribes: myrcinae berg, orthotestimoninae berg, and eugeniinae berg. the latter tribe includes the genus hexachlamys (legrand, 1962) that presents approximately 10 species distributed in south america (cruz et al., 2011), among which is h. edulis. authors cited the species h. edulis with several names and also including it in different genera (table 1). psidium amygdalinum (hooker and arnott, 1833) is the oldest available name for this species. sometime later the species was named myrcianthes edulis by o. berg, and then bentham and hooker rename this taxon to eugenia. this species became known as eugenia edulis until niedenzu showed that the name already existed and introduced eugenia myrcianthes nied. finally, kausel and legrand pub­ lished the new nomenclature as hexachlamys (proença, 2006). the name h. edulis was first published in 1950 and since 1968 the genus hexachlamys was indepen­ dent (mcvaugh, 1968). hexachlamys was differenti­ ated from eugenia by berg based on the number of chalice pieces and the embryo morphology. flowers of eugenia gender have tetrameric chalice while those of hexachlamys have pentameric or hexameric calix, and the embryo of hexachlamys have hypocotyl visible and exserted and cryptic in eugenia (da cruz et al., 2013); however, some authors considered this argument inconsistent to separate the genus (ciarlante, 2003; da cruz, 2012). other names used for the species were luma grisebachii, luma myrcianthes, myrtus excelsa, calomyrtus excelsa, myrcia gemmiflora (eds), campomanesia cagaiteira, myrcia sparsifolia, hexachlamys excelsa, eugenia montevidensis, eugenia edulis ex grisebac, myrciaria edulis skeels, myrciaria plicatocostata o. berg, eugenia plicato‐ costata glaz, marlierea edulis nied, plinia anonyma sobral, plinia edulis vell, plinia plicatocostata o. berg amshoff (rotman, 1982; borges et al., 2014). since the publication of mattos (1995) and landrum and kawasaki (1997) it was discussed whether hexachlamys should be considered as a dif­ ferent genus or as a synonym of eugenia. in addition, new phylogenetic molecular analysis have revealed that hexachlamys species do not form a monophyletic clade, so the hexachlamys proposal as a synonym for eugenia was corroborated (cruz et al., 2011; da cruz, 2012; da cruz et al., 2013; mazine et al., 2014, 2016, 2018). mazine et al. (2018) suggested more consistent and stable classification: hexachlamys as a subgenus of eugenia. this last denomination, h. edulis, is the most currently used as is observed in figure 2 (proença, 2006; gbif, 2019). different authors (niedenzu, 1893; mattos, 1995; landrum and kawasaki, 1997; sobral, 2003; proença, 2006; da cruz, 2012; mazine et al., 2014, 2016; table 1 ­ different names assigned to the species by botanists over the years year botanists hooker & arnott o. berg bentham & hooker niedenzu kausel & legrand 1833 psidium amygdalinum 1857 myrcianthes edulis 1865 eugenia edulis 1893 eugenia myrcianthes 1950 hexachlamys edulis fig. 2 ­ different names used to classify hexachlamys edulis. figure taken from gbif (2019). https://www.sciencedirect.com/science/article/pii/s0308814617311007?pes=vor#bb0240 adv. hort. sci., 2021 35(4): 389­397 392 wcsp, 2020) argue that it is correct to accept the synonymy between h. edulis (berg) kausel & legrand and eugenia myrcianthes nied. morphological characterization hexachlamys edulis is a fruit tree with globular treetop (fig. 3a), with an altitude up to 10 meters according to rotman (1982), 12 meters according to legrand and klein (1977), cardoso marchiori and santos (2010) and 15 meters by dematté (1997). persistence of the foliage is contradictory (carrere, 2008). rotman (1982) consider this species as evergreen but in uruguay it shows leaf fall from late may to late august and blooming at the begin­ ning of the spring without leaves. foliage unfolds later, when the anthesis declines and vegetative growth stops in early december (gonzález, 2003). a more exhaustive observation allowed determining that this species is phanerophytes ­ by the aerial loca­ tion of its buds (raunkiaer, 1934) ­ and deciduous with concomitant foliar renewal and that apparently low temperatures accelerate defoliation (gonzález, 2003). leaves are described as petiolated, with a coria­ ceous leaf blade, pubescent, oval or elliptic­oblong, briefly acuminate, obtuse base, (3­) 5­9 cm of length by (1­) 2.5­3.4 cm, reticulate venation, with the medi­ an nerve prominent on the abaxial face; pubescent fig. 3 ­ hexachlamys edulis grown in argentina. (a) tree grown in parque nacional el palmar, entre ríos; (b) cluster of flowers; (c­d) inmature fruit; (c) globose; (d) piriform; (e­f) mature fruit; (f) disected fruit with seeds (s). bars = 1 cm. povilonis et al. ‐ hexachlamys edulis. a review 393 petioles, (3­) 7­10 mm of length (rotman, 1982). lorca et al. (1995) described polygonal to sinuous cells of various sizes and sparse trichomes stand out in the adaxial epidermis; the cuticle is thick and stri­ ated. in their abaxial epidermis they describe abun­ dant single celled trichomes and clustered and raised stomata. stomata are paracytic, that is, they have 2 adjoining cells arranged parallel to the occlusive cells. also, the presence of hypodermis towards the adaxi­ al face, palisade parenchyma 1­stratified and spongy parenchyma 5­6­stratified, with large intercellular spaces stand out. the central rib has flabeliform xylem and phloem in bundles located on the abaxial and adaxial sides of the xylem. sclerenchymal sheath interrupted on the sides. collenchyma present on the abaxial side and 3­4 hypodermis stratified on the adaxial side. flowers are typical of myrtaceae; appear in the leaf axils in apical position, are white, solitary and hermaphroditic (fig. 3b). rotman (1982) describes clusters with 2­4 flowers with pubescent pedicels from 1 to 22 mm in length, puberulous, linear lanceo­ late, up to 5 mm in length; pubescent hypanthus; sepals (4­) 5, puberulous, deltoid, acute, persistent, 3­5 mm in length; petals (4­) 5, obtuse, ciliolate, pubescent on the outer, face spacedly hairy on the inner face, 5­9 mm length; stamens 5­9 mm in length, pubescent staminal disc; 2­3 locular ovary with axillary placentation. although there are no morphological references of nectariferous glands, fagundez (2011) cites h. edulis as a native pollen­ nectariferous species of interest. flowering season is variable according to diferent the researchers. flowering, occurs from august to january in northeastern argentina, in the department of artigas, uruguay, and in asunción, paraguay by rotman (1982). instead, for dematté (1997) it hap­ pens between august and november, that is, it reduces the range, without specifying the location, while vignale and bisio (2005) reports a shorter flow­ ering period, during september, in salto, uruguay. in the same locality, gonzález (2003) observes that in early october flowering declines and ends entirely in the middle of the month. for plants grown in the locality of lajeado, brazil, guizzo and jasper (2005) recorded flowering from august to september. finally, fagundez (2011) found that h. edulis reaches full bloom during the month of october in diamante, argentina. fruit is a globose drupe (lughadha and proenca, 1996) with a yellow color, about 5 cm in maximum equatorial diameter (lorenzi et al., 2000) and up to 50 g of fresh weight in the mature state. fruit set and ripening occur in a few weeks, staggered from mid october to the end of november when the harvest can be done (vignale and bisio, 2005). rotman (1982) describes an edible fruit, globose or piriform (fig. 3c­ d), up to 9 cm long. fruits are yellow, up to 4 cm in diameter according to gonzález (2003). it is a barely fluffy fruit, with an orange flesh, very juicy, with a slightly sour taste and a pungent odor when fully ripe (www.descubriendocorrientes2012.com) (fig. 3e­f). also, it has been described as sweet sour, pleasant, and a quickly maturing (gonzález, 2003) and as a very acidic fruit (chebez and masariche, 2010). however, other authors have characterized the ripe fruit by a disgusting smell, although it would seem that there is great diversity in the fruits between genotypes (vignale and bisio, 2005), to which carrere (2008) attributes that more or less fruits could be found pleasant to smell depending on the subjectivity of those who perceive it. regarding to associated pathologies, gonzález (2003) found that most of the fruits had insect bites, but without specifications; only rossini et al. (2015) cites h. edulis as host of anastrepha fraterculus (wiedemann). the seed is exalbuminated, globose and during germination it remains enclosed by a woody struc­ ture that some authors interpreted as endocarp (rotman, 1982) (fig. 3f); but it has not been anatomi­ cally corroborated (franceschini, 2000). it has a single embryo (lughadha and proenca, 1996) solid, globose and glabrous (franceschini, 2000) with fully welded cotyledons. germination is hypogeal and cryptocoti­ lar. the seedling is completely covered with simple hairs, undeveloped hypocotyl, light green and cylin­ drical epicotyl. alternate membranous cataphylls have acute apex, obtuse base and entire margin. simple, alternate or opposite nomophiles; petiole ribbed on adaxial face; elliptical blade, chartaceous, light green, sharp apex and base and entire margin (franceschini, 2000). chemistry composition researchers promote the consumption of diets rich in fruits and vegetables due to its health benefi­ cial effects, such as lowering the risk of cardiovascu­ lar disease, cancer and conditions associated with aging and oxidative stress (gomez da silva et al., 2019). studies show that these foods can prevent dis­ 394 adv. hort. sci., 2021 35(4): 389­397 eases and disorders due to the presence of bioactive compounds with antioxidant properties. these com­ pounds interfere with biological mechanisms such as the protection against free radicals, cellular signaling mediated for free radicals, inflammation, allergies, platelet aggregation, ulcers, viruses, tumors and hepatotoxicity (denardin et al., 2015). several studies have shown that the fruits of myrtaceae have antioxidant activity (borges et al., 2014) due to the presence of anthocyanins, flavonoids, carotenoids or other secondary metabo­ lites. in particular, h. edulis fruit would have great potential as a functional food due to its compositional profile, outstanding nutritional value (vignale and bisio, 2005), very favorable for health, and considering that its vitamin content has been highlighted (cecotto et al., 2007). in addition, fruit has vitamin c quantified in 75.1 mg/100 g of fresh matter (rozycki et al., 1997). also, different phenolic compounds (2181.42 mg gae/100 g of dry matter), carotenoids (242.53 µg/g of dry matter) and antioxidant activity (153.09 µmol trolox equivalent/g of dry matter) have been quanti­ fied (branco et al., 2016). it also stands out for its pro­ tein content of 8.05% in dry matter, and good relative amounts of zinc and boron minerals (kinupp and de barros, 2008). hexachlamys edulis was noted for its high content of total pectin (403.5 mg/100 g) in fresh fruits tissue (rozycki et al., 1997). this high content explains the excellent quality of the fruit jelly of this species cited by kinupp and de barros (2008). all these data show the potential of this fruit tree, especially for the fruit pulp, juice and jelly agroindus­ try. however, this species is not considered yet by brazilian fruit producers and researchers (kinupp and de barros, 2008), unlike the manifest interest observed in other countries of south america. on the other hand, the leaf of the species belong­ ing to the myrtaceae is the most used plant organ in south america for medicinal purposes (camelo munevar, 2016), and perhaps for this reason, the chemical composition of the leaves has been the most studied. h. edulis leaves contain steroid triter­ penes, low molecular weight terpenes, tannins, polyphenols, saponins, alkaloids, flavonoids, and gly­ cosides (bertucci et al., 2008). flavonoid glycosides: myricetin 3­o­rhamnoside; myrcetin 3­o­pentoside; quercetin 3­o­pentoside; quercetin 3­o­rhamnoside and myricetin deoxyhexoside­gallate (schmeda­ hirschmann, 1995; schmeda­hirschmann et al., 1996; celli et al., 2010; takao et al., 2015). essential oils have a predominance of sesquiterpenes, of which β­ selinen (16.1%), β­caryophyllene (8.3%) and δ­ cadinene (8.3%) are the most abundant (apel et al., 2005). also, borges et al. (2014) found β­caryophyl­ lene, its oxide and caryophyllene oxide (39.3%). ethnobotany according to legrand and klein (1977), h. edulis is a species frequently cultivated in rio grande do sul (brazil), for its tasty and fragrant fruits. fruits are consumed fresh, in jams, juices, jellies (da cruz, 2012) and ice creams have been made with great acceptance. kozel (1991) describes that consumption of h. edulis fruits favor fights bladder stones and nephritic stones. also, two mbyá guaraní communi­ ties of the san rafael park reserve harvest the fruit for food (dujak et al., 2015). the fruit is considered by some people to be slightly laxative (lorenzi et al., 2000). leaves are widely used as infusions in brazil for the treatment of bronchitis, cough, whooping cough (camelo munevar, 2016) and diabetes (rodriguez et al., 1992). h. edulis leaf extract causes a significant response in blood glucose levels due to its rich con­ tent of flavonoids and tannins. in addition, lorca et al. (1995), highlight its use for the treatment of dia­ betes in the form of herbal teas or with mate. consumption was reaffirmed by pirondo et al. (2011), due to the supply of the product in the markets of corrientes, argentina. other authors highlighted the benefits of the leaves and their medical use to reduce uric acid due to the inhibition of the enzyme xanthine oxidase, producer of uricate, whose excess is considered to cause hyperuricemia in gout (a form of inflammatory arthritis) (lio et al., 1985). theoduluz et al. (1988) analyzed h. edulis leaves and it was proved to be the most active among the 15 species in a comparative study. other possible secondary uses are the production of vinegar from the fruit (carrere, 2008) or produc­ tion of essential oils from its flowers (cecotto et al., 2007). also, its wood is useful (molina, 2016) due to it is moderately heavy, hard, compact and resistant, suitable for common carpentry, tool handles, turning (cardoso marchiori and santos, 2010) and its bark can be used in the chemical industry for the prepara­ tion of white ink and tannins (carrere, 2008). it is used as alternative forage in paraguarí, paraguay (benítez and bertoni, 2015) or can also be used for reestablishing native woodland or forest gardens povilonis et al. ‐ hexachlamys edulis. a review 395 (lorenzi, 2002). chebez and masariche (2010) includes h. edulis in their list of healing trees. h. edulis is known by many vernacular names such as “ubajay” and due to its similarity to the peach (prunus persica l. batsch.), it is called “duraznero de monte” (in argentina and uruguay) (hanelt et al., 2001). also, h. edulis was named “iba hay”, “ibajai”, “igu jhay mi”, “uva jy” (legrand, 1941; rotman, 1982) and “clagye locoic” in mocovic lenguage (rosso and scarpa, 2012). in brazil it is known as “cereja do rio grande”, “ocorocil lo”, “cerejeira”, “ivai”, “pessegueiro bravo”, “pêssego do mato”, “ubajaí” and “ibajaí” (romagnolo and souza, 2004; proença, 2006). in paraguay it is known as “yvahái” (dujak et al., 2015). 4. conclusions despite the ecological and economic importance, little is known about this species of the genus hexachlamys, so it is important to revalue its use. h. edulis (o. berg) kausel & d. legrand, “ubajay” research could reveal and record its ethnobotanical potential based on the chemical composition of its fruits and other organs. at the same time, we refer to a species with the possibility of cultivation that offers the opportunity to diversify production, in this 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(*) corresponding author: s.bouzroud@um5r.ac.ma citation: benjelloun j., bouzroud s., zaid y., guedira a., smouni a., 2023 ­ warm stratification combi‐ ned with organic manure application enhances seed germination and improves cycas revoluta growth and development ­ adv. hort. sci., 37(4): 353­365. copyright: © 2023 benjelloun j., bouzroud s., zaid y., guedira a., smouni a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 march 2023 accepted for publication 21 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14544 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 353­365 354 1. introduction cycas revoluta is one of the common ornamental trees, grown in temperate, subtropical and tropical areas of the world, more precisely in miyazaki and kagoshima prefectures in kyushu district down to the ryukyu islands, okinawa prefecture in japan (dehgan et al., 1994; zarchini et al., 2011). taxonomically known as the foremost primitive species among the living cycads, this species has been used as an indoor and outdoor landscape plant over decades (jones, 1994). cycas revoluta can either be propagated from seeds or from vegetative offshoots (demıray et al., 2017). cycas propagation through seeds is extremely hard due to the physical dormancy imposed by the presence of hard coat (frett, 1987; zarchini et al., 2011; ullah et al., 2020). moreover, seeds also showed a rapid loss of viability along with a low mor­ phogenic potential, which delays their germination and thus limits their use for rapid and effective prop­ agation (naderi et al., 2015; demıray et al., 2017). to overcome these limitations, different pre­germina­ tion treatments have been applied to accelerate the germination process where several studies have sug­ gested that germination can be improved by mechanical or chemical scarification (frett, 1987; zarchini et al., 2011; fallahabadi et al., 2012; ullah et al., 2020). warm stratification has also been pro­ posed as an efficient strategy to enhance seed germi­ nation (benjelloun et al., 2021). besides the problem encountered with seeds’ propagation, cycas revoluta is a slow growing species that requires up to 10 years to reach the reproduc­ tive maturity (frett, 1987; rinaldi, 1999). nutritional management through organic manures is useful to enhance plant growth, yield and quality (el­sherbeny et al., 2012; marak et al., 2020). organic manures are, by definition, derived from animals, plants and microorganisms. a large panel of manures are nowa­ days available in the local markets at affordable prices (khairnar and kaur, 2022). organic fertilizers act as slow­release fertilizers, providing nutrients in lower amounts over an extensive time period (shaji et al., 2021). they are considered as natural source of nutrient supply in the soil and ensure the return of essential macronutrients such as nitrogen (2.42%), phosphorus (1.51%) and potassium (0.41%) as well as micronutrients including calcium, magnesium, man­ ganese and sulphur (parham et al., 2002; wang et al., 2010; khaitov et al., 2019). organic amendments such as animal, green or composted farmyard manures enhance soil’s physical properties by reduc­ ing bulk density, improving soil water­holding capaci­ ty and increasing infiltration rates (tester, 1990; werner, 1997; gopinath et al., 2008). they also heighten the existing soil nutrients, resulting in the improvement of plant growth by increasing nutrient availability (shaji et al., 2021). organic manures have broadly been used to enhance plant growth in many crop species, including wheat (triticum aestivum), sugarcane (saccharum officinarum), rice (oryza sativa) and maize (zea mays) and in ornamental plants such as marigolds, gladiolus (gladiolys grandiflorus) and roses (shanmugam and veeraputhran, 2000; attiyeh et al., 2002; singh et al., 2006; aziz et al., 2010; abbas et al., 2012; soomro et al., 2013; idan et al., 2014; baruati et al., 2018). we thought to investigate the effect of organic, sheep and horse manure, on cycas revoluta growth and develop­ ment by assessing morphological, biochemical and physiological analyses. besides, as mentioned above, warm stratification enhances cycas revoluta seed ger­ mination by accelerating embryos development. however, the beneficial effect of warm temperature on seeds’ germination is still not fully understood. thus, we focused on the histological and the biochem­ ical changes occurring in the warm stratified seeds in comparison with the control, to explain the positive effect of warm stratification on seed’s germination. 2. materials and methods plant material freshly harvested seeds collected from 50 years old female mature plants grown in the garden of the faculty of sciences, mohammed v university, morocco were used in this study. effect of warm stratification on embryos develop‐ ment warm stratification of cycas revoluta seeds was applied as described by benjelloun et al. (2021). warm treatment was applied after the mechanical removal of the sacrotesta. two treatments have been applied: the first treatment (t1) consisted on seed storage at 25°c for 2 months. the second treat­ ment (t2) consisted on seed storage at 30°c for 2 months. meanwhile, control plot (c) was not subject­ ed to any treatment. the embryos from the control plot are 0 month­old. benjelloun et al. ‐ cycas revoluta growth and development 355 zygotic embryos length and width measurements. zygotic embryos (ze) were isolated according the protocol described by benjelloun et al. (2021). zes length and width were measured in each condition and the mean was calculated from at least 12 biologi­ cal replicates. zygotic embryos germination. seeds subjected to the three different conditions (c, t1 and t2) were planted in bins containing sterilized soil at 2 5 cm depth. cultures were incubated at 25±2°c, with a photoperiod of 16 hours of light and 8 hours of dark­ ness and watered daily depending on soil moisture. daily observations were performed and seed emer­ gence was recorded. percentage of germination was then calculated. microscopic observation of zygotic embryos. microscopic observation of zygotic embryos from c and t2 treatments (2 months­old) was conducted using epson light microscope equipped with an imag­ ing software. for that, zygotic embryos were fixed using a mixture of 95°c ethanol and acetic acid (3:1) for 24 hours as described by brhadda and abousalim (2007). zes were dehydrated by passing through a series of alcohol baths (70°, 95° and 100°c). after complete dehydration, zes were transferred to two successive bath of toluene for 24 hours. samples inclusion in paraffin was performed in three succes­ sive bath of paraffin maintained at 80°c, each bath lasting 60 minutes. the 10­15 µm thick sections, made with a microtome, were spread on perfectly degreased slides. sections were stained with 1% tolu­ idine blue and viewed with. qualitative assay for alpha‐amylase activity. the presence of alpha­amylase activity was assessed according to the method of xie et al. (2007). zygotic embryos and embryoless half­seeds were placed on 2% agar in 9­cm petri dishes. the agar plates included 0.2% of soluble potato starch, 20 mm cacl2 and 20 mm sodium succinate ph 5.0. the petri dishes were then incubated at 28°c for 48 hours. after incuba­ tion, i2/ki solution (2.8 mm i2+ 43.4 mm ki in 0.2 n hcl) was added to the plates. after 5 minutes, the reaction between starch and iodine turned the agar plates to blue­purple. the agar around zes or the half­seeds with alpha­amylase activity remained colourless due to starch hydrolysis triggered by alpha­amylase activity. quantitative assay for alpha‐amylase activity. alpha­amylase activity was quantitatively determined according to a slightly modified version of the method of miller (1959) as described by liu et al. (2018). isolated zygotic embryos were collected, ground and mixed with 100 ml of chilled distilled water. the mixture was soaked in a cooling bath (4°c) for 10 minutes. the mixture was filtered and the extract was then collected and centrifuged at 12000 rpm for 10 minutes at 4°c. the recovered supernatant was heated for 15 min at 70°c. 1 ml of embryos extract was then mixed with 1 ml of 1% sol­ uble starch dissolved in sodium acetate buffer ph 5.6. the mixture was incubated for 15 minutes at 40°c ad then boiled for 5 minutes in the presence of 2 ml of 3,5­dinitrosalicylic acid. the amount of released reducing sugar was measured using a spectropho­ tometer at 540 nm using maltose as the reducing sugar standard. phytohormones quantification in zygotic embryos. abscisic acid (aba) and gibberillic acid (ga3) quantifi­ cation in zygotic embryos was performed on a finnigan lc­ms/ms system (thermo electron, san jose, ca, usa) consisting of a surveyor autosampler, a surveyor ms pump and a finnigan ltq linear ion trap mass spectrometer equipped with an esi source that was operated in negative mode. the data acqui­ sition software used was xcalibur. the lc separation was carried out by an hiq sil c18 column (250 mm × 4.6 mm i.d., 5 m). the two phytohormones were eluted isocratically with methanol/water containing 0.2% formic acid (50:50, v/v) at the flow­rate of 1.0 ml min­1. the injector volume selected was 25 l. lc­ ms/ms conditions were as follows: esi spray voltage, 4 kv; sheath gas flow­rate, 70 arb; auxiliary gas flow rate, 20 arb; capillary voltage, ­38 v; capillary tem­ perature, 350°c and tube lens, 95 v. the srm mode was used for the determination of the phytohor­ mones. ga3 and aba were monitored at m/z transi­ tions of 345→239 and 263→153, 219, respectively. the optimized collision energies for ga3 and aba were 21 and 20 ev, respectively. selected ion moni­ toring (sim) mode was used for the determination of istd. istd was monitored at m/z 121. effect of soil amendments on plant growth and devel‐ opment plant material and soil treatments. cycas revoluta plants, coming from seeds were grown in green­ house, in 3l pots. after one year of culture, plants were randomized and divided based on them into three similar groups (corresponding to each one of adv. hort. sci., 2023 37(4): 353­365 356 the three soil­substrates­treatments). the three treatments were as follows: (i) plants grown soil sub­ strate only, (ii) plants grown on soil substrate, mixed with sheep manure and (soil: sheep manure = 90:10) (iii) plants grown on soil substrate, mixed with horse manure (soil: horse manure=90:10). sheep and horse manures were produced by sardi breed and arabic breed, respectively. the chemical composition of soil and organic manure are represented in table 1. in each of the three described treatments, 20 plants­ replicates (one plant per pot) were included. during the experiment that lasts six months, all the plants were tri­weekly irrigated with distilled water. plant growth data recording. at the end of the experiment, number of leaves, plant height, stip length and width, root length and density, the num­ ber of nodules per plant, their length and width were measured. leaf, stip and root nutrient contents. at the end of the experimental period (6 months), cycas revoluta leaves, stips and roots were washed and dried. they were then ground to a fine powder, to pass a 30­ mesh screen. 0.5 g of the fine powder of each sample was dry­ashed at 515°c in a muffle furnace, for 5 hours. the ash was dissolved with 3 ml of 6 n hcl and diluted with double distilled water up to 50 ml. the concentrations of p, na, k, fe, cu and zn were deter­ mined using dtpa method as described by lindsay and norvell (1978). nitrogen content was estimated using the kjeldahl method. macronutrient (p, na, k and n) were expressed in % dw, while micronutri­ ents (fe, cu and zn) amounts were expression in mg/kg dw. chlorophyll fluorescence. chlorophyll fluorescence measurements were performed using a pulse­modu­ lated fluorometer (os30p, opti­sciences, hudson, nh, usa). fluorescence measurements were assessed in dark­adapted leaves, using the leaf­clips which were put on the adaxial leaf blades away from the leaf vein. two measurements were made on each pot. the following chlorophyll fluorescence parame­ ters were determined: maximal photochemical effi­ ciency of psii (fv/fm), the maximum quantum yield of primary photochemistry (fv/f0) and quantum photosynthetic yield of psii. chlorophyll a, b and total chlorophyll contents. chlorophyll content was performed as described by bassa et al. (2012) with a slight modification. 0.25 mg of fresh leaves were randomly taken for each treat­ ment. the fresh tissue was fine grounded in a mortar and pestles in the presence of 80% of acetone. the mixture was then centrifuged in 10000 rpm for 1 minute. samples were analyzed by spectrophotome­ try at two wavelengths, 645 and 663 nm, using 80% acetone as the blank. the chlorophyll a, b and total chlorophyll contents were calculated according to the following equations: chl a=0.999a663­ 0.0989a645; chl b= 0.328a663+1.77a645 and total chlorophyll content=20.2*chl a +8.02*chl b. statistical analysis all the analysed parameters have been compared using a fixed model of analysis of variance (anova). for each parameter and condition, means and stan­ dard deviation were calculated based on at least twelve biological replicates (except for α­amylase, aba and ga3 amounts for which means were calcu­ lated based on three independent biological repli­ cates). in case of significant difference between groups, a tukey test was used for means separation, at risk of 0.05. the relationship between parameters was observed using pearson coefficient. a principal component analysis was also launched to determine which parameter contribute most to the variation in data. 3. results and discussion warm stratification affects alpha‐amylase activity, ga3 and aba contents and enhances zygotic embryos development and seed germination seed germination partially relies on the degrada­ tion of storage reserves in mature seeds. sugars from starch hydrolysis are the major source of energy required for seedling emergence (beck and ziegler, 1989). alpha amylase is the major enzyme involved in table 1 ­ chemical composition of soil and organic amendments before assays launch soil horse manure sheep manure ph 7.64±0.05 8.50±0.01 8.3±0.01 cec (ms/cm) 0.21±0.02 2.13±0.08 2.15±0.04 potassium (ppm) 120.02±12.50 612.91±5.56 512.38±2.58 phosphorus (ppm) 58.45±0.71 330.97±0.99 329.16±0.98 nitrogen (%) 0.038±0.004 1.87±0.01 2.55±0.01 organic matter (%) 2.88±0.91 44.8±1.2 46.12±1.07 dry matter (%) 91.13±0.26 97.08±0.03 98.335±0.06 magnesium (%) 7.25±0.04 0.65±0.01 0.7±0.01 carbon (%) 1.81±0.005 105.23±0.01 106.12±0.005 sodium (m eq/100 g) 1.29±0.06 7.35±0.05 7.65±0.15 benjelloun et al. ‐ cycas revoluta growth and development 357 starch mobilization and its degradation into small organic molecules to provide energy and nutrient indispensable for seed germination and seedling emergence (ali and elozeiri, 2017). quantification in treated (t2) and untreated embryos (c) revealed a huge difference in alpha amylase activity between the two treatments. we observed an increase in alpha amylase activity in 30°c warm stratified embryos (t2), as compared to the untreated plot (c) (fig. 1b). this finding was also confirmed by the quali­ tative data (fig. 1a). the colourless areas around embryoless half­seeds derived from untreated (c) seeds were much smaller than those stored at 30°c for 2 months (t2). previous work showed that alpha amylase activity substantially increased with the increase of temperature. indeed, salisbury and ross (1995) demonstrated that some enzymes like alpha amylase reactions and thus activities increased with temperature increases from 0°c to 35°c. however, above 40°c, enzyme activities decreased due to their denaturation (salisbury and ross, 1995). sari (2021) showed that the highest alpha amylase activities were recorded in rice seeds (oryza sativa var cisokan) at 30­40°c. they noticed the absence or the decrease in alpha­amylase activity in temperature below 28°c and above 40°c, respectively (sari, 2021). here, we reported a notable and significant increase in alpha­amylase activity in warm treated seeds (t2), which can be explained by the beneficial effect of moderate temperatures (30°c in our case) on alpha­ amylase enzyme. seed dormancy and germination are mainly regu­ lated by two major antagonist phytohormones, abscisic acid (aba) and gibberellin (ga). aba positive­ ly regulates the induction and the maintenance of seed dormancy while ga enhances germination (tuan et al., 2018). investigating aba and ga3 amounts in pre­treated (t2) and untreated (c) embryos of cycas revoluta revealed a notable differ­ ence in aba and ga3 amounts. untreated embryos (c) accumulated more aba than warm stratified embryos (t2) (table 2). however, ga3 amount in warm treated (t2) seeds was higher than in the con­ trol (c). this difference was although statistically insignificant. it was previously reported that the bal­ ance of seed aba/ga levels is a pivoting regulatory mechanism underlying the maintenance and release of seed dormancy. seeds ‘dormancy studied in arabidopsis thaliana revealed that the suppression of ga3 biosynthesis and aba catabolism inhibited seeds ‘ germination and resulted in dormancy implementa­ tion (chen et al., 2020). the improvement of rice seed germination was linked to the trigger of the gly­ colytic metabolism and the restoration of ga/aba balance in seeds (yang et al., 2022). warm stratified embryos accumulated more ga3 than aba. besides, as mentioned above, warm treated embryos showed higher alpha­amylase activity, reflecting a strong gly­ colytic activity. thus, the lower aba content along with the increased ga3 levels and alpha­amylase activity suggest that those embryos can easily germi­ table 2 ­ ga3 and aba contents in untreated (c) and warm strat­ ified (t2) embryos values are mean ±sd of three independent biological replicates. (*) showed statistical differences according to t­student test (p<0.05). fig. 1 ­ qualitative (a) and quantitative (b) determination of α­ amylase activity in untreated (c) embryos and those stored at 30°c for 2 months (t2). the presence of α­amy­ lase was qualitatively determined in embryos and embry­ oless half­seeds. the quantitative determination of α­ amylase activity was performed on untreated and treat­ ed embryos. values are mean ± sd of three biological replicates. asterisk (*) showed statistical significance according to t­student test (p<0.05). treatments ga3 aba untreated embryos (c) 68.91±1.35 (*) 42.09±0.44 (*) warm stratified (t2) embryos 65.15±0.81 (*) 30.11±1.29 (**) 358 adv. hort. sci., 2023 37(4): 353­365 nate and explained the stimulatory effect of warm stratification on embryos germination that has been previously reported by benjelloun et al. (2021). cycas revoluta seeds have not the ability to imme­ diately germinate after seed shed. this finding was attributed to embryo immature stage as previously reported in cycas rumphii (de silva and tambiah, 1952), cycas revoluta (dehgan and schutzman, 1989) and eucephalartos natalensis (woodenberg et al., 2014). in this study, histological analysis showed that untreated embryos displayed a rudimentary struc­ ture subtended by a long suspensor as expected (fig. 2a). treated embryos (t2) was although observed to undergo considerable growth and development which confirmed our earlier observation (fig. 2b) (benjelloun et al., 2021). microscopic observation of embryos from control plot (c) or subjected to treat­ ment 2 (t2) thin longitudinal sections stained with toluidine blue revealed that untreated embryos were at early stage of development, which corresponds with early stage of globular embryo (fig. 2). the embryo tissue had no intercellular spaces. cells were bounded by thin walls with prominent nuclei. warm stratified embryos showed several morphological, histological, and cellular differentiation (fig. 2). those embryos were able to reach cotyledonary stage in only 2 months. shoot and root meristems can be dif­ ferentiated. shoot meristem is well developed and flanked by cotyledonary protuberances. procambium tissue, a meristematic tissue concerned with provid­ ing the primary tissues concerned with providing the primary tissues of the vascular system, was well developed. this phenomena has been earlier explained by devillez in 1976 in taxus baccata by the fact that warm stratification prompted after­ripening in underdeveloped embryos along with the suppres­ sion of the morphological dormancy (devillez, 1976). chien et al. (1998) have found that warm stratifica­ tion promoted embryos development in taxus species, the embryos reached the double of their size after six months of warm stratification (chien et al., 1998). this finding is consistent with our previous observation, in which we reported that 2 and 4 months’ exposure to warm treatment significantly increases cycas revoluta embryos’ length (benjelloun et al., 2021). seed germination in dormant seeds gen­ erally occurs as a result of the metabolic activation (bewley and black, 2013), supplying cells with the energy required for cell differentiation, expansion and development. for instance, woodenberg et al. (2013) showed that encephalartos natalensis devel­ oped embryos (at the cotyledonary stage) accumulat­ ed high amounts of starch compared to the other stages. they also suggested that the accumulated starch serves more as carbohydrate reserve during germination and seedling establishment than during the embryo growth in the ovule (woodenberg et al., 2014). given the beneficial effect of warm treatment on zygotic embryos germination, we thought to examine growth parameters (length and width) of zygotic embryos deriving from seeds stored at 25°c (t1) or 30°c (t2) for 2 months, in comparison with untreated seeds (c) isolated directly after seed shed (0 month). our results showed that seed storage for 2 months at 25°c (t1) or 30°c (t2) significantly increased zygotic embryos’ length and width, compared to the control. we noticed that zes length increased by 126% and 221% with t1 and t2 treatments, respectively (table 3). similarly, we were able to record a 5 to 7 times increase in zes width, when seeds were stored at 25°c or 30°c respectively. germination percentage fig. 2 ­ histology of (a) embryos from the control plot (0 month) (c) and (b) embryos stored at 30°c for 2 months (t2). the embryos were stained darkly with toluidine blue. bar, 200 µm. observations have been made for the control, using embryos isolated immediately after seed shed. for those subjected to t2 treatment, the observations were performed after 2 months of storage at 30°c. untreated embryos (c) displayed a rudimentary developmental stage, cells of the embryo are thin, showed small size in comparison with cells of the suspensor (longer cells). warm stratified embryos (t2) showed a differentiated structure with two apparent cotyledons, shoot and root meristem can be easily identified. benjelloun et al. ‐ cycas revoluta growth and development 359 was also stimulated with the application of warm treatment. a significant increase in the percentage of germination was reported with the application of t2 treatment. indeed, the highest germination percent­ age of 49.33% was recorded with seeds pre­treated at 30°c for 2 months. meanwhile, no significant dif­ ferences were detected between the control plot (25.33%) or t1 treatment (28.00%). these data strongly showed the stimulatory effect of warm treatment on zes development and germination, thereby confirming our earlier observations (benjelloun et al., 2021). organic amendments stimulate cycas revoluta growth investigating the effect of soil organic amend­ ments on cycas revoluta growth in greenhouse con­ ditions revealed a stimulatory effect of organic manures on plant growth (fig. 3). all fertilization treatments increased plant growth attributes except for root length. we found that both horse and sheep manures significantly increased plant height and stip length, but no significant differ­ ences were recorded between the different manures (table 4). regarding root development, a significant increase in root length and root density was observed with the use of sheep manure. nodules’ number also increased in a significant way with sheep manure amendment, while comparable values were recorded in the control treatment and in the presence of horse manure (table 4). besides nodules ‘number, nodules’ length and width was notably vari­ able. significant differences in nodules’ number have been recorded between the three culture conditions. table 3 ­ effect of warm stratification on zygotic embryos length, width and germination values are mean ± sd of at least 12 biological replicates. for each analysed parameter, different letters indicate statistical difference according to tukey test (p<0.05). parameters control (c) 25°c (t1) 30°c (t2) zes length 3.46 ± 1.14 a 7.84 ± 1.29 b 11.14 ± 1.20 c zes width 0.09 ± 0.02 a 0.51 ± 0.04 b 0.68 ± 0.03 c germination percentage 25.33 ± 1.63 a 28.00 ± 1.60 a 49.33 ± 1.70 b fig. 3 ­ effect of organic amendments on cycas revoluta growth. (control) one­year­old cycas plant grown in soil substrate only, (hm) one­year­old cycas plant grown in a mixture of soil and horse manure and (sm) one­year­old cycas plant grown in the presence of mixture of soil and sheep manure. scale bar = 13 cm. parameters soil soil­horse manure soil­ sheep manure number of leaves per plant 1.69 ± 0.48 a 2.94 ± 0.99 b 2.73 ± 0.47 b plant height (cm) 20.17 ± 2.40 a 57.43 ± 4.53 b 58.14 ± 2.91 b stip length (cm) 3.19 ± 0.32 a 4.35 ± 0.34 b 4.34 ± 0.42 b stip width (cm) 3.52 ± 0.34 a 4.15 ±0.38 b 4.17 ± 0.32 b root length (cm) 21.14 ± 1.68 a 21.11 ± 3.60 a 32.02 ± 2.51 b root density 1.13 ± 0.23 a 1.23 ± 0.20 ab 1.49 ± 0.29 b number of nodules per plant 3.18 ± 1.93 a 4.31 ± 1.19 a 12.67 ± 2.05 b nodules length (cm) 0.81 ± 0.12 a 0.95 ± 0.19 b 1.22 ± 0.21 c nodules width (cm) 0.84 ± 0.12 a 1.39 ± 0.30 b 1.35 ± 0.35 b table 4 ­ effect of organic amendments (horse and sheep manures) on the agro­morphological parameters of cycas revoluta plants values are mean ± sd of at least twelve independent replicates. for each parameter, values with different letters indicate statistical differences according to tukey test (p<0.05). adv. hort. sci., 2023 37(4): 353­365 360 the highest nodules’ length values were recorded in the presence of sheep manure. regarding nodules’ width, the statistical analysis revealed a significant difference between the control and the different manures treatments. the highest width values were interestingly reported with the use of horse manure. the cross­section of these nodules revealed the pres­ ence of blue­green halo, which can potentially indi­ cate the presence of cyanobacteria (fig. 4). further analysis should be conducted to confirm these obser­ vations. organic amendments have the ability of binding minerals like, magnesium, potassium and calcium in a colloidal form (humus and clay), which can promote the formation of stable aggregate of soil particles at desired porosity to support plant growth (azarmi et al., 2009; chang et al., 2010). here, we found that organic manures (sheep or horse manures) signifi­ cantly promoted plant height, number of leaves per plant and stip length. indeed, manure supplementa­ tion increases macro and micronutrient contents as well as soil physico­chemical properties, which can ultimately lead to a better vegetative growth (adekiya et al., 2020). root development can be sig­ nificantly influenced by soil mineral composition. the presence of sufficient nutrients prompted the devel­ opment of root system. gregory (1994) compared the influence of fertilization on root growth to its beneficial effect on shoot growth. smith showed that additional nitrogen levels can result in a better leaf growth and number of another cycadales species; zamia integrifolia (smith, 1978). here, we found that sheep manure allowed a better development of root system, as evaluated by root length and density (gregory, 1994). in quinoa (chenopodium quinoa), kakabouki et al. (2019) have linked the better devel­ opment of root system in plants grown in a fertilized soil with the presence of high amounts of nitrogen (kakabouki et al., 2019). sheep manure used in this study displayed the highest nitrogen content (2.55 ± 0.01%) (table 1), thus explaining the better plant development recorded. besides promoting shoot and root development, sheep manure supplementation resulted in a significant increase in the number of nodules per plant (table 4). it has been previously reported that cycas revoluta forms beneficial associ­ ation with the blue green algae, ensuring nitrogen fixation. this symbiotic nitrogen fixation, occurring in cycads coralloid roots has been reported to arise nitrogen at a significant rate (halliday and pate, 1976; grove et al., 1980). nitrogen fixation by coral­ loid roots was estimated to be comprised between 18.8 kg n/ha and 35 kg n/ ha (smith, 1978). dehgan (1983) showed that nitrogen fixation in the coralloid roots contributes significantly to plant growth (dehgan, 1983). the presence of great number of nodules per plant in cycas plants grown in the presence of sheep manure could indicate a better nitrogen fixation potential, which can increase plant supplying with sufficient amount of nitrogen. besides cyanobacteria ­ cycas roots association, previous reports showed also the presence of endophytes in regular and coral­ loid roots of cycas bifida (zheng et al., 2018). endophytic bacteria have been associated with the growth promotion of several crop species such as maize (alkahtani et al., 2020), wheat (khan et al., 2017), tomato (solanum lycopersicum) (amaresan et al., 2012), rice (oryza sativa) (khan et al., 2020) and chilli (capsicum annuum) (amaresan et al., 2012). the plant growth is promoted through improved nutrient acquisition, including nitrogen fixation and the production of plant growth promoting sub­ stances such as indole acetic acid and cytokinins (miliute et al., 2015). therefore, it could be more interesting to investigate the presence of these bene­ ficial microorganisms in the observed nodular struc­ tures, reported in this work. fig. 4 ­ cycas revoluta plant presenting a globular­like structures or nodules in the underground part of the plant. (a) cycas revoluta root system with several nodular struc­ ture, (b) a single nodule and (c) isolated nodule present­ ing a blue­green halo which indicates the presence of cyanobacteria. black narrow pointing the blue­green halo. scale bar = 1 cm. benjelloun et al. ‐ cycas revoluta growth and development 361 organic manures have a positive effect on cycas rev‐ oluta seedlings photosynthetic apparatus chlorophyll a, b and total chlorophyll contents sig­ nificantly increased with the application of different organic manures (fig. 5). an average increase (2.11 and 2.67 mg/g fw) of chlorophyll a content was recorded with the application of horse manure and sheep manure respectively. similar trend was also observed with chlorophyll b (3.61 and 4.61 mg/g fw) and total chlorophyll (71.06 and 82.98 mg/g fw) con­ tents. regarding the chlorophyll fluorescence, our results showed an increase in chlorophyll fluores­ cence attributes mainly the potential activity of psii, the maximum quantum yield of primary photochem­ istry and effective quantum yield of psii with the application of either horse or sheep manure (fig. 5). note that no significant difference has been observed in the potential activity of psii between the control and plants grown in horse manure­soil mixture. it is well established that organic fertilizers improved plant growth and development, by improv­ ing soil physico­chemical properties and nutrient availability to plants (eneji et al., 2001; azarmi et al., 2009; osama et al., 2016). this latter seems to direct­ ly affected photosynthesis process (osama et al., 2016). several studies conducted on different plant species such as sugarcane (saccharum officinarum), soybean (glycine max), potato (solanum tuberosum) and kiwifruit (actinidia deliciosa) have underlined the positive effect of organic amendments on chlorophyll content (ghosh et al., 2004; bokhtiar and sakurai, 2005; najm et al., 2012; sharma et al., 2022). sharma et al. (2022) associated this beneficial effects on pho­ tosynthetic properties with the presence of high nitrogen amounts conferred by organic manure sup­ plementation (sharma et al., 2022). moriwaki et al. (2019) explained the positive effect of nitrogen on photosynthetic attributes (photosynthetic quantum yield) by the increase in thylakoid density, which can enhance green light absorption (moriwaki et al., 2019). in cycas revoluta, we found that organic manures, more precisely sheep manure, significantly improve plant photosynthetic attributes (fv/fm and φpsii). this can likely be attributed to the high nitro­ gen content of sheep manure along with the pres­ ence of great number of nodules per plant. organic manures modify mineral allocation in cycas revoluta plants the mineral contents of cycas revoluta botanical parts (leaf and root) were determined after six months of culture. our results showed a huge differ­ ence in macronutrients and micronutrients amounts between the control plants and those grown in the presence of either horse or sheep manure (table 5). regarding sodium content, the highest and lowest sodium amounts was recorded in roots grown in the presence of sheep manure and leaves collected from the control plants. potassium content was highly vari­ able between the three different treatments. the highest potassium content of 15.02±2.18% was detected in roots isolated from plants grown in the presence of sheep manure. meanwhile, the lowest potassium content of 3.2±0.64% was this time recorded in leaves under sheep manure treatment. the highest nitrogen content was recorded in roots of the control plants and leaves collected from plants grown in the presence of sheep manure. however, no significant differences have been recorded in phosphorus amount. for iron, the highest and the lowest amounts of 7.02±0.39 mg/kg and 11.83±0.50 mg/kg were observed in leaves collected from plants grown in the presence of sheep manure and roots of the control plants, respectively. the highest copper and zinc contents were recorded in leaf under horse manure treatment and root of the control plants respectively while the lowest amounts were found in roots of the control plots and roots under horse manure treatment, respectively. organic fertilization is known to affect the concen­ trations and the uptake of several macro and fig. 5 ­ effect of organic amendments ­ horse manure (hm) and sheep manure (sm) ­ on the photosynthetic attributes. (a) chlorophyll a content, (b) chlorophyll b content, (c) total chlorophyll content, (d) the maximum quantum yield of primary photochemistry (fv/fm), (e) potential activity of psii (fv/fo) and (f) effective quantum yield of psii (φpsii) of cycas revoluta plants. values are mean ± sd of at least six independent replicates. for each param­ eter, values with different letters indicate statistical dif­ ferences according to tukey test (p<0.05). adv. hort. sci., 2023 37(4): 353­365 362 micronutrients such as nitrogen, potassium and phosphorus by plants, independently from the irriga­ tion system (yang et al., 2004). the highest nitrogen amounts were recorded in the aerial parts of cycas plants grown in the presence of sheep manure, thus explaining the positive effect of sheep manure sup­ plementation on vegetative growth and photosyn­ thetic attributes. potassium concentration was signif­ icantly more in the roots isolated from plants grown in the presence of either sheep or horse manure than in plants grown without organic manure addition. maximum increase of leaf k amount was detected with the addition of horse manure. the increase in k concentration, resulting from organic matter addition might be attributed to k concentration in the organic fertilizers (table 1), as previously shown for zea mays (aziz et al., 2010) and brassica juncea (aziz et al., 2006). the interrelationship between plant morphologi­ cal, biochemical, and physiological attribute using the pearson’s correlation matrix revealed the existence of a significant positive correlation of potassium con­ tent in roots with leaves number (r=0.89), stip width (0.75), nodules’ number (r=0.72), nodules’ length (r=0.59), nodules’ width (r=0.54), chlorophyll a (r=0.77), chlorophyll b (r=0.81) and total chlorophyll (r=0.78) contents. similarly, root sodium content was positively correlated with root length (r=0.81), nod­ ules’ number (r=0.80), their length (r=0.61) and root potassium amount (r=0.68). root sodium amounts were also positively correlated with photosynthetic attributes, more precisely with fv/f0 (r=0.84), fv/fm (r=0.76) and psii (r=0.79) (fig. 6a). interestingly, root zinc amounts were negatively correlated with stip length (r=­0.78) and width (r=­0.59), leaf length (r=­ 0.9), chlorophyll a (r=­0.84), chlorophyll b (r=­0.96) and total chlorophyll contents (r=­0.87). pca (principal component analysis) showed that variables explained 66.9% of the variation in the first two axes (fig. 6b), which is why 42.6% and 24.3% variances were accounted respectively, for the first and second principal components. the first principal component counted more attributes than the second principal component. results from this study strongly suggest that the application of sheep manure significantly affected the plant root growth (root length), the pho­ tosynthetic attributes (fv/fo, psii, fv/fm) and also root sodium content. horse manure seems to strong­ ly affected copper amounts in both leaves and roots. table 5 ­ mineral composition of roots and leafs of cycas revoluta plants subjected to the three following treatments (control "c", horse manure "hm" and sheep manure "sm") for six months values are mean ±sd of three independent biological replicates. values with different letters indicate statistical differences according to tukey test (p<0.05). treatments nitrogen (%) phosphorus (%) sodium (%) potassium (%) iron (mg/kg) cupper (mg/kg) zinc (mg/kg) root c 1.75±0.09 a 0.33±0.05 a 1.50±0.36 a 4.55±0.15 cd 11.56±0.21 a 46.33±2.08 d 338.66±9.08 a hm 1.44±0.09 c 0.36±0.08 a 1.95±0.26 a 11.42±1.11 b 10.99±0.38 ab 49.83±2.83 d 89.90±3.34 d sm 1.60±0.14 ab 0.43±0.08 a 4.03±0.77 b 15.02±2.18 a 11.15±0.52 ab 47.50±3.44 d 168.0±15.93 c leaf c 1.19 ±0.07 d 0.43 ±0.05 a 1.12 ±0.13 a 4.55 ±0.05 cd 7.92 ±0.72 c) 67.0 ±2.00 bc 282.50 ±4.5 b hm 1.01 ±0.11 de 0.38 ±0.09 a 1.40 ±0.20 a 10.50 ± 1.21 b 8.12 ±0.59 c 85.00 ±3.60 a 166.33 ±18.55 c sm 1.77 ±0.18 a 0.35 ±0.05 a 2.31 ±0.10 a 2.68 ±0.08 d 7.02 ±0.37 d 61.00 ±1.82 c 245.50 ±24.49 b fig. 6 ­ pearson correlation (a) and principal component analysis (b) gathering all the agro­morphological and physiologi­ cal parameters that have been used for this study. benjelloun et al. ‐ cycas revoluta growth and development 363 4. conclusions here, we demonstrated that warm stratification significantly enhanced cycas revoluta seeds’ germi­ nation by promoting embryos’ length. in the present work, we found that warm stratification increased α­ amylase (key enzyme involved in starch mobilization and degradation) activity triggered by the moderate temperature imposed during the warm treatment application. decreased aba content was also detect­ ed in warm stratified embryos, thus explaining the highest germination percentage recorded in the pre­ vious work (benjelloun et al., 2021). at the histologi­ cal level, we found that warm stratification triggers cell differentiation and embryos development to reach cotyledonary stage after 2 months of treat­ ment only. plant growth was highly stimulated by the application of organic manures, more specifically by sheep manure application. indeed, sheep manure application improves plant growth, namely plant height, root length and density, the number of nod­ ules per plant, nodules ‘length and width, nitrogen allocation and photosynthetic attributes. overall, these data strongly recommend the use of warm stratification to enhance cycas revoluta seed’s germi­ nation followed by sheep manure application to accelerate cycas revoluta growth, thereby offering new insights on the use of biological agriculture inputs for sustainable production of horticultural plants. acknowledgements the authors are grateful to dr. h el ouardi for hplc analysis and to m. a. el mouttaqi for his assis­ tance during statistical analysis performance. references abbas g., khattak j., mir a., ishaque m., hussain m., wahedi h., ahmed m., ullah a., 2012­ effect of organic manures with recommended dose of npk on 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microbiol., 56: 337­345. impaginato 83 adv. hort. sci., 2024 38(1): 83­96 doi: 10.36253/ahsc­14691 efficacy of active and passive modified atmosphere packaging on quality preservation and storage life of pome­ granate fruit and arils: a review f. moradinezhad 1 (*), a. ranjbar 1 department of horticultural science, college of agriculture, university of birjand, birjand, iran. 2 pistachio research center, agriculture research education and extension organization (areeo), horticultural science research institute, rafsanjan, iran. key words: fruit quality, minimal processing, nutritional characteristics, post­ harvest, ready­to­eat pomegranate, storability. abstract: pomegranate has nutritional value and health benefits due to its bioactive compounds and antioxidant properties. fruit consumption is strongly recommended due to its high content of vitamins, fiber, minerals, and polyphe­ nols. supplying ready­to­eat pomegranate can be a beneficial technique to increase consumption with regard to its nutritional properties. however, main­ taining nutritional quality and preventing microbial spoilage is a major chal­ lenge. fruit quality is lost with visible symptoms such as weight loss, shriveling, husk scald, chilling injury, fungal rot, aril color degradation, and off­flavor dur­ ing long­term storage. therefore, it is very important to use appropriate strate­ gies to maintain pomegranate whole fruit and aril quality. gases around the product create a suitable environment for oxidative reactions and aerobic microorganism growth. therefore, changing the atmosphere around the prod­ uct can help maintain its quality. one of the effective methods to increase the postharvest life of products is to use modified atmosphere packaging (map), which reduces microbial spoilage and chilling injury, preserves the quality, and extends the shelf life by reducing the respiration rate. modified atmosphere packaging, which uses natural atmospheric components (o2, co2 and n2), has been widely accepted due to the lack of toxic residues on the product. this review discusses on recent research in terms of map application on quality properties and postharvest life of pomegranate fruit and arils during storage. 1. introduction regarding botanical classification, pomegranate belongs to the angiospermae category, dicotyledoneae subcategory, myrtales order, lythraceae family, punica genus, and p. granatum species. p. granatum species is diploid (16x=2 n=2). it has four subspecies: plenty‐flora, spinisia, nana, and sativa. edible pomegranate is in the subspecies of sativa (*) corresponding author: fmoradinezhad@birjand.ac.ir citation: moradinezhad f., ranjbar a., 2024 ­ efficacy of active and passive modified atmosphere packaging on quality preservation and storage life of pomegranate fruit and arils: a review. ­ adv. hort. sci., 38(1): 83­96. copyright: © 2024 moradinezhad f., ranjbar a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 may 2023 accepted for publication 8 november 2023 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-14691 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 83­96 84 (jalikop, 2010). this fruit is mainly grown in spain, turkey, egypt, tanzania, saudi arabia, azerbaijan, pakistan, afghanistan, india, and china. among these countries, india, iran, china, and turkey are the main pomegranate producers (ramezanian and erkan, 2017). pomegranate has many nutritional properties and bioactive compounds with anti­inflammatory, antioxidant, anticancer, antihypertensive, antidiabet­ ic and liver damage­reducing effects (kalaycıoglu and erim, 2017; khajebishak et al., 2019; sohrabet al., 2019; barati boldaji et al., 2020; firdous et al., 2023). the pomegranate is considered a non­climacteric fruit, and is harvested at the optimal maturity stage for storage, which has optimal organoleptic charac­ teristics. the harvest index is the ratio of sugar to acid, and the standard index for harvesting is differ­ ent depending on the cultivar (artés et al., 2000). post­harvest quality loss due to weight loss, harden­ ing of the husk, cracking husk, chilling injury (ci) symptoms and fungal diseases limit its storage potential (pareek et al., 2015; porat et al., 2016; ranjbari et al., 2018; candir et al., 2019; lufu et al., 2020) (fig. 1). moreover, ready­to­eat pomegranate aril is very perishable and rapidly lost its quality dur­ ing storage. the most important goal of the posthar­ vest industry is to maintain the quality during trans­ portation and storage (el­ramady et al., 2015; moradinezhad and dorostkar, 2021). reducing postharvest losses leads to more available food, reducing cultivated areas, and preserving natural resources. therefore, it is necessary to use tech­ niques to maintain the fruit quality after harvest and during storage. changing the atmosphere around the fruit through controlled atmosphere (ca) or modified atmosphere packaging (map) is a reliable and safe approach (caleb et al., 2012; caleb et al., 2013 a, b). in map, the gas composition inside the package is obtained based on the gas exchange through the semi­permeable layer and fruit respiration rate (caleb et al., 2018). although the respiration rate of pomegranate fruit is slow in cold temperatures, how­ ever, during the respiration process, oxygen (o2) is consumed and dioxide carbon(co2) is produced, which changes the composition of the gas inside the package (passive map). in addition to passive map, the initial modification of respiratory gases (active fig. 1 ­ chilling injury development in pomegranate cultivars stored at cold storage. a) the external appearance and weight loss of ‘wonderful’pomegranate fruit after 4, 8, and 12 weeks of storage at 7°c, photo caption from left to right, respectively (adetoro et al., 2020), b) the external and internal appearance of ‘mengzi sweet’ pomegranate fruit after 8 weeks of storage at 2°c (chen et al., 2021), c) husk scald and chilling injury in pomegranate ‘wonderful’ fruit skin after 120 days of storage at 3.5°c (maghoumi et al., 2022), d) husk scald symptoms of ‘wonderful’ pomegranate fruit after 12 weeks of storage at 7°c (li et al., 2016), e) the internal appearance and discoloration of the texture of ‘wonderful’ pomegranate fruit after two weeks of storage at 1°c (kashash et al., 2019), f) hard husk and shriveling symptoms of ‘hicaznar’pomegranate fruit after 6 months of storage at 6°c (candir et al., 2019). moradinezhad and ranjbar ‐ efficacy of map on the shelf life of pomegranate fruit and arils 85 map) based on the physiology of the product, envi­ ronmental conditions and the properties of the pack­ aging materials has a significant effect in reducing respiratory activity and increasing the shelf life (opara et al., 2015; opara et al., 2017; belay et al., 2018; dorostkar and moradinezhad, 2022). despite the advantages of map, ultra­low or high concentrations of gases inside packages may cause damage to the texture. an excessive increase in o2 concentration increases the production of radicals that damage the cytoplasm, such as superoxide (o−2), hydrogen peroxide (h2o2), and hydroxyl (oh−), conse­ quently reducing the quality by inhibiting some meta­ bolic activities (choudhury et al., 2017). the reduc­ tion of o2 below the critical limit causes the initiation of anaerobic respiration and fermentation, resulting in an unpleasant aroma and taste (li et al., 2014). also, excessive accumulation of co2 can lead to a decrease in quality by accelerating color changes and increasing the hydrolysis of pectin compounds (teixeira et al., 2016). with the increasing demand for map application, it is necessary to understand the role of gases and their effect mechanism on product quality. therefore, a simplex lattice design approach was con­ sidered to select and identify the optimal gas compo­ sition to maximize the quality parameters of pome­ granate aril cv. wonderful under modified atmos­ phere conditions (belay et al., 2019 a). the partial pressure of gases as visual quality, physicochemical characteristics, antioxidant properties and volatile organic compounds (vocs) were selected as response variables. the results showed that co2 was the most important factor affecting color, texture firmness and volatile organic compounds (aldehydes, ketones, monoterpenes) of the wonderful cultivar (li et al., 2018; li et al., 2020). o2 had the greatest effect on color, organic acid, decay development and alco­ holic volatile organic compounds. the maximum con­ centration of sugars, organic acids, total soluble solids (tss), and color using a gas mixture (6­7 kpa o2+ 7­8 kpa co2),and the maximum release of volatile compounds responsible for the taste of arils was obtained using a gas mixture (2 kpa o2 + 18 kpa co2 + 80 kpa n2) (belay et al., 2019 a). low o2 inhibits the rate of oxidation by reducing the rate of respiration and delaying fruit ripening (li and zhang, 2015; teixeira et al., 2016). the concen­ tration of super atmospheric o2was effective in inhibiting microbial growth and reducing decay by preventing anaerobic respiration on minimally processed pomegranate arils (cv. wonderful) (belay et al., 2017 b). the qualitative changes of the fruit are related to the change of different metabolic path­ ways which are presented in the modified atmos­ phere by determining the genomic interpretation and their transcription frequency under the influence of packaging conditions (rosales et al., 2016). the response of fruits to gas concentration is character­ ized by the profile of primary metabolite (respiration rate) and secondary metabolite (fermentative metabolites and volatile compounds) (blanch et al., 2015). despite the advantages of the modified atmos­ phere in increasing the shelf life of the product, reducing storage losses without preservatives appli­ cation, accurate control of storage temperature due to the effect of temperature on the permeability of used films, respiration rate, and solubility of gas in the aqueous phase of the food and the nutrient leak­ age, and determination of the specific gas composi­ tion for each product should be investigated. considering the importance of the storage envi­ ronment, especially the concentration of o2 and co2 in the occurrence of injury symptoms and the shelf life of products, this review aimed to investigate the efficacy of map on the overall quality of the whole pomegranate fruit and arils during cold storage. 2. influence of map on quality traits of pomegran­ ate chilling injury, weight loss, and overall quality one desirable approach to minimize weight loss in a modified atmosphere is to reduce respiratory activ­ ity, which substantially reduces transpiration (belay et al., 2018). therefore, in the map, it is recommend­ ed to choose the appropriate gas composition to con­ trol the weight loss of the product. in the investiga­ tion of the suitable gas composition to reduce the respiration rate, the concentration of o2 (2, 10 and 21 kpa) and co2 (2, 10 and 20 kpa) on pomegranate arils cv. hicaznar (ersan et al., 2009), and the concen­ tration o2 (5, 21, and 30 kpa) and co2 (0, 10, and 40 kpa) on the pomegranate arils cv. wonderful (banda et al., 2015) stored at 5°c showed that low o2 con­ centration significantly decreases the respiration rate. also, the concentration of o2 2­4 kpa is recom­ mended to maintain the quality of pomegranate arils cv. mollar de elche (lópez­rubira et al., 2005). in a study on pomegranate arils cv. wonderful in modi­ adv. hort. sci., 2024 38(1): 83­96 86 fied atmosphere (4.67kpa o2, and 12.67kpa co2) packed with propafilm and nature flex showed that arils packed in propafilm had lower mass loss than natureflex, due to the film’s lower water vapor transmission rate (wvtr) (belay et al., 2018). high gas barrier properties propafilm, even at high rela­ tive humidity, lead to the potential for shelf­life extension. weight changes are related to changes in respiration and transpiration, which are influenced by the difference in diffusion resistance and the sur­ face to volume ratio of pomegranate arils (khorshidi et al., 2011). long­term storage of pomegranate arils causes more weight loss due to higher enzyme activi­ ty and lower resistance of the cell membrane against water loss (belay et al., 2018). modified atmosphere packaging reduces the vapor pressure difference between the surface and environment of the product by maintaining the relative humidity around the fruit, accordingly reducing the water loss of the product (ngcobo et al., 2013). water loss of whole pomegranate fruit causes husk browning at the storage (nerya et al., 2006). also, enzymatic browning after microbial infection is the main cause of quality reduction (ioannou and ghoul, 2013) that polyphenol oxidase (ppo) and per­ oxidase (pod) activity increases the brown superficial discoloration of pomegranate fruits (xie et al., 2019; baghel et al., 2021). storage of pomegranates cv. mollar de elche in controlled atmospheres (10kpa o2 and 5kpa co2; 5kpa o2 and 5kpa co2; 5kpa o2 and 10kpa co2;or 5kpa o2 and 0kpa co2) for 8 weeks at 5°c showed that all treatments except 10kpa o2 and 5kpa co2 reduced weight loss, fungal rot and chilling injury symptoms (husk scald) and were efficient for increasing the quality and extending the shelf life of pomegranate fruits (artés et al., 2000). moreover, storage of pomegranate fruit in a controlled atmos­ phere (1 kpa o2 +15 kpa co2 or 5 kpa o2 +15 kpa co2) significantly reduced botrytis rot and scald for up to 6 months at 7°c (defilippi et al., 2006; palou et al., 2007). pomegranate fruits stored in a modified atmosphere (5kpa co2 + 3kpa o2) for three months at 5°c had wrinkle­free husk and smoother, less chilling injury, fewer disease symptoms and, as a result, bet­ ter quality compared to fruits stored in a normal atmosphere (sidhu et al., 2019). pomegranate fruits stored in a modified atmosphere (5­10 kpa co2 + 3­5 kpa o2) increases shelf life due to reduced weight loss, decay and injury symptoms (selçuk and erkan, 2015; porat et al., 2016; maghoumi et al., 2022). high co2 concentration is effective in maintaining the activity of antioxidant enzymes such as catalase (cat), superoxide dismutase (sod) and ascorbate peroxidase (apx) (song et al., 2013), and on the other hand, due to the lower o2 concentration, pod and ppo enzymes do not catalyse the oxidation of phenols (ali et al., 2019). also, increasing the concen­ tration of co2 and decreasing the concentration of o2 will inhibit fungal contamination by suppressing res­ piration (almenar et al., 2006). as mentioned, water stress, oxidative stress, lipid peroxidation and cell membrane instability are key factors in burn develop­ ment (singh et al., 2018). map and ca prevent husk scald by limiting oxygen access, oxidative stress and water loss prevention. it has been proved that map is effective in maintaining the external and internal quality of pomegranate fruit by controlling weight loss, and preventing fungal decay and husk scald dur­ ing cold storage (selçuk and erkan, 2015; porat et al., 2016). fruit weight loss increases ci symptoms by destroying the membrane integrity (opara et al., 2015; maghoumi et al., 2023). the decrease in unsat­ urated fatty acid content and membrane fluidity causes damage to the membrane structures and a lack of resistance to cold (casares et al., 2019). it has been reported that ci symptoms coincide with the leakage of electrolytes in the pomegranate peel (casares et al., 2019). oxidative damage, membrane chilling injury and electrolyte leakage in pomegranate peel are indicated as a function of o2 levels in the first days of storage (valdenegro et al., 2018). in the modified atmosphere condition, the stability of sod and cat enzymes leads to less accumulation of h2o2 and malondialdehyde (mda), more integrity of the membrane and therefore less electrolyte leakage (li et al., 2016; valdenegro et al., 2022), and a higher pal/ppo ratio reduces oxidative damage (baghel et al., 2021). researchers have studied extensively the effect of a low­oxygen atmosphere on the quality characteristics of whole pomegranate fruit or arils cv. primosole (d’aquino et al., 2010), cv. acco and herskawitz (caleb et al., 2013 a, b), cv. wonderful (banda et al., 2015), cv. hicaznar (candir et al., 2018), cv. shishe­kab (moradinezhad et al., 2013, 2019), and it has been found that the atmosphere with low oxygen has the potential to prevent weight loss, chill­ ing injury, decay and delay in post­harvest ripening (table 1). firmness the firmness reduction is related to water loss, moradinezhad and ranjbar ‐ efficacy of map on the shelf life of pomegranate fruit and arils 87 cell membrane deterioration and senescence (díaz­ mula et al., 2012; hussein et al., 2015). the effect of map on maintaining fruit firmness is related to the control of weight loss, which has an important effect on postharvest management (jouki and khazaei, 2014). also, maintaining post­harvest firmness is related to the control of biochemical processes (activities of pectinsterase and polygalacturonase enzymes) (fagundes et al., 2015; bang et al., 2019) and the prevention of ethylene synthesis under a modified atmosphere (akbudak et al., 2012). high co2 inhibits ethylene production and delays ripening (kader and watkins, 2000). a similar effect of high co2 and super atmospheric o2 has been reported on firmness of aril cv. wonderful that arils stored under super atmospheric o2 (70%) showed a slight increase in the firmness compared to low o2 treatment (5 and 10%) (belay et al., 2017 b). a low respiration rate lim­ its the activity of cell wall­degrading enzymes (such as pectinase and cellulase) and preserves firmness during storage (fagundes et al., 2015; bessemans et al., 2016), and as a result delayed ripening (mahajan et al., 2014; el­eryanet al., 2020). map can lead to structure preservation, less tissue damage and shelf­ life quality of aril due to increased vapor pressure and reduced cell wall polysaccharides degradation (zhao et al., 2019). color characteristics fruit color is related to the breakdown of chloro­ plasts, chromoplasts and the change of natural pig­ ments (chlorophylls, anthocyanins, carotenoids, flavonoids) that are affected by packaging and stor­ age conditions (yin et al., 2016). l*, a*, b* values rep­ resent the lightness, redness and yellowness. chroma (c*) and hue angle (h°) describe the color intensity and purity, respectively. l*, c* and h° indices reflect the intensity of the color. a slight decrease in c* and an increase in h° indicates the loss of color intensity of pomegranate arils during storage (palma et al., 2015). loss of the color intensity during map can be controlled by regulating enzymatic and non­enzymat­ ic activities through decreasing o2 concentration or reducing water loss (belay et al., 2018). high co2 con­ centration prevents enzymatic browning by reducing phenolic substrate and ppo activity (manolopoulou and varzakas, 2013). belay et al. (2017 b) reported that map, storage time and their interaction had a significant effect on color intensity of pomegranates cv. wonderful stored at 5°c. the highest c* was observed under a low o2 atmosphere (5kpa), while the super o2 atmosphere (70kpa) maintained initial c* values during storage (belay et al., 2017 b). titratable acidity (ta) and total soluble solids (tss) the reduction in ta of pomegranate juice cv. mollar de elche without changes in tss was observed under uv­c treatment and super atmospheric o2 conditions, which is related to metabolic activities and increased catabolism of organic acids in the res­ piration process (maghoumi et al., 2013). on the other hand, increasing ta of arils cv. kingdom and mr­100 under the passive modified atmosphere at 5°c were due to fermentation, which was confirmed by the growth of total aerobic bacteria, yeasts, and molds (adiletta et al., 2017). changes in gas composi­ tion (increase and/or decrease o2 or co2) hydrolyze polysaccharides to sugars (sucrose, glucose, and fruc­ tose) by changing the activity of carbohydrate biosyn­ thesis enzymes and sugar compound metabolism. the active modified atmosphere provides high non­ reducing sugars at the end of storage, which can affect the chemical, sensory and quality characteris­ tics of pomegranate arils during storage (patanè et al., 2019; moradinezhad et al., 2020). in addition, an increase in sugar content (fructose, glucose, and sucrose) of pomegranate cv. wonderful was observed in 4.6kpa o2 and 12.65kpa co2 (belay et al., 2018), likely because that exposure to co2 preserves energy reserves. the reduction of tss in the super­ atmospheric is due to the reduction of carbohy­ drates, pectin, partial hydrolysis of protein and breakdown of glycosides into constituent units during respiration (blanch et al., 2015). the effect of map on organic acids and sugars of pomegranate fruit report­ ed as inconsistent and were mainly depended on the cultivar and also the duration of storage, as the tss value on cv. mridula increased (barman et al., 2011), while on cv. ruby decreased (fawole and opara, 2013), and on cv. mollar remained unchanged (sayyari et al., 2011). therefore, the control of respi­ ration rate (rr) and transpiration rate (tr) is crucial to preserve ta and tss values during storage as much as possible, in order to get a higher tss to ta ratio index. ascorbic acid, antioxidant and anthocyanin content the reduction of ascorbic acid (aa) was observed in arils cv. malese saveh stored under super atmos­ pheric o2 (70kpa) for 14 days at 4°c (maghoumi et al., 2014). in investigating the effect of the modified atmosphere, low o2 (5 or 10kpa o2, 10kpa co2), super 88 adv. hort. sci., 2024 38(1): 83­96 atmosphere (70kpa o2, 10kpa co2) and normal atmosphere on the aa content of pomegranate cv. wonderful at 5°c, it was found that oxidation aa was associated with the presence of o2. as a result, the content of aa decreased in the super atmosphere and normal atmosphere (belay et al., 2017 b). however, super atmospheric o2 has beneficial effects on other quality characteristics. excessive amounts of o2 and co2 may cause the oxidation of aa through increasing oxidative stress on plant tissues (belay et al., 2017 b). besides the atmosphere, the nature of the fruit also affects the concentration of aa during storage, so acidity levels are one of the factors affect­ ing the stability of aa during storage (wahyuningsih et al., 2017). the reduction of aa as an antioxidant agent is due to its use as an electron donor to oxi­ dants for neutralizing free radicals is attributed to fruit respiration and sensitivity to chilling injury (artés et al., 2006). the effect of packaging with different gas compo­ sitions (5 kpa o2 + 10 kpa co2 + 85 kpa n2; 10 kpa o2 + 5 kpa co2 + 85 kpa n2; 70 kpa o2 + 10 kpa co2 + 20 kpa n2; 21 kpa o2 + 0.03 kpa co2 + 78 kpa n2) was investigated on the physicochemical characteristics, nutrient and volatile organic compounds of aril cv. wonderful for 12 days at 5°c. it was observed that arils packed with low o2(5 kpa o2 + 10 kpa co2 + 85 kpa n2) have more nutrients content (belay et al., 2017 b). higher values of aa, anthocyanin and phe­ nolic compounds were observed in pomegranate cv. wonderful stored in low o2 concentration (5 kpa o2+ 10 kpa co2 + 85 kpa n2 and 10 kpa o2 + 5 kpa co2 + 85 kpa n2) at 5°c. also, maintaining low o2 concen­ tration using low permeability polypropylene film preserved pomegranate anthocyanin and improved sensory quality (banda et al., 2015). decreasing the respiration rate reduces the amount of carbohy­ drates, and the carbohydrates that accumulate in the tissue are used in the production of phenolic com­ pounds (wang et al., 2017). increasing the activity of antioxidant enzymes, such as sod, cat, and apx removes oxygen free radi­ cals and reduces the activity of ppo and pod enzymes involved in the browning of arils cv. purple queen was packed in semipermeable film, which had higher polyphenol and anthocyanin content (adiletta et al., 2019). also, heat treatment, uv­c and super atmospheric o2 packaging delayed the ppo and glu­ tathione peroxidase (gpx) activity of pomegranate arils cv. malese­saveh and maintained the antioxi­ dant concentration (maghoumi et al. , 2013). accumulation of phenolic compounds exposed to high o2 can be a physiological stress response, and stimulates phenylalanine ammonia­lyase (pal) activi­ ty during minimal processing (baenas et al., 2014). the increase of o2 in the first days of storage may increase the antioxidant activity, but in the long­ term, it reduces the main antioxidants including anthocyanins and phenolic compounds due to the oxidation stimulated by o2 (maghoumi et al., 2014). increasing reactive oxygen species (ros) causes the oxidation of phenolic compounds due to the increase in ppo activity and loss of membrane compartmen­ talization (cisneros­zevallos et al., 2014). at the end of the storage of pomegranate arils cv. wonderful the lowest anthocyanin concentration was observed n high o2 atmospheres (30kpa o2 and 10kpa co2) (banda et al., 2015), which could be due to the oxida­ tion of aa (maghoumi et al., 2014). palma et al. (2015) related the changes in anthocyanin content to the presence of organic acids (e.g. ascorbic acid) and titratable acidity, which provide the carbon skeleton for the synthesis of secondary metabolites (e g. anthocyanins) during storage (palma et al., 2015). changes in anthocyanin content can be attributed to the interaction of arils with gas composition, biosyn­ thesis and stability of individual anthocyanins (palma et al., 2015; moradinezhad et al., 2020). due to the inhibition of anthocyanin biosynthesis in high co2, the anthocyanins of pomegranate cv. wonderful stored in atmospheres enriched with co2 (10­20 kpa) were lower compared to fruit stored in air (holcroft et al., 1998). higher levels of co2 in x5 and x12 pack­ ages probably delayed anthocyanin synthesis and reduced the intensity of aril color during storage by reducing anthocyanin and phenol (selcuk and erkan, 2015; tzoumaki et al., 2009). the reduction in antho­ cyanin content, which affects the color of arils, is a disadvantage of storage with high co2 levels (table 1). volatile organic compounds (vocs) the identified vocs comprised five compound groups (aldehyde, ketone, alcohol, ester and monoterpene), ester compounds were dominant, fol­ lowed by ketones and aldehydes, whereas, alcohol and monoterpenes were the least abundant (belay et al., 2018). increasing vocs are related to the acceler­ ation of metabolism in response to the atmosphere, which can lead to stress and disruption of enzyme systems (giuggioli et al., 2015). increased vocs at low o2 stimulate the production of fermentative moradinezhad and ranjbar ‐ efficacy of map on the shelf life of pomegranate fruit and arils 89 table 1 a ­efficacy of modified atmosphere packaging (map) on whole pomegranate fruit and arils pomegranate cultivar treatment whole fruit/aril storage time (days) outcomes reference hicaznar passive modified atmosphere using xtend® and zoepac whole fruit 210 increase of polyphenols, anthocyanins, antioxidant activity, delay in color change and maintain appearance quality up to day 120, maintain physiological and biochemi­ cal properties up to day 180. (selcuk and erkan, 2015) hicaznar passive modified atmosphere using xtend® whole fruit 180 maintaining husk color, titratable acidity, and ascorbic acid content, and reducing weight loss and husk scald. (candir et al., 2018) shishe­kab pre­treatment with short­term high co2 and packaging in polyeth­ ylene bags, nano­bags and decoo magic bag whole fruit 90 reducing respiration rate, weight loss, decay, and chilling injury, and maintaining organoleptic properties (moradinezhad et al., 2018) afganski, crab, cranberry, entek­ habi­saveh modified atmosphere packaging (5 kpa co2 + 3 kpa o2) whole fruit 90 the fruit had a wrinkle­free skin, less chill­ ing injury, less disease symptoms and bet­ ter quality (sidhu et al., 2019) succary passive modified atmosphere using high ethylene absorption (hea), perforated polyethylene (ppe), polyethylene (pe) fi lm, stretchable cling film, poly vinyl whole fruit 90 the fruit had a less chilling injury, lower changes in acidity and soluble solid con­ tent, and increased antioxidant activity. (serry, 2019) wonderful passive modified atmosphere using non­perforated ‘decco’ and ‘zoe’, micro­perforated xtend®, micro and macro perforated high density polyethylene (hdpe) whole fruit 84 packaging whole fruit with micro­ and macro­ perforation reduced post­harvest losses by minimizing moisture condensa­ tion, fruit rot and shriveling. (lufu et al., 2021) wonderful passive modified atmosphere using micro­perforated xtend® and macro­perforated high­densi­ ty polyethylene whole fruit 42 fruits packaged in the micro­perforated xtend® had least weight loss, lowest respi­ ration rates, highest total soluble solids and no fungal decay. (kawhena et al., 2022) wonderful passive modified atmosphere using xtend™ bags whole fruit 120 increasing the concentration of antho­ cyanin in the husk and arils, delaying the symptoms of chilling injury up to 120 days (valdenegro et al., 2022) wonderful passive modified atmosphere using 100% cellulose­based film natureflex (nf), bi­axial­oriented polypropylene (bopp)­based film propafilm (pf), nf­pf (66:33%) film, and pf­nf (33:66%) film aril 9 pakage nf­pf (66:33%) film, and pf­nf (33:66%) film resulted in lowest in­package water vapour condensation and mold growth, and maintained the quality of arils at storage. (belay et al., 2018) purple queen passive modified atmosphere using micro­perforated (mpp) and semipermeable (sp) films aril 16 arils packaged in the sp system had high polyphenols, anthocyanins contents, antioxidant activity (superoxide dismutase, catalase, and ascorbate peroxidase) and low polyphenol oxidase and peroxidase activity. (adiletta et al., 2019) wonderful passive modified atmosphere using xtend bag, polyethylene bag, polypropylene bag, and silver nano bag aril 18 silver nano bag maintained the taste, aroma and overall acceptability, antho­ cyanin, vitamin c and antioxidant activity and reduced pectinase activity. (el­eryan, 2020) wonderful the nitrogen and argon­based map treatment (map ar) aril 16 arils packaged in the (map ar) had high sugar/acid ratio, and desired sensory quali­ (tinebra et al., 2021) adv. hort. sci., 2024 38(1): 83­96 90 compounds (zhang et al., 2013 a; cortellino et al., 2015) and induce cell damage and senescence by producing anaerobic metabolism (li and zhang, 2015). super atmospheric o2 affects the synthesis and accumulation of some vocs related to respirato­ ry metabolism (such as acetaldehyde, ethanol, and ethyl esters). accumulation of acetaldehyde is the first indicator of fermentation metabolism, which is rapidly converted to ethanol by the enzyme alcohol dehydrogenase (adh) and negatively effects on sen­ sory properties (thewes et al., 2015; manolopoulou and varzakas, 2013). the highest amount of vocs was observed in arils stored under super atmospheric o2 and enriched co2 (70kpa o2, 10kpa co2) and the low­ est amount was observed in arils stored in the normal atmosphere at the end of storage (belay et al., 2017 a). increasing synthesis of vocs in response to wound (amaro et al., 2012), or high co2 concentration leads to disruption of enzymatic systems, such as the lipoxygenase pathway (giuggioli et al., 2015) which catalyzes the oxidation of unsaturated fatty acids. microbial load fungi (yeasts and molds) are important pathogen­ ic microorganisms that are resistant to acid condi­ tions (jacxsens et al., 2001; firdous et al., 2023). yeasts are facultative anaerobes, and in contrast, molds are aerobes, which has been observed high co2 (>10%) inhibits mold growth (molin, 2000). the reduction count of mesophilic bacteria has been reported in minimal processing pomegranate cv. hicaznar under a high o2 atmosphere (70 kpa) com­ pared to low o2 and normal atmosphere at 5°c (ayhan and estürk, 2009). a high o2 atmosphere is used in fresh­cut due to its ability to prevent anaero­ bic fermentation, enzymatic discoloration and micro­ table 1 b ­efficacy of modified atmosphere packaging (map) on whole pomegranate fruit and arils pomegranate cultivar treatment whole fruit/aril storage time days outcomes reference bhagwa passive modified atmosphere using transparent high­density aril 5 increasing titratable acidity, anthocyanins reducing sugars, and total soluble solids, (rokalla et al., 2022) rabbab passive modified atmosphere using polyethylene+ polyester (pe+pes) and biaxial oriented polypropylene (bopp) film aril 15 pe+pes film caused delay in decreasing the trend of total antioxidant activity and had the lowest number of aerobic mesophilic bacteria and psychrophilic bacteria. (ranjbar and ramezanian, 2022) wonderful active modified atmosphere based on high o2 aril 12 the gas mixture containing 30 kpa o2 + 10 kpa co2 + 60 kpa n2 had lower aerobic mesophilic bacteria counts, higher sensory scores and long­term shelf life. (banda et al., 2015) wonderful active modified atmosphere based on low o2 and enriched co2 aril 9 the gas mixture containing 12.67–18 kpa co2, 2–4.67 kpa o2 and 80 − 82.67 kpa n2 reduced microbial count. (belay et al., 2017 a) wonderful active modified atmosphere based on low o2 and super­atmos­ pheric o2 aril 12 the gas mixture containing 5 kpa o2 + 10 kpa co2 + 85 kpa n2 and 10 kpa o2 + 5 kpa co2 + 85 kpa n2 maintained phytonutrient content, 70 kpa o2 + 10 kpa co2 + 85 kpa n2 had low aerobic mesophilic bacteria, yeast and mold counts. (belay et al., 2017 b) cv. wonderful active modified atmosphere based on low o2 aril 9 the gas mixture containing 2 kpa o2 +18 kpa co2 + 80 kpa n2 leads to the accumula­ tion of ethanol, increase in respiration quo­ tient and oxidation of organic acids. (belay et al., 2019 b) moradinezhad and ranjbar ‐ efficacy of map on the shelf life of pomegranate fruit and arils 91 bial growth (jacxsens et al., 2001) and it is effective by increasing the lag phase of growth and reducing the growth of bacteria and yeast in arils pomegran­ ate (belay et al., 2017 a; moradinezhad et al., 2020). the inhibitory effect of the high o2 is due to the toxi­ city of oxygen, which causes damage to the antioxi­ dant system, dna and nucleoproteins of microor­ ganisms by ros (o2−, h2o2 and oh−) produced at a partial pressure of o2 (tomas­callejas et al., 2011). pre­storage short­term high co2treatment signifi­ cantly reduced the decay of pomegranate fruits dur­ ing cold storage (moradinezhad et al., 2018). high co2 reduces the microbial load of fruit by penetrat­ ing the microbial membrane, changing intracellular ph or forming carbonic acid, which has bacteriostat­ ic effects (zhang et al., 2013 b; banda et al., 2015; belay et al., 2017 b; ranjbari et al., 2018; van de velde et al . , 2019, 2020; moradinezhad and dorostkar, 2020). high co2 pretreatment has signifi­ cant potential to prevent water loss, oxidative dam­ age, and control decay. it seems to be related to the induction of specific defense proteins, including dehydrins and pathogenesis­related proteins, as well as endogenous protective osmolytes (vazquez­ hernandez et al., 2018). at ambient air temperature, the active modified atmosphere affected on the chemical and qualitative characteristics of pome­ granate arils, which were related to the reduction of microbial load, safety and high organoleptic proper­ ties (rokalla et al., 2022). 3. conclusions and future prospects post­harvest loss is one of the main problems in the pomegranate industry worldwide. since the qual­ ity of the fruit is determined by internal and external characteristics, it is necessary to maintain the overall quality of the product for supply to consumers. considering that pomegranate fruit is non­climac­ teric, the use of map polymer films may have a good potential for the maintenance of its quality.in this study, the mechanism of the effects of map on the physicochemical and qualitative characteristics of whole and minimally processed arils of pomegranate werereviewed, and indicated that map had a signifi­ cant effect to prevent chilling injury and maintain fruit quality. several studies have reported the advantages of the modified atmosphere in extending the shelf life based on low o2 concentration and enriched co2. map and vacuum packaging compris­ ing optimal concentrations o2 and co2depends on physiology cultivar is a valuable technique to main­ tain nutritional quality, and antioxidant activity, reduce weight loss, and control the storage diseases and disorders of whole pomegranate fruit and arils.however, there is a need for extensive studies to develop the map system for pomegranate arils and fruit in different commercial cultivars. references adetoro a.o., opara u.l., fawole o.a., 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feasibility of vis/nir spectroscopy to detect and estimate fungicide residues on intact lettuces a.j. steidle neto (*), d.c. lopes, w.a. silva federal university of são joão del‐rei, campus sete lagoas, sete lagoas, minas gerais, brazil. key words: dithiocarbamate, lactuca sativa l., multivariate data analysis, spec­ tral reflectance. abstract: pesticides are applied repeatedly to grain, fruit, and vegetable crops for protection against pathogens, pests, and weeds, in short periods of time before harvest. the effective and fast monitoring of chemical residues in agri­ cultural products is important for the assurance of healthy food. this study was accomplished to evaluate the feasibility to detect and estimate the concentra­ tion of dithiocarbamate fungicide (mancozeb) residues on intact lettuce leaves based on vis/nir spectral reflectance measurements and multivariate data analysis. in the pre­harvest interval, a high initial rate of decline of dithiocarba­ mate residues was observed between one and seven days after pesticide spray­ ing (decrease of 90.3%), while a slower decline was verified from seventh to fourteenth day (decrease of 8.7%). the usefulness of this spectrometric method has been evidenced by determination of dithiocarbamate residues at concen­ trations between 0.23 and 10.3 mg cs2 kg­1, with detection and quantitation limits of 0.49 and 1.41 mg cs2 kg­1, respectively. vis/nir spectral reflectance combined to the partial least square analysis have potential to be applied for estimating dithiocarbamate concentrations on intact lettuce leaves, presenting advantages such as real­time measurements and the possibility to be built into the industrial processing lines. 1. introduction consumers demand grain, fruits, and vegetables with high sensorial and nutritional qualities, but without pesticides. although the correct use of fungicides does not cause problems of public concern in health and environmental areas, undesirable residues can remain on agricultural products after harvest if inappropriate or abusive treatments are applied without respecting safety recommendations indicated by the specialized agencies and manufacturers (lópez­fernández et al., 2013). in modern agriculture, pesticides are applied repeatedly to grain, fruit, and vegetable crops for protection against pathogens, pests, and weeds, in short periods of time before harvest (jankowska et al., 2019). the main exposure to pesticides for humans is via food, especially by consumption of agricultural products. (*) corresponding author: antonio@ufsj.edu.br citation: steidle neto a.j., lopes d.c., silva w.a., 2020 ­ feasibility of vis/nir spectroscopy to detect and estimate fungicide residues on intact lettuces ‐ adv. hort. sci., 34(2): 223­232. copyright: © 2020 steidle neto a.j., lopes d.c., silva w.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 february 2020 accepted for publication 13 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 223­232 224 introduced between 1940 and 1980, dithiocarba­ mate fungicides still represent an important class of pesticides widely used in agriculture. they are char­ acterized by a broad spectrum of activity against vari­ ous plant pathogens, low acute mammal toxicity, and low production costs (crnogorac and schwack, 2009). however, laboratory studies showed that dithiocar­ bamates can result in neuropathology, thyroid toxici­ ty, and developmental toxicity to the central nervous system (ipcs, 1993; caldas et al., 2006). mancozeb is an ethylene­bis­dithiocarbamate used as fungicide to protect fruit and vegetable crops from a range of fungal diseases (pereira et al., 2014) and was considered a multipotent carcinogenic agent in a long­term study (belpoggi, 2002). this fungicide is registered to 38 food crops, occupy the third place in the ranking of the most commercialized pesticides in brazil in terms of tones of active ingredient (anvisa, 2018; ibama, 2018). caldas et al. (2006) reported data obtained from the program on pesticide residue analysis in food (para), coordinated by the brazilian national sanitary surveillance agency (anvisa), about dithio­ carbamate residues on 34% of lettuce samples in a total of 297 analyzed, based on vegetable samples collected from 2001 to 2004. in another brazilian study, accomplished from 2005 to 2015, jardim et al. (2018) showed that 14.6% of 1483 lettuces presented dithiocarbamate residues. lópez­fernández et al. (2013) verified the presence of mancozeb and other dithiocarbamate residues on 72% of lettuce samples collected at spain, with some samples containing residues three times higher than the maximum limit (5 mg kg­1). various methods have been improved for the determination of dithiocarbamate residues, including gas and/or liquid chromatography, often in conjunc­ tion with mass spectrometry (crnogorac and schwack, 2009). these current methods requiring sample preparation (destructive), time consuming, and laboratory labor demanding, well­trained per­ sonnel and relatively expensive process chemistry. they also produce chemical and sample waste, which adversely affects product traceability by preventing real­time decision­making (salguero­chaparro et al., 2013; steidle neto et al., 2017). although more sensi­ tive, these methods are suitable for spot checks. the reasons for vis/nir spectroscopy great suc­ cess as one of the most important and versatile tech­ niques in analytical chemistry include its speediness and easiness to handle and provide molecular specif­ ic information for different types of samples in any physical state, with little or no previous chemical treatment (gonzálvez et al., 2011). however, one possible drawback is the range of concentration of the target analyte. pesticide residues tend to have very small concentrations in foods. despite this, pre­ vious studies proved the feasibility of spectroscopy to detect and quantify low concentrations of analytes in fruits and vegetables (saranwong and kawano, 2005; gonzálvez et al. , 2011; acharya et al. , 2012). nevertheless, very few published studies have addressed the use of vis/nir spectroscopy for pre­ dicting pesticides residues in harvested intact sam­ ples. the scientific researches of sánchez et al. (2010), for peppers, and jamshidi et al. (2016), for cucumbers, reported estimates of pesticides without pre­treatment or preparation of samples. this advan­ tage allows that spectroscopy technique can be built into the processing line, enabling large­scale individu­ al analysis and real­time decision­making. another recent technique for trace level detection of pesti­ cides is the surface enhanced raman spectroscopy (sers), that use noble metal nanostructures (e.g. gold) to increase the weak signals from analytes. but, sers requires spectrometer, laser source, probe, sample holder, and substrates that are more expen­ sive when compared with spectral reflectance equip­ ment. the present scientific research was carried out to evaluate the feasibility to detect and estimate the concentration of dithiocarbamate fungicide residues on intact lettuce leaves based on vis/nir spectral reflectance measurements and multivariate data analysis. 2. materials and methods lettuce cultivation lettuce (lactuca sativa l. cv. regina) with plain green leaves, was cultivated under organic conditions in a certified farm located at the capim branco city, minas gerais state, brazil (19° 34’ s latitude, 44° 10’ w longitude, and 816 m a.s.l.). according to köppen classification, the region climate is cwa (warm tem­ perate ­ mesothermal), with dry winter and rainy summer. the lettuce seeds were planted in plastic trays containing organic substrate. seedling production occurred under a low­density polyethylene cover (suncover av blue 120 μm, ginegar polysack, são paulo, brazil), which allowed a better irrigation con­ trol and was internally coated with a photoselective steidle neto et al. ‐ spectroscopy to estimate fungicide on lettuces 225 shading mesh (chromatinet raschel red 35%, ginegar polysack, são paulo, brazil), capable of reducing the percentage of beam solar radiation on the plants, also increasing the fresh mass production. the vigorous and healthy seedlings were individu­ ally transplanted to plastic pots containing a thin gravel layer overlapped by soil mixed with organic compound (cattle manure and vegetable biomass). the crop irrigation was performed by a drip system, controlled by a digital timer. the experiment consist­ ed of 500 lettuce plants. however, 355 plants were effectively used as experimental units and 145 plants were cultivated for boundary effects. some days before plants reach the physiological maturation, the lettuces were transported to a greenhouse with the objective of performing the fungicide spraying. the greenhouse is located at the campus sete lagoas of the federal university of são joão del­rei, which is distant from the organic farm about 22 km. this greenhouse also was covered and coated with the same polyethylene film and photo selective shading mesh described above. fungicide spraying a non­systemic fungicide (mancozeb) was used, which is classified in the alkylenebis group (dithiocar­ bamate). the active ingredient of this pesticide is a carbon disulphide (cs2) precursor and is registered in the brazilian ministry of agriculture, livestock, and food supplies (mapa) for application on some crops. however, the dithiocarbamate (mancozeb) is not authorized by the brazil ian national sanitary surveillance agency (anvisa) for lettuce crop. this fungicide was chosen based on reports of the brazilian monitoring program for pesticides residues in food, developed by anvisa, that mentioned the indiscriminate use of this pesticide on lettuces and other crops by some brazilian farmers. contrarily, this fungicide is authorized for lettuce crop by other agencies and committees, such as the european food safety authority and the fao codex alimentarius. the solubilization of mancozeb in water was done to provide a sufficient volume for uniform and homo­ geneous application on lettuces. the dosage pre­ scribed in the brazilian package leaflet for other green leafy vegetables, such as cabbage, was adopt­ ed (2­3 kg of fungicide ha­1). this dose is recommend­ ed to control the mildew (peronospora parasítica) and the pod spot (alternaria brassicae). excepting for five plants (control units), which were randomly selected in the greenhouse, the application of man­ cozeb to lettuces was performed using an electric sprayer (droplets with average diameter of 29 µm). the application time and distance from the sprayer nozzle to the plants were standardized. similar to the dosage, the pre­harvest interval for consumption of green leafy vegetables informed in the brazilian package leaflet (14 days) was consid­ ered in this study. samplings for the spectral reflectance measurements and laboratory analyzes were carried out on alternate days during the pre­ harvest interval, totaling 7 days and starting one day after the fungicide spraying. daily 10 samples were collected, each one weight­ ing more than 500 g and corresponding to five let­ tuce plants randomly selected. the fresh mass was determined using a precision balance and discarding the roots. therefore, 50 plants were used per day, totaling 350 plants during the sampling period. the statistical design was entirely randomized with 7 treatments (alternate days after spraying) and 10 repetitions (samples). reflectance measurements spectral reflectance was measured by a miniature and hand­held spectrometer (jaz­el350, ocean optics, dunedin, usa), coupled to a tungsten­halo­ gen light source, and preconfigured to acquire and store reflectance data from 350 to 1000 nm, with spectral resolution of 1.3 nm. a specific clip probe (spectroclip­r, ocean optics, dunedin, usa) was used to collect reflected light from the lettuce leaves (fig. 1). this probe contains an integrating sphere that captures diffuse reflected light more efficiently fig. 1 ­ hand­held spectrometer, clip probe, and diffuse reflec­ tance standard used to collect reflected light from the lettuce leaves. adv. hort. sci., 2020 34(2): 223­232 226 than lens­based collection optics. the active illumi­ nated leaf area in the clip probe is 5 mm diameter. two premium fibers vis/nir (600 μm) interconnect­ ed the spectrometer and the light source to the clip probe. a diffuse reflectance standard with spectralon™ was used as a reference to measure spectral reflectance. after the warm up time of the light source, the reference standard measurements were made before the spectral reflectance measurements on let­ tuce leaves. data acquisitions were performed in a temperature­controlled environment with the pur­ pose of avoiding the overheating of the light source and the spectrometer detector due to the extended using time. the reflectance values were calibrated by means of the software (oceanview, ocean optics, dunedin, usa) and expressed as a relative percent­ age of the reference standard (xing et al., 2006): rλ cal = [(rλ leaf ‐ rλ dark)/(rλ ref ‐ rλ dark)] x 100 (1) where rl cal is the calibrated spectral reflectance from the leaves (%), rl leaf is the spectral reflectance from the leaves (dimensionless), rl dark is the spectral reflectance considering light absence (dimension­ less), and rl ref is the spectral reflectance from the dif­ fuse reflectance standard (dimensionless). three leaves of each plant were randomly select­ ed from the external, middle, and central parts of the lettuce head. three separate measurements on stan­ dardized and equidistant locations of the adaxial sur­ face were performed in each leaf, avoiding its central vein and boundaries. thus, 3195 spectral signatures were obtained during the sampling period, consider­ ing the 350 sprayed plants and the five control let­ tuces (without dithiocarbamate). the electronic files containing the spectral signa­ tures were stored in a memory card and later trans­ ferred to a notebook for analyzes performed with electronic spreadsheets. during the analyzes, spec­ tral signature averages were obtained for each let­ tuce sample. after this, data was stored in an exter­ nal hard disk. dithiocarbamate analytical determination samples were quartered, milled in an electric grinder, placed in hermetic packages, and frozen at ­ 30°c in an ultrafreezer for minimizing the degrada­ tion and metabolization of the dithiocarbamate. the analytical determination of the concentration of dithiocarbamate was performed based on the method proposed by cullen (1964) and improved by keppel (1971). mancozeb residues were measured by the spectrophotometric determination of the cupric complex formed with the cs2 evolved from the acid decomposition of dithiocarbamate in the presence of stannous chloride as a reducing agent (caldas et al., 2004). the solution of the complex formed from the reaction between cs2 and copper (ii) acetate mono­ hydrate was measured at 435 nm in uv/vis spec­ trophotometer (cary 50, varian, agilent technologies inc., usa). at the end of the laboratory analyzes, 70 reference measurements were obtained, corre­ sponding to 10 values for each treatment. data analysis the partial least squares (pls) multivariate analy­ sis was applied with the purpose of developing a mathematical model capable of predicting the dithio­ carbamate concentrations based on lettuce pre­ treated spectral signatures. thus, a response matrix, composed by the dithiocarbamate concentrations obtained by laboratory analytical measurements, was correlated with a spectral matrix, containing the average reflectance measurements. an orthogonal basis of latent variables was constructed one by one in such a way that they were oriented along the directions of maximal covariance between the two original spaces (response and spectral matrices), try­ ing to achieve an optimal prediction for new data (wold et al., 2001; anderson, 2009; cozzolino et al., 2011). the latent variables were calculated by iterative methods as linear combinations of the original inde­ pendent variables (spectral reflectances) and the dependent ones (dithiocarbamate concentrations). new variables were found, representing estimates of the latent variables or their rotations. these new variables were called x­scores and were predictors of the response ones. a weight matrix was also calculat­ ed so that each of their elements maximized the covariance between response variables and the cor­ responding latent variable scores. the unexplained part of the predictor variables was represented by the deviations between the measured and predicted responses, which were also calculated and called y­ residuals (wold, 2001; lopes and steidle neto, 2018). the detrending pre­treatment was applied to the spectral signatures prior to the model calibration and external validation. the detrending algorithm cor­ rected scatter and simple deformations of the spec­ tra baseline as vertical shift and slope (barnes et al., 1989; steidle neto et al., 2016). according to moura et al. (2016), this was the most effective pre­treat­ ment for removing irrelevant information which could not be handled by the regression technique and principal component analysis. steidle neto et al. ‐ spectroscopy to estimate fungicide on lettuces 227 as recommended by huang et al. (2008), two thirds of the data for each response variable were used as the calibration with cross­validation set, whereas one third of the data as the external valida­ tion set. the calibration set was used for developing the model and the external validation set was employed for assessing the calibrated model predic­ tion performance. this procedure was applied for predicting independent data, not related with those included in the calibration with cross­validation set (agelet and hurburgh, 2014). during calibration the leave­group­out cross­validation technique was applied to data. this sampling plan also agrees with the suggested by kramer (1998), who affirmed that the number of data in the calibration set should be more than 10 times the number of variable compo­ nents in the experiment. in this study the dithiocar­ bamate residues represented the independent source of significant variation in the data. the software spectox was specifically developed for this study with the purpose of assisting in the spectral reflectance data processing and the multi­ variate analysis (calibration and validation), also allowing the performance evaluation of the predic­ tion model for dithiocarbamate concentrations. the spectox was written in java language, by using the free netbeans ide (apache software foundation, maryland, usa). additional algorithms of the pre­ treatments and multivariate methods were included in the scilab software (scilab enterprises, versailles, france). the optimal number of latent variables was deter­ mined as recommended by jha (2010), considering the minimum value for the root mean square error to avoid over fitting (eq. 2). rmse = ∑ (yo ­ yp)² (2) n where rmse is the root mean square error (mg kg­1), yo are the values measured by the uv/vis spec­ trophotometer (mg kg­1), yp are the values predicted by the model (mg kg­1), and n is the number of sam­ ples (dimensionless). the calibration and cross­validation processes were evaluated by the root mean square errors for calibra­ tion (rmsec) and cross­validation (rmsecv) sets, respectively. additionally, the predictive capacity of the adjusted model regarding external validation was evaluated by the statistical parameters mean absolute error (mae), mean bias error (bias), coefficient of determination (r2), and index of agreement (d). willmott and matsuura (2005) pointed out that mae (eq. 3) is unambiguous and the most natural measure of the mean error magnitude. these authors considered that mae should be used as the basis for all dimensioned evaluations and inter­com­ parisons of model performance. the bias (eq. 4) rep­ resents the average difference between measured and predicted data. thus, values close to zero indi­ cate low systematic error between the measured and predicted values (high accuracy of the model). also, negative bias values indicate underfitting, while posi­ tive bias values reveal overfitted predictions (steidle neto et al., 2016). the coefficient of determination (eq. 5) represents the proportion of explained vari­ ance of the response variable in the validation set, with results varying from 0 to 1, and the maximum value reflecting a perfect agreement between mea­ sured and predicted data (steidle neto et al., 2017). finally, index of agreement (eq. 6) varies from 0 to 1, where the maximum value reflects a perfect agree­ ment between measured and predicted data. as affirmed by willmott (1981), this is an important index since it is not a measure of correlation or asso­ ciation in the formal sense but rather a measure of the degree to which the model’s predictions are error free. mae = ∑│yp ­ yo│ (3) n bias = ∑(yo ­ yp) (4) n r² = [∑(yp ­ ȳp)(yo ­ ȳo)]² (5) ∑(yp ­ ȳp)²∑(yo ­ ȳo)² d = 1­ ∑(yp ­ yo)² (6) ∑(│yp ­ ȳo│+│yo ­ ȳo│)² where mae is mean absolute error (mg kg­1), bias is the mean bias error (mg kg­1), r2 is the coefficient of determination (dimensionless), and d is index of agreement (dimensionless). the limit of detection (lod) and limit of quantita­ tion (loq) are frequently used to describe the small­ est concentrations of a sample that can be reliably measured by an analytical procedure. the lod corre­ sponds to the lowest analyte concentration at which detection is feasible, while the loq is the lowest con­ centration at which the analyte can be effectively quantified. thus, loq tends to be equivalent or high­ er than lod (armbruster and pry, 2008). in this study, the statistical lod and loq determinations were applied (clsi, 2004; jeon et al., 2007). that is, 228 adv. hort. sci., 2020 34(2): 223­232 the spectra of five representative blank lettuce sam­ ples (containing no dithiocarbamate residues) were measured, following the same procedures used dur­ ing the calibration and external validation of the pls model. the mean and standard deviation of the pre­ dicted cs2 concentrations were calculated based on these spectra, which were used as input for the developed model. the lod and loq were equal to the mean of the predicted cs2 concentrations plus three times or ten times the standard deviation of the mean, respectively. according to clsi (2004) and armbruster and pry (2008), although the blank sam­ ples are devoid of analyte, they can produce an ana­ lytical signal that might be consistent with a low con­ centration of dithiocarbamate. 3. results figure 2 presents the degradation curve of dithio­ carbamate residues on lettuce leaves during the pre­ harvest interval, obtained from average values of lab­ oratory analytical measurements. the estimated half­ life of the dithiocarbamate residues was 5­7 days. the rates of decline of dithiocarbamate concentration in two­day intervals were quite variable from first to seventh day after spraying (4.7, 3.2, and 1.4 mg cs2 kg­1), corresponding to a decrease of 90.3%. after the seventh day, the rates of decline were 0.3 mg cs2 kg­1, indicating a decrease of 8.7%. although very small concentration residues persisted at the final of pre­ harvest interval, the results indicate that after this period the pesticide metabolization is advanced, assuring reliability for lettuce consumption. the pls model for dithiocarbamate concentration on lettuces was more precise and accurate when detrending pre­treatment was applied to spectral sig­ natures, compared with predictions obtained from spectra without pre­treatment or treated with other methods (centering, standardization, first and second derivatives). despite nir bands were more sensible, all wavelengths (350­1000 nm) presented potential to explain the dithiocarbamate concentration residues on lettuce leaves from spectral reflectance, contributing to the good performance of the predic­ tion pls model. table 1 shows the statistical results for the dithio­ carbamate concentration model, considering the cali­ bration with cross­validation and external validation datasets. the data processing showed that the use of more than four latent variables for dithiocarbamate concentrations resulted in an over­fitting, character­ ized by a slight divergence in the downward trend of the rmse, which continued to decrease for calibra­ tion (rmsec), but almost established for cross­valida­ tion (rmsecv). the proposed pls model was satisfactory since presented low rmsec, rmsecv, rmse, mae, and bias when compared to the range values (table 1). additionally, the performance of the model for the external validation presented high index of agree­ ment (0.94), reflecting a good accuracy for indepen­ dent predictions of the dithiocarbamate concentra­ fig. 2 ­ dithiocarbamate concentration decay (mg cs2 kg­1) on lettuce leaves at seven intervals after mancozeb spraying. vertical bars represent the standard error of the average values. table 1 ­ statistical parameters of calibration with cross­validation and external validation processes for the estimation model of dithio­ carbamate concentration on lettuces calibration external validation number of latent variables 4 r2 (dimensionless) 0.87 rmsec (mg cs2 kg­1) 1.86 rmse (mg cs2 kg­1) 1.41 rmsecv (mg cs2 kg­1) 2.74 mae (mg cs2 kg­1) 1.24 range (mg cs2 kg­1) 0.23­10.3 bias (mg cs2 kg­1) ­0.37 d (dimensionless) 0.94 steidle neto et al. ‐ spectroscopy to estimate fungicide on lettuces 229 tions on lettuces. the negative bias value indicated a majority tendency of underfitting predictions by the model, mainly from 2.3 mg cs2 kg­1 (fig. 3). also, low bias value represented small systematic error (table 1). regarding the differences among statistics pre­ sented in table 1, the spectral measurements and predictions of external validation may deviate from the calibration as they originate from different sam­ ple sets. as mentioned by liu and ying (2005), in this way, the ability of the calibration model to withstand unknown variability is assessed. the correlations between values determined by the reference analytical method and those predicted by the external validation of the pls model are pre­ sented in figure 3. prediction performance resulted in good agreement between reference and estimated values, with r2 of 0.87 (table 1), indicating that 87% of the measured values were accurately represented by the model. the comparison of parameters (lod, loq, and range) associated to different methods of determina­ tion of dithiocarbamates on lettuces are showed in table 2. the lod and loq values verified in this study were higher than those obtained with the gas or liquid chromatography, as well as the spectropho­ tometric method. the usefulness of the methodology presented in this study has been evidenced by the determination of dithiocarbamate residues on lettuce samples at concentrations between 0.23 and 10.3 mg cs2 kg­1. this range can be considered appropriated when the results of previous studies are used as reference (table 2). further, values between these lower and upper limits are sufficient to measure a wide range of dithiocarbamate concentrations on lettuces. 4. discussion and conclusions past studies showed dithiocarbamate degradation profiles for lettuces similar to those found in this study. yip et al. (1971) found that the dithiocarba­ mate concentration (maneb) from a single spray on lettuces declined from 45 mg kg­1 initially to about 5 mg kg­1 after 15 days (decrease of 89%). on the other hand, hughes and tate (1982) monitored mancozeb residues on lettuces and verified a reduction of 90 mg kg­1 after a 14­day interval (decrease of 72%). these authors also reported a high initial rate of decline in dithiocarbamate concentration during the first seven days after spraying, confirming the high degradation of analyte. despite of the dithiocarbamates with active ingre­ dient based on mancozeb are not authorized by anvisa for lettuces in brazil, the european food safety authority (efsa) allows the use of this pesti­ cide on lettuce crop in the countries that integrate the european union, considering a maximum residue limit of 5 mg kg­1 (efsa, 2013). in addition to the dietary risk, the edafoclimatic differences and dietary patterns of the countries justify the distinct positions between anvisa and efsa. based on the results, let­ tuces presented dithiocarbamate concentrations fig. 3 ­ external validation of the pls model for estimating dithiocarbamate concentrations (mg cs2 kg­1) on lettuce leaves. table 2 ­ limit of detection, limit of quantitation, and range (expressed in mg cs2 kg­1) for different methods of determination of dithio­ carbamates on lettuces method limit of detection limit of quantitation range reference gas chromatography 0.004 0.013 0.04­5.0 česnik and gregorčič (2006) liquid chromatography 0.04 0.11 0.50­9.3 lópez­fernández et al. (2012) gas chromatography 0.02 0.05 0.04­1.0 pizzutti et al. (2017) spectrophotometric 0.28 0.40 0.20­4.5 pizzutti et al. (2017) spectrometric 0.49 1.41 0.23­10.3 present study adv. hort. sci., 2020 34(2): 223­232 230 mancozeb has 2 nh and 2 ch2, and that the nh bond position is unhindered within the chemical structure, there is a high likelihood it will produce a sharp and strong absorption band, which should improve detec­ tion of mancozeb. the mean spectral signatures of intact lettuce leaves with absence and presence of fungicide pre­ sented differences mainly in nir region, with fungi­ cide­contaminated samples resulting in greater absorbance than that in the fungicide­free lettuce leaves. according to that reported by sánchez et al. (2010) and jamshidi et al. (2016), increase of the absorbance in nir region after 900 nm could be due to the c­h absorption. based on the results, it can be said that the vis/nir spectroscopy combined to the multivariate data analysis have potential to be applied as an alter­ native method to estimate dithiocarbamate concen­ trations on lettuce leaves. however, the success and widespread adoption of this method also depends of suitable measurement practices. it is important that measurements are performed using a spectrometer with high spectral resolution, after the time required to warm­up the light source, and after adequate spectrometer calibration (proper sampling of refer­ ence standard). additionally, important factors to achieve good results include the standardization of the measurement points in the samples, the homo­ geneity of the target area, and the positioning of samples on a black non­reflective panel with the pur­ pose of prevent the light reflection going through the leaf. another essential aspect is related to the inci­ dence angle of the light bunch, which is emitted by the light source over the sample and directly influ­ ences the light reflection by sample (steidle neto et al., 2017). although the effects of the abovemen­ tioned individual error sources may appear small, their combined presence may result in measure­ ments with significant errors, influencing the spectral reflectance independently of dithiocarbamate con­ centrations, as well as, the model predictions. vis/nir spectral reflectance measurements com­ bined to the partial least square analysis showed to be feasible and effective as a promising method for estimating concentration of dithiocarbamate fungi­ cide residues on intact lettuce leaves. the developed pls model allowed the detection and quantification of the dithiocarbamate without any preparation and/or processing of lettuce samples. this method offers advantages over traditional laboratory meth­ ods, such as real­time measurements and the possi­ bility to be built into the industrial processing lines, lower than the maximum residue limit established by efsa from the fourth day after spraying. according to lopes and steidle neto (2018) detrending, which was the most appropriated spec­ tral pre­treatment in this study, is usually used to remove specific data offsets that are not related to the chemical or physical properties of interest for the chemometric modeling. sánchez et al. (2010) also developed pls models for predicting pesticide residues on intact peppers using near­infrared reflectance spectroscopy, applying detrending method for scatter correction in data, and obtaining good results. steidle neto et al. (2016) affirmed that detrending was the best spectra pre­treatment when predicting chlorophyll content in lettuces, helping to remove non­linear trends in spectroscopic data, and consequently correcting scatter. the number of latent variables considered ade­ quate for predicting dithiocarbamate concentrations on lettuce leaves in this study agree with cozzolino et al. (2011), who affirmed that if more than optimum number of latent variables is used, the solution can become over­fitted and the model will be very dependent on the dataset, giving poor predictions. otherwise, using less than the optimum number of latent variables will cause under­fitting and the model will not be accurate enough to capture the variability in the data. this result also agrees with other researchers who applied spectroscopy and pls models to non­destructively predict of pesticide con­ centrations. jamshidi et al. (2016) showed that 5­7 latent variables were required for pls models when predicting diazinon residues on cucumbers. although the proposed model tended to underes­ timate the pesticide concentrations, slight overesti­ mates were verified for low dithiocarbamate concen­ trations, evidenced in this study until the fifth day after spraying. the method based on spectral reflectance provid­ ed sufficient sensibility for detecting and quantifying concentrations lower than the maximum residue limit allowed by the european food safety authority for mancozeb­based dithiocarbamates on lettuces (5 mg kg­1). makino et al. (2009) used spectral reflectance for detection and quantification of chlor­ pyrifos residues on apple surface, also demonstrating the feasibility of spectroscopy for estimating low con­ centrations of pesticide residues in fruits. according to acharya et al. (2012), the spectral features can be assigned to overtone and combina­ tion bands of various c­h and n­h bonds within these molecules. considering that the chemical structure of steidle neto et al. ‐ spectroscopy to estimate fungicide on lettuces 231 caldas e.d., tressou j., boon p.e., 2006 ­ dietary expo‐ sure of brazilian consumers to dithiocarbamate pesti‐ cides ‐ a probabilistic approach. ­ food. chem. tox., 44: 1562­1571. česnik h.b., gregorčič a., 2006 ­ validation of the method for the determination of dithiocarbamates and thiuram disulphide on apple, lettuce, potato, strawber‐ ry and tomato matrix. ­ acta chim. slov., 53: 100­104. clsi, 2004 ­ protocols for determination of limits of detec‐ tion and limits of quantitation (guideline). document ep17. ­ clinical and laboratory standards institute (clsi), wayne, usa. cozzolino d., cynkar w.u., shah n., smith p., 2011 ­ multivariate data analysis applied to spectroscopy: potential application to juice and fruit quality. ­ food res. 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agrotóxi‐ cos. ­ inst. brasileiro do meio ambiente e dos recursos naturais renováveis, brasília, distrito federal, brasil. ipcs, 1993 ­ mancozeb pesticide residues in food: 1993 evaluations part ii toxicology . ­ international programme on chemical safety (ipcs), us environmental protection agency, washington, usa. jamshidi b., mohajerani e., jamshidi j., 2016 ­ developing a vis/nir spectroscopic system for fast and non‐destructive pesticide residue monitoring in agricul‐ tural product. ­ measurement, 89: 1­6. jankowska m., lozowicka b., kaczyński p., 2019 ­ comprehensive toxicological study over 160 processing factors of pesticides in selected fruit and vegetables after water, mechanical and thermal processing treat‐ ments and their application to human health risk assessment. ­ sci. tot. environ., 652: 1156­1167. jardim a.n.o., mello d.c., brito a.p., van der voet h., boon p.e., caldas e.d., 2018 ­ probabilistic dietary enabling large­scale individual analysis in 100% of the 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solanum lycopersicum. abstract: salicylic acid (sa) and ferulic acid (fa) are considered phenolic com­ pounds that act as elicitors due to their regulatory functions on plant growth, development, metabolic and physiological responses in plants. the aim of this research was to evaluate the effect of sa and fa on growth, fruit quality and synthesis of secondary metabolites in tomato (solanum lycopersicum cultivar santa clara). the experiment was conducted in pots in a greenhouse. the appli­ cation of sa and fa was performed at concentration of 1.0 mmol l­1 alone and in combination, with water treated plants as control. exogenous application of sa and fa either alone or in combination (sa + fa) resulted in increases in bio­ mass accumulation and chlorophyll contents in tomato plant; and soluble sugar, total polyphenol, flavonoids, lycopene and β­carotene contents in fruits. it was concluded that application of sa and fa resulted in higher production and concentration of secondary compounds in tomato. 1. introduction tomato (solanum lycopersicum l.) belongs to the solanaceae family and it is considered as low­calorie food. it is a good source of vitamins a, c and e, and mineral salts (adalid et al., 2010; kazemi, 2014). in addition to vitamins and minerals, tomato has a high lycopene and β­carotene content, which has antioxidant and anticancer properties. carotenoids is naturally present in many fruits and vegetables and plays an important role as a functional food when consumed as part of the diet, producing specific health benefits such as reducing the risk of various diseases (martínez­hernández et al., 2016; mehta et al., 2018) and polyphenols (flavonoids, flavanones and flavones) are also present in significant amount in tomato acting as antioxidant, anti­mutagenic, anti­proliferati­ ve, anti­inflammatory and anti­atherogenic activities (martí et al., 2016; chaudhary et al., 2018). salicylic acid (sa), a natural plant hormone, act as an important sig­ nalling molecule triggering tolerance against abiotic and biotic stresses (*) corresponding author: pgorni@gmail.com citation: gorni p.h., da silva cornelissen b., pereira a.a., 2021 ­ exogenous salicylic acid and ferulic acid improve growth, phenolic and carotenoid content in tomato. ­ adv. hort. sci., 35(4): 335­ 341. copyright: © 2021 gorni p.h., da silva cornelissen b., pereira a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 march 2020 accepted for publication 3 september 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8295 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 335­341 336 (hernández­ruiz and arnao, 2018; gorni et al., 2020; gorni et al., 2021). sa plays a significant role in many physiological and biochemical processes of the plant, being able to act as growth regulators and also as an abiotic elicitor capable of increasing the synthesis of secondary compounds beneficial to human health (javanmardi and akbari, 2016). ferulic acid (fa) is a phenolic compound syn­ thetized from the metabolism of phenylalanine and tyrosine in plants. it is an important biological and structural component of the plant cell wall and it accumulates in soil and influences plant growth (li et al., 2013; paiva et al., 2013). fa is considered as non­ enzymatic antioxidant and acts under stress to elimi­ nate free radicals produced in plants (andreasen et al., 2001; engwa, 2018). studies on the exogenous application of fa in plants have demonstrated that this compound acts as an important regulator of sev­ eral physiological processes related to plant growth and mitigating stress, such as, stomatal closure, cell division, membrane permeability, photosynthesis, respiration and many other metabolic processes (santos et al., 2008; li et al., 2013; singh and deen, 2014; hussain et al., 2017; cheng et al., 2018). however, exogenous sa (kazemi, 2014) and fa (singh and deen, 2014) applications were effective in inducing the growth and formation of secondary metabolites in tomato plants. therefore, it is suggest­ ed that the application of sa and fa may result in combined effects of growth promotion and induction of biosynthetic pathways of secondary metabolism. previous studies have revealed the hormonal and eliciting action of sa and fa (1.0 mmol l­1) in tomato plants (hussain et al., 2017; kumar et al., 2017). in this context, the application of sa and fa can improve the productive performance and the biosyn­ thesis of secondary compounds in tomato, making possible an increase in the commercial value of this crop, reaching greater market competitiveness. in this study we evaluated the effect of leaf spray of sa and fa either alone and in combination on the growth, yield and secondary compounds in tomato cultivar santa clara. 2. materials and methods the experiment was conducted under greenhouse (without temperature and humidity control) covered with a 50% solar radiation shade located in gammon colleges, paraguaçu paulista (22°41’76” s, 50°58’33” w, 517 a.s.l.), sao paulo, brazil. trademark tomato cultivar santa clara seeds were placed in a germination tray and after the training period (30 days) they were planted in 18 l pots con­ taining soil. soil samples were collected and submit­ ted to chemical analysis according to van raij et al. (2001) (table 1) and was corrected by applying lime­ stone to increase the saturation of bases to 80% (40.0 g pots­1), potassium chloride (3.0 g pots­1) and simple super phosphate (20.0 g pots­1). for fertiliza­ tion with micronutrients it was added yoorin master 1 si® (granulated) (si: 10%, b: 0.1%, mn: 0.3%, cu: 0.05% and zn: 0.55%) (1.5 g pots­1), according to the recommendations of bulletin 100 (iac) for tomato species. the pots were irrigated by sprinklers per day at 8 a.m. and 5 p.m. in order to keep the soil mois­ ture and ensure the availability of water throughout the experimental period. the application of salicylic acid (sa: 138.121 g mol­ 1) and ferulic acid (fa: 194.18 g mol­1) was made for 3 consecutive days after having reached 20 days from transplant. foliar applications of sa and fa at a dose of 1.0 mmol l­1 alone and in combination, and water­ treated plants were used as control. sa and fa treat­ ments were carried out by spraying the shoots of the plants with water­based solutions supplemented with agral® (50 μl l­1 of solution) until the drop point (10 ml per plant), as follows: t1 ­ plants sprayed only with water (control); t2 ­ applications of 1.0 mmol l­1 sa (sa); t3 ­ applications of 1.0 mmol l­1 fa (fa); and t4 ­ applications of 1.0 mmol l­1 sa + 1.0 mmol l­1 fa (sa + fa) by spraying the plants for three consecutive days. plants were harvested 90 days after transplanting the seedlings by collecting leaves, roots and fruits. plant growth the effect of sa and fa on the plants were evalu­ table 1 ­ chemical analysis of the soil used in the experiment ph organic matter g dm­3 h + al mmolc dm­3 ca mmolc dm­3 mg mmolc dm­3 k mmolc dm­3 p mg dm­3 4.4 6.0 20.0 7.0 2.0 0.8 6.0 gorni et al. ‐ salicylic acid and ferulic acid elicitation in tomato 337 ated based on the fallowing: leaf area (cm2), number of leaves per plant, number of fruits per plant, plant height (cm), dry weight of shoot and roots (g plant­1) and mean fruit weight (g plant­1). the leaf area was assessed using imagej® software (powerful image analysis) with 5 plants per treatment. the number of leaves and fruit per plant was determined by manual counting, considering fully expanded leaves and fruits at harvest point. the plant height was deter­ mined by measuring tape. the shoot and root dry weight were determined after drying in an oven with air circulation at 70°c until constant weight. chlorophyll content chlorophyll content (μg ml­1) was determined spectrophotometrically following extraction in aceto­ ne, according to the method of lichtenthaler (1987). fresh leaf tissue (0.6 g) was added in 7 ml of 80% acetone, the tubes were shaken and left to stand for 30 minutes. the readings were performed in a spec­ trophotometer at λ 663 and λ 645 nm. total soluble sugar content total soluble sugar content in fruit was determi­ ned according to the method described by dubois et al. (1956) using glucose as the standard. fresh tissue (0.1 g) was added in 10 ml of 80% ethanol, and mac­ erated in a mortar and left to rest for 30 min. the analyzes followed with the addition of phenol (5%) and sulfuric acid (98 n). the readings were per­ formed in a spectrophotometer at λ 490 nm. preparation of fruit tomato extract determinations of total polyphenolic compounds and total flavonoid were obtained from the tomato juice. the fruits were ground in a low speed food pro­ cessor (3000 rpm) for two minutes and passed in 2 mm sieves. total polyphenols contents total polyphenols contents were determined spectrophotometrically (λ 765 nm) by folin­ciocalteu method with modification described by stagos et al. (2012). the total polyphenols contents were report­ ed based on micrograms of gallic acid equivalents per milliliters (μg gae ml­1). gallic acid solutions were prepared with concentrations of 25 to 500 μg ml­1 in absolute ethyl alcohol. the assays were performed in triplicate. total flavonoid content total flavonoid content was determined spec­ trophotometrically (λ 510 nm) by the method of yao et al. (2013). the total flavonoids contents were reported based on micrograms of rutin equivalents per milliliters (μg re ml­1). rutin solutions were pre­ pared with concentrations of 25 to 500 μg ml­1 in absolute ethyl alcohol. the assays were performed in triplicate. carotenoid content carotenoid content were extracted from fresh tomato samples after homogenisation of whole fruits. the homogenised sample was mixtured with chloroform/acetone/ethanol (2:1:1, v/v/v) (sadler et al., 1990). for the determination of lycopene and β­ carotene, the absorbance was read at λ 470 and λ 450 nm. carotenoid content were determined according to the equation described by craft and soares (1992), using the molar extinction coefficient of 3450 for lycopene, and 2592 for β­carotene and expressed in micrograms per grams (µg g­1). the assays were performed in triplicate. statistical analysis the experiment was arranged in a completely ran­ domized design with four treatments and five repli­ cations per treatment. the shapiro­wilk test was used to ensure the normality assumption and the homogeneity of variances. the data were submitted to analysis of variance (p≤0.05) and tukey test (p≤0.01) using the sisvar software. the results were presented by means of the treatments and standard error. 3. results the results indicated that sa and fa both caused a significant increase of growth in tomato (table 2). the exogenous application of 1.0 mmol l­1 sa resul­ ted in increasing in the accumulation of root dry weight by 61% and total dry weight by 41.3% compa­ red to control plants. the application of 1.0 mmol l­1 fa resulted in increasing in shoot dry mass by 52.3% and total dry mass by 52% compared to control plants. however, the interaction between sa + fa resulted in increasing in shoot dry mass by 51.5%, root dry mass by 146.37% and total dry mass by 66.6%, when compared to control plants. fruit weight increased by 16.5% in plant treated with 1.0 mmol l­1 fa. the exogenous application of sa + fa resulted in increasing the leaf area by 47% and number of leaves by 204.3% compared to control plants (table 3). application of sa and fa showed increasing of 101.8 adv. hort. sci., 2021 35(4): 335­341 338 and 167.5% for the number of leaves, 70.2 and 68.3% for the number of fruits, when compared with con­ trol plants (table 3). the plant height treated with sa and fa was not changed in relation to control plants. chlorophyll a content (table 4) observed in toma­ to plants treated with sa, fa and the interaction with sa + fa were significantly higher by 26, 12 and 27.6%, respectively compared to control. chlorophyll b and total chlorophyll decreased with the applica­ tion the elicitors (table 4). the total soluble sugar content in fruits, increased up to 36% in 1.0 mmol l­1 sa and 163% in 1.0 mmol l­1 fa treated plants when compared to the control. the exogenous application of sa, fa and the inter­ action with sa + fa resulted in increasing in the accu­ mulation of total polyphenols of 20.4, 110.3 and 12%, in relation to control plants (table 5). total flavonoid content in tomato were 81, 107 and 37% higher in sa, fa and the combined with sa + fa treatments when compared to control plants, respectively. in relation to carotenoid content plants treated with sa also presented increases of 157.3% (lycopene), and 157.7% (β­carotene). the combination of sa+fa showed increases of 65.25% (lycopene), and 65.3% (β­carotene), respectively, in comparison to the con­ trol (table 5). table 3 ­ effect of salicylic acid and ferulic acid on leaf area, number of leaves per plant, number of fruits per plant and plant height in tomato table 4 ­ effect of salicylic acid and ferulic acid on the chlorophyll and fruit total soluble sugar contents in tomato table 2 ­ effect of salicylic acid and ferulic acid on the shoot, root, total dry weight and fruit weight in tomato different letters indicate significant differences by tukey’s test (p≤0.01). treatments shoot dry weight (g plant­1) root dry weight (g plant­1) total dry weight (g plant­1) fruit weight (g plant­1) control 24.01±1.11 c 4.55±0.83 c 28.57±1.60 c 524.46±2.07 b sa 33.06±1.59 abc 7.32±0.36 b 40.38±1.60 b 554.29±7.44 b fa 36.57±2.35 a** 6.84±0.42 bc 43.41±1.86 ab 610.78±11.94 a** sa + fa 36.38±3.99 a 14.02±1.72 a** 47.59±4.57 a** 525.40±20.23 b cv (%) 15.78 15.08 12.04 5.39 treatments leaf area (cm²) number of leaves number of fruits plant height (cm) control 7955.4±466.9 b 65.2±3.69 c 20.8±0.86 b 77.4±2.67 ns sa 8739.6±1054.1 ab 131.6±16.02 b 35.4±3.81 a ** 79.6±2.75 fa 10209.8±850.6 ab 174.4±10.49 ab 35.0±3.20 a 88.2±7.20 sa + fa 11691.6±577.1 a * 198.4±10.09 a ** 28.6±0.40 ab 77.0±1.04 cv (%) 16.11 16.71 19.89 10.75 different letters indicate significant differences by tukey’s test (p≤0.01). treatments chlorophyll a (µg ml­1) chlorophyll b (µg ml­1) total chlorophyll (µg ml­1) fruit total soluble sugar (mg g­1) control 9.66±0.23 c 21.02±0.13 a ** 30.68±0.14 a ** 22.63±0.96 c sa 12.13±0.07 a 15.69±0.02 c 27.88±0.04 c 30.77±1.38 b fa 10.82±0.04 b 13.03±0.08 d 23.90±0.05 d 59.47±1.92 a ** sa + fa 12.33±0.07 a ** 16.05±0.02 b 28.45±0.04 b 26.89±0.21 bc cv (%) 1.73 0.73 0.59 7.30 different letters indicate significant differences by tukey’s test (p≤0.01). gorni et al. ‐ salicylic acid and ferulic acid elicitation in tomato 339 4. discussion and conclusions tomato plants treated with sa and fa presented higher total biomass (table 2). the effect of elicitors plays a key role in growth regulation, plant develop­ ment and these findings can be associated with the changes in hormone functions or the improvement of photosynthesis and carbohydrate accumulation in plants (hussain et al., 2017; gorni et al., 2020). the foliar application of sa and fa trigger a greater cellu­ lar activity that result in a larger number of leaves and leaf area, thus presenting a larger photosynthetic surface, improving the physiological processes (gorni and pahceco, 2016; hussain et al., 2017). regarding the increase of leaf area, number of leaves, number of fruits and fruit weight, our results showed that the application of sa and fa can alter the growing pat­ tern, in tomato plants (table 3). the application of sa and fa increased the growth of leaves and roots, wit­ hout changing the plant height (khan et al., 2003; de carvalho et al., 2020). biomass gains as an effect of sa application were also observed in achillea millefo‐ lium (gorni and pacheco, 2016), foeniculum vulgare (gorni et al., 2017) and mentha spicata (kundu et al., 2018). increase in biomass, were also observed after fa application in plants of arabidopsis thaliana (reigosa and pazos­malvido, 2007), pisum sativum (orcaray et al., 2011) and cucumis sativus (li et al., 2013). pigments are the essential components for growth and development and they can determine the health status of plants (hussain et al., 2017; gorni et al., 2020). based on our results, the applica­ tion the elicitors sa and fa alone or in combination suggest an increase in chlorophyll a (table 4). on the other hand, the application of sa and fa did not affect the chlorophyll b and total chlorophyll content, which directly reflected in the plant height values table 5 ­ pair­wise genetic distance estimates based on observed phenotypes of 21 amaranthus accessions treatments polyphenols (µg gae ml­1) flavonoid (µg re ml­1) lycopene (µg g­1) β­carotene (µg g­1) control 245.58±7.21 c 272.33±4.81 d 7.54±0.16 c 10.03±0.2 c sa 295.58±12.02 b 493.16±2.40 b 19.4±0.16 a** 25.85±0.2 a** fa 516.42±0.00 a** 564.00±4.81 a** 6.96±0.16 c 9.25±0.2 c sa + fa 274.75±4.81 b 372.33±4.81 c 12.46±0.00 b 16.58±0.0 b cv (%) 2.60 1.85 2.39 2.17 different letters indicate significant differences by tukey’s test (p≤0.01). (table 3). anway, other studies conducted on diffe­ rent species reported increases in chlorophyll a, chlo­ rophyll b and total chlorophyll contents consequen­ ces of sa treatment (kazemi, 2014; chakraborty et al., 2016) and in plants sprayed with fa (zhu and wakisaka, 2018). positive effects of sa and fa on the physiological processes of tomato suggest that these elicitors may be associated with the regulation of several essential primary metabolic processes, inclu­ ding synthesis of chlorophylls (zhu and wakisaka, 2018; gorni et al., 2020). the positive effects of the application of sa and fa on the physiological processes of tomato plants were also reflected in higher soluble sugar content (table 4). sugar is one of the ingredients in tomato (hafeznia et al., 2014), and it is an important organic solute with low molecular weight in higher plants. the sugars which are accumulated under stress con­ ditions or as consequence of treatments with sa or fa, keeping osmotic regulation and turgor inside the plant (orcaray et al., 2011; gorni et al., 2020). results show that application of sa also resulted in increa­ sing the total soluble sugar content in tomato plants (kazemi, 2014), and chamomile (zarinkamar et al., 2013); similarly to application of fa in pea (orcaray et al., 2011) and soybean (ferrarese et al., 2001). polyphenols and flavonoids provide many physio­ logical functions for plant survival and are of funda­ mental importance for plants adaptation (verma and shukla, 2015; mehta et al., 2018). our results suggest that the application of sa and fa may modulate and alter the concentration of polyphenolic compounds (table 5) in tomato plants. studies report that the application of sa and fa stimulates the phenyl­ propanoid pathway, increasing the accumulation of polyphenols and flavonoids in plants (salvador et al., 2013; gorni et al., 2021). tomato plants have a good free radical scaven­ ging capacity due to the high concentration of lyco­ zhu m.x., zhao c.f., bai j.g., 2018 ­ ferulic acid pre‐ treatment alleviates heat stress in blueberry seedlings by inducing antioxidant enzymes, proline, and soluble sugars. ­ biol. plant., 62(3): 534­542. craft n.e., soares jr. j.h., 1992 ­ relative solubility, sta‐ bility and absorptivity of lutein and beta‐carotene in organic solvents. ­ j. agr. food chem., 40(3): 431­434. de carvalho j.s.b., da silva j.p.r., batista r.d.c.m., 2020 ­ use of salicylic acid as an attenuator to the effects of water deficit on basil plants. ­ diversitas j., 5(3): 1561­1574. dubois m., gilles k.a., hamilton j.k., rebers p.t., smith f., 1956 ­ colorimetric method for determina‐ tion of sugars and related substances. ­ anal. chem., 28(3): 350­356. engwa g.a., 2018 ­ free radicals and the role of plant phytochemicals as antioxidants against oxidative stress‐related diseases. phytochemicals: source of anti‐ oxidants and role in disease prevention. ‐ bod­books on demand, 7: 49­74. ferrarese m.l.l., souza n.e., rodrigues j.d., ferrare­ se­filho o., 2001 ­ carbohydrate and lipid status in soybean roots influenced by ferulic acid uptake. ­ acta physiol. plant., 23: 421­427. gorni p.h., brozulato m.o., lourenção r.s., konrad e.c.g., 2017 ­ increased biomass and salicylic acid elici‐ tor activity in fennel (foeniculum vulgare miller). braz. j. food technol., 20: e2016172. gorni p.h., pacheco a.c., 2016 ­ growth promotion and elicitor activity of salicylic acid in achillea millefolium l. ­ afr. j. biotechnol., 15(16): 657­665. gorni p.h., pacheco a.c., lima moro a., silva j.f.a., moreli r.r., de miranda g.r., pelegrini j.m., spera k.d., bronzel jr j.l., silva r.m.g., 2020 ­ salicylic acid foliar application increases biomass, nutrient assimila‐ tion, primary metabolites and essential oil content in achillea millefolium l. ­ sci. hort., 270:109436. gorni p.h., pacheco a.c., lima moro a., silva j.f.a., moreli r.r., de miranda g.r., pelegrini j.m., zani­ boni c.b., spera k.d., bronzel jr j.l., silva r.m.g., 2021 ­ elicitation improves the leaf area, enzymatic activities, antioxidant activity and content of secondary metabolites in achillea millefolium l. grown in the field. ­ j. plant growth, 40(4): 1652­1666. hafeznia m., mashayekhi k., ghaderifar f., mousavi­ zadeh s.j., 2014 ­ tomato morphological and bioche‐ mical characteristics in response to foliar applying of salicylic acid. ­ int. j. biosci., 5(9): 237­243. hernández­ruiz j., arnao m., 2018 ­ relationship of melatonin and salicylic acid in biotic/abiotic plant stress responses. ­ agronomy, 8(4): 33. hussain i., singh n.b., singh a., singh h., singh s.c., yadav v., 2017 ­ exogenous application of phytosynt‐ hesized nanoceria to alleviate ferulic acid stress in solanum lycopersicum. ­ sci. hortic., 214: 158­164. javanmardi j., akbari n., 2016 ­ salicylic acid at differ‐ 340 adv. hort. sci., 2021 35(4): 335­341 pene and β­carotene. this pigment, it is the most effective antioxidant which gives color to tomato fruit as maturity progresses (kumar et al., 2017). polyphenolic compounds and carotenoid act as anti­ oxidants because they remove singlet oxygen and other free radicals in cells (shahidi and ambigaipalan, 2015) due to their ability to donate hydrogen from hydroxyl groups positioned along the aromatic ring to prevent oxidation by radicals free from lipids and other biomolecules (sousa et al., 2007). researchers showed an increase in carotenoid content during ripening due to the gradual degrada­ tion of chlorophyll (adalid et al., 2010). our results showed that the application of sa and fa may modu­ late and alter the concentration of lycopene and β­ carotene (table 5) and chlorophyll a contents (table 4) in tomato plants. thus, our study evidenced that foliar application of sa and fa in tomato crop could enhance its antioxidant activity due to the higher concentration of these bioactive compounds. our results corroborate with those found by kumar et al. 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j., wang y., 2013 ­ in vivo and in vitro antioxidant activity and α‐glucosidase, α‐ amylase inhibitory effects of flavonoids from cichorium glandulosum seeds. ­ food chem., 139(1­4): 59­66. zarinkamar f., saderi z., soleimanpour s., 2013 ‐ excluder strategies in response to pb toxicity in matricaria chamomilla. ­ environ. ecol. res., 1(1): 1­11. zhu j., wakisaka m., 2018 ­ growth promotion of euglena gracilis by ferulic acid from rice bran. ­ amb express, 8(1): 16. impaginato 329 adv. hort. sci., 2023 37(3): 329­341 doi: 10.36253/ahsc­15064 characterization of italian honeys: integrating volatile and physico­ chemical data c. taiti 1, g. guardigli 1, s. babbini 1, e. marone 2, e. masi 1, d. comparini 1 (*), s. mancuso 1, 3 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 facoltà di bioscienze e tecnologie agro‐alimentari e ambientali, università degli studi di teramo, campus coste s. agostino, via r. balzarini, 1, 64100 teramo, italy. 3 fondazione per il futuro delle città, via boccaccio, 50, 50133 firenze, italy. key words: honey, honey characterization, honey origin, honey properties, honey volatiles, monofloral honey, proton transfer reaction time­ of­flight mass spectrometer. abstract: this article focuses on the comprehensive characterization of italian honeys using various physico­chemical analyses and their volatile organic com­ pounds (vocs) fingerprint obtained through the ptr­tof­ms technology. honey characteristics, including ph, electrical conductivity, moisture content, hydroxymethylfurfural (hmf), and sugar content, were analyzed to assess their quality and origin. honey samples from different flowers, including acacia, chestnut, citrus, linden, and multifloral, were collected and investigated. furthermore, a few aged honeys were collected and analyzed and compared with the fresh ones. physico­chemical analysis revealed that chestnut honey is characterized by high ph and ec values. acacia honey has a higher fructose con­ tent, while aging appears to influence hmf levels, a vital indicator of honey quality, with aged samples exhibiting significant increases in hmf content. the voc profiles have been found to vary among different honey types, suggesting that vocs could be used as indicators of honey origin. multivariate statistical analyses, such as partial least squares discriminant analysis (pls­da), have been applied to the vocs data to differentiate honey types based on their volatile profiles. acacia honey exhibited different physicochemical parameters but on the contrary, in the vocs analysis, it displayed similarities with the lin­ den honey due to their shared low emissions of volatile compounds. citrus honey had similar chemical parameters to linden and multifloral honeys, but its distinctive vocs emission allowed for a more accurate identification. in conclu­ sion, the analysis performed with the ptr­tof­ms was successful in obtaining specific volatile fingerprints of those samples and was effective for improving the characterization of honeys. 1. introduction honey is a natural product known and used by humans since antiquity (*) corresponding author: dr.comparini@gmail.com citation: taiti c., guardigli g., babbini s., marone e., masi e., comparini d., mancuso s., 2023 ­ characterization of italian honeys: integrating volatile and physico‐chemical data. ­ adv. hort. sci., 37(3): 329­341. copyright: © 2023 taiti c., guardigli g., babbini s., marone e., masi e., comparini d., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 august 2023 accepted for publication 20 october 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15064 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 329­341 330 (nikhat and fazil, 2022). the italian legislation, trans­ posing directive 2001/110/ec, defines honey as “the natural sweet substance produced by apis mellifera bees from the nectar of plants or from secretions of living parts of plants or excretions of plant sucking insects on the living parts of plants, which the bees collect, transform by combining with specific sub­ stances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature”. honey characteristics, such as flavour and physico­chemical properties, can vary substantially depending on botanical and geographical origin (zhou et al., 2002; warui et al., 2019). the italian legislation (d. lgs. 21/05/2004, n. 179) has established thresholds and values for physico­chemical criteria, including mois­ ture, electrical conductivity, hydroxymethylfurfural (hmf), sugar, and others, to evaluate the marketabil­ ity and quality of honey, which were added to the aromatic profile of honey. nonetheless, there are roughly 320 distinct types of honey available on the market, which can be grouped into monofloral and multifloral varieties (vîjan et al., 2023). in italy, there is a rich assortment of honeys, and this diversity is the result of the unique combination of regional pro­ duction, climate conditions, and a multitude of floral sources (castiglioni et al., 2017). monofloral honey is obtained from bees that have mainly visited a unique botanical species, these honeys are particularly valu­ able on the market (schuhfried et al., 2016). as reported by ismea, the cost of multifloral honey dif­ fers from the cost of monofloral honey (ismea, 2023). however, european legislation does not speci­ fy the properties of monofloral honey, so countries like italy imposed a national regulation with a mini­ mum percentage of pollen required for the identifica­ tion as monofloral, which varied from floral origin depending on the pollen production, position and flower structure of each botanical species (tedesco et al., 2022). on the other hand, multifloral honey is produced from several types of flowers, and its char­ acteristics and properties can differ greatly depend­ ing on the visited flowers and the geographical origin. melissopalynological analysis is the official method for identifying the botanical and geographical origin of honey (aronne and de micco, 2010). however, this analysis is time­consuming and cannot be applied to filtered honey. moreover, the execution requires palynological competence, which is a limiting factor (mureșan et al., 2022). in previous studies, ptr­tof­ ms has been used for the categorization of honey types based on their aromatic profiles, such as the monofloral classification (kuś and van ruth, 2015; schuhfried et al., 2016) and for the discrimination of their botanical origin (ballabio et al., 2018). thus, the primary objective of this study was to comprehensively characterize italian honeys by employing a combination of volatile compound analysis, alongside conventional physico­chemical analyses. by integrating volatile profiling using prt tof­ms with established analytical techniques, the aim was to determine whether this analysis could serve as an additional, complementary or substitute method for discriminating different botanical origins of italian honey. 2. materials and methods sample collection honey samples were gathered in 2022 from may to august directly from beekeepers from different natural geographical macro­areas (districts) of italy to have a variety of sources that include region, province, altitude, and botanical origins. a total of 84 samples of honey were collected, 78 of these were obtained in 2022, and 6 were collected between 2020 and 2021 (aged samples). each sample was stored in the dark in a cool and dry place. the collec­ tion focused mostly on italian artisan­produced honey as reported in table 1. in addition, to achieve even more powerful results, we collected 12 italian commercial samples. the types of honey were 49 multifloral (of which 6 commercial), 16 acacia (of which 2 commercial), 11 chestnut, five citrus (of which 4 commercial) and three lindens. physico‐chemical analysis all the physico­chemical analysis were performed according to the guidelines of the italian regulation dm 25/07/2003 gu number 185 (gazzetta ufficiale, 2003). determination of ph. to assess the ph, 10 g of sample was thoroughly mixed in 40 ml ultrapure dis­ tilled water (dilution 1:5) from a millipore milli­q lab water system. the resulting solution was measured using a phm 210 standard ph meter (meterlab, radiometer copenhagen), which was previously cali­ brated with standard ph 4 and ph 7 solutions. electrical conductivity. the ec of honey was obtained from the same diluted solution used to assess the ph. the measurement was done using a conductometer (conductimeter glp 31 crison) cali­ taiti et al. ‐ characterization of italian honeys 331 brated with appropriate standard solutions. the results were expressed in ms/cm. the maximum ec for honey is 0.8 ms/cm according to italian law (directive 2001/110/ec), while for the honeydew, multifloral/mixed, and chestnut honey the ec values must be greater than 0.8 ms/cm. moisture content. the water content of the honey samples was determined with a handheld refrac­ tometer (hhtec) with automatic temperature com­ pensation. the samples were measured as­is, and the results are expressed as moisture content percent. the legal threshold for selling honey is 20%, but in competitions for premium/quality honeys, the limit is usually lowered to 18%. hydroxymethylfurfural (hmf) and furfural (f) quantification by hplc. the hmf and f were quanti­ fied following the hplc method, which had been pre­ viously described in other studies with a few adjust­ ments in accordance with italian legislation guide­ lines (fallico et al., 2004; truzzi et al., 2012). briefly, 5 g of honey was diluted with ultrapure distilled water (1:5) and mixed. then, within 12h, samples have been filtered on a 0.45 μm syringe filter and 20 μl were injected into the hplc system (azura, knauer, berlin, germany) coupled to a uv detector (analytical uv flow cell detector uvd 2.1s, knauer). the chro­ matographic column was eurospher ii 100­5 c18 150 x 4 mm, and the analysis conditions were: isocratic mobile phase, water­methanol 90:10 v/v; flow rate 0.6 ml/min; column temperature 30°c. the detector wavelength was fixed at 285nm, the identification of hmf and f was done by comparing the retention time of standard solution, and the quantification was done using a calibration curve specific for each mole­ cule (fig. 1 a). the calibration curve for hmf was made with five solutions at different concentrations (0.0005, 0.005, 0.01, 0.05, 0.1 mg/ml), while the f calibration curve was 0.0006, 0.001, 0.002, 0.006, 0.01 mg/ml. according to the law, the results were expressed in mg/kg, and the legal limit for hmf in commercial honey is 40 mg/kg. sugars determination brix determination. brix degrees of the honey samples was measured with the same refractometer of moisture content measurement. brix degrees rep­ resent the percentage of sugar content in honey by weight, with 1 brix degree equivalent to 1 g of sucrose in 100 g of solution (geană et al., 2020). sugar quantification by hplc. hplc coupled to a refractive index detector was used for the qualitative botanical origin region province production source harvest year no. of samples acacia tuscany firenze beekeeper 2022 5 acacia tuscany livorno beekeeper 2022 1 acacia tuscany arezzo beekeeper 2022 1 acacia abruzzo pescara beekeeper 2022 1 acacia tuscany ­ commercial 2022 1 acacia italy ­ commercial 2022 1 acacia lombardy cremona beekeeper 2022 1 acacia abruzzo teramo beekeeper 2022 1 acacia tuscany prato beekeeper 2022 1 acacia piedmont torino beekeeper 2022 1 acacia tuscany pisa beekeeper 2022 1 acacia emilia­ romagna forlì beekeeper 2022 1 chestnut tuscany firenze beekeeper 2022 4 chestnut tuscany livorno beekeeper 2022 1 chestnut tuscany arezzo beekeeper 2022 1 chestnut lombardy cremona beekeeper 2022 1 chestnut piedmont torino beekeeper 2022 1 chestnut tuscany pisa beekeeper 2022 1 chestnut emilia­ forlì beekeeper 2022 1 chestnut campania salerno beekeeper 2022 1 citrus calabria ­ commercial 2021 1 citrus italy ­ commercial 2021 1 citrus italy ­ commercial 2022 2 citrus sicily ragusa beekeeper 2022 1 linden tuscany firenze beekeeper 2022 1 linden lombardy cremona beekeeper 2022 1 linden emilia­ forlì beekeeper 2022 1 multifloral tuscany firenze beekeeper 2020 1 multifloral tuscany firenze beekeeper 2021 1 multifloral tuscany firenze beekeeper 2022 21 multifloral tuscany prato beekeeper 2021 1 multifloral tuscany livorno beekeeper 2022 1 multifloral tuscany arezzo beekeeper 2022 3 multifloral abruzzo pescara beekeeper 2022 1 multifloral italy ­ commercial 2021 1 multifloral italy ­ commercial 2022 5 multifloral lombardy cremona beekeeper 2022 4 multifloral abruzzo teramo beekeeper 2022 1 multifloral sicily ragusa beekeeper 2022 3 multifloral piedmont torino beekeeper 2022 1 multifloral umbria todi beekeeper 2022 1 multifloral tuscany pisa beekeeper 2022 1 multifloral lazio roma beekeeper 2022 2 multifloral campania salerno beekeeper 2022 1 total 84 table 1 ­ description of the traits of the samples analyzed, con­ sidering the different botanical origin, geographical area, and year of production adv. hort. sci., 2023 37(3): 329­341 332 and quantitative analysis of sugars (azura rid 2.1l, knauer, berlin, germany). the chromatographic col­ umn was eurospher ii 100­3 nh2 150 x 4 mm, employing a mobile phase 80:20 of acetonitrile­water and an isocratic flow rate of 1.5 ml/min at 35°c. honey samples were prepared by placing 0.5 g in 10 ml of h2o (1:20), mixing for 12­24h, then filtering and diluting 1:1 using the same solution as the mobile phase. calibration curves were prepared using fructose, glucose, sucrose, and maltose stan­ dards for quantification, and retention times were used for identification (fig. 1 b). six distinct solutions, each with a different concentration, were employed to construct the calibration curve. the concentra­ tions used were 0, 2.5, 3.75, 5, 7.5, 10, and 15 mg/ml. defect identification by sensory analysis before to test, each samples were homogenized by mixing with a glass rod, filtered and left until com­ pletely clear, after which they were subjected to organoleptic analysis (consistency, color, smell and taste) according to the national standard sr 784­ 3:2009 (council european union, 2001). particular attention was directed towards identifying any potential defects in the honey samples, with a specif­ ic focus on the detection of fermentation. to confirm the conformance of honeys and eventually exclude samples with imperfections, the odour, colour, taste, and texture of honey were assessed. all the samples were found to be conforming and free from defects based on that assessment. ptr‐tof‐ms measurements and data analysis using a ptr­tof­ms 8000 (ionicon analytik gmbh, innsbruck, austria) with h3o+ as the reagent ion and over the mass range of m/z 20­250, volatile finger­ prints of 84 samples were acquired. the benefits of the ptr­ms technology are fully and completely described in a previous study (blake et al., 2009). volatile headspace from each sample was ana­ lyzed follow the setup previously proposed by schuhfried et al. (2016) with some modification. in short, 5 g of honey (±0.1 g) were placed into a 250 ml glass jar with two teflon septa on the cap’s opposing sides for the vocs analysis. then, each jar has been sealed and fluxed with clean air for 60 seconds before the incubation time, in order to remove all the vocs accumulated during the sample preparation. subsequently, the samples have been incubated at 37°c for 30 min in order to allow vocs to fill the head­space. finally, the volatile compounds were analyzed using the ptr­tof­ms in its standard config­ uration. the zero air­generator (peak scientific instruments) supplied clean air at a flow rate of 0.5 lpm (lpm = liter per minute) to the entry of the sam­ pling device during all analyses, and the same flow rate was set for the ptr­ms inlet flow. to prevent the systematic memory effect, clean air was fluxed for five minutes in the tool apparatus between mea­ surements. for each sample run, 120 s worth of mass spectra were captured. the instrument’s settings of 2.20 mbar for the drift­tube pressure, 60°c for the drift temperature, and 550 v for the drift voltage pro­ duced an electric field strength to number density ratio (e/n) of 120 td. every sample was examined twice. internal calibration of tof spectra was per­ formed off­line after dead time correction in order to achieve high mass resolution (cappellin et al., 2011). the ptr­tof­ms’s better resolution offers a sum formula and a rough identification of each mass peak found. the tofdaq programme (tofwerk ag, thun, switzerland) was used to collect, record, and analyze the data. data were expressed in ppbv using a process outlined by lindigner and jordan (lindinger and jordan, 1998). finally, all the voc data were fil­ tered using a threshold of 0.50 ppbv and by eliminat­ ing any signals that may be attributed to the chem­ fig. 1 ­ chromatographic profiles of analyzed compounds (a) chromatogram representing the analysis of hydrox­ ymethylfurfural (hmf) and furfural (f) content in honey samples. the retention times and identified compound names are indicated. (b) chromatogram illustrating the sugar analysis in honey samples. retention times and identified sugar names are provided. taiti et al. ‐ characterization of italian honeys 333 also calculated. pls­da analysis was performed using pls­toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab r2015b (mathworks inc., natick, ma, usa). in addition, to study the relationships between the different samples as a function of different physi­ co­chemical variables, a factor analysis (fa) was applied, considering as factors the content of the dif­ ferent analyzed sugars (glucose, fructose, sucrose, maltose), ph, electrical conductivity (ms/cm), and hmf (5­hydroxymethylfurfuraldehyde) level. this last parameter is essential to evaluate the compliance of honey with current legislation. the level of hmf is used as an indicator of the heating or high tempera­ ture storage of the honey. in fact, it is generally not present in fresh honey, while its content increases during conditioning and storage (zappalà et al., 2005). furthermore, it is inversely proportional to the fructose content and the fructose/glucose ratio (kesić et al., 2014). factor analysis (fa) allows to visualize variables and samples simultaneously in a two or three­dimen­ sional space and to study the relationships between the observations (honey samples) and the variables (greenacre, 1984; escofier and pagès, 1992). computations were performed by xlstat version 2014.5.03. 3. results and discussion ph the ph values of the examined honey samples fell within the acidic range, varying between 3.5 and 6, reported in table 2 as the mean and the standard deviation (mean ± sd). acacia honey exhibited the lowest average ph of 3.77 ± 0.13, closely followed by citrus honey with an average ph of 3.90 ± 0.51. istry of the water or to interfering ions, which are thought to be challenging to precisely quantify. statistics were applied to the filtered data. statistical analysis multivariate partial least square­discriminant analysis (plsda) (supervised method) was applied to the spectra obtained from 84 honey samples pro­ duced by different genotypes, comprehensive of 38 protonated masses, for exploring the possibility of correctly classifying the botanical origin of the hon­ eys (acacia, chestnut, citrus fruits, linden, and wild­ flower, this last coming from a mix of species). as a pre­processing step, data were submitted to logarith­ mic transformation and auto­scaling. the whole data set was split into training and validation subset, opti­ mally chosen with the euclidean distances based on the algorithm of kennard and stone (1969). the training set consisted of about 85% of the samples, used for selection of the optimal number of latent variables (lvs), model calibration and cross validation (internal validation). the test set, used to predict the class membership (external validation), included 15% of samples removed from the data set. the training set was used to build a model based on venetian blinds cross validation procedures, evaluated by the number of correct predictions and the root­mean­ square error of cross­validation (rmsecv), subse­ quently validated with the removed samples (exter­ nal validation set). external validation of the model was quantified by the root­mean­square error of pre­ diction (rmsep). the optimal number of lvs was selected as those associated to the minimum error and misclassification rate of the calibration dataset. confusion matrices were used to study the reliability of the models. the threshold to assign a sample to a class was chosen minimizing the number of false pos­ itives and false negatives (bayes theorem). variable importance in projection (vip) scores (p = 0.01) were ph ec (ms/cm) moisture content (%) hmf (mg/kg) f (mg/kg) acacia 3.77 ± 0.13 0.27 ± 0.10 16.44 ± 0.74 0.95 ± 0.45 0.95 ± 0.54 chestnut 5.19 ± 0.49 1.58 ± 0.33 16.60 ± 0.73 1.78 ± 0.48 2.21 ± 1.15 citrus 3.90 ± 0.51 0.57 ± 0.54 17.20 ± 0.86 3.50 ± 1.64 2.74 ± 1.38 linden 4.16 ± 0.06 0.92 ± 0.12 15.33 ± 1.15 2.40 ± 0.20 3.70 ± 1.53 multifloral 4.12 ± 0.36 0.80 ± 0.42 16.16 ± 1.12 1.67 ± 0.56 1.53 ± 1.30 aged 4.00 ± 0.19 0.70 ± 0.51 16.48 ± 1.85 6.82 ± 3.04 2.96 ± 1.17 table 2 ­ physico­chemical honey characteristics. the table reports the value of ph, electrical conductivity (ec), moisture content, hydroxymethylfurfural (hmf), and furfural (f) data are reported as the mean ± standard deviation. 334 adv. hort. sci., 2023 37(3): 329­341 linden and multifloral honeys demonstrated slightly higher average ph values of 4.16 ± 0.06 and 4.12 ± 0.36, respectively. as expected, multifloral honeys displayed a considerable range of ph values (3.44 to 5.05), reflective of their inherent compositional diversity. in contrast, chestnut honey exhibited the highest ph value of 5.19 ± 0.49 among the tested varieties. notably, the ph of aged honey, at 4.00 ± 0.19, aligned closely with the ph values of other hon­ eys of corresponding botanical origins, such as citrus and multifloral. the acidic nature of the honey sam­ ples has implications for their antimicrobial activity (acquarone et al., 2007). the observed ph variations align with previous findings, with acacia and citrus honeys consistently displaying lower ph levels while chestnut the highest (bertoncelj et al., 2011; živkov­ baloš et al., 2018). the near 4 ph level observed in other honey types is consistent with results reported in earlier studies (truzzi et al., 2014). electrical conductivity (ec) the ec of honey samples ranged between 0.1 and 2 ms/cm, depending by the botanical origin (table 2). acacia honeys were characterized by a considerably low ec (with an average value of 0.27 ± 0.10 ms/cm); in contrast, chestnut honeys had high ec values, with an average of 1.58 ± 0.33 ms/cm and a maximum value of 1.96 ms/cm. citrus showed a mean of 0.57 ± 0.54 ms/cm, and linden of 0.92 ± 0.12 ms/cm. on the other hand, multifloral honeys exhibited a wide range of values, from 0.21 ms/cm to 1.86 ms/cm, the overall mean was 0.80 ± 0.42 ms/cm. additionally, aged honey samples did not show significant differ­ ences when compared to honey of the same botani­ cal origin (0.69 ± 0.51 ms/cm). the ec values of most honey samples fell within the standard limit (table 2), with the exception of two honeys. notably, linden honey exhibited an average electrical conductivity (ec) that exceeded the established threshold. however, when considering a limit of 0.8 ms/cm, the ec values for linden honey (0.92 ms/cm) remained acceptable, thanks to an exemption stated in d.lgs. 179/04, which allows ec levels above 0.8 ms/cm. nevertheless, ec is considered a reliable indicator of the botanical origin of honey. chestnut honey is char­ acterised by a high ec value, followed by linden honey, which also displayed a relatively high value, as reported in other studies (truzzi et al., 2014; živkov­ baloš et al., 2018). excluding the outlier value of cit­ rus honey, the average ec of the samples was similar to findings in other studies (0.24 ms/cm) (di marco et al., 2017; di rosa et al., 2019). conversely, multi­ floral honeys showed a wide range of ec values, ranging from 0.21 ms/cm to 1.86 ms/cm, reflecting the variation in floral sources visited by the bees. moisture content the moisture content of all the honey types ranged from 13.4% to 19.6%, and all the samples were under the maximum limit of 20% (table 2). moreover, 94% of the samples meet the criteria for quality competitions (value ≤ 18%). the highest aver­ age value was 17.20 ± 0.86% of citrus honey, while the lower was 15.33 ± 1.15% of linden honey. acacia, chestnut, multifloral, and aged honeys had similar values of 16.44 ± 0.74%, 16.60 ± 0.73%, 16.16 ± 1.12%, and 16.48 ± 1.85%, respectively. honey mois­ ture content is an important factor and a parameter used to evaluate the product’s quality. values that are too low can cause processing problems, while values that are too high could lead to the onset of fermentation processes, altering its quality, shelf life, taste, and composition (el sohaimy et al., 2015). there was no discernible difference between the water content of various varieties of honey when the samples were compared, despite the significant vari­ ety and botanical origin of the samples. indeed, there is a relationship between moisture content and honey maturation, production season, ventilation of the beehive, meteorological conditions and work processes (kirs et al., 2011; escuredo et al., 2014; de sousa et al., 2016; lazarević et al., 2017). hydroxymethylfurfural (hmf) and furfural (f) the hplc quantification of hmf (hydroxymethyl­ furfural) showed that all samples had hmf content within the standard thresholds. acacia honey exhibit­ ed an average hmf content of 0.95±0.45 mg/kg, while chestnut honey showed a higher value of 1.78 ±0.48 mg/kg of hmf. citrus honey recorded an even higher content, with 3.50±1.68 mg/kg of hmf with the higher value represented from the aged commer­ cial sample. samples of linden honey displayed an average hmf content of 2.40±0.20 mg/kg, while mul­ tifloral honey showed a mean value of 1.67±0.56 mg/kg of hmf. however, the main difference was highlighted between fresh and aged honey. indeed, samples of aged honey showed a considerable rise in hmf content, with an average of 6.82 ± 3.04 mg/kg. all honey harvested in 2022 had values from 0.5 to 3 mg/kg, while aged honey had significantly higher val­ ues from 4.6 to 12.12 mg/kg (table 2). these results clearly indicate that ageing process can significantly influence hmf levels, which serve as taiti et al. ‐ characterization of italian honeys 335 an important indicator of honey quality and fresh­ ness. indeed, as reported in previous studies, these compounds are related to the heating practices and preservation conditions of honey and derive from the degradation of fructose (aronne and de micco, 2010; tedesco et al., 2022). in the same chromatographic run of hmf, furfural (f) data was also obtained. acacia honey showed a mean of 0.95 ± 0.54 mg/kg, representing the honey with the lowest average f content, ranging from 0 to 1.89 mg/kg. multifloral honey followed with a con­ tent of 1.53­1.30 mg/kg. chestnut honey exhibited an average f content of 2.21 ± 1.15 mg/kg. both citrus and aged honey displayed similar values of f: 2.74 ± 1.38 mg/kg and 2.96 ± 1.17 mg/kg, respectively. the highest mean value was found in linden honey (3.70 ± 1.53 mg/kg); however, the sample with the highest f content was multifloral honey with 6.74 mg/kg. also, if neither restrictions nor indications are report­ ed for furfural in the legislation, it is related to stor­ age and of honey, since both f and hmf are usually produced by the maillard reaction (zhang et al., 2009). however, the average furfural content in the different honey types was in line with other studies (gaspar and lopes, 2009; apriceno et al., 2018; tedesco et al., 2022). brix the degree brix analysis, representing the total sugar content in honey, revealed that all honey sam­ ples exhibited values ranging from 78.8% to 85.5%. among the varieties, citrus honey displayed the low­ est mean value (81.10±0.84%), while linden honey showcased the highest (82.93 ± 1.10%) (table 3). similarly, in line with previous studies, our brix values were found to be comparable to those reported, reaffirming the absence of significant distinctions in sugar content among different honey botanical ori­ gins. however, similar to what was reported in earlier studies that also found similar brix values, the study of sugars using the refractometer did not reveal any appreciable differences between different types of honey (oroian and ropciuc, 2017; geană et al., 2020). sugar quantification quantitative analysis of fructose, glucose, sucrose, and maltose was conducted using hplc, with results expressed as percentages (g/g). comprehensive data, including the sum of fructose and glucose, as well as individual sugar levels, are presented in table 3. acacia honey exhibited the highest fructose content at 49.08 ± 1.71%, while aged honey displayed the lowest fructose content (39.18 ± 3.50%). the fructose content across all samples ranged from 33.1% to 52.8%. glucose content ranged from 22% to 40.2%, with chestnut honey demonstrating the lowest aver­ age value (27.24 ± 2.93%) and aged honey the high­ est (33.71 ± 4.76%). the range of maltose concentra­ tion was 0.9% to 4.8%, with chestnut honey having the highest level and linden honey the lowest (table 3). additionally, according to council directive 2001/110/ce of december 20, 2001, in unadulterat­ ed honeys, the sum of glucose and fructose should not fall below 60 g/100g for nectar honey, while sucrose must not exceed 5 g/100g. more specifically, 5g/100g for acacia (robinia pseudoacacia), lucerne (medicago sativa), banksia (banksia menziesii), sulla (hedysarum coronarium), eucalyptus (eucalyptus camaldulensis), not more than 10 g/100 g for citrus (citrus spp.) honey, and no more than 15 g/100 g for lavender (lavandula spp.) and borage (borago offici‐ nalis). in reference to these criteria, all 84 samples were unadulterated, in accordance with the legisla­ tion. the sum of glucose and fructose ranged from 63.80 to 84.90%, affirming the high quality of the honey samples, while sucrose was always lower than brix fructose glucose maltose g + f f/g acacia 81.90 ± 0.72 49.08 ± 1.71 30.05 ± 2.08 3.12 ± 0.45 79.12 ± 2.68 1.64 ± 0.13 chestnut 81.71 ± 0.77 45.34 ± 2.43 27.24 ± 2.93 3.36 ± 0.82 72.58 ± 4.76 1.68 ± 0.15 citrus 81.10 ± 0.84 42.65 ± 2.57 32.79 ± 5.24 2.90 ± 1.02 75.44 ± 6.74 1.32 ± 0.17 linden 82.93 ± 1.10 43.28 ± 2.02 28.39 ± 8.25 2.48 ± 0.80 71.66 ± 8.22 1.61 ± 0.42 multifloral 82.19 ± 1.16 43.85 ± 3.71 30.26 ± 3.79 2.98 ± 0.87 74.11 ± 5.25 1.47 ± 0.23 aged 82.24 ± 1.89 39.18 ± 3.50 33.71 ± 4.76 2.74 ± 0.62 72.89 ± 7.81 1.17 ± 0.10 table 3 ­ the table reports the value (average and standard deviation) of degree brix of honey (%) that represents the total sugar con­ tent and fructose, glucose, and maltose expressed as percentages (g/g). in the table it is also reported the sum of glucose and fructose (g+f) and the fructose glucose ratio (f/g) adv. hort. sci., 2023 37(3): 329­341 336 1.8%, detected in only 15 samples of the total, with a maximum value of 1.8% (7 acacia, 1 citrus and 7 mul­ tifloral). moreover, the fructose/glucose (f/g) ratio was calculated. the f/g ratio determines whether honey may crystallise; therefore, a ratio higher than 1 suggests a fluid honey, whereas a ratio lower than 1 indicates honey crystallizing more quickly (geană et al., 2020). the highest values were found in acacia, chestnut, and linden honeys (1.64, 1.67, and 1.60, respectively), indicating honey’s ability to remain liq­ uid for a longer amount of time. citrus, multifloral flowers, and aged honeys, on the other hand, showed lower ratios (1.32, 1.47, and 1.17, respective­ ly). additionally, no samples had a value lower than 1, however aged honey with an f/g ratio of 1.17 is most likely to have crystallized. in the current study, the fructose and glucose values found across differ­ ent honey samples are, on average, higher compared to those reported in other studies. however, the f/g ratio for acacia, citrus, and multifloral honeys remains consistent with literature values (oddo and piro, 2004; geană et al., 2020). factor analysis (fa) in figure 2, the fa biplot simultaneously repre­ sents the relationship between the different sugars analyzed (glucose, fructose, sucrose, maltose), the level of hmf, the ph and the electrical conductivity, highlighting the relative distances among the 84 honey samples. the first axis explains 37.18% of the total variability in the spectral data, the second axis 18.01%. from the fa graph, some groups of samples emerge which seem to be related to a compound or to a specific physico­chemical characteristic of the honey. in particular, the wildflowers are concentrat­ ed on the hmf vector, and, in a diametrically oppo­ site position, the samples of acacia honey are grouped very close along the fructose axis. this is in accordance with the fact that hmf is formed through the degradation of fructose, thus establishing a nega­ tive correlation between these two parameters. citrus and linden honeys are situated in the lower region of the graph, indicating slightly higher glucose values for citrus honey and higher hmf values for both honeys. the ph values are also significantly higher in chestnut samples, although maltose seems to somehow influence its distribution. ptr‐tof‐ms results data on the emissions of volatile organic com­ pounds (vocs) from the five honey groups ­ multifloral, acacia, chestnut, citrus, and linden­ are presented in table 4. all signals have been separated according to their respective molecular weights and are expressed as the mean concentration in parts per billion by volume (ppbv). the table shown a subset of 33 compounds obtained upon filtering the data (were eliminated all signals with an average concen­ tration below to 1 ppbv). from our study on 84 dif­ ferent honey samples a total of 37 different com­ pounds with an average value higher than 1ppbv were found. among these, the peaks with the higher emission were detected at 33.034, 45.033, 47.010, 59.049, which corresponding to the following com­ pounds methanol, acetaldehyde, formic acid and ace­ tone. similar results were obtained from other stud­ ies on honey samples from different botanical origins (kuś and van ruth, 2015; schuhfried et al., 2016). the average of total emission recorded for each honey botanical origin varies from a minimum value of 265.2 ppbv for acacia and 336.1 for linden honey to a maximum value of 1971.8 for citrus honey (table 4). acacia and linden honey showed a rather similar volatile profile characterized by both a lower level of emission and a lower number of signals (30 and 28 respectively) compared to the other botanical ori­ gins. citrus honey emerged both for a higher emis­ sion of methanol, acetaldehyde and acetone com­ pounds as well for a higher emission of terpene com­ pounds (mz 111.101, 121.101, 135.116, 137.132) compared to the other botanical origins. chestnut samples are characterized by large signals of com­ pounds detected at m/z: 69.033 (c4h5o+, tentatively identified as furan), 83.086 (c6h11+, ti as c6 compounds) and 105.069 (c5h13s+, ti pen­ fig. 2 ­ biplot from factor analysis. relationships between 84 honey samples and different physicochemical parame­ ters. ac = acacia, ci = citrus fruits, ch = chestnut, wf = wildflower, li = linden. taiti et al. ‐ characterization of italian honeys 337 table 4 ­ number of signals detected, chemical formula, tentative identification, vip score and average amount (ppbv) of each com­ pound detected from different honey samples n° of compounds mz chemical compound tentative identification* multifloral (n=49) acacia (n=16) chestnut (n=11) citrus (n=5) linden (n=3) 1 27022 c2h3+ acetylene 33.49 10.80 30.91 99.50 11.85 2 33033 ch5o+ methanol 189.78 61.30 61.15 243.54 49.04 3 41038 c3h5+ alkyl fragment 22.58 5.39 11.16 50.39 19.03 4 43018 c2h3o+ aceton or acetate fragments 44.33 14.86 47.49 87.52 23.39 5 45033 c2h5o+ acetaldehyde** 369.53 88.24 506.01 884.92 106.64 6 47010 ch3o2+ formic acid/formats** 25.21 7.05 41.97 180.30 7.90 7 49011 ch5s+ s compound (methanethiol) 3.23 2.10 2.93 8.96 2.00 8 53038 c4h5+ cyclobutadiene 2.48 2.03 2.21 2.79 2.00 9 55054 c4h7+ alkyl fragment 6.43 4.09 5.93 18.59 3.68 10 57069 c4h9+ alcohol fragments 13.25 5.02 3.02 6.33 2.59 11 59049 c3h7o+ acetone** 119.70 42.56 210.57 284.23 100.21 12 61028 c2h5o2+ acetic acid 12.25 7.25 11.14 15.98 5.94 13 63033 c2h7s+ s compound (dimethyl sulfide) 13.07 4.07 17.29 25.27 2.15 14 65.00 c5h5+ s compound 2.59 tr 2.35 3.63 tr 15 67050 c5h7+ 3­penten­1­yne/terpene fragment tr tr tr 2.65 tr 16 69033 c4h5o+ furan 5.33 2.93 16.02 5.84 tr 17 71086 c5h11+ alcohol compounds 2.24 2.77 2.24 1.10 1.10 18 73054 c4h9o+ butan­2­one 13.60 5.54 10.70 20.21 3.48 19 75044 c3h7o2+ isobutanol 2.67 2.71 2.58 2.64 2.33 20 77038 c6h5+ alkyl fragment 2.11 2.03 2.06 2.14 1.01 21 79049 c6h7+ benzene/terpene fragment 2.88 2.72 2.52 3.34 0.00 22 83086 c6h11+ c6 compounds 2.35 2.02 6.92 2.87 1.10 23 85059 c5h9o+ (e)­2­pentenal 3.14 2.17 2.14 3.21 0.00 24 87044 c4h7o2+ 2,3­butanedione 7.59 3.65 10.10 15.91 3.81 25 93069 c7h9+ terpene fragments tr tr tr 3.23 tr 26 95011 c2h7o2s+ dimethyl sulfone 7.34 4.53 3.52 6.44 3.82 27 97033 c5h5o2+ furfural 6.09 3.26 4.30 8.49 2.63 28 99044 c5h7o2+ furfuryl alcohol tr tr tr tr tr 29 101060 c5h9o2+ dihydro­methyl­furanone tr tr 0.00 0.00 0.00 30 103080 c5h11o2+ 2­/3­methylbutyric acid tr 0.00 0.00 2.71 0.00 31 105069 c5h13s+ pentanethiol** tr 0.00 4.26 1.89 tr 32 107086 c8h1 1+ 1,3­dimethylbenzene/terpenes fragments tr tr 1.87 1.58 0.00 33 109070 c7h9o+ benzyl alcohol tr 0.00 tr 1.12 1.41 34 111101 c8h15+ terpenes fragments** tr 0.00 tr 1.88 0.00 35 121101 c9h1 3+ terpenes fragments tr 0.00 1.12 2.54 0.00 36 135116 c10h15+ terpenes** tr 0.00 tr 2.10 0.00 37 137132 c10h17+ terpenes tr 0.00 0.00 2.08 0.00 total emission average 889.30 265.28 996.51 1971.86 332.26 total signals detected 37 30 34 36 28 * each value has been tentatively assigned to compounds, based on ptr­honey literature data (kuś and van ruth, 2015; schuhfried et al., 2016; ballabio et al., 2018). ** compounds with the highest vip score. tr means trace and these compounds have been identified in at least 1 sample per group but with an overall average value below 1 ppbv. adv. hort. sci., 2023 37(3): 329­341 338 tanethiol) in agreement with ballabbio et al. (2018). as can be seen from the data shown in the table 4, the average volatile profile of multifloral honey showed an average emission for many signals and trace compounds in large numbers (identified only in some samples). no significant differences were observed between the volatile organic compound (voc) emissions of commercial honey and those produced by beekeep­ ers. pls‐da analysis with the aim to get an overview of the voc data collected, a pls­da analysis was applied on the whole dataset obtained from 37 different vocs data collect­ ed from 84 samples. it emerges that the honey sam­ ples distance themselves from each other according to their botanical origin. multifloral honey seem to show a variable trend probably linked to its botanical origin. to provide a more detailed characterization of the vocs emitted by different honey samples, vip scores higher than 1 and their possible identification on the basis of literature data were reported in table 4 (marked by two asterisks). the volatile compounds with higher vip value could be good candidates for the honey species identification. in particular, the chemical species with the higher significance were detected at mz 45.033(ti acetaldehyde), 47.01 (ti formic acid/formates), 59.049 (ti acetone), 105.069 (ti pentanethiol), 111.10 (ti terpenes fragments), 135.116 (ti terpenes). pls­da approach was applied to find vocs able to discriminate among species. by applying the model developed by the pls­da on honey samples of differ­ ent botanical origin, a correct distinction of the taxo­ nomic category of two/five different groups was achieved. indeed, the multifloral honey could be obtained by honeybees from the nectar of different flowers. score plot from the pls­da model is shown in figure 3. the global quality of the model, evaluated by its performances indicators (table 5), resulted robust enough to discriminate the botanical origin of the citrus and chestnut samples compared to the others in the calibration/validation data set, and in the independent test set. indeed, the pls­da three­ component model successfully classified 100% of fig. 3 ­ score plot (lv1, lv2) of the pls­da model. samples of different botanical origin are highlighted. red = acacia; green = citrus fruits; blue = chestnut; light blue = wild­ flower; lilac = linden. statistics y­blocks class 1 ­ acacia class 2 – citrus fruits class 3 ­ chestnut class 4 ­ wildflower class 5 ­ linden sensitivity (se) (cal) 1.000 1.000 1.000 0.721 1.000 specificity (sp) (cal) 0.800 1.000 1.000 0.724 0.643 sensitivity (se) (cv) 0.833 1.000 1.000 0.744 1.000 specificity (sp) (cv) 0.800 0.970 1.000 0.552 0.643 sensitivity (se) (p) 1.000 0.000 0.000 0.000 1.000 specificity (sp) (p) 0.875 1.000 0.833 0.182 0.583 class. error (cal) 0.100 0.000 0.000 0.277 0.178 class. error (cv) 0.183 0.015 0.000 0.352 0.178 class. error (pred) 0.062 0.500 0.583 0.909 0.208 rmsec 0.319 0.104 0.169 0.414 0.161 rmsecv 0.328 0.159 0.199 0.464 0.166 rmsep 0.387 0.296 0.719 0.674 0.044 table 5 ­ pls­da statistics for the honey samples for the five botanical origins: 1 = acacia, 2 = citrus fruits, 3 = chestnut, 4 = wildflower, 5 = linden. sensitivity (se), specificity (sp), class. error, rmsec, rmsecv, rmsep, for calibration (cal), cross validation (cv), and prediction (pred), respectively taiti et al. ‐ characterization of italian honeys 339 honey samples from classes 2­chestnut and 3­citrus into their respective taxonomic categories during fit­ ting, cross­validation (internal validation), and predic­ tion (external validation), while the acacia and linden honey samples are confused with the multifloral samples. 4. conclusions in this study, a comprehensive analysis of various physico­chemical properties and volatile organic compounds (vocs) present in italian honeys was con­ ducted. it was revealed that the quality of the honey sold is excellent, as legal limits were adhered to for all samples (except for few ec values). the electrical conductivity (ec) values demonstrated significant variability, with chestnut and linden honeys standing out due to their high and relatively high ec values, respectively. the observed ph variations among dif­ ferent honey types were consistent with their botani­ cal origins. furthermore, parameters such as sucrose content and the fructose­to­glucose ratio, indicative of potential adulterations, remained within legal lim­ its for both commercial and beekeeper honey. moreover, within this context, the honey varieties were discerned based on their distinctive characteris­ tics. aged honey, as expected, was characterized by a high hmf level; however, it remained within legal thresholds. acacia honey, characterized by its high fructose content, exhibited low ph and ec values. interestingly, in voc analysis, it displayed similari­ ties with linden honey due to their shared low emis­ sions of volatile compounds. chestnut honey, which had high ph and ec values, was easy to differentiate from other types of honey using both conventional metrics and ptr­tof­ms­based voc analyses. its distinctive profile made it simple to classify. citrus honey displayed physicochemical characteristics similar to linden and multifloral honeys, but its dis­ tinctive voc emissions allowed for a more accurate identification. conversely, linden and multifloral honeys shared close resemblances in chemical and physical analyses, and the significant variability in the multifloral variety’s composition due to its diverse floral sources hindered differentiation through vocs. factor analysis provided insights into the rela­ tionships between different sugars, hmf, ph, and electrical conductivity, highlighting distinct group­ ings of honey samples. finally, vocs analysis revealed a diverse range of compounds, with noticeable variations attributed to the botanical ori­ gin of the honey. partial least squares­discriminant analysis (pls­da) facilitated discrimination among different honey types based on their voc profiles highlighting the potential for this method in distin­ guishing honey types based on voc profiles. the analysis of vocs has the advantage of being a faster alternative to pollen analysis, providing an efficient means of differentiating between honey samples with varying botanical sources. for this reason, ptr­ tof­ms­based voc analysis serves as a valuable tool to complement or even replace melissopalyno­ logical analysis, as demonstrated by the effective combination of voc analysis and pls­da for distin­ guishing honeys of different botanical origins. in conclusion, the collective application of physi­ co­chemical and voc analyses yielded a compre­ hensive means of effectively characterizing honey varieties. this integrated approach underscores the robustness of employing multiple techniques for a thorough understanding of honey attributes. acknowledgements this research is part of the beewin project: beekeepers weather indexed insurance project funded by mipaaf 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morocco 3 department of life sciences, bioengineering unit, walloon agricultural research centre (cra‐w), chaussée de charleroi 234, p.o. box 5030, gembloux, belgium. key words: adventitious root, argania spinosa, auxins, stem cuttings. abstract: argania spinosa (l.) is an endemic tree species of south­western morocco; it plays a very important socio­economic and environmental role. however, the vegetative propagation of the argan tree by traditional cuttings is limited by the difficulty of rooting and survival during transplantation in the field. considering these facts, this study intended to investigate the rooting ability and growth performance of argan tree cuttings, collected from four élite trees rated asoc1, asoc2, asoc3 and asoc4, and treated with four concentra­ tions (0, 1000, 3000 and 5000 mgl­1) of iba, naa and iaa. the results revealed cuttings of asoc2 and asoc3 genotypes were relatively less responsive than asoc1 and asoc4, this genotype effect was more pronounced in auxin treated cuttings. treatment of cuttings by iba was more effective than treatments by either naa or iaa. among all the media tested, 3000 mgl­1 of iba with asoc1 resulted in higher sprouting (81.75%), rooting (60.75%) and survival rates (96.25%). however, with the increase of iba concentration levels (>3000 mgl­1), adventitious roots and sprouts performances decreased in all the genotypes. argania spinosa could be successfully propagated by cuttings from selected élite trees. 1. introduction the argan tree ­ argania spinosa (l.) skeels ­ is a monoecious tree species, evolving in arid and semiarid areas and belonging to the tropical sapotaceae family (m’hirit et al., 1998). conventionally, argan plants are propagated by seeds. however, this method is not adequate for argan trees domestication. this species is allogamous and shows extreme vari­ ability and heterogeneity (nouaim et al., 2002; alouani and bani­aameur, (*) corresponding author: meriem.malaoui@gmail.com citation: benbya a., mdarhri alaoui m., gaboun f., delporte f., chlyah o., cherkaoui s., 2019 ­ vegetative propagation of argania spinosa (l.) skeels cuttings: effects of auxins and genotype. ­ adv. hort. sci., 33(4): 519­527. copyright: © 2019 benbya a., mdarhri alaoui m., gaboun f., delporte f., chlyah o., cherkaoui s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 june 2019 accepted for publication 30 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 519­527 520 2004; msanda et al., 2005). in addition, seed propa­ gation does not ensure preserving selected genetic characters for the next generation and could also result in a loss or dilution of favorable mother trees genes (hartmann et al., 2002). however, vegetative propagation techniques (cuttings, layering, division or separation, budding, grafting and tissue culture) are not used for clonal forestry purposes because the argan tree is a hard­to­root species (nouaim et al., 2002). thus, the development of an efficient vegeta­ tive propagation technique will provide an opportuni­ ty to facilitate domestication, improvement and mass multiplication of élite trees of this species (leakey et al., 1990; benbya et al., 2018). the most common vegetative propagation methods that have the high­ est short­term potential for large scale production of woody plants are micropropagation (tissue culture) and macropropagation (rooted cuttings) (duryea and dougherty, 1991). the success of cutting techniques depends on several factors that influence rooting efficiency (hartmann et al., 2002). adventitious root formation (arf) is a synchronized developmental process involving various biochemical, physiological and histological events in the induction, initiation, expression and elongation phases of adventitious roots (nemeth, 1986; soundy et al., 2008). the large variation in adventitious root formation in a. spinosa species is attributed to genotype (nouaim et al., 2002). in fact, the loss of ability to regenerate roots and shoots by cuttings can be described in terms of ontogenetic stage, physiological and chronological ages, which may eliminate the possibility of success­ ful propagation of selected trees (rasmussen et al., 2014). the physiological age may also depend on environmental growth conditions and plant respons­ es to stress (greenwood et al., 2001; rasmussen et al., 2014). among external factors, the most impor­ tant role for adventitious root formation is ascribed to growth regulators, which have been successfully employed in many plant species to improve the root­ ing ability of cuttings (singh et al., 2011; sağlam et al., 2014). the balance of plant hormones in the cut­ ting could affect the development of root primordia, initial root development, root elongation, hardening and further development of the rooted cutting (jaenicke and beniest, 2003). in fact, adventitious root induction in cuttings is promoted by high auxin levels and low cytokinin levels in the rooting zone (de klerk et al., 2001). auxin, the first identified plant hormone, is involved in various plant growth and development processes, including embryogenesis, organogenesis, vascular tissue patterning (xylem and phloem), flower development, fruit setting, ripening and senescence (vanneste and friml, 2009; davies, 2010). auxin homeostasis within plant tissues is regu­ lated by the interplay of biosynthesis, conjugation, transport, and signaling pathways (zhao, 2010). the endogenous auxin, indole­3­acetic acid (iaa), plays a central role in adventitious rooting of cuttings (de klerk et al., 1999). the difference in rooting ability between easy and hard­to­root cuttings can be attrib­ uted to iaa transport and accumulation (ford et al., 2002). the endogenous auxin increased up to fifteen days to decline thereafter, and exogenous auxin application increased the indole/auxin content (kochhar et al., 2008). many studies have shown that application of exogenous auxin results in an increased rooting initiation and development. the most common root­promoting compound in the nursery industry is indole­3­butyric acid (iba) (hartmann et al., 2002). in addition to enhancing the rate of adventitious root development, exogenous auxin application has been found to reduce rooting development time, to increase the number of roots per plant and root system uniformity (leakey et al., 1990; overvoorde et al., 2010). the present study was undertaken to investigate the influence of geno­ type, auxin type and its concentration, and their combined interaction effect on adventitious rooting capacity and vegetative growth attributes of mature semi­hardwood cuttings of argania spinosa. a low­ cost technology of non­mist propagation system was used for all the experiments. 2. materials and methods experimental site and selection of plant material this study was conducted under an experimental greenhouse at the biotechnology unit of the regional center of agricultural research of rabat, morocco. cuttings grew from may 2014 to september 2015. argania spinosa cutting materials were collected from trees of oued cherrat forest arboretum, benslimane province, morocco (33°81’96” n ; 7°11’03” w; 45 m altitude), which is located within 2000 m of the moroccan atlantic coast and with an average annual rainfall of 460 mm.yr­1. the selection of a healthy mature candidate plus tree (cpt) was based on general growth, phenology (leafing, initia­ tion of flowering, initiation of fruiting, fruiting period, maturation of fruiting, fruit shape and caliber), high biomass (crown diameter) and regeneration ability. the four genotypes (stock plants) were tagged for benbya et al. ‐ vegetative propagation of argaria spinosa. effects of auxins 521 identification and were pruned regularly (thrice a year) to encourage production of good shoots and maintain juvenility of the trees. after one­year, semi­ hardwood cuttings were collected sequentially from shoots located in the middle­part of the tree crown in the months of april to june. the cuttings were taken early in the morning by using sterile pruning scissors and the collected shoots were kept under shade. after harvesting, these shoots were kept in perforated plastic bags inserted in a cool box to mini­ mize a possible desiccation effect during collection and transportation. at the laboratory, the cuttings were kept in a cold room (4°c) for 48 hours. preparation of cuttings and application of auxins cuttings were screened by using a calibrated elec­ tronic digital vernier caliper for desired and uniform size of (5±1) mm width, (10±0.5) cm length, with 4­6 leaves and 7­10 nodes per cutting, after removal of the apices. each cutting was granted by two vertical cuts below the node on the basal end and a slanting cut above the node on the apical portion. the lower thorns and leaves (50% of total leaves) of each cut­ ting were removed, keeping intact leaf buds in each cutting. the base of each cutting was freshly trimmed by 0.5 mm, then it was immersed in a 0.2% fungicide solution (dithane with active ingredient mancozeb 750 g/kg) for 10 min and washed thoroughly with dis­ tilled water. the apical cut ends of the treated cut­ tings were sealed with tree wound dressings to reduce water loss, prevent diseases and decay. the lower 5 cm portion of the cuttings was dipped for 5 min in a concentrated auxin solution prepared by dis­ solving the hormone powder into 10 ml of ethanol (95%), and then sterile distilled water was added to a final volume of 1l. the concentrations of auxin solu­ tions were 1000, 3000 and 5000 mgl­1 which corre­ sponds to (5.71, 17.12, and 28.54) mm iaa, (5.37, 16.11 and 26.85) mm naa and (4.92, 14.76 and 24.60) mm iba respectively. untreated cuttings (cut­ tings were dipped in distilled water) were considered as a control set. experimental growth conditions cuttings were initially raised in a non­mist green­ house to allow root initiation to take place. the tem­ perature of the greenhouse was 32±2°c, with a 16h/8h photoperiod and 80% humidity. in the green­ house, the basal cut portion was inserted vertically according to the positive polarity in 1000 cc poly­ styrene pots (two cuttings per pot). the pots perfo­ rated at the bottom were filled with a rooting medi­ um of sterilized and sieved fine river sand. holes were punched into the rooting medium to allow the insertion of the cuttings without damaging the cam­ bium or removing the rooting hormone. after sticking of the cuttings, the rooting medium was pressed slightly around them. cuttings were irrigated regular­ ly to field capacity by tap water every two days. the rooting experiment was conducted for several weeks until the cuttings initiated roots. then, rooted cut­ tings were removed from pots and substrate was carefully washed away from the root system. cuttings with roots (≥ 1 mm) were considered as rooted and were included for calculating the rooting ratio. root shoots (≥ 1 cm) were considered for calcu­ lating mean number of roots. the root systems were handled carefully so that no visible damage occurred during transplantation. after data recording, these plantlets were transferred to larger black polyethyl­ ene pots (20 cm diameter, 20 cm depth) containing a mixture of sterilized forest soil, peat moss (ph of 6, water retention of 800 ml/l and organic matter con­ tent of 20%) and sieved fine river sand rooting medi­ um (1:1:1 v/v). these pots were placed in the green­ house with full sunlight, at a spacing of 20 cm × 20 cm. a hoagland nutrient solution (hoagland and arnon, 1950) was also used once a week to provide the nutritional needs of plantlets. the cuttings were recorded after 48 weeks, a period that was consid­ ered sufficient to measure the survival of rooted cut­ tings, following a preliminary study in the laboratory. experimental design and treatments the experiment was conducted in greenhouse using a randomized complete block design (rcbd) with four replications to study the effect of four genotypes (asoc1, asoc2, asoc3 and asoc4), three auxin types (iba, iaa and naa), four concentrations (control, 1000, 3000 and 5000 mgl­1) and their inter­ actions. measured parameters eight morphological characteristics per cutting, including the number of leaves (ln), leaf size in cm2 (ls), number of sprouts (sn), sprout length in cm (sl), sprouting rate (sp), number of roots (rn), longest root length in cm (rl), and rooting rate (rp) were measured 12 weeks after planting. the survival rate (sr) was recorded 48 weeks after rooting induction. statistical analysis the data was submitted to tests of analysis of variance (anova) for treatment effects using the general linear model (glm) procedure of sas pro­ gram version 9.1 (sas institute, cary, nc, usa), for all adv. hort. sci., 2019 33(4): 519­527 522 the evaluated parameters. comparisons between treatments were performed by using duncan’s multiple range test (dmrt) at p<0.05 level of signifi­ cance. values were means ± standard deviation (se). data given in percentages was subjected to arcsine √x transformation before statistical analysis. 3. results the results have shown that sprouting preceded rooting initiation on the cuttings. indeed, cuttings developed leaflets after about ten days, sprouts with­ in six weeks and there was no rooting till twelve weeks after planting (fig. 1), although there were sig­ nificant differences between genotypes treated with different auxin types and concentrations in the process of adventitious root development and shoot growth of argania spinosa cuttings (table 1). effect of auxin type, concentration and genotype on the number of leaves and leaf size per cutting the number and size of leaves followed the same pattern; they have a significant response (p<0.05) to auxin type, concentration and for the different geno­ types studied. the interaction between auxin type, concentration and genotype was also significant on the mean number of leaves and leaf size (table 1) the results show that genotypes responded to all treatments including low concentrations (1000 mg l­1). the highest numbers of leaves values (35.75 and table 1 ­ three­way analysis of variance (anova) for effects of genotype (asoc1, asoc2, asoc3 and asoc4), auxin type (iaa, naa and iba), concentration (0; 1000; 3000; 5000) and their interactions on measured parameters of a. spinosa cuttings df= degrees of freedom; level of significance p<0.05; sprouting rate, rooting rate and survival rate were subjected to arcsine √x transformation before statistical analysis. source of variance dependent variable df f­value p­value genotype no. of leaves 3 99.127 0.000 leaf size (cm²) 3 66.694 0.000 no. of sprouts 3 56.110 0.001 sprout length (cm) 3 10.321 0.000 no. of roots 3 11.500 0.000 root length (cm) 3 12.100 0.000 sprouting rate (%) 3 343.16 0.000 rooting rate (%) 3 109.75 0.000 survival rate (%) 3 57.643 0.000 auxin no. of leaves 2 15.180 0.000 leaf size (cm²) 2 9.750 0.000 no. of sprouts 2 1.295 0.274 sprout length (cm) 2 3.084 0.046 no. of roots 2 33.270 0.000 root length (cm) 2 38.420 0.000 sprouting rate (%) 2 65.200 0.000 rooting rate (%) 2 861.00 0.000 survival rate (%) 2 1125.5 0.000 concentration no. of leaves 3 148.10 0.000 leaf size (cm²) 3 165.34 0.000 no. of sprouts 3 512.55 0.000 sprout length (cm) 3 480.47 0.000 no. of roots 3 281.49 0.000 root length (cm) 3 252.28 0.000 sprouting rate (%) 3 4638.9 0.000 rooting rate (%) 3 2830.6 0.000 survival rate (%) 3 8245.3 0.000 genotype x auxin x concentration no. of leaves 18 6.832 0.000 leaf size (cm²) 18 8.511 0.000 no. of sprouts 18 00.164 1.000 sprout length (cm) 18 8.425 0.000 no. of roots 18 57.646 0.000 root length (cm) 18 14.281 0.000 sprouting rate (%) 18 3.681 0.000 rooting rate (%) 18 4.715 0.000 survival rate (%) 18 1.692 0.047 fig. 1 ­ vegetative propagation of argania spinosa through mature semi­hardwood cuttings. (a) selected plus tree in natural conditions (month of may). (b) cuttings in polyethylene pots according to a randomized complete block design (rcbd). (c) leaf initiation (10 days) and sprouts elongation (6 weeks). (d) root primordia initia­ tion and adventitious root development (12 weeks). (e) high rate of adventitious roots from semi­hardwood cut­ tings treated with 3000 mgl­1 iba (48 weeks). (f) plant produced from cuttings transplanted in black polyethyle­ ne pots under non­mist greenhouse conditions (3 years). benbya et al. ‐ vegetative propagation of argaria spinosa. effects of auxins 523 of asoc1 with 3000 mgl­1 iba enhanced the number of sprouts (1.81 times) and sprout length by 15.94 cm, in comparison with the control. thereby, naa and iaa applications also increased the number and length of sprouted cuttings but were less effective than iba. indeed, by using 1000 mgl­1 naa treatment on asoc1 cuttings, the number of sprouts was 1.63 times greater and sprouts were longer by 12.38 cm than the control set, while by using 5000 mgl­1 iaa treatment, the greatest number of sprouts was 1.69 times greater for asoc4 and the highest sprouts length was 11.19 cm longer for asoc1 (fig. 3). effect of auxin type, concentration and genotype on the number of roots and longest root length per cut‐ ting the analysis of variance indicated that there were significant differences between auxin type and con­ centration as well as genotype on the number of roots produced and root length formed on a. spinosa cuttings. the interactions between auxin type and its concentration with genotype influenced (p<0.05) root number and root length (table 1). it may be inferred from above that there is a synergism between the number of roots and length of roots in all genotypes studied. indeed, the more there are roots per cuttings and the more there will be longer roots. the adventitious root production increased at lower concentrations of iba and began to level off between 1000 and 3000 mgl­1, whereas with bigger hormone concentration levels (>3000mgl­1), the number and length of roots were smaller. similar fig. 3 ­ effects of auxin type, concentration and genotype on sprouts growth (mean values of sprouts number and sprout length) of a. spinosa cuttings. within each treat­ ment, the mean values followed by the same letter are not significantly different (duncan’s multiple range test, p<0.05, mean± se, n = 32). fig. 2 ­ effects of auxin type, concentration and genotype on leaves development (mean values of leaves number and leaf size cm2) of a. spinosa cuttings. within each treat­ ment, the mean values followed by the same letter are not significantly different (duncan’s multiple range test, p<0.05, mean± se, n = 32). 34.56) were recorded from asoc1 treated respec­ tively with 3000 and 5000 mgl­1 iba, while the lowest number of leaves (13.31) was observed for asoc2 cuttings treated with 1000 mgl­1 iaa (fig. 2). it was noticed that the number of leaves of asoc1 cuttings, exposed to 3000 mgl­1 iba, is 77% higher compared to the control group. the number of leaves for asoc1 cuttings ­ that received 1000 mgl­1 naa is 73% greater than the con­ trol group, while it is 52% higher for the 5000 mgl­1 iaa application. the mean number of leaves pro­ duced in the control group of asoc1 is 19.29 leaves per cutting (fig. 2). according to the results, the high­ est leaf size mean (32.42 cm2) was observed in asoc1 treated by 3000 mgl­1 iba and it is 3 times greater than the control group. similarly, leaf size (27.29 cm2) of asoc1 cuttings receiving 1000 mgl­1 naa is 2.87 times higher than leaf size in the control set. while, the cuttings of asco1 that received 5000 mgl­1 iaa developed a leaf size 1.52 times bigger than control. whereas the minimum leaf size (6.82 cm2) was recorded for the asoc2 cuttings treated with 1000 mgl­1 iaa (fig. 2). effect of auxin type, concentration and genotype on the number of sprouts and sprout length per cutting the comparison between various treatments revealed that genotype, auxin type, concentration and their interaction had a significant influence (p < 0.05) on sprout length per cutting. however, the mean number of sprouts is influenced only by geno­ type and auxin concentration (table 1). the number of sprouts and sprout length were induced by all the treatments except control (fig. 3). the pretreatment 524 adv. hort. sci., 2019 33(4): 519­527 rooting response was observed with naa hormonal treatments (fig. 4). according to the results of the study, iba significantly promoted the number and length of roots in comparison with naa and iaa. genotypes asoc2 and asoc3 were relatively less responsive than genotypes asoc1 and asoc4 in term of root number and root length per cutting. the cut­ tings in the control group and cuttings treated with 1000 mgl­1 iaa failed to produce roots, while the cut­ tings belonging to asoc1 receiving a 3000 mgl­1 iba application developed a maximum number of roots (41) with an average length of 22.94 cm. effect of auxin type, concentration and genotype on sprouting, rooting and cuttings survival rates sprouting, rooting and survival ratios of a. spinosa cuttings were significantly influenced (p<0.05) by the genotype from which cuttings were collected, as well as by the type and amount of auxin applied (table 1). most cuttings that rooted in the non­mist green­ house survived and ultimately produced sprouts as a sign of their successful regeneration. the results indi­ cate that an application of iba, naa and iaa increased sprouting, rooting and survival rates, and iba was the most effective auxin. when treated with concentrations >3000 mgl­1 sprouting, rooting and survival rates began to decrease rapidly in all cuttings treated with naa and iba (fig. 5). the results indi­ cate that cuttings collected from asoc1 and treated with 3000 mgl­1 iba had the highest sprouting per­ centage (81.75%), rooting percentage (60.75%) and survival percentage (96.25 %) per rooted cutting, fol­ lowed by asoc4 which had a sprouting ratio of 74%, a rooting ratio of 55.5% and a survival ratio of 94.5%. among the different concentrations of naa used, the maximum sprouting percentage (71.5%), rooting per­ centage (46.5%) and survival percentage (94.5%), were obtained in asoc1 cuttings treated with 1000 mgl­1. besides, compared with the control, all iaa treatments enhanced the rooting rate of a. spinosa cuttings but were less effective than iba and naa. indeed, the highest sprouting ratio (63.25%), rooting ratio (27.75%) and survival ratio (94.0%) with iaa treatment was obtained with asoc1 by using 5000 mgl­1 iaa. 4. discussion and conclusions the results of the present study indicate that there is a large amount of variability between the genotypes in their sprouting, rooting and survival measurements. these results fit well with those obtained by nouaim et al. (2002), which reported that the rooting capacity of argania spinosa cuttings has proven to be difficult and strongly genotype dependent. the significant variation among geno­ types could be due to considerable genetic variation (prat et al., 1998). it could be possible that some of the variation for root response results from the dif­ ferences in age or in physiological states of initial cut­ ting sources. it has also been reported that cuttings time of collection in different seasons could also fig. 4 ­ effects of auxin type, concentration and genotype on growing roots (mean values of roots number and mean longest root length) of a. spinosa cuttings. within each treatment, the mean values followed by the same letter are not significantly different (duncan’s multiple range test, p<0.05, mean ± se, n = 32). fig. 5 ­ effects of auxin type, concentration and genotype on sprouting, rooting and survival rate of a. spinosa cut­ tings. within each treatment, values marked by the same letter are not significantly different (duncan’s multiple range test, p<0.05, mean ± se, n = 32). mean values of sprouting percentage, rooting percentage, and survival rate were subjected to arcsine √x transformation before statistical analysis. benbya et al. ‐ vegetative propagation of argaria spinosa. effects of auxins 525 affect the rooting potential in clonal propagation. in fact, the varied effectiveness of auxin concentrations among genotypes and species is likely to be related with differences in the amount of endogenous hor­ mone and associated to root co­promoters in the plant tissues at the time of severance (hartmann et al., 2002). otherwise, the decreasing in rooting ability could be assigned to low sensitivity of the tissues to auxin or by secondary metabolites accumulation, causing oxidation and inactivation of enzymes and phytohormones which inhibit regenerative potential­ ities of tissues (wilson, 1994; husen and pal, 2007). similar observations have been reported by mabizela et al. (2017), who concluded that four studied geno­ types of honey bush (cyclopia subternata) had signifi­ cant differences in rooting potential. considering the above results, sprouting, rooting and survival ability of argania spinosa cuttings were significantly higher (p> 0.05) in cuttings treated with exogenous auxins. the fact that exogenous auxin treatment exhibited a greater potential for adventitious rooting as well as a rapid growth of cuttings is widely recognized on dif­ ferent plant species (hunt et al., 2011; gehlot et al., 2014). the effect of auxin on adventitious roots growth and development can be explained by its role in wound healing through the inhibition of iaa­oxi­ dase activity and the activation of the enzymatic antioxidant defense system, which helps to restore the redox balance and protect tissues from oxidative damage, particularly during the different steps of adventitious root development (rout, 2006). while in contact with the basal cell, the auxin pool may have an indirect influence by promoting activity of starch hydrolytic enzymes and enhancing the translocation speed of carbohydrates to the cuttings base (haissig, 1974; aminah et al., 1995). consequently, it supplies the cutting with the required energy for hastening cell differentiation of root primordia, growth and development via cell divisions and elongation (husen, 2008). auxin could also act through selective proteol­ ysis and cell­wall loosening (schopfer, 2001). hence, it regulates the organ growth and development, pro­ motes emergence of shoot buds, sprout length and consequently resulting in a better overall growth of the cuttings (schroeder and walker, 1990). in the present study, cuttings treated with distilled water without growth regulators did not show any response on rooting and sprouting. thus, it has been proved that the iba application is more effective than naa and iaa. in most cases, 3000 mgl­1 of iba was the most effective concentration for promoting sprouting, rooting and survival rates of all studied genotypes. higher efficiency of iba at inducing adventitious rooting may be explained by higher chemical stability against catabolism and inactivation by conjugation, nontoxic over a wide concentration range, low mobility and available over a longer peri­ od of time in the plant tissue (barrel et al., 2001; ludwig­muller, 2000; hartmann et al. , 2002). whereas, the higher concentration of iaa stimulates ethylene production in plant cells, which is known to inhibit root induction and elongation (mulkey et al., 1982). similar to our results, kesari et al. (2009) found a relatively poor rooting yield with iaa treated stem cuttings of pongamia pinnata in comparison to iba. the result shows that iba had a stronger effect on sprouting and rooting than naa, the reason may be that naa is very stable and more persistent than other auxins and remains present in the tissue in its free form (dunlap et al., 1986). thus, naa decreases the level of nutrients mobi­ lization and translocation to the root primordia and blocks the roots outgrowth (husen and pal, 2006). for most genotypes, auxin responses are concentra­ tion dependent and tissues react in a distinct manner to varying amounts of exogenous auxins. thereby, adventitious root production decreased at very low concentrations of iba (<1000 mgl­1) and increased to levels between 1000 and 3000 mgl­1. however, with the increase in hormone concentration levels (>3000 mgl­1), the number and length of roots decreased. in accord to our results, akakpo et al. (2014) showed a decline in rooting rates of vitellaria paradoxa stem cuttings for iba concentrations exceeding 3000 mgl­1. this result confirms the findings of hartmann et al. (2002), who demonstrated that too little iba can decrease rooting and concentrations substantially higher than those normally found in plant tissues may be inhibitory, phytotoxic or even cause cell death. the results of our study revealed that iba con­ centrations and genotype strongly influence the sprouting and rooting ability of a. spinosa, which confirms previous studies indicating that the optimal iba concentrations for suitable adventitious root responses vary according to different species. indeed, singh and rawat (2017) achieved a maximum increase in sprouting and rooting on semi­hardwood cuttings of zanthoxylum armatum treated with 3000 mgl­1 iba. besides, tsipouridis et al. (2003) observed a maximum rooting rate in prunus persica treated with 2000 mgl­1 iba. husen (2008) reported that cut­ tings of dalbergia sissoo treated with 2000 mgl­1 iba induced a stronger rooting system. also, in aesculus indica, the highest rooting rate was recorded in stem adv. hort. sci., 2019 33(4): 519­527 526 cuttings treated with 4000 mgl­1 iba (majeed et al., 2009). the maximum rooting rate for tectona gran‐ dis was obtained with 4000 mgl­1 iba as compared to the other treatments (husen and pal, 2007). however, in pongamia pinnata, iba at 1000 mgl­1 was found to be the most effective concentration for rooting ratio and root number (kesari et al., 2009). the results of this study revealed that adventi­ tious root development and shoot growth of argania spinosa semi­hardwood cuttings were significantly influenced by auxin type, concentration, genotype and their interactions. there was a variation among the different genotypes studied, indeed cuttings taken from asoc1 and asoc4 were rooted and sprouted better than those from asoc2 and asoc3. it was observed that almost all the treatments, except the control set, were able to induce sprouting and rooting in cuttings, and the iba application is more effective than naa and iaa. iba application at a concentration of 3000 mgl­1 to asoc1 cuttings seemed to be the best treatment in terms of sprout­ ing, rooting and survival rates, and also for the num­ ber of leaves, leaf size, number of sprouts, sprout length, number of roots, and root length per cuttings. the present study indicated that a. spinosa can be propagated through semi­hardwood cuttings of élite trees and can be usefully applied to promote the role of vegetative propagation of this species through for­ est restoration and for breeding programs. thus, fur­ ther experiments should be conducted to extend the techniques to induce rooting of cuttings from select­ ed élite genotypes used as a source material for argan tree orchards. acknowledgements this work is supported by public funds from the ministry of agriculture through the national institute for agronomic research inra (institut national de la recherche agronomique), morocco. references akakpo d.b., amissah n., yeboah j., blay e., 2014 ­ effect of indole 3‐butyric acid and media type on adven‐ titious root formation in sheanut tree (vitellaria para­ doxa c.f. gaertn.) stem cuttings. ­ am. j. plant sci., 5(3): 313­318. alouani m., bani­aameur f., 2004 ­ argan (argania spi­ nosa (l.) skeels) seed germination under nursery condi‐ tions: effect of cold storage, gibberellic acid and moth‐ er‐tree genotype. ­ ann. for. sci., 61(2): 191­194. aminah h., dick j.m., leakey r.r.b., grace j., smith r.i., 1995 ­ effect of indole butyric acid (iba) on stem cut‐ tings of shoreale prosula. ­ forest ecol. manag., 72(2­ 3): 199­206. barrel b., le clere s., magidin m., zolman b.k., 2001 ­ inputs to the active indole‐3‐acetic acid pool: de novo synthesis, conjugate hydrolysis, and indole‐3‐butyric acid β‐oxidation. ­ j. plant growth regul., 20: 198­216. benbya a., mdarhri alaoui m.m., gaboun f., delpor­ te f., cherkaoui s., 2018 ­ vegetative propagation of argania spinosa (l.) skeels cuttings: effects of nutrient solution. ­ int. j. environ. agric. biotechnol., 3(4): 1369­ 1381. davies p.j., 2010 ­ the plant hormones: their nature, occurrence, and functions, pp. 1­15. ­ in: davies p.j. 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(amaryllidaceae) and stored at -20°c. pollen culture was performed using 1% (w/v) agar, medium that contained 0.01% (w/v) cacl 2 , 0.01% (w/v) h 3 bo 3 , 0.0007% (w/v) kh 2 po 4 , 10% (w/v) sucrose, and 0.01% (w/v) yeast extract at ph 5.8 (hirano and hoshino, 2009, 2010). the culture medium was sterilized by autoclaving at 121°c for 15 min. effects of different wavelengths of led light on pollen germination and direction of pollen tube elongation in cyrtanthus mackenii y. hoyo*, k. fujiwara**, y. hoshino*(1) * field science center for northern biosphere, hokkaido university, kita 11, nishi 10, kita-ku, sapporo 060-0811, japan. ** graduate school of agricultural and life sciences, the university of tokyo, 1-1-1 yayoi, bunkyo, tokyo 113-8657, japan. key words: cyrtanthus mackenii, direction of pollen tube elongation, germination rate, light emitting diode, light wavelength. abstract: the effects of different light wavelengths on pollen germination and determination of the direction of pollen tube elongation of cyrtanthus mackenii was examined in vitro using light-emitting diodes (led) with five peak wavelengths: 405 nm (violet), 465 nm (blue), 630 nm (orange), 660 nm (red), and 735 nm (far-red). pollen grains were cultured on a medium solidified with agar, and maintained at 21°c in an incubator under 3-4 h of continuous lighting or darkness. neither the pollen germinated under led lighting and dark conditions nor the rate of pollen germination differed among light conditions, including darkness. however, pollen tubes elongated less in the direction toward light under led, and elongated with no directional trend in darkness. moreover, pollen tubes did not elongate toward far-red leds. these results suggest that light exerts effects on the factors determining the direction of pollen tube elongation, but not on those controlling pollen germination. adv. hort. sci., 2014 28(4): 190-194 (1) corresponding author: hoshino@fsc.hokudai.ac.jp received for publication 17 september 2014 accepted for publication 3 october 2014 191 the effect of light condition on pollen germination rate five spots of culture medium with a diameter of 1 cm were put on a glass slide at ca. 5 mm intervals. a tiny amount of frozen pollen grains were sown on each coagulated spot of medium using a paintbrush. two or three prepared slides were horizontally positioned in a petri dish, which was kept at high humidity (fig. 1a), with toothpicks between the slides to avoid overlapping. samples were then cultured at 21°c in an incubator (biotron lph200, nk system, japan) for 3-4 h under different light conditions. the incubator was divided into five rooms fitted with overhead led panels (40×10 cm) fabricated using 2,800 indicator-type leds of 3 mm diameter (fujiwara et al., 2011; yano and fujiwara, 2012). the leds were of five types: violet (l405r-36; epitex inc., kyoto, japan), blue (l460-36; epitex inc., kyoto, japan), orange-red (l630-36; epitex inc., kyoto, japan), red (srk3-3a80-le; toricon co., shimane, japan), and farred (l735-36au; epitex inc., kyoto, japan). each led emitted a specific peak wavelength (λ p ) of light: 405 nm, 460 nm, 630 nm, 660 nm, and 735 nm, respectively. the petri dishes were set 2.5 cm below the led panels. in addition to these treatments, lightproof petri dishes wrapped in aluminum foil were set in a room to confirm whether pollen germination was stimulated or suppressed under dark conditions. five replicated culture slides were prepared per treatment (fig. 1b). after culturing, all slides were observed using a microscope (axiovert 40 cfl, carl zeiss, germany), and germinated pollen grains were counted. germinated pollen grains with pollen tubes that were shorter than the pollen grain diameter were not counted. we repeated the experiment three times under the same conditions, and are referred to as g1, g2, and g3. the effects of light condition on the direction of pollen tube elongation cover glasses placed over spots of medium with a diameter of 1 cm were prepared. frozen pollen grains were sown on the spots of medium in the same manner as for the pollen germination experiment. five cover glasses were vertically set in a humid petri dish using a paper stand to prevent the glasses from sticking. the petri dishes were wrapped in aluminum foil with a slit (8 cm × 0.5 cm) on the upper side for led light irradiation, and were then cultured at 21°c in the previously described incubator for 3-4 h. to verify the effects of light intensity on pollen tube elongation, two petri dishes were prepared for each light condition: one was placed 3 cm below the led panel (strong light) and the other was placed 22 cm below (weak light). after culturing, the direction of pollen elongation was observed using the previously described microscope. the direction of pollen tube elongation was determined as follows: 1. elongation of the pollen tube tip within a 60° arc centered on the line connecting the led panel, pollen grain, and floor, and widening in a direction toward the led light, was defined as “light” (under led lighting conditions) or “upward” (under dark conditions) (fig. 1c); 2. “dark” or “downward” defined the occurrence of pollen tube elongation in the opposite direction of the light or upward response; 3. pollen tube elongation toward a direction that was neither light/upward nor dark/downward was defined as “vertical.” 4. this experiment was also repeated three times under the same experimental settings and is referred to as d1, d2, and d3. statistical analysis all statistical analyses were conducted with the statistical package r (v.3.0.2) (r foundation for statistical computing, 2014). a generalized linear mixed-effects model (glmm) with an offset term was used to investigate determinants on the number of germinated pollen grains, assuming poisson distribution. the light condition, triple experiment design, and subsequent interactions were explanatory variables in the model, and an individual spot of medium was a random factor that considered effects seen on an individual spot compared to other spots on the same slide. the total number of pollen grains per medium was fig. 1 diagrammatic representation of the experimental systems and germinated pollen. a, petri dish set in prepared slides for pollen germination experiment. b, petri dishes set in cover glasses and then wrapped in aluminum foil with a slit on the upper side for pollen tube elongation experiment. c, criterion for determining of the direction of pollen tube elongation. 192 an offset term. the akaike information criterion (aic) was used to select the best models of the glmms. to estimate the effects of light intensity and light wavelengths on the direction of pollen tube elongation, multinomial logistic regression (venables and ripley, 2002) was performed using the r package’s vector generalized linear and additive model (vgam). the direction of pollen tube elongation was considered a categorical response variable (light/upward, vertical, dark/downward). the explanatory variables were distance from led panel (long or short); wavelength (405, 460, 630, 660, or 735 nm); and triple experiment design in the case of led lighting conditions. in the case of analysis for dark conditions, the experiment was the explanatory variable. 3. results the effect of light condition on pollen germination rate the total number of c. mackenii pollen grains sown on spots of medium were 9 017 (g1), 6 769 (g2), and 4 257 (g3). the pollen germinated under both led lightning and dark conditions (fig. 2). the mean and standard deviation of germination rate under each light condition were 0.30±0.015 at 405 nm, 0.38±0.019 at 465 nm, 0.28±0.018 at 630 nm, 0.34±0.020 at 660 nm, and 0.31±0.023 at 735 nm peak wavelength leds, and 0.37±0.018 in darkness. the germination rate varied among the three experiments, but did not differ among the five types of led and darkness (table 1). the effect of light condition on the direction of pollen tube elongation the total number of pollen tubes elongated in the three defined directions as detailed in fig. 1c for the three experiments was 1 410 for light, 2 994 for vertical, and 1 223 for dark under led lightning, and 187 for upward, 421 for vertical, and 161 for downward in darkness (fig. 3). under led lightning, the probability of pollen tube elongation classified as light or vertical was higher than that classified as dark (table 2). however, under darkness, the probability of pollen tube elongation classified as upward was equivalent to that of downward, although the vertical classification was higher. variability among the three experiments was not found in light and upward pollen tube elongation, but was found in vertical elongation. there was no effect of the distance from led panel on pollen tube elongation. under lighting from 735 nm peak wavelength leds, the number of light pollen tube elongations was lower than for dark pollen tube elongations. 4. discussion and conclusions in this study, light neither inhibited nor promoted cyrtanthus pollen germination, and the different wavelengths table 1 parameters estimated by glmm to predict number of geminated pollen in different light conditions for three times of germination experiment. the light condition was discarded explanatory variable by aic coefficient z score intercept -1.19 -15.98 *** experiment g2 -0.37 -3.40 *** experiment g3 0.33 3.06 ** significance is determined by z scores. ***: significantly different from g1 at p < 0.001 and ** p < 0.01. fig. 2 relationships between light condition and each germination experiment and germination rate of pollen. germinated pollen per total sown pollen on each medium spot under each irradiated leds and darkness: 405 nm (squares), 465 nm (circles), 630 nm (triangles), 660 nm (diamonds), 735 nm (upside-down triangles) peak wavelength of the led, and darkness (small circles). three lines show the estimated mean number of germinated pollen for three experiments by glmm. results of glmm are shown in table 1. fig. 3 rates of pollen tube elongation in three directions for each irradiated leds for the two distances (3 and 22 cm) from led panels and for shading. the three directions are expressed as follows: toward light or upward (light or upward, grey culms), away from light or downward (dark or downward, black culms), and in a direction that is neither light/upward nor dark/downward (vertical, white culms). the rates are calculated using sum of the number of pollen tubes in five replications of all experiments. the width of culm shows the number of pollen tubes. 193 table 2 coefficients estimated by multinomial logistic regression models that predict probability of pollen tube elongation toward to the light/ upward and vertical directions based on dark/downward direction in short distance from 405 nm peak wavelength led panel in d1 leds irradiation darkness direction light vertical upward vertical intercept 0.23* 0.72*** -0.02 n.s 0.59 ** experiment d2 0.05 n.s 0.30*** 0.19 n.s 0.42 n.s d3 0.03 n.s 0.11 n.s 0.27 n.s 0.54 * distance long -0.007 n.s -0.02 n.s wavelength 465 nm -0.09 n.s 0.14 n.s 630 nm -0.05 n.s 0.08 n.s 660 nm -0.10 n.s 0.03 n.s 735 nm -0.37** 0.10 n.s significant effects of distance and type of led lights on the direction of pollen tube elongation are determined by z scores. ***: significantly different at p < 0.001, **p < 0.01, *p < 0.05 and ns: non-significant. of led light also did not affect the pollen germination rate. however, pollen tubes were less likely to elongate toward the opposite direction of light, although they elongated with no directional trend in darkness. these results suggest that light has some effect on the factors determining the direction of pollen tube elongation, but not on the factors controlling pollen germination. the growth direction of a pollen tube is continuously reoriented by external signals and physical obstacles (cheung and wu, 2008). without any chemotropic attractants or physical directional control caused by configuration of female tissue or gametophytes, pollen tubes elongated randomly in vitro (horade et al., 2013). however, the pollen tubes did not show negative phototropism under led lighting from only one direction in the present study. therefore, it can be assumed that there is an integrative mechanism that causes unequal elongation of the pollen tube tip, e.g. perception of the light environment (casal, 2013), transcription factors such as phytochrome interacting factors (pifs) (chen and chory, 2011), or hormones and growth-related genes such as for hypocotyl cells (braidwood et al., 2014). in a unique manner, the pollen tubes did not elongate toward light from 735 nm peak wavelength leds. this result was consistent with previous studies that examined the effect of different light wavelengths in combination with hormones on pollen tube growth in arachis hypogaea (chhabra and malik, 1978) and pinus roxburghii (dhawan and malik, 1981). considering the red:far-red ratio, phytochrome undergoes a conformational change from the active far-red form into the red-light-absorbing, active red form, and derepresses pif activity, resulting in an increase in cell elongation. if the phytochrome exists in pollen tube tips and causes local cell elongation at the point irradiated with far-red light, the phenomenon of pollen tube elongation away from light may be explained. in conclusion, our results demonstrate that the different wavelengths of led light affected the direction of pollen tube elongation, but not pollen germination, in an in vitro experimental system. further studies are necessary to clarify the influence of light wavelength on determination of the direction of pollen tube elongation. acknowledgements this study was supported by grants from a grant-inaid for scientific research from the ministry of education, culture, sports science and technology (mext), japan. references braidwood l., breuer c., sugimoto k., 2014 my body is a cage: mechanisms and modulation of plant cell growth. new phytologist, 201(2): 388-402. campbell a.w., griffin w.b., burritt d.j., conner a.j., 2001 the importance of light intensity for pollen tube growth and embryo survival in wheat × maize crosses. annals of botany, 87(4): 517-522. casal j.j., 2013 photoreceptor signaling networks in plant responses to shade. annual review of plant biology, 64: 403-427. cerny t.a., faust j.e., layne d.r., rajapakse n.c., 2003 influence of photoselective films and growing season on stem growth and flowering of six plant species. j. amer. soc. for hortic. sci., 128(4): 486-491. chen m., chory j., 2011 phytochrome signaling mechanisms and the control of plant development. trends in cell biology, 21(11): 664-671. cheung a.y., wu h.m., 2008 structural and signaling networks for the polar cell growth machinery in pollen tubes. annual review of plant biology, 59: 547-572. chhabra n., malik c.p., 1978 influence of spectral quality of light on pollen tube elongation in arachis hypogaea. annals of botany, 42(5): 1109-1117. dhawan a.k., malik c.p., 1981 effect of growth regulators and light on pollen germination and pollen tube growth in pinus roxburghii sarg. annals of botany, 47(2): 239-248. fujiwara k., yano a., eijima k., 2011 design and development of a plant-response experimental light-source 194 system with leds of five peak wavelengths. j. of light & visual environ., 35(2): 117-122. godo t., fujiwara k., guan k., miyoshi k., 2011 effects of wavelength of led-light on in vitro asymbiotic germination and seedling growth of bletilla ochracea schltr. 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118(3): 733-741. palmer j.m., short t.w., briggs w.r., 1993 correlation of blue light-induced phosphorylation to phototropism in zea mays l. plant physiology, 102(4): 1219-1225. r foundation for statistical computing, 2014 r: a language and environment for statistical computing. r development core team. r foundation for statistical computing vienna, austria. venables w.n., ripley b.d., 2002 modern applied statistics with s. fourth edition. springer, tokyo, japan, pp. 183-210. yano a., fujiwara k., 2012 plant lighting system with five wavelength-band light-emitting diodes providing photon flux density and mixing ratio control. plant methods, 8: 46. impaginato 293 adv. hort. sci., 2022 36(4): 293­301 doi: 10.36253/ahsc­13824 an analysis on the impacts of cryogenic freezing on raspberry quality o. gales, j. jones, n. swarts tasmanian institute of agriculture, university of tasmania, private bag 98, hobart, 7001 tasmania, australia. key words: berries, liquid nitrogen, rubus ideaus, shelf life, storage. abstract: counter­season supply of horticultural products is of increasing demand. consumers are demanding annual supply of raspberries, which has historically been challenging due to their seasonal summer supply and charac­ teristically high metabolism resulting in a short shelf life and limited period of availability. however, the development of freezing technologies for increasing the length of storage of raspberries offers an opportunity for continual supply of premium quality raspberries. we investigated berry quality after freezing whole fresh raspberries, comparing conventional freezing methods with a mod­ ern cryogenic freezing method over a period of six months. significant increases in total soluble solids, titratable acidity, hue and chroma were found when raspberries were frozen compared to fresh raspberries. no overall difference in berry quality was observed between freezing methods for any parameter assessed. when assessed at time intervals, total soluble solids, ph, titratable acidity, chroma and hue were consistent between freezing methods for all durations of time frozen. these findings provide decision support for producers and distributors pursuing a novel counter season supply chain. 1. introduction raspberries (rubus ideaus l.) are a perennial plant that produce a compound fruit composed of many drupelets (wang et al., 2009). raspberries grown for fresh production typically have a unique bright red colour with distinctive aroma and taste, as well as health benefits due to their antioxidant properties (liu et al., 2002). raspberries are a non­cli­ macteric fruit with a high metabolism, making them perishable with a very short shelf life (tezotto­uliana et al., 2014). the ability to maximise fruit quality whilst harvesting and during post­harvest is imperative to commercially viable raspberry production. the development and adoption of innovative technologies has allowed conventional agricultural systems to pursue increased efficiency, yield, quality, food safety, and sustainability with reduced costs (sunding and zilberman, 2001). in the raspberry industry, freezing technologies for post­harvest storage offer producers the ability to extend the market win­ dow whilst preserving or increasing fruit quality standards. additionally, freezing horticultural products allows access to diversified markets and (*) corresponding author: oliver.gales@seh.ox.ac.uk citation: gales o., jones j., swarts n., 2022 ­ an analy‐ sis on the impacts of cryogenic freezing on rasp‐ berry quality. ­ adv. hort. sci., 36(4): 293­301. copyright: © 2022 gales o., jones j., swarts n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 september 2022 accepted for publication 4 november 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13824 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(4): 293­301 294 supply chains, creating increased economic feasibility and prosperity (bora et al., 2021; safari et al., 2021). demand by consumers for out­of­season supply of produce has driven research into the impacts of dif­ ferent storage methods on fruit and berry quality parameters (boorse et al., 1998; gonzález et al., 2002; chassagne­berces et al., 2010; ceballos et al., 2012; alhamdan et al., 2018). the perishable charac­ teristics of raspberries caused by high rates of metab­ olism and limited seasonal production, makes counter season supply problematic for producers (adobati et al., 2015). despite extensive literature investigating variances in quality parameters for hor­ ticultural products post conventional and cryogenic (liquid nitrogen) freezing, there is currently a gap in research comparing the impacts of the two freezing methods on the quality of whole fresh raspberries (alhamdan et al., 2018; otero et al., 2000). some notable issues with freezing of berry fruit have been identified including ice crystal nucleation and subse­ quent cell membrane rupture and electrolyte leakage which compromises the eating quality of frozen fruit (silva et al., 2008). the mouth feel of frozen fruit is related to cell turgor pressure which is driven by each cell having a separate functional semi permeable membrane that splits when ice nucleation occurs, resulting in soft textures in comparison to crisp fresh fruit (grout et al., 1991). to reduce the damage to cell membranes by crystal nucleation during the freezing of berries, there have been several develop­ ments in freezing methodologies. liquid nitrogen (~­ 80°c) applied to each berry individually reduces the thermal gradient and creates a snap freeze process that reduces the loss of quality through cell mem­ brane damage caused by slower and higher tempera­ ture conventional freezing, resulting in a higher quali­ ty product (ceballos et al., 2012). studies have demonstrated that the loss of quality, particularly tis­ sue damage and subsequent texture and colour from freezing, is impacted by the rate of freezing. fruit frozen at lower temperatures (­40°c) have been shown to display better quality parameters than fruits frozen at higher temperatures (­14°c) (chassagne­berces et al., 2010). whilst the demand for frozen horticultural prod­ ucts has seen considerable investment into studies on the impacts of freezing on vegetables and fruits, there is comparatively limited evidence on the specif­ ic effects of freezing on raspberry quality to support industry investment. gonzález et al. (2002) found that berry variety, harvest timing and quality at har­ vest are critical factors influencing the impact freez­ ing has on raspberry fruit quality. de ancos et al. (2000) showed an increase in anthocyanin content during freezing; however, this conclusion was deter­ mined to be a result of anthocyanin extraction effica­ cy after ice nucleation, rather than a biological increase in anthocyanin content. conversely, kampuse et al. (2001) highlighted a decrease in anthocyanins in frozen raspberries, with varietal dif­ ferences influencing the degree of anthocyanin depletion. cryogenic freezing technology has the potential to improve the quality of frozen berries. this study investigated if the application of freezing technolo­ gies can lead to improvements in raspberry fruit quality when frozen. specifically, using a traditional commercially grown variety and a new propriety vari­ ety, we asked ­ does freezing method (conventional and cryogenic) and freezing duration (2, 4, and 6 months) influence raspberry quality? the findings of this research are discussed in the context of industry pursuing continuous supply to a market that demands high quality and affordable produce throughout the year. 2. materials and methods experimental site the experimental site was located at the westerway raspberry farm (42° 47’ 26.34” s, 146° 47’ 26.34” e), an established 60­acre raspberry and mixed berry farm at westerway, in southern tasmania. the westerway raspberry farm is situated on an alluvial soil deposit adjacent to the tyenna river. the raspberry canes utilised for the experi­ ment were oriented in a ne sw direction and were trained in a commercial vertical trellis system. westerway receives an average annual rainfall of 764 mm (bom, 2019). the traditional and popular ‘willamette’ raspberry variety and a newly developed proprietary variety were investigated for the purpose of this experiment. the proprietary variety has been developed for the purpose of machine harvesting, with characteristics of diseases resistance, fruit firm­ ness and low release force from plant. sample collection harvest coincided with the commercial raspberry season on 27 december 2018. fruit was hand har­ vested from randomly selected rows within the block gales et al. ‐ cryogenic freezing on raspberry quality 295 with berries collected randomly along each row. consistent with commercial practice, samples were collected into individually labelled commercial plastic 400 g punnets and stored in a commercial cool room (7°c) on site for five hours until transportation. treatments and storage conditions for both raspberry varieties there were two dif­ ferent treatments (post­harvest storage technique and duration frozen) with five replicates for each treatment combination. treatments were estab­ lished as a fully factorial trial with two freezing tech­ niques (conventional and cryogenic) and three lengths of storage (2, 4 and 6 months) tested with two varieties of raspberries. punnets containing fruit were immediately frozen on site in either a commercial conventional ­8°c freezer (gunter, germany) or snap frozen in a liquid nitrogen (ln) tunnel at ­80°c (linde cryoline mt 5­ 600 quick­freezing (iqf) tunnel, munich, germany) depending on treatment. for ln tunnel freezing, the berries were tipped out of the punnets and passed through the ln tunnel individually on the inbuilt con­ veyor belt and collected into labelled punnets imme­ diately after freezing. all berries from both treat­ ments were then stored in the commercial ­8°c freezer for the duration of the storage period. during storage, all samples were stored in the commercial grade plastic punnets with lids and stacked into crates holding 50 punnets per crate, aligned with industry practice. frozen samples remained in the freezer for three prescribed periods of up to six months. laboratory analysis samples were transported from the farm freezing facilities back to the laboratory at the tasmanian institute of agriculture (university of tasmania) for analysis in an ice chilled eski. the samples were then stored in a cool room (4°c) during the laboratory analyses, which were completed within 48 hr after being removed from the freezer. the ph for every replicate was measured using a diluted sample in an auto­titrator (metrohm 702 sm titrino, herisau, switzerland). a subsample of 50 berries from each sample was juiced by squeezing berries through a microporous cloth (30­micron mesh filter cloth; allied filter fabrics p/n m0032, sydney, australia). a sample of the juice (5 ml) was pipetted (brantech 10 ml transfer pipette s, san francisco, america) into a 25 ml beaker and com­ bined with 15 ml of distilled water. the solution was analysed for initial ph by electrode probes used in the auto­titration. the same solution used for ph was analysed for titratable acidity by the metrohmauto­ titrator (702 sm titrino). the solution was mixed using a magnetic stirrer and combined with 1m sodi­ um hydroxide to determine the titratable acidity (titration to ph = 8.2). berry titratable acidity was expressed in g/l of citric acid. total soluble solids (tss) was determined using a portion of the juice (~3 ml) and a digital refractometer (atago pocket refractometer pal­1). the sample of juice was analysed immediately after juicing. using a hand dis­ posable plastic pipette, the sample was placed into the measuring chamber and analysed with the tss figure (%) recorded manually. the chamber was cleaned between each sample using paper towels and distilled water to remove any residue. the total anthocyanin content (mg cyanidin­3­glu­ coside equivalents/g berry fresh weight) was deter­ mined using the ph differential method (lee et al., 2005). subsamples (30 berries) were homogenised for 30 seconds at 7000 rpm using a retsch grindomix gm200 (haan, germany). homogenised subsamples of 10 g were weighed into 50 ml centrifuge tubes and combined with 40 ml of acidified 70 % methanol [700 ml methanol, 300 ml distilled h2o, 0.1 ml con­ centrated hcl (0.01%v/v)]. the tubes were then placed into an ultrasonic bath (grant xuv digital ultrasonic bath (royston, uk) in darkness for 30 min­ utes at 10°c. the tubes were then dried and placed into a hettich benchtop centrifuge (universal 320 r model tuttlingen, germany) and centrifuged for 10 minutes at 1520 g and 4°c. a 0.5 ml sample of the clear supernatant solution was then mixed with each of the buffers; buffer 1 solution being 0.025 m potas­ sium chloride and buffer 2 being 0.4 m sodium acetate. the dilution used was a 1:10 dilution (0.5 ml supernatant to 4.5 ml of buffer). the solutions were then left to equilibrate for 30 minutes. using a benchtop spectrophotometer (thermo scientific genesys 10s uv­vis, waltham, america), the absorp­ tion of each solution was measured at 530 nm and 700 nm. this allowed the absorbance of the diluted sample to be calculated using the following equation (lee et al., 2005): absorbance = (a530 ­ a700) ph1 ­ (a530 ­ a700) ph 4.5 where a530 is the spectral wavelength absorption measurement at 530 nm, and a700 is the spectral wavelength absorption at 700 nm. adv. hort. sci., 2022 36(4): 293­301 296 using the calculated absorbance value, the monomeric anthocyanin content was calculated using the equation (lee et al., 2005): monomeric anthocyanin pigment (mg/l) = (a x mw x df x 1000)/ (e x 1) where a = absorbance, mw = 449.2 g/mol for cyani­ din­3­glucoside, df = dilution factor determined (1 in 10 dilution), 1000 = factor for conversion from g to mg, e = 26,900 molar extinction coefficient, in l x mol­1 x cm­1, for cyd­3­glu and 1 = path length in cm. max used e = 30200. the final value from the monomeric anthocyanin pigment (mg/l) was converted to the commonly expressed mg per 100 g fresh weight unit. this con­ version was completed by knowing the total amount of anthocyanin in a litre and the initial fresh mass of raspberry sample used. anthocyanin (mg per 10 g fresh weight) = (0.04 x anthocyanin mg/l value) x 100 g/fresh weigh (g) where 0.04 = proportion of 40 ml of a litre, 100 g = final unit, anthocyanin mg/l value = answer from monomeric equation and fresh weight = 10 g weighed initially using the homogenised samples, homogenate colour was measured using a colourimeter (konica minolta chroma meter cr­400, new york, usa) (edgley et al., 2019). spectrophotometer tubes were filled with homogenate from each sample and placed in the colourimeter. the values for l*, a* and b* were recorded manually and gave a three­dimensional colour space with each value interpreted as: l* a measure from opaque (0) to completely black (100), a positive a* indicates redness whilst negative a* indicates greenness and a positive b* indicates yel­ lowness whilst negative b* indicates blueness on the hue­circle (voss, 1992; gonçalves et al., 2007). chroma was obtained by the formula: chroma = (a*2 + b*2)2 and hue from the formula: hue= arctg b*/a* (gonçalves et al., 2007). statistical analysis analysis of variance and statistical significance of quality parameters (tss, ph, titratable acidity, antho­ cyanins, hue and chroma) between treatment inter­ actions and treatment main effects were analysed using the 2019 ibm spss statistics package. analysis of mean variance was determined using univariate linear models for each differing continuous variable. post hoc tests were completed using tukey’s range test of statistical significance. 3. results there were no three­way interactions between variety, freezing method and length of duration frozen for all quality parameters assessed. however, there were two­way interactions for freezing method and variety, duration frozen and variety, and dura­ tion frozen and freezing method for a range of the quality parameters assessed. freezing method and variety raspberries of both proprietary and ‘willamette’ varieties had significantly (p<0.05) increased tss val­ ues after freezing, regardless of method, in compari­ son to their fresh tss values (fig. 1a). conventionally frozen proprietary berries had significantly (p<0.05) lower ph values in comparison to fresh, however they were not significantly (p>0.05) different to cryo­ genically frozen proprietary berries (fig. 1b). cryogenically frozen ‘willamette’ berries and frozen fig. 1 ­ tss (a), ph (b), titratable acidity (c), anthocyanin content (d), hue (e) and chroma (f) values for fresh and frozen propriety (grey bars) and ‘willamette’ raspberries (black bars). errors bars denote two times standard error. letters above bars indicate significant differences from the mean at p<0.05. gales et al. ‐ cryogenic freezing on raspberry quality 297 proprietary berries had statistically similar ph values in comparison to their respective fresh berries (fig. 1b.) freezing both ‘willamette’ and proprietary rasp­ berries significantly increased (p<0.05) titratable acidity (g/l citric acid), whilst the method of freezing made no significant (p>0.05) difference for either variety (fig. 1c). anthocyanin content of berries of both varieties were the same whether fresh or frozen, regardless of freezing method (fig. 1d). freezing both ‘willamette’ and the proprietary vari­ ety significantly increased (p<0.05) both the hue and chroma values for berry colour, whilst the method of freezing did not have a significant impact on each variety individually (fig. 1e and 1f). proprietary rasp­ berries exposed to cryogenic freezing however had significantly greater (p<0.05) hue and chroma values than ‘willamette’ exposed to cryogenic and conven­ tional freezing (figs. 1e and 1f). duration frozen and variety the tss (%) for the proprietary raspberry variety was not significantly influenced (p>0.05) within the first two months of freezing, however tss increased significantly (p<0.05) after four and six months frozen compared to the fresh samples (fig. 2a). the ‘willamette’ variety had significantly increased (p<0.05) tss two and six months after freezing, how­ ever tss for the four­month frozen sample was not significantly different (p>0.05) from the fresh sam­ ples (fig. 2). once frozen, there was no significant dif­ ference (p>0.05) in tss for berries frozen for two, four or six months for either variety. the ‘willamette’ variety had no significant (p>0.05) change in ph for the duration frozen. ph of the proprietary variety berries continuously declined at each assessment date but was only significantly lower after being frozen for six months when com­ pared to fresh berries (fig. 2b). ta (g/l citric acid) of both the ‘willamette’ and proprietary variety significantly (p<0.05) increased once frozen but did not significantly change through the duration of freezing (two, four and six months) (fig. 2c). the change in titratable acidity to tss ratio was statistically similar across duration frozen. the colour (hue units) of both ‘willamette’ and the proprietary variety raspberries also significantly (p<0.05) increased when frozen (fig. 2d). once frozen, hue of the proprietary variety did not change for the duration of frozen storage, whilst hue for the ‘willamette’ berries significantly (p<0.05) reduced at the six­month assessment compared to the second month assessments (fig. 2d). duration frozen and freezing method berries that were initially cryogenically frozen had significantly (p<0.05) higher tss values at four months compared to fresh raspberries, but no signifi­ cant (p>0.05) difference at six months in comparison to fresh berries (fig. 3a). for berries that were initial­ ly frozen in a conventional freezer, the two­ and four­ month frozen treatments were not significantly dif­ ferent from fresh raspberries in tss, but at six months there was significantly (p<0.05) greater tss fig. 2 ­ brix (a), ph (b), titratable acidity (c) and hue (d) for both the proprietary (black bars) and ‘willamette’ (grey bars) variety of raspberry after being frozen for up to six months. errors bars denote two time standard error. letters above bars indicate significance differences from the mean at p<0.05 of time frozen values. fig. 3 ­ tss (%) (a), ph (b), titratable acidity (c) and hue (d) for both the conventional (grey bars) and cryogenic (black bars) methods of freezing raspberries. error bars denote two times standard error. letters above bars indicate significance differences from the mean at p<0.05. 298 adv. hort. sci., 2022 36(4): 293­301 compared to fresh berries (fig. 3a). cryogenically frozen berries had no significant (p>0.05) differences in ph value for the duration frozen in comparison to fresh berries (fig. 3b). however, conventionally frozen berries at six months had significantly (p<0.05) lower ph compared to fresh raspberries. there was a significant increase in ta once frozen, but at each assessment interval (two, four and six months), there was no significant difference in freez­ ing method on the ta concentration (fig. 3). for both conventional and cryogenic freezing, there was no significant change in ta between two, four and six months frozen (fig. 3). for hue, there was no significant difference between freezing method at any single duration measured for the duration of the experiment (fig. 3). both freezing methods did exhibit a significant increase in hue colour units initially after freezing, increasing by ~25 % between 0 and two months frozen (fig. 3). 4. discussion and conclusions consumer demand for counter season supply of premium horticultural products is driving the need and innovation of storage techniques whilst main­ taining premium quality produce (kuchler and arnade, 2015; martindale and schiebel, 2017). the results of this study found significant differences in quality parameters for whole raspberries after they were frozen compared to fresh berries. measurements of total soluble solids, titratable acidi­ ty, hue and chroma all significantly increased when berries were frozen. this study found no difference however in berry quality once frozen between freez­ ing methods for any parameter assessed. total solu­ ble solids, ph, titratable acidity and hue were consis­ tent between freezing methods for all durations of time frozen. these results improve the understand­ ing of the impacts of post­harvest storage on rasp­ berry quality and provides critical information and decision support for producers to ensure optimal raspberry quality for consumers. raspberries are no exception in the unrelenting demand from consumers for premium quality counter season supply of horticultural products, however, the metabolic characteristics that result in raspberries being highly perishable makes the long­ term storage of raspberries comparatively difficult (gonçalves et al., 2018). for both the ‘willamette’ and proprietary varieties, tss increased once frozen regardless of freezing method. this finding of initial tss increase after freezing is consistent with gonzález et al. (2002) who showed a similar initial increase in tss after freezing ‘heritage’, ‘autumn bliss’, ‘zeva’ and ‘rubi’ raspberries. the treatments of two, four­ and six­months freezing duration showed differences for tss between varieties when fresh, with the ‘willamette’ variety having greater than 20% higher tss than the proprietary variety. varietal difference as shown here is consistent with shamaila et al. (1993) who found differences for fresh berries for the quality parameters ph, tss, ta and anthocyanins between ‘chilcotin’, ‘chilliwack’, ‘meeker’, ‘skeena’ and ‘tulameen’ varieties. this sig­ nificant difference between varieties persisted for all freezing periods. however, once frozen, tss values did not significantly change for both varieties for the duration of the trial, and the freezing method, con­ ventional vs cryogenic, also had no impact on tss. this finding contrasts with the gonzález et al. (2002) study described earlier, which showed that tss con­ tinued to significantly increase during duration frozen. the findings of consistency in tss levels once frozen for the proprietary and ‘willamette’ variety suggest the quality of these varieties once frozen is more stable compared to varieties such as ‘heritage’, ‘zeva’ and ‘rubi’ used by gonzález et al. (2002). this finding is likely a result of differing tss profiles between varieties having differing stability when frozen, resulting in a variation in tss level changes. titratable acidity contributes to the mouth feel a consumer experiences when stimulated by the multi­ ple acids including citric, ascorbic and phosphoric acid found in raspberries (haffner et al., 2002; marsh et al., 2004). freezing is the most common form of long term storage for fresh produce, with the reduc­ tion in metabolism and respiration when frozen allowing raspberries to be stored for long periods of time (chaves and zaritzky, 2018). cryogenic freezing facilitates the rapid transition into the frozen state which reduces membrane damage (and therefore maintains raspberry quality) from slow water crystal­ lization and subsequent membrane damage that occurs during conventional freezing (cao et al., 2018). consistent with findings for changes in tss, there was a significant increase in titratable acidity for frozen berries compared to fresh berries for both varieties, but there was no significant difference resulting from freezing method for either variety. gales et al. ‐ cryogenic freezing on raspberry quality 299 titratable acidity significantly increased when initially frozen but did not change with length of duration frozen. these results are consistent with findings of increased total and conjugated acid in frozen rasp­ berries by mullen et al. (2002). however, de ancos et al. (2000) who quantified ellagic acid concentrations, found a significant decrease (14­21%) in ellagic acid content when raspberries were frozen, highlighting that the increase in titratable acidity in this investiga­ tion may be due to increase in other acids found in raspberries. raspberry quality is derived from multiple para­ meters with each contributing to the consumer’s sen­ sory experience and as such, the ratio of tss with ta is a major contributor to consumer experience. despite possible variances of specific acids during freezing, it is the ratio of sugar to acid that influences the consumer’s tasting experience (shamaila et al., 1993). the increase in sugar when frozen is propor­ tional to the increase in acidity and showed no signif­ icant difference between freezing methods and dura­ tion, and it is this consistency in ratio that is critical to consumer experiences. the consistency in the ratio between tss and titratable acidity whilst frozen is also broadly consistent with research by gonzález et al. (2002) who used different raspberry varieties and durations frozen. no physiological mechanism or chemical changes induced by freezing/thawing has been described for the increase in titratable acidity post­harvest for raspberries. the measurement of increased titratable acidity in this study may be a physiological response to storage or an increase in extraction efficiency post storage, and until further investigation quantifies the reason for the change, the implications cannot be fully understood. in straw­ berries, retention of quality parameters including acids, colour and anthocyanin content was associat­ ed with the thawing process, where rapid thawing was shown to be more favourable (holzwarth et al., 2012). the impact of freezing method and duration on the colour of raspberries is important for influencing consumer demand. a colour that is perceived to be preferable by the consumer increases both the accep­ tance and perception of other quality parameters such as taste and texture (clydesdale, 1993). both varieties had similar hue and chroma values when fresh, however once frozen, colour values increased significantly. when frozen conventionally, both vari­ eties had similar coloured fruit; however, when frozen cryogenically, the proprietary variety had significantly higher hue and chroma unit values than ‘willamette’ raspberries (fig. 1). this was the only quality parame­ ter analysed that showed similarities between vari­ eties for fresh berries but when frozen showed a sig­ nificant difference between varieties. this highlights a difference in the impacts of freezing methods between the varieties and the subsequent impact on colour. the contrasting influence of freezing between varieties is consistent with findings by other researchers. gonzález et al. (2002), for example, showed that for the varieties ‘heritage’ and ‘autumn bliss’, chroma and hue was not different when fresh, but after six and nine months of being frozen, hue and chroma values differed between varieties. the significant change in hue and chroma between fresh and frozen berries was not accompa­ nied by a change in anthocyanin levels. this is incon­ sistent with findings by han et al. (2004), who attrib­ uted the darker colour of frozen raspberries to anthocyanin synthesis. however, for strawberries, holcroft and kader (1999) showed that ph affects the colour expression of anthocyanins, concluding that the red flavylium cation only remains stable in acidic conditions, and strawberries lose their red colour (become pale) when the ph increases. therefore, if the anthocyanin stability and subsequent redness of raspberries is associated with the amount of acids present (acidity), the increase in titratable acidity observed in this study, may be associated with increasing the amount of stable red flavylium cations, causing the raspberries to be darker when frozen. this investigation used commercial standards of raspberry quality as a benchmark to compare innova­ tive freezing methods for post­harvest management of raspberry fruit. in this investigation, consistent quantitative results for the quality parameters, benchmarking the commercial standard, provides support for the application of new technology in pro­ ducing a consistent high­quality product. however, the commercial implications of a non­consistent (sig­ nificantly different) result are not known, as it may be of better or worse quality in the perception of the consumer. therefore, results that highlight significant (p<0.05) differences present opportunities for further studies and evaluations of quality as well as potential for changing practices. the results of this investigation broadly demon­ strated consistent quality parameters between con­ ventional freezing and the modern, innovative and comparatively expensive technique of cryogenic freezing. we suggest that based on these findings, adv. hort. sci., 2022 36(4): 293­301 300 there is evidence­based support for the commercial retention of the cheaper conventional freezing over cryogenic freezing for post­harvest storage of rasp­ berries. the study has provided critical decision sup­ port for post­harvest management of commercial raspberry production. this is particularly important for the uptake of horticultural technology that aims to access new markets and innovate logistics in sup­ ply chains. the findings of this study provide the framework and basis for the uptake and refinement of innovative technologies used in the raspberry industry, and allows freezing method choices to be evidence­based. the changes in titratable acidity dur­ ing the freezing duration trial is unexplained here nor any published literature to date. determining whether a physiological increase of titratable acidity occurs or if an increase in extraction efficiency of titratable acidity is the reason behind the results of this study would provide additional insights into the impacts of freezing on raspberries. the quantitative results of the current study also provide impetus for a sensory analysis to investigate consumer percep­ tions in parallel with the quantitative laboratory evi­ dence provided in this study. acknowledgements this research did not receive any specific grant from funding agencies in the public, commercial, or not­for­profit sectors. the assistance of professor dugald close, dr max edgeley and ms caroline claye from the university of tasmania was greatly appreci­ ated. the authors greatly appreciated the support of the westerway raspberry farm (tasmania) for this research. references adobati a., limbo s., uboldi e., piergiovanni l., 2015 ­ active packaging in master bag solutions and shelf life extension of red raspberries (rubus ideus l.): a reliable strategy to reduce food loss. ­ italian j. food sci., 1: 107­110. alhamdan a., hassan b., alkahtani h., abdelkarim d., younis m., 2018 ­ cryogenic freezing of fresh date fruits for quality preservation during frozen storage. ­ j. saudi soc. agric. sci., 17: 9­16. bom, 2019 ­ daily rainfall ‐ westerway (leesons road) ­ bureau of meterology, australian government, http://www.bom.gov.au/jsp/ncc/cdio/weatherdata/av ?p_nccobscode=136&p_display_type=dailydatafile&p _startyear=&p_c=&p_stn_num=095075. boorse g.c., bosma t.l., meyer a.c., ewers f.w., davis s.d., 1998 ­ comparative methods of estimating freezing temperatures and freezing injury in leaves of chaparral shrubs. ­ int. j. plant sci., 159: 513­521. bora j., tongbram t., yaseen m., malik m., hanan e., 2021 ­ characterization of modern cold storage for hor‐ ticulture crops, pp. 1­26. ­ in: alam t. 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gutiérrez et al., 2021). some genera of malvaceae with ornamental potential are pavonia, lecanophora, (*) corresponding author: aguti@criba.edu.ar citation: gutierrez a., monzón p., micheletto s., marinangeli p., 2023 ­ reproductive biology of sphaeralcea species with ornamental interest. ­ adv. hort. sci., 37(4): 345­352. copyright: © 2023 gutierrez a., monzón p., micheletto s., marinangeli p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 august 2022 accepted for publication 30 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13616 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 345­352 346 modiolastrum, rynchosida and sphaeralcea (ponce et al., 2006; torres et al., 2008; masini and rovere, 2015; gutierrez et al., 2021). in argentina, the sphaerlacea genus have native and herbaceous species with attractive characteristics for ornamental cultivation, such as s. australis, s. crispa, s. mendoci‐ na and s. bonariensis (sriladda et al., 2012; gutierrez et al., 2021). they are adapted to semi­arid condi­ tions, and have tolerance to water stress, high and low temperatures and high insolation, which make them good candidates for breeding programs in urban ecosystems adapted to extreme weather con­ ditions and for sustainable landscaping. moreover, the use of native germplasm in breeding programs contributes to the conservation of biodiversity (masini and rovere, 2015). in general, native plants make more efficient use of environmental factors such as water and other climate factors, as well as edaphic and biological variables, which result in a lower maintenance demand and in a good perfor­ mance under the restrictive local conditions. knowledge of the plants reproductive biology is essential for classical breeding programs, because it allows better orientation and planning of the crosses. in the case of the species under study, their repro­ ductive biology is unknown. this knowledge is essen­ tial to be used in pollinations, and to increase the chance of successful fertilization. some of these aspects are pollen viability, stigma receptivity, polli­ nation type and combining ability since these depend on successful reproduction. pollen viability and stig­ ma receptivity are aspects that plays an important role in successful hybridizations (figueiredo et al., 2020). pollen viability is a measure of male fertility (liu et al., 2021), viable pollen is critical to the process of reproduction and pollen longevity can be affected by temperature and relative humidity (ren et al., 2019). stigma receptivity is the ability to receive the pollen, therefore it directly affects the plant life cycle, allowing the pollen to adhere, hydrate and germinate (shivanna and sawhney, 1997). a detailed knowledge of these features will determine the best moment for pollination, to enable successful controlled pollination in breeding programs. stigma receptivity is related to the activity of enzymes such as peroxidase, esterase and dehy­ drogenase (galen and plowright, 1987). receptive stigmas have high enzyme activity, which can occur in different phases of flower development. the obser­ vation of the activity of these enzymes can be used to characterize stigma receptivity (zhang et al., 2021). for the pollination process to occur, the trans­ fer of pollen to the stigma must happen during the period in which the stigma is receptive, otherwise, pollen cannot adhere and germinate. the aim of this study was to evaluate the repro­ ductive biology of four native species of the genus sphaeralcea, determining pollen viability, timing of stigma receptivity, pollination type and combining ability. the hypothesis we followed was that it is pos­ sible to determine aspects of the reproductive biolo­ gy of the native germplasm of sphaeralcea, with the ultimate objective to develop new ornamental vari­ eties. 2. materials and methods collection area the pampas region is an extensive plain located to the east of argentina between 31° and 39° south lati­ tude. aliaga et al. (2017) characterized the pampas general climate, considering rainfall, air temperature, humidity, and wind speed as well as the altitude and the alternation between dry and wet events in the area. based on these elements, they categorized the southwest of buenos aires and the southeast of la pampa as semi­arid region. sphaeralceae seeds were collected between december 2020 and march 2022 in different sites of this semi­arid region (fig. 1). the sites were characterized by the occurrence of long periods of drought and isolated floods together with windy periods which affect severely the water avail­ ability (aliaga et al., 2017). fig. 1 ­ geographic distribution and collection sites of studied populations of sphaeralcea australis, s. crispa, s. men‐ docina and s. bonariensis in central argentina, south america. for labels see the table. gutiérrez et al. ‐ reproductive biology of sphaeralcea species 347 plant material and experimental design the plant material was collected in the indicated area (fig. 1) as seeds and preserved at 4°c dry with silica gel. the seeds were germinated after pre­ger­ minative treatments to break dormancy (gutierrez et al., 2019). after scarification, the seeds were placed in petri dishes on filter paper moistened with distiller water in a germination chamber at 20°c (ista, 2019) with a 12 h photoperiod previously used in other malvaceae (erickson et al., 2016; leperlier et al., 2020). five plants of each sphaeralcea species were grown in pots with commercial substrate (growmix multipro®) in the greenhouse of the center of renewable natural resources from the semi­arid region (cerzos, conicet ­ uns), under controlled temperature (18­28°c), irrigation and relative humid­ ity (55­85%). during the day, when the flowers were in anthe­ sis, pollen viability and stigma receptivity were stud­ ied at 8:00 h, 10:00 h, 12:00 h in the morning and at 2:00 h, 4:00 h, and 6:00 h in the afternoon. the same experimental designs were used to eval­ uate pollen viability and stigma receptivity. four species (treatments), five plants per species (replica­ tions) and three flowers per plant, were used to eval­ uate a total of 60 anthers and 60 stigmas in a com­ pletely randomized experiment. stigma receptivity the osborn method was used to evaluate the stig­ ma receptivity, based on the reaction of the peroxi­ dase enzyme. the stigma is classified as receptive when placing a drop of hydrogen peroxide at 40% on the flowers stigmas a bubble production is observed (osborn et al., 1988). the reaction of stigma receptiv­ ity was examined with a stereomicroscope. pollen viability the estimation of viable pollen was carried out with the alexander technique (alexander, 1980). the grain dyed in an intense violet color was taken as viable and the one that was colored green was taken as non­viable. pollen grain counts were performed in an optical microscope of four random fields per preparation to estimate the percentage of pollen via­ bility (%) = [(number of viable pollen grains/number of total pollen grains) x 100]. mating system the experimental trial to test the reproductive system in sphaeralcea genus was based on four experiments and is seen in table 1. in the green­ house, the plants of experiments 2, 3, and 4 were iso­ lated with a fine mesh in a cage that excludes poten­ tial pollinators. the plants of experiment 1 were used as control and located outside the cage. for experiment 1, five plants of each species (s. australis, s. crispa, s. mendocina and s. bonariensis) were used and three flowers of each plant were marked the day before anthesis. the flowers were allowed to develop normally without any manipula­ tion, as control. the developed fruits were properly identified and covered until harvest. for experiment 2, five plants of each species were used, and three flowers of each plant were marked the day before anthesis. the fruits were properly identified and covered until fully developed and har­ vest. for experiment 3, five plants of each species were used, and three flowers of each plant were marked the day before anthesis. the flowers were allowed to develop normally and without any type of manipula­ tion to evaluate natural self­pollination. the devel­ oped fruit were properly identified and covered until harvest. for experiment 4, inter­specific reciprocal and intra­specific crossing were performed between s. australis, s. crispa, s. mendocina and s. bonariensis table 1 ­ experimental management on sphaeralcea australis, s. crispa, s. mendocina and s. bonariensis. normal seed set “+” and great­ ly reduced or zero seed set “­” (modified from simpson, 2019) experimental management seed production 1. flowers left to develop normally, as control. + fertile − infertile 2. isolated flowers, then self­pollinated by hand. + self­fertile − not self­fertile 3. flowering plants in caged, then left freely. + self­pollinating − not self­pollinating 4. isolated flowers, then emasculated and outcrossed. + outcrossing − not outcrossing adv. hort. sci., 2023 37(4): 345­352 348 (table 2). five plants of each species were used, and three flowers for each plant were marked on the day before anthesis. the flower buds were emasculated, the anthers were removed prior to pollen release and reciprocal outcrossing were made once a day during the flowering period. other flowers were used as male parent. after pollination, the flowers were properly identified and covered until fruit harvest. the fruiting and fertility results of the four experi­ ments were assessed by counting the seed set in relation to the number of pollinated flowers [fructification (%) = (number of fruits produced / number of pollinated flowers) x 100] and assessment the full seed in relation to the total seed [fertility (%) = (number of full seeds/total number of seeds (full + empty)) x 100]. a classification range based on fructi­ fication and fertility percentage was used, therefore 0 to 35% was considered low, 36 to 65% intermedi­ ate and 66 to 100% high. combining ability the full seeds of the intra­ and interspecific cross­ es of the previous experiments were subjected to mechanical scarification because native sphaeralcea species present physical dormancy in its seeds (gutierrez et al., 2019). scarified seeds were then germinated in a culture chamber. the seeds that ger­ minated were sown in seedling trays with commer­ cial growmix multipro® substrate and cultivated in the greenhouse under controlled light (shading net 50% of light extinction), temperature (18­28°c) and humidity (55­85%) conditions (early growth stage). the seedlings that developed three to four true leaves were transplanted into 7x7x9 cm pots with a substrate composed of 50% sandy soil, 35% peat, 10% perlite and 5% compost (advanced growth stage). survival of germinated seeds and seedlings from intraspecific offspring (siblings) and reciprocal interspecific offspring (hybrids) were evaluated to quantify combining ability at each stage of develop­ ment (early growth stage and advanced growth stage). a classification range was used for the per­ centages of germination and seedling survival as low (0 to 35%), intermediate (36 to 65%) and high (66 to 100%). 3. results stigma receptivity the four species of sphaeralcea had different behaviors in terms of stigma receptivity, reaching dif­ ferent maximum percentages and at different times of the day. s. bonariensis showed high values at 8:00 am and sustained over time until 2:00 pm when it was 100%. s. australis and s. crispa had similar behaviors with two high peaks of receptivity, at 8:00 am and the maximum at 2:00 pm (99% s. australis and 93% s. crispa). s. mendocina during the morning hours showed a different behavior from the rest, with very low stigma receptivity values with an exponential growth between 12:00 to 2:00 pm where it reached the highest percentage of receptivity (92%). after 2:00 pm, when all the species had their maximum peaks of stigma receptivity, the values began to decrease in different ways. for s. australis and s. bonariensis the decrease was marked, reaching val­ ues of 0% at 6:00 pm. for s. crispa and s. mendocina it was gradual until 6:00 pm, when receptivity was null (fig. 2). pollen viability the four species of sphaeralcea obtained high percentages of pollen viability, although these values varied throughout the day and between species. the highest values were recorded at 2:00 pm for s. aus‐ tralis (99%), s. bonariensis (99%) and s. crispa (98%), with no statistically significant differences between table 2 ­ combinations of interspecific reciprocal and intraspe­ cific crosses that originated the hybrid and sibling off­ spring, respectively female parent (♀) x male parent (♂) interspecific reciprocal crosses s. australis s. crispa s. australis s. mendocina s. australis s. bonariensis s. bonariensis s. crispa s. bonariensis s. australis s. bonariensis s. mendocina s. crispa s. australis s. crispa s. mendocina s. crispa s. bonariensis s. mendocina s. crispa s. mendocina s. australis s. mendocina s. bonariensis intraspecific crosses s. australis s. australis s. bonariensis s. bonariensis s. crispa s. crispa s. mendocina s. mendocina gutiérrez et al. ‐ reproductive biology of sphaeralcea species 349 them, and at 4:00 pm for s. mendocina (99%). at 12:00 pm the percentages were also high for all species and the lowest values were recorded at 6:00 pm for all species (fig. 3). mating system all combinations, inter and intraspecific crosses, managed to form fruits (table 3) except s. mendocina x s. bonariensis. in the case of self­pollinations, fruits were not observed. the species used as female parent produced dif­ ferences in the percentage of fruit production. the values were low when s. crispa was used as female (7 to 33%), intermediate when it was s. mendocina (47 to 53%) and high (67 to 100%) with s. australis and s. bonariensis. in the intraspecific crosses, the same pattern was repeated, showing low fruiting percent­ ages for s. crispa (13%), intermediate in s. mendocina (40%) and high in s. australis (67%) and s. bonariensis (87%). regarding fertility, most of the interspecific cross­ table 3 ­ number of pollinated flowers (npf), number of fruits produced (nfp), fructification percentage (fp), number of full seeds (nfs), number of empty seeds (nes) and seed fertility percentage (sfp) for intraspecific and interspecific reciprocal crosses between s. australis (sa), s. bonariensis (sb), s. crispa (sc), and s. mendocina (sm) fig. 2 ­ stigma receptivity percentage in sphaeralcea australis (sa), s. bonariensis (sb), s. crispa (sc) and s. mendocina (sm) flowers in function of time of the day. fig. 3 ­ pollen viability percentage in sphaeralcea australis (sa), s. bonariensis (sb), s. crispa (sc) and s. mendocina (sm) flowers at different times of the day. means with differ­ ent letters indicate significant differences with the fisher's lsd test, p<0.05. each vertical bar represents mean ± standard error. (♀ x ♂) npf nfp fp (%) nfs nes sfp (%) interspecific reciprocal crosses saxsb 15 15 100 277 43 83.1 saxsc 15 15 100 188 43 78.3 saxsm 15 10 66.7 90 68 55.7 sbxsm 15 8 53.3 18 115 4.5 sbxsc 15 15 100 367 43 89.5 sbxsa 15 10 66.7 179 14 90.2 scxsb 15 2 13.3 14 0 78.6 scxsm 15 1 6.7 1 9 10 scxsa 15 5 33.3 68 6 90.5 smxsc 15 7 46.7 30 8 78.9 smxsa 15 7 46.7 58 12 79.7 intraspecific crosses saxsa 15 10 66.7 121 74 62.1 sbxsxb 15 13 86.7 283 17 94.3 scxsc 15 2 13.3 21 9 70 smxsm 15 6 40 63 8 86.7 350 adv. hort. sci., 2023 37(4): 345­352 es yielded high percentages of full seed production with values between 78 to 91%. however, in the cross of s. australis x s. mendocina it was intermedi­ ate (56%) and it was low for s. bonariensis x s. men‐ docina (4%) and s. crispa x s. mendocina (10%). for intraspecific crosses, fertility was high for s. bonar‐ iensis (94%), s. mendocina (89%) and s. crispa (70%) and intermediate for s. australis. combining ability the germination percentage after mechanical scarification was high for all crosses except for s. aus‐ tralis x s. australis, which showed intermediate val­ ues (table 4). seedling survival decreased throughout development in all descendants, with high and inter­ mediate values predominating in the first growth stage (plant tray) and the majority being low in the advanced stage of development (pot). in the case of the hybrid, the offspring from s. australis x s. bonar‐ iensis showed the highest values of final survival (56%). in the intraspecific crosses, the highest values of descendant survival were from s. australis x s. aus‐ tralis (66%). the crosses that failed to develop live seedlings were s. bonariensis x s. 4. discussion and conclusions the methods used by osborn et al. (1988) and alexander (1980) to evaluate stigma receptivity and pollen viability, respectively, were effective to achieve successful crosses in the genus sphaeralcea. stigma receptivity is a highly variable trait among species of the plant kingdom. there are species such as carica papaya l. where the flowers are receptive before the floral opening and until the closing (parés et al., 2002), others such as passiflora edulis are receptive during anthesis until flowers closed (ángel coca et al. , 2011). our results for the genus sphaeralcea showed that s. australis, s. crispa and s. bonariensis had high stigma receptivity at flowers opening, except for s. mendocina, which obtained positive results after they opened, and the bubbling was null before flower closure (6:00 pm) for all species. ambient heat can serve to attract insects to an open flower through volatilization of floral scent during anthesis, and also helps to maintain a period of maximum stigma receptivity (consiglio and bourne, 2001). in our results, stigma receptivity was high between 12:00 pm and 2:00 pm which are coinci­ table 4 ­ number of full seeds (nfs), germination percentage (gp), number of seedlings in early growth stage (nsegs), survival percen­ tage of early growth stage (spegs), number of seedlings in advanced growth stage (nsags) and survival percentage of advan­ ced growth stage (spags) for intraspecific and interspecific reciprocal crosses between s. australis (sa), s. bonariensis (sb), s. crispa (sc), and s. mendocina (sm) (♀ x ♂) nfs gp (%) nsegs spegs (%) nsags spags (%) interspecific reciprocal crosses saxsb 277 88.8 181 73.6 102 56.4 saxsc 188 83.5 139 88.5 12 8.6 saxsm 90 100 90 100 35 38.9 sbxsm 18 100 9 50 0 sbxsc 367 80.5 186 67.4 70 37.6 sbxsa 179 100 77 43 7 9.1 scxsb 14 100 13 92.9 3 23.1 scxsm 1 100 0 0 ­ scxsa 68 69.1 30 63.8 0 smxsc 30 71.4 7 35 1 14.3 smxsa 58 92.6 20 40 0 intraspecific crosses saxsa 121 63.6 64 85.3 42 65.6 sbxsxb 283 82.3 120 51.5 47 39.2 scxsc 21 100 11 55 0 0 smxsm 63 73.8 42 93.3 6 14.3 gutiérrez et al. ‐ reproductive biology of sphaeralcea species 351 dent with the time of day when the maximum ambi­ ent temperatures were recorded, with an average of 30.4°c (national meteorological service, https://www.smn.gob.ar/). in some species high temperatures affect pollen viability (rao et al., 1992; radice et al., 2020; iovane et al., 2022). in sphaeralcea, there is still no evidence of how environmental factors affect pollen viability, but our results are indirect evidence that pollen would not be affected by high summer temperatures. one possible explanation for these results is that they are native species adapted to local climate con­ ditions and therefore high temperatures do not gen­ erate the thermal stress that affects viability during pollen development or in its mature state. the results of this research indicate that the pollinations that take place between 12:00 pm and 2:00 pm have a greater probability of generating fruits and seeds, since it is when most of the open flowers are recep­ tive, and the viability of pollen is optimal. these species demonstrated to be self­incompati­ ble and allogamous, with different degrees of repro­ ductive compatibility and combining ability between them. the s. mendocina x s. bonariensis cross pro­ duced aborted fruits and were not able to produce offspring. the crosses s. bonariensis x s. mendocina, s. crispa x s. mendocina, s. mendocina x s. australis and s. crispa x s. crispa managed to produce viable seeds that germinated, but with no descendants since the seedlings were not fully develop. these effects are probably the product of reproductive incompatibility between the species since they have different chromosome numbers, s. mendocina is 2n = 30 and the rest of the sphaeralcea are 2n = 10 (krapovickas, 1949). it would be interesting to achieve offspring with the germplasm of s. mendoci‐ na since it has very attractive and particular orna­ mental features such as the color of the leaves with shades in the range of gray and pink flowers (gutiérrez et al., 2021). the null survival of the intraspecific crosses for s. crispa could be due to the rapid loss of vigor of the seeds, since at the time of germination they were smaller plants with a very weak appearance. except for s. bonariensis x s. mendocina and s. australis x s. mendocina crosses, which were not compatible due to chromosomal differences; the crosses with the best combining ability were those that had s. australis and s. bonariensis as maternal parent with good fruit production. regarding com­ bining ability, the crosses with the best survival off­ spring were s. australis x s. bonariensis and s. aus‐ tralis x s. australis. both produced the greatest quan­ tity and quality of descendant plants that prospered over time and had adequate growth and develop­ ment. these novel results will allow us to improve the pollination efficiency and to design a strategic plan for the ornamental improvement of sphaeralcea genus our study provides the first data on the reproduc­ tive biology and mating system of sphaeralceae genus belonging to four native species, which pro­ vides valuable information for the formulation and implementation of new approaches for genetic improvement programs. this facilitates the develop­ ment of new varieties of sphaeralcea hybrids with ornamental qualities. acknowledgements authors thank to national research council of argentina (conicet) and department of agronomy, national university of the south. this work was sup­ ported by the national university of the south (uns) [grant number peu 237/19]. references alexander m., 1980 ­ a versatile stain for pollen, fungi, yeast and bacteria. ­ stain technol., 55: 13­18. aliaga v., ferrelli f., piccolo c., 2017 ­ regionalization of climate over the argentine pampas. ­ int. j. climatol., 37: 1237­1247. ángel coca c., nates g., ospina r., melo c., amaya m., 2011 ­ biología floral y reproductiva de la gulupa passiflora edulis sims f. edulis. ­ caldasia, 33: 433­451. consiglio t., bourne g., 2001 ­ pollination and breeding system of a neotropical palm astrocaryum vulgare in guyana: a test of the predictability of syndromes. ­ j. trop. ecol., 17: 577­592. erickson t., merritt d., turner s., 2016 ­ overcoming physical seed dormancy in priority native species for use in arid‐zone restoration programs. ­ austral. j. bot., 64: 401­416. figueiredo m., passos a., hughes f., santos k., dos silva a., soares t. 2020 ­ reproductive biology of physalis angulata l. 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gramaje et al., 2018). underperforming vines have been found to be affected by trunk and root diseases, disturbing physiological processes such as carbohydrate metabolism, defense responses, and photosynthetic rate (fontaine et al., 2015; akgül and ahioğlu, 2019). in this context, fusarium spp. have been associated with the failure or poor establishment of the vineyards, mainly in tropical regions (halleen et al., 2003; garrido et al., 2004; król, 2006; ziedan et al., 2011; cruz et al., 2014; abdullah et al., 2015; markakis et al., 2017; ghuffar et al., 2018; reveglia et al., 2018; akgül and ahioğlu, 2019). fusarium spp. are soil­inhabiting pathogens that affect many plant (*) corresponding author: alessandrarussi@yahoo.com.br citation: cavião h.c., russi a., schwambach j., 2024 ­ biocontrol of fusarium spp. in vitro and in vine cuttings using bacillus sp. f62. ­ adv. hort. sci., 38(2): 189­196. copyright: © 2024 cavião h.c., russi a., schwambach j. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 november 2023 accepted for publication 26 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15354 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 189­196 190 species, including grapevines (sotoyama et al., 2016). these phytopathogens infect the vines through wounds in the root system, causing root rot. subsequently, the pathogen promotes xylem obstruction, vascular injuries, and plant wilting due to the interruption of water and nutrients transportation to the shoots (brum et al., 2012; eljounaidi et al., 2016; markakis et al., 2017). besides, this pathogen can be transmitted through pruning and grafting, infecting the rootstock, graft union, and scion (akgül and ahioğlu, 2019). fusarium wilt mainly affects susceptible vine rootstocks belonging to the berlandieri‐riparia family, including the varieties so4, kobber 5bb, and solferino. although the ‘so4’ rootstock exhibits high adaptability to different soils and climate conditions, ensuring good vineyard yield and fruit quality, it is highly susceptible to fusarium wilt (vilvert et al., 2016). given the difficulties in managing soil­borne pathogens, the limited efficacy, and the environmental risks of synthetic fungicides (armengol and gramaje, 2016; gramaje et al., 2018), the use of antagonistic bacteria such as bacillus spp. represent an alternative in the control of fusarium wilt. these rhizobacteria can colonize plant tissues and vessels, suppressing the proliferation of vascular pathogens (eljounaidi et al., 2016). in addition, rhizobacteria can promote plant growth and enhance crop yield (legein et al., 2020; morales­cedeño et al., 2021). in previous research, the rhizobacterium bacillus sp . strain f62 demonstrated the potential to suppress black foot disease by 24.6% in ‘so4’ (vitis berlandieri x v. riparia) and by 29.5% in ‘1103p’ (vitis berlandieri x v. rupestris) rootstock plants obtained through micropropagation. considering these findings, the present study aimed to evaluate the ability of bacillus sp. f62 suspension to inhibit the mycelial growth of three isolates of fusarium spp. and investigate its biocontrol activity against fusarium sp. isolate fusa06­18 in stem wounds in the susceptible rootstock ‘so4’. 2. materials and methods microorganism isolates three isolates of fusarium spp. (fusa97­11, fusp08­10, and fusa06­18) were isolated from symptomatic grapevines from brazilian vineyards (table 1). the rhizobacterium bacillus sp. strain f62 was obtained from the soil in caxias do sul, rio grande do sul state, brazil. all microorganisms were preserved in the collection of the laboratory of biological plant disease control at the university of caxias do sul, brazil. molecular identification of the rhizobacterium was performed by amplifying the 16s rdna gene with primers for bacteria domains, according to sterky and lundeberg (2000). the sequence exhibited 100% similarity to a pre­existing sequence in the national center for biotechnology information (ncbi) of bacillus sp. f62 with accession number nr 102783.2. antagonism on mycelial growth of the pathogen the antagonistic effect of bacillus sp. f62 against fusarium spp. was assessed in two different assays: antagonism through volatile and diffusible compounds. these experiments followed the methodology described by russi et al. (2020). initially, a single colony­forming unit (cfu) of bacillus sp. f62 was cultured in a flask containing 10 ml of potato dextrose (pd) broth. the incubation was conducted on a rotary shaker at 150 rpm and 30 ± 2°c for 12 h. subsequently, this pre­inoculum was transferred to an erlenmeyer flask with 100 ml of pd broth and maintained under the same incubation conditions for 24 h. afterwards, the bacterial suspension was centrifuged (3,500 × g) at 23°c for 5 min. the supernatant was discarded, and the pellet was washed twice with sterile water and resuspended in a 0.85% nacl solution. the bacterial concentration was adjusted to 1 × 106 cfu ml­1 for in vitro antagonism and 1 × 108 cfu ml­1 for in vivo assay. mycelial discs (5 mm in diameter) of the pathogen isolates were obtained from 10­day­old colonies grown in potato dextrose agar (pda) medium at 25 ± 2°c, with a 12 h light/12 h dark cycle. in the antagonism through diffusible compounds, a mycelial disc was placed in a pda medium plate, and after 24 h, four drops of a bacterial suspension (1 × 106 cfu ml­1) were inoculated around the fungal mycelium. for the antagonism through volatile table 1 ­ isolates of fusarium spp. used in the assays isolates origin (city/country) grapevine variety fusa97­11 alto feliz, brazil isabella fusp08­10 caxias do sul, brazil isabella fusa06­18 caxias do sul, brazil yves cavião et al. ‐ biocontrol of fusarium spp. using bacillus sp. f62 191 compounds, a mycelial disc of the pathogen colony was inoculated in the center of a plate containing pda medium. in another plate with the same medium, 100 µl of bacillus sp. f62 suspension (1 × 106 cfu ml­1) was uniformly spread. subsequently, the plates were affixed together and sealed to prevent the loss of the bacterial metabolites. plates inoculated with the pathogen isolates served as a control. all plates were incubated at 25±2°c with a 12 h light/12 h dark cycle for 14 days. the experiment was performed using a completely randomized design, with ten replicates for each fungal isolate. measurements of the colony diameter were performed using a digital caliper, and the data were used to determine the mycelial growth rate (mgr), according to the formula: mgr = σ [(d ­ dp) / n] where d represents the mean of the colony diameter at the present day, dp represents the mean of the colony diameter from the previous day, and n represents the number of days of plate incubation. the mycelial growth inhibition (mgi) was also determined on the 14th day of the experiment according to mgi = [(dc ­ dt) / dc] × 100 where dc and dt represent the mean of the colony diameters of control and treated groups, respectively, as described by oliveira et al. (2016). biocontrol on rootstock cuttings four­year­old dormant cuttings of ‘so4’ were obtained from vineyards at embrapa grape and wine, bento gonçalves, rio grande do sul state, brazil. after hydration in distilled water for 24 h, the cuttings (30.0 cm in height) were subjected to hot water treatment at 50°c for 30 min, as described by lerin et al. (2017). four cuttings were arranged in each plastic pot containing 500 ml of autoclaved substrate (90% sphagnum peat and 10% vermiculite), ph 5.5, amended with 5 g l­1 of gradual release fertilizer (5­6 months). the isolate of fusarium sp. fusa06­18 was selected for the in vivo assay due to its intermediate behavior in bacillus sp. f62 antagonism. the experiment was carried out using a completely randomized design, with 60 rootstock cuttings per treatment, according to haidar et al. (2016 a), with modifications. rootstock cuttings were subjected to surface disinfection with 70% (v/v) ethanol by rubbing with cheesecloth, and then cuttings were wounded with a scalpel above the first basal bud (4 mm in diameter). the trial consisted of applying the following treatments at the wounds: control (40 μl of sterile water), bac (40 μl of bacillus sp. f62 suspension containing 1 × 108 cfu ml­1), bac + fus (40 μl bacillus sp. f62 suspension and a mycelium disc of fusa06­18), and fus (mycelium disc of fusa06­18). the wounds were covered with plastic film, and the cuttings were maintained in a growth chamber, at 26±2°c, under a 12 h light/12 h dark photoperiod provided by cool white fluorescent tubes. the relative humidity was maintained at 70%. during a 60­day experiment, the cuttings were watered three times a week with sterile water, at 80% of the maximum water holding capacity. the following morphophysiological responses were assessed after 30 and 60 days: bud number (budn), leaf number (leafn), inflorescence number (infln), and shoot length (shootl, cm). the pathogen re­isolation frequency (fpr, %) was also determined at the experiment’s end. the stems were debarked for pathogen re­isolation, and four fragments were collected 1 cm above and below the inoculation site. these fragments were surface disinfected by sequential immersion in 70% (v/v) ethanol for 30 sec and 3% (v/v) sodium hypochlorite for 1 min. subsequently, the stem fragments were rinsed three times with sterilized water and then inoculated in plates containing pda medium. the plates were incubated at 25°c for 10 days. the frequency of fusarium spp. re­isolation was recorded compared to the total number of fragments obtained from each rootstock cutting. statistical analysis the dataset was subjected to shapiro­wilk and levene’s tests to assess the normality and homoscedasticity, respectively. in the in vitro antagonism and the assay with rootstock cuttings, parametric data underwent one­way anova followed by the tukey test and non­parametric data were analyzed using the kruskal­wallis test followed by the dunn­bonferroni test. the frequency of pathogen re­isolation (fpr) between the treatments fus and bac + fus was evaluated using the mann– whitney u­test. all analyses were performed with spss 22.0 software (spss inc. chicago, il), and the threshold for statistical significance was set at p<0.05. adv. hort. sci., 2024 38(2): 189­196 192 3. results bacterial antagonism on mycelial growth the inhibitory potential of bacillus sp. f62 was determined against three isolates of fusarium spp. (fusa97­11, fusp08­10, and fusa06­18) using diffusible and volatile compounds assays. in the antagonism through diffusible compounds, the bioagent exhibited statistically significant suppression of all isolates of fusarium spp., reducing the mycelial growth rate compared to the control (table 2, fig. 1). among the pathogenic strains evaluated, fusarium sp. isolate fusa97­11 demonstrated the highest mycelial growth rate. the mycelial growth inhibition (mgi), determined on the last day of the assay, ranged from 30.4% (fusa06­18) to 47.1% (fusp08­10). in the assessment of bacterial antagonism through volatile compounds, there was a statistically significant difference between the treatments (fus and bac + fus). the volatile organic compounds led to a reduction in mycelial growth rate in all the fusarium spp. isolates (table 2, fig. 2). the mycelial growth inhibition (mgi) ranged from 8.2% (fusa97­ 11) to 14.3% (fusp08­10). although volatile compounds exhibited lower effectiveness in inhibiting the radial growth compared to diffusible compounds, these volatile metabolites not only affected the radial growth of the pathogen but also caused modifications in mycelial morphology (fig. 3). regarding the antagonism of bacillus sp. f62 against fusarium spp., both diffusible and volatile compounds promoted a higher suppression against the pathogenic isolate fusp08­10. fig. 1 ­ mycelial growth of three fusarium spp. isolates during 14 days of incubation in the antagonism assay through dif­ fusible compounds. a) fusa97­11, b) fusp08­10, and c) fusa08­16 table 2 ­ mycelial growth rate (mgr, mm/day) of three fusarium spp. isolates subjected to the following treatments: fusarium spp. (fus) and bacillus sp. f62 + fusarium spp. (bac + fus), in the antagonism through diffusible and volatile compounds. the mycelial growth inhibition (mgi, %) was determined on the last day of the experiment *statistical analysis was performed separately in the antagonism through diffusible and volatile compounds. **equal lowercase letters indicate no statistically significant difference between the treatments (fus and bac + fus) using t­test (p<0.05). equal uppercase letters indicate no significant difference among the fungal isolates, using anova followed by tukey’s test (p<0.05). treatments fusa97­11 fusp08­10 fusa06­18 mean antagonism through diffusible compounds fus 10.8 ± 0.4 aa 10.4 ± 0.2 ab 10.7 ± 0.2 aab 10.6 ± 0.3 a bac + fus 6.5 ± 1.3 ba 5.5 ± 0.8 ba 6.1 ± 1.3 ba 6.0 ± 1.1 b mgi (%) 39.8 47.1 30.4 39.1 antagonism through volatile compounds fus 7.3 ± 0.1 ab 7.0 ± 0.2 ac 8.1 ± 0.2 aa 7.5 ± 0.2 a bac + fus 6.7 ± 0.8 bab 6.0 ± 0.6 bb 7.1 ± 0.5 ba 6.6 ± 0.6 b mgi (%) 8.2 14.3 12.3 11.6 cavião et al. ‐ biocontrol of fusarium spp. using bacillus sp. f62 193 biocontrol on rootstock cuttings the bioagent was applied for the biocontrol of fusarium spp. isolate fusa06­18 in stem wounds of ‘so4’ cuttings (table 3). while the inoculation of bacillus sp. f62 in wounds did not improve the growth promotion responses evaluated in rootstock cuttings, it reduced the frequency of the pathogen re­isolation from 81.7% in the fus treatment to 63.3% in the bac + fus treatment (reduction of 22.5% in the fusarium wilt incidence). fig. 2 ­ mycelial growth of three fusarium spp. isolates during 14 days of incubation in the antagonism assay through volatile compounds. a) fusa97­11, b) fusp08­10, and c) fusa08­16. fig. 3 ­ morphology of colonies of fusarium sp. isolate fusa06­ 18 in the antagonism assay with volatile compounds syn­ thesized by bacillus sp. f62 after 14 days of incubation. the control treatment is on the upper left side of the photograph. treatments 30 days post­inoculation 60 days post­inoculation bud number leaf number inflorescence number shoot length bud number leaf number inflorescence number * inflorescence number fpr ** control 0.5 ± 0.3 7.3 ± 0.9 2.3 ± 0.6 9.9 ± 4.4 0.6 ± 0.2 9.5 ± 2.3 1.9 ± 0.6 19.8 ± 7.2 ­ bac 0.6 ± 0.2 6.7 ± 1.3 2.2 ± 0.9 9.1 ± 2.5 0.6 ± 0.2 10.5 ± 1.6 1.9 ± 0.4 20.5 ± 4.2 ­ bac + fus 0.6 ± 0.2 7.1 ± 1.9 2.2 ± 0.6 9.5 ± 2.9 0.6 ± 0.1 10.5 ± 1.3 1.70 ± 0.8 21.7 ± 5.2 63.3 ± 2.1 b fus 0.6 ± 0.1 6.9 ± 1.1 2.3 ± 0.9 10.3 ± 3.2 0.7 ± 0.1 10.3 ± 1.7 1.8 ± 0.5 22.7 ± 6.1 81.7 ± 1.5 a table 3 ­ morphophysiological responses in rootstocks cuttings of ‘so4’: bud number (budn), leaf number (leafn), inflorescence num­ ber (infln), shoot length (shootl, cm), and frequency of pathogen re­isolation (fpr, %), subjected to the treatments: control, bacillus sp. f62 (bac), bacillus sp. f62 + fusa06­18 (bac + fus) e fusa06­18 (fus). the responses were assessed in two different periods: 30 and 60 days post­inoculation * different letters indicate statistically significant difference using anova followed by the tukey’s test (p<0.05), except for inflorescence number (infln) analyzed using the kruskal­wallis test followed by the dunn­bonferroni test (p<0.05). ** frequency of pathogen re­isolation (fpr) was subjected to the mann­whitney u­test (p<0.05). 194 adv. hort. sci., 2024 38(2): 189­196 4. discussion and conclusions the antagonism activity of the rhizobacterium bacillus sp. f62 against fusarium spp. was evaluated in two experiments, in vitro and in vivo, with vine cuttings of the rootstock ‘so4’. in the dual culture assay, the bioagent inhibited the growth rate of fusarium spp. through the release of antimicrobial compounds and competition for space and nutrients. this finding is consistent with the observations of nourozian et al. (2006), who reported that two strains of b. subtilis inhibited the mycelial growth of fusarium graminearum by 97%. similarly, ziedan et al. (2010) found that seven strains of streptomyces spp. exhibited notable antagonistic activity in vitro against f. oxysporum. santos et al. (2016) also observed that a commercial product containing b. subtilis (rizolyptus®) reduced the mycelial growth of six isolates of dactylonectria macrodidyma by approximately 41%. however, the volatile metabolites produced by bacillus sp. f62 did not suppress fungal growth in vitro. this is in line with the findings of nigris et al. (2018), who reported that b. licheniformis gl174 did not control the mycelial growth of phaeoacremonium aleophilum, botryosphaeria spp., and botrytis cinerea through volatile compounds, while diffusible compounds inhibited the colonies growth by 60%. likewise, gao et al. (2018) observed that volatile molecules synthesized by b. subtilis cf­3 did not suppress the development of macrophoma kuwatsukai and penicillium expansum, causal agents of apple diseases. in contrast, rocha and moura (2013) observed that volatile compounds of streptomyces sp. dfs1315 and b. subtilis reduced the colony diameter of fusarium oxysporum f. sp. lycopersici by 18.1% and 17.5%, respectively. regarding the biocontrol potential of bacillus sp. f62 against fusarium sp., our experiments demonstrated a reduction in the percentage of pathogen re­isolation. likewise, haidar et al. (2016 a) reported that eight bacterial strains isolated from french vineyards effectively controlled p. chlamydospora, reducing the frequency of pathogen re­isolation from 31.4 to 38.7% compared to the control. additionally, several bacterial strains, especially pantoea agglomerans, significantly reduced the length of necrosis caused by n. parvum by 32.3% and 43.5% on grapevine cuttings (haidar et al., 2016 b). wicaksono et al. (2017) also observed that two isolates of pseudomonas sp. inoculated onto wounds in grapevine cuttings cv. sauvignon blanc inhibited two botryosphaeriaceous species, neofusicoccum luteum and n. parvum, and reduced lesion length caused by 32­52% compared to the untreated control. numerous studies have reported the ability of rhizobacteria to improve plant growth through nutrient solubilization, production of siderophores and phytohormones, such as auxins, gibberellins, and cytokinins (olanrewaju et al., 2017). rolli et al. (2017) tested the potential of fifteen rhizobacteria obtained from grapevines, olive trees, and pepper plants to enhance the growth of ‘syrah’ grafted on ‘1103p’ rootstock and ‘cabernet sauvignon’ grafted on ‘so4’ rootstock in the field. the results demonstrated rapid colonization of the rhizoplane and root system of grapevine by the rhizobacteria. moreover, bacterized plants showed longer shoots, larger diameters, and higher number of nodes on shoots. in the current study, the application of bacillus sp. f62 in artificially induced injuries did not increase plant growth of ‘so4’ cuttings. nevertheless, the inoculation of this same bacterium by soil drenching in cuttings of ‘so4’ improved plant development by increasing the length of the primary shoot, the number of nodes in the primary shoot, and the total number of nodes (russi et al., 2020). wicaksono et al. (2017) evaluated the efficacy of two methods for bioagent inoculation: stem wounding and soil drenching. the authors found that pseudomonas sp. colonized internal tissues of ‘sauvignon blanc’ cuttings when inoculated by wounding, but the bacterial proliferation failed when soil inoculated. as a result, plant morphological barriers and released toxins can prevent tissue colonization by some bacterial strains, reducing their effect in the phyllosphere (balmer et al., 2012). according to compant et al. (2010), tissue colonization is influenced by several factors, such as the pattern of plant exudates, nutrient availability, rhizobacteria growth rate, bacterial­host interactions, stress conditions, and plant genotype, which may explain the differences observed among these studies. furthermore, the plant tissue inoculated and the phytopathogen strain can influence the antagonistic potential of rhizobacteria (haidar et al., 2016 b). in summary, this study demonstrated the effectiveness of bacillus sp. strain f62 in controlling three isolates of fusarium spp., inhibiting mycelial growth through volatile and diffusible compounds. moreover, the rhizobacterium reduced the incidence of fusarium wilt in ‘so4’ vine cuttings that were cavião et al. ‐ biocontrol of fusarium spp. using bacillus sp. f62 195 artificially infected with the pathogen. consequently, bacillus sp. f62 holds promising potential as a biocontrol agent for suppressing fusarium spp. in susceptible vines. acknowledgements the authors thank to coordination of superior level staff improvement (capes) for providing funding for this research. references abdullah s.k, al­samarraie m.q., al­assie a.h., 2015 ­ fungi associated with grapevine (vitis vinifera l.) decline in middle of iraq. ­ egypt. acad. j. biol. sci., 7(1): 53­59. akgü d.s., ahioğlu m., 2019 ­ fungal pathogens associated with young grapevine decline in the southern turkey vineyards. ­ bio web of conferences, 15: 01027. armengol j., gramaje d., 2016 ­ soil borne fungal pathogens affecting grapevine rootstocks: current status and future prospects. ­ acta horticulturae, 1136: 235­238. balmer d., planchamp c., mauch­mani b., 2012 ­ on the move: induced resistance in monocots. ­ j. exp. bot., 64: 1249­1261. brum m.c.p., araújo w.l., maki c.s., azevedo, j.l., 2012 ­ endophytic fungi from vitis labrusca l. 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streptomyces alni as a biocontrol agent to root‐rot of grapevine and increasing their efficiency by biofertilisers inocula. ­ arch. phytopathol. plant prot., 43(7): 634­646. impaginato 367 adv. hort. sci., 2023 37(4): 367­376 doi: 10.36253/ahsc­13749 yield related traits in some persian wal­ nut cultivars: analysis of genetic and genetic by environment interaction a. soleimani 1 ,2 (*), v. rabiei 1 (*), d. hassani 2 (*), m.r. mozaffari 3, r. dastjerdi 2 1 department of horticulture, faculty of agriculture, university of zanjan, zanjan, iran. 2 temperate fruits research center (tfrc), horticultural science research institute (hsri), agricultural research, education, and extension organization (areeo), karaj, iran. 3 agricultural and national resources research centre of kerman, kerman, iran. key words: climatic conditions, cultivar, heritability, juglans regia l., stability. abstract: the most important trait in tree species, including walnut, is the yield. in this study, the effect of genotype and their interaction with year on nut weight, kernel weight, kernel percentage, fruit set, nuts number on scaffold (canopy) cross area (sca), nut weight on sca and kernel weight on sca were evaluated on caspian, persia, alvand, and chaldoran walnut cultivars. the results showed that the effects of year, genotype, and year × genotype interac­ tion on all traits were significant. the results showed that alvand had the high­ est number of nuts (41.8 per m2) and nut weight (472.1 g/m2) on (sca). heritability (h2 b) for kernel weight and kernel percentage, were estimated 0.75 and 0.80, respectively. the lowest value of h2 b (0.36) was belong to fruit set. the analyses of genetic and phenotypic correlations between traits showed that, the nut weight had (rg = 0.31, rp = 0.27) a moderate correlation with sca same as kernel weight (rg = 0.34, rp = 0.29). the gge biplot analysis explained most of the existing variations (>90%). the genetic effect (pc1) for all traits were higher respect to the genetic × environment interaction (pc2), especially for the kernel percentage (94.4%) and number and weight of nut and kernel on sca (>90%). the lowest value of the pc1 was related to the fruit set (65.6%), which indicates the trait was more affected by genetic × environment interac­ tions (21.8%). so, this result showed that the yield­related traits in walnut is highly relevant to environment(year in this study) and evaluation of the new cultivars needs careful attention in this case. 1. introduction the accurate identification of genotypes is a basic requirement for appropriate utilization of germplasm in practical breeding programs. the diverse climatic conditions, environment, and their interactions with (*) corresponding author: rabiei@znu.ac.ir d.hassani@areeo.ac.ir citation: soleimani a., rabiei v., hassani d., mozaffari m.r., dastjerdi r., 2023 ­ yield rela‐ ted traits in some persian walnut cultivars: analysis of genetic and genetic by environment interaction. ­ adv. hort. sci., 37(4): 367­376. copyright: © 2023 soleimani a., rabiei v., hassani d., mozaffari m.r., dastjerdi r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 september 2022 accepted for publication 1 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13749 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 367­376 368 genetic are the most important factor determining the performance of the cultivars (fehr, 1987). therefore, the genetic and environment implies the differential performance of genotypes that rises from the variations in the genotype’s sensitivities to the environmental conditions (rawandoozi et al., 2021). change of climate not only affect the phenology of tree species, but also affects its production. so, with considering the climate changes and abiotic stresses, walnut production in the world has encountered challenges more than ever before. on the other hand, selection for fruit quality traits is complex; because the most of these traits are often controlled by several loci that are also influenced by the envi­ ronment (bliss, 2009). nut and kernel weight as well as fruit set percentage could be considered as walnut yield components, while the yield efficiency could include nut and kernel weights produced on trunk cross area (tca) or scaffold cross area (sca) (mahmoodi et al., 2015; dogra et al., 2018; hassani et al., 2020 b). these traits can be affected by envi­ ronmental conditions in several ways. for example, the climatic factors affect the receptivity period of walnut pistillate flowers and therefore affect the fruit set percentage and yield of walnut trees (mariana and sina niculina, 2017). dogra et al. (2018) calculated phenotypic and genetic broad sense heritability of walnut yield relat­ ed traits. based on their study the pistillate flower density, fruit set percentage, circumference and cross section of tree trunk showed the highest corre­ lation with the yield. some walnut trees somewhat show different alternate bearing habits, so the yield is affected by the crop load of the previous year (mahmoodi et al., 2015). marrano et al. (2019) reported that lateral bearing habit have a significant influence on yield of walnuts. besides the leafing date had high heritability (88%) and was therefore recommended as a reliable character for improve­ ment of new cultivars. combining analysis of variance and stability analy­ sis could determine the contribution of genetic, envi­ ronment and their interactions in traits. in spite of cli­ mate change is becoming a bigger challenge every day, determining the genetic and environmental effects can led to understand the response of the cul­ tivars to different environments and select the appropriate cultivars for specific environments and eventually to deal better with changing climate (bliss, 2009; rawandoozi et al., 2021). research with number of genotypes evaluated in different locations and years, makes the genetic × environment analysis a major contest. the gge biplot analysis is a beneficial tool for data analyzing in multi environment trials (yan and tinker, 2006). rawandoozi et al. (2021) estimated the variance components, genetic × environment interaction and heritability of fruit quality related traits in nine peach and nectarine low to medium chill f1 full­sib families together with their parents in two locations. based on their research the ripe date and fruit development period had high narrow sense heritability. fruit weight and shape showed the lower heritability. scariotto et al. (2013) based on budburst percent­ age and fruit­bearing shoot formation, evaluated the compatibility and stability of peach genotypes in four years. arji (2018) investigated the stability of yield components of olive cultivars for three years. despite the high priority for data availability regarding the climatic adaptability of walnut cultivars, there is need for a continuous basis research with the newly released cultivars. therefore, this study is con­ ducted to evaluate the adaptability of some new persian walnut cultivars to determine the variance components and cultivars adaptability affecting the yield components and yield efficiency traits, especially with increasing the climate change challenge. 2. materials and methods plant materials and location walnut yield component together with the yield efficiency traits in four newly released cultivars (i.e., caspian, persia, alvand, and chaldoran) (hassani et al., 2020 b) with chandler and jamal as reference cul­ tivars, were evaluated in three consecutive years (2015­2017). the cultivars, grafted on persian walnut seedlings rootstocks, were planted in karaj in 2006 (35.76031 n, 50.96833 e; elevation: 1240 m a.s.l.; mean annual temperature: 15.8°c; and mean annual precipitation; 247 mm). evaluated traits the data were recorded on yield component traits including nut and kernel weight and fruit set percent­ age together with the yield efficiency traits such as: nut and kernel weights produced on scaffold cross area (sca). to estimate the number of pistillate flow­ ers and fruits on experimental trees, pistillate flowers and fruits were counted in sample branches and then were used to predict the whole trees using regression. to measure scaffold cross area (sca), the tree’s canopy diameter was measured. the sca was then soleimani et al. ‐ genetic and environmental interactions on walnut yield 369 estimated using the canopy and the circle approxima­ tion. for nut and kernel traits, 30 samples in each treatment were evaluated. the tree nut and kernels’ yield were obtained from the number of nuts per tree multiplied per average nut and kernel weights. next, the nut and kernel yield of trees were divided by the corresponding sca’s, for estimating yield effi­ ciencies based on nut and kernel (hassani et al., 2014). to calculate the fruit set percentage, the fruit number in sample branches were divided by the cor­ responding number of pistillate flowers. statistical analysis the combined analysis of variances was carried out using general linear model (glm) procedure. means were separated by duncan’s multiple range test (dmrt) and least significant difference (lsd). phenotypic (σ2 p), genetic (σ2g) and genetic × year interaction (σ2 gy) variances were obtained from their corresponding expected mean square in anova table. heritability in the broad sense (h2 b) was esti­ mated using the genetic and phenotypic variances (visscher et al., 2008). the phenotypic and genetic correlations were estimated using the variances and variance­covariance matrices of traits (dogra et al., 2018; marrano et al., 2019). the gge biplot analysis was employed to determine the year and genotype interaction, besides the combining analysis of vari­ ances (yan and tinker, 2006). 3. results yield‐related traits variability and analysis the descriptive statistics of the traits were report­ ed in table 1. the nut weight varied from 8­15.3 g with the average of 11.2 g, while the kernel weight average was 5.8 g varying from 3.9­8.3 g. though the variation in kernel percentage range were 39.4­ 66.7%. the fruit set average was 48.7%, with a wide range variation (14­82%) in different cultivars. mean number of nuts on sca were 26.8 with a range of 2.7­ 64.7. moreover, the average of nut and kernel weight on sca were 295.1 and 157.4 g/m2, respectively. nut weight on sca ranged 28.8­782.9 g/m2, while the ker­ nel weight on sca ranged 12.1­409.7 g/m2. in gener­ al, a high variation was observed for the evaluated traits in different cultivars. the three years combined analysis of variance and genetic variance components for the studied traits are shown in table 2. the effect of the year was sig­ nificant on fruit set percent, the nut number on sca table 1 ­ descriptive statistics of the traits evaluated in walnut cultivars evaluated traits min max range mean variance nut weight (g) 8 15.3 7.3 11.2 2.9 kernel weight (g) 3.9 8.3 4.4 5.8 1.3 kernel percentage 39.4 66.7 27.3 52.1 54.5 fruit set percentage (%) 14 82 68 48.7 264.1 nut number on scaffold cross area (no./m2) 2.7 67.4 64.7 26.8 271.6 nut weight on scaffold cross area (g/m2) 28.8 782.9 754.1 295.1 29655.1 kernel weight on sca (g/m2) 12.1 409.7 397.6 157.4 9448.5 **, * and ns show statistical significance at the probability level of 1%, 5% and not significant, respectively. sca = scaffold cross area; cv = coefficient of variance; h2 b = broad­sense heritability and se = standard error of h2 b. table 2 ­ combined analysis of variance, genetic variance components and broad­sense heritability (h2 b) of the traits in six walnut culti­ vars (2015­2017) variance component df nut weight mean squares kernel weight kernel percentage fruit set percentage nuts number on sca nut weight on sca kernel weight on sca year 2 10.3 ns 0.99 ns 16.8 ns 995.4 * 1149.8 * 159004 * 47838 * replication (year) 6 1.3 0.18 17.1 142.8 70.1 5671.1 2319.3 genotype 5 16.2 ** 10.9 ** 430.9 ** 1197.8 * 1056.4 * 141510 * 46238 * year x genotype 10 3.17 ** 0.71 ** 21.8 ** 330.3 ** 239.9 ** 30441 ** 10044 ** error 30 0.47 0.19 6.1 95.3 76.7 7912.8 2300.1 cv (%) 6.1 7.5 4.7 20.1 22.9 30.1 30.5 h2 b ­ 0.41 0.75 0.80 0.36 0.42 0.44 0.45 se ­ 0.1 0.025 0.015 0.13 0.11 0.104 0.11 adv. hort. sci., 2023 37(4): 367­376 370 and the nut and kernel weight on sca (p≤0.05). however, it was not significant on nut weight, kernel weight and kernel percent. the effects of genotype and year × genotype interaction was statistically sig­ nificant in all traits (table 2). a high broad­sense heritability (h2 b) obtained for kernel weight (0.75) and kernel percentage (0.80). the lowest value of h2 b (0.36) was belong to fruit set (table 2). based on the analysis of results for determining the effect of different years the highest fruit set per­ centage was observed in 2016 and 2015 with the average of 52.4% and 51%, respectively, although, the lowest fruit set percentage was 42.7% in 2017 (fig. 1). the highest number of nuts on sca; and nut and kernel weight on sca were attained in 2016 with the corresponding averages of 34.3 nuts per m2; and 317.4 and 210.8 g per m2. evaluation of traits in different genotypes during three experimental years showed that the average of nut weight varied from 9.2 g in caspian to 13.2 g in chaldoran. kernel percentage varied from 42.2% in chandler to 60.1% in persia. fruit set ranged from 33.8% in persia to 62.7% in jamal. furthermore, fruit set was significantly lower in late leafing cultivars and genotypes such as persia, chandler, and caspian (marrano et al., 2019, hassani et al., 2020 a) com­ pared with early to medium leafing ones (33.8­43.5% and 55.6­62.7%, respectively) (fig. 2). based on the results a wide range of differences was observed in yield efficiency traits (number of nuts per m2 sca and weight of nut and kernel per m2 sca). the highest number of nuts per m2 sca was observed in alvand with an average of 41.8 nuts/m2, while the lowest amount was recorded in jamal with 9.1 nuts/m2. moreover, the highest nut weight on sca were observed in alvand with 472.1 g/m2. alvand and chaldoran had the highest kernel weight on sca with 239.7 and 218.6 g/m2, correspondingly. the lowest nut and kernel weight on sca with 108.2 g/m2 and 50.4 g/m2 belonged to jamal (fig. 2). genetic and genetic per environment (gge) analysis according to statistically significant interactions between years and genotypes, the studied cultivars showed different responses to years. analyzing the effect of genotypes and genotype × year interaction on the studied traits have been shown in gge biplot diagrams in figure 3. in gge biplot diagrams, the hor­ izontal axis (pc1) shows the effects of genotypes and the vertical axis (pc2) shows the interaction of geno­ type per year (environment). according to the results for nut weight (fig. 3 a), the pc1 and pc2 explained respectively 79.5% and 19.2% of variability, with the fig. 1 ­ mean comparisons for the effect of years on fruit set per­ centage (a), nut number on scaffold cross area (sca) (b), nut weight on sca (c), and kernel weight on sca (d). soleimani et al. ‐ genetic and environmental interactions on walnut yield 371 98.7% of total variability. the biplot 2a was divided into four sectors with a principal sector grouping the years 2015 and 2017, together with chaldoran and alvand with higher nut weight. while in the second sector, the year 2016 was grouped with persia. for the percentage of kernel (fig. 3 b), the pc1 and pc2 explained respectively 94.4% and 4.7% of variability, with 99.1% of total variability in five sectors. the principal sector grouped the years 2015 and 2017. persia with high kernel percentage was included in this sector. the second sector were grouped the year 2016 together with chaldoran and caspian. these cultivars had greater kernel percentage than general average. in figure 3 c the pc1 and pc2 explained respectively 65.6% and 21.8% of variability, with 87.4% of total variability about fruit set percentage. the biplot for fruit set percent was divided into five sectors, too. the principal sector grouped the years 2015 and 2017, and jamal with higher fruit set. the second sector grouped the year 2016 and chaldoran. this cultivar had fruit set greater than average. for the fruit number on sca (fig. 3 d), the pc1 and pc2 explained 94.2% and 5.5% (99.7% of total) of variabil­ ity correspondingly. the gge biplot was divided into four sectors. in the principal sector the years 2015 and 2016, and the cultivars alvand, caspian and chaldoran were classified together with higher fruit number on sca. in the second sector, the year 2017 and persia were grouped together. similar results were obtained for nut weight on sca (fig. 3 e). for the kernel weight on sca (fig. 3 f), the pc1 and pc2 explained respectively 90% and 9.4% of variability fig. 2 ­ mean comparisons of the effect of the cultivar and year × cultivar on fruit weight (a), kernel percent (b), fruit set (c), number of fruits on sca (d), fruit weight on scaffold cross area (sca) (e), and kernel weight on sca (f) using duncan multiple range test for genotypes and least significant difference (lsd) test for interaction of year × cultivar. 372 adv. hort. sci., 2023 37(4): 367­376 and 99.4% of total variability. the gge biplot for ker­ nel weight on sca was also divided into five sectors. the principal sector grouped the years 2015 and 2016, and the cultivars alvand and chaldoran with higher kernel weight on sca. the second sector grouped the year 2017 and persia that had greater kernel weight on sca. the figure 4 shows scattering of walnut cultivars based on the yield efficiency traits in a biplot. in fig­ ure 4b chandler, persia, chaldoran and alvand had the highest nut weight on sca and also nut weight. the same results on figure 4 c with chaldoran and alvand which had the highest kernel weight on sca too. according to figure 4 d the highest fruit set per­ cent and nut weight on sca also was belong to alvand and chaldoran. genetic and phenotypic correlations the genetic and phenotypic correlations between the traits are reported in table 3. these results showed that the nut weight did not considerably cor­ relate with kernel percentage and number of nuts on sca. nut weight had a moderate impact on fruit weight on sca (rg = 0.31, rp = 0.27) and kernel weight on sca (rg = 0.34, rp = 0.29). as expected, a high genetic and phenotypic correlation was observed between the number of nuts on sca and nut weight on sca (rg = 0.95, rp = 0.95) as well as kernel weight on sca (rg = 0.90, rp = 0.91) (table 3). kernel weight on sca was significantly correlated with most of the traits, but the highest genetic and phenotypic corre­ lations were observed between this trait and fruit weight on sca (rg = 0.97, rp = 0.97). fig. 3 ­ genotype and genetic × environment (gge) biplot of six walnut cultivars over three years (2015­2017) for nut weight (a), kernel percentage (b), fruit set percentage (c), nut number on scaffold cross area (sca) (d), nut weight on sca (e) and kernel weight on sca (f). soleimani et al. ‐ genetic and environmental interactions on walnut yield 373 4. discussion and conclusions the significant effects of year, genetic and genetic × year interaction showed that cultivar and its inter­ action with environmental conditions as the main determinants of cultivar’s adaptability. therefore, stable and compatible cultivars should be found and introduced for appropriate climate(s) (rawandoozi et al., 2021). the low fruit set in 2017 caused the fruit production to be significantly lower compared to other two years, while there were no significant dif­ ferences in pistillate flowers (data not shown). understanding the genotype and genetic × envi­ ronment interaction also is important for increasing fig. 4 ­ biplot of regression coefficients against average yields of walnut cultivars (the horizontal solid line represents the mean coeffi­ cient of regression and the vertical solid line denotes the average fruit weight on scaffold cross area (sca). the standard error (±1se) was included and represented by the dotted lines for both yield efficiency and regression coefficients) (a), biplot of nut weight with nut weight on sca (b), kernel weight with kernel weight on ca (c), and percentage of fruit set with nut weight on sca (d). solid lines in graphs show the average of each trait. table 3 ­ genetic and phonotypic correlations of different traits g and p are the genetic and phenotypic correlations, respectively. sca = scaffold cross area. kernel (%) fruit set (%) number of nuts on sca2 nut weight on sca kernel weight on sca nut weight g1 0.19 0.72 ­0.02 0.31 0.34 p1 0.12 0.53 ­0.03 0.27 0.29 kernel (%) g 1 ­0.34 0.25 0.31 0.55 p ­0.26 0.26 0.30 0.52 fruit set (%) g 1 ­0.29 ­0.09 ­0.14 p ­0.12 0.03 0.01 number of nuts on sca g 1 0.95 0.90 p 0.95 0.91 nut weight on sca g 1 0.97 p 0.97 kernel weight on sca g 1 p adv. hort. sci., 2023 37(4): 367­376 374 the gain in cultivar improvement programs. the genotype’s main effect nd especially it’s g x e interac­ tion implies the different performance of genotypes across environments that arises from the various sensitivities to the different environments (rawandoozi et al., 2021). for all of the yield related traits, gge biplot have described most of the existing variations (more than 90 %), which indicates the rela­ tive validity of the biplot in explaining the variations of genotypes and genetic × environment interaction (yan and tinker, 2006). the effect of genotype in determining the walnut yield efficiency has been demonstrated by dogra et al. (2018). based on our results high amounts of pc1 has been recorded for kernel percentage (94.4%) as well as number and weight of nut and kernel on sca (more than 90%). so, a high and stable production will be expected by selecting cultivars with higher yield efficiencies and more compatible with environmental conditions. the results on nut number and nut weight in cultivars are consistent with the findings of mahmoodi et al. (2016). fruit set is another important trait affecting the number of nuts in tree (mcgranahan and leslie, 2009; sarikhani khorami et al., 2014; khadivi­khub et al., 2015). it is clear that, number of pistillate flowers is relatively lower in cultivars with terminal bearing habit compared to lateral bearing ones (mcgranahan and leslie, 2009; hassani et al., 2020 b). in terminal bearing cultivars like jamal, the higher fruit set usual­ ly could compensate the production to some extent. among the yield related traits, the lowest value of the pc1 was belong to fruit set (65.6%), which indi­ cates this trait is more affected by genetic × environ­ ment interaction (21.8%). the results of this study present the clear effect of genotype on fruit set (fig. 2 c) which is consistent with kumar et al. (2005). due to the fact that the amount of pistillate flowers pro­ duced in walnut is lower than pome and stone fruit trees, higher fruit set (50­90%) is necessary in order to produce an adequate yield. in addition to genetic, genetic × environment interaction plays a very impor­ tant role in pollination and fruit set of cultivars (cosmulescu et al., 2010; mariana and sina niculina, 2017). according to the results, there was a signifi­ cant negative correlation between bud break and fruit set (r = ­0.54), so that with delayed leafing, the fruit set decreased. it is clear that the late leafing cul­ tivars deal better with late spring frosts (mcgranahan and leslie, 2006; hassani et al., 2013; hassani et al., 2020 b), but the pollination and fruit set were not the same in late and early leafing walnut cultivars. to obtain a sufficient fruit set, care must be taken regarding producing a sufficient pollen volume with an adequate overlap of pollen­shedding for the receptivity period of pistillate flowers. the response of cultivars could be affected by different climatic conditions in different years especially at leafing time and time of pollination (cosmulescu et al., 2010; sarikhani khorami and vahdati, 2019; cao et al., 2020). temperature is one of environmental factors influences the percentage of fruit set by influencing pollination factors, such as pistillate flowers receptiv­ ity and pollen­shedding period. in late leafing culti­ vars the environmental factors such as high tempera­ tures at the pollination time, could lead to lower effective pollination period and pistillate flower receptivity period, that could reduce the fruit set (ramos, 1997). high broad­sense heritability relative to kernel weight and kernel percentage also indicated that these traits are less affected especially by genetic × environment interaction. conversely, low­moderate heritability and high ratio of genetic × environment interaction for fruit set indicated substantial environ­ mental effects on this trait. the heritability values in the present study were somewhat lower than what reported by eskandari et al. (2006), dogra et al. (2018), and marrano et al. (2019). in terms of yield stability, it seems that the genet­ ics, environment and their interaction could con­ tribute to various characteristics such as: fruit­bear­ ing habit, growth vigor, nut weight, kernel weight, kernel percentage, previous year crop load, pollina­ tion, fruit set and late spring frosts (cosmulescu et al., 2010; asma, 2012; sarikhani khorami et al., 2014; dogra et al., 2018; cao et al., 2020; hassani et al., 2020 a). generally, in low­yielding cultivars such as jamal, year­by­year variations of traits were low. however, they were higher in cultivars with more production such as chaldoran and alvand (hassani et al., 2020 a). some studies have reported significant alternate bearing in walnut cultivars (asma, 2012; hassani et al., 2014; mahmoodi et al., 2016). so, alternate bearing, opposed to genetic stability, is affecting the fruit production trends of walnut culti­ vars in different years (amiri et al., 2010). mahmoodi et al. (2015), reported the presence of 2­15% of alter­ nate bearing among different walnut cultivars. in majority of high­yielding cultivars, a heavy crop load is followed by a low fruit production in the subse­ quent year. asma (2012) found that the yield is influ­ enced mostly by leafing time, fruit­bearing habit, tree soleimani et al. ‐ genetic and environmental interactions on walnut yield 375 size, nut and kernel weights, and kernel percentage. moreover, dogra et al. (2018) reported that yield is controlled polygenically and is influenced by environ­ mental conditions. they stated that pistillate flower density, fruit set, trunk section area, trunk circumfer­ ence, tree height, shoot length, pollen­shedding peri­ od, fruit weight, kernel percentage, and shell thick­ ness had affected the yield of walnut trees, which were in part consistent with the findings of the pre­ sent study. high variation was observed in yield components and yield efficiency traits with different environments and cultivars, and it was found that genetic by envi­ ronment interaction are the most important factors determining yield variations. understanding the con­ tribution of genetics and genetic × environment interaction is very important. the genetic × environ­ ment interaction in fruit set was more than other yield­related traits, while the broad sense heritability (h2 b = 0.36) was the lowest value. therefore, fruit set is most affected by variation of environmental condi­ tions, so that under undesirable climatic conditions it will be yield determining factor especially in late leaf­ ing walnut cultivars. regarding the nut weight, the effect of genetic by environment interaction was strong while heritability was greatly affected by the environmental conditions. the contribution of genet­ ic × environment interaction on other traits related to yield efficiency was estimated to be less than 10%. genetic and phenotypic correlation also indicated that nut weight, kernel weight and kernel percentage had a low­moderate correlation with nut and kernel weight on sca. on the contrary, the nut number on sca had the highest genetic and phenotypic correla­ tion with nut and kernel produced on sca. the results showed that in walnuts, that is a nut tree species well adapted to temperate climate, the vari­ ability of yield related traits over environments (years), was highly significant. so, the change in cli­ mate in one hand and the scarcity of the resources 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whereas in one location organic grapes were judged not edible. results showed a higher nutritional value in grapes obtained with the organic farming system although in terms of visual quali­ ty, storability and yield, conventional fruit had a better performance. 1. introduction more than 403’000 hectares of organic grapes are grown worldiwide, (*) corresponding author: giancarlo.colelli@unifg.it citation: amodio m.l., colelli g., 2020 ­ physio‐chemi‐ cal quality attributes of ‘italia’ grapes from orga‐ nic and conventional farming at harvest and during storage. ­ adv. hort. sci., 34(1s): 109­115 copyright: © 2020 amodio m.l., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 january 2020 accepted for publication 24 august 2020 ahs advances in horticultural science https://it.wikipedia.org/wiki/carl_von_linn%c3%a9 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 109­115 110 constituting 5.7 percent of the world’s grape­growing area (7.1 million hectares in 2016, according to fao­ stat). in europe, over 340’000 hectares (8.7 percent of the harvested grape area) are organic; spain and italy, cultivated more than 100’000 hectares of organic grapes, followed by france with over 78’000 hectares. the italian national information system on organic agriculture (sinab) reported for the 2012 an increase of 6.4% of the organically cultivated land compared to the previous year, with a total of 49,709 organic operator. regions in southern italy have the largest organic areas (sicilia, puglia and calabria) and the largest number of organic farms with an increase of operators (20.3%) in puglia region, while most of the processors are located in northern italy (especial­ ly emilia romagna and lombardia). as known, grapes contain a wide range of nutritional and functional component such as vitamins, minerals, organic acids, enzymes as well as phytochemicals (walzem, 2008), among which phenolics, particularly flavonoids, anthocyanins and resveratrol, are the most impor­ tant because are held accountable for their health benefits (yang et al., 2009). however, as known, both pre­ and postharvest practices may affect the amount of these nutritional and functional com­ pounds as well as many other elements of horticul­ tural crops (lee and kader, 2000). among the pre­ harvest conditions, genotype, environmental condi­ tions, cultural practices, and maturity at harvest influence quality attributes of grapes such as the con­ centration of phenolic compounds (sellappan et al., 2002). particularly the application of organic and con­ ventional agricultural techniques may affect the table grapes quality. generally organic agriculture opti­ mizes the health and productivity of interdependent communities of soil life, plants animals and humans. in fact, organic agriculture does not use synthetic pesticides and fertilizers (briar et al., 2007), but only ecological products. several studies showed that the use of organic or conventional techniques significant­ ly influenced the production, in terms of number of bunches on the vine stock and the average weight of the bunch (detoni et al., 2007). however, in the last years researchers focused their attention on the influence of organic cultivation on the content of sec­ ondary metabolites although no clear behaviors were observed. higher concentration of bioactive com­ pounds in plants grown with the organic system were reported by several authors, which were considered as the results of the plant exposure to situation that leads to an increase of natural defenses (winter and davis, 2006). dani et al. (2007) reported that organic crop influenced the phenolic content and the antioxi­ dant activity of white and purple grape juices, but for some study difference observed 1 month before har­ vest, were not observed at the harvest time (mulero et al., 2010). regarding differences observed during storage, thompson seedless grapes from organic orchard showed more desirable color and lower browning index, and generally higher nutritional con­ tent than conventional grapes, with similar decay incidence (zahedipoura et al., 2019), but there is not much literature to this regard. based on the above considerations, this paper had the principal aim of comparing the physio­chemical attributes at harvest and during storage of table grapes cultivated with organic and conventional techniques also taking into account the effect of two different production areas in puglia region (southern italy). 2. materials and methods in this experiment two organic farms were chosen in 2 different locations of the puglia region, one at castellaneta (78 m above sea level) in province of taranto (loc1) and one in adelfia (151 m on sea level) in province of bari (loc2). the climate for both loca­ tions is mediterranean semi­arid, characterized by hot and dry summers and moderately cold and rainy win­ ter seasons, with annual mean temperature of 14­ 15°c and mean annual rainfall within 450­500 mm. for each location a conventional farm, with similar charac­ teristics, and in the same area (within 1 km) was cho­ sen as control, resulting in a comparison among 2 dif­ ferent organic with 2 conventional farms. in each loca­ tion, 6 plants were used as replicate, and 3 bunches for each replicate were collected in the same day, at the commercial maturity stage, with a total of 18 bunches per field. soils were composed by 63.2% sand; 22.1% clay; and 14.7% silt for bari location and 69.8% sand; 15.1% silt and 15.1% clay for taranto. the conventional vineyard was managed according to common viticultural practices for the growing area, including winter mineral nutrition, spring­summer fer­ tigation, and irrigation with seasonal volume of about 2000 m3/ha by drip irrigation. the organic farming was managed according (ec) reg. 834/07 and reg. 889/08. at harvest bunches were weighed, closed in a sealed container to measure respiration rate, before to detach 30 berries per bunch. on these 30 berries per bunch biometrical attributes were evaluated, including berry weight and dimensions (major and amodio and colelli ‐ quality of ‘italia’ organic grapes at harvest and during storage 111 minor axes), and peel thickness. following, bunches from each replicate were kept in individual 15­l jars at 0°c and connected to a humidified flow of air for the entire duration of the experiment. after 7 and 14 days of storage, respiration rate, weight loss, physical and nutritional attributes were also monitored on 20 berries per bunch. after berry detachment stalks were protected by excessive dehydration by using adhesive tape around the berry abscission zone. quality indexes initially and after 6 and 14 days, bunches were individually scored using a 5 to 1 subjective scale, with 5 = excellent, no defects, 4 = very good, minor defects, 3 = fair, moderate defects, 2 = poor, major defects, 1 = inedible. a score of 3 was considered as the limit of marketability and a score of 2 as the limit of edibility. then 20 berries for each bunch were used the fol­ lowing quality assessment: ­ peel color using a spectrophotometer (konica minolta cm 2600d, japan) in the cie l*a*b* mode, and then calculating hue angle and chroma values; ­ flesh firmness with a manual firmness tester by measuring force required by a 2­mm probe to penetrate the tissue for 5 mm in two opposite locations; ­ titratable acidity on 4g of grape juice for each replicate, using an automatic titrator (crison, titromatic 1s, barcelona, spain) with 0.1 n naoh solution to ph 8.1 and reported as percent of tar­ taric acid; ­ soluble solids content (ssc) using a refractometer (atago, pr­32; tokyo, japan); for respiration rate 3 bunches of each plant were closed in a sealed container to let co2 accumulate. samples of gas (0.1 ml) were collected through a rubber septum and injected into a gas chromato­ graph (shimadzu, model 17a, kyoto, japan) equipped with a thermal conductivity detector (230°c). separation of co2 was achieved on a carboxen 1006 plot (30 m x 0.53 mm, supelco, bellefonte, pa usa), with a column flow of 7 ml min­1, and oven tempera­ ture of 180 °c; the difference in concentration was then referred to the sample weight, to the elapsed time, and to the head space volume. the sample weight at each storage time was also used to calcu­ late weight loss. nutritional quality after previous determinations, part of the berries were peeled, and the skin was frozen for the analysis of phenols and antioxidant activity whereas the juice was used for sugars composition and vitamin c. ascorbic and dehydroascorbic acid were analysed on fresh samples by high performance liquid chro­ matography (hplc agilent 1200 series, waldbronn, germany) equipped with a binary pump, an autosam­ pler, and a photodiode array detector (dad) (zapata and dufour, 1992). the following determinations were performed on the frozen samples: ­ organic acid and sugar composition by using the hplc equipped with the (dad) array and a refrac­ tometric detector (pérez et al., 1997). ­ total phenolics of grape skins by the folin­ ciocalteu reagent (singleton and rossi, 1965), dilutions were carried out in duplicate and calcu­ lated using a calibration curve obtained with gallic acid, reading the absorbance at 575 nm. ­ antioxidant activity by spectrophotometer using the dpph (2,2 diphenyl1­1­picrylhydrazil) method (brand­williams et al., 1995). ­ phenolic composition using the same extract for total phenolics analysed with hplc procedure (bonilla et al., 1999). statistical analysis on the data collected after harvest, a split plot design for the location and treatment was run, whereas on the whole data set a split plot with loca­ tion as main plot, treatment as subplot, and time as third plot was assessed. finally, due to the presence of significant 3th order interaction, a split plot for each location (for treatment and time of storage) was run. 3. results and discussion the effect of location (loc1 and loc2), and culti­ vation system (organic and conventional) on quality attributes is shown in tables 1 (physical and physio­ logical attributes including productivity) and 2 (chem­ ical composition) at harvest. both area of cultivation and cultivation system showed a significant effect (p<0.05) on most of physical attributes (table 1), with significant interaction only in the case of firmness, and chroma, but generally the effect of the cultiva­ tion system was the same in both locations. particularly conventionally grown grapes showed higher visual quality, firmness, and production per plant, which in turn induced lower dimensions of the berries. at the same time loc2 (bari) also induced adv. hort. sci., 2020 34(1s): 109­115 112 higher productivity, higher visual quality and firm­ ness compared to loc1 (taranto). respiration rate was higher in organic grown grapes than in conven­ tional grapes and in loc2 compared to loc1, indicat­ ing a higher metabolic activity for these fruit. as shown in table 2, total phenolics, flavonols and antioxidant activity were higher in organic grapes and in loc1, compared to respectively conventional grapes and loc2. for soluble solids content and titratable acidity there was a significant interaction, but looking to sugar and acid composition (data not shown), no significant differences due to the cultiva­ tion system were found for total organic acids, with significant highest amount of tartaric acid and glu­ cose, among sugars, in organically grown grapes and in loc1. differences among conventional and organic grapes were maintained, for each location, during storage at 0°c. conventional grapes maintained a higher visual quality during storage, resulting below the limit of marketability (score 3) but above the edi­ bility limit (score 2) at 14 days, whereas in loc1 (taranto) at that time organic grapes were judged not edible (data not shown). these results may be explained with the higher metabolic activity of organ­ ic table grapes, which reduce their storability. firmness was higher in conventional grapes than in organic grapes for samples grown in taranto area while no differences were observed for grapes grown in bari. also in another study the authors reported higher firmness for conventionally grown grapes than for organic, being related to the higher thickness of table 1 ­ productivity, morphological indexes, physical and physiological quality attributes at harvest of organic and conventional grapes grown in the locations of taranto (loc1) and bari (loc2) in the apulia region (italy) table 2 ­ chemical composition at harvest of organic and conventional grapes grown in the locations of taranto (loc1) and bari (loc2) in the puglia region (italy) treatment location location x treatmentorganic conventional loc1 loc2 production per plant (kg) 23.5 b 31 a 25 b 29 a ns visual score 4.1 b 4.5 a 3.9 b 4.7 a ns * hue angle 102.3 ns 103.7 ns 102.4 ns 103.7 ns ns chroma 7.5 ns 7.4 ns 8.1 a 6.9 b * respiratory activity 3.8 a 2.6 b 1.1 b 5.3 a ns morphological indexes berry lenght (mm) 29.6 a 27.4 b 27.8 b 29.3 a ns berry width (mm) 24 a 22.4 b 27.9 b 23.7 a * berry weight (g) 10.9 a 9.5 b 27.10 b 10.8 a ns peel thickness 0.99 ns 1.04 ns 27.11 ns 1.13 ns ns different letters indicate significant differences among mean values according to tukey’s test (𝑃 value≤0.05). ns= not significant. different letters indicate significant differences among mean values according to tukey’s test (𝑃 value≤0.05). ns= not significant. treatment location location x treatmentorganic conventional loc1 loc2 titratable acidity (ta) 0.4 b 0.5 a 0.5 a 0.4 b * soluble solids content (ssc) 1.70 ns 16.7 ns 16.9 ns 16.8 ns * ssc/ta ratio 41.4 a 37.6 b 38 ns 41 ns * flavan­3­oli 47.4 ns 48.5 ns 42.5 b 53.5 a * hydrocinammic deriv. 4.3 ns 4.6 ns 4.9 a 3.9 b ns flavonols 0.015 a 0.005 b 0.014 a 0.006 b ns total phenols 387.6 a 307.3 b 437.7 a 257.2 b ns antioxidant activity 940 a 641 b 987 a 594 b ns ascorbic acid 0.4 ns 0.4 ns 0.2 b 0.6 a ns dehydroascorbic acid 2.8 ns 2.8 ns 2.8 ns 2.8 ns ns amodio and colelli ‐ quality of ‘italia’ organic grapes at harvest and during storage 113 phenolic content of organic table grapes was, in gen­ eral, higher than that of conventional samples proba­ bly because the conventional growing practices uti­ lize levels of pesticides that can result in a disruption of phenolic metabolites in the plant that have a pro­ tective role in plant defense mechanisms (macheix et al., 1990). particularly, in the moment of harvest a phenolic content of 505.1±52.4 mg gallic acid 100 g­1 for organic sample and 369.8±57.8 mg gallic acid 100 g­1 for conventional table grapes grown in taranto were observed (p<0.05) (fig. 1); nevertheless, during storage this difference was progressively reduced becoming not significant after 6 days of storage. at harvest significantly higher values for antioxidant activity were observed for organic grapes (1210.7±134.3 mg trolox 100 g­1 fw) than in conven­ tional ones (763.4±97.6 mg trolox 100 g­1 fw ) (fig. 1) as well as after 6 and 14 days of storage at which organic samples showed an antioxidant activity of 992.5 mg trolox 100 g­1 fw and conventional samples of 850.9 mg trolox 100 g­1 fw. for samples grown in bari area significant differ­ ences were maintained between the phenolic con­ tent and antioxidant activity of organic and conven­ tional up to 6 days of storage (fig. 2) although, in this case antioxidant activity was much lower than in loc1. phenolic content of ‘italia’ grapes was higher com­ pared with those reported in literature studying the antioxidant and phenolic composition of different grape cultivars grown with conventional system ranged between 148.5 mg gallic acid 100 g­1 for ‘chasselas dorè’ and 123.1 mg gallic acid 100 g­1 for ‘nepoca’ grapes (mulero et al., 2010). similar differ­ ences were observed in some diffferent harvests (macheix et al., 1990). on the other hand, other authors pointed out that the concentration of pheno­ lic compounds of the skin changes greatly depending on the variety and also on the grape ripening stage (riu­aumatell et al., 2002). also in the case of antioxi­ dant activity values were higher in comparison with the results of reported on literature. values in the range of 55.7­274.2 µg g­1 extract in grape skins on four varieties are reported (anastasiadi et al., 2010), while the antioxidant activity was higher for organic grapes (5.70 mm trolox g­1) compared with conven­ tional grapes (4.40 mm trolox g­1) (mulero et al., 2010). as reported from several authors, high vari­ ability of phenols content in grapes may be caused by many factors including genotype, ripening stage, environmental and growing condition. among these organic farming have shown in different studies on the epicuticular layer (zahedipoura et al., 2019). similar results were also observed for organic kiwifruits (amodio et al., 2007), where firmness was not related to the thickness of the skin, being con­ ventional fruit firmer despite the thin skin and the more advanced maturity stage. in the present study, the slight differences in skin thickness resulted not significant, and difference observed for one location could be due to berry dimensions and water turgor. moreover, firmness of fruit can be affected by sever­ al agricultural practices such as sunlight exposure and (sams, 1999) fertilization. mineral content of soil and plant was not assessed in this experiment, but amodio et al. (2007) shown a possible effect of differ­ ent mineral composition of organic and conventional fruit. figures 1 and 2 show the changes of phenolic con­ tent and antioxidant activity of grape samples as a function of storage time for both conventional and organic production obtained in taranto and bari. fig. 1 ­ evolution of the antioxidant activity and phenol content during storage of organic and conventional table grapes grown in loc1 (taranto). different letters indicate signifi­ cant differences among mean values according to tukey’s test (𝑃 value ≤0.05). fig. 2 ­ evolution of the antioxidant activity and phenol content during storage in organic and conventional table grape grown in loc2 (bari). different letters indicate significant differences among mean values according to tukey’s test (𝑃 value ≤0.05). 114 adv. hort. sci., 2020 34(1s): 109­115 grapes (zahedipour et al., 2019) and other fruit as kiwifruits (amodio et al., 2007), and raspberry (ponder and hallmann, 2019) to positively affect antioxidant compounds, and particularly phenolics, representing a defense mechanism released from plant cells in response to biotic and abiotic environ­ mental stress (macheix et al., 1990; zhang et al., 2011). in organic farming, synthetic pesticides are banned, therefore, plants need to create their own defence mechanisms against pests and diseases (young et al., 2005). an increase of vitamin c and antioxidant enzymes activity was also observed in passion fruit (de oliveira et al., 2017). in terms of vit­ amin c content at harvest and during storage organic and conventional table grapes presented almost the same values without any significant differences. particularly, when the organic system was applied the vitamin c values were in the range of 1.46­3.12 mg 100 g­1 in taranto and 2.18­3.36 mg 100 g­1 in bari while for conventional samples vitamin c values were between 1.01­2.94 mg 100 g­1 in taranto and 1.90­ 3.22 mg 100 g­1 in bari (data not shown). many fac­ tors are responsible for the wide variation in vitamin c content of fruits and vegetables at harvest. maturity at harvest, harvesting method, and posthar­ vest handling conditions also affect the vitamin c content of fruits and vegetable (lee and kader, 2000). 4. conclusions results showed a higher phenolic content and antioxidant activity for grapes obtained with the organic farming system although in terms of visual quality, and yield performance conventional fruit had a better performance, resulting in a higher storability. these results confirmed previous finding on different antioxidant properties of organic fruit, but pointed out to the necessity of increasing their shelf­life in order to allow a better distribution, also considering international request of organic grapes. references amodio m.l., colelli g., hasey j.k., kader a.a., 2007 ­ a comparative study of composition and postharvest performance of organically and conventionally grown kiwifruits. ­ j. sci. food agric., 87: 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comparative study on quali‐ ty attributes and physiological responses of organic and conventionally grown table grapes during cold storage. ­ sci. hortic., 247: 86­95. zapata s., dufour j.p., 1992 ­ ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse phase ion interaction hplc. ­ j. food sci., 57(2): 506­511. zhang j., wang x., yu o., tang j., gu x., wan x., fang c., 2011 ­ metabolic profiling of strawberry (fragaria×ananassa duch.) during fruit development and maturation. ­ j. exper. bot., 62: 1103­1118. impaginato 273 adv. hort. sci., 2024 38(3): 273­279 doi: 10.36253/ahsc­16128 https://oaj.fupress.net/index.php/ahs pruning date and hydrogen cyanamide effects on growth and yield of grapevine var. cabernet sauvignon n. ghimire (*), p. sapkota, p. poudel, r. sapkota, k.c. dahal institute of agriculture and animal science, tribhuvan university kathmandu, nepal. key words: budbreaker, budburst, hydrogen cyanamide, pruning, viticulture. abstract: harvesting the grapes before the monsoon season is crucial to ensure the quality of berry and bunches. this study aims to identify the optimal win­ dow for pruning and hydrogen cyanamide (hc) application to prepone the berry harvesting. the experiment was conducted in randomized complete block design with five treatments and four replications. treatments were five differ­ ent pruning dates in 2021: jan. 17, jan. 24, jan. 31, feb. 7 and feb. 14, followed by 5% hc application one week after pruning. annual growth stages of grapevine were recorded by using modified e­l growth stage; reproductive attributes recorded during flowering; and vine yield and berry quality attributes recorded at harvesting. the earlier pruning resulted earlier budburst compared to late pruning. vines prunefed after feb. 7 had <50% budburst, while vines pruned before feb. 7 reached 50% budburst, exhibiting no differences in num­ ber of days needed to achieve it. jan. 17 pruning had the highest budburst (%) and bud fruitfulness (%), where pruning on feb. 7 had lower values. average bunch weight did not differ while berry quality attributes differed between treatments for the same day harvest. the negative responses in late pruned and hc treated vines, potentially attributed to the phytotoxic effect of hc on tender buds near the natural time of dormancy break. the early pruning and subsequent application of hc triggered earlier budburst, and advances flower­ ing and harvesting of berries in grapevine. this research demonstrated a poten­ tial techniques for advancing harvesting time (2­3 weeks) in grapevine. 1. introduction grape (vitis vinifera l.) is a non­climacteric berry fruit of the deciduous woody vines belonging to the family vitaceae, indigenous to eurasia (this et al., 2006). grapes can be consumed in fresh or processed products like juice, wine, vinegar, jelly, jam, grape seed oil, and raisins. cabernet sauvignon is a widely cultivated grape variety for wine due to its small berries with a higher concentration of tannin and coloring pigment (robinson et al., 2012). grape cultivation in nepal was started about 70 (*) corresponding author: ghimirenishes@gmail.com citation: ghimire n., sapkota p., poudel p., sapkota r., dahal k.c., 2024 ­ pruning date and hydrogen cyanamide effects on growth and yield of grape‐ vine var. cabernet sauvignon. ­ adv. hort. sci., 38(3): 273­279. orcid: ng: 0009­0004­9674­7420 ps: 0009­0006­7058­2108 pp: 0000­0002­0855­9103 rs: 0000­0002­7342­1071 kcd: 0000­0001­5147­8037 copyright: © 2024 ghimire n., sapkota p., poudel p., sapkota r., dahal k.c. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwisenoted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interest. received for publication 9 may 2024 accepted for publication 5 august 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16128 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0004-9674-7420 http://orcid.org/0009-0006-7058-2108 http://orcid.org/0000-0002-0855-9103 http://orcid.org/0000-0002-7342-1071 http://orcid.org/0000-0001-5147-8037 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 273­279 274 years ago but there was no significant expansion of viticulture farms (dahal et al., 2017). currently, few commercial vineyards are producing mainly wine grapes while table grapes are in small quantities. the estimated production of nepal was 76 t from 20 ha area with a productivity of 8.5 t ha­1 (atreya et al., 2015) but the demand of grape has been increasing manifolds in recent years (acharya et al., 2023). it shows there is a huge demand for grapes along with bottlenecks of nepalese viticulture. the main chal­ lenge is harvesting time coincides with the rainy sea­ son causing a high risk of fungal diseases and insect infestation during ripening resulting in inferior quality berries and bunch (shrestha, 1998). such problems are also common in humid subtropical areas in other region too. grapevine dormant buds starts the resumption of annual growth cycle after winter in most of the sub­ tropical and temperate climates. chilling require­ ment and breaking dormancy are crucial affecting temperate fruit trees when grown in tropical climates (botelho and müller, 2007). various chemicals, such as mineral oil (black, 1936), dinitro­ortho­cresol (erez and sur, 1981), thiourea (blommaert, 1965), garlic extract (botelho and müller, 2007), and hc have been used to break dormancy in grapevine. among these chemicals, hc has great efficiency in bud break­ ing (nir and levee, 1993) as well as enhances uniform and rapid bud breaking (mccoll, 1986; halaly et al., 2008). however, the effect of hc depends upon the time and concentration used. hydrogen cyanamide breaks endo­dormancy by respiratory disturbance, hormonal signaling, and oxidative stress (liang et al., 2019). this research was purposed to advance the natural budburst period, which might lead to bunch­ es being harvested before monsoon. to harvest the crop before monsoon, breaking of bud dormancy earlier than natural time is required. in the warmer climates, pruning followed by hc are employed to induce budburst. aiming to prepone natural budburst and to allow the berry harvesting before the heavy rainfall or monsoon. 2. materials and methods the experiment was conducted from january 17 to july 04, 2021, at the commercial vineyard (27°44’ n, 85°6’ e) in dhading, on a south­facing plot with a gentle slope at an altitude of 800 meters above mean sea level. the grapevine cv. cabernet sauvignon (6 years old) grafted on 5c rootstock, was selected for the research. five pruning dates followed by hc application treatments was arranged on a randomized complete block design with five replica­ tions considering a vine as replication (table 1). to differentiate treatments, vines within replica­ tion were tied with different colored ribbons. one­ year­old vines were spur­pruned, leaving three basal buds per spur. ten spurs from each vine were select­ ed and tagged with different colored threads. buds in spur were marked as 1, 2, and 3 from the basal to distal. hence, 30 buds were marked per vine. phenological observations using the modified eichhorn and lorenz (e­l) grapevine growth stages scale began on february 16, 2021, and carried out in every four days interval until april 26, 2021. the growth stages, reproductive attributes during flower­ ing, vine yield, and berry quality attributes were recorded at harvest. development parameters such as budburst, fruitfulness in total buds, and fruitful­ ness in burst buds were calculated using the follow­ ing formulas: budburst (%) = number of burst buds/total buds x 100 observed fruitfulness (%) = number of buds with inflorescence(s)/ total buds x 100 fruitfulness in burst buds (%) = number of buds with inflores­ cence(s) / total burst buds x 100 all bunches of each treatment were harvested on the same day, july 4, 2021 to ensure that the mini­ mum standard quality berries and bunches harvested before the monsoon arrives. quantitative attributes were measured from randomly selected 10 bunches. the qualitative attributes (total soluble solid and total titratable acidity) were assessed by randomly selected 10 berries from each selected bunch (dahal et al., 2019). data recorded from the field were table 1 ­ description of treatment details and coding of treat­ ment practiced in a commercial vineyard, dhading, nepal, 2021 treatments pruning date 5% hc application date treatment code t1 january 17 january 24 j17j24 t2 january 24 january 31 j24j31 t3 january 31 february 07 j31f07 t4 february 07 february 14 f07f14 t5 february 14 february 21 f14f21 ghimire et al. ‐ pruning and hydrogen cyanamide effect on grapevines 275 entered, tabulated and analyzed using ms excel 12 and genestat version 18.1. 3. results phenological observations annual growth stages of grapevine. considerable variation in average e­l growth stages among differ­ ent treatments were observed throughout the exper­ imental period as shown in table 2. lower values were recorded in later treated vines (f07f14 and f14f21) while early treated (j17j24 and j24j31) vines had higher values of e­l stages. at the last date of observation (26th april), the average e­l stage of j17j24 and f07f14 were 19.69±6.27 and 3.21±2.98, respectively. number of days to budburst. the number of days to first budburst differed significantly between treat­ ments while treatments did not significantly differ in days to 50% budburst (table 3). budburst was earlier d= date on phenological observation was done. d0 = 16th feb.; d4 = 20th feb.; d8 = 24th feb.; d12 =28th feb.; d16 =4th mar.; d20 = 8th mar.; d24 = 12th mar.; d28 = 16th mar.; day32= 20th mar.; d36 =24th mar.; d40 = 28th mar.; d46 = 3rd apr.; d49 =6th apr.; d51= 8th apr.; d55 = 12th apr.; d59 = 16th apr.; d65 = 22nd apr.; d69= 26th apr.; values are µ ± se where µ = mean stage; lsd = least significance difference; ** highly significant at α =5%; ns = not significant. table 2 ­ average e­l stage of grapevine buds in different dates of pruning and hc application, dhading, nepal, 2021 observation day average e­l growth stage in treatments statistical analysis j17j24 j24j31 j31f07 f07f14 f14f21 grand mean lsd (α =5) d0 1.39±0.38 c 1.07±0.11 b 1.03±0.07 b 1.01±0.04 a 1.01±0.04 a 1.10 0.105 ** d4 1.73±0.52 d 1.33±0.24 c 1.16±0.16 b 1.01±0.04 a 1.01±0.04 a 1.25 0.101 ** d8 2.23±0.8 d 1.71±0.45 c 1.31±0.26 b 1.02±0.06 a 1.01±0.05 a 1.47 0.279 ** d12 3.26±1.17 d 2.39±0.76 c 1.73±0.38 b 1.05±0.10 a 1.03±0.07 a 1.89 0.514 ** d16 4.97±1.69 d 3.76±1.4 c 2.71±0.88 b 1.1±0.18 a 1.09±0.15 a 2.72 0.836 ** d20 6.11±1.92 c 5.08±1.84 c 4.14±1.55 b 1.17±0.33 a 1.17±0.24 a 3.53 1.044 ** d24 7.74±2.34 c 6.57±2.29 bc 5.65±2.11 b 1.27±0.46 a 1.43±0.45 a 4.53 1.392 ** d28 8.7±2.55 c 7.25±2.48 bc 6.08±2.24 b 1.39±0.61 a 1.78±0.73 a 5.07 1.527 ** d32 9.35±2.76 c 8.05±2.73 bc 6.86±2.52 b 1.51±0.77 a 2.14±0.95 a 5.61 1.735 ** d36 10.85±4.75 c 8.9±3.02 b 8.11±2.91 b 1.71±1.04 a 2.85±1.39 a 6.48 1.895 ** d40 11.16±3.26 c 9.93±3.38 bc 8.48±3.02 b 1.91±1.29 a 3.36±1.71 a 6.97 2.161 ** d46 12.43±3.63 c 11.1±3.78 bc 9.69±3.46 b 2.12±1.54 a 4.25±2.27 a 7.92 2.510 ** d49 13.79±4.04 c 11.98±4.13 bc 10.58±3.93 b 2.31±1.8 a 4.87±2.62a 8.13 3.017 ** d51 15.1±4.56 b 12.76±4.53 b 12.57±4.52 b 2.50±2.03 a 5.49±3.02 a 9.70 3.525 ** d55 16.09±5 b 13.82±5.02 b 13.61±5.02 b 2.69±2.28 a 6.21±3.49 a 10.48 3.725 ** d59 16.9±5.28 b 14.72±5.32 b 14.63±5.35 b 2.87±2.5 a 6.97±3.97 a 11.21 4.140 ** d65 18.33±5.74 b 16.26±5.92 b 16.00±5.86 b 3.05±2.75 a 7.83±4.52 a 12.25 5.081 ** d69 19.69±6.27 b 17.65±6.46 b 17.53±6.46 b 3.21±2.98 a 8.62±5.06 a 13.29 5.679 ** table 3 ­ effect of pruning date followed by hc application on number of days to 1st and 50% budburst, dhading, nepal, 2021 mean with the same letter(s) within the column do not differ significantly by dmrt at 5%. values are µ±se where µ = mean and se = standard error. lsd=least significance difference. cv = coefficient of variance. na= not applicable. treatments days to the first budburst days to 50% budburst j17j24 0.8 ±0.8 a 16.8 ±2.8 j24j31 8.0 ±1.26 ab 17.2 ±0.8 j31f07 10.4 ±1.6 b 19.0±1.03 f07f14 26.4 ±3.7 c na f14f21 24.8 ±4.08 c na grand mean 14.1 17.67 lsd 7.93 na f­probability <0.001 na cv% 42.0 19.1 adv. hort. sci., 2024 38(3): 273­279 276 in the early pruned vine as compared to late pruned grapevines. early pruned (jan. 17) grapevine burst their first bud in 0.8±0.8 days while late pruned vines (feb. 7) took 26.4±3.7 days after hc application. growth and development observations budburst percentage. the overall budburst was less than 50% for all treatments. significant differ­ ences between treatments were found in different observation dates after treatment application. early pruned and hc treated vines (j17j24, j24j31, and j31f07) were not only early in budburst, but they also had higher budburst (%) compared to late pruned and hc treated vines. late pruned and hc treated vines had lower budburst (%) even around 10% as shown in figure 1. hc application close to nat­ ural budburst time damages the buds and buds did not sprout (<4 e­l growth stage), hence late pruned and hc treated (f07f14 and f14f21) vines did not reach the 50% budburst. observed fruitfulness in total buds. the overall flowering of all treatments was less than 40%, how­ ever, significant difference was observed between treatments on different dates of observations (fig. 2). flowering (%) variation among treatments follows a similar trend to that of budburst percentage as shown in figure 1 and figure 2. late pruned and hc treated vines had lower flowering (%) and delayed in flowering. vine took 40­49 days to flower after bud­ burst. late pruned and hc treated vines started to flower after 49 days while earlier treated vines start­ ed flowering after 40 days. fig. 3 ­ effect of different timing of pruning followed by hc appli­ cation on observed fruitfulness. means are separated with different letter(s) for the respective day of observa­ tion using duncan’s multiple range test at 5%. fig. 2 ­ effect of different timing of pruning followed by hc appli­ cation on observed fruitfulness. means are separated with different letter(s) for the respective day of observa­ tion using duncan’s multiple range test at 5%. fig. 1 ­ effect of different date of pruning followed by hc appli­ cation on budburst (%). means are separated with differ­ ent letter(s) for the respective day of observation using duncan’s multiple range test at 5%. fruitfulness in burst buds. flowering percentage in burst bud was insignificant between treatments from the last date of bud observation (fig. 3). on april 26, the flowering (%) observed in the burst bud was 82.6% with the death of average 10% buds and the remaining bud did not reach the flowering stage. treatments j17j24, j24j31, j31f07, f07f14, and f14f21 had bud death percentage as 14.28%, 13.41%, 6.98%, 6.25%, and 4% of total burst buds, respectively. yield attributes. the tss and ta of berries were significantly different between treatments (table 4). early pruned and hc treated vines produced berries with higher tss and lower tta. tss and tta of j17j24, j24j31, j31f07, and f07f14 were statistically similar to each other while f14f21 had a higher tta and lower tss value. the average bunch weight was similar for all treatments (table 4). ghimire et al. ‐ pruning and hydrogen cyanamide effect on grapevines 277 4. discussion and conclusions this study showed the growth and phenological stages were significantly influenced by the date of pruning followed by 5% hc application in grapevine cv. cabernet sauvignon. budburst depends on warm or forcing conditions in some cultivars after endo­ dormancy (keller and tarara, 2010). the general threshold temperature for shoot development is 10°c. to some extent, the budburst date corresponds to the cumulated temperatures above this threshold (lebon et al., 2004). martin and dunn (2000) found that hc did not significantly affect the times of the onset of budburst, 60% budburst, anthesis or verai­ son, or fruit maturity at harvest, but interacted signif­ icantly with later pruning to delay fruit maturity. martin and dunn (2000) reported that six­week dif­ ferences in pruning time resulted in 5 days differ­ ences in budburst time in the cultivar cabernet sauvignon in melbourne, australia. hc treatment prepone budburst of vine due to increased accumula­ tion of h2o2, soluble sugar/starch ratio, iaa, and cytoplasmic protein­tyrosine kinase concentration with decreased aba concentration (liang et al., 2019). delayed winter pruning postponed 10­11 days for budburst which reported a possible solution to prevent spring freeze damage (persico et al., 2021). in late pruned and hc treated vine, dramatic decrease in budburst was observed. low budburst is potentially linked to phytotoxic effects caused by late application of hc. as the natural budburst time approaches, buds are succulent and vulnerable to the toxic effect of hc. george and nissen (1988), george et al. (1988), and shulman et al. (1983) also reported the drying out of young shoots due to too early or late application of hc. early burst bud may dry out as succulent and young shoots have to face the frost. in most fruit trees, the biggest effect of hc reported when applied few weeks before the natural budburst (pontikis, 1989). while the late application (f14f21) may not have an impact because the chemical resis­ tance reduces quickly after being released from endo­dormancy (snir, 1988; klinac et al., 1991). using two different hc application dates (mid­dec. and mid­jan.), or et al. (1999) reported that there was no discernible difference in the budburst (%). in both dates, the budburst was found to be 50% after four weeks and an additional 20% within the following two weeks. cabernet sauvignon has a shorter dura­ tion of budburst to flowering period as compared to merlot and cabernet franc in bordeaux, france (leeuwan et al., 2004). further they reported that the low yield was typically associated with low bud­ burst rates, while hc influence on grapevine yield has been attributed to its impact on budburst. a high level of budburst would result in an increased shoot number and, therefore, a high yield (or et al., 1999). the phytotoxic effect of hc acting upon naturally burst tender buds has been reported since its early application (shulman et al., 1983). lower budburst and flowering in late pruned and hc treated vines result in lower fruitfulness. bud fruitfulness depends upon climatic variables mainly sunshine and daily maximum temperature between 82°f and 90°f and water availability (williams, 2000). unfavorable climatic conditions hasten diseases and insect infestation such as anthracnose, downy mildew, mealy bugs, thrips, and leaf hoppers that result in burst bud mortality (somkuwar et al., 2021). vine productivity is a distinguishing feature of a variety that fluctuates depending on several parame­ ters, such as rootstock, and vine management. the significance of pruning in the grapevine is relatively consistent (rives, 2000). martin and dunn (2000) found that the earlier pruned (7 july) vines matured earlier than the later pruned (17 august) vines, and the mean tss of the berry was 0.91°b lower for the later pruned vines. dhakal (2021) found insignificant difference between the average bunch weight of vine pruned at different times followed by hc application table 4 ­ effect of pruning date followed by hc application on yield attributes of grapevine, dhading, nepal, 2021 mean with the same letter(s) within the column do not differ significantly by dmrt at 5%. values are µ±se where µ = mean and se = standard error. lsd=least significance difference. cv = coefficient of variance. na= not applicable. treatments total soluble solid (°b) tta (g/l tartaric acid) average bunch weight j17j24 18.96±0.55 b 9.93±0.81 a 69.56±14.31 j24j31 19.26±0.59 b 9.93±0.32 a 62.86±8.81 j31f07 18.84±0.89 b 10.27±0.94 a 70.06±6.98 f07f14 17.98±0.43 b 10.87±0.57 a 74.92±12.04 f14f21 16.08±0.71 a 12.72±0.3 b 70.05±6.70 grand mean 18.22 10.75 69.5 lsd (5% level) 1,829 1,537 na f­probability 0.013 ** 0.00 ** 0.96 ns cv (%) 7.5 10.7 35.50 278 adv. hort. sci., 2024 38(3): 273­279 in kirtipur, nepal. this research demonstrated the advancing bud­ burst date through pruning and hc application ulti­ mately advances the harvesting time. thus, it can be a potential and viable strategy to address the chal­ lenges of monsoon coinciding with harvesting time in grapevine growing areas. this study demonstrated that shoot pruning during second fortnight of january followed by 5% hc application advances 2­3 weeks in harvesting of grapevine without compromising in minimum acceptable berry quality. particularly, in the subtropical conditions of nepal, it is recommend­ ed to apply hc before feb. 7th to optimize its effects with 5% hc application. acknowledgements authors express gratitude to the tu, directorate of research (crg grape) for funding this research. additionally, we are thankful to mr. kumar karki, ceo of kewalpur agro farm located in thakre­10, dhading, for all possible arrangement to conduct this trial. references acharya a.k., acharya, s., kushwaha, a., dahal k.c., 2023 ­ understanding bud fruitfulness and and impor‐ tance of gibberellic acid (ga3) application(s) in success‐ ful grapevine cultivation. ­ proceeding 2nd international conference on horticulture, 3­4th april, lalitpur, nepal. atreya p.n., lamicchane m., kafle k., 2015 ­ commercial grape producton: technical bullettin. ­ fdd/doa/moa/government of nepal, 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(ed.) raisin production manual. university of california, agriculture and natural resources, ucanr publications, california, usa, pp. 295. impaginato 11 adv. hort. sci., 2021 35(1): 11­19 doi: 10.36253/ahsc­9548 improving ‘piyarom’ date palm fruit quality with fruit thinning and bunch covering treatments b. madani 1 (*), a.m. dastjerdy 2 (*), a. shahriyari 1 1 horticultural crops research department, natural resources research and education center of hormozgan, areeo, bandar abbas, iran. 2 department of horticultural science, faculty of agriculture and natural resources, university of hormozgan, bandar abbas, iran. key words: khalal, kimri, phoenix dactylifera l., quality, tamar. abstract: ‘piyarom’ dates are one of the most important commercial date culti­ vars grown in iran but fruit quality and postharvest losses can be a problem. thinning and the use of bunch covers were examined to improve the date fruit quality. three different levels of fruit thinning were applied (without thinning, removal of one third of total strands from terminal tips during pollination and removal of one third of terminal tips of central strands in early kimri). in addi­ tion, the effects of bunch covers were assessed with the use of polyethylene bunch covers that were applied in the early stages of khalal. the results showed that bunch covering reduced the lightness of the fruit but increased bunch weight, fruit length, total antioxidant activity and total phenolic com­ pounds compared to the control. thinning during pollination increased the weight of the fruit compared to the control. the thinning during kimri stage increased the percentage of tamar and ascorbic acid content. these results show that the use of covers play a positive role in increasing quality and bunch weight and thinning at both pollination and kimri ripening stage is recommend­ ed to produce high quality ‘piyarom’ dates. 1. introduction date palms (phoenix dactylifera l.) are an ancient horticultural crop cultivated for its sweet fruit which are also rich in nutrients, antioxidants and phenolic compounds (hussain et al., 2016). iran is one of the largest producing countries of dates growing 171,647 hectares of date palms with an annual production of 1,204,158 tons (fao, 2018). date fruit has five stages of growth and ripening; (1) hababouk (cell division and elongation), (2) kimri (firm full colored), (3) khalal (physiolog­ ically mature with a hard and crisp texture and a moisture content between 50­85%), (4) rutab (partially browned, reduced moisture con­ tent 30­45%, fibres softened, perishable), and (5) tamar (fruit colour from amber to dark brown with a moisture content further reduced ­ below 25% down to 10% and less, and the texture is soft pliable to firm to hard) (*) corresponding author: babakmadani2010@gmail.com mirzaalian@hormozgan.ac.ir citation: madani b., dastjerdy a.m., shahriyari a., 2021 ­ improving ‘piyarom’ date palm fruit qua‐ lity with fruit thinning and bunch covering treat‐ ments. ­ adv. hort. sci., 35(1): 11­19 copyright: © 2021 madani b., dastjerdy a.m., shahriyari a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2020 accepted for publication 24 december 2020 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9548 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 11­19 12 (awad and al­qurashi, 2012). date palm (phoenix dactylifera l.) cv. piyarom is one of the most impor­ tant commercial semi­dry dates in iran which is con­ sumed at tamar ripening stage and fruit reaches full maturity. it is widely grown in hormozgan province of iran with increasing export production. in com­ mercial production, larger fruit with higher quality are more marketable and therefore fruit thinning is one of the main agronomic methods to improve fruit size and quality. fruit thinning is widely used in horti­ cultural production which increases fruit quality, reduces bi­annual bearing and ensures a physiologi­ cal balance between vegetative and reproductive parts due to reduced competition for water and food absorption (slatnar et al., 2020). there are different times and methods for date palm thinning. for exam­ ple, thinning 1/3 of the bunch at pollination period or 1/3 terminal tips of central strands in early kimri stage of fruit development, as has been described by el­badawy et al. (2018) and moustafa et al. (2019). however some date palm cultivars can react contrari­ ly to the different treatments of thinning for exam­ ple, awad and al­qurashi (2015) reported that thin­ ning 5­10 cm bunch four weeks after pollination in ‘barhee’ date did not affect the qualitative characters of fruit. however, ahmed et al. (2019) showed that thinning 1/3 terminal tips of central strands in early kimri stage of fruit development of ‘zaghlool’ decreased titrable acidity and increased soluble solid content. however there has been no research on the effects of different thinning options on the quality of ‘piyarom’ date where most farmers in southern iran currently use bunch thinning only at pollination peri­ od. pre­harvest covering of fruit is practiced in many crops and its usage is increasing in many countries to improve fruit quality. however, the effects of fruit covering on final fruit quality is variable and reflect differences in the covering type, fruit age at covering and cultivar response (sharma et al., 2014). for example kassem et al. (2010) stated that ‘zaghlool’ dates covering with dark polyethylene at kimri stage did not affect total acidity (ta) and total soluble solid content (tss). however, awad and al­qurashi (2012) concluded that bunch craft bagging after pollination increased bunch weight, ta and tss, ascorbic acid and decreased total phenols and rutab percentage of ‘barhee’ date. harhash and al­obeed (2010) showed that bunch bagging increased fruit and bunch weight and quality of ‘succary’ and ‘khalas’ dates. harhash et al. (2020) further showed that bunch weight was greatly increased by covering bunches with white paper and white and brown cloth as compared to control. fruit weight was increased remarkably by using white or kraft paper and white or brown cloth in covering bunches of ‘barhee’ date palm while tss was increased in the fruit which were produced from bunch covering with white or kraft paper and white or brown cloth over control. due to its high consumer appeal, ‘piyarom’ date is a promising cultivar for export but a more consistent high fruit quality is required. therefore, this study assessed the effects of both date fruit thinning and the use of bunch covers on the bunch weight and quality of ‘piyarom’ dates over two seasons. 2. materials and methods experimental procedure fruits selected for this trial were from ‘piyarom’ date palms of the same age (18 years old) and size which were grown on sandy loam soil and drip irri­ gated at the hajiabad agricultural research station in hormozgan province in iran for two years during 2018­2019. all regular cultural practices were con­ ducted according to the normal commercial sched­ ules. pollination was conducted using a local ‘green’ male cultivar to manually pollinate the female flow­ ers in april each year with a total of eight 8 bunches were left on each experimental tree. this trial was a factorial trial with fruit thinning and bunch covers as the two factors. within the fruit thinning treatment, three levels of thinning were examined; (1) removal of one third of the end of total strands from terminal tips of bunch at the time of pollination, (2) removal of one third of terminal tips of central strands in early kimri (performed in june), and (3) untreated flowers (control). the second fac­ tor of experiment was bunch covering at two levels (uncovered bunches and bunch covering with poly­ ethylene mesh which consisted on a 100 cm long and 80 cm wide bag with mesh holes (diameter of each hole is about 2 mm) which were applied in early stage of khalal. the thinning treatments and bunch coverings were performed on four bunches in each palm tree, and fruit sampling of these bunches was performed at harvest. in november on each tree, all bunches were harvested and bunch weight per palm was recorded. the tamar percentage was also calcu­ lated. fruit samples were collected at the tamar stage for physicochemical characteristics measure­ madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 13 ments. physico‐chemical characters at harvest time (tamar stage), 100 fruit subsam­ ples from each replicate were sampled from palm trees and the factors related to the colour of the fruit measured by minolta (cr­300, minolta corp, osaka, japan) colorimeter and expressed as l*, c* and h◦. fruit weight, length and diameter of each fruit and seed were measured on 25 fruit per replicate. fruit and seed length and diameter were measured by a digital caliper and the weight of the fruit and seed were measured by a digital scale. fruit total sol­ uble solid content (tss), ascorbic acid, moisture con­ tent, total phenolic compounds and total antioxidant activity were measured on a composite sample of 20 fruit per replicate. tss was measured as brix % with a digital refractometer (dbr95, taiwan). the spec­ trophotometric method was used for measuring ascorbic acid content (etemadipoor et al., 2019). ten ml of distilled water used for homogenizing one gram of fruit tissue. then, the solution filtered and 0.1 ml of it was added to 1% metaphosphoric acid. then, solution combined with 9 ml of 2, 6­dichloro indophenol (0.0025%). after that, the mixture absorbance was evaluated by spectrophotometer (cecil, ce2501, england) at 517 nm. the results were reported accordingly (mg/100 g fresh fruit tissue). for moisture content 20 g of fruit sample from each replicate was cut into small pieces by a sterilized knife. then, the fresh weight of each sample was measured. then, dried to a constant weight in oven at 70◦c. dry weight was measured and fruit moisture expressed as percentage (marzouk and kassem, 2011). total antioxidant activity (taa), total phenolic con‐ tent (tpc) measuring taa involved adding 1 ml 2,2­ diphenyl­1­picryl­hydrazyl (dpph) (0.1 mm) to 0.1 ml methanolic extract. this solution was subsequently mixed with 1 ml of tris­hcl (ph 7.5). the absorption value of each sample was determined at 517 nm wavelength via spectrophotometer (cecil, ce2501, england) (etemadipoor et al., 2020). the antioxidant activity was determined as follows: antioxidant activity (%) = [1 ­ (abs sample /abs control) × 100] the folin­ciocalteu reagent was used for measur­ ing tpc (ehteshami et al., 2019). briefly, fruit tissue (0.5 g) was squashed and mixed into methanol (3 ml, 85%). the resultant solution was centrifuged at 12,000 × g at 4°c for 20 min. the extract was consid­ ered as supernatant. then, 300 μl of extract was combined with sodium carbonate (1.2 ml, 7%). this mixture was stored at ambient temperature for 5 min. after that folin­ciocalteu (10%, 1.5 ml) was added. the absorbance was determined at 760 nm and tpc was presented as mg gae 100 g­1 fw. statistical analysis of data the experimental was as factorial in a randomized complete block design with three replicates (block) and each replicate consisted of two palm tree. two factors included bunch thinning and bunch covering. bunch thinning consisted of 3 levels and bunch cov­ ering consisted of 2 levels. the treatment unit con­ sisted of four bunches on each of the two tree treat­ ment blocks. data were analyzed by statistical analysis system version 9.1 (sas institute inc., cary, nc, usa). the means were compared using lsd at a significance level of p= 0.05. 3. results and discussion bunch weight and tamar percentage the results of this experiment showed that the coverage did not have a significant effect on the per­ centage of tamar, but date palm fruit thinning in the kimri and pollination stages significantly increased tamar percentage of both years (table 1). the tamar percentage was approximately 94.25­113.75% higher than the control group and occurred as a result of thinning in the kimri stage during both years of experiment. also, the bunch weight was significantly higher in the control as compared to bunch weights that occurred after date palm fruit thinning at the pollination and kimri stages, for both years (table 1). in this research, the tamar percentage was not affected by covering. in the available literature, there are inconsistent results on the use of covering for fruit ripening. for example, awad and al­qurashi (2012) stated that bunch bagging of ‘barhee’ date can eventually reduce the percentage of rutab in comparison with the control. however, kassem et al. (2011) stated that spathe­bagging increased fruit ripening in ‘zaghlool’ date, as compared to the con­ trol. tamar is the last stage of ‘piyarom’ date fruit maturity. however, date fruit do not ripen evenly, and even in a bunch rutab and khalal can be observed at harvest. this causes economic loss of fruit. therefore, increasing tamar percentage in the bunch can rise the commercial value of product. the adv. hort. sci., 2021 35(1): 11­19 14 increase in the percentage of tamar, as a result of the thinning treatment, can be due to a lowered level of competition between fruit for the absorption of water and nutrients, thereby accelerating fruit matu­ rity and increasing the percentage of tamar (radwan, 2017). the results of this study on increasing bunch weight of the control, as compared to the date palm fruit thinning treatment, are consistent with previous results (moustafa et al., 2019) on the khadravi culti­ var. however, the primary goal is to determine an optimum method of date palm fruit thinning so as to improve fruit quality, obtain a reasonable bunch weight and thus enhance marketability. taa, tpc the antioxidant properties of date fruit differ depending on the amount of phenolic compounds in the fruit (hussain et al., 2016). phenolic compounds account for most of the antioxidant properties of dates. they exhibit a range of biological effects such as the prevention of nucleic acid damage. there has been growing interest in the topic of antioxidants, regarding the ability of antioxidants to scavenge free radicals associated with various diseases (aleid, 2014). thus, dates can be used as antioxidative func­ tional food ingredients (aleid, 2014). fruit of differ­ ent date palm cultivars have different total phenolic contents and antioxidant activities (al­turki et al., 2010). in the current study, comparison of the main effects of date palm fruit thinning on total antioxi­ dant activity (taa) showed that date palm fruit thin­ ning during the kimri phase increased the taa as compared to the control (table 2). also, date palm fruit thinning during the kimri phase increased the tpc as compared to the control for both years. moreover, bunch covering increased taa and tpc table 1 ­ main effects of bunch date palm fruit thinning and covering on bunch weight and tamar percentage of ‘piyarom’ date z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. treatment bunch weight (kg) tamar (%) 2018 thinning control 15.49 az 29.16 c removal of one third of total strands from terminal tips during pollination 13.44 b 49.33 b removal of one third of terminal tips of central strands in early kimri 11.37 c 62.33 a lsd 4.89 3.82 bunch covering control 13.09 b 47.66 a bunch covering 13.77 a 46.22 a lsd 3.99 3.12 thinning x bunch covering ns y ns 2019 thinning control 15.51 a 32.00 c removal of one third of total strands from terminal tips during pollination 14.05 b 45.66 b removal of one third of terminal tips of central strands in early kimri 11.00 c 62.16 a lsd 3.23 5.10 bunch covering control 13.29 b 46.33 a bunch covering 13.75 a 46.88 a lsd 2.64 4.16 thinning x bunch covering ns ns table 2 ­ effect of fresh organic amendments on nematode population, gall number and galling index treatments taa (%) tpc (mg gae 100 g fw) 2018 thinning control 49.66 c z 130.16 b removal of one third of total strands from terminal tips during pollination 67.26 b 135.45 ab removal of one third of terminal tips of central strands in early kimri 70.43 a 138.21 a lsd 2.89 5.73 bunch covering control 58.73 b 131.23 b bunch covering 66.17 a 137.98 a lsd 2.36 4.68 thinning x bunch covering ns y ns 2019 thinning control 48.18 c 126.51 b removal of one third of total strands from terminal tips during pollination 67.83 b 141.16 a removal of one third of terminal tips of central strands in early kimri 71.10 a 140.06 a lsd 2.21 4.15 bunch covering control 57.31 b 134.28 b bunch covering 67.43 a 138.21 a lsd 1.80 3.39 thinning x bunch covering ns ns z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 15 compared to the control for both years (table 2). little research exists on the effects of bunch thinning and covering on taa and tpc of date. hussain et al. (2016) stated that thinning treatments of ‘hillawi’ and ‘khadrawi’ increased tpc in comparison with the control, probably because of a greater light exposure to the fruit in response to less fruit density in each bunch. indeed, light plays an important role in processes that are responsible for the accumulation of phenolic compounds. on the other hand, in fruit of the control group, there were lower amounts of light and air circulation available to the fruit, thereby affecting the rate of photosynthetic carbon assimila­ tion rate and tpc (hussain et al., 2016). the contra­ dictory effects of covering on phenolic­compound might be due to differences in the covering material, exact time and period of covering, cultivars and cli­ matic conditions (sharma and sanikommu, 2018). chen et al. (2012) stated that fruit bagging reduced the concentration of phenolic compounds in ‘golden delicious’ apple, whereas griñán et al. (2019) report­ ed that pomegranate fruit bagging increased the antioxidant content. physiochemical characteristics comparison of main effects of coverage on color lightness, chroma and hue showed that coverage sig­ nificantly reduced the lightness and chroma, while at the same time increased the hue in comparison with the control for both years (table 3). also, the com­ parison of main effects of thinning on lightness, chro­ ma and hue showed that thinning in pollination and the early kimri stage significantly reduced the light­ ness and chroma, compared to the control. in addi­ tion, this treatment increased the hue in fruit, com­ pared to the control for both years (table 3). the colour of date fruit is one of the most important fac­ tors that can largely influence customers and can determine prices. so far, the available literature does not include measurements of lightness, chroma and hue when bunch covering and thinning are used as treatments for date palm. the positive correlation table 3 ­ main effects of bunch date palm fruit thinning and covering on colour, tss, ascorbic acid and moisture of ‘piyarom’ date z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. treatment l (*) c (*) h (◦) tss brix (%) ascorbic acid (mg 100 g­1) moisture (%) 2018 thinning control 30.31 a z 11.99 a 40.83 c 61.11 b 6.06 c 12.13 a removal of one third of total strands from terminal tips during pollination 25.66 b 9.45 b 46.98 b 63.11 b 11.25 b 11.25 a removal of one third of terminal tips of central strands in early kimri 22.58 c 7.12 c 51.80 a 66.05 a 13.46 a 11.86 a lsd 2.19 1.49 2.60 2.41 1.11 1.19 bunch covering control 27.50 a 10.19 a 45.21 b 63.01 a 10.68 a 11.95 a bunch covering 24.84 b 8.85 b 47.86 a 63.84 a 9.83 a 11.54 a lsd 1.79 1.22 2.13 1.97 0.90 0.97 thinning x bunch covering ns y ns ns ns ns ns 2019 thinning control 28.75 a 11.14 a 44.71 c 62.01 c 6.81 c 10.76 a removal of one third of total strands from terminal tips during pollination 24.06 b 8.05 b 50.30 b 64.88 b 12.13 b 11.60 a removal of one third of terminal tips of central strands in early kimri 21.90 b 6.36 c 55.73 a 67.45 a 13.66 a 10.41 a lsd 2.41 1.25 3.34 2.26 1.28 1.61 bunch covering control 25.90 a 9.17 a 47.87 b 64.96 a 11.34 a 11.09 a bunch covering 23.91 b 7.86 b 52.62 a 64.60 a 10.40 a 10.75 a lsd 1.96 1.02 2.72 1.85 1.04 1.31 thinning x bunch covering ns ns ns ns ns ns 16 adv. hort. sci., 2021 35(1): 11­19 between taa and hue has been shown in table 4. based on our results, bunch covering increased taa which can be effective in improving the colour of the fruit (siddiq et al., 2013). the mechanism involves the formation of dark pigments on fruit, thereby reducing the brightness of the fruit and increasing hue in fruit (siddiq et al., 2013). on similar accounts, covering has reportedly improved the colour of apple (sharma and pal, 2012). a more pronounced fruit colouring is one of the most important goals of thin­ ning. the increase in fruit colour, as a result of the date palm fruit thinning treatment, is due to the absorption of more nutrients in the remaining fruit and also due to the release of more sugars that lead to antioxidant compounds and dark pigments in fruit (hussain et al., 2016). these results are comparable with previous results reported by ahmed et al. (2019) where date palm fruit thinning increased the colour index in ‘zaghlool’ date fruit. the results of statistical compound analysis showed that bunch covering did not affect significantly on soluble solid content (table 3). comparison of mean values of the main effects of thinning on tss showed that thinning in the early kimri caused a significant increase in tss compared to thinning at the pollination stage and in the control group (table 3). with the softening of the fruit, astringency is lost and tss increases, thereby sweet­ ening the fruit (serrano et al., 2001). the increase in tss due to thinning usually occurs because of the fact that fruit use more leaf area for the production of photosynthetic materials, soluble carbohydrates and soluble solids as the fruit ripens (moustafa et al., 2019). the main effects of bunch covering on ascor­ bic acid content showed that bunch covering did not affect this variable compared to the control. moreover, comparison of the mean values of the main effects of date palm fruit thinning on ascorbic acid content showed that date palm fruit thinning in the early kimri caused a significant increase in ascor­ bic acid compared to the control and also compared to thinning at the pollination stage (table 3). moreover, ahmed et al. (2019) reported that date palm fruit thinning increased ascorbic acid in “zaghlool” dates. also, awad and al­qurashi (2012) reported that bagging ‘barhee’ dates increased ascorbic acid content. however, the mechanism by which bunch covering affects ascorbic acid is not clear. the main effects of bunch date palm fruit thin­ ning and covering on the percentage of fruit moisture were not significant (table 3). the percentage of fruit moisture is usually affected by many environmental factors and tree management. in some cultivars, thinning of dates reduces the percentage of moisture in the ‘saidy’ date palms fruit (samouni et al., 2016). however moustafa et al. (2019) showed that date palm fruit thinning does not affect the moisture con­ tent of ‘khadrawi’ date fruit. the results of statistical compound analysis showed significant effects of bunch date palm fruit thinning on fruit length, fruit weight and seed length. comparison of mean values on the main effects of covering bunches revealed that fruit length increased as compared to the control for both years (table 5). mean comparison of the main effects showed that date palm fruit thinning caused a significant increase in the length and weight of fruit in comparison with the control for both years. also, date palm fruit thin­ ning at the time of pollination significantly increased the diameter of fruit and the length of the seeds compared to the control and thinning in the early kimri stage for both years. thinning did not affect the weight and diameter of the seeds for both years (table 5). the effects of coverage on different fruit have been investigated. however, there are contra­ dictory reports on the effects of covering on fruit size. for example, in mango, covering increased the size of the fruit (chonhenchob et al., 2011). in anoth­ er study, bagging reduced fruit size in bananas (hasan et al., 2001). fruit weight and size usually affect the marketing of dates. larger sizes have bet­ ter marketability (al­qurashi and awad, 2011). thinning can be an important treatment in the orchard which improves the quantitative and qualita­ tive properties of fruit for export. in dates, date palm fruit thinning can be performed until the middle of the kimri phase. growth occurs during the cell divi­ sion and enlargement stages. cell division occurs after fertilization and continues until the end of the table 4 ­ pearson correlation between taa, lightness, chroma and hue **= correlation is significant at the 0.01 level. correlation taa lightness chroma hue taa 1 ­0.88** ­0.84** 0.82** madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 17 kimri phase. then, cellular growth ensues. a proper distribution of nutrients during cell division and enlargement increases the size of the fruit and caus­ es better marketability. in the process of fruit growth and development, there is competition for water and nutrients. thinning reduces competition between fruit for their absorption of water and nutrients, while providing adequate conditions for growth. this reduces the number of fruit and increases the length of the fruit. these results agree with the results of ahmed et al. (2019) that thinning in ‘zaghlool’ dates at an early stage of fruit development can have a greater effect on fruit size due to a more comprehen­ sive allocation of water and nutrients. 4. conclusions the results of this study showed that covering the date bunch reduced the brightness of the fruit, but increased the bunch weight, fruit length, total antioxi­ dant activity and total phenolic compounds. thinning at the pollination stage increased the weight of the fruit compared to the control. thinning at the kimri stage increased the percentage of tamar, ascorbic acid content, and total antioxidant activity. we con­ clude that bunch covering at the pre­harvest stage is a simple, grower­friendly method. it is safe to use and has several beneficial effects on the physiochemical traits of fruit. this approach can be an integral part of fruit production in orchards. moreover, fruit thinning and covering increased total antioxidant activity and total phenolic compounds of fruit which are important quality factors. also, thinning is recommended for ‘piyarom’ dates at both pollination and kimri stages. acknowledgements the authors would like to thank prof. john b. golding for critical review and revising the manu­ script. table 5 ­ main effects of bunch date palm fruit thinning and covering on fruit and seed length, diameter and weight of ‘piyarom’ date treatments fruits length (cm) fruits diameter (cm) fruits weight (g) seed length (cm) seed diameter (cm) seed weight (g) 2018 thinning control 3.42 c z 1.78 b 6.75 c 2.30 b 1.06 a 0.99 a removal of one third of total strands from terminal tips 4.95 a 2.76 a 10.46 a 3.10 a 1.08 a 1.05 a removal of one third of terminal tips of central strands in 4.30 b 1.95 b 8.77 b 2.10 b 1.00 a 1.11 a lsd 0.42 0.52 1.45 0.32 0.21 0.18 bunch covering control 3.97 b 2.33 a 8.34 a 2.50 a 1.11 a 1.06 a bunch covering 4.48 a 2.00 a 8.98 a 2.50 a 0.98 a 1.04 a lsd 0.34 0.42 1.18 0.26 0.17 0.15 thinning x bunch covering ns y ns ns ns ns ns 2019 thinning control 3.75 b 1.96 b 7.08 c 2.23 b 0.75 a 0.98 a removal of one third of total strands from terminal tips 4.75 a 3.31 a 11.50 a 3.25 a 0.81 a 0.99 a removal of one third of terminal tips of central strands in 4.66 a 2.26 b 8.73 b 2.06 b 0.78 a 1.05 a lsd 0.31 0.45 1.04 0.36 0.20 0.18 bunch covering control 4.05 b 2.41 a 8.90 a 2.62 a 0.82 a 0.95 a bunch covering 4.72 a 2.62 a 9.31 a 2.41 a 0.74 a 1.06 a lsd 0.25 0.37 8.85 0.29 0.16 0.14 thinning x bunch covering ns ns ns ns ns ns z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. adv. hort. sci., 2021 35(1): 11­19 18 references ahmed b.h., mehana s.a., zagzog o.a., qauod e.m., 2019 ­ improvements of yield and fruits quality of zaghlool date palm by using the different thinning treatments. ­ j. product. dev., 4: 743­756. aleid s.m., 2014 ­ date fruits processing and processed products, pp. 171­202. ­ in: siddiq m., aleid s.m., kader a.a. 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(ed.) pre‐harvest modulation of postharvest fruits and vegetable quality. academic press, london, uk, pp. 516. siddiq m., aleid s.m., kader a.a., 2013 ­ dates: posthar‐ vest science, processing technology and health benefits. ­ john wiley & sons, usa, pp. 326. slatnar a., mikulic­petkovsek m., stampar f., veber­ ic r., marsic n.k., 2020 ­ influence of cluster thinning on quantitative and qualitative parameters of cherry tomato. ­ eur. j. hort. sci., 1: 30­33. impaginato 89 adv. hort. sci., 2022 36(2): 89­96 doi: 10.36253/ahsc­12403 postharvest application of calcium chloride and 1­methylcyclopropene for quality conservation on organic ripe fig m.b.d. tofanelli 1 (*), f.l. cuquel 1, j.w. de o. d’angelo 2, j.g.s. medeiros 3 1 department of plant science, federal university of paraná, 80350‐050 curitiba‐pr, paraná, brazil. 2 national rural apprenticeship service, 80010‐010 curitiba‐pr, paraná, brazil. 3 federal institute of education, 68740‐970 castanhal, pará, brazil. key words: brown turkey fig, ficus carica, fruit conservation, shelf life. abstract: the postharvest phase is an important step in the fruit production chain. fig is an especially perishable fruit, which has encouraged researchers to study the effects of various substances on the postharvest life of this commodi­ ty. the objective of the present work was to evaluate the effects of calcium chloride (cacl2) and 1­methylcyclopropene (1­mcp) on the postharvest quality of the ‘roxo­de­valinhos’ fig cultivar. this study aimed to verify the effects of applying a 4% solution of cacl2 and a 1% solution of 1­mcp to figs and evaluat­ ing at four different storage times (0, 2, 4, and 6 days). the results showed that a 4% solution of cacl2 promoted better firmness, and when cacl2 at 4% solution was applied in combination with 1­mcp at 10 μg l­1, the maturation index increased. in contrast, the 1­mcp treatment alone did not improve the posthar­ vest quality of ‘roxo­de­valinhos’ ripe fig. we conclude that application of 4% solution of cacl2 and 1­mcp at 10 μg l­1 promote firmness and increase matura­ tion index of ‘roxo­devalinhos’ figs. 1. introduction fig (ficus carica l.) is an important crop globally, particularly in southeast europe, the middle east, north africa, and the americas, where the usa and brazil are highlighted for fig production (rosianski et al., 2016; uzundumlu et al., 2018; paolucci et al., 2020). in brazil, 19.6 tonnes were harvested from 2.114 hectares in 2020 (ibge, 2022), where fig trees are cultivated mainly in the southeast and south regions where ripe figs are harvested for consumption and unripe figs for industrializa­ tion (tofanelli et al., 2018). although fig tree cultivation has the potential to escalate, some pro­ duction bottlenecks have inhibited this expansion, such as lack of com­ mercial cultivars, unfamiliarly about ripe fig as excellent fruit for food from consumers, relative high price of ripe figs in the market, inefficient fresh ripe fig distribution and difficult to conserve quality of ripe figs after (*) corresponding author: mbrasildt@ufpr.br citation: tofanelli m.b.d., cuquel f.l., de o. d’angelo j.w., medeiros j.g.s., 2022 ­ postharvest appli‐ cation of calcium chloride and 1‐methylcyclopro‐ pene for quality conservation on organic ripe fig. ­ adv. hort. sci., 36(2): 89­96. copyright: © 2022 tofanelli m.b.d., cuquel f.l., de o. d’angelo j.w., medeiros j.g.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 december 2021 accepted for publication 4 february 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12403 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 89­96 90 harvested. thus, one of the principal challenges is the high perishability of ripe fig after it has been harvest­ ed (mirshekari et al., 2020), especially in brazil where the main cultivar used is ‘roxo­de­valinhos’, also known as ‘brown turkey’, which produces highly­per­ ishable fruit (ferraz et al., 2020). ‘roxo­de­valinhos’ figs have a very short postharvest life of 1 to 2 days when stored under environmental conditions of mar­ ket shelf, or 4 to 10 days under refrigerated storage (lakshmi et al., 2018). several studies have been conducted on the effects of postharvest treatments on figs to extend their storage duration. the majority of research has focused on substances such as calcium chloride (cacl2), 1­methylcyclopropene (1­mcp), fungicides, and sodium hypochlorite (irfan, 2013; song et al., 2019; jusoh et al., 2020). cacl2 has been applied to fruits both in preharvest and postharvest to promote quality conservation (li et al., 2014). this nutrient has been considered the most consequential for fruit quality promotion through its contributions to decreased softening, browning, and senescence of fruits, thereby extend­ ing their postharvest life (suriati et al., 2021). 1­mcp has an unsaturated cyclic olefin that acts as a com­ petitive ethylene antagonist and blocks ethylene receptors, consequently inhibiting maturation processes such as ethylene production, respiration rate, browning, and softening, and extending the shelf life of fruits (sozzi et al., 2005; watkins, 2008; freiman et al., 2012). the response of cacl2 or 1­mcp application on fruits varies according to the application form, fruit species, cultivar, maturation, and ripening stage of the fruit (sozzi et al., 2005; watkins, 2006; irfan, 2013). currently, there are few reports applying cacl2 and 1­mcp to figs to evaluate their effects on fruit quality. therefore, the objective of this study was to eval­ uate the effects of the application of cacl2 and 1­mcp on quality of fresh ripe fig ‘roxo­de­valinhos’ after harvested to extend their shelf life. 2. materials and methods plant materials the experiment was conducted at the federal university of paraná state. figs were harvested from plants grown in the orchard located in the canguiri experimental farm (pinhais county, paraná state, brazil, 25°26ʹ s and 49°16ʹ w, 947 m above sea level), considered cfb by the köppen climate classification, with annual maximum and minimum temperatures of approximately 24°c and 11°c, respectively, and annual average rainfall of 1,500 mm. fig trees used were 5­year­old roxo­de­valinhos cultivars, also known as ‘brown turkey’. the first har­ vest was done when plants were 2 years old. plants were cultivated in an organic plantation system, sub­ jected to heavy annual pruning, and spaced at 1×2.5 m. fertilization was done using cattle manure at 60 l per plant annually, separated into three 60 days intervals beginning in august. phytosanitary manage­ ment was performed by spraying a 2% lime sulfur solution after pruning and a 0.2% bordeaux mixture (biweekly in rainy period and monthly in dry period) from the vegetative and production stages of the fig trees to two weeks prior to harvest, mainly to pre­ vent rust (cerotelium fici). to control pests, such as tree borer (azochis gripusalis) and borer beetle (coleobogaster cyanitarsis), a 1% solution of oil extracted from neem (azadirachta indica a. juss) was sprayed on plants when these pests were detected damaging the fig trees. mechanical weeding in crop rows and mowing between rows were used to con­ trol weeds, integrated with black oat (avena strigosa l.) planted in july in 2012 and 2013. fruit materials figs were hand­harvested on april 1, 2014. overall, this period is the late season for harvest of ripe fig in brazil. fruits were harvested when they showed at least a 50% change to their skin color, from green to reddish brown at stage 6 (freiman et al., 2012) by visual evaluation. this maturation stage is usually better accepted by brazilian market. after harvesting, figs were immediately transported to the postharvest laboratory and located in the same experimental farm for experimental proceedings. figs were divided into lots containing 8 figs each lot for storage during 0, 2, 4, and 6 days, which were then placed in a paper box commonly used in ripe fig mar­ kets. postharvest treatments after figs were harvested, they were treated with calcium chloride (cacl2) (oliveira junior et al., 2018) and 1­methylcyclopropene (1­mcp) (tofanelli et al., 2018). half of the selected figs were dipped in the tofanelli et al. ‐ application of cacl2 and 1‐mcp for ripe fig quality 91 solution of cacl2 at 4%, and the other half were dipped in distilled water and immersed for 15 min. after that, figs were taken from the treatments to dry their surface by natural air­drying under the labo­ ratory bench for 30 min. after drying, figs were placed in a specific com­ mercial paper box that fit 8 figs each. the packed figs were placed into a plastic container (70 l) when they were ready to be treated with 1­mcp. smartfreshò powder (0.14% active ingredient) was used to pre­ pare the 1­mcp solution. this powder was measured with a precision balance and dissolved in distilled water to create a concentration of 10 μg l­1 inside the container, where figs were kept after treatment for 24 h. untreated figs (control) were compounded only with distilled water and stored under the same con­ ditions. thus, 1­mcp powder was introduced into a syringe and supplemented with water until a specific volume was reached. the 1­mcp solution and control were injected into the respective plastic containers, which were immediately blocked. therefore, both untreated figs and those treated with cacl2 were also treated with 1­mcp or the control (distilled water), resulting in a combination of 2 levels of cacl2 (0 and 4%) vs. 2 concentrations of 1­mcp (0 and 10 μg l­1). after 1­mcp treatment, packed figs were removed from boxes and stored at 4±1°c and 90­95% rh for 6 days, when they were removed to proce­ dure analyses. fruit quality parameters fig quality conservation was evaluated using firm­ ness, total soluble solids (tss) concentration, titrat­ able acidity (ta), maturation index (mi), and weight loss. the samples were withdrawn from the refrigera­ tor and 2 figs per replicate were analyzed every two days. fruit­peel firmness was determined on the equator of the fig using a manual penetrometer (ptr­ 100) with a 7.9­mm­diameter tip and expressed in terms of lb force. fruit juice was extracted from the stored figs using a centrifuge in order to assess tss and ta. tss was measured with a handheld refractometer using a drop of juice and expressed as a percentage while ta was expressed as % citric acid and measured using 100 ml of the solution (10 ml of juice + 90 ml distilled water) that was immediately titrated with 0.1 n naoh using 3 drops of phenolphthalein as an indica­ tor. mi was determined by dividing tss by ta (tss/ta ratio). weight loss of stored figs was obtained using the relative weight variation (%) of 2 figs per replicate during a storage period of 6 days. the figs were weighed at harvest time and at every storage period of 2 days, and weight loss was determined. fig weights were assessed using an electronic balance. experimental design three replicates of samples with 8 figs per repli­ cate for each treatment were used for physical and chemical quality analyses; 2 figs were analyzed immediately after 1­mcp proceedings and the other 6 figs were stored. four storage periods (0, 2, 4, and 6 days) were used to evaluate postharvest quality conservation. theses samples of figs were random­ ized selected avoiding to select damage figs by pests and mechanical injury. the experimental design was completely random­ ized. treatments included the application or absence of 1­mcp (10 μg l­1) combined with the application or absence of cacl2 (4%) and with no storage (0) and three storage times (2, 4, and 6 days). thus, the experiment was designed using a 23 factorial arrange­ ment (2´2´4): (2) cacl2 at 2 levels (0 and 4%), (2) 1­ mcp at 2 levels (0 and 10 μg l­1), and (4) storage peri­ od at four levels (0, 2, 4, and 6 days). statistical procedures analyses were carried out with analysis of vari­ ance (anova) using the sisvar statistical program (version 5.3) (ferreira, 2011). treatments that showed a significant effect were subjected to multi­ ple comparisons of the mean using the tukey test at a 5% probability level. 3. results results from anova showed that cacl2 promoted significant effect on firmness and soluble solids of the ‘roxo­de­vlinhos’ ripe figs, that storage periods had significant influence on soluble solids, titratable acidi­ ty, weight loss, and maturation index, whereas 1­ mcp only affected significantly the maturation index (table 1). the exclusive interaction that showed a sig­ nificant effect was cacl2 x 1­mcp on the fig ratio. as expected, storage influenced the quality parameters evaluated in the ripe figs, except in terms of firm­ ness. there was no significant effect of cacl2 and 1­ mcp on all parameters evaluated in this present work after storage of 6 days (table s1). adv. hort. sci., 2022 36(2): 89­96 92 except for effect of the interaction between cacl2 x 1­mcp on the maturation index of figs, we could also observe from anova that there was no signifi­ cant effect of interactions between the factors on parameters evaluated in this present work (table 1). when ripe figs were post­harvest treated with cacl2, they demonstrated more firmness than untreated figs (3.37 lb and 1.91 lb, respectively) (table 2). cacl2 postharvest treatment also influ­ enced tss on ripe figs, where untreated fruits showed a higher value (8.70%) compared to the treated figs (7.98%). figs stored for 4 (8.74%) and 6 days (8.81%) showed higher tss than non­stored fruits (7.47%) (table 3). the titratable acidity was higher after 6 days of storage (2.58%), when compared with no storage (1.98%) and 2 days of storage (2.07%) (table 3). the weight loss of ripe figs was significantly table 1 ­ analysis of variance (anova) for parameters evaluated on storage of ripe figs treated with calcium chloride (cacl2) and 1­ methylcyclopropene (1­mcp) cc= calcium chloride; mcp= methylcyclopropene; sp= storage periods; cv= coefficient of variation; fi= firmness; ss= soluble solids; ta = titratable acidity; wl= weight loss; mi= maturation index; ms= mean square; f= f test; ** significant at p≤0.01; * significant at p<0.05. ns = not significant. table 2 ­ effects of calcium chloride (cacl2) treatments on quality parameters of ripe figs stored over different periods * standard deviation. values followed by the different lowercase letter in a column are significantly different from each other (p<0.05). table 3 ­ effects of storage periods on quality parameters of ripe figs * standard deviation. values followed by the same lowercase letter in a column are not significantly different from each other (p<0.05). source of variation df fi (lb) ss (°brix) ta (%) wl (%) mi (ratio) ms f ms f ms f ms f ms f cc 1 25.37 17.06 ** 6.31 9.84 ** 0.02 0.09 ns 102.11 0.36 ns 404.26 1.70 ns mcp 1 2.34 1.57 ns 0.08 0.13 ns 0.64 3.15 ns 28.29 0.11 ns 1029.53 4.33 * sp 3 3.94 2.65 ns 4.57 7.14 ** 1.04 5.09 ** 97.99 104.25 ** 765.64 3.22 * cc´mcp 1 0.07 0.05 ns 0.61 0.95 ns 0.81 3.97 ns 2.53 1.69 ns 987.36 4.15 * cc´sp 3 0.48 0.33 ns 1.01 1.57 ns 0.18 0.86 ns 0.04 0.14 ns 9.85 0.01 ns mcp´sp 3 1.39 0.93 ns 0.86 1.35 ns 0.09 0.44 ns 0.09 0.05 ns 342.21 1.44 ns cc´mcp´sp 3 3.90 2.62 ns 0.58 0.91 ns 0.46 2.27 ns 0.33 0.39 ns 84.59 0.36 ns cv (%) 46.2 9.6 19.9 13.2 22.2 fruit parameter cacl2 firmness (lb) soluble solids (%) untreated 1.91 ± 1.34* b 8.70 ± 1.00 a treated 3.37 ± 1.29 a 7.98 ± 0.91 b coefficient of variation (%) 46.2 9.6 storage (days) fruit parameters soluble solids (%) titratable acidity (%) weight loss (%) maturation index 0 7.47 ± 0.99* b 1.98 ± 0.35 b 0 ± 0.00 d 69.22 ± 12.56 ab 2 8.35 ± 0.68 ab 2.58 ± 0.73 b 7.67 ± 0.86 c 62.22 ± 16.49 b 4 8.74 ± 0.82 a 2.07 ± 0.47 ab 13.67 ± 2.84 b 80.47 ± 21.62 a 6 8.81 ± 1.02 a 2.46 ± 0.22 a 18.75 ± 3.96 a 65.23 ± 11.54 ab coefficient of variation (%) 9.6 19.9 27.3 22.2 tofanelli et al. ‐ application of cacl2 and 1‐mcp for ripe fig quality 93 enhanced as storage duration increased, as expected. the greatest weight loss was 18.75% after 6 days of cold storage. the maturation index (tss/ta ratio) was the para­ meter that showed a significant interaction effect (table 1). when ripe figs were treated with cacl2 after with 1­mcp, they showed a higher ratio (75.55) than those treated only with cacl2 (57.22) (table 4). 4. discussion and conclusions cacl2 has been widely used for food conservation, mainly because it can maintain firmness during the shelf life of vegetables, especially in fruits immersed in calcium chloride solution after harvest (li et al., 2014). calcium plays an important role in maintaining the structure of the cell wall, since its cation binds to pectins producing calcium pectates that structure the cell wall, thereby inhibiting enzymes such as poly­ galacturonase and polymethylesterase. irfan et al. (2013) recorded the lowest sugar con­ tent when figs were treated with cacl2 at 4%, as reported before when we showed that figs treated with cacl2 obtained lower sugar content against untreated figs. these authors concluded that this likely occurred because calcium caused a delay in fruit ripening through the inhibition of certain events such as starch hydrolysis and formation of sugars, a response to the high amounts of calcium deposition in the fruit cell wall of the treated figs. these authors also mentioned that components such as pectic sub­ stances, calmodulin, and hemicellulose polysaccha­ rides are the most likely binding sites for calcium, preserving the texture and stability of the treated fruit and promoting higher fruit resistance by main­ taining high calcium concentration in the cytosol, as well as less free sugars released during storage. storage having no influence on firmness likely relates to the figs being harvested at a relatively advanced ripening stage, when they showed at least a 50% change in skin color, from green to reddish brown at stage 6. in brazil, the late season to harvest ripe figs for table takes place from late march to early april, when they usually get better prices in the mar­ ket. thereby, it would be interesting whether grow­ ers had a technique that was able to extend shelf life of ripe figs in order to add value to their product. thus, at this harvest time, figs already displayed pre­ harvest firmness reduction; therefore, the collected data of firmness from harvest to last storage period did not show a significant difference. according to freiman et al. (2012), postharvest treatment on figs at the maturation stage can be ineffective for their quality conservation or may have a slight effect on softening, promoting its retardation, especially when figs are collected at an advanced stage. these authors mentioned that marketable ripe figs are defined as commercially mature when they have changing skin coloration from 20 to 70% of their surface. tofanelli et al. (2018) also showed tss increasing during storage in ‘roxo­de­valinhos’ figs collected at stage 5 (freiman et al., 2012) and treated with 1­ mcp. according to those authors, this tss increase likely occurs either due to the hydrolysis of several polysaccharides, such as pectins, starch, and other oligosaccharides in the cell wall, which become part of the cellular juice when they are solubilized in the aqueous phase, or due to starch accumulation during fruit maturation, which is degraded into sugars by the action of enzymes such as α­amylase, β­amylase, and starch phosphorylase, consequently increasing the tss concentration. although the acidity of figs tends to decrease dur­ ing the storage period (irfan et al., 2013; song et al., 2019), it has been observed that in the initial storage time, acidity tends to increase for a short period in the first few days. gözlekçi et al. (2008) evaluated the effect of 1­mcp on the quality of figs stored dur­ ing three different periods (5, 10, and 15 days) and table 4 ­ effects of calcium chloride (cacl2) and 1­methylcyclopropene (1­mcp) treatments on the ratio (%) of ripe figs * standard deviation. values followed by the same uppercase letter in a column and lowercase letter in a row are not significantly diffe­ rent from each other (p<0.05). cacl2 1­mcp untreated treated untreated 72.09 ± 11.65* aa 72.28 ± 18.67 aa treated 57.22 ± 11.10 bb 75.55 ± 20.28 aa coefficient of variation (%) 22.2 94 adv. hort. sci., 2022 36(2): 89­96 observed that fruit acidity slightly increased until the first storage period. in addition, álvarez­herrera et al. (2016) studied the effect of 1­mcp on the posthar­ vest conservation of pitahaya (selenicereus megalan‐ thus) during seven storage periods (4, 8, 12, 16, 20, 24, and 28 days), and discovered an increase in acidi­ ty in the treated pitahayas until the second storage period (8 days). in the present work, the last evaluat­ ed storage period was 6 days; therefore, it was expected that the acidity of ripe figs would increase during the entire experimental period. fruit transpiration and several physiological disor­ ders of figs, such as skin side cracking and ostiole­end cracking/splitting occur during storage, and these could promote increasing levels of ta and tss, as well as the maintenance of several organic acids dur­ ing the storage period (byeon and lee, 2020). in our study, 1­mcp treatments only affected on maturation index (ratio). cantín et al. (2020) also did not obtain improvement on shelf life of ‘cuello dama negro’ dark­skin commercial figs treated with 1­mcp at 1 μl l ­1 and according to these authors the advance maturity stage of the figs at the moment of harvest could be promoted a high increase in ethyl­ ene production even under 1­mcp treatment. they also highlighted that, although the best moment to harvest fresh­market figs is variable according to each cultivar or variety, it is usually done as early as possible. in our work, studied figs were harvested at maturation stage 6 (fig. 1b), thus an early harvest (fig. 1c) could be promoted different results, what we would release here as a challenge for future stud­ ies, as well as evaluate how these figs harvested on early maturation stage would be taken up by the market. however, figs may be a climacteric or non­ climacteric fruit due to their maturation shows rapid changes of compositional features that are typical of climacteric fruit, whereas figs are not capable to keep ripening after harvested (d’aquino et al., 2015). thus, that is a good question when would be the bet­ ter time to harvest ripe figs for post treatment in order to enlarge their shelf life as well as promote their quality conservation. fig fruits are highly perishable products in which physiological events such as transpiration, respira­ tion, and degradation occur quickly (gözlekçi et al., 2008; ozkaya et al., 2014; byeon and lee, 2020). it is interesting to consider the unique morphological structure of figs, as they have a thin skin over the entire fruit with a distal end orifice called the ostiole where water loss through evaporation occurs by exu­ dation of a syrupy liquid (d’aquino et al., 2003; freiman et al., 2012). cacl2 combined with 1­mcp applied on posthar­ vest ripe figs likely promoted sugar content accumu­ lation and was not favorable to acidity oscillation of fruits during storage. some structural effects on figs have been observed when they are treated with calcium, such as mechanical strength improvement and increased resistance of fruit tissue due to absorbed calcium in the apoplast primarily complexing with the cell wall, where the plasma membrane works as a cementing material. calcium is also involved in the maintenance of the cell wall structure by interacting with pectic acid and forming calcium pectate (irfan et al., 2013). thus, in the present study, cacl2 application likely promoted these events in figs, resulting in sugar con­ tent accumulation and acidity stabilization, conse­ quently enhancing the maturation index (ratio). in addition, when 1­mcp was applied after calci­ um on the ripe figs, the mi ratios increased, poten­ tially due to ethylene inhibition, decreased fruit res­ piration, and glucose and fructose stabilization during cold storage (ozkaya et al., 2014; song et al., 2019). overall, the results showed that a 4% cacl2 solu­ tion was capable of promoting higher firmness and sugar content stabilization, whereas when cacl2 was applied in combination with 1­mcp at 10 μg l­1, the maturation index increased. these results reinforce fig. 1 ­ figs of roxo­de­valinhos harvested at different stages of ripening. a ­ advanced maturation; b ­ maturation at 50% change skin color; c ­ maturation at 25% change skin color, d ­ maturation at 10% change skin color. tofanelli et al. ‐ application of cacl2 and 1‐mcp for ripe fig quality 95 that calcium really may maintain more stable the structure of the cell wall, seeing that when it was combined with 1­mcp there was no effect on firm­ ness. an explanation in this case, may be because humidity into the container increased softly due to 1­ mcp solution applied indoor, what could be sufficient to promote moisture saturation from cacl2 reducing its absorbing capacity. the present work encourages the development of further investigation in order to verify the effects of both cacl2 and 1­mcp applied at numerous different concentrations on quality conservation of ‘roxo­de­ valinhos’ ripe figs harvested at earlier maturation stage and during a longer storage period than stud­ ied here. in conclusion, this study showed the effect of cal­ cium chloride and 1­methylcyclopropene on con­ serve quality of ‘roxo­de­valinhos’ ripe figs to market for table. the calcium chloride solution applied at 4% improves firmness and promotes soluble solid stabi­ lization of ripe figs stored for 6 days in a refrigerator, whereas 1­mcp treatment at a dose of 10 μg l­1 does not contribute for maintaining postharvest quality of ripe fig, nor its shelf life. however, treatment with 4% cacl2 combined with 1­mcp 10 μg l­1 increases the maturation index (ratio) of fresh ripe figs. finally, a better understanding of harvest seasons, maturation stages and postharvest treatments on ripe figs is essential for crop fig, grower and market. references álvarez­herrera j.g., deaquiz y.a., herrera a.o., 2016 ­ effect of different 1‐methylcyclopropene doses on the postharvest period 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10.13128/ahsc­9764 climatic and physiological parameters related to the progress and prediction of apple sunburn damage in a neotropical climate v. severino 1 (*), m. arias­sibillotte 1, s. dogliotti 1, e. frins 2, j. gonzalez­ talice 1, j.a. yuri 3 1 facultad de agronomía, universidad de la república (udelar), garzón 780, cp 12900 montevideo, uruguay. 2 facultad de ingeniería, udelar, julio herrera y reissig 565, cp 11300 montevideo, uruguay. 3 centro de pomáceas, universidad de talca, 1 poniente 1141, talca, chile. key words: hydric potential, malus domestica, proline, spectroradiometry, reflectance indices. abstract: apple production in neotropical climate is affected by sunburn and the high interannual variability in meteorological conditions makes prediction and management of damage difficult. non­destructive methods associated with physiological variables are keys to monitoring but their development is still incipient. in our study occurrence of sunburn, meteorological conditions and physiological parameters was monitored throughout four crop cycles. fruit visu­ al assessment and reflectance measures in field, as well as, pigments, proline and hydric potential in laboratory, were accomplished. the results show that the availability of water in the soil was more related to the evolution of sun­ burn than air temperature. plant senescence reflectance index (non­destruc­ tive predictor) discriminated between healthy and damaged fruits and fruit hydric potential and proline content were good indicators of sunburn, although such variables are determined when damage has already occurred. our results suggest focusing future research on the water balance of the system and on the physiological indicators of osmotic stress as a way to predict damage. 1. introduction fruit sunburn has been reported since 1870, and although several studies have touched upon the matter since the early 20th century (racsko and schrader, 2012), it is still a cause of significant economic loss in apple production (reig et al., 2019). although some expressions of damage may be easily perceived in the field, on occasions the symptoms of sunscald are imperceptible and only appear after months of cold stor­ age (usually after three months), which makes the damage difficult to control (yuri et al., 2000). symptoms appear as brown stains on the fruits’ (*) corresponding author: vseverin@fagro.edu.uy citation: severino v., arias­sibillotte m., dogliotti s., frins e., gonzalez­talice j., yuri j.a., 2020 ­ climatic and physiological parameters related to the progress and prediction of apple sunburn damage in a neotropical climate. ­ adv. hort. sci., 34(4): 431­440 copyright: © 2020 severino v., arias­sibillotte m., dogliotti s., frins e., gonzalez­talice j., yuri j.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 september 2020 accepted for publication 30 november 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 431­440 432 exterior, being granny smith the most susceptible reported cultivar (felicetti and schrader 2008; hernandez et al., 2014). there is consensus that sunburn is related to the combination of high temperature and irradiance (uv­ b range is thought to be essential) during the fruit growth period (yuri et al., 2000, 2010; racsko and schrader, 2012; torres et al., 2013; darbyshire et al., 2015; torres et al., 2016 a, b), however, there is a limited understanding of the physiological aspects of the changes in the fruit’s internal quality (racsko and schrader, 2012) and generation of sun­related physi­ ological disorders in fruit. this problem requires fur­ ther research into the environmental and physiologi­ cal processes that occur prior to and during sun injury development and more importantly, biochemi­ cal changes that may contribute to resistance to envi­ ronmental conditions that cause sun­related disor­ ders in fruit (morales­quintana et al., 2020). stress adaptation mechanisms of plants, such as chlorophyll reduction (ballester et al., 2017), dissipa­ tion of excitation energy, increase of solutes of low molecular weight (wen and moriguchi, 2015) and changes in pigmentation (merzlyak et al., 2003) have been previously studied in relation to sunburn in apple fruit. based on these results, in the last decade, work has been done on the development of non­ destructive methods to predict and detect sun dam­ age based on the composition and location of skin pigments as well as the optical properties of the underlying fruit tissue (solovchenko et al., 2010; torres et al., 2016 a, b). in this direction, sunburn has been related to: fruit reflectance values in the visible and near­infrared (nir) spectra (solovchenko et al., 2010; torres et al., 2016 a), crop water stress index and chlorophyll fluorescence (torres et al., 2013, 2016 b). about temperature effect, it has been reported that increases in fruit temperature above a certain limit may cause enzymes denaturation and protein coagulation, leading to tissue damage (yuri et al., 2010). studies performed in cv. fuji fruits showed a highly susceptible caused by excessive heat and did not sustain damage when exposed to uv radiation only (yuri et al., 2000). air temperatures of 38­42°c increases the heat­shock proteins induction (woolf and ferguson, 2000) and sunburn symptomatology appear with fruit temperature of 46°c and higher (racsko and schrader, 2012). the water status of the plant and fruit has also been related to sunburn, although fewer studies have focused attention on this aspect. recent work in chile analyses the association of acclimation events with fruit water relations and osmoregulation occur­ ring in sun­exposed fruit tissue (torres et al., 2013). studies in japan and south africa discuss the effect of foliar aba on antioxidant levels and the incidence of sunburn with variable results (mupambi et al., 2018). antioxidant system plays a crucial part in the elimina­ tion of free radicals under stress conditions (chen and murata, 2002). compatible solutes such as pro­ line, betaine and polyols are accumulated in response to abiotic stress (suzuki, 2015). these solutes affect the osmotic balance and the mem­ brane stability and have been proven to maintain tur­ gor pressure, cellular volume and electrolyte concen­ tration (roberts, 2005). proline is known to be a sta­ bilizer of sub­cellular structures (kautz et al., 2015) and although many studies have established a con­ nection between proline and antioxidant activity in apple plant leaves and xylem under abiotic stress (šircelj et al., 2005; nemeskéri et al., 2015; afonso et al., 2017) no relationship between proline and sun­ burn has yet been reported. most of the existing research has been carried out in latitudes similar to that of the present study but in more arid climates such as, chile, australia, and south africa (southern hemisphere) or spain, turkey, and washington state (north hemisphere), however, few studies have addressed sunburn in humid growth­season conditions like in eastern new york state (reig et al., 2019). the region where the study was conducted, defined as neo­tropical (bernardi et al., 2016), has been considered restrictive for apple quality in relation to sunburn aspects (fao­mgap, 2013) due to the occurrence of high temperatures during the fruit growth period. changes in el niño evolution after 1976 may have played a role in alter­ ing the relationship between temperature extreme events in uruguay and the atmospheric circulation (renom et al., 2011). the average maximum temper­ atures of the summer period show a high iner­annual variability (71­86%) and lower variability in the medi­ um (10 years) or long­term (>30 years) components, 23% and 6% respectively (tiscornia et al., 2016), so it is expected that the climate in the region will contin­ ue to be favorable to the occurrence of burning. the aims of this work were to study apple sunburn progress and its relation to meteorological variables in a neo­tropical climate, and to establish correla­ tions between fruit physiological parameters and reflectance index. severino et al. ‐ apple sunburn in a neotropical climate 433 2. materials and methods plant material the experiment was conducted during the 2012/2013 to 2015/2016 crop cycles (hereinafter, cycles 1 to 4), on a granny smith/m7 plantation established in 2003. the crop is located in uruguay (southeastern of south america) with the coordi­ nates of 34°38’18’’ s and 56°40’06’’ w and 45 meters above sea level.the climate of this regional ecotone is classified by bernardi et al. (2016) as neo­tropical. crop had planting distance of 4x1.5 m, rows arranged from n to s and trained in central leader system. the soil types are mainly argiudolls and hapluderts and a drip irrigation system with a maximum daily watering capacity of 4.5 mm is installed. three fruits per tree from ten trees per row, in a total of five rows were selected between 40 and 50 days after full bloom (dafb) in the four evaluated cycles. trees and rows were randomly marked, and 150 fruits exposed to radiation were classified by visu­ al assessment of different external initial conditions: a) 50 fruits with no visible sunburn (hf=healthy fruits); b) 50 fruits with red color (rf=red fruits); c) 50 fruits with an early degree of sunburn (sbf=sunburn fruits) [sunburn browning, according to the classifica­ tion of racsko and schrader (2012)] as indicated in fig­ ure 1. the flowering dates for cycles 1 to 4 were, september 27 (cycle 1), october 28, 3 and 14, to cycles 2, 3 and 4 respectively. the exposed side of each fruit was defined as the one directly exposed to sunlight, facing the space between rows, and the internal side as the one facing the trunk, with no direct exposition to solar radiation. field tests the sunburn progress in each marked fruit was assessed by observation. its frequency varied between 1 week and 1 month, with weekly observa­ tions predominating. in each observation, fruits were reclassified according to the categories mentioned above (hf, rf, sbf). reflectance measurements of the exposed side of ten fruits of each condition were recorded with an ilt 950 spectroradiometer (international light technologies, usa) at 91, 99 and 154 dafb in cycle 4. percent reflectance was calculat­ ed based on a dark spectrum and a reference spec­ trum from a white reference standard. the content of chlorophyll (chl), anthocyanins (ant), carotenoids (car), flavonoids (fla) and senescence indexes were calculated based on the reflectance measurements. chlorophyll was calculated according to the following indexes: chl1, chl2 (mullan, 2013) rarsa, rarsb, pssra, msr, cl1, cl2 (solovchenko et al., 2010) and anthocyanins, according to the anthocyanin reflectance index (ari) (solovchenko et al., 2010). carotenoids were calculated according to the following indexes: rarsc (mullan, 2013), cri1 and cri2 (solovchenko et al., 2010) and flavonoids according to the flavonoid reflectance index (fri) (solovchenko et al. , 2010). the normalized phaeophytization index (npqi), the pigment simple ratio (psr), the normalized difference pigment index (ndpi), the structural independent pigment index (sipi) (solovchenko et al., 2010) and the plant senescence reflectance indexes (psri480 psri500) (mullan, 2013) were also calculated (table 1). an automated meteorology station located 1900 m from the crop recorded the maximum temperature (tmax) (°c) and rainfall (rf) (mm) variables in the fruit’s growth period. the soil water balance (swb) for each growth cycle was calculated. plot characteristics and local and regional meteorology stations were used. local variables used were irrigation (mm), daily rain­ fall (mm), root deep (m), phenological stages (days) and soil texture. the eto (reference evapotranspira­ tion) (mm) was recorded at the meteorology station of inia las brujas using penman­monteith (allen et al., 1998). crop coefficient (kc) was adjusted to reflect the wetting frequency of soil surface and local climatic conditions according to allen et al. (2006): kcini = kcini (*) + (i­10) [kcini (**) ­ kcini (*)] (40­10) kcmid = kcmid (tab) + [0.04 (u2 ­ 2) ­ 0.004 (rhmin ­ 45)] ( h ) 0.3 3 where: kcini(*): value for kc ini from figure 29 in allen et al. (2006). kcini(**): value for kc ini from figure 30 in allen et al. fig. 1 ­ examples of fruit categories. rf=red fruits, hf=healthy fruits, sbf=sunburn fruits. adv. hort. sci., 2020 34(4): 431­440 434 (2006). i: average infiltration depth (mm). kc mid (tab): value for kc mid taken from table 12 in allen et al. (2006) apples, cherries and pears crops with active ground cover without frosts. u2: mean value for daily wind speed at 2 m height over grass during the mid­season growth stage (m s­ 1), for 1 m s­1<u2<6 m s­1. rhmin: mean value for daily minimum relative humidity during the mid­season growth stage (%), for 20%<rhmin< 80%. h: mean plant height during the mid­season stage (m) for 0.1 m < h < 10 m. laboratory five fruits of each damage category were sampled on the same dates on which the reflectance mea­ surements were carried out (91, 99 and 154 dafb) and in addition to the 112, 142 and 170 dafb of cycle 4 for laboratory evaluations. hydric potential of the fruits’ tissue (ѱf) was determinate with wp4c dew­ point potentiometer (decagon devices, usa) on the exposed side (ѱfe) and the internal side (ѱfi). each measurement was made on 3 cm2 of epidermis with 1 mm of sub­epidermal tissue. to determine chl, car and proline content (pro), 0.5 g of epidermis without sub­epidermal tissue was removed. the sam­ ple was macerated with 0.5 ml of a methanol­chloro­ form­water compound (mcw, 12:5:1), producing two phases. in phase 1, pigments chla, chlb and car were determined, recording absorbances at 665.6, 647.6 and 480 nm, respectively (wellburn, 1994). in phase 2, after reacting to a one­hour immersion at 90°c with acid ninhydrin and the addi­ tion of toluene, pro was determined with spec­ trophotometry, recording absorbance at 515 nm (troll and lindsley, 1955; charest and phan, 1990). to determine ant, 0.2 g tissue was incubated in methanol­acid for 48 h, recording absorbance values at 530 and 657 nm (wellburn, 1994). statistical analysis the contrastive analysis of the three fruit condi­ tions regarding pigment quantification and assess­ ment, pro, ѱf and senescence indexes were done with non­parametric methods, using the kruskal­ wallis test to compare medians and the kruskal nemenyi post­hoc test for the multiple comparisons of pairs, with the r statistical software. differences were assessed at p≤0.05. pearson’s correlation coeffi­ cient was determined for the proportion of sun­dam­ aged fruits and the climatic variables, maximum tem­ perature (tmax) and soil water balance (swb). the same analysis was performed for the pigment con­ tent tested in the laboratory and those estimated by spectroradiometry in the field, as well as for pro and ѱf. best fit regressions for variables with higher cor­ relation coefficients were estimated. table 1 ­ indexes calculated by reflectance in the field name index index calculation parameter source reflectance ratio chl1 r750/r550 chlorophyll braun and payne, 2013 chl2 r750/r700 braun and payne, 2013 ratio analysis of reflectance spectrum (chla) rarsa r675/r700 chlorophyll a braun and payne, 2013 ratio analysis of reflectance spectrum (chlb) rarsb r675/(r650*r700) chlorophyll b braun and payne, 2013 ratio analysis of reflectance spectrum (carotenoids) rarsc r760/r500 carotenoids braun and payne, 2013 pigment­specific simple ratio pssra r800/r675 chlorophyll a braun and payne, 2013 normalized phaeophytization index npqi (r415­r435)/(r415+r435) chlorophyll degradation braun and payne, 2013 modified spectral ratio msr (r750­r445)/(r705­r445) chlorophyll concentration braun and payne, 2013 pigment simple ratio psr r430/r680 carotenoid­chlorophyll ratio braun and payne, 2013 normalized difference of pigment ratio ndpi (r680­r430)/(r680+r430) carotenoid­chlorophyll ratio braun and payne, 2013 structural independent pigment index sipi (r800­r435)/(r415+r435) carotenoid­chlorophyll ratio braun and payne, 2013 chlorophyll index cl1 (r700 ­1­r800 ­1)* r800 chlorophyll (solovchenko et al., 2010) cl2 (r640 ­1­r800 ­1)*r800 chlorophyll (solovchenko et al., 2010) anthocyanin reflectance index ari (r550 ­1­r700 ­1)* r800 anthocyanin (solovchenko et al., 2010) plant senescence reflectance index psri480 psri480=(r678­r480)* r­1800 carotenoid­chlorophyll ratio (solovchenko et al., 2010) psri500 psri500=(r678­r500)* r­1800 carotenoid­chlorophyll ratio (solovchenko et al., 2010) flavonoid reflectance index fri (r410 ­1­r460 ­1)* r800 flavonoids (solovchenko et al., 2010) carotenoid reflectance index cri1 (r520 ­1­r700 ­1)* r800 carotenoids (solovchenko et al., 2010) cri2 (r520 ­1­r550 ­1)* r800 (solovchenko et al., 2010) severino et al. ‐ apple sunburn in a neotropical climate 435 3. results sunburn progress in the four crop cycles showed a high inter annual variability, with differences in the incidence values (% of damaged fruits) and the moment when the maximum occurs. maximum val­ ues of 70% and 62% of sunburnt fruits were recorded in late december and early january for cycles 2 and 3 respectively (approximately 12 and 14 weeks after full bloom). for cycles 1 and 4, maximum values under 45% were recorded between mid­february and early march, at 19 and 22 weeks after full bloom (fig. 2). from the analysis of the meteorological records of the four cycles, it can be highlighted that, in cycle 2, 11 days were recorded with maximum temperatures above 35°c, starting at 68 dafb and reaching a maxi­ mum of 38.9°c at 77 dafb. cycles 1 and 3 respective­ ly recorded 1 and 0 days with maximum tempera­ tures above 35°c. in cycle 4, 5 days had a maximum higher than 35°c with only 1 peak above 38°c (fig. 3). fig. 3 ­ air maximum temperature (tmax) by days after full bloom (dafb) for four production cycles. a. cycle 1; b. cycle 2; c. cycle 3; d. cycle 4. fig. 2 ­ proportion of sunburn fruits and soil water balance (swb) by number of days after full bloom (dafb) for four production cycles. a, cycle 1; b, cycle 2; c, cycle 3; d, cycle 4. the error bars represent standard deviation. lowest values of swb during the first 80 dafb (about 11 weeks), in the period when damage appears, were recorded in cycles 2 and 3 showing minimum values of ­123 and ­46 mm respectively. on the other hand, in cycles 1 and 4, swb decreases from week 12 (fig. 2). pearson’s correlation coefficient between the pro­ portion of sun­damaged fruits and the soil water bal­ ance (swb) was ­0.41, with variations between ­0.37 and ­0.7 depending on the cycle. the correlation with maximum temperature was 0.28, with a variation of between ­0.1 and 0.51 for the different cycles (fig. 4). variables such as chl, car, ant, pro, ѱf and stress index, showed significant differences for at least one of the three kinds of fruit (hf, sbf, rf) when measured in the laboratory and/or when esti­ mated in the field, except for ѱfi and chlb content measured by spectrophotometry (rarsb). ant con­ centration, measured in laboratory, and msr (field fig. 4 ­ correlations between proportion of sunburn fruits and climatic variables. a. soil water balance (swb); b. air maximum temperature (tmax). colors represent each cycle: black for cycle 1, red for cycle 2, green for cycle 3, 436 adv. hort. sci., 2020 34(4): 431­440 chl estimate), differed significantly between the three types of fruit. ѱfe was significantly different between sbf and the other two conditions (rf and hf). remaining variables differed significantly between hf and rf or sbf, but not between rf and sbf. higher pro concentrations were measured in rf and sbf than hf fruits (fig. 5, table 2). pearson correlation coefficients calculated for the different variables assessed reached a maxi­ mum of ­0.75 between ѱfe and pro. chl concen­ tration measured in the laboratory had negative correlations of 0.42 with ѱf and 0.57 with pro (fig. 6). the relationship between ѱfe and pro, chla and ѱfe, ndpi and psri480, 860900 and chla, and ndpi and chla have the best fit with a 2nd degree polynomial regression (fig. 7) and a maximum r2 of 0.60. 4. discussion and conclusions sun damage incidence values reported in our trial reached a maximum of 70%. this was higher than reported by racsko and schrader (2012) in warmer climates of australia, south africa, spain, turkey and fig. 6 ­ correlations between variables with greater discrimina­ tion capacity for the different fruit conditions [proline (pro), carotenoids (car), chlorophylls (chl)] measured by spectrophotometry in the laboratory, ndpi, 860­900, cri1, sipi, fri, rarsb, msr, psri480, cri2, cl1, rarsc, pssra and chl2, calculated based on reflectance measu­ rements in the field. fig. 5 ­ boxplot of variables with greater discrimination capacity for the three types of fruit (hf= healthy fruit; sbf= sun­ burnt fruit; rf= red fruit). a) anthocyanin concentration; b) hydric potential of the exposed side of the fruit; c) msr (modified spectral ratio, chlorophyll concentration estimate by reflectance); d) proline concentration; e) psri480 (plant senescence reflectance index); f. sipi (structural independent pigment index). table 2 ­ median of anthocyanins (ant), chlorophyll (chl), carotenoids (car) and proline (pro) concentration, hydric potential of the exposed side of the fruit (ѱfe), reflectance ratio (chl2), chlorophyll concentration estimate by reflectance (msr, modified spectral ratio), structural independent pigment index (sipi) and plant senescence reflectance index (psri480), for the three types of fruit (healthy fruit, sunburnt fruit, and red fruit) sunburnt fruit red fruit healthy fruit ant (ng.mg­1 dw) 29.871 b 23.438 a 0 c chl (µg.mg­1 dw) 0.034 b 0.041 ab 0.045 a car (µg.mg­1 dw) 0.009 b 0.011 ab 0.012 a pro (ng.mg­1 dw) 0.724 a 0.788 a 0.64 b ѱfe (mpa) ­1.705 a ­1.54 b ­1.475 b chl2 1.808 b 1.785 b 2.658 a msr 1.660 a 1.716 b 2.504 c sipi 8.239 b 8.052 b 16.012 a psri480 ­0.843 b ­1.534 b 3.65 a different letters in rows indicate significant difference between types of fruit severino et al. ‐ apple sunburn in a neotropical climate 437 chile, and higher than the 40% reported by yuri and bailey (2015) for chilean crops. similar sunburn val­ ues were reported during a heat wave in southeast australia in 2009 (darbyshire et al., 2015). all cycles differed, both in how the damage progressed and at the time when the maximum damage occurred. these differences can be clarified by analysing the meteorological variables, which, as expected, were also highly variable (tiscornia et al., 2016). the corre­ lation of sun damage with swb was moderate to strong (0.37 to 0.7 depending on the cycle) and high­ er than that presented with maximum temperatures (fig. 4). in cycle 2, 11 days with maximum tempera­ tures above 35°c along with the swb progress, could explain the occurrence of sunburn. however, in cycle 3, only the early decrease in swb seems to be the explanatory variable of a high damage incidence (figs. 2 and 3). in four cycles studied, sunburn reached maximum values at 19, 12, 14 and 22 weeks after full bloom (fig. 2). in all cases, minimum fruit diameter reported as necessary to absorb enough solar radiation to increase the temperature and result in damage (45 mm 7 weeks) (racsko and schrader, 2012) had reached. fruit surface temperatures between 46 and 49°c, depending on the cultivar, cause browning, while temperatures over 52°c cause necrosis (schrader et al., 2001). this is in accordance with fruit temperatures recorded by darbyshire et al. (2015) for australia, as well as other reports suggesting that the temperature of the exposed side of the fruit may be 12 to 15°c higher than air temperature (woolf and ferguson, 2000). fruit temperatures over 52°c were recorded in our trials with thermal camera. on days with average air temperatures above 38°c (in cycle 2) were reached fruit temperatures up to 62°c (data not shown). variations in ѱf and pigment concentration (labo­ ratory measurements or estimates with reflectance in the field), measured in three types of fruit (hf, rf, sbf) (fig. 5), have been reported in previous works by solovchenko et al. (2010) and torres et al. (2016 a, b). symptoms of sun damage were associated with increase in car and reduction in chl and ѱf, as reported by felicetti and schrader (2009), torres et al. (2013, 2016 b) and yuri et al. (2000). chlb esti­ mated by reflectance did not decrease. ѱfe was the only discriminating variable in sbf vs hf and rf (fig. 5), in accordance with torres et al. (2013). increase of 860­900 nm reflectance, reported as a consequence of structural differences between damaged and undamaged tissue (torres et al., 2016 a), could not be confirmed by spectroradiometry in our work. a 0.42 correlation between ѱfe and reflectance values in the 860­900 nm range were obtained (fig. 6). although it has been widely reported that the content of pro in vegetables tissues is an indicator of stress (suzuki, 2015), correlations between pro, chl and ѱf (fig. 6) in apples have not been reported. research on apple trees under hydric stress conduct­ ed by šircelj et al. (2005) does not report consistent patterns of change of individual free amino acids. these authors, however, mention the lack of agree­ ment between those results and their own previous studies, as well as those by chandel and chauhan in 1991, which report significant increases in pro foliar, glu, orn, arg and total free amino acids under stress, evidencing active osmoregulation (šircelj et al., 1999). our results showed an increase in pro con­ tent in sunburn fruits. more studies should be carried out to evaluate the possibility of using this amino acid as an early indicator of sun damage. total amino acid content and its variations should be analyzed to elucidate between de novo pro synthesis and simple proteolysis (šircelj et al., 2005; arias­sibillotte et al., 2019). in accordance with the results of felicetti and fig. 7 ­ regressions between laboratory variables. colors repre­ sent condition of fruit: green for healthy fruit (hf), red for red fruit (rf), and black for sunburnt fruit (sbf). symbols represent days after full bloom (dafb): ○ for 91; δ for 99; + for 154. adv. hort. sci., 2020 34(4): 431­440 438 schrader (2009), the correlations found between chl and car concentrations were significant and nega­ tive, whereas the correlation between the values of pigments measured in the laboratory and those esti­ mated by reflectance reached a maximum of 0.25 (fig. 6). plant senescence index psri480 was the spectroradiometry indicator with the best capacity to discriminate between fruit types (fig. 5). correlation between psri480 and the best­performing laboratory indicators (pro y ѱf) was 0.15 (fig. 6). pigment con­ centration expressed on the basis of surface area were lower than range cited by solovchenko et al. (2010) in a 1/50 ratio, but similar to those recorded by yuri et al. (2010), expressed as dry weight concen­ tration. this difference could explain the weak corre­ lation between the laboratory tests of pigments and field estimates in our trials. high inter­annual variability of sunburn in apple fruit was observed in terms of magnitude and moment of occurrence. the inter­annual variability of rainfall combined with insufficient irrigation to meet crop water demand was more associated with sun­ burn during the period studied than the maximum temperature. regarding non­destructive prediction, the psri480 senescence reflectance index was the best discriminator between undamaged and dam­ aged fruits, however, like other recent studies, we were unable to devise a method of non­destructive prediction that could be used for commercial produc­ tion. ѱfe is the main variable that discriminates the degree of damage to the fruits, so it could be the consequence of the effects of high irradiance and temperature. the association between low hydric potential and pro 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the cul‐ tivar and areas of the fruit. ­ j. food. agric. environ., 8: 920­925. yuri j.a., torres c., bastías r., neira a., 2000 ­ golpe de sol en manzanas. ii. factores inductores y respuestas bioquímicas. ­ agro­ciencia, 16: 23­32. impaginato 343 adv. hort. sci., 2021 35(4): 343­349 doi: 10.36253/ahsc­10983 reflective benches for improving ligh­ ing in residential basil cultivation d. ferreira cavalcante 1, e. pradi vendruscolo 1 (*), a. seleguini 2, c. de castro seron 1, e. costa 1, l. leandro pires 3 1 mato grosso do sul state university, rod. ms 306, km 6,4 cep 79540‐ 000, cassilândia, mato grosso do sul, brazil. 2 triângulo mineiro federal university, rio paranaíba avenue, cep 38280‐000, iturama, minas gerais, brazil. 3 agronomy school, goiás federal university, samambaia campus, esperança avenue, cep 74690‐900, goiânia, goiás, brazil. key words: cultivation technology, light supplementation, ornamental species, urban cultivation. abstract: light­reflecting covers can help plant development in shaded environ­ ments, improving growing conditions in small residential spaces, encouraging the practice of small cultivations of food and ornamental species. thus, the objective of this work was to evaluate the effect of reflective bench covers on the growth and development of different cultivars of basil, in pot and shaded environment. the treatments consisted of three reflective materials (aluminum thermal reflective mesh, shiny red and white coating on laminates), arranged on the cultivation benches, and two basil cultivars (green and purple). after 22 days under these environments, the number of leaves and nodes, stem diame­ ter, plant height, relative chlorophyll content, fresh and dry masses of the aeri­ al and roots parts were determined. the highest value of reflective photosyn­ thetically active radiation was shown when using white laminate, followed by the red laminate and thermal reflective mesh. the green basil varieties showed higher values for number of leaves, stem diameter, plant height, fresh shoot weight and number of nodes, compared to the purple variety; however, in rela­ tion to the relative chlorophyll content, the purple variety was superior. the bright white and red laminate covers positively influence environmental condi­ tions, increasing the reflectance of photosynthetically active radiation, and the development of basil plants in conditions of greater shading. 1. introduction in view of the urban valorization, the well­lit spaces that could be reserved for the cultivation of ornamental plants, fruits, vegetables, or medicinal plants, have been extinguished in the residences of modern society. with the reduction of the sizes of urban lots, the practice of culti­ vation continues in inadequate spaces, with little or no direct sunlight, limited in containers, arranged in corridors, balconies, garages and even indoors. (*) corresponding author: agrovendruscolo@gmail.com citation: ferreira cavalcante d., pradi vendruscolo e., seleguini a., de castro seron c., costa e., leandro pires l., 2021 ­ reflective benches for improving lighting in residential basil cultivation. ­ adv. hort. sci., 35(4): 343­349. copyright: © 2021 ferreira cavalcante d., pradi vendruscolo e., seleguini a., de castro seron c., costa e., leandro pires l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 february 2021 accepted for publication 2 august 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10983 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 343­349 344 in the last decades the consumer has been look­ ing for novelties in the area of ornamental plants (noordegraaf, 2000) and has been attracted by the use of species with unusual aesthetic potential for cultivation in pots. however, the lack of information for the cultivation of some species in residential spaces, causes the inadequate development of plants to occur and culminate in loss of stimulus on the part of the owners. basil (ocimum basilicum l.) belong to the lamiacea family and it’s economic value have being increased due to its multiple uses as aromatic and seasoning species, on pharmaceutical industry, and essential oil production, (pereira and moreira, 2011). this species is a vigorous shrub, with potential to also be used as an ornamental, due to its characteristics as the shape of the plant, leaves, flowers, and inflo­ rescences, added to the pleasant aroma and the pos­ sibility of direct consumption (frança et al., 2017). studies with basil have been developed to identify agronomic aspects, chemical compounds for the pharmaceutical and cosmetics industry, among oth­ ers (pereira and moreira, 2011). however, research that seeks to assess the potential of culture for the home environment with some light restriction is incipient. light is the main element for photosynthesis to occur, enabling the conversion of light energy into organic energy. therefore, plant growth and develop­ ment are affected in a complex way by solar radiation (taiz et al., 2017). researches have been carried out to evaluate the responses of plants to light intensity, using parameters such as height, diameter of the neck, dry matter of the aerial part and roots. in a study by chang et al. (2008) with four levels of solar radiation on basil, there was a reduction in height, weight, leaf area and an intense drop in photosyn­ thesis under 75% of shading. in the presence of light, the responses of the plants are mediated by changes depending on the intensity, quality, direction, and duration of the solar incidence and are controlled by specialized photore­ ceptors (kami et al., 2010). photoreceptor proteins have a small cofactor or chromophore molecule, which capture and act at specific wavelengths of light over a specific spectrum (burgie et al., 2014). the development of some plants common to domestic cultivation, such as basil, is effectively affected by both the spectrum and the intensity of light that falls on the plant, which can enhance the biometric development and the chemical composi­ tion of plant organs (stagnari et al., 2018). thus, the implementation of techniques to obtain better condi­ tions for the development of plants is necessary, since shading interferes with the use of solar radia­ tion causing losses in the development of some species (conforto et al., 2011). aiming to obtain concise data and to stimulate the practice of small domestic crops of food and ornamental species cultivation, in environments with low solar incidence, the objective of this work was to evaluate the effect of reflective materials on the growth and development of two basil cultivars pot­ ted in a shaded environment. 2. materials and methods location and characterization of the experimental area the work was carried out in an urban residential area in the space of a corridor formed between the wall and the house, in the municipality of cassilândia ­ ms (19° 10’ 81” s; 51° 73’ 44” w and average alti­ tude of 540 m), from october to december 2019. according to the köppen climate classification, the region’s climate is humid tropical (aw) with an aver­ age annual temperature of 29°c. during the conduc­ tion of the experiment, the climatic data were col­ lected in a meteorological station present in the municipality (fig. 1). the space presented between the wall (2.20 m high) and the house wall (6.50 m high) in the dimen­ sions (1.45 m wide and 6.20 m long) forms an open corridor, which receives at least 3 hours of sunlight during the morning (fig. 2). this environment does not include a source of artificial lighting, the color of the walls of the house being white and the ceramic brick wall in natural color. fig. 1 ­ summary of climatic conditions during the experiment. ferreira cavalcante et al. ‐ reflective benches for residential basil cultivation 345 experimental design a completely randomized design was used to evaluate the reflective materials, with six treatments and eight repetitions, each pot being one repetition. the treatments were composed of three reflective materials arranged on the cultivation bench (thermal reflective mesh, bright red laminate sheet; bright white laminate sheet) and two varieties of basil ocimum basilicum l. (sweet basil ­ green leaf and purple basil ­ purple leaf), both from the manufactur­ er feltrin®. implementation and conduction of the experiment the experiment was installed and conducted on benches made with reused wood pallets, in the dimensions of 0.40 m width, 1.20 m length and 0.90 m height, spaced 0.70 m apart from each other and covered with the three different reflective materials. these same materials were fixed on the walls of the house, in a vertical direction, with dimensions 0.50 m x 1.20 m. the seedlings were produced in expanded poly­ styrene trays of 200 cells, filled with commercial sub­ strate carolinasoil® at the experimental area. following the recommendations of the manufactur­ er, the depth for sowing was 0.5 cm, both for green and purple basil. the emergence occurred 5 days after sowing, thus thinning the seedlings leaving only one per cell and 26 days after sowing the transplant was carried out to the black flexible pvc pots with a capacity of 1 l, being manually irrigated two times a day. after 22 days, the relative chlorophyll content in the leaves, the number of leaves, plant height, stem diameter, the number of nodes, fresh and dry weight of the aerial part and roots were evaluated. the plant height, in centimeters, was determined from the level of the substrate in the pots to the inflection of the highest leaf. the stem diameter, in millimeters, was measured from the base of the stem using a digital caliper. the relative chlorophyll con­ tent was obtained using a portable digital chlorophyll meter spad­502 (minolta camera co. ltd.), deter­ mined from the third or fourth mature leaf and com­ pletely expanded, starting from the apex of each plant. to obtain fresh weight, the plants were cut close to the substrate, divided into aerial part and roots and weighted. the determination of the dry weight of the aerial part and root the plants were dried in an oven with forced air circulation at 65°c for 72 hours. microclimatic data of the cultivation environment were collected between 9 am and 10 am on different sunny days, with the apogee model mp­200 measur­ ing the reflected par (µmol m­2 s­1) of each material (treatment) with the sensors facing the bench at a height of 30 cm and the incident environmental par (µmol m­2 s­1). statistical analysis the data were submitted to analysis of variance (test f) and the averages were compared by the tukey test, at 5% probability. the analyzes were per­ formed using the statistical software sisvar® version 5.6 for windows (ferreira, 2014). 3. results and discussion it was found that the reflected radiation was high­ er when using white laminate sheet (fig. 3), which reflected about 12.67% of the total radiation, fol­ lowed by the thermal reflective mesh (trm) and red fig. 2 ­ location of the residential corridor used for the domestic cultivation of basil (ocimum basilicum l.) plants in a pot according to the solar orientation. fig. 3 ­ reflected radiation of with different bench covers, from an environmental incident radiation of 1910 μmol m­2 s­1. adv. hort. sci., 2021 35(4): 343­349 346 laminate sheet, with 7.01% and 6,09%, respectively. the interaction between the two factors studied (cultivars x bench covers) on the growth attributes of basil was not verified (p>0.05). however, there was a significant effect, both for the type of bench used (p> 0.05) and for the varieties (p>0.05). regardless of the cultivars, basil plants grown on thermal reflective mesh had lower number of leaves, stem diameter, plant height, relative chlorophyll con­ tent, fresh and dry aerial weight than plants grown using white and red laminate sheets (fig. 4). however, the dry root mass did not vary between dif­ ferent materials. proportionally, these reductions were on average 33.48%, 15.32%, 12.88%, 20.70%, 13.98% and 35.67% respectively, for the number of leaves, stem diameter, plant height, relative chloro­ phyll content, fresh weight and dry weight of aerial parts, compared to the combined average of white and red laminate sheets (fig. 4). the gains in biometric characteristics when using bright white laminate sheet may be related to the quality of light, antonopoulou et al. (2004) state that white light is a rearrangement of colors including blue and red. these wavelengths are those used by plants for the process of photosynthesis, as well as physiological process. the number of leaves was affected by cover mate­ rial used, so that plants grown in the environment with bright white and red laminate sheets had a high­ er number of leaves in relation to the thermal reflec­ tive mesh. therefore, it can be inferred that basil plants are responsive to the quality of light under the conditions evaluated. in the cultivation of pereskia aculeata, vieira (2017) obtained a lower number of leaves under red mesh in relation to the pearl mesh, which is a varia­ tion of the white color, showing different results from that found in the present work. differences were verified for the stem diameter as a function of the materials used. the thermal reflec­ tive mesh provided the lowest value, when compared to the bright white and red laminate sheets. larger stem diameter is a desirable characteristic in seedlings because it guarantees greater support for the aerial part, when these plants are exposed to adverse wind or intense rain conditions (souza et al., 2014). the growth pattern of basil plants varied accord­ ing to the environments to which they were submit­ ted. plants exposed to colored laminate sheets had greater height when compared to those on thermal reflective mesh but there was no significant differ­ ence between white and red laminate sheet. in a study by souza et al. (2014) evaluating the growth of rosemary plants under colored meshes and in full sun, found a greater height of plants when cul­ tivated in full sun, attributing to this the high lumi­ nous intensity and the characteristics of the species. this corroborate with what was observed in the pre­ sent study, when the high radiation reflected by the white laminate sheet was verified, increasing the total radiation on plant tissues (fig. 4). although the red laminate sheet reflected a lower percentage of radiation, it yielded results similar to those of the white laminate sheet, which possibly has a relation­ ship with the quality of the reflected light. the growth in terms of stem diameter and plant height and higher chlorophyll content in the leaves showed greater gains in the colored laminate sheets, compared to the thermal reflective mesh, indicating that there was no etiolation. according to taiz et al. (2017), etiolated plants have long hypocotyls, a hooked apex, and non­photosynthetic leaves. henrique et al. (2011), studying the development of coffee seedlings under cover with colored meshes, blue, white, gray, black and red, with shading of 50%, found that the red one was more efficient in promot­ ing growth in plant height, area and leaf dry matter mass and total dry matter mass. in the present study with basil, it was possible to observe that, there was fig. 4 ­ number of leaves (a), stem diameter (b), plant height (c), relative chlorophyll content (d) fresh weight of the aerial part (e) and dry weight of roots (f) of green and purple basil (ocimum basilicum l .), grown in pots in a domestic environment, with different bench covers. ferreira cavalcante et al. ‐ reflective benches for residential basil cultivation 347 no difference between the white and red laminate sheets, in the variable plant height. for the relative chlorophyll the plants placed on the bench covered with thermal reflective mesh had a lower value, which indicates the low capacity of this material to reflect the frg. the spad index (soil plant analysis development) is used to diagnose the nitrogen status of crops, an element related to plant growth and productivity (pôrto et al., 2011) and related to the photosynthetic condition (dinh et al., 2019). costa et al. (2012), evaluating the vegetative growth of peppermint seedlings in five environ­ ments, full sun and under black, aluminized, blue and red meshes, all with 50% irradiance, observed superi­ or gains in leaf dry weight, stem and shoot biomass under full sun, red and black mesh respectively, when compared to other cultivation environments. paulus et al. (2016) found similar results when culti­ vating basil under a photoconverting mesh, finding a greater accumulation of fresh and dry mass of the aerial part in full sun, in relation to the red and alu­ minized mesh. divergent results were found in the present study, which showed a greater accumulation of fresh weight of the aerial part in plants grown on white and red laminate sheet. basil is a heliophyte species and according to silva (2014) it is considered efficient at high radiation intensities, which promotes better photosynthetic activity. the reflected radiation when using white laminate sheet was higher than that observed for the red one, however, no results were obtained in the accumulation of fresh biomass from the aerial part, which may be also associated with the quality of the light reflected by the materials used. the aerial part dry weight of the basil plants was affected by the use of the cover materials (fig. 4f). plants grown with the use of bright white and red laminate sheets showed a higher dry weight of the aerial part, in relation to the thermal reflective mesh. melo and alvarenga (2009), evaluating the effect of radiation in full sun condition and by covering with red, blue and black meshes, in catharanthus roseus l. g. don vinca plants, found greater increases in dry weight using the red material. sunlight is like a shower of photons and plays an essential role in photosynthesis, the process of trans­ forming light energy into chemical energy. wave spectra have different roles in plants. blue light (400­ 500 nm) promotes root growth and intense photo­ synthesis. the red light that comprises the light range between (600­700 nm, provides increases in the accumulation of dry mass, lengthening of the stem and expansion of the leaf area in addition to improvements in photosynthetic activity. the pho­ toreceptors that promote morphogenic changes in plants are those capable of to absorb blue and red light, with phytochrome being responsible for the absorption of these spectra (taiz et al., 2017). despite the lower proportion of radiation reflect­ ed by the red laminate sheet, this did not prevent the plants grown in this environment from accumulating an equal amount of dry weight from the aerial part to that of the plants when grown on white laminate sheet. this demonstrates that plants have the ability to modify their development model in response to the luminous environment (larcher, 2004). plants have a specificity regarding the wavelength received due to the absorption spectrum determined by the photosynthetic pigments present in the chloroplast. the absorption spectrum determines the amount of light energy captured or absorbed by a molecule or substance as a function of the wave­ length received. in this sense, the reflective materials arranged on the benches promote better use of radi­ ation by reflecting the luminosity of the environment on the leaf’s abaxial face (lima et al., 2018). studying the effect of red light emitting diode (led) lamps on grapes, poudel et al. (2008) found greater optimization of photosynthesis, directly influ­ encing the height of the aerial part, length of intern­ odes and rooting frequency, however, it is notewor­ thy the need for blue light for chlorophyll synthesis and stomatal activity. lima et al. (2010) evaluating the growth of anthurium andraeanum apalai under colored meshes with 70% shading found that the black mesh provided better growth conditions in relation to the blue, red and thermal reflective mesh. among basil varieties, it was observed that for the variables of leaf number, stem diameter, plant height, fresh shoot weight and number of nodes, the plants of the green variety were 144.20% higher, 34.95%, 35.59%, 4.76% and 20.91%, respectively, in relation to the purple variety. however, in relation to the relative chlorophyll content, the purple variety was 12.0% higher (fig. 5). lin et al. (2020) investigating the response of pur­ ple and green basil when subjected to different pro­ portions of red, blue and green light, found a greater increase in height, leaf area and stem diameter in green basil plants compared to purple. this shows the influence of the environmental conditions 348 adv. hort. sci., 2021 35(4): 343­349 imposed on the growth and development of the plants. the results found by the authors are similar to the present study, where there was interference from the environment and the genotype in the growth and development of plants. the accumulation of dry weight in the aerial part and roots was also higher in plants of the green vari­ ety, about 52.51% and 83.64%, respectively, in rela­ tion to plants of the purple variety (fig. 6). the differ­ ences between the genotypes of basil used in the present study were expected due to species great genetic diversity, not only morphological, but also in terms of the characteristic compounds of the species, such as essential oils (blank et al., 2010; veloso et al., 2014; castro et al., 2016). the cultivation of plants in homes is a custom passed down from generation to generation. however, due to the growth of urban areas and the consequent reduction of spaces for cultivation, there is a need for innovative techniques that provide bet­ ter conditions for the development of plants, especial­ ly in environments with less incidence of light. in this sense, the use of colored materials on countertops in cultivation environments is a technique that has been studied and has shown positive results in this new model, as demonstrated in the present study. the use of colored benches in low solar radiation places provides better conditions for the growth and development of basil plants very similar and/or supe­ rior to the cultivation carried out in the absence of shading, according to a study by souza et al. (2011). according to the authors, the accumulation of dry weight of plants grown with substrate in full sun was less than the values of the present study. based on the results of the present study, and in view of the characteristics of cultivation in domestic spaces, it is inferred preference for the use of white coloring, due to the characteristic of providing greater luminosity to the environment, in addition to the aesthetic enhancement of the vegetables, with contrast provided between the plants and the bench. in addition, concerning the cultivation of basil, the use of a white or red cover favors the development of plants due to the greater reflectance of photosyn­ thetically active radiation. in this way, both colors can be used for the cultivation of this species in shad­ ed environments. references antonopoulou c., dimassi k., therios i., chatzissav­ vidis c., 2004 ­ the influence of radiation quality on the in vitro rooting and nutrient of peach rootstock. ­ biologia plantarum, 48(4): 549­553. blank a.f., de souza e.m., de paula j.w., alves p.b., 2010 ­ comportamento fenotípico e genotípico de pop‐ ulações de manjericão. ­ horticultura brasileira, 28: 305­310. burgie e.s., bussell a.n., walker j.m., dubiel k., vier­ stra r.d., 2014 ­ crystal structure of the photosensing module from a red/far‐red light‐absorbing plant phy‐ tochrome. ­ proceedings of the national academy of sciences, 111(28): 10179­10184. castro h.g., veloso r.a., cardoso d.p., chagas fig. 5 ­ number of leaves (a), stem diameter (b), plant height (c), relative chlorophyll content (d), aerial fresh weight (e) and number of nodes (f) between green and purple basil cultivars (ocimum basilicum l.) grown in pots in a domestic environment, with different bench covers. fig. 6 ­ dry weight of the aerial part and roots between green and purple basil cultivars (ocimum basilicum l.), cultiva­ ted in pots in a domestic environment, with different bench covers. ferreira cavalcante et al. ‐ reflective benches for residential basil cultivation 349 júnior a.f., chagas l.f.b., 2016 ­ characterization botanical‐agronomic and essential oil of basil acces‐ sions . ­ revista verde de agroecologia e desenvolvimento sustentável, 11(4): 1­6. chang x., alderson p.g., wright c.j., 2008 ­ solar irra‐ diant ce level alters the growth of basil (ocimum basilicum l.) and its content of volatile oils. ­ environ. exper. bot., 63: 216­223. conforto e.c., bittencourt júnior n.s., scaloppi júnior e.j., moreno r.m., 2011 ­ comparação entre folhas sombreadas de sete clones adultos de seringuei‐ ra. ­ revista ceres, 58(1): 29­34. costa a.g., chagas j.h., pinto j.e.b.p., bertolucci s.k.v., 2012 ­ crescimento vegetativo e produção de óleo essencial de hortelã‐pimenta cultivada sob malhas. ­ pesquisa agropecuária brasileira, 47(4): 534­ 540. dinh t.h., takaragawa h., watanabe k., nakabaru m., kawamitsu y., 2019 ­ leaf photosynthesis respon‐ se to change of soil moisture content in sugarcane. ­ sugar tech, 21(6): 949­958. ferreira d.f., 2014 ­ sisvar ‐ a guide for its boots rap pro‐ cedures in multiple comparisons. ­ ciência e agrotecnologia, 38(2): 109­112. frança m.f.m.s., vilela m.s., costa a.p., nogueira i., pires m.c., souza n.o.s., 2017 ­germination test and ornamental potential of diferente basil cultivars (ocimum spp.). ­ ornamental horticulture, 23(4): 385­ 391. henrique p.c., alves j.d., deuner s., goulart p.f.p., livramento d.e., 2011 ­ aspectos fisiológicos do desenvolvimento de mudas de café cultivadas sob telas de diferentes colorações. ­ pesquisa agropecuária brasileira, 46(5): 458­465. kami c., lorrain s., hornitschek p., fankhauser c., 2010 ­ light‐regulated plant growth and development. ­ current topics in developmental biology, 91: 29­66. larcher w., 2004 ­ ecofisiologia vegetal. ­ vol. i são carlos, sp, rima artes e textos, pp. 531. lima j.d., nomura e.s., fuzitani e.j., silva s.h.m.g., 2010 ­ variáveis fisiológicas de antúrio cultivado sob diferentes malhas de sombreamento. ­ scientia agraria, 11(3): 193­200. lin k.h., huang m.y., hsu m.h., 2020 ­ morphological and physiological response in green and purple basil plants (ocimum basilicum) under different proportions of red, green, and blue led lightings. ­ scientia horticulturae, 275(3). melo a.a.m., alvarenga a.a., 2009 ­ sombreamento de plantas de catharanthus roseus (l.) g. don ‘pacifica white’ por malhas coloridas: desenvolvimento vegeta‐ tivo. ­ ciência e agrotecnologia, 33(2): 514­520. noordegraaf c.v., 2000 ­ an approach to select new ornamental crops. ­ acta horticulturae, 541: 75­78. paulus d., valmorbida r., ferreira s.b.; 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(liliaceae) is a rare and endangered ornamental plant grown in mountain regions and zagros altitudes, ilam province, iran. this species is in danger of extinction due to invasive collection. plant regeneration was done by organogenesis from bulb scales as explants cultured on murashige and skoog (ms) media fortified with different concentrations of kinetin (kin, 0.00, 0.50, 1.00 and 2.00 mg l­1) and α_naphthaleneacetic acid (naa, 0.00, 0.50, 1.00 and 2.00 mg l­1), either individually or in combination. the largest number of leaf (3.80), root (5.86) and callus (8.16) per explant was regenerated on the medium containing 0.50 mg l­1 kin and 1.00 mg l­1 naa. maximum viability per­ centage (96.66%) was obtained in medium supplemented with 1.00 mg l­1 kin. in vitro regenerated plantlets were cultivated in plastic pots containing peat moss and perlite (1:1). the plantlets were successfully acclimatized in an adap­ tation greenhouse with a survival rate of 95% exhibiting normal developmental patterns. 1. introduction fritillaria (liliaceae) is a genus of about 100 species of bulbous peren­ nials found in a range of habitats, from woodland to open meadows and high screes, distributed throughout the temperate regions of the n. hemi­ sphere, particularly the mediterranean, s.w. asia and w. north america. each bulb of fritillaria has 2 or more scales, and sometime abundant basal bulblets. fritillaria imperialis l. (crown imperial or tears of mary) is a perennial plant with high medicinal and ornamental importance (wang et al., 2005). the 14 important species of f. imperialis l. are native to iran (de hertogh and le nard, 1993). wild populations of f. imperialis are mostly found in high altitudes (>2,000 m) of western parts of iran, partic­ ularly in three provinces, chahar mahal­va­bakhtiari, kohkyluyeh­va­ bouyrahmad and ilam. the leaves are usually lance­shaped or linear and the flowers, borne in spring or early summer, are usually pendulous and solitary, or in terminal racemes or umbels, and have 6 tepals. f. imperialis (*) corresponding author: sedaghathoor@yahoo.com citation: seydi s., sedaghathoor s., kaviani b., 2019 ­ plants regeneration by organogenesis from bul‐ bous explants in fritillaria imperialis l., a wild rare ornamental species at the risk of extinction. ­ adv. hort. sci., 33(4): 503­510. copyright: © 2019 seydi s., sedaghathoor s., kaviani b. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 february 2019 accepted for publication 2 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 503­510 504 l. has been used either as pot plant for designing landscape or cut flower. thanks to attractive red and yellow flowers, this plant reveals a great commercial potential (de hertogh and le nard, 1993). the bulbs of f. imperialis contain alkaloids, non­alkaloid and high starch content (li et al., 2000; wang et al., 2005). in iran, wild populations of two important species, f. imperialis and f. persica, are at the risk of extinc­ tion, because of many harvesting, lack of protecting rules, changing the pastures to dry farmlands, and pest and pathogens invasions. the natural prolifera­ tion rate of fritillaria is relatively low that hampers the large­scale cultivation of this plant. f. imperialis cannot efficiently propagate by traditional methods such as cutting, bulb scale and seed, because of small numbers of scales (3­5) per bulb and restricted amount of meristematic cells (de hertogh and le nard, 1993). plant proliferation takes 5­7 years through the seed, also seeds have physiological dor­ mancy. in addition, seedlings are weak, survival rate and growth are low, and produced plants by seed are not true­to­type; because of cross­pollinate nature of fritillaria (de hertogh and le nard, 1993; baskin and baskin, 2004). limited availability of bulblets from nature is another problem. these limitations suggest the need to develop alternative propagation meth­ ods for commercial production of these elite species. the application of biotechnology especially in vitro proliferation is a suitable method for reproduc­ tion of rare and endangered species with difficult propagation and mass production of valuable geno­ types (vetchinkina et al., 2012). this method is becoming increasingly important for conservation of rare and endangered plant species (almeida et al., 2005). in vitro propagation is an effectively alterna­ tive means for rapid multiplication of species, in which conventional methods have problems and limi­ tations. tissue culture using bulb scale segments, the most commonly used explants in tissue culture of bulbous plants including liliaceae family (mirici et al., 2005), and other explants such as foliar and flower explants has been reported for some cultivars of f. imperialis (paek and murthy, 2002). two basic morphogenetic ways leading to the regeneration of the whole plant from somatic tissues are organogenesis and embryogenesis. both ways of morphogenesis can occur as direct (without passing callus phase) and indirect (with passing callus phase). the wild population of f. imperialis is highly het­ erozygote and non­uniform, because of its self­ incompatibility nature. there are a few reports on direct and indirect organogenesis in f. imperialis (witomska and lukaszewska, 1997; witomska, 2000; paek and murthy, 2002; subotić et al., 2010; kizil and khawar, 2014). this paper describes a protocol for rapid in vitro multiplication of f. imperialis l. by bulb scales as explant and kin and naa as plant growth regulators (pgrs) that could be helpful for large­scale production for field culture. 2. materials and methods the bulbs of fritillaria imperialis l. were harvested from natural habitat (mountain regions and zagros altitudes, ilam province, iran) (figs. 1, 2a) and used as the starting material for the establishment of in vitro culture. bulbs were transferred to the plant tissue culture and biotechnology laboratory, amol, mazandaran province located in the northern part of iran. in laboratory, bulbs (fig. 2b) were washed under running tap water (fig. 2c) for half an hour to remove mud and dirt. the bulbs were put into a ves­ sel filled with water and a few drops of dish­washing liquid for 10 min (fig. 2d). then, these were washed in running tap water for half an hour, again. cleaned bulbs were decontaminated with a fungicide (0.10 mg l–1 benomile + carbendazim, rural t.s.) for 20 min followed by once washing for 10 min. in distilled water. the bulbs were sterilized by immersing in 0.01 g l–1 mercuric chloride (hgcl) for 15 min with continu­ ous stirring using magnetic stirrer, then by 20% sodi­ um hypochlorite (naocl) solution (commercial bleach) for 15 min followed by three times rinsing (each for 10 min.) in sterile distilled water and finally fig. 1 ­ fritillaria imperialis l. growing at its natural habitat (mountain regions and zagros altitudes, ilam province, iran). seydi et al. ‐ in vitro propagation of fritillaria imperialis l. 505 the bulbs were dipped in 70% ethanol for 60 sec. the scales were rinsed in sterile distilled water for three times. dried bulbs (by placing on paper for 5 min.) were vertically cut into 10 × 10 mm under aseptic condi­ tions to obtain twin scales as explants to induce cal­ lus and shoots for in vitro propagation (fig. 2e). outer scales directly in contact with disinfectant during sterilization were removed before obtaining double scale explants (fig. 2f). the medium used was ms (murashige and skoog, 1962) with 3.00% sucrose and 0.80% agar­agar. the ph of the medium was adjusted to 5.80. all media were autoclaved at 104.00 kpa and 121°c for 20 min. the scales were cultured on media fortified with naa and kin (0.00, 0.50, 1.00 and 2.00 mg l­1 from each) to induce shoots from bulb scales. callus was formed on bulb scales cultured in media containing pgrs. in vitro regenerated shoots were grown in these media to find the suitable regeneration potential for shoot and root production. all experiments were per­ formed in 280 ml­jam glasses (6 cm diameter × 10 cm height) and each glass contained 50 ml medium. all cultures were incubated at 24­26°c, 70­80% rh under light intensity of 50 µmol m­2 s­1 from white flu­ orescent lamps with 16 h light photoperiod per day. data were recorded after 65 days of culturing. the parameters: leaf length, leaf diameter, leaf number, root length, root number, callus number and viability percentage were assessed. in vitro produced plantlets were taken out from culture vessels and washed thoroughly under running tap water to remove adherent nutrient and trans­ ferred to plastic pots containing peat moss and per­ lite (1:1). all the pots were then transferred to the adaptation greenhouse with temperature of 24±2°c to 20±2°c day/night, light intensity of 8000 lux, rh of 80­85% and 14­h photoperiod for acclimatization. after 30 days all plantlets were acclimatized. the experiments were carried out in a completely randomized design with three replicates per treatment and four scales per replicate (totally; 192 explants). pgrs­free ms medium is used as control in the experi­ ments. the results are expressed as mean ± sd of the experiments. data pertaining to plantlets growth and development were subjected to analysis of variance (anova) and means were compared by the lsd test at p < 0.05 using the spss ver. 17 (spss inc., usa). 3. results the twin bulb scale explants (10×10 mm) of f. imperialis l. cultured on ms media containing differ­ ent concentrations of kin and naa showed variation in the frequency of callus formation, shoot regenera­ tion and root formation (fig. 3, tables 1­4). leaf induction and proliferation callus was induced on bulb scales (fig. 3a). leaves were produced from callus through indirect organo­ genesis (figs. 3b, c). the minimum leaf length (1.26 cm) from twin scale explants was measured on ms fig. 2 ­ sterilization of bulbs and preparation of scales explants. a) fritillaria imperialis l. eradicated from the soil. b) separated bulbs. c) washing the bulbs under running tap water. d) bulbs ready for sterilization. e) to cut cleaned bulbs as slices (scales). f) cultivation of scales as explant into the culture vessels (scale bar = 10 mm). fig. 3 ­ micropropagation process of fritillaria imperialis l. a) callus production on bulb scales. b) leaves produced from callus by indirect organogenesis. c) developed lea­ ves before planting in greenhouse condition. d) regenerated plantlets from callus on media containing different concentrations of kin and naa. e) rooted plan­ tlets. f) regenerated leaves in media supplemented with different concentrations of kin and naa. g) acclimatization of plantlets grown into the pots filled with peat moss and perlite (1:1) in an adaptation greenhouse (scale bar = 10 mm). adv. hort. sci., 2019 33(4): 503­510 506 medium without pgrs (table 4). the maximum num­ ber of 3.16 leaves per explant on twin scale explants was measured on ms medium containing 1.00 mg l­1 kin and 0.50 mg l­1 naa (figs. 3d, f). differences of leaf length in samples grown under different concen­ trations of kin and kin in combination with naa were significant (p≤0.01) (table 1). no significant difference was seen between naa and leaf length. there was no any positive effect between increasing leaf length and increasing kin and naa concentrations (tables 2­4). among all concentrations of kin used singularly, max­ imum and minimum leaf length (2.41 and 1.80 cm, respectively) was induced in bulbs treated with 1.00 mg l­1 and control (table 2). on the other hand, among all concentrations of naa used as singular pgr, maximum and minimum leaf length (2.24 and 1.85 cm, respectively) was induced in bulbs treated with 2.00 and 1.00 mg l­1 (table 3). there was no sta­ tistically significant difference among different con­ centrations of kin + naa and leaf diameter (table 1). the data clearly show that leaf number is strongly affected by treatments of kin (p≤0.01), naa (p≤0.01) and kin + naa (p≤0.01) (table 1). the largest number of leaf (3.80 per explant) was calculated in bulbs grown on medium enriched with 0.50 mg l­1 kin along with 1.00 mg l­1 naa (table 4; figs. 3d, f). the smallest number of leaf (1.66 per explant) was obtained in bulbs grown on medium with 2.00 mg l­1 kin along with 1.00 mg l­1 naa. all treatments con­ taining 2.00 mg l­1 kin in combination with all con­ centrations of naa produced less than 2.00 leaves per explant (table 4). of all concentrations of kin, the largest and smallest number of leaf (3.33 and 1.85, respectively) was induced in explants grown on medium enriched with 0.50 and 2.00 mg l­1 (table 2). also, differences in leaf number between all concen­ trations of naa were not noticeable. root induction and growth the maximum average of the root length per table 3 ­ mean comparison of the effect of different concentrations of naa on measured characters of fritillaria imperialis l. table 1 ­ analysis of variance of the effect of different concentrations of kin and naa on measured characters of fritillaria imperialis l. table 2 ­ mean comparison of the effect of different concentrations of kin on measured characters of fritillaria imperialis l. *, ** = significant at the 0.05 and 0.01 probability level, respectively, ns= not significant at p=0.05. source of variations df mean of squares leaf length leaf diameter leaf number root length root number callus number viability percentage kin 3 0.877 ** 0.020 ns 4.580 ** 4.820 ** 4.201 ** 7.440 ** 413.80 ** naa 3 0.379 ns 0.041 * 0.690 ** 1.023 ** 1.397 ns 2.087 * 113.80 ns kin × naa 9 0.686 ** 0.030 ns 0.356 ** 1.059 ** 1.815 ** 5.270 ** 67.50 ns error 32 0.216 0.014 0.099 0.226 0.554 0.636 50.00 cv (%) 22.40 15.80 12.10 12.90 18.03 18.00 8.35 kin (mg l­1) leaf length (cm) leaf diameter (mm) leaf number root length (cm) root number callus number viability (%) 0.00 1.800 b 0.7666 a 2.750 b 2.833 c 3.483 b 3.491 b 78.33 b 0.50 1.933 b 0.8083 a 3.333 a 3.700 b 4.866 a 5.266 a 89.16 a 1.00 2.416 a 0.7083 a 2.450 c 4.375 a 4.341 a 4.858 a 90.00 a 2.00 2.158 ab 0.7666 a 1.850 d 3.766 b 3.616 b 4.116 b 80.83 b means with different letters on the same column are significantly different (p<0.05) based on lsd test. naa (mg l­1) leaf length (cm) leaf diameter (mm) leaf number root length (cm) root number callus number viability (%) 0.00 2.008 a 0.750 ab 2.80 a 3.266 b 3.74 b 4.458 ab 85.00 ab 0.50 2.200 a 0.700 b 2.31 b 3.791 a 4.33 ab 4.333 ab 80.83 b 1.00 1.858 a 0.758 ab 2.79 a 3.950 a 4.48 a 4.975 a 84.16 ab 2.00 2.241 a 0.841 a 2.47 b 3.666 a 3.95 ab 3.966 b 88.33 a means with different letters on the same column are significantly different (p<0.05) based on lsd test. seydi et al. ‐ in vitro propagation of fritillaria imperialis l. 507 and 3.48) was counted in media fortified with 0.50 mg l­1 kin and control, respectively (tables 2, 3). analysis of variance (anova) test demonstrated that concentrations of kin and kin in combination with naa were significant with respect to number of roots produced from scale sections (table 1). callus induction current investigation demonstrated variable fre­ quency of callus induction on bulbs scales at various concentrations of kin and naa in the culture medi­ um. callus was emerged after 30 days of culture (fig. 3a). callus formation started at the margins of bulbs scales. callus induction was evident in response to the presence of both kin and naa. maximum (8.16) and minimum (3.00) callus number per explant was observed on explants cultured on medium supple­ mented with 0.50 mg l­1 kin plus 1.00 mg l­1 naa and medium without pgrs (control), respectively (table 4). viability percentage viability percentage of the bulbs scales in the media was changed significantly with the use of dif­ ferent pgrs (table 4). viability percentage of the bulbs scales in ms medium supplemented with 1.00 mg l­1 kin without naa (96.66%) was the maximum. viability percentage in medium supplemented with 1.00 mg l­1 naa without kin (73.33%) was the mini­ mum (table 4). statistically significant differences were recovered between the means for viability per­ centage and kin (table 1). explant (5.50 cm) was calculated with treatment of 1.00 mg l­1 kin and 1.00 mg l­1 naa (table 4). the minimum average of the root length per explant (2.30 cm) was measured with treatment without kin and naa (control). concerning the root length induced by various concentrations of kin, the maxi­ mum and minimum length (4.37 and 2.83 cm) was obtained in media containing 1.00 mg l­1 and control, respectively (table 2). concerning the root length induced by various concentrations of naa, the maxi­ mum and minimum length (3.95 and 3.26 cm) was obtained in media containing 1.00 mg l­1 and control, respectively (table 3). there was statistically signifi­ cant difference among different concentrations of kin, naa also kin in combination with naa and root length (p≤0.01). kin in combination with naa was superior in induction of root and on the medium fortified with kin (0.50 and 1.00 mg l­1) + naa (0.50 and 1.00 mg l­1) optimal of over than 5 roots were produced per explant on ms medium (table 4). the largest num­ bers of roots (5.86 per explant) was formed in ms medium supplemented with 0.50 mg l­1 kin plus 1.00 mg l­1 naa (table 4; fig. 3e). bulbs scales cultured on ms medium without any pgrs produced least roots (2.83). the root number (5.40 and 5.00 per explant) in media containing 0.50 mg l–1 kin plus 0.50 mg l­1 naa and 1.00 mg l­1 kin plus 0.50 mg l­1 naa was proper (table 4). concerning the root number pro­ duced using various levels of kin and naa, each one as singularly; the largest and smallest number (4.86 table 4 ­ mean comparison of the effect of different concentrations of kin and naa on measured characters of fritillaria imperialis l. kin (mg l­1) naa (mg l­1) leaf length (cm) leaf diameter (mm) leaf number root length (cm) root number callus number viability (%) 0.00 0.00 1.266 ±0.208 d 0.733±0.0289 abcd 2.700±0.000 cd 2.300± 0.200 f 2.836±0.252 h 3.000±0.854 e 76.66±2.887 de 0.00 0.50 1.366±0.208 d 0.533±0.0577 d 2.266±0.058 ef 2.933± 0.115 f 3.233±0.153 e­h 4.000±0.854 cde 80.00±0.000 c­e 0.00 1.00 2.200±0.781 bc 0.900±0.1000 a 3.233±0.252 c 2.766±0.208 f 3.700±0.200 d­h 3.333±0.473 e 73.33±5.774 e 0.00 2.00 2.366±0.513 bc 0.900±0.1000 a 2.500±0.100 e 2.833f ± 0.153 4.733±0.451 3.033±0.462 e 83.33±5.774 b­e 0.50 0.00 1.933±0.493 bcd 0.800±0.1000 abc 3.700±0.000 ab 2.933±0.058 f 4.400±1.015 b­f 3.133±0.666 e 90.00±10.000 abc 0.50 0.50 1.933±0.153 bcd 0.766±0.0577 abc 2.500±0.200 e 4.666±0.058 b 5.400±0.656 ab 5.033±1.514 bc 86.66±15.275 abcd 0.50 1.00 1.700±0.458 cd 0.800±0.1000 abc 3.800±0.100 a 3.366±0.153 ef 5.866±1.422 a 8.166±0.702 a 86.66±5.774 a 0.50 2.00 2.166±0.321 bc 0.866±0.0577 ab 3.333±0.153 bc 3.833±0.289 cde 3.800±0.781 c­h 4.733±1.106 cd 93.33±11.547 ab 1.00 0.00 2.333±0.802 bc 0.666±0.2082 bcd 2.533±0.603 e 3.466±0.208 def 3.333±0.473 f­h 6.233±0.850 b 96.66±5.774 a 1.00 0.50 3.166±0.416 a 0.666±0.1528 bcd 2.566±0.473 de 4.300±0.100 bc 5.000±1.803 abc 3.933±0.153 cde 80.00±5.000 c­e 1.00 1.00 1.700±0.458 cd 0.700±0.1732 abcd 2.466±0.751 e 5.500±0.854 a 4.666±0.777 a­e 4.866±0.603 bcd 90.00±5.000 abc 1.00 2.00 2.466±0.252 abc 0.800±0.2000 abc 2.233±0.451 ef 4.233±0.451 bc 4.366±0.493 b­f 4.400±0.721 cde 93.33±2.887 ab 2.00 0.00 2.500±0.721 ab 0.800±0.1732 abc 1.966±0.058 fg 3.866±0.862 cde 3.766±0.115 d­h 4.86±0.635 bcd 76.66±5.774 de 2.00 0.50 2.333±0.153 bc 0.833±0.1155 abc 1.933±0.115 fg 3.266±0.666 ef 4.100±0.173 c­g 4.366±0.850 cde 76.66±5.774 de 2.00 1.00 1.833±0.351 bcd 0.633±0.0577 cd 1.666±0.231 g 4.166±0.850 bcd 3.700d±0.265 ­h 3.533±0.666 de 86.66±5.774 a­d 2.00 2.00 1.966±0.416 a­d 0.800±0.0000 abc 1.833±0.153 fg 3.766±0.702 cde 2.900±0.100 gh 3.700±0.800 cde 83.33±5.774 b­e means with different letters on the same column are significantly different (p<0.05) based on lsd test (mean ± sd). 508 adv. hort. sci., 2019 33(4): 503­510 plantlets were successfully acclimatized in an adaptation greenhouse and recorded 95% survival rate after 30 days in pots filled with perlite: peat moss (1:1 v/v) (fig. 3g). there were no visual mor­ phological abnormalities in the micropropagated plantlets. 4. discussion and conclusions fritillaria is a rare and critically threatened genus due to the large­scale eradication, irregular grazing and lack of protecting rules. this plant reveals a great commercial potential, therefore, the plants in danger of extinction, like fritillaria, should be protected. in vitro propagation is an effective method for conser­ vation and rapid multiplication of the species in dan­ ger of extinction like the members of liliaceae family including fritillaria due to limitations in conventional methods of propagation. various factors influence the process of micro­ propagation from bulb scales and in vitro regenerat­ ed bulblet explants, especially type, concentration and combination of pgrs particularly auxins and cytokinins. optimal selection of pgrs is especially important when the amount of the original plant material of rare and endangered plant species is lim­ ited (kulkhanova et al., 2015). for f. imperialis, in vitro propagation is carried out by bulb scales using mainly naa, iaa and ba (witomska and lukaszewska, 1997; lukaszewska et al., 1998; witomska et al., 1998; mohammadi­ dehcheshmeh et al., 2006; petrić et al., 2013). for most members of the genus fritillaria, micropropaga­ tion is done by bulb scales using mainly ba, bap, tdz, naa, iaa and 2,4­d (petrić et al., 2013; kulkhanova et al., 2015). the concentrations of 0.10­4.00 mg l­1 of naa in combination with 0.10­2.00 mg l­1 kin has been applied using bulb scales for in vitro propaga­ tion of some species of the genus fritillaria, such as f. anhuiensis (xue et al., 2008), f. camtschatcensis (okawa and nishino, 2000), f. roylei hook (joshi et al., 2007), f. thunbergii (seon et al., 1999), and f. ussuriensis (sun and wang, 1991), not for f. imperi‐ alis. naa is the most effective auxin in inducing the in vitro formation of bulblets from the segments of bulb scales of f. sonnikovae, and the maximal regenera­ tion was obtained combining 1.62 μm naa and 4.65 μm kin also 5.00 μm bap and 2.00 μm naa (kulkhanova et al., 2015). the use of bap in combina­ tion with iaa was effective for micropropagation of f. unibracteata (gao et al., 1999). in our study, the use of 0.50 mg l­1 kin and 1.00 mg l­1 naa was useful for shoot and root production through indirect organo­ genesis. cytokinins are generally known to promote the formation of buds in many excised and in vitro tissue cultured organs. the medium containing 2.20 µm ba was most effective for shoot formation on bulb scales of lilium longiflorum (han et al., 2004). similar results were also reported by others (naik and nayak, 2005). bulblets regeneration and shoot multi­ plication on bulb scale explants were observed in some liliaceae members, like f. thunbergii and ornithogalum ulophyllum (paek and murthy, 2002; ozel et al., 2008). ipek et al. (2006) applied different concentrations of bap, naa and kin to obtain bul­ blets from immature embryos of ornithogalum platy‐ phyllum. naik and nayak (2005) induced direct induc­ tion of bulblets on the bulb scales grown on the ms media enriched with 1.00 mg l­1 naa and 2.00 mg l­1 ba. naik and nayak (2005) showed a plant regenera­ tion procedure in ornithogalum virens through direct shoot bud formation and indirect organogenesis using bulb scale as explant cultured on ms medium containing 1.00 mg l­1 naa and 2.00 mg l­1 ba. paek and murthy (2002) revealed that the maximum bul­ blet regeneration and leaf production in f. thunbergii using bulb scale segments as explants were obtained in ms medium supplemented with a combination of kin and naa. current investigation is consistent with this finding. our study showed the same importance of kin and naa for shoot regeneration. in agreement with us, study of kukulczanka et al. (1989) on f. meleagris l. demonstrated that the highest percent­ age of the regenerating explants, was obtained in case of joint action of cytokinin and auxin. in f. aurea schott, increased tdz concentrations increased leaf number. the highest regeneration rate was obtained from combination of cytokinin and auxins (kizil et al., 2016). in f. imperialis, the number of regenerated shoots was the highest on ms medium supplement­ ed with 0.50 mg l­1 tdz and the number of roots was the highest on ms medium supplemented with 0.20 mg l­1 naa (rahimi et al., 2014). we observed variable frequency of callus induction at various concentrations of kin­naa in the culture medium. similar findings were reported by others using bap­naa (nayak and sen, 1995; naik and nayak, 2005; ozel et al., 2008). malabadi and van staden (2004) induced shoot bulblets and embryogenic calli in ornithogalum longibracteatum on medium containing kin. in the study of çakmak et al. (2016) on f. persica, all explants including bulb scales showed competency seydi et al. ‐ in vitro propagation of fritillaria imperialis l. 509 to regenerate callus and bulblets using 2,4­d and kin, or tdz. bulblet explants responded fairly well to a higher level of tdz in order to obtain maximum shoot regeneration. callus induction was evident in response to high tdz concentration. current study showed that the maximum root formation was obtained on medi­ um containing both kin and naa. similar to our find­ ing, ozel et al. (2008) successfully rooted bulblets regenerated on 2.00 mg l­1 bap and 0.50 mg l­1 naa. in f. aurea schott, maximum rooting was noted on 0.50 mg l­1 iba (kizil et al., 2016). in conclusion, f. imperialis, bulbous cultivated as ornamental cut flower and garden plant, has impor­ tant also as medicinal plant and it is at the risk of extinction. traditional propagation is l imited, because production of bulbs is poor and seeds have a low germination rate. therefore, in vitro propagation by direct and indirect organogenesis and embryogen­ esis is a proper approach. the success of these proce­ dures highly depends on type and concentrations of auxins and cytokinins applied in culture medium, sin­ gular or in combination. in our study, 0.50 mg l­1 kin along with 1.00 mg l­1 naa successfully induced leaf and root formation. references almeida r., goncalves, s., romano a., 2005 ­ in vitro micropropagation of endangered rhododendron pon­ ticum l. sub sp. baeticum (boissier and reuter) handel‐ 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thakur et al., 2024). beginning publicly in the early 1960s, tomato grafting became an (*) corresponding author: sunny.29533@lpu.co.in citation: thakur v., kumar p., sharma s., 2025 ­ insight to the conventional and biotechnological approaches in tomato on potato grafting (pomato): a review. ­ adv. hort. sci., 39(3): 213­226. orcid: tv: 0000­0002­3994­5496 kp: 0000­0002­3343­0504 ss: 0000­0002­5813­5210 copyright: © 2025 thakur v., kumar p., sharma s. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. credit author contribution conceptualization: v.t., p.k, and s.s., writing ­ original draft prepara­ tion, v.t., s.s., writing ­ review and editing, v.t., and s.s., funding acquisition, v.t., and s.s. received for publication 17 february 2025 accepted for publication 2 august 2025 ahs advances in horticultural science review paper ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15798 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-3994-5496 http://orcid.org/0000-0002-3343-0504 http://orcid.org/0000-0002-5813-5210 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 231­244 232 indispensable technique for tomato cultivation worldwide (lee and oda, 2003). the primary rationale for tomato graft ing was to use this approach instead of methyl bromide to control soil­ borne pathogen incidence under protected conditions (rivard et al., 2010; mcavoy et al., 2012; rana et al., 2024). grafting is currently being used on a large scale to combat abiotic stresses, alike low and high temperature stresses, heavy metals, salinity and water stress, as well as to improve quality and yield (venema et al., 1999; abdelmageed and gruda, 2009; flores et al., 2010; turhan et al., 2011; colla et al., 2013; bhatt et al., 2015; kumar et al., 2015; wudu and kassaw, 2022). because of this, the major purpose of adopting grafting techniques in tomatoes is to boost output while preserving nutritional value and minimizing the harmful effects of environmental conditions. the success rate of the grafted plant is determined by a variety of factors, but genomic considerations may be found to be the most important among all others because they confirm the interoperability percentage between the scion and rootstock (thakur et al., 2022). various studies have verified that grafting can have either positive or negative effects in terms of performance and yield (huh et al., 2003; yetisir et al., 2003; davis et al., 2008 a). furthermore, conflicting changes in the transplanted union may occur as a result of a sensitive transplanted union, physical incompatibility, grafted seedling collapse, and toxic compound accumulation (davis et al., 2008 b; villyáni et al., 2024). however, graft compatibility is more likely if the scion and rootstock are taxonomically close to each other (wang, 2011; farooque et al., 2024). numerous reports have predicted that grafting can be performed intraspecifically and interspecifically among crops in solanaceous crops (petran and hoover, 2014; chaudhari et al., 2016). tube and cleft grafting are the furthermost common techniques used in tomato grafting (lee and oda, 2003; thakur et al., 2024), but amongst these two, tube grafting is found to be the most uti l ized technique by accelerated farmers worldwide (hanna, 2012; vu et al., 2015). tube grafting is known to produce strong, raised grafts because it confirms a robust vascular attachment amid the union of scion and rootstock (bausher, 2013; rana et al., 2024). successful adaptation, which is the process of rehabilitation and densification of transplanted seedlings before polytunnel or field seeding, is another factor that guarantees successful graft establishment (lee and oda, 2003). some of the countries, such as china, south korea, japan, spain, italy, france, canada, turkey, the usa, mexico, india, and other countries (israel, netherlands, egypt, and brazil) are the major contributors in the vegetable grafting market (nawaz et al., 2017). oscar soderholm presented the basic concept of conceptualizing the tomato onto a potato, i.e., tomapotato, in 1930. the max planck institute for developmental biology in tübingen, germany, initially conceived this grafted pairing in 1977; nevertheless, the resulting grafts failed to produce fruit and tubers. moreover, the institute of plant biotechnology research in koln, germany, made a successful attempt at tomato/potato grafts in 1977, which produced fruits and tubers on the plant mixture (reinhard, 2008; bahadur et al., 2020; thakur et al., 2022, thakur et al., 2024). peres et al. (2005) studied the combination by grafting tomato on top of a potato plant: a method for studying leaf­ derived signaling on tuberization. the study revealed that tomato and potato seedlings were crossbred when they were 20 days old to maintain nursery stability and yield. tomato and potato crossbreds can yield up to 5 kg of tomato and 0.5 kg of potato, which is comparable to the yield of the control plant. kiambu prison made another attempt, according to another piece of literature cited by lubbock online news in 2002. in 2013, a uk­based horticultural mail company, thompson and morgan, sold grafts of the “tomtato” plant. the next year, new zealand’s incredible edible nursery announced a grafted plant called double up potato tom (gillies, 2013; kumar et al., 2015). bari and badc are currently producing pomato plants for production purposes (nusrat, 2014). in india, csk, hpkv palampur began research on the tomato plant in 2015 under protected conditions, while icar­ i ivr varanasi made a successful attempt in open field conditions for the first time in 2013 (kumar et al., 2015; bahadur et al., 2020). several successful reports have been documented by various researchers until 2021 that claim the pomato plant’s success story is not only one of comparable yield production but also matches the required quality attributes. besides this, it can also help in combating biotic and abiotic stresses. pomato is a chimeric plant and the result of hetero­grafting, in which a tomato scion is grafted onto a potato rootstock from two different species. as a result, a plant combination is developed that may produce potato tubers below ground and thakur et al. ‐ pomato grafts: bridging conventional and modern techniques 233 utilize the balcony and backyard space. as the population grows, the effects of urbanization, industrialization, and industrialization on agricultural lands will exacerbate, making agricultural lands more scarce. so, to fulfill the need for food availability, these kinds of combinations can benefit more and more in the future. in another case study conducted by villyáni et al. (2024) studied the influence of tomato grafted onto potato tubers on skin colour and the metabolome of the produce. the outcome of the study confirmed the significant variation in the studied quality traits, skin colour, and metabolome of the harvested produce. meanwhile, out of the one hundred twelve identified metabolites of the tubers, all three cultivars showed a consistent trend in the amounts of twelve chemicals. each cultivar showed a rise in tuber starch content relative to the self­ grafted control, except ‘white lady’ and ‘hópehely,’ where protein levels were found to be unchanged. the formerly oval tubers become more circular. there was a correlation between the enhanced anthocyanin content of ‘hópehely’ and ‘désirée’ tuber skins and the elevation of stan1 expression, which was caused by the tomato scion. this research shows that tomato scion significantly affects potato tuber quality measures. 2. developmental procedure of pomato plants using the vegetable grafting method grafting tomato scion onto potato rootstock resulted in pomato plants. firstly, for the preparation of scion tomato seeds were grown in protrays filled with a commercial mixture under protected cultivation. potato tubers were cut into two pieces and stored for at least 7 days at 18°c with 80% rh before developing scion seedlings. grafting was done when the potato plant reached a graftable size of 15­ 20 cm (thakur et al., 2022). for successful graft union establishment, the polyhouse should be kept at the optimal temperature and humidity. graft ing vegetables is a cost­effective method for developing viable tomato grafts. for a strong union between the scion and rootstocks, there is a need for skilled workers and optimum climate conditions (rana et al., 2024). the detailed procedure for graft development is shown in figures 1 and 2. detailed instructions for the development of grafts were provided by kumar et al. (2015), negi et al. (2016), arefin et al. (2019), islam et al. (2019) and kumar et al. (2021). pomato tomato fruits on stems on the same plant (albacete et al., 2015). because crossbreeding cannot be used to develop pomato grafts, grafting is the only viable and possible method (arefin et al., 2019). pomato plant yields, tomato fruits per plant about 2.72 kg, while potato tuber yield per plant was 211 g, with the number of fruits and tubers obtained from a double harvest plant estimated to be 35 fruits and 4 to 5 tubers (islam et al., 2019). based on the dry matter and mineral distribution process of tomato and potato plant products, nitrogen and phosphorus are distributed along with, but to a lesser extent than, dry matter. meanwhile, potassium distribution was found to be more liberal than dry matter, whereas magnesium and calcium showed a different pattern than n, p, k, and dry matter content (bünemann and grassia, 1973). the combination of the tomato hybrid sweet million f1 and red potato variety memphis had a significantly lower response for the degree of fruit binding than the control in a behavioral study of the tomato plant (giosanu et al., 2020). conferring to the report of negi et al. (2016), the combination of gs­600 grafted on kufri himalini using the cleft grafting technique resulted in the highest survival rate and grafting success rate. the production of tomato fruits and potato tubers from the pomato plant can also be affected by the scion age; the scion was 25 days old (arefin et al., 2019). when compared to individual tomato and potato crop returns of 1:1.93 and 1:0.26, respectively, the pomato plant ensures higher returns of about 1:2.12 (negi et al., 2017). because tomato is a double­ harvest crop, the demand for fertilizer can also double. there is no standard fertilizer application recommendation for the tomato plant. to know the fertilizer’s application doses, experimentation was conducted on the tomato plant by kumar et al. (2021). they suggested using the necessary amount of fertilizer as well as fertigation twice a week to meet the grafted plant’s nutrient demand. the recommended dose was 75% rdf with fertigation (19:19:19) 4.56 g m2 and 100% rdf with fertigation (19:19:19) 4.56 g/m2 (19:19:19); 6.84 g/m2 (kumar et al., 2021; thakur et al., 2024). furthermore, the plastids of tomato and potato belong to the same family and contain a single species of dna, the size and density of which (156 kbp and 1.697 g/cm3) are comparable to those of higher plants’ mtdna. so, from the standpoint of utilizing vertical space, the primary need for developing this type of dual­crop potato plant is one of the most feasible ways to adv. hort. sci., 2025 39(3): 231­244 234 grafting, which unites a tomato plant (aerial portion) with a potato plant (subterranean portion), often employs a grafting clip or silicone tube to tightly bind the grafted stems throughout the healing phase (thakur et al., 2024). an acute grafting knife is important for executing precise, congruent incisions on both the scion (tomato) and rootstock (potato) stems, often using a “splice” or “cleft” grafting method. complementary equipment, such as grafting tape or a film, could be employed to secure and safeguard the graft union, hence reducing transpiration and infection. a humidity structure or grafting chamber maintains optimal humidity and temperature, facilitating graft union development. these instruments facilitate alignment, sterility, and healing, which are essential for the effective cultivation of pomato plants (thakur et al., 2024). farooque et al. (2024) successfully grafted two different varieties of tomato, red cherry tomato and bari tomato­15, onto potato rootstock, namely diment. the results confirmed that the success rate of grafted plants was 94 percent, and also improved fig. 2 ­ pomato development a) selection of scion; b) scion preparation; c: grafting; d,e,f) clipping; g) sucession of graft; h) yielding (source: personal communication). fig. 1 ­ sequential procedure for the successful pomato graft development. thakur et al. ‐ pomato grafts: bridging conventional and modern techniques 235 the growth and yield attributes of harvested produce, i.e., tomato and potato. meanwhile, another researcher had confirmed that the grafting success rate among the tomato variety “mersa” grafted onto the potato rootstock variety “belete” was 64 percent (wudu and kassaw, 2022). 3. differential responses of tomato/potato grafting on fruiting, quality, and abiotic stress yield improvement the pomato plant’s proliferation and production characteristics were significantly improved. (singh et al., 2020). the application of gs­600 to kufri himalini improved tuber yield (rootstock) and fruit yield (scion) per grafted plant (negi et al., 2016). there is a significant improvement in growth and yield attributes in grafted tomato plants between bari tomato­11 (scion) and two potato rootstocks, namely asterix and cardinal (arefin et al., 2019) (table 1). moreover, among grafting techniques in pomato plants, cleft and tongue grating showed better results in terms of yield­related aspects such as tuber weight, tubers per plant, tuber yield/plant and meter square, and in tomato for least days to first flowering and harvest, longest harvest duration, fruit length, width, and plant height (kumar et al., 2016). in some graftings, due to excessive uptake of nitrogen in grafted plants, there was accelerated vegetative growth, resulting in delayed maturity, tuberization, and a reduction in potato tuber quality (zebarth and rosen, 2007). also, it is observed that key qualities to growth and yield are affected by fertigation treatments and fertilizer effects. treatments affected the no. of shoots per plant, length of the harvest, the time it took to reach graftable shoots, the time it took for the plant to flower, and yield attributes (kumar et al., 2021). from the above­discussed finding, it has been concluded that the tuber and fruit yield obtained from tomato and potato grafted plants produced a comparable yield as solely grown potato and tomato yield per plant, which could assure a fair means of production and be economically beneficial (table 2). meanwhile, in the year 2024, one of the researchers confirmed that the pomato plant can produce more yield than of individual plant of tomato and potato. apart from this, a single graft union of pomato, from the aerial part of tomato, had the 103.87 g of average fruit weight, and fruit yield per plant, i.e., 4.11 kg (thakur et al., 2022). from the same plant, potato had the average tuber weight of 175.83 g, where the tuber yield per plant was 908.11 g (thakur et al., 2024). takeuchi et al. (2022) made a reverse combination of potato onto tomato to breed potato cultivars through the conventional method. this combination has been made to resolve the flower initiation and seed setting issue in potatoes because the tuber and flowering will occur simultaneously. the outcomes of the results suggested that the fruit formation rate of potatoes was increased by up to 19% in potato/tomato grafts than of non­grafted potato plants with 1.1% success. so, this procedure can ease in development of null sergeant progenies by crossing the mutant lines of potatoes. quality focuses on nutritional content numerous contradictory reports have been published concerning the quality of fruits (rouphael et al., 2010). limited research work has been done on tomato/potato plant quality analysis because the material is scarce for this aspect. a tomato cultivar grafted on four different potato cultivars showed an increase in vitamin c, tss, and soluble sugars in tomato fruits, while in potatoes, only the reduced sugar content had been leveled up (zhang and guo, 2018). among the various grafting techniques, tongue graft ing was found to be significantly beneficial in tomato grafts for aspects of potato starch content, pericarp thickness, tss, and tomato ascorbic acid content. maximum tss was observed in potatoes during cleft grafting (kumar et al., 2016). however, a different grafted­splice method combination of yuvraj scion onto k. jyoti and k. pukhraj had a significant effect on the biochemical compositions of tomato plant products. so, as per the assurance of the results, the pomato plant might be used as an alternative strategy to get the best quality product along with a double harvest (diwan and sharma, 2021). furthermore, the quality aspects related to the fruits and tubers of the pomato plant influenced it in a positive direction (panahandeh et al., 2020). table 2 lists a recent achievement in the quality aspects of the tomato harvest. the quality aspects of pomato are questionable among growers and consumers. as discussed above, quality attributes such as tss, ascorbic acid, total sugars, and soluble sugars of pomato products have been enhanced along with other yield traits. this assures 236 adv. hort. sci., 2025 39(3): 231­244 table 1 ­ recent achievements in terms of yield, quality and tolerance of pomato plant origin of scion and rootstock targeted traits references gs­600 onto kufri himalini tuber yield/plant and marketable fruit yield/plant negi et al., 2016 zy988 onto ls6, qs9, hz88 and dts1 tomato: vitamin c, tss, soluble sugars, fruit number and size; potato: tuber sprouting, reducing sugar zhang and guo, 2018 bari tomato­11 onto asterix and cardinal morphological traits: plant height, branches, leaves, and clusters yielding: fruits per plant, size of tuber, fruit weight, and yield per plant; arefin et al., 2019 avinash­2 onto kufri jyoti fruiting attributes nd tuber yield/plant islam et al., 2019 moneymaker × san marzano (f1) onto agria chlorophyll index, total soluble solids, dry weight, and titratable acidity panahandeh et al., 2020 moneymaker × san marzano (f1) onto agria foliage fresh, fruit weight, and plant height, panahandeh et al., 2020 kufri pukhraj onto pusa ruby and kufri bahar onto cherry tomato number of fruits per plant, average fruit weight, days to harvest (tubers), tuber yield per plant singh et al., 2020 cherry tomato onto potato tomato­fruit yield and potato­tuber yield (0.5­0.8 kg per plant) bahadur et al., 2020 lakshmi, aviral, yuvraj onto kufri jyoti and k. pukhraj ascorbic acid content, acidity %, total sugar% %, reducing sugar %, and non­reducing sugar %. diwan and sharma, 2021 palam tomato hybrid­1 onto kufri himalini morphological traits like height, number of tubers per plant, tuber yield, tuber weight, marketable fruits, and fruit yield per plant, and tomato fruit weight kumar et al., 2021 ikram to charlotte (cv) salinity tolerance parthasarathi et al., 2021 marsa (tomato scion) grafted onto belete (potato rootstock) improved number of fruits per plant (18), average fruit weight (53 g), fruit yield per plant (954 g), number of tubers per plant (4­5), tuber yield per plant (219.27 g) wudu and kassaw, 2022 avtar on kufri pukhraj highest fruit yield per plant thakur et al., 2022 avtar onto kufri jyoti minimum days to first flowering, days to first harvest thakur et al., 2022 avtar (f1 hybrid) onto kufri pukhraj maximum average fruit weight, fruit yield per square meter, and fruit yield quintal per hectare, tuber yield per plant and quintal per hectare, tuber equivalent yield, b: c ratio, thakur et al., 2024 red cherry tomato and bari tomato­15 grafted onto diamant maximum plant growth, tomato and potato yield per plant farooque et al., 2024 potato scion qingshu no. 9 grafted onto tomato rootstock zhongyan 988 improved pollen viability up to 15­20 percent, identified 13 degs (differentially expressed genes associated with gametophyte, pollen development, protein processing, and carbohydrate metabolism. zhang et al. 2024 thakur et al. ‐ pomato grafts: bridging conventional and modern techniques 237 table 2 ­ germination performance parameters of shallot seeds under different boron concentrations s. no. aim methodology characterization of materials results obtained references 1 rejuvenation and genomic count determination in potato and tomato somatic hybrids by iso­electric focusing of rubpcase subunits protoplast fusion incubation of protoplasts in the existence of polyethylene glycol containing a high concentration of ca2+ ions rubpcase can be utilized to show that the plants are hybrids due to the change in chromosomal number from 48 to 50 in three hybrids and near to 70 in the fourth hybrid. melchers and sacristan, 1978 2 chilling resistance protoplast fusion an aminco dw­2a dual­wavelength spectrophotometer monitored callus transfer and cytochrome reduction. all four tomato­potato hybrids had intermediate chilling resistance between tomato and potato. smillie et al., 1979 3 ribulose bisphosphate carboxylase small subunit somatic hybrid peptide mapping protoplast fusion ribulose bisphosphate carboxylase isoelectric focusing pattern as a nuclear and protoplast genome phenotypic marker. four somatic hybrids had ribulose bisphosphate carboxylase oligomers with tomato and potato genomes. potato­tomato somatic hybrids have functioning tomato dna and tyrosine­containing poulsen et al., 1980 4 restriction endonuclease analysis of fused hybrid plastids dna, potato, and tomato callus fusion 12 somatic inter­generic hybrid progenies of dihaploid potato and tomato each species carries ptdna of tomato and potato at a 0.1 to 3 % level of detection schiller et al., 1982 5 steroidal glyco­alkaloids analysis in tomato and potato somatic hybrids protoplast fusion (pomato with the plastid of potato and topato with the plastid of tomato) protoplasts from dihaploid potato liquid cultures and tomato mesophyll fusion somatic hybrids had 98 percent potato alkaloid, while tubers had 60–70% tomatine. roddick and melchers, 1985 6 somatic hybrid identification by pollen, anther protein of somatic hybrids of potato and tomato plants protoplast fusion extraction of proteins from pollen and anthers of somatic hybrids. pollen viability test by fluoro­chromatic reaction. protein estimation by the lowry method protein extraction by isoelectric focusing in polyacrylamide gel for hybrid identification by pollen and anther proteins chen and ninnemann, 1990 7 chloroplast and mitochondrial dna triploid and tetraploid tomato­potato somatic hybrids protoplast fusion southern blotting technique with four mtdna­ specific probes dna reduction. the 18s + 5s rrna genes in tomato and potato mt dnas may be connected to coxii genes. wolters et al., 1991 9 gus activity, total genomic dna content, and chloroplast type, shoot regeneration potential, expression of potato iso­enzymes, and relative genomic composition asymmetric somatic hybridization protoplast fusion flurometric β­glucuronidase assay, flow cytometric analysis, dna isolation, dna probes, southern blot, and dot blot analysis no viable plants were obtained; calli were highly polyploid, and hybrids expressed gus activity schoenmakers et al., 1994 10 mitotic and meiotic irregularities in somatic hybrids protoplast fusion, root tip meristems culture genomic in situ hybridization, cytological analyses for chromosome counts, and karyotype analysis exclusively sterile pollens wolters et al., 1994 11 regeneration, alien chromosome identification through rflp, and gish protoplast fusion cytological technique for meiosis study, fertility, crossability, and embryo rescue technique, starch composition determination all fusion hybrids were sterile, the hexaploids produced stainable pollen and berries with badly developed seeds jacobsen et al., 1992 that the quality of pomato products can be improved through graft ing; satisfactory results were documented, which match the needs of growers and consumers. salinity tolerance according to various case studies, the potato genome is vital for improving the yield and quality of tubers by the tomato/potato graft (sue et al., 2010; arefin et al., 2019). however, the graft of transmissible ribonucleic acid from the inverse combination of potato onto tomato articulated the phenotype of the scion (kudo and harada, 2007). salinity tolerance was tested in the tomato scion cv. ikram on the potato cv. charlotte plant. the results showed that using potato rootstock as a substitute approach to assure irrigation water salinity tolerance and boost the quality and quantity of fruits and tubers harvested from the grafted plant may be useful. the improved characteristics include higher total dry mass, varied root characteristics, equal mineral distribution across the entire plant, and higher water productivity than non­grafted plants (parthasarathi et al., 2021). table 2 gives an illustration of salinity tolerance. only a few attempts have been made recently by researchers to manage abiotic stress, namely using a graft combination of tomato and potato plants. with the studies mentioned above, it is evident that abiotic stresses like salinity, temperature, and mineral stresses can be managed by utilizing tolerant and resistant rootstocks. 4. developmental procedure of hybrid fusion through somatic hybridization for the isolation of plant protoplasts, 50 ml of a three­ to four­day­old suspension culture was centrifuged at 600 rpm for five minutes. the pallets were then resuspended in 30 ml of am media. after centrifuging the filtrate suspension, the protoplast pellets were re­suspended in bm media and centrifuged at 600 rpm for five minutes. the centrifuged protoplast pellets were resuspended again in bm media. using a nylon fi lter and a centrifuge set at 1000 rpm for 10 minutes, the protoplasts were separated from cell debris. the protoplast was rinsed in as media (1 fold), excluding enzymes, and in bs media (2 folds). before electro­ fusion, a protoplast mixture (1:1) was made, and 0.4 adv. hort. sci., 2025 39(3): 231­244 238 millilitre of the combination was added to a fusion chamber before electrodes were affixed to a glass petri dish. an alternating current field was useful to the allied protoplasts, followed by a reduction in the ac field to zero. the protoplasts were cultured in tm2g media at a density of 2.5 x 105 protoplasts per ml. the details of the procedure were explained by roddick and melchers (1985), schoenmakers et al. (1994), and wolters et al. (1995). effect of somatic hybridization on various traits of pomato fusion hybrid a few scientists initiated the research work for developing the tomato plant via protoplast fusion. in 1955, a potato + tomato fusion hybrid was developed by researchers through protoplast fusion (jacobsen et al., 1992). a first attempt had been made to develop a fusion hybrid plant by melchers and sacristan in 1978. roddick and melchers (1985) carried out the research on the creation of somatic fusion hybrids of tomato and potato, with topato having tomato plastids and pomato comprising potato plastids. smillie et al. (1979) investigated chilling resistance in various somatic fusions of tomato and potato and discovered that the chilling resilience of tomato­potato somatic hybrids was intermediate between that of tomato and potato. these somatic fusions may be beneficial for relocating genes for chilling resistance to cultivated tomatoes, to know the potential and confines of asymmetric somatic hybridization among tomato and potato plants. the two mutation products of the nitrate reductase­deficient mutant of tomato are isolated and assessed, and they might be used as selectable markers in potato somatic hybrid fusion assessment. the outcome showed that several hybrid fusion plants were developed, but plant regeneration did not take place, as mentioned by schoenmakers (1993). wolters et al. (1995) demonstrated that mtdna segregation occurs independently in somatic fusions from chloroplast dna (cpdna). it is concluded that the mtdna of both tomato and potato may contain the coxii gene, which is closely associated with the 18s and 5s rrna genes. a protein of pollen and other sources is used for inter­specific assessment, while chen and ninnemann (1990) confirmed the presence of an intermediate protein band pattern in somatic hybrids of tomato and potato. besides this, a report by schoenmakers et al. (1994) mentioned that an excessive variation had been seen among all the thakur et al. ‐ pomato grafts: bridging conventional and modern techniques 239 fusion hybrids for all studied traits, while the expression of gus activity was also reported in somatic fusion. in another case study, the chromosome count had been analyzed at the mitotic and meiotic stages of division in 107 somatic hybrids of tomato + potato. although about 79% of fusion combinations are found to be aneuploid with the absence of one or two chromosomes, among the five studied hybrids, 46 chromosomes were found in hybrid k2h2­ic, with the highest percentage. even though all the microspores degenerated with immediate effect after the tetrad stage, this resulted in pollen sterility (poulsen et al., 1980; wolters et al., 1991; wolters et al., 1994). table 1 summarises attempts made in the early 1900s to develop fusion hybrid lines through somatic hybridization. to reduce labor and maintenance costs, there is an urgent need to focus on developing the tomato onto potato plants via somatic hybridization. tissue culture is the only method through which these grafts can be developed. because seeds can’t be obtained from a pomato plant due to a lack of skilled labor and knowledge about the procedure of grafting, growers cannot perform this at the field level. somatic hybrids can resolve this issue of plant material availability at the market level. 5. signaling mechanism involved in pomato potato tuberization can be affected by photomorphogenesis and hormones, while the tomato scion does not succeed in producing these substances, which leads to converting stolen into tubers in an established strong source­sink relationship. the source­sink relationship is important in potato tuberization because cytokinin affects tuberization and is thought to be a major contributor (roitsch and ehneb, 2000). thus, the involvement of genes can also contribute to altering the tuberization success, as the over­expression of the “knox” gene can diminish the level of gibberellic acid and level up the cytokinin production that ultimately leads to augmented tuberization (sakamoto et al., 2001; frugis et al., 1999; rosin et al., 2003). in recent case studies, results revealed that the “ipt gene” exhibits a better ability for potato tuber formation (galis et al., 1995). as a result, the diverse range of homoerotic tomato mutants can be used as an effective means of targeting the substances involved in the conversion of stolon into tuber formation, which forms a strong sink. however, the elimination of phyb through a gene, i.e., anti­ sense phyb, resulted in tuber formation in short and long­day conditions, while the over­expression of this gene improved the inhibition effect of long days on tuber formation (aksenova et al., 2002). kudo and harda (2007) carried out experiments on hetero­ grafting using potato and tomato as scion and rootstock, respectively. the combination had been tested to ensure that the rna molecule responsible for altering the leaf shape could function transversely in grafted seedlings. the study found no significant changes in the potato scion as a result of rna molecule transmission in the grafted plant. but this can be utilized in further cultivar development programmes in vegetable crops. however, in another case study, nielsen and stitt (2001) confirmed that a sufficient amount of chimeric transcript can be supplied from the rootstock without leaves to modify the leaves of the scion. even though the pfp enzyme works well in young leaves, it is distributed evenly throughout the plant. meanwhile, this pfp transcript can also actively participate in the root system. so, this methodology has now been used in vegetable production to combat the various biotic and abiotic stresses. several reports confirm the utility of pfp transcripts in the grafted plant by altering the characters of the scion (ohata, 1991; taller et al., 1999) and may lead to the transportation of gene transcripts as per the report of liu (2006). a recent investigation initiated on the tomato plant revealed that potato rootstock expresses merely a negligible phenotypic alteration in tomato scion, but has shown a minute impact on differential expression genes. on the other hand, in the meantime, tomato scion has shown a strong impact on the rootstock of potatoes, leading to the expression of thousands of differential genes, some of which are concerned with hormone pathways and their signaling (zhang et al., 2019). due to the presence of two dissimilar hormone­regulated signaling systems, the yield aspect of the pomato plant would be challenged in numerous tomato­ potato combinations (peres et al., 2005). in the meantime, changes in hormone signaling, either for fruit setting or tuberization, may occur in pomato plants, leading to a downplaying of the antagonistic interactions of hormones such as gibberellic acid and cytokinins (yasinok et al., 2009). approximately 209 genes related to the synthesis of starch and sucrose were identified as being upregulated in the tomato scion. the tomato scion had less of an effect on the adv. hort. sci., 2025 39(3): 231­244 240 development of the signaling substance involved in tuberization, but it did initiate the development of stolons and an aerial stem portion during germination. based on rna­sequencing, upregulated and downregulated genes were identified based on a count of 1529 and 1329, respectively, amongst st‐sw and st‐r. a few of them took part in hormone signaling transduction via st della (receptor) and stgid 1 (protein) (zhang et al., 2019). the graft union establishment is affected by the signaling mechanisms of the scion and rootstock. further research is required to improve plant survival rates and to understand the actual mechanism of the available hormonal pathway between the scion and rootstock combination. this type of investigation has the potential to broaden the research area in grafted vegetable plants. this can also ensure the quality aspects that are in dispute. 6. movement of small rna molecules in grafted plants the transfer of genetic information in the form of small rnas is a critical problem that has received a lot of attention in the literature over the last decade. so, to accomplish this, grafting has been widely utilised in plant physiology and biology to discover mobile molecules that include small rna, mrna, and proteins that influence significant facets of plant growth and development. in higher plants, the phloem transports amino acids, carbohydrates, proteins, vital nutrients, and certain rna molecules (wu et al., 2006). grafting has been employed in several experiments to demonstrate that messenger rna molecules move throughout plants via the phloem (turnbull and lopez­cobollo, 2013). it has been suggested that phloem sap includes real, tiny regulatory rnas because small rnas match multiple potential target genes (yoo et al., 2004). yoo et al. (2004) also found the same in the phloem sap of cucumber, pumpkin, and castor beans, where they found the existence of an endogenous population of small rna species with 18 to 25 nucleotides. ruiz­ medrano et al. (1999) confirmed the presence of several mrnas in the cdna clones obtained from the phloem sap of pumpkin. recently, it was proposed that ft (flowering locus t) mrnas, which are synthesised in the leaves and transported by the phloem to the shoot apex, interact with another transcription factor to form the flowering locus t protein (biazquez, 2005). as a result, in reaction to particular environmental factors, the long­distance rna translocation mechanism seems to influence the development of the entire plant. when me­like potato leaves appeared to have been induced by the transcript transported through the graft junction from the me tomato rootstock, kudo and harda (2007) demonstrated the presence of chimeric transcripts. pfp promoter activity was seen in the lower sections of the plant, notably in the root system, in addition to the identification of pfp transcripts in the sap from the cut surface. genetic material has recently been reported to be horizontally transmitted between the two grafted partners, either as dna or plastids, by stagemann and bock (2009). later in 2012, stagemann and his coworkers verified that n. benthamiana had successfully received the chloroplast dna of n. tabacum (a chloroplast transgenic strain) through a grafting junction. small rnas, such as phosphorus deficiency­induced mirnas, have been demonstrated to transfer from shoot to root systems through the use of micrografting studies (pan et al., 2008). according to bhogale et al. (2014), additional mirnas like mir156, mir172, and mir395 may transfer from the scion to the rootstock. since it is thought that srnas move through the phloem and plasmodesmata, it has been demonstrated that srna mobility within grafted plants is more effective when srnas are produced in the scion and move towards the rootstock rather than vice versa (melnyk et al., 2011). tomato graft unions were developed using non­transgenic scions and transgenic rootstocks (silenced fatty acid desaturase gene). the silencing of the fatty acid desaturase gene (lefad7) and the presence of sirna in grafted scions suggest that both were transferred to the scion via genetically engineered rootstock. it is conceivable that sirnas generated from the rootstock might similarly go in that direction among these compounds and highlight the considerable phenotypic alterations seen in the scion. in addition, it has been demonstrated that the tomato scion receives viral resistance from the rootstock. the findings of an experiment conducted by spano et al. (2015) demonstrated that when resistant tomato rootstock varieties with higher rna interference were grafted onto resistant tomato scion varieties that accumulate less viral rna, the grafted plants displayed the expression of crucial rnai mechanism genes. the roots of resistant grafted plants showed upregulation of genes such as thakur et al. ‐ pomato grafts: bridging conventional and modern techniques 241 agronaute (ago) and rdr. it has also been proven that self­grafted plants showed stronger rnai silencing, and even grafting itself can provoke the activation of resistance mechanisms. 7. future prospects vegetable grafting provides a new perspective to broaden the research area, increase yield, and improve quality attributes associated with vegetable crops. this can be used as a backup method to ensure food availability. because variety development takes eight to ten years, grafting can compensate for production losses and aid in the resistance to biotic and abiotic stresses during this time. however, the basic requirement for grafting is the development of resistant or tolerant rootstock material on the ground level. this is a farmer­friendly technique because it is environmentally friendly. the pomato, also known as the “horticultural wonder plant,” is the best example of vegetable grafting. two products can be obtained from a single plant, namely tomato fruit and potato tubers. more research on pomato plants is required because only a few reports have been documented to date. the quality aspects of this plant product are always in doubt, which could be a major hurdle that must be overcome so that farmers can also grow these profitable plants with high­quality produce. aside from the manual graft ing of tomato plants, researchers should concentrate on using biotechnological techniques to create tomato/potato fusion hybrids, such as somatic hybridization. so, because the major downside of developing a pomato plant is that it cannot be grown from seeds, the only way to develop these grafts is through vegetable graft ing. to modernize the development process and ensure the availability of grafts on the market, these grafts must be developed using the tissue culture technique. 8. conclusions the primary goal of developing pomato plants is to ensure maximum space utilization and combat environmental challenges. a few inconsistent findings on the fixable effects of graft union on horticultural and quality aspects have also been reported. pomato plants can be grown in two ways: through grafting or somatic hybridization. several attempts were made in the 1900s to create hybrid mutants through somatic hybridization. later, manual grafting was used to develop tomatoes onto potato grafts. now that only grafting is used on commercial­level tomato plants, there is an urgent need to focus on developing a hybrid tomato/potato graft. to be aware of the utility and benefits of this dual­harvest plant, researchers must focus on the start of additional research work. many aspects of its biochemical and physiological properties have yet to be studied, particularly to make this grafted plant commercially available to growers. this grafted plant may also be considered an alternative way to produce clean potato seed tubers, especially in hydroponics and aeroponics systems, and in the future, it might be proven to be one of the approaches worth considering. so, there is a need to work on this aspect to get more benefits in terms of quality and quantity. however, several successful reports regarding the tomato/potato plant were documented, so the possible information has been reviewed to learn more about the successful experimentation. acknowledgements we heartily thank cskhpkv palampur and lpu phagwara for assisting in compiling the subjective material for writing this manuscript. references abdelmageed a.h.a., gruda n., 2009 ­ influence of graft ing on growth, development and some physiological parameters of tomatoes under controlled 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responsible for its characteristic aroma (fischer et al., 2011) and flavor as a condiment, which along with color and freshness determine its commercial value. essential oil aromatic compounds and productive behavior are affected by the environment, genotype and agronomic techniques. some chemotypes from different geographic origins have been classified based on the aroma profiles of essential oil (suppakul et al., 2003). another classification of o. basilicum cultivars has been made according to morphology, height, leaf color, dimension and flower color (darrah, 1980). yield and essential oil composition are remarkably variable between purple and green genotypes (marotti et al., 1996; sajjadi, 2006) which also differ in biomass yield (hochmuth and leon, 1999). basil is cultivated under a range of conditions but temperate climates are the most suitable for the crop. chang et al. (2005) stated that the maximum dry matter content was obtained with temperatures of 30°c. putievsky (1983) reported that increasing daytime temperatures between 21°c and 30°c enhanced plant height. light influences essential oil composition and productive behavior. when the irradiance level decreases the methyl-eugenol content increases, plants are smaller, have thinner leaves, less dry and fresh weight, sprouts and foliar area, whereas with high irradiance levels, linalool, eugenol and the total content of essential oil rise and photosynthesis and growth rate increase. under high irradiance conditions, more photosynthates are biosynthesized and a greater amount of secondary metabolites accumulate (chang et al., 2008). although this aromatic crop is grown in open field and greenhouse conditions, hydroponic basil cultivation in a protected environment is an efficient commercial alternative, most importantly for those areas with limited agricultural soils and dependant on irrigation (hassanpouraghdam et al., 2010). the benefits of this system include high quality plants, rapid growth, off-season and agronomic performance and essential oil composition of ocimum basilicum l.: effect of genotype and date of harvest a. vazquez(1), e. sanchez*, c. van baren**, d. frezza* * cátedra de horticultura, facultad de agronomía de la universidad de buenos aires, av. san martin 4453 c1417dse, buenos aires, argentina. ** cátedra de farmacognosia, facultad de farmacia y bioquímica de la universidad de buenos aires, junin 954, 2° piso, c1113aad, buenos aires, argentina. key words: green basil, purple basil, soilless culture, volatile oils, yield. abstract: an experiment was conducted to assess the agronomic performance and essential oil composition of ocimum basilicum l. (basil) with two genotypes during autumn-winter cycle, in a hydroponic system in greenhouse. genotypes (i.e. green and purple) were provided to study productive parameters. two harvest dates and both genotypes formed treatments to investigate oil composition and its stability. one pruning was made before the last harvests. results of fresh and dry weight (g. plant-1), absolute growth rate (g. day-1) and relative growth (g. g-1.day-1) and yield (g. plant-1) showed great differences in comparison with the optimal growing season values. although pruning encourages new growth, it was strongly reduced in purple basil. essential oil composition varied for both genotypes and between harvest dates. linalool prevailed at the first harvest date whereas methyl-eugenol increased towards the second harvest date, and significantly in purple basil. radiation and temperature data showed a downward trend during the cycle which influenced biomass production and essential oil composition. green basil had better productive behavior than the purple variety. essential oil stability between harvest dates varied for both genotypes. pruning strongly affected purple basil growth which altered essential oil composition. the findings presented in this study confirm that it is possible to grow basil in autumn-winter season in greenhouse. although yield slightly decreases in comparison with optimal growing season, high quality aromatic plants can be obtained. adv. hort. sci., 2013 27(4): 166-172 (1) corresponding author: avazquez@agro.uba.ar received for publication 13 september 2013 accepted for publication 21 january 2014 167 all-year-round production, maximizing the benefits for producers. on the other hand, in a pure hydroponic system the nutritive solution is recycled, reducing the environmental impact and with minimal groundwater contamination (resh, 2001). nutritive solution management is important to obtain plants with high yield and good quality. studies regarding electrical conductivity demonstrated that the highest fresh weight (g.plant-1) was obtained with 1.5 ds.m-1 and it did not affect essential oil concentration (carrasco and izquierdo, 1996), while values above 3 ds.m1 affected plant growth. considering environmental conditions, the production system and the different varieties of ocimum basilicum l., the aim of this work was to evaluate the agronomic performance and essential oil composition of green and purple genotypes during the autumn-winter cycle. 2. materials and methods the trials were carried out in the experimental fields of the horticultural department of agriculture college of the university of buenos aires, in a polyethylene-metallic greenhouse. seeds of two varieties of basil, purple and green (ocimum basilicum var. violeto and ocimum basilicum var. genovese) were obtained from zorzi, di hortus sementi srl. seeds were sown, at the beginning of autumn to finish the crop cycle in winter, in expanded polystyrene growing trays on a soilless media mix (vermiculite, peat moss, perlite and fertilizer npk with micro elements 1.3 g l-1,, ph 5.5-6.5, with fine structure).the trays were located in a hydroponic floating system until the seedlings had two to three pairs of unfolded leaves. plants were transplanted into a closed hydroponic nft (nutrient film technique) system. a low polyamide tunnel was built to avoid frost damage and it was used from late afternoon to early morning each day. the nutrient solution was composed of ammonium nitrate 5.625 g, potassium nitrate 75 g, calcium nitrate 93.75 g, mono potassium phosphate 28.13 g, magnesium sulphate 33.75 g and micro elements 18.75 c.c. crop density was 25 pl. m-2. electrical conductivity and ph of the nutrient solution were measured three times a week. environmental temperature (°c), radiation (w.m-2), relative humidity and nutrient solution temperature (°c) were measured using a data logger (hobo). thermal time was calculated using base temperature for basil (t base =10.9°c), a. plant growth fresh and dry, aerial and root plant weight (g.plant-1), number of leaves, root density (g.cm-3), plant height (cm), absolute growth rate (agr g.d-1), relative growth rate (rgr g.g-1.d-1) and yield (g. m-2) were measured throughout the cycle. b. identification and quantification of volatile oils oil extraction. essential oil analysis was carried out with leaves harvested on two harvest dates with an interval of 37 days between them for both genotypes. basil samples were collected during a period of 92 days in the autumn-winter season and two harvests were made with a pruning between them: sample 1 (5 days after transplant), sample 2 (13 days after transplant), sample 3 (21 days after transplant), sample 4 (29 days after transplant), sample 5 (first harvest and 50 days after transplant), pruning, sample 6 (second harvest and 92 days after transplant). fresh leaf material (250 g per sample) was subjected to a 2-h water distillation using a clevenger type apparatus where material and distilled water where located. a refrigerant attached to the distillation balloon allowed accumulation and separation of the essential oil from the condensed mixture. the oils obtained were dried over anhydrous sodium sulfate. identification of volatile oils. the essential oils were analyzed by cg-fid-ms, with perkin elmer gc equipment model clarus 500. chromatograph operating conditions with cg-fid-ms were: helium as a carrier gas at a constant flow rate of 1.87 ml/min, and an auto sampler connected to an injector split (split rate: 1:100) in turn connected to a flux divisor of two fused silica capillary column (polar and no polar). the temperature parameters were t. initial: 90°c; ramp (3°c /min); t. final: 225°c (15 min); t. injector: 255°c; t. detector: 275°c; final run time 70 min; mass range scanned 40-400 m/z. the injected samples consisted of 0.2 μl in a dilution of 10% ethanol. the components of the essential oil were identified by comparing their retention times obtained from the two columns of different polarity with those of authentic samples and/or data in the literature, and comparison with the mass spectra in the database of the pharmacognosy department of the university of buenos aires and other commercial sources. the relative percentage amounts of the volatile oil constituents were evaluated from total peak area (tic). statistical analysis. the experiment was conducted in a complete randomized block design repeated over time, with three replications. the treatments for growth stage were green genotype and purple genotype. the treatments for essential oil analysis were according to harvest date (first and second) and genotype: green genotype, 92 days after transplant (dat); purple genotype, 92 dat; green genotype, 50 dat; purple genotype, 50 dat. data was analyzed by anova and means were compared by tukey test at the 0.05 probability level. 3. results and discussion plant growth fresh and dry aerial weight. the aerial fresh weight was significantly different between genotypes (p<0.0001) and harvest dates (p<0.0001). on the third sample date, the differences between genotypes began to be greater. on the fifth sample date the difference for green genotype climbed up to a 47%, a result that coincided with other authors who obtained 91% more biomass for green genotypes than purple (neikin and schuch, 2010). the data from the present study contrast with a crop 168 grown in optimum season, as reported by some authors for green basil with values between 64.44 and 110.33 g.plant-1 (benito and chiesa, 2000; carrasco et al., 2007) and fresh weights of 57.84 g.plant-1 and 41.50 g.plant-1 for purple basil (krizaj, 2010). differences in aerial dry weight between genotypes (p<0.0001) and date of harvest (p<0.0001) were significant. aerial dry weight followed the same trend as aerial fresh weight (fig. 1). the values recorded for 50 dat (table 1) were lower than those registered in the same productive system in optimum season, with 10% fewer days of cycle; green and purple genotype with 6.72 g.plant-1 and 4.26 g. plant-1 (krizaj, 2010) respectively. an increase of 92% and 184% compared to values reported in this experiment. fresh and dry root weight. there were statistical differences in fresh and dry root weight (table 1) between genotypes (p fresh = 0.0007 and p dry = 0.0024) and date of harvest (p fresh <0.0001 and p dry <0.0001). aerial and root dry matter aerial dry matter differed significantly between genotypes (p=0.0339) and sample dates (p=0. 0169). the difference increased greatly for green basil at the third sample date. at the first harvest (50 dat), dry matter percentage for purple basil was 19% lower than the green basil value (table 2). the data obtained was similar to other authors’ results, in protected environments and soilless systems, for green (11.67%) and purple basil (9.85%) (cenóz and burgos, 2010). these authors also concluded that dry matter (%) was effectively reduced in protected environment systems compared with other systems (cenóz and burgos, 2010). however, other experiments did not show significant differences in dry matter between genotypes in optimum season for nft system (krizaj, 2010). the present study was carried out in autumn-winter and both genotypes performed differently which would suggest a noticeably genotypic effect when the season is not optimal. root dry weight was not significantly different between dates of harvest and genotypes. root density, plant height, leaf number and leaf apparition rate root density was significantly different between genotypes (p<0.0001) and sample dates (p<0.0001). root density was markedly higher in purple basil at all sample dates except sample date 2, however root weight (dry and fresh) were not higher due to the high content of water and the lower percentage of dry matter. significant differences were detected for plant height values between sample dates (p<0.0001) and genotypes (p<0.0115). from the third sample date, plant height was markedly higher for green basil until the end of the study. in optimal season studies (benito and chiesa, 2000) with similar growing cycle duration, taller plants were obtained with 133% more height in 56 days (79.8 cm). leaf number also showed significant differences between sample dates (p<0.0001), however this difference was not significant between genotypes. in a shorter cycle (10% fewer days) in optimum season, 124% more leaves were obtained (120 leaves.plant-1)(krizaj, 2010); 208% more leaves.plant-1 in nft in greenhouse (carrasco et al., 2007). parameter values are presented in table 3. leaf apparition rate 50 dat was 1.074 leaves.day-1 for green and 21% lower for purple basil (0.84 leaves.day-1). absolute and relative growth rates green basil maintained a higher absolute growth rate (agr) over almost all the cycle period, and reached the maximum (1.86 g.day-1) on the fourth sample date (29 dat) as did purple basil (0.74 g.day-1). on the contrary, the highest relative growth rate (rgr) for both genotypes was reached with the first sample date (0.2 g.g-1.d-1). both genotypes presented a downward trend over the study petable 1 growth parameters for both genotypes 50 days after transplant. average values and standard error growth parameters (g.plant-1) genotype green purple aerial fresh weight 31.14±0.23 16.5±2.18 root fresh weight 18.13±0.23 6.5±1.80 aerial dry weight 3.5±0.01 1.5±0.10 root dry weight 0.7±0.02 0.43±0.12 table 2 aerial and root dry matter values 50 days after transplant for both genotypes. mean values and standard error genotype areal dry matter (%) root dry matter (%) green 11.24±0.1 3.86±0.05 purple 9.26±1.92 7.33±3.68 35 30 35 25 30 35 25 30 35 e i g h t 20 25 30 35 s h w e i g h t a n t 1 ) 15 20 25 30 35 a l f r e s h w e i g h t ( g . p l a n t 1 ) green15 20 25 30 35 e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) green purple 10 15 20 25 30 35 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) green purple 5 10 15 20 25 30 35 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) green purple 0 5 10 15 20 25 30 35 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 a e r i a l f r e s h w e i g h t ( g . p l a n t 1 ) sample dates green purple fig. 1 fresh aerial weight (g.plant-1) over the whole growth cycle (5th sample date = first harvest; 6th sample date = second harvest) for both genotypes. 169 riod (figs. 2 and 3). results obtained 50 dat were notably low, supporting another author’s findings (krizaj, 2010). as expected, plant response to decreasing radiation and temperature was translated into lower parameter values in comparison with those of an optimal season. in general, until the fourth sample date, temperature and radiation allowed moderate photosynthesis and growth. from the beginning of the study, radiation declined 50% (from 406 w.m-2 to 197 w.m-2). pruning between the first and second harvest stressed plants, however it caused a different effect in purple basil, which had a slower regrowth compared with the green variety. purple genotype showed a lower leaf apparition rate than the green genotype during the cycle. this was added to the adverse environmental conditions after pruning, which led to a slower recovery. average temperature during the study was inferior to optimal for the species and showed a downward trend, furthermore it was outside the range for maximum dry matter accumulation. the data revealed a genotypic effect in growth response in the autumn-winter season that does not occur in an optimal season (krizaj, 2010). the contrast in behavior between genotypes was noted when environmental conditions began to be adverse. relative humidity, nutritive solution temperature, electrical conductivity and nutritive solution ph did not show great variations that could influence growth parameters. although thermal time 50 dat was 50% lower than thermal time achieved in optimal season for the same duration and crop conditions (krizaj, 2010), it was possible to grow basil under protection in the autumn-winter season. biomass yield: descriptive analyses biomass yield for green basil, 50 dat, was 778.5 g.m-2 and 412.5 g.m-2 for purple basil. in contrast, greater values were obtained in an optimal season in open field, with yields between 1000 and 1500 g.m-2 for green genotype (gill and randhawa, 1996), 29% and 92% more respectively than found in the present investigation. also, in greenhouse and nft system and in optimal season, 86% and 151% more biomass yield was obtained for green and purple basil respectively (krizaj, 2010). at the fifth sample date (first harvest) fresh weight yield per square meter was 778.5 g.m-2 for green basil and 47% lower (412.5 g.m-2) for purple basil. at the second harvest date, after pruning, green and purple genotype yielded 630.5 g.m-2 and 227 g.m-2 respectively. volatile oil analysis essential oil composition and genotype effect. gcms analyses identified 32 aromatic compounds in green and 30 aromatic compounds in purple basil. in both cases the identified compounds account for 94% of the total. the composition is expressed relative to 100%, as each peak has an area and the total of areas is 100%. the essential oils from o. basilicum show significant differences between genotypes: linalool (p= 0.0001), eugenol (p=0.0457), methyleugenol (p=0.0001), alpha transbergamotene (p= 0.0251), 1.8 cineol (p= 0.0113) and tau cadinol (p= 0.0253). the other components were not considtable 3 growth parameters at each sample date for both genotypes. mean values and standard error parameter genotype 1 2 3 4 5 root density (g.cm-3) green 3.7±0.6 3.1±1.7 1.1±1 1.1±5 1.2±1.1 purple 4.4±0.0 2.9±1.3 1.2±4.3 1.3±0.01 1.2±1 height (cm) green 10.6±0.4 7.7±1.5 13.7±3.5 25.6±3.8 34.3±1.2 purple 10.1±1.6 9.8±1 15.7±2.5 22.5±0.5 23.3±0.5 leaves number green 4±0.5 11±3.6 25±4.7 44±10.9 54±1.5 purple 5±1.1 10±3.7 21±11.9 43±1.5 42±1.7 20 15 20 10 15 20 t e 5 10 15 20 w t h r a t e d a y 1 ) 5 10 15 20 g r o w t h r a t e f w . d a y 1 ) green 0 5 10 15 20 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) green purple -5 0 5 10 15 20 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) green purple -5 0 5 10 15 20 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) green purple -10 -5 0 5 10 15 20 0 1 2 3 4 5 6 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) green purple -10 -5 0 5 10 15 20 0 1 2 3 4 5 6 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) sample dates green purple -10 -5 0 5 10 15 20 0 1 2 3 4 5 6 a e r i a l g r o w t h r a t e ( g f w . d a y 1 ) sample dates green purple fig. 2 absolute growth rate (agr) for aerial fresh weight (g fw.day-1) for both genotypes during the cycle. values represent the mean. 0 250,25 0,2 0,25 e 0,15 0,2 0,25 h r a t e 1 0 1 0,15 0,2 0,25 r o w t h r a t e . d a y 1 0 05 0,1 0,15 0,2 0,25 i v e g r o w t h r a t e g f w . d a y 1 green 0,05 0,1 0,15 0,2 0,25 l a t i v e g r o w t h r a t e g f w . d a y 1 green purple 0 0,05 0,1 0,15 0,2 0,25 r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple -0,05 0 0,05 0,1 0,15 0,2 0,25 0 1 2 3 4 5 6 sample dates r e l a t i v e g r o w t h r a t e g f w . d a y 1 green purple fig. 3 relative growth rate (rgr) for aerial fresh weight (g fw.g-1. day-1) for both genotypes during the cycle. values represent the mean. 170 ered in the statistical analyses as they were only detected in some samples and with extremely low values. values for the main compounds are shown in figure 4. date of harvest effect the mean percentages of linalool (p=0.0001) and methyl-eugenol present significant differences between dates of harvest (p=0.0019) whereas eugenol, alpha transbergamotene, 1.8 cineol and tau cadinol values did not show significant variation between dates. the mean contents for linalool and methyl eugenol for both genotypes varied as follows: linalool from 41.8% at the first harvest date to 25.7% at the second; methyl-eugenol from 5.78% at the first harvest date to 18.78% at the second. linalool significantly decreased at the second harvest while methyl eugenol increased, however the content was lower than linalool as seen in figures 5 and 6. different radiation led to essential oil variations. it could be that the highest radiation level before the first harvest led to higher rates of linalool. radiation decreased over the period of the experiment: at the second harvest date lower levels of methyleugenol were found. this might be explained by the fact that in this moment radiation was lower. unlike other authors’ findings (chang et al., 2008), in this study no differences in eugenol values between dates of harvest were found. temperature influences aromatic compounds and metabolic activity of plants. the effect of temperature at 25°c, in which the highest contents of linalool, 1.8 cineol and eugenol are obtained, was reported (chang et al., 2005). although is known that geranial pyrophosphate is precursor of both linalool and 1.8 cineol, and the enzymes linalool synthetase and 1.8 cineol synthetase were identified, the environmental effects on the enzymes activity is not clear at the moment. (chang et al., 2005). in this study, linalool percentages in the two successive harvests (36.6% and 22.7%) were higher than values reported by other authors in hydroponic systems (fernandes et al., 2004). higher radiation and moderate temperatures before the first harvest date might explain the higher content of linalool for both genotypes. eugenol is metilated to methyleugenol by the enzyme eugenol-o-methyl-transferase (lewinsohn et al., 2000; 41,17 35 40 45 41,17 26,33 20,85 22,35 20 25 30 35 40 45 n t a g e % 41,17 26,33 20,85 12,23 2 22 22,35 6,48 9,77 4,48 6,64 4,45 2 71 10 15 20 25 30 35 40 45 p e r c e n t a g e % 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 p e r c e n t a g e % 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p p e r c e n t a g e % 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds 41,17 26,33 20,85 12,23 2,22 22,35 6,48 9,77 4,48 6,64 4,45 2,71 0 5 10 15 20 25 30 35 40 45 g p g p g p g p g p g p linalool eugenol m.eug. a.t.berg. 1,8 cineol tau cadinol p e r c e n t a g e % aromatic compounds fig. 4 percentage composition of the main aromatic compounds of basil essential oil for both genotypes (g= green, p= purple). mean value of the two harvest dates (methyl eugenol= m.eug, alpha-trans-bergamotene= a.t.berg.). 47,53 34 8045 50 55 first harvest 47,53 23,17 34,80 18,53 30 35 40 45 50 55 e % first harvest second harvest 47,53 23,17 34,80 18,53 7 27 4 5015 20 25 30 35 40 45 50 55 r c e n t a g e % first harvest second harvest 2 97 47,53 23,17 5,70 4 40 6,00 34,80 18,53 3,77 7,27 4,50 10 15 20 25 30 35 40 45 50 55 p e r c e n t a g e % first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1 8 i l li l l l t b t di l p e r c e n t a g e % first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 2,97 47,53 23,17 0,67 5,70 4,40 6,00 34,80 18,53 3,77 7,27 4,50 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest fig. 5 percentages (relative to total in essential oil= 100%) of the main aromatic compounds of basil essential oil for green genotype at first and second harvest. mean values ± standard error (methyl eugenol= m.eug, alpha-trans-bergamotene= a.t.berg.). 171 robison and barr, 2006). methyleugenol is in the group of the alikobenzenes together with iso-eugenol, eugenol, estragol and safrol, which are considered carcinogenic following the tested effects on rats and mice after intake of high doses. the effect in human beings generates certain concern, however the doses at which humans are exposed through diet (mainly via intake) are very low (robison and barr, 2006). methyleugenol content is related to vegetal tissue age. greater enzyme activity in young leaves was reported (lewinsohn et al., 2000). this supports other authors who state that enzyme activity is significantly greater in young and developmental leaves than in mature leaves, because as the leaf develops it produces glandular trichomes with high levels of this enzyme (gang et al., 2002). in young leaves there are more trichomes per unit area before cell expansion (gang et al., 2001) but when glands reach maturity, enzyme levels decrease. in totally mature plants of o. basilicum var. genovese methyleugenol was not found (marotti et al., 1996). in mature hydroponic plants in greenhouse a percentage similar to this study was found for o. basilicum var. genovese (0, 6% methyl-eugenol in leaves) (hassangpouraghdam et al., 2010). in the present investigation, agronomic techniques (pruning) and environmental conditions may have influenced the content of this compound. pruning after the first harvest decreased sinks, caused regrowth and the development of abundant young tissue, which might have influenced the rise in glandular trichome density, and as a consequence, the higher amount and activity of the eugenol-o-methyl-transferase at the sixth sample date (second harvest), methyl-eugenol content increased in the essential oil of both genotypes. the increase was remarkably higher in purple basil (33.80%). this result could be due to the slower regrowth of this variety, with a lower foliar apparition rate throughout the cycle (at sixth sample date foliar apparition rate was 1.01 leaves.day-1 in purple basil and 1.52 leaves.day-1 in green basil) which led to a greater amount of young and developmental leaves with elevated density of trichomes and eugenol-o-methyl-transferase enzyme activity. 4. conclusions the agronomic performance of green basil was greater throughout the growing cycle. this genotype showed greater fresh and dry weight (aerial and root), dry matter percentage, number of leaves, plant height and total biomass yield than purple basil. the environment could have influenced agronomic performance and volatile oils composition. hence, it can be concluded that variations in aromatic compounds and the productive behavior were affected by temperature and radiation. differences in volatile oils between dates of harvest and genotypes was clear. at the fifth sample date (first harvest) linalool prevailed in green and purple basil, but in green basil it was significantly higher. at the sixth sample date (second harvest) methyleugenol content increased in both genotypes but the increase was markedly higher in purple basil. pruning after the first harvest might have promoted sprouts, but in purple basil it also might have decreased yield because regrowth after the cut is more difficult for this variety. this difficulty was also enhanced by the environmental conditions (sub-optimal radiation and temperatures). it is also concluded that this agronomic technique could have altered the methyleugenol content. references benito a.p., chiesa a., 2000 parámetros fisiológicos y productivos en cultivares de albahaca (ocimum basilicum l.). fave, 14(1): 19-28. carrasco g., izquierdo j., 1996 la empresa hidropónica de mediana escala: la técnica de la solución nutritiva recirculante (“nft”). ed. univ. de talca, talca, chile, pp. 62. carrasco g., ramírez p., vogel h., 2007 efecto de la conductividad eléctrica de la solución nutritiva sobre el 33 8 50 55 first harvest s d h t 36 07 33,8 45 50 55 first harvest second harvest 36,07 33,8 40 45 50 55 % first harvest second harvest 36,07 33,8 35 40 45 50 55 e % first harvest second harvest 36,07 16,9 33,8 25 30 35 40 45 50 55 e n t a g e % first harvest second harvest 36,07 15 87 16,9 33,8 20 25 30 35 40 45 50 55 r c e n t a g e % first harvest second harvest 36,07 15,87 10,90 9 63 16,9 33,8 9 9 1 20 25 30 35 40 45 50 55 p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 6 55 16,9 8,6 33,8 9,9 15 20 25 30 35 40 45 50 55 p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3 17 6,55 16,9 8,6 33,8 9,9 2 25 10 15 20 25 30 35 40 45 50 55 p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1 8 cineol linalool eugenol m eug a t berg tau cadinol p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest 6,73 36,07 15,87 10,90 9,63 3,17 6,55 16,9 8,6 33,8 9,9 2,25 0 5 10 15 20 25 30 35 40 45 50 55 1,8-cineol linalool eugenol m.eug. a.t.berg. tau-cadinol p e r c e n t a g e % aromatic compounds first harvest second harvest fig. 6 percentages (relative to total in essential oil= 100%) of the main aromatic compounds of basil essential oil for purple genotype at first and second harvest. mean values ± standard error (methyl eugenol= m.eug, alpha-trans-bergamotene= a.t.berg.). 172 rendimiento y contenido de aceite esencial en albahaca cultivada en nft. idesia, arica, 25(2): 59-62. cenóz p., burgos a., 2010 influencia de la fertilización nitrogenada en el rendimiento de la albahaca (ocimum basilicum l.). boletín hortícola, fac. cs. agr. y forestales, univ. nac. de la plata, 8(27): 4-7. chang x., alderson p.g., wright c., 2005 effect of temperature integration on the growth and volatile oil content of basil (ocimum basilicum l.). j. hort. sc. bio., 80(5): 593-598. chang x., alderson p.g., wright c., 2008 solar irradiance alters the growth of basil (ocimum basilicum l.) and its content of volatile oils. env. exp. bot., 63(1-3): 216-223. darrah h.h., 1980 the cultivated basils. buckeye printing, independence. fernandes p.c., facanali f., teixeira j.p.f., furlani p.r., marques m.o.m., 2004 cultivo de manjericão em hidroponia e em diferentes substratos sob ambiente protegido. hort. bras., brasília, 22(2): 260-264. fischer r., nitzan n., chaimovitsh d., rubin b., dudai n., 2011 variation in essential oil composition within individual leaves of sweet basil (ocimum basilicum l.) is more affected by leaf position than by leaf age. j. agric. food chem., 59(9): 4913-4922. gang d.r., lavid n., zubieta c., chen f., beuerle t., lewinsohn e., joseph n., pichersky e., 2002 characterization of phenylpropene o-methyltransferases from sweet basil. plant cell, 14(2): 505-519. gang d.r., wang j., dudareva n., nam h.k., simon j.e., lewinsohn e., pichersky e., 2001 an investigation of the storage and biosynthesis of phenylpropenes in sweet basil. plant phys., 125(2): 539-555. gill b., randhawa g., 1996 effect of different transplanting dates and harvesting stages on the quality of french basil oil. j. herbs, spices med. plants, 4(3): 35-42. hassanpouraghdam m.b., gohari r.g., tabatabaei j.s., dadpour r.m., 2010 inflorescence and leaves essential oil composition of hydroponically grown ocimum basilicum l. j. serbian chem. soc., 75(10): 1361-1368. hochmuth r.c., leon l.l., 1999 evaluation of six basil cultivars grown in a vertical hydroponic production system inside a greenhouse. univ. of florida, flo, nfrec-sv research report, 99-06, p. 2. krizaj c., 2010 comportamiento agronómico-productivo de albahaca (ocimum basilicum l.) verde y morada para consumo en fresco. facultad de agronomía universidad de buenos aires, buenos aires, trabajo de intensificación para acceder al título de ingeniero agrónomo, p. 25. lewinsohn e., ziv-raz i., dudai n., tadmor y., lastochkin e., larkov o., 2000 biosynthesis of estragole and methyl-eugenol in sweet basil (ocimum basilicum l.): developmental and chemotypic association of allylphenol omethyltransferase activities. plant science, 160(1): 27-35. marotti m., piccaglia r., giovanelli e., 1996 differences in essential oil composition of basil (ocimum basilicum l.) italian cultivars related to morphological characteristics. j. agric. food chem., 44(12): 3926-3929. neikin j.b., schuch u.k., 2010 retractable roof greenhouse production of basil (ocimum basilicum and lemon grass (cymbopogon citrates) in a semi-arid climate). acta horticulturae, 659: 113-120. putievsky e., 1983 temperature and day length influences on the growth and germination of sweet basil and oregano. j. hort. sc., 58(4): 583-587. resh h.m., 2001 cultivos hidropónicos. nuevas técnicas de producción. mundi-prensa, madrid, españa. robison s.h., barr d., 2006 use of biomonitoring data to evaluate methyl eugenol exposure. env. health persp., 114(11): 1797-1801. sajjadi e.s., 2006 analysis of the essential oils of two cultivated basil (ocimum basilicum l.) from iran. daru, 14(3): 128-130. suppakul p., miltz j., sonneveld k., bigger s.w., 2003 antimicrobial properties of basil and its possible application in food packaging. j. agric. food chem., 51(11): 3197-3207. 147 1. introduction the strawberry (fragaria x annanassa duch.), a member of the rose family, is not really a berry but a false fruit and consists of many tiny individual fruits embedded in a fleshy scarlet receptacle. the brownish or whitish specks, commonly considered seeds, are the true fruits known as achenes. strawberries are an excellent source of vitamin c, a good source of folate and potassium, and are relatively low in calories. strawberry is one of the most widely appreciated fruits and it has attained a premier position in the fresh fruit market and processing industries of the world (sharma and sharma, 2003). initially grown in temperate zones of india, its cultivation has now become possible in the sub-tropical zones as well with the introduction of day neutral cultivars (asrey and singh, 2004). strawberry offers quicker returns on capital outlay than any other fruit crop since under special methods of cultivation a crop can be picked as early as the first summer after planting. the fruit is the first of the home-grown supplies to reach the market. strawberry is one of the most important high value cash crops around the world. in india, it is cultivated commercially in hamachal pradesh, utter pradesh, maharashtra, west bengal, nilgiri hills, delhi, haryana, punjab, rajasthan and jammu and kashmir. its cultivation can be extended successfully to other suitable areas of the world where irrigation and transportation facilities can be assured. strawberry is an attractive, luscious, tasty and nutritious fruit with a distinct and pleasant aroma and flavor. it has a unique place among cultivated berry fruits in world. rich in vitamin c and iron, the principal demand for cultivated strawberry is from the processing and baking industries around the world as suggested by childs (1937) that the strawberry is a delectable fruit that is highly prized by almost everyone. impact of integrated nutrient management on growth, yield and quality of strawberry (fragaria x annanassa duch.) cultivation in india rayees a. wani*(1), sunam sheema**, t.h. malik**, seerat geelani*, sabiya bashir*, n.a. dar*, v. m. prasad*** * department of horticulture, sam higginbottom institute of agriculture technology and science, allahabad, u.p. 211007, india. ** department of agriculture jammu, jammu and kashmir, india. *** department of horticulture shiats allahabad. (1) present address: dry land agriculture research station budgam, skuast-k srinagar, kashmir india. key words: inorganic fertilizers, integrated nutrient management, organic fertilizers, strawberry. abstract: modern agricultural practices are mostly directed toward high application of commercial fertilizers to achieve high yield. it is widely recognized that application of fertilizer (especially nitrogen) can cause ground water pollution by nitrate leaching through the soil profile. a new approach to farming is often referred to as sustainable agriculture and it seeks to introduce friendlier agricultural practices to the environment and maintains the long term ecological balance of the soil ecosystem. hence investigations were carried out to develop nutrient management for strawberry cultivar sweet charley subjected to various treatment combinations of organic and inorganic fertilizers. traits such as plant growth characteristics (leaves/plant and plant spread), yield characteristics (flower buds, fruits per plant and fruit yield tons/ha) and quality characteristics (juice content, total sugar content, vitamin c and specific gravity) were observed. the runners of strawberry cv. sweet charley were planted in the first week of november with a spacing of 30 x 60 cm. the investigation was laid out in a randomized block design with five treatment combinations replicated thrice. the data regarding the different growth parameters observed at 30, 45, 60, 90, 105, 120, yield parameters at 45, 60, 90, 120, 135, 150 days after planting and their quality parameters clearly indicate that the application of integrated sources of nutrients significantly affect the vegetative, reproductive and yield characteristics of the strawberry plant. however the manure fertilizer combination under treatment t4 (75% organic fertilizers + 25% inorganic fertilizers) was found to be the best treatment with regard to integrated and combined application of nutrient resources for strawberry cultivation in india. adv. hort. sci., 2013 27(4): 147-151 received for publication 16 november 2013 accepted for publication 30 december 2013 148 among the various factors which contribute towards the growth, yield and quality of strawberry, nutrition is the most important and it has direct bearing on crop production (umar et al., 2008). integrated nutrient management includes the use of inorganic and organic sources of nutrients to ensure balanced nutrient proportions by enhancing nutrient response efficiency and maximizing crop productivity of desired quality. it also helps to minimize the existing gap between nutrient removal through continuous use of chemical fertilizers and supply through slow release of fertilizers. it is widely reported that the extensive use of chemical fertilizers adversely affects soil health and results in decreased crop productivity and quality (macit et al., 2007). even with the application of recommended doses, yield potential of strawberry cropping systems has reduced to a plateau because soil health has deteriorated drastically and especially its organic matter content has depleted significantly. as intensive agriculture is becoming more and more necessary to meet the needs of the population, the soil nutrient balance is becoming increasingly negative and thus requiring appropriate supplement through integrated nutrient management. the use of organic and inorganic nutrient sources not only helps to increase crop yields but also helps to storehouse nutrients for successive crops in addition to improving the physical condition of the soil. bioorganic nutrition also improves the yield and quality of product. organically produced fruit fetches higher prices compared to products grown using inorganic fertilization. the practical approach will be organic farming to the most responsive fruit crops and to encourage integrated nutrient supply for tapping the potential yield of crops. as reported by hennion et al. (1999), “strawberry cultivation is highly nutrition responsive” and therefore the present experiment was undertaken to manage the growth, yield and quality of strawberry through an integrated approach. 2. materials and methods the present investigation was carried out at experimental fields of the horticulture department shiats, allahabad during spring season 2009-10. the experimental field is located at an elevation of 78 m above sea level and latitude 81.15o e and longitude 28.87 o n. the soil of the experimental field was sandy loam in texture, poor in nitrogen (0.24%), comparatively richer in potassium (0.57%) and phosphorus (0.62%) and slightly acidic (ph=6.60) in nature. the strawberry (cv. sweet charley) was procured from wimco seedling ltd, baghwale rudrapur (u.s.n) uttar pradesh, india. the experiment was comprised of the following five treatment combinations: 1. 100% recommended dose of inorganic fertilizers +0% recommended dose of manures (t 1 ); 2. 5% recommended dose of inorganic fertilizers + 25% recommended dose of manures (t 2 ); 3. 50% recommended dose of inorganic fertilizers + 50% recommended dose of manures (t 3 ); 4. 25% recommended dose of inorganic fertilizers +75% recommended dose of manures (t 4 ); 5. 0% recommended dose of inorganic fertilizers + 100% recommended dose of manures (t 5 ). the manures used in this experiment were farmyard manure and forest litter. the experiment was laid out in a randomized block design with four replications. the observations recorded were on growth parameters (leaves/ plant and plant spread), yield characteristics (flower buds, fruits per plant and fruit yield tons/ha) and quality characteristics (juice content, total sugar content, total soluble solids, vitamin c, ph and specific gravity). 3. results and discussion all the treatments in the present investigation had significant impact for all observed traits. however, treatments differed significantly from one another at various time intervals (tables 1 and 2; figures 1-8). the highest vegetative growth (15.25 mean leaves/plant and 21.50 cm mean plant spread) was recorded for treatment t 4 . observations revealed that plant spread and leaves/plant coincided with peak flower buds/plant, which within one month produced the highest fruits/plant. afterwards production declined drastically and fewer fruits/plant were produced towards the lag end of growth (i.e. mid april). overall, treatment t 4 which contained a greater amount of organic manures and less inorganic fertilizers was judged superior to the other treatments. this treatment showed 45.61% superiority over treatments t 5 and t 1 (control) in average leaves/plant and 61.53% superiority over t 1 in average plant spread/plant. recently it was revealed that available npk and micronutrients increased significantly with organic sources of nutrients either alone or in combination with inorganic fertilizers over inorganic fertilizers used alone, thus improving vegetative growth. similar results were also reported by klaas (2000), funt and blerman (2000), prasad et al. (2002), gajbhiye et al. (2003), arancon et al. (2004) and singh and dwivedi (2011). hence, taking these findings as reference, strawberry cv. sweet charley responded positively to the manure and fertilizer combination applied in treatment t 4 . eight pickings were carried out starting from 60 days after planting to 150 days after planting (i.e. from end of december to mid april). treatment t 4 produced 2.5 mean flower buds/plant more than t 1 (control) whereas in the number of fruits/plant, treatment t 4 produced 5.75 average number of fruits more than treatment t 1 with greater length/diameter ratio of 1.43, compared to 1.25 for treatment t 1 . thus, treatment t 4 showed 61.53%, 80.95% and 14.4% superiority over treatment t 1 in average flower buds/plant, average number of fruits per plant and length/ diameter ratio of fruits, respectively. also fruit yield per plant was highest (482.6 g/plant=26.81 tons/ha) with treatment t 4 and lowest with treatment t 5 (351.5 g/plant=19.55 149 tons/ha); treatment t 4 showed 37.14% superiority over treatment t 5 in fruit yield tons/ha. kopanski and kawecki (1994), gajbhiye et al. (2003), asrey and singh (2004), umar et al. (2008), singh and singh (2009), singh et al. (2010) and yadav et al. (2010) all reported the superiority of organic manures compared to npk inorganic fertilizers in producing higher yields. the superiority of treatment t 4 in producing a maximum number of fruits/plant may be due to a greater growth and reproductive capacity of plants as influenced by a mixture of manures and inorganic fertilizers applied in treatment. as far as fruit quality parameters are concerned, treatment t 4 showed overall significant superiority to treatments t 1 and t 5 . the juice content of fruits increased with increasing manure content and decreasing npk inorganic fertilizer content although reduced slightly when manures were used alone (table 2; table 1 impact of integrated nutrient management on growth and development of strawberry cv. sweet charley in india treatments average leaf size/plant average plant spread (cm) average flower buds per plant average fruits per plant days after transplanting days after transplanting days after transplanting days after transplanting 30 45 60 90 120 30 45 60 90 105 45 60 90 120 135 60 90 120 135 150 t 1 3.25 3.50 4.25 6.75 10.75 9.25 11.25 11.75 14.75 14.25 1.0 2.00 2.25 3.25 3.25 1.25 3.00 3.75 4.5 5.25 t 2 3.75 4.25 4.50 7.25 11.25 9.5 11.25 12.00 15.25 15.00 0.75 2.00 2.50 4.00 4.0 1.00 3.50 4.50 5.25 5.75 t 3 4.25 4.50 5.00 7.50 11.75 10.0 11.75 12.50 15.75 15.50 1.0 2.00 2.00 4.50 4.5 1.00 3.75 5.25 5.75 6.50 t 4 5.75 6.00 7.75 11.2515.25 11.0 13.0 16.25 21.50 20.75 1.25 3.00 3.75 5.25 5.25 1.75 4.00 8.00 9.0 9.50 t 5 3 3.50 4.00 6.50 10.75 9.0 11.25 11.50 14.75 14.50 1.0 2.25 1.75 3.75 3.75 0.50 3.25 3.75 4.0 6.25 se± 0.97 0.91 1.36 1.73 1.69 0.70 0.67 1.89 2.57 2.41 0.37 0.40 0.69 0.55 0.68 0.63 0.35 1.57 1.75 1.48 c.d at 5% 0.66 0.39 0.78 0.55 0.55 0.48 0.77 0.50 0.57 0.57 0.55 0.55 0.10 1.52 0.71 0.67 0.98 1.14 2.43 2.15 table 2 impact of integrated nutrient management on yield and quality characteristics of strawberry cv. sweet charley in india treatments length/ diameter ratio of fruits total fruits/ plant fruit yield tonnes /ha juice content (%) acidity content (%) tss 0b total sugar (%) ascorbic acid content (mg/100 g) ph specific gravity t 1 1.25 23.75 21.13 82.10 0.16 9.00 13.50 35.55 3.01 0.80 t 2 1.30 25.25 22.53 84.50 0.15 10.00 14.25 38.56 2.96 1.00 t 3 1.35 27.75 24.93 89.10 0.14 11.00 14.95 42.74 2.91 1.10 t 4 1.43 29.50 26.81 93.20 0.12 13.00 15.00 49.78 2.87 1.30 t 5 1.26 24.50 19.55 91.40 0.11 14.00 15.25 53.60 2.81 0.90 se± 0.06 2.14 2.60 4.16 0.019 1.85 0.63 6.75 0.06 0.17 c.d at 5% 1.17 5.55 9.08 19.54 0.48 7.40 4.09 7.32 1.01 0.84 fig. 1 effect of different integrated nutrient management on yield (tons/ha) of strawberry cv. sweet charley in india. fig. 2 effect of different integrated nutrient management on t.s.s. (°b) of strawberry cv. sweet charley in india. 150 fig. 6 effect of different integrated nutrient management on acidity (%) of strawberry cv. sweet charley in india. fig. 4 effect of different integrated nutrient management on vitamin c (mg/100 g) of strawberry cv. sweet charley in india. fig. 5 effect of different integrated nutrient management on fruit juice (%) of strawberry cv. sweet charley in india. fig. 7 effect of different integrated nutrient management on ph value of strawberry cv. sweet charley in india. fig. 8 effect of different integrated nutrient management on specific gravity of strawberry cv. sweet charley in india. fig. 3 effect of different integrated nutrient management on total sugar (%) of strawberry cv. sweet charley in india. 151 figs. 1 and 5). the maximum juice content (93.2%) was recorded for treatment t 4 and the minimum (82.10%) for treatment t 1 . the increase in juice content may be due to an interactional effect of mixture of manures and inorganic npk applied through treatment t 4 which might have improved cell elongation, cell thickening and fruit development, resulting in better ripening with more succulent fruits. several reports have indicated that manure and fertilizer combinations in strawberry have led to the production of softer fruits (neuweiler, 1997; ghaderi and talaie, 2008; singh and singh, 2009). the maximum tss (14%) was found in treatment t 5 and the minimum (9%) in treatment t 1 , likewise total sugar and vitamin c content was highest in treatment t 5 and lowest in treatment t 1 (table 2 and figure 2, 3, 4). on the other hand, both acidity (%) and ph were highest in treatment t 1 and lowest in treatment t 5 while treatment t 4 exhibited the highest specific gravity of fruits and treatment t 1 the lowest, as shown in table 2 and figures 6, 7 and 8. thus strawberry cv. sweet charley responded negatively to npk inorganic fertilizers and positively to higher manure levels under the experimental conditions of this study. present findings are in close conformity with the results presented by dradi and faedi (1995), bergamaschi et al. (1995), prasad et al. (2002), nazir et al. (2006) and singh and dwivedi (2011). 4. conclusions it can be concluded from the present study that an integrated nutrient management combination such as treatment t 4 (25% recommended dose of inorganic fertilizers +75% recommended dose of manures) is advisable for maximum returns from strawberry. acknowledgements the authors are thankful to the head of the department of horticulture and vice chancellor sam higginbottom institute of agriculture technology and science for their continuous support during this research work. references arancon n.q., edwards c.a., bierman p., welch c., metzger j.d., 2004 influences of vermicompost on field strawberries: 1. effects on growth and yields. bioresource technology, 93: 145-153. asrey r., singh r., 2004 evaluation of strawberry varieties under semi-arid irrigated region of punjab. indian journal of horticulture, 61(2): 122-124. bergamaschi m., faedi w., lavarone e., lelli a., longoni f., lovati f., lucchi p., pianezzola a., testoni a., 1995 aspetti qualitativi e nutrizionali di diverse varietà di fragola. l’informatore agrario, li(44): 29-36. childs w.h., 1937 seasonal distribution of yield and size variation for ever bearing strawberry varieties. proc. amer. soc. for hortic. sci., 33: 364-367. dradi r., faedi w., 1995 qualitative variation of strawberry fruits during harvesting. south indian horticulture, 23: 37-39. funt r.c., blerman p., 2000 composted yard waste improves strawberry soil quality and soil water relation. acta horticulture, 517: 37-39. gajbhiye r.p., sharma r.r., tewari r.n., 2003 effect of biofertlizer on growth and yield parameters of tomato. indian journal of horticulture, 60(4):368-371. ghaderi n., talaie a.r., 2008 influence of manure and urea on yield and some other fruit characteristics in strawberry cv. kurdistan. iranian j. hortic. sci., 39(1): 99-107. hennion b., vaysse p., verpont f., 1999 fraisier. fertillisation et qualité du fruit. infos-ctifl, 154: 36-39. klaas l., 2000 effect of manure to the growth and forming runners in strawberry. proceedings of the international conference on fruit production and fruit breeding, tatru, estonia, 12-13 september. kopanski k., kawecki z., 1994 wplyw nawozenia azotem i obornikiem na wzrost i plonowanie dwoch odmian truskawek w warunkach zulaw wislanych. roczniki gleboznawcze, 45(1/2): 66-75. abstract in english. macit i., koc a., guler s., deligoz i., 2007 yield, quality and nutritional status of organically and conventionally grown strawberry cultivars. asian journal of plant sciences, 6(7): 1131-1136. nazir n., singh s.r., aroosa k., masarat j., shabeena m., 2006 yield and growth of strawberry cultivar sena-sengana as influenced by integrated organic nutrient management system. environment and ecology, 243(3): 651-654. neuweiler r., 1997 fertilizer combinations produce softer fruits in strawberry. punjab journal of horticulture, 12: 17-19. prasad v.m., das k.s., dashrath y., 2002 effect of biofertilizers on growth and quality of tomato. bioved, 13(1/2): 125-127. sharma v.p., sharma r.r., 2003 the strawberry. indian council of agricultural research, new delhi, pp. 166. singh a., singh j.n., 2009 effect of biofertilizer and bioregulators on growth, yield and nutrient status of strawberry cv. sweet charlie. indian journal of horticulture, 66(2): 220-224. singh n., dwivedi h., 2011 studies on the different mulches on vegetative growth of strawberry (fragaria x ananassa duch.) cv. chandler. progressive horticulture, 43(1): 134-136. singh s.r., zargar m.y., singh u., ishaq m., 2010 influence of bio-inoculants and inorganic fertilizers of yield, nutrient balance, microbial dynamics and quality of strawberry (fragaria x annanassa) under rain fed conditions of kashmir valley. indian journal of agricultural sciences, 80(4): 275-281. umar i., wali v.k., kher r., sharma a., 2008 impact of integrated nutrient management on strawberry yield and soil nutrient status. applied biological research, 10: 22-25. yadav s.k., khokhar u.u., yadav r.p., 2010 integrated nutrient management for strawberry cultivation. indian journal of horticulture, 67(4): 445-449. impaginato 529 adv. hort. sci., 2019 33(4): 529­535 doi: 10.13128/ahsc­8178 effect of different nutrient solution and irrigation regimes on growth of lily (la hybrid ‘fangio‘) z.s.n. mohajer 1, m.h. asil 1 (*), j.­a. olfati 1, m.r. kaledian 2 1 department of horticultural, faculty of agricultural sciences, guilan university, rasht, iran. 2 department of water engineering, faculty of agricultural sciences, guilan university, rasht, iran. key words: nutrition, soilless culture, vase life, water consumption. abstract: a better understanding of the effects of nutrients element and irriga­ tion levels on production of lily (lilium la hybrid fangio) can lead to optimal uses of nutrients and water. plant growth is strongly correlated with the amount of irrigation and fertilization. in this regard, a greenhouse experiment was carried out to evaluate the effect of different nutrient solution viz. high concentration of elements (s1), medium concentration of elements (s2), and high concentration of elements (s3) under different irrigation regimes (100, 90, 80 and 70 % of field capacity (fc) in soilless culture. in well­watered treatments (100% fc), s3 enhanced the vase life by 17% compared to s1. the maximum leaf number was observed in the interaction of s3 and 90% fc, whereas its min­ imum was found in the interaction of s1 and 70% fc. under 70% fc, s3 increased the leaf length by 6% in comparison with s1. leaf width was altered by simultaneous use of nutrient solution and irrigation, ranging from s2 and 80% fc (13.3 mm) to s1 and 70% fc (9 mm). in s3, 70% fc decreased the bud length by 9% relative to 100% fc. the days until flowering varied from the interaction of s2 and 70% fc (4.1 days) to s1 and 100% fc (6 days). under s1 treatments, 70% fc decreased the flower number by 18% compared to 100% fc. the highest weight of daughter bulb was observed in interaction of s2 and 80% fc. in contrast, the lowest weight of daughter bulb was found in s2 and 90% fc. 1. introduction lilies have become economically important, mainly because of beauti­ ful, large, attractive and fascinating form of flowers, long vase life and capacity to rehydrate after long transportation. bulbs are produced com­ mercially for use in the cut flower and potted plant industries (aslam et al., 2013; al­allaq et al., 2014). the importance of this genus in the world flower market is due to the existence of a wide variety of hybrids and numerous commercial cultivars (dhyani et al., 2009). in recent decades, supplying the nutrient solution to plants in order to optimize crop nutrition has been widely used in both soil and soilless cul­ (*) corresponding author: asilhassanpour@yahoo.com citation: mohajer z.s.n., asil m.h., olfati j.­a., kale­ dian m.r., 2019 ­ effect of different nutrient solution and irrigation regimes on growth of lily (la hybrid ‘fangio‘). ­ adv. hort. sci., 33(4): 529­ 535. copyright: © 2019 mohajer z.s.n., asil m.h., olfati j.­a., kaledian m.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 august 2019 accepted for publication 30 august 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 529­535 530 ture under greenhouse conditions (savvas et al., 2013). nutrient solution management can provide a sustainable and effective schedule in floriculture by applying different horticultural practices such as water use efficiency. it has been estimated that 100­ 350 l of water are needed to produce 1 kg of plant dry matter, and this may vary with species and vari­ ety, cultivation system and plant growing season (cassaniti et al., 2012). the lack of dependable sup­ plies of good quality water in many regions of the world has become a concern among agricultural, urban, industrial and environmental components (valdez­aguilar et al., 2009). optimal irrigation sched­ uling could lead to higher water use efficiency. it is also very important since it influences the rhizos­ phere environment, media water potential, and salt accumulation, which in turn affects plant growth, photosynthesis and consequently crop production and quality (tsirogiannis, 2010). appropriate use of water supply and nutrients results in better water use efficiency, stressful situations, and control pro­ duction (raviv and blom., 2001). since in soilless cul­ ture systems the water is generally distributed in excess, and consequently the nutrients are removed from the substrate by drainage water and accumu­ late in the recirculating nutrient solution, which has to be flushed out regularly, representing the negative environmental impacts (rouphael and colla., 2009; wortman, 2015). in bulbous ornamental plants, the flower production and bulb yield are remarkably important in their profitability (de vroomen, 1993; maroyi, 2016). an accurate schedule on nutrients and water supplies may decrease production costs and the risk of water pollution (dufour and guérin., 2005). hence, it is essential to have a good knowl­ edge of the plant’s mineral requirements in order to avoid nutrient waste. on the other hand, it is neces­ sary to limit mineral imbalance in the medium by assuring a minimal leaching of excess nutrient solu­ tion (chang et al., 2010). recently, some nutritional solution formulas and irrigation levels have been used to increase the pro­ duction efficiency, which the suitability of these treatments should be considered with appropriate levels of nutrients and irrigation (grewal and maheshwari, 2011; waraich et al., 2011; grzebisz et al., 2013; quaggio et al., 2019). the intent of these studies was to develop the optimal irrigation and nutrient systems that could meet the high productivi­ ty of plants with explaining different levels of nutri­ ents and water supply. to our knowledge, there is no published document on the water use and nutrients application on the growth of lily. therefore, the pur­ pose of present study was to assess the simultaneous application of different irrigation regimes and nutri­ ent solutions on growth of lily (lilium la sp. cv. fangio). 2. materials and methods plant material and growing conditions the experiment was carried out during 2016­ 2017, under greenhouse conditions at the research unit greenhouse of islamic azad university, gorgan, iran, for three months. inside the greenhouse, venti­ lation was provided automatically when the air tem­ perature exceeded 26˚c. average day and night tem­ perature were 25˚c and 14˚c, respectively, and aver­ age maximum and minimum relative humidity was 70% and 50%, respectively. uniform sized bulbs of la lilium (lilium la. hybrid) cv. ‘fangio’ were obtained from a commercial importer (circumference 18­20 cm) and was immersed in a fungicide drench for 10 seconds. the bulbs were cultured in the medium con­ taining cocopeat and perlite (2:1) with ec of 2.95 ds m­1, ph of 5.9, and bulk density of 0.17 g cm­3. the bulbs were cultured in the plastic pots (15 cm height, 17 cm diameter, and 3 liters) on september 2016. nutrient solution and irrigation regimes the study was designed as factorial based on completely randomized design (crd) with three lev­ els of nutrient solution and four levels of irrigation in six replications. the nutritional solutions contained a low concentration of elements (s1), medium concen­ tration of elements (s2) and high concentration of elements (s3). the nutrient solutions were prepared based on modified quick nutrient solution (olfati, 2015). all chemical substrate (solution or elements) used in the study were purchased from merk (darmstadt, germany). water regimes were applied at 100, 90, 80 and 70% field capacity (fc). it was con­ ducted based on weighing method as 200 ml for well­ watered treatment (100% fc) followed by 180, 160, and 140 ml for 90, 80 and 70% fc, respectively. the nutrient solutions used in the experiment are shown in table 1. the concentrations of ions in the irrigation water were expressed as mgl­1 (table 1). micronutrients were added to the plants as iron chelate (eddha) (0.075 ppm), manganese sulfate 0.001, zinc sulfate 0/01, copper sulfate 0.03, edta molybdenum 0.001, and boric acid (h3bo4) (0.02 per­ cent. two weeks after culturing the bulbs (when the mohajer et al. ‐ effect of different nutrient solution on growth of lily 531 bulbs grew in 10 cm), feeding was carried out manu­ ally according to the plant’s water requirement. growth parameters at the end of experiment, the bud length, leaf length, leaf width, number of leaves, plant height, number of flowers, time of flowering, number and size of daughter bulb were measured. flowers were harvested when the first flower in plants was bloomed. subsequently, vase life was determined. the vase life of individual flowers in an inflorescence was evaluated according to their appearance as the number of days since bud opening till the appear­ ance of deformation because of petal wilting. the vase life of the inflorescence was determined as the number of days from the beginning of the experi­ ment up to the fading of the second flower. the end of vase life was determined by flower wilting, tepal abscission or color change in tepals, while in case of leaves, by color fading of blade, yellowing or dying on 30% of the leaf surface. statistical analysis the data (n=6) were subjected to one­way analy­ sis of variance (anova) and using the sas software package for windows (sas, version 9.3, sas institute, cary, nc). when statistical significance (p<0.05) was detected, the mean values subjected to duncan’s multiple range tests. 3. results vase life and plant height vase life of lily was affected by nutrient solution (p≤0.05, table 2). in well­watered treatments (100% fc), s3 enhanced the vase life by 17% compared to s1 (table 3). plant height was influenced by the nutri­ ent solution (p≤0.05, table 2). plant height was dif­ ferent between treatments, ranging from 18.8 cm in table 1 ­ the concentration of elements used in the nutritional solution nutrient solutions (mg l­1) potassium nitrate dipotassium hydrogen phosphate potassium dihydrogen phosphate calcium nitrate ammonium nitrate sodium chloride magnesium sulfate ec (ds m­1) ph nutrient solution 1 (s1) 631.3 43.5 102 512.5 150 14.6 230.6 1.04 5.8 nutrient solution 2 (s2) 757.55 52.2 122.4 615 180 17.5 276.7 2.38 5.4 nutrient solution 3 (s3) 883.8 60.9 142.8 717.5 210 20.4 322.8 2.74 5.7 table 3 ­ effect of nutrient solutions and irrigation regimes on post vase life, plant height, leaf number and size of lily (lilium la sp. cv. fangio) nutrient solutions irrigation regime vase life (day) plant height (cm) leaf number leaf length (mm) leaf width (mm) nutrient solution 1 (s1) 100% 14.6±1.8 c 113.0±2.1 c 92.5±2.5 c 101.5±1.9 de 10.6±1.2 fg 90% 15.1±1.1 a­c 113.5±3.3 c 90.5±2.4 cd 94.8±3.5 f 11.6±1.1 d­f 80% 15.0±1.4 a­c 114.8±2.5 bc 93.5±3.6 c 96.1±4.1 f 9.8±0.5 gh 70% 14.0±1.8 ab 112.8±3.7 c 87.5±1.9 d 90.3±4.2 g 9.0±0.8 h nutrient solution 2 (s2) 100% 16.5±1.7 ab 115.5±2.1 a­c 96.6±1.9 b 101.5±1.4 de 11.8±0.8 c­e 90% 16.1±1.4 ab 116.8±1.9 a­c 94.0±3.2 bc 109.5±5.1 a 12.8±0.7 a­c 80% 17.0±2.1 a 116.5 ±2.3 a­c 105.0±2.6 a 104.1±2.1 b­d 13.3±0.7 a 70% 15.6±1.2 a­c 116.3.5±4.1 a­c 90.0±1.9 cd 100.2±2.3 e 11.5±0.8 d­f nutrient solution 3 (s3) 100% 17.0±1.4 a 115.8±3.1 a­c 96.8±1.5 b 107.5±2.1 ab 12.1±0.6 b­d 90% 16.1±1.5 ab 118.8±3.3 a 104.6±2.6 a 105.1±2.3 bc 13.7±0.8 a 80% 15.8±0.5 a­c 118.8±3.2 a 93.5±2.5 bc 102.8±1.8 c­e 13.1±0.6 ab 70% 16.8±1.2 a 118.0±3.4 ab 90.1±3.8 cd 96.8±2.7 f 10.8±0.8 e­g table 2 ­ analysis of variance for the studied traits of lily (lilium la sp. cv. fangio) s.o.v df vase life plant height leaf number leaf length leaf width days until flowering flower number bulb number bulb length bulb width nutrient solution (ns) 2 22.87 ** 117 ** 229.4 ** 488 ** 35.95 ** 4.05 ns 0.29 ns 10.68 ns 263 ** 0.96 ns irrigation regime (ir) 3 1.03 ns 11.2 ns 240.7 ** 235 ** 16.52 ** 57.27 ns 11.12 ** 0.12 ns 326 ** 9.14 ns ns×ir 6 2.13 ns 3.24 ns 137.1 ** 64.2 ** 2.22 * 38.27 ns 0.81 ns 0.4 ns 27.7 ns 8.58 * error 55 2.63 8.2 9.05 8.62 0.83 40.8 0.85 0.77 14.6 3.76 cv 10.24 2.4 3.18 2.93 7.81 8.61 10.56 17.32 3.7 17.81 adv. hort. sci., 2019 33(4): 529­535 532 plants supplied with the interactions of s3 and 90% fc as well as s3 and 80% fc to 112.8 cm in plants treated with the interaction of s1 and 70% fc (table 3). leaf number and size the number, length and width of leaves were sig­ nificantly changed by nutrient solution, irrigation regimes, and their interaction (p≤0.01, table 2). the maximum leaf number was observed in the interac­ tion of s3 and 90% fc (104.6 leaf), whereas its mini­ mum was found in the interaction of s1 and 70% fc (table 3). under 70% fc, s3 increased the leaf length by 6% in comparison with s1 (table 3). leaf width was changed by simultaneous application of nutrient solution and irrigation, ranging from s2 and 80% fc (13.3 mm) to s1 and 70% fc (9 mm). bud length, days until flowering, and flower number bud length was affected by nutrient solution and irrigation regime (p≤0.01, table 4). in plants supplied with s3, 70% fc decreased the bud length by 9% rela­ tive to 100% fc (table 4).the days until flowering was not significantly influenced by nutrient solution and irrigation regimes (p≥0.05, table 2). under the appli­ cation of both nutrient solution and irrigation regimes, the days until flowering varied from the interaction of s3 and 70% fc (69.1 days) to s1 and 90% fc (77.8 days) (table 4). flower number was sig­ nificantly affected by irrigation regime (p≤0.01, table 2). under s1 treatments, 70% fc decreased the flower number by 18% compared to 100% fc (table 4). the number and weight of daughter bulbs daughter bulb number was not significantly influ­ enced by nutrient solution and irrigation regimes (p≥0.05, table 2). under the application of both nutrient solution and irrigation regimes, bulb number varied from the interaction of s3 and 100% fc (4.1 bulbs) to s1 and 100% fc (6 bulbs) (table 4). daughter bulb weight was affected by the interaction of nutrient solution and irrigation regime (p≤0.01, table 2). the highest weight of bulb was observed in interaction of s2 and 80% fc. in contrast, the lowest weight of daughter bulb was found in s2 and 90% fc (table 4). 4. discussion and conclusions the results showed that high concentration of the elements (s3) improved the flower and leaf attribut­ es of lily. treder (2005) reported an improvement of bud length and leaf size under the mixture of multi­ cote and liquid fertilization. the results of morpho­ logical parameters such as leaf size, number of leaves per plant, and flower number in our study are sup­ ported by khosa et al. (2011), as they observed an increase in growth and flowering with the increasing fertilization level of macronutrients. also, the results of the previous studies have shown that the height of some cultivars might not be affected by fertilization (treder, 2003). therefore, it can be concluded that in different cultivars of the lily, the height and some vegetative growth characteristics are influenced by cultivars (treder, 2003). our results are also consis­ tent with devecchi and remotti (2003), who studied calla (zantedeschia aethiopica) in which different doses of nitrogen and potassium did not significantly affect the length of the floral stem. moreover, the results of plant height are well­supported by treder (2005) as the application of multicote at three levels and water­soluble fertilizer for some cultivars of lily at during the vegetation period did not significantly affect the maximum plant height in ‘acapulco’, rela­ table 4 ­ effect of nutrient solutions and irrigation regimes on the flower and bulb properties of lily (lilium la sp. cv. fangio) nutrient solutions irrigation regime bud length (mm) days until flowering flower number bulb number bulb weight (gr) nutrient solution 1 (s1) 100% 98.9±2.5 c­e 71.5±6.1 ab 9.50±0.9 a 6.0±0.8 a 10.7±2.4 ab 90% 101.7±2.7 b­d 77.8±6.5 a 8.16±0.8 bc 5.8±0.6 a 10.1±3.1 ab 80% 98.4±2.9 c­e 74.6±6.9 ab 9.00±1.1 a­c 5.6±0.7 ab 12.1±2.8 ab 70% 92.7±2.5 f 72.0±3.9 ab 8.00±0.6 bc 5.5±0.7 a­c 10.4±3.4 ab nutrient solution 2 (s2) 100% 109.1±3.2 a 73.6±5.4 ab 9.16±0.8 ab 5.1±0.8 a­d 11.8±2.1 ab 90% 105.6±2.5 ab 73.3±4.7 ab 8.83±0.9 a­c 5.1±0.4 a­d 8.3±2.6 b 80% 103.1±3.5 bc 74.0±4.6 ab 9.66±0.7 a 4.6±0.6 b­d 12.5±1.1 a 70% 99.2±2.8 c­e 74.6±6.6 ab 7.83±0.9 c 5.1±1.1 a­d 11.7±3.2 ab nutrient solution 3 (s3) 100% 103.7±2.4 bc 76.1±6.8 ab 9.66±0.7 a 4.1±0.6 d 10.9±1.9 ab 90% 108.9±3.3 a 77.5±5.8 ab 7.83±0.8 c 4.3±0.4 d 11.1±2.1 ab 80% 103.4±2.8 bc 76.8±4.4 ab 9.50±1.0 a 4.5±0.4 cd 10.1±1.9 ab 70% 95.2±2.7 ef 69.1±5.1 b 7.83±0.6 c 4.6±0.1 b­d 10.7±2.2 ab mohajer et al. ‐ effect of different nutrient solution on growth of lily 533 reported, which is probably due to k role in water relations, turgor maintenance, and cell expansion (mengel and kirkby, 2001). thus, an ample supply of k is recommended for leaf development, which will contribute to plant quality since the foliage is consid­ ered an important quality factor to lilium (mckenzie, 1989). thus, lilium can be considered as a species that is compatible with a wide range of nutrient con­ centrations. nitrogen is an essential component of chlorophyll, in this connection, the nitrogen content of leaf correlates positively with the leaf chlorophyll content (loh et al., 2002). kang et al. (2016) showed that an increase in fertilization can reduce the severi­ ty of chlorosis in pansy. they pointed out that leaf chlorophyll concentration increased asymptotically with fertilization rate, which finally increase the leaf size and plant height. in order to reducing over watering, we investigat­ ed the water use efficiency and nutrient elements in different nutrient solution and irrigation regimes to ensure adequate water for plants during the growth periods. outcomes of the present study indicated that lilium la hybrid ‘fangio’ can be properly grown in medium levels of irrigation with no significant change in its morphological properties. 70% fc slight­ ly changed the plant growth particularly leaf size and bud length. hence, to optimum use of water, we can reduce the water amount to 80% fc even 70% with high use of nutrients concentration. references al­allaq h., ali h., hazzim y., 2014 ­ hybrid lilies under bulb removal stress. ­ agri. research, 8: 631­637. asker h.m., 2015 ­ hydroponic technology for lily flowers and bulbs production using rain water and some com‐ mon nutrient solutions. ­ afr. j. biotechnol., 14(29): 2307­2313. aslam f., naz s., tariq a., ilyas s., shahzad k., 2013 ­ rapid multiplication of ornamental bulbous plants of lilium orientalis and lilium longiflorum. ­ j. botany., 45(6): 2051­2055. bernstein n., ioffe m., luria g., bruner m., nishri y., philosoph­hadas s., salim s., dori i., matan e., 2011 ­ effects of k and n nutrition on function and pro‐ duction of ranunculus asiaticus pedosphere. ­ soil. sci. soc. china, 21(3): 288­301. cassaniti c., romano d., flowers t.j., 2012 ­ the response of ornamental plants to saline irrigation water, pp. 131­158. ­ in: garcia­garizabal i., and r. abrahao (eds.) irrigation. water management, pollu‐ tion and alternative strategies. intech europe, rijeka, croatia, pp. 234. tive to other cultivars of lily. similar to our results, in sandersonia aurantiaca, the number of flowers was not affected by various nutrient treatments (bernstein et al., 2011). also, yield, quantified by the mean number of bud per plant, did not differ between the three nutrient solutions or between the four different levels of irrigation tested. the increase of morphological properties of lily fertilization is due to the significant role of essential element presented in the nutrient solution on plant growth. for example (n), nitrogen is a major constituent of most impor­ tant plant substances. n compounds comprise 40% to 50% of the dry matter of protoplasm, and it is a constituent of amino acids, the building blocks of proteins. it is also an essential constituent of chloro­ phyll. n deficiency most often results in stunted growth, slow growth, and chlorosis. potassium (p) regulates the opening and closing of the stomata by a potassium ion pump, which has a significant role on leaf and flower growth. additionally the outstanding role of magnesium in plant nutrition is as a con­ stituent of the chlorophyll molecule (vatansever et al., 2017). vegetative characteristics of plants are subjective to genetic and environmental factors cou­ pled with optimum fertilizer application and water use method (lucidos et al., 2013). marin et al. 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acidity, boron concentration, flowering, mangifera indica l., tss. abstract: imbalance uptake of boron disturbs the process of pollination that eventually decrease the flowering, fruit setting and yield. its deficiency also deteriorates the quality of fruit by increasing fruit acidity. therefore, source, balanced application, method of application and optimum uptake of b is an important aspect and need keen scientific attention. so, a field study was con­ ducted with the hypothesis that foliar application of b would be an effective technique to improve the yield and quality of mango cv. summer bahisht (sb) chaunsa. the source of b was boric acid (ba) applied twice as foliar spray i.e., 0, 0.1, 0.2 and 0.3%. results confirmed that as compared to control, a significant improvement in fruit weight at ripening and harvesting stages (36.9%), fruit length (21.9%), fruit width (10.1%), flower (22.1%) and fruit terminals m­2 (40.0%) confirmed the effectiveness of t4 (ba= 0.3%). a significant improve­ ment in average yield (78.6%) validated the efficacious functioning of boric acid (0.3%). in conclusion, boric acid is an important and effective source of b to improve the quality and yield of mango cv. sb chaunsa. similarly, ba (0.3%) is a better option than 0.2 and 0.1% ba to improve the quality and yield of mango. 1. introduction mango (mangifera indica l.) belongs to the genus mangifera and fami­ ly anacardiaceae which has 74 genera and 600 species (mitchell and mori, 1987; tian et al., 2010). it is known as ‘king of fruits’ due to its sweetness, fragrance and nutritional status (sharma and singh, 2009). mango is becoming popular in the western countries and it is originated from indian sub­continent (yadav and singh, 2017). however, in recent years, the productivity of mango has been reduced due to deficiency of micronutrients especially boron (b) and other environmental stresses (saran and kumar, 2011; adak et al., 2017; ahmad et al., 2018). boron deficiency is quite common after zinc micronutrient especially in (*) corresponding author: niaz.ahmad@bzu.edu.pk citation: haider z., ahmad n., danish s., iqbal j., arif ali m., khalid chaudhry u., 2019 ­ effect of foliar application of boric acid on fruit quality and yield traits of mangor. ­ adv. hort. sci., 33(4): 457­464. copyright: © 2019 haider z., ahmad n., danish s., iqbal j., arif ali m., khalid chaudhry u. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 march 2019 accepted for publication 8 july 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 457­464 458 arid and semi­arid regions (zhang et al., 2015). the defi­ ciency of b is ubiquitous in acidic sandy, alkaline soils and sandy soils (camacho­cristóbal et al., 2018). boron deficiency is one of the key factors for a reduction in the yield of fruit crops (davarpanah et al., 2016; karlidag et al., 2017). its deficiency symptoms show their effect on younger plant parts, while toxic effects are on older parts of plants (fernández­escobar et al., 2016). the deficiency of b in mango plant results in deformed leaves hooked and pre­matured dieback of inflores­ cence, loss of apical dominance, death of apical bud, swelling at internode and retorted growth (litz, 2009). contemporary agricultural practices include the additional supply of fertilizers that enhanced the growth of plants and resulted in an improvement of yield (barker and pilbeam, 2006). micronutrients espe­ cially b plays an indispensable role in the growth and development of fruit trees (davarpanah et al., 2016). mango fruit drop before maturity is a common issue in most of the mango growing areas (murti et al., 2008). on the other hand, the potential of foliar application of b has been well documented as an effective amend­ ment for the improvement in mango fruit formation (saran and kumar, 2011). boron is intrinsic for fruit trees in forming higher germinating pollen and elon­ gated pollen tube which sets fruit (el­sheikh et al., 2007). it is also indirectly involved in the activation of plant hormones and dehydrogenase enzyme (marschner, 2012). boron has low adsorption capacity and leaches at a high rate in soil (raja et al., 2005). foliar application of fertilizers is more convenient and effective as compared to soil application (fernández et al., 2013). it has been observed that foliar application of fertilizers moreover confers quick response and alleviate the deficiency symptoms lead­ ing to fruitful returns (obreza et al., 2010). scientists have also documented that foliar application of b at different rates enhance mango fruit setting panicle­1, fruit weight and volume (zhong and dong, 2000; bibi et al., 2019). therefore, the current study was conducted with the aim to examine the effectiveness of the vari­ ous application of boric acid as a source of b on growth and yield of mango. it is hypothesized that foliar appli­ cation of boric acid as a source of b would be an effec­ tive technique for improvement in growth and yield of mango. 2. materials and methods experimental site the study was planned to evaluate the influence of foliar applied boron on yield and quality of mango cultivar summer bahisht chaunsa during the year 2016­17 at experimental mango orchard near bahauddin zakariya university, multan (fig. 1). fifteen to twenty years old mango trees were select­ ed for the experimental purpose. the study was car­ ried out in accordance with rcbd design (randomized complete block design) with four treat­ ments and four replications. treatment plan there were four different levels of boric acid (ba) i.e., t1 (control) = no boric acid, t2 = 0.1% ba, t3 = 0.2% ba and t4 = 0.3% ba which were applied as foliar application. each tree was taken as one replica of each treatment (consist of four trees). all treat­ ments were applied twice in a year i.e., firstly at inflo­ rescence stage and secondly at the marble stage (pea size of fruit). physio‐chemical analysis soil sampling. the data regarding physio­chemical composition was recorded at harvest stage on ripened fruit. composite soil samples (0­30 cm) were collected from experimental mango orchard. this depth for soil sampling was selected because the majority of mango feeding roots are present in the depth of 0­30 cm. sample preparation. soil samples were initially air dried, after that grinded and finally sieved through 2 mm sieve in the laboratory of department of soil fig. 1 ­ gps locations of area. haider et al. ‐ boron improves mango fruits quality and yield 459 science, faculty of agricultural sciences and technology bahauddin zakariya university multan, pakistan for determination of different physio­chemi­ cal characteristics of the soil. soil characterization. for determination of soil texture, hydrometer method was used (bouyoucos, 1962). soil ph and electrical conductivity (ec) was assessed by using jenway 3510 ph and bante dds­ 12dw microprocessor ec meter (abid et al., 2017). soil cation exchange capacity (cec) was calculated according to richards (1954) and rhoades (1982). for determination of soil organic matter (som) walkley black method was followed (jackson, 1975). olsen et al. (1954) method was adopted for the analysis of soil available p while for soil extractable k rowell (1994) method was used. calcium carbonate (lime) was determined according to allison et al. (1965). all characteristics of the soil are provided in table 1. boron determination in soil and leaves leaves samples for b determination were collect­ ed twice during 2016­17. samples were collected prior to foliar application of boric acid and after spraying the mango orchard. boron (b) concentration from soil was assessed by hcl extraction as described by ponnamperuma et al. (1981), while for leaves samples method of gaines and mitchell (1979) was followed. growth and yield attributes different mango parameters were recorded by selecting 15 panicles and labelled on each tree prior to measurements in an experimental mango garden. flower terminal m­2, fruit terminal m­2, total number of flowers per panicles was observed with respect to each treatment and average was computed among all the treatments. the total number of flowers from the selected panicles (in case of each tree) was calcu­ lated to calculate the average one. fruit weight (fw) after harvest and ripening was measured by selecting 15 fruits from each treatment. yield of fruits the average yield tree­1 was assessed at the time of harvest by weighing out all fruits of tree regarding each treatment and their average was calculated by multiplying average fruit weight with total number of fruits. fruit weight after harvest was noted soon after harvesting when fruit was not ripen. when the fruit was ripened, again the weight of fruit was noted and referred to as fruit weight after ripening. maturity days maturity days were recorded by estimation of days from flowering period to harvest stage. length and diameter of fruits fruit length and diameter were assessed by digital vernier caliper after randomly selecting 5 fruits from each treatment. total soluble solids the total soluble solid percentage was calculated by using a digital refractometer as described by aoac (2005). acidity of fruits the acidity of mango was estimated followed by the method as proposed in souza et al (2015), one drop (mango juice) was retained on the mirror regarding digital refractometer and reading was observed in case of brix on the screen. sugar contents total sugar contents were recorded according to the titrimetric method as described by raganna (1986). the method proposed by rusk (1961) was used to estimate the vitamin c content from the juice of mango pulp. pulp recovery (%) was determined by the following formula pulp recovery = (peel weight – stone weight / fruit weight) × 100 statistical analysis for statistical analysis standard statistical proce­ dure was adopted (steel et al., 1997). the signifi­ cance of treatments was analyzed through analysis of variance (anova). tukey’s test was applied at p ≤ 0.05 for comparison of treatments by using statistical software “statistix 8.1”. textural class depth (cm) ph ece (ds m­1) sar (mmol l­1)1/2 caco3 (%) organic matter (%) boron concentration in soil (mg kg­1) boron concentration in leaves (mg kg­1) phosphorus in soil (mg kg­1) potassium in soil (mg kg­1) loam 0­30 8.4 2.04 2.3 6 0.31 0.36 20.49 13 218 table 1 ­ pre­experimental characteristics of soil adv. hort. sci., 2019 33(4): 457­464 460 3. results fruit weight effect of various foliar application rates of boric acid remained significant (p≤0.05) for fruit weight at ripening and harvesting stages. no significant change in fruit weight was observed at ripening and harvest stages among control t3 and t2. however, the appli­ cation of t4 was significantly better as compared to control for fruit weight at ripening and harvesting stages (table 2). the maximum increase of 36.9% in fruit weight was noted where t4 was applied as com­ pared to control at ripening and harvesting stages. fruit length and width effect of various foliar application rates of boric acid remained significant (p ≤ 0.05) for fruit length and width. application of t2, t3 and t4 were statisti­ cally alike to each other but performed significantly better as compared to control for fruit length and width. the maximum increase of 21.9 and 10.1% in fruit length and width was noted respectively where t4 was applied as compared to control. average yield effect of various foliar application rates of boric acid remained significant (p ≤ 0.05) for average yield. application of t3 and t2 did not differ significantly for the average yield of mango fruit as compared to con­ trol. however, the application of t4 was significantly better as compared to control for average yield (table 2). the maximum increase of 7.86% in average yield was noted where t4 was applied as compared to control. peel and stone weight effect of various foliar application rates of boric acid was significant (p ≤ 0.05) for stone and peel yield. application of t3 and t2 did not differ signifi­ cantly for stone and peel weight of mango fruit as compared to control. however, the application of t4 significantly decreased stone and peel yield as com­ pared to control (table 2). the maximum decrease of 22.2 and 15.0% in peel and stone yield was noted where t4 was applied as compared to t3 and t2 respectively. flower and fruit terminal m‐2 effect of various foliar application rates of boric acid was significant (p ≤ 0.05) for flower and fruit ter­ minals m­2. it was observed that t4 and t2 did not dif­ fer significantly but differed significantly better as compared to control for flower terminals m­2. application of t3 also performed significantly better for flower terminals m­2 as compared to control (table 3). for fruit terminals, m­2 t3 and t4 were sta­ tistically alike to each other but differed significantly as compared to control. application of t4 also dif­ fered significantly better for fruit terminals m­2 as compared to control (table 3). the maximum increase of 22.1 and 40.0% in flower and fruit termi­ nals m­2 was noted respectively where t4 was applied as compared to control. total number of flowers, male and hermaphrodite flower effect of various foliar application rates of boric acid was significant (p≤0.05) for total number of flowers, table 2 ­ effect of various levels of boric acid on yield attributes of mango table 3 ­ effect of various levels of boric acid on flowering related attributes of mango treatments fruit weight after ripening (g) fruit weight after harvest (g) fruit length (cm) fruit width (cm) average yield (kg tree­1) peel weight (g) stone weight (g) t1 (control) 250.72 b 270.93 b 10.46 b 5.74 b 132.25 b 40.48 ab 51.18 ab t2 0.1% boric acid 281.62 b 301.74 ab 11.82 a 6.10 a 135.75 b 47.20 a 58.30 a t3 0.2 % boric acid 304.29 ab 334.28 ab 12.01 a 6.11 a 138.43 ab 47.72 a 51.27 ab t4 0.3 % boric acid 343.34 a 371.16 a 12.75 a 6.32 a 142.65 a 37.15 b 49.53 c mean values followed by the different letter in the same column are statistically different (p ≤ 0.05). mean values followed by the different letter in the same column are statistically different (p ≤ 0.05). treatments flower terminal m­2 fruit terminal m­2 total number of flowers panicle­1 male flower (%) hermaphrodite flowers (%) maturity days t1 (control) 21.50 c 7.32 c 895.43 c 548.475 b 346.525 ab 195 b t2 0.1% boric acid 25.08 ab 8.75 b 933.54 b 618.45 ab 314.55 b 197 a t3 0.2 % boric acid 24.25 b 9.25 a 1017.5 a 633.225 ab 384.275 a 196 b t4 0.3 % boric acid 26.25 a 10.25 a 1001.75 a 672.525 a 329.225 b 198 a haider et al. ‐ boron improves mango fruits quality and yield 461 results are in accordance with silva et al. (2014) that supply of boron is intrinsic to enhance the length and diameter of mango fruit with foliar application. moreover, it was revealed that boron is less mobile in the plant, therefore, it piles up in older plant leaves and becomes unable for the sturdy growth of fruit development (oldoni et al., 2018). contrarily male and hermaphrodite flower. application of t3 and t4 did not differ significantly with each other but remained significant for total number of flowers in mango as com­ pared to control (table 3). however, the application of t2 also significantly improved total number of flowers in mango as compared to control. in the case of male flow­ ers, only t4 remained significantly better from control (table 3). for hermaphrodite flowers, application of t3 remained significantly better as compared to control. the maximum increase of 11.9, 22.6, and 22.2% in total number of flowers, male and hermaphrodite flower was noted where t4, t4 and t3 were applied as compared to control, control and t2 respectively. maturity days effect of various foliar application rates of boric acid was significant (p≤0.05) for fruit maturity days. application of t2 and t4 remained statistically alike to each other but remained significantly better as com­ pared to control for maturity days. no significant change was noted among t3 and control for maturity days. the maximum increase of 1.51% in maturity days was noted where t4 was applied as compared to control. acidity and boron concentration the foliar application was effective in case of quality traits of mango. it was observed that with the applica­ tion of boric acid treatments acidity of fruit was decreased. minimum acidity was noted in t3 treatment while t2 and t4 treated plots exhibited the same response for the acidity of fruit (fig. 2). the pulp recov­ ery, vitamin c, total soluble solids and total sugar con­ tents tend to increase with an increasing application rate of foliar boric acid (0.3% boric acid) (fig. 3). boron concentration in leaves foliar application of b was found effective in term of improvement in leaves b concentration (fig. 4). higher application of foliar b 0.3% (t4) enhanced the b concentration in leaves as compared to t1. lowest b concentration was noted in t1. no significant change was noted among t4 and t3 for b concentra­ tion in leaves. similarly, t3 and t2 were statistically alike to each other for b concentration in leaves. 4. discussion and conclusions the current study depicted the beneficial role of foliar application of boric acid as a source of b for improving mango yield and quality traits. similarly, the effective role of foliar application has been reported previously (anees et al., 2011; singh et al., 2017; ahmad et al., 2018; oldoni et al., 2018). our fig. 2 ­ effect various levels of boric acid on acidity of mango fruit. fig. 3 ­ effect of various levels of boric acid on pulp recovery, vitamin c, total soluble solids and total sugar contents in mango fruits fig. 4 ­ effect of various levels of boric acid on boron concentra­ tion in mango leaves. 462 adv. hort. sci., 2019 33(4): 457­464 foliar application was reported to supplement boron supply to the growing plant organs which ultimately enhanced mango fruit diameter (bhatt et al., 2012). it was further strengthened by the findings of singh et al. (2017) that b application increases fruit size due to the better mobilization of food material from pro­ duction sites to storage organs and rapidly fruit development. it was noted that foliar application was significantly effective for improvement in mango fruit weight. our results were inlined with findings of dutta (2004). the possible reason behind the increase in fruit weight might be due to increased cell expansion and cell division. moreover, boron contri­ bution in hormonal metabolism and boron is a key player in the rapid mobilization of sugar and water in fruits (haq et al., 2013). increase in yield after boron application was correlated to an increase in carbohy­ drates metabolism (perica et al., 2001 a). the higher yield was also associated with the greater number of flower formation due to boron absorption and they also set a greater number of fruits (usenik and stampar, 2007; sarrwy et al., 2012). the similar result regarding an increase in fruit yield was also reported in almond (nyomora et al., 1999), kinnow (ullah et al., 2012), guava (rawat et al., 2010), persimmon (khayyat et al., 2007) and peach (ali et al., 2014). mango flowering is influenced by physiological events taking place throughout the course of its growth. we observed that improvement in flowering is due to the synthesis of flower promoters synthe­ sized in leaves and their translocation to sprouts via phloem (ramírez and davenport, 2012). foliar appli­ cation increases bud formation by the synthesis of essential hormones and metabolite translocation to the bud of the tree (usenik and stampar, 2002). perica et al. (2001 b) reported the similar findings that boron foliar spays resulted in a higher percent­ age of perfect flowers. it was well acknowledged that boron concentration obtained higher in pollen grains and flowers as compared to leaves. this improved flowering is due to the readily available boron required for reproductive organs (dell and huang, 1997; blevins and lukaszewski, 1998). therefore, these results are in agreement with other fruits, where boron concentration at bud initiation, repro­ ductive tissues, flowers and fruits were favourable (nyomora et al., 1999). it was well documented the role of boron application considerably enhanced the emerging flowers and fruits (perica et al., 2001 b). mango quality traits were improved due to boron application was reported (ahmad et al., 2018). increase in sugar contents due to boron was also reported from earlier studies (hassan, 2000; shaaban, 2010). it is due to the development of stor­ age and accumulation in sugar content with the con­ version of polysaccharide and starch into simple sugar (kahlon and uppal, 2005). it is evident that boron is responsible for declining acidity of fruit (baiea et al., 2015). this result was further supported by (anees et al., 2011; sarker and rahim, 2012; el­ razek et al., 2013). other quality traits improvement reports were found accordingly with our results (bhatt et al., 2012; haq et al., 2013). the higher rate of boron accumulation in leaves is due to the direct absorption by the leaves (khan et al., 2012). foliar application increased boron concentration with high­ er application rate (perica et al., 2001 b). this result was endorsed by previous studies in olive (hegazi et al., 2018), apricot (karlidag et al., 2017) and straw­ berry (kitir et al., 2018). it was concluded that foliar application of boric acid is an effective source to alleviate b deficiency in mango trees. in addition, the application of boric acid also improves the yield and fruit quality of mango. furthermore, 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(ed.) new trends in postharvest management of fresh produce ii. fresh produce 4 (special issue 1). kagawa ken: global science books, pp. 127. ullah s., khan a.s., malik a.u., afzal i., shahid m., razzaq k., 2012 ­ foliar application of boron influences the leaf mineral status, vegetative and reproductive growth, yield and fruit quality of ‘kinnow’mandarin (citrus reticulata blanco.). ­ j. plant nutr., 35(13): 2067­ 2079. usenik v., stampar f., 2007 ­ effect of late season boron spray on boron accumulation and fruit set of ‘summit’ and ‘hedelfinger’ sweet cherry (prunus avium l.). ­ acta agric. slov., 89: 51­58. yadav d., singh s.p., 2017 ­ mango: history origin and distribution. ­ j. pharmacogn. phytochem., 6(6): 1257­ 1262. zhang y., hu c., tan q., nie z., zheng c., gui h., sun x., zhao x., 2015 ­ soil application of boron and zinc influ‐ ence fruit yield and quality of satsuma mandarin in acidic soils. ­ agronomy j., 107(1): 1­8. 15900 table s1.xlsx height stem       inl  sg  ll  lw  nolp la  spad ldw sla dtfba  (days) dtfb (days) dof      (days) fd          (cm) ffw         (g) sl                (cm) not        (no.) tw         (g) growing media (gm) 0.003 0.000 0.000 0.003 0.005 0.067 0.000 0.897 0.000 0.000 0.000 0.000 0.299 0.000 0.000 0.001 0.023 0.001 shade levels (sl)  0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.772 0.001 0.502 0.001 gm x sl  0.776 0.000 0.073 0.000 0.000 0.174 0.000 0.516 0.000 0.000 0.000 0.000 0.977 0.066 0.995 0.795 0.595 0.001 significance of fixed factors (p‐value) treatments table s1 ‐ significance levels of main effects and interactions for cutting survival, rooting, growth and quality characteristics for  dahlia x hybrida  variety ‘babylon lila’ inl:  internodal length; sg: stem girth; ll: leaf length; lw: leaf width; nolp: number of leaf pairs; la: leaf area; ldw: leaf dry weight; sla: specific leaf area; dtfba: days to first bud appearance; dtfb: days to full bloom; dof:  duration of flowering; fd: flower diameter; ffw: flower fresh weight; sl: stem length; not: number of tubers per plant; tw: tuber weight.  flower characteristics tuber characteristicsvegetative characteristics leaf characteristics 5 1. introduction in stone fruit trees, the cycle of shoot growth is marked by the development of two bud types: the vegetative and reproductive buds. vegetative buds comprise bud scales, leaf primordia, and the shoot apical meristem. reproductive buds have a more complex structure made up of bud scales and flower primordia represented by pistil and stamens. the flower buds of temperate-zone fruit trees are initiated during the previous growing season and several critical steps during bud morphogenesis were identified, from floral induction up to complete bud differentiation (ryugo, 1990). during the autumn-winter season, the plant elaborates mechanisms for survival under unfavorable growing conditions by adopting a dormancy strategy to cold temperature acclimation. meristem activity becomes insensitive to growth-promoting signals (rohde and bhalerao, 2007) preserving the buds in a quiescent state under potentially damaging environmental conditions (ćechová et al., 2012). bud dormancy starts with the perception by the plant of rest-signals under the influence of short and cool days; this process finishes after an accumulation of chilling temperatures. the conventional terminology identifies dormancy evolution as: paradormancy, when growth is inhibited by endogenous factors outside the dormant structure; endodormancy, when growth is regulated by physiological factors within the dormant structure; and ecodormancy, when the inability to grow is imposed by environmental factors (lang et al., 1987). in trees, winter dormancy means endodormancy: during this phase the chilling temperatures are accumulated, and flower buds are unable to respond to warm temperatures required for dormancy breaking (fig. 1). when the chilling requirement (cr) is fulfilled, buds are in ecodormancy and can react to warm temperatures, but remain in an apparent quiescent state due to persistent low temperatures (horvath et al., 2003). the release of dormancy is genetically controlled and regulated by complex phenomena affected by various integrated elements whose interaction(s) determine the point in time when release of bud dormancy occurs (faust et al., 1997). enapricot flower bud dormancy: main morphological, anatomical and physiological features related to winter climate influence r. viti*, s. bartolini**, l. andreini* * dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. ** istituto di scienze delle vita, scuola superiore sant’anna, piazza martiri della liberta, 33, 56127 pisa, italy. key words: budbreak, climate, endodormancy process, prunus armeniaca l. abstract: this review examines recent advances regarding flower bud dormancy in apricot, focusing on biological, anatomical, and physiological processes which occur during the induction and depth of dormancy. in a scenario of global climate change, the relationship between endodormancy and winter climate influence is discussed. dormancy regulation is a complex process necessary for plant survival and development. in fruit species, the knowledge of mechanisms controlling dormancy and establishing its release appears crucial for successful yields. specific studies have suggested that, when the flower buds are apparently inactive, slow and gradual changes occur in the whorls: organogenesis, such as microsporogenesis processes and vascular connections take place during the entire dormancy period. it has been indicated that an asynchronism between biological (i.e. endodormancy release, microsporogenesis evolution), anatomical (i.e. xylem vessel differentiation) and biochemical (i.e. changes in metabolic compounds and enzymes) events could represent further causes determining an inconstant rate of blooming. temperature is the main factor involved in dormancy triggering and releasing. in the perspective of global warming, mild winter temperatures could greatly impact apricot ecological cropping systems. phenological process-based models are considered to be the best tool to study the climatic changes and subsequent expected phenology variation (dormancy and flowering). a new model, calibrated and validated on apricot cultivars, is proposed to predict the dormancy release date in a future scenario. adv. hort. sci., 2013 27(1-2): 5-17 the authors equally contributed to the manuscript. received for publication 21 december 2012 accepted for publication 2 april 2013 6 vironmental factors (temperature, light, relative humidity, etc.) are directly related to biochemical changes involved in bud dormancy release. in fruit species, understanding the mechanisms controlling dormancy and establishing its release appear crucial for successful yields. apricot (prunus armeniaca l.) is characterized by a restricted spread in the mediterranean basin and its adaptability to specific environments is strongly influenced by the ability of certain cultivars to overcome flower bud dormancy (viti et al., 2010). climatic-environmental factors may heavily influence flower bud dormancy breaking, affecting the entity of appearance of floral anomalies and determining irregular and/or insufficient fruit bearing (clanet and salles, 1972; legave et al., 1984; viti and monteleone, 1995). cold winter temperatures influence the time of bud dormancy release and, consequently, the cr of a genotype (garcia et al., 1999). most of the known apricot cultivars are characterized by a high cr (table 1); more than 1000 chill units (cu) are required to overcome bud dormancy (guerriero et al., 2006). in mediterranean apricot growing areas, characterized by a mild-winter climate, many cultivars have an inadequate satisfaction of cr. during a short or mild rest season (garcia et al., 1999), the unsatisfactory cr can determine an incomplete release of flower bud dormancy, a late bud break with scanty blooming and a high flower bud drop (viti and monteleone, 1991; erez, 2000). thus, cr is a key factor for breaking dormancy and knowledge about it has practical significance and economic impact on the control, maintenance and production of woody plants (fennell, 1999). this statement appears crucial for the crop management of apricot cultivars in most cultivation areas. the present paper examines the dormancy process of apricot flower buds, focusing on biological, anatomical, and physiological changes that occur during the endodormancy phase in relation to winter temperature influence within the context of global climate change. 2. morphological and anatomical features the pattern of apricot floral organogenesis after initiation is typical of other prunoideae. the first floral organs to appear are sepals and petals followed by the stamen and pistil; the most internal whorls of the pistil derive from the gradual evolution of the receptacle (monet and bastard, 1968; bartolini et al., 2013). this process requires about two months and is normally completed before leaf fall but not all buds reach the stage of pistil appearance (legave, 1975). apricot flower bud evolution was described for the first time by baggiolini (1952). the classification has been revised to describe accurately the progress of flower bud phenological development by the addition of sub-phases within each phenological stage (bartolini et al., 2004). figure 2 depicts the evolution of flower bud phenological stages from deep dormancy (stage a) until petal fall (stage h). the stages related to the dormancy process are referred to as: 1) stage a 0 -a 2 during endodormancy; 2) stage b 1 e 2 from ecodormancy to bud swelling; and 3) stage f 1 -h from beginning of flowering to petal fall. dormant buds (stages a 0 , a 1 , a 2 ) are characterised by a conical shape, rounded at the base, with the brown bud scales tightly closed (a 0 ) or table 1 chilling units (cu) required to induce break of endo-dormancy in flower buds of several apricot cultivars. starting date and amount (%) of flowering are reported cultivars cu flowering starting date % san castrese 870 1 mar 57 goldrich 950 7 mar 18 sarritzu i° 950 7 mar 51 alessandrino 1000 7 mar 10 baracca 1000 14 mar 21 d’alessandria 1000 10 mar 5 bebeco 1030 7 mar 15 canino 1030 2 mar 34 moniqui 1125 5 mar 2 aurora 1140 29 feb 2 amabile vecchioni 1140 2 mar 30 s.nicola grosso 1140 9 mar 25 bergeron 1225 17 mar 1 rapareddu 1250 7 mar 23 polonais 1300 9 mar 5 orange red 1450 14 mar 1 stark early orange not defined not defined 0 mean 1100 sd 151 fig. 1 schematic representation of apricot flower bud growth during the annual cycle. 1) paradormancy: from flower bud initiation to beginning of dormancy; 2) endodormancy: from deep dormancy to bud swelling; 3) ecodormancy: from beginning of flowering to petal fall. solid and dotted lines represent the flower bud length (mm) and the flower bud fresh weight (mg/ fb), respectively. evolution of the main phenological stages of flower buds are illustrated. 7 more enlarged (a 1 and a 2 ); at the beginning of bud swelling (stages b 1 and b 2 ) the pink sepal tips appear; at stages c 1 and c 2 sepals are clearly visible; at stages d 1 and d 2 full bud swelling takes place with the appearance of the white petal tips; at stages e 1 and e 2 petals are clearly visible; at stages f 1 and f 2 full flowering occurs; at stages g and h petal fall begins with the end of flowering. during endodormancy, the accumulation of chilling temperatures occurs and it is usually believed that flower bud development is arrested because no clear signs of growth are evident. however, specific studies have shown that bud growth is hardly perceptible, although constant (alonso et al., 2005). the buds are metabolically active and continue their development during the entire winter, leading to bud break. in peach flower buds continuous anatomical development during the late autumn and winter dormancy period were observed, without macroscopic changes (reinoso et al., 2002). apricot flower buds also have a gradual and prolonged development during morphogenesis since their organogenesis is not generally completed until just before anthesis (erez and couvillon, 1987). during the first stage (a) a minimal variation in bud size and weight was detected. indeed, during december and january, when buds are apparently still dormant, it was possible to detect a continuous growth in weight and size. the parameters of weight increase and height/ width ratio have revealed a statistically appreciable slow and progressive buds growth from the middle of december (guerriero et al., 1986; scalabrelli et al., 1991). during this apparent rest period, anatomical observations revealed that processes of microsporogenesis, macrosporogenesis and xylogenesis take place. as regards microsporogenesis, the following phases have been identified (fig. 3) in relation to flower bud development (nyújtó and banai, 1975; viti and scalabrelli, 1988; viti and monteleone, 1991): a) sporigen cells differentiation (sticked cells), during the paradormancy period; b) pollen mother cells (diploid microsporocytes differentiated from sporogenous cells that become spherical and separated; c) tetrads appearance (four haploid microspores produced by meiosis of the pollen mother cells and surrounded by a callose wall) during the endodormancy period; d) young pollen grains (isolated microspores produced when the callose wall disappears) at the overcoming of endodormancy; e) mature pollen grains (full development of the wall made up of two layers, exine and intine) after the resumption of growth. the tetrad stage has been considered a signal marking the end of endodormancy (bordeianu et al., 1962; szabò et al., 2002). nevertheless, other studies have shown that tetrads occur at a late stage of morphological bud growth and they are not closely linked to chilling accumulation, because the meiosis process took place also in buds exposed to high temperature (martìnez-tellez et al., 1982; felker et al., 1983; viti and scalabrelli, 1988). contradictory studies have shown that winter dormancy sets a boundary between the development of the sporogenous tissue and further microspore development (julian et al., 2011). these authors observed that, in autumn, stamens develop until the differentiation of sporogenous tissue, remaining in this anatomically quiescent stage during the three months of winter; microspore development took place only after dormancy. concerning gyneceum development and the related macrosporogenesis, a later evolution than stamen and microsporogenesis process was observed. morphologically, the pistil has a lengthwise growth that can be divided into different phases: intensive growth during paradormancy; slow growth during endodormancy; and increased growth during ecodormancy, at first very slowly then followed by a significant rapid rise a few weeks before flowering (szalay and nemeth, 2010). anatomical observations showed that the first signs of the ovary can be detected in october. several studies have found a well-developed embryo sac at anthesis; egea and burgos (1995) found a frequent presence at anthesis of ovules without the sac being formed. in apricot, two to three ovules/ovary are usually noted, but at anthesis they are frequently malformed and quickly degenerate. ovule fig. 2 evolution of flower bud phenological stages in apricot: deep dormancy (stage a), pink sepal tips appearance (stage b), sepals clearly visible (stage c), achievement of bud swelling (stage d), petals clearly visible (stage e), anthesis (stage f), end of flowering (stage g) and petal fall (stage h). from bartolini et al. (2004). fig. 3 main microsporogenesis phases (x 400). a) pollen mother cells (pmc); b), b1) tetrads (t) and c) young pollen grains (ypg). 8 number seems to be related to agronomic and climatic conditions, rather than genetics (egea and burgos, 1995; burgos and egea, 1993; albunquerque et al., 2002). at the end of the endodormancy process (end of winterbeginning of spring), a rapid weight increase and the complete development of floral verticils at the transition from phenological stage b (visual beginning of bud swelling) up to stage d (appearance of the white petal tip) have been observed. in this period, elongation of pistil and stamen filaments occurs while mature pollen grains and complete gynaecium development take place only few days before blossoming (luna et al., 1990). in concomitance with the effective warm temperatures, female and male gametophytes become functional. during the temporary delay of visible bud growth, the process of xylem development within the flower bud axis occurs. the bud, during early growth, is connected to the stem through a parenchymatous zone, traversed by a procambial strand. in several prunus species, it was observed that, during winter, vascular tissues are not completely differentiated and the connection between the flower primordium and the bud axis is formed only by the procambium (ashworth and rowse, 1982). this tissue, which constitutes the vascular strands, is made up of elongated cells and contains densely stained cytoplasm and lacks lignified secondary wall thickenings (esau, 1965). the differentiation process consists of the transition from this meristematic tissue to xylem cells, i.e. dead cells with lignified walls producing an empty conduit through which water flows. in apricot, gradual xylem development was observed during winter, when no bud growth changes were visible. the acropetal progression of primary xylem differentiation along the flower bud axis was defined by five stages according to bartolini and giorgelli (1994): stage 1 = at the base of the axis; stage 2 = at ½ of the axis; stage 3 = at ¾ of the axis; stage 4 = at the base of the ovary; stage 5 = inside the pistil (fig. 4). stage 3 appears to be the most significant with regard to breaking dormancy because it is in concomitance with the first morphological sign of bud growth resumption. a good relationship between an advanced xylem differentiation (‘stage 3’) and endodormancy release was observed in cultivars with a low chilling requirement (i.e. ‘san castrese’). the availability of nutritional elements throughout xylem supply in correspondence with bud growth reactivation could be a factor determining flowering regularity. a correlation between an increase of certain elements (i.e. potassium and boron) by the xylem acropetal transport and bud swelling has been observed (hanson and breen, 1985; essiamah and eschrich, 1986; bartolini and giorgelli, 1995). on the other hand, in cultivars with a high chilling requirement (i.e. ‘orange red’), xylem ‘stage 3’ occurs when flower buds are still in endodormancy. this early anatomical trait of flower buds does not coincide with the reactivation of bud growth; it could be hypothesized that the newly formed vessels might preserve the ability to function in water transport. this feature was confirmed by a recent research where a good syncronism between overcoming dormancy, xylogenesis and microsporogenesis, was found in cultivars with a low cr, while in cultivars with a medium or high cr an asynchrony between such processes was observed (bartolini et al., 2006 a), leading to inconstant blooming and fruit yield. 3. physiological features control of plant dormancy is the result of multifactorial regulatory networks in which nutrients, phytohormones, genes, proteins and climatic factors (namely temperature and photoperiod) are involved at some point in time (chao and anderson, 2010). light and temperature climate parameters are important determinants in several aspects of dormancy. in northern temperate regions, the stimulus for induction of growth cessation and dormancy has been considered to be primarily controlled by short photoperiod (allona et al., 2008). increasingly, several authors have shown that temperature may replace or strongly mediate this short photoperiod dormancy response in woody species (kalcsits et al., 2009; tanino et al., 2010). the onset and release of dormancy state might begin with the perception of a signal by the plant upon exposure to chilling temperatures (or et al., 2002). then, it would be followed by transduction of this signal via a cascade of biochemical events to the stage where it imposes or releases repression of bud meristematic activity (faust et al., 1997). many cellular activities leading to morphological, physiological and biochemical changes take place inside the bud during the transition period from endodormancy to active bud growth, including respiratory rate, reserve carbohydrate mobilisation, water content increase, energy transport, and gene expression. fig. 4 representation of xylem vessel differentiation along the flower bud axis, from bartolini et al., 2006: stage 1 (at the base of the axis); stage 2 (at ½ of the axis); stage 3 (at ¾ of the axis); stage 4 (at the base of the ovary); stage 5 (inside the pistil). 9 ‘sink strength’ and carbohydrates considering that the bud of a woody plant is morphologically complex and constituted by organs differing in structure and physiology, the bud anabolic potential is partially regulated by the “sink strength” of dormant and nondormant tissues influencing the subsequent capacity to accumulate metabolites (crabbé and barnola, 1996). during early bud growth, the parenchymatous zone, which represents the connection between bud and stem, is fed through the symplasm of this region (pétel and gendraud, 1996). thus, plant dormancy and dormancy breaking appear to depend on peculiar short distance relationships between the bud and its underlying tissues (champagnat, 1973; gendraud and pétel, 1990). in peach, during the dormant period, the parenchyma shows a strong atpase activity driving a powerful proton extrusion, with a consequent ph cytoplasmatic alkalisation linked to enzyme activity (pétel et al., 1992; petél and gendraud, 1996). a relationship between changes in intracellular ph (phi) and bud dormancy release has been observed, first, in jerusalem artichoke and peach vegetative buds (marquat et al., 1996; aue et al., 2000), and subsequently in apricot buds (zanol and bartolini, 2003), suggesting the involvement of phi changes as signalled by growing evidence (zimmermann et al., 1999). in particular, when apricot flower buds were still in deep dormancy, an increase of phi in the flower primordia tissues was found, just before the first sign of growth reactivation, usually denoted by a significant bud weight increase (fig. 5). the changes in phi values might be useful to detect in advance bud growth capacity, showing the potential competitive sink for nutrients between the different bud tissues from dormancy to growth resumption (zanol and bartolini, 2003). the phi measurement is a good parameter to estimate ‘sink strength’ determining the nutrient fluxes (gendraud and pétel, 1990; bonhomme et al., 1999; robert et al., 1999). the energy source for budbreak comes mainly from the mobilization of products stored in the perennial parts of the tree. carbohydrates are the main source of energy for the metabolic changes that occur during the dormant period and for spring sprouting and blooming (flore and layne, 1996; sherson et al., 2003). changes in the content of carbohydrates in different tissues from vegetative and reproductive structures were found during dormancy, associated with chilling temperatures (wang and faust, 1987; valentini et al., 2006). there was a significant decrease in starch concentration in the bark tissue of prunus sp. due to exposure to chilling temperatures (gonzález-rossia et al., 2008). the effect of low temperatures on starch and sugar concentration during the rest period can be explained: amylase activity is induced by cold temperature, increasing starch hydrolysis and, consequently, sugar concentration (elle and sauter, 2000; bonhomme et al., 2005). soluble sugars, important signalling molecules involved in many processes in the life-cycle of plants, are also related to the dormancy period and involved in increased frost resistance (tabuenca, 1975; sheen et al., 1999; smeekens, 2000). in particular, starch levels were negatively correlated with hardiness but most soluble sugars were positively correlated (jones et al., 1999). glucose, fructose, and sorbitol were the main sugars in the bark tissues of peach, nectarine, plum and apricot; sorbitol concentrations varied significantly with chilling accumulation (bonhome et al., 2005; gonzález-rossia et al., 2008). in other sorbitol-synthesizing plants, such as sweet cherry (prunus avium l.), sucrose is the most predominant soluble carbohydrate during dormancy (keller and loescher, 1989). in peach primordia, especially the floral ones, very high concentrations of transport forms of carbohydrates (sucrose and sorbitol), imported during growth capacity recovery, were found (bonhomme et al., 2005). from autumn to mid-winter, a significant relationship between total sugars and starch concentrations, with a marked increase in amounts of sorbitol, fructose, glucose and sucrose, was found in the bark tissue of stone and pome fruits, coinciding with a decrease in starch content (wang and faust, 1987; gonzález-rossia et al., 2008). free radicals and antioxidant mechanisms studies have shown that free radicals, activated oxygen species (aos), implicated in a number of biological phenomena, are produced in dormant buds of some fruit species, where their removal seems to be associated with bud break as a result of changes in antioxidant systems (wang et al., 1991). the generation of aos, particularly h 2 o 2 , during stress has been proposed as part of the signalling cascade leading to plant response (anderson et al., 1998). the plant’s antioxidant defence system, via enzymatic and non-enzymatic mechanisms (i.e. amino acids, glutathione, acid ascorbic, carotenoids, α-tocopherol), provides protection against high levels of free radicals responsible for the fig. 5 intracellular ph at different phenological stages (from st a 0 to st c). changes in different tissues: cushion, vegetative bud (vb), flower bud (fb) axis, flower bud (fb) primordial. 10 peroxidation of membrane lipids and the destruction of proteins (de kok and stulen, 1993). antioxidative enzymes, individually or cooperatively, have been viewed as a defensive team that protect cells from active oxygen damage performing a detoxifying function (kranner and grill, 1996). superoxide dismutase (sod), catalase (cat), ascorbate peroxidase (apx), guaiacol peroxidase (gpx) and glutathione reductase (gr) are the main antioxidative enzymes involved in the reduction of h 2 o 2 to h 2 o (schmidt and kunert, 1986; noctor and foyer, 1998; rojas-beltran et al., 2000). maximum h 2 o 2 accumulation could act as a signaling molecule to trigger the sequence of reactions to break endodormancy (kuroda et al., 2002). during the dormancy process, biochemical analysis conducted on apricot flower buds revealed cat and gpx as the most involved enzymes, with significant increases of activity at the release of endodormancy, particularly in cultivars with a low or medium cr (viti et al., 2013). other metabolites such as glutathione and antioxidant proteins were associated with increased capacity for free radical scavenging (siller-cepeda et al., 1991). glutathione (γ-glutamyl-cysteinyl-glycine) is generally considered to be a ubiquitous sulfhydryl-containing tripeptide in living cells (fahey et al., 1975), being the main reserve and long-distance transport form of reduced sulfhydryls which are indispensable for protein synthesis (rennemberg, 1982). it is an important metabolite in stabilizing the cell redox state during the cold hardening process (de kok and stulen, 1993). in particular, in stressed plants, reduced glutathione (gsh) protects protein thiol groups from auto-oxidation (kranner and grill, 1996). gsh is oxidized to glutathione disulfide (gssg) and, under normal conditions, is reduced efficiently back to gsh by the action of glutathione reductase (foyer et al., 2001). the major portion of the glutathione in the cell is maintained in the reduced state, and a high reduced/oxidized ratio (gsh/ gssg) is necessary for numerous physiological functions. glutathione was suggested as one of the strong factors in controlling bud dormancy, first, in grape (tohbe et al., 1998); later, in apricot flower buds a significant relationship between the end of endodormancy and the increase of gsh/gssg ratio was found (bartolini et al., 2004). in cultivars with low-medium cr the reduced and oxidized glutathione ratio (gsh/gssg) increased in accordance with the end of endodormancy, while in cultivars with high cr the gsh/gssg ratio was kept low during the rest period. this could suggest that a minimum threshold in antioxidant activities could be crucial for scavenging free radicals during the rest season. hormonal involvement the endogenous system of hormonal regulation mediates the annual transition from growth to dormancy due to the shortened length of autumn days and reduction of daily mean temperatures (thimann, 1985). knowledge of hormonal regulation of dormancy processes has become increasingly more complex, particularly with recent findings of auxin-and ethylene-triggered abscisic acid induction revealing more responses mediated by abscisic acid (aba) than originally considered (tanino, 2004). aba has long been studied as a potential mediator of short-induced cessation of growth and initiation of bud dormancy in trees (guak and fuchigami, 2001). an increase in its activity or its accumulation in the fall is an indispensable condition for the onset of apical growth inhibition, and an essential prerequisite for emergence of the dormant state and the ability to adapt to winter frosts in apricot tress (kuzina and kalinina, 1993). kawamata et al. (2002) found that free aba levels in buds increased suddenly at onset of dormancy and decreased afterward. chen et al. (2002) suggested that changes in dormancy status are more closely related to changes in aba receptivity than to changes in aba levels. aba affects dormancy progression through its action on dehydrins or membrane permeability (jacobsen and shaw, 1989). moreover, the regulatory effect of aba in growth inhibition, dormancy, and thermoregulation in woody plants is realized only with the action of other phytohormones: auxins, gibberellins, and cytokinins (back and richmond, 1971). gibberellin ga 3 was tested in peach buds to promote bud burst under conditions of prolonged dormancy (erez et al., 1971). the comparatively high levels of ga 3 found in mid-winter could be one of the factors that control the process of anther and gynoecium development (basconsuelo et al., 1995). molecular features population and quantitative genetics studies indicate that phenological traits such as time to bud set, chilling requirement and time to bud flush show significant genetic variation, and that such traits are often controlled by multiple genes exhibiting small effects (howe et al., 2003; rohde et al., 2011). dormancy signals, impacting numerous physiological processes, involve changes in the expression patterns of numerous regulatory genes that could play a key role in dormancy transition (horvath et al., 2008). in contrast to paradormancy, the molecular aspects of endodormancy are poorly understood. the molecular biology of endodormancy has been analysed in several recent studies by global approaches where an initial set of candidate genes involved in coldor light-induced dormancy in tree species were described (bielenberg et al., 2008). gyllenstrand et al., (2007) found a significant and close correlation between growth rhythm (both bud set and bud burst) and the expression pattern of an ft (flowering locus t) homologue, suggesting that ft is a key integrator of photoperiodic and thermal signals in the control of growth rhythms in gymnosperms. additionally, the substitution of a single amino acid can transform an ft protein from an activator into a suppressor of flowering. thus, the limited but tantalizing linkage between the floral regulatory machinery and seasonal growth cessation and bud set, through regulation of ft and ft-like genes, suggests a general model for endodormancy regulation (horvath, 2009). flowering locus c (flc)-like genes have been shown to be regulated differentially during the satisfaction of 11 cr in vegetative buds of poplar (chen and coleman, 2006). furthermore, bielenberg et al. (2008) revealed a cluster of six mads-box transcription factors (named dormancy-associated mads-box or dam genes) as candidate genes for the regulation of terminal bud formation in evergrowing peach. the expression of two of these genes, dam5 and dam6, is suppressed by chilling temperatures and inversely correlated with bud break rate in peach (jimenez et al., 2010), whereas dam4 and dam6 expression is promoted by short photoperiods (li et al., 2009). similar genes were expressed differentially during dormancy induction, maintenance and release also in apricot. in prunus mume, yamane et al. (2008) have generated two ssh/mos (subtractive hybridization supplemented with mirror orientation selection) libraries containing gene pools that are expressed preferentially in endodormant buds in comparison with paradormant or ecodormant buds to search for the genes that are upregulated by endodormancy induction or down-regulated by endodormancy release. differential screening and sequencing indicated that genes involved in gibberellin metabolism, stress resistance, cell wall modification, and signal transduction, such as transcription factors, are upregulated in endodormant buds. at transition from dormancy to active bud growth, genes related to carbohydrate and energy metabolism have been specifically identified in prunus armeniaca l. céchová et al., (2012) have observed strong expression of xyloglucan endotransglycosylase/hydrolase (xth) and exgta1 (endoxyloglucan transferase) in the week before, and during, the exit of apricot flower buds from endogenous dormancy. research is still in progress to study changes in the expression in regulatory genes involved in numerous physiological signals related to the dormancy process. 4. influence of mild temperatures during the dormancy process the complex process of dormancy is affected by a close interaction between genotype and environment, where photoperiod and temperature are the main factors involved in triggering and releasing. their individual and combined effects change during the transition from a dormant to a non-dormant state (caffarra et al., 2011). apricot culture is greatly restricted by climatic conditions, with a decisive influence on development and productivity (quamme et al., 1982; guerriero and bartolini, 1991). it is well known that some cultivars are closely linked to their geographical origin and, consequently, have a low adaptability to other climatic conditions (bassi et al., 2006). autumn-winter temperature trends seem to be the main cause of this low plasticity related to the need for adequate and specific satisfaction of cr for dormancy breaking (viti et al., 2010). a warm climate may prevent or delay this process. in fact, during winter mild temperatures have a negative impact on endodormancy release due to an unfulfilled cr. in this situation, apricot is frequently affected by the appearance of floral anomalies, e.g. pistil abortion and/or browning and/or necrosis of flower buds, which are usually attributed to unfulfilled cr. however, a correlation between flower anomalies and cr satisfaction was not always achieved (guerriero and bartolini, 1991; viti and monteleone, 1993; legave, 2002). at present, knowledge about the mechanisms and nature of anomalies is still scant but, on several genotypes, a genetic determinism has been found through analysis of different apricot progenies (legave et al., 2006). from a physiological point of view, climatic conditions affect inductive signals regarding metabolism and phytohormones involved in the control of several events such as primary vascular differentiation microand macrosporogenesis (aloni, 1980; creber and chaloner 1984; fukuda, 1996). in apricot flower buds, xylem differentiation was found to be slow when the winter minimum mean temperatures were predominantly below zero (bartolini et al., 2006 a). moreover, during the autumn-winter season, after a minimum threshold of chilling amount, the supply of constant warm temperatures stimulated the development of vascular elements, at least in cultivars with a low-medium cr (bartolini and giorgelli, 1995). as regards the microsporogenesis process, the post-meiotic phase is crucial because temperature may influence the transition from tetrads to pollen grains (viti and scalabrelli, 1988). a lack of synchronism between dormancy release, xylem differentiation and microsporogenesis evolution has recently been demonstrated under warm winter conditions (bartolini et al., 2006 b; andreini et al., 2012). the negative role of certain temperatures is confirmed also during the ecodormancy stage. in particular, temperature fluctuations affect different stages of reproductive development and this stress might lead to developmental asynchrony in pollen–pistil–ovule functioning, leading to reduced fertilization levels (hedhly, 2011). considering such events, determining the effectiveness of temperature regimes on dormancy release has, for a long time, been a focus of many studies. several models have been proposed to predict the response of buds to chilling, establishing the cr of each specific genotype. the most simple one was introduced by weinberger (1950) who simply defined the ‘chilling hours’ as the number of hours at or below 7°c. the method of bidabé (1965) calculates the effect of given temperatures either for chilling or heat requirements by exponential models. the utah model weights the efficiency of different temperatures for cr fulfillment (richardson et al., 1974 and 1975). this model is the best tailored for cool and temperate regions (seeley, 1996), while under warm conditions (i.e. the subtropical regions) erez et al. (1990) proposed the ‘dynamic model’ as a better indicator for peach cr estimation. the model assigned negative values to high temperatures (negation of rest) during endodormancy (allan et al., 1993). bonhomme et al. (2010), indicate a simplified smoothed utah model as the best for french conditions, introducing a more broad range of effective temperatures to break dormancy. 12 …and what about dormancy processes with regard to climatic changes? global warming of the climate system is unequivocal from observations of increases in average air temperatures in many parts of the world (legave et al., 2009). since the end of the 1980s, this temperature change has influenced plant phenology, and in the future further climate changes will probably have an impact on crop yields (chuine and cour, 1999; chmielewski et al., 2004). mean temperatures will probably rise between 1.8 and 4.0°c by the end of the 21st century, according to ipcc reports. over the period 1910-2003, climate warming was already in motion with a minimum temperature increase of 0.25°c per decade, as reported for the californian san joaquin valley (baldocchi and wong, 2007). the most striking feature of climate change in eastern asian countries during the past century may be the remarkable winter-season warming (kwon et al., 2008). as a consequence, significant impact can be expected on winter dormancy and spring bud-burst for crops and natural vegetation in this region, as has been observed in europe (chmielewski et al., 2004). in the mediterranean area, the winter climate shows a tendency to become progressively milder (guerriero et al., 2010; menzel et al., 2011). in this context, certain years (i.e. 2006-2007) were characterized by constantly very mild autumn and winter temperatures: the minimum values occasionally went below 0°c, while the maximum values often exceeded 15°c (luterbacher et al., 2007; viti et al., 2010). thus, over the past twenty years a progressive reduction of chilling amount was recorded (fig. 6). in the global warming scenario, phenological process-based models are considered the best tool to study the climatic changes and subsequent expected phenology alteration (chuine et al., 2003). brin is a recent phenological model, calibrated for grapevine, able to predict, simultaneously, timing of budburst and flowering (garcía de cortázar-atauri et al., 2009). this model computes the dormancy period using bidabe’s cold action model (bidabé, 1965), and the post-dormancy period by the sum of hourly temperatures (growing degree hours-gdh, method of richardson et al., 1974). recently, the brin model has been calibrated and validated also for apricot cultivars under the climatic conditions of southern france allowing an effective prediction of dormancy release date (andreini et al., 2013). as a consequence, brin model could be applied in a “a1b” future scenario taking into account two aspects valuable for all cultivars: a delayed dormancy break and an early flowering. advances in modeling will be made using the experimental data of dormancy release to calibrate forecast models that can reproduce the physiological behaviour of the three. according to bonhomme et al. (2010) further study will consist in testing a wide panel of data and also assessing the introduction of the optimized endodormancy release model as sub-model into bud break/ bloom phenological model. this will likely improve them and could be very interesting for phenological predictions in the global change context. concluding remarks and perspectives survival and competitive success of woody plants depend on a precise timing of growth, winter dormancy in synchrony with seasonal changes in temperature. in particular, apricot is a species sensitive to satisfying the cr and thus achieving the release of dormancy and bud break. several cultivars of different geographical origins are heavily affected by the problem of dormancy release, which is one of the main causes of inconstant yields. the effect of temperature on the induction and depth of dormancy may explain some of the observed annual variation in dormancy and cr for its release. the picture emerging from this work points to a complex relationship between winter temperatures and several biological processes in response to overcoming dormancy in flower buds. in particular, it has been demonstrated that climatic conditions characterized by mild autumn-winter seasons can affect the regular development of flower bud organs, leading to the appearance of anomalies. moreover, several studies have indicated that an asynchronism among biological (i.e. endodormancy release, microsporogenesis evolution), anatomical (i.e. xylem vessel differentiation) and biochemical (i.e. changes in metabolic compounds and enzymes) events could represent further causes for an inconstant rate of blooming. at this time, knowledge of the mechanisms involved in dormancy is still fragmentary although recent studies of global gene expression in diffig. 6 chilling units accumulation from 1991 to 2012 : a) during winter season, b) on 31 january. data recorded under the climatic conditions of the tuscany coastal area. 13 ferent species have 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bakri et al., 2013). extracellular enzymes are thought to be particularly important cwdes in interactions between fusarium spp. and their hosts (ahmed et al., 2012). much research work is still needed to fully understand the degradation process, and particularly the enzymes and other metabolites secreted by the fungi during infection. information on these aspects could help to elucidate the biochemical mechanisms of wheat infection by fusarium diseases and consequently facilitate fungal strain selection for industrial applications. however, studies of enzyme production by a phytopathogenic fungus are complicated by the presence of the plant, particularly by the presence of plant enzymes and microbial enzyme inhibitors that occur in plants. the most practical way to study the production of enzymes by a fungus is therefore to study the production of its enzymes on artificial growth media that contain no plant or enzyme inhibitors. solid-state fermentation (ssf) is considered an attractive alternative method for the production of industrially demanded enzymes that employ microorganisms (krishna, 2005). ssf was defined by pandey (2003) as a fermentation process involving solids in the absence (or near absence) of free water. among the microorganisms that are capable of growing on solid substrates, only filamentous fungi can grow to a significant extent in the absence of free water (guimaraes et al., 2006). the present study was undertaken to assess the potentialities of several fusarium spp. in the production of industrially relevant enzymes under ssf conditions. 2. materials and methods isolation of fusarium species fusarium spp. have been isolated from infected wheat seeds showing disease symptoms, and 21 were screened out of 105 strains for their host-pathogen interactions (alazem, 2007). seeds were sterilized to remove all microbial epiphytes by soaking them in 1:5 dilutions of naocl (sodium hypochlorite) solution for 15 min. they were then rinsed in sterile distilled water and dipped in 70% ethanol for 10 min. subsequently, the seeds were washed with distilled water, dried between sterilized filter paper, and incubated in 9-cm petri dishes containing potato dextrose agar (pda, difco, detroit, mi. usa) for 10 days, at 23±1°c in the dark to allow mycelial growth. all strains were identified morphologically according to nelson et al. enzymatic activity of the endophytic fusarium species strains isolated from wheat y. bakri (1), m. jawhar, m.i. e. arabi department of molecular biology and biotechnology, aecs, damascus, syria. key words: fusarium spp., hydrolytic enzymes, wheat. abstract: fusarium is a genus of fungi that cause some of the most important plant diseases affecting agricultural and horticultural crops. members of the genus establish an endophytic role inside the tissue of plants and produce a wide range of biologically active metabolites and enzymes. in the present study, the enzymatic profiles of several dominant fusarium spp. were determined under solid state fermentation and activities were detected for xylanase, lipase, amylase, polygalacturonase, filterpase, and carboxy-methyl cellulase. each fusarium spp. showed a wide range of enzyme activities and protein contents. the ability to produce these enzymes was distributed amongst the strains tested, however amylase and xylanase f. solani sy7 was found in a high percentage of strains. this study provides additional information to support future research about the industrial potential of these enzyme-producing species. adv. hort. sci., 2014 28(3): 129-132 (1) corresponding author: ascientific@aec.org.sy received for publication 12 june 2014 accepted for publication 29 september 2014 130 (1983). the fusarium spp. strains used in the study are listed in table 1. the cultures were maintained on silica gel at 4°c until needed. extraction of enzymes from solid-state cultures enzyme production by the fusarium spp. strains was carried out in 250 ml erlenmeyer flasks containing 5 g of solid substrate and nutrients (based on 100 ml of liquid medium) plus distilled water to adjust the moisture content to 75%. fresh fungal spores were used as inoculums and 1 ml spore suspension (containing around 106 spores/ml) was added to sterilized medium and incubated at 30°c. flasks were removed after cultivation and the enzyme was extracted by adding distilled water containing 0.1% triton x 100 to make the in-flask volume equivalent to 100 ml. flask contents were stirred for 1.5 h on a magnetic stirrer. the clear supernatant was obtained by centrifugation (5000 x g for 15 min) followed by filtration (whatman no. 1. paper). carboxy methyl-cellulase and filter paperase (fpase) activity extra cellular enzymes were extracted by filtering the culture through whatman no. 1 filter paper. the cmcase and fpase activity were measured using the methods described by refaz et al. (2013). one unit of enzyme activity (iu) was defined as the amount of enzyme that released 1 μmol of glucose per ml per minute. amylolytic activity α-amylase activity was determined as described by okolo et al. (2001). the reaction mixture consisted of 1.25 ml of 1% soluble starch, 0.25 of 0.1 m acetate buffer (ph5.0), 0.25 ml distilled water and 0.25 ml of crude enzyme extract. after 10 min of incubation at 50°c, the liberated reducing sugars (glucose equivalent) were estimated by the dinitrosalicylic acid method of miller (1959). one unit (iu) of α-amylase is defined as the amount of enzyme that releases 1 μmol of glucose equivalent per min under the assay conditions. lipolytic activity lipase activity was determined using 1 ml sunflower oil, 5 ml of 50 mm phosphate buffer ph 7.0 and 1 ml enzyme solution. the assay was carried out according to the method of park et al. (1988). one unit of lipase activity was defined as the amount of enzyme liberating 1μ mol of fatty acid per min under the experimental conditions. pectinolytic activity assay of polygalacturonase (pgase) activity was carried out according to marcia et al. (1999). pg activity was determined by measuring the release of reducing groups using the dinitrosalicylic acid reagent (dns) assay (miller, 1959). the reaction mixture containing 0.8 ml of 1% citric pectin with 67% of metoxilation in 0.2m citratephosphate, ph 6.0 buffer and 0.2 ml of culture supernatable 1 enzyme activity (u/g) of the 21 strains of fusarium species used in this study strain xylanase carboxy-methyl cellulase fpase polygalacturonase amylase lipase f.culmorum sy1 20.3 3.31 2.46 23.52 45.5 61.28 2 96.36 6.67 3.64 38.72 55.36 0.30 3 163.69 3.89 2.03 70.96 54.6 0.90 6 131.93 2.78 2.03 80.4 51.13 117.04 9 12.16 1.76 2.03 15.68 52 65.04 12 115.92 2.32 2.03 74.24 66.8 81.28 13 90.64 2.87 2.03 23.92 47.4 0.50 14 19.52 2.09 2.03 41.04 51.4 0.10 f.solani sy7 757.2 5.27 2.03 67.92 118.35 81.28 11 112.16 2.66 2.03 22.72 40.4 0.70 20 234.96 1.43 2.03 61.04 39.25 0.90 35 125.6 2.39 2.03 43.76 41.7 57.52 f.verticillioides sy15 61.92 1.79 2.03 32 44.8 0.40 16 16.56 3.01 2.03 31.44 54.3 0.20 19 108.56 5.74 2.03 97.92 70.95 87.6 27 129.92 8.92 2.03 35.52 76.13 73.76 29 138.72 2.47 2.03 80 38.7 60.4 31 151.92 2.14 2.03 41.52 43.5 54.8 f.equiseti sy41 93.2 2.41 2.03 29.52 33.6 0.60 42 84.64 4.92 2.03 20.56 45.1 0.40 43 122.24 0 2.03 68 58.8 82.48 lsd= least significant difference at p < 0.05. 131 tant, was incubated at 40ºc for 10 min. one unit of enzymatic activity (u) was defined as 1 µmol of galacturonic acid release per minute. xylanolytic activity xylanase activity was measured with the optimized method described by bailey et al. (1992), using 1% birchwood xylan as substrate. the solution of xylan and the enzyme at appropriate dilution were incubated at 55°c for 5 min and the reducing sugars were determined by the dinitrosalicylic acid method described by miller (1959), with xylose as standard. the released xylose was measured spectrophotometrically at 540 nm. one unit of xylanase is defined as the amount of enzyme required to release 1 µmol of reducing sugar as xylose equivalent per min under the above assay conditions. protein determination total proteins were determined for their importance when purifying an enzyme, since purity depends on the removal of unwanted proteins, and can be assessed by relating the activity to total protein present. the protein content in the enzyme preparation was determined according to the method of lowry et al. (1951). statistical analysis all the experiments were performed in triplicate and the means were analyzed statistically with the analysis of variance (anonymous, 1988) using the stat-itcf computer package to test for differences in enzyme production among fusarium spp. strains. 3. results all 21 strains of endophytic fusarium spp. tested were able to produce one or the other extracellular enzymes (table 1); some of the strains were able to produce all six enzymes tested. the profiles of extracellular enzymatic activities varied among fusarium spp., but some general features were noted. significant differences (p<0.05) in the mean yield values were detected among strains, with high values being consistently higher in the strain f. solani7 for xylanase and amylase activities with mean values of 757.2 and 118.35 u/g, respectively. some authors have suggested that the absence of catabolic repression in ssf systems is due to several factors collectively, including the slow and low processes of diffusion in solid state cultures due to low water activity (krishna, 2005). on the other hand, cmcase and fpase activity were detected in the lowest quantity, in terms of total units per culture (table 1). whereas, f. verticillioides strain sy19, showed the highest pgase activity with a mean value 97.92 u/g. hoondal et al. (2002) reported that degradation of host tissue by phytopathogens generally begins with the production of pectinolytic enzymes, which are the major enzymes involved in plant attack. the effect of different carbon sources on pectinase synthesis by fungi in ssf have been studied and it is generally agreed that the optimum medium for the enhanced production of extracellular pectinase contains pectic materials as an inducer (yadav et al., 2005). additionally, the results show that fusarium spp. strains varied in lipase activity (table 1), which could be related to a differential capacity to infect wheat. pritsch et al. (2000) suggested that lipases might have participated, to a certain extent, in prior degradation of the cuticle. on the other hand, protein contents also showed significant (p<0.05) differences among fusarium spp. (fig. 1). the increase in protein content in some strains f. solani sy7, f. culmorumsy2 and f. verticillioides sy27 that produced high enzymatic activity suggests the growth of fungi contributing to the fungal protein by utilizing available nutrients in the substrate, making it possible to metabolize the available protein in the substrate. consumption of substrate protein the production of biomass protein are reported by scopes (1993). 4. discussion and conclusions the fusarium spp. strains studied here produced significant levels of enzyme activity in vitro. the results might indicate that this activity influences the aggressiveness of the strains of fusarium spp. towards wheat plants. kang and buchenauer (2000) showed that fusarium culmorum infects the wheat ovary usually through the junctions between the epidermal cell walls. these junctions may be a more preferable site for entry of the pathogen, allowing a quicker establishment of infection. however, priest (1984) showed that there are several possible regulatory mechanisms in enzyme production including induction. the action of such enzymes gives rise to the possibility that the “genetic recombination” of the endophyte with the host which occur in evolutionary time. this may be the reason why some endophytes fusarium spp. can produce some photochemicals originally characteristic of the host. promputtha et al. (2007) provided phylogenetic evidence indicating that the endophytes produced the same degrading enzymes as their saprobic counterparts. however, the capability of studied endophytes, such as the fusarium spp. in this work, to produce different enzymes should have an important role as saprobes since they do not decompose the host living tissue. these degrading enfig. 1 total protein of fusarium spp. strains under ssf. 132 zymes are important factors which affect the lifestyle of these species to become pathogens on wheat plants. indeed, fusarium species strains produced in vitro enzymes, a good indication that they may also do so under natural conditions. schwarz et al. (2002) reported that fusarium can produce various cwdes in vitro and analyzed their regulation, suggesting that the initial infection depends of the secretion of these enzymes. the present study demonstrates that fusarium spp. are able to produce quite a good source of different types of industrially important enzymes. knowledge of the types and amounts of enzymes produced by these species would be useful for the selection of strains best suited for industrial requirements. there are currently no reported studies on xylanase, lipase, amylase, polygalacturonase, filterpase, and carboxy-methyl cellulase enzymes to the authors’ knowledge and thus there is a need for further in-depth studies on these isolated bioactive fusarium spp. strains. acknowledgements the authors thank the director general of aecs and the head of the molecular biology and biotechnology department for their continuous support throughout this work. thanks are also extended to dr. b. al-safadi for critical reading of 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infection of wheat spikes by fusarium graminearum. mol. plantmicrobe interact., 13: 159-169. promputtha i., lumyong s., dhanasekaran v., mckenize e.h.c., hyde k.d., jeewon r., 2007 a phylogenetic evaluation of whether endophytes become saprotrophs at host senescence. microbial. ecol., 53: 579-590. refaz a.d., saba i., shahnawaz m., sangale m.k., ade a.b., rather s.a., oazi p.h., 2013 isolation, purification and characterization of carboxymethyl cellulase (cmcase) from endophytic fusarium oxysporum producing podophyllotoxin. adv. enz. res., 4: 91-96. schwarz p.b., jones b.l., steffenson b.j., 2002 enzymes associated with fusarium infection of barley. j. amer. soc. br. chem., 60: 130-134. scopes r.k., 1993 protein purification, principles and practice. new york: springer-verlag. summerell b.a., leslie j.f., backhouse d., bryden w.l., burgess l.w., 2001 fusarium: paul e. nelson memorial symposium. 1st ed. american phytopathological society press, st. paul minnesota, usa. tajehmiri a., aliabadi m.a., darsanaki r.k., 2014 occurrence of deoxynivalenol in cereals and cereal based products: a short review. sci. j. biol. sci., 3: 1-5. yadav s., shastri n.v., 2005 partial purification and characterization of a pectin lyase produced by penicillium oxalicum in solid-state fermentation (ssf). indian j. biotechnol., 4: 501-505. impaginato 117 adv. hort. sci., 2023 37(1): 117­122 doi: 10.36253/ahsc­14103 sanitization system in horticultural sector l. buglia fruit control equipments srl, via r. luxemburg, 55, 20085 locate di triulzi (mi), italy. key words: fruit preservation, ionization, sanitation, sanitization, storage. abstract: the food industry has recognized the importance of environmental sanitation, and fruit control, a renowned leader in controlled atmosphere, has invested in sanitation through the use of ionizers to eliminate microorganisms in agri­food environments. this report presents results of tests conducted on various products, both in experimental and real scenarios in fruit and vegetable distribution platforms, to evaluate the effectiveness of ionization on vegetable products. the report covers three different situations: the first two focused on the effects of ionization on radish and table grape cells, while the last test veri­ fied the impact of ionization on a distribution platform that processes and mar­ kets various types of fruits and vegetables, such as pepper, tropical fruits, blue­ berry, apples, pears, table grape, chicory, and more. 1. introduction the preservation of fruit and vegetables has always gone hand in hand with the need to ensure the healthiness of the stored products. in fact, it is necessary that the products are stored in the best conditions, from the organoleptic but also from the health point of view (gnanasekharan et al., 1992; karabulut et al., 2004; del­valle et al., 2005). lately this aspect has become increasingly important, especially for greater protection for con­ sumers using physical means such as irradiation (γradiation), ultraviolet­c light, sub­atmospheric pressure, high hydrostatic pressure and ionized gases (gas plasma) (romanazzi et al., 2001; moreau et al., 2008; tappi et al., 2014; papoutsis et al., 2019). ionization consists in the generation of one or more ions due to the removal or addition of electrons from a neutral molecular entity which can be caused by collisions between particles or by absorption of radia­ tion (lin and lin, 2017; baggio et al., 2020; tanaka, 2022) the atoms or molecules that have a number of electrons lower than the atomic number remain positively charged and are called “cations”; those that have a number of electrons greater than the atomic number, remain negatively charged and are called “anions” (forney et al., 2001; fan et al., 2002; lin and lin, 2017; tanaka, 2022). (*) corresponding author: buglia@fruitcontrol.it citation: buglia l., 2023 ­ sanitization system in horticul‐ tural sector. ­ adv. hort. sci., 37(1): 117­122. copyright: © 2023 buglia l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 december 2022 accepted for publication 13 april 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14103 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 117­122 118 many tests and researches have been done in col­ laboration with research institutes and universities on many fruit and vegetables (i.e. mango, melon, let­ tuce, apple, kiwifruit) taking into consideration multi­ ple parameters (acidity, hardness, color, etc.) (perni et al., 2008; tamaki and uyama, 2008; bernardinelli et al., 2012; tappi et al., 2014; ramazzina et al., 2015; tappi et al., 2016, woo et al., 2017). this report is a demonstration of some of the tests done in last years, especially at industrial level, and they take in consideration the results relating to the phytosanitary aspects, the subject of this symposium. the tests were conducted with the equipment of our production called “ionny”. ionny is produced in dif­ ferent versions and sizes depending on the volume of the cells in which it is installed. there are also cabinet versions intended for research institutes. the tests carried out and reported are essentially aimed at verifying the elimination of micro­organisms during the conservation and processing of fruit and vegetables, with the aim of verifying whether ioniza­ tion can actually be considered a valid system for preventing rotting and sanitizing such products. the machines used were sized according to the volumes of the cells and the environments in which they were installed. 2. materials and methods plants material and experimental setup three separate tests were conducted: first test done in cold room containing radish (cicorium intybus) using a ionny 400; second test done in cold room containing table grape (vitis vinifera, cv. italia) with ionny 400; third test done in a cold warehouse (ce.di.or, zelo buon persico, lodi, italy). inside this cold ware­ house three different areas were tested (cold rooms, processing, and shipping rooms) using ionizers mod duct as compared to control (without ionizer). radish test the test was carried out at geofur, gallese verona, italy, in a storage warehouse where radishes were stored in a refrigerated cell with a volume of 400 m3. the ionization process was performed using an ionny 400 and the storage room was maintained at a temperature of ­1°c and a relative humidity of 95%. the radishes were stored in bins measuring 110 cm x 11 cm x 56 cm. the ionny 400 ionizer was capa­ ble of covering a volume of up to 400 m3, making it suitable for the test. table grape test to ionize table grapes cv italia that were stored in a refrigerated cell measuring 400 m3, an ionny 400 (mod. industrial, noacoop ­ noicattaro ba ­ italy) was used. the ionizer effectively covered the entire volume of the cell. the grapes were harvested from the same orchard and stored in wooden boxes weighing 15 kg each. the storage warehouse had a volume of 300 m3 and was maintained at a tempera­ ture of 4°c and a relative humidity of 95%. warehouse platform test the experiment was carried out in a storage ware­ house located in zelo buon persico lo, italy, called ce.di.or. the warehouse stored and processed vari­ ous types of fruits and vegetables under real condi­ tions. to ensure the ionizator machines had the appropriate capacity for each area, one or more units (model duct) were installed in each room without any construction work needed. the machines were fixed in the upper part of the rooms, near the evapo­ rator positioned in the suction area. the products were stored in different types of packaging, such as bins, wooden boxes, and cardboard boxes of varying sizes. the storage temperature ranged between 0­ 10°c. specifically, during the experiment, the tem­ perature was between 1­4°c in the cold storage and 10­15°c in the processing and shipping areas of the logistic structure ce.di.or. the humidity level was between 85­90% in the cold rooms and 75­89% in the processing and shipping areas. additionally, during the experiment, the amount of ozone generated (tested only for this experiment with a manual ana­ lyzer) did not exceed 0.06 ppm inside the room, even after 8 hours of operation. petri dishes containing tryptic soy contact agar + lt + icr were positioned in the storage room and other areas at ground level, specifically in the corner of the cold room store (fig. 1): cold room (which was totally full) for test 1 and 2; cold room, processing area, and shipping room for test 3. these petri dishes were placed at a height of approximately 70 cm. the air sampling procedure and incubation conditions of the plates were carried out as described below. microbial analysis during storage for each of the tests described above, petri dishes (plates) (90 mm diameter) containing tryptic soy con­ tact agar + lt + icr (merck, darmstadt, germany) buglia l. ‐ nanosponges and cppu in postharvest 119 were positioned by hand (with the use of gloves in order to ensure absolute non­contamination by the operator) in the area of interest according to the test performed in each case investigated. air samples were taken before the use and after the operation of the ionizers used in each case. during air sampling, plates remained open for 1 min and then closed again. incubation of the plates took place at 18­22°c for 7 days and the colonies developed in each petri dish were counted, after dividing them into fungi (black­grey), bacteria (yellow) and yeasts (white). for the determination of total yeast and mold counts, plates were incubated at 20­25°c for 5­7 days (for all the test done). results were calculated as colony forming units (cfu) per m3 using the following equa­ tion proposed by omelyansky (1940), n(cfu m­3)=5a x 104 (bt)­1 , where: a is the number of colonies count­ ed per petri dish, b the petri dish area (in cm2), and t the exposure time (in min) and further expressed as cfu m­3 x 103 (viani et al., 2020). ion cluster generator and its principle when a voltage is applied to electrode plate with the air between them, if the applied voltage exceed a certain level, the air layer is ionized to flow the cur­ rent between electrode plates, creating, so­called, an arc discharge. if electrode plates are separated by 1 cm, when at 30.000 volts is applied, a discharge occurs (in the air, if the intensity of electric field is more than 30 kv/cm, a discharge occurs). at this time, ions are created, they are bonded with surrounding water molecules to produce, so­called, ions. there is no other fluid used except air. ventilation is due to evaporator ventilation (ducted model) or internal fan (in case of industrial model). 3. results and discussion the effects of ionny operation in a cold room for radish storage and in a general purpose fruit and veg­ etable distribution platform (industrial test) are pre­ sented in figure 2. not using ionny in a cold room containing radishes, bacterial counts ranged between 7.07 and 20.43 cfu m­3 x 103, while fungi populations reached up to 2.36 cfu m­3 x 103. the use of ionny resulted in a dramatic decrease of both bacteria and fungi, with numbers found at levels of 0.79 cfu m­3 x 103 for both. overall, the reduction of air microorgan­ isms (fungi and bacteria) inside the cold rooms con­ taining radishes with the use of ionny is clearly indi­ cated in figure 2. this microbial reduction might be attributed to the formation of hydroxyl radical (oh٠) during air ionization. this radical can compromise the function of the microbial membranes due to oxida­ tion of the unsaturated fatty acids in the membranes (zhang et al., 2019). in addition, the different struc­ ture of the bacterial and fungal cell wall could also influence the susceptibility to ionization, as it has been reported that chitin the main component of fig. 1 ­ cedior logistic platform, position of petri dishes (violet) and model of ionny used. fig. 2 ­ microorganism inside a cold room during storage of radish without (a) and with (b) ionny use after 48h from switching on of ionizator. tnt: not treated commodity; ion: ionny use; 1­5: positions inside the cold room where the petri dishes were placed. adv. hort. sci., 2023 37(1): 117­122 120 fungal cell wall is a more rigid material compared to peptidoglycan (liang et al., 2012). a diversity among bacteria, fungi and yeasts pop­ ulations was observed in different areas of a cold storage in a distribution platform (industrial test) when the ionny was not used (for 48 h) (fig. 3a). however, the use of ionny for 48 h resulted in signif­ icant decrease of the tested microorganisms. in this case, the total effectiveness of ionny was equal to approx. 70% and the effect of this reduction was found similar to all the investigated microorganisms (fungi, bacteria and yeasts). these findings are not in accordance with a previous study were cold atmo­ spheric plasma treatment (created by dielectric barri­ er discharge­dbd) on ginseng seed surface presented great bactericidal and fungicidal activity, with fungi found to be more susceptible than bacteria (lee et al., 2021). this difference might be attributed to the time/duration of application, the type of ionizer used and the microorganisms exposed; among other fac­ tors (arnold et al., 2004; tappi et al., 2014; zhang et al., 2019). the effectiveness of ionny on the different areas examined (cold room, processing room and shipping room) was more or less similar. moreover, fungi were absent and/or in low populations after the treatment with ionny (except in some cases) (fig. 3b). these findings suggest that the use of ionny can lower the risk of fresh fruit and vegetables contami­ nation in an industrial level. a previous study showed a reduction of airborne and surface salmonella enteritidis artificially (generated aerosol) ranging from 72 up to 98% when exposed to negative air ions (seo et al., 2001). the differences between the microorganisms examined inside the three investi­ gated areas in the distribution warehouse especially prior the use of ionny might be attributed mainly to the different actions that take place there. for instance, more movement and increased air exchange might be observed more frequently in the processing and shipping room compared to the cold room. also, the different temperatures of the areas might also affect the air’s microbial load i.e. cold room temperature: 1­4°c, whereas processing and shipping room temperature: 10­15°c. the use of ionny during cold storage of table grapes for 21 days, showed a decrease in molds pop­ ulations inside the room after 10 and 21 days, com­ pared to non­treated room (fig. 4a). this phe­ nomenon was more evident on the 21st day of stor­ age. when high voltage atmospheric cold plasma was applied against aspergillus flavus spores, inactivation of spore forms was observed as well as degradation of fungal culture and its mycotoxin (deoxynivalenol) (ott et al., 2021). moreover, in the present work yeasts population was found to decrease in a room used for cold storage of table grapes when ionny was applied, 10 and 21 day after application (fig. 4b). the numbers of yeasts were decreased during the 21 days of storage for both treated and un­treated with ionny samples, with greater decrease been seen on the 21st day with the ionny use (1.02 and 1.57 cfu m­3 x 103, respectively). kikuchi et al. (2020) showed that dielectric barrier dischargers presented great antifungal activity against an airborne fungi (penicillium italicum) (up to 2.5 log decrease), whilst spores adhered to the ioniz­ ers were not affected. the antimicrobial effects of ionization on molds and yeasts might be attributed to the ionization of hydroxyl groups, atomic oxygen and nitrogen and the subsequent production of reactive oxygen and nitrogen species (ros and rns) i.e. ozone (o3), nitrogen dioxide (no2), nitrate (no3), dini­ fig. 3 ­ bacteria, fungus and yeasts inside a cold room without (a) and with (b) ionny use for a period of 48 h inside dif­ ferent areas of a commercial distribution warehouse platform. 1­20: positions inside the warehouse where the petri dishes were placed. buglia l. ‐ nanosponges and cppu in postharvest 121 trogen tetroxide (n2o4), and dinitrogen pentoxide(n2o5) (ott et al., 2021). these oxidizing forms have been previously mentioned to interfere and decrease cordyceps pruinosa spore viability, by damaging the fungal cell wall (deformation and dete­ rioration), increasing permeability and cell compo­ nents leakage (kim et al., 2016). in table 1, different ionny sanitization system models are available for different uses are shown. 4. conclusions ionization is a valid alternative sanitizing, physical method for the reduction of microorganisms (bacte­ ria, fungi and yeasts), which can replace and elimi­ nate the use chemical and other sanitizing systems in the food sector. this method does not cause harm to human health; neither does damage metallic sur­ faces. these benefits among others suggest that ion­ ization can be used in a continuously manner without endangering the environment. in fact, if the ion source is properly sized relatively to the application volume, recommendations and limits issued by the usa agency for safety and health at work (osha) 0.06 ppm (50 ppb) for a continuous 8 h/5 days expo­ sure (mac 8 hrs) 0.3 ppm (300 ppb) for a 15 min exposure (mac15min), can be completely respected ensuring human health and protection. ionny reduces ethylene, molds, fungi, virus, yeast and air born bacteria, extending extends the fruit shelf life, preserving value of the produce and lower­ ing costs with economic benefits for the producer(buglia et al., 2013; fadanelli et al., 2017). references arnold l.w., boothe d.h., mitchell b.w., 2004 ­ use of negative air ionization for reducing bacterial pathogens and spores on stainless steel surfaces. ­ j. appl. poult. res., 13(2): 200­206. baggio a., marino m., innocente n., celotto m., maifreni m., 2020 ­ antimicrobial effect of oxidative technologies in food processing: an overview. ­ eur. food res. technol., 246(4): 669­692. berardinelli a., vannini l., ragni l., guerzoni m.e., 2012 ­ impact of atmospheric plasma generated by a dbd device on quality‐related attribute of “abate fetel” pear fruit), 457­467. in: machala z., hansel k., aki­ shev y. (eds.) plasma for bio‐decontamination, medicine and food security. nato series, springer, dordrecht, the netherlands, pp. 496. buglia l., fadanelli l., mattè p., pedrazzini a., 2013 ­ ionny the new innovative storage method without chemical treatments. ­ ca.ma., trani, italy. del­valle v., hernández­muñoz p., guarda a., galotto m.j., 2005 ­ development of a cactus‐ mucilage edible coating (opuntia ficus indica) and its application to extend strawberry (fragaria ananassa) shelf‐life. ­ food chem., 91(4): 751­756. fadanelli l., turrini l., zeni f., mattè p., buglia l., 2017 ­ new scientific knowledge in kiwifruit storage using sanitization systems (ionny®) avoiding posthar‐ table 1 ­ different ionny sanitization system models are avail­ able for different uses fig. 4 ­ populations of molds (a) and yeasts (b) inside a table grapes room without and with ionny use for a period of 21 days. model area coverage fluid used ionny 150 home and office up to 150 m3 air ionny 300 home and office up to 300 m3 air ionny 400 up to 400 m3 air ionny evo 1000 up to 800 m3 air ionny evo 2000 up to 1600 m3 air ionny ducted 1 up to 1200 m3 air ionny ducted 2 up to 2400 m3 air ionny ducted 4 up to 4800 m3 air ionny ducted 6 up to 7200 m3 air ionny ducted 8 up to 9600 m3 air 122 adv. hort. sci., 2023 37(1): 117­122 vest chemical treatments ­ acta horticulturae, 1218: 511­516. fan l., song j., hildebrand p.d., forney c.f., 2002 ­ interaction of ozone and negative air ions to control microorganisms. ­ j. appl. microbiol., 93(1): 144­148. forney c.f., fan l., hildebrand p.d., song j., 2001 ­ do negative air ions reduce decay of fresh fruits and veg‐ etables. ‐ acta horticulturae, 553: 421­424. gnanasekharan v., shewfelt r.l., chinn m.s., 1992 ­ detection of color changes in green vegetables. ­ j. food sci., 57: 149­154. karabulut o.a., gabler f.m., mansour m., smilanick j.l., 2004 ­ postharvest ethanol and hot water treat‐ ments of table grapes to control gray mold . ­ postharvest biol. technol., 34: 169­177. kikuchi t., guionet a., takahashi k., koichi t., ishida s., terazawa t., 2020 ­ elimination effect of airborne fungi using dielectric barrier discharges driven by a pulsed power generator. ­ plasma med., 10(3): 169­ 180. kim j.y., lee i.h., kim d., kim s.h., kwon y.w., han g.h., cho g., choi e.h., lee g.j., 2016 ­ effects of reactive oxygen species on the biological, structural, and optical properties of cordyceps pruinosa spores. ­ rsc adv., 6(36): 30699­30709. lee y., lee y.y., kim y.s., balaraju k., mok y.s., yoo s.j., jeon y., 2021 ­ enhancement of seed germination and microbial disinfection on ginseng by cold plasma treat‐ ment. ­ j. ginseng res., 45(4): 519­526. liang y., wu y., sun k., chen q., shen f., zhang j., yao m., zhu t., fang j., 2012 ­ rapid inactivation of biolog‐ ical species in the air using atmospheric pressure non‐ thermal plasma. ­ environ. sci. technol., 46(6): 3360­ 3368. lin h.f., lin j.m., 2017 ­ generation and determination of negative air ions. ­ j. anal. test, 1(1): 1­6. moreau m., orange n., feuilloley m., 2008 ­ non‐ thermal plasma technologies: new tools for bio‐decon‐ tamination. ­ biotechnol. adv., 26(6): 610­617. omelyansky vl., 1940 ­ manual in microbiology. ­ ussr academy of sciences moscow, leningrad. ott l.c., appleton h.j., shi h., keener k., mellata m., 2021 ­ high voltage atmospheric cold plasma treat‐ ment inactivates aspergillus flavus spores and deoxyni‐ valenol toxin. ­ food microbiol., 95: 103669. papoutsis k., mathioudakis m.m., hasperué j.h., zio­ gas, v., 2019 ­ non‐chemical treatments for preventing the postharvest fungal rotting of citrus caused by penicillium digitatum (green mold) and penicillium italicum (blue mold). ­ trends food sci. technol., 86. 479­491. perni s., liu d.w., shama g., kong m., 2008 ­ cold atmospheric plasma decontamination of the pericarps of fruit. ­ j. food prot., 71: 302­308. ramazzina i., berardinelli a., rizzi f., tappi s., ragni l., sacchetti g., rocculi p., 2015 ­ effect of cold plas‐ ma treatment on physico‐chemical parameters and antioxidant activity of minimally processed kiwifruit. ­ postharvest biol. technol., 107: 55­65. romanazzi g., nigro f., ippolito a., salerno m., 2001 ­ effect of short hypobaric treatments on postharvest rots of sweet cherries, strawberries and table grapes. ­ postharvest biol. technol., 22: 1­6. seo k.h., mitchell b.w., holt p.s., gast r.k., 2001 ­ bactericidal effects of negative air ions on airborne and surface salmonella enteritidis from an artificially gener‐ ated aerosol. ­ j. food prot., 64(1): 113­116. tamaki m., uyama k., 2008 ­ effect of air ion supply on quality of lettuce during storage. ­ environ. control. biol., 46(1): 57­60. tanaka t., 2022 ­ plasma thermal and nonthermal tech‐ nologies, pp. 13­24. ­ in: horikoshi s., g. brodie, k. takaki, and n. serpone (eds.) agritech: innovative agriculture using microwaves and plasmas. thermal and non‐thermal processing. springer, dordrecht, the netherlands, pp. 349. tappi s., berardinelli a., ragni l., dalla rosa m., guarnieri a., rocculi p., 2014 ­ atmospheric gas pla‐ sma treatment of fresh‐cut apples. ­ innov. food sci. emerg. technol., 21: 114­122. tappi s., gozzi g., vannini l., berardinelli a., romani s., ragni l., rocculi p., 2016 ­ cold plasma treatment for freshcut melon stabilization. ­ innov. food sci. emerg. technol., 33: 225­233. viani i., colucci m.e., pergreffi m., rossi d., veronesi l., bizzarro a., capobianco e., affanni p., zoni r., saccani e., albertini r., pasquarella c., 2020 ­ passive air sampling: the use of the index of microbial air contamination. ­ acta biomed., 91(suppl 3): 92­105. woo c.g., kim h.j., kim y.j., han b., 2017 ­ enhanced antimicrobial activity on non‐conducting and conduct‐ ing air filters by using air ions and grapefruit seed extract. ­ aerosol air qual. res., 17(7): 1917­1924. zhang k., perussello c.a., milosavljević v., cullen p.j., sun d.w., tiwari b.k., 2019 ­ diagnostics of plas‐ ma reactive species and induced chemistry of plasma treated foods. ­ crit. rev. food sci. nutr., 59(5): 812­ 825. impaginato 221 adv. hort. sci., 2023 37(2): 221­229 doi: 10.36253/ahsc­13608 impact of exogenous pre and posthar­ vest salicylic acid applications on md2 pineapple quality d.m. cano­reinoso 1, c. wibowo 2 (*) 1 department of agrotechnology, faculty of agriculture, jenderal soedirman university, purwokerto, jl dr. suparno, karangwangkal 53123, indonesia. 2 department of food science and technology, faculty of agriculture, jenderal soedirman university, purwokerto jl dr. suparno, karangwangkal 53123, indonesia. key words: antioxidant, browning, concentration, enzyme, translucency. abstract: salicylic acid (sa) is a natural plant compound that has been proven to enhance the quality of fruits; therefore, its impact on pineapple should be further studied, especially in the most marketable hybrids. this study aimed to evaluate the effect of sa treatments on md2 pineapple quality. the experiment consisted of two parts with applications pre and postharvest following the next treatments, control: no use of sa, and 5, 7 and 9 mm of sa. the total soluble solids, total acidity, ascorbic acid content, respiration rate, together with the severity and incidence of internal browning and flesh translucency were deter­ mined, after 40 days of cold storage. the treatment using 9 mm of sa in pre and postharvest delivered the best results, having the most elevated ascorbic acid (526.75 mg kg­1) and total acidity (0.8%), the lowest severity and incidence outcomes of internal browning and flesh translucency (0% in both cases), with the most reduced respiration rate values during postharvest. in conclusion, sa treatments with concentrations of 9 mm applied in pre and postharvest on md2 pineapple can improve its quality after 40 days of cold storage. 1. introduction pineapple is a fruit characterized by its rich source of sugars, organic acids, fibers, minerals, vitamins, flavonoids, and carotenoids (de ancos et al., 2017). these are essential food properties for healthy human nutri­ tion. nowadays, low acid hybrids like md2 are the most exported by the industry worldwide (hossain, 2016; cano­reinoso et al., 2022 a). this hybrid is known for its bright­gold colour, sweeter taste, high ascorbic acid (asa) content, and uniform size (bin thalip et al., 2015; cano­reinoso et al., 2022 a). nevertheless, md2 is susceptible to physiological disorders like flesh translucency and internal browning, which are major problems that negatively impact its quality (chen and paull, 2017; paull and chen, 2018). (*) corresponding author: condro.wibowo@unsoed.ac.id citation: cano­reinoso d.m., wibowo c., 2023 ­ impact of exogenous pre and postharvest salicylic acid applications on md2 pineapple quality. ­ adv. hort. sci., 37(2): 221­229. copyright: © 2023 cano­reinoso d.m., wibowo c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 august 2022 accepted for publication 9 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13608 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 221­229 222 currently, the use of natural compounds to deal with these disorders has become a trend (lu et al., 2011; goñi et al., 2017). because of the reduced neg­ ative impact these compounds cause to the environ­ ment and human health, multiple studies have been implemented by producers and growers (ponce et al., 2011; goñi et al., 2017). in this context, salicylic acid (sa) has been investigated as a potential treat­ ment to decrease physiological disorders due to its positive impact on fruit metabolism (hayat et al., 2010; goñi et al., 2017). this secondary metabolite has been proved to enhance ion uptake and trans­ port, disease resistance, ripening delay, and control postharvest quality and shelflife of horticultural prod­ ucts (asghari and aghdam, 2010; goñi et al., 2017). for instance, sa has demonstrated outstanding results in reducing the fruit softening rates and the degrading of the sugar and acid content during postharvest (asghari and aghdam, 2010; goñi et al., 2017). also, sa can mitigate the cell wall degrading and polygalacturonase (pg) enzyme activity, phe­ nomena highly associated with translucency and internal browning in pineapple (goñi et al., 2017; paull and chen, 2018; cano­reinoso et al., 2021). previous studies in pineapple fruit demonstrated that postharvest sa treatments (in solutions with concentrations between 5 and 9 mm), reduced the internal browning severity and translucency occur­ rence without affecting the total soluble solids (tss) and total acidity (ta) negatively (lu et al., 2010, 2011; cano­reinoso et al., 2022 b). besides, it was determined that in pineapple sa could cause a posi­ tive effect on the antioxidant content with a reduc­ tion of the peroxidase (pod), polyphenol oxidase (ppo) and phenylalanine ammonia­lyase (pal) enzyme activities, concomitantly with a low respira­ tion rate (lu et al., 2010, 2011; cano­reinoso et al., 2022 b). however, despite the previous information described currently, there are no sufficient studies on pineapple fruit that document the impact of sa, especially complementing postharvest with prehar­ vest applications. for example, concerning the pre­ harvest stage only two experiments have reported the use of sa, obtaining contradictory results, and mostly implementing just low concentrations (around 2 mm), like in lu et al. (2011) and cano­reinoso et al. (2022 b). on top of that, some of these former researches have mainly focused on acid hybrids. therefore, this issue open questions concerning the determination of the exact impact of sa on low acid hybrids like md2, primordially with a complementary administration, including pre and postharvest. as a result, this study aims to evaluate the impact of exogenous pre and postharvest sa applications on md2 pineapple quality. 2. materials and methods preparation of the experiment this research was implemented in pineapple fields in lampung, a place located in south sumatra indonesia, from january to march 2020, employing the md2 pineapple hybrid. in order to fulfil the objective of the research, the experiment was divid­ ed into two parts. the first part regarding preharvest applications of sa, while the second part concerned the postharvest administration of this natural com­ pound. preharvest treatments implementation regarding the first part of the experiment, a com­ pleted randomized block design was used with four replications, having 20 fruits each of the replications. the fruits were harvested between 144­147 days after flowering, when it has been determined that md2 can expose the most optimal quality character­ istics for commercial consumption (bin thalip et al., 2015; ding and syazwani, 2016). besides, for this part of the research, four rows in each block were pre­ pared with a width and length of 0.4 and 3.75 m, respectively. pineapple plants were organized in two lines of ten plants with a separation of 0.25 m. for this stage the treatments implemented were; con­ trol: no use of sa, 5, 7 and 9 mm of sa. furthermore, the administration of sa was done using sprayings with their respective solutions until the fruits were wet to runoff. sa solutions were mixed with 1% (v/v) of ethanol and 0.01% (v/v) of tween 20 (emulsifier) before their application. the sprayings were performed at eight, six, four and two weeks before harvest at night, in the fruit shell and crown. previous experiment of cano­reinoso et al. (2022 b) demonstrated that two applications prior to harvest (six and three weeks) can cause a positive effect on pineapple quality. therefore, this research aimed to increase the frequency of application pre­ harvest in order to enhance the influence on the fruit quality. moreover, the fruit shell and crown were selected as adequate spots to be used based on the findings about mineral mobility in pineapple plants cano‐reinoso and wibowo ‐ salicylic acid application on md2 pineapple 223 described by vásquez­jiménez and bartholomew (2018) and murai et al. (2021). they demonstrated that through these plant structures there was miner­ al assimilation when foliar fertilizations were carried out after flowering. the soil, where the pineapple plant was cultivat­ ed, was fertilized previously with 200 kg ha­1 di­ ammonium phosphate, 1000 kg ha­1 of k2so4 and 200 kg ha­1 kieserite crystal. three months after plating 700 kg ha­1 of urea were administered by sprayings; also, 700 kg ha­1 of (nh₄)₂so₄, 1000 kg ha­1 of k2so4, 170 kg ha­1 of mgso4, 60 kg ha­1 feso4, 60 kg ha­1 znso4, were applied in intervals of 30 days. besides, borax was sprayed in doses of 30 kg ha­1 at flower induction. climatological conditions were determined where the plants were cultivated with a weather sta­ tion (lsi lastem; equipped with a cr6 data logger from campbell scientific; italy). an average of 70.60% of relative humidity (rh), 22.43°c, 10.18 w m­1 of solar radiation, and rainfall of 353.10 mm were detected. the physical and mineral composition of the soil of the first part of the experiment is present­ ed in table 1. postharvest treatments implementation concerning the second part of the experiment, randomly ten fruits per replication were selected at harvest, organized according to their respective treatments and replications inside a cold storage dur­ ing 40 days (8°c and 90% rh), and analyzed in inter­ vals of eight days. the treatments implemented in this stage of the research were; control: no use of sa, 5, 7 and 9 mm of sa. all the treatments, including the control, received fungicide and waxing applica­ tions in postharvest; those materials, following that order, were administrated in dipping applications for ten seconds, just after the dipping on sa. the fungi­ cide product used was prochloraz in doses of 2 cc l­1, while the waxing material employed was sta­fresh 2952 in doses of 74 g l­1. furthermore, the sa concen­ trations were dissolved in a water container of 25 l; besides, in this case sa was also mixed with 1% (v/v) of ethanol and 0.01% (v/v) of tween 20 (emulsifier). the dipping in sa was done for five minutes. this dip­ ping time and the concentrations implemented were selected based on lu et al. (2011) and cano­reinoso et al. (2022 b). they proved that postharvest dipping applications around five minutes should have a mini­ mum concentration of 2 mm to cause a positive impact on pineapple fruit. finally, the summary of the treatments implemented pre and postharvest in this research are presented in table 2. fruit quality evaluation total soluble solids (tss) and total acidity (ta) in the fruit the total tss and ta content was calculated in every fruit selected of every treatment from each replication, employing the method described in shamsudin et al. (2020). tss was measured using a hand­held refractometer (master­53 α; atago; japan), while ta was determined by titration to ph 8.1 with 0.1 m naoh employing phenolphthalein indicator and expressed as a percentage of citric acid. table 1 ­ physical and mineral composition of the soil in the first part of the experiment the n, p, k, ca, mg and na represent the available mineral con­ tent in the soil. properties content texture clay (%) 9.00 loam (%) 36.56 sand (%) 50.42 chemical properties ph (h20) 7.62 c (%) 4.60 n (mg kg­1) 853.00 p (mg kg­1) 5.29 k (mg kg­1) 9.68 ca (mg kg­1) 76.39 mg (mg kg­1) 109.00 na (mg kg­1) 12.84 table 2 ­ summary of the treatments implemented in pre and postharvest stage of the experiment * preharvest salicylic acid (sa) was sprayed at 8, 6, 4 and 2 weeks before harvest until fruits were wet to runoff. postharvest sa was administrated with dipping of 5 minutes, and comple­ mented with fungicide and waxing applications. pre and posthar­ vest sa solutions were mixed with: 1% (v/v) ethanol, and 0.01% (v/v) tween 20 (emulsifier). treatment description a control: no use of sa b 5 mm sa pre/postharvest c 7 mm sa pre/postharvest d 9 mm sa pre/postharvest adv. hort. sci., 2023 37(2): 221­229 224 fruit respiration rate the respiration rate was calculated in a fruit selected from eight until 40 days of cold storage in each replication of every treatment implemented. a similar method previously reported in bhande et al. (2008) and cano­reinoso et al. (2022 b) was imple­ mented. for this case, changes in the co2 concentra­ tion were measured in a sealed glass container hav­ ing 31 cm height x 24 cm wide with 9 l of capacity. the device used for this procedure was a portable az7788a carbon dioxide detector (co2 range: 0­5000 mg kg­1, 10­95 % rh, 0­50°c; az instrument corp; taiwan). moreover, before beginning the procedure, the fruit weight was calculated using a weighing scale, as described in shamsudin et al. (2007). once finished this process, the co2 detector and the fruit were arranged inside the container, avoiding any air introduction or scape. the changes in the co2 con­ centration were measured during one hour in every fruit, and after that, the respiration rate was deter­ mined using the following expression: (1) where, respiration of the fruit (rf) is the respiration rate in ml co2 kg­1·· h­1, fco2 is the co2 gas concentra­ tion in ml l­1, h is the storage time in hours, δt the time difference between two co2 gas measurements, v is the free volume of the container in l and fw is the weight of the fruit in kg. the free volume of the container was obtained as the total volume of the container minus the volume occupied by its content at the moment of the measuring, using a water dis­ placement method as described in bhande et al. (2008). figure 1 shows a picture of the arrangements carried out to implement the respiration rate method of this experiment already described. ascorbic acid (asa), translucency and internal brown‐ ing incidence determination these variables were measured in each of the fruits per replication of every treatment. the asa content was calculated using dye, 2,6­dichlorophe­ nol­indophenol titration method described in ding and syazwani (2016) and ojukwu and nwobi (2017). first, diluted pineapple juice was pipetted into a coni­ cal flask and mixed with glacial acetic acid, titrating the solution until faint permanent pink colour. next, the titrated value was recorded; then, the titration was repeated, boiling and cooling with distilled water for a blank and a standard ascorbic acid solution. the titrations obtained were repeated twice, and the average value was calculated and expressed as mg kg­1 fruit fresh weight. on the other hand, the translucency internal browning incidence was obtained accounting number of fruits affected from the total examined, since har­ vest until 40 days of cold storage. the results are expressed in percentage. statistical analysis the statistical analyses were performed using spss version 22.0 software (spss inc., chicago, il, usa). all data were analysed by an analysis of vari­ ance of one­way (anova). mean significant differ­ ences at p<0.05 were determined by duncan’s multi­ ple range tests. 3. results fruit physicochemical quality the tss, ta and tss/ta ratio did not show signifi­ cant differences after 40 days of cold storage in the implemented treatments. the tss and ta content was on average 15 and 0.70%, respectively; mean­ fig. 1 ­ picture showing the respiration rate method employed. 1) co2 detector used, 2) cable of electricity to activate the detector introduced by the top of the container, 3) fruit and 4) sealed container. cano‐reinoso and wibowo ‐ salicylic acid application on md2 pineapple 225 while, for the tss/ta the mean values ranged around 21 (table 3). for these variables, high sa concentra­ tions were not clearly associated with more elevated mean results. on the other hand, the asa results did not demonstrate significant differences in this experi­ ment after 40 days of cold storage. nevertheless, it is important to notice that the treatment using 9 mm of sa in pre and postharvest obtained the highest mean value (526.75 mg kg­1), which was linked to the more superior content of ta (0.80%) (table 3). moreover, in figure 2 it is possible to observe the trend of asa through the postharvest time of the experiment. asa continually elevated and reduced its level, with the treatment employing 9 mm of sa in pre and postharvest having the most inferior peak change, especially between 32 and 40 days of cold storage. furthermore, in the case of the respiration rate, this variable exposed significant differences after 40 days of cold storage. the control treatment (no use of sa) provided the highest value, while the treat­ ment utilizing 9 mm of sa in pre and postharvest had the most reduced one (8.24 and 6.32 ml co2 kg­1·h­1, respectively). figure 3 shows the respiration rate trend during postharvest. in this figure, it is evi­ denced that the treatments employing a higher sa concentration, like the treatments with 7 and 9 mm of sa in pre and postharvest, had a steadier trend and lower change during cold storage. on the other hand, the control treatment and the one using 5 mm of sa in pre and postharvest suffered a remarkable increase, primordially in 16 days, suggesting a repre­ sentative metabolic change at that moment in the fruit. on top of that, high concentrations of sa were associated with a lower respiration rate and more elevated asa content at harvest. internal browning and flesh translucency in the fruit the internal browning and flesh translucency pro­ vided significant differences in the results obtained. regarding the internal browning, this had its most elevated severity and incidence in the treatments using 5 and 7 mm of sa in pre and postharvest (3.75 and 6.25%, respectively), while the control treatment fig. 2 ­ effect of the treatments implemented on the ascorbic acid (asa) content during 40 days of cold storage. a) con­ trol [no use of salicylic acid (sa)], b) 5 mm sa pre/postharvest, c) 7 mm sa pre/postharvest, and d) 9 mm sa pre/postharvest. values are the mean four repli­ cates, and vertical bars represent ± se. fig. 3 ­ effect of the treatments implemented on the fruit respi­ ration rate during 40 days of cold storage. a) control [no use of salicylic acid (sa)], b) 5 mm sa pre/postharvest, c) 7 mm sa pre/postharvest, and d) 9 mm sa pre/posthar­ vest. values are the mean four replicates, and vertical bars represent ± se. table 3 ­ impact of the treatments implemented on the fruit quality characteristics after 40 days of storage * mean values ± se in each column followed by the same lower­case letters are not statistically different by duncan’s multiple range test, and kruskal­wallis test (for the internal browning and translucency data) (p < 0.05). treatment tss (%) ta (%) tss/ta asa (mg kg­1) respiration rate (ml co2 kg­1 h­1) browning (%) translucency (%) severity incidence severity incidence a 14.80 ± 0.22 a 0.70 ± 0.07 a 21.73 ± 1.85 a 496.25 ± 62.86 a 8.24 ± 0.40 a 0.00 b 0.00 b 1.85 ab 16.67 a b 15.40 ± 0.50 a 0.67 ± 0.07 a 24.10 ± 3.65 a 402.50 ± 71.81 a 8.23 ± 0.92 a 3.75 a 6.25 a 0.64 b 8.33 b c 15.80 ± 0.29 a 0.75 ± 0.05 a 21.43 ± 1.53 a 492.50 ± 65.97 a 6.83 ± 0.65 ab 3.75 a 6.25 a 0.35 b 4.17 b d 15.15 ± 0.31 a 0.80 ± 0.06 a 19.13 ± 1.23 a 526.75 ± 23.47 a 6.32 ± 0.53 b 0.00 b 0.00 b 0.00 b 0.00 c 226 adv. hort. sci., 2023 37(2): 221­229 and the one employing 9 mm of sa in pre and postharvest did not expose representative symptoms after 40 days of cold storage (0% for the severity and incidence in both cases) (table 3). moreover, this lowest browning severity and incidence outcome was related to the most inferior respiration rate and the highest asa content at harvest, primordially in the treatment utilizing 9 mm of sa in pre and posthar­ vest. concerning the flesh translucency, the out­ comes of this experiment also revealed that this pre­ vious treatment is the one causing the lowest severi­ ty and incidence (0% in both cases), while the control treatment was the least efficient in this aspect (1.85 and 16.67%, respectively) (table 3). similar to the internal browning results, the most elevated asa content and lowest respiration rate at harvest in the treatment employing 9 mm of sa in pre and posthar­ vest were linked to the most inferior translucency incidence and severity. 4. discussion and conclusions typically, md2 pineapple displays 12% or higher values for tss during cold storage (paull and chen, 2018; cano­reinoso et al. , 2021). the values obtained in this research were higher than 14%, especially for those using sa applications. tss repre­ sents the sucrose content in pineapple fruit primor­ dially; also, a change in its content commonly is asso­ ciated with modifications in the cell wall invertase (cwi) activity (saradhuldhat and paull, 2007; paull and chen, 2018). the results of this experiment infer that the pre and postharvest treatments employed did not cause a representative impact on the sucrose content and the sugar metabolizing enzymes, essen­ tially a negative impact, to cause a change in the tss level at the end of the cold storage. in the case of the ta, due to its low acid proper­ ties, md2 has evidenced ta values among 0.4­0.7% at harvest, which is much lower than acid hybrids like smooth cayenne (ding and syazwani, 2016; paull and chen, 2018). nevertheless, a small increase in this range has been reported during cold storage of pineapple due to the rise of the citric acid content (mandal et al., 2015; ding and syazwani, 2016). citric acid is considered a source for several metabolizing process in fruits, like antioxidant scavenging and res­ piration; also, it is the main acid influencing the ta measuring in pineapple (paull and chen, 2018; yang et al., 2019). therefore, in concomitance with the fruit decay, this acid could has speed up its accumula­ tion as a reflect of this physiological deterioration, influenced by the need of a scavenger agent to cope with this condition. this fact can explain why despite the lack of significant differences, in mostly all the treatments, ta had higher values than the minimal recommended for md2, after 40 days of cold stor­ age. on top of that, as also the control treatment (no use of sa) had an ideal ta outcome, it could be sug­ gested that the waxing formula used was ideal to decrease the fruit metabolism in order to avoid a high consumption of citric acid as a substratum of several physiological process, generating the respec­ tive ta content. furthermore, a small increase in the tss and ta content has been observed by sa applications in pineapple (lu et al., 2011; mandal et al., 2015). moreover, some studies have demonstrated that pre and postharvest implementations of sa tend to sup­ press the tss and ta degrading, especially during postharvest, like in mango and grape (champa et al., 2014; hong et al., 2014). this information could explain why the tss and ta values were in the opti­ mal content for consumption or slightly superior in almost all the treatments employing sa. besides, md2 has more elevated tss/ta ratios than other hybrids, especially because of its lower acid content, ranging between 20­30 in mostly all cases. (chen et al., 2009; ding and syazwani, 2016). overall, values between this range were observed in the results of this experiment (table 3). on the other hand, regarding asa, this is consid­ ered a powerful plant antioxidant and one of the most representative in pineapple fruit (kongsuwan et al., 2009; noichinda et al., 2017). this information infers that the treatment utilizing 9 mm of sa in pre and postharvest can provide a more elevated antioxi­ dant production, as it impacts the asa level positively and the previous described ta content associated with citric acid. md2 delivers asa values as minimal as 300 mg kg­1 at harvest, much higher than pineap­ ple acid hybrids (lu et al., 2014; paull and chen, 2018). this value tends to increase during posthar­ vest to maintain optimal physiological conditions, essentially when a stress factor affects the fruit (lu et al., 2011; mandal et al., 2015). the variability of the asa exposed in figure 2 could be associated with the activity of the ascorbic peroxidase (apx) and mon­ odehydroascorbate reductase (mdhar) enzymes. for example, apx is an optimal scavenger of h2o2, which is generated with the progress of the fruit cano‐reinoso and wibowo ‐ salicylic acid application on md2 pineapple 227 senescent. however, this enzyme can cause the oxi­ dation of asa into monodehydroascorbate (mdha). therefore, mdha have to be recycled into asa by the glutamic acid pathway employing mdhar as cofac­ tor, in order to maintain an ideal level of asa to cope with impact of the fruit decay. as a result, these two enzymes can be used as an indicator of the asa con­ tent and metabolization (gallie, 2013; akram et al., 2017). based on that, the treatment working with 9 mm of sa in pre and postharvest could be considered the one causing more influence on these enzymes, catalyzing their activities during cold storage. similar patterns in the asa production during postharvest has been reported in pineapple by lu et al. (2010) and cano­reinoso et al., (2022 b), where higher sa concentrations were associated with a more superior asa content. in the case of the respiration rate, values between 5 and 20 ml co2 kg­1·h­1 have been reported for pineapple in previous studies (lu et al., 2011; hu et al., 2012; cano­reinoso et al., 2022 b). the outcomes obtained in this experiment provided results among that recommended range. on the other hand, sa has been associated with an increase in antioxidant scav­ enger enzymes like catalyze (cat) and superoxide dismutase (sod), which, together with the apx, reduced the impact of the fruit decay symptoms dur­ ing postharvest, eliminating singlet of oxygen­derived from reactive oxygen species (ros), essentially the already mentioned h2o2 (goñi et al., 2017; noichinda et al., 2017). on top of that, lu et al. (2011) and cano­reinoso et al. (2022 b) demonstrated that sa solutions with concentrations higher than 5 mm, essentially with postharvest implementations, can generated a low respiration rate, with a similar trend pattern of this experiment. besides, they determined that this low respiration rate was associated with a more superior activity of the cat, sod and apx, especially between 16 and 24 days of cold storage. that period of time was regarded as the moment when more ros were produced in the fruit; there­ fore, the higher activity of these enzymes to cope with the damage caused. according to that, in this experiment the treatments using 7 and 9 mm of sa in pre and postharvest could be suggested as the ones causing a more elevated scavenger activity of cat, sod, apx, generating less ros, decreasing the respiration rate, and promoting an ideal fruit physio­ logical condition. regarding the internal browning, this physiologi­ cal condition in fruits is associated with a high activity of phenylalanine ammonia­lyase (pal) and polyphe­ nol oxidase (ppo) enzymes during postharvest, con­ comitant with an elevated level of ros and low antioxidant content (youryon et al., 2018; paull and chen, 2019). this finding suggests that sa solutions with concentrations of 5 and 7 mm applied pre and postharvest can catalyze pal and ppo activity, increasing the ros content, creating higher browning symptoms in the flesh. in the case of the treatment employing 9 mm of sa in pre and postharvest, its lowest internal browning severity and incidence was linked to the lowest respiration rate and the most elevated antioxidant content. this fact infers that those high concentrations of sa (9 mm), on the con­ trary, can inhibit the pal and ppo activity, reduce the ros level, generating positive effect on the fruit metabolism. on the other hand, these results contradict the outcomes of lu et al. (2011) and mandal et al. (2015), where concentrations of 5 mm or higher applied pre and postharvest caused the lowest inter­ nal browning incidence and severity, as in this experi­ ment the control treatment, produced positive results in this matter. furthermore, despite having a high respiration rate, the control treatment delivered an ideal asa and ta content. this situation exposes that the fruits of this treatment were harvested with optimal physiological characteristics, necessary to delay any internal browning symptoms until the last day of cold storage. besides, some authors have sug­ gested that md2 pineapple, due to its higher antioxi­ dant content, could extend its postharvest shelflife up to 45­50 days, depending on the fruit quality con­ dition collected from the field (paull and chen, 2018, 2019). conversely, translucency is a physiological disor­ der of pineapple fruit characterized by water soaking symptoms, being md2 a susceptible hybrid (chen and paull, 2017; paull and chen, 2018). furthermore, the low calcium content in the fruit and possible low assimilation of ca2+ into the cell wall matrix have been linked to this physiological disorder’s exhibition (paull and chen, 2015, 2018). these facts suggest that sa applications employed in pre and posthar­ vest, essentially at high concentrations like in the treatment working with 9 mm of sa in pre and postharvest, could enhance the calcium ion assimila­ tion and concentration in flesh tissues, reducing the exhibition of translucency. these results agree with adv. hort. sci., 2023 37(2): 221­229 228 the findings of mandal et al. (2015), where imple­ menting sa with concentrations of 5 mm after 15 days of cold storage got to reduce translucency inci­ dence, compared to treatments without sa. on top of that, similar with the internal browning, the lowest translucency severity and incidence was associated with the most inferior respiration rate and highest asa content. as a result, after analyzing all the vari­ ables measured in this experiment, it is possible to suggest that this treatment could be considered as the more appropriate to obtain an ideal pineapple quality after the 40 days of cold storage. finally, the current available information about the sa effect on pineapple exposes that low prehar­ vest concentrations, between 2 and 5 mm, mixed with more elevated ones in postharvest, provide the best results regarding the fruit quality (lu et al., 2010, 2011; cano­reinoso et al. , 2022 b). nevertheless, this experiment got to demonstrated that elevated concentrations of sa employed prehar­ vest could also benefit the fruit. for example, a more superior calcium uptake, and asa level and recycling can be encourage with those high sa concentrations (primordially between 7 and 9 mm). because of that, future experiments in pineapple should be focus on these concentrations, prioritizing the impact on the fruit mineral status and antioxidant content. also, deeper researches concerning the determination of the ideal pre harvest frequency of sa application should be done, as former studies still lack on this matter. these preliminary results demonstrated that four preharvest applications can be more beneficial than two, complementing former experiments. in conclusion, sa treatments applied pre and postharvest affected the md2 pineapple quality. the treatment employing sa solutions with a concentra­ tion of 9 mm pre and postharvest provided the best results, essentially because it had the lowest and reg­ ular postharvest trend and outcome of respiration rate, and the highest value of asa and ta content. also, this treatment obtained the most reduced severity and incidence of internal browning and flesh translucency after 40 days of cold storage. acknowledgements the authors would like to express their sincere thanks to pt great giant pineapple and their research department in lampung indonesia for the technical support during the research period. references akram n.a., shafiq f., ashraf m., 2017 ­ ascorbic acid‐a potential oxidant scavenger and its role in plant devel‐ opment and abiotic stress tolerance. ­ front. plant sci., 8: 1­5. asghari m., aghdam m.s., 2010 ­ impact of salicylic acid on post‐harvest physiology of horticultural crops. ­ trends food sci. technol., 21: 502­509. bhande s.d., ravindra m.r., goswami t.k., 2008 ­ respiration rate of banana fruit under aerobic condi‐ tions at different storage temperatures. ­ j. food eng., 87: 116­123. bin thalip a.a., tong p.s., casey ng., 2015 ­ the md2 “super sweet” pineapple (ananas comosus). ­ utar agric. sci. j., 1: 14­17. cano­reinoso d.m., kharisun k., soesanto l., wibowo c., 2022 a ­ effect of calcium and silicon fertil‐ ization after flowering on pineapple mineral status and flesh translucency. ­ plant physiol. rep., 27: 96­108. cano­reinoso d.m., soesanto l., kharisun wibowo c., 2021 ­ effect of pre‐harvest fruit covers and calcium fertilization on pineapple thermotolerance and flesh translucency. ­ emir. j. food. agric., 33: 834­845. cano­reinoso d.m., soesanto l., kharisun wibowo c., 2022 b ­ effect of pre‐and postharvest treatments with salicylic acid on physicochemical properties of pineapple cv. md2. ­ cmuj. nat. sci., 21: e2022039. champa w.a.h., gill m.i.s., mahajan b.v.c., arora n.k., 2014 ­ preharvest salicylic acid treatments to improve quality and postharvest life of table grapes (vitis vinifera l.) cv. flame seedless. ­ j. food sci. technol., 52: 3607­3616. chen n.j., paull r.e., 2017 ­ production and postharvest handling of low acid hybrid pineapple . ­ acta horticulturae, 1166: 25­34. chen n.j., paull r.e., chen c.c., saradhuldhat p., 2009 ­ pineapple production for quality and postharvest handling. ­ acta horticulturae, 822: 253­260. de ancos b., sánchez­moreno c., gonzález­aguilar g.a., 2017 ­ pineapple composition and nutrition, pp. 221­239. ­ in: lobo m.g., and r.e. paull (eds.) handbook of pineappple technology.production, postharvest sciences, processing and nutrition. john wiley and sons, ltd, chichester, uk, pp. 270. ding p., syazwani s., 2016 ­ physicochemical quality, antioxidant compounds and activity of md‐2 pineapple fruit at five ripening stages. ­ int. food res. j., 23: 549­ 555. gallie d.r., 2013 ­ l‐ascorbic acid: a multifunctional mole‐ cule supporting plant growth and development. ­ scientifica, 2013: 1­25. goñi m.g., quirós­sauceda a.e., velderrain­ rodríguez g.r., ovando­martínez m., roura s.i., gonzález­aguilar g.a., pareek s., 2017 ­ salicylic acid treatments, pp. 119­148. ­ in: pareek s. 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calcium gluconate peduncle infiltrations alleviate the internal browning of queen pineapple in refrigerated storage. ­ hortic. environ. biotechnol., 59: 205­213. impaginato 81 adv. hort. sci., 2020 34(1): 81­91 doi: 10.13128/ahsc­8404 salicylic acid effects on some physio­ chemical properties and secondary metabolite accumulation in mentha piperita l. under water deficit stress g. abdi 1 (*), l. karami 1, 2 1 department of biotechnology, persian gulf research institute, persian gulf university, 7516913817 busheher, iran. 2 department of horticultural sciences, faculty of agriculture and natural resources, university of persian gulf, busheher, iran. key words: mentha piperita l., persian gulf, secondary metabolites, water deficit stress. abstract: salicylic acid (sa) play important roles in different physiological processes of plants such as plant growth, stress response, plant adaptation and secondary metabolite accumulation. this study was conducted to evaluate the effect of exogenous sa applications on the growth measurements such as fresh and dry weight of aerial part and lead dry weight, biochemical properties (mem­ brane permeability, lipid peroxidation, proline content and ros scavenger enzymes) and secondary metabolite accumulation (total phenolic and the flavonoid and essential oil content) in peppermint (mentha piperita l.) plants grown at different levels of water deficit stress (field capacity: fc). for this aim, three different water deficit stress [no stress (100% fc), mild stress (75% fc) and moderate stress (50% fc)] and four different sa concentrations (0, 1, 2 and 2.5 mm) were applied to peppermint plants. results showed that all of the measured parameters were affected by the water deficit stress and sa application. by ele­ vating the level of water deficit stress, fresh and dry weights of aerial parts and dry leaf weight decreased. increasing in the water deficit stress level from mild to moderate stress resulted to reduce the essential oil content while proline, lipid peroxidation, total phenolic contents, flavonoid content, and antioxidant enzyme activities increased depending on water deficit stress. exogenous application of sa obstructed the negative effects of water deficit stress by decreasing the lipid peroxidation and membrane permeability and improving the antioxidant enzymes activities. essential oil content increased significantly in plant treated with sa grown under water deficit stress conditions. application of 2 or 2.5 mm of sa enhanced the plant growth and development without any toxic effects and increased significantly the total phenolic content, dry leaf weight, and the essen­ tial oil content in stressed and even in control (100% fc) peppermint plants. 1. introduction peppermint (mentha piperita l.) is considered as one of the most important medicinal and aromatic herb in the world cultivated in many (*) corresponding author: abdi@pgu.ac.ir citation: abdi g., karami l., 2020 ­ salicylic acid effects on some physiochemical properties and secon‐ dary metabolite accumulation in peppermint (mentha piperita l.) under water deficit stress. ­ adv. hort. sci., 34(1): 81­91. copyright: © 2020 abdi g., karami l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 march 2019 accepted for publication 17 december 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 81­91 82 countries such as india, italy, france, china, hungary and united states among others (lawrence, 2007). during the last two decades peppermint cultivation has experienced a remarkable increase, mainly due to the rediscovery of medicinal properties of pepper­ mint essential oil, which has a lower risk of side effects compared to synthetic drugs (kumar and kumar­gupta, 2008; herro and jacob, 2010). furthermore, m. piperita is used as flavoring agent for beverages (phenolic and flavonoids compounds group, including eriocitrin, hesperidin and luteolin 7­ o­rutinoside) (mckay and blumberg, 2006; hossain et al., 2010) and food industry (essential oil and differ­ ent type of plant exteract), as a fragrance (higher amounts of high­volatile monoterpenes such as men­ thone and isomenthone (rohloff, 1999), as insecti­ cide (karamaouna et al., 2013) and fungicide in many industrial products. essential oil of peppermint con­ tains very important monoterpenes (essential oil of peppermint contain menthofuran, menthol, men­ thone, isomenthone, pulegonecaryophyllene, betacaryophyllene, neomenthol, 1,8­cineole, sabinene and limonene) with antibacterial, antifungal (edris and farrag, 2003), antioxidant and cytotoxic activities (mimica­dukic et al., 2003; hussain et al., 2010). due to markedly importance of peppermint, maintaining a high and constant essential oil produc­ tion for industries requirements and supplying mar­ ket demands (silva, 2002) under unfavorable condi­ tions such as water deficit stress is very important. water deficit stress is the major yield limiting fac­ tor for many agriculture crop plants affecting many biochemical, morphological and physiological para­ meters. many research studies indicated that drought stress influenced the growth, yield, sec­ ondary metabolite production and composition in different aromatic and medicinal plants. furthermore, between the abiotic stresses, drought stress can exacerbates the effect of the other stress­ es in plants and increase the effects of other stresses such as salinity, cold or hot stress (khalid, 2006; azhar et al., 2011; verma and shukla, 2015). in order to reduce the harmful effects and to increase the resistance of plants to the drought stress, some exogenous plant growth regulator applications such as salicylic acid (sa) can be used (lee et al., 2019). salicylic acid, as a plant messenger molecule, plays a non­enzymatic antioxidant role, regulates many physiological and biochemical mechanisms as cell expansion, vascular differentiation, vegetative and reproductive development, seed germination, flow­ ering, fruit set and secondary metabolite accumula­ tions during stress occurrence (arfan et al., 2007; hayat and ahmad, 2007). this compound belongs to the group of the phytochemicals with beneficial effects on human health (hayat and ahmad, 2007). in addition, sa is a phenolic compound that has a cru­ cial role in plant defense against pathogenic agents (dempsey and klessing, 1994; hayat et al., 2010). apart from growth­promoting and health related effects of sa in different plants for instance in artemisia annua l. (aftab et al., 2010), cuminumcy minum (rahimi et al., 2013) sweet basil (ocimum basilicum l.) and marjoram (majorana hortensis) (gharib, 2007) and salvia macrosiphon (rowshan et al., 2010), sa is also reported to confer resistance to plants against various stresses e.g. in matricaria chamomilla (kovácik et al., 2009), and vigna radiate (khan et al., 2014 a). this experiment was conducted to determine the effect of different concentrations of exogenous sa applications on some physiochemical properties and secondary metabolite accumulation in m. piperita plants grown under different level of water deficit stress. 2. materials and methods plant materials, sa and water deficit stress treat‐ ments plants were initiated from rhizome segments of m. piperita obtained from a commercial nursery located in alborz province, karaj. the plants were grown in a greenhouse at the university of persian gulf in pots with a diameter of 10 cm and irrigated every 2 days during the first 30 days. the daily tem­ perature inside the greenhouse was within optimal ranges for peppermint growth (20­25°c). fertilization was carried out at 15 and 25 days after planting. each pot was fertilized with a solution containing ca(no3)2 (1.12 g/l), mgso4 (0.45 g/l), kno3 (0.35 g/l), kh2po4 (0.30 g/l), ferric edta (0.06 g/l), and mnso4 (0.01 g/l). thirty­day old seedlings were transferred to plastic pots with a diameter of 30 cm filled with soil, cocopeat and perlite (1:1:1; v/v) and kept in an incu­ bation room where the temperature was 25°c ± 1; the relative humidity was 60­80% and photoperiod of 16 h/d. the first sa (synonym: 2­hydroxybenzoic acid; linear formula: c7h6o3 (cas 69­72­7), sigma­ aldrich) treatments (0, 1, 2 and 2.5 mm) was applied to 35 days old plants by spraying aerial parts of the plants. the stock solutions were prepared by dissolv­ ing sa in etoh and final volume was maintained by karami and abdi ‐ salicylic acid effects on peppermint 83 distilled water containing 0.02% of tween 20 as sur­ factant. control plants were sprayed with distilled water including 0.02% of tween 20 and etoh (100%) as in sa solutions. the 2th, 3th and 4th sa application were repeated at days 50 and 65 and 80 days after transplanting on peppermint plants exposed in three different irrigation (100, 75 and 50 fc) treatments as fc (100% field capacity ­ fc), mild stress (75% fc), and moderate stress (50% fc). throughout cultiva­ tion period, moisture levels in the growth media were controlled by daily weighting following the pro­ cedure of yadav et al. (2014). briefly, to calculate the amount of water necessary to bring each soil to determined fc, a 50 g soil sample from randomly chosen pots were collected and the water content was determined by drying at 100°c at 24 h after the pots were watered. the percentage of soil water con­ tent was calculated according to yadav et al. (2014) method. nutrient and water leaching from pots was captured in dish placed under each pot and the leachate was returned to the soil before the addition of any water. at the end of the experiment, all plants within each pot were harvested and then analyzed. determination of biochemical and physical properties the growth response of the plants to elicitor treatments and water deficit stress were determined by measuring the fresh and dry weights of aerial parts per plant. lutts et al. (1996) method was used for determin­ ing the membrane permeability of the excised leaves at the end of the experiment by using a conductivity meter. after harvesting (from three plants per treat­ ment), 5 leaves (randomly chosen full developed leaves from upper part of the plant) were cut into 1 cm segments. leaf segments were washed with three changes of deionized water to remove surface adhered electrolytes. samples were placed in 20 ml vials contain deionized water and incubated at 25°c on a rotary shaker (100 rpm). after 24 h incubated at 25°c, the electrical conductivity of the bathing solu­ tion (l1) was determined by using a conductivity meter. then samples were autoclaved at 120°c for 20 min and the electrical conductivity (l2) was obtained. the membrane permeability was obtained using l1/l2. all measurements were made in tripli­ cate. lipid peroxidation in leaves was determined by estimating the malondialdehyde (mda). for malondi­ aldehyde (mda) determination, 1 g leaf sample (full developed leaves were collected from the upper part of each plant) was homogenized in 5 ml 1% trichloro­ acetic acid and centrifuged at 10000 g for 10 min. the amount of mda in the supernatant was estimat­ ed by the thiobarbituric reaction as described by madhava et al. (2000). mda concentration was calcu­ lated from the absorbance at 532 nm by using the extinction coefficient of 155 mm cm­1. all measure­ ments were made in triplicate. proline content of treated plant was determined as described by bates et al. (1973). a 1:1:1 solution of proline, ninhydrin acid and glacial acetic acid was incubated at 100°c for 1 hour. for proline colorimet­ ric determinations, the reaction was arrested in an iced bath and the cromophore was extracted with 4 ml toluene and its absorbance at 520 nm was deter­ mined in a biomate spectrophotometer (thermo­ spectronic). after the analyses, following equation: (g proline in extract/115.5) g­1 sample = mol g­1 fw) was used for calculation the proline concentration from a standard curve. enzyme assays at the end of experiment, leaves were collected and frozen in liquid nitrogen and stored at ­80°c before enzyme extraction. ros scavenger enzymes were extracted as described by zhang and kirkham (1996) with some modifications. all operations were carried out at 4°c. intact leaves were ground using mortar and pestle under liquid nitrogen in cold 50 mm sodium phosphate solution (ph 7.5) containing 250 mm sucrose, 10 mm kcl, 1 mm mgcl2, 1.0 mm edta, 0.5 mm 0.1 mm dithiothreitol, 0.1 mm phenyl­ methylsulfonyl fluoride, and 1% (w/v) polyvinyl­ polypyrrolidone in a 6:1 proportion (w/v). the homogenate was then filtered and centrifuged at 25,000 g for 20 min at 4°c. then solid ammonium sulfate (nh4)2so4 added to the supernatant to make up 80% saturated solution and allowed to stir gently for several hours at 4°c. after centrifugation (28,000 g for 45 min at 4°c), pellets, were resuspended in a small volume of 50 mm of sodium phosphate (ph 7.5) and used for enzyme assays. the superoxide dismutase (sod) activity was assayed by observing the inhibition of photochemical reduction of nitro blue tetrazolium according to krishnan et al. (2002) protocol. one ml of assay mix­ ture consisted of 75 μm nitro blue tetrazolium, 2 μm riboflavin, 50 mm na­p buffer (ph 7.8) 0.1 mm edta, 13 mm methionine, and enzyme extract. the sam­ ples were kept 30 cm under a light source (4000 lux) for 15 min and the reaction started during this time. the reaction was stopped by switching off the light. a non­irradiated reaction mixture, served as a control which was run in parallel. the absorbance was read at 560 nm. adv. hort. sci., 2020 34(1): 81­91 84 the ascorbate peroxidase (apx) activity (ε= 2.8 mm­1 cm­1) was determined from the decrease in a290, due to the h2o2­dependent oxidation of ascor­ bate using procedure of zhang and kirkham (1996). one ml reaction mixture contained 50 mm na­p (ph 7.0), 0.5 mm ascorbic acid, 0.1 mm edta, 0.1 mm h2o2, and enzyme. the catalase (cat) activity (ε h2o2 = 39.4 mm­1 cm­1) were assayed spectrophotometrically according to zhang and kirkham (1996) method by monitoring the change in a240 due to the decreased absorption of h2o2. the reaction mixture contained enzyme extract, 50 mm na­p, ph 7.0, and 15 mm h2o2 (in 1 ml final volume). the reaction was initiated by addi­ tion of h2o2. secondary metabolites determination at the end of the experiment, in all plants within each pot leaves (1 g full developed leaves were col­ lected from the upper part of the each plant) were collected and frozen in liquid n2 immediately and stored at ­80°c before analysis. folin­ciocalteu colourimetric method was used for determining the total phenolic content of the peppermint extract (singleton and rossi, 1965). samples were trans­ ferred into the different test tube and mixed thor­ oughly with folin­ciocalteu reagent (5 ml). after 5 mins, 4 ml of 7.5% sodium carbonate (na2co3) was added and allowed at room temperature to react for 2 hrs. the absorbance was measured using spec­ trophotometer at 765 nm. samples were measured in three replicates. the flavonoid content was deter­ mined according to liu et al. (2002) by aluminium trichloride method using catechin. a volume of 125 μl of extract is added to 75 μl of a 5% nano2 solu­ tion. the mixture was allowed to stand for 6 min, and then 150 μl of aluminium trichloride (10%) was added and incubated for 5 min, followed by the addi­ tion of 750 μl of naoh (1m). the final volume of the solution was adjusted to 2500 μl with distilled water. after 15 min of incubation the mixture turned to pink and the absorbance was measured at 510 nm. total essential oil content hydro distillation method was used.in this method, at least 10 g of dried m. piperita shoot relat­ ed to the treatment immersed in 150 cc water were submitted to hydro­distillation with a clevenger­type apparatus for 3 h (until no more essential oil was obtained). the essential oil was collected, dried with anhydrous sodium sulfate, and stored at 4°c until used. statistical analysis this experiment was performed in factorial arrangement based a completely randomized design with 3 replicates per treatment and one plant per replicates in which pots were placed on the benches in the incubation room. the factors included differ­ ent sa concentration (0, 1, 2 and 2.5 mm) and water deficit stress in 3 levels. data were analyzed by spss 19 software, and differences among treatments were determined using tukey’s test (p<0.05). 3. results and discussion growth measurement foliar application of salicylic acid (sa) and water deficit stress affected peppermint plants in different ways, including physiological and biochemical prop­ erties. some physiological disorders such as leaf yel­ lowing and abscission were observed in plants at sever water deficit irrigation. also, the high concen­ tration of sa caused burning and drying the edge of some mature leaves in plants (data not shown). the limited water supply is one of the major abiotic fac­ tors that adversely affect agricultural crop production worldwide. water deficit stress significantly reduced the growth measurements (fresh and dry weights of aerial parts of plants and leaf dry weight) in pepper­ mint plants at 50% fc (table 1). however, sa treat­ ments were not caused any growth reduction but in plants under water deficit stress, prevented the growth reduction rate. highest growth values were table 1 ­ effect of foliar spray of sa on some growth parameters * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm ) fresh weight of aerial parts (g) dry weight of aerial parts (g) dry weight of leaf (g) control 0 30.74 b 5.47 b 2.61 bc 1 31.31 b 7.15 a 3.45 b 2 42.28 a 8.40 a 4.33 a 2.5 41.24 a 8.34 a 4.08 a fc (75%) 0 20.92 cd 4.56 c 1.93 d 1 22.25 cd 4.75 bc 2.13 cd 2 26.66 cde 4.84 bc 2.83 d 2.5 27.71 c 4.65 c 1.91 d fc (50%) 0 19.25 cde 4.44 c 1.58 d 1 19.92 cde 4.26 c 1.62 d 2 18.32 e 3.95 c 1.92 d 2.5 19.21 de 3.91 c 1.64 d karami and abdi ‐ salicylic acid effects on peppermint 85 more than 2­fold compared to the untreated plants in the presence of moderate water deficit stress. responses of plant to environmental stress such as drought stress in terms of volatile emissions are species specific and depend on the duration and intensity of the stress period (jord´anm et al., 2003). eo yield increased under mild water stress in this study. in medicinal and aromatic plants, the effect of obtained from the plants treated only with 2 mm of sa. khorasaninejad et al. (2011) also observed signifi­ cant reduction in growth parameters of peppermint (m. piperita l.) under water stress. sa treatment in non­stress and even in stress conditions showed pos­ itive effect on the growth measurements in compari­ son to the control (table 1). different concentrations of sa (from 10­5 to 10­3 m) improved growth parame­ ters (plant height, number of branches, nodes, and leaves per plant, as well as leaf area) in basil (ocimum basilicum) and marjoram (origanum majo‐ rana) plants (gharib, 2007) and these authors sug­ gested that such effects could be related to increased photosynthetic activity. the beneficial effects of sa on the growth measurements especially in term of dry weight of mint in this study paralleled with the results of khodary (2004) and hayat and ahmad (2007). this growth enhancement suggests that sa affects various physiological and biochemical processes, including photosynthesis, water process­ es, ion homeostasis, antioxidant capacity generates and a wide array of metabolic responses in plants (hayat and ahmad, 2007). also, sa modulates several physiological responses such as maintain indole‐3‐ acetic acid (iaa, 3‐iaa) and gibberellin levels, inhibit ethylene biosynthesis and prevent auxin oxidation in plants (pierpoint, 1994), which could be one reason for the sa­enhanced vegetative growth of pepper­ mint in our study. essential oils peppermint essential oil (eo) is a rich source of monoterpenes such as menthol and menthone used in pharmaceutical, food, cosmetic and cleaning industries (kamatou et al., 2013) and its commercial value depends on the essential oil contents. in this study, essential oil content was affected with both water stress and sa application (table 2). essential oil content increased from 1.146% (control plants) to 1.192% (mild stress treated plant) and decreased to 0.782% in moderate stress treated plant (table 3). exogenous application of sa alleviated this adverse effect especially in moderate stress condition and also sa application enhanced the eo yield in mild stress condition. in control irrigation conditions, 2 mm sa treatment increased total essential oil con­ tent to 1.256% while application of sa at higher con­ centrations caused a reduction in total essential oil content. under water deficit stress conditions, sa sig­ nificantly increased the total essential oil content when compared to the untreated plants. particularly, 2 and 2.5 mm of sa increased essential oil content table 2 ­ effect of foliar spray of sa on biochemical properties of peppermint plants grown at different level of fc table 3 ­ effect of foliar spray of sa on phenolic and essential oil contents of peppermint plants grown at different level of fc * differences between means indicated by the same letters are not statistically significant (p≤0.05). * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm) membrane permeability (s) lipid peroxidation (nm g­1) proline (mol g­1) control 0 37.11 def 5.81 d 0.58 ab 1 27.43 efg 6.35 d 0.42 bc 2 28.32 efg 5.38 d 0.28 c 2.5 21.43 fg 6.05 d 0.66 ab fc (75%) 0 41.02 cd 14.48 a 0.67 ab 1 53.74 c 12.01 a 0.59 ab 2 50.26 cd 6.09 d 0.4 bc 2.5 25.49 fg 7.11 cd 0.62 ab fc (50%) 0 87.04 a 11.71 ab 0.83 a 1 41.52 cde 11.29 ab 0.79 a 2 74.28 b 8.66 c 0.59 ab 2.5 49.91 cd 7.03 cd 0.76 a irrigation treatment sa (mm) total phenolic content (mg g­1) flavonoid content (mg gae/g) total essential oil content (%) control 0 8.37 e 17.7g 1.146 d 1 16.51 d 22.2e 1.153 d 2 14.28 de 38.7d 1.256 c 2.5 14.32 de 34.81e 1.092 e fc (75%) 0 28.08 ab 37.2d 1.191 d 1 22.71 bc 42.3c 1.201 d 2 18.08 cd 47.4b 1.418 b 2.5 17. 94 cd 48.2b 1.482 b fc (50%) 0 29.39 ab 40.7c 0.782 f 1 31.99 a 55.3a 1.010 d 2 18.28 cd 53.2a 1.597 a 2.5 33.65 a 55.7a 1.575 a 86 adv. hort. sci., 2020 34(1): 81­91 water deficit stress on essential oil yield is variable and depends on plant species. for example, drought stress had a negative effect on the essential oil yield of m. piperita (khorasaninejad et al., 2011). by con­ trast, water stress had a positive effect on salvia officinalis and showed a higher concentration of monoterpenes such as cineole, camphor and α/ß­ thujone (nowak et al., 2010). increasing in essential oil yield under stress condition may be related to induction changes in morphological or physiological traits such as high trichome density and smaller leaf size that prevent excessive water loss and allow them to survive in arid or semi­arid environmental condi­ tions (nobel, 1999). foliar spray with triazole (a tria­ zole refers to any of the heterocyclic compounds with molecular formula c2h3n3, having a five­mem­ bered ring of two carbon atoms and three nitrogen atoms) decreased the leaf area in m. pulegium (hassanpour et al., 2012) and increased trichome density in bougainvillea spectabilis (mansouri and kurup, 2009). the results suggest that the stimulation of essential oil production under drought stress could be due to low allocation of carbon to the growth and high terpene concentrations under stress conditions. plant growth regulators (pgrs) favorably affect the yield and quantity of essential oil in lemon grass, rose grass, peppermint, spearmint, and sage (shukla and farooqi, 1990; khan et al., 2014 b, 2015). among dif­ ferent concentration of sa tested on m. piperita in none stress condition, 2 mm of sa showed the most effective in improving the essential oil content. the positive effect of sa on essential oil content and essential oil yield may be ascribed to the improve­ ment in overall plant growth and metabolism by sa. it seemed that sa, considered to be a signaling mole­ cule, might be involved in the signal transduction sys­ tem, leading to the balance and improved quantity of the secondary metabolites, i.e., essential oil (hayat and ahmad, 2007). thus, sa­mediated enhanced plant growth, photosynthesis, and the overall plant metabolism might account for oil accumulation in the present study. these results corroborate with the findings on artemisia annua, salvia macrosiphon, cumium cyminum, ocimum basilicum, and majorana hortensis (gharib, 2007; aftab et al., 2010; rowshan et al., 2010; rahimi et al., 2013). moderate stress sig­ nificantly decreased essential oil content in current study (table 3). similarly, khorasaninejad et al. (2011) reported significant decreases in total essen­ tial oil content in water stressed peppermint plants. these results suggest that a high level of water stress can suppresses the essential oil biosynthesis pathway in peppermint. reduction in photosynthesis rate and/or any changes in metabolic pathway could be caused some disorders of oil biosynthesis resulted in reduction in oil content (srivastava et al., 1998). in control irriga­ tion condition, foliar application of sa at 2 mm con­ centration resulted to increase in essential oil con­ tent of plants compared to the control plants while higher concentrations of sa decreased the oil con­ tent. another interesting result is that sa foliar appli­ cations increased the essential oil content in water stressed plants. under water deficit stressed condi­ tions, essential oil contents of peppermint plants treated with sa were higher in comparison with the group exposed to water stress alone. the highest oil content was found in plants treated with sa at 2.5 mm concentration grown under moderate water stress. in this condition, oil content was more than 2 fold greater than that of plants under combination of moderate water stress and 0 mm of sa. according to the commercial importance of peppermint, our results represented that peppermint plants can be grown in mild or in moderate stress in the presence of sa applications. membrane permeability, lipid peroxidation and pro‐ line content both water deficit stresses resulted to a significant increase in the levels of membrane permeability, a sign of injury to the cells (table 2). the maximum increase in membrane permeability was obtained in plants subjected to 50% fc and sa treatments reduced significantly this increase was in under water stress. the maximum reduction in membrane perme­ ability was observed in plants treated with 2.5 mm sa. our results indicated that water stress increased the membrane permeability levels of peppermint plants and they were alleviated significantly by foliar applications of sa. water stress significantly increased the lipid per­ oxidation compared to the control irrigation plants (table 2). the maximum lipid peroxidation content was recorded in plants grown under high level of water deficit stress. however, sa treatments, espe­ cially at 2 mm concentration, significantly overcame the toxicity generated by water stress alone and almost leveled the values with those of control. however, sa did not affect the lipid peroxidation level in control irrigation plants. the proline content increased by increasing the water stress but foliar spraying of sa at 2 mm con­ centration upon water stressed plants reduced the http://https://en.wikipedia.org/wiki/molecular_formula karami and abdi ‐ salicylic acid effects on peppermint 87 proline content. ion leakage is known as a index for evaluating the cell wall damage caused from stress. membrane per­ meability increased in line with the elevating levels of water deficit stress in this study. also, there is a close relationship between producing oxidative damage under water stress condition of cell membrane per­ meability. however sa significantly decreased the membrane permeability in plants under water deficit stress (table 4). this result can approve the capability of sa spraying treatment in negative effect of drought stress. water deficit stress resulted to the displacement of membrane proteins, cellular com­ partmentalization disruption, loss of membrane selectivity, integrity and a loss of activity of enzymes (mahajan and tuteja, 2005). malondialdehyde is one of the main known indi­ cator or reliable biomarker for lipid peroxidation, which is part of the oxidative damage at cell level. malondialdehyde increase the cell permeability by deterioration of membrane integrity. our results showed that there is a significant relationship between lipid peroxidation and water deficit stress (table 4). under water deficit stress conditions the amount of lipid peroxidation increased in peppermint plants, moreover, it was determined that mda level increased as a result of deterioration of the cell struc­ ture. karray­bouraouia et al. (2009) and karlıdag et al. (2011) reported a positive correlation between stress condition and lipid peroxidation. water deficit stress caused considerable membrane injuries lead­ ing to membrane lipid peroxidation. foliar spray of sa did not show any significant changes in lipid per­ oxidation content in control irrigation condition while it significantly decreased lipid peroxidation content in water stress­treated plants. the results showed that sa had an important role in membrane integrity and maintenance of membrane structure via preventing damages caused by water deficit stress. low mem­ brane lipid peroxidation in drought­exposed and sa (0.5 mm)­supplemented t. aestivum reported by kang et al., (2012). amino acids play an important role in plant devel­ opment and metabolism. proline, an amino acid, plays main roles in plants exposed to stressful condi­ tions (like water deficit stress, low temperature, salinity, heavy metal exposure and uv radiations, etc) such as osmoprotective, protein compatible hydrotrope, scavenging free radicals and buffering cellular redox potential, energy supply, stabilize the function and structure of protein and dna, alleviat­ ing cytoplasmic acidosis and maintaining appropriate nadp+/nadph ratios compatible with metabolism, metal chelator, antioxidant properties and a signaling molecule (naidu et al., 1991; bassi and sharma, 1993; schat et al., 1997; hare et al., 1998; rhodes et al., 2002; kavi kishor et al., 2005; munns, 2005; sharma and dietz 2006; ashraf and foolad, 2007; chookhampaeng, 2011). so, overproduction of pro­ line is a common physiological response of plants against stressful environment. in this study, proline contents were increased with increasing water deficit stress (table 4). so, more proline accumulation has been associated with increasing the stress tolerance of plant. in general, results showed that drought stress affected the proline biosynthesis in the leaves. the foliar spray of sa on stressed plants cause in reduction of proline content but these declines were not statistically significant in some concentration. it could be due to positive effects of sa in maintaining the membrane maintaining cell turgor or osmotic balance as an osmotic regulator (wang and zeng, 1993) or in stabilizing cell membranes, preventing electrolyte leakage and bringing concentrations of ros within normal ranges. phenolic and flavonoids contents total phenolic and total flavonoid contents were increased under water deficit stress significantly as compared to control irrigation (100% fc) (table 3). minimum phenolic content was measured in control plants while its level increased with increasing the table 4 ­ effect of foliar spray of sa on antioxidant enzyme acti­ vities of peppermint plants grown at different level of fc * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm) sod (unit mg protein­1) cat (mol min­1 mg protein­1) apx (mol min­1 mg protein­1) control 0 25.17 cd 6.44 cde 311.99 c 1 17.47 ef 3.11 e 153.71 d 2 12.31 f 6.63 cde 189.48 d 2.5 21.19 de 4.15 de 279.11 c fc (75%) 0 30.88 bc 15.92b 392.95 b 1 21.17 de 8.52 c 158.21 d 2 23.46 d 7.43 cd 264.01 c 2.5 21.13 de 5.41 cde 314.64 c fc (50%) 0 41.79 a 21.33 a 501.28 a 1 19.94 de 16.21 b 183.23 d 2 31.95 b 9.15 c 331. 53 c 2.5 47.61 a 9.58 c 483.12 a adv. hort. sci., 2020 34(1): 81­91 88 water deficit stress intensity and sa application. foliar application of sa (2 mm) under control irriga­ tion increased total phenolic content less than two times compared to controls. moreover, not only water stress but also sa applications enhanced total phenolic contents compared to controls. the plants under moderate stress were sprayed with a solution of 2.5 mm sa showed the highest phenolic contents. phenolic compounds as stress markers accumulate under stressful environment. it is believed that solu­ ble phenolics compounds act as scavengers of ros and membrane stabilizer during abiotic stress (moyer et al., 2002; taiz and zeiger, 2006). the increase in total phenolic contents in our study is similar to that observed by el­danasouryet al. (2010) in a study with mentha spicata and queslati et al. (2010) with mentha pulegium under salinity stress. also, foliar sa application resulted to increase in total phenolic con­ tents in control plants. foliar sa application at 2 mm was the most effective dose under stress free condi­ tions for peppermint plants. in water stressed plants, foliar spry of sa generally have positive effects on phenolic accumulations. especially at moderate stress and 2 and 2.5 mm concentrations of sa showed the highest amount of phenolics. pepper­mint treated with sa or drought stress showed an increase in total phenolic and flavonoid content in our study. this effect was previously reported in ginger (zingiber officinale) plants, where the total phenolic content increased by approximately 20% after treatment with 10­3 m sa (ghasemzadeh and jaafar, 2012). also, figueroa­pérez et al. (2014) previously reported in peppermint plants, where the flavonoids, increased by approximately 93%, 100% and 56% treated with 0.5, 1.0 and 2.0 mm sa, respectively, when compared with the control. flavonoids were increased in water deficit stress condition and sa treated plants in our study. flavonoids are the main bioactive secondary metabolites in plants and they can serve as scav­ engers of ros by locating and neutralizing radicals before they damage the cells and are thus important for plants under adverse environmental conditions (such as wounding, drought, metal toxicity, and nutri­ ent deprivation) (løvdal et al., 2010; agati et al., 2012). plants often respond to environmental stress­ es with an increase in the endogenous sa level (janda et al., 2014). sa is a promising compound for the reduction of stress sensitivity in the practical agricul­ ture (horváth et al., 2007). ros enzyme activity the activities of sod, cat and apx enzymes were investigated in the leaves of peppermint plants. sod, cat and apx enzymes activities significantly increased in parallel to the water stress in the present study. activity of sod was found at 25.17 unit/mg protein, 30.88 unit mg protein­1 and 41.79 unit mg­1 protein in plants under control, mild and moderate stress, respectively (table 4). in general, foliar application sa on stressed and non­stressed plants alleviated the activity of sod. however, 2.5 mm of sa increased the sod activity in plants under control and moderate stress condition. also, cat activity increased in the plants under water deficit stress (table 4). in control irrigation plants, foliar spray of sa treatments did not show a significant change in cat activity whereas they substantially decreased the cat activity in mild and moderate stress. apx activity almost showed the simi­ lar trend with sod activity and its activity increased with water deficit stress, gradually (table 4). sa appli­ cation at 2.5 mm concentration had no significant effect in terms of the reducing the apx activity in con­ trol and moderate stress treatments. the maximum apx activities were found in under moderate stress without sa application or with 2 mm sa while the highest reductions in apx activity content were recorded in plants treated with 1 mm sa in both mild and moderate water stress treatments. developing enzymatic and non­enzymatic antioxidant defense mechanisms against reactive oxygen species is the main strategy of plants in stressful condition (noctor et al., 2002; gong et al., 2005; cheeseman, 2007). in this study, ros enzyme activities enhanced by increasing the intensity of water stress and apx enzyme activity increased with increasing stress con­ ditions and the highest increases occurred in plants under moderate stress condition (table 4). 4. conclusions according to the obtained results, it may be con­ cluded that prolong water stress especially under moderate water deficit stress had negative effect on peppermint. under water deficit stress, peppermint plants showed a significant decline in growth mea­ surement in term of fresh and dry weights of aerial parts and leaf dry weight while increased antioxidant enzyme activities, the membrane permeability and lipid peroxidation. in addition, water stress enhanced total phenolic and flavonoids contents while signifi­ cantly decreased the total essential oil content in moderate stress condition (mild stress increased the total essential oil content but, it was not significant). karami and abdi ‐ salicylic acid effects on peppermint 89 however, the findings of the present study showed that foliar spray of sa increased grown parameters in non­stress plants while there were no significant effects of sa on water stressed plant. severity of water stress has a great impact on the physiological and biochemical process of plants. sa applications in this study reduced the severity of both water stress condition and decreased the level of grown parame­ ters reduction rate. sa significantly reduced the water stress­induced negative effects by improving the antioxidant enzyme activities and decreasing the lipid peroxidation and membrane permeability. in terms of the secondary metabolites, sa treatment increased the total phenolic contents in control plants while, especially in plants exposed to water stress, total essential oil content increased. sa con­ tributed to the plant growth and development with­ out any toxic effects and 2 mm of sa significantly increased the essential oil content in non­stressed plants. also, it was determined that 2 mm of sa for control irrigation and 2 and 2.5 mm of sa for both water stress condition were the optimum concentra­ tions in terms of dry leaf weight, total phenolic con­ tents and essential oil content in peppermint plants. acknowledgements the author wishes to thank dr. m. maffeifor pro­ viding the facilities and technical support. references aftab t., khan m.m,. idrees a.m., naeem m., moinud­ din m., 2010 ­ salicylic 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peppermint http://refhub.elsevier.com/s0926-6690(16)30206-0/sbref0160 http://refhub.elsevier.com/s0926-6690(16)30206-0/sbref0230 untitled 99 innovative techniques for pre-multiplication of grapevine clones: the ce.pre.ma.vi. experience f. mannini*, l. nervo** * istituto di virologia vegetale, cnr unità di grugliasco, via leonardo da vinci 44, 10095, grugliasco (to), f.mannini@ivv.cnr.it. ** vivaio cooperativo vivalb, fraz. vaccheria, 12051 alba (cn). the ivv-cnr unit of grugliasco (to) has selected and registered in the national grapevine cultivar catalogue more than 100 clones belonging to 25 cultivars of north-west italy. the primary sources of these clones are preserved and pre-multiplied by the grapevine premultiplication centre of piedmont (cepremavi), based in alba (cn) and managed by vivalb on behalf of the region of piemonte. premultiplication supplies the commercial nurseries with propagation material of the “basic” category, i.e. selected and virus-free. in the 2013 campaign cepremavi produced more than 37,000 “basic” grafted rootlings. healthy primary sources, in accordance with official regulations, are kept individually potted inside a screen-house, where each clone is represented by three plants (on their own roots). isolation from the soil and from the outside, thanks to a double insect-proof net, safeguards the original sanitary status of the clones from both virus and phytoplasma. the “initial material” collected from the primary sources is then used to establish mother plant vineyards (mpv) suitable for the production of ‘basic’ material. in recent times, due to the spread of phytoplasma diseases (flavescence dorée and bois noir), cepremavi has undertaken a new approach in the production of “basic” clonal material, introducing complete isolation from the ground (except for roots) and the outside, also for mpv. the aim is to prevent, at the highest level, the propagated material from any possible virus and phytoplasma infection. the new technique consists of planting the mpv inside insect-proof tunnels provided with a double room entrance and with complete plastic mulching of the ground. each plant row is isolated from the others. along the rows a metallic structure is designed to support the net coverage as well as the wire sets to uphold the plant canopy. a plastic pipe runs through the upper part of the tunnels with sprinklers for fungicide and insecticide sprays. vineyard management (i.e. the entry of workers inside the tunnels) is limited to green pruning and to routine sanitary inspections. during winter the nets are removed from the supports and rolled up on the ground near the rows. in order to maintain the optimal sanitary status throughout the pre-multiplication steps, the woody propagation materials collected from the covered mpv are processed by hot water treatment at 50°c for 45 min before grafting. in addition, beginning with the 2013-14 campaign, the traditional open-field nursery will be eliminated. after the callusing period, the grafted cuttings will be potted (biodegradable pot) and maintained for about 20 days in a greenhouse, then transferred under a wide tunnel, ground-mulched and covered by an insect-proof net, where they will complete seasonal growth until fall. thanks to the new technique, cepremavi will be able to produce “basic” propagation material with the maximum degree of protection from virus and phytoplasma infection. adv. hort. sci., 2013 27(3): 99 impaginato 81 adv. hort. sci., 2021 35(1): 81­89 doi: 10.13128/ahsc­8107 genetic variability and relationship among different accessions of froriepia subpinata bail (gijavash) an endan­ gered medicinal plant from iran revealed by issr and irap markers a. jorkesh 1, y. hamidoghli 1, j. olfati 1, h. samizadeh 2, d. bakhshi 1 1 department of horticultural sciences, faculty of agricultural sciences, university of guilan, rasht, iran. 2 department of plant biotechnology, faculty of agricultural sciences, university of guilan, rasht, iran. key words: endemic plant, genetic conservation, threatened species. abstract: the genetic variability of froriepia subpinata ledeb. bail., an endan­ gered iranian endemic species, has been estimated with a total of 52 accessions using 20 markers including issr and irap. the results showed the polymorphic band produced by primers was 82.3%. the best mean values of genetic diversi­ ty parameters observed in issrs markers, being ubc873, ubc811, and ubc873 the best primers tested. the similarity range among accessions was 34.45% to 93.3%. the cluster analysis classified the accessions into five main groups that in totally, accessions with similarity in region generally were clustered in the same group. overall, present study could provide elementary information for formulation of conservation strategies and invaluable elementary genetic infor­ mation for next breeding or designing conservation programs. 1. introduction froriepia subinata ledeb. bail. syn= buplerum subinatum, froriepia nuda is a biennial medicinal and aromatic plant, locally known as gijavash, that belong to the apiaceae family. it is a self­pollinated plant with white flowers and small achene fruits. gijavash leaves are used in diet people and have antimicrobial, antifungal properties and high antiox­ idant activity (salmanian and sadeghi, 2012). this species is the only endemic threatened one of froriepia genus in northern iran. in this genus, somatic chromosome number ranged between 2n= 14 and 2n=16. some of its phonological characteristics like irregular and delay germina­ tions and also excessive and improper harvest have exposed it to mortali­ ty and annihilation (mozaffarian, 2015). nowadays, due to increased human activities and excessive growth in residential area, plants habitats have been destroyed. so, investigation and study on each threatened plant for identifying best way to protect should be considered. although pastures protection ways as well as molecular finger­ (*) corresponding author: a.jorkesh@gmail.com citation: jorkesh a., hamidoghli y., olfati j., samiza­ deh h., bakhshi d., 2021 ­ genetic variability and relationship among different accessions of froriepia subpinata bail (gijavash) an endange‐ red medicinal plant from iran revealed by issr and irap markers. ­ adv. hort. sci., 35(1): 81­89 copyright: © 2021 jorkesh a., hamidoghli y., olfati j., samizadeh h., bakhshi d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 september 2020 accepted for publication 12 february 2020 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8107 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 81­89 82 printing, micropropagation and other biotechnological methods and beneficial techniques (glover and abbott, 1995; sudha et al., 1998) are the most important method for conservation of rare and endangered plants. therefore, genetic diversity of endangered species is the first step in conservations strategies and selection for domesticating process (vicente et al., 2011). moreover, recently an increasing number of stud­ ies for plant conservation biology, especially in rare endemic species have demonstrated the value of genetic data (gaudeul et al., 2000; bellusci et al., 2008; gonzález­pérez et al., 2009). one of the most important features for long­term survival and adaptation to environment conditions of population or species is genetic variation within their taxon (frankham, 2010).having information on genetic diversity of a plant species is very necessary for its conservation (höglund, 2009; frankham, 2010; laikre, 2010) as losses of genetic diversity are likely to have consequences for plant fitness (reed and frankham, 2003; dostálek et al., 2010). preserving rare species endangered especially those which have restricted geographic distributions is main concern of scientist because of their habitat destruction and fragmentation. on the other hand, losing allelic rich­ ness or genetic diversity in fragmented populations due to their genetic drift and inbreeding depression have increased population differentiation (buza et al., 2000; tomimatsu and ohara, 2003). thus, an accurate estimate on the level and distri­ bution of genetic diversity of threatened and endan­ gered species seems necessary for designing conser­ vation programs (smith and wayne, 1996; höglund, 2009). in addition, understanding the chance of species survival in the short­term, formulation of conservation strategy for long­term survival need to population genetic information (cires et al., 2013). meanwhile, knowledge of population genetic struc­ ture can provide important information to under­ stand the evolution of rare and endangered different species. for example, by identifying populations of greatest evolutionary potential as well as populations best suited for source material for ex situ preserva­ tion or reintroduction (furches et al., 2009). according to the fragmentation distribution and the endangered status of this endemic species, there are no population genetic studies and conservation management plans. as an initial step in developing such plan, we have assessed the genetic variability of 52 natural populations of f. subpinata using inter simple sequence repeat (issr) and inter­retrotrans­ poson amplified polymorphism (irap).these popula­ tions were naturally grown in their own habitat. to avoid from possibility pollination between different populations, the distance between the populations was considered and tried to elect population which had more difference between each other due to mor­ phological and environmental characteristics. issr and irap were chosen because of their advantages over other dna polymorphism analysis methods, as they do not require prior sequence knowledge, cloning procedures or characterized probes. it is also generally accepted that they have a comparatively high reproducibility (jones et al., 1997). therefore, both techniques have been suc­ cessfully used in plant population genetic studies, especially for endangered species (li and jin, 2007; gong et al., 2010; noroozisharaf et al., 2015). froriepia subpinata is commonly used in tradition­ al foods in iran for its bioactive compounds and antioxidant potential. however, the destruction of its natural habitats by human activity has put a strain on its survival. therefore, the study of genetic diversity among accessions collected from different areas of iran would be very useful in the biodiversity manage­ ment and conservation plans organization. 2. materials and methods plant material fresh leaves of gijavash (f. subpinata) accessions were gathered from 52 different localities of guilan province, iran. to accurate estimate the genetic vari­ ability, based on local people’s knowledge and distri­ bution of the plant, we elected 52 locations through­ out several cities (fig. 1). fig. 1 ­ geographical map of 52 f. subpinata accessions location collected from guilan province, iran. jorkesh et al. ‐ genetic variation of froriepia 83 all the accessions are listed in table 1 with the location and the altitude of each one. plants were randomly selected from two or three individuals of each site. to reduce the probability duplicate sam­ pling fresh leaves were taken from each individual separated at least 3 m apart. samples were immedi­ ately frozen in liquid nitrogen and kept at ­80°c for genomic dna extraction. in addition, voucher speci­ mens were collected, dried by pressing in absorbent paper, stored at room temperature, and lodged at the herbarium of the university of guilan. dna extraction dna extraction was carried out using the ctab method described by (doyle, 1990) with minor modi­ fications as follow: approximately 50­70 mg leaf material was ground in liquid nitrogen, then 600 µl of hot (65°c) extraction buffer 2x (100 mm tris hcl, ph 8; 20 mm edta; 1.4 m nacl; 2% ctab; 1% pvp) was added. subsequently, an equal volume of cold chloroform/isoamyl alcohol (24:1) was added and mixed by gentile inversion of the tube until a light green single phase emulsion is performed. in the next step, the emulsion centrifuged at 10000 rpm for 10 min. then, the aqueous phase transferred into a clean tube and addition 100 µl of ctab solution (10% ctab, 0.7 m nacl) and the extraction was repeated. this step may takes several times until no precipitate can be detected at phenol/aqueous layer interface. the aqueous phase is removed and the rest mixed with an equal volume of hot (65°c) ctab precipita­ tion buffer (50 mm tris hcl, ph 8.5, 10 mm edta, 1% ctab). the solution mixed gently and incubated every 3­5 min at room temperature for 30 minutes. the resulting ctab/dna complex is immediately plat­ ted by centrifugation at 12000 rpm for 10 min. the resulting pellet resuspended in 650 µl high salt buffer (10 mm tris hcl, ph 8; 1 mm edta; 1 m nacl) and the dna precipitated by addition 1300 µl of cold 100% ethanol. the precipitation gently mixed and incubated every 3­5 min on ice for 30 min. then, it centrifuged at 12000 rpm for 10 min. the pellet was washed three times with 1 ml of cold 70% ethanol, and then dried at room temperature. finally, pellet was resuspended in 100 µl te buffer (10 mm tris hcl, ph 8; 1mm edta). extracted dna was qualified using 1% (w/v) agarose gel electrophoresis. afterwards, the dna concentration and contamina­ tion rate was evaluated by nanodrop spectropho­ tometers (thermo fisher scientific, 5225 verona rd, usa). for pcr reaction, only template of dna was used which had a purity of 2 in a dilution of 15 ng/ml. table 1 ­ different heat treatments used to enhance seed ger­ mination of cycas revoluta accession name gathered site voucher number altitude (m) g1 poushal apf53098 ­14 g2 reshvandeh apf53099 ­11 g3 lashkam apf54000 ­20 g4 loskehkelayeh apf54001 ­18 g5 pasgahfarhad apf54002 ­5 g6 kisom apf54004 2 g7 touchipaybast apf54005 1 g8 seghaleksar apf54007 35 g9 lakan apf54008 62 g10 selkisar apf54011 90 g11 aziz kiyan apf54013 174 g12 hasan kiadeh apf54014 ­22 g13 kiashahr forest apf54015 ­22 g14 kiashahr apf54016 ­24 g15 koshkbijar apf54017 ­25 g16 ghasabmahaleh apf54018 ­17 g17 goharsara apf54019 145 g18 sheykhan bar apf54020 124 g19 toustan apf54022 ­12 g20 salkuyeh apf54024 ­19 g21 taleshmahaleh apf54025 73 g22 langrud1 apf54026 4 g23 langrud2 apf54027 8 g24 chafjir apf54028 ­25 g25 sahnehsara apf54030 ­15 g26 karaj posht apf54031 ­21 g27 chinijan apf54029 ­21 g28 rudsar apf54032 ­20 g29 darehposht apf54033 89 g30 saravan park apf54035 141 g31 tekhsem apf54036 114 g32 saravan apf54037 93 g33 tahergurab apf54038 24 g34 ziabar apf54039 31 g35 shanderman apf54034 49 g36 sheykhneshin apf54040 43 g37 someehsara apf54038 3 g38 fuman1 apf54041 35 g39 sehpiranpayin apf54042 20 g40 dobakhshar apf54044 98 g41 kohnehgurab apf54043 14 g42 fuman2 apf54046 41 g43 shaft apf54045 43 g44 khartum apf54047 71 g45 mozhdeheh apf54048 83 g46 shah khal apf54049 48 g47 dastkhat chamacha apf54050 122 g48 dozdak apf54052 51 g49 ezberem apf54051 62 g50 bidrun apf54053 24 g51 siyah kal1 apf54054 37 g52 siyah kal2 apf54055 42 adv. hort. sci., 2021 35(1): 81­89 84 pcr amplification pcr reactions were done in 1500 µl reaction vol­ umes containing 750 µl of sterile double distilled water, 150 µl of taq polymerase reaction buffer (10×), 1 mm mgcl2, 150 µl of dntps (10 mm), 100 µl of each primer at 5 mm, 0.5 unit of taq dna poly­ merase, and 200 µl of plant dna. the planning of thermal cycling was as follows: initial template denaturation at 94°c for 4 min, 35 cycles of denatu­ ration 94°c for 1 min, annealing at 42­50°c (depend­ ing on primer used) (table 2) for 1 min, extension at 72°c for 90s, and final extension at 72°c for 5 min. the pcr products were loaded on 1.5% (w/v) agarose gel in 1× tae buffer at voltage of 70 for 90 min. the gel’s images were captured using the biometra gel documentation system (whatman biometra, gottingen, germany). the produced frag­ ments size in comparing to size marker was distin­ guished (generuler 1 kb dna ladder, sm0241, fermentase, ontario, canada). data analysis in all, 20 individual issr and irap primers with their combinations were used (table 2). only repro­ ducible and well clear bands in the replications were considered as potential polymorphic markers. it was assumed that each band represented the phenotype at a single biallelic locus, because the issr and irap markers are dominant (williams et al., 1990). amplified fragments were scored for presence (1) or absence (0) of homologous bands. according to pcr banding patterns, a data matrix was created for each reaction. polymorphism information content (pic), effective multiplex ratio (emr) and marker index (mi) were calculated (smith and wayne, 1996). effective number of alleles (ne), nei’s gene diversi­ ty (nei, 1972) and shannon’s information index (shannon and weaver, 1949) were estimated for total accessions using popgene software version 1.31 (yeh, 1999). similarity matrix based on simple matching coefficient was constructed from the issr and irap table 2 ­ polymorphism detected with issr and irap marker in 52 germplasm accession marker type no. primer name annealing temperature (˚c) sequence (5ʹ­3ʹ) no. of bands no. of poly­ morphic bands % of polymorphic bands irap 1 toc­1 48.8 tgttgggaatagtcccaca 9 7 77.77 2 toc­2 45.2 tgttgaatagttccacatt 7 7 100.00 mean 8 7 88.88 issr 3 ubc808 47.4 (ag)8 c 8 6 75.00 4 ubc811 41 (ga)8 c 10 9 90.00 5 ubc812 41.2 (ga)8 a 5 4 80.00 6 ubc813 42.8 (ct)8t 8 7 87.5 7 ubc816 49.2 (ca)8t 9 8 88.88 8 ubc817 48.8 (ca)8a 7 5 71.42 9 ubc824 46.6 (tc)8g 8 8 100.00 10 ubc825 50 (ac)8t 6 6 100.00 11 ubc826 50 (ac)8 c 8 6 75.00 12 ubc873 45.8 (ag)8ctt 7 7 100.00 mean 7.6 6.6 86.78 issr+issr 13 ubc808+ubc817 45.2 (ag)8c+ (ca)8a 6 6 100.00 14 ubc812+ubc813 41 (ga)8a+ (ct)8t 7 5 71.42 15 ubc811+ubc813 42 (ga)8c+ (ct)8t 6 5 83.33 16 ubc816+ubc817 46 (ca)8t+ (ca)8a 7 4 57.14 17 ubc825+ubc826 47 (ac)8t+ (ac)8c 9 5 55.55 mean 7 5 73.48 issr+irap 18 ubc817+toc­1 45.8 (ca)8a+ tgttgggaatagtcc­ 5 4 80.00 19 ubc812+toc­2 42.8 (ga)8 a+ tgttgaatagttcca­ 7 5 71.42 20 ubc813+toc2 44.2 (ct)8t+ tgttgaatagttcca­ 8 7 87.50 mean 6.66 5.33 79.64 total mean 7.3 5.98 82.19 total 147 121 82.31 jorkesh et al. ‐ genetic variation of froriepia 85 data. it was used for the cluster analysis and construc­ tion of dendrogram through unweighted pair­group method using arithmetic average (upgma), per­ formed by ntsys­pc software (rohlf, 2000). in order to evaluate fitness between the dendrogram and simi­ larity matrix, the cophenetic correlation coefficient was calculated. principal coordinate analysis (pcoa) was accomplished using genstat (genstat v12, vsn international ltd, uk) on a similarity matrix. 3. results twenty individuals issr and irap and their combi­ nations (issr+issr; issr+irap) produced 147 distin­ guish­able fragments out of which 121 (82.31%) were polymorphic. the polymorphic rang was from 4 in ubc812 to 9 in ubc811 with an average number of 6.05 polymorphic bands per primer. the products number varied from 5 in ubc812 to 10 in ubc811. the mean of polymorphic band percent were 88.88, 86.78, 73.48 and 79.64 for iraps, issrs, issr+issr and issr+irap, respectively. the toc­2, ubc824, ubc825, ubc873 and ubc808+ubc817 primers had the maximum of polymorphic bands (100%). the minimum of polymorphic bands produced by ubc825+ubc826 primer (55.55%) (table 2). the means of pic value for the amplification prod­ ucts was 0.30 (table 2). ubc816+ubc817 and ubc873 showed the lowest (0.19) and the highest (0.45) pic values, respectively. the means of pic for iraps, issrs, issr+issr and issr+irap were 0.27, 0.32, 0.27 and 0.26, respectively (table 3). on the whole, among the 20 used primers, maxi­ mum of the emr, mi, ne, h and i recorded in ubc811 (8.1), ubc873 (3.16), ubc825 (1.75), ubc825 (0.41) and ubc825 (0.60), respectively .also it must be con­ sidered that the total mean of emr, mi, ne, h and i were 5.11, 1.59, 1.49, 028 and 0.43 respectively (table 3). pic= polymorphism information content; emr= effective multiplex ratio; mi= marker index; ne= effective number of alleles; h= nei’s gene diversity; i= shannon’s information index. table 3 ­ genetic diversity detected with issr and irap markers in 52 germplasm accessions marker type no. primer name pic emr mi ne h i irap 1 toc­1 0.26 5.44 1.46 1.50 0.29 0.43 2 toc­2 0.28 7 1.98 1.57 0.34 0.52 mean 0.27 6.22 1.72 1.53 0.31 0.47 issr 3 ubc808 0.23 4.5 1.06 1.52 0.28 0.41 4 ubc811 0.34 8.1 2.77 1.56 0.32 0.47 5 ubc812 0.36 3.2 1.16 1.54 0.31 0.46 6 ubc813 0.32 6.12 2.00 1.46 0.23 0.38 7 ubc816 0.35 7.11 2.55 1.60 0.34 0.51 8 ubc817 0.26 3.57 0.94 1.53 0.29 0.42 9 ubc824 0.31 8.00 2.54 1.38 0.24 0.40 10 ubc825 0.35 6.00 2.10 1.75 0.41 0.60 11 ubc826 0.27 4.5 1.22 1.40 0.24 0.38 12 ubc873 0.45 7.00 3.16 1.60 0.36 0.55 mean 0.32 5.81 1.95 1.53 0.30 0.45 issr+issr 13 ubc808+ubc817 0.35 6.00 2.11 1.73 0.40 0.58 14 ubc812+ubc813 0.22 3.57 0.78 1.40 0.24 0.36 15 ubc811+ubc813 0.38 4.16 1.58 1.45 0.28 0.44 16 ubc816+ubc817 0.19 2.28 0.44 1.25 0.16 0.26 17 ubc825+ubc826 0.22 2.77 0.61 1.30 0.18 0.28 mean 0.27 3.75 1.10 1.43 0.25 0.38 issr+irap 18 ubc817+toc­1 0.23 3.2 0.76 1.55 0.31 0.45 19 ubc812+toc­2 0.26 3.57 0.93 1.29 0.18 0.29 20 ubc813+toc2 0.30 6.12 1.84 1.48 0.28 0.43 mean 0.26 4.29 1.17 1.43 0.25 0.39 total mean 0.30 5.11 1.59 1.49 0.28 0.43 86 adv. hort. sci., 2021 35(1): 81­89 in addition, there was a significant correlation at p≤0.01 probability level between most of these indices, so that only between emr and ne as well as emr and h were significant at p≤0.05 level of proba­ bility (table 4). principal coordinate analysis (pcoa) was con­ structed based on simple matching coefficient of sim­ ilarity. the results showed that the first twelve princi­ pal coordinates account for 70.29% of total variation. the first and second extracted component accounted for 26.43% and 9.11% of the variation, respectively (fig. 2). to draw cluster analysis for 52 gijavash acces­ sions, the obtaining data from issr and irap analysis were used. figure 3 presents the dendrogram of genetic relationships among the accessions as revealed by the upgma method. the 52 accessions of gijavash classified into 5 main groups. the similari­ ty coefficient range varies from34.45% to 93.27%. the highest similarity was related to g4 and g6 and the lowest similarity observed between g26 and g38 (fig. 3). also, high amount of calculated cophenetic correlation coefficient (r=95.2%) showed that upgma method was useful in the clustering plant accessions. 4. discussion and conclusions using dominant molecular markers for assessing genetic diversity is usually similar and directly compa­ rable (nybom, 2004). so that, these dominant mark­ ers widely have been used for earning genetic infor­ mation in large number of endemic and endangered species from different plant families (jeong et al., 2010; brütting et al. , 2012; cires et al. , 2013; noroozisharaf et al., 2015) and also for formulation and implementation conservation strategies, along with testing genetic relationships between species (gonzález­pérez et al., 2009). we applied 20 issr and irap primers to examine the genetic diversity of 52 accessions from the natural distribution of wild f. subpinata. the results showed that a high genetic diversity has been achieved in this species (h= 0.28, i= 0.43) in comparison with the corresponding genet­ ic coefficients of other endangered species (hamrick and godt, 1996; nybom, 2004; zheng et al., 2008). fig. 3 ­ upgma dendogram of issr and irap analyses on 52 germplasm accessions of froripia subpinata ledeb. bail based on simple matching coefficient. fig. 2 ­ principal coordinate analysis (pcoa). the plot shows the first two principal components (coord. 1 and coord. 2). accessions codes are identified in table 1. table 4 ­ correlation between genetic diversity parameters pic emr mi ne h i pic 1 emr 0.59 ** 1 mi 0.81 ** 0.93 ** 1 ne 0.58 ** 0.49 * 0.56 ** 1 h 0.64 ** 0.52 * 0.61 ** 0.98 ** 1 i 0.70 ** 0.60 ** 0.69 ** 0.96 ** 0.98 ** 1 pic= polymorphism information content; emr= effective multi­ plex ratio; mi= marker index; ne= effective number of alleles; h= nei’s gene diversity; i= shannon’s information index. ** p ≤0.01; * p≤0.05 according to tukey test. jorkesh et al. ‐ genetic variation of froriepia 87 the results of issr and irap markers demonstrat­ ed similar overall trends for genetic diversity. nevertheless, the genetic diversity indices from irap approximately are lower than those from issr due to irap tending to produce somewhat low estimates of within­population variation (nybom, 2004). according to the attributes of f. subpinata acces­ sions (i.e. fragmented, endemic) it could expected that there should be low genetic diversity, but in gen­ eral, it seems that the total genetic diversity based on issr and irap markers is similar to, or slightly higher, than most of those used by different authors in other plants l ike primula heterochroma, bupleurum rotundifolium, changium smyrnioide, cycas guizhouensis. nei’s genetic diversity accounts in other issr and irap studies ranged from 0.10­0.28 (qiu et al., 2004; wu et al., 2004; xiao et al., 2004; shao et al., 2009; jeong et al., 2010; brütting et al., 2012; noroozisharaf et al., 2015). base on this result and high polymorphism rate (82.31%), our research has manifested the potential of issr and irap mark­ ers, reproducible and useful methods for classifying different accessions. principal coordinate (pcoa) showed that acces­ sions were divided into two groups, (i) the first group of accessions who collected from east of guilan province and (ii) the second group belong to west of guilan province origination. many biological factors can influence both the species genetic diversity and its distribution among populations. among these, the geographic distribution has been considered as one of the most important (hamrick and godt, 1990). in contrast, in another study the geographical range had no significant influence on genetic diversi­ ty (nybom, 2004). our finding may be related to self­ pollination character of this plant, that cause, acces­ sions with less distance from each other had more genetic similarity. the result of cluster analysis (fig. 2) also showed that accessions with same region had more similarity to each other, so that maximum of similarity (93.27%) was between g4 and g6, and also the low­ est of similarity (34.45%) was between g26 and g38. the g26 and g38 accessions originated from the east (rudsar city) and west (fuman city) of guilan province, respectively (fig. 1) and it could confirm the relative between genetic similarity and geograph­ ic distance in this research. overall, present study could provide invaluable elementary genetic information for next breeding plan. genetic diversity of different gijavash accessions was analyzed using issr and irap molecular markers for the first time. results revealed that using of issr and irap mark­ ers had high efficiency for differentiating among the various accessions. among all used primers, the high­ est pic value, emr and mi was belonging to ubc873, ubc811 and ubc873, respectively. the maximum of ne, h and i observed in ubc825. with respect to these findings the ubc873, ubc811, ubc873 and specially ubc825 were the most informative primers which could be used to determine the diversity of gijavash accessions. acknowledgements we gratefully acknowledge the guilan science and technology park, rasht, iran and guilan university for funding this study. special thanks to prof. dr. seyed hasan zali for guidance to identify the species and mr. hossein asa’di the head of plant herbarium of agriculture faculty of guilan university. references bellusci f., palermo a.m., pellegrino g., musacchio a., 2008 ­ genetic diversity and spatial structure in the rare, endemic orophyte campanula pseudostenocodon lac.(apennines, italy), as inferred from nuclear and plastid variation. ­ plant biosyst., 142: 24­29. brütting c., meyer s., kühne p., hensen i., wesche k., 2012 ­ spatial genetic structure and low diversity of the rare arable plant bupleurum rotundifolium l. indicate fragmentation in central europe. ­ agric., ecosyst. environ., 161: 70­77. buza l., young a., thrall p., 2000 ­ genetic erosion, inbreeding and reduced 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jokhan, r.d. gopalan, r.d. singh, a.v. prasad school of agriculture, geography, environment, ocean and natural sciences, the university of south pacific, suva, fiji islands. key words: antioxidant properties, climate, fruit production, morinda citrifolia, photosynthesis. abstract: noni (morinda citrifolia) fruit juice is widely used as a strong antioxi­ dant nutritional supplement. with its demand for supplementary products globally, commercial noni farming is now increasing in the pacific islands. information on its growth performance and fruit quality under variable climatic condition is limited. this study aimed to establish the climatic requirements and identify the agro­climatic zone in fiji that provides for increased antioxi­ dant levels in fruits in addition to optimal plant growth and physiological per­ formance. the study investigated plant growth, photosynthetic performance, fruit yield and antioxidant properties of plants that were cultivated under rain fed conditions in the dry, wet and intermediate agro­climatic zones in fiji islands. the physiological performance was significantly influenced by the soil moisture, sunshine hours and soil nutrients. physiological performance includ­ ing fruit yields were the highest in the intermediate zone which was character­ ized by a moderate rainfall and fairly good soil properties while it was lowest in the dry zone. highest fruit antioxidant properties occurred in the dry zone fol­ lowed by wet zone. the study implies that under cultivation, moderate abiotic stress can enhance the antioxidant properties of noni. 1. introduction noni (morinda citrifolia l.) is a tropical evergreen perennial plant with large elliptical leaves and a compound fruit (nelson and elevitch, 2006). noni is native to is native to southeast asia and australia and has a pantropical distribution (nelson, 2003; pandiselvi et al., 2019) the plants are significant source of traditional polynesian medicine. the fruits range from 4­10 cm in length and about 3­4 cm in diameter and are the most commonly used parts. the fruit has a broad range of nutraceutic and therapeutic potentials (chan­blanco et al., 2006; mahantesh et al., 2018; almeida et al., 2019; inada et al., 2020). noni fruit products have become quite popular in the area of health care due to its biologically active com­ (*) corresponding author: reema.prak@gmail.com citation: prakash r., jokhan a.d., gopalan r.d., singh r.d., prasad a.v., 2024 ­ physiological perfor‐ mance and fruit quality of noni (morinda citrifolia l.) cultivated in different agro‐climatic zones of fij. ­ adv. hort. sci., 38(2): 177­187. copyright: © 2024 prakash r., jokhan a.d., gopalan r.d., singh r.d., prasad a.v. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2023 accepted for publication 5 february 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14019 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 177­187 178 pounds and high antioxidant potential. commercial noni farming occurs in most tropical countries and is now growing in the pacific islands. growing noni under rain fed conditions are highly beneficial in tropical countries where rainfall is abun­ dant with good volcanic soil (nelson and elevitch, 2006). most countries, however, have their own agro­climatic zones where certain crops perform well. agro­climatic zone is the characterization of an area based on its climatic parameters that are suit­ able for agriculture (parry et al., 1988). farming in agro­climatic zones ensures that a crop is ecologically viable together with being economically profitable. amin et al. (2004), lists some agro­climatic parame­ ters as rainfall, maximum and minimum tempera­ ture, humidity, evapotranspiration, maximum possi­ ble sunshine hours and wind speed. crop growth and agricultural productivity are primarily affected by all climatic elements, whether its effects occur singly or in combination (parry et al., 1988; mittler, 2006). precipitation, temperature and solar radiation have a direct effect on key plant metabolic processes such as photosynthesis and respiration while humidity is crucial for regulating transpiration and plant water balance. sub­optimal values of precipitation, temper­ ature, light intensity and relative humidity can result in crop yield reduction and product quality (ferrante and mariani, 2018). noni is renowned for tolerating a wide range of climatic conditions in its habitats. according to nelson (2003), noni is found from 1 m to 800 m above sea level growing in a wide range of soils (infertile soils, acidic and alkaline soils) in its natural habitats. the mean annual rainfall range is from 250­ 4000 mm, and mean annual temperature range is from 20oc to 35oc. noni plants can survive dry sea­ sons with less than 40 mm of rainfall for at least 3 ­ 4 months depending on plant size and age and its sur­ rounding temperature and humidity (nelson, 2003). according to nelson and elevitch (2006), in cultiva­ tion, about 500­1500 mm annual rainfall that evenly spreads over the year is ideal for obtaining high yields. in wet areas where the annual rainfall is up to 4000 mm/year, the yield of noni is high, but the fruits are usually very watery, less sweet and tend to have much slower and uneven ripening while in drier areas fruits are much sweeter with rapid and even ripening (nelson and elevitch, 2006). under cultivation, noni has been found to be a very low­maintenance plant which requires low irrigation and fertilizer. since noni’s commercial market firmly bases its adverts on high antioxidant properties, noni growers must ensure that good plant growth and high fruit yield also accompanies fruits with high antioxidant properties. antioxidants can be enhanced in fruits by changing some cultivation practices once the specific environmental effects on fruits are known (dumas et al., 2003; wang, 2006). major antioxidants, both enzymatic and non­enzymatic protect higher plant cells from oxidative stress damage that usually occurs when the plants undergo environmental stresses. antioxidant properties of noni are highly associated with the non­enzymatic phenolic compounds (dussossoy et al., 2011). the non­enzymatic antioxi­ dants in plants are mainly comprised of ascorbic acid, glutathione, α­tocopherol, carotenoids, phenolics, flavonoids, and amino acid cum osmolyte proline. due to their essential role in protection and develop­ ment, high levels of non­enzymatic antioxidants are usually expected in plants undergoing adverse envi­ ronmental stress hence noni’s antioxidants may be elevated under stressful conditions. growers often make the cultivation environments ideal and stress­free for plants which are likely to lower the antioxidant capacity of the fruits. hence apart from increasing the plant growth and fruit yield, it is also important to know what kind of envi­ ronmental conditions would enhance the antioxidant levels in the noni fruits. this study examined the growth and physiological performance of noni plants cultivated in fiji’s three different agro­climatic zones and compared its growth, fruit production and yield together with its antioxidant properties. the study implicates how noni’s cultivation environment can be altered to enhance the antioxidant production together with maintaining the overall productivity. 2. materials and methods plant growth and physiological performance of noni cultivated in dry, intermediate and wet climate zones of viti levu, fiji were studied for two years from january 2016 to january 2018. experimental sites the main centers of dry (nadi ­ 17°45’15”s, 177°28’3”e), wet (suva ­ 18°14’80” s, 178°44’76’’e) and intermediate (sigatoka ­ 18°06’05”s, 177°32’13”e) agro­climatic zones on the island of viti levu, fiji were chosen as the experimental sites (fig. 1). prakash et al. ‐ effect of climate on physiological performance and fruit quality of noni 179 data on rainfall and temperature for 2016 and 2017 was obtained from the fiji meteorological service office for the three sites (table 1). soil sam­ ples were collected randomly three times during the experiment duration, and the nutrient content was analyzed for the three cultivation sites. cultivation of noni four­month old noni plants were established in ground plots located in the dry, intermediate and wet agro­climatic zones of the island of viti levu, fiji (fig. 1). a total of 20 plants were transplanted with a spac­ ing of 2 m. the transplanted plants were watered for the first month on a 3­day interval and on a weekly interval for the second and third month. starting from the third month of establishment, the plants were left to be rain­fed. balanced nitrogen, phospho­ fig. 1 ­ average annual rainfall distribution in viti levu and the locations of the study sites in dry zone (nadi), intermedi­ ate zone (sigatoka) and wet zone (suva) (adapted and modified from printemps, 2008). table 1 ­ weather and soil attributes of the experimental sites for the two­year growth period (2016 and 2017) mean ± se are shown, n=24 (weather attributes) and n= 6 (soil attributes). according to kruskal­wallis test at p<0.05, there was a signifi­ cant difference in mean rainfall per month (p= 0.0332, h= 6.810), rainfall per wet month (p=0.0664, h= 5.423), rainfall per dry month (p=0.0030, h= 11.63), maximum temperature (p=0.0032, h=11.50), minimum temperature (p= <0.000, h= 20.82), sunshine hours per month (n=24, p=<0.0001, h=23.63). there is no significant difference in monthly temperature (p =0.4951, h= 1.406). soil attributes were also significantly different using kruskal­wallis test at p<0.05, soil ph (p<0.0001, h=15.51), electrical conductivity (p=<0.0001, h=16.11), total nitrogen (p=0.0001, h=12.48), phosphorus (p=<0.0001, h=15.3), potassium (p=0.0004, h= 11.69), calcium (p=<0.0001, h= 15.25) and magnesium (p<0.0001, h=15.25). means not sharing the same letter are significantly different using the mann­whitney test at p<0.05. note: ideal ranges for fiji soil mineral nutrient content include 0.3­0.6% nitrogen, 20­30 mg/kg phosphorous, 117­234 mg/kg potassium, 400­2000 mg/kg calcium, 122­366 mg/kg magnesium. source: koronivia research station, fiji. data nadi (dry zone) sigatoka (intermediate zone) suva (wet zone) total rainfall (mm) 2237.3 ± 92.9 1645.5 ± 107.9 3102 ± 299.1 total rainfall wet months (mm) 1936.2 ± 184.1 1091.7 ± 34.8 2119 ± 128.3 rainfall dry months (mm) 301.3 ± 90.8 553.5 ± 72.8 982.8 ± 427.4 mean monthly rainfall (mm) 186.4 ± 41.6 ab 139.3 ± 24.4 b 258.6 ± 37.5 a mean rainfall wet months (mm) 322.7 ± 59.5 ab 201.4 ± 38.76 b 353.3 ± 56.55 a mean rainfall dry months (mm) 50.2 ± 18.4 a 77.2 ± 16.8 a 163.8 ± 32.3 b mean temperature (oc) 26.3 ± 1.4 a 25.8 ± 1.7 a 26.4 ± 1.6 a mean max temperature (oc) 30.5 ± 1.2 a 30.7 ± 1.7 b 29.2 ± 1.8 c mean min temperature (oc) 22.0 ± 1.7 a 20.9 ± 1.9 b 23.5 ± 1.4 c mean sunshine hours 7.0 ± 0.4 a 5.9 ± 0.4 b 4.9 ± 0.7 c mean soil ph 5.7 ± 0.1 a 6.8 ± 0.1 b 7.2 ± 0.1 c mean electrical conductivity (ms cm­1) 0.01 ± 0 a 0.09 ± 0.01 b 0.27 ± 0.06 c mean total nitrogen (n) (%) 0.13 ± 0.03 a 0.19 ± 0.01 b 0.20 ± 0.02 c mean available phosphorous (p) (mg/kg) 3.7 ± 0.6 a 36.3 ± 0.6 b 10.6 ± 2.9 c mean potassium (k) (mg/kg) 66.5 ± 39.1 a 518.7 ± 19.3 b 520.0 ± 92.2 c mean calcium (ca) (mg/kg) 221.3 ± 23.4 a 5607.3 ± 89.5 b 7568.7 ± 401.1 c mean magnesium (mg) (mg/kg) 35.4 ± 4.4 a 872.7 ± 28.9 b 619.8 ± 7.4 c adv. hort. sci., 2024 38(2): 177­187 180 rus and potassium (n.p.k) fertilizer was applied in equal amounts (2 g) to each of the plants at the end of the 2­month period. after that, n.p.k fertilizer in a 13:13:21 ratio was applied in equal amounts (2 g) at three months’ interval up to 8 months (the end of 5 months and 8 months). fertilizer treatment was then stopped, and the plants were left to grow in its culti­ vated environment. determination of plant survival and growth rate plant survival rates were calculated by counting the number of noni plants that had survived until the end of the two­year period divided by the number of plants that initially planted (20 plants). the growth rates of plants at the three sites were determined using the plant height. initial plant height was mea­ sured on the day of transplanting, and the final height was measured at the end of the experiment. the plant growth rate in cm per day was calculated by dividing the change in height by the number of days. gas exchange measurements photosynthesis rate (an), transpiration (e) and stomatal conductance (gs) were measured using the portable photosynthesis meter (lcpro­sd by adc bioscientific) connected to a broad leaf chamber. at the cultivation plot, mature leaf (fifth fully expanded leaf) from each plant was selected for gas exchange and transpiration measurements. over the two­year period, gas exchange measurements were done 17 times at random intervals starting from the 5th month of growth at the dry, intermediate and wet sites. the instantaneous leaf water use efficiency (wuei) was calculated as the an/e ratio. determination of changes in fruiting, fruit yield and total soluble solids the plants started flowering and fruiting by eighth month of growth. once all plants at the three sites had fully developed fruits, number of fruits (both young and mature) on each plant were counted ran­ domly on eight occasions. any mature fruit (hard and whitish in colour) present at the time of observation was collected weighed using a top pan balance. the weight of the fruit was recorded as yield in grams. the total soluble solids (tss) in obrix was measured using the hand­held refractometer (atago). a total of 10 fruits were randomly collected and sampled. determination of antioxidant capacity of fruits the total antioxidant activity of fruits from differ­ ent locations was estimated by the 2, 2­diphenyl­1­ picrylhydrazyl (dpph) method, as described by yang et al. (2011). a total of 10 fruits from each site were used to find out antioxidant capacity where 1 fruit was used as an individual sample. a solution of dpph was made in methanol using 0.025 g dpph in 1 l of methanol. noni fruit was ground, and its juice was extracted using a muslin cloth. diluted noni fruit extracts (2 µl, 5 µl, 10 µl, 20 µl, 30 µl and 40 µl) were added to the 3 ml dpph solution and incubated at room temperature for about 40 minutes. after 40 minutes, the absorbance of the mixture was mea­ sured at 515 nm with a spectrophotometer (ce1021 uv­vis). inhibitions of dpph radicals in percentage were calculated as follows: % inhibition = [(acontrol ­ asample)/acontrol] x 100 where: acontrol is the absorbance value of the control reaction (containing all reagents except for the test­ ed fruit extracts) and asample is the absorbance value of fruit extracts. the 50% radical scavenging activity was deter­ mined by calculating the half­maximal inhibitory con­ centration (ic50). the ic50 value was calculated by plotting the percentage inhibition against the con­ centrations of fruit extracts. the concentration that provided 50% inhibition was noted as the ic50 value. the noni fruit extract concentration at ic50 was expressed as ascorbic acid equivalent antioxidant capacity (aeac) in mg/ 100 g fresh weight (fw) of fruits. to find out the aeac, a standard curve was prepared using ascorbic acid at 1mg/ml concentra­ tion at various concentrations of 2 µg/ml, 3 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml. the antioxidant activity of noni fruits was expressed as aeac per 100 g of fresh weight (aeac/100 g fw): aeac = ic50 (ascorbic acid)/ ic50 (sample) × 105 (velde et al., 2013) determination of total phenol content in fruits total phenol content (tpc) of noni fruits were determined with folin­ciocalteu reagent as described by yang et al. (2011). from each site, 10 ripe fruits were collected, ground, and the juice was extracted using a muslin cloth. for each individual sample, 1 fruit was used. exactly 20 µl of noni fruit extract was mixed with 1.58 ml of distilled water. to this mix­ ture, 100 µl of folin­ciocalteu reagent and 300 µl of 20% na2co3 was added. the mixture was incubated prakash et al. ‐ effect of climate on physiological performance and fruit quality of noni 181 at 40oc for 30 minutes. after 30 minutes, the absorbance was measured at 765 nm with the spec­ trophotometer. a standard curve of total phenols was prepared using gallic acid at various concentra­ tions (1 mg/ml, 2 mg/ml, 4 mg/ml, 6 mg/ml, 7 mg/ml, 10 mg/ml and 20 mg/ml). the equation of the standard curve was used to determine the tpc and it was expressed as mg of gallic acid equivalent (gae) per 100 g of fresh weight (mg gae/100 g of fw). statistical analysis analysis of data collected was performed by using graph pad prism version 7.0 (graphpad software inc, san diego, california, usa). shapiro­wilk test was used to determine the normality of the data. the assumption of homogeneity of variances was tested using bartlett’s test and brown­forsythe test at 95% significance level. one­way anova and kruskal­ wallis test at 95% significance level were used to test the significant differences among the results obtained for the three sites. significant differences among the treatments were compared using tukey’s test and mann­whitney test at p<0.05. 3. results weather and soil attributes of dry, intermediate and wet cultivation sites the total amount of rainfall for the two­year peri­ od was highest for suva (wet zone) and lowest for sigatoka (intermediate zone) (table 1). a distinct dif­ ference in rainfall was observed during the dry months (may to october). highest rainfall level was recorded for suva area and the lowest level was for nadi area while sigatoka area received rainfall in moderate amounts (table 1). the average tempera­ ture and humidity were comparable for the three sites during the two­year study period. considerable differences were seen in the average sunshine hours for the three sites. lowest sunshine hours were recorded for the wet zone, followed by the interme­ diate zone while highest sunshine hours occurred in the dry zone. considerable differences were also observed for the soil quality among the three sites (table 1). soil moisture was highest in the wet site, while it was lowest for the dry site ranging. the wet site had the highest average soil ph and highest ec while the dry site had the lowest. major mineral nutrition content levels were also different for the three sites. the dry zone site had considerably low nutrient content, average soil total n, available p and k were lowest. for the intermediate site, nutrient levels were adequate. for the wet zone site, n and k levels were adequate. however, available p, howev­ er, was quite low. plant growth survival rate was high in the wet zone and the intermediate zone while it was lowest in the dry zone (table 2). decrease in survival rate was due to death of plants that occurred by the end of 9 months. plant growth rate was highest in the intermediate zone and lowest in the dry zone (table 2). gas exchange attributes net photosynthesis (an), transpiration (e), instan­ taneous water use efficiency (wuei) and stomatal conductance (gs) (fig. 2) were significantly different among the cultivation zone. all three physiological parameters were significantly higher in plants grown in intermediate and wet zones compared to the plants that grew in the dry zone. dry zone plants had the lowest mean an (fig. 2a) while plants in the inter­ mediate zone had the highest an. wet zone plants had comparable mean an rate to intermediate zone. similar patterns were also observed for e, gs and wuei. mean e recorded was highest for plants grow­ ing in the wet zone (fig. 2b). plants in the dry area had the lowest mean e. for plants growing in the intermediate e was comparable to the wet zone. mean gs of plants was also significantly different among the three zones (fig. 2d). lowest mean gs was recorded for the plants in the dry zone while plants in the intermediate and wet zone plants had compara­ ble mean gs. wuei was also lowest for plants in the table 2 ­ the survivability and growth attributes in noni plants in the dry, intermediate and wet cultivation zone mean ± se are shown, n=9 (dry zone), n=15 (intermediate and wet zones). mean growth rate is significantly different (kruskal­ wallis test (p<0.0001). for the given cultivation zones, means not sharing the same letter are significantly different using the mann­whitney test. cultivation zones studied survival rate (%) growth rate (cm/day) dry 45% 0.09 ± 0.009 a intermediate 85% 0.23 ± 0.006 b wet 80% 0.15 ± 0.011 c 182 adv. hort. sci., 2024 38(2): 177­187 dry zone (fig. 2c). plants in the intermediate zone had the highest wuei. fruit attributes fruit numbers were comparable in the intermedi­ ate and wet zone while plants in the dry zone had significantly lower number of fruits (table 3). fruit weights were also comparable in wet and intermedi­ ate zone while it was lowest in the dry zone (table 1). fruit tss was significantly higher in the dry zone when compared to intermediate and wet zone (table 3). there was no significant difference in the fruit tss between intermediate and wet zones. fruit antioxidant properties antioxidant activity (aeac) in fruits was signifi­ cantly higher in dry and wet zones compared to the intermediate zone (table 4). aeac was comparable for dry and wet zones aeac while it lowest in the intermediate zone. there was with no significant dif­ ference in the mean ic50 value between the wet and the dry zone as well (table 4). tpc in fruits was signif­ icantly different at the three sites (table 4). highest mean tpc was obtained for fruits in dry location fol­ lowed by fruits in the wet zone. tpc was lowest for fruits from the intermediate zone. 4. discussion and conclusions when taking into consideration the climate (rain­ fall, temperature, humidity and sunshine hours) and the soil features over the two­year experimental period for the three different sites (table 1), differ­ table 3 ­ mean number, weight and total soluble solids (tss) of fruits from noni plants in dry, intermediate and wet cultivation zones mean ± se is shown, n=9 (dry zone), n=15 (intermediate and dry zones). there is significant difference in fruit numbers per plant anova (f df (2, 33) = 0.6480, (p<0.0001), fruit weight anova (f df (2, 42) = 11.75, (p<0.0001) and fruit sweetness anova (f df (2, 36) = 0.7563, (p<0.0001). for given cultivation zones, means not sharing the same letters are significantly different using tukey's test. fig. 2 ­ net photosynthesis rate (a), transpiration rate (b), water use efficiency (c) and stomatal conductance (d) of noni plants growing in dry, intermediate and wet cultivation zones. mean ± se is shown, n=17. there is a significant difference in an anova (f df (2, 48) = 53.95) p<0.0001, e anova (f df (2, 48) = 14.77) p<0.0001, wuei anova (f df (2, 45) = 5.05) p<0.0104 and gs anova (f df (2, 48) = 5.807) p<0.0001. for given cultivation zones, means not sharing the same letters are significantly different using tukey’s test. cultivation zones studied fruit num­ bers per plant fruit weight (g) tss °brix dry 3 ± 0.25 a 74.7 ± 3.8 a 8.4 ± 0.1 a intermediate 8 ± 0.28 b 299.8 ± 41.5 7.9 ± 0.1 b wet 7 ± 0.30 b 271 ± 19.7 b 7.7 ± 0.1 b prakash et al. ‐ effect of climate on physiological performance and fruit quality of noni 183 ences in plant growth rates and the physiological per­ formance is clearly due to the differences in water and nutrient availability. water availability, together with most required essential nutrients such as n, k, ca, and mg was sufficient in the intermediate and wet zones compared to the dry zone. highest yielding plants that also maintained high­ er photosynthesis, transpiration and water use effi­ ciency occurred at the intermediate zone. in compar­ ison to dry and wet zones, this cultivation zone had moderate amount of rainfall and good soil quality overall. highest plant growth rate (table 2), high gas exchange (fig. 2) and high fruit yields (table 3) in the intermediate zone showed that water availability and nutrients were not an issue for this zone. even though the annual total rainfall in this zone was less than the dry zone, the dry months (may to october) here had considerably higher rainfall compared to the dry zone and lower rainfall compared to wet zone (table 1). during the dry months, the zone’s total precipitation was 553.5 mm which falls in the range required to grow high yielding noni plants. nelson and elevitch (2006), also stated that for high yields, noni cultivation site should receive moderate rainfall preferably 500­1500 mm annually that is dis­ tributed evenly throughout the year. higher growth in the intermediate zone was complemented by the high mineral nutrient content of the soil. major essential mineral nutrients required for plant growth such as n, k, ca, mg including p levels were consider­ ably higher than their ideal range in the soil at this site (table 1). water availability was highest in the wet cultivation zone together with adequate soil nutrients (table 1). this site had the highest mean rainfall within the two­year period (table 1). the soil n and k content were comparable to the intermedi­ ate zone. ca, and mg were considerably higher in this zone (table 1). p content, however, was lowest in the wet zone. with generally good soil properties plus plentiful of water, the growth and physiological per­ formance of noni in the wet zone were expected to be much higher. sunshine hours of 4.9 hours (table 1) in the wet zone was lowest compared to the other zones as the area had cloudy and rainy days more often. similarly, transpiration rates and wuei were also comparable between the two zones. one crucial factor that may have influenced the physiological performance of noni plants in this zone by limiting photosynthetic activity is the much lower soil p con­ tent (table 1). short­term inadequate supply of p lim­ its the photosynthesis rates due to restriction of pho­ tophosphorylation during the light reactions (rychter and rao, 2005). however, the effect of low p content on the physiology of noni is not that strongly evident. phosphorous deficiency symptoms were not seen on the leaves as well. noni may have some adaptive mechanism for surviving in p limited soils. mo et al. (2019), showed that some tropical lowland forest plants are able to acclimatize to low p levels by changing the foliar p allocation to fulfil the p demand for photosynthesis. acclimatization of noni plants to low phosphorous levels requires further investiga­ tion. the dry cultivation zone had significantly lower water and nutrient availability (table 1). the com­ bined effects of water deficit and nutrient deficiency stress lowered the growth of noni in the dry zone. low photosynthesis and transpiration rates, together with low wuei of plants (fig. 2) in this agro­climatic zone can also be attributed to both water and nutri­ ent deficiency. the climatic conditions on days of measurement, especially during the dry season were optimal (i.e. fine sunny days) for high transpiration rates at the dry site, but the rates were significantly low. this was mainly due to partial stomatal closure as indicated by the low stomatal conductance (fig. 2d). low stomatal conductance indicates a low degree of stomatal opening, resulting in low rates of incoming co2 and outgoing water vapour. the stom­ atal conductance is usually higher in k deficient plants (as was the case here) under drought stress since k deficiency impairs stomatal function by sig­ nalling for ethylene production which in turn inhibits the action of abscisic (aba) on stomata delaying the closure (wang et al., 2013). however, low k levels in the dry zone did not appear to influence the stomatal closure. during drought stress in a drought­resistant table 4 ­ total phenol content (tpc), radical scavenging activity (ic50) and antioxidant activity (aeac) in fruits from dry, intermediate and wet cultivation zones mean ± se is shown, n = 10. there was a significant difference in tpc anova (f df (2, 21) = 0.2139, (p<0.0001), ic50 values anova (f df (2, 21) = 3.760, (p<0.0001) and aeac content anova (f df (2, 21) = 17.58, (p<0.0001). for given cultivation zones, means not sharing the same letters are significantly different using tukey's test. cultivation zones studied fruit num­ bers per plant fruit weight (g) tss °brix dry 3 ± 0.25 a 74.7 ± 3.8 a 8.4 ± 0.1 a intermediate 8 ± 0.28 b 299.8 ± 41.5 7.9 ± 0.1 b wet 7 ± 0.30 b 271 ± 19.7 b 7.7 ± 0.1 b adv. hort. sci., 2024 38(2): 177­187 184 plant, aba level initially increases leading to stomatal closure, but as the drought stress is continued, aba levels decrease markedly, and stomatal closure becomes water potential­driven (brodribb and mcadam, 2013). this strategy allows the plants to respond to any rainfall quickly and open the stomata much faster for gas exchange than a non­drought tol­ erant plant. drought tolerant plants are also able to maintain gas exchange to gain carbon for a longer period of time during drought (tardieu and davies, 1993). for drought tolerant plants, complete stom­ atal closure is avoided or delayed during drought stress, this helps to maintain the carbon balance (brodribb and mcadam, 2013). this appeared to be the case for noni plants growing in the dry zone as the stomatal conductance measurements of noni leaves over the two­year period did not show com­ plete closing of stomata in the dry zone. noni has also been claimed to be a drought­tolerant plant (nelson, 2003; singh and rai, 2007). being drought­tolerant, wuei in noni plants from the dry area was also expected to be comparable to wet and intermediate zone plants. however, the wuei was significantly lower in the dry zone (fig. 2c) indicating that carbon gain per water loss was low, which resulted due to the low photosynthesis rates and subsequently smaller plants. nutrient deficiency stress being an add­on to water stress in the dry zone limited photosynthesis activity leading to low wuei, hence overall productivity of the plant was low. a decrease in photosynthesis was also due to low amounts of co2 entering the leaves as a result of low stomatal conductance. in addition, an increase in leaf temperature may have enhanced the suppression of photosynthesis in the dry zone. according to haldimann et al. (2008), reduced stomatal conduc­ tance and reduced transpiration rate raise leaf tem­ perature by several degrees resulting in suppression of photosynthesis due to reversible inactivation of rubisco. there was no considerable difference between the mean air temperature and the maxi­ mum air temperature between the three zones (table 1). however, an increase in leaf temperature in the dry zone may have resulted from significantly longer sunshine hours (table 1). marias et al. (2017), showed that an increase in leaf temperature due to reduced stomatal conductance could be very dramat­ ic in the full sun compared to partial sun. high fruit yield in the intermediate zone (table 3) also showed that climate and soil features of the intermediate zone were ideal for growing noni under rain fed conditions. effects of water stress on fruit production and yield is highly evident as seen from the lowest yield in the dry zone. similar results under drought conditions have been reported for apples (yao et al., 2001), peach (rahmati et al., 2018), citrus (huang et al., 2000), oranges (perez­perez et al., 2009) and tomatoes (sivakumar and srividhya, 2016. according to rahmati et al. (2018), fruit sizes during drought stress decrease because of reduced water flow to the fruits due to fruit stomatal closure, devel­ opment of thick cuticles or due to reduction in micro­ crack occurrences. nutrient deficiency can also be a factor contributing to fruit size as fruit size is also depended on the number of cells at anthesis (bohner and bangerth, 1988). ca and k, which have crucial roles in cell division and stomatal conductance respectively are lower in the dry zone leading to smaller fruit size. despite being smaller in size and producing a low number of fruits per plant, fruit sweetness (in terms of tss) was not low as expected. interestingly, it was significantly higher compared to the intermediate and the wet zones (table 3). increase in fruit sweetness under moderate water stress has been reported for tomatoes (veit­kohler et al., 1999; bertin et al., 2000; nahar and ullah, 2018), peach (kobashi et al., 2000), plums (maatallah et al., 2014) and nectarines (thakur and singh, 2012). sugar levels notably increase under drought stress to affect osmotic potentials and high sugar levels in cell vacuoles helps to produce high turgor pressure (ma et al., 2017). despite excellent physiological performance and yield, fruit antioxidant properties were lowest in the intermediate zone (table 4), which clearly indicated that plants in this zone were not significantly affected by any abiotic stress conditions. significantly higher tpc and acea in dry and wet zones (table 4) indicat­ ed considerable abiotic stress. low antioxidant prop­ erties of fruits in the intermediate zone on the other hand clearly indicated that the plants at the interme­ diate zone were not significantly affected by any abi­ otic stress conditions. antioxidant compounds in plants increase in response to abiotic stress. phenolic compounds are powerful antioxidants that protect plants from oxidative stress by scavenging harmful reactive oxygen species (ros) under different abiotic and biotic stresses (balasundram et al., 2006; lattanzio et al., 2006; li et al., 2012; kulbat, 2016; naiko et al., 2019; samec et al., 2021;). highest phe­ nol content in the dry zone is undoubtedly due to antioxidant defense activation. an increase in pheno­ prakash et al. ‐ effect of climate on physiological performance and fruit quality of noni 185 lic compounds under drought stress has been also reported for fruits such as mulberry (khamjad et al., 2021), grapevine (irani et al., 2021), pomegranate (farji et al., 2020) and strawberries (unal and okatan, 2023). the increase in antioxidant properties may also be in response to high oxidative stress caused by adverse environmental conditions created by nutri­ ent deficiency, high temperature and high uv light (high sunshine hours) (das and roychoudhury, 2014). comparable and high antioxidant properties in the wet and dry zone (table 4) indicated that plants were under considerable environmental stress. abiotic stress in the wet zone can be attributed to either excessive precipitation or low phosphorous availabili­ ty, as discussed earlier. alfaro et al. (2013) reported an increase in polyphenol content and antioxidant activity in murtilla fruits with an increase in rainfall. extreme precipitation in the area may have also resulted in soil compaction lowering the oxygen lev­ els in the soil leading to activation of the antioxidant defense system. ros generation also occurs during oxidative stress due to hypoxia (lack of oxygen) which can result from soil compaction (blokhina et al., 2003; ali and alqurainy, 2006). vergara et al. (2012), also found activation of the antioxidant defense sys­ tem in grapevines in response to hypoxia. p deficien­ cy stress, as discussed earlier, maybe the second rea­ son for noni plant stress and high antioxidant produc­ tion in the wet zone. increase in antioxidants in response to phosphorous deficiency has been report­ ed by tewari et al., (2007); zhang et al., (2014) and joel et al. (2017). mineral deficiency stress can also be added to the list of stresses producing high antiox­ idants in fruits from the dry zone. tewari et al. (2007), showed that antioxidant activity increased in mulberry plants under n, p and k deficiencies. in conclusion, this study showed that the physio­ logical performance, fruit yield and antioxidant prop­ erties of cultivated noni were significantly influenced by water availability and soil nutrient content. the physiological performance including fruit yield was optimum in the intermediate zone which was charac­ terized by a moderate rainfall and fairly good soil properties while poorest physiological performance was in the dry zone. fruit yield was highest in the intermediate zone with lowest antioxidant properties while it was lowest with highest antioxidant proper­ ties in the dry zone. overall the physiological perfor­ mance plus fruit yield and antioxidant properties were greatest in the wet zone where plants appear to have a moderate level of abiotic stress. this study also implies that under cultivation, moderate abiotic stress can enhance the antioxidant properties of noni. acknowledgements this work was supported by the school of agriculture, geography, environment, ocean and natural sciences, the university of south pacific, suva and the research division, ministry of agriculture, fiji. references alfaro s., mutis a., palma r., quiroz a, seguel i., scheuermann e., 2013 ­ influence of genotye and harvest year on polyphenol content and antioxidant. ­ journal od soil ali a.a., alqurainy f., 2006 ­ activities of antioxidants in plants under environmental stress, pp. 187­256. ­ in: motohashi n. 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ii) monitoring and optimization of procedures in the grapevine production chain; iii) experimental trials to verify the effectiveness of trichoderma spp. treatments of grafts and rootlings; iv) cleaning of multiplication material by chemical or alternative agents; v) hot water treatments; and vi) molecular characterization of a. vitis strains in order to establish molecular markers for traceability of infection sources. hot water treatments, unfortunately, are completely ineffective in obtaining bacterium-free rootlings (lucchetta et al., 2013), while initial analyses of soils and mother plants in the verona area have been encouraging; cleaning trials with acidic water and virkon need further study. other experimental trials are ongoing and initial results will be outlined in the presentation. acknowledgements this work is financed by agro.free project (psr regione veneto 2012-14), ersa, miva and vcr. we are grateful to the cra-vit staff, the nurserymen involved and cbc europe for the collaboration. references lucchetta g., miotti l., forte v., sartori e., angelini e., 2013 per il tumore della vite la termoterapia non funziona. l’informatore agrario, 17: 55-58. adv. hort. sci., 2013 27(3): 109 impaginato 3 adv. hort. sci., 2023 37(1): 3­13 doi: 10.36253/ahsc­13902 fruit maturity and antioxidant activity affecting superficial scald development in ‘abate fétel’ pears a. bonora 1 (*), a. venturoli 1, 2, m. venturi 1, a. boini 1, l. corelli grappadelli 1 1 department of agricultural and food science, university of bologna, viale giuseppe fanin, 46, 40127 bologna, italy. 2 current affiliation: terremerse soc. coop., via cà del vento, 21, 48012 bagnacavallo (ra), italy. key words: antioxidant capacity, fruit quality, preharvest factors, pyrus commu­ nis, superficial scald, total phenolic content. abstract: superficial scald (ss) is one of the main physiological disorders affect­ ing postharvest of pears. its onset is linked to oxidative processes. antioxidant compounds such as ascorbic acid and phenolics could play a key role in pre­ venting ss. growing environment and fruit quality also have an influence on ss symptoms occurrence. the aim of this project is to understand the relationship between antioxidant activity, phenolic content, and development of ss in ‘abate fétel’ pear. moreover, the effect on ss of fruit maturity at harvest was assessed using multivariate statistical approach. data were collected in thirty orchards in the emilia­romagna region (italy) in three seasons (2018, 2019 and 2020), and the fruit were stored in a regular atmosphere for 120 days. antioxidant capacity was determined by 2,2­diphenyl­1­picrylhydrazy (dpph) method and total phenol content by folin­ciocalteau colorimetric protocol. the results showed that 340 mg of ascorbate/100 g of fw and 300 mg of gallic ac./100 g of fw at least provide good protection against ss. multivariate analy­ sis indicated that pulp firmness and index of absorbance difference (iad) seem to keep low the ss occurrence, when at harvest are higher than 6.3 kg and 1.9, respectively. in conclusion, it would be possible to build a forecasting model to control ss that considers pre­harvest data and content of antioxidants in differ­ ent orchards, to improve the postharvest management of ‘abate fétel’. 1. introduction superficial scald (ss) is one of the main physiological storage disorders of european pears (pyrus communis l.). ss is a skin disorder that appears as brown or black patches on the fruit. ss is considered a chilling injury which induces a damage and death within the surface layers of cells in localized regions (lurie and watkins, 2012). during ss development necrosis of the hypodermal cortical tissue seems to be induced by oxida­ tion products of the sesquiterpene (e, e)­α­farnesene (bain and mercer, 1963; rowan et al., 2001). α­farnesene, accumulates at a relatively high (*) corresponding author: a.bonora@unibo.it citation: bonora a., venturoli a., venturi m. boini a., corelli grappadelli l., 2023 ­ fruit maturity and antioxidant activity affecting superficial scald development of ‘abate fétel’ pears. ­ adv. hort. sci., 37(1): 3­13. copyright: © 2023 bonora a., venturoli a., venturi m. boini a., corelli grappadelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 october 2022 accepted for publication 17 november 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13902 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 3­13 4 level in the fruit peel during low­temperature storage (whitaker et al., 2009; yazdani et al., 2011; lu et al., 2013; calvo et al., 2015). the observation that ss could be inhibited by certain antioxidant treatments and low oxygen in the storage rooms atmosphere has provided evidence that development of the disorder was associated with oxidative processes (huelin and coggiola, 1970; whitaker, 2004; vanoli et al., 2015). thus, the conjugated trienols (ctols) that result from the oxidation of α­farnesene are assumed to play a causal role in the occurrence of ss (whitaker, 2007; giné bordonaba et al., 2013). nevertheless, it is gen­ erally accepted that the accumulation of both α­far­ nesene and ctols may be mediated by ethylene which is effectively correlated with ss development (bai et al., 2009; lu et al., 2013; xie et al., 2014; yazdani et al., 2011). therefore, it has been suggest­ ed that α­farnesene oxidations is a direct conse­ quence of free radical reactions occurring during chilling injury and α­farnesene is not always required for the induction of ss but rather in aggravating the symptoms in fruit already compromised by oxidative stress (rao et al., 1998; rupasinghe et al., 2000). in this context, it has been suggested that superficial scald mainly results from an imbalance between the fruit capacity to generate antioxidants and the reac­ tive oxygen species (ros) produced during cold stress (ahn et al. , 2007; guerra et al. , 2012; ju and bramlage, 2019). nevertheless, the antioxidant sys­ tem in fruit includes an enzymatic and a non­enzy­ matic component that play an important role modu­ lating oxidative damage to cell walls (ahn et al., 2007; lurie and watkins, 2012; li et al., 2016). furthermore, non­enzymatic antioxidants can pre­ vent oxidation­linked damages responsible for super­ ficial scald through biosynthesis of phenolics that are involved in protective redox­linked pathways under cold stress (larrigaudière et al., 2016; sarkar et al., 2018). the nonenzymatic scavengers of reactive oxy­ gen species include low molecular mass antioxidants with high­reducing potentials, such as ascorbic acid (aa) and glutathione (gsh). ascorbic acid acts as an antioxidant compound since it can protect fruit mem­ branes from lipid peroxidation (shewfelt and del rosario, 2000) and acts against reactive o2 species in concert with α­tocopherol (jimenez et al., 1997). nevertheless, aa tends to decrease during storage and processing of fruit and vegetables (haffner et al., 1997). a relationship was found between aa content and the susceptibility to browning during experimen­ tal storage under various brown core­inducing condi­ tions (pintó et al., 2001). in pears the antioxidant capacity is well explained by phenolics content (galvis sánchez et al., 2003). several studies have demonstrated that these compounds are associated with resistance to ss development in apples and pears (ju et al., 1996; zhao et al., 2016). phenolic compounds are particularly sensitive to storage fac­ tors such as controlled atmosphere (amiot et al., 1993). variability of phenolics in plant tissues depends on many pre­harvest factors, such fruit maturity and environmental conditions, including temperature, uv light, and nutrition (markham et al., 1998; rivero et al., 2001; rühmann et al., 2002). casero et al. (2004) used the partial least squares regressions (pls), a multivariate technique, and found correlations between fruit quality attributes, such as fruit acidity and firmness, and storage disor­ ders with nutrients such as calcium, potassium and phosphorus, both in the leaf and fruit. moreover, pca biplots were helpful in showing the segregation between ss classes and their associations with the various physicochemical attributes (cronje et al., 2015). in pear, pulp firmness is one of the most rele­ vant quality parameters (saquet, 2019). softer fruit had rounder cells separated by larger intercellular spaces than firmer fruit. on the other hand, firmer fruit have smaller cells with less interspace which means denser tissues and longer storage than soft fruit (johnston et al., 2002). moreover, the da­ meter, a handheld device that measures chlorophyll concentration several millimetres into the flesh of fruit providing the index of absorbance difference (iad) (ziosi et al., 2008), can discriminate the ripening stage of climacteric fruit for postharvest tailored cold storage (bonora et al., 2013; gagliardi et al., 2014; sadar and zanella, 2019). fruit ripeness is also well predicted by starch degradation using a multivariate statistical approach (zude­sasse et al. , 2002). conversely, in ‘abate fétel’ pear fruit the starch index is not always employed even if some studies have reported the use of this procedure to predict pear storability and postharvest issues (kingston, 1992; le lezec and belouin, 1994; agar et al., 1999; calvo et al., 2011). in pears starch pattern degrada­ tion can be influenced by environmental and man­ agement factors such as temperatures, harvest date and deficit irrigation affecting the kinetics of starch accumulation and degradation (watkins et al., 1982; kramer, 1983; lopez et al., 2013; lindo­garcía et al., 2019). total sugar content is an internal fruit quality trait that is crucial for consumer acceptance (osorio bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 5 and fernie, 2014). total soluble solids in ‘abate fétel’ and ‘forelle’ pear are mainly fructose, glucose and sucrose (mesa et al., 2016), and they increase in con­ centration after storage since starch is converted via hydrolysis into sugars over time (visser et al., 1968; crouch and huysamer, 2011; rizzolo et al., 2015). additionally, sorbitol accumulates in the fruit still attached to the tree (mesa et al., 2016), acting as cry­ oprotectant in cellular structures during cold storage by preventing dehydration of membranes and pro­ teins through an osmotic adjustment process (busatto et al., 2018). therefore, the aim of this work was to research relations between antioxidant activi­ ty, phenolic content, and ss development on ‘abate fétel’ pears. furthermore, preharvest maturity and non­destructive postharvest quality parameters, as well as antioxidant activity and phenolic content, influencing the occurrence of superficial scald using multivariate analysis and regression trees were inves­ tigated to develop new reliable hypotheses of their effects in ss development, without compromising consumer acceptance and nutritive value. 2. materials and methods fruit material and superficial scald evaluation fruit were harvested during three consecutive seasons (2018, 2019 and 2020) from different ‘abate fétel’ orchards located in the emilia­romagna region, italy. fruit from 30 and 23 farmers were col­ lected and their maturity assessed in 2018 and in seasons 2019 and 2020, respectively. the farmers were indicated by three digit­numbers. in all seasons, two orchards with historical higher ss and two with lower ss were subjected to biochemical analysis at harvest and during storage. in 2018, eighteen 15 kg boxes for each farm were placed in a regular atmo­ sphere (0.5°c and >90% of relative humidity ­ rh). after 3 (t1), 4 (t2), and 5 months (t3) of storage, the room was opened, following the calendar normally applied by the company. in 2019 and 2020 only six 15 kg boxes per orchard were harvested and placed with a regular atmosphere in a cold room which was opened after 4 months (t2). afterwards, the pres­ ence of superficial scald was assessed in 30 fruits per farm. we defined four classes depending on the severity of symptoms in the skin of pears: class 0 where there was no peel browning, class 1 from 0% to 25% fruit peel showing ss, class 2 from 25% to 50% ss, and class 3 over 50% ss after shelf life. a ss index was computed as follows (bonora et al., 2021): 4 ss index = ∑ (index level) x (fruit at this level) 0 total number of fruit analysis of the physical characteristics in all seasons, 30 fruits per orchard at harvest (t0) were subjected to qualitative analysis such as fruit size, index of absorbance difference (iad), pulp firm­ ness, soluble solid content and starch content. moreover, non­destructive fruit quality such as size and iad after 4 months (t2) of cold storage were con­ sidered. weight and dimensions (diameter and height) of each fruit were measured with an auto­ matic caliper (s_cal work, sylvac, switzerland) and an electronic balance (kb 1200­2n, kern, germany) connected to a notebook. individual fruit ripeness expressed as iad was measured with the da­meter 53500 (sinteleia, bologna, italy) on the fruit side most exposed and less exposed to the sun. individual fruit flesh firmness (fff) was determined by fta (fruit texture analyser, güss instruments, strand, western cape, south africa) fitted with an 8 mm diameter tip, after removing the fruit peel from opposite sides at 180°. the mean value of fruit ripeness and firmness, from the two sides, was calcu­ lated. soluble solid concentration (ssc;°brix) was determined by measuring the refractive index of the juice for each fruit with a digital refractometer (pal­ 1, atago). the stage of starch hydrolysis was deter­ mined by dipping half­cut pears into a lugol solution and scoring the fruit according to the ctifl­eurofru scale (1­10; 1 = minimum, 10 = maximum starch hydrolysis) (planton, 1995). finally, at harvest (t0) and during storage (t1, t2, t3) pieces of the same size with pulp and peel of fruit from all the orchards in 2018 and from four representative farmers in 2019 and 2020 were frozen in liquid nitrogen and stored at ­80°c. these plant materials have been used for quantification of antioxidant activity and total pheno­ lic content. quantification of antioxidant activity to estimate the antioxidant activity, the 2,2­ diphenyl­1­picrylhydrazy (dpph) method was used (adapted from brand­williams et al., 1995). the dpph working solution was prepared in 70% acetone (v/v), with a final concentration of 0.02 mg/ml (w/v) and stored at 4°c until needed. afterwards, antioxi­ dant compounds from 0.5 g of pear (flesh and peel) were extracted in 10 ml of acetone 70%. the frozen adv. hort. sci., 2023 37(1): 3­13 6 material (0.5 g of pear) was homogenised in a ultraturrax (ika t25 digital ultra­turrax) with 10 ml of extraction solution (acetone 70%) for 2 min­ utes on ice. after vortexing, the tubes were sonicated in a bath­type sonicator for 15­20 minutes and the homogenates were centrifuged at 1,500 x g for 20 minutes at 5°c. fruit extracts (0.1 ml) were allowed to react with 3.9 ml of the dpph solution for 30 min­ utes in the dark, and the absorbance at 515 nm by uv­vis spectrophotometer (libra s80pc vbw uv/vis, biochrom), was measured. the dpph working solu­ tion was considered as the blank and the calibration curve was made using ascorbic acid. total phenolic content phenolic compounds quantification was per­ formed using the folin­ciocalteau colorimetric method (adapted from vieira et al., 2009). total phe­ nolics from 0.5 g of pear (flesh and peel) were extracted in 10 ml of 70% acetone. the frozen mate­ rial (0.5 g of pear) was homogenised in a ultraturrax (ika t25 digital ultra­turrax) with 10 ml of extrac­ tion solution (acetone 70%) for 2 minutes on ice. after vortexing, the tubes were sonicated in a bath­ type sonicator for 15­20 minutes. the homogenates were centrifuged at 1,500 x g for 20 minutes at 5°c. 250 μl of supernatant were added to 2 ml of deion­ ized water and 250 μl of folin reagent. after mixing, samples were incubated for 5 min and 5 ml of sodi­ um carbonate (na2co3) and 5 ml of distilled water were added. following 1 h incubation in the dark, absorbance was measured at 750 nm by uv­vis spec­ trophotometer (libra s80pc vbw uv/vis, biochrom). the phenolic concentrations were determined using gallic acid as a standard. data treatment and statistical analysis all the results of antioxidants and phenolics were statistically evaluated by analysis of variance (anova). furthermore, these data were presented considering four key producers at harvest (t0), after 3 (t1), 4 (t2) and 5 months (t3) of regular air storage. these producers were selected according to the inci­ dence of ss: two had a high incidence of ss (131 and 432) and the others had a low development of ss (272 and 351). moreover, the fruit quality data were subjected to multivariate analysis to highlight which among the factors considered appears to be more related to the onset of superficial scald. multivariate statistical analyses, such as canonical correspon­ dence analysis (cca) and recursive partitioning and regression trees (rpart) analysis, were performed using the statistical software r (r core team, 2020), by addition of packages “vegan” (oksanen et al., 2019) and “rpart” (therneau and atkinson, 2019). cca was used to estimate the interactions between the frequencies of ss classes and the numeric vari­ ables. the blue vector indicates the increase of the factors in a certain direction (ss class). finally, we considered the total variability explained by two components (cca1 and cca2) and how each variable affects the first and the second component. therefore, maturity data at harvest and ss after 4 months in all seasons were considered to elaborate the overall picture. finally, rpart analysis was applied to detect which factors could contribute more to ss and to understand their thresholds. green and red lights indicate a decrease or an increase in ss index, respectively. 3. results in 2018 antioxidant capacity in fruit during stor­ age decreased significantly (fig. 1 and table 1). regarding phenolic compound content in fruit of dif­ ferent producers, the differences were not statistical­ ly significant at harvest and during conservation (table 1). this can be explained looking at the differ­ ent producers’ behaviour (fig. 2). indeed, two differ­ ent trends can be observed during the first 3 months of storage: in 272 and 351 phenols tend to increase, while in 131 and 432 they decrease. thereafter, phe­ nols in 131, 432 and 351 increase from t1 to t2 fig. 1 ­ evolution of antioxidant capacity in season 2018 (mg ascorbic acid/100 g of fresh fruit) of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). points followed by the same letter in every sampling point are not signifi­ cantly different from each other. mean separation by lsd test (p≤0.05). bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 7 before decreasing notably again. on the other hand, in 272 we note only a slightly decrease from t1 to t2. in our study there is a clear distinction between t1, t2 and t3 in terms of ss occurrence in the first season (table 1). in addition, figures 3, 4, and 5 con­ firms the great variability of the incidence of ss among the different producers in t2 in all seasons. the evolution of ss index in 2018 of the 30 producers is also shown in table 1. at t1 the index is low while there is a considerable increase of ss incidence at t2 and at t3, while antioxidants decrease significantly. among the key producers of the first season in figure 3, two farmers had a higher ss index (131, 432), while two producers had a lower ss index (272, 351). in detail, the results show that the producers with the lowest ss (351, 272) are those in which phenols increase during the first three months of storage (fig. 2). therefore, has been hypothesized that fruit were able to initially react and use these substances to protect themselves from oxidative stress. epochs ss index antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) total phenolic content (mg gallic acid/100 g of fresh fruit) t0 mean / 480.92 a 281.99 sem / 19.04 15.91 t1 mean 5.89 b 370.40 b 312.62 sem 0.80 11.62 17.85 t2 mean 35.46 a 300.38 c 299.37 sem 2.52 7.85 12.90 t3 mean 43.08 a 264.41 c 263.16 sd (%) 2.66 13.05 18.95 significance (p<0.05) *** *** ns levene test ns ns ns table 1 ­ mean and standard error of the mean (sem) of ss index, antioxidant capacity, total phenolic content in season 2018 at harvest (t0), after 3 months (t1), 4 months (t2), 5 months (t3) in cold storage data represent the average of fruit quality of 30 producers between epochs for each variable. values followed by the same letter in colu­ mns are not significantly different from each other. means separation by lsd test (p<0.05). *** significant at p≤0.001; ns = not significant. fig. 2 ­ evolution of total phenolic content in season 2018 (mg gallic acid/100 g of fresh fruit) of four selected farms (131, 272, 351, 432) and their average trend. bars repre­ sent standard error of the mean (±sem). values followed by the same letter in every sampling point are not signifi­ cantly different from each other. mean separation by lsd test (p≤0.05). fig. 3 ­ evolution of superficial ss index of four selected farms (131, 272, 351, 432) and their average trend during stor­ age in 2018. bars represent standard error of the mean (±sem). values followed by the same letter in every sam­ pling point after harvest are not significantly different from each other. mean separation by lsd test (p≤0.05). 8 adv. hort. sci., 2023 37(1): 3­13 particularly, 351 accumulated phenols till 4 moths which drop from t2 to t3 even below 431, probably, consuming their reducing power instead of antioxi­ dants avoiding polyphenol oxidase activity and browning. on the other hand, the producers (131, 432) with the greatest ss are those in which the phe­ nols drop during the first three months of storage, even if they rise again in the following months (fig. 2). probably, the damage caused by oxidative stress is already underway. notably, we found a drastic decrease of antioxidants between t1 and t2 in pro­ ducer 272, even if denoted the highest initial antioxi­ dant values at harvest (fig. 1). nevertheless, 272 had a low incidence of ss and this could be explained by the fact that during the first three months the antiox­ idants were high, and phenols increase reaching and keeping a certain threshold value till t3. weather and physiological factors in the second and the third season appear to also influence the average nonenzymatic scavengers’ level and the ss occurrence (fig. 4 and fig. 5). thus, we found a gen­ eral high presence of antioxidants and low ss in 2019, characterized by a rainy and cold season. on the contrary, the protective compounds decreased, and ss increased in all producers in 2020 when the temperatures and yields were higher. moreover, the data shows that antioxidants drop in the first three months of storage in all the four producers consid­ ered (fig. 4 and fig. 5). however, in both seasons the incidence of ss in producers 131 and 432 was higher when the antioxidants decrease drastically after 3 months of cold storage, regardless of the level at har­ vest. in figure 6 and figure 7, cca and rpart analysis are applied to study the effects of maturity of ‘abate fétel’ pear at harvest and during storage against ss development at t2 during three consecutive seasons (2018, 2019 and 2020). the multivariate model fig. 4 ­ evolution of antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) and ss index after 4 months of cold storage (t2) in seasons 2019 of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). values followed by the same letter between four producers are not significantly different from each other considering dpph values at t0 and t1 or ss index during storage. mean separation by lsd test (p ≤ 0.05). fig. 5 ­ evolution of antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) and ss index after 4 months (t2) of cold storage in seasons 2020 of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). values followed by the same letter between four producers are not significantly differ­ ent from each other considering dpph values at t0 and t1 or ss index during storage. mean separation by lsd test (p≤0.05). fig. 6 ­ canonical correlation analysis (cca) of superficial scald classes in ‘abate fétel’ pear after 4 months of cold stor­ age (clas0 0%, clas1 1%­25%, clas2 26­50%, and clas3 51­ 100% of peel symptoms) against qualitative orchard fea­ tures at harvest during three seasons 2018, 2019 and 2020 (blue vectors) and the scores of producers (black circles). total variability explained (53%): cca1 (90%); cca2 (8%). the following abbreviations have been used: weight of the fruit at harvest (sizehrv), weight of the fruit after 4 months of cold storage (sizet2sl), pulp firm­ ness at harvest (firmhrv), soluble solid content at har­ vest (brixhrv), iad­meter values at harvest (iadhrv), iad values after 4 months of cold storage (iadt2sl), starch pattern index at harvest (spihrv). bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 9 explains 27% of the observed ss variability (cca1 89% and cca2 7%). in our study we found that flesh firmness at harvest can prevent ss after cold storage considering all seasons and its contribution to com­ ponent 1 is 0,95 against ss (fig. 6). the orchards (23%) with pulp firmness at harvest higher than 6.3 kg developed low ss (7.2 ss index), while the ss index increased three times in the farms (67%) which, at harvest, scored less than 6.1 kg of firmness (fig. 7). however, in figure 6 we noted that bigger fruit at harvest and after storage are more prone to ss (its contribution to principal component is 0.11 at harvest and 0.40 after storage towards ss). in our research starch content at harvest in different pro­ ducers and seasons influences ss during cold storage with an important contribution to component 1 and component 2 (0.43 and 0.51 respectively towards class 3 after 4 months). the non­destructive iad­ meter values also contribute to preventing ss (fig. 6), although its contribution to component 1 is lower than firmness and spi (0.30 and 0.25, at harvest and during storage respectively against ss). furthermore, in figure 7 we found a specific value of iad which con­ tributed to ss occurrence for three consecutive years. among the farms which scored firmness value lower than 6.1 (67%), a fraction (13%) with iad higher than 1.9 developed an average ss index of 17. the 54% with firmness and iad lower than 6.1 and 1.9 respectively denoted a ss index higher than 33. moreover, we found that °brix promotes resistance to ss during storage of ‘abate fétel’ pears in emilia romagna (fig. 6) and its contribution to component 1 is remarkable (0.20 against ss). 4. discussion and conclusions as shown in our research, several studies confirm that antioxidant capacity, in particular ascorbic acid, drops during storage (lee and kader, 2000; franck et al., 2003), promoting a variable ss development in pear between orchards located in different environ­ ment (bonora et al., 2021). indeed, silva et al. (2010) reported that storage reduced differences in antioxi­ dant capacity between producers at harvest. about phenolic content, fruit may react and produce more phenols when stored for few months. this behaviour is reported in apples by leja et al. (2003) who showed that phenolic compounds are synthesised during storage. moreover, calvo et al. (2015) high­ lighted that in addition to the initial value of antioxi­ dants, it is important the level of protective com­ pounds be maintained. regarding quality factors affecting ss, wang and arzani (2019) also reported a good and negative cor­ relation between high flesh firmness at harvest and ss development in ‘d’anjou’ pears. nevertheless, fruit with a high flesh firmness are more unripe (stow, 1988) and more prone to contain less antioxi­ dants (kaur et al., 2021). furthermore, larger fruit generally ripe faster and are characterised by lower firmness and dry matter after storage, by probably increased respiration rate, oxidative stress, and water loss as consequence (gwanpua et al., 2013). accelerated senescence, and increased susceptibility to chilling injury have been reported to result from weight loss (prange and wright, 2023). on the other hand, the higher surface­volume ratio of larger fruit seems to prevent ss by a reduced evapotranspiration and weight loss during storage (pasquariello et al., 2013). although stow (1988) described starch pattern index as an unreliable method to determine opti­ mum harvesting date of pears, szczesniak and ilker (1988) reported that parameters influencing storabil­ ity and fruit textural characteristics of ‘forelle’ pears include the starch content. in contrast with our study, the incidence of superficial scald in apple fig. 7 ­ recursive partitioning and regression tree (rpart) analy­ sis, correlation between quality factor and scald index. towards green point hypothesis (firmhrv≥ 6.1; firmhrv≥6.3; iadhrv≥1.9) is confirmed, to red point is not satisfied. numbers in the circle represent scald index and the percentage of producer that are included in that value of scald index. the colour of the boxes represents the severity of ss: low ss (scald index: 0­15; most fruits do not show ss or show slight symptoms), medium to severe ss (scald index: 16­30; occurrence of progressive­ ly more severe symptoms), severe ss (scald index: >30; most fruit show severe symptoms and other very severe symptoms). the following abbreviations have been used: pulp firmness at harvest (firmhrv), index of absorbance difference at harvest (iadhrv). adv. hort. sci., 2023 37(1): 3­13 10 declines when the starch pattern index advances (watkins et al., 1982; mditshwa et al., 2015). concerning iad meter values, a three­year study by delong et al. (2014) to develop optimal harvest time for ‘honeycrisp’ in nova scotia (canada) led to fruit with a low incidence of disorders after 3 months of storage. indeed, ‘abate fétel’ pears with higher iad values at harvest ripen less over 6 months of cold air storage (rudell et al., 2017). in fact, the content of primary photoassimilates certainly supports the pro­ duction of secondary metabolites such as antioxi­ dants (mellidou et al., 2021). to conclude, the development of ss seems to be the consequence of the occurrence of many quality and biochemical traits. therefore, it is important to highlight that it is not possible to consider only one variable at a time to find a solution in pears. we explored the possibility to use multivariate analyses to help understand the relationships between all the factors that may influence ss. antioxidant capacity is essential in ‘abate fétel’ pear to prevent ss occur­ rence. moreover, good pulp firmness, increased iad values, high total soluble solids and low starch degra­ dation at harvest seems to have a positive impact on ss development. furthermore, rpart analysis of fruit maturity at harvest confirms the importance of reaching threshold values, as indicators of potential fruit susceptibility to ss during storage, in addition to the absolute trends in multivariate analysis. therefore, pre­harvest quality and antioxidant values at harvest can be compared with threshold values to discriminate batches of fruit based on their potential to develop ss symptoms. however, it is important to consider that for application purposes it would be necessary to develop faster systems for the quantifi­ cation of fruit maturity and antioxidant capacity at harvest in the orchards or during storage, using reli­ able, non­destructive methods. accordingly, the fruit industry may consider a predictive software to help manage the storage, minimising ss in pears and improving cold room fulfilment and energy efficiency, by recording at harvest 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postharvest biol. technol., 25(2): 123­ 133. impaginato 239 adv. hort. sci., 2024 38(3): 239­248 doi: 10.36253/ahsc­15528 https://oaj.fupress.net/index.php/ahs assessment of genetic parameters and heritability of dendrobium species section spatulata native to indonesia e.w. tini 1, p. widodo 2, sugiyono 2, z. ulinnuha 1 (*) 1 jenderal soedirman university, faculty of agriculture, department of agrotechnology, purwokerto, indonesia. 2 jenderal soedirman university, faculty of biology, department of biology, purwokerto, indonesia. key word: diversity, morphological traits, orchidaceae, plant breeding. abstract: being one of the most abundant genera of orchids, dendrobium pre­ sents a valuable genetic resource for hybridization programs. morphological characterization and assessment of genetic parameters plays a crucial role in establishing genetic connections among orchid species within the same genus. the study aims to discern the morphological traits of five indonesian dendrobium species, intending to evaluate their potential as candidates for crossbreeding programs. the materials used in this study were d. antennatum, d. discolor, d. stratiotes, d. lineale, d. gouldii, and d. sylvanum. this research examines 21 quantitative traits and 21 qualitative morphological traits of the studied dendrobium. the findings reveal variations in characters related to flowers, leaves, and pseudobulbs. the analysis of genetic parameters indicates the presence of genetic diversity in traits such as flower stalk length, length of inflorescence, flower series length, flower length, flower width, dorsal sepal width, lateral sepal width, petal length, petal width, labellum length, labellum width, and the number of florets. all observed traits demonstrate high heri­ tability. so, the characters that have high genetic variability and heritability are valuable in selection criteria for plant breeding. 1. introduction indonesia possesses abundant biodiversity, encompassing a variety of orchids. out of the approximately 30,000 orchid species worldwide, around 5,000 are present in indonesia, distributed across diverse regions, with certain species being endemic to the country (puspitaningtyas, 2020). dendrobium as the most plentiful genus of orchids globally, boast­ ing approximately 1,600 species and holding the title of the largest genus within the orchidaceae family. the collective number of hybrids, derived from both natural variations and crossbreeding, exceeds 20,000 and encompasses single and multi­genera varieties (hartati et al., 2021). indonesia also known for hosting a significant variety of dendrobium (*) corresponding author: zulfaulinnuha@unsoed.ac.id citation: tini e.w., widodo p., sugiyono, ulinnuha z., 2024 ­ assessment of genetic parameters and heritability of dendrobium species sec‐ tion spatulata native to indonesia. ­ adv. hort. sci., 38(3): 239­248. orcid: ewt: 0000­0001­5122­5507 pw: 0000­0002­2203­7168 s: 0000­0003­2002­9196 zu: 0009­0003­8675­9252 copyright: © 2024 tini e.w., widodo p., sugiyono, ulinnuha z. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 december 2023 accepted for publication 13 june 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15528 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-5122-5507 http://orcid.org/0000-0002-2203-7168 http://orcid.org/0000-0003-2002-9196 http://orcid.org/0009-0003-8675-9252 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 239­248 240 orchids, contributing to the overall diversity of orchid species in the region (rahayu and yusri, 2022). schuiteman (2012) has identified 20 sections of dendrobium, and one of these is the spatulata sec­ tion. the spatulata species within the section display significant diversity in flower characters, encompass­ ing variations in colors, labellum shapes and colors, horn shapes and colors, as well as the duration of flower bloom shelf life. therefore, it is essential to assess the genetic variation of these species to offer fundamental genetic insights and facilitate genetic enhancements within the spatulata orchid section (purwantoro et al., 2023). to discern the variations among species, it is essential to employ a characteri­ zation method. utilizing morphological features such as leaves, stems, tubers, fruits, and roots for charac­ terization is anticipated to facilitate the identification and understanding of the specific utility of these characterized plants (de et al., 2015). observing mor­ phological characters is visually straightforward, allowing for a swift assessment of their diversity in comparison to other traits. furthermore, employing morphological characterization proves valuable in evaluating the relationships among orchids, which is crucial for conservation initiatives and enhances the practicality of plant genetic resources (vo et al., 2015). while the assessment of genetic relationships through morphological characters can be significantly affected by environmental factors, it remains essen­ tial to thoroughly characterize these traits. this thor­ ough characterization is necessary to ease the utiliza­ tion of germplasm by breeders (aloysius et al., 2017). the characterization of plants plays a crucial role in evaluating the genetic proximity between orchid species within the same genus, influencing the effec­ tiveness of plant crossbreeding. a more intimate genetic connection enhances the likelihood of suc­ cessful crosses. this characterization serves not only to craft plant descriptions but also to ascertain the genetic relationships among different species (mursyidin et al., 2021). in genetics studies of quanti­ tative traits, the primary role of heritability lies in its predictive function, indicating the dependability of phenotypic value as a predictor of breeding value (ponzi et al., 2018). genetic variability in plant breed­ ing refers to the range of genetic differences or varia­ tions that exist among individuals within a population of plants. it is a key concept in plant breeding because this variability is the raw material that plant breeders work with to develop new and improved crop varieties (yani et al., 2018). by assessing genetic variability, plant breeders can identify traits that exhibit variation within a population. this informa­ tion is crucial for selecting traits that are desirable and heritable, meaning they can be passed on to future generations (wirasti and purwantoro, 2018). traits with high heritability are more likely to respond positively to selection, making them prime candidates for improvement through breeding (george et al., 2020). the objective of this research is to characterize the dendrobium section spatulata orchid from indonesia and investigate its genetic diversity and heritability as part of a plant breeding initiative. 2. materials and methods experimental location the research was carried out in banjarsari village, sumbang district, banyumas regency, central java province, indonesia 7.3576° s, 109.2445° e. the research was carried out from march to june 2023. experimental materials the characterization was conducted on the fol­ lowing dendrobium orchids: d. antennatum, d. dis‐ color, d. stratiotes, d. lineale, d. gouldii, and d. syl‐ vanum. these plants were sourced from farmers and orchid collectors in indonesia, and they represent mature specimens in the flowering stage. a total of three plants per species were analyzed. the tools used are digital calipers, rulers, digital single­lens reflex (dslr) cameras, and writing instruments. cultivation methods dendrobium was cultivated in a screen house with 50% shade. air temperature at daytime temperature around 27­32°c and nighttime temperature around 22­25°c, and humidity levels between 50­80%. plants are watered once a day or according to plant needs. if the media humidity is still high, the plants can be watered every two days. the medium used for culti­ vation is charcoal. fertilization is carried out once a week with npk 20:20:20 fertilizer with follicular application. regularly inspect dendrobium orchids for pests. additionally, monitor the orchids for signs of fungal or bacterial diseases and take necessary pre­ cautions to prevent spread. characterization procedure the characterization procedure is based on the tini et al. ‐ genetic parameters of indonesian dendrobium section spatulata 241 orchid characterization guidelines published by the indonesian ornamental plant research institute which was adapted from the dendrobium characterization guidelines by the international union for the protection of new varieties of plants (upov). variables observed and analyzed were nature of stem, leaf shape, apexes of leaf, apexes of dorsal sepal, apex of lateral sepal, apex of petal, petal curva­ ture, lip shape, lip margin, dorsal sepal shape, lateral sepal shape, petal shape, dorsal sepal cross section, lateral sepal cross section, petal cross section, lip overlapping of basal part, lip shape of lateral lobe, lip shape of apical lobe, lip type of curving, lip shape of eye, color of anther cap; plant height (cm), pseudob­ ulb diameter (cm), internode length (cm), leaf length (cm), leaf width (cm), leaf area (cm2), peduncle­ovary length (cm), length of inflorescence (cm), length of flower arrangement (cm), leaf thickness (mm), flower length (cm), flower width (cm), dorsal sepal length (cm), dorsal sepal width (cm), lateral sepal length (cm), lateral sepal width (cm), petal length (cm), petal width (cm), labellum length (cm), labellum width (cm), number of flowers per spike. the tools used are digital calipers, rulers, digital single­lens reflex (dslr) cameras, and writing instruments. statistical analysis to evaluate the performance of observed traits, conducted a variance analysis. the estimation of genetic parameters such as coefficient of genetic varibility (cgv), coefficient of phenotypic variability (cpv) and heritability followed jambormias (2014) steps. 3. results dendrobium species origin dendrobium antennatum is native to papua (indonesia) (table 1, fig. 1). these orchids thrive on tall tree branches within coastal forests, mangrove swamps, and rainforests, typically below 1200 meters in elevation. dendrobium discolor var. tanimbar is native to the tanimbar islands. it thrives in warm to hot cli­ mates, growing both as an epiphyte and a lithophyte. it can be found in various habitats, including man­ grove forests along the coast, behind sand dunes where it may experience salt spray, as well as on cliffs and rock faces up to an elevation of 550 meters. dendrobium stratiotes is native to the moluccas (including halmahera and morotai), the sunda islands, and sulawesi, this orchid thrives at lower ele­ vations. it typically grows as a medium to large­sized epiphyte, preferring warm to hot conditions, often forming clustered groups. dendrobium lineale is native to papua, this orchid species grows as a large to giant­sized plant, thriving in warm to hot climates as either an epiphyte or fig. 1 ­ six species of dendrobium section spatulata native to indonesia studied. table 1 ­ dendrobium species used in the study name of germplasm source of germplasm dendrobium antenna‐ tum papua dendrobium discolor var. tanimbar tanimbar island, maluku dendrobium stratiotes western papua, the moluccas (halmaheira and morotai), the sunda islands and sulawesi dendrobium lineale papua dendrobium gouldii papua dendrobium sylvanum papua, new guinea adv. hort. sci., 2024 38(3): 239­248 242 lithophyte. it is typically found at elevations of up to 800 meters, often near streams and coastal areas. dendrobium gouldii is native to papua. this orchid species grows as a large to giant­sized plant, thriving in warm to hot climates as either an epiphyte or lithophyte. it can be found in riverine forests, coastal forests, swamp forests, beaches, and plantations, typically at altitudes ranging from sea level to 700 meters. dendrobium sylvanum is native to papua new guinea in lowland areas, this orchid species grows as a large to giant­sized epiphyte, thriving in warm cli­ mates. morphological characters most diversity was found in the characters of leaves (leaf shape, apexes of leaf), pseudobulb (nature of stem), flowers (apexes of dorsal sepal, apex of lateral sepal, apex of petal, petal curvature, lip shape, lip margin, dorsal sepal cross­section, later­ al sepal cross­section, petal cross­section, lip overlap­ ping of basal part, lip shape of lateral lobe, lip shape of apical lobe, lip type of curving, and lip shape of eye) (table 2). table 2 ­ qualitative characters of dendrobium studied flowers in this study, all the flower parts of dendrobium that were observed had diverse characters, even though they were still in one section, namely the spatulata section. the section spatulata includes several species known for their characteristic flat­ tened or spatula­shaped lip petals. this diversity shows differences in the shape of the petals, dorsal sepals, lateral sepals and labellum. the spatulata section’s characters are the dorsal, lateral sepal and petal shapes were categorized as narrow elliptic. however, there is diversity in petal curvature, namely, d. antennatum and d. statiotes exhibit a spiral pattern. conversely, in d. discolor, d. gouldii, and d. sylvanum, the curvature is deflexed, while in d. lineale, it remains straight. the diversity in the labellum lies in the lip shape of the lateral lobe, namely in d. antennatum, d. sylvanum, and d. lineale it is triangular, while in d. discolor it is broadly trapezoidal, and in d. gouldii and d. stratiotes it is ovate. variability is also found in the lip shape of the apical lobe, namely in d. antennatum, d. stratiotes, d. discolor, d. gouldii it is rhombic, while in d. lineale it is reniform, and in d. no. characters d. antennatum d. discolor d. stratiotes d. lineale d. gouldii d. sylvanum 1 nature of stem cane cylindric cane cylindric cane cane cylindric cane cylindric cane cylindric 2 leaf shape lenceolate lanceolate lanceolate elliptic ovate lanceolate 3 apexes of leaf acute obtuse acute acute acute obtuse 4 apexes of dorsal sepal acuminate obtuse acuminate acute obtuse obtuse 5 apex of lateral sepal acuminate obtuse acuminate acute obtuse obtuse 6 apex of petal acuminate obtuse acuminate acute obtuse obtuse 7 petal curvature spiral deflexed spiral straight deflexed deflexed 8 lip shape ovate undulate ovate ovate oblong obovate 9 lip margin entire undulate entire undulate undulate undulate 10 dorsal sepal shape narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic 11 lateral sepal shape narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic 12 petal shape narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic narrow elliptic 13 dorsal sepal cross section narrow elliptic moderately convex strongly concave strongly concave strongly concave moderately convex 14 lateral sepal cross section narrow elliptic moderately convex strongly concave strongly concave strongly concave flat 15 petal cross section narrow elliptic straight strongly concave strongly concave strongly concave moderately concave 16 lip overlapping of basal part present absent present present absent absent 17 lip shape of lateral lobe tringular broad trapezoid ovate tringular ovate triangular 18 lip shape of apical lobe rhombic rhombic rhombic reniform rhombic elliptic 19 lip type of curving type i type 1 type 1 type i type vi type iv 20 lip shape of eye type i type 1 type 1 type i type ii type i 21 color of anther cap yellow yellow yellow yellow yellow yellow tini et al. ‐ genetic parameters of indonesian dendrobium section spatulata 243 sylvanumit is elliptic (table 2). there was diversity in the quantitative flower parameters observed (table 3). the length of the flowers in d. sylvanum is smaller compared to other species, measuring only 2.36 cm, but the width of the flowers reaches 5.00 cm. this indicates that the petals and sepals extend outward. this flower type is also similar to d. gouldii, characterized by a length of 3.43 cm and a width of 5.26 cm. d. antennatum and d. stratiotes have petal shapes resembling antlers, resulting in a longer length than width. besides that, in d. antennatum, the length is 4.75 cm, and the width is 2.15 cm. for d. discolor and d. lineale, the proportions of flower length and width are more or less the same. the flower size of dendrobium studied showed that d. stratiotes had the largest flower length, namely 9.33 cm compared to other species. this is related to the longer petals on d stratiotes, namely 6.23 cm. besides that, in d. discolor, the length is 4.50 cm, and the width is 4.00 cm. in d. lineale, the length is 5.15 cm, and the width is 6.16 cm. leaves leaves characterization included leaf shape, apex­ es of leaf, leaf length (cm), leaf width (cm), and leaf area (cm2). the variability in the leaf shape of dendrobium was d. antennatum, d. discolor, d. stra‐ tiotes, and d. sylvanum, being lanceolate. in contrast, d. lineale has an elliptic shape, and d. gouldii has an ovate shape. variability in the apexes of leaves in d. antennatum, d. stratiotes, d. linelae, d. gouldii is acute, d. sylvanum and d. discolor are obtuse. variability in leaf length is not wide, namely around 10.93 ­ 13.67 cm. the narrowest leaf width is d. antennatum, which is 2.50 cm, while the widest is d. discolor, which is 4.13 cm. the difference in leaf width affects the leaf area, the largest leaf on d. dis‐ color is 47.30 cm2, while the smallest leaf is d. anten‐ natum, namely 25.13 cm2. pseudo bulb there is variation in the height among the observed dendrobium species. specifically, in d. stra‐ tiotes and d. gouldii, the height exceeds 100 cm, reaching 108.50 cm and 156 cm, respectively. in con­ trast, d. lineale reaches a height of 90 cm, while d. antennatum and d. discolor have heights of 51.33 cm and 57.33 cm. the lowest height is recorded in d. syl‐ vanum at 34.60 cm. additionally, the length of pseu­ do bulb internodes varies across different table 3 ­ quantitative characters of dendrobium species studied no. characters d. antennatum d. discolor d. stratiotes d. lineale d. gouldii d. sylvanum 1 plant height (cm) 51.33 57.33 108.5 90.00 156.00 34.6 2 pseudobulb diameter (cm) 38.3 43.00 45.00 25.2 21.67 22.5 3 internode length (cm) 8.1 12.80 11.6 4.13 5.13 4.4 4 leaf length (cm) 12.2 13.6 13.26 10.93 13.67 12.00 5 leaf width (cm) 2.5 4.13 2.96 3.83 2.83 3.5 6 leaf area (cm2) 25.13 47.3 34.2 36.00 35.96 33.6 7 peduncle­ovary length (cm) 2.66 2.2 3.4 2.06 2.4 1.6 8 length of inflorescence (cm) 12.00 32.00 20.00 58.00 50.00 20.00 9 length of flower arrangement (cm) 5.00 26.00 14.5 48.00 41.00 14.00 10 leaf thickness (mm) 2.3 1.03 1.7 1.43 1.9 1.2 11 flower length (cm) 4.75 4.5 9.33 5.15 3.43 2.36 12 flower width (cm) 2.15 4.00 2.7 6.16 5.26 5.00 13 dorsal sepal length (cm) 2.00 2.5 1.98 2.1 1.6 1.56 14 dorsal sepal width (cm) 0.4 0.7 1.00 1.3 0.83 0.6 15 lateral sepal length (cm) 1.85 2.5 2.6 2.9 1.9 2.36 16 lateral sepal width (cm) 0.65 0.6 0.93 1.16 0.56 0.63 17 petal length (cm) 3.8 4.3 6.23 4.4 3.00 2.93 18 petal width (cm) 0.15 0.7 0.35 0.8 1.06 0.8 19 labellum length (cm) 1.65 2.5 3.7 2.7 2.23 2.26 20 labellum width (cm) 1.05 1.00 0.96 1.5 0.7 0.63 21 number of flowers per spike 6.00 13.00 8.3 25.00 19.67 12.00 244 adv. hort. sci., 2024 38(3): 239­248 dendrobium species. the lengths of d. discolor and d. stratiotes measure 12.80 cm and 11.60 cm, respec­ tively. d. antennatum exhibits a length of 8.10 cm, while the shortest internodes are observed in d. lin‐ eale, d. gouldii, and d. sylvanum at 4.13 cm, 5.13 cm, and 4.40 cm, respectively. this suggests that plant height does not necessarily corellate to longer pseudobulb internodes in dendrobium species. besides that, the diameter of the pseudobulb varies among different dendrobium species. in d. antenna‐ tum, d. discolor, and d. stratiotes, the pseudobulb diameter reaches 38.30 cm, 43.00 cm, and 45.00 cm, respectively. meanwhile, for d. lineale, d. gouldii, and d. sylvanum, it is around 25.20 cm, 21.67 cm, and 22.50 cm. genetic parameters the analysis of variance revealed a significant effect of dendrobium species on all observed traits. a low coefficient of variation indicates that the variation in the data from the average is relatively small compared to the average value (table 3). this early finding suggested the presence of genetic diversity within the dendrobium species. the variance attributed to genotypes was highly significant for all the studied characteristics, indicating that the selected genotypes were genetically different (table 4). genetic parameter estimation was conducted to verify the presence of genetic variability within the dendrobium section spatulata were observed. the high values of broad­sense heritability (hbs) and genotypic coefficient of variation (gcv) suggest a substantial genetic influence on phenotypic variability. the genetic variability coefficient (table 5) showed that the flowering characters, namely flower stalk length (24.86), length of inflorescence (56.43), flower series length (74.40), flower length (47.91), flower width (36.08), dorsal sepal width (38.99), lateral sepal width (31.29), petal length (29.39), petal width (51.72), labellum length (27.12), labellum width (31.38), number of florets (50.10) are included in the high genetic variability category. other vegetative characters are leaf width (45.43), leaf thickness (28.24), leaf area (51.77), plant height (57.66), stem diameter (42.72) also characterized by high genetic variability. besides that, the length of dorsal sepal (17.63) and lateral sepal (17.07) was categorized as moderate genetic variability, whereas the length of leaf (8.43) was categorized as low genetic variability. table 4 ­ analysis of variance (mean square) for quantitative characters in dendrobium species studied characters mean square cv (%) replication genotype error peduncle­ovary length (cm) 0.035555556 1.116888889 0.058222222 10.10063 length of inflorescence (cm) 1.791666667 989.3 0.691666667 2.585478 length of flower arrangement (cm) 0.1666666667 885.125 0.166666667 1.768585 leaf length (cm) 0.157222222 3.527555556 0.136555556 2.930227 leaf width (cm) 0.093888889 16.32588889 0.041888889 3.991354 leaf thickness (mm) 0.002222222 0.659222222 0.050888889 14.14823 leaf area (cm2) 21.86166667 2476.621333 11.763 6.194563 plant height (cm) 141.5555556 6649.747222 57.32222222 9.313237 pseudobulb diameter (mm) 15.73388889 130.8982222 2.509888889 10.34715 internode length (cm) 0.035 0.569 0.055 5.350284 flower length (cm) 0.223888889 17.07922222 0.392222222 12.72345 flower width (cm) 0.292638889 7.282138889 0.345305556 13.94501 dorsal sepal length (cm) 0.000688889 0.358688889 0.000555556 1.203245 dorsal sepal width (cm) 0.010555556 0.300555556 0.004555556 8.378672 lateral sepal length (cm) 0.005138889 0.504138889 0.020138889 6.03166 lateral sepal width (cm) 0.00125 0.171916667 0.002916667 7.121693 petal length (cm) 0.010555556 4.404888889 0.025222222 3.863069 petal width (cm) 0.001116667 0.335383333 0.00145 5.9037 labellum length (cm) 0.000416667 1.396583333 0.00775 3.509664 labellum width (cm) 0.000416667 0.28458333 0.00375 6.280743 number of flowers per spike 8.166666667 152.5333333 4.9 15.81139 tini et al. ‐ genetic parameters of indonesian dendrobium section spatulata 245 all traits observed in this study had high heritability (table 6). if genetic factors rather than environmental influences primarily determine a trait, it is more likely to have high heritability. traits controlled by a few genes with large effects, known as major genes, are often highly heritable. table 6 ­ heritability of in the dendrobium species studied table 5 ­ coefficient of genetic variability (cgv), and coefficient of phenotypic variability in the dendrobium species studied characters range mean cgv category cpv category peduncle­ovary length (cm) 1.60 ­ 3.40 2.38 24.86 high 26.84 high length of inflorescence (cm) 12.50 ­ 59.00 32.17 56.43 high 56.49 high length of flower arrangement (cm) 5.00 ­ 48.00 23.08 74.4 high 74.42 high leaf length (cm) 10.80 ­ 14.00 12.61 8.43 low 8.92 low leaf width (cm) 2.40 ­ 9.00 5.12 45.43 high 45.6 high leaf thickness (cm) 0.80 ­ 2.40 1.58 28.24 high 31.58 high leaf area (cm2) 23.70 ­ 112.40 55.37 51.77 high 52.14 high plant height (cm) 24.60 ­ 156.00 81.29 57.66 high 58.41 high pseudobulb diameter (cm) 7.30 ­ 28.60 15.31 42.72 high 43.96 high internode length (cm) 3.50 ­ 5.40 4.38 9.44 low 10.85 moderate flower length (cm) 2.30 ­ 10.00 4.92 47.91 high 49.57 high flower width (cm) 2.00 ­ 7.50 4.21 36.08 high 38.68 high dorsal sepal length (cm) 1.60 ­ 2.50 1.95 17.63 moderate 17.67 moderate dorsal sepal width (cm) 0.30 ­ 1.30 0.8 38.99 high 39.88 high lateral sepal length (cm) 1.60 ­ 3.00 2.35 17.07 moderate 18.1 moderate lateral sepal width (cm) 0.50 ­ 1.20 0.75 31.29 high 32.09 high petal length (cm) 2.80 ­ 6.30 4.11 29.39 high 29.64 high petal width (cm) 0.10 ­ 1.10 0.64 51.72 high 52.06 high labellum length (cm) 1.50 ­ 3.80 2.5 27.12 high 27.35 high labellum width (cm) 0.60 ­ 1.60 0.97 31.38 high 32 high number of flowers per spike 6.00 ­ 25.00 14.00 50.1 high 52.54 high characters σ2e σ2g σ2f h2bs category peduncle­ovary length (cm) 0.0582 0.3528 0.4111 85.83 high length of inflorescence (cm) 0.6916 329.53 330.22 99.79 high length of flower arrangement (cm) 0.1667 294.98 295.15 99.94 high leaf length (cm) 0.1365 11.303 12.668 89.22 high leaf width (cm) 0.0418 5.428 54.698 99.23 high leaf thickness (cm) 0.05 0.202 0.253 79.93 high leaf area (cm2) 11.763 821.61 833.38 98.58 high plant height (cm) 57.32 2197.475 2254.797 97.45 high pseudobulb diameter (cm) 2.509 42.796 45.306 94.46 high internode length (cm) 0.055 0.1713 0.2263 75.69 high flower length (cm) 0.392 5.562 5.954 93.41 high flower width (cm) 0.3453 23.122 26.575 87 high dorsal sepal length (cm) 0.0005 0.1193 0.1199 99.53 high dorsal sepal width (cm) 0.0045 0.0986 0.1032 95.58 high lateral sepal length (cm) 0.02 0.161 0.181 88.9 high lateral sepal width (cm) 0.0029 0.056 0.059 95.07 high petal length (cm) 0.0252 14.598 14.851 98.3 high petal width (cm) 0.00145 0.11311 0.11276 98.71 high labellum length (cm) 0.00775 0.4629 0.4706 98.35 high labellum width (cm) 0.00375 0.0936 0.0973 96.14 high number of flowers per spike 4.9 49.21 54.11 90.94 high adv. hort. sci., 2024 38(3): 239­248 246 4. discussion and conclusions morphological characteristics in the context of orchids, flowers typically consist of three outer floral parts known as sepals and three inner floral parts called petals. the petals are often more colorful and visually striking than the sepals. they play a crucial role in attracting pollinators, such as insects, and contribute to the overall aesthetic appeal of the orchid flower. the arrangement and characteristics of petals are important features used in the identification and classification of orchid species (dirks­mulder et al., 2017) in addition to attracting pollinators, orchid petals, along with other floral parts, may also have specialized structures or markings that aid in the orchid’s reproductive process, such as facilitating the transfer of pollen. orchids are known for their intricate and diverse floral structures, and the characteristics of their petals contribute significantly to their overall beauty and ecological function (li et al., 2021). the labellum is a specialized petal in orchids that stands out from the other floral parts due to its distinct shape, size, and often elaborate structure. it is the modified third petal of the orchid flower, differentiating from the two lateral petals and three sepals (dalayap et al., 2011). the diversity in the part of dendrobium flower studied, can be a source of diversity in plant breedingaimed at increasing the aesthetic value of hybrid dendrobium orchids. studying leaf shape contributes to understanding the phenotypic variation within a species. this information is essential for describing the diversity and range of characteristics exhibited by plants. considerable genetic diversity exists in both the size and shape of leaves among different species and populations within the same species. this diversity was influenced by robust heredity, carrying both genetic and environmental information that contributes to variations (ren et al., 2020). pseudo bulb is a modified form of stem in several types of orchid plants. a pseudobulb in orchids is a specialized, swollen, or bulbous structure that serves as a storage organ for water and nutrients. this structure is a key adaptation to various environmental conditions, particularly in epiphytic and lithophytic orchids (zhang et al., 2018). the pseudobulbs on dendrobium orchids belong to the homoblastic type, characterized by two or more internodes of the same or varying lengths (ng and hew, 2000). furthermore, the nature of stem (pseudobulbs) can vary significantly among different dendrobium species. d. antennatum, d. discolor, d. lineale, d. gouldii, and d. sylvanum typically exhibit cylindrical canes. in contrast, d. stratiotes stands out with its cane­shaped stem, representing a distinctive morphological trait within the genus. the size of the pseudobulb indicates the large carbohydrate reserves in the organ. according to ng and hew (2000), carbohydrate reserves in orchid pseudobulbs are an important part in the initiation of new seedling growth. the large size of the pseudobulb also functions to support the growth of new shoot and flower development. genetic parameters genetic diversity plays an important role in the development of dendrobium breedings with high economic value. it serves as the main germplasm in plant breeding. greater genetic diversity increases the potential for enhancing plants in accordance with the desired traits. this variability offers ample opportunities for plant breeders to choose superior genotypes for improving crops (swarup et al., 2021). high genetic diversity in plants reflects substantial genetic variation, signifying numerous genetic differences between individual plants. this diversity holds promising potential for more effective plant breeding endeavors. moniruzzaman et al. (2012) supports this, emphasizing the critical role of high genetic diversity in breeding programs, particularly in the development of new crop varieties featuring improved traits like exotic flower diversity. genetic variation offers breeders a wide genetic reservoir, enabling the selection and crossbreeding of individuals to produce desirable hybrids. in this study, high cgv values indicate that genetic factors have a significant influence on the observed traits. on the other hand, it is also known that there is a difference in value between cpv and cgv for each lower trait. these findings suggest that the environment has comparatively minor influence on the observed traits. this is in accordance with research of malek et al. (2014) stated that narrow distinction between cpv and cgv in the majority of traits suggests a minimum impact of environmental factors on the manifestation of these traits, increasing the probability of achieving significant selection gains. the observation results showed that all the observed characters showed high broad sense tini et al. ‐ genetic parameters of indonesian dendrobium section spatulata 247 heritability (table 6). this suggests that the variability in these traits is primarily influenced by genetic factors rather than environmental factors (swarup et al., 2021). in the research of singh et al. (2018) on dendrobium orchid, heritability estimates were identified for characteristics such as plant height, leaf count per shoot, quantity of aerial roots, length and thickness of aerial roots, shoot thickness, internodal length, leaf length, and leaf area. heritability values represent the degree of genetic impact on a trait (hadi et al., 2019). heritability of quantitative traits are influenced by multiple genes as well as environmental factors. if a trait is primarily determined by genetic factors rather than environmental influences, it is more likely to have high heritability. traits controlled by a few genes with large effects, known as major genes, are often highly heritable. the potential for improvement through direct selection was indicated by high heritability with high genetic variability flower stalk length, length of inflorescence, flower arrangement length, flower length, flower width, dorsal sepal width, lateral sepal width, petal length, petal width, labellum length, labellum width, number of florets. when heritability is high in plants, it indicates that a significant genetic variation is a primary factor in the observed traits. high heritability typically indicates that the majority of phenotypic variation in a trait can be attributed to genetic factors, and this is frequently associated with the influence of additive genes (amien et al., 2021). additive genes contribute cumulatively to the phenotypic expression of a trait, and when these genes are predominant, heritability tends to be high (beavis et al., 2021). therefore, high heritability is commonly regarded as an indication that genetic factors, especially additive genes, exert a substantial influence on the plant’s traits (karavolias et al., 2020). high gcv and hbs values increase the chances of obtaining dendrobium orchids have superior characters that can be inherited in their phylogeny. genetic variability plays a crucial role in selecting parents for hybridization and breeding initiatives effectively (mazid et al., 2013; mai et al., 2021). acknowledgements we extend our appreciation to research and community service institutions of jenderal soedirman university, purwokerto, indonesia for pro­ viding the necessary financial support that enabled the realization of this project. references aloysius s., purwantoro a., dewi k., semiart i., 2017 ­ improvement of genetic variability in seedlings of spathoglottis plicata orchids through x‐ray irradiation. ­ biodiversitas, 18(1): 20­27. amien s., maulana h., ruswandi d., nurjanah s., 2021 ­ genetic gain and relationship of yield and yield attributes of mutant and cross‐bred stevia (stevia rebaudiana) genotypes. ­ biodiversitas, 22(8): 3119­ 3126. beavis w., merrick l., meade k., campbell a., muenchrath d., fei s.­z., 2021 ­ inheritance of quan‐ titative traits. ­ https://pbea.agron.iastate.edu dalayap r.m., torres m.a.j., demayo c.g., 2011 ­ landmark and outline methods in describing petal, sepal and labellum shapes 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bulb quality in onions. a field experiment was conducted to determine the optimum n rate and application frequency for high onion productivity. treatments com­ prised levels of nitrogen (n): 50, 100, 150, 200, or 250 kg ha­1, and nitrogen application frequency: all at once, twice, three times, or four times. the n rate and application frequency affected growth performance, bulb characters, yield, and bulb quality of onions. the supplement of 150 kg ha­1 n at three times the application frequencies generated the highest marketable yield (46.5 t ha­1) with the highest net benefit (626317 etb ha­1). application of 150 kg ha­1 of inorganic nitrogen with three times the application frequency improves the marketable yield of onions with the highest and most acceptable net benefit. the intensive and economical use of inorganic nitrogen and its frequency of application increased the growth and economic yield of onions in field condi­ tions. 1. introduction intensive nutrient management for onions (allium cepa l.) involves using fertilizer as efficiently as possible. the principle behind nutrient management is balancing soil nutrient inputs with crop requirements (fekadu and dandena, 2006). nitrogen (n) is an essential nutrient whose deficiency limits crop productivity. compared to other vegetables, onions require high amounts of nitrogen and are applied at different times dur­ ing the growth (geisseler et al., 2022). the recovery of fertilizer can be low, 30 to 40% (halvorson et al., 2002; sharma et al., 2012), due to the shallow onion root system. low n fertilizer recovery in onions is, in part, due to the variable amounts of mineral n present in the soil before sow­ ing (brewster, 2008). appropriate n fertilizer management requires knowledge of crop demand and the time it is needed (ipni, 2012). a low rate of n causes a low yield of onions due to a shortage of n required for the chlorophyll pigment that is responsible for photosynthe­ (*) corresponding author: hussien@hu.edu.et citation: dinega t.m., haile a., beshir h.m., 2023 ­ improving onion productivity and producer inco‐ me through nitrogen management. ­ adv. hort. sci., 37(3): 317­327. copyright: © 2023 dinega t.m., haile a., beshir h.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. ethical approvals: this study does not involve experiments on ani­ mals or human subjects. funding: the research was conducted as a work product of the employee, alage teachers vocational education technical college. competing interests: the authors declare no competing interests. received for publication 14 january 2023 accepted for publication 21 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13944 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 317­327 318 sis (jilani et al., 2004; khan et al., 2021). excessive n is hazardous to the environment, weaken the foliage and predispose the plants to pathogenic diseases (geary et al., 2015). these factors decreased the potential of processing photosynthesis that resulted in yield reduction due to a decrease in assimilates in plant leaves (reay et al., 2012). the frequency of n application affects the produc­ tivity of onions (grant et al., 2012). when applying n fertilizer all at once, more n is susceptible to denitri­ fication, leaching, or volatilization. when n fertilizer is applied faster than plants can use it, soil bacteria convert it to nitrate and decrease n use efficiency (zhang et al., 2013). the majority of small­scale farmers use less nitro­ gen and apply it all at once, which is sub­optimal (shura et al., 2022). there is often excessive applica­ tion of n fertilizer by large­scale commercial farmers near harvest. it is assumed that synchronization of crop demand for n fertilizer and time of application with sufficient amounts improves yield of onions and reduces waste of n, reducing production costs and environmental pollution due to unused excess n leaching in different forms (geisseler et al., 2022). however, the optimum amount of n that matches the crop demand during the growth period is little explored. we hypothesized that the low n use effi­ ciency of the onion due to its shallow and sparse root system can be improved by controlled application of n fertilizer during the growth period. therefore, this work was conducted to determine the optimum n rate and proper application frequency during the growth period for economically feasible onion pro­ duction. 2. materials and methods site description the study was conducted at alage agricultural technical vocational educational and training college, near bulbula town, ethiopia, under supple­ mental irrigation (when precipitation is lacking) from june to october 2021. the area is situated between 7°65” n latitude and 38°56” e longitude at 1600 m above sea level in the dry plateau of the southern part of the ethiopian rift valley system. the area is characterized by a bimodal rainfall pattern where a short rainy season occurs during march and april and the main rain starts in june and extends to september, with high rainfall in july and august. the mean annual rainfall is 800 mm, and the annual mean minimum and maximum temperatures are 11 and 29°c, respectively. the soil of the area ranges from sandy loam to sandy clay loam, with some clay loam and a few clay soils, and is slightly alkaline, ph 7.8 (alemayehu and bewket, 2016). physico‐chemical properties of the experimental field soil before planting, five soil samples were randomly taken from the field at a depth of 0 to 20 cm in a zigzag pattern using an auger. samples were mixed to produce a representative composite sample of 1 kg. the soil sample was air­dried and ground to pass 2 and 0.5 mm (for total n) sieves and analyzed for total p, total n, ph, organic carbon (oc), exchangeable cations, and physical properties at the batu agricultural research centre soil laboratory. the soil was a silty clay loam with 18.0% sand, 50.5% silt, and 31.5% clay. the soil was slightly alka­ line in reaction, with a ph (h2o 1:2.5) of 7.82, which is within the range of ideal soil ph for onion bulb pro­ duction (graham et al., 2004). total n, available p, organic carbon (oc), and cec of the soil before plant­ ing were 0.12%, 11.48 mg kg­1, 1.39%, and 31.74 cmol (+) kg­1, respectively. the total n content of the soil was within the range of low, according to havlin et al. (1999). the cation exchange capacity (cec, 31.74 meq/100 g) of the soil was high according to the rat­ ing of jackson (1975). the carbon­to­nitrogen ratio (c:n) was 11.5%. planting materials the onion, cv. bombay red, was used. it is adapt­ ed to areas of 700 to 2000 m above sea level. the size of the bulb of this variety ranges from 85 to 90 g, with a yield potential of 25 to 30 t ha­1 under research conditions (lemma and shimeles, 2003). sources of fertilizers were urea (46% n) and triple super phosphate (tsp) (46% p2o5) for n and phos­ phorus, respectively. the nitrogen fertilizer was applied immediately after weeding. onion seeds were sown 56 days before transplanting on a 1 m wide seed bed 10 m in length (area 10 m2). seedlings were grown under suitable conditions of fertilization, weeding, and pest control. seven days before trans­ planting, seedlings were gradually exposed to field conditions and withheld from the water supply (hard­ ened off) in the nursery, and then manually trans­ planted at 56 days old. the tsp was applied during transplantation. all cultural practices and crop pro­ tection measures (diseases and insect control) were dinega et al. ‐ onion productivity through nitrogen management 319 carried out uniformly for all plots (earo, 2004). treatments, design and experimental procedure treatments were arranged in factorial combina­ tions in a randomized complete block design with three replications. while transplants were being developed, the soil was manually pulverized twice with an oxen­driven plough. after the soil was pulver­ ized, levelling and ridge preparation were done with hoes and spades. rates of n were: 50, 100, 150, 200, or 250 kg ha­1 in band application according to appli­ cation frequencies, which were: naf1 (all at once 1 week after transplanting), naf2 (half of the n at 1 week after transplanting, another half of the n at 21 days after transplanting), naf3 (one­third of the n at 1 week after transplanting, one­third of the n at 21 days after transplanting, and one­third of the n at 42 days after transplanting), or naf4 (one­fourth at 1 week after transplanting, one­fourth of the n at 21 days, one­fourth of the n at 42 days after transplanti­ ng, and one­fourth of the n at 63 days after trans­ planting). treatment combinations were assigned randomly to experimental units within each block. the national blanket recommendation of n fertilizer for onion production is 100 kg ha­1, which could be considered a control treatment. double row planting was done by hand on ridges about 20 cm high at a spacing of 40 cm for water furrows, 20 cm between rows on raised beds, and 5 cm between plants within rows. there were 60 plots corresponding to the 20 treatment combinations with three replications. the unit plot size of the experiment was 2 x 2.5 m (5 m2). blocks were separated by 1.5 m, and the space between each plot within a block was 1 m. in each plot, 10 rows were prepared, and in each row, 50 seedlings were manually planted. generally, 500 onion seedlings were planted per plot. the outer 2 rows on both sides of the plot and the 2 plants at both ends of the rows were border plants. the plants in the six central rows were used for measurements. data collection ten plants were randomly selected from each plot’s central six rows, and data on growth perfor­ mance, quality indicators, and yield components were recorded for each of them. for the data on bulb yield, all of the plants in each plot were harvested. thus, the following information was collected: maturity and growth parameters days to maturity were recorded as the number of days from seedling transplanting to a day at which more than 80% of the plants in each plot showed yel­ lowing of leaves or attained physiological maturity. plant height was measured from the ground to the tip of the leaves on 10 randomly selected plants from the central rows in each plot at maturity. leaf length was recorded at physiological maturity from the sheath to the tip of the leaf from the third youngest leaves of ten representative plants, which was used to count the number of leaves per plant using a ruler. leaf diameter was measured from the third­youngest leaves at the bottom, middle, and tip parts of the leaves from ten randomly selected plants using a veneer caliper. leaf number per plant was counted as the total number of leaves from 10 randomly selected plants at maturity, and the average of the ten plants was taken. the aboveground biomass was harvested by cutting the plant at the crown part, dry­ ing it in an oven at 650°c until a constant weight was attained, and the shoot dry matter was determined and expressed in grammes at harvest. additionally, the total dry biomass was determined by summing the shoot and bulb dry weights of the sample. yield and yield related parameters the mean bulb diameters of ten sample bulbs were measured at the maximum wider portion of matured bulbs using calipers. the bulb length of ten sample bulbs was measured along the length of the bulb from the basal end to the top end, at which the bulb neck was removed from matured bulbs using calipers after harvest. the average fresh weight of ten randomly taken mature bulbs was measured using a sensitive balance and finally expressed in grammes. fir bulb dry weight ten bulbs were ran­ domly taken from each plot and chopped, mixed thoroughly, placed in an aluminum paper bag, and put in the oven to dry at 650°c until a constant dry weight was attained. then each sample was immedi­ ately recorded as a bulb’s dry weight. the bulb dry matter concentration (%) was determined by ran­ domly selecting ten bulbs from each plot and chop­ ping them, mixing them thoroughly, then weighting them and recording the fresh weight. then each sub­ sample was placed in an aluminum paper bag and put in an oven at 650°c until constant dry matter was attained. each sub­sample was then immediately weighed and recorded as a dry matter yield. the dry matter concentration was determined using the loss weight, and the fresh sample was weighed to the nearest gramme using the formula set by ruck (1969) and dantata (2014): adv. hort. sci., 2023 37(3): 317­327 320 marketable bulb yield (t ha­1) was determined from the weight of healthy and marketable bulbs that range from 20 g to 160 g in weight (lemma and shimeles, 2003). the marketable yield was deter­ mined from the net plot at the final harvest. unmarketable bulb yield (t ha­1) was measured as the total weight of unmarketable bulbs that are under­ sized (<20 g), diseased, decayed, and bulbs from plants with physiological disorders such as thick neck and split were measured from a net plot at final har­ vest. undersized bulb yield (t ha­1) was determined by taking under sized bulbs (<20 g) as unmarketable bulbs per net plot and converted to t ha­1 as deter­ mined. total bulb yield (t ha­1): the total bulb yield was measured from the total harvest of net plot as a sum weight of marketable and unmarketable yields that was measured in kg per plot and finally convert­ ed into t ha­1. harvest index (%) was expressed as the ratio of total bulb dry weight to the total biomass dry weight and expressed in percentage. the tss was determined at harvesting time from ten randomly selected bulbs per plot using the proce­ dures described by waskar et al. (1999). aliquot juice was extracted using a juice extractor, and 50 ml of the slurry was centrifuged for 15 minutes. the tss was determined by a hand refractometer (atago tc­ 1e) with a range of 0 to 32 brix and resolutions of 0.20 brix by placing 1 to 2 drops of clear juice on the prism, washing it with distilled water, and drying it with tissue paper. data analysis data were subjected to analysis of variance using stat­8 software (ver. 8.1.1, analytical software, tallahassee, fl). assumptions of anova were tested, and no violation was observed. the data analysis was done with a generalized linear model. the n rate and application frequency were fixed effects, and the block was random. if the interaction was significant, it was used to explain the results. if the interaction was not significant, the main effects were separated using lsd (walter and duncan, 1969). economic analysis a partial budget analysis was made to determine the exact rate of return that producers gain on their investment by changing existing cultural practices to alternative ones. the potential response of added fertilizer corresponding to labor costs and the price of fertilizers (variable costs for urea fertilizer at 16.2 birr) throughout the crop growing season was evalu­ ated. the birr is the local currency. 1 birr is equal to 0.031 us during the growth period. the economic outcome was analyzed using agronomic indices for n and its frequencies. the economic analysis was com­ puted using accepted procedures (cimmyt, 1988), where: avy (gross average bulb yield, average yield of each treatment); ajy (adjusted yield, the average yield adjusted downward by 10% to reflect the differ­ ence between experimental yield and yield of the farmers); gfb (gross field benefit determined by mul­ tiplying field price that farmers receive for the crop by adjusted yield); nfb (net field benefit calculated by subtracting total costs from gross field benefit; gfb for each treatment); and mrr (marginal rate of return %) calculated by dividing change in net benefit by the marginal cost reflecting change in cost (cim­ myt, 1988). 3. results and discussion the main effects of n rate and application fre­ quency influenced plant height, leaf length, leaf width, and number of leaves. the interaction effect of two factors was not significant for these parame­ ters. plant height increased with increasing n rate from 50 to 250 kg ha­1 n. the tallest and shortest onion plants were at 250 kg ha­1 and 50 kg ha­1, respectively. onions treated with n at 250 kg ha­1 exceeded their mean height by 34.23% compared with the 50 kg ha­1 n­treated onions (fig. 1a). in response to an n application frequency, the tallest plants were for 3 time’s n application (table 1). application of n at all frequencies (once, twice, and four times) was not different for plant height. the increase in plant height with the addition of higher n fertilizer could be attributed to the increased avail­ ability of n for growth as a result of protein synthesis and the accumulation of carbohydrates (rizk, 2012). the tallest plants recorded at three applications of n may be because of increased n use efficiency, decreasing n loss by leaching and volatilization (rizk, 2012). application of n three times better matched the availability of n with crop n demand compared to application all at once, or 2 or 4 applications of n (brewster, 2008). this result is consistent with the findings of morsy et al. (2012) and nasreen et al. (2007), who reported onion plant height increased as dinega et al. ‐ onion productivity through nitrogen management 321 n fertilizer rate increased. shorter leaves were from the 50 kg ha­1 n that decreased by about 31.56% and 33.95% compared to values obtained from 200 and 250 kg ha­1 n, respec­ tively (fig. 1). the effects of 100 and 150 kg ha­1 n were similar. the influence of n rate at 200 and 250 kg ha­1 was not significant for leaf length. the longest and shortest leaves were when n fertilizer was applied three times and all at once, respectively (fig. 1b). these values agree with rao et al. (2013), who reported that higher n fertilization increased onion leaf length, and khan et al. (2002), where the longest leaves were when n was applied three times. increased leaf length at three times n applications could be due to increased recovery of n by onions and decreased n loss (ali and ceyhan, 2001). the narrowest leaves were from plants treated with 50 kg ha­1 n. leaf diameter increased with increased n, from 50 to 150 kg ha­1. the n rates of 150, 200, and 250 kg ha­1 had similar leaf diameters (fig. 1a). leaf diameter was influenced by the fre­ quency of n applications. three times n application increased leaf diameter by 38.99% compared to applying n all at once (fig. 1b). the increase in leaf diameter with an increase in n rate from 50­150 kg means followed by the same letters within a column are not significantly different at p<0.05. table 1 ­ interaction effects of n rate and application frequency on bulb size, marketable bulb yield, total bulb yield, bulb dry weight, total dry biomass, and total soluble solids of onions fig. 1 ­ the effect of nitrogen rate (a) and application frequency (b) on plant height, leaf length, leaf diameter and leaf number of onion. treatments average bulb size (g plant­1) marketable bulb yield (t ha­1) total bulb yield (t ha­1) bulb dry weight (g plant­1) total dry biomass (g plant­1) total soluble solids (°brix)nitrogen rate (kg ha­1) nitrogen applica­ tion frequency 50 once 31.55 k 25.75 j 28.00 k 5.01 i 3.65 i 9.27 i twice 40.23 j 31.17 hi 33.17 ij 6.12 h 3.83 h 10.17 h three times 44.25 ij 33.58 gh 35.00 hij 7.08 g 4.58 gh 10.47 fg four times 43.44 ij 30.43 i 32.38 j 7.45 g 4.94 g 10.03 gh 100 once 53.00 hi 29.52 i 32.33 j 8.72 f 6.00 f 10.73 efg twice 56.27 gh 34.42 fg 36.00 ghi 8.95 f 6.15 ef 11.05 def three times 57.88 fgh 39.48 de 41.12 de 10.15 e 6.92 de 11.59 cd four times 63.52 defg 36.27 fg 38.00 fg 9.19 f 6.13 ef 11.0 2def 150 once 62.92 efgh 30.78 hi 33.32 ij 10.42 e 7.37 cd 10.62 fg twice 82.33 b 45.17 bc 46.97 b 12.01 cd 8.83 b 11.06 def three times 94.00 a 46.95 a 48.84 a 13.95 a 9.79 a 12.30 b four times 66.39 cdefg 43.5 bc 45.16 bc 11.39 d 7.85 c 11.14 def 200 once 69.67 cde 33.75 fgh 36.18 gh 11.67 d 7.87 c 10.42 fg twice 75.35 bc 42.5 bcd 44.37 bc 12.15 cd 8.19 bc 11.36 de three times 70.29 cde 42.50 bc 45.08 bc 12.95 b 8.81 b 12.36 b four times 70.79 cde 43.83 bcd 45.57 bc 12.79 bc 8.25 bc 11.37 de 250 once 66.00 defg 36.73 ef 39.33 ef 11.93 cd 7.87 c 10.73 efg twice 67.92 cdef 42.92 bc 39.32 ef 12.74 bc 8.19 bc 11.15 def three times 73.51 bcd 47.88 bcd 45.04 bc 12.77 bc 8.81 b 13.13 a four times 69.59 cde 43.92 ef 43.60 cd 12.72 bc 8.25 bc 12.24 bc lsd 10.28 3.124 2.84 0.9 0.9 0.74 α ** ** ** * ** ** cv (%) 9.88 5.02 4.33 5.17 7.64 4.02 322 adv. hort. sci., 2023 37(3): 317­327 ha­1 and 3 applications of n could be associated with a better supply of n and better n use efficiency. application of n with three or more applications and a higher supply of n could increase leaf thickness, capture resources for photosynthesis, and promote better growth and development. the lowest leaf diameter was due to the low n rate applied to onions (abdissa et al., 2011; woldeyohannes et al., 2013; seid et al., 2014). increasing n from 50 to 150 kg ha­1 increased leaf number. beyond 150 kg ha­1, the number of leaves decreased. onion leaf number increased as the n fer­ tilizer rate increased from 50 to 150 kg n ha­1 (fig. 1a). increasing n frequency from one to three appli­ cations increased leaf number. at 4 n applications, leaf number decreased (fig. 1b). this may be due to a lack of enough nitrogen at the early growth stage at which leaf formation was initiated. when n is applied four times during the growth period, a quarter of it will not be used by the onion plants for the initiation of leaf formation due to late application (geisseler et al., 2022). increases in the number of leaves with a further increase in the rate of n could be attributed to enhanced photo­assimilate production, cell divi­ sion, and vegetative growth (suthar, 2009). the n plays a role in leaf production and vegetative growth (nasreen et al., 2007). increases in the number of leaves per plant with up to 3 applications of n may be attributed to increasing n use efficiency (geisseler et al., 2022). this indicates that one­third of the n fertilizer in the first application was enough for leaf number production and vigorous vegetative growth, which agrees with mengel et al. (2006). bulb diameter and bulb length were influenced by n rate and n application frequency, but the interac­ tion of n rate and frequency was not significant. increasing the n rate from 50 to 150 kg ha­1 increased bulb diameter by 43.66%. increasing the n rate beyond 150 kg ha­1 did not increase bulb diameter (fig. 2b). the widest bulbs were for the 3­times n application (fig. 2b), which agrees with nasreen et al. (2007). development of wider bulbs with increasing frequency and rate of n fertilizer could be associated with the availability of more growth resources due to efficient n use as bulbs develop. increasing n rate and split application are associated with promoting cell elongation, above­ground vegetative growth, and the synthesis of chlorophyll, resulting in dark green leaves (geisseler et al., 2022). these results agree with soleymani and shahrajabian (2012) and ghaffoor et al. (2003). increasing the rate of n application from 50 to 150 kg ha­1 increased bulb length. at n rates of 150, 200, or 250 kg ha­1 bulb lengths were similar (fig. 2a). increasing application frequency up to three times increases bulb length. when all n fertilizer was applied at once, bulb length was reduced (fig. 2b). the reason for the longest bulbs at 150 kg ha­1 n could be the supply of optimum n (fageria and baligar, 2005). the increase in bulb length at 3 n applications might be due to the recovery of n by onions and its subsequent use for growth (singh and chaure, 1999; bahadur and singh, 2005; mengel et al., 2006). bulb neck thickness was only affected by the n rate. the widest bulb necks were due to the applica­ tion of 250 kg ha­1 n, and the narrowest bulb neck diameter was 50 kg ha­1 n (fig. 2a). the reason for the widest neck diameter at 250 kg ha­1 n rate might be due to high n that resulted in excessive vegetative growth and delayed maturity, resulting in a large neck size (grant et al., 2012; morsy et al., 2012). the increase in neck diameter might show its involve­ ment in the synthesis of amino acids, as they link together to form proteins and make up metabolic processes required for plant growth, including neck thickening (jilani, 2004). fig. 2 ­ the effect of nitrogen rate (a) and application frequency (b) on bulb diameter, bulb length and bulb neck of onion. dinega et al. ‐ onion productivity through nitrogen management 323 the main effects of n rate and n application fre­ quency influenced the shoot dry matter weight and harvest index of onion plants. the interaction effect of an n application and its application frequency was not significant. the unmarketable yield was only affected by the n rate. bulb dry matter, dry total bio­ mass weight, bulb fresh weight, marketable bulb yield, total soluble solids, and total bulb yield were affected by the interaction effect of n rate and appli­ cation frequency in addition to the main effects. the highest shoot dry matter weight was 250 kg ha­1 n. as the n rate increased from 50 to 250 kg ha­1, the shoot dry matter of onions increased by 50.51% (fig. 3a). this might be due to excessive vegetative growth that resulted from the application of excess n (kandil et al., 2013). the lowest shoot dry weight yield on 50 kg ha­1 n might also be because of n defi­ ciency, which limits cell division and expansion, chloroplast development, chlorophyll concentration, and enzyme activity (soleymani and shahrajabian, 2012). the highest shoot dry matter weight per plant was at 3 n applications. the lowest shoot dry matter weight was from treatment at n applied once (a). shoot dry matter yield from 3 applications of n increased by 17.42% compared to shoot dry matter produced when n was applied at one time (fig. 3b). three applications of n fertilizer increased the n use efficiency of onions (sharma, 1992). the present find­ ing agrees with nasreen et al. (2007), who indicated the shoot dry matter weight increased with an incre­ ment of n fertilizer rate applied three times. the harvest index was increased from 63.74 to 71.51% as n was increased from 50 to 150 kg ha­1. beyond 150 kg ha­1 n, the harvest index decreased (62.2% at 250 kg ha­1 n) due to increase above­ ground dry biomass compared to the bulb weight of onions. the harvest index recorded at 50 kg ha­1 n was similar to that recorded at 200 and 250 kg ha­1 n (fig. 3a). the average harvest index across all n rates was 66.2%. the highest harvest index at 150 kg ha­1 n might be due to increased bulb weight due to enough n sufficient for photosynthesis and assimilate pro­ duction that increased bulb dry weight with optimum above ground biomass resulting in a higher harvest index (geisseler et al., 2022). the low harvest index from treatment with 50 kg ha­1 was due to low bulb dry matter weight due to n deficiency (nasreen et al., 2007). the low results obtained from 200 and 250 kg ha­1 might be because of etiolated growth of above­ ground biomass and low bulb growth performance resulting from the application of excess n (negash et al., 2009; abdissa et al., 2011). with increasing frequency of n up to 3 applica­ tions, the unmarketable bulb yield of onions decreased. the highest value of unmarketable bulb yield was all n applied once. this was followed by the 4n applications. the lowest unmarketable bulb yield was 3 n applications (fig. 3b). the unmarketable yield from the application of all n at once was exceeded by the unmarketable yield from three applications of n. this may be due to the loss of n because of volatilization and leaching of n, which decrease n use efficiency if n supply is sufficient. sustainable application of n during the active growth period can decrease unmarketable bulb yield (balemi et al., 2007; biesiada and kołota, 2009; soleymani and shahrajabian, 2012). increasing the rate of nitrogen application up to 150 kg ha­1 increased the average bulb weight by increasing the frequency of nitrogen application up to three times. the highest average fresh bulb weight was recorded at an n rate of 150 kg ha­1 applied three times (table 1). this treatment approximated the expected bulb weight for the cultivar. the lowest bulb size was for the 50 kg ha­1 n all applied at once (table 1). application of 150 kg ha­1 n three times exceeded the lowest fresh bulb weight recorded at 50 kg ha­1 n all applied at once by 66.4%. the increase in fresh bulb weight with an increase fig. 3 ­ the effect of nitrogen rate (a) and application frequency (b) on shoot dry matter, harvest index and unmarketable yield of onion. adv. hort. sci., 2023 37(3): 317­327 324 in nitrogen fertilizer rate might be due to a sufficient supply of nitrogen that enhances cell division and expansion, chloroplast development, chlorophyll concentration, and enzyme activity. the increase in fresh bulb weight with increasing nitrogen applica­ tion frequency up to 3 times might be due to the increasing matching of the crop demand for n with the plant requirement for n (khan et al., 2002; yadav et al., 2003). when all of the nitrogen is supplied ahead of crop growth, more of that nitrogen is sus­ ceptible to denitrification, leaching, or volatilization (brady, 1985). increasing the n from 50 to 150 kg ha­1 increased the production of marketable bulbs across the board by increasing the n application frequency up to three times. beyond that, marketable bulb yield was not increased. the highest marketable bulb yield was from onions, which provided 150 kg ha­1 n in 3 appli­ cations. the lowest marketable bulb yield was in response to 50 kg ha­1 n applied at once (table 1). this might be attributed to an optimum rate of n fer­ tilizer and sustained application of n in relation to the demand of the crop, with reduced loss of n. split applications can be timed to match the n available with crop demand. this reduces the residence time of fertilizer n in the soil and the risk of n being lost. the marketable bulb yield of onions per unit area is a function of the n dose supplied and the application frequency of n fertilizer (naik and hosamani, 2003; latif et al., 2010). a higher marketable bulb yield was achieved at a 150 kg ha­1 rate of n fertilization applied three times (balemi et al., 2007; soleymani and shahrajabian, 2012). total bulb yield increased in response to increas­ ing n rates up to 150 kg ha­1 across increasing fre­ quency of n application up to 3 times. the highest total bulb yield was obtained from onion plants at 150 kg ha­1 n and 3 applications (table 1). the lowest total bulb yield was in response to the application of n at 50 kg ha­1 all at once. the increased total bulb yield in response to 150 kg ha­1 n and 3 applications might be due to plants receiving enough n. high n use efficiency and crop recovery occurred when n was applied 3 times during active growth (tsai et al., 2012). the decrease in total bulb yield with applica­ tion of n beyond 150 kg ha­1 might be due to luxury consumption of n that affects onion plant metabo­ lism by decreasing the ability of the root surface to absorb phosphorus and decreasing the assimilate preparation (mahdieh et al., 2012). the highest bulb yield due to the application of 150 kg ha­1 n applied three times agrees with gebremedhin et al. (2018). enhanced leaf number and length may lead to increased assimilate production and increased bulb yield (geisseler et al., 2022). the dose of n up to 120 kg ha­1 increased total bulb yield, but below this rate, total bulb yield decreased (jilani et al., 2004). a low rate of n applied all at once produced lower total yields compared to higher n doses applied in 3 splits (balemi et al., 2007; soleymani and shahrajabian, 2012). plants treated with 150 kg ha­1 n applied three times produced the highest total dry biomass. plants treated with 50 kg ha­1 n all at once produced the lowest dry total biomass weight (table 1). the total dry biomass from plants treated with 150 kg ha­1 n applied three times was about 64.09% higher than the lowest total dry biomass weight produced by onion plants treated with 50 kg ha­1 n applied all at once (table 1). an increase in total dry biomass in response to an increasing rate of n may be associat­ ed with sufficient supply and efficient use, which enhance vegetative growth and contribute to an improved rate of photosynthesis and assimilate pro­ duction (nasreen et al., 2007; sikder et al., 2010; daniel et al., 2021). total soluble solids increased with increasing n across the frequency of application. the highest total soluble solids were for plants grown at 250 kg ha­1 n with 3 applications. the lowest total soluble solids were for plants grown at 50 kg ha­1 n applied all at once (table 1). total soluble solids from treatment with 250 kg ha­1 n applied three times exceeded total soluble solids obtained at 50 kg ha­1 n applied all at once by about 29.4% (table 1). the possible reason for increasing total soluble solids with a higher applica­ tion rate of n along with increasing n application fre­ quency might be increased chlorophyll content and dry weight per plant (naik and hosamani, 2003; mengel et al., 2006; moursy et al., 2007; morsy et al., 2012). the minimum acceptable mrr is 100% (cimmyt, 1988). the labor cost for applying n fertilizer was increased depending on the labor required for each n fertilizer application frequency. the cost of labor for n fertilizer application was 100, 200, 300, and 400 birr ha­1 for 1, 2, 3, or 4 applications of n fertilizer, respectively. the field price of onions during harvest­ ing was 15 birr per kg. all total variable costs were subtracted from the gross benefit to obtain the net benefit (table 2). dinega et al. ‐ onion productivity through nitrogen management 325 partial budget analysis indicated the highest mrr of 7.295% was from an application of 50 kg ha­1 n rate in 2 applications. for every 1 birr invested in 100 kg ha­1 n applied three times, growers can expect to recover the 1 birr and obtain an additional 7.295 birr. the higher net benefit with an acceptable mrr of 240.50% was from 150 kg ha­1 n applied three times (table 2). the most attractive combinations for farm­ ers were in response to an application of 150 kg ha­1 n, which provided three times the highest mar­ ketable yield and net benefit. onion producers may maximize their net benefit by using 150 kg ha­1 of n applied three times. 4. conclusions the interaction of n rate and application frequen­ cy resulted in the highest total bulb yield (48.84 g plant­1), marketable bulb yield (46.95 t ha­1), total dry biomass weight (13.5 g plant­1), and bulb dry biomass yield (9.7 g plant­1), when the combination of n rate at 150 kg ha­1 and three times application frequencies of n was realized. the highest value of total soluble solids (13.13°brix) was recorded at the treatment combination of 250 n kg ha­1 with three times the application frequency. the partial budget analysis revealed that the highest net benefit of birr 628317 with an acceptable mrr of 240.5% was obtained from the application of n at 150 kg ha­1 and three fre­ quencies of n application. generally, the treatment combination of an n rate of 150 kg ha­1 with three times the n application frequency can be recom­ mended to achieve a high bulb yield of onions with the highest net benefit. acknowledgements the authors acknowledge the management in table 2 ­ cost benefit analysis(z) of nitrogen and its application frequencies (z) the analysis was performed according to accepted procedures (cimmyt, 1988). (y) adj. yield = adjusted marketable yield downward by 10%. (w) d = dominated (any treatment that has net benefits that are less than or equal to those of a treatment with lower costs that vary is dominated; cimmyt, 1988). n = non­dominated, *= the exchange rate of the us dollar to birr was 38 birr in 2021. nitrogen rate nitrogen application frequency average marketable yield adjusted marketable yield (y) gross field benenit nitrogen cost labour cost for nitrogen application total variable cost net benefit dominance marginal rate of return (%) 50 once 25.75 23.18 347700 1761 100 1861 345839 ­ ­ 50 twice 31.17 28.05 420750 1761 200 1961 418789 n 729.50 50 three times 33.58 30.22 453300 1761 300 2061 451239 n 324.50 50 four times 30.43 27.39 410850 1761 400 2161 408689 d 100 once 29.52 26.57 398550 3522 100 3622 394928 d 100 twice 34.42 30.98 464700 3522 200 3722 460978 n 5.90 100 three times 39.48 35.53 532950 3522 300 3822 529128 n 681.50 100 four times 36.27 32.64 489600 3522 400 3922 485678 d 150 once 30.78 27.43 411450 5283 100 5383 406067 d 150 twice 45.17 40.65 609750 5283 200 5483 604267 n 45.24 150 three times 46.95 42.26 633900 5283 300 5583 628317 n 240.50 150 four times 43.5 39.15 587250 5283 400 5683 581567 d 200 once 33.75 30.38 455700 7044 100 7144 448556 d 200 twice 42.5 38.25 573750 7044 200 7244 566506 d 200 three times 42.97 38.673 580095 7044 300 7344 572751 d 200 four times 43.83 39.45 592750 7044 400 7444 585306 d 250 once 36.73 33.06 495900 8805 100 8905 486995 d 250 twice 42.92 38.63 579450 8805 200 9005 570445 d 250 three times 42.5 38.25 573750 8805 300 9105 564645 d 250 four times 41.25 37.125 556875 8805 400 9205 547670 d adv. hort. sci., 2023 37(3): 317­327 326 charge of the alage teachers vocational education technical college, which partly supported this research by providing field space. reference abdissa y., tekalign t.s., pant l.m., 2011 ­ growth, bulb yield and quality of onion (allium cepa l.) as influenced by nitrogen and phosphorus fertilization on vertisol i. growth attribute biomass 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national academy of science usa, 110: 8375­8380. https://www.sciencedirect.com/science/article/pii/s2666154322000540?via%3dihub" /l "! https://www.sciencedirect.com/science/article/pii/s2666154322000540?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2666154322000540?via%3dihub#! https://www.sciencedirect.com/journal/journal-of-agriculture-and-food-research https://www.sciencedirect.com/journal/journal-of-agriculture-and-food-research https://www.sciencedirect.com/journal/journal-of-agriculture-and-food-research impaginato 281 adv. hort. sci., 2023 37(3): 281­287 doi: 10.36253/ahsc­14018 container volume and doses of maxi­ mum technical efficiency of controlled­ release fertilizer on cordia alliodora seedlings a.g. souza 1 (*), o.j. smiderle 2 1 centro university of maringá, maringá, brazil. 2 brazilian agricultural research corporation embrapa roraima, boa vista, brazil. key words: dickson index, freijó, forest nursery, nitrogen. abstract the objective of this study was to determine the correlation between the morphological characteristics of cordia alliodora seedlings produced as a function of container volume and controlled­release fertilizer (crf) doses under nursery conditions in northern amazon. the experimental design was a 2 x 6 factorial scheme, corresponding to two container volumes (1.8 and 2.2 l) and six doses of forth cote® (0, 1, 2, 4, 8, and 12 g l­1 in medium sand), with five replicates. the evaluations were: height (h), stem diameter (sd), shoot dry mass (sdm), root dry mass (rdm), total dry mass, increment in stem diameter (δsd) and increment in height (δh) obtained from the data collected every fif­ teen days, from transplanting, encompassing the period of plant growth until the end of the experiment (three months), in addition to dickson quality index (dqi). container volume of 2.2 l is suitable for the formation of good­quality cordia alliodora seedlings at 90 days after transplanting. controlled­release fer­ tilizer doses from 8.0 g l­1 are not indicated to obtain seedlings of this species in the northern region of brazil, with quality, regardless of the container volume. 1. introduction the forest­based sector can be described as an important component of the brazilian economy, because it contributes significantly to the gen­ eration of products, taxes, jobs and income (smiderle et al., 2021 a). the growing expansion of this sector has driven forest investors to opt for the cultivation of native species in the state of roraima (smiderle et al., 2022). in this scenario, there arises a challenge to meet the demand for seedlings of high­quality standard and adequate nutritional status for the implementation of reforestation for economic purposes. among the potential species for the implementation of reforestation in the northern region of brazil, cordia alliodora ruiz & pavon (boraginaceae), popularly known as ‘freijó’ and ‘louro­freijó’, component (*) corresponding author: alinedasgracas@yahoo.com.br citation: souza a.g., smiderle o.j., 2023 ­ container volume and doses of maximum technical effi‐ ciency of controlled‐release fertilizer on cordia alliodora seedlings. ­ adv. hort. sci., 37(3): 281­ 287. copyright: © 2023 souza a.g., smiderle o.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 november 2022 accepted for publication 30 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14018 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 281­287 282 of the local flora of the state of roraima, stands out (smiderle and souza, 2022). the species lacks infor­ mation in the national and international literature that can provide important methods and techniques for producing seedlings in quantity and quality (massad et al., 2017). regarding the growth of cordia alliodora , smiderle and souza (2022) reported that under favorable conditions plants in the seedling stage can reach growth in height of approximately two meters in the first year in the field. in turn, height incre­ ments occur between the second and tenth year, and maturity is reached between 5 and 10 years (smiderle and souza, 2022). smiderle et al. (2021 b), in studies with native forest species of roraima, determined that their initial growth is slow and they require more time in the nursery to reach desirable minimum size, which in turn induces the use of larger containers, as well as the addition of adequate fertil­ ization. controlled­release fertilizers (crf) have been described as an alternative to conventional fertilizers for fertilization of seedlings in the nursery phase, because they induce rapid initial growth (wang et al., 2016; ao et al., 2018; shi et al., 2019) and favor sur­ vival and vigor after the field planting phase (fu et al., 2017; shi et al., 2019). currently, research has shown a positive effect of controlled­release fertiliz­ ers on the production of native seedlings in northern brazil. for instance, smiderle et al. (2020) worked with crf and container size and concluded that the crf dose of maximum technical efficiency of 4.71 g l­ 1 in medium sand substrate under container volume of 2.2 l promotes greater increments of shoots, stem diameter and biomass of agonandra brasiliensis miers ex benth. & hook. f. seedlings. for some species native to northern brazil, con­ trolled­release fertilizer has shown negative influ­ ence, as observed by smiderle et al. (2022) when evaluating hymenaea courbaril l. seedlings in a screened nursery. these authors reported that con­ trolled­release fertilizer doses greater than 6.0 g l­1 are not indicated to obtain seedlings suitable for planting in the field and with quality. likewise, mota et al. (2021) also revealed that crf doses above 8.0 g l­1 induce reduction in the variables related to the root system of pau­marfim plants. studies of this nature indicate the need for research related to the appropriate doses of crf, in npk 18­05­09 formulation, as well as the appropriate container volume for producing seedlings of native forest species of roraima, which need to be deter­ mined. in view of the above, the objective was to correlate the morphological characteristics of cordia alliodora seedlings produced as a function of contain­ er volume and controlled­release fertilizer doses under nursery conditions in northern amazon, aim­ ing to obtain quality seedlings. 2. materials and methods the seeds of cordia alliodora used to obtain the seedlings were collected from trees located at embrapa roraima (2°45’22” north latitude, 60°43’55” west longitude and altitude of 80 m), located beside the br­174 highway, km 8, in the municipality of boa vista, state of roraima, brazil. after obtaining the seeds, they were manually processed and then sown in a bed containing washed sand of medium particle size as substrate for seedling emergence. the moisture of the sand substrate was maintained through automated irrigation, with four daily waterings. to irrigate the plants, the field capac­ ity for the amount of substrate was determined before the experiment (1.8 and 2.2 litres); this was then taken as the reference for maintaining the sup­ ply of water to the plants throughout the experimen­ tal period. approximately 12 days after sowing, the seedlings began to emerge and, as soon as they homogeneous­ ly reached an approximate height of 5.0 cm, they were transplanted to polyethylene bags containing medium sand as substrate (inert material) and con­ trolled­release fertilizer (forth cote®), in npk 18­05­ 09 formulation, was incorporated into the surface, according to treatment. then, the plants were arranged in a screened nursery with 50% shading and maintained under sprinkler irrigation three times a day for periods of 5 min. the experimental design adopted was completely randomized in a 2x6 factorial scheme, corresponding to two container volumes (1.8 and 2.2 l of substrate) and six doses of forth cote® (0; 1; 2; 4; 8 and 12 g l­1 of fertilizer), with five replicates, each consisting of five seedlings (one in each container). the morphological attributes evaluated at 90 days after transplantation were: stem diameter (sd, in mm) (5 cm from the plant collar, determined with a digital caliper), shoot height (h) (from the sand level to the seedling apex, measured with a graduated ruler, in cm), and survival rate (%). souza and smiderle ‐ efficiency of controlled‐release fertilization of cordia alliodora 283 subsequently, the plants were collected and divid­ ed into roots and shoots (stem and leaves), and then dried in an oven with forced air circulation, at 70± 5°c, until reaching constant weight. after drying, the dry mass of the different plant parts was individually determined: shoot dry mass (sdm, g plant­1) and root dry mass (rdm, g plant­1), which were summed to obtain the total dry mass (tdm, g plant­1). the results allowed the calculation of the dickson quality index (dqi), following the formula proposed by dickson et al. (1960). dqi = tdm (g) [(h/sd)+ (sdm/rdm)] the increment in stem diameter (δsd) and the increment in height (δh) were obtained from the data collected every fifteen days, during the growing period of the plants, from transplanting to the end of the experiment. possible differences between treatments were checked by analysis of variance (anova) in factorial scheme. variables that showed significant differences were subjected to regression analysis in order to assess the growth response of the plants as a func­ tion of the increasing doses of crf for the two con­ tainer volumes. the dose of maximum technical effi­ ciency (dmte) was calculated using the equation pro­ posed by tiesdale et al. (1993) (y=ax2+bx+c) and by the mathematical model x = ­b/2a. data analysis was performed with sisvar statistical software (ferreira, 2014). 3. results and discussion at the end of the experiment (90 days after trans­ planting), the survival rate of cordia alliodora seed‐ lings was 100% for all treatments. all morphological variables evaluated showed quadratic behavior in the fit of the regression equa­ tions. figure 1a shows the height (h) of seedlings cul­ tivated with the crf dose of maximum technical effi­ ciency of 4.54 g l­1, corresponding to 24.30 cm, rep­ resenting an increase of 17.7% when compared to h of plants cultivated without the addition of crf. smiderle et al. (2020), in a study conducted with pau­ marfim (agonandra brasiliensis) seedlings in sub­ strate, also recorded positive quadratic response for all morphological variables evaluated according to the increase in crf doses up to dose of maximum technical efficiency (dmte), suggesting that the application of doses higher than dmte does not guarantee the absorption and mainly utilization of nutrients by the plant. in addition, dmte for the 1.8 l container was 2.98 g l­1 of crf, resulting in a height of 22.46 cm (fig. 1b). according to smiderle et al. (2022), shoot height combined with stem diameter is one of the most important morphological parameters to estimate the growth of native forest seedlings in northern brazil, after definitive planting in the field. for stem diameter (sd), the maximum estimated value was 4.35 mm at the dmte of 3.37 g l­1 of crf incorporated into the medium sand substrate in the container with volume of 2.2 l (fig. 1b). in turn, the 1.8 l container led to the largest stem diameter (3.95 mm) of cordia alliodora seedlings at the dmte of 5.38 g l­1 of crf (fig. 1b). conversely, without addi­ tion of crf (control) the stem diameter was equal to 3.73 mm in the 1.8 l container. thus, 3.37 g l­1 of crf in the 2.2 l container (fig. 1b) resulted in stem diam­ eter 0.4 mm (10%) higher than the value found in the fig. 1 ­ combinations of crf doses and container volume on the height (a) and stem diameter (b) of cordia alliodora seedlings produced under nursery conditions, boa vista, rr. adv. hort. sci., 2023 37(3): 281­287 284 1.8 l container, which even with 2.01 g l­1 more was not able to promote the same diameter, with a value 0.62 mm higher than that obtained without applica­ tion of crf (3.73 mm), an increase of 16%. determining the volume of container and the dmte of fertilizers allows obtaining the ideal sd for planting the field in a shorter time, so it becomes of paramount importance for the production of cordia alliodora seedlings, both to reduce the period of obtaining commercial seedlings, which is long in the traditional method, and to achieve efficiency in the use and utilization of fertilizer by the plant, thus ensuring maximum increment of plant organs in a short time when performing this management. the highest increment in height ­ δh (fig. 2a) occurred at the estimated dmte of 4.70 g l­1 in the 2.2 l container, corresponding to a height of 21.92 cm, which represents an increase of 22.45% com­ pared to the control treatment (substrate without addition of crf), at 90 dat (fig. 1a). certainly the increment in height was due to the higher dose of the controlled­release fertilizer, as well as the vol­ ume of the container, with a combination between the continuous supply of nitrogen (n) to the plant and factors such as solar radiation and temperature, thus resulting in greater photosynthetic efficiency and the production of new tissues in the plant organs. in addition, the dmte for δsd was 4.64 g l­1, corresponding to a value of 3.45 mm (fig. 2b) in the 2.2 l container, values similar to those reported by mota et al. (2020), who worked with agonandra brasiliensis seedlings under different doses of con­ trolled­release fertilizer and containers of different sizes in substrate and obtained seedlings similar to those of the present study. conversely, cordia alliodora plants produced in the 1.8 l container showed lower δsd (2.94 mm) with the dmte of 5.76 g l­1 compared to those grown in the 2.2 l container at the dmte of 4.64 g l­1 (fig. 2b). high n doses in 1.8 l containers affected the physiological quality of plants, causing negative effects on their development, especially those relat­ ed to seedling diameter (menegatti et al., 2022). mota et al. (2020) commented that the n supply in containers with a volume of less than 1.8 l can easily have a negative effect on native forest species, which is not common with other nutrients. however, a positive response was found for the 2.2 l container, for instance in shoot dry mass (sdm), which had dmte of 5.82 g l­1 of crf, with 24.6% gain in the sdm of cordia alliodora when compared with the container volume of 1.8 l, with dmte of 5.80 g l­1 of crf (fig. 3a). this result is probably related pro­ portionally to the volume of the container, as well as the availability of greater space for root growth, thus ensuring greater expansion of the root system and utilization of nutrients. according to damasceno et al. (2019), shoot dry mass indicates the rusticity of a seedling, and the highest values, obtained in plants grown in the 2.2 l container, with the dmte of 5.82 g l­1 of crf (fig. 3a), represented more lignified and rustic seedlings, with greater guarantee for establishment and sur­ vival in the field. according to figure 3b, the maximum value (3.17 g plant­1) of root dry mass was obtained at the dmte of 5.54 g l­1 of crf in the 2.2 l container, which rep­ resents a gain in root dry mass of 13.0%, compared with the 1.8 l container at the dmte of 3.50 g l­1 of crf. chu et al. (2019) related the decrease in growth characteristics related to the root system to exces­ fig. 2 ­ increments in height (δh) (a, cm) and stem diameter (δsd) (b, mm) of cordia alliodora seedlings as a function of the dose of controlled­release fertilizer, in two vol­ umes of container, produced under nursery conditions, boa vista, rr. souza and smiderle ‐ efficiency of controlled‐release fertilization of cordia alliodora 285 sive and toxic absorption of nutrients. for fu et al. (2017), higher fertilization rates lead to a reduction in variables related to the root system of plants, due to the reduction in ph, which reduces the availability of some nutrients indispensable to the photosynthetic apparatus, such as phosphorus and magnesium, and favors excessive solubilization of elements, such as aluminum, making the substrate highly saline and toxic, especially to the organ in direct contact, the roots. menegatti et al. (2020) tested different crf doses and also found higher production of root dry mass in prunus persica seedlings up to dmte of 4.82 g l­1 of crf; above the dose of maximum technical efficien­ cy, the other treatments became inferior to the con­ trol, that is, there was an inhibitory effect on the ini­ tial growth of the seedlings. regarding the total dry mass (tdm), a gradual increase was observed as a function of the doses up to dmte of 5.75 g l­1 of crf, followed by a reduction with the dose of 8 g l­1 of crf, assuming a logistic form (fig. 4a), regardless of the volume of the con­ tainer in which cordia alliodora seedlings were grown. according to the results obtained for tdm, the increase in the crf doses used caused reduction in its values, indicating a dmte equal to 5.75 g l­1, since positive responses in the gain of tdm were obtained at the dose of 4.0 to 5.75 g l­1 of substrate until 90 days of growth of cordia alliodora seedlings. the quality of cordia alliodora seedlings was esti­ mated using dickson quality index, and the highest estimate was obtained for plants grown in the 2.2 l container, with dmte of 6.24 g l­1 of crf (fig. 4b) incorporated into the medium sand substrate. therefore, the seedlings of this treatment were again considered superior, with greater growth balance. according to smiderle et al. (2021 b), this quality index is a good indicator of initial survival of seedlings in the field, because it considers important character­ istics for evaluating the quality of the seedlings to be fig. 3 ­ shoot dry mass (a, g plant­1) and root dry mass (b, g plant­1) of cordia alliodora seedlings as a function of the dose of controlled­release fertilizer and the container volume, produced under nursery conditions, boa vista, rr. fig. 4 ­ total dry mass (a, g plant­1) and dickson quality index (b) of cordia alliodora seedlings as a function of doses of controlled­release fertilizer and container volume, pro­ duced under nursery conditions, boa vista, rr. 286 adv. hort. sci., 2023 37(3): 281­287 transplanted, considering their robustness and bal­ ance of biomass distribution. according to souza et al. (2018), results obtained for native species of northern amazon are of great interest to producers of seedlings of forest species in the region, since there is an increase in the quality of seedlings produced, which is an advantage at the time of planting, since seedlings with better quality tend to have faster establishment and their growth is favored also in the field, in addition to contributing to minimizing the time of establishment. according to table 1, there was a positive and strong correlation (0.92) between the variables h and sd of cordia alliodora seedlings, which can be attrib­ uted to their cultivation in containers, since the vol­ ume and depth of soil to be explored are limited, making the expenditure of energy and nutrients for root growth in length, as occurs in field growth, unnecessary. for smiderle et al. (2021 a), the positive and strong correlation between h and sd demon­ strates the balance of growth between the height and stem diameter of seedlings. there was also a positive and weak correlation between sd and δh; the correlation is considered weak when it has a coefficient of variation of 0.1≤ p < 0.5 (santos, 2010). the estimate of correlation between tdm and dqi at 90 days after transplanta­ tion was 0.95, a correlation considered positive and strong according to the criterion of santos (2010), with coefficient of variation of 0.8≤p<1. considering the results obtained in this study, it is possible to obtain cordia alliodora seedlings with high quality standard with crf incorporated into the substrate and the container volumes used. in general, the correlation between the dose of maximum technical efficiency and container volume found in this study can be described as dependent on the container volume and crf dose. all this informa­ tion, if considered jointly, allows suggesting the improvement of the traditional system for the produc­ tion of cordia alliodora seedlings in suitable contain­ ers, through the use of fertilization of plants in nursery phase, considering the nutritional efficiency as a func­ tion of the container volume, aiming at better use of the input and reduction in the time for production. 4. conclusions container volume of 2.2 l with controlled­release fertilizer in npk 18­05­09 formulation is suitable for the formation of good­quality cordia alliodora seed­ lings at 90 days after transplanting. controlled­release fertilizer at the maximum tech­ nical efficiency dose of 4.64 g l­1 in 2.2 l container is indicated to obtain cordia alliodora seedlings with greater increment in stem diameter. container volume of 2.2 l at the maximum techni­ cal efficiency dose of 5.75 g l­1 of controlled­release fertilizer led to higher biomass in cordia alliodora seedlings at 90 days after transplanting. controlled­release fertilizer doses from 8.0 g l­1 are not indicated to obtain cordia alliodora seedlings in the northern region of brazil, with quality, regard­ less of the container volume. acknowledgements we thank the national council for scientific and technological development (cnpq) for granting the scientific initiation scholarship (cnpq/embrapa ­ process: 122545/2021­4) to the first author and the research productivity grant to the second author. table 1 ­ correlation matrix between phytotechnical variables, plant height (h), stem diameter (sd), increments in height (δh) and stem diameter (δsd), shoot dry mass (sdm), root dry mass (rdm), total dry mass (tdm) and dickson quality index (dqi) of cordia alliodora seedlings as a function of the doses of controlled­release fertilizer and container volume under nursery conditions, boa vista, rr variables sd δh δsd sdm rdm tdm dqi h 0.92 * 0.87 * 0.58 * 0.83 * 0.79 * 0.83 * 0.77 * sd 0.54 * 0.72 * 0.84 * 0.73 * 0.74 * 0.73 * δh 0.68 * 0.80 * 0.74 * 0.72 * 0.54 * δsd 0.79 * 0.81 * 0.87 * 0.56 * sdm 0.72 * 0.87 * 0.71 * rdm 0.90 * 0.82 * tdm 0.95 * souza and smiderle ‐ efficiency of controlled‐release fertilization of cordia alliodora 287 references ao y., hirst p.m., li g., zhang, r., 2018 ­ combined effects of provenance and slow‐release fertilizer on nursery and field performance of yellowhorn seedlings. ­ silva fennica, 52: 110­117. chu x., wang x., zhang d., wu x., zhou z., 2019 ­ responses of taxus chinensis and phoebe chekiangen­ sis seedlings to controlled‐release fertilizer in various formulations and application rates . ­ iforest ­ biogeosciences forestry, 12: 254­261. damasceno a.s.s., boechat c.l., morais j., gonçalves b.p.s., arauco a.m.s., 2019 ­ soil classes and regional organic residues affect nutrition, morpho‐ physiology and quality of copaiba seedlings. ­ cerne, 25: 131­139. dickson a., leaf a.l., hosner j.f., 1960 ­ quality appraisal of white spruce and white pine seedling stock in nurseries. ­ for. chron., 36: 10­13. ferreira d.f., 2014 ­ sisvar: a 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comparison of 18 iranian caprifig culti­ vars based on some morphological and biochemical parameters b. jamali 1 (*), h. amin 2 1 department of agriculture, minab higher education center, university of hormozgan, bandar abbas, iran. 2 department of plant production, college of agriculture and natural resources of darab, shiraz university, darab, iran. key words: antioxidants, characterization, essential elements, fig, screening. abstract: caprifig is a valuable candidate for fig breeding programs as it typically grows naturally under non­optimal conditions. the present study was carried out to evaluate the biochemical/morphological characteristics of 18 caprifig cul­ tivars indigenous to the darab region/southern iran with 4 replications in a completely randomized block design. from each cultivar, healthy uniform leaf samples and spring fruits were taken and analyzed. our results showed that ‘naneghasem’ had the highest leaf α­tocopherol and polyphenol concentration. the highest leaf ascorbic acid concentration was obtained from the gol khengi cultivar (17.03 μg g­1 fresh weight). also, the contents of chlorophyll, carotenoids, and anthocyanins were significantly different among the studied cultivars. various cultivars had different absorption potentials for essential ele­ ments as macro and micronutrients concentration in the leaves were statistical­ ly different in various caprifigs; ‘naneghasem’ had the highest ca (4.46 mg g­1 dry weight) and fe (67.71 mg kg­1 dry weight) concentration and the highest leaf k concentration (22.46 mg g­1 dry weight) was observed in ‘mahali layzengan’. in conclusion, ‘naneghasem’ was evaluated as a cultivar which seems to be more morphologically­ and biochemically­distant from other studied caprifig varieties and probably more adaptable/tolerant to environmental conditions. 1. introduction the fig, a deciduous tree of the moraceae family and native to the southern arabian peninsula in the subtropical region, is an important hor­ ticultural crop that is grown for dry and fresh consumption. dried figs are an excellent source of amino acids, vitamins, minerals, polyphenols, and crude fiber (5.8%, w/w) (pourghayoumi et al., 2016). satisfactory dry fig production is highly dependent on sufficient pollination. caprifig is the only fig species that has both pistillate and staminate flowers. growers collect the fruits of caprifigs, place them in cans, and hang the cans on smyrna­type figs; blastophaga psenes wasps, which live in the syconium of caprifigs, leave the profichi and enter the female flowers of the edible (*) corresponding author: babakjamali@ymail.com citation: jamali b., amin h., 2022 ­ comparison of 18 iranian caprifig cultivars based on some morpho‐ logical and biochemical parameters. ­ adv. hort. sci., 36(4): 303­314. copyright: © 2022 jamali b., amin h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 july 2022 accepted for publication 14 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13349 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(4): 303­314 304 figs and pollinate their pistillate flowers (rahemi and jafari, 2008). in recent years, fig cultivation has developed rapidly due to the wide adaptability, early fruit and high yield, fewer pests and diseases, and simple man­ agement of this crop in various parts of the world (zhang et al., 2020). this increases the importance of protecting and developing fig germplasm. maintaining possible local varieties for commercial cultivation requires characterization of the available germplasm. this type of thermophilic tree could be useful to adapt to the consequences of global warming, as fig trees can grow in dry regions where many other species may not survive (sugiura et al., 2007). fig trees have not been subjected to extensive breeding programs; consequently, many fig populations have rich diversity that cannot be fully exploited until cor­ rectly identified (perez­jiménez et al., 2012). one of the first steps in breeding is the selection of promising species/cultivars. previous studies show differences between various common fig cultivars (aradhya et al., 2010; perez­jiménez et al., 2012; zhang et al., 2020). these differences suggest varied levels of acclimation and/or tolerance potential to abiotic stress conditions. the plant’s enzymatic and non­enzymatic antioxidant potential plays a vital role in this regard (bonyanpour and jamali, 2020). maintenance of high antioxidant capacity to scav­ enge toxic reactive oxygen species (ros) has been associated with better adaptation of plants to envi­ ronmental conditions (sharma et al., 2012). in addi­ tion, different cultivars have different abilities to uptake nutrients. the concentration of macro­ and micronutrients in plant organs and tissues correlates with the plant’s potential to tolerate prevailing envi­ ronmental conditions (jamali et al., 2016; jamali and bonyanpour, 2017; tian et al., 2021). iran has one of the richest fig germplasm resources in the world; however, the majority of pre­ vious studies have focused on edible commercial cul­ tivars, while studies on caprifig varieties are rare. caprifig can be a valuable resource for breeding pro­ grams because it grows naturally under less than optimal conditions and is not domesticated. strengthening research on caprifig germplasm’s genetic/chemical diversity is of scientific importance for germplasm conservation, efficient breeding, and satisfactory production. typically, cultivar characterization studies involve determining a vast range of morphological, biochemi­ cal, molecular, etc., parameters to find promising varieties for future breeding projects. as part of a series of similar investigations on caprifig cultivars, the present study aimed to compare 18 cultivars indigenous to the darab/southern iran region, focus­ ing on phonological parameters and biochemical characteristics associated with non­optimal growing conditions. 2. materials and methods plant material uniform and healthy plants of eighteen mature autochthonous caprifig cultivars were selected. they were planted and grown in a completely randomized block design with 4 replications; each replication had 4 plants with a row spacing of 2.5 m and an inter­row spacing of 3.5 m in a rain­fed collection orchard in the darab region, fars province, southern iran. average annual climate parameters in the experi­ mental region were: precipitation: 200 mm, relative humidity max: 55%, min: 18%, temperature max: 42°c, min: 4°c. the soil of the orchard was sampled and analyzed for soil texture, mineral content, organ­ ic matter, ph, and ec (table 1). cultivars included: 1­ mashgholamrezai (mgr), 2­darabi (drb), 3­abbasali (asa), 4­khazraie (kzr), 5­ieji (ij), 6­gol khengi (gk), 7­rastaghi (rs), 8­naneghasem (ngm), 9­mahali layzengan (mlz), 10­cheshmbolboli (cmb), 11­ pouzehdonbali (pzd), 12­danesibi (ds), 13­pasras (pr), 14­johari (jr), 15­maseeh (ms), 16­cho (co), 17­pasbehdari (psd), and 18­suzu (sz). leaf samples were taken from different orientations (north, south, west, and east) of the trees; 25 fully expanded mature leaves from each side of all trees (100 leaves per tree as bulk samples) were transported to the table 1 ­ analysis of soil samples in the experimental region soil depth (cm) soil texture soil mineral content (mg kg­1) organic carbon (%) ec (ds m­1) nitrate ca mg k fe zn mn 0­50 loamy clay 37 1110 170 220 7.00 1.7 5.50 0.70 0.72 jamali and amin ‐ characterization of caprifig cultivars 305 laboratory. leaves were of spring bloom, the middle third of the branch, at the height of 1­1.5 m, includ­ ing the petiole. leaves with abnormal symptoms, including mechanical lesions caused by diseases or pests as well as chlorosis, were avoided. routine cul­ tural practices were carried out during the experi­ mental period. measurements the following parameters were determined in caprifig cultivars for two consecutive years, with an average reported. leaf dry matter content three uniform leaves were washed with tap and distilled water. after drying with a clean towel, they were weighed with a digital scale to get fresh weight (fw) and then dried in an oven at 70°c for 72 hours and re­weighed to get dry weight (dw). the percent­ age of leaf dry matter was calculated according to the following formula: leaf dry matters (%) = [leaf dw (g)/leaf fw (g)] × 100 leaf relative water content (lrwc) ten leaf discs from each treatment were weighed (fw), then hydrated to saturation (constant weight) for 48 hours at 5°c in darkness (turgid weight, tw). leaf discs were dried in an oven (dw). the relative water content was calculated according to the fol­ lowing formula (jamali and eshghi, 2015): lrwc (%) = (fw­dw)/(tw­dw) × 100 leaf electrolyte leakage leaf electrolyte leakage (el) was determined by recording leaf leachate’s electrical conductivity (ec) in double­distilled water at 40 and 100°c. leaf sam­ ples were cut into uniform­sized disks and placed in test tubes containing 10 ml of double­distilled water. the test tubes were kept at 40°c for 30 minutes and at 100°c for 15 minutes, and their respective electri­ cal conductivities (ec1 and ec2) were measured using a conductivity­meter (metrohm conductometer 644, switzerland): electrolyte leakage (%) = (ec1/ec2) × 100 leaf soluble carbohydrates leaf samples of 0.5 g (dw) were homogenized in 5 ml ethanol (95%) and centrifuged at 4500 × g for 15 min. the supernatant was removed from the sample, and the residue was resuspended in 5 ml ethanol (70%). the supernatant was centrifuged for final extraction. both supernatants were combined. the anthrone sulfuric acid assay was used for determina­ tion (irigoyen et al., 1992). an aliquot of 100 μl was added to 3 ml of the anthrone­sulfuric acid solution; the mixture was shaken, heated in a boiling water bath for 10 min, then cooled at 4°c. the absorbance at 625 nm was determined spectrophotometrically. glucose (0­100 mg/l, merk) was used as a standard (jamali and bonyanpour, 2017). leaf chlorophyll and carotenoids concentration leaf discs of 0.25 g were extracted in 2.5 ml of acetone (80%) and then centrifuged at 6,000 × g for 10 min. the supernatant was used to prepare a final volume of 50 ml of leaf extract. extraction of the leaf tissue with the buffer was continued until decol­ oration. the absorbance of the extract was measured at 470, 645, and 663 nm spectrophotometrically. acetone (80%) was used as a blank. finally, the con­ tent of chlorophyll and carotenoids was calculated using the following equations (lichtenthaler, 1987): leaf total chlorophyll concentration (mg. g­1 fw)= [(7.15a663 + 18.71a645) × v /1000 × w] carotenoids (mg. g­1 fw): 1000a470­ 1.82chla­ 85.02chlb / 198 a = absorbance at ƛ (nm), w = sample weight. leaf anthocyanins concentration total anthocyanins in the leaves were determined by the ph differential method using a spectropho­ tometer and two buffer systems: potassium chloride buffer, ph 1.0 (0.025 m), and sodium acetate buffer, ph 4.5 (0.4 m). 0.25 g leaf samples were extracted with 1 ml methanol: water: concentrated hcl solu­ tion (80:20:1 v/v/v). 0.4 ml of leaf extract was mixed with 3.6 ml of corresponding buffers and read against water as blank at 510 and 700 nm. absorbance (a) was calculated as a = (a515 ­ a700) ph 1.0 ­ (a510 ­ a700) ph 4.5 then total leaf anthocyanins concentration was cal­ culated using the following equation: anthocyanin (μg. g­1 fw) = (a × mw × df × 1000) / e where a is the absorbance of the diluted sample, df is the dilution factor (10), mw is the molecular weight of cyanidin­3­glucoside (449.2), and e = 26,900 l/mol.cm, the molar extinction coefficient of cyani­ din­3­ glucoside. leaf α‐tocopherol concentration the α­tocopherol was extracted using chong et al. (2004) method. 200 mg lyophilized sample was homogenized in 1 ml acetone at 4°c with a pre­ chilled mortar and pestle. 0.5 ml hexane was added, and the homogenate was first vortexed for 30 s, cen­ trifuged at 1000 × g for 10 min. the upper hexane adv. hort. sci., 2022 36(4): 303­314 306 layer was removed while the acetone layer contain­ ing α­tocopherol remained in the vial. a second aliquot of 0.5 ml hexane was added, and the extrac­ tion process was repeated three times. α­tocopherol was determined by the method of kanno and yamauchi (1997). to 0.2 ml of pooled extract a 0.4­ml aliquot of 0.1% (w/v) 3­(2­pyridyl)­5,6­ diphenyl­1,2,4­triazine was added. the volume was made up to 3 ml with absolute ethanol, 0.4 ml 0.1% (w/v) ferric chloride (fecl3.6h2o) was added, and the content was gently mixed under dim light in a dark room to avoid photo­ chemical reduction. after a 4­minute reaction at room temperature, 0.2 ml 0.2 m orthophosphoric acid was added, and the mixture was left for another 30 min. absorbance was determined spectrophoto­ metrically at 554 nm and reported as μg g­1 fw. the blank was prepared the same way; absolute ethanol was used instead of the sample. α­tocopherol (sigma chemical) was used as a standard. glutathione concentration two hundred mg of tissue was homogenized in 2 ml of ice­cold 5% tca. the homogenate was cen­ trifuged at 17,000 × g for 30 min at 4°c. a volume of 75 μl of the supernatant was added to a cuvette con­ taining 300 μl of phosphate buffer (0.2 m, ph 8.0) and 750 μl of 0.6 mm dtnb (5,5ˊ­dithiobis­ 2­ nitrobenzoic acid) in phosphate buffer. the absorbance at 412 nm was read, and glutathione concentration was derived against a standard curve prepared with known amounts of gsh in 5% tca (moron et al., 1979). leaf total polyphenols this parameter was determined using the folin­ ciocalteu reagent. one gram of freeze­dried leaf samples was placed in an eppendorf tube, mixed with 1 ml of methanol (80%), ground at 4°c, then centrifuged at 10000 × g for 15 minutes. the extract was mixed with 0.5 ml of folin­ciocalteu reagent; diluted 1:1 with water, and then 1 ml of a 5% sodium carbonate solution was added. absorbance was mea­ sured at 725 nm and expressed as mg per g fw after 30 minutes. gallic acid was used as the standard phe­ nolic compound (bonyanpour and jamali, 2020). lipid peroxidation a sample of 0.2 g was homogenized in 2 ml 0.1% trichloroacetic acid (tcaa) solution and then cen­ trifuged at 15,000 × g for 10 min at 4°c, the 0.5 ml of supernatant was added to 1.5 ml 0.5% tba in 20% tcaa, followed by incubation of the mixture at 95 °c in a shaking water bath for 30 min. the tubes were placed in an ice­water bath to stop the reaction. the samples were re­centrifuged at 10,000 × g for 5 min, and the absorbance of the supernatant was deter­ mined spectrophotometrically at 532 nm and cor­ rected for non­specific turbidity by subtracting the absorbance at 600 nm. the malondialdehyde (mda) concentration was calculated using the extinction coefficient of 155mm­1 cm­1 (cakmak and horst, 1991). leaves proline concentration leaf samples were homogenized with 3% sulfosal­ icylic acid. the homogenate was centrifuged at 2500 × g for 20 min. the supernatant was treated with acid ninhydrin and acetic acid, boiled for 60 minutes, and then the absorbance of the solution was record­ ed at 520 nm. leaves proline contents were expressed as μmol·g­1 fw. proline amino acid was used as standard (bates et al., 1973). leaf ascorbic acid concentration this parameter was measured by the method of omaye et al. (1979). to 1 g of freeze­dried leaf sam­ ple, 10% ice­cold tca was added and centrifuged at 3500 × g for 20 min at room temperature. the super­ natant (1 ml) was mixed with 0.2 ml of dtc reagent and incubated at 37°c for 3 h. then 1.5 ml of ice­cold 65% sulfuric acid was added and mixed, and the solu­ tions were allowed to stand at room temperature for another 30 min. the color developed was read at 520 nm spectrophotometrically and reported as μg g­1 fw. macro and micronutrients’ concentration oven­dried leaf samples (0.5 g) were ground and ashed at 550°c in a porcelain crucible for seven h. the white ash was mixed in 2 m hot hcl, filtered, and finally made up to 50 ml with distilled water. nitrogen (n) concentration was measured using the kjeldahl digestion method. potassium (k) concentra­ tion was determined using the flame emission method using a sherwood scientific ltd model 360 flame photometer. an atomic absorption spec­ trophotometer (aa 6200, double beam atomic absorption spectrophotometer shimadzu, kyoto, japan) was used to determine ca, mg, and micronu­ trient element, including fe, zn, and mn concentra­ tions. phosphorus (p) concentration was determined colorimetrically (kalra, 1998). leaf biometrics leaf area was determined using a leaf area meter jamali and amin ‐ characterization of caprifig cultivars 307 (kaiser rs1) and expressed as mm2; leaf and petiole length by using a digital caliper and expressed as mm. fruit fresh and dry weight spring fruits were harvested and weighed using a digital scale and reported as gram. then they were oven­dried and weighed again. experimental design and statistical analysis the experiment was carried out in a completely randomized block design with four replications. data were analyzed by anova test using spss (ver. 9.1); means were compared using duncan’s multiple range test at 5% probability level. cluster analysis was also performed according to ward’s minimum­variance method using spss (ver. 9.1) to classify the cultivars. 3. results the leaf glutathione concentration in ngm (67.01 μg g­1 fw) was nearly 50% higher than ms (32.32 μg g­1 fw). co (64 μg g­1 fw) was not statistically differ­ ent when compared with ngm. the leaf mda con­ centration in kzr (12.12 μg g­1 fw) was lower than ms and co cultivars. other caprifig trees were not statistically different (table 2). the leaf ascorbic acid concentration in the gk cul­ tivar (17.03 μg g­1 fw), was 59% higher than drb (7.14 μg g­1 fw). rs, cmb, pzd, jr, ms, and co culti­ vars were not statistically different in comparison with gk. the leaf proline concentration in ms (6.77 μg g­1 fw) was not statistically different in compari­ son with gk, ngm, cmb, pr, jr, and ms cultivars. the leaf total polyphenols concentration in ngm (26.71 mg g­1 fw), was 56% higher when compared with the mgr cultivar. pzd, jr, and psd were not sta­ tistically different compared to the ngm cultivar (table 2). the leaf dry weight in ngm was 26% higher when compared with mgr. cmb, pzd, ms, co, psd, and sz cultivars were not statistically different in comparison with ngm. leaf total sugars were not statistically dif­ ferent in all cultivars. lrwc in the ngm cultivar was 8.4% higher when compared with ms . ij, gk, mlz, ds, co, and sz were not statistically different in com­ parison with ngm. leaf el in mgr (22.63%) was sig­ nificantly higher than ds (17.62%). this parameter was not statically different in other cultivars (table 3). leaf nitrogen concentration was not statistically different in all cultivars. the leaf p concentration in ds (3.32 mg g­1 dw) was higher in comparison with pr, jr, and co; other cultivars were not different. table 2 ­ the concentration of some non­enzymatic antioxidants and mda in leaves of studied cultivars z means followed by the same letters within columns are not different at 5% probability using duncan’s test. cultivar glutathione (µg g­1 fresh weight) mda (µg g­1 fresh weight) α­tocopherol (µg g­1 fresh weight) ascorbic acid (µg g­1 fresh weight) proline (µg g­1 fresh weight) polyphenol (mg g­1 fresh weight) mgr 45.02 fg z 15.00 abc 92.92 d 7.40 c 2.83 d 17.06 e drb 44.30 fg 16.00 abc 110.41 ab 7.14 c 3.65 cd 17.17 e asa 53.3 de 14.07 bc 102.13 bc 8.88 c 3.33 d 18.19 e kzr 57.20 cd 12.12 c 69.82 e 10.12 bc 4.41 bcd 18.88 e ij 50.10 ef 17.07 abc 97.04 cd 10.21 bc 3.45 cd 22.09 bcd gk 61.11 bc 15.19 abc 72.20 e 17.03 a 5.55 abc 18.29 e rs 53.44 d 15.41 abc 105.45 bc 12.00 abc 4.14 bcd 17.21 e ngm 67.01 a 12.14 c 122.18 a 8.22 c 6.71 a 26.71 a mlz 48.15 ef 13.25 c 114.12 a 9.06 c 2.92 d 19.82 de cmb 40.22 gh 17.15 abc 115.45 a 16.33 a 6.20 ab 18.85 e pzd 52.36 de 18.02 abc 105.31 bc 12.14 abc 4.38 bcd 25.42 ab ds 61.10 bc 14.14 bc 100.05 bc 7.63 c 2.78 d 23.41 bc pr 45.16 fg 13.14 c 77.14 e 10.47 bc 5.61 abc 22.22 bcd jr 37.35 hi 14.20 bc 98.32 cd 12.42 abc 5.99 ab 24.56 ab ms 32.32 i 20.37 ab 77.11 e 15.15 ab 6.77 a 20.37 cde co 64.00 ab 21.07 a 100.07 cd 15.71 ab 4.70 abcd 20.17 cde psd 40.05 gh 15.3 abc 105.50 bc 9.16 c 3.77 cd 20.22 abc sz 41.07 gh 13.33 c 115.00 a 8.05 c 3.42 cd 22.15 bcd 308 adv. hort. sci., 2022 36(4): 303­314 the leaf k concentration in mlz (22.46 mg g­1 dw) which was not statistically different compared with asa, kzr, gk, rs, ngm, pzd, ds, pr, jr, ms, co, psd, and sz. the leaf ca concentration (4.46 mg g­1 dw) in ngm was 25% higher than pr. cmb had the highest leaf mg concentration (2.48 mg g­1 dw); kzr had 42% lower leaf mg concentration. other cultivars were not statistically different (table 4). the leaf fe concentration in ngm (67.71 mg kg­1 dw) was significantly higher than kzr, mlz, pr, jr, ms, psd, and sz. the leaf zn concentration in ngm (27.31 mg kg­1 dw) was not statistically different in comparison with drb, mlz, psd, and sz. leaf mn concentration was not statistically different in all cul­ tivars (table 5). the leaf total chlorophyll concentration in ngm (1.43 mg g­1 fw) was not statistically different com­ pared with drb, asa, kzr, mlz, ds, ms, psd, and sz (fig. 1). the leaf concentration of carotenoids in psd was significantly higher than asa, kzr, gk, cmb, pzd, rs, jr, and ms. other cultivars were not statistically different in comparison with psd (fig. 2). the concen­ tration of leaf anthocyanins in mgr was significantly higher than psd. this parameter was not significantly different in other cultivars (fig. 3). table 3 ­ leaf relative water content, electrolyte leakage, dry matter, and total sugars in studied caprifig cultivars z means followed by the same letters within columns are not dif­ ferent at 5% probability using duncan’s test. cultivar leaf dry matter (%) leaf total sugars (mg g­1 dry weight) leaf relative water content (%) electrolyte leakage (%) mgr 26.22 b z 27.33 a 77.92 d 22.63 a drb 28.30 b 26.05 a 81.21 abc 22.04 ab asa 27.33 b 27.17 a 78.89 bcd 21.87 ab kzr 26.55 b 29.55 a 77.92 cd 19.12 ab ij 27.44 b 26.77 a 83.04 a 20.00 ab gk 28.15 b 27.37 a 80.08 abcd 22.03 ab rs 27.61 b 28.47 a 79.52 bcd 21.43 ab ngm 33.05 a 29.14 a 83.60 a 17.96 ab mlz 28.15 b 30.13 a 81.11 abc 19.06 ab cmb 30.31 ab 28.45 a 79.63 bcd 22.33 ab pzd 29.55 ab 29.02 a 77.32 d 22.34 ab ds 27.22 b 27.37 a 80.29 abcd 17.62 b pr 29.71 ab 28.02 a 77.14 d 20.77 ab jr 28.44 b 30.00 a 78.32 cd 19.72 ab ms 29.81 ab 28.88 a 77.11 d 19.75 ab co 28.88 ab 30.11 a 81.00 abc 21.72 ab psd 29.02 ab 27.14 a 79.67 bcd 19.55 ab sz 28.87 ab 28.64 a 82.22 ab 19.15 ab table 4 ­ leaf macronutrients in studied caprifig cultivars z means followed by the same letters within columns are not different at 5% probability using duncan’s test. cultivar n (mg g­1 dry weight) p (mg g­1 dry weight) k (mg g­1 dry weight) ca (mg g­1 dry weight) mg (mg g­1 dry weight) mgr 22.12 a z 2.60 ab 16.33 c 3.76 bc 2.00 ab drb 20.13 a 3.10 ab 17.16 bc 3.77 bc 2.22 ab asa 20.15 a 3.05 ab 19.65 abc 3.97 abc 2.02 ab kzr 21.41 a 2.52 ab 18.27 abc 4.44 a 1.55 b ij 21.05 a 2.97 ab 17.05 bc 3.65 bc 1.95 ab gk 21.77 a 2.55 ab 21.20 abc 3.80 bc 1.77 ab rs 22.30 a 2.92 ab 18.85 abc 4.20 ab 1.67 ab ngm 22.12 a 3.00 ab 20.38 abc 4.46 a 2.35 ab mlz 19.64 a 3.14 ab 22.46 a 3.95 abc 2.20 ab cmb 20.51 a 2.46 ab 16.44 c 3.67 bc 2.48 a pzd 22.35 a 2.81 ab 18.71 abc 3.72 bc 2.36 ab ds 19.19 a 3.32 a 18.22 abc 3.90 abc 1.83 ab pr 22.66 a 2.45 b 19.70 abc 3.55 c 2.11 ab jr 20.44 a 2.37 b 19.00 abc 3.63 bc 2.07 ab ms 20.11 a 2.88 ab 20.77 abc 4.18 ab 1.61 ab co 22.00 a 2.44 b 18.00 abc 3.76 bc 2.18 ab psd 21.02 a 3.11 ab 21.56 ab 4.02 ab 2.14 ab sz 21.48 a 3.23 ab 22.07 ab 3.89 abc 2.25 ab jamali and amin ‐ characterization of caprifig cultivars 309 the highest fruit fw and dw were obtained from ds. ngm had the highest leaf length (154.66 mm). pr, ms, pzd, cmb, rs, and kzr were not statistically different. the leaf area (11581.2 mm2) and petiole length (66.69 mm) in ngm were significantly higher than all other cultivars (table 6). the cluster analysis based on ward’s method divided the cultivars into three major clusters, includ­ ing 8 cultivars in cluster 1, 9 cultivars in cluster 2, and 1 cultivar in cluster 3. the first cluster consisted of two subclusters: subcluster 1 (mgr, ij, asa, and ds) and subcluster 2 (drb, psd, sz and mlz). cluster 2 also had 2 subclusters: subcluster 1 (kzr, ms, and rs) and subcluster 2 (gk, co, cmb, jr, pzd, and pr). cluster 3 had only one cultivar: ngm (fig. 4). 4. discussion and conclusions non­enzymatic antioxidants were statistically dif­ ferent among investigated cultivars in our study, which was consistent with previous studies. jamali fig. 2 ­ concentration of leaf carotenoids in studied caprifig cul­ tivars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=4). fig. 3 ­ concentration of leaf anthocyanins in studied caprifig cultivars. columns with the same letters are not statisti­ cally different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=4). fig. 1 ­ total chlorophyll concentration in studied caprifig culti­ vars. columns with the same letters are not statistically different at 5% probability using duncan’s test. vertical bars indicate standard errors (n=4). table 5 ­ leaf micronutrients in studied caprifig cultivars z means followed by the same letters within columns are not dif­ ferent at 5% probability using duncan’s test. cultivar fe (mg kg­1 dry weight) zn (mg kg­1 dry weight) mn (mg kg­1 dry weight) mgr 60.66 ab z 23.33 bcd 47.22 a drb 60.47 ab 26.50 ab 50.16 a asa 62.23 ab 22.74 cd 49.44 a kzr 58.36 b 22.46 cd 50.33 a ij 63.33 ab 23.00 bcd 49.31 a gk 64.44 ab 21.20 d 48.56 a rs 60.96 ab 22.85 cd 47.91 a ngm 67.71 a 27.31 a 48.05 a mlz 57.22 b 25.44 abc 50.20 a cmb 61.22 ab 22.07 cd 50.27 a pzd 63.42 ab 23.08 bcd 49.22 a ds 60.75 ab 22.77 cd 48.71 a pr 59.29 b 23.12 bcd 48.05 a jr 57.33 b 21.90 cd 49.66 a ms 59.40 b 22.35 cd 50.23 a co 60.05 ab 22.68 cd 51.44 a psd 58.63 b 24.88 abcd 50.63 a sz 59.34 b 25.14 abc 47.33 a adv. hort. sci., 2022 36(4): 303­314 310 and bonyanpour (2017) determined leaf mineral composition and also some biochemical parameters, i.e., concentration of non­enzymatic antioxidants in leaves, in seven iranian pomegranate cultivars for selecting probable more tolerant cultivars. they found that cultivars with a higher concentration of non­enzymatic antioxidants (e.g., glutathione, α­ tocopherol, proline, etc.) had better adaptability to the prevailing environmental conditions. as men­ tioned earlier, various species and cultivars have inherently different potential to tolerate non­optimal growth conditions. one of the possible reasons for this difference is their enzymatic/non­enzymatic antioxidant responses and/or their ability to absorb macro/micro nutrients. therefore, determination of these characteristics can be used as biochemical markers for cultivar comparison/screening. gholami et al. (2012) compared four fig cultivars (deyme ahvaz, sabz estahban, siah, and shahanjir) under normal, drought, and recovery conditions. according to their results, some of the non­enzymat­ ic and enzymatic antioxidants in leaves differed sig­ nificantly under optimal conditions. their findings show that the fig cultivars demonstrated a clear dif­ ference in their response to water stress and recov­ ery. they evaluated deyme ahvaz as a more tolerant cultivar than sabz estahban. deyme ahvaz had higher leaf concentration of glutathione and anthocyanin in comparison to sabz estahban under non­stress condi­ tions. samec et al. (2021) reviewed the role of phenolic compounds in inducing tolerance to non­optimal conditions in plants. they reported that the potential tolerance of a plant species to stress conditions can be associated with this diverse family of chemicals which is universally present in plants and includes fig. 4 ­ cluster analysis dendrogram of 18 iranian caprifig culti­ vars based on ward's method. table 6 ­ fruit fresh and dry weight and leaf and petiole length, and leaf area in studied caprifig cultivars z means followed by the same letters within columns are not different at 5% probability using duncan’s test. cultivar fruit fresh weight (g) fruit dry weight (g) leaf length (mm) leaf area (mm2) petiole length (mm mgr 4.77 hi z 0.90 fg 78.97 g 1879.5 j 23.45 h drb 5.77 gh 1.09 ef 89.70 fg 3217.3 efg 29.36 fgh asa 6.48 efg 1.20 de 83.54 g 2232.6 hij 23.08 h kzr 7.60 cde 1.21 de 121.63 bc 6352.9 b 37.46 cd ij 4.94 hi 0.95 fg 98.97 ef 2005.9 ij 28.60 fgh gk 8.06 cd 1.48 bc 107.57 de 4433.1 d def 10/33 rs 8.64 c 1.76 a 118.04 bcd 7090.9 b 50.72 b ngm 6.63 efg 0.94 fg 154.66 a 11581.2 a 66.69 a mlz 4.01 i 0.72 g 79.28 g 2814.7 fgh 26.57 gh cmb 7.12 def 1.09 ef 116.12 bcd 3698.2 def 38.30 cd pzd 5.92 fgh 0.96 f 117.25 bcd 4373.1 d 35.95 de ds 12.40 a 1.71 a 98.90 ef 2522.8 ghij 30.79 efg pr 6.39 efg 1.03 ef 113.67 bcd 4554.1 d 37.77 cd jr 6.37 efg 1.07 ef 99.53 ef 3980.3 de 32.84 defg ms 10.88 b 1.58 ab 125.09 b 6518.2 b 42.68 c co 6.76 efg 1.02 ef 112.09 cd 5459.2 c 43.36 c psd 6.67 efg 1.36 cd 81.50 g 3334.1 efg 28.24 fgh sz 4.94 hi 0.9 fg 87.78 fg 2965.6 fgh 29.02 fgh jamali and amin ‐ characterization of caprifig cultivars 311 more than 8000 molecules. phenolic compounds play an essential role in plant responses, especially in defense mechanisms. they have strong antioxidant properties and neutralize the detrimental effects of reactive oxygen species (balasundram et al., 2006). the absolute concentrations of these chemicals in plant organs vary significantly depending on the determination method, cultivar, or tissue, but it can be concluded that higher phenol concentrations cor­ relate with increased stress tolerance. arteaga et al. (2020) have proposed the use of proline as a biochemical marker for rapid and simple large­scale screening of different genotypes for drought and salt tolerance. transgenic plants, espe­ cially those overexpressing genes for proline accumu­ lation, show higher adaptation to abiotic stresses (el moukhtari et al., 2020; ghosh et al., 2022. similarly, endogenous proline concentration has been linked to a relatively higher stress tolerance when comparing different cultivars by various authors in different species (kapuya et al., 1985; misra and gupta, 2005; goharrizi et al., 2020). the lower mda content in ngm, rs, or mlz may be due to the higher content of antioxidants such as α­tocopherol in these two caprifigs. this is consistent with previous studies (assaha et al., 2015; amoah et al., 2019; nawaz and wang, 2020). except for leaf n and mn concentration, other macro and micronutrients were significantly different in various caprifig cultivars. this was in agreement with previous studies on different fig cultivars (anac et al., 1982; aksoy et al., 1987; askin et al., 1998; hakerlerler et al., 1998; bougiouklis et al., 2020). the higher leaf dw or lrwc in ngm could be attributed to the higher leaf concentration of k, ca, fe, or zn in this caprifig in comparison with other studied cultivars. this was in agreement with previ­ ous studies as various cultivars absorb macro/micronutrients differently. hegwood (1972) reported a significant varietal effect on leaf mineral composition during full bloom and harvest in eleven snapbean cultivars. jordão et al. (1999) studied 15 olive cultivars and found that the mean effect of cul­ tivar on leaf concentrations of essential elements, including n, p, k, ca, mg, s, mn, zn, and b, was signif­ icant. there are also many other examples of the effects of cultivar or root system of different species on leaf mineral composition (tsipouridis and thomidis, 2005; north and cook, 2006; tomala et al., 2008; kviklys et al., 2012). this difference in endogenous macro/micronutri­ ent composition can affect numerous aspects of plant growth and development. in other words, the presence of a particular macro­ or micronutrient (e.g., ca) above a critical concentration can alter the plant’s response to non­ optimal growing conditions (pilbeam and morley, 2007; taiz and zeiger, 2010). for instance, fageria (2013) reported an increased root dw in 12 lowland rice genotypes after adding k fertilizer. they found that increase in root dw at high k levels was 246% compared to the low level of k. leaf chlorophyll and carotenoids concentration were significantly different in studied cultivars. chlorophyll concentration is a sensitive biochemical marker indicating cellular metabolic state (chutipaijit et al., 2011). previous studies have shown that chlorophyll concentration and stability correlate with plants’ high tolerance potential against abiotic stress­ es such as salinity or drought (hasanuzzaman et al., 2013). carotenoids protect the photosynthesis appa­ ratus by scavenging free radicals keeping its integrity against photo­oxidative damages (dall’osto et al., 2007; andrade­souza et al., 2011). as an aba precur­ sor, higher carotenoid concentration means lower photo­oxidative damage and elevated potential for regulating plant growth under stress conditions (götz et al., 2002; han et al., 2008). caprifig cultivars in our study were clustered into three main groups indicating lower level of variability within clusters and higher variability levels between clusters. morphological and biochemical markers have long been used for screening and characterizing different genotypes, as they are the first steps in describing/classifying any germplasm (cantini et al., 1999). previous studies on figs show the usefulness of these markers in documenting variability among genotypes (salhi­hannachi et al., 2006; saddoud et al., 2008; podgornik et al., 2010). ngm had the highest leaf glutathione, α­ tocopherol, proline, polyphenols, ca, and zn concen­ trations. leaf length, leaf area, and petiole length were the highest in this cultivar. cluster analysis indi­ cated that ngm was the only cultivar in cluster 3. in conclusion, ngm was evaluated as a genotype which seems to be more distant (morphologically and bio­ chemically) from other studied caprifigs and probably more adaptable/tolerant. our findings can be used as an overture for complementary studies for selecting promising genotypes for breeding and introducing new tolerant fig cultivars. adv. hort. sci., 2022 36(4): 303­314 312 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doi: 10.36253/ahsc­9945 postharvest quality of pepino melon (solanum muricatum aiton) as influ­ enced by npk fertilizer rates, growing environment and storage temperature c. mutua (*), r. gesimba, j. ogweno department of crops, horticulture and soils, egerton university, p.o. box 536, 20115 egerton, kenya. key words: firmness, shelf life, total soluble solids, weight loss. abstract: the present study evaluated the effect of npk fertilizer (17:17:17) rates (0, 100, 200, 300 and 400 kg ha­1) on the postharvest quality of field and greenhouse grown pepino melons (solanum muricatum ait.) stored at room temperature (15­22°c) and at low temperature (7°c). the study was carried out in randomized complete block design with fruits from the field and greenhouse, five npk fertilizer rates as treatments and the two storage temperatures repli­ cated three times. data were collected on percentage fruit weight loss (pwl), total soluble solids (tss), firmness and shelf life. results indicated that green­ house and field grown fruits from the control and plants supplied with 100 kg npk ha­1 had low pwl at both storage temperatures. field grown fruits from the control stored at room temperature had the highest tss and were firmer after 28 days of storage. field grown fruits not supplied with fertilizer and stored at low temperature had a shelf life of 27 and 26 days in trial one and two respectively. application of 100 kg npk ha­1 and storage of pepino melon fruits at low temperature can be used to enhance quality and shelf life. 1. introduction pepino melon (solanum muricatum aiton) is a little­known vegetable crop which belongs to the family solanaceae. it originated from the tropi­ cal and subtropical region of andes and is grown for its edible fruits (heiser, 1964). pepino melon fruits are aromatic, juicy, scented, mild sweet, and vary in size, shape and colour depending on the cultivar (martinez­romero et al., 2003). the fruits mature 30 to 80 days after pol­ lination and the skin is usually golden yellow with purple stripes (nuez and ruiz, 1996). several studies have reported significant losses in horti­ cultural produce after harvest (toktam et al., 2019). such losses are caused by dehydration, decay, and physiological disorders during posthar­ vest handling. fresh fruits and vegetables also undergo rapid transforma­ tion in nutritional and sensory quality after harvest, some of which con­ tribute to loss of market value (ahmad and siddiqui, 2015). the losses can (*) corresponding author: carolmutua713@yahoo.com citation: mutua c., gesimba r., ogweno j., 2021 ­ postharvest quality of pepino melon (solanum muricatum aiton) as influenced by npk fertilizer rates, growing environment and storage tempe‐ rature. ­ adv. hort. sci., 35(1): 21­32 copyright: © 2021 mutua c., gesimba r., ogweno j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 october 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9945 http://www.fupress.net/index.php/ahs http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 21­32 22 be reduced through good management of pre­ and postharvest factors (toktam et al., 2019). postharvest quality is also affected by climatic fac­ tors such as temperature and light intensity, and other pre­harvest factors like soil type, fertilization, irrigation, mulching, and other cultural practices (toktam et al., 2019). temperature affects growth and development of fruits and vegetables as well as cellular compounds, their structure and this in turn affects produce firmness (toktam et al., 2019). fertilizers have also been shown to influence postharvest quality of most fruits and vegetables. the type of fertilizer used and the amount applied will dictate the quality of the resulting vegetables (arah et al., 2015). application of potassium fertiliz­ ers on tomato has been shown to improve fruit colour, reduce the occurrence of yellow shoulder and enhance titratable acidity (passam et al., 2007). on the other hand, application of high doses of nitroge­ nous fertilizers to greenhouse grown tomatoes reduces fruit quality by reducing total soluble solids (passam et al., 2007). temperature management between the time of harvesting and consumption has been shown to be effective in maintaining the quality of harvested veg­ etables. high temperatures increase metabolic activi­ ties and ethylene production but this is dependent on other factors like oxygen or carbon dioxide levels, time of exposure and the ripening stage (de wild et al., 2003). storage of vegetables at low temperature slows down metabolic processes and hence extends the shelf life of horticultural produce (arah et al., 2015). the present study sought to investigate the effect of npk fertilizer rates, growing environment and storage temperature on the postharvest quality of pepino melon. 2. materials and methods experimental site description the experiment was conducted at the horticulture research and teaching field, egerton university, njoro. the field lies at a latitude of 0° 23’ south, longitudes 35° 35’ east in the lower highland iii agro ecological zone (lh3) at an altitude of approximately 2,238 m above sea level. average maximum and minimum temperatures range from 19°c to 22°c and 5°c to 8°c, respectively, with a total annual rainfall ranging from 1200 to 1400 mm. the soils are predominantly mollic andosols (jaetzold and schimdt, 2006). the greenhouse used was 8 m by 60 m and the covering material was polythene with a thickness of 12×150 microns purchased from amiran kenya ltd. the mean monthly temperatures in the greenhouse and field during the experiment are pre­ sented in table 1. plant material and experimental design pepino seedlings (ecuadorian gold variety) were obtained from garlic and pepino farm, nakuru and planted in the field and greenhouse. the experimen­ tal design was randomized complete block design (rcbd) with fruits from the five npk fertilizer treat­ ments, the two growing environments and two stor­ age temperatures replicated three times. the five npk fertilizer treatments were [0, 100, 200, 300 and 400 npk (17:17:17) kg ha­1], two growing environ­ ments (field and greenhouse) and two storage tem­ peratures (room and low temperature). mature green pepino fruits were harvested from the field and greenhouse experiments and stored at low tem­ perature (7°c) in a refrigerator and at room temper­ ature (15­22°c) in the biotechnology laboratory of table 1 ­ average monthly field and greenhouse temperature (°c) in trial one and two experimental conditions temperature 2018 2019 trial one nov dec jan feb mar apr may field 20.9 19.7 20.9 21.7 22.8 22.6 21.2 greenhouse 30.3 21.0 33.4 30.2 29.4 34.0 35.8 2019 2020 trial two july aug sept oct nov dec jan field 19.1 19.2 20.5 19.3 19.3 18.9 19.1 greenhouse 18.5 29.4 30.0 28.0 32.0 28.0 35.3 mutua et al. ‐ pepino melon postharvest studies 23 egerton university. the experimental design was rcbd consisting of fruits from the five treatments replicated three times. the experiment therefore comprised of five treatments similar to those of the field and greenhouse experiments each replicated three times giving a total of thirty experimental units each represented by a plastic tray. each experimen­ tal unit comprised of twenty pepino fruits randomly selected from the harvest of the individual respec­ tive treatments in the field and greenhouse experi­ ments. data collection and analysis data were collected and recorded on percentage fruit weight loss (pwl), firmness, total soluble solids (tss) and shelf life. to determine pwl, five fruits in each replication for each treatment were marked before storage and weighed using a digital balance (hangping ja 12002, japan). the same fruits were weighed at the beginning of the experiment and weighing continued at an interval of 7 days for 28 days. the results were expressed as the percentage loss of initial weight using the formula: percentage weight loss = initial weight­final weight × 100 initial weight fruit firmness was determined using hand held penetrometer (model 62/dr, uk) from the begin­ ning of the experiment and continued at an interval of 7 days for 28 days. the results were reported in kg force. tss was determined on the same fruits used for determination of firmness using a hand held refractometer (0­30°brix) (rhw refractometer, optoelectronic technology company ltd, uk) was used as per the procedure described by tigchelaar (1986). results were expressed as °brix. this was done at the beginning of the experiment and con­ tinued at an interval of 7 days for 28 days. the shelf life of pepino fruits was determined by counting the number of days at which at least 50% of the fruits had reached senescence and were not marketable (too soft, wrinkled or with fungal rots). quality evaluation was done using a rating scale of 1­5 (miguel and marita, 1996). data collected were subjected to analysis of variance (anova) and significant means separated using tukey’s honestly signif icant difference (tukey’s hsd) test at p≤0.05. the sas statistical package (sas institute, 2005) was used for data analysis. 3. results percentage weight loss (pwl) npk fertilizer rates, growing environment and storage temperature had a significant effect at p≤ 0.05 on pwl of pepino fruits after 28 days of storage in trial one. during this trial, highest pwl of 10.863% and 15.77% were recorded in greenhouse grown fruits from plants supplied with 300 and 400 kg npk ha­1 during production as well as in field grown fruits from plants supplied with 400 kg npk ha­1 regardless of the storage temperature (table 2). the lowest pwl was, on the other hand, recorded in greenhouse and field grown fruits without npk fertilizer applica­ tion (control) stored at low temperature although the difference in weight loss for this treatment was not significantly different from that of other treatment combinations. in trial two, npk fertilizer rates, grow­ ing environment and storage temperature had a sig­ nificant effect on pwl from day 7 to day 28 of the study. on day 28, greenhouse and field grown fruits from plants supplied with 400 kg npk ha­1, stored at room temperature had the highest pwl of 19.38% and 15.54% respectively (table 2). it was noted that as the fertilizer rates increased the pwl also increased in both growing environ­ ments and storage temperatures in both trials. generally, fruits stored at low temperature had lower pwl compared to those stored at room temperature in both trials. greenhouse grown fruits also had a higher pwl compared to field grown fruits in both trials. total soluble solids (tss) npk fertilizer rates, growing environment and stor­ age temperature had a significant effect at p≥0.05 on tss of pepino fruits from day 7 to day 28 in both tri­ als. in trial one, field grown fruits from plants which were not supplied with fertilizer (control) and stored at room temperature had the highest tss from day 7 to day 28. the highest tss was recorded after 28 days of storage where field grown fruits from plants not supplied with npk fertilizer and stored at room tem­ perature had a tss of 8.67 °brix which was significant­ ly higher than that recorded from fruits from all other treatment combinations. greenhouse grown fruits from plants supplied with the highest fertilizer rate of 400 kg npk ha­1 and stored at low temperature had the lowest tss of 4.40°brix after 28 days (table 3). in trial two, field grown fruits from plants not supplied with npk fertilizer (control) and stored at room tem­ adv. hort. sci., 2021 35(1): 21­32 24 * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. storage temperature environment fertilizer (kg ha­1) weight loss day 7 day 14 day 21 day 28 trial one room temperature field 0 1.067 2.050 2.837 3.987def 100 1.423 2.800 3.620 5.303 def 200 1.980 3.580 4.713 6.383 cdef 300 2.500 5.147 5.717 9.120 bcd 400 4.417 7.280 11.353 12.457 ab greenhouse 0 1.213 1.840 2.820 3.413 ef 100 2.037 4.317 5.590 7.047 cde 200 2.673 5.540 7.160 8.913 bcd 300 3.167 6.400 8.720 10.863 abc 400 5.810 9.327 11.410 15.770 a low temperature field 0 0.523 1.043 1.283 1.557 f 100 0.917 1.720 1.997 4.300 def 200 1.057 2.170 2.763 2.310 ef 300 1.740 3.883 4.040 7.120 cde 400 2.737 5.497 8.020 15.123 a greenhouse 0 0.543 1.087 1.630 2.173 ef 100 1.063 1.353 2.070 2.633 ef 200 1.543 2.417 2.867 4.012 def 300 2.240 2.977 5.080 3.183 ef 400 3.950 6.107 9.067 8.630 bcd trial two room temperature field 0 0.940 ij 1.173 kl 1.443 kl 4.187 fghi 100 1.893 fghi 2.633 fghijk 3.123 fghij 7.100 defg 200 2.507 efgh 3.130 defghi 4.590 cdefg 8.723 cde 300 3.667 bcd 4.773 cde 5.653 cd 11.690 bc 400 4.550 b 7.363 b 10.883 b 15.543 ab greenhouse 0 1.156 hij 1.670 ijkl 1.943 ijkl 3.360 fghi 100 3.067 cdef 3.077 efghi 4.230 defgh 7.293 def 200 3.703 bcd 4.333 cdef 5.327 cd 9.290 cd 300 4.113 bc 4.813 cd 6.190 c 11.600 bc 400 6.617 a 10.477 a 14.703 a 19.397 a low temperature field 0 0.663 j 0.917 l 1.283 l 1.653 i 100 1.047 ij 1.420 ijkl 1.997 ijkl 2.243 hi 200 1.970 efghi 2.173 hijkl 2.783 hijkl 2.487 hi 300 2.720 defg 2.887 fghij 3.533 efghi 3.017 ghi 400 4.127 bc 3.997 cdefg 4.740 cdef 5.967 defgh greenhouse 0 1.607 ghij 1.337 jkl 1.720 jkl 2.190 hi 100 2.313 efgh 2.473 ghijkl 2.993 ghijk 4.593 efghi 200 3.117 cde 3.520 defgh 4.223 defgh 5.330 defghi 300 3.693 bcd 4.163 cdefg 4.823 cde 4.880 efghi 400 4.473 b 5.487 c 6.20 c 5.930 defgh table 2 ­ effect of npk fertilizer rates, growing environment and storage temperature on percentage weight loss of pepino fruits in trial one and two mutua et al. ‐ pepino melon postharvest studies 25 storage temperature environment fertilizer (kg ha­1) tss (°brix) day 0 day 7 day 14 day 21 day 28 trial one room temperature field 0 4.00 6.00 a 6.93 a 7.83 a 8.67 a 100 4.00 4.37 cde 5.73 b 6.13 c 6.40 bcde 200 4.00 4.53 cde 4.97 c 5.73 cd 6.13 cdef 300 4.00 4.53 cde 4.90 c 5.47 de 5.80 defg 400 4.00 4.17 de 4.67 cde 4.80 ghi 5.20 ghi greenhouse 0 4.00 5.00 b 6.00 b 6.77 b 7.07 b 100 4.00 4.40 cde 4.90 c 5.47 de 5.80 defg 200 4.00 4.47 cde 4.90 c 5.23 ef 5.67 fgh 300 4.00 4.17 de 4.63 cde 4.86 fghi 5.10 ghij 400 4.00 4.10 e 4.30 ef 4.53 hijk 4.80 ij low temperature field 0 4.00 5.00 b 5.63 b 6.00 c 6.83 bc 100 4.00 4.60 bcd 4.83 c 5.13 efg 5.77 efg 200 4.00 4.43 cde 4.63 cde 4.97 fg 5.27 ghi 300 4.00 4.30 de 4.40 def 4.73 ghij 4.97 hij 400 4.00 4.10 e 4.13 f 4.33 jk 4.56 ij greenhouse 0 4.00 4.77 bc 5.00 c 5.90 c 6.50 bcd 100 4.00 4.60 bcd 4.77 cd 4.93 fghi 5.23 ghi 200 4.00 4.47 cde 4.63 cde 4.80 ghi 4.97 hij 300 4.00 4.30 de 4.33 ef 4.50 ijk 4.73 ij 400 4.00 4.17 de 4.10 f 4.20 k 4.40 j trial two room temperature field 0 4.00 5.60 a 6.70 a 7.60 a 8.133 a 100 4.00 4.50 cde 5.36 c 5.67 d 6.10 cd 200 4.00 4.33 defg 4.97 cde 5.26 ef 5.77 def 300 4.00 4.17 efg 4.53 fghi 4.80 ghi 5.00 hij 400 4.00 4.60 g 4.13 ijk 4.27 j 4.50 jkl greenhouse 0 4.00 4.70 bc 5.23 c 6.07 c 6.50 bc 100 4.00 4.50 cde 4.80 def 5.10 efg 5.50 efgh 200 4.00 4.33 defg 4.97 cde 4.87 ghi 5.13 ghi 300 4.00 4.13 fg 4.53 fghi 4.60 ij 4.90 ijk 400 4.00 4.03 g 4.07 k 4.27 j 4.47 kl low temperature field 0 4.00 5.27 a 5.83 b 6.47 b 6.93 b 100 4.00 4.60 bcd 5.00 cde 5.37 de 5.83 def 200 4.00 4.30 defg 4.70 efg 4.93 fghi 5.43 fgh 300 4.00 4.13 fg 4.50 fghij 4.70 hi 5.00 hij 400 4.00 4.07 fg 4.10 jk 4.27 j 4.60 jkl greenhouse 0 4.00 4.87 b 5.17 cd 5.67 d 6.00 cde 100 4.00 4.50 cde 4.77 defg 5.00 efgh 5.53 efg 200 4.00 4.30 defg 4.53 fghi 4.73 ghi 5.00 hij 300 4.00 4.13 fg 4.37 ghijk 4.60 ij 4.83 ijk 400 4.00 4.03 g 4.13 ijk 4.23 j 4.30 l table 3 ­ effect of npk fertilizer rates, growing environment and storage temperature on tss (°brix) of pepino melon fruits in trial one and two * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p ≤ 0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. perature had the highest tss from day 14 to day 28. on day 7, field grown fruits from plants not supplied with npk fertilizer and stored at room or under low temperature had the highest tss of 5.6 and 5.27 °brix, respectively (table 3). the highest tss was recorded after 28 days of storage where field grown fruits from plants not supplied with npk fertilizer and stored at room temperature had a tss of 8.13 °brix which was significantly higher than for greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored 26 adv. hort. sci., 2021 35(1): 21­32 at low temperature with a tss of 4.3°brix. it was observed that tss increased as the storage time pro­ gressed and decreased as the fertilizer rates increased regardless of the environment under which the fruits were produced and the temperature during storage. generally, field grown fruits had higher tss compared to greenhouse grown fruits regardless of the storage temperatures in both trials. on the other hand, fruits stored at low temperature had lower tss values com­ pared to those stored at room temperature in both trials. firmness npk fertilizer rates, growing environment and storage temperature had a significant effect on firm­ ness of pepino melon fruits on day 7 and day 28 in trial one, and day 21 and 28 in trial two. in day 7 of trial one, field grown fruits from plants which were not supplied with npk fertilizer and stored at either room or low temperature had the highest firmness of 4.67 kg f and 4.83 kg f, respectively. however, this was not significantly different from the firmness of 4.57 kg f recorded for greenhouse grown fruits har­ vested from plants not supplied with npk fertilizer and maintained under low temperature during stor­ age. on day 28, the highest firmness was recorded in field grown fruits from plants not supplied with npk fertilizer and stored at low temperature with a firm­ ness of 3.83 kg f. the lowest firmness of 0.52 kg f was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature after 28 days of storage (table 4). in trial two, npk fertilizer rates, growing environ­ ment and storage temperature had a significant effect on firmness of pepino melon fruits at day 21 and day 28 of storage. in day 21 of storage, field grown fruits from plants not supplied with npk fertilizer and stored at low temperature had the highest firmness of 4.13 kg f but this was not significantly different from the firmness of 3.93 kg f recorded from green­ house fruits from plants not supplied with npk fertiliz­ er and stored at low temperature, field grown fruits from plants not supplied with npk fertilizer stored at room temperature with a firmness of 3.70 kg f and field grown fruits from plants supplied with 100 kg npk ha­1 maintained under low temperature with a firmness of 3.67 kg f (table 4). the lowest firmness of 1.70 kg f was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 stored at low temperature but this was not significantly differ­ ent from the other treatment combinations. generally, it was observed that field grown fruits were firmer compared to greenhouse grown fruits and fruits stored at low temperature were firmer compared to those stored at room temperature. firmness also decreased as the fertilizer rates and storage days increased. shelf life npk fertilizer rates, growing environment and storage temperature had a significant effect (p≤0.05) on the shelf life of pepino melon fruits in both trials. in trial one, field grown pepino fruits from plants which were not supplied with npk fertilizer (control) and stored at low temperature (7°c) had the longest shelf life of 27 days. field grown fruits from plants supplied with 100 kg npk ha­1 stored at low tempera­ ture had shelf life of 22 days but this was not signifi­ cantly different from the shelf life of greenhouse grown fruits from plants not supplied with npk fertil­ izer and those supplied with 100 kg npk ha­1 and maintained at low temperature with a shelf life of 21 and 19 days respectively (table 5). the lowest shelf life of 11 days was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature although this was not significantly different from that of field grown fruits from plants supplied with 300 and 400 kg npk ha­1 and stored at room temperature, greenhouse grown fruits from plants supplied with 200 and 300 kg npk ha­1 and stored at room temperature and field grown fruits from plants supplied with 400 kg npk ha­1 and stored at low temperature. in trial two, field grown pepino fruits from plants not supplied with npk fertilizer and stored at low temperature had the longest shelf life of 26 days, fol­ lowed by field grown fruits from plants supplied with 100 kg npk ha­1 stored at low temperature with a shelf life of 21 days. the shelf life recorded for fruits from this treatments was, however, not significantly different from that of greenhouse grown fruits from plants not supplied with fertilizer, those supplied with 100 kg npk ha­1 and stored at low temperature and field grown fruits from the control and stored at room temperature (table 5). the lowest shelf life was recorded in greenhouse grown fruits from plants sup­ plied with 400 kg npk ha­1 stored at room tempera­ ture with a shelf life of 10 days but this was not signif­ icantly different from the shelf life recorded for green­ house grown fruits from plants supplied with 200 and 300 kg npk ha­1 stored at room temperature and that of field grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature. generally, it was observed that fruits stored at mutua et al. ‐ pepino melon postharvest studies 27 low temperature had a longer shelf life than those stored at room temperature. field grown fruits had a longer shelf life compared to greenhouse grown fruits. the shelf life decreased as the npk fertilizer rates increased. 4. discussion and conclusions there was a progressive increase in percentage fruit weight loss as the storage days advanced. field and greenhouse grown fruits from plants which table 4 ­ effect of npk fertilizer rates, growing environment and storage temperature on firmness (kg) of pepino melon fruits in trial one and two storage temperature environment fertilize (kg ha­1) firmness (kg) day 0 day 7 day 14 day 21 day 28 trial one room temperature field 0 5.00 4.67 a 4.03 3.60 3.03 b 100 5.00 3.90 c 3.27 3.03 2.77 bc 200 5.00 3.57 cde 3.20 2.87 2.37 cdef 300 5.00 3.03 ef 2.57 2.00 1.57 hijk 400 5.00 2.83 f 2.33 1.50 1.02 kj greenhouse 0 5.00 3.97 bc 3.57 2.90 2.47 cdef 100 5.00 3.73 cd 3.13 2.23 2.07 efgh 200 5.00 3.43 cdef 2.83 2.13 1.80 ghij 300 5.00 2.93 ef 2.20 1.83 1.50 ijk 400 5.00 2.17 g 1.87 1.27 0.52 l low temperature field 0 5.00 4.83 a 4.77 4.33 3.83 a 100 5.00 3.97 bc 3.67 3.37 2.70 bcd 200 5.00 3.83 cd 3.40 3.07 2.50 bcde 300 5.00 3.57 cde 3.20 2.83 2.17 defg 400 5.00 3.33 cdef 2.97 2.17 1.50 ijk greenhouse 0 5.00 4.57 ab 4.40 3.30 3.03 b 100 5.00 3.53 cde 3.30 2.73 2.27 cdefg 200 5.00 3.20 def 2.93 2.40 1.93 fghi 300 5.00 3.00 ef 2.57 2.13 1.57 hijk 400 5.00 2.87 f 2.20 1.80 1.27 jk trial two room temperature field 0 5.00 4.63 4.07 3.70 abc 2.73 bc 100 5.00 3.90 3.67 3.10 def 2.60 bc 200 5.00 3.80 3.17 2.87 efgh 2.13 def 300 5.00 3.43 2.87 2.17 ijk 1.87 fg 400 5.00 3.00 2.43 1.90 jk 1.20 ij greenhouse 0 5.00 4.27 3.80 3.07 def 2.47 cde 100 5.00 3.93 3.27 2.80 fgh 2.03 efg 200 5.00 3.60 3.00 2.40 hij 1.70 fgh 300 5.00 3.13 2.63 2.00 jk 1.30 hi 400 5.00 2.90 2.20 1.70 k 0.85 j low temperature field 0 5.00 4.70 4.37 4.13 a 3.83 a 100 5.00 4.40 3.93 3.67 abc 2.83 bc 200 5.00 4.13 3.60 3.33 cde 2.93 b 300 5.00 3.93 3.37 3.07 def 2.63 bc 400 5.00 3.63 3.07 2.77 fgh 2.07 defg greenhouse 0 5.00 4.27 4.07 3.93 ab 3.40 a 100 5.00 4.03 3.87 3.50 bcd 2.83 bc 200 5.00 3.83 3.20 2.97 efg 2.50 bcd 300 5.00 3.43 2.87 2.53 ghi 2.10 def 400 5.00 3.20 2.53 2.17 ijk 1.63 ghi * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. adv. hort. sci., 2021 35(1): 21­32 28 received 400 kg npk ha­1 and stored at room temper­ ature had the highest pwl. similar results were reported in sweet potato in which excessive applica­ tion of nitrogen led to an increase in percentage weight loss during storage (mark et al., 2003). nitrogen fertilizer rates affect the rate of water loss in fruits and vegetables (warner et al., 2004). transpiration is the main cause of deterioration because it results in direct loss of weight. weight loss is the major cause of softening and shriveling of fruits and vegetables damaging the appearance of fruits and loss of market value (wilson et al., 1999). the quality of most fruits and vegetables is affected by weight loss but this depends on the temperature and humidity during storage (perez et al., 2003). storage of pepino fruits at room temperatures (15­22°c) could also have resulted in production of high levels of ethylene, increased respiration and subsequent weight reduction. high temperatures during storage leads to increased water loss resulting to shriveling and loss of fresh appearance of the fruits (wills et al., 1989). fruits lose weight when metabolic activities increase and this is accelerated by an increase in temperature around the produce resulting in loss of water. weight loss is mainly as a result of water loss and as temperature increases the rate of water loss also increases. in this study, pepino melon fruits were harvested when green mature and as ripening progressed there was an increase in ethylene produc­ tion which led to senescence and shriveling of the fruits during storage (wills et al., 1989). greenhouse fruits had a higher percentage weight loss probably because of the high preharvest temperature (table 1). at room temperature the temperatures were higher than at low temperature (7°c) and this could have resulted to faster ripening, increased respira­ tion rates and hence high pwl. fruits stored at low temperature had lower pwl compared to those stored at room temperature. vanitha and mehalai (2016) also reported that pepino fruits stored at room temperature had a higher weight loss com­ pared to those stored at low temperature. temperatures above 20°c can lead to abnormal physiological processes in fresh produce, respiration occurs and water is lost to the surrounding environ­ ment and hence reduction in weight. although there was an increase in pwl as the storage days increased, the rate was much lower in pepino fruits stored at low temperature. loss in weight could also be due to activity of polygalacturonase which increases cell wall permeability and hence increase in transpiration. low temperature reduces respiration and metabolic processes thereby slowing down the rate of fruit weight loss during storage. low tempera­ ture also reduces the sensitivity of fruits to ethylene and senescence is reduced (wills et al.,1989). in both trials, field and greenhouse grown pepino fruits sup­ plied with the highest npk fertilizer rate had the highest pwl. this is in agreement with the findings of hailu et al. (2008) and mark et al. (2003) where appli­ cation of highest nitrogen fertilizer rates had the highest physiological weight loss of carrots and sweet potatoes during storage. the increased pwl due to increased level of nitrogen supply may be attributed to the higher moisture content in the fruits which may lead to decreased shelf­life due to rapid meta­ bolic activity, moisture loss and shrinkage in storage (el­tantawy and el­beik, 2009). on the other hand, fruits from the control (no npk fertilizer) had the lowest pwl which could be attributed to low mois­ ture content in the fruits, slowed metabolic activities and hence reduced moisture loss. the pwl decreased as the phosphorous and potassium rates table 5 ­ effect of npk fertilizer rates, growing environment and storage temperature on shelf life (days) of pepino melon fruits in trial one and two * means followed by the same letters are not significantly diffe­ rent according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. storage temperature environment fertilizer (kg ha­1) shelf life (days) trial 1 trial 2 room temperature field 0 18 cd * 20 bcd (15­22°c) 100 17 cde 18 cdef 200 15 defg 16 fghij 300 14 fghi 14 ijk 400 12 hi 11 lm greenhouse 0 18 cd 17 efgh 100 16 defg 14 hijk 200 13 ghi 12 klm 300 12 hi 10 lm 400 11 i 10 m low temperature (7°c) field 0 27 a 26 a 100 22 b 21 b 200 18 cd 17 defg 300 16 def 15 ghij 400 14 fghi 13 jkl greenhouse 0 21 b 21 bc 100 19 bc 19 bcde 200 17 cdef 16 efghi 300 16 defg 15 ghij 400 15 efgh 14 ijk mutua et al. ‐ pepino melon postharvest studies 29 in the npk fertilizer increased. this could be due to the fact that potassium plays a role in maintaining fruit firmness but high rates do not result to further increase in firmness. the firmer the fruit the less the pwl and reduction in firmness results to more pwl. on the other hand, high nitrogen levels coupled with high phosphorous levels reduce fruit quality because most of the carbohydrates are translocated to the shoots rather than to the developing fruits resulting to dense vegetative growth. fruits produced by plants which have dense vegetative growth tend to be less firm resulting to high pwl while fruits from plants with less vegetative growth are firmer and hence low pwl. in summary, pwl progressively increased with increase in storage time in both room and low temperatures. as the fruit continues to ripen the rate of respiration also increases and this also leads to increase in weight loss. however, low tem­ perature leads to delayed ripening and hence reduced respiration resulting to low pwl compared to ambient room temperatures. tss increased as the storage days increased. our results are in agreement with harman et al. (1986) and hailu (2016) who reported that as pepino melon and mango fruits mature tss increases significantly during maturation and ripening. the increase in tss might be due to alteration of cell wall structure and the breakdown of complex carbohydrates into simple sugars. at room temperature, the temperatures were high and this led to an increase in metabolic process­ es, respiration and ripening resulting to high tss. increase in tss could also be due to excessive mois­ ture loss of fruits which led to increased concentra­ tion of pepino fruits stored at room temperature (nath et al., 2011). at high temperatures the rate of ripening is higher than at low temperatures and this increases tss. field grown pepino fruits had a higher tss compared to greenhouse grown pepino fruits in both storage temperatures. the high tss recorded in field grown pepino fruits could be due to high light intensity and thus high photosynthesis leading to more accumulation of sugars in the fruit compared to greenhouse grown fruits where the light intensity was low leading to reduced photosynthesis and hence low accumulation of sugars in the fruits (beckmann et al., 2006). any factor that interferes with photosynthesis will affect glucose and sucrose accumulation in the fruit and thus alter tss (rana et al., 2014). high relative humidity in the greenhouse may also have led to reduced transpiration and this enhances flow of water in the xylem vessels and this is good for the fruits because fruits act as drains for high concentrations of organic molecules leading to low water potential (bertin et al., 2000). the low water potential in the fruits promotes absorption of water by the fruits leading to “dilution effect” making the fruits to have low tss compared to those grown in the field (rana et al., 2014). the low tss recorded in greenhouse grown fruits could also be due to the fact that high temperatures during ripening of pepino melon reduce sugar content of the fruits (pluda et al., 1993). fruits stored at low temperature had a lower tss compared to that of fruits stored at room tem­ perature. this could be due to delayed fruit ripening and slow conversion of carbohydrates into simple sugars. during ripening there is breakdown of com­ plex carbohydrates into simple sugars and this increase tss. at high temperatures the conversion of carbohydrates into simple sugars is accelerated and this results to high tss whereas at low temperature ripening is delayed and the hydrolysis of carbohy­ drates to sugars is slower, resulting to low tss. in the present study, tss ranged from 4.00­7.07 and 4.00­ 8.13 °brix in trial one and two respectively. other studies have reported lower tss of pepino melon in the range of 4.91­5.40 °brix (kola, 2010) and 5.04­ 5.46 °brix (maruapey and yuwono, 2016). the low tss could be attributed to high water content in pepino fruits in the range of 90­92% (gonzalez et al., 2000) and the fact that the quality of pepino melon fruits is greatly influenced by the environment in which these studies were conducted which is quite different from the environment in this study. tss decreased as the fertilizer rates increased in both growing environments and storage temperatures in both trials. field grown fruits from control plants had the highest tss and this could be due restriction of vegetative growth because no npk fertilizer was applied and thus the fruits became the only sink for sugars and hence increase in tss (pluda et al., 1993). greenhouse grown fruits from plants supplied with the highest fertilizer rate of 400 kg npk ha­1 had the lowest tss and this might be due to excessive vegeta­ tive growth of both the main and side shoots there­ fore most of the photosynthates were directed to the young developing shoots rather than to the fruits leading to low sugar concentration in the fruits (pluda et al., 1993). excess nitrogen fertilizers make plants be more succulent, thus fruits from plants sup­ plied with 400 kg npk ha­1 had a high water content and this might have led to dilution of sugars in the fruit resulting to low tss. adv. hort. sci., 2021 35(1): 21­32 30 fruit firmness decreased as the storage days increased. decrease in firmness is strongly related to increased weight loss because as the fruits lose weight they become soft hence decreased firmness. in this study, firmness decreased as the npk fertilizer rates increased. fruits from plants which were sup­ plied with 400 kg npk ha­1 had the lowest firmness and this could be due to the fact that plants with dense vegetative growth are less firm than those with low or moderate vegetative growth (toktam et al., 2019). fruits which were not supplied with npk fertilizer (control) were firmer due to decreased veg­ etative growth. loss of moisture and enzymatic changes results to change in firmness (ball, 1997). hemicelluloses and pectins become more soluble and this causes changes and loosening of the cell wall (paul et al., 1999). in the present study, both trials field grown pepino fruits were firmer than green­ house grown fruits. this could be due to the fact that lower temperature during the growing season increases firmness (anagnostou and vasilakakis, 1995). in the greenhouse the temperatures (table 1) were high and it has been reported that high pre­ harvest temperatures tend to decrease firmness (paul et al., 1999). previous studies reported that loss of firmness in pepino melon is due to softening which is caused by breakdown of structural cell wall carbo­ hydrates and an increase in soluble pectic substances during storage (heyes et al., 1994). increase in pectic substances leads to weakening of cell walls and reduction of cohesive forces binding cells together resulting to loss of firmness (heyes et al., 1994). in summary, fruit softening is caused by structural as well as compositional changes in various components of the cell wall carbohydrates partly as a result of fruit softening enzymes (abbasi et al., 2011). other studies have reported that fruit softening is as a result of cell wall digestion by pectinesterase, poly­ galacturonase and other enzymes and this is increased by an increase in storage temperature (ahmed et al., 2009). low temperature storage main­ tained firmness of pepino melon fruits. pepino fruits stored at low temperature had a longer shelf life compared to those stored at room temperature. this could be attributed to reduced ethylene production, respiration, ripening, weight loss, senescence, retention of firmness and reduction of other metabolic activities and this enhances shelf life and quality of produce (lei yi et al., 2019). on the other hand, pepino fruits stored at room tempera­ ture had a shorter shelf life because high tempera­ ture results to increased ethylene production, respi­ ration, ripening, weight loss, senescence, loss of firm­ ness and other metabolic processes and this reduced shelf life (mutari and debbie, 2011). field grown fruits had a longer shelf life compared to greenhouse grown fruits. this could be attributed to lower tem­ perature in the field during the growing season (table 1) as low temperatures have been reported to increase firmness (anagnostou and vasilakakis, 1995). in the greenhouse the temperatures were high and it has been reported that high temperatures tend to decrease firmness (paul et al. , 1999). therefore, field grown fruits remained firmer than greenhouse grown fruits and hence the former had a longer shelf life. fruits from the control had the longest shelf life and this could be attributed to low nitrogen levels and low water content in this fruits hence they remained firmer. on the other hand, fruits from plants supplied with high npk fertilizer rates had a short shelf life and this might be due to high water content in the fruits due to excess nitro­ gen which also leads to postharvest decay especially fruits which were stored at room temperature. based on the foregoing results and discussion, we conclude that application of high amounts of npk fer­ tilizer leads to increased weight loss, less firm fruits and low tss of pepino fruits stored at room tempera­ ture. storage of pepino melon fruits at 7°c maintains quality through reduced weight loss and maintaining firmer fruits. we therefore recommend application of 100 kg npk ha­1 for both field and greenhouse grown pepino melon and storage at low temperature (7°c) for enhanced quality and shelf life of the fruits. acknowledgements the authors would like to express their sincere appreciation and thanks to world bank through the centre of excellence in sustainable agriculture and agribusiness management (cesaam), egerton university for the financial support of this research. thanks to the biotechnology laboratory and department of crops, horticulture and soils of egerton university, kenya for hosting this research. references abbasi k.s., anjum n., sammi s., masud t., ali s., 2011 ­ effect of coatings and packaging material on the 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(ed.). breeding vegetable crops. intercept, andover, uk. toktam t., siddiqui r., laban k r., 2019 ­ the effect of preharvest factors on fruits and nutritional quality in strawberry. strawberry pre‐ and post‐harvest manage‐ ment techniques for higher fruit quality. ­ intech. 84619 pp. 1­23. vanitha t., mehalai v., 2016 ­ studies on quality changes of pepino (solanum muricatum) fruit during storage. ­ int. j. sci. technol., 4(3): 138­143. warner j., zhang t.q., hao x., 2004 ­ effects of nitrogen fertilization on fruit yield and quality of processing tomatoes. ­ can. j. plant sci., 84: 865­871. wills r.b.h., mcglasson w.b., graham d., lee t.h., hall e.g., 1989 ­ postharvest. an introduction to the physiology and handling of fruit and vegetables. ­ van nostrands reinhold, new york, nw, usa, pp. 17­52. wilson l.g., boyette m.d., estes e.a., 1999 ­ postharvest handing and cooling of fresh fruits, vegeta‐ bles and cut flowers for small farms. ­ north carolina state university horticulture information leaflets available at http://www.ces.ncsu.edu/depts /hort/hil/hil­800.html (accessed on 3rd june 2020). impaginato 41 adv. hort. sci., 2023 37(1): 41­48 doi: 10.36253/ahsc­13910 quality of cold stored lemon fruit from orchards consociated to ancient olive trees m. allegra (*), f. ferlito, b. torrisi, s. trovato, g. cicciarello, m.c. strano consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (crea), centro di ricerca olivicoltura, frutticoltura e agrumicoltura, corso savoia, 90, 95024 acireale (ct), italy. key words: agroecology, agroforestry, cold storage, consociation, lemon fruit, postharvest quality. abstract: in the hilly area of gioia tauro (calabria, southern italy), lemon orchards are grown in consociation with centuries­old olive trees. lemons are partially shaded by olive canopies and the microclimate at the level of their canopies is suitable for plants growth and quality productions. under these conditions, lemon trees are grown even without irrigation, providing, despite this limitation, a quality product. this study aimed to i) investigate the qualita­ tive characterisation of two clonal selections of the lemon cultivar femminello, f. siracusano (s) and f. zagara bianca (zb), from the described intercropping, on irrigated (i) and non­irrigated (ni) crops; ii) assess the quality preservation during cold storage, in order to evaluate the availability of lemons for market­ ing in a period of shortage such as the summer season. fruits were harvested at commercial maturity, and cold stored at 10±1°c and rh 85­90%, for 60 days. decay incidence, physiological disorders, weight loss, and the main physical­ chemical parameters were assessed at harvest (t0) and every 15 days (t15, t30, t45, t60). the absence of decay and physiological disorders was observed throughout the 60­day storage period, in both clonal selections under the two management conditions. the weight loss was greater in fruits from irrigated lemon groves of both s and zb. s_i showed significantly lower fruits weight and higher titratable acidity than s_ni. total soluble solids and titratable acidity were statistically lower for zb_i than for ni fruits. 1. introduction several research have highlighted that some agri­food models are not sustainable from an economic, social and environmental point of view (ipes, 2016; fao, 2017). for many local agricultural contexts, the main perspective to survive is the design and set up of agricultural systems more at the service of the right to food (food sovereignty), enhancing ter­ ritoriality and reducing ecosystems degradation (loker and francis, 2020; ciaccia et al., 2021). in some cases these objectives are reached by the cultural consociation, adopted among crops with the aim of fostering (*) corresponding author: maria.allegra@crea.gov.it citation: allegra m., ferlito f., torrisi b., trovato s., cicciarello g., strano m.c., 2023 ­ quality of cold stored lemon fruit from orchards consociated to ancient olive trees. ­ adv. hort. sci., 37(1): 41­ 48. copyright: © 2023 allegra m., ferlito f., torrisi b., trovato s., cicciarello g., strano m.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 28 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13910 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 41­48 42 agro­ecological services thus providing both environ­ mental and economic benefits (las casas et al., 2022). this represents an agroforestry model, mixed and multifunctional by definition, that should be pro­ moted, empowering marginalized actors and farmers by creating something different (rossi, 2020) and fea­ sible only by a holistic approach that embrace a long­ term vision, such as agroecology (barrios et al., 2020). these models are not purely productive, as the presence of trees also provides environmental services such as soil improvement, surface runoff reduction, and conservation of biodiversity. at the same time, agroforestry systems provide aesthetic services as well (katsoulis, 2022). in the hilly area of gioia tauro (calabria, southern italy), lemon orchards are planted in consociation with centuries­old olive trees over 20 meters high. the general distance between olives is wide (10x10 m) according to their large and rarely pruned canopy, ensuring that the lemons are planted both in intra­ and inter­rows, in a 5x5 m design. with this regular agroforestry model, olives play a key role in terms of the agroecological service, characterizing the land­ scape and preserving the fragile area, preventing soil erosion, and increasing biodiversity. moreover, thanks to its long history in the area and manage­ ment, compared to the other intensive woody crops, does not require high external inputs, thus contribut­ ing to reducing environmental pollution (de graaff and eppink, 1999). the microclimate and the ade­ quate lighting conditions at the level of the lemon canopy is suitable for the growth of this culture and for obtaining high quality productions. under these conditions, lemon trees can be grown rainfeed, although irrigation may influence fruit quality at har­ vest and during the postharvest process (asrey et al., 2018; tadayon and hosseini, 2020). to the best of our knowledge no studies were carried out about the effect of the consociation of olive­lemon cultivations and on postharvest performance of lemon fruit, which quality could also be prolonged by proper cold storage (strano et al., 2022, 2023). the extension of the availability to market of high quality fruit, in a period of shortage such as the summer season, would allow to obtain the maximum economic profit for the growers. this study aimed to assess i) the physical­chemical parameters of two clonal selec­ tions of lemon cv femminello: f. siracusano (s) and f. zagara bianca (zb), with high commercial value, consociated with olive cultivations, from irrigated (i) and non­irrigated (i.e., rain­fed, ni) crops; ii) the qual­ itative response to the cold storage of lemons com­ ing from the two different management techniques. 2. materials and methods study site and fruit sample fruits of lemon (citrus limon (l.) osbeck) cultivar femminello, clones siracusano (orchard 1) and zagara bianca (orchard 2), both grafted on sour orange (c. aurantium l.), planted in a 5x5 m design on a loam soil, from irrigated and non­irrigated crops of the corresponding orchard, were grown in the hills of gioia tauro (calabria, italy; lat. 38,32 n; long. 15,97 e; elevation 200 m a.s.l.). according to the usda (2017) texture triangle, soil of the two orchards had a loam texture (orchard 1: 462 g kg­1 sand, 317 g kg­1 silt, 222 g kg­1 clay; orchard 2: 602 g kg­1 sand, 231 g kg­1 silt, 167 g kg­1 clay), and an organic matter content of 97 (orchard 1) and 88 g kg­1 (orchard 2). plants of irrigated crops were fully irrigated, corresponding to 95­98% of crop demand, while non­irrigated crops had no irrigation system, relying on rainfall for water (ferlito et al., 2014). fruits were harvested at commercial maturity in april 2022, transported to crea laboratories (acireale, sicily, italy), selected accordingly to their uniformity in size, absence of defects and alterations (lesions and/or rot symptoms) and, washed with tap water. three replicates of 100 fruits for each sample, were randomly placed in plastic boxes, for a total of 300 fruits, then air­dried at 20°c and stored for 60 days at 10±1°c and 85­90% relative humidity (rh). physical‐chemical changes fruits were evaluated at harvest (t0), for the fol­ lowing parameters: weight, rind thickness, carpel axis, peel and pulp color, firmness, juice yield, total soluble solids (tss), ph, titratable acidity (ta), matu­ rity index (tss/ta) and, after 15 (t15), 30 (t30), 45 (t45) and 60 (t60) days of storage, also for weight loss, decay incidence and severity of physiological disorders (chilling injury and aging). fruit weight loss, expressed as percentage, was evaluated on 30 fruits per sample (10 fruits/replicate) by weighting the same fruit at t0 and at each control. the results were calculated as follows: = [(m0­m1) / m0] × 100; where: m0 = the initial weight; m1 = the weight measured at each control. the juice was extracted using an electric citrus fruit squeezer and the pooled juice of five fruits per replicate was analyzed. the juice yield was calculated allegra et al. ‐ lemon‐olive tree consociation: effect of lemon quality 43 as follows: juice yield (%) = (juice weight/fruit weight) x 100. tss content was determined with a digital refractometer (atago rx­5000, atago, japan) and results expressed as °brix. titratable acidity (ta), expressed as % (w/v) of citric acid equivalent, was determined by potentiometric titration (t50 automatic titrator, mettler toledo) with 0.1 n sodi­ um hydroxide solution (aoac, 1995; ladaniya, 2008). vitamin c (ascorbic acid, mg·100 ml­1) concentration was determined by liquid chromatography (rapisarda and intelisano, 1996). the technological index ‘ti’ was calculated according to kluge et al. (2003) by the equation: ti = (total soluble solids x juice percentage) / 100. ti is an important variable linked to the quality of the juices destined for the citrus industry. higher ti val­ ues correspond to better quality (chitarra and chitarra, 1990). decay incidence (%) was determined by a visual evaluation of fruit infected by rots. the severity of chilling injury was determined by the visual examina­ tion of the fruit pericarp, using a four­grade scoring system to estimate the damage of the rind surface: 0 = none, 1 = less than 5% (light), 2 = 5% to 25% (mod­ erate), and 3 = over 25% (severe). the incidence of the fruits affected by aging (%) was determined by the presence of fruits with a necrotic area of rind tis­ sue around the stem­end button. firmness measurement firmness was tested by a texture analyser (zwick/roell do­fb0.5 ts model 2002, genoa, italy) using an 8 mm flat probe (mitcham et al., 1996). two measurements were made on two opposite of the equatorial zones of 15 fruits per sample. the results were reported as the peak force in newton (n) (nasrin et al., 2020). colour evaluation fifteen fruits for each sample were taken, at each time interval, for the determination of peel and pulp colour by cielab coordinates l* (lightness), a* (red­ green component) and b* (yellow­blue component) using a minolta spectrophotometer cm­2500d (minolta, milan, italy). three color measurements were made for each sample fruit and the results were expressed as citrus colour index (cci) = (1000 × a*)/(l* × b*) (jiménez­cuesta et al., 1981). the effect of cold storage at 10°c on the color of lemon fruits was also evaluated, at each time inter­ val, through the chroma (c*), which defines the color intensity (higher c* values indicate brighter yellow color); the hue angle (h°), with values closer to 90° for the more yellow fruits and increasing for the greener fruits; and browning index (bi), both for peel and pulp (kluge et al., 2003). the browning index (bi) was used as an indicator of the intensity of brown discoloration. bi was calculated as follows (palou et al., 1999; olivas et al., 2007): bi = [100(x−0.31)] / 0.172 where: x = [(a* +1.75)] / [5.646 l + a* ­3.012 b*]. statistical analysis one­way and factorial analysis of variance (anova) was performed using statistica 6.0 soft­ ware (stat soft italia srl) according to a completely randomized experimental design. data are means of three independent determinations. means compar­ isons were performed by tukey’s hsd test at p≤0.05, 0.01, 0.001, based on the f­test significance. 3. results physical‐chemical changes observation of the fruits revealed the absence of decay and physiological disorders in both clonal selections under the two management conditions (i and ni) throughout the 60­day storage period (data not showed). as shown in figure 1, the weight loss was signifi­ cantly greater in fruits from irrigated lemon groves of both clonal selections, with average values of 23.7% fig. 1 ­ weight loss percentage of 30 lemon fruits from each clonal selection (femminello siracusano and f. zagara bianca) and irrigation management (i: irrigated; ni: non­ irrigated) monitored during cold storage at 10±1°c and rh 85­90%. textured bars represent total weight loss from t0 to t60 (p≤0.001 ***). means of the same time interval indicated by different letters are significantly dif­ ferent (lowercase p≤0.05, uppercase p≤0.001) based on tukey’s hsd test. error bars show the standard deviation. adv. hort. sci., 2023 37(1): 41­48 44 (i) vs 12.9% (ni), in the interval from t45 to 60 days of cold storage, although it remained 3.6% (i) and 3% (ni) up to t45. the response to irrigation management resulted clonal selection­specific. the response of each clonal selection over time is reported. the width of the carpellar axis did not reveal significant differences in the comparison between the irrigation managements of each clonal selection, except at t15 in the case of s, and at t30 in the case of zb, in favor of ni (fig. 2). no significant difference between managements in the percentage of juice and tss for s. only at t60, the juice yield of zb_i was significantly higher than zb_ni, while tss already differed at t0 up to t30 in favor of zb_ni (fig. 2). the titratable acidity had a dif­ ferent behavior for the two clonal selections, with i statistically higher for s at t15, t45 and t60, and ni for zb at t15 and t45 (fig. 2). tss/ta was statistically higher for s_ni, compared to s_i, while no difference was recorded for zb (fig. 2). peel percentage and peel to pulp ratio did not show significant differences. only at the end of the storage (t60) zb_ni showed significantly higher val­ ues (fig. 3). fig. 3 ­ response of the physicochemical parameters (peel, % w/w; peel to pulp ratio; technological index, ti; peel and pulp citrus color index, cci) monitored during cold stor­ age at 10±1°c, rh 85­90%, of lemon fruits from two clon­ al selections (femminello siracusano and f. zagara bian­ ca) to irrigation management (i= irrigated; ni= non­irri­ gated). means of the same time interval indicated by dif­ ferent letters are significantly different (lowercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. fig. 2 ­ response of the physicochemical parameters (carpellar axis, mm; firmness, n; juice, %; total soluble solids, tss °brix; titratable acidity, ta %; tss/ta ratio) monitored during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano and f. zagara bianca) to irrigation management (i= irrigated; ni= non­irrigated). means of the same time interval indi­ cated by different letters are significantly different (low­ ercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. allegra et al. ‐ lemon‐olive tree consociation: effect of lemon quality 45 vitamin c values were significantly higher for s, while no significant differences emerged between i and ni management (fig. 4). 4. discussion and conclusions the modern woody crops systems are generally characterized for a strong modification on agro­ ecosystem structure and functioning. in a climate change scenario also a modification of the related fig. 5 ­ response of the colour parameters of fruit peel and pulp (chroma, c*ab; hue angle, h°; browning index, bi) moni­ tored during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano and f. zagara bianca) to irrigation manage­ ment (i= irrigated; ni= non­irrigated). means of the same time interval indicated by different letters are significant­ ly different (lowercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. fig. 4 ­ variation of the vitamin c content during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano ‘s’ and f. zagara bian­ ca ‘zb’) and two irrigation management (i= irrigated; ni= non­irrigated). means observed at each time interval relative to each irrigation management of the clonal selections indicated by different letters are significantly different (p≤0.001) based on tukey’s hsd test. textured bars represent the results of the factorial anova (p≤0.05*; ns, no significant differences). error bars show the standard deviation. firmness measurement no significant difference during storage regarding firmness for zb, while s showed higher values for i as early as t0 (fig. 2). colour evaluation while the color parameters and indices relating to the pulp very rarely showed significant differences between irrigation managements i and ni (fig. 5), those relating to the peel show in some cases signifi­ cant, and often antithetical, differences between the two clonal selections. this is the case of cci, chroma and bi, for which s_ni had statistically higher values than s_i, while the opposite occurred for zb. the peel h° had a specular behavior with respect to what was described for the other indices (fig. 5). differences at harvest (t0) occurred in the case of s, for firmness (fig. 2), peel cci (fig. 3) and peel bi (fig. 5) with higher values for i, which was probably later in ripening, as the lower tss/ta value seems to demonstrate (fig. 2). zagara bianca showed differences already at har­ vest (t0) in tss (fig. 2) and peel h° (fig. 5), higher in ni, while peel cci (fig. 3), c* and bi (fig. 5) were higher in i. 46 adv. hort. sci., 2023 37(1): 41­48 agronomical processes such as the water and mineral availability, the solar radiation and the spontaneous flora growth could be affected (ciaccia et al., 2019). in the present work the response of the particular consociation between the lemon and the old­century olives” in calabria was investigated, assuming that it could allow to lengthen the lemon quality traits and shelf life throughout the response to long periods of cold storage (fung et al., 2019). furthermore, the response of irrigated and non­irrigated lemon groves was compared. understanding these linkages, the obtained data can predict how the effect of a single agronomic practice at the micro and macro­area scales can reduce the water input for irrigation. moreover, the lemon storage could drastically reduce the lengthen of the irrigation season. moreover, from the econom­ ical point of view, the proposed study can be an evaluable way for the marketing of fresh lemons. in fact, as observed by ciriminna et al. (2020), the fresh lemons have a constant demand from consumers which in some seasons cannot be entirely satisfied by italian production. finally, the consociated model could be diversified also adopting other species that are well adapted to shade such as berries (cicala et al., 2002). the microclimatic effects induced by the presence of the olive trees are reflected on the preservation of the fruit quality, on the protection from natural adversities such as excessive insolation or hail, and seems to result in a postponement of ripening, par­ ticularly in non­irrigated lemon groves (brunori et al., 2019). the results of this study seem to support the hypothesis of the effectiveness of the shading effect and of the microclimate induced by the presence (cover, protection) of the olive trees, on the quality of the production of both clonal selections studied, and on the sustainability of non­irrigated crops, given the comparability of many of the qualitative parame­ ters with those of irrigated crops. therefore, despite the considered period is not too long to draw general conclusions the preliminary results of the research show that the agroecological system and the use of the non­economical valuable olive as an agro­ecolog­ ical service crop could realize a mutual relationship into the agroecosystem. storage at 10°c avoided the development of decay and physiological disorders regardless of clonal selection and irrigation management. management strategies that increase the produc­ tivity of existing agricultural land are increasingly needed (salmon et al., 2015). the diffusion of the consociation here studied could allow the exploita­ tion of large tracts of land that host centuries­old olive groves, which must be preserved for their envi­ ronmental and cultural importance, but no longer usable at a production level, given the very high man­ agement and harvest costs (brunori et al., 2019). additionally, the possibility of eliminating or reducing the water supply makes this intercropping particular­ ly interesting. increased agricultural productivity is generally achieved by increasing inputs. irrigation, which cur­ rently accounts for about 70% of global freshwater withdrawals, is one of the most important and wide­ spread means of achieving this goal (lobell et al., 2009). rain­fed croplands, also called dryland farm­ ing, include all cropland where no water from any storage or delivery mechanism is utilized, but crops are not flooded, and where harvesting occurs at least once per year (salmon et al., 2015). as a result of the present work, the comparable quality of the fruits at harvest, both with and without irrigation, and their qualitative maintenance with cold storage would allow to have a good product on the market even in periods of shortage. the comparison with lemon groves without olive trees will be investigated in order to estimate any qualitative improvement induced by the two crops consociation. references aoac, 1995 ­ official methods of analysis. ­ in: cuniff p. 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techniques affect vigour and flower formation of ukrainian sweet cherry cultivars p. bondarenko 1, 2 (*), o. alekseeva 3, v. senin 2, p. kondratenko 4 1 laimburg research centre, 39040 auer/ora (bz), italy. 2 melitopol fruit growing research station named after m.f. sydorenko, 72311 melitopol, ukraine. 3 dmytro motornyi tavria state agrotechnological university, 69600 zaporizhzhia, ukraine. 4 national academy of agrarian sciences of ukraine, 01010 kyiv, ukraine. key words: canopy, flower, heading cut, pruning severity, shoot, stub, thinning cut. abstract: excessive tree vigour and late entrance into full production, inherent to sweet cherry trees, are major challenges in the intensive cultivation of this crop. possible ways to reduce the vigour and stimulate flower induction include shifting the term of pruning and reducing its severity. however, the reaction of the trees may differ depending on specific cultivar, soil and climatic conditions. therefore, the aim of the study was to determine the effect of various tech­ niques and terms of pruning on young sweet cherry trees in order to adapt the intensive cultivation technology to the arid conditions of southern ukraine. the results showed a strong cultivar­specific reaction to various pruning treat­ ments. pruning young sweet cherry trees in late summer contributed to a reduction of trunk and canopy indices by 11­22% on one of the cultivars and an increase in the number of flowers per tree by 1.4­1.7 times on both cultivars, compared to dormant pruning. low severity pruning reduced 1­year­old shoot length by 9­25% and increased the number of flowers by 1.5­2.5 times com­ pared to more aggressive pruning. the effect of pruning treatments on tree vigour was more pronounced during the first and second year of their applica­ tion. 1. introduction sweet cherry (prunus avium l.) is one of the most important stone fruit crops in the world, its annual gross production has increased by 1.4 times over the last 20 years and reached 2.6 million t in 2020 (fao, 2020). ukraine is one of the main sweet cherry producing countries, with annual production volume ranging between 60­85 thousand t, with 60% of indus­ trial production concentrated in the south­eastern region (state statistic service of ukraine, 2020). there is a well­developed extensive traditional (*) corresponding author: pavlo.bondarenko@laimburg.it citation: bondarenko p., alekseeva o., senin v., kondratenko p., 2023 ­ pruning terms and techniques affect vigour and flower formation of ukrainian sweet cherry cultivars. ­ adv. hort. sci., 37(3): 271­280. copyright: © 2023 bondarenko p., alekseeva o., senin v., kondratenko p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 september 2022 accepted for publication 16 june 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13827 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 271­280 272 cultivation technology of the crop in the region that utilizes mahaleb seedlings as rootstock, a central leader training system, and plant density of 200­ 350 trees ha­1 (rulyev, 2003). the main drawback of such cultivation technology is late entry of the trees into production and, as a result, late return of initial capital investment. additionally, modern export markets for ukrainian sweet cherry show increased requirements for the size and overall quality of the fruits. it is well known that the highest quality cherries are formed on young wood (dolya, 2011; claverie and lauri, 2005). trees with large canopies, characteristic for traditional cul­ tivation technology, are not always able to ensure timely fruiting wood renewal, and thus, good fruit quality. these factors contribute to the fact that sweet cherry cultivation technology in ukrainian orchards is being intensified. orchards with trees grafted on dwarfing rootstocks are not always commercially suc­ cessful due to the low adaptivity of those rootstalks to the arid continental climate of southern ukraine. therefore, new orchards utilising interstems and rootstocks of medium and high vigour with a density of 667­1200 trees ha­1 to promote precocity may be a better alternative (kishchak et al., 2020; bondarenko, 2018). it should be noted that currently there are no well­developed pruning technologies for such orchards in ukraine, and direct use of foreign tech­ niques is ineffective and requires adaptation to the specific climate and soil conditions in the cultivation region. the main challenge when cultivating high density orchards is controlling the vigour and the size of tree canopies. aggressive annual pruning in order to keep tree canopies within the limits of the planting scheme can cause strong vigour reaction, reduce flower bud initiation, and delay fruiting (lang, 2005). different agronomic techniques can be used in order to reduce the vigour of sweet cherry trees. application of growth regulators contributes to reduction in shoot length and increases the number of generative buds on a tree (jacyna et al., 2012; elfving et al., 2003). root pruning can also be effec­ tive in reducing vigour (pal and mitre, 2016; webster et al., 1997). another promising and easily applied method is shifting the term of tree pruning. in ukraine, pruning in the second half of the growing season is being actively used in apple orchards (melnyk and mulienok, 2020; chaploutskyy and melnyk, 2015), and in the world ­ for sweet cherry as well (blažková and drahošová, 2012). in addition to potential decrease in vigour due to removing a part of assimilation area prematurely, pruning trees in august­september, compared to traditional pruning during dormancy, has an advantage that wounds from the cuts heal faster and trees are more resistant to pathogens, especially those of bacterial aetiology (spotts et al., 2010; colhoun et al., 2015). while sci­ entific data on the degree of spread of bacterial dis­ eases in ukrainian orchards is insufficient (patyka et al., 2016), farmers report visual manifestations of bacterial diseases in most sweet cherry plantations (pers. comm.), underlining the importance of using agronomic measures, including pruning, to contain the infection. it is also indicated that summer pruning can reduce winter frost damage to generative organs for certain sweet cherry cultivars (vaszily et al., 2011). another challenge in intensive sweet cherry culti­ vation is the need of regular fruiting wood renewal in order to maintain yields and fruit quality. inherently, sweet cherry has low regeneration and shoot­forma­ tion ability, further complicating this process for the farmer. therefore, it is often recommended to avoid thinning cuts on 1­year­old wood and to instead use stub cutting to preserve a bigger number of growth points on the tree. also, in order to stimulate spur formation, heading cuts can be avoided. that, how­ ever, reduces the number of new shoots in the sub­ sequent season and worsens their position on a tree (long et al., 2015; mika, 2006). in general, optimal techniques of pruning for intensive sweet cherry orchards in ukraine are not yet fully determined. the objective of this study was to determine the reaction of the young sweet cherry trees of different cultivars to various techniques and terms of pruning in order to adapt the intensive cultivation technology to the arid conditions of southern ukraine. 2. materials and methods site description the study was conducted in a commercial sweet cherry orchard located near the city of melitopol, south­eastern ukraine (46°80’n, 35°34’e, 38 m a.s.l.). the climate is moderately continental, the mean daily air temperature in january is ­3.1°c and in july is +22.8°c; the average annual amount of precipitation is 475 mm. the soil of the experimental site is south­ ern chernozem (black soil), with loam soil texture. bondarenko et al. ‐ vigour and flowering of cherry depending on pruning 273 experimental design the orchard was planted in late october 2014 using 4.5×2 m planting scheme (1111 trees ha­1) with 1­year­old maiden trees without lateral branches. trees were grafted on colt rootstock and trained as spindle canopies with a single central leader. bud scoring and branch bending were applied to the trees during the first 2 years after planting, where neces­ sary. different pruning treatments were applied in the orchard starting from the 3rd leaf, and the results of the study in 2017, 2018 and 2019 present the reaction of the trees after 1, 2 and 3 pruning cycles, respectively. the orchard is drip irrigated. plant pro­ tection and fertilisation were carried out in accor­ dance with the recommendations for sweet cherry cultivation in the region. ‘krupnoplidna’ and ‘melitopolska chorna’ culti­ vars were chosen for the study as they are the main cultivars in commercial orchards in southern ukraine and have distinct growth habits, not similar to each other. both cultivars are late ripening and were bred in melitopol fruit growing research station named after m.f. sydorenko, melitopol, ukraine (quero­ garcía et al., 2017). two pruning terms were studied: during dorman­ cy and in late summer. dormant pruning is traditional for sweet cherry cultivation in ukraine, and in the conditions of our experiment was performed in the second half of february in dry weather. late summer pruning was performed between the 25th and 31st of august. while it is sometimes advised to prune trees earlier, even immediately after harvest (ayala and lang, 2017), our previous experience suggested that pruning as late as the 15th of august can cause regrowth, so the term was shifted to eliminate this risk. three different pruning severities with various techniques were studied: ­ high severity (control). traditional style of pruning for sweet cherry in ukraine. heading cuts are applied to most 1­year­old shoots (either by removing one third of the shoot length or pruning it back to 60 cm if removing one third of the shoot length still leaves more than 60 cm of length); thinning cuts are applied to undesirable 1­year­ old shoots that grow straight up or down, overly thicken the canopy, intertwine with other shoots, or hinder tractor movement between the rows. on average, 45­50% of a tree’s 1­year­old wood is removed by pruning. ­ medium severity. heading cuts are applied only to strong 1­year­old shoots longer than 60 cm; stub cuts are applied to undesirable 1­year­old shoots, with stub length of 15­20 cm; thinning cuts are not used. on average, 35­40% of a tree’s 1­year­ old wood is removed by pruning. ­ low severity. heading or thinning cuts are not used; stub cuts are applied to undesirable 1­year­ old shoots, with stub length of 15­20 cm. on aver­ age, 25­30% of a tree’s 1­year­old wood is removed by pruning. in all variants of the experiment, the extension shoot of the central leader was headed to 80 cm every year. for the sake of the experiment, cuts on 2­ year­old and older wood were avoided unless abso­ lutely necessary. pruning in all variants was done manually. the following variants of the length of the stubs on annual shoots were studied: short stubs with 1­2 buds and long stubs with a length of 20 cm. those lengths were chosen as the ones that are easy to apply to pruning in industrial orchards (20 cm is roughly the length of the pruning shears). every combination of cultivar, pruning term, prun­ ing severity, and stub length was replicated 3 times with 3 trees in each replication. the experiment was arranged using randomized block design. measurements tree canopy parameters, trunk cross­sectional area (tcsa), and number and length of 1­year­old shoots were measured before pruning each year on the 15th of august. canopy volume was calculated as the volume of a cone using the following formula: canopy volume = 1/3 (h­0.6) π (w1 + w2) 2 4 where h = height of the tree, m; 0.6 = distance between the ground and first lateral branch, m; w1 = maximum width of the tree in the row, m; w2 = maxi­ mum width of the tree across the row, m. tcsa was measured 30 cm above the grafting point. all 1­year­old shoots longer than 10 cm were measured. shoots shorter than 10 cm were consid­ ered spurs and not included into calculations of the number of 1­year­old shoots per tree and mean shoot length. number of flowers per tree was counted during full bloom (15th­25th of april during the years of the research). in the 3rd leaf (2017), both cultivars had only up to 20­30 flowers per tree, so the flowering adv. hort. sci., 2023 37(3): 271­280 274 data for this year was excluded. spring frosts during flowering (minimal air temperature in the orchard reached ­4.3 c in april 2018 and ­7.8 °c in april 2019) damaged up to 95% of pistils, which led to a poor fruit set and a very low number of fruits per tree. thus, the data on yield and fruit quality was also excluded. data analysis statistical analysis of the results was conducted using the software minitab 19 (minitab inc., state college, pa). since the studied cultivars reacted dif­ ferently to the treatments, a two­way analysis of variance was performed separately for each cultivar, with tukey’s range test with an accuracy of 0.05 car­ ried out post hoc to determine the significant differ­ ences between the means. in addition, the cultivars were compared using a one­way analysis of variance with the same post hoc test. the exception was the reaction of trees to stub cutting, which was more uni­ form among the cultivars, and a single combined analysis of variance was performed. in order to determine the relationship between the indices, pearson’s correlation was used. 3. results trunk cross‐sectional area and canopy volume the results of the experiment indicate different reactions by sweet cherry cultivars to pruning treat­ ments. ‘krupnoplidna’ trees inherently have a more spreading growth habit, wider crotch angles with a tendency to form round canopies, and better shoot formation ability at a young age. ‘melitopolska chor­ na’ trees are more upright, with narrow crotch angles, and produce fewer shoots. as a result, low pruning severity with no heading cuts allowed ‘krupnoplidna’ trees to increase their canopy volume faster, exceeding the variant with high pruning severity by 35% in 2017 and 12­16% in 2018 and 2019 (table 1). for ‘melitopolska chorna’ trees, pruning severity had no effect on canopy vol­ ume. pruning severity had a different effect on tree trunks depending on cultivar. ‘krupnoplidna’ trees had higher tcsa and annual increase in tcsa when pruning severity was low, while for ‘melitopolska chorna’ those indices were the highest with aggres­ sive high­severity pruning. table 1 ­ influence of pruning term and severity on trunk cross­sectional area and canopy volume of sweet cherry trees tcsa = trunk cross­sectional area. different letters within the same group indicate significant difference between the means according to tukey’s test (p<0.05). variant tcsa in 2019 (cm2) annual increase in tcsa (cm2) canopy volume (m3) 2017 2018 2019 'krupnoplidna' pruning severity high (c) 80.8 b 23.2 b 4.8 b 7.5 b 10.4 b medium 90.3 ab 26.2 ab 6.0 a 7.4 b 11.5 ab low 95.2 a 28.3 a 6.5 a 8.4 a 12.1 a pruning term dormancy 93.3 a 26.6 a 5.9 a 8.7 a 11.6 a late summer 84.2 b 25.2 a 5.6 a 6.8 b 11.0 b 'melitopolska chorna' pruning severity high (c) 92.0 a 27.9 a 3.3 a 5.2 a 9.8 a medium 80.6 b 23.9 b 3.3 a 5.2 a 9.7 a low 76.8 b 22.2 b 3.4 a 5.4 a 9.8 a pruning term dormancy 83.3 a 24.6 a 3.2 a 5.2 a 9.7 a late summer 83.0 a 24.8 a 3.4 a 5.4 a 9.7 a cultivar comparison krupnoplidna 88.7 a 25.9 a 5.8 a 7.8 a 11.3 a melitopolska chorna 83.2 a 24.7 a 3.3 b 5.3 b 9.7 b bondarenko et al. ‐ vigour and flowering of cherry depending on pruning 275 pruning ‘krupnoplidna’ trees in late summer decreased tree vigour, with an 11% decrease in tcsa and lower canopy volume, most notably by 22% in 2018, compared to pruning during dormancy. these indices were not significantly different when compar­ ing the effect of different pruning terms on ‘melitopolska chorna’ trees. due to growth habit differences, ‘melitopolska chorna’ trees had more compact canopies compared to ‘krupnoplidna’, especially at a younger age: the difference of canopy volumes between the cultivars was 1.8 times in 2017, 1.5 times in 2018 and 1.2 times in 2019. cultivars had no significant influence on tsca of trees in the trial. 1‐year‐old shoot parameters it was determined that low and medium pruning severity led to an 11­23% increase in the number of 1­year­old shoots per ‘krupnoplidna’ tree in the first two years of the research (table 2). after 3 cycles of pruning, however, this indicator levelled off among all variants. pruning severity had no effect on the number of shoots on ‘melitopolska chorna’ trees. low pruning severity without heading or thinning cuts decreased the mean length of 1­year­old shoots by 9­25% on both studied cultivars, compared to tra­ ditional pruning techniques. for ‘krupnoplidna’ trees, this effect appeared most strongly in the year follow­ ing the first application of such pruning, while for ‘melitopolska chorna’ the shoot length decrease was more apparent starting from the second pruning cycle. pruning term had little effect on the number of shoots per tree, regardless of cultivar. the only statis­ tically significant difference was observed for ‘melitopolska chorna’ in 2019, indicating that trees pruned during dormancy retained more vigour. late summer pruning reduced the mean shoot length of ‘krupnoplidna’ trees by 10­12% during the first two years of the research. in general, the cultivar comparison highlights that ‘krupnoplidna’ trees formed more new shoots per tree, especially in the first years after planting, whereas ‘melitopolska chorna’ trees formed more vigorous longer shoots. flower formation both the severity and term of pruning had a signif­ icant effect on flower formation in the orchard. in the case of ‘krupnoplidna’, both low and medium prun­ different letters within the same group indicate significant difference between the means according to tukey’s test (p < 0.05). table 2 ­ influence of pruning term and severity on shoot parameters of sweet cherry trees variant number of 1­year­old shoots per tree mean length of a 1­year­old shoot (cm) 2017 2018 2019 2017 2018 2019 ‘krupnoplidna' pruning severity high (c) 62 b 131 b 239 a 77.7 a 51.1 a 51.4 a medium 73 a 146 ab 218 a 69.9 a 45.2 ab 44.3 b low 71 a 162 a 218 a 60.9 b 42.4 b 45.0 b pruning term dormancy 66 a 136 b 232 a 73.2 a 49.1 a 46.6 a late summer 71 a 157 a 218 a 65.8 b 43.4 b 47.3 a ‘melitopolska chorna' pruning severity high (c) 39 a 80 a 206 a 77.5 a 64.8 a 62.2 a medium 38 a 78 a 185 a 75.4 a 61.3 a 59.0 a low 36 a 78 a 180 a 70.2 b 48.9 b 49.4 b pruning term dormancy 37 a 79 a 212 a 73.7 a 57.8 a 58.6 a late summer 38 a 79 a 169 b 75.0 a 58.8 a 55.2 a cultivar comparison krupnoplidna 68 a 146 a 225 a 69.5 b 46.2 b 46.9 b melitopolska chorna 38 b 79 b 190 b 74.3 a 58.3 a 56.9 a 276 adv. hort. sci., 2023 37(3): 271­280 ing severity increased the number of flowers per tree on average by 1.5­1.7 times compared to high prun­ ing severity (fig. 1). for ‘melitopolska chorna’, only low pruning severity with no heading and thinning cuts affected the number of flowers per tree. the increase, however, was more significant ­ by 2.5 times on average. pruning the orchard in late summer promoted flower formation for both cultivars. summer­pruned trees on average formed 1.4 and 1.7 times more flowers for ‘krupnoplidna’ and ‘melitopolska chorna’ cultivars respectively, compared to dormant pruning. it should be noted that regardless of pruning treat­ ments, based on the number of flowers per tree, ‘krupnoplidna’ trees entered production during the 4th leaf (2018) while ‘melitopolska chorna’ trees still could not be considered bearing even in the 5th leaf (2019). stub length stub length of 1­year­old shoots significantly influ­ enced the proportion of stubs that produced new shoots in the subsequent season. this index was 1.2 times higher on long stubs compared to short stubs (table 3). long stubs also formed 1.6 times more shoots per stub, which can be explained by a much higher number of buds on them compared to shorter stubs with only 1­2 buds. stub length did not affect mean length of the new shoots on stubs, while prun­ ing term did not significantly influence any of the studied parameters. table 3 ­ reaction of sweet cherry trees to stub cutting different letters within the same group indicate significant difference between the means according to tukey’s test (p<0.05). fig. 1 ­ influence of pruning term and severity on the number of flowers formed on sweet cherry trees of ‘krupnoplidna’ (a) and ‘melitopolska chorna’ (b) cultivars. note the dif­ ference of scale between the graphs. different letters within the same group indicate significant difference between the means according to tukey’s test (p<0.05). variant proportion of stubs that produced shoots next year (%) number of 1­year old shoots per stub mean length of a 1­year­old shoot on a stub (cm) stub length short (1­2 buds) 82 b 1.4 b 62.7 a long (20 cm) 96 a 2.2 b 67.7 a pruning term dormancy 89 a 1.8 a 63.4 a late summer 90 a 1.8 a 67.0 a tree age 3rd leaf (2017) 93 a 1.8 a 75.7 a 4th leaf (2018) 90 ab 1.8 a 58.1 b 5th leaf (2019) 85 b 1.8 a 61.8 b cultivar krupnoplidna 85 b 1.9 a 62.3 b melitopolska chorna 94 a 1.7 a 68.1 a bondarenko et al. ‐ vigour and flowering of cherry depending on pruning 277 the proportion of stubs that produced new shoots decreased over the duration of the trial. as trees got older and more points of growth were formed throughout the tree canopy, new stubs were slightly less likely to form new growth. the number of shoots per stub, however, was not influenced by the tree age. mean shoot length on the stubs followed the same tendencies as this index for the whole tree, being the highest in 2017 and decreasing by 18­23% in each subsequent year. stub cuts on ‘melitopolska chorna’ trees had a higher chance to produce new growth the next year, and those shoots were longer, compared to ‘krupnoplidna’ trees. 4. discussion and conclusions one of the main takeaways of our study is a strong cultivar­specific reaction to various pruning treatments. most of the vigour indices were affected by pruning more significantly when it was applied to the trees of the cultivar ‘krupnoplidna’, which is char­ acterized by spreading round canopies and a higher ability to produce new shoots compared to the more compact upright canopies and fewer shoots formed per tree of ‘melitopolska chorna’ cultivar. this proves the importance of a cultivar­based approach to the choice of optimal training systems, plant den­ sity and pruning measures in intensive sweet cherry orchards (long et al., 2021). the effect of different pruning severities on trunk growth was inconsistent among the studied cultivars. other research on this topic shows similar results: exposure of trees to low severity pruning or no prun­ ing at all can lead, depending on the cultivar studied, to tcsa decrease, increase, or no change in trunk parameters (usenik et al., 2008; radomirska and domozetova, 2017; zec et al., 2020). summer prun­ ing decreased tcsa and canopy volume for ‘krupnoplidna’ trees, in comparison with dormant pruning. this can be explained by the decreased length of the shoots in this variant, leading to more compact canopies. a similar effect was observed in other studies for sweet cherry (blažková and drahošová, 2012), sour cherry (gonda, 2006), peach (ikinci et al., 2014), but not plum (sosna, 2010). the number of shoots formed on the tree was largely influenced by cultivars and tree age. an initial increase in shoot formation observed on ‘krupnoplidna’ trees in variants with low and medi­ um severity appeared mainly in multiple new shoots forming near the terminal end of the previous­year shoots. these types of branches with long sections of spurs and new growth only at terminal points may be problematic from an agronomic point of view, as sweet cherry spurs become less productive and die relatively quickly with age, especially in suboptimal lighting conditions, resulting in blind wood (ayala and lang, 2017; bondarenko and alekseeva, 2020). renewing such branches by stub cutting can also be ineffective, particularly in the lower zones of the canopy and when trees are older (stan, 2015; hansen and black, 2019). our study observed a reduction of shoot length on the trees pruned less severely, which was also documented in other studies (usenik et al., 2008; villasante et al., 2012), and may be explained by bet­ ter nitrogen use efficiency by extension shoots on pruned branches (ayala et al., 2018). ‘krupnoplidna’ trees also had decreased values of this indicator when pruned in late summer compared to dormancy. a similar effect was observed for peach, where shift­ ing the pruning term further (june ­ july ­ august ­ september) progressively decreased both the diame­ ter and mean length of new shoots in the subsequent season (ikinci et al., 2014). it should be noted that the effect of pruning treat­ ments on most of the parameters of tree vigour was more pronounced during the first and second cycles of pruning. during the third year of the research, those indices were either statistically non­significant among treatments, or, at least, less pronounced due to the trees adapting to the treatments and exhibit­ ing their inherent growth habit. so, from an agro­ nomic point of view, if the vigour in the orchard is excessive, shifting the pruning to late summer or removing less wood during pruning may be a valu­ able short­term technique in reducing tree vigour and increasing precociousness. in the long term, however, vigour is more dependent on specific root­ stock and cultivar combination than on pruning mea­ sures. both late summer pruning and lower pruning severity positively contributed to flower formation on sweet cherry trees. in the case of the pruning term, it is reported that pruning in august led to an increase in carbohydrate content in sweet cherry flower buds in the outer and upper part of the tree canopy, compared to dormant pruning (vosnjak et al., 2021). late summer pruning also shifts source­ sink relations in the tree, promoting flower bud initi­ adv. hort. sci., 2023 37(3): 271­280 278 ation, as there is a strong adverse correlation between bud initiation and vigour (flore and layne, 1999). lower pruning severity with fewer heading cuts and no thinning cuts increased the number of flowers, which can be attributed to an overall increase in the points of growth on the trees, as well the fact that weaker, less vigorous shoots were formed in these variants. these points are supported by the fact that in the conditions of our trial, a nega­ tive correlation was observed between the mean shoot length in the previous growing season and the number of flowers per for both ‘krupnoplidna’ (r= ­ 0.623 in 2018 and r= ­0.526 in 2019) and ‘melitopolska chorna’ (r = ­0.550 in 2018 and r = ­ 0.683 in 2019) cultivars. for ‘krupnoplidna’, a posi­ tive correlation was also found between the number of shoots in the previous growing season and number of flowers per tree (r= 0.717 in 2018 and r = ­0.593 in 2019). no such relationships were observed for other studied vigour indices. the results of this study are consistent with other trials on sweet cherry that report a yield increase when trees were unpruned or lightly pruned (villasante et al., 2012; claverie and lauri, 2005). however, it should be noted that when trees enter full bearing, low pruning severity has a negative effect on fruit size and thus marketability of the yield (gonkiewicz, 2011; von bennewitz et al., 2011; ayala et al., 2018), so more aggressive pruning may be need­ ed to manage crop load and maintain fruit quality. cultivar genotype had a bigger effect on the num­ ber of flowers on the tree than any pruning treat­ ments, further proving that a cultivar­based approach is essential for the cherry cultivation tech­ nology to be successful. the reaction of the trees to stub cutting was more uniform among the studied cultivars compared to other treatments in this study. in general, both short and long stubs consistently produced new shoots in the subsequent season and mean shoot length was not affected by stub length. as the trees got older, the share of the dead shoots increased, mostly for short stubs, but was still minor. another study, in which trees were grafted on a dwarfing rootstock, indicates that removing a large portion of the current year’s shoots can lead to much larger proportion of dead shoots ­ up to 50­70% (usenik et al., 2008). our further research of stub cutting of 1­year­old shoots will focus on the effect of this technique in different parts of the canopy (stubs in lower and upper zones of the tree, on the central leader and on lateral branches), as stubs of different length often pro­ duced mixed results depending on the shoot position in the tree. in summary, we can conclude that pruning young sweet cherry trees in late summer and reducing pruning severity leads to a decrease in vigour, mani­ festing itself mostly in the reduction of 1­year­old shoot length, and has a positive effect on flower bud initiation and precociousness. the effect of pruning treatments on tree vigour was more pronounced during the first and second year of their application. stub cutting of 1­year­old wood is a valuable alterna­ tive to thinning cuts: one can preserve more points of growth on the tree, and new shoots form more con­ sistently in the desired locations of the canopy. the specific cultivar’s growth habit should always be con­ sidered when choosing pruning strategies in the orchard. acknowledgements this research did not receive any specific funding. the authors thank i. hryntsiv for providing the orchard for the trial, o. nosachenko, m. shevchenko and v. topov for their help with pruning and field data collection, and r. williams for improving the quality of english in the manuscript. p.b. is personally grateful to laimburg research centre staff for providing him shelter during the russian aggression against ukraine and making the preparation of this article possible. references 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horticulturae, 451: 643­652. zec g., milatović d., boškov đ., čolić s., đorđević b., đurović d., 2020 ­ influence of pruning on biological properties of sweet cherry cultivars grafted on ‘oblačinska’ sour cherry. ­ acta horticulturae, 1289: 105­110. 152 1. introduction ‘kinnow’ mandarin occupies a prime position amongst the citrus fruits grown in india. it can be used for processing into a variety of beverages, as well as industrial and medicinal uses due to its attractive colour, distinctive flavour and rich source of vitamin ‘c’, vitamin ‘b’, β-carotene, calcium and phosphorous (sogi and singh, 2001). despite its attributes and commercial importance, ‘kinnow’ cannot be enjoyed for long periods due to its poor shelf life. the aggregate post-harvest losses from orchards to consumers in ‘kinnow’ range from 15 to 22% (gangwar et al., 2007). storage at low temperature is one of the potential options to extend the avaibility of many fruits and vegetables (lei et al., 2012). however refrigeration facilities are not generally within the reach of a majority of growers and pathological disease occurrence in ‘kinnow’ is very high in cold storage (singh and jain, 2004). edible coatings are promising postharvest treatments to extend the self-life of many fruits as reported in mango (abbasi et al., 2011; singh et al., 2012), strawberry (del valle et al., 2005), custards apple (el-monem and el-mayeed, 2003) and sweet orange (shahid and abbasi, 2011). both semperfresh tm and sta-fresh 960 are commercial edible coating materials, the former is a sucrose-fatty acid ester-based wax while the later is a paraffin polyethylene-based wax. another important technology used for extending shelf-life of fresh fruits and vegetables is modified atmosphere packaging (map) (ladaniya, 2001; wasker and gaikward, 2005; sharma et al., 2012). also in ‘kinnow’ various postharvest treatments such as waxing (ahmad et al., 2005), map packaging (ahmad et al., 2005; jawandha et al., 2012), bavistin dip (sonkar et al., 2008) and a combination of these treatments are reported to extend the shelf-life during storage and transportation. however, there are few works on edible coating, map packaging and low cost storage systems such as zero energy cool chamber (zecc) for ‘kinnow’ fruits and the subject calls for further investigation. other workers have reported that zecc may be an alternative low cost storage system (roy and khurdiya, 1986; pal et al., 1997) however the treatments must be hazard free and eco-friendly (siddiqui and dhua, 2010). considering these factors, in the present study ‘kinnow’ fruits were treated with different coating materials to evaluate their performance under ambient, zecc and cold storage conditions. 2. materials and methods raw material and treatments mature ‘kinnow’ fruits were procured from the regional horticulture research station, dhaulakuan (hp) and brought to the postharvest technology laboratory, uhf, nauni, solan immediately after harvest. sound and unblemished fruits were treated with different waxing postharvest treatments for preserving quality of ‘kinnow’ fruit under different storage conditions m.s. ahmad*(1), k.s. thakur**, m.w. siddiqui* * department of food science and technology, bihar agricultural university, sabour, bhagalpur (bihar), india. ** department of food science and technology, dr. y.s. parmar university of horticulture and forestry, nauni, solan (hp), india. key words: edible coating, kinnow, quality, shelf-life, zero energy cool chamber. abstract: ‘kinnow’ mandarin is an attractive and nutritious fruit available only for a short period due to its poor shelf life. the effect of different postharvest treatments and storage conditions on the postharvest quality of ‘kinnow’ up to 60 days was examined. with progression of the storage period, tss and total sugars tended to increase whereas acidity, ascorbic acid, juice content, and overall acceptability decreased. fruits stored at low temperature (4±1°c, rh 85-95%) and zero energy cool chamber (zecc) (12-22°c, rh 85-95%) showed a slower rate of physico-chemical changes compared to ambient conditions (18-32°c, rh 45-65%). both waxing and pe-packaging maintained the external appearance of fruits irrespective of storage systems. however, off-flavour development was noticed in pe-packed fruits after 15 days at room temperature and 40 days in cold storage and zecc. waxing of ‘kinnow’ mandarin with undiluted sta-fresh 960 along with low temperature and low cost storage (zecc) may be recommended to extend the availability of fruits. adv. hort. sci., 2013 27(4): 152-158 1 corresponding author: shamsher73@gmail.com received for publication 15 august 2013 accepted for publication 25 november 2013 153 materials as follows: t 1 = semperfresh (0.5%), t 2 = semperfresh (1.0%), t 3 = semperfresh (1.5%), t 4 = sta fresh 960 (100%), t 5 = sta fresh 960 (50%), t 6 = rice starch (3%)+bavistin (0.05%), t 7 = rice starch (6%)+bavistin (0.05%), t 8 = rice starch (3%)+bavistin (0.05%)+guar gum (2%), t 9 = rice starch (6%)+bavistin (0.05%)+guar gum (2%), t 10 = bavistin (0.05%)+ packing of four fruits in a 150 gauze polyethylene film, t 11 = control. storage conditions treated and air-dried fruits from all treatments with their replications were divided into three lots and stored in plastic crates with paper moulded trays under ambient (18-32°c, rh 45-65%), zero energy cool chamber (12-22°c, 80-95% rh) and cold store (cs, 4±1°c, 80-90% rh) conditions. chemical analysis different biochemical parameters of the juice were analyzed at fortnightly intervals. total soluble solids (tss) were estimated by hand refractometer (0-32°b). the readings obtained were calibrated against a standard temperature at 20°c as per the international temperature correction table and expressed as °brix. acidity and ascorbic acid were determined by standard method (aoac, 1990) and results were expressed as percentage citric acid and mg/100 ml of juice respectively. total sugars were estimated by the lane and eynon volumetric methods (aoac, 1990). physical analysis the juice was extracted with the help of an electrically operated citrus juice extractor. the fruits were first weighed and then cut into halves and the cut portion of each half was placed on the revolving ridge knob of the extractor till only the skin part remained and all the segments were crushed and pressed; the juice was collected in the bottom of the juice extractor. sensory evaluation sensory evaluation of samples was conducted by a panel of judges (consisting of teachers, students, and staff) at periodic intervals of storage. the judges were given coded samples consisting of whole and cut fruits for evaluation regarding overall acceptability of the fruits on the basis of appearance, color, taste and defects if any. the evaluation consisted of a 9-point hedonic scale for each attribute (wills et al., 1980). statistical analysis interactions among treatments, storage conditions and biochemical attributes were assessed by completely randomized design whereas, sensory attributes were assessed by the randomized block designed using the statistica v. 8.0 (stat soft, tulsa, ok, usa) package. 3. results and discussion total soluble solids (tss) it was observed that tss in general increased as the storage period progressed under all treatments and storage conditions (table 1). among the fruits kept at ambient temperature, the highest mean tss contents (14.04°b) were recorded in t 11 (control), whereas the lowest mean tss contents (12.84°b) were found in treatment t 4 (100% sta-fresh 960), which was closely followed by t 10 and t 5 , respectively. the control fruits also exhibited the maximum increase of tss under zecc and cold storage conditions, whereas it was usually minimum in response to t 4 followed by t 10 . table 1 effect of postharvest treatments on total soluble solids* (b) of ‘kinnow’ fruits under different storage systems during 60 days of storage storage systems (s) treatments (t) storage intervals (days) 15 30 45 60 mean room temperature (18-32°c, rh 45-65%) t 1 11.90 12.59 13.82 14.69 13.25 t 2 11.84 12.59 13.57 14.10 13.02 t 3 11.84 12.36 13.41 14.45 13.01 t 4 11.76 12.31 13.22 14.09 12.84 t 5 11.78 12.35 13.29 14.49 12.98 t 6 12.04 12.87 14.18 15.81 13.72 t 7 12.02 12.84 14.09 15.37 13.58 t 8 12.00 12.79 14.05 15.50 13.58 t 9 11.98 12.73 14.92 15.33 13.49 t 10 11.77 12.31 13.16 14.15 12.85 t 11 11.12 13.46 14.45 16.15 14.04 mean 11.91 12.65 13.74 14.92 13.03 zero energy cool chamber (12-22°c, rh 80-95%) t 1 11.53 11.82 12.12 12.43 11.98 t 2 11.50 11.80 13.23 13.24 12.44 t 3 11.49 11.84 12.62 13.25 12.30 t 4 11.45 11.65 12.06 12.88 12.01 t 5 11.48 11.87 12.61 13.15 12.28 t 6 11.57 12.10 12.95 13.40 12.50 t 7 11.56 11.88 12.20 12.53 12.04 t 8 11.55 11.86 12.18 12.92 12.13 t 9 11.54 11.95 12.13 12.83 12.20 t 10 11.46 11.68 11.91 12.84 11.97 t 11 11.59 12.94 13.30 14.68 13.12 mean 11.52 11.98 12.48 13.10 12.31 cold storage (4±1°c, rh 85-95%) t 1 11.49 11.73 12.00 12.30 11.88 t 2 11.48 11.72 12.23 12.23 11.82 t 3 11.46 11.67 12.15 12.15 11.80 t 4 11.40 11.56 11.91 11.91 11.65 t 5 11.47 11.67 11.94 11.94 12.04 t 6 11.54 11.89 12.47 12.47 12.01 t 7 11.53 11.80 12.41 12.41 11.96 t 8 11.51 11.78 12.35 12.35 11.92 t 9 11.51 11.77 12.32 12.32 11.91 t 10 11.42 11.58 11.93 11.93 11.67 t 11 11.65 11.96 12.62 12.62 12.13 mean 11.50 11.73 11.98 12.23 cd 0.05 storage systems (s)0.10, sxt0.33, sxi0.19, sxtxi0.66. *initial total soluble solids (tss) of ‘kinnow’ = 11.35°b. 154 the tss content increased due to hydrolysis of insoluble polysaccharides into sugars at a faster rate at high temperature (ambient) and at a slower rate at lower temperatures, i.e. in cold storage and in zecc (siddiqui, 2008; siddiqui et al., 2011; jawandha et al., 2012). the higher value of tss in control fruit might be due to a higher concentration of sugars because of higher transpiration losses as these fruits were not covered, which could impede the movement of water out of the fruits. on the other hand, waxing and pe-packing might have reduced moisture losses to a maximum extent as the combination offers excellent moisture barrier properties (ben-yehoshua, 1985). waxing treatments can act as an additional barrier to moisture loss but are less effective because waxes are more permeable to moisture and gases. however, it is a well established fact that wax materials are capable of delaying ripening process by maintaining slow degradation of polysaccharides as observed in mango (abbasi et al., 2011) and kinnow mandarin (chaudhary et al., 2004). shahid and abbasi (2011) also reported less change compared to control in tss in stored sweet orange fruits treated with bee’s wax and paraffin wax coatings throughout the storage period. manzano and diaz (2001) and hayat et al. (2005) found the similar results in apple after pe-packing and waxing treatments. titratable acidity a gradual decline in titratable acidity contents (table 2) was observed with an increase in storage duration under all three storage conditions during the entire 60-day storage period. 100% sta-fresh 960 (t 4 ) retained the highest mean ta (0.93%) under ambient conditions whereas t 4 and t 3 presented the maximum values under zecc (1.02%) and cs followed by t 10 . at the same time, the control treatment (t 11 ) had the lowest mean titratable acidity (0.64, 0.91, and 0.93%) under ambient, zecc and cs conditions, respectively. the faster rate of decline in acidity at room temperature could be due to faster metabolic reactions leading to earlier senescence at higher temperature. among metabolic reactions in fruits, respiration is an important process which may utilize organic acids as substrate for the production of energy resulting in a decrease in acidity during prolonged storage (sharma et al., 2012). the organic acids involved in the respiratory process are not oxidized at a faster rate at lower temperature, and therefore their levels remained high. furthermore, polyethylene and wax materials slow down the metabolism of fruits and vegetables as these have been reported to maintain higher co 2 and lower o 2 inside the coated/pe-packed fruits (kader et al., 1989): this might explain the higher acid levels in waxed and pe-packed fruits. these findings are further supported by the findings of bisen and pandey (2008) and siddiqui (2008) in guava and mango, respectively. total sugars total sugar increased significantly (table 3) throughout the storage period with the increase being faster under ambient storage and slower under cold storage. under ambient conditions, t 4 (100% sta-fresh 960) proved to be the most effective in delaying the increase in total sugars up to 60 days, whereas, under the other two storage conditions t 10 proved to be the best during the same period. at the end of the study period, the mean maximum total sugar contents (7.74%) was found in treatment t 11 (control) whereas the minimum average sugar contents were recorded for treatments t 4 and t 10 (6.93% and 6.96%, respectively). table 2 effect of postharvest treatments on titratable acidity* (as % citric acid) of ‘kinnow’ fruits under different storage systems during 60 days of storage storage systems (s) treatments (t) storage intervals (days) 15 30 45 60 mean room temperature (18-32°c, rh 45-65%) t 1 1.00 0.86 0.79 0.63 0.82 t 2 1.00 0.96 0.84 0.68 0.84 t 3 1.01 0.95 0.88 0.67 0.88 t 4 1.03 0.98 0.93 0.76 0.93 t 5 1.00 0.95 0.90 0.70 0.89 t 6 0.96 0.87 0.74 0.74 0.83 t 7 0.98 0.88 0.53 0.53 0.78 t 8 1.00 0.87 0.54 0.54 0.79 t 9 1.00 0.89 0.54 0.54 0.81 t 10 1.01 0.96 0.72 0.72 0.89 t 11 0.80 0.64 0.60 0.52 0.64 mean 0.98 0.89 0.80 0.64 0.82 zero energy cool chamber (12-22°c, rh 80-95%) t 1 1.04 1.02 0.99 0.96 1.01 t 2 1.04 1.02 1.00 0.97 1.01 t 3 1.04 1.03 1.01 0.98 1.02 t 4 1.04 1.03 1.01 0.96 1.02 t 5 1.02 1.02 1.01 0.98 1.00 t 6 1.02 0.96 0.91 0.85 0.94 t 7 1.02 0.99 0.95 0.90 0.92 t 8 1.03 1.00 0.97 0.93 0.98 t 9 1.03 1.00 0.98 0.94 0.98 t 10 1.04 1.02 1.01 0.98 1.01 t 11 1.00 0.94 0.87 0.82 0.91 mean 1.03 1.00 0.95 0.93 0.98 cold storage (4±1°c, rh 85-95%) t1 1.04 1.02 1.00 0.97 1.01 t2 1.04 1.02 1.01 0.99 1.02 t3 1.04 1.03 1.02 1.00 1.03 t4 1.04 1.03 1.02 1.01 1.03 t5 1.03 1.03 1.02 1.00 1.02 t6 1.02 0.98 0.95 0.91 0.97 t7 1.02 1.00 0.97 0.93 0.98 t8 1.03 1.01 0.99 0.96 0.99 t9 1.04 1.02 1.00 0.97 1.00 t10 1.04 1.03 1.02 1.01 1.02 t11 1.01 0.96 0.91 0.85 0.93 mean 1.03 1.01 0.99 0.96 1.00 cd 0.05 , storage systems (s) 0.007, sxt0.02, sxi0.01, sxtxi0.05. *initial titratable acidity (%) of ‘kinnow’ = 1.05%. 155 the greater increase in sugar contents under ambient conditions may be due to rapid hydrolysis of insoluble polysaccharides into sugars (siddiqui et al., 2011; jawandha et al., 2012). the great content of sugars in control fruit might be due to greater transpiration losses. pe-packing and waxing have been reported as excellent moisture barriers which reduced moisture losses in fruits. moreover, both pe-packing and waxing can produce modified atmosphere by increasing co 2 and decreasing o 2 concentration. ascorbic acid the ascorbic acid content showed a general declining trend in all treatments and storage conditions. however, the decrease was more pronounced under ambient conditions as compared to the other two storage systems (table 4). the slow degradation rate and consequently higher retention of ascorbic acid under cold storage condition table 3 effect of postharvest treatments on total sugar* (%) of ‘kinnow’ fruits under different storage systems during 60 days of storage storage systems (s) treatments (t) storage intervals (days) 15 30 45 60 mean room temperature (18-32°c, rh 45-65%) t 1 6.81 7.37 8.27 9.87 8.08 t 2 7.10 7.25 7.95 9.25 7.88 t 3 6.71 7.11 7.71 8.70 7.55 t 4 6.76 7.07 7.49 8.19 7.37 t 5 6.69 7.17 7.84 8.85 7.63 t 6 6.90 7.81 9.07 10.65 8.60 t 7 6.87 7.80 8.91 10.35 8.48 t 8 6.84 7.75 8.75 10.35 8.42 t 9 6.81 7.71 8.75 10.45 8.43 t 10 6.72 7.17 7.67 8.40 7.49 t 11 7.00 8.30 10.49 11.60 9.34 mean 6.83 7.50 8.44 9.69 8.11 zero energy cool chamber (12-22°c, rh 80-95%) t 1 6.68 6.75 6.82 6.97 6.80 t 2 6.67 6.72 6.80 6.93 6.78 t 3 6.66 6.71 6.78 6.86 6.75 t 4 6.66 6.72 6.75 6.82 6.73 t 5 6.65 6.68 6.76 6.84 6.73 t 6 6.69 6.85 6.95 7.09 6.89 t 7 6.71 6.81 6.93 7.06 6.87 t 8 6.70 6.80 6.92 7.04 6.86 t 9 6.68 6.76 6.90 7.02 6.84 t 10 6.64 6.68 6.74 6.81 6.71 t 11 6.73 6.86 7.03 7.21 6.95 mean 6.67 6.76 6.81 6.90 6.77 cold storage (4±1°c, rh 85-95%) t 1 6.66 6.73 6.80 6.90 6.77 t 2 6.64 6.70 6.78 6.86 6.74 t 3 6.65 6.69 6.74 6.80 6.72 t 4 6.64 6.67 6.71 6.75 6.69 t 5 6.64 6.68 6.74 6.80 6.71 t 6 6.70 6.80 6.91 7.03 6.86 t 7 6.70 6.79 6.85 7.00 6.83 t 8 6.46 6.68 6.88 6.98 6.75 t 9 6.68 6.76 6.86 6.96 6.81 t 10 6.63 6.66 6.70 6.75 6.68 t 11 6.72 6.84 6.99 7.15 6.92 mean 6.64 6.72 6.81 6.90 6.77 cd 0.05 , storage systems (s)0.01, sxt0.06, sxi0.03, sxtxi0.12. *initial total sugar content of the fruit = 6.51%. table 4 effect of postharvest treatments on ascorbic acid contents (mg/100 ml juice) of ‘kinnow’ fruits under different storage systems during 60 days of storage storage systems (s) treatments (t) storage intervals (days) 15 30 45 60 mean room temperature (18-320c, rh 45-65%) t 1 24.78 24.31 23.84 23.35 24.07 t 2 24.79 24.33 23.84 23.35 24.08 t 3 24.81 24.37 23.93 23.49 24.15 t 4 24.84 24.43 24.02 23.61 24.23 t 5 24.80 24.35 23.90 23.45 24.12 t 6 24.65 24.05 23.45 22.82 23.74 t 7 24.69 24.13 23.57 23.04 23.86 t 8 24.66 24.07 23.48 22.88 23.77 t 9 24.65 24.05 23.45 22.85 23.75 t 10 24.85 24.45 23.72 23.65 24.17 t 11 24.64 24.03 23.42 22.78 23.72 mean 24.74 24.23 23.69 23.21 23.96 zero energy cool chamber (12-22°c, rh 80-95%) t 1 25.08 24.78 24.53 24.27 24.66 t 2 25.10 24.79 24.55 24.30 24.68 t 3 25.10 24.82 24.59 24.35 24.71 t 4 25.15 24.91 24.74 24.55 24.84 t 5 25.11 24.84 24.62 24.39 24.74 t 6 25.01 24.66 24.35 24.03 24.51 t 7 25.05 24.72 24.44 24.15 24.59 t 8 25.03 24.71 24.38 24.07 24.55 t 9 25.03 24.69 24.40 24.11 24.56 t 10 25.16 24.94 24.76 24.56 24.85 t 11 25.02 24.64 24.29 24.94 24.47 mean 25.08 24.77 24.51 24.25 24.52 cold storage (4±1°c, rh 85-95%) t 1 25.12 24.86 24.65 24.43 24.76 t 2 25.12 24.90 24.71 24.51 24.81 t 3 25.14 24.94 24.77 24.54 24.86 t 4 25.16 24.98 24.83 24.67 24.91 t 5 25.18 24.90 24.71 24.51 24.82 t 6 25.16 24.82 24.59 24.35 24.71 t 7 25.10 24.86 24.65 24.43 24.76 t 8 25.12 24.84 24.62 24.39 24.74 t 9 25.11 24.80 24.56 24.31 24.69 t 10 25.09 25.00 24.86 24.71 24.94 t 11 25.19 24.76 24.49 24.21 24.63 mean 25.07 24.88 24.68 24.46 24.78 cd 0.05 , storage systems (s)0.01, sxt0.06, sxi0.03, sxtxi0.12. *initial ascorbic acid content of the fruit = 25.25 mg/100 ml. 156 and in cool chamber might be due to a reduced metabolic rate at lower temperature. greater ascorbic acid content under low temperature might be due to a reduced rate of fruit metabolic activities, mainly respiration. these results are in accordance with the findings of wills et al. (2007) and worawaran et al. (2013). among the treatments, pepacked fruits (t 10 ) and undiluted sta-fresh 960 (t 4 ) had the highest average ascorbic acid during the 60-day storage. the better retention of ascorbic acid in fruits of both the treatments might be due to modifications in the atmosphere immediately surrounding the fruits. pe packing and waxing have been reported to retain higher ascorbic acid (bayindirli et al., 1995; kaushal and thakur, 1996). juice content the juice content of ‘kinnow’ fruit was highest in zecc (table 5) followed by cs; the lowest juice content was found in fruits kept under ambient conditions. in the present study it was also observed that the juice content (initially 40.18%) increased under all treatments and storage conditions at the early sampling dates and then declined as the storage period progressed. maximum juice contents (43.51%) were recorded in treatment t 10 followed by t 5 and t 4 , in comparison to the control fruits which yielded only 40.05 percent at 60 days storage. these findings might be due to a greater moisture loss at higher temperature coupled with the lower humidity conditions under ambient conditions than zecc and cs. among the treatments, higher juice recovery was recorded in pe-packed fruits (t 10 ) followed by the fruits with 100% sta-fresh 960 (t 4 ). this might be due to less water loss in pe-packaging and waxing treatments as the combination acts as a barrier to moisture loss. similar results were also obtained by chaudhary et al. (2004) in kinnow mandarin and bisen and pandey (2008) in kagzi lime. sensory quality a perusal of data in table 6 indicates that the storage temperature had a profound influence on the overall acceptability of the fruits. cold-stored fruits were the most acceptable after 60 days storage, followed by fruits from zecc while those stored at ambient conditions were the least acceptable. up to 60 days storage, fruits from t 4 outscored all other treatments under all three storage conditions, followed by 50% sta-fresh 960 (t 5 ) fruits. at the end of the storage period (60-days), the maximum acceptability (8.01, 7.90 and 6.70) was observed in response to t 4 followed by t 5 (7.85, 7.65 and 6.50), t 3 (7.85, 7.65 and 6.05), t 2 (7.70, 7.45 and 5.90 ) and t 10 (7.67, 7.35 and 5.82) in cold store, zecc and under room temperature respectively. better acceptability of cold-stored fruits is understandable as low temperature storage of ‘kinnow’ fruit helps maintain storage quality, thereby increasing acceptability. pe packing and waxing creates beneficial effects and these conditions are more effective in retaining fruit quality at higher temperature (kader et al., 1989; ladaniya and sonkar, 1997; ladaniya, 2007). the fruits treated with sta-fresh 960 (t 4 ) registered overall good acceptability at the end of 60 days. the present results show similarity with the findings of ladaniya (2001) who demonstrated that taste scores were highest in ‘musambi’ sweet orange (citrus sinensis) fruits treated with sta-fresh 451 wax, and wang et al. (2004) who revealed that due to the waxing, eating quality was good without an unpleasant taste in fruits of jincheng orange variety. similar results were also reported by mahajan et al. (2005) in ‘kinnow’ fruits. table 5 effect of postharvest treatments on juice content* (%) of ‘kinnow’ fruits under different storage systems during 60 days of storage storage systems (s) treatments (t) storage intervals (days) 15 30 45 60 mean room temperature (18-32°c, rh 45-65%) t 1 44.68 46.02 39.54 35.59 41.47 t 2 43.01 43.01 40.17 36.17 40.59 t 3 43.78 41.76 40.11 36.11 40.44 t 4 40.44 44.20 44.30 43.01 42.98 t 5 42.88 44.47 42.82 38.82 42.24 t 6 44.45 45.24 37.12 33.12 39.98 t 7 42.95 42.17 39.75 35.72 40.14 t 8 40.41 45.43 36.77 32.94 38.89 t 9 43.24 43.17 35.78 32.94 38.78 t 10 40.92 42.16 46.70 42.70 43.12 t 11 40.73 39.16 35.43 29.76 36.27 mean 42.68 43.34 38.96 36.08 40.44 zero energy cool chamber (12-22°c, rh 80-95%) t 1 41.45 43.76 44.02 44.79 43.50 t 2 41.38 41.78 43.69 44.14 42.74 t 3 40.98 40.07 44.07 48.11 43.31 t 4 40.35 42.00 44.50 44.45 42.85 t 5 40.75 43.20 45.00 48.82 44.44 t 6 42.08 47.12 46.05 43.12 44.59 t 7 40.18 42.15 42.17 43.75 42.06 t 8 42.90 41.75 43.75 40.94 42.33 t 9 40.05 43.75 45.55 41.76 42.78 t 10 40.28 43.15 45.15 46.68 43.82 t 11 40.30 44.75 42.15 40.76 41.99 mean 40.97 43.04 43.39 44.30 43.08 cold storage (4±1°c, rh 85-95%) t 1 41.75 43.22 40.75 44.75 42.62 t 2 42.48 41.78 41.78 49.70 43.93 t 3 40.35 41.20 40.80 49.43 42.94 t 4 41.78 41.83 42.65 44.45 42.68 t 5 41.08 41.35 43.33 48.30 43.52 t 6 40.47 42.35 41.25 44.10 42.04 t 7 40.70 40.80 42.80 44.80 42.27 t 8 41.25 42.15 43.25 48.55 43.80 t 9 40.25 41.19 42.25 45.50 42.45 t 10 41.20 42.45 44.35 46.48 43.63 t 11 40.75 40.35 42.25 44.25 41.90 mean 41.09 41.69 42.12 46.14 42.88 cd 0.05 , storage systems (s)0.37, sxt-1.23, sxi0.74, sxtxi2.47. * initial juice content of fruit = 40.18%. 157 4. conclusions the result of this investigation showed that using ecofriendly edible coating along with zero energy cool chamber, the shelf life of ‘kinnow’ fruits can be increased substantially. among the treatments, waxing with undiluted sta-fresh 960 along with low temperature storage and 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(18-32°c, rh 45-65%) t 1 6.80 5.80 5.40 3.80 5.45 t 2 7.20 6.20 5.60 4.60 5.90 t 3 7.20 6.40 6.00 4.60 6.05 t 4 7.60 7.20 6.80 5.20 6.70 t 5 7.40 6.80 6.40 5.40 6.50 t 6 7.80 5.00 3.80 2.80 4.60 t 7 7.20 5.60 4.60 4.00 5.35 t 8 7.00 5.40 4.20 3.40 5.00 t 9 6.80 5.00 3.80 2.40 4.50 t 10 8.00 6.40 5.70 3.20 5.82 t 11 6.00 4.80 3.00 1.80 3.90 mean 7.18 5.87 5.03 3.75 5.43 zero energy cool chamber (12-22°c, rh 80-95%) t 1 8.20 7.60 7.20 6.60 7.40 t 2 7.80 7.80 7.40 6.80 7.45 t 3 8.00 7.80 7.60 7.20 7.65 t 4 8.20 8.00 7.80 7.60 7.90 t 5 8.00 7.80 7.60 7.20 7.65 t 6 7.80 7.60 7.00 6.40 7.20 t 7 7.80 7.80 7.20 6.60 7.35 t 8 7.80 7.60 7.00 6.60 7.25 t 9 7.60 7.00 5.80 5.60 6.50 t 10 8.60 8.40 6.80 5.60 7.35 t 11 7.60 6.40 5.80 5.40 6.30 mean 7.95 7.62 7.02 6.51 7.27 cold storage (4±1°c, rh 85-95%) t 1 8.20 8.00 7.80 7.20 7.80 t 2 8.20 7.80 7.60 7.20 7.70 t 3 8.20 8.00 7.80 7.40 7.85 t 4 8.40 8.20 8.00 7.80 8.10 t 5 8.20 8.00 7.80 7.40 7.85 t 6 8.00 8.00 7.60 7.00 7.65 t 7 8.00 7.80 7.40 6.80 7.50 t 8 7.80 7.80 7.20 7.00 7.45 t 9 7.80 7.40 6.80 6.40 7.10 t 10 8.80 8.60 7.10 6.20 7.67 t 11 7.80 7.40 7.00 6.40 7.15 mean 8.12 7.90 7.46 6.98 7.62 cd 0.05 , storage systems (s)0.09, sxt0.31, sxi0.18, sxtxi0.62. *initial score for overall acceptability = 8.60. 158 application and packaging on quality of stored nagpur mandarins. indian j. agric. sci., 67: 500-503. ladaniya m.s., 2007 quality and carbendazim residues of nagpur mandarin fruit in modified atmosphere package. j. food sci. technol., 44: 85-89. lei j., pang j., li s., xiong b., cai l.g., 2012 application of new physical storage technology in fruit and vegetable industry. afr. j. biotechnol., 11: 6718-6722. manzano j.e., diaz a., 2001 effect of storage time, temperature and wax coating on the quality of fruits of ‘valencia’ orange (citrus sinensis l.). proceedings international society for tropical horticulture, 44: 24-29. mahajan b.y.c., dhatt a.s., sandhu k.s., 2005 effect of different post harvest treatments on the storage life of ‘kinnow’. j. food sci. technol., 42: 296-299. pal r.k., roy s.k., srivastava s., 1997 storage performance of ‘kinnow’ mandarin in evaporative cool chamber and ambient condition. j. food sci. technol., 34: 200-203. roy s.k., khurdiya d.s., 1986 studies on evaporative cooled zero-energy input cool chamber for the storage of horticultural produce. indian food packer, 40: 26-31. shahid m.n., abbasi n.a., 2011 effect of bee wax coating on physiological changes in fruits of sweet orange cv. blood red. sarhad j. agric., 27(3): 385-394. sharma r.r., pal r.k., rana v., 2012 effect of heat shrinkable films on storability of ‘kinnow’ fruits under ambient condition. indian j. hort., 69: 404-408. siddiqui m.w., 2008 studies on some aspects of mango ripening. m.sc. thesis, bidhan chandra krishi viswavidyalaya, mohanpur, nadia, west bengal, india. siddiqui m.w., bhattacharjya a., chakraborty i., dhua r.s., 2011 6-benzylaminopurine improves shelf life, organoleptic quality, and health-promoting compounds of fresh-cut broccoli florets. j. sci. indus. res., 70: 461-465. siddiqui m.w., dhua r.s., 2010 eating artificially ripened fruits is harmful. curr. sci., 99: 1664-1668. singh d., jain r.k., 2004 post harvest losses in distance marketing of kinnow. plant dis. res., 19: 36-39. singh a.k., singh c.p., kushwaha p.s., chakraborty b., 2012 effect of postharvest treatments on fruit marketability and physico-chemical characteristics of dashehri mango. prog. hort., 44: 215-219. sogi d.s., singh s., 2001 studies on bitterness development in kinnow juice ready-to-serve beverage, squash, jam and candy. j. food sci. technol., 38: 433-438. sonkar r.k., sarnaik d.a., dikshit s.n., saxena r.r., singh v.k., 2008 wrapping of kinnow mandarin with ldpe film under ambient storage. 11th international citrus congress (isc congress) wuhan china, pp. 333. wang r.k., shao p.f., zhou l., zhu r.g., 2004 effect of fruit waxing agent a on the commodity quality of jincheng orange variety. south china fruits, 29: 13-15. waskar d.p., gaikward r.s., 2005 effect of various postharvest treatments on extension of shelf-life of kesar mango fruits. indian j. agric. res., 39: 95-102. worawaran r., nithiya r., noppol l., danai b., 2013 influence of storage conditions on physico-chemical and biochemical of two tangerine cultivars. j. agric. sci., 5: 70-84. wills r., mcglasson b., graham d., joyce d., 2007 postharvest: an introduction to the physiology and handling of fruit, vegetables and ornamentals. second edition, university of new south wales press, sydney, australia. wills r.b.h., bam b.p.a., scott k.j., 1980 use of flesh firmness and their objective tests to determine consumer acceptability of delicious apple. austral. j. expt. agric. anim. husb., 20: 252-256. 33 1. introduction endodormancy is defined as a phase of suspended growth in the meristematic part of a plant structure that is controlled from within the same plant structure (lang et al., 1987). endodormancy thus characterizes not a whole plant but individual plant structures (buds, cambial zones, seeds), and the endodormancy release enabling this meristematic part to recover full growth ability needs to be understood at this individual structure level. this allows for heterogeneity between buds in the tree structure regarding endodormancy status, which is not taken into account in standard phenological models. however, studies on temperate fruit tree species cultivated in tropical or subtropical conditions, i.e. experiencing limiting chilling conditions, have widely reported strong heterogeneity in bud break timing and spacing attributed to heterogeneity in endodormancy release (crossa-raynaud, 1955; guerriero and scalabrelli, 1982; dennis, 1987; bernardi, 1988; mauget and rageau, 1988; lam yam, 1990; zguigal, 1995). this heterogeneity in endodormancy release has been shown to result in major impairment of bud break/blooming patterns, including bud-breaking rate with extended and heterogeneous bud breaking delay which in turn results in low leafing rates, the production of ‘rosettes’, and branching deficiency (zguigal, 1995). in light of current global warming, such disturbances could potentially extend to new areas, starting in the south of the current temperate zone. this hypothesis is supported by recent observations: since 1988 (the start of continuous increase of yearly mean temperature in france) bloom date has advanced, compared to the 1976-1988 period, whereas endodormancy release date has tended to retreat, most visibly in southern europe (chmielewski et al., 2004; legave et al., 2008) and japan (honjo, 2007; primack et al., 2009). in order to model blooming/bud-breaking date in a context of global change, an accurate endodormancy release module is needed, requiring greater knowledge of chilling signal evidence for non-occurrence of node-to-node or stem-to-bud transfer of chilling temperature signal for dormancy release m. bonhomme *, **, andré lacointe *, **, rémy rageau *, ** * inra, umr547 piaf, 5 chemin de beaulieu, f-63100 clermont-ferrand, france. ** clermont université, université blaise pascal, umr547 piaf, f-63000 clermont-ferrand, france. key words: chilling perception, endodormancy release, prunus persica, temperature sensing. abbreviations: cu: chilling unit, gdh: growing degree hour, mtb: mean time until bud break, pcu: positive chilling unit. abstract: in the current context of global changes, phenology is expected to be one of the major processes affected by temperature increase, notably through the dynamics of endodormancy release. however, the actual impact on bud break pattern is difficult to predict due to poor knowledge about the spatial extent of chilling sensing, which likely affects bud break heterogeneity. indeed, contrary to a widely held opinion, the strictly local perception of the chilling air signal has never been demonstrated. the present experiment addresses this issue through local chilling or heating of selected nodal groups of buds on shoots of peach tree under endodormancy. a temperature-conditioned localizable air-jet device was designed to provide the sharpest possible temperature contrast between selected treated buds and the ‘not-treated’ rest of the tree structure, including adjacent axis tissue. different chilling doses were tested over two experimental seasons, and a heat treatment was applied on single nodes in a cold environment. chilled vegetative buds did not break when the local chilling dose received was less than 50% of the requirements even though neighboring axis tissue received the standard chilling dose. the maximum rate of bud break reached 80% at chilling completion and most of the broken buds produced long shoots. we conclude that temperature sensing occurs on a very local scale with the signal not reaching neighboring untreated buds, meaning that endodormancy release should be considered a very local process. the local response permits analysis of the intra-canopy heterogeneity of bud break and the possible relationship between bud status and intra-canopy heterogeneity of bud temperature. adv. hort. sci., 2013 27(1-2): 33-43 (1) corresponding author: marc.bonhomme@clermont.inra.fr received for publication 14 january 2013 accepted for publication 21 may 2013 34 sensing, transduction and response. as a step towards understanding and integrating bud break heterogeneity into relevant models, it is important to identify how locally the chilling air temperature signal is perceived and whether it is transduced between structures (buds). dormancy release has been investigated at more macroscopic scales, ranging from a twig up to a whole branch. coville (1920) chilled or warmed a whole branch of blueberry plants, timmis and worrall (1974) chilled a whole branch of douglas fir, krassnosselskaya and richter (1942) applied a warm bath on sections of poplar and ash branches, and witkowska-zuk (1970) applied a warm bath on a section of populus x berolinensis branch. in reviews covering older works, nooden and weber (1978) and saure (1985) concluded that the influence of endodormancy release factors appears confined to the buds of the treated parts; however treatments were not applied at bud scale and, consequently, did not permit identification of exactly which structure (buds, inter-node, or branch axis) hosted the actual perception zones sensing the endodormancy releasing factors (mainly temperature signal). in his review, perry (1971) concluded that ‘although there is much evidence for the concept of a control center of dormancy processes in buds and leaves, the possibility that other plant parts may be involved is not excluded’. this issue could be partially resolved by investigations at a smaller spatial scale. witkowska-zuk (1970) applied warm baths to terminal buds of long shoots of populus x berolinensis. other studies used endodormancy-releasing chemicals on single buds on the shoot: denny and stanton (1928) applied ethylene chlorohydrin on lilac; wang and faust (1987) applied thidiazuron on apple. when plants or branches were subsequently exposed to mild temperatures, only the treated buds broke, prompting the conclusion that buds were effective perception zones of these endodormancy breaking factors and that only the treated bud was able to receive the signaling process borne in it. however, with warm bath treatments it is impossible to distinguish the effects of temperature, tissue moistening and their interaction, such as the oxygen limitation that saure (1985) highlighted as an important factor. in addition, perry (1971) pointed out that ‘all that the treatments used for breaking dormancy are severe. often the temperature or chemical concentrations used border on being lethal. many of the chemicals used are poisonous at relative low concentrations’. so the interpretation of such results remains questionable, even in the most convincing case of lilac bud treatment by denny and stanton (1928). surprisingly, to date no experimental study at bud level has used chilling temperature, although it is undoubtedly the main natural factor driving bud endodormancy release at bud level. the temperature signal was also investigated by authors working on vernalization signal, but the treatment was not applied strictly to the bud; for example metzger (1988) chilled the upper part of the twig. furthermore, the process investigated is not exactly the same because, in the vernalization process, leaves are involved (crosthwaite and jenkins, 1993). thus, contrary to a widely held opinion, a strictly local perception of the chilling air signal has never been demonstrated. the present experiment addresses this issue through local application of different temperatures to either buds or neighboring non-bud areas in an attempt to answer the following questions: 1) which structures are able to perceive the chilling signal inducing dormancy release bud only, non-bud area only, or both; 2) is the chilling signal applied to one bud able to break dormancy in other buds on the twig; 3) is the signal applied to non-bud area able to break dormancy in buds; 4) is non-bud chilling required in addition to bud chilling for dormancy release? 2. materials and methods the experiment was conducted on ‘redhaven’ peach trees (three years old in 2003) grown outside in 200-l containers filled with a peat-local soil mixture. thermal conditioning was applied locally to parts of one-year-old shoots at bud scale, with two contrasting levels: either (i) target buds were submitted to chilling temperature while the rest of the tree structure, including shoot tissues adjacent to these buds, remained at non-chilling temperature (t>15°c in a greenhouse, experiments 1 and 2); or (ii) a symmetrical treatment, with target buds kept at non-chilling temperature while the rest of the tree structure remained at chilling temperature in a cold chamber (experiment 3). as the focus of the study was on endodormancy release in relation to outgrowth and branching pattern, the term ‘target buds’ refers to vegetative buds only; the one or two floral buds that could be on either side of the single vegetative bud on each node, sharing the same thermal conditions, were not taken into account. conditioning device local thermal conditioning was achieved by a thermostated air-jet (fig. 1). low-pressurized air was circulated in plastic pipes from a thermostated water bath and delivered through nozzles (modified pipette tip cones) directly blowing onto node buds. the neighboring shoot axis structures were kept isolated from the air jet by a plastic deflector set up at the base of each air-chilled or air-warmed bud group. fig. 1 schematic diagram and picture of the device developed for selectively chilling or heating buds (for the warming treatment, warm water replaced cooled water in the water bath). 35 bud temperature was monitored by thermocouples inserted between the basal scales of one floral bud (fig. 2), and internode temperature corresponded to ambient temperature. thermocouples inserted into the bark of the shoot axis 3-5 mm away from the bud point connection made it possible to verify that adjacent axis tissues (aat), very close to the buds but protected by the deflector (fig. 1), were not significantly affected by the cooled air jet. this device allowed efficient local, differential thermal control of buds and the non-bud area (fig. 3). thermocouple data were recorded on a delta t logger (delta-t-device-siis, cambridge, uk) every 10 min. ambient temperature, corresponding to the inter-node temperature, was recorded (time lapse: 10 min) through a thermistor (hobo temp pro 64k, prosensor). selective chilling of buds on cold-deprived trees under endodormancy (experiments 1 and 2) trees were cold-deprived from the time of deepest endodormancy onwards by transferring them to a greenhouse kept at a temperature that was high enough to prevent any chilling effect (heating threshold: 15°c) and moderate enough to prevent heat shocks (cooling threshold: 25°c). experiment 1. in 2003, six trees were transferred from outside into the greenhouse on october 14. due to technical constraints, two 80-cm-long shoots were selected on one of the six trees for the cooling treatment. on each shoot (sx), eight bud groups at nx node position from the terminal (one vegetative bud with one or in most cases two floral buds) were chilled under the cooled air-jet from november 18 onwards. on december 23, a thermocouple was inserted under the bark of the shoot axis at mid-internode between nodes n14 (not chilled) and n15 (chilled) of shoot s2. the data (not shown) revealed that during the cooling period, mid-internode temperature was very close to the ambient temperature as recorded with the thermistor. this enabled us to map the temperature dynamics of each shoot section (buds, internodes) of the treated zone. the buds of nodes n6 and n9 on shoot s1 fell at the beginning of december due to mechanical injury while setting up the air device. they were replaced by two new groups, i.e. nodes n5 and n7 on s1. on december 30, the cooling systems on all buds were stopped. we noticed damage on the buds at node n5, which were consequently discarded from the analyses. consequently, this treatment actually had two levels: a ‘short chilling’ treatment (buds of node n7 on s1) and a ‘long chilling’ treatment (the other buds). experiment 2. in order to investigate the impact of insufficient chilling on dormancy release and bud breaking patterns, a second experiment, similar to experiment 1, was conducted with low and medium chilling doses. in 2004, six trees were transferred from outside into the greenhouse on october 10. four one-year-old shoots on one of the six trees and six bud groups were selected on each shoot. air jet cooling started on 11 february 2005 (fig. 2). it was terminated on march 15 for shoots s1 and s4 (‘short chilling’ treatment) and on april 6 for shoots s2 and s3 (‘long chilling’ treatment’). on march 15, when switching off the nozzles, we noticed damage on the buds at node n12 on s1; the corresponding data were discarded. calculating chilling and heating doses. the chilling doses received by different parts of the treated shoots were calculated from the thermocouple (cooled buds) or thermistor data (other parts of the shoot) by applying the classical utah model (cu) from richardson et al. (1974), the positive utah chill units (pcu) model (linsley-noakes et al., 1994) and the dynamic model (fishman et al., 1987 a, b). the pcu model was chosen because it appears more relevant in warm country (with october 1 as start date and hourly temperature data) and the dynamic model because it is, described as the most accurate model for walnut by luedelling et al. (2009 a, b; 2011). for each experiment, we calculated the heat unit doses between the end of the bud cooling phase and the mean date of bud break. we used the classical growing degree hours (gdh) model, with 4.5°c as base temperature (richardson et al., 1975). selective warming of buds on chilled trees under endodormancy (experiment 3) our objective was to condition targeted buds at temperatures unable to release endodormancy (>15°c) while the rest of the tree structure, including shoot tissues adjacent to these buds, remained at chilling temperatures. two trees were cold-deprived by transferring them on 15 september 2008 to a greenhouse maintained at a temperature that was high enough to prevent any chilling effect on the trees (heating threshold: 15°c). on 17 october 2008, they were transferred into a cold chamber set around 8°c with fig. 2 schematic diagram of the experimental design for the treatment of bud groups and for collecting the temperature data for treated bud groups and the corresponding adjacent axis tissues. fig. 3 temperature applied at stem and bud level in experiment 1 (partial recording as example). 36 a short photoperiod (8 h) for 52 days, which corresponded to the target accumulation of ca. 1000 cu calculated with the pcu or utah models as chilling requirements were estimated as 870 cu by richardson et al. (1974, 1975) and 1180 cu by werner et al. (1988) for redhaven peach trees. the corresponding value for the dynamic model was initially set as 75 ‘portions’ for this variety (erez et al., 1988) but later revised to 45 ‘portions’ (erez, personal communication) and optimized by bonhomme et al. (2010) at 48 ‘portions’. during this period, the same bud conditioning device was used to provide warm air (>15°c) to selected buds on six twigs of two trees. finally, the trees were transferred on december 8 into a greenhouse where the temperature was kept above 15°c until bud break. biological observations dormancy assessment. to ascertain if vegetative buds were endodormant at the start of the chilling or warming treatments, we characterized dormancy status using the “single node cuttings” forcing test at 25°c (rageau, 1978; dennis, 2003) on buds from non-treated shoots. mean time to bud break (mtb) was then calculated. at the end of both treatments, mtb was calculated to verify that endodormancy had not been released in experiments 1 and 2 for non-chilled buds but had been released in experiment 3 for non-warmed buds. bud phenology and shoot growth. each of the buds of treated shoots was checked twice a week after the end of the cooling or warming treatment (i.e. non-chilling); the buds, like the other parts of the trees, were kept under the forcing temperatures (>15°c) of the greenhouse. we watched for bud break in vegetative buds, at “green tip” stage, #09 in bbch scale (meier, 2001). after the cooling treatment, shoot length was measured several months after bud break, in late may and june in experiments 1 and 2, respectively, when growth had almost completely stopped, in order to check for alterations in shoot growth. the same control checks were done in june for experiment 3. statistical analysis bud breaking rates obtained for the different levels of chilling doses were compared with fisher’s exact test. the relationship between bud break delay (db) and chilling doses received, and between the length of shoots (l) and chilling doses were analyzed by linear correlation. the effect of year (experiment) on db and l was tested using the non-parametric kruskal-wallis test. all the analyses were performed using the r software package (r development core team 2011; r foundation, http://www.r-project.org/). 3. results chilling doses received by the different parts of the shoots accumulated chilling doses (in cu, pcu and portions) received by the different parts of the shoots were computed according to experiment and chilling treatment duration (table 1). table 1 chilling amounts (computed with pcu and cu and dynanic models) received by different organs and tissues: all internodes and at cooled and non-cooled node buds according to experiments 1 and 2 on shoots (si) and nodes (nj) buds at cooled nodes endodormancy release model shoot node class of chilling amounts positive cu (pcu) utah (cu) dynamic (portions) experiment 1 “short chilling” treatment s1 n5 medium 622 537 24 s1 n7 medium 620 540 20 “long chilling” treatment s1 n11 high 1060 970 37 s1 n12 high 1078 993 38 s1 n13 high 1060 970 37 s1 n14 high 1060 970 37 s1 n15 high 1060 970 37 s1 n17 high 991 906 37 s2 n6 high 1061 976 38 s2 n8 high 1060 970 37 s2 n9 high 1060 970 37 s2 n11 high 1083 998 37 s2 n13 high 1060 970 37 s2 n15 high 1060 970 37 s2 n16 high 1060 970 37 s2 n19 high 1079 993 37 chilling amounts at all internodes and non-cooled-node buds very low 97 0 3 experiment 2 “short chilling” treatment s1 n8 low 469 334 30 s1 n10 low 500 370 30 s1 n15 medium 629 499 32 s1 n17 low 531 400 31 s1 n20 medium 652 523 32 s4 n9 low 451 313 30 s4 n12 low 440 300 30 s4 n17 low 378 231 28 s4 n21 low 528 386 30 s4 n24 low 440 300 30 s4 n29 low 399 249 29 “long chilling” treatment s2 n10 low 558 410 29 s2 n11 medium 750 610 29 s2 n13 medium 674 534 29 s2 n17 medium 711 571 29 s2 n20 medium 862 726 30 s2 n23 medium 750 610 29 s3 n9 medium 761 626 30 s3 n11 medium 773 639 30 s3 n16 medium 887 982 30 s3 n19 high 1025 756 31 s3 n24 medium 806 667 30 s3 n29 medium 810 730 30 chilling amounts at all internodes and non-cooled node buds very low 129 0 11 for non-thermocoupled buds, chilling amounts were estimated as means (rounded to the nearest 10 pcu or cu) of all experimental values from the same class of chilling on the same shoot in the same experiment and indicated in italics 37 based on the recorded thermistor data, some chilling units were accumulated during experiments 1 and 2 (97 and 129 units, respectively, which is very low compared to the chilling requirements as mentioned above) at the inter-node tissues as well as at buds and aat at non-cooled nodes. about half of this chilling occurred before transfer to the greenhouse, the rest occurred before the cooling treatment. at the cooled nodes, chilling doses were computed, either from actual data for the thermocoupled buds or non thermocoupled buds, as means of all experimental values of the same chilling class from the same experiment (table 1). four classes of chilling dose were defined: ‘very low’ (<150 pcu), ‘low’ (350-550 pcu), ‘medium’ (600-900 pcu) and ‘high’ (>950 pcu). internodes and non-cooled buds received very low doses (table 1), mainly before the transfer into the greenhouse. in experiment 3, the accumulated chilling dose of nonwarmed buds reached 973 pcu (table 2). as the temperature was around 8°c, almost the same value was obtained with the classical utah model, thus meeting the chilling requirements of the redhaven peach bud (≈900 cu). for warmed buds, the pcu dose was very low (32 pcu maximum, i.e. only 3% of chilling requirements, 1 portion i.e. around 2% of chilling requirements for dynamic model) for the coldest bud, and zero for the warmest bud. the classical utah model yielded zero cu for all warmed buds. endodormancy status after trees were transferred to the greenhouse, the mtb values of non-chilled vegetative buds decreased over the first two to three months and leveled down to ca. 22.5±0.5 days in experiment 1, and 21.5±0.5 days in experiment 2 (data not shown). at periods marking the start of bud cooling, mtb was 31.5±0.5 d in experiment 1 and 21.5±0.5 d in experiment 2, thus indicating that vegetative buds were actually endodormant [mtb threshold between endodormancy and ecodormancy for vegetative ‘redhaven’ peach tree buds is cited as 12 days by bonhomme et al. (2000)]. in experiment 3, at the beginning of the treatment, the mtb value of the non-chilled vegetative buds was 32±10 d (data not shown), thus indicating that the vegetative buds were actually endodormant and close to the maximum of endodormancy. when trees were transferred from the cold chamber to the greenhouse for bud break forcing, the mtb value was 12±1.5 d, indicating endodormancy release for these chilled buds. it was impossible to determine mtb of the warmed buds due to insufficient replications. nevertheless, bud break did not occur, which clearly indicated that endodormancy was not released. vegetative bud response vegetative bud behavior resulting from chilling dose in experiments 1 and 2. table 3 presents the vegetative bud responses to the chilling doses received: break, abscission, or no detected event. neither very low (<115 pcu) nor low (mean: 469 pcu) doses resulted in bud break. consequently the bud break rate obtained with medium chilling dose is significantly higher (p<0.0001) than very low and low rates. almost no buds fell after very low doses, but 20% of buds fell after low doses. medium and high doses (mean: 745 and 1057 pcu, respectively) both resulted in about 10% bud fall and in 69 and 80% bud break, respectively, values which were not statistically different. on the shoots with medium and high doses, none of the non-cooled vegetative buds broke (fig. 4a, 4b), either on the equipped tree or on the other five trees kept under the greenhouse. figure 5a shows that within each experiment, the individual delay to bud break (db), i.e. the time between the end of cool air application and bud break, was not dependent on the pcu received by the bud (for experiment 1, r2 = 0.22, p = 0.12; for experiment 2, r2 = 0.10, p = table 2 chilling amounts (computed with pcu and cu and dynamic models) received by different organs and tissues: all internodes and at non-cooled node buds according to experiment 3; at warmed nodes, shoots (si) and nodes (nj) buds at cooled nodes endodormancy release model shoot node class of chilling amounts positive cu (pcu) utah (cu) dynamic (portions) experiment 3 “warming” treatment s1 n16 very low 0 0 0 s1 n17 very low 18 0 0 s1 n21 very low 0 0 0 s1 n24 very low 6 0 0 s2 n16 very low 30 0 1 s2 n21 very low 27 0 1 s2 n27 very low 24 0 1 s3 n16 very low 0 0 0 s3 n21 very low 0 0 0 s3 n24 very low 0 0 0 s3 n28 very low 0 0 0 s3 n29 very low 0 0 0 s4 n16 very low 0 0 0 s4 n18 very low 0 0 0 s4 n20 very low 0 0 0 s5 n13 very low 0 0 0 s5 n20 very low 0 0 0 s5 n24 very low 0 0 0 s6 n14 very low 3 0 0 s6 n18 very low 14 0 0 s6 n23 very low 32 0 1 s6 n24 very low 14 0 0 s6 n28 very low 6 0 0 chilling amounts at all internodes and non-warmed node buds high 973 941 41 for non-thermocoupled buds, chilling amounts were estimated as means (rounded to the nearest 10 pcu or cu) of all experimental values from the same class of chilling on the same shoot in the same experiment and indicated in italics. 38 0.41; and if both experiments 1 and 2 were analyzed as one single dataset, r2 = 0.08, p = 0.21). there was a clear experiment effect on the mean db (p < 0.001): 35.5 d in experiment 1 and 28.0 d in experiment 2. this difference could be mainly explained based on mean temperature under the greenhouse after the end of bud cooling, which was higher in experiment 2 (19.1°c) than experiment 1 (16.2°c). in addition, figure 5b shows that the length of a growth unit sprouted from a broken bud was not dependent on the cumulated pcu that had been received by that bud (for experiment 1, r2 = 0.05, p = 0.51; for experiment 2, r2 = 0.08, p = 0.47; and if both experiments 1 and 2 were analyzed as one single dataset, r2 = 0.008, p = 0.79). the fig. 4 bud break in the localized-chilled experiment (a and b) and localized-warmed experiment (c). fig. 5 responses of vegetative buds according to chilling dose. a: individual delay to bud break (db); b: individual final length (l) of shoots in experiments 1 (☐) and 2 (▲). table 3 vegetative bud responses to the received chilling doses buds at cooled nodes endodormancy release model shoot node class of chilling amounts positive cu (pcu) utah (cu) dynamic (portions) experiment 3 “warming” treatment s1 n16 very low 0 0 0 s1 n17 very low 18 0 0 s1 n21 very low 0 0 0 s1 n24 very low 6 0 0 s2 n16 very low 30 0 1 s2 n21 very low 27 0 1 s2 n27 very low 24 0 1 s3 n16 very low 0 0 0 s3 n21 very low 0 0 0 s3 n24 very low 0 0 0 s3 n28 very low 0 0 0 s3 n29 very low 0 0 0 s4 n16 very low 0 0 0 s4 n18 very low 0 0 0 s4 n20 very low 0 0 0 s5 n13 very low 0 0 0 s5 n20 very low 0 0 0 s5 n24 very low 0 0 0 s6 n14 very low 3 0 0 s6 n18 very low 14 0 0 s6 n23 very low 32 0 1 s6 n24 very low 14 0 0 s6 n28 very low 6 0 0 chilling amounts at all internodes and non-warmed node buds high 973 941 41 for very low chilling level of the non-treated shoots in experiments 1 and 2, the number (nb) of non-broken buds was not exactly determined (>1000) but corresponded to all the buds (100%) of the six trees placed under the greenhouse. significantly different rates, as assessed by fisher’s exact test, are indicated by different letters. 39 experiment effect on sprouted shoot length was not significant (p = 0.06). it can be concluded that selective chilling of a nodal group of buds was effective in releasing the endodormancy of its vegetative bud. no endodormancy release of buds of any other node was observed, which shows that the signal is not transmitted. vegetative bud behavior resulting from the warming treatment in experiment 3. with a few exceptions at the stem bases, almost all buds chilled in the cold chamber broke, corresponding to the classical bud break pattern observed in temperate climates (82% of bud break on the control twigs). on the treated tree, not a single warmed vegetative bud broke, indicating that the chilling signal received at the stem was not transmitted to non-chilled buds (fig. 4c). for non-warmed trees, bud break was observed on 9 january 2009 for terminal buds and 16 january 2009 for axillary buds, corresponding to 8419 and 10479 gdh (with the 4.5°c base) after chilling, respectively, i.e. around 10% higher than was calculated for axillary buds in experiments 1 and 2. mean temperature in the greenhouse over the 39-day period between the end of chilling and axillary bud break was 15.8°c. 4. discussion and conclusions vegetative bud behavior resulting from chilling dose in experiments 1 and 2 contrasting with medium and high chilling doses, the low chilling dose treatment allowed some events to take place in the buds during further forcing, that ultimately led to abscission of a significant percentage of these buds. the selective chilling of a nodal group of buds was effective in releasing endodormancy of its vegetative bud, and did not result in endodormancy release of buds of any other node. nevertheless, some other aspects of the results were unexpected. medium chilling doses (620-887 pcu) were almost as effective as high doses (991-1083 pcu): the percentage of broken buds was only slightly lower (table 3) and the shoots borne from the broken buds showed normal growth. the chilling requirement for redhaven peach trees is cited at 870 cu (utah model) by richardson et al. (1975), 1180 cu by werner et al. (1988) and 45 portions in the dynamic model (erez, personal communication), optimized to 48 portions (bonhomme et al., 2010) from one-node cutting test results. this points out the three critical elements in the models: (1) the determination of the starting date for chilling accumulation, (2) taking into account (or not) the negative temperatures and (3) the reality and intensity of the reverse effect of mild temperatures and the duration during which the reversion is possible (a short period in dynamic model, without limits in utah model, 24 h in pcu). the computed heat requirement for bud break (around 9000 gdh) was much higher than the value given by richardson et al. (1975) for ‘redhaven’, i.e. 4922 gdh (for the same 4.5°c baseline) for floral buds (full bloom) with 870 cu of prior chilling, and closer to that found by scalabrelli and couvillon (1986), i.e. 8000 gdh (same base) for vegetative bud break with 900 cu of prior chilling. the heat requirements would, surprisingly, have been quite similar for buds under medium chilling doses (8900 gdh) and buds under high chilling doses (9200 gdh). this suggests that chilling requirements may have been overestimated and/or that these buds were in deeper endodormancy in experiment 1 than in experiment 2 when chilling started. although some shoots had not totally stopped growing when the shoot lengths were recorded, figure 5b shows that the length of a growth unit borne from a broken bud was not dependent on the cumulated chilling dose received by this bud. vegetative bud behavior resulting from warming treatment in experiment 3 excluding some buds at the base of the twigs, almost all the buds chilled in the cold chamber broke, thus reproducing the classical bud break pattern observed in temperate climates. not a single warmed vegetative bud broke, indicating that the chilling signal received at the stem was not transmitted to the non-chilled buds. normal bud break occurred at a chilling mean temperature of around 9.3°c, showing once again that the weinberger model is not accurate, even under temperate climates. in this range of chilling temperatures, the utah and pcu models gave the same cumulated chilling (990 cu), close to the chilling requirement cited by richardson et al. (1974). the heat requirement for bud break estimated in this experiment was a little higher than that found by scalabrelli and couvillon (1986), i.e. 8000 gdh (same base) for vegetative bud break with 900 cu of prior chilling, and also than the amounts deduced from experiments 1 and 2. the small difference (2-3 days) could probably be attributed to the uncertainty in determining bud break date or starting date (i.e. endodormancy release date). all these results indicate that the temperature for endodormancy release has to be applied at the level of the structure, where the cause of growth blockage is located, i.e. the bud itself, according to the definition of endodormancy given by lang et al. (1987). accuracy of the chilling and heat requirements these requirements are very approximate determinations that are not fully suitable for modeling the impacts of global changes. figures on chilling requirements for endodormancy release are often confounded by the chilling received during the period between growth arrests or leaf fall and bud break. heat requirements given in the literature are also approximations, as the thermal threshold permitting growth is generally unknown. moreover, heat and chilling action could combine at mild temperature 40 (around 10°c), and determination of the start date for heat action remains problematic. further, the delay to bud break (or bloom) and the heat requirements calculated may also be dependent on factors other than the sole bud response to temperature, such as bud water and nutrient uptake rate and, consequently, the temperature of the roots as revealed in young et al. (1987). thus, the questions about chilling and heat requirements will probably have to be revisited before chilling and heat requirement figures can be used to model climate change impacts. temperature signal transfer from the literature, the most clear response is that the chilling (temperature) signal is not transferred from one terminal bud of a branch to the terminal bud of another branch. regarding vernalization process studies, it is clear that a signal is transferred from the leaves of sugar beet to the terminal meristem (crosthwaite and jenkins, 1993) but this signal could be a thermic signal or a secondary biochemical signal. this signal could also be originated in roots (metzger et al., 1992). some grafting approaches (metzger, 1988) seem to show that the vernalized condition is not transferred to other buds but it is not exactly the same process (transfer of a status and not simultaneous transfer of the signal received by one bud to another bud). moreover, vernalization seems to be correlated with the presence of dividing cells and the dormancy release process occurs on buds with their cell cycle blocked in g1 phase (cottignies, 1987). therefore, even if vernalization and dormancy release seem to be very similar (metzger, 1996), the literature does not make it possible to conclude about the absence of transfer for the chilling signal from the close part of the twig to the bud or from one bud to another bud located on the next internode or the complementarity of both tissues. synthesis: interpretation in terms of chilling signal transfer candidates for the chilling receptive zone are the different nodal bud groups and the axis; candidates for the meristematic targets are the vegetative buds of different nodal groups (only two groups represented). with regard to possible paths, we considered 1) ‘univocal’ paths, i.e. endodormancy release response can only result from the signal coming from one sensing zone; 2) ‘parallel’ paths, i.e. each of the signals borne in either a bud group or an axis results in effective endodormancy release in a given bud; 3) ‘cooperative’ paths, i.e. effective endodormancy release in a given bud needs signals from both bud and axis; 4) ‘short’ paths, i.e. the signal reaching a bud comes from its bud group, and 5) ‘long’ paths, i.e. the signal reaching a bud comes from another group. this can be translated into different hypotheses: hypothesis 1: a local application of chilling or warming induces a local response for dormancy release. as chilled buds broke and warmed buds did not, the present results are consistent with this hypothesis. hypothesis 1’: the chilling (or warming) signal can be transmitted to another bud. as on a twig in warm conditions only chilled buds broke, this hypothesis has to be rejected. hypothesis 2: a chilling signal applied to a stem could reach the bud and permit bud break. as warmed buds on stems placed in a cold environment did not break, this hypothesis has to be rejected. hypothesis 3: chilling applied to buds does permit bud break even if parallel warming on the stem is applied. as chilled buds broke on a twig in warm conditions, the present results are consistent with this hypothesis. the reciprocal hypothesis chilling applied to a twig does permit bud break even if parallel warming on the bud is applied is rejected consequent to the rejection of hypothesis 2. hypothesis 3’: the chilling signal applied to a bud could be transmitted to other buds, permitting bud break, even if a parallel warm signal is applied to the stem. as only chilled buds broke on a twig in warm conditions, this hypothesis has to be rejected. hypothesis 4: chilling applied to buds needs additional chilling on the stem to permit bud break. as chilled buds broke on a twig in warm conditions, this hypothesis has to be rejected. hypothesis 4’: chilling applied to buds could be transmitted to other buds if additional chilling is applied to the stem. since in a cold environment (i.e. not-warmed buds and twig are chilled), warmed buds did not break, this hypothesis has to be rejected. figure 6 summarizes the different hypotheses tested and the possible “chilling receptive zone  vegetative meristematic target” pathways for endodormancy release signal. in conclusion, our results validated hypotheses 1 and 3; the other cases are to be rejected. thus, chilling clearly has to be applied to the given bud to be effective for its subsequent endodormancy release. chilling the stem without chilling the bud is not effective for endodormancy release. chilling on the bud does not need additional chilling on the stem. consequently, the physiological processes involved in chilling-temperature response for endodormancy release have to be investigated at bud level. this is not necessarily (and probably not actually) the case for primordial growth in buds during the ecodormancy phase, as water and nutrient uptake fluxes from roots are needed. acknowledgements this work was supported by the inra (institut national de la recherche agronomique), environment and agronomy department. we thank c. bodet, m. crocombette and j.p. richard for technical assistance and att for language editing of the manuscript. 41 fig. 6 schema of the different hypotheses tested for “chilling perception”  “meristematic response” spatial paths in the endodormancy release response to chilling; 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investigations on the bud dormancy of populus x berolinensis dipp. v. relation between the growth apexes on neighbouring shoots. acta soc. bot. pol., 39: 285-296. young e., motomura y., unrath r., 1987 influence of root temperature during dormancy on respiration, carbohydrates, and growth resumption in apple and peach. j. amer. soc. hort. sci., 112: 514-519. zguigal y., 1995 evolution et caracteristiques de la dormance des bourgeons de pommier (malus domestica borkh., cv golden delicious) dans un climat à hiver doux (région de meknes, maroc). phd thesis, institut agronomique et veterinaire hassan ii (ma). impaginato 149 adv. hort. sci., 2025 39(2): 149­162 doi: 10.36253/ahsc­16803 https://oaj.fupress.net/index.php/ahs physiological tolerance of shallot varieties to airborne salinity in coastal sandy soils saparso 1 (*), a. sudarmaji 1, m. bachtiar musthafa 1, e. wukir tini 1, f. pramana putra 2, r. raditya kurniawan 1 1 agrotechnology department, agriculture faculty, universitas jenderal soedirman, jl. dr. soeparno no. 63, purwokerto 53122, central java, indonesia. 2 agriculture department, faculty of animal and agricultural sciences, universitas diponegoro, jl. prof. sudarto no. 13, semarang 50275, cental java, indonesia. key words: resistant varieties, salinity tolerance index, seasonal shoreline wind. abstract: shallot as a horticultural crop has various benefits and important uses as a provider of nutritional needs. its uniqueness in aroma and flavor makes it commonly used as a seasoning so that it has a good economic value as an increase in farmers’ income. sandy land on the coast has the potential for shallot cultivation. the presence of wind that airborne salinity on coastal land requires the selection of tolerant varieties and knowledge of the level of airborne salinity concentration that shallot plants can tolerate. experiments have been conducted from july to december 2023 in the screenhouse and horticultural agronomy lab, faculty of agriculture, jenderal soedirman university, purwokerto (7°24’27.7”s, 109°15’19.1”e). treatments consisted of the use of shallot varieties bali karet (b1) and bima brebes (b2), with the application of several concentrations of airborne salinity consisting of 0, 6, 12, and 18 ms cm­1. the bali karet variety excels in plant height and root dry weight morphologically. physiologically, bima brebes has higher levels of chlorophyll a and stomatal density, while bali karet is superior in chlorophyll b. harvest results show bima brebes produces more tubers, while bali karet produces higher fresh tuber weight per clump. morphological parameters (plant height, root dry weight), physiology (chlorophyll a, chlorophyll b, stomatal aperture, stomatal density), and yield showed the highest value at the lowest air salinity concentration (0 ms cm­¹). both varieties increased proline as a tolerance mechanism to 18 ms cm­¹ air salinity. the best interaction occurred between bali karet and 0 ms cm­¹ salinity on stomatal opening, and between bima brebes and 0 ms cm­¹ salinity on stomatal density. both varieties were classified as having moderate tolerance to 18 ms cm­¹ salinity, but total chlorophyll was very sensitive to this salinity concentration. 1. introduction horticultural crops play an important role in providing food nutrition (*) corresponding author: saparso@unsoed.ac.id citation: saparso, sudarmaji a., bachtiar musthafa m., wukir tini e., pramana putra f., raditya kurniawan r., 2025 ­ physiological tolerance of shallot varieties to airborne salinity in coastal sandy soils. ­ adv. hort. sci., 39(2): 149­162. orcid: s: 0000­0002­4289­6920 sa: 0000­0002­3068­7996 bmm: 0000­0002­9658­3401 wte: 0000­0001­5122­5507 ppf: 0000­0001­6778­0775 rkr: 0009­0007­0174­7738 copyright: © 2025 saparso, sudarmaji a., bachtiar musthafa m., wukir tini e., pramana putra f., raditya kurniawan r. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 15 november 2024 accepted for publication 9 june 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16803 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-4289-6920 http://orcid.org/0000-0002-3068-7996 http://orcid.org/0000-0002-9658-3401 http://orcid.org/0000-0001-5122-5507 http://orcid.org/0000-0001-6778-0775 http://orcid.org/0009-0007-0174-7738 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 149­162 150 as well as increasing farmers income. horticultural development continues as technology advances. horticultural products are an important source of valuable nutritional and nutraceutical compounds as nutrients needed by humans (durazzo and lucarini, 2022). horticultural crops include fruit plants, medicinal plants, vegetable plants, plantation plants, spices, and ornamental plants, playing an important role in the economic development and prosperity of a country (kour et al . , 2022). the export of horticultural crop commodities provides a great opportunity globally in increasing the country’s income and the welfare of farmers. one of the potential commodities in horticultural production activities is shallots. shallot (allium ascalonicum l.) as a commodity type of horticulture with high economic potential for farmers’ income. shallot cultivation plays an important role in the national economy and globally. there is a high market demand for shallots domestically and internationally, increased production and technological development are required, contributing to food security. shallot production reached 1.985 million tons in 2023, marking a 0.14% increase (2.87 thousand tons) compared to 2022. household consumption of shallots in 2023 decreased by 4.07% (33.83 thousand tons), totaling 797.32 thousand tons compared to the previous year. the import value of shallot in 2023 reached us $1.82 million, increased of 21.94% (us $327.46 thousand) from 2022. the consumption needs of shallots by households in indonesia have fluctuated in the last five years, respectively in 2019 by 750.63 thousand tons; 2020 by 729.82 thousand tons; 2021 by 790.63; 2022 by 831.14 thousand tons; and 2023 by 797.32 thousand tons (badan pusat statistik, 2024). based on the high interest and potential, a strategy is needed to increase shallot productivity. as an archipelago, indonesia has many islands spread across its territory. indonesia as an archipelago consists of 17,504 islands, has a coastal area of 95,118 kilometers (syamsuddin et al., 2019). the amount of sandy beach land is a potential in increasing agricultural land for farmers. sand land is one of the potentials to overcome the problem of agricultural land conversion, as well as in horticultural development (fikri, 2021). seeing the increasingly limited cultivation land provides a highlight of the potential of coastal land as a feasible marginal land utilization effort. extensification activities on coastal land can significantly increase the total shallot planting area, thereby increasing total production in an area. one of the efforts to meet shallot production needs is done with off season cultivation (susanawati and fauzan, 2019). off season shallot cultivation can be done on coastal sand land (fauzan, 2020). different soil and climatic conditions make coastal land a challenge in conducting shallot cultivation activities. however, coastal sand land is easy to cultivate because of its loose texture so that it can save time and cost of land treatment and land is relatively safe from disease (iriani, 2013). another major problem that needs to be considered in cultivation on coastal land is the presence of airborne salinity. a simple sensor exposure method with a wet sponge in coastal areas showed air salinity of 19.69 ms at 6 hours and 151.19 ms at 24 hours (saparso et al., 2023). this shows that the air salinity in coastal areas is very high as indicated by the salt particles captured on the wet sponge. evaporation that occurs in the sea around the coast causes salt particles to be carried into the atmosphere. winds in coastal areas carry water vapor that has a certain level of salinity originating from the sea area. when carried inland on agricultural land, water vapor with a certain level of salinity can affect plants. deposition of salt particles on the surface of leaves and other organs, allowing uptake by plants. growth reduction due to high salinity results from a combination of osmotic stress causing water deficit and the impact of excess na⁺ and cl­ ions on crucial biochemical processes (munns and tester, 2008). nacl in high concentrations is toxic when accumulated in plant tissues. high concentrations of na⁺ disrupt the uptake of k⁺ and ca²⁺ nutrients, while high concentrations of cl ­ decrease photosynthetic capacity due to chlorophyll degradation (tavakkoli et al . , 2010). salinity stress in plants influences numerous cellular mechanisms, such as disturbing cellular homeostasis, hindering photosynthesis, affecting mrna processing, transcription, and protein synthesis, as well as disrupting energy metabolism, amino acid biosynthesis, and lipid metabolism (hameed et al., 2021). salinity stress can cause a reduction in photosynthesis efficiency, chlorophyll, total protein, biomass, stomatal closure and increasing the oxidative stress (gupta and huang, 2014). salinity stress in plants increases the production saparso et al. ‐ tolerance shallot variety on airborne salinity 151 aims to determine the impact of airborne salinity on the morphology, physiology, and yield of shallot plants in two different varieties on coastal land. 2. materials and methods experimental design experiments have been conducted from july to december 2023 in the screenhouse and horticultural agronomy lab, faculty of agriculture, jenderal soedirman university, purwokerto (7°24’27.7”s, 109°15’19.1”e). screenhouse microclimate with daytime peaks of 33.17°c (36,7% rh) under solar radiation and nighttime lows of 27.03°c (55,74% rh) due to radiative cooling. experiment with factorial research with a two­ factor completely randomized design (crd) instrument. the first factor shallot varieties consisted of karet bali (b1) and bima brebes (b2), the second factor airborne salinity at a concentration of 0 ms cm­ 1 (a0), 6 ms cm­1 (a1), 12 ms cm­1 (a2), and 18 ms cm­1 (a3). there were 8 treatment combinations with 3 replications, there are 24 units, with 5 polybags each, making a total of 120 polybags. plant material the shallot variety bima brebes originates from brebes. the plant starts flowering in 50 days and can be harvested in 60 days. it reaches 34.5 cm in height and produces 7­12 bulbs per clump. the leaves are green, cylindrical, and 14­50 in number. dry tuber production reaches 9.9 tons per hectare. this variety is quite resistant to tuber rot but susceptible to leaf tip rot. the tubers are oval and pink in color, suitable for lowlands (annex to the regulation of the indonesian minister of agriculture number: 594/kpts/tp.240/8/1984 dated: august 11, 1984). the bali karet (batu ijo) variety of shallots originates from batu, malang. plants start flowering in 45­50 days and are harvested in 55­60 days in the lowlands or 65­70 days in the highlands. it is between 45­60 cm tall and produces 2­6 bulbs per clump. the leaves are dark green, cylindrical, and number 45­50. the dry tuber production reaches 18.5 tons per hectare. the tubers are round and pink in color, and this variety is well adapted to areas with an altitude of 50­1000 meters above sea level (annex to the regulation of the indonesian minister of agriculture number: 366/kpts/lb.240/6/2004 dated: june 2, 2004). of reactive oxygen species (ros) through oxidative stress mechanisms. ros are normal products of cell metabolism, but environmental stress increases their production excessively, damaging biomolecules and organelles. the role of ros as signals or stressors is determined by the balance between their formation and elimination by the antioxidant system, and disruption of this balance leads to oxidative stress (hasanuzzaman et al., 2021). due to the presence of high salinity there is a water deficit and an increase in free radicals that damage cell structures, plants respond by synthesizing osmolytes such as proline and sugar. proline has antioxidant activity, activates the detoxification system, contributes to cellular homeostasis by protecting redox balance, and serves as a protein precursor and energy source in the recovery process from stress (mansour and ali, 2017). proline is able to minimize damage from ros thereby reducing lipid peroxidation, which results in protection of the photosynthetic apparatus in various plant species (ashraf and foolad, 2007; wani et al., 2012). each crop variety has a different genetic makeup that determines its adaptability to environmental stress, such as salinity. research shows that allium species, including shallots, are plants that are quite sensitive to salinity stress (kadayifci et al., 2005; kiremit and arslan, 2016). to investigate the effect of salinity on shallot, two different varieties were used: bima brebes and bali karet. genetic differences in shallots of bima brebes and bali karet varieties cause differences in morphology, physiology, and yield in plants. alavan et al. (2015), stated that different varieties affect the diversity of plant appearance, due to differences in plant traits (genetic) or environmental influences. the results of research by karo and manik (2020), showed that differences in shallot varieties had a significant effect on the number of flowers with the highest value being the pancasona variety 2.93 stalks and the lowest birma 0.07 stalks. according to azmi et al. (2011), that several varieties planted on the same land have different bulb sizes for each variety. to improve productivity on land with exposure to airborne salinity, it is necessary to select varieties that can adapt to salinity exposure. this selection of plant varieties is based on morphological, physiological and molecular markers (soltabayeva et al., 2021). currently, there is still no information and research on the impact of airborne salinity on shallots grown on the coast. therefore, this study adv. hort. sci., 2025 39(2): 149­162 152 agronomic variables plant height (cm) was determined from the soil surface to the uppermost shoot. the roots were dried in an air­circulated oven at a constant temperature of 70°c until constant weight (72 hours). root dry weight was then weighed using an analytical balance with an accuracy of 0.01 g and expressed in grams (g) per plant. counting the number of tubers per clump was done at harvest time. uniform and healthy sample plants were uprooted along with the tubers. after being cleared of soil, the clumps of tubers were manually separated from the remains of dried roots and leaves. each bulb in a clump was counted manually, and the results were expressed as the number of bulbs per clump (bulbs per clump). fresh bulb weight per clump was measured at harvest. each whole clump was directly weighed using an analytical balance (accuracy 0.01 g). measurement results were expressed in grams per clump (g). assessment of leaf greenness data on the greenness value of shallot leaves were observed in the late vegetative and late generative phases 34 and 47 days after planting, respectively. leaf greenness value was determined with the sp3 leaf chlorophyll meter on the spad­502 plus device. data on chlorophyll content in the leaves were taken randomly in the sample unit. the leaf greenness each leaf sample observed was then taken as the average value. the results of the average value of spad­502 plus as sample data are processed. data collection in sunny weather to increase the accuracy of data collection. assessment of chlorophyll content chlorophyll concentration was determined using the modified international rice research institute (irri) method (alsuhendra, 2004). a total of 0.01 g of shallot leaves were weighed on a balance sheet, pulverized in a mortar with the addition of 10 ml of 80% acetone. leaves that have been pulverized, filtered with filter paper. the shallot leaf extract was analyzed for chlorophyll content on a spectrophotometer, 663 and 645 nm wavelengths. chl content (mg l­1) = (20.2 x a645) + (8.02 x a663) (1) a663 = absorbance at 663 nm wavelength a645 = absorbance at 645 nm wavelength assessment of stomatal characteristics stomatal opening was quantified by identifying epidermal impressions which were obtained from the abaxial leaf surface using clear nail polish. after application, transparent adhesive tape was pressed onto the coated section and carefully peeled to transfer the imprint. the tape­mounted impression was then affixed to a glass slide for stomatal aperture observation at 400× magnification. imprints were examined under a compound light microscope equipped with a calibrated ocular micrometer. stomatal opening width (μm) was measured as the maximum pore distance between guard cells. stomatal density was quantified by counting stomata within a defined microscopic field of view (area = 0.1589 mm² at 400× magnification). the density was calculated using the formula: density = number of stomata/field of view area proline content determination proline (μmol g­1 fresh weight) was determined based on the technique (bates et al., 1973), in 0.5 g fresh leaves that have been mashed given 10 ml of 3% 5­sulfosalicylic acid, then filtered. the filtrate was then given 2 ml ninhydrin (2,2­dihydroxyindane­1,3­ dione) and 2 ml glacial acetic acid, put in a tube, for one hour heated at 100°c (212.0°f) with the addition of 4 ml toluene. the extract solution turned dark red indicating proline content, measured by milton roy 2d spectrophotometer, wavelength 520 nm. the value on the spectrophotometer was calculated by the formula: proline content (μmol g­1 fresh weight) = (64.3649 x absorbance) + (­5.2987 x 0.347) (2) 64.3649 = the slope value of the standard curve, which indicates the increase in proline content (μmol g­¹) per unit increase in absorbance. absorbance = spectrophotometric measurement value that is directly proportional to the concentration of proline in the sample. assessment of stress tolerance index (sti) the stress tolerance index (sti) quantifies shallot yield under salinity stress relative to yield under normal conditions. this index was calculated using the formula established by hooshmandi (2019): sti = (hp × hs) / (h ̄p)² (3) where sti is stress tolerance index, hp= yield of a genotype under non­stressed conditions, hs the yield saparso et al. ‐ tolerance shallot variety on airborne salinity 153 of a genotype under stressed conditions, and h ̄p is mean yield of all genotypes under non­stressed conditions. data analysis analysis of variance (anova), was used in data analysis. duncan’s multiple range test (dmrt) was then used on data significantly different at 5% standard error. statistical data were processed using spss 26 supported by microsoft excel. 3. results the results show that salinity in several levels affects the morphological variables of shallots (table 1, fig. 1) . plant height and root dry weight of shallots of bali karet varieties are 58.53 cm and 0.11 g plant­1 higher than bima brebes by 20.16% and 120%. while the leaf greenness of both varieties is not significantly different. bali karet variety is higher than bima brebes in all morphological parameters, indicating it is more tolerant to salinity stress. airborne salinity treatment significantly reduces plant height, leaf greenness, and root dry weight variables with the highest values of 57.69 cm; 48.16; table 1 ­ varietal effect and air salinity on shallot morphology data are expressed as the mean of determination ± sd in 3 replicates. means followed by the same letter in one column are not significantly different (p<0.05). treatment plant height (cm) leaf greenness root dry weight (g plant­1) varieties (b) bali karet (b1) 58.53±2.55 a 43.67±6.24 a 0.11±0.06 a bima brebes (b2) 48.71±4.68 b 44.18±3.36 a 0.05±0.01 b airborne salinity (a) 0 ms cm­1 (a0) 57.69±5.18 a 48.16±2.43 a 0.12±0.08 a 6 ms cm­1 (a1) 53.98±6.01 ab 45.00±4.17 ab 0.08±0.03 ab 12 ms cm­1 (a2) 52.81±6.06 b 42.93±4.26 bc 0.08±0.04 ab 18 ms cm­1 (a3) 49.99±6.49 b 39.57±4.71 c 0.06±0.03 b varieties (b) x airborne salinity (a) b1a0 61.01±2.44 a 49.00±2.86 a 0.18±0.08 a b1a1 59.30±1.60 ab 45.45±5.23 abc 0.10±0.03 b b1a2 58.03±2.15 ab 42.53±5.96 bcd 0.10±0.04 b b1a3 55.80±1.05 b 37.68±6.34 d 0.09±0.03 b b2a0 54.37±5.30 b 47.34±2.35 ab 0.06±0.02 b b2a1 48.68±1.76 c 44.56±3.93 abc 0.06±0.01 b b2a2 47.60±2.34 c 43.34±3.06 bcd 0.06±0.02 b b2a3 44.19±1.75 c 41.47±2.16 cd 0.05±0.00 b fig. 1 ­ effects of using different varieties (b1= bali karet, b2= bima brebes) on plant height (a) and root dry weight (b). data are expressed as the mean of determination ± sd in 3 replicates. 154 adv. hort. sci., 2025 39(2): 149­162 and 0.12 g plant­1 at 0 ms cm­1 (a0), respectively, with differences reaching 15.40%; 21.71%; and 100% at 18 ms cm­1 (a3). the analysis of two shallot varieties at several levels of airborne salinity shows that bali karet and bima brebes varieties are slightly tolerant to airborne salinity and both have the same decreasing trend in morphology (plant height, leaf greenness, and root dry weight) as airborne salinity increases (fig. 2); however, both varieties have different mechanisms to salinity stress. in table 2 and figure 3 can be observed that the shallot variety bima brebes has a value of 9.33 mg l­1 13.69% greater than the value of chlorophyll a bali karet. in contrast, the bali karet variety has values of 6.85 mg l­1 and 16.19 mg l­1 respectively 86.14% and 11.58% greater than the chlorophyll b and total values of bima brebes. physiological characteristics were significantly affected by the level of airborne salinity in chlorophyll a, b, and total variables (fig. 4) with the highest values of 12.28 mg l­1; 8.54 mg l­1; and 20.83 mg l­1 at 0 ms cm­1 (a0), these values were 57.44%; 288.18%; and 108.09% higher than the 18 ms cm­1 treatment (a3). considering the results of the two varieties under escalating airborne salinity, bali karet and bima brebes deploy contrasting chlorophyll strategies. bali karet boosts chlorophyll b to maximize light harvesting for growth, while bima brebes prioritizes chlorophyll a to protect fig. 2 ­ effects of airborne salinity (0, 6, 12, and 18 ms) on plant height (a), leaf greenness (b), and root dry weight (c). data are expressed as the mean of determination ± sd in 3 replicates. table 2 ­ varietal effect and air salinity on shallot physiology (chlorophyll) data are expressed as the mean of determination ± sd in 3 replicates. means followed by the same letter in one column are not significantly different (p<0.05). treatments chlorophyll a (mg l­1) chlorophyll b (mg l­1) total chlorophyll (mg l­1) varieties (b) bali karet (b1) 9.33±2.35 b 6.85±4.52 a 16.19±5.13 a bima brebes (b2) 10.81±2.61 a 3.68±2.58 b 14.51±4.40 a airborne salinity (a) 0 ms cm­1 (a0) 12.28±2.33 a 8.54±4.22 a 20.83±2.70 a 6 ms cm­1 (a1) 10.88±1.66 ab 5.54±3.76 ab 16.43±2.56 b 12 ms cm­1 (a2) 9.32±1.41 bc 4.78±3.63 ab 14.12±3.19 bc 18 ms cm­1 (a3) 7.80±2.47 c 2.20±1.40 b 10.01±2.54 c varieties (b) x airborne salinity (a) b1a0 10.88±2.49 bc 11.13±2.50 a 22.01±1.20 a b1a1 10.25±1.88 bcd 7.04±5.30 b 17.29±3.49 bc b1a2 8.71±1.65 bcd 6.17±5.19 b 14.89±4.63 cd b1a3 7.50±2.71 d 3.07±1.19 bc 10.58±2.74 de b2a0 13.68±1.22 a 5.97±4.27 b 19.65±3.56 ab b2a1 11.52±1.47 ab 4.05±0.69 bc 15.57±1.44 bc b2a2 9.94±1.07 bcd 3.40±0.54 bc 13.35±1.47 cde b2a3 8.11±2.75 cd 1.34±1.11 c 9.45±2.76 e saparso et al. ‐ tolerance shallot variety on airborne salinity 155 photosynthetic reaction centers. this reflects a fundamental trade­off between photon capture (bali karet) and photochemical resilience (bima brebes), a divergence critical for variety­specific airborne salinity adaptation. table 3 shows that the use of different varieties on stomatal physiology impacts only density of stomata, with the variety of bima brebes having a stomatal density of 55.55 stomatal mm­2 greater 17.79% than the variety of bali karet. no significant differences are found on stomatal opening and proline content. nevertheless, it can be noticed that bali karet variety has a value of 3.58 µm 4.68% greater than bima brebes on stomatal opening, and that bali karet variety has a value of 1.15 μmol g­1 fresh weight 19.01% lower than bima brebes on proline content. several treatments at the airborne salinity level had an effect on decreasing stomatal opening and density and increasing proline. the highest value of stomal opening and stomatal density are 4.66 µm and 58.70 stomatal.mm­2 at 0 ms cm­1, 100% and 36.61% surpassed the 18 ms cm ­1 treatment. in opposite, the highest value on proline 2.24 μmol g­1 fresh weight fresh leaves at 18 ms cm­1 on proline up to 397.78% greater than the control (0 ms cm­1). in the physiological characteristics, the interaction between the use of different varieties and the level of airborne salinity influenced considerably stomatal mechanism of stomatal opening and stomatal density with the highest values of 5.33 µm and 54.50 stomatal mm­2 (b1a0), respectively 128.76% and 52.96% surpassed b1a3 and b2a3 on stomatal opening and b1a3 on stomatal density (fig. 5). although there was no interaction on proline between the use of two shallot varieties and airborne salinity at several levels, it can be observed that the bima brebes variety accumulated higher proline than bali karet with the same increasing trend. this shows the type of adaptation of bima brebes on cellular adaptation, compared to bali karet which focuses on growth optimization (fig. 6). data are expressed as the mean of determination ± sd in 3 replicates. means followed by the same letter in one column are not significantly different (p<0.05). table 4 shows that there is an influence of both varieties on yield characteristics, variable number of bulbs per clump bima brebes 6.66 pieces greater 55.61% than bali karet. in fresh bulb weight per clump on the contrary, bali karet has a value of 45.38 fig. 3 ­ effects of using different varieties (b1= bali karet, b2= bima brebes) on chlorophyll a (a) and chlorophyll b (b). data are expressed as the mean of determination ± sd in 3 replicates. fig. 4 ­ effects of airborne salinity (0, 6, 12, and 18 ms) on chlorophyll a (a), chlorophyll b (b), total chlorophyll (c). data are expressed as the mean of determination ± sd in 3 replicates. g, 95.09% greater than bima brebes. the yield parameter in the airborne salinity treatment in the research conducted had no effect. the interaction of two shallot varieties at several levels of airborne salinity was not significant on yield. bali karet variety adv. hort. sci., 2025 39(2): 149­162 156 fig. 5 ­ effects of airborne salinity (0, 6, 12, and 18 ms) on stomatal opening (a), stomatal density (b), and proline (c). data are expressed as the mean of determination ± sd in 3 replicates. fig. 6 ­ interaction of different varieties (b1= bali karet, b2= bima brebes) with different levels of airborne salinity (0, 6, 12, and 18 ms) on stomatal opening (a) and stomatal density (b). data are expressed as the mean of determination ± sd in 3 replicates. table 3 ­ varietal effect and air salinity on shallot physiology (stomatal and proline) data are expressed as the mean of determination ± sd in 3 replicates. means followed by the same letter in one column are not significantly different (p<0.05). treatment stomatal opening (µm) stomatal density (stomatal mm­2) proline (μmol g­1 fresh weight) varieties (b) bali karet (b1) 3.58±1.31 a 47.16±8.69 b 1.15±0.78 a bima brebes (b2) 3.42±0.90 a 55.55±6.48 a 1.42±0.92 a airborne salinity (a) 0 ms cm­1 (a0) 4.66± 0.82a 58.70±6.50 a 0.45±0.38 c 6 ms cm­1 (a1) 4.16±0.41 a 52.41±8.59 ab 0.95±0.32 bc 12 ms cm­1 (a2) 2.83±0.41 b 51.36±2.57 b 1.47±0.44 ab 18 ms cm­1 (a3) 2.33±0.52 b 42.97±8.36 c 2.24±0.87 a varieties (b) x airborne salinity (a) b1a0 5.33±0.58 a 54.50±3.63 abc 0.21±0.05 d b1a1 4.00±0.00 abc 46.12±7.26 c 0.91±0.33 bcd b1a2 2.66±0.58 cd 52.41±3.63 bc 1.53±0.30 abc b1a3 2.33±0.58 d 35.63±3.63 d 1.94±0.76 ab b2a0 4.00±0.00 abc 62.89±6.29 a 0.71±0.42 cd b2a1 4.33±0.58 ab 58.70±3.63 ab 1.00±0.37 bcd b2a2 3.00±0.00 bcd 50.31±0.00 bc 1.42±0.61 bc b2a3 2.33±0.58 d 50.31±0.00 bc 2.55±1.03 a saparso et al. ‐ tolerance shallot variety on airborne salinity 157 has an escape response shown in fresh bulb weight per clump which is higher than bima brebes, although bima brebes is higher in the number of bulbs per clump due to the defense response from airborne salinity stress (fig. 7). table 5 show that shallot varieties bali karet and bima brebes were medium tolerant variety (mt) on 6, 12, and 18 ms cm­1 airborne salinity. this shows the ability of both varieties to tolerate salinity stress, but have different response mechanisms. the responses of the two varieties to physiology, morphology, and yield are shown in tables 1­4. 4. discussion and conclusions salinity on certain levels can affect morphology, physiology and yield in plants. according to shokat and großkinsky (2019), salinity stress is one of the major problems in agriculture studied globally. dry weight loss is one of the signs that plant growth is affected by salinity (suharjo et al., 2021). the results in table 1 show that the higher the airborne salinity, the lower the morphological variables in shallots. salinity determines the ability of plants to grow because it can damage cells. high salinity levels affect water uptake by plants due to salt around the plant roots, which causes oxidative stress (anwar et al., 2024), prolongs shoot emergence, slows leaf growth, reduces plant height, changes the form of tubers, and reduces their overall mass and size (alam et al., 2023). table 4 ­ varietal effect and air salinity on shallot yield treatment number of bulbs per clump fresh bulb weight per clump (g) varieties (b) bali karet (b1) 4.28±0.77 b 45.38±11.15 a bima brebes (b2) 6.66±1.23 a 23.26±10.46 b airborne salinity (a) 0 ms cm­1 (a0) 4.80±0.49 a 41.17±22.01 a 6 ms cm­1 (a1) 5.90±1.52 a 32.78±14.57 a 12 ms cm­1 (a2) 5.47±2.15 a 33.08±15.50 a 18 ms cm­1 (a3) 5.73±1.85 a 30.27±9.15 a varieties (b) x airborne salinity (a) b1a0 4.40±0.35 b 54.68±12.97 a b1a1 4.67±0.42 b 43.85±12.53 abc b1a2 3.73±1.21 b 45.49±10.19 ab b1a3 4.33±0.92 b 37.51±5.36 abcd b2a0 5.20±0.00 b 27.65±22.26 bcd b2a1 7.13±1.01 a 21.71±2.34 cd b2a2 7.20±1.06 a 20.66±5.88 d b2a3 7.13±1.36 a 23.03±4.81 cd data are expressed as the mean of determination ± sd in 3 replicates. means followed by the same letter in one column are not significantly different (p<0.05). table 5 ­ varieties effect and airborne salinity on stress tolerance index stress tolerance index <0.5 sensitive variety (tt), 0.5­1.0 medium tolerant variety (mt). sti >1 tolerant variety (t) (saparso et al., 2024). variables varieties airborne salinity (ms cm­1) 6 12 18 stress tolerance index bali karet 0.802 (mt) 0.832 (mt) 0.686 (mt) bima brebes 0.785 (mt) 0.747 (mt) 0.833 (mt) fig. 7 ­ effects of using different varieties (b1= bali karet, b2= bima brebes) on number of bulbs per clump (a) and fresh bulb weight per clump (b). data are expressed as the mean of determination ± sd in 3 replicates. and 58.70 stomatal.mm­2 at 0 ms cm­1, 100% and 36.61% surpassed the 18 ms cm ­1 treatment. accordingly, the results of research by fakhri and ekawati (2020), explained that different salinities had a significant effect on the chlorophyll a content in dunaliella sp., an increase in salinity from 15 to 35 ppt caused a 32.65% decrease in chlorophyll a content with the highest concentration (11.27 mg l­1) produced at 15 ppt salinity. salinity inhibits the osmotic uptake of water, which negatively affects the carbon assimilation process, salinity decreases photosynthetic rate, transpiration, stomatal conductance and chlorophyll levels in plants, affecting the ability of plants to photosynthesize optimally (ashraf and ali, 2008). salinity stress lowers the osmotic potential of the soil solution reducing the availability of water for plants and increasing the concentration of ions that are toxic to plants (anugrah et al., 2022). plants have mechanisms to deal with stress. exposed to salinity stress on plants, stomatal will be closed to protect against water loss, leading to increased leaf temperature, salinity­ induced stress resulting in stomatal regulation, with strategies to cope with ionic and osmotic pressures induced by nacl (orzechowska et al., 2021). accumulations of cytotoxic­dependent toxic ions such as na+ and cl­ and formation of reactive oxygen species (ros), can occur due to salinity stress disrupting plant development and growth through water stress (isayenkov, 2012). under conditions of oxidative stress, changes in cell metabolic processes occur, causing the production of ros to increase excessively, damaging proteins, fats, nucleic acids, and can cause plant cell death (ahmad et al., 2019). plants activate antioxidants (sod, cat) and accumulate compatible solutes (proline, glycine betaine) for mitigation (hasegawa et al., 2000). according to the research saparso et al. (2023), higher proline content makes plants more tolerant of air salinity stress, proline content in the plant increases the higher the level of air salinity applied, where the highest proline content of corn plants treatment of 18 ms air salinity, which is 3.58 μmol g­1 and the lowest proline content in the treatment of 0 ms air salinity, which is 1.75 μmol g ­1. this is consistent with the results of this study, that increased exposure to airborne salinity increases proline levels. the proline functions as an osmolyte helping to maintain osmotic balance in plant cells, at high salinity water tends to escape from cells due to differences in ion concentration, the presence, adv. hort. sci., 2025 39(2): 149­162 158 this research found that higher airborne salinity reduced morphological characteristics such as plant height, leaf greenness, and root dry weight. those effects may be explained by the fact that saline environments generally have the same or even higher osmotic pressure than in plant cells, which can inhibit water from entering plant cells. water flows from areas of low osmotic pressure to areas of higher osmotic pressure, causing plants in saline conditions to experience water stress. in allium cepa, stress inhibits cell division so that the number of new cells is reduced and the meristem shrinks in size (kielkowska, 2017). osmotic pressure also affects the speed at which cells absorb nutrients (zainuddin et al., 2017). compared to the control, root fresh weight in pomegranate cultivars decreased by 46.3%, 57.4%, and 66% under 6, 9, and 12 ds m⁻ ¹ salinity treatments, respectively, while root dry weight decreased by 45.4%, 52.5%, and 59% at the same salinity levels (jadidi et al., 2020). salinity stress significantly reduced pepper plant height (badem and söylemez, 2022), leaf greenness values were lower under higher nacl stress (rustikawati et al., 2023). according to research by kul et al. (2021), water salinity caused 22.0% decrease in root fresh weight and 36.0% decrease in root dry weight of tomato compared to non­saline control and unamended control. plants have evolved biochemical and molecular mechanisms, which work in sync as an integrated physiological response to soil salinity (ruiz­lozano et al., 2012). high salinity reduces crop production, subsequent growth, and cause physiological defects threatening global food security and prosperity (balasubramaniam et al., 2023). furthermore, high salinity environments can damage plant membranes and chlorophyll in zea mays and cyperus rotundus, causing disturbances in nutrient absorption due to disturbed ion balance in plant roots (pranasari et al., 2012). the accumulation of na⁺ and cl­ in tissues disrupts enzyme function, photosynthesis, and cell division, especially in young leaves (munns and tester, 2008). other responses in plants include selective buildup or exclusion of salt ions as maintenance on the photosynthesis process to reach adequate values for plant growth, changes in membrane structure, and phytohormone synthesis (türkan and demiral, 2009). the present study shows that as increasing airborne salinity from 0 to 18 ms cm­1 decreased total chlorophyll by 20.83, 16.43, 14.12, 10.01 mg l­1 recpectively. the highest value of stomal opening and stomatal density are 4.66 µm saparso et al. ‐ tolerance shallot variety on airborne salinity 159 accumulation of proline so that plant cells can draw water in, prevent dehydration and maintain cell turgor. according to khanna­chopra et al. (2019), plants produce proline and accumulate in the cytosol, in response to stresses such as salinity, to modify the osmotic properties of the cytoplasm thereby increasing tolerance in plants. however, it is also known that proline can increase the resistance and growth ability of plants under stressful conditions, such as high salinity. increase in proline under salinity stress as extra nitrogen (n) and energy storage achieved through salinity­induced growth reduction for plant survival and growth under stress conditions (kubala et al., 2015). table 3 and 5 indicate that the increase in proline due to exposure to 6 ms cm ­1 to 18 ms cm ­1 represents the ability of both varieties to maintain cell osmoregulators so as not to cause physiological and metabolic plant stress. according to ayub et al. (2015), high proline in plants tolerant of environmental stress plays a role in regulating plant cell osmoregulators. the defense mechanism from cell damage due to ros as free radicals, plants respond through the antioxidant defense system (denaxa et al., 2020). proline plays a very important role in reducing the negative effects of plant salinity stress by neutralizing free radicals formed due to increased ros. plants have enzymatic and non­ enzymatic antioxidant defense systems, which play an important role in detoxifying ros generated under stress conditions, it is known that proline acts as an enzyme protector and ros antioxidant. (khatun et al., 2020). according to silva­ortega et al. (2008), proline accumulates dominantly in leaves to maintain chlorophyll levels and cell turgor pressure, which is crucial for preserving photosynthetic productivity when facing salinity stress. accumulation of proline in stressed plants occurs both through induction of proline bio­synthesizing gene expression (p5cr and p5cs) and by inhibition of genes associated with the degradation pathway. under osmotic stress conditions, proline synthesis is mediated by the enzymes encoded by the p5cs and p5cr genes in most plants (furlan et al., 2020). salinity has three effects on crop growth and yield in the form of ion unbalance, ionic and osmotic stress (anshori et al., 2018). however, experiment results reported in this study showed that airborne salinity had no effect on shallot production. there was a 19.38% decrease in the number of bulbs per clump in the comparison of control and 18 ms cm­1 treatment, in addition to the fresh bulb weight per clump control was 26.48% greater than 18 ms cm ­1 treatment, but both parameters were not significantly different. this is in line with research saparso et al. (2024), in cauliflower and cabbage, unlike the physiological response, plants in the air salinity level treatment had no impact on yield. salinity conditions affect plant nutrient uptake due to the presence of excess na+ and cl­ ions that prevent the uptake of no3 ­, ca2 +, and k+ ions respectively (kharisun et al., 2022), the decrease may be due to the fact that these elements are very important in the initiation of bulbs (mardhiana et al., 2018). in research, some crops showed a decrease in yield due to salinity. tomato yield decreased by 7.2% at 5 ms cm­1 salinity and increased at higher salinities (zhang et al., 2016). research on onion granex 33 variety against 6 nacl concentrations showed that increasing nacl concentration resulted in a decrease in the fresh weight of mature plant bulbs, even plants could not survive at 125 mm nacl concentration (ratnarajah and gnanachelvam, 2021). according to research by syamsiyah et al. (2020), high salinity levels did not significantly affect yield components such as growth, yield, number of tubers, fresh and dry tuber weight of local shallots of brebes and purbalingga which varieties are tolerant of salinity up to salinity levels of 3 ms cm­1. meanwhile, this study shows that exposure to airborne salinity at the highest level up to 18 ms cm ­1 (a3) does not significantly affect the yield of shallots bima brebes and bali karet on the variable number of bulbs per clump and fresh bulb weight per clump. in this study, it can be said that airborne salinity stress does not affect the yield of shallot varieties of bima brebes and bali karet because both varieties are tolerant and able to adapt to certain levels of salinity. this adaptability allows them to maintain physiological and morphological stability, resulting in consistent yields despite saline conditions. bali karet variety has an increased response on growth variables, while bima brebes has a response on physiological variables. the results of research by hadianti and damanhuri (2019), that the six varieties of shallots: bima brebes, bauji, super philip, tajuk, katumi, and trisula are tolerant of high salinity concentrations, at ppm 12,000 experiencing severe stress. sidabariba and sudjatmiko (2023), stated that with its advantages, the bali karet (batu ijo) variety can adapt well to its growing environment. a schematic representation of the different adv. hort. sci., 2025 39(2): 149­162 160 acknowledgements gratitude to the institute for community research and development, jenderal soedirman university, which is stated in the applied funded research budget contract for the fiscal period 2023. references ahmad r., hussain s., anjum m.a., khalid m.f., saqib m., zakir i., hassan a., fahad s., ahmad s., 2019 ­ oxidative stress and antioxidant defense mechanisms in plants under salt stress , pp. 191­205. ­ in: hasanuzzaman m., k.r. hakeem, k. nahar, and h.f. alharby (eds.) plant abiotic 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pertumbuhan dan biopigmen dunaliella salina. ‐ jurnal enggano, 2(1): 46­ 57. zhang p., senge m., dai y., 2016 ­ effects of salinity stress on growth, yield, fruit quality, and water use efficiency of tomato under hydroponics system. ­ rev. agric. sci., 4: 46­55. impaginato 93 adv. hort. sci., 2025 39(2): 93­100 doi: 10.36253/ahsc­16106 https://oaj.fupress.net/index.php/ahs lettuce postharvest quality: affordable packaging and storage durations s.m. shamsi, d. el pebrian (*), s. mustaffha, h. sulaiman, m.k. zahari, n.a.a. ghazali faculty of plantation and agrotechnology, universiti teknologi mara, melaka branch, jasin campus, 77300 merlimau, melaka, malaysia. key words: lettuce, packaging, postharvest, shelf life. abstract: the affordability of packaging materials and proper storage facilities to preserve the quality of lettuce (lactuca sativa l.) has become a problematic issue for small­scale farmers in malaysia, who have limited resources and rely on their crops for income. this study compares the post­harvest quality preservation of lettuce using plastic bags and newspapers as cost­effective packaging materials under storage durations of 0, 3, 6, 12, and 15 days. the lettuce quality parameters measured were chlorophyll, sucrose, weight loss, and overall visual quality (ovq). the results showed newspapers had the highest mean weight loss (7.91 g), while plastic had the lowest one (5.81 g). however, chlorophyll content did not significantly differ between the two packaging types. lettuce packaged in plastic bags had a significantly lower total soluble solids (tss) content mean value (2.89%) compared to newspaper (3.89%). in addition, the plastic bag materials gave a better ovq than that of the newspaper. generally, the use of plastic bags as a packaging option for small­scale lettuce farmers in malaysia is affordable and readily available. 1. introduction lettuce (lactuca sativa l.) is a widely consumed vegetable known for its high nutritional value, low calorie content, and potential health benefits. it is a valuable source of vitamins, minerals, and dietary fiber, which are essential for maintaining good health and preventing chronic diseases (shatilov et al., 2019). the increasing demand for organic lettuce in malaysia has led to more farmers adopting organic farming practices. this crop, along with tomatoes, brassicas, cabbage, and cucumbers, ranked in the top 5 among vegetables with the highest production in the country. faostat (2025) reported that lettuce production from malaysian farmers was recorded at 101,680.39 metric tons in 2023. such production gave a share of 8.99% of the total 1,130,287.58 metric tons’ production of all 30 types of cash crops and vegetables in the country. being a perishable crop, lettuce requires proper post­harvest handling and storage conditions, such as optimal temperature and humidity levels, (*) corresponding author: darius@uitm.edu.my citation: shamsi s.m., el pebrian d., mustaffha s., sulaiman h., zahari m.k., ghazali n.a.a., 2025 ­ lettuce postharvest quality: affordable packing and storage durations. ­ adv. hort. sci., 39(2): 93­100. orcid: ssm: 0000­0002­4820­1931 epd: 0000­0001­9139­9755 ms: 0000­0002­5373­8573 sh: 0000­0003­3329­3383 zmk: 0000­0001­5687­2519 copyright: © 2025 shamsi s.m., el pebrian d., mustaffha s., sulaiman h., zahari m.k., ghazali n.a.a. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 1 may 2024 accepted for publication 27 may 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16106 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-4820-1931 http://orcid.org/0000-0001-9139-9755 http://orcid.org/0000-0002-5373-8573 http://orcid.org/0000-0003-3329-3383 http://orcid.org/0000-0001-5687-2519 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 93­100 94 are critical to ensure the quality and freshness of lettuce. the optimal storage temperature ranges of 0ºc to 5°c is crucial to maintain the quality and freshness of lettuce (gross et al., 2016). farmers need to get the vegetables to the market or supplier as quickly as possible to maintain their freshness and quality. once lettuce has lost its freshness, it cannot be sold, leading to further losses and waste (ravula et al., 2020). unfortunately, many small­scale farmers in malaysia sti l l lack access to adequate storage facilities, and they often lack the equipment and know­how necessary to preserve their harvests, which can lead to large losses. when lettuce is stored improperly, it might wilt, become brown, and show indications of decomposition, which will make it unsellable. inadequate storage practices can cause farmers to lose 20­50% of their harvest, resulting in waste and financial losses (nkolisa et al., 2018). due to their limited resources and reliance on their crops for revenue, small­scale farmers are most affected by this issue. previous studies reported that polymeric films were commonly used as packaging materials for lettuce preservation. according to lee and chandra (2018), packaging films have shown beneficial effects in maintaining sensory, nutritional, and microbial qualities in many fresh or fresh­cut products. however, to the best of our knowledge, little is known about the benefits of plastic bags and newspapers, which have been identified as the most used packaging materials for preserving post­harvest vegetables among small­scale lettuce farmers in malaysia. these materials were affordable and accessible for packaging options for preserving post­ harvest vegetables among farmers. therefore, this study compared newspaper and plastic bags as economical packaging materials for preserving the post­harvest quality of lettuce under varied storage durations. by systematically comparing these factors, this study offers novel insights into how different packaging materials influence key postharvest qualities such as weight loss, chlorophyll content, and total soluble solids (tss), which sheds new light on their impact on the nutritional value and visual appeal of lettuce. building on these findings, this study addresses a critical gap in understanding the role of effective and affordable packaging materials in maintaining lettuce quality during storage, with practical implications for small­scale lettuce farmers. 2. materials and methods the lettuce was grown in a greenhouse at the universiti teknologi mara (uitm) farm, jasin campus, melaka, malaysia, was harvested and transported to the postharvest laboratory within one h. leaves that were overly mature, uneven, or abnormal, as well as those that were damaged or physically injured, were removed from the samples and discarded. the lettuce was chosen based on size, color, and absence of defects. the plastic bag used in the experiment was made of low­density polyethylene (ldpe), measuring 15 cm (w) × 30 cm (l) + 4 cm (gusset). this type of plastic is specifically designed for packaging vegetables and fruits, and it is commonly used by the farmers for lettuce packaging. the plastic film has a thickness of 0.05 mm, with an oxygen transmission rate (otr) of approximately 40,000 ml m­2 day­1 and a water vapor transmission rate (wvtr) of around 9 g m­2 day­1 under standard conditions. the samples were then stored in a 5°c cooling room unti l the end of their shelf l ife. this temperature was selected as the optimal lettuce storage temperature range. this is in line with the fda (2010), which advised that cut­leafy greens must be maintained at temperatures of 5°c or less during cold storage and display. in this study, the growth parameters of chlorophyll, sucrose, weight loss, and visual quality (ovq) were measured. weight loss evaluation the lettuce was weighed after being stored. using an analytical balance (elt602, sartorius, gottingen, germany), the weight loss percentage was computed by subtracting the current sample weight from the initial sample weight (waghmare and annapure, 2015). the percentage of weight loss was computed using the formula in equation (1): weight loss (%) = (wi − wt)/wi x 100 (1) where wi represents the initial sample weight (g) at day 0 and wt represents the most recent sample weight (g) at day t. chlorophyll evaluation the chlorophyll content of the lettuce leaves was measured using a spad 502 plus chlorophyll meter (minolta camera co., osaka, japan). the chlorophyll shamsi et al. ‐ postharvest lettuce: packaging and storage 95 the appearance of lettuce, including its color, texture, and overall freshness. ovq was evaluated using a 9­ point scale adapted from wheeler et al. (2015): 9= excellent, 7= good, 5= acceptable, 3= poor, and 1= unusable. the overall visual quality was evaluated on the day of processing and every three days until the end of the shelf life during 5°c storage. factorial experiment in the ibm spss software was used to statistically analyze the effects of different packing materials and storage durations on mean weight loss, chlorophyll, and tss content. the interaction between packaging materials and storage duration as factors towards the respective mean weight loss, chlorophyll, and tss were evaluated. an analysis of variance (anova) was applied to test the mean significance between the storage duration and the quality of lettuce stored under the respective plastic and newspaper packaging. tukey’s range test was used to compare the differences between treatments. 3. results and discussion weight loss as shown in table 1, both packaging type and storage duration had a significant effect on weight loss. the packaging type showed a statistically significant difference with a p­value of 0.021, indicating that the choice of packaging material influenced the extent of weight loss. this is further supported by the data in table 2, where lettuce stored in newspaper packaging experienced a higher mean weight loss (7.91 g) compared to plastic packaging (5.81 g), suggesting that newspaper is less effective in preserving the product and minimizing weight loss. the p­value of less than 0.001 for the duration of content was measured by placing the meter’s measuring head on the adaxial side of five randomly selected leaves from each treatment. the spad values were recorded, and the mean value for each treatment was calculated. the chlorophyll concentration was expressed in terms of spad units (su). total soluble solids analysis the total soluble solids (tss) of the lettuce samples were determined using a brix meter (brand huiley, model ht113atc) in accordance with the instructions provided by the manufacturer. the sampled lettuces awas washed, cut into small pieces, and homogenized briefly. before each measurement, the brix meter was calibrated with distilled water. the measurement range of the brix meter was 0­ 32%, and each sample was replicated three times. the measurements were conducted on days 0, 3, 6, 12, and 15 of storage. overall visual quality (ovq) the ovq rating is a measure of the lettuce’s condition during storage. it considers factors such as appearance, texture, and color to determine the overall quality of the lettuce. these variables can be affected by a variety of factors, such as lettuce storage duration, storage temperature, and the storage method employed. a panel comprised of three individuals from the university’s farm was chosen to evaluate the ovq rating. one man and two women, ages 20 to 23, were selected as the panelists. before the test, the panelists were trained to recognize and evaluate the lettuce’s ovq. this training is vital because it ensures that panel members can evaluate samples of lettuce consistently and accurately. the ovq score considers table 1 ­ analysis of variance of weight loss * significant at the 0.05 probability level. ** significant at the 0.01 probability level. source of variation degree of freedom sum of squares mean square f value significant packaging 1 39.543 39.543 261.927 * .021 duration 5 398.222 79.644 6.107 ** <.001 packaging x duration 5 38.158 7.632 12.301 .349 error 24 155.396 6.475 total 36 2327.250 adv. hort. sci., 2025 39(2): 93­100 96 storage indicated that the storage duration of lettuce had a significant effect on weight loss. this suggests that the longer the product was stored, the higher the weight loss would be. however, the p­value of 0.349 for the interaction between packaging type and storage duration suggested that there was no significant difference in the effect of storage duration on weight loss between plastic and newspaper packaging. this implies that both types of packaging were equally affected by storage duration; therefore, storage duration is an important factor to consider for both types of packaging. lettuce undergoes weight reduction during storage mostly because of transpiration and respiration. respiration is the mechanism by which lettuce utilizes oxygen and emits carbon dioxide to produce energy, resulting in the decomposition of carbohydrates and the release of water. furthermore, enzyme activity, subject to the influence of storage conditions, plays a role in these physiological alterations (escalona et al., 2006; yang et al., 2024; nitu et al., 2025). overall, the study findings suggested that plastic packaging was more effective in preventing weight loss in lettuce compared to newspaper packaging. the lower weight loss in plastic packaging may be attributed to its better barrier properties, which can limit the exchange of gases, such as oxygen and carbon dioxide, between the product and its environment. these gases can promote respiration and accelerate the metabolic processes in lettuce, leading to weight loss (semco, 2014). moreover, plastic packaging can prevent moisture loss and maintain high humidity around the product, which can help reduce water loss and keep the product fresh for a longer period. chlorophyll content the results in table 3 show that the type of packaging material used had a significant effect on the spad value for chlorophyll, with a p­value of 0.021. this effect is further illustrated in table 2, where the mean spad value for lettuce packaged in plastic (13.39 su) was higher than that for lettuce packaged in newspaper (12.81 su), indicating a higher concentration of chlorophyll in plastic­packaged lettuce. the duration of storage had a significant effect on the spad value for chlorophyll, with longer storage durations leading to lower chlorophyll content. the findings of this study are consistent with previous research on the degradation of chlorophyll during storage. the interaction between packaging type and storage duration was not significant, suggesting that regardless of the type of packaging material used, the degradation of chlorophyll will table 3 ­ analysis of variance of chlorophyll content * significant at the 0.05 probability level. ** significant at the 0.01 probability level. source of variation degree of freedom sum of squares mean square f value significant packaging 1 39.543 3.121 5.054 * .021 duration 5 398.222 30.139 48.807 ** <.001 packaging x duration 5 38.158 1.591 2.577 .349 error 24 155.396 0.618 total 36 2327.250 table 2 ­ mean comparisons of the quality of lettuce with different packaging materials means in the same column followed by the same letters are not significantly different at the 0.05 probability level. packaging material quality of lettuce weight loss (g) chlorophyll content (su) tss content (%) control 3.15 c 13.23 a 4.72 a newspaper 7.91 a 12.80 a 3.89 b plastic 5.81 b 13.39 a 2.89 c shamsi et al. ‐ postharvest lettuce: packaging and storage 97 packaging type and storage duration indicates that the influence of storage time on tss content differs depending on the packaging material used. overall, these findings emphasize the importance of both packaging type and storage duration in determining the tss content of lettuce packaging materials play a crucial role in maintaining the freshness and quality of lettuce by providing a barrier against moisture loss and physical damage during storage and transportation. the use of newspaper packaging may lead to higher sucrose levels in lettuce due to its absorbent nature, which can draw moisture from the vegetables. tss content is a key component of the flavor and texture of fruits and vegetables, and excess moisture can lead to an increase in tss production in lettuce. however, the absorbent nature of newspaper packaging can also increase the risk of moisture loss and spoilage in lettuce. in contrast, plastic packaging can provide a more effective barrier against moisture loss and protect the lettuce from external factors that may affect its quality. this can lead to a lower concentration of sugars in the lettuce compared to newspaper packaging. it is important to use appropriate packaging materials that balance the need for moisture retention with the need for protection and safety. the findings of this study suggest that different packaging materials may have different effects on the tss content levels of lettuce, and that storage duration is an important factor to consider when selecting the appropriate packaging. based on the comparison of mean quality values for lettuce packed with different materials (table 5), it appears that the newspaper had the highest mean value (7.91 g) while the plastic had a lower mean value (5.81 g) for weight loss. although there occur over time, resulting in a decrease in the spad value for chlorophyll in lettuce as stated by mattos et al. (2013). while, in addition, ferrante and maggiore (2007) stated that as lettuce is a non­durable vegetable, the degradation of leaf pigments such as chlorophylls and carotenoids occurs during storage, leading to tissue browning. degl’innocenti et al. (2005) and ferrante and maggiore (2007) added that a common indicator of these processes is browning and discoloration resulting from damage to the leaf surface. the loss of color occurs due to the degradation of chlorophylls because of vegetable aging during storage. in response to the above findings, the appropriate selection of packaging materials is crucial to maintaining the quality and freshness of lettuce during storage and transportation. the use of plastic packaging can result in higher spad values for chlorophyll in lettuce compared to newspaper packaging, but this effect may be limited over time due to the natural degradation of chlorophyll. therefore, it is important to balance the need for optimal packaging materials with the limitations of prolonged storage durations. total soluble solids content as shown in table 4, both the type of packaging and the duration of storage had a statistically significant effect on the total soluble solids (tss) content of lettuce (p<0.001). table 2 further i l lustrates this effect, with lettuce stored in newspaper packaging exhibiting higher tss levels (3.89%) than those stored in plastic packaging (2.89%). in addition, tss levels increased progressively with longer storage durations, suggesting that extended storage promotes sucrose accumulation. the significant interaction between table 4 ­ analysis of variance of tss content ** significant at the 0.01 probability level. source of variation degree of freedom sum of squares mean square f value significant packaging 1 9.000 9.000 32.400** <.001 duration 5 21.222 4.244 15.280** <.001 packaging x duration 5 11.667 2.333 8.400** <.001 error 24 6.667 .278 total 36 462.000 98 adv. hort. sci., 2025 39(2): 93­100 is no specific research on the effect of packaging materials between paper and plastic on the quality and shelf l i fe of lettuce, some studies have investigated the effect of packaging on the shelf life of other fruits and vegetables. a study on tomato storage conditions and packaging materials found that hdpe packaging material resulted in the least weight loss when stored in a refrigerator (sualeh et al., 2016). for the chlorophyll content, the mean value for the lettuce stored in plastic packaging is 13.39 su, and for the lettuce stored in newspaper packaging, the mean value is 12.80 su (table 2), whereas there is no significant difference between these two packaging due to their falling into the same grouping. the chlorophyll content was not significantly different between the two types of packaging. this could be because chlorophyll is relatively stable and is not affected by moisture or air. however, it is important to note that the chlorophyll content can be affected by other factors, such as light exposure and temperature. while for tss content, the mean value for the lettuce packed with plastic has the lowest value, which is 2.89%, compared with the newspaper, whose mean value is 3.89% (table 2). tss corresponding to sucrose, which is a disaccharide composed of glucose and fructose and is commonly known as table sugar. the query suggests that the lower sucrose content in plastic packaging could be due to its hygroscopic nature, which leads to the absorption of moisture. water vapor permeation through the package material occurs in three steps: water vapor adsorption, diffusion, and desorption from the package. this process is driven by transferring from high to low water activity or relative humidity (rh) (sand, 2021). however, plastic packaging has lower moisture permeability, which could prevent the loss of moisture and the absorption of sucrose, resulting in lower levels of sucrose in plastic­packaged products. table 5 shows the weight loss and chlorophyll content of lettuce stored for 0 days in plastic and newspaper packaging are significant with the storage duration of 3, 6, 9, 12, and 15 days. this occurs due to the shock of the environment from a normal temperature to a cold temperature. however, there is no significant difference in tss content between the storage durations of lettuce under plastic packaging. when the lettuce was stored under newspaper packaging, it was found that there were significant differences between the storage durations of 0, 3, 6, 9, 12, and 15 days. overall visual quality the findings of this study, as presented in figure 1, indicate that the overall visual quality (ovq) of lettuce stored at 5°c can be effectively maintained for up to 12 days with the use of plastic bags. this is evidenced by the consistency of ovq scores of 7 or table 5 ­ mean comparisons of the quality of lettuce with different storage durations under both packaging materials means in the same column followed by the same letters are not significantly different at the 0.05 probability level. packaging quality of lettuce weight loss (g) chlorophyll content (su) tss content (%) plastic day 0 0.00 b 16.23 a 3.00 a 3 8.95 a 13.97 b 2.67 a 6 6.54 ab 13.77 b 2.67 a 9 5.93 ab 13.00 bc 2.67 a 12 5.04 ab 12.30 bc 3.00 a 15 8.44 ab 11.10 c 3.33 a newspaper day 0 0.00 c 17.47 a 2.67 b 3 8.59 ab 13.77 b 3.33 b 6 7.78 b 13.10 bc 3.00 b 9 8.10 b 11.80 bc 3.00 b 12 9.93 ab 11.33 cd 5.00 a 15 13.07 a 9.37 d 6.33 a shamsi et al. ‐ postharvest lettuce: packaging and storage 99 higher, which exceed the marketability cut­off score of 6. however, when stored in newspaper packing, the ovq scores of lettuce samples deteriorated rapidly, falling below the marketability cut­off score after just 6 days. the results suggest that the choice of packaging material is a critical factor in maintaining the quality of lettuce during storage, with plastic bags proving more effective than newspaper in this regard. this may be attributed to plastic bags’ superior ability to prevent moisture loss and inhibit microbial growth, which can contribute to the deterioration of lettuce quality and appearance. additionally, the findings indicate that storage temperature plays a crucial role in preserving lettuce quality, with lower temperatures being more effective in achieving this objective. the significant effect of storage duration on weight loss highlights the importance of proper storage conditions in preserving the quality and shelf­ l ife of lettuce. low temperatures, high humidity, and adequate ventilation are some of the essential storage conditions that can help slow down the metabolic processes and reduce weight loss in lettuce. the lack of significant interaction between packaging type and storage duration suggests that both types of packaging can benefit from proper storage conditions to minimize weight loss. figure 2 shows the physical appearance of lettuce in 15 days using plastic in 5°c. overall, the study suggests that small­scale lettuce farmers in malaysia may benefit from using plastic fig. 2 ­ the physical appearance of lettuce in 15 days using plastic in 5°c. packaging to preserve the quality and freshness of their produce. however, the choice of packaging material should also consider factors such as cost and accessibility to ensure it is both effective and practical for small­scale operations. additionally, to align with sustainable development goals, the environmental impact of using recycled materials such as newspapers should be an important consideration when selecting packaging options. it has been proven that newspaper as packaging material aligns with environmental sustainability goals due to its biodegradable nature and origin from renewable resources. newspapers are also easily recycled or used for renewable energy, which helps reduce reliance on fossil resources. fig. 1 ­ overall visual quality of leaf lettuce packaged with plastic bags and newspapers during storage at 5°c for up to 15 days. 4. conclusions the selection of appropriate packaging materials is crucial to maintaining the quality and freshness of lettuce during storage and transportation. the study found that plastic packaging was more effective in preventing weight loss and maintaining higher concentrations of chlorophyll in lettuce than newspaper packaging. however, newspaper packaging resulted in higher tss levels compared to plastic packaging. the length of time the product was stored also had a significant effect on weight loss, chlorophyll degradation, and tss levels. the study highlights the importance of balancing the need for optimal packaging materials with the limitations of prolonged storage durations. further studies are recommended to explore the use of alternative recycled packaging materials that are not only effective and environmentally friendly adv. hort. sci., 2025 39(2): 93­100 100 but also affordable for small­scale farmers. this research could focus on identifying cost­effective and locally available options, such as biodegradable or compostable materials. additionally, investigating the feasibility of a shared packaging system among small­scale farmers may offer a practical solution to reduce costs and enhance access to sustainable packaging options. acknowledgements the authors are very grateful to the universiti teknologi mara (uitm) for funding this research under the uitm grant myra science technology no. 600­rmc/gpm st 5/3 (039/2021). references degl’innocenti e., guidi l., pardossi a., tognoni f., 2005 ­ biochemical study of leaf browning in minimally processed leaves of lettuce (lactuca sativa l. var. acephala). ­ j. agric. food chem., 53(26): 9980­9984. escalona v.h., verlinden b.e., geysen s., nicolaï b.m., 2006 ­ changes in respiration of fresh‐cut butterhead lettuce under controlled atmospheres using low and superatmospheric oxygen conditions with different carbon dioxide levels. ­ postharvest bio. tech., 39(1): 48­55. faostat, 2025 ­ crops and livestock products (malaysia). crops and livestock products. ‐ https://www.fao.org/faostat/en/#data/qcl accessed on 16 may 2025. fda, 2010 ­ program information manual retail food protection: recommendations for the temperature control of cut leafy greens during storage and display in retail food establishments. the united states food and drug administration (fda), usa, pp. 1­5. ferrante a., maggiore t., 2007 ­ chlorophyll a fluorescence measurement to evaluate storage time and temperature of valeriana leafy vegetables. ­ postharvest bio. tech., 45(1): 73­80. gross k.c., wang c.y., saltveit m., 2016 ­ the commercial storage of fruits, vegetables, and florist and nursery stocks. ­ usda handbook, united states dept. agric., pp. 386­387. lee j.s., chandra d., 2018 ­ effects of different packaging materials and methods on the physical, biochemical and sensory qualities of lettuce. ­ j. food sci. technol., 55(5): 1685­1694. mattos l.m., moretti c.l., silva e.y.y., 2013 ­ effects of modified atmosphere packaging on quality attributes and physiological responses of fresh‐cut crisp head lettuce. ­ cyta j. food., 11(2013): 392­397. nitu o.a., ivan e.ş., tronac a.s., arshad a., 2025 ­ variety‐specific lettuce responses to oxygen‐enriched water and led light in a controlled greenhouse: a multivariate analysis across two cycles. ­ int. j. plant biol., 16(1): 29. nkolisa n., magwaza l.s., workneh t.s., chimpango a., 2018 ­ evaluating evaporative cooling system as an energy‐free and cost‐effective method for postharvest storage of tomatoes (solanum lycopersicum l.) for smallholder farmers. ­ sci. hort., 241(2018): 131­143. ravula p., kasala k., ray s., 2020 ­ impact of covid‐19 on food security: insights from telangana, india. ­ agric. econ. res. rev., 33(167): 167­168. sand c.k., 2021 ­ controlling moisture in foods using packaging. ‐ ifi. institute of food technologists, food technology magazine, september 1. semco, 2014 ­ post‐harvest cooling and storing of lettuce. ­ semco/semcold llc, texas, usa, pp. 1­2. shatilov m.v., razin a.f., ivanova m.i., 2019 ­ analysis of the world lettuce market. ­ iop conference series: earth and environ. sci., 395(1). sualeh a., daba a., kiflu s., mohammed a., 2016 ­ effect of storage conditions and packing materials on shelf life of tomato. ­ food sci. qual. manag., 56(2016): 60­67. waghmare r.b., annapure u.s., 2015 ­ integrated effect of sodium hypochlorite and modified atmosphere packaging on quality and shelf life of fresh‐cut cilantro. ­ food packaging shelf life, 3(2015): 62­69. wheeler l., kitinoja l., barrett d., 2015 ­ use of insulated covers over product crates to reduce losses in amaranth during shipping delays. ­ agric., 5(4): 1204­ 1223. yang j., song j., liu j., dong x., zhang h., jeong b.r., 2024 ‐ prolonged post‐harvest preservation in lettuce (lactuca sativa l.) by reducing water loss rate and chlorophyll degradation regulated through lighting direction‐induced morphophysiological improvements. ­ plants(basel), 3(18): 2564. impaginato 119 adv. hort. sci., 2024 38(2): 119­127 doi: 10.36253/ahsc­14965 phenological and yield response of primed carrot (daucus carota l.) seeds under deficit irrigation s.h. muhie 1 (*), f. akele 1, t. yeshiwas 2 1 department of plant science, college of agriculture, wollo university, dessie, ethiopia 2 department of horticulture, bahir dar university, bahir dar, ethiopia key words: carrot, irrigation interval, marketable yield, physiological changes, priming. abstract: seedling emergence and stand establishment of carrot seeds are often slow and erratic which results in low productivity. poor seed quality together with lack of pre­sowing seed treatments and improper irrigation man­ agement can be mentioned as the major factors that influence the yield and productivity of carrot. the present study was carried out with the objective to evaluate the effects of different seed priming techniques on early seedling establishment, growth and yield of carrot (daucus carota l. cultivated variety nantes) exposed to different irrigation intervals, under field conditions at gerado, south wollo, ethiopia. four seed priming treatments (no priming, hydro priming, halo priming and hormonal priming) and three irrigation inter­ vals (4, 7 and 10 days) were combined as factorial rcbd in split plot arrange­ ment with three replications. the irrigation intervals were assigned to main plots and the seed priming techniques to sub plots. result indicated that the interaction effects of priming techniques and irrigation intervals significantly affected the phenological and yield parameters. distilled water treatment in seven and ten days irrigation interval recorded the highest marketable carrot root yields of 33.73 t h­1 and 30.63 t h­1, respectively. hence, hydro priming and seven days irrigation interval can be recommended for the production of carrot in the study area and similar agro­ecologies. given the promising results obtained, further repetitions of the study are recommended to validate the use of these techniques in further locations and in different seasons. 1. introduction carrot (daucus carota l.) is a crop that belongs to the family apiaceae previously umbelliferea (thiviya et al., 2021). carrot is among the top ten most economically important vegetable crops in the world in terms of areas of production and market value (simões et al., 2010). its yield can range from 30 to 100 t ha­1 in major carrot growing countries of the world (tegen and jembere, 2021). carrot is one of the major root vegetable crops produced in ethiopia. it (*) corresponding author: hamidashm@gmail.com citation: muhie s.h., akele f., yeshiwas t., 2024 ­ phenological and yield response of primed carrot (daucus carota l.) seeds under deficit irrigation. ­ adv. hort. sci., 38(2): 119­127. copyright: © 2024 muhie s.h., akele f., yeshiwas t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 july 2023 accepted for publication 31 january 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14965 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 119­127 120 is nowadays cultivated predominantly as a cash crop throughout the country, for sale to urban markets, including hotels and restaurants as indicated by getachew and mohammed (2012). according to csa (2022) about 6,759.92 ha has been covered by car­ rots with a total production of 31,671.6 tons and pro­ ductivity of 4.7 t ha­1. in 2022, the area coverage of carrots increased by 30% compared to the produc­ tion year of 2021 whereas, in the amhara region about 1,619.58 ha has covered by carrots with a total production of 9,145.8 tons and productivity of 5.6 t ha­1. the productivity of carrots in ethiopia is very low (4.7 t ha­1) compared to other countries which can be up to 100 t ha­1 in major carrot­growing coun­ tries of the world as reported by csa (2022). carrot has remarkable nutritional and health value and is considered as a rich source of carotenoids, phenolic compounds, polyacetylenes, and vitamins (alhariri and boras, 2020; glowka et al., 2021). the health benefits and public awareness of nutritional health security are positively influencing the demand and consumption of carrots by con­ sumers and by the nutraceutical­based industry (selvakumar et al., 2019). heterogeneous, asynchronized and lower rates of seed germination are major challenges in apiaceae (umbelliferae) family and have a major impact on final yield and quality especially for vegetable crops established by direct seeding such as carrot and pars­ ley (mozumder and hossain, 2013). improvement in seeds’ homogenous, longevity and germination speed are major concerns for carrot growers and the seed agribusiness sector. recently, different pre­sow­ ing seed treatments have been proposed to improve seed vigour, uniformity and seedling emergence (sagvand et al., 2022). the crop is usually established by direct seeding and therefore, low soil moisture content may lead to poor crop stand (mahmood­ur­ rehman et al., 2020). low soil moisture content delay or inhibit seed germination in the field, reduce uniformity of seedling performance, total stand establishment and ultimately reduce the yield of car­ rot. in addition, before sowing the seeds, it is essen­ tial to ensure that the soil is adequately moist. this can be achieved by irrigating the field a few days before planting. the moisture content should be suf­ ficient for the seeds to absorb water and germinate. the production and productivity of carrot in the study area is low mainly due to low quality seeds and poor cultural practices including improper irrigation intervals. carrot producers mainly use seeds stored for long periods which hardly germinate in the field. the germination is not uniform and fails to establish a homogeneous crop stand especially under limited irrigation intervals. different seed priming techniques help to enhance seed quality thereby improving seedling emergence percentage and uniformity even under stress conditions. even though a number of researches have been done on seed priming (pereira et al., 2009; paparella et al., 2015), there is no single method of seed priming technique that can be best suited to all crops. thus, it’s of paramount impor­ tance to examine the different priming techniques to enhance the quality of carrot seeds. further, water requirement studies for proper irrigation scheduling of carrot also has great importance. however, infor­ mation regarding the efficient use of irrigation water for improved growth and yield of carrot has also lack­ ing in ethiopia. scarcity of irrigation water is an acute problem for successful crop production in ethiopia. hence the need for more efficient utilization and management of scarce irrigation water is crucial. therefore, this research was initiated to investigate the use of different seed priming techniques on seedling emergence, growth and yield of carrot under different irrigation frequencies in south wollo, ethiopia. 2. materials and methods description of experimental site the experiment was conducted at gerado, mid­ altitude of south wollo administrative zone, amhara regional state, ethiopia during 2020/21 cropping sea­ son. the study area is geographically situated 11 12’ 00’’ north and 39 42’ 00’’ east with an elevation between 2200­2800 m a.s.l. (fig. 1) the general cli­ mate is midland with a mean annual rainfall ranging fig. 1 ­ geographical location of the study area. muhie et al.‐ carrot yield and quality under water deficits 121 from 900 mm to 1000 mm and mean annual temper­ ature between 15°c to 20°c (tamru, 2006). the major soil type in the area is sandy­loam. gerado has three permanent rivers which have the potential to irrigate throughout the year. these are yito medehanialem, negewoliy and gerado. in the study area the most cultivated crops are maize, teff, wheat and barley. vegetable crops such as carrot, cabbage, tomato, and potato are cultivated using irrigation water from the nearby rivers (bahir et al., 2015). experimental material carrot (daucus carota var. nantes) seed (250 g), were obtained from vegetable seed providing enter­ prise in dessie city andused as a test crop since it is adapted to the experimental area and preferred by most small­ holder farmers and consumers. nantes variety has orange colour and cylindrical roots with a blunt end and strong leaves. it has a wide adaptabili­ ty and grows well at altitudes ranging from 1600­ 2400 m a.s.l with annual precipitation of 760­1010 mm and can be produced all year round both under rain­fed and irrigation condition (hubbard et al., 2012). treatment and experimental design this study applied two treatments concerning priming techniques and irrigation levels. in total four techniques of priming; none primed/control (np), potassium nitrate (kno3), gibberelic acid (ga3), and distilled water (dw) where seeds were dipped in the respective solutions 12 priming hours. irrigation treatment consisted of three irrigation intervals: four days interval (i4), seven days interval (i7) and ten days interval (i10).kno3 was used at a concentration level of 50mm (lara et al., 2014) and ga3 was used at a concentration level of 0.05 mm (kwon et al., 2020). the ratio (w/v) of seed: priming solutions was 1:5 (g/ml) (ruan et al., 2002). the experiment was laid out in randomized complete block design (rcbd) in split plot arrange­ ment with three replications. there were 3 main plots in each replication and each main plot was fur­ ther divided into 4 sub plots where irrigation inter­ vals assigned as main plots and the priming tech­ niques was assigned to sub­plots. the gross area of the experimental site was 240 m2 (30x8) and the net area was 103.68 m2. the area of a single plot was 2.88 m2 (1.6x1.8 m). spacing between main plots and sub­plots were 1m and 0.5m respectively. blocks were separated by 1.5 m. the seeds were planted on rows with spacing of 5 cm between plants and 20 cm between rows. each experimental plot had 7 rows with 36 plants per row. the outer single rows at both sides of the plot and one plant at both ends of the rows were considered as border plants. all manage­ ment activities except irrigation and priming tech­ niques were applied uniformly in all plots of the experiment. experimental procedure and management of plants at first, seeds were superficially sterilized with sodium hypochlorite solution (2%) for five minutes to sterilize fungal agents and were then thoroughly wash with distilled water and air dried (farooq et al., 2005). seed samples of nantes variety was then divided in to three for each priming media. after that, seeds except the non ­ primed (control) were fully immersed in different priming medias (distilled water, ga3 and kno3) on petri dishes for 12 priming hours (abnavi and ghobadi, 2012). after the priming application, all seeds were removed from the priming media at the same time and then rinse thoroughly with distilled water, dried on paper towels at room temperature and ventilated until they regained their original weight (giri and schillinger, 2003). cropwat version 8.0 was used to calculate the amount of water required by carrot crop and water was applied using calibrated watering can to bring the water requirement of each interval days for the treatments. crop water requirement (cwr) is the total water needed for evapotranspiration, from planting to harvest for a given crop in a specific cli­ mate regime when soil does not limit plant growth and crop yield (ouda et al., 2015). considering this the cwr was determined by the formula developed by fao as follows. etc = kc x et0 eq. 1 where etc is crop potential evapotranspiration (mm/unit time), kc is crop coefficient (influence of crop type and growth stage) and eto is reference evapotranspiration (mm/unit time) [influence of cli­ mate]. the effect of climate on crop water requirements is given by the reference crop evapotranspiration (eto) which is defined as the rate of evapotranspira­ tion from an extensive surface of 8 to 15 cm tall, green grass cover of uniform height, actively growing, completely shading the ground and not short of water. fao (2005) defined the crop potential evapo­ transpiration (eto) for a given crop as: adv. hort. sci., 2024 38(2): 119­127 122 et0 eq. 2 where eto is the reference evapotranspiration (mm day­1), rn is the net radiation (mj m­2 day­1), g is the soil heat flux density (mj m­2 day­1 ), t is the mean daily air temperature at 2 m height (oc ) d is the slope of the saturated vapour pressure curve (kpa oc­1 ), g is the psychrometric constant (66 pa oc ­1), es is the sat­ urated vapour pressure at air temperature (kpa), ea is the prevailing vapor pressure (kpa), and u2 is the wind speed measured at 2 m height (m s­1). based on the above formula water requirement of carrot was 402.4 liter per m2 of land for all growing season. the gross and net plot sizes were 2.88 m2 and 2.63 m2, respectively. therefore, the total water requirement of each plot was 1,158.912 liters. each treatment was irrigated with equal amount of total irrigation water during the growth period, which was calculated based on cropwat 8 software. accordingly, the amount of irrigation water applied by three levels irrigation intervals was different, as the irrigation interval increases, the frequency of water application decreases leading to lesser amount of water applied and vice versa. thus, 46.36 litres of water were supplied within the irrigation interval of 4 days, 82.79 litres in the irrigation interval of 7 days, and 115.89 litres in the irrigation interval of 10 days. the amount of water required for each interval was obtained by dividing the total water requirement of a single plot by the number of irrigation applications over the total growing season). to determine the baseline of irrigation interval to set up the treatment, input data for the cropwat model were obtained from the national meteorological services agency, soil laboratory results and fao publications. metrological data of ten years which were collected from ethiopian metrology agency, north eastern region, kombolcha station were used. soil analysis of the experimental site soil samples were collected from the experimen­ tal site before treatment application at a depth of 30 cm in a zigzag pattern from nine randomly selected points by using auger. the collected samples were air dried, mixed and made a composite representative sample of 1 kg for analysis. the composite sample was packed in a polythene bag, labelled and taken to dessie soil testing laboratory. soil analysis data on soil texture, ph, organic matter, field capacity, per­ manent wilting point and organic carbon of the soil were analyzed as shown in (table 1). data collection and statistical analysis phenological, growth, yield related parameters and yield were collected from net plot area using standard procedures. data were subjected to two­ way analysis of variance (anova) using r­software based on agricole statistical package. the mean sepa­ ration was carried out using least significant differ­ ence (lsd) at probability level based on the results of anova analysis. the detailed procedures were as fol­ lows: emergence percentage (%). after emergence of first seedling of every treatment, the numbers of emerged seedlings were counted daily up to 14 days after sowing. emergence percentage was calculated as: emergence (%) = total number of emerged seedling x 100 total seeds sown mean emergence time (count). it was calculated according to (ellis and roberts, 1980): met= (σn x d)/σn where n is the number of seeds emerged at day d, and d the number of days since the start of emer­ gence test (sowing). days to 90 % maturity (days). it was measured by counting the number of days elapsed from date of sowing to the date when 90% of the plant in each plot attained physiological maturity and used for fur­ ther analysis. carrot plants were physiologically matured when the leaf color turns in to yellow and roots are at harvestable size and the crown attained a table 1 ­ physical and chemical properties of the soil in the study area soil properties data organic matter (%) 4.13 organic carbon 2.4 ph 7.02 texture sand (%) 45 silt (%) 13 clay (%) 42 class sandy clay field capacity (vol. %) 39 permanent wilting point 27 muhie et al.‐ carrot yield and quality under water deficits 123 diameter ranging from 2­3.8 cm diameter as described by unece (2018). marketable root yield (t ha‐1). carrot roots, which are free from mechanical damages, disease and insect pest attack and sizes (>50 g) were considered as marketable (unece, 2018). the weight of such car­ rots harvested from the net plot area was weighed using scale and expressed as ton per hectare. unmarketable root yield (t ha‐1). carrot roots which are diseased, insect pest damaged, cracked and under sized (<50 g) was considered as unmar­ ketable as described by unece, 2018. the weight of such carrots harvested from the net plot area was weighed using scale and expressed as ton per hectare. total root yield (t ha‐1). it was obtained by sum­ mation of marketable and unmarketable yields and then converted to hectare basis and expressed in t ha­1. 3. result and discussion phenology emergence percentage. increasing the frequency of irrigation as well as treating seeds with different priming solution increased the emergence percentage of carrot plants. in the interaction effect, the results showed that, decreasing irrigation interval along with priming techniques improved the emergence per­ centage of carrot plants. therefore, the highest emer­ gence percentage of carrot (83%) was observed by the treatment combination of four days irrigation interval and distilled water treatment. the lowest ep values were obtained using 10 days of irrigation inter­ val for all the priming treatments, with minimum value in non­primed seeds (46.67%) (table 2). the highest emergence percentage of carrot plants in treatments receiving short irrigation inter­ vals combined with pre­sowing seed treatment might be due to priming­induced changes in biochemical contents of the seeds, membrane integrity and enhanced physiological activities during seed germi­ nation and available moisture which is required for germination (alam et al., 2013). on the other hand, the lowest emergence percentage recorded from longer intervals might be due to moisture deficiency. the result of this study was in agreement with selvarani and umaran (2011), who found that hydro priming is the best priming technique for daucus carota seeds. dessalew et al. (2022) also reported that halo, hydro and hormonal­priming techniques improved germination, seedling growth, seedling vigour and seed yield of carrot (daucus carota). table 2 ­ interaction effect of seed priming and irrigation interval on phonological parameters of carrot means with the same letter/s in column are not significantly different; * = significant (p<0.05); cv = coefficient variance; lsd = least significant difference; se= standard error, dm= days to 90% maturity (days). treatment combinations emergence (%) mean emergence time (days) dm irrigation interval priming treatment 4 days gibberelic acid 77.67 b 11.33 f 99.33 f distilled water 83.00 a 10.7 f 97.00 f none primed 76.33 c 12.96 bc 104.57 cde potassium nitrate 77.67 b 12.3 d 103.90 de 7 days gibberelic acid 69.00 bc 13.00 bc 103.37 de distilled water 75.67 ab 11.40 ef 102.20 e none primed 56.00 d 13.10 b 105.40 bcd potassium nitrate 66.33 c 12.33 cd 104.80 bcd 10 days gibberelic acid 57.00 d 13.10 b 107.00 abc distilled water 55.33 d 13.0 b 106.57 bc none primed 46.67 e 13.93 a 109.00 a potassium nitrate 55.33 d 13.16 b 107.37 ab lsd (5%) 0.07 0.66 2.41 cv (%) 6.50 3.40 1.40 se+/­ 0.03 0.42 1.40 124 adv. hort. sci., 2024 38(2): 119­127 similarly, mehri (2005) reported that the maximum germination percentages were obtained from water treatment while the least value was recorded from non­primed seeds. mean emergence time. treating seeds with differ­ ent soaking chemicals as well as frequent irrigation generally reduced the spread of emergence times. the different priming techniques significantly decreased the mean emergence time across the increasing frequency of irrigation as compared to control. the shortest mean emergence time (10.70 days) was recorded from seeds treated with distilled water and irrigated at four days interval. however, the combined effect of longest irrigation interval (ten days) with unprimed seeds recorded the longest mean emergence time (table 2). the significant reduction in emergence time derived from the combination of seed priming with frequent irrigation intervals might be due to the com­ pletion of pre­germinative metabolic activities during the priming process (bourioug et al., 2020). primed seeds germinated soon after planting compared to untreated dry seeds since seed priming stimulates an array of biochemical changes such as hydrolysis of starch, activation of enzymes and breaking dormancy in the seed (patel and rai, 2018; tania et al., 2020). furthermore early reserve breakdown and reserve mobilization might also be the cause of significant reduction in emergence time due to readily available assimilate during germination (farooq et al., 2005; farooq et al., 2006). the results are in line with alhariri and boras (2020) and mahmood­ur­rehman et al. (2020) who reported that seeds treated with different chemicals germinated faster than untreated seeds (control). moreover, alam et al. (2013) found that unprimed spinach seeds took longer to emerge compared to primed seed. synchronized emergence of primed seeds can insure a vigorous and better crop stand with rapid canopy development, giving plants a pre­ liminary advantage over weeds resulting in increased weed competitiveness (dhage and anishettar, 2007; raj and syriac, 2017; juraimi et al., 2020). moreover, safiatou (2012) reported that seed priming improved the competitive ability of a crop against weeds, and faster emergence along with increased vigour of a primed stand is the key factors for tolerating weeds. days to 90% maturity. seed soaking in different priming chemicals along with frequent irrigation resulted in lowest mean number of days to reach 90% maturity. the result of interaction effect showed that, the earliest days to maturity (97.00 days) was recorded from the treatment combination of four days irrigation intervals with distilled water treat­ ment which was statistically similar to the combined effect of gibberelic acid seed treatment and four days irrigation interval (99.33 days). the longest days to maturity (109.00 days) was recorded on unprimed seeds with the longest irrigation interval (table 2). the accelerated maturity of carrot roots irrigated with frequent irrigation interval might be associated with easily uptake of nutrients as the available water helps the plant to dissolve the nutrients and move through transpiration pull which in turn helps carrot roots to mature early. in addition, early emergence due to priming can contribute to early maturity. plants which emerge early can reach maturity earlier. the results of the present study are in agreement with the findings of reid and gillespie (2017), who observed accelerated maturity of carrot roots with frequent irrigation intervals. similarly, safiatou (2012), singh et al. (2015) and aluko et al. (2020) also reported that compared to unprimed seeds, primed seed took significantly fewer days to emerge and reach maturity. yield marketable yield. distilled water treatment con­ sistently increased marketable yield of carrot similar­ ly, increasing the duration of irrigation interval up to seven days intervals hasincreased the marketable yield of carrot. in the interaction effect, the highest marketable yield (33.73 t ha­1) was recorded from seeds soaked in distilled water and irrigated in every seven days interval followed by seeds primed with distilled water and irrigated at ten days interval with the values of (28.30 t ha­1) which was statistically sim­ ilar with that of plants grown in the treatment combi­ nation of four days irrigation interval with distilled water treatment (27.96 t ha­1). on the other hand the lowest (16.53 t ha­1) marketable carrot yield was obtained from the combination of unprimed seeds with ten days irrigation interval (table 3). the reason for higher marketable yield from primed seeds and irrigated at seven days irrigation interval might be due to uniform and vigorous seedling growth, well­developed root system and efficient subsequent growth with lesser competition for nutrient and water that eventually led to higher yield. the lower marketable yield resulted from non­ primed seeds might be associated with suboptimal uniformity at emergence resulting in poor uniformity muhie et al.‐ carrot yield and quality under water deficits 125 in plant size. on the other hand longer irrigation interval with non­ primed seed resulted in carrot roots which are unfit for the market. similar studies were made by knox et al. (2012) who reported that supply of adequate water was critical for high quality vegetable production. the current results are sup­ ported by the findings of govinden­soulange and levantard (2008), alam et al. (2013) and castañares and bouzo (2018) who reported that seed priming is a mean to improve early flowering, maturity time and yield of a crop due to early seedling growth. total carrot root yield. the interaction effect of irrigation interval and priming techniques significant­ ly impacted total carrot root yield. the highest total fresh root yield of carrot (35.23 t ha­1) was recorded from distilled water seed treatment combined with seven days irrigation interval which was statistically similar with total yield obtained from the combina­ tion of ten days irrigation interval and distilled water treatment (32.53 t ha­1) followed by the average fresh total root yield obtained from the interaction effects of gibberelic acid with seven days irrigation interval (29.53 t ha­1). on the other hand the lowest (19.33 t ha­1) total fresh root yield of carrot was recorded for plants produced from non­primed seeds and irrigated at ten days interval (table 3). the increased total root yield due to priming and seven days irrigation interval might be due to early seedling growth, and improved plant stands with the benefits of priming. moreover, seven days irriga­ tion interval produced higher yield because of suffi­ cient soil moisture in the root zone which enhanced the uptake and assimilation of nutrients. this result was in agreement with the finding of hamma et al. (2012) who reported that optimum irrigation inter­ val significantly produced higher plant height, weight of individual root and yield per hectare of carrot. 4. discussion and conclusions the results of the present study showed that phe­ nological and yield related parameters of carrot plant were significantly influenced by both seed priming techniques, irrigation intervals and their interaction. frequent irrigation intervals led to higher marketable root yield due to the increment in the percentage emergence, decrease in the mean emergence time and in the number of days to reach 90% maturity. the highest marketable carrot root yield 33.73 t ha­1 was obtained combining distilled water treatment and seven days irrigation interval. based on the results of the present study, plants table 3 ­ interaction effect of seed priming techniques and irrigation interval on yield parameters of carrot treatment combinations total root yield (t ha­1) marketable root yield (t ha­1)irrigation intervals priming techniques four days intervals gibberelic acid 29.03 cd 26.76 cd distilled water 29.23 bc 27.03 bcd none primed 23.33 f 20.66 f potassium nitrate 27.73 cde 25.03 cde seven days intervals gibberelic acid 29.53 bc 27.96 bc distilled water 35.23 a 33.73 a none primed 24.46 ef 22.06 ef potassium nitrate 28.90 cd 27.36 bcd ten days intervals gibberelic acid 27.63 cde 25.63 cde distilled water 32.53 ab 30.63 ab none primed 19.33 g 16.53 g potassium nitrate 25.73 def 24.03 def lsd (5%) 3.08 3.23 cv (%) 5.40 6.30 se+/­ 1.51 1.62 means with the same letter/s in column are not significantly different; * = significant (p<0.05); cv = coefficient variance; lsd = least significant difference; se= standard error. adv. hort. sci., 2024 38(2): 119­127 126 obtained from distilled water primed seeds and irri­ gated at seven days interval recorded the shortest days to mean emergence time, day to maturity and, highest marketable root yield of carrot which can be recommended for economical 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(ed.) major crops and water scarcity in egypt. springer, cham, switzerlan, pp. 132. paparella s., araújo s.s., rossi g., wijayasinghe m.a.l.a.k. a., carbonera d., balestrazzi a., 2015 ­ seed priming: state of the art and new perspectives. ­ plant cell reports, 34: 1281­1293. patel y.k., rai p.k., 2018 ­ effect of seed priming on seed quality of tomato (solanum lycopersicum l.). ­ pharma innovation j., 7(2): 264­267. pereira m.d., dias d.c.f.d.s., dias l.a.d.s., araújo e.f., 2009 ­ primed carrot seeds performance under water and temperature stress. ­ scientia agricola, 66: 174­ 179. raj s.k., syriac e.k., 2017 ­ weed management in direct seeded rice: a review weed management in direct seed‐ ed rice: a review. ­ agricultural reviews, 38(1): 41­50. reid j.b., gillespie r.n., 2017 ­ yield and quality respons‐ es of carrots (daucus carota l.) to water deficits. ­ new zealand j. crop hortic. sci., 45(4): 299­312. ruan s., xue q., tylkowska k., 2002 ­ the influence of priming on germination of rice (oryza sativa l.) seeds and seedling emergence and performance in flooded soils. ­ seed sci. technol., 30: 61­67. safiatou s.s., 2012 ­ effect of different seed priming methods on germination, seedling establishment and vigour in sorghum (sorghum bicolor (l.) moench) and bambara groundnut (vigna subterrenea (l.) verdc.). ­ m.sc. thesis school of graduate studies, kwame nkrumah university of science and technology, kumasi, ghana. sagvand m., esfahani m.n., hadi f., 2022 ­ pre‐sowing enrichment of echinacea angustifolia seeds with macronutrients improved germination performance and early seedling growth via stimulating the metabolism of reserves. ­ industrial crops products, 188: 115614. selvakumar r., kalia p., raie r.s., 2019 ­ genetic analy‐ sis of nutritional traits in tropical carrot (daucus carota l.). ‐ genetika, 51(2):641­660. selvarani k., umarani r., 2011 ­ evaluation of seed priming methods to improve seed vigour of onion (allium cepa cv. aggregatum) and carrot (daucus carota). ­ j. agric. technol., 7(3): 857­867. shafiei abnavi m., ghobadi m., 2012 ­ the effects of source of priming and post‐priming storage duration on seed germination and seedling growth characteristics in wheat (triticum aestivem l.). ­ j. agric. sci., 4(9): 256­268. simões s., calinas r., vieira m.t., vieira m.f., ferreira p.j., 2010 ­ in situ tem study of grain growth in nanocrystalline copper thin films. ‐ nanotechnology, 21: 1­12. singh h., jassal r.k., kang j.s., sandhu s.s., kang h., grewal k., 2015 ­ seed priming techniques in field crops ‐ a review. ­ agric. reviews, 36(4): 251­264. tania s.s., rhaman m.s., hossain m.m., 2020 ­ hydro‐ priming and halo‐priming improve seed germination, yield and yield contributing characters of okra (abelmoschus esculentus l.). ­ tropical plant res., 7(1): 86­93. tegen h., jembere m., 2021 ­ influences of spacing on yield and root size of carrot (daucus carota l.) under ridge‐furrow production. ­ open agriculture, 6(1): 826­ 835. thiviya p., gamage a., piumali d., merah o., madhu­ jith t., 2021 ­ apiaceae as an important source of antioxidants and their applications. ­ cosmetics, 8(4): 111. unece, 2018 ­ the unece standard ffv‐10 concerning the marketing and commercial quality control of carrots. ­ united nations economic commission for europe, geneve, switzerland, pp. 8. 88 book reviews il tempio della notte. architettura ipogea nei giardini paesaggistici. breda m.a. giardini e paesaggio, vol. 35. leo s. olschki, florence (italy), 2012, pp. 112. isbn 978-88-222-6194-6. € 23.00. in this 35th volume in the “giardini e paesaggio” series maria chiara zerbi, in her presentation, underlines that the book is the result of many years of enthusiastic work and that through it the reader can appreciate these “architecturally secret places that were found in what were once private gardens but are now part of public lands, neglected and in need of restoratotion and consolidation to restore the original quality”. the author brings to this work many years of experience as a historian of garden architecture and history and treats in this book a unique theme: the temple of the night, an underground architectural element, or rather an environment within a grotto found in englishstyle gardens in italy between the end of the 18th and beginning of the 19th centuries, and probably influenced by germany models. the book deals with two cases studied in depth by the author and highlights her ability to observe and compare, to recognize different situations for what they are, and to bring these aspects together through systematic study. she successfully presents the data through drawings and photographs, which make the book pleasant to read and easy to understand. the first of the two cases, the temple created within one of the artificial grottoes in the park of the conte ambrogio uboldo at cernusco sul naviglio, is known to specialists but has not been studied before. the second is a previouslyunknown temple uncovered by chance in the park of villa batthyany near gorla (in the province of milan). this is an interesting contribution – both scientifically and historically-culturally – to the vast literature in landscape studies. the reader will discover new and significant knowledge through this unique book that escapes easy definition. indeed, its originality renders the book special for the specific topic, and especially as a way to know more about hidden architecture that until a few years ago essentially unknown. francesco ferrini pietro porcinai a pistoia e in valdinievole. bucelli c.m., and c. massi (eds.). giardini e paesaggio, vol. 34. leo s. olschki, florence (italy), 2012, pp. xiv+376. isbn 978-8822-6162-5. € 39.00. pietro porcinai, one of the great landscapists, worked in pistoia and the valdinievole area from the early 1930s to the 1980s. as explained in the presentation of this volume, his concern for the social aspect of gardens as a healthy environment for recreation and sport, as a place to play, as well as for urban living and as an extension for the suburbs is particularly evident around pistoia. examination of his masterpieces in the province of pistoia is fundamental in the study of his vast and important production. this volume aims to gain greater insights into the link between the artist and the pistoia landscape, with the idea that the matrices and points of reference for many of his projects in the area come from the atmosphere around him, and is a further testament to the role porcinai’s work has played, and continues to play in garden design. supported by the experience and careful attention of the two editors, the book is a valuable guide to the work of this landscapist. through its interesting text, personal stories, drawings, past and present images, the volume is also a fascinating work of art in itself and will be appreciated as an enrichment of the current literature on this topic, meeting the needs of technicians and students and an ever-increasing number of enthusiasts. clearly, the meticulous genius of porcinai shines through, demonstrating that there is still much to know about him and his important work. francesco ferrini impaginato 295 adv. hort. sci., 2023 37(3): 295­305 doi: 10.36253/ahsc­13849 response of hydroponic baby lettuce to uv­b radiation exposure during the growing period a.c. silveira 1, l. rivera marchant 2, v.h. escalona 2 (*) 1 poscosecha de frutas y hortalizas, dpto. producción vegetal, facultad de agronomía, avda. garzón 780, montevideo, uruguay. 2 centro de estudios postcosecha, facultad de ciencias agronómicas, universidad de chile, avda. santa rosa 11315, la pintana, santiago, chile. key words: carotenoids, chlorophyll, dry matter, leaf area, polyphenols. abstract: lettuce (lactuca sativa l.) has a nutritional contribution comparable to other vegetables. it is produced in soil and hydroponics systems, outdoors or indoors, and in some cases, with the management of radiation. uv­b radiation exposure can influence the functional quality of vegetables and is becoming more frequent. cultivars kristine rz and versaï rz were exposed to four radia­ tion doses: uv­b0 (0 μw·cm­2), uv­b16 (16 μw·cm­2), uv­b33 (33 μw·cm­2) and uv­b58 (58 μw·cm­2), during 30 min for 10 days. lettuce leaves were harvested twice. the leaf area of ‘versaï rz’ was not affected by radiation in the first har­ vest, while the high doses (33 and 58 µw·cm­2) reduced the leaf area of ‘kristine rz’ between 15­30%, respectively. the radiation did not significantly impact the percentage of dry matter and the color parameters. however, functional com­ pounds were affected. in general, the cv. kristine rz responded positively to the dose of 16 µw·cm­2 while ‘versaï rz’ to 58 µw·cm­2. an increase in the con­ tent of functional compounds was also observed in ‘versaï rz’ in the second harvest, and a reduction in the levels measured in ‘kristine rz’ indicated a dif­ ferent adaptation to uv­b radiation that must be studied individually. 1. introduction lettuce (lactuca sativa l.) is one of the most consumed leafy vegeta­ bles worldwide. within the species, there are four botanical varieties with different characteristics: a) l. sativa var. capitata; b) l. sativa var. longifo‐ lia; c) l. sativa var. crispa; and d) l. sativa var. acephala (kim et al., 2016). it has a high water content (~95%), and despite its vast consumption, it is not considered an essential source of nutrient supply. however, its nutri­ tional contribution is comparable to that of other vegetables because it is consumed raw, implying that the cooking processes do not affect its com­ position (xiao et al., 2012). lettuce is low in calories, fat, and sodium and provides minerals, fibers, provitamin a or β­carotene, vitamins c, k, and folate (vitamin b9), and phenolic compounds to the diet, among others (*) corresponding author: vescalona@uchile.cl citation: silveira a.c., rivera marchant l., escalona v.h., 2023 ­ response of hydroponic baby lettuce to uv‐b radiation exposure during the growing period. ­ adv. hort. sci., 37(3): 295­305. copyright: © 2023 silveira a.c., rivera marchant l., escalona v.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 25 july 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13849 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 295­305 296 (kim et al., 2016). the plasticity of the crop determines that it can be produced in soil and hydroponics systems. in addi­ tion, it can be harvested with different degrees of development ranging from the first leaves (cotyle­ dons), seedlings (baby leaf), or fully developed plants (xiao et al., 2012). hydroponics is a production system in which nutrients are supplied to plants artificially through water (sharma et al., 2018). three large hydroponic cultivation systems are differentiated into substrate, water, and air (aeroponics). the most used are water crops, which include two types: floating root system, where the crop is in continuous contact with the nutrient solution, and which have the advantages of being easy to perform, low cost, and do not require extra energy use; and the nutrient film technique (nft), which is a closed system where plants grown in a constant recirculation of a thin layer solution through the roots, with no loss or leakage of nutrient solution (magwaza et al., 2020). in addition to being the essential energy source for photosynthesis, light is one of the environmental factors that determine plants’ growth, development, morphology, and synthesis of secondary metabolites. the relationship between light and plants has differ­ ent conceptions and levels of complexity that involve aspects such as quality and quantity of light, which directly influence photosynthesis, but also the responses of plants to environmental stimuli. in this sense, many works have been developed on the abili­ ty of plants to detect and respond to the moment, duration, wavelength, dose, and direction of light, which involves the processes of photoperiodicity, phototropisms, and the photomorphogenesis (robson et al., 2015). the visible spectrum region, which goes from 400­ 700 nm, corresponds to the range of emissions they use and is known as photosynthetically active radia­ tion (par). however, plants require a broader range for their development, which goes from 300 to 800 nm, which includes, in addition to par radiation, uv, and far red (li and kubota, 2009; chory, 2010). one of the components of light is uv radiation, which according to its wavelength, is divided into uv­c (100­280 nm), uv­b (280­315 nm), and uv­a (315­ 400 nm). uv­c radiation and much of uv­b radiation (wavelengths less than 290 nm) do not reach the earth. uv­a radiation contributes approximately 5% of the photons in the photosynthetically active radia­ tion (400­700 nm, par); it is highly variable, consti­ tuting no more than 0.33% of the photons in par. although it represents a tiny fraction of the radiation that reaches the earth, it plays a fundamental role in regulating metabolic pathways in the development of the associated specific photomorphogenic responses (robson et al., 2015; robson et al., 2019). the study of the relationship of plants with uv­b radiation allowed the identification of a specific photorecep­ tor, the uv resistance locus 8 (uvr8), which allowed a substantial advance in the understanding of signaling and response processes (rai et al., 2021). exposure to uv­b radiation has a negative effect on photosynthesis due to damage at the level of dna, proteins, and especially in the photosystems (psi and psii) and the light­harvesting complexes, which result from the increase in the levels of ros. however, it is an effective elicitor to increase the content of bioactive compounds since one of the responses to exposure to uv­b radiation involves the induction and biosynthesis of phenolic compounds, including flavonoids, which act as uv protection com­ ponents, and have antioxidant potential (neugart and schreiner, 2018). different works mentioned increases in the concentrations of individual phenyl­ propanoids, such as hydroxycinnamates and flavonoids, in plants exposed to uv­b radiation. these changes are generally believed to positively impact the antioxidant capacity and uv protection (moreira­rodríguez et al., 2017 a, b; dou et al., 2019; rodríguez­calzada et al., 2019; castillejo et al., 2021; loconsole and santamaria, 2021). in the last 20 years, the consumption of vegetables has focused on the contribution of compounds of high nutritional value. vitamins (e, c); hundreds of chemical com­ pounds, such as sulfur and selenium; polyphenols such as flavonoids, stilbenes, and ellagic acid; and carotenoid compounds, such as lycopene, lutein, and ß­carotene among others are included in this group (kyriacou et al., 2016). consumers are looking for new products that promote health and longevity combined with gastronomic delight. consequently, the way is opened to develop exceptional products that may be new, as in the case of microgreens or traditional products whose production systems have been modified (light management, for example) to influence their functional quality positively. advances in the knowledge of physiological processes mediat­ ed by light have allowed the safe, healthy, and sus­ tainable production of different plant species within controlled environments known as plant factories (sharathkumar et al., 2020; yoon et al., 2022). based silveira et al. ‐ uv‐b during lettuce growing 297 on the above, the objective of this work was to evalu­ ate the effect of the application of ultraviolet­b (uv­ b) radiation under greenhouse conditions on the antioxidant characteristics of red and green “baby” lettuce leaves grown in a hydroponic system. 2. materials and methods plant material production the study was carried out in a greenhouse at the centro de estudios de postcosecha (cepoc), at the facultad de ciencias agronómicas de la universidad de chile (32°40’ south latitude and 70°32’ west longitude and 625 m a.s.l. altitudes, santiago, chile). lettuce (lactuca sativa l.), cultivars ‘kristine rz’ and ‘versaï rz’, both of oak leaf, green and red respectively, were used for the experiment. the sowing was carried out at the end of autumn in alveolate trays of 200 units, with a substrate of perlite with rock wool in a 1:1 ratio. at the first stages of the seedling, irrigation was made by tap water, depending on the requirements of the crop. upon reaching the phenological stage of the first true leaf, the seedling was watered with a hoagland ii­ modified nutrient solution, diluted to 50% with a ph between 5.5 and 5.8 measured with a potentiometer (hi99301, hanna instruments, usa). before sowing, a germination test was carried out according to ista standards, obtained 96.7% for ‘kristine rz’ and 100% for ‘versaï rz’. when the lettuces reached the stage of the third to fourth true leaf, they were transplanted to a 1.5x7 m nft table with 8 profiles, a slope of 2.5%, and the height of the nutrient solution sheet of 0.005 m. plant density was 53 plants m­2. once the transplant was carried out, the crop was irrigated continuously with tap water for 5 days to reduce the stress of the transplant. when plants were in the phenological stage of the four to fifth true leaf, they were irrigated with a hoagland ii­ modified nutrient solution diluted to 50%. during cultivation, ph conditions were between 5.5 and 5.8, measured with a potentiometer (hi99301, hanna instruments, usa). uv‐b irradiation treatments before treatments applying, photosynthetically active radiation (par, μmol m­2s­1) was measured with a par radiation meter (fieldscout, model 3415, spectrum technologies, inc., i l l inois, usa) to determine the best time for uv­b irradiation. the chosen time was at 7:00 p.m., which corresponded to the moment closest to the point of l ight compensation of sun plants (20­30 μmol m­2 s­1); where the photon flux in the net exchange of the leaf is zero, equalizing the rates of production and consumption of co2 (yin et al., 2011). uv­b radiation was applied with 13 uv radiation lamps (q­panel 313, cleveland, usa), arranged in a steel structure of 1.5x1.8 m. the lamps were covered with a 0.11 mm mica (socomish, santiago, chile) to isolate any other type of radiation other than uv­b radiation. four radiation doses corresponding to: uv­ b0 (0 μw·cm­2), uv­b16 (16 μw·cm­2), uv­b33 (33 μw·cm­2) and uv­b58 (58 μw·cm­2) were evaluated. radiation was applied for 30 min daily for 10 days when the lettuces reached the fifth to sixth true leaf. greenhouse growth conditions were 22.4±4.5°c mean daily temperature and 48.8±6.5% mean daily relative humidity. after 10 days, when the lettuces were in the phenological stage of 8th to 9th true leaf, 7 to 8 outer leaves were harvested, leaving 2 to 3 leaves per plant. these plants remained in growing conditions until they reached 5 to 6 fully extended leaves the moment they were irradiated. when plants reached the same condition as in the first harvest, they were harvested again (second harvest). the time difference between the first and second harvests was 19 days. at each moment of analysis, the following determinations were made: leaf area (cm2). measured in a total of 30 leaves per variety, with a leaf area determiner (area meter, li­cor 3000, usa). fresh and dry matter of the aerial part (g). weight of whole leaves was determined with a precision balance (radwag, as 100/c/2, poland) and corresponded to the fresh matter. after that, leaves were dried in an oven (labtech, ldo­150f, korea) with forced air ventilation at 70°c until constant mass to obtain the dry mass. values were expressed in percentages. colour. measured in the adaxial part of the distal sector of the lamina in 30 leaves per variety, using a compact tristimulus colorimeter (minolta chroma meter, cr­300, ramsey, nj, usa) with a d65 light source, an angle observed from 0° and calibrated with a white standard, using the cielab system. parameter values were expressed as hue (hab), chroma (c*), and lightness (l*). chlorophyll a, b, and carotenoids determination adv. hort. sci., 2023 37(3): 295­305 298 (mg g‐1). it was carried out according to the methodology proposed by lichtenthaler and wellburn (1983). for the extraction, 0.4 g of the distal part of the leaf blades were weighed (radwag, as 100/c/2, poland), and 15 ml of 80% (v/v) acetone were added. subsequently, the mixture was homogenized at 3,500 rpm for 30 s (ika t18 basic, ultra turrax, wilmington, usa), filtered with gauze, and centrifuged (hermle labortechnik, z326k, wehingen, germany) for 15 min at 3,630 gx. the determinations were made in the supernatant that was measured in a plate spectrophotometer (asys, uvm340, eugendorf, austria) at 470, 646, and 663 nm. for the quantification, the following expressions were used: ca = 12.25 a663 ­ 2.79 a646 cb = 21.5 a646 ­ 5.1 a663 cx+c= 1000 a470 ‐ 1.82 ca ‐ 85.02 cb 198 where ca is the chlorophyll a content, cb the chlorophyll b content and cx+c, the carotenoid content. extraction of bioactive compounds. it was made following the methodology proposed by swain and hillis (1959) with some modifications. for this, 5 g of sample were weighed (radwag, as 100/c/2, poland), mixed with 20 ml of methanol, and homogenized at 3,500 rpm for 45 s (ika t18 basic, ultra turrax, wilmington, usa). the homogenate was stored at 5°c for 24 h. subsequently, it was filtered with gauze and centrifuged (hermle labortechnik, z326k, wehingen, germany) for 20 min at 3,630 x g . measurements were made on the supernatant. total phenolic compounds (µg gae g‐1). it was determined according to the colorimetric method of folin ciocalteu (singleton and rossi, 1965), placing 19.2 µl of extract/blank, together with 29 µl of folin­ cioclateu reagent (1:8 v/v with distilled water) in each cell of the elisa plate. after 3 min, 192 µl of 1n na2co3 were added, and 10 min after, the time at which the reaction was complete as previously determined; absorbance was measured at 750 nm. for the calculation, a calibration line was made with gallic acid (r2= 0.9958). the values were expressed in µg of gallic acid equivalent (gae) g­1 of fresh weight. antioxidant capacity by dpph (µg et∙g‐1). it was determined according to the methodology of brand­ williams et al. (1995), placing 21 µl of sample and 194 µl of dpph solution in each cell (previously adjusted to 1.1 absorbance at 515 nm). after 2 h, at which time the reaction was complete, the absorbance was measured. for the calculation, a calibration curve was made with trolox (r2= 0.9992). the results were expressed as µg trolox equivalent (te) g­1 fresh weight. antioxidant capacity by frap (µg et∙g‐1). for the analysis, the methodology proposed by benzie and strain (1996) was followed. to 6 µl of sample, 198 µl of frap reagent was added (buffer acetate 300 mm ph 3.5 + ferric chloride 20 mm aqueous solution + 2,4,6­tripyridyl­s­triazine 10 mm in hcl 40 mm). after 30 min, time in which the reaction stabilized, the absorbance at 593 nm was measured. a calibration curve was made with trolox (r2= 0.9951) to express the results as µg trolox (et)·g­1 fresh weight. statistical analysis the experimental design was a 4x2 factorial, completely randomized with 3 repetitions. the factors corresponded to the level of uv­b radiation and cultivar, being distributed randomly within each repetition. the experimental unit used was 8 plants per replicate and cultivar. an analysis of variance (andeva) was performed, and when statistically significant differences were found, tukey’s multiple range comparison test was used, with a significance level of 5%. the percentage values were corrected prior to statistical analysis using the following formula: where y is the percentage values (0 to 100). 3. results the foliar leaf area showed differences between genetic materials and treatments in the first harvest (fig. 1a). the foliar leaf of cv. ‘kristine rz’ was more significant than cv. ‘versaï rz on control and lower uv­b radiation (16 µw.cm­2). when supplemented with uv­b radiation, no response was found in the cv. versaï rz. however, the higher doses of radiation (33 and 58 µw·cm­2) reduced the leaf area of ‘kristine rz’. nevertheless, uv­b radiation doses applied to each cv. in the second harvest did not differ. differences were only observed between ‘kristine rz’ and ‘versaï rz’, being the leaf area higher in the first one (fig. 1b). silveira et al. ‐ uv‐b during lettuce growing 299 the kristine rz cultivar dry matter did not show significant differences between the intensities of uv­ b radiation in the first harvest, with an average of 6.4% (fig 2a). in ‘versaï rz,’ differences were only observed between the control and 58 µw·cm­2. the cultivar effect was expressed in a higher dry matter/fresh matter ratio by the cultivar ‘versaï rz’ compared to ‘kristine rz’ in the uv­b radiation inten­ sities of 16 and 58 µw·cm­2; while at 0 and 33 µw·cm­ 2, there were no significant differences between culti­ vars. in the second harvest, the cv. kristine rz pre­ sented a higher percentage of dry material (~13%) compared to ‘versaï rz’ (~11%), without differences related to the intensity of the radiation being observed in any of them (fig. 2b). uv­b radiation did not significantly affect the dif­ ferent color parameters of the evaluated lettuce (table 1). the ‘kristine rz’ presented hab values of 120° corresponding to green coloration, while ‘versaï rz’ presented values of 90° indicating red­yellowish coloration. the cv. kristine rz did not show variations in this parameter either in the first or the second har­ vest. in the case of ‘versaï rz,’ the behavior was each value was indicated by mean±standard error (n=8). different letters indicate significant differences by tukey's multiple test with a significance level of 0.05. table 1 ­ lettuce cultivars used in this experiment fig. 1 ­ effect of uv­b radiation on leaf area (cm2) of baby lettuce 'kristine rz' and 'versaï rz' in a hydroponic system, on first (a) and second harvest (b). values are means (n= 10). different letters, uppercase for cultivars and lower­ case for treatments, indicate significant differences according to tukey's test (p≤0.05). treatment first harvest second harvest 'kristine rz' 'versaï rz,' 'kristine rz' 'versaï rz,' l* control 121.81 ± 1.17 aa 72.14 ± 0.88 ba 117.85 ± 1.57 aa 30.92 ± 1.78 bc 16 121.89 ± 1.38 aa 67.03 ± 1.13 bb 117.41 ± 1.14 aa 25.96 ± 0.67 bd 33 122.53 ± 1.06 aa 72.41 ± 0.88 ba 117.81 ± 1.27 aa 33.42 ± 0.23 bb 58 121.47 ± 1.04 aa 67.52 ± 0.96 bb 117.69 ± 1.65 aa 37.66 ± 0.18 ba hab control 41.25 ± 0.57 aa 8.45 ± 1.11 ba 45.94 ± 1.63 aa 7.33 ± 0.95 bb 16 40.12 ± 0.72 aab 8.33 ± 0.88 ba 46.73 ± 0.16 aa 7.99 ± 1.16 bab 33 38.41 ± 2.69 abc 7.95 ± 0.66 ba 45.44 ± 2.38 aab 8.58 ± 1.21 bab 58 37.72 ± 1.23 ac 8.09 ± 0.85 ba 44.14 ±0.28 ab 9.06 ± 0.28 ba c* control 60.79 ± 0.53 aa 34.32 ± 0.47 ba 64.78 ± 2.16 a ns 32.01 ± 3.74 b ns 16 59.77 ± 0.81 aab 33.74 ± 0.72 bab 64.02 ± 2.51 a 32.42 ± 2.27 b 33 57.31 ± 1.55 ab 33.66 ± 1.63 bb 62.81 ± 1.27 a 32.31 ± 3.27 b 58 57.16 ± 1.38 ab 33.11 ± 0.16 bb 64.14 ± 1.72 a 32.72 ± 2.69 b fig. 2 ­ effect of uv­b radiation on dry matter (%) of baby let­ tuce 'kristine rz' and 'versaï rz' in a hydroponic system, on first (a) and second harvest (b). values are means (n= 10). different letters, uppercase for cultivars and lower­ case for treatments, indicate significant differences according to tukey's test (p≤0.05). 300 adv. hort. sci., 2023 37(3): 295­305 quite erratic since, in the first harvest, the leaves of the control and the 33 µw·cm­2 treatment were the least red. compared to the first harvest, in the sec­ ond, the leaves of ‘versaï rz’ were redder, with val­ ues between 25­35, especially those of the control and the 16 µw·cm­2 treatment. regarding saturation expressed by c*, ‘kristine rz’ presented higher values than ‘versaï rz’ in both harvests, indicating more vivid colors. it also present­ ed greater luminosity (l*). higher uv­b radiation intensities in the first harvest in ‘kristine rz’ generally reduced saturation and lightness (c* and l* respec­ tively). while in ‘versaï rz,’ only a decrease in lumi­ nosity was observed. in the second harvest, the behavior of ‘kristine rz’ was like that of the first. however, in ‘versaï rz,’ the effect was the opposite since the greater intensity of radiation determined a greater saturation. however, the parameter l* was not affected by radiation in either of the two culti­ vars. chlorophyll a content showed differences between genetic materials and radiation levels. in the first harvest, ‘kristine rz’ presented about 10 times more than ‘versaï rz’ in all treatments. in ‘kristine rz,’ the highest values were measured in control and 16 µw.cm­2 , while the lowest was in those with higher radiation intensities, with no differ­ ences between them. on the contrary, the treat­ ments did not affect chlorophyll’s a level of ‘versaï rz.’ (fig. 3a). in the second harvest, the behavior was practical­ ly the opposite. in most treatments, ‘kristine rz’ pre­ sented lower levels of chlorophyll than cv. versaï rz. no response to treatments was found in ‘kristine rz,’ while in ‘versaï rz,’ there was an increase in treat­ ments of 33 and 58 µw·cm­2, respectively (fig. 3b). comparing the values measured in the first and sec­ ond harvest, in the cv. kristine rz, the values were practically halved. at the same time, in ‘versaï rz,’ it increased between 3 and 9 times, indicating a very different response to radiation linked not only to the genotype but also to the age of the plant. chlorophyll b values measured at the first harvest in cv. kristine rz were around 10 higher than those of ‘versaï rz.’ in both, an effect of uv­b radiation levels was observed. in ‘kristine rz,’ the 16 µw·cm­2 treat­ ment determined an increase, while in ‘versaï rz,’ the increase was observed in the 58 µw·cm­2 treat­ ments (fig. 4a). in the second harvest, only differ­ ences between varieties were observed (fig. 4b). however, contrary to the first, the values measured in ‘kristine rz’ were around half of those measured in ‘versaï rz.’ also, in the case of carotenoids, both in the first and in the second harvest, differences were found between genetic materials. while, in the first harvest, ‘kristine rz’ presented around 10 times more, in the second, it presented between 37­62% less (fig. 5a and b). regarding the effect of the intensity of the radiation, in the first harvest the two highest doses of uv­b radiation had a negative effect on the carotenoids of ‘kristine rz’, while the intensity did not affect the carotenoid levels of ‘versaï rz’. on the fig. 3 ­ effect of uv­b radiation on chlorophyll a content (mg·g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydro­ ponic system, on first (a) and second harvest (b). values are means (n= 3). different letters, uppercase for culti­ vars and lowercase for treatments, indicate significant differences according to tukey's test (p≤0.05). fig. 4 ­ effect of uv­b radiation on chlorophyll b content (mg·g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydro­ ponic system, on first (a) and second harvest (b). values are means (n= 3). different letters, uppercase for culti­ vars and lowercase for treatments, indicate significant differences according to tukey's test (p≤0.05). silveira et al. ‐ uv‐b during lettuce growing 301 contrary, in the second harvest the radiation did not cause differences in the content measured in ‘kristine rz’ but determined in ‘versaï rz’ lettuces exposed to 33 and 58 µw·cm­2 an increased. at both harvest times, ‘versaï rz’ presented about 70­80% more total phenolic compounds than ‘kristine rz.’ in ‘versaï rz’ from the first harvest, the radiation intensity did not affect the values (fig. 6a). in the second harvest, an increase in phenolic com­ pounds was observed in both cultivars, independent of the radiation dose (fig. 6b). regarding cat measured by the dpph method, both in the first and second harvest, ‘versaï rz’ sur­ passed ‘kristine rz,’ which presented between 3 and 5 times fewer antioxidant compounds (fig. 7). in the first harvest, there was no response to radiation in the case of ‘kristine rz,’ while in ‘versaï rz’ the intensities of 16 and 33 µw.cm­2 reduced the levels of these compounds (fig. 7a). however, in the treat­ ment of 58 µw.cm 2, there were no differences with the control. in the second harvest, the response to the dose was somewhat erratic in ‘kristine rz’. at the same time, in ‘versaï rz’, the control treatment pre­ sented the lowest levels while the radiation deter­ mined an increase, reaching the highest values at 16 µw·cm­2 (fig. 7b). on the other hand, when cat was measured by the frap method, both in the first and in the second harvest, differences were observed between genetic materials, with ‘versaï rz’ being superior to ‘kristine rz’ (fig. 8). 4. discussion and conclusions there are reports that the application of supple­ mental uv­b radiation has a negative effect on vege­ tative growth in general and consequently on the yield of different plant products. the most frequently reported alterations include a decrease in the leaf area and/or an increase in the thickness of the leaves. plant exposure to uv­b radiation, both in the field and in controlled environments, reduced leaf development in lettuce, peas, corn, and sweet pep­ per, among others (choudhary and agrowal, 2014; fig. 5 ­ effect of uv­b radiation on carotenoid contend (mg·g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydro­ ponic system, on first (a) and second harvest (b). values are means (n= 3). different letters, uppercase for culti­ vars and lowercase for treatments, indicate significant differences according to tukey's test (p≤0.05). fig. 6 ­ effect of uv­b radiation on total polyphenol content (µg gae. g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydroponic system, on first (a) and second harvest (b). values are means (n= 3). different letters, uppercase for cultivars and lowercase for treatment, indicate signifi­ cant differences according to tukey's test (p≤0.05). fig. 7 ­ effect of uv­b radiation on total antioxidant capacity by dpph method (µg te·g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydroponic system, on first (a) and sec­ ond harvest (b). values are means (n= 3). different let­ ters, uppercase for cultivars and lowercase for treat­ ments, indicate significant differences according to tukey's test (p≤0.05). adv. hort. sci., 2023 37(3): 295­305 302 fina et al., 2017; rodríguez­cazalda et al., 2019). although alterations occur because of exposure to uv­b radiation, it is mentioned that they could be transitory since, once the acclimatization stage has been overcome, which involves processes such as the positive regulation of ros elimination, the detection uv and dna repair capabilities; any interruption in leaf development is overcome. this may allow leaf development to resume its original pattern, or even produce a compensatory response whereby greater expansion is matched by reduced division (héctors et  al., 2010; robson et al., 2015). this could be explain­ ing the behavior observed in the second harvest. on the other hand, and contrary to what was found in this work, it is indicated that exposure to uv­b radiation reduces plants’ biomass due to the lower capacity for photosynthesis, mainly due to the effect that uv­b radiation could have. b on photosys­ tem ii (psii) (bornman, 1989; mittal et al., 2021). another factor involved is the reduction in the amount of chlorophylls that negatively affects bio­ mass (kataria and guruprasad, 2012) as well as the lower turgor pressure that prevents cells from increasing their water content (choudhary and agrawal, 2014; fina et al., 2017). more recently, rizi et al. (2021) pointed out that exposure to uv­b rays negatively affects many compounds and biochemical processes in plants, including chlorophyll content and photosynthesis, which reduces carbohydrate produc­ tion, with the consequent adverse effect on growth and biomass. the changes observed in color are related to those observed in the different compounds, both pig­ ments, chlorophyll a, b, and carotenoids, as well as phenolic compounds, which include others also linked to color, such as anthocyanins (goto et al., 2016; sytar et al., 2018; gurdon et al., 2019). the dif­ ferential response found in the varieties studied was also observed by other authors. uv­b radiation induces physiological, biochemical, and morphologi­ cal stress responses in plants, which are species­spe­ cific and even differ between cultivars. in a study where two blueberry cultivars (legacy and bluegold) were analyzed, a different response was found where in legacy (resistant to uv­b radiation) there was an increase in photoprotective pigments during the first week of exposure (19 µw·cm­2) and from the second there was a reprogramming of its metabolism that determined an increase in phenolic compounds and its antioxidant capacity (luego escobar et al., 2017). the differences found between the first and sec­ ond harvests may be linked to the lettuce varieties presenting differential acclimatization mechanisms. in this sense, ‘kristine rz’ has an immediate response, but tolerates low levels of radiation. on the other hand, ‘versaï rz’ takes longer to adapt to uv­b radiation. however, it is capable of responding to higher radiation doses. the differential response is because after exposure to radiation, plants need to reprogram their metabolism to alleviate stress (barnes et al., 2015; wargent et al., 2015). on the other hand, the answer will depend on the type of pigment being considered, which in the case of carotenoids comprises different molecules with dif­ ferent sensitivity to uv­b radiation (badmus et al., 2022). in a study carried out on broccoli sprouts, the application of 0.042 w·m­2 for 4h + 24h of adaptation did not determine variations in carotenoids or chlorophylls (mewis et al., 2012). however, when broccoli sprouts were treated with 7.16 w·m­2 for 120 min, photoreceptor pigments were differentially affected, determining increases in carotenoids, lutein and mainly neoxanthin, and in chlorophyll a, in rela­ tion to control (moreira­rodríguez et al., 2017 a). according to león­chan et al. (2017), the daily expo­ sure of pepper, during growth (days), to 72 kj·m­2 for 6 h, did not alter the levels of chlorophyll a and b in relation to the control. however, it determined a notable increase in carotenoids (from 0.02 to 2.18 mg·100 g­1 fw). fig. 8 ­ effect of uv­b radiation on total antioxidant capacity by frap method (µg te·g­1) of baby lettuce 'kristine rz' and 'versaï rz' in a hydroponic system, on first (a) and sec­ ond harvest (b). values are means (n= 3). different let­ ters, uppercase for cultivars and lowercase for treat­ ments, indicate significant differences according to tukey's test (p≤0.05). silveira et al. ‐ uv‐b during lettuce growing 303 to protect themselves from the damage generat­ ed by uv­b radiation, plants activate their defense mechanisms to avoid excess ros and maintain the stability of their cellular structures. the biosynthesis of antioxidant compounds, among which are those of a phenolic nature (phenolic acids, flavonoids, among others) as well as vitamins, is one of the defense mechanisms (león­chan et al., 2017; moreira­ rodríguez et al., 2017b; neugart and schreiner, 2018). in a study carried out with basil exposed to different doses of uv­b radiation (8.5, 34, 68, 102 kj m­2 day­1), it was found that discontinuous applica­ tions for long periods (about 6 days) determined an increase in phenolic compounds without altering the photosynthetic process, directly proportional to the dose of radiation used (mosadegh et al., 2018). in a similar work with purple and green basil exposed to 18.7 kj m­2 h­1 for different exposure times, increases in the concentrations of anthocyanin, phenols, and flavonoids were found that even reached 169% (dou et al., 2019). therefore, it is expected that exposure to uv­b radiation will increase, as observed in the let­ tuce varieties studied. castillejo et al. (2021) applied doses of 5, 10, and 15 kj·m­2 to kale sprouts during germination at 3.5, 7, and 10 days (25% of the dose at each moment) and found variations in the levels of antioxidant compounds. doses of 10 and 15 kj .m­2 increased phenol levels by 30%. in addition, tac experienced increases of 10% (measured by dpph) and 20% (measured by frap) because of the protec­ tion mechanism of plants against the stress factor constituted by uv­b radiation. in work carried out by hao et al. (2022) in pak choi, an increase in the amount of phenolic com­ pounds measured by dpph and frap was found, depending on the applied radiation dose. doses of 0.7 w.m­2 for 4 and 8h determined increases. however, no response was observed when the radia­ tion increased to 1.4 w·m­2 or the exposure time was greater than 8 h. the authors attribute this to the fact that different signaling pathways are activated depending on the dose. a response linked to genetic characteristics and plant age was also observed, but it did not follow the same pattern as for chlorophylls and carotenoids. in this case, both varieties took longer to acclimatize, so the most critical response corresponded to the sec­ ond harvest. in this sense, rizi et al. 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using optical simulation with three‐dimensional plant models in plant factories. ­ biosyst. eng., 221: 1­18. 159 1. introduction in recent years, consumers have paid increasing attention to the health and nutritional aspects (vitamins contents, mineral elements and antioxidants) of horticultural products (scalzo et al., 2005). and fruits are generally beneficial to human health, conferring not only nutritive value but also physiological and biochemical benefits. they are also excellent functional foods, contributing to the prevention of degenerative diseases. these beneficial properties have been associated to the presence of bioactive compounds such as phenolics, carotenoids, tocopherols and ascorbic acid (soobrattee et al., 2005). fruit phenolic compounds are relevant in terms of quality, as they have a role in visual appearance (pigmentation and browning), taste (astringency), and health-promoting properties (free-radical scavengers) (tomás-barberán and robins, 1997). the flavonoids are a large group of phenolic compounds ubiquitously distributed in the plant kingdom, and they exhibit diverse biological activities (erlund, 2004; spencer et al., 2004). many of these biological functions have been attributed to their radical scavenging and antioxidant activity (soobrattee et al., 2005) because they are highly reactive as hydrogen or electron donors (amić et al., 2003). the phenolic content in plants varies among genotypes (tomás-barberán et al., 2001), environmental conditions, nutrient availability, agricultural practices, and postharvest conditions (giorgi et al., 2005; chludil et al., 2008). in fruits, the phenolic composition varies greatly among cultivars and, generally, skin fruit tissues contain larger amounts of phenolics, anthocyanins and flavonols than flesh tissue (wang et al., 1996). due to chemical structure, these compounds are able to react with many active substances in the human body, showing high antioxidant activity (amić et al., 2003). peach is one of the most popular fruits in the world due to its high nutrient level and pleasant flavor. in addition to vitamins and carotenoids (gil et al., 2002) peach contains important phytonutrients such as phenolic acids and flavonoids (prior and cao, 2000; tomás-barberán et al., 2001; remorini et al., 2008). it is known that the antioxidant activity of peach fruit is dependent on rootstock/genotype combination, ripening time and post-harvest preservation (di vaio et al., 2001; scalzo et al., 2005). worldwide, peaches are still principally produced by grafting selected varieties onto rootstocks. in addition to conferring resistance to diseases and tolerance to stressed soil conditions, rootstocks differentially influence tree physiology resulting in differences in growth and vigor (layne, 1994). moreover, the effects of rootstock type on the mineral composition and sugar and organic acid content of the fruit have been reported (di vaio et al., 2001). nevertheless, present knowledge of rootstock effects on peach fruit quality, and particularly pre and post-harvest management affects functional quality of peach (prunus persica l.) cv. flavorcrest g.b. corbino, g. sánchez, j. gonzález, r.e. murray, j. gabilondo, g.h. valentini, l.e. arroyo estación experimental agropecuaria san pedro, instituto nacional de tecnologia agropecuaria, inta, ruta 9, km 170, san pedro, buenos aires, argentina. key words: antioxidants, dpph, fertilization, heat shock, peach, rootstock. abstract: the effect of rootstock, fertilization and post-harvest heat treatments on the antioxidant capacity and total phenolic content in fruits of peach cultivar flavorcrest was studied. `flavorcrest´ grafted on `mrs. 2/5´ and `flordaguard´ rootstocks produced fruits with the highest antioxidant capacity while the activity in the fruit skin was around ten times higher than in the flesh. treatment without fertilization produced the highest antioxidant capacity in fruit flesh while the fruit skin showed no significant differences between treatments. a moderate heat shock (34 and 42°c), evaluated at 24 h post-harvest, improved the antioxidant capacity of fruits but after keep them for 72 h at 20°c, the values were similar to those observed in untreated fruit. pre-harvest (rootstocks and fertilization) and post-harvest (heat shock) treatments influenced the functional quality of ‘flavorcrest’ peach cultivar fruits. adv. hort. sci., 2013 27(4): 159-165 received for publication 9 january 2014 accepted for publication 20 february 2014 160 on nutritional attributes of the fruit, is generally limited (giorgi et al., 2005). stresses such as drought, extreme temperatures, low soil quality, nutrient levels and/or the presence of herbicides and pathogens have direct consequences in the proportion of secondary metabolites produced by plants. often, plants growing in poor nutrient habitats or under stressful soil conditions contain a greater proportion of secondary metabolites (tang et al., 1995) but there is scarce information about the effects of fertilizers on the production of phenolic compounds by plants. peaches ripen and deteriorate quickly at ambient temperature. cold storage has always been used as the main method to slow these processes as well as the development of decay (wang et al., 2006). on the other hand, heat treatments have been used in postharvest fruit technology for insect disinfestations, decay control, ripening delay and modification of fruit responses to other stresses (lurie, 1998; paull and chen, 2000). high-temperature stress induces biosynthesis of phenolic compounds such as flavonoids and phenylpropanoids (wahid et al., 2007). ‘flavorcrest’ is a common yellow-flesh mid season peach variety, widely cultivated in argentina. the aim of this study was to determine the effect of rootstock, fertilization and post-harvest heat treatments on the antioxidant capacity and total phenolic contents of fruits of this cultivar. 2. materials and methods chemicals 2, 2-diphenyl-1-picrylhydrazyl (dpph), ascorbic acid, folin-ciocalteu reactive and anhydrous sodium carbonate was obtained from sigma-aldrich (argentina); chlorogenic acid from fluka (argentina). fruits samples (fruit) were obtained from plants grown in controlled experimental plots at the inta san pedro agricultural experimental station (san pedro, buenos aires, 33° 44´ 34.7´ s, 59° 47´ 34.4´´ w). the number of replicates is described in each different experiment. after harvest, fruits were immediately transported to the laboratory. pre-harvest assays cultivar/rootstock assay. the influence of genotype/ rootstock combination was evaluated on fruits of the ‘flavorcrest’ cultivar grafted on a) ‘mr. s 2/5’ (natural hybrid of prunus cerasifera); b) ‘flordaguard’ (a sixth generation descendant from the cross ‘chico 11’ x prunus davidiana (carr.) franch, c-26712. ‘chico 11’ was a seedling of ‘shau thai’, pi 65821) (sherman et al., 1991); and c) ‘cuaresmillo’ (a selection of prunus persica (l.) batsch, from seedlings of a population grown in mountainous regions of western argentina) (valentini et al., 2003). a randomized block design was used for the experiment, with five replications per treatment and three trees per replication. twenty fruits per cultivar/rootstock combination were harvested at commercial maturity stage. ten fruits were selected for chemical evaluation. fertilization assay. evaluation of the fertilization effects was carried out on fruit harvested from trees growing on soils belonging to the order of mollisols, great group argiudoles, sub-group vertico (ramallo series). soils of this series are fertile, lightly acidic in the surface, with a good content of organic matter and silty clay loam texture. the transition to the b2t horizon is gradual. the study was carried out on trees planted in june of 2005. the experiment was comprised of a randomized block design with 12 plants, three plants per block. the assay consisted of four treatments: n, nk, np, and npk. peach trees without fertilizer were used as control (c). phosphorus, as calcium triple superphosphate (46-48% p 2 o 5 ), 60 g/plant, and potassium, as potassium chloride (60% k 2 o), 100 g/plant were applied after planting. nitrogen, as calcium nitrate (15.5% n), 20 g/ plant, was applied after planting in four different moments: november and december 2005, and september and october 2006. the data regarding tree vigor (trunk diameter and cumulative weight of pruned wood) have been published previously by gonzález and del pardo (2011). post-harvest assay heat shock assay. heat treatments were applied inside an adapted walk-in cooler (frutitec, río negro, argentina) provided with refrigeration, heating and humidification systems. the fruit was heated to 20, 34, and 42°c (±1°c), 90% rh, and kept at these conditions for 24 h. another batch of fruit was cooled to 0°c±0.5°c and kept in cold storage for 24 h. a pool of fruit without treatment was used as control (c). the fruit was evaluated after 24 h and then kept at 20°c for 72 h. fruit quality parameters flesh firmness (ff) was measured on two opposite sides in the equatorial zone of individual fruits with an effegi 327 fruit pressure tester (milano, italy) and expressed as kg/cm2. total soluble solids (tss) were determined in juice from the longitudinal side opposite the suture, with an n1 atago hand refractometer (osaka, japan) and reported as obrix. color determination was performed with a minolta chroma meter cr-300 (osaka, japan). results were expressed as l*, c* [(a2 + b2) ½] and ho (tan-1 b/a) color units calculated from a* (green chromaticity) and b* (yellow chromaticity). sample extractions extractions were carried out using 3 g of fresh fruit skin or flesh homogenized in 15 ml of 7% acetic acid in methanol. tubes were stored for 24 h at 4°c. they were then centrifuged (10 min at 2000 g), filtered and stored at 4°c in darkness until use. ten fruits per treatment were processed for chemical analyses. 161 fruit functional quality assay of dpph radical scavenging activity. the dpph method was adapted from brand-williams et al. (1995). a total of 20 µl of peel extract or 200 µl of flesh extract were diluted to 1 ml with methanol. the diluted sample reacted with 2 ml of dpph●+ (150 μm in methanol) at 30°c. decrease in absorbance was measured at 517 nm after 30 min. results were expressed as μmols of ascorbic acid equivalents/g of fresh weight (μmol aeac/g fw). total phenolic content. total phenolic content (tpc) was determined by the folin-ciocalteu method (swain and hillis, 1959) using chlorogenic acid as standard for the calibration curve. results were expressed as μmols of chlorogenic acid equivalent/g of fresh weight (cae/g fw). the sample (20 µl of peel extract or 200 µl of flesh extract) or standard (0, 50, 100, 200, 300, 400, 500 µl of 0.4 g/l chlorogenic acid) were diluted with water to a final volume of 4.45 ml. 50 µl of folin-ciocalteu reagent (2 n) were added. after 3 min sodium carbonate (0.1 n) was added. results were read at 725 nm after 1 h. statistical analyses data were submitted to analysis of variance and duncan tests were conducted to identify differences among means. pearson correlation test was used to determine the correlations among means. statistical significance was declared at p<0.05. 3. results and discussion fruit quality parameters cultivar/rootstock assay. rootstock influence was found to not be significant for firmness (6.8-7.5 kg/cm2) and soluble solid content (10.9-11.3°brix). while fruit skin lightness (l*) and hue angle (hº) values were significantly higher in ‘flavorcrest’ fruits grafted on ‘mr.s 2/5’ and ‘flordaguard’ rootstock, while chroma values (c*) were not significantly affected by rootstock (table 1). fertilization assay. fruit from np and npk treatments presented the greatest firmness with no significant differences in comparison to control. although fertilizer affected soluble solid content, it did not have pronounced effects. fruit skin color characteristics, lightness (l*), hue angle (hº) and chroma (c*) values showed no significant differences with fertilizer treatments (table 1). heat shock assay. in general, fruit firmness decreased after storage at 20°c for 72 h. flesh lightness (l*) and hue angle (hº) were not modified by treatments. chroma (c*) values decreased with 34 °c treatments (24 h) with respect to control (table 1). table 1 firmness (ff), total soluble solids (tss) and color (lch system) of ‘flavorcrest’ peach fruit grafted on ‘mr.s 2/5’, ‘flordaguard’ and ‘cuaresmillo’ rootstocks included in fertilization and heat shock assays treatment ff tss l c h peel color characteristics rootstock mr. s 2/5 6.81 a 11.31 a 69.03 a 46.64 a 85.63 a flordaguard 7.41 a 11.20 a 66.86 a 45.85 a 79.44 a cuaresmillo 7.21 a 10.88 a 62.34 b 44.86 a 71.18 b fertilization c 5.95 a 12.16 a 55.67 a 43.55 a 61.48 a n 4.77 b 11.13 b 59.38 a 45.44 a 63.95 a np 5.77 a 11.96 ab 57.81 a 45.58 a 61.14 a nk 3.93 c 11.40 ab 56.84 a 45.17 a 59.95 a npk 5.16 ab 11.97 b 55.15 a 45.23 a 58.04 a heat shock flesh color characteristics   control 7.87 a 11.02 ab 73.94 a 49.64 a 99.80 a 0°c 7.84 a 10.96 ab 74.16 a 46.88 ab 98.92 a 20°c 7.42 a 10.70 b 73.63 a 46.35 ab 98.43 a 34°c 7.70 a 11.20 ab 74.57 a 44.13 b 99.49 a 42°c 8.12 a 12.45 a 75.15 a 45.94 ab 97.55 a control + 3d 5.92 a 11.44 a 73.83 a 48.72 a 96.49 a 0°c + 3d 5.88 a 11.20 a 71.44 a 46.67 a 96.87 a 20°c + 3d 2.79 b 11.28 a 72.99 a 46.56 a 95.99 a 34°c + 3d 4.47 ab 11.58 a 72.33 a 45.25 a 94.86 a 42°c + 3d 2.01 b 12.05 a 70.96 a 47.45 a 92.72 a values are the mean of 30 replications. means followed by the same letters are not significantly different (p= 0.05). 162 fruit functional quality cultivar/rootstock assay. total antioxidant capacity was determined in fruit flesh and skin. the fruit flesh of ’flavorcrest’ grafted on ‘flordaguard’ and ‘mr. s 2/5’, both middle vigor rootstocks, presented the highest aeac/g fw values, with 1.77 and 1.65 μmols of ascorbic acid equivalents/g of fresh weight, respectively (fig. 1). previous studies have shown that total antioxidant capacity changes as a function of the rootstock. remorini et al. (2008) demonstrated that ‘mr.s 2/5’ produced fruits with the highest total antioxidant capacity, attributing this to low-vigor properties. despite these results, they did not find a link between rootstock vigor and total antioxidant capacity. on the other hand, scalzo et al. (2005) observed higher antioxidant capacity values with vigorous rootstock. light has been reported to be one of the major environmental factors that affect phenolic production (par and bolwell, 2000). fruits of dense foliage trees receive less light and this could affect the phenolic content. phenolics are the major antioxidant compounds in peach fruits (tomás-barberan et al., 2001). fruit skin total antioxidant capacity was approximately five times higher (8.8-10.6 μmols aeac/g fw) than that of flesh and showed no significant differences between rootstocks (fig. 1). the effect of rootstock on flesh total phenolic content was significantly different. ‘flavorcrest’ grafted on ‘flordaguard’ (1.14 µmol cae/g fw) and ‘mr.s 2/5’ (1.02 cae/g fw) showed the highest values. fruit skin tpc was higher (ten times) than that of flesh and no differences were observed between rootstocks. other authors also found a higher phenolic content in fruit skin compared to flesh (tomás-barberán et al., 2001; remorini et al., 2008), reporting values two to four times higher. total antioxidant capacity and total phenolic content were positively correlated in flesh (r= 0.8052) and peel (r= 0.8190), which suggests that phenolic compounds greatly contribute to the total antioxidant capacity (fig. 2). fertilization flesh from control fruits (without fertilization) had the highest antioxidant capacity (2.2 μmols aeac/g fw), whereas the antioxidant capacity decreased (with respect to control) when n (1.55 μmols aeac/g fw), np (1.75 μmols aeac/g fw) and npk (1.79 μmols aeac/g fw) treatments were applied, and even more so with nk (1.32 μmols aeac/g fw). fruit skin showed no significant differences (fig. 3). fig. 1 total antioxidant capacity determined by dpph assay in flesh and peel of fruits of flavorcrest cultivar grafted on ‘mr.s 2/5’, ‘flordaguard’ and ‘cuaresmillo’ rootstocks. values are means (± s.e.) of 10 replicates. inside each group (flesh or peel), means followed by the same letters are not significantly different (p= 0.05). fig. 2 correlation between total phenolic content (µmol ca/g fw) and total antioxidant capacity (µmol aeac/g fw) of flesh a) and b) fruit skin of flavorcrest cultivar, grafted on ‘mr.s 2/5’, ‘flordaguard’ and, ‘cuaresmillo’ rootstocks. 163 according to the ‘c/n balance theory’, when n is readily available, plants will primarily synthesize compounds with high n content (e.g. protein to growth). instead when n availability is limited, metabolism changes towards carbon-containing compounds such as starch, cellulose, and non n-containing secondary metabolites such as phenolics and terpenoides (haukioja et al., 1998). in plants, it has been shown that competition between protein and phenolic synthesis exists for the common precursor lphenylalanine (riipi et al., 2002). the relative differences in the release of nutrients from various fertilizers could lead to different c/n ratios in plants and this in turn leads to a difference in the production of secondary metabolites (brandt and molgaard, 2001). fertilization treatments were found to not significantly affect the vegetative variables (trunk cross sectional area and pruned wood) (gonzález and del pardo, 2011). environmental stresses including nutrient deficiency are known to activate the biosynthesis of phenylpropanoid compounds (dixon and paiva, 1995), which could explain why the highest antioxidant activity was found without fertilizer treatment. the highest total phenolic content in flesh (3.37 µmol cae/g fw) and fruit skin (13.34 µmol cae/g fw) was obtained in plants without fertilization (c), which differed significantly from the rest of the treatments (fig. 4). flesh total phenolic content showed a low correlation with antioxidant capacity (r= 0.48). heat shock treatments in this assay, the effect of post-harvest temperature on the functional quality of fruit flesh was evaluated. total antioxidant capacity was significantly different between fruit flesh evaluated at 24 h and fruit held at 20°c for 72 h. the moderate heat shock treatments (34°c and 42°c) at 24 h improved the antioxidant capacity (0.76 μmols and 0.84 μmols aeac/g fw, respectively) in comparison to control (0.48 μmols aeac/g fw), 0°c (0.49 μmols aeac/g fw) and 20°c (0.52 μmols aeac/g fw). aeac (µmol/g fw) showed no significant differences between treatments after keeping fruits for 72 h at 20°c. comparing fruits evaluated at 24 and 72 h, the total antioxidant capacity was significantly increased in control, 0°c and 20°c treatments and significantly decreased in 34°c and 42°c treatments (fig. 5). total phenolic content in the flesh, evaluated 24 h after treatment applications, was significantly higher at 34°c (0.41 cae/g fw) and 42°c (0.49 cae/g fw) than control fig. 3 total antioxidant capacity determined by dpph assay in flesh and fruit skin of flavorcrest cultivar under fertilizer n, nk, np and npk. values are means (±s.e.) of 10 replicates. in bars corresponding to flesh values, means followed by the same letters are not significantly different (p= 0.05). fig. 4 total phenolic content determined by folin-ciocalteau assay in flesh and fruit skin of flavorcrest cultivar under fertilizer n, nk, np and npk. values are means (±s.e.) of 10 replicates. fig. 5 total antioxidant capacity determined by dpph assay in flesh of flavorcrest cultivar submitted to different temperature treatments: 0, 20, 34, and 42°c. fruit was evaluated at 24 h (dark grey bars) and after 72 h (light grey bars) from treatment application. each bar indicates the mean (±s.e.) of 5 replications. 164 (0.30 cae/g fw), 0°c (0.30 cae/g fw) and 20°c (0.34 cae/g fw), following the same behavior as antioxidant capacity. after 72 h at 20°c, all treatments differenced of control. when treatments for the two evaluation periods (24 and 72 h) were compared, the only heat treatment that showed a significant difference was 42°c (fig. 6). there was a positive correlation between total antioxidant capacity and total phenolic content (r=0.67) (fig. 7). heat treatment affects several aspects of fruit ripening such as ethylene production and cell wall degradation (lurie, 1998). thermal stress enhances activities of oxidative stress enzymes and induces the accumulation of phenolic compounds like flavonoids and phenylpropanoids (wahid et al., 2007). a previous study on peach cultivars showed that heat treatments promoted the development of red color in the fruit flesh (budde et al., 2002) which could link these phenomena to an increased synthesis of phenolic compounds. 4. conclusions the results of this study show that pre-harvest (rootstocks and fertilization) and post-harvest (heat shock) treatments influence the functional quality of ‘flavorcrest’ peach fruits. ‘mrs. 2/5’ and ‘flordaguard’ rootstocks produced fruits with the highest antioxidant capacity and phenolic content, whereas ‘cuaresmillo’, the most commonly used peach rootstock in our peach growing area, showed the lowest. although these results could be attributed to vigor it is not possible to determine a general behavior; assays with other rootstocks could be useful. it has been reported that soluble phenolics are the principal contributors to the total antioxidant capacity. the accelerated plant growth induced by fertilization may cause a reduction in concentrations of phenylpropanoids (haukioja et al., 1998), resulting in the lowest antioxidant capacity observed in fertilized treatments. heat stress causes accumulation of secondary metabolites of a multifarious nature in plants (wahid et al., 2007). while higher antioxidant capacity was observed in heattreated fruit at 24 h, the total antioxidant capacity values were similar to those observed in non heat-treated fruit after they were held for 72 h at 20°c. acknowledgements the authors thank the instituto nacional de tecnología agropecuaria (inta), pnfru 3191 and aeta 2682 projects by financial support. references amić d., davidovié-amić d., beslo d., trinajstić 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traditional accessions of potato a. elbitar 1, a. chehade 1, f. kanj 2, s. yahfoufi 2 (*) 1 lebanese agricultural research institute, tal amara station, department of plant biotechnology, plant tissue culture unit, po box 287, zahleh, lebanon. 2 lebanese university, faculty of agronomy and veterinary sciences, department of environment, dekwaneh, lebanon. key words: mannitol, micropropagation, sodium chloride, solanum tuberosum l., temperature. abstract: abiotic stresses, including heat, drought, and salinity, pose severe threats to agricultural yields, globally affecting essential crops like potatoes. the aim of this study is to establish an in vitro culture system for three potato accessions: tal amara 1 (ta1), tal amara 2 (ta2), and tal amara 3 (ta3) and to quantify their tolerance to temperature, drought, salinity, and combined stresses. the results demonstrated that ms0 (devoid of growth regulators) medium was the best for culture initiation, with a percentage of reactive meristems of 82.22%, whereas ms1 (0.35 mg l­1 kin + 0.2 mg l­1 iaa + 0.1 mg l­1 ga3) medium resulted in the highest multiplication rate of 5.5. the most heat tolerant accession was ta1, with shootlets lengths ranging from 2 cm to 4.4 cm at temperatures of 4°c and 38°c respectively. concerning the effect of combined drought and temperature stresses, ta1 and ta3 showed tolerance to the different mannitol concentrations. likewise, the most prominent accession in terms of combined salinity and temperature tolerance was ta2, with shootlets lengths of 3.2 cm (60 mm nacl, 22°c), 2.03 cm (60 mm nacl, 4°c) and 1.6 cm (60 mm nacl, 38°c). 1. introduction belonging to the family of solanaceae, potato (solanum tuberosum l.) is considered as the most commonly cultivated tuber crop and is ranked the fourth most important food crop in the world, after wheat, rice and maize (hussen, 2021). potato is recognized as a crop of future and possesses a strong link in sustaining the global food security (bakhsh et al., 2023). it is cultivated in more than 158 countries worldwide (muthoni and shimelis, 2020). considering its global importance, it is essential to (*) corresponding author: suzanne.yahfoufi@st.ul.edu.lb citation: elbitar a., chehade a., kanj f., yahfoufi s., 2024 ­ non in vitro propagation and shootlets assessment for drought and salinity tolerance of traditional accessions of potato. ­ adv. hort. sci., 38(4): 351­362 orcid: ys: 0009­0005­7542­6561 copyright: © 2024 elbitar a., chehade a., kanj f., yahfoufi s. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 20 june 2024 accepted for publication 18 october 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16275 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0005-7542-6561 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 351­362 352 maintain its yield and sustain its productivity (bakhsh et al., 2023). potatoes grow perfectly in excellent climatic conditions with an optimal growth and yield at a temperature range of 15­20°c and ideally at a minimum rainfall 750­1000 mm. however, potato tuber growth is strongly affected by temperature fluctuations <5°c and >30°c. temperatures above 30°c can negatively impact the potato production, especially by reducing, the tuber growth starch partitioning and dormancy and increasing disease incidences. such increase in temperature can adversely reduce the growth performance, the yield of crops and thus the weight of tubers. moreover, at shallow temperatures, potatoes become susceptible to frost damage and this in turn, causes reduced growth and damage to tubers (mwakidoshi et al., 2021). besides, drought stress delays the emergence, slows the plant development, and reduces the plant mass weight as well as the tuber number, size and yield (zaki and radwan, 2022). alternatively, salt stress negatively impacts crop yield by changing the plant metabolism and inducing substantial alterations in both biochemical and molecular processes (abdelsalam et al., 2021). seed production of potato is normally vegetatively propagated through the use of potatoes that have been previously propagated by harvesting and replanting the tubers in the field (singh et al., 2012; shiwani et al., 2021). however, this conventional seed plant potato production has proved to be prone to pests and disease infestations where fungal, bacterial and viral disease agents can be transmitted easily through the tubers (morais et al., 2018; shiwani et al., 2021). in this event, plant tissue culture techniques and more specifically micropropagation offers a great potential to complement conventional breeding methodology for potato improvement and production (singh et al., 2012). micropropagation is generally referred to the production of a large number of in vitro plants on a defined nutrient media under aseptic conditions within a limited space and time. this term includes the use of different techniques in potato production such as, shoot­tip culture, meristem culture, single­ node culture and micro­tuberization (shiwani et al., 2021). potato production in lebanon is very important for sustaining the food security from one side, and as a source of revenue in rural areas (dalleh et al., 2023). this strategic crop, covers around 19,000 ha in the bekaa plain, with a production reaching 300,000 tonnes per year, and is considered as the greatest field crop tonnage in lebanon (choueiri et al., 2017; dalleh et al., 2023). seventy percentage of the lebanese total potato cultivated area is concentrated mainly in the bekaa valley at 900­1000 m above sea level (dalleh et al., 2023). its cultivated for direct consumption and processing product with a part being exported. despite its importance, a number of production constraints are hindering the full export potential of potato production in lebanon such as, climate change, the use of low yielding varieties and the occurrence of bacterial diseases and viral infections (choueiri et al., 2017). furthermore, potatoes during the summer are vulnerable to drought and salinity stresses due to insufficient irrigation water and temperature extremes (verner et al., 2018). therefore, the need for identification of heat, drought and salinity tolerant potato genotypes for breeding by early selection is immense. the aim of this present investigation is to establish a micropropagation system and screen in vitro initial explants of three potato accessions for temperature, drought and salinity tolerance: tal amara 1 (ta1), tal amara 2 (ta2) and tal amara 3 (ta3). 2. materials and methods plant material this study for in vitro micropropagation was conducted at the lebanese agricultural research institute (lari, tal amara station). clean tubers of three high yielding potato accessions namely tal amara 1 (ta1), tal amara 2 (ta2) and tal amara 3 (ta3) were kept under heating room conditions at 38°c for 30 days and used as a source for explants throughout the experiment. four­weeks old healthy sprouts extracted from the three accessions, were surface sterilized using 70% ethanol for only 1 minute then dipped in 20% (v/v) sodium hypochlorite for 10 min. following that, explants were rinsed four times with sterile distilled water for 20 min. in vitro propagation meristem tips of the three potato accessions were dissected from apical and lateral buds of the disinfected sprouts. the size of the meristem ranged from 0.5 to 1 mm. the dissected meristems were placed on petri dishes containing three ms basal elbitar et al. ‐ in vitro propagation and stress tolerance in potato 353 media (murashige and skoog, 1962); “ms0” without growth regulators, “ms1” containing kinetin (kin) 0.35 mg l­1 in combination with indole­3­acetic acid (iaa) 0.2 mg l­1 and gibberellin (ga3) 0.1 mg l­1 and “ms2” containing 6­benzylaminopurine (bap) 1 mg l ­1 and gibberellin (ga3) 0.5 mg l ­1 (salem and hassanein, 2017). the three ms media contained ms macroelements, ms microelements, ms vitamins and ms ferrous with 30 g l­1 of sucrose and 7.6 g l­1 agar. all the prepared media were adjusted to a ph 5.7­ 5.75, then were autoclaved at 121°c and 0.103 mpa pressure for 20 minutes and cooled to 60°c. each treatment was performed in ten replicas with 15 meristems per replica for testing. the cultures were then placed in the culture growth room under the following conditions (temperature 22°c, photoperiod 16:8 h light:dark, relative humidity 50%, illumination of 3000­4000 lux). thirty days later, the number of reactive meristems was recorded. shoots derived from meristems were further multiplied by nodal cuttings. nodal segments were cultured on fresh media using the same three prepared ms media (ms0, ms1 and ms2). eight shoots, derived from nodal segment, per jar were inoculated and five replications for each treatment were conducted. every 30 days, shoots were aseptically taken out and inoculated on the multiplication medium marking a new subculture, three subcultures were examined. for every subculture the following parameters were registered: the multiplication rate (number of new shootlets/number of initial shootlets) and shootlets height. the heights of shootlets were measured from their base to the tip. sanitary control for each accession, 30 samples of shootlets of the third subculture were tested for six potato viruses, pvs, pvm, pvx, pvy, pva and plrv at the plant protection laboratory (lari tal amara) by using bioreba kit of double antibody sandwich­enzyme linked immuno sorbent assay (ren et al., 2022). screening for stress tolerance plants from four subcultures on ms0 medium were used in the following experiments: heat, drought, salinity, and combined drought­heat and salinity­heat tolerance of the tested accessions were screened. heat tolerance assay. individual nodal segments were cultured in test tubes each containing ms0 medium with 30 g l­1 sucrose. this in vitro assay employed three temperature treatments t1 (22°c control, tal amara culture room, 16:8 h photoperiod and 3000­4000 lux), t2 (4°c, tal amara fridge room) and t3 (38°c, tal amara heating room). drought adaptive screening. in order to assess drought tolerance, nodal segments were cultured on ms0 medium supplemented with mannitol. four replications were conducted for each treatment in test tubes, with three nodal segments per replicate. mannitol concentrations (c1: 403, c2: 807, c3: 1210 mm) were added to the media to reduce the water potential of the media to ­1, ­2 and ­3 mpa. the water potential was calculated according to van’t hoff equation: π=i×m×r×t, where π is the osmotic potential of the media, i is the van’t hoff factor for solute (mannitol), m is the molarity of the solution, r is the gas constant (=0.0083 mpa g/l­1 k­1), and t is the temperature in kelvin (pant et al., 2014). all subcultures were maintained under 22°c with 16:8 h photoperiod and 3000­4000 lux, as a light intensity. salinity tolerance evaluation. to screen for salt tolerance, nodal segments were grown in test tubes on an ms0 medium supplemented with various concentrations of nacl (c1: 40 mm and c2: 60 mm) at 22°c for 3 weeks (garramone et al., 2023) with four replications per treatment and three nodal segments per replicate. combined drought‐temperature stress. in order to induce a combined drought and heat stress, nodal segments were placed in test tubes on an ms0 medium containing the three different concentrations of mannitol mentioned above (c1: 403, c2: 807, c3: 1210 mm) at temperatures of 4 and 38°c for 3 weeks (handayani and watanabe, 2021) with four replications per treatment and three nodal segments per replicate. combined salt‐temperature stress. coupled treatments of salt and heat stresses were induced and single nodes of each accession were placed in test tubes on an ms0 medium supplemented with the two concentrations of nacl mentioned above (c1: 40, and c2: 60 mm) at temperatures of 4 and 38°c for 3 weeks (nahar et al., 2022). similarly, four replications per treatment were conducted and three nodal segments per replicate were used. statistical analysis four replications per accession were studied using a completely randomized design (crd) and evaluated under each treatment for stress tolerance. growth and morphological changes as a result of the adv. hort. sci., 2024 38(4): 351­362 354 different stresses in culture were observed and recorded after 30 days of in vitro propagation and 3 weeks of stress tolerance assays. data for various shoot and root characteristics were recorded based on the study of albiski et al. (2012): shootlets height (cm), number of shootlets leaves, number of shootlets roots, shootlets fresh and dry weights (oven dried plants at 70°c for 48h) (g) and plant water content (pwc%) = [(fresh weight ­ dry weight)/fresh weight] x 100. all experimental results were expressed as mean values ± standard deviation. the data were tested for normal distribution using shapiro­wilk test, an analysis of variance (anova) was calculated to assess accessions and treatments effects and interactions, and means were compared pairwise by tukey tests at p<0.05 using the extension xlstat 2016 from microsoft excel (addinsoft, 2016). 3. results and discussion in vitro propagation isolated meristems from the three potato accessions, ta1, ta2 and ta3, were subjected to three different media, ms0, ms1 and ms2. among the three media, ms0 demonstrated a superior effect on meristem culture with a percentage of reactive meristems ranging between 71.83% to 82.22%, while on ms1 the percentage of reactive meristems ranged between 54.44% and 78%, and on ms2 between 34.44% and 63.33% (fig. 1). among the three accessions, ta2 (82.22%) exhibited the best response when established in the control media. there were no significant differences observed in the number of reactive meristems on ms0 and ms2 among the accessions (except for ta3). these findings are similar to those reported by dalleh et al. (2023) where the highest percentage of reactive meristems in spunta variety occurred on an ms hormone­free media. xhulaj and gixhari (2018) also reported that potato explants established on ms media supplemented with phytohormones resulted in high proliferation rates which is consistent to our results, where ms1 and ms2 supplemented with hormones also yielded high rates of reactive meristems, reflecting the importance of using murashige and skoog medium during the establishment phase of potato. established shootlets of ta1, ta2 and ta3 were transferred onto the three fresh ms media (ms0, ms1 and ms2) previously uti l ized for the cultures’ establishment. detailed data on the multiplication rate and shootlets’ height was recorded over three subcultures as presented in table 1. the highest multiplication rate (8 shootlets) was observed with ta1 using ms1 medium while the lowest (2.27 shootlets) was observed with ta3 using ms2 medium along the 1st subculture. however, regarding the shootlets height, ta2 demonstrated the highest shootlets height on ms1 (8.85 cm) at the 3rd subculture, while the lowest height (1.94 cm) was also observed by ta2 at the 1st subculture on ms2 (fig. 2). besides, ms1 resulted in the highest multiplication rates among the three accessions during the three subcultures, and achieved the highest shootlet height during the first and third subcultures. these results are in accordance with dalleh et al. (2023) where the highest number of shootlets per plant was obtained on a medium containing 0.4 mg l­1 kin, 0.5 mg l­1 ga3 and 0.5 mg l­1 iba for the spunta potato variety. emaraa et al. (2017) also reported that the highest multiplication rate of the lady­rosetta potato variety was obtained on an ms media supplemented with kin 0.2 mg l­1 in combination with naa 0.2 mg l­1. on the other hand, xhulaj and gixhari (2018) demonstrated the importance of combining ga3 and bap to improve the number of shoots of bergerac potato cultivar. similarly, dessoky et al. (2016) revealed that ms medium containing 3 mg l­1 ga3 and 0.1 mg l­1 kin resulted in the highest multiplication rate in diamant potato cultivar. fig. 1 ­ percentage of reactive meristems of the three potato accessions ta1 (tal amara1), ta2 (tal amara2) and ta3 (tal amara3) during culture establishment after 30 days on the different ms media (ms0: 0 hormones, ms1: kinetin 0.35 mg l­1+ iaa 0.2 mg l­1 + ga3 0.1 mg l­1 and ms2: bap: 1 mg l­1 + ga3 0.5 mg l­1). histograms sur­ mounted by same letters are not significantly different (p<0.05) according to tukeys’ test. elbitar et al. ‐ in vitro propagation and stress tolerance in potato 355 effect of accession evaluating the effect of potato accession on multiplication rate and shootlets’ height, the results are depicted in figure 3. when combining subcultures and media together, no statistically significant distinctions were noted in multiplication rate and shootlets’ height across the three tested potato accesssions (ta1, ta2 and ta3). tal amara 1 showed the highest multiplication rate (4.66) followed by tal amara 3 and tal amara 2. ta1 also exhibited the highest average shootlets’ height at 4.93 cm, followed by ta2 at 3.64 cm and ta3 at 3.17 cm. table 1 ­ effect of the three media (ms0: 0 hormones, ms1: kinetin 0.35 mg l­1 + iaa 0.2 mg l­1 + ga3 0.1 mg l­1 and ms2:bap:1 mg l­1+ ga3 0.5 mg l­1 ) on the multiplication rate and shootlets height measured during in vitro propagation of the three potato accessions; tal amara 1 (ta1), tal amara 2 (ta2) and tal amara 3 (ta3) along 3 subcultures data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). fig. 2 ­ shootlets proliferation in a: tal amara1, b: tal amara2 and c: tal amara3 accessions of potato cultured on dif­ ferent ms media (ms0: 0 hormones, ms1: kinetin 0.35 mg l­1+iaa 0.2 mg l­1+ ga3 0.1mg l­1 and ms2: bap: 1 mg l­1+ ga3 0.5 mg l­1). fig. 3 ­ effect of potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) on the multiplication rate and shootlets height of plants; media and subcultures are combined. same letters indicate not significantly differ­ ent (p<0.05) according to tukeys’ test. treatments multiplication rate shootlets height (cm) ta1 ta2 ta3 ta1 ta2 ta3 subculture 1 ms0 2.53±0.66 d 2.64±0.78 d 2.33±0.65 d 4.19±1.82 abc 2.62±1.61 d 2.87±1.21 cd ms1 8.00±0.63 a 3.46±0.51 c 6.20±0.78 b 5.75±1.48 a 2.60±1.04 d 3.18±0.96 bcd ms2 5.85±0.89 b 2.38±0.50 d 2.27±0.46 d 5.02±0.52 ab 1.94±0.97 d 2.50±0.77 d subculture 2 ms0 3.97±0.75 de 3.37±0.49 f 3.43±0.50 ef 7.80±1.48 a 6.06±1.58 b 6.62±1.31 b ms1 4.91±0.73 bc 5.37±0.50 ab 5.55±0.51 a 4.37±1.68 c 4.43±1.76 c 3.87±0.95 c ms2 3.88±0.76 def 4.68±0.74 c 4.33±0.49 cd 2.55±0.75 d 2.31±1.09 d 2.20±0.74 d subculture 3 ms0 3.60±0.50 d 3.66±0.66 d 4.26±0.45 c 6.87±1.20 b 4.67±1.25 cde 5.83±1.45 bcd ms1 4.91±0.84 ab 5.40±0.50 a 4.83±0.78 abc 8.57±2.68 a 8.85±1.81 a 4.88±1.91 cde ms2 4.33±0.48 bc 3.38±0.50 d 3.42±0.51 d 6.40±2.31 bc 4.32±1.56 de 3.78±1.03 e 356 adv. hort. sci., 2024 38(4): 351­362 these results contradict with asnake et al. (2023) and tessema et al. (2021) who reported that variety significantly influence the growth parameters of potato, and this discrepancy could be correlated to the duration between subcultures and nutrient media used effect of culture media the effect of culture medium on the multiplication rate and height of potato shootlets is illustrated in figure 4. when combining accessions and subcultures, noteworthy is the absence of any significant differences in shootlets’ height among the three tested media. however, the control medium (ms0) yielded the greatest shootlets’ height at 5.28 cm, succeeded by ms1 at 5.16 cm and ms2 at 3.44 cm. ebad et al. (2015) reported that ms medium supplemented with vitamins without exogenous plant growth regulators can be used for mass propagation of potatoes. the highest multiplication rate was observed with ms1 medium (5.4) followed by ms2 and control (ms0). hajare et al. (2021) also reported that the highest multiplication rate was obtained in ms medium containing kinetin (2.5 mg l­1). moreover, emaraa et al. (2017) revealed that the highest multiplication rate was noticed on ms media supplemented with 0.2 mg l­1 naa together with 0.2 mg l­1 kin while, othman et al. (2016) reported that medium augmented with 2.0 mg l­1 ba and 0.250 mg l­1 naa was the most favorable for the multiplication of lady balfour and bellini cultivars. this thus elucidates the importance of both kinetin ba and bap in the multiplication of potatoes. effect of subculture sequential to the systematic exploration of factors influencing shootlets characteristics, this study transitions to investigate the effect of subculture on multiplication rate and shootlets’ height. the experimental findings, regardless of accessions, are graphically represented in figure 5. by combining accessions and media, no statistically significant difference of multiplication rate was observed between subcultures. subculture 3, however, showed the highest multiplication rate (5.19). a noticeable upward trend in shootlets’ height was evident with successive subcultures, reaching a significant value of 6.01 cm at the 3rd subculture. these findings align with the results of muthoni et al. (2014) where there was an increase in the multiplication rate of all potato cultivars with subcultures. he also noted that subculture 3, gave more cuttings than the first two. sanitary control a total of ninety plant samples belonging to the three potato accessions (ta1, ta2 and ta3) were fig. 4 ­ effect of culture media (ms0: control, ms1: kinetin 0.35 mg l­1 +iaa 0.2 mg l­1+ ga3 0.1 mg l­1 and ms2: bap: 1 mg l ­1+ ga3 0.5 mg l ­1) on multiplication rate and shootlets height of potato plants; accessions and subcul­ tures are combined. fig. 5 ­ effect of subcultures (sub1, sub2 and sub3) on multiplica­ tion rate and shootlets height of potato plants; media and accessions are combined. same letters indicate not significantly different (p<0.05) according to tukeys’ test. elbitar et al. ‐ in vitro propagation and stress tolerance in potato 357 similarly, plants subjected to the control treatment (22°c) and high temperature treatment developed more roots per shootlet ranging from 6 roots (ta3) to 13.5 roots (ta2) and 5.5 roots (ta2) to 8.5 roots (ta1) respectively as compared to the low temperature which indicated a number of roots ranging from 2.5 roots (ta3) to 4.5 roots (ta1). on the other hand, no significant percentage of the plant water content was recorded. it is fluctuated between 91.07% and 94.17% at high temperature, between 91.67% and 92.16% at low temperature, compared to the control (93.41% to 94.09%). these observations demonstrate that the greatest tolerance under both high and low temperature conditions was exhibited by ta1 potato accession, with ta3 showing the next highest tolerance at high temperature. the distinct responses of potato accessions to varying temperatures indicate genotype­specific differences in growth parameters, consistent with studies reporting enhanced growth at higher temperatures (mohamed et al., 2016). the reduction in nutrient absorption through the roots influence the shoot development under suboptimal temperature conditions, resulting in an immediate impact of temperature on shoot growth. furthermore, leaf growth reacts promptly with various environmental stresses; including low and high temperatures; which could generally explain the decline in leaf number at low temperature associated with the hindrance of leaf initiation rates, leading to a direct reduction in both leaf cell division and elongation. the overall root system encountered likewise a significant decrease under low temperatures compared to high temperatures table 2 ­ effect of in vitro heat stress on shootlets height, number of leaves, number of roots and plant water content percentage of 3 potato accession (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). tested for potato viruses; pvs, pvm, pvx, pvy, pva and plrv; using das­elisa. it was shown that all the in vitro shootlets were 100% free from the 6 tested viruses, and no significant differences were observed between the three accessions. these findings are consistent with several studies showing that the meristem culture method is effective in producing disease­free plants in potato (spunta) and other crops (pradhan et al., 2016; dalleh et al., 2023). additionally, the size of the meristem explant is important for the efficient elimination of viruses (azad et al., 2020). temperature treatments the in vitro effect of temperature stress on the growth and development of three potato accessions revealed that high and low temperatures had a significant impact on the evaluated growth parameters, such as shootlets height, the leaf and root number, and the plant water content percentage (table 2). low temperature (4°c) and high temperature (38°c) treatments indicated significant low values of shootlets height ranging from 1.6 (ta3) to 2 cm (ta1) at 4°c and 2.50 (ta2) to 4.40 cm (ta1) at 38°c when compared to the control treatment (22°c, ranging from 4.9 for ta2 to 10.9 cm for ta1). shootlets at the control treatment (22°c), presented a greater number of leaves (ranging between 6 leaves and 11.5) compared to those exposed at high temperature (from 4 to 7.5 leaves) and low temperature (ranging 2 and 4.5 leaves), with the exception for the accession ta3, where the high temperature showed more leaves (7.5) than the control treatment (6.0). accession temperature (°c) shootlets height (cm) no of leaves no of roots plant water content pwc (%) ta1 t0 22 (control) 10.90±0.28 a 11.00±0.00 a 9.50±0.70 ab 93.41±0.08 a t1 4 2.00±0.70 c 4.50±0.70 b 4.50±2.12 cd 92.16±1.93 a t2 38 4.40±0.14 b 7.00±2.82 ab 8.50±0.70 bc 94.17±1.79 a ta2 t0 22 (control) 4.90±0.14 b 11.50±0.70 a 13.50±0.70 a 94.09±1.48 a t1 4 1.75±0.07 c 3.50±0.70 b 3.50±0.70 d 91.67±0.82 a t2 38 2.50±0.70 c 4.00±0.00 b 5.50±0.70 bcd 91.07±1.56 b ta3 t0 22 (control) 5.20±0.35 b 6.00±1.41 ab 6.00±0.00 bcd 94.09±0.59 a t1 4 1.60±0.14 c 2.00±1.41 b 2.50±0.70 d 92.02±0.96 a t2 38 4.25±0.35 b 7.50±2.12 ab 6.50±2.12 bcd 93.08±0.28 a adv. hort. sci., 2024 38(4): 351­362 358 in shootlets’ height, leaf number, and root development as mannitol concentrations increased across the three tested temperatures (22, 4, and 38°c) (table 3, table 4 and table 5). however, a significant reduction in plant water content percentage was observed with escalating mannitol concentrations at the tested temperatures. at 22°c the highest shootlets’ height (1.95 cm), leaf (4) and root number (3), and plant water content percentage (81.06%) were observed by ta2 at the low mannitol concentration (c1: 403 mm), followed by ta1 and then ta3 (table 3). however, with the increase in mannitol concentrations reaching c3 of 1210 mm, ta3 presented a stabilized response of 0.90 cm height, average number of leaves and roots of 1 and 59.55% plant water content. ta1 and ta2 on other hand didn’t develop any roots at c3, due to attributed to the cessation of all root growth and developmental parameters. similarly, chen et al. (2024) described the effect of low temperature on hindering the growth of potatoes, where a temperature below 7°c can cease the seedlings growth, which illustrates the low shootlets height, low number of leaves and roots observed at 4°c. besides, at elevated temperatures, plants tend to close their stomata to minimize the water loss (marchin et al., 2021; reddy et al., 2021). this phenomenon elucidates the relatively high percentage of plant water content observed in ta1 and ta3 at high temperatures. drought treatments the response of potato accessions to drought stress, indicated the absence of significant difference table 3 ­ effect of in vitro drought stress on shootlets height, number of leaves, number of roots and plant water content of 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at normal laboratory temperature (22±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). accession 22°c mannitol concentration (mm) shootlets height (cm) number of leaves number of roots plant water content pwc% ta1 c1 403 1.53±0.11 abcd 1.66±0.57 a 2.00±0.00 ab 76.14±1.69 abcde c2 807 1.10±0.00 bcd 1.00±0.00 a 1.66±1.52 ab 70.87±2.67 defgh c3 1210 1.00±0.42 ab 1.00±0.70 a 0.00±0.00 b 59.50±2.36 j ta2 c1 403 1.95±0.07 cd 4.00±1.41 a 3.00±1.41 ab 81.06±1.77 ab c2 807 1.60±0.07 abcd 1.50±0.00 a 0.00±0.00 b 69.75±0.17 efgh c3 1210 1.20±0.14 bcd 1.00±0.00 a 0.00±0.00 b 60.04±2.12 j ta3 c1 403 1.20±0.14 bcd 1.00±0.00 a 2.00±1.41 ab 75.92±0.49 abcde c2 807 1.10±0.21 abc 1.00±0.00 a 1.00±1.41 ab 67.14±0.05 ghi c3 1210 0.90±0.26 abcd 1.00±0.00 a 1.00±0.57 ab 59.55±1.24 j table 4 ­ effect of in vitro drought stress on shootlets height, number of leaves, number of roots and plant water content of 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at low temperature (4±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). accession 4 °c mannitol concentration (mm) shootlets height (cm) number of leaves number of roots plant water content pwc % ta1 c1 403 1.60±0.14 abcd3 2.00±0.00 a 2.00±0.00 ab 81.56±1.35 ab c2 807 1.60±0.07 abcd 1.50±0.70 a 2.00±0.00 ab 74.10±0.50 cdef c3 1210 1.20±0.07 bcd 1.00±0.00 a 1.00±1.41 ab 64.75±0.88 hij ta2 c1 403 1.75±0.21 ab 3.00±1.41 a 0.00±0.00 b 82.35±0.57 a c2 807 1.50±0.14 abcd 3.50±0.70 a 0.00±0.00 b 68.81±0.39 efgh c3 1210 1.35±0.21 abcd 2.50±0.70 a 0.00±0.00 b 67.83±4.24 fgh ta3 c1 403 1.10±0.05 bcd 1.00±0.00 a 1.66±0.57 ab 81.41±0.66 ab c2 807 1.10±0.63 bcd 1.50±0.70 a 1.50±0.70 ab 72.89±0.61 defg c3 1210 0.90±0.28 d 1.50±0.70 a 1.50±0.70 ab 59.74±0.03 j elbitar et al. ‐ in vitro propagation and stress tolerance in potato 359 contributed to the enhancement of the tolerance mechanisms in potato plants, which is in accordance with other findings that demonstrated the positive impact of combined stresses on plants’ tolerance (rafique et al., 2019). notably, the drop in the plant water content, that appeared in response to increasing mannitol concentrations, highlights the water stress that is simulated when an osmotic agent is introduced to the growth medium. this acts at reducing the availability of nutrients that are crucial to plant growth and hinders the absorption of water through the roots (tican et al. , 2021). drought stress additionally reduce the number, mass, and growth of roots, which in turn limits the availability of nutrients and water for the plant shoots (jafari et al., 2019). salinity treatments at 22°c, ta2 exhibited superior performance in terms of plant water content (%pwc), shootlets’ impact of elevated drought on the inhibition of the key physiological and biochemical processes (gervais et al., 2021). transitioning to low and high temperature conditions at 4°c and 38°c, the trend observed at 22°c persisted (table 4, 5). ta2 continued to exhibit the highest tested parameters, except for the number of roots at low temperature and high concentration of mannitol. concurrently, ta1 and ta3 exhibited a constant response in the measured parameters with the increase in concentration of mannitol. this indicates that the shootlets height, number of leaves, root development, and plant water content percentage were significantly affected by drought stress for all three potato accessions. the results are consistent with other studies that show a decrease in spunta shoot length and roots at mannitol concentrations of 200 mm and above (sattar et al., 2021). the concurrent interplay between mannitol and temperature stresses, table 5 ­ effect of in vitro drought stress on shootlets height, number of leaves, number of roots and plant water content of 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at high temperature (38±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). table 6 ­ effect of in vitro salinity stress on shootlets’ height, number of leaves, number of roots and plant water content of 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at normal laboratory temperature (22±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). accession 38 °c mannitol concentration (mm) shootlets height (cm) number of leaves number of roots plant water content pwc% ta1 c1 403 1.65±0.07 abc 2.50±0.70 a 2.00±0.00 ab 76.80±2.00 abcd c2 807 1.33±0.07 abcd 1.50±0.70 a 2.00±0.00 ab 70.18±1.61 defgh c3 1210 1.20±0.00 bcd 1.50±0.70 a 2.50±0.70 ab 64.95±2.42 hij ta2 c1 403 1.96±0.25 a 4.00±1.00 a 3.66±1.15 a 79.30±0.81 abc c2 807 1.95±0.14 a 3.50±2.12 a 0.00±0.00 b 69.46±1.19 efgh c3 1210 1.65±0.07 abc 2.50±0.70 a 0.00±0.00 b 65.33±0.47 hij ta3 c1 403 1.75±0.07 ab 1.00±0.00 a 2.00±0.00 ab 75.26±0.96 bcde c2 807 1.20±0.00 bcd 1.50±0.70 a 2.00±0.00 ab 72.53±0.80 cdefg c3 1210 1.10±0.28 bcd 2.00±0.00 a 2.00±0.00 ab 60.75±0.12 ij accession 22°c concentration of nacl (mm) shootlets height (cm) number of leaves number of roots pwc% ta1 c1 40 2.50±0.14 abcd 9.50±0.70 abc 6.00±0.00 abc 88.67±0.30 ab c2 60 2.00±0.14 cde 4.50±2.12 abcde 2.50±2.12 cde 87.06±1.50 ab ta2 c1 40 3.45±0.35 a 11.50±2.12 a 7.00±1.41 ab 89.85±0.20 a c2 60 3.2±0.07 cde 8.50±3.53 abcd 4.50±0.70 abcd 88.53±0.18 ab ta3 c1 40 3.40±0.28 ab 9.00±0.00 abc 6.00±1.41 abc 88.82±2.26 ab c2 60 2.70±0.00 abc 8.50±3.53 abcd 3.50±0.00 bcde 88.28±0.73 ab adv. hort. sci., 2024 38(4): 351­362 360 height, and the number of leaves and roots at both nacl concentrations (c1 and c2)(table 6). specifically, with the increase in nacl concentration a reduction was experienced in growth, where at c2, ta2 achieved the highest %pwc at approximately 88.53%, surpassing ta3 (88.28%) and ta1 (87.06%), shootlets height of 3.2 cm with an average number of 8.5 leaves and 4.5 roots. as the temperature dropped to 4°c , ta2 and ta3 showed a stabilized effect to the increase in nacl concentrations (table 7). notably, ta2 continued to have the maximum numbers of leaves and roots as well as the highest shootlets height of 2.03 cm at c2. on the other hand, ta1 reported the greatest pwc% at c2 (87.80%), followed by ta2 (87.05%) and ta3 (85.07%), the highest shootlets height (1.6 cm), number of leaves (5) and roots (3), when subjected to a temperature of 38°c (table 8). the exposure to salinity stress resulted in reductions in shootlets height, the number of leaves and roots, and the percentage of plant water content (% pwc). these reductions could be correlated to the modifications induced in terms of balance, water status, mineral nutrition as well as efficiency of photosynthesis (abdelsalam et al., 2021). these findings also align with prior research, indicating that spunta exhibited growth variations with the escalating nacl concentrations (40 to 80 to 120 mm), and the growth of various potato cultivars was affected, showing a decrease in both shoot and root length (khenifi et al . , 2011). notably, ta2 demonstrated resilience to salinity stress at both nacl concentrations at 22°c. under low­temperature conditions, ta3 exhibited the highest water content preservation at the elevated salt concentration, while ta2 displayed notable tolerance in terms of shootlets’ height and the number of leaves and roots compared to other accessions. nevertheless, at higher temperature and salt concentrations, ta1 maintained the water content whereas ta2 displayed highest values in leaves, roots, and height. these results demonstrate the tolerance displayed by ta2 table 8 ­ effect of in vitro salinity stress on shootlets height, number of leaves, number of roots and plant water content 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at high temperature (38±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). table 7 ­ effect of in vitro salinity stress on shootlets’ height, number of leaves, number of roots and plant water content of 3 potato accessions (ta1: tal amara1, ta2: tal amara2 and ta3: tal amara3) at low temperature (4±2°c) data are expressed as the mean of the determinations ± s.d. means followed by the same letter within a column are not significantly different (p<0.05). accession 4°c concentration of nacl (mm) shootlets height (cm) number of leaves number of roots pwc% ta1 c1 40 1.60±0.14 de 1.00±0.00 e 0.00±0.00 e 90.92±0.16 a c2 60 1.50±0.14 de 1.00±0.00 e 0.00±0.00 e 87.94±0.51 ab ta2 c1 40 2.35±0.21 cd 2.50±0.70 cde 2.00±0.00 cde 89.30±0.85 ab c2 60 2.03±0.05 cde 2.00±0.00 cde 1.33±1.15 de 88.53±1.48 ab ta3 c1 40 1.55±0.07 de 1.50±0.70 de 1.50±0.70 de 89.09±0.36 ab c2 60 1.50±0.49 de 1.33±0.70 de 1.00±0.70 e 88.59±0.24 ab accession 38°c concentration of nacl mm shootlets height (cm) number of leaves number of roots pwc% ta1 c1 40 2.45±0.35 bcd 7.50±0.70 abcde 3.50±0.70 bcde 88.75±1.46 ab c2 60 1.30±0.07 e 3.00±0.70 cde 2.00±0.00 cde 87.80±0.35 ab ta2 c1 40 2.20±0.00 cde 7.50±0.70 abcde 2.00±0.00 cde 89.01±0.51 ab c2 60 1.60±0.14 de 5.00±0.00 abcde 3.00±1.41 bcde 87.05±1.82 ab ta3 c1 40 1.65±0.07 de 1.50±0.70 de 2.00±1.41 cde 86.48±2.03 ab c2 60 1.35±0.63 e 3.50±0.70 bcde 2.00±0.00 cde 85.07±1.80 b elbitar et al. ‐ in vitro propagation and stress tolerance in potato 361 when subjected to single salinity stress or combination of salinity­heat stresses. 4. conclusions in conclusion, medium devoid of hormones (ms0) was an optimal medium for initiation of potato. ms1 demonstrated efficacy in achieving substantial multiplication rates. importantly, ta1 showed the best tolerance to high and low temperature treatments. ta2 exhibited tolerance to low drought stress (low concentration of mannitol), while the results of ta1 and ta3 indicated more stability in their tolerance at different concentrations of mannitol. ta2 also showed remarkable resilience under salinity and combined salinity­temperature stresses followed by ta3. these results highlight that ta1 accession is more relevant during temperature stress with no humidity stress, while during low drought stress ta2 performs the best and ta3 and ta1 show constant response when subjected to increasing drought stress. moreover, ta2 is well suited in conditions of salinity and temperature stresses. this study emphasizes the importance of selecting resilient potato accessions to govern sustainable seed production, focusing on the interrelations that exist between abiotic stresses and the growth factors of potato, and underscores the importance of ongoing research to integrate laboratory findings with practical field assessments. acknowledgements this study was conducted at the tissue culture unit, department of plant biotechnology and department of plant 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induced with different osmotic agents. ­ studia universitatis “vasile goldis” arad. seria stiintele vietii (life sciences series), 31(2): 78­84. verner d., ashwill m., christensen j., mcdonnell r., redwood j., jomaa i., saade m., massad r., chehade a., bitar a., treguer d., 2018 ­ droughts and agriculture in lebanon: causes, consequences, and risk management. ­ world bank, pp. 26. xhulaj d.b., gixhari b., 2018 ­ in vitro micropropagation of potato (solanum tuberosum l.) cultivars. ­ agric. forestry, 64(4): 105­112. zaki h.e.m., radwan k.s.a., 2022 ­ response of potato (solanum tuberosum l.) cultivars to drought stress under in vitro and field conditions. ­ chem. biol. technol. agric., 9(1). https://chembioagro.springeropen.com/articles/10.1186/s40538-021-00266-z#auth-haitham_e__m_-zaki-aff1-aff2 https://chembioagro.springeropen.com/articles/10.1186/s40538-021-00266-z#auth-khlode_s__a_-radwan-aff3 untitled 104 updated knowledge of the vine rootstocks m. bornice, g. ferroni, g. scalabrelli department of agriculture, food and environment, university of pisa, via del borghetto, 80, 56124 pisa, marcello.bornice@for.unipi.it for some time, the use of rootstocks in viticulture is no longer considered only as a means of agronomic method to avoid the phylloxera damages (daktulosphaira vitifoliae, fitch, 1856), but as a tool to be able to influence the physiology of the cultivars and to adapt the vine’s behavior to the different soil conditions, the climatic characteristics and the cultivation techniques (ferroni and scalabrelli, 1995; intrieri et al., 1999; di collalto et al., 2001; scalabrelli et al., 2001). in particular, it is increased the need to use the rootstock as a means to control the physiological processes of the plant, being the root activity closely related to that of the canopy, by exchanging signals and metabolites. the selection of the vine rootstocks started at the end of 1800, originated the main part of the rootstocks currently used in viticulture, using v. riparia and v. berlandieri. subsequently, through the use of other genotypes were obtained new hybrids even more complex. currently, in italy there are 39 varieties of vine rootstock recognized on the national register of grapevine varieties (agg. dm 23/03/2012, rev. 24/07/2012, http://catalogoviti.politicheagricole.it/catalogo. php) but are essentially five, those used on large surfaces: kober 5bb, so4, 140 ru, 1103 p, 110 r (bavaresco, 1998). in the past, for the diffusion has had an important role the need for nurserymen, who tended to favor rootstocks with excellent performance in propagation. while today there is a greater interest to satisfy the needs of viticulture’s techniques also as a consequence of the changed cultivations’ conditions. in fact, we need of new rootstocks able to adapt to environmental stresses caused by climate change in progress (mainly droughts and changes in the distribution of rainfall) and/or linked to new growing environments which have restricting factors for the vines. moreover, the need to obtain specific qualitative characteristics (polyphenols content, grapes’ acidity, etc..), suggest to deepen even more the aspects that can influence the wine quality. at the same time, it is necessary to reduce the period of selection (a new rootstock from crossing takes between 20 to 25 years), which through the use of molecular markers could also be reduced by half. in this paper we provided an overview about the behavior of some rootstocks tested in tuscany during the last years. in particular, the survey covered the vigor, the productivity, and the characteristics of grapes at maturity, including the phenolic content. the rootstocks considered were: 420a, kober 5bb, so4, 161-49, 140 ru, 1103p, 110r, 779p, 775p, 3309c, 101.14, fercal, gravesac and 41b (scalabrelli et al., 2003). references bavaresco l., 1998 scelta del portinnesto in viticoltura. l’informatore agrario, 38: 53-59. di collalto g., scalabrelli g., ferroni g., 2001 osservazioni preliminari sull’attività vegeto-produttiva del “sangiovese” innestato su tredici portinnesti nella zona di produzione del “morellino di scansano”. in: arsia. il sangiovese, atti del simposio internazionale, firenze, 15-17 february, 2000, arsia, regione toscana, pp. 259-264. ferroni g., scalabrelli g., 1995 effect of rootstock on vegetative activity and yield in grapevine. acta horticulturae, 388: 37-42. fregoni c., 2012 il portinnesto ideale. vq in vigneto, 5: 24-26. intrieri c., silvestroni o., vespignani g., filippetti i., 1999 indagini poliennali sull’impiego dei portinnesti diversi per il vitigno lambrusco grasparossa. vignevini, 3: 49-54. scalabrelli g., ferroni g., d’onofrio c. di collalto g., venerini f., 2003 trials with ‘sangiovese’ grafted on several grapevine rootstocks in two different areas of tuscany. acta horticulturae, 617: 73-83. scalabrelli g., loreti f., pisani p.l., bandinelli r., porcinai s., 2001 comportamento bio-agronomico del “sangiovese” su diversi portainnesti nel chianti classico. in: arsia. il sangiovese, atti del simposio internazionale, firenze, 15-17 februart, 2000, arsia, regione toscana, pp. 289-294. adv. hort. sci., 2013 27(3): 104 55 1. introduction honey is a natural substance formed when the nectar and sweet deposits from plants are gathered, modified and stored in honeycomb by honey bees (azeredo et al., 2003; wei et al., 2010). however, honey needs to satisfy numerous quality and certification criteria before commercialization (devillers et al., 2004; laube et al., 2010). different methods based on parameters, such as nutritious, prophylactic properties, pollen and unique flavors analyses were applied to specify the quality of honeys. although these methods have many advantages, they are not recommended for the fast routine procedure because their applications required for highly specialized personnel; furthermore, they are laborious and time-consuming (chudzinska and baralkiewicz, 2010; wei et al., 2010). several researchers found the physicochemical analysis method as a most prevalent tool that could be used for detecting the origin of honey (adebiyi et al., 2004; felsner et al., 2004; serrano et al., 2004; corbella and cozzolino, 2006; cantarelli et al., 2008). for instance, lachman et al. (2007) classified czech republic honey samples by combining between the mineral content and the electrolytic conuctivity analyses. the elemental content of honeys is closely related to the soil and vegetation in the area where the raw material for honey was collected (caroli et al., 1999; bilandžić et al., 2012). for instance, tuzen et al. (2007) determined the levels of several trace elements in honey from different botanical origins in turkey and established a correlation between the content of trace elements and the botanical and geographical origin of honey. pisani et al. (2008) showed the influence of botanical origin on the chemical composition of honey through analysis of various elements in 51 italian honey samples. likewise, grembecka and szefer (2012), using flame atomic absorption spectrometry, estimated honey quality from different locales in poland and europe in light of their mineral composition. syria has various flora-rich regions that have been considered suitable for apiculture. unfortunately, data dealing with element concentrations in syrian honeys has been ignored. thus, the present work focuses on determining several elements in different types of honey from different natural and artificial sources using a dry ashing method for x-ray fluorescence (xrf) analysis. in addition, cluster analysis (ca) is also applied in the present study to group the analyzed samples with regard to their botanical origin. the ability of ca to discriminate between natural honeys and those produced from bees fed with sugar was also studied. 2. materials and methods honey samples a set of 24 syrian natural honey samples were analyzed. in addition to the local samples, three jujube honey samples (200 g each) imported from india (two samples) trace and minor elements in bee honeys produced in syria a. khuder* (1), m. ahmad*, r. hasan *, g. saour** * department of chemistry, atomic energy commission of syria, p.o. box 6091, damascus, syria. ** department of biotechnology, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: cluster analysis, honey, minerals, x-ray fluorescence. abstract: eleven minerals (k, ca, ti, cr, mn, fe, ni, cu, zn, rb, and sr) in 31 honey samples, including 24 syrian, three imported and four honeys produced from bees fed with sugar were quantified using a dry ashing method for x-ray fluorescence analysis. the search for natural groups in the honey samples was carried out by cluster analysis, using complete linkage and euclidean distance. the syrian and imported samples clustered into three honey groups: 1) poor in element concentrations (citrus honey); 2) rich in minor and trace element concentrations (wild plants and jujube honeys); and 3) moderate mineral concentrations (multiflora, eucalyptus, crataegus, and sunflower honeys). results are discussed in terms of the mineral concentrations in syrian honeys and in comparison with international values. adv. hort. sci., 2013 27(1-2): 55-60 (1) corresponding author: scientific@aec.org.sy. received for publication 5 june 2012 accepted for publication 28 march 2013 56 and pakistan (one sample) were used for comparison purposes. the syrian honey samples (500 g each) were collected directly from sedentary beehives in different parts of syria during the late spring and early summer months. all samples were collected in clean, closed glass jars and immediately transferred to the laboratory. the samples were unpasteurized, stored in glass bottles and kept at 4-5°c in the dark until analysis. the syrian honey samples under study belonged to six representative honey types: citrus (c, n=6), multiflora (m, n=6), eucalyptus (eu, n=5), crataegus (crat, n=3), sunflower (sun, n=2) and wild plants (w, n=2). the botanical origin of some honey samples was confirmed by pollen analysis, according to louveaux et al. (1978). additionally, two types of artificial honey samples based on honeys produced from bees fed with sugar were collected after feeding a sugar solution to bees in one apiary (one beehive). the two sugar solutions were prepared as follows: the first solution (his) was prepared by mixing sugar and water on a 1:1 basis. a total of 1.5 kg commercial sugar was completely dissolved in 1.5 l hotultrapure water (18.2 mωcm specific resistivity) using an electrical heating plate at 60°c. the obtained solution was cooled at room temperature. then, the following chemical salts were dissolved in the sugar solution: (0.7259 g) fecl 3 .6h 2 o, (0.0372 g) ni(no 3 ) 2 .6h 2 o, (0.0424 g) cucl 2 .2h 2 o, (0.1563 g) zncl 2 ; the solution was mixed with a glass rod. the final volume of the obtained sugar solution was 2480 ml. the second sugar solution (hsb) was prepared in the same way but no chemical salts were added. for statistical analysis, two indipendent his and hsb sugar solutions were prepared. feeding to bees of the sugar solutions was carried out at seven-day intervals. reagents and solutions all aqueous solutions and dilutions were prepared with ultrapure water obtained from a water purification system (new human power ii, south korea) with 18.3 mωcm specific resistivity. the solutions of 14 n hno 3 ‘analar’ (bdh) were used for the honey ash dissolutions. the stock standard solutions of k, ca, ti, cr, mn, fe, ni, cu, zn, rb, and sr with concentrations of 1000 μg ml−1 each were used for the preparation of the multi-element reference targets for xrf calibration. a pure cellulose powder (ag) for analysis from seelze (hannover/germany) was used as a binder for preparation of the xrf targets. analytical procedures eleven elements (k, ca, ti, cr, mn, fe, cu, ni, zn, rb, and sr,) were determined in the honey samples using a dry ashing method for xrf analysis (khuder et al., 2010). ten g of each honey sample were put in a 50-ml crucible and dried in an oven at 105°c for 72 h, covered, cooled in a desiccator and weighed. each crucible-held dried sample was subjected to ashing in an electrical furnace. the temperature was increased in three steps: 200, 300, and 550°c; where the first and second steps lasted for 20 min each, while the third step lasted for 16 h. the ash of each honey sample was weighed and kept in the desiccator. each obtained ash was dissolved in 1-ml volume of 6 n hno 3 then removed to a small 5-ml volume vial. a volume of 100 μl of suspended cellulose solution (0.120 g ml-1) was added to each dissolved ash. the obtained mixtures were thoroughly shaken using an electrical shaker (ks 125 basic, ikalabortechnik co., japan) for 5 min, then removed to xrf spectro-cups with surface area of 4.91 cm2 each, and dried under ir lamp. finally, each obtained honey target was weighed and subjected to xrf analysis using mo-secondary target for the determination of fe, ni, cu, zn, rb, and sr, and cu-secondary target for the determination of k, ca, ti, cr, and mn. instrumental measurements the xrf measurements were performed using an energy dispersive x-ray fluorescence instrument equipped with a 2 kw mo tube and a si (li) semiconductor detector (pgt co.) with an energy resolution of 140 ev at 5.9 kev. the operating conditions were differed, depending on the mode of the x-ray excitation: 7 ma and 17 kv, and 5 ma and 45 kv by using cuand mo-secondary targets, respectively. the live time was 1000 s for both of the x-ray excitation modes. the peak areas in the obtained spectra were evaluated using the axil-qxas software package (iaea, 2005). the xrf results were compared with those obtained by standardized aas method using hollow cathode lamps (rashed and soltan, 2004 ). the accuracy, precision, and limits of detection (lod) of the xrf were estimated using the method described by khuder et al. (2010). statistical analysis basic statistics were carried out using the statistica 6.0 statistical package for windows (statsoft). prior to chemometric processing, the root square of data was carried out in order to stabilize the variance. ca was used to group the analyzed honey samples with regard to their botanical origin. the euclidean distance was used to measure the similarity as clustering method single linkage. 3. results xrf analysis a typical xrf spectrum of a honey sample excited by means of cuand mo-secondary targets is shown in figure 1. the spectra confirmed the presence of k, ca, ti, cr, mn, fe, ni, cu, zn, rb, and sr in the analyzed samples. the detected elements were calibrated by constructing sensitivity curves (fig. 2) and quantified using the axilqxas program. the validity of xrf was examined by estimating the precision, accuracy, and lod parameters (table 1). the lod of k, ca, ti, cr, and mn were 0.40, 0.09, 0.06, 0.01, and 0.01 µg g-1, respectively; while those of fe, ni, cu, zn, rb, and sr were 0.050, 0.032, 0.031, 0.030, 0.009, and 0.007 µg g-1, respectively. a comparison 57 between xrf and aas methods revealed a correlation coefficient above 0.990, indicating that the xrf was reliable and suitable to determine most of the elements in honey samples. honey analysis chemical analysis data (table 2) concerning the studied honey samples differentiated two mineral groups: the most abundant and the trace elements. the first group was composed of k and ca, having concentrations of more than 10 µg g-1. the second mineral group comprised the trace elements: fe, cu, zn, rb, ti, cr, mn, ni, and sr. of these, two subgroups were noted: the trace elements fe, cu, zn, and rb with concentrations in the 1-10 µg g-1 range, and a second subgroup (ti, cr, mn, ni, and sr) with concentrations < 1.0 µg g-1. the data were clustered in order to find the similarities between analyzed honey samples (fig. 3) and also the elements (fig. 4). 4. discussion and conclusions the honey samples in the present work were subjected to an ashing process in order to increase the sensitivity of the xrf analysis, and the concentrations of the elements were much higher than the obtained lod values. table 1 determination of different elements in multi-element standard sample using cuand mo-secondary targets for xrf analysis (z) secondary target elements (means±sd) (y) a (%) (x) rsd (%)(w) cu k 9.68±0.35 -3.2 ±3.62 ca 10.25±0.25 2.5 ±2.44 ti 10.22±0.30 2.2 ±2.94 cr 9.66±0.35 -3.4 ±3.62 mn 9.72±0.33 -2.8 ±3.40 pooled (rms)(v) 2.85 mo fe 9.95±0.66 -0.5 ±6.63 ni 9.50±0.55 -5 ±5.79 cu 10.44±0.82 4.4 ±7.85 zn 10.42±0.52 4.2 ±4.99 rb 9.92±0.11 -0.8 ±1.11 sr 9.95±0.08 -0.5 ±0.80 pooled (rms)d   3.24   (z) the ‘dark matrix’ entered for running qxas-axil program with cu-secondary target mode was c (5%) and h by difference; while, that for mo-secondary target was si (1%) and o by difference. (y) xrf results (µg) were obtained by measuring the multi-element standard three times; sd is the standard deviation. (x) a is the accuracy calculated by the difference between the obtained and the used amounts (absolute amount is 10 µg). (w) rsd is the relative standard deviation; rsd=(sd x mean ˉ¹) x 100. (v) is the root mean square of elemental accuracy. fig. 1 typical spectra of a honey sample excited by x-ray mo tube with (a) cu-, and (b) mo-secondary targets. element atomic no cu target mo target k 19 533000 c 20 917000 252000ca 20 917000 252000 ti 22 1700000ti 22 1700000 cr 24 3260000cr 24 3260000 mn 25 4190000mn 25 4190000 fe 26 6560000 1020000 7,00e+06ni 28 1390000 6 00e+06 7,00e+06 cu 29 1540000 z 30 1760000 6,00e+06 7,00e+06 zn 30 1760000 rb 37 3360000 5,00e+06 6,00e+06 7,00e+06 rb 37 3360000 sr 38 3730000 4 00e+06 5,00e+06 6,00e+06 7,00e+06 v i t y sr 38 3730000 4,00e+06 5,00e+06 6,00e+06 7,00e+06 s i t i v i t y 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 s e n s i t i v i t y cu target mo target 1 00e 06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 s e n s i t i v i t y cu target mo target 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 s e n s i t i v i t y cu target mo target 0,00e+00 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 s e n s i t i v i t y cu target mo target 0,00e+00 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 15 20 25 30 35 40 s e n s i t i v i t y cu target mo target 0,00e+00 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 15 20 25 30 35 40 s e n s i t i v i t y atomic number cu target mo target 0,00e+00 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 15 20 25 30 35 40 s e n s i t i v i t y atomic number cu target mo target 0,00e+00 1,00e+06 2,00e+06 3,00e+06 4,00e+06 5,00e+06 6,00e+06 7,00e+06 15 20 25 30 35 40 s e n s i t i v i t y atomic number cu target mo target fig.2 . calibration sensitivity curves obtained by x-ray excitation of elements i c d m dusing cuand mosecondary targets. fig. 2 calibration sensitivity curves obtained by x-ray excitation of elements using cuand mosecondary targets. 58 table 2 element concentrations in syrian honeys from different botanical origins determined by using x-ray fluorescence analysis botanical origins element concentrations (µg.g-1) k ca ti cr mn fe cu ni zn rb sr m mean 138 76.1 0.212 0.029 1.04 5.87 2.34 0.23 1.21 1.03 0.63 min. 56.2 44.6 0.102 0.017 0.36 4.0 0.83 0.08 0.21 0.44 0.41 max. 183 118 0.285 0.036 1.69 9.42 4.53 0.39 3.82 2.2 1.03 eu mean 121 92.6 0.292 0.016 1.74 8.35 3.82 0.18 2.44 1.05 0.64 min. 66.7 50.6 0.145 0.011 0.36 5.72 1.59 0.17 0.53 0.43 0.47 max. 228 127 0.401 0.024 3.16 12.4 6.09 0.20 7.16 1.78 0.79 c mean 40.3 45.5 0.085 0.026 0.50 1.74 1.14 0.13 2.42 0.32 0.31 min. 5.7 7.3 0.052 0.011 0.15 1.04 0.62 0.11 1.01 0.07 0.14 max. 84.6 78.5 0.099 0.054 1.06 2.43 1.59 0.14 4.30 0.94 0.70 crat mean 125 43.3 0.103 0.028 0.90 7.57 2.95 0.20 3.50 1.57 0.48 min. 47.5 39.1 0.071 0.014 0.46 3.83 1.66 0.15 1.41 0.26 0.44 max. 179 46.1 0.125 0.055 1.34 13.1 4.39 0.25 7.33 2.53 0.50 sun mean 107 34.4 0.142 0.024 0.32 4.83 3.10 0.162 3.83 0.81 0.65 min. 98.3 33.6 0.115 0.019 0.2 4.68 2.67 0.135 3.75 0.65 0.32 max. 115 35.1 0.168 0.029 0.42 4.98 3.53 0.188 3.91 0.98 0.99 w mean 198 50.3 0.30 0.029 2.54 17.0 3.4 0.265 2.88 4.52 0.38 min. 187 49.1 0.18 0.024 1.81 14.2 1.06 0.109 1.38 1.57 0.36 max. 208 51.4 0.42 0.034 3.26 19.7 5.73 0.420 4.37 7.47 0.41 j mean 250 58.5 0.17 <0.01 3.37 5.40 1.46 0.083 0.65 3.97 0.49 min. 219 38.7 0.12 0.60 4.98 0.83 0.081 0.27 2.69 0.13 max. 280 78.3 0.22 6.13 5.82 2.08 0.084 1.03 5.24 0.85 hsb mean 4.1 16.1 0.05 <0.01 0.11 1.35 1.34 0.069 3.19 0.08 0.09 min. 3.9 15.3 0.03 0.10 1.28 1.27 0.064 3.03 0.07 0.08 max. 4.3 16.9 0.07 0.20 14.2 1.41 0.074 3.35 0.08 0.10 his mean 5.1 17.0 0.05 <0.01 0.09 22.0 3.29 1.36 21.6 0.05 0.07 min. 4.8 16.1 0.03 0.08 20.9 3.12 1.28 20.5 0.04 0.06   max. 5.4 17.9 0.07 0.12 23.1 3.46 1.44 22.7 0.05 0.07 m, eu, c, crat, sun, w, j, correspond to multiflora, eucalyptus, citrus, crataegus, sunflower, wild plants and jujube, honeys respectively. hsb and his were honeys produced from bees fed with sugar alone and sugar enriched with salts, respectively.fig. 3 dendrogram of cluster analysis of elements in different types of honeys (root square of data was treated by the linkage method with euclidean distance as measure of similarity) his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce data was treated by the linkage method with euclidean distance as measure of similarity). his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce his and hsb (sugar-feed honeys), c (citrus), j (jujube) w (wild plants), sun (sunflower), crat (crataegus) eu (eucalyptus ) m (multiflora) his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce crat (crataegus), eu (eucalyptus ), m (multiflora). his hsb c j w sun crat eu m 30 60 90 li nk ag e di st an ce fig. 3 dendrogram of cluster analysis of elements in different types of honeys (root square of data was treated by the linkage method with euclidean distance as measure of similarity). his and hsb= honeys produced from bees fed with sgar enriched with salts and sugar alone, c= citrus, j= jujube, w= wild plants, sun= sunflower, crat= crataegus, eu= eucalyptus, m= multiflora. fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fig. 4 dendrogram of 11 analyzed elements in different types of honey samples (root square of dat treated by the linkage method with euclidean distance as measure of similarity) fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce treated by the linkage method with euclidean distance as measure of similarity). fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fe zn cu rb mn cr sr ni ti ca k 0 30 60 90 120 li nk ag e di st an ce fig. 4 dendrogram of 11 analyzed elements in different types of honey samples (root square of data was treated by the linkage method with euclidean distance as measure of similarity). 59 based on our data the minerals in syrian natural, artificial, and imported honeys fell into two groups: the first group was composed of k and ca, while the other included the trace elements fe, cu, zn, rb, ti, cr, mn, ni, and sr. potassium represented the most abundant element in the syrian honey samples with a mean concentration of 107 µg g-1. this finding coincides with most other authors who consider this element to be the most quantitatively important in honey (terrab et al., 2004; fernandez-torres et al., 2005; nozal nalda et al., 2005; pisani et al., 2008). the highest and lowest k concentrations were found in jujube (250 µg g-1) and citrus (40.3 µg g-1) honeys, respectively; the mean concentrations of k in syrian honeys were very similar to those in honeys from brazil (sodré et al., 2007) and less than values obtained for turkish honeys (cantarelli et al., 2008). calcium in syrian honeys was the second most abundant element with a mean concentration of 61.9 µg g-1; the values found for this element in syrian honeys were similar to those of jujube honeys imported from pakistan and india, as well as for honeys from other countries (cantarelli et al., 2008; chudzinska and baralkiewicz, 2010). the mean concentration of fe and cu in syrian honeys was estimated to be 6.33 and 2.49 µg g-1, respectively. our value for fe is very similar to that obtained by cantarelli et al. (2008) for turkish honeys, but higher than for honey from argentina, brazil and switzerland (bogdanov et al., 2007; sodré et al., 2007; cantarelli et al., 2008). it is worth mentioning that the concentrations of cu in syrian honeys were much higher than those for honeys from poland, switzerland, the czech republic, brazil and argentina (bogdanov et al., 2007; lachman et al., 2007; sodré et al., 2007; cantarelli et al., 2008; chudzinska and baralkiewicz, 2010). the mean concentration of zn and rb was 2.27 and 1.50 µg g-1, respectively; the value for the former was comparable to those obtained for the imported honeys (1.89 μg g-1), as well as for honeys from poland, argentina, spain, italy and turkey (cantarelli et al., 2008; chudzinska and baralkiewicz, 2010). the mean concentration of rb in brazilian honey was much lower than that obtained in the present work (sodré et al., 2007). latorre et al. (1999) reported a mean value of 1.5 µg.g-1 in spain, which is similar to the mean rb concentration found in syrian honeys. titanium was found in the honey samples with a mean value of 0.184 µg g-1, and cr was identified in 20 samples (87% of the total) with a mean concentration of 0.025 µg g-1. sodré et al. (2007) found ti in brazilian honey samples (mean value 0.112 µg g-1) while the mean concentrations of cr in different honeys from switzerland, chile, and brazil were 0.005, 0.070 and 0.038 µg g-1, respectively (fredes and montenegro, 2006; bogdanov et al., 2007; sodré et al., 2007). manganese and ni were found in 21 and 14 honey samples, respectively, with mean concentrations of 1.29 and 0.204 µg g-1. the mean concentrations of mn in honeys from argentina and the czech republic varied from 0.33 to 2.87 µg g-1 (lachman et al., 2007; cantarelli et al., 2008), and a mean value of 1.0 µg g-1 was found in turkish honey (tuzen et al., 2007). concentrations of ni in syrian honeys were comparable to those obtained for honeys from chile, the czech republic and switzerland (fredes and montenegro, 2006; bogdanov et al., 2007; lachman et al., 2007). strontium was verified in all samples with a mean concentration of 0.518 µg g-1. our data showed that the highest sr concentration (1.03 µg g-1) came from a multiflora honey obtained from a beehive placed near highways. results similar to ours have been recorded previously: fredes and montenegro, (2006) found that the highest concentrations of sr in chilean honeys were noted in honeys harvested from beehives close to roads and highways. cluster analysis was applied to the data of the 11 elements in the nine types of studied honeys. at a similarity level of 60%, the natural honey samples were grouped into three clusters (fig. 3). the first cluster contained only the citrus honey, the second jujube and wild plant honeys, and the third the remaining honey types (sunflower, crataegus, eucalyptus, and multiflora). at the same similarity level, the two artificial honey samples (his and hsb) were well discriminated in two clusters. the hierarchical dendrogram (fig. 4) discriminated between the elements according to their concentrations. the elements zn, fe, rb, cu, mn, ni, sr, cr, and ti represented a group of elements with concentrations ≤10 µg g-1; while k and ca represented a group of elements with concentrations ≥10 μg g-1. the hierarchical dendrogram shown in figure 4 could suggest a potential relationship between the samples’ origin and the clusters of particular elements, which reflects the chemical composition of the botanical origin of honey. in conclusion, cluster analysis grouped the natural honeys into different clusters with regard to their botanical origin. citrus honeys, which were poor in element concentrations, formed an individual group while wild plants and jujube honeys formed the second group, which were rich in the determined minor and trace elements. samples of mutiflora, eucalyptus, crataegus, and sunflower honeys formed the third natural group with moderate elemental concentrations. ca technique also revealed a very good discrimination between natural honeys and those produced from bees fed with sugar. acknowledgements we would like to thank the general director of aecs, prof. i. othman, and the 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469-479. untitled 95 system application and the availability of vine germplasm g. tempesta consorzio italiano vivaisti viticoli ampelos, via tebano, 45 faenza (ra) 48018. info@ampelositalia.com gftempesta@vivaioenotria.it. viticulture systems are based, obviously, on the vine, man, his history, his knowledge and the environment, which combine to form the various “terroir” or denomination of origin (do). grapevine, by its nature, has a long lifespan and generational rhythms. knowledge about the area, the variety and the characteristics of clones come from everyday life and an accumulation of understanding. different vine-growing areas claim historical and synergic structures for processing and marketing systems. while they are linked to tradition, innovation is necessary to respond to new lifestyles and preferences that reward typologies that are often based on new varietal ranges and clones. this innovation, which has led to “vigneto italia”, has been favored by public projects such as feoga 1970-1975 and incentives for renovation in 2000-2006. today, 15,000 to 18,000 hectares of the estimated 600,000 hectares of total cultivation area undergo renewal or replacement annually. this translates into a nursery demand for an estimated 50-60 million rootlings that must follow the rapid changes and stimuli of the market. a striking example of this are the varieties prosecco and pinot grigio: the former has gone from two million grafts to nearly 18 million, and the latter from one million to the current 12 million. similar significant variations have taken place for low-productivity sangiovese clones or others that are utilized for the production of “commodity” wines. while modern viticulture is based on tradition, at the same time, it requires nurseries to respond rapidly to changes in a hard-to-predict market. nurseries attempt to face this challenge thanks to vast varietal and clonal offerings but to fully succeed in satisfying the variations in the market, a genetic patrimony that is ten times greater than what is currently available would be necessary. greater availability could also compensate for declassifications resulting from material that is no longer suitable in sanitary or genetic terms. however, the economic commitment necessary to sustain public research bodies in their quest for new genetic material is hard to come by and the spread of already available initial and basic material is not always optimal. adv. hort. sci., 2013 27(3): 95 179 1. introduction the utilization of light-emitting diodes (leds) is increasing because of their technological improvements and reduction in prices. because leds emit monochromatic light, studying the effects of light quality on plant growth has become important for efficient utilization of leds in horticultural production. plant factories are a promising system of horticultural production where the completely artificial environment may lead to efficient photosynthesis, year-round production unaffected by climate, farming without insecticides, and effective utilization of resources such as water (kozai, 2013). however, plant factories consume large amounts of energy for lighting and air conditioning, which may result in global warming and high fuel costs and electricity bills. using leds, which consume less electricity and have a long life, may reduce energy consumption and production costs. leds emit little thermal irradiation and can be used in close proximity with plants, making them suitable for multistage production to increase unit production (goto, 2012). leds are also expected to be useful in lighting culture, where flowering time is controlled by night-time lighting for cut flower production in greenhouses, because incandescent lamps, which have been commonly used for longday treatments, are characterized by high energy consumption and short life. chrysanthemum morifolium (hereby referred to as chrysanthemum), the most commonly used cut flower in japan, is a short-day plant; it is produced year-round by lighting culture, repressing flowering by long-day treatments. long-day cut flowers can also be produced under short-day conditions during autumn and winter by long-day treatments to promote flowering. the effects of monochromatic light quality on plant growth, morphogenesis, and metabolism need to be investigated for the development of horticultural production using leds. blue, red, and far-red lights are already known to be effective in plant morphogenesis and flowering, and molecular mechanisms underlying the regulation of flowering by each light color have been proposed in studies using the model plant arabidopsis thaliana, which is a long-day plant. far-red light or a low red/far-red ratio, which promotes flowering in arabidopsis, reportedly promotes flowering in long-day cut flowers such as eustoma grandiflorum and gypsophila paniculata (baby’s breath) (yamada et al., 2008, 2009; nishidate et al., 2012). however, studies have not focused on the effects of blue light on flowering; blue light could also be important in timing of flowering. therefore, this review describes flowering response to blue light and its molecular mechanisms in arabidopsis and horticultural plants with the aim of advancing research on the utilization of leds in horticultural production. 2. flowering response to light quality and its molecular mechanisms flowering is triggered upon the expression of flowering locus t (ft), which is a key flowering integrator, in flowering response to blue light and its molecular mechanisms in arabidopsis and horticultural plants t. shibuya, y. kanayama* school of agricultural science, tohoku university, 1-1 tsutsumidori-amamiyamachi, aoba-ku, sendai 981-8555, japan. key words: cut flower, light-emitting diode, lighting culture, plant factory, vegetable. abstract: using light-emitting diodes (leds) in plant factories and protected horticulture is expected to decrease energy costs and environmental burden. because leds emit monochromatic light, detailed knowledge of plant responses to light quality is essential for efficient and appropriate utilization of leds in horticultural production. timing of flowering is important in cut flower and fruit/vegetable production, and it is often affected by light quality. red/far-red light is well known to be effective in flowering, and there is abundant knowledge about the effects of red/far-red ratio on flowering of horticultural plants as well as of the model plant arabidopsis thaliana. however, studies have not focused on the effects of blue light on flowering. therefore, this review describes the progress in the promotion of flowering by blue light and its molecular mechanisms in arabidopsis and horticultural plants. adv. hort. sci., 2014 28(4): 179-183 (1) corresponding author: kanayama@bios.tohoku.ac.jp received for publication 17 september 2014 accepted for publication 2 october 2014 review article 180 the phloem of leaves induced by constans (co) under long-day conditions in the photoperiodic flowering pathway proposed for arabidopsis (an et al., 2004). co expression is regulated downstream of the circadian clock, and co mrna is expressed during the dark period under short-day conditions and during the light period (late afternoon) under long-day conditions (suârez-lôpez et al., 2001). because co protein translated from co mrna is degraded through ubiquitination by an e3 ubiquitin ligase, constitutive photomorphogenesis 1 (cop1), during the dark period, the expression of ft and consequent induction of flowering are not triggered under short-day conditions (jang et al. 2008; liu et al. 2008 b). in arabidopsis, flowering is promoted under long-day conditions with blue and far-red lights but not with red light (eskins, 1992). blue light may induce ft expression by stabilizing co, as described below. far-red and blue light signaling through phytochrome a and through cryptochrome 1 (cry1) and cry2, respectively, stabilize co protein by an antagonistic function to red light signaling through phytochrome b that induces co degradation (mockler et al., 1999, 2003; valverde et al., 2004). as a result, the accumulation of co protein increases during the light period (late afternoon), and the expression of ft and consequent induction of flowering are triggered under long-day conditions. ft protein expressed in leaf phloem moves to the apical meristem through sieve tubes (corbesier et al., 2007) and activates floral identity genes such as apetala1 and fruitful by interacting with flowering locus d (fd), a bzip transcription factor (abe et al., 2005; wigge et al., 2005). in addition, ft activates suppressor of overexpression of constans 1 (soc1) to promote flowering (yoo et al., 2005). 3. molecular mechanism underlying the promotion of flowering mediated by blue light signaling via fkf1 flavin-binding kelch repeat f-box 1 (fkf1), which has flavin mononucleotide as a chromophore, and gigantea (gi) mediate co transcription in the photoperiodic flowering pathway (fig. 1) (nelson et al., 2000; imaizumi et al., 2003; martin-tryon et al., 2007). they are important for the promotion of flowering because in the co promoter region, fkf1 forms a complex with gi in a blue light-dependent manner, induces the degradation of cycling dof factor 1 (cdf1) to repress co transcription, and promotes co transcription (fig. 2) (imaizumi et al., 2005; sawa et al., 2007). in detail, the f-box domain of fkf1 interacts with arabidopsis skp1-like proteins to form the e3 ubiquitin ligase complex, and this complex induces cdf1 degradation by ubiquitination (yasuhara et al., 2004; imaizumi et al., 2005; sawa et al., 2007). arabidopsis has multiple cdf family proteins, which redundantly repress co, and the degradation of another cdf family protein cdf2 is also induced by the interaction of fkf1 with gi in a blue lightdependent manner (fornara et al., 2009). in addition, fkf1, gi, and cdf1 are proposed to regulate ft transcription in the same manner as co transcription regulation (sawa and kay, 2011; song et al., 2012). furthermore, fkf1 stabilizes co by interaction of the lov domain of fkf1 with co protein, which is enhanced by blue light (song et al., 2012). thus, fkf1 transduces blue light signals to promote flowering in a complex manner. 4. molecular mechanism underlying the promotion of flowering mediated by blue light signaling via cry2 cry is a blue/uva photoreceptor in plants (cashmore et al., 1999). in arabidopsis, the expression and function of cry2 in vascular bundles regulates flowering (endo et al., 2007). cop1 controls the accumulation of gi and co proteins, that is, cop1-mediated degradation of gi and co proteins during the dark period is repressed by cry2 during the light period to promote flowering (fig. 1) (jang et al., 2008; liu et al., 2008 b; yu et al., 2008). cop1mediated degradation of co is repressed by blue lightdependent interactions between cry2, cop1, and suppressor of phya-105 1 (spa1) (zuo et al., 2011). cryptochrome-interacting basic-helixloop-helix 1 (cib1) has been isolated as an interacting factor of cry2, and its interaction with cry2 is promoted by blue light. cib1 induces ft transcription to promote cry2-dependent floral initiation (liu et al., 2008 a). cib1 is believed to bind to e-box (canntg) elements in the promoter region of ft and stimulate ft mrna expression (liu et al., 2008 a). cib family proteins, containing cib1, form heterodimers, and notably, in comparison with homodimers, some of these heterodimers have higher binding fig. 1 proposed model for the induction of ft expression by blue light through two photoreceptors (jang et al., 2008; liu et al., 2008 a, b; sawa and kay, 2011; zuo et al., 2011; song et al., 2012). fkf1 stabilizes co protein to activate ft expression in a blue light-dependent manner. cry2 activates cib1 as a transcription factor to induce ft expression and inhibits cop1, inducing the degradation of co protein during the light period in a blue light-dependent manner. *see figure 2. 181 affinity to e-box elements. this suggests the importance of the interaction between cib proteins and e-box elements in the induction of ft expression (liu et al., 2013 b). although the interaction of cry2 with cib1 plays a role in ft expression in response to blue light, the stabilization of cib1 protein may be controlled by other blue light photoreceptors such as zeitlupe (ztl) and lov kelch protein 2 (lkp2). ztl and lkp2, belonging to the lov domain of blue light receptors containing fkf1, are required for suppressing the degradation of cib1 protein by blue light (liu et al., 2013 a). 5. flowering response to blue light in long-day horticultural plants in some long-day horticultural plants, similar in behavior to arabidopsis, the promotion of flowering by blue light under long-day conditions has been reported. flowering is promoted by inducing the expression of fbp28, a petunia homolog of the flowering accelerator soc1 in petunia, an important potted and bedding plant (fukuda et al., 2011). the flowering of a popular cut flower e. grandiflorum can be promoted under short-day conditions by a night break with blue light as well as with far-red light or a low red/far-red ratio (yamada et al., 2011). the effect of light quality on flowering has also been investigated in long-day fruit/vegetable production. ever-bearing strawberry cultivars (fragaria x ananassa) are long-day plants in contrast with june-bearing strawberry cultivars, which are short-day plants. similar to arabidopsis, the flowering of ever-bearing strawberry cultivars is promoted by longday treatments with blue as well as far-red light (nishiyama and kanahama, 2009; yoshida et al., 2012). these reports indicate that the flowering of various long-day horticultural plants is promoted under long-day conditions with blue light. however, flowering is not promoted by long-day treatments with blue light, but it is promoted with far-red light in g. paniculata ‘bristol fairy,’ which is a popular cut flower frequently used in flower arrangements (hori et al., 2011; nishidate et al., 2012). in a study conducted by hori et al. (2011), flowering in arabidopsis was reportedly induced with soc1 expression by long-day treatments with far-red light, whereas it was induced with both ft and soc1 expression by long-day treatments with blue light. in contrast, although flowering in g. paniculata ‘bristol fairy’ was induced with the expression of the soc1 homolog of g. paniculata (gpsoc1) by long-day treatments with farred light, flowering was not induced because of the low expression of the ft homolog of g. paniculata (gpft) and gpsoc1 by long-day treatments with blue light. to the best of our knowledge, there has been no report of blue light receptors fkf1 and cry in these horticultural plants. further studies are needed to understand the molecular mechanisms underlying the diversity in flowering response to blue light. 6. flowering response to blue light in short-day and day-neutral horticultural plants in short-day horticultural plants, flowering is usually repressed by a night break with red light under short-day conditions; there are only a few studies on flowering response to blue light in short-day horticultural plants. in chrysanthemum, a major short-day cut flower, flowering is repressed by a 4-h night break with blue light or far-red light under short-day (12 h) conditions with blue light (higuchi et al., 2012). however, flowering of chrysanthemum is not repressed by 4-h end-of-day irradiation with blue light under 11-h photoperiods with mixed red and blue lights (jeong et al., 2014). these studies suggest that in chrysanthemum, blue light affects flowering under some limited conditions, but red light plays a major role in the repression of flowering. fig. 2 proposed model for the induction of co and ft expression by blue light through fkf1 (yasuhara et al., 2004; imaizumi et al., 2005; sawa et al., 2007; sawa and kay, 2011; song et al., 2012). cdf1 represses co and ft expression under short-day conditions. fkf1 forms a complex with gi and cdf1 on co and ft promoters in a blue light-dependent manner in the late afternoon under long-day conditions, and then induces the degradation of cdf1 protein by ubiquitination. as a result, the expression of co and ft are induced because of the absence of cdf1. 182 solanum lycopersicum (tomato), the most important fruit/vegetable, is well known as a day-neutral plant. tomato plants flower after vegetative growth regardless of photoperiods, and their flowering is believed to be unaffected by photoperiods. however, the node position of the first flower truss under a lower blue/red ratio has been reported to be lower than that under a higher blue/red ratio (nanya et al., 2012); the constitutive expression of the cry2 homolog of tomato lecry2 delays flowering (giliberto et al., 2005), suggesting that blue light represses flowering in tomato plants. it is important to investigate the tomato homologs of genes that play roles in light signaling in the photoperiodic flowering pathway because the ft homolog of tomato sft is a flowering activator, and heterozygosity for loss-of-function alleles of sft increases the yield (krieger et al., 2010). 7. concluding remarks similar to arabidopsis, blue as well as far-red light-dependent promotion of flowering in long-day horticultural plants has been reported. on the other hand, flowering was not promoted by blue light, but by far-red light in a cultivar of another long-day plant, g. paniculata. this diversity in flowering response to blue light is notable, and gaining knowledge about it is important for promoting the utilization of leds in plant factories and lighting culture. the flowering of arabidopsis is promoted by blue light in a complex manner through fkf1 and cry. there is a paucity of information on the molecular mechanisms underlying blue light signaling in the promotion of flowering in horticultural plants, although there are few studies on the relationship between flowering and expression of flowering-related genes under long-day conditions with blue 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antioxidant enzyme activities of tomato plants under drought stress a. farzane 1, h. nemati 1 (*), m. shoor 1, h. ansari 2 1 department of gardening, faculty of agriculture, ferdowsi university of mashhad, iran. 2 department of water engineering, faculty of agriculture, ferdowsi university of mashhad, iran. key words: catalase, evapotranspiration, kcl, polyphenol oxidase, superoxide dismutase. abstract: water stress negatively affects productivity in crops, while the foliar application of potassium­containing compounds may be helpful in reducing the drought effects. this study evaluated the efficacy of foliar applied potassium chloride (control ­ distilled water spray ­, 3 and 6 mm­1) on tomato plants under drought stress. three irrigation levels were maintained at 100, 75 and 50% according to evapotranspiration designated as well watered, moderate and severe drought stressed. increasing drought stress significantly reduced plant growth and yield. the foliar applied kcl produced maximum leaf area, stem diameter and length, plant yield under each drought stress conditions compa­ red to control. the minimum of growth factors were obtained by control under severe stress. highest yield per plant was also recorded for foliar applied kcl under moderate condition than other treatments. foliar applied kcl alone decreased the sod, cat and ppo in well­watered condition but kcl application on tomato plants under drought stress induced the antioxidant enzyme activi­ ties more than control well­watered treatment. 1. introduction the major limitation for plant growth and crop production in arid and semi­arid regions is soil water availability. plants that are continuously exposed to drought stress can form ros (reactive oxygen species), which leads to leaf damage (foyer et al., 2002; oerke and dehne, 2004; cakmak, 2005) and, ultimately, decreases crop yield. the decrease in soil water potential causes alteration in minerals uptake by plant roots and reduc­ tion in leaf expansion under drought stress conditions (kaminek et al., 1997; pospisilova et al., 2000). drought is becoming a serious threat to crop production worldwide resulting in 67 and 82% reduction in k uptake under mild and severe water stress conditions (baque et al., 2006). during drought stress, root growth and the rates of k+ diffusion in the soil towards the roots were (*) corresponding author: nemati@um.ac.ir citation: farzane a., nemati h., shoor m., ansari h., 2021 ­ foliar application of potassium on antioxi‐ dant enzyme activities of tomato plants under drought stress. ­ adv. hort. sci., 35(1): 3­9 copyright: © 2021 farzane a., nemati h., shoor m., ansari h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2020 accepted for publication 24 dcember 2020 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8468 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 3­9 4 both restricted, thus limiting k acquisition. the result ing lower k concentrations can further depress the plant resistance to drought stress, as well as k absorption, which ultimately leads to reduction in the fruit size, plant yield, lack in red color of tomato and fruit quality (bidari and hebsur, 2011; afzal et al., 2015), and deteriorated photo­ synthesis, enzyme activation in plants (marschner, 1995; garg et al. , 2004; afzal et al. , 2015). maintaining adequate plant k is, therefore, critical for plant drought resistance. when plants were supplied with different k+ con­ centrations and then subjected to drought stress, their stomatal conductance was more markedly reduced in normal k plants than in low k plants (wang et al., 2013). during drought stress, the sto­ mata cannot function properly in k+­deficient plants, resulting in greater water loss. drought stress did not decrease water use efficiency (wue), whereas it did increase wue by rapid stomata clo­ sing during water deficit (egil la et al. , 2005). adequate levels of k nutrition enhanced plant drou­ ght resistance, water relations, wue and plant growth under drought conditions (wang et al., 2013). besides various adaptive mechanisms; potas­ sium (k) sprayed under drought condition can improve the tolerance of crop plants to various types of abiotic stresses, and it also improved sub­ sequent growth and yield. mengel and kirkby (2001) reported that k improves physiological processes by the regulation of turgor pressure and photosynthe­ sis; translocation of cations and enzymes activation, while, cakmak (2005) also observed that plant suf­ fering from drought stress required more internal k. yield limiting effect of water deficit could be over­ come by increasing k supply (damon and rengel, 2007). in legumes, devastating effects of drought can be alleviated by rich k supply (sangakkara et al., 2000). a close relationship between k nutritional status and plant drought resistance has been demonstrated. the bottom lines of the reviewed results in this section indicate that under drought stress conditions, yield losses can be minimized by the sufficient supply of k. however, its application effect at tomato growth stages is not well understood yet. the objec­ tive of present work was to study the possible role of k applied on tomato plant under drought, in mitiga­ tion of stress in terms of physiological components and antioxidant enzyme values. 2. materials and methods this experiment was designed to observe the effect of kcl on tomato (solanum lycopersicum l.) seedlings under drought stress. seeds of tomato were sown in plastic trays and maintained in a greenhouse up to 4 real leafy stages, at department of horticulture, faculty of agriculture, ferdowsi university of mashhad, iran. the experimental design was a split plot design with three water stress plot as main plot and 3 kcl treatments (0, 3 and 6 mm­1) as subsidiary plot with 3 replications. the subsidiary plot area was 1 m2 (1×1 m) and consisted of four plants per plot. after 70 days from sowing, seedlings with uniform growth were transplanted to an experi­ mental field with a 50 cm inter­seedling spacing. according to evapotranspiration (etc = crop evapo­ transipration), the water stress was conducted on tomato plants at three levels well­watered (100% etc), moderate and severe drought stress (75 and 50% etc, respectively). according to penman­ monteith equation, the crop evapotranspiration, etc (formula 1), is calculated by multiplying the referen­ ce crop evapotranspiration, eto, by a crop coeffi­ cient, kc and eto (formula 2) is calculated by ep and kp factors. etc = kc eto [1] where etc= crop evapotranspiration [mm d­1], kc= crop coefficient [1.15 for tomato], eto= crop evapo­ transpiration [mm d­1]. eto= ep. kp [2] where kp= pan coefficient (0.77), ep0 pan evapora­ tion. the foliar spray was applied five times (during two month) to tomato plants during growth and fruit set with kcl at 0, 3 and 6 mm­1 dose. growth and plant analysis plant height and stem diameter were measured at the end of the harvesting season and presented as cm and mm respectively. the total yield of tomato fruits were measured by gram scales (g) in different harvesting times per plant. the leaf area (la) measu­ red by windias (delta­t co, england) and presented as (mm2). the integrated water­use efficiency (formula 3) is typically defined as the ratio of biomass produced (d, kg h­1) to the rate of total water irrigation (wi, m 3ha­1) and rainfall (wp, mm) during the drought stress treat­ https://en.wikipedia.org/wiki/penman%e2%80%93monteith_equation https://en.wikipedia.org/wiki/penman%e2%80%93monteith_equation https://en.wikipedia.org/wiki/biomass farzani et al. ‐ tomato antioxidant enzyme activities 5 ment. wue= d/(wp + wi) [3] assays of enzymatic and non‐enzymatic antioxidants fruits were randomly selected from each treat­ ment, at the end of the experiment. total soluble proteins were quantified by following the protocol devised by bradford (1976). for determination of enzymatic antioxidants, fruit samples were extracted in 50 mm phosphate buffer (ph 7.8). the extract was centrifuged at 15,000 rpm 4°c and the supernatant was used for further assay of catalase (cat) chance and maehly, 1955 and poly phenol oxidase (ppo) (siriphanich and kader, 1985) and super oxide dismu­ tase (sod) activities (giannopolitis and ries, 1977). tomato juice was squeezed from the fresh tomatoes onto a digital refractometer (pr­100, atago co. ltd., tokyo, japan) to measure total soluble solids (tss) and the results were expressed in brix according to aoac method 932.12 (2005). for measured the proli­ ne content, leaves were randomly selected and expe­ rimented according to bates et al. (1973) method. statistical analysis effects of water stress treatments; kcl and corre­ sponding interactions were determined by analysis of variance according to the general linear model proce­ dure of sas (version 8.2; sas institute, cary, n.c.). means were compared using least significant difference (lsd, p≤0.05) according to method descri­ bed by tukey hsd. analysis showed a significant inte­ raction between kcl and watering treatment for the some measured parameters. the graphs draw by using excels 2010 software. 3. results increasing drought stress (ds) levels significantly reduced plant growth and yield, but foliar applica­ tions of kcl improved the harmful effects of drought stress. comparison of means indicate that leaf area (la) was gradually decreased with increasing drought stress (table 1). the minimum leaf area was obtained under extreme drought conditions (50% etc). water stress decreased la but exogenous application of kcl ameliorated the negative effects of water stress significantly than water spray only (fig. 1a). however, highest la in each treatment was recorded for foliar applied kcl than control under well­watered, mode­ rate and severe drought stress. a minimum la was recorded under severe drought stress, especially con­ trol × 50% etc treatment (fig. 1a). the increasing water stress decreased stem length (fig. 1b). a maximum stem length was observed under well­watered conditions by exogenous applica­ tion of 6 mm­1kcl while in moderate and severe drought, foliar applied kcl had no significant effects. minimum stem length was noted under extreme water stress without foliar kcl application. stem dia­ meter decrease in response to ds (table 1). foliar kcl application helps to plant tolerance with increasing stem diameter under ds condition. data showed (fig. 1c) the increasing in kcl doses (0 up to 6 mm­1) lead to increasing in stem diameter in all of treatments. the tomato yield displayed a significant reduction in response to the increasing levels of water deficit treatments. for example, under effect of 50% etc condition, the yield decreased by 21% (table 1) com­ pared to control. in the other hand, plants had more vegetative growth and less yield in well­watered con­ dition (100% etc) than moderate ds (75% etc). in non­ds condition, foliar spray of kcl showed similar fruit yield under well­watered, moderate and severe drought conditions. but foliar kcl application had effective enhancement on plant yield under drought stress (fig. 1d). tomatoes irrigated with 50% etc without kcl foliar application also produced fruits with significantly higher juice brix value than other table 1 ­ effects of different drought stress levels on tomato plants means in each column followed by similar letters are not significantly different at 0.05 probability level, using lsd (least significant difference) test. drought la (mm2) st. length (cm) st. diameter (mm) wue yield plant ­1 brix 0 proline (µg/g.fw) protein (mg g­1 fw) sod (iu mg­1 min­1 protein) cat (iu mg­1 min­1 protein) ppo (iu mg­1 min­1 protein) control 7555.156 a 52.66 a 14.69 a 4.360 b 1225.861 a 7.00 b 6734.877 c 1.405 a 967.28 a 178.35 c 80.777 b 75% etc 5464.41 b 47.44 b 14.41 a 6.187 a 1404.72 a 7.11 b 9468.355 a 1.540 a 1240.31 a 196.63 b 142.66 b 50% etc 3294.32 c 42.66 c 11.82 b 2.462 c 963.62 b 7.722 a 8161.222 b 1.382 a 1133.79 a 207.83 ab 258.11 a adv. hort. sci., 2021 35(1): 3­9 6 treatments (fig. 1e). the results in figure 1 (f) revealed that ds on tomato had higher significantly record in water use efficiency than those normally irrigated. because of fewer yield in control treatment than 75% etc, the results affirmed that the treatment 75% etc and then control showed the highest water use efficiency than 50% etc treatments. foliar application of kcl had no significant effects of wue under non­ds condition. finally, the foliar application of kcl × 75% etc showed the highest significant record in wue in plants subjected to ds, respectively. meanwhile, the treat­ ment 6 mm­1 kcl × 75% etc recorded the highest significant wue compared to other studied treat­ ments under well­watered and sever ds. leaf proline concentration responded differently to drought and k supply. drought stress (table 1) increased proline concentration in leaves but exoge­ nous kcl decreased the proline content under none ds conditions (table 2). the tomato leaves were sprayed with kcl had more proline content under drought stress than control conditions (fig. 2 a). at 0 mm­1kcl level with no ds, the proline content in lea­ ves was 1168.4 µm/g f.wt. which increased to 13368.5 and 12111.5 µm/g f.wt. with increasing ds stress in 50% etc with 3 and 6 mm­1 kcl foliar applica­ tion, respectively. increase in kcl doses enhanced the total soluble proteins in tomato fruits (table 2). maximum fruit protein content was evident at moderate stress with 3 mm­1kcl followed by 0 mm­1kcl with minimum value under well­watered conditions (fig. 2b). foliar spray with kcl had impressive effects on protein con­ tents under well­watered and drought stress. however, the exogenous application of kcl perfor­ med better than control for fruit total soluble pro­ tein. a rise in drought stress also amplified the antioxi­ dants status of tomato fruits (fig. 2c, d, e). results showed that the enzymatic antioxidant contents in tomato fruits were sprayed by kcl, were decreased. so, the maximum sod with no significant differences were recorded for foliar applied 0 and 3 mm­1kcl under severe drought and 0 mm­1kcl under modera­ te conditions (fig. 2c). increased cat activities with no significant differences were found under medium and severe drought stress (fig. 2d). decreasing water level increased ppo contents for all treatments (fig. 2e) and the highest value of ppo was obtained under sever ds but increasing in foliar application of kcl doses from 0 to 6 (mµ­1) caused to decrease the ppo fruit content (fig. 2e). fig. 1 ­ effect of exogenous application of different kcl level on leaf area (a), stem length (b), stem diameter (c), yield (d), brix (e), and wue (f) of tomato under drought condi­ tions. (lsd, p≤0.05). table 2 ­ foliar application effects of different kcl levels on tomato plants means in each column followed by similar letters are not significantly different at 0.05 probability level, using lsd (least significant difference) test. kcl la (mm2) st. length (cm) st. diameter (mm) wue yield plant ­1 brix 0 proline (µg/g fw) protein (mg g­1 fw) sod (iu mg­1 min­1 protein) cat (iu mg­1 min­1 protein) ppo (iu mg­1 min­1 protein) control 4836.40 b 43.11 b 12.23 c 4.014 a 1168.46 a 7.777 a 8953.16 a 1.286 b 1539.26 a 230.56 a 167.44 ab 3 5197.64 b 46.55 b 13.88 b 4.426 a 1211.41 a 6.777 ab 7590.68 c 1.562 a 761.61 b 196.29 ab 204.66 a 6 6279.84 a 53.11 a 14.81 a 4.014 a 1214.32 a 7.277 b 7820.60 b 1.479 ab 1040.52 ab 155.96 b 109.44 b farzani et al. ‐ tomato antioxidant enzyme activities 7 4. discussion and conclusions increasing water stress has direct impact on crop growth and yield reduction and similar observations for reduction in growth and fruit yield of tomato were found in present study. reduction in tomato yield under restricted water availability might have been due to reduction in photosynthetic area such as leaf area and leaf number (chaves et al., 2011; khan et al., 2015). in the present study, yield, leaf area and stem length were drastically reduced due to drought effect, whilst kcl foliar application improved these characters in tomato plants. it has been reported that the foliar application of kcl improved the growth factors in agronomy crops (ahmad and jabeen, 2005; yasmeen et al., 2013). besides adaptation role, positive effect of kcl on pro­ tein contents was also pronounced under drought stress. proline is a well­known amino acid that gene­ rally accumulates when plants are exposed to envi­ ronmental stresses (kavi­kishor et al. , 2005). enhanced proline synthesis is a common response of tomato plants to drought and may determine the stress tolerance (doan and maurel, 2005; khan et al., 2015). proline is believed to acts as a signaling mole­ cule that initiates adaptation to the stress (maggio et al., 2002), acts as osmolyte for osmotic adjustment (hayat et al. , 2012), helps in stabilizing membranes/proteins and scavenges free radicals (ashraf and foolad, 2007). thus, it decreases the adverse effects of cytoplasmic acidosis and maintains proper nadp+ /nadph ratios (liang et al., 2013). in plants grown under drought conditions, proline indu­ ces the expression of drought stress responsive genes and, thus, decreases the damage due to exces­ sive na+ ions accumulation (chinnusamy et al., 2005). proline act as a compatible solute in the plants (mansour, 2000) and, generally, increases with increase in both the salinity stress and drought stress duration (kishor and sreenivasulu, 2014). thus, it is likely to observe enhanced proline synthesis with increasing drought stress duration. antioxidant enzy­ mes activities were considered as indicators of sca­ venging ros and reducing oxidative stress (dionisio­ sese and tobita, 1998; lin and kao, 2002). for exam­ ple, sod may convert superoxide radicals into h2o2 and h2o2 was further decomposed by cat and pod (redman et al., 2011). in this study, drought stress significantly increased the antioxidant enzymes acti­ vities. the results of many researchers’ studies pro­ ved that adequate external k supply significantly decreased antioxidant enzymes activities and proline in drought stressed plants might be caused by enhan­ cing plant physiological metabolism and reducing ros production and mda content (wei et al., 2013; yasmeen et al., 2013; bahrami­rad and hajiboland, 2017). in the other hand, the combination of drought stress and exogenous kcl application improved the antioxidant activities and proline content than well­ watered conditions. so, rise in drought stress with exogenous application of kcl induce tolerance in crops (yasmeen et al., 2013). a similar trend was fol­ lowed by all the treatments in case of antioxidant enzyme activities under each water stress treatment. the results of this study clearly demonstrated that water deficit at any critical crop growth stage seve­ rely affected the physiological and antioxidant and non­antioxidant parameters of tomato. foliar appli­ cation of k on a drought stressed plants at all growth stages improved the physiological performance and plant tolerance but reduced antioxidant enzyme acti­ vities. all these findings lead us to recommend that for tomato crop under drought farmers should spray the crop with 6 (mµ­1) kcl to minimize the negative effect of drought. this can have a dual benefit: fig. 2 ­ effect of exogenous application of different kcl levels on enzymatic and non­enzymatic antioxidants; proline (a), total soluble protein (b), superoxide dismutase (c), cata­ lase (d) and polyphenol oxidase (e) of tomato fruits under drought conditions. 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abstract: to investigate the effect of some pulsing and holding solutions on the quality of carnation cv. turbo cut flowers, a laboratory experiment was con­ ducted in the agricultural research center and cairo university, egypt during 2020 and 2021 seasons. in this regard distilled water, silver thiosulfate (sts) at 0.4 ppm + sucrose 10% (ps1) and agno3 at 10.0 ppm + sucrose 10% (ps2) were employed as a pulsing solution for 15 min while distilled water, sucrose 4% (hs1), boric acid (boa) at 200 ppm + sucrose 4% (hs2), 8­hydroxyquinoline sul­ fate (8­hqs) at 300 ppm + sucrose 4% (hs3), amino­oxyacetic acid (aoa) at 250 ppm + sucrose 4% (hs4), 8­hqs + aoa + sucrose 4% (hs5), boa + 8­hqs + sucrose 4% (hs6), boa + aoa + sucrose 4% (hs7), boa + 8­hqs + aoa + sucrose 4% (hs8), rosemary extract at 25% + sucrose 2% (hs9) and thyme extract at 25% + sucrose 2% (hs10) were used as holding solutions. regarding pulsing solutions, ps2 and ps1 exhibited a positive effect on all studied traits, while the mastery was to hs8 concerning the effect of holding solutions. pulsing cut car­ nations in a solution containing ps2 followed by holding in hs8 resulted in the highest values in terms of vase life, water balance, chlorophyll a, carotenoids and total sugars, while the highest water uptake and loss and chlorophyll b were obtained by pulsing in ps1 followed by holding in hs8. it is recommended to pulse carnation cv. turbo cut flowers in agno3 at 10.0 ppm + sucrose 10% solution for 15 min followed by holding in boa + 8­hqs + aoa + sucrose 4% for getting the longest vase life, enhancing water uptake and maintaining water balance. additionally, this preservative solution effectively reduces chlorophyll degradation and preserves the content of carbohydrates throughout the postharvest period. (*) corresponding author: koki_assem@yahoo.com citation: sarhan a.m.z., heikal a.a.m., saadawy f.m., noor el­deen t.m., abd elkareem k.m., 2023 ­ effect of some chemical and natural preservative solutions on vase life, water relations and some chemical composition of dianthus caryophyllus l. cut flowers. ­ adv. hort. sci., 37(3): 255­269. copyright: © 2023 sarhan a.m.z., heikal a.a.m., saadawy f.m., noor el­deen t.m., abd elkareem k.m. this is an open access, peer reviewed article publi­ shed by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 july 2022 accepted for publication 19 may 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14247 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 255­269 256 1. introduction carnation (dianthus caryophyllus l.; fam. caryophyllaceae) is native to southern europe and the mediterranean region and is a half­hardy peren­ nial flowering plant with a wide range of colours. each stem of the carnation forms a terminal flower; hence inflorescence is generally a terminal cyme. the flowering shoots can be single or multiple (stem sprays) (ponnuswami and sowmeya, 2015). moreover, the exceptional keeping qualities of carna­ tions make them an excellent choice for cut flowers. they possess remarkable longevity, the ability to withstand long­distance transportation, and an exceptional capacity to rehydrate even after pro­ longed shipping (panwar et al., 2022). there is a wide range of techniques applied to extend flower preservation, including the use of flower preservatives, inhibitors of ethylene action, growth regulators, calcium and the control of tem­ perature and flower dehydration (finger and barbosa, 2006). pulsing solutions are used basically to provide sugars (sucrose or glucose at 2­20%) and silver compounds (sts) (armitage and laushman, 2003). pulsing solutions are used on freshly harvest­ ed flowers that are in a bud stage where a short peri­ od (or pulse) in a high­sugar solution will extend the vase life or open buds. sugar is the main ingredient while sts is used to reduce ethylene sensitivity (jones, 2001). to promote water uptake, the cut flower’s stems are placed in a holding solution which contains an acidifier for hydration, a biocide of bacte­ rial control and an energy/food source, which is typi­ cally sugar. the flowers usually stay in these solutions for one to several days as they are transported to local distributors and retailers and are used for retail displays (dole and faust, 2021). a lot of chemicals are used in formulations of pulsing or/and holding solutions e.g. sts, agno3, 8­ hqs, aoa and boric acid. ebrahimzadeh et al. (2008) concluded that sts is used to inhibit harmful effects of ethylene and prolong vase life in many ornamen­ tals including carnation, agno3 inhibits ethylene syn­ thesis and action and is used as an antimicrobial agent, 8­hqs is an antimicrobial additive in preserva­ tives and is ethylene synthesis inhibitor, aoa inhibits the biosynthesis of ethylene, and boric acid used as anti­ethylene synthesis. various authors have demonstrated the beneficial effects of the aforemen­ tioned chemicals, integrated either individually or in combinations into preservative solutions, on carna­ tion cut flowers cvs. dolce vita, amstel, monte lisa, aliceo, and paola (wawrzynczak and goszczynska, 2003), cvs. kristina, aleda, master, and vienna (krishnappa and reddy, 2004), cvs. nelson, dream, and delphi (lopez et al., 2008), cv. optima (karimi et al., 2012), cv. charmant (darwish et al., 2014), cv. felice (madhuri et al., 2016), and cv. mirella (adam and eldeeb, 2021). certain natural materials e.g. plant extracts and essential oils are used instead of chemicals in cut flowers’ preservative solutions due to the harmful effects of such chemicals (particularly those based on silver compounds) on human health. a lot of studies were carried out to investigate the effect of these natural materials. in this regard, hashemabadi et al. (2021) on dianthus caryophyllus l. cv. yellow candy stated that both vase life and solution uptake were increased by using dill essential oil in the solution compared to distilled water. numerous studies have provided evidence of the impact of employing natur­ al substances in preserving solutions for carnation cut flowers. for example, investigations have been conducted on the utilization of dill, clove, and corian­ der oils on cvs. farida and madam collate (shanan et al., 2010); extracts of lupin and clove and juice of the lemon on cv. domingo (el­ashwah, 2011); essential oils of dill, geranium and caraway on cv. yellow candy (rad, 2018) and clove essential oil on cv. cinderella (eldeeb and adam, 2021). such materials were used in these reported studies either individual­ ly or in combination with other post­harvest chemi­ cals. therefore, the present study was carried out to investigate the effect of different pulsing (mainly sts, agno3 or distilled water) and holding (boric acid, 8­ hqs, aoa, rosemary and thyme water extracts) solu­ tions on vase life, water relations, pigments content and total sugars of carnation cut flowers with the possibility to get the longest vase live with the best quality. 2. materials and methods a laboratory experiment was carried out in the post­harvest lab., ornamental plants and landscape gardening res. dept., horticulture res. inst., agricultural research center, giza, egypt and ornamental horticulture dept., faculty of agriculture, cairo university, egypt during 2020 and 2021 seasons with the aim to study the effect of some pulsing and holding solutions (containing some natural extracts) on the postharvest quality of carna­ sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 257 tion cut flowers. plant materials fresh cut flowers of carnation (dianthus caryophyllus l.) cv. turbo, at the paintbrush stage with red color were obtained from a local commer­ cial greenhouse farm in giza, egypt, in the first week of january each season. flowers’ stem lengths were adjusted to 60 cm with 3.0­3.5 cm flower diameter, while fresh weight ranged from 18 to 20 g. immediately, after transferring to the laboratory under dry conditions, about 5 cm of stem bases were recut under water and 4 pairs of leaves were left on each stem and then rapidly precooled by placing them in cold water for three hours. experiment treatments pulsing solutions these precooled cut flowers were distributed in 500 ml jars (3 flowers/jar) and then equally divided into three groups, each one containing 300 ml from one of the following applied pulsing solutions: 1. distilled water 2. sts at 0.4 ppm + sucrose at 10% (ps1) 3. agno3 at 10.0 ppm + sucrose at 10% (ps2) sts (silver thiosulfate; 1agno3.4na2s2o3·5h2o) solution was prepared according to gorin et al. (1985). in this regard, both agno3 (0.079 g) and na2s2o3·5h2o (0.462) were dissolved separately in 500 ml deionized water, then agno3 solution was poured into na2s2o3·5h2o solution with continuous stirring. while the agno3 solution was prepared by dissolving 0.01 g of agno3 in deionized distilled water to prepare a 10­ppm concentration. holding solutions each group of the pulsed cut flowers were divided into 11 subgroups (3 flowers/500 ml jar containing 300 ml of different holding solutions) and kept under lab conditions (light intensity at 1000 lux supplied by fluorescent lamps, the average temperature at 18­ 20°c and relative humidity at 50­55%) as follows: 1. control (distilled water) 2. sucrose at 4% (hs1) 3. boric acid at 200 ppm (boa) + sucrose at 4% (hs2) 4. 8­hydroxyquinoline sulfate at 300 ppm (8­hqs) + sucrose at 4% (hs3) 5. amino­oxyacetic acid at 250 ppm (aoa) + sucrose at 4% (hs4) 6. 8­hqs + aoa + sucrose at 4% (hs5) 7. boa + 8­hqs + sucrose at 4% (hs6) 8. boa + aoa + sucrose at 4% (hs7) 9. boa + 8­hqs + aoa + sucrose at 4% (hs8) 10. rosemary extract at 25% (re) + sucrose at 2% (hs9) 11. thyme extract at 25% (te) + sucrose at 2% (hs10) extracts preparation both thyme (thymus vulgaris l.) and rosemary (rosmarinus officinalis l.) extracts were prepared by water extraction. in this concern, 250 g of the dry herb was extracted in 250 ml of distilled water with boiling at 100°c and stirring for 30 minutes. after that, the solution was filtered using filter paper, and the remaining solution was completed to 1000 ml with distilled water. afterwards, 250 ml of each solu­ tion was poured into 1000 ml of distilled water to get a 25% concentration. experiment design this experiment was laid out as a complete ran­ domized design (crd) in a factorial experiment. factor (a) was represented by 3 levels of pulsing solutions, while factor (b) was represented by 11 lev­ els of holding solutions. thus, a total of 33 treat­ ments were utilized. each treatment consisted of 3 replicates, with each replicate containing 3 jars. within each jar, there were 3 flowers, resulting in a total of 27 flowers per treatment. data collection vase life vase life was determined as the number of days to the beginning of flowers wilting. water relations the total water uptake (g/flower) was calculated by subtracting the weight of water at the end of the experiment from the initial weight. the total water loss (g/flower) was determined by measuring the difference between the weight of jars with spikes and solution at the beginning of the experiment and the weight of jars with spikes and solution at the end of the experiment. the total water balance (g/flower) was obtained by subtracting the total water loss from the total water uptake. determination of pigments and sugars at the end of flower longevity, pigment and sugar contents were measured on the attached leaves. contents of chlorophylls a, b and carotenoids were determined colourimetrically in fresh leaves according to the method described by wellburn and lichtenthaler (1984). total sugars in the dry leaves were determined adv. hort. sci., 2023 37(3): 255­269 258 colourimetrically according to dubois et al. (1956). statistical analysis the obtained data were statistically analyzed as a factorial experiment using mstat computer program (mstat development team, 1989). duncan’s multi­ ple range test (duncan, 1955) was used to compare the means between various treatments. 3. results and discussion vase life the data reported in table 1 clearly show that ps2 significantly increased the vase life of carnation cut flowers to 18.4 and 18.7 days in the first and second seasons, respectively. conversely, pulsing the cut flowers in distilled water solution recorded only 16.3 and 16.5 days in both seasons, respectively. preserved carnation cut flowers in different hold­ ing solutions showed a significant influence on vase life (days) as presented in table 1. the highest num­ ber of days was recorded by holding in hs8 (24.3 and 24.6 days) in the first and second seasons, respec­ tively. the lowest values were obtained by holding the cut flowers in hs9 (12.6 and 12.9 days), hs10 (12.7 and 13.0 days) and distilled water (12.8 and 12.6 days) in both seasons, respectively. comparing to the control treatment (dw), it was evident that all combined treatments involving both pulsing and holding solutions led to a substantial improvement in the vase life of cut carnation flowers. the key technique employed to achieve this outcome involved pulsing the flowers in a ps2, along with table 1 ­ effect of pulsing and holding solutions and their interaction on vase life (days) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 13.67 o­q 14.18 op 10.67 t 12.84 f hs1 15.69 l­n 15.98 lm 14.69 no 15.45 e hs2 18.04 ij 18.02 ij 16.71 kl 17.59 d hs3 21.44 de 23.11 bc 17.73 jk 20.76 b hs4 19.09 hi 22.13 c­e 16.38 lm 19.20 c hs5 17.60 jk 18.00 ij 15.60 mn 17.07 d hs6 21.87 de 21.24 ef 20.33 fg 21.15 b hs7 20.11 gh 19.36 gh 19.42 gh 19.63 c hs8 23.98 b 26.67 a 22.40 cd 24.35 a hs9 12.67 qr 13.29 pq 11.84 rs 12.60 f hs10 13.09 pq 11.04 st 14.00 op 12.71 f mean (a) 17.93 b 18.46 a 16.34 c second season (2021) control (dw) 13.00 p­r 14.23 n­p 10.83 s 12.69 f hs1 15.45 l­n 16.00 k­m 14.90 m­o 15.45 e hs2 18.23 g­i 18.10 g­i 17.13 i­k 17.82 d hs3 21.68 de 23.63 bc 17.80 h­j 21.04 b hs4 19.51 fg 22.20 c­e 16.50 j­l 19.40 c hs5 17.93 h­j 18.87 gh 16.33 k­m 17.71 d hs6 22.30 cd 21.50 de 20.80 ef 21.53 b hs7 20.80 ef 19.53 fg 19.50 fg 19.94 c hs8 24.57 b 27.00 a 22.23 c­e 24.60 a hs9 13.43 p 13.60 op 11.83 q­s 12.96 f hs10 13.23 pq 11.63 rs 14.33 n­p 13.07 f mean (a) 18.19 b 18.75 a 16.56 c sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 259 holding them in hs8. this combination resulted in vase lives of 26.6 and 27.0 days in both seasons, respectively. the lowest number of days of vase life in both seasons was recorded by pulsing and holding in dis­ tilled water only (10.6 and 10.8 days), pulsing in ps2 + holding in hs10 solution (11.0 and 11.6 days) and pulsing in distilled water and holding in hs9 solutions (11.8 and 11.8 days), respectively. the aforementioned findings were consistent with the results obtained in previous studies on carnation e.g. serrano et al. (2001), lopez et al. (2008), hashemabadi (2014), liu et al. (2018) and gocan et al. (2021). similar outcomes were observed on roses cut flowers (elgimabi, 2014; kumar et al., 2017), ger­ bera (bhanushree and rao, 2015; jafarpour et al., 2015), hydrangeas (kazaz et al., 2020; suntipabvivat­ tana et al., 2020), cymbidium (usha et al., 2014). kabari and soleimandarabi (2019) focused on alstroemeria cut flowers, while ichimura et al. (2009) examined cut eustoma, delphinium, and snapdragon flowers. in this regard, darwish et al. (2014) reported that using a solution of 300 ppm 8­hqs + 40 g/l sucrose and 0.4 mm sts + 50 g/l sucrose significantly increased vase life of carnation cut flowers cv. felice. also, badawy et al. (2016) revealed that agno3 showed the longest vase life of chrysanthemum cut flowers cv. royal accent. water relations water uptake data reported in table 2 show that ps1 solution significantly enhanced water uptake of carnation cut flowers resulting in the highest values in both sea­ sons, (48.2 and 49.9 g/flower in 2020 and 2021, ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. table 2 ­ effect of pulsing and holding solutions and their interaction on water uptake (g/flower) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 36.52 mn 36.55 mn 32.06 p 35.04 g hs1 47.74 h­j 36.20 mn 34.01 op 39.32 f hs2 57.35 d 50.23 fg 46.81 ij 51.46 c hs3 52.18 ef 48.95 gh 48.85 g­i 49.99 d hs4 50.57 fg 40.68 l 40.96 kl 44.07 e hs5 59.70 c 57.10 d 54.11 e 56.97 b hs6 56.49 d 46.68 j 46.54 j 49.91 d hs7 46.71 j 42.77 k 45.90 j 45.12 e hs8 67.61 a 65.47 b 60.15 c 64.41 a hs9 29.31 q 37.80 m 37.53 m 34.88 g hs10 26.79 r 34.56 no 29.95 q 30.43 h mean (a) 48.27 a 45.18 b 43.35 c second season (2021) control (dw) 38.42 mn 38.46 mn 34.10 p 36.99 g hs1 49.42 h­j 38.23 mn 36.11 o 41.25 f hs2 58.75 d 51.85 fg 48.53 ij 53.04 c hs3 53.74 ef 50.60 gh 50.50 g­i 51.61 d hs4 52.17 fg 42.58 l 42.85 kl 45.87 e hs5 61.03 c 58.51 d 55.61 e 58.39 b hs6 57.92 d 48.41 j 48.27 j 51.53 d hs7 48.43 j 44.60 k 47.64 j 46.89 e hs8 68.71 a 66.63 b 61.47 c 65.60 a hs9 31.55 q 39.78 m 39.53 m 36.95 g hs10 29.11 r 36.64 no 32.17 pq 32.64 h mean (a) 49.93 a 46.94 b 45.16 c 260 adv. hort. sci., 2023 37(3): 255­269 respectively). the lowest significant values were recorded when the cut flowers were pulsed in a dis­ tilled water solution. (43.3 and 45.1 g/flower in 2020 and 2021, respectively). all applied holding solutions positively affect the water uptake of cut carnations as shown in table 2. the holding solution containing hs8 resulted in the highest water uptake (64.4 and 65.6 days in 2020 and 2021, respectively). on the other hand, holding in solutions containing either rosemary or thyme extracts adversely affected water uptake of carnation cut flowers (34.8 and 36.9 g/flower for rosemary and 30.4 and 32.6 g/flower for thyme extract in 2020 and 2021, respectively). furthermore, it is noteworthy that when the flowers were held in a distilled water solution alone, it exhibited a similar effect to holding them in a solution containing rosemary extract, with no significant difference observed (35.0 and 36.9 g/flower in the first and second seasons, respectively. with regard to the interaction between pulsing and holding solutions, it can be observed that pulsing in ps1 solution followed by holding in hs8 recorded the highest significant values in both seasons (67.6 and 68.7 g/flower, respectively in 2020 and 2021). water uptake of carnation cut flowers was decreased by all applied pulsing solutions when combined with holding solutions containing either rosemary, thyme or only distilled water. the lowest values in this regard were obtained by pulsing in ps1 in addition to holding in hs10 solutions (26.7 and 29.1 g/flower in 2020 and 2021, respectively). water loss as shown in table 3, the greatest water loss was observed when the flowers were pulsed in ps1 in both seasons. the water loss values recorded were table 3 ­ effect of pulsing and holding solutions and their interaction on water loss (g/flower) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 38.23 ij 41.25 f­h 37.46 jk 38.98 e hs1 49.26 d 33.77 l­n 33.21 mn 38.75 e hs2 54.14 bc 46.64 e 42.28 f­h 47.69 b hs3 47.32 de 42.61 fg 43.50 f 44.48 c hs4 46.57 e 35.72 kl 37.80 jk 40.03 de hs5 59.81 a 53.39 c 53.53 c 55.58 a hs6 51.81 c 42.04 f­h 39.90 h­j 44.58 c hs7 42.71 fg 38.23 ij 40.46 g­i 40.46 d hs8 60.64 a 56.01 b 51.78 c 56.15 a hs9 35.58 k­m 43.42 f 42.50 fg 40.50 d hs10 29.50 o 41.67 f­h 32.84 n 34.67 f mean (a) 46.87 a 43.16 b 41.39 c second season (2021) control (dw) 39.40 l­n 43.28 hi 42.09 i­k 41.59 d hs1 50.21 c­e 31.40 t 33.73 rs 38.45 e hs2 50.79 cd 43.83 hi 40.89 j­l 45.17 c hs3 42.20 ij 40.25 j­m 42.19 ij 41.55 d hs4 44.50 h 33.28 r­t 35.40 p­r 37.73 e hs5 58.60 a 52.08 c 55.00 b 55.22 a hs6 48.63 d­f 37.05 o­q 37.57 n­p 41.08 d hs7 40.89 j­l 36.97 o­q 38.37 m­o 38.74 e hs8 54.72 b 46.74 fg 47.01 fg 49.49 b hs9 39.95 k­m 48.30 ef 45.33 gh 44.53 c hs10 32.58 st 46.85 fg 35.17 qr 38.20 e mean (a) 45.68 a 41.82 b 41.16 b sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 261 46.9 and 45.7 g/flower, respectively in 2020 and 2021. the lowest values were recorded when the flowers were pulsed in a distilled water solution (41.4 and 41.2 g/flower in 2020 and 2021, respectively). there was no significant difference between pulsing the flowers in a solution of ps2 (41.8 g/flower) and using distilled water in the second season only. regarding the effect of holding solution, it can be said that holding in hs8 or hs5 solutions resulted in the highest water loss in both seasons. only in the first season, there was no significant difference between them, while in the second one, hs5 was sig­ nificantly higher than hs8. in the first and second seasons, the results were 56.1 and 49.4 g/flower for hs8 and 55.5 and 55.2 g/flower for hs5 respectively. as for the effect of interaction, the highest water loss was obtained by pulsing in ps1 then holding in either hs8 (60.6 and 54.7 g/flower) or hs5 (59.8 and 58.6 g/flower in 2020 and 2021, respectively). however, in the first season, there was no significant difference between these two combined treatments. in contrast, in the second season, when combined with a holding solution containing hs5, it resulted in a significantly higher water loss compared to the combination of hs8. the lowest significant water loss was obtained by pulsing in ps1 followed by holding in hs10 in the first season (29.5 g/flower) and ps2 fol­ lowed by hs1 only in the second one (31.4 g/flower). water balance regarding the impact of pulsing solutions on the water balance of carnation cut flowers, the data pre­ sented in table 4 revealed that ps2 exhibited superi­ ority in this regard, recording the highest values of ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. table 4 ­ effect of pulsing and holding solutions and their interaction on water balance (g/flower) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) ­1.71 m ­4.69 n ­5.40 no ­3.93 f hs1 ­1.52 lm 2.43 j 0.79 k 0.57 e hs2 3.21 h­j 3.59 g­j 4.53 f­i 3.78 d hs3 4.87 e­g 6.34 c­e 5.35 d­f 5.52 b hs4 4.00 f­i 4.95 e­g 3.17 ij 4.04 cd hs5 ­0.11 kl 3.71 g­j 0.58 k 1.39 e hs6 4.68 f­h 4.65 f­i 6.64 cd 5.32 b hs7 4.00 f­i 4.54 f­i 5.44 d­f 4.66 bc hs8 6.97 bc 9.45 a 8.37 ab 8.26 a hs9 ­6.27 op ­5.62 n­p ­4.97 no ­5.62 g hs10 ­2.70 m ­7.11 p ­2.89 m ­4.23 f mean (a) 1.40 b 2.02 a 1.96 a second season (2021) control (dw) ­0.98 m ­4.82 o ­7.99 q ­4.60 e hs1 ­0.79 m 6.83 ij 2.38 k 2.81 d hs2 7.96 gh 8.01 gh 7.63 gh 7.87 c hs3 11.54 c 10.35 e 8.31 g 10.07 b hs4 7.67 gh 9.29 f 7.46 hi 8.14 c hs5 2.44 k 6.43 j 0.61 l 3.16 d hs6 9.29 f 11.35 cd 10.70 de 10.45 b hs7 7.54 h 7.63 gh 9.27 f 8.15 c hs8 13.98 b 19.89 a 14.46 b 16.11 a hs9 ­8.40 q ­8.52 q ­5.80 p ­7.57 g hs10 ­3.46 n ­10.20 r ­2.99 n ­5.55 f mean (a) 4.25 b 5.11 a 4.00 c adv. hort. sci., 2023 37(3): 255­269 262 2.0 g/flower and 5.1 g/flower in 2020 and 2021, respectively. pulsing solution containing distilled water occupied the second position without signifi­ cant differences in the first season only (1.9 g/flower). regarding the effect of holding solutions, hs8 resulted in the highest significant positive values in both seasons (8.2 and 16.1 g/flower in 2020 and 2021, respectively). the lowest values were obtained when using hs9, which resulted in negative values of ­5.6 g/flower and ­7.5 g/flower in the first and second seasons, respectively. a significant interaction between pulsing and holding solutions was observed. pulsing in ps2 then holding in hs8 seemed to be the most effective treat­ ment resulting in the highest positive values (9.4 and 19.8, in 2020 and 2021, respectively. the lowest val­ ues, on the other hand, were obtained when using any pulsing solution followed by hs9, hs10, or hs1. specifically, pulsing the flowers in ps2 followed by holding them in hs10 resulted in the lowest values in this regard, measuring ­7.1 g/flower and ­10.2 g/flower in the first and second seasons, respectively. in this regard, bhanushree and rao (2015) con­ ducted a research on gerbera jamesonii cv. lomborgini and reported that the application of agno3 at 20 ppm resulted in an increase in water uptake and water loss. khella et al. (2018) reported that water uptake of limonium sinuatum cv. girlie wings cut flowers was enhanced by sts at 500 mg/l for 1/2 h or by sts at 500 mg/l for 1/4 h followed by agno3 at 500 mg/l for 1/2 h. in a study conducted by kazaz et al. (2020) on cut hydrangeas, it was found that the application of 8­hqs at 200 mg/l resulted in an improvement in solution uptake compared to the control group. these findings align with the results reported by elgimabi (2014) on rosa damascena cv. trigintipetala and usha et al. (2014) on cymbidium hybrid cv. red princess. determination of pigments and sugars pigments content the pulsing solution containing ps1 recorded the highest values of chlorophyll a (0.349 and 0.354 mg/g f.w.), chlorophyll b (0.234 and 0.241 mg/g f.w.) and carotenoids (0.184 and 0.182 mg/g f.w.) in both sea­ sons, respectively. the lowest values for chlorophyll a, chlorophyll b, and carotenoids were observed when using distilled water, with recorded values of 0.293 and 0.297, 0.218 and 0.214, and 0.133 and 0.143 in the first and second seasons, respectively (tables 5, 6, and 7). concerning the effect of holding solutions, hs8 resulted in the highest values of chlorophyll a (0.626 and 0.641 mg/g f.w.), chlorophyll b (0.435 and 0.434 mg/g f.w.) and carotenoids (0.236 and 0.247 mg/g f.w.) in both seasons, respectively. it could be noticed that the lowest values in terms of chlorophyll a (0.167 and 0.171 mg/g f.w.) and carotenoids (0.110 and 0.116 mg/g f.w.) in both 2020 and 2021seasons were obtained by using a holding solution containing hs9, while hs10 produced the lowest values in case of chlorophyll b (0.105 and 0.116 mg/g f.w. in 2020 and 2021 respectively). in general, all holding solu­ tion formulations involving hs9, hs10 or distilled water resulted in the lowest values for the measured parameters. combined treatment of ps2 in addition to hs8 resulted in the highest significant values in terms of chlorophyll a (0.642 and 0.655 mg/g f.w.) and carotenoids (0.238 and 0.255 mg/g f.w.) and occu­ pied the second rank in case of chlorophyll b with values of 0.468 and 0.466 in 2020 and 2021, respec­ tively. on the other hand, the highest values of chlorophyll b were obtained when using ps1 in com­ bination with hs8 (0.481 and 0.485 mg/g f.w. in 2020 and 2021, respectively). ps1 combined with hs9 gave the lowest values in terms of chlorophyll a, while pulsing and holding in distilled water resulted in the lowest values of chlorophyll b (0.052 and 0.059 mg/g f.w.) and carotenoids (0.062 and 0.060 mg/g f.w.) in both sea­ sons. in a similar context, badawy et al. (2016) conduct­ ed a study on chrysanthemum cut flowers cv. royal accent and reported that agno3 exhibited the least decrease in chlorophyll content. khella et al. (2018) reported that sts at 500 mg/l for 1/2 h or sts at 500 mg/l for 1/4 h followed by agno3 500 mg/l for 1/2 h enhanced pigments content of limonium sinuatum cv. girlie wings cut flowers. the same results were reported by elgimabi (2014) on rosa damascena cv. trigintipetala. total sugars content the data presented in table 8 clearly show that pulsing cut carnations in ps1 resulted in the highest percentage of sugars, with recorded values of 28.0% and 28.5% in 2020 and 2021, respectively. following closely in the second position, a solution of ps2 showed a percentage of 27.7% in 2020 (not being sig­ nificantly different from the highest) and 28.0% in sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 263 2021 (being significantly different from the highest). conversely, the lowest values were observed when using distilled water, with percentages of 25.8% and 26.0% in 2020 and 2021, respectively. when considering the influence of holding solu­ tions, it was evident that hs8 outperformed other treatments in terms of total sugars, resulting in the highest values in both seasons (47.5% and 48.3% in 2020 and 2021, respectively). conversely, hs9 exhib­ ited a negative effect, leading to the lowest sugar content (11.4% and 11.7%). regarding the combined treatments, ps2 in addi­ tion to hs8 produced the highest values, recording 52.0% and 54.6% in 2020 and 2021, respectively. on the other hand, when distilled water was combined with hs9, the lowest values were observed, with per­ centages of 10.5% and 11.1% in 2020 and 2021, respectively. these results align with the findings of badawy et al. (2016), who reported that agno3 exhibited the highest total carbohydrate content in chrysanthemum cut flowers cv. royal accent. additionally, khella et al. (2018) demonstrated that sts at 500 mg/l for 1/2 h enhanced the total carbo­ hydrate percentage of limonium sinuatum cv. girlie wings cut flowers, followed by the treatment with sts at 500 mg/l for 1/4 h and agno3 at 500 mg/l for 1/2 h. similarly, chore et al. (2020) investigated gladiolus grandiflorus l. cv. fado and found a signifi­ cant increase in total soluble sugars in spikes pulsed with 600 ppm 8­hqs + 5% sucrose compared to the control. these results are consistent with the findings table 5 ­ effect of pulsing and holding solutions and their interaction on chlorophyll a (mg/g f.w.) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 0.250 t 0.281 r 0.143 z 0.225 h hs1 0.267 s 0.323 o 0.247 t 0.279 g hs2 0.353 l 0.383 k 0.314 p 0.350 e hs3 0.480 d 0.463 e 0.327 no 0.423 b hs4 0.395 j 0.440 f 0.288 q 0.374 d hs5 0.329 mn 0.398 j 0.187 w 0.305 f hs6 0.416 h 0.429 g 0.430 g 0.425 b hs7 0.408 i 0.410 i 0.333 m 0.383 c hs8 0.636 b 0.642 a 0.601 c 0.626 a hs9 0.120 [ 0.212 u 0.170 y 0.167 j hs10 0.186 w 0.196 v 0.178 x 0.187 i mean (a) 0.349 b 0.380 a 0.293 c second season (2021) control (dw) 0.255 r 0.279 p 0.139 y 0.224 h hs1 0.264 q 0.334 m 0.243 s 0.280 g hs2 0.355 l 0.403 k 0.330 m 0.363 e hs3 0.483 d 0.454 e 0.334 m 0.424 b hs4 0.406 jk 0.458 e 0.295 o 0.386 c hs5 0.329 m 0.417 gh 0.190 v 0.312 f hs6 0.420 g 0.413 hi 0.440 f 0.424 b hs7 0.405 jk 0.408 ij 0.323 n 0.379 d hs8 0.650 b 0.655 a 0.618 c 0.641 a hs9 0.131 z 0.213 t 0.169 x 0.171 j hs10 0.197 u 0.199 u 0.184 w 0.193 i mean (a) 0.354 b 0.385 a 0.297 c adv. hort. sci., 2023 37(3): 255­269 264 of elgimabi (2014) on rosa damascena cv. trigintipetala and bhanushree and rao (2015) on gerbera jamesonii cv. lomborgini. the present study exhibited the beneficial role of certain chemicals used in pulsing or holding solutions of carnation cut flowers either individually or in com­ binations. the application of these chemicals resulted in a significant increase in the vase life of cut flowers, more than doubling its duration. furthermore, these treatments also had a positive impact on water rela­ tions and chemical composition, including chlo­ rophylls a, b, carotenoids, and total sugars. it is well known that the vase life of cut carnation is conside­ red one of the most vital traits for florists (panwar et al., 2022). finger and barbosa (2006) summarized the factors affecting the longevity of cut flowers as (1) their tender nature, (2) a lot of stresses leading to water uptake reduction, stored carbohydrates exhau­ stion and respiration increment (3) the harmful effects of ethylene. otherwise, the vase life of cut flowers is affected basically by ethylene which enhan­ ces the senescence of many cut flowers as well as microorganisms which reduce the amount of water uptake by causing a vascular blockage (zencirkiran, 2010). halevy (1987) reported that carnation cut flowers are highly sensitive to ethylene either endo­ genously produced or exogenously applied. carnation is known to be highly susceptible to the buildup of microorganisms in the vase solution or at the cut ends of the flower stems. this accumulation can result in blockage of the vascular system and ulti­ mately reduce the vase life of the flowers (van doorn table 6 ­ effect of pulsing and holding solutions and their interaction on chlorophyll b (mg/g f.w.) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 0.105 x 0.174 r 0.052 \ 0.110 j hs1 0.156 t 0.184 q 0.154 t 0.165 h hs2 0.206 p 0.251 n 0.260 m 0.239 f hs3 0.396 d 0.423 c 0.294 l 0.371 b hs4 0.217 o 0.357 ef 0.222 o 0.266 e hs5 0.185 q 0.300 k 0.143 u 0.209 g hs6 0.362 e 0.316 i 0.338 g 0.339 c hs7 0.294 l 0.309 j 0.332 h 0.312 d hs8 0.481 a 0.468 b 0.356 f 0.435 a hs9 0.079 [ 0.163 s 0.119 w 0.120 i hs10 0.088 z 0.098 y 0.128 v 0.105 k mean (a) 0.234 b 0.277 a 0.218 c second season (2021) control (dw) 0.127 n­p 0.181 j­m 0.059 q 0.122 h hs1 0.172 k­n 0.181 j­m 0.168 k­n 0.174 g hs2 0.208 i­k 0.260 gh 0.240 g­i 0.236 e hs3 0.398 bc 0.437 ab 0.285 fg 0.373 b hs4 0.230 h­j 0.368 cd 0.215 h­k 0.271 d hs5 0.188 j­l 0.284 fg 0.144 l­o 0.205 f hs6 0.367 cd 0.324 d­f 0.324 d­f 0.338 c hs7 0.291 e­g 0.337 de 0.316 d­f 0.315 c hs8 0.485 a 0.466 a 0.350 cd 0.434 a hs9 0.088 pq 0.171 k­n 0.124 n­p 0.128 h hs10 0.095 o­q 0.130 m­p 0.123 n­p 0.116 h mean (a) 0.241 b 0.285 a 0.214 c sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 265 et al., 1991). hence, eliminating ethylene production and microorganism accumulation is a vital procedure to prolong the vase life of cut carnations. all solu­ tions containing sucrose showed a great influence compared to control (distilled water only) or solu­ tions with only 2.0% sucrose. this observation aligns with the well­known fact that sucrose, as a source of sugar, plays a crucial role in preservative solutions. the use of sucrose in pulsing solution or as a consti­ tuent of vase solution may extend the vase life of the flowers by improving the water balance, stimulating flower opening or by delaying the senescence due to lower synthesis of ethylene, as observed in cut carna­ tion (finger and barbosa, 2006). this study clearly highlights the significance of using integrated silver thiosulfate (sts) in post­harve­ st solutions. in this regard, treatment with silver thio­ sulfate complex (sts) delayed the senescence of atta­ ched and detached petals of dianthus caryophyllus cv. barbara (ichimura and niki, 2014). sts is highly mobile in the xylem of carnation flowers and may become a practical treatment for carnation flowers (reid and kofranek, 1980). hashemabadi (2014) revealed that sts treatment extended the longevity of cut carnation ‘tempo’ flowers by reducing oxidati­ ve stress, improving the antioxidant system, reducing bacterial populations and delaying flowering. chemicals such as sts, are also effective at the recep­ tor level and prevent the binding of ethylene (ebrahimzadeh et al., 2008). using agno3 in a pulsing solution of carnation cut flowers showed a great influence. in this regard, table 7 ­ effect of pulsing and holding solutions and their interaction on carotenoids (mg/g f.w.) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 d.w. mean (b) first season (2020) control (dw) 0.139 n 0.179 k 0.062 t 0.127 i hs1 0.179 k 0.207 i 0.123 pq 0.170 h hs2 0.189 j 0.211 hi 0.131 o 0.177 f hs3 0.230 de 0.237 ab 0.133 o 0.200 c hs4 0.210 hi 0.232 b­d 0.125 p 0.189 e hs5 0.188 j 0.214 h 0.119 q 0.174 g hs6 0.234 a­d 0.226 ef 0.232 cd 0.231 b hs7 0.222 fg 0.219 g 0.148 m 0.196 d hs8 0.236 a­c 0.238 a 0.234 a­d 0.236 a hs9 0.091 s 0.172 l 0.065 t 0.110 j hs10 0.110 r 0.135 no 0.086 s 0.110 j mean (a) 0.184 b 0.207 a 0.133 c second season (2021) control (dw) 0.129 n 0.201 i 0.060 r 0.130 h hs1 0.182 j 0.207 h 0.127 n 0.172 g hs2 0.200 i 0.218 g 0.148 l 0.189 e hs3 0.216 g 0.230 de 0.142 m 0.196 c hs4 0.199 i 0.235 c 0.143 m 0.192 d hs5 0.182 j 0.235 cd 0.131 n 0.182 f hs6 0.225 f 0.216 g 0.246 b 0.229 b hs7 0.228 ef 0.209 h 0.158 k 0.198 c hs8 0.236 c 0.255 a 0.249 b 0.247 a hs9 0.089 p 0.179 j 0.079 q 0.116 i hs10 0.118 o 0.141 m 0.093 p 0.118 i mean (a) 0.182 b 0.212 a 0.143 c adv. hort. sci., 2023 37(3): 255­269 266 agno3 is used as an antimicrobial, since the ag+ ion replaces the hydrogen cations (h+) on surface pro­ teins in the cell membranes of bacteria, which leads to loss of membrane integrity and causes cell death (feng et al., 2000). this study exhibited a positive influence of 8­hqs addition to the holding solution in extending vase life, enhancing water relations (uptake and balance) and chemical constituents of cut carnation flowers, this effect may be explained by the positive role of 8­hqs as a germicide agent. preservative solutions of carna­ tion cut flowers containing 8­hqs showed a strong inhibitory effect on fungi, yeasts and bacteria (el­ ashwah, 2011). numerous authors have corroborated this fact, supporting the use of 8­hydroxyquinoline sulfate (8­hqs) in vase solutions to effectively reduce microbial counts. one such study by madhuri et al. (2016) demonstrated that the addition of 8­hqs resul­ ted in the lowest microbial counts in vase solutions. kabari and soleimandarabi (2019) on alstroemeria cut flowers observed the lowest bacterial population in vase solution in the treatment of 200 8­hqs mg/l. in addition, 8­hqs is considered an ethylene synthesis inhibitor (ebrahimzadeh et al., 2008). to explain the positive role of aoa in the present study, son et al. (1995) reported that aoa appeared to inhibit the activities of arginine decarboxylase and acc synthase. ethylene production was significantly decreased by aoa at concentrations over 100 mg/l, the decline in acc content was observed after using 100 or 150 mg/l aoa (karimi et al., 2012). amino­ oxyacetic acid (aoa) inhibits the synthesis of ethyle­ ne by reducing the competitive and irreversible acti­ vity of 1­aminocyclopropane­1­carboxylic acid (acc) table 8 ­ effect of pulsing and holding solutions and their interaction on total sugars (%) of dianthus caryophyllus cv. turbo cut flowers during 2020 and 2021 seasons ps1= sts at 0.4 ppm + sucrose 10%, ps2= agno3 at 10.0 ppm + sucrose 10%, hs10 sucrose 4%, hs2= boric acid at 200 ppm + sucrose 4%, hs3= 8­hqs at 300 ppm + sucrose 4%, hs4= aoa at 250 ppm + sucrose 4%, hs5= 8­hqs + aoa + sucrose 4%, hs6= boric acid + 8­hqs + sucrose 4%, hs70= boric acid + aoa + sucrose 4%, hs8= boric acid + 8­hqs + aoa + sucrose 4%, hs9= rosemary extract at 25% + sucrose 2%, hs10= thyme extract at 25% + sucrose 2%. holding solutions (b) pulsing solutions (a) ps1 ps2 dw mean (b) first season (2020) control (dw) 20.43 o 18.89 p 9.83 t 16.38 i hs1 23.29 n 22.69 n 23.30 n 23.09 h hs2 29.19 jk 23.43 n 29.95 j 27.52 f hs3 40.87 d 39.72 de 30.28 ij 36.96 b hs4 30.16 ij 33.40 g 27.68 l 30.41 e hs5 25.79 m 28.75 kl 21.26 o 25.27 g hs6 35.63 f 31.12 hi 39.01 e 35.25 c hs7 31.72 h 30.00 ij 32.91 g 31.55 d hs8 47.70 b 52.01 a 42.89 c 47.53 a hs9 11.02 s 12.82 r 10.55 st 11.46 k hs10 13.04 r 12.24 r 16.22 q 13.83 j mean (a) 28.08 a 27.73 a 25.81 b second season (2021) control (dw) 21.39 o 19.20 p 9.98 u 16.86 h hs1 24.71 lm 23.77 mn 22.75 no 23.74 g hs2 31.08 hi 24.35 l­n 31.10 hi 28.84 e hs3 42.73 c 40.24 de 31.64 g­i 38.20 b hs4 29.13 jk 33.06 g 29.10 jk 30.43 d hs5 25.64 l 27.55 k 21.87 o 25.02 f hs6 34.95 f 31.76 g­i 38.94 e 35.22 c hs7 30.38 ij 28.63 k 32.61 gh 30.54 d hs8 48.48 b 54.67 a 41.86 cd 48.34 a hs9 11.23 s­u 12.88 rs 11.10 tu 11.74 j hs10 14.43 qr 12.06 st 16.01 q 14.17 i mean (a) 28.56 a 28.02 b 26.09 c sarhan et al. ‐ preservative solutions’ effect on carnation‐cut flowers 267 synthase, reducing the amount of substrate for acc oxidase, and therefore the conversion of acc to ethylene (finger and barbosa, 2006). boric acid also showed a promising influence when it was integrated into the holding solution of cut carnation flowers, this could be explained by its role in preventing the early rise in ethylene produc­ tion and considerably improving carnation vase life (serrano et al., 2001). while many studies have highlighted the positive impact of natural extracts as a component in preser­ vative solutions, such as el­ashwah (2011) on carna­ tion cv. domingo, khenizy et al. (2014) on gypsophila paniculata l. “perfecta,”, zaky et al. (2014) on carna­ tion cv. america, and hashemabadi et al. (2017) on carnation cv. white liberty, our study did not obser­ ve a similar effect. the lack of satisfactory results in our study regarding the effectiveness of rosemary and thyme extracts may be attributed to the fact that these extracts were used in combination with sucro­ se at 2.0% only, without the addition of any germici­ dal agents to the solution. the presence of sugar in the solution without germicidal agents can promote the growth of microorganisms, since these extracts alone may not possess sufficient biocidal properties to effectively control microorganisms (armitage and laushman, 2003). therefore, the outcomes of using natural extracts in our study were not as promising. in future research, it is recommended to consider incorporating germicidal agents along with these extracts, particularly when using extracts obtained through water extraction methods. this approach can help enhance the antimicrobial efficacy and ove­ rall performance of natural extracts as preservatives in cut flower solutions.adam in conclusion, it is highly recommended to pulse carnation cv. turbo cut flowers in a solution contai­ ning agno3 at 10.0 ppm + sucrose 10% for a duration of 15 minutes, followed by holding them in a solution of boa + 8­hqs + aoa + suc 4% to extend the vase life of the flowers, enhancing water uptake and main­ taining water balance. additionally, this preservative solution effectively reduces chlorophyll degradation and preserves the content of carbohydrates throu­ ghout the postharvest period. references adam a.i., eldeeb m.m., 2021 ­ effect of some biocides on the vase life of carnation (dianthus caryophyllus l.) cut flowers. ­ scientific j. flowers ornam. plants, 8(2): 209­ 221. armitage a.m., laushman j.m., 2003 ­ specialty cut flowers, the production of annuals, perennials, bulbs, and woody plants for fresh and dried cut flowers. ­ timber press, oregon, usa, pp. 586. badawy n.m., hassan s.e., el­shoura h.a.s., el­shreif m.h.m., el­quesni f.e.n.m., 2016 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analyses. the bacteria had morphological and colony characteristics resembling those of luteibacter species. colonies were white to yellow, round, and slightly raised, with the entire margin in nutrient agar medium. the bacterial isolates were gram­negative based on the potassium hydroxide test (koh) test. phylogenetic analysis of the 16s ribosomal gene region grouped the three isolates in the luteibacter yeojuensis clade. the three luteibacter yeojuensis isolates were not pathogenic to banana ‘grand nain,’ ‘lakatan,’ and ‘saba’ in both wounded and unwounded assays conducted in controlled assays. no stunting, wilting, and corm tissue browning were observed 14 days post­inoculation when the bacteria were inoculated on tissue­cultured plants; two of the three isolates significantly increased plant height of cv. lakatan (p<0.05) and one isolate, l. yeojuensis gn11­20, enhanced shoot proliferation in cv. grand nain. the study reports l. yeojuensis as an endophytic bacterium with growth­promoting activity in tissue­cultured banana plants. the endosymbiotic association of l. yeojuensis in bananas could enhance plant growth and resistance to banana diseases. 1. introduction banana (musa spp.) is a high­value fruit and cash crop widely grown as one of the staple foods in asia (rossman et al., 2012). the philippines remains the top producer of bananas in asia and the second largest exporter globally, following ecuador (fao, 2023). there are three major varieties of banana produced in the country: cavendish (51%), saba (29%), and lakatan (10%) (anzures et al., 2022). data from the philippine statistics authority (2023) show that the davao region is the top producer of bananas, with 868.19 thousand metric tons (mt) or 37.4% of the total banana production in the last quarter of 2023. this was followed by northern mindanao with 431.86 thousand mt (19%) and soccsksargen region with 279.20 thousand mt (12.3%) shares in production, respectively (philippine statistics authority, 2023). (*) corresponding author: mark.angelo.balendres@dlsu.edu.ph citation: cruz m.a., alcasid c., balendres m.a., 2025 ­ endophytic luteibacter yeojuensis strains stimulate banana plant growth. ­ adv. hort. sci., 39(1): 1­10 orcid: bma: 0000­0001­8739­5858 copyright: © 2025 cruz m.a., alcasid c., balendres m.a. this is an open access, peer reviewed article publi­ shed by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 30 september 2024 accepted for publication 17 january 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16663 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-8739-5858 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 1­10 2 cultivated banana genotypes are triploids that are generally sterile and seedless (uma, 2021). thus, conventional production of banana planting materials is made through sword suckers. however, these vegetative materials may also harbour plant pathogens. they may subsequently result in the carry­over of diseases in succeeding planting materials; hence, plant tissue culture is a widely practiced method as an alternative for banana production. aside from preventing the carry­over of diseases, the technology allows the plants to aseptic production with uniform genotypes in a relatively shorter period (souza et al., 2000). the technology only requires a small portion (1­3 mm2) of the plant parts (meristems) for in‐vitro mass production and germplasm conservation (agbadje et al., 2021); this allowed bananas rapid propagation from a single corm with favourable pests and pathogen­free genotypes (agbadje et al., 2021). however, contamination with microorganisms that may act as endophytes, saprophytes, or asymptomatic pathogens is commonly observed (cassells, 1991). contamination may emanate from the plant’s plant tissues (endophytes) and surfaces (nair and padmavathy, 2014). proper growth media and explant sterilizations can easily control microbes on the explant’s surface and those carried from the environment (sivanesan et al., 2021). in contrast, endophytes are challenging to control because they are inside the plant tissue and are tolerant to surface sterilization (hardoim et al., 2015). endophytes l ike bacteria, fungi, and actinomycetes can colonize healthy living tissues and establish a symbiotic relationship with the host plant (nair and padmavathy, 2014). host plants benefit from endophytes through plant growth promotion (afzal et al., 2019), pathogen and insect attack defence (sturz and matheson, 1996; azevedo et al., 2000; pieterse et al., 2014; martínez­hidalgo et al., 2015; oukala et al., 2021), and increased tolerance to abiotic factors including salinity (ali et al., 2014), low temperature (subramanian et al., 2015), and heavy metals (rajkumar et al., 2009). bacterial endophytes of bananas are known plant growth promoters and biocontrol agents. for instance, several bacterial endophytes from diverse communities form an antagonistic relationship against fusarium oxysporum f.sp. cubense (foc) (jie et al., 2009). plant growth promotion in banana cv. prata ana has also been demonstrated in shoot tip cultures colonized with endophytic klebsiella pneumoniae (fernandes et al., 2013). thus, the utilization of endophytes could improve the banana production system. however, any new endophytes found from a plant must undergo pathogenicity testing to ensure that they do not cause infection to the host once removed from their natural system (within the host). this study identified and characterized an endophytic bacterium isolated from healthy tissue­ cultured banana ‘grand nain’ and ‘saba.’ we hypothesize that endophytes from healthy banana plants do not harm host plants but function as plant growth promoters. thus, these endophytes may be used directly or indirectly as potential bioinoculants under a green and sustainable agriculture production system. 2. materials and methods isolation and storage of the bacterial contaminants white­to­yellow pigmented bacteria were observed in multiple shoot cultures of tissue­cultured banana cultivars grand nain and saba. a loopful of bacterial cells growing from the stems was transferred onto a nutrient agar (na) medium. plates were stored at room temperature (28­30°c) for two days (with 14 hours of light in 24 hours cycle) (cruz and balendres, 2021). the bacterium was then purified and further characterized (see succeeding section). a loopful of the bacterium from a 48­hour­ old culture was transferred to a fresh na plate and incubated using the abovementioned conditions. cultures were stored in microcentrifuge tubes containing 1 ml of sterile distilled water. the cultures were deposited at the bacteria repository of the institute of plant breeding, agriculture and food science college, university of the philippines los baños, laguna, philippines. morphocultural characterization and pcr assay the bacterial morphology of 48 to 72­hour­old cultures was assessed under a light microscope (olympus cx23, japan), and the colony characteristics were recorded. the bacterial genomic dna was extracted using chen and kuo’s procedure (chen and kuo, 1993) for molecular analyses. the isolated genomic dna was standardized to 30 ng/μl and was subsequently used as a template for the succeeding polymerase chain reaction (pcr) assay, which amplifies the 16s ribosomal gene region. the pcr assay was performed in mycycler™ thermal cruz et al. ‐ endophytic luteibacter yeojuensis 3 cycler (bio­rad, usa) in a 15­μl reaction volume (cruz and balendres, 2021). the pcr cocktail mix consisted of 1x pcr buffer (invitrogen), 2.0 mm mgcl2 (invitrogen), 0.2 mm dntps (invitrogen), 0.2 μm each of the forward (27f, 5’­ agagtttgatcctggctcag­3’) and reverse (1492r, 5’­ggttaccttgttacgactt­3’) primers (lane, 1991), one u taq dna polymerase (invitrogen), one μl of the bacterial genomic dna, and depc­water to volume. the thermal cycling conditions were as follows: initial denaturation at 95°c for two min, followed by 30 cycles of denaturation at 95°c for 30 s, annealing at 55°c for 30 s, and extension at 72°c for two min, and final extension at 72°c for seven min. the pcr products were resolved by gel electrophoresis in 1.0% agarose (vivantis) and 0.5x tris­acetate­edta (tae) buffer and were sent to apical scientific sdn. bhd. (malaysia) for dna sequencing. molecular characterization and phylogenetic analysis a consensus dna sequence was derived from the resultant forward and reverse sequences using geneious software. sequence similarity analysis was performed in the ncbi blastn program (zhang and madden, 1997). sequences were analysed based on the highest percent similarity, e­value, and query cover. the authentic 16s rdna sequences of five species of luteibacter available in genbank (table 1) were compared with the consensus sequences of the three luteibacter yeojuensis sbm36c, gn11­20, sabam36a isolates obtained from this study. the phylogenetic distance of the three bacterial isolates to eight luteibacter yeojuensis isolates from other table 1 ­ luteibacter species with the closest similarity to the 16s rdna region of the bacterium were isolated in this study ihb= institute of himalayan bioresource technology, post box no. 6, palampur, himachal pradesh 176061, india; jw= nanjing agricultural university, tongwei road 6, nanjing, jiangsu 210095, china; ccug= culture collection university of gothenburg, department of clinical bacteriology; lj= department of bacteriology, danish veterinary institute, buelowsvej 27, dk­1790, denmark; mah= food and nutrition, chung­ang university, 4726, seodong­daero, daedeok­myeon, anseong­si, gyeonggi­do 17546, south korea; lmg= ghent university, belgium. species strain source locality 16s genbank accession reference luteibacter yeojuensis ihb b 6856 aquilaria agallocha india kf668474.1 ncbi genbank luteibacter jiangsuensis jw­64­1 soil china nr_132709.1 wang et al. (2011) luteibacter anthropi ccug 25036 human blood sample sweden nr_116911.1 kampfer et al. (2009) luteibacter rhizovicinus lj96 hordeum vulgare denmark nr_042197.1 johansen et al. (2005) luteibacter pinisoli mah­14 soil south korea ky964279.1 huq and akter (2017) burkholderia vietnaminensis lmg 10929 oryza sativa vietnam nr_041720.1 lipuma et al. (1999) table 2 ­ luteibacter yeojuensis strains from other countries compared with the strains isolated in this study ihb= institute of himalayan bioresource technology, post box no. 6, palampur, himachal pradesh 176061, india; jw= nanjing agricultural university, tongwei road 6, nanjing, jiangsu 210095, china; ccug= culture collection university of gothenburg, department of clinical bacteriology; lj= department of bacteriology, danish veterinary institute, buelowsvej 27, dk­1790, denmark; mah= food and nutrition, chung­ang university, 4726, seodong­daero, daedeok­myeon, anseong­si, gyeonggi­do 17546, south korea; lmg= ghent university, belgium. species strain source locality 16s genbank accession reference luteibacter yeojuensis ihb b 6856 aquilaria agallocha india kf668474.1 ncbi genbank luteibacter yeojuensis t­79 curcuma longa india km589043.1 kandan et al. (2014) luteibacter yeojuensis nbrc 106387 not available japan ab682403.1 nakagawa et al. (2011) luteibacter yeojuensis hbu 72524 not available china mw365223.1 lv (2020) luteibacter yeojuensis r2a16­10 soil korea nr_043618 kim et al. (2006) luteibacter yeojuensis rt27 oryza sativa china mk014251.1 ncbi genbank luteibacter yeojuensis osenb_alm_b9 oryza sativa india mn889326.1 kumar et al. (2020) luteibacter yeojuensis z51 rock china km019785.1 zhang (2014) burkholderia vietnaminensis lmg 10929 oryza sativa vietnam nr_041720.1 lipuma et al. (1999) adv. hort. sci., 2025 39(1): 1­10 4 countries (table 2) was further determined. burkholderia vietnaminensis (lmg 10929) was used as the outgroup in all analyses (lipuma et al. 1999). the generated 16s rdna sequences were aligned using clustalw in mega x software (kumar et al. 2018). the phylogenetic trees were constructed using the tamura nei model (tamura and nei 1993) with uniformly distributed rates and 1,000 bootstrap replicates. gram test using koh a potassium hydroxide (koh) test was performed following buck’s procedure (buck, 1982) to identify the gram reaction of the bacterial isolates. therefore, a micropipette placed 10 μl of 3% koh on the top of a clean microscope slide. a loopful of bacteria from a forty­eight to 72­hour­old was then transferred to the drop of koh using a sterile wire loop. the mixture was constantly stirred for 15 seconds and observed for viscosity and formation of mucoidal string. gram­negative bacteria turn vicious and form a mucoidal string. in contrast, gram­positive bacteria do not turn viscous with no formation of mucoidal string. three replicates were used for each isolate. in‐vitro pathogenicity testing and morphometric assessment the pathogenicity of the bacterial isolates in tissue­cultured banana plantlets was assessed in vitro using a pseudostem injection assay; the four­week­ old banana ‘lakatan’ (aa), ‘grand nain’ (aaa), and ‘saba’ (abb) were surface­disinfected using 70% ethanol and air­dried. pseudostems were cut and inoculated by injecting 200 μl of bacterial suspension (0.5, od600) in wounded and unwounded tissues. the pseudostems were injected with sterile distilled water (sdw) for the control treatment. treated plantlets were maintained in the plant regeneration medium (basal murashige and skoog, 3 mg·l­1 6­ benzylaminopurine, 3% sucrose, 7 g·l­1 agar, ph 5.7) and exposed to 14­hour fluorescent light cycle at 20±5℃ temperature (murashige and skoog, 1962). four replicate plantlets were used for each isolate and variety. symptom development was assessed at 14 days post­ inoculation. morphometric characteristics ­ plant height, number of shoots, and roots formed ­ were evaluated from all treatments. analysis of variance (anova) was performed using the irri statistical tool for agricultural research (star nebula) software with a 95% confidence. the experiment was performed twice. 3. results identity of the bacterial contaminants bacteria that are white to yellow were isolated from heathy­looking tissue­cultured bananas cv. grand nain and saba (fig. 1a to 1c). colonies are white to yellow in color, round, and slightly raised, with the entire margin in na medium. cell shapes were bacilli, 1­2 μm x 7­10 μm size, containing monotrichous, amphitrichous, or lophotrichous flagella (fig. 1d to 1f). the isolate colony and morphology resembled that of luteibacter spp. the isolates were gram­negative based on the koh test (fig. 1 g to 1i). the bacterial isolates’ identity was further validated through molecular analysis of the 16s ribosomal dna region. the bacterial isolates had high similarity (>99%) to luteibacter yeojuensis in blastn analysis and were grouped within the luteibacter yeojuensis clade in the constructed phylogenetic tree (table 3, fig. 2). a distance tree also revealed that the three philippine isolates have the closest similarity to luteibacter yeojuensis strain ihb b 6856 from india (fig. 3). fig. 1 ­ morphological and cultural characteristics of the bacterial contaminants. cultural characteristics of gn11­ 20 (a), sbm36a (b), and sbm36c (c) strains of 48­hour­ old cultures grown in nutrient agar (na) medium. the white bar represents 2 cm. bacterial cell morphology for each isolate was also shown (d, e, and f). the black bar at the upper left represents 10 µm. gram reaction of the bacterial contaminants using potassium hydroxide (koh) test (g, h, and i) indicated the isolates were gram­ negative through viscous string formation. cruz et al. ‐ endophytic luteibacter yeojuensis 5 morphometric characters of the banana plants results demonstrated the potential of l. yeojuensis for increased plant height and shoot proliferation on three. both l. yeojuensis gn11­20 and l. yeojuensis sbm36a increased plant height in ‘lakatan’ plants (table 4, fig. 4). more shoots were consistently recorded in banana cv. grand nain inoculated with the three l. yeojuensis isolates compared to the control treatment. one isolate, luteibacter yeojuensis gn11­20, significantly increased shoot proliferation of tissue­cultured ‘grand nain’, the cultivar where the bacterium was initially isolated (table 4, fig. 4). on the other hand, no significant differences were observed in the number of shoots formed in banana ‘lakatan’ and ‘saba’ were inoculated with the three l. yeojuensis isolates compared to the control treatment. the plant height, the number of shoots, and the roots of the three genotypes inoculated with l. yeojuensis sbm36c were not significantly different from the control treatment. non‐pathogenicity of bacteria to tissue‐cultured bananas in‐vitro pathogenicity tests showed that the three luteibacter yeojuensis strains were non­pathogenic to the three banana cultivars (fig. 4 and 5) in wounded and unwounded assays. there were no adverse effects observed in plants inoculated with luteibacter yeojuensis sbm36c, gn11­20, and sbm36a as compared to the control treatments (fig. 5). no stunting and wilting in any of the test banana plants were observed. when corm tissues were dissected, there was no browning in any inoculated plants, and the appearance of the corm was similar to that of the control treatment. 4. discussion and conclusions little is known about the endophytes of banana plants. this study isolated bacterial endophyte l. fig. 2 ­ the phylogenetic position of the 16s rdna of the three luteibacter yeojuensis strains was isolated in this study with other bacterial species. the tree was constructed using the tamura­nei model (tamura and nei, 1993) with 1,000 bootstrap replicates. burkholderia viet­ namiensis (lmg 10929) served as an outgroup. fig. 3 ­ the distance tree of the 16s rdna sequences of luteibacter yeojuensis gn11­20, sbm36a, and sbm36c was isolated in this study with other strains from asia. the tree was constructed using the tamura­nei model (tamura and nei, 1993) with 1,000 bootstrap replicates. burkholderia vietnamiensis (lmg 10929) served as an outgroup. table 3 ­ percentage similarities of the three bacterial isolates associated with tissue­cultured banana based on blastn search isolate species 16s rrna (accession) sbm36a luteibacter yeojuensis ihb b 6856 99.09% (kf668474.1) gn11­20 luteibacter yeojuensis ihb b 6856 99.64% (kf668474.1) sbm36c luteibacter yeojuensis ihb b 6856 99.16% (kf668474.1) 6 adv. hort. sci., 2025 39(1): 1­10 yeojuensis isolates sbm36c, gn11­20, and sbm36a from healthy tissue­cultured banana cultivars saba and grand nain. while the colony characteristics (size and pigmentation) of the three bacterial isolates varied among the isolates, all three isolates belonged to the same species, as confirmed by the 16s rdna fig. 4 ­ pathogenicity of luteibacter yeojuensis sbm36c, gn11­ 20, and sbm36a strains isolated in this study at 14 days post­inoculation (dpi). data shows two plantlets for each treatment. control plantlets (treated with sterile distilled water) were also shown (d). the bar at the upper left represents 2 cm. fig. 5 ­ morphometric characteristics of tissue­cultured banana plantlets inoculated with luteibacter yeojuensis sbm36a, gn11­20, and sbm36c strains isolated in this study. plantlets inoculated with sterile distilled water served as control. the plant height (a), number of shoots that emerged (b), and number of roots formed (c) of the three genotypes were collected 14 days post­inoculation. different letters in each bar indicate significant differ­ ences (p<0.05). plantlets inoculated with water served as control. asterisks (*) indicate significant differences between the treatments and the corre­ sponding control by least significant difference (lsd) test at ɑ= 0.05; ns= not significant. different letters in the mean values for each genotype indicate significant differences between the treatments. genotype treatment plant height (cm) number of shoots emerged number of roots formed mean±sd p­value mean±sd p­value mean±sd p­value grand nain sbm36c 2.7 ± 0.5 0.7855 ns 4.3± 2.5 ab 0.0278 * 2.5 ± 1.9 0.4148 ns gn11­20 2.4 ± 0.6 7.3 ± 0.5 a 2.5 ± 1.3 sbm36a 2.4 ± 0.4 3.0 ± 2.7 b 4.3 ± 1.7 control 2.7 ± 0.7 2.5 ± 1.7 b 2.5 ± 1.9 lakatan sbm36c 2.3 ± 0.6 b 0.0224 * 5.0 ± 3.4 0.6799 ns 2.0 ± 0.8 0.0659 ns gn11­20 3.2 ± 0.2 a 3.8 ± 1.0 5.0 ± 0.8 sbm36a 3.5 ± 0.3 a 3.5 ± 0.6 2.5 ± 1.3 control 2.9 ± 0.7 ab 5.0 ± 2.7 3.8 ± 2.5 saba sbm36c 2.9 ± 0.5 0.5947 ns 4.3 ± 0.5 0.0851 ns 2.8 ± 1.7 0.2367 ns gn11­20 2.4 ± 0.3 2.5 ± 1.7 2.5 ± 1.3 sbm36a 2.6 ± 0.8 4.3 ± 0.5 2.0 ± 1.2 control 2.5 ± 0.6 2.8 ± 1.3 1.3 ± 0.5 table 4 ­ effect of inoculation of luteibacter yeojuensis sbm36c, gn11­20, sbm36a strains on in­vitro shoot and root production of cruz et al. ‐ endophytic luteibacter yeojuensis 7 sequence analyses. bacterial species under the order xanthomonadales, such as luteibacter sp., are gram­ negative, aerobic, and carotenoid­producing species that provide the yellow­orange­red colour of the cultures (saddler and bradbury, 2005). the carotenoid pigments in xanthomonadales are lipid­ soluble and play a significant role in culture survival under low­temperature conditions and against uv radiation (azman et al., 2018). the bacterial isolates from this study were obtained from different sources of banana genotypes: gn11­20 was isolated from ‘grand nain,’ sbm36c and sbm36a were isolated from ‘saba,’ respectively. these genotypes were of different ages from the time of isolation of the endophytes. hence, they had a potentially varying exposure to temperature and uv radiation. this genotypic and environmental variation might have affected the levels of carotenoids found in the endophytes isolated in this study, thereby affecting pigmentation despite belonging to the same species (dieser et al., 2010). this study also highlights the importance of molecular assays, analyzing the 16s rdna sequences, in identifying species when phenotypes of the bacteria are influenced by their response to the environment. none of the l. yeojuensis isolates from this study resulted in infection in tissue­cultured banana ‘lakatan,’ ‘grand nain,’ and ‘saba.’ however, there were recorded differences in the morphometric characteristics of the three banana cultivars as influenced by the inoculation of three endophytes. both l. yeojuensis gn11­20 and l. yeojuensis sbm36a have growth­promoting properties in tissue­ cultured banana plants at 14 days post­inoculation; this positively affects plants and may increase if treatments are extended for a longer incubation time (e.g., a month). the study supports the hypothesis that bacterial endophytes from healthy plants benefit their host plant. nevertheless, not all endophytes within the same species have plant growth­ promoting properties, as demonstrated by l. yeojuensis sbm36c. two isolates ­ l. yeojuensis sbm36a and l. yeojuensis gn11­20 ­ significantly enhanced plant height in ‘lakatan’. the l. yeojuensis gn11­20 further improved shoot emergence in ‘grand nain’ plants. these results suggest a symbiotic relationship between the two luteibacter yeojuensis isolates and bananas. on the other hand, l. yeojuensis sbm36c did not significantly affect the growth of banana plants regarding plant height, number of shoots, and roots. several factors may have affected the performance of the three bacterial isolates or the endophytes’ successful colonization in the plant host. these factors include plant genotype and tissue type (hardoim et al., 2015). the longevity of exposure to the endophyte could also have an effect. extending the inoculation period from 2 weeks to 4 weeks might increase the plant growth­promoting activity of the endophyte. none of the l. yeojuensis isolates increased the number of roots in the three genotypes tested. previous reports have recorded the potential of luteibacter rhizovicinus for enhanced root development in barley, specifically leading to higher weight and length of the roots (guglielmetti et al., 2013). hence, investigating the potential of the endophytes isolated for root development might also lead to a further understanding their plant growth­ promoting activities. the improved plant height and shoot production by l. yeojuensis strains isolated in this study might be stimulated by the increased iaa synthesis (pieterse et al., 2009). luteibacter sp., as an endohyphal bacterium (endophyte that forms a symbiotic relationship with a fungus) of platycladus orientalis, increased indole­acetic acid (iaa) production, resulting in significantly higher seedling and root length (hoffman et al., 2013). the antagonistic property of l. yeojuensis to plant pathogens has also been associated with high indolic compound production by l. yeojuensis. however, further investigation is needed since endophytic bacteria can utilize several other mechanisms (e.g., phosphate solubilization, gelatinase, and chitinase production) for plant growth improvement (liu et al., 2017; tang et al., 2020). several luteibacter species are endophytes of economically important crops such as rice (raj et al., 2019), and apple (piagnini et al., 2007) acting either as plant­growth promoters or biocontrol agents. in bananas, luteibacter sp. has been previously detected as an endophyte of ‘gros michel’ (köberl et al. , 2015) and was later found to have an antagonistic relationship with fusarium oxysporum f. sp. cubense tr4 (foc tr4) that causes severe wilt disease in bananas (köberl et al., 2017; nakkeeran et al., 2021). this study isolated and identified three strains of luteibacter species, specifically l. yeojuensis, from healthy banana plants. this is the first confirmed report of l. yeojuensis as an endophyte of banana ‘lakatan’, ‘grand nain’, and ‘saba’. the cultivar grand nain, a cavendish adv. hort. sci., 2025 39(1): 1­10 8 azevedo j.l., maccheroni w. jr., pereira j.o., de araújo w.l., 2000 ­ endophytic microorganisms: a review on insect control and recent advances on tropical plants. ­ elect. j. biotech., 3(1): 15­16. azman a.s., mawang c.i., abubakar s., 2018 ­ bacterial pigments: the bioactivities and as an alternative for therapeutic applications. ­ nat. product comm., 13(12): 1748. buck j.d., 1982 ­ nonstaining (koh) method for determination of gram reactions of marine bacteria. appl. environ. microbiol., 44(4): 992­993. cassells a.c., 1991 ­ problems in tissue culture: culture contamination, pp. 31­44. ­ in: debergh p.c., and r.h. zimmerman (eds.) micropropagation: technology and application. springer, dordrecht, netherlands, pp. 484. chen w.p., kuo t.t., 1993 ­ a simple and rapid method for the preparation of gram‐negative bacterial genomic dna. ­ nucleic acids res., 21(9): 2260. cruz m.a., balendres m.a., 2021 ­ dragon fruit calli development and fungal contamination as influenced by activated charcoal. ­ j. professional assoc. cactus devel., 23: 58­73. dieser m., greenwood m., foreman c.m., 2010 ­ carotenoid pigmentation in antarctic heterotrophic bacteria as a strategy to withstand environmental stresses. ­ arctic antarctica alpine res., 42: 396­405. fao, 2023 ­ banana market review. preliminary results 2023. ­ fao, rome, italy, pp. 1­12. fernandes t.p., nietsche s., costa m.r., xavier a.a., pereira d.f.g.s., pereira m.c.t., 2013 ­ potential use of endophytic bacteria to promote the plant growth of micropropagated banana cultivar prata anã. ­ african j. biotechn., 12(31): 4915­4919. guglielmetti s., basilico r., taverniti v., arioli s., piagnani c., bernacchi a., 2013 ­ luteibacter rhizovicinus mimr1 promotes root development in barley (hordeum vulgare l.) under laboratory conditions. ­ world j. microbiol. biotech., 29: 2025­ 2032. hardoim p.r., van overbeek l.s., berg g., pirttilä a.m., compant s., campisano a., döring m., sessitsch a., 2015 ­ the hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. ­ microbiol. mol. biol. rev., 79: 293­320. jie l., zifeng w., lixiang c., hongming t., patrik i., zide j., shining z., 2009 ­ artificial inoculation of banana tissue culture plantlets with indigenous endophytes originally derived from native banana plants. ­biol. control, 51: 427­434. johansen j.e., binnerup s.j., kroer n., molbak l., 2005 ­ luteibacter rhizovicinus gen. nov., sp. nov., a yellow‐pigmented gammaproteobacterium isolated from the rhizosphere of barley (hordeum vulgare l.). ­ int. j. syst. evol. microbiol., 55(pt 6): 2285­2291. kampfer p., lodders n., falsen e., 2009 ­ luteibacter banana cultivar group member, is susceptible to foc tr4. hence, it would be worthwhile to test the bioactivity of the three l. yeojuensis strains in this study to foc tr4 strains found in the country. this study reports three luteibacter yeojuensis strains as banana endophytes for the first time. it further demonstrates the growth­promoting potential of two l. yeojuensis strains in three tissue­ cultured banana plants ‘grand nain’, ‘saba’, and ‘lakatan’. the bacterial strains could be used to develop bioinoculants to improve plant growth in the future. however, its effect on other banana cultivars not used in this study should be further explored. investigating the role of isolated endophytes as potential biological agents of diseases in bananas would be worthwhile research. acknowledgements the authors thank fe dela cueva, yron retuta, diane biglete, pamela quintos, and ms. loida pascual for their assistance. we also thank flor cuevas, rochelle delgado of the fruit crops section, olivia damasco, and michelle lyka valle­descalsota of the national plant genetic resources 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methamidophos‐degrading bacterium isolated from a methamidophos‐ manufacturing factory. ­ curr. microbiol., 62(1): 289­ 295. zhang j., madden t.l., 1997 ­ powerblast: a new network blast application for interactive or automated sequence analysis and annotation. ­ genome res., 7(6): 649­656. 143 1. introduction strawberry (fragaria x ananassa duch.), a small fruit crop and a hybrid of two highly variable octoploid species, has adapted to extremely different environmental conditions (rieger, 2005). prerequisites for successful strawberry growing are suitable climate, cultivars, soil and nutrition (almaliotis et al., 2002). seed priming enhances seed performance by rapid and uniform germination in normal and vigorous seedlings, leading to faster and better germination in different crops (cantliffe, 2003). priming is responsible for the repair of age-related cellular and sub cellular damage of low vigour seeds that may accumulate during seed development. priming of seed promotes germination by repairing the damaged proteins, rna and dna (koehler et al., 1997). several treatments have been used with the aim of improving strawberry achene (seed) germination: humidity (guttridge and bright, 1978); exposure to red light (iyer et al., 1975); and osmotic pre-treatment (hanke, 1993). with the rapid growth of nanotechnology, there are increasing concerns about the potential adverse impact of engineered nanoparticles (enps) in the environment. however, our understanding of how enps may affect organisms within natural ecosystems lags far behind our rapidly increasing ability to engineer novel nanomaterials (bernhardt et al., 2010). on the other hand, lin and xing (2007) studied the positive effects of suspensions of nanoparticles on seed germination and root growth of six different crop species [radish (raphanus sativus), rape (brassica napus), rye grass (lolium perenne), lettuce (lactuca sativa), corn (zea mays) and cucumber (cucumis sativus)]. also, the effects of nano-tio2 (rutile) and nonnano-tio 2 on the germination and growth of naturally aged spinach seeds were studied and an increase of these factors was observed at 0.25-4‰ nano-tio 2 treatment (zheng et al., 2005). feizi et al. (2011) reported that nano-tio 2 at suitable concentration could promote the seed germination of wheat in comparison to bulk tio 2 while at high concentrations it had an inhibitory or no effect on the crop. limited studies have been done on the effects of nanoparticles on crops and thus, we decided to investigate the phytotoxicity or positive effects of different concentrations of nano-tio 2 on seed germination and seedling growth of strawberry. 2. materials and methods in order to evaluate the effect of nano priming on the quality of seedling production during germination in strawberry (fragaria ananassa l.), this experiment was conducted in 2013 at shahid chamran university ahvaz in iran using a factorial design with eight replications. seeds of strawberry (fragaria ananassa cv. queen eliza) were from the gene bank of iran, at seed and plant improvement institute, tehran (karaj). the factors studied included different concentrations of tio 2 (control, 3.5, 5.5, 7.5 and 9.55%) and time (24 and 48 h). in order to prepare nano anatase solutions a stock solution with the highest application of anatase nanoparticles (tio2) on strawberry seed germination (fragaria ananassa l.) h.r. hosseini*, m. chehrazi*, e.h. dehkourdi*, m. hosseini** * department of horticultural sciences, faculty of agricultural sciences, university of shadid chamran ahvaz, khuzestan, iran. ** department of management sciences, university of vali asr, kerman, iran. key words: anatase, germination, nano, strawberry,tio 2. abstract: priming enhances germination, establishment and yields in a range of crops in many diverse environments. this experiment evaluated the effects of soaking strawberry seeds in different concentrations (0, 3.5, 5.5, 7.5 and 9.5 percentage) of nano anatase on germination parameters (germination percentage, germination rate index, radicle and plumule length, fresh weight of seedlings and vigor index) using a factorial design with eight replications. results showed that an increase in the concentration of nano anatase led to significant differences in the percentage of germination, germination rate index, root and shoot length, fresh weight and vigor index of seedlings. the best nano anatase concentration was found to be 7.5%. adv. hort. sci., 2013 27(4): 143-146 received for publication 10 december 2013 accepted for publication 14 january 2014 144 concentration (9.5%) was fixed and then with dilution of this stock solution, various concentrations of nano anatase were obtained. strawberry seeds were sterilized using sodium hypochlorite solution (1%) for 10 min, then washed several times with distilled water, and soaked in nano-tio 2 solutions at different concentrations. seeds were placed in disinfected petri dishes, each dish contained 100 seeds. all of the petri dishes were irrigated using distilled water. seeds were allowed to germinate at 25±3°c for 14 days. every day the number of seeds with visible radicle were counted and recorded as sprouted seeds; the length of seedling shoot and root were also measured. germination rate [eq. (1)] and germination percentage [eq. (2)] were calculated using the following formula (hosseini et al., 2013): gr= ∑ n ∑ dn (1) where gr is the germination rate, σn is the number of seeds germinated on the day, and σdn is the number of days from the start of experiment. gp= ∑ n n ×100 (2) where gp is the germination percent, σn is the number of seeds germinated until the last day of experiments, and n is the total number of seeds. seed vigor indices [eq. (3)] were calculated according to the following formula (hosseini et al., 2013): vi= (rl+pl) ×gp (3) where vi is the seed vigor index, rlis radicle length, pl is the plume length, and gp the germination percentage. statistical analysis the data were analyzed using sas 9.1 software. the significant levels of difference for all measured traits were calculated, and the means were compared by the multiplerange duncan test at 1% level. 3. results and discussion analysis of variance showed that treatment of nano anatase led to significant differences in germination percentage, germination rate, root and shoot length, allometric index and vigor index of the seedlings. time treatment was not significant for root, shoot, seedling length and seedling allometric coefficient. interaction of anatase nanoparticles and time treatment was not significant for root and shoot length and seedling allometric coefficient (table 1-3). the interaction of anatase nanoparticles and time showed the lowest percentage of germination in the treattable 1 analysis of variance of strawberry seed germination indices affected by nano anatase sources of variation df percentage of germination germination rate shoot length root length seedling length allometric vi nano-tio 2 4 2260.6 * 0.184 * 11.077 * 15.366 * 0.525 * 3.33 * 103028.5 * time 1 136.9 * 1.089 * 0.196 ns 0. 016 ns 0.0022 ns 0.049 ns 5712.1 * nano*time 4 76.9 * 0.078 * 0.179 ns 0.0053 ns 0.001 ns 0.087 ns 3868.1 * error 30 13.7 0.180 0.148 0.145 0.004 0.069 578.23 *= significant at 1% level. ns= non-significant. table 2 effect of nano anatase on seed germination of strawberry after 24 hours concentration germination (%) germination rate shoot length (cm) root length (cm) fresh weight (g) 0 % 10.97 e 0.60 d 1.78 e 2.0 d 0.012 d 3.5 % 40.10 d 0.60 c 8.50 d 4.5 c 0.021 c 5.5 % 68.80 ab 0.67 b 9.10 c 4.4 c 0.029 b 7.5 % 92.46 a 1.20 a 12.24 a 6.0 a 0.034 a 9.5 % 74.64 b 1.00 ab 10.00 b 4.8 b 0.030 b means with different letters at each column are statistically different at 1% level. table 3 effect of nano anatase on seed germination of strawberry after 48 hours concentration germination (%) germination rate shoot length (cm) root length (cm) fresh weight (g) 0 % 0 e 0 e 0 e 0 d 0 d 3.5 % 23.2 d 0.17 d 3.5 d 2.5 c 0.011 c 5.5 % 40.6 b 0.26 b 5.3 c 2.9 c 0.017 b 7.5 % 87.6 a 0.56 a 10.3 a 5 a 0.030 a 9.5 % 46.3 bc 0.35ab 7.1 b 3.3 b 0.025 b means with different letters at each column are statistically different at 1% level. 145 ment without nanoparticles and the highest at a concentration of 7.5% of nanoparticles for 48 h (fig. 1). zheng et al. (2007) reported that the significant effect of nano-sized tio 2 on germination is probably due to the small particle size which allows penetration into the seed during the treatment period, exerting its enhancing functions during growth. results showed that the lowest germination rate resulted from treatment without nanoparticles while the highest rates were obtained at a concentration of 7.5% of nanoparticles in 24 and 48 h. the key to increase seed geermination is the penetration of nanomaterial into the seed (hashemi and mousavi, 2013) (fig. 2). khodakovskaya et al. (2009) mentioned that the carbon nanotubes can effectively penetrate through the seed coat, thus influencing seed germination. exposure of tomato seeds to carbon nanotubes (cnts) resulted in enhanced seed germination and growth rate. among the different nanoparticle tio 2 concentrations, 7.5% for both times tested (24 and 48 h) showed the maximum seedling vigor index while the control showed the lowest (fig. 3). effects positive of nano anatase are reported that nano-tio 2 (anatase) improved plant growth by enhanced nitrogen metabolism (yang, 2006) that promotes the absorption of nitrate in spinach and, accelerating conversion of inorganic nitrogen into organic nitrogen, thereby increasing the fresh weights and dry weights. studies also showed the effects of nitrogen photoreduction on the improved growth of treated spinach plant (yang, 2007). effects of nano-tio 2 (anatase) on the content of light harvesting complex ii (lhc ii) on thylakoid membranes of spinach was studied and it showed an increase in lhc ii content (mingyu, 2007 a). these promote energy transfer and oxygen evolution in photosystem ii (ps ii) of spinach (hong, 2005). it has also been found that nano anatase tio 2 promoted antioxidant stress by decreasing the accumulation of superoxide radicals, hydrogen peroxide, malonyldialdehyde content and enhance the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase and thereby increase the evolution oxygen rate in spinach chloroplasts under uvb radiation (mingyu, 2007 b). the results indicated that nano sized tio 2 in an appropriate concentration could promote the seed germination and seedling growth of strawberry. the results show that with increasing concentration of nano anatase to 7.5% increased germination parameters. the seedlings grown with nano anatase increased length when compare to the control seedlings. effect of nano anatase on root, shoot and seedling strawberry may be due to early emergence induced by nano anatase treatment as compared to control seeds. rapid embryo growth resulted when the obstacle to germination was removed. although the concentration of 9.5% caused lower germination parameters toward concentration of 7.5%, showed more desirable effects of the concentration of 5.5% nano anatase and control. in all treatments, the better result was obtained in time of 48 hour. in order to understand the possible benefits of applying nanomaterials in agriculture, it is important to analyze penetration and transport of nanoparticles in the plants. references fig. 1 effect of interaction nano anatase and time on germination percentage of strawberry seeds. fig. 2 effect of interaction nano anatase and time on germination rate of strawberry seeds. fig. 3 effect of interaction nano anatase and time on vigor index of strawberry seeds. 146 almaliotis d., velmis d., bladenopoulou s., karapetsas n., 2002 leaf nutrient levels of strawberries (cv. tudla) in relation to crop yield. acta horticulturae, 567: 447-450. bernhardt e., colman b., hochella m., cardinale b., nisbet r., 2010 an ecological perspective on nanomaterial impacts in the environment. journal of environmental quality, 39: 1954-1965. cantliffe d.j., 2003 seed enhancements. acta horticulturae, 607: 53-59. feizi h., razavi p., shahtahmaseb n., fotovat a., 2011 impact of bulk and nanosized titanium dioxide (tio 2 ) on wheat seed germination and seedling growth. biol. trace elem. res., 146: 101-106. guttridge c., bright s., 1978 accelerating and synchronizing germination of strawberry seeds by osmotic pretreatments. euphytica, 3: 843-848. hanke v., 1993 untersuchungen zur keimung von achänen bei erdbeere. erwerbsobstbau, 35(4): 105-109. hashemi e., mousavi m., 2013 effect of anatase nanoparticles (tio 2 ) on parsely seed germination (petroselium crispum) in vitro. biol. trace elem. res., 155: 283-286. hong f., 2005. effect of nano tio 2 on spectral characterization of photosystem particles from spinach. 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(eds.) introduction to fruit crops. haworth food & agricultural products press, new york, pp. 462. yang f., 2006 influences of nanoanatase tio 2 on the nitrogen metabolism of growing spinach. biol. trace elem. res., 110: 179-190. yang f., 2007 the improvement of spinach growth by nanoanatase tio 2 treatment is related to nitrogen photoreduction. biol. trace elem. res., 119: 77-88. zheng l., hong f., lu s., liu c., 2005 effect of nanotio 2 on strength of naturally aged seeds and growth of spinach. biology trace elemental reserch, 105: 839-843. zheng l., minguy s., xiao w., chao l., chunxiang q., liang c., hung h., xiaoqing l., hong f., 2007 effect of nano anatase on spectral characteristics and distribution of lhcll on the thylakoid memberance of spinach. biol trace elem res., 120: 273-280. impaginato 113 adv. hort. sci., 2025 39(2): 113­123 doi: 10.36253/ahsc­17221 https://oaj.fupress.net/index.php/ahs effect of partial­extreme root restriction and nutrient solution concentration on the performance of hydroponically grown tomato a.w. ayarna 1, s. tsukagoshi 2, h.o. sintim 3 (*), j. ulzen 1, j.n. amissah 4, m. bukari 3, s.a. poku 5, m. takagaki 2, n. lu2, s. adjei­nsiah 1, g.o. nkansah 1 1 forest and horticultural crops research centre, school of agriculture, college of basic and applied sciences, university of ghana, legon, p.o. box lg 25 accra, ghana. 2 center for environment, health and field sciences, chiba university, kashiwanoha 6‐2‐1, kashiwa, chiba 277‐0882, japan. 3 institute of applied science and technology, college of basic and applied sciences, university of ghana, legon, p.o. box lg 25, accra, ghana. 4 department of crop science, college of basic and applied sciences, university of ghana, legon, p.o. box lg 25, accra, ghana. 5 department of plant and environmental biology, college of basic and applied sciences, university of ghana, legon, p.o. box lg 25, accra, ghana. key words: fruit quality, hydroponics, nutrient mix strength, photosynthesis, root confinement, water uptake. abstract: tomato is a valuable agricultural commodity widely used across africa with the potential to contribute to food and nutritional security. however, its yield, quality, and profitability are hindered by several challenges. the study evaluated the impact of partial­extreme root restriction and no root restriction on the performance of jaguar tomato cultivar in two different nutrient solution concentrations: standard (2.4 ds m­1) and half concentration (1.2 ds m­1). the cultivation spanned three months using a recirculating hydroponic system arranged in a 2 x 2 factorial in a randomized complete block design with three replications. data were collected on physio­morphological responses, yield, fruit quality, and water uptake. plant growth, leaf gas exchange, yield, fruit quality, total water uptake, and root growth were significantly influenced by the nutrient solution concentration with root restriction. particularly, plant growth, photosynthesis, total water use (52­ 62%), and yield were significantly reduced but fruit quality was improved by 25% compared to previous findings in ghana. conversely, the standard nutrient solution concentration without root restriction recorded the highest yield of 32.4 kg m­2y­1. these findings can serve as a manipulative hydroponic tool to increase tomato productivity and resource­use efficiency, especially in regions with limited water availability. (*) corresponding author: hosintim@ug.edu.gh citation: ayarna a.w., tsukagoshi s., sintim h.o., ulzen j., amissah j.n., bukari m., poku s.a., takagaki m., lu n., adjei­nsiah s., nkansah g.o., 2025 ­ effect of partial‐extreme root restriction and nutrient solution concentration on the performance of hydroponically grown tomato. ­ adv. hort. sci., 39(2): 113­123. orcid: aaw: 0000­0001­7189­4302; ts: 0000­0002­ 2891­1530; sho: 0000­0003­4173­4089; uj: 0000­0001­7378­1002; ajn: 0000­0003­4520­ 4958; bm: 0009­0001­4606­6633; psa: 0009­ 0001­0260­6933; tm: 0000­0002­3200­7587; ln: 0000­0001­7585­4279; ans: 0000­0002­7394­ 4913; ngo: 0009­0006­9794­9117 copyright: © 2025 ayarna a.w., tsukagoshi s., sintim h.o., ulzen j., amissah j.n., bukari m., poku s.a., takagaki m., lu n., adjei­nsiah s., nkansah g.o. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. author contribution: conceptualization: awa, hos, st, ju, mb, ans, jna, sap; methodology: awa, hos, st, gon, mt, ju, sap, nl, mb, ans; formal analysis and inve­ stigation: awa, hos, st, gon, ju, mt, nl, mb, ans; writing ­ original draft preparation: awa, hos, st, gon, mt, nl, mb, ans, jna, ju; writing ­ review and editing: awa, hos, st, gon, mt, nl, mb, ans, jna, ju; funding acquisition: awa, hos, st, gon, mt, nl, mb, ans, jna, ja; resources: awa, st, mt, nl, jna, ju, ans; supervision: awa, st, gon, jna, mt, nl, hos, ju, ans. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 24 january 2025 accepted for publication 30 april 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17221 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-7189-4302 http://orcid.org/0000-0002-2891-1530 http://orcid.org/0000-0002-2891-1530 http://orcid.org/0000-0003-4173-4089 http://orcid.org/0000-0001-7378-1002 http://orcid.org/0000-0003-4520-4958 http://orcid.org/0000-0003-4520-4958 http://orcid.org/0009-0001-4606-6633 http://orcid.org/0009-0001-0260-6933 http://orcid.org/0009-0001-0260-6933 http://orcid.org/0000-0002-3200-7587 http://orcid.org/0000-0001-7585-4279 http://orcid.org/0000-0002-7394-4913 http://orcid.org/0000-0002-7394-4913 http://orcid.org/0009-0006-9794-9117 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 113­123 114 1. introduction tomato (solanum lycopersicum l.) is a globally important crop, which is widely grown using various cultivation systems in different countries. the crop is cultivated for its edible fruits, which are used as a vegetable, for medicinal purposes, among others (quinet et al., 2019). the current target for growing tomatoes among industrialized countries is to meet medicinal and nutritional needs. one of the most efficient but cost­effective cultivation system as adopted in some industrialized countries including japan is the ‘low node­order pinching at high­density planting’ (ln&hd) (watanabe, 2006; takahashi et al., 2012; kinoshita et al., 2014). this system adopts a low substrate volume at high­density cultivation plus pinching (topping) between the first and the fourth truss. with the low substrate volume, the plants are subjected to root restriction. vegetable production using root restriction is becoming popular especially where there is a need to adapt to adverse growing conditions, such as space constraints, limited water availability, and extreme temperatures (shi et al., 2008; yamaura et al., 2020). root restriction is a cultivation strategy that involves deliberately confining plant roots within smaller container sizes with low substrate volume, thereby limiting their natural expansion. this technique influences root architecture and physiological processes, subsequently influencing overall plant growth, development, and resource allocation. root restriction affects the physiology of grown plants (peterson and krizek, 1992; salisu et al., 2018). the findings of shi et al. (2008), mugnai and al­debei (2011), and campany et al. (2017) revealed that root restriction impairs the photosynthetic process due to a reduction in stomatal conductance. a reduction in photosynthesis in root­restricted plants might also be attributed to the physiological downregulation of photosynthetic activities due to high carbohydrate accumulation in the shoots of the plants (pezeshki and santo, 1998). however, other authors have indicated no significant differences in photosynthetic rate, stomatal conductance, intercellular co₂ concentration, and transpiration between root­ restricted and control plants (kharkina et al., 1999; zakaria et al., 2020). an earlier study by hieke et al. (2002) showed that there was no inhibition of photosynthesis once there was new plant shoot regrowth. root restriction has also been reported to reduce plant growth (ismail and noor, 1966; mugnai and al­ debei, 2011; ayarna et al., 2021). bihmidine et al. (2013) revealed that photosynthates were rather translocated into the stems of root­restricted pepper plants when the reproductive sinks were limited, leading to a yield reduction of 23%. root restriction has also been reported to reduce fruit yield in tomato (saito et al., 2008). root restriction reduces water uptake (saito et al., 2008), leading to a subsequent reduction in transpiration (bar­tal et al., 1994). using root restriction can increase the sugar content of tomato (li et al., 2022). root restriction has been reported to increase total sugar content due to reduced water uptake (zakaria et al., 2020). however, saito et al. (2008) reported that root restriction did not affect the sugar content of tomato but reduced its water uptake. conventional root restriction confines the root system within the grow pot throughout the plant’s growth cycle, limiting any further root expansion. in contrast, partial­extreme root restriction (characterized by a very low substrate volume, such as 0.25 l) utilizes a small (such as 0.25 l capacity) grow pot with an open base, initially imposing spatial confinement before allowing root extension beyond the restricted volume. after an initial phase of extreme root restriction in the small pot, this approach is expected to promote continuous root proliferation and growth, enhancing resource use efficiency and overall plant performance. partial root restriction in tomato has been reported by ayarna et al. (2021), who revealed that partially root­restricted plants produced more fine young roots, which were more efficient in the uptake of water and nutrients, subsequently increasing tomato yield. in hydroponic cultivation systems, nutrients are supplied as a nutrient solution for plant uptake and utilization. hoagland (1929) and schwarz et al. (2002) emphasized that nutrient solution formulation should be synchronized with the cultivation system as well as the associated crop. many nutrient solution formulations with appropriate concentrations have been developed to provide adequate nutrients for plant use (jones, 1982; sakamoto and suzuki, 2020). however, improper or disproportionate formulation of nutrient solutions can adversely affect crop yield at any growth stage. ayarna et al. ‐ partial extreme root restriction 115 2. materials and methods experimental materials and procedures the study was conducted between february and april 2024 at the university of ghana’s forest and horticultural crops research centre at kade, ghana (43vx+ggg), in a greenhouse. jaguar, a tropical tomato cultivar (technisem savanna seed company limited­france) was used for the study. the greenhouse daily ambient temperature and humidity were recorded using thermorecorder­tr­72wb (t&d holdings, inc., tokyo, japan). two factors, namely root restriction of tomato and varied nutrient solutions of standard and halve concentrations, were evaluated. the tomato plants were subjected to partial­extreme root restriction (fig. 1) as the main treatment, which was compared to the control treatment, with no root restriction. the adoption of extreme root restriction with a 0.20 l substrate volume in this study followed the method of zhang et al. (2015), who subjected tomato plants to extreme root restriction using a 0.25 l extreme­ low substrate volume in d­trays, geared toward improving fruit quality. however, the pattern of partial­extreme root restriction was after ayarna et al. (2021). the cultivation of plants was carried out in many growers have attempted to use high amounts of fertilizer to achieve higher yields, but this practice has resulted in poor performance, with reduced yield and fruit quality (zhang et al., 2017). nutrient solutions in hydroponic systems have low buffer capacity (agius et al., 2022), which can negatively impact plant growth. to prevent this, lu et al. (2022) emphasized the need for judicious nutrient solution management. when the nutrient solution concentration (nsc) is relatively low (1.5 ds m⁻¹) in unrestricted root conditions, nutrient availability is inadequate, reducing fruit quality (cliff et al., 2012; beesigamukama et al., 2020) and causing low nutrient stress, which hampers plant growth, photosynthesis, and stomatal conductance (beesigamukama et al., 2020; lu et al., 2022). conversely, while a high nsc (4.5 ds m­¹) leads to excess nutrient availability, causing stress and weakening plant growth (anjum et al., 2011; rosadi et al., 2014), it can also improve fruit quality by enhancing photosynthetic rate, transpiration rate, and stomatal conductance (wang, 2017; yang et al., 2017). meanwhile, tomato yield is not adversely affected at moderate nsc levels ranging from 1.5 to 2.4 ds m⁻¹ (veit­köhler et al., 1999). depending on the grower’s objectives, nutrient solutions are maintained at 1.2 ds m⁻¹ or higher. the enshi nutrient solution recipe has been formulated for the cultivation of all vegetable crops at a standard concentration of 2.4 ds m⁻¹. a half­concentration of the enshi recipe (1.2 ds m⁻¹) has been employed for the cultivation of root­restricted tomatoes in japan with success. in general, extreme root restriction and higher nutrient solution concentration (nsc) are strategies specifically aimed at enhancing tomato fruit quality, albeit at the expense of yield. ghana consistently records low tomato yields and a low sugar content of 3.5­5.6% brix (nkansah et al., 2003), making it crucial for implementing effective strategies to improve tomato yield and sugar content in the country. there are relatively few studies or reports on the effects of nutrient solution concentration on tomatoes grown under extreme but partially restricted­root conditions. the objective of this study was to evaluate the effect of partial­ extreme root restriction and nutrient solution concentration on the performance of tomato, with the expectation that this approach would enhance the yield and fruit quality of tomato. fig. 1 ­ (a) schematic representation of partial­extreme root restriction in hydroponic tomato cultivation. (b) zones of extreme but partial root restriction and root proliferation. adv. hort. sci., 2025 39(2): 113­123 116 a recirculating hydroponic system, using cocopeat as the substrate. a substrate volume of 1.0 l was used per plant in all treatments. in the root restriction treatment, the substrate was segmented into 0.20 l (pot) plus 0.80 l (trough). in the 0.20 l pot, the plants were subjected to an initial extreme root restriction; after which finer young roots were expected to proliferate into the 0.80 l trough for further water and nutrient absorption. the control treatment (unrestricted plant) was grown directly in the trough, containing 1.0 l of cocopeat. a nutrient solution with a concentration of 2.4 ds m­1 was prepared following the enshi recipe (hori, 1966) as shown in table 1. this concentration was then halved through dilution to 1.2 ds m ­1. the nutrient solutions (1.2 and 2.4 ds m ­1) were maintained within a ph of 5.5­6.5 and were delivered to the root zone of each plant using a drip system, in accordance with the adopted treatments. tomato seeds from the evaluated cultivar were sown in cell trays filled with cocopeat as the sowing medium. the seeds were then watered; and placed in a dark chamber under greenhouse conditions until they germinated. the germinated seedlings were supplied daily with a nutrient solution concentration of 0.5 ds m⁻¹ using the nutrient film technique until the third week, when they were ready for transplanting. twenty seedlings were transplanted into each treatment on the third week after seed germination at a spacing of 0.2 by 1.2 m. the set­up used an automated irrigation system to supply the tomato plants with nutrient solutions (1.2 or 2.4 ds m⁻¹) for 24 minutes daily, following treatment conditions from transplanting to harvest. after anthesis, 1.0 ml l­1 4­chlorophenoxyacetic acid was sprayed on the flowers every other day to enhance fruit set. plants in each treatment were pinched at the last three leaves above the third truss to terminate further growth. fifteen plants were tagged for data collection in each treatment. data collection and experimental design data were collected on the following parameters: morphological and physiological responses, yield, and water use efficiency. morphological responses which were collected at 74 days after transplanting included: plant height, girth (measured below the third truss), and number of leaves per plant. physiological parameters were measured between 12:30 p.m. and 1:30 p.m. on the second and sixth weeks after transplanting. these included photosynthetic rate (pr), transpiration (tr), stomatal conductance (gs), and the intercellular co2 concentration (ci) using the li­6400 portable photosynthesis system (li­cor, lincoln, nebraska, usa). the leaves immediately below the first and third trusses were measured for photosynthetic parameters in the second and sixth weeks after transplanting, respectively. all mature ripe fruits were harvested and counted to determine total and average fruit weights. the average fruit weight was determined as the ratio of the total weight to the total number of harvested fruits. total sugar content (brix %) of the blended tomato juice was determined using the atago™ pocket refractometer. plant water uptake was measured as the difference between total volume of nutrient solution supplied and the volume of nutrient solution left in the reservoir 14 h after daily irrigation. the total water used was measured as the total amount of nutrient solution absorbed per plant in the cultivation period (74 d). water use efficiency was determined as the fruit yield per total water used per plant. after harvest, the fresh roots were cautiously extracted, wiped with a soft face towel and weighed. additionally, portions of the root, which proliferated beyond the zone of extreme root restriction were table 1 ­ characterization of the adopted nutrient recipe in terms of macro­ and micronutrients concentration nutrients standard nutrient solution concentration (2.4 ds m­1) macro‐nutrients mm nh4­n 1.3 no3­n 16 po4­p 1.3 k 8.0 ca 4.0 mg 2.0 so4­s 2.0 micro‐nutrients ppm fe 3.0 mn 0.5 cu 0.02 zn 0.05 mo 0.01 b 0.5 ayarna et al. ‐ partial extreme root restriction 117 transplanting) as shown in figure 4. the nsc had a significant impact on the physiological parameters of tomato during both the vegetative and reproductive growth stages (figs. 3 and 4). the interaction effect of root restriction and nsc significantly affected the physiological response of tomato at the two stages of growth. the standard nutrient solution concentration with root restriction markedly reduced the pr, ci, and gs compared to the unrestricted roots during the reproductive phase of growth (fig. 3). in contrast, during the vegetative stage, root restriction under half­strength nsc increased pr, while ci, gs, and tr decreased relative to the no­restriction treatment (fig. 3). collected and weight as the portion involved with water and nutrient uptake. the experiment was laid out in a 2 x 2 factorial in a randomized complete block designed with three replications. data collected were analyzed with the sisvar version 5.6 (ferreira, 2008) while the tukey’s honestly significant difference (hsd 0.05) was used to separate the means at p<0.05. grouped graphs were constructed using graphpad prism version 8.0 for windows, graphpad software, san diego, california usa. 3. results greenhouse ambient temperature and humidity the greenhouse ambient humidity and temperature recorded during the study are shown in figures 2 a and b. plant morphological and physiological responses partial­extreme root restriction (r) and nutrient solution concentration (nsc) significantly p<0.05 affected the growth of tomato (table 2). plant height, girth, and leaf number decreased with root restriction compared to the unrestricted roots. plant height and leaf number were significantly higher with the standard nsc compared to the half concentration. according to figure 3, the photosynthetic rate (pr) of the jaguar tomato was not affected by root restriction at both nutrient concentrations, even though the stomatal conductance (gs), transpiration (tr) and intercellular co2 concentration (ci) decreased significantly (p<0.05) at the vegetative phase (second week after transplanting) in the restricted treatment. compared to the control, root restriction reduced pr, gs, tr and ci significantly at the reproductive phase (sixth week after small letters compare means within root restriction, while capital letters compare means within nutrient solution concentration (nsc). values in the same column or row followed by the same letters indicate no significant difference according to tukey hsd (p<0.05). table 2 ­ morphological response of tomato to partial­extreme root restriction and nutrient solution concentration at 74 days after transplanting nutrient solution concentration root restriction plant height (cm) stem girth (mm) leaf number standard restricted 116.3 ± 0.88 aa 8.7 ± 0.07 ab 11.7 ± 2.19 bb unrestricted 117.3 ± 5.04 ab 11.7 ± 0.03 aa 20.0 ± 1.15 aa half concentration restricted 102.7 ± 2.30 bb 10.3 ± 0.07 aa 14.3 ± 0.33 ba unrestricted 161.0 ± 6.81 aa 10.0 ± 0.06 aa 17.0 ± 0.41 ab p­values <0.01 0.045 0.048 fig. 2 ­ greenhouse ambient humidity (a) and temperature (b) during the experiment. 118 adv. hort. sci., 2025 39(2): 113­123 yield and sugar content root restriction and the nsc did not significantly (p<0.05) affect the number of fruits produced per plant as shown in table 3. the yield, average fruit weight, and sugar content (brix%) were markedly affected by root restriction and the nsc. partial fig. 3 ­ physiological response of tomato to nsc and root restriction 2 weeks after transplanting. lowercase letters compare means within nsc, while uppercase letters compare means among nsc and levels of r. fig. 4 ­ physiological response of tomato to nsc and root restriction 6 weeks after transplanting. lowercase letters compare means within nsc, while uppercase letters compare means among nsc and levels of r. ayarna et al. ‐ partial extreme root restriction 119 extreme root restriction with the standard concentration of nutrient solution significantly reduced the yield and the average fruit weight of the cultivar compared to the other treatments. unrestricted roots grown in the standard nsc had the highest yield of 8.1 kg m­2 with a low sugar content of 4.6%. conversely, extreme partially restricted roots grown in the same nsc recorded the highest sugar content of 6.9% but with the lowest yield of 3.63 kg m­2. water uptake trend and water use efficiency, and root growth characteristics figure 5 illustrates that the trend of water uptake among the treatments were similar between day 1 and 28 but diverged on the 30th day after transplanting. water uptake in the restricted roots cultivated in the standard concentration was generally lower than the other treatments throughout the cultivation period. however, peak of water uptake in the other treatments was observed between the 33rd and 56th day after transplanting. the total water use (twu) and its efficiency were significantly (p<0.05) affected by root restriction as shown in table 4. total water use was markedly reduced in restricted roots compared to unrestricted roots, leading to a higher water use efficiency in the former. the amount of water used was influenced significantly by the concentration of nutrient solution. standard nsc reduced water uptake than the half concentration. root growth (fresh weight) was markedly affected by root restriction and nsc. root growth was markedly reduced by root restriction compared to the unrestricted. the standard nsc had a significant reducing effect on root growth than the half concentration. root proliferation (zone of root growth beyond the zone of extreme partial root restriction) was reduced with the standard (high) nsc. 4. discussion and conclusions plant root restriction is a cultivation technique in horticulture where roots are confined within a limited space and this subsequently limits the plant growth potential. numerous reports indicate that this technique generally results in improved fruit quality (sugar accumulation, anthocyanin enrichment) despite the reduction in photosynthesis (wu et al. 2018; zakaria et al., 2020). the adoption of high nutrient solution concentrations in soilless culture has been used singly as efforts to increase fig. 5 ­ influence of partial root restriction and nutrient solution concentration on the trend of tomato daily water uptake. table 3 ­ influence of root restriction and nutrient solution concentration on tomato yield, yield components, and sugar content small letters compare means within root restriction, while capital letters compare means within nsc and levels of root restriction. values in the same column or row followed by the same letters indicate no significant difference according to tukey hsd (p<0.05). parameter nutrient solution concentration restricted (mean ± se) unrestricted (mean ± se) p­value fruit number per plant standard 11.3 ± 0.5 aa 13.0 ± 0.6 aa 0.1963 half­conc 13.3 ± 0.9 aa 12.9 ± 0.129 aa yield (kg m⁻²) standard 3.88 ± 0.3 bb 8.08 ± 0.22 aa <0.01 half­conc 7.32 ± 0.19 aa 7.08 ± 0.13 ab average fruit weight (g) standard 76.2 ± 0.30 bb 149.6 ± 0.22 aa 0.0001 half­conc 140.6 ± 0.19 aa 130.9 ± 0.12 bb brix (%) standard 6.97 ± 0.09 aa 4.57 ± 0.03 bb <0.001 half­conc 4.43 ± 0.2 ab 4.2 ± 0.06 ab adv. hort. sci., 2025 39(2): 113­123 120 fruit quality but usage coupled with root restriction remains unclear hence this study tried to obtain plausible explanations to the mechanisms that influences plant growth, yield, fruit quality and water uptake under these two factors i.e., root restriction and nutrient solution concentration. physio‐morphological responses to plant root‐ restrictions and nsc in this study, plant root­restriction reduced the growth of tomato, which has been confirmed in the finding of kasai et al. (2012). the work of zakaria et al. (2020) on chili pepper reported a 14% reduction in plant height due to root restriction. however, the findings of this work showed that partial­extreme root restriction of tomato reduced plant height by 21% compared to the control. although these comparisons are from two different crops, the trends of the impact of root restriction are similar. the differences in absolute percentage change might be due to the extent of root mass reduction because of the initial extreme restriction imposed on tomato roots. tomato plants subjected to root restriction under the standard concentration also showed reduced plant growth in terms of height, girth, and leaf number. a high nsc is known to impair water uptake (ding et al., 2018), and in partial extreme root­restricted conditions this might have influenced the growth reduction. the tomato plants with extreme partially restricted roots showed decreased leaf gas exchange compared to the control plants six weeks after transplanting. an impairment in photosynthetic rate due to a reduction in stomatal conductance in root­ restricted plants has been confirmed in the works of shi et al. (2008), mugnai and al­debei (2011), and campany et al. (2017). nevertheless, the outcomes of this study differed from zakaria et al. (2020) and santos et al. (2022) findings, which indicated that leaf gas exchange of chili pepper and jenipapo was not significantly affected by root restriction. the standard nutrient solution concentration, with or without root restriction, induced a reduction in the photosynthetic rate of tomato compared to the half­strength, despite an increase in the tr, gs, and ci. this finding could be attributed to downregulation of photosynthetic rate due to water stress, particularly in the partially extreme root­ restricted plants, which demonstrated sink limitation resulting from a decrease in root mass. the findings table 4 ­ influence of root restriction and nutrient solution concentration (nsc) on water uptake and water use efficiency small letters compare means within root restriction, while capital letters compare means within nsc and levels of root restriction. values in the same column followed by the same letters indicate no significant difference according to tukey hsd (p<0.05). nutrient solution concentration restricted unrestricted total water uptake (l plant ‐1) standard 13.9 ± 0.058 bb 36.93 ± 0.14 aa half concentration 28.9 ± 0.46 ab 31.0 ± 0.48 ba p­value <0.001 water use efficiency (g fresh fruit weight l‐1) standard 259.2 ± 0.058 aa 218.8 ± 0.145 aa half concentration 253.3 ± 0.463 aa 228.5 ± 0.481 aa p­value 0.563 root fresh weight (g plant ‐1) standard 31.67 ± 1.67 bb 62.67 ± 1.15 ba half concentration 56.00 ± 1.73 ab 70.00 ± 1.45 aa p­value 0.002 proliferation root mass (% fresh weight plant‐1) standard 5.00 ± 1.15 b (16.1%) half concentration 12.67 ± 0.33 a (22.6%) p­value 0.034 ayarna et al. ‐ partial extreme root restriction 121 of beesigamukama et al. (2020) suggested that a low nsc induces nutrient stress hence, photosynthetic rate is significantly reduced. the findings of this study, however, showed that the photosynthetic rate of tomato could be reduced by 41% when grown in the standard nutrient solution concentration of 2.4 ds m­1. furthermore, the findings of the study revealed that partial extreme root­restricted tomato plants, which were grown in the standard nutrient solution concentration showed a 65­70% reduction (downregulation) in the photosynthetic rate at the generative phase. in other studies, lu et al. (2022) reported that a nsc of 1.5 ds m ­1 could induce nutrient stress, reducing the rate of photosynthesis of cherry tomato, but they reiterate that the rate of photosynthesis can only be maintained at a concentration of 3.0­5.0 ds m­1. on the other hand, this present study found that a nsc of 1.2 ds m­1 was sufficient to provide necessary nutrients to plants without causing any nutrient stress, as plants in the partially extreme restricted root treatment did not show signs of nutrient deficiency. additionally, when the 1.2 ds m­1 nsc is doubled, the cultivar turned out with a divergent response when the roots were restricted. yield, fruit quality, and water uptake under plant root‐restriction and nsc the tomato yield decreased significantly by 35.4% due to partial extreme root restriction. this restriction inhibited root growth, leading to a diminished sink capacity and ultimately inducing a downregulation of photosynthesis. bihmidine et al. (2013) found that pepper experienced a 23% decrease in yield due to root restriction. other studies (saito, et al., 2008; ayarna et al., 2021) have also reported yield reductions in tomatoes due to root restriction. partial extreme root restriction, in this study, increased the fruit quality (sugar content) of tomato by 52%, which is a 25% improvement over values previously recorded in ghana (nkansah et al., 2003). the findings of li et al. (2022) also reported that root restriction increased the sugar content of tomatoes. tomato fruits from the standard nsc had higher sugar content without affecting yield. this finding aligns with previous studies by veit­köhler et al. (1999) and wang (2017). findings from this study revealed that partial­extreme root restriction with the standard nsc increased the sugar content of tomato while the yield was markedly reduced. these two technical hydroponic tools could be employed to increase the sugar content of tomatoes, especially, the cherry type. furthermore, the synergistic effect of root restriction and standard nutrient solution concentration generally reduced water uptake in the tomato cultivar. the reduction in water uptake was markedly lower at the generative phase of growth. osmotic stress in the root environment most probably accounts for the remarkable reduced water uptake in the tomato plants that were subjected to root restriction in the standard nsc. after the initial extreme root restriction, subsequent root proliferation produced a smaller root mass with a higher absorptive surface area per unit due to the presence of finer, younger roots. however, these roots remain generally disadvantaged by a reduced overall absorptive capacity compared to unrestricted roots. under these conditions, an nsc of 2.4 ds m⁻¹ was sufficient to induce water stress, leading to reduced water uptake. this observation is consistent with the findings of saito et al. (2008) and liu et al. (2023), who reported that root restriction under high nsc conditions enhanced tomato fruit quality but reduced fruit size due to water stress. partial­ extreme root restriction of tomato reduced total water uptake by 37% compared to the control plants. water use was more efficient in the partially extreme root­restricted plants however, the yield was negatively affected because of a reduced photosynthetic rate with low dry matter production. the findings of ismael and dalia (1995) and bar­tal et al. (1994) confirmed that water uptake reduces with root restriction in tomato. a high nsc significantly decreased the uptake of water, and yield of tomato when roots are extremely confined (restricted). in an environment with extreme partial root restriction, the standard nsc, which denotes a higher nutrient solution concentration, induced a significant reduction in root growth. this suggests that the concentration of the nutrient solution has a notable impact on root growth in such conditions. in the environment with extreme root restriction, our observations indicate that 16.1% portion of root mass was present in the standard concentration of the nutrient solution, while 22.6% was evident in the half concentration. under extreme partial root­ restricted conditions, only 16­22% of the root mass was found to be most probably actively involved in adv. hort. sci., 2025 39(2): 113­123 122 water and nutrient uptake. reduced root growth indicates a decrease in the plant sink structure (capacity), which influenced the downregulation of photosynthesis and yield reduction. under normal growth conditions, slight changes in nsc may not have an adverse effect on tomato performance, unless the associated roots are restricted. when roots are extremely but partially restricted in their growth under the same nutrient solution concentration, there is a trade­off between sugar content and yield. in the low node order pinching at high density planting, partial­extreme root restriction and nsc are effective manipulative hydroponic tools for comparatively increasing the yield and fruit quality of tomato while conserving water. in general, the productivity of tomatoes could be improved at a cost­effective level since the cultivation system involves the use of low substrate volume. this growing system can allow four cultivation cycles of tomato per year. the sugar content of tomatoes grown in ghana could be improved by 25% with a corresponding yield of 14.5 kg m­2 y­1. cherry 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(in chinese). watanabe s., 2006 ­ new growing system for tomato with low node‐order pinching and high‐density planting. ­ proc. veg. tea sci., 3: 91­98. wu y., zhou j., wang j., men x., liu n., chen f., 2018 ­ effect of root restriction on quality and yield of watermelon cultivated in soilless facilities. ­ jiangsu agric. sci., 46: 113­115. yamaura h., fujita s., ikeda k., takano n., kanno k., matsuo s., zhao t., nakano a., iwasaki y., 2020 ­ year‐round production of tomato fruits by low node‐ order pinching and high‐density planting in subtropical area in japan. ­ acta horticulturae, 1296: 457­462. yang p., chen r., li j., wu y., zheng z., 2017 ­ effects of different irrigation limits and nutrient solution concentrations on tomato cultivated in substrate. ­ jiangsu agric. sci., 45: 126­128. zakaria n.i., ismail m.r., awang y., megat wahab p.e., berahim z., 2020 ­ effect of root restriction on the growth, photosynthesis rate, and source and sink relationship of chilli (capsicum annuum l.) grown in soilless culture. ­ biomed res. int., 2020. zhang p., senge m., yoshiyama k., ito k., dai y., zhang f., 2015 ­ effects of low salinity stress on growth, yield, and water use efficiency of tomato under soilless cultivation. ­ j. irrigation, drainage rural eng., 85: 15­21. untitled 100 preservation and premultiplication of selected grape material in trentino: collaboration between fems. michele all’adige and trentino grape-nurseries avit-consortium u. malossini*, l. gretter** * edmund mach foundtion, istituto agrario di s. michele all’adige, via edmondo mach, 1, 38010 san michele all’adige (tn), umberto.malossini@fmach.it. ** avit, consorzio vivaisti viticoli trentini, via r. guardini, 73, 38121 trento. in trentino, the edmund mach foundation-san michele all’adige (fem) is the institution responsible for the selection of grape propagation material for the wine industry, serving not only the local area. the premultiplication material, selected and maintained by fem, is released commercially in the vine-nursery chain in collaboration with the trentino grape nurseries consortium (avit). fem has made significant investments for the qualification of sanitary selection. in particular, the already existing structures (screen-house of approx. 500 m2) have been totally renovated and new openfield greenhouses/tunnels (with anti-insect nets) have been realized. special attention was paid to scientific training and updating of the personnel responsible for the control and treatment of clonal material primary sources. the personnel involved in the selection programs is also qualified thanks to participation in activities and projects carried out at national level, in collaboration with other premultiplication centres and scientific institutions, for the study and validation of official protocols of selection and/or premultiplication. active participation in these initiatives has enabled a constant updating of procedures for internal audit on selected materials. the preservation of “primary sources” of registered clones takes place both under screen house and in a micropropagation laboratory (tissue culture), allowing the possible “sanitation” of special accessions. this service is carried out also for other breeders. annually fem manages at their farm more than 3.5 hectares of surface area for “initial”, “basic” and “certified” categories of v. vinifera and rootstock propagation material an additional approx. 1.5 hectares of vineyards, obtained with fem selections but commercially classified in the “standard” category, is also under fem control. the “initial” materials of 84 accessions of v. vinifera and vitis hybrid rootstock clones are preserved under screen-house. these materials belong to clones registered by fem, some of them in association with other institutions, or preserved on behalf of third parties. each accession or clone is present with an average of five or more plants (grown in 70-l containers), depending on the “commercial” interest. the yield of buds or cuttings to supply grafting activity is significant. the two tunnel-like structures in the field (one for the production of rootstock cuttings and the other of v. vinifera scions) cover a total area of approx. 2,000 m2 with a defined mapping of every single vine. most of the materials under the tunnels belong to the “basic” category. in addition to vineyard management, a strict sanitary control is carried out by means of field surveys and visual monitoring of the main disease symptoms. plants are routinely sampled for diagnostic virus assays, particularly for the official verifications required by certification services. all the operations are carefully recorded as required for genuine traceability of nursery materials and as a guarantee in the subsequent stages of propagation. since 2008, avit acts as the only authorized nursery for management and marketing of “basic” category grafted rootlings of the fem clones. avit, along with its administrative office in collaboration with nucleo di premoltiplicazione viticola delle venezie, arranges reservations, production and distribution of selected rootlings. avit has recently been recognized as a co-breeder proponent, along with fem, of seven new clones (registered in the national catalogue). in 2012, a consortium was formed by fem and civit avit with the aim of pursuing a path of mutual interest, in the wake of innovation in the field of viticulture and wine making. adv. hort. sci., 2013 27(3): 100 25 1. introduction bud dormancy in temperate-zone deciduous fruit trees is an adaptive mechanism to survive unfavourable conditions during the winter (faust et al., 1997) and it is classified into three different stages: paradormancy, endodormancy and ecodormancy (lang, 1987). in autumn, buds enter a dormant state known as endodormancy after the trees stop growing and the leaves fall. during this state the trees cannot start bud growth even if the environmental conditions are favourable. endodormancy is broken by accumulation of low temperatures, known as chilling requirement (cr), and it depends on the species and cultivar (westwood, 1978; saure, 1985). however, a lack of adequate chilling to satisfy the cr to break endodormancy due to climate change (especially global warming) in recent years inhibits the normal growth of new organs in spring (sugiura et al., 2007). japanese pear [pyrus pyrifolia (burm. f.) nakai] is one of the most important fruits for the japanese and the earliest records of pear cultivation in japan date back 1300 years. commercial japanese pear cultivation increased with the development of a consumer society during the edo period (1600-1868). since then, japanese pear production underwent from 1920 to 1930, then again from 1950 to a peak in 1970; cultivation is now stable (tamura, 2006). figure 1 and table 1 show the main pear growing areas in japan. the average annual temperatures of these areas range from 12 to 17°c, and the average growing season temperature (april to september) is 19 to 23°c. in recent years, japanese pear cultivation has significantly increased in warm-winter regions (chen et al., 1995). in countries such as new zealand (kingston et al., 1990; klinac and geddes, 1995) and brazil (petri and herter, 2002; petri et al., 2002), a lack of winter chilling has caused problems in promoting bud break in pear during spring. several recent studies have focused on the breaking of dormancy in grapes (or et al., 2000, 2002; pang et al., 2007; halaly et al., 2008) by using hydrogen cyanamide (hc) as a tool to modify the breaking of endodormancy. in grapes, treatment of buds with hc increased bud dormancy in japanese pear y. takemura, f. tamura faculty of agriculture, tottori university, koyama, tottori 680-8553, japan. key words: budbreak, chill units, chilling requirements, climatic change, global warming, japanese pear, phytohormone. abstract: in this paper, after reviewed recent advances in research of endodormancy, research on japanese pear were reported. in the case of japanese pear, endodormancy was induced by low temperature of 5oc, without effect of day length. chilling requirement (cr) of japanese pear for completion of leaf bud endodormancy shows wide range from below 800 to 1800 chilling units (cu). we investigated the budbreak percent in taiwanese pear yokoyama, in japanese pear strain th3 and their f1 for 3 years. the percentage of budbreak in th3 was lower than yokoyama on any observational days and it gradually increased from early december to early january. the percentage of budbreak in f1 plants was widely distributed between that of yokoyama and th3 on all observational days. from results of chi-square test, it was suggested that pear plant had quantitative trait loci (qtl) as genetic factor to decide chilling requirement (cr) for breaking endodormancy. expression levels of gene encoding gast-like gibberellin (ga) regulated protein increased with development of endodormancy in japanese pear cultivars. aba concentrations in bud of japanese pear in open-field were increased with the induction of endodormancy by chilling. however, the aba concentrations in un-chilled plants were lower. adv. hort. sci., 2013 27(1-2): 25-32 corresponding author: takemura_yoshihiro67@yahoo.co.jp received for publication 28 november 2012 accepted for publication 19 february 2013 fig. 1 main producing prefectures in japan. 26 hydrogen peroxide concentration and inhibited catalase activity (perez et al., 2008). this is the first step in a cascade that up-regulates several signaling proteins such as transcription factors, protein phosphatases, and protein kinases (neill et al., 2002). application of hc has also been shown to result in transcriptional up-regulation of grape dormancy-breaking related protein kinase (gdbrpk), a sucrose non-fermenting protein kinase (snf-like protein), pyruvate decarboxylase (pdc), alcohol dehydrogenase (adh), thioredoxin h (trxh), glutathione s-transferase (gst), ascorbate peroxidase (apx), glutathione reductase (gr), and sucrose synthase (susy) (or et al., 2000; pérez and lira, 2005; keilin et al., 2007; halaly et al., 2008; pérez et al., 2008). however, the mechanism regulating induction and breakage of endodormancy in japanese pear remains unknown. in this paper, we review the progress of our research on endodormancy related problems, e.g. cr of cultivars, genetic factors of cr, endodormancy induction, gene expression in japanese pear over the past 10 years, and future trends. 2. materials and methods environmental factors of endodormancy induction the effects of temperature and day length during autumn on induction of endodormancy in japanese pear were examined. the experiment was conducted using two japanese pear cultivars [(pyrus pyrifolia (burm.f.) nakai)], ‘nijisseiki’, grafted onto p. betulaefolia bunge seedlings planted at tottori university, tottori, japan (35.5°n, 134.2°e). potted ‘nijisseiki’ pear trees were placed before endodormancy induction in a greenhouse kept at a minimum temperature above 18oc in september 2004 or in an open field; the photoperiod was controlled for 16 hrs or kept under natural conditions in each place, respectively. then, one year old shoot was collected and the percentage of budbreak was defined. the apical flower bud was cut, and shoots were divided into five-node cuttings containing five continuous lateral leaf or floral buds. the basal part of the cuttings was submerged in 0.03% (v/v) aluminum sulfate and 0.3% (v/v) 8-hydroxyquinoline. the cuttings were then maintained in a growth chamber at 23±1°c and 24-h photoperiod for four weeks. bud break is defined as a developmental stage characterized by swelling of the buds and the emergence of a green tip between scales (tamura et al., 1992). the incidence of bud break in each bud type was determined on five shingle shoots having five buds for 28 days. in addition to this experiment, ‘nijisseiki’ pear shoots were collected in late september 2005. shoots were submerged in 0.003% (v/v) aluminum sulfate and 0.3% (v/v) 8-hydroxyquinoline and treated at 5 and 15oc for 5, 7 and 14 days. after that bubreak percentage was defined. crs and forecasting model for endodormancy breaking in order to estimate the cr for breaking endodormancy in wild pear species and pear cultivars, cultivars grown in the orchard of tottori university and tottori horticultural experiment station (35.5°n, 133.7°e) during the 20082009 season were used. leaf bud break on the cuttings prepared from these trees was determined as previously described. the cr was calculated as chill unit (cu) values using the saitama method (asano and okuno, 1990) as described by tamura et al. (1997). effective chilling hours were calculated after october 31 when the largest negative accumulation was attained. in addition to this experiment, we also investigated the budbreak percentage in taiwanese pear yokoyama, japanese pear strain th3 which is a selected strain from the seedlings of self-pollinated ‘osa-nijisseiki’ having homozygote of s4sm gene and their f1 for three years (2008, 2009 and 2010 seasons). mechanisms involved in induction and breaking of endodormancy we isolated the candidate gene related to endodormancy breaking by suppression subtractive hybridization (ssh) method. total rna was isolated from ‘nijisseiki’ pear buds on 12 november 2005 (deepest period of endodormancy: dp) and 12 january 2006 (breaking period of endodormancy: bp). ssh was carried out between bud in bp (‘tester’) and bud in dp (‘driver’) using the pcrselect subtractive hybridization kit (clontech, palo alto, ca, usa) according to the manufacturer’s instructions, except for the modification of the firstand second-round pcr conditions. genes isolated as candidate genes related to bud endodormancy were analyzed by northern blot analysis using total rna isolated from buds of ‘nijisseiki’ and ‘kousui’ pear. 3. results and discussion endodormancy is induced in buds of deciduous fruit trees in autumn, and then it is broken by the accumulation of low temperature in winter. the report focused on endodormancy induction are few, but the necessary environment condition to induce endodormancy is reported to be short day condition (kawase, 1961) or low temperature (tohbe et al., 1998). in ‘delaware’ grape before endodormancy induction, buds treated long-days inhibited the induction of endodormancy than buds on natural condition (horiuchi et al., 1981). on the other hands, tamura et al. (1997) showed that the induction period of endodormancy in japanese pear varies greatly as calendar days from year to year. thus, the effects of temperature and day length during autumn on induction of endodormancy in japanese pear were tested. as a result, green house grown ‘gold nijisseiki’ trees kept high level of budbreak even though on mid-december, with no effect of photoperiod (table 2) (takemura et al., 2011). in contrast, the percentage of budbreak of trees decreased in both open field. thus, a lack of chilling by the heating treatment above 18oc inhibited endodormancy induction, but a 16 hours photoperiod did not prevent it. in addition to this experiment, we collected ‘nijisseiki’ pear shoots on late september, and then were 27 exposed at 5 and 15 oc for 5, 7 and 14 days. then percentage of budbreak was detected. as a result in the experiment, it was cleared that temperature of 5oc was effective for inducing bud endodormancy in the cuttings, whereas a temperature of 15oc was ineffective (fig. 2) (takemura et al., 2011). from these results, the endodermancy of the japanese pear was induced by low temperatures in autumn rather than short-day. the cu and developmental index (dvi) models were earlier developed to predict the day of breaking bud endodormancy in the japanese pear (asano and okuno, 1990; sugiura and honjo, 1997; tamura et al., 1997). the start day of the calculation and the low temperature accumulation on both models are decided as the day perceived low temperature or calendar day. to decide environmental factors of endodormancy induction is important to reduce the gap of start day in both models by climate change in the future. next, the effects of temperature and day length during autumn on induction of endodormancy in japanese pear was tested. from these results, the chilling requirement for completion of leaf bud endodormancy in pear plants ranged from below 400 to 1800 cu (table 3) (takemura et al., 2013). among the pear plants examined, ‘yokoyama’, which is originated in taiwan, had the lowest chilling requirement (below 400 cu). in wild pear species, p. fauriei showed the lowest chilling requirement (400-800 cu), followed by p. calleryana and p. dimorphophylla. in addition, akibae cultivar had the lowest cr among the japanese pear cultivars examined, and their cr was between 800 and 1000 cu (table 3) (takemura et al., 2013). this cultivar has the same level of cr of ‘hosui’ and ‘ninomiya’, the lowest cu cultivar observed among japanese pear varieties (tamura et al., 2001). therefore, these three cultivars may serve as suitable cultivars for propagation in warm-winter areas. on the other hand, there is no japanese pear which required low-chilling as shown in ‘anna’ apple, having 300 cu as chilling requirement. from these results, ‘yokoyama’ was selected as gene resource for breeding japanese pear requiring low-chilling. in 2003, ‘yokoyama’ pollen was pollinated with the flower of japanese pear strain ‘th3’ just after castration. ‘th3’ is a s1 of ‘osa-nijisseiki’, which has homozygous of s4sm as the gene of selfcompatibility haplotype. using the seedlings obtained, the budbreak percentage in the parents, ‘yokoyama’ and ‘th3’, and their f1 was investigated for 3 years. table 3 chilling requirement for breaking leaf bud endodormancy in pyrus plants evaluated by the seasonal changes in percent leaf budbreak (takemura et al., 2013) chilling requirement (cu) registered name -400 ‘yokoyama’ 400-800 p. fauriei 800-1000 p. calleryana, p. dimorphophylla, ‘ci li’, ‘akibae’, ‘hosui’, ‘ninomiya’ 1000-1200 p. betulaefolia, ‘kosui’, ‘wasekouzo’ 1200-1400 p. pyrifolia, ‘qui bai li’, ‘ya li’, ‘chojuro’, ‘hakko’, ‘niitaka’, ‘nijisseiki’, ‘taihaku’, ‘yakumo’ 1400-1600 p. longipes, ‘choju’, ‘hattastu’, ‘imamuraaki’, ‘kikusui’, ‘kumoi’, 1600-1800 p. aromatica, p. communis, ‘bai li’, ‘beijing bai li’, ‘akaho’, ‘amanogawa’, ‘doitsu’, ‘kimitsukawase’, ‘okusankichi’, ‘shinsetsu’, ‘shinsui’ table 2 effect of temperature and photoperiod on endodormancy induction of leaf bud in ‘gorld-nijisseiki’ pear (areas in japan) (takemura et al., 2011) temperature conditions photoperiod conditions bud break (%) sampling date/days of forcing 24 oct. 10 dec. 14 days 21 days 28 days 14 days 21 days 28 days greenhouse (18oc) 16 hr. 18.0 az 40.0 a 60.0 a 36.0 a 50.0 a 60.0 a natural 18.0 a 34.0 a 44.0 b 22.0 a 50.0 a 64.0 a open field 16 hr. 0.0 b 14.0 b 16.0 c 0.0 b 8.0 b 8.0 b natural 0.0 b 18.0 b 18.0 c 0.0 b 6.0 b 16.0 b z different letters within the same column show a significant difference at p < 0.05 by t-test. fig. 2 effect of temperature on endodormancy induction of leaf bud in ‘gorld-nijisseiki’ pear (takemura et al., 2011). (z) 28 days after forcing at 23oc. 28 the data obtained showed that the percentage of budbreak in ‘yokoyama’ was higher than 60% on all observational days. the percentage of budbreak in ‘th3’ was lower than in ‘yokoyama’ on all observational days, and it gradually increased from early december to early january. the percentage of budbreak in f1 plants was widely distributed between that of ‘yokoyama’ and ‘th3’. on 8 of all observational days, the average percentage of budbreak in f 1 plants was near to that of ‘th3’ rather than ‘yokoyama’ (fig. 3) (takemura et al., 2012). thus, we formulated the hypothesis that ‘th3’ is homozygous for a dominant gene involved in the depth of endodormancy, but the hypothesis was rejected based on a chi-square test. therefore, it was suggested that pear plant had quantitative trait loci (qtl) to decide cr as the genetic factor. it has recently been reported that the fifth chromosome of apricots or ninth chromosome of apples possesses qtl that influence the cr for breaking endodormancy (van dyk et al., 2010; campoy et al., 2011). in addition to these researches, it has been investigated about biochemical or molecular biological changes during induction or breaking periods of endodormancy in the buds of deciduous fruit trees. previously researches about coldhardening during winter in many higher plants shown the change of the lipid composition within cellular membrane (uemura and steponkus, 1994; uemura et al., 1995) or the accumulation of compatible solute within the cell (koster and lynch, 1992; wanner and junttila, 1999; kamata and uemura, 2004). in the peach buds, the accumulating period of total soluble sugar contents and water contents during endodormancy seasons were different between two cultivars having difference of crs, even though plants were of the same specie (yooyongwech et al., 2009). moreover, yooyongwech et al. (2009) have shown that expression levels of pp-pip1 and pp-γtip1 genes encoding aquaporin regulating water transport in tonoplast and plasma membrane increased in the peach buds of high-chilling cultivars than that of lowchilling cultivars before endodormancy breaking. in addifig. 3 distribution of percentage budbreak on f1 seedlings of ‘th3’ × ‘yokoyama’ (takemura et al., 2012). (z) 28 days after forcing at 23°c. vertical bars indicate mean of percentage budbreak on f1 seedlings. : budbreak of ‘th3’ and ‘yokoyama, respectively. 29 tion, it has been reported that the change of dehydrin levels has been associated with cold hardiness, endodormancy and the content/state of water in the tissue of tree (arora et al., 1997, 2003; erez et al., 1998; karlson et al., 2003). yamane et al. (2006) reported that the role of dehydrin in the bark tissue during the dormant season is common to all prunus species from research compared dehydrin expression level between evergreen and deciduous peach genotypes. however, yakovlev et al. (2008) found that the expression of some dehydrin genes in norway spruce (betula pubescens ehrh.) gradually decreased when approaching bud burst, and suggested that the observed changes cannot be related to winter dormancy. from results in our research using bud in japanese pear, it was not clear that dehydrin genes is involved in development of endodormancy because the results cannot be find the common pattern in two cultivars having difference crs (fig.4; a13f). on the other hands, expression levels of gene encoding gast-like gibberellin (ga) regulated protein increased with development of endodormancy in both two japanese pear cultivars (fig.4; b9c). in previous research, there is a lot of reports focused on association between endodormancy breaking and phytohormone, including ga (erez et al., 1979), ethylene (wang et al., 1985), auxin (nakano et al., 1980), cytokinin (broome and zimmerman, 1976; sterrett and hipkins, 1980) and abscisic acid (aba) (corgan and peyton, 1970; seeley and powell, 1981; tamura et al., 1992, 1993). among them, aba is termed ‘dormin’ or ‘dormancy inductor’ (addicott, 1983), and considered the most important growth inhibitor. on endodormancy induction period, it has been thought that the accumulation of aba in shoots of grape controlled the induction or development of endodormancy (düring and alleweldt, 1973). in addition, the contents of aba within leaf bud in japanese pear on mid-november increased in only the open field condition which confirmed inducing endodormancy (fig. 5) (takemura et al., 2011). moreover, tamura et al. (1992) reported the decrease of aba contents and the increase of gas contents on endodormancy breaking period in leaf bud of japanese pear. the vernalisation has been controlled by gas interacting closely with aba and assessed similarities with dortable 4 results of sequence homology search for the candidate genes related to endodormancy reference clone cdna size (bp) best database match e-value a2g 596 proline-rich protein 1 (vitis vinifera; aal02329.1) 7.00e-41 a13f 826 dehydrin 1 (eriobotrya japonica ; acl01288.2) 2.00e-16 b9c 468 putative gast-like gibberellin regulated protein (prunus dulcis; abr13302.1) 1.00e-23 b11d 794 predicted: early light-induced protein, chloroplastic (vitis vinifera; xp_002283398.1) 2.00e-59 c3c 927 cysteine protease inhibitor cystain (malus x domestica; aao19652.1) 2.00e-137 c11f 503 predicted: uncharacterized rna-binding protein c23e6.01c-like isoform 1 (vitis vinifera; xp_002285479.1) 3.00e-25 d4a 1049 unnamed protein product (vitis vinifera; cbi31586.3) 3.00e-18 fig. 4 northern blot analysis of the candidate genes is shown in table 4 in japanese pear buds of ‘kousui’ and ‘nijisseiki’. cu is shown as number in parentheses on the right side of calendar day. fig. 5 the change of aba contents on induction period of endodormancy in the leaf bud of japanese pear. n.c. and l.c. are shown as natural day-length condition and long-day condition, respectively (takemura et al., 2011). 30 mancy release (chouard, 1960). in arabidopsis, the vernalisation after prolonged exposure to low temperatures was regulated by flowering locus c (flc) which is mads-box gene (sung and amasino, 2005). in evergrowing peach, bielenberg et al. (2008) reported a cluster of six mads-box transcription factors (dormancy-associated mads-box: dam) as candidate genes for the regulation of terminal bud formation. the expression of two of these genes, dam5 and dam6, is suppressed by chilling temperatures and inversely correlated with bud break rate in peach (jimenez et al., 2010 b), whereas dam4 and dam6 expression is promoted by short photoperiods (li et al., 2009). recent studies have also shown that dam genes are differentially expressed in response to seasonal dormancy transitions in other plant, inclucing raspberry (rubus idaeus l.) (mazzitelli et al., 2007), japan apricot (prunus mume) (yamane et al., 2008), peach (jimenez et al., 2010 a; leida et al., 2010) and pear (ubi et al., 2010). 4. conclusions endodormancy in temperate-zone deciduous fruit trees is an essential mechanism to defend buds from unfavourable conditions during winter. to predict the day of breaking bud endodormancy is very important for cultivating. especially on the forcing culture, a lack of cr for breaking endodormancy caused growth inhibitor as non-germination during spring. therefore, molecular markers which can estimate whether plants are broken to the endodormancy or not should be developed. until now, detail of mechanism regulating the induction and the breaking of endodormancy is still unknown. in the future, protein analysis related endodormancy in addition to these genetic approaches will lead us the final conclusion of the mechanisms. on the other hands, it is suggested that pear and other plants had qtl to decide cr as the genetic factor. to provide for global warming in the future, deciduous fruit tree having lower cr should be bread in the future. the data of the present paper showed that endodormancy in japanese pear was induced by low temperature, without effect of day length. japanese pear had quantitative trait loci (qtl) as genetic factor to decide chilling requirement (cr) for breaking endodormancy. the increase of abscisic acid (aba) and gibberellin (ga) concentrations in the bud of japanese pear is related with induction and breaking of endodormancy. references addicott f.t., 1983 abscisic acid in abscission, pp. 269300. in: addicott f.t. 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westwood m.n., 1978 temperate-zone pomology, pp. 199-303. w.h. freeman and co., san francisco, ca, usa, pp. 428. yakovlev i.a., asante d.k.a., fossdal c.g., partanen j., junttila o., johnsen o., 2008 dehydrins expression related to timing of bud burst in norway spruce. planta., 228: 459-472. yamane h., kashiwa y., kakehi e., yonemori k., mori h., hayashi k., iwamoto k., tao r., kataoka i., 2006 differential expression of dehydrin in flower buds of two japanese apricot cultivars requiring different chilling requirements for bud break. tree physiol., 26: 1559-1563. yamane h., kashiwa y., ooka t., tao r., yonemori k., 2008 suppression subtractive hybridization and differential screening reveals endodormancy-associated expression of an svp/agl24-type mads-box gene in lateral vegetative buds of japanese apricot. j. am. soc. hortic. sci., 133: 708-716. yooyongwech s., sugaya s., sekozawa y., gemma h., 2009 differential adaptation of highand low-chill dormant peaches in winter through aquaporin gene expression and soluble sugar content. plant cell rep., 28: 1709-1715. impaginato 83 adv. hort. sci., 2023 37(1): 83­87 doi: 10.36253/ahsc­13856 image analysis to predict the maturity index of strawberries a. corvino 1, r. romaniello 2, m. palumbo 1, 2, i. ricci 1, m. cefola 1, s. pelosi 1, b. pace 1 1 institute of sciences of food production, national research council (cnr) c/o cs‐dat, via michele protano, 71121 foggia, italy. 2 department of agriculture, food and natural resources and engineering, university of foggia, via napoli, 25, 71122 foggia, italy. key words: computer vision system, cv. sabrosa, fragaria × ananassa duch, harvest, multivariate analysis, ripening. abstract: traditionally, strawberries are harvested manually when the typical colour of the cultivar does not reach at least 80% of the surface. the focus of this research activity is to develop an automatic system based on image analy­ sis in order to objectively define the optimal harvest time. strawberries (cv. sabrosa), with different degrees of maturation, were analyzed in four different harvesting periods and subsequently selected and classified, based on the ripe­ ning percentage, in three maturity classes: r0­25, r50­70 and r75­100. each class of 10 strawberries, evaluated in triplicate, was subjected to image analysis and physiological and qualitative evaluation by measuring the following para­ meters: respiration rate, ph, total soluble solids content, and titratable acidity. the images, captured by a digital camera, were processed using matlab® software and all the data found were supported by multivariate analysis. the image processing has made it possible to create an algorithm measuring objec­ tively the percentage and the saturation level of red assigning the fruit to each class. principal component analysis (pca) shows that discriminating parameters are the chroma and the red area, then used in a partial least square regression (plsr) model to predict the tss/ta ratio with r2 of 0.7 and 0.6 for calibration and validation set, respectively. 1. introduction strawberries are fruit, belonging to the family of the rosaceae and genus fragaria. only at the end of 1600 strawberry is no longer consid­ ered an ornamental plant, but rather a fruit to be cultivated and market­ ed for its delicacy. fragaria chiloensis, coming from chile, arouses the interest of many farmers for its unusual size differing from other straw­ berry species. the current strawberry, called f. ananassa, comes from the random hybridization, that occurred in the second decade of 1700, of f. virginiana (coming from the eastern united states) with f. chiloensis (coming from the chilean coasts of the pacific) (angelini, 2010). the (*) corresponding author: antonia.corvino@ispa.cnr.it citation: corvino a., romaniello r., palumbo m., ricci i., cefola m., pelosi s., pace b., 2023 ­ image analysis to predict the maturity index of strawberries. ­ adv. hort. sci., 37(1): 83­87. copyright: © 2023 corvino a., romaniello r., palumbo m., ricci i., cefola m., pelosi s., pace b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 october 2022 accepted for publication 26 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13856 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 83­87 84 obtained species is consumed and appreciated all over the world for sensorial and nutritional quality. strawberries are usually consumed as fresh fruit and they represent a healthy food choice for their rich­ ness in vitamin c, micronutrients and bioactive com­ pounds, mostly natural antioxidants such as phenols known even for anti­inflammatory action. this fruit, expressing better its potential, needs to be harvested at the right ripening because it is not climacteric. in general ripening influences the appearance, texture, flavour, and aroma due to physiological, biochemical and structural modifications. strawberry is hand­ picked evaluating visually the product when the char­ acteristic colour is reached. as consequence, the mis­ take of collecting an overmature or immature fruit, by presenting a poor product on the market, is very likely to occur. generally, the qualitative parameters of fresh products are determined by destructive analytical techniques which involve a sample preparation phase, time­consuming and can be performed on a limited number of samples, often reducing their rep­ resentativeness. in addition, the environmental impact, and the contact of the operator with the chemicals should not be overlooked, especially if they are not properly trained and experienced. for these reasons, it is important to consider eco­friendly and objective non­destructive methods that can quickly assess the proper harvest time by evaluating the quality of the product at hand. numerous studies have investigated various non­destructive techniques and their applicability in the field for the determina­ tion of the main qualitative parameters of fruit and vegetables. image analysis (ia) has proven to be a successful contactless tool in fruit and vegetables quality assessment. this technology captures images in the electromagnetic spectrum and extracts the most discriminating external characteristics (shape, colour and defects) and the next phase of data pro­ cessing can allow, through predicting models, the estimation of chemical and physical properties of samples (palumbo et al., 2022). the objectives of this study were (1) to implement a standardized comput­ er vision system to characterize quantitatively colour changes during the ripening of strawberries using the l*, a*, b* colour space, (2) to identify features of interest that can be related with ripening stages, such as colour saturation (chroma) and hue angle, (3) to develop a statistical model using selected features to identify the ripening stages of strawberries from sam­ ples previously classified by expert visual inspection. 2. materials and methods plant material candonga strawberries (fragaria × ananassa duch.) var. sabrosa, which have different degrees of ripeness, were provided by a cooperative company of fresh fruit (apofruit italia soc. coop., scanzano jonico, italy) in four different consecutive harvest times from february to may (one harvest per month) called h1, h2, h3 and h4. then, they were transport­ ed in cold conditions to the postharvest laboratory of cnr­ispa of foggia to be processed. fruits were selected by eliminating damaged sample and were grouped into three classes, based on the visual evalu­ ation of colour: r0­25 (from 0 to about 25% of red colour on fruit surface), r50­70 (from 50 to about 70% of red colour on fruit surface) and r75­100 (from 75 to about 100% of red colour on fruit sur­ face) (fig. 1). each class, consisting of 10 strawber­ ries, was evaluated in triplicate; each replicate was subjected to ia analysis, physiological (respiration rate) and physical­chemical (ph, total soluble solids and titratable acidity) characterization as below reported. computer vision system digital camera ap­3200t­pge (jai ltd., yokohama, japan), positioned inside a photo studio box hpb­60d (havox®, vendôme, france), was used to image a batch containing 10 strawberries for each replicate. in total, for each class, three replicates were consid­ ered, for a total of 30 berries. the camera sensor was an rgb cmos type, providing a spatial resolution of 3.2 mp at 2 fps and a colour depth of 24 bit/pixel. the lens used was a 12 mm focal length and f1.8 (kowa lens mod. lm12nc3 1/2) allowing a field of view (fov) of (35 × 30 cm). the lighting was supplied by two led handrails consisting of 20 diodes (havox hpb­60, 5500k, 13,000 100 lumen cri 93+). a colour checker passport photo 2 (x­rite italy srl, prato italy) fig. 1 ­ maturity class selected for the experiment. corvino et al. ‐ image analysis for ripeness evaluation of strawberry 85 with 24 known colour stains was placed in the cam­ era’s fov as a chromatic reference. the images cap­ tured by the digital camera were processed using matlab® r2021b (mathworks inc., natick, ma, usa). image segmentation each raw image of the strawberry was separated from the background, generating a binary image. in detail, the algorithm processed the raw images by cropping the unnecessary image border and separat­ ing the three colour­components: red, green and blue (rgb). the background was thresholded using the r image, since showing the highest contrast between the object of interest (strawberry) and the background. the coarse segmentation of the straw­ berries was carried out by a threshold method (gonzalez et al., 2004). on the resulting binary images, a morphological filter was applied to erode the strawberry rim and a flood­ filling operation was carried out to overcome the threshold defects. using this primary mask (binary image), the total area and the red area of each strawberry were calculated to get the percentage of red coverage. in the red area, colour features have been extracted to get informa­ tion on chroma and hue angle needed to correlate them with the analytical data. destructive chemical analysis titratable acidity (ta) and ph using a semi­auto­ matic titrator/ph meter (ph­burette 24 ­ crison instrument, barcelona, spain), were measured on about 100 g of homogenized strawberries (for each class and replicate) as reported by cozzolino et al. (2021). similarly, the total soluble solids value (tss) was determined using a digital refractometer (dbr35­xs instruments, carpi, italy) and results were expressed in °brix. the maturity index (mi) was calcu­ lated as the ratio of tss and ta for each class (melgarejo et al., 2017). respiration rate the respiration rate (rr) of strawberries was determined at 4°c using a closed system as reported by kader (2002). thoroughly, each replicate, about 250 g of product, was put into a 3.6 l sealed plastic container to let co2 accumulate up to 0.1% as the co2 standard concentration. at regular time intervals co2 concentration was monitored until the reference value is reached. a gas sample (1 ml) was drained from the headspace through a rubber septum and injected into the gas chromatograph (p200 micro­gc­ agilent, santa clara, ca, usa) equipped with dual columns and a thermal conductivity detector. carbon dioxide was analysed with a retention time of 16 s and a total run time of 120 s on a 10­m porous poly­ mer (ppu) column (agilent, santa clara, ca, usa) at a constant temperature of 70°c. the rr was expressed as ml co2/kg h. statistical analysis the data obtained were analyzed by multifactor anova for p ≤ 0.05 to evaluate the effects of the maturity class and harvest time (fixed factors) on ph, total soluble solid, titratable acidity, colour parame­ ters and rr (variables). parameters affected only by maturity class were subjected to a post­hoc test (fisher), using statgraphics (version 18.1.12, warrenton, va, usa). a principal component analysis (pca) was per­ formed using the software statistica version 6.0. (statsoft inc., tulsa, ok, usa) (jolliffe, 2022) with the aim of selecting the parameters able to discriminate the maturity classes. based on the results obtained a partial least square regression (plsr) was applied to develop a predictive method using the unscrambler 10.0 software (camo software, oslo, norway). in detail, 70% of the data was used in the calibration step and the remaining 30% was used to validate the obtained model. 3. results and discussion among the analytical data, rr and the mi were affected by the interaction of the two factors (matu­ rity class x harvest time) as reported in figure 2. mi showed all classes were different at first harvest with values of 6.61 (± 1.22), 7.38 (± 0.09) and 9.02 (± 0.84) for r0­25, r50­70 and r75­100, respectively; at the second harvest r75­100 reported higher value than the other samples, and this difference was measured also in the last two harvests (fig. 2a). a similar trend was observed for the rr (fig. 2b); in detail, at the first harvest time r0­25, r50­70 and r75­100 report­ ed values of 5.12 (± 0.06), 7.73 (± 0.45) and 12.30 (± 0.45) ml co2/kg h, respectively. then, at the second harvest, the rr increased at values around 10 ml co2/kg h for the fruit coming from r0­25 and r50­70 maturity classes, remaining almost constant in the last two harvests. on the other hand, for the full maturity class (r75­100), an increase in rr was found during the harvest time reaching at the last the val­ ues of about 20 ml co2/kg h (fig. 2b). regarding the classes r50­70 and r75­100, similar results were found by cozzolino et al. (2021) on strawberries (cv. adv. hort. sci., 2023 37(1): 83­87 86 4. conclusions results demonstrated that is possible to predict tss/ta index starting by colour parameters extracted by ia on strawberry (cv. sabrosa), collected in four consecutive harvests. the performance of the predic­ tive model obtained might be improved by increasing the number of samples and extending the analysis sabrosa) collected at two different ripening stages, namely half­red (in ripening phase, fully expanded and 50% red) and red (in ripening phase, fully expanded and 100% red) in three consecutive har­ vests. the image processing allowed us to measure the percentage of red (area red) and the colour parame­ ters chroma and hue angle, which enabled the three classes’ differentiation as indicated in figure 3. since, harvest time affected only two quality parameters, data coming from the different harvests were collected and used for the multivariate analysis (pca and plsr). regarding pca, also confirming data of anova analysis, area red and chroma were able to discriminate the maturity class on the first compo­ nent, which accounted for 94% of the variability (fig. 4a). thus, these two parameters were used as pre­ dictors of maturity index tss/ta, in a resulted pls model, which showed r2 of 0.7 in calibration and 0.6 in validation (fig. 4b). on the basis of these findings, an algorithm was developed using the matlab® software, for the objec­ tive measurement of the percentage of red (red area) and the saturation level (chroma) of a straw­ berry starting from the acquired images to automati­ cally and non­destructively attribute the fruit to each class, applying the plsr model. fig. 2 ­ effect of the maturity class and harvest time on maturity index (tss/ta) (a) and respiration rate (ml co2/kg h) at different harvest time (b). fig. 3 ­ strawberries image, mask, and correlated colour data (area red , hue angle and chroma). different letters indi­ cate statistically significant differences according to the least significant difference (lsd) fisher’s test. fig. 4 ­ principal component analysis (pca) (a), and partial least square regression (plsr) (b) model. corvino et al. ‐ image analysis for ripeness evaluation of strawberry 87 also to other cultivars trying to build an algorithm available for handheld devices used in the field or in general for applications available to consumers to consciously buy the product. acknowledgements the authors thank giuseppe sicuro and gianluca faliero of apofruit italia soc. coop. agricola for the agronomy support during fruit harvest, massimo franchi of cnr­ispa for the technical support, and joseph massaro for reviewing the english form of the paper. this research was funded by the project pon «r&i» 2014­2020­azione ii ­ “e­crops­ technologies for digital and sustainable agriculture” funded by the italian ministry of university and research (mur) under the pon agrifood program (contract ars01­ 01136). references angelini r., 2010 ­ la fragola. ‐ script, bologna, italy, pp. 548. cozzolino r., pace b., palumbo m., laurino c., pica­ riello g., siano f., de giulio b., pelosi s., cefola m., 2021 ­ profiles of volatile and phenolic compounds as markers of ripening stage in candonga strawberries. ­ foods, 10(12): 3102. gonzalez r.c., woods r.e., eddins s.l., 2004 ­ digital image processing using matlab. ­ pearson prentice hall, upper saddle river, nj, usa. jolliffe i.t., 2002 ­ principal component analysis. ­ springer series in statistics, 2nd ed. springer, new york, ny, usa. kader a.a., 2002 ­ methods of gas mixing, sampling and analysis, pp. 145­148. ­ in: kader a.a. (ed.) postharvest technology of horticultural crops. university of california agriculture and natural resources, oakland, ca, usa, pp. 535. melgarejo p., legua p., pérez­sarmiento f., martínez­font r., martínez­nicolás j. j., hernán­ dez f., 2017 ­ effect of a new remediated substrate on fruit quality and bioactive compounds in two strawber‐ ry cultivars. ­ j. food nutr. res., 5(8): 579­586. palumbo m., cozzolino r., laurino c., malorni l., picariello g., siano f., stocchero m., cefola m., corvino a., romaniello r., pace b., 2022 ­ rapid and non‐destructive techniques for the discrimination of ripening stages in candonga strawberries. ­ foods, 11: 1534. impaginato 307 adv. hort. sci., 2023 37(3): 307­315 doi: 10.36253/ahsc­14158 growth and yield performance of carrot (daucus carota l.) as influenced by plant population density under irrigation condition s.h. muhie 1 (*), h.s. yimer 2 1 department of plant science, college of agriculture, wollo university, dessie, ethiopia. 2 dessie city agricultural office, dessie, ethiopia. key words: cv. nantes, marketable yield, plant spacing, row distance, taproot, unmarketable yield. abstract: poor agronomic practices, such as inadequate plant density, can result in suboptimal carrot (daucus carota l.) yield and quality in some regions. in 2020, a field experiment was conducted under irrigation conditions in gerado, south wollo administrative zone, ethiopia, using the nantes variety as a test crop, to investigate the impact of inter­row and intra­row spacing on car­ rot yield. the treatments involved three inter­row (row) spacing levels (10, 15, and 20 cm) and three intra­row (plant) spacing levels (5, 10, and 15 cm) in a randomized complete block design with three replications. row and plant spac­ ing significantly affected (p<0.05) total yield, plant height, leaf fresh weight, root length, root diameter, and root fresh weight. the highest marketable yield (490.4 q ha­1) was achieved with a plant density of 20 x 5 cm. in contrast, the highest unmarketable yield (36.3 q ha­1) was obtained with a spacing of 20 x 15 cm. hence, a plant density of 20 x 5 cm is recommended for optimal mar­ ketable carrot yield in the study region and similar agroecologies, although fur­ ther research across multiple locations and seasons is necessary to validate the results. 1. introduction carrot (daucus carota l.) is a short duration vegetable crop. in terms of production areas and market value, it is among the top ten most eco­ nomically significant vegetable crops in the world. carrots are widely cul­ tivated because they offer a low­cost source of vitamins (particularly vitamin a), minerals, and fibre in the human diet (nuez and prohens, 2008). the taproot contains high amount of carotene (10 mg per 100 g), thiamine (0.04 mg per 100 g) and riboflavin (0.05 mg per 100 g). additionally, it contains protein, fat, minerals and vitamin c. due to these several uses, carrot consumption has increased from time to time (tegen and jembere, 2021). (*) corresponding author: hamidashm@gmail.com citation: muhie s.h., yimer h.s., 2023 ­ growth and yield performance of carrot (daucus carota l.) as influenced by plant population density under irri‐ gation condition. ­ adv. hort. sci., 37(3): 307­315. copyright: © 2023 muhie s.h., yimer h.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress. net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribu­ tion, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. author contribution: the corresponding author, seid hussen muhie developed the research idea, drafted the propo­ sal, and wrote the manuscript the co­author, hussen seid yimer, developed the proposal, conducted the research, did data col­ lection and analysis, and wrote the draft manu­ script. competing interests: the authors declare no competing interests. received for publication 1 january 2023 accepted for publication 30 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14158 http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 307­315 308 in the world’s main carrot­growing nations, yields of carrots can range from 30 to 100 t ha­1. carrot yields per unit area in the majority of developing nations like ethiopia (whose average fresh carrot yield per ha is 5.6 t) continue to be below the global average (kassa et al., 2018). numerous factors, such as poor production techniques, a lack of technical inputs, pests, and postharvest losses, are linked to low productivity (tegen and jembere, 2021; tschirley et al., 2004). abiotic stress can also contribute to the decline in quantity and quality of horticultural prod­ ucts, such as carrot (muhie et al., 2021). one of the key elements affecting marketable car­ rot root yield and root size is plant population density (lana, 2012). in previous research investigations, it was reported that plant population of 450,000 and 300,000 is ideal for fresh market and processing car­ rots, respectively (tegen and jembere, 2021). in addi­ tion, it was also revealed that narrow spacing result­ ed in a higher marketable carrot root yield (da silva et al., 2008; shiberu and tamiru, 2016). on the other hand, another group of researchers reported that crops planted with wider spacing produced the high­ est total yield. this discrepancy between the result findings of researchers came from the purpose of production of carrots (for fresh market or for indus­ trial use), as each purpose has its own specific root size range (kabir et al., 2013; lana and carvalho, 2013). researchers also looked into the possibility of producing baby carrots that are more suited for com­ mercialization through the use of high population density cropping and early harvesting. farmers typi­ cally sow carrots by broadcast at a rate of 4­5 kg/ha, although some of them prefer inter­row spacing ranging from 20 to 30 cm and intra­row spacing of 10 to 20 cm. crops such as carrot (daucus carota) and chinese jute (abutilon theophrasti) exhibit plasticity in their morphology and modular growth, making it challenging to determine a suitable unit for popula­ tion density (wang et al., 2017; ford and sorrensen, 2018), a crucial variable that connects individuals to crops. some authors have provided details on agro­ nomic practices used in carrot production (bender et al., 2020; reginaldo et al., 2021). however, there is limited information available on the plant population density of irrigated carrots that can ensure an opti­ mal marketable yield. farmers in the study area use broadcasting method of sowing under rainfed pro­ duction system. recent research finding also recom­ mended the need to investigate the effects of plant density on carrot (biratu et al., 2022). the objective of the current study was to identify the optimum population density by adjusting inter­ and intra­row spacing for marketable root yield and root size of car­ rot under irrigation. 2. materials and methods descriptions of the study area the experiment was conducted at gerado, south wollo administrative zone, northeastern amhara region, ethiopia in 2020 cropping season under irriga­ tion. the area is located at distance of 401km away from the capital to the northeastern part of the country. geographically, the study area is found at the intersection of 11°8ʹ n and 39°38ʹ e (fig. 1). it falls within semi­arid climatic zone with an average monthly minimum and maximum temperature of 12.37°c and 26.27°c, respectively. the area receives an annual rainfall amount of 1291.3 mm/year with erratic nature. due to this the area is characterized as moisture deficit unless there is supplementary irriga­ tion. the soil type of the area is sandy­loam. it has three permanent rivers which have the potential to irrigate throughout the year. experimental design and treatments the experiment included nine treatments involv­ ing three different inter­row distances (10, 15, and 20 cm), also referred to as row distance (rd), and three different intra­row distances (5, 10, and 15 cm), also referred to as plant distance in the row (pd). the experiment was arranged in a randomized complete block design with three replications following the procedures of gomez and gomez (2010). the treat­ fig. 1 ­ map of the study area. muhie and yimer ‐ plant population density of carrot under irrigation 309 ments were assigned randomly to the experimental plots within a block. experimental materials and procedures carrot, cv. nantes was used as a test crop for this experiment. this cultivar of carrot is well adapted in the study areas. the land was well prepared to a fine tillage to a depth of 30 cm following the conventional tillage practice, using oxen to plough. thereafter, a field layout was prepared, and each treatment was assigned randomly to the experimental plots. seeds were sown in raised beds with 20 cm height at a spacing based on treatment assigned to the plot. carrot seeds were sown by drilling in 1.2 m x 1 m long rows in each plot. the complete amount of phosphorus (175 kg p2o5 ha­1) was applied at once, while the nitrogen in the form of urea (150 kg ha­1) was applied in two parts: half of the amount was applied during sowing, and the remaining half was manually top­dressed in the inter­row spaces during the mid­tillering crop stage, which occurred 35 days after emergence (dae). irrigation and other required cultural practices were applied equally to all plots. during the experimental periods, a successful crop was produced by applying furrow irrigation at seven days interval and consistently performing all recom­ mended cultural practices. weeds were manually removed and collected from the crop fields, while harvesting was carried out at crop maturity using a hand hoe. data collection phenological data. data such as days to 50% emergence and days to 90% physiological maturity were recorded by counting the number of dates to the respective phenological parameters. growth parameters. plant height, leaf number, leaf fresh weight, and canopy cover were recorded appropriately using five randomly selected plants. canopy cover was determined as the perimeter of the plant at its widest horizontal plane. it typically assumes that there are a few minor gaps in the leaves and that an average crown perimeter will smooth out any uneven edges. yield parameters. root length and root diameter were measured from five randomly selected plants using a calliper. the fresh weight of roots per plant was determined by measuring the weight of five ran­ domly selected plants using a sensitive balance and the average value was calculated and used for analy­ sis. the yield of marketable roots was calculated per unit plot excluding border effects. the yield per unit area was converted to marketable yield per hectare. the unmarketable roots were identified based on cracked, branched, small size with diameter of approximately 1­1.5 cm and rotten. then, the unmar­ ketable roots were calculated per unit plot excluding border rows. the yield per unit area was converted to unmarketable yield per hectare. statistical analysis the collected data underwent analysis of variance (anova) using sas 9.1, which was appropri­ ate for the design of the experiment. the means of significant treatment effects were separated using the least significant difference (lsd) test at a 5% level of significance. 3. results and discussion phenology plant density is considered as one of the most important factors affecting crop phenology (shafi et al., 2012; khan et al., 2017). in the present experi­ ment, plant spacing did not significantly (p≤0.05) affect days to 50% emergence. this could be attribut­ ed to the fact that the viability of the seed, moisture availability, and air conditions are the essential ele­ ments required for germination, rather than spacing. similarly, tesfu and charles (2010) found that neither sowing date nor planting density significantly affect­ ed the number of days required for 50% crop emer­ gence. in general, carrot plants grown with a narrower row distance tended to mature faster than those grown with wider spacing. the fastest time to reach maturity for carrot plants (73.3 days) was observed with a spacing of 10 x 5 cm, while the slowest time (134.3 days) was observed with a spacing of 20 x 15 cm, followed by a spacing of 20 x 10 cm (121.3 days) (table 1). this suggests that row spacing has a more significant effect than plant spacing on maturity in this specific case study. indeed, when the distance between rows was increased from 10 cm to 20 cm, the number of days to reach physiological maturity increased by 34 days. other investigations have also found that plant density has a significant impact on the time it takes to reach 90% maturity (da silva et al., 2008; tegen et al., 2021). tesfu and charles (2010) proposed that lower plant density may allow for more space and resources per plant, which could lead to extended vegetative growth and a longer adv. hort. sci., 2023 37(3): 307­315 310 time to reach maturity. growth and yield the result of analysis of variance (anova) showed that all growth and yield parameters of nantes carrot were significantly affected (p <0.05) by plant and row distances and their interaction (plant spacing). plant height it is well known that growth parameters, such as plant height, can be influenced by plant population density (abuzar et al., 2011; rahman et al., 2011). based on our results, the highest plant height of car­ rot (73.0 cm) was recorded from a spacing of 20 x 15 cm, followed by 15 x 15 cm spacing (60.0 cm). conversely, the shortest plant height (17.3 cm) was recorded when carrots were sown with a spacing of 10 x 5 cm (table 2). increasing the distance between rows from 10 cm to 20 cm resulted in a 26.7 cm increase in plant height. this may be due to the availability of essential resources necessary for growth and development, as well as the presence of adequate free space between plants to reduce competition in the higher spacing. these findings are consistent with previous research, such as dawuda et al. (2011), who reported that taller plants were observed at higher spacing, and kabir et al. (2013), who reported that taller plants were observed at a spacing of 30 x 20 cm compared to a spacing of 20 x 10 cm. according to kabir et al. (2013), plants sown in higher spacing had enough space for vegetative growth and experienced less competition for nutrients compared to those sown in lower spacing treatments, such as 20 x 10 cm and 25 x 15 cm. when crops have to compete with their neighbouring plants for soil nutrients and sunlight, their health and growth can be negatively impacted. poorly functioning plants will not attain their desired height or canopy and their roots will have to com­ pete not only for nutrients and water but also for space. furthermore, high planting density can inhibit photosynthesis. leaf number, leaf weight and canopy cover plant density have been reported to affect leaf number, canopy development, plant architecture, early ground cover and competitive ability of crops with weed (bonaparte and brawn, 1976; deressegn and telele, 2017; hou et al., 2019; bernhard and below, 2020). moreover, leaf weight can be affected by the accumulation and the partitioning of synthe­ sized food to non­photosynthetic parts (halford, 2010; osorio et al., 2014). the highest values of leaf count (21.0), leaf weight table 2 ­ mean plant height (cm) of carrot as influenced by inter and intra row spacing mean values within rows and columns followed by different letter(s) are significantly different at 5% probability level. cv= coefficient of variation. table 1 ­ mean days to maturity of carrot as influenced by plant spacing row distance (cm) plant distance in the row (cm) mean 5 10 15 10 73.33 e 81.67 de 87.67 d 80.89 c 15 82.00 de 89.67 d 108.33 c 93.33 b 20 89.67 d 121.33 b 134.33 a 115.11 a mean 81.67 c 97.56 b 110.11 a cv (%) = 7.44% row distance (cm) plant distance in the row (cm) mean 5 10 15 10 17.33 f 23.67 ef 31.33 de 24.11 c 15 21.17 ef 41.60 cd 60.00 b 40.92 b 20 27.33 ef 52.00 bc 73.00 a 50.78 a mean 21.94 c 39.09 b 54.78 a cv (%) = 17.81% mean values within rows and columns followed by different letter(s) are significantly different at 5% probability level. cv= coefficient of variation. muhie and yimer ‐ plant population density of carrot under irrigation 311 (15.0 g) and canopy spread (52.7 cm) were observed at the widest plant spacing (20 x 15 cm), while the lowest values (6.3, 9.1 g, and 14.3 cm, for leaf number, leaf weight and canopy cover, respectively) were found with the narrowest spacing of 10 x 5 cm (table 3). when the row distance was increased from 10 cm to 20 cm, it was observed that approximately seven additional leaves could develop, indicating that wider spacing can result in higher leaf area for maximum assimilate synthesis. this could be attributed to the greater free space available for plant growth between rows, which reduces competition for nutri­ ents. this finding is consistent with previous studies by dawuda et al. (2011) and kabir et al. (2013), which reported that wider spacing can lead to more leaves and larger canopies, potentially intercepting more light for better growth and yield. lower crop popula­ tion density, as reported by demisie and tolessa (2018) and van delden et al. (2021), may allow foliage to receive maximum photosynthetically active radiation (par) and synthesize assimilates, ultimately contributing to greater leaf growth. an increase in canopy size is likely to enhance photosynthesis, lead­ ing to the production of more leaves, which has been supported by the findings of appiah et al. (2017) and tesfu and charles (2010) in other plants. similar find­ ings have also been reported in carrot (alam et al., 2020) and radish (sandipan and rawat, 2020). root length, root diameter and root weight the number of plants per unit area can influence the yield and quality of horticultural crops (rodriguez et al., 2007; lencha and buke, 2017; demisie and tolessa, 2018; sinta and garo, 2021; tegen et al., 2021). in the current investigation, the maximum root length (22.2 cm), root diameter (5.9 cm) and root weight (136.3 g) were recorded at spacing of 20 x 15 cm followed by 15 x 15 cm spacing (19.1 cm for root length, 5.2 cm for root diameter and 115.4 g for root weight). on the contrary, the lowest values (13.7 cm, 3.4 cm, and 64.2 g for root length, diameter and weight, respectively) were recorded when plants were cultured in rows spaced 10 cm apart with 5 cm between plants within rows (table 4). increasing plant spacing resulted in longer roots having larger diameter, thus in higher plant yield, potentially due table 3 ­ mean leaf number, leaf weight and canopy cover of carrot (daucus carota l.) plants as influenced by inter and intra row spacing table 4 ­ mean root length, root diameter and root weight of carrot (daucus carota l.) plants as influenced by inter and intra row spacing distances (cm) leaf number (n) leaf weight (g) canopy cover (cm) row distance (rd) 10 8.22 c 11.23 c 27.89 c 15 12.44 b 12.51 b 33.89 b 20 15.33 a 14.15 a 39.67 a plant distance in the row (pd) 5 8.89 c 11.18 c 24.56 c 10 11.33 b 12.94 b 31.44 b 15 15.78 a 13.81 a 45.44 a plant spacing (rd × pd) 10 × 5 6.33 g 9.06 e 14.33 f 10 × 10 8.00 gf 12.19 cd 31.33 de 10 × 15 10.33 def 12.57 cd 38.00 c 15 × 5 9.33 ef 12.57 cd 27.33 e 15 ×10 12.00 cd 12.36 cd 28.67 de 15 × 15 16.00 b 13.89 ab 45.67 b 20 × 5 11.00 de 12.19 bc 32.00 cde 20 × 10 14.00 bc 14.27 ab 34.33 cd 20 × 15 21.00 a 14.98 a 52.67 a cv (%) 11.74% 5.74% 10.37% mean values within rows and columns followed by different let­ ter(s) are significantly different at 5% probability level. cv= coefficient of variation. distances (cm) root length (cm) root diameter (cm) root weight (g) row distance (rd) 10 15.51 c 3.97 c 79.14 c 15 17.03 b 4.37 b 104.60 b 20 19.31 a 4.87 a 114.46 a plant distance in the row (pd) 5 15.34 c 3.68 c 85.26 c 10 17.36 b 4.43 b 98.03 b 15 19.06 a 5.09 a 114.92 a plant spacing (rd × pd) 10 × 5 13.77 h 3.40 f 64.23 f 10 × 10 16.57 ef 4.33 cd 80.23 ef 10 × 15 16.20 f 4.18 d 92.98 de 15 × 5 14.73 g 3.63 ef 96.09 cde 15 ×10 17.23 de 4.30 cd 102.26 bcd 15 × 15 19.10 b 5.17 b 115.45 b 20 × 5 17.53 cd 4.02 de 95.45 de 20 × 10 18.23 c 4.65 c 111.62 bc 20 × 15 22.17 a 5.93 a 136.32 a cv (%) 2.83% 5.87% 28.6% mean values within rows and columns followed by different let­ ter(s) are significantly different at 5% probability level. cv= coefficient of variation. 312 adv. hort. sci., 2023 37(3): 307­315 to the availability of sufficient resources for root growth and development, and reduced competition for available soil resources. the results of our study are in line with previous research, which suggests that wider spacing of plants can lead to increased nutrient uptake and photosyn­ thesis rates, resulting in improved areal and root growth as well as fresh root weight in carrot produc­ tion (kabir et al., 2013; d’hooghe et al., 2018; appiah et al., 2021). kharsan et al. (2019) reported a gradual increase in root diameter with increasing spacing, observing a 1.4 cm increase in root diameter when plant spacing was increased from 5 cm to 15 cm. similar results and arguments were reported more recently also by tegen and jembere (2021), kwiatkowski et al. (2022) and searight et al. (2022). it was suggested that plants sown with wider spacing had more room to develop their roots in the soil, leading to an increase in root diameter compared to those planted with lower spacing. marketable and unmaketable root yield (q ha‐1) according to pant and sah (2020) and sandhu et al. (2021), the success of crop establishment, yield, and profitability are all affected by plant density. poor plant stand is a major factor in reducing yield, and increasing planting density can further exacer­ bate this problem by decreasing the plant’s net pho­ tosynthetic rate (pn), stomatal conductance (gc), and leaf chlorophyll content, ultimately leading to decreased yield (zhang et al., 2021). the highest marketable yield (490.4 q ha­1) was recorded at spacing of 20 x 5 cm followed by 15 x 5 cm spacing (359.5 q ha­1), as the smallest root mar­ ketable yield (83.4 q ha­1) was recorded at 20 x 15 cm spacing (table 5). thus, the highest marketable root yield of carrot per hectare was obtained with the smallest plant distance within the row and the largest distance between rows, while the carrot yield was the lowest when plants were grown using the largest intra­ and inter­row spacings. this result is supported by appiah et al. (2021), who observed that narrow spacing resulted in small and uneven root sizes which are rejected from the market. the reason for the lower marketable yield resulting from wider spacing can be indirectly attributed to the number of plants per unit area. each plant has the chance to produce marketable root. hence, the density of plants per unit area has a direct impact on the avail­ able number of roots, which ultimately affects the yield. moreover, an increase in row spacing causes excessive branching and cracking of the roots, mak­ ing them less desirable to consumers or in the mar­ ket, as stated by connors (2022) and searight et al. (2022). according to haque and sakimin (2022), exceed­ ing a certain planting density threshold can lead to decreased yield and quality due to inadequate resource supply, resulting in produce that is unsuit­ able for the market. in our experiment, larger plant­ ing distances of 20 x 15 cm and 15 x 15 cm resulted in the highest unmarketable yields of 36.3 q ha­1 and 17.9 q ha­1, respectively. conversely, reducing the dis­ tances to 10 x 5 cm minimized the unmarketable yield to just 2.7 q ha­1, as shown in table 5. dawuda et al. (2011) also reported maximum unmarketable yield from plants that were grown adopting wide spacing up to 30 × 5 cm. in agreement with the present finding, adem seid et al. (2019) reported a decline in unmarketable yield as plant spacing increased. it was suggested that percentage of root cracking might increase in the wider spacing due to more fluctuation of available soil moisture as table 5 ­ marketable and unmarketable root yield of carrot (daucus carota l.) as influenced by inter and intra row spacing distances (cm) marketable yield (qha­1) unmarketable yield (qha­1) row distance (rd) 10 166.98 c 5.85 c 15 225.67 b 9.66 b 20 286.10 a 16.97 a plant distance in the row (pd) 5 338.11 a 5.61 c 10 227.74 b 9.29 b 15 112.90 c 17.58 a plant spacing (rd × pd) 10 × 5 164.42 def 2.70 d 10 × 10 185.98 cde 6.38 cd 10 × 15 150.55 def 8.47 cd 15 × 5 359.48 b 9.72 c 15 ×10 112.82 cd 11.29 bc 15 × 15 104.70 ef 17.99 b 20 × 5 490.43 a 4.40 cd 20 × 10 284.43 bc 10.21 bc 20 × 15 83.43 f 36.29 a cv (%) 25.74% 32.89% mean values within rows and columns followed by different let­ ter(s) are significantly different at 5% probability level. cv= coefficient of variation. muhie and yimer ‐ plant population density of carrot under irrigation 313 absorbed by the plants. according to some authors (merfield, 2006; adem seid et al., 2019; tegen and jembere, 2021; mahaffee et al., 2023), the only dis­ advantages of high­density plantings include produc­ ing fewer jumbo carrots and lack of airflow through the field that can increase the incidence of foliar dis­ eases, but this can be managed via integrated pest management. in a previous investigation on carrot cultivation, it has been reported that the yield of boxed sized root increases with plant density to a maximum and then decreases, being maximum yield achieved with higher plant density (tegen and jembere, 2021). 4. conclusions the growth and productivity of carrots are signifi­ cantly affected by agronomic practices, with plant population being a vital management factor. results from our experiment showed that spacing had a sig­ nificant influence on all parameters except for days to emergence. the lowest sowing density with a spacing of 20 x 15 cm resulted in the highest values of plant height (73.0 cm), number of leaves (21.0 cm), diameter of root (3.4 cm), and length of root (22.2 cm). however, the highest root yield per hectare (490.4 q ha­1) was recorded from a spacing of 20 x 5 cm, while the lowest yield (83.4 kg ha­1) was from the widest spacing of 20 x 15 cm. although wider spacing resulted in greater root length, leaf and root fresh weights, and plant height, it also led to maximum unmarketable yield, demonstrating the significance of plant distance on carrot productivity. thus, our research suggests that the optimal plant density for maximum marketable yield of carrots in similar agroclimatic conditions and irrigated produc­ tion systems is 5 cm intra­row spacing and 20 cm inter­row spacing. going beyond this optimum level may cause branching, cracking, and unsuitability of the carrot roots for the market. however, it’s impor­ tant to note that these findings are from a single sea­ son and location, and further research across various locations and seasons is necessary for more reliable recommendations. references abuzar m.r., sadozai g.u., baloch m.s., baloch a.a., shah i.h., javaid t., hussain n., 2011 ­ effect of plant population densities on yield of maize. ­ j. animal plant sci., 21(4): 692­695. adem seid m., gedamu f., mohammed d., 2019 ­ effects of blended nps fertilizer rates and plant spacing on growth, yield and quality of carrot (daucus carota l. subsp. sativus) in tehuledere district, northeastern ethiopia. ­ haramaya university, etiopia. appiah e.a., kugbe j.x., rufai a.m., 2021 ­ maize growth and yield response to incremental rates of phosphorus in p‐depleted lixisols of nothern ghana. ­ inter. j. agric. envir. biores., 6(3): 257­275. bender i., edesi l., hiiesalu i., ingver a., kaart t., kaldmäe h., talve t., tamm i., luik a., 2020 ­ organic carrot (daucus carota l.) production has an advantage over conventional in quantity as well as in quality. ­ agronomy, 10(9): 1420. bernhard b.j., below f.e., 2020 ­ plant population and row spacing effects on corn: phenotypic traits of posi‐ tive yield‐responsive hybrids. ­ agron. j., 112(3): 1589­ 1600 bonaparte e.e.n.a., brawn r.i., 1976 ­ effects of plant density and planting date on leaf number and some developmental events in corn. ­ canadian j. plant sci., 56(3): 691­698. biratu w., molla b., abebe h., gebremeskel h., 2022 ­ study growth, root yield and yield related character of carrot (daucus carota) varieties under highland areas of southern tigray region, northern ethiopia. ­ int. j. life sci., 10(4): 317­323. connors n.a., 2022 ­ addressing weed and soil manage‐ ment trade‐offs in vegetables through integrated cul‐ tural and mechanical strategies. ­ phd thesis, michigan state university, michigan, usa, pp. 100. da silva j.b.c., vieira j.v., lana m.m., 2008 ­ processing yield of the carrot cultivar esplanada as affected by harvest time and planting density. ­ scientia hortic., 115(3): 218­222. dawuda m.m., boateng p.y., hemeng o.b., nyarko g., 2011 ­ growth and yield response of carrot (daucus carota l.) to different rates of soil amendments and spacing. ­ j. sci. techn. 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gixhari and ramadani, 2016). in europe also, there is a lack of information regarding its geographical distribution and for this reason, in the iucn red list of threatened species (iucn, 2019), it is (*) corresponding author: valbona.sota@fshn.edu.al citation: sota v., benelli c., çuko b., kongjika e., 2021 ­ effect of a double phase culture system and acti‐ vated charcoal on in vitro propagation of malus sylvestris (l.) mill. ­ adv. hort. sci., 35(4): 361­ 369. copyright: © 2021 sota v., benelli c., çuko b., kongjika e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 august 2021 accepted for publication 11 november 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-11825 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 361­369 362 included in the category of ‘data deficient’ species. beside this, it is also reported as endangered in some european countries (larsen et al., 2006; wagner et al., 2014). the specific need for agricultural yield is one of the key reasons for the development of tissue culture technology worldwide. indeed, plant micro­ propagation is an efficient method of propagating disease­free, genetically uniform and massive amounts of plants under in vitro conditions (gupta et al., 2020). over the years, the number of plant species which have been clonally propagated through tissue culture was increased and the most commer­ cially important species have been studied. in vitro propagation techniques have found wide use because of their effectiveness in terms of the high­ quality product obtained and of reduced cost (jain and ishii, 2003; debnath et al., 2006; damiano et al., 2008; lambardi et al., 2013). clonal propagation cre­ ates the possibility of obtaining a large quantity of homogeneous plant material, which can be con­ served for short/mid­term periods through minimal growth methods, or long­term period, through cryop­ reservation (kameswara, 2004; day and stacey, 2007; benelli et al., 2012). for all these advantages, and also because of the current situation on the geo­ graphical distribution and importance of m. sylvestris, it is of strategic importance to develop an effective micropropagation protocol, to obtain signifi­ cant numbers of clonal plantlets which can be used for ex situ conservation strategies or other purposes. in all micropropagation methods, the main goal is to optimize a successful protocol that ensures a rapid clonal propagation and results also as a time­saving technique (lambardi et al., 2013). in most reports, the protocols implemented for malus sp. microprop­ agation are based on conventional micropropagation systems in semisolid culture media which typically include explants inoculation/proliferation, subcul­ ture, and rooting steps. several authors mentioned the effective stabilization of wild apple micropropa­ gation using conventional micropropagation system consisting in a monophasic/agarized medium (modgil et al., 1999; boudabous et al., 2010; dobránszki et al., 2011; kereša et al., 2012; zhang et al., 2020). but teixeira da silva et al. (2019), in a review regarding tissue culture of malus sp., mentioned that most reports aimed to find alternative gelling agents other than agar, in order to reduce costs. although agarized media are successfully used for plant micropropagation, nowadays it has become absolutely important to improve the productivity and uniformity of valuable vegetal materials with eco­ nomic values by reducing the cost of production, space, time or, optimizing other issues related to micropropagation coefficient, rooting index, etc. in addition to solid media, several techniques have been successfully practiced for the micropropagation of economically important plants such as 1) the use of liquid cultures for the micropropagation of two apple rootstocks (mehta et al., 2014), pineapple (dal vesco et al., 2001) and dioscorea sp. (jova et al., 2011); 2) the use of continuous immersion bioreac­ tors for apple rootstock (chakrabarty et al., 2003), eucalyptus (mendonça et al., 2016), chestnut (vidal et al., 2017), hybrid chestnut (cuenca et al., 2017); 3) the use of temporary immersion bioreactors for wild apple (sota et al. , 2021), apple rootstocks (chakrabarty et al., 2003; zhu et al., 2005), and oak (gatti et al., 2017). an alternative for improving in vitro micropropa­ gation protocols is the use of double­phase nutrient media (dps). in this method, the explants are fixed in a solid medium, while the liquid medium is periodi­ cally added during the culture, therefore eliminating the need for subcultures. in this way, the propaga­ tion costs and the chances of contamination are reduced (senapati, 2015). there are very few reports on the use of double­phase media for in vitro propa­ gation of plants (scherwinski­pereira et al., 2012; lopez and suarez, 2018). in most reports, the same culture medium was used in solid and liquid phases (except agar presence) to increase the plantlets’ mass production during the cultures. but it would also be useful and interesting to test various media for propagation and rooting in the same culture con­ tainer. in this case, the solid phase would have a hor­ monal content effective for rooting induction, while the liquid one would have a hormonal content to induce lateral shoots development. furthermore, the addition of activated charcoal (ac) in the culture media enhances in vitro rooting induction/develop­ ment in some fruit­trees species (wang and huang, 1976; thomas, 2008), and also acts as an adsorbent of phenolic compounds to avoid oxidation phenome­ na (boudabous et al., 2010; shinde et al., 2010). this study aimed to evaluate the efficiency of dps (solid/liquid culture system) in comparison with the conventional cs (solid) propagation system for improving in vitro shoots regeneration of malus sylvestris (l) mill. in addition, the effect of various concentrations of ac in both systems on rooting abili­ ties was evaluated. sota et al. ‐ micropropagation of wild apple in a double‐phase system 363 2. materials and methods plant material and micropropagation systems axillary buds of wild apple [m. sylvestris (l.) mill.] were excised from scions of the population of maminas at durrës county in western albania and were used as initial explants. the explants were disin­ fected with 70% ethanol for 3 min, followed by the treatments with 0.2% of 50% carbendazim (bavistine) for 7 min and 0.01% hgcl2 for 10 min., and multiple rinses with sterile distilled water were performed. in this research, two micropropagation systems for in vitro regeneration of m. sylvestris plantlets were compared: ‐ conventional micropropagation system (cs), con­ sisting in the explants culture on solid medium (monophase system); ‐ double phase system (dps), consisting in the explants culture on solid medium plus a liquid phase at the same time. for dps micropropagation systems, the liquid medium was added every week in the culture ves­ sels, specifically 1 ml in the test tubes (proliferation stage) and 3 ml in erlenmeyer flasks (rooting stage). all procedures were performed under aseptic condi­ tions. media composition and culture conditions in vitro shoots proliferation. ms medium (murashige and skoog, 1962) was used, supplement­ ed with 1 mg l­1 6­benzylaminopurine (bap) and 0.1 mg l­1 α­naphtalenacetic acid (naa) for both liquid and solid phase. rhizogenesis induction. the liquid phase was the same as in the proliferation stage, while for both sys­ tems under study the solid medium was supplement­ ed with 1 mg l­1 naa and different concentrations (0, 250, 500, 1000 mg l­1) of activated charcoal (ac). in all cases, in the medium was added sucrose at 3%, while, for solid medium preparation, 7 g l­1 of agar (sigma­aldrich) was also supplemented. the ph was adjusted to 5.7 before medium autoclaving at 120°c for 20 min. the cultures were maintained in the growth chamber at 25±2°c in a 16 h/8 h light/dark regime with cool, white fluorescent light of intensity 43.4 mmol m­2 s­1. data elaboration leaves number, shoots number, shoots length, roots number and roots length, were evaluated after 42 days of culture for each micropropagation stage. experimental data were elaborated by tukey­kramer test, and the analysis of variance (anova) with jmp 7.0 statistical software. 3. results and discussion in vitro regeneration wild apple shoots in the dps and cs systems after 42 days of in vitro culture, the growth dynamic of wild apple explants in the cs and dps micropropagation systems was evaluated. during proliferation, for both systems, the pgrs ratio was such that induced lateral shoots regeneration. the comparative growth dynamic between the two prop­ agation systems for leaves number, shoots number and shoots length, is presented in the variability charts (fig. 1), and it clearly shows that the micro­ propagation system highly affected growth parame­ ters (fig. 2 a­e). the contact of the explants with the liquid medium (fig. 2 c), facilitated and increased the amount of nutrients absorbed by the explants while in the solid medium (fig. 2 a; b), the solid consistency itself slowed down the rate of absorption. in our findings, the number of leaves for shoot in both systems, was high, specifically 18.75 during the culture in the cs system, and 19.25 in the dps system (fig. 1 a). fig. 1 ­ variability chart: leaves number (a); shoots number (b); shoots length (cm) (c), in the dps and cs micropropaga­ tion systems. adv. hort. sci., 2021 35(4): 361­369 364 dps showed a higher efficiency for shoots number and shoot length, 2.85 and 3.39 cm, respectively, compared to cs, with 2.10 shoots number and 2.77 cm shoots length (fig. 1 b; c). overall, the explants grown in the cs propagation system, showed a lower regeneration potential for the monitored biometric parameters, which can be related to the absorption of substances from the nutrient medium. the greater thickness of the shoots cultivated in the dps system was evident during the proliferation phase (fig. 2 d; e). the efficiency of the dps system due to the pres­ ence of the liquid phase that enhanced the contact area of the explant with the nutrient medium, lead­ ing to an increase in the rate of diffusion, absorption and, continuous replacement of nutrients consumed over the days of culture is reported by various authors stressing the efficiency of the double phase micropropagation system (moraes et al., 2004; pullman and skryabina, 2007; scherwinski­pereira et al., 2012; dorić et al., 2014; senapati, 2015). in this regard, rodriguez et al. (1991) found that the micro­ propagation rate of pyrus communis l., especially in terms of axillary shoots formation, was higher when a liquid medium was added onto the jellified one. the superiority of dps compared with cs system is also reported by oliveira et al. (2013) on in vitro propagation of vanilla. in this species, axillary shoot multiplication was greatest in dps, with an increase over 2.5­fold in comparison to the solid medium sys­ tem, after 90 days of cultivation. similarly, couselo et al. (2006) noted that the micropropagation rates of albariño plants were significantly higher when cultur­ ing in a dps system with the same concentration of ba (8.88 μm) in both phases, in comparison to a monophasic one. the same trend on the efficiency of dps cultivation system was reported for the micro­ propagation of arrow cane (lopez and suarez, 2018) ananas (scherwinski­pereira et al., 2012), pyrus sp. (moraes et al., 2004), and rauwolifa serpentine (senapati, 2015). on the contrary, barceló­muñoz et al. (1999) during micropropagation of avocado, reported that continuous culturing under dps condi­ tions induced succulence in shoot bases and hyperhy­ dric of the cultures. together with the effectiveness for the growth dynamic, dps has another advantage due to the peri­ odic addition of the liquid medium. in this condition, the period from one subculture to the other can be longer than in a monophasic liquid or solid medium, where this period is up to 3­4 weeks, as reported also by mahmad et al. (2014). the cs system needs to change the medium in culture vessels after some time to avoid nutrient deficiencies. all this requires a series of laborious operations in terms of costs, both of hand labor and chemicals. moreover, there is more working time in the cabinet laminar flow which creates possibilities for increasing the percentage of culture contamina­ tion. so, it can be said that in dps propagation sys­ tem, the cost of plant production is reduced. this finding is also accurately reported by lopez and suarez (2018) who calculated the costs of production per plant and found that, for arrow cane, the cultiva­ tion in the dps system reduced the micropropagation costs by 20%. a similar estimation in terms of production cost was also realized by senapati (2015) who found that dps system was much more effective due to the costs reduction specifically with 33.36% on the nutrients used, 39.28% on the energy used and 33.33% on the labor costs. optimization of such technique that, in addition to being low cost, also provides rapid in vitro plantlets regeneration, is of great interest for commercial use. biomass production during the rooting stage after the proliferation stage, the shoots were transferred in dps system and cs system for rooting. the first system had in the liquid phase the same type and concentration of pgrs of the proliferation stage, to promote the shoots development and at the same time, the solid phase was prepared to give fig. 2 ­ in vitro regeneration of wild apple shoots in cs micropro­ pagation system (a, b) and in dps micropropagation system (c, d, e). sota et al. ‐ micropropagation of wild apple in a double‐phase system 365 a rhizogenic induction. the medium of cs system was supplemented with rooting induction hormone (iaa). the biometric parameters were greatly affected, dur­ ing this stage, not only by the propagation system but also by the concentration of activated charcoal in the media (table 1). rhizogenesis induction from a comparison between the two propagation systems for the same concentration of ac, in general, the best result was achieved in the dps propagation system. overall, rooting induction was highly affected by the concentration of ac in the culture media, and it was observed a positive correlation between the concentration of ac in the media and the rhizogene­ sis rate (table 1; fig. 3 a, b). for roots length parameter, during the dps culture, the best result (13.16 cm) was obtained at 500 mg l­1 of ac concentration; while the higher roots number (9.80) was showed at 1000 mg l­1 of ac concentration. in the cs, for both parameters, the best results were achieved at 1000 mg l­1 of ac, respectively with 10.40 cm for roots length and 7.53 for roots number. there were no significant differences for the roots number parameter in cs propagation system between culture media supplemented with 1000 or 500 mg l­1 of ac. from an overall evaluation and comparison for the rooting response depending on both propagation system and ac concentration, the optimal condition for in vitro rooting of wild apple shoots was on dps propagation system supplemented with 1000 mg l­1 or 500 mg l­1 of ac. on culture medium supplemented with 250 mg l­1 of ac or ac­free medium, the rooting response did fig. 3 ­ one­way anova analysis of roots number (a) and roots length (cm) (b) depending on the activated charcoal (ac) concentration and micropropagation system. table 1 ­ the effect of the micropropagation system and activated charcoal concentration on roots and shoots development during the rooting phase of m. sylvestris data represents mean ± standard error. within each column, data followed by different letters are significantly different at p≤0.05 by tukey­kramer test. propagation system activated charcoal (mg l­1) roots shoots length (cm) number length (cm) number dps 0 2.46 ± 0.16 e 2.66 ± 0.21 e 3.40 ± 0.15 c 3.53 ± 0.38 e 250 5.23 ± 0.27 d 4.86 ± 0.31 d 4.61 ± 0.62 b 5.61 ± 0.36 b 500 13.16 ± 0.67 a 8.73 ± 0.51 b 5.58 ± 0.17 a 7.46 ± 0.48 a 1000 11.03 ± 0.30 b 9.80 ± 0.29 a 4.76 ± 0.17 b 5.40 ± 0.32 bc cs 0 2.22 ± 0.11 e 2.46 ± 0.24 e 2.74 ± 0.11 d 2.40 ± 0.19 f 250 4.81 ± 0.22 d 4.33 ± 0.28 d 3.18 ± 0.14 c 3.93 ± 0.31 de 500 9.93 ± 0.33 c 7.33 ± 0.39 c 3.37 ± 0.12 c 4.60 ± 0.25 cd 1000 10.40 ± 0.39 bc 7.53 ± 0.29 c 3.02 ± 0.12 cd 4.26 ± 0.30 de not show significant differences between dps and cs. this indicated that ac, with concentrations ˃ than 250 mg l­1, was the determining factor to improve rooting response. in this propagation stage, also dps system resulted most advantaged in comparison to cs one. new shoots regeneration during the rooting stage since the liquid phase in the dps had a hormonal ratio improving the development of lateral buds, at this stage also the response of plants in terms of mass formation of new shoots was evaluated. the 366 adv. hort. sci., 2021 35(4): 361­369 solid phase in both cs and dps was optimized only for the induction of rhizogenesis. as it can be seen from data presented (table 1; fig. 4 a, b and fig. 5) the differences regarding shoots length and number between dps and cs cultivation systems are highly significant. the comparison between the dps and cs propa­ gation systems showed the considerable dps efficien­ cy for shoots length and shoots number. in particular, in ac­free media, the differences were highly signifi­ cant, 3.40 cm (shoots length) and 3.53 (shoots num­ ber) for dps in comparison with 2.74 cm and 2.40 shoots, respectively, in the cs. obviously, dps was more effective due to the supplementation of liquid phase with hormones responsible for shoots prolifer­ ation. also, this proved further that the double phase system was effective for the growth of plantlets and at the same time for their rooting. during the rooting stage, the dps allowed the simultaneous formation of roots and new shoots at a higher rate, thus leading to the possibility to reduce the micropropagation cost by combining in a single one the last stage of subculture and rooting. the presence of ac supported also the develop­ ment of additional shoots, in both propagation sys­ tems, but in dps this effect was more pronounced and significant in comparison to cs. moreover, the ac influence increased until 500 mg l­1 concentration, and, indeed at this ac concentration, the dps showed the higher rate of shoots number (7.46) and shoots length (5.58 cm). the same tendency was observed even in cs where the concentration of ac at 500 mg l­1 gave higher efficiency, with 3.37 cm of the shoots length and 4.60 for the shoots number. the results indicated that ac concentration in the media was an important factor that highly affected micropropagation rate. roots and new shoots forma­ tion responses were positively correlated and were dependent on the ac concentration in the nutrient media. in ac­free media, the roots formation was not at a high rate in comparison to the other ac concen­ trations. addition of activated charcoal in culture medium may affect in vitro plants growth, in terms of multipli­ cation ratio, shoot elongation, rooting and embryo­ genesis (pan and van staden, 2001; thomas, 2008; abdulwahed, 2013). most publications have focused on the effects of activated charcoal on tissue response during in vitro propagation, and it was shown that its use may either promote or inhibit growth under in vitro conditions, depending on dif­ ferent factors. boudabous et al. (2010) reported that the use of ms­half medium supplemented with 200 mg l­1 of ac and 3.0 mg l­1 of iba, was highly effective on in vitro rhizogenesis of apple. on the other hand, magyar­tábori et al. (2002), in their study didn’t find any favourable effect of activated charcoal on rooting characteristics of apple, but the plants originated from media that contained activated charcoal grew more vigorously during rooting and acclimatization. in cotton (gossypium hirsutum), the addition of ac in the medium enhanced shoots and roots induc­ fig. 4 ­ one­way anova analysis of shoots number (a) shoots length (cm) (b) depending on the activated charcoal (ac) concentration and micropropagation system. fig. 5 ­ rhizogenesis induction and mass plantlets production during the rooting stage: culture in ac­free media (a); wild apple cultures grown in various concentrations of ac (b); roots measurements (c); mass production of roo­ ted plantlets (d). sota et al. ‐ micropropagation of wild apple in a double‐phase system 367 tion as well as shoots length from split embryo axes as compared to ms basal medium (hazra et al., 2002). also, dev et al. (2015) mentioned that the use of 200 mg l­1 of ac, significantly improved in vitro multiplication of some grape genotypes, meanwhile hassan et al. (2011) found that the presence of ac in the medium enhanced microtuberization and in vitro regeneration of potato plantlets. moreover, it is widely accepted that some of the beneficial effects of activated charcoal can be attributed to the removal of inhibitory substances from the nutrient medium. this phenomenon is mostly considerable for m. sylvestris in vitro propagation, because shoot explants of this plant species even after establish­ ment and several subcultures shows browning at the shoot base (sota et al., 2021). roots grow according to negative phototropism and, in many cases, light is considered as a stress fac­ tor for roots induction (silva­navas et al., 2015) and it is reported that the combination of darkness and exogenous auxins enhance rooting response (monteuuis and bon, 2000; lópez­pérez and martínez, 2015). in our research, the presence of ac in the nutrient medium created a state of darkness, thus enabling the formation of a well­developed roots system. this ensured a higher absorption of the nutrients, which can lead also to new shoots forma­ tion and development. furthermore, the presence of a higher concentration of cytokinins vs. auxins in the liquid phase also was responsible for lateral shoots development. in this context, in our study, both the propagation system types and the presence of ac in the media, highly affected the micropropagation rate and the quality of regenerated plantlets. moreover, another advantage of dps in compari­ son to cs system that can explain enhanced growth is the weekly addition of the liquid media into the ves­ sel during the culture period, since the plantlets did not suffer mineral deficiencies. 4. conclusions malus sylvestris, wild apple, is a very important plant species and is properly considered a threatened one; in such situation, the constant optimization of the micropropagation protocols is a necessity. the specific type of propagation system plays a key role in enhancing growth parameters. in the present study, our findings sufficiently indicated that the use of the dps propagation system in comparison to cs one was highly effective during in vitro shoots’ regen­ eration and rooting stage. moreover, the addition of activated charcoal for rooting induction obviously improved not only roots formation but also the growth of wild apple shoots. among various concen­ trations of ac examined, the addition of 500 mg l­1 ac in culture medium was found to be the best concen­ tration for this process. the obtained results demon­ strated a direct correlation between the propagation system used and the concentration of ac in the cul­ ture media. the application of dps propagation sys­ tem for in vitro propagation of wild apple, therefore represents more time­saving and a low­cost tech­ nique in comparison to the cs. these findings could provide a platform for progressively improving the clonal propagation of wild apple plantlets grown under in vitro conditions, leading so to the possibility for the effective use of these plantlets for its ex situ conservation or other scientific or practical purposes. references abdulwahed m.s., 2013 ­ identification of the effect of different levels of activated charcoal and sucrose on multiplication shoots of date palm phenix dactylifera l. cv. sufedy in vitro. ­ j. hortic. forestry, 5(9): 139­145. barceló­muñoz a., encina c.l., simón­pérez e., pliego­alfaro f., 1999 ­ micropropagation of adult avocado. ­ plant cell tiss. organ cult., 58: 11­17. benelli c., ozudogru e.a., lambardi m., dradi g., 2012 ­ in vitro conservation of ornamental plants by slow growth storage. ‐ acta horticulturae, 961: 89­93. boudabous m., mars m., marzougui n., ferchichi a., 2010 ­ micropropagation of apple (malus domestica l. cultivar douce de djerba) through in vitro culture of axillary buds. ­ acta bot. gallica, 157: 513­524. chakrabarty d., hahn e.j., yoon y.j., paek k.y., 2003 ­ micropropagation of apple rootstock m.9 emla using bioreactor. ­ j. hort. sci. biotech., 78(5): 605­609. couselo j.l., varela p., rey m., 2006 ­ effect of benzyla‐ denine concentration and double‐phase culture system on in vitro multiplication of adult albari plants. ­ amer. j. enol. vitic., 57: 109­112. cuenca b., sánchez c., aldrey a., bogo b., blanco b., correa b., vidal n., 2017 ­ micropropagation of axil‐ lary shoots of hybrid chestnut (castanea sativa x c. cre­ nata) in liquid medium in a continuous immersion sys‐ tem. ‐ plant cell tiss. organ cult., 131: 307­320. dal vesco l.l., de almeida pinto a., zaffari g.r., nodari r.o., dos reis m.s., guerra m.p., 2001 ‐ improving pineapple micropropagation protocol through explant size and medium composition manipu‐ lation. ­ fruits, 56(3): 143­154. adv. hort. sci., 2021 35(4): 361­369 368 damiano c., arias padro m.d., frattarelli a., 2008 ­ propagation and establishment in vitro of myrtle (myrtus communis l.), pomegranate (punica granatum l.) and mulberry (morus alba l.). ­ propag. ornam. ­ plants, 8(1): 3­8. day j.g., stacey g.n., 2007 ­ cryopreservation and freeze‐ drying protocols. ­ second edition. humana press, totowa, new jersey, usa, pp. 365. debnath m., malik c.p., bisen p.s., 2006 ­ micropropagation: a tool for the production of high quality plant‐based medicines. ­ curr. pharmac. biotechn., 7(1): 33­49. dev r., singh s.k., singh a.k., verma m.k., 2015 ­ comparative in vitro multiplication of some grape (vitis vinifera) genotypes. ­ ind. j. agr. sci., 85: 1477­1483. dobránszki j., magyar­tábori k., tombácz e., 2011 ­ comparison of the rheological and diffusion properties of some gelling agents and blends and their effects on shoot multiplication. ­ plant biotechnol. rep., 5: 345­ 352. dorić d., ognjanov v., ljubojević m., barać b., dulić j., pranjić a., dugalić k., 2014 ­ rapid propagation of sweet and sour cherry rootstocks. ­ not. bot. horti. agrobo., 42(2): 488­494. fao, 2015 ­ global forest resources assessment 2015. ­ food and agriculture organization of the united nations, rome, itay, pp. 245. gatti e., sgarbi e., ozudogru e.a., lambardi m., 2017 ­ the effect of plantformtm bioreactor on micropropaga‐ tion of quercus robur in comparison to a conventional in vitro culture system on gelled medium, and assess‐ ment of the microenvironment influence on leaf struc‐ ture. ­ plant biosyst., 151: 1129­1136. gixhari b., ramadani m., 2016 ­ agrobiodiversity in fushë arrëz. ­ monograph published within the project founded by un “increasing the capacity of the community of fushë­arrëz for the preservation of autochthonous agricultural, nut and fruit seeds”. 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temporary immersion princi‐ ple. ­ plant cell tiss. organ cult., 81: 313­318. https://www.researchgate.net/journal/revista-brasileira-de-fruticultura-0100-2945 https://www.researchgate.net/journal/revista-brasileira-de-fruticultura-0100-2945 https://www.researchgate.net/journal/revista-brasileira-de-fruticultura-0100-2945 http://www.ncbi.nlm.nih.gov/pubmed/?term=pullman%20gs%5bauthor%5d&cauthor=true&cauthor_uid=17235557 http://www.ncbi.nlm.nih.gov/pubmed/?term=skryabina%20a%5bauthor%5d&cauthor=true&cauthor_uid=17235557 http://www.ncbi.nlm.nih.gov/pubmed/17235557 http://link.springer.com/journal/11240 http://link.springer.com/journal/11240/109/2/page/1 http://www.euforgen.org/fileadmin/templates/euforgen.org/upload/publications/technical_guidelines/922_technical_guidelines_for_genetic_conservation_and_use_for_wild_apple_and_pear__malus_sylvestris_and_pyrus_pyraster_.pdf http://www.euforgen.org/fileadmin/templates/euforgen.org/upload/publications/technical_guidelines/922_technical_guidelines_for_genetic_conservation_and_use_for_wild_apple_and_pear__malus_sylvestris_and_pyrus_pyraster_.pdf http://www.euforgen.org/fileadmin/templates/euforgen.org/upload/publications/technical_guidelines/922_technical_guidelines_for_genetic_conservation_and_use_for_wild_apple_and_pear__malus_sylvestris_and_pyrus_pyraster_.pdf http://www.euforgen.org/fileadmin/templates/euforgen.org/upload/publications/technical_guidelines/922_technical_guidelines_for_genetic_conservation_and_use_for_wild_apple_and_pear__malus_sylvestris_and_pyrus_pyraster_.pdf http://www.euforgen.org/fileadmin/templates/euforgen.org/upload/publications/technical_guidelines/922_technical_guidelines_for_genetic_conservation_and_use_for_wild_apple_and_pear__malus_sylvestris_and_pyrus_pyraster_.pdf https://en.wikipedia.org/wiki/european_forest_genetic_resources_programme https://en.wikipedia.org/wiki/european_forest_genetic_resources_programme https://en.wikipedia.org/wiki/european_forest_genetic_resources_programme https://www.infona.pl/contributor/0@bwmeta1.element.elsevier-114b857b-6d32-3b39-9507-87a6a5c67e12/tab/publications https://www.infona.pl/contributor/1@bwmeta1.element.elsevier-114b857b-6d32-3b39-9507-87a6a5c67e12/tab/publications https://www.infona.pl/contributor/2@bwmeta1.element.elsevier-114b857b-6d32-3b39-9507-87a6a5c67e12/tab/publications https://www.researchgate.net/journal/biotechnology-advances-0734-9750 untitled 101 propagation of endangered grapevine cultivars: some reasons to recover and protect this patrimony e. dallavalle*, e. triolo** * agronomo libero professionista, bologna, elisabettadallavalle@libero.it ** presidente tos.co.vit., s. piero a grado (pi). endangered grapevine plants of uncertain origin may represent a target for safeguarding. current regulations guarantee the propagation of selected plant material following clonal procedures with the aim of excluding the most dangerous plant viruses. thus, rare grapevine plants that survived the nineteenth-century plagues may be characterized by interesting epigenetic features. their progeny could take on an important role with regard to today’s viticulture: consider how these individuals could represent an important source of genetic material that not only possesses biological, historical, scientific and educational value, but also the qualitative potential to attract interest from the most demanding markets. moreover, in recent years, the recovery of ancient grapevines has been reassessed with the use of plants more than a hundred years old to produce new wines, and for which novel characteristics may be described on the label. in this context, in the last few years, intense research has been undertaken to evaluate forgotten plants and as a way to utilize old plantings. the ‘associazione partriarchi’ of forli was the first to apply this approach to save a group of ancient vines that had been abandoned for decades. ‘uva caveccia’ and ‘uva morta’ were the vines involved: these very old vines have close ties to the romagna plain in italy where they were cultivated in connection with maple or elm, and often with almond, pear or plum. in another case, the trunk of a century-old, white grape vine has surfaced recently near aosta (moriondo et al., 2010). genetic analysis was undertaken to determine its parentage: a descendant of prié. the historical data, morphological measurements and parentage with prié lead to the conclusion that the surviving plant belongs to the blanc comun variety. these are not singular examples of the use of old vines from the pre-phylloxera period. for instance, there has been interest in valdobbiadene toward 100-150 year old plants located on steeper slopes between farra di soligo and valdobbiadene. the senarum vinea project has made it possible to discover, also in tuscany, ancient examples of century-old autochthonous/minor vines that have survived, essentially forgotten, up to the current day. among the identified vines, we can mention ‘gorgottesco’, ‘tenerone’, ‘salamanna’, ‘occhio di pernice’ and ‘rossone’. in addition, another interesting application for old grapevines may derive from their use as ornamental plants, as suggested by some examples within the monastery of s. maria maddalena in bologna or in the olivetani cloister of the church of s. maria in regola in imola. references moriondo g., sandi r., vouillamoz j., 2010 identificazione del neret di saint-vincent, antico vitigno valdostano. l’informatore agricolo, 5. adv. hort. sci., 2013 27(3): 101 untitled 113 competitiveness of the wine sector: considerations on future scenarios g. scalabrelli, a. toffanin department of agriculture, food and environment, university of pisa, via del borghetto 80, 56124 pisa, italy, giancarlo.scalabrelli@unipi.it for a critical assessment of the issues relating to the future viticulture nursery system in order to identify the strengths and weaknesses is essential to examine the relationship between factors of the supply chain and the extrinsic factors able of modulating the interactions between the whole wine system. the uncertainty of the evolution over time of the forecasts suggests examining these aspects for hypothetical scenarios which involves the following interfaces: consumer (market)/ producer, grower/nursery, research/nursery/grower/consumer. other aspects might modulate the above relations: social ethics, energy cost, globalization, economic situation, climate change, pathogens emergencies. consumer (market)/producer. what is certain is that enlargement of the european community to the east and the lack of restrictions on the spread of viticulture in new emerging countries (latin america, south africa and oceania), will pose competition problems on traditionally wine countries such as italy and france which will fight fiercely to defend the current position revenue linked to the prestige of wine-growing areas. there is no doubt that the new emerging countries, at least for several years, will base their market strategy on the best ratio between cost and price, with condition that it will be possible to maintain rapid and streamlined trades between countries, leaving the market share of high-quality wines. the dominant effects of the recession could lead to the reduction in the purchasing power of the population and thus to decrease the consumption of luxury goods, within the framework of the poorer population groups. the decrease in consumption will vary depending on the role attributed to the wine (food or hedonistic) and the ability to reduce other purchases luxury or essential goods, although the pleasure being part of main needs (but individual) will favors the consumption of high quality wine. new dietary needs could emerge, to supplement the daily diet, and especially locally or from neighboring basins (localism, joint buying groups). environmental sensitivity could increase the need for produce in an environmentally-friendly way (scalabrelli, 2010) and with less wasted energy and co 2 production due to long-range transport. there is, however, to take into account many motivational aspects regarding the future evolution of wine consumption. we already feel the first symptoms of new trends that tend to favor wines with special features of healthiness, peculiarity and uniqueness. in fact, a part of the population does not seek more homogeneous characteristics and wines already granted who find themselves generally in varietal wines produced in a specified area or a specific brand. the uncertainty on these issues lies in sizing the scope of this question. grower/nursery. the expected decline in demand should not have immediate effects on the structural consistency of the companies, although it might affect the renewal of new vineyards and delay the activation of new business ventures. consequently, with the exception of the physiological abandonment of vineyard areas, it could be envisaged a slight decline in demand for new plantations with possible surplus of material produced. in a first stage the survival of nursery companies will not be at risk, but those more efficient and better organized will have more chances of remain on the market. in a longer period, the continuing demand for wine (or diversification), might request to redirect the offer to new sales channels (or production), although it is hardly predictable as the globalized market might move and what changes might be required in order to produce and sell. companies will have to carefully follow the market and consumer demands, develop new solutions to meet the new population requirements. for example, produce in an environmentally-friendly, organic, biodynamic will be no longer just slogans, if the research will have clarified and deepened aspects and come up with appropriate techniques, but the needs imposed by the consumer. for productive diversification, there is currently a clear differentiation between traditional varieties and the advent of new varieties obtained by genetic improvements. the tendency to preserve the local varieties and to use the traditional ones appears a goal practiced by small farm meanwhile the possible use of new varieties obtained by genetic improvement will be probably a strategy of the globalized system. of course biodiversity conservation would not be exhaustive unless their characteristics and performance will be known in view to utilise this source for possible direct cultivation or for genetic improvement. in this contest the nurseries could have new opportunities either on the production of autochthon varieties or propagating new material obtained by genetic improvement. research/nursery/grower/consumer. the critical economic situation it is not expected to influence promptly the work in progress for the clone selection, previously started, altogether new projects initiatives could be restricted unless, a reversal trend towards investment of funds for research will take place. the local germplasm collection and its use are adv. hort. sci., 2013 27(3): 113-114 114 prospected although this aspect is not universally shared, as worldwide is focusing attention on few international grapevine varieties. in any case the biodiversity of grapevine germplasm is considered a resource (scalabrelli, 2007; d’onofrio and scalabrelli, 2010). another area of great scientific interest is the study of varietal behavior, in view of climate change and, above all, thanks to the progress made with the grape genome sequencing (jaillon et al., 2007) genetic improvement to obtain varieties more resistant to biotic and abiotic stress, can take advantages of the assisted breeding techniques. we could have, therefore, on world market engineered plants produced exclusively from several countries or companies that can find spread depending on the acceptance or rejection by the consumers. it is clear that a fundamental part of research will be carried out to clarify whether gmos can pose risks to health or the environment. on this basis a possible clarification it might also change the current hostile attitude of some countries or part of the population. the strategy choice to cultivate or not genetically modified vines could also result in disruption in the spread of viticulture in different countries, but this could not occur before ten years. in our opinion, it should be advisable the introduction of specific genes within the genus euvitis. moreover another field of research is the intensification of the studies to understand the mechanisms that regulate the plants functioning and the role of symbiotic microorganisms which could facilitate the cultivation and the development of environmentally friendly techniques (biodynamic method?). beneficial microorganisms, genetically modified or not, will be useful for finding biotechnological solutions, especially to control biotic and abiotic stress and for a biofertilization in balance with the aimed productions. in presence of a high critical situation of funding we need to wisely manage the ethical aspects of research and ensure adequate and rational fund investment. it would be desirable for local projects carry out a “shared research”, involving the various components of the “wine chain”, a new need this to satisfy, especially if the state and the regions will not be able to devote resources to this sector, as committed to supporting consumption and to invest on social safety nets references jaillon o., aury j.-m., noel b., policriti a., clepet c., casagrande a., choisne n., aubourg s., vitulo n., jubin c., vezzi a., legeai f., hugueney p., dasilva c., horner d., mica e., jublot d., poulain j., bruyère c., billault a., segurens b., gouyvenoux m., ugarte e., cattonaro f., anthouard v., vico v., del fabbro c., alaux m., di gaspero g., dumas v., felice n., paillard s., juman i., moroldo m., scalabrin s., canaguier a., le clainche i., malacrida g., durand e., pesole g., laucou v., chatelet p., merdinoglu d., delledonne m,, pezzotti m., lecharny a., scarpelli c., artiguenave f., pè m.e., valle g., morgante m., caboche m., adam-blondon a.-f., weissenbach j., quétier f., wincker p., the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. nature, 449: 463-467. d’onofrio c., scalabrelli g., 2010 un database viticolo universale. italus hortus, 17(3): 328-333. russo a., carrozza g.p., vettori l., felici c., cinelli f., toffanin a., 2012 plant beneficial microbes and their application in plant biotechnology, pp. 57-72. in: agbo e.c. (ed.). innovations in biotechnology. intech europe, rijeka, croatia, pp. 474. scalabrelli g., 2007 i vitigni autoctoni. locus. felici editore, pisa, 7: 45-52. scalabrelli g., 2010 quale viticoltura per il futuro? italus hortus, 17(3): 680-686. 195 1. introduction cyclamen is an herbaceous perennial used as a flowering potted plant (karlsson and werner, 2001; elmer and mcgovern, 2004). in the genus cyclamen, cyclamen persicum is the major species used for commercial cultivation (ishizaka et al., 2002). it has a longer growing season and temperature is one of the most important environmental factors that affect the growth, development and distribution of this plant (yesson and culham, 2006). cyclamen is highly susceptible to various temperature conditions and the recommended temperature for cyclamen production from seeding to appearance of flower bud is 20°c (ball, 1991). currently, due to global warming, summer temperatures are increasingly elevated. however, garden type cyclamen is intolerant to heat stress, which severely affects growth and development of this plant in southwest japan during summer (goto et al., 2011). on the other hand, in summer, heat is not the only stress factor, but pathogens also pose a threat. pathogenesis infection impacts commercial cyclamen production. although various diseases are reported, anthracnose is especially destructive disease worldwide. anthracnose is responsible for colletotricum gloeosporioides (cg) and it causes extensive lesions on aerial plant parts. today, cost effective and eco-friendly control strategies against heat and anthracnose are needed. arbuscular mycorrhizal fungus (amf) has drawn attention by crop researchers for its benefits to host plants. improving nutrient uptake, especially of phosphate, of the host plant is well known as one of the benefits of arbuscular mycorrhizal symbiosis (marschner and dell, 1994). additionally, several investigators have reported a higher resistance of mycorrhizal plants to biotic and abiotic stresses (garmendia et al., 2006 ; wu et al., 2006; li et al., 2010). as for cyclamen, mycorrhizal plants showed growth promotion under heat stress (30°c) (maya and matsubara, 2013 a) and higher tolerance to anthracnose caused by cg (maya and matsubara, 2013 b). however, growth improvement under extreme shock heat stress, resistance to anthracnose under such heat stress and proteomic changes through heat stress and anthracnose in mycorrhizal plants are yet to be revealed. when plants suffer to heat stress and pathogenesis infection, reactive oxygen species (ros) generate in cells and the oxidative stress to intercellular structures they cause are major damaging factors in plants (wahid et cross-protection against anthracnose with heat stress, antioxidative changes and proteomic analysis in mycorrhizal cyclamen s. watanabe* y.-i. matsubara**(1) * graduate school of applied biological sciences, gifu university, gifu 501-1193, japan. ** faculty of applied biological sciences, gifu university, gifu 501-1193, japan. key words: colletotricum gloeosporioides, dpph, growth promotion, shock heat stress, sod, symbiosis. abstract: cross-protection against anthracnose with shock heat stress, antioxidative changes and proteomic analysis in mycorrhizal cyclamen were investigated. eleven weeks after arbuscular mycorrhizal fungus (amf, glomus fasciculatum) inoculation, cyclamen (cyclamen persicum, cv. pastel) plants were acclimated under 20°c for 4 days, followed to 35°c (shock heat stress, shs) for 7 days, and inoculated with colletrichum gloeosporioides (cg) as anthracnose pathogen. seven days after shs treatment, dry weights of leaves, bulbs and roots increased in mycorrhizal plants, thus, growth promotion appeared. in addition, mycorrhizal plants showed higher resistance to anthracnose compared to non-mycorrhizal control plants. regarding antioxidative activity, superoxide dismutase (sod) activity increased in roots of mycorrhizal plants under 7d after shs. dpph radical scavenging activity increased in some parts of the mycorrhizal plants under shs and cg-inoculated conditions. as for proteomic analysis, totally 29 spots changed in mycorrhizal plants through shs and cg-inoculated conditions. in this case, the spot of 20.4kda was detected only in mycorrhizal plots, furthermore, 4 spots intensively appeared in mycorrhizal plots through shs and cg conditions. from these findings, amf could alleviate heat shock stress with promoting host plant growth and induce resistance to anthracnose under heat stress. in addition, it supposed that antioxidative modification would have cross association with the resistance to heat shock and anthracnose, and the symbiosis-specific changes in some proteins might have concern with the cross protection. adv. hort. sci., 2014 28(4): 195-201 (1) corresponding author: yamatsu@gifu-u.ac.jp received for publication 17 september 2014 accepted for publication 2 october 2014 196 al., 2007). in order to cope with these toxic ros, plants have antioxidants as detoxification factors (kuzniak and sklodowska, 2004; wu et al., 2006). antioxidative defense mechanisms consist of antioxidative enzymes such as superoxide dismutase (sod), ascorbate peroxidase (apx) and catalase (cat) and nonenzymatic antioxidants such as polyphenols and ascorbic acid (asada, 1999). enhancement of antioxidative activity in mycorrhizal plants has been already reported (lambais et al., 2003). physiological changes in plants due to heat stress have not yet been revealed. however, plants have shown different responses under heat acclimation and heat shock (durand et al., 2012). according to li et al. (1984), heat shock caused stronger growth inhibition than heat acclimation. although heat shock is thought to be a stronger heat stress treatment, some researchers reported that heat shock (around 45°c) induced tolerance against aboveground diseases such as anthracnose to host plants (teruya et al., 2012; yoshino et al., 2012). therefore, heat shock could be considered as a trigger defense system toward pathogens. on the other hand, amf symbiosis also causes alteration of gene expression in leaves and roots (liu et al., 2007). campos et al. (2012) reported that expression of some defense related genes such as transcriptional factors, regulators of ca2+ signaling pathway, mapks and npr1 (nonexpressor of pr genes) etc. had been up-regulated in mycorrhizal rice leaves. yet, the influence of amf on protein expression in cyclamen under heat stress and cg inoculation have not been revealed. the aim of this study was to determine cross-protection against heat stress and anthracnose in mycorrhizal cyclamen in association with antioxidative changes and proteomic analysis. 2. materials and methods plant material and mycorrhizal inoculation cyclamen (cyclamen persicum, cv. pastel) seedlings (three months of age) were replanted in plastic pots containing autoclaved (121°c, 1.2 kg/cm2, 15 min) commercial potting media (sogemix sm-2). in the meantime, plants were inoculated with amf (glomus fasciculatum) inoculum for mycorrhizal (amf) plants and autoclaved inoculum for non-mycorrhizal (control) plants. in both cases, inocula (3 g/plant) were placed 4 cm below the bulbs. commercial amf inoculum supplied by idemitsuagri co. ltd, tokyo, japan was used in this study: the spore density was unknown. plants were transferred into poly silver pot (9 cm diameter) after 11 weeks of amf inoculation. plants were fertilized with slow release granular fertilizer (n:p:k= 5:10:15, ube industries ltd.) once a month and raised in a greenhouse. treatment with shock heat stress (shs) eleven weeks after amf inoculation, the plants are transferred to a growth chamber (20°c constant, 12 hr daylength, rh 60%) for 4 days (d) as acclimation under optimum environmental conditions. after 4 days at 20°c, the temperature was suddenly increased to 35°c (constant) as shock heat stress treatment (shs). the plants were grown for 7 d under shs. inoculation of colletrichum gloeosporioides the isolate (maff744024) of colletrichum gloeosporioides (cg) was collected from the ministry of agriculture, forestry and fisheries, japan. it was grown in potato dextrose agar (pda) medium and was incubated at 25°c for two weeks in dark conditions to prompt sporulation. the spore concentration was adjusted to 105 cfu using sterile distilled water. the shoots of each plant were sprayed at 7 d after shs with 10 ml of cg suspension. symptoms of anthracnose were evaluated 9 d after cg inoculation. a completely randomized design with five replicates was used. disease incidence and index of anthracnose disease symptoms were checked 9d after cg inoculation. the disease severity in individual plants was rated visually on a scale of 0-5. 0= no symptoms (healthy plants); 1= <20% disease symptoms (small discolored leaf lesions covering less than 20% of total leaves of a plant); 2= 20-40% disease symptoms (minor small discolored lesions covering 20-40% of leaves); 3= 40-60% disease symptoms (moderate brown lesions in 40-60% of leaves and 15% defoliation); 4= 60-80% disease symptoms (mild wilt discolouration covering 60-80% of leaves and more than 50% leaf defoliation in case of fusarium wilt); 5= 80-100% disease symptoms (stems and leaves severely affected). the disease index for cg was calculated using the following formula: disease index= [σ (number of diseased plants x severity level)] x 100/ (total number of plants x maximum level of disease severity) plant growth and mycorrhizal colonization dry weight of leaves, bulbs and roots of five plants which were grown at 20°c for 4 d, under shs for 2 d, 7 d and for 9 d after cg inoculation was determined after plant material was dried at 100°c for 24 h. the lateral roots of each plant were sampled and used to check the level of amf colonization according to phillips and hayman (1970). lateral roots were sampled in 70% ethanol and later washed with distilled water. they were then ground in 10% naoh and autoclaved (121°c, 1.2 kg/cm2, 10 min) and subsequently washed with distilled water and stained with trypan blue. the ratio of am fungal colonization was checked in 1-cm segments of lateral roots and approximately 60 samples of 1-cm segments were checked per plant. the average was calculated from the values of five plants for each time. 197 antioxidative analysis superoxide dismutase (sod) as enzymatic antioxidative activity and dpph radical scavenging activity as nonenzymatic antioxidative activity were analyzed. plants which were sampled 4 d after 20°c, 2 d and 7 d after shs, and 9 d after cg inoculation preserved with liquid nitrogen were used for each analysis. total sod activity was measured according to beauchamp and fridovich (1971). dpph radical scavenging activity test was carried out as described by burits and bucar (2000). all experiments were replicated three times. proteomic analysis protein extraction. one g of frozen extended leaves was homogenized in liquid nitrogen with mortar and pestle. powdered leaf sample was mixed with 2 ml of extraction buffer containing 7 m urea, 2 m thiourea, 4% chaps and 50 mm tris (ph 7). the mixture was then centrifuged (14,000 rpm, 4°c, 60 min), and the collected supernatant was mixed with 400 ml of 50% tca. this mixture was centrifuged (14,000 rpm, 4°c, 10 min) and supernatant was discarded. one ml of 100% acetone was added to the pellet and this mixture was centrifuged (14,000 rpm, 4°c, 10 min); the supernatant was discarded. these steps were repeated three times. after acetone was dried at rt, 200 µl of dissolution buffer (8 m urea, 2% nonidet p-40, 1% dithioslatol) was added to the pellet and centrifuged (14,000 rpm, 4°c, 5 min). the supernatant was sampled and mixed with 20 µl of 1m iodoacetamide. electrophoresis. the first dimension ief was run using wse 1500 (atto corp.) according to the manufacture’s instruction manual with some modifications (300v, 210 min). after electrophoresis, the gel was soaked in 100 ml of 2.5% tca for 5 min. then, it was washed with distilled water for 2 h. for the second dimension (sds-page), it was soaked in 100 ml of sds equilibration buffer a (50 mm tris-hcl, 2% sds, 0.0001% bpb, 0.001% dtt), shaken for 10 min and subsequently shaken in sds equilibration buffer b (50 mm tris-hcl, 2% sds, 0.0001% bpb, 0.001% iodoacetamide) for 10 min. sds-page was run on ae-6500 (atto corp.) using 12.5% polyacrylamide gel (epagel e-d12.5l, atto corp.) according to the manufacture’s instructions (100v, 20 ma, 150 min). finally, proteins were visualized by cbb staining. 3. results seven days after shs treatment, dry weights of leaves, bulbs and roots increased in mycorrhizal plants (fig. 1 and 2). in addition, leaf yellowing and browning caused by shock heat stress were alleviated in mycorrhizal plants compared to non-mycorrhizal control plants. the amf colonization level reached around 50% through all the treatments and did not differ among the treatments (data not shown). with regard to anthracnose symptoms, greater fig. 1 growth of mycorrhizal cyclamen under shock heat stress (shs) and colletotricum gloeosporioides (cg) inoculation. fig. 2 dry weight of mycorrhizal cyclamen before and after shs and cg inoculation. ● control; o amf (glomus fasciculatum). shs, cg, see figure 1. 20°c 4d, 4 days after 20°c; 35°c 2d, 2 days after shs; 35°c 7d, 7 days after shs; cg 9d, 9 days after cg inoculation. bars represent standard errors (n=5). ns, non-significant. *, significantly different between c and amf by t-test (p<0.05). 198 severity appeared in non-mycorrizal plants and mycorrhizal plants showed less severity and lower indices (fig. 3). differences in antioxidative activity occurred between amf and control plants during the experimental period. as for sod activity, significant change was not detected in leaves and bulbs but sod activity was enhanced in roots at 20°c, 2 d and 7 d after shs (fig. 4a). however, no difference in sod activity appeared under cg inoculation. dpph radical scavenging activity as non-enzymatic antioxidative activity increased roots of mycorrhizal plants through the experimental period, and some parts of mycorrhizal plants also showed higher dpph levels compared to control (fig. 4b). as a result of proteomic analysis, overall the expressions of 29 spots were detected as different spots in mycorrhizal plants compared to control through shs and cg inoculation (table 1). four days after 20°c, differences in expression of 11 spots between mycorrhizal and non-mycorrhizal plants appeared. in this case, although six increased spots and three newly appeared spots were detected in mycorrhizal plants, two spots slightly decreased in contrast to fig. 3 disease incidence and index of anthracnose in mycorrhizal cyclamen 9 days after cg inoculation under shs. shs, cg, c, amf, see figure 2. ratio of diseased leaves to the total in each plant. -20% ; 20-40% ; 40-60% ; -80%. fig. 4 sod activity, dpph radical scavenging activity in mycorrhizal cyclamen before and after shs and cg inoculation. control ; amf. shs, cg, see figure 2. bars represent standard errors (n=3). ns, non-significant. *, significantly different between c and amf by t-test (p<0.05). table 1 expression levels in 29 proteins in leaves of mycorrhizal cyclamen under shs and cg inoculation spot id estimated mw (kda) 20°c 4 d 35°c 7 d cg 9 d c amf c amf c amf a 68.0 + + b 68.0 + + + c 87.1 ++ ++ ++ + d 33.4 + ++ + ++ e 29.9 + f 26.9 ++ g 20.4 n n n h 19.4 ++ + + ++ i 37.5 n n d j 36.7 n n d k 24.3 + l 27.3 + + + m 27.7 + n 19.7 + + ++ o 19.5 + + + p 32.2 + ++ q 36.0 r 36.0 s 36.0 t 21.0 + u 27.0 ++ v 16.9 + ++ + ++ w 47.4 ++ x 20.7 ++ y 36.3 + ++ + ++ z 28.5 ++ ++ α 26.9 + ++ β 19.1 ++ ++ γ 19.1 ++ ++ shs, cg, c, amf, see figure 2. + = slightly increased spot in expression; ++ = greatly increased; = slightly decreased; -= greatly decreased; n= new spot; d= disappeared spot. in c plots, marks represent changes in expression level compared to former treatment. in amf plots, marks mean changes in comparison to c under same treatment. 199 control. seven days after shs, expression in 17 spots changed compared to 4 d after 20°c in control (fig. 5a). as for mycorrhizal plants, 14 spots showed higher expression levels than non-mycorrrhizal plants at 7 d after shs (fig. 5b). after cg inoculation, expression levels of a total of 23 spots changed compared to 7 d shs. in the control, eights spots increased in expression but six spots decreased and two spots disappeared compared to 7 d shs (fig. 5c). regarding amf, 13 spots increased in expression and two new spots appeared compared to control (fig. 5d). spot g (20.4kda) was mycorrhizal-specific expression through shs and cg treatments. the spots increased in expression by both shs and cg were l (27.3kda), o (19.5kda), v (16.9kda), y (36.3kda). in this case, especially, v and y increased in mycorrhizal plants compared to the control. 4. discussion and conclusions mycorrhiza-induced growth promotion in cyclamen under heat stress (30°c) and anthracnose were reported in previous study (maya and matsubara, 2013 a, b). however, growth improvement under shock heat stress (35°c), resistance to anthracnose in light of cross-protection under heat stress, and proteomic changes through heat stress and disease in mycorrhizal plants have not been clarified. in the present study, growth of mycorrhizal cyclamen under shs was greater than control, and amf plants showed greater increases in dry weight of leaves than other parts. this phenomenon might be caused by an allocation of carbohydrate by amf in shoots that was proportionally greater than tuberous tissues and below ground tissues (shokri and maddi, 2009). generally, temperature influences mycorrhizal symbiosis and growth promotion (zhu et al., 2011) i.e. high temperature caused negative effect or no effect on mycorrhizal colonization (li et al., 2008; compant et al., 2010). in the present study, no significant differences occurred in amf colonization levels, meaning heat stress might affect mycorrhizal symbiosis less. although many reports suggest that amf had induced disease resistance to host plants, most of them refer to soilborne disease interactions (whipps, 2004; li et al., 2006). in contrast to soil-borne diseases, shoot disease resistance induced by amf is rarely reported. however, some study have shown amf alleviated shoot disease and induced systemic acquired resistance (sar) (campos et al., 2012). as for anthracnose caused by colletotricum gloeosporioides, one study demonstrated that amf symbiosis suppressed anthracnose in cyclamen (maya and matsubara, 2013 b). in this experiment, mycorrhizal cyclamen plants also showed resistance to anthracnose even under the shock heat stress condition. hence, it is supposed that amf induced cross protection to heat stress and anthracnose in the cyclamen examined in this study. biotic (pathogen, nematode etc.) and abiotic stresses (heavy metal, drought, high temperature etc.) cause reactive oxygen species (ros) production in plant tissues. although ros have a positive role as a signal which triggers stress responses, it causes destructive damage to organs in cells and a loss of homeostasis. in order to limit oxidative damage and remove ros, plants have developed a detoxification system through changing antioxidative activity. antioxidative activity includes enzymatic and non-enzymatic antioxidants where sod is a key enzyme in the enzymatic antioxidation system, and non-enzymatic antioxidation is also an effective defense system, because it efficiently prevents accumulation of ros under stress conditions. there are reports suggesting that antioxidative modifications in mycorrhizal plants refer to amf-induced stress resistance (nahiyan and matsubara, 2012; lambais et al., 2003). however, as for antioxidative changes in host plants, both increases and decreases in activity has been reported (roldan et al., 2008). generally, ros is produced in roots under heat stress (kolupaev et al., 2013). increases in sod activity in roots of mycorrhizal cyclamen under heat stress have also observed (maya and matsubara, 2013 a). in this study, the same phenomenon occurred in mycorrhizal cyclamen. in addition, as for dpph radical scavenging activity that means total activity of non-enzymatic antioxidants: ascorbic acid, polyphenols, flavonoid etc., dpph radical scavenging activity increased in roots of mycorrhizal plants in this experiment. some investigafig. 5 2-d electrophoresis gel with leaf protein in mycorrhizal cyclamen 7 days after shs and 9 days after cg inoculation. a (control), b (amf) in 7 days after shs. c (control), d (amf) in 9 days after cg inoculation. shs, cg, see figure 2. letters show spot id in table 1. in a and c, black circle, expressionincreased spots compared to former treatment; white circle, decreased spots; black arrows, newly appeared spots; white arrows, disappeared spots. in b and d, black circle, expressionincreased spots compared to control under same treatment; white circle, decreased spots; black arrow, newly appeared spot. 200 tions have shown that amf-induced increases in dpph radical scavenging activity under salinity stress and pathogen infection (hichem et al., 2009; nahiyan and matsubara, 2012). although decreases and increases occurred in leaves and bulbs in this experiment, the decrease might be caused by the resolution of ros by such increased antioxidants. some investigators have reported that amf has enhanced activity of various defense mechanisms as well as antioxidants. campos et al. (2012) reported that various genes which regulate stress response in plants had were up-regulated in mycorrhizal rice (oryza sativa l.), and suggested that salicylic acid (sa)-dependent and jasmonic acid (ja)-dependent mechanisms associate with amf-induced disease resistance. they also implied that systemic acquired resistance (sar) is caused in mycorrhizal plants. in the present experiment, expression of many protein spots changed following heat stress and cg inoculation. moreover, expression levels of some spots also changed by amf. in this case, 4 d after 20°c, an intensive increase in six spots and three newly appeared spots (g, h and i) were recognized in amf plants. these spots may supposed to be associated with growth promotion through symbiosis. although these changes might be defense response to amf colonization itself, which is caused temporary in initial phase of amf colonization (campos et al., 2010). at 7 d after shs, an intensive increase in 12 spots and decrease in three spots (36.0 kda) was visible in control plants compared to 20°c. spots i and j, which were visible in the amf treatment at 20°c, were expressed by shs treatment. in mycorrhizal plants, 13 spots increased and especially six spots showed greater increase; spot g (20.4 kda) was still visible at shs treatment. after cg inoculation, eight spots increased intensively in non-mycorrhizal plants. compared to control plants, mycorrhizal plants showed considerable increase in 13 spots and spot g was still detected. in this study, proteins which were up-regulated under both shs treatment and cg inoculation were l (27.3 kda), o (19.5 kda), v (16.9 kda) and y (36.3 kda). especially, v and w showed greater expression levels in mycorrhizal plants than in the control under both stress conditions. yoshino et al. 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(japan), 11(1): 121-126. zhu x.c., song f.b., liu s.q., liu t.d., 2011 effects of arbuscular mycorrhizal fungus on photosynthesis and water status of maize under high temperature stress. plant soil, 346: 189-199. impaginato 89 adv. hort. sci., 2023 37(1): 89­99 doi: 10.36253/ahsc­14180 ethanol fermentation­ and ethylene physiology­related gene expression profiles in red delicious apples stored under variable hypoxic conditions and protocols e. salamé 1, s. brizzolara 1 (*), m. rodrigues 2, m. iob 3, p. tonutti 1, b. ruperti 2 1 crop science research center, scuola superiore sant’anna, piazza martiri della libertà, 33, 56127 pisa, italy. 2 department of agronomy, food, natural resources, animals and environment, via dell’università, 16, 35020 legnaro (pd), italy. 3 marvil engeneering, zona produttiva schwemm, 8, 39040 magré sulla strada del vino (bz), italy. key words: dynamic controlled atmosphere, erf, low oxygen, malus domestica, postharvest. abstract: dynamic controlled atmosphere (dca) is beneficial in maintaining specific quality parameters but, due to the extreme oxygen levels applied, can cause adverse effects on the fruit by inducing excessive anaerobic metabolism and the production of off­flavors. the metabolic adaptation and responses of apples (malus domestica borkh.) cv. red delicious to static or dynamic oxygen concentrations (0.3 and 0.8%, with sequential shifts) during cold storage for 7 months were studied by monitoring quality parameters and the expression of genes involved in sugar, fermentative metabolism, and ethylene physiology. ethanol content reached the highest levels (around 400 mg/kg fw) under 0.3% oxygen concentration and fruit firmness appeared to be reduced in samples accumulating the highest levels of ethanol. the oxygen switch was effective in reducing the ethanol concentrations with timing­dependent variable effects. the expression of fermentative (alcohol dehydrogenase, lactate dehydroge‐ nase, pyruvate decarboxylase) and sugar metabolism (β‐amylase; phosphofruc‐ tokinase; sucrose synthase) genes resulted to be differently affected by the hypoxic conditions imposed, in particular during the early stages of storage. sucrose synthase expression appeared to be highly sensitive to changes in low oxygen concentration. ethylene biosynthesis (acc synthase and oxidase) genes showed marked differences in their expression in relation to the static and dynamic protocols and the hypoxic conditions, as well as six ethylene responsive factors (erf) genes, some of them possibly involved in the oxygen sensing mechanism operating in fruit tissues. (*) corresponding author: stefano.brizzolara@santannapisa.it citation: salamé e., brizzolara s., rodrigues m., iob m., tonutti p., ruperti b., 2023 ­ ethanol fermentation‐ and ethylene physiology‐related gene expression profiles in red delicious apples stored under variable hypoxic conditions and protocols. ­ adv. hort. sci., 37(1): 89­99. copyright: © 2023 salamé e., brizzolara s., rodrigues m., iob m., tonutti p., ruperti b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 january 2023 accepted for publication 18 february 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14180 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 89­99 90 1. introduction dynamic controlled atmosphere (dca) represents one of the latest technical innovations for the long storage of apples (and few other fruit crops) (tonutti, 2015). with this technology, fruits are kept under extremely low oxygen concentrations (0.4 kpa or lower) that are beneficial for maintaining specific quality parameters (e.g., flesh firmness, acidity). however, by activating anaerobic metabolism, an accumulation of ethanol takes place. low concentra­ tions of ethanol are desirable in terms of improving organoleptic traits, reducing the incidence of chilling injuries (e.g., superficial scald) and limiting ethylene biosynthesis (dixon and hewett, 2000; scott et al., 2000; weber et al., 2020). yet, the accumulation of excessive ethanol results in the appearance of off­fla­ vors and physiological disorders (pedreschi et al., 2009). thus, based on different stress indicators (chlorophyll fluorescence ­cf­, respiratory quotient ­ rq­, and ethanol concentration), oxygen must be promptly adjusted (increased) to reach “safe” con­ centrations. the imposed extreme hypoxic conditions induce selective responses of apple tissues starting from the modulation of gene expression involved in particular in primary metabolism and hormone (mainly ethylene) physiology (cukrov et al., 2019). in granny smith, one of the apple cultivar most fre­ quently stored in dca, differential expression of sucrose synthase (susy), alcohol dehydrogenase (adh) and pyruvate­related metabolism (lactate dehydrogenase, ldh, pyruvate decarboxylase, pdc, and alanine aminotransferase, alaat) genes was detected when comparing 0.4 with 0.8 kpa oxygen concentration (cukrov et al., 2016). when, according to the dca protocol, oxygen level is increased from the lowest applied concentrations, molecular and metabolic rearrangements rapidly occur, with changes in both primary and secondary metabolism (brizzolara et al., 2019), indicating that highly reac­ tive mechanisms and oxygen sensors are present in apple cortex. the expression of genes involved in fer­ mentative metabolisms (e.g., adh), in secondary metabolism (e.g., phenylpropanoid pathway), hor­ monal responses and regulatory mechanisms (ethyl­ ene biosynthesis, erfs) resulted to be affected by the oxygen switch. the duration of the storage and the oxygen con­ centrations applied obviously play a key role in deter­ mining the fruit metabolic responses and the dynam­ ics of fermentative metabolite accumulation. in addi­ tion, apple varieties react differently to extremely low oxygen conditions during storage, in particular in terms of fermentation, ethanol production and accu­ mulation (thewes et al., 2019; brizzolara et al., 2020; thewes et al., 2021 a; park et al., 2022). zanella and stürz (2015) showed that, differently from eight other varieties, ‘red delicious’ apples react signifi­ cantly and accumulate higher ethanol levels under hypoxia, and in a specific comparison between granny smith and red delicious (brizzolara et al., 2017), it was reported that the latter considerably accumulated ethanol under both ulo (0.9 kpa oxy­ gen) and dca (0.2­0.55 kpa oxygen) conditions, as also observed by lumpkin et al. (2014). among important commercial apple varieties, the responses of red delicious to controlled atmosphere (ca) and dca still need to be compared and clarified, which makes this cultivar a genotype of interest in terms of both applied aspects and physiological stud­ ies related to dca conditions and different oxygen regimes and concentration adjustment protocols. 2. materials and methods experimental design and sampling organic apple (malus domestica borkh., cv. red delicious) fruit were harvested in correspondence of an average tss value of 10.8°brix. fruit were selected for their uniformity and absence of physical defects/decay and then kept for 3 days of acclima­ tion at low temperature (0°c). control atmosphere storage was applied by divid­ ing the fruit into two groups in two different cold chambers. the first group (about 300 fruit) was ini­ tially stored under oxygen concentration of 0.3% (0.3ox) while the second group (60 fruit) was stored under the safer oxygen concentration of 0.8% (0.8ox) for a total period of 218 dia (days in atmosphere). samples were collected at harvest and t0 sampling was carried out after 24h in atmosphere (1 dia). to simulate the dynamic changes in oxygen concentra­ tions applied during a dca storage, at t1 (10 dia) 60 fruit originally stored under 0.3ox were shifted to 0.8% oxygen level and kept under these conditions for the whole period of storage (218 dia). these sam­ ples were called shift 1 and were sampled successive­ ly at the following time points. at t2 (20 dia), anoth­ er 60 fruit were moved from 0.3% to 0.8% oxygen and were called shift 2. shift 3 was performed at t3 (31 dia) and shift 4 took place at t4 (110 dia). a salamé et al. ‐ dca storage of red delicious apples 91 schematic diagram of the experimental design is reported in figure 1. at each sampling point, three biological replicates taken from three different fruit for each treatment were considered. samples of cor­ tex tissue were collected, immediately frozen in liq­ uid nitrogen, and stored at ­80°c. flesh firmness, total soluble sugars content and ethanol quantification flesh firmness was measured at harvest (t0) and at the end of the storage at t5 (218 dia). measurements were taken at the two opposite sides of the equatorial part using a fruit penetrometer (mod. ft 327, 3­27 lbs) with a large plunger tip (11 mm­diameter) after removing 1 mm of the peel. total soluble solid content (tss, °brix) was deter­ mined in the flesh juice of apples using a portable refractometer (sinergica soluzioni, pescara, italy); measurements were performed at harvest and the end of the storage at t5 (218 dia) on pulp juice sam­ ples taken from the opposite sides of the fruit. ethanol content was measured by using a tectronik (tectronik, padova, italy) senzytec analyser, following the instructions of the manufacturer and using 100µl of juice obtained by collectively pressing portions (approximately 1/3) of the cortex of three apples rep­ resenting the biological replicate. rna extraction and cdna synthesis total rna was isolated from cortex tissue using ‘sigma ­aldrich’ rna extraction kit following the manufacturer instructions. total rna was quantified (ng/μl) using uv spectrophotometry calibrated with rnase­free water. rna purity was assessed by evalu­ ating the absorbance ratio at 260/280 and 260/230 nm. ribosomal rna bands integrity was verified using gelred™­stained 1% agarose gel (aranda et al., 2012). rna was reverse­transcribed into first­strand cdna using 4 µl readyscript™ (sigma, rdrt­ 500rxn), starting from 400 ng of total rna in a final volume of 20 µl using depc treated water. the reac­ tion was incubated at 25°c for 5 minutes, then at 46°c for 20 min and then heated at 95°c for 1 min and finally at 4°c for 15 min. the synthetized cdna was diluted 1:5 by adding sterile water. gene expression analysis by real‐time pcr quantitative real­time pcr was performed using three biological replicates and two technical repli­ cates for each sample. based on the paper by cukrov et al. (2016), primer pairs of genes related to sucrose/starch metabolism (β‐amylase, mdbam; phosphofructokinase, mdpfk; sucrose synthase, mdsusy), the fermentative/pyruvic acid metabolism (alcohol dehydrogenase, mdadh; lactate dehydroge‐ nase, mdldh; pyruvate decarboxylase, mdpdc; ala‐ nine aminotransferase, mdalaat), ethylene biosyn­ thesis (acc synthase, mdacs; acc oxidase, mdaco), and 6 ethylene responsive factors (erfs) were used (table 1). actin was used as a housekeeping gene. reaction mixtures were prepared, under sterile conditions, for the target and reference genes, con­ taining each 5 μl of 2xsybr® green qpcr readymix™ (sigma), 1 μl of each primer (forward and reverse) (10 µm), 2 µl rnase­free water and 1 μl of cdna. the automated thermal cycler was programmed according to the following conditions: initial denatu­ ration of 95°c for 30 sec followed by 40 cycles of: denaturation at 95°c for 10 sec, primer annealing (according to primer tm) for 30 sec and extension at 72°c for 30 sec. finally, melt­curve stage at 65°c for 0.5 sec followed by 95°c for 0.5 sec. the ct­values generated were used to evaluate the results of the gene expression levels comparing the expression of each target gene to the housekeep­ ing gene (actin). for samples under static 0.8ox, data were expressed with the 2­δδct method (livak and schmittgen, 2001) and normalized to the correspond­ ing sample at t1 (1 dia). data of samples under 0.3ox and the shifts were expressed as fold change of the expression level using the formula: fc=log2(2‐δδct) normalized to the corresponding sample of 0.8ox at each time point starting from 10 dia. statistical analysis for gene expression, data analysis was performed on six replicates (3 biological x 2 technical replicates). rstudio was used with external package “pcr” to cal­ culate the relative expression and fold change. for fig. 1 ­ red delicious dca storage experimental design. the numbers indicate the days in atmosphere (dia), ▽ indicates the sampling with color coded for different samples, 0.3ox (blue), 0.8ox (purple), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue). adv. hort. sci., 2023 37(1): 89­99 92 fruit firmness, tss and ethanol content 3 biological replicates were used. internal statistical functions and external package “agricolae” were used to ana­ lyze the data (kronthaler and zoellner, 2021). all data were analyzed using t‐test for samples at 1 dia to compare 0.3 to 0.8ox samples. one­way anova and mean comparison with least significant differ­ ence (lsd) post­hoc test (p≤0.05) was used to com­ pare different samples of shifts to the corresponding 0.8ox at each time point starting from 10 dia. a kruskal­wallis test was applied to non­parametric data (p≤0.05). 3. results flesh firmness, tss and ethanol production at the end of the storage period and after five days of shelf life at room temperature no physiologi­ cal disorders or external/internal defects were observed in all analysed samples collected from the different protocols. concerning technological parameters, table 2 reports apple firmness and tss values for samples taken at harvest (t0) and at the end of the trial at 218 dia. results showed that samples stored for 30 table 1 ­ primers pairs used in the rt­qpcr analysis the table reports the genes nomenclature according to https://iris.angers.inra.fr/gddh13/, the primers sequence, length, gc content and melting temperature (tm) genes primer sequence 5'­3' length (bp) gc content (%) tm (°c) acc synthase md15g1302200 f aagtggcgaactggagtcga 20 55 67.2 r ggtttgatgggttcgtgacc 20 55 66.4 acc oxidase md10g1328100 f cagtcggatgggaccagaa 19 57.8 66.3 r gcttggaatttcaggccaga 20 50 66.3 pyruvate decarboxylase md04g1160100 f caaggcagtaaagccggtta 20 50 63.9 r aaatcggtccagcaaacaag 20 45 63.9 alcohol dehydrogenase md10g1014200 f ggaagcactgaagccatgat 20 50 64.2 r ctccacgacagagggaatgt 20 55 64.2 lactate dehydrogenase mdp0000143956 f cataaaactccttcaggctcca 22 45.4 64.1 r gtgsggtcttgggtgaggat 22 55 63.9 beta‐amylase 6 md09g1103800 f ctatgtgccgatcttcgtga 20 50 63.8 r actgcttgaaacacgctcct 20 50 63.9 sucrose synthase md15g1223500 f tccgtgttcactgctacgag 20 55 64.1 r gcctcaagaaggtccaacag 20 55 63.8 phosphofructokinase md04g1042400 f agtcgtggagtggtggaatc 20 55 64.1 r tagagggtgagggcttcaga 20 55 63.9 alanine aminotransferease md09g1173000 f tgctgtccgaggtgaaatcgtc 22 54.5 70.6 r agcccggattcgcctttaactc 22 54.5 69.9 ethylene response factor md11g1306500 variant f cggtggtgctataatctccg 20 55 64.2 r ggaattgagtcggtgtgagtagtt 24 45.8 64.3 ethylene response factor md11g1306500 f ctcccttcgccaagttcg 18 61.1 65.9 r ttgagtcggtgcgattaacc 20 50 64.9 ethylene response factor md16g1162900 f ccagaagcccaaaccatcag 20 55 66.7 r ttcctcggcggtgttgta 18 55.5 65.4 ethylene response factor md13g1163300 f ggtggggaaatgtatgctaaga 22 45.4 63.7 r gtcatccagcatccacagg 19 57.8 64.4 ethylene response factor md17g1152400 f cttctgcaaagcgttctgtg 20 50 63.7 r ggcaggatcggatggag 17 64.7 64.5 ethylene response factor md09g1174400 f ttctgcaaagcgttccatc 19 47.3 64 r ttcattggcagggaaggtg 19 52.6 66 actin md04g1127400 f tgaccgaatgagcaaggaaatta 25 40 67.4 r tactcagctttggcaatccacatc 24 45.8 68 salamé et al. ‐ dca storage of red delicious apples 93 (sh3) and 110 (sh4) days under 0.3% oxygen before being shifted to 0.8% oxygen showed the lowest val­ ues of firmness at the end of the trial. while 0.8ox, sh1 and sh2 samples maintained firmness values not significantly different from those detected in t0 sam­ ples. no significant difference (p=0.414, alpha level ≤ 0.05) was recorded for tss levels over time or between the different applied protocols. ethanol levels have been monitored along the entire experimental period to assess the activation of fermentative metabolism under static and dynamic ca storage (fig. 2). 0.3ox samples already showed higher levels than those of the 0.8ox sample at 10 dia, and a further increase from 10 to 20 dia with values around 400 mg/kg fw up to 110 dia (last sampling time for this specific treatment). on the other hand, 0.8ox samples showed a similar trend in terms of ethanol accumulation but with significantly lower amounts compared to 0.3ox samples and a decreasing trend after the highest concentrations detected at 20 and 31 dia. samples subjected to par­ tial re­oxygenation at different time points (sh1, sh2, sh3 and sh4) showed a different behaviour: sh1 did not show significant difference from 0.3ox at 20 dia , but displayed a reduced amount at 31 and at 110 dia as also observed for sh2. interestingly, at 110 dia ethanol levels were simi­ lar in all shifted and in 0.8ox samples. this was also observed at 218 dia, except for sh4 apples that dis­ played still significant higher levels (fig. 2). effect of different protocols on sugar metabolism‐ and fermentation‐related gene expression for gene expression data analysis, 0.8ox samples, kept under a static concentration throughout the experiment (from 1 to 218 dia), were considered as a reference for the other storage protocols (0.3ox, sh1, sh2, sh3 and sh4). consequently, the gene expres­ sion levels of these latter samples were expressed as fold change in relation to 0.8ox. considering sugar metabolism, the expression lev­ els of three genes were monitored throughout stor­ age (fig. 3). in 0.8ox samples, mdbam gene revealed a significant up regulation, compared to t0, at 31 and 110 dia. a significantly lower expression level was recorded in 0.3ox samples at 10 and 110 dia. sh1, sh2, sh3 and sh4 samples had significantly lower lev­ els of expression at 110 dia. at the last sampling time, 218 dia, all shifted samples, except sh3, revealed significantly higher expression values com­ pared to 0.8ox. mdpfk gene expression showed a steady state in samples stored at 0.8ox. a lower expression level of this gene was detected at 10 dia in 0.3ox samples. sh1 samples showed higher expres­ sion at 31 dia, sh2 had lower expression level at 31 dia and higher at 110 and 218 dia, while sh3 sam­ ples showed higher expression levels at 218 dia. considering mdsusy, in 0.8ox apples the expression showed a significant induction at 10 and 20 dia, fol­ lowed by a basal expression level at all the other sampling points. samples stored under 0.3ox revealed two marked and significant peaks of induc­ tion, at 10 and 110 dia. interestingly, sh1 showed significantly reduced levels of expression at 20 dia, while sh2 and sh4 revealed significantly higher expression at 218 dia. concerning the gene related to the fermenta­ tive/pyruvic acid metabolism (fig. 4), mdldh expres­ sion showed, in 0.8ox samples, a significant increase fig. 2 ­ ethanol content (mg/kg fw). samples stored in 0.3ox (blue), 0.8ox (purple), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) analysed from 10 to 218 days in atmosphere (dia). different letters indicate sig­ nificant differences among samples (anova, lsd post­ hoc test (p ≤ 0.05). different letters indicate significant differences among samples (anova, lsd post­hoc test (p≤0.05). table 2 ­ firmness and total soluble solids (tss). mean values (±se) of apple samples at harvest (t0) and after 218 dia of static (0.8ox) and dynamic atmosphere storage (shift 1­4) are reported in the table samples dia firmness (n) tss (°brix) at harvest 0 75.10 ± 0.96 a 10.85 ± 0.45 static 0.8ox 218 67.67 ± 2.85 ab 11.40 ± 0.06 shift 1 218 62.52 ± 3.51 ab 11.76 ± 0.32 shift 2 218 67.91 ± 0.73 ab 11.53 ± 0.24 shift 3 218 58.10 ± 6.76 b 10.86 ± 0.13 shift 4 218 61.30 ± 5.83 b 11.40 ± 0.06 94 adv. hort. sci., 2023 37(1): 89­99 of expression only at 10 dia. compared to 0.8ox sam­ ples, 0.3ox treatment induced higher expression at 10 and 110 dia, and a general up­regulation in shift­ ed samples at 218 dia. mdpdc, involved in the pro­ duction of acetaldehyde, increased its expression at 20 and 31 dia in 0.8ox samples. 0.3ox apples revealed an earlier increase in the expression of mdpdc gene, significant at 10 dia. among samples that underwent partial re­oxygenation, a general lower expression, always compared to 0.8ox, was observed at 110 dia, followed by increased levels at 218 dia. acetaldehyde can be further converted to fig. 4 ­ relative expression of genes related to fermentative/pyruvic acid metabolism, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase and alanine aminotransferase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant dif­ ferences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p ≤ 0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. fig. 3 ­ relative expression of genes related to sugar metabolism and energy production β­amylase, phosphofructokinase, and sucrose synthase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line repre­ sents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. salamé et al. ‐ dca storage of red delicious apples 95 ethanol under low oxygen levels by the enzyme coded by mdadh genes, and this is a crucial reaction in apple tissue under hypoxia. compared to t0 sam­ ples, the selected mdadh gene showed a significant increase only at 218 dia in 0.8ox sample. the applica­ tion of 0.3% oxygen resulted in general higher levels of expression until 31 dia, significant at 10 and 31 dia. at 110 dia, a significant decrease in mdadh expression was recorded for 0.3ox apples as well as for all shifted samples. mdalaat is involved in the reversible transfer of an amino group from glutamate to pyruvate, which in turn forms 2­oxoglutarate and alanine. in 0.8ox sam­ ples, this gene showed a peak of expression at 110 dia. the expression in 0.3ox samples is highly induced at 10 dia, while it strongly decreased at 110 dia, similarly to sh2 and sh3. higher expression of this gene was detected at 218 in sh4 sample (fig. 4). ethylene biosynthesis and erfs gene expression the expression of two genes involved in ethylene biosynthesis, namely mdacs and mdaco, has been analysed (fig. 5). these two genes appeared to be highly affected during storage under 0.8% oxygen concentration. the expression of both genes increased with time, constantly for mdaco, which also reached the highest recorded levels of expres­ sion, while, in the case of mdacs, a peak at 110 dia was detected. regarding mdacs gene expression, 0.3ox samples showed increased values (compared to 0.8ox) at 10 dia, but lower levels at 20, 31 and 110 dia, when also sh2 and 3 showed low expression lev­ els. mdaco gene revealed marked higher levels in 0.3 ox samples at 10 dia and in sh1 samples at 20, 31, and 110 dia. lower expression levels were detected in 0.3ox apples at 20, 31 and 110 dia. interestingly, sh1 samples showed higher levels of expression, compared to 0.8ox, at 20, 31, and 110 dia. all shifted samples had lower expression level than 0.8ox apples at 218 dia. the ethylene signalling and response pathway includes ethylene response factors (erfs), which belong to the transcription factor family apetala2/erf that plays important roles in stress­ related responses. the effect of the different applied storage protocols on the expression level of six erf genes has been investigated throughout the experi­ ment (fig. 6). in general, samples showed relatively similar responses in terms of erf expression. overall, considering 0.8ox samples a general increase of erf genes expression was observed up to 110 dia, which was significant at different time points for the differ­ ent analysed erfs (md09g1174400 gene at 110 dia; md13g1163300 gene at 10, 20 and 31 dia; md11g1306500 gene at 10, 20 and 31 dia; md16g1162900 gene at 10, 31 and 110 dia; md11g1306500 variant gene at 10, 20 and 31 dia; fig. 5 ­ relative expression of ethylene biosynthesis genes acc­synthase and acc­oxidase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤ 0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. adv. hort. sci., 2023 37(1): 89­99 96 md17g1152400 gene at 10 and 110 dia). the expression of erf genes in 0.3ox samples was instead characterized by significant lower levels at the different time points with only two exceptions: md09g1174400 gene at 10 dia and md11g1306500 gene at 20 dia, when significantly higher levels of expression than those of 0.8ox apples were recorded. on the other hand, considering apples subjected to partial re­oxygenation samplings, lower levels of expression were generally detected up to 31 dia, with only two exceptions (md11g1306500 and md16g1162900) concerning sh2 samples at 31 dia, when a significantly higher level of expression was observed. after 110 dia, the different samples revealed vari­ able patterns. concerning md09g1174400 and md13g1163300 genes, sh1 and sh3 showed signifi­ cantly lower levels of expression, while sh2 had sig­ nificantly higher levels. for these two genes at 218 dia only sh2 revealed significantly higher levels of expression compared to 0.8ox samples, despite a general increasing trend in all apples subjected to partial re­oxygenation. as far as the md11g1306500 gene is concerned, significant higher expression was detected for both sh1 and sh2 at 110 dia, while only sh3 had signifi­ cantly higher levels at 218 dia. md16g1162900 gene expression in shifted samples revealed significant lower levels at 110 dia, while only sh4 had signifi­ cantly higher levels at 218 dia. in the case of md11g1306500 variant gene sh1 and sh2 had higher expression levels at 110 dia, while only sh2 and sh4 showed significantly higher levels at 218 dia. lastly, md17g1152400 gene had a significantly high­ er level of expression in samples of sh1 and sh2 at 110 dia, while sh3 apples had significantly lower fig. 6 ­ relative expression of genes belonging to ethylene response factor (erf) gene family. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8%ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. salamé et al. ‐ dca storage of red delicious apples 97 expression for this gene. on the other hand, all shift­ ed samples showed significantly higher expression of this gene at 218 dia compared to 0.8ox apples. a general trend was identified for all samples subjected to partial re­oxygenation: in general the expression level of the erf gene was induced at 218 dia. 4. discussion and conclusions an in­depth understanding of low oxygen responses in fruits is of fundamental importance for the optimisation of storage approaches and for the development of protocols aimed at maintaining opti­ mal quality while preventing the occurrence of physi­ ological disorders associated with long term storage. metabolic adaptation responses to hypoxic condi­ tions have only recently started to be clarified in apple fruits and are gaining increasing interest since apples are routinely stored for very long periods of time thanks to the adoption of low oxygen (0.8 kpa oxygen, ultra low oxygen, ulo) or dynamic con­ trolled atmosphere (dca, 0.4 or lower kpa oxygen) protocols. primary metabolism and ethylene physiol­ ogy are markedly affected by hypoxia with differ­ ences depending on the oxygen concentration and modulation, and the genotype. in this study we char­ acterized the ethanol accumulation and the expres­ sion pattern of sugar/fermentative metabolism­ and ethylene physiology­related genes of red delicious apples in ca/dca storage. our goal was that of better understanding the behaviour and the responses (also in terms of specific quality parameters) of this apple variety in relation to two levels of low oxygen con­ centration and the variable (in terms of timing) switch from 0.3 to 0.8% oxygen levels. the storage under 0.3% oxygen resulted in an early significant accumulation of ethanol already at 10 dia and that further increased at 20 dia. this level remained rather stable as long as the fruit were kept at 0.3% oxygen atmosphere until 110 dia. although to a lesser extent, ethanol content also increased in 0.8ox samples, confirming what observed by brizzolara et al. (2017). in these apples, ethanol con­ tent levelled off after three months of storage. the metabolization of ethanol seemed to be more sensi­ tive to re­oxygenation when apples had experienced a shorter period of dca. in fact, the longest storage under 0.3% oxygen resulted in more stable ethanol levels in the cortex at the end of the storage period (218 dia, sh4 in fig. 2). considering one of the main parameters dictating the commercial life of apples, the samples sh3 and sh4 showed the lowest values of flesh firmness at the end of the trial. this behaviour could be associated to the highest levels of ethanol accumulated in these samples. in braeburn apples ethanol production exceeding 472 μl·l­1 and the overproduction of anaerobic metabolites in royal gala resulted in a decrease of flesh firmness (weber et al., 2020; thewes et al., 2021 b). in persimmon, it has been observed that accelerated loss of flesh firm­ ness during storage was induced by ethanol treat­ ments applied to reduce astringency (vilhena et al., 2022). these authors observed that this event is closely related to greater parenchyma degradation during storage caused by ethanol treatment. if this cellular event also occurs in apple fruit accumulating high ethanol levels following hypoxic storage condi­ tions remains to be elucidated. it is interesting to note that even in the samples with the highest ethanol content (0.3ox) no internal physiological disorders (e.g., flesh breakdown) were detected. the gene expression data confirmed that the molecular regulation of hypoxic responses is overall conserved among apple varieties: the up­regulation of mdpdc, mdadh and mdalaat in response to extreme levels of hypoxia (0.3 oxygen concentration) is readily activated and peaks at 10 dia, after which is promptly and progressively levelled down until 110 dia, when a general low level of expression (com­ pared to 0.8ox samples) is present in 0.3ox and shift­ ed samples. this possibly suggests a negative feed­ back exerted by ethanol on its own synthesis. in agreement with these findings, one of the genes encoding group vii ethylene response factors (md09g1174400), with similarity to rap2 proteins involved in low oxygen signalling in model systems (licausi et al., 2011; gibbs et al., 2011), displayed an expression pattern overlapping with that of the fer­ mentative metabolic genes with a transient up­regu­ lation at 10 dia and low expression levels at 110 dia. it is well known that under energy shortage condi­ tions, such as those induced by low oxygen condi­ tions, plant tissues and organs (including fruit) instead of using invertases and hexokinases to pro­ duce hexose­phosphates to form sucrose, an atp consuming process, can use sucrose synthase as alternative energy saving pathway (mustroph et al., 2014). the activation under hypoxic conditions of sucrose synthase was already reported by cukrov et al. (2016) in granny smith apples, and our expression adv. hort. sci., 2023 37(1): 89­99 98 data (showing a high induction at 10 dia in 0.3ox and a prompt reduction of expression level in sh1 apples at 31 dia) confirm that this gene can be considered highly sensitive to oxygen levels also in cv. red delicious. a cultivar­specific behaviour is, instead, observed regarding beta­amylase and phosphofruc­ tokinase. in fact, these genes in granny smith apples follow a similar expression pattern compared to mdsusy (cukrov et al., 2016), not observed in the present trials on red delicious. as far as ethylene biosynthesis is concerned, the expression pattern of acc oxidase detected in 0.8ox red delicious samples mirrors that observed in granny smith (cukrov et al., 2016), while the tran­ sient higher expression levels observed for both acc synthase and oxidase at 10 dia under 0.3% oxygen appear to be a specific response of cv. red delicious apple. interestingly, the transcription of both genes appeared to be re­activated exclusively in the first and second shift to 0.8ox (sh1 and sh2), performed after 10 and 20 dia, respectively, and showed a peak at 31 dia followed by lower expression levels. however, the increase of mdacs and mdaco tran­ script following the oxygen resupply reached a level significantly lower than that reached by apples that had been constantly kept at 0.8ox. it could be hypothesised that this may be due to the higher lev­ els of ethanol accumulated in the pulp of dca stored apples, which might exert a suppressive action on ethylene biosynthesis as previously shown by some authors in different apple varieties (pesis et al., 2005; thewes et al., 2019; weber et al., 2020; thewes et al., 2021 a). concluding, the recovery from anoxia in apple fruits is dependent on the length of exposure to the anoxic stress (wood et al., 2022). our data on ethanol accumulation and ethylene­related gene expression are in line with these findings, showing that longer periods of exposure to 0.3% oxygen result in the maintenance of higher levels of ethanol and on the prevention of transcription of ethylene biosyn­ thetic genes. the effect of low oxygen storage of red delicious apples on transcript abundance of several important genes related to hypoxic stress response in apple fruit revealed both similarity with granny smith apples stored under the same ca protocols, and spe­ cific responses of cv. red delicious. in both red delicious and granny smith apples two phases can be recognized in relation to fermentative metabolism: a first phase characterised by the activation of fermen­ tative pathways, and a second phase (from two months onward) in which a generalized de­activation of fermentative metabolism is observed. acknowledgements this study was carried out by pt and sb within the agritech national research center and received funding from the european union next­ generationeu (piano nazionale di ripresa e resilienza (pnrr) – missione 4 componente 2, investimento 1.4 – d.d. 1032 17/06/2022, cn00000022). this manuscript reflects only the authors views and opinions, neither the european union nor the european commission can be consid­ ered responsible for them. sb was supported by the special project of scuola superiore sant’anna isvstrategico2019. this work has been supported by the networking activities “oxygen sensing a novel mean for biology and technology of fruit quality” (ca:18210) which is implemented under the cost action “roxy­cost,” funded by the european cooperation in science & technology (2019­2023). funding was provided by marvil engeneering srl through project “rupe comm17_01” and by university of padova project bird173975/17. references aranda p.s., lajoie d.m., jorcyk c. l., 2012 ­ bleach gel: a simple agarose gel for analyzing rna quality. ‐ elect., 33(2): 366­369. brizzolara s., cukrov d., mercadini m., martinelli f., ruperti b., tonutti p., 2019 ­ short‐term respons‐ es of apple fruit to partial reoxygenation 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ca: fruit physiology controls atmosphere composition during storage. ­ acta horticulturae, 1071: 59­68. https://www.sciencedirect.com/science/article/pii/s0925521422002058 https://www.sciencedirect.com/science/article/pii/s0925521422002058 impaginato 169 adv. hort. sci., 2024 38(2): 169­176 doi: 10.36253/ahsc­15659 the effect of thymol and carvacrol rich­ plant essential oils on controlling postharvest decay molds in orange fruit l. mokhtarnejad 1 (*) , m. farzaneh 2 1 plant protection research department, west azarbaijan agricultural and natural resources research center, areeo, urmia, iran. 2 department of agriculture, medicinal plants and drugs research institute, shahid beheshti university, evin, tehran, iran. key words: antifungal effect, satureja spp., spoilage, thymus spp. abstract: the antifungal activity of essential oils of thymus daenensis, thymus vulgaris, satureja hortensis and satureja khuzistanica as well as their major compounds were studied against mold decays of orange fruit. according to gc­ ms analysis, the major compounds of t. danensis essential oil were thymol (65.5%) and alpha­terpinene (11.9%) whereas t. vulgaris was rich in thymol (59%) and p­cymene (15.6%). carvacrol (88.4%) in s. khuzistanica oil and car­ vacrol (51%), gamma­terpinene (20.8%) and p­cymene (13.7%) in s. hortensis oil were charecterized as major compounds. the oil of s. khuzistanica and its major compound carvacrol exhibited the strongest fungicide activity against penicillium digitatum, rhizopus stolonifer and colletotrichum gloeosporioides at 300 µl/l. the results on orange fruits exhibited that the use of s. khuzistani‐ ca and s. hortensis eos as spraying and dipping treatments could considerably reduce spoilages decays in the fruit. 1. introduction post­harvest diseases of fruits are mainly caused by fungal species such as botrytis spp., colletotrichum spp., aspergillus spp., alternaria spp., rhizopus spp. and penicillium spp. (agrios, 2005). the fruit decay caused by post­harvest diseases is usually more than what is thought, because with the decrease in yield the price of damaged fruits (wills and golding, 2016). citrus fruits, especially oranges, are among the fruits that are highly sensitive to fungal infections. the use of fungicides, such as benomyl, thiabendazole and imazalil, is the most common method of controlling post­harvest decays of citrus fruits. these fungicides have health and environmental problems such as cumulative and carcinogenic properties in living organisms and acute or chronic poisoning effects. in addition, resistance to these fungicides is increasing in the population of pathogens (sharifi­tehrani and farzaneh, 2018). anyway, the increase in global demand for providing sufficient and healthy food, based on health standards, along with the policies of the world food and agriculture (*) corresponding author: l.mokhtarnejad@gmail.com citation: mokhtarnejad l., farzaneh m., 2024 ­ the effect of thymol and carvacrol rich‐plant essential oils on controlling postharvest decay molds in orange fruit. ­ adv. hort. sci., 38(2): 169­176. copyright: © 2024 mokhtarnejad l., farzaneh m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2022 accepted for publication 2 february 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15659 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 169­176 170 organization (fao) and the environmental protection organization (epo) has caused extensive research to be carried out. according to the food and drug administration (fda), the essential oils (eos) of some medicinal plants are known as natural and healthy alternatives to chemical fungicides and are more acceptable to the public (brun et al., 2003; carvalho de sousa et al., 2004; nazzaro et al., 2017). eos are volatile and natural complex compounds that are characterized by their sharp and strong smell and are formed as secondary metabolites in aromatic plants. some eos that have antiseptic properties (antibacterial, antiviral and antifungal properties) are used in food and pharmaceutical industries (burt, 2004; bolouri et al., 2022). in nature, eos play an important role in protecting plants against bacteria, viruses, fungi and insects (regnault­roger et al., 2012; zitzelsberger and buchbauer, 2015). they may also attract a number of insects to disperse pollen and seeds (bakkali et al., 2008). medicinal plant eos not only have no side effects (at the right concentra­ tion), but due to their antioxidant properties, may increase the quality and storage time of fruits (arras and usai, 2001; anthony et al., 2003; plotto et al., 2003; plaza et al., 2004). research has shown that aromatic plants belonging to the lamiaceae and asteraceae families are rich in antimicrobial and antioxidant compounds (barroso and ruberto, 1998; farzaneh et al., 2006 a, b; farzaneh et al., 2015). the antifungal property of eos is also related to some of their compounds such as carvacrol, menthol, cymene, thymol, cinnamaldehyde, eugenol, pinene, and linalool, which are known as compounds with high antifungal effect (cimanga et al., 2002). the purpose of this research is to investigate the potential of eos of plants rich in thymol and car­ vacrol, such as thymus danensis, thymus vulgaris, satureja hortensis, and satureja khuzistanica in pre­ venting rot and decay of orange fruit caused by colletotrichum gloeosporioides, aspergillus niger, rhizopus stolonifer and penicillium digitatum. 2. materials and methods inoculum preparation of pathogens four fungi that cause post­harvest decay of orange fruit, including c. gloeosporioides, a. niger, r. stolonifer, and p. digitatum, were obtained from the mycology collection of the department of plant pro­ tection, agriculture and natural resources campus, university of tehran. in order to prepare the pathogen inoculum, 5 ml of distilled sterile water containing 0.05% tween 80 was added to the seven­ day old culture of each fungus on pda medium and the surface of the colony was scraped to provide spores and mycelia suspension. the resulting suspen­ sions were passed through four­layer cheesecloth, and then the spore population was adjusted to a con­ centration of 1*105 spores per milliliter using a hemacytometer. plant eos and their major compounds the aerial parts of tow thyme species, t. danensis and, thymus vulgaris, at the flowering stage were collected from semirom region of isfahan province, while the aerial parts of two savory species, s. hort‐ ensis and s. khuzistanica were collected from pol­ dokhtar and majin regions of lorestan province, respectively. the collected plant parts were delivered to the medicinal plants and drugs research institute (mpdri), shahid beheshti university (sbu) in tehran. after confirming the identity, the plants were dried at room temperature and shade. each sample was pow­ dered using a mill, and then their eos was extracted by distillation with water in a clevenger according to the method recommended in the british pharmacopoeia (1988). the standard compounds of thymol, carvacrol, para­cymene and gamma­ter­ pinene were purchased from sigma­aldrich co. analysis and identification of eos compounds the eo obtained from each plant was identified with gas chromatography (gc) and gas chromatogra­ phy coupled with mass spectrometry (gc­ms) meth­ ods. first, one microliter of eo extracted from each plant was injected into the tracetm gc 2000 gas chromatograph (thermoquest italia s.p.a., rodano, milan, italy) with a flame ionization detector (fid) and fused silica capillary db­1 column (60 m × 0.25 mmi.d.; film thickness= 0.25 μm). injector and detec­ tor temperatures were 250˚c and 300˚c, respective­ ly. helium was used as the carrier gas at a flow rate of 1.1 ml/min; oven temperature was programmed from 60˚c to 250˚c at the rate of 4˚c/min, and finally held isothermally for 10 min. gc­ms analysis was also performed by using a thermoquest finnigan trace gc/ms (thermoquest italia s.p.a., rodano, milan, italy), equipped with a db­1 column (60 m × 0.25 mmi.d.; film thickness= 0.25 μm). gas chromato­ graphic conditions and the thermal programming were as given for gc. helium was used as carrier gas mokhtarnejad and farzaneh ‐ carvacrol/thymol rich oil control mold decays in storage orange 171 with ionization voltage of 70 ev. ion source and inter­ face temperatures were 200˚c and 250˚c, respective­ ly. mass range was from m/z 43­456. identification of individual compounds was done by comparison of their mass spectra with those of similar compounds from a database (wiley/nbs library) or with authen­ tic compounds and confirmed by comparison of their retention indices with authentic compounds or with those of reported in the literature. the percentage of each compound was determined according to its rel­ ative area percentages obtained by fid, without using correction factors (adams, 2007). in vitro antifungal assay the main ingredients of eos, including thymol, carvacrol, paracymene and gamma terpinene, were obtained from the phytochemistry department of mpdri. antifungal effect of eos and main com­ pounds was investigated against four post­harvest decay fungi of fruit by mixing eo with pda solid cul­ ture medium (farzaneh et al., 2006 b). in short, petri dishes containing concentrations of 75, 150, 300, 600 and 1200 microliters of eo/standard major ­com­ pound per liter of culture medium were prepared and after placing a fungal disk (with a diameter of 5 mm) in center of petri dishes, they were kept at a temperature of 25 °c in darkness. the growth of each fungus colony was measured daily until the surface of control petri dishes was completely occupied by the fungus. the percentage of growth inhibition was cal­ culated. the minimum inhibitory concentration (mic) of the eos was calculated to prevent the growth of fungi. to investigate whether the eo shows fungicidal or fungistatic activity, the fungal disk of the treat­ ments without fungal growth, was re­cultured on the pda culture medium, and the growth or not growth of the fungus on the pda was investigated after one week to calculate the minimum fungicidal concentra­ tion (mfc). in addition, the ec50 value (effective con­ centration causing 50% inhibition of mycelial growth) was calculated from the data by probit analysis. antifungal assay on orange fruit the healthy orange fruits (thomson cultivar) free of any chemical and physiological treatment and same in size and ripeness index were provided from citrus and subtropical fruits research center, ramsar, mazandaran province, iran. after disinfect­ ing the fruits surface by 70% ethanol for one minute, a wound of 1 mm in diameter and 2 mm in depth (limited to the albedo part in the equatorial region of the fruit) was created on each fruit in sterile condi­ tion. then the fruits were treated with the concentra­ tion of 1/1000 (1000 ppm) eo by two methods; dip­ ping and spraying. then treated fruits were inoculat­ ed by spraying of a suspension of 1×105 spores per milliliter. control treatments included dipping and spraying of fruits with tween 80 solution (0.05%) and thiabendazole fungicide. in this experiment, each treatment contained of 4 replicates and each repli­ cate consisted of 8 experimental units (fruits). the surface of treated fruits was dried under air flow for 2 h and then they arranged on special fiber plates before transferring to a storage room of 25°c and darkness. after the storage period (10 days), the diameter of the decay area on orange fruit was mea­ sured using a caliper. the efficacy of the eos was determined by the formula: ip =(c–t/c)*100), where ip is the inhibitory percentage of the of spoilage decay, and c and t are the spoilage decay area in control and treatment, respectively. statistical analysis to analyze the data, three software were used. at first, the normality of the data was normalized by mini­tab software version 17.1 (minitab inc. state college, pa). then sas software version 9.1.3 (sas institute inc., cary, nc) with glm method was used for variance analysis. after analysis the variance, the mean of the data was compared using duncan’s multi­range test at the 5% level. the ec50 values were calculated from the data subjected to probit analysis using ibm spss statistics version 26 (ibm corp. chicago, il). 3. results the main compounds of eos the main compounds in t. danensis eo included thymol (65.5%), alpha­terpinene (11.9%) and para­ cymene (7.5%) (table 1). thymol (59%), paracymene (15.6%) and gamma­terpinene (4.2%) were the main compounds identified in the eo of t. vulgaris (table 1). the main compounds in the eo of the s. khuzis‐ tanica included carvacrol (88.4%), para­cymene (3%) and gamma­terpinene (4.5%). carvacrol (51%), gamma­terpinene (20.8%) and para­cymene (13.7%) were the main compounds identified in the eo of the s. hortensis species (table 2). adv. hort. sci., 2024 38(2): 169­176 172 antifungal effect of eos the results of the antifungal effect of the eos on the growth of fungi are shown in table 3. in general, the more the concentration of eo increased, the more antifungal activity was seen. in addition, the intensity of the eos inhibitory effects against c. gloeosporioides and r. stolonifer was more evident (table 3). according to the results (table 3), for controlling the a. niger growth, only the eo of s. khuzistanica showed the highest antifungal activity with the mic 300 μl/l. to inhibit the growth of p. digitatum, all eos showed significant antifungal activity with the table 1 ­ the major constituents (%) of chemical composition of thymus daenensis and t. vulgaris essential oils * retention indices relative to c6­c24 n­alkanes on the db­1 column. no. compound retention indices t. daenenis (%) t. vulgaris (%) 1 alpha­thujene 925 0.8 1.8 2 alpha­pinene 933 1.6 1.6 3 beta­pinene 974 0.7 2.6 4 myrcene 981 1.1 1.9 5 alpha­phelandrene 999 0.2 1.7 6 para­cymene 1014 7.5 15.6 7 gamma­terpinene 1053 ­ 4.2 8 alpha­terpinene 1080 11.9 ­ 9 thymol 1266 65.5 59.0 10 carvacrol 1282 0.1 3.1 11 carvacryl acetate 1345 2.5 2.0 12 beta­caryophyllene 1424 3.8 1.5 13 beta­bisabolene 1501 1.3 0.9 total ­ 97.0 95.9 table 2 ­ the major constituents (%) of the chemical composi­ tion of satureja khuzistanica and satureja hortensis essential oils * retention indices relative to c6­c24 n­alkanes on the db­1 column. no. compound retention indices s. khuzistanica s. hortensis (%) 1 alpha­thujene 925 ­ 2.5 2 alpha­pinene 933 ­ 2.9 3 beta­pinene 974 0.2 1.1 4 myrcene 981 0.2 1.5 5 para­cymene 1014 3.0 13.7 6 1.8­cineole 1023 0.7 1.0 7 gamma­terpinene 1053 4.5 20.8 8 carvacrol 1282 88.4 51.0 9 carvacryl acetate 1345 0.1 1.3 total ­ 97.1 96.7 table 3 ­ the inhibitory activity (%) of four plant essential oils at different concentrations against spoilage fungi of citrus fruit by poi­ sonous pda medium method essential oil concentration (µl/l) inhibitory activity (%) a. niger p. digitatum c. gloeosporioides r. stolonifer t. daenensis 75 22.16 34.20 11.13 24.96 150 57.35 56.33 34.20 96.05 300 90.11 85.71 100 100 600 100 100 100 100 1200 100 100 100 100 t. vulgaris 75 20.94 27.11 3.38 0.60 150 65.45 55.55 31.77 90.22 300 75.01 73.34 82.29 100 600 100 100 100 100 1200 100 100 100 100 s. hortensis 75 16.22 28.92 2.49 14.25 150 27.94 39.11 58.33 51.55 300 88.32 78.92 100 100 600 100 100 100 100 1200 100 100 100 100 s. khuzistanica 75 24.10 11.50 26.32 25.81 150 67.91 60.00 80.77 96.26 300 100 90.66 100 100 600 100 100 100 100 1200 100 100 100 100 mokhtarnejad and farzaneh ‐ carvacrol/thymol rich oil control mold decays in storage orange 173 mic 600 μl/l., whereas the eos of three species, including s. hortensis, s. khuzistanica and t. danensis showed the great antifungal activity against c. gloeosporioides with the mic 300 μl/l. to inhibit r. stolonifer, all four eos with the mic 300 μl/l showed the noticeable antifungal activity. the results (table 4) obtained from the re­culture of fungal disks, in the treatments which no fungal growth was observed, showed that none of the eos had fungicide properties on the a. niger. two eos of s. khuzistanica and s. hortensis at a concentration of 1200 μl/l showed fungicidal properties against p. digitatum, whereas t. daenensis and t. vulgaris eos showed the mfc values more than 1200 μl/l. essential oil of s. khuzistanica showed mfc against c. gloeosporioides at mfc 600 μl/l, while s. hortensis and t. danensis eos exhibited mfc of 1200 μl/l of culture medium. howevere, t. daenensis oil didn’t show mfc value at the maximum concentra­ tion. to control of r. stolonifer, the eos of s. khuzis‐ tanica and s. hortensis showed mfc at the concen­ trations 300 μl/l and 600 μl/l, respectively, while both t. vulgaris and t. danensis eos exhibited mfc at the concentration of 1200 μl/l (table 4). antifungal properties of the main components of eos in general, by increasing the concentration of the eo/standard main­component its antifungal activity increased (table 5). among the main compounds, carvacrol exhibited the highest antifungal activity. the mic of carvacrol against the growth of r. stolonifer was 150 μl/l. carvacrol at a concentration of 300 μl/l prevented the growth of other fungi as well. thymol was anoth­ er main compound in the eos, especially thyme, which showed considerable antifungal activity. thymol at the mic of 300 μl/l completely prevents table 4 ­ minimum fungicidal concentration (µl/l) of four essen­ tial oil against citrus fruit spoilage fungi. the experi­ ments were carried out in vitro by poisonous pda medium method fungi s. khusiztanica s. hortensis t. danensis t. vulgaris a. niger >1200 >1200 >1200 >1200 p. digitatum 1200 1200 >1200 >1200 c. gloeosporioides 600 1200 1200 >1200 r. stolonifer 300 600 1200 1200 table 5 ­ the inhibitory activity (%) of four major compounds of essential oils at different concentrations against spoilage fungi of citrus fruit by poisonous pda medium method. the percentage of inhibition in each treatment corresponds to 4 repetitions (4 petri dishes with a diameter of 8 cm) compound concentration (µl/l) inhibitory activity (%) a. niger p. digitatum c. gloeosporioides r. stolonifer thymol 75 10.20 6.70 14.65 14.50 150 55.48 55.75 66.30 58.86 300 95.34 100 100 100 600 100 100 100 100 1200 100 100 100 100 carvacrol 75 22.71 29.63 31.88 36.40 150 64.95 65.50 88.84 100 300 100 100 100 100 600 100 100 100 100 1200 100 100 100 100 para­cymene 75 0 0 0 0 150 30.87 28.44 36.50 33.96 300 61.54 55.89 69.74 62.94 600 93.38 94.61 100 100 1200 100 100 100 100 gamma­terpinene 75 0 0 0 4.69 150 19.85 15.32 26.58 27.63 300 63.35 88.84 89.12 85.38 600 96.48 100 100 100 1200 100 100 100 100 174 adv. hort. sci., 2024 38(2): 169­176 the growth of three fungi; c. gloeosporioides, r. stolonifera, and p. digitatum whereas the growth rate of a. niger was inhibited by 95.3%. para­cymene had also showed antifungal activity that was able to inhibit the growth of all the fungi at the concentra­ tion of 1200 μl/l. among the fungi, r. stolonifer and c. gloeosporioides were more sensitive to para­ cymene and their growth was completely inhibited at the concentration of 600 μl/l. it is also necessary to mention that this compound did not show any signifi­ cant antifungal effect against any of the fungi at the low concentrations (<150 μl/l). gamma­terpinen is one of the main components of eos, especially in the savory plants that at the mic concentration of 600 μl/l caused a complete inhibition of the growth of all fungi except a. niger. in the other hand, the fungus a. niger was the most resistant fungus to this com­ pound, whose mic was 1200 μl/l. low concentra­ tions of this compound did not show the inhibitory effect on the growth of the fungi (table 5). the results of the mfc indicated that gamma ter­ pinene at any of the concentrations did not cause the death of the fungi. in addition, carvacrol and thymol showed strongest fungicidal activity with mfc 600 μl/l against r. stolonifer. carvacrol also exhibited strong fungicidal activity (mfc 600 μl/l) against c. gloeosporioides. however, a. niger had the highest resistance to the compounds, and its mfc value was often more than 1200 μl/l (table 6). in addition, the antifungal potency of each eo and its main compound was determined according to ec50 value as well (table 7). the lower the ec50 indicates the less the concentration of antifungal compound that is required to inhibit 50% of fungal growth. in general, the lowest ec50 values were achieved by s. khuzistanica eo that showed ec50 values of 95.14, 108.00, and 120.93 μl/l against r. stolonifer, c. gloeosporioides, and a. niger, respectively. in confir­ mation of it, carvacrol showed the lowest ec50 values of 80­124 μl/l against four citrus fruit spoilage fungi. spoilage decay control on fruit in general, the application of eos by dipping method showed the greatest effect in reducing spoilage and fruit rot, whereas the spraying method also had significant effect. in addition, s. khuzistanica essential oil was the most effective oil to reduce a. niger (95.4%) and p. digitatum (86.8%) decays area on the fruit in dipping method. the eo of s. khuzis‐ tanica had the greatest effect against r. stolonifer and c. gloeosporioides decays on the fruits by both methods of dipping and spraying of the fruit which could completely (100%) inhibit the both decays table 6 ­ minimum fungicidal concentration (µl/l) of major compounds of essential oils; thymol, carvacrol, para­ cymene and gamma­terpinene; against spoilage fungi of citrus fruit. the experiments were carried out in vitro by poisonous pda medium method fungi thymol carvacrol para­ cymene gamma­ terpinene a. niger >1200 >1200 >1200 >1200 p. digitatum 1200 1200 >1200 >1200 c. gloeosporioides 1200 600 >1200 1200 r. stolonifer 600 600 1200 >1200 table 7 ­ the ec50 value (effective concentration causing 50% inhibition of mycelial growth) of each essential oil and its major com­ pounds against spoilage fungi of citrus fruit on pda (µl/l) calculated by probit analysis essential oil/compound a. niger p. digitatum c. gloeosporioides r. stolonifer t. danensis 154.40 (118.69­197.18) (z) 151.86 (97.52­219.75) 164.02 (151.83­178.53) 95.84 (88.81­103.094) t. vulgaris 172.02 (80.34­324.16) 179.65 (108.88­283.32) 210.16 (194.97­226.88) 119.23 (111.86­125.91) s. hortensis 191.92 (176.59­209.02) 186.28 (136.57­256.56) 141.95 (134.20­150.60) 143.68 (132.68­156.79) s. khusiztanica 120.93 (111.04­131.95) 160.21 (118.11­213.54) 108.00 (99.28­117.03) 95.14 (88.11­102.40) thymol 156.31 (131.03­187.02) 143.14 (133.79­154.54) 128.59 (119.27­139.13) 135.97(125.77­147.96) carvacrol 124.70 (114.56­136.16) 119.49 (109.03­131.12) 97.53 (89.30­105.78) 80.92 (78.78­83.06) para­cymene 289.02 (191.82­459.22) 230.03 (171.91­329.54) 296.60 (213.73­432.76) 245.27 (183.04­365.97) gamma­terpinene 284.61 (213.66­403.15) 220.44 (206.91­234.68) 207.22 (193.60­221.86) 207.85 (193.04­224.10) (z) numbers in parentheses indicate 95% confidence limits determined by probit analysis. mokhtarnejad and farzaneh ‐ carvacrol/thymol rich oil control mold decays in storage orange 175 (table 8). in addition, s. hortensis could completely inhibit r. stolonifera decay. however, t. danensis and t. vulgaris couldn’t completely inhibit of the any fruit fungal decay and exhibited weak fungicide activity on the fruit. in addition, fungicide tiabendazole could completely control r. stolonifera decay. it seems that p. digitatum and a. niger are the most resistance fungi to these eos on the orange fruit. 4. discussion and conclusions in our study, all four plants eos (belong to thymus and satureja geniuses, lamiaceae) exhibited considerable antifungal activity against postharvest spoilage fungi. it has been found that some medicinal plants of the lamiaceae family have high antifungal properties (bakkali et al., 2008; adeyinka and richard, 2015). thymol was included the main part (more than 50%) of t. danensis and t. vulgaris eos whereas s. khuzistanica and s. hortensis eos were rich in carvacrol (more than 50%). in addition, their major compounds and specially thymol and carvacrol resulted in strong fungictatic and fungicide activities. however, the lowest mfc and ec50 values were obtained by s. khuzistanica oil and carvacrol. the antibacterial and antimicrobial properties of the main components of eos such as cinnamaldehyde, eugenol, thymol and carvacrol have been identified in several studies (bakkali et al., 2008; adeyinka and richard, 2015). the antimicrobial and antifungal activity of the eo may be due to the characteristics of terpenes/terpenoids compounds, which, due to their high lipophilic nature and low molecular weight, that enable them destroying cell membranes, and inhibiting spore germination (bakkali et al., 2008; nazzaro et al., 2017). however, the dominant composition of the eo may cause the antifungal activity of the eo alone or in synergic manner with other compounds (plotto et al., 2003). therefore, in our study, the antifungal property of these eos can be contributed to their thymol or carvacrol content, although other eo constitutes may act synergistically and increase the antifungal activity of the main compound. research has shown that aromatic plants belonging to the families lamiaceae and asteraceae are rich in antimicrobial and antioxidants compounds and increase the quality of the fruit and the length of its storage period as well (tajkarimi et al., 2010; hyldgaard et al., 2012; gyawali and ibrahim, 2014). in addition, eos could control post­ harvest diseases due to their antifungal effects on the both vapor and non­vapor phases (tripathi et al., 2008). in our study, the application of eos by dipping method showed the more fungicide activity than the spraying method in terms of reducing spoilage and fruit rot. none of the four eos and their dominant compounds at the maximum concentration studied in this research (1200 µl/l) could completely controlled a. niger in vitro and on fruit conditions, which indi­ cates the high tolerance of this fungus to eo com­ pounds. in addition, although both savory oils could completely kill p. digitatum by 1200 µl/l in vitro, they couldn’t completely inhibit the p. digitatum decay on fruit. on the other hand, the sensitivity of p. digita‐ tum to eo would be reduced on fruit. however, savory oils could completely inhibit r. stolonifer and c. gloeosporioides decays on fruit. although, the significant in vitro antifungal activity of the eos studied in this research depended on the content of carvacrol and thymol, the eos of both table 8 ­ the control of orange fruit fungal decays by four medicinal plants essential oils (1 per 1000) trough spraying and dipping meth­ ods, after 10 days’ incubation in the dark condition at 25°c. an= aspergilus niger; pd= penicillium digitatum; cc= colletotrichum gloeosporioides; rs= rhizopus stolonifer. means followed by the same letter within a column are not significantly different at p≤0.05. treatment disease incidence (%) spraying method dipping method a.n p.d c.c r.s a.n p.d c.c r.s t. daenensis 25.8 c* 38.8 b 9.4 ef 11.0 e 16.3 d 17.6 d 4.5 fg 7.2 f t. vulgaris 25.4 c 33.5 b 13.7 de 12.4 de 16.1 d 25.4 c 5.0 fg 6.6 f s. hortensis 9.6 ef 27.2 c 0.0 g 3.3 fg 7.2 f 16.3 d 0.0 g 0.0 g s. khuzistanica 7.2 f 22.0 c 0.0 g 0.0 g 4.5 fg 12.5 de 0.0 g 0.0 g tiabendazole 7.6 f 21.5 c 3.8 fg 3.3 fg 3.5 fg 7.8 f 3.3 fg 0.0 g infected control 97.5 a 97.5 a 95.0 a 95.0 a 97.5 a 95.0 a 95.0 a 95.0 a adv. hort. sci., 2024 38(2): 169­176 176 savory species (rich in carvacrol) were more effective than thyme species oils (rich in thymol) in terms of controlling fungal decays on fruit. finally, plant eos rich in carvacrol are introduced as promising candi­ dates for the commercial production of natural fungi­ cides to disinfection and management of post­har­ vest decay molds of citrus fruits. references adams r.p., 2007 ­ identification of essential oils compo‐ nents by gas chromatography/quadrupole mass spectrometry. 4th edition. ­ allured publishing, carol stream, il, usa, pp. 804. adeyinka a., richard f., 2015 ­ application of phyto‐ chemical extracts and essential oils in food products. ‐ int. j. biotechnol. food sci., 3(3): 31­35. agrios g.n., 2005 ­ plant pathology. 5th edition. 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n.k., shukla a.k., 2008 ­ use of some essential oils as post‐harvest botanical fungicides in the management of grey mold of grapes caused by botrytis cinerea. ­ world j. microbiol. biotechnol., 24: 39­46. wills r.b.h., golding j.b., 2016 ‐ postharvest: an introduction to the physiology and handling of fruit and vegetables. 6th edition. ­ university of new south wales press, sydney, pp. 293. ziztzelsberger c., buchbauer g., 2015 ­ essential oils as “a cry for help. ­ rev. nat. prod. commun., 10: 1127­ 1138. 137 1. introduction grapevine phylloxera (daktulosphaira vitifoliae) is a tiny aphid-like insect that feeds on grapevine (vitis vinifera l.) roots and leaves, leading to stunted growth or death. it is considered the most destructive grapevine pest (vidart et al., 2013). in syria, there are more than 70,000 ha of grapevine with an estimated 540,000 ton annual production (statistics of syrian agriculture ministry, 2011). however, phylloxera causes millions of dollars in losses in grapevine production annually. grapevine phylloxera forms damaging root galls which are metabolically active organs suited to meet the nutritional requirements of phylloxera and support its generation with high reproductive rates, making this pest capable of destroying the root system of v. vinifera vines. root injuries reduce the vines’ ability to absorb nutrients and water, causing a decline in vigor and productivity. as a consequence, weakened plants probably become more susceptible to secondary infections by fungal diseases and other insects and are also vulnerable to environmental stresses (granett et al., 2001). the use of resistant rootstocks is considered the most common and effective means to control phylloxera in the field. the vast majority of these rootstocks have been durably resistant for a long period. in syria, the widely used resistant rootstocks are ru140 (v. rupestris x v. berlandieri), r99 (v. rupestris x v. berlandieri), and 3309c (v. riparia michaux x v. rupestris) and b41 (v. vinifera x v. berlandieri) (makee et al., 2003). it is important to note that some rootstocks are more resistant than others to grapevine phylloxera. however, for yet unknown reasons, some rootstocks may lose their resistance to phylloxera. for example, axr#1 (vitis vinifera x v. rupestris scheele hybrid) has failed to resist phylloxera in several parts of the world after many years of use (granett et al., 1983). likewise, rootstock b41 has remained resistant in france while it is not resistant in californian vineyards, therefore farmers have to replant their vineyards with the appropriate resistant rootstocks (song and granett, 1990; de benedictis and granett, 1993). plants have active defense mechanisms against pathogen attacks. a group of microorganisms referred to as plant growth-promoting rhizobacteria (pgpr) are able to reduce disease through the induction of systemic resistance (isr) that renders the host plant more resistant to further pathogen ingress (pieterse et al., 2002). this phenomenon can occur in many plant species and was demonstrated to be effective against a broad spectrum of fungal, bacterial and viral diseases beside its effect on insect and nematode pests (van loon et al., 1998; ramamoorthy et al., 2001; durrant and dong, 2004; verhagen et al., 2010; weller et al., 2012). in addition to eliciting isr against pathogens, protective effects of pgpr against insects have been noted (zehnder et al., 1997 a, b; zehnder et al., 2001; kloepper et al., 2004; vijayasamundeeswari et al., 2009; valenzuela-soto et al., 2010). however, to our knowledge no studies have been carried out to assess in vitro the effects of pgpr on grapevine phylloxera. in this context, a non-pathogenic pseudomonas putida btp1 strain has shown enhancement of the level of resistance in cucumber, bean and tomato against the fungal pathogens pythium aphanidermatum and botrytis in vitro pseudomonas putida btp1-induced systemic resistance in grapevine rootstocks against phylloxera (daktulosphaira vitifoliae) a. adam (1), i. idris, z. ayyoubi department of biotechnology, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: b41, grapevine phylloxera, isr, pgpr, pseudomonas putida btp1, ru140. abstract: this study investigates the systemic resistance induced by pseudomonas putida strain btp1 against phylloxera using an in vitro model in ruggeri (ru140) and b41 roostocks. significant differences were found with regard to matured females, fecundity and oviposition period between untreated and bacteria-treated plants in both rootstocks. treated ru140 rootstocks were more resistant than treated b41 ones. btp1 impacted negatively on the ability of phylloxera to develop, indicating an increase in grapevine resistance and tolerance toward this pest in bacteria-treated plants. this is the first known study of biocontrol of phylloxera in grapevine rootstocks by non-pathogenic p. putida strain btp1 in vitro. adv. hort. sci., 2013 27(4): 137-142 (1) corresponding author: ascientific@aec.org.sv received for publication 16 september 2013 accepted for publication 10 december 2013 138 cinerea, respectively (ongena et al., 1999; ongena et al., 2004; adam et al., 2008). in a previous study performed on fresh roots from local grape variety helwani (v. vinifera), we demonstrated the influence of p. putida btp1 on reproduction and development of grapevine phylloxera (adam et al., 2012). implementation of in vitro dual culture assay has been used to evaluate the phylloxera/grapevine interaction (forneck et al., 1996; makee et al., 2003; vidart et al., 2013). this method has several advantages for our designed experiments such as providing optimal conditions for phylloxera infestation, conducting experiments in small space, preventing the spreading of phylloxera and rhizobacteria, as well as reliable results in a relatively short period. the present work aims to demonstrate the isr-related protective effect triggered by p. putida btp1 in vitro in ru140 and b41 rootstocks against grapevine phylloxera. the percentage of mature females, fecundity and oviposition period of phylloxera were determined. 2. materials and methods establishment of the phylloxera colony grapevine phylloxera was originally collected from field-infested roots of the local grapevine varieties in southern parts of syria. the phylloxera colony was established following similar procedures to those mentioned by makee et al. (2003). fresh and healthy pieces of roots (4-7 mm in diameter and 5-7 cm long) of local grapevine cultivar helwani (v. vinifera) were taken and washed with tap water. each piece was wrapped with moist cotton wool around one end, and then 10 to 15 phylloxera eggs were placed on each piece. the infested root pieces were then placed on a wet filter paper disk inside a plastic petri dish (12 cm diameter). each dish had three to four root pieces. for ventilation purposes the petri dish lid was modified with a 1-1.5 cm cloth-screened hole. the edges of the dishes were sealed with parafilm and they were kept in plastic boxes with tightly fitting lids and incubated at 25±1°c, 70±5% rh and 24 h darkness. the root pieces were replaced when they desiccated, rotted or the phylloxera became crowded. microbial strain and inoculum preparation p. putida strain btp1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron transport (jacques et al., 1995; ongena et al., 2002). it was maintained and prepared for use in the isr assays as previously described by ongena et al. (2002). for the bioassays, btp1 strain was grown in erlenmeyer flasks (250 ml) containing 100 ml of casamino acids medium (caa) for 24 h on a rotary shaker (150 r.p.m.) at 28ºc. cells were removed by centrifugation at 16500 g for 15 min at 4ºc and washed in sterile nacl (5 g l–1). the final pellet was resuspended in an adequate volume of sterile distilled water to obtain a bacterial suspension at 108 cfu ml-1. in vitro culture of grapevine plants for in vitro culture of grapevine plants, we used a protocol described by makee et al. (2010). wood cuttings having four to five nodes of ru140 and b41 rootstocks were collected from the field while the buds were still dormant. all cuttings were washed in water, and then treated with gentamicine sulphate 160 mg l-l. thereafter, they were incubated in 0.5 g l-1 carbamate fungicide [methyl-1-(butylcarbamoyl)2-benzimidazole-carbamate 50%] (bell®) for 24 h, and then grown in sterilized water at 25±1°c under 16 h photoperiod (140-150 μmol m-2 s-1) from daylight fluorescent tubes (philips tld 38/54). shoots were grown in glass jars (1000 ml) when they became about 8 cm long; buds of 4 mm length were taken from the middle of each stem. these buds were dipped in a solution of 70% ethanol for 3 min, 1.5% commercial bleach for 15 min followed by 0.7% commercial bleach for 5 min (charbaji and nabulsi, 1999). after sterilization, they were washed three times with sterile water and planted in tubes containing 20 ml dsd1 medium (da silva and doazan, 1995). the dsd1 media contains 100 mg l-1 nh 4 no 3 , 1000 mg l-1 kno 3 , 180 mg l-1 mgso 4 . 7h 2 o, 100 mg l-1 kh 2 po 4 , 500 mg l-1 ca (nh 3 )4h 2 o, 27.5 mg l-1 mnso 4 .7h2o, 37.5 mg l-1 na 2 edta, 0.025 mg l-1 cuso 4 5h 2 o, 0.025 mg l-1 cacl 2 .6h 2 o, 1 mg l-1 h 3 bo 3 , 1 mg l-1 znso 4 7h 2 o, 27.5 mg l-1 fe so 4 7h2o, 10 mg l-1 myoinositol, 1 mg l-1 acid nicotinic, 1 mg l-1 thiamine, and 1 mg l-1 pyrodoxine. the ph of the medium was adjusted to 6.4 before adding agar and it was then autoclaved at 116°c for 25 min. the tubes were closed using cellophane paper and the edge of the tubes was sealed with parafilm to avoid contamination. all tubes were then incubated as described above. experimental design six-week-old grapevine plants were used to induced resistance; plantlets with two or three roots were selected. due to the lack of phylloxera to infest the roots in the medium and to avoid the interaction between phylloxera and btp 1 , one root of each plants was pulled out of the medium but kept within the tube while the other root remained in the medium. the second root was treated with 1 ml of bacterial suspension (108 cfu ml-1) of p. putida btp1 on the root surface and inside the medium, or by distilled water for the control plantlets. the tubes were closed again as described above and incubated at 25°c under 16 h photoperiod. seven days later, the second root was infested with sterile eggs of phylloxera according to makee et al. (2003). three-day-old eggs were taken from the colony and placed into 1.5 ml eppendorf tubes for sterilization of the egg cuticle. one ml of formaldehyde (2.5%) was added to the eggs, gently shaken for 10 min, and left for 20 min. the sterilizing solution was then removed with a micro-pipette and the eggs were extracted and placed on sterile filter paper. the sterile eggs were gently transferred and spread on the non-inoculated roots of in vitro cultured plants by using a 10 ml sterile loop (kendall, usa). for each rootstock, five treated and five untreated plantlets were infested with 25 surface-sterile phylloxera eggs. the tubes were resealed with parafilm to 139 prevent contamination and to avoid the escape of phylloxera crawlers, and were then incubated at 25±1°c under 16 hr photoperiod (140-150 μmol m-2 s-1) from daylight fluorescent tubes (philips tld 38/54). evaluation procedure stereo microscope inspection was carried out daily on treated and untreated plantlets maintained in closed tubes to observe distribution of the eggs. the number of eggs hatched, feeding nymphs and adults were recorded to determine the mean developmental time (egg to egg) for each tested plant. five random of root-feeding phylloxera females in each tube were inspected to determine the mean of oviposition period and the mean of fecundity (total number of eggs) of phylloxera. thus, 25 females were examined on each plantlet. all eggs laid by each female were observed daily and counted till the female’s death. egg distribution during oviposition period (number of eggs per day), fecundity (total number of eggs) and female longevity were determined. statistical analysis all statistical analyses were performed using graphpad prism 5 program at 5% level (p= 0.05). data were subjected to analysis of variance (anova) for the determination of differences in means between tested plants of each treatment. differences between means were tested for significance using tukey hsd test. 3. results effect of p. putida btp1 on grapevine resistance against phylloxera percentage of matured females the result showed significant difference in phylloxera egg numbers that were able to hatch and develop to reach adult stage (matured females) on both rootstocks ru140 and b41 (f= 79.6; df=3, 16; p<0.001) (fig. 1). p. putida btp1treated plants emerged significantly percentage decreased of matured females in both rootstocks comparing to control plants. however, there was no significant difference in the percentage of emerged matured females between treated plants of b41 rootstock and plants of rootstock ru140 no treated (fig. 1). the percentage of matured females of phylloxera on treated b41 was significantly greater (33%) than that on treated ru140 (16%) (fig. 1). fecundity there was a significant difference in the mean of fecundity between ru140 and b41 rootstocks (f= 140.8; df=3.96; p<0.001) (fig. 2), with it resulting greater (19.4 eggs) in ru140 than in b41 (13.7 eggs). when plants were treated with btp1, the mean number of eggs laid significantly decreased in both rootstocks, pointing to a significant decrease in the mean of fecundity of phylloxera in both rootstocks. however the decrease in this parameter was greater in treated ru140 (5.4 eggs) than treated b41 rootstocks (9.9 eggs) (fig. 2). oviposition period a significant difference was observed in the mean oviposition period of phylloxera between ru140 and fig. 1 effect of p. putida btp1 on percentage of matured females of phylloxera in vitro in b41 and ru140 rootstocks in comparison with control plants. each column represents data from 25 samples. data were subjected to anova analysis and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). fig. 2 effect of p. putida btp1 on fecundity of phylloxera in vitro in b41 and ru140 rootstocks in comparison with control plants. each column represent data from 25 samples. data were subjected to anova analysis and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). 140 b41 rootstocks (f= 38; df=3.96; p<0.001) (fig. 3). the oviposition period was 7 and 6 days in ru140 and b41, respectively. however, when plants were treated with p. putida btp1, the oviposition period decreased in a significant way only in ru140. no significant differences were observed in the mean oviposition period between treated and untreated b41 plants (fig. 3). the mean oviposition period of phylloxera on treated b41 was significant: lasting one day longer than treated ru140 (6 and 5 days respectively) (fig. 3). 4. discussion and conclusions a recent study carried out on healthy pieces of roots of local grapevine cultivar helwani showed the influence of non-pathogenic p. putida btp1 on reproduction and development of grapevine phylloxera (adam et al., 2012). however, in the present work, the aim was to investigate the ability of this bacteria to induce systemic resistance in two grapevine rootstocks against phylloxera by using in vitro cultured plants. for our module this approach provided a strict separation condition between the inducer (bacteria) and the pathogen or pest (phylloxera) to induce systemic resistance (ongena et al., 2002; bakker et al., 2007). in agreement with a previous study (ongena et al., 2002), the bacteria did not migrate through the plants, suggesting the observed decrease in the life cycle of phylloxera was due to induction of systemic resistance in the host plant. the present study confirmed that p. putida btp1 had a protective effect on ru140 and b41 rootstocks against phylloxera. the means of fecundity and oviposition period and emerged mature female percentage decreased significantly in both btp1-treated rootstocks in comparison with control plants. these results are consistent with similar previous studies that demonstrated the ability of some strains of pgpr to induce systemic resistance in tomato against whitefly, where the percentage of matured females decreased in treated plants (hanafi et al., 2007; valenzuela-soto et al., 2010). in addition, similar results were reported when cucumber beetles and american bollworm fed on pgpr-treated cucumber plants and cotton bolls, respectively (zehnder et al., 1997) (a, b; vijayasamundeeswari et al., 2009). other studies also indicated that changes in the feeding behavior of leaffolder and decreases in the weight of larvae and pupae were observed in rice plants treated with rhizobacteria (radjacommare, 2002). on the other hand, our results showed that there was a significant difference in reproduction and development of grapevine phylloxera between btp1-treated b41 and ru140 rootstocks. in comparison, the percentage of matured females and the means of fecundity and oviposition period decreased significantly by up to 50%, 45% and 12% respectively in treated ru140 rootstock versus treated b41 rootstock (figs. 1, 2 and 3). this is consistent with results of previous studies indicating the presence of a type of gradient from the resistant plant to sensitive plant (granett et al., 1983; makee et al., 2010). these results show that phylloxera laid a large number of eggs on sensitive varieties, more than on resistant varieties. it is believed that poor nutrition or the inability to colonize good locations for feeding could directly affect the number of eggs and ultimately the ratio of hatching. thus, the resistance of grapevine to phylloxera could be a reflection of the strong relationship between poor nutrition and a decline in the productivity of the insect (granett et al., 1983). in addition, the mechanism of defense in these rootstocks may be due to toxic effects against phylloxera, such as the accumulation of some phenolic compounds in the cells of resistant plants leading to an increase in the death rate (omer et al., 1999). other workers illustrated that there is a positive relationship between resistance/susceptibility characteristics against aphids and flavonoid glycoside content (quercetin and isorhamentin) of cowpea lines as these compounds possess a good inhibitory rate for aphid reproduction (lattanzio et al., 2000). therefore, the resistance of ru140 and b41 rootstocks to phylloxera may be attributed to an ability to produce such toxic phenolic compounds. in conclusion, understanding the mechanisms of defense induced by some strains of pgpr in plants is very important to develop systemic resistance in plants. the current study provides evidence that p. putida strain btp1 has the ability to stimulate a systemic resistance in grapevine rootstocks against phylloxera. we suggest that p. putida btp1 treatment leads to an alteration in the plant’s metabolic pathway eliciting the induction of plant defense compounds. these substances would have a negative influence on phylloxera feeding and development in treated plants. however, more research in the field must be done before implementing this technique on a large scale. fig. 3 effect of p. putida btp1 on oviposition period of phylloxera in vitro in b41 and ru140 rootstocks in comparison with control plants. each column represent data from 25 samples. data were subjected to anova analysis and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). 141 to our knowledge, this work is the first study interested in biocontrol of phylloxera in grapevine by pgpr strains in vitro. furthermore, this investigation supplies important information about the possibility of implementing this strain to stimulate systemic resistance against plant pests. moreover, this study illustrates the effectiveness of using in vitro dual culture in evaluating the phylloxera/grapevine and grapevine/rhizobacteria interactions. in fact, this testing system could be considered a very promising tool to: i) examine the phylloxera resistance of newly developed rootstocks; ii) prevent the spread of phylloxera; iii) study phylloxera genetic variation, biology and control method; and iv) study the mechanisms of defense induced in plants by rhizobacteria against pests. acknowledgements the authors thank prof. i. othman (atomic energy commission of syria) and dr. n. mirali (department of biotechnology) for their help. we thank prof. p. thonart and dr. m. ongena of the university of liège, who provided us with the p. putida btp1 strain. references adam a., makee h., idris i., 2012 the influence of a non-pathogenic pseudomonas putida strain btp1 on reproduction and development of grape phylloxera. adv. hort. sci., 26(2): 75-80. adam a., ongena m., duby f., dommes j., thonart p., 2008 systemic resistance and lipoxygenase-related defence response induced in tomato by pseudomonas putida strain btp1. bmc plant biology, 8: 113. bakker p.a.h.m., pieterse c.m.j., van loon 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j.f., sikora e.j., kloepper j.w., 2001 application of rhizobacteria for induced resistance. eur. j. plant pathol., 107: 39-50. impaginato 543 adv. hort. sci., 2019 33(4): 543­552 doi: 10.13128/ahsc­8187 study on relationship between morphological and physiological traits with resistance to rust fungus (puccinia allii) in iranian garlic clones a. anjomshoaa 1, h. jafary 2, m.r. hassandokht 3 (*), m. taheri 2 , v. abdossi 1 1 department of horticulture science and agronomy, science and research branch tehran, islamic azad university, iran. 2 plant protection research department, zanjan agricultural and natural resources research and education center, areeo, zanjan, iran. 3 university college of agriculture and natural resources, university of tehran, karaj, iran. key words: allium sativum l., garlic rust, combined variance analysis, infection frequency. abstract: in the present study we collected 12 clones of garlic from different geographical origin in iran. the clones were sown in a field trial under natural infection of the rust fungus during two consecutive years. after 210 days, the reactions of the clones to the disease as well as the morphological features of the clones were evaluated. the results of analysis of variance on morphological traits showed a significant difference among the clones in terms of bulb weight, mean clove weight, number of bulb skin, number of cloves in the bulb, leaf temperature and the percentage of clove dry weight, and nutrient uptake for n,p,k, mn and zn. the results showed a positive and significant correlation between the leaf temperature, photosynthesis, nitrogen and manganese uptake and percentage of leaf infection at 1% probability level. the results of the infection frequency showed that the clones ‘gilvan1’ and ‘lalejin’ had the lowest percentage of infection and were identified as resistant clones to the rust disease. the results also showed that garlic clones reacted differently to the rust fungus and are separated into resistant, semi resistant, semi­suscepti­ ble and susceptible clones. 1. introduction garlic (allium sativum l.) is one of the most important vegetable crops in the world. asian countries such as china, india, afghanistan and iran are known as the main growing area of this vegetable. currently, garlic cultivation and production has been developed with a wide variety from east asia to south america. due to the commercial, economic and phar­ maceutical­industrial importance of garlic, the interest in increasing pro­ (*) corresponding author: mrhassan@ut.ac.ir citation: anjomshoaa a., jafary h., reza hassan­ dokht m., taheri m., abdossi v., 2019 ­ study on relationship between morphological and phy‐ siological traits with resistance to rust fungus (puccinia allii) in iranian garlic clones. ­ adv. hort. sci., 33(4): 543­552. copyright: © 2019 anjomshoaa a., jafary h., reza hassandokht m., taheri m., abdossi v. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 august 2019 accepted for publication 9 september 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 543­552 544 duction, yield and development of new varieties is rapidly growing (cunha et al., 2012). the world aver­ age yield of garlic in the last 20 years has been around 10 tons per hectare, while in recent years it increased to 18 tons per hectare (faostats, 2018). the iranian plateau is one of the most important cen­ ters of vegetation diversity in the world (mousavi, 1994) and among scientists, it is known as the arc of garlic distribution. morphological and molecular studies have indicated a wide variety of iranian garlic clones distributed all over the country (baghalian et al., 2005; vafaee, 2007). despite the long history of forced apomixis in the garlic, its cultivars show a great diversity of morphological, physiological and biochemical differences (etoh and simon, 2002; ammarellou et al., 2014). garlic colonies have a wide variety in vegetative traits, taste and flavor, bolting and fertility capacity. the cultivar characteristics dif­ fer significantly according to the location of cultiva­ tion and climate, and the weather conditions has a significant effect on cloves, flowering and taste of garlic. all garlic colonies are sterile, and therefore genetic variation in this plant may only be due to ran­ dom mutations or non­sexual variation and to the introduction of new genetic variation using modern molecular techniques. biochemical and molecular studies indicate that the highest level of heterogene­ sity occurs within the genetic reserves of central asia, which may include favorite genes for use in future genetic studies, as well as the improvement of plant programs. kallo and bergh stated that cloves’ mor­ phological traits, in particular number and size of leaves, height of the flowering stem, number of bulb shells, reaction to temperature, seed dormancy, quality of maintenance, vegetative and maturity peri­ od duration, are genetically controlled and these are polygenic traits (kallo and bergh, 1993). one of the most important problems faced by farmers in relation to garlic cultivation is the out­ break of fungal rust disease due to high humidity of cultivated areas. most herbaceous fungi are destroyed by fungicides, but for many of them there is still no proper and effective fungicide. in addition, the use of fungicides generally causes pollution of the environment, groundwater resources, and prod­ ucts themselves. the cause of garlic rust is the fungus puccinia allii that is an airborne fungus, and its release of spores is very fast (michael and sarah, 1995); spores are commonly transported by the wind and causes the disease characterized by a sore or pustules at the leaf level. the teliospores live in non­ crop seasons in the soil and plant infected residuals. the disease develops at temperatures below 10 °c and is destroyed by frozen dew. if the garlic is sub­ jected to drought stress or excess water or con­ densed due to excessive consumption of nitrogen fertilizer, it will become susceptible to the disease (houshiarfard and pourabdollah, 2015). in the world, an overgrowth of rust on garlic has been reported in many cases (schwartz and mohan, 1999). for example, garlic rust is one of the major problems in garlic cultivation in the united states. studies show that the clones do not have complete resistance to garlic rust, they may be tolerant showing (on average) 51% reduction in the product (coviello, 2007). selection of resistant clones from the genetic complex of iranian local clones is one of the pre­ breeding research priorities of the country in iran. identification of resistant crop cultivars has many advantages over the use of chemicals and other con­ trol methods. since the economic importance of garlic rust has been grown up in recent years, few studies have been focused on the identification of morpho­ logical traits of garlic related to the yield, but none of the studies addressed on the relationship between morphological, physiological component and the resistance to the rust fungus. this study aimed to investigate the physiological and morphological fea­ tures in the resistant and the susceptible iranian garlic clones, with the ultimate scope to achieve resistant colonies to the garlic rust in selected clones. 2. materials and methods plant materials based on geographical distribution of garlic in iran, we indicated the main cultivation regions with long crop history throughout the country. in each region, we collected one sample which represented the main geographical characteristic of that region to avoid any doubling in sampling and to collect as much as distinct clones. in total we collected 12 superior clones from all over the country (table 1). the cloves were planted after disinfection with fungi­ cide carbendazim and confidor insecticide in a field trial in chavarzagh tarom zanjan (48 46’ 43.34” e ­ 36 59’ 51.33” n) and altitude of 484 meter from sea level and temperate­semi­arid climate in a complete­ ly randomized block design with three replications during two consecutive years. soil texture of cultiva­ tion site was clay loam with ph= 7.6 and electrical conductivity of 0.8 ds/m, percentage of neutralizing matter was 6.2%, and available phosphorus and anjomshoaa et al. ‐ iranian garlic resistance to rust fungus 545 potassium in the soil were 3.8 and 261 mg/kg, respectively. in the annual cultivation process, the cloves of each clone were grown on 35 cm stacks with cultivation depth of 3 cm and 10 cm spacing. due to possible genetic variation on resistance to the rust fungus within each clone, the most resistant seedlings of clones were selected for diseases resis­ tance evaluation in the second year. at garlic harvest­ ing stage, the bulbs were harvested at the time of physiological maturity, when the leaves fell and 70% dried (rubatzky and yamaguchi, 1997). evaluation of morphological traits the morphological traits were evaluated accord­ ing to the descriptor recommended by the international plant genetic resources institute (ipgri, 2000). the evaluated morphological traits included bulb weight, bulb diameter, number of leaves, mean clove weight, number of bulb sheet, number of cloves in a bulb, color of clove, bulb and leaf, side view of bulb, shape of mature bulb and type of bulb structure. leaf color and number were evaluated when the plants were fully developed, while the traits of bulb were studied after complete maturation of the plant and bulb harvesting. evaluation of physiological traits at the end of march each year, the leaf chloro­ phyll content was estimated by measuring 10 leaf samples per replication and estimation of the mean data was done with the help of spad device. other physiological measurements on leaf such as pure photosynthesis, stomatal conductance of h2o, leaf surface temperature and transpiration sub stomatal co2 at the full leaf growth stage were performed by uk manufacturing adc device (adc bioscientific lci analyser serial no. 32648). sampling of green leaves was done at full leaf growth stage to estimate the rate of nutrients absorption and dry matter percent­ age of the leaf and immediately transferred to labo­ ratory of zanjan agricultural research center. the percentage of dry weight of cloves and leaves was calculated and recorded by placing a sample of cloves or leaves of each treatment for 24 hours in oven at 70°c and measuring the weight of samples before and after drying. the nutrient uptake was evaluated in water and soil laboratory of zanjan agriculture research center. after sampling the plant leaves, they were immediately transferred to the laboratory and after washing they were dried immediately in a 70° c oven for 48 hours. then, the samples were digested with acid and the plant extract was pre­ pared. finally, macro elements such as nitrogen, potassium, phosphorus and micro elements such as zinc, copper and manganese were measured by atomic absorption and calorimetric (emami, 1996). evaluation of resistance to the rust fungus in order to evaluate the frequency of leaf infection by rust fungus among the clones, the numbers of pus­ tules were recorded at 4 stages (20 april, 5 may, 20 may and 5 june). the evaluation method of infection frequency of the disease was as follows: 10 plants per plot were randomly selected. the average of number of pustules on leaves of 10 plants was determined as the percentage of rust infection in each clone (clifford and jones, 1983; dhingra and sinclair, 1995). methods and tools for analyzing the data in this study, statistical software sas and spss was used to analyze the statistical data. analysis of vari­ ance of traits and their mean comparison were done using duncan test at 1% level. the correlation between traits was measured by pearson’s two­ table 1 ­ origin specifications of iranian garlic clones row clone country province city/region longitude latitude altitude 1 dezfol iran khozestan dezfol 48° 25' 52.57" e 32° 22' 59. 08" n 143 3 sahneh iran kermanshah sahneh 47° 41' 41.08" e 34° 28' 26. 53" n 1354 4 lalejin iran hamedan lalejin 48° 28' 34.42" e 34° 58' 25. 22" n 1700 5 azar shahr iran azarbayejan sharghy azar shahr 45° 58' 59.78" e 37° 44' 1. 64" n 1415 6 lahijan iran gilan lahijan 50° 0' 12.07" e 37° 12' 25. 46" n 4 7 khaf iran khorasan razavi khaf 60° 8' 50.48" e 34° 34' 18. 53" n 975 8 sojas iran zanjan sojas 48° 33' 04.45" e 36° 14' 24. 14" n 1774 9 hesar iran zanjan hesar 47° 43' 7.24" e 36° 57' 26. 62" n 1158 10 gilvan 1 iran zanjan gilvan 1 49° 4' 48.88" e 36° 48' 10. 53" n 340 11 gilvan 2 iran zanjan gilvan 2 49° 7' 51.54" e 36° 47' 10. 94" n 340 12 chavarzagh iran zanjan chavarzagh 48° 46' 43.34" e 36° 59' 51. 31" n 484 adv. hort. sci., 2019 33(4): 543­552 546 domain test with the spss software. at the end of the second year, combined variance analysis was per­ formed. 3. results analysis of variance the results of two­year combined analysis of vari­ ance of morphological traits showed a significant dif­ ference between the clones in terms of bulb weight, mean clove weight, number of bulb skin, number of cloves in the bulb at 1% level, while there was no sig­ nificant difference between the bulb diameter and number of leaves (table 2). the results of combined analysis of variance for two years from physiological traits showed a signifi­ cant difference between the studied clones in leaf surface temperature and clove dry weight percent­ age at 1% probability level. in terms of stomatal con­ ductance and photosynthesis, the difference was sig­ nificant at 5% probability level, while there was no significant difference between substomatal co2, tran­ spiration, chlorophyll and leaf dry weight percentage among the clones (table 3). combined analysis of variance in terms of nutrient uptake of n, p, k, mn, and zn showed that a signifi­ cant difference was found between the clones at 1% level and in terms of cu uptake at 5% probability level (table 4). the results of combined analysis of variance in terms of the resistance of the clones to the rust fun­ table 2 ­ results of combined analysis of variance for morphological characteristics in some iranian garlic clones s.o.v df ms bulb weight bulb diameter mean clove weight number of bulb skin number of cloves bulb leaf number year 1 11295.04 ** 5.24 * 2.41 ns 0.12 ns 0.05 ns 0.00 ns rep/year 4 120.94 0.46 1.68 0.06 3.81 0.44 treat 11 351.53 ** 0.59 ns 5.77 ** 22.31** 32.77 ** 0.46 ns treat × year 11 363.21 ** 0.57 ns 0.53 ns 0.42 ns 0.38 ns 0.00 ns error 44 103.00 0.57 0.95 0.87 1.84 0.56 cv% 17.48 15.30 25.46 18.73 11.41 10.83 table 3 ­ results of combined analysis of variance for physiological characteristics in some iranian garlic clones *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. s.o.v df ms clove dry weight leaf dry weight chlorophyl sub stomatal co2 stomatal conductance of h2o photosynthesis transpiration leaf surface temperature year 1 590.64 ** 0.61 ns 123.76 ns 9531.20 ns 0.0013 ns 233.24 ** 160.68 ** 894.64 ** rep/year 4 0.21 1.84 39.75 2517.86 0.0036 0.37 1.70 7.26 treat 11 8.55 ** 1.89 ns 39.51 ns 455.23 ns 0.0032 * 10.53 * 0.38 ns 3.14 ** treat × year 11 13.66 ** 1.44 ns 62.30 ns 1061.43 * 0.0032 * 3.74 ns 0.58 ns 1.74 ** error 44 0.84 2.48 39.16 581.20 0.001 5.21 0.51 0.52 cv% 3.11 12.29 11.18 10.05 17.50 19.38 11.23 1.96 table 4 ­ results of combined analysis of variance for physiological characteristics in some iranian garlic clones s.o.v df ms nitrogen phosphorus potassium manganese zinc copper year 1 26/21 ** 0/026 ns 0/0004 ns 445/80 ns 3786/84 ** 1/85 ns rep/year 4 0/064 0/009 0/199 25/19 32/23 2.45 treat 11 0/24 ** 0/0071 ** 1/326 ** 67/36 ** 56/87 ** 1/28 * treat × year 11 0/098 ** 0/0075 ns 0/429 ** 22/86 ns 15/60 ns 2/53 ** error 44 0/017 0/0006 0/09 15/32 9.81 0/61 cv% 2.9 7.31 7.32 10.27 11.08 9.39 *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. anjomshoaa et al. ‐ iranian garlic resistance to rust fungus 547 among the physiological traits was related to photo­ synthesis (19.38). among the morphological traits, the highest coefficient of variation was related to the mean weight of the clove (25.46). in the study of the percentage of infection of garlic to the rust fungus, the highest coefficient of variation appeared at the second stage of evaluation in the early may (33.60). regarding to significance of analysis of variance among the clones, for some traits the mean compari­ son was done using duncan’s multi­domain test. the results of the two­year mean comparison of morpho­ logical traits showed that among the studied clones, the lowest weight was related to the clones ‘chavarzagh’ (49.84) and ‘lahijan’ (51.47) and the high­ est was related to the clones ‘sahne’ (79.40) and ‘sojas’ (61.11). in terms of the mean weight of the clove, the lowest weight belonged to the clone ‘khaf’(1.97) and the highest weight belonged to the clones ‘sahne’ (5.14) and ‘gilvan1’ (4.98). the mean comparison of the studied clones showed that the lowest number of bulb skin was for the clones ‘chavarzagh’ and ‘lahijan’ (2.33) and the highest was for the clone ‘azar shahr’ (7.16). in terms of number of cloves in a bulb, the lowest number was related to the clone ‘hesar’ (8.66) and the highest was related to the clone ‘khaf (17.33) (table 6). gus at four stages of measurement showed that the difference at the first stage was significant at 1% level. at the next three stages, the difference in resis­ tance between the clones was significant at 5% level (table 5). the effect of year was also significant in terms of the leaf surface temperature, photosynthesis, tran­ spiration, percentage of clove dry weight, absorption of nutrients zn and n and bulb weights at 1% level. in term of resistance to garlic rust, the effect of the year was significant at 4 stages at 1% level. the effect of year on the treatment showed that the leaf surface temperature, n, p, k and copper nutrient uptake percentage, percentage of dry weight cloves, bulb weight, and percentage of resistance gar­ lic rust were significant at the first and second stages of growth at 1% level and substomatal co2, stomatal conductance, and percentage of infection of garlic rust were significant at the 3rd and 4th stages of growth. other physiological traits including photosyn­ thesis, transpiration, chlorophyll content, dry matter percentage, manganese and zinc intake, and morpho­ logical traits including the bulb diameter, mean bulb weight, number of the bulb skin, number of cloves in the bulb and number of leaves did not showed signifi­ cant differences. the highest coefficient of variation table 5 ­ results of combined analysis of variance for resistance to rust in some iranian garlic clones s.o.v df ms 20/04/2019 05/05/2019 20/05/2019 05/06/2019 year 1 491.42 ** 651.86 ** 2604.74 ** 2947.88 ** rep. year 4 0.35 4.65 24.94 11.23 treat 11 1.38 ** 15.19 * 75.02 * 144.83 * treat × year 11 1.38 ** 20.63 ** 84.50 * 119.35 * error 43 0.35 6.29 44.57 63.58 cv% 22.85 33.60 32.58 20.29 table 6 ­ mean comparison of morphological traits in some iranian garlic at two years *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. treatment bulb weight (gr) bulb diameter (mm) mean clove weight (gr) number of bulb skin number of cloves per bulb leaf number per plant gilvan 1 60.13 b 4.96 a 4.98 a 5.83 abc 12.00 cd 6.66 a gilvan 2 56.65 b 5.07 a 4.69 a 4.83 c 12.33 bc 6.66 a azar shahr 55.08 b 5.05 a 3.15 bcd 7.16 ab 13.16 bc 6.66 a hesar 52.77 b 4.77 a 3.85 abc 5.66 bc 8.66 f 7.33 a lahijan 51.47 b 4.96 a 3.68 abc 2.33 d 12.00 cd 7.33 a khor va biabanak 54.22 b 4.88 a 4.07 ab 2.83 d 11.33 cde 7.33 a sojas 61.11 b 5.27 a 2.28 cd 6.66 ab 9.66 def 6.66 a dezfol 56.41 b 4.80 a 4.47 ab 2.66 d 14.33 b 7.00 a sahneh 79.40 a 5.36 a 5.14 a 6.33 abc 11.50 cde 7.00 a khaf 58.45 b 4.10 a 1.97 d 5.83 abc 17.33 a 7.00 a chavarzagh 49.84 b 4.93 a 3.89 abc 2.33 d 11.33 cde 7.00 a lalejin 60.85 b 5.08 a 3.94 abc 7.33 a 9.33 ef 6.66 a in each column different letters mean significant differences between samples. 548 adv. hort. sci., 2019 33(4): 543­552 the results of the two­year mean comparison of physiological traits showed that among the studied clones in terms of the leaf surface temperature, the clone ‘gilvan1’ had the highest leaf surface tempera­ ture (37.98 ℃) while the clone ‘azar shahr’ had the lowest one (35.53 ℃). stomatal conductance was highest in the clone ‘azar shahr’ (0.23) while the low­ est value was found for the clone ‘gilvan1’ (0.15). the clone ‘azar shahr’ had the highest photosynthe­ sis rate (13.35) while the clone ‘gilvan1’ had the low­ est one (8.80). in terms of percentage of the clove dry weight, the highest was related to the clone ‘lahijan’ (31.92) and the lowest value was related to the clone ‘azar shahr’ (28.11) (table 7). the mean comparison of the studied clones in terms of nutrient uptake showed that the highest nitrogen uptake was related to the clones ‘dezful’ (4.83) and ‘chavarzagh’ (4.77) and the lowest was related to the clones ‘hesar’ (4.22) and ‘khaf’ (14.32). in terms of phosphorus uptake, the highest uptake was related to the clone ‘lalejin’ (0.44) and the low­ est was related to the clone ‘sojas’ (0.30). in terms of potassium and manganese uptake, the highest uptake was related to the clone ‘dezful’ in potassium (4.85) and manganese (42.77), and the lowest was related to the clone ‘lalejin’ in potassium (3.62) and manganese (30.89). in terms of zinc and copper uptake, the highest uptake was related to the clone ‘lalejin’ in zinc (35.78) and copper (9.21), the lowest zinc uptake was for the clone ‘gilvan1’ (24.61) and the lowest copper uptake was for the clone ‘khaf’ (7.55) (table 8). it is worth noting that all 12 clones were infected with garlic rust, although showing different reaction to the fungus. in two­year mean comparison, it was found that the observation of rust symptoms began from the second half of april, and disease progressed along with the growth of the plants. at the first stage table 7 ­ mean comparison of physiological traits in some iranian garlic at two years in each column different letters mean significant differences between samples table 8 ­ mean comparison of physiological traits in some iranian garlic at two years treatment leaf surface temperature °c sub stomatal co2 stomatal conductance of h2o photo­ synthesis transpiration chlorophyl leaf dry weight clove dry weight gilvan 1 37.98 a 236.33 a 0.15 b 8.80 b 5.96 a 49.45 a 13.33 a 28.83 c gilvan 2 36.65 ab 224.75 a 0.20 ab 13.16 a 6.08 a 58.53 a 13.42 a 29.44 bc azar shahr 35.53 c 231.83 a 0.23 a 13.35 a 6.37 a 55.76 a 12.17 a 28.11 c hesar 36.61 bc 233.50 a 0.22 a 12.85 a 6.46 a 57.26 a 13.05 a 28.66 c lahijan 37.03 ab 249.50 a 0.21 ab 10.18 ab 6.31 a 58.70 a 12.38 a 31.92 a khor va biabanak 37.30 ab 236.50 a 0.19 ab 11.47 ab 6.29 a 58.96 a 12.55 a 29.39 bc sojas 36.98 ab 252 a 0.21 ab 10.78 ab 6.43 a 56.78 a 13.35 a 30.86 ab dezfol 35.68 c 247.48 a 0.23 a 12.29 ab 6.28 a 55.45 a 12.32 a 28.76 c sahneh 36.60 bc 250 a 0.23 a 12.27 ab 6.61 a 55.26 a 12.60 a 29.62 bc khaf 37.65 ab 243.83 a 0.21 ab 11.50 ab 6.74 a 54.23 a 13.91 a 28.75 c chavarzagh 37.28 ab 232.25 a 0.22 a 12.36 ab 6.80 a 55.13 a 12.43 a 31.29 a lalejin 37.25 ab 238.08 a 0.19 ab 12.35 ab 6.15 a 55.68 a 12.40 a 28.76 c treatment nitrogen phosphorus potassium manganese zinc copper gilvan 1 4.73 ab 0.32 bcd 4.52 ab 42.53 a 24.61 c 8.06 bc gilvan 2 4.60 ab 0.35 bc 4.47 abc 37.81 abc 28.69 bc 8.72 ab azar shahr 4.67 abc 0.35 bc 4.30 bcd 40.58 ab 31.04 ab 8.52 abc hesar 4.22 e 0.35 bc 3.84 ed 36.22 abc 24.80 c 8.71 ab lahijan 4.76 ab 0.38 b 4.83 a 35.11 bc 29.03 bc 8.31 abc khor va biabanak 4.66 abc 0.35 bc 4.01 cde 40.27 ab 29.48 bc 7.84 abc sojas 4.47 cd 0.30 d 3.64 e 36.69 abc 26.63 bc 7.70 bc dezfol 4.83 a 0.35 bc 4.85 a 42.77 a 26.98 bc 8.55 abc sahneh 4.33 de 0.38 b 3.84 de 37.55 abc 28.53 bc 8.00 bc khaf 4.32 de 0.35 bc 3.62 e 36.79 abc 26.71 bc 7.55 c chavarzagh 4.77 ab 0.35 bc 4.51 abc 39.83 ab 26.74 bc 8.49 abc lalejin 4.48 cd 0.44 a 3.62 e 30.89 c 35.78 a 9.21 a in each column different letters mean significant differences between samples anjomshoaa et al. ‐ iranian garlic resistance to rust fungus 549 of growth, the highest percentage of infection was related to the clone ‘lahijan’. at the second stage, the highest percentage of infection was related to the clone ‘khor va biabanak and the lowest was relat­ ed to the clone ‘hesar’. at the third and fourth stages, the clones ‘gilvan1’ and ‘lalejin’ had the low­ est percentage of infection and were identified as resistant to rust. this may indicate that garlic clones behave in different way during seedling and adult plant stages and therefore they have different genet­ ics for resistance to the garlic rust fungus in different growth stages. the most susceptible clone was ‘chavarzagh’ showing the highest percentage of infection (table 9). the first uredospores of garlic rust appeared in the first half of april each year, and the first teliospores appeared two weeks later. the onset and spread of infection completely follow the progressive growth pattern and with the help of ure dospores and in the absence of fungicides, the level of plant infection can reach from over 1% to more than 80% within two months. the results of this study showed that garlic clones respond differently to the rust and are separated into resistant, semi resistant, semi­susceptible and sus­ ceptible clones (table 10). in sum, combined mean comparison of garlic clones in terms of resistance to disease showed that gilvan1 cultivar had distinct traits compared to other clones. morphologically, to color pf this clone is violet with code 5, 6­8 skins, side view shape of the compound bulb is broadly ovate, basal plate event, shape of matured bulb is globe and type of bulb structure is regular with two­fan group of cloves. the distinction of this clone with other vio­ let clones is in the number of skins. the next resistant clone, according to the field conditions of the present research, was ‘lalejin’ from hamadan province, which has white stripes bulb with code 4 and white clove with code 1. the number of skins is 5­8 and side view shape of the compound bulb is broadly ovate, basal plate event, shape of matured bulb is broadly oval, and type of bulb structure is regular multi­shelled. table 9 ­ mean comparison of resistance to rust in some iranian garlic clones in two year (difference in days to maturity) treatment percentage of the disease 20/04/2019 05/05/2019 20/05/2019 20/06/2019 gilvan 1 1.72 b 2.53 bc 3.97 c 5.83 c gilvan 2 1.58 b 2.59 bc 4.65 abc 6.42 abc azar shahr 1.72 b 2.99 abc 4.66 abc 6.30 bc hesar 1.72 b 2.36 c 4.21 abc 6.25 bc lahijan 2.01 a 2.86 abc 5.04 a 6.57 abc khor va biabanak 1.72 b 3.32 a 4.92 ab 6.52 abc sojas 1.72 b 2.45 bc 4.00 bc 5.97 bc dezfol 1.72 b 3.00 abc 4.94 a 6.67 ab sahneh 1.72 b 2.85 abc 4.29 abc 6.04 bc khaf 1.72 b 3.03 ab 4.24 abc 5.95 bc chavarzagh 1.72 b 2.88 abc 4.80 abc 7.08 a lalejin 1.72 b 2.71 abc 4.55 abc 5.85 c in each column different letters mean significant differences between samples. table 10 ­ reaction of 12 iranian garlic clones to rust fungus in a two years field traits under natural infection clones grouping of resistance resistant semi resistant semi susceptible susceptible gilvan 1 x lalejin x khaf x sojas x sahneh x hesar x azar shahr x gilvan 2 x khor va biabanak x lahijan x dezfol x chavarzagh x adv. hort. sci., 2019 33(4): 543­552 550 traits’ correlation the correlation coefficients between physiological traits and resistance to rust are shown in table 11. the results of the study showed a positive and signifi­ cant correlation between the leaf surface tempera­ ture, photosynthesis, nitrogen and manganese uptake and percentage of leaf infection at the proba­ bility level of 1%. the relationship between the leaf surface temperature and infection frequency indi­ cates that by increasing the leaf temperature, the percentage of infection will be increased. this result is reasonable according to other researchers’ reports that the percentage of rust damage has only a direct correlation with the mean temperature. in addition, a positive and significant correlation was observed between percentage of rust infection and photosyn­ thesis and nitrogen, manganese and zinc. also, a neg­ ative and significant correlation was found between percentage of rust infection and transpiration rate at the level of 1%. the correlation coefficients between morphologi­ cal traits and resistance to rust are shown in table 12. a negative and significant correlation was observed between traits such as transpiration rate, bulb weight, bulb diameter, number of bulb skins and type of bulb structure and rust infection percentage at the 1% level. this relationship indicates that garlic rust is effective on the factors affecting the garlic yield, such as the bulb weight, bulb diameter and by increasing the percentage of infection, the value of these traits are reduced. results of the infection frequency in four growth stages (fig. 1) showed that the clones with high infec­ tion frequency at the early stages of growth had a higher disease symptomps at the final stages of growth. therefore, the presence of infection at the early stages reduced the growth, yield and quality of the product. however, those that were resistant to some extent at the early stages of growth had rela­ tively low infection and slow spread of disease, and with better growth of the clones. 4. discussion and conclusions this is the first study on the identification of resis­ tant clones to rust on garlic through the study of morphological and physiological traits collected on treat percentage of the disease percentage of the disease 1 bulb weight ­0.462 ** bulb diameter ­0.285 ** mean clove weight 0.068 number of bulb skin ­0.404 ** number of cloves per bulb ­0.007 leaf number 0.128 color bulb 0.183 color clove 0.009 color leaf 0.096 side view of bulb ­0.118 shape of mature bulb ­0.098 type of bulb structure ­0.526 ** table 12 ­ correlation coefficients between morphological traits and resistance to rust (puccinia allii) in iranian garlic clones measured by pearson's two­domain test table 11­ correlation coefficients between physiological traits and resistance to rust (puccinia allii) in iranian garlic clones measured by pearson's two­domain test *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. treat percentage of the disease percentage of the disease 1 leaf surface temperature ­ 0.602 ** sub stomatal co2 ­ 0.206 stomatal conductance of h2o 0.059 photosynthesis 0.411 ** transpiration ­ 0.520 ** chlorophyl 0.217 * leaf dry weight 0.059 clove dry weight 0.160 nitrogen 0.625 ** phosphorus ­ 0.085 potassium 0.142 manganese 0.298 ** zinc 0.469 ** copper 0.19 *, **, ns= significant at 5%, 1% and not significant probability levels, respectively. fig. 1 ­ correlation coefficients between morphological traits and resistance to rust (puccinia allii) in iranian garlic clo­ nes measured by pearson's two­domain test anjomshoaa et al. ‐ iranian garlic resistance to rust fungus 551 12 iranian clones from iran during two crop years. the clones ‘gilvan1’ and ‘lalejin’ had the lowest number of stomata in the leaf area, because the fun­ gus of the garlic rust through the stomata comes in to the plant, the low number of stomata reduces the possibility of pathogen entry and may be the most important feature of resistance in these clones. the results of two­year combined analysis of vari­ ance of morphological traits are consistent with the research report of nourbakhshian et al. (2007). the results of their research showed that a significant dif­ ference was found between the number of cloves in the bulb, clove weight, length and diameter of the clove and amount of dry matter per unit area (nourbakhshian et al., 2007). vafaee and colleagues (2009), in a study on the genetic diversity of iranian garlic clones using mor­ phological traits and aflp as molecular markers, clas­ sified iranian garlic clones in 6 main groups, while according to morphological traits, the total clones were classified into 4 general groups. the results of this study showed that garlic clones respond differently to the rust. the results of a study on resistance to garlic rust in pakistan showed that rust severity of 0.8% was recorded on variety hazro (alam et al., 2007). a study in the us state of california regarding the susceptibility of the tested clones to garlic rust showed that none of the cultivars including selective later and early clones, and spanish and chinese cultivars showed complete resistance and were usually tolerant (coviello, 2007). another california breeding program to test the resistance to garlic rust on three genotypes pe493096, pe540315, w12820 showed that all of them were infected with garlic rust and only about 1% of them had less than 26% of the infection (davis, 2007). the results of correlation coefficients between morphological traits and resistance to rust are consis­ tent with nourbakhshian’s report on factors affecting garlic yield and bulb weight (nourbakhshian et al., 2008). the results of this study are consistent with the results of other researchers regarding the superi­ or clones’ traits (baghalian et al., 2005). kallo report­ ed that a clove weight, number of cloves and bulb diameter had the most direct effect on garlic yield, and stated that the selection of the clones based on these traits would improve the yield potential of gar­ lic (kallo, 1988). the use of commercial clones carrying rust resis­ tance genes is the most efficient, economical and environmental friendly method of rust disease con­ trol. using morphological trait for early screening of the clones for resistance to garlic rust can be very important. the results of the infection frequency showed that the clones ‘gilvan1’ and ‘lalejin’ had the lowest percentage of infection and were identified as resistant clones to the rust. we found resistant clones with the introduction of each morphological trait. it should be noted that since the intensity measurements of infection were carried out at four stages, it was found that the earli­ est symptoms of garlic rust began in early may, and then the growing trend was accompanied by the growth of garlic. two years of field trails lead us to achieve resistance components and to find resistant clones of garlic to the rust pathogen, however in order to get more in detail on mechanism and genet­ ics of resistance we are following genetics studies on resistance and susceptible clones. by this research we will find the diversity of genes involved in resis­ tance to rust pathogen in garlic and we will explore some more details on mechanisms of resistance in the future. references alam s.s., ahmad m., usman a., ahmad a., naveedul­ lah a., 2007 ­ variation in garlic varieties for reaction to natural infection of ‘puccinia porii’ and alternaria porri. ­ sarhad j. agric., 23(1): 149­152. ammarellou a., kazemitabar k., najafei zarreini h., 2014 ­ effects of genetic and environmental conditions on bulbing quality of 38 iranian garlic (allium sativum l.) cultivars. ­ 3th national congress on medicinal plants, mashhad, iran, pp. 331. baghalian k., ziai s.a., naghavi m.r., naghdi abadi h., khalighi a., 2005 ­ evaluation of alicin content and botanical traits in iranian garlic (allium sativum) eco‐ types. ­ sci. hort., 103: 155­166. clifford b.c., jones d.g., 1983 ­ cereal diseases. ­ basf uk, pp. 309. coviello r.l., 2007­ us ipm pest management guidelines: onion and garlic‐insects section. ­ us anr publication 3453, statewide ipm program, agriculture and natural resources, university of california, usa. cunha c.p., hoogerheide e.s.s., zucchi m.i., mon­ teiro m., pinheiro b., 2012 ­ new microsatellite markers for garlic, allium sativum l. (alliaceae). ­ amer. j. bot., 99(1): e17­e19. davis r.m., 2007­ us ipm pest management guidelines: onion and garlic‐diseases section. ­ us anr publication 3453. statewide ipm program, agriculture and natural resources, university of california, usa. dhingra o.d., sinclair j.b., 1995 ­ basic plant pathology methods. ­ crc press, boca raton, florida, usa, pp. adv. hort. sci., 2019 33(4): 543­552 552 448. emami e., 1996 ­ the instructions of plant decomposition methods . ­ agricultural and natural resources research center of iran, (1)982. etoh t., simon p.w., 2002 ­ diversity, fertility and seed production of garlic, pp. 101­11. ­ in: rabinowitch h.d., and l. currah (eds.) allium crop sciences: recent advances. cab international, egham, surrey, uk, pp. 528. faostat, 2018 ­ http://www. fao.org/ faostat/en/# data/qc. houshiarfard m., pourabdollah s.h., 2015 ­ garlic rust, civilica.com/r/5920. ipgri, 2000 ­ descriptors for allium. ­ international plant genetic resources institute, rome, italy, pp. 43. kallo g., 1988 ­ vegetable breeding. ­ crc press, boca raton, florida, usa, inc., pp. 239. kallo g., bergh b.o., 1993 ­ genetic improvement of vegetable crops. ­ pergamon press, oxford, uk, pp. 846. michael j.c., sarah c., 1995 ­ the fungi: a microbiologi‐ cal perspective. 2nd edition ­ academic press, pp. 635. mousavi a., 1994 ­ study the ecophysiological character‐ istics of tareh irani. ­ ms. c. thesis in horticultural sci­ ence, faculty of agriculture, tehran university, iran, pp. 87. nourbakhshian s.j., mousavi s.a., bagheri h.r., 2007 ­ evaluation of agronomic traits and path coefficient analysis of yield for garlic cultivars. ­ pajouhesh and sazandegi, 77:10­18 (in farsi). rubatzky v.e., yamaguchi m., 1997 ­ world vegetables. second edition, chapman and hall, usa, pp. 417. schwartz h.f., mohan s.k., 1999 ­ compendium of onion and garlic diseases . ­ the american phytopathological society, pp. 70. vafaee y., dashti f., mardi m., ershadi a., 2009 ­ a detection of genetic diversity among iranian garlic clones (allium sativum l.) via morphological characters and aflp markers. ­ iranian journal of agricultural sciences, 40: 13­22 (in farsi). impaginato 107 adv. hort. sci., 2022 36(2): 107­117 doi: 10.36253/ahsc­12041 postharvest quality responses of pome­ granate fruit (cv. shishe­kab) to ethanol, sodium bicarbonate dips and modified atmosphere packaging m. dorostkar, f. moradinezhad (*) department of horticultural science, university of birjand, birjand, iran. key words: anthocyanins, decay, sensory quality, shelf­life, vacuum packaging. abstract: pomegranate fruit is very popular due to its high commercial impor­ tance and health benefits. this experiment aimed to evaluate the sensory qual­ ity, color, and biochemical properties (tss, ta, tss/ta, anthocyanin content and total antioxidant capacity) of pomegranate fruit under post­harvest treat­ ments, included ethanol (etoh), sodium bicarbonate (sbc), and different pack­ aging. experimental treatments included: 10% (v/v) etoh, 1% (w/v) sbc, and the type of packaging (passive­map and vacuum). fruit were then stored at 5±1°c and 90% relative humidity for ten weeks. the peel and aril color evalua­ tions indicate that etoh treatment and vacuum packaging (vp) improved the quality of pomegranate color by increasing a* and decreasing l*. these treat­ ments made the skin color and aril color lighter and redder in pomegranate. in addition, the treatments reduced decay and maintained total soluble solids (tss), and titratable acidity (ta). interestingly, etoh treatment improved fruit nutritional quality as it increased total antioxidant capacity and anthocyanin content by 20% and 50%, respectively, compared to the control. the sensory analysis indicated that treated fruit with etoh and vp scored higher in taste, color, texture, and appearance, and showed the best acceptability from the panelists’ viewpoint. in conclusion, etoh and vp significantly improved pome­ granate fruit quality during cold storage since preserved sensorial quality and bioactive compounds and reduced decay. 1. introduction pomegranate is mainly confined to the tropics and subtropics and grows well in arid and semi­arid climates. the edible portion of pome­ granates (arils) is about 55 to 60% of the total fruit weight, and contains 80% juice and 20% seeds (erkan and kader, 2011). the fresh juice con­ tains 85% water and 15% sugars, pectins, ascorbic acid, polyphenolic flavonoids, anthocyanins, and amino acids (erkan and kader, 2011). the amount of these compounds vary with pomegranate variety, maturity, and environmental and cultivation conditions. the statistics on acreage and production of pomegranate are not available with food and (*) corresponding author: fmoradinezhad@birjand.ac.ir citation: dorostkar m., moradinezhad f., 2022 ­ postharvest quality responses of pomegranate fruit (cv. shishe‐kab) to ethanol, sodium bicarbo‐ nate dips and modified atmosphere packaging. ­ adv. hort. sci., 36(2): 107­117. copyright: © 2022 dorostkar m., moradinezhad f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2022 accepted for publication 25 february 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12041 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(2): 107­117 108 agriculture organization at the global level. however, the estimated global cultivated area of pomegranate is around three hundred thousand hectares, with the production of three million tones (venkitasamy et al., 2019). pomegranate is classified as a non­climacteric fruit due to low respiration and ethylene production rates after harvest (kader et al., 1984). despite its non­climacteric nature, the fruit still undergoes both qualitative and quantitative losses during postharvest handling and storage, resulting in chilling injuries, husk scald, weight loss, and decay (opara et al., 2015). postharvest losses of fresh horticultural prod­ ucts occur from harvest and continue during the han­ dling and storage period, estimated more than 20% of production in developed countries and about 40­ 50% in developing countries (watkins, 2020). to reduce quantitative and qualitative losses during the supply chain and increase food availability, posthar­ vest decay control is one of the significant factors to be considered. because most of the commercial pomegranate cultivars are susceptible to chilling injury when they are stored at low temperatures less than 5°c, commonly held at 5°c or higher tempera­ ture (moradinezhad et al., 2020). as a result, postharvest decay occurs by different fungi. various fungicides have been used traditionally to control postharvest decay in fresh fruits. however, there is public concern about food safety regarding fungi­ cides application in pre and postharvest stages (tzatzarakis et al., 2020). chemical treatments should be compounds with known and minimal toxicological effects on mammals and impact on the environment. as substances that will be in contact with fresh produce, they should be affirmed as generally recognized as safe (gras) by the united states food and drug. ethanol, carbon­ ates, and bicarbonates belong to the gras group (palou, 2018). previous reports (teksur, 2015; droby et al., 2016; dukare et al., 2019) focused on using environmentally friendly, effective, and safe alterna­ tive control methods to fungicides to reduce posthar­ vest decay of fresh fruits. safe inorganic compounds have shown antimicrobial activity (deliopoulos et al., 2010). successful control of postharvest decay in var­ ious fresh fruits was indicated using different safe chemical compounds applications such as ethanol (etoh), sodium carbonate (sc), sodium bicarbonate (sbc), and ozone (nunes, 2010). etoh is a volatile organic compound with antimicrobial potential, widely used as a disinfectant. the positive effects of etoh application have been indicated in fresh fruits like, grapes, peaches, oranges, nectarines, strawber­ ries, apples, and chinese bayberries (dao and dantigny, 2011). sbc is a common food additive that has been used in the food industry as a safe and effective chemical for controlling fungi growth as a bio fungicide. it is cheap, readily available, and a low risk of injury to the fresh fruit (vilaplana et al., 2018). the beneficial effects of modified atmosphere packaging (map) have been demonstrated on differ­ ent fruits and vegetables. the results of various stud­ ies of map in different cultivars of pomegranate showed significant improvement in quality mainte­ nance and extension of the storage life of fruit (moradinehzad et al., 2018; sahel et al., 2018; venkataramudu et al., 2018; candir et al., 2019; moradinehzad et al., 2019, 2020). map also reduces the growth of pathogens and consequently posthar­ vest decay (pareek et al., 2015; teksur, 2015; rodriguez and zoffoli, 2016; ansarifar and moradinezhad, 2021). despite map has been widely applied to pomegranate; however, the literature review shows that no research focused on the effect of pre­storage etoh or sbc dips and their combina­ tion with modified atmosphere packaging on postharvest decay control and quality attributes of the pomegranate fruit. therefore, this study aimed to assess the efficacy of pre­storage etoh or sbc dips and map on physiological responses and quality of pomegranate fruit cv. shishe­kab during postharvest storage. 2. materials and methods fruit preparation and treatments about 200 fully mature pomegranate fruit cv. shishe­kab were harvested from a commercial orchard in south khorasan province, birjand, iran, in october 2019. pomegranate fruits were harvested and placed in carton boxes. a row of fruits was placed in each box so that the fruits would not be damaged. the fruits were transported to the postharvest lab of the university of birjand, iran, immediately after harvest. uniform fruits (267­320 g) free of defects were selected and were then dipped in 200 ppm sodium hypochlorite solution for 1 min for surface disinfection. before applying the treat­ ments on fruits, five fruits were peeled and their juice was taken for initial analysis (color properties, tss, ta, tss/ta, anthocyanin content and total dorostkar and moradinezhad ‐ response of pomegranate to chemicals and map 109 antioxidant capacity). thereafter , f ru i ts were dipped in chemical solutions including 10% (v/v) etoh or 1% (w/v) sbc for 2 minutes. control fruit were immersed in distilled water (20°c). all the chemicals used in this experiment were obtained from the merck company (germany). fruit were then air­dried and placed into low­density polyethylene (ldpe) bags (carton plast co., iran) (3 treatments × 2 packages × 3 replications) with 0.05 mm thickness (ten fruit per bag). bags were sealed after removal of air by a vacuum pump to make vacuum packaging (vp) or sealed without removal of air (passive map) and then stored at 5±1°c and 85±5% relative humidi­ ty. physico­chemical and sensory quality attributes of fruits were determined after 10 weeks of cold stor­ age. fruit quality assessments color attributes. aril and peel color of fruit were evaluated in all treatments. the color was deter­ mined in terms of l*, a*, and b* values using a col­ orimeter (tes­135 a, country of manufacture taiwan). chroma and hue were obtained with the fol­ lowing equations (1 and 2): chroma= [a(0.5+ )b(0.5]2 (1) hue angle (h) = tan­1 (b/a) (2) measurements were made at three different points on the skin of each fruit. three fruits in each replicate were used. determination of total soluble solids (tss), titrat‐ able acidity (ta), and tss/ta ratio. to prepare the juice, we first separated the pomegranate arils from fruit’ peel. then pomegranate juice was prepared by hand pressure, and at the end it was filtered using a thin cloth. to prepare juice, three fruits selected from each replication. for higher accuracy in the experiment, the average of three samples was pre­ sented as one replication (juice was prepared sepa­ rately from each fruit). tss in the extracted juice of each slice (one center section) was measured by a hand­held refractometer (rf 10, °brix, 0­32%, extech co., usa). to measure titratable acidity, 5 ml of extracted fruit juice titrated with 0.1n sodium hydroxide. the ta was calculated as a percentage of citric acid. the tss to ta ratio cal­ culated by dividing tss to ta in each replication of treatments. determination of anthocyanin content. the total anthocyanin content of juice determined by the ph­ differential method using two buffer systems com­ prised of potassium chloride (ph 1, 0.025 m) and sodium acetate (ph 4.5, 0.4 m). one ml of juice sam­ ple mixed with 10 ml of buffer, and the absorbance (a) measured at 510 and 700 nm using a spectropho­ tometer (unico 2100, china) (wagner, 1979). determination of total antioxidant capacity.to determine the total antioxidant activity, dpph radical inhibitor activity method was used. the dpph radical­ scavenging activity of the samples was evaluated according to the method described by turkmen et al. (2005) dissolved in distilled water at different concentrations. pomegranate juice samples were mixed with 1 ml of a freshly made methanol solution of dpph radical (100 µm). the contents were vigorously incorporated and incubated at room temperature in the dark for 20 min, and the absorbance was read at 517 nm. methanol solutions of tested extracts and dpph were used as blank and control measurements, respectively. all experiments were carried out three times on two separate occasions. the percentage of total antioxidant activity (taa) of the tested extracts was calculated according to the following equation (3). % radical scavenging activity = [(absorption control­absorp­ tion sample)/ absorption control] × 100 (3) sensory quality and decay percentage. sensory evaluation of samples was done by a panel of ten trained members, based on a 5­point hedonic scale at the end of the cold storage period. in all treat­ ments, aril was first isolated from the peel and the panelist evaluated and scored three fruits from each treatment. mean scores was considered as one repli­ cation. after tasting each sample by the evaluators, we asked them to drink some water. all the treat­ ments were randomized at room temperature (22°c), and panelists rated the appearance, taste, color, tex­ ture, and acceptance of pomegranate arils on a five­ point scale, 1= the extremely bad, and 5= extremely good (3≤ acceptable) as described by moradinezhad et al. (2018). the decay evaluated visually during the storage time. fruit were examined daily and considered infected when a visible lesion was observed (such as surface mycelia, slimy patches, bruises, and blemish­ es). results expressed as the percentage of infected fruits. in fact, upon observing the first effects of decay on the fruit, that fruit considered as a percent­ age of decay in the whole box. in other words, in a package of ten fruits, if one fruit has decay symp­ adv. hort. sci., 2022 36(2): 107­117 110 toms, 10% of decay is reported for that box. statistical analysis the recorded data were subjected to a two­way analysis of variance (anova) with two factors, pre­ storage treatments and packaging methods in three replications using the genstat program (version 12, 2010, vsn international, ltd., uk). lsd test at 1% level of probability (p≤0.01) was used to compare means of different treatments. 3. results there were no significant interactive effects of chemical treatments × packaging type on all evaluat­ ed traits (data not shown). therefore, only the simple effects presented in the tables. aril and peel color attributes the aril color analysis showed that the l*, hue angle (h°), and chroma (c) values in all treatments decreased compared to the fruit at harvest, while a* value increased (table 1). as shown in table 1, post­ harvest application of etoh and sbc had a significant effect on l* and a* of aril. the highest l* value after 10 weeks of storage was recorded in treated fruit with etoh (20.2), and the lowest l* obtained in con­ trol fruit (11.6). the highest a* value (28.1) obtained from etoh treatment, and the lowest (24.3) observed in control. however, etoh and sbc treatments had no significant effect on b*, chroma, and hue parame­ ters. the results showed that treatments had a signif­ icant impact on the l* and a* parameters of pome­ granate peel. in fact, these treatments caused to a lighter and redder both the peel and aril color. as shown in table 2, all the color parameters of aril and peel fruit were significantly different com­ pare to fruit at harvest. most of the parameters (except a*) were reduced. in addition, the type of packaging had a significant effect on the l* and a* values. the highest l* (18) and a* (25.7) were obtained in vacuum­packed fruit. however, the type of packaging had no significant effect on b*, hue table 1 ­ effect of etoh and nahco3 dipping on color properties of pomegranate fruit (cv. shishe­kab) aril and peel after 10 weeks of cold storage at 5°c means ± se followed by different letters in the same column for the same evaluated parameter are significantly different (p≤0.01) accor­ ding to the lsd test. sbc= sodium bicarbonate. table 2 ­ effect of different ma packaging on color properties of pomegranate fruit (cv. shishe­kab) aril and peel after 10 weeks of cold storage at 5°c means ± se followed by different letters in the same column for the same evaluated parameter are significantly different (p ≤ 0.01) according to the lsd test. pre­storage treatments aril peel l* a* b* h° c* l* a* b* h° c* at harvest 23.2±1.7 a 22.5±1.4 d 6.7±0.1 a 19.2±1.1 a 28.8±2.2 a 44.6±3.1 a 37.4±2.8 c 13.4±1.2 a 19.8±1.4 a 46.7±3.9 a control 11.6±0.8 d 24.3±2.0 c 7.6±0.8 a 16.7±0.6 b 26.2±1.7 b 35.4±3.4 c 43.6±2.7 b 15.1±1.9 a 18.7±1.2 a 45.7±3.7 a ethanol (10%) 20.2±1.9 b 28.1±1.6 a 7.4±0.8 a 17.7±1.3 b 25.0±1.8 b 39.4±2.9 b 45.4±4.5 a 14.3±1.0 a 18.1±1.9 a 46.4±4.1 a sbc (1%) 18.7±1.2 c 26.1±0.9 b 6.8±0.4 a 17.9±1.2 b 25.3±1.9 b 39.2±3.1 b 43.2±2.8 a 16.9±1.5 a 19.3±1.1 a 46.9±3.4 a level of sig. ** ** ns * ** * ** ns ns ns lsd 5.46 6.52 3.62 4.65 6.52 5.04 5.02 5.47 4.21 4.57 packaging aril peel l* a* b* h° c* l* a* b* h° c* at harvest 23.6±1.4 a 23.5±1.8 c 7.5±0.8 a 21.7±1.1 a 27.8±2.0 a 48.0±2.5 a 32.4±2.9 b 16.9±1.8 a 23.1±1.6 a 35.9±3.4 b passive map 16.3±1.3 c 24.4±1.9 b 6.8±0.5 b 20.5±1.9 b 23.0±1.8 b 40.3±2.8 b 44.2±3.8 a 14.8±0.9 b 20.9±1.9 b 47.8±3.5 a vacuum 18.0±2.1 b 25.7±2.1 a 6.1±0.9 b 20.4±1.7 b 25.4±1.4 b 37.7±3.7 b 44.0±3.4 a 15.2±1.5 b 18.7±1.2 b 46.9±3.9 a level of sig. ** ** * ** ** ** * ** ** * lsd 4.46 5.32 1.45 6.06 5.32 4.12 4.10 4.38 5.95 3.73 dorostkar and moradinezhad ‐ response of pomegranate to chemicals and map 111 angle, and chroma of pomegranate aril (table 2). after 10 weeks of cold storage, passive map and vac­ uum packaging had no significant effect on the peel color attributes of the pomegranate fruit. total soluble solids (tss), titratable acidity (ta), tss/ta ratio the tss and tss/ta ratio in all treatments increased compared to fruit at harvest time. however, ta value in all treatments decreased com­ pared to fruit at harvest (table 3). data analysis showed that the highest content of tss and tss/ta found in control (18.54 °brix, and 18.05, respective­ ly), and the lowest content obtained from treated fruit with etoh (16.31°brix, and 9.24, respectively). also, the highest and lowest ta values obtained from etoh (1.94%) and control (1.02%) respectively. in addition, the evaluation of ma packaging showed that the highest content of tss and tss/ta were related to passive map (respectively, 18.40 °brix, and 16.78), and the lowest content obtained from vacu­ um packaging (respectively, 17.05°brix, and 9.34). also, the highest amount of ta observed in vacuum­ packed fruit (1.94 %) (table 4). anthocyanin content according to table 3, anthocyanin at harvest time was 21.51 mg l­1. after 10 weeks of storage, its value in the control group was 23.19 mg l­1 (about 8% more than harvest time). while, anthocyanin content was higher (34.27 mg l­1) in etoh­treated fruits (about 40% higher than harvest time). also, antho­ cyanin in the sbc treatment was significantly higher than in the control. however, the highest amount of anthocyanin was obtained from ethanol treatment after 10 weeks of storage, and the lowest amount was observed in control. as shown in table 4, the anthocyanin content at harvest time was 22.41 mg l­1. after 10 weeks of storage, the amount of antho­ cyanin significantly increased than harvest time. however, there was no significant difference between different packaging treatments. table 3 ­ effect of etoh and nahco3 dipping on biochemical attributes of pomegranate fruit (cv. shishe­kab) after 10 weeks of cold storage at 5°c means ± se followed by different letters in the same column for the same evaluated parameter are significantly different (p≤0.01) accor­ ding to the lsd test. sbc= sodium bicarbonate. tss (°brix) ta (%) tss/ta anthocyanin (mg l­1) antioxidant activity (%) at harvest 15.74±0.98 d 2.42±0.03 a 7.21±1.82 d 21.51±2.03 d 61.7±5.24 c control 18.54±1.51 a 1.02±0.01 d 18.05±1.28 a 23.19±1.17 c 68.2±6.50 b ethanol (10%) 16.31±0.84 c 1.94±0.01 b 9.24±1.87 c 34.27±1.41 a 77.3±5.74 a sbc (1%) 17.72±1.22 b 1.26±0.04 c 13.28±1.09 b 26.88±1.26 b 70.9±6.01 b level of sig. ** ** ** ** ** lsd 0.96 0.12 2.13 4.07 9.42 table 4 ­ effect of different ma packaging on biochemical attributes of pomegranate fruit (cv. shishe­kab) after 10 weeks of cold stor­ age at 5°c means ± se followed by different letters in the same column for the same evaluated parameter are significantly different (p≤0.01) accor­ ding to the lsd test. packaging tss (°brix) ta (%) tss/ta (°brix/%) anthocyanin (mg l­1) antioxidant activity (%) at harvest 16.02±1.78 c 2.28±0.01 a 7.13±1.48 c 22.41±2.07 b 63.5±4.54 b passive map 18.40±1.28 a 1.12±0.04 c 16.78±0.98 a 28.63±1.35 a 79.1±4.91 a vacuum 17.05±1.47 b 1.94±0.02 b 9.34±1.04 b 29.84±1.09 a 77.2±5.20 a level of sig. ** ** ** * ** lsd 0.78 0.09 1.74 3.33 7.68 112 adv. hort. sci., 2022 36(2): 107­117 the total antioxidant capacity the antioxidant activity of pomegranate fruit was measured by neutralizing free radicals. in general, in all treatments antioxidant activity was higher com­ pared to fruit at harvest (tables 3 and 4). the highest and lowest antioxidant activity recorded from etoh and control treatments, respectively. however, sbc had no significant effect on antioxidant activity. also, there was no significant difference between vacuum packaging and passive map. sensorial quality the appearance of the product is effective in con­ sumer preference. figure 1 a, and b shows the effect of treatments on the scores of acceptance, taste, tex­ ture, appearance, and color of arils during storage at 5°c. etoh treatment had the best sensory quality as scored higher by panelists. however, sbc treatment also achieved acceptance scores. besides, sensory properties were improved in vacuum­packed fruit. interestingly, passive map fruit scored acceptable quality by panelists. decay as shown in figure 2, etoh treatment significantly controlled fruit decay. the percentage of fruit rot under etoh treatment was 12.57%, while in control samples was 36.21% (3­fold higher than etoh). also, vacuum packaging reduced the fruit decay more effectively than passive map. 4. discussion and conclusions the l* represents lightness changes from 0, which has no lightness (absolute black) to 100, which is maximum lightness (absolute white) (morales et al., 2020). a* varies between green and red, negative values of a* indicate green colors, and positive values represent red colors. b* varies between blue and yel­ low, negative values of b* indicate blue colors and positive values represent yellow colors. hue is the color tone, or color name of a color. chroma is the amount of saturation of a color. colors of high chro­ ma are said to be clear, bright or brilliant. dull (pas­ tel) colors have a low chroma (morales et al., 2020). jin et al. (2013) reported that the application of fig. 2 ­ effect of different chemical treatments and ma packa­ ging on decay of pomegranate arils (cv. shishe­kab) after 10 weeks of cold storage at 5°c. error bars represent the error (percentage). identical letters mean there is no significant difference between them, at p≤0.01 (lsd). sbc= sodium bicarbonate. fig. 1 ­ effect of different chemical treatments (a) and ma packaging (b) on sensorial assessments of pomegranate arils (cv. shishe­kab) after 10 weeks of cold storage at 5°c. sbc0= sodium bicarbonate. dorostkar and moradinezhad ‐ response of pomegranate to chemicals and map 113 etoh on melon sweet fruit preserves the color and freshness of the fruit. they showed that after 16 days of storage, the highest l* value related to etoh 0.5 ml treatment, which is inconsistent with the results of the present report. the results showed that during the storage period, the l* of fruit peel decreased or darkened, which indicates a decrease in the peel quality compared to harvest time. the researchers showed that the synthesis of ethylene, followed by senescence, causes the enzymes associated with oxidative reactions and ultimately leads to darkening (low l* value) of the color (hasan et al., 2018; morales et al., 2020). in line with the findings of abdi et al. (1998), our results showed that the highest l* value obtained in etoh treatment, maybe due to the inhibition of ethylene biosynthesis which delaying fruit senescence and changes of senescence­related pigments. the results of this study on fruit color char­ acteristics are also in line with the findings of ponzo et al. (2018) on guava fruit. moradinezhad and dorostkar (2020) found that fresh jujube fruit under vacuum packaging had a higher l* value. they stated that vacuum packaging might inhibit enzymatic reac­ tions and delay the darkening of fruit color. similar results were reported by moradinezhad et al. (2019) on pomegranate fruit. they showed that passive map and vacuum packaging did not have a significant effect on the pomegranate peel color attributes. therefore, it concluded that the treatments used in the present study did not have adverse effects on the peel of pomegranate fruit and did not reduce the marketable value of the fruit. the increase in a*, is related to the increased biosynthesis and accumulation of anthocyanin pig­ ments, which are responsible for the intense red color of ripe pomegranate fruit (lyu et al., 2020). moradinezhad et al. (2019) reported that the a* value of pomegranate peel in vacuum­packed fruit was higher than the control. they stated that vacuum packaging modified the atmosphere around the fruit, extending its shelf­life and improving the color of the fruit, which is similar to the results of the present study. vacuum­packed fruits had the lowest respira­ tion rate, probably because of reduced o2 concentra­ tion and ethylene removal from the intercellular spaces (rana et al., 2018). in most fruits and vegetables, sugar makes up the main component of tss, which is thus a reasonable indicator of the values sugar levels (huang et al., 2021). as the results showed, the tss content of pomegranate fruit increased slightly after 10 weeks of storage compared to harvest time, because pome­ granate fruit is classified as a non­climatic fruit. generally, most non­climacteric fruits have a minor change on tss during the storage period, mainly due to low starch accumulation content during growth and development. however, one of the most signifi­ cant changes that occur during fruit ripening is the hydrolysis of starch to sugar, which changes the taste and texture of the product. likely, any treatment that delays maturation reduces soluble solids content. previous studies have shown that etoh inhibits the production and action of ethylene (podd and van staden, 1998) as a ripening hormone. etoh prevent­ ed the conversion of acc to ethylene (podd and van staden, 1998). this compound reduces ethylene syn­ thesis by reducing acc synthase activity (a key enzyme in ethylene synthesis). in the present study, we found that 10% etoh preserves tss of the pome­ granate fruit. liu et al. (2019) showed that post­har­ vest application of 50% etoh in cassava for 12 and 24 hours reduced tss compared to control samples. this decrease in tss is probably due to a reduction in the respiration rate. a similar result has been reported on blueberry (ji et al., 2021). the organic acids present in foods influence the flavor, color, microbial stability keeping quality (jawad et al., 2020). citric acid is the predominant acid in pomegranate fruit (tozzi et al., 2020). however, inorganic acids such as phosphoric and car­ bonic acids (arising from carbon dioxide in solution) often play an important and even predominant role in food acidulation (jawad et al., 2020). in plant tis­ sue, etoh and acetaldehyde can be converted to each other. therefore, external application of etoh increases the amount of acetaldehyde inside the fruit tissue, which causes production of more etoh in the presence of oxygen (podd and van staden, 1998). but, in the process of etoh production, carbon diox­ ide is also produced as a byproduct (podd and van staden, 1998). increased carbon dioxide inhibits eth­ ylene synthesis and reduces the respiration rate (park et al., 2021). as carbon dioxide increases, the synthesis of sugars (especially glucose) also increas­ es, and also organic acids are not consumed in cellu­ lar respiration (park et al., 2021). therefore, the use of etoh causes the accumulation of organic acids, as a result, increasing ta. according to the proposed mechanism, we found that the post­harvest applica­ tion of 10% etoh preserves the ta in pomegranate fruit. our finding is in consistent with the report of shao et al. (2020) on wampee fruit. adv. hort. sci., 2022 36(2): 107­117 114 the results also indicated that the lowest tss/ta ratio obtained from etoh treatment and vacuum packaging. it can be concluded that these treatments inhibit respiration rate and maintain the acidity of the pomegranate fruit. selcuk and erkan (2016) in turkey found similar results on sweet pomegranate fruit. the reduction of tss /ta ratio using map indi­ cates a delay in the ripening of pomegranate fruit. this results are consistent with the findings of venkatachalam and meenune (2015) on longkong fruit. anthocyanins are commonly found in plant cell vacuoles in the form of glycosides (i.e., the combina­ tion of anthocyanins with simple sugars such as glu­ cose, galactose, etc.) (podd and van staden, 1998). the presence of sugar in the structure of antho­ cyanins makes them soluble in water. interestingly, we found that 10% etoh treatment increased the anthocyanin content by 50% compared to the con­ trol. tzortzakis and economakis (2007) showed that post­harvest application of etoh (vapor) to tomatoes increases glucose, fructose and total soluble solids values. they found the increase in sugar was due to the weight loss of the tomatoes. our findings support the above­mentioned study. similarly, on bayberries, wang et al. (2010) found that etoh (22.32 μmol l­1) increased anthocyanins. they stated that this increase in anthocyanin content was related to increased antioxidant activity. liu et al. (2019) showed that treatment of 50% etoh for 24 hours on cassava was able to increase the anthocyanin content by about 14% compared to control. they stated that this increase in anthocyanin content could be related to the effect of etoh on reducing free radicals and increasing the activity of antioxidant enzymes. similar results were reported by el kereamy et al. (2002) on the grapes and huang et al. (2015) on the blueberry. as presented in table 4, the type of packaging did not affect the anthocyanin content of pomegranate fruit. an unavoidable consequence of aerobic metabo­ lism is reactive oxygen species (ros). environmental stresses lead to enhanced generation of ros in plants due to disruption of cellular homeostasis (wang et al., 2021). all ros are highly harmful to organisms at high concentrations. when the level of ros exceeds the defense mechanisms, a cell is in a state of “oxidative stress” (tang and vashisth, 2020). plant possess complex antioxidative defense systems comprising of non­enzymatic and enzymatic compo­ nents to scavenge ros (wang et al., 2021). the antioxidants are substances capable of delaying or inhibiting oxidation processes (hudson, 2012). it has been shown that pomegranate juice, and even fermented pomegranate juice have high antiox­ idant activity (li et al., 2006). these activities may be related to diverse phenolic compounds present in pomegranate juice, including punicalagin isomers, ellagic acid derivatives and anthocyanins (delphini­ din, cyanidin and pelargonidin 3­glucosides and 3,5­ diglucosides). these compounds are known for their properties in scavenging free radicals and inhibiting lipid oxidation in vitro (li et al., 2006). our results showed that postharvest use of etoh maintained the antioxidant activity of pomegranate fruit. in line with these results, wang et al. (2015) showed that etoh (300 µl l­1) increased h2o2 (a type of free radical). they stated that due to the balance between free radicals and antioxidants, the antioxidant content of loquat fruit increased. natural antioxidants may exhibit one or more of the following roles: free radi­ cal scavenger, reducing agent, and quencher of sin­ glet oxygen formation (kim et al., 2007). moreover, anthocyanidins also have the potent antioxidant capacity and possible protective effects on human health (kim et al., 2007). anthocyanins have greater antioxidant activity than either vitamin c or e (kim et al., 2007). these reports are in accordance with the results of anthocyanin content in the present study, which indicate the relationship between antioxidant activity and anthocyanin content in pomegranate aril. suzuki et al. (2004) showed that broccoli packaged with etoh pads in a perforated film maintained their green color longer than control. lurie et al. (2006) suggested etoh as a promising alternative to so2 in grapes since it prevented the occurrence of quality decay associated with loss of freshness, glossy appearance, and browning. similar to our results, liu et al. (2012) reported that etoh (6 ml/kg of fruit weight) improved the sweet melons sensory proper­ ties. they stated that the enhanced sweet melons’ sensory properties might be more dependent on the etoh treatment than the process of ripening and senescence. as mentioned before, pomegranate fruits are harvested during commercial maturity, so they have the highest sensory and taste quality at harvest time. according to table 3, tss and tss/ta ration in ethanol treated fruit changed less than harvest time (compared to control and sbc). therefore, ethanol treatment preserved the taste of treated fruit. in addition, the results of table 3 in terms of tss and tss/ta ratio are consistent with the results of evalua­ dorostkar and moradinezhad ‐ response of pomegranate to chemicals and map 115 tors. also, as shown in figure 1, vacuum packaging better retained the taste of pomegranate fruit, which is in line with the results of tss, and ta in the present study. similar results have been reported on the effect of vacuum packaging on preserving the sensory properties of jujube (moradinezhad and dorostkar, 2020) and litchi (shah and nath, 2006) fruit. researchers speculate that etoh could kill the mitochondrial inner membrane of fungal spores, thereby preventing the spread of infection in the fruit (gabler et al., 2004). similarly, in a recent study by ji et al. (2019) the effect of etoh on the physical prop­ erties of blueberries investigated. they found that etoh treatment (1000 µl l­1) significantly reduced fruit contamination (rotting was observed about 4% in etoh treatment and 40% in control). this reduc­ tion in etoh­induced decay may be due to the preservation of cell membranes in treated samples. since low concentrations of etoh can lower the tem­ perature at which phospholipids undergo a phase change (rowe, 1983), the increases in the spore mor­ tality and decay control following the addition of etoh may have resulted from a lowering of the phase­change temperature of mitochondrial mem­ branes of the spores under these conditions (margosan et al., 1997). also, possibly external appli­ cation of etoh has been shown to increase acetalde­ hyde produced by the alcohol dehydrogenase. acetaldehyde directly attacks pathogens. etoh has also been shown to increase resistance to other envi­ ronmental stresses (such as chilling in cucumber) in addition to controlling diseases (especially fungal) (frenkel and erez, 1996). it had been proven that low oxygen in vacuum treatment significantly decreased ethylene production (min et al., 2019). on the other hand, a treatment that reduces ethylene synthesis has a good effect on infection control (min et al., 2019). low ethylene production in a vacuum treat­ ment may result in lower cell wall enzyme activity and cell integrity maintenance, considering the fact that cell wall enzymes are activated by ethylene. this will probably help maintain the strength of the cell wall and reduce decay (ntsoane et al., 2019). similar results were obtained by moradinezhad and dorostkar (2020) on fresh jujube fruit. the results of presented in this report show that the use of etoh (10%) or vacuum packaging alone minimize decay, and maintain color, total soluble solids and titratable acidity of pomegranate fruit during storage. also, these treatments increased bio­ chemical properties such as anthocyanin content and antioxidant capacity and improved sensory qual­ ity. overall, it can be concluded that etoh has the potential to control decay, enhance antioxidant sys­ tems, and extend the shelf­life of the pomegranate fruit. references abdi n., mcglasson w.b., holford p., williams m., mizrahi, y., 1998 ­ responses of climacteric and sup‐ pressed‐climacteric plums to treatment with propylene and 1‐methylcyclopropene. ­ postharvest biol. technol., 14: 29­39. ansarifar e., moradinezhad f., 2021 ­ preservation of strawberry fruit quality via the use of active packaging with encapsulated thyme essential oil in zein nanofiber film. ­ int. j. food sci. technol., 56: 4239­4247. candir e., özdemi̇r a.e., aksoy m.c., 2019 ­ effects of modified atmosphere packaging on the storage and shelf life of hicaznar pomegranate fruits. ­ turk j. agric. for., 43: 241­253. dao t., dantigny p., 2011 ­ control of food spoilage fungi by ethanol. ­ food control., 22: 360­368. deliopoulos t., kettlewell p.s., hare m.c., 2010 ­ fungal disease suppression by inorganic salts: a review. ­ crop prot., 29: 1059­1075. droby s., wisniewski m., teixidó n., spadaro d., jijak­ li m.h., 2016 ­ the science, development, and commer‐ cialization of postharvest biocontrol products. ­ postharvest biol. technol., 122: 22­29. dukare a.s., paul s., nambi v.e., gupta r.k., singh r., sharma k., vishwakarma r.k., 2019 ­ exploitation of microbial antagonists for the control of postharvest dis‐ eases of fruits: a review. ­ crit. rev. food sci. nutr., 59: 1498­1513. el kereamy a., chervin c., souquet j.m., moutounet m., monje m.c., nepveu f., roustan j.p., 2002 ­ ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening. ­ plant sci., 163: 449­454. erkan m., kader a.a., 2011 ­ pomegranate (punica granatum l.) , pp. 287­313. ­ yahia e.m. 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environmental science, shimane university, 2059 kamihonjo, matsue, shimane 690‐1102, japan. 2 department of crop botany, faculty of agriculture, bangladesh agricultural university, mymensingh‐2202, bangladesh. 3 the united graduate school of agricultural sciences, tottori university, koyama‐cho, minami tottori, tottori 680‐8553, japan. 4 department of agriculture and food technology, faculty of bioenvironmental sciences, kyoto university of advanced science, 1‐1 nanjo otani, sogabe, kameoka, kyoto 621‐8555, japan. 5 laboratory of horticultural science, department of agricultural science and technology, faculty of agriculture, setsunan university, 45‐1, nagaotogecho, hirakata, osaka, 573‐0101, japan. key words: artificial lighting, controlled environment agriculture, non­renewed solution, root exudates, successive cultures. abstract: lettuce grown under recycled hydroponics ensures efficient water and nutrient utilization. however, lettuce yield is often reported to be declined from successive cultures for accumulating phytotoxic root exudates. degrading toxic exudates by titanium­based electrode and increasing photosynthetic efficiency by adding green light would improve lettuce yield. alternate current electro­degradation (ac­ed) was applied along with addition of green light in light spectrum to enhance lettuce yield. lettuce seedlings were grown in plant factory using half­strength of enshi solution. three consecutive cultures were performed under three combinations of leds [red (r):green (g): blue (b) viz. 235:00:59, 211:30:53 and 187:60:47 μmol m­² s­1] using renewed (rw), non­ renewed (nr) and ac­ed applied non­renewed (nr+ac­ed) nutrient solutions. results showed that in subsequent cultures, lettuce yield declined in nr solution under 187:60:47 of r:g:b. contrarily, nr+ac­ed solutions showed maximum lettuce growth and enhanced about 30% of yield under 30 μmol m­² s­ 1 of green light addition. however, addition of 60 μmol m­² s­1 of green light showed lower yield under all nutrient solutions. nutritional quality of lettuce was not varied by nutrient solutions and leds. our study recommends applying ac­ed for reutilizing fertigation water and addition of 30 μmol m­² s­1 green light for higher lettuce yield under successive cultivation. (*) corresponding author: toshiki.asao@setsunan.ac.jp citation: razzak m.a., asaduzzaman m., asao t., 2024 ­ titanium based electro‐degradation of nutrient solution and green light improve autotoxicity, growth and yield of lettuce grown in recycled hydroponics. ­ adv. hort. sci., 38(4): 393­405 orcid: rma: 0000­0002­7334­8337 am: 0000­0001­5516­4262 copyright: © 2024 razzak m.a., asaduzzaman m., asao t. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 22 may 2024 accepted for publication 1 august 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16177 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-7334-8337 http://orcid.org/0000-0001-5516-4262 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 393­405 394 1. introduction now­a­days commercial production of vegetables and fruits cultivated hydroponically are getting popularity compared to open field cultivation due to high temperatures, resurgences of insect pests and diseases as well as shortage of labor. in non­recycle system, culture solutions fully drain out to start next culture after harvesting of first culture. the damped nutrient solutions may cause environmental pollution and reduce water use efficiency from hydroponic cultivation systems. while in recycled hydroponics, nutrient solutions are reused repeatedly for several cultures by adjusting the strength of nutrient solution. recycled hydroponics increase the water and nutrient use efficiency and avoid the cost of addition and disposal of nutrient solution. in single pass hydroponic systems, in addition to the benefits of supply management, re­collection and recycling of irrigation water or fertigation water can result in significant savings. reuse of water is an excellent option for advanced controlled­environment agriculture (cea) systems (kozai et al., 2015). recycling of nutrient solutions used in cea would reduce the amount of nutrients that enter freshwater bodies like ponds, lakes, rivers etc. these benefits of recycled hydroponics are helpful for sustainable crop production by maximizing water and ferti l izer use efficiency as well as reducing environmental pollution. this in turn helps achieve the targets of sustainable developmental goals in respect of clean water and sanitation (sdg 6) as well as climate actions (sdg 13). however, in commercial cultivation, reuse of culture solutions negatively affects the yield of crop. under this situation, farmers fully drain out single used solutions before starting new cultivation thus, requiring more water and fertilizer, ultimately increasing production cost. it is well documented that, cultures without replacement of nutrient mixture accompanies the accumulation of phytochemicals secreted by plants possibly through autotoxicity phenomena (tang and young, 1982; asao et al., 1998; singh et al., 1999; asao et al., 2003; asao et al., 2004; kitazawa et al., 2005; asao et al., 2007; asaduzzaman et al., 2012), or allelopathy­like mechanism (nakahisa et al., 1994). under plant autotoxicity, plants release chemical compounds into their rhizosphere via a variety of mechanisms like leaching, volatilization, root exudation. asaduzzaman and asao (2012) reported some allelochemicals in different vegetables and ornamental plants like lactic acid, benzoic acid, succinic acid, adipic acid, hydroxybenzoic acid, vanillic acid etc. to address the issue of autotoxicity, researchers have used a variety of detoxification methods for allelochemicals, like adsorption by amberlite xad­4 (lee et al., 2006) and activated charcoal (kitazawa et al., 2005), addition of amino acids (mondal et al . , 2013) and auxin (kitazawa et al., 2007), and electro­degradation of allelochemicals (asaduzzaman et al., 2012; talukder et al., 2019 a). among all above mentioned methods, electro­degradation is the simplest and cost­effective method. the electro­degradation (ed) machine is a portable electric device that allows culture solution to pass through the titanium electrode, where electrochemical degradation occurs. on the other hand, one of the most important factors in crop production is light. supplying an adequate amount of artificial light is important for sustainable crop production through hydroponics in a vegetable factory. additionally, release of growth inhibitors, such as secondary metabolites associated with photosynthesis, may be influenced by light conditions (darko et al., 2014). generally, crops are cultivated using fluorescent light in cea. however, for higher light spectral emissions and higher amount of electricity usage of fluorescent light, now­a­days light emitting diodes (led) is getting popularity. a narrow wavelength range of high­quality light produced by led is suitable for plant growth and development (carvalho and folta, 2014). red and blue light is commonly used in plant factories considering their higher photosynthetic efficiency. led lights in red (660 nm) and blue (450 nm) are commonly used to grow a variety of crops including lettuce, spinach and radish (yanagi et al., 1996; hanyu and shoji, 2002). red and blue light, as well as their combinations, are the most effective in promoting plant growth and development and changing their architecture in the visible light spectrum (naznin et al., 2019). since the plants grown under combination of single­band blue and red light displays purplish­gray shade, it is difficult to monitor the health status of plants especially leaves with insect and disease infections. but when examined in a full spectrum of light environment by adding green light (550 nm) with red and blue, the color of the plant could be observed as green which largely improves the working conditions (razzak et al., 2022; kim et al., 2004). besides that, at low photosynthetic photon flux density (ppfd), green razzak et al. ‐ titanium based ac‐ed and green light enhance lettuce yield 395 light has a lower quantum yield than red and blue light due to its lower absorptance, but at high ppfd, red and blue light have a lower quantum yield than green light due to uniform dispersion of the of light in lower leaves and into the plant canopy (sun et al., 1998; evans and vogelmann, 2003; terashima et al., 2009). additionally, applying green light creates a full spectrum of white light which makes a congenial working environment in plant factory. we hypothesized that application of ac­ed and supplementation of green led would prevent the retardation of growth and enhance the yield of lettuce. the present study was executed to investigate the effects of ac­ed in non­renewed nutrient solution for successive lettuce cultures under recycled hydroponics and also to observe the influence of supplementation of green light to red and blue light in vegetable factories. 2. materials and methods planting materials and process of lettuce cultivation lettuce seeds (lactuca sativa cv. souther) were sown in vermiculite (1­5 mm size) ­filled cell trays and placed in a growth chamber under the following conditions: temperature: 25/20°c (day/night), relative humidity: 60%, fluorescent light intensity: 140­160 mol m­2 s­1, photoperiod: 16 hrs. light and 8 hrs. dark , and co2: 1000 ppm. in the first 7 days, only tap water was supplied in the tray and from 8th day 50% enshi nutrient solution (hori, 1966) was added. the chemical compositions of the enshi solution were presented in table 1. after 21 days, seedlings were planted in a vegetable factory in three steps in vertical growing beds (125 cm × 90 cm × 10.5 cm). each growing bed had 18 lettuce seedlings held in place by urethane cubes (23 mm× 23 mm × 27 mm). fifty percent of the enshi nutrient solution was pumped into three beds, each with a 50­l capacity and a 300­l saving tank. a timer (ks­1500, iuchi, osaka, japan) and an automatic pump (kp­101, koshin, kyoto, japan) were set to recycle the nutrient solutions at 55/5 min (recycled/stopped). except for light conditions, the plant factory maintained similar environmental conditions to the ambient room conditions. experimental treatments and data measurement during harvest lettuce seedlings were planted in three types of nutrient solutions under three led light conditions with and without addition of green light in light spectrum. the solutions were considered as renewed (r), non­renewed (nr), and nr+ac­ed (alternate current electro­degradation). there were three extents of green light supplementation to red and blue with total light intensity (µmol m­2 s­1) as r:g:b = 235:00:59 (g0), r:g:b = 211:30:53 (g30), and r:g:b = 187:60:47 (g60) (fig. 1 a). plants cultivated under each lighting conditions were separated from other plants by a silver sheet which spread from light sources up to the bottom of the growth beds to avoid mix up effects of led treatments. the ppfd of the led combinations were measured by mq­200 quantum sensor with par (photosynthetically active radiation) meter (apogee instruments, inc. logan, ut, usa) at five points above the plant canopy, 20 cm from the led panels. the spectral profiles of different led conditions employed in this experiment are presented in figure 1 (b, c, d). electro­degradation was applied continuously during the experiment in case of ac­ed treatment. the size electric current and voltage of the electric supply was set to 1.5. for lighting with leds, the pulse­wide modulation at 550 hz under 50% duty was employed. cultivation and harvest of lettuce plants were consecutively repeated for three cycles in the above­ mentioned culture solutions under leds. nine plants from each replication of each treatment were sampled for comparison. data on growth traits, and yield were measured at harvest. relative chlorophyll contents were measured using a spad meter (spad­502 plus, konica minolta, tokyo, japan). management of nutrient solution in renewed treatment, nutrient solution was replaced by freshly prepared 50% enshi nutrient table 1 ­ chemical compositions of “enshi” nutrient solution (half­strength) (hori, 1966) chemicals amounts (g/1000 l) ca (no3)2.4h2o 475 kno3 405 mgso4.7h2o 250 nh4h2po4 77.5 h3bo3 3 znso4.7h2o 0.22 mnso4.4h2o 2 cuso4.5h2o 0.05 na2moo4.2h2o 0.02 na fe­edta 25 adv. hort. sci., 2024 38(4): 393­405 396 solution maintaining the electrical conductivity (ec) of 1.42 dsm­1. on the other hand, nutrient solution was not renewed throughout the experiment from culture i to culture iii in case of non­renewed and non­ renewed + ac­ed treatments. all types of nutrient solutions were fresh during starting culture cycle i (ci), but solutions were once used and twice used in culture cycle ii (cii) and culture iii (ciii) except renewed treatment. however, almost similar ec level (1.42 ds m ­1) of the nutrient solutions were maintained by weekly­based supplementation of the nutrient stock solutions. the ec of the culture solution was measured (before and after nutrient adjustment) by ec meter (es­51, horiba, ltd., kyoto, japan) and ph values were checked by ph meter (d­12, horiba, ltd., kyoto, japan) at 7 days intervals and the range observed was 7.00­7.20. the ec values of nutrient solutions during weekly supplementation of nutrient stock solutions was presented as supplementary information in figure 1s. the ec and ph of used tap water was 0.22 ds m­1 and 8.0, respectively. components of electro‐degradation system and its working principle an ac­type electrode (yonago shinko co., ltd., tottori, japan) was used to degrade the accumulated autotoxic chemicals. the ac­ed electrode had a middle core made of titanium with a surface area of 53.1 cm2 (anode/cathode) surrounded by a cylindrical tube of 95.5 cm2 (cathode/anode) titanium. the solution was able to pass through the electrode where degradation occurred. the electrodes were connected with a digital ac power provider (ad­8735d, and, japan). in an electro­degradation machine, the culture solution can pass through the electrode where allelochemicals degradation take place. different components of ac­ed system were shown in figure 2. at the anode of the electrode, autotoxic compounds are oxidized by hydroxyl radicals (oh­) developed fig. 2 ­ schematic diagram of an electrode utilized in electro­ degradation. there are several different parts, including a pump (1), a plastic tube linking the pump and electrode (2), an anode (3), a cathode (4), a central ferrite core (5), a titanium pipe that is cylindrical (6), and a flow of nutri­ ent solution (7) (asaduzzaman et al., 2012). fig. 1 ­ lettuce cultivated under different combinations of red, green and blue led lights (a) using different nutrient solutions (picture from culture cycle i) along with the spectral distribution (relative intensity) of red (r), green (g) and blue (b) light (b, c, d, respectively) used in the experiment. the wavelength of emission peaks of red, green and blue light was observed at 660 nm, 520 nm and 445 nm, respectively. the par meter was used to quantify the photosynthetic photon flux density (mean ppfd, µmol m­2 s­1) of led combinations at five sites at the plant canopy, 20 cm from the led panels (n=4). http://oaj.fupress.net/index.php/ahs/article/view/16177/13161 razzak et al. ‐ titanium based ac‐ed and green light enhance lettuce yield 397 from water and combustion of adsorbed chemicals by forming carbon dioxide (co2) (fleszar and poszyńska 1985; comninellis and pulgarin 1991; feng and li 2003). mineral nutrient analysis in shoots and roots shoots and roots that had been separated upon harvest were dried at 80°c for 72 hours in oven (dkn812, yamato scientific co. ltd., japan), and ground into powder using a mixer (national mx­x53, japan). microwave­assisted digestion was performed for 0.25 g of ground materials suspended in 8 ml of 60% hno3 (ethos1, milestone s.r.l., bergamo, italy). following the digestion, the sample volume was adjusted to 50 ml by adding distilled water, and then the solution was filtered through a filter paper (advantec grade no. 131, 185 mm). the resultant fluid was subjected to analysis of element compositions with an atomic absorption spectrophotometer (z­ 2310, hitachi high technologies corporation, tokyo, japan). determination of ascorbic acid in lettuce leaves the ascorbic acid content in leaves was determined after freezing in a freezer (at ­30°c). stored samples were removed from the freezer and pressed to extract enough juice for analysis. the ascorbic acid content was determined using spectrophotometer colorimetry with 2,4­dinitrophenylhydrazine (dnp) at 520 nm wavelength (razzak et al., 2022). experimental design and statistical analysis three independent experiments were implemented following completely randomized design (crd) of three replications in the plant factory. the collected data were analyzed for two­way anova by the statcel 4 software (oms publication, tokorozawa, saitama, japan). for mean differences tukey­kramar test was done at p<0.05. 3. results yield performance of lettuce cultivated in different nutrient solutions under supplemented green light in three successive culture cycles (ci ‐ciii) first culture cycle (ci) in first culture cycle (ci), lettuce showed lower yield (shoot fresh weight­ sfw) (54.1 g plant­1) in nr solution when lettuce cultivated under g60 leds compared to the rw (70.6 and 69.5 g plant ­1, respectively) and ac­ed solution (66.9 and 67.4 g plant­1, respectively) under the g0 and g30 leds (fig. 3a). the reason for lowering yield performance might be due to the addition of higher amount of green light and also for autotoxicity in nr solution. shoot dry weights were almost similar in treatment combinations except nr solution which was lower under g60 compared to rw solution under g30 only. second culture cycle (cii) in second culture cycle (cii), it was noted that higher sfw of lettuce was found in rw (66.3 g plant­ 1) and once used ac­ed treated (65.7 g plant ­1) solutions under g30 leds which was similar under g0 (fig. 3b). the lower sfw were recorded in the once used nr solution under the led of g0 (50.7 g plant­1) and g60 (43.3 g plant­1). in addition, shoot dry weight was declined in rw and once used nr solution under fig. 3 ­ shoot fresh weight (sfw) and shoot dry weight (sdw) of lettuce cultivated using different nutrient solutions in three culture cycles [a(ci), b(cii) and c(ciii)] under sup­ plemented green light. nutrient solutions were fresh at starting, once used and twice used in ci, cii and ciii, respectively. ac­ed= alternate current electro­degrada­ tion. photosynthetic photon flux density from r (red), g (green), and b (blue) of led combinations (mean ppfd, µmol m­2 s­1). standard error of the mean (se) is shown as a bar (n = 9). according to the tukey­kramar test at p <0.05, different letters indicate significant differences between treatments. 398 adv. hort. sci., 2024 38(4): 393­405 g60 compared to ac­ed treated solution under g30. final culture cycle (ciii) in the final culture cycle (ciii), lettuce yield (sfw) was negatively affected in twice used nr solution and the lowest sfw was recorded when lettuce plants exposed to g60 leds (fig. 3c). on the other hand, the higher lettuce sfw was found in both rw and twice used nr+ac­ed solution under the led of g30 which was insignificant with the led of g0 using the same solutions. besides that, lower shoot dry weight was measured in rw, twice used nr and ac­ed treated solutions under g60 along with nr solutions under g0 and g30 leds. variation in leaf and root parameters of lettuce cultivated using different nutrient solutions under supplemented green light in three successive culture cycles (ci ‐ciii) leaf number and leaf relative chlorophyll content of lettuce plants showed no variation among the nutrient solutions and led treatments in cii and ciii however, in first culture cycle (ci), lettuce plants growing in the rw solution under the g30 and g60 leds exhibited fewer leaves compared to some of the other treatments (table 2). on the other hand, ac­ed treated nr solutions showed lower relative chlorophyll content under all led conditions and nr solutions under g30 and g60 (table 2). additionally, in subsequent cultures (cii and ciii), leaves of plants growing in the nr solution were smaller (length and width) when exposed to the g0 and g60 leds and rw solutions under g60 (table 2). lettuce plants developed longer roots under ac­ ed treated nr solutions under all leds whereas rw and nr showed shorter roots under g0 and g60 leds in ci. but in cii and ciii rw produced smaller roots under all leds (table 3). in case of root dry weight, nr solution given lower root mass under g60 in all culture cycles (ci, cii and ciii) (table 3). though lower root length was observed in rw solutions under all leds but root dry weight was higher. the reason might be producing higher root numbers with shorter length per plant. nutritional quality of lettuce shoots and roots cultivated using different nutrient solutions under supplemented green light in three successive culture cycles (ci ‐ciii) ascorbic acid (vitamin c) content in lettuce leaves did not showed variation among different nutrient solutions and leds in 3 subsequent lettuce cultures (ci­ciii) (table 3). among the mineral nutrients content in lettuce shoots, ca and na content varied in the final culture (ciii) and fe in cii as well as zn in ci whereas other nutrients did not showed variation among the nutrient solutions and leds in different culture cycles (table 4 a, b). compared to rw solution under g30, ca content in lettuce shoot was reduced in twice used nr and ac­ed treated solutions in ciii. the fe content in lettuce shoot was determined lower in once used ac­ed treated solution under g30 and once used nr solution under g0 and g30 leds in cii. moreover, zn content in lettuce shoot in ci was declined in ac­ed solution under g0; nr solution under g0 and g30 as well as rw solution under all led conditions. contrarily, in lettuce roots, ca content in the culture cycle ci was measured minimum in nr+ac­ed solution under g60 leds (table 5 a, b). however, in lettuce roots, lower k was recorded in rw solution under the g30, and g60 leds compared to nr solution under g60 leds. the zn content in lettuce root was estimated higher in nr solution compared to other treatment combinations when lettuce plant cultivated under g60 leds. in the second cycle (cii), the amount of k in lettuce roots was reduced when cultivated using rw solutions by exposing to g30 and g60 leds compared to once used nr and ac­ed treated solutions under g60 leds. additionally, na content in roots was decreased in rw and once used ac­ed treated solutions under g0 and g30 whereas lower fe was found in once used ac­ed treated solution under g30 and g60 leds. in the final culture cycle (ciii), higher amount of ca in root was determined in nr solution under g30. however, lower k content was observed in twice used nr under g0 and ac­ed treated solutions under g0 and g60 leds. comparatively lower zn content in roots was recorded in rw solutions under all leds. 4. discussion and conclusions in recycled hydroponic systems, crop cultivation by using same culture solutions several times is limited for developing some autotoxic chemicals which are released from plant roots. many researchers reported such type of autotoxicity phenomenon when crop cultivated hydroponically using unchanged culture solutions in hydroponic like in lettuce (lee et al., 2006), in strawberry (kitazawa ra zz ak e t a l. ‐ t ita ni um b as ed a c‐ ed a nd g re en li gh t e nh an ce le tt uc e yi el d 39 9 ta bl e 2 ­ le af c ha ra ct er ist ic s o f l et tu ce c ul tiv at ed in re ne w ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt so lu tio ns u nd er su pp le m en te d gr ee n lig ht i n th re e su cc es siv e cu ltu re c yc le s ( ci ­c iii ) n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z le af n um be r p la nt �1 m ax im um l ea f l en gt h (c m ) m ax im um l ea f w id th (c m ) re la tiv e ch lo ro ph yl l c on te nt (% ) ci w ci i ci ii ci ci i ci ii ci ci i ci ii ci ci i ci ii re ne w ed 23 5: 00 :5 9 13 .1 a ­c 10 .7 a 10 .7 a 20 .8 a b 22 .0 a b 18 .5 b c 18 .5 a b 17 .9 b c 15 .3 a ­c 34 .9 a 30 .5 a 35 .3 a 21 1: 30 :5 3 12 .7 b c 10 .8 a 10 .5 a 21 .6 a b 21 .0 a ­d 19 .2 a ­c 17 .9 a ­c 16 .6 c d 14 .7 a ­c 32 .9 a ­c 30 .2 a 33 .8 a 18 7: 60 :4 7 11 .9 c x 10 .9 a 10 .2 a 21 .7 a b 19 .9 c 18 .4 b c 18 .3 a b 16 .0 d 12 .2 d 32 .5 a ­c 33 .1 a 33 .4 a n on ­r en ew ed 23 5: 00 :5 9 13 .9 a b 10 .8 a 10 .7 a 19 .8 b 19 .5 d 17 .7 c 16 .4 b c 16 .9 b ­d 14 .1 b ­d 33 .6 a b 30 .5 a 32 .9 a 21 1: 30 :5 3 14 .3 a 10 .9 a 11 .2 a 21 .2 a b 20 .9 b ­d 18 .4 b c 17 .8 a ­c 16 .2 d 14 .8 a ­c 30 .2 b ­d 29 .1 a 32 .7 a 18 7: 60 :4 7 13 .2 a ­c 10 .7 a 10 .0 a 19 .7 b 17 .8 e 16 .9 c 15 .6 c 14 .1 e 13 .6 c d 29 .0 c d 33 .7 a 32 .0 a n on ­r en ew ed + ac ­e dy 23 5: 00 :5 9 13 .4 a b 10 .9 a 10 .7 a 22 .1 a 21 .0 a ­c 20 .9 a 19 .1 a 18 .4 a b 16 .8 a 28 .0 d 30 .3 a 31 .0 a 21 1: 30 :5 3 13 .7 a b 11 .3 a 10 .8 a 22 .6 a 22 .5 a 21 .3 a 20 .3 a 19 .7 a 15 .8 a b 29 .1 c d 30 .3 a 31 .5 a 18 7: 60 :4 7 14 .0 a b 10 .5 a 10 .6 a 22 .3 a 22 .2 a b 20 .5 a b 18 .0 a ­c 17 .1 b ­d 15 .3 a ­c 29 .8 b ­d 33 .7 a 32 .8 a si gn ifi ca nc e le ve l * n s n s * * * * * * * n s n s w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. ta bl e 3 ­ ro ot c ha ra ct er ist ic s a nd q ua lit y of le tt uc e cu lti va te d in re ne w ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt so lu tio ns u nd er su pp le m en te d gr ee n in th re e su c­ ce ss iv e cu ltu re c yc le s ( ci ­c iii ) w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z ro ot l en gt h (c m ) ro ot d ry w ei gh t ( g pl an t­1 ) as co rb ic a ci d (m g/ 10 0g f w ) ci w ci i ci ii ci ci i ci ii ci ci i ci ii re ne w ed 23 5: 00 :5 9 22 .0 b x 44 .9 b 36 .0 c 0. 17 a b 0. 20 a 0. 18 a 9. 67 a 8. 5 a 9. 3 a 21 1: 30 :5 3 23 .1 a b 44 .4 b 36 .2 c 0. 20 a 0. 17 a b 0. 16 a b 7. 16 a 9. 6 a 10 .1 a 18 7: 60 :4 7 22 .6 b 45 .0 b 36 .6 c 0. 16 a b 0. 12 b c 0. 14 a b 8. 38 a 8. 5 a 11 .3 a n on ­r en ew ed 23 5: 00 :5 9 21 .4 b 62 .6 a 55 .1 a b 0. 15 a b 0. 12 b c 0. 10 a b 9. 50 a 11 .6 a 11 .4 a 21 1: 30 :5 3 24 .3 a b 61 .3 a 56 .8 a b 0. 15 a b 0. 12 b c 0. 10 a b 7. 92 a 10 .6 a 11 .2 a 18 7: 60 :4 7 20 .1 b 54 .4 a b 50 .5 b 0. 12 b 0. 10 c 0. 08 b 8. 78 a 11 .0 a 11 .8 a n on ­r en ew ed + ac ­e d y 23 5: 00 :5 9 29 .8 a 50 .6 a b 61 .9 a 0. 16 a b 0. 15 a b 0. 18 a 7. 23 a 9. 5 a 9. 7 a 21 1: 30 :5 3 34 .7 a 58 .3 a b 53 .6 a b 0. 23 a 0. 16 a b 0. 14 a b 6. 10 a 9. 2 a 9. 9 a 18 7: 60 :4 7 26 .4 a b 49 .6 a b 59 .9 a 0. 16 a b 0. 13 b c 0. 12 a b 7. 59 a 8. 5 a 12 .7 a si gn ifi ca nc e le ve l * * * * * * n s n s n s ad v. h or t. sc i., 2 02 4 38 (4 ): 39 3­ 40 5 40 0 ta bl e 4 a ­ m ac ro nu tr ie nt s co nt en t in le tt uc e sh oo ts c ul tiv at ed in r en ew ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt s ol ut io ns u nd er s up pl em en te d gr ee n in t hr ee su cc es siv e cu ltu re c yc le s ( ci ­c iii ) w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z ca lc iu m (m g/ 10 0 gf w ) m ag ne siu m (m g/ 10 0 gf w ) po ta ss iu m ( m g/ 10 0 gf w ) ci w ci i ci ii ci w ci i ci ii ci w ci i ci ii re ne w ed 23 5: 00 :5 9 52 .7 a 70 .6 a 61 .3 a b 15 .6 a 15 .8 a 15 .9 a 40 5. 7 a 55 0. 7 a 46 2. 7 a 21 1: 30 :5 3 64 .7 a 62 .6 a 86 .5 a 20 .6 a 14 .8 a 18 .4 a 46 5. 3 a 42 5. 8 a 48 8. 5 a 18 7: 60 :4 7 73 .3 a 57 .4 a 67 .8 a b 22 .3 a 14 .2 a 16 .1 a 48 8. 7 a 50 7. 3 a 45 4. 6 a n on ­r en ew ed 23 5: 00 :5 9 57 .8 a 51 .6 a 48 .6 a b 18 .7 a 15 .5 a 14 .4 a 42 8 a 46 9. 5 a 39 0. 2 a 21 1: 30 :5 3 60 .1 a 57 .8 a 52 .5 a b 19 .3 a 16 .8 a 12 .0 a 41 8. 2 a 47 4. 7 a 34 3. 6 a 18 7: 60 :4 7 55 .4 a 64 .1 a 39 .6 b x 17 .0 a 17 .6 a 12 .3 a 37 8. 3 a 48 4. 1 a 34 5. 5 a n on ­r en ew ed + ac ­e dy 23 5: 00 :5 9 65 .5 a 62 .3 a 38 .0 b 19 .5 a 17 .2 a 12 .6 a 44 8. 2 a 44 1. 7 a 36 9. 0 a 21 1: 30 :5 3 85 .1 a 52 .0 a 62 .1 a b 26 .8 a 15 .0 a 13 .2 a 51 6. 5 a 42 8. 5 a 34 4. 5 a 18 7: 60 :4 7 49 .1 a 54 .7 a 64 .7 a b 14 .9 a 16 .4 a 14 .1 a 38 4. 2 a 48 4. 1 a 42 7. 4 a si gn ifi ca nc e le ve l n s n s * n s n s n s n s n s n s ta bl e 4 b ­ m ic ro nu tr ie nt s co nt en t in le tt uc e sh oo ts c ul tiv at ed in r en ew ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt s ol ut io ns u nd er s up pl em en te d gr ee n in t hr ee su cc es siv e cu ltu re c yc le s ( ci ­c iii ) w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z so di um (m g/ 10 0 g fw ) iro n (m g/ 10 0 g fw ) zi nc ( m g/ 10 0 g fw ) ci w ci i ci ii ci w ci i ci ii ci w ci i ci ii re ne w ed 23 5: 00 :5 9 7. 5 a 7. 9 a 6. 7 ab 0. 42 a 0. 50 a 0. 61 a 0. 14 e 0. 25 a 0. 15 a 21 1: 30 :5 3 7. 7 a 6. 1 a 7. 8 a 0. 41 a 0. 39 a b 0. 59 a 0. 15 d e 0. 17 a 0. 23 a 18 7: 60 :4 7 7. 4 a 7. 0 a 6. 4 ab 0. 42 a 0. 44 a b 0. 52 a 0. 19 c ­e 0. 20 a 0. 16 a n on ­r en ew ed 23 5: 00 :5 9 7. 1 a 7. 2 a 5. 9 ab 0. 50 a 0. 35 b 0. 37 a 0. 21 b ­d 0. 16 a 0. 22 a 21 1: 30 :5 3 8. 0 a 6. 6 a 5. 0 b 0. 46 a 0. 34 b 0. 33 a 0. 23 b c 0. 19 a 0. 18 a 18 7: 60 :4 7 6. 8 a 8. 5 a 5. 0 b 0. 49 a 0. 38 a b 0. 36 a 0. 24 a ­c 0. 26 a 0. 19 a n on ­r en ew ed + ac ­e dy 23 5: 00 :5 9 7. 8 a 6. 7 a 5. 1 b 0. 48 a 0. 39 a b 0. 65 a 0. 27 a b 0. 26 a 0. 20 a 21 1: 30 :5 3 8. 5 a 6. 3 a 5. 2 b 0. 63 a 0. 35 b 0. 39 a 0. 31 a 0. 23 a 0. 15 a 18 7: 60 :4 7 6. 7 a 6. 9 a 5. 8 ab 0. 39 a 0. 39 a b 0. 48 a 0. 19 c ­e 0. 23 a 0. 17 a si gn ifi ca nc e le ve l n s n s * n s * n s * n s n s ta bl e 5 a ­ m ac ro nu tr ie nt s st at us in le tt uc e ro ot s cu lti va te d in r en ew ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt s ol ut io ns u nd er s up pl em en te d gr ee n in t hr ee su cc es siv e cu ltu re c yc le s ( ci ­c iii ) w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. ta bl e 5 b ­ m ic ro nu tr ie nt s st at us in le tt uc e ro ot s cu lti va te d in r en ew ed , n on ­r en ew ed , a nd n on ­r en ew ed + a c­ ed a pp lie d nu tr ie nt s ol ut io ns u nd er s up pl em en te d gr ee n in t hr ee su cc es siv e cu ltu re c yc le s ( ci ­c iii ) w c i ( cu ltu re c yc le i) = fr es h nu tr ie nt so lu tio n; c ii (c ul tu re c yc le ii )= o nc e us ed n ut rie nt so lu tio n an d ci ii (c ul tu re c yc le ii i)= tw ic e us ed n ut rie nt so lu tio n. x a cc or di ng to th e tu ke y­ kr am ar te st a t p < 0. 05 , d iff er en t l et te rs in a c ol um n ha ve su bs ta nt ia lly d iff er en t, an d 'n s' de no te s n on ­s ig ni fic an t d iff er en ce s. y a c­ ed = a lte rn at e cu rr en t e le ct ro ­d eg ra da tio n. z m ea n ph ot os yn th et ic p ho to n flu x de ns ity (m ea n pp fd , μ m ol m ­² s­ 1 ) f ro m c om bi na tio ns o f r ed (r ), gr ee n (g ), an d bl ue (b ) l ed s. ra zz ak e t a l. ‐ t ita ni um b as ed a c‐ ed a nd g re en li gh t e nh an ce le tt uc e yi el d 40 1 n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z ca lc iu m (m g/ 10 0g fw ) m ag ne siu m ( m g/ 10 0g fw ) po ta ss iu m ( m g/ 10 0g fw ) ci w ci i ci ii ci w ci i ci ii ci w ci i ci ii re ne w ed 23 5: 00 :5 9 17 .1 a 5. 9 a 5. 4 b 3. 0 a 1. 6 a 1. 7 a 68 a ­c 92 .0 a ­c 88 .0 a 21 1: 30 :5 3 16 .6 a 8. 6 a 7. 8 ab 2. 0 a 1. 6 a 1. 4 a 58 .2 c 84 .9 c 88 .0 a 18 7: 60 :4 7 9. 9 bc x 8. 2 a 5. 3 b 3. 0 a 1. 8 a 1. 2 a 61 .3 b c 86 .5 c 86 .2 a n on ­r en ew ed 23 5: 00 :5 9 11 .9 a ­c 10 .6 a 11 .3 a b 2. 7 a 1. 7 a 1. 5 a 89 .1 a b 88 .8 b c 60 .8 b 21 1: 30 :5 3 8. 5 bc 10 .4 a 18 .6 a 2. 8 a 1. 6 a 3. 8 a 84 .0 a ­c 94 .5 a ­c 75 .8 a b 18 7: 60 :4 7 8. 7 bc 6. 8 a 6. 2 b 2. 5 a 1. 8 a 1. 7 a 94 .6 a 10 8. 8 ab 93 .8 a n on ­r en ew ed + ac ­e dy 23 5: 00 :5 9 13 .6 a b 4. 7 a 4. 6 b 3. 1 a 1. 7 a 1. 5 a 76 .5 a ­c 92 .0 a ­c 59 .2 b 21 1: 30 :5 3 7. 7 bc 5. 3 a 7. 5 b 3. 7 a 1. 9 a 2. 6 a 74 .3 a ­c 10 3. 9 a­ c 76 .0 a b 18 7: 60 :4 7 6. 9 c 5. 9 a 5. 5 b 3. 3 a 1. 6 a 1. 9 a 88 .6 a ­c 11 1. 9 a 64 .1 b si gn ifi ca nc e le ve l * n s * n s n s n s * * * n ut rie nt so lu tio ns a nd l ed s ( r: g :b )z so di um (m g/ 10 0g fw ) iro n (m g/ 10 0g fw ) zi nc ( m g/ 10 0g fw ) ci w ci i ci ii ci w ci i ci ii ci w ci i ci ii re ne w ed 23 5: 00 :5 9 2. 3 a 2. 0 d 3. 1 a 2. 8 a 2. 3 a­ d 1. 8 a 0. 15 b 0. 13 a 0. 07 b 21 1: 30 :5 3 2. 3 a 2. 8 b­ d 3. 5 a 3. 5 a 2. 6 a 1. 8 a 0. 17 b 0. 14 a 0. 08 b 18 7: 60 :4 7 2. 1 a 4. 4 a 2. 4 a 2. 9 a 2. 5 ab 1. 1 a 0. 14 b 0. 17 a 0. 06 b n on ­r en ew ed 23 5: 00 :5 9 2. 3 a 4. 2 ab 2. 8 a 2. 5 a 2. 5 a­ c 0. 9 a 0. 16 b 0. 15 a 0. 08 a b 21 1: 30 :5 3 2. 2 a 3. 2 a­ d 3. 3 a 2. 5 a 2. 5 a­ c 1. 6 a 0. 18 b 0. 17 a 0. 12 a 18 7: 60 :4 7 2. 3 a 3. 9 a­ c 3. 0 a 2. 4 a 2. 3 a­ d 1. 2 a 0. 27 a 0. 16 a 0. 09 a b n on ­r en ew ed + ac ­e dy 23 5: 00 :5 9 3. 0 a 2. 0 d 1. 8 a 3. 8 a 1. 9 b­ d 1. 1 a 0. 15 b 0. 15 a 0. 10 a b 21 1: 30 :5 3 2. 3 a 2. 5 cd 2. 6 a 3. 3 a 1. 8 d 1. 7 a 0. 11 b 0. 15 a 0. 10 a b 18 7: 60 :4 7 2. 5 a 2. 9 a­ d 2. 6 a 2. 5 a 1. 9 cd 2. 1 a 0. 12 b 0. 14 a 0. 08 a b si gn ifi ca nc e le ve l n s * n s n s * n s * n s * 402 adv. hort. sci., 2024 38(4): 393­405 et al., 2005), in cucumber (asao et al., 1998; yu and matsui, 1994), in many leafy vegetables (asao et al., 2004) as well as different ornamental plants (asao et al., 2007). we observed in our research that during the initial culture (culture i­where nutrient solutions were newly prepared at starting the lettuce culture) lettuce yield was not shown distinct variation among the nutrient solutions under different leds (fig. 3a) though lower leaf dimension and root dry weight were recorded in nr solution under g60 and fewer leaves was recorded in rw solution under the same leds (table 2 and table 3). however, nr+ac­ed solutions produced comparatively longer roots than nr and rw solutions under all leds (table 3). from second culture, growth (leaf length, width, root dry weight) and yield performance were demonstrated declining trend in nr solutions under g60 but opposite trend was observed in case of root length and that declining trend was more prominent in that solutions and leds in third culture (table 2, table 3 and fig. 3b). reduction in growth some features and yield in successive lettuce cultures under different leds might be attributed to the accumulation of higher allelochemicals in the nutrient solutions in later cultures (culture cycle ii and iii) in nr solution, as solutions were not changed or renewed throughout the experimental period. lee et al. (2006) reported that the intensity of allelochemicals increased due to repeated use of culture solutions. similar results of lowering yield performance in lettuce in closed hydroponic systems using non­ renewed solutions were pointed out by talukder et al. (2019 b). allelochemicals amount enhanced in frequently used nutrient solutions resulted in synergistic effects that inhibited development of plant (inderjit, 1996). several plant physiological processes such as photosynthesis, respiration, water and nutrient absorption, gene expression, phytochrome metabolism etc was influenced by the allelochemicals released from root exudates (inderjit and duke, 2003; blum and gerig, 2005). allelochemical can act on oxidative damage by enhancing the activities of reactive oxygen species scavenging enzymes and augmenting the membrane lipid peroxidation levels (baziramakenga et al., 1995; politycka 1996; yu et al., 2003; ye et al., 2004; lara­ nuñez et al., 2006; ye et al., 2006). besides that, autotoxic chemicals can modify genome­wide gene expression which trigger death of root cells (bais et al., 2003). we observed comparatively lower root dry matter in nr solutions (table 3). root is the first plant parts which suffer more in allelochemical stresses in hydroponic systems. roots damaged by autotoxic chemicals hamper nutrient and water absorption in plants. though root dry weight was lower in case of once and twice used nutrient solutions, root length was recorded higher in those solutions compared to renewed solution. the possible reason for increasing root length might be for absorbing nutrient elements by longer roots because in some roots nutrient uptake might be affected by allelochemicals accumulated in the nutrient solutions in later cultures. these effects are ultimately responsible for lowering in shoot fresh weight and other growth parameters. in the case of led conditions, the amount of red and blue light supplementation was greatly reduced under g60 leds considering leds of each solution, which may have hampered the photosynthetic rate. kim (kim et al., 2004) reported that plant growth was reduced by supplementing more than 75 μmol m­2 s­1 of green light. plant growth and development enhanced under red and blue light and their combinations in the artificial l ighting (naznin et al . , 2019). in our experiment higher performance of lettuce was observed under g30 which was similar with to g0 using rw and ac­ed treated nutrient solutions. this yield enhancement under g30 could be attributed to the involvement of green light in increasing total photosynthetic efficiency of the plant by maximizing light utilization in both upper and lower leaves. green light penetrates the leaf layers more deeply than red or blue light, scattering into cellular parts of the leaf and increasing the photosynthetic efficiency of lower chloroplasts (terashima et al., 2009; brodersen and vogelmann, 2010). moreover, by incorporating green light in the light spectrum, light environment turned into white which was congenial for workers to implement the necessary management practices of lettuce plants. for overcoming the inhibitory effect of allelochemicals in recycled hydroponics it is necessary to degrade or detoxify the accumulated chemicals in the nutrient solutions. for degrading chemicals from the solutions electro­degradation mechanism can be utilized where in the anode of electrode autotoxic chemicals can degrade into carbon dioxide (co2) (fleszar and poszyńska, 1985). electro­degradation technique was successfully uti l ized in strawberry (asao et al . , 2008; asaduzzaman et al., 2012; talukder et al., 2019 a) and lettuce (talukder et al., 2019 b). in subsequent 403 razzak et al. ‐ titanium based ac‐ed and green light enhance lettuce yield performance in all types of nutrient solutions specifically once or twice used nr solutions. therefore, ac­ed would be applied along with 30 μmol m ­² s ­1 of green light supplementation for improving lettuce growth and yield cultivated in recycled hydroponics. acknowledgements we would like to thank japan aerospace exploration agency (jaxa) and faculty of life and environmental sciences, shimane university for fig. 5 ­ amount of macronutrient (a) and micronutrients (b) and (c) used by lettuce plants grown under different types of nutrient solutions throughout the experiment (culture cycle ci ­ ciii). fig. 4 ­ water utilization rates (l/plant) by lettuce plants grown under different types of nutrient solutions throughout the experiment (culture cycle, ci ­ ciii). lettuce cultures (culture cycle ii, iii), we recorded higher growth and yield performance in ac­ed applied solution once or twice used culture solutions under g30 (table 2, table 3, fig. 3b, c). the lettuce performance in this treatment was similar to renewed treatment under the same light condition. this yield recovery phenomenon might be due to the degrading the allelochemicals accumulated in the solutions by the electrode of ac­ed as well as enhancing photosynthetic efficiency under g30 for maximum use of energy from light by both lower and upper leaves. additionally, for producing lettuce in hydroponics, water and fertilizers requirement was lower in non­ renewed solutions compared weekly renewal systems (fig. 4, 5). however, hydroponic farmers’ need to renew nutrient solution due to accumulation of autotoxic chemicals exudates from plant roots. if farmers can continuously use the nutrient solutions through overcoming autotoxicity from the hydroponic systems by using ac­ed, it would be possible to minimize the water and fertilizer requirements in hydroponics cultivation which ultimately reduce environmental risk that may occur through the release of nutrient solutions into environment. lettuce growth and yield performance declined in non­renewed solution cultivated under g60. decreasing trend of yield reduction was more prominent in subsequent cultures (cii and ciii) using same nutrient solutions. ac­ed enhanced about 30 % of lettuce yield and successfully recovered the retarded lettuce growth under g30 of leds. there were no substantial differences in lettuce sfw between g0 and g30 in rw and ac­ed treated nutrient solutions. however, addition 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2019 b ­ application of alternating current electro‐ degradation improves retarded growth and quality in impaginato 357 adv. hort. sci., 2020 34(4): 357­372 doi: 10.13128/ahsc­9425 management of root knot nematodes (meloidogyne sp.) and enhancing growth yield of greenhouse produced tomatoes by using fresh plant derived soil amendments p.c. otieno (*), r.m.s. mulwa, j. otieno ogweno egerton university, department of crops, horticulture and soils, p.o. box 536, 20115 egerton, kenya. key words: lippia kituensis, meloidogyne sp., ocimum gratissimum, organic amendments, tomato yield. abstract: production of greenhouse tomato is hampered by myriad of chal­ lenges emanating from growth medium in the sub­saharan africa (ssa), which has led to instability in the production trend. a greenhouse experiment was conducted to study the effect of soil amendment with fresh plant biomass from lippia kituensis vatke and ocimum gratissimum l. aimed at managing root knot nematodes (rkn) and enhancing tomatoes yield. the amendments were applied at 0 (soil negative control), lippia and ocimum, each at 200 g, 400 g % and 800 g in 10 kg potted soil mixes, singly and in all possible combinations. azadirachtin (0.3 w/w) was also used as a positive control. the mixtures were treated inoculums carrying 1000 second instar meloidogyne sp. juveniles. an unbalanced factorial in a randomized complete block design with 3 replica­ tions was used. the parameters measured were nematode populations, root gall numbers, galling index, tomato growth, development and yield. results indicated that interactive effect of soil amendment at 800 g of both lippia and ocimum, significantly (p<0.05) reduced the rkn population by 82.1% compared to the non­amended soil. at same rates, galls were reduced by 95.5% while galling index by 83.3%, compared to non­amended treatment. in plant develop­ ment same amendment rates demonstrated higher vegetative growth. for fruit number and marketable yield, 76.7% and 82.2% more fruits per plant were recorded from 800 g lk+ og at 800 g and azadirachtin respectively, compared to non­amended soil. based on the results, lippia and ocimum may be poten­ tial sources for nematicidal plant products for greenhouse tomato production. 1. introduction tomato is among the leading greenhouse vegetable crops grown in kenya in both soil and soilless media (hcd, 2012). tomato constitutes 7% of the total horticultural produce in kenya and 14% of all the entire veg­ etable produce (ochilo et al., 2019). in terms of production in the year (*) corresponding author: pcotieno@egerton.ac.ke citation: otieno p.c., mulwa r.m.s., otieno ogweno j., 2020 ­ management of root knot nematodes (meloidogyne sp.) and enhancing growth yield of greenhouse produced tomatoes by using fresh plant derived soil amendments. ­ adv. hort. sci., 34(4): 357­372 copyright: © 2020 otieno p.c., mulwa r.m.s., otieno ogweno j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 july 2020 accepted for publication 28 september 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(4): 357­372 358 2018, kenya was rated 7th amongst the leading coun­ tries in sub­saharan africa with 599,458 tonnes per year, though the trend has not been stable (fao­ stat, 2018). to stabilize the trend of production, greenhouse technology is seen as one of the solu­ tions in tomato production, due to high levels of effi­ ciency and the potential to support sustainable socio­ economic development. however, greenhouse toma­ to production has not been vibrant in kenya as it should be in the tropical humid region (sanzua et al., 2018). this is because under soil­based production, there is nutrient depletion through plant uptake and leaching beyond the root­zone of vegetable crops (kirimi et al., 2011), resulting in reduced yield in the following season. besides, the continuous growing of tomato in the greenhouse soil leads to accumulation of soil borne pests and pathogens especially bacterial wilt ralstonia solanacearum, fusarium wilt, and nematodes (meloidogyne sp.), which has forced most farmers to abandon greenhouse tomato pro­ duction (hcd, 2017). studies have shown that soil based media with appropriate amendments combined with other addi­ tives may effectively manage soil­born pests and dis­ eases (mcsorley, 2011). among the pests root­knot nematodes (rkn) meloidogyne spp are the most damaging nematodes in tomatoes grown in the trop­ ics (walker, 2007). infection of roots with meloidogyne initiates a series of events that changes the entire physiology of the host plant. root galls result when nematodes penetrate the cells of the cortex and pericycle the endodermis and reach the stale. about 5­7 cells surrounding the nematode’s head enlarged to become a specialized giant cells, much larger than others. the thick nuclei enlarge, become polyploidy and undergo series of synchro­ nized division, (mai and mullin, 1996). root symp­ toms may appear as root knots (root galls), root lesions, excessive root branching (ogallo et al., 1997) as root tips are injured and roots rot when nematode is accompanied by plant pathogenic or saprophytic bacteria of fungi (cerkaukas, 2004). these nema­ todes are prevalent in the greenhouse condition and invade almost all vegetable crops resulting in sub­ stantial yield losses (stirling and stirling, 2003). bekal and becker (2000) observed that at peak, 100 cm3 of soil contained 1000 nematodes. this declines with weather variation to approximately 50 nematodes per 100 cm3 of soil. according to stirling and kopittke (2000), the economic threshold of rkn on most crops range between 2 and 10. on yield various authors have reported on reduction in tomato due to rkn meloidogyne spp ranging from 28% to 68% (safiuddin shahab et al., 2012). under soil based greenhouse tomato production, nematode infestations are a serious constraint leading to a yield reduction (pakeerathan et al., 2009). suppressed plant growth and yield has been observed in nematode infested fields (vovlas et al., 2005). many crops grown as vegetables are sus­ ceptible to m. incognita and m. javanica particularly tomato, aubergine, okra, cucumber, melon, carrot, gourds, lettuce and peppers (varela et al., 2003). it has been suggested by mcsorley (2011) that reduction of nematodes in fields treated with plant biomass wastes results in improved soil structure, fertility and improvement of plant resistance from nematode toxins. similarly, it may increase fungal and bacterial parasites population in the soil or other nematode antagonistic agents. apart from nematode control, these plant based biomass also provide essential nutrients (such as n and p), which help to rebuild soil organic matter contents, and aid in the re­establishment of beneficial microbial populations (suresh et al., 2004; allen et al., 2007; dauda et al., 2008). additionally, higher organic matter content increases soil water holding capacity and supports thriving communities of decomposers and predators in the soil system. the nematicidal properties of plants may be a contribution from extracted biomol­ ecules resident in the plant bodies. the major classes of compounds with proven nematicidal activity include alkaloids, fatty acids, glucosinolates, isothio­ cyanates, phenols, diterpenes and a variety of essen­ tial oils (chitwood, 2002). neem cake, known to be rich in azaderachtin is also associated with strong nematicidal activity (abbasi et al., 2005). laboratory extracted essential oils have also been reported to affect development of nematode eggs and second juvenile stage (j2) under in vitro conditions (onifade, 2007). other extracts with strong pesticidal proper­ ties include rotenone, nicotine and pyrethrins (berger, 1994). in verbenaceae, lippia is among the genus of aro­ matic plants due to their essential oils (kosgei et al., 2014). they are shrubs or woody herbs, leaves oppo­ site or verticilate, glandular. flower is pedunculate, crowded spikes; corolla obscurely 2­ lipped, fruits are 2 dry mericarps each with very small seed. various species in kenya include; l. dauensis (chiov.) chiov, l. grandifolia a. rich, l. javanica (birm. f.) spreng, l. kituensis vatke, l. somalensis vatke (beentje, 1994). otieno et al. ‐ management of meloidogyne in tomato by soil amendments 359 the species l. kituensis vatke has opposite leaves, ovate or elliptic; flowers white with yellow throat. in a previous study by (kosgei et al., 2014), phytochemi­ cal analysis from methanol extracts of lippia kituen‐ sis vatke were done and reported to possess monoterpenes, sesquiterpenes, diterpenes and other essential oil which were found to be effective against larvae of rhipicephalus appendiculatus. some of the these essential oils included alpha­pinene (­)­, camphene, sabinene, beta­myrcene, l­phellandrene, dl­limonene, gamma­terpinene, trans­sabinene hydrate, alpha­terpinolene, neo­allo­ocimene, camphor (1s 4s)­(­)­, camphore, borneol (=endo­bor­ neol), 4­methyl­1­(1­methylethyl)­ 3­cyclohexen­1­ol, 4­terpineol. in the same extract, sesquiterpenes yielded were; beta­bourbonene, isopropyl­5­methyl­ 9­methylene­ bicyclo[4.4.0]dec­1­ene, germacrene d, gamma­cadinene, 2­isopropyl­5­methyl­9­methyl­ ene­ bicyclo[4.4.0]dec­1­ ene. duschatzky et al. (2004) reported that nematicidal activity of the essential oils isolated from lippia juneliana and l. turbinata, which were evaluated using in vitro experiments. in another study, the oils of l. juneliana and l. turbinata showed the highest nematicidal activity among the tested oils, killing more than 80% of the juveniles of the meloidogyne sp. analysis of the oils revealed that l. juneliana contain; piperitenone oxide (36.5%), limonene (23.1%), camphor (8%) and spathulenol (6.5%) and l. turbinata has l imonene (43.3­60.6%) and piperitenone oxide (39.3­ 17.8%). the genus ocimum l. (lamiaceae) comprises 30­ 160 annual and perennial herbs and shrubs, collec­ tively called basil. species of this genus are popular sources of essential oils and aromatic compounds, of condiments, and ornamental plants (nagai et al., 2011). the most cultivated species worldwide are o. africanum lour. o. americanum l., o. basilicum l., o. gratissimum l., o. minimum l. and o. tenuiflorum l., mainly due to their economic and medical impor­ tance (carović­stank et al., 2010). essential oils in ocimum incude linalool, eugenol, methyleugenol, trans­α­bergamotene, p­cresol, 2, 6­di­tert­butyl, δ­ cadinene and (z, e)­α­farnesene (nagai et al., 2011). sifola and barbieri (2006) reported that o. basilicum essential oil is constituted of phenylpropanoids, like eugenol, chavicol and its derivatives, and terpenoids; limonene, linalool and methyl cinnamate. masi et al. (2006), studying nine different cultivars of o. basilicum commonly utilized, were able to identify five distinct chemotypes based on the main essential oil constituent; iso­pinocamphone (35.1%) and car­ vone (39.7%) which were the predominant compo­ nents of the essential oil in cultivated o. basilicum. this was also confirmed in another study by almeida et al. (2010). several species of ocimum have been reported to yield oils of diverse nature (matasyoh et al., 2007; ogendo et al., 2008). other studies have also shown that the leaf extract of ocimum gratissimum contain potential bioactive components of essential oils. these are made up of eugenol, citrol linalol, charvi­ col, thymol, gerianol, triterpenoids saponins and alkaloids (atuboyedia et al., 2010). it was also report­ ed by echeverrigaray et al. (2010) that monoter­ penoids significantly reduce the hatching of eggs and mobility of j2 of meloidoyne spp. onifade (2007), in the study to find the effect of essential oils from five ocimum sp. on the pathogenicity of pratylenchus brachyurus (godfrey) in tomato, reported that in vitro at 25­100 µg ml­1, the oils of o. gratissimum and o. basillicum completely inhibited egg hatching and larval survival of nematodes after 24 hours. more study is still needed to explore the potential of essen­ tials and other compunds involved in the nematode control in the two plant families verbenaceae and lamiaceae. the current work was therfore focused on investigating the potency of lippia kituensis vatke and ocimum gratissimum l. on the management of root knot nematodes meloidogyne spp. in this study we report results of a greenhouse pot experiment to evaluate management of root knot nematodes using fresh plant derived soil amendments with lippia kituensis vatke. and ocimum gratissimum l. and their effects on tomato yield. 2. materials and methods experimental site the study was conducted in two growing seasons at the horticulture research and teaching field, egerton university, kenya between july 2015 and may 2016. the site received a mean rainfall of 1012 mm with a mean day temperature of 22°c and night ranges of 5­10°c (jaetzold et al., 2012). the pot experiment was conducted in a polytunnel greenhouse measuring 8 m wide × 60 m length and 3 m height, covered with uv stabilized polythene sheet gauge 200 μm. plant materials leafy twigs of l. kituensis vatke. and o. gratissi‐ mum l. were collected in the wild around egerton university at flowering stage, when essential oil was adv. hort. sci., 2020 34(4): 357­372 360 at its peak in the plants in the month of july 2015 (fig. 1). these two shrubs flower all the year around visited by bees hovering over the flowers to collect nectar from scent produced by the plants. the mate­ rials were chopped into aggregates approx. 0.5 cm; the aggregates were incorporated in various propor­ tions (0 ­negative control, 200 g, 400 g and 800 g) in 10 kg of potted solarized forest soil, singly and in all possible combinations. crop establishment the planting material used in the study was toma­ to seedlings ‘rio grande’ rio grande is a determinate tomato cultivar with a high yielding potential thus preferred by many farmers. this vigorous variety is well adapted to extreme temperatures. due to the potential of heavy crops and to keep the fruit clean and easy to pick, it is recommended to support plants with stakes or cages. seeds used to raise the tomato seedlings were obtained from simlaws seed company in nakuru (kenya) and established in a nursery for 5 weeks before transplanting. five weeks old tomato seedling were transferred to the sub­ strates in the pots in the greenhouse to develop (fig. 2). when plants reached a height of 30 cm, they were supported using sisal twines tied on a binding wire trellis at a height of 150 cm above the bed. calcium ammonium nitrate (can, 26% n) was applied at the rate of 10 g per pot 21 days after transplanting (dat) to maintain growth. plants were pruned to maintain two stems per plant and water­ ing was done continuously during the growing period with rates being adjusted according to plant growth phases. in the first 30 dat, 2 l of water was applied per plant per day and thereafter, the rate was increased to 4 l to 42 dat per day. from 43 dat water supply was increased to 5 l per day as the plants developed, according to the work by pires et al. (2011). nematode augmentation, extraction and inoculum preparation nematodes were collected from a field previously grown with infested tomatoes and augmented on two weeks old potted tomato seedlings established in a greenhouse following the method of siddiqui and akhtar (2007). specifically, galls were extracted from the roots of infested tomatoes, chopped and mixed with the native soil. the mixture was added to pots planted with 2 week old tomato seedlings and the inoculum allowed to infest and multiply for 8 weeks. after augmentation period, nematode egg masses were extracted from the heavily galled tomato roots by chopping the roots to lengths of 0.5 cm and mac­ erating the tissues to release egg masses. these were placed in 15 cm diameter sieves of 1 mm pore size, lined with cross­layered tissue papers and incubated at 27°c to hatching in glass petri­dishes containing distilled water. after hatching, the second instar juve­ niles (j2) were transferred into 2 l conical flasks. quantification of juveniles was done under a stereo­ scope with gridded petri dishes. ten 1 ml replicate samples were drawn from the well mixed suspen­ sions to establish the average number of juveniles per ml. the determined quantity was 20 juveniles per ml. finally, the nematode inoculum suspension sam­ ples were adjusted to contain approx. 1000 juveniles in 50 ml of distilled water. nematode inoculation and determination of infesta‐ tion parameters the 50 ml j2s inoculum suspensions were added to pots containing the various plant biomass amend­ ed media planted with 28 day old tomato trans­ plants. the inoculum was allowed to develop under normal tomato culture conditions in a polytunnel greenhouse. destructive sampling of tomato plants to determine nematode infestation was conducted 100 dat. four plants from each replicate were sam­ fig. 1 ­ a. lippia kituensis vatke and b. ocimum gratissimum l.; c. chopping of the fresh ocimum gratissimum plants into smaller pieces; d. potting of the mixture (soil amend­ ment process). fig. 2 ­ transplants on treatment pots in the greenhouse. otieno et al. ‐ management of meloidogyne in tomato by soil amendments 361 pled at the peak of flowering. determination of macro nutrient from the tomato leaves nitrogen (kjeldahl) and phosphorus. tomato leaves tissue were analyzed at 49 dat for macro ele­ ments n, p, k, mg and ca. from the top of the plant, leaves were taken from the third and fourth leaflet per treatment. the sample materials were chopped into aggregates approx. 0.5 cm and oven dried at 70°c until constant weight. the oven dry plant sam­ ples were ground and wet digested by a sulfuric ­ percloric acid mixture as described by cottenie et al. (1982). nitrogen and phosphorus contents of vegeta­ tive samples were measured in the digesting extract according to the methods of aoac (2012). calcium and potassium content was determined in vegetative sample by ashing dry sample as described by chapman and pratt (1978) extract method. analysis of potassium, calcium and magnesium. a substrate sample weighing 0.3 g was digested in digestion tubes using a digestion mixture comprising of hcl, hno3, hf and h3 bo3. the temperatures in the block was maintained at 360°c for two hours then samples cooled and transferred to 50 ml volumetric flasks and volume made to the mark. calibration was done for each element using certified standards. samples were analyzed using atomic absorption spectrophotometer (aas), varian spectra aa10 aas machine. the determination of these elements in the substrate was done using double acid method of table 1 ­ treatment combinations and description of soil amendment rates used in nematode management og= ocimum gratissimum l. (g/10 kg soil); lk = lippia kituensis vatke (g/10 kg soil), are soil organic amendments; 0 = no amendment; azad = positive control of commercially know pesticide azadirachtin. extraction. aas was used for estimation of these available elements in the tested substrate. this fol­ lowed the procedure of okalebo et al. (2002). experimental design and treatment layout the experimental design used was a factorial embedded in a randomized complete block design and in total there were 17 treatments. there were three blocks and pots arranged in rows spaced at 0.6 m between and 0.4 m within the rows. there were 4 levels (0 g, 200 g, 400 g, and 800 g of organic amend­ ments from each of the plant species, replicated 3 times. a negative control of solarized non­amended soil and a positive control of 0.3% w/w azadirachtin, a farmer’s standard commercially known organic based neem extract were included. in total there were 17 treatment plots of 6 potted tomato plants in each block (table 1). nematodes evaluation nematodes population. to determine the nema­ tode population in the biomass amended pot soil treatments, second stage juveniles (j2) were extract­ ed from 100 cm3 composite sample of soil from each replicate, using the method described by kimenju et al. (2010). specifically, at 100 dat, the soils from each of the four pots were sampled by taking 100 cm3 of sample. the samples were placed in 9 cm diameter sieves with pore diameters of 1 mm lined with double layered tissue paper. the sieves were half immersed in metallic troughs containing 250 ml soil amendments treatments (g/pot) description of treatments 0 0% w/w lippia kituensis and 0% w/w ocimum gratissimum (control) 200 og 2% w/w ocimum gratissimum 400 og 4% w/w ocimum gratissimum 800 og 8% w/w ocimum gratissimum 400 lk 4% w/w lippia kituensis 200 lk +200 og 2% w/w lippia kituensis and 2% w/w ocimum gratissimum 200 lk +400 og 2% w/w lippia kituensis and 4% w/w ocimum gratissimum 200 lk +800g 2% w/w lippia kituensis and 8% w/w ocimum gratissimum 400 lk 4% w/w lippia kituensis 400 lk+ 200 og 4% w/w lippia kituensis and 2% w/w ocimum gratissimum 400 lk+ 400 og 4% w/w lippia kituensis and 4% w/w ocimum gratissimum 400 lk+ 800 og 4% w/w lippia kituensis and 8% w/w ocimum gratissimum 800 lk 8% w/w lippia kituensis 800 lk +200 og 8% w/w lippia kituensis and 2% w/w ocimum gratissimum 800 lk +400 og 8% w/w lippia kituensis and 4% w/w ocimum gratissimum 800 lk +4 og 8% w/w lippia kituensis and 8% w/w ocimum gratissimum azad commercial (0.3% azadirachtin) control 362 adv. hort. sci., 2020 34(4): 357­372 of distilled water to allow nematode migration into the water underneath for 24 hours. nematode counts were determined in 10 replicate samples of 1 ml for each soil sample as previously described. gall number and galling index. for gall assess­ ments, plants were gently uprooted and their roots thoroughly washed under tap water to remove all the adhering soil. galling was determined by counts of galls size 1 mm diameter and above by a light microscope using the lowest objective lens x 4. galled roots were spread in on plastic petri dish made of 1 cm2 grids, numbered at the base. the galls were scored from each square to get the summation per plant. the galling index was scored on a scale of 1­10, where 0= no gall, 1= 1­50 galls, 2= 51­100 galls, 3= 101­150 galls, 4= 151­200 galls, 5= 201­250 galls, 6= 251­300 galls, 7= 301­350 galls, 8= 351­400, 9= 401­450 and 10= 451 and above (kimenju et al., 2010). the scores were converted into numerical entries and their means worked out for analysis of variance. tomato growth evaluation number of leaves. leaf count data was collected from 4 plants in each plot. leaf count data collection was commenced 21 dat and continued at intervals of 14 days up to 91 dat. at each instance of data col­ lection the mean number of leaves per plant from each replicate was computed. the mean number of leaves per treatment was determined by computing the means. plant height. plant height data was collected from 4 plants in each plot. this started at 21 dat and con­ tinued at 14 days interval up to 77 dat. at each instance of data collection the mean height per plant from each replicate was computed. the mean height per treatment was determined by computing the means. root volume. root volume data was determined by carefully removing the plants from the pot, shak­ ing off the soil, and washed in running water on the trough at 100 dat. root volume was determined water displacement method in a one liter plastic measuring cylinder. the cylinder was filled to 500 ml mark, then the roots dipped carefully until the water just covered all the roots on the 4 plants used for root length determination and means computed to get the means. shoot and root dry weights. from the 4 plants used for root length and volume determination, shoots were separated from the roots at the collar. these were individually placed in kaki paper bags and dried in an oven at 70°c to constant weights. both parts were weighed separately and means of the weights computed as above. physiological parameter stomatal conductance and chlorophyll content. leaf stomatal conductance (mmol·m­2·s­1) was mea­ sured on four tomato tagged plants from each treat­ ment. using a steady state leaf porometer (sc­1, decagon devices, pullman, wa), stomatal conduc­ tance was measured on a 2 weeks interval from 21 days after transplanting (dat). since tomato plants are hypostomatous, stomatal conductance was mea­ sured only on the abaxial leaf surface on 3 leaves on the upper parts of the plant and the average was computed. leaf chlorophyll content was taken from the same leaves used for stomatal conductance. the instrument used was a chlorophyll content meter (ccm­200 plus, opti­sciences, tyngsboro, ma) and measurement in chlorophyll concentration index units (ccis), as an estimate of chlorophyll content on leaves. yield number of fruits per plant. weekly piece meal harvesting of pink stage tomato fruits from the 4 tagged plants in each treatment was done. at each harvest, the number and weight of fruits were recorded for each treatment. marketable and non‐marketable fruit yield. physiologically mature fruits (at pink stage) were har­ vested from the 4 tagged plants in each treatment. harvesting was piece meal on weekly basis. at each harvest, fruits were sorted into marketable and non­ marketable (kg/plant) separately and their weight determined and recorded. fruit weight was taken from mature marketable fruits (at pink stage), har­ vested from the 4 tagged tomato plants from each treatment. these were weighed using a spring bal­ ance (atz, shangai precision and scientific instrument co., shangai, china) at each harvest and later summed up to give the total marketable weight (kg/plant). data analysis data for the two trials were pooled since there was no statistical difference between them. it was then subjected to analysis of variance (anova) and means separated by the tukey’s hsd using the sas statistical package version 9. the model fitted for this experiment was; yijk= μ + βi + αj + γk + αγjk + εijk where yijkl = tomato response, μ = overall mean, βi = effect of the ith block, αj = effect of the jth level of otieno et al. ‐ management of meloidogyne in tomato by soil amendments 363 lippia kituensis vatke ,γk = effect of the kth level of ocimum gratissimum l., αγjk = interaction effect of the jth level of lippia kituensis vatke and kth level of ocimum gratissimum l. εijk = random error compo­ nent term which are normally and independently dis­ tributed about zero means with a common variance σ2. 3. results effect of soil organic amendment on nematode popu‐ lation, gall numbers and galling index the different levels of amendments with lippia kituensis vatke and ocimum gratissimum l. biomass and their combinations significantly (p<0.05) influ­ enced nematode populations in the treatments (table 2). various rates of treatment reduced the juvenile populations when compared with the con­ trol. in single treatments of lippia at 200 g, 400 g and 800 g, nematode numbers was reduced to 38.88 (25.9%), 19.29 (63.3%), and 20.58 (60.8%) respective­ ly, compared to the non­amended treatments. ocimum had similar trend with reduction to 47.38 (9.75%), 34.58 (34.1%) and 30.63 (41.7%), respective­ ly (table 2). however, the interactive effect of the two plant biomass from 400 g and above produced better nematode reduction than single treatments alone compared to soil. gall numbers and galling index were determined 100 dat and were significantly p<0.05) influenced by organic amendments (table 2). there was a general decrease of gall numbers in roots of tomato plants with increased levels of lippia and ocimum biomass in the potting soil. application of lippia singly at rates of 200 g, 400 g, and 800 g per pot reduced gall num­ bers to 190 (66.9%), 155 (73.0%) and 125.60 (78.1%) respectively compared to soil with ocimum following the same trend with reduced galls numbers to 470.67 (18.1%), 422.5 (26.5%) and 175.3 (69.5%) respective­ ly. interactive effect of the two plant biomass above at the rates of 200 g was better reduction of gall numbers up to 25.17 galls (95.5%) and no significant differences (p<0.05) were evident among the various combinations. the efficacy of the fresh plant biomass materials in managing nematode proliferation on tomato roots was also evident in the galling index scores from the various treatments. in general the gall index showed a reducing trend with increasing levels of lippia and ocimum biomass amendments. the interactive effect of the two plant species both at 800 g produced tomatoes with vigorous, fibrous root system with very few galls only observed under light microscope. table 2 ­ effect of fresh organic amendments on nematode population, gall number and galling index og = ocimum gratissimum l.; lk = lippia kituensis vatke; 0 = no amendment (soil). * means followed by the same letter series within a column are not significantly different according to tukey’s honestly significant diffe­ rence (thsd) at p≤0.05. amendments g/10 kg) no. of nematodes (per 100 cm3 soil) no. of galls/plant galling index 0 52.50 a * 574.67 a 10.00 a 200 og 47.38 b 470.67 b 10.00 a 400 og 34.58 d 422.50 c 9.00 a 800 og 30.63 ef 175.30 de 5.00 c 200 lk 38.88 c 190.00 de 7.30 b 400 lk 19.29 g 155.00 defgh 5.70 c 800 lk 20.58 g 125.60 defgh 3.00 de 200 lk + 200 og 38.21 c 211.00 cd 4.67 c 200 lk + 400 og 33.29 de 197.90 de 5.00 c 200 lk + 800 og 28.33 f 123.40 defgh 3.67 cd 400 lk + 200 og 17.50 gh 155.60 defgh 4.50 c 400 lk + 400 og 12.25 i 130.17 defgh 3.00 de 400 lk + 800 og 10.38 i 85.00 fgh 2.00 ef 800 lk + 200 og 12.25 i 100.00 fgh 3.67 cd 800 lk + 400 og 9.38 i 80.67 fgh 2.00 ef 800 lk + 800 og 9.42 i 25.67 gh 1.67 ef azadirachtin 4.17 j 13.17 h 1.00 f adv. hort. sci., 2020 34(4): 357­372 364 comparatively, those roots of plants grown in non­ amended treatments (soil) had numerous galled roots which were less fibrous (fig. 3). effect of fresh plant organic amendments on tissue of greenhouse tomato the different levels of amendments of lippia and ocimum soil amendments had significant (p<0.05) influence on tomato plant tissue nutrients. the plant macro nutrient analyzed included n, p, k, ca and mg, which are the most essential element in tomato pro­ duction. the interactive effect of the lippia and ocimum biomass were significantly higher in the tomato leaf tissues from both amendment rates above 400 g/10 kg of the substrate (table 3). in single state, both plant biomass registered significantly higher n content at 800 g only. number of leaves tomato leaf numbers were significantly influ­ enced by the use lippia and ocimum as soil organic amendments (table 4). there were significantly (p<0.05) higher leaf number at 21 dat in soil alone and 200g pots of both lippia and ocimum levels than 400g and above. however trend changed as from 35 dat as from 35 dat on 400 g of both species having the highest leaf numbers. at 49 dat there were no significant difference (p<0.05 on treatment of both species from rates above as single or in combination 4 non­amended soil had the least number of leave except for 21 dat. compared to the positive con­ trols, azadirachtin treated had relatively lower num­ ber leaves than 800 g of both lippia and ocimum combined, however this was higher than the un­ amended soil. plant height the result showed that the height of tomato plant was significantly (p<0.05) influenced by the lippia and ocimum levels (table 5). plants amended with 400 g of lippia or ocimum were taller than those of the lower levels of amendment and azadairachtin. as in the leaf numbers plant height differences were observed 49 dat were no difference were from each treatment up to the highest rate. root volume the organic amendments significantly influenced the development of the total root volume of the tomatoes grown in the pots during the production seasons (table 6). in root volume interactive effect fig. 3 ­ the effect of lippia and ocimum on the tomato root system. a) lk+ og at 800 g show fibrous root system, b) lk+og at 200 g with fewer galls, and c) non­amended soil high nematodes infestation. table 3 ­ effect of fresh plant organic amendments on tissue analysis of greenhouse tomato og = ocimum gratissimum l.; lk = lippia kituensis vatke; 0 = no amendment (soil). * means followed by the same letter series within a column are not significantly different according to tukey’s honestly significant diffe­ rence (thsd) at p≤0.05. amendments (g/10 kg) n (%) p (%) k (%) ca (%) mg (%) 0 2.95 d * 0.23 e 2.82 g 0.68 l 0.28 f 200 og 3.35 bcd 0.33 cd 3.12 ef 0.98 k 0.33 ef 400 og 3.45 abc 0.30 cde 2.92 fg 1.18 i 0.32 ef 800 og 3.35 bcd 0.32 cd 3.12 ef 1.68 f 0.36 de 200 lk 3.55 ab 0.33 cd 3.32 de 1.08 j 0.42 cd 400 lk 3.75 ab 0.43 b 3.42 cd 1.58 g 0.31 ef 800 lk 3.85 a 0.56 a 3.72 ab 2.08 b 0.49 abc 200 lk + 200 og 3.35 bcd 0.34 cd 3.32 de 1.88 d 0.36 de 200 lk + 400 og 3.75 ab 0.35 bc 3.72 ab 1.38 h 0.36 de 200 lk + 800 og 3.55 ab 0.60 a 3.12 ef 1.78 e 0.38 de 400 lk + 200 og 3.85 a 0.38 bc 3.52 bcd 1.88 d 0.35 def 400 lk + 400 og 3.65 ab 0.37 bc 3.82 a 1.98 c 0.37 de 400 lk + 800 og 3.65 ab 0.55 a 3.72 ab 2.38 a 0.42 cd 800 lk + 200 og 3.65 ab 0.61 a 3.62 abc 2.38 a 0.46 bc 800 lk + 400 og 3.55 ab 0.59 a 3.72 ab 2.08 b 0.52 ab 800 lk + 800 og 3.65 ab 0.58 a 3.82 a 2.38 a 0.54 a azadirachtin 3.05 cd 0.26 de 2.92 fg 1.08 j 0.28 f otieno et al. ‐ management of meloidogyne in tomato by soil amendments 365 was observe in the amendment at 400 g of both species with highest of 312.3 cm3. azadirachtin treat­ ed soils recorded relatively lower with root volume of 89.00 cm3, which was not significantly different from the non­amended soil. root and shoot dry weight as observed in the root volume, root and shoot dry weight showed effect by lippia and ocimum lev­ els in the treatments for the two seasons in a similar trend (table 6). interactive effect of the two plant table 4 ­ effect of fresh plant biomass on tomato leaf numbers og = ocimum gratissimum l.; lk = lippia kituensis vatke; 0 = no amendment (soil). dat = days after transplanting. * means followed by the same letter series within a column are not significantly different according to tukey’s honestly significant diffe­ rence (thsd) at p≤0.05. amendments g/10 kg) leaf numbers 21 dat 35 dat 49 dat 63 dat 77 dat 0 12.00 ab * 13.06 gh 20.83 cd 21.83 fg 22.67 k 200 og 11.39 abc 12.61 h 20.94 bcd 22.67 ef 23.67 j 400 og 10.28 cdef 14.61 cde 21.94 abcd 25.61 cd 26.20 fg 800 og 0.28 cdef 14.78 cd 25.39 abc 27.33 abc 27.73 de 200 lk 12.21 a 13.28 fgh 21.83 abcd 26.61 c 27.66 de 400 lk 10.06 defg 13.5 efgh 16.94 d 20.11 g 24.67 i 800 lk 11.22 abc 14.28 cdef 24.89 abc 22.67 ef 27.20 e 200 lk + 200 og 9.50 efgh 14.28 cdef 23.28 abc 26.83 bc 27.32 de 200 lk + 400 og 11.22 abc 14.94 bcd 24.39 abc 25.79 cd 27.33 de 200 lk + 800 og 10.94 bcd 15.22 abc 23.39 abc 26.72 c 27.27 e 400 lk + 200 og 10.28 cdef 15.22 abc 24.11 abc 29.28 ab 29.53 ab 400 lk + 400 og 9.28 fgh 14.06 defg 21.17 abcd 23.83 de 26.53 ef 400 lk + 800 og 10.78 cd 16.21 a 23.50 abc 27.11 bc 29.60 a 800 lk + 200 og 10.61 cde 15.39 abc 24.22 abc 26.18 cd 28.60 bc 800 lk + 400 og 8.61 h 16.00 ab 24.56 abc 26.33 cd 29.73 a 800 lk + 800 og 9.06 gh 16.33 a 27.39 a 29.83 a 29.90 a azadirachtin 12.33 a 13.56 efgh 23.89 abc 27.17 bc 28.13 cd table 5 ­ effect of fresh plant biomass from lippia kituensis vatke and ocimum gratissimum l. on tomato plant height og = ocimum gratissimum l.; lk = lippia kituensis vatke; 0 = no amendment (soil). dat= days after transplanting. * means followed by the same letter series within a column are not significantly different according to tukey’s honestly significant diffe­ rence (thsd) at p≤0.05. amendments g/10kg) plant height 49 dat 63 dat 77 dat 0 48.78 fg * 91.56 f 91.56 f 200 og 49.33 f 94.00 ef 94.00 ef 400 og 71.72 ab 99.00 cde 99.00 cde 800 og 70.11 abc 98.93 cde 98.93 cde 200 lk 58.22 def 98.60 cde 98.60 cde 400 lk 60.06 cde 102.80 abcd 102.80 abcd 800 lk 73.67 a 108.40 a 108.40 a 200 lk + 200 og 66.61 abcd 97.47 de 97.47 de 200 lk + 400 og 72.83 a 99.73 bcde 99.73 bcde 200 lk + 800 og 67.06 abcd 97.80 de 97.80 de 400 lk + 200 og 62.22 bcde 101.27 abcde 101.27 abcde 400 lk + 400 og 72.83 a 105.73 abcd 105.73 abcd 400 lk + 800 og 69.67 abc 108.27 ab 108.27 ab 800 lk + 200 og 65.78 abcde 100.13 abcde 100.13 abcde 800 lk + 400 og 66.50 abcd 106.53 abc 106.53 abc 800 lk + 800 og 76.00 a 105.47 abcd 105.47 abcd azadirachtin 69.28 abc 88.40f 88.40 f adv. hort. sci., 2020 34(4): 357­372 366 were significantly higher weight as from 400 g and above. as was observed in the root volume, highest root dry weight of 53.1 g was not significantly differ­ ent from azadirachtin treated soils 51.7 g but signifi­ cantly different from the control soil 24.1 g. similarly interactive effect of lk and og registered 101 g, sig­ nificantly higher than azadirachtin treated soils 86.2 g and soil 46.1 g in lk 800 g combined with 800 g og, azadirachtin treated soils and soil alone respectively. effect of organic amendments on physiology response of tomatoes chlorophyll content and stomata conductance organic amendments levels influenced chloro­ phyll content in the tomato plant positively as shown in figure 4. for both season, interactive effect of both plant species biomass were significantly higer than most of the single rates. soil had the least chlorophyll content compared to the treated soils. the stomatal conductance of the leaves from the rates mentioned were affected in similar manner (fig. 5). however azadirachtin treated soil was not significantly differ­ ent from highest combination of the biomass in stomatal conductance. generally single treatments showed lower physiological process in the leaves. number of fruits per plant lippia and ocimum, significantly (p<0.05) influ­ enced fruit number per plant (fig. 6). interactive effect of the two plants had mean of 62.72 fruits, fig. 4 ­ effect of fresh plant organic amendments on tomato plant chlorophyll content taken after two week interval. means followed by the same letter in a letter series within a column per season are not significantly different according to tukey’s honestly significant difference (thsd) at p≤0.05. og =ocimum gratissimum l. and lk = lippia kituensis vatke, are soil organic amendments in g/10 kg soil, 0 = no amendment and azad = positive con­ trol of commercially know pesticide azadirachtin. fig. 5 ­ effect of fresh plant organic amendments on tomato plant stomatal conductance taken after two week inter­ val. means followed by the same letter in a letter series within a column per season are not significantly different according to tukey’s honestly significant difference (thsd) at p ≤ 0.05. og =ocimum gratissimum l. and lk = lippia kituensis vatke, are soil organic amendments in g/10kg soil, 0= no amendment (soil), azad= azadirachtin. table 6 ­ effect of fresh plant biomass on tomato leaf numbers og = ocimum gratissimum l.; lk = lippia kituensis vatke; 0 = no amendment (soil). * means followed by the same letter series within a column are not significantly different according to tukey’s honestly significant diffe­ rence (thsd) at p≤0.05. amendments (g/10 kg) root volume (cm­3) root dry weight (g) shoot dry weight (g) 0 69.33 i * 24.17 h 46.12 j 200 og 135.33 h 28.50 h 57.92 i 400 og 84.6 7i 44.17 cdef 73.86 fg 800 og 281.00 d 39.83 defg 69.95 fgh 200 lk 132.00 h 39.67 defg 71.42 fg 400 lk 249.67 e 39.58 efg 77.01 e 800 lk 282.33 cd 38.58 fg 86.56 c 200 lk + 200 og 286.67 bcd 38.00 g 66.47 h 200 lk + 400 og 229.33 e 43.00 cdefg 66.53 h 200 lk + 800 og 279.67 d 46.50 bc 75.66 ef 400 lk + 200 og 194.67 f 45.17 cde 86.10 c 400 lk + 400 og 165.67 g 45.25 cd 96.68 b 400 lk + 800 og 312.00 a 53.08 a 99.78 ab 800 lk + 200 og 248.67 e 56.08 a 97.67 b 800 lk + 400 og 310.00 ab 53.17 a 98.78 b 800 lk + 800 og 306.00 abc 53.00 a 100.85 a azadirachtin 89.00 i 51.75 ab 86.23 cd otieno et al. ‐ management of meloidogyne in tomato by soil amendments 367 while non­amended control had 22.24 tomatoes per plant for season 1 and 2. azadirachtin had 32.50 tomatoes per plant. effect of fresh plant biomass on marketable yield and non‐marketable yield there was a general increase of marketable toma­ to fruits with the increase of the rates of plant organ­ ic amendment (fig. 7a). the interaction between the especially above in lippia or ocimum 400 g produced tomato with higher t/ha than single treatments and soil media. azadirachtin treated soil had better yield though significantly lower than those in 800 g of both species, which were rated as marketable.. non­mar­ ketable showed a reverse trend on the fruit weight (fig. 7). there was an opposite trend in the effect of the amendment from that of marketable. any combi­ nation biomass lower than 400 g did not produce marketable but poor qualities fruits represented as blossom end rot, blotch ripening, puffiness, gold flex, car face, sunscald and very small size stony fruits, which rendered them non­marketable. (fig. 7b1, 7b2, 7b3). 4. discussion and conclusions the practice of adding organic matter to soil for management of soil pest and increase yield is as old as the agriculture (akhtar and alam, 1993) and this has been successfully explored to control some plant parasitic nematodes (ferraz and freitas, 2004; lopes, 2011). this study revealed positive interactive effects of lippia kituensis vatke and ocimum gratissimum l. as fresh biomass for the control of nematodes (meloidogyne spp.) in greenhouse tomatoes. in over­ all, the results indicated effective nematode control in the tomato crop treated with the plant biomass compared to the control treatments where no amendments were applied. additionally, it was gen­ erally observed that interactive effect of biomass treatments of the two species at the higher rates (400 g and 800 g) was more effective than the single treatments. in line with this study, oka et al. (2007) reported sensitivity of plant­parasitic nematodes to plant derived amendments, however, they indicated that the effect varied with the nematodes species targeted and the rates applied. our results further revealed that the second instar juveniles (j2) of meloidogyne spp were more susceptible to higher rates of these treatments and effectively suppressed the nematodes in the media. several postulations on the mechanisms of action of these fresh biomass materials have been put for­ ward. it has been reported that during the decompo­ sition of these organic materials, volatile fatty acids, ammonia and hydrogen sulphide gases are released (mcsorley, 2011) and these may enhance nematode control. alternatively, other authors have explained the mechanisms of nematode population reduction by soil amendments with organic matter to involve fig. 6 ­ effect of fresh plant biomass from the plant species lippia kituensis and ocimum gratissimum. on tomato plant fruit per plant. means followed by the same letter in a letter series within a column per season are not significantly different according to tukey’s honestly signi­ ficant difference (thsd) at p ≤ 0.05. og =ocimum gratis‐ simum l. and lk = lippia kituensis vatke, are soil organic amendments in g/10 kg soil, 0 = no amendment and azad = positive. fig. 7 ­ a. effect of fresh plant biomass on marketable yield and fruit quality. means followed by the same letter in a let­ ter series within a variable are not significantly different according to tukey’s honestly significant difference (thsd) at p≤0.05. og =ocimum gratissimum l. and lk = lippia kituensis vatke, are soil organic amendments, 0 = no amendment and azad = azadirachtin. (2% og= 200 g og, 4% og= 400 g og 8% og= 800 g og; 2% lk= 200g lk, 4% lk= 400 g lk, 8% lk= 800 g lk /10 kg). b. the picture shows difference in the quality of tomato at different rates of lippia and ocimum amendments b1= lk+og at 800 g; b2= 0 lk+200 g og; b3= non­ amended soil. adv. hort. sci., 2020 34(4): 357­372 368 stimulation of antagonistic microorganisms, libera­ tion of secondary volatile or nonvolatile phytochemi­ cals with nematicidal properties (lopes et al., 2011). as earlier reported by chavarría­carvajal and rodríguez­kábana (1998), the amendments may improve the growth of the plants and hence increase the tolerance and plant resistance to nematodes. in this study l. kituensis and o. gratissimum biomass additions to soil probably proved toxic to meloidogyne spp. under greenhouse conditions, even at low rates of two species when combined at 200 g per pot of 10 kg of soil (table 2). these results concur with the study of lopes et al. (2011), who reported that soil amendment with the aerial portion of cer­ tain plant species has nematicidal properties. similar results have been reported by kagai et al. (2012), working with selected plant biofumigants in the man­ agement of plant parasitic nematodes in asclepias tubaerosa l. in line with this study, onifade (2007) earlier reported that essential oils from basil (ocimum basillicum) had nematicidal effect on para­ sitic nematodes, especially meloidogyne spp and pratylenchus penetrance which is root lesion nema­ tode. the second possible mechanisms for nematode suppression by these organic amendments could be direct inhibition or reduced infectivity of nematodes on the plant host. this may also be speculated that the use of lippia and ocimum as fresh soil organic amendment enhances antagonism in the soil mixes by increasing the abundance of other competing ben­ eficial organisms, thus reduces the chances rkn sur­ vival. these results are in concurrence with a study by claudius­cole et al. (2010), which reported reduc­ tion of meloidogyne incognita on cow pea vigna unguiculata (l) walp using plant extract. besides, in agreeement with the present study hasabo and noweer (2005), earlier reported that the extract of ocimum reduced nematode population on eggplant, and increased resultant fruit yields. in the present study, different rates of amend­ ments with l. kituensis vatke and o. gratissimum l. biomass significantly influenced gall numbers and galling index in the treatments especially when the two plant biomass interacted (table 2). this was demonstrated when galling index was drastically reduced. various studies have shown similar observa­ tions in using organic amendments to control rkn. breakdown of plant organic material releases nemati­ cidal substances that may contribute to nematode control (chen et al., 2000). akhtar and malik (2000) also reported that crops and weeds release biochem­ icals that counteract the activities of nematodes. this has also been confirmed by mcsorley (2011) that nematicidal compounds released from decomposing materials can stimulate the natural enemies of nema­ todes and improving plant tolerance. in line with the present study, lippia and ocimum have been report­ ed to yield essential oils of diverse nature (atuboyedia et al., 2010). laboratory analysis of ocimum yielded eugenol, citrol linalol, charvicol, thy­ mol, gerianol, triterpenoids, saponins and alkaloids (matasyoh et al., 2007; ogendo et al., 2008). based on the findings of the present investigation, it is plau­ sible to suggest that these biomolecules extracted during decomposition of the plants biomasses helped to inhibit nematode activity in the amended soil, leading to low galling index. it also concur with observations by onifade (2007) indicated that use of essential oils of o. gratissimum and o. basillicum in vitro at rates which completely inhibited egg hatch­ ing and larval survival of nematodes and this proba­ bly caused reduction of gall number. from the present results on macro element analy­ sis of the amended media, it is clear that besides act­ ing as a nematicide for the management of rkn, lippia and ocimum also acted as plant nutrient source for tomato growth and yield. mostly these element were significantly higher in the both amend­ ment rates above 400g and above per 10 kg of the substrate (table 3). nitrogen, p, k mg and ca are essential macro element, important in entire plant growth and development. in particular, nitrogen is mostly required by plants to achieve high rates of growth and yield of tomato. the presence of these elements may promote physical and physiological changes in the plant and mostly related to photosyn­ thesis, whereas mg also plays a big role in chlorophyll structure (taiz and zieger, 2002). nitrogen is a criti­ cal macronutrient influencing processes growth and development directly on source­sink relations, alter­ ing the distribution of assimilates between the vege­ tative and the reproductive part resulting into yield (zuba et al., 2011). phosphorus is for root develop­ ment, flower initiation, seed and fruit development. unlike n and p, k does not form any vital organic compounds in the plant, however, its presence is vital for plant growth, being known to be an enzyme activator that promotes metabolism (silva and uchida, 2000). the effect of lippia and ocimum rates and their interaction on vegetative phase, increased leave number and height was revealed in this study (table 4). with increased rates of organic amendment in the combination, it is probable that npk levels in the soil otieno et al. ‐ management of meloidogyne in tomato by soil amendments 369 may also enhance growth, leading to the increase in leaf number. leaf number is a function of n in plant (otieno et al., 2019) and this is very key for the high­ er number of leave observed at the rates of 800 g lippia and 800 g of ocimum combined. at dat 21 there were more leaves in the non­amended soils compared to amended soil in seasons 1 and 2 (table 4). this was probably due to the loss of nutrient especially n from the decomposing fresh organic amendments by microorganisms involved. the microorganisms involved in the decomposition possi­ bly out­competed the tomatoes in the used the n available for the plant and this may have led to reduction in growth rate. however, this trend was changed as from 35 dat upwards indicating that both lippia and ocimum had started releasing nutri­ ent from decomposition process for tomato use. this is in conformity with observation made by pakeerathan et al. (2009) in the management of meloidogyne incognita using different green leaf manures on tomato under field conditions, where n contributed more toward the vegetative components (leaves and stems) of the plant than reproductive components. the height of tomato was also influenced by the higher rates lippia and ocimum (table 5) and proba­ bly this was a function of k in the organic amend­ ments rates applied as observed in table 3. this is in consistence with el­nemr et al. (2012) who reported potassium (k) concentration as among the plant macronutrients that affected these growth parame­ ters. in another study, faruk et al. (2011) reported similar observation on the effect of poultry organic amendment on root knot nematode management and its influence on the height of greenhouse grown tomatoes. the ability of plants to obtain water and mineral nutrients from the soil is related to their capacity to develop extensive roots and root hairs (taiz and zieger, 2002). as in table 6, there was a significant increase in root volume with increased rates of lippia and ocimum especially when the two species were combined above 400 g, compared single rates and the controls. for root growth and development, organic amendments in the soil has been known to increase the bulk density of the growing media (otieno et al., 2020), giving room for root system to explore wider range of the soil environment for more nutrients (faruk et al., 2011; otieno et al., 2019). at lower amendment level, fewer root were observed, resulting in low root volume. in contrary, roots pro­ duced from 800 g of lippia and ocimum produced higher volume of fibrous roots. from this study it may be speculated that the root system of the toma­ toes from highly amended media probably were affected in two ways; either by enhancing soil struc­ ture in favour of the roots growth (otieno et al., 2019), or reduction of nematodes population in the soil or both. increasing amendments to the soil may alter many factors that affect root development in the rhizosphere. these include soil structure, particle aggregation, ph, salinity, level of carbon dioxide, oxygen and other chemicals (akhtar and malik, 2000). in this way, this probably increased the roots’ ability to increase in dry weight and subsequently shoot dry weight. similarly, the current result concurs yadessa et al. (2010), whose findings showed that 10% of fym produced significantly higher shoot and root dry weight compared to non­amended. leaf chlorophyll content (ccl) and stomatal con­ ductance responded positively to increasing rates of these organic amendments. at 49 dat, it was marked with maximum physiological processes in the crop (figs. 4 and 5), indicating higher formation of chloro­ phyll content on the tomato crop at higher rates of lippia and ocimum. nitrogen is an essential nutrient for normal growth and development of a plant as it is an integral part of the chlorophyll molecule (kitonga­ mwanza, 2011), together with mg; the principle site of light absorption necessary for photosynthesis. stomatal conductance on the other hand was similar­ ly influenced by both lippia and ocimum rates in the organic amendments (fig. 4). mostly k is involved in stomata closing and opening, therefore when galls have interfered with root system, the stomatal func­ tion may have been negatively affected at lower rate of amendments showing lower rate of stomatal con­ ductance. this suggests that apart from the influence of k in the organic amendments, nematodes also played a part by the interference of the plant root system (mai and mullin, 1996), reducing the flow of water and minerals upward the plant. from the current study, amendments rates influ­ enced yield differently among the individual rates (fig. 6). highest yield in terms of fruit numbers regis­ tered per plant was observed in those with 400 g and above in rates of lippia and ocimum amendments, indicating that the production of fruits was probably from primary plant nutrients as reflected in tomato tissue analysis (table 3). this observation is in agree­ ment with that made by walker (2007), where, the effect of organic amendments, fertilizers and fenamiphos was reported on reduction of parasitic and free­ living nematodes as well as increased yield adv. hort. sci., 2020 34(4): 357­372 370 of tomato. a number of authors have reported that adequate k nutrition is linked with increased yields (kanai et al., 2007; afzal et al., 2015), which further confirm the current findings plant derived organic amendments and their impacts on tomato produc­ tion. finally, the marketable yield of tomato is basically dependent on regular nutrient and moisture avail­ ability in growing media for plant use. moisture in particular is essential for nutrient movement, where­ as its irregular flow in the plant system may cause blossom end rot of (ber) in tomato. this scenario is more pronounced irregular calcium mobility in both growing media and the plant system. as for the cur­ rent study, application of both lippia and ocimum at 800 g level per 10 kg pot seemed to have increased water holding capacity of the amended soil since organic matter enhances the moisture availability in the media leading to more nutrient availabilty hence higher marketable yield. in concurrence to the pre­ sent study, akhtar and malik (2000) 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including u.k., germany, denmark, norway, france (ebben and last, 1973), and italy (cappelli et al., 2004). in japan, the disease was first reported in 1985 in squash root which was the rootstock of a cucumber (cucumis sativa) (hashimoto and yoshino, 1985). since then, the disease has also been found in melon (cucumis melo), watermelon (citrullus lanatus), pumpkin (cucurbita maxima), and other cucurbits, threatening the production of these major cucurbit crops with severe economic damage. shishido et al. (2006) identified the causal agent of this disease as p. sclerotioides kesteren through both morphological and phylogenetical analyses. because of recent development in dna sequence comparisons, asexual state of genus, phomopsis, has connected to sexual state, diaporthe, which has priority over phomopsis, and should be the generic name adopted for these taxa (udayanga et al., 2012; gomes et al., 2013), and thus i use diaporthe sclerotioides as the name of the pathogen of this disease in this article. to control black root rot of cucurbits, solarization with a combination of soil fumigants such as chloropicrin has been widely applied in greenhouses, especially in warmer climate regions (kobayashi et al., 1997). however, the disease has been consistently spreading in cucumber production areas in northern parts of japan, where climate conditions are not warm enough to apply such temperature-dependent measures. in addition, the disease has not yet been contained in the southern parts of japan even though the incidence is sporadic and inconsistent (shishido, 2006). the present paper offers a concise review of recent developments in knowledge about black root of cucurbit crops as well as control measures against this disease in japan. 2. taxonomy of diaporthe sclerotioides morphology the telemorphic stage of d. sclerotioides has not yet been discovered, while anomorphic stages of diaporthe species are often characterized with specific pycinidia. d. sclerotioides forms pycinidium which are mostly subglobose or spherical under the epidermis (van kesteren, 1966). in our experiment, pycnidia were found submersed in sterilized beanpods after three weeks of incubation, mostly globose and varying in size (200-500 μm in diameter) (fig. 1a). the internal cavity of a pycnidium was often divided by protrusions of the proliferous layer (fig. black root rot caused by diaporthe sclerotioides threatens cucurbit cultivation in japan m. shishido graduate school of horticulture, chiba university, 648 matsudo, matsudo-shi, 271-8510 japan. key words: black root rot, cucurbitaceae, diaporthe sclerotioides, abstract: in japan, since black root rot of cucurbitaceous crops was found more than 30 years ago, the disease has caused severe economic losses to the cucurbit crop industry. subsequent to the pathogen being correctly identified as diaporthe sclerotioides based on morphology and dna sequence, knowledge about dna sequences has developed making technical tools to detect and quantify the pathogen in natural samples of plants and soils available. in addition to chemical soil disinfectants, solarization and biological soil disinfestation have been developed as environment-friendly methods to effectively control this disease. although it is difficult to apply such temperature-dependent methods in open-fields especially under cool climate conditions, an alternative approach, which changes soil ph to weak alkaline with amending steel converter slag, has also proved effective against the disease. in this mini-review, the process of d. sclerotioides identification, detection and quantification methods developed for this fungus, host specificity, and disease control measures available and practiced in japan are discussed concisely. adv. hort. sci., 2014 28(4): 208-213 (1) corresponding author: shishido@faculty.chiba-u.jp received for publication 17 september 2014 accepted for publication 5 november 2014 mini review 209 1b). numerous pycnospores (fig. 1c), mostly ellipsoidal to ovoid, hyaline, 7-12 × 3-6 μm in size, usually with two guttules, were observed in the pycnidium (fig. 1d) and apparently were produced on conidiogenous cells (fig. 1e). conidiophores filiform, hyaline, septate at the base, rarely branched, up to 25 μm, were formed from the inner cells of the locular walls. these only α-type conidiospores were unlike other diaporthe species and no β-type were observed. although we found that an isolate produced pycnidia and conidiospores, the event was very rare and no such organs have been reported in the natural environment. therefore, we suspect that conidiospores are not the primary source of inoculum of this pathogen. on the other hand, d. sclerotioides easily forms dull, grayish-brown mycelial mats on common agar media including potato dextrose agar (shishido et al., 2006). the mycelium consisted of thin hyaline hyphae (2-5 μm in diameter) and thick hyaline to dark brown hyphae (10-20 μm in diameter) (fig. 1f). a layer of the thick hyphae and dark brown, thick-walled cells formed a small sclerotium (fig. 1g), and later became superficial or submersed pseudo-sclerotia, mostly flattened, of various sizes (fig. 1h). because these pseudo-sclerotia are commonly observed on diseased roots as well, they are likely the primary inocula of black root rot of cucurbit crops. molecular phylogeny phylogenic analyses of the genus diaporthe have been conducted in a number of studies (rehner and uechker, 1994; zhang et al., 1998; kanematsu et al., 2000; farr et al., 2002; murali et al., 2006; shishido et al., 2006; santos et al., 2010; udayanga et al., 2012; gomes et al., 2013). dna sequences of the its regions of d. sclerotioides isolated from various parts of japan formed a single distinct clade without differing from the ex-type d. sclerotioides (cbs 296.67) by even a single nucleotide (shishido et al., 2006). the close kin species of d. sclerotioides were d. columnaris (farr et al., 2002) and d. strumella var. longispora (udayanga et al., 2012). these species differ 11 and 26 bases of the dna sequences in their its regions out of 329 bases in total (its 1 and 2). the relationships detected with the dna sequences of the its region were also found in other loci including elongation factor 1-α (udayanga et al., 2012) and a mating type gene, mat1-1-1 (santos et al., 2010). interestingly, we found no host-specific dna sequences within the its regions among the japanese isolates of d. sclerotioides that originated from four different host species: melon, watermelon grafted on bottlegourd, pumpkin, and cucumber. rehner and uecker (1994) argued that the host-based species concept was not reliable for diaporthe because of the genetic diversity among isolates of this genus from various hosts. kanematsu et al. (2000) supported this hypothesis by demonstrating that the phenotypic divergence of phomopsis species, i.e. w type (mainly white colonies, weakly virulent, bearing both αand β-type conidia) and g type (mainly gray colonies, highly virulent, bearing only α-type conidia) was dependent on their its sequences rather than on the host species. since the japanese d. sclerotioides isolates of black root rot are all g type, it may not be surprising that these isolates create a single clade in the phylogenetic tree. 3. methods for detection and quantification of diaporthe sclerotioides plant pathogenic fungi are usually detected by their growth on selective media or by biochemical, chemical, and immunological analyses. however, none of these conventional techniques are available for d. sclerotioides. moreover, morphological identification of these fungi on nonselective media is time-consuming and requires expert knowledge of classical taxonomy. in recent years, pcrfig. 1 typical features of diaporthe sclerotioides grown under gnotobiotic conditions. a, pycnidia on a sterilized beanpod. b, vertical section of pycnidium. c, numerous pycnospores mechanically released from pycnidium. d, pycnospores. e, pycnophores on the internal wall of pycnidium. f, thick-walled, crenate aerial mycelium commonly found in various agar cultures. g, masses of dark-colored mycelium, later become pseudomicrosclerotia. h, small, brownish-black pseudomicrosclerotia on a boiled cucumber leaf. reprinted from reference shishido et al. (2006) with permission from the publisher. 210 based molecular techniques have contributed to the detection and identification of various types of plant pathogens. real-time pcr techniques combine the sensitivity of conventional pcr with the generation of a specific fluorescent signal. this signal can be measured throughout the procedure, providing real-time analysis of the reaction kinetics, and thereby allowing for quantification of specific dna targets (schena et al., 2004). shishido et al. (2010) constructed conventional and real-time pcr primers to detect d. sclerotioides, utilizing a dna sequence in the its region specific to this fungus. the designed primers, cps-1 (forward) and cps-2 (reverse), successfully detected the fungus in soil and plant samples collected from fields naturally infested with the disease. furthermore, the cps-1/cps-2 primer pair was applied to a real-time pcr assay with sybr green i, and the protocol achieved a linear response with a high correlation coefficient between input dna and cycle threshold. however, because sybr green i is a dna-intercalating dye without sequence specificity (wittwer et al., 1997), and because dna in natural samples usually contains unknown sequences, the amount of dna will only be accurate if no presence of other dna similar to this fungus is confirmed in the sample. this problem was solved using a taqman probe-based real-time pcr assay, which is highly specific, sensitive and quantitative (shishido et al., 2013). in addition, the taqman probe-based protocol allows multiplex real-time pcr, and thus using internal standard dna such as gfp for soil samples (klerks et al., 2004) and cox for plant samples (weller et al., 2000), quantification of the fungal dna should be more accurate than the mono-plex counterparts. table 1 summarizes primer sequences that can be used for detecting and quantifying d. sclerotioides dna in natural samples. 4. host range and specificity of diaporthe sclerotioides in general, it is important in breeding programs to know if there are specific interactions between pathogenic microorganisms and host species. although d. sclerotioides can cause black root rot only in cucurbit species, until recently little has been known about the degrees of either the host susceptibility to this disease or host specificity of the pathogen. shishido et al. (2014) hypothesized that d. sclerotioides isolates were more infective and virulent to the cucurbit species from which the pathogens were originally isolated than to other host species. they conducted cross-inoculation experiments using cucumbers, melons, pumpkins, watermelons, and bottlegourd (lagenaria siceraria var. gourda), by inoculating 12 d. sclerotioides isolates from these cucurbit species. the virulence of the isolates was evaluated as the area under the disease progress curve (audpc). all cucurbit species were susceptible to each isolate, but audpcs were significantly different among the hosts as melon > cucumber ≥ watermelon ≥ bottlegourd ≥ pumpkin. the infectiveness of isolates, on the other hand, was assessed as the quantity of d. sclerotioides dna detected in the hypocotyls of seedlings two weeks after inoculation using the taqman-based real-time pcr protocol described above. the fungal dna quantities varied among the species in the same order as the audpcs. orthogonal contrasts indicated no specificity in either the fungal virulence or infectiveness between d. sclerotioides isolates and the cucurbit hosts from which these isolates originated (fig. 2). based on these results, the original hypothesis was refuted and they concluded that though host susceptibility to black root rot varies among cucurbit species, d. sclerotioides isolates are unlikely to have specificity to the host table 1 pcr protocols for detecting and quantifying diaporthe sclerotioides in natural samples detection of d. sclerotioides (shishido et al., 2010) cps-1 (forward) cps-2 (reverse) 5´-gcctcggcgcaggccggcctcacc-3´ 5´-ggggccttccagaacgaaatataattt-3´ note: not recommended for real-time pcr, expected amplicon size: 392 bp detection and quantification of d. sclerotioides (shishido et al., 2013) cps2f (forward) cps2r1 (reverse) cps2t (probe) 5´-actgcttggtgttggggcacc-3´ 5´-tccagaacgaaatataatttactacgct-3´ 5´[fam]-aaagggcgggccctgaaatctagtggcga-[tamra] -3´ note: applicable with sybr green i instead of taqman probe, expected amplicon size: 101 bp internal standard of soil samples (klerks et al., 2004) fpgfp (forward) rpgfp (reverse) pyygfp (probe) 5´-tggccctgtccttttaccag-3´ 5´-ttttcgttgggatctttcgaa-3´ 5´[vic]-aaccattacctgtccacacaatctgccc-[tamra] -3´ note: applicable with sybr green i instead of taqman probe as an external standard. internal standard of plant samples (weller et al., 2000) cox-f (forward) cox-r (reverse) cox-p (probe) 5´-cgtcgcattccagattatcca-3´ 5´-caactacggatatataagagccaaaactg-3´ 5´[vic]-agggcattccatccagcgtaagca-[tamra] -3´ note: applicable with sybr green i instead of taqman probe as an external standard. 211 species in terms of either virulence or infectiveness. this research implies that in practice seedlings and soils from infested areas should be handled carefully because the pathogen may spread to various cucurbit species irrespective of the original host. interestingly, the relationship between the audpc and d. sclerotioides dna quantity in hypocotyls provided evidence that the virulence of this pathogen was not highly correlated with its infectiveness, although some degree of fungal invasion was obviously required for disease development (fig. 3). the correlation analyses also showed that, although for most of the cucurbit species there were statistically significant correlations between audpcs and d. sclerotioides dna quantities in the hypocotyls, their relatively low coefficients of determination indicated limited associations for these variables, therefore suggesting that the virulence of d. sclerotioides may be due to factors in addition to infectiveness. 5. control measures against black root rot of cucurbit crops van kesteren (1966) indicated that cucurbita ficifolia was tolerant to the disease despite apparent root infection by d. sclerotioides. however, no true resistant variety or r-genes as such has been reported to black root rot. it is possible to use c. ficifolia for rootstocks of cucumber, and in fact it used to be a common practice in cucumber production in japan. unfortunately, the rootstock of c. ficifolia causes a fine white powder on the surface of the cucumber fruits, called “bloom”, primarily composed of silica (mitani et al., 2011). in the late 1980s, cucumber without any bloom (bloomless cucumber) became popular in japan because of its more attractive and distinctly shiny appearance; and thus c. ficifolia has no longer be used as rootstock of cucumber in the commercial production. because d. sclerotioides is a soil-borne pathogen, soil disinfestation is a common control measure against black root rot of cucurbits. although chloropicrin appears to be the most effective among disinfectant chemicals, its high toxicity to humans and unpleasant odor limits its popularity in practical applications (shishido and takeuchi, fig. 2 the area under the disease progress curve (audpc) for black root rot in cucumber, melon, pumpkin, watermelon, and bottlegourd plants (a), and the quantity of diaporthe sclerotioides dna detected in the hypocotyls of these plants (b) after root inoculation with 12 isolates of d. sclerotioides. in both charts, orthogonal contrasts were tested between original host-fungal isolate combinations and non-original host-fungal isolate combinations with p values showing type i error probabilities of null hypotheses. the same letters indicate no significant difference between plant species as determined by tukey’s hsd test (p < 0.05). error bars denote the standard error of the mean. reprinted from reference shishido et al. (2014) with permission from the publisher. fig. 3 correlations between the area under the disease progress curve (audpc) and the quantity of diaporthe sclerotioides dna detected in the hypocotyls of all the cucurbit species combined (a), cucumber (b), melon (c), pumpkin (d), watermelon (e), and bottlegourd (f) after root inoculation with 12 isolates of d. sclerotioides. reprinted from reference shishido et al. (2014) with permission from the publisher. 212 2005). soil solarization is an alternative and environmentally-healthy measure against the disease since the pathogen is known to be heat-sensitive (kobayashi et al., 1997). one of the problems in soil solarization is to maintain high temperatures (37.5°c for 2 days or 35.0°c for 6 days) throughout the root range of soil depth, ca. 30 cm. in recent years, biological soil disinfestation (bsd), or anaerobic soil disinfestation (asd) has been successfully applied to control some soil-borne pathogens and pests including fusarium oxysporum, ralstonia solanacearum, and parasitic nematodes (momma et al., 2006; lamers et al., 2010). bsd increases the effect of solarization by amending organic substances such as wheat bran or green manure crops under anaerobic conditions. yokoyama et al. (2012) demonstrated that bsd with a low concentration of ethanol (0.5%-1.0%) was sufficiently effective to control black root rot of cucumber. they recommend a low concentration of ethanol instead of wheat bran for the soil amendment because the former produces almost the same level of disease control efficacy to the latter with little unpleasant odor during the anaerobic process. although bsd may be a promising control measure against black root rot, the practice may only be applicable in protected facilities such as greenhouses and walk-in tunnels in relatively warm climate regions. therefore, other control measures are needed that are applicable in vast, unprotected fields especially in cool climate regions. iwadate (2012) demonstrated that changing soil ph to weak alkaline, i.e. ph=7.5, with amending steel converter slag, significantly reduced the disease severity of cucumber in unprotected fields. all of these cucumber fields were in iwate prefecture, located in the north-eastern part of japan, an area known for its cool climate. although the mechanisms of disease suppression of this method have not yet been elucidated, it is certainly interesting since steel converter slag is easily available at a low cost as a byproduct of steel manufacturing. to date, only a few studies of biological control have been conducted regarding black root rot, e.g. gliocladium roseum by moody and gindrat (1977) and pseudomonas sp. by fuchs and defago (1991). nonetheless, because d. sclerotioides is a slow growing fungus compared with other major soil-borne fungal pathogens such as fusarium, rhizoctonia, and pythium, the fungus may be less competitive in searching for nutrients and habitats in soil. interestingly, hypovirulent elements such as double-stranded rna are also known to infect diaporthe species (ghabrial, 2013). although the detailed mechanism has not been elucidated, some isolates of d. sclerotioides significantly reduce in virulence (shishido et al., 2014). therefore, biological control including hypoviruses may have potential for controlling black root rot of cucurbit crops in future. 6. conclusions in japan, more than 30 years have passed since black root rot was first reported in cucumber. since then, the fungal isolates were correctly identified as diaporthe sclerotioides based on their morphology and dna sequence. in addition, the knowledge of dna sequence has developed technical tools for detecting and quantifying the pathogen in natural samples of plants and soils. on the other hand, aside from chemical soil disinfestation, environmentfriendly control measures have also been developed by applying solarization as well as biological soil disinfestation. although such temperature-dependent methods may not properly be applicable to out-fields especially in cool climate regions, an alternative measure by changing soil ph to weak alkaline with amending steel converter slag has proved effective to control this disease. nonetheless, the area of its infestation is still expanding to northern parts of japan. to prevent further damage to cucurbit production from black root rot and reducing the infestation of d. sclerotioides, studies are needed especially in the area of breeding of resistant varieties and understanding the ecology of the pathogen to develop more promising and effective measures to control of this disease. references cappelli c., stravato v.m., carannante g., parisella r., 2004 first report of cucumber black root rot caused by phomopsis sclerotioides in italy. plant dis., 88: 425. ebben m.h., last f.t., 1973 cucumber black root rot caused by phomopsis sclerotioides. ann. appl. biol., 73: 259-267. farr d.f., castlebury l.a., rossman a.y., putnam m.l., 2002 a new species of phomopsis causing twig dieback of vaccinium vitis-idaea (lingonberry). mycol. res., 106: 745-752. fuchs j., defago g., 1991 protection of cucumber plants against black root rot caused by phomopsis sclerotioides with rhizobacteria, pp. 57-62. in: keel c., b. koller, and g. defago (ed.) plant growth-promoting rhizobacteria-progress and prospects (iobc/wprs bull xiv/8). iobc/wprs, interlaken, switzerland. ghabrial s.a., 2013 advances in virus research, mycoviruses. academic press, oxford, uk. gomes r.r., glienke, c., videira, s.i.r., lombard, l., groenewald, j.z., crous, p.w. 2013 diaporthe: a genus of endophytic, saprobic and plant paghogenic fungi. persoonia, 31: 1-41. hashimoto k,, yoshino m., 1985 a new soil-borne disease, phomopsis black root rot of grafted cucumber caused by phomopsis sp. plant prot., 39: 570-574. iwadate y., 2012 suppression of cucumber black root rot by application of converter slag. jpn. j. phytopathol., 78: 288. kanematsu s., minaka n., kobayashi t., kudo a., ohtsu y., 2000 molecular phylogenetic analysis of ribosomal dna internal transcribed regions and comparison of fertility in phomopsis isolates from fruit tree. j. gen. plant pathol., 66: 191-201. klerks m.m., zijlstra c., van bruggen a.h.c., 2004 comparison of real-time pcr methods for detection 213 of salmonella enterica and escherichia coli o157:h7, and introduction of a general internal amplification control. j. microbiol. methods, 59: 337-349. kobayashi m., uekusa h., orihara n., udagawa a., 1997 control of black root rot of melon by solarization and chemicals. proc. kanto-tosan plant prot. soc., 44: 79-81. lamers j.g., runia w.t., molendijk l.p.g., bleeker p.o., 2010 perspectives of anaerobic soil disinfestation. acta horticulturae, 883: 277-284. mitani n., yamaji n., ago y., iwasaki k., ma j.f., 2011 isolation and functional characterization of an influx silicon transporter in two pumpkin cultivars contrasting in silicon accumulation. plant j., 66: 231-240. momma n., yamamoto k., simandi p., shishido m., 2006 role of organic acids in the mechanisms of biological soil disinfestation (bsd). j. gen. plant pathol., 72: 247252. moody a.r., gindrat d., 1977 biological control of cucumber black root rot by gliocladium roseum. phytopathology, 67: 1159-1162. murali t.s., suryanarayanan t.s., geeta r., 2006 endophytic phomopsis species: host range and implications for diversity estimates. can. j. microbiol., 52: 673-680. rehner s.a., uecker f.a., 1994 nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete phomopsis. can. j. bot., 72: 1666-1674. santos j.m., correia v.g., phillips a.j.l., 2010 primers for mating-type diagnosis in diaporthe and phomopsis: their use in teleomorph induction in vitro and biological species definition. fungal biol., 114: 255-270. schena l., nigro f., ippolito a., gallitelli d., 2004 real-time quantitative pcr: a new technology to detect and study phytopathogenic and antagonistic fungi. eur. j. plant pathol., 110: 893-908. shishido m., 2006 black root rot of cucurbits caused by phomopsis sclerotioides. crop prot., 59: 583-586. shishido m., kubota i., ohashi t., usami t., 2013 comparison of pcr assays for detection and quantification of phomopsis sclerotioides in plant and soil. j. gen. plant. pathol., 79: 8-17. shishido m., ohashi t., momma n., 2014 diaporthe sclerotioides exhibits no host specificity among cucurbit species. plant pathol., in press. shishido m., sato k., yoshida n., tsukui r., usami t., 2010 pcr-based assays to detect and quantify phomopsis sclerotioides in plants and soil. j. gen. plant pathol., 76: 21-30. shishido m., takeuchi t., 2005 analysis of questionnaire survey on sudden wilt of watermelon and disease management. crop prot., 59: 65-68. shishido m., yoshida n., usami t., shinozaki t., kobayashi m., takeuchi t., 2006 black root rot of cucurbits caused by phomopsis sclerotioides in japan and phylogenetic grouping of the pathogen. j. gen. plant pathol., 72: 220-227. udayanga d., xingzhong l., crous p.w., mckenzie e.h.c., chukeatirote e., hyde k.d., 2012 a multi-locus phylogenetic evaluation of diaporthe (phomopsis). fungal divers, 56: 157-171. van kesteren h.a., 1966 “black root rot” in cucurbitaceae 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2023 37(3): 289­293 doi: 10.36253/ahsc­14608 new mutations of flower shape in nigella damascena l., its pleiotropic effects and patterns of inheritance v. lyakh 1, 2 (*), a. soroka 2 1 zaporozhye national university, 66 zhukovsky street, 69600 zaporozhye, ukraine. 2 institute of oilseed crops, national academy of agrarian sciences of ukraine, institutskaya street 1, 70417 zaporozhye, ukraine. key words: flower shape, inheritance, mutant, nigella damascena, pleiotropic effect, shortened sepal. abstract: two mutants with short sepals were identified after ethyl methane­ sulfonate treatment of nigella damascena seeds. in one of them (“shs1” gene = short sepal 1), isolated from the line with double flowers, the sepals, in addi­ tion to reduced size, were divided into several rounded lobes, which granted the flower an original rose­like appearance of ornamental value. another mutant with reduced sepals (“shs2” gene = short sepal 2) was isolated from the line with simple flowers. the allelism test showed that these two genes were non­allelic. both mutants as pollen parents were crossed with the same line with single flowers. in a dihybrid cross, simple flower, non­reduced sepals (wild type) × double flower, reduced sepals (“shs1” gene) f1 hybrids demonstrated a wild phenotype. f2 progeny, in addition to two parental classes, showed two recombinant classes in a 9:3:3:1 ratio, indicating that flower shape and sepal size were inherited monogenously and independently, and the plant with rose­ like flowers was a double recessive homozygote. reduced sepals (“shs2” gene) in crosses with the single flower line of wild type were inherited as a mono­ genic recessive trait, showing a 3:1 segregation ratio in f2. both mutant genes had a number of similar pleiotropic effects, which, however, were different in strength. thus, both mutant genes shortened leaf segments, divided the cotyle­ don leaves into several lobes, and caused disturbances in the female generative sphere, leading to a lack of seed setting. at the same time, the identification of mutants as early as at the cotyledon stage, due to the pleiotropic effect, makes it possible to select and maintain them, especially with regard to the mutant with rose­like flowers, which is highly decorative. 1. introduction nigella damascena l. is an annual herbaceous plant of the buttercup family (ranunculaceae). this is a crop of wide application, the products of which are used in medicine, food industry, and perfumery. its seeds con­ tain about 50% fats, which consist mainly of unsaturated fatty acids, up to (*) corresponding author: lyakh@iname.com citation: lyakh v., soroka a., 2023 ­ new mutations of flower shape in nigella damascena l., its pleiotro‐ pic effects and patterns of inheritance. ­ adv. hort. sci., 37(3): 289­293. copyright: © 2023 lyakh v., soroka a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 april 2023 accepted for publication 1 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14608 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(3): 289­293 290 20% protein, 2­3% essential oil, enzymes, more than 20 macro­ and microelements, including essential ones (riaz et al., 1996). nigella is best known for the fact that nigedase is obtained from its seeds, a lipolytic enzyme prepara­ tion that is widely used in medical practice. the absence of animal protein and bile components in its composition permits to prescribe this enzyme prepa­ ration for allergies, as well as in cases where the presence of bile acids is highly undesirable. the liter­ ature contains information on the pharmacological activity of other biologically active compounds isolat­ ed from this plant, in particular, fatty oils and a num­ ber of essential oil components (helvacıoğlu et al., 2021; salehi et al., 2021; benazzouz­smail, 2023). the variety of applications of nigella has initiated research to develop various in vitro biotechnologies for this crop from callus culture to protoplast culture (klimek­chodacka et al., 2020). in addition to the above, nigella damascena has long been among the highly ornamental annual plants. its high decorativeness is granted by rather large petal­ like sepals of white, different shades of blue, purple, pink and even red colors. in floriculture, the shape of the flower is no less valuable than the color of the flower for giving the appearance to an ornamental plant. the presence of floral dimorphism in nigella damascena, which ensures the shape of single or dou­ ble flowers, and a variety of sepal colors allowed breeders to create a series of wonderful varieties. it has long been shown that the floral dimorphism is monogenically controlled, with the ‘single’ morph being dominant and the ‘double’ morph being reces­ sive (toxopeus, 1927). in recent years, flower dimor­ phism and different types of petal modifications in nigella damascena have served as the basis for using this plant as a model for elucidating the molecular control of floral dimorphism and identifying genes expressed during petal development (jabbour et al., 2015; zhang et al., 2020, galipot et al., 2021). as a result of studies on induced mutagenesis in nigella, we have identified two mutations affecting the size of the sepals, which ultimately alters the shape of the flower. these mutations, as well as their pleiotrop­ ic effects, are described in this article, which also pre­ sents the inheritance patterns for the mutant traits. 2. materials and methods in our studies on chemical mutagenesis, two mutations with a similar phenotypic appearance, expressed in the deformation of the sepals, were identified in nigella damascena. in one case the mal­ formation was manifested in shortening the sepal and rounding its edge, so that the sepal instead of a pointed shape had an oval shape. it was by the pres­ ence of a shorter sepal and its rounded edge that this mutation was originally isolated. another mutation was only designated by shortened sepals. both muta­ tions were identified in m3 generation. the first mutant was isolated from a variety with double flow­ ers after seed treatment with ethyl methanesul­ fonate at the concentration of 0.01% and exposure for 16 hours, the other was found from a variety with single flowers as a result of seed treatment with the same mutagen at the same concentration for 6 hours. in order to check whether these two mutations are allelic, an allelism test was performed. to study the inheritance of the mutant traits, both mutants, using them as pollen parents, were crossed with the same line with single flowers. f1 hybrids were self­pollinated and in f2 families the segregation ratios were analyzed. in the cross combination “sin­ gle flower, non­reduced sepals × double flower, reduced sepals”, four classes were considered, and in the combination “single flower, non­reduced sepals × single flower, reduced sepals”, the f2 population was divided into two classes. to test if the observed frequencies of plants in f2 populations correspond the expected ones a chi­ square test was used (griffiths et al., 2004). 3. results and discussion flowers of two mutants with deformed sepals are shown in figure 1. the flower of the mutant isolated as a result of mutagenic treatment of seeds of the double­flowered nigella plant is shown in figures 1a and 2b. as can be seen from the figures, the mutant, in contrast to the usual double flower (2d), was char­ acterized by shortened and more rounded sepals (“shs1” = shortened sepals with rounded edges). the incompletely opened flower of the mutant plant resembled the shape of a rose flower. in a cross combination of “single flower, non­ reduced sepals (wild type) × double flower, reduced sepals (mutant type)”, f1 hybrids had a single flower and non­reduced elongated sepals like the wild type parent. that is, a single flower completely dominated lyakh and soroka ‐ new mutations of flower shape in nigella damascena 291 the double one, and non­reduced sepals over reduced ones (“shs1”). in this cross combination the parents differed by two genes and, if these genes are inherited independently, we have to obtain a typical dihybrid pattern with the four unique phenotypes in a 9:3:3:1 ratio in f2. two f2 families showed a segre­ gation ratio where, in addition to the parental classes of single flower, non­reduced sepals (2a) and double flower, reduced sepals (2b), two recombinant classes appeared ­ single flower, reduced sepals (2c) and double flower, non­reduced sepals (2d) in approxi­ mately equal proportions (table 1, fig. 2). in both f2 families, there was a complete correspondence of the observed segregation ratios to the theoretically expected frequencies. the identified segregation model indicated an independent combination of flower morph and sepal shape traits and, conse­ quently, the absence of linkage between the genes that determine those traits. the flower of another mutant with deformed sepals, isolated after mutagenic treatment of seeds of a plant with simple flowers, in contrast to the first mutant, was characterized by a stronger shortening of the sepals and the absence of roundness at their ends (“shs2” = shortened sepals) (fig. 1b). the reduc­ tion in the sepal length was accompanied by a signifi­ cant deformation of the flower pistil, which was visu­ ally revealed in the strong shortening of the sty­ loides. some flowers of this mutant lacked them alto­ gether. sepals reduced in length (“shs2”) in crosses with the single flower line of wild type (with non­reduced sepals) were inherited in a monogenic recessive pat­ table 1 ­ f2 segregation for sepal shape and floral morph in cross of single flower, elongated sepals (wild type) and double flower, oval sepals (mutant type) plants in n. damascena fig. 1 ­ flowers of two nigella damascena mutants with reduced sepals: a) rose­like double flower at the beginning of opening (shs1 gene); b) single flower with reduced sepa­ ls (shs2 gene). f1 phenotype total f2 plants f2 phenotypes segregation ratio tested χ2 (p value) single flower, non­ reduced sepals single flower, reduced sepals double flower, non­ reduced sepals double flower, reduced sepal single flower, non­reduced sepals 100 61 17 16 6 (3:1) × (3:1) = 9:3:3:1 0.29 (0.59) single flower, non­reduced sepals 68 40 9 13 6 (3:1) × (3:1) = 9:3:3:1 1.39 (0.24) χ205 (d.f. 3) = 7.82. fig. 2 ­ phenotypic classes in f2 nigella damascena cross combi­ nations single flower, non­reduced sepals (wild type) × double flower, reduced sepals (shs1 mutant): a) single flower, non­reduced sepals; b) double flower, reduced sepals; с) single flower, reduced sepals; d) double flower, non­reduced sepals. adv. hort. sci., 2023 37(3): 289­293 292 tern, showing complete dominance of the wild type over the mutant in f1, and a 3:1 segregation ratio in f2 (table 2). the allelism test performed showed that these two genes, which determine the shortening of the sepals, are non­allelic. however, they have a number of similar pleiotropic effects. both genes, without affecting plant height, cause shortening of true leaf segments. the bracts of both mutants are also short­ ened and more densely attached to the ripening boll than in the wild type. it should be noted that the shs2 gene as compared with the shs1 gene causes stronger changes (fig. 3). shortening the leaves and bracts changes the habit of the plant, making it more compact. both mutant genes affect not only true leaves, but also cotyledons, causing them to be dissected into lobes. the division of one or two cotyledons into two lobes is characteristic of the mutant with shs2 gene (fig. 4 b), while the multi­lobed state of both cotyle­ dons is inherent for the mutant carrying shs1 gene (fig. 4 a). the negative effect of both mutant genes on the main function of the flower, which is reproduction, was also noted. the mutants were successfully used in various crosses as a source of pollen, but their involvement in hybridization as female parents was problematic. sometimes such crossings were success­ ful with the shs2 mutant when using late flowers, but it was not possible to obtain seeds from the shs1 mutant even after free pollination. this indicates seri­ ous disturbances in the female generative sphere of the flowers of both mutants. previously, in our studies with linum grandiflorum desf., a mutant with short petals, resembling a wild carnation flower, was identified (lyakh, 2018). after mutagenic treatment of immature sunflower embryos, a mutant with shortened petals (ray flow­ ers) was also obtained (soroka and lyakh, 2009). in both cases, as for nigella, ethyl methanesulfonate was used. the mutation identified in sunflower had a strong pleiotropic effect, affecting the stem, leaf, and even cotyledons. at the same time, true and cotyle­ don leaves had, in contrast to the elongated, round­ ed end of the leaf blade. two­locus genetic control of petal shape was revealed in linum grandiflorum and sunflower, where a shortened petaled plant is a double reces­ sive homozygote (soroka and lyakh, 2017; lyakh, 2018). in turn, a simpler genetic system is known that controls the shape of plant organs, in particular leaves. thus, it was found that the shape of the leaflet in cowpea is monogenously controlled, with the lanceolate leaflet shape dominant over the ovoid one (nwofia, 2014). the same monogenic control of leaf shape, but with a co­dominant interaction of table 2 ­ f2 segregation for sepal shape in cross of single flowered plants with non­reduced (wild type) and reduced sepals (mutant type) in n. damascena χ205 (d.f. 1) = 3.84 f1 phenotype total f2 plants f2 phenotypes segregation ratio tested χ2 (p value)non­reduced sepals reduced sepals non­reduced sepals 62 51 11 3:1 1.74 (0.19) non­reduced sepals 59 50 9 3:1 2.94 (0.09) fig. 3 ­ bracts and cаpsules of two nigella damascena mutants with reduced sepals compared to the wild type: a) shs1 mutant, b) shs2 mutant, c) wild type (single flower). fig. 4 ­ сotyledons of two nigella damascena mutants: a) shs1 mutant, b) shs2 mutant. lyakh and soroka ‐ new mutations of flower shape in nigella damascena 293 alleles, was found in caladium (deng and harbaugh, 2006). as noted above, both mutations caused a partial reduction in the size of sepals of nigella flowers. there is an opinion that the size of the flower organs is controlled by one genetic program, while the num­ ber of flower organs is determined by another genet­ ic system, independent of the first one. at the same time, they both regulate the size of the generative organ itself, the flower (weiss et al., 2005). our data on the independent combination of genes that deter­ mine the number of sepals and their size support the above judgment. of the two mutations of reduced sepals identified in nigella, only one (shs1 gene) affected the shape of the flower, turning an ordinary double flower into a rose­like flower with a decorative value. 4. conclusions the preservation and reproduction of plants with such a flower shape for ornamental use in the usual way is problematic due to the inferiority of the female generative sphere. a partial way out of this problem could be the use of the pleiotropic effect detected at the cotyle­ don leaf stage. then the screening of the offspring of heterozygous plants that are the part of a self­ pollinated family, carrying the rose­like flower gene, and a subsequent elimination of seedlings with wild­ type cotyledons, will not only preserve but also allow to use this unique genotype in ornamental floriculture. references benazzouz­smail l., achat s., brahmi f., bachir­bey m., arab r., lorenzo j.m., benbouriche a., boudiab k., hauchard d., boulekbache l., madani kh., 2023 ­ biological properties, phenolic profile, and botanical aspect of nigella sativa l. and nigella dama­ scene l. seeds: a comparative study. ­ molecules, 28(2): 571. deng z., harbaugh b.k., 2006 ­ inheritance of leaf shape and main vein color in caladium. ­ j. amer. soc. hortic. sci., 131: 53­58. galipot p., gerber s., le guilloux m., jabbour f., damerval c., 2021 ­ micro‐and macroscale patterns of petal morphogenesis in nigella damascena (ranunculaceae) revealed by geometric morphometrics and cellular analyses. ­ frontiers plant sci., 12: 2486. griffiths a.j.f., wessler s.r., lewontin r.c., 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2023 37(1): 101­109 doi: 10.36253/ahsc­14258 application of antiperspirants to improve the condition of ornamental plants subject to medium­ and long­dis­ tance transport in refrigerated container l. ghiselli, d. bonetti, d. prisa, s. nin, g. burchi (*) council for agricultural research and economics, research centre for vegetables and ornamental crops, via dei fiori, 8, 51012 pescia (pt), italy. key words: cold storage, nursery industry, post­harvest physiology, potted shrubs. abstract: the ornamental nursery sector sells and delivers its products not only within the european union but throughout the world, thus shipping for long distances has become commonplace in the industry. extended transport times may result in loss of quality and reduced longevity. consequently, an effective logistics strategy is of competitive importance for nursery production. this research was carried out with the aim of improving long­distance transport conditions (up to 6 weeks) of ornamental plants produced in the nurseries of the pistoia district. phenotipic and physiological parameters of plants during transport were studied, testing three biodegradable antiperspirants and a biodegradable microfilm to protect plants on five important pot ornamental species: maple (acer palmatum), cypress (cupressocypari leylandii), privet (ligustrum texanum), nandina (nandina domestica) and viburnum (viburnum tinus). plant tolerance to storage conditions in refrigerated cell or container (t° = 8­12°c) varied considerably according to the considered species, with cypress resulting extremely tolerant and maple and nandina very sensitive. treatments with antiperspirants did not exhibit particularly evident effects on the tested species. the use of biodegradable film was inadequate to protect plant quality during long­distance shipments. even in cases of total or partial loss of leaves by species such as maple and nandina, an optimal recovery of vegetative devel­ opment was highlighted once these species were relocated in outdoor cultiva­ tion. among physiological parameters, mda and phenols contents were the most stress­related variables, being negatively correlated to the quality decay of plants transported in dark refrigerated cells for 2­6 weeks. 1. introduction the nursery district of pistoia (tuscany) is the heart of italian orna­ mental production and leader in europe. this activity covers over 5200 ha, with about ha 1000 of pottery, 1500 companies, over 5500 direct employees (in addition to the related industries) and a gross saleable (*) corresponding author: gianluca.burchi@crea.gov.it citation: ghiselli l., bonetti d., prisa d., nin s., burchi g., 2023 ­ application of antiperspirants to impro‐ ve the condition of ornamental plants subject to medium‐ and long‐distance transport in refrigera‐ ted container. ­ adv. hort. sci., 37(1): 101­109. copyright: © 2023 ghiselli l., bonetti d., prisa d., nin s., burchi g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 january 2023 accepted for publication 27 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14258 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 101­109 102 production over 300 m €, of which 160 m € are exported. the distribution of cultivation is as follows: evergreen trees and shrubs 1600 ha, conifers 1350 ha, ornamental deciduous trees 1420 ha, deciduous shrubs 350 ha, creepers and other shrubs 380 ha, roses 100 ha (marzialetti, 2015). the ornamental nursery sector sells and delivers its products not only within the european union but throughout the world, thus shipping, often for long distances, has become commonplace in the industry. plant transfer, mainly carried by road, rail or some­ times by sea, and in crowded, stifling hot or refriger­ ated truck, can also be very long. the extended ship­ ping and/or storage times may result in loss of quali­ ty. consequently, an effective logistics strategy is of competitive importance for companies to make sure plants are delivered on time and in the best condi­ tions. plants travelling long distances are frequently negatively affected by the critical environmental con­ ditions during transport, such as exclusion from light in closed containers, exposure to harmful gases and temperature extremes, poor air ventilation, high rela­ tive humidity (rh) and vibration. these conditions can lead to deterioration of even the highest quality plants. further, the environmental and physical stresses imposed upon plants during transfer are worsened if plants are improperly produced, incor­ rectly packaged and/or mishandled during shipping or upon receipt. thus, keeping the quality of potted ornamental plants is an essential condition for their commercial success and for promoting trust in cus­ tomer relationships. the main quality parameters for leafy pot plants are the size and the green colour of the leaves (wang et al., 2005). biotic and abiotic stresses during ship­ ping lead to several physiological disorders with numerous negative effects, such as: leaf yellowing due to decreased photosynthesis (starman et al., 2007), leaf and flower abscission with slowed growth and uptake of water and nutrients, color loss of flow­ ers and leaves, damage to cell membrane phospho­ lipids with increased lipid peroxidase (mittler, 2002). phenolic compounds including flavonoids play a role in plant defense against various oxidative stresses, with antioxidant and free radical scavenging activity thus improving plant tolerance to stresses (trchounian et al., 2016; tohidi et al., 2017). the accumulation of these various secondary metabolites has been shown to be influenced by interactions between plant genotype (species, and variety within species) and environmental factors, including cultiva­ tion technique, season, abiotic and biotic stress, and nutrient status (dixon and paiva, 1995; vyn et al., 2002; downey et al., 2006; ksouri et al., 2007). this research was carried out within the in.tra.viva project, funded by the tuscany region, with the aim of improving long­distance transport conditions (up to 6 weeks) of ornamental plants pro­ duced in the nurseries of the pistoia district and to reduce the die­off of potted plants during transport (up to 30%), mainly caused by the fall of the leaves and the inability to recover the vitality that the same plants had on departure. the commitment of crea­ of pescia to the project includes the following research activities: i) monitoring the phenotypic and physiological behaviour of plants during transport; ii) testing new biodegradable antiperspirant products to increase the resistance duration of plants; iii) testing a biodegradable microfilm to protect plants during transport. 2. materials and methods five popular pot plant species, commonly grown for outdoor use, were tested for their tolerance to long distance shipping: maple (acer palmatum), cypress (cupressocypari leylandii), privet (ligustrum texanum), nandina (nandina domestica) and vibur­ num (viburnum tinus). thirty plants of each species were provided by giorgio tesi group, pistoia, at the end of march: the plants were 4 years old, grown in 9 l pots ø 24 cm (maple and viburnum) or 3 l pots ø 18 cm (cypress, privet and nandina). medium and long­distance transport simulation tests were carried out in refrigerated cells (t° = 10°c) at the experimental farm and laboratories of crea research centre for vegetable and ornamental crops (pescia, pt) during spring 2021. the spring sea­ son is the most important and critical season for the farmers, both from an economic and a physiological point of view since the plants are in full vegetation or yet at the beginning of the flowering stage. five plants of each species were placed in the nursery, in open air (oa), thus acting as an untreated control not stored in a refrigerated cell. the remaining 25 plants of each species were transferred in the laboratories on april 2nd, measured, treated with antiperspirants and then placed in a refrigerated cell simulating a medium­distance transport (t1 = 2 weeks, from april 14th to april 28th) and a long­distance transport (t2 = 6 weeks, from april 14th to may 26th) at t° = 10°c. the ghiselli et al. ‐ long‐distance transport of ornamentals 103 plants were subjected to the following treatments: i) spraying with ‘barzaghi­a 10%’ (a) and ii) with ‘barzaghi­b 10%’ (b), two experimental and biodegradable antiperspirants based on car­ boxymethylcellulose; iii) spraying with vapor gard® 5% (v), a commercial antiperspirant based on pino­ lene 96% (di­l­para­menthene); iv) wrapping in a her­ metically sealed experimental biodegradable film (p) provided by lampo (department of chemistry, university of milan); v) spraying with tap water (c), considered as the stored control treatment. antiperspirants (a) and (b) were provided by barzaghi speciality chemicals srl, arluno (mi). the percentages of the active ingredients are not dis­ closed to the public as these ingredients are protect­ ed by patent (n.1428533/15.5.2017). the manufac­ turer claims about the effectiveness of these prod­ ucts based on specific private research. this study could demonstrate the efficacy of this biodegradable antiperspirant and the manufacturer could use this information to market the product to consumers. phenotypical data of all plants (height and diame­ ter) were measured at the start of the trial (april 2nd to 14th), at t1 (april 28th) and t2 (may 26th). leaf physiological measurements (chlorophyll a, chloro­ phyll b, phenols, carotenoids and malondialdehyde content) were carried out on leaf samples collected from all plants of the three evergreen species cypress, privet and viburnum at t1 and t2. on the other hand, semi­evergreen nandina and deciduous maple shrubs lost all their leaves during their stay in the cell, hence pigment analysis and estimates of malondialdehyde levels were not performed on these species. at the end of the cold storage experiment (end of may), all plants were moved to the nursery in open air and placed together with the non­stored control plants (oa). malondialdehyde content (mda), the final prod­ uct of the lipid peroxidation process, is a widely used marker of oxidative lipid injury caused by environ­ mental stress (kong et al., 2016). mda content was measured by 2­thiobarbituric acid (tba) reaction as reported by li et al. (2010). the absorbance of the aqueous phase was detected at 450, 532 and 600 nm. mda content was calculated based on the fol­ lowing formula: c (µmol/g weight) = (6.45 x (a532 ­ a600) ­ 0.56 x a450) / w (sample weight g) leaf chlorophyll, carotenoid and phenol contents were analyzed following the method reported by lichtenthaler and buschmann (2001) on fresh frozen (­80°c) leaf discs obtained by excising 5­6 fully expanded leaves collected from the middle portion of the plants grown in container at t1 and t2. the absorbances of chlorophyll a and b were assessed spectrophotometrically (thermo evolution 300 uv­ visible spectrophotometer) at 665.2 nm, 652.4 nm, and 470 nm, respectively, while carotenoid and phe­ nol absorbances were read at 260 nm and 530 nm, respectively. collected data were subjected to the analysis of variance (anova) to determine the significance level of the different sources of variation: treatment (tr = 5 levels = a, b, v, c, oa), storage time (st = 2 levels = t1 and t2) and tr x st interaction. differences between means were tested using duncan’s multiple comparison test with a confidence level of 95%. the statistical analysis packages used for processing were ibm spss statistics for windows, version 28.0 (ibm corp., armonk, ny). 3. results and discussion no perceptible increments in plant growth (height and diameter) were detected in all refrigerated plant species during the time interval from t0 to t1 and t2, regardless of the type of antiperspirant treatment and biodegradable film used. conversely, control plants kept in open air (oa) grew and developed as expected (data not reported). plant protection with the biodegradable parafilm proved to be ineffective for the purpose. at t1 the biodegradable film resulted perforated in several points by the twigs of the plants, piled up into the refrigerated cell (fig. 1), while at t2 the film was rot­ ten due to the contact with the humidity of the leaves because of their transpiration. thus, the parafilm treatment was not considered in the statisti­ cal analysis. after fifteen days of refrigeration, nandina and maple plants lost part of the leaves, whereas at the end of the refrigerated storage, regardless of the treatment, all the leaves had fallen or rotted on the plant (fig. 2). all nandina plants, when moved out­ doors at the end of may, resumed their vegetative activity, reaching development rates comparable to control plants at the end of october (fig. 3 a). on the contrary, the plants of maple not treated with antiperspirants (c, p and oa) started regularly to veg­ adv. hort. sci., 2023 37(1): 101­109 104 etate again in the nursery but all those treated with antiperspirants (a, b and v) died (fig. 3 b). this phe­ nomenon has to be further investigated but it is pos­ sible that the antiperspirants reduced transpiration of maple and thus the plants suffered a too high level of humidity in the pot substrate. it is very important to avoid excessive wetting of pot substrate before loading the plants into the container and to irrigate the plants a couple of days prior to scheduled ship­ ment, so that the excess water can drain completely. in this trial, maybe the humidity level of the soil sub­ strate resulted correct for the control plants but too high for the plants treated with antiperspirants. no phenotypical differences were observed among cypress plants stored for 15 days and 6 weeks in the refrigerated cells and control plants main­ tained in the field (fig. 4) (data not reported). once moved outdoors in the field, cypresses began to fig. 4 ­ regardless of the type of spraying treatment used, the cypress plants that were kept in refrigerated cells for 6 weeks did not exhibit any damage. fig. 1 ­ the biodegradable parafilm proved to be ineffective for the purpose: at long storage time (t2 = 6 weeks), , rela­ tive humidity levels were too high and there was lack of air circulation; plant transpiration caused the humidity around the leaves to be saturated with water vapor and the pellicle to rot. fig. 2 ­ after 6 weeks of refrigerated storage, all the leaves of nandina (2 a) and maple (2 b) had fallen or rotted on the plant. fig. 3 ­ all nandina plants resumed their vegetative activity once they were relocated in the field reaching development rates comparable to control plants after 5 months of cul­ tivation in open air (3 a). maple plants that were not treated with antiperspirants started regularly to vegetate again in the nursery (3 b, on the left), while those treated with antiperspirants died (3b, on the right). ghiselli et al. ‐ long‐distance transport of ornamentals 105 sprout into new vegetation as the control plants. concerning physiological responses of cypress plants during refrigerated storage, st had a signifi­ cant effect on all considered parameters, except for carotenoid content, while tr x st interaction signifi­ cantly influenced all parameters of cypresses except for mda content (table 1). only mda was significant­ ly affected by plant treatment with antiperspirants. more specifically, plants sprayed with tap water only (c) showed significantly higher level of mda (190.60 µmol/g dw) than those treated with barzaghi biodegradable antiperspirants (a and b) and vapor gard® (v), evidencing a higher level of stress of the untreated plants. mda values were lowest in b and v treated plants (114.97 and 109.76 µmol/g dw, respectively), highlighting some protective action of these antiperspirants on plants subjected to trans­ port stress, even if the phenotypic analyses did not show significant differences among treatments. in general, cypress plants kept in open air (oa) showed the highest values of phenols (fig. 5), carotenoids, chlorophyll a and b compared to stored plants. these plants suffered a late spring frost in mid­april (­0.9°c to ­3.3°c from h 4:00 am to h 9:00 am on april 8, 2021). since plants exposed to various abiotic stress conditions produce many secondary metabolites, including phenolic compounds and carotenoids, in higher concentrations (yeshi et al., 2022), it can be hypothesized that in our experiment plants may have produced high amounts of phenols and carotenoids in response of spring frost hazard. moreover, chlorophyll a and b decreased significantly from t1 to t2, indicating a reduction in plant photo­ synthetic activity during transport over a long period. indeed, it is well known that stressed plants reduce plant metabolism, especially photosynthetic activity, in order to resist adverse conditions (starman et al., 2007). in privet (fig. 6 a) and in viburnum (fig. 6 b), no apparent differences were observed in the growth of plants stored in the refrigerated cell and of control plants maintained in the field (data not reported). about plant development, it was noted that all plants treated with antiperspirants (a, b, v) were character­ ized by new shoot sprouting, which was absent in all plants not sprayed with antiperspirants (c, p, oa) (fig. 6 a, b), the meaning of this phenomenon should be furtherly analysed. moreover, as it was noted also on cypress, all plants of privet and viburnum treated with vapor gard® had shinier and brighter green leaves: this was due to the oily matrix of the product which creates this pleasant optical effect. all cold table 1 ­ effect of storage time (st) and treatment (tr) on oxidative stress (mda), phenols, carotenoids, and chlorophyll contents of pot­ ted cypress ** significant at p≤001; * significant at p≤005; ns= not significant. mean values within each column followed by the same letter are not significantly different at 5% level according to duncan’s multiple range test. fig. 5 ­ effect of treatment x storage time interaction on phenols content of potted cypress. error bars indicate the stan­ dard error of the mean. t1= medium distance transport, 2 weeks; t2 = long distance transport, 6 weeks. source of variation mda µmol/g dw phenols mg/g fw carotenoids µg/g fw chlorophyll a µg/g fw chloropyll b µg/g fw storage time (st) ** ** ns ** * t1 = 2 weeks 162.59 a 196.83 a 0.048 0.277 a 0.195 a t2 = 6 weeks 119.27 b 116.77 b 0.03 0.169 b 0.122 b treatment (tr) ** ns ns ns ns barzaghi­a 141.79 b 139.14 0.025 0.191 0.152 barzaghi­b 114.97 c 118.5 0.023 0.148 0.114 vapor gard ® 109.76 c 130.68 0.026 0.199 0.158 control 190.60 a 126.64 0.031 0.186 0.123 open air control 151.31 b 269.05 0.081 0.391 0.248 st x tr ns ** ** ** * 106 adv. hort. sci., 2023 37(1): 101­109 stored plants recovered after being transferred to the open field at the end of may, resulting in final growth developmental patterns like control plants at the end of october (data not reported). in privet, a, b and v antiperspirant treated plants showed a significantly lower phenol content, while storage time significantly affected carotenoids con­ tent (table 2). in addition, a statistically significant interaction between these factors was found for phe­ nols and chlorophyll a. the untreated oa and c plants showed the highest phenol values: privet out­ door plants experienced spring frost disturbance in mid­april, as described for cypress, while the higher phenols content in c plants suggests that untreated plants get stressed by transport conditions more than plants treated with antiperspirants (a, b and v) (fig. 7). carotenoid content raised in cold stored priv­ et plants from t1 to t2, indicating that plant stress table 2 ­ effect of storage time (st) and treatment (tr) on phenols, carotenoids, and chlorophyll contents of potted privet ** significant at p≤001; * significant at p≤005; ns = not significant. mean values within each column followed by the same letter are not significantly different at 5% level according to duncan’s multiple range test. fig. 6 ­ in privet (6 a: left, vapor gard®; right, control) and in viburnum (6 b: left, vapor gard®; right, control), it was noted that all plants treated with antiperspirants were characterized by new shoot sprouting; moreover, all plants treated with vapor gard® had shinier and brighter green leaves. fig. 7 ­ effect of treatment x storage time interaction on phenols content of potted privet. error bars indicate the standard error of the mean.t1= medium distance transport, 2 weeks; t2 = long distance transport, 6 weeks. source of variation phenols mg/g fw carotenoids µg/g fw chlorophyll a µg/g fw chloropyll b µg/g fw storage time (st) ns ** ns ns t1 = 2 weeks 132.3 0.104 b 0.518 0.324 t2 = 6 weeks 139.5 0.166 a 0.619 0.276 treatment (tr) ** ns ns ns barzaghi­a 119.78 bc 0.142 0.577 0.282 barzaghi­b 116.90 c 0.125 0.514 0.275 vapor gard ® 124.61 bc 0.11 0.479 0.256 control 150.17 ab 0.171 0.717 0.366 open air control 168.06 a 0.128 0.555 0.322 st x tr ** ns * ns ghiselli et al. ‐ long‐distance transport of ornamentals 107 increased during transport with increasing st (table 2). on the contrary, phenols content in oa plants reached the highest value in april (t1) due to late spring frost damages, but thereafter levels were cut down to a normal range within 6 weeks (fig. 7). chlorophyll pigment molecules play a key role in pho­ tosynthesis; plants use chlorophyll to absorb light and convert it into chemical energy (bollivar, 2006). in privet plants, chlorophyll content increased from t1 to t2 in both treated (a, b, and v) and untreated (c) plants maintained inside the cold container, while the untreated open­air (oa) plants showed an oppo­ site trend (fig. 8). in this context, it is probably realis­ tic to assume that the increase in chlorophyll content might be related to water loss occurring in leaves during prolonged storage or transportation rather than to an actual increase in photosynthetic activity (ferrante et al., 2015). the data regarding mda anal­ ysis were not considered for privet. in fact, the method used to assess mda was the thiobarbituric acid (tba) reactive substance assay. this analysis is simple and quick, but it was found to be ineffective for privet species as pointed out by wang et al. (2013). indeed, it seems that there are substances present in the leaves of this species that interfere with the tba reagent. in viburnum species, st significantly affected carotenoids and chlorophylls contents (table 3), with highest values (more shiny leaves) found in plants at t1, contrary to what was recorded for privet. mda levels were influenced by both antiperspirant treat­ ment and tr x st interaction. indeed, mda values were highest in the control and in plants treated with antiperspirants a and b after 6 weeks of cold storage (t2), indicating that viburnum shrubs get more stress with increasing storage time (fig. 9). on the other hand, vapor gard® (v), seemed to exert some pro­ tective action on viburnum plants over time. as expected, plants maintained in open air showed no table 3 ­ effect of storage time (st) and treatment (tr) on oxidative stress (mda), phenols, carotenoids, and chlorophyll contents of viburnum privet ** significant at p≤001; * significant at p≤005; ns = not significant. mean values within each column followed by the same letter are not significantly different at 5% level according to duncan’s multiple range test. fig. 8 ­ effect of treatment x storage time interaction on chloro­ phyll a content of potted privet. error bars indicate the standard error of the mean.t1= medium distance trans­ port, 2 weeks; t2 = long distance transport, 6 weeks. fig. 9 ­ effect of treatment x storage time interaction on malon­ dialdehyde content of potted viburnum. error bars indi­ cate the standard error of the mean.t1= medium dis­ tance transport, 2 weeks; t2 = long distance transport, 6 weeks. source of variation mda µmol/g dw carotenoids µg/g fw chlorophyll a µg/g fw chloropyll b µg/g fw storage time (st) ns ** ** ** t1 = 2 weeks 136.22 0.233 a 0.918 a 0.438 a t2 = 6 weeks 151.77 0.043 b 0.226 b 0.117 b treatment (tr) ** ns ns ns barzaghi­a 223.97 a 0.15 0.555 0.261 barzaghi­b 141.15 b 0.124 0.513 0.242 vapor gard ® 122.73 b 0.101 0.501 0.286 control 150.78 b 0.145 0.626 0.302 open air control 81.34 c 0.169 0.665 0.296 st x tr ** ns ns ns adv. hort. sci., 2023 37(1): 101­109 108 signs of stress over the long term. it is likely that dark conditions and lack of water over a 6­week period are not limiting factors for the tested species once the optimal conditions of tem­ perature and humidity are met into the container, as it was during our trials. potted plants must be ade­ quately prepared and carefully handled before long­ distance transport to overcome problems of this transitory phase by reducing both the plant’s metabolism and normal physiological processes. in general, even if phenotypical data did not show evident differences for plant growth between long term stored and not stored plants, the physiological analyses on cypressus, privet and viburnum showed interesting significant differences for mda, phenols and carotenoids: these parameters seem to be corre­ lated to abiotic storage stress of plants and thus could be useful in further studies to monitor the quality of plants before, during and after storage for short, mid and long times in refrigerated cells. this could also help various sectors of the post­harvest ornamentals supply chain to: i) assess the potential quality of plants before shipment; ii) improve plant transport conditions; iii) monitor plant quality throughout the various stages of shipping “from farm to buyer”, through the various steps with other com­ ponents of the supply chain (transporters, whole­ salers, markets); iv) understand whether any deterio­ ration in the quality of the plants at the end of the travel was perhaps due to non­maintenance of the optimal conditions envisaged during transport, due to negligence by the operators. 4. conclusions plant tolerance to storage conditions in refrigerat­ ed cell or container (t° = 8­12°c) varied considerably according to the considered species. cypress proved to be extremely tolerant to storage conditions over long periods. maple and nandina, on the contrary, resulted the most sensitive species to medium­ and long­distance transport with a high percentage of fallen or rotten leaves occurring during spring storage in refrigerated cells. treatments with antiperspirants did not exhibit particularly evident effect on quality value (plant growth and aesthetic appearance of the leaves) in plants kept in the dark in a cold room or container. only the antiperspirant vapor gard® seemed to improve the aesthetic appearance of cypress, viburnum, and privet with shinier and brighter green leaves, probably due to the oily matrix of the product. furthermore, even in cases of total or partial loss of leaves by species such as maple and nandina, an optimal recovery of vegetative develop­ ment was highlighted once these species were relo­ cated in outdoor cultivation. the use of the tested biodegradable film was inadequate to protect plant quality during long­distance shipments, thus, further research is needed to improve microfilm perfor­ mances by changing its thickness and composition. among physiological parameters, mda, phenols, and carotenoids contents were the most stress­related variables, being negatively correlated to the quality decay of plants transported in dark refrigerated cells for 2­6 weeks. it is a preliminary study and some uncertainty and/or not complete discussion are due to the lack of some measurement (i.e. leaf colour)., however these parameters could be useful in further studies to monitor the quality of plants before, dur­ ing and after storage for short­, mid­ and long­term transport in refrigerated containers. acknowledgements this research was carried out within the in.tra.viva project, funded by the tuscany region (psr 2014­2020 ­ measure 16.2 ps­go 37/2017). references bollivar d.w., 2006 ­ recent advances in chlorophyll biosynthesis. ­ photosynth. res., 89: 1­22. dixon r.a., paiva n.l., 1995 ­ stress‐induced phenyl‐ propanoid metabolism. ­ plant cell, 7: 1055­1097. downey m.o., 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abiotic stresses has potential application in pharmaceu‐ tical product development. ­ molecules, 27(1): 313. impaginato 245 adv. hort. sci., 2025 39(3): 245­257 doi: 10.36253/ahsc­18140 https://oaj.fupress.net/index.php/ahs achillea millefolium l.: a comprehensive review of its phytochemistry and pharmacological properties d. prisa 1 (*), a. jamal 2 1 crea research centre for vegetable and ornamental crops, council for agricultural research and economics, via dei fiori 8, 51012 pescia, pt, italy. 2 department of soil and environmental sciences, faculty of crop production sciences, the university of agriculture, peshawar 25130, pakistan. key words: antioxidant activity, essential oils, ethnopharmacology, flavonoids, wound healing. abstract: achillea millefolium l., commonly known as yarrow, is a perennial herb traditionally used in various cultures for its therapeutic properties. belonging to the asteraceae family, it has gained attention for its rich phytochemical composition, including flavonoids, terpenoids, sesquiterpene lactones, phenolic acids, and essential oils. these bioactive compounds contribute to a wide range of pharmacological activities such as anti­ inflammatory, antimicrobial, antioxidant, antispasmodic, and wound­healing effects. ethnobotanical use of yarrow includes treatment of gastrointestinal disorders, skin injuries, menstrual irregularities, and respiratory infections. modern preclinical studies have substantiated many of these traditional claims, although clinical validation remains limited. additionally, its essential oil components show potential in cosmetic and food industries due to their preservative and aromatic properties. despite its therapeutic promise, concerns related to allergic reactions and toxicity at high doses, particularly from thujone­containing oils, warrant further safety assessment. this review aims to consolidate current scientific knowledge on a. millefolium, highlighting its pharmacological relevance, phytochemical diversity, and future potential in evidence­based herbal medicine and natural product development. 1. introduction achillea millefolium l., commonly known as yarrow, is a herbaceous perennial species belonging to the asteraceae family, historically recognized for its extensive use in traditional medicine across europe, asia, and north america. references to its medicinal applications date back to ancient greece, where the warrior achilles was said to use the (*) corresponding author: domenico.prisa@gmail.com citation: prisa d., jamal a., 2025 ­ achillea millefolium l.: a comprehensive review of its phytochemistry and pharmacological properties. ­ adv. hort. sci., 39(3): 245­257. orcid: pd: 0000­0001­8213­8895 ja: 0000­0001­8518­3130 copyright: © 2025 prisa d., jamal a. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. credit author contribution received for publication 1 july 2025 accepted for publication 25 august 2025 ahs advances in horticultural science review paper ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-18140 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-8213-8895 http://orcid.org/0000-0001-8518-3130 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 245­257 246 plant to treat battlefield wounds­hence its genus name achillea (applequist and moerman, 2011). traditionally, yarrow has been employed to treat a wide array of conditions including gastrointestinal disturbances, menstrual irregularities, respiratory infections, wounds, and skin inflammations (falk et al., 1975). these uses have prompted growing scientific interest, especially as research has begun to validate many of its ethnomedicinal claims through the lens of modern pharmacology. phytochemical investigations reveal that a. millefolium is rich in secondary metabolites such as flavonoids (e.g., apigenin, luteolin), phenolic acids (e.g., caffeic and chlorogenic acids), sesquiterpene lactones (e.g., achillicin), and essential oils including monoterpenes (e.g., camphor, 1,8­cineole) (falconieri et al., 2011; judzentiene, 2016). these constituents have demonstrated diverse biological activities in vitro and in vivo, including antioxidant, anti­inflammatory, antimicrobial, antispasmodic, wound­healing, and neuroprotective effects (montanari et al., 1998; amirghofran and karimi, 2002; keser et al., 2013; dias et al., 2013). however, while these preclinical studies provide a strong pharmacological basis for traditional claims, the translation of these findings into clinical practice remains inconsistent and underdeveloped. a critical issue is the disconnect between laboratory evidence and human trials. many clinical studies on yarrow suffer from limitations such as small sample sizes, poor extract characterization, variable dosages, and lack of placebo control. for example, clinical trials investigating its efficacy in dysmenorrhea or functional gastrointestinal disorders have shown promise but lack the methodological rigor required for medical validation (madisch et al . , 2004; benedek et al . , 2005). moreover, the high variability in phytochemical content across different ecotypes and growing conditions complicates standardization, which is essential for reproducibility and regulatory approval (georgieva et al., 2015). underexplored but promising therapeutic areas include neuroprotection, metabolic regulation, and dermatological applications. animal studies have highlighted gabaergic modulation and acetylcholinesterase inhibition by flavonoids, suggesting anxiolytic and cognitive­supportive effects, yet robust human data are absent (falk et al., 1975; ivancheva et al., 2002). similarly, studies in diabetic rats show hypoglycemic and lipid­lowering effects (judzentiene and mockute, 2010), warranting further investigation in metabolic syndrome and type 2 diabetes. in addition to its pharmacological significance, a. millefolium’s ecological resilience and adaptability to various climates offer potential for sustainable cultivation and industrial use. recent efforts in optimizing cultivation conditions, such as soil composition, irrigation regimes, and genetic selection, show promise in enhancing biomass yield and phytochemical uniformity (stojanović et al., 2005; benedek et al., 2007). incorporating these agronomic insights into large­scale production models could support supply chain consistency for pharmaceutical and nutraceutical development. furthermore, to ensure safe integration into therapeutic systems, clearer safety profiles and potential interactions must be addressed. while generally considered safe, compounds like thujone and sesquiterpene lactones require careful dosing and labeling. regulatory frameworks, including ema and commission e monographs, support traditional use but demand further toxicological evaluation, especially for high­dose or long­term applications. this review aims to synthesize current scientific knowledge on a. millefolium , emphasizing standardized extraction methods, quantification of bioactive constituents, clinical applicability, and sustainability. by bridging traditional use with scientific rigor, a. millefolium can emerge as a credible, multifunctional agent in herbal therapeutics and commercial natural product development. 2. taxonomy and botanical description recognized both in traditional medicine and botany, achillea millefolium l. (yarrow) is a taxonomically diverse member of the asteraceae family, one of the largest plant families globally with over 1,600 genera and 23,000 species (khan and gilani, 2011) (fig. 1). the genus achillea comprises over 100 species, primarily distributed across europe, temperate asia, and north america. among these, a. millefolium is the most widely known and studied due to its medicinal and ecological importance (pires et al . , 2009). botanically, a. millefolium is a rhizomatous, herbaceous perennial that typically grows between 30 and 90 cm in height. it features a highly branched, erect stem covered with fine hairs (babaei et al., 2007). the leaves are alternate, prisa and jamal ‐ yarrow phytochemistry review 247 ecotypes. molecular studies using dna barcoding and issr markers have revealed significant intraspecific diversity, especially among populations in europe and north america (dalsenter et al., 2004; benedek et al., 2007). ecologically, a. millefolium thrives in a variety of habitats, including meadows, grasslands, roadsides, and disturbed soils. it is tolerant to drought and poor soils, making it a resilient species in both wild and cultivated environments (stojanović et al., 2005). its robust root system and allelopathic properties also make it useful in soil stabilization and ecological restoration projects (lazarevic et al., 2010). 3. phytochemical composition achillea millefolium l. is a chemically rich plant whose therapeutic properties are largely attributed to its diverse array of bioactive secondary metabolites (table 1) (georgieva et al., 2015). these include flavonoids, terpenoids, phenolic acids, sesquiterpene lactones, coumarins, tannins, and alkaloids, among others. both the aerial parts and essential oils of the plant have been extensively studied, revealing a complex chemical profile that varies depending on geographical origin, harvesting time, plant part, and extraction method (usmanghani et al., 1997; falconieri et al., 2011; keser et al., 2013). flavonoids are one of the major phytochemical classes in a. millefolium, known for their antioxidant, anti­ inflammatory, and vasoprotective effects (dall’acqua et al . , 2011). common flavonoids identified include: apigenin; luteolin; quercetin; kaempferol; rutin. these compounds are present mainly as glycosides and aglycones in the leaves and inflorescences. luteolin and apigenin have demonstrated anti­ inflammatory activity via inhibition of cytokine release and cox­2 expression (lopes et al., 2005). phenolic acids are potent antioxidants that contribute to free radical scavenging and metal ion chelation. in a. millefolium , the following are commonly found: caffeic acid; chlorogenic acid; ferulic acid; p­coumaric acid. chlorogenic acid, in particular, is known for its hepatoprotective and anti­ diabetic effects (judzentiene and mockute, 2010). the essential oil composition of a. millefolium bipinnate to tripinnate, finely dissected, and resemble feathers­hence the species epithet millefolium, meaning “thousand leaves” in latin (li et al., 2011). these leaves emit a strong, characteristic aroma when crushed, attributed to its volatile oil content (jaimand et al., 2006). the inflorescence is a compound corymb composed of numerous small capitula, each containing both central disc florets and peripheral ray florets. flower colors range from white and pale pink to purple, depending on the variety and environmental conditions (applequist and moerman, 2011). the plant flowers from late spring to early fall and is an important nectar source for pollinators. achillea millefolium exhibits considerable morphological and genetic variability, which has led to taxonomic complexities and ongoing debate regarding its classification into subspecies and fig. 1 ­ achillea millefolium: taxonomy and botanical description. adv. hort. sci., 2025 39(3): 245­257 248 varies widely depending on chemotype but typically includes: monoterpenes: camphor, 1,8­cineole (eucalyptol), α­ and β­pinene, sabinene; sesquiterpenes: β­caryophyllene, chamazulene, germacrene d; thujone (α­ and β­thujone): a controversial component due to its potential neurotoxicity at high concentrations. these volatile compounds confer antimicrobial, spasmolytic, and carminative properties (goldberg et al . , 1969). the oil is typically blue due to the presence of chamazulene, formed from matricin during distillation (dalsenter et al., 2004). found primarily in the aerial parts, sesquiterpene lactones such as achillicin and millefolide are known for their anti­inflammatory and cytotoxic properties. these compounds act by inhibiting nf­κb signaling pathways and interfering with cell proliferation (applequist and moerman, 2011). coumarins such as umbelliferone and scopoletin have been reported in small amounts and are recognized for their anti­coagulant and anti­microbial effects. tannins contribute to the plant’s astringent and wound­healing activities, useful in topical formulations for cuts and abrasions. although present in lower concentrations, alkaloids such as stachydrine and betonicine have been detected and may require caution, particularly during pregnancy. polysaccharides and sterols, such as β­sitosterol, have also been isolated from a. millefolium , contributing to its immunomodulatory and cholesterol­lowering properties (lazarevic et al., 2010). 4. pharmacological activities achillea millefolium l. exhibits a broad spectrum of pharmacological effects, many of which are consistent with its extensive traditional use in herbal medicine. modern phytopharmacological studies have confirmed numerous biological activities, largely attributed to its rich phytochemical composition, including flavonoids, terpenoids, phenolic acids, sesquiterpene lactones, and essential oils (fig. 2) (kristoffersen et al., 2022). the following sections summarize key pharmacological properties with supporting scientific evidence. anti‐inflammatory activity the anti­inflammatory effects of a. millefolium are among its most studied properties. extracts, particularly those rich in flavonoids (e.g., apigenin, luteolin), and sesquiterpene lactones, have table 1 ­ phytochemical composition of achillea millefolium phytochemical class main compounds location in plant biological activities flavonoids apigenin, luteolin, rutin, quercetin, kaempferol leaves, flowers antioxidant, anti­inflammatory, spasmolytic, vasoprotective phenolic acids caffeic acid, chlorogenic acid, ferulic acid leaves, stems, flowers antioxidant, hepatoprotective, antimicrobial sesquiterpene lactones achillicin, millefolide, proazulene derivatives aerial parts (mainly flowers) anti­inflammatory, cytotoxic, antimicrobial essential oils camphor, 1,8­cineole, thujone, borneol, chamazulene flowers, leaves antimicrobial, anti­inflammatory, carminative tannins hydrolyzable and condensed tannins whole plant astringent, wound healing, antimicrobial coumarins umbelliferone, scopoletin stems, flowers anticoagulant, anti­inflammatory alkaloids stachydrine, betonicine leaves, roots (in trace) uterotonic, cardiotonic (possible) sterols β­sitosterol whole plant anti­inflammatory, cholesterol­lowering polysaccharides arabinogalactans, pectins aerial parts immunomodulatory, healing support prisa and jamal ‐ yarrow phytochemistry review 249 demonstrated significant inhibitory effects on pro­ inflammatory mediators such as prostaglandins, cytokines (e.g., tnf­α, il­6), and nitric oxide (no) (amirghofran and karimi, 2002). mechanism of action: inhibition of cyclooxygenase (cox) and lipoxygenase (lox) enzymes; suppression of nf­κb and mapk signaling pathways; reduction in leukocyte infiltration and edema in animal models (falconieri et al., 2011). these findings support its traditional use for inflammatory conditions such as arthritis, menstrual cramps, and gastrointestinal inflammation. antimicrobial and antiviral effects essential oils and extracts from a. millefolium show broad­spectrum antimicrobial activity against bacteria, fungi, and some viruses. volati le constituents like camphor, 1,8­cineole, and α­thujone are largely responsible for these effects (baretta et al., 2012). reported effects: inhibition of staphylococcus aureus, escherichia coli, candida albicans, and helicobacter pylori (falconieri et al., 2011; gharibi et al., 2011); suppression of biofilm formation and quorum sensing (guo et al., 2008); moderate activity against herpes simplex virus in vitro (falk et al., 1975). these properties may support the external and internal use of yarrow for infections, wounds, and gastrointestinal disturbances. antioxidant activity achillea millefolium has potent antioxidant properties due to its high content of polyphenolic compounds, including caffeic acid, chlorogenic acid, flavonoids, and chamazulene (candan et al., 2003). effects observed: strong free radical scavenging (dpph, abts assays); chelation of metal ions (e.g., fe²⁺); protection against oxidative damage in lipid and protein models (keser et al., 2013; judzentiene, 2016). antioxidant activity is believed to underlie many of its protective effects in cardiovascular, neurodegenerative, and metabolic disorders. wound healing and hemostatic effects historically known as “soldier’s woundwort,” yarrow has long been applied to cuts and wounds (anne et al., 2006). preclinical studies confirm its effectiveness in accelerating wound closure and improving tissue regeneration (falconieri et al., 2011). mechanisms involved: stimulation of fibroblast proliferation and collagen synthesis; anti­ inflammatory and antimicrobial protection of the wound site; local vasoconstriction and astringent action facilitating hemostasis (borrelli et al., 2012). a topical gel containing a. millefolium extract significantly improved healing in full­thickness wounds in rats, supporting its use in dermatological applications (applequist and moerman, 2011). gastroprotective and antispasmodic activity achillea millefolium is traditionally used to treat dyspepsia, ulcers, and irritable bowel syndrome. studies in animal models demonstrate: reduction in gastric ulceration and acidity; relaxation of intestinal smooth muscle (antispasmodic); inhibition of h. pylori and pro­inflammatory cytokines in gastric tissues (gharibi et al., 2011). these actions are linked to flavonoids and fig. 2 ­ graphical abstract of the pharmacological activities of achillea millefolium. this infographic illustrates the primary pharmacological properties of achillea millefolium (yarrow), supported by preclinical and limited clinical evidence. the central botanical drawing is surrounded by key bioactivities, including anti­ inflammatory, antimicrobial, antioxidant, gastroprotective, hepatoprotective, wound healing, spasmolytic, and anxiolytic effects. these therapeutic actions are attributed to the plant's diverse phytochemical constituents, such as flavonoids, sesquiterpene lactones, and essential oils. 250 adv. hort. sci., 2025 39(3): 245­257 table 2 ­ toxicity data summary essential oil components that modulate gut motility and inflammation. metabolic and antidiabetic effects recent studies suggest that a. millefolium may have antidiabetic and lipid­lowering potential. judzentiene and mockute (2010) reported that ethanolic extracts: lowered blood glucose and total cholesterol in diabetic rats; enhanced antioxidant enzyme activity (sod, cat); increased collagen synthesis and improved skin morphology. such findings indicate potential for a. millefolium in metabolic syndrome and diabetes­related complications. neuroprotective and anxiolytic effects preliminary studies indicate that achillea millefolium may exert beneficial effects on the central nervous system. its mild anxiolytic and sedative properties, observed in animal models, are primarily attributed to the presence of flavonoids and monoterpenes (falk et al., 1975). additionally, the plant has demonstrated acetylcholinesterase inhibitory activity, suggesting potential for cognitive enhancement and neuroprotection (keser et al., 2013). these findings highlight the plant’s promise as a candidate for further investigation in the context of neurodegenerative diseases and stress­related disorders. anticancer activity some sesquiterpene lactones and flavonoids from a. millefolium have shown cytotoxic activity against cancer cell l ines, including breast, colon, and leukemia cells (amirghofran and karimi, 2002). mechanisms may involve: induction of apoptosis; cell cycle arrest; anti­angiogenic effects. however, clinical relevance is yet to be established. 5. toxicity and safety while achillea millefolium is widely regarded as a safe medicinal plant in traditional medicine and herbal formulations, its long­term safety and toxicity profile warrant careful examination, particularly due to certain bioactive constituents such as thujone, sesquiterpene lactones, and potential allergenic compounds (table 2) (kazemi, 2015). understanding the safety of yarrow is essential for its proper therapeutic use and formulation in nutraceuticals, cosmeceuticals, and pharmaceuticals. general safety and traditional use yarrow has a long history of traditional use, including as a tea, tincture, topical poultice, and essential oil. it is included in several national pharmacopeias (e.g., british herbal pharmacopoeia, european pharmacopoeia) for its wound­healing and digestive properties. at traditional dosages, it is generally well tolerated with low acute toxicity (falk et al., 1975). thujone content and neurotoxicity risk one of the main safety concerns associated with a. millefolium is the presence of α­thujone and β­ thujone, monoterpene ketones found in the plant’s essential oils (farasati far et al., 2023). these compounds are known to be neurotoxic in high aspect evidence summary acute toxicity low; ld₅₀ > 2,000 mg/kg in rodents (oral extracts) chronic toxicity limited data; high doses may stress liver/kidney function allergenicity moderate risk in sensitive individuals (sesquiterpene lactones) thujone neurotoxicity present in essential oils; not recommended for internal use in concentrated form teratogenicity not well studied; avoided during pregnancy due to uterine stimulation drug interactions possible with anticoagulants, sedatives, nsaids prisa and jamal ‐ yarrow phytochemistry review 251 doses, capable of causing: seizures; restlessness and hallucinations; convulsions in extreme cases. thujone acts as a gaba_a receptor antagonist, reducing inhibitory neurotransmission in the central nervous system. the european food safety authority (efsa) has established a maximum daily intake level of 0.1 mg/kg body weight/day for thujone (efsa, 2012). although thujone levels in commercial yarrow preparations are generally below this threshold, concentrated essential oils may exceed it if improperly dosed or ingested (dalsenter et al., 2004). allergic reactions and contact dermatitis as a member of the asteraceae family, yarrow contains sesquiterpene lactones, which are known sensitizers and can cause: allergic contact dermatitis; photosensitivity reactions; skin rashes and itching, especially with topical use. individuals allergic to other asteraceae species (e.g., chamomile, ragweed, daisies) are more likely to react adversely to yarrow (lazarevic et al., 2010). patch testing is advisable before widespread dermal application. reproductive and pregnancy concerns although traditionally used to regulate menstruation, achillea millefolium has emmenagogue and uterotonic properties, which may stimulate uterine contractions (hajhashemi et al., 2016). animal studies have shown that high doses can potentially influence reproductive hormones or embryo implantation (amirghofran and karimi, 2002). therefore: not recommended during pregnancy, especially the first trimester; avoid use in women with heavy menstruation or bleeding disorders. safety during lactation has not been adequately studied, and caution is advised. hepatic and renal effects limited evidence suggests that chronic high­dose exposure may exert stress on liver enzymes and renal markers in animals. however, these effects are dose­ dependent and typically associated with non­ standardized or excessive use of extracts or essential oils (gharibi et al., 2011). no hepatotoxicity has been reported in traditional doses or short­term clinical use. drug interactions yarrow contains compounds that may interact with conventional medications (ghavami et al., 2010), such as: anticoagulants (due to coumarins and flavonoids) ­ increased risk of bleeding; sedatives or cns depressants ­ possible additive effects; nsaids or corticosteroids ­ overlapping anti­inflammatory actions may enhance effects or side effects. patients on medication should consult a healthcare provider before using a. millefolium. clinical and regulatory observations the german commission e monograph considers achillea millefolium safe when used for dyspepsia and loss of appetite, with minor side effects such as photosensitivity reported rarely. the european medicines agency (ema) includes it in the list of herbal substances for traditional use, acknowledging its safety in specified indications and dosages (gervais, 1977). no major adverse effects were reported in small­ scale clinical trials involving topical or oral use (applequist and moerman, 2011). 6. clinical evidence and applications despite its long­standing use in traditional medicine and promising preclinical findings, clinical evidence for achillea millefolium (yarrow) remains limited but steadily growing. most human data come from small­scale trials, ethnopharmacological surveys, and herbal combination studies, rather than large randomized controlled trials (rcts) (fierascu et al., 2015). nevertheless, these studies offer valuable insights into the plant’s therapeutic potential across various systems, especially in gastrointestinal health, wound healing, gynecological care, and cosmeceutical applications (fig. 3). gastrointestinal disorders the most well­supported clinical applications of a. millefolium involve its spasmolytic, carminative, and anti­inflammatory properties in the treatment of functional dyspepsia, gastritis, and irritable bowel syndrome (ibs) (eghdami and sadeghi, 2010). in a randomized clinical trial, a combination herbal formulation containing a. millefolium, mentha piperita, and matricaria chamomilla significantly reduced symptoms of indigestion, bloating, and abdominal pain in patients with functional dyspepsia (madisch et al., 2004). these effects are attributed to the plant’s ability to relax smooth muscle, reduce gastric inflammation, and modulate gut motility. adv. hort. sci., 2025 39(3): 245­257 252 fig. 3 ­ graphical abstract of the clinical evidence and applica­ tions of achillea millefolium. this infographic summarizes the primary clinical applications of achillea millefolium supported by traditional use and emerging scientific evi­ dence. highlighted therapeutic areas include gastroin­ testinal disorders (e.g., functional dyspepsia, irritable bowel syndrome), gynecological health (e.g., dysmenor­ rhea, menstrual regulation), wound healing (topical use for skin inflammation), and cosmeceutical applications (e.g., treatment of dermatitis and inclusion in skincare formulations). these uses reflect the plant’s anti­inflam­ matory, spasmolytic, and tissue­repairing properties. wound healing and dermatology topical applications of a. millefolium have been investigated for accelerating wound healing, particularly due to its astringent, antimicrobial, and anti­inflammatory properties (tozyo et al., 1994). in a clinical case series, a yarrow­based cream applied to superficial skin wounds and minor cuts resulted in faster re­epithelialization and lower infection rates compared to standard treatment (csupor et al., 2009). another clinical study reported that a topical gel containing yarrow extract improved skin hydration and reduced inflammation in patients with mild eczema (applequist and moerman, 2011). these outcomes are consistent with earlier in vivo findings demonstrating increased fibroblast activity, collagen deposition, and wound closure speed (konyalioglu and karamenderes, 2005). gynecological health traditionally used for regulating menstruation and relieving menstrual cramps, yarrow has also shown promise in early clinical studies (de souza et al., 2011). a randomized trial in iran found that a. millefolium extract capsules reduced dysmenorrhea pain intensity and duration in adolescent girls compared to placebo, likely due to the plant’s antispasmodic and anti­inflammatory constituents (benedek et al., 2005). additionally, herbal vaginal washes containing yarrow are used in some clinical settings for vaginitis and vaginal irritation, with anecdotal evidence of symptom relief and microbial balance restoration. cosmeceutical and topical use achillea millefolium is increasingly featured in cosmetic and dermatological formulations due to its anti­inflammatory, antioxidant, and astringent properties (farooq et al., 2012). clinical­grade formulations such as creams, lotions, serums, and cleansing gels are used for: acne and oily skin regulation; soothing inflamed or sensitive skin; enhancing skin tone and elasticity. preliminary human­use studies report good tolerability and efficacy, though large­scale dermatological trials are still lacking (falk et al., 1975). metabolic and cardiovascular effects (emerging area) while human data remain sparse, animal studies showing antidiabetic, antihyperlipidemic, and antioxidant effects of a. millefolium have encouraged nutraceutical exploration (gadgoli and mishra, 2007). a pilot human study using a multi­herbal formula containing yarrow in type 2 diabetic patients showed modest improvements in fasting glucose and lipid profi le, though it was not placebo­controlled (judzentiene and mockute, 2010). the presence of flavonoids and phenolic acids may contribute to insulin sensitization and lipid­lowering effects, indicating future potential pending more rigorous trials. traditional and integrative medical use yarrow is officially recognized in several european herbal monographs (de santanna et al., 2009), including those from: german commission e, which approves it for gastrointestinal spasms and minor inflammation; european medicines agency (ema), which lists it as a traditional herbal remedy for dyspeptic complaints and minor skin inflammations prisa and jamal ‐ yarrow phytochemistry review 253 it is also included in integrative and naturopathic protocols, often combined with other herbs like peppermint, calendula, or chamomile for synergistic effects. 7. industrial and cosmetic applications achillea millefolium l. has garnered increasing industrial interest due to its rich phytochemical profile, which includes volatile oils, flavonoids, phenolic acids, and sesquiterpene lactones (costescu et al., 2014). these constituents contribute to the plant’s antimicrobial, anti­inflammatory, antioxidant, and astringent properties—traits that are highly valued in the cosmetic, pharmaceutical, nutraceutical, and agricultural industries. yarrow is used in a wide range of commercial products from topical skin formulations to botanical extracts in eco­ friendly agriculture (candan et al., 2003). cosmetic industry applications the skin­soothing and antimicrobial qualities of yarrow make it a valuable ingredient in the cosmetic and personal care industries (boskovic et al., 2005). it is commonly incorporated into products designed for sensitive, inflamed, or acne­prone skin, owing to its natural anti­ inflammatory and wound­healing activities. common cosmetic uses: facial cleansers and toners: used for its astringent action that tightens pores and reduces oiliness; moisturizers and serums: yarrow extracts are included to reduce redness, irritation, and signs of inflammation in sensitive or damaged skin; anti­aging formulations: its antioxidant compounds (e.g., flavonoids, phenolic acids) combat oxidative stress and may protect against premature aging; aftershave and soothing balms: yarrow’s cooling and antimicrobial effects aid in reducing razor burn and skin irritation; scalp and hair care: infusions or extracts are used in shampoos to treat dandruff and soothe itchy or inflamed scalps. in a 2021 study, topical application of a yarrow­ containing cream led to significant improvements in skin hydration, elasticity, and reduction of inflammatory skin symptoms, such as in mild cases of eczema and dermatitis (applequist and moerman, 2011). pharmaceutical and herbal formulations industrially, yarrow is processed into standardized extracts, tinctures, capsules, and teas that are distributed through herbal supplement markets. it is especially popular in european phytotherapy and is listed in the european pharmacopoeia (david et al., 2010). key pharmaceutical uses include: digestive aids: as an ingredient in herbal bitters, often in combination with gentian, peppermint, and fennel, for bloating, dyspepsia, and poor appetite; topical antiseptic agents: creams and gels containing yarrow extract are used for minor wounds, burns, and abrasions, owing to its antibacterial and wound­ healing properties; women’s health: included in herbal combinations for menstrual regulation and pain relief. industrial formulations often standardize yarrow preparations to their flavonoid or essential oil content, ensuring consistent therapeutic outcomes. nutraceuticals and functional foods with growing consumer interest in natural health products, yarrow is emerging in the functional food and beverage sector (bimbiraite et al., 2008), especially in: herbal teas and infusions: dried aerial parts of the plant are used in herbal blends marketed for digestion, relaxation, and detox; functional beverages: combined with other herbs to create health­promoting drinks that claim anti­ inflammatory or digestive benefits; botanical dietary supplements: in capsule or powder form, yarrow is marketed for gastrointestinal support and immune modulation. a recent trend in europe includes the incorporation of yarrow extracts into fortified waters and botanical sodas, emphasizing their antioxidant potential and natural origin (keser et al., 2013). essential oil and fragrance industry yarrow’s essential oil, obtained through steam disti l lation of the flowering tops, is used in (cavalcanti et al., 2006): aromatherapy: for stress relief, minor wound care, and muscle relaxation; perfume and fragrance formulations: due to its herbal, sweet, and slightly camphoraceous scent; massage oils: blended with carrier oils to relieve muscle tension and improve circulation. its striking blue color, due to chamazulene formed during distillation, enhances its visual and marketing appeal in natural product lines. ecological and agricultural applications yarrow also plays a role in sustainable agriculture adv. hort. sci., 2025 39(3): 245­257 254 and ecological industries (benetis et al., 2008) natural pest repellent: volatile oils act as a mild insect deterrent; pollinator support: its extended flowering season attracts bees and beneficial insects, enhancing crop pollination; soil stabilization and phytoremediation: its deep roots help prevent erosion and assist in soil recovery in degraded environments (lazarevic et al., 2010). yarrow is increasingly used in organic farming as a companion plant and in biodynamic preparations to improve soil vitality and compost activity. industrial processing considerations for commercial applications, the plant is typically harvested during full bloom, when phytochemical content is at its peak. it is processed into: dried herb (cut or powdered); aqueous, ethanol, or co₂ extracts; steam­distilled essential oil; freeze­dried powders for encapsulation or food formulation standardization and quality control are critical for maintaining efficacy and ensuring regulatory compliance, particularly for exports in the eu and north american markets (niu, 2020). 8. discussion and conclusions despite the wealth of traditional knowledge and preclinical data supporting the pharmacological efficacy of achillea millefolium, its integration into modern evidence­based medicine is constrained by several gaps (akram, 2013). 1. clinical vs. preclinical gap: while many laboratory and animal studies demonstrate significant pharmacological activities, translation into clinical application is weak. most clinical studies suffer from small sample sizes, lack of placebo­controlled designs, and inconsistent dosing protocols. for instance, while yarrow showed strong spasmolytic and anti­inflammatory activity in models of irritable bowel syndrome and gastritis, clinical trials validating these effects are limited and methodologically fragile (madisch et al., 2004; eghdami and sadeghi, 2010) 2. phytochemical variability and standardization: one major challenge in assessing efficacy across studies is the phytochemical variability of a. millefolium. composition can fluctuate based on environmental conditions, harvesting time, and extraction method (georgieva et al., 2015). this affects reproducibility of results and complicates formulation standardization, especially when active constituents such as flavonoids or thujone­containing essential oils vary widely in content. future studies should prioritize well­characterized, standardized extracts with quantification of key bioactives. 3. conflicting and inconclusive evidence: in several areas, research findings either conflict or remain inconclusive. for example, while some studies show notable anxiolytic or cognitive­enhancing effects, others do not observe such benefits, likely due to variability in extract types and dosing. a comparative analysis of different extracts and their phytochemical profiles may clarify these discrepancies. 4. underexplored therapeutic areas: neuro­ protective and metabolic effects of a. millefolium represent two of the most promising yet under­ investigated fields. while gaba modulation and acetylcholinesterase inhibition have been observed (falk et al., 1975; keser et al., 2013), no human studies have evaluated its impact on anxiety or cognitive decline. similarly, animal models show anti­ diabetic and lipid­lowering potential, but these effects remain untested in robust clinical trials (judzentiene and mockute, 2010). 5. ecological resilience and industrial supply: achillea millefolium ’s ecological adaptability, including drought tolerance and allelopathic behavior, enhances its suitability for sustainable cultivation (lazarevic et al., 2010). this resilience makes it a strong candidate for commercial exploitation in phytopharmaceutical and cosmeceutical industries. however, standardized cultivation practices and genetic profi l ing are essential to maintain consistent therapeutic quality. 6. recommendations for future research: conduct large­scale, placebo­controlled clinical trials using chemically standardized yarrow extracts; include pharmacokinetic and toxicological assessments in long­term human studies; investigate phytochemical variability across ecotypes to link specific bioactive profiles with therapeutic efficacy; explore integrative applications in neuro­degeneration, metabolic syndrome, and dermatology; clearly delineate areas where findings converge, confl ict, or remain uncertain. achil lea millefolium l. is a botanically and pharmacologically significant species with deep roots in traditional medicine and growing relevance in modern therapeutic, cosmetic, and industrial applications. its rich phytochemical composition­ including flavonoids, sesquiterpene lactones, phenolic acids, and essential oils­forms the basis for prisa and jamal ‐ yarrow phytochemistry review 255 a broad spectrum of biological activities, notably antioxidant, anti­ inflammatory, antimicrobial, antispasmodic, and wound­healing effects. these pharmacological actions validate many of the plant’s historical uses and support its continued inclusion in herbal pharmacopeias and integrative medicine. however, several challenges remain. clinical data are still sparse and often lack methodological rigor, emphasizing the need for larger, well­controlled trials using standardized extracts. toxicological assessments indicate that while the plant is generally safe in traditional doses, components like thujone and sesquiterpene lactones necessitate cautious use, particularly in essential oi l formulations and during pregnancy. additionally, the taxonomic complexity and phytochemical variability across populations underscore the importance of genetic characterization and quality control in both research and industry. looking forward, achillea millefolium presents exciting opportunities for new drug development, phytocosmetics, and evidence­based herbal therapies. bridging the gap between traditional knowledge and scientific validation will be key to ful ly unlocking the plant’s therapeutic and commercial potential. collaborative research, regulatory standardization, and interdisciplinary innovation are essential to ensure the safe, effective, and sustainable use of this valuable medicinal herb. acknowledgements the authors would like to express his heartfelt gratitude to colleagues and providers of plants and to all other sources for their cooperation and guidance in writing this article. references akram m., 2013 ­ mini review on achillea millefolium linn. ­ j. membrane biol., 246: 661­663. amirghofran z., karimi m.h., 2002 ­ cytotoxic activity of thymus vulgaris, achillea millefolium and thuja orientalis on different growing cell lines. ‐ med. j. islam. repub. iran, 15: 149­154. anne o., elmar a., ain r., 2006 ­ phytochemical analysis of the essential oil of achillea millefolium l. from various european countries. ­ nat. prod. res., 20: 1082­ 1088. 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application dates were 28 january, 25 february, and 25 march. bud break was evaluated throughout spring. during harvest, number and weight of clusters were recorded. berry samples were analyzed for ph, °brix, and titratable acidity (ta). pruning weights and number of clusters of the 2009 treated vines were recorded in march and august 2010, respectively. in the 2009 field experiment, oil and naa at 1000 mg/l significantly delayed bud break by two to six days, compared to the control. pruning weights were not significantly affected by the treatments. in 2010, oil applications significantly delayed bud break by eight to 12 days compared to the control and no significant differences were found between naa at 1500 and 1000 mg/l. in both years, treatments had no significant effects on yields, cluster weights, berry weights, °brix, ph or ta. adv. hort. sci., 2013 27(1-2): 18-24 received for publication 4 january 2013 accepted for publication 30 april 2013 19 break on fruit yield and characteristics such as juice ph, °brix, and titratable acidity (ta). 2. materials and methods field experiment 2009 the research was conducted during the 2009 winter season at james arthur vineyards located in raymond, nebraska within lancaster county. treatments were applied on 12-year-old vines. the vines are trained according to the geneva double curtain trellis system. planting distances are 2.44 m between plants and 3.66 m between rows. rows are oriented north to south. the experiment consisted of five treatments: naa (500, 750, and 1000 mg/l) purchased from phyto technology laboratories (shawnee mision, ks), amigo oil (loveland industries, greely, co) applied at 10% v/v which consisted of 9.3% oil and 0.7% emulsifier, and the control which was not sprayed. a randomized complete block design was used with three blocks of 20 vines each. there were four application dates: 6 january, 3 february, 3 march, and 1 april 2009. most canes were pruned to leave the five proximal buds before applying treatments. the remaining distal portion of the canes was removed by the normal pruning practices employed at james arthur vineyards. thus, the canes studied in the first year all had the same number of buds (i.e. five) and the total number of buds on vines were approximately the same. the whole vine was sprayed using a hand sprayer with each vine receiving approximately 0.33 l. after spraying, two canes per vine, each having five buds, were randomly selected and labeled. in the spring of 2009, vines were visually evaluated for bud break. bud break was determined as stage five of the eichhorn and lorenz (1977) scale of grapevine development, where bud scales have expanded to the point at which a green shoot is visible. bud break was evaluated day by day throughout the spring until each cane reached 60% bud break (three buds opened out of the five left after pruning). the number of julian days starting from 1 january 2009 until achieving 60% bud break was used as the basis of calculating number of days for bud break. harvest date was determined by taking random samples of berries and measuring their °brix with a refractometer. on 14 august 2009 the number and weight of clusters from the two selected canes were recorded. from the total clusters, 50 berries were randomly counted, placed in a plastic storage bag, and placed at 0°c until berry sample analysis could be conducted. on 14 september 2009 berry samples were analyzed for ph, °brix, and titratable acidity (ta). the 50 berries/vine were weighed, allowed to thaw to reach room temperature, wrapped in cheese cloth, and crushed manually using a mortar and pestle. the extracted juice was poured into test tubes to conduct the analyses. juice ph was measured with a pope ph/ion meter model 1501. soluble solids (°brix) content was measured using an atago pr-101 digital refractometer purchased from nova-tech international, inc. (houston, tx). ta was determined by titration with naoh, using the procedure of dharmadhikari and wilker (2001). on 18, 22, and 25 march 2010 pruning weights were recorded using an upright balance scale. in order to obtain data regarding cumulative effects of the treatments on fruiting the following year, the total number of clusters/vine were counted on 10 august 2010. temperatures for raymond, nebraska throughout 2009 were obtained from the high plains regional climate center, university of nebraska, lincoln. all statistical analyses were performed using sas/stat version 9.2 and analysis of variance was conducted by the proc glimmix procedure. field experiment 2010 treatments were applied on 13-year-old vines. these vines were not the ones sprayed in 2009. the experiment consisted of five treatments: naa (500, 1000, and 1500 mg/l), amigo oil applied at 10% v/v, and the control which was not sprayed. a randomized complete block design was used with three blocks of 15 vines each. there were three application dates: 28 january, 25 february, and 25 march 2010. the entire unpruned vines were sprayed and each vine received approximately 1l of treatment solution. the treated vines were then pruned on 30 march 2010. the total number of buds/vine was recorded to determine when 50% of the total buds showed bud break and not 60% as in 2009 because of the differences in treatment procedures. in the spring of 2010, vines were visually evaluated for bud break, which was determined as previously mentioned. the total number of buds/vine was counted and bud break was evaluated day by day throughout the spring until each vine reached 50% bud break of the total number of buds that were recorded in march. the number of julian days starting from 1 january 2010 until 50% bud break was achieved was used as the basis to calculate the number of days for bud break. on 11 august 2010 the number of clusters and weight of clusters/vine were recorded. from the clusters, 30 berries were randomly counted, placed in a plastic storage bag, and placed in the freezer until berry sample analysis could be conducted. on 18 august 2010 berry samples were analyzed for ph, °brix, and titratable acidity (ta) using the same procedures as described for the 2009 field experiment. similarly to 2009, temperatures for raymond, nebraska throughout 2010 were obtained from the high plains regional climate center, university of nebraska, lincoln. all statistical analyses were performed using sas/stat version 9.2 and analysis of variance was conducted by the proc glimmix procedure. 3. results and discussion field experiment 2009 as revealed from the analysis of variance, there was a significant treatment by month interaction for bud break 20 and ph at (p≤ 0.05) (table 1). similar interactions were found by dami and beam (2004) in ‘chancellor’ and ‘chambourcin’ but not in ‘chardonel’. due to interaction effects, month effects within treatments and treatment effects within the month are presented in table 2. delaying pruning until march had a significant effect in delaying bud break by two days compared to vines pruned in february (table 2). while a difference between the pruning carried out in february and march was detected, that difference does not exist when considering pruning in january and april. therefore, the treated vines in 2010 study were pruned on the same date (30 march 2010) to avoid pruning effects. the idea of pruning, then applying treatments was to maintain apical dominance since pruning can signal vine bud growth, increase the effectiveness of spraying since ‘edelweiss’ exhibits a vigorous growth, and reduce chemical applications since the canes will eventually be headed back to a certain number of buds in the pruning season. from the results of naa applications, it seems that auxin applications failed to maintain apical dominance and inhibit lateral bud growth because grapevines exhibit such a strong dominance (friend et al., 2001). no significant differences were found among naa 500, 750, and 1000 ppm between the months (table 2). regarding oil, no significant differences were found within months except in march (table 2). this was due to the improper mixing of the oil with water because the oil was mistakenly frozen on the day of spraying. with regard to the effects of treatments within months, it appeared that desirable results were achieved by oil and naa at 1000 ppm treatments in all months (table 2). except in march, oil applications significantly delayed bud break by five days compared to the control (table 2). oil and naa at 1000 ppm were only significantly different in january and april (table 2). overall, there were no significant differences between the control, naa at 500, and 750 ppm. table 1 reveals neither a treatment by month interaction nor a treatment effect, but it does show a month effect at (p≤ 0.05) regarding the number of clusters in 2009. the largest average number of clusters per cane was found in april-treated vines but they were not significantly different from averages for vines treated in march and january (table 3). the average number of clusters was lowest in february-treated vines and significantly different from the other three treatment dates (table 3). most importantly, no treatment effect was detected. the number of clusters per shoot ranged from 5.5 to 8.2 (data not shown). five buds were retained after pruning. usually, each bud can produce one to two clusters (personal observation), hence ten clusters would have been an optimum production in this case. nevertheless, the above averages were acceptable to james arthur vineyards and the average difference between apriland februarytreated vines, although statistically different, is only 1.5 clusters (table 3). this difference could be explained by cluster characteristics of ‘edelweiss’ which are known to be very loose and many clusters can simply fall down on the ground by any table 1 analysis of variance table for the experiment conducted in 2009 source of variance df bud break no. of clusters produced in summer 2009 cw bw °brix ph ta pw no. of clusters produced in summer 2010 treatment 4 < 0.0001 0.49 0.18 0.68 0.60 0.29 0.16 0.88 0.98 month 3 0.60 0.02 0.32 0.44 0.51 0.38 0.63 0.53 0.43 interaction 12 0.02 0.63 0.68 0.47 0.63 0.0039 0.38 0.34 0.47 cw=cluster weight, bw=berry weight, ta=titratable acidity, pw=pruning weight. table 2 pruning time and spray treatment effects on average days to show 60% bud break in 12-year-old ‘edelweiss’ grapevines pruning time spray treatments control naa 500 ppm naa 750 ppm naa 1000 ppm oil january 125.0 ab (c) 126.0 a (c) 126.5 a (bc) 128.0 a (b) 130.0 a (a) february 124.0 b (c) 124.5 a (bc) 126.0 a (b) 129.0 a (a) 129.0 a (a) march 126.0 a (b) 126.0 a (b) 127.0 a (ab) 128.3 a (a) 126.5 b (ab) april 125.5 ab (c) 125.0 a (c) 126.0 a (bc) 127.7 a (b) 129.7 a (a) different letters in a column indicate significant differences at p≤0.05 according to fisher’s protected lsd. the lower case letters in parenthesis are related to treatment effects within months. table 3 least significant difference test (lsd) for average number of clusters per cane of 12-year-old ‘edelweiss’ grapevines pruning time average number of clusters per cane april 7.3 a march 7.2 a january 7.0 a february 5.8 b different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 21 means of physical contact (swenson et al., 1980; brooks and olmo, 1997; smiley et al., 2008). no significant interaction, month, or treatment effect was found in average weights of ‘edelweiss’ clusters at (p≤ 0.05) (table 1). cluster weights of the two selected canes ranged from 1.33 to 2.22 kg (data not shown). this supports the lack of differences found regarding the average number of clusters per cane since cluster weights were recorded as averages of the two canes selected and the differences found in average number of clusters per cane is attributed to the looseness characteristic of ‘edelweiss’ clusters. also, no significant interaction, month, or treatment effect was found in weights of 50 samples of ‘edelweiss’ berries (p≤ 0.05) (table 1). once again, this will supports neglecting the differences detected in the average number of clusters per cane. the 50 berry sample weights ranged from 116.0 to 125.2 g (data not shown). a treatment by month interaction was only found in ph analysis at (p≤0.05) (table 1). due to interaction effects in ph analysis, month effects within treatments and treatment effects within the month are presented in table 4. naa at 750 and 1000 ppm showed no differences in ph values for all four pruning times (table 4). meanwhile, oil treatment gave a significantly lower ph for april pruning, as did the control in january, although the latter was not significantly different from february’s result (table 4). more obvious differences were observed with naa at 500 ppm for the four pruning times (table 4). regarding treatment effects within months, no significant differences were found among treatments within march (table 4). the control was significantly different from all other treatments in january (table 4) and the oil treatment was significantly different from the control and naa at 750 ppm in april. from the results in table 4 and table 1, it seems that differences in ph values are not due to the delay in bud break but to environmental conditions. in this regard, creasy and creasy (2009) mentioned that berry characteristics are totally dependent on environmental conditions, especially the microclimate (climate within canopy). this was also confirmed by huck (2009). in her study, training systems influenced sunlight penetration, canopy structure and thus fruit composition of ‘frontenac’ was totally dependent on climate within the canopy. the vines showed vigorous vegetative growth in 2010 with a low number of clusters. weaver (1976) noted that calyptras may not fall in cold rainy weather and this will reduce the amount of fruit set. the °brix ranged from 12.3 to 13.2, ph values were 3.14 to 3.27, and ta values were 0.83 to 1.13 g/100 ml (data not shown). regarding harvest parameters, dharmadhikari and wilker (2001) mentioned that optimum ranges for white wine would be 21-22%, 3.2-3.4, and 0.70.9 g/100 ml for the total soluble solids, ph, and the ta, respectively. ‘edelweiss’ is purposely harvested at an earlier stage regarding °brix and then chaptalized. swenson et al. (1980) mentioned that ‘edelweiss’ juice is relatively low in acidity (0.6-0.8%) and has moderate soluble solids (14-16%), and recommended that for wine making it should be picked at an early mature stage (14°brix). harvest parameter results of the 2009 study were in the recommended ranges, except for soluble solids. lower °brix values than the preferable ranges (14-16%) of the samples harvested in 2009 were due to the cooler july temperatures (fig. 1). higher temperatures during that month would have been preferable for the vines to produce more photosynthates and accumulate more sugar. weaver (1976) and winkler et al. (1974) mentioned that an optimum temperature for photosynthesis ranges from 25 to 30°c. in addition, lower nighttime temperatures would have been preferable to reduce respiration rates and breakdown the accumulated sugars. winkler et al. (1974) mentioned that 4.4°c halves the respiration rate and is an advantage to suppress fungal disease. table 4 pruning time and spray treatment effects within month on ph of fruit harvested in 2009 from 12-year-old ‘edelweiss’ grapevines treated with naa and “amigo oil” pruning time spray treatments control naa 500 ppm naa 750 ppm naa 1000 ppm oil january 3.14 b (b) 3.26 ab (a) 3.26 a (a) 3.22 a (a) 3.26 a (a) february 3.21 ab (ab) 3.27 a (a) 3.23 a (ab) 3.17 a (b) 3.25 a (a) march 3.23 a (a) 3.18 c (a) 3.20 a (a) 3.21 a (a) 3.22 a (a) april 3.25 a (a) 3.20 bc (ab) 3.24 a (a) 3.20 a (ab) 3.14 b (b) different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. the lower case letters in parenthesis are related to treatment effects within months. fig. 1 the maximum and minimum monthly average temperatures for raymond, nebraska in 2009. source: high plains regional climate center. 22 the treatments had no effect on pruning weights taken in winter 2010 as shown in table 1 at (p≤ 0.05). this shows that naa and oil applications had no negative effect on vegetative growth during spring, summer, and fall seasons after bud break. pruning weights ranged from 1.05 to 1.43 kg (data not shown). cultural practices such as mounding that tend to increase cold hardiness can result in higher pruning weights (gu, 2003). in his study, mounding protected ‘gewürztraminer’ vines from the cold winter and significantly increased pruning weights. although cold hardiness was not measured in this study, the experimental applications had no negative effects on grapevine vegetative growth and such delays in bud break should be of no concern. regarding the concerns of grape growers on cumulative effects, especially with applications of plant growth regulators, the number of clusters per vine ranged from 12 to 16 (data not shown). analysis of variance in table 1 showed that there were no such effects regarding the number of clusters produced in the following harvest year 2010 (p≤ 0.05). field experiment 2010 unlike the study in 2009, there was no significant treatment by month interaction, but a significant pruning time and spray treatment effect was present (p≤ 0.05) (table 5). the oil treatment significantly delayed bud break by eight days compared to the control. furthermore, it significantly delayed bud break by nearly four and five days compared to naa 1500 ppm and 1000 ppm, respectively (table 6). delaying bud break up to 12 days can encourage grape growers to use oil as an effective method to delay bud break and avoid spring frost injury. “the probability of freezing temperatures occurring decreases as spring progresses. therefore, cultural methods that delay the onset of bud break will decrease the risk of frost damage” (friend et al., 2001). furthermore, delaying pruning until march was very effective in improving results compared to the study in 2009 regarding delaying bud break. it is suggested that grape growers should delay pruning as much as possible and if this strategy is to be adopted to reduce frost risk, pruning should be done late in the pruning season especially for cultivars that exhibit early bud break. although there was no month effect on bud break, overall march applications delayed by one to two days more than february and january (data not shown). table 5 shows no effects on the total number of clusters per vine in 2010 (it was cluster per cane in 2009) (p≤ 0.05). the total number of clusters per vine ranged from 12 to 19 (data not shown), which is similar to the range found in 2009. in addition, similar analysis of variance results were obtained compared to the 2009 study regarding treatment by month interaction and treatment effects. the only difference was a month effect present in the 2009 study which was not present in 2010. totally different weather conditions prevailed in 2009 compared to 2010 (fig. 2). according to the high plains regional climate center, normal precipitation for raymond, nebraska is 121 mm and 95 mm for may and june, respectively. in 2010, monthly precipitation in june was 249 mm, almost three times the monthly average. this had a negative impact on ‘edelweiss’ fruit set. in fact, ‘edelweiss’ yields harvested at james arthur vineyards were 8 tons/acre in 2009 and table 5 analysis of variance table for the experiment conducted in 2010 source of variance df bud break no. of clusters produced in summer 2010 cw bw °brix ph ta treatment 4 < 0.0001 0.90 0.91 0.76 0.89 0.81 0.64 month 2 0.26 0.26 0.28 0.34 0.84 0.03 0.07 interaction 8 0.20 0.94 0.92 0.10 0.81 0.23 0.82 cw=cluster weight, bw=berry weight, ta=titratable acidity, pw=pruning weight. fig. 2 the maximum and minimum monthly average temperatures for raymond, nebraska in 2010. source: high plains regional climate center. table 6 least significant difference test (lsd) for average number days of bud break for 13-year-old ‘edelweiss’ grapevines in 2010 treatment average number days to bud break oil 122 a naa 1500 ppm 118 b naa 1000 ppm 117 b naa 500 ppm 115 c control 114 c different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 23 only 3 tons/acre in 2010. no significant effects (p≤ 0.05) were found in ‘edelweiss’ cluster weights (table 5), which ranged from 2.26 to 3.65 kg (data not shown). analysis of variance for berry weights showed a similar trend in 2010 as in 2009. no significant effects were present at p≤ 0.05 (table 5). berry sample weights ranged from 97.94 to 104.45 g (data not shown). similar trends were present in 2010 °brix and ta results with no significant effects at p≤ 0.05. regarding ph, unlike the results of 2009 where a treatment by month interaction was present, the 2010 study showed a month effect at (p≤ 0.05). °brix ranged from 12.7 to 13.5, ph values were 3.26 to 3.41, and ta values were 1.1 to 1.4 g/100 ml (data not shown). °brix ranges were higher in 2010, which was expected since average july temperatures were 27.2°c and 28.8°c in 2009 and 2010, respectively. regarding ph and ta, the 2010 results for the former are in the same range as recommended (3.2-3.4) by dharmadhikari and wilker (2001), but ta ranges were a little higher than the recommendation of 0.7-0.9%. the analysis of variance table for ph results showed a significant month but not a treatment effect at (p≤ 0.05). absence of a treatment effect on berry characteristics is important for recommendation purposes. however, only slight differences were found between months (table 7). 4. conclusions based on the 2010 results, delaying pruning until march, especially for cultivars that show early bud break such as ‘edelweiss’, will delay bud break. amigo oil did not exhibit the 20-day delay reported by dami and beam (2004) in french-american hybrids and naa did not exhibit the 16to 27-day delay that was obtained in the study with cut stems taken from ‘aramon’ (vitis vinifera) vines (nigond, 1960). amigo oil gave better performance compared to naa in both years, even at higher naa concentrations. it delayed bud break slightly longer (four to five days) and did not affect either the quantity or quality of fruit produced. naa at 1000 and 1500 ppm showed a potential bud break delay similar to that of amigo oil. oil applications and naa at 1000 to 1500 ppm in march and up to early april could provide grape growers with an acceptable delay of bud break. this is based on the performance of both oil and naa applications in the field experiments. delaying bud break shows no negative impact on berry characteristics. as a result of this research, it can be recommended to use amigo oil at 10% or naa at 1000 to 1500 ppm from march to april for sites that are prone to frost events such as in southeastern nebraska and on cultivars that show early bud break such as ‘edelweiss’. any resulting delay in bud break will decrease the possibility of frost injury. furthermore, this study opens the door to future studies regarding the value of repeated spraying (qrunfleh, 2010) or mixing amigo oil with naa. furthermore, investigation of any phytotoxicity damage to buds caused by oil applications that could possibly occur under vineyard conditions is warranted. references brooks r., olmo h., 1997 the brooks and olmo register of fruit and nut varieties. third edition. ashs press, alexandria, va, usa, pp. 744. call r., seeley s., 1989 flower bud coatings of spray oils delay dehardening and bloom in peach trees. hortscience, 24(6): 914-915. creasy g., creasy l., 2009 grapes. crop production science in horticulture. 16. cabi, cambridge, uk, pp. 312. dami i., beam b., 2004 response of grapevines to soybean oil application. am. j. enol. vitic., 55: 269-275. dharmadhikari m., wilker k., 2001 micro vinification: a practical guide to small-scale wine production. missouri state fruit experiment station, mountain grove, missouri, usa, pp. 145. eichhorn k., lorenz d., 1977 phänologische entwicklungsstadien der rebe. nachrichtenbl. deut. pflanzenschutz, 29: 119-120. friend a., stushnoff c., creasy g., trought m., 2001 manipulating bud break date in grapevines. proceedings of the asev 52nd anniversary annual meeting, pp. 16. gu s., 2003 rootstock and mounding effect on growth and cold hardiness of ‘gewürztraminer’ (vitis vinifera) and bud dormancy of ‘lacrosse’ and ‘chambourcin’ (vitis spp.). ph d. dissertation, university of nebraska, lincoln, usa. guinan p., 2007 understanding and preventing freeze damage in vineyards. workshop proceedings, university of missouri extension, pp. 7-12. huck c., 2009 training system effects on sunlight penetration, canopy structure and fruit composition of ‘frontenac’ grape (vitis spp.). m.sc. dissertation, university of nebraska, lincoln, usa. loomis n., 1939 note on grape foliation as affected by time of pruning. proc. amer. soc. for hort. sci., 37: 653-654. nigond j., 1960 delaying bud break in vines by the use of α-naphthaleneacetic acid and defense against frost. compt. rend. acad. agr. france, 46: 452-457. qrunfleh i., 2010 delaying bud break in ‘edelweiss’ grapevines to avoid spring frost injury by naa and vegetable oil applications. ph d. dissertation, university of nebraska, lincoln, usa. smiley l., domoto p., nonnecke g., miller w., 2008 table 7 least significant difference test (lsd) for ph of berry samples in 2010 month average ph february 3.37 a january 3.34 ab march 3.29 b different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 24 cold climate cultivar. a review of cold climate grape cultivars. iowa state university, ames, ia, usa. swenson e., pierquet p., stushnoff c., 1980 ‘edelweiss’ and ‘swenson red’ grapes. hortscience, 15(1): 100. weaver r., 1976 grape growing. wiley-intescience, a john wiley & sons, inc. publication, ames, ia, usa, pp. 374 wiggans c., 1926 a study of the relative value of fruiting shoots arising from primary and secondary buds of the ‘concord’ grape. proc. amer. soc. for hort. sci., 23: 293-296. winkler a.j., cook j.a., kliewer w.m., lider l.a., 1974 general viticulture. university of california press, usa, pp. 710. 67 1. introduction onion (allium cepa l.) belongs to the family alliaceae in the genus allium (friensen, et al., 2006; brewster, 2008). in nigeria, onion is grown mainly for its bulb which is used in virtually every home on a daily basis to flavour and season a wide variety of dishes. as a constituent of a meal, both the green leaves and bulbs can be eaten raw or cooked in soups and salads. it is claimed to minimize high blood pressure and other heart diseases due to its favourable action on the elasticity of blood vessels. as an item of world trade, onion ranks second in importance after tomatoes among vegetables in nigeria, where annual production stands at 621 000 t, making it the 36th highest producing country in the world. the genus allium constitutes “bulb crops” and these are weak competitors with weeds which emerge among the crops (boydston and seymour, 2002). weeds are known to reduce available moisture, nutrients, sunlight and growing space needed by crop plants. subsequently, weeds can reduce growth, quality and yield of crops and make harvesting difficult. onion seedlings are weak competitors with weeds because of their slow growth, small stature, shallow roots and thin canopy. in addition, their cylindrical upright leaves do not shade the soil enough to suppress weed growth. weed interference was observed to be more devastating than insect (thrips tabaci) infestations on onion dry bulb yield (ghosheh and al-shannag, 2000). herbicides may be applied before planting or after planting. pre-plant application of a soil residual herbicide such as oxadiazon has proved effective for weed control in onion (amrutkar et al., 2002). oxyfluorfen and pendimenthalin were reported to have significantly reduced the weed population and increased onion yield to levels comparable to yields of weed control in a relay cabbage-onion cropping system (sanjeev et al., 2003). onions are sown at densities that exceed 20 plants/m2, and hand weeding operations are practiced with great care. on the other hand, high costs, lack of manpower and low economic returns are currently pushing onion farmers to consider herbicides as an alternative weed management option in onion production. the reliance on unstructured or non-sequenced weed management programs by farmers, especially in a country like nigeria where agricultural commodities are still inexpensive, is also very common. therefore, the objective of this study was to evaluate the efficacy of weed control using single herbicide applications. although not all the herbicides used in this experiment were registered for onion production, producers might be tempted to use those available and at the lowest cost. 2. materials and methods field experiments were conducted during the 2006/2007 and 2007/2008 dry seasons at the irrigation research station, kadawa, of the institute for agricultural research, screening of herbicides for weed control, growth and yield of irrigated onion (allium cepa l.) in tropical savanna climate d.i. adekpe, j.a.y. shebayan, c.p. shinggu, l. aliyu * department of agonomy, faculty of agriculture, institute for agricultural research, ahmadu bello university, p.m.b. 1044, zaria, nigeria. key words: herbicides, hoe weeding, onion, weed control. abstract: field experiments were conducted during the 2006/2007 and 2007/2008 dry seasons at sudan savanna nigeria to study the effects of herbicides on weeds and growth of irrigated onion (allium cepa l.). the field was laid out in a randomized complete block design (rcbd) and the treatments included four rates (with and without supplementary hoe weeding shw) of oxadiazon 25 ec, butachlor 50 ec, pendimenthalin 50 ec, bullet 700 sc (atrazine + acetochlor + terbuthylazine), eight rates (with or without shw) of oxyfluorfen 41 fw, hoe weeded at 3 and 6 weeks after transplanting (wat) and untreated control. the treatments were replicated three times. the results indicated that application of oxadiazon 25 ec at 4 l/ha and oxyfluorfen 41fw at 6 l/ha both followed by shw gave low weed dry weight, larger bulb diameter, higher mean bulb weight and bulb yield per hectare compared to the hoe-weeded control. adv. hort. sci., 2013 27(1-2): 67-72 corresponding author: idokodav@yahoo.com received for publication 18 february 2013 accepted for publication 15 may 2013 68 ahmadu bello university, zaria, nigeria (11o 39’n, 08o 02’e and 500 m above sea level). meteorological data on temperature, relative humidity and sunshine hours, as well as physical and chemical characteristics of the soil of the experimental site were collected december 2006-april 2007 and december 2007-april 2008 (tables 1, 2 and 3). land preparation involved ploughing, harrowing and ridging (75 cm apart). the plot size was 12 m2 (4 x 3 m) representing the planting basin. planting was done in rows at spacing of 15 x 30 cm within and between rows. the onion variety used, d77, has a maturity period of 120 to 130 days and potential yield of 5-15 t/ha. plots were irrigated by surface flooding before transplanting a day after herbicide application. subsequent irrigation was applied once a week by surface irrigation of the basin to field capacity, resulting in approximately 15-20 applications to supply about 350-550 mm of moisture as needed by the plant per growing season. in the two seasons, single super phosphate (ssp) fertilizer at a rate of 19.8kg p/ha and muriate of potash (potassium) at 10.4 kg k/ha were incorporated into the basin as basal fertilizer before transplanting. nitrogen (45 kg n/ha) was applied by broadcasting table 1 meteorological data showing mean minimum and maximum temperatures, relative humidity and sunshine hours during 2006/2007 dry season at kadawa month days air temperature (oc) relative humidity (%) sunshine hours min. max. december 2006 1-10 14.0 33.5 25.1 7.2 11-20 13.2 32.6 27.2 7.4 21-31 13.5 33.0 27.4 7.0 january 2007 1-10 11.7 31.6 28.3 6.8 11-20 14.9 35.7 29.5 7.0 21-31 13.4 34.3 29.8 6.1 february 2007 1-10 16.2 36.3 32.0 6.1 11-20 19.7 35.9 34.1 5.8 21-29 19.5 37.1 35.8 5.0 march 2007 1-10 19.2 35.8 37.1 6.2 11-20 19.4 38.3 36.1 6.8 21-3 20.4 41.4 36.7 5.7 april 2007 1-10 19.6 40.7 36.9 5.4 11-20 18.6 39.1 37.5 5.6 21-30 22.3 40.9 37.6 6.0 total 255.6 546.2 491.1 95.1 average 17.04 36.42 32.74 6.34 source: meteorological unit of the institute for agricultural research, samaru, zaria. table 2 meteorological data showing mean minimum and maximum temperatures, relative humidity and sunshine hours during 2007/2008 dry season at kadawa month days air temperature (oc) relative humidity (%) sunshine hours min max dececember 2007 1-10 13.6 35.0 26.9 6.0 11-20 14.0 33.3 23.4 6.4 21-31 14.5 32.0 27.2 5.4 january 2008 1-10 15.1 34.3 27.9 5.0 11-20 13.6 32.7 28.0 5.7 21-31 na na 30.1 6.0 february 2008 1-10 17.3 38.3 34.3 5.0 11-20 15.6 34.7 30.8 4.5 21-29 14.8 35.9 32.0 4.5 march 2008 1-10 15.7 34.5 29.9 5.0 11-20 18.9 32.3 32.3 6.1 21-31 23.3 39.4 36.3 6.8 april 2008 1-10 25.7 42.8 38.3 5.4 11-20 23.9 41.1 36.9 6.0 21-30 25.7 41.2 37.6 6.0 total 251.7 507.4 471.9 83.8 average 18.00 36.24 31.46 5.59 source: meteorological unit of the institute for agricultural research, samaru, zaria. na= not available. table 3 physico-chemical properties of the 0-20 cm depth of soil at the experimental site during the 2006/2007 and 2007/2008 dry seasons at kadawa composition dry season 2006/2007 dry season 2007/2008 physical properties sand (%) 56.0 58.0 silt (%) 30.0 30.0 day (%) 14.0 12.0 textural class sandy-loam sandy-loam chemical properties ph in h 2 o 5.70 5.80 ph in 0.01 m cacl 2 (1:2.5) 5.10 5.32 organic carbon (g/kg) 0.58 0.56 total nitrogen (g/kg) 1.20 1.30 available phosphorus 0.90 1.20 exchangeable cations (cmd/kg soil) calcium 1.88 0.60 sodium 0.81 0.73 potassium 0.21 0.37 magnesium 3.13 2.97 c.e.c. 6.70 6.90 69 using urea (46%w) as split doses at two and six weeks after transplanting (wat). treatments consisted of the following rates, each applied with and without supplementary hoe weeding (shw) for a total of four treatments per herbicide: oxadiazon 25 ec (4 and 6 l/ha), butachlor 50 ec (5 and 6 l/ha), pendimenthalin 50 ec (5 and 6 l/ha) and bullet 700 sc 9 (1.04 and 1.4 l/ha) (atrazine + acetochlor + terbuthylazine), eight treatments (4, 6, 8 and10 l/ha with and without shw) of oxyfluorfen 41 flowable (fw), and an untreated control; hoe weeding was carried out at 3 and 6 weeks after transplanting (wat). these treatments were laid out in a randomized complete block design (rcbd) with three replications. all herbicides were applied a day before transplanting using a conventional cp3 knapsack sprayer with a green deflector nozzle at a pressure of 2.1 kg/m2 to deliver a spray volume of 200 l/ha. supplementary hoe weeding was imposed at 6 wat on some plots as indicated in the treatment because some herbicides have short persistence in the soil and therefore cannot exhibit long season weed control. in the nigerian savanna, despite high labour requirements and cost of inputs, some workers have emphasized the need to supplement pre-emergence herbicide treatments with hoe weeding for long season weed control for increased yields in various crops (adigun et al., 1987; magani, 2008; oluwafemi, 2013). onion bulbs were harvested manually at 16 wat. data on weed dry weight, mean bulb diameter, bulb weight and bulb yield per hectare were assessed and subjected to statistical analysis of variance (anova) as described by snedecor and cochran (1967). differences between treatment means were compared using duncan multiple range test (dmrt) (duncan, 1955) at 5% level of probability. 3. results weather and soil conditions meteorological data on temperature, relative humidity and sunshine hours during the study periods (2006/2007 and 2007/2008) are shown in tables 1 and 2 respectively, while physical and chemical characteristics of the soil of the experimental sites for the same period are presented in table 3. the average maximum temperature was 36.42 and 36.24°c in 2006/2007 and 2007/2008 respectively, while for the same periods the relative humidity was 32.74 and 31.46% and sunshine hours were 6.34 and 5.59. with regard to the soil of the experimental sites, it was classified as sandy loam in both study periods, and the organic carbon, total nitrogen and available phosphorus were, respectively, 0.58, 1.20 g/kg-1 and 0.90 mg/kg-1 in 2006/2007 and 0.56, 1.30 g/kg-1 and 1.20 mg/kg-1 in 2007/2008. weed dry weight the untreated control had significantly (p≤m0.05) higher weed dry weight than all the other treatments except for oxadiazon at 4 and 6 l/ha in 2006/2007 and for the combined mean (table 4). the hoe-weeded control in the 2006/2007 study period resulted in significantly lower weed dry weight, comparable to the application of oxadiazon at 4 l/ha followed by supplementary hoe weeding (fbshw), oxyfluarfen at 10 l/ha fbshw, butachlor at 6 l/ ha fbshw and pendimenthalin at 5 l/ha fbshw (table 4). in 2007/2008, all the herbicides and their rates, including the untreated control, gave statistically comparable weed dry weight. the application of oxadiazon at 6 l fbshw had significantly (p<0.05) higher weed dry weight than butachlor at 6 l/ha and the hoe-weeded control which gave statistically similar weed dry weights (table 4). table 4 effects of rates of herbicides on weed dry weight in onion during dry seasons of 2006/2007, 2007/2008 and combine at kadawa rates (l/ha) weed dry weight (kg/ha) combined mean herbicides 2006/2007 2007/2008 oxadiazon 4 101.70 a 17.47 a-c 59.58 ab 4 fbshw 34.60 l-g 11.23 a-c 22.93 ef 6 82.07 a 15.90 a-c 49.00 a-c 6 fbshw 38.60 c-f 21.00 a 29.85 d-f oxyfluorfen 4 56.37 b-e 8.57 a-c 30.08 c-f 4 fbshw 41.8 c-f 13.96 a-c 27.85 d-f 6 57.70 b-e 14.90 a-c 36.32 c-f 6 fbshw 38.10 c-f 15.10 a-c 28.60 d-f 8 45.97 l-f 13.96 a-c 29.97 d-f 8 fbshw 49.59 b-c 9.60 a-c 29.55 d-f 10 62.33 b-e 16.37 a-c 39.35 c-e 10 fbshw 33.90 d-g 17.7 a-c 25.80 d-f butachlor 5 45.57 c-f 14.30 a-c 29.93 d-f 5 fbshw 38.40 c-f 14.46 a-c 26.43 d-f 6 48.37 b-f 6.13 bc 27.25 d-f 6 fbshw 21.27 fg 14.97 a-c 18.12 fg bullet 1.04 57.20 b-e 13.03 a-c 35.12 c-f 1.04 fbshw 42.60 c-f 10.53 a-c 26.57 d-f 1.4 46.80 c-f 14.20 a-c 30.50 d-f 1.4 fbshw 29.17 e-g 17.70 a-c 23.43 e-f pendimenthalin 5 67.70 b-d 19.43 a-c 43.57 b-d 5 fbshw 33.47 d-g 21.30 a-c 27.40 d-f 6 69.27 bc 20.20 a-c 44.73 a-d 6 fbshw 62.33 b-e 14.46 a-c 38.55 c-e hoe weeded 3 + 6wat 3.70 g 5.00 c 6.13 g untreated check 109.03 a 12.66 a-c 60.85 a se(±) 10.203 3.847 5.533 means followed by same letter(s) within a column are not significantly different at 5% (p=0.05) using dmrt. fbshw= followed by supplementary hoe weeding. wat= weeks after transplanting. 70 mean bulb diameter table 5 shows mean bulb diameter for the herbicide treatments. the results reveal a larger bulb size in the first study season compared to the second season. the application of oxyfluorfen at 4 l/ha produced larger bulbs for all treatments in 2006/2007 and for the combined mean (2006/2007-2007/2008); the values were comparable with the hoe-weeded control in both cases. in 2007/2008, the use of oxadiazon at 6 l/ha fbshw resulted in bigger bulbs compared with those produced by oxadiazon at 4 l/ha (with or without shw), pendimethalin at 6 l/ha without shw and the hoe-weeded control. oxadiazon at 6 l/ha and the untreated control gave comparable but smaller onion bulbs for the combined mean (table 5). mean bulb weight mean bulb weight appeared higher in the 2006/2007 dry season compared with the following year (table 6). in the former, hoe weeded control yielded a significantly (p<0.05) higher mean bulb weight, compared with oxadiazon at 4 l/ha (with or without shw) and bullet (atrazine + acetochlor + terbuthylazine) at 1.04 l/ha fbshw. the untreated control gave the lowest mean bulb weight, although it was comparable to some herbicide treatments. the application of oxadiazon at 6 l/ha followed by supplementary hoe weeding (fbshw) in 2007/2008 and combined mean resulted in the highest mean bulb weight, but it was comparable to oxadiazon at 4 l/ha (with and without table 5 effects of rates of herbicides on mean bulb diameters in onion during dry seasons of 2006/2007, 2007/2008 and combine at kadawa herbicides rates (l/ha) mean bulb diameter (cm) combined meandry season 2006/2007 dry season 2007/2008 oxadiazon 4 19.52 ab 9.49 b-d 14.51 a-d 4 fbshw 19.13 ab 9.36 b-d 14.64 a-d 6 14.85 b-f 7.99 d 11.42 d 6fbshw 17.68 a-e 16.40 a 17.04 ab oxyfluorfen 4 21.60 a 12.88 a-d 17.24 a 4 fbshw 16.93 a-f 15.75 ab 16.17 a-c 6 14.43 b-f 11.87 a-d 13.15 a-d 6fbshw 15.75 b-f 10.65 a-d 13.20 a-d 8 13.37 ef 13.12 a-d 12.75 b-d 8fbshw 13.53 l-f 10.77 a-d 12.15 cd 10 15.79 b-f 10.95 a-d 13.37 a-d 10fbshw 17.99 a-c 14.13 a -d 16.06 a-c butachlor 5 13.19 d-f 13.22 a-d 13.23 a-d 5 fbshw 17.37 a-f 12.25 a-d 14.81 a-d 6 14.76 b-f 9.97 a-d 12.36cd 6 fbshw 17.55 a-f 14.18 a-d 15.87 a-c bullet 1.04 15.67 b-f 8.49 cd 12.06 cd 1.04 fbshw 19.54 ab 13.23 a-d 16.38 a-c 1.4 16.92 a-f 12.43 a-d 14.67 a-d 1.4 fbshw 16.60 a-f 13.00 a-d 14.80 a-d pendimenthalin 5 16.52 a-f 11.15 a-d 13.84 a-d 5 fbshw 19.15 a-c 14.64 a-c 16.90 ab 6 17.02 a-f 9.76 b-d 13.34 a-d 6 fbshw 18.08 a-c 12.19 a-d 15.14 a-d hoe weeded 3 + 6wat 18.96 a-d 9.78 b-d 14.37 a-d untreated check 11.87 f 10.05 a-d 10.90 d se (±) 1.673 1.903 1.269 means followed by same letter(s) within a column are not significantly different at 5% (p=0.05) using dmrt. fbshw= followed by supplementary hoe weeding. wat= weeks after transplanting. table 6 effects of rates of herbicides on mean bulb weight of onion during dry seasons of 2006/2007, 2007/2008 and combine at kadawa herbicides rates (l/ha) mean bulb weight (g) combined meandry season 2006/2007 dry season 2007/2008 oxadiazon 4 77.48 a-c 20.72 c 49.10 a-f 4 fbshw 86.03 ab 35.63 a-c 60.83 a-d 6 48.34 c-h 19.38 c 33.89 fg 6fbshw 68.26 b-e 71.03 a 69.65 a oxyfluorfen 4 33.51 f-h 43.41 a-c 38.46 c-g 4 fbshw 58.97 b-g 64.45 ab 61.71 a-c 6 37.38 e-h 33.08 a-c 35.23 fg 6fbshw 50.88 c-h 31.59 bc 41.23 c-g 8 27.65 gh 43.70 a-c 35.68 e-g 8fbshw 35.77 f-h 27.61 bc 31.69 fg 10 52.89 c-h 25.65 c 39.27 c-g 10fbshw 62.05 b-f 50.05 a-c 56.05 a-f butachlor 5 41.59 d-h 45.48 a-c 43.53 b-g 5 fbshw 48.60 c-h 48.27 a-c 48.44 a-f 6 43.57 d-h 36.86 a-c 40.21 c-g 6 fbshw 47.36 c-h 45.87 a-c 46.62 a-f bullet 1.04 50.18 c-h 21.95 c 36.06 d-g 1.04 fbshw 77.25 a-c 43.19 a-c 60.22 a-e 1.4 44.89 d-h 37.96 a-c 41.43 b-g 1.4 fbshw 68.58 b-e 38.24 a-c 53.41 a-f pendimenthalin 5 55.15 c-g 24.67 c 39.91 c-g 5 fbshw 52.96 c-h 55.03 a-c 53.00 a-f 6 60.68 b-f 22.81 c 43.75 b-g 6 fbshw 69.19 b-d 33.68 a-c 51.44 a-f hoe weeded 3 + 6wat 103.06 a 28.46 bc 65.77 ab untreated check 22.87 h 20.03 c 21.45 g se (±) 9.126 11.171 7.189 means followed by same letter(s) within a column are not significantly different at 5% (p=0.05) using dmrt. fbshw= followed by supplementary hoe weeding. wat= weeks after transplanting. 71 supplementary hoe weeding), oxyfluorfen at 4 and 10 l/ ha fbshw, pendimenthalin at 5 and 6 l/ha fbshw and the hoe-weeded control (table 6). the untreated control consistently maintained the lowest mean bulb weight in both years of study and in the combine mean. onion bulb yield in 2006/2007, the use of oxadiazon at 4 l/ha fbshw, oxyfluorfen at 6 l/ha and pendimentalin at 5 and 6 l/ha fbshw resulted in statistically similar but significantly (p≤0.05) higher bulb yield compared with oxyfluorfen at 4, 8 and 10 l/ha and the untreated control. the differences in bulb yield between all other herbicides and their rates including the untreated control were not significant (p≤0.05) statistically (table 7). the effect of the herbicides and their rates on onion bulb yield was not significant in 2007/2008. the application of pendimenthalin at 5 l/ha fbshw resulted in significantly (p≤0.05) higher bulb yield compared with oxyfluorfen at 8 and 10 l/ha and the untreated control for the combined mean. all the other herbicides and their rates gave statistically similar bulb yields (table 7). 4. discussion and conclusions weed dry weights were very high in the untreated control compared to the herbicide-treated plots in 2006/2007 and combined means, except for oxadiazon at 4 and 6 l/ ha without fbshw. this could be due to a higher weed intensity resulting from no hoe weeding and herbicide treatment. lower weed dry weights and herbicide suppression of weeds in crops has been demonstrated in many studies (ibrahim, 2001; ishaya, 2004; adekpe et al., 2007). the statistically similar weed dry weight found in the present study for hoe weeded control and oxadiazon at 4 l/ha fbshw, oxyfluorfen at 10 l/ha fbshw, butachlor at 6 l/ha fbshw and pendimnthalin (5 l/ha fbshw) demonstrates herbicide competitiveness with conventional hoe weeding in weed management practices. similar observations have been made in other related studies (adekpe et al., 2007; shinggu et al, 2009; oluwafemi, 2013). bulb size was higher with the application oxyfluorfen at 4 l/ha compared to to hoe-weeded control in both 2006/2007 and combined data. numerical differences in bulb size (diameter) were observed and in some cases not statistically significant, reflecting the within treatment variability encountered in the study. the use of oxyfluorfen has been reported to increased onion performance (ghosheh, 2004) onion mean bulb weight and bulb yield (t/ha) were observed to be better with oxyfluorfen (6 l/ha fbshw) and pendimenthalin at 5 and 6 l/ha fbshw. this observation may not be unconnected with the observed favourable weed control, higher bulb diameter influenced by these herbicides. the weed control must have helped in the reduction of competition for both the above and underground growth factors by the weeds, thereby providing an adequate environment for the growth and bulb yield of onion. the high total nitrogen and available phosphorus found in the soil of the experimental sites might have contributed to high bulb yield in these low weed-infested plots. the use of oxyfluorfen, oxadiazon and pendimethalim has been reported to improve performance in some bulb and root vegetable crops (amrutkar et al., 2002; sanjeev et al., 2003; adekpe et al., 2007; adekpe et al., 2012). it can be concluded from the present study that the use of oxadiazon at 4 and 6 l/ha fbshw and oxyfluorfen at 6 l/ha fbshw gives the lowest weed dry weight, higher bulb diameter, higher mean bulb weight and bulb yield per hectare compared to the hoe-weeded control. table 7 effects of rates of herbicides on onion bulb yield during dry seasons of 2006/2007, 2007/2008 and combine at kadawa herbicides rates (l/ha) onion bulb yield (t/ha) combined meandry season 2006/2007 dry season 2007/2008 oxadiazon 4 6.00 a-c 0.73 3.37 a-d 4 fbshw 6.93 a 0.83 3.88 a-c 6 3.78 a-d 0.66 2.22 a-d 6fbshw 4.30 a-d 2.66 3.44 a-d oxyfluorfen 4 2.17 b-d 2.27 2.22 a-d 4 fbshw 3.06 a-d 2.60 2.83 a-d 6 7.05 a 1.13 4.09 ab 6fbshw 3.90 a-d 0.70 2.30 a-d 8 1.78 cd 2.13 1.96 b-d 8fbshw 3.68 a-d 0.93 2.30 a-d 10 2.05 cd 0.88 1.46 cd 10fbshw 5.22 a-d 1.90 3.58 a-c butachlor 5 3.22 a-d 2.07 2.65 a-d 5 fbshw 5.05 a-d 2.13 3.59 a-c 6 3.22 a-d 1.90 2.58 a-d 6 fbshw 3.58 a-d 1.40 2.48 a-d bullet 1.04 3.45 a-d 0.92 2.19 a-d 1.04 fbshw 5.39 a-d 1.20 3.30 a-d 1.4 2.83 a-d 2.13 2.48 a-d 1.4 fbshw 4.22 a-d 1.00 2.61 a-d pendimenthalin 5 4.61 a-d 0.47 2.54 a-d 5 fbshw 6.56 a 2.93 4.75 a 6 4.00 a-d 0.53 2.27 a-d 6 fbshw 6.92 a 1.20 4.06 ab hoe weeded 3 + 6wat 5.78 a-c 0.47 3.12 a-d untreated check 1.22 d 0.73 0.98 d se (±) 1.279 0.762 0.737 means followed by same letter(s) within a column are not significantly different at 5% (p=0.05) using dmrt. fbshw= followed by supplementary hoe weeding. wat= weeks after transplanting. 72 acknowledgements the authors wish to acknowledge the permission of the director, institute for agricultural research, ahmadu bello university, zaria to use its facilities in carrying out this work. references adekpe d.i., essien j.e., shebayan j.a.y., ishaya d.b., shinggu c.p., 2012 evaluation of herbicides for weed management in irrigated carrot (daucus carota l.) at samaru, zaria, nigeria. conference of the weed science society of nigeria (wssn), ahmadu bello university, zaria, 18-22 november. adekpe d.i., shebayan j.a.y., chiezey u.f., mike s., tunku p., 2007 effects of weed control, date of planting and intra-row spacing on weeds and bulb yield of garlic (allium sativum l.) at kadawa, nigeria. adv. hort. sci., 21(3): 165-171. adigun j.a, lagoke s.t.o., karikari s.k., 1987 herbicides evaluation studies in transplanted chilli pepper (capsicum frutescens l.) in the nigerian savanna. crop protection, 6(4): 15-19. amrutkar s.d., patil b.m., karunakar a.p., jiotode d.j., 2002 effect of various herbicides on yield and uptake of nutrients in onion (allium cepa l.). res. crops, 3: 659-661. boydston r.a., seymour m.d., 2002 volunteer potato (solanum tuberosum l.) control with herbicides and cultivation in onion (allium cepa l.). weed technology, 16: 620-626. brewster j.l., 2008 onions and other vegetable allium. 2nd edition, bddles ltd, king’s lynn, uk, pp. 432. duncan d.b., 1955 duncan multiple range and multiple f. test. biometris, 11: 1-42. friensen n., fritsch r., blattner f.r., 2006 phylogeny and new intragenetic classification of allium (alliaceae) based on nuclear ribosomal dna and its sequences, pp. 372395. in: columbus j.t. (ed.) monocots iii. proceedings of the third international conference on the comparative biology of monocotyledons, claremart, california, usa. ghosheh h.z., 2004 single herbicide treatments for control of broadleaved weeds in onion (allium cepa l.). crop protection, 23: 539-542. ghosheh h.z., al-shannag h.k., 2000 influence of weeds and onion thrips, thrips tabaci (thysanoptera) on onion bulb yield in jordan. crop protection, 19: 175-179. ibrahim. d.d., 2001 effects of rates of nitrogen, method of weed control and intra-row spacing on the growth and yield of irrigated paper (capsicum annuum l.) at samaru zaria, nigeria. m.sc seminar pepper, department of agronomy, ahmadu bello university, zaria, p. 28. ishaya d.b., 2004 evaluation of weed control treatment and rice varieties on weeds, growth, and yield of rice/sorghum mixture. phd seminar paper, department of agronomoy, ahmadu bello university, zaria, pp. 34. magani i.e., 2008 weed control in sorghum groundnut mixture in the simultaneous farming system of southern guinea savanna zone of nigeria. journal of animal and plant sciences, 1(1): 3-8. oluwafemi a.b., 2013 evaluation of weed management strategies in cocoyam (colocasia esculentus (l.) schott) production in ado-ekiti, ekiti state, nigeria. international research journal of agricultural science and soil science, 3(2): 38-42. sanjeev a, sandhu k.s., ahuja s., 2003 weed management through the use of herbicides in cabbage onion relay cropping system. annal of biology, 19: 27-30. shinggu c.p., dadari s.a., shebayan j.a.y., adekpe d.i., ishaya d.b., 2009 effects of variety, crop arrangement and period of weed interference on the performance of maize grown in mixture in northern guinea savannah of nigeria. arpn jounrnal of agricultural and biological science, 4(2): 47-51. snedecor g.w., cochran w.g., 1967 statistical methods. 6th edition, iowa state university press, usa. 141 1. introduction kiwifruit (actinidia deliciosa) is cultivated mainly due to its sensory properties and its ability for prolonged cold storage. this latter attribute enables consumption of kiwifruit throughout the year in many parts of the world (minas et al., 2010). the most widely grown actinidia cultivar is the a. deliciosa cv. hayward. commercial production of this variety has spread to many countries because of its distinctive characteristics, including fruit size, high productivity, and sufficient storageability (ferguson, 1999). kiwifruit is a good source of natural antioxidant substances, in particular vitamin c (nishiyama et al., 2004). in fact, the content of vitamin c in kiwifruit ranges between 25 and 155 mg/100 g of fresh weight (fw) of fruit (tavarini et al., 2009), making it higher than that determined in orange, strawberry, lemon and grapefruit. beever and hopkirk (1990) showed that vitamin c content in kiwifruit was tenfold higher than the same content found in apple and peach. esti et al. (1998) have observed that the vitamin c content of kiwifruit depends on genotype, ripening degree, storage and the analysis method utilized. kiwifruit’s strong antioxidant capacity is due to a wide number of phytonutrients including carotenoids, lutein, phenolics, flavonoids and chlorophyll (kaya et al., 2008). in fact, during fruit ripening, several biochemical, physiological and structural modifications occur and these changes determine the final fruit quality attributes (lee and kader, 2000; ayala-zavala et al., 2004). during postharvest storage of horticultural crops, important changes in antioxidant status can occur (ayala-zavala et al., 2004). kiwifruit is rich in bioactive compounds, especially polyphenols (park et al., 2009). polyphenolic compounds are a complex group of substances that have gained enormous attention in recent years, especially within the analytical chemistry field because they exhibit important quality properties and antioxidant activity (escarpa and gonzález, 2001). the antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (osawa, 1994). the dark green color of kiwifruit, due to the chlorophylls in plastids in the pericarp cells of the flesh (talens et al., 2002), can be modified during storage. most fleshy fruits are green only during the earlier stages of development: they undergo dramatic changes in chemical composition and ultra-structure during maturation and ripening. associated with tissue softening and the changes in carbohydrate and organic acid metabolism there is conversion of chloroplasts into chromoplasts and a concomitant loss of chlorophyll, often accompanied by accumulation of carotenoids (montefiori et al., 2009). the pigments responsible for the flesh color in ripe fruit of a. deliciosa have already been described (mcghie and ainge, 2002; nishiyama et analytical statistical interpretation of relationship between different parameters of kiwifruit (actinidia deliciosa cv. hayward) during cold storage sh. shahkoomahally (1), a. ramezanian department of horticultural science, faculty of agriculture, shiraz university, shiraz, iran. key words: ascorbic acid, correlation, kiwifruit, total antioxidant activity, total phenolic compounds. abstract: physicochemical and metabolic changes of kiwifruit characteristics were studied during storage. parameters, such as total phenolic compounds (tpc), color (l*, hue, chroma), total antioxidant activity (taa) and ascorbic acid (aa) content, were evaluated at harvest and every 15 days during fruit storage at 0ºc and 90% ± 5 rh. correlations between different parameters were also evaluated. the results of this study suggest that high antioxidant capacity in kiwifruit is due to a strong association (r2˃0.90) between aa and tpc, and that phenolic compounds and aa are the major contributors to the antioxidative activities of actinidia deliciosa cv. hayward. color parameters were found to have weak correlation coefficients with tpc, taa and aa. it is worth noting that the physical-chemical parameters do not have a relationship with the chromatic parameters. adv. hort. sci., 2014 28(3): 141-145 (1) corresponding author: shirin.shakoomahally@yahoo.com received for publication 25 june 2014 accepted for publication 17 september 2014 142 al., 2005); the color is mainly due to chlorophylls a and b (nishiyama et al., 2005). the changes in bioactive compounds of kiwifruit that occur during cold storage have not previously been studied, thus in this paper we describe changes in pericarp color, ascorbic acid (aa), total antioxidant activity (taa) and total phenolic contents (tpc) in the fruits with different storage times. the differences and correlations between these parameters are also examined. 2. materials and methods plant material mature, unripe kiwifruit (actinidia deliciosa cv. hayward) of medium size (80-120 g), free from visible defects or decay, were harvested from a commercial kiwifruit orchard in gorgan, iran with average firmness of 10 (kg/ cm2) and 7% °brix. fruits were immediately transferred to the postharvest laboratory at shiraz university. kiwifruits were individually labeled and packaged into ventilated bags, then stored for four months at 0±1°c and 90±5% relative humidity (rh). samples were taken at monthly intervals during storage for quality evaluation and analyses. physical and physicochemical assays total phenolic contents.the total phenolic contents of each extract were determined according to the method of gutfinger (1981). extracts (1 ml) at 1 mg/ml concentration were mixed with 1 ml of 2% na 2 co 3 . after standing for 3 min, 0.2 ml of 50% folin-ciocalteu reagent was added to the mixture and left to stand for 30 min. the mixture was centrifuged at 13 400×g for 5 min. the absorbance was measured at 750 nm and tpc are expressed as gallic acid quivalents (gae). color. color was determined using digital imaging (afshari-jouybari and farahnaky, 2011). fruit was photographed in a chamber; angle light with the horizontal surface of the images was 45 degrees. after transferring the images to a computer, photoshop image processing software was performed. individual l*, a* and b* parameters were recorded: l* is lightness, a* [-greenness to +redness) and b* (-blueness to + yellowness) are chromacity coordinates. the a* and b* values were converted to chroma (c* = (a*2 + b*2)1/2) and hue angle (h° = tan-1(b*/a*)]. dpph radical scavenging activity (rsa). the free radical scavenging activity was measured by 2,2-diphenyl-2picrylhydrazil (dpph) on the basis of the method of brand-williams et al. (1995) with minor modifications. for this determination, aliquots (0.1 ml) of the extract were mixed with 1 ml of a dpph solution (500 μm) in 80% ethanol. the mixture was incubated at room temperature for 30 min. solution absorbance was determined at λ =515 nm. the dpph radical concentration was calculated using the following equation: scavenging effect (taa %) = (1 af/ao) × 100; ao stands for the absorbance of the control sample and af for the absorbance in the presence of the sample. l-ascorbic acid was used for the calibration curve, and the results are expressed as mg l-ascorbic acid equivalent. 100 g-1 fw (fresh weight). ascorbic acid. ascorbic acid was measured by the oxidation of ascorbic acid with 2, 6-dichlorophenol endophenol and the results are expressed as mg/100 g fresh weight (rangana, 1977). statistical analysis. four replicates of each sample were used for statistical analysis. correlation analyses between different parameters were carried out using the correlation and regression programme in minitab 16. correlations were obtained by pearson’s correlation coefficient (r) in bivariate linear correlations. all statistical analyses and correlations were carried out with sas software package v. 9.1 for windows. differences at p<0.05 were considered to be significant. 3. results and discussions ascorbic acid ascorbic acid (aa) content first increased up to 30 days (47.74 mg/100 g-1) and then decreased (table 1) and could be a result of its synthesis during the initial storage period. the observed variation (increase) of aa content is due to fruit weight loss by dehydration (hence due to a higher concentration) or actually to aa ex novo synthesis. utilization of aa during later storage periods may be the reason for its decreased amounts. the accumulation of aa during ripening depends on the type of fruit. lee and kader (2000) reported that aa content increased with ripening in apricot, peach and papaya, but decreased in apple and mango. generally, when fruits become overripe, vitamin c table 1 changes in aa, tpc, taa and chromacity of kiwifruit during storage storage time (day) ascorbic acid (mg/100 g fw) total phenolic compounds (mg/100 g dw) total antioxidant activity (mg/ 100 g fw) color parameters l* hue chroma 0 40.17±1.53 b 45.75 ±0.99 c 489.17±42.01 bc 51.9 ± 0.83 a 66.92±1.01 a 48.42±1.75 a 30 47.74 ±1.38 a 57.9±1.91 a 602.17±43.97 a 46.07±0.55 b 64.63±2.95 ab 45.49±1.19 b 60 40.97±2.17 b 50.65±3.35 b 556.20±70.33 ba 41.34±0.78 c 62.20±0.73 bc 39.61±0.77 c 90 35.68±2.11 c 43.3±2.56 c 434.34±47.60 dc 35.35±1.50 d 61.82±1.33 bc 35.68±1.16 d 120 27.44±1.89 d 30.45±1.65 d 354.18±40.84 d 39.92±0.82 c 59.76±1.41 c 32.62±1.73 e means within each column with different superscript letters are significantly different (p < 0.05) for each sampling. 143 content declines concurrently with the degradation of fruit tissues (kalt, 2006). in persimmon fruits, aa and tpc content showed a linear relationship with a positive correlation coefficient of r2=0.976 (fig. 1). other authors found strong correlations between aa and tpc in different fruits (gonçalves et al., 2004; serrano et al., 2005). ascorbic acid content was positively correlated with antioxidant activity (r2 = 0.944), suggesting that aa makes a significant contribution to the total antioxidant capacity of kiwifruit (fig. 2). total phenolic compounds total phenolic compounds of fruits increased up to 30 days (57.9 mg/100 g-1) and then decreased (table 1). during storage, tpc increased initially, probably due to synthesis from sugars, and decreased later due to its participation or utilization in other metabolic processes. tavarini et al. (2008) reported that tpc may increase or decrease in fruits and vegetables, depending on the storage conditions. the increase in tpc during storage may be the result of fruit damage and tissue disruption during storage. phenolic compound synthesis in response to wounding has been reported (saltveit, 2000). all o-quinone molecules are highly reactive and may interact with other phenols or other substances, co-polymerise and thus produce compounds, which determine undesired fruit browning, or oxidise further compounds, reduce to original phenols, or react with different nucleophile compounds such as amines, thiols, imidazole, and indole. generally there is a positive correlation between tpc availability and vulnerability to ppo attack (ramírez et al., 2003). phenolic compounds represent the main substrates used by oxidative enzymes, having consequences in terms of color and quality changes, as well as being associated with plant defense mechanisms against stress situations that can affect the postharvest period (tomás‐ barberán and espín, 2001). good correlation between tpc and aa was observed with a high significance level (p<0.001), and a similar relationship (0.97) was also obtained between tpc and taa (table 2). this positive and significant relationship between tpc and taa was greater compared to aa and taa. the results indicate strong association between antioxidative activities and phenolic compounds, suggesting that the latter are probably responsible for the antioxidative activities of kiwifruit. phenolic compounds are also effective hydrogen donors, making them good antioxidants (rice-evans et al., 1995). reports in literature on the relationship between tpc and taa are contradictory; some authors have observed a high correlation (proteggente et al., 2002; tsao et al., 2003; khanizadeh et al., 2008). total antioxidant activity taa increased and peaked over the course of one month (57.9 mg/100 g-1) then decreased toward the end of the storage period (table 1). there is debate in the literature about the influence of vitamin c on the antioxidant capacity of fruits and vegetables (guo et al., 2003). however, it is also known that fruits with high antioxidant capacity generally contain more antioxidants and most of these antioxidants have been shown to be phenolic compounds, in particular flavonoids (guo et al., 2003). for example in pomegranate, ascorbic acid and phenolic compounds are responsible for the taa, alone or in combination (kulkarni and aradhya, 2005). examining the entire storage period, it can be observed that changes in antioxidant activity table 2 correlation matrix (pearson correlation coefficients) tpc aa taa chroma hue l* tpc 1.00 aa 0.98*** 1.00 taa 0.97** 0.97** 1.00 chroma ns ns ns 1.00 hue ns ns ns 0.97** 1.00 l* ns ns ns 0.89* 0.86* 1.00 ns= not significant. * significant to 0.05 p level. ** significant to 0.01 p level. *** significant to 0.001 p level. fig. 1 correlation between taa and aa in kiwifruit. fig. 2 correlation between taa and total phenolic compounds or ascorbic acid in kiwifruit. 144 were very similar to phenolic compounds (lemoine et al., 2009). taa was highly and positively related to aa (r2 = 0.944) (fig. 2). however, in the present study, the best correlation (r2 > 0.96) was observed between tpc and rsa (fig. 2). this fact probably indicates that the antioxidant capacity of kiwifruits is primarily due to tpc and aa. color a significant decrease in hue angle was observed during the storage of fruits at 0°c, indicating continued ripening during cold storage. the initial l* value was 51.9. internal lightness decreased sharply within 90 days of storage and then increased significantly until the end of storage (table 1). the chroma (c*) value was initially 48.42 (table 1). chroma values (internal) of fruits decreased during the entire storage period and reached their minimum values at the end of storage. changes in c* result principally from a loss of chlorophyll content, mostly chlorophyll a which decreases during storage (fuke et al., 1985). in kiwifruit, lightness and chroma values significantly decreased during cold storage and shelf-life, indicating less color intensity (koukounaras and sfakiotakis, 2007). when fruits darken, skin color becomes less chromatic and surface browning develops. diminished red skin and darkening due to oxidative browning reactions have been found to be more marked in ripe strawberries that suffer greater moisture loss during storage (nunes et al., 2005). correlation analyses showed that in the fruit tissues, most of the correlation coefficients were lower, positive and not significant. however, the correlation between chroma of kiwifruit and tpc, taa and aa was weakly positive and significant (table 2). these results indicate that the physical-chemical parameters do not have a relationship with the chromatic parameters and taa, tpc and aa in kiwifruit. similar results were reported by drogoudi et al. (2008) and vieira et al. 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r.d.d.m.c., denardi f., fett r., 2009 physico-chemical and antioxidant properties of six apple cultivars (malus domestica borkh) grown in southern brazil. scientia horticulturae, 122: 421-425. impaginato 269 adv. hort. sci., 2021 35(3): 269­275 doi: 10.36253/ahsc­10193 effect of vine and fruit pruning on yield attributes of two watermelon (citrullus lanatus) cultivars a. ndereyimana 1 (*), b.w. waweru 1, b. kagiraneza 1, a.n. niyokuri 2, p. rukundo 1, g. hagenimana 1 1 rwanda agriculture and animal resources development board (rab), agriculture innovation and technology transfer department, p.o. box 5016, kigali, rwanda. 2 department of crop sciences, college of agriculture, animal sciences and veterinary medicine, university of rwanda, p.o. box 210, musanze, rwanda. key words: apical shoot pinching, cucurbits, fruit pinching, fruit size, hybrid. abstract: this study was carried out to determine the effect of vine and fruit pruning on watermelon (citrullus lanatus) yield. five pruning methods: p1=no pruning at all, p2=pruning to four vines with two fruits per vine, p3=pruning to four vines with one fruit per vine, p4=pruning to three vines with two fruits per vine and p5=pruning to three vines with one fruit per vine were evaluated on two watermelon cultivars: ‘sugar baby’ and ‘julie f1’ under a factorial random­ ized complete block design with three replications. investigations were carried out in the seasons 2017a (short rains) and 2017b (long rains) at karama and rubona experimental sites belonging to rwanda agriculture and animal resources development board. the obtained results indicated a significant dif­ ference among the different cultivars and pruning methods tested during both seasons and at two sites. generally, all studied parameters recorded higher val­ ues during season 2017b than in season 2017a at rubona site. a similar trend was recorded at karama site except that the fruit yield per plant and per hectare for plants which were pruned to three vines with one fruit reduced dur­ ing season 2017b as compared to season 2017a. the highest number of fruits per plant, fruit weight, fruit yield per plant and per hectare was recorded in ‘julie f1’ compared to ‘sugar baby’ at both sites and during both seasons. higher fruit weight was obtained when both cultivars were pruned to three or four vines with one fruit per vine. higher number of fruits per plant and higher fruit yield per plant was observed under pruning to four vines with two fruits per vine at rubona site; while at karama site, higher fruit yield per plant was recorded under pruning to three vines with one fruit or two fruits per vines and pruning to four vines with two fruits per vine. a similar trend was observed in fruit yield per hectare. based on results of the current study, cultivation of the hybrid ‘julie f1’ and pruning to three vines with one fruit per vine is recom­ mended for optimum watermelon yield with big­sized fruits. 1. introduction watermelon is a crop belonging to the cucurbitaceae family that has gained a great economic importance due to its delicious fruits that are also rich in various nutritional compounds (kong et al., 2017). during the (*) corresponding author: assinapol.ndereyimana@rab.gov.rw citation: ndereyimana a., waweru b.w., kagiraneza b., niyokuri a.n., rukundo p., hagenimana g., 2021 ­ effect of vine and fruit pruning on yield attributes of two watermelon (citrullus lanatus) cultivars. ­ adv. hort. sci., 35(3): 269­275 copyright: © 2021 ndereyimana a., waweru b.w., kagiraneza b., niyokuri a.n., rukundo p., hagenimana g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 december 2020 accepted for publication 16 july 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10193 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 269­275 270 year 2018, the worldwide watermelon production was 103,931,337 t harvested over an area of 3,241,239 ha with the yield of 32.1 t ha­1 (fao, 2020); while in east africa, it was 230,729 t from an area of 12,110 ha with the yield of only 19.1 t ha­1. the yield gap of 13 t ha­1 compared to the world’s yield can be addressed by improving production practices, includ­ ing regulation of number of vines and fruits per plant (oga and umekwe, 2016; dube et al., 2020). as other cucurbits, watermelon is also a crop with vines on which female flowers appear after about every five male flowers (dube et al., 2020). the num­ ber of vines per plant is an important parameter determining the performance of cucurbitaceae crops, including watermelon (gomes et al., 2019). on the other side, the number of fruits per vine is also an important parameter that determines fruit size, mass and yield (lins et al., 2013). therefore, due con­ sideration given to these aspects of cucurbit manage­ ment is of utmost importance. in cucurbits, regula­ tion of number of vines per plant and fruits per vine can be achieved through different methods including vine and fruit pruning (campos et al., 2019). pruning is a special horticultural practice that is carried out by removing some parts of plant to boost flowering and subsequent fruiting. this leads to improved yield with enhancement in features required by consumers, such as fruit size and appear­ ance (oga and umekwe, 2016). the main purpose of pruning is to promote balance between vegetative growth and fruit load (anwar et al., 2019). pruning has been reported to increase yield of cucumber (nayak et al., 2018) and butternut (eve et al., 2016). pruning is also a common practice in watermelon production that can lead to synchronization of har­ vesting period and production of uniform fruits (oga and umekwe, 2016). in east african countries, limited investigations have been carried out on watermelon as compared to legume and cereal crops (dube et al., 2020). besides, east african farmers rarely practice water­ melon pruning due to limited knowledge on appro­ priate pruning method. the study was carried out to determine the effect of vine and fruit pruning on yield attributes of two watermelon cultivars. 2. materials and methods sites and seasons of study field experiment was carried out at karama and rubona experimental sites belonging to rwanda agriculture and animal resources development board (rab). karama site is located in eastern province, bugesera district, on longitude 02°23’15”s, latitude 30o11’27”e, and at an altitude of 1524 m above sea level (ndabamenye et al., 2013; kabirigi et al., 2017). its annual average rainfall is 850­1100 mm and the average temperature is 20­21oc (verdoodt and van ranst, 2003). rubona site is located in southern province, huye district, on longitude 029o46’475”e, latitude 02o29’327”s, and at an altitude of 1727 m above sea level (ndabamenye et al., 2013; kabirigi et al., 2017). the annual average rainfall at rubona is 1100­1400 mm and the average tempera­ ture is 17­20oc (verdoodt and van ranst, 2003). at both sites, the study was conducted in two cropping seasons: season 2017a (short rain season) covering the period from september to december 2016 and season 2017b (long rain season) from february to may 2017. study design and treatments the study was conducted in a factorial randomized complete blocks design (rcbd) where treatments were replicated thrice. the treatment structure con­ sisted of five different pruning methods (factor one) evaluated on two watermelon cultivars (factor two). the five pruning methods were designed as follows: p1=no pruning at all (control), p2=pruning to four vines with two fruits per vine, p3=pruning to four vines with one fruit per vine, p4=pruning to three vines with two fruits per vine, and p5=pruning to three vines with one fruit per vine. the two studied watermelon cultivars were c1: ‘sugar baby’ and c2: ‘julie f1’. these cultivars were selected because of their high yield potential under breeder’s conditions. moreover, ‘julie f1’ is a popular hybrid among farm­ ers in east african region. in total, ten treatment com­ binations (cultivar x pruning method) were studied. trial establishment and maintenance field was ploughed twice at an interval of two weeks and was subdivided into plots of 6 m x 3 m that were mulched using dry grass. there were ten plots in each replication; and each plot received a treatment combination of cultivar and pruning method. watermelon seedlings for each cultivar were raised in biodegradable pots and transplanted 30 days after sowing at a spacing of 3 m x 2 m. pruning was achieved by pinching the apical shoot of watermelon vine after six nodes, about 18 days after ndereyimana et al. ‐ effect of vine and fruit pruning on yield attributes of two watermelon cultivars 271 transplanting (anwar et al., 2019). then, at 15 days after pinching, four vines were maintained for p2 and p3 treatments while three vines were maintained for p4 and p5. thereafter, two fruits were maintained per vine for p2 and p4 while one fruit was maintained per vine for p3 and p5. for all the treatments where pruning was carried out, one of the maintained vines was not allowed to have fruits for it to support other in feeding their fruits. apart from watermelon cultivars and pruning methods, other crop management practices such as fertilization, watering, weeding, pests and diseases management were carried out as recommended. fertilization was conducted by applying organic manure and the inorganic fertilizer n:p:k at the rates of 25 t ha­1 and 90:60:60 kg ha­1, respectively. the inorganic fertilizer npk 17­17­17 was used to supply 60:60:60 kg ha­1, which was given in two equal splits (at transplanting and at one month after transplanti­ ng); while additional 30 kg of n ha­1 was supplied in form of urea 46% and was given at two months after transplanting. where necessary, copper oxychloride and carbendazim pesticides were sprayed to control diseases while lambda­cyhalothrin and imidacloprid were used against pests. data collection and analysis observations were carried out on four randomly selected plants per each treatment combination. data were recorded on number of fruits per plant, fruit weight, fruit yield per plant and fruit yield per hectare. the number of fruits per plant was obtained at harvesting through counting all available fruits on four selected plants per treatment combination fol­ lowed by calculating the average per plant. fruit weight (kg) was achieved as the average of five fruits randomly selected from the fruits harvested on the four pre­selected observational plants. fruit yield per plant (kg) was recorded as the average weight of all fruits harvested from four observational plants in the plot under consideration. fruit yield per hectare was derived from computation using the data on fruit yield per plant. analysis of variance for the collected data was performed using genstat 14th edition soft­ ware package and the level of significance was set at p<0.05. least significant difference (lsd) test was conducted for pair­wise comparisons of means. 3. results the number of fruits per plant differed significant­ ly (p<0.05) between cultivars in both seasons (2017a and 2017b) at karama site. significant differences among pruning methods were recorded only in sea­ son 2017a at karama and in both seasons at rubona. between the two cultivars, the highest number of fruit per plant was recorded in ‘julie f1’ (3.5­4.0 fruits per plant) compared to ‘sugar baby’ (3.4­3.7 fruits per plant). for pruning method, the highest number of fruit per plant (4.1­4.8 fruits per plant) at karama site was recorded when both watermelon cultivars were pruned to four vines with two fruits per vine (table 1). a similar trend was also observed at rubona where both watermelon cultivars were pruned to four vines with two fruits per vine record­ ed the highest number of fruits per plant (4.8­5.0) (table 2). there was no significant interaction among cultivars and pruning methods on the number of fruits per plant. fruit weight differed significantly (p<0.05) between cultivars and among pruning methods. the highest fruit weight was recorded in plots where ‘julie f1’ was planted at both sites and it varied between 3.8­3.9 kg per fruit at karama site (table 1) and 3.4­3.8 kg per fruit at rubona site (table 2). among pruning methods, the highest fruit weight (4.2­4.3 kg at karama and 3.7­4.1 kg at rubona) was obtained when both cultivars were pruned to three vines with one fruit per vine during season 2017a and 2017b (table 1 and 2). the interaction of culti­ vars and pruning methods on fruit weight in both 2017a and 2017b seasons at rubona and at karama during the season 2017a was significantly different. however, it was not significantly different in the sea­ son 2017b at karama site. higher fruit weight (4.1 4.6 kg per fruit) was obtained under ‘julie f1’ pruned to three vines with one fruit per vine, followed by the same cultivar pruned to four vines with one fruit per vine (3.7­4.2 kg per fruit) (tables 1 and 2). fruit yield per plant and per hectare was signifi­ cantly (p<0.05) different between cultivars and among pruning methods and their interaction in both seasons 2017a and 2017b, and at karama and rubona sites was not relevant. ‘julie f1’ recorded sig­ nificantly higher fruit yield per plant (11.9­15.4 kg) and per hectare (59.6t ha­1 ­76.9t ha­1) compared to sugar baby which recorded 9.5­11.2 kg per plant and 47.5t­59.1t per hectare. among the pruning meth­ ods, the highest fruit yield per plant (13.9­15.9 kg) and yield per hectare (69.3 t ha­1 ­78.0 t ha­1) was observed under pruning to four vines with two fruits per vine at rubona site (table 2). at karama site, adv. hort. sci., 2021 35(3): 269­275 272 higher fruit yield per plant (15.8 kg) and per hectare (78.8 t ha­1) was recorded under pruning to three vines with one fruit per vine during season 2017 a (tables 1) while during season 2017b, higher fruit yield per plant and per hectare was recorded on watermelon plants that were pruned to three vines with one fruit per vine or four vines with one fruit or two fruits per vine (14.2­15.8 kg per plant and 71.1 t ha­1 ­78.9 t ha­1) (table 1). 4. discussion and conclusions among others, crops of cucurbitaceous family can be manipulated by altering the source: sink ratio through stem and fruit pruning, which affects the number of leaves per plant and consequently leaf area (source) and demand for photoassimilates (sink) (queiroga et al., 2008). in this family, more pruning studies have been carried out on other crops such as cucumber (nayak et al., 2018) and butternut (eve et al., 2016); there is limited information on this aspect as far as watermelon is concerned. therefore, the current study contributed valuable knowledge on watermelon management in a view to optimize its production. heavier fruits obtained under watermelon plants (julie f1) pruned to three or four vines with one fruit per vine fall in line with lins et al. (2013) who report­ ed that fruit thinning allows larger amounts of pro­ duced photo­assimilates to be used by few fruits causing them to attain a size demanded on market. similar to results of the current study, dhillon et al. (2017) also obtained heavier fruits on pruned cucum­ table 1 ­ performance of watermelon as affected by cultivars and pruning method, karama site c1=sugar baby, c2=julie f1, p1=no pruning, p2= pruning to 4 vines with 2 fruits per vine, p3= pruning to 4 vines with 1 fruit per vine, p4= pruning to 3 vines with 2 fruits per vine, p5= pruning to 3 vines with 1 fruit per vine, 2017a=short rains, 2017b=long rains. sed= standard error difference. * and ** =significant difference at 5% and 1% respectively, ns=not significantly different (p>0.05). means within each column followed by the same letter are not significantly different as per lsd test at p<0.05. treatments no. of fruits per plant fruit weight (kg) fruit yield (kg per plant) fruit yield ( t ha­1) season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b factor 1: cultivar (c) c1 3.4 b 3.7 b 3.2 b 3.2 b 10.8 b 11.2 b 54.1 b 59.1 b c2 3.8 a 4.0 a 3.8 a 3.9 a 14.8 a 15.4 a 74.0 a 76.9 a significance *** *** *** *** *** *** *** *** sed 0.095 0.159 0.060 0.063 0.439 0.644 2.19 3.22 factor 2: pruning method (p) p1 3.0 c 3.4 2.8 d 2.9 e 8.6 c 10.0 c 43.2 c 49.7 c p2 4.1 a 4.8 3.2 c 3.3 d 13.5 b 15.8 a 67.3 b 78.9 a p3 3.5 b 3.4 3.8 b 3.8 b 13.4 b 13.2 b 67.0 b 65.7 b p4 3.6 b 4.0 3.5 c 3.5 c 12.8 b 14.2 ab 63.8 b 71.1 ab p5 3.7 ab 3.5 4.2 a 4.3 a 15.8 a 14.9 ab 78.8 a 74.5 ab significance *** ns *** *** *** *** *** *** sed 0.151 0.251 0.094 0.099 0.694 1.019 3.47 5.09 interaction (c x p) c1p1 2.8 3.1 2.7 f 2.7 7.7 8.5 38.3 42.4 c1p2 4.0 4.7 3.6 cd 2.9 11.5 13.7 57.4 68.3 c1p3 3.3 3.4 3.4 de 3.4 11.1 11.7 55.5 58.6 c1p4 3.3 3.8 3.1 ef 3.1 10.3 11.9 51.5 59.7 c1p5 3.6 3.4 3.8 bcd 3.9 13.5 13.3 67.7 66.6 c2p1 3.3 3.8 2.9 ef 3.0 9.6 11.4 48.0 57.0 c2p2 4.3 4.9 2.9 ef 3.6 15.4 17.9 77.2 89.5 c2p3 3.7 3.4 4.2 ab 4.2 15.7 14.6 78.5 72.9 c2p4 3.9 4.2 3.9 bc 3.9 15.2 16.5 76.2 82.6 c2p5 3.9 3.6 4.6 a 4.6 18.0 16.5 90.0 82.4 significance ns ns *** ns ns ns ns ns sed 0.213 0.355 0.133 0.140 0.981 1.441 4.91 7.20 ndereyimana et al. ‐ effect of vine and fruit pruning on yield attributes of two watermelon cultivars 273 ber plants compared to unpruned ones. these find­ ings could be further explained by the fact that plants with less branches allow more light interception and this leads to improved photosynthesis, more accu­ mulation of carbohydrates, and thus overall improved crop performance as compared to plants with relatively higher number of branches (feng et al., 2008). in agreement with the findings of current study, ali et al. (2016) also reported interaction effect of cultivars and pruning on yield of bottle gourd (lagenaria siceraria). in this study, fruit and vine pruning have been observed to significantly affect watermelon yield. this would be attributed to the fact that stem and fruit density is among agronomic variables associated with yield performance of vegetable crops (ayala­ tafoya et al., 2019). the number of stem per plant contributes to plant density, which affects distribu­ tion and utilization of soil nutrients and solar energy (rahmatian et al., 2014). according to campos et al. (2019), efficient solar radiation and production of photo­assimilates are the important pre­requisites for optimum watermelon production. the number of vines per plant also affects the root volume and plant vigour, which in turn influences water and nutrients’ uptake and availability. viana et al. (2008) reported that the lower the number of vine per plant, the higher the root volume and plant vigour and finally the higher yield as a result of improved nutrient and water uptake. the lower yield recorded on watermel­ on plants without pruning could result from competi­ tion for water, nutrients and light (gomes et al., 2019). similarly, muñoz­rengifo et al. (2018) argued that since watermelon has naturally many branches, table 2 ­ performance of watermelon as affected by cultivar and pruning method, rubona site c1=sugar baby, c2=julie f1, p1=no pruning, p2= pruning to 4 vines with 2 fruits per vine, p3: pruning to 4 vines with 1 fruit per vine, p4= pruning to 3 vines with 2 fruits per vine, p5= pruning to 3 vines with 1 fruit per vine, 2017a=short rains, 2017b=long rains. sed= standard error difference. * and **=significant difference at 5% and 1% respectively, ns=not significantly different (p>0.05). means within each column followed by the same letter are not significantly different as per lsd test at p<0.05. treatments no. of fruits per plant fruit weight (kg) fruit yield (kg per plant) fruit yield ( t ha­1) season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b factor 1: cultivar (c) c1 3.4 3.6 2.8 b 3.1 b 9.5 b 11.1 b 47.5b 54.3b c2 3.5 3.8 3.4 a 3.8 a 11.9 a 14.2 a 59.6a 69.9a significance ns ns *** *** *** *** *** *** sed 0.198 0.182 0.051 0.057 0.617 0.643 3.08 3.10 factor 2: pruning method (p) p1 3.0 b 3.2 b 2.5 e 2.8 e 7.6 c 9.0 c 38.0 c 44.0 c p2 4.8 a 5.0 a 2.9 d 3.2 d 13.9 a 15.9 a 69.3 a 78.0 a p3 3.0 b 3.2 b 3.4 b 3.7 b 10.1 b 12.1 b 50.5 b 59.0 b p4 3.5 b 3.7 b 3.1 c 3.4 c 10.9 b 12.9 b 54.3 b 63.7 b p5 3.0 b 3.2 b 3.7 a 4.1 a 11.2 b 13.3 b 55.8 b 65.9 b significance *** *** *** *** *** *** *** *** sed 0.312 0.288 0.081 0.090 0.975 1.016 10.25 4.90 interaction (c x p) c1p1 2.7 2.9 2.4 f 2.7 f 6.5 7.7 32.3 37.4 c1p2 5.0 5.0 2.6 ef 2.9 ef 12.9 14.3 64.3 69.9 c1p3 3.0 3.2 3.0 d 3.3 d 9.0 10.8 45.0 52.5 c1p4 3.3 3.6 2.7 e 3.0 e 9.1 10.8 45.5 52.9 c1p5 3.0 3.2 2.4 c 3.7 c 10.1 12.1 50.5 59.0 c2p1 3.3 3.6 2.6 ef 2.9 ef 8.7 10.4 43.7 50.6 c2p2 4.7 5.0 3.2 cd 3.6 cd 14.9 17.6 74.3 86.1 c2p3 3. 3.2 3.7 b 4.2 b 11.2 13.4 56.0 65.5 c2p4 3.7 3.9 3.4 c 3.8 c 12.6 14.9 63.2 74.5 c2p5 3.0 3.2 4.1 a 4.5 a 12.2 14.6 61.0 72.9 significance ns ns * * ns ns ns ns sed 0.442 0.407 0.115 0.127 1.379 1.437 6.90 6.93 274 adv. hort. sci., 2021 35(3): 269­275 pruning is advised to keep adequate number of branches, leaves and fruits to enable them to share efficiently the plant resources. in agreement with findings of the current study, douglas et al. (2001) and palada and chang (2003) also reported a signifi­ cant effect on yield of cucumber and bitter gourd through pruning by removal of lateral shoots. based on results of the current study, pruning to three vines with one fruit per vine is therefore, recom­ mended for optimum yield with big­sized fruits of both watermelon cultivars (‘julie f1’ and ‘sugar baby’). acknowledgements we thank the government of rwanda for sup­ porting this study through the project “development of market­responsive plant varieties and seed sys­ tems to reduce dependence on importation”. authors are thankful to dative niyomufasha, innocent ndutiye, eric sibomana, immaculée mukabanana, and raurence mukanziza for their enthusiastic involvement in field work. references ali m.r., halim g.m.a., mehrajc g.h.h., 2016 ­ stages of vine pruning for vine production of bottle gourd vari‐ eties and lines in summer season. ­ j. biosci. agric. res., 9(01): 792­795. anwar n.a., gad a.a., bardisi a., zyada h.g., 2019 ­ 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a. tehranifar, h. nemati department of horticultural science and landscape, ferdowsi university of mashhad, iran. key words: antioxidant activity, hollyhock, trace elements, water stress. abstract: water scarcity and dwindling natural resources due to global warm­ ing are negatively impacting ornamental plant survival. soil fertility remains a problem in arid and semiarid regions. in this study, the effects of four media (arable soil, arable soil + cow manure, arable soil + rice hull, arable soil + wheat straw) on macronutrient content and quantitative characteristics of alcea rosea l. under drought stress were investigated. application of organic amendments mitigated the negative effects of drought in the soil and increased the available organic macronutrients. the application of organic amendments increased the total n, p, and k content in the soil and leaves of hollyhock. total soluble sugars (by 11.9%), rwc (by 8.75%) and phenolics (by 36.4%) of hollyhock were signifi­ cantly improved by the application of organic amendments at 80% fc. the amended soil (soil + cow manure) increased the activities of superoxide dismu­ tase and ascorbate peroxidase at 80% fc. moreover, the soil + cow manure proved to be the best supplement to improve leaf area and dry weight. in con­ clusion, the application of organic amendments can be successfully used as a cost­effective management method to improve soil fertility and crop produc­ tion in arid and semi­arid areas. 1. introduction considering the increasing population of the planet earth, stability in green space has particular importance (wolch et al., 2014). exacerbation of environmental stressors lays the groundwork for the loss of ornamen­ tal plants. therefore, one of the critical goals of plant producers and breeders is providing quality and stress­resistant plants for green space (anguelovski et al., 2020). among ornamental plants, alcea, commonly known as hollyhock, is a perennial plant of the malvaceae family, with decorative and medicinal importance. people use the flowers of this plant to produce medicinal tea due to pigments (shehzad et al., 2020). in addi­ tion, the antibacterial, anticancer (lim, 2012), antioxidant (ahmed et al., 2016), anti­depressant, anti­inflammatory (ahmadi et al., 2012), anti­ fatigue, febrifuge, mouth washing (burt and reinders, 2003), and blood circulation enhancer (lim, 2012) characteristics of this plant have been proven. regarding the climatic changes and the resistance of this plant to adverse conditions, it can be a desirable plant in the green space (oraee (*) corresponding author: shoor@um.ac.ir citation: oraee a., shoor m., oraee t., tehranifar a., nemati h., 2022 ­ organic amendments role in reducing drought stress in alcea rosea l. ­ adv. hort. sci., 36(3): 201­214. copyright: © 2022 oraee a., shoor m., oraee t., tehranifar a., nemati h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 november 2021 accepted for publication 1 july 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12185 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 201­214 202 et al., 2019). among the environmental factors, drought is the most critical factor limiting growth in many areas (toscano et al., 2019; bhusal et al., 2020). based on the scientistsʼ prediction, global temperature could rise by 3 to 9°c with far­reaching effects and signifi­ cantly increase the dryness of the arable lands (haile et al., 2020). regarding the increase in global tem­ perature by 1.5°c, plants selection with high toler­ ance to drought stress to ensure the survival and sta­ bility of green space is an essential strategy (seleiman et al., 2021). in such conditions, in addition to the skill and accuracy of using and consuming these water resources, recommending tolerant plants, determining the drought tolerance threshold, and increasing soil fertility has become more neces­ sary (banks et al., 2019; du et al., 2019). the elements required in organic matter can increase soil fertility and plantʼs yield in agriculture (ukalska­jaruga et al., 2020). organic fertilizers pro­ vide nutrient balance and increase soil nutrient avail­ ability (verma et al., 2020). these organic substances have benefits, including reducing leaching and wastage of nutrients (quynh and kazuto, 2018) and helping the release elements (mupambwa and mnkeni, 2018). they enhance root growth due to improved soil structure, increase the amount of organic matter and soil exchange capacity, and ulti­ mately serve as a source for the growth of soil organ­ isms and increase soil yield (juriga et al., 2018; chew et al., 2019). some studies documented the reduced element transfer in plants under soil drought stress (liu et al., 2018; qi et al., 2019). in water shortage conditions, organic matters can conserve water in the soil and prevent the destructive effects of drought stress on plant yield (kaya et al., 2020). also, khosravi shakib et al. (2019) reported that the total dry weight and water use efficiency were about 3­fold higher in marigold (calendula officinalis l.) grown in 30% manure compost substrate compared to the control plants. different plant species show a wide range of drought resistance mechanisms, including antioxi­ dant activity and osmoregulatory adaptations (siddique et al., 2018; khan et al., 2020). there should be reconsideration of the plants type grown in arid and semi­arid regions. also, some plants with high water requirements should be replaced with the drought­tolerant plant (de souza aguiar et al., 2020). hollyhock is a low­expectation plant that grows well in natural and marginal areas that can be considered suitable for cultivation in low­input systems. this is a first work useful to develop a suitable methodology for studying the use of soil organic amendments. also, this study was conducted to identify the drought tolerance threshold of hollyhock and identi­ fy different growth mediums on drought tolerance of hollyhock. 2. materials and methods plant material the experiment was conducted in the research greenhouse of ferdowsi university in mashhad, iran. seeds were sown in august in the greenhouse at an average temperature of 20±1°c in plug trays contain­ ing a mixture of coco peat, perlite and peat (1:1:0.5 v/v/v). plants grew at 400 μmol m­2 s­1 combined with a photoperiod of 14 and 10 h d­1. then, all plants at the 5­6 leaf stage were transferred to pots (18 cm high and 8 cm in diameter) containing arable soil. in may, the plants were transferred to pots of size 20 with a soil mixture of four different substrates (arable soil, arable soil + manure, arable soil + rice hull, and arable soil + wheat straw). experimental design and treatments the experiment consisted of two factors (organic supplements and irrigation regime). the organic sup­ plements and irrigation regimes had four and three levels, respectively. three sources of organic supple­ ments were used (manure, rice hulls, and wheat straw). the pot experiment (one plant per pot) was conducted factorially in a randomised complete block experiment with three replicates under greenhouse conditions. the mixtures between the soil and cow manure, rice hull and wheat straw were prepared in a 50:50 ratio (50% w/v soil and 50% w/v organic amend­ ments). the soil from the four treatments (control soil and the three additives) was taken to the laboratory for chemical analyses after the mixtures were pre­ pared (as described above). the physicochemical properties of the pot samples are explained in table 1. drought stress treatments plants were subjected to drought stress during flowering on june 5. soil volumetric water content was measured during the flowering period using the oraee et al. ‐ organic amendments and drought stress in hollyhock 203 time domain reflectometry (tdr) instrument (trike­ fm, england). plants were exposed to drought stress for one month. plants were irrigated through a poly­ ethylene piping network with volumetric meters at 80, 60, and 40% fc. foliar nutrient analysis foliar nitrogen, potassium, and phosphorus were measured at the end of the experiment. leaf samples (0.2 g) were heated to 400°c with 10 mg sulfuric acid and a catalyst mixture in a digestion apparatus, and then nitrogen content was measured using a kjeldahl apparatus (kjeldal, 1998). leaf potassium was mea­ sured using the flame photometer method (tandon, 1998). phosphorus content was determined by the colorimetric method (yellow molybdate vanadate) at 470 wavelengths (rayan et al., 2001). determination of electrolyte leakage leaf electrolyte leakage was measured according to the method of reddy et al. (2004). tissue samples (0.2 g) from the first true leaf of 9­month­old plants were placed in 50 ml of distilled water. the samples were stored at laboratory temperature for 24 hours and the conductance of the solution was measured using a conductivity meter (jenway model). all leaves were autoclaved for 20 minutes to measure the final leakage. electrolyte leakage was calculated as: el% = ec1/ec2 × 100. total soluble sugars assay spectrophotometry at 620 nm was used to ana­ lyze total soluble sugars using the anthron method (mccready et al., 1950). total soluble sugars were extracted by homogenizing the plant material in 80% ethanol. samples were centrifuged at 3500 rpm for 10 min. anthrone solution (10 ml 0.15%) was added to 1 ml of the solution. the samples were then heat­ ed to 95°c and immediately transferred to an ice bath. the total sugar concentration of the samples was calculated using the standard glucose curve based on mg g­1 dry weight (ebell, 1969). determination of proline and relative water content proline content and relative water content (rwc) were determined according to the procedure described by bates et al. (1973) and turner (2018), respectively. chlorophyll determination chlorophyll was determined using the method of arnon (1949). for this purpose, 0.2 g of fresh leaves were ground with 80% acetone. the resulting solu­ tion was centrifuged at 4000 rpm for 10 minutes. the optical absorbance of the supernatant was measured using a spectrophotometer (shimadzu uv­160a) to determine the amount of chlorophyll at 645 and 663 nm. phenol assay in this study, total phenol was determined using the folin­ciocalteu reagent (fcr) according to the method of singleton and rossi (1965). an amount of 4.5 ml of distilled water and 0.1 ml of folin­ciocalteu reagent were added to 0.1 ml of the methanolic extract. after 3 minutes, 2% sodium bicarbonate solution was transferred to 0.3 ml solution. the dif­ ferent concentrations of gallic acid were used to con­ struct the standard curve and measure the absorbance using a spectrophotometer (shimadzu uv­160a) at 760 nm. total phenol was calculated based on mg gae g­1 dry weight. protein and enzyme activity assays bradfordʼs method (1976) with slight modification was used to measure total protein . to assay protein, 50 mg of fresh leave was ground in liquid nitrogen. then 50 mg of polyvinylpolypyrrolidone, 495 μl of extraction buffer solution (including 40 mm hydrogen tris­chloride buffer, 2% sodium dodecyl sulfate, 20% glycerol, and 60 mm dithiothreitol), and 5 μl of phenylmethyl methanol to 200 ml of phenylmethyl methanol were added to each solution. the samples were centrifuged at 8000 rpm at 4°c for 15 minutes. then 300 μl of the supernatant with 900 μl of ace­ tone containing 10% trichloroacetic acid and 0.07% table 1 ­ physicochemical properties of the soils used in the experiment growth medium n (%) p (mg kg­1) k (mg kg­1) ph ec (ds/m) soil 0.065 23 235 7.15 1.93 soil + cow manure 0.42 2214 1592 7.76 2.53 soil + rice hull 0.23 271 343 7.26 1.77 soil + wheat straw 0.21 117 1572 6.68 3.13 adv. hort. sci., 2022 36(3): 201­214 204 dithiothreitol was added and placed at ­20°c. 100 μl of adsorption buffer was added to the supernatant. nakano and asadaʼs method (1981) was used to measure the activity of ascorbate peroxidase. the reaction mixture consisted of 20 μl of enzyme extract, 770 μl of 50 mm phosphate buffer, 100 μl of 0.1 mm edta, 100 μl of 5 mm ascorbate, and 10 μl of 0.1 mm hydrogen peroxide. the absorption rate of the reac­ tion was read by a spectrophotometer at 290 nm. superoxide dismutase activity (sod) was mea­ sured according to the sairam et al. (2002) method with slight changes. the enzymatic reaction mixture consisted of 935 μl of 50 mm phosphate buffer con­ taining 0.1 mm edta, 13 mm methionine, and 75 mm nitroblue tetrazolium (nbt), 15 μl of 0.12 mm riboflavin, and 50 μl of enzymatic extract. after preparing the control and blank samples to measure enzymatic activity, the blank sample was placed in the dark for 15 minutes, and the control and enzyme extract samples were shaken for 15 minutes in a shaker at 25°c with two 20 w fluorescent lamps at 100 rpm. the absorbance was read at 560 nm using a spectrophotometer (shimadzu uv­160a). aebiʼs method (1984) was used to measure the activity of catalase (cat) enzyme, where 20 μl of enzymatic extract mixed with 50 mm phosphate buffer containing ten mm hydrogen peroxide and their adsorption changes were recorded at 240 nm by spectrophotometer (shimadzu uv­160a). glutathione reductase was measured by sofo et al. (2004). determination of growth parameters leaf area of hollyhocks was determined using a delta­t leaf area meter (device ltd., cambridge, uk). plants were dried at 60°c and weighed to calcu­ late their respective dry weights. statistical analysis statistical analysis was performed using sas 8.1 software. data are presented as mean ± se of three replicates. means were also compared using the lsd test at a probability level of 5%. 3. results foliar nutrients (n, k, and p) and soil nutrients (n and k) were significantly (p≤0.05) affected by organic amendment and drought. in the desiccated soils, n content increased significantly with the application of manure amended soil. foliar n content was 50% and 14% higher in the manure­amended and rice hull­ amended soils, respectively, than in the control at 80% fc. finally, n foliar content was higher in soils enriched with manure in all irrigation regimes, fol­ lowed by soils enriched with rice hulls at 80 and 60%, respectively fc (fig. 1a). similar to n content, leaf p content was also higher in soils fertilized with 80% fc. overall, drought stress (60% fc) had no effect on p content in soils enriched with manure and rice hulls (fig. 1b). the magnitude of the increase in k content was more significant in plants under 80% fc than in plants under 40% fc, and it was more pronounced in the amended soils than in the control. drought stress significantly affected k content, decreasing it slightly (by 4.74%) in manure amended soils under 40% fc compared to 80% fc (fig. 1c). leaf nutrient contents analyzed were similar com­ pared with those in soils. in the unamended soils subjected to different irrigation regimes, soil n con­ fig. 1 ­ leaf n (a), leaf p (b), and leaf k (c) of hollyhock plants. values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). oraee et al. ‐ organic amendments and drought stress in hollyhock 205 tent decreased significantly. compared to the con­ trol, organic soil amendments caused a less dramatic increase in n content. n content in soils amended with manure was higher in soils amended with 80, 60, and 40% fc than in other treatments (fig. 2a). p contents in desiccated soils decreased progressively, and these values (p≤0.01) were significantly lower than those determined for well­watered soils. soil p content was consistently higher in soils amended with manure and rice hulls than in control soils. application of organic amendments to desiccated soils (40 and 60% fc) also increased k content. k accumulation was lower in soils enriched with wheat straw than in soils enriched with manure (fig. 2b). organic amendment significantly affected ph index (p≤0.01), but ec was affected by organic amendment and drought stress. the ph index in soil amended with manure increased significantly (by 5.57%) compared to the control. overall, the ec was higher in soils with 40% fc than in the corresponding soils with 80% fc. the ec was 22.9% higher in the manure amended soils than in the control. organic amendments and drought stress significantly (p≤0.05) affected rwc, el %, and phenolic content. rwc decreased slightly in amended soils with 40% fc compared to soils with 80% fc. conversely, 40% fc decreased rwc in unamended soils (fig. 3a). el % in unamended soils was higher at 40% fc than in well irrigated soils, while for rice hull and wheat straw there was no difference in this variable at 40% fc (fig. 3b). total soluble sugar and proline were signifi­ cantly (p≤0.01) affected by the interaction between drought stress and organic amendments. in well­ watered soils, soluble sugar and proline contents were consistently higher in amended than in control soils. in leaf tissues of plants treated with 40% fc, total soluble sugar content increased by 6.17% in amended soils compared with unamended soils, and this index remained unchanged in leaf tissues of soils amended with rice hull and wheat straw at 60 and 40%, respectively fc (fig. 3c). proline tissues were also dependent on drought stress, and a maximum increase of 177% was observed in the soils amended with slurry at 40% fc compared to the control (fig. 3d). chlorophyll a, b, and total chlorophyll were signifi­ cantly (p≤0.05) affected by the interaction between drought stress and organic amendments. it was found that chlorophyll content increased in plants grown with organic amendments under drought stress. the highest chlorophyll a content was found in soils fertilized with 80% cow manure fc (fig. 4a). soils fertilized with cow manure showed a significant increase in chlorophyll b under drought stress (fig. 4b). between treatments, the highest average total chlorophyll (3.18 mg g­1 fw) was measured in soils fig. 2 ­ the soil­n (a) and soil­k (b). values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). fig. 3 ­ electrolyte leakage (a), rwc (b), proline (c), and total soluble sugar (d) in hollyhock plants under different medium. values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). 206 adv. hort. sci., 2022 36(3): 201­214 amended with cow manure at 80% fc, while the low­ est total chlorophyll (1.37 mg g­1 fw) was measured in control plants at 40% fc (fig. 4c). changes in phe­ nolic content of hollyhocks showed similar trends for both soils (wheat straw enriched soils), such that phenolic content increased under severe water stress, but this index in plant tissues grown in manure and pod enriched soils showed no difference under all irrigation regimes (fig. 4d). the interaction of drought stress and organic amendments significantly affected antioxidant activi­ ty (p≤0.01). glutathione reductase levels were higher in rice­treated soils than in other treated soils. drought stress significantly increased this index in amended soils. however, glutathione reductase in leaves of hollyhocks grown in manure amended soils (40% fc ) increased significantly by 28% compared to control plants (80% fc). there was no significant dif­ ference between glutathione reductase in manure and wheat straw ­amended soils at 40% fc (fig. 5a). in addition, significant improvement of sod and apx was observed under severe drought stress. the maxi­ mum value of sod was measured in the amended and unamended soils in 40% fc treatments (fig. 5c). the activity of apx was lower in the unamended soils than in the corresponding soils in amended soils, while there was no significant difference between soil irrigation regimes. overall, apx activity was high­ er in manure amended soils at 60 and 40% fc than in the corresponding soils at 80% fc (fig. 5d). no differ­ ences were observed in cat values between manure­ amended and non­manure­amended soils at 40% fc, while a slight increase was measured in manure­ amended soils (fig. 5d). leaf area and dry weight of hollyhock (p≤0.05) were significantly affected by drought and amended soils. leaf area at 40% fc was generally lower than that of the control throughout the evaluation period. leaf area at 80% fc was higher in manure amended soils than in nonamended soils (fig. 6a). in well­ watered soils, dry weight was consistently higher in amended soils than in control soils. in general, this index was lower in soils at 60 and 40% fc than at 80% fc. drought (40% fc) had a negative effect on dry weight, especially in the unworked soils. at 80% fc, this index was lower in soils with rice hull admix­ ture than on wheat straw medium (fig. 6b). 4. discussion and conclusions the results of a recent experiment show that a suitable nutrient medium reduces the negative effects of drought stress. the results show that organic amendments in soils improved macroele­ ments in leaves and soil. our results are in agree­ ment with those of banik et al. (2006), who showed an increase in nitrogen, phosphorus, and potassium in rice (oryza sativa l.) after the application of vari­ ous organic matter such as animal and poultry manure and rice. the researchers observed the high­ est phosphorus and potassium content in rice grain fig. 4 ­ chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), phenol (d) in hollyhock plants under different medium. values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). fig. 5 ­ gr (a), cat (b), sod (c), and apx (d) in hollyhock plants under different medium. values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). oraee et al. ‐ organic amendments and drought stress in hollyhock 207 and rice branches, respectively, under cow manure. he and dijkstra (2014) found that nitrogen and phosphorus concentrations decreased under drought stress. a significant decrease in nitrogen, phosphorus and potassium uptake in maize (zea mays l.) branch­ es was found to be twofold under drought stress con­ ditions (alizadeh, 2010). in the studies of ghazi (2017), increasing drought stress from 50% to 100% fc reduced nitrogen, phosphorus and potassium uptake in maize leaves. under drought stress, the average potassium concentration in the roots of treated apocynum plants (apocynum venetum l.) decreased by 40%, but in the leaves of this plant, the amount of potassium remained constant and increased in the stem. the result suggests that the plant stores large amounts of potassium to maintain osmotic adjustments (cui et al., 2018). leaf potassium content decreased under severe drought stress because nutrient transport and uptake depend on soil moisture. decreased element transfer in plants under drought stress conditions has been documented in many studies (ahangar et al., 2016; li et al., 2021). hollyhock plants were able to maintain potassium content in leaves up to 60% fc, which was consistent with the results of qi et al. (2019), which showed that the accumulation of potassium in the plant was higher under non­stress conditions than under stress conditions, in addition, further reduction of irrigation under severe stress reduced the amount of potassium in the leaves of plants. the reduction in transpiration rates and changes in membrane trans­ porters under drought conditions due to water deficit, which lead to reduced mineral nutrition in plants, showed that phosphorus uptake in rice increases with increasing moisture content (roy, 2018). the highest phosphorus and potassium uptake occurred due to mineralization of fertilizer elements in the soil. application of nitrogen and phosphorus fertilizers in tortoiseshell bamboo (phyllostachys edulis) increased soil phosphorus, nitrogen, and leaf phosphorus (wu et al., 2018). there is a positive cor­ relation between soil macroelements and leaf ele­ ments (table 2). numerous studies have shown that organic fertilizer increases soil ph (liu et al., 2010; han et al., 2016). organic fertilizers increase calcium carbonate, which is believed to increase buffering and thereby improve soil ph (whalen, 2000). electrical conductivity can serve as a critical indicator of nutrient and water uptake (dhaliwal et al., 2019). application of poultry, cattle, and goat manure signif­ icantly increased soil electrical conductivity, and the potential for manure­induced soil salinity was rela­ tively high for poultry and goat manure (azeez and van averbeke, 2012). the increased electrical con­ ductivity of soil is due to the release of salts from fer­ tilizers. as a result of the decomposition of organic matter in the soil, the ions obtained from the decom­ position entered the soil solution and consequently increased the salinity of the soil, which was consis­ tent with roy and kashem (2014). an increase in drought stress resulted in an increase in electrolyte loss, such that electrolyte loss increased in leaves of maize (mozdzen et al., 2021) and tomato (ors et al., 2021) compared to the con­ trol under drought conditions. in addition, nitrogen uptake occurs by mass uptake and potassium and phosphorus uptake occurs by diffusion (li bot et al., 2021). in the absence of water, the uptake of nitro­ gen and phosphorus is reduced. in addition, the pres­ ence of potassium under drought stress conditions maintains turgor pressure and osmotic adjustments of cells (singh et al., 2021). in turtle shell bamboo, phosphorus and nitrogen addition to plants under drought stress decreased malondialdehyde and reduced electrolyte loss compared to the control (wu et al., 2018). in the current experiment, due to fig. 6 ­ leaf area (a), and dry weight (b) in hollyhock plants under different medium. values in columns followed by the same letter are not significantly different at the 0.05 level (p≤0.05). adv. hort. sci., 2022 36(3): 201­214 208 the increase of nitrogen, potassium, and phosphorus in the soil and the increase of these elements in the leaves of the irrigated plants by 80% fc, the rate of electrolyte loss in these plants decreased compared to other substrates. researchers showed that k+ efflux could play an essential role in anabolic reac­ tions by stimulating catabolic processes and saving “metabolic” energy for adaptation and repair needs in plants (demidchik et al., 2014). in our study, an increase in drought stress also decreased the amount of chlorophyll in leaves, which is consistent with the opinions of others (khayatnezhd and gholami, 2021). in peach plants, the use of phosphorus increased chlorophyll content, but reducing phosphorus decreased protein and chlorophyll content (dutt et al., 2013). the use of nitrogen­ and phosphorus­containing fertilizers effec­ tively increased the chlorophyll and carotenoid con­ tent of apples because an increase in nitrogen pro­ moted the formation of photosynthetic pigments by thylakoid and stomatal proteins, which also increased the formation of chloroplasts in growing leaves (jahan et al., 2020; siddiqui et al., 2021). the biochemical and biosynthetic properties of photosyn­ thetic pigments require phosphorus as well as nitro­ gen (da silva tavares et al., 2020). in the recent experiment, there was a positive correlation between nitrogen and potassium with chlorophyll (table 2). the amended soil has increased the leaf elements by increasing the number of ele­ ments in the soils under 80% fc. in hyssop (hyssopus officinalis), the effects of drought stress and potassi­ um fertilizer on chlorophyll content were significant, such that the amount of total chlorophyll increased with increasing potassium and irrigation regime (lopo de sa et al., 2014). in plants under higher drought stress, nutrient deficiencies and reduced energy absorption of sunlight lead to damage to the photosynthesis and chlorophyll systems because nutrients play an essential role in the electron trans­ fer system and carbon metabolism (xu et al., 2020; ma et al., 2021), which is consistent with the study results because the lowest chlorophyll in drought treatment of 40% fc was recorded in the soil sub­ strate. with increasing drought stress from 80 to 40% fc, the plantʼs rwc decreased. however, organic fertiliz­ ers reduced the adverse effects of drought stress on the rwc. there have been numerous reports of changes in relative water content and osmotic adjustments in leaves occurring under drought stress, and that these variations were different depending on cultivar, species, duration, and intensity of stress (kizilgeci et al., 2020; zhu et al., 2020). the main rea­ table 2 ­ correlation coefficients among different parameters of hollyhocks el= electrolyte leakage, rwc= relative water content, gr= glutathione reductase, cat= catalase, apx= ascorbate peroxidase, sod= superoxide dismutase, * p≤0.05; ** p≤0.01; ns= not significant. soil n soil p soil k leaf n leaf p leaf k chloro­ phyll el rwc proline total soluble sugars gr cat apx sod leaf area dry weight soil n 0.82 ** 0.90 ** 0.92 ** 0.55 ** 0.74 ** 0.47 ** ­0.31 ** 0.71 ** 0.89 ** 0.71 ** 0.84 ** 0.06 ns 0.75 ** 0.30* 0.89 ** 0.95 ** soil p 0.65 ** 0.97 ** 0.52 ** 0.66 ** 0.46 ** ­0.25 * 0.57 ** 0.81 ** 0.57 ** 0.81 ** 0.17 ns 0.59 ** 0.49 ** 0.71 ** 0.82 ** soil k 0.82 ** 0.45 ** 0.78 ** 0.41 ** ­0.25 * 0.67 ** 0.94 ** 0.72 ** 0.81 ** 0.02 ns 0.72 ** 0.28 * 0.79 ** 0.56 ** leaf n 0.47 ** 0.67 ** 0.47 ** ­0.42 ** 0.77 ** 0.87 ** 0.71 ** 0.71 ** 0.02 ns 0.66 ** 0.31 ** 0.79 ** 0.89 ** leaf p 0.68 ** 0.42 ** ­0.70 ** 0.64 ** 0.42 ** 0.36 ** 0.49 ** 0.19 ns 0.52 ** 0.25 * 0.76 ** 0.61 ** leaf k 0.31 ** ­0.42 ** 0.54 ** 0.71 ** 0.61 ** 0.86 ** 0.01 ns 0.87 ** 0.37 ** 0.91 ** 0.82 ** chlorophyll ­0.52 ** 0.81 ** 0.19 ns 0.15 ns 0.08 ns 0.36 * 0.06 ns ­0.36 ** 0.57 ** 0.51 ** el ­0.73 ** ­0.18 ns 0.16 ns ­0.20 ns ­0.24 * ­0.34 ** 0.26 * 0.61 ** 0.48 ** rwc 0.56 ** 0.31 ** 0.43 ** 0.22 ns 0.37 ** 0.05 ns 0.76 ** 0.78 ** proline 0.83 ** 0.86 ** ­0.06 ns 0.72 ** 0.40 ** 0.71 ** 0.86 ** total soluble sugars 0.78 ** ­0.13 ns 0.70 ** 0.58 ** 0.55 ** 0.65 ** gr ­0.02 ns 0.86 ** 0.54** 0.74 ** 0.80 ** cat ­0.02 ns ­0.1 ns 0.20 ns 0.12 ns apx 0.60 ** 0.74 ** 0.73 ** sod 0.26 * 0.31 ** leaf area 0.89 ** dry weight oraee et al. ‐ organic amendments and drought stress in hollyhock 209 son for the improvement in drought stress relief in the presence of potassium is due to the osmotic adjustments and the preservation of the rwc (ibrahim et al., 2020). fahri et al. (2021) reported an increase in the rwc of two oilseeds with potassium fertilizers under drought stress. similar responses to potassium fertilizers were reported in corn (khadem et al., 2010) under drought stress. also, in the study, a positive and significant correlation was recorded between leaf nitrogen and potassium content with the rwc of leaves (table 2), and the lowest leaves relative water content was recorded in soil­substrate plants with the lowest amount of nutrition. drought stress leads to biochemical and physio­ logical changes that lead to osmotic changes and decreased turgor pressure in the cell. these changes degrade nitrate assimilation by reducing nitrate reductase activity (zhang et al., 2009). plants use osmotic adjustments to reduce the effects of drought stress due to nitrate assimilation, in which proline accumulation plays a vital role in osmotic adjustment (ozturk et al., 2021). zhang et al. (2014) showed that the accumulation of osmotic compounds such as pro­ line, betaine, and potassium ion in two maize culti­ vars was higher in more resistant cultivars than in more sensitive cultivars under drought stress. moreover, potassium maintains the turgor pressure by reducing the leaf water potential (da silva et al., 2021). potassium application causes proline accumu­ lation by maintaining osmotic adjustments (aksu and altay, 2020). in the present experiment, the proline increased with increasing drought stress. leaf analy­ sis showed that the potassium in leaves of plants in animal manure culture is higher than other sub­ strates, which increases the accumulation of proline in osmotic adjustments. although its exact mecha­ nism is unknown, it seems to be due to the role of potassium in amino acid metabolism or the effect of potassium on the proline cycle (zhu et al., 2019). drought stress not only increases the stomatal resis­ tance of plants but also causes the accumulation of osmotic substances (zahoor et al., 2017). by increasing water uptake and reducing water loss, plants under drought stress prevent further damage (closing stomatal and smaller leaves) (hill et al., 2020). when photosynthesis is not responsive to the plant, carbohydrates break down, maintaining osmotic adjustment in the cell. the sucrose signifi­ cantly decreased under drought stress (yang et al., 2019). nutrients play an important role in drought stress on total soluble sugars as an osmotic adjust­ ment and mitigate drought stress. the potassium accumulation in the vacuole and sucrose maintains the turgor and osmotic pressure and effectively increases water uptake in plants (sardans and peñuelas, 2021). in addition, drought stress condi­ tions prevent chloroplast damage by maintaining high cellular ph (cakmak, 2005). drought stress treat­ ment on apple (malus domestica borkh.) plants showed that plant sucrose decreased at the begin­ ning of stress but increased over time. glucose and fructose also had an increasing trend at all times. the process of reducing sucrose reduces vegetative growth in plants under drought stress. the leaf nitro­ gen decreased with decreasing the amount of applied irrigation water, and with re­irrigation, the nitrogen increased. phosphorus levels also showed a downward trend in plant parts under drought stress (jie et al., 2010). in a recent experiment, plants in soil + manure substrates kept the total soluble sugars constant in irrigation at 40% fc compared to 80% fc by absorb­ ing nutrients from the soil. the correlation between the total soluble sugars with nitrogen and potassium proves these results (table 2). in addition, in the treatment of 40% fc, plants used the total soluble sugars increase for osmotic adjustment and as a sys­ tem to resist drought stress. in the present experiment, antioxidant activity was increased under drought conditions. several studies reported that the antioxidant enzymes activi­ ties increased under stress (hasan et al., 2021; sepahvand et al., 2021). our results were in line with safari et al. (2021) and babaei et al. (2021), who showed that drought stress increased ascorbate per­ oxidase activity in impatiens and marigold, respec­ tively. applying nitrogen and phosphorus fertilizers sig­ nificantly increased the activity of the peroxidase enzyme under drought­stressed plants when com­ pared to the non­application of these two elements (wu et al., 2018). using vermicompost organic fertil­ izer under drought stress conditions in lettuce (lactuca sativa l.) increased the activity of superox­ ide dismutase and catalase enzymes as an enzyme system, reducing the adverse effects of stress. the correlation between growth traits and the activity of these enzymes under stress showed that the vermi­ compost application increased plant growth and drought tolerance. the results also showed that the nitrogen and organic matter in vermicompost soil was higher, which led to increased plant growth adv. hort. sci., 2022 36(3): 201­214 210 (kiran, 2019). in plants treated with 80% fc in soil substrate + animal manure with higher leaf elements, enzymatic antioxidant activity increased. moreover, enzyme activity were positively correlated with elements such as nitrogen and potassium in the leaves (table 2). the plant needs potassium to maintain enzyme activity and protein synthesis because protein struc­ ture need high k+ in cystocele (blevins, 1985). a reduction in leaf area and dry weight under drought stress is attributed to photosynthesis and carbon assimilation. in the present experiment, the plant leaf area decreased with increasing drought stress, and the dry leaf weight decreased with decreasing leaf area, consistent with the results of khaleghnezhad et al. (2021). in the low moisture con­ ditions, the plants reduce the leaf area to decrease the level of respiration. in a recent experiment, plant leaves fell under severe drought stress. to this aim, the leaf area was reduced relative to sufficient mois­ ture conditions, and this may be a positive adapta­ tion to acclimatization to dehydration (isa et al., 2021). chekanaia et al. (2018) showed that the appli­ cation of nitrogen (ammonium phosphate) and phos­ phorus (superphosphate) in beans doubled the dry weight and plant yield in both treatments compared to the control. lubis et al. (2021) stated that dry weight of rice and plant height that received ele­ ments increased under manure medium. potassium reduces the negative effect of drought stress on leaf area index and plantʼs dry weight by increasing the ability of photosynthesis, the rate of co2 stabilizing with interference in osmosis, the activity of the enzyme rubisco, and improving the synthesis and transport of dry matter (wang et al., 2013). the data from the present experiment show that potassium increased plant resistance to drought stress, for which researchers published many reports on the subject (aksu and altay, 2020; aqaei et al., 2020). in the present experiment, the negative correla­ tion between plant dry weight and electrolyte leakage (table 2) indicates that dry weight plants decreased in severe drought stress with increasing electrolyte leak­ age, which was consistent with the opinion of khan et al. (2021). they showed that the plants dry weight decreased with increasing electrolyte leakage due to the increase of malondialdehyde in severe drought stress. also, the results showed a positive correlation between chlorophyll content and plant dry weight (table 2). xiao et al. (2008) showed that chlorophyll depletion occurred under drought stress due to chlorophyll decomposition. this is the first work aimed at developing an appropriate methodology to study the use of organic soil amendments for plants such as hollyhock. the application of organic amendments also efficiently improved the uptake of n, p and k from the soil under water stress. drought stress negatively affect­ ed plant traits, while soil amendments in planting beds improved plant performance and promoted plant resistance to drought stress. based on the results of this study, soil with cow manure is suggest­ ed as an efficient soil amendment that is statistically better than rice husks and wheat straw for minimiz­ ing water requirements and improving plant toler­ ance to drought in a potted hollyhock. due to the availability of cow manure compared to other sub­ strates, the use of this material under potting soil conditions is recommended as these amounts cannot be used in the field. further 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from the lowest areas in the mountains to the mountain zone in the tohoku district (northeastern region of japan); from the mountain zone to the alpine region in the chubu district (central region of japan); and in the alpine regions of the kinki, chugoku, and shikoku districts. it is thought that this species is also present in a limited area of the alpine regions in the kyushu district, but it has not yet been discovered around mt. aso, which is consistent with the fact that we could not find any pressed leaf specimen in the universities located in the kyushu distinct. it is noteworthy that this species is not distributed in south korea, china, and neighboring countries; including far eastern russia. however, it has been confirmed that yamabudou grows naturally in the south chishima and sakhalin districts (horikawa, 1972). sankakuzuru (gyojanomizu), v. flexuosa thunb. (fig. 2) this species is distributed in the mid regions of the yamabudou (fig. 1) and ebizuru (fig. 3) ranges, overlapwild grape germplasms in japan h. yamashita*, r. mochioka** * faculty of life and environmental sciences, university of yamanashi, 4-4-37, takeda, kofu, yamanashi, japan. ** faculty of agriculture, kagawa university, 2393 ikenobe, miki-cho, kita, kagawa, japan. key words: anthocyanins, breeding, classification, geographic distribution, growth cycle, vitis. abstract: in japan, seven species and eight varieties of wild grapes were identified, among which the main species are vitis coignetiae pulliat, v. flexuosa thunb., and v. ficifolia bunge var. lobata (regel) nakai (syn. v. thunbergii sieb. et zucc.). this paper summarizes the identification and classification of wild grapes native to japan based on the past reports. their distributions in japan and physiological and ecological traits are also reviewed for effective practical use for grape breeding in the future. adv. hort. sci., 2014 28(4): 214-224 received for publication 17 september 2014 accepted for publication 14 november 2014 215 table 1 systematic and geographical distribution of wild grapes native to japan (nakagawa et al., 1991) species or varieties japanese name locality where grown vitis coignetiae pulliat yamabudou hokkaido, honshu, shikoku vitis coignetiae pulliat var. glabrescens hara takeshimayamabudou hokkaido, honshu vitis flexuosa thunb. sankakuzuru (gyojanomizu) honshu, shikoku, kyusyu vitis flexuosa thunb. var. rufo-tomentosa makino kesankakuzuru southern honshu, shikoku vitis flexuosa thunb. var. tsukubana makino usugesankakuzuru northern honshu vitis flexuosa thunb. var. crassifolia hara atsubasankakuzuru shikoku vitis saccharifera makino amazuru (otokobudou) southern honshu, shikoku vitis yokogurana makino yokogurabudou shikoku (kochi pref.) vitis ficifolia bunge var. lobata (regel) nakai (vitis thunbergii sieb. et zucc.) ebizuru (z) all over japan vitis ficifolia bunge var. izu-insularis hara shititouebizuru izu islands vitis ficifolia bunge var. sinuata hara kikubaebizuru southern honshu, shikoku, kyusyu vitis ficifolia bunge var. ganebu hatusima ryuukyuuganebu amami islands, okinawa islands, yaeyama islands vitis austrokoreana hatusima kenashiebizuru tsushima islands vitis kiusiana momiyama kumagawabudou kyushyu (kumamoto pref., kagoshima pref.) vitis shiragai makino shiragabudou honshu (okayama pref.) vitis sp. shiohitashibudou (tentative) kyusyu (kagoshima pref.) (z) used in some classifications as a species (thunbergii). fig. 1 geographic distribution of vitis coignetiae pulliat (nakagawa et al., 1986). fig. 2 geographic distribution of vitis flexuosa thunb. (nakagawa et al., 1986). 216 ping with the two species, and is found in slightly lower altitude areas than yamabudou (fig. 1). we can usually find this species from the lowlands to the mountainous area of the tohoku district or the chubu district; it does not grow naturally in hokkaido. ebizuru, v. ficifolia bunge var. lobata (regel) nakai (fig. 3) this species is one of the most widespread vitis species in japan. its distribution extends from the southern hokkaido region to the flatlands and mountainous terrain in the okinawa district; it can be found in a wide variety of habitats, including both the seashore and urban districts. this species is considered to be highly adaptable to the environment, thus it has a wide distribution compared with other vitis species. as a result, many variants of morphological and physiological traits are found in this species as a result of adaptation to local climates. ryuukyuuganebu, shichitouebizuru, and kikubaebizuru are varieties belonging to v. ficifolia. kenashiebizuru is also closely related to v. ficifolia, although its scientific name is given as v. auskoreana hatusima. these grapes are generally included in the v. ficifolia group. other species (fig. 4) as shown in table 1 and figure 4, in addition to these, many species and varieties are spread in various districts of japan. takeshimayamabudou, a variety of yamabudou (v. coingnetiae), was discovered in hokkaido (around lake akan) and nagano prefecture. it has no hairiness on the lower leaf surface and a thinner leaf compared to yamabudou. kumagawabudou has prickly shoots and ovoid leaves; one wild grape (v. davidii) with prickly shoots grows naturally in china, however, it differs significantly from kumagawabudou in its morphological characteristics. shichitouebizuru grows in seaside areas of seven islands of izu. moreover, its fruit-set is the highest among all wild grapes native to japan. 3. the classification of japanese wild grapes as genus vitis is mainly classified by morphology, some taxa may be taken as different classifications even if they are the same grape. for example, shiragabudou has two scientific names, vitis shiragai makino (makino, 1918) and vitis amurensis rupr. (ohwi, 1953). in this section, wild grapes native to japan are classified concisely based on chemical, biochemical and genetic knowledge. morphological classifications classification by leaf structure. galet (1979) tried to classify genus vitis through ampelographic measurements fig. 3 geographic distribution of vitis ficifolia bunge var. lobata (regel) nakai (nakagawa et al., 1986). fig. 4 geographic distribution of wild grapes native to japan (nakagawa et al., 1986). 217 of the leaf (fig. 5, table 2). nakagawa et al. (1991) coded the characteristics of grape leaves and the result is presented in table 3. code numbers of vein length rations (abc) for the five basic leaf shapes are as follows (galet, 1979): cordiform: 357 to 468, cuneiform: 135 to 247, truncate: 045 to 247, orbicular: 015 to 136, reniform: 014 to 136. according to this method, code numbers are relatively near for close species (e.g. v. coignetiae and v. amurensis) (table 3). classification by pollen ultrastructure. mochioka et al. (1993) observed ultrastructures of mature pollen grains of wild grapes native to japan, korea and china using a scanning electron microscope and reported that the pollen could be classified as one of three types by the lumina forms in muri (fig. 6). they also reported the table 2 the code number of the galet (1979) ruler for the values of a, b, c, r, s 1 and s 2 code number vales of a, b and c vales of r vales of s 1 values of s 2 0 0.91~1.00 ≤0.80 ≤ 70° ≤ 100° 1 0.81~0.90 0.81~0.90 71°~80° 101°~110° 2 0.71~0.80 0.91~1.00 81°~90° 111°~120° 3 0.61~0.70 1.01~1.10 91°~100° 121°~130° 4 051~0.60 1.11~1.20 101°~110° 131°~140° 5 0.41~0.50 1.21~1.30 111°~120° 141°~150° 6 0.31~0.40 1.31~1.40 121°~130° 151°~160° 7 0.21~0.30 1.41~1.50 131°~140° 161°~170° 8 0.11~0.20 141°~150° 171°~180° 9 0.00~0.10 ≥151° ≥181° a= l 2 length/l 1 length; b= l 3 length/l 1 length; c= l 4 length/l 1 length, where l 1 = midwein; l 2 = superior lateral vein and l 3 = inferior lateral vein; l 4 = petiolar vein. r= leaf length/leaf width. s 1 = α + β; s 2 = α + β + r where α= angles between l 1 and l 2 . β= angles between l 2 and l 3 . and r= angles between l 3 and l 4 . fig. 5 the general morphology of a mature grape leaf. al.= apical lobe; ii= lateral lobe; pl.= proximal lobe; ss.= superior sinus; is.= inferior sinus; ps. petiolar sinus; pe.= petiol; mv.= midvein; sv.= superior lateral vein; iv.= inferior lateral vein; pv.= petiolar vein; vl.= veinlet; se.= serration; lm= leaf margin. (nakagawa et al., 1991). table 3 the code number of various wild grapes obtained by using the galet’s method (nakagawa et al., 1991) species abc-r-s 1 s 2 vitis coignetiae 146-4-24 vitis amurensis 146-3-24 vitis flexuosa 357-7-01 vitis ficifolia var. lobata 246-3-13 vitis shiragai 136-3-13 vitis sp. (daisankakuzuru) 146-3-12 vitis ficifolia bunge var. ganebu 135-3-24 vitis sp. (shiohitashibudo) 257-5-02 vitis ficifolia var. izu-insularis 146-3-01 vitis kiusiana 368-7-01 fig. 6 scanning electron microphotographs of grape exine. scale bars= 1 µm. type-1 a pollen has perforations in the lumina; type-1 b pollen has perforations and corrugation in the lumina, and type-ii pollen has granules in the lumina (mochioka et al., 1993). 218 pollen ultrastructures of related species belonged to the same type (table 4). chemotaxonomic classifications classification by anthocyanins in grape skin. mochioka et al. (1995) analyzed by hplc anthocyanins in the berry skin of 10 wild grapes (four species, five varieties and one unidentified type) native to japan. the dendrogram, showing phylogenetic relationships, was drawn from the pairwise comparison of matching coefficients based on anthocyanin table 4 morphological characteristics of pollen grains of the wild grapes native to japan, korea and china (mochioka et al., 1993) species or cultivars pollen size (µm) l/w ratio no. of colpi pollen exine typelength (l) width (w) japan vitis coignetiae pulliat ♂ 20.0±0.2 (z) 19.4±0.2 1.03±0.01 3 i a ♀ 22.0±0.2 20.7±0.2 1.06±0.01 0 i a vitis flexuosa thunb. ♂ 20.3±0.5 19.6±0.3 1.04±0.03 3 i a ♀ 20.6±0.2 20.1±0.2 1.03±0.01 0 i b, ii vitis ficifolia bunge var. lobata (regel) nakai ♂ 20.0±0.2 19.2±0.2 1.04±0.01 3 ii ♀ 20.9±0.2 20.0±0.2 1.05±0.01 0 ii vitis ficifolia bunge var. izu-insularis hara ♂ 20.9±0.3 20.1±0.4 1.04±0.01 3 ii ♀ 21.4±0.2 20.2±0.2 1.06±0.01 0 ii vitis ficifolia bunge var. ganebu hatusima ♀ 21.4±0.3 20.3±0.2 1.05±0.01 0 i a vitis shiragai makino ♂ 21.1±0.2 20.7±0.2 1.02±0.01 3 i a ♀ 22.6±0.2 21.1±0.3 1.08±0.01 0 i a vitis kiusiana momiyama ♀ 20.4±0.2 19.5±0.2 1.05±0.01 0 i b vitis sp. (provisional name: shiohitashibudo) ♀ 21.3±0.3 20.0±0.3 1.06±0.01 0 ii korea vitis amurensis rupr. ♂ 20.6±0.3 20.1±0.2 1.03±0.01 3 i a ♀ 23.4±0.2 22.1±0.2 1.06±0.01 0 i b vitis sp. (provisional name: daisankakuzuru) ♀ 21.4±0.2 20.3±0.2 1.06±0.01 0 i b china vitis amurensis rupr. ♂♀ 21.5±0.2 20.7±0.2 1.04±0.01 3 i b vitis flexuosa thunb. ♂ 19.8±0.3 19.0±0.3 1.04±0.01 3 i a vitis ficifolia bunge ♂ 20.6±0.3 19.0±0.3 1.04±0.01 3 ii vitis adstricta hance ♂ 21.5±0.2 21.2±0.2 1.02±0.01 3 ii vitis adstricta hance var. ternata w.t. wang ♂ 22.3±0.3 21.0±0.4 1.06±0.01 3 ii vitis bellula (rehd.) w.t. wang ♂ 19.3±0.3 18.8±0.2 1.03±0.01 3 ii vitis davidii (roman.) foëx ♂ 23.1±0.2 22.4±0.2 1.03±0.01 3 i b ♂♀ 20.4±0.2 19.9±0.1 1.03±0.01 3 i b vitis pseudoreticulata w.t. wang ♂ 21.3±0.2 20.8±0.2 1.03±0.01 3 i a vitis hancokii hance ♂ 20.1±0.2 19.6±0.1 1.03±0.01 3 i b vitis chugii metcalf. ♂ 19.9±0.2 19.3±0.2 1.03±0.01 3 i b vitis chunganensis hu ♂ 19.9±0.2 19.7±0.2 1.01±0.01 3 i a cultivar vitis vinifera l. ‘muscat of alexandria’ ♂♀ 23.2±0.4 22.4±0.4 1.03±0.01 3 i b vitis lambrusca l. ‘concord’ ♂♀ 23.3±0.3 22.9±0.3 1.02±0.01 3 i b vitis lambruscana bailey ‘delaware’ ♂♀ 21.3±0.3 20.1±0.3 1.06±0.01 3 i b ‘campbell early’ ♂♀ 24.6±0.4 23.4±0.4 1.05±0.01 3 ii ‘kyoho’ (tetraploid) ♂♀ 28.2±0.3 26.9±0.3 1.05±0.01 3.4 i a (z) each valure represents the mean of 20 individual measurements ±se. 219 components, and agrees well with the morphological taxonomy (fig. 7, table 5) (mochioka et al., 1995). in this study 19 anthocyanins were identified, and there were more kinds of anthocyanins in the berry skins of wild grapes distributed in southern regions than those of wild grapes distributed in northern regions (table 5). classification by isozyme and dna analysis. speciesspecificity was observed in the alleles dominated by gpi2 and pgm-2 gene loci (fig. 8, table 6) (ohmi et al., 1991). the f band of gpi-2 and the a and the c bands of pgm-2 existed only in wild grapes native to east asia (table 6). while restriction fragment length polymorphism (rflp) and random amplified polymorphic dna (rapd) analyses were used to analyze the relationships among wild and cultivated grapes, a phenogram of rapd data obtained showed a clear separation between wild and cultivated grapes (goto-yamamoto et al., 1998). fig. 7 dendrogram of berry skin anthocyanin phenotypes of wild and cultivated varieties (mochioka et al., 1995). table 5 percentage of anthocyanin composition in grape berry skins analyzed by hplc (z) (mochioka et al., 1995) species or cultivars peak no (y) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 wild grape vitis coignetiae 1 4 66 1 4 2 1 18 2 v. amurensis 2 2 81 v. flexuosa 5 4 4 57 7 4 1 12 2 v. flexuosa var. rufo-tomentosa 2 1 28 3 3 2 1 4 1 38 2 8 v. ficifolia var. lobata 6 2 10 6 44 2 2 1 7 5 10 12 1 v. ficifolia var. izu-insularis 15 3 17 5 27 3 1 3 12 5 2 2 4 v. ficifolia var. ganebu 8 4 9 8 10 28 2 4 3 4 1 3 2 9 1 v. ficifolia var. sinuata 10 5 39 13 8 9 4 3 6 1 2 1 v. shiragai 11 7 5 13 15 26 2 1 3 4 2 2 2 4 v. kiusiana 13 10 28 19 11 1 1 6 3 3 2 1 v. sp. (tentative name: shiohitashibudou) 6 3 2 10 15 37 2 6 1 1 4 1 1 2 2 v. sp. (tentative name: daisankakuzuru) 5 3 28 17 12 7 7 4 3 2 3 2 2 3 v. sp. (tentative name: taiwan kesankakuzuru) 5 2 24 6 36 1 8 3 3 2 2 2 3 cultivars ‘delaware’ 44 26 4 3 9 4 ‘muscat bailey a’ 3 21 4 32 23 2 2 1 2 2 1 2 ‘campbell early’ 6 6 12 46 6 2 7 6 (z) absorbance at 520 nm. (y) peak no. 3= delphinidia 3-monoglucoside, no. 5= cyaniding 3-monoglucoside; no. 9= petunidin 3-monoglucoside; no. 16= malvidin 3-monoglucoside. fig. 8 gpi and pgm isozymes bands coded by each alleles at gp1-2 (left) and pgm-2 (right) loci (ohmi et al., 1991). 220 classification by general judgments since the past horticultural plant classification was qualitatively performed considering a small number of characteristics, a different result for some researchers might be found (e.g. shiragabudou). therefore, using plural classification methods is desirable because just one method might induce the wrong result. what follows is a brief discussion of judgments about some japanese wild grapes with questionable taxonomic points. shiragabudou. shiragabudou was discovered in okayama prefecture, honshu and was first named by makino (1918). as the leaf shape of this wild grape resembles that of v. amurensis rupr. ohwi (1953) changed its scientific name to vitis amurensis rupr. the leaf morphology and pollen ultrastructures of these two wild grapes are in the same group (table 3, 4), but anthocyanin composition in berry skins (table 5) and species-specific alleles at 2 loci (table 6) are apparently different. furthermore, ecological differences exist between shiragabudou and v. amurensis. shiragabudou is distributed over the warm lowland from 20 to 240 m above sea level in okayama while v. amurensis has a growth area in the cold districts at 40 to 50° n latitude. the cross section morphology of shiragabudou shoots is hexagonal, while that of v. amurensis is circular. these differences show that shiragabudou and v. amurensis are not the same species, thus vitis shiragai makino should be used as the scientific name for shiragabudou. ebizuru and its varieties. ebizuru is distributed widely in japan. there are a number of varieties and ecotypes in the ebizuru group, and morphological differences are various. even if they are the same species, there are several synonyms for this group. even now, vitis ficilolia, v. ficifolia var. lobata, and v. thunbergii are used as scientific names for ebizuru. an isotype of v. ficifolia is the wild grape native to china. there are definitely differences in leaf morphology and bearing habit between chinese ficifolia and japanese ebizuru. natural hybrids. different vitis species can be hybridized easily with each other, so there are many natural hybrids. v. yokogurana is supposed to be a hybrid of v. flexuosa and v. saccharifera (makino, 1918); v. flexuosa var. tukubana is supposed to be a hybrid of v. flexuosa and v. ficifolia var. lobata (murata, 1971). yamabudou. yamabudou (v. coignetiae) must be a species related to v. amurensis, but there is no report that both these two species are simultaneously distributed over the same regions in japan or other countries. shiohitashibudou. (tentative name): shiohitashibudou is an unidentified species, and it was discovered in kagoshima prefecture, kyushu (nakagawa et al., 1991). its leaf morphology is definitely different from that of other japanese wild grapes. shiohitashibudou might be a related species or natural hybrid of ebizuru because its flowering habit is ever-bearing and the pollen ultrastructure is type ii. its bud endodormancy is deeper, and the soluble solid content of its juice is higher than other japanese wild grapes (mochioka, 1996). ryuukyuuganebu. ryuukyuuganebu is distributed over amami, ryukyu and yaeyama islands, and is supposed to be a variety of v. ficifolia. however its leaf shape is different from v. ficifolia var. lobata (fig. 9, table 3), and its pollen ultrastructure is also different (table 4). ryuukyuuganebu is ever-green in its habitat. 4. physiological and ecological traits of wild grapes native to japan four species [v. coignetiae, v. flexuosa, v. shiragai, and shiohitashibudou (tentative name. vitis sp.)] and two varieties (v. ficifolia var. lobata and v. ficifolia var ganebu ) of wild grapes native to japan, and two species [chosen yamabudou (v. amurensis) and daisankakuzuru (tentative name. v. sp.)] grown in korea, and ‘delaware’ (v. labrustable 6 species-specific alleles at 2 loci in grape (ohmi et al., 1991) locus allele species (z) gpi-2 a vin. b vin., amur. (?) c lab., rip. (?) d vin. e lab., shir. f thun., shir. g vin., amur. h lab., aest. (?), vulp (?) i lab., rup., champ., linc. (?), coig., shir. j bourq. and/or lab. k champ. l vulp. and/or lab. pgm-2 a shir. b vin. c amur., coig., thun., shir. d vin. e vin. f lab. and/or linc. g lab., aest. (?) h vin., rup., champ., lab., coig., aest. (?), bourq. (?), rip. (?), vulp. (?) i vin. j rup. k champ. (z) abbreviations: amur.= vitis amurensis; aest.= v. aestivalis; bourq.= v. aestivalis var. bourquiana; champ.= v. champini; lab.= v. labrusca; linc.= v. lincecumii; rip.= v. riparia; rup.= v. rupestris; shir.= v. shiragai; tun.= v. thunbergii (=v. ficifolia var. lobata); vin.= v. vinifera; vulp.= v. vulpina. 221 cana bailey) (table 7) were cultivated in the horticultural experiment field at osaka prefecture university (sakai, osaka) and their physiological and ecological traits were compared (nakagawa et al., 1986). here, chosen yamabudou is a wild grape grown naturally in the northern and northeastern regions of china, northern region of korea and southeastern region of the former soviet union. the results are illustrated below. growth cycle bud burst. bud burst of yamabudou and chosen yamabudou occurred earlier among the wild grapes, followed by sankakuzuru, ebizuru, and daisankakuzuru. shiohitashibudou showed the latest bud burst in this study (table 7). ‘delaware’ broke bud later than all wild grapes except for shiohitashibudou. full bloom (50% cap off). in grape cultivation, early bud burst does not always mean early bloom. indeed, the orders of bloom date differed from those of bud burst (from early to late bloom): yamabudou, sankakuzuru ebizuru/shiragabudou, daisankakuzuru, shiohitashibudou, and ryuukyuuganebu. here, ebizuru blooms in the same period as shiragabudou, and the bloom date of sankakuzuru was comparable to that of ‘delaware’ (table 7). veraison (berry coloring begins). the skin color of yamabudou changed earliest among the wild grapes. changes in skin color in chosen yamabudou and ‘delaware’ occurred on the same date. skin color change occurred the latest in daisankakuzuru, shiohitashibudou, and ryuukyuuganebu; and sankakuzuru, shiragabudou and ebizuru were the next to latest (table 7). maturity. maturity is denoted when berry weights and soluble solids attain maximum maturation. ‘delaware’ matured in mid-august, which was earlier than the studied wild grapes. sankakuzuru and ryuukyuuganebu matured in mid-september and daisankakuzuru in early-october (table 7). thus, wild grapes tend to have medium to late maturation in osaka. defoliation. defoliation indicates the date when all leaves (from basal to tenth leaf) fall completely. defoliation in sankakuzuru and daisankakuzuru took place in early-november, yamabudou and chosen yamabudou shed their leaves in mid-november, and shiohitashibudou, ebizuru, and shiragabudou in late-november. interestingly, ryuukyuuganebu, a subtropical grape, showed extremely late defoliation; in some cases, leaves did not fall until january. characteristics of organ shoot. observations of shoot growth in summer enabled us to classify the wild grapes into three types: continuous, subcontinuous, and discontinuous. the continutable 7 growth cycle of wild grapes native to japan at sakai osaka (nakagawa et al., 1986) species and varieties bud burst full bloom veraison harvest leaf fall chosen yamabudou (v. amurensis rupr.) 3/27 5/14 8/1 9/29 11/14 yamabudou (v. coignetiae pulliat) 3/27 5/12 7/26 9/29 11/13 sankakuzuru (v. flexuosa thunb.) 3/29 5/26 8/6 9/14 11/7 ebizuru [v. ficifolia bunge var. lobata (regel) nakai] 3/29 6/4 8/11 9/11 11/27 shiragabudou (v. shiragai makino) 3/29 6/4 8/6 9/15 11/23 daisankakuzuru (tentative vitis sp.) 3/29 6/10 9/4 10/6 11/4 shiohitashibudou (tentative vitis sp.) 4/10 6/13 9/4 9/22 11/25 ryuukyuuganebu (v. ficifolia bunge var. ganebu hatusima 4/3 6/19 8/31 9/20 after end of dec. delaware (v. labruscana bailey) 4/6 5/27 7/30 8/20 11/8 fig. 9 standard design of mature leaf of various wild grapes (├─┤ 1 cm) (nakagawa et al., 1991). 222 ous group had constant growth of some shoots in summer; shiohitashibudou and daisankakuzuru were included in this type. the subcontinuous type exhibited slight growth of some shoots in summer; ryuukyuuganebu, sankakuzuru, ebizuru, and shiragabudou corresponded to this type. finally, the discontinuous group stopped shoot growth in summer, for example yamabudou and chosen yamabudou in this study. tendril placement of all vitis species and varieties native to japan is intermittent. inflorescence. wild grapes native to japan (seven species and eight varieties) are dioecious, meaning that they contain imperfect individual male and female plants. three types of fruiting habits were found (fig. 10). “a” type: as is the case of yamabudou, chosen yamabudou, sankakuzuru, daisankakuzuru, shiragabudou, and kumagawabudou, with two to four inflorescences at the basal part on each shoot without any inflorescences on lateral shoots. “b” type: as seen in shiohitashibudou, shichitouebizuru, and some shiragabudou, there are two to four inflorescences at the basal part on each shoot with some inflorescences on lateral shoots. “c” type: ebizuru and ryuukyuuganebu belong to this type, with two to six inflorescences at the basal part and the upper part of each shoot, having contiguous inflorescences from the base to the top of lateral shoots. grape comparison and fruit quality (table 8) sugars. shiohitashibudou attained a sugar concentration of 17.7%, which is the highest among the other species (measured as 12-14%) except ebizuru and ryuukyuuganebu, which had sugar concentrations of around 8%. almost all studied species have high glucose content, especially kumagawabudou, shiohitashibudou, and daisankakuzuru, which contained two to three fold more glucose than fructose. acids. kumagawabudou and daisankakuzuru contained about 0.7 and 0.8% of organic acids, while almost all other species contained about 0.5%; shiragabudou showed the lowest level of organic acids (0.36%) (table 8). amino acids. the concentration of amino acids varied widely from 50 mg% (ebizuru) to 294 g% (shiohitashibudou). anthocyanins. northern species such as yamabudou and chosen yamabudou showed small amounts of anthocyanin, while the southern species, ryuukyuuganebu, contained a larger amount of this component (table 8). dormancy and cold hardiness dormancy. although all species have a dormancy trait, its intensity depends on the species. ryuukyuuganebu showed a short dormancy period, while it was generally longer in kumagawabudou and shiohitashibudou (nakagawa et al., 1986; nakagawa, 1989). cold hardiness. cold hardiness varied markedly among the wild grapes. for example, ryuukyuuganebu and kumagawabudou were very susceptible to severe damage at -3°c. on the contrary, yamabudou, ebizuru, shiragabufig. 10 fruiting habit of wild grapes native to japan. table 8 berry composition of wild grapes native to japan (nakagawa et al., 1986) species and varieties reducing sugars (%) glucose (%) fructose (%) glucose/ fructose organic acid (%) amino acid (mg %) anthocuanin (od 537 nm) chosen yamabudou (v. amurensis rupr.) 12.7 9.2 3.5 2.6 0.50 191.2 0.12 yamabudou (v. coignetiae pulliat) 12.3 5.2 7.1 0.7 0.50 155.3 0.21 sankakuzuru (v. flexuosa thunb.) 14.2 8.0 6.2 1.3 0.52 214.0 0.30 ebizuru [v. ficifolia bunge var. lobata (regel) nakai] 8.1 4.1 4.0 1.0 0.51 50.7 0.30 shiragabudou (v. shiragai makino) 12.0 6.7 5.3 1.3 0.36 222.6 0.45 daisankakuzuru (tentative vitis sp.) 12.3 8.1 3.9 2.2 0.81 250.8 0.42 kumagawabudou (v. kiusiana momiyama) 12.0 9.2 2.8 3.3 0.72 180.5 0.49 shiohitashibudou (tentative vitis sp.) 17.7 12.7 5.0 2.5 0.48 294.5 0.41 ryuukyuuganebu (v. ficifolia bunge var. ganebu hatusima 7.8 3.7 4.1 0.8 0.51 138.0 0.70 delaware (v. labruscana bailey) 16.8 7.5 9.3 0.8 0.77 220.8 0.05 223 dou, shichitouebizuru, and chosen yamabudou showed moderate cold hardiness; their survival has even been reported at -10°c (nakagawa, 1989). 5. value and use of wild grape germplasms in japan wine between the 1960s and 1980s, yamabudou was successfully cultivated in commercial vineyards for winemaking in the town of ikeda (hokkaido); its cultivation has attracted attention as a means to revitalize towns in japan. in china, v. quinquangularis is processed into an excellent wine (li et al., 1992). kumagawabudou, which is thought to be the same species as v. quinquangularis (li et al., 1991), therefore, may be an important resource for wine making. breeding the major cultivars, bred using wild grapes, are ‘sawanobori waingurando’ and ‘yama sauvignon’ in japan, both of which are used for wine production. ‘sawanobori waingurando’ is a cross seedling of v. amurensis × (seibel 13053 × nakajima no.1, a strain of yamabudou) and it was released in 1998. by contrast, ‘yama sauvignon’ is a progeny of yamabudou × ‘cabernet sauvignon’, which was released in 1990 by dr. yoshihide yamakawa at the university of yamanashi. ‘yama sauvignon’ has the following superior characteristics: 1) no cracking of berry; 2) resistance to ripe rot, downy mildew, and gray mold; 3) adaptability to the prevailing weather conditions in japan; 4) high productivity; and 5) high quality wine with typical aroma and taste (yamakawa et al., 1989). considering the potential use of wild grapes as breeding material, these grapes have the following notable characteristics: drought resistance, cold hardiness, salt tolerance, water logging tolerance, heat tolerance, disease resistance, high concentration of important substances, ever bearing, and short dormancy. four characteristics are especially promising: i) ever bearing, ii) short dormancy, iii) salt tolerance, and iv) heat tolerance. therefore, we explain the usefulness of these characteristics for grape breeding programs in more detail. i) ever bearing strains of ryuukyuuganebu and ebizuru bloom and fruit as long as the growth of axillary buds continues. this trait makes it possible to carry out year-round culture and/ or culture using a factory system, like some vegetables, using artificial light and controlled irrigation. however, the major gene related to this trait has not yet been identified. ii) short dormancy ryuukyuuganebu can be released from dormancy after being subjected to low temperature for extremely short periods. this trait may be profoundly related to ever bearing. thus, it may be possible to force culture inside greenhouses due to the low cost, for year-round culture and/or culture using a factory system. iii) salt tolerance salt accumulation through the use of chemical fertilizers causes serious problems which sometimes result in the loss of plants. shiohitashibudou is excellent in its resistance to salts and ryuukyuuganebu grows naturally along the seashore. although the mechanism underlying salt tolerance of this grape has not yet been fully elucidated, this trait could be useful not only for scion but also for root stock. iv) heat tolerance as global warming progresses, fruit skin, including grape, shows poor coloration, which leads to a defective appearance and reduced commercial value. poor coloration also affects wine production. interestingly, the coloration of wild grapes native to japan is very high, even under high temperature conditions during their ripening season. in grape, since myb is involved in red skin coloration, it is valuable to compare myb genes between wild grapes and primary japanese cultivars such as ‘kyoho’ and ‘aki queen’. thus, this trait could be useful for sustainable grape production with high quality and high adaptability in the production area. 6. conclusions wild grapes native to japan have been actively studied over a long period, but in recent years attention has declined. however, wild grapes native to japan can offer many useful characteristics, such as short dormancy, ever bearing, heat and salt tolerance. these traits are very attractive, not only for their use as rootstock, but also in terms of breeding material or for genetic studies. we have recently begun a breeding study using ryuukyuuganebu. it is expected that some novel grapes will be bred to withstand increasing global temperatures or for use in grape cultivation factories. references galet p., 1979 a practical ampelography: grapevine identification. cornell university press, new york, usa, pp. 24-47. goto-yamamoto n., mochioka r., lin b., hashizume k., umeda n., horiuchi s., 1998 rflp and rapd analysis of wild and cultivated grapes (vitis spp.). j. japan. soc. hort. sci., 67: 483-490. horikawa y., 1972 yamabudo. atlas of the japanese flora: an introduction to plant sociology of east asia. gakken co., ltd., tokyo, japan, pp. 208. horiuchi s., matsui h., 1996 nihon budougaku. yokendo, tokyo, japan, pp. 58-102. li s.c., horiuchi s., mochioka r., matsui h., 1992 some issues on wild grapes native to east asia (2). agric. hortic. (nogyo-oyobi-engei), 67: 1277-1280. li s.c., horiuchi s., mochioka r., yamazawa m., matsui h., 1991 comparison between wild grapes native to japan and china. 2. study on morphological character224 istics of v. kiusiana and v. quinquanglaris. j. japan. soc. hort. sci., 60(suppl. 1): 132-133. makino t., 1918 a contribution of the knowledge of the flora of japan. j. jpn. bot., 1: 31-33. mochioka r., 1996 taxonomic classification and utilization on wild grapes native to japan based on their various characteristics. ph. d. thesis, osaka prefec. university, sakai, japan. mochioka r., horiuchi s., li s.c., matsui h., murai y., 1993 pollen morphology of wild grapes native to eastern asia. jpn. j. palynol., 39: 11-20. mochioka r., yamaguchi m., horiuchil s., matsui h., kurooka h., 1995 chemotaxonomic classification of native wild japanese grapes by anthocyanins in berry skin. j. japan. soc. hort. sci., 64: 463-470. murata g., 1971 vitaceae, pp. 237-241. in: kitamura s., and g. murata (eds.) genshoku-nippon-no-shokubutsuzukan (mokuhonhen i). hoikusha, osaka, japan. nakagawa s., 1989 studies on collection, preservation and usage of gene resorces in fruit trees. report for grants in aid for scientific research, pp. 10-20. nakagawa s., horiuchi s., matsui h., yuda e., 1986 studies on the cultivation and use of the wild grapes native to japan. report for grants in aid for scientific research, pp. 1-49. nakagawa s., horiuchi s., matsui h., yuda e., yamada s., murai y., komatsu h., 1991 distribution and leaf morphology of wild grapes native to japan. j. japan. soc. hort. sci., 60: 31-39. ohmi c., wakana a., shiraishi s., 1991 phylogenic implication of genus vitis on the basis of isozyme analysis. j. japan. soc. hort. sci., 60(suppl. 2): 62-63. ohwi j., 1953 vitis linn. flora of japan. shibundo, tokyo, japan, pp. 760-762. yamakawa y., moriya m., anamizu h., 1989 characterization of hybrid new red-wine grape cultivar ‘yama sauvignon’. j. inst. enol. vitic. yamanashi univ., 24: 15-24. impaginato 101 adv. hort. sci., 2025 39(2): 101­112 doi: 10.36253/ahsc­17292 https://oaj.fupress.net/index.php/ahs impact of chitosan­aloe vera gel with coconut oil coating on postharvest quality and antioxidant of ‘gopalbhog’ mango at ambient storage r.s. amin 1, m. rukunuzzaman 1, m. ahmed 2, m.a. khatun 1, m.a. rahman 1, m.g. saroare 1, m.t. islam 1 (*) 1 department of horticulture, faculty of agriculture, hajee mohammad danesh science and technology university (hstu), dinajpur‐5200, bangladesh. 2 department of food processing and preservation, hajee mohammad danesh science and technology university, dinajpur‐5200, bangladesh. key words: antioxidant enzymes, coatings, mango, quality, storage. abstract: mangoes are valuable fruits because of their nutritional value and commercial significance. it ripens rapidly but deteriorates in quality while stored. therefore, natural and biodegradable materials must be used in post­ harvest management to reduce crop losses. aloe vera (avg), chitosan (cts), and coconut oil (co) either alone or in combinations were tested on mango postharvest features during 12 days’ storage at ambient conditions (27±2°c and 80­85% rh). at the end of storage, coatings (avg+cts) reduced weight loss (20.02, 37.88%) and decay (9.52, 18.46%) compared to controls while enhancing fruit quality, especially firmness (3.21, 1.44 kg cm­2), ascorbic acid (9.25, 5.89 mg 100 g­1), tss (10.77, 19.5°brix), acidity (0.46, 0.41%) and ph. furthermore, coated fruits’ total phenol content and antioxidant activity were substantially higher than those of uncoated fruits. control fruits exhibited the least activity of cat and pod enzymes during storage, while coated fruits had the lowest ppo activity. the coated fruit peels discoloured less after storage than the control fruits. co treatment had a deleterious effect on various measures, possibly due to its high concentration. these findings suggested that the cts+avg coating may be efficient at the right dose to retain bioactive components and mango (cv. gopalbhog) fruit quality after harvest. 1. introduction mango (mangifera indica l.) is widely traded due to its versatility, taste, smell, and dietary content (hossain, 2016). its excellent nutritional value and abundance of vitamins and minerals make it extremely valuable (athoo et al., 2024). bangladesh is the seventh­largest mango producer; (*) corresponding author: tariqul.hrt@tch.hstu.ac.bd tariqulhstu@gmail.com citation: amin r.s., rukunuzzaman m., ahmed m., khatun m.a., rahman m.a., saroare m.g., islam m.t., 2025 ­ impact of chitosan‐aloe vera gel with coconut oil coating on postharvest quality and antioxidant of ‘gopalbhog’ mango at ambient storage. ­ adv. hort. sci., 39(2): 101­112. orcid: ars: 0009­0007­4262­8399 rm : 0009­0007­5478­3134 am: 0009­0009­5229­2942 kma: 0000­0002­6175­8531 rma: 0009­0008­1154­0471 smg: 0009­0005­2177­769x imt: 0009­0005­1543­3786 copyright: © 2025 amin r.s., rukunuzzaman m., ahmed m., khatun m.a., rahman m.a., saroare m.g., islam m.t. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 9 february 2025 accepted for publication 19 june 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17292 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0007-4262-8399 http://orcid.org/0009-0007-5478-3134 http://orcid.org/0009-0009-5229-2942 http://orcid.org/0000-0002-6175-8531 http://orcid.org/0009-0008-1154-0471 http://orcid.org/0009-0005-2177-769x http://orcid.org/0009-0005-1543-3786 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 101­112 102 production currently occupies around 123,997.70 hectares, yielding over 1,482,937.04 mt which contributes 34.33% of the area and 26.21% of the production of total fruit crops in bangladesh (bangladesh bureau of statistics, 2024). in bangladesh, there is a huge number of mango varieties; among these is gopalbhog. this variety is an early well­liked type grown primarily in the north. the fruits produced by this variety are attractive, clean, juicy, fibreless, sweet and weigh 200­300g and pest­free thus, they demand a premium price in the market. mangoes are climacteric fruits that mature, soften, and ripen quickly, and are prone to mechanical damage that causes significant postharvest losses (lawson et al., 2019). due to their climacteric nature, mangoes cannot be stored at room temperature for extended periods, as they mature between 2 and 10 days after harvesting (kumar et al., 2023), with shelf life varying from a few days at ambient temperature to up to three weeks in cold storage at 13°c. fruit maturation involves a series of metabolic activities, including loss of weight, increased respiration, structural polysaccharide changes, chlorophyll degradation, carotenoids biosynthesis, starch hydrolysis to sugars, and fruit ripening to acceptable quality (khanum et al., 2020). a number of factors contribute to post­ harvest losses, including the use of improper harvesting, handling and transportation equipment, unsuitable packaging materials, temperature control, rough handling of fresh fruits, and inadequate road infrastructure (kefas et al., 2024). a variety of fungi attacks mango fruits once they reach maturity and are harvested from the tree. postharvest losses of mangoes in bangladesh are estimated to be around 35% (alom et al., 2019). many harvested mangoes never reach consumers due to these losses (giovannoni et al., 2017). less shelf life and postharvest monitoring have hampered mango deliveries to distant markets. therefore, maintaining mango fruit quality and extending post­ harvest life requires effective strategies. mangoes’ postharvest life is artificially prolonged by chemicals that harm the environment and human health. recently, several treatments that are nontoxic and non­harmful have been employed to enhance the postharvest quality of mangoes, including edible coatings (liu et al., 2020; perez­vazquez et al., 2023; aaqil et al., 2024), essential oils, or nanoparticles (kanwar et al., 2024). applying chitosan exogenously improves antioxidant activity, maintains firmness, reduces transpiration rate, and enhances fruit quality overall (wang et al., 2021). being a naturally occurring compound with antibacterial capabilities, it delays fruit deterioration by making mangos more durable and preventing microbial attacks (parvin et al., 2023). chitosan can form layers and is safe and recyclable (kumar et al., 2021). it could treat mango fruit postharvest diseases as an antibacterial agent (shah et al., 2020). as stated by silva et al. (2017), chitosan has already extended the shelf life of lemon, papaya, and mango following harvest. according to eshetu et al. (2019), chitosan, either by itself or in combination, lowers respiration, softening of tissue, loss of weight, disease, and more. aloe vera is an environmentally safe postharvest treatment that researchers are interested in. it is frequently added to edible coatings to enhance their antibacterial and moisturizing qualities. aloe vera gel improves fruit preservation by increasing coating flexibility and barrier properties (ahmed, 2024). researchers have extensively studied it as an edible covering material to enhance food quality and safety. however, studies indicate that aloe vera gel, either by itself or mixed with other ingredients in edible coatings, can minimise lipid oxidation, slow respiration, soften cell walls, promote weight loss, and prevent fruit decay. this effect prolongs the shelf life of mangoes (amin et al., 2021), table grapes (ayyub et al., 2024), apples (kaur et al., 2024), tomatoes (tobing et al., 2023), and apricots (farooq et al . , 2023), while maintaining other quality characteristics. coconut oil, a tasty fruit coating, reduces respiration, transpiration, and ethylene production. it’s rich in lauric acid. this acid may be endogenously converted to monolaurin, which has antimicrobial properties (liberman et al., 2006). in order to restrict respiration, transpiration, and microbial activity, coconut oil encircles stomata and lenticels (bisen and patel, 2012). hence, this study set out to analyze the impact of varying chitosan­aloe vera gel with coconut oil coating concentration on ‘gopalbhog’ mango quality and shelf life at ambient storage. the aim of this study was to apply chitosan and aloe vera gel as environmentally friendly preservation coatings for decreasing fruit softening, maintaining postharvest mango quality, and prolonging the commercial storage period. amin et al. ‐ mango postharvest quality improved by edible coating 103 preparation of coconut oil‐aloe vera gel coating (co+avg) in order to create a translucent liquid, co and avg (1:1 v/v) were pooled in a beaker and heated in a hot water bath. weight loss following the usual technique, the weight loss % was calculated: loss of weight (%)= [initial fruit weight (g)­ fruit’s weight on the observation day(g)]/[initial fruit weight(g)] ×100 fruit firmness, tss and ph pressure testers measured fruit firmness in kg cm­2. a 2 mm stainless­steel spherical probe entered the fruit sample. three measures calculated the average stiffness. a digital refractometer calculated the tss (obrix). ph was measured with a chinese digital ph meter (hi 2211 ph/orp). vitamin c vitamin c was assessed as described by mchenry and graham (1935) with slight modification. briefly, whatman no.1 filtered 5g of mango pulp with 5 ml of a 20% metaphosphoric acid solution. five ml of fi ltrate was agitated with two drops of phenolphthalein solution in a small beaker and titrated with touching 2, 6­indophenol till pink. vitamin c (mg/100 g) = (titrate × factor of dye (0.5) × prepared volume)/(filtrate volume taken × weight of sample) titratable acidity (ta) to test titratable acidity (citric acid %), 5 ml of the juice was titrated with naoh (0.1 n) and phenolphthalein until it turned bright pink (ph = 8.0). color we used a cr­2000 japan chroma meter to quantify the color of the mango skin on two different sides of the fruit. the results were shown as l* values (positive means brightness, negative means darkness), a* values (negative means green, positive means red), and b* values (negative means blue, positive means yellow). decay incidence the following equation is used to identify rotten fruits: decay (%) = (number of decayed fruits )/(number initial fruits ) ×100 2. materials and methods fruit material mature ‘gopalbhog’ mangoes (peel turned yellow at the bottom and green at the top) from the center of the plant canopy were collected from an orchard near the hstu (lat. 25°38’11.6664’’ n and long. 88°38’10.9592’’ e) in bangladesh. we used local producers’ harvest stages fruits, which were uniform in size and shape, had no damage and/or microbial infection, and were attractive in colour (green peel and yellow from bottom to top when mature). fruits were transferred to the laboratory within 2 hours. 72 physiologically mature mangoes (4 fruits per replication) were cleaned with a sodium hypochlorite solution (1% v/v) and dried at room temperature before use. when mango peels turned yellow at the bottom and green at the top, they were harvested. treatments and storage the following six treatments were randomly assigned to six lots of fruits (12 fruits each lot): control (distilled water), aloe vera gel (avg 1:1 v/v), chitosan solution (cts, 1.5% w/v), coconut oil (co 1:1 v/v), avg+cts, and avg+co. fruits were coated by brushing for five minutes, then air­dried for 2 hours at room temperature. finally, treated fruits were stored at ambient conditions (27±2°c and 80­ 85% rh). quality assessments were done every four days, from day 0 to day 12 of storage. aloe vera gel extraction the picked aloe vera leaves were peeled. the parenchyma, after being homogenised into a mucilaginous jelly, had to be filtered to get rid of fibrous materials (song et al., 2013). chitosan solution preparation (cts) in 100 ml of 1% aqueous lactic acid (v/v), 1.5 grams of chitosan powder were dissolved in 1 ml of glycerin. to homogenize the solution, a magnetic stirrer was used for four hours at 25°c. the solution was filtered through three layers of muslin cloth. preparation of chitosan‐aloe vera gel coating (cts+avg) a magnetic stirrer was used to mix cts and an avg (1:1 v/v) at the ambient temperature for four hours. adv. hort. sci., 2025 39(2): 101­112 104 total phenolic content (tpc) total phenolic compounds (tpc) were determined using the method described by singleton and rossi (1965) with a few adjustments. methanol (10 ml) was used to extract and strain 1 g of fruit pulp. an aliquot (1 ml) was mixed with 0.5 ml of folin­ciocalteu reagents (sigma aldrich) and 7.5% (w/v) aqueous na2co3. distilled water was added to bring the volume up to 10 ml. the samples were vortexed for 35 minutes at ambient temperature, then centrifuged for 10 minutes at 4000 rpm. absorbance at 765 nm was checked using a uv 1800 shaanxi, china spectrophotometer. the tpc was measured as mg of gae (gallic acid equivalent) per 100­gram fruit pulp. antioxidant activity by dpph scavenging ten milliliters of methanol and a gram of fruit pulp were mixed to determine the antioxidant activity of the treatment. whatman no. 1 filter paper was used to filter the mixture. after that, 0.1 ml of extract and 1.9 ml of dpph solution (0.3 mm) were added to a falcon tube, which was vortexed for half an hour. trolox solutions (te) were used to create the standard curve. a spectrophotometer (uv 1800 shaanxi, china) was used to detect absorbance at 517 nm compared to a blank. assaying enzymes the fruit pulp (0.2 g) was mashed in 3 ml phosphate buffer (100 mm, ph 7, and 4% polyvinylpolypropylene) and centrifuged at 12000 rpm for 15 minutes, aliquoted, and stored at 4°c for future use. polyphenol oxidase (ppo) enzyme extract (600 μl), 1200 μl phosphate buffer solution (100 mm, ph 7), and 600 μl catechol (100 mm,) were mixed together. activity was validated as u g­1 fw after 2 minutes of 410 nm absorbance. catalase (cat) the mixture included k2so4 buffers (700 μl, 50 mm, ph 7), h2o2 (100 μl), and 100 μl of edta (100 μl, 2.5 mm). a spectrophotometer recorded absorbance at 240 nm for two minutes. u g­1 fw referred to catalase activity. peroxidase (pod) to observe the peroxidase activities, we used enzyme extract (100 μl), h2o2 (100 mm), guaiacol (20 mm), edta (2.5 mm), and phosphate buffer (600 μl, 100 mm, ph 7. color development was measured at 470 nm absorbance for 2 minutes, and the results were shown as u g­1 fw. sensory evaluation the coated fruits and control were evaluated for sensory attributes, including color, flavor, texture, sweetness, appearance, and overall expression, using a nine­point hedonic scale. 1 ­ extreme dislike, 2 ­ dislike very much, 3 ­ moderate dislike, 4 ­ slight dislike, 6 ­ like slightly, 7 ­ like moderate, 8 ­ like very much, 9 ­ like extremely (salehin et al., 2025). statistical analysis data was evaluated using a completely randomized design with three replications and two factorial designs. statistical tool for agricultural research (star, version 2.0.1; irri, laguna, philippines) and r (version 3.4.2; r core team, 2017) with one­way anova were used to analyse the data. lsd estimated mean value differences (p<0.05). pca found likely associations between variables. 3. results water loss influences fruit postharvest quality. storage times and treatments significantly reduced fruit weight. figure 1a shows that fruits lost weight as storage time increased. control fruits lost the maximum weight (29.24%) during storage, while avg+cts dropped the least (20.02%). cts, avg, co, and co+avg treated fruits lost 26.80%, 29.37%, 35.31%, and 26.07%, respectively. storage duration and treatments considerably influenced firmness. throughout the storage period, the firmness gradually decreased regardless of treatment. for control and cts+avg, initially, the mango firmness values were 5.36 and 5.9 kg cm­2, which dropped significantly at 12 days of storage, reaching values of 1.44 and 3.21 kg cm­2, respectively (fig. 1b). storage times and coatings significantly affected the mango fruit’s mean total soluble solid (tss) content. figure 2a shows that fruit tss increased significantly with increasing storage periods. the control had the maximum tss concentration (19.50°brix), while cts+avg and avg­treated fruits had the lowest (10.77 and 11.0°brix, respectively) at the end of the storage. amin et al. ‐ mango postharvest quality improved by edible coating 105 fig. 1 ­ mango weight loss (a) and fruit firmness (b) after 12 days of storage at 27±2°c and 80­85% relative humidity due to coatings and storage intervals. the vertical line shows the standard error of the means of three replicates. control: distilled water; cts= chitosan; avg= aloe vera gel; co= coconut oil; cts+avg= chitosan+ aloe vera gel; co+avg= coconut oil+ aloe vera gel. fig. 2 ­ mango tss (a) and decay incidence (b) after 12 days of storage at 27±2°c and 80­85% relative humidity due to coatings and storage intervals. the vertical line shows the standard error of the means of three replicates. see figure 1. from figure 2b, it is observed that fruit degradation began after 4th day of storage in the control group, whereas treated fruits began to deteriorate on the 8th day of storage. compared to the control fruit (18.46%), the mango fruits coated with cts+avg showed the least deterioration (9.52%) after 12 days of storage. color attracts customers’ preferences. coated treatments maintained color, whereas control fruits changed faster. table 1 provides the color parameters l*, a*, and b*. all samples lost l* values during storage. control and treated fruit differed greatly. the l* value of the control fruits was considerably lower (30.30) than that of the coated fruits during storage. higher l* values (41.18) were recorded during storage for fruits covered with cts+avg. after storage, control fruit exhibited higher a* values (­1.31) than coated fruits. the control and treated mangoes initially showed a rise in b* values, which decreased until the end of storage. however, the higher b* values (32.61) were found in cts+avg­ treated fruits when compared to the control. since fruit color affects product quality and fresh market value, customer approval is essential. mango fruit ascorbic acid content was significantly affected by storage and treatment. due to physiological metabolism and ascorbic acid oxidation, treated and control mangoes lost vitamin c during storage (fig. 3a). at 12 days, mean ascorbic acid was 7.56 mg 100 g­1. cts+avg­treated fruits had a higher ascorbic acid level (9.25 mg 100g­1), while control fruits had less (5.89 mg 100g­1) after the storage compared to the other treatments. acidity decreased during storage. the mean acidity was 0.53% after 12 days storage. in figure 3b, the control had the least acidity (0.41%) while the cts+avg treatment had the highest (0.64%). cts+avg­treated fruits exhibited a lower ph 3.39 while control showed higher ph value 4.65 at the end of storage (fig. 3c). during storage, treated and control fruits significantly lost phenolic compounds. at 12 days, cts+avg had higher phenolic content (126.34 mg 100 g­1), compared to the control (84.13 mg 100g­1) (fig. 4a). figure 4b shows that there was a significant decrease in antioxidant capacity in treated and control fruits after storage. at the conclusion of storage, cts+avg fruit samples showed the highest dpph activity (293.51 μmol g­1), followed by cts 106 adv. hort. sci., 2025 39(2): 101­112 (244.29 μmol g­1), avg (276.12 μmol g­1), co (222.79 μmol g­1), and co+avg (227.04 μmol g­1). the control group had the lowest dpph activity (182.65 μmol g­1) compared to the coated groups, which may help the fruit generate antioxidant molecules and activate antioxidant defense enzymes. fig. 3 ­ mango ascorbic acid (a) acidity (b) and ph after 12 days of storage at 27±2°c and 80­85% relative humidity due to coatings and storage intervals. the vertical line shows the standard error of the means of three replicates. see figure 1. table 1 ­ mango peel color (l*, a, and b) after 12 days of storage at 27±2°c and 80­85% relative humidity due to coatings and storage intervals values are means with three replicates ± se. means followed by different letters (s) indicate significant differences within the columns or rows. control= distilled water; cts= chitosan; avg= aloe vera gel; co= coconut oil; cts+avg= chitosan+ aloe vera gel; co+avg= coconut oil + aloe vera gel. treatments storage periods (days) mean (treatments)0 4 8 12 l* control 43.09±3.29 def 37.53±0.32 hi 31.96±0.4 j 30.30±0.35 j 35.72 c cts 45.33±2.73 cde 43.35±0.32 def 40.69±0.11 fgh 37.35±0.41 i 41.68 b avg 50.40±0.55 ab 45.40±1.07 cde 42.77±0.20 def 40.96±0.47 fg 44.88 a co 47.80±0.17 bc 43.02±1.94 ef 40.86±0.20 fg 38.96±0.07 ghi 42.66 b cts+avg 53.08±1.37 a 43.44±0.63 def 42.47±0.05 ef 41.18±0.11 fg 45.04 a co+avg 46.28±1.38 cd 44.73±0.80 cde 43.62±0.01 def 40.82±0.26 fg 43.86 a mean (storage periods) 47.66 a 42.92 b 40.59 c 38.26 d a* control ­7.73±0.58 hij ­4.23±1.27 cde ­3.91±0.35 b ­1.31±0.06 ab ­2.07 a cts ­8.04±0.13 jk ­7.10±0.66 ghij ­5.47±0.07 efg ­3.40±0.06 cd ­6.00c d avg ­8.57±0.45 jk ­6.13±0.15 fgh ­5.08±0.10 def ­3.00±0.03 c ­5.70 c co ­7.93±1.25 ijk ­6.07±0.32 fgh ­4.08±0.68 b ­2.74±0.42 a ­3.15 b cts+avg ­9.55±0.54 k ­6.71±0.06 fghi ­6.02±0.29 fgh ­4.09±0.33 cde ­6.59 d co+avg ­88.84±0.31 jk ­7.76±0.37 hij ­4.22±1.48 b ­1.53±0.33 ab ­3.82 b mean (storage periods) ­8.44 d ­6.33 c ­2.63 b ­0.81a b* control 15.12±0.50 l 20.57±1.67 k 30.37±0.48 fcde 28.26±0.53 efghi 23.58 d cts 25.41±0.58 ij 27.09±0.73 ghij 30.91±1.40 cde 30.57±0.52 cdef 28.49 c avg 25.78±0.64 hij 27.58±1.02 fghi 30.90±0.13 cde 30.12±0.37 cdefg 28.59 bc co 28.82±2.12 efgh 32.25±0.78 cd 36.17±1.60 ab 33.13±0.58 bc 32.59 a cts+avg 24.04±2.57 j 28.64±0.90 efgh 38.81±1.65 a 38.95±0.09 a 32.61 a co+avg 29.00±0.72 efg 29.64±0.31 defg 30.69±0.38 cdef 29.77±0.29 defg 29.77 b mean (storage periods) 24.69 d 27.63 c 32.98 a 31.80 b post­harvest treatments and storage durations significantly (p< 0.05) influenced mango fruit antioxidant enzymes. as demonstrated in figure 5a the control group had the highest polyphenol peroxidase (ppo) activity (7.17 u g­1) after 12 days, while cts+avg­treated fruits had the lowest (3.95 u amin et al. ‐ mango postharvest quality improved by edible coating 107 g­1). as shown in figure 5b treated fruit samples and control­maintained catalase activity (cat) throughout storage. after 12 days of storing, the cts+avg group had higher cat activity (0.47 u g­1) than the control group (0.33 u g­1). figure 5c shows the maximum pod activity (0.55 u g­1) in cts+avg­ treated fruits after 12 days of storage. pod activity at 12 days of storage was in cts (0.48 u g­1), avg (0.51 u g­1), co (0.50 u g­1), and co+avg (0.49 u g­1) while the control (0.48 u g­1) exhibited lower pod activity. mango fruits treated with cts + avg had superior overall impression (7.71) compared to the control (7.01). cts + avg improved the mango fruit’s flavor (7.97), sweetness (7.33), taste (7.63) and color (7.90) at 8 days of storage (fig. 6). principal component analysis (pca) pcas examined biochemical properties and antioxidant enzymes after postharvest treatment. two pcas explained 88.4% of pca variation. pc1 (dim 1) explained 80.1% of the dataset variation and pc2 (dim 2) explained 8.3%. aa, tpc, dpph, ff, l* values, pod, and cat enzymes strongly connect with pc1, while tss, wl, ppo enzyme, and a* values negatively correlate. pc2 was linked positively with b* values but negatively with ta and ph (fig. 7). fig. 5 ­ mango ppo (a), cat (b) and pod (c) activity after 12 days of storage at 27±2°c and 80­85% relative humidity due to coatings and storage intervals. the vertical line shows the standard error of the means of three replicates. see figure 1. fig. 4 ­ mango total phenol content (a) and dpph activity (b) after 12 days of storage at 27±2 °c and 80­85% relative humidity due to coatings and storage intervals. the vertical line shows the standard error of the means of three replicates. see figure 1. adv. hort. sci., 2025 39(2): 101­112 108 4. discussion and conclusions mango ripening is associated with changes in color, texture, and flavor, resulting from modifications in the fruit’s chemical composition. these include the conversion of starch to sugars, a reduction in acidity, and the generation of new volatile compounds (taiti et al., 2015). fruit weight loss increases due to moisture evaporation and metabolic activity. ncama et al. (2018) reported that water loss during postharvest storage leads to weight reduction, changes in texture and appearance, and shriveling. however, water loss can be reduced when fruit surfaces are covered with semipermeable edible coatings. shah and hashmi (2020) also discovered that cts + avg coatings reduce the weight of mango fruit. cts+avg significantly maintained the firmness of mango fruits. according to rukunuzzaman et al. (2025), mangoes treated with cts + avg decreased mango softening by reducing the breakdown of cell wall polymers, keeping the fruit firm. our study also confirmed rastegar and atrash (2021) that cts+avg delayed mango fruit firmness. total soluble solids were significantly increased by moisture loss and the breakdown of carbohydrates into soluble sugars. however, edible coatings may limit respiration and exchange of gases in coated fruits by closing stomata, preventing an increased rate of tss content (chavan et al., 2023). cts+avg­ treated mangoes showed lower tss during storage, as described by yu et al. (2021). fig. 7 ­ principal component loading plot of physiochemical and antioxidant enzymes activities of mango fruit during storage. fig. 6 ­ mango color (a), flavor (b), sweetness (c), taste (d) and overall impression (e) at 8 days of storage at 27±2°c and 80­85% relative humidity due to coatings treatment. the vertical line shows the standard error of the means of three replicates. see figure 1. amin et al. ‐ mango postharvest quality improved by edible coating 109 the chitosan coating significantly decreased the frequency of disease­ induced mango fruit deterioration while preserving fruit quality in agricultural commodities (hasan et al., 2020). similar to this, avg coating can prolong the shelf life of apricots by postponing microbial deterioration, whether it is applied alone or in conjunction with other treatments (nourozi and sayyari, 2020). in this study, the use of cts either alone or in combination with avg significantly decreased the incidence of mango fruit deterioration during storage. changes of color in climacteric fruits occur because of the alteration of chlorophyll to other pigments. kaur et al. (2024) stated that cts + avg coatings slow down metabolic and pigment changes by regulating gas exchange in fruits while they are being stored. due to the lower availability of oxygen and higher carbon dioxide in the internal microenvironment, it causes slower ripening (paul et al., 2019). the l* value decreased across all treatments, although it declined more slowly in treated fruits than in the controls. the lower a* values of the coated samples, which are more green than red throughout storage, may suggest a delay in mango fruit ripening. according to a previous study by abdelshafy et al. (2023) and begum et al. (2023), edible coatings can influence epidermal permeability, gas exchange, and oxidation. seyed et al. (2021) found that mango fruits stored with aloe vera gel and chitosan changed color less than control fruits. ascorbic acid, a powerful antioxidant, scavenges free radicals and reactive oxygen species during fruit ripening (fenech et al., 2019). the cts + avg coating in mango fruits might limit the permeability of oxygen and carbon dioxide on its surface and reduce the loss of ascorbic acid content. our results were confirmed by shah and hashmi (2020), who reported higher levels of vitamin c in mango fruits treated with cts+avg. acidity presumably decreased due to widespread catabolisation of organic acids to sugars. a sudden reduction in ta indicated senescence in mango fruit (shah and hashmi, 2020). seyed et al. (2021) found that aloe vera gel with chitosan­treated mango fruits had the highest ta. fruit coated with cts+avg has the lowest ph. in line with our findings, amin et al. (2021) discovered that cts+ avg reduces mango ph. according to sogvar et al. (2016) who reported that strawberry’s ph raised during storage in both control and aloe vera gel­coated fruits; whereas control fruits had a higher ph value. retaining the fruit’s nutritional quality (color, bitterness, astringency, acidity, and taste) during storage requires retaining phenolic chemicals, which decrease with ripening. phenolic molecules, secondary plant metabolites, scavenge ros to increase fruit antioxidants (swallah et al., 2020). an edible coating may reduce phenolic component oxidation in mangoes. agreeing with seyed et al. (2021), the current work determined that chitosan and aloe vera gel coatings improve the phenolic retention of mangos during storage. the treated fruits’ antioxidant capacity, which is linked to total phenolic content, may boost the activity of dpph scavenging. aloe vera coatings­ maintained antioxidant (dpph) activity throughout storage (khaliq et al., 2019 a or b). mango fruit treated with aloe vera and chitosan during postharvest storage period showed improved dpph scavenging activity, as reported by begum et al. (2023). the fruit’s antioxidant system’s genes for ppo, pod, and cat increase throughout ripening, defending against ros accumulation (loay and el­ezz, 2021, and yu et al., 2021). most horticultural crops discolor when ppo oxidizes phenolic compounds, altering them to quinones. the coating reduced ppo activity, which may have activated defence­related enzymes to prevent browning of mango fruit and extend storage (adiletta et al., 2019). ppo and enzymatic browning may have been inhibited by fruit surface chitosan coating co2, o2, and ethylene (romanazzi et al., 2018). these findings are consistent with previous findings in strawberry (petriccione et al., 2015), where chitosan coatings significantly reduced ppo activity and fruit discoloration. cat activity reduces o2 and h2o2. shah and hashmi (2020) discovered that mango fruit chitosan coating boosts cat activity. pod, a fruit­ specific oxyradical detoxifying enzyme, may reduce oxidative damage and a complex covering of chitosan and cinnamon oil increased pod activity in jujube, promoting storage disease resistance (xing et al., 2020). according to these findings, using aloe vera gel along with chitosan was the most effective way to mitigate decay symptoms and lessen the physicochemical alterations in mango fruit. mangoes treated with avg coatings, either separately or in combination, exhibited improved sensory quality, adv. hort. sci., 2025 39(2): 101­112 110 according to khaliq et al. (2019 a, b). this study presented that chitosan and aloe vera coatings may increase mango storage l i fe by minimizing weight loss, reducing postharvest deterioration and retaining ascorbic acid, titratable acidity, f irmness, and peel color throughout storage. mango fruit with coatings had higher total phenol and antioxidant levels than the control and ripened more slowly. these coatings may boost cat and pod antioxidant enzymes and lower ppo during storage. given human health concerns, edible coatings l ike chitosan­aloe vera may improve mango storage quality. natural edible coatings of aloe vera, chitosan, and coconut oil may affect antioxidant enzyme activity. in order to make mango fruit appealing to consumers and enhance its storability with edible coatings, further research is needed. acknowledgements this work was supported by irt, hstu (ey: 2023­ 24). we also acknowledge the ministry of science and technology for funding the nst scholarship, which made study possible. 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college of natural and computational science, mekdela amba university, tuluawlia, ethiopia. key words: hydroponics, production system, soilless farming, sustainability, urban agriculture. abstract: new constraints such as urbanization, food security, farmland scarcity, and escalating greenhouse gas emissions underscore the importance of vertical farming. this eco­friendly method offers a promising solution to traditional farming, aiding a growing global populace in securing sustenance. resource use efficiency of vertical farming and the ability to produce premium agricultural goods are driving its global appeal. particularly beneficial in areas with limited soil and water resources, vertical farming could play a vital role in sustaining fruit and vegetable production. vertical agriculture emphasizes the critical need for urban centers to combat pollution and escalating food expenses by prioritizing self­reliance through local food production. advanced cultivation techniques like hydroponics and aeroponics make vertical farming viable for urban environments, requiring minimal oversight and yielding higher outputs. despite its potential, vertical farming encounters obstacles such as steep upfront and operational costs, complexity, and maintenance demands for optimal growth conditions. to promote urban agriculture, there is a need for enhanced extension services to educate and train growers and farmers on vertical farming techniques for producing diverse horticultural yields. 1. introduction what is urban horticulture? with each one­degree centigrade increase in temperature, an estimated 10 percent of the current cropping area is predicted to become unusable (bouteska et al., 2024). this scenario raises concerns about the potential scarcity of land for farming to adequately feed the global population shortly (clemson university, 2011). moreover, research indicates that roughly 80 percent of the currently cultivated land worldwide is actively being utilized (clemson university, 2011). urban horticulture, involving the precise cultivation of crops for the production, processing, and distribution of food and other goods within urban and peri­urban settings, offers a solution to this challenge. definitions of (*) corresponding author: jemalseid780@gmail.com citation: hussen j.s., ahmed g.e., 2025 ­ role of vertical farming for sustainable urban horticulture: a review. ­ adv. hort. sci., 39(1): 69­80 orcid: hjs: 0009­0000­4631­2761 copyright: © 2025 hussen j.s., ahmed g.e. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 20 july 2024 accepted for publication 13 december 2024 ahs advances in horticultural science review paper ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16549 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0000-4631-2761 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 69­80 70 urban and horticultural practices vary across nations, regions, and local cultural contexts (cdrf, 2010). horticulture involves creating gardens and growing plants, while agronomy focuses on managing forests, trees, and their products, along with the cultivation of cereals, animal feed, and other agricultural activities (abegunde, 2014). horticulture, which encompasses the production of crops for food, medicine, or aesthetics, falls under the umbrella of agriculture. conversely, agronomy, primarily concerned with field cropping, centers around producing durable goods like fiber, oilseeds, and legumes. urban horticulture encompasses the cultivation of edible and medicinal plants such as fruits, vegetables, herbs, and mushrooms in urban areas and their environs, especially in land­constrained settings (orsini et al., 2013). its applications range from landscaping public spaces to therapeutic uses for individuals with disabilities (moustier and danso, 2006). this practice varies in scale and scope, spanning from high­tech intensive methods to small­ scale soilless cultivation with localized irrigation systems (drescher et al., 2006). in developed nations, urban horticulture often occurs on small state­ owned, private, or public plots like community gardens, offering not just fresh produce but also social engagement and recreational benefits (van leeuwen et al., 2017; nugent, 2000). conversely, in developing countries, urban horticulture serves as a means to establish self­sustaining enterprises, generating income and fostering social stability (van leeuwen et al . , 2017). furthermore, urban horticulture enhances access to fresh, nutritious foods through local gardens and plays a significant role in combating obesity and related health issues. by cultivating fruits and vegetables locally, urban communities can increase their intake of nutritious foods. this approach not only offers scarce food resources to migrant communities but also fosters a sense of community and belonging among residents through urban horticulture projects. community gardens in particular, provide opportunities for social interaction, shared responsibility, and the establishment of support networks, which can contribute to overall well­being and healthier lifestyles. these advancements address malnutrition prevalent in many urban settings (lovell, 2016). urban horticulture plays a vital role in stimulating employment opportunities within agriculture, marketing, and processing domains, thereby enhancing livelihoods and boosting supplemental income for all essential participants within the horticultural value network (orsini et al., 2013). local food production minimizes intermediaries, transportation, storage costs, and handling, thereby reducing the economic and environmental footprint of food production (fao, 2014). furthermore, urban horticulture can help alleviate food deserts in underserved communities and provide affordable food options (de leever, 2010). by offering recreational spaces, preserved environments, and biodiversity, urban horticulture enhances the quality of life for urban residents (van leeuwen et al., 2017). pace et al . (2018) observed that concerning postharvest storage, lettuces grown in soilless conditions demonstrated superior qualitative and microbiological performance compared to those cultivated in soil. specifically, the soilless growing system enhanced the storability of lettuces and facilitated the production of uncontaminated raw materials. what is vertical farming? vertical farming, initially implemented by american geologist gilbert ellis bailey in 1915 (kretschmer and kollenberg, 2011) and it was revolutionized by patrick blanc, who invented and patented a geometric approach that maximizes production efficiency within a compact footprint. this method entails vertically aligning animals and plants for food or other purposes in a soil­free, space­saving design (anirudh, 2014) that capitalizes on acreage volume, expanding development possibilities in three dimensions compared to traditional farming practices. vertical farming systems can range from stacked containers to interconnected networks sprawled over multiple acres, adaptable for indoor and outdoor settings. utilizing biodegradable and reusable mediums, these systems can be powered by alternative energy sources for pumps and sustainably nourished with organic materials. the definition of vertical farming varies based on factors like size, density, control level, layout, structure, location, and objectives, leading to diverse stakeholder perspectives, from viewing it as a minor crop activity to a pivotal component for future food security. the interchangeable use of “vertical farming” as both an activity and a term further complicates its perception (waldron, 2018). essentially, “vertical farming” refers to cultivating plants in multiple layers to maximize yield within a hussen and ahmed ‐ vertical farming sustain urban horticulture 71 limited surface area. chin et al. (2017) note that vertical gardening serves various purposes, including ecological and financial benefits. for plant enthusiasts, growing plants vertically in confined spaces provides an engaging activity for both plant enthusiasts and experts. for educators, vertical garden cultivation offers a unique and stimulating opportunity for teachers to practically impart science, basic arithmetic, and finance concepts. for health­conscious consumers, vertical planting, based on organic principles, yields cleaner, fresher fruits, and vegetables with minimal chemical input, appealing to those focused on healthy eating. for business­oriented individuals, vertical farming provides businesses with a sustainable and efficient approach to food production, creating valuable opportunities for growth and innovation. this controlled setting offers numerous benefits, such as decreased risks of disease and pest outbreaks, reduced environmental impact, and the ability to uphold produce quality while conserving water (van der schans et al., 2014). vertical farming systems minimize water usage and streamline food sales and distribution within urban areas, lowering associated costs. vertical gardening acts as a sustainable substitute for conventional crop cultivation, aiding in the conservation of land for traditional farming methods. recognized as a cost­effective food production method (jansen, 2017), vertical farming proves invaluable in land­scarce scenarios, making it an ideal solution for terrace kitchen gardens. furthermore, vertical farming plays a significant role in promoting organic farming practices (anirudh, 2014), allowing growers to sidestep land­related challenges like pollution, environmental rehabilitation, and zoning issues through the use of indoor vertical farming techniques (anderson et al., 2012). moreover, this technique would reduce the reliance on synthetic pesticides (garg and balodi, 2014). other advantages of vertical farming include year­ round produce distribution, immunity to yield losses due to adverse weather, job creation in urban settings, water conservation, and reuse, and decreased food spoilage risks from mishandling. the utilization of controlled environments in vertical farming enhances its resilience against the effects of climate change (germer et al., 2011). crops in vertical gardens are typically more manageable for cultivation and harvesting, with lower susceptibility to diseases and pests (utami et al., 2012). the absence of soil in vertical farming systems generally prevents weed growth, reducing labor costs in this aspect. plants receive optimal aeration, and the visual appeal of the garden is enhanced through vertical farming practices. comparatively, higher yields are often achieved with this method than with traditional farming practices. for example, chinese cabbage yield using vertical farming reached 45 tons per hectare, surpassing the 10 to 25 tons per hectare range seen in field cultivation of the same crop (utami et al., 2012). according to the opinion of kannaujia et al. (2021), the benefits of vertical farming encompass increased net returns per unit area, accelerated growth leading to higher yields, reduced water and fertilizer usage, job opportunities, enhanced farmer incomes, continuous year­round production, environmentally friendlier processes compared to traditional methods, decreased risks of crop failures, minimized reliance on pesticides for pest control, progress towards nutritional security, and immunity to adverse environmental conditions. vertical structures primarily cultivate plants that grow upright, are not sprawling, and bear fruits along their stems. these include vegetables like lettuce, tomatoes, cucumbers, bell peppers, mint, cilantro, spinach, strawberries, various herbs, and certain medicinal plants that demand minimal vertical space (kannaujia et al., 2021). 2. vertical farming types, techniques, and production systems outdoor vertical farming type vertical farming outdoors entails cultivating vegetables using vertical support systems constructed from readily available materials, such as bamboo structures supplemented with wires to assist the vegetables in climbing upwards (kannaujia et al., 2021). indoor vertical farming type indoor vertical farming involves a range of permanent structures that support plant growth, typically within polyhouses, net houses, shade nets, and bamboo structures covered with plastic sheets. this method encompasses hydroponics, aeroponics, and aquaponics (kannaujia et al., 2021). an indoor multilayer plant cultivation system meticulously adv. hort. sci., 2025 39(1): 69­80 72 manages all growth factors like temperature, light, carbon dioxide levels (co2), humidity, water, and nutrients to yield abundant, high­quality fresh produce consistently throughout the year, independent of natural light or outdoor conditions (sharathkumar et al., 2020). vertical farming involves growing crops or vegetables in vertically stacked layers or on vertical structures that promote upward growth, typically within a controlled environment tailored for optimal plant growth. it frequently employs soilless farming methods like hydroponics, aquaponics, and aeroponics (kannaujia et al., 2021). hydroponics hydroponics entails cultivating plants without soil, with their roots submerged in nutrient­rich liquid solutions comprising vital macronutrients like nitrogen, phosphorus, and potassium, along with trace elements such as iron, manganese, and zinc. inert materials like gravel, sand, sawdust etc. are used to support the roots in hydroponic systems (fig. 1). by maintaining controlled environments and adhering to strict certification standards, hydroponic farms present a pragmatic method for sustainable food production by eradicating the necessity for harmful chemicals like pesticides. this innovative farming method is not just a concept but is actively being integrated into sustainable agriculture practices to meet the increasing global food demand (debangshi, 2021). hydroponics involves plants growing without soil, instead utilizing inert mediums like coco peat and nutrient­rich water solutions to support and nourish the roots, and can conserve about 70 percent more water than traditional farming methods (bhanu murthy et al., 2022). it presents a viable production alternative that vegetable growers can explore, with setups possible even in small spaces like a 400 ft² area or a small shed (usda, 2016). in hydroponic systems, plant roots are immersed in a nutrient solution tailored to meet their nutrient requirement (despommier, 2014). aquaponics aquaponics represents a type of hydroponics where plants are cultivated alongside aquatic organisms in a closed­loop system that emulates natural processes. a device for solids removal filters the nutrient­rich wastewater from fish tanks before channeling it to a biofilter, where harmful ammonia is converted into nutrient­rich nitrate. the plants absorb these nutrients, cleansing the wastewater before it returns to the fish tanks. additionally, plants can absorb carbon dioxide (co2) from the air to perform photosynthesis, which is essential for their growth. in an aquatic environment like a fish tank or aquaponic system, fish release carbon dioxide into the water through respiration (fig. 2). therefore, in fig. 1 ­ a model of hydroponics. adapted from: https://images. app.goo.gl/jshegzqxe9uqnuk3a fig. 2 ­ a model of aquaponics (adapted from: https://images. app.goo.gl/6skpjmtefi1z4mgz7). http://images.app.goo.gl/jshegzqxe9uqnuk3a http://images.app.goo.gl/jshegzqxe9uqnuk3a http://images.app.goo.gl/jshegzqxe9uqnuk3a http://images.app.goo.gl/6skpjmtefi1z4mgz7 http://images.app.goo.gl/6skpjmtefi1z4mgz7 http://images.app.goo.gl/6skpjmtefi1z4mgz7 hussen and ahmed ‐ vertical farming sustain urban horticulture 73 an aquaponic system or a similar setup where fish and plants coexist, it is important for the plants to uptake co2 from the fish to help maintain the balance of gases in the water and promote the well­ being of both the aquatic life and the plants. despite its aquacultural aspect, aquaponics is not as commonly employed as traditional hydroponics in commercial vertical farming setups, which typically focus on cultivating select fast­growing vegetable crops (debangshi, 2021). aquaponics functions as an ecosystem built on mutual relationships between plants and fish. the concept revolves around establishing a semi­self­sustaining system where fish waste nourishes plants in a vertical farming environment (diver, 2021). notably, aquaponics utilizes only 2 percent of the water required in traditional farming, and operates as a closed­loop system with recyclable outputs, thereby generating minimal waste (mccollow, 2014). for instance, aerofarms in new jersey reportedly consumes 95 percent less water compared to field cultivation to produce equivalent volumes of leafy greens (peters, 2015). filtration poses a significant challenge for aquaponic businesses, as the accumulation of debris can endanger the fish and cause a chemical imbalance in water which is crucial for supporting plant growth (mccollow, 2014). aeroponics aeroponics originated from nasa’s project in the 1990s, aiming to develop an efficient method for plant cultivation in space. unlike hydroponics and aquaponics, aeroponics operates without a solid or liquid medium for plant growth. however, a liquid nutrient solution is being sprayed on plant roots in sprinkler form. it stands out as the most environmentally sustainable soilless cultivation technique, requiring no medium replenishment and consuming up to 90 percent less water than highly efficient traditional hydroponic systems. no growing medium is necessary for aeroponics, as the system is designed vertically, leading to energy savings as excess liquid naturally drains away due to gravity, unlike traditional horizontal hydroponic systems that often rely on water pumps to manage surplus solutions (fig. 3). while aeroponic systems are not prevalent in current vertical farming practices, they are gradually gaining popularity (debangshi, 2021). aeroponics supports the growth of vegetables, flowers, and fruits, offering potentially higher nutrient quality and absorption of vitamins and nutrients compared to other methods (birkby, 2016). despite the use of modern pumps and control systems, aeroponics requires less automation. these systems are favored over hydroponics for their water conservation benefits and reduced disease susceptibility (boston, 2014). the vertical growth pattern in aeroponics makes it ideal for producing a large volume of plant materials in limited spaces, with plants either growing vertically in a tube­like structure or suspended in containers. fig. 3 ­ a model of aeroponics (adapted from: https://images.app.goo.gl/9jcb4ergypya8vsa8) http://images.app.goo.gl/9jcb4ergypya8vsa8 74 adv. hort. sci., 2025 39(1): 69­80 3. what are the opportunities and importance of vertical farming for sustainable urban horticulture? globally, urban regions exhibit higher population densities compared to rural areas, accommodating 54 percent of the total population in 2014 (fig. 4). the rapid economic growth over the last thirty years has coincided with a significant increase in the urban population. in 1950, urban dwellers constituted 30 percent of the global populace; projections indicate that by 2050, this figure will swell to 66 percent. with the world’s population expected to approach nine billion by 2050, the role of vertical farming in fostering sustainable urban horticulture and ensuring food security has emerged as a pivotal topic for discussion. urbanization entails a demographic shift where a larger share of a nation’s populace resides in cities due to migration from rural areas to regions that subsequently witness declining populations, leading to escalated land utilization (satterthwaite et al., 2010). the repercussions of urbanization on agriculture are poised to revolutionize how food is distributed and marketed (kennedy et al., 2004). notably, urban locales are witnessing a rising inclination towards the consumption of fresh produce such as fruits, vegetables, meat, and dairy (agnes, 2014). forecasts suggest that urbanization will amplify job opportunities in food processing, transportation, and retail sectors while diminishing agricultural employment (cohen and garret, 2010). consequently, urban residents will be compelled to offer high­quality products for consumption or commercial purposes through urban agriculture to sustain their livelihoods (redwood, 2009). in the contemporary era, we are witnessing a swift surge in global population, particularly in developing regions like africa, asia, and south america. projections by united nations indicate that by 2100, the global population is expected to surpass 21 billion individuals. un estimations suggest that by 2050, 66 percent of the world’s populace will reside in urban areas, with europe reaching 82 percent urbanization (united nations, 2017). the rapid expansion of the population invariably translates into heightened food requirements. presently, 80 percent of the world’s arable land is under cultivation, while the remaining 20 percent lies barren, its agricultural potential nearly squandered due to inadequate land management in recent decades (eigenbrod and gruda, 2015). the global population is swift ly escalating, alongside the pace of urbanization. un projections from 2015 forecast a population increase exceeding double by 2050, urbanization trends continue to evolve, with more people moving to cities globally. by 2050, the united nations projects that nearly 68% of the world’s population will live in urban areas. as urban regions contribute to more than 70 percent of worldwide co2 emissions, resulting in pollution and negative environmental consequences, ensuring food security emerges as a critical issue in urban environments. as urban populations swell, the conventional cultivation of fresh fruits and vegetables faces challenges due to nutrient­deficient soil and limited usable land, posing a significant threat to the supply of fresh produce (zaręba et al., 2021). vertical farming, a soilless agricultural technique for cultivating fresh vegetables, is swiftly gaining traction and holds a promising future. researchers and scientists are exploring this method of farming as a viable option for urban dwellers. despite its longstanding presence, vertical farming remains relatively unknown to many, with only a minority having harnessed its potential for growing fresh produce. this innovative approach has the potential to significantly enhance food production while reducing the environmental impact of agriculture by utilizing less land, water, pesticides, and fertilizers, thereby enhancing overall efficiency (barui et al., 2022). the rising popularity of vertical farming globally is attributed to its efficient resource management and ability to yield high­quality foods. particularly in regions with limited access to soil and water resources, vertical farming could revolutionize fruit and vegetable production. across all crops, the fig. 4 ­ world urban and rural population, 1950­2050. source, nwosisi and nandwani (2018). hussen and ahmed ‐ vertical farming sustain urban horticulture 75 use of vertical farming technology results in a substantial increase in yields. however, the degree of improvement varies significantly, with crops like potatoes and tomatoes showcasing the most potential for yield enhancement. peas and spinach reflect minimal benefits from the vertical farming technology (table 1). to combat issues like congestion, pollution, and escalating food prices, urban areas facing scarcity of land and high real estate costs must generate sufficient food to sustain their populations (mir et al., 2022). factors such as rapid urbanization, natural calamities, climate change, and indiscriminate use of chemicals and pesticides have collectively contributed to deteriorating soil ferti l ity. consequently, soil productivity has plummeted, soil health has deteriorated, and the available land per person has dwindled (lal, 2015). projections suggest that by 2050, with the global population expected to reach 8.9 billion, there will be a 50 percent surge in food demand, necessitating additional arable land that is simply not accessible (fao, 2011). the anticipated arable land per capita by 2050 is forecasted to be less than 0.20 hectares, marking a decline to less than one­third of the 1970 levels (fao, 2011) (fig. 5). the challenges posed by these issues pose significant threats to conventional soil­based agricultural systems, rendering food production a formidable task in the present day. to address these challenges, traditional soil­based farming methods need to be augmented with more efficient and environmentally sustainable modern farming practices (lehmann, 2010). in transitioning to fig. 5 ­ scenario of global population and cultivable land with their projection up to 2050. source: fao (2011). table 1 ­ estimated yield of a vertical farm compared to traditional farming. source: barui et al., 2022 crops yield in vertical farming (tons/ha) field yield (tons/ha) carrots 58 30 radish 23 15 potatoes 150 28 tomatoes 155 45 pepper 133 30 strawberry 69 30 peas 9 6 cabbage 67 50 lettuce 37 25 spinach 22 12 total (average) 71 28 innovative vertical farming techniques, factors such as diminishing soil productivity, limited soil nutrient reserves, insufficient irrigation water availability, and the impacts of climate change must all be taken into account. vertical farming methods, serving as an alternative to conventional soil­based farming systems, have the potential to serve as a supplementary solution in mitigating the scarcity of fertile arable lands and water resources (texier, 2013). the human population, grappling with the impacts of climate change, must forge a new realm of spaces that ensure environmental sustainability and foster sustainable urban horticulture. in the future, the rise of vertical urban farms will increase crop production, lower costs, and improve the quality of food in urban areas. this will aid in reducing the harmful environmental effects of urbanization (thomaier et al., 2015). vertical urban farms offer numerous advantages for the natural environment, including the autonomy of vertical crops from the polluted urban surroundings, aligning with the principles of sustainable development for environmental well­ being, minimal water usage that enhances sustainable water management, and the potential utilization of organic waste generated (despommier, 2013). crucially, advanced vertical farming surpasses traditional farming in food production. recent plant factory technologies can cultivate fresh, safe, and nutrient­rich produce year­round within cities, despite climate fluctuations, exhibiting high water and land efficiency while requiring less labor than traditional methods (kozai and niu, 2015). the proliferation of horticultural offerings in urban adv. hort. sci., 2025 39(1): 69­80 76 spacing (burdon and chivers, 1982). therefore, the quest for maximizing crop production per unit area must strike a balance between plant density and avoiding overcrowding for optimal crop growth and health. even traditional open­field growers face mounting pressure to enhance productivity. the planet is showing signs of strain, with escalating disease pressures underscoring the critical need for sustainable solutions like vertical farming in regions striving for self­reliance. as we face the daunting challenge of feeding a burgeoning global population over the next two decades, the strategy involves harnessing space efficiently, expanding skyward, and repurposing existing urban areas (kumar et al., 2018). urban food security hinges on factors such as food availability, accessibility, and quality, all of which stand to benefit from the implementation of urban vertical farming techniques. various gardening methods can significantly bolster the food security of communities and households. whether at a personal or communal scale, cultivating food can enhance food and nutrition security across all economic strata (kortright and wakefield, 2011). the integration of urban horticulture in future city planning holds the promise of elevating food security levels, fostering a more balanced food distribution between rural and urban regions. despite the advancement of global urban horticulture, rural agriculture will continue to play a pivotal role in ensuring global food security (dubbeling et al., 2010). vertical farming epitomizes sustainability, with the ability to stack farms vertically to amplify productivity per unit of land, thereby conserving arable land for alternative uses. moreover, these farms eliminate transportation costs by bringing crops closer to urban markets, while also facilitating the recycling of energy and water within their structures. additionally, the utilization of methane digesters can convert organic waste into energy, powering the operations within the building itself (kumar et al., 2018). 4. challenges in vertical farming systems the study conducted by jasim et al. (2016) revealed that the majority of vertical farms interviewed lacked systematic tracking of key metrics such as monthly energy and water usage, precise yield, waste management, air quality, and the extent of pesticide or fertilizer application. these deficiency poses significant obstacle in establishing a settings grants individuals more opportunities to engage with greenery and fosters community bonds. many city dwellers, for instance, partake in activities like tending flower beds to enhance their city’s green spaces and landscapes, showcasing how plants can cultivate communal ties. moreover, these undertakings not only enhance the aesthetic appeal of cities but also significantly impact the well­being and quality of life of participants (wakefield et al., 2007). vertical farming emerges as a promising strategy to address a range of sustainable development goals (sdgs) established by the united nations general assembly in 2015, covering “good health and well­ being, sustainable cities and communities, responsible consumption and production” (united nations, 2020). nature­related hobbies such as gardening and horticultural pursuits are renowned for their stress­reducing effects. recent research underscores that engaging in gardening or horticultural activities can alleviate stress (van den berg and cluster, 2011), boost self­esteem, nurture social connections (cammack et al., 2002), and enhance cognitive well­being (cimprich, 1993). vertical farming is swiftly gaining prominence as a preferred cultivation method among various urban agricultural practices (agrilyst, 2017). vertical farming represents an advanced form of soilless cultivation that pushes the boundaries of traditional agriculture, evolving into a fusion of urban and rural practices. these vertical farms serve as hubs for the self­ production of food, recreating ideal climatic conditions inside structures to support the growth of diverse plant varieties. here, vegetables thrive without soil, drawing nutrients from water solutions under led lighting systems. supporters of vertical farming and urban horticulture emphasize that by functioning within controlled environments, these farms can protect crops from insect infestations and diseases, eliminating the necessity for pest control measures (despommier, 2013). a key objective of vertical farming is to maximize crop yields per unit of land area utilized. however, when determining the optimal number of plants to cultivate within a given building space, careful attention must be paid to plant spacing. the density of plants is a critical factor in managing pests and diseases, as it affects the microclimate around the plants (burdon and chivers, 1982). achieving complete prevention of disease transmission would require impractically large plant hussen and ahmed ‐ vertical farming sustain urban horticulture 77 in vertical farms. companies specializing in vertical farming technologies play a key role in developing unique designs tailored to accommodate the specific needs of diverse crops in vertical farming systems. 5. summary and future line of works vertical farming involves cultivating crops in confined areas where plants receive regular nutrients and water. quick­growing green leafy vegetables such as mint, amaranthus, and lettuce thrive in vertical systems due to the accurate nutrients and water they get. this efficient growth results in high­ quality produce and good yields, making vertical farming a promising solution for urban areas facing land scarcity and high food demand, aiming to combat pollution and escalating food prices. the outlook for vertical farming seems promising, particularly with the uti l ization of advanced technologies like hydroponics, aeroponics, and aquaponics. these contemporary farming techniques are ideal for urban settings as they boost productivity significantly while vertical farms often uti l ize automation technologies such as sensors, artificial intelligence, and robotic systems to monitor and control factors like lighting, temperature, humidity, and nutrient delivery. this automation can reduce the need for constant manual intervention and monitoring. the decreasing availability of cultivable land in urban areas has worsened the difficulty of supplying fresh, high­quality vegetables. hence, vertical farming emerges as an ideal alternative for cultivating fresh leafy vegetables and fruits in the times ahead. further research into vertical farming is crucial to enhance production and lower operational expenses compared to traditional soil­based agriculture. to sustain urban horticulture, there is a pressing need to focus on extension programs to train farmers on vertical farming techniques and cultivate various crops in vertical structures. this education will be crucial in encouraging the extensive uptake of vertical farming techniques for a more sustainable urban food production system. references abegunde a.a., 2014 ­ urban horticulture and community 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solutions supporting a healthy society living in the urban environment. ­ resources, 10(11): 109. https://www.researchgate.net/journal/international-journal-of-chemical-studies-2321-4902?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6inb1ymxpy2f0aw9uiiwicgfnzsi6inb1ymxpy2f0aw9uiiwicg9zaxrpb24ioijwywdlsgvhzgvyin19 https://www.researchgate.net/journal/international-journal-of-chemical-studies-2321-4902?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6inb1ymxpy2f0aw9uiiwicgfnzsi6inb1ymxpy2f0aw9uiiwicg9zaxrpb24ioijwywdlsgvhzgvyin19 https://www.researchgate.net/journal/international-journal-of-chemical-studies-2321-4902?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6inb1ymxpy2f0aw9uiiwicgfnzsi6inb1ymxpy2f0aw9uiiwicg9zaxrpb24ioijwywdlsgvhzgvyin19 173 1. introduction tomato as a plant indigenous to tropical regions is susceptible to chilling injury when subjected to low temperature storage (saltveit, 2001). chilling injury limits tomato storage life and leads to significant degradation of fruit quality and decreases the market value. it can increase membrane permeability, and a resultant increase in leakage of ions from cell membrane, surface pitting, susceptibility to decay and diseases, weight losses, abnormal ripening, change in respiration, ethylene production and senescence. chilling injury symptoms mainly develop during shelf life following cold storage (candan et al., 2007). numerous attempts, such as breeding for increased chilling tolerance, genetic engineering, modifying crop management practices and application of chemicals, have been made to increase chilling tolerance and avoid chilling injury (baninasab, 2009). chilling alleviation in fruits and vegetables has been attributed to several factors including accumulation of polyamines, nitric oxide and proline (aghdam and bodbodak, 2013). over the years, studies have shown the involvement of polyamines (pas) in a wide array of processes in plants, ranging from triggering organogenesis to protecting against stress (walden et al., 1997). pa accumulation occurs under abiotic stresses including drought, salinity, extreme temperatures, uv-b, heavy metals, mechanical wounding and herbicide treatment (hussain et al., 2011). amongst different kind of pas, the diamine putrescine (put), triamine spermidine (spd), and tetramine spermine (spm) are the most common pas in plant cells, while others are of more limited occurrence (galston and sawhney, 1990; valero et al., 2002). distribution of these biogenic amines differs between species with put and spd being particularly abundant and spm the least abundant in plant cells. these amines are important for cell viability and their intracellular levels are tightly regulated, making it difficult to characterize individual effects of put, spd and spm on plant growth and developmental processes (mattoo et al., 2010). it has been reported that exogenous pa application leads to an inhibition of ethylene emission rate in the climacteric fruits (valero et al., 2002) and delays ripening and fruit abscission (paksasorn et al., 1995). on the other hand, pas are precursors of many important secondary metabolites and changes in pa biosynthesis appear to have a reciproca1 effect on ethylene biosynthesis (walden et al., 1997). accumulation of put in tissues seems to be a general response of plants to chilling temperatures (faust and wang, 1992). accordingly, we hypothesized that exogenous application of pas may have effects on the postharvest physiology of fruits, especially on chilling tolerance. an experiment was arranged to study the effects of exogenous pa application post-storage quality and physiological responses of tomato fruits treated with polyamines j. javanmardi(1), m. rahemi, m. nasirzadeh department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. key words: chilling injury, electrolyte leakage, fruit quality, vitamin c. abstract: two greenhouse f1 tomato cultivars, m19 and m79, were grown hydroponically and the mature green fruits were harvested and subjected to eight polyamine (pa) treatments including 1 and 2 mm putrescine (put), spermidine (spd) and their combination before being placed at 3°c for 15 and 25 days. electrolyte leakage, weight loss, fruit firmness, decay percentage, chilling injury index, titratable acidity, total soluble solid content and ascorbic acid content were then measured after keeping at 20°c for 3 days and compared to control. the put:spd (2:2 mm) treatment decreased electrolyte leakage (over 50%), chilling injury index and fruit decay percentage. combinations of pas caused greater total soluble solids and greater effect on decreasing weight loss during storage when compared to their sole pa application. pas caused a net increase in fruit firmness during post-harvest life. titratable acidity increased with increasing duration of low temperature storage for all treatments. ascorbic acid in fruits stored at low temperature for 25 days was greater than those stored for 15 days. the effects of exogenous pas on reducing chilling-related disorders decreased with time. correlations among weight loss, electrolyte leakage, chilling injury, decay percentage and fruit firmness during low temperature storage were positive and significant, but they were non-significant or significantly negative when compared against ascorbic acid, titratable acidity and tss. adv. hort. sci., 2013 27(4): 173-181 (1) corresponding author: javanm@shirazu.ac.ir received for publication 4 january 2014 accepted for publication 7 march 2014 174 on the level of chilling injury, ripening, shelf life and some quality factors of tomato fruit (as a model plant) after a period of low temperature storage. another goal of the study was to determine which polyamine, concentration and/or their combination is more effective. two greenhouse tomato cultivars were selected to evaluate whether pas play the same role in different cultivars or not. 2. materials and methods plant material and polyamine treatments f1 hybrid seeds of tomato (solanum lycopersicom l.) cultivars m19 and m79 (tropica seeds, indosem ltd. india) were sown in 30 × 30 × 60 mm/cell plastic plug trays filled with peat and perlite (1:1 v:v). seedlings were grown for five weeks in a polycarbonate greenhouse (25/21°c and 60-70% relative humidity). plugs were then transplanted into 12-l pots filled with a peat:perlite (60:40 v/v) mixture. pots were kept in the same greenhouse with an average 60% relative humidity and 25±5°c temperature until the end of the experiment. cultural practices consisting of standard recommendations for growing tomato seedlings and plants in a hydroponic system were according to papadopoulos (1991). fruits for study were harvested at mature green stage based on the “color classification requirement in united states standards for grades of fresh tomatoes” chart, published by the usda. eight polyamine treatments consisted of immersing same-sized, selected mature green fruits in 1 and 2 mm putrescine (put), spermidine (spd) and their combination for 4 min as indicated by mirdehghan et al. (2007 a). immersion in distilled water for the same period was taken as control (table 3). fruits were then held at 3°c in a temperature-controlled chamber in darkness with relative humidity of 90%. after 15 and 25 days, five fruits from each treatment replicate were sampled and stored at 20°c for 3 days. electrolyte leakage, weight loss, fruit firmness, decay percentage, chilling injury index, titratable acidity, total soluble solid content (tss) and ascorbic acid content of fruits were measured as described below. electrolyte leakage electrolyte leakage was used to assess membrane permeability. the procedure was based on lutts et al. (1996) with slight modification. briefly, five tomato fruit pericarp discs (10 mm diameter) per replicate from randomly chosen fruits were taken and placed in test tubes containing 10 ml of distilled water followed by three washes with distilled water to remove surface contamination. samples were incubated at room temperature on a shaker for 24 h. electrical conductivity (ec) of the bathing solution (ec1) was read after incubation. the samples were then placed in a boiling water bath for 20 min and the second reading (ec 2) was determined after cooling the solution to room temperature. the electrolyte leakage was calculated using ec1/ec2 and expressed as a percentage. all leakage data were expressed as a percentage of the total electrolyte readings. weight loss weight loss during postharvest storage of individual replicates was determined by subtracting sample weights on sampling dates (day 15 or 25 of low temperature storage) from their initial weight on day 0 and presented as percent of weight loss compared to initial weight. fruit firmness fruit firmness was determined according to ben-yehoshua et al. (1983). a compression tester using a 1 kg weight centered over a locule on the equatorial region of each tomato fruit was used. full deformation was measured 30 s after exerting the force on the fruit, then the weight was removed and residual deformation was measured 15 s later. lower readings denoted firmer fruit. five fruits were measured for each treatment replicate. firmness was expressed as mm deformation. chilling injury index chilling injury (ci) was evaluated according to ding et al. (2002) at 20°c for 3 days, following 15and 25day low temperature storage period. briefly, tomato fruit surface pitting was considered as ci symptom. the severity of ci symptoms was assessed visually according to a four-stage scale: 0= no pitting; 1= a few scattered pits; 2= pitting covering up to 5% of the fruit surface; 3= extensive pitting covering 5-25% of the fruit surface, and 4= extensive pitting covering more than 25% of the fruit surface. the average extent of ci damage was expressed as a ci index and calculated using the following formula: ci index = ∑(injury classification level x number of fruit at that level) total number of friut at the treatment decay percentage extent of decay was assessed according to gonzálezaguilar et al. (2000), based on the area of decay and the surface area with microorganisms growing on it. decay was rated for each replicate of treatments at the end of the 15 and 25-day cold storage periods after an additional 3 days at 20°c. the values were expressed as decay percentage. total soluble solids, titratable acidity and ascorbic acid content total soluble solids concentrations (tss) of fruit juice were determined using a digital refractometer (pal-3, atago co., ltd. tokyo, japan) at 20°c and presented as °brix. fruit juice titratable acidity (ta) was determined by titration of 1 ml juice in 25 ml distilled water with 0.1n naoh until the ph reached 8.1, according to el ghaouth et al. (1992). results were expressed as gram of citric acid equivalent per 100 g fresh weight (g cae/100 g fw). fruit ascorbic acid content was determined according to the method described by the aoac (1984). statistical analysis the experiment was arranged in a completely random175 ized design. each polyamine treatment consisted of three replicates; each replicate consisted of 10 plants. data were analyzed separately for tomato cultivars and days of low temperature incubation using one-way analysis of variance. means for each low temperature storage duration and cultivar were compared separately using the least significant differences (lsd) test at p≤0.01. all data analyses including correlation analysis were performed using spss21 (spss inc., chicago, il) computer software for windows. 3. result and discussions one-way analysis of variance showed significant differences for all measured characteristics in both cultivars except for ascorbic acid content in cv. m79 after 15 days of low temperature storage (table 1 a, b, and table 2 a, b). electrolyte leakage application of pas decreased electrolyte leakage of low-temperature incubated tomato fruits. the highest table 1 a one way analysis of variance for characteristics of polyamine-treated tomato cultivar m19 after 15 and 25 days storage at 3°c sov df mean squares electrolyte leakage weight loss fruit firmness chilling injury 15 25 15 25 15 25 15 25 polyamines 8 88.692** 163.37** 5.193** 3.055** 0.755** 0.87** 114.593** 65.167** error 18 3.751 3.731 0.161 0.283 0.051 0.047 3.625 16.759 total 26 29.887 52.848 1.709 1.136 0.268 0.3 37.767 31.654 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. table 1 b one-way analysis of variance for characteristics of polyamine-treated tomato cultivar m19 after 15 and 25 days storage at 3°c sov df mean squares decay percentage tss titratable acidity ascorbic acid 15 25 15 25 15 25 15 25 polyamines 8 370.37 ** 902.759 ** 0.832 ** 0.592 ** 0.028 ** 0.033 ** 1.697 ** 15.319 ** error 18 37.037 65.852 0.167 0.079 0.002 0.003 0.185 0.999 total 26 139.601 323.362 0.369 0.236 0.01 0.012 0.650 5.406 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. table 2 a one-way analysis of variance for characteristics of polyamine-treated tomato cultivar m79 after 15 and 25 days storage at 3°c sov df mean squares electrolyte leakage weight loss fruit firmness chilling injury 15 25 15 25 15 25 15 25 polyamines 8 48.792** 101.07** 4.593** 4.708** 0.564** 0.749** 80.624** 135.995** error 18 0.948 0.673 0.034 0.087 0.017 0.046 5.701 10.417 total 26 15.669 31.564 1.437 1.509 0.185 0.262 28.754 49.056 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. table 2 b one-way analysis of variance for characteristics of polyamine-treated tomato cultivar m79 after 15 and 25 days storage at 3°c sov df mean squares decay percentage tss titratable acidity ascorbic acid 15 25 15 25 15 25 15 25 polyamines 8 193.667** 424.833** 0.431** 0.951** 0.021** 0.029** 1.043ns 3.987** error 18 13.852 29.667 0.026 0.029 0.003 0.002 0.989 0.822 total 26 69.179 151.256 0.150 0.313 0.009 0.01 1.005 1.795 ns, *, ** non-significant and significant at 0.05 and 0.01, respectively. 176 electrolyte leakage was observed in control treatments as almost 100% greater than those treated with the highest pa concentrations in combined treatments on both cultivars (table 3). pretreatment of cucumber plants with pas diminished the increased electrolyte leakage caused by chilling in the leaves (gill and tuteja, 2010). the exogenous application of polyamines on pomegranate (punica granatum l.) protected the membrane lipid from being converted from liquid crystalline to a solid-gel state (induced by chilling) through preventing lipid peroxidation (mirdehghan et al., 2007 b). previously, put has been reported to act as protective toward cold stress in tomato plants, since reduced cold-induced electrolyte leakage in leaves due to its application was observed (kim et al., 2002). in addition, pa application induced cold acclimation through maintenance of membrane fluidity at low temperatures and reduced electrolyte leakage, skin browning, and thus the severity of ci symptoms (mirdehghan et al., 2007 a). in general, the effect of spd on decreasing electrolyte leakage was significantly greater than put but the differences were not significant when they were applied in combinations (table 3). in agreement with our results, gill and tuteja (2010) found different patterns of put and spd action in different cucumber cultivars. accordingly, it seems the mode of action of pas may differ within species. a large amount of evidence showed that exogenous application of pas plays a role in stabilizing plant cell membranes and protecting them from damage under stress conditions (liu et al., 2007; he et al., 2008; gill and tuteja, 2010). pas in their free forms have been described as anti-senescence agents (valero et al., 2002) due to their capacity to preserve membrane stability, which is crucial in plant adaptation to temperature stresses (oufir et al., 2008). their attachment to membranes by way of phospholipids results in altered patterns of solute permeation through those membranes and decreased fluidity of membrane components (galston and sawhney, 1990). they are involved in the regulation of many basic cellular processes, including cellular cation-anion balance and membrane stability (gill and tuteja, 2010). in the present experiment, tomato cv. m19 showed greater electrolyte leakage for all pa and low temperature storage treatments compared to cv. m79 (table 3). possibly the pas pattern of action in the studied tomato cultivars was at different rates, as previously found for cucumber cultivars (gill and tuteja, 2010). increasing the duration of low temperature storage from 15 to 25 days increased electrolyte leakage in both cultivars (table 3). this could be an indicator that the effects of exogenous pas on lowering electrolyte leakage decrease with time. weight loss it has been reported that tomatoes at room temperature showed greater weight loss than those stored in cold storage (javanmardi and kubota, 2006). at least 50% greater weight loss was found in control treatments compared to those with pa applied in both cultivars and low temperature durations (table 3). transpiration has been considered the main cause of weight loss during tomato storage (javanmardi and kubota, 2006). reduction in weight loss and respiration rate due to put and spd application in mango has been reported (malik and singh, 2005). the differences between combined pa treatments were not significant in the studied cultivars for the two low temperature durations, however they showed less weight loss when pas were applied singularly (table 3). it has been reported that chilled fruits had a greater weight loss rate than non-chilled fruits after transfer to non-chilling conditions. this is due to the development of microscopic cracks in peel tissue (cohen et al., 1994), cellular breakdown and loss of membrane integrity which have an important role in water exchange through the rind (gonzález-aguilar et al., 2000). storage conditions or treatments that reduce fruit water loss have been shown table 3 effect of polyamine application and duration (15 and 25 days) of low-temperature (3°c) storage on electrolyte leakage and fruit weight loss in tomato cultivars m19 and m79 treatment electrolyte leakage weight loss m 19 m 79 m 19 m 79 15 25 15 25 15 25 15 25 control 30.06 a 42.43 a 24.40 a 35.64 a 5.48 a 5.22 a 5.27 a 5.87 a put 1 mm 19.55 b 24.96 b 15.43 b 20.13 b 3.13 b 3.27 b 2.52 b 3.22 b put 2 mm 18.50 b 22.77 bc 14.20 b 19.78 b 2.54 bc 2.90 bcd 2.40 b 2.86 bc spd 1 mm 16.43 bc 22.98 bc 14.20 b 19.47 bc 2.40 cd 3.17 bc 2.14 bc 2.96 b spd 2 mm 14.93 bc 21.83 bc 13.91 b 17.73 bc 1.92 cde 2.48 bcd 1.90 cd 2.40 cd put 1 mm + spd 1 mm 15.56 bc 21.31 bc 13.13 bc 18.81 bc 1.69 de 2.23 cd 1.59 de 2.20 de put 1 mm + spd 2 mm 14.98 bc 21.99 bc 12.97 bc 17.74 bc 1.61 de 2.13 d 1.61 de 2.00 de put 2 mm + spd 1 mm 11.45 c 18.19 c 10.32 c 17.31 c 1.19 e 2.17 cd 1.19 e 1.93 de put 2 mm + spd 2 mm 13.01 c 18.05 c 11.41 c 17.30 c 1.40 e 1.97 d 1.27 e 1.79 e lsd value 0.01 4.55 4.54 2.29 1.92 0.94 1.25 0.43 0.69 means in columns followed by the same letter are not significantly different, p ≤ 0.05, lsd test. means for each column were compared separately. 177 to alleviate ci (wang, 1993). in our experiment, pa application resulted in less membrane permeability (less electrolyte leakage) and therefore less water loss than control fruits. also storage duration affected weight loss: the longer fruits remained in low-temperature storage, the greater their weight loss. fruit firmness fruit firmness was affected by pa application (table 4). all pa-treated fruits (except for put 1 mm after 15 days of storage in m19) showed firmer fruit (less compression) than control fruits (table 4). the differences between combined pa treatments in each low temperature storage duration were not significant. however, when compared to the control they showed at least 43 and 69% greater firmness in m19 tomato, and 80 and 45% in m79 tomato for 15 and 25 days storage, respectively. it has been shown that fruits and vegetables infiltrated with pas had a net increase in firmness during post-harvest life. this effect of pas on fruit firmness has been attributed to the cross linking to the coogroup of the pectic substance and changes in polygalacturonic acids in the cell wall (valero et al., 2002). retarded fruit softening due to put and spd application in mango has been reported (malik and singh, 2005). although it is believed that the overall softening process results from a number of changes in turgor pressure, cell wall and membrane composition and degradation, but cell wall modifications have been implicated to be the major determinant of fruit softening (smith et al., 2002). put and spd have anti-senescence properties (saftner and baldi, 1990), and are able to retard the maturation process (including softening) in a wide range of climacteric and non-climacteric fruits (valero et al., 2002). the ethylene production in tomato fruits, enhances softening, but its effect may decrease due to increased polyamine level (tiecher et al., 2013). increased duration of low temperature storage decreased fruit firmness for all treatments. fruit firmness of control plants m19 and m79 showed 52 and 56% decrease, respectively, when 15 days of low temperature storage values were compared width 25 days. the values obtained from 2.2 mm, put:spd tratment were 108 and 148% for m19 and m79, respectively (table 4). pas with higher number of available cations have greater effect on fruit firmness, as spm+4>spd+3>put+2 (valero et al., 2002). according to our results, the pa concentration would also affect the fruit firmness. chilling injury index chilling injury index drastically increased with time of low temperature storage in both cultivars (table 4). the increased percentages of 25 days compared to 15 days of low temperature storage for the control treatment were 64 and 102% in m19 and m79, respectively. the combined pa treatments resulted in greater ci index (but less than control) after 25 days of low temperature storage than 15 days, especially when 2:2 mm put:spd was used (table 4). the effects of pa on 15 days low temperature stored tomatoes showed significant decrease in ci index, but its impact for a longer period (25 days) was not significant, except for 1 mm put (table 4). the lowest ci index after 15 days of low temperature storage was found in put:spm (2:2 mm) treated tomatoes in both cultivars (table 4). it is possible that the impact of pas on lowering ci index in tomato are time as well as species dependent. pre-storage application of pas improved shelf-life of pomegranate (punica granatum l.) stored at chilling temperature by increasing endogenous polyamine levels (mirdehghan et al., 2007 a). although increased endogenous put in tomato due to low temperature storage is considered to act as protective toward cold stress, the reported mechanism is unclear (gonzalez-aguilar et al., 1998). the involvement of polyamines in reducing chilling injury has been related to reducing oxidative damage via increases of antioxidant or reducing the activity of oxidative enzyme (oufir et al., 2008). table 4 effect of polyamine application and duration (15 and 25 days) of low-temperature (3°c) storage on fruit firmness and chilling injury index in tomato cultivars m19 and m79 treatment fruit firmness (mm deformation) chilling injury index (%) m 19 m 79 m 19 m 79 15 25 15 25 15 25 15 25 control 2.41 a 3.67 a 2.19 a 3.43 a 19.17 a 31.50 a 16.42 a 33.33 a put 1 mm 2.04 ab 2.95 b 1.69 b 2.85 b 16.67 ab 25.67 ab 13.33 ab 20.00 b put 2 mm 1.82 b 2.48 c 1.45 c 2.52 bc 15.50 b 24.17 abc 9.17 bc 16.67 bcd spd 1 mm 1.88 b 2.49 c 1.54 bc 2.79 b 9.17 c 21.67 bc 10.00 bc 17.50 bcd spd 2 mm 1.68bc 2.24 cd 1.40 c 2.37 cd 5.83 c 19.18 bc 7.67 cd 14.17 bcd put 1 mm + spd 1 mm 1.68bc 2.16 cd 1.21 d 2.35 cd 6.67 c 22.50 bc 4.17 def 15.00 bcd put 1 mm + spd 2 mm 1.16 c 1.94 d 1.03 d 2.04 de 1.75 d 15.83 c 2.50 ef 11.67 cd put 2 mm + spd 1 mm 1.18c 1.99 d 1.04 d 2.19 cde 0.75 d 21.33 bc 6.67 cde 14.17 bcd put 2 mm + spd 2 mm 0.92 c 1.92 d 0.78 d 1.94 e 0.42 d 17.67 bc 0.0f 10.83 d lsd value 0.01 0.53 0.49 0.31 0.37 4.47 9.62 5.61 7.85 means in columns followed by the same letter are not significantly different, p ≤ 0.05, lsd test. means for each column were compared separately 178 decay percentage decay incidence in all control treatments was significantly greater than in pa-treated fruits (table 5). prolonging low temperature storage from 15 to 25 days resulted in greater decay incidence. although the differences between pa-treated fruits were not significant, the combined pa treatments could be considered more effective since 0% decay decreases possible further contamination and decay. as a susceptible crop to chilling injury, tomato shows increased susceptibility to decay when stored at low temperatures after harvest (ding et al., 2002). in the present study, treatment with put:spd (2:2 mm) was very effective in alleviating chilling injury and decreasing the incidence of decay in tomato fruits (tables 4, 5). this result suggest that pa application enhances the natural resistance of the fruits to chilling injury and decaying agents. taken together, results obtained in this study indicate that the higher levels of pas reduce ci and decay of tomato fruit. the reduction in ci symptoms by pas has been related to their capacity to preserve membrane integrity, both by lowering the membrane phase-transition temperature fluidity and by retarding lipid peroxidation, resulting in increased cell viability (gonzález-aguilar et al., 2000). total soluble solids and titratable acidity in all cases the highest values for tss were observed in put:spd (2:2 mm), however the differences among other pa combinations were also not significant (table 5). the differences in tss between 15 and 25 days of low temperature storage were not significant in the two cultivars (data not shown). the earlier experiment showed no significant changes in tss between room temperature and low temperature stored tomatoes (javanmardi and kubota, 2006). it has been reported that tss remains unchanged after chilling and reconditioning (luengwilai and beckles, 2010), however it is possible that individual sugars and the sugar-acid balance may be adversely affected (beckles, 2012). harvesting riper fruit (i.e. those with already welldeveloped sugar profiles) would reduce the harm caused by chilling injury due to lower temperature (beckles, 2012). titratable acidity in combined pa treatments for fruits stored 15 days at low temperature were significantly greater than other treatments in both cultivars. treated fruits with put:spd (2:2 mm) showed the highest titratable acidity after 25 days of storage (table 6). titratable acidity increased with increasing duration of low temperature storage for all treatments (table 6). it is reported that increased endogenous pas, spermine and spermidine, through transgenic manipulation of tomato showed similar levels of juice tss, ph and titratable acidity in transgenic, azygous, and wild-type fruits (mehta et al., 2002). application of put and spd on apricot did not affect tss and ta (koushesh saba et al., 2012). ascorbic acid content all treated fruits showed greater ascorbic acid in fruits stored for 25 days than 15-day low temperature storage group (table 6). the pattern of changing ascorbic acid level in tomato fruit varies with the physiological ripening stages as it increases slowly reaching a maximum and then declines slowly coinciding with the initiation of ripening, as indicated by color change, and an increase in the activity of ascorbate oxidase (yahia et al., 2001). in this experiment the mature green fruits treated and stored at low temperature showed the same increasing pattern until 25 days of storage. at that time the color had not started to change. most likely pa application does not change the ascorbic acid pathway at least until ripening symptoms (color change) appear. it is reported that during the ripening process, the levels of spd and spm decline table 5 effect of polyamine application and duration (15 and 25 days) of low-temperature (3°c) storage on fruit decay percentage and total soluble solid content in tomato cultivars m19 and m79 treatment decay percentage total soluble solid content (°brix) m 19 m 79 m 19 m 79 15 25 15 25 15 25 15 25 control 33.33 a 60.00 a 25.00 a 40.00 a 4.01 b 4.28 b 4.80 b 4.13 c put 1 mm 13.33 b 16.33 b 9.67 b 9.67 b 4.50 b 4.85 ab 5.18 ab 4.73 bc put 2 mm 6.67 b 11.37 b 3.33 b 4.00 b 5.43 ab 5.13 ab 5.43 ab 5.20 b spd 1 mm 3.33 b 12.33 b 3.33 b 6.33 b 5.35 ab 5.08 ab 5.25 ab 5.00 bc spd 2 mm 0.00 b 10.00 b 2.33 b 5.00 b 5.55 a 5.25 ab 5.33 ab 5.55 ab put 1 mm + spd 1 mm 0.00 b 6.00 b 0.00 b 3.33 b 5.35 ab 5.30 a 5.35 ab 5.50 ab put 1 mm + spd 2 mm 0.00 b 5.00 b 0.00 b 1.67 b 5.60 a 5.50 a 5.73 a 5.63 ab put 2 mm + spd 1 mm 0.00 b 6.67 b 0.00 b 6.67 b 5.28 ab 5.48 a 5.68 a 5.60 ab put 2 mm + spd 2 mm 0.00 b 5.67 b 0.00 b 3.33 b 5.75 a 5.68 a 5.88 a 5.85 a lsd value 0.01 14.3 19.07 8.74 12.78 0.96 0.66 0.38 0.40 means in columns followed by the same letter are not significantly different, p≤ 0.05, lsd test. means for each column were compared separately. 179 in fruits (mattoo and handa, 2008). the ripening process has been shown to be delayed in tomato fruits by infusing them with put (saftner and baldi, 1990). polyamines and ethylene are known to have opposite effects in relation to fruit ripening and senescence (saftner and baldi, 1990). free polyamines inhibit ethylene production in a variety of tissues (suttle, 1981) and the elevated level of free polyamines may be responsible for the reduction in both ethylene production and ripening processes of tomato fruits (saftner and baldi, 1990). elevated levels of polyamines help maintain cellular vitality and longer life of ripening tomato (mattoo et al., 2010). correlations among fruit characteristics the correlations among weight loss, electrolyte leakage, chilling injury, decay percentage and fruit firmness for 15 and 25 days of low temperature storage in both cultivars were positive and significant, but they were either non-significant or significantly negative when analyzed against ascorbic acid, titratable acidity and tss (tables 7-10). the greatest impact of electrolyte leakage was found on weight loss and decay percentage for the studied table 6 effect of polyamine application and duration (15 and 25 days) of low-temperature (3°c) storage on fruit juice acidity and ascorbic acid content in tomato cultivars m19 and m79 treatment titratable acidity (g cae/100 g fw) ascorbic acid (mg/100ml) m 19 m 79 m 19 m 79 15 25 15 25 15 25 15 25 control 0.34 b 0.43 c 0.37 bc 0.47 b 9.09 b 11.01 c 11.88 a 12.07 c put 1 mm 0.35 b 0.41 c 0.31 c 0.37 c 9.74 b 11.97 bc 10.53 a 12.12 c put 2 mm 0.41 b 0.44 c 0.31 c 0.42 c 9.81 b 11.02 c 11.35 a 13.19 bc spd 1 mm 0.37 b 0.43 c 0.36 bc 0.39 c 9.60 b 11.97 bc 11.25 a 12.25 c spd 2 mm 0.39 b 0.50 b 0.40 bc 0.44 bc 11.01 a 12.44 bc 10.63 a 13.07 bc put 1 mm + spd 1 mm 0.52 a 0.56 b 0.48 ab 0.53 b 10.40 a 13.47 b 11.39 a 15.00 a put 1 mm + spd 2 mm 0.57 a 0.63 a 0.43 abc 0.69 a 11.32 a 17.91 a 12.40 a 15.60 a put 2 mm + spd 1 mm 0.54 a 0.60 a 0.46 ab 0.54 b 11.05 a 12.06 bc 11.88 a 13.62 abc put 2 mm + spd 2 mm 0.58 a 0.70 a 0.56 a 0.65 a 11.80 a 13.60 b 12.15 a 14.03 ab lsd value 0.01 0.11 0.13 0.13 0.10 1.01 2.35 2.29 2.13 means in columns followed by the same letter are not significantly different, p ≤ 0.05, lsd test. means for each column were compared separately. table 7 correlation analysis between fruit characteristics of tomato cv. m19 treated with different polyamines after 15 days storage at 3°c weight loss electrolyte leakage chilling injury decay percentage fruit firmness ascorbic acid titratable acidity electrolyte leakage 0.927 ** chilling injury 0.745 ** 0.623 ** decay percentage 0.769 ** 0.803 ** 0.694 ** firmness 0.753 ** 0.761 ** 0.741 ** 0.699 ** ascorbic acid -0.332 ns -0.367 ns -0.258 ns -0.340 ns -0.380 ns titratable acidity -0.679 ** -0.629 ** -0.680 ** -0.555 ** -0.726 ** 0.377 ns tss -0.701 ** -0.719 ** -0.642 ** -0.701 ** -0.618 ** 0.083 ns 0.421 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. table 8 correlation analysis between fruit characteristics of tomato cv. m79 treated with different polyamines after 15 days storage at 3°c weight loss electrolyte leakage chilling injury decay percentage fruit firmness ascorbic acid titratable acidity electrolyte leakage 0.950 ** chilling injury 0.716 ** 0.737 ** decay percentage 0.894 ** 0.909 ** 0.735 ** firmness 0.861 ** 0.825 ** 0.806 ** 0.815 ** ascorbic acid -0.018 ns 0.023 ns -0.162 ns 0.003 ns -0.166 ns titratable acidity -0.353 ns -0.378 ns -0.664 ** -0.314 ns -0.617 ** 0.291 ns tss -0.642 ** -0.633 ** -0.632 ** -0.624 ** -0.806 ** 0.415 * 0.548 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. 180 cultivars and low temperature durations. 4. conclusions the results of this experiment indicate that exogenous put and spd application on tomato fruit could maintain or even improve fruit quality during low temperature storage. the combined application of put with spd (2:2 mm) could be recommended for low temperature and long duration storage of tomato fruits. references aoac, 1984 official methods of analysis. association of official agricultural chemists, 14th ed., washington, dc, usa. aghdam m.s., bodbodak s., 2013 physiological and biochemical mechanisms regulating chilling tolerance in fruits and vegetables under postharvest salicylates and jasmonates treatments. scientia horticulturae, 156: 73-85. baninasab b., 2009 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characteristics of tomato cv. m19 treated with different polyamines after 25 days storage at 3°c weight loss electrolyte leakage chilling injury decay percentage fruit firmness ascorbic acid titratable acidity electrolyte leakage 0.954 ** chilling injury 0.625 ** 0.714 ** decay percentage 0.912 ** 0.859 ** 0.536 ** firmness 0.849 ** 0.845 ** 0.721 ** 0.731 ** ascorbic acid -0.346 ns -0.287 ns -0.539 ** -0.400 * -0.576 ** titratable acidity -0.485 * -0.532 ** -0.600 ** -0.464 * -0.664 ** 0.545 ** tss -0.837 ** -0.806 ** -0.624 ** -0.710 ** -0.821 ** 0.428 * 0.583 ** ns, *, ** non-significant, significant at 0.05 and 0.01, respectively. table 10 correlation analysis between fruit characteristics of tomato cv. m79 treated with different polyamines after 25 days storage at 3°c weight loss electrolyte leakage chilling injury decay percentage fruit firmness ascorbic acid titratable acidity electrolyte leakage 0.946 ** chilling injury 0.884 ** 0.884 ** decay percentage 0.860 ** 0.912 ** 0.764 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113: 1009. wang c.y., 1993 approaches to reduce chilling injury of fruits and vegetables. horticultural reviews, 15: 63-95. yahia e.m., contreras-padilla m., gonzálezaguilar g., 2001 ascorbic acid content in relation to ascorbic acid oxidase activity and polyamine content in tomato and bell pepper fruits during development, maturation and senescence. lwt. food science and technology, 34: 452-457. impaginato 75 adv. hort. sci., 2024 38(1): 75­81 doi: 10.36253/ahsc­14920 comparison quality parameters of saf­ fron (crocus sativus l.) produced in herat, afghanistan and torbat heydarieh, iran r. nazarian 1 (*), m. nasiri mahalati 2, h. sahabi 3, h. feizi 3 1 agronomy department, faculty of agriculture, herat university, afghanistan. 2 department of agrotechnology, faculty of agriculture, ferdowsi university of mashhad, iran. 3 department of plant production, saffron institute, university of torbat heydarieh, iran. key words: crocin, picrocrocin, safranal, secondary metabolites, spectrophoto­ metric analysis. abstract: saffron crocus sativus l. (iridaceae) is one of the most valuable and expensive medicinal plants in the world. in order to compare the quality charac­ teristics of saffron in afghanistan and iran, samples of dried c. sativus from dif­ ferent saffron­producing regions of herat province (afghanistan) and torbat heydarieh county (iran) were collected in the year 2021. the experiment was analyzed in glm format and nested method with three replications. the samples of saffron produced from seven different districts of herat province in afghanistan, including injil, karukh, guzara, pashtun zarghun, zendeh jan, ghoryan, obeh and nine saffron­producing villages (fakhrabad, kadkan, nasar, ghaleno, feizabad, khorgh, abrood, benhang, shadmehr) of torbat heydarieh county in iran were examined. the results showed that the effect of country and region (districts and villages) on the quality traits of saffron (crocin, picrocrocin and safranal) was significant. moreover, the comparison of the mean values indi­ cated the presence of significant differences in the qualitative characteristics of saffron among the different investigated villages of torbat heydarieh. the saf­ fron produced in afghanistan had the highest amount of crocin (on average 279.1 ɷ 440nm) and picrocrocin (on average 101 ϕ 257 nm), while the highest value of safranal (on average 34.2 θ 330 nm) was observed in saffron samples produced in iran. the highest amount of the above quality traits belonged to the saffron of zendeh jan herat region 5 (af5, crocin: 303 ɷ 440 nm, picrocrocin: 106 ϕ 257 nm, safranal: 33 θ 330 nm), while the five torbat heydarieh villages had the lowest quality of saffron (ir5, crocin: 164 ɷ 440nm, picrocrocin: 71 ϕ 257 nm, safranal: 34 θ 330 nm). in the comprehensive analysis of saffron­producing regions in afghanistan and iran, in terms of the qualitative attributes (crocin, picrocrocin, and safranal) it was evident that regions 1 to 6 in herat, afghanistan (af1, af2, af3, af4, af5, af6) and the abrood village of torbat heidarieh, iran (ir7) formed a distinct cluster, demonstrating superior quality compared to other regions. furthermore, with the exception of the feizabad village of torbat heydarieh (ir5), all examined samples surpassed the saffron iso international standard and were categorized as first­grade quality. the exceptional quality of herat saffron from afghanistan is likely attributable to the unique geographical features, virgin lands, and specific climatic conditions across diverse cultivation areas. (*) corresponding author: ra.nazarian@alumni.um.ac.ir citation: nazarian r., mahalati m.n., sahabi h.,feizi h., 2024 ­ comparison quality parameters of saf‐ fron (crocus sativus l.) produced in herat, afghanistan and torbat heydarieh, iran. ­ adv. hort. sci., 38(1): 75­81. copyright: © 2024 nazarian r., mahalati m.n., sahabi h., feizi h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. authors contributions: design of experiment and data analysis: r. nazarian and m. nasiri mahalati. laboratory measurements: h. sahabi and h. feizi. collecting of samples: r. nazarian, h. sahabi, h. feizi. writing and revising the manuscript: r. nazarian, m. nasirimahalati, h. sahabi, h. feizi. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 july 2023 accepted for publication 29 november 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14920 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 75­81 76 1. introduction saffron (crocus sativus l.) is one of the world’s most valuable and expensive medicinal and industri­ al plants. originally confined to limited geographic habitats, its significance has led to its widespread cultivation in various parts of the world (mathew, 1999). in addition to iran, saffron is also cultivated in other countries such as afghanistan, india, spain, italy, morocco, turkey and greece. in 2019, afghanistan, as the fourth producer in the world, exported about six tons of saffron (asyb, 2021), while iran was the world largest producer with 281 tons of saffron in 2021 (sci, 2021). due to the high economic value of saffron, its cultivated area is expanding rapidly in the neighboring countries of iran, such as afghanistan and pakistan (fao, 2018). recently, afghan farmers have developed saffron cultivation in herat province. also, the afghan gov­ ernment is encouraging farmers to grow saffron instead of poppy (papaver samniferum l.) to improve their livelihoods. herat province is geo­ graphically located in the neighborhood of khorasan province in iran. in addition khorasan is the largest saffron production center in the world (nazarian et al., 2018). khorasan­razavi and south khorasan provinces in iran are considered the main saffron production pole in the world (mollafilabi and khorramdel, 2016). herat province of afghanistan is the main center of saffron production in the coun­ try, so farmers are engaged in saffron production in almost all the cities of this province (nazarian et al., 2018). the production method of saffron is slightly different in iran and afghanistan. compliance with the technical principles of cultivation, the use of inputs, and the method of drying saffron are not the same in the two aforementioned countries. so that these factors may have been able to affect the quantity and quality of saffron production. the stigma of three branches of saffron, which has a red color, contains three main compounds: crocin, picrocrocin and safranal (tajik et al., 2012). saffron is widely used in the food and pharmaceuti­ cal industries, and due to its therapeutic effects on cancer and depression, it is receiving increasing attention from consumers and processing industries (lechtenberg et al., 2008). the medicinal and nutri­ tional importance of saffron is related to the exis­ tence of a set of secondary metabolites in the stig­ ma. due to the presence of these metabolites and high amounts of crocin, picrocrocin, and safranal, saffron has the ability to provide color, a special taste and a pleasant aroma in food products. limited spices have a similar ability to simultane­ ously provide color, aroma and flavor in the food product to which they are added (valle garcia­ rodriguez et al., 2014). considering the importance of secondary metabolites such as crocin, picro­ crocin, and safranal in determining the nutritional and medicinal value of saffron; the measurement of these three parameters can be used as a suitable indicator of quality (zalacain et al., 2005). to deter­ mine the quality of saffron in iran, in addition to using the iso international standard (iso/ts 3632­1 and 3632­2), the national standard (no. 1/259 and 2/259) is also used. saffron is widely used in the food and pharmaceutical industries. saffron quality is usually determined by three important secondary metabolites in the stigma i.e. crocin, picrocrocin and safranal, known as effective substances. it has been confirmed that temperature is the main factor con­ trolling the flowering behavior of saffron (koocheki et al., 2009). in a prior study conducted by feizi and moradi (2019), it was revealed that the saffron stig­ ma yield exhibited a positive correlation with regional rainfall, organic matter, nitrogen, phospho­ rus, and soil sand percentage. conversely, there was a negative correlation with average temperature and soil clay percentage. the authors also highlight­ ed that altitude above sea level serves as a signifi­ cant spatial parameter influencing the production of secondary metabolites. additionally, their findings indicated positive correlations between altitude and picrocrocin, safranal and crocin. similarly, kaveh and salari (2018) reported a positive correlation between crocin and both picrocrocin and safranal. conversely, both crocin and safranal exhibited a negative correlation with altitude. moreover, it has been stated that soil ph within the neutral to slight­ ly alkaline range is positively correlated with saffron quality (gresta et al., 2008). in general, the crop quality is influenced by various factors, exhibiting variation across different regions, such as agricul­ tural practices, nutrition, soil type, water manage­ ment and air conditions, and ultimately the method employed for saffron drying. on this basis, the pur­ pose of this research was to compare the quality characterist ics of saffron produced in herat province of afghanistan and torbat heydarieh coun­ ty of iran. nazarian et al. ‐ saffron quality comparison 77 2. materials and methods in order to compare the qualitative characteristics of saffron in afghanistan and iran, samples of dried saffron from seven different districts of herat province (injil, karukh, guzara, pashtun zarghun, zendeh jan, ghoryan, obeh) of afghanistan and nine saffron­producing villages (fakhrabad, kadkan, nasar, ghaleno, feizabad, khorgh, abrood, benhang, shadmehr) of torbat heydarieh county of iran (fig. 1) were collected in the year 2021. the experimental data underwent analysis through the general linear model (glm) procedure. within the glm, seven sam­ ples from afghanistan (af) and nine samples from iran (ir) were defined, each with three replications, and the saffron samples were nested over countries. unlike a factorial model, the nested model differs in that the levels of the second factor (saffron samples) vary within each level of the first factor (country), making it unfeasible to estimate interaction effects in this model. mean values of qualitative characteristics were compared using the fisher lsd test. to assess similarities both between and within samples from the two countries, the collected data underwent clus­ ter analysis. all statistical analyses were conducted using minitab®21 (https://www.gmsl.it/minitab­anal­ isi­dati/). the quality characteristics of the saffron samples were determined by the laboratory of the saffron institute of university of torbat heydarieh. to deter­ mine the amount of crocin, picrocrocin and safranal, the spectrophotometer method (ultraviolet­visible spectrophotometry) and the iso/ts 3632 standard were used. for this purpose, 500 mg of each saffron sample was ground and after transferring to a 1000 ml volumetric flask, 900 ml of distilled water was added to it. then it was stirred by a magnetic stirrer for one hour at a speed of 1000 rpm. the volume of the resulting solution was brought up to 1000 ml with distilled water and again placed in the mixer to obtain a uniform solution. the amount of 20 ml of the resulting solution was brought to a volume of 200 ml and stirred until a uniform solution was formed. the solution was filtered away from light, and after obtaining a clear solution, the amount of light absorption in the wavelengths of 200 to 700 nm was recorded compared to the reference (distilled water). the absorption value in the range of 257 nm was related to picrocrocin, in the range of 330 nm to safranal, and in the range of 440 nm to crocin. the reported number was obtained from the following equation (kaveh and salari, 2018): e = 𝐷 x 1000 /m (100−𝐻) where e is the value of each color compound, d is the absorption value (the number recorded in the spec­ trophotometer), m is the weight of the saffron sam­ ple in grams, and h is the weight percentage of mois­ ture and volatile substances in the sample. minitab software was used for statistical analysis, and the means were compared by lest significant dif­ ference test (lsd). 3. results and discussion the results of nested analysis of variance showed that the effect of country and regions on quality traits of saffron (picrocrocin, safranal, and crocin) fig. 1 ­ map and geographical characteristics of the studied regions in herat province of afghanistan and torbat heydarieh county of iran. adv. hort. sci., 2024 38(1): 75­81 78 was significant (table 1). moreover, the comparison of mean values indicated the presence of significant differences in the qualitative characteristics of saf­ fron among the investigated regions of herat, and torbat heydarieh (table 2). the saffron produced in afghanistan had the highest amount of crocin (with an average of 279.1 ɷ 440 nm) and picrocrocin (with an average of 101 ϕ 257 nm), while the highest amount of safranal (with an average of 34.2 θ 330 nm) was observed in the samples of saffron produced in iran. the highest amount of crocin (303 ɷ 440 nm) and picrocrocin (106 ϕ 257 nm) belonged to the saf­ fron sample of zendeh jan region and the highest amount of safranal (39 θ 330 nm) was observed in the 6thsaffron sample of torbat heydarieh (ir6) (table 2). in the cluster analysis of qualitative traits of saf­ fron produced in different regions of herat, four clus­ table 2 ­ mean comparison of quality parameters of saffron from different regions of herat province ­ afghanistan and torbat heydarieh, iran means with the same letters are not significantly different based on the lsd test at 1% probability level p≤0.01. table 1 ­ analysis of variances (ms) for saffron quality parameters in different regions of herat province – afghanistan and torbat heydarieh, iran ** and * are significant at the 0.01 and 0.05 levels of probability, respective. source of variation df picrocrocin (ϕ 257 nm) safranal (θ 330 nm) crocin (ɷ 440 nm) country 1 1547.15 ** 89.76 ** 20539.1 ** region 14 168. 05 ** 21.47 ** 2545.5 ** error 32 0.98 0.95 0.98 total 47 country region name picrocrocin (ϕ 257 nm) safranal (θ 330 nm) crocin (ɷ 440 nm) afghanistan 101 a 31.4 b 279.1 a iran 89.6 b 34.2 a 237.4 b lsd 0.05 0.291 0.285 0.291 af1 injil 103 c 30 f 281 d af2 karukh 104 bc 29 f 289 b af3 guzara 101 d 34 cd 270 f af4 pashtun zarghun 104 bc 29 f 283 c af5 zendeh jan 106 a 33 de 303 a af6 ghuryan 97 e 29 f 275 e af7 obeh 92 gh 36 b 253 i ir1 fakhrabad 93 fg 30 f 252 i ir2 kadkan 88 k 36 b 236 j ir3 nasar 91 hi 33 de 259 h ir4 ghaleno 90 ij 32 e 236 j ir5 feizabad 71 m 34 cd 164 m ir6 khorgh 89 jk 39 a 227 k ir7 abrood 105 ab 35 bc 289 b ir8 bengang 94 f 34 cd 265 g ir9 shadmehr 85 l 35 bc 209 l lsd 0.05 0.816 0.799 0.816 nazarian et al. ‐ saffron quality comparison 79 ters were observed. the highest amount of the above qualitative traits belonged to zone 5 of zendeh jan (af5, crocin: 303 ɷ 440 nm, picrocrocin: 106 ϕ 257 nm, safranal: 33 θ 330 nm), and the lowest amount was belonged to zone 7 of obeh (af7, crocin: 253 ɷ 440 nm, picrocrocin: 92 ϕ 257 nm, safranal: 36 θ 330 nm). also, the above qualitative traits in saffron were obtained from torbat heydarieh region of iran in four different clusters including (fakhrabad: ir1, nasar: ir3, benhang: ir8), (kadkan: ir2, ghaleno: ir4, khorgh: ir6, shadmehr: ir9), abrood: ir7 and feizabad: ir5, so that region 5 had the lowest quality of saffron (ir5, crocin: 164 ɷ 440 nm, picrocrocin:71 ϕ 257 nm, safranal:34 θ 330 nm). it should be noted that in terms of quality, the different saffron producing regions of torbat heydarieh were ranked after the saffron of different regions of herat. in general, in the examination of the regions of the two countries in terms of the three qualitative attributes of picro­ crocin, crocin, and safranal, it was observed that regions 1, 2, 3, 4, 5 and 6 of herat (af1, af2, af3, af4, af5, af6) and region abrood of torbat heydarieh (ir7) was in a cluster and was superior to other regions (fig. 2). also, all the studied samples, except for torbat heydarieh feizabad (ir5), were qualitatively higher than the iso international standard and placed in the first place among first­class saffron (table 3). in the review and comparison of the quality of saf­ fron produced in the major production centers of razavi and south khorasan provinces, it was reported that out of 14 production centers (safi abad, qaynat, kashmar, torbathydarieh, khalil abad, bardeskan, shahn­abad, chakhmaq, torbat­jam, feiz­abad, sabzevar, sarayan, taghi­abad and birjand) the amount of safranal was lower than the iso and national standards of iran in all the investigated vil­ lages. for picrocrocin, all regions had values higher than the iso standard, but regions 2, 10, 11, and 12 could not achieve the minimum national standard, and the product produced in regions 5, 8, and 9 was within the standard limit. region 12 (ir5: feizabad) had the lowest amount of crocin, which was lower than the iso standard. regions 2, 5, 10 and 14 also had crocin values lower than the national standard of iran (kaveh and salari, 2018). the study of the quality characteristics of saffron in herat­afghanistan showed that the difference between picrocrocin and safranal was significant in different regions. the high­ est amount of picrocrocin (104/50 ϕ 257 nm) and safranal (34/95 θ 330nm) was observed in the saffron table 3 ­ sample classification based on iso 3632/1­2 normative (iso, 2003) fig. 2 ­ dendogram for hierarchical cluster analysis of 16 studied locations based on crocin, picrocrocin and safranalin heart, afghanistan and torbat heydarieh, iran. ir1­ ir9 (ir1: fakhrabad, ir2: kadkan, ir3: nasar, ir4: ghaleno, ir5: feizabad, ir6: khorgh, ir7: abrood, ir8: benhang, ir9: shadmehr) are 9 saffron producing regions of torbat heydarieh, iran and 7 different district of heart, afghanistan are: af1: injil, af2: karukh, af3: guzara, af4: pashtun zarghun, af5: zendeh jan, af6: ghuryan, af7: obeh. accordingly, to the absorbance readings at different wavelengths of solutions of the same concentration e1% (w.v­1) at 257 and 440 nm. source: gresta et al., 2008. iso category e 1% 257 nm e 1% 440 nm i grade 70 190 grade ii 55 150 grade iii 40 100 80 adv. hort. sci., 2024 38(1): 75­81 samples of the saffron­producing regions, while the highest amount of crocin (236/95 ɷ 440nm) was recorded in the saffron samples of pashtun zarghun city. also, by comparing the studied samples with the international saffron standard (iso 3632), it was observed that all the samples were higher than the group grade 1 (nazarian et al., 2021). in the world, there are many differences in prod­ uct quality parameters from one region to another. the reported values for crocin range from 0.85 to 32.4 percent of dry matter (alonso et al., 1999), safranal from 0.026 to 0.29 mg/dry weight (hadizadeh et al., 2006), picrocrocin 2.18 to 6.15 per­ cent of dry matter (straubinger et al., 1998) has been reported for iranian saffron. the results showed that there is a positive correlation between picrocrocin with safranal, crocin and altitude above sea level (kaveh and salari, 2018). the correlation of traits with altitude was consistent with the results of (lage and cantrell, 2009). studies conducted on the corre­ lation of saffron quality and geographical conditions of various factors such as soil, altitude above sea level and temperature have been reported as factors with positive or negative correlation. in a study on the qualitative characteristics of saffron in morocco, it was reported that the quality of the soil had no effect on the quality of saffron (lage and cantrell, 2009). in another study, it has been stated that soil acidity in the range of neutral to slightly alkaline has a positive correlation with saffron quality (gresta et al., 2008). in many countries, researchers reported that the chemical composition of saffron samples can be strongly influenced by weather conditions, agricul­ tural management practices, harvesting methods, and methods used to dry saffron stigma (lozano et al., 2000; zareena et al., 2001; kanakis et al., 2004; behdani and fallahi, 2015). based on the above reports, it can be said that the amount of saffron compounds in different countries may be very differ­ ent. during the last two decades, saffron cultivation in afghanistan has developed significantly, and it has been promoted in most provinces of the country. afghan farmers do not pay enough attention to saf­ fron nutrition management due to the bad economic situation. therefore, they use the least amount of animal manure and chemical fertilizers in saffron cul­ tivation. the saffron samples examined in this research were selected from different regions of herat province with different geographical character­ istics (altitude, amount of rainfall, latitude, etc.). the results showed that these different geographical characteristics can affect the quality of saffron pro­ duced. zendeh jan region with an altitude of 835 m and an annual rainfall of 201 mm was produced the best quality of saffron, while the quality of saffron decreased with the increase of the altitude and the amount of rainfall, so that obeh region in herat and samples of saffron produced in torbat heydarieh were have the lowest quality of saffron. on the other hand, due to the simultaneous growth of saffron with winter rainfall, the water required by saffron can be supplied by irrigating 2 to 3 times in this way. the method of drying saffron, which affects the quality of saffron, is completely traditional in afghanistan, and without the use of machines. therefore, according to the prosperity and development of saffron cultivation in different regions and the virgin lands of herat province, which have not been under the cultivation of this new and economic product, the qualitative superiority of saffron produced in different regions of herat compared to the samples of torbat heydarieh county, for the amount of secondary metabolites such as crocin, picrocrocin and safranal does not seem unlikely. 4. conclusions the chemical composition of saffron stigma can be strongly influenced by weather conditions, alti­ tude, farm management practices, harvesting meth­ ods, and the approach to stigma drying. in general, the samples from regions 1 to 6 (anjil, karukh, guzarah, pashtun zarghun, zendeh jan and ghoryan) of herat province, afghanistan and abrood region (ir7) of torbat heydarieh, iran, exhibited higher levels of crocin and picrocrocin compared to the iso stan­ dard.these samples not only secured the first­place ranking but also 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although up-take depends on environmental conditions (berrie, 1960). multiple applications of sugars to foliage on tomato in a dark room increases growth rate and especially when application is at fairly high temperatures (went and carter, 1948). tomato plants treated with a 10% aqueous sucrose solution daily up to three days before transplanting, produced more adventitious roots (smith and zink, 1951). application of 70 g·l-1 sucrose resulted in improved establishment in the field and shoot growth as a result of producing more adventitious roots (percival and fraser, 2005). in other investigations on non-vegetable plants such as young trees of silver birch, cherry, and red oak, applications of sugar ≤ 50 g·l-1 in water as a root drench significantly enhanced root vigor by week 12 (percival, 2004). sucrose injection to soybeans increased leaf area and pod numbers but suppressed photosynthesis (abdin et al., 1998). there is very little information in the literature on the application of sucrose on transplants to improve chilling resistance and other transplant quality characteristics. the present study was undertaken to investigate effects of sucrose application on tomato transplants, as a commercially important vegetable established from transplants, and evaluate effects on increasing chilling resistance and other traits under field conditions. foliar application was chosen because roots take up very little sugar, stems take it up mainly through wounds, but intact leaves absorb it readily; apparently through the whole surface (went and carter, 1948). 2. materials and methods the study was carried out in a 200 m2 area, at an altitude of 1810 m above sea level. the regional mean relative humidity was 46% and the mean monthly minimum and maximum temperatures during the growing season were 15.6 and 30.7 °c, respectively. soil samples were analyzed prior to the start of the experiment. the loamy soil had an ec of 2.41 mmhos·m-1 and a ph of 7.6 (soil extract 1:2). the soil was plowed and disked to turn under existing plant application of sucrose on tomato seedlings improves transplant quality, crop establishment, cold and dark hardiness jamal javanmardi (1), shandiz emami department of horticultural sciences, faculty of agriculture, shiraz university, shiraz, iran. key words: chilling tolerance, survival in dark, transplant establishment, transplant hardening, transplanting. abstract: transplant production is an important part of the vegetable production process. therefore, improving transplant quality and resistance to adverse environmental conditions is important. effects of sucrose solutions (0, 5, 10, 15, 20 and 25%), applied to the foliage of tomato (solanum lycopersicum l.) cultivars calj-n3 and rio-grande were studied. treatments were applied to developing seedlings at every other irrigation for six weeks. application of 25% sucrose increased fresh and dry weights of shoots, fresh weight of roots, and shoot and root dry weight percentages. application of 25% sucrose led to a 13 and 18% higher survival to chilling temperature in ‘calj-n3’ and ‘rio-grande’ tomato seedlings, respectively. the highest transplant survival percentages in darkness were found at 10% sucrose and higher. seedlings sprayed with 15% sucrose solution had the highest transplant establishment in the fi eld and fl owering was approximately four to fi ve days earlier. a 15% sucrose treatment is thus preferred. adv. hort. sci., 2013 27(3): 122-126 1 corresponding author: javanm@shirazu.ac.ir received for publication 11 march 2013 accepted for publication 8 july 2013 123 material. fertilization was according to soil test result and crop need to supply nutrient requirements for tomato production (jones, 2007). the tomato cultivars calj-n3 and rio-grande were used. seeds were sown in 300 ml plastic pots filled with peat and perlite (1:1 v:v) and irrigated every other day. plantlets emerged after one week. pots were irrigated with water for the first two weeks after sowing. afterwards, they were fertigated with a complete npk fertilizer as recommended by papadopoulos (1991) for tomato transplant production. greenhouse conditions for transplant production were 24±4°c and 45±5% relative humidity. seedling foliage was treated with six sucrose solutions (0, 5, 10, 15, 20 and 25% concentrations) starting two weeks after emergence. one drop of tween 20 was added to each solution to increase leaf surface absorption. application covered all leaf surfaces at every other irrigation interval until seedlings were moved to the field at the sixth week after seed sowing. leaves were washed using a mist system between each sucrose application. for the field experiment, six-week-old seedlings were transplanted into the field at a 40 60 cm spacing in rows covered with black polyethylene mulch. plots were fertigated through tape irrigation with starter fertilizer 10-1010 npk for the first week after transplanting to help stand establishment according to masson et al. (1991). cultural practices until the end of the experiment were carried out as described by papadopoulos (1991). the field experiment was repeated in two subsequent years. seedlings at the end of the sixth week from seed sowing were divided into four groups. group a was used to determine vegetative characteristics including leaf chlorophyll content (saini et al., 2001), transplant shoot and root fresh and dry weights, and shoot and root dry matter percentage. group b was stored in darkness at 20°c for 96 h to determine effects of treatment on darkness survival. group c was placed in an incubator at 0°c for 96 h to determine effects of sucrose on resistance to chilling. group d was transplanted into the field to determine degree of field establishment, leaf number preceding first flower and the days from transplanting to first flower formation. group a, b and c experiments were arranged in a completely randomized design; group d (factors after transplanting into field) was arranged in a completely randomized block design over two years. all treatments consisted of six replicates of 10 plants. data analysis was performed using the spss12 (spss inc., chicago, il) computer software for windows. if differences were significant, means were separated with least significant difference (lsd) test. 3. results and discussion the effects of treatments on all measured traits were always significant except for leaf number, leaf chlorophyll content, fruit set percent and yield; for group d the effects of year and its interaction with treatments for all measured traits were not significant (data not shown). fresh and dry weights of shoots and roots, shoot and root dry matter percentage increased with increasing sucrose concentration in both cultivars (table 1). the highest values were for the 25% sucrose concentration. similar results in terms of increased dry matter of shoots and roots were found in seedlings of a non-vegetable plant, birch (betula pendula roth.), after a root drench application of sucrose and fructose (percival and fraser, 2005). accordingly, it seems that sucrose could be absorbed by aerial and underground plant organs and used in metabolism for growth and development. ritchie (1987) reported that root growth potential is not a physiological process. however, it integrates many important physiological processes in seedlings and has become a popular and useful indicator of seedling vigor. table 1 vegetative characteristics of two tomato cultivars, sprayed with aqueous sucrose solution for a 6-week transplant raising period tomato cultivar sucrose concentration (%) shoot fresh weight (g) shoot dry weight (g) shoot dry weight percentage root fresh weight (g) root dry weight (g) root dry weight percentage leaf number preceding the first flower days from transplanting to first flower calj-n3 0 4.25±0.20 e 0.33± 0.01 e 7.83±0.19 f 0.21±0.01 g 0.018±0.048 a 8.55±0.05 ef 9.67±0.33 a 27.33±0.88 a 5 4.57±0.23 de 0.35±0.03 e 7.55±0.29 fg 0.24±0.02 ef 0.021±0.002 a 8.79±0.12 de 9.33±0.33 ab 26.33±0.33 b 10 4.74±0.20 cd 0.52±0.02 c 10.89±0.15 cd 0.42±0.03 cd 0.037±0.002 a 8.86±0.12 cd 8.67±0.33 c 24.67±0.33 d 15 4.72±0.24 cd 0.52±0.03 c 10.94±0.23 bcd 0.50±0.05 bc 0.046±0.005 a 9.27±0.11 ab 7.33±0.33 ef 23.33±0.33 ef 20 5.02±0.04 c 0.59±0.01 b 11.73±0.31 ab 0.61±0.04 a 0.057±0.005 a 9.40±0.15 a 7.00±0.00 f 23.00±0.00 ef 25 6.22± 0.15 a 0.74±0.02 a 11.97±0.18 a 0.64±0.02 a 0.060±0.003 a 9.36±0.07 ab 7.33±0.33 ef 22.67±0.33 f rio-grande 0 3.24±0.19 g 0.22±0.02 f 6.70±0.26 h 0.11±0.01 h 0.008±0.001 b 7.52±0.19 h 9.67±0.33 a 27.33±0.33 a 5 3.55±0.16 fg 0.24±0.03 f 6.84±0.49 gh 0.15±0.02 gh 0.012±0.002 ab 7.96±0.30 g 9.33±0.33 ab 25.67±0.33 bc 10 3.84±0.16 f 0.38±0.02 de 10.00±0.26 e 0.31±0.03 e 0.026±0.002 a 8.26±0.09 f 9.00±0.00 bc 25.00±0.57 cd 15 4.26±0.20 e 0.42±0.02 d 9.78±0.74 e 0.40±0.05 d 0.036±0.005 a 8.95±0.04 cd 8.00±0.00 d 23.67±0.33 e 20 5.02±0.09 c 0.51±0.03 c 10.19±0.50 de 0.51±0.03 b 0.046±0.004 a 9.10±0.12 bc 7.67±0.33 de 23.33±0.33 ef 25 5.54±0.19 b 0.62±0.02 b 11.15±0.58 bc 0.53±0.01 b 0.048±0.001 a 9.12±0.05 abc 7.33±0.33 ef 23.33±0.33 ef lsd value 0.37 0.053 0.81 0.075 0.042 0.28 0.59 0.86 data are mean ± s.e. of six replicates. different letters indicate significant differences for each column at p≤ 0.05 by lsd test. 124 any physiological problem affecting seedlings should show up as a decrease in the seedling’s ability to produce roots. thus, sucrose spray is an applicable method to increase root fresh weight and thereby increase transplant vigor. survival in dark tomato growers often purchase seedlings from distant transplant producers, risking deterioration of transplants during transportation. transportation conditions, status of flower development at time of transport, planting conditions after transport, and unfavorable combinations of these conditions often result in flower abortion and delayed fruit development of the first truss (kubota et al., 2004). low-temperature storage in darkness has been reported as a way to preserve seedling quality (leskovar and cantliffe, 1991; kaczperski and armitage, 1992; kaczperski et al., 1996). nearly all plant food reserves are stored as starch or sugars. these are produced by photosynthesis and consumed by respiration to sustain plant growth and metabolism. cold storage affects photosynthesis and respiration in two ways. first, absence of light interrupts photosynthesis, and second, low temperature decreases respiration rate. the net effect is that seedlings very slowly burn up their supply of reserve carbohydrates in storage (ritchie, 1987). therefore, survival in darkness and seedling quality are important for transplant transportation. low temperature and dim light during transportation maintain transplant quality at an acceptable level (kubota and kroggel, 2006). our results showed that by increasing sucrose concentration, survival of transplants in darkness was increased. the highest transplant survival percentage in darkness was found with 10% concentration of sucrose and was not cultivar dependent (fig. 1). kubota et al. (1997) reported reductions in levels of soluble sugars and starch in dark-stored broccoli (brassica oleracea l. botrytis ‘group green duke’) plantlets. in spite of interruption of the supply of photosynthates in darkness, amounts of soluble sugars increase through degradation of starch in shoots (sato et al., 2004). accordingly, it is concluded that foliar application of sugars can be used as an energy source for respiration in darkness with low, or no, decrease in transplant quality. chilling temperature survival seedling survival percentage increased as sucrose concentration increased in both cultivars (fig. 2). application of 25% sucrose solution produced the best survival in response to chilling. compared to controls there was 13 and 18 times greater chilling resistance in ‘calj-n3’ and ‘rio-grande’ seedlings, respectively. since carbohydrate reserves undergo a net loss during low temperature storage and hardiness development requires an expenditure of metabolic energy (ritchie, 1987), providing sucrose as an available source of energy through foliar application can help transplants survive low temperatures. sucrose has been reported as a metabolite for stabilizing membranes in plant tissues during chilling (king et al., 1988). sugar is able to maintain dry mass of plantlets under a wider range of environmental conditions during low temperature storage (kubota, 2005). leaf chlorophyll content leaf chlorophyll content was not different in cultivars due to treatment (data not shown). the same result was observed in birch (percival and fraser, 2005) where sugar feeding at 25 g·l-1 level had no significant effect on carotenoid and chlorophyll concentrations. field establishment efficacy high transplant establishment percentage in the field results from quality transplants and successful transplanting. tomato seedlings treated with 15 or 25% sucrose had the highest and similar transplant establishment rates (fig. 3). this could be related to the greater root systems produced by those plants. root growth is considered a useful indicator of seedling vigor (ritchie, 1987). number of leaves and days to first flower a standard indicator for flowering time is the number of leaves produced on the primary shoot before first flowers are initiated (koornneef et al., 1991). in tomato, the number of leaves produced before flowers is genetically controlled, but mediated by environmental conditions. usually only from six to 11 leaves are required below the first inflorescence in tomato (kinet and peet, 1997), with fig. 1 effect of sucrose foliar spray on the percent of survived ‘caljn3’ and ‘rio-grande’ tomato transplants in darkness. fig. 2 effect of sucrose foliar spray on the percent of ‘calj-n3’ and ‘rio-grande’ tomato cultivar transplants that survived chilling temperatures. 125 the lower number being found in an early crop. the ‘caljn3’ and ‘rio-grande’ seedlings treated with 15 and 20% sucrose solution, respectively, produced flowers sooner than other treatments (table 1). in those concentrations there were fewer leaves than in the controls. it may be that sucrose application provides additional carbohydrates required for flower initiation. flowering was observed to occur approximately four to five days earlier in cultivars treated with ≥15% sucrose (table 1). fruit set percent and yield analysis of variance for both cultivars over two years did not show differences for fruit set percent and fruit yield (data not shown). it seems the effect of sucrose application on seedlings might not extend beyond flowering. fruit set (and eventually yield) is affected by environmental factors and plant growth regulators during the fruit set period (kinet and peet, 1997). 4. conclusions overall, transplant quality of horticultural crops is generally defined by physiological potentials (growth and developmental characteristics and photosynthetic ability), visual quality (color and morphology), genetic uniformity, and pathogen status (kubota et al., 2002). in the present study, in most cases the 15-25% sucrose treatment gave the best results. the main detracting factor for the 25% sucrose concentration was sticky leaf surfaces after application, which may attract insects. washing leaves to remove stickiness and applying this amount of sugar is probably not economical. therefore, the 15% level is recommended, which can be applied through overhead mist or irrigation system in commercial transplant production operations. overhead irrigation systems wash excesses sucrose off leaves, reducing problems. the results from this investigation can be useful for transplant production companies and growers. adverse environmental conditions do not always allow immediate transplanting into the field and sucrosetreated seedlings can be held for a few days, thus allowing flexibility in crop scheduling and labor management. references abdin a.o., zhou x., coulman b.e., clautier d., faris m.a., donald l., 1998 effect of sucrose supplementation by stem injection on the development of soybean plants. j. exp. bot., 49(329): 2013-2018. berrie a.m.m., 1960 the effect of sucrose sprays on the growth of tomato. physiologia plantarum, 13(1): 9-19. jones j.b., 2007 tomato plant culture: in the field, greenhouse, and home garden. crc press, taylor and francis group, boca raton, fl, usa, pp. 400. kaczperski m.p., armitage a.m., 1992 short-term storage of plug-grown bedding plant seedlings. hortscience, 27(7): 798-800. kaczperski m.p., armitage a.m., lewis p.m., 1996 performance of plug-grown geranium seedlings preconditioned with nitrogen fertilizer or low-temperature storage. hortscience, 31(3): 361-363. kinet j.m., peet m.m., 1997 tomato, pp. 207-258. in: wien h.c. (ed.) the physiology of vegetable crops. cab international, wallingford, uk, pp. 663. king a.i., joyce d.c., reid m.s., 1988 role of carbohydrates in diurnal chilling sensitivity of tomato seedlings. plant physiol., 86(3): 764-768. koornneef m., hanhart c.j., veen j.h., 1991 a genetic and physiological analysis of late flowering mutants in arabidopsis thaliana. mol. gen. genet., 229(1): 57-66. kubota c., 2005 low temperature storage of plants under dim light, pp. 205-212. in: kozai t., f. afreen, and s.m.a. zobayed (eds.) photoautotrophic (sugar-free medium) micropropagation as a new micropropagation and transplant production system. springer verlag, the netherlands, pp. 316. kubota c., kroggel m., 2006 air temperature and illumination during transportation affect quality of mature tomato seedlings. hortscience, 41(7): 1640-1644. kubota c., kroggel m., solomon d., benne l., 2004 analyses and optimization of long distance transportation conditions for high quality tomato seedlings. acta horticulturae, 659: 227-234. kubota c., rajapakse n.c., young r.e., 1997 carbohydrate status and transplant quality of micropropagated broccoli plantlets stored under different light environments. postharvest biology and technology, 12(2): 165-173. kubota c., seiyama s., kozai t., 2002 manipulation of photoperiod and light intensity in low temperature storage of eggplant plug seedlings. scientia horticulturae, 94(1-2): 13-20. leskovar d.i., cantliffe d.j., 1991 tomato transplant morphology affected by handling and storage. hortscience, 26(11): 1377-1379. masson j., tremblay n., gosselin a., 1991 effects of nitrogen fertilization and hps supplementary lighting on vegetable transplant production. ii. yield. j. amer. soc. hort. sci., 116(4): 599-602. papadopoulos a.p., 1991 growing greenhouse tomatoes in soil and in soilless media. agriculture canada publication, ottawa, canada. percival g.c., 2004 sugar feeding enhances root vigor of young trees following containerization. j. of arboric., 30(6): 357-364. fig. 3 effect of sucrose foliar spray on the percent of successful stand establishment of ‘calj-n3’ and ‘rio-grande’ tomato cultivar transplants. 126 percival g.c., fraser g.a., 2005 use of sugars to improve root growth and increase transplant success of birch (betula pendula roth.). j. of arboric., 31(2): 66-77. ritchie g.a., 1987 some effects of cold storage on seedling physiology. tree planters’ notes, 38: 11-15. saini r.s., sharme k.d., dhankhar o.p., kaushik r.a., 2001 laboratory manual of analytical techniques in horticulture. agrobios, india. sato f., yoshioka h., fujiwara t., higashio h., uragami a., tokuda s., 2004 physiological responses of cabbage plug seedlings to water stress during lowtemperature storage in darkness. scientia horticulturae, 101(4): 349-357. schrader w.l., 2000 using transplants in vegetable production. the regents of the university of california, anr publication, 8013: 5. shukry w.m., el-otaby l.m., 2011 increased chilling tolerance and altered protein patterns in tomato seedlings following hardening of seeds or system in application. acta botanica hungarica, 53(3-4): 389-408. smith p.g., zink f.w., 1951 effect of sucrose foliage sprays on tomato transplants. proceedings amer. soc. hort. sci., 58: 168-178. went f., carter m., 1948 growth response of tomato plants to applied sucrose. american journal of botany, 35(2): 95-106. impaginato 63 adv. hort. sci., 2024 38(1): 63­74 doi: 10.36253/ahsc­14148 in vitro salt stress tolerance of ‘sahand’ cultivar grafted on two wild almond rootstocks: an evaluation of physiologi­ cal and biochemical traits between rootstocks m. asadi zargh abad, a. shekafandeh (*) department of horticultural science, college of agriculture, shiraz university, p.o. box 65186, 71441 shiraz, iran. keywords: antioxidant enzymes, glycine betaine, grafting combination, proline. abstract: the low salinity tolerance of almond cultivars can cause a significant setback in almond production. therefore, selecting suitable cultivars and root­ stocks in salinity­affected areas can facilitate sustainable crop production. in this research, the effects of two wild almond species, badamkohiand arjan as rootstocks on the salinity tolerance of ‘sahand’ as a scion were investigated through in vitro culture. a factorial experiment of 2 (species) × 4 (levels of salin­ ity) was conducted in a completely randomized design (crd) with 4 replica­ tions. the results showed that ‘sahand’ grafted on badamkohi had the higher fresh and dry weight than grafted on arjan in all level of salinity. the na+ and cl­ ions contents inthe shoots and root of both micrografting combinations increased with increasing salinity. however, their amount in the shoot and the root of ‘sahand’/arjan plants were significantly higher than those ions in ‘sahand’/badamkohi plants at 80 and 120 mm nacl. the amount of total chlorophyll in ‘sahand’ grafted on badamkohi was 0.68 mg g­1 fw which was significantly higher than the total chlorophyll of the same scion grafted on arjan rootstock (0.51 mg g­1 fw) at 120 mm nacl. the highest leaf cell electrical leakage occurred in ‘sahand’ grafted on arjan which was significantly higher than leaf electrical leakage of the same scion grafted on badamkohi at 120 mm nacl. the grafting combination of ‘sahand’/badamkohi showed a higher pro­ line and glycine betaine content, compared to the grafting combination of ‘sahand’/arjan. the shoot and root antioxidant enzyme activities (sod, pox and cat) in micrografting combination of ‘sahand’/ badamkohi were also sig­ nificantly higher than those in ‘sahand’/arjan. it can be concluded that ‘sahand/badamkohi combination is a suitable choice for the regions with late spring frost and saline conditions. 1. introduction almond (prunus dulcis mill.) is one of the most important nut crops (ansari and gharaghan, 2019), with particular importance in the world. it (*) corresponding author: shekafan@shirazu.ac.ir citation: asadi zargh abad m., shekafandeh a., 2024 ­ in vitro salt stress tolerance of ‘sahand’ cultivar grafted on two wild almond rootstocks: an eva‐ luation of physiological and biochemical traits between rootstocks. ­ adv. hort. sci., 38(1): 63­ 74. copyright: © 2024 asadi zargh abad m., shekafandeh a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 january 2023 accepted for publication 17 january 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14148 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 63­74 64 is characterized by a high storage capacity, low degree of waste, ease of processing and transporta­ tion. the feasibility of its economic production has led to an increase in the total area of almond orchards (bybordi, 2013). salinity still remains the major abiotic stress that limits agricultural produc­ tion (seleiman et al., 2020). almonds can be affected by salinity stress through osmotic mechanisms and by enhanced levels of osmotic potential in the soil solution (shrivastava and kumar, 2015). high salinity in root zone not only reduces water uptake and tree growth, but also can cause nutritional imbalances and toxicity effects of the major saline ions (na+ and cl­). meanwhile, the low salinity tolerance of almond trees can cause a significant setback in almond pro­ duction (kaundal et al., 2019). therefore, selecting suitable cultivars and rootstocks in salinity­affected areas can facilitate sustainable crop production. the dynamic nature of salinity with respect to time and space, as well as limited experimental designs restrict the complete study of genotype­envi­ ronment interactions (sauvage et al. , 2014). therefore, the crop breeding program can be com­ plemented with a suitable management option, such as grafting on appropriate salt tolerant rootstocks (cuartero et al., 2006). grafting has been reported as a rapid method for enhancing salt tolerance (singh et al., 2020) that counteracts the salinity effects by maintaining low na+/k+ ratios in the shoot and improves leaf stomatal conductance (wei et al., 2017). the behavior of the rootstock in different plant species influences the metabolic processes of the scion leading to tolerance. rootstocks are an essential component in modern fruit production (shahkoomahally et al., 2020) and can provide sever­ al traits that may be absent in the scion, such as resistance to soil pest and disease, better root sys­ tems, enhanced nutritional uptake, better tolerance to soil salinity and water scarcity (kumar et al., 2017). the cultivation of grafted plants has gradually increased in recent years since grafting enables the plant system to control important agronomic traits and offers a flexible pattern to the growth of a partic­ ular scion (kumar et al., 2017). grafting a scion on a suitable rootstock generally allows extensive use of the rootstocks (gainza et al., 2015). while consider­ ing a wide range of salt­tolerant genotypes in the genus prunus, the selection of more tolerant species as rootstock can lead to sustainable solutions in han­ dling commercial cultivars of almond and peach (najafian et al., 2008). the use of wild almond species as rootstocks, has been considered feasible especially in arid and semi­arid regions (karimi et al., 2015). using these rootstocks can highlight the strat­ egy of allowing plants to overcome environmental stress, because of their adaptability and stimulated growth. moreover, rootstocks affect the nutritional status of the scion and plant height (aras and eşitken, 2019). trees have lengthy biological cycles, which implies prolonged time lapses until plants are produced for study, as well as evaluations that can last for the entire growing season or even more than one season (bado et al., 2015). this is of particular importance in plant breeding programs where thousands of plants are handled yearly and the staff and land are restrict­ ed. however, the long juvenile periods and cost of maintaining all the seedlings until they are grown trees have encouraged researchers to develop early studies in young plants to discard those genotypes which do not fit in the breeding goals and minimize the cost and time of field evaluations (vives­peris et al., 2017). in recent years, tissue culture and in vitro selec­ tion have emerged as an effective tool in the further­ ance of efforts to develop stress­tolerant plants. in vitro cultivation techniques can largely assist with the study and selection of plant species, because more control is exerted on plant growth compared to the outside environment, and evaluations are usually conducive to good results in a confined space (ghaleb et al., 2010; rai et al., 2011). when the sta­ bility of medium culture enables a controlled condi­ tion, a uniform application of stress to all explants can create reliable results, because other intervene factors are eliminated (seth and kendurkar, 2015). the in vitro system can characterize the degree of salt tolerance of different genotypes at their primary growth phase within a short time, limited space and low cost (ghaleb et al., 2010). this method has been applied for screening salt tolerant genotypes of some fruit species including cherry rootstocks (erturk et al., 2007), citrus rootstocks (ghaleb et al., 2010), grape rootstocks (alizadeh et al., 2010), fig cultivars (abdoli nejad and shekafandeh, 2014), apple varieties (shibli et al., 2000), kiwifruit (sotiropoulos and dimassi, 2004) and pear (sotiropoulos et al., 2006). the aim of this research was to study the influence of badamkohi and arjan as rootstocks on the salinity tolerance of ‘sahand’ (a late­bloom almond cultivar) as a scion through certain morphological and bio­ http://https://www.frontiersin.org/articles/10.3389/fpls.2020.595055/full#ref43 asadi zargh abad and shekafandeh ‐ in vitro salt tolerance of different almond graft combinations 65 chemical responses in vitro condition. 2. materials and methods establishment of in vitro micrografting according to asadi and shekafandeh (2021) pro­ cedure, the mature seeds of naturally grown wild almond trees, prunus scoparia (c. schneider) and prunus elaengnifolia (e. murrary) named badamkohi and arjun respectively, grow in arid and semi­arid regions were grown in vitro to produce seedlings. after removing the endocarps, they were surface­ sterilized by immersion in 70% alcohol for 1 min and then in 20% whitex solution (sodium hypochlorite 5%) for 10 min. subsequently, they were rinsed three times with sterile distilled water. the sterilized seeds were cultured on ms medium (murashige and skoog, 1962) supplemented with 30 g l­1 sucrose and 8 g l­1 agar as gelling agent (fig. 1 a, b). for preparation of the micro­scion, young shoots of the late­blooming ‘sahand’ were disinfected and cultured on ms medi­ um supplemented with 2.2 μm benzyladenine (ba) and 0.54 μm naphthaleneacetic acid (naa). after two weeks, the young offshoots have reached a suitable size to be used as scions (fig. 1 c). then, the two weeks old in vitro produced seedlings were decapi­ tated and by the help of a sharp scalpel a vertical slit (0.5 cm) was created on top of the stump (fig. 1 d). the scion was cut into a “v” shaped wedge (fig. 1 e) and inserted into the rootstock to form a micrograft (fig. 1 f). the jars containing explants were maintained at 25±1°c under a 16 h photoperiod (light intensity of 4000 lux). salinity treatments the graft combinations were allowed to grow for 4 weeks, and then they were cultured on agar free ms medium with a bridge paper and different con­ centrations of nacl (0, 40, 80 and 120 mm). after 4 weeks of culture, certain morpho­physiological and biochemical characteristics of graft combinations were measured as follow. fresh and dry weight of scion and rootstock after measuring the fresh weight of the shoot (scion) and the root (rootstock), they were dried in an oven for 24 h at 60°c and then re­measured for their dry weight (in mg). sodium (na+) and chlorine (cl‐) the samples (1 g) were dried in an oven at 500 to 550°c for 5 h, then reduced to ash. to each sample in the crucible was added 5 ml of 2 normal hcl. after passing the solution through filter paper, the filtered solution was transferred to a jug balloon. the volume of each sample was made up to 50 ml with hot dis­ tilled water and then sodium was measured using a flame photometer (model jenway pfp7 bibby scientific ltd, staffordshire, uk) and calculated in mg g­1 dry weight. to determine the chlorine of each sample, accord­ ing to the method of chapman and pratt (1961), one gram of sample was poured into a chinese mortar and 250 mg of calcium oxide was added to each and was kneaded with distilled water. they were then placed in a kiln at 250°c for one h to remove all the soot from the initial burning. after, the kiln tempera­ ture was slowly raised to 550°c to reduce the sam­ ples to ashes. then 15 ml of hot distilled water was added to the samples. after cooling, 5 drops of 5% potassium chromate were added to the solution and titrated with 0.05 n silver nitrate (2.12 g of silver nitrate in 250 ml of distilled water) to observe a red brick­colored precipitate. finally, chlorine was calcu­ lated as mg g­1 dry weight. total chlorophyll (chl) fresh leaf samples (0.1 g) were placed in test tubes and added 7 ml of dimethyl sulfoxide, then they were placed in an incubator for 30 minutes at 65°c. after extraction, the volume of extracts was made up to 10 ml by adding dimethyl sulfoxide. fig. 1 ­ performing in vitro micrograft. germinated seeds of arjan (a) and badamkohi (b) using as rootstock. proliferated shoots of ‘sahand’ cv. on ms medium sup­ plemented with 2.2 μm ba and 0.54 μm naa using as scion (c). an arjan rootstock ready to be grafted (d). a scion ready for grafting (e). a micrograft combination of ‘sahand’/arjan (f). adv. hort. sci., 2024 38(1): 63­74 66 finally, the absorbance of the extracts at wave­ lengths of 645 and 663 nm was read using a spec­ trophotometer (usa epoch microplate, biotek instruments, inc) (gross, 1991). chlorophyll content was determined as follows: chl (mg g­1) fw = [20.2 (od645nm) + 8.02 (od663nm)] × v/fw × 1000 where v is final volume of solution (ml), fw the leaf fresh weight (mg), and od the optical density. electrolyte leakage(el) el was determined according to the method of gulen and eris (2004). ten discs were cut from the fully developed leaves of the plants in each replica­ tion. then, they were transferred to vials containing 5 ml deionized water and kept at 10°c for 24 h. after measuring their electrical conductivity (ec1) using a conductometer (metrohm 644, awess), the samples were then placed in a water bath at 95°c for 20 min, and after cooling at 25°c the electrical conductivity (ec2) was re­measured. the el was calculated with the following formula: el (%) = (ec1/ec2) × 100 proline contents according to the modified method of bates (1973), the proline content was determined. the leaf sample (0.5 g) was grinded in 10 ml sulfosalicylic acid (3%). the mixture was then centrifuged at 10,000×g for 10 min. two ml of the supernatant was added to each test tube which contained freshly prepared acid­ninhydrinas a diluted solution (2 ml). the tubes were incubated in a water bath at 90°c for 30 min. ultimately, the reaction ended in an ice bath. the reaction mixture was extracted using toluene (5 ml) and was vortexed for 15 s. the tubes were stored in darkness at room temperature for 20 min, thereby allowing the separation of toluene from the aqueous phase. the toluene phase was then carefully collect­ ed and the absorbance was measured at 520 nm by a spectrophotometer (model t60 usa). glycine betaine glycine betaine was measured by the method of grattan and griere (1985). the powdered sample of leaf (0.5 g) was mixed in a mortar with 20 ml of ion­ ized water. the specimens were placed on a shaker for 48 h at 25°c. they were passed through a whatman filter paper ‘g42’ and diluted in a 1:1 ratio with two­molar sulfuric acid. then, 0.5 ml of this solution was removed and poured into the eppendorf tube. after cooling the samples for 2 h, 0.2 ml of potassium tri­iodide solution was added to each. then, they were centrifuged for 20 min at 15,000 rpm at 0°c. the top phase discarded and the periodontal crystals were dissolved in the bottom of the container in 9 ml of 1­2 dichloroethane. then, the absorption of the samples was measured at 365 nm with a spectrophotometer. enzyme activity in order to estimate the enzymes activities, the leaf samples (0.5 g) were first homogenized in 50 mm potassium phosphate buffer (ph 7.8) containing 1 mm edta, 3 mm 2­mercaptoethanol, and 2% (w/v) polyvinyl polypyrrolidone (pvpp) in a chilled mortar. the homogenate was then centrifuged at 16000 g for 30 min at 4°c and the supernatant was used for enzyme assays. superoxide dismutase (sod) the sod (ec 1.15.1.1) can be measured by deter­ mining its ability to halt the photochemical reduction of nitro blue tetrazolium chloride (nbt) in the pres­ ence of light. in this method, the reaction mixture (3 ml) contained 50 μl enzyme extract, 50 mm potassi­ um phosphate buffer, 13 mm l­methionine, 75 μm nbt, 0.1 mm edta and 4 μm riboflavin. the reaction mixture was shaken and placed in a light chamber for 15 min to allow the reaction to take place. eventually, the absorption rate of each specimen was recorded at 560 nm using a spectrophotometer (biochrom wpa biowave ii uv/visible spectrophoto­ meter, england) against the non­irradiated blank (dhindsa and motowe, 1981). catalase (cat) the determination of the activities of cat (ec 1.11.1.6) was based on the rate of h2o2 decomposi­ tion as measured by decreasing the absorbance at 240 nm (dhindsa and motowe, 1981). while the reaction mixture contained 50 ml potassium phos­ phate buffer (ph 7) and 15 mm hydrogen peroxide (h2o2), the reaction started by adding 1000 μl of the enzyme extract. one unit of activity is the amount of enzyme that could decompose 1 mm of h2o2 in 1 min. peroxidase (pox) peroxidase enzyme activity was read at 470 nm, based on an enhanced degree of light absorption as a result ofguaiacol oxidation in the presence of peroxi­ dase hydrogen. this was carried out by a spectropho­ asadi zargh abad and shekafandeh ‐ in vitro salt tolerance of different almond graft combinations 67 tometer (jenway model 7315 uk) in 1 min with a time interval of 10 s (ozden et al., 2009). the activity of the enzyme was calculated based on the oxidized μmol of guaiacol per min and per g of fresh leaf weight. statistical analyses the experiment was carried out as a factorial 2 (species) × 4 (levels of salinity) in a completely ran­ domized design (crd) with 4 replications and 4 micrografted plants per replicate. a total of 122 micro­grafting combination of ‘sahand’/argan and ‘sahand’/badamkohi were used in this experiment. data were analyzed using sas 9.4 software and mean values were compared using lsd test (p≤0.05). 3. results the results of analysis of variance showed that the interaction between species and salinity was sig­ nificant at 5% or 1% level in all measured traits. so, all the results were presented by the interactions. effects of salinity on scion‐rootstock combination growth the results showed that, both rootstocks (badamkohi and argan) had significant difference in the root length of control (free salt medium). badamkohi showed the highest root length of 263 mm that was significantly higher than arjan (122 mm) (table 1). this showed that they have different growth habit. however, in both rootstocks with increasing salinity to 120 mm in culture medium the root length decreased significantly. this reduction was 50% and 18% for badamkohi and arjan respectively compared to their controls. the highest length of scion (64 mm) was related to the salt­free treatment on badamkohi rootstock which showed a significant difference with the same scion on arjan rootstock (41 mm) in the same treatment. with increasing the concentration of sodium chlo­ ride from 0 (control) to 120 mm, the scion (‘sahand’) length on both rootstocks was significantly reduced (fig. 2 b, c, e, f), this reduction was 50.6% and 49% on badamkohi and arjan respectively. in both rootstocks, root fresh and dry weight decreased with increasing nacl concentrations, how­ ever fresh weight loss in badmkohi and arjan was 62% and 76%, respectively, and also dry weight loss was 55% in badamkohi and 51% in arjan (fig. 3 a and c). fresh and dry weight of ‘sahand’ grafted on badamkohi at all salinity levels (except scion dry weight in 120 mm salt) was significantly higher than fresh and dry weight of ‘sahand’ grafted on arjan (fig. 3 b and d). in both rootstocks, increasing the salt from 0 to 120 mm reduced the fresh weight of the scion by about 60% while this reduction in the dry weight of scion was 62% on badamkohi rootstock and 55% on arjan rootstock (fig. 3 b and d). effect of salinity on na+ and cl‐ contents in micro‐ grafting combinations the results showed that the na+ and cl­ contents in the roots and the shoots of both micrografting combinations increased with increasing salinity (fig. 4 a, b, c, d,). however, the amount of na+ and cl­ in the shoot and the root of ‘sahand’/arjan plants were sig­ fig. 2 ­ growth of a micrografted combination of ‘sahand’/ badamkohi in control (a) and in 120 mm salinity (b and c); a micrografted combination of ‘sahand’/arjan in con­ trol, (d) and 120 mm salinity (e and f). table 1 ­ effect of sodium chloride on scion and root length in micrografting combinations of ‘sahand’/badamkohi and ‘sahand’ /arjan micrografting combination nacl mm root length (mm) shoot length (mm) ‘sahand’/badam kohi 0 263 a 64 a 40 202 b 44 b 80 148 c 38 bc 120 130 d 30 d ‘sahand’/arjan 0 122 ed 41 b 40 114 edf 33 cd 80 106 ef 29 d 120 100 f 21 e in each column, means with the same letters are not significantly different at 5% probability level using lsd test. 68 adv. hort. sci., 2024 38(1): 63­74 nificantly higher than these ions in the shoot and the root of ‘sahand’/badamkohi plants at 80 and 120 mm nacl. total chlorophyll (chl) the chl of ‘sahand’ grafted on both rootstocks decreased with increasing salt concentration. the highest amount of chl was obtained in ‘sahand’ (as scion) leaf grafted on badamkohi rootstock in unsalt­ ed medium (control) which showed a significant dif­ ference compared to the same scion grafted on arjan rootstock. the amount of chlin ‘sahand’ grafted on badamkohi was 0.68 mg g­1 fw which was significant­ ly higher than the chl of the same scion grafted on arjan rootstock (0.51 mg g­1 fw) at 120 mm nacl (table 2). at the highest salinity level, the leaf chloro­ phyll content of ‘sahand’ either grafted on badamkohior arjan was decreased by 51% and 63% respectively compared to their controls. electrolyte leakage (el) in all levels of salinity, the el of ‘sahand’ grafted on arjan rootstock was significantly greater than the same cultivar grafted on badamkohi. the highest leaf cell el (72.29%) occurred in ‘sahand’ grafted on arjan rootstock which was significantly higher than leaf el of the same scion grafted on badamkohi rootstock at 120 mm nacl (table 2). proline and glycine betaine (gb) in micrografting combination increasing the level of salinity caused a significant rise in the proline content of both grafting combina­ tions (‘sahand’/badamkohi and ‘sahand’/arjan) (table 3). the grafting combination of ‘sahand’/ badamkohishowed a higher proline content (in both parts, scion and rootstock) compared to the grafting combination of ‘sahand’/arjan in all salinity levels. the results also indicated that by increasing the salt concentration from 0 to 120 mm, the amount of gb increased in both micrografting combinations. the shoot and the root in micrografting combination of ‘sahand’/badamkohi showed significantly greater gb than those in micrografting combination of ‘sahand’/arjan in all level of salinity (except for root gb in salt free medium of both rootstocks). fig. 3 ­ the effect of different concentrations of nacl on fresh and dry weight of micrografting combinations of ‘sahand’/ badamkohi and ‘sahand’/arjan. different let­ ters indicate significant difference at p ≤ 0.05 level of probability using lsd test. table 2 ­ effects of sodium chloride on leaf total chlorophyll and electrolyte leakage in micrografting combinations fig. 4 ­ the na+ and cl­ contents of both rootstocks (a, b) and scion (c, d) in grafting combinations of ‘sahand’ /badamkohi and ‘sahand’/arjan in different concentra­ tion of nacl. different letters indicate significant differ­ ence at p ≤ 0.05 level of probability using lsd test. micrografting combination nacl mm total chlorophyll (mg g­1 fw) electrolyte leakage (%) ‘sahand’/badam kohi 0 1.45 a 23.28 e 40 1.42 ab 31.32 de 80 0.95 bc 38.05 cd 120 0.68 d 43.86 c ‘sahand’/arjan 0 1.33 b 38.15 cd 40 1.25 bc 43.77 c 80 0.77 d 55.15 b 120 0.51 e 72.29 a in each column, means with the same letters are not sig­ nificantly different at 5% probability level using lsd test. asadi zargh abad and shekafandeh ‐ in vitro salt tolerance of different almond graft combinations 69 superoxide dismutase (sod) sod activity in both‘sahand’/badamkohi and ‘sahand/arjan plants increased with increasing the levels of salinity (table 4). the highest increase in the activity of sod was obtained in the ‘sahand’ /badamkohi, combination with 101.27 and 103.30 (u g­1 fw min­1) in shoot and root respectively at 120 mm salt, which was significantly higher than those in ‘sahand/arjan plants. peroxidase (pox) in the shoots and in the roots of both micrograft combinations, pox activity increased with increasing salt concentrations in the medium. although, at all salinity levels, pox activity was higher in the shoot and the root of micrografting combination of ‘sahand’/bada kohi than ‘sahand’/ajan, however the difference was only significant at 120 mm nacl. catalase (cat) both micrografting combinations showed the enhancement cat activity in response to increasesalt concentrationfrom 0 to 120 mm nacl. the highest cat activity occurred in the shoot (63.70 u g­1 fw min­1) androots (59.70 u g­1 fw min­1) of ‘sahand’/ badamkohi plants at 120 mm nacl which was signifi­ cantly higher than the activity of this enzyme in the ‘sahand’/arjan plants (table 4). table 4 ­ effects of sodium chloride on the shoot and root enzymes activities (sod, pox and cat) in micrografting combinations of ‘sahand’/ badamkohi and ‘sahand’/arjan table 3 ­ effects of sodium chloride on the amounts of proline and glycine betaine (gb) in the shoots and roots of micrografting combi­ nations micrografting combination nacl levels (mm) root gb µmol g­1 dw shoot gb µmol g­1 dw root proline µmol g­1 fw shoot proline µmol g­1 fw ‘sahand’/bdam kohi 0 10.55 ef 6.88 cd 24.93 e 20.64 d 40 14.60 d 7.48 c 38.03 c 27.73 c 80 19.00 b 8.90 b 46.70 b 40.88 b 120 22.60 a 11.00 a 62.40 a 55.38 a ‘sahand’/arjan 0 9.30 f 5.70 e 14.80 f 13.22 e 40 11.47 e 6.30 de 21.70 ef 18.97 de 80 14.02 d 6.80 cd 28.9 de 24.06 cd 120 16.50 c 7.60 c 34.50 cd 30.40 c in each column, means with the same letters are not significantly different at 5% probability level using lsd test. micrografting combination nacl (mm) sod shoot u g­1 fw min­1 sod root u g­1 fw min­1 pox u g­1 fw min­1shoot pox root u g­1 fw min­1 cat shoot u g­1 fw min­1 cat root u g­1 fw min­1 ‘sahand’/badam kohi 0 90.10 e 76.60 c 31.62 ed 51.60 fe 40.60 cd 32.50 ef 40 92.90 d 77.30 bc 48.81 bc 60.30 cde 41.80 cd 44.90 bc 80 96.10 c 80.00 bc 50.70 b 69.80 bc 46.90 bc 48.70 b 120 101.27 a 103.30 a 76.99 a 95.00 a 63.70 a 59.70 a ‘sahand’/arjan 0 87.60 f 72.10 c 23.40 e 48.10 f 39.60 d 31.30 f 40 92.20 d 75.20 c 36.60 cd 58.80 de 41.10 cd 38.60 de 80 95.20 c 78.00 c 45.60 bc 67.20 bcd 43.60 bcd 42.40 cd 120 97.60 b 85.60 b 47.80 bc 73.50 b 50.50 b 46.20 bc in each column, means with the same letters are not significantly different at 5% probability level using lsd test. adv. hort. sci., 2024 38(1): 63­74 70 4. discussion and conclusions performance of grafted plants compared to non­ grafted or self­grafted plants under a stressful condi­ tion is often dependent on the rootstock’s root sys­ tem characteristics. a vigorous root system could be the most important criterion for increasing salt toler­ ance (balliu et al., 2007). in this research, badamkohi showed the highest root length of 263 mm that was significantly higher than arjan (122 mm). the root­ stock’s root systems architecture specified by root length and density, root hairs and root surface area play a critical role in ion and water uptake, thus determining salt tolerance of grafted plants (colla et al., 2010). a vigorous root system, for instance, pro­ duced more cytokinins and transported water to the shoot system by xylem sap, which positively affected plant growth and crop yield (oztekin and tuzel, 2011). furthermore, hydraulic conductivity of the roots may control plant growth by manipulation of the water supply to epigeous plant parts (gregory et al., 2013). the most immediate effect of salinity on plants is the inhibition of root and shoot development. due to the imbalance of water potential between the apoplast and simplast, the osmotic potential decreas­ es and the absorption of water is hampered. ultimately, this reduces plant growth by closing the stomata and weakening photosynthesis (dustgeer et al., 2021). fresh and dry weight of plants decrease with increasing salinity, which is usually due to ion toxicity and water stress (arif et al., 2020; corell et al., 2020). salinity has the effects on metabolic activi­ ty and reduces the division of new cells and disrupt cellular processes, thus reduces plant growth com­ pared to normal conditions (carillo et al., 2019). in this study, as mentioned before, the root system of badamkohi was stronger than arjan. however, in high level of salinity, the shoot length of the same scion (‘sahand’) on badamkohi was greater than on arjan. it seems that the arjan rootstock has a dwarf­ ing effect on the scion. on the other hand, at all salin­ ity levels, the fresh and dry weight of the scion on badamkohi was more pronounced than on arjan. this means that badamkohi supports scion growth better than arjan. it has been also reported some rootstocks are more capable of inducing tolerance to the scion against salt stress (zrig et al., 2016; aras and eşitken, 2018). in this regard, adding sodium chloride to the growth medium of two graft combinations (‘sahand’/badamkohi and ‘sahand’/arjan) led to a decrease in scion growth, however this growth reduction of ‘sahand’ subjected to increasing concen­ trations of nacl was more acute when the rootstock was arjan. this result is in consistence with the results of sweet almond grafted on different root­ stocks (zrig et al., 2016). sodium ion (na+) is toxic to cellular metabolism and affects the activity of some enzymes, and high concentrations of na+ causes ion imbalance (roy et al., 2014). the ability of almond rootstocks varies in absorb­ ing or transferring sodium to the scion and there is a very close relationship between tolerance to salinity and the amount of sodium transferred to the leaves (mickelbart and arpaia, 2002). in the present research, na+ concentration in the aerial parts was lower in ‘sahand’/ badamkohi compared to ‘sahand’/arjan. such a prohibiting mechanism may explain, the higher shoot length and biomass of ‘sahand’/badamkohithat observed in our study. a higher na+ concentration in the environment of root can depress k+/na+ ratios in the plant, thereby, the plant becomes susceptible to specific ion injury as well as to nutritional disorders which may affect growth and yield. the exclusion of cl­ from shoots is related to the ability of cell membranes to restrict the movement of cl­ through the root to vascular tis­ sue and the degree of cl­ accumulation in the roots (walker and douglas, 1983). in this experiment, badamkohi rootstock was able to slow the accumula­ tion of cl­ in the leaves. similar results have been reported by garcía­sánchez et al. (2002) in which the cleopatra rootstock reduced the accumulation of cl­ in the scion compared to the carrizo rootstock. the finding of this experiment showed that badamkohi rootstock hold up more the chloroplast integrity of ‘sahand’ than arjan rootstock. chlorophyll depletion in salt stress can be linked to factors such as structural damage of chloroplasts due to the formation of reactive oxygen species and photo oxidation of chlorophyll (taïbi et al., 2016), the destruction of chlorophyll synthesis precursors, the inhibition of biosynthesis of new chlorophylls, and hormone disorders (sabzmeydani et al., 2020). this reduction could also be due to the increase in the activity of the enzyme chlorophyllase or to the insta­ bility of the protein pigment complexes by the ions (saha et al., 2010). surendar et al. (2013) reported a decrease in chlorophyll content under stress was caused to the destruction of the chloroplast mem­ asadi zargh abad and shekafandeh ‐ in vitro salt tolerance of different almond graft combinations 71 brane with increasing phosphatase activity, which is located on the membrane. plasma membranes are the primary site of ion­ specific salt injury. undesirable performance of the cell’s metabolism during periods of abiotic stress leads to the stimulation of reactive oxygen species which would damage the cell membrane and increase electrolyte leakage. therefore, electrolyte leakage from plasma membranes is reported as one of the most important selection criteria for identifica­ tion of salt­tolerant plants (besma and denden, 2012). in this study, especially at higher levels of salinity, the ‘sahand’ grafted on arjan experienced a greater damage to the cell membrane of its leaves, than grafted on badamkohi. this indicates badamkohi rootstock’s ability to maintain the integri­ ty of scion cell membrane in salt stress conditions. in accordance with our finding, colla et al. (2012) reported that in cucumber grafted plants, the amount of ion leakage in salinity stress is reduced compared to non­grafted plants and rootstock helps the maintenance of membrane function. in this study, the grafting combination of ‘sahand’/badamkohi accumulated more proline and glycine betaine in the root and shoot, compared to the same scion grafted on arjan. proline accumulation in salinity condition can play a role in stress tolerance mechanismsby stabilizing proteins at high ionic con­ centrations (krasensky and jonak, 2012). it also reduces damages caused by salinity via the preserva­ tion of water in cells and by diluting salts in the plant (gulen et al., 2018). proline has essential functions by osmoregulation, reducing the undesirable effects of ross under salinity stress.therefore, higher proline accumulation can induce higher tolerance against salinity by the plant (akbari et al., 2018). the proline content of micrografting combination significantly increased in response to an increase in the salinity level, in this regards ‘sahand’/badamkohi micrograft combination was more prominent than ‘sahand’/arjan. our finding is in agreement with other reports on in vitro salt tolerance of pistachio (raoufi et al., 2020) and fig (abdoli nejad and shekafandeh, 2014). glycine betaine was also found to play signifi­ cant roles in enhancing salt tolerance (wei et al., 2017). it can maintain the osmotic regulation, improve the production, nutrients and water absorption, there­ by photosynthetic proteins are produced and mem­ brane peroxidation is reduced (dustgeer et al., 2021). in this research, the grafting combination of ‘sahand’/badamkohi accumulated more glycine betaine in the root and shoot, compared to the graft­ ing combination of ‘sahand’/arjan, that means badamkohi protects cell osmotic pressure, enhancing cell membrane integrity as well as maintaining photo­ synthetic apparatus (niazian et al., 2021). as mentioned above, with increasing salinity lev­ els, badamkohi rootstock enhanced sod, pox and cat activity in ‘sahand’ scion more than arjan root­ stock. sod plays a major role in ros scavenging in plants and is considered as the first line of defense against the toxic effects of elevated ros (hou et al., 2019). sod catalyzes the dismutation of superoxide radicals to h2o2 and o2 (feng et al., 2015). increasing the level of superoxide dismutase activity is impor­ tant for protecting chloroplasts and mitochondria from the stress of reactive oxygen species. in fact, under stress conditions, the chloroplast is where the majority of active oxygen species are produced and where is caused the highest degree of damage (sofo et al., 2005; kuşvuran et al., 2016). according to the results of the present study, salinity stress caused the sod activity to increase sig­ nificantly in both micrograft combinations (‘sahand’/badamkohi and ‘sahand’/arjan). nonetheless, badamkohi rootstock enhanced the sod activity in ‘sahand’ more than arjan rootstock. enhanced activities of sod enzyme usually reflect defensive responses to cellular damage induced by higher nacl concentrations in the culture medium (akbari et al., 2018; kuşvuran et al., 2021). the enzyme hydrogen peroxidase (pox) also reduces oxidative stressby protecting the metabolic enhancers that sustain cell and plant survival (aliakbarkhani et al, 2017). the results of this study showed that by increasing salinity levels in the growth medium of the two grafted combinations, there was an increase in the activity of peroxidase enzyme and this increment was more obvious in ‘sahand’ grafted on badamkohi rootstock. according to fayek et al. (2018), the activity of pox enzyme changes with different scions graftedon different rootstocks. an increase in pox activity in grafted plants could be an indicator that the grafting process can rapidly induce a higher capacity to breakdown h2o2 in plant cells (elsheery et al., 2020). catalase is one of the most important enzymes that can inhibit ros activity. it converts hydrogen peroxide to water and oxygen in the mitochondria, peroxisomes and cytosol (acosta­motos et al., 2017). based on the results of this study, with increasing salinity levels, the activity of catalase increased in adv. hort. sci., 2024 38(1): 63­74 72 both grafted combinations showing that tolerance to salinity corresponds with an increase in catalase enzyme of the plants, although badamkohi rootstock was more tolerant than arjan. the greater availability of cat can enable the plant defense mechanism to increase the capability of eliminating reactive oxygen species (madadkhah et al., 2018). our finding corre­ sponds with those reported previously in pistachio (akbari et al., 2018) and cherry (chatzissavvidis et al., 2008). in all levels of salinity ‘sahand’as scion grafted on badamkohi had more shoot length, fresh and dry weight than grafted on arjan. badamkohi restricted the absorption of na+ and cl­ ions from the root medi­ um and reduced their transportation to aerial parts. the ‘sahand’ leaf chlorophyll depletion and el were higher on arjan rootstock than on badamkohi in all level of salinity condition. badamkohi protected cell osmotic pressure, enhancing cell membrane integrity than arjan by inducing more proline and gb as osmo­ protectants in the shoots. badamkohi rootstock also enhanced the activities of antioxidant enzymes in ‘sahand’ more than arjan rootstock which reflect defensive responses to cellular damage induced by reactive oxygene species in higher nacl concentra­ tions in the culture medium. in conclusion, badamkohi could be a more suitable rootstock than arjan for ‘sahand’ scion under salinity conditions. reference abdolinejad r., shekafandeh a., 2014 ­ responses of two figs (ficus carica l.) cultivars under salt stress via in vitro condition. ­ a. sci. dev., 3: 194­199. acosta­motos j.r., ortuño m.r., bernal­vicente a., diaz­vivancos p., sanchez­blanco m.j., hernan­ dez j.a., 2017 ­ plant responses to salt stress: adaptive mechanisms. ­ agronomy, 7(1): 1­18. akbari m., mahna n., ramesh k., bandehagh a., maz­ zuca s., 2018 ­ ion homeostasis, osmo regulation, and physiological changes in the roots and leaves of pista‐ chio rootstocks in response to salinity. ­ protoplast, 255(5): 1349­1362. aliakbarkhani s.t., farajpour m., asadian a.h., aali­ far m., ahmadi s., akbari m., 2017 ­ variation 479 of nutrients and antioxidant activity in seed and exocarp layer of some persian pistachio genotypes. ­ ann. agri. sci., 62(1): 39­44. alizadeh m., singhs.k., patelv.b., bhattacharya r.c., yadav b.p., 2010 ­ in vitro responses of grape root‐ stocks to nacl. ­ biol. plant., 54(2): 381­385. ansari a., gharaghani a., 2019 ­ a comparative study of genetic diversity, heritability and inter‐relationships of tree and nut attributes between prunus scoparia and p. elaeagnifolia using multivariate statistical analysis. ­ inter. j. hort. sci. techn., 6(1): 137­150. aras s., eşitken a., 2018 ­ physiological responses of cherry rootstocks to short term salinity. ­ erwerbs­ obstbau, 60: 161­164. arif y., singh p., siddiqui h., bajguz a., hayat s., 2020 ­ salinity induced physiological and biochemical changes in plants: an omic approach towards salt stress tolerance. ­ plant physiol. biochem., 156: 64­77. asadi m., shekafandeh a., 2021 ­ in vitro grafting of ‘sahand’ cultivar on two wild almond rootstocks and evaluation of its some physiological and biochemical traits vis‐a‐vis different rootstocks. ­ plant cell tissue org. cult., (pctoc) 145: 507­516. bado s., forster b.p., nielen s., ali a.m., lagoda p.j.g., till j., laimer m., 2015 ­ plant mutation breed‐ ing: current progress and future assessment, pp. 23­87. ­ in: janick j. 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http://https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=chang%2c+yuru http://https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=brecht%2c+jeffrey+k http://https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=sarkhosh%2c+ali http://https://onlinelibrary.wiley.com/journal/20487177 impaginato 257 adv. hort. sci., 2024 38(3): 257­272 doi: 10.36253/ahsc­15664 https://oaj.fupress.net/index.php/ahs morphological and molecular characterization of some chrysanthemum (dendranthema grandiflora) cultivars y.i. el­nashar ornamental plants and landscape gardening research department, alexandria 21554, horticultural research institute, a.r.c., giza, egypt. key words: flower yield traits, gas exchange, pigments, ssr markers, vegetative growth traits. abstract: the diversity and genetic relationships among seven commercial chrysanthemum cultivars were analyzed using morphological and molecular markers. vegetative growth, flowering, flower yield, and flower quality parameters were evaluated to assess genetic variability across the cultivars. cultivars crystal red, kodiack, and crystal white exhibited superior vegetative growth, while abrun, crystal red, and kodiack displayed better flowering characteristics, particularly in terms of the number of inflorescences per plant and mass of colored flowering. crystal white, coca bleach, and crystal red cultivars demonstrated the highest inflorescence stalk length, while cvs. crystal red, crystal yellow, crystal pink, and kodiack yellow recorded the maximum number of ray floret inflorescences. other quality parameters such as inflorescence diameter and ray floret length were found to be optimal in kodiack, crystal white, crystal pink, coca bleach, and crystal yellow cultivars. simple sequence repeat (ssr) markers were employed to distinguish and identify standard­type chrysanthemum cultivars, utilizing twelve ssr markers from the chrysanthemum ssr database. the results suggest that these ssr markers hold promise for identifying additional chrysanthemum cultivar types and assessing genetic relationships among them. association studies combining morphological and molecular data offer a valuable approach to identifying informative markers for plant breeding purposes. 1. introduction chrysanthemum (mums) (dendranthema grandiflora tzvelev, formally, chrysanthemum morifolium ramat.) is one of the most important ornamental crops grown worldwide. it belongs to the family compositae (asteraceae) and has been commonly cultivated in gardens for more than 2500 years (bose et al., 2003). it is produced on a large scale as a cut flower or as a potted plant due to its commercial significance (van der (*) corresponding author: yelnashar@hotmail.com citation: el­nashar y.i., 2024 ­ morphological and mole‐ cular characterization of some chrysanthemum (dendranthema grandiflora) cultivars. ­ adv. hort. sci., 38(3): 257­272. orcid: yie­n: 0000­0002­3417­5664 copyright: © 2024 el­nashar y.i. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 7 january 2024 accepted for publication 18 july 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15664 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-3417-5664 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 257­272 258 ploeg and heuvelink, 2006). analyzing genetic variability in chrysanthemum is essential for breeding programs as it can provide data on genetic relationships among different genotypes of this genus. among the available strategies for assessing genetic variability, molecular markers are the most widely applicable, as they are best suited for understanding the genome and can be used for genetic variability characterization, paternity testing, elucidating genetic relationships between genotypes, developing methods for maintaining genetic variability in germplasm banks, and identifying genes or combinations of features related to key biological and agronomic traits (hayden et al., 2010). another approach to studying genetic variability is the analysis of morphological and phenotypic characters, as these methods are relatively simple to perform. however, analysis based solely on morphological features may not be conclusive due to the limited number of characters and the strong influence of plant development stage and environmental factors. morphophenological characterization does not replace molecular analyses but may complement both characterization and genetic variability studies and cultivar development (fufa et al., 2005). in contrast, molecular markers based on dna sequence polymorphisms are unaffected by environmental factors and exhibit high rates of polymorphism. while morphological markers reflect variation in the coding regions of the genome, dna­based molecular markers represent variations occurring in various regions of the genome, including coding and non­coding regions. thus, molecular markers provide a rapid and reliable method to estimate genetic relationships between genotypes (tatikonda et al., 2009). molecular characterization has been widely used to quantify genetic variability among different accessions comprising germplasm banks (glaszmann et al., 2010), enabling researchers to elucidate the genetic structure and diversity in a wide range of plant species (kilian et al., 2007; leišová et al., 2007). various methods have been employed to evaluate genetic diversity, with morphological character measurement being a commonly used index due to its simplicity in quantifying genetic variation while simultaneously assessing genotype performance under normal growing conditions (fu et al., 2008). however, investigating morphological traits is labor­ intensive, and the phenotypic plasticity of plants poses challenges due to environmental variation (van beuningen and busch, 1997). in contrast, molecular markers offer several advantages over morphological measurement for assessing genetic diversity. assessing genetic variability is crucial in breeding programs, with molecular markers providing a direct means to access genome sequences and enabling the isolation of genetic differences from environmental influences (ferrão et al., 2007). simple sequence repeats (ssr) markers have shown potential in assessing genetic diversity among chrysanthemum species, cultivars, and germplasm bank collections, as well as determining geographical origin, level of domestication, dispersal history, species and cultivar identification, and genealogy (lopez­gartner et al., 2009; hong et al., 2013; hong et al., 2016). ssr markers offer several advantages over other markers such as rapd, aflp, srap, and issr, including co­dominance, multi­allelic nature, abundance, and wide distribution across the genome, making them easy to score (powell et al., 1996; feng et al., 2016). various ssr databases have been constructed and utilized for purposes such as cultivar identification, seed purity tests, and determining parent­offspring relationships in crops like citrus and pear (kim and nou, 2016; nguyen et al., 2019). recent studies have also employed ssr markers for variety identification in chrysanthemum and other crops (caramante et al., 2011; zhang et al., 2014). chrysanthemum, particularly standard­type cultivars with long, sturdy stems and large flowers, is a commercially significant crop, valued highly as cut flowers and for flower arrangements. therefore, accurate genetic identification and fingerprinting of these cultivars are crucial for safeguarding breeders’ intellectual property rights (manjulatha et al., 2020). the promising potential of ssr markers in assessing chrysanthemum diversity has prompted this research endeavor. the objectives of this study were to: (a) compare morphological analysis and molecular markers (ssr) of seven commercial chrysanthemum cultivars and provide molecular data to assess genetic relationships among accessions, and (b) declare the genetic diversity among cultivars. 2. materials and methods plant material and experimental site seven commercial chrysanthemum (dendran‐ el‐nashar ‐ phenological and molecular diversity on chrysanthemum 259 thema grandiflora tzvelev) cultivars were selected for morphological and molecular characterization: c1 ­ crystal pink (violet), c2 ­ crystal white (white), c3 ­ crystal yellow (yellow), c4 ­ crystal red (dark purple red), c5 ­ kodiack (yellow), c6 ­ coca bleach (brown red), and c7 ­ abrun (violet) (table 1 and fig. 1). the investigation was conducted in a greenhouse at the nursery of the plant production department, college of food and agriculture sciences, king saud university, riyadh, saudi arabia, during the period 2019­2020. planting method and experimental design uniform rooted cuttings of the seven chrysanthemum cultivars, each measuring 7 cm in height with 5­6 true leaves, were selected. nine rooted cuttings per cultivar were then planted on february 2nd, 2019 (first growing season), and february 4th, 2020 (second growing season), by placing them in 6­inch diameter plastic pots (one cutting per pot) filled with a mixture of peat and perlite growing media (2:1 by volume). the minimum day and night temperatures in the greenhouse were 18°c, with the ventilators opening at 22°c. short days (10 hrs light) and daytime relative humidity were set at 70%, and the area was maintained shaded with black polythene sheets. once the cuttings were established, they were decapitated (pinched) above the 3rd ­ 4th leaf from the base to encourage the production of lateral shoots. fig. 1 ­ standard­type chrysanthemum cultivars used in this study. table 1 ­ list of chrysanthemum cultivars used in this study and their inflorescence colors no. code cultivars royal horticultural society color chart no. inflorescence color 1 c1 crystal pink 77d violet, light center 2 c2 crystal white n155d white, yellow center 3 c3 crystal yellow 5c yellow 4 c4 crystal red 53a dark purple red 5 c5 kodiack 4b yellow 6 c6 coca bleach 179a brown red, yellow green center 7 c7 abrun n78a dark violet, yellow green center the pinching procedure ensured that the apical meristems of all plants started active growth at the same time under the same conditions, increasing the uniformity of flowering (cockshull, 1976). a chemical growth retardant, b­nine (crompton uniroyal chemical co., washington, dc, usa), was applied as an aqueous solution with concentrations of 2000 ppm via foliar spray until runoff. this treatment was repeated three times at one­week intervals, starting three weeks after planting on february 23rd, march 2nd, and march 9th, respectively. a slow­release fertilizer, osmocote (the scotts co., marysville, oh, usa), was applied at the rate of 140 mg kg­1 soil of media, which contained nitrogen (n), phosphorus (p2o5), and potassium (k2o), in the ratios of 17:11:10, respectively (el­ nashar, 2013). adv. hort. sci., 2024 38(3): 257­272 260 the experiment was conducted using seven chrysanthemum cultivars in a completely randomized design (crd) with three replications. each cultivar represented one treatment, and each replication included three plants of a cultivar. morphological characteristics plant characterization took place in late april, and the morphological evaluation was carried out when the plants were in full bloom. observations regarding the color of the flowers were recorded with the assistance of the royal horticultural society (rhs) color chart (rhs, 1966; dorling, 2008). all cultivars were observed and divided into two parts: the vegetative parts and the inflorescence parts. the number of characters that differed from each other was scored to determine the distinctiveness and uniformity of the plant under investigation. the data from all plants were compared to identify any variations between cultivars. plant development and growth were recorded per pot/plant unit during both the 2019 and 2020 growing seasons by measuring the following parameters: plant height (cm) (ph), number of branches (nl), number of leaves (nl), leaf area (cm2) (la), using a leaf area meter (li­cor, lincoln, 404, ne), shoot fresh and dry weights (g) (sfw and sdw), root fresh and dry weights (g) (rfw and rdw), root length (cm) (rl), leaf area of one leaf 10 cm from plant height (cm2) (la10), leaf width (cm) (lw), and leaf length (cm) (ll). additionally, flower production per pot/plant unit was monitored, taking into account the following traits: number of inflorescences (flower yield) (ni), inflorescence diameter (cm) (id), total inflorescences fresh and dry weights (g) (ifw and idw), inflorescence stalk length (cm) (isl), number of inflorescences per branch (nib), number of ray florets per inflorescence (nrfi), length of ray florets per inflorescence (cm) (lrfi), and fresh and dry weights of a single inflorescence (g) (sfwi and sdwi) using a precision balance (kern, 440­47n, balingen, southern germany). fresh weight was carefully recorded after removing the plants (88 days from planting). dry weight trait was determined after drying the plant material in a dry oven for 48h at 70°c until the weight became constant. photosynthetic pigments extraction of photosynthetic pigments (chlorophyll (chl.) a and b) from leaves were implemented using n, n­ dimethylformamide (dmf) method. chls a, b, chls a+ b concentrations in μmol l­1 and chls ratio were then estimated utilizing the equations of porra et al. (1989) as follows: chl a = 13.43 w 663.8 ­ 3.47 w 646.8 chl b = 22.90 w 646.8 ­ 5.38 w 663.8 total chlorophyll (chl a+b) = 19.43 w 663.8 ­ 8.05 w646.8 chls a and b concentrations were estimated spectrophotometrically using uv spectrophotometer (pharmacia biotech ultrospec 2000). moreover carotenoid = (1000 w 480 − 0.89 chl a − 52.02 chl b)/245 (wellburn, 1994; vicaş et al. 2010) and anthocyanin = w 530 ­ 0.25 w 657 were taken into account (mancinelli, 1994). gas exchange the assessment of the leaves’ gas exchange was conducted using a portable photosynthesis system, known as the li­cor 6400, manufactured by li­cor inc., based in lincoln, u.s.a. the evaluation of net photosynthetic rate (pn), stomatal conductance of water (gs), transpiration rate (e), and the intercellular co2 concentration (ci) was carried out on fully expanded fourth leaves between 10:20 and 11:30 am on a sunny day with a humidity level of approximately 60±5%. the measurements were taken at an ambient temperature of 27°c and under a photosynthetic photon flux density (ppfd) of about 720 μmol m­2 s­1. the co2 concentrations were compared to the reference levels present in the growth chamber. dna extraction fresh young leaf tissues were collected from all chrysanthemum cultivars, then frozen in liquid nitrogen in a mortar, and stored at ­80°c. the genomic dna was extracted utilizing the dneasy plant mini kit, manufactured by qiagen in germany. subsequently, the quality of the dna was assessed through electrophoresis on a 1% agarose gel, while the dna concentration was determined using quick drop, a product of molecular devices in the united states. the dna was then appropriately diluted to a concentration of 25 ng μl−1 and employed for ssr analysis. ssr analysis seven standard­type chrysanthemum cultivars were classified and identified using a total of twelve ssr markers. detailed information about these el‐nashar ‐ phenological and molecular diversity on chrysanthemum 261 markers is provided in table 2. each ssr marker was amplified separately in a reaction volume of 25 µl. this reaction volume included 8 μmol of each forward (5’ fam labelled) and reverse primer, 50 ng of total genomic dna, 2 mm mgcl2, 10 mm tris­hcl (ph = 7.5), 50 mm kcl, and 0.3 u/μl of taq dna polymerase in 1 x pcr supplied buffer. the pcr reaction took place in a thermocycler (applied biosystems, veriti, c.a., u.s.a.) following these cyclic parameters: one cycle of 3 min at 94°c, 45 cycles of about 1 min at 94°c, 1 min at 50 to 60°c, 2 min at 72°c, and a final extension for 10 min at 72°c. the pcr product was then analyzed by checking 2 µl of it on a 2% agarose gel electrophoresis. to detect the product, dna loading star (dyne bio, s. korea) was used. images were captured and photographed following the application of ethidium bromide stain on a gel deposition apparatus. data analysis concerning morphological and physiological analyses, the average and standard deviation values were calculated by one­way analysis of variance (anova) using the statistical analysis software computer program (sas institute inc., cary, nc). the least significant difference (lsd) procedure was used to determine significant differences among the means of cultivars at the 0.05 significance level (steel et al., 1997). regarding genetic data analyses, power marker was used to calculate the total number of alleles, genetic diversity, heterozygosity, allele frequency, and polymorphism information content (pic) for each ssr locus (liu and muse, 2005). the ssr amplification bands were assigned a score of 0 for absence and 1 for presence. a simple matching similarity index was utilized to calculate the similarity of the qualitative data. the genetic similarity data were subjected to cluster analysis using the unweighted pair group method of arithmetic averages (upgma), and a dendrogram was generated using dendroupgma software. principal component analysis (pca) was conducted using the software past (version 3.14). 3. results plant vegetative growth significant differences were observed among the various cultivars in terms of plant height, number of leaves per plant, leaf area, and leaf length in both seasons (table 3). during the first season, the mean plant height ranged from 18.03 to 23.76 cm, while in the second season, it ranged from 17.98 to 23.03 cm. the cultivar crystal red recorded the highest plant height, followed by coca bleach, while crystal pink exhibited the shortest plant height, followed by ‘brun’ (table 3). the mean number of branches per plant and leaf width was not significantly affected by the compared cultivars in both seasons. the number of leaves per plant ranged from 27.33 to 33.07 for cv. coca bleach and 52.02 to 60.00 for cv. crystal red respectively. ‘abrun’ exhibited the lowest leaf area (231.39 and 230.02 cm2) in both seasons, while the largest leaf area was detected for cv. crystal red (390.53 and table 2 ­ list of twelve ssr primers that were screened to distinguish the seven standard­type chrysanthemum cultivars s. no. ssr primers primer sequence forward reverse 1 xcfd1 5' accaaagaacttgcctggtg 3' 5' aagcctgacctagcccaaat 3' 2 xgwm205 ' cgacccggttcacttcag 3'5 5' agtcgccgttgtatagtgcc 3' 3 xgwm133 5' atctaaacaagacggcggtg 3' 5' atctgtgacaaccggtgaga 3' 4 xcfd9 5' ttgcacgcacctaaactctg 3' 5' caagtgtgagcgtcgg 3' 5 xcfd46 5' tggtggtatagtcgttggagc 3' 5' ccacacacacacaccatcaa 3' 6 xgwm181 'tcattggtaatgaggagaga 3'5 5' gaaccattcatgtgcatgtc 3' 7 xcfd49 5' tgagttcttctggtgaggca 3' 5' gaatcggttcacaagggaaa 3' 8 xgwm174 5' gggttcctatctggtaaatccc 3' 5' gacacacatgttcctgccac 3' 9 xcfd18 5' catccaacagcaccaagaga 3' 5' gctactactatttcattgcgacca 3' 10 xcfd183 5'acttgcacttgctatacttacgaa3' 5' gtgtgtcggtgtgtggaaag 3' 11 xgwm210 5' tgcatcaagaatagtgtggaag 3' 5' tgagaggaaggctcacacct 3' 12 xcfd66 5' aggtcttggtggttttggtg 3' 5' ttttcacatgcccacagttg 3' 262 adv. hort. sci., 2024 38(3): 257­272 455.31 cm2) in both seasons. ‘crystal red’ recorded the maximum leaf area followed by cv. crystal white, while the least leaf area was recorded in ‘abrun’ followed by ‘coca bleach’. leaf length ranged from 6.90 to 6.80 cm for ‘crystal pink’ and 9.30 to 9.23 cm for ‘crystal red’ respectively (table 3). the mean values of the leaf area of one leaf 10 cm from the plant height did show significant differences among plant cultivars in both seasons (table 4 and fig. 2). ‘crystal pink’ exhibited the lowest leaf area (9.34 and 13.10 cm2) in both seasons, while the largest leaf area was detected in ‘crystal red’ (19.99 and 20.46 cm2) in both seasons. the mean values of the compared cultivars did not show any significant table 3 ­ plant height, number of branches, number of leaves, leaf area, leaf width and leaf length of the seven studied chrysanthemum cultivars cultivars vegetative growth character plant heigh (cm) branches (no.) leaves (no.) leaf area (cm2) leaf width (cm) leaf length (cm) 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 crystal pink 18.03 c 17.98 b 3.73 a 3.37 a 37.67 bc 42.07 308.80 256.10 c 4.71 a 4.80 a 6.90 bc 6.80 d crystal white 20.77 bc 20.83 ab 4.37 a 4.73 a 42.01 ab 42.03 380.88 a 377.17 4.43 a 5.11 a 8.20 ab 8.83 ab crystal yellow 18.23 c 20.23 ab 3.74 a 3.73 a 38.67 bc 44.34 bc 244.65 b 289.96 3.91 a 4.60 a 6.71 c 7.43 bcd crystal red 24.43 a 23.03 a 4.36 a 4.77 a 52.02 a 60.00 a 390.53 a 455.31 a 5.23 a 4.97 a 9.30 a 9.23 a kodiack 22.43 ab 20.27 ab 3.43 a 4.03 a 49.01 ab 36.67 cd 380.24 a 313.16 4.67 a 4.96 a 6.86 bc 7.10 cd coca bleach 23.76 a 22.93 a 3.07 a 2.76 a 27.33 c 33.07 d 252.00 b 270.12 4.43 a 4.73 a 7.1 bc 8.66 abc abrun 19.01 c 18.04 b 3.70 a 3.37 a 47.32 ab 49.77 b 231.39 b 230.02 c 4.56 a 4.23 a 7.5 bc 8.01 a­d values in each column followed by the different letter(s) are significantly different at p≤0.05. least significant difference. table 4 ­ leaf area, shoots fresh weight, shoots dry weight, root fresh, root dry weight and root length of the seven studied chrysanthe­ mum cultivars la10= leaf area measured 10 cm from plant height; sfw= shoot fresh weight per pot unit; sdw= shoot dry weight per pot unit; rfw= root fresh weight per pot unit; rdw= root dry weight per pot unit. values in each column followed by the different letter(s) are significantly different at p≤0.05 (least significant difference). cultivars vegetative growth characters la10 (cm2) sfw (g) sdw (g) rfw (g) rdw (g) root length (cm) 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 crystal pink 9.34 e 14.78 bc 17.17 a 16.27 a 1.72 a 1.67 a 4.21 b 4.95 b 0.74 a 1.02 a 22.43 a 22.63 a crystal white 17.98 ab 19.91 a 19.80 a 18.92 a 2.15 a 2.02 a 5.17 b 5.50 b 1.39 a 1.06 a 22.14 a 21.60 a crystal yellow 11.13 de 13.75 bc 16.67 a 19.77 a 1.74 a 2.61 a 9.94 a 8.94 a 1.71 a 2.13 a 24.63 a 26.20 a crystal red 19.99 a 20.46 a 18.53 a 22.65 a 2.15 a 2.46 a 5.42 b 5.80 b 1.21 a 1.45 a 22.40 a 24.67 a kodiack 15.52 bc 18.49 ab 17.87 a 14.76 a 2.04 a 1.88 a 4.11 b 4.48 b 0.84 a 0.99 a 24.11 a 25.80 a coca bleach 14.71 17.67 15.17 a 20.31 a 1.75 a 2.40 a 3.65 b 4.46 b 0.82 a 1.14 a 24.13 a 19.41 a abrun 12.01 13.10 c 17.41 a 18.16 a 2.24 a 2.71 a 6.33 b 6.25 b 1.31 a 1.45 a 18.53 a 20.04 a fig. 2 ­ leaf morphology of the studied chrysanthemum cultivars. 1) ‘crystal pink’, 2) ‘crystal white’, 3) ‘crystal yellow’, 4) ‘crystal red’, 5) ‘kodiack yellow’, 6) ‘coca bleach’, 7) ‘abrun’. refer to table 1 cultivars el‐nashar ‐ phenological and molecular diversity on chrysanthemum 263 differences in plant shoot fresh and dry weights per plant in both seasons (table 3). regarding the cultivar’s effect on root characteristics in both seasons, the highest root fresh weight was recorded for cv. coca bleach (3.65 and 4.46 g), whereas the lowest root fresh weight (9.94 and 8.94 g) was detected for cv. crystal yellow. the mean values of the compared cultivars did not show any significant differences in root dry weight and root length per plant in both seasons. flower characteristics the plants comparison cultivars had highly significant effects on number of inflorescence per plant, inflorescence diameter and inflorescence stalk length in both seasons. in the first season, the mean number of inflorescence per plant varied from 10.07 to 29.40, while the mean number height varied from 11.06 to 26.71 in the second season. the cv. abrun recorded maximum number of inflorescence per plant followed by crystal red and crystal pink cultivars recorded the least height followed by cv. crystal white (table 5 and fig. 3). the inflorescence diameter ranged from cv. abrun (6.07 and 6.13 cm) to cv. kodiack (9.93 and 10.47 cm), respectively (fig. 3). a highest inflorescence stalk length was recorded at cv. crystal white (6.37 and 6.77 cm), whereas the shortest inflorescence stalk length (2.77 and 2.43 cm) was detected at the cv. crystal yellow. insignificant differences were detected between the first and second seasons in fresh inflorescence weight. significant differences in chrysanthemum inflorescence dry mass per plant were detected in the second season. the lower value resulted in an increase in inflorescence dry weight with cv. crystal pink (1.36 g). on the other hand, the highest value of inflorescence dry weight was observed with cv. abrun (2.86 g). no significant differences were detected among the first season in chrysanthemum inflorescence dry weight per plant (table 5). the compared cultivars had highly significant effects on the number of inflorescences per branch, number of ray florets per inflorescence, ray floret length, and one inflorescence fresh weight in both seasons. in the first season, the mean number of table 5 ­ number of inflorescences, inflorescence diameter, inflorescence stalk length, inflorescences fresh weight and inflorescences dry weight of the seven studied chrysanthemum cultivars fig. 3 ­ standard­type inflorescences of chrysanthemum cultivars used in this study. 1) crystal pink, 2) crystal white, 3) crystal yellow, 4) crystal red, 5) kodiack yellow, 6) coca bleach, 7) abrun. refer to table 1 cultivars. cultivars flower characteristics inflorescences (no.) inflorescences diameter (cm) inflorescence stalk length (cm) inflorescences fresh weight (g) inflorescences dry weight (g) 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 crystal pink 10.07 c 11.06 b 9.33 a 9.37 ab 5.20 abc 3.90 cd 25.36 a 23.04 a 1.58 a 1.36 c crystal white 11.03 c 13.46 b 9.01 a 9.97 ab 6.37 a 6.77 a 20.00 a 23.27 a 1.85 a 1.89 bc crystal yellow 12.13 bc 12.40 b 7.23 bc 7.70 c 2.77 d 2.43 d 23.04 a 28.22 a 1.75 a 2.65 ab crystal red 24.07 a 22.03 a 6.17 c 6.13 d 4.46 bcd 4.23 bc 17.18 a 26.47 a 1.53 a 3.01 a kodiack 14.77 bc 12.73 b 9.93 a 10.47 a 3.63 cd 2.96 cd 20.70 a 21.10 a 2.01 a 2.03 abc coca bleach 17.03 b 14.77 b 8.60 ab 8.87 bc 6.23 ab 5.57 ab 14.03 a 22.24 a 1.11 a 2.05 abc abrun 29.40 a 26.71 a 6.07 c 6.13 d 4.1 cd 4.40 bc 19.10 a 23.76 a 2.20 a 2.86 ab values in each column followed by the different letter(s) are significantly different at p≤0.05 (least significant difference). adv. hort. sci., 2024 38(3): 257­272 264 inflorescences per branch varied from 4.37 to 9.73, while the mean height varied from 3.80 to 8.73 in the second season. the cultivar crystal red recorded the maximum number of inflorescences per branch followed by cv. abrun; however, crystal yellow cv. recorded the least height followed by cv. kodiack (table 5). the number of ray florets per inflorescence ranged from cv. coca bleach (32.33 and 28.30) to cv. crystal red (121.00 and 126.66) in both seasons, respectively. the ray floret length ranged from cv. abrun (2.03 and 2.30 cm) to cv. kodiack (4.13 and 4.83 cm). the one inflorescence fresh weight ranged from (0.92 and 0.95 g) in cv. abrun to (5.94 and 4.26 g) in cv. crystal pink on the other hand, one inflorescence dry weight, as affected by the compared cultivars, showed negative effects in both seasons (table 6). photosynthetic pigments the mean values of leaf chlorophyll contents are presented in figure 4a, while figure 4b shows the mean values of carotenoid and anthocyanin contents in leaves. the levels of chl a and chl b showed negligible effects, but significant changes were observed in total chlorophyll and the chl a/b ratio. moreover, there were significant differences in carotenoid and anthocyanin contents. ‘crystal pink’ showed an increase in total chlorophyll content, while cv. kodiack exhibited a significant reduction in total chlorophyll content compared to all other cultivars. in the present study, the levels of carotenoid and anthocyanin in the compared cultivars increased in cv. crystal pink, whereas cv. kodiack had lower levels of carotenoid and anthocyanin. gas exchange gas exchange parameters [net photosynthetic rate (pn), stomatal conductance (gs), transpiration (e), and intercellular co2 concentration (ci)] of the seven chrysanthemum cultivars under study showed significant variation and are depicted in figure 5. table 6 ­ number of inflorescences per branch, number of ray floret per inflorescences, ray floret length, one inflorescence fresh weight and one inflorescence dry weight of the seven studied chrysanthemum cultivars nib= number of inflorescences per branch; nrfi= number of ray florets per inflorescence; lrfi= length of ray florets per inflorescence; sfwi= fresh weight of a single inflorescence; sdwi= dry weights of a single inflorescence. values in each column followed by the different letter(s) are significantly different at p≤0.05 (least significant difference). cultivars flower characteristic nib nrfi lrfi (cm) sfwi (g) sdw (g) 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 crystal pink 4.47 c 4.70 bc 108.67 ab 115.00 ab 3.87 ab 4.30 ab 5.94 a 4.26 a 0.40 a 0.38 a crystal white 6.36 bc 6.73 ab 84.65 c 91.33 c 4.06 a 3.93 bc 3.32 b 3.41 b 0.32 a 0.35 a crystal yellow 4.37 c 3.80 c 114.68 a 106.32 bc 3.20 b 3.47 c 2.79 c 3.36 b 0.47 a 0.35 a crystal red 9.73 a 8.73 a 121.00 a 126.66 a 2.43 c 2.83 d 2.01 d 1.89 c 0.23 a 0.24 a kodiack 4.40 c 4.03 c 98.64 b 100.67 bc 4.13 a 4.83 a 2.08 d 3.83 ab 0.21 a 0.34 a coca bleach 6.03 bc 5.66 bc 32.33 d 28.30 d 3.53 ab 3.93 bc 2.16 d 1.69 cd 0.26 a 0.17 a abrun 8.13 ab 8.41 a 33.32 d 30.34 d 2.30 c 2.03 e 0.92 e 0.95 d 0.23 a 0.25 a fig. 4 ­ leaf pigments of the chrysanthemum cultivars under study a) chlorophyll (chl) a, b, total and a/b chls con­ tent; b) carotenoid and anthocyanins content. means are given with standard error. el‐nashar ‐ phenological and molecular diversity on chrysanthemum 265 cultivar ‘crystal red’ had the highest pn, while ‘abrun’ had the lowest pn compared to other cultivars (fig. 5a). ‘crystal yellow’ exhibited the lowest gs and e, whereas ‘kodiack yellow’ had the highest values under the given conditions (figs. 5b and c). ci increased in response to ‘crystal yellow’, while it decreased in ‘kodiack yellow’, compared to other cultivars (fig. 5d). cultivars showed significant differences in net photosynthetic activities; however, these variations were only evident under controlled conditions. ssr analysis pcr amplification of dna using 12 primers for ssr analysis resulted in a total of 40 amplified bands, all of which were polymorphic bands with a 100% polymorphism rate (table 7; figs. 6a and b). these results also demonstrated the presence of uniquely amplified bands in the genomic dna of the seven chrysanthemum cultivars, which were used as molecular markers to identify each of these seven different chrysanthemum cultivars. the number of amplified bands varied from two in primers xgwm205, xgwm133, xgwm181, and xgwm210, three in primers xcfd49, xcfd18, and xcfd66, four in primers xcfd9, xcfd46, and xgwm174, and five in primer xcfd1, with a total of 40 bands and dna lengths ranging from 100 to 600 bp. additionally, six bands were found in primer xcfd183, with dna lengths ranging from 900 to 1000 bp. the results obtained from the phylogenetic tree, based on twelve ssr primers as displayed in figure 7, indicated that the seven different chrysanthemum cultivars were separated into two main clusters. cluster a consisted of the c1 (crystal pink) and c5 (kodiack yellow) cultivars, whereas cluster b was further divided into two sub­clusters. sub­cluster b1 contained only the c2 (crystal white) cv., while sub­ cluster b2 was also divided into two sub­clusters. the first sub­cluster included the c6 (coca bleach) and c7 (abrun) cultivars, forming a closely related group, whereas the second sub­cluster consisted of the c3 (crystal yellow) and c4 (crystal red) cultivars. the similarity matrix indicated a range of values from 0.18 to 0.39, with crystal white standing out as a distinct cultivar among the seven cultivars analyzed. 4. discussion and conclusions vegetative growth parameters such as plant fig. 5 ­ gas exchange parameters of the chrysanthemum culti­ vars under study (a) net photosynthesis rate, (b) stomatal conductance to h2o, (c) transpiration rate, and (d) intercellular co2 concentration). means are given with standard error. adv. hort. sci., 2024 38(3): 257­272 266 height, number of branches, number of leaves per plant, leaf area, and dry weight accumulation play a crucial role in determining the overall crop yield. in this study, cultivars crystal red, coca bleach, kodiack, and crystal white exhibited vigorous growth, while abrun and crystal yellow cultivars displayed medium growth, and crystal pink was characterized by dwarfism, recording the shortest plant height. the observed variations in plant height among the cultivars may be attributed to a combination of genetic factors, environmental conditions during growth, and plant management practices (sirohi and behera, 2000; gharge et al., 2009). the increased number of leaves per plant in these cultivars was associated with higher plant height and number of branches per plant. similar results were reported by tarannum and naik (2014) and prasanth et al. (2020). these variations in growth characteristics may contribute to higher leaf area and ultimately increased dry weight production per plant table 7 ­ total numbers of amplified fragment and polymorphic fragments generated by pcr using ssr primers fig. 6 ­ banding of ssr patterns of seven chrysanthemum cultivars using twelve selected random primers, c1 ­ crystal pink, c2 ­ crystal white, c3 ­ crystal yellow, c4 ­ crystal red, c5 ­ kodiack yellow, c6 ­ coca bleach, c7 ­ abrun. a) first six primers, and b) second six primers of the list primers used in this study. s. no. primer name total number of bands monomorphic bands polymorphic bands unique bands percent of polymorphism % 1 xcfd1 5 0 5 0 100 2 xgwm205 2 0 2 0 100 3 xgwm133 2 0 2 0 100 4 xcfd9 4 0 4 0 100 5 xcfd46 4 0 4 0 100 6 xgwm181 2 0 2 0 100 7 xcfd49 3 0 3 0 100 8 xgwm174 4 0 4 0 100 9 xcfd18 3 0 3 0 100 10 xcfd183 6 0 6 (1) 900­1000 bp c6 100 11 xgwm210 2 0 2 0 100 12 xcfd66 3 0 3 0 100 total 40 0 40 1 100 el‐nashar ‐ phenological and molecular diversity on chrysanthemum 267 in superior cultivars. these findings align with the conclusions of barigidad et al. (1992) and yoon­jung et al. (2013) in chrysanthemum. the differences in growth characteristics between genotypes may be attributed to their inherent genetic traits, as all plants were subjected to similar practices under the same environmental conditions; baskaran et al. (2016) also reported comparable findings. flower yield is a crucial factor in determining the suitability of specific genotypes for commercial cultivation, which directly affects the cost of cultivation. the maximum number of inflorescences per plant was recorded in the cultivars abrun and crystal red, while crystal pink and crystal white cultivars exhibited the lowest numbers. the study revealed that larger leaf area, more number of leaves and branches per plant, along with increased dry weight accumulation, resulted in higher photosynthetic activity, contributing to the production of more and larger flowers. these results are consistent with the findings of tarannum and naik (2014), reddy et al. (2016), palai et al. (2018), singh et al. (2019), and prasanth et al. (2020). flower stalk length is a critical quality characteristic that influences the quality of chrysanthemum cut flowers and extends their post­ harvest life. the variation in stalk length among genotypes may be attributed to inherent genetic factors and growing environmental conditions, as reported by dalal et al. (2009) and tarannum and naik (2014). flower diameter, being a genetically controlled trait, was found to be superior in cultivars kodiack yellow, crystal pink, crystal white, and coca bleach, possibly due to the presence of more petals per inflorescence. however, abrun cultivar produced smaller­sized flowers, which may be attributed to the fewer number of ray florets in its flower buds. the variation in inflorescence size could be attributed to the genetic makeup of the genotypes (reddy et al., 2016; neelam et al., 2018; prabhu et al., 2018). cultivar crystal red exhibited superiority in terms of inflorescence dry weight, followed by abrun, while it was lower in cultivar crystal pink. variations in inflorescence weight could be expected among different cultivars due to differences in genetic structure (gharge et al., 2009). carbohydrates serve as an energy source for growing buds, inflorescence opening, and longevity, ultimately resulting in strong and long inflorescence stalks and large­sized buds or inflorescences. these variations may be attributed to varietal characteristics, as reported by halvey and mayak (1979). similar variations have been observed in chrysanthemum and carnation by several researchers, such as sirohi and behera (2000), singh and sangama (2003), and uddin et al. (2015). the yield and growth of any flower crop are influenced by various factors, including environment, season, and varieties. among these factors, varieties play a significant role in the evolution of any flower crop, particularly in selecting varieties with high inflorescence production. therefore, the selection of appropriate varieties is crucial for successful floriculture cultivation (palai and rout, 2011). photosynthetic pigments the values of chlorophyll, carotenoid, and anthocyanin contents in the leaves of chrysanthemum cultivars are presented. results clearly distinguish cultivars with high pigment content (crystal pink, crystal white, crystal red, and crystal yellow) from those with low pigment content (kodiack yellow, coca bleach, and abrun). photosynthesis in plants relies on capturing light energy using the pigment chlorophyll (blankenship, 2014). differences in chlorophyll a, b, carotenoid, and anthocyanin contents are indicators of damage to the photosynthetic apparatus, stress, or senescence and affect the normal course of plant biological processes (filimon et al., 2016). having a higher amount of chlorophyll could lead to increased light absorption, which is advantageous for photosynthesis in rosa hybrida (terfa et al., 2013). the genotype of a plant affects pigment fig. 7 ­ dendrogram of relationship between seven chrysanthe­ mum cultivars using jaccard’s (1908) index for ssr primers. c1 ­ ‘crystal pink’, c2 ­ ‘crystal white’, c3 ­ ‘crystal yellow’, c4 ­ ‘crystal red’, c5 ­ ‘kodiack yellow’, c6 ­ ‘coca bleach’, c7 ­ ‘abrun’. adv. hort. sci., 2024 38(3): 257­272 268 accumulation by influencing the morphology and anatomy of the leaves (hopkins and hüner, 2009). leaf area has been identified as a factor that can limit the photosynthetic capacity of plants, as reported by petrie et al. (2000). however, it is important to note that the intensity of net photosynthesis (pn) is not necessarily correlated with chlorophyll content, with differences potentially arising from variations in intracellular spaces and gaseous conductivity (patakas et al., 2003). chlorophyll loss is often linked to environmental stress, and changes in the chlorophyll/carotenoid ratio can serve as an indicator of stress in plants (netto et al., 2005). the specific cultivar of a plant can also impact the accumulation of photosynthetic pigments by influencing the morphology and anatomy of the leaves, including factors like mesophyll thickness, area, and perimeter (salem­fnayou et al., 2011). gas exchange light affects not only the photosynthetic rates but also the stomatal function. previous research has focused on studying the impact of long­term acclimation to specific wavelength light on stomatal morphology, density, and opening rates, as demonstrated by studies conducted by wang et al. (2016) and zheng and van labeke (2018). numerous studies have also shown that light has the ability to induce stomatal opening, as observed in research conducted by shimazaki et al. (2007). stomatal conductance (gs) is influenced by the density of stomata on the leaf surface as well as how wide the stomata are open. when plants have abundant water, high gs levels can lead to more transpiration, resulting in reduced leaf water content. closing stomata can help maintain leaf water content by reducing transpiration. in the case of crystal yellow cv. leaves, the low stomatal conductance and leaf transpiration can mainly be attributed to a decrease in stomatal conductance compared to other cultivars. the impact of different types of light on the process of photosynthesis and transpiration in chrysanthemums at various stages of growth is uncertain. scientists conducted experiments to measure the exchange of co2 and h2o in the leaves and entire plants of chrysanthemums under long­day and short­day conditions. it was observed that all l ight sources effectively stimulated leaf photosynthesis, regardless of whether it was a long or short day (leonardos et al., 2019). molecular analysis chrysanthemum cultivars pose challenges in terms of genetic backgrounds and similar morphological features, making it difficult to distinguish among them. various molecular markers like sequence­related amplified polymorphism (srap) (fei et al., 2011), inter simple sequence repeats (issr) (shao et al. , 2010), and simple sequence repeats (ssr) (chang et al., 2018) have been employed to identify and classify chrysanthemum cultivars. between these markers, ssrs offer advantages such as co­dominance, high variability, and reproducibility. ssr markers have also been utilized in the construction of molecular maps, analysis of genetic diversity, and assessment of intellectual property rights in different plants (feng et al., 2016; mekapogu et al., 2020). previous studies have employed ssrs for genetic analysis of chrysanthemum and related genera (chang et al., 2018). this investigation introduces a method for identifying standard­type seven cultivars in chrysanthemum. previous research has already established a database consisting of ssr markers that can be used to identify different cultivars of chrysanthemum. in two separate studies conducted by shim et al. (2015) and olejnik et al. (2021), a total of 28 ssr markers from the chrysanthemum dna profile database were utilized to analyze the genetic relationship among a vast number of chrysanthemum cultivars, specifically 147 and 97, respectively. however, it is worth noting that very few studies have delved into the potential use of ssr markers for distinguishing standard­type chrysanthemum cultivars, as highlighted by han et al. (2018) and thakur et al. (2023). in the current study, a set of twelve ssr markers was employed to distinguish and classify seven different standard­type chrysanthemum cultivars. it was determined that out of the twelve ssr markers utilized, there was noticeable genetic variation observed in the seven standard­type cultivars. the evaluation of genetic relationships between populations serves as the foundation for both selective breeding and cultivar identification. by analyzing the ssr data and constructing a upgma­ based dendrogram, it was observed that the tested chrysanthemum cultivars could be divided into two main groups. the similarity matrix indicated a range of values from 0.18 to 0.39, with crystal white standing out as a distinct cultivar. among the seven el‐nashar ‐ phenological and molecular diversity on chrysanthemum 269 cultivars analyzed (crystal pink, crystal white, crystal yellow, crystal red, kodiack yellow, coca bleach, and abrun) which formed cluster i, coca bleach and abrun cultivars showed a moderate level of distinction and displayed complete genetic similarity. this suggests that there is a relatively low genetic diversity between these cultivars and that they may have been developed from a limited genetic background. in this study, it was found that kodiack yellow and crystal white cultivars exhibited genetic divergence, which aligns with the findings of shim et al. (2015), olejnik et al. (2021), and thakur et al. (2023), who also observed a distant relationship between kodiack yellow and crystal white cultivars. ssr genetic diversity does not necessarily match morphological differences. however, in the present investigation, to a certain extent, the clustering of genotypes in the sub­clusters seemed to correspond with a few phenotypic traits. crystal pink and kodiack, with the same shape of inflorescences (spoon­type) and without disk florets; the leaves are three­lobed, and both have an average inflorescence diameter, root fresh weight per pot unit, length of ray florets per inflorescence, and number of inflorescences per branch, formed sister relationships within cluster a. within the second sub­ cluster b2, two genotypes with the same shape of inflorescences (reflex­type); the leaves are five­lobed, and both have a light fresh weight of a single inflorescence, length of ray florets per inflorescence, number of ray florets per inflorescence, and inflorescence diameter (crystal yellow and crystal red), formed the sister relationships. coca bleach and abrun have the same shape of inflorescences (single/sami­double­type) and disk florets color (yellow­green center), and both have a low number of ray florets per inflorescence, leaf area, and leaf length, forming sister relationships within the first sub­cluster b1. the genotype crystal white remained as a single cultivar in a separate sub­cluster within the major (b), which is the only white color cultivar in the group (b). inflorescence was the type of irregular­ type chrysanthemums. buldewo et al. (2012) performed clustering based on spathe color in anthurium andraeanum to group phenotypic traits/colors. dai et al. (2012) used ssr markers to classify chrysanthemum germplasm based on inflorescence. the economic uses of various cultivars were observed to be linked with different clusters within the primary groups. in a study by minano et al. 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years the range of in vitro propagated species grew and, in addition to other peach rootstocks, self-rooting peach, apricot, kiwi, artichoke and pear rootstocks started being multiplied. today, micropropagation is the most advanced agamic multiplication technique allowing an elevated number of genetically identical plants to be obtained in a short time due to an exponential growth rate of the number of individuals with each subculture step. in vitro culture offers the advantage of high quality and health safety of the plant material as it not subject to an accumulation of pathogens or viruses over the years, as can occur with other agamic in vivo propagation techniques. moreover, in vitro procedures pass on to plants a physiologic equilibrium that is comparable to that of seed-grown individuals, demonstrating good vigor of the aerial structures and notable rhizogenic exploratory ability in roots. these behaviors can be associated with a “rejuvenation” of the plant material. for example, the majority of grapevine cultivars have been, over the millennia, vegetatively propagated and for this reason they present important problems from a health standpoint. toward the end of the 1980’s prof. carmine liuni, director of the viticulture institute of turi (bari), proposed a collaboration with vitroplant for in vitro propagation with the aim of resolving some of the numerous problems found in viticulture nurseries. initially the idea was to use this propagation technique to multiply some known rootstocks and new seedless table grape varieties. the results obtained from field tests were very satisfactory. the micropropagated plants were very vigorous and demonstrated excellent development of rooting structures, highlighting the potential of this propagation technique also in grapevine. this type of development, comparable to seed-grown plants, is not due to genetic mutation but rather to phenotypic manifestations that come from the rejuvenation of in vitro-produced plants. juvenile phases are recognizable in some arboreal species thanks to specific morphological, cellular or physiological traits such as a particular leaf size, shape or phyllotaxis, the presence of thorns or ability to root. plants do not however undergo genetic variations as they pass from the juvenile to the adult phase, instead these morpho-physiologic differences are expressions of the genotype. juvenile as well as adult tissues can be present on the same plant at the same time, a phenomenon called heteroblasty, and leaves on a single plant can have different forms. juvenile tissues are generally found in the lower portion of the trunk and branches. to lower the point of transition from juvenile to adult tissues, it is possible to make a cut near ground-height, avoiding the risk of juvenile tissues in the shoots intended for production. micropropagation has shown to be an essential technique for the production of healthy material in quantity and rapidly, two aspects that are very useful for clonal selection or genetic improvement programs, providing nurseries with sufficient starting material in a timely manner. micropropagated plants, as self-rooted plants not needing rootstock, can be subject to phylloxera attack. with careful attention to the selection of soil for mother plant fields and suitable preventative measures, it is possible to keep this pathogen under control. in any case, after five or six years micropropagated mother plants have fulfilled their purpose: they have provided quickly healthy buds for propagation. today it is increasingly difficult to keep mother plants healthy in open fields and a reduction of their useful period with frequent renewal can be predicted for the future. a new frontier for nurseries in coming years may be the use of rootstocks amenable to genic silencing to induce resistance to viruses or other pathogens of the varieties onto which they are grafted. unfortunately considerable difficulty can be expected with regard to the spread of this technique in europe where there is deep aversion toward biotechnology, while in other emerging, future-competitor grape-growing countries the technique has a better chance of taking hold. adv. hort. sci., 2013 27(3): 105 impaginato 11 adv. hort. sci., 2025 39(1): 11­20 doi: 10.36253/ahsc­16960 https://oaj.fupress.net/index.php/ahs preliminary evaluation of nematode community responses to ground covers in jute leaf cultivation b.d. waldo 1 (*), c.g. arlotta 2, m.l. richardson 2 ** 1 mycology and nematology genetic diversity and biology laboratory, usda, ars, northeast area, 10300 baltimore ave, beltsville, md 20705, maryland, usa. 2 center for urban research, engagement and scholarship, university of the district of columbia, washington dc 20008, usa. ** current address: usda forest service, northern research station, silver spring, md 20902, maryland, usa. key words: compost, ethnic crop, landscape fabric, mulch, straw, wood chips. abstract: jute leaf (corchorus olitorius) is an emerging ethnic crop in the mid­ atlantic united states. no information is available on nematode associations and nematode community responses to jute leaf grown with ground cover. we conducted a preliminary field study in the summer of 2023 in beltsville, maryland to evaluate the responses of endemic nematode communities to three cultivars of jute leaf (firebird, molokhia, and usda pi 404029) and four ground cover treatments (compost, compost and landscape fabric, compost and straw mulch, and compost and wood mulch). we extracted nematodes from soil samples collected before planting, at midseason, and at harvest. by the end of the season, plots with straw had higher counts of prismatolaimus, mononchus and total plant­parasites and plots with wood chips had higher counts of helicotylenchus. structure index and maturity index 2­5 were also higher in plots with straw at the end of the season. cultivar had a relatively small impact on the nematode community with usda pi 404029 plots having the highest plectus counts and firebird plots having the highest predator counts at midseason only. the channel and enrichment indexes showed a shift occurred with all four treatments: the soil environment became dominated by bacterial decomposition pathways with nutrient enriched conditions. however, plant biomass was not different between treatments. these results suggest ground covers can influence soil nematode communities in jute leaf production. 1. introduction ethnic foods are increasingly in demand in the united states (us). metropolitan areas such as washington, dc are home to growing populations of ethnically diverse residents with a broad range of dietary preferences (mangan et al., 2008; govindasamy et al., 2022). many (*) corresponding author: benjamin.waldo@usda.gov citation: waldo b.d., arlotta c.g., richardson m.l., 2025 ­ preliminary evaluation of nematode com‐ munity responses to ground covers in jute leaf cultivation. ­ adv. hort. sci., 39(1): 11­20. orcid: wbd: 0000­0002­5465­5462 acg: 0009­0007­1881­3586 rml: 0000­0001­9769­1669 copyright: © 2025 waldo b.d., arlotta c.g., richardson m.l. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 2 december 2024 accepted for publication 24 february 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16960 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-5465-5462 http://orcid.org/0009-0007-1881-3586 http://orcid.org/0000-0001-9769-1669 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 11­20 12 tropical crops from africa and asia can be grown as annuals in the summer months in the mid­atlantic us (mangan et al., 2010). local production of ethnic crops on small­scale farms can increase accessibility of products that might otherwise be difficult to obtain at high quality due to long­distance transport (trobe, 2001). jute leaf (corchorus olitorius) is an important crop in tropical countries in africa and asia and is in demand in the eastern us (govindasamy et al., 2007). it is primarily grown as a leafy vegetable and the leaves are often used to add flavor and thicken soups and stews (islam, 2013). roots can be used for medicinal purposes and stems can also be used for fiber production (islam et al., 2013; nyadanu et al., 2017). a management practice useful for crop production is using ground covers and mulching. physical barriers laid on the soil surface can create an impediment for weed development and aids in conserving moisture and regulating soil temperature (flint, 2012; richardson et al. , 2023, 2024). sustainable ground covers derived from organic materials used to suppress weeds can contribute to higher soil moisture content and higher yields of vegetables in the northeastern us (gheshm and brown, 2018; larkin, 2020; richardson et al., 2023). urban centers produce large quantities of organic waste, some of which can be repurposed as compost or ground covers for agricultural use (arcas­pilz et al., 2023). wastes such as biosolids, yard waste, and food waste can provide nutrients to crops as compost, ground covers, or amendments (wang et al., 2008; splawski et al., 2016; shrestha et al., 2020). ground cover applications can have cascading effects on the soil ecosystem. nematodes are microscopic worms that are ubiquitous in soil environments and are sensitive to additions of organic matter­based mulches and amendments (mcsorley and gallaher, 1996; forge and kempler, 2009; waldo et al., 2024). plant­parasitic nematodes feed on plant roots and can negatively affect plant health and reduce yields in leafy vegetable crops, including jute leaf (atungwu et al., 2013; mbogoh et al., 2013; kimaru et al., 2014). yield of jute leaf in nigeria and india has been reduced by 52% and 68%, respectively, due to feeding injury from meloidogyne incognita (saikia and phukan, 1986; adepoju and oluwatayo, 2016). organic amendments can contribute to suppression of undesirable plant­ parasitic nematodes by introducing and enhancing natural antagonistic organisms including predatory/ parasitic fungi, collembola, tardigrades, mites and protozoa as well as releasing lethal compounds such as ammonia and organic acids that are byproducts of decomposition (akhtar and malik, 2000; thoden et al., 2011; timper, 2014; rosskopf et al., 2020). some microorganisms such as bacillus spp., pastueria spp., pochonia spp, and trichoderma spp. have shown promise at reducing numbers of plant­parasitic nematodes and have been further studied as potential biocontrol agents of plant­parasitic nematodes in cropping systems (meyer and roberts, 2002; pires et al., 2022). other nematode groups feed on a range of soil microflora and microfauna and can have positive effects on soil health (yeates et al., 1993; neher, 2001). bacterivore nematodes rapidly increase following additions of organic matter in response to bacterial blooms (ferris and bongers, 2006). bacterivores contribute to nutrient cycling by culling bacteria, which releases carbon (c) and nitrogen (n) back into the soil that may otherwise be respired or immobilized during periods of high microbial activity (akhtar and malik, 2000; wang and mcsorley, 2005). omnivorous and predatory nematodes also play important roles in soil ecosystems as biological indicators of food web structure. increases in predatory nematode abundance are common in response to applications of organic materials, which is desirable in agricultural systems (forge et al., 2003; oka, 2010; mcsorley, 2011). predatory nematodes consuming plant­ parasitic nematodes and opportunistic nematodes can act as a check on unregulated population growth that could otherwise occur under nutrient enriched conditions (bongers and bongers, 1998; ferris, 2010). as farmers look for opportunities to diversify their crop production and reach underserved ethnic markets, they need an understanding of best practices for cultivating ethnic crops. however, it is unknown how ground covers influence yield of jute leaf and associated nematode communities in the mid­atlantic us. information on impacts of ground covers on the nematode community in maryland may help identify the structure of the soil food web and potential risks of plant­parasitic nematodes to jute leaf. to fill this gap in knowledge, we conducted a preliminary investigation to ascertain how ground covers influenced yield of jute leaf and the nematode community. we used four ground cover treatments (compost, compost and landscape fabric, compost and straw, and compost and wood mulch) to grow three cultivars of jute leaf (firebird, molokhia, and waldo et al. ‐ nematode responses to groundcovers 13 usda pi 404029) in maryland. 2. materials and methods study site we established experimental plots in may 2023 at the university of the district of columbia’s (udc) firebird research farm (39°3’11.1492 n, 76°52’52.716 w). the soil was classified as a russett­ christiana complex, with a fine loamy, mixed, semiactive, mesic aquic hapludults for the russett series, and a fine, kaolinitic, mesic aquic hapludults as part of the christiana series. soil texture was 39% sand, 36% silt, and 25% clay. plots were solarized prior to planting to kill weeds. plots were hand weeded as necessary during the study and no pesticides or fertilizers were applied. treatments we used four ground cover treatments and three jute leaf cultivars in the study. ground cover treatments were applied to 0.9 m by 9 m plots and were arranged as a randomized complete block design in a 4×3 factorial with four replicates (supplementary materials ­ sm ­ figs. 1s ­ 2s). each ground cover plot was subdivided into three 0.9 m by 3 m subplots, with each subplot randomly being assigned one of the three cultivars. fifteen centimeters of mushroom compost was applied on top of the ground of the entire study area with a c:n ratio of 9:1. the four ground cover treatments chosen were: mushroom compost only (compost), landscape fabric (fabric), straw, or wood chips. each treatment was placed on top of the 15 cm of mushroom compost by the beginning of june (5 june 2023). mushroom compost and straw were purchased from purple mountain organics (takoma park, md). landscape fabric used in the study was sunbelt black ground cover 3.2oz (dewitt, sikeston, mo). wood chip mulch was produced in 2019 from a mixture of local softwood and hardwood trees felled from firebird farm and chipped for use as mulch. a compost sample was sent to waypoint analytical inc. (leola, pa) to determine c:n content (peters et al., 2003). the three jute leaf cultivars included firebird (developed at udc), molokhia (egyptian spinach) (kitazawa seed co, oakland, ca), and usda pi 404029 (usda germplasm repository). seeds were soaked overnight and planted in fort vee potting mix (vermont compost, montpelier, vt) in 50 cell trays and grown under high tunnel conditions for 30 days prior to transplanting. soil sampling and data collection soil samples were collected after a soil solarization (pre­treatment) in early may (8 may 2023). we collected midseason and final samples on 10 august and 29 september, respectively. the experiment lasted a total of 144 days from soil pre­ treatment to collection of final samples. we collected six 3 cm × 20 cm soil cores from the center of each plot. the six cores were combined and homogenized into a single composite sample per plot, placed in individual polyethylene sample bags, and then placed into a cooler for transportation to the lab where they were stored at 4.5°c. soil cores were homogenized and nematodes were extracted from 100 cm3 soil using centrifugal sugar floatation (jenkins, 1964). nematodes were fixed in 2% formalin and the genera of a subset of 100 nematodes were identified from each sample using an inverted microscope (zeiss, oberkochen, germany). relative abundance was determined by multiplying the proportion of each genus in the sample by the total number of nematodes in the sample. nematodes were categorized into functional groups, based on their diet, and colonizer­persister (cp) groups (bongers, 1990; yeates et al., 1993). the cp numbers assigned to genera reflect life history traits. numbers near one correspond with r strategists that are associated with nutrient enriched and disturbed ecosystems and numbers near five correspond to k strategists that are associated with stable ecosystems (bongers, 1990; yeates et al., 1993; ferris et al., 2001). we calculated ecological indexes from nematode counts using the nematode indicator joint analysis (ninja) online tool (sieriebriennikov et al., 2014). we also measured fresh aboveground plant biomass at the end of the season by cutting plant stems 7.5 cm above the soil surface and measuring the mass of each plant. statistical analysis we compared the effects of ground cover, cultivar, and the interaction of ground cover and cultivar on nematode abundance and index means using analysis of covariance (ancova). log10 transformations were performed on data prior to analysis to improve normality and homogeneity of variance. mean relative abundance of nematodes from midseason and final sampling dates were each http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 adv. hort. sci., 2025 39(1): 11­20 14 analyzed individually using pre­treatment counts as a covariate. ancova was selected to help account for seasonal variation by including pre­treatment counts as a covariate. means of plant biomass across ground cover treatments and cultivars were compared using analysis of variance (anova). significant results (p≤0.05) were separated with tukey’s hsd. significant differences presented at ≤0.05 occurred within an individual sampling date (midseason or final). statistical analyses were conducted in r using ‘agricolae’ package (r core team, 2019; de mendiburu, 2021). graphics were generated using ggplot2 (wickham, 2016). 3. results we processed 144 soil samples during the study. twenty­seven nematode genera were identified, with six classified as plant­parasitic nematodes, ten as bacterivores, five as fungivores, four as omnivores, and two as predators (table 1). bacterivores were table 1 ­ nematode genera and proportion of all 144 soil samples that contain each genus from the study in beltsville, md functional group (z) genus cp or pp value (y) proportion of samples genus was identified plant­parasite criconemella 3 <0.01 helicotylenchus 3 0.94 heterodera 3 <0.01 hoplolaimus 3 0.03 paratylenchus 2 0.28 pratylenchus 3 0.04 bacterivore acrobeles 2 0.03 alaimus 4 0.15 butlerius 1 0.45 cephalobus 1 0.44 diploscapter 1 0.10 eucephalobus 1 0.80 panagrolaimus 1 0.03 plectus 2 0.26 prismatolaimus 3 0.60 rhabditis 1 1.00 fungivore aphelenchoides 2 0.22 aphelenchus 2 0.79 diphtherophora 3 0.05 ditylenchus 2 0.44 tylenchus 2 1.00 omnivore aporcelaimus 5 0.04 dorylaimoides 4 <0.01 eudorylaimus 4 <0.01 prodorylaimus 4 0.02 predator ironus 4 0.10 mononchus 4 0.27 (z) functional groups assigned to genera based on nematode genus primary dietary preference (yeates et al., 1993). (y) colonizer­persister (cp) or plant­parasite (pp) number according to (bongers, 1990). cp and pp numbers near one correspond with r strategists that are associated with nutrient enriched and disturbed ecosystems and numbers near five correspond to k strategists that are associated with stable ecosystems (ferris et al., 2001). waldo et al. ‐ nematode responses to groundcovers 15 the most abundant functional group followed by fungivores (sm tables 1s, 2s). the most prominent genera were rhabditis (58% of total nematodes) and tylenchus (25% of total nematodes). helicotylenchus was the most abundant plant­parasitic nematode genus, representing 93% of all plant­parasitic nematodes. paratylenchus was the second most abundant genus, representing 6% of the total plant­ parasitic nematodes. ground cover effects nematode counts and ecological indexes were not different among ground cover treatments at midseason sampling, but were during the final sampling period (figs. 1, 2). total plant­parasitic nematodes numbers were more than two times as abundant in straw plots than compost only plots (p= 0.02). plots with wood chips had greater helicotylenchus abundance than plots with compost only (p= 0.03). straw plots had the highest counts of prismatolaimus (p= 0.02) and cp 3 nematodes (p= 0.02) compared to fabric plots as well as the highest counts of mononchus (p= 0.03), total predators (p= 0.03), and cp 4 nematodes (p= 0.04) compared to wood chip plots. whereas, the range of count means for rhabditis greatly increased from pre­treatment samples (55­150) to midseason samples (671­1044) (p= 0.7) and final samples (682­1004) (p= 0.6), no differences occurred among treatments (sm table 1 s). maturity index 2­5 (p= 0.03) and structure index (p= 0.05) were also greater in plots with straw compared to those with fabric at the final sampling date (fig. 2). across all treatments at the end of the season, the range of enrichment index means were near the upper limit of possible values at the end of the study (87­91) (p= 0.4) and the range of channel index means were approaching the lowest limit of possible values at the end of the study (9­15) (p= 0.6), but no differences occurred among treatments for either index (fig. 2). mean biomass for the four ground covers ranged 1.39­1.69 kg, but was not statistically different among ground cover treatments (p= 0.4). effects of plant cultivars plectus and the predator functional group differed across cultivars (fig. 3). plectus counts at midseason were higher in plots with usda pi 404029 than molokhia or firebird (p= 0.01). predators were more numerous at midseason in firebird plots than fig. 1 ­ mean number of nematodes categorized by functional group or genus, with standard errors of the mean, across three sampling periods (pre­treatment, midseason, and final) and four ground cover treatments: compost only; compost+fabric; compost+straw; compost+wood chips. different letters indicate differences between treatments within a sampling date (tukey hsd, p≤0.05). ppn = plant­ parasitic nematodes. http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 16 adv. hort. sci., 2025 39(1): 11­20 molokhia and usda pi 404029 (p= 0.02). no ecological indexes differed across cultivars (p>0.05) (sm table 2s). mean biomass ranged 1.42­1.72 kg, but the effect of cultivar was not significant (p= 0.2). fig. 2 ­ mean number of nematodes that were categorized as colonizer persisters (cp) or ecological indexes. means, with standard errors of the mean, were presented across three sampling dates (pre­treatment, midseason, and final) and four ground cover treatments: compost only; compost+fabric; compost+straw; compost+wood chips. different letters indicate differences between treatments within a sampling date (tukey hsd, p ≤ 0.05). cp 3 = colo­ nizer­persister group 3; cp 4 = colonizer­persister group 4; mi2­5 = maturity index colonizer­persister groups 2­5; si = structure index. colonizer­persister groups are based on life history traits with values approaching one repre­ senting r selection strategists and values approaching five representing k selection strategists. mi2­5 is a mea­ sure of environmental disturbance with values approach­ ing zero indicating high disturbance and values approach­ ing five indicating low disturbance. si is a measure of food web complexity with values approaching zero indi­ cating low food web complexity and values approaching 100 indicating high food web complexity. fig. 3 ­ mean relative abundance of nematode plectus (a) and predatory nematodes (b), with standard errors of the mean, across three sampling periods (pre­treatment, midseason, and final) and three cultivars of jute leaf: firebird; molokhia; and usda pi 404029. different letters indicate differences between treatments within a sam­ pling date (tukey hsd, p≤0.05). 4. discussion and conclusions in this study we demonstrate how ground cover treatments on jute leaf, has a direct impact on the nematode community. no single ground cover enhanced abundance of all free­living nematodes, but nematode abundance differed across the treatments. plots with straw had higher counts of the free­living bacterivore prismatolaimus and predator mononchus by the end of the season than the others treatment. higher counts of prismatolaimus contributed to the increase of cp 3 nematodes and structure index in straw plots at the end of the study. differences in abundance of mononchus largely contributed to increases in cp 4 nematodes, predators, and maturity index 2­5 in straw plots at the end of the study. organic mulch applications can stimulate increases of bacterivore and predatory nematode abundances (ferris and bongers, 2006; http://oaj.fupress.net/index.php/ahs/article/view/16960/13342 waldo et al. ‐ nematode responses to groundcovers 17 wang et al., 2008; pavao­zuckerman and sookhdeo, 2017). prismatolaimus and mononchus are indicators of preliminary structure development and become more common as enriched conditions transition to more stable conditions (ferris et al., 2001). use of amendments with high carbon content such as straw can result in greater free­living nematode abundance compared to using nitrogen rich sources such as manures (liu et al., 2016). soil amended with products that include straw can have positive effects on physical properties of soil that are favorable for nematodes such as increased moisture retention and enhanced soil porosity as well as providing high levels of carbon that stimulates growth of microbial food sources (zhao et al., 2009). elevated nutrient levels occurring following applications of straw mulch can lead to an increase of prismatolaimus abundance and omnivore­predator metabolic activity (song et al., 2020). predator abundance has been shown to increase following application of compost mixtures with straw and leaf litter mulches in barley and tomato production systems (renčo et al., 2010; petrikovszki et al., 2021). helicotylenchus was the dominant plant­parasitic nematode in our study in line with what already presented in natural environment by other studies (babatola, 1983; atungwu et al., 2013). abundance of helicotylenchus numerically declined across all ground cover treatments from pre­treatment to midseason, and remained low at final sampling. compost only plots had lower abundance of helicotylenchus than straw and lower total plant­ parasitic nematode abundance than wood chips at the end of the season. materials with high c:n content such as straw or wood chips can be less effective at suppressing plant­parasitic nematodes than low c:n content mulches like compost or manure (liu et al., 2016; hornung et al., 2020). low c:n content materials may better facilitate growth of nematode antagonists or make soil conditions less favorable by altering ph through soil acidification, though these mechanisms need further evaluation (liu et al., 2016; ye et al., 2018; martinez et al., 2023). despite the differences in plant­parasite abundance, no differences occurred in plant yield. higher helicotylenchus abundance in plots with wood chips was accompanied by zero presence of predatory nematodes. the lack of predatory nematodes may have resulted in less top­down regulatory pressure on helicotylenchus reproduction in plots with wood mulch compared to other treatments. enriched conditions dominated by bacterial composition with low community structure were observed in this study, which are commonly associated with addition of organic amendments (thoden et al., 2011). bacterial feeders in the genus rhabditis greatly increased following applications of all ground cover treatments. the enrichment and channel indexes were strongly influenced by the drastic change in rhabditis abundance. rhabditis nematodes are categorized as cp 1 bacterial feeding nematodes which are opportunistic and respond quickly to nutrient enriched conditions (bongers and bongers, 1998; ferris and bongers, 2006). flushes of bacterial growth in response to compost applications provides abundant resources for r­strategists like rhabditis which often drives rapid population growth (bulluck et al., 2002; ferris and bongers, 2006; fengjuan et al., 2020). in our study, densities of plectus were greatest in usda pi 404029 plots and predator densities were greatest in firebird plots at midseason sampling. the counts were very low among the three cultivars at midseason and impacts were temporary and did not last to the end of the season. plant species diversity can impact nematode abundance and diversity (yeates 1999; porazinska et al., 2003; de deyn et al., 2004). for example, bacterivore abundance generally increases as plant species diversity increases, but responses of predatory nematodes can be more variable and take place more gradually (viketoft et al., 2011; kostenko et al., 2015; cortois et al., 2017; dietrich et al., 2021). impacts of intraspecific plant diversity on functional groups of soil invertebrates are less well studied, but increases of plant diversity can influence soil communities, particularly at lower trophic levels (koricheva and hayes, 2018; yan et al., 2021). for example, omnivore­predator abundance varied between individual genotypes of phragmites australis, whereas bacterivore nematode abundance was affected by the overall genetic diversity of p. australis genotypes (yan et al., 2021). the mechanism of how plant genetic diversity impacts free­living nematodes is not clearly understood. resource quantity (plant biomass) and root quality (c:n ratio) may play a role in influencing nematode communities, especially where levels of organic matter in soil are low (bezemer et al., 2010; cortois et al., 2017; dietrich et al., 2021). secondary compounds produced by roots may also influence microbial communities and could also shape adv. hort. sci., 2025 39(1): 11­20 18 env., 905: 167193. atungwu j.j., dairo o.s., olabiyi t.i., kehinde l.o., 2013 ­ nematodes associated with three indigenous leafy vegetables commonly grown in inland valley ecology in nigeria. ­ arch. phytopathol. pflanzenschut, 46: 1564­1570. babatola j.o., 1983 ­ nematode problems of local leaf vegetables in south western nigeria. ­ acta horticulturae, 123: 247­256. bezemer t.m., fountain m.t., barea j.m., christensen s., dekker s.c., duyts h., van hal r., bezemer t.m., fountain m.t., barea j.m., christensen s., dekker s.c., duyts h., van hal r., harvey j.a., hedlund k., maraun m., mikola j., mladenov a.g., robin c., de ruiter p.c., scheu s., setälä h., smilauer p., van der putten w.h., 2010 ­ divergent composition but similar function of soil food webs of individual plants: plant species and community effects. ­ ecol., 91: 3027­3036. bongers t., 1990 ­ the maturity index: an ecological measure of environmental disturbance based on nematode species composition. ­ oecologia, 83: 14­19. bongers t., bongers m., 1998 ­ functional diversity of nematodes. ­ appl. soil ecol., 10: 239­251. bulluck l.r. iii, barker k.r., ristaino j.b., 2002 ­ influences of organic and synthetic soil ferti l ity amendments on nematode trophic groups and community dynamics under tomatoes. ­ appl. soil ecol., 21(3): 233­250. cortois r., veen g.f., duyts h., abbas m., strecker t., kostenko o., eisenhauer n., scheu s., gleixner g., de deyn g.b., van der putten w.h., 2017 ­ possible mechanisms underlying abundance and diversity responses of nematode communities to plant diversity. ­ ecosphere, 8: 01719. de deyn g.b., raaijmakers c.e., ruijven j., berendse f., w.h. putten, 2004 ­ plant species identity and diversity effects on different trophic levels of nematodes in the soil food web. ­ oikos, 106: 576­586. de mendiburu f., 2021 ­ agricolae: statistical procedures for agricultural research . ­ https://cran.r­ project.org/package=agricolae. dietrich p., cesarz s., liu t., roscher c., eisenhauer n., 2021 ­ effects of plant species diversity on nematode community composition and diversity in a long‐term biodiversity experiment. ­ oecologia, 197: 297­311. fengjuan p.a.n., xiaozeng h.a.n., na l.i., jun y.a.n., yanli x.u., 2020 ­ effect of organic amendment amount on soil nematode community structure and metabolic footprints in soybean phase of a soybean‐ maize rotation on molli‐sols. ­ pedosphere, 30: 544­ 554. ferris h., 2010 ­ contribution of nematodes to the structure and function of the soil food web. ­ j. nematol., 42: 63­67. nematode communities from the bottom up (bezemer et al., 2010). cultivar effect in our study could have been more pronounced earlier in the season, but likely had a lesser overall impact on the nematode community. effects from organic matter decomposition from mulches may have been limited earlier in the season, but became more pronounced as nematode populations may have responded to changes in the availability of resources. our findings indicate that ground covers derived from organic materials and urban wastes can influence nematode communities in plots of jute leaf in maryland. our study was limited to a single year because of insufficient funding and capacity to continue for additional years. future research incorporating multi­season trials in a broader geographic area could validate these findings and help growers select a ground cover that promotes nematode communities beneficial to soil health. information is also needed on ecological impacts of producing other ethnic crops on the soil community in the mid­atlantic us. acknowledgements the authors would like to thank dr. oumoule ndiaye and mr. roy lycorish for their assistance in setting up plots and mr. shiguang li for technical assistance in nematode sampling and extractions. mention of trade names or commercial products in this publication is solely for the purpose of 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2009 ­ the effects of two organic manures on soil properties and crop yields on a temperate calcareous soil under a wheat‐maize cropping system. ­ eur. j. agron., 31: 36­ 42. impaginato 129 adv. hort. sci., 2024 38(2): 129­139 doi: 10.36253/ahsc­15671 salicylic acid and iron­oxide nano­ particles improved the growth and productivity of ajowan under salt stress k. ghassemi­golezani (*), s. abdoli department of plant ecophysiology, faculty of agriculture, university of tabriz, tabriz, iran. key words: foliar application, root growth, salinity, seed filling, yield parameters. abstract: two factorial experiments with randomized complete block design in three replicates were conducted in a greenhouse at the university of tabriz to investigate the individual and combined effects of sa and fe2o3­nps spray (1 mm and 3 mm, respectively) on cations contents, root and shoot growth, seed filling and yield parameters of salt­stressed ajowan plants (0, 4, 8 and 12 ds m­1 nacl; as non­saline and low, moderate and high salinities, respectively). salt stress enhanced na+ contents and reduced k+ and ca2+ contents, and k+/na+ and ca2+/na+ ratios, leading to a reduction in root and shoot growth, particularly under high salinity. reduction in plant growth parameters under salt stress had a negative impact on yield components and seed yield of ajowan. these deleterious impacts of salinity on plants were largely overcome by foliar treatments, particularly by sa + fe2o3­nps. the improvement of seed yield by these treatments was highly correlated with enhanced root and shoot growth, seeds per plant, and 1000­seed weight, especially under moderate and high salinities. thus, the simultaneous application of sa and fe2o3­nps was the best foliar treatment for enhancing the growth and productivity of ajowan plants under normal and saline conditions. 1. introduction growth and development of plants are constantly influenced by various environmental stresses including salinity (sarker and oba, 2019). salt stress triggers many cellular events, causing physiological, biochemical and eventually morphological alterations. this stress chiefly causes ionic toxicity by enhancing na+ concentration in plant cells, which ultimately prevents the acquisition of essential nutrients, ionic homeostasis and cell metabolism (nikpour­rashidabad et al., 2022). salinity not only causes cellular water imbalance, osmotic stress and abscission, but also significantly influences different photosynthetic enzymes and gas exchange parameters (lotfi et al., 2020; rasheed et al., 2020). salt toxicity may also lead to oxidative stress due to the physiological imbalance between the generation and scavenging of (*) corresponding author: golezani@gmail.com citation: ghassemi­golezani k., abdoli s., 2024 ­ salicylic acid and iron‐oxide nanoparticles impro‐ ved the growth and productivity of ajowan under salt stress. ­ adv. hort. sci., 38(2): 129­139. copyright: © 2024 ghassemi­golezani k., abdoli s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 january 2024 accepted for publication 10 march 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15671 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 129­139 130 reactive oxygen species (ros) (ghassemi­golezani and abdoli, 2022 a). elevated ros may cause the oxidation of proteins and membrane lipids and also impair cellular redox homeostasis. a stress­induced decline in plant growth and productivity is a common phenomenon in many plant species, which is most likely attributed to the changes in plant physiology, and metabolism (ghassemi­golezani et al., 2021; ghassemi­golezani and rahimzadeh, 2022) and phenology attributes (kazan and lyons, 2016). the major negative impacts of salinity on root growth have been formerly confirmed in portulaca oleracea (kafi and rahimi, 2011), mentha spicata (chrysargyris et al., 2019), oryza sativa (chang et al., 2019) and jatropha curcas (abrar et al., 2020), brassica napus (ghassemi­golezani and abdoli, 2022 b) plants. therefore, salt toxicity is an escalating problem in different agricultural systems worldwide. the emerging roles of plant hormones and nanoparticles in modulating various abiotic stresses have been extensively evaluated (ghassemi­golezani and abdoli, 2021; singh et al., 2021 b). salicylic acid (sa) as a naturally phenolic hormone has effectual roles in numerous metabolic processes and regulates photosynthesis and antioxidant activities, redox and osmotic hemostasis, ionic uptake and secondary metabolite synthesis in plants exposed to salinity (abdoli and ghassemi­golezani, 2021; hussain et al., 2021). salicylic acid can reverse the ethylene­induced detrimental impacts via modulating the transcription of acs, nhx, sos1, hkt1 and hkt2 genes, and improving antioxidants capacity, leading to an increase in shoot and root growth, leaves per plant, leaf area and plant productivity (rao et al., 2021). simultaneous application of sa and nanoparticles can be more effective in augmenting the ameliorative effects of sa under salt stress (mozafari et al., 2018; ghassemi­golezani and abdoli, 2021). nanoparticles (nps) rapidly penetrate the plant cell due to their small size and high solubility, thereby compensating the nutrient deficiencies. through phloem vessels and plasmodesmata, foliar applied nanoparticles can be transferred into the cells (knoblauch and oparka, 2012). the bond between carrier proteins and nanoparticles facilitates the entry of nanoparticles into the cells through ion channels, aquaporin, and endocytosis (nair et al., 2010). since the uptake of most micronutrients is reduced under salinity, supplying nano­forms of these elements not only reduces nutritional imbalance, but also helps the plants to cope with stress through various physiological and metabolic changes. for instance, silica nps boost salt tolerance by regulating ion homeostasis, osmotic adjustment and chlorophyll content, which recover plant growth and productivity (alsaeedi et al., 2019). iron oxide nps may have critical roles in different biochemical synthesis, antioxidant activity and genes expression (moradbeygi et al., 2020). the fe2o3­nps induced salt tolerance in moldavian balm plants was due to augmenting dpph radical scavenging activity, biochemical compounds accumulation and stimulating expression of genes involved in the bio­ synthesis pathway of important phenolic acids such as rosmarinic acid (moradbeygi et al., 2020). recent investigations indicated that foliar spray of fe2o3­nps on plants under salt stress notably increased chlorophyll concentration, carbohydrate content (i.e., sugars), and enzymatic defense capacity. moreover, it decreased lipid peroxidation and ros generation (singh et al., 2021 a). according to dola et al. (2022) application of 200 ppm iron­oxide nanoparticles resulted in an improvement of plant growth, relative water content chlorophyll content, 100­seed weight, seed yield, and protein and oil contents of soybean. adding fe2o3­nps to the soil enhanced leaf area, leaf number per plant, shoot length, and shoot and root weights of tomatoes (el­ desouky et al., 2021). improving plant growth by iron oxide nanoparticles was also observed by several studies on wheat (rizwan et al., 2019; manzoor et al., 2021; el­saber et al., 2021). ajowan (trachyspermum ammi l.) is a medicinal plant belonging to the apiaceae family.it is well known for its essential oil (up to 5%), particularly in seeds (minija and thoppil, 2002). due to numerous pharmacological properties of ajowan essential oil including stimulant, antiseptic, anesthetic, antimicrobial, antiviral, antiulcer, antihypertensive, antitussive, antihyperlipidemic and bronchodilatory, this plant is widely employed (bhadra, 2020). in our previous reports (abdoli et al., 2020; ghassemi­ golezani and abdoli, 2021) the mechanisms of improving salt tolerance in ajowan plants by salicylic acid and iron oxide nanoparticles were discussed in details. in addition to the reported results, this research aimed at evaluating the growth responses of ajowan to these treatments focusing on root and shoot growth, and yield­related traits under salt stress. ghassemi‐golezani and abdoli ‐ improving ajowan performance by foliar treatments 131 2. materials and methods experimental conditions and treatments two pot experiments with a factorial arrangement based on a randomized complete block design in three replicates were set up in a greenhouse at the university of tabriz, iran, to investigate the effects of individual and simultaneous application of sa (1 mm) and fe2o3­nps (3 mm) on sodium, potassium and calcium contents, root and shoot growth, seed filling and yield parameters of salt­stressed (0, 4, 8 and 12 ds m­1 nacl; as non­saline and low, moderate and high salinities, respectively) ajowan plants. the salinity (nikpour­rashidabad et al., 2022) and foliar spray (hussain et al., 2019; ghassemi­golezani and farhadi, 2022) levels were selected according to previous reports. the average temperatures of day and night, relative humidity and light intensity in the greenhouse were 29°c, 25°c, 35­40%, and 141 wm­2 (about 780 µmol m­2s­1), respectively. this research was performed with 52 pots (48 pots for sowing and 4 unsown pots for checking the water status). ajowan seeds (30 seeds per pot) were sown in each pot in 1 cm depth of a mixture of perlite and cocopeat to keep long­term moisture in the substrate. the tested salt solutions were added to the substrate of the pots up to 100% field capacity (fc). the emerged seedlings were reduced to keep 10 plants per pot. the water loss from the pots was compensated by tap water or hoagland solution (ec=1.3 ds m­1, ph=6.7­7.2) up to 100% fc. toprevent excess increment of ec in the substrate due to hoagland addition, the perlite + cocopeat within all pots were washed slowly every 30 days by pouring water into the pots and draining from the lower holes of the pots. the ec of draining water was measured frequently and when the pouring and draining waters showed similar ecs, washing was stopped and then re­treated with salt solutions. the sa, fe2o3­nps and tap water were sprayed on plants at two different stages (7 leaves and flowering), by a two­liter manual sprayer. estimation of na+, k+ and ca2+ contents the sodium, potassium and calcium contents in plant tissues were determined by a flame photometer (corning flame photometer, 410). the samples of ajowan plants were reduced to dry ashes in an electric furnace at 500℃ for 7 h, and the carbon­free residue was then dissolved in 1 n hcl. the na+, k+ and ca2+ contents were determined as milligrams per gram dry weight. measurement of root and shoot parameters two plants from each pot were removed at maturity. the roots were cut from the crown and thoroughly washed and air­dried. then, the root and shoot lengths, root diameter, branches per plant and leaves per plant were recorded. subsequently, the samples of roots and shoots were separately dried at 75℃ for 48 h and weighed. seed filling during seed filling in 2018, two plants from each pot were harvested at 10 days intervals, beginning 20 days after flowering and then seeds were removed from the plants and weighed at five stages. yield parameters the seeds of two plants from each pot were separated and then the number of umbels per plant, seeds per umbel, seeds per plant, 1000­seed weight and seed yield were determined. the harvest index was calculated as: harvest index = (seed yield/shoot + seed mass) × 100 statistical analysis all collected data in this study were subjected to a two­way analysis of variance (anova) using mstat­ c, and means were compared by duncan ̓ s multiple range test at p ≤ 0.05. the mean data were presented as means ± standard error. pearson correlation coefficient was used to analyze the relations between morphological and yield­related traits of ajowan plants, using spss 16. 3. results the na+, k+ and ca2+ contents the na+, k+ and ca2+ contents and k+/na+ and ca2+/na+ ratios were significantly affected by salt stress and foliar treatments (p ≤ 0.01). the k+ and ca2+ contents and k+/na+ and ca2+/na+ ratios were decreased, while the na+ content was increased by the increment of salt toxicity. the 4 ds m­1 nacl had no significant impact on ca2+ content. foliar treatments, particularly sa + fe2o3­nps, reduced na+ content and enhanced k+ and ca2+ contents and k+/na+ and ca2+/na+ ratios. differences between sa and sa + fe2o3­nps treatments in na+ and ca2+ adv. hort. sci., 2024 38(2): 129­139 132 contents and ca2+/na+ ratio were not statistically significant. all of these parameters (except k+/na+ ratio) were similarly affected by sa and fe2o3­nps treatments (table 1). root growth significant interaction of salt stress and foliar treatments were observed for root growth parameters (p≤0.01). the length, weight and diameter of ajowan roots were decreased as salinity increased. no differences among foliar treatments in root parameters were recorded under low salinity. nonetheless, foliar sprays significantly promoted root growth of plants at different saline conditions, especially under moderate and high salinities. the sa and sa + fe2o3­nps treatments were the superior treatments in improving root growth (table 2). in most cases, the differences betweenhormonal and nutritional treatments were not statistically significant (table 2). shoot parameters a significant interaction of salinity and foliar applications was observed for shoot mass and length, branches and leaves per plant (table 3). increasing salinity significantly decreased shoot parameters. the shoot length of treated and untreated plants was not significantly varied under non­saline conditions. however, foliar treatments significantly increased shoot length under all salinity levels. the shoot mass, and branches and leaves per plant were enhanced by different foliar treatments, especially by sa + fe2o3­ nps, under saline and non­saline conditions. the differences among sa, fe2o3­nps and sa + fe2o3­nps treatments in shoot mass under 4 ds m­1nacl, in shoot length under 4 and 8 ds m­1 nacl and in branches per plant under 8 ds m­1 nacl were not significant (table 3). seed filling the dry weight of ajowan seeds was gradually enhanced with seed development up to about 50 days after flowering and afterwards no significant changes occurred. seed dry weight was not significantly affected by low salinity (4 ds m­1 nacl), but further increment of salt stress, particularly high salinity, caused a significant decrease in the dry weight of ajowan seeds at later stages of seed development under all foliar treatments. application of sa, fe2o3­nps individually and in combination form increased seed weight under moderate and high salinities. this improvement was mainly due to increasing seed filling rate rather than seed filling duration (fig. 1). yield components the interaction of salinity and foliar treatments was significant for umbels per plant, seeds per plant, 1000­seed weight, seed yield and harvest index (table 4). seeds per umbel were only affected by salt stress. low salinity had no significant effect on seeds per umbel. however, further increment in salinity table 1 ­ the na+, k+ and ca2 + contents (mg g­1 dry weight) of ajowan plants affected by foliar treatments under saline and non­saline conditions different letters in each column indicate significant differences at p≤0.05; ** = significant at p≤0.01. fe2o3­nps= iron­oxide nanoparticles; sa= salicylic acid. treatments na+ k+ ca2+ k+/na+ ca2+/na+ salinity conditions non­saline 9.29 ± 0.45 d 45.17 ± 1.8 a 13.94 ± 0.24 a 5.05 ± 0.38 a 1.53 ± 0.06 a 4 ds m­1 nacl 15.14 ± 1.17 c 40.86 ± 1.8 b 13.19 ± 0.43 a 2.86 ± 0.23 b 0.92 ± 0.08 b 8 ds m­1 nacl 27.57 ± 0.99 b 30.72 ± 1.3 c 10.63 ± 0.55 b 1.15 ± 0.08 c 0.40 ± 0.03 c 12 ds m­1 nacl 32.35 ± 1.51 a 28.01 ± 1.2 d 7.82 ± 0.72 c 0.90 ± 0.07 d 0.25 ± 0.03 d f test 479.64 ** 220.88 ** 60.57 ** 679.22 ** 369.80 ** foliar treatment water 26.39 ± 3.52 a 28.36 ± 1.74 c 9.91 ± 1.19 c 1.53 ± 0.33 d 0.57 ± 0.14 c sa 19.53 ± 2.72 b 37.97 ± 2.49 b 11.86 ± 0.64 ab 2.82 ± 0.61 b 0.86 ± 0.17 ab fe2o3­nps 19.30 ± 2.47 b 37.16 ± 2.22 b 11.23 ± 0.81 b 2.56 ± 0.46 c 0.78 ± 0.15 b sa+fe2o3­nps 19.14 ± 2.60 b 41.28 ± 2.29 a 12.58 ± 0.58 a 3.04 ± 0.63 a 0.89 ± 0.16 a f test 52.44 ** 101.29 ** 10.15 ** 82.99 ** 22.85 ** ghassemi‐golezani and abdoli ‐ improving ajowan performance by foliar treatments 133 considerably reduced this parameter. the plants grown under high salinity had the lowest seeds per umbel compared to unstressed plants (fig. 2). rising salinity significantly reduced the other yield parameters and harvest index. foliar treatments had no significant effects on 1000­seed weight under non­saline conditionsand on umbels per plant, seeds per plant and harvest index under non­saline and low salinity. however, hormonal and nutritional treatments enhanced these parameters under saline conditions. this improvement was more evident under moderate and high salinities. in these levels of salinities, the sa and sa + fe2o3­nps were the best treatments for improving yield parameters, followed by fe2o3­nps (table 4). correlations all morphological and yield parameters of ajowan had asignificant positive correlation with each other (table 5). root and shoot masses and lengths were highly related toeach other and with leaves per plant (r≥0.85**). the root and shoot parameters as well as yield components and harvest index were positively and significantly correlated with seed yield per plant. however, the highest relations with seed yield were recorded for shoot mass and seeds per plant, followed by shoot and root lengths, leaves per plant and 1000­seed weight (table 5). 4. discussion and conclusions the na+ toxicity negatively influenced different aspects of plant growth such as root and shoot parameters, resulting in less seed production. the salt­treatedajowan plants responded to foliar applications, particularly to sa and sa + fe2o3­nps treatments, as demonstrated by higher k+ and ca2+ table 2 ­ combined analysis of variance of the data for root growth parameters of ajowan affected by foliar treatments under saline and non­saline conditions in 2018 and 2019 different letters in each column indicate significant differences at p≤0.05; ns, *, **= no significant and significant at p≤0.05 and p≤0.01, respectively. fe2o3­nps= iron­oxide nanoparticles; sa= salicylic acid. salinity foliar treatments root mass (g) root length (cm) root diameter (mm) non­saline water 1.88 ± 0.02 abc 32.98 ± 0.05 abc 2.63 ± 0.17 a sa 1.97 ± 0.04 a 34.28 ± 0.6 ab 2.51 ± 0.11 a fe2o3­nps 1.94 ± 0.05 ab 34.48 ± 0.5 a 2.37 ± 0.11 ab sa+ fe2o3­nps 1.99 ± 0.05 a 34.23 ± 0.4 ab 2.57 ± 0.12a 4 ds m­1 nacl water 1.74 ± 0.05 ef 28.95 ± 0.6 d 2.41 ± 0.08 ab sa 1.85 ± 0.03 bcd 32.95 ± 0.5 abc 2.43 ± 0.08 ab fe2o3­nps 1.83 ± 0.04 cde 31.83 ± 0.8 c 2.38 ± 0.7 ab sa+ fe2o3­nps 1.90 ± 0.04 abc 32.20 ± 0.8 bc 2.48 ± 0.08 a 8 ds m­1 nacl water 1.23 ± 0.05 h 17.12 ± 0.7 hi 1.88 ± 0.06 def sa 1.76 ± 0.02 de 24.02 ± 0.8 ef 2.13 ± 0.06 bcd fe2o3­nps 1.64 ± 0.03 f 22.47 ± 0.8 f 2.31 ± 0.05 abc sa+ fe2o3­nps 1.80 ± 0.03 cde 25.75 ± 1.0 e 2.32 ± 0.10 abc 12 ds m­1 nacl water 0.83 ± 0.06 i 14.37 ± 0.6 j 1.62 ± 0.06 f sa 1.50 ± 0.04 g 19.03 ± 0.4 gh 2.05 ± 0.07 cd fe2o3­nps 1.21 ± 0.06 h 16.48 ± 0.5 i 1.72 ± 0.09 ef sa+ fe2o3­nps 1.48 ± 0.02 g 19.90 ± 0.6 g 2.00 ± 0.06 de source of variation year (y) ns ns ns salinity (s) ** ** ** foliar treatments (f) ** ** * y x s * * ns y x f ns ns ns s x f ** ** * y x sxf ns ns ns f test 17.14** 4.39** 2.36* 134 adv. hort. sci., 2024 38(2): 129­139 contents in plant tissues, root and shoot growth and yield­related traits. a high concentration of na+ ions in the substrate led to an increase in na+ uptake and accumulation in plant tissues and a decline in k+ and ca2+ uptake, and k+/na+ and ca2+/na+ ratios (table 1). this imbalance in the nutrient status of tissues might be due to an injury in the cell membrane and specific ion channels (jayakannan et al., 2013). however, the reduction of na+ accumulation by foliar spray, particularly by sa + fe2o3­nps, enhanced the uptake of essential nutrients. this improvement could be related to the activation of h+­atpase and h+­ppase pumps by sa and fe2o3­nps treatments that induces na+ secretion in vacuoles (ghassemi­golezani and abdoli, 2021) and helps plants cope withsalt toxicity.our results suggest that the enhancement of root growth by these treatments (table 2) is effective in improving nutrient availability to the plants.the manganese­iron nanoparticles have been reported to increase the efflux of h+ and influx of k+, leading to high k+/na+ ratio (wang et al., 2022). the roots play an imperative part in plant establishment in the soil and water and nutrient absorptions. a comprehensive understanding of root response to the foliar spray of sa and fe2o3­nps can more likely provide useful information for improving table 3 ­ combined analysis of variance of the data for shoot mass, shoot length, brunches and leaves of ajowan plants affected by foli­ ar treatments under saline and non­saline conditions in 2018 and 2019 salinity foliar treatments shoot mass (g) shoot length (cm) branches per plant leaves per plant non­saline water 15.48 ± 0.18 b 99.87 ± 0.72 abc 11.85 ± 0.74 d 58.47 ± 1.10 ab sa 16.08 ± 0.20 a 101.1 ± 0.78 ab 13.82 ± 0.29 ab 57.33 ± 0.88 b fe2o3­nps 15.97 ± 0.22 a 99.93 ± 0.70 abc 12.33 ± 0.48 cd 59.10 ± 0.53 ab sa+fe2o3­nps 16.17 ± 0.22 a 101.7 ± 0.96 a 14.62 ± 0.44 a 60.78 ± 0.91 a 4 ds m­1 nacl water 14.22 ± 0.15 d 95.22 ± 0.70 e 10.50 ± 0.36 e 45.00 ± 0.86 de sa 14.73 ± 0.22 c 96.93 ± 0.99 de 13.40 ± 0.43 bc 51.33 ± 0.95 c fe2o3­nps 14.70 ± 0.20 c 99.03 ± 0.64 bcd 12.40 ± 0.34 cd 47.10 ± 0.80 d sa+fe2o3­nps 14.90 ± 0.21 c 97.78 ± 0.61 cd 14.17 ± 0.37 ab 50.77 ± 0.68 c 8 ds m­1 nacl water 8.63 ± 0.27 g 68.67 ± 0.71 gh 8.85 ± 0.31 fg 31.67 ± 0.80 i sa 12.42 ± 0.27 e 79.22 ± 0.67 f 11.72 ± 0.37 d 41.57 ± 0.69 fg fe2o3­nps 11.35 ± 0.34 f 78.82 ± 0.66 f 11.75 ± 0.45 d 39.47 ± 0.46 g sa+fe2o3­nps 12.49 ± 0.25 e 80.87 ± 0.68 f 11.67 ± 0.37 d 42.67 ± 0.56 ef 12 ds m­1 nacl water 4.52 ± 0.27 j 61.23 ± 0.61 i 8.17 ± 0.42 g 24.00 ± 0.89 j sa 7.54 ± 0.12 h 69.03 ± 0.79 g 9.05 ± 0.28 fg 35.33 ± 0.84 h fe2o3­nps 6.25 ± 0.23 i 66.27 ± 0.60 h 8.48 ± 0.38 g 30.70 ± 0.63 i sa+fe2o3­nps 7.73 ± 0.09 h 68.80 ± 0.98 g 9.72 ± 0.33 ef 34.67 ± 0.92 h source of variation year (y) ns ns * ns salinity (s) ** ** ** ** foliar treatments (f) ** ** ** ** y ´ s ns ns * ns y ´ f ns ns ns ns s ´ f ** ** ** ** y ´ s ´ f ns ns ns ns f test 21.99** 11.64** 3.05** 7.81** different letters in each column indicate significant differences at p≤0.05; ns, *, **= no significant and significant at p≤0.05 and p≤0.01, respectively. fe2o3­nps= iron­oxide nanoparticles; sa= salicylic acid. ghassemi‐golezani and abdoli ‐ improving ajowan performance by foliar treatments 135 crop productivity in saline soils. the length, mass and diameter of roots in salt­stressed plants, especially under moderate and high salinities, were limited due to nutritional (table 1) and hormonal imbalances (zhang et al., 2018), that limit cell elongation and division (yang et al., 2019). the gia roots analysis alsorevealed a decrease in the penetration and distribution of plant roots under different levels of salinities, particularly under high saline conditions (ghassemi­golezani and abdoli, 2022 b). these negative impacts of salinity on root growth were relieved by foliar treatments of sa and fe2o3­nps (table 2). the cross­talk of sa with auxins, cytokinins and gibberellins (shakirova et al., 2003; agami and mohamed, 2013; miura et al., 2013) can potentially promote cell elongation and division. in addition, the expression of auxin biosynthesis genes might be regulated by fe status of cells (sun et al., 2017). iron­ nps may enhance root growth by inducing oh radical generation and demolition of cell wall polysaccharides (kim et al., 2014). stimulation of sa synthesis in plants by fe2o3­nps related treatments (abdoli et al., 2020) is also effective in improving plant growth. reduction in shoot mass and length, and branches and leaves per plant due to salinity (table 3) is related to na+ toxicity and competition of plants for nutrients (table 1) and water (abdoli et al., 2020). this is a mechanism for minimizing energy losses and maintaining plant survival chance under stress conditions. it has been confirmed that salt stress leads to growth reduction, which is more pronounced in leaf area (acosta­motos et al., 2015), branches and leaves per plant and shoot length and mass (table 3). foliar treatments promoted shoot growth by enhancing root growth (table 2), and improving k+ and ca2+ contents in plant tissues bylimiting na+ absorption by the plants (table 1). the effectiveness of salicylic acid inpromoting cell division and enlargement is also supported by a previous report on wheat plants (agami and mohamed, 2013). inhibition of ethylene synthesis by sa (khan et al., 2014) can enhance the plant growth duration. a decline in abscisic acid contentdue to iron nanoparticlesmay also promote growth and retard the senescence of plants (rui et al., 2016). moreover, iron nanoparticlesareinvolved in protein synthesis and enzymes activation, which can promote the plant growth and reduce the senescence especially under stressful conditions (sheykhbaglou et al., 2018). wang et al. (2022) suggested that cytokinin level, scftir1/afb‐aux/iaa signaling pathway, atp synthesis, cell elongation and plant biomass could be enhanced by iron nanoparticles. decreasing root­ and shoot­related traits (tables 2 and 3) due to salt stress reduced yield parameters including seed filling rate (fig. 1), seeds per plant, 1000­seed weight and seed yield (table 4; fig. 2). these reductions are most likely attributed to the enhanced vegetative and reduced reproductive periods under salinity (ghassemi­golezani and farhangi­abriz, 2021). retarding flowering due to salinity reduced umbels and seeds per plant, 1000­ seed weight, and consequently seed yield (table 4). the reduction in photosynthetic efficiency (ghassemi­golezani et al., 2021) and allocation of assimilates to the seeds (kafi et al., 2013) might be the main reasons for yield losses under moderate and high salinities. the sa + fe2o3­nps treatment alleviated these detrimental impacts of salinity on plant productivity through the improvement of nutrient availability (table 1), root (table 2) and shoot (table 3) growth, photosynthetic potential (ghassemi­golezani and farhadi, 2022) and stimulation of flower­inducing factor (hayat et al., 2007). in a study on salt­stressed pennyroyal plants, the sa application enhanced rubisco activity, a fig. 1 ­ changes in mean seed weight during seed development in response to salinity and foliar treatments. fe2o3­nps= iron oxide nanoparticles, sa= salicylic acid. adv. hort. sci., 2024 38(2): 129­139 136 critical enzyme in photosynthetic machinery.the enhanced seed­filling rate (fig. 1) by simultaneous application of sa and fe2o3­nps under moderate and high salinities resulted in the production of larger seeds (table 4). the high correlation of root and shoot parameters, seeds per plant and 1000­seed weight with seed yield (table 5) suggests that improving these traits by breeding or foliar treatments can potentially increase crop productivity under normal and stressful conditions.our results indicated that foliar spray of sa and sa + fe2o3­nps often similarly improves salt tolerance and seed yield of ajowan, so application of sa and/or sa + fe2o3­nps treatments can be cost­effective in reducing salinity­ induced losses in large­scale production systems. salinity remarkably limited the root and shoot salinity foliar treatments umbels per plant seeds per plant 1000­seed weight (mg) seed yield (g plant­1) harvest index (%) non­saline water 50.67 ± 0.42 a 6367.9 ± 56.7 a 865.7 ± 3.7 ab 5.51 ± 0.07 b 35.63 ± 0.06 a sa 51.00 ± 1.24 a 6498.5 ± 110.0 a 888.3 ± 6.0 a 5.77 ± 0.07 a 35.88 ± 0.19 a fe2o3­nps 52.00 ± 0.36 a 6577.6 ± 30.5 a 870.0 ± 6.8 ab 5.72 ± 0.07 a 35.84 ± 0.16 a sa + fe2o3­nps 51.18 ± 0.76 a 6527.8 ± 119.1 a 882.2 ± 7.0 a 5.75 ± 0.07 a 35.59 ± 0.14 a 4 ds m­1 nacl water 45.68 ± 0.92 b 5760.1 ± 47.0 b 829.3 ± 9.2 cd 4.78 ± 0.08 d 33.60 ± 0.28 b sa 45.22 ± 0.23 b 5751.6 ± 89.0 b 881.7 ± 4.7 a 5.07 ± 0.07 c 34.43 ± 0.17 b fe2o3­nps 45.83 ± 0.54 b 5874.3 ± 71.0 b 854.3 ± 8.2 bc 5.02 ± 0.07 c 34.14 ± 0.17 b sa + fe2o3­nps 46.83 ± 0.31 b 5919.4 ± 85.5 b 866.0 ± 3.6 ab 5.12 ± 0.06 c 34.40 ± 0.24 b 8 ds m­1 nacl water 33.72 ± 0.77 e 3953.6 ± 144.3 e 669.3 ± 9.6 f 2.64 ± 0.06 g 30.63 ± 0.45 d sa 39.35 ± 0.83 c 4789.9 ± 97.4 c 827.5 ± 7.0 d 3.96 ± 0.07 e 31.92 ± 0.31 c fe2o3­nps 36.65 ± 0.78 d 4437.6 ± 106.2 d 780.3 ± 6.9 e 3.46 ± 0.06 f 30.55 ± 0.53 d sa + fe2o3­nps 39.78 ± 1.00 c 4852.7 ± 120.6 c 836.2 ± 12.8 cd 4.05 ± 0.08 e 32.45 ± 0.30 c 12 ds m­1 nacl water 19.57 ± 0.72 g 1946.3 ± 108.7 h 605.0 ± 2.2 h 1.18 ± 0.07 j 26.10 ± 0.51 f sa 31.75 ± 0.37 e 3326.2 ± 117.0 f 687.7 ± 12.8 f 2.27 ± 0.03 h 30.19 ± 0.18 d fe2o3­nps 27.18 ± 1.04 f 2820.9 ± 87.3 g 630.0 ± 2.6 g 1.78 ± 0.05 i 28.47 ± 0.30 e sa + fe2o3­nps 33.32 ± 0.44 e 3471.7 ± 102.6 f 686.7 ± 8.8 f 2.38 ± 0.05 h 30.79 ± 0.42 d source of variation year (y) ns ns ns ns ns salinity (s) ** ** ** ** ** foliar treatments (f) ** ** ** ** ** y ´ s ns ns ns ** ns y ´ f ns ns ns ns ns s ´ f ** ** ** ** ** y ´ s ´ f ns ns ns ns ns f test 13.19** 12.73** 12.92** 32.35** 9.78** different letters in each column indicate significant differences at p≤0.05; ns, **= no significant and significant at p≤0.01, respectively. fe2o3­nps= iron­oxide nanoparticles; sa= salicylic acid. table 4 ­ combined analysis of variance of the data for yield parameters of ajowan affected by salinity and foliar treatments in 2018 and 2019 fig. 2 ­ changes in seeds per umbel of ajowan in response to salinity. the data represents the average of three repli­ cates in two years ± standard errors. different letters indicate significant differences at p≤0.05. ghassemi‐golezani and abdoli ‐ improving ajowan performance by foliar treatments 137 growth of ajowan, leading to lower seed yield.negative impacts of salt stress on plant growth and productivitycould be considerably alleviated by exogenous salicylic acid and fe2o3 nanoparticles, particularly in combined form. these beneficial effects were more pronounced under severe salinity. the ameliorative effects of sa and sa+ fe2o3­nps on seed yield of salt­subjected ajowan plants were mostly related to enhancing root and shoot growth, seeds per plant, seed filling rate, and 1000­seed weight. future works may reveal other beneficial effects of different hormones and/or nanoparticles on crops under various environmental conditions. acknowledgements we appreciate the financial support of this research by the university of tabriz and the iran national science foundation. references abdoli s., ghassemi­golezani k., 2021 ­ salicylic acid: an effective growth regulator for mitigating salt toxicity in plants. ­ j. plant physiol. breeding, 11: 1­15. abdoli s., ghassemi­golezani k., alizadeh­salteh s., 2020 ­ responses of ajowan (trachyspermum ammi l.) to exogenous salicylic acid and iron oxide nanoparticles under salt stress. ­ environ. sci. pollut. res. int., 27(29): 36939­36953. abrar m.m., saqib m., abbas g., atiq­ur­rahman m., mustafa a., 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is mainly attributed to inadequate pollination resulting from pistillate flower abscission (pfa) and adverse climatic conditions during pollen shedding and stigma receptivity. apart from these factors, dichogamy is also a main cause for low yields in walnut which was steady and biological characteristics of walnut (akca and sen, 1997). the phenomenon of dichogamy involves development on the same plant of male and female organs at different times. ‘protandry’ refers to the shedding of pollen prior to stigma receptivity and ‘protogyny’ the reverse sequence (gleeson, 1982). the mating system with both protandrous and protogynous plants within the species has been classified as heterodichogamy (gleeson, 1982; luza and polito, 1988). the extent of heterodichogamy varies from almost complete overlap (homogamy) to complete separation of male and female bloom periods (mcgranahan and leslie, 1991). the degree and nature of dichogamy is a varietal character, but it is also greatly affected by the age of the tree, climate and geographic location (wood, 1932). walnut variety or locality is free from dichogamy. on the basis of these data it is possible to make combinations of cultivars, which ensure pollinating and enable regular harvest. the present work deals with protandrous-protogynous dimorphism in 20 walnut cultivars/selections. to overcome the problem of low yields and to identify suitable cultivars that can overlap in blooming time, the present study was conducted at the university of horticulture and forestry, nauni-solan for three consecutive years. on this basis we can define the most suitable combinations of cultivars for north-western region of india. protandrous-protogynous dimorphism in indigenous selections from north western india and some exotic cultivars of persian walnut (juglans regia l.) a. kumar*, n. sharma** * division of fruit science, sher-e-kashmir university agricultural sciences and technology, kashmir, shalimar, srinagar, jammu and kashmir, india. ** division of fruit science, sher-e-kashmire university agricoltural sciences and tecnology, jammu, chatta, jammu, jammy and kashmir, india. key words: dichogamy, low productivity, pollen shedding, stigma receptivity, walnut. abstract: walnut (juglans regia l.) is one of the most important temperate nuts grown worldwide. in india, however, most of the produce comes from age-old trees of unknown origin. apart from non-adoption of standard farm practices, certain inherent problems like dichogamy are the major factors of low productivity. the problem of dichogamy is further aggravated by short period of pollen shedding and stigma receptivity. dichogamy in walnut prevents self-pollination and necessitates cross-pollination to set fruits. for optimum fruit set and consequent yield, an attempt was made with 20 cultivars/selections grafted on seedling walnuts for three consecutive years to determine the nature and degree of dichogamy. observations were recorded on the time of male and female flowering, duration of pollen shedding and stigma receptivity, nature and degree of dichogamy. results indicate that out of 20 cultivars/selections fourteen have protandrous nature, ‘gobind’ in the first and third year, ‘kx giant’ in second and third year and ‘plant no. 45’ in the second year have homogamous nature and only ‘gobind’ have protogynous nature in the second year. degree of dichogamy varies from zero to 100 per cent among various cultivars/selections. findings of the present study emphasized on the interplanting of protandrous and protogynous cultivars or homogamous cultivars to ensure adequate pollination to obtain higher nut yields in walnut. adv. hort. sci., 2013 27(1-2): 61-66 received for publication 20 march 2013 accepted for publication 24 april 2013 62 2. materials and methods the present study was carried out over a three-year period (2006-2008) in the walnut germplasm collection block of the department of fruit breeding and genetic resources, dr. y s parmar university of horticulture and forestry, nauni-solan, himachal pradesh. the walnut block is located at an elevation of 1225 m a.m.s.l. and between 31on latitude and 77oe longitude. the mean average rainfall of the study area during the flowering season (february to april) ranges from 125 to 200 mm. the minimum and maximum temperatures of the field area during the flowering season range between 10 and 30oc. the experiment was conducted with the following 20 cultivars/selections: ‘aco 38853’, ‘blackmore’, ‘gobind’, ‘hartley’, ‘plant no.10’, ‘netar akhrot’, ‘roopa akhrot’, ‘kx giant’, ‘rattan akhrot’, ‘lake english’, ‘kandaghat selection’, ‘payne’, ‘inder akhrot’, ‘plant no. 32’, ‘xenia’, ‘plant no. 45’, ‘plant no. 46’, ‘plant no. 47’, ‘solding selection’ and ‘luxmi akhrot’. these cultivars/selections were grafted on walnut seedlings and the age of the planting material during the study was 18-20 years old. the grafted plants were planted at a distance of 7x7 m and standard practices of orchard management were followed. observations were taken with regard to time of male and female flowering, duration of pollen shedding and stigma receptivity, nature and degree of dichogamy. nature of dichogamy was determined as protandry: maturation of male catkins prior to stigma receptivity of female flower protogyny: stigma receptivity prior to maturation of male catkins homogamy: maturation of male catkins coinciding with stigma receptivity in female flowers. degree of dichogamy was calculated according to the formula suggested by solar et al. (1997). degree of dichogamy (%) = = 1 no. of days when male and female flowering coincides x 100 number of days of female flowering 3. results and discussion the results obtained from the three consecutive years of study showed variation with respect to time of male and female flowering, duration of pollen shedding and stigma receptivity, nature and degree of dichogamy. the earliest emergence of catkins in all three years was recorded in ‘plant no. 45’ (i.e. 12 march, 17 march and 15 march in the first, second and third years, respectively) along with ‘luxmi akhrot’ (12 march) in the first year, and ‘netar akhrot’ and ‘inder akhrot’ (17 march) in the second year (fig. 1). late emergence of catkins was observed in ‘solding selection’ (25 march) in the first year; in the second and third years late emergence was observed in ‘gobind’ (7 april and 28 march, respectively). ‘plant no. 45’ showed early female flowering (15 march in the first year, 22 march in second and 17 march in the third), whereas late emergence of female flowering in the first year was observed in ‘rattan akhrot’ (31 march), in the second year in ‘plant no. 32’ (8 april) and in the third in ‘aco 38853’ and ‘luxmi akhrot’ (1 april). early pollen shedding in 2006 and 2008 was recorded in ‘plant no. 45’ (17 march and 20 march, respectively) along with ‘hartley’ and ‘luxmi akhrot’ in 2006, however, it was recorded in ‘netar akhrot’ in 2007 (24 march) (fig. 2). as for late pollen shedding the following observations were made: ‘solding selection’ (30 march 2006) and ‘gobind’ (10 april 2007 and 1 april 2008). in all three years ‘plant no. 45’ had early stigma receptivity (19 march, 25 march and 21 march, respectively). however, there was late stigma receptivity in ‘rattan akhrot’ in the first year (5 april), ‘plant no. 32’ and ‘xenia’ in the second year (12 april) and ‘aco 38853’, ‘blackmore’ and ‘luxmi akhrot’ in the third year (5 april). regarding the nature of dichogamy, it is clear from table 1 that ‘gobind’ was found to be homogamous in the first and third years, ‘kx giant’ in the second and third years and ‘plant no. 45’ in the second year. however, during the study period only ‘gobind’ showed a protogynous nature in the second year. while ‘kx giant’, ‘plant no. 32’ and ‘plant no. 45’ in 2006, ‘netar akhrot’ and ‘xenia’ in 2007 and again ‘plant no. 45’ in 2008 revealed a slight protandrous nature. all the other cultivars were of protandrous nature in all years. ‘aco 38853’, ‘blackmore’, ‘hartley’, ‘roopa akhrot’, ‘lake english’, ‘inder akhrot’, ‘plant no. 47’ and ‘luxmi akhrot’ were found to have a 100% degree of dichogamy during the course of study; ‘kx giant’ and ‘plant no. 45’ in the second year, and ‘gobind’ in the third year showed a 0% degree of dichogamy. other cultivars/selections had a wide range of degrees of dichogamy during the study. from the present study it is clear that persian walnut has a large variation with regard to nature and degree of dichogamy. the degree of dichogamy in various cultivars/ selections varies from zero to 100%. it is generally assumed that protandrous cultivars are the most numerous, followed by protogynous types, while homogamous types are quite rare (mcdaniel, 1957; majackaja, 1969; germain et al., 1981) as also revealed in the present study. the trend here is towards protandry with as many as fourteen protandrous cultivars/selections (‘aco 38853’, ‘blackmore’, ‘hartley’, ‘plant no. 10’, ‘roopa akhrot’, ‘rattan akhrot’, ‘lake english’, ‘kandaghat selection’, ‘payne’, ‘inder akhrot’, ‘plant no. 46’, ‘plant no. 47’, ‘solding selection’ and ‘luxmi akhrot’) as demonstrated from the extent of synchronization of male and female flowering, and especially the time of pollen shedding and stigma receptivity. ‘gobind’ in the first and third years, ‘kx giant’ in second and third and ‘plant no. 45’ in the second year had a homogamous nature, whereas only ‘gobind’ showed a protogynous nature in the second year. however, the trend towards protandry was also reported by earlier researchers. germain et al. (1983 a) found among 63 64 65 table 1 nature and degree of dichogamy in different walnut cultivars/selections cultivar/selection first year second year third year nature degree nature degree nature degree aco 38853 protandrous 100 protandrous 100 protandrous 100 blackmore protandrous 100 protandrous 100 protandrous 100 gobind homogamous 14.28 protogynous 50 homogamous 0 hartley protandrous 100 protandrous 100 protandrous 100 plant no. 10 protandrous 100 protandrous 83.33 protandrous 83.33 netar akhrot protandrous 62.50 slightly protandry 33.33 protandrous 57.14 roopa akhrot protandrous 100 protandrous 100 protandrous 100 kx giant slightly protandry 33.33 homogamous 0 homogamous 14.28 rattan akhrot protandrous 100 protandrous 60 protandrous 57.14 lake english protandrous 100 protandrous 100 protandrous 100 kandaghat selection protandrous 71.43 protandrous 100 protandrous 100 payne protandrous 80 protandrous 60 protandrous 50 inder akhrot protandrous 100 protandrous 100 protandrous 100 plant no. 32 slightly protandry 28.57 protandrous 100 protandrous 42.86 xenia protandrous 83.33 slightly protandry 33.33 protandrous 50 plant no. 45 slightly protandry 16.67 homogamous 0 slightly protandry 14.28 plant no. 46 protandrous 66.67 protandrous 100 protandrous 71.42 plant no. 47 protandrous 100 protandrous 100 protandrous 100 solding selection protandrous 42.86 protandrous 60 protandrous 42.86 luxmi akhrot protandrous 100 protandrous 100 protandrous 100 20 french cultivars 16 protandrous, three protogynous and one homogamous. among 100 cultivars studied by yadrov (1982) the majority was protandrous (approximately 60%), while approximately 30% were protogynous and only 10 % were homogamous. working on 10 cultivars of french and american origin aleta and ninot (1987) identified six absolutely protandrous cultivars, one protogynous and three partly homogamous. korac et al. (1989) found among 14 cultivars, 11 protandrous, two protogynous and one partly homogamous. the occurrence of heterodichogamy in persian walnut (i.e. protandry, protogyny and homogamy) as revealed here, is reported also by several other workers (cheng, 1978; radicati, et al., 1983; cerovic et al., 1995). the present investigation on the nature of dichogamy is primarily based upon the overlap between pollen shedding and stigma receptivity and not on the conventional method of classifying varieties on the basis of time of emergence of male and female flowers. the degree and nature of dichogamy is a varietal character, but it is also greatly affected by tree age, climate and geographic location (wood, 1932). thus it is concluded that dichogamy is genotype-specific as shown from the considerable variation observed in the 20 cultivars/selections studied. to achieve optimum fruit set and consequent yield, near homogamous types (‘gobind’ or ‘kx giant’) may be planted in single cultivar walnut orchards, or a mixture of cultivars can be interplanted to allow sufficient overlap between pollen shedding and stigma receptivity. on the basis of the present study on protandrous-protogynous dimorphism, the mentioned possible pollinators in table 2 are suggested as suitable cultivars/ selections for growing in north-western india. references akca y., sen s.m., 1997 the relationship between dichogamy and yield-nut characteristics in juglans regia l. acta horticulturae, 442: 215-216. aleta n., ninot a., 1987 el nogal. un cultivo tradicional con futuro. fruticultura professional, 11: 55-59. bauckmann m., 1974 the length of male flowers (catkins) on grafted walnut trees. mitt. rebe wein, obstbau freuchterwert, 24: 463-466. cerovic s., korac m., todorovic j.n., 1995 dichogamy in walnut (juglans regia l.). jugoslovensko vocarstvo, 29(3/4): 21-25. cheng w.c., 1978 juglans regia l. the editorial commission of chinese flora of trees. the planting technology of the main forest trees in china, pp. 1342. forde h.i., 1977 walnuts, pp. 439-455. in: janick j. and j.n. moore (eds.). advances in fruit breeding. purdue university press, west lafayette ind., usa. germain e., jalinat j., marchou m., 1981 divers aspects de la biologie florale de noyer, pp. 13-27. in: bergougnoux f., and p. grospierre(eds.) le noyer. invuflec, paris france. germain e., jalinat j., leglise p., masseron a., tronel c., chartier a., 1983 a le noyer, resultants de 20 ans experimentation. arb. fruit., 356: 55-60. 66 gleeson s.k., 1982 heterodichogamy in walnuts. inheritance and stable ratios. evolution, 36(5): 892-902. korac m., cerovic s., mitroviac m., kuzmanovski i., jovaneeviac d., solar a., 1989walnut production, population variability and breeding results achieved in yugoslavia. first int. symp. on walnut production, budapest, hungary, 25-29 sept., pp. 9. luza j.g., polito v.s., 1988 microsporogenesis and anther differentiation in juglans regia l.; a developmental basis for heterodichogamy in walnut. bot. gaz., 149(1): 30-36. majackaja a.d., 1969 dichogamy and fruiting in walnuts. lesn. hoz., 2: 32-35. manning w.e., 1938 the morphology of the flowers of juglandaceae. i. the inflorescence. american journal of botany, 25: 407-419. mcdaniel j.c., 1957 the pollination of juglandaceae varieties – illinois observations and review of earlier studies. nut growers assn. annual rep., 48: 89-93. mcgranahan g.h., leslie c., 1991 walnuts (juglans), pp. 907-951. in: ballington j.r., and j.n. moore(eds.) genetic resources of temperate fruit and nut crops. ishs secretariat, the netherlands, vol. ii, pp. 980. radicati l., me g., sacerdote s., vallania r., 1983 prime valutazioni di cultivars di noci americana e francesi per le zone montana. frutticoltura, 45(3-4): 19-23. solar s., stamper f., smole j., 1997 the degree of heterodichogamy of some walnut cultivars (juglans regia l.) in slovenia. acta horticulturae, 442: 217-224. wood m.n., 1932 dichogamyan important factor affecting production in the persian walnut. proceedings of american society for horticultural science, 34: 160-164. yadrov, a.a. 1982. dichogamy and fruit production in walnut. byulleten gosudarstvennogo nikitskogo botanicheskogo sada, 49: 68-72. table 2 suitable pollinators for different walnut cultivars/selections main cultivars/selections suitable pollinators aco 38853 plant no. 32 blackmore gobind, xenia gobind xenia, plant no. 32 hartley gobind, xenia, plant no. 32 plant no. 10 gobind, xenia, plant no. 32 netar akhrot roopa akhrot, inder akhrot, plant no. 45, lake english roopa akhrot kx giant, plant no. 32, plant no. 46 kx giant kandaghat selection, plant no. 10 rattan akhrot plant no. 32, plant no. 46 kandaghat selection plant no. 32, xenia payne plant no. 32, xenia inder akhrot plant no. 32, kx giant, solding selection plant no. 32 gobind xenia gobind plant no. 45 roopa akhrot, netar akhrot plant no. 46 gobind, xenia plant no. 47 solding selection, gobind, aco 38853 solding selection plant no. 32, kx giant luxmi akhrot plant no. 32, solding selection, kx giant, aco 38853 123 1. introduction persian walnut (juglans regia l.) is one of the most important nut crops grown in temperate regions and it produces edible nuts of high nutritional value. in india there are no systematic orchards of walnut containing standard cultivars. although some efforts have been made in this direction in the last two decades and the superior selections from the seedling populations were selected through field surveys in different walnut growing districts of himachal pradesh (thakur, 1993; gupta, 1999; sharma, 1999, 2002; thakur et al., 2005). however, a large number of exotic accessions have also been introduced from abroad and a germplasm collection block has been maintained after propagation of these selections and exotic accessions on the seedling rootstock. therefore characterization and evaluation of this germplasm is important for future use and a wider divergence is a prerequisite for breeding purposes. information about the extent of genetic divergence is critical for the improvement of any crop in order to have heterotic responses and desirable segregants. furthermore, information on the nature and degree of genetic divergence present in the collected germplasm could help for further improvement through hybridization. 2. materials and methods the present investigation was undertaken on threeto four-year-old plants for a total of 54 indigenous selections collected through field survey at different locations in himachal pradesh and exotic cultivars of persian walnut introduced directly from abroad (table 1). these were propagated on seedling rootstocks and are presently growing at oachghat block of dr. y.s. parmar university of horticulture and forestry, nauni-solan (himachal pradesh) located around 1275 m above mean sea level and between 31on latitude and 77oe longitude. data for various growth [plant height (m), trunk girth (cm2), plant spread (cm), tcsa (cm2) and plant volume (cm3)] and foliage characters [leaf length (cm), leaf width (cm), leaf area (cm2), leaflet length (cm), leaflet width (cm) and number of leaflets] were recorded. ten compound leaves were taken from each individual tree for foliage characters and average means were calculated. standard procedures were followed to calculate the mean, standard deviation and coefficient of variation for various characters as described by panse and sukhatme (1995). the genetic divergence among the accessions was estimated by mahalanobis d2 statistic as suggested by rao (1952). genetic divergence studies regarding different growth and foliage characters of walnut (juglans regia l.) germplasm s. sharma*, k. kumar** (1), a. kumar*** * division of fruit science, skuast, jammu, jammu and kashmir, india. ** department of fruit science, uhf, nauni-solan, himachal pradesh, india. *** division of fruit science, skuast-kashmir, jammu and kashmir, india. key words: divergence, exotic cultivars, growth, local selections, walnut germplasm. abstract: genetic divergence of 29 exotic walnut cultivars introduced from abroad and 25 local selections from seedling population was studied. the analysis of variance revealed significant differences among accessions for each character under study. based on mahalanobis d2 values the accessions were grouped into eight clusters. cluster i had a maximum of 12 accessions followed by cluster ii with 11 accessions. the mean intra and inter cluster distance (d) revealed that cluster vii had the highest intra cluster distance (2.171), while the inter cluster distance was the greatest between cluster ii and iv (10.528). characters like plant height, trunk girth, plant spread, plant volume, leaf length, leaf width and number of leaflets contributed the most to total divergence. adv. hort. sci., 2014 28(3): 123-128 (1) corresponding author: khokherak@rediffmail.com received for publication 15 august 2013 accepted for publication 11 september 2014 124 all the accessions were grouped into clusters according to the tocher’s method described in rao (1952). 3. results and discussion the analysis of variance revealed significant differences among the genotypes for each character, indicating the existence of variability among the genotypes for such characters. on the basis of the relative magnitude of d2 values, 54 accessions were grouped, using tocher’s method, into eight clusters. cluster i was the largest with 12 accessions followed by cluster ii having 11 accessions, and cluster v having 10 accessions; clusters iii, iv, vi, vii, viii had 5, 1, 9, 4, 2 accessions, respectively (fig. 1 and table 2). the highest intra cluster distance was registered in cluster vii (2.171) followed by cluster ii (1.951), while the minimum intra cluster value was exhibited by cluster iv (0.000). the minimum inter-cluster d2 value was recorded between cluster i and v (1.821), indicating a close relationship and similarity of most traits of the accessions. hence, selection of parents from these clusters should be avoided. however, the highest inter-cluster d2 value was observed between clusters ii and iv (10.528), followed by clusters i and iv (10.411), confirming a wide genetic distance between these groups (table 3). since these clusters exhibited the greatest inter-cluster distances, selection of parents from such clusters for hybridization programmes could help to develop novel hybrids. a similar trend of clustering patterns has been reported by barua and sharma (2003) in apple, thakur et al. (2005) in almond., kaushal and sharma (2005) in pecan, and sharma and sharma (2005), pandey and tripathi (2007), and sharma et al. (2010) in walnut. the mean value, standard deviation and co-efficient of variability on various growth and foliage traits of cluster is presented in table 4. cluster iv had the highest mean values for characters plant height (5.50 m), trunk girth (33.00 cm) and tcsa (86.55 cm2), while cluster viii exhibited the highest mean values for plant spread (110.00 cm), plant volume (1.71 cm3), leaf length (56.50 cm), leaf width (30.25 cm), leaf area (57.35 cm2), leaflet length (16.17 cm). maximum mean values for leaflet width (7.19 cm) and number of leaflets (9.73) was recorded in clusters vi and ii, respectively. clusters iv and viii can be expected to give promising and desirable recombinations in segregating generations because they comprise desirable features as revealed from their cluster means. similarly, maximum co-efficient of variability for plant height (40.00%), trunk girth (33.11%), plant spread (42.08%), tcsa (65.21%), plant volume (87.50%) and number of leaflets (23.12%) was observed in cluster ii, however, cluster i had the highest co-efficient of variability for traits leaf area (32.86%), leaflet length (13.46%) and leaflet width (19.79%). in the principal component analysis presented in table 5, the first vector shows the highest eigen value (4.332) table 1 different exotic and indigenous walnut accessions and their place of origin s. no. accession place of origin exotic cultivars 1. adams 10 usa 2. aksu≠24 china 3. aksu≠71 china 4. aksu≠81 china 5. aksu≠210 china 6. aksu≠417 china 7. aksu hun ≠85 china 8. bulgaria 3 bulgaria 9. chandler usa 10. chico usa 11. cisco usa 12. conway mayette usa 13. corne france 14. graves franquette usa 15. hotien 8 china 16. howard usa 17. howe usa 18. mayette usa 19. meylannaise france 20. nn 88 godyn poland 21. parisienne france 22. placentia usa 23. ronde de montignac france 24. scharsch franquette usa 25. serr usa 26. shinrei china 27. xin zad fen china 28. xin zheng zhu china 29. zhong lin≠3 china indigenous selections 30. brij lal selection h.p. (india) 31. chamba selection-20 h.p. (india) 32. chamba selection-32 h.p. (india) 33. chamba selection-60 h.p. (india) 34. chamba selection-101 h.p. (india) 35. chamba selection-123 h.p. (india) 36. daulat ram selection h.p. (india) 37. jaunaji selection -2 h.p. (india) 38. jaunaji selection –4 h.p. (india) 39. jaunaji selection –6 h.p. (india) 40. jaunaji selection-7 h.p. (india) 41. jaunaji selection-10 h.p. (india) 42. jaunaji selection-12 h.p. (india) 43. jogindernagar selection-23 h.p.(india) 44. jogindernagar selection-39 h.p. (india) 45. jogindernagar selection-61 h.p. (india) 46. jogindernagar selection-122 h.p. (india) 47. nauradhar selection-10 h.p. (india) 48. nauradhar selection-36 h.p. (india) 49. nauradhar selection-53 h.p. (india) 50. rajgarh selection-1 h.p. (india) 51. rajgarh selection-8 h.p. (india) 52. rajgarh selection-11 h.p. (india) 53. selection no. 8 h.p. (india) 54. selection no. 51 h.p. (india) 125 and accounts for 39.38% of the total variation. the first vector is the combination of leaf length, trunk girth, plant height and leaflet length. the second vector has an eigen value of 3.008 and explains 27.34% of total variation, and this factor is mainly the combination of number of leaflets and trunk girth. the third vector has an eigen value of 1.155 and total variation of 10.50%, from the combination of leaf width and plant spread. the fourth vector has an eigen value of 1.047 and total variation of 9.52%, with the maximum contribution of trunk girth. vector fifth has an eigen value of 0.424 and total variation of 3.85%, mainly a combination of plant volume and plant spread. the sixth vector has an eigen value of 0.347 with total variation of 3.15 % and the highest contributable 2 distribution of 54 walnut accessions into various clusters clusters number of accessions accessions i 12 aksu≠210, chamba selection-32, chamba selection-60, conway mayette, hotien 8, jaunaji selection-2, jaunaji selection-12, jogindernagar selection-61, nauradhar selection-10, parisienne, ronde de montignac, xin zheng zhu ii 11 adams 10, aksu≠24, chamba selection-101, jaunaji selection-7, jaunaji selection-10, jogindernagar selection-39, jogindernagar selection-122, meylannaise, nauradhar selection-36, nauradhar selection-53, selection no. 8. iii 5 aksu≠81, cisco, daulat ram selection, jaunaji selection-4, scharsch franquette iv 1 howard v 10 aksu≠71, aksu≠417, bulgaria 3, graves franquette, howe, jaunaji selection-6, mayette, rajgarh selection-1, selection no. 51, xin zad fen vi 9 aksu hun≠85, brij lal selection, chico, corne, jogindernagar selection-23, nn 88 godyn, serr, shinrei, zhong lin ≠3 vii 4 chamba selection-20, placentia, rajgarh selection-8, rajgarh selection-11 viii 2 chamba selection-123, chandler fig. 1 dendrogram showing different clusters of genotypes, grouped by tocher’s method, in walnut accessions. 126 table 4 mean, standard deviation and coefficient of variation for various clusters on the basis of growth and foliage characters of walnut germplasm s. no. characters parameters clusters i ii iii iv v vi vii viii 1 plant height (m) mean 1.12 1.00 2.98 5.50 1.36 1.77 1.91 2.65 sd 0.27 0.40 0.85 0.00 0.52 0.49 0.61 0.21 cov 24.11 40.00 28.52 0.00 38.23 27.68 31.94 7.92 2 trunk girth (cm) mean 7.15 7.55 10.40 33.00 9.45 10.75 12.12 16.00 sd 2.05 2.50 2.25 0.00 2.60 1.70 3.12 2.83 cov 28.67 33.11 21.63 0.00 27.51 15.81 25.74 17.69 3 plant spread (cm) mean 31.71 36.36 44.00 50.00 39.50 51.94 70.00 110.00 sd 11.48 15.30 13.87 0.00 12.95 17.36 17.80 14.14 cov 36.20 42.08 31.52 0.00 32.78 33.42 25.43 12.85 4 tcsa (cm2) mean 4.38 5.03 8.91 86.55 7.61 8.27 12.28 20.74 sd 2.17 3.28 3.29 0.00 4.56 2.99 6.10 7.25 cov 49.54 65.21 36.92 0.00 59.92 36.15 49.67 34.96 5 plant volume (cm3) mean 0.07 0.08 0.32 0.72 0.15 0.30 1.57 1.71 sd 0.06 0.07 0.21 0.00 0.11 0.25 0.76 0.57 cov 85.71 87.50 65.62 0.00 73.33 83.33 48.41 33.33 6 leaf length (cm) mean 33.46 46.73 35.00 40.00 27.05 39.33 32.62 56.50 sd 5.47 3.98 2.89 0.00 3.46 4.84 6.94 6.36 cov 16.35 8.52 8.26 0.00 12.79 12.31 21.27 11.26 7 leaf width(cm) mean 18.55 24.97 18.95 20.50 15.50 22.58 18.06 30.25 sd 2.90 2.26 2.15 0.00 2.41 3.97 1.88 0.35 cov 15.63 9.05 11.34 0.00 15.55 17.58 10.41 1.16 8 leaf area (cm2) mean 29.73 56.69 33.34 38.39 26.28 48.00 38.10 57.35 sd 9.77 8.90 8.12 0.00 6.25 8.64 9.60 2.96 cov 32.86 15.70 24.35 0.00 23.78 18.00 25.19 5.16 9 leaflet length (cm) mean 10.70 14.57 10.79 12.50 8.81 13.58 10.64 16.17 sd 1.44 1.15 1.02 0.00 1.13 1.56 1.32 0.23 cov 13.46 7.89 9.45 0.00 12.83 11.49 12.41 1.42 10 leaflet width (cm) mean 4.95 6.93 5.21 5.46 4.55 7.19 5.58 7.00 sd 0.98 0.68 0.82 0.00 0.65 0.55 1.05 0.00 cov 19.79 9.81 15.74 0.00 14.28 7.65 18.82 0.00 11 number of leaflets mean 9.29 9.73 8.80 9.00 7.65 8.17 8.00 8.00 sd 1.32 2.25 0.45 0.00 0.82 0.87 1.15 1.41 cov 14.21 23.12 5.11 0.00 10.72 10.65 14.37 17.62 table 3 average interand intra-cluster distance among various clusters in walnut germplasm clusters i ii iii iv v vi vii viii i 1.658 ii 3.535 1.951 iii 2.295 3.975 1.497 iv 10.411 10.528 8.795 0.000 v 1.821 4.953 2.505 10.048 1.396 vi 3.159 2.115 2.689 9.396 3.875 1.657 vii 3.870 4.898 2.984 9.037 3.536 3.388 2.171 viii 7.505 5.835 6.356 8.920 7.931 5.081 5.220 1.373 bold figures represent intra-cluster distances. 127 tion came from plant height, while the minimum eigen roots value 0.045 and percent contribution towards diversity (0.41%) was observed for the eleventh component, with a maximum contribution from tcsa. the percent variation explained by the first seven components was 95.95 (table 5). the genetic diversity among genotypes could be due to various factors, like heterogeneity, genetic architecture of populations and developmental traits, as described by (murty and arunachalam, 1966). rao et al. (2003) reported that geographical distribution and genetic diversity are correlated and concluded that eco-geographically different cultivars also differ from each other genetically. a clustering pattern similar to our pattern was reported in walnut (pandey and tripathi, 2007) and hazelnut (srivastava et al., 2010). sardana et al. (1997) observed that cluster means reveal the inner diversity in the material under study. de et al. (1988) proposed that traits contributing the most towards the d2 values should be given priority in choosing a cluster for further selection and choice of parents for hybridization. based on the present findings of genetic divergence and its component analysis it can be concluded that intercrossing between genotypes of genetically diverse clusters that show a superior mean outcome may be helpful for obtaining desirable segregates. in the case of walnut germplasm the highest genetic diversity was registered between clusters ii and iv. comparison of the cluster means for 11 characters indicated that the studied traits considerably differed between the clusters. therefore, it is suggested to cross ‘howard’ with ‘chamba selection 123’ and with ‘chandler’ as these accessions are among the most distant clusters, with a high mean outcome, in order to get desirable transgressive segregates. references barua u., sharma r.k., 2003 estimation of genetic divergence in apple (malus x domestica borkh.). indian journal of plant genetic research, 16: 4-6. de r.m., seetharaman r., sinha m.t., banarjee s.p., 1988 genetic divergence in rice. indian journal of genetics, 48: 189-194. gupta s.k., 1999 characterization and evaluation of walnut (j. regia l.) germplasm accessions. m.sc. thesis, dr. y.s. parmar university of horticulture and forestry, naunisolan, india. kaushal r.k., sharma s.d., 2005 non-hierarchical euclidean cluster analysis studies in seedling trees of pecan [carya illinoensis (wang) k. koch.] growing in himachal pradesh. acta horticulturae, 696: 97-101. murty b.r., arunachalam v., 1966 the nature of divergence in relation to breeding system in crop plants. indian journal of genetics, 26: 188-198. pandey g., tripathi a.n., 2007 estimation of genetic divergence in walnut. indian journal of horticulture, 64(4): 399-401. panse v.g., sukhatme p.v., 1995 statistical methods for agricultural workers. icar pub., new delhi, pp. 296. rao c.r., 1952 advanced statistical method in biometrical research. john wiley & sons, new york, pp. 390. rao e.s., verma v.k., munshi a.d., 2003 breeding potential of cucumber (cucumis sativus l.) genotypes using d2 analysis. indian journal of horticulture, 60(1): 53-58. sardana s., borthakur d.n., laakanpal t.n., 1997 genetic divergence in rice germplasm of tripura. oryza, 34: 201-208. sharma o.c., 1999 studies on variability and selection of superior persian walnut (juglans regia l.) seedling trees in himachal pradesh. ph.d. thesis, dr. y.s. parmar univertable 5 eigen vectors, eigen roots and percent variation explained by growth and foliage characters of walnut germplasm s. no. characters eigen roots per cent variation eigen vectors 1 2 3 4 5 6 7 8 9 10 11 1 plant height (m) 4.332 39.38 0.131 0.180 0.239 0.147 0.192 0.415 0.419 0.404 0.432 0.369 0.035 2 trunk girth (cm) 3.008 27.34 0.458 0.490 0.293 0.456 0.295 -0.145 -0.126 -0.195 -0.169 -0.196 -0.163 3 plant spread (cm) 1.155 10.50 -0.202 -0.228 0.424 -0.383 0.496 -0.128 0.018 -0.037 -0.042 0.068 -0.557 4 tcsa (cm2) 1.047 9.52 -0.059 -0.132 0.371 -0.170 0.326 0.211 0.099 -0.153 -0.129 -0.354 0.697 5 plant volume (cm3) 0.424 3.85 -0.006 -0.018 -0.379 0.051 0.579 -0.023 -0.483 0.308 -0.160 0.337 0.225 6 leaf length (cm) 0.347 3.15 0.818 -0.293 -0.075 -0.391 -0.025 -0.076 0.057 -0.158 0.037 0.213 0.066 7 leaf width(cm) 0.243 2.20 -0.054 0.125 0.512 -0.070 -0.326 -0.468 -0.111 0.276 -0.231 0.418 0.270 8 leaf area (cm2) 0.191 1.74 -0.199 0.026 -0.206 0.179 0.242 -0.426 0.469 -0.506 0.101 0.357 0.180 9 leaflet length (cm) 0.118 1.07 0.110 -0.047 -0.209 -0.024 0.141 -0.452 0.446 0.565 -0.110 -0.432 -0.002 10 leaflet width (cm) 0.091 0.83 -0.013 0.060 0.073 -0.090 0.049 -0.362 -0.354 0.001 0.813 -0.217 0.126 11 number of leaflets 0.045 0.41 -0.060 0.742 -0.192 -0.627 0.024 0.058 0.074 -0.056 -0.056 -0.003 0.021 percent variation explained by first 7 components = 95.9517 128 sity of horticulture and forestry, nauni-solan, india. sharma o.c., 2002 selecting walnuts from seedling tress populations in some villages of district sirmour (h.p.). m.sc. thesis, dr. y.s. parmar university of horticulture and forestry, nauni-solan, india. sharma o.c., murkute a.a., kanwar m.s., 2010 assessing genetic divergence in seedling trees of persian walnut (juglans regia l.). indian journal of agricultural sciences, 80(5): 360-363. sharma o.c., sharma s.d., 2005 intra specific divergence in seedling trees of persian walnut (juglans regia) for metric tree, foliage and floral characters in himachal pradesh, india. plant genetic resources newsletter, 144: 45-53. srivastava k.k., zargar k.a., singh s.r., 2010 genetic divergence among corylus colurna genotypes based on morphological characters of hazelnut. biodiversity research and conservation, 17: 13-17. thakur b.s., sharma o.c., mehta k., sharma d.p., rehalia a.s., 2005 genetic divergence in almond (prunus amygdalus batsch) growing in kinnaur, himachal pradesh acta horticulturae, 696: 69-74. thakur d., 1993 genetic variability in bearing seedling walnuts (j. regia l.) in kullu valley. m.sc. thesis, dr. y.s. parmar university of horticulture and forestry, nauni-solan, india. impaginato 249 adv. hort. sci., 2024 38(3): 249­256 doi: 10.36253/ahsc­15711 https://oaj.fupress.net/index.php/ahs effects of foliar application of two forms of selenium on anatomic features of thymus daenensis celak z. abravesh 1, a. iranbakhsh 1 (*), h. mirzaie­nodoushan 2, m. ebadi 3, z. oraghi ardebili 4 1 department of biology, science and research branch, islamic azad university, tehran, iran. 2 biotechnology department, research institute of forests and rangelands, teheran, iran. 3 department of biology, damghan branch, islamic azad university, damghan, iran. 4 department of biology, garmsar branch, islamic azad university, garmsar, iran. key words: medicinal plant, nanoparticle, thymus daenensis. abstract: to investigate selenium effects on anatomic features of thymus daenensis, three concentrations of bulk and nanoparticles of the element (2, 4 and 8 ppm) along with distilled water, as a control, were sprayed on foliar of young seedlings of the plant species for six times with 2 weeks intervals. transverse sections of stems, roots and leaves of the treated plants were manually produced and stained by carmine and methylene blue. average of 5 readings was recorded for each treatment on several anatomic traits. statistical analysis of the data revealed that the effects of selenium treatments were significantly different, at 1% of probability, on all of the studied anatomic characters. the first level of nanoparticles showed the most positive effects on several root characteristics. the highest level of selenium nanoparticles (8 ppm) showed the most positive effects on stem diameter and stem vascular cylinder diameter. the last two levels of nanoparticles had similar and the largest effect on stem epidermis thickness. the lowest level of bulk selenium (2 ppm) showed the most increment on leaf characteristics. application of a low concentration of selenium by foliar spraying on young plants of the species is suggested for more vegetative growth and higher photosynthetic efficiency. 1. introduction thymus daenensis celak related to lamiaceae family is endemic to western part of iran, known as a medicinal herb, with high concentration of thymol, and high antimicrobial activities on human pathogenic strains (zarshenas and krenn, 2015). the species is regarded as a valuable spice (*) corresponding author: iranbakhsh@iau.ac.ir citation: abravesh z., iranbakhsh a., mirzaie­ nodoushan h., ebadi m., oraghi ardebili z., 2024 ­ effects of foliar application of two forms of selenium on anatomic features of thymus daenensis celak. ­ adv. hort. sci., 38(3): 249­256. orcid: za: 0009­0005­6874­8060 ai: 0000­0001­5879­0613 hm­n: 0000­0002­1021­9220 me: 0000­0002­5664­357x zoa: 0000­0002­1705­3796 copyright: © 2024 abravesh z., iranbakhsh a., mirzaie­ nodoushan h., ebadi m., oraghi ardebili z. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 23 january 2024 accepted for publication 17 july 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15711 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0005-6874-8060 http://orcid.org/0000-0001-5879-0613 http://orcid.org/0000-0002-1021-9220 http://orcid.org/0000-0002-5664-357x http://orcid.org/0000-0002-1705-3796 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 249­256 250 plant, growing mainly on zagros mountain chain of the country. it is a perennial shrub which was traditionally used as a tonic or herbal medicine to treat gastrointestinal disorders, headache and cold. numerous studies indicated that environmental factors such as nutrient deficiency, highly affect biological properties of medicinal plants, including their anatomic features. on the other hand, anatomic studies have found a vast application domain, such as identifying fragmentary plant organs as well as providing characters of taxonomic significance, in medicinal plants. this technique could be used as a proper tool to identify the exact effects of environmental factors such as fertilizers and chemical substances on plant ultrastructure. in other words, the anatomical features are supposed to be good indicators of environmental fluctuation effects. also in areas with high levels of toxic elements, a sensitive plant species such as thymus daenensis have potential to be used as indicator of the environmental conditions. selenium is rarely found in its elemental form in our living environment, it is found in several other forms such as selenide, selenite and selenate oxidation status in soil and water (gomez­ariza et al., 1998). however, a suitable level of selenium plays a significant role in plants, animal and human health in direct or indirect ways. so that in selenium deficient areas there may be a health risk for plants, animal and human population. selenium is identified as affecting plant growth by causing physiological, morphological and anatomical alterations in plant tissues. some researches indicated that alterations depend on the concentration of the applied nanoparticle treatment (tymoszuk and kulus, 2020). in fact, as a part of selenium role in plant, antioxidant enzymes are activated, minerals are balanced, chlorophyll is also increased, and the plant would be more resistant to various stresses (raina et al., 2021). selenium also increases plant resistance against oxidative stress, which is caused by free oxygen radicals. it delays senescence of higher plants, increases their tolerance to uv­induced oxidative stress, and promotes seedling growth. it is also shown that selenium regulates water status of plants under drought conditions (kuznetsov et al., 2003). although utilizing proper concentration of selenium have positive effects such as increasing crop yield, but adverse effects of some nanoparticles have also been reported on growth characteristics of some plant species (singla et al., 2019; gao et al., 2023). plants can absorb chemical substances in nanoparticle form, better than other forms. achievements of nanofertilizers are mainly due to very small particles that are easily absorbed by plant root system. it is also due to their large surface area that increases the plant sorption effectiveness by three folds (el­ saadony et al., 2021). selenium used to be considered as a toxic element for human being. whereas, its role has been changed during the past 40 years. it contributes to healthy growth, as well as reducing the toxicity of elements such as mercury. maximum limit of selenium intake for human being is suggested to be 400 μg/day, over which negative effects of the element are expected (arthur, 1991). however, daily selenium intake of 50­200 μg/day is recommended to overcome deficient effects of the element (navarro­ alarcon and lopez­martinez, 2000). although selenium may be available at toxic level in some areas, which is harmful for grazing livestock, but as a result of soil acidity, it is not available enough in some areas, resulting to low selenium intake in human at the final food chain. in other words, suitable level of selenium supplementation seems to be beneficial for plants, animals and human being. in areas with selenium deficit in soil, fertilizers are recommended to be supplemented with some selenium forms such as sodium selenite. this research was conducted mainly to investigate the possible effects of exogenous application of bulk and nanoparticles of selenium on anatomic structure of thymus daenensis, as well as finding a safe dosage of the element to optimize foliar application of selenium for the species. 2. materials and methods seed germination and plant establishment a trial was performed at research institute of forests and rangelands of iran, to study influence of selenium on growth and anatomic structures of a medicinal plant species named thymus daenensis celak. seeds of the species were first washed with tap water and soaked in distilled water for two days. then they were first scattered on wet filter paper and kept at 4°c for 72 hours.then they were sown in planting trays. the new grown seedlings were transplanted to plastic pots containing peat moss growing media and kept in a greenhouse. abravesh et al. ‐ effects of selenium on anatomic features of thymus daenensis 251 treatment application by adaptation of the seedlings to the greenhouse conditions, the pots were arranged in five replications with three pots per each plot. exogenous application of selenium treatments was conducted on the plantlets, as follows: three concentrations of selenium nanoparticles (2, 4, and 8 ppm of nanose), and sodium selenate as the bulk form (2, 4, and 8 ppm of sose), along with distilled water as a control for the experiment, were applied on the plantlets by 6 times foliar spraying with intervals of two weeks, starting at four leave stage of the plantlets based on a completely randomized design, with five replications. the concentrations of selenium treatments were determined based on a preliminary experiment in the greenhouse. microscopic sample preparation three days after the last application of the treatments, several leaf, stem and root samples were randomly taken from each plot and fixed in a mixture of ethanol 96% and glycerin (1:1). transverse sections of the fixed samples of stems, roots and leaves were performed manually to produce slices in about 20 µm thickness and washed by distilled water. to distain the samples, they were socked in 10% sodium hypochlorite solution for 10 minutes and washed thoroughly by distilled water. for staining the sections, a combination of carmine (for 20 min) and methylene blue (for 30 sec.) were used to make the tissues with wooden skeleton, in green, and tissues with cellulosic skeleton in pink (sotoodehnia­ korani et al., 2020). then the sections were washed by distilled water again. the process was completed with the samples assembled on glass slides. the slides were observed under an olympus microscope coupled with digital camera. five readings and measurements were recorded per each experimental unit. several anatomic characteristics were recorded as indicated in table 1. statistical analysis the mean values of the data recorded on the anatomic features of the experimental units, affected by the seven different selenium treatments, were subjected to analysis of variance, after testing the homogeneity of their error variance using sas software. in other words, regarding five replications and seven treatments of the experiment, 35 mean values were analyzed for each anatomic characteristic, based on a completely randomized design. duncan multiple range test was used to classify the treatment means on the studied characteristics. 3. results and discussion the statistical analysis of the data revealed that the selenium treatments had significant effects on all of the studied anatomic characteristics at 1% of probability (table 1). classified means of the studied characteristics are presented in table 2. the lowest level of nanoparticles (2 ppm of nanose) promoted root growth by increasing root diameter (595 µm), root epidermis (75.5 µm) and root vascular cylinder diameter (38 µm) (table 2). therefore, addition of 2 ppm nanose promoted the root growth and its vascular cylinder increment. this is in accordance with the results of domokos­ szabolcsy et al. (2012). root sections formed concentric cylinders and deformation was not observed by the studied treatment levels. meanwhile, root diameter was negatively affected by the treatments, so that the smallest diameter table 1 ­ results of analysis of variation on the data recorded on the effects of seven different selenium treatments on anatomic characteristics of roots, stems and leaves of thymus daenensis (z) **= significant at 1% level of probability. (y) seven treatments and five replications, resulted in 35 data values for each trait. source of variation treatment z error y traits (df=6) (df=28) root diameter 17078.5 ** 479.6 root vascular cylinder diameter 7580.9 ** 453.9 root epidermis thickness 748.6 ** 119.4 stem diameter 36951.9 ** 5871.4 stem vascular cylinder diameter 4679.7 ** 681.2 stem epidermis thickness 3138.7 ** 305.3 stem xylem diameter 2.8 ** 0.07 leaf thickness 8944.2 ** 666.6 leaf upper cortex thickness 58.0 ** 8.8 leaf main vascular thickness 1372.0 ** 56.0 leaf lower cortex thickness 63.1 ** 11.8 leaf spongy mesophyll thickness 141.5 ** 5.8 leaf palisade mesophyll thickness 104.1 ** 6.7 leaf upper epidermis thickness 2.5 ** 0.5 leaf lower epidermis thickness 1.2 ** 0.2 leaf number of vascular bundles 3.0 ** 8.8 adv. hort. sci., 2024 38(3): 249­256 252 belonged to the highest level (8 ppm) of both nanose and sose (table 2). therefore, it may be concluded that the application of a proper quantity of selenium, in either form of nanose or sose, would improve organogenesis and root growth of thymus daenensis. zsiros et al. (2019) had the same conclusion on tobacco. however, high concentrations of the element would not be recommended. zayed et al. (1998) concluded that plants accumulate more selenium in their shoots and leaves than in their root tissues. regarding their conclusion roots of the plant species studied in this research could have been less affected by the treatments than its stems and leaves. regarding the effects of the treatments on appearance of the root transverse sections, all the treatments had similar effects, so that all of the sections of the different treatments looked circular (fig. 1). transverse sections of the stems were not completely quadrangular for the studied treatments (fig. 2). meanwhile, there were several fluctuations due to the treatments effects. two to four layers of collenchyma cells were located at the corners of the (z) mean of each trait and treatment, resulted from 5 data values obtained by five replicates. (y) means with similar letters in the same raw are not significantly different at 5% level of probability. table 2 ­ classification of the anatomic characteristics of thymus daenensis, affected by seven different concentrations of sodium sele­ nate as the bulk selenium (sose), and nanoparticles of selenium (nanose) treatments z control y 2 mg/l of sose 2 mg/l of nanose 4 mg/l of sose 4 mg/l of nanose 8 mg/l of sose 8 mg/l of nanose traits root diameter (μm) 520.2 b 480.0 c 595.0 a 518.7 b 450.1 d 465.4 cd 415.8 e root vascular cylinder diameter (μm) 286.6 de 326.4 b 381.6 a 316.5 bc 295.8 cd 275.7 de 263.8 e root epidermis thickness (μm) 36.3 c 52.2 b 75.5 a 60.3 b 65.3 ab 51.8 b 56.8 b stem diameter (μm) 734.0 b 618.6 c 662.3 bc 685.2 bc 747.2 b 711.3 bc 888.9 a stem vascular cylinder diameter (μm) 323.2 ab 258.7 c 294.4 b 321.9 ab 290.0 bc 304.0 b 355.4 a stem epidermis thickness (μm) 112.8 bc 91.8 cd 99.3 cd 75.8 d 148.3 a 87.8 d 127.0 ab stem xylem diameter (μm) 8.1 a 6.1 d 7.7 b 6.4 cd 6.7 c 6.8 c 7.6 b leaf thickness (μm) 259.8 c 260.5 c 293.0 bc 268.1 c 319.5 b 327.7 b 373.1 a leaf upper cortex thickness (μm) 30.7 a 22.4 c 23.1 bc 23.6 bc 29.5 a 26.8 ab 23.1 bc leaf main vascular thickness (μm) 138.7 d 170.4 b 155.1 c 185.5 a 147.7 cd 174.6 b 171.0 b leaf lower cortex thickness (μm) 27.8 b 32.5 a 24.2 bc 24.2 bc 22.3 c 25.6 bc 22.7 c leaf spongy mesophyll thickness (μm) 100.6 c 108.9 a 106.1 ab 103.7 bc 94.2 d 97.0 d 106.0 ab leaf palisade mesophyll thickness (μm) 90.6 c 98.1 a 92.5 bc 87.5 de 89.3 cd 84.8 e 95.3 ab leaf upper epidermis thickness (μm) 6.0 ab 6.1 a 5.0 bc 5.6 ab 5.0 bc 4.2 c 6.0 ab leaf lower epidermis thickness (μm) 4.2 bc 4.9 a 3.7 c 4.4 ab 4.1 bc 3.5 d 4.5 ab leaf number of vascular bundles 5.0 a 5.0 a 4.0 b 5.0 a 3.0 c 5.0 a 4.2 ab fig. 1 ­ transvers sections of roots of thymus daenensis affected by seven different selenium treatments. a) 8 ppm of sodium selenate (sose), b) 4 ppm of sose, c) 2 ppm of sose, d) 8 ppm of nanoparticles of selenium (nanose), e) 4 ppm of nanose, f) 2 ppm of nanose, g) control, h) 8 ppm of sose, i) 8 ppm of nanose, e: epiderm, en= endo­ dermis, ph= phloem, vc= vascular cylinder, xy= xylem, bars = 50 µm. stems. five to six layers of roundish parenchyma cells formed the cortex of the stems. two opposite sides of the quadrangular stems were convex (fig. 2a), abravesh et al. ‐ effects of selenium on anatomic features of thymus daenensis 253 whereas, the other two sides were concave. the mentioned cavity was deeper in the selenium treatments (fig. 2c and 2e). epidermis, exodermis and endodermis were all single layer. epidermis with one layer of cells covered by cuticle and various trichomes, were similar for the studied treatments. within the range of studied concentrations of selenium nanoparticles (nanose), the largest stem xylem diameter was obtained by control of the experiment (no selenium in both forms). the highest level of the nanoparticles showed the most effects on stem diameter (888.9 µm) (table 2) and stem vascular cylinder diameter (355.4 µm). four ppm concentration of selenium nanoparticles had the largest effect on stem epidermis thickness (148.3 µm) (table 2). according to aly et al. (2023) there is a significant correlation between nutrients content, growth parameters and nanoparticles concentrations. they came to this conclusion that some nanoparticles were more efficient by low concentrations, whereas, others were more efficient by higher concentrations. we came to this conclusion that even in a single nanofertilizer, some part of the plant species, lower concentration of selenium was more effective (leaf mesophyll) (table 2), whereas, in other parts of the same plant, higher concentration of the element was more effective (stem diameter and leaf thickness). this study confirmed that an appropriate concentration of nanose enhanced the vascular cylinder and epidermis of both roots and stems of the plant species. two ppm of bulk selenium showed the largest increments on leaf characteristics such as leaf upper and lower epidermis thickness, leaf spongy and palisade mesophyll thickness (table 2). leaf main vascular thickness was mainly affected by 4 ppm concentration of bulk selenium treatment (fig. 3). whereas, the highest level of nanoparticles (8 ppm) showed the same trend but weaker effects on majority of the mentioned leaf characters (table 2). epidermis of the leaves, along with cuticle, provide protection for plants, thickening of epidermis on both upper and lower epidermis of the leaves fig. 3 ­ transvers sections of the leaves of thymus daenensis affected by seven different selenium treatments. a, b and c) 8 ppm of sodi­ um selenite (sose), d) 2 ppm of sose, e) 4 ppm of nanose, f) 2 ppm of sose, g) control, ab­ep= abaxial epidermis, ab­par= abaxi­ alparechyma, ad­ep= adaxial epidermis, ad­par= adaxial parenchyma, co= collenchyma, gt= glandular trichome, m: main middle vein, ph: phloem, pp= palisade parenchyma, sc= sclerenchyma, sp= spongy parenchyma, snv= small netted vein, tr= trichome, vb= vascular bundle, xy= xylem, bars = 100 µm. fig. 2 ­ transvers sections of the stems of thymus daenensis affected by seven different selenium treatments. a) control, b) 4 ppm of sodium selenate (sose), c) 2 ppm of sose, d) 8 ppm of nanoparticles of selenium (nanose), e) 4 ppm of nanose, f) 2 ppm of nanose, cc= concave, co= collenchyma, cp= cortex parenchyma, cv= convex, e= epi­ derm, en= endodermis, pi= pith, ph= phloem, xy= xylem, bars = 100 µm. 254 adv. hort. sci., 2024 38(3): 249­256 generated by selenate sodium (sose) may alleviate the negative and diverse effects of biotic and abiotic stresses. in fact, both forms of selenium, increased leaves upper and lower epidermis thickness, thereby leaf heat load is reduced, causing leaf internal temperature and resultantly transpiration rate to be reduced. application of nanoparticles of other elements such as silicates in k2sio3 and casio3 forms also showed positive results on banana, by higher stomatal density and smaller stomatal size (asmar et al., 2015). both upper and lower epidermises may act as a barrier to excessive transpiration, therefore, the thicker epidermis observed on selenium containing treatments, can better protect the plant leaves against water loss as well as protecting the plant tissues against biotic and abiotic stresses. abbas­ azimi et al. (2020) also came to this conclusion that some plant species change their anatomic features to combat dehydration. application of either form of selenium has thicken both mesophyll tissue layers (spongy and palisade parenchyma). it can be noticed that the leaves under either form of selenium treatments, have wider palisade and spongy mesophyll compared to untreated leaves. since mesophyll tissues are the main container of chloroplasts, thickness increment of the mesophyll layers would lead to increment of chlorophyll in the plant tissues, which in turn leads to improvement of photosynthetic efficiency. smrkolj et al. (2006) also showed that selenium induced higher respiratory potential in the leaves of foliar treated potato plants. nanose also negatively affected the number of vascular bundles, compared to the control of the experiment. whereas, the three levels of bulk selenium had the same effect as that of the control. it means selenate sodium (sose) treatment does not have significant effect on the number of vascular bundles. it should be noticed that according to yang et al. (2022), selenium content is higher in actively plant tissues and younger leaves, but higher buildup of the element occurs in aging leaves. therefore, selenium toxic effects may be seen in older leaves, whereas its deficient symptoms appear on new leaves. therefore, the exact toxic effects of the used dosage of the element could be examined on the older leaves of the plants under study. however, this study suggested that the leaf anatomic characteristics of thymus daenensis are highly affected by different concentrations of both forms of selenium. different plant species respond differently to selenium fertilization. plants are able to transform selenite into organic selenium by foliar spraying or fertilization. meanwhile, plant species have different capabilities to absorb and maintain selenium in their tissues. most plant species contain 1­2 mg/kg of selenium, but there are some species with ability to accumulate a larger amount of the element (juhaszne toth and csapo, 2018). for instance, brassicaceae and fabaceae families are able to accumulate much more selenium in a kilogram of dry matter than other plant species (ellis and salt, 2003). these species may be used for soil purification purposes in toxic soils. in this study, the highest level of selenium did not favor several studied characteristics such as root diameter, root vascular cylinder diameter, root epidermis thickness and leaf lower and upper cortex thickness; accordingly, the species under study seems to be sensitive to the large quantities of selenium. according to tymoszuk and kulus (2020), treating plants with nanoparticles, particularly high level of the particles, may even cause genetic variation, depending on their size, type and concentration. therefore, using the right concentration of the element is the first step to be taken. this was done in this research by a preliminary experiment to select the appropriate concentrations as the treatments of the experiment. the interaction between plant cells and nanoparticles may also cause positive or negative morpho­physiological alterations, depending on several factors such as size, shape, concentration, surface covering and mode of nanoparticle application as well as plant age, genotype, and developmental phase (rajput et al., 2021). however, utilizing appropriate form and concentration of selenium by foliar spraying results in anatomical and structural changes of thymus daenensis, that seems beneficial to the species. moderate concentrations of selenium acts highly positive in relation to different anatomic characteristics of the foliar parts of the plant species. root and stem epidermis thickness and vascular cylinder diameter, are favored by foliar addition of selenium in moderate concentrations of either nanoparticle or bulk forms. spongy and palisade mesophyll thickness of leaves of the plant species are also favored by the foliar spraying of selenium especially when 2 ppm of nanoparticles of the element or 8 ppm of bulk selenium is used. the lower abravesh et al. ‐ effects of selenium on anatomic features of thymus daenensis 255 level of nanoparticles and higher examined level of bulk selenium also showed their positive effects on increasing upper and lower leaf epidermis thickness as well as the number of leaf vascular bundles. therefore, foliar spraying on the young plants of the species by a moderate concentration of selenium, within the range of studied concentrations of the element is suggested for more vegetative growth and higher photosynthetic efficiency. acknowledgements the authors would like to highly appreciate the authorities of research institute of forests and rangelands of islamic republic of iran, for their generosity in financial supports and logistic helps. majority of biotechnology department staff of the institute helped us to accomplish this work, for which we would like to express our sincere thanks and appreciations. the authors also would like to thank dr. abbas­azimi for 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lemos de carvalho 1, i. de barros 2, l. marques de carvalho 1, e.a. girardi 3, o. sampaio passos 3, w. dos santos soares filho 3 1 embrapa tabuleiros costeiros, avenida beira mar, 3250, bairro jardins, aracaju, 49025‐040 sergipe, brazil. 2 embrapa gado de leite, rua eugênio do nascimento, 610, 36038‐330 juiz de fora, minas gerais, brazil. 3 embrapa mandioca e fruticultura, rua embrapa sn, 44380‐000 cruz das almas, bahia, brazil. key words: alternate bearing, citrus sinensis, fruit quality, scions, vegetative growth, water deficit, yield. abstract: citrus orchards in northeastern brazil are mostly rainfed and com­ prised basically of ‘pera cnpmf d­6’ sweet orange budded on ‘rangpur’ lime, for the drought tolerance and productivity imparted by this rootstock. therefore, the selection of new varieties is needed to broaden the genetic basis of citrus cultivated in this region. accordingly, this study compared vegetative, productive, and fruit quality traits of eight sweet orange scions grafted on ‘rangpur’ lime over eleven years under the tropical rainfed conditions of north­ eastern brazil. ‘kona’ trees excelled in yield performance associated with bulk canopy, precocity, sweet fruit with intermediate acidity, and high vitamin c contents in spite of proneness to alternate yields and low ratio (maturity index). ‘valencia montemorelos’ and ‘rubi’ trees, in turn, had high yield perfor­ mances coupled with intermediate canopies, sweet fruit, intermediate acidity (‘rubi’) and vitamin c contents, low propensity for yield fluctuation (‘valencia montemorelos’), and high precocity (‘rubi’), albeit low ratio. overall, our results emphasize ‘kona,’ ‘valencia montemorelos,’ and ‘rubi’ as superior sweet orange varieties for diversification of tropical rainfed orchards for their outstanding yield performance and good fruit quality. 1. introduction brazil is the largest producer of sweet oranges [citrus sinensis (l.) osbeck] worldwide, with 578,057 ha and 16.21 million tons of fruit har­ vested in 2021 (fao, 2021). the country is also the world’s top exporter of orange juice. most orchards are rainfed, and the southeast and north­ east regions are the main producers nationwide, with 421,171 ha and 98,475 ha, respectively. however, the yields in the northeast (11.40 t·ha­1) (*) corresponding author: adenir.teodoro@embrapa.br citation: teodoro a.v., lemos de carvalho h.w., de barros i., marques de carvalho l., girardi e.a., sampaio passos o., dos santos soares filho w., 2023 ­ superior sweet oranges for varietal diversification of tropical rainfed orchards. ­ adv. hort. sci., 37(2): 141­148. copyright: © 2023 teodoro a.v., lemos de carvalho h.w., de barros i., marques de carvalho l., girardi e.a., sampaio passos o., dos santos soares filho w. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 january 2023 accepted for publication 21 march 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14173 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 141­148 142 are only a third of those in the southeast (ibge, 2019). lower yields in northeastern brazil stem mainly from soils with fertility restrictions and hardsetting layers that impair drainage and root development in addition to water deficits due to irregular rainfall dis­ tribution, low technology adoption, improper man­ agement practices, and aging plants (gomes et al., 2017; carvalho et al., 2020; martins et al., 2020). despite water deficit negatively affecting citrus yields, citriculture in brazil is predominantly rainfed (carvalho et al., 2019 a, 2020, 2022). water stress linked to climate change is predicted to increase and affect the citrus industry worldwide (fares et al., 2017). in addition to these constraints, most citrus orchards in this region comprise ‘pera cnpmf d­6’ sweet orange (referred to as ‘pera’) budded on ‘rangpur’ lime (c. limonia osbeck), which is a graft­ compatible rootstock that confers good tolerance to drought, quality to fruits, and yield to scions (carvalho et al., 2020). ‘pera’ has a medium­sized canopy and is classified as a mid­season maturating variety beginning in july with fruit suited for both in natura consumption and juice production (carvalho et al., 2019 b). however, broadening the genetic diversity of scions could increase the much­needed fruit yield and quality to enhance the competitiveness of farms in this region. accordingly, in 2008, the brazilian agricultural research corporation (embrapa) estab­ lished a comprehensive research project aimed at varietal diversification with scion­rootstock combina­ tions for rainfed citrus orchards in the coastal table­ lands of northeastern brazil. as a result, new combi­ nations of sweet oranges and rootstocks have been selected for cultivation in this area (carvalho et al., 2019 b, 2020, 2022). herein, we recommend new varieties of sweet oranges for the diversification of tropical rainfed orchards. to this end, we conducted comparative vegetative, productive, and fruit quality trait assessments among eight sweet orange scions grafted on ‘rangpur’ lime over eleven years in brazil’s northeastern region. 2. materials and methods site description and experimental design the study was conducted from 2008 to 2019 at the experimental station of embrapa in umbaúba (11° 22’ 37’’ s, 37° 40’’ 26’’ w; 109 m a.s.l.), sergipe state, in the coastal tablelands of northeastern brazil. the study site soil is an haplic acrisol (ultisol), which is a reddish­yellow acid soil with medium texture, a clay­rich b horizon (kaolinite). according to soil ana­ lyses at 0­20 cm depth, the ph is 6.72, phosphorus 13.5 mg⋅dm3, organic matter 21.2 g.kg1, potassium 0.23 g.kg1, calcium 2.22 g.kg1, magnesium 0.86 g.kg1, and base saturation (74%). the climate is classified as “as” according to köppen­geiger, with a rainy period from may to september. rainfall was recorded over the study period, with an annual mean of 1309 (±275) mm. the yearly rainfall and potential evapo­ transpiration patterns over the study period are shown in figure 1 a. the experimental orchard consisted of eight sweet orange scions grafted onto ‘rangpur’ lime in a randomized complete block design, with three repli­ cates and three trees per plot. the orchard was planted at a density of 416 plants ha­1 (6.0×4.0 m) under rainfed conditions, but plants received 6 l of water weekly in the driest months. the orchard was annually fertilized according to the recommendation for sweet oranges in this region (carvalho et al., 2022). pest and weed control treatments with regis­ tered pesticides as well as pruning were also conduc­ ted whenever necessary. the orchard was fertilized twice a yer based on soil analysis and soil acidity was corrected by the application of dolomitic limestone. the scions were ‘kona’, ‘rubi’, ‘valencia monte­ morelos’, ‘pera’, ‘natal cnpmf­112’, ‘sukkari’, ‘lima verde’ and ‘lima’, and were obtained from the embrapa mandioca e fruticultura breeding program. these varieties hold potential for diversifying fig. 1 ­ water balance measured by the difference between rain­ fall and potential evapotranspiration (pet) (a) and box­ plot chart of citrus yield for all observations during the experimental period (b) (2008­2019). teodoro et al. ‐ superior sweet oranges 143 orchards in the study region that are dominated by ‘pera’ sweet orange, therefore their agronomical per­ formances under rainfed field conditions were assessed. vegetative performance and fruit yields vegetative growth of scions was evaluated in 8­ year­old trees in 2016 by recording plant height (ph, in m), rootstock (rd, in m) and scion diameters (sd, in m), and by estimating canopy volume (cv, in m3) as per zekri (2000). productive performance was assessed by fruit yield (fy, in t·ha­1) from the first harvest in 2011 to 2019, and yield efficiency (ye, in kg·m­3) was estimat­ ed in 2016 by the quotient between per plant fruit production and canopy volume. the alternate bear­ ing index (abi) was estimated using fy from 2011 to 2019 using the following formula (monselise and goldschmidt, 1982): |yi­yi­1| abi= ∑n (i=2) (yi+1 +yi­1 ) equation (1) n­1 where n denotes years and yi is the yield in year i. precocity (prec., in %) estimates considered the ratio between fy in the first two harvests and the cumula­ tive yield (cy; 2011 to 2019). fruit quality the quality was appraised in nine randomly cho­ sen fruits per plant from the 2015 and 2016 harvests as follows: fruit weight (fw, in g·fruit­1), diameter (fd, in mm), and height (fh, in mm), as well as rind thickness (rt, in mm) as measured by a caliper, juice content (jc, in g·100 g­1 of fruit mass), total titratable acidity (tta) was measured with 0.1 mol l­1 naoh as titrant and given in g of citric acid per 100 ml of juice, total soluble solids (tss, in °brix) using a refrac­ tometer, and vitamin c content (vit. c, in mg·100 ml­ 1 of juice) as measured by the oxidation­reduction volumetric technique using potassium iodate solu­ tion. the ratio, or maturity index, was estimated as the quotient between tss and tta. all measure­ ments followed the methods described by frança et al. (2016). statistical procedures data were subjected to anova, and means were grouped by scott­knott analysis. multivariate analy­ ses were also performed using xlstat to identify homogenous groups of scions, considering only the variables that were significant in univariate analyses. briefly, a principal component analysis (pca) was used to shorten the dataset into synthetic and uncor­ related variables, that is, the first principal compo­ nent (carvalho et al., 2019 a). afterwards, the scions were grouped by agglomerative hierarchical cluster­ ing analysis (ahc) applied to the pca scores that complied with the kaiser criterion, that is, those whose eigenvalues were ≥1.0. euclidean distance was used as a measure of dissimilarity, and ward’s mini­ mum variance was used to identify clusters. the automatic truncation option was used for cluster splitting. this approach creates homogenous groups based on the largest decrease in shannon’s entropy between a node and the next one. the resulting clus­ ters were interpreted using pca results and put into perspective with the results of univariate analyses of variance (carvalho et al., 2019 a). 3. results vegetative performance ph, rd, sd, and rd/sd girth ratio were not influ­ enced by scions (data not shown, p < 0.05). although not significantly different, ph varied from 2.5 m (‘lima’) to 3.5 m (‘kona’), and rd/sd girth ratio from 1.19 (‘valencia montemorelos’) to 1.68 (‘lima’). however, ‘kona’ trees were characterized by the largest cvs (26.3 m3); ‘valencia montemorelos’ (19.0 m3), ‘rubi’ (18.5 m3), and ‘sukkari’ (17.1 m3) had intermediate values, while ‘pera’ (15.5 m3), ‘lima verde’ (15.1 m3), ‘natal cnpmf­112’ (14.3 m3), and ‘lima’ (11.4 m3) presented the smallest canopy vol­ umes (p<0.0001). fruit yields the annual water balance over the experimental period was predominantly negative, reaching deficits of 586 and 543 mm in the driest years of 2012 and 2016, respectively (fig. 1 a). considering yield perfor­ mance, the production peak of sweet oranges was generally achieved in the fifth harvest (2015), decreased until 2017, and stabilized thereafter (fig. 1 b, table 1). ‘kona’ trees exhibited the highest mean yields over nine years, followed by ‘valencia montemorelos’ and ‘rubi’. ‘pera’ which is the main sweet orange grown in the study region, had inter­ mediate yields while ‘lima’ and ‘lima verde’ were the least productive (table 1). except for ‘lima verde’ with the lowest values, all varieties had similar yield efficiencies. ‘lima verde’, natal cnpmf­112’, adv. hort. sci., 2023 37(2): 141­148 144 ‘pera’, ‘sukkari’ and ‘valencia montemorelos’ were less prone to alternate bearing. ‘rubi’ appeared to have the highest precocity (prec.) for the ratio between the first two harvests and the cy. however, ‘kona’ and ‘rubi’ presented the highest absolute yields in 2011­2012 with more than 30 t·ha­1 (table 1). fruit quality regarding fruit quality, ‘lima’ had a smaller fruit than the other scions. ‘rubi’ fruit had the thickest rind in contrast to ‘lima verde’, which had the thinnest. fruit of ‘natal cnpmf­112’ and ‘valencia montemorelos’ showed the highest citric acid con­ tent, followed by those of ‘kona’, while ‘lima’, ‘lima verde’ and ‘sukkari’ exhibited the least acid fruit. ‘kona’, ‘lima’, ‘natal cnpmf­112’, ‘rubi’, ‘sukkari’ and ‘valencia montemorelos’ produced sweeter fruit than the remaining scion varieties (table 2). additionally, ‘kona’ and ‘lima’ had the highest vita­ table 2 ­ attributes of fruit quality of eight sweet orange varieties budded on ‘rangpur’ lime (average 2015­2016) z means in the same column followed by the same letter are not significantly different according to the scott­knott analysis (p<0.05). tta= total titratable acidity; tss = total soluble solids. table 1 ­ yield performance of eight sweet orange varieties budded on ‘rangpur’ lime (2008­2019) z means in the same column followed by the same letter are not significantly different according to the scott­knott analysis (p<0.05). y yield efficiency; x alternate bearing index; w precocity. fruit yield (t·ha­1) yey abix prec.w (%)2011 2012 2013 2014 2015 2016 2017 2018 2019 mean kona 12.1 c z 23.5 a 11.7 b 19.1 b 72.5 a 46.4 a 14.0 b 29.8 a 33.2 a 29.2 a 4.27 a 0.33 c 13.6 c lima 4.3 f 4.7 e 10.1 b 12.0 d 34.4 c 17.6 d 7.4 d 13.8 d 13.9 d 13.1 e 3.73 a 0.27 b 7.6 e lima verde 4.2 f 4.8 e 12.2 b 11.2 d 31.8 c 17.5 d 8.9 d 12.6 d 10.9 d 12.7 e 2.79 b 0.24 a 7.9 e natal cnpmf­112 13.5 b 7.6 d 10.3 b 15.2 c 20.1 d 24.8 c 11.6 c 13.2 d 23.2 b 15.5 d 4.26 a 0.20 a 15.2 b pera cnpmf d­6 8.0 d 15.4 b 13.8 a 15.9 c 39.4 b 25.4 c 15.5 a 14.0 d 12.8 d 17.8 c 3.98 a 0.19 a 14.6 b rubi 11.6 c 23.0 a 15.6 a 20.8 a 40.7 b 26.8 c 12.7 c 24.7 b 15.2 d 21.2 b 3.53 a 0.26 b 18.1 a sukkari 6.5 e 11.8 c 12.2 b 13.3 d 34.8 c 29.9 b 13.6 b 25.3 b 20.0 c 18.6 c 4.37 a 0.22 a 10.9 d valencia 15.2 a 8.9 d 15.0 a 22.5 e 40.8 b 30.1 b 15.9 a 20.7 c 25.9 b 21.7 b 3.83 a 0.21 a 12.3 c cv (%) 10.9 14.7 15.4 8.9 9.9 5.8 8.7 10.7 17.4 4.3 13.5 8.6 7.8 f 51.3 50.4 3.32 24.8 44.2 99.7 23.7 32.3 15.6 135.0 2.99 14.9 40.5 p­value <0.0001 <0.0001 0.027 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.038 <0.0001 <0.0001 orange varieties mean fw (g·fruit­1) fruit diameter (mm) fruit height (mm) rind thickness (mm) juice content (g·kg­1) tta (g·100 ml­1) tss (°brix) vitamin c (g·100 g­1) ratio tss/tta kona 190 76.3 71.0 a z 3.81 b 545 0.798 b 11.5 a 60.0 a 14.5 c lima 193 70.0 60.8 b 3.34 c 544 0.128 d 11.3 a 58.3 a 88.0 b lima verde 190 74.6 72.7 a 2.60 d 579 0.097 d 8.9 b 40.6 d 93.2 a natal cnpmf­112 180 72.9 69.8 a 3.59 b 572 1.208 a 12.3 a 52.3 b 10.4 c pera cnpmf d­6 205 74.9 75.6 a 3.12 c 548 0.670 c 8.9 b 43.5 d 14.7 c rubi 187 75.9 70.0 a 4.15 a 539 0.617 c 11.3 a 49.2 c 18.7 c sukkari 189 72.9 67.0 a 3.08 c 544 0.114 d 10.5 a 54.1 b 93.7 a valencia montemorelos 188 72.1 69.6 a 2.99 c 549 1.180 a 11.0 a 53.4 b 9.6 c cv (%) 8.2 3.5 5.9 6.0 4.2 11.0 7.2 7.1 8.2 f 0.63 2.01 3.38 18.62 1.15 142.1 7.56 10.00 397.2 p­value 0.723 0.126 0.025 <0.0001 0.385 <0.0001 0.0007 0.0002 <0.0001 teodoro et al. ‐ superior sweet oranges 145 min c contents, whereas ‘lima verde’ and ‘pera’ had the lowest values. ‘sukkari’ and ‘lima verde’ fol­ lowed by ‘lima’ had the highest values of ratio, a proxy for fruit maturity (table 2). fw, fd, and jc were not affected by scions (table 2). multivariate analyses multivariate analysis helped to identify scion groups that performed homogeneously, considering the universe of all significant attributes. the first two pca principal components explained more than 66% of the total observed variability, and the square cosine of the variables showed that, while average yield, cv, tta, prec., rt, tss, and ratio were mostly associated with pc1, fh, alternate bearing, and vita­ min c contributed to most of the variation along pc2 (fig. 2 a). multi­correlation analysis indicated that average yield correlated positively with cv and that prec. exhibited a positive correlation with total acidity, but both correlated negatively with ratio. in addition, while alternate bearing correlated positively with vit­ amin c, it had a negative correlation with fh (fig. 2 a). agglomerative hierarchical clustering analysis (ahc) using shannon entropy for grouping all obser­ vations that showed similar results for the entire set of variables identified three distinct clusters (fig. 2 c) with the following characteristics, as observed through the visual inspection of the observation cloud projection in the plane of the first two principal components of the pca (fig. 2 b). the heatmap of the results for all variables is shown in figure 2 c. the first cluster grouped all observations of ‘kona’ and its main characteristics were high and alternate yields associated with bulk canopy and high vitamin c content; intermediate values for tss, rt, prec., acidi­ ty, fh, and low ratio. the second cluster included the observations of ‘valencia montemorelos’, ‘rubi’, ‘pera’ and ‘natal cnpmf­112’. in general, these varieties showed intermediate values for all evaluated variables except ratio, which were mostly low for this cluster. finally, the third cluster encompassed all observa­ tions of ‘sukkari’, ‘lima verde’, and ‘lima’. in contrast to the first and second clusters, the main characteris­ tic was that the ratio was high for all observations and average yield, cv, rt, and acidity were predomi­ nantly low. intermediate values were observed for alternate bearing, vitamin c, tss, and fh. 4. discussion and conclusions here, we comparatively assessed vegetative, pro­ ductive, and fruit quality traits among eight sweet orange scions grafted on ‘rangpur’ lime for enhan­ cing orchard varietal diversification under the tropi­ fig. 2 ­ principal component and agglomerative hierarchical clustering analysis. (a) correlation circle of the variables; (b) score plot of the observations in the plane of the two first principal components and (c) heatmap of the rela­ tive values of all observations for all variables and group separation by the agglomerative hierarchical clustering analysis (ahc). the first two axes (pc1 and pc2) account­ ed for 66.82% of the total variance. arrows in green rep­ resent variables associated with pc1 whereas those in blue are associated with pc2 in figure a. observations belonging to different groups by the ahc analysis were delineated (boxes) in figure b. cv: canopy volume; ay: average yield; abi: alternate bearing index; vit.c: vitamin c content; tss: total soluble solids content; rt: rind thickness; prec.: precocity; tta: total titratable acid­ ity; fh: fruit height. 146 adv. hort. sci., 2023 37(2): 141­148 cal rainfed conditions of northeastern brazil. overall, the varieties reached peak yields in the fifth harvest, and subsequently decreased. the same pattern was observed for ‘sincora,’ ‘valencia tuxpan,’ and ‘pineapple’ sweet oranges in the same study region (carvalho et al., 2019 a; martins et al., 2020). as the experimental orchard was cultivated under rainfed conditions, predominantly negative yearly water bal­ ances possibly interfered with the productive poten­ tial of the different varieties. major drought spells occurred in 2012, 2016, and 2018, with water deficits exceeding 480 mm. water stress strongly impairs growth and development of citrus trees and sweet orange varieties commonly present water deficiency symptoms throughout the study region even when scions are grafted on drought­tolerant ‘rangpur’ lime, which emphasizes the severe seasonal drought rainfed orchards face (soares et al., 2015; carvalho et al., 2016). as we did not specifically evaluate drought tolerance, further studies are needed to shed light on the susceptibility of sweet orange varieties to drought. comparatively, ‘kona’ was the most productive variety, with an annual average of 29.2 t·ha­1 of fruit. ‘valencia montemorelos’ (21.7 t.ha­1) and ‘rubi’ (21.2 t·ha­1) also had remarkable yield performances. the high fruit yield of ‘valencia tuxpan’ grafted on ‘santa cruz rangpur’ lime was also verified by carvalho et al. (2019 a). similarly, ‘cnpmf 003 rangpur’ lime and ‘santa cruz rangpur’ lime rootstocks conferred high yields to ‘valencia’ in brazil’s southeastern state of são paulo (fadel et al., 2018). the dominantly grown ‘pera’, however, was characterized by intermediate yields. ‘kona’ trees produced 72.5 t·ha­1 at its peak, and alongside ‘rubi’ excelled in precocity, with yields that surpassed 23 t·ha­1 in the second harvest. apart from ‘lima verde’ with lower values, all varieties had similar yield efficiencies despite the sharp differences in canopy volumescvs. for instance, ‘kona’ trees had the largest canopies (26.3 m3) in contrast with ‘lima’ (11.4 m3) and ‘pera’ (15.5 m3). this is consistent with similar yield efficiencies of ‘pineapple’ sweet orange irrespective of the grafted rootstock in the same study region (martins et al., 2020). it is noteworthy that after the two driest years (2012 and 2016), the most productive varieties were ‘pera’ and ‘valencia montemorelos’. melgar et al. (2010) showed that ‘valencia’ sweet orange trees [grafted on ‘swingle’ citrumelo c. paradisi macfad. x poncirus trifoliata (l.) raf.] that experienced drought for a hundred days and were well irrigated in subsequent months pro­ duced more fruit than those that were not subjected to water stress. alternate bearing is a widespread phenomenon among fruit trees in which high yield in one harvest is followed by low production in the subsequent har­ vest (monselise and goldschmidt, 1982). alternate bearing is an undesirable trait from an economic standpoint, especially for mandarins and tangerines, but generally a minor to moderate problem for sweet oranges such as ‘valencia’ (monselise and goldschmidt, 1982; abobatta, 2019). alternate bear­ ing in citrus is caused by fruit load inhibiting return flowering (abobatta, 2019) and seemed here to be variety­specific, as ‘kona’ possessed high propensity for yield alternation in contrast with less­prone ‘lima verde’, ‘natal cnpmf­112’, ‘pera’, ‘sukkari’ and ‘valencia montemorelos’. ‘lima’ and ‘rubi’, in turn, had an intermediate degree of susceptibility to yield alternation. nutrition, hormones, and abiotic stresses ranging from soil fertility and physical restrictions to drought susceptibility might also have played a role in yield fluctuation (monselise and goldschmidt, 1982; abobatta, 2019; carvalho et al., 2019 a, 2020). fruit quality is expressed by several parameters including the amount of juice, tss content, acidity level, and the amount of vitamin c (frança et al., 2016; lado et al., 2018; tirado­corbalá et al., 2020). moreover, the tss content is the basis for the pay­ ment of a premium price differential for high­quality fruit (zhang and ritenour, 2016). the ratio, or maturi­ ty index, expresses fruit ripeness and is also an indi­ cator of flavor (lado et al., 2018; ribeiro et al., 2020). these fruit quality traits may be influenced by the scion variety, management practices, maturity level, climate, and rootstocks (al­mohuei and choumane, 2014; carvalho et al., 2020; ribeiro et al., 2020; tirado­corbalá et al., 2020). here, we showed that fw, fd, and juice yield did not differ among the sweet orange varieties. however, ‘lima’ trees pro­ duced smaller fruit than the other varieties. ‘rubi’ produced the thickest and ‘lima verde’ the thinnest fruit rinds. ‘lima verde’ and ‘pera’ produced the least sweet fruit, while ‘natal cnpmf­112’ and ‘valencia montemorelos’ were the most acidic. the high fruit acidity of ‘valencia’ grafted on ‘santa cruz rangpur’ lime was also demonstrated by rodrigues et al. (2019). there is evidence that rainfed orchards generally produce sweeter fruit, as lower juice yields related to water deficits favor sugar concentration (lado et al., 2018). this is in line with higher fruit sugar contents teodoro et al. ‐ superior sweet oranges 147 of ‘valencia’ trees subjected to deficit irrigation treat­ ments in italy (mossad et al., 2020). the ratio, or maturity index, was highest for ‘lima verde’ and ‘sukkari’, which can be related to their lower acidity levels. ‘kona’ and ‘lima’ fruit had the highest con­ tents of vitamin c as opposed to the lowest values for ‘lima verde’ and ‘pera’. generally, the juice yields, tss, and ratio values obtained here were with­ in the minimal requirements for brazilian fresh orange markets. multivariate analyses showed that fruit quality traits were mostly associated with pc1 (rt, tta, tss and ratio) while fh and vit. c were strongly related to pc2. ratio was negatively related to rt and espe­ cially to tta. tss, in turn, was associated with the majority of fruit quality traits, being negatively rela­ ted to fh (smaller fruit, higher tss) and positively with rt, tta and vit. c. however, tss was not related to ratio. pc2, which accounted for 25% of variability, was negatively related with fh and vit. c contents, suggesting that smaller fruit concentrate more vit. c. collectively, our results obtained in northeastern brazil highlight ‘kona’, valencia montemorelos’ and ‘rubi’ as superior sweet orange varieties for diversifi­ cation of tropical rainfed orchards. ‘kona’ excelled in productive performance combined with a volumi­ nous canopy, precocity, sweet fruit with moderate acidity, and high levels of vitamin c despite the propensity for alternate yields and low ratio. ‘valencia montemorelos’ and ‘rubi’ had high yield performances coupled with intermediate canopies, sweet fruit, moderate acidity (‘rubi’) and vitamin c levels, low proneness for yield fluctuation (‘valencia montemorelos’), and high precocity 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sign to initiate the plowing of paddy fields for rice cultivation in china. therefore, chinese people take an interest in looking at plum flowers, which is the reason why many cultivars of chinese plum have been developed. the chinese plum blooms in early february in japan. japanese farmers pay attention to the flowering of flowering cherry because it blooms at the end of march (fig. 1). as japan is located at the northern limits of the rice cultivation area, without a calendar, the timing to start rice cultivation was very important to identify the cultivation period and secure the best yield for farmers. rice cultivation started by plowing paddy fields, then about a month later sowing rice seeds on the paddy. growth of rice plants was restricted to the period between may and october (ca. 160 days) both in china and japan. farmers had to harvest the rice grains before snow at the end of october. as the rice yield was the basis of japanese hierarchy, everyone in japan, not only farmers, has been interested in the flowering of flowering cherry. more than 250 cultivars of flowering cherry have been selected or bred from wild cerasus species in japan (kawasaki, 1991). farmers both in china and japan started rice cultivation by ploking paddy fields at the end of march (yellow circle). ‘somei-yoshino’ a single cultivar, prunus yedoensis (matsum.) a.v. vassil. ‘somei-yoshino’ (iketani et al., 2006), comprises more than 80-90% of flowering cherry trees planted in parks, and along roads and rivers across japan, except in okinawa and hokkaido islands. as cerasus species have complete self-incompatibility, ‘somei-yoshino’ has been propagated as a clone using grafting techniques. this paper discusses three topics in terms of the origin of diversity and breeding of flowering cherry in japan i. nakamura*(1), h. takahashi*, y-i. sato** * graduate school of horticulture, chiba university, 648 matsudo, matsudo, 271-8510 chiba, japan. ** future center, kyoto sangyo university, motoyama, kamigamo, kita-ku, 603-8555 kyoto, japan. key words: genetic diversity, hanami, horticulture, origin of ‘somei-yoshino,’ pola1, prunus. abstract: in early spring, the flowering of cherry trees is taken as a good sign for farmers to initiate rice cultivation in japan. in the era before calendars, the timing of cultivation was very important for growing rice plants in temperate regions. nine native species of flowering cherry (prunus subgenus cerasus) are present in japan and they are classified into three groups: yamazakura, miyamazakura, and edohigan. more than 250 cultivars of japanese flowering cherry have been selected or bred from these wild species. two species, oshimazakura and edohigan, have specially contributed to the breeding of flowering cherry cultivars. while edohigan is distributed in most areas of japan, oshimazakura (of the yamazakura group) is an endemic species found around the izu and boso peninsulas. ‘somei-yoshino,’ prunus × yedoensis, is the most popular cultivar and now comprises 80-90% of all flowering cherry trees planted in japan. ‘someiyoshino’ was probably created through hybridization between edohigan and oshimazakura in the edo era. in this paper, the diversity and breeding of japanese flowering cherry, including the origin of ‘somei-yoshino,’ are described along with the political and horticultural backgrounds. adv. hort. sci., 2014 28(4): 236-243 (1) corresponding author: inakamur@faculty.chiba-u.jp received for publication 17 september 2014 accepted for publication 30 october 2014 fig. 1 changes of average temperature in nanjin (china) and tokyo (japan). 237 ‘somei-yoshino’: 1. genetic variations of cerasus species in japan, 2. political and horticultural backgrounds, 3. dna analysis to reveal the origin of ‘somei-yoshino’ (fig. 2). 2. genetic variations of flowering cherry in japan wild cerasus species in japan the subgenus cerasus of the genus prunus includes more than 50 species, most of which are distributed in temperate areas in the northern hemisphere, especially in china, where 33 wild species occur (yu and li, 1986). in japan, nine native species have been recorded (table 1): yamazakura (p. jamasakura sieb. ex koidz.), oyamazakura (p. sargentii rehder), kasumizakura [p. verecunda (koidz,) koehne], mamezakura (p. incisa thumb. ex murray), takanezakura (p. nipponica matsum.), chojizakura [p. apetala (sieb. et zucc.) fr. et sav.], oshimazakura [p. lannesiana (carr.) wilson var. speciosa (koidz.) makino], and edohigan [p. pendula f. ascendens (makino) ohwi]. in addition, three wild species, karamizakura (p. pseudo-cerasus lindl.), himarayazakura (p. cerasoides d. don) and kanhizakura (p. campanulata maxim.), have been popularly cultivated since their introductions from china, taiwan, and nepal, respectively (kawasaki, 1991) (fig. 3). distribution of wild cerasus species in japan two wild species, yamazakura and its close relative oyamazakura, are broadly distributed throughout japan (fig. 4). while edohigan is distributed from kyushu island to the tohoku region (northern area of mainland japan), this species is absent from some large peninsulas, such as kii, noto and boso. as edohigan blooms faster than yamazakura and other japanese cerasus species, it was possibly spread by farmers along with rice cultivation from the south to the north of japan. mamezakura and its variant kinki-mamezakura are restricted to the mountainous area in the middle of the mainland. the wild population of oshimazakura is endemic to the izu and boso peninsulas and to oshima islands. as the flowers of osfig. 2 flowering cherry ‘somei-yoshino’. image credit: http://iko4wd. blog.so.net.ne.jp/2008-03-25. table 1 wild species of the subgenus cerasus in japan yamazakura group yamazakura p. jamazakura ooyamazakura p. sargentii kasumizakura p. verecunda oshimazakura p. iannesiana mamezakura group mamezakura p. incisa takanezakura p. nipponica chojizakura group chojizakura p. apetala edohigan group edohigan p. pendula miyamazakura group miyamazakura p. mazimowiczii karamizakura group karamizakura p. pseudo-cerasus (china) kanhizakura group kanhizakura p. campanulata (taiwan) himarayazakura group himarayazakura p. cerasoides (nepal) fig. 3 cerasus species. flowering of different cultivars. top left ‘edohigan’, top middle ‘yamazakura’, top right ‘oshimazakura’, bottom left ‘chojizakura’, bottom right ‘kanhizakura’. courtesy of mr. makoto tsuruta. fig. 4 distribution of wild cerasus species in japan (kawasaki, 1991). 238 himazakura have large white petals with a pleasant fragrance, it has been used as a parent to develop various cultivars of flowering cherry. its green leaves are also used to decorate cakes. the flowers of edohigan bloom faster than its leaves extrude. in contrast, flowers and leaves appear at the same time in yamazakura and other species. wild edohigan and yamazakura trees reach a height of 20-25 m, while mamezakura and oshimazakura have a relatively short stature (ca. 10 m). cultivars of flowering cherry the history of the breeding of flowering cherry in japan can be classified into three phases: selection phase (ancient to azuchi-momoyama era), mutant phase (first half of edo era), and cross-hybridization phase (last half of edo era to present). two important incidents, the establishment of the tokugawa government (1603) and the practice of the kyoho reforms (1716), are related in the division of the three phases (fig. 5). before the azuchimomoyama era, about 20 cultivars were selected from the native population of flowering cherry. ‘ukon,’ with yellowish petals, and ‘shidare zakura,’ with droopy branches, were representative cultivars in the selection phase. during the first half of the edo era, because shoguns, especially the 3rd shogun tokugawa iemitsu, asked daimyos to donate fantastic or unusual flowers and trees, many ornamental cultivars of more than 100 species were brought together at edo castle. about 30 cultivars of flowering cherry with mutant phenotypes, such as many petals ‘ito kukuri’ and dwarf stature ‘asahiyama’ were collected in the mutant phase. during the last half of the edo era, flowering cherry cultivars were developed using natural or artificial cross-hybridization. in the cross-hybridization phase of the last half of the edo era, more than 80 cultivars, such as ‘ichiyo’ with a leafy stigma and ‘surugadainioi’ with a fragrance, were created by hybridization with oshimazakura as parent (fig. 6). 3. political and horticultural backgrounds 260-year peace during edo period ieyasu tokugawa (1542-1616), who was the 1st shogun, struggled to establish the tokugawa government (16031868). it is thought to be the longest period of peace in the world at that time. the 260 years of peace under the tokugawa government was an important background to the development of various cultures, including horticulture. takatora todo (1556-1630) was probably a key person for the establishment of the tokugawa government and for the basis of the origin of ‘somei-yoshino.’ he constructed more than 20 castles, including edo castle. and he once owned kisyu kokawa-han and visited mt. yoshino, which was famous for its large yamazakura population. later, takatora moved to ise-han where both iga and koga ninja lived. thus, takatora organized the ninja into a cia (central intelligence agency, usa)-like ‘onmitsu’ system in order to keep an eye on the behavior of daimyos for the tokugawa government. his house was located in ‘somei’ village of edo city and the name of his gardener was ito ihei, who was probably a secret manager of ‘onmitsu’ under the control of the todo clan. after ieyasu died, takatora constructed ‘kan-eiji’ temple for ieyasu’s grave in 1625. iemitsu tokugawa (3rd shogun) and takatora directed the transplantation of wild yamazakura trees from mt. yoshino to ‘kan-eiji’ temple. as a result, ‘kan-eiji’ temple became a famous ‘hanami’ place in edo city. note: ‘hanami’ means looking at blossoms whilst eating and drinking collection of cultivars and mutants during the lifetime of iemitsu tokugawa (1623-1651) of the 3rd shogun of the tokugawa government, most social systems, including transport and the economy, were established. iemitsu, who was rather eccentric and an enthusiast of ornamental flowers and trees, asked daimyos to donate fantastic or unusual plants, and as a result many mutant cultivars of more than 100 plant species were brought to edo castle. these collected plants were later fig. 5 three phases in the breeding of flowering cherry. fig. 6 cultivars (cross hybridization phase) during last half of edo era. courtesy of mr. makoto tsuruta.. 239 given to the ito ihei clan through the todo clan. hybridizations among the collected genetic resources led to the development of many horticultural cultivars, such as morning glory, japanese azalea, camellia, orchid, and fern, by the ito ihei clan (gardeners) and people in edo city. they frequently held competitive exhibitions of the cultivars that they had developed. for example, an unusual phenotype ’shiro saizaki botan’ of morning glory appeared at a frequency (one out of 20,000 seeds), corresponding to recombination among 6-7 recessive alleles. thus, japanese gardeners and others might have recognized a principle of inheritance before mendel (1865) (fig. 7). accumulation of horticultural knowledge during the period of peace, not only gardeners but also enthusiasts in edo city enjoyed breeding ornamental flowers and trees. much knowledge and many horticultural techniques were accumulated during the process of developing the new cultivars of ornamental plants. many books and illustrations describing the knowledge and developed cultivars were published during the last half of the edo era. for example, kan-en iwasaki published a book ‘somoku sodate-gusa’ (plant breeding) in 1818. in this book, he illustrated six different grafting, four cutting and three layering techniques, which are comparable to the present horticultural techniques. in those days, as more than 80 cultivars of flowering cherry had been developed by using natural or artificial cross-hybridization, this horticultural knowledge and these techniques must have contributed to the development of new cultivars of flowering cherry (fig. 8, 9). ‘ippon-zakura’ to ‘gun-zakura’ yoshimune tokugawa (1684-1751) used to be a daimyo in wakayama, close to mt. yoshino, which was famous for its beautiful scenery covered with yamazakura trees. after he became the 8th shogun, he enacted the ‘kyoho reforms’ to rebuild the finances of the tokugawa government. also he opposed the powers of the todo clan and kan-eiji temple. yoshimune abolished ‘onmitsu’ under the control of the todo clan and then organized his subordinates into the ‘oniwaban,’ which was also a cialike system. this change meant that ito ihei might lose his salary as a secret manager of the ‘onmitsu.’ against the kan-eiji temple, yoshimune tried to develop new ‘hanami’ places because ‘kan-eiji’ was the famous ‘hanami’ place in edo city. he wanted to reconstruct the scenes of flowering cherries on mt. yoshino in edo city. therefore, he directed transplantation of a group of wild yamazakura trees, propagated by grafting in edo castle, to several new ‘hanami’ places, such as shinagawa, sumida-gawa and asuka-yama. then, he encouraged people to eat and drink under blossoms because he knew the starting time of rice cultivation was very important for farmers to achieve the best rice yield. fig. 7 ‘shiro saizaki botan’, morning glory (6-7 recessive allele). courtesy of dr yoshiaki yoneda http://protist.i.hosci.ac.jp/asago/yoneda_db/j/menu.html. fig. 8 illustrations of six different grafting techniques. image credit: http://dl.ndl.go.jp/info:ndljp/pid/2569455. fig. 9 illustrations of 4 cutting (left) and 3 layering (right). 240 during the first five to ten years, people probably enjoyed ‘hanami’ under blossoms but after 15 years, the wild yamazakura trees grew to be over 15 m high. yoshimune did not realize that the tree height of flowering cherry was so important because people were able to look at flowers closely in spite of the height of the trees on the slopes on mt. yoshino (fig. 10). in flat places of edo city, the wild trees of yamazakura grew too high to look at the blossoms closely from under the trees but no one complained for fear that yoshimune would punish them. nowadays, all yamazakura trees, planted by yoshimune, have been replaced by ‘somei-yoshino.’ before the mid-edo era, people enjoyed seeing a distant scene of a single big flowering cherry ‘ippon-zakura.’ on the contrary, yoshimune introduced the concept of a group of flowering cherry ‘gun-zakura’ to people in edo city. involvement of masatake ito ihei masatake (1676-1757) of 4th ito ihei and his father sannojo (?-1719) were excellent plant breeders who developed many ornamental cultivars, japanese azalea, camellia and sweet flag. as the ito ihei clan probably earned a salary from the todo clan as managers of the ‘onmitsu,’ they enjoyed plant breeding and gardening as a hobby. sannojo and masatake wrote many horticultural books, such as ‘kadan-chikin-syo’ (1695) and ‘koeki -chikin-syo’ (1719). sannojo illustrated 120 plant species of ornamental flowers and trees including 46 cultivars of flowering cherry. masatake also illustrated 197 species of ornamental plants, however, there was no description of flowering cherry. it is very unlikely that masatake was uninterested in the breeding of flowering cherry. he probably excluded descriptions of flowering cherry from his books in order to avoid conflict with yoshimune who initiated the ‘kyoho reforms’ in 1716. masatake might have found that wild yamazakura trees were not suitable to ‘gun-zakura’ but he could not blame yoshimune because yoshimune might not just punish masatake, he might wipe out the entire todo clan. thus, masatake may have tried to develop a new cultivar for ‘gun-zakura’ in secret within kan-eiji temple. 4. dna analysis to reveal the origin of ‘somei-yoshino’ ‘somei-yoshino’ was first identified and named by yorinaga fujino in 1900 and dr. jinzo matsumura of tokyo university registered it as prunus x yedoensis at 1901. dr. ernest h. wilson (1916) proposed, through morphological observations, the hypothesis that ‘somei-yoshino’ is a hybrid of oshimazakura and edohigan. dr. kaname takenaka (1962, 1965) confirmed wilson’s hypothesis by observing morphologies of the hybrids, izu-yoshino and amagiyoshino, produced through artificial hybridization between the two species. however, these hybrids show higher stature and whiter petals compared with ‘somei-yoshino.’ analyses of restriction fragment length polymorphism of chloroplast dna (kaneko et al., 1986) and plastid subtype identity sequence (ohta et al., 2006) clearly indicate that the maternal lineage of ‘somei-yoshino’ is edohigan. in contrast, nuclear dna analysis of flowering cherry is difficult because cerasus species have complete self-incompatibility. we found that sequence variations of pola1 gene are useful for analyzing phylogenetic relationships in rice and wheat (takahashi et al., 2009; rai et al., 2012). pola1 is a single copy nuclear gene encoding the largest subunit of rna polymerase i complex. thus, dna fragments containing intron 19 and exon 20 sequences of pola1 gene were amplified by pcr using template dnas from wild strains of cerasus species in japan (fig. 11). pcr products were purified and analyzed by direct sequencing technique. sequence analysis of exon 20 indicates that fig. 10 sitting person’s view of flowering cherry on the slopes of mt. yoshino (a) and on flat places in edo city (b). fig. 11 sequence analyses of intron 19 and exon 20 of pola1 gene in cerasus species. 241 yamazakura and edohigan can be clearly distinguished (fig. 12). a particular insertion of 24 bp was found in the yamazakura group including oshimazakura as well as mamezakura and chojizakura groups (table 1). this data indicates that these three groups share the same ancestor. the sequence analysis of intron 19 shows a similar result to that of exon 20 (fig. 12), however oshimazakura is clearly distinguished by three single nucleotide polymorphisms (snps) from yamazakura and other species. sequence analysis of intron 19 shows that ‘somei-yoshino’ contains a haplotype containing the three snps at the positions of 25 101, and 171, which are specific to oshimazakura (fig. 13). this result is very important for considering the origin of ‘somei-yoshino’ because oshimazakura is an endemic species to the izu and boso peninsulas (fig. 4). another haplotype of ‘somei-yoshino’ is identical to that of the wild edohigan except for one snp at the position of 392. the same snp was found in a haplotype of ‘komatsu-otome.’ ‘komatsu-otome’ is a cultivar of edohigan with a short stature (ca. 6 m) and is found as an original tree in ueno park, which was in the precincts of kan-eiji temple in the edo era. these results suggest that ‘somei-yoshino’ originates from hybridization between a maternal parent, a semi-dwarf cultivar closely related to ‘komatsu-otome,’ and a paternal parent, a cultivar of oshimazakura (fig. 14). also ‘somei-yoshino’ could originate from hybridization between hybrids containing each haplotype. fig. 12 relationships of exon 20 (a) and intron 19 (b) sequences of pola1 gene among cerasus species, in japan. fig. 13 comparison of two allelic intron 19 sequences within pola1 gene among ‘somei-yoshino,’ edohigan, ‘komatsuotome,’ oshimazakura, and yamazakura. fig. 14 schematic representation of the origin of ‘somei-yoshino’ (nakamura et al., 2007). 242 ‘komatsu-otome’ (tree no. 135, assigned by ueno park) was grown around the ruins of the bell tower in kan-eiji temple (fig. 15). now, a total of ten trees related to edohigan grow around the bell tower. four trees (133, 134, 136, 138) have been identified as ‘someiyoshino.’ sequence analysis of chloroplast dna shows that the maternal lineage of all ten trees is edohigan. and sequence analysis of the pola1 gene indicates that three trees (141, 142, 144) and ‘somei-yoshino’ contain a haplotype (o) specific to oshimazakura. one tree (145) is homozygous of the haplotype (k), which is shared by ‘komatsu-otome’ and ‘somei-yoshino.’ in addition, one haplotype (t) found to be specific to the chloroplast dna of ‘some-yoshino’ (ando et al., unpublished) is shared with another tree (142). these results suggest that flowering cherry trees around the bell tower were developed by artificial hybridizations between edohigan and oshimazakura, and that there were sufficient genetic resources to develop ‘somei-yoshino’ and ‘komatsuotome.’ dna analysis of offshoots of four ‘somei-yoshino’ clones revealed that these clones were propagated by using layering or grafting with a weak stock. this result suggests the action of a professional gardener. the purpose of planting the four clones together might be related to the evaluations of the ‘gun-zakura’ by buddhists of the kan-eiji temple. masatake ito ihei (1676–1757) lived at the same time as yoshimune tokugawa (1684–1751). masatake had the ability to develop ‘somei-yohsino’ because he had developed many ornamental cultivars of various plant species (iwasaki, 1989, 1991). masatake might be the person who planted hybrids of flowering cherry around the bell tower because kan-eiji temple had a close relationship with the todo clan and because the precinct of kan-eiji was an extraterritorial place from the tokugawa government. except for masatake, there was no reason why anyone would record the origin of ‘somei-yoshino.’ after the meiji restoration ken-eiji temple was destroyed during the ‘boshin’ war (1868) and the temple grounds were established as ueno park (1873). the meiji government held japanese industrial exhibitions several times in ueno park in order to promote japanese industry. ‘somei-yoshino’ shoots were massively propagated using grafting with a strong stock by gardeners in ‘somei’ village, and then the shoots were probably sold in the exhibitions. the oldest known ‘somei-yoshino’ tree is one of the 1,000 shoots that were planted within hirosaki castle in 1882, the year after the 2nd japan industry exhibition (1881). as no one could buy the 1,000 shoots without seeing the real ‘somei-yoshino,’ it was probably planted within ueno park at that time. at present, many ‘somei-yoshino’ trees are beginning to show gaps in their stems, which seems to support the idea that ‘somei-yoshino’ has a 60-year life span. as these gaps are caused by imbalanced growth between the scion and stock after grafting, if clones of ‘someiyoshino’ are propagated using grafting with a weak stock, they would be able to survive for hundreds of years. 5. conclusions there is no record of the origin of ‘somei-yoshino’ but the results presented in this paper suggest that ito ihei masatake, or someone else, developed ‘somei-yoshino’ in secret as a suitable cultivar for ‘gun-zakura’ within the grounds of kan-eiji temple. ‘somei-yoshino’ was created by horticultural techniques developed during the long period of peace and economic stability of the edo era. ‘somei-yoshino’ has been a parent of more than 100 cultivars since the meiji restoration. dr. yozaburo shirahata (2000) suggested that ‘hanami’ consisting of ‘gunzakura,’ (‘eating and drinking’, and ‘mass gathering’) is a popular culture unique to japan, and the japanese love of ‘hanami’ is showing no sign of diminishing. acknowledgements we would like to express our sincere thanks to professor drs. toshio ando and masahiro mii, graduate school of horticulture, chiba university and dr. akira kobayashi of management office of tokyo metropolitan parks for their kind assistance during the course of this research. the authors dedicate this paper to the memory of mr. masayuki hashimoto, who was a great breeder of ornamental flowers and flowering cherry in japan. references iketani h., katsuki t., kawahara t., 2006 prunus yedoensis cv. somei-yoshino, a correct cultivar name for yoshino cherry. j. jap. bot., 81: 123-125. ito i., 1695 kadan-chikin-syo. katyuken uhei, tokyo, japan, pp. 106. fig. 15 haplotypes of intron 19 in pola1 gene and of chloroplast (cp) dna analyzed for ten trees around the ruins of the bell tower (b) in kan-eiji temple. 243 ito i., 1719 koeki-chikin-syo. vol. 8. suharaya mohei, tokyo, japan, pp. 621 iwasaki f., 1989 bibliographic studies on the origin of the flowering cherry, somei-yoshino (prunus yedoensis matsumura). bull. agr. for. res. univ. tsukuba, 1: 85-103. iwasaki f., 1991 origin of ‘somei-yoshino’ and wild status of the related species. bull. agr. for. res. univ. tsukuba, 3: 95-110. iwasaki k.e., 1818 somoku-sodate-gusa. yamashiroya sahei, tokyo, japan, pp. 106. kaneko t., terachi t., tsunewaki k., 1986 studies on the origin of crop species by restriction endonuclease analysis of organellar dna. restriction analysis of ctdna of 11 prunus species. jpn. j. 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(ed.) flora reipublicae popularis sinicae 38. science press, beijing, rpc. 44 1. introduction although olives are grown in other world regions, such as california, australia and argentina, the most important production areas are found in the mediterranean basin where olive finds its best growing conditions, in particular in spain, portugal, italy, greece, albania, north africa and the middle east. olive tree has had traditional importance in the mediterranean region since ancient times (loumou and giourga, 2003) and its cultivation retains importance in this area for its social, environmental and economic value. currently a large part of the total world olive plantations are found in the mediterranean basin. the olive tree defines the mediterranean region within the holarctic kingdom (ubaldi, 2003) and it is considered one of the most typical species of this area, where it represents a very important element in defining the “identity” of the rural landscape. the ecological function of rural landscapes and the promotion of multifunctional agriculture is an important topic in agricultural and agri-environmental policy within the european union (gerowitt et al., 2003). vegetation related to olive orchards plays an important ecological function that can be efficiently used to improve the multifunctional role of olive growing in the mediterranean region (margaris, 1980). for instance, saavedra (1998) reports over 500 species in the olive area of córdoba province. in a selection of plantations in western andalucía, 75 plant species were recorded prior to the spring cultivation (rodenas et al., 1977). in greece, traditionally managed olive groves have been identified as important habitats that often support a rich ground flora, which may include species with habitats threatened by land-use changes (allen et al., 2006). surveys in several olive orchards of south-western albania report more than 80 species belonging to 14 botanical families (huqi et al., 2009). in italy, viggiani (2009) reports more than 50 species as typical of olive groves, most of them having also ethno-botanical importance. the number could be even higher considering the species that can be found along field margins, i.e. roads, stone walls and other traditional human infrastructures typical of olive orchard landscapes. the presence of a significant number of plant species in olive groves offers favourable conditions for a multitude of animals such as arthropod fauna, reptiles, mammals and birds (beaufoy, 2000; loumou and giourga, 2003). this is due not only to primary production in the food chain, but also to the provision of cover and reproduction sites (marshall et al., 2003). potts et al. (2006) assessed the biodiversity value of six common habitats on the greek island of lesvosmic. they found that manground cover management strategies in an apulian oil-producing olive grove: agronomic and ecological assessment proposals m. fracchiolla, d. caramia, c. lasorella, p. montemurro dipartimento di scienze agroambientali e territoriali, università degli studi di bari, via g. amendola, 165/a, 70126 bari, italy. key words: chopping, cover crop, herbicides, olea europaea l., soil management, weeds. abstract: several studies have pointed out that ground flora in olive groves, such as in any orchard, should ideally combine adequate positive effects on the agro-environment with only marginal negative competitive effects on the olive plants. this paper reports the results of an experiment carried out in an irrigated olive orchard (cv. leccino), located in the area of savelletri, puglia (southern italy), regarding the effects of ground flora as a consequence of different management techniques. an aggregate index is proposed, able to provide a comprehensive evaluation of flora from both an ecological and agronomic point of view. four different weed control strategies were compared: a) seeding, every other year, of a cover crop (vicia sativa l.) chopped in springtime; b) weed control using a mixture of a systemic herbicide and a residual herbicide; c) weed control using a systemic herbicide only; d) chopping. the results revealed that the different management practices largely influenced the ground cover values in each study year, but not the yield. ground cover features, assessed both from an agronomic and ecological point of view varied in particular, as was well reflected by the applied index, which proved to easily and effectively describe the flora features in different plots. adv. hort. sci., 2013 27(1-2): 44-54 received for publication 27 february 2013 accepted for publication 21 march 2013 45 aged olive groves had the highest diversity of bees, comparable with natural habitats such as oak woodlands and pine forests. natural flora in olive orchards and the related fauna are also important sources of food for many species of birds, with consequent internationally important effects related to the migration of these animals (guzman alvarez, 1999). for example, in southern italy about 6% of olive groves are included in “natura 2000 habitats” (birds directive 92/43/eec). from an agronomic point of view, natural flora is often able to enhance pest control because it can be an alternative host or direct food source for beneficial organisms (marshall et al., 2003; norris, 2005). in addition, ground cover can positively affect the diversity of soil biota, improving the soil ecosystem function. this effect was shown in a trial conducted in a rain-fed olive orchard, located in south-eastern spain by moreno at al. (2009) in which covered soils exhibited greater bacterial biomass and diversity, as well as higher microbial functional diversity than non-covered soils. conservative flora management can also increase co 2 fixation and enhance the capacity of olive orchards to accumulate significant amounts of biomass and humus (sofo et al., 2004; palese et al., 2005). vegetation cover also has an important function in significantly reducing soil erosion (hernandez et al., 2005), one of the most serious and widespread environmental problems in many areas of the mediterranean region (pastor muñoz-cobo and castro, 1995) where olive groves are often located in marginal soils and on steep slopes (gomez et al., 2003; francia martínez et al., 2006). however, olive tree vegetation and yield can be significantly damaged if weed flora is not correctly managed, especially under rain-fed conditions. in addition to competing with olive for water, nutrients and at early crop stages even for light, weeds may also hamper olive picking. moreover, during summer, dead weed residues can catch fire and seriously damage olive plants in cases where the residues are abundant. traditional soil management is based on tillage, keeping the soil bare of vegetation all year round. this practice, in addition to undoing the potential benefits of natural flora, is also labour-intensive and expensive, hence it must be considered not sustainable. several experiments show that it is possible to obtain the same or better productive results by adopting practices, such as chemical weeding or mowing, reducing or eliminating soil tillage and maintaining weed flora density at a level that is not dangerous for olive plants. some significant results are reported by huqi et al. (2009) in albania, montemurro and mastropirro (1995), montemurro et al. (2002) and toscano et al. (2004) in southern italy, hernandez et al. (2005) in central spain, pastor muñoz-cobo (1990; 1991) in spain, and kabourakis (1999) in greece. these alternative strategies could represent a remarkable sustainable approach for the maintenance of the environment both in intensive systems, mitigating the environmental impact of olive growing, and in low-intensity farming systems located in marginal areas. in these latter areas, reduced tillage could reduce management costs and contribute to preventing abandonment of these groves and preserve natural and cultural resources (duarte et al., 2008). the effects could be beneficial on a large portion of the european territory; olive groves occupy approximately 5.4 million hectares, or about 4% of the utilisable agricultural area (source: european community). each soil management system provides different conditions of the growth for weeds. tillage destroys the annual flora, but can also create favourable conditions for new germinations and, moreover, it benefits perennial weeds by fragmenting and scattering vegetative reproductive organs such as rhizomes, tubers, bulbs and stolons. foliar herbicides, such as glyphosate, are able to control both annual and perennial plants, but they could exert a selection pressure on tolerant or resistant species. residual herbicides, such as oxyfluorfen, keep the soil weed-free for a longer period of time. generally, chemical weed control can cause a simplification of the flora spectrum with fewer species that are often more problematic to manage. mowing can encourage those species that are able to re-sprout after cutting. cover crop (living mulches) may contribute significantly to weed suppression providing early soil coverage and reducing the number of established weed seedlings. research proposal weed flora, also as a consequence of different management practices, can have both positive and negative effects on olive orchards as well as on the agro-ecosystem. several studies have focused specifically on the ecological importance of natural flora in olive groves; other experiments have been performed in order to suggest the best control practices and to reduce the negative effects of weeds. it is reasonable to suppose that ground flora in olive groves, such as in any orchard, should ideally combine adequate positive effects on the agro-environment with only marginal negative competitive effects on the olive plants. the objective of the current work is to report data on flora communities established as a consequence of different management techniques and to suggest an aggregate index able to give a comprehensive evaluation of flora, both from the ecological and agronomic point of view. the effects on olive production and oil yield are also considered. 2. materials and methods the experimentation was carried out between november 2005 and december 2010 in an irrigated olive orchard located in the area of savelletri (puglia -southern italy) made up of 11-year-old cv. leccino plants, vase shape trained and spaced 7 x 7 m. the soil that hosted the orchard was loamy-textured (16.9% clay 35.8% 46 silt 47.3% sand), with a moderate presence of shallow pebbles (7.5 to 25 cm in size). the trial involved the comparison of the following four different weed control strategies: i) seeding, every other year, of a cover crop (vicia sativa l.) chopped in springtime. the subsequent infestation was controlled by chopping (ve); ii) weed control using a mixture of a glyphosate-based systemic herbicide and a residual herbicide containing oxyfluorfen, at a rate of 1.08 and 0.12 l ha-1 respectively (gly + ox); iii) weed control using glyphosate only at a rate of 1.08 l ha-1 (gly); iv) chopping (tri). herbicides were diluted in a water volume of 400 l ha-1 and applied with a hand-pump spray bottle, equipped with flat fan nozzles. weeds were chopped using a shredder. the vetch was sown broadcast at the rate of 80.0 kg ha-1, burying the seed by a shallow harrowing. the different management strategies of ground flora were applied following the general principle of applications so as to keep the orchard fully free of natural flora in the peak vegetative growth period, i.e. in spring-summer, when the flora reached a mean height of about 10-15 cm. the dates of weeding operations for each treatment are detailed in table 1. the other agronomic and plant protection practices were applied using the techniques commonly used in the research area. the experimental plots, covering an area of 441.00 m2 (21 x 21 m), were arranged in the field following a randomised block design, with four replicates; for flora surveys a central test area of 196.0 m2 was used including the four plants on which vegetation production surveys were conducted. flora surveys and data processing plot flora surveys were run in april and october of each year and in the two peak growth periods of weeds. in these surveys, made prior to the execution of scheduled weed control operations, species were divided into the following two groups: a) species distributed uniformly in the test area; b) species represented either by solitary plants or distributed in restricted patches. afterwards, for each species of the first group, a percent ground cover value related to the reference test area was estimated. these data have been used to calculate the specific contribution (cs), dividing the ground cover of each single species by the total cover (sum of the covers attributed to each single species) and multiplying it by 100. moreover, the presence of each botanical family was obtained by summing the percent ground covers of each species belonging to it. nomenclature refers to pignatti (1982). to provide an estimate of the ground cover features of each treatment, an index defined as ground cover quality index (gcqi) was proposed. this index is calculated by the following formula: gcqi = [ ∑ i (csi x v i ) ]/6, where v i is a total score assigned to each species having a uniform distribution in the test area, calculated by summing the values assigned to the following parameters: ability to cover the soil and protect it from erosion processes (0 = negligible; 1 = average; 2 = good); general ability to improve/preserve the chemical and physical soil properties through biomass production, nitrogen fixation or development of bunched roots (e.g. grass plants) that increase its porosity (0 = negligible; 1 = good); competitive ability against the orchard (0 = very competitive; 1 = normal; 2 = negligible); flammability in summer periods (0 = plant that leaves much dry biomass easily flammable; 1 = thin plant that produces little biomass potentially flammable or whose biomass is easily degraded prior to the warm season or that remains green in summer periods). it follows that the value of <v i > could range between 0 and 6 and that, by the indicated formula, the specific contribution (cs) of each species, based on their morphologic and eco-physiological features, can take a weight varying between 0 and 6 times its value. the specified parameters are proposed as a general indication, based on the specific needs of the test area. this does not exclude the possibility of using other ones based on other needs related to different conditions (e.g. aesthetic contribution, ability to be intermediate hosts of predators or of hyper pests, etc.). this work presents only the flora data and the relevant analyses for 2006, 2008 and 2010, i.e. only in the presence of vetch (ve treatment) and in heavy years, considering olive alternate bearing. surveys on olive plants for each of the four plants included in the test area, plants were tested for mean shoot growth recorded between april and october of each year, by selecting four shoots per plant arranged along the four cardinal directable 1 calendar of applied practices in different ground cover management strategies strategies practices 2005 2006 2007 2008 2009 2010 nov april oct april july oct april oct april oct nov april oct 1) ve sowing of vicia sativa x x x chopping x x x x x x x x 2) gly + ox chemical weeding x x x x x x x x x x 3) gyi chemical weeding x x x x x x x x 4) tri chopping x x x x x x x x x x x x 47 tions. the data concerning each plot was thus obtained as an average of 16 values (four shoots per plant x four plants). at the beginning and end of the trial, the trunk diameter of each plant included in the plot area was also measured, and the mean growth occurred in that period was obtained by difference. olive harvesting was carried out in alternate years, i.e. in december 2006, 2008 and 2010. the fruits produced by the four plants of the test area were weighed. the oil yield was measured on a randomly chosen 2 kg sample from all olives harvested in each plot. the applied procedure complied with the guidelines of annex xv of the ec reg. no 2568/1991. all data were submitted to variance analysis and the means were compared using duncan’s test. climate pattern figures 1 and 2 show the climate pattern observed during the experiment. each year, in accordance with the climate pattern of the test area, the hottest months were june, july and august, whereas the coldest ones were december, january and february. the highest positive deviations (from +2.0 to +3.6°c) were recorded for the mean temperatures of january, june, july and august of 2007, january 2008 and november 2010; the highest negative difference was observed in february 2009 (-2.0°c compared to the plurennial mean). as to rainfall, the rainiest years were 2009 and 2010 with values exceeding the plurennial mean of the area (577.2 mm) by 243.5 mm and 162.1 mm, respectively. in those years, the months that deviated most from the average were january (+110.7 mm), march (+57.5 mm) and october (+64.6 mm) in 2009, and may (+72.9 mm) and october (+147.3 mm) in 2010. 3. results table 2 lists the species found during the experiment. a total of 60 were identified; only 34 had a uniform distribution in the plots. the results obtained with regard to the uniformly distributed species are addressed in this section. spring flora surveys in 2006 (table 3), the statistically lowest total ground cover values were observed in treatments gly and gly+ox with 35.6 and 33.9%, respectively, followed by vetch-sown plots with 91.5%. the highest mean number of species, equal to 23.0, was recorded in vetch-sown areas, whereas the highest number of families (10.0) was observed in the plots subjected to chopping. table 4 reveals that in the tri treatment the statistically highest mean ground cover values were found for gramineae, compositae and leguminsae, equal to 113.9-12.8 and 12.5% respectively, whereas the lowest values were recorded in treatment gly (10.2% graminae and 0.4% leguminosae), gly+ox (13.3% graminae, 5.7% compositae and 0.1% leguminosae) and ve (6.1% compositae). as to single species (table 5), the highest specific contributions were calculated in chemically weeded plots for malva sylvestris l. with 16.7% in treatment gly+ox and 12.8% in gly; these values were significantly higher than those observed in the plots of treatments ve and tri. for avena sterilis l., bromus sterilis l. and lolium rigidum gaudin, in chopped or vetch-sown plots higher specific contributions were observed than in chemically weeded plots. on the contrary, the specific contribution of hordeum murinum l. and setaria verticillata (l.) beauv. was significantly higher in the gly+ox treatment. with regard to the ground cover quality index in treatments tri and ve, the observed values (52.4 and 55.1 respectively) were shown to be statistically higher than those calculated for chemically weeded treatments (table 3). in 2008, the highest values of total infestation and mean number of species (table 3) were found in chopped (83.7% 18.0) and vetch-sown plots (85.3% 18.0), whereas the number of families was lower in treatment ve. the family gramineae had a ground cover equal to 63.8% in treatment tri, which is statistically higher than fig. 1 mean monthly temperatures recorded during the trial and pluriannual means (1951-2001). fig. 2 monthly rainfall recorded during the trial and pluriannual means (1951-2001). 48 table 2 species found in experimental plots (z) treatments ve gly+ox gly tri 2006 2008 2010 2006 2008 2010 2006 2008 2010 2006 2008 2010 adonis aestivalis l. + anagallis arvensis l. x x x x x x anthemis arvensis l. x x x x x arum italicum miller. + + + asparagus acutifolius l. + + + + + + + + + + + + asphodelus fistulosus l. + aster squamatum (sprengel) hieron. + + + + + + + + + + avena sterilis l. x x x x x x x x x x x x bellardia trixago (l.) ali. x x x x x x x x briza maxima l. + bromus sterilis l. x x x x x x x x x x x x calendula arvensis l. x x x x x x x x capsella bursa-pastoris (l.) medicus x x x x x catapodium rigidum (l.) hubbard + + + + cerinthe major l. + chrysanthemum segetum l. x x x x x x x x x x x x convolvulus arvensis l. x x x x x x conyza canadensis (l.) cronq. x x x x x x x x x x x cynodon dactylon (l.) pers. x x x x x x x x x x digitaria sanguinalis (l.) scop. + + + + diplotaxis erucoides (l.) dc. x x x x x x x x x x x x diplotaxis muralis (l.) dc. x x x dittrichia viscosa greuter. + + erodium malacoides (l.) l’hér. x x x x x x x x x x x x euphorbia chamaesyce l. + + + + + + + galactites tomentosa moench. + + + + geranium molle l. + + + + + heliotropium erupaeum l. x x x x x x x x hippocrepis unisiliquosa l. + hordeum murinum l. x x x x x x x x x x x x lactuca serriola l. + lamium purpureum l. x lolium rigidum gaudin x x x x x x x x x x x x lotus ornithopodioides l. + + malva sylvestris l. x x x x x x x x x x x x medicago hispida gaertner x x x x x x x x x x x x melilotus indica (l.) all. + mercurialis annua l. + + muscari neglectum guss. + + + + ononis natrix l. + + + + oxalis pes-caprae l. x x x x x x x x x x x papaver rhoeas l. x x x x x x x x x x phalaris paradoxa l. x x x x x x x x portulaca oleracea l. x x x x raphanus raphanistrum strobl. + + + + + scorpiurus muricatus l. x x x x serapias sp. + + setaria verticillata (l.) beauv. x x x x x x sherardia arvensis l. + + + solanum nigrum l. x x x x sonchus oleraceus l. x x x x x x x x x x x x sonchus tenerrimus l. x x x x tetragonolopus purpureum moench. + + trifolium campestre shreber x x x x x x x trifolium fragiferum l. + + + + + + trifolium repens l. x x x x x x x x x x x x trifolium scabrum l. x x x x x x x x x x trifolium tomentosum l. x x x x x x x x x x valerianella eriocarpa desv. + + + + + + + + + + verbascum sinuatum l. + + + + + total species with uniform distribution (n.) 27 22 22 25 18 22 24 21 25 26 24 25 total others (n.) 6 6 12 5 5 10 7 8 11 9 9 15 (z) x = uniformly distributed species; + = others: solitary plants or restricted to patchy areas. 49 table 4 ground cover (%) of the botanical families found in spring surveys weeds crop year 2006 crop year 2008 crop year 2010 gli gl+ox tri ve gly gliy+ox tri ve gly gly+ox tri ve gramineae 10.2 c 13.3 c 113.9 a 68.5 b 22.7 d 40.0 c 52.4 b 63.8 a 23.2 b 22.4 b 30.0 b 51.9 a compositae 9.1 b 5.7 c 12.8 a 6.1 c 8.2 a 7.4 a 7.3 a 5.9 b 21.5 a 20.0 a 6.7 b 6.6 b leguminosae 0.4 c 0.1 c 12.5 a 7.3 b 1.4 d 2.9 c 16.2 a 6.7 b 12.0 b 11.1 b 63.5 a 10.5 b cruciferae 1.6 a 1.6 a 1.0 b 0.6 b 1.6 b 1.7 ab 0.5 c 1.9 a -- - -primulaceae 2.6 a 0.0 c 1.9 b 0.0 c - ---4.5 b 0.0 d 5.7 a 1.9 c scrofuliaraceae 0.0 b 0.0 b 2.0 a 0.1 b 0.0 0.1 0.6 a 0.0 1.9 a 0.5 b 0.5 b 0.5 b convolvulaceae 5.1 4.9 4.9 4.7 2.3 a 2.2 a 0.0 b 0.0 b -- - -geraniaceae - - - - 2.0 b 1.9 b 2.9 a 2.6 ab -- - -boraginaceae - - - - 0.9 b 1.7 a 2.4 a 1.9 a -- - -labiatae 0.0 0.5 0.0 0.0 - ----- - -malvaceae 4.5 b 5.6 a 3.2 c 0.0 d 5.7 a 2.6 b 1.0 c 0.6 c 7.0 a 3.6 b 0.5 c 2.5 b papaveraceae 0.1 c 0.3 c 1.3 b 3.0 a 0.4 b 0.0 b 0.4 b 2.0 a 4.0 a 0.5 b 0.5 b 0.0 b portulacaceae 1.9 1.8 2.0 1.5 - ----- - -(z) values that do not have a letter in common are significantly different at 0.05 p (duncan’s test). table 3 total ground cover, number of families and species, agro-ecological indices in spring surveys ground cover management strategies (2) crop year 2006 crop year 2008 crop year 2010 gly gly+ox tri ve gly gly+ox tri ve gly gliy+ox tri ve total ground cover (%) 35.6 c 33.9 c 155.7 a 91.5 b 45.3 c 61.5 b 83.7 a 85.3 a 74.2 b 58.2 c 107.5 a 74.5 b botanical families (n.) 8.5 b 8.0 b 10.0 a 7.7 b 9.0 a 9.0 a 9.0 a 8.0 b 7.0 a 4.5 c 5.5 b 6.0 b species 16.0 c 15.5 c 20.7 b 23.0 a 17.0 b 17.0 b 18.0 a 18.0 a 16.2 a 12.5 b 15.7 a 15.7 a gcqi 53.2 b 49.6 c 52.4 a 55.1 a 53.1 c 53.9 c 61.0 a 54.6 b 57.5 b 58.4 b 80.9 a 57.3 b (z) values that do not have a letter in common are significantly different at 0.01 p (capital letter) or at 0.05 p (small letter) (duncan’s test). the values recorded for the other strategies under consideration (table 4). the leguminosae, instead, showed the highest cover value (16.2%) in chopped plots. the data presented in table 5 point out that the statistically highest cs value of a. sterilis was found in vetch plots ve (18.1%), whereas that of b. sterilis was higher with only chopping, where the lowest specific contribution of l. rigidum was also calculated. within leguminosae, the most represented species were medicago hispida gaertner, trifolium repens l. and trifolium tomentosum l., whose specific contributions were higher in chopped plots. conyza canadensis (l.) cronq. and m. sylvestris, instead, showed the significantly highest cs in the experimental plots weeded only by glyphosate. as for the ground cover quality index (table 3), the highest statistical value was calculated in chopped plots (61.0), followed by treatment ve (54.6). in 2010, the statistically highest total cover percentage (table 3) was observed in the chopped plot (107.5%), followed by vetch plots (74.5%). the lowest mean number of families and species (4.5 and 12.5, respectively) was recorded in ox treatment. the data included in table 4 point out that the most significant cover value of graminae and leguminosae was found, respectively, in the plots with vetch (51.9%) and in tri treatment (63.5%). as for single species (table 5), a. sterilis and b. sterilis showed the statistically highest mean values of cs in the ve treatment (21.3 and 22.0% respectively); the cs of l. rigidum and trifolium campestre shreber were found to be, instead, the lowest in statistical terms in treatments tri (4.2%) and gly+ox (0.0%). with regard to the gcqi (table 3), the statistically highest mean value (80.9) was observed in chopped plots among all compared treatments. autumn flora surveys in 2006, the statistically highest infestation value (table 6) was observed in chopped plots (34.6%). the statistically lowest mean number of species and families, 13.0 and 9.0 respectively, was recorded in the tri treatment. in tri and ve treatments the mean cover values of gramineae species were 22.4 and 21.5% respectively, statistically higher values compared to treatments gly and gly+ox (table 7). as to specific contributions, table 8 shows that l. rigidum is the species with the highest mean data of all the monitored species; more specifically, it accounted for 63.5% of cover in the ve treatment and 61.1% in tri, both values being significantly higher than those observed in chemically weeded plots. the highest gcqi (table 6), 50 equal to 54.9, was calculated in the plot submitted to chopping; this data is statistically different from that observed in the other treatments. in 2008, the statistically highest values of total cover percentage and number of species (table 6) were observed in chopped plots (49.6% and 13.7%, respectively). for the number of families (table 6), which varied between 6.0 and 6.5, no sharp differences were found. the statistically highest mean cover values of gramineae (21.8%), leguminosae (5.0%) and compositae (14.5%) were recorded in the tri treatment (table 7). the data shown in table 8 point out that the highest specific contributions were found for: a) a. sterilis in the treatment weeded by the mixture of glyphosate and oxyfluorfen (22.9%); b) b. sterilis (12.6%) and oxalis. pes-caprae l. (14.6%) in ve; c) c. canadensis (47.0%) in the plots weeded by glyphosate only; d) heliotropium erupaeum l. in both chemically weeded plots; and e) m. hispida in the plots submitted to chopping table 5 specific contributions (%) calculated for the species found in spring surveys weeds crop year 2006 crop year 2008 crop year 2010 gli gli+ox tri ve gli gli+ox tri ve gli gli+ox tri ve anagallis arvensis l. 7.3 a (z) 0.0 c 1.2 b 0.0 c ----6.1 a 0.0 c 5.3 ab 2.6 b anthemis arvensis l. 0.0 b 0.0 b 1.2 a 0.0 b ----4.8 a 0.9 b 0.4 b 3.3 a avena sterilis l. 3.3 c 3.9 c 20.2 a 17.1 b 0.5 c 2.1 c 13.0 b 18.1 a 0.6 c 5.1 b 3.8 b 21.3 a bellardia trixago (l.) ali. 0.0 c 0.0 c 1.3 a 0.1 b 0.0 c 0.2 b 0.7 a 0.0 c 2.7 0.9 0.5 0.7 bromus sterilis l. 3.0 c 3.9 c 19.9 a 16.5 b 23.5 c 25.6 b 29.2 a 17.9 d 9.4 c 12.7 b 14.3 b 22.0 a alendula arvensis l. ----0.3 b 0.2 b 0.2 b 1.1 a ----capsella bursa-pastoris (l.) medicus 0.0 b 0.0 b 0.0 b 0.2 a 0.2 0.2 0.1 0.2 ----chrysanthemum segetum l. 3.2 b 4.8 a 1.3 c 1.0 c 5.9 a 4.4 b 3.2 c 3.2 c 6.2 a 8.5 a 1.1 b 4.0 a conyza canadensis (l.) cronq. 9.8 a 11.0 a 2.9 b 0.5 c 3.2 a 0.4 b 0.0 b 0.0 b 12.7 b 16.2 a 0.9 c 0.9 c convolvulus arvensis l. 14.3 a 14.7 a 3.2 b 5.1 b 5.1 a 3.7 b 0.0 c 0.0 c ----cynodon dactylon (l.) pers. 14.8 a 14.4 a 3.1 b 0.2 c 3.2 a 2.5 b 0.4 d 2.1 c ----diplotaxis erucoides (l.) dc. 4.4 a 4.8 a 0.6 b 0.5 b 4.4 a 3.1 b 3.5 b 3.0 b ----hordeum murinum l. 0.3 d 6.3 a 2.1 c 3.5 b 2.1 2.8 2.8 2.2 2.6 bc 0.8 c 4.6 b 5.4 a lamium purpureum l. 0.0 b 1.3 a 0.0 b 0.0 b 1.3 c 14.0 a 13.6 ab 12.9 b ----lolium rigidum gaudin 5.6 c 6.4 c 27.1 b 34.7 a 24.9 a 24.8 a 6.9 b 25.8 a 16.7 a 17.8 a 4.2 b 20.0 a malva sylvestris l. 12.8 b 16.7 a 2.1 c 0.0 c 12.7 a 4.3 b 1.2 c 0.7 c 9.5 a 6.3 a 0.5 b 3.4 a medicago hispida gaertner 0.7 b 0.1 b 1.9 a 0.0 b 1.9 d 3.9 b 6.6 a 2.9 c 4.1 7.9 5.2 2.0 papaver rhoeas l. 0.3 b 0.9 b 0.8 b 3.3 a 1.0 b 0.0 c 0.5 bc 2.3 a 5.5 a 0.8 b 0.4 b 0.0 b phalaris paradoxa l. 0.0 b 0.0 b 0.0 b 0.1 a 0.0 b 0.0 b 0.0 b 0.1 a 1.6 a 1.7 a 0.9 b 1.6 a portulaca oleracea l. 5.5 a 5.3 a 1.3 b 1.7 b --------setaria verticillata (l.) beauv. 1.7 b 4.3 a 0.7 b 2.4 b --------scorpiurus muricatus l. 0.0 c 0.0 c 0.2 b 1.1 a ----0.7 0.0 0.4 0.0 sonchus oleraceus l. 12.5 a 1.1 d 2.9 c 5.1 b 8.8 a 6.9 b 5.4 c 2.5 d 5.5 b 8.5 a 3.8 b 1.0 c trifolium campestre shreber 0.3 b 0.1 b 1.5 a 1.8 a ----3.4 a 0.0 b 5.2 a 3.4 a trifolium repens l. 0.1 b 0.1 b 1.3 a 1.4 a 1.1 c 0.8 c 5.2 a 1.8 b 0.6 c 1.2 c 11.8 a 3.4 b trifolium scabrum l. 0.1 b 0.1 b 1.6 a 1.9 a 0.0 c 0.0 c 1.4 a 0.9 b 1.9 b 2.7 b 21.9 a 2.0 b trifolium tomentosum l. 0.1 b 0.1 b 1.5 a 1.8 a 0.0 c 0.0 c 6.3 a 2.2 b 5.5 b 7.8 b 14.9 a 3.4 b (z) values that do not have a letter in common are significantly different at 0.05 p (duncan’s test). table 6 total ground cover, number of families, number of species and agro-ecological indices in autumn surveys crop year 2006 crop year 2008 crop year 2010 gli gli+ox tri ve gli gli+ox tri ve gli gli+ox tri ve total ground cover 23.4 c (z) 23.6 c 34.6 a 31.9 b 19.1 c 20.7 c 49.6 a 39.8 b 8.2 b 9.1 b 24.0 a 22.0 a n° of families 10.0 a 10.0 a 9.0 b 9.7 a 6.5 6.5 6.0 6.2 6.7 ab 5.7 bc 5.5 c 7.5 a n° of species 14.0 a 14.0 a 13.0 b 13.7 a 8.7 c 11.5 b 13.7 a 11.7 b 9.7 7.7 8.2 10.7 gcqi 53.3 b 52.7 b 54.9 a 53.6 b 42.2 c 55.7 a 54.2 a 52.6 b 58.8 62.0 58.4 56.8 (z) values that do not have a letter in common are significantly different at 0.05 p (duncan’s test). 51 (10.1%). the gcqi with the lowest statistical value was in the treatment weeded by glyphosate only (42.0), whereas the values calculated in the other treatments were not statistically different from each other (table 6). in 2010, the highest total cover values were observed in tri and ve treatments, with 24.0 and 22.0%, respectively (table 6). the highest mean number of families (7.5) was recorded for treatment ve. as to the number of species, statistical analysis did not point out any reliable difference between the values of different strategies that ranged between 7.7 for treatment gly+ox and 10.7 for ve. the data in table 7 show that graminae and compositae had a statistically higher mean cover value in tri and ve plots, whereas the cover values of leguminosae species did not show any remarkable difference between each other. as to single species (table 8), the highest cs values in statistitable 7 ground cover (%) of the botanical families found in autumn surveys weeds crop year 2006 crop year 2008 crop year 2010 gli gli+ox tri ve gli gli+ox tri ve gli gli+ox tri ve graminaceae 11.1 b (z) 11.6 b 22.4 a 21.5 a 1.1 d 6.3 c 21.8 a 9.3 b 1.3 b 1.7 b 4.3 a 5.9 a compositae 2.5 a 2.7 a 2.9 a 1.5 b 10.8 b 6.1 c 14.5 a 11.5 b 2.2 b 3.5 b 6.6 a 6.6 a leguminosae 1.2 c 1.1 c 3.3 a 1.7 b 0.1 c 0.3 bc 5.0 a 0.6 b 0.9 1.0 1.5 1.2 cruciferae 2.1 a 2.2 a 1.3 c 1.9 b 0.3 b 0.5 b 0.2 b 6.0 a 0.1 0.1 0.2 0.7 convolvulaceae 2.6 2.4 2.4 2.3 --------geraniaceae 0.5 b 0.5 b 0.5 b 1.0 a 0.0 0.0 0.0 0.1 1.0 0.2 0.4 1.0 boraginaceae 0.6 0.6 0.8 1.0 5.2 b 5.1 b 5.1 b 6.5 a ----malvaceae 1.3 a 1.0 ab 0.9 b 0.7 b 0.1 0.1 0.0 0.0 1.6 a 0.4 b 0.2 b 0.7 b oxalidacee 1.0 a 1.0 a 0.1 b 0.0 b 1.5 d 2.2 c 2.9 b 5.8 a 0.6 d 2.1 c 10.2 a 5.5 b primulaceae --------0.3 0.3 0.5 0.4 solanacee 0.4 a 0.5 a 0.2 b 0.2 b --------(z) values that do not have a letter in common are significantly different at 0.05 p (duncan’s test). table 8 specific contributions (%) calculated for the species found in autumn surveys weeds crop year 2006 crop year 2008 crop year 2010 gli gli+ox tri ve gli gli+ox tri ve gli gli+ox tri ve anagallis arvensis l. --------4.1 0.5 1.9 2.0 avena sterilis l. ----0.0 c (z) 22.9 a 5.0 b 3.7 b ----bromus sterilis l. ----0.0 c 1.3 b 11.6 a 12.6 a 2.4 c 1.5 c 13.3 b 25.0 a calendula arvensis l. ----8.9 b 1.7 c 10.6 b 16.3 a 4.3 b 1.1 b 20.1 a 16.0 a chrysanthemum segetum --------3.8 0.0 0.9 2.5 conyza canadensis (l.) cronq. 6.5 b 7.6 a 4.4 c 1.4 d 47.0 a 3.6 c 8.6 b 0.0 c 11.0 a 12.9 a 0.1 b 1.6 b convolvulus arvensis l. 10.9 a 10.3 a 6.9 b 7.2 b --------cynodon dactylon (l.) pers. 5.4 a 5.3 a 3.6 b 3.9 b 5.9 a 5.3 a 2.2 b 0.4 c 3.5 0.0 1.1 0.0 diplotaxis erucoides (l.) dc. 4.9 a 5.0 a 3.3 b 5.5 a 1.4 2.4 0.3 13.8 1.5 1.2 0.9 3.2 diplotaxis muralis (l.) dc. 4.2 a 4.1 a 0.4 b 0.5 b 0.1 bc 0.0 c 0.2 b 1.3 a ----erodium malacoides (l.) l’hér. 2.1 b 2.0 b 1.4 c 3.1 a 0.1 0.0 0.0 0.1 12.4 a 2.5 b 1.7 b 4.6 b heliotropium europaeum l. 2.7 2.7 2.4 3.1 27.5 a 24.5 a 10.3 c 16.4 b ----hordeum murinum l. ----0.0 b 0.7 b 10.1 a 0.1 b ----lolium rigidum gaudin 42.3 b 43.9 b 61.1 a 63.5 a 0.0 b 0.2 b 9.6 a 0.2 b 10.0 ab 16.5 a 3.7 bc 1.6 c malva sylvestris l. 5.4 a 4.1 b 2.5 c 2.4 c 0.5 0.7 0.0 0.0 19.9 a 4.1 b 1.1 b 3.2 b medicago hispida gaertner 0.9 c 0.9 c 7.0 a 2.0 b 0.3 c 1.4 b 10.1 a 1.6 b 12.2 12.0 6.1 4.2 oxalis pes caprae l. 4.3 a 4.1 a 0.0 b 0.3 b 7.7 c 10.9 b 5.9 c 14.6 a 8.5 c 25.0 b 42.6 a 25.8 b setaria verticillata (l.) beauv. ----0.0 b 0.0 b 5.6 a 6.3 a ----sonchus oleraceus l. ----0.5 d 24.2 a 10.1 c 12.6 b 2.9 5.4 0.9 4.0 sonchus tenerrimus l. 4.2 4.1 3.8 3.1 ----3.3 b 16.9 a 5.2 b 5.2 b solanum nigrum l. 1.9 a 2.2 a 0.6 b 0.4 b --------trifolium campestre shreber 4.3 3.7 2.5 3.5 --------(z) values that do not have a letter in common are significantly different at 0.05 p (duncan’s test). 52 cal terms were observed: a) for b. sterilis (25.0%) in the plots of treatment ve; b) for calendula arvensis l. both in ve (16.0%) and tri (20.1%); c) for c. canadensis in both chemically weeded plots; and d) for o. pes-caprae in the chopped treatment (42.6%).the gcqi (table 6) ranged between 62.0 in treatments gly+ox and 56.8 in treatment ve and did not show any significant differences between the values of the strategies being compared. finally, table 2 lists the other species found during the experiment, with low cover percent values and a non-uniform distribution. the occurrence of these 26 species increased during the years for all treatments, although none of them attained a uniform distribution over time. vegetation surveys shoot growth and trunk diameter did not show any significant differences between the mean values measured for the compared treatments (table 9) during the study three years. olive production and oil yield as shown by the data in table 10, no statistical differences were observed between the mean recorded values during the study period for the different strategies being compared with regard to olive production and oil yield per plant. 4. discussion and conclusions in this work diachronic analysis was not carried out given the limited number of years under study, however synchronic comparisons have supplied data that can lead to some interesting conclusions. the different management practices employed in the study largely influenced the ground cover values in the various years, both quantitatively and qualitatively, but not the yield. in all surveys, the most represented families, both in terms of ground cover and number of species, were graminae, leguminosae and compositae. with regard to single species, significant specific contributions were recorded for a. sterilis, b. sterilis, c. canadensis, l. rigidum, m. sylvestris, o. pes-caprae and trifolium spp. the largest differences, in terms of total ground cover, were observed between the chemically weeded plots and plots submitted to chopping only or sown with vetch. moreover, at the time of surveys, no important differences were found between the treatment with the systemic herbicide only and the plot supplied also with the residual herbicide; this is presumably due to the low application rate of oxyfluorfen. in particular, in spring and autumn surveys, the highest total infestation was found in chopped and vetchsown plots. the latter, although covering the whole plot area at spring surveys, has only partially limited the growth of weeds, especially grasses (graminae). the number of species having a uniform distribution in springtime in the plots controlled by chopping or through the sowing of the cover crop rarely exceeded the value observed under different management practices. the number calculated in autumn, instead, was virtually equal for all strategies. the species that showed the highest specific contributions in chemically weeded plots include c. canadensis and m. sylvestris, which might be related to the fact that those species are tolerant to the applied rates of herbicides or maybe, for c. canadensis, resistant to glyphosate (montemurro, 2008; herbicide resistance action committee, 2012). in the two other conditions, in general, no single species was found to be markedly present. the most influenced families in spring surveys were leguminosae and graminae; in particular, the latter seemed to be facilitated by vetch sowing, whereas the former was aided by chopping, conditions that were more evident in spring than in autumn surveys when all differences in general seemed to be less marked. table 9 shoot and trunk growth measured during the trial treatments shoots (cm) trunk diameter (cm) april-october 2006 april-october 2008 april-october 2010 april 2006-october 2010 ve 15.9 14.2 15.0 3.0 gli + ox 13.5 16.6 14.0 3.5 gli 16.4 17.3 13.4 3.9 tri 13.1 16.6 14.8 3.1 table 10 olive production per plant and oil yield production per plant (kg) oil yield (% ) 2006 2008 2010 2006 2008 2010 gli 13.6 11.7 14.8 18.0 19.1 21.6 gli+ox 13.9 10.8 15.0 19.0 20.0 21.7 tri 15.8 11.1 15.6 19.0 19.0 22.3 ve 12.8 10.9 15.1 17.9 20.1 20.9 53 the ground cover quality index calculated in spring 2006 and 2008 was on average higher in the chopped or vetch-sown plots. in 2010 the value calculated in chopped plots was markedly higher than in the other treatments, which, instead, did not show any differences for this parameter. this would suggest a shifting, over time, of vegetation towards a higher quality composition which was more accentuated in the case of the strategy involving chopping only. these effects are well summarized by the applied index, which indicates that for the same weed species assortment influenced the ground cover quality. differences in the flora composition, both quantitatively and qualitatively, did not affect olive yield or vegetation, since weeds were however controlled during the plants’ critical periods. what varied was above all the ground cover features, assessed both from an agronomic and ecological point of view. from our perspective, this feature is well reflected by the applied index, which easily and effectively described the flora features in different plots. in this regard it should be said that this index is obviously influenced by the value attributed to each species that may vary in relation to the objectives of weed management, as previously mentioned. in our case emphasis was placed on competition, protection from erosion and on the capacity to preserve or even increase fertility: these features coincide with the objectives that olive growers normally try to achieve, especially in our areas. in other situations different parameters could be applied, namely by varying the index numerically while still keeping its functional meaning. moreover, in the case under study, the gcqi value was largely influenced by the total ground cover because none of the weeds found in the trial was assigned a zero score (v i ). in the event that undesired species were found in among the cover composition, the index would certainly have been less dependent on total ground cover. finally, since different cover crop and ground flora management practices seemed to give results, in terms of yield, that were not different from each other, a long-term approach could be applied for their selection. currently it seems possible to prefer a ground cover management strategy that enables a sustainable use of olive agro-ecosystems and emphasizes the different roles of wild flora, including landscaping. this keeps in mind the fact that in 2006 the puglia regional government enacted a law regarding the protection and enhancement of monumental olive trees and of the olive agro-ecosystems of its region (l.r. n. 39 del 03/10/2006) and that the location where the trial was conducted falls within the areas of highest density of ancient and traditional olive tree landscapes. the value of this area was also further declared by its inclusion among the “high nature value farmland” areas (european environment agency, 2004). references allen h.d., randall r.e., amable g.s., devereux b.j., 2006 the impact of changing olive cultivation practices on the ground flora of olive groves in the messara and psiloritis regions, crete, greece. land degrad. develop., 17: 249-273. beaufoy g., 2000 the environmental impact of olive oil production in the european union: practical options for improving the environmental impact. report produced by the european forum on nature conservation and pastoralism and the asociación para el análisis y reforma de la política agro-rural. published by 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viggiani p., 2009 la flora spontanea. in: pisante m., p. inglese, and g. lercker l’ulivo e l’olio. coltura & cultura. collana ideata e diretta da angelini renzo. bayer crop science. art servizi editoriali s.p.a., bologna, italy, pp. 784. untitled 110 how information technology can support regulations and best practices for the management of health status of grapevine and product safety a. luvisi department of agriculture, food and environment, university of pisa, via del borghetto, 80, 56124 pisa, italy, aluvisi@agr.unipi.it the need for knowledge about the origins and qualitative characteristics of food products or plants that are commercialized worldwide has increased due to consumer demands. this fact is due in part to recent negative events related to food production and has, in turn, led to stricter regulations to safeguard public health and ecosystems from the spread of pathogens. in an essential step to guarantee quality, beginning in the 1960s the former european economic community was involved in defining legal regulations regarding the health status of grapes and their production (68/93/eec). the regulation of grapevine identity, health and production continued to develop until the last decade in europe (2005/43/ce) and italy (dm 13/12/2011), revealing its importance over the past 50 years. regulations have followed the general trends in agriculture during the past century: not only with regard to new farming approaches and consequential environmental impact, but also the globalized trade of products which poses new challenges to import/ export regulations. food safety, market protection, property rights and ecological conservation are common themes of “global” consumers and governmental agencies. these concepts have been reinforced by the eu through ‘‘the european white paper on food safety”. nowadays, many foods and agricultural products have to carry identifying labels or documents, as required by legal regulations (e.g. 2000/13/ec), to establish a safe traceability system. in the eu, grapevines in the certified category must be in line with the most recent directive (2005/43/ce), and associated labels have to report essential data such as the nursery where they were produced. plant traceability, as in foods, can be supported by information technology (it) and can be considered a best practice in agriculture, as is the case for livestock. the it revolution, exemplified by the internet, has made traceability and monitoring economically feasible and enabled traceability of food products through the labyrinth of the agricultural product supply chain. with regards to food plants, the implementation of it solutions to trace the plant-to-food chain seems to be possible only in fruit trees, including grapevine, due to the difficulties in labeling and/or tracking herbaceous plants. the wine production line is characterized by many effective or potential it innovations as technology has been able to permeate nearly every production step. with regard to the first step (i.e. selection and registration of a grapevine clone), an online database gives breeders, researchers and stakeholders an easy and lasting consulting system to share information regarding available clones. several databases are available in europe, such as the italian “catalogo istituzionale del registro nazionale delle varietà di vite” or “italian vitis data base”, the french “base de données du réseau français des conservatoires de vignes”, the german “the european vitis database” or “vitis international variety catalogue”. as for specific aims, some of these databases are more genetic-oriented than exhaustive digital archives, while others monitor production phases of premultiplication material. in any case, if the knowledge about varieties and clones is well supported by it, the situation changes on farms or in vineyards where few it solutions are available for health and quality management as most of them are in the prototype phase. in contrast to the situation with livestock, where technology plays an important role with electronically labeled and checked animals, farms generally have a low level of computerization, due to both the costs involved and the lack of urgency to shift to a more in-depth traceability system (luvisi et al., 2012). however, available technology can satisfy various needs. radio-frequency identification (rfid) microchips can represent a safe tool to identify plants and foods that are protected by rights or subjected to specific regulations. the initial tests in grapevine by the associazione toscana costitutori viticoli (tos.co.vit.) were carried out in 2006 on clones and involved the use of microchips implanted within the pith of rootstock (bandinelli et al., 2009). this technology, if appropriately supported by information management systems, can support health controls and be a useful tool for managing risks related to environmental impacts of production systems, chemical residues and the worldwide spread of plant pathogens. in certified plant propagation and breeding programs, risk management may be a sufficient reason to change to rfid systems. similar technology can be implemented in order to tracking the application of agrochemicals (peets et al., 2009), the virtualization of vineyards by combining gps technology (luvisi et al., 2011), and the management of widespread monitoring stations using mobile devices adv. hort. sci., 2013 27(3): 110-111 111 (cunha et al., 2010). finally, collaborative web 2.0-based workspaces can be used to support sampling for health checks and the exchange of information between users and laboratories (luvisi et al., 2012). it can also offer real options for wine cellar management and bottling. electronic labeling of wine for high value products using rfid systems and for the fight against forgery are principal areas for application, but do not involve cross reference to information about plant health or identity or the previously mentioned databases. in conclusion, even if it solutions can support management procedures with regard to the spread of pathogens in plant material and fight forgery, much still has to be done in order to create a virtual environment for grape and wine production, changing this fragmented agricultural “internet of things” into a coherent “internet of trees”, in which regulations and best practices may converge in harmonized electronic labeling and databases, without losing the link between plants and food. indeed, the relationship between plants and food is not just a simple question of input/output, but rather a complex system in which plant pathogens and their control play an important role. this link is promoted by the european food safety authority (efsa), the agency that provides scientific advice and communication on existing and emerging risks associated with the food chain and the authority’s work covers all matters with a direct or indirect impact on food safety, including plant protection and plant health as included in the general objective and mission of the efsa. references bandinelli r., triolo e., luvisi a., pagano m., gini b., rinaldelli e., 2009 employment of radiofrequency technology (rfid) in grapevine nursery traceability. adv. hort. sci., 23(2): 75-80. cunha c.r., peres e., morais r., oliveira a.a., matos s.g., fernandes m.a., ferreira p.j.s.g., reis m.j.c.s., 2010 the use of mobile devices with multi-tag technologies for an overall contextualized vineyard management. computers and electronics in agriculture, 73: 154-164. luvisi a., pagano m., bandinelli r., rinaldelli e., gini b., scartòn m., manzoni g., triolo e., 2011 virtual vineyard for grapevine management purposes: a rfid/gps application. computers and electronics in agriculture, 75: 368-371. luvisi a., panattoni a., triolo e., 2012 electronic identification-based web 2.0 application for plant pathology purposes. computers and electronics in agriculture, 84: 7-15. peets s., gasparin c.p, blackburn d.w.k., godwin r.j., 2009 rfid tags for identifying and verifying agrochemicals in food traceability systems. precision agriculture, 10: 382-394. impaginato 215 adv. hort. sci., 2025 39(3): 215­230 doi: 10.36253/ahsc­17150 https://oaj.fupress.net/index.php/ahs improving drought tolerance of leucophyllum frutescens through the application of paclobutrazol and cycocel plant growth regulators m.a. el­ashwah, t.m. noor­el­deen*, w.m. bazaraa ornamental plants and landscape gardening research department, horticulture research institute, agricultural research center, giza, egypt. key words: cycocel, paclobutrazol, stress indices, texas­sage, water stress. abstract: leucophyllum frutescens is an evergreen shrub renowned for its drought tolerance. paclobutrazol (pbz) and cycocel (ccc) growth retardants were applied as foliar spray on l. frutescens plants cultivated in pots to assay the possibility of increasing these plants’ tolerance to water stress while maintaining high quality. the experiment was accomplished under full sun in the open field of the nursery of ornamental plants and landscape gardening res. dept., hort. res. inst., arc, egypt during the 2022 and 2023 seasons. different concentrations of pbz at 50, 100 and 150 ppm and ccc at 1000, 2000 and 3000 ppm were combined with 100, 75 and 50% pot capacity (p.c.) irrigation water levels, and some morphological, chemical and tolerance indices were examined. the obtained results showed a great reduction in all studied parameters except for proline content due to reduced irrigation levels. all growth retardants applied increased the values over control except for plant height and fresh weight of 10 flowers. regarding the interaction treatments, the highest concentration of pbz (150 ppm) and ccc (3000) produced the highest values in most cases when combined with both 75 and 50% irrigation water levels. such treatments increased the number of main and lateral branches/plant, fresh and dry weights of vegetative growth and roots and root length, while mediated values were obtained for chemical constituents. water use efficiency, relative stress index and stress tolerance index are also, greatly enhanced by such treatments. cycocel at 3000 ppm could be recommended to treat leucophyllum frutescens plants cultivated in 30 cm pots and subjected to only 50% irrigation water level as this treatment demonstrated good performance with high stress tolerance index to drought. 1. introduction leucophyllum frutescens (syn., leucophyllum texanum) is native to southwestern texas and northeastern mexico so it is known as texas (*) corresponding author: taiar2005@yahoo.com citation: el­ashwah m.a., noor­el­deen t.m., bazaraa w.m., 2025 ­ improving drought tolerance of leucophyllum frutescens through the application of paclobutrazol and cycocel plant growth regulators. ­ adv. hort. sci., 39(3): 215­230. orcid: ema: 0009­0002­9214­9680 ntm: 0000­0002­9438­8995 bwm: 0009­0000­8926­2872 copyright: © 2025 el­ashwah m.a., noor­el­deen t.m., bazaraa w.m. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 11 january 2025 accepted for publication 22 september 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) https://doi.org/10.36253/ahsc-17150 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0002-9214-9680 http://orcid.org/0000-0002-9438-8995 http://orcid.org/0009-0000-8926-2872 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 215­230 216 ranger or texas­sage. it belongs to the family scrophulariaceae and is considered the largest species in the genus leucophyllum. the plant is an evergreen shrub with silver­grey foliage that reaches 2.0 meters in height and 2.5 meters in spread. the foliage is soft to the touch, and densely clustered along spreading branches. the leaves are small measuring 2.50 × 1.25 cm and broaden at the tip. the flowers are bell­shaped and range from white to pink to purple with up to 2.5 cm long. the plants bloom well in summer when the temperature and humidity are high. texas ranger is a popular choice for hedges, specimens, visual screening and wind control and could certainly be used as an accent plant (mielke, 1993). leucophyllum frutescens has recently been cultivated extensively in egypt due to its reputation for drought and salinity tolerance, although more research is needed to substantiate this. younis et al. (2017) found that, compared to other landscape plants, l. frutescens shows strong potential as it requires minimal water. in this context, ashour and el­attar (2017) concluded that l. frutescens can be irrigated with tap water every 10 days, and saline water at 2000 ppm can be used every 4 days without an obvious reduction in plant growth and quality. water insufficiency is a serious challenge affecting many countries worldwide. to address this issue the strategy of minimizing water use could be adopted, particularly in areas where it is not essential, such as the irrigation of green open spaces and landscape plants. deister (2013) reported that, in egypt, open and privately owned areas consume very high amounts of irrigation water (4.76­7.14 l/m²/day and 7.14 ­11.9 l/m²/day, respectively) compared with other countries. to reduce the water consumption for open areas and landscape plants, selecting species with low water requirements or increasing the tolerance of the cultured species by different available means is required. research has demonstrated that plant growth regulators (pgrs), particularly growth­retarding types, can significantly improve drought tolerance in various plant species through multiple physiological mechanisms. these compounds help plants withstand water deficit conditions by: (1) strengthening antioxidant defenses against oxidative stress caused by drought (abbasi et al., 2015; singh et al., 2016 ); (2) controlling stomatal function (marshall et al., 1991; dehghanzadeh and adavi, 2023); (3) facil itating osmotic adjustment via accumulation of protective compounds like proline (waqas et al., 2017) and (4) improving water acquisition through enhanced root growth relative to shoot biomass (fletcher et al., 2000). paclobutrazol (pbz) inhibits gibberellin biosynthesis and subsequently limits stem elongation (tesfahun, 2018). pbz has been shown to help safeguard various crops against a range of environmental stresses, such as drought. paclobutrazol (pbz) has proven effective for drought mitigation in plants across multiple studies: robinia pseudoacacia (thakur et al., 2000); hybrid poplar and birch (zhongzhu and xiangwei, 2006); lonicera implexa (navarro et al., 2008); bougainvillea spp. (ting et al., 2014); odontonema strictum (rezazadeh et al., 2016); petunia (hatamifar and samani, 2017); sequoia sempervirens (shu­ming et al., 2020); and salvia officinalis (maghsoudi et al., 2023). cycocel (ccc), a plant growth regulator valued for its low toxicity and broad crop effectiveness, can be absorbed by both roots and leaves. cycocel is used to restrict plant growth while enhancing the harvest index through increased enzymatic activity, leaf thickness, pigment levels, and assimilation efficiency. spraying cotton with 500 ppm of ccc resulted in higher chlorophyll and relative water content, along with improved nutrient uptake and a reduction in leaf size (dhopte and lall, 1987). drought­protective effects of ccc have been documented in: sunflower (kumari and bharti, 1992); groundnut (mathew and pandey, 2006); barley (sharif et al., 2007; afkari and ghaffari, 2018); japanese mint (mathur and farooqi, 2009); olive (memari et al., 2011; akbari et al., 2015); safflower (partovian et al., 2013); basil (estakhroeih and babaei, 2016); chickpea (safari and azadikhah, 2021); and wheat (dehghanzadeh and adavi, 2023). this study hypothesized that foliar application of paclobutrazol (pbz) and cycocel (ccc) at optimized concentrations would enhance water stress tolerance in potted leucophyllum frutescens, while preserving morphological and physiological quality markers. this improvement is anticipated to be evident across varying irrigation regimes, with pbz and ccc mitigating drought­induced damage through growth regulation and osmotic adjustment mechanisms. 2. materials and methods the experiment was accomplished on leucophyllum frutescens (berland.) i.m. johnst. shrubs in the open field under full sun at the nursery el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 217 experimental layout a factorial experiment with two factors in a randomized complete block design (rcbd) as reported by gomez and gomez (1984) was utilized to lay out this pot experiment. three irrigation levels at 100, 75 and 50% of pot capacity were represented as factor a, while factor b included seven foliar spray treatments, three concentrations of paclobutrazol (pbz: 50, 100, and 150 ppm), three concentrations of cycocel (ccc: 1000, 2000, and 3000 ppm), and an untreated control (0 ppm pbz or ccc). consequently, the present experiment contained 21 treatments (3 irrigation levels × 7 foliar spraying with growth retardants), each treatment was replicated three times, and each replicate contained 5 pots. irrigation water treatments different water irrigation levels were applied one month after transplantation (on the first week of april each season) as a percentage (100, 75, 50%) of pot capacity (the equivalent of field capacity for soils in pots). the weighing method described by brown of ornamental plants and landscape gardening res. dept., hort. res. inst., arc, egypt, during the spring to autumn of 2022 and 2023 seasons to assay the possibility of increasing these plants’ tolerance to water stress by applying foliar spraying with paclobutrazol (pbz) and cycocel (ccc) at different concentrations under different irrigation water levels. plant material in the first week of march each season, well­ developed healthy transplants of l. frutescens (30­35 cm height and 3 main branches) were acquired from a private nursery at shibin el qanater, qalyubia governorate, egypt. the transplants were planted in 30­diameter plastic pots (1 plant/pot) filled with 5.5 kg loamy soil. the analysis of the experiment’s growing medium and meteorological parameters are shown in tables 1 and 2. for complete establishment, the plants were left for about one month at the nursery to supply them with the original water amount and required maintenance. table 1 ­ physical and chemical analyses of the used soil soil type particle size distribution (%) s.p. (%) e.c. (ds/m) ph cations (meq/l) anions (meq/l) sand silt clay ca++ mg++ na+ k+ hco3 cl­ so4 ­­ loamy 48.0 35.5 16.5 44.0 1.36 8.28 3.5 2.5 6.63 0.65 0.5 7.5 5.28 table 2 ­ some meteorological parameters at giza governorate during the experiment period of 2022 and 2023 seasons rh= relative humidity at 2 meters; ws= wind speed at 2 meters; precip.: precipitation. power data access viewer program of nasa was utilized to collect daily data, and then the data were averaged monthly to fit the experiment period (https://power.larc.nasa.gov). months 2022 2023 temperature (°c) r.h. (%) w.s. (m/s) precip. (mm/d) temperature (°c) r.h. (%) w.s. (m/s) precip. (mm/d)avg. max. min. avg. max. min. march 13.39 28.98 2.40 59.98 3.02 1.30 17.46 32.65 6.97 54.11 2.67 0.19 april 21.82 40.00 8.51 44.97 3.20 0.00 21.03 36.58 8.64 45.51 2.88 0.10 may 25.06 41.25 11.75 39.75 3.37 0.00 25.08 38.39 11.69 42.51 3.43 0.03 june 29.24 44.30 17.36 42.11 3.24 0.00 29.30 42.56 18.42 39.58 3.50 0.04 july 29.91 40.61 19.26 42.45 3.02 0.06 31.52 43.89 20.49 40.54 3.23 0.00 august 30.32 41.90 21.25 46.09 3.06 0.06 30.86 43.53 20.66 44.56 3.21 0.00 septemb 28.43 39.48 18.87 49.15 3.12 0.13 29.62 43.48 19.82 45.29 2.87 0.00 october 23.80 39.56 15.76 57.68 2.72 0.18 25.10 35.81 17.19 58.10 2.59 1.71 s.p. = saturation percentage; e.c. = electrical conductivity. adv. hort. sci., 2025 39(3): 215­230 218 (2002) was adopted to determine water pot capacity. the plants were irrigated three times a week. the amount of water applied/pot/irrigation and the total water applied/pot/season (6 months/season) is shown in table 3. foliar spraying with growth retardants both paclobutrazol (pbz) and cycocel (ccc) were dissolved in distilled water to present solutions with the applied concentrations (50, 100 and 150 ppm for pbz and 1000, 2000 and 3000 ppm for ccc). in this study, plants that did not receive foliar spray application of growth retardants served as the control treatment. plants received six foliar applications of pbz or ccc (at specified concentrations) at monthly intervals, with each treatment applied to runoff (defined as the first appearance of solution droplets from leaf margins, indicating full surface coverage and absorption saturation). the first application was done in the first week of april each season. paclobutrazol (c15h20cln3o, mw: 293.80) and cycocel (chlormequat chloride; c5h13cl2n, mw: 158.10) were obtained from a local company in egypt. data recorded at the beginning of october each season the following data and measurements were done: vegetative growth and flowers. plant height (cm), number of main branches/plant (emerged on main stems), number of lateral branches/plant (emerged on main branches), vegetative growth fresh and dry weights (g) and fresh weight of 10 flowers (g). root system parameters. at the end of this study, plants were extracted by tilting the pots and tapping the bases until the root balls released. roots were cleaned through sequential shaking, low­pressure washing, and soft brushing to remove all soil while preserving root architecture. the following parameters were recorded on the clean extracted roots: root system length (cm) and fresh and dry weights of the root system (g). chemical composition analysis. chlorophylls a+b (mg/g f.w.) and carotenoids (mg/g f.w.) in fresh leaves according to wellburn and lichtenthaler (1984), total carbohydrates (%) in dry leaves according to herbert et al. (1971), proline (mg/g d.w.) in dry leaves according to bates et al. (1973). these determinations were done in the second season only. water use efficiency and stress indices. water use efficiency (wue) g/l according to karkanis et al. (2011), relative stress index (rsi) according to fischer and wood (1979), and stress tolerance index (sti) according to fernandez (1993). the following equations were adopted based on the aerial parts dry mass of plants subjected to different treatments: wue (g/l) = d/a. rsi = (dp/ds)/(xs/xp) sti = (dp × ds)/xp2 where d: dry mass of aerial parts (g) whether under normal or water stress conditions, a: amount of water applied per pot (l) during the experiment, dp: dry mass of aerial parts of plants of each treatment under normal water conditions, ds: dry mass of aerial parts of plants of each treatment under water stress conditions, xp: mean dry mass of aerial parts for all plants under normal conditions and xs: mean dry mass of aerial parts for all plants under stress conditions. statistical analysis mstat computer program (mstat, 1989) was used to statistically analyze the obtained data by applying an anova test for the two­factor experiment in rcbd (gomez and gomez, 1984). the means were compared using duncan’s multiple range test as described by (duncan, 1955). 3. results vegetative growth and flower parameters: different irrigation water levels showed a significant influence (tables 4 and 5). reducing water levels gradually caused a reduction in all studied vegetative growth and flowers. the lowest values were obtained by the lowest irrigation water level for plant height (86.00 and 77.05 cm), number of main branches/plant (12.86 and 15.62), number of lateral table 3 ­ amount of water applied for each pot (l/pot) irrigation level (% of pot capacity) for each irrigation (l/pot) total water applied each season (l/pot) 100% 0.820 59.040 75% 0.615 44.280 50% 0.410 29.520 el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 219 branches/plant (33.05 and 37.48), vegetative growth fresh weight (131.50 and 139.20 g), vegetative growth dry weight (32.09 and 35.83 g) and fresh weight of 10 flowers (1.08 and 1.19 g) in both seasons, respectively. foliar spraying with paclobutrazol and cycocel at various concentrations generally reduced plant height, except for paclobutrazol at 50 ppm, which unexpectedly insignificantly increased plant height in the first season (117.80 cm) compared to the control untreated plants (115.90 cm). notably, cycocel at 3000 ppm resulted in the lowest plant height values for both seasons, measuring 83.33 and 84.33 cm, respectively. regarding other vegetative growth characteristics, cycocel at 3000 ppm, followed by paclobutrazol at 150 ppm, resulted in the highest table 4 ­ effect of water irrigation levels and two plant growth retardants on plant height (cm), number of main branches/plant and number of lateral branches/plant of leucophyllum frutescens during two seasons of 2022 and 2023 treatments plant height (cm) number of main branches/plant number of lateral branches/plant 1st season 2nd season 1st season 2nd season 1st season 2nd season water irrigation levels 100% p.c. 117.30 a 114.60 a 16.00 a 19.24 a 47.90 a 52.90 a 75% p.c. 100.30 b 90.50 b 14.33 b 17.00 b 41.29 b 45.81 b 50% p.c. 86.00 c 77.05 c 12.86 c 15.62 c 33.05 c 37.48 c growth retardants control 115.90 a 110.30 a 6.78 f 8.67 f 19.67 f 20.89 e pbz 50 ppm 117.80 a 100.70 b 10.33 e 13.22 e 30.33 d 31.78 d pbz 100 ppm 102.30 c 94.33 c 15.44 c 16.67 c 45.67 c 52.44 c pbz 150 ppm 94.44 d 91.89 d 19.67 a 23.22 a 54.33 b 61.33 b ccc 1000 ppm 108.40 b 90.00 e 12.33 d 14.67 d 26.67 e 31.56 d ccc 2000 ppm 88.22 e 86.78 f 16.78 b 20.78 b 47.11 c 51.22 c ccc 3000 ppm 81.33 f 84.33 g 19.44 a 23.78 a 61.44 a 68.56 a p.c. = pot capacity. means within a season followed by the same letter are not significantly different (p>0.05) according to duncan (1955). table 5 ­ effect of water irrigation levels and two plant growth retardants on vegetative growth fresh weight (g), vegetative growth dry weight (g) and fresh weight of 10 flowers (g) of leucophyllum frutescens during two seasons of 2022 and 2023 p.c. = pot capacity. means within a season followed by the same letter are not significantly different (p>0.05) according to duncan (1955). treatments vegetative growth fresh weight vegetative growth dry weight (g) fresh weight of 10 flowers (g) 1st season 2nd season 1st season 2nd season 1st season 2nd season water irrigation levels 100% p.c. 194.10 a 206.10 a 50.83 a 58.36 a 1.36 a 1.51 a 75% p.c. 159.70 b 172.80 b 42.64 b 47.08 b 1.24 b 1.35 b 50% p.c. 131.50 c 139.20 c 32.09 c 35.83 c 1.08 c 1.19 c growth retardants control 99.98 e 109.20 f 24.78 f 27.04 g 1.08 d 1.22 d pbz 50 ppm 125.40 d 138.00 e 33.19 d 38.54 e 1.09 d 1.23 d pbz 100 ppm 166.40 c 179.50 d 44.02 c 51.66 d 1.22 c 1.31 c pbz 150 ppm 204.10 ab 213.70 b 50.28 b 58.11 b 1.30 b 1.42 ab ccc 1000 ppm 127.40 d 138.60 e 29.97 e 34.35 f 1.28 b 1.37 bc ccc 2000 ppm 199.80 b 208.80 c 49.83 b 55.58 c 1.28 b 1.40 b ccc 3000 ppm 209.40 a 221.20 a 60.93 a 64.33 a 1.35 a 1.48 a 220 adv. hort. sci., 2025 39(3): 215­230 values. however, an exception occurred with paclobutrazol at 150 ppm, which produced the highest number of main branches per plant (19.67) in the first season only, surpassing cycocel at 3000 ppm (19.44 main branches/plant). the highest values obtained by foliar spraying with cycocel at 3000 ppm were 23.78 for number of main branches/plant (in the second season only), then 61.44 and 68.56 for number of lateral branches/plant, 209.40 and 221.20 g for vegetative growth fresh weight, 60.93 and 64.33 g for vegetative growth dry weight and 1.35 and 1.48 g for fresh weight of 10 flowers in both seasons, respectively (tables 4 and 5). regarding the interaction between irrigation water levels and plant growth retardants, there was a great influence on vegetative growth and flowering traits (tables 6 and 7). the different treatments greatly influenced plant height. the tallest plants were obtained either by the control (without foliar spraying with growth retardants) or with paclobutrazol at 50 ppm, when both of them were combined with the 100% irrigation water level. in the first season, these two combined treatments resulted in plant heights of 140.30 and 136.70 cm, respectively, while in the second season, the heights were 150.30 and 127.80 cm, respectively. the shortest plants were obtained by cycocel at 2000 ppm (71.00 and 71.50 cm in both seasons, respectively) and cycocel at 3000 ppm (72.00 and 96.17 cm in both seasons, respectively) when combined with the lowest water level (50%). despite plant height, cycocel at 3000 ppm resulted in the highest values on the other characteristics when combined with the irrigation water level of 100% table 6 ­ effect of interaction between water irrigation levels and two plant growth retardants on plant height (cm), number of main branches/plant and number of lateral branches/plant of leucophyllum frutescens during two seasons of 2022 and 2023 p.c. = pot capacity. within each season, values sharing the same letter across water levels and growth retardant treatments indicate no significant difference growth retardants water irrigation levels 100% p.c. 75% p.c. 50% p.c. 100% p.c. 75% p.c. 50% p.c. 1st season 2nd season plant height (cm) control 140.30 a 115.70 cd 91.63 hi 150.30 a 98.17 f 82.33 kl pbz 50 ppm 136.70 a 114.00 cd 102.70 f 127.80 b 93.67 g 80.67 lm pbz 100 ppm 117.70 bc 100.30 fg 89.00 h­j 110.50 c 92.00 gh 80.50 lm pbz 150 ppm 110.00 de 93.00 hi 80.33 k 106.00 d 90.50 hi 79.17 m ccc 1000 ppm 123.00 b 107.00 ef 95.33 gh 105.00 d 89.00 ij 76.00 n ccc 2000 ppm 105.30 ef 88.33 ij 71.00 l 102.20 e 86.67 j 71.50 o ccc 3000 ppm 88.00 ij 84.00 jk 72.00 l 100.30 ef 83.50 k 69.17 o number of main branches/plant control 7.67 k 6.67 k 6.00 k 9.33 j 9.00 jk 7.67 k pbz 50 ppm 14.00 gh 9.67 j 7.33 k 16.00 gh 12.33 i 11.33 i pbz 100 ppm 17.67 de 15.33 fg 13.33 h 20.67 ef 14.67 h 14.67 h pbz 150 ppm 20.33 a 19.67 a­c 19.00 a­d 24.33 ab 23.33 b­d 22.00 de ccc 1000 ppm 13.67 gh 12.33 hi 11.00 ij 16.67 g 16.00 gh 11.33 i ccc 2000 ppm 18.33 b­e 16.67 ef 15.33 fg 22.67 cd 20.00 f 19.67 f ccc 3000 ppm 20.33 a 20.00 ab 18.00 c­e 25.00 a 23.67 a­c 22.67 cd number of lateral branches/plant control 29.33 kl 18.00 o 11.67 p 27.33 hi 23.00 i 12.33 j pbz 50 ppm 39.00 hi 33.00 jk 19.00 no 40.33 fg 33.67 gh 21.33 i pbz 100 ppm 57.00 cd 45.33 g 34.67 ij 64.33 bc 50.00 d 43.00 ef pbz 150 ppm 62.33 ab 58.00 b­d 42.67 gh 69.67 b 64.67 bc 49.67 de ccc 1000 ppm 29.67 j­l 27.00 lm 23.33 mn 34.67 g 33.67 gh 26.33 i ccc 2000 ppm 50.67 ef 46.00 fg 44.67 g 53.33 d 52.67 d 47.67 de ccc 3000 ppm 67.33 a 61.67 bc 55.33 de 80.67 a 63.00 bc 62.00 c el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 221 table 7 ­ effect of interaction between water irrigation levels and two plant growth retardants on vegetative growth fresh weight (g), vegetative growth dry weight (g) and fresh weight of 10 flowers (g) of leucophyllum frutescens during two seasons of 2022 and 2023 growth retardants water irrigation levels 100% p.c. 75% p.c. 50% p.c. 100% p.c. 75% p.c. 50% p.c. 1st season 2nd season vegetative growth fresh weight (g) control 134.40 g 100.90 h 64.70 j 141.60 h 112.20 i 73.72 k pbz 50 ppm 165.50 f 130.70 g 80.14 i 180.40 f 139.40 h 94.35 j pbz 100 ppm 196.00 cd 172.20 f 131.20 g 221.20 cd 174.80 f 142.70 h pbz 150 ppm 234.60 b 205.60 c 172.10 f 250.30 b 228.40 c 162.50 g ccc 1000 ppm 128.30 g 127.20 g 126.70 g 141.00 h 137.40 h 137.30 h ccc 2000 ppm 241.70 b 187.80 de 169.90 f 242.70 b 202.00 e 181.90 f ccc 3000 ppm 258.50 a 193.90 cd 175.80 ef 265.80 a 215.60 d 182.30 f vegetative growth dry weight (g) control 32.10 i 22.63 lm 19.62 m 34.21 ij 24.36 l 22.54 l pbz 50 ppm 50.73 ef 29.95 ij 18.90 m 58.40 d 32.68 ij 24.55 kl pbz 100 ppm 52.53 d­f 48.37 f 31.15 ij 66.15 bc 53.11 ef 35.74 i pbz 150 ppm 57.62 c 56.00 cd 37.22 h 68.35 b 64.89 bc 41.08 h ccc 1000 ppm 37.90 h 27.07 jk 24.94 kl 43.39 h 31.63 j 28.03 k ccc 2000 ppm 55.05 c­e 51.97 d­f 42.48 g 62.78 c 56.63 de 47.32 g ccc 3000 ppm 69.90 a 62.53 b 50.35 f 75.24 a 66.22 bc 51.52 f fresh weight of 10 flowers (g) control 1.28 e­g 1.07 l 0.89 m 1.48 bc 1.18 ij 0.99 k pbz 50 ppm 1.25 f­h 1.10 kl 0.93 m 1.39 c­f 1.23 g­i 1.07 jk pbz 100 ppm 1.33 de 1.21 g­i 1.13 j­l 1.41 b­e 1.32 d­h 1.21 hi pbz 150 ppm 1.36 b­d 1.34 c­e 1.19 h­j 1.53 ab 1.43 b­e 1.30 e­i ccc 1000 ppm 1.39 b­d 1.25 f­h 1.19 h­j 1.52 bc 1.35 d­g 1.25 g­i ccc 2000 ppm 1.42 b 1.32 d­f 1.08 kl 1.54 ab 1.44 b­d 1.22 g­i ccc 3000 ppm 1.50 a 1.41 bc 1.15 i­k 1.66 a 1.52 b 1.27 f­i p.c. = pot capacity. within each season, values sharing the same letter across water levels and growth retardant treatments indicate no significant difference (p>0.05) according to duncan's multiple range test (duncan, 1955). p.c., this was followed significantly or insignificantly with other treatments e.g. cycocel at 3000 ppm + 75% water level, paclobutrazol at 150 ppm + 100% water level and paclobutrazol at 150 ppm + 75% water level. in this regard, cycocel at 3000 ppm + 75% water level resulted in intermediate values 20.00 and 23.67 for number of main branches/plant, 61.67 and 63.00 for number of lateral branches/ plant, 193.90 and 215.60 g for vegetative growth fresh weight, 62.53 and 66.22 g for vegetative growth dry weight and 1.41 and 1.52 g for fresh weight of 10 flowers in both seasons, respectively. the lowest values (except for plant height) were obtained by the lowest irrigation water level (50%) when the plants were deprived of growth retardants or sprayed with paclobutrazol at 50 ppm. although plants treated with the highest concentrations of pbz (150 ppm) and ccc (3000 ppm) under the lowest irrigation level (50%) did not achieve the highest values for vegetative growth and flower parameters, these values were still relatively high compared to plants exposed to the three irrigation levels without foliar application of pbz and ccc, except for plant height and the fresh weight of 10 flowers. root system parameters table 8 showed that both irrigation levels at 100 and 75% resulted in the longest roots without significant differences between them (48.88 and 55.79 cm for 100% and 47.74 and 55.91 cm for 75%, in both seasons, respectively). the highest water level (100%) produced the highest fresh weight of root system (47.40 and 54.83 g) and dry weight of root system (16.30 and 17.77 g) in both seasons, respectively. the lowest irrigation water level (50%) the lowest values were recorded. as shown in table 8, cycocel at 3000 ppm followed significantly by paclobutrazol at 150 ppm produced the highest values in terms of root system characteristics (62.74 and 73.89 cm for root system length, 62.47 and 72.98 g for fresh weight of root system and 23.52 and 24.55 g for dry weight of root system, in the first and second seasons, respectively). control plants (deprived of plant growth retardants) recorded the lowest values for all studied root system characteristics. data presented in table 9 cleared that there were no significant differences between foliar spraying with cycocel at 3000 ppm when combined with the highest irrigation water level (100%) and the medium one (75%) in case of root length (63.53 and 62.70 cm in the first season and 74.23 and 74.17 cm in the second one for these two treatments, respectively), fresh weight of root system (65.95 and 62.69 g in the first season for these two treatments, respectively) and dry weight of root system (24.08 and 23.81 g in the first season, 25.69 and 25.62 g in the second season for these two treatments, respectively), while a significant difference was observed between these two combined treatments in the second season only in the case of fresh weight of root system (76.30 and 71.90 g, for cycocel at 3000 ppm + 100% water level and cycocel at 3000 ppm + 75% water level, respectively). although plants treated with the adv. hort. sci., 2025 39(3): 215­230 222 highest concentrations of pbz (150 ppm) and ccc (3000 ppm) under the lowest irrigation level (50%) did not achieve the highest values for root system parameters, these values were still relatively high compared to plants exposed to the three irrigation levels without foliar application of pbz and ccc. chemical composition analysis the highest level of irrigation water (100%) as shown in table 10 resulted in the highest values in case of chlorophylls a+b (1.88 mg/g f.w.), carotenoids (0.51 mg/g f.w.) and total carbohydrates (27.14%) and the lowest value of proline (0.97 mg/g d.w.). the highest proline content was obtained by water irrigation level of 50% (1.70 mg/g d.w.). the two growth retardants also had a significant, though varying, impact on the biochemical variable evaluated in this study (table 10). cycocel at 3000 ppm seems to be more effective than other treatments on chlorophylls a+b, total carbohydrates and proline as produced the highest values (1.98 mg/g f.w., 26.75% and 1.62 mg/g d.w., respectively). the same treatment recorded the lowest carotenoids content (0.41 mg/g f.w.). control plants deprived of spraying with growth retardants produced the highest value for carotenoids and the lowest values in terms of chlorophylls a+b (1.38 mg/g f.w.), total carbohydrates (21.30%) and proline (1.08 mg/g d.w.). regarding the combined treatment effects on chlorophyll (a + b) content, the highest concentration (2.18 mg/g fresh weight) was table 8 ­ effect of water irrigation levels and two plant growth retardants on root system length (cm), root system fresh weight (g) and root system dry weight (g) of leucophyllum frutescens during two seasons of 2022 and 2023 p.c. = pot capacity. means within a season followed by the same letter are not significantly different (p>0.05) according to duncan (1955). treatments root system length (cm) root system fresh weight (g) root system dry weight (g) 1st season 2nd season 1st season 2nd season 1st season 2nd season water irrigation levels 100% p.c. 48.88 a 55.79 a 47.40 a 54.83 a 16.30 a 17.77 a 75% p.c. 47.74 a 55.91 a 42.58 b 47.56 b 14.74 b 16.01 b 50% p.c. 43.49 b 49.18 b 36.85 c 43.13 c 12.02 c 13.45 c growth retardants control 33.71 e 42.40 d 23.70 f 29.77 f 7.92 f 10.06 d pbz 50 ppm 39.69 d 44.57 d 30.68 e 36.00 e 9.28 e 10.13 d pbz 100 ppm 46.21 c 53.02 c 38.93 d 45.43 c 13.00 c 13.98 c pbz 150 ppm 53.97 b 60.00 b 55.21 b 59.08 b 18.17 b 19.59 b ccc 1000 ppm 38.99 d 43.32 d 32.19 e 38.73 d 10.88 d 12.47 c ccc 2000 ppm 51.61 b 58.19 b 52.77 c 57.56 b 17.73 b 19.44 b ccc 3000 ppm 62.74 a 73.89 a 62.47 a 72.98 a 23.52 a 24.55 a el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 223 table 9 ­ effect of interaction between water irrigation levels and two plant growth retardants on root system length (cm), root system fresh weight (g) and root system dry weight (g) of leucophyllum frutescens during two seasons of 2022 and 2023 water irrigation levels growth retardants 100% p.c. 75% p.c. 50% p.c. 100% p.c. 75% p.c. 50% p.c. 1st season 2nd season root system length (cm) control 38.63 h­j 35.43 j 27.07 k 44.50 ij 42.13 i­k 40.57 j­l pbz 50 ppm 42.67 gh 41.90 g­i 34.50 j 49.97 fg 45.33 hi 38.40 kl pbz 100 ppm 47.63 d­f 47.00 ef 44.00 fg 53.17 ef 55.00 e 50.90 fg pbz 150 ppm 55.97 b 56.03 b 49.90 c­e 59.27 cd 65.20 b 55.53 de ccc 1000 ppm 41.93 g­i 37.93 ij 37.10 j 48.80 gh 44.37 ij 36.80 l ccc 2000 ppm 51.80 b­d 53.17 bc 49.87 c­e 60.57 c 65.20 b 48.80 gh ccc 3000 ppm 63.53 a 62.70 a 62.00 a 74.23 a 74.17 a 73.27 a root system fresh weight (g) control 27.17 j 26.11 j 17.82 k 33.35 hi 33.23 hi 22.73 j pbz 50 ppm 39.21 gh 26.71 j 26.11 j 43.72 f 33.23 hi 31.04 i pbz 100 ppm 43.55 f 41.73 fg 31.51 i 52.07 e 43.85 f 40.37 fg pbz 150 ppm 63.80 a 56.27 cd 45.55 f 69.30 b 56.93 d 51.00 e ccc 1000 ppm 36.60 h 31.81 i 28.15 ij 43.83 f 36.58 gh 35.77 h ccc 2000 ppm 55.53 cd 52.70 de 50.07 e 65.23 c 57.17 d 50.28 e ccc 3000 ppm 65.95 a 62.69 ab 58.77 bc 76.30 a 71.90 b 70.73 b root system dry weight (g) control 9.04 jk 8.09 kl 6.62 l 11.58 g­i 9.56 h­j 9.05 ij pbz 50 ppm 13.33 gh 8.04 kl 6.45 l 14.05 fg 8.42 j 7.91 j pbz 100 ppm 15.68 f 15.17 fg 8.16 kl 17.16 de 16.39 ef 8.39 j pbz 150 ppm 20.63 bc 19.11 cd 14.75 fg 22.09 b 20.23 bc 16.44 ef ccc 1000 ppm 12.40 hi 10.88 ij 9.35 jk 13.48 g 12.25 gh 11.66 g­i ccc 2000 ppm 18.93 cd 18.11 de 16.15 ef 20.35 bc 19.63 b­d 18.34 c­e ccc 3000 ppm 24.08 a 23.81 a 22.67 ab 25.69 a 25.62 a 22.35 b p.c. = pot capacity. means followed by the same letter are not significantly different (p>0.05) according to duncan (1955). p.c. = pot capacity. within each season, values sharing the same letter across water levels and growth retardant treatments indicate no significant difference (p>0.05) according to duncan's multiple range test (duncan, 1955). treatments chlorophylls a+b (mg/g f.w.) carotenoids (mg/g f.w.) total carbohydrates (%) proline (mg/g d.w.) water irrigation levels 100% p.c. 1.88 a 0.51 a 27.14 a 0.97 c 75% p.c. 1.74 b 0.39 c 23.82 b 1.24 b 50% p.c. 1.52 c 0.49 b 19.15 c 1.70 a growth retardants control 1.38 g 0.51 a 21.30 e 1.08 g pbz 50 ppm 1.58 f 0.49 b 22.00 d 1.13 f pbz 100 ppm 1.68 e 0.49 b 22.82 c 1.26 d pbz 150 ppm 1.80 c 0.46 c 24.21 b 1.39 c ccc 1000 ppm 1.73 d 0.45 c 22.58 cd 1.24 e ccc 2000 ppm 1.85 b 0.44 d 23.92 b 1.42 b ccc 3000 ppm 1.98 a 0.41 e 26.75 a 1.62 a table 10 ­ effect of water irrigation levels and two plant growth retardants on chemical composition analysis of leucophyllum frutescens during the second season 2023 adv. hort. sci., 2025 39(3): 215­230 224 observed under the 3000 ppm cycocel treatment with 100% irrigation. this was followed by the 2000 ppm cycocel + 100% water level treatment (2.02 mg/g fresh weight). total chlorophyll concentrations were relatively lower with both paclobutrazol at 150 ppm + water level at 100% and cycocel at 3000 ppm + water level at 75%, without significant differences between them. these four treatments also yielded the highest total carbohydrate contents: cycocel at 3000 ppm + 100% water level (31.74%), cycocel at 2000 ppm + 100% water level (27.42%), paclobutrazol at 150 ppm + 100% water level (28.14%), and cycocel 3000 ppm + 75% water level (26.37%). the highest carotenoid concentration (0.58 mg/g f.w.) was observed in control plants at 50% irrigation, with progressively lower values under paclobutrazol treatments at 100 ppm (0.57 mg/g f.w.) and 150 ppm (0.56 mg/g f.w.) combined with full irrigation (100% water level). data presented in table 11 demonstrated that proline was enhanced by cycocel at 3000 ppm when combined with the lowest water level (50%) as the highest value was recorded (2.01 mg/g d.w.). the lowest chlorophylls a+b (1.23 mg/g f.w.), carotenoids (0.32 mg/g f.w.), total carbohydrates (17.21%) and proline (0.82) were obtained by control + 50% water level, cycocel at 3000 ppm + 75% water level, paclobutrazol at 50 ppm + 50% water level and control + 100% water level, respectively. wue and stress indices although the lowest water level (50%) produced the lowest dry mass, as shown in figure 1 and table 12 this was accompanied by the highest water use efficiency (wue) obtained (1.09 and 1.21 g/l for both seasons, respectively). on the other hand, cycocel at 3000 ppm enhanced wue in both seasons as the highest values (1.43 and 1.51 g/l, respectively) were obtained. in the case of the combined treatments, the superiority was observed by cycocel at 3000 ppm + 50% irrigation water level (1.71 and 1.75 g/l in both seasons, respectively), this was followed significantly by cycocel at 2000 ppm + 50% water level (1.44 and 1.60 g/l) then cycocel at 3000 ppm + 75% water level (1.41 and 1.50 g/l) in both seasons respectively. water stress caused by reducing irrigation water levels was measured by the relative stress index (rsi) and presented in figure 2. at 150 ppm, paclobutrazol reduced water stress effects most effectively under 75% irrigation, showing the lowest rsi values (1.23 and 1.31 in both seasons, respectively), meaning that these plants treated with this treatment experienced minimal water stress and are in good condition. this was followed by paclobutrazol at 100 ppm, cycocel at 2000 ppm and 3000 ppm resulting in low rsi values and mitigated water stress at 75% level compared with 50% level. it is worth mentioning that the highest water stress experienced by plants was observed with the application of pbz at 50 ppm growth retardants water irrigation levels 100% p.c. 75% p.c. 50% p.c. chlorophylls a+b (mg/g control 1.57 jk 1.33 m 1.23 n pbz 50 ppm 1.71 hi 1.66 i 1.38 m pbz 100 ppm 1.79 ef 1.73 gh 1.53 kl pbz 150 ppm 1.97 bc 1.85 d 1.59 j ccc 1000 ppm 1.94 c 1.78 efg 1.48 l ccc 2000 ppm 2.02 b 1.83 de 1.69 hi ccc 3000 ppm 2.18 a 1.99 bc 1.77 fg carotenoids (mg/g control 0.43 f 0.53 b 0.58 a pbz 50 ppm 0.51 cd 0.42 f 0.53 b pbz 100 ppm 0.57 a 0.39 g 0.50 d pbz 150 ppm 0.56 a 0.35 h 0.46 e ccc 1000 ppm 0.46 e 0.38 g 0.51 cd ccc 2000 ppm 0.50 d 0.36 h 0.46 e ccc 3000 ppm 0.52 bc 0.32 i 0.38 g total carbohydrates control 23.70 f 22.13 h 18.06 kl pbz 50 ppm 25.61 e 23.17 f­h 17.21 l pbz 100 ppm 27.11 b­d 23.20 fg 18.15 kl pbz 150 ppm 28.14 b 25.40 e 19.10 jk ccc 1000 ppm 26.25 de 22.29 gh 19.21 ij ccc 2000 ppm 27.42 bc 24.16 f 20.18 i ccc 3000 ppm 31.74 a 26.37 c­e 22.13 h proline (mg/g d.w.) control 0.82 q 1.08 m 1.33 g pbz 50 ppm 0.84 p 1.14 jk 1.40 f pbz 100 ppm 0.90 o 1.15 j 1.73 d pbz 150 ppm 0.96 n 1.31 h 1.91 b ccc 1000 ppm 0.89 o 1.10 l 1.73 d ccc 2000 ppm 1.13 k 1.32 gh 1.80 c ccc 3000 ppm 1.29 i 1.56 e 2.01 a table 11 ­ effect of interaction between water irrigation levels and two plant growth retardants on some chemical constituents of leucophyllum frutescens during the second season of 2023 p.c. = pot capacity. values sharing the same letter across water levels and growth retardant treatments indicate no significant difference (p>0.05) according to duncan's multiple range test (duncan, 1955). el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 225 under an irrigation level of 50%, as the highest rsi levels were recorded (4.24 and 3.87 in both seasons, respectively). under moderate and low irrigation water levels, the stress tolerance index was calculated to determine how far the applied plant growth retardants can enhance the plants under these stress conditions (fig. 2). cycocel at 3000 ppm significantly enhanced water stress tolerance, particularly under 75% and 50% irrigation levels. this treatment yielded the highest values in both seasons, as recorded 1.70 and 1.36 (season 1) and 1.47 and 1.14 (season 2), fig. 2 ­ effect of water irrigation levels and two plant growth retardants on relative stress index (rsi) and stress tolerance index (sti) of leucophyllum frutescens during two seasons of 2022 and 2023. fig. 1 ­ effect of water irrigation levels and two plant growth retardants on water use efficiency (g/l) of leucophyllum frutescens during two seasons of 2022 and 2023. means within a season followed by the same letter are not sig­ nificantly different (p > 0.05) according to duncan (1955). table 12 ­ effect of interaction between water irrigation levels and two plant growth retardants on water use efficiency (g/l) of leucophyllum frutescens during two seasons of 2022 and 2023 growth retardant water irrigation levels 100% p.c. 75% p.c. 50% p.c. 100% p.c. 75% p.c. 50% p.c. 1st season 2nd season control 0.54 jk 0.51 k 0.66 i 0.58 m 0.55 m 0.76 kl pbz 50 ppm 0.86 h 0.68 i 0.64 i 0.99 ij 0.74 l 0.83 k pbz 100 ppm 0.89 gh 1.09 de 1.06 ef 1.12 gh 1.20 e­g 1.21 ef pbz 150 ppm 0.98 fg 1.26 c 1.26 c 1.16 fg 1.47 cd 1.39 d ccc 1000 ppm 0.64 i 0.61 ij 0.85 h 0.74 l 0.71 l 0.95 j ccc 2000 ppm 0.93 gh 1.18 cd 1.44 b 1.06 hi 1.28 e 1.60 b ccc 3000 ppm 1.18 cd 1.41 b 1.71 a 1.27 e 1.50 c 1.75 a p.c. = pot capacity. within each season, values sharing the same letter across water levels and growth retardant treatments indicate no significant difference (p>0.05) according to duncan's multiple range test (duncan, 1955). adv. hort. sci., 2025 39(3): 215­230 226 respectively. however, paclobutrazol at 150 ppm enhanced the tolerance to water stress at 75% level (1.25 and 1.30, in the first and second seasons, respectively). 4. discussion and conclusions the findings of the present study revealed a marked reduction in all assessed parameters at 50% irrigation level, except proline content and water use efficiency (wue), both of which increased under severe water deficit. application of paclobutrazol (pbz) and cycocel (ccc) significantly enhanced drought tolerance in leucophyllum frutescens under these conditions, with the highest concentrations of each compound producing the most pronounced effects. although both growth regulators led to a significant reduction in plant height, this effect was accompanied by notable increases in the number of branches and the fresh and dry biomass of shoots and roots, suggesting an overall improvement in plant vigor. furthermore, ccc demonstrated greater efficacy than pbz in enhancing growth­related parameters, indicating its superior potential for mitigating the adverse effects of severe water limitation. regarding the effect of irrigation levels, the results obtained were consistent with similar trials i.e. ashour and el­attar (2017) who reported that water deficiency induced by prolonged irrigation intervals reduced survival percentage, plant height, number of branches/plant, root length, and fresh and dry weights of vegetative growth and roots of leucophyllum frutescens . multiple studies demonstrate consistent physiological responses to water deficit across diverse plant species. research by noor el­deen (2020) on duranta erecta l. ‘golden edge’, noor el­deen et al. (2018) on zinnia elegans, and khalil et al. (2012) on jatropha curcas l. revealed a common pattern where reduced irrigation significantly decreased most growth parameters (plant height, biomass), while simultaneously increasing proline accumulation ­ a key osmoprotectant. similar findings were reported by amin (2013) in pinus radiate and robinia pseudoacacia transplants, where extended irrigation intervals reduced shoot and root biomass. closing stomata, inefficient carbon fixation process, photosynthesis rate reduction and decreasing formation and expansion of leaf tissues are the main disorders of water deficit (pallardy, 2008). on the other hand, dhopte and ramteke (2017) divided the effects of drought into physiological (desiccation of protoplasm which causes inhibition of cell division, elongation and differentiation as well as reduction of water absorption and dehydration, stopping stem and leaf elongation) and biochemical (proteolysis and protein synthesis retardation, nucleic acid synthesis failure, effect on enzyme synthesis, cytokinin levels are low, toxic products accumulation like ammonia­a main cause of drought injury, starch synthesis inhibition and aba level in roots are increased) effects. under drought conditions, the excessive production of reactive oxygen species (ros) and the generation of radical­scavenging compounds like ascorbate and glutathione further intensify the negative effects (deotale, 2017). ros­generated compounds cause oxidative damage to plant cells by restraining membrane attributes (singh et al., 2016). in this study, wue was calculated as the ratio of aerial biomass (shoot dry weight) to total water applied during the experimental period under each level of water. the values obtained showed that wue in drought­stressed plants (50%), relative to 100% pc­grown plants, was improved. this result suggests that the reduction in biomass (due to water deficit) was proportionally smaller than the 50% reduction in water application, so the value was high in this case. our study demonstrated that the highest concentrations of both pbz and ccc significantly improved drought tolerance in leucophyllum frutescens under severe water deficit (50% irrigation level). regarding pbz, our results agreed with thakur et al. (2000) on robinia pseudoacacia seedlings, who found that paclobutrazol improved water relations, enhanced drought tolerance, and increased proline and soluble sugar contents. also, this fits zhongzhu and xiangwei (2006) on two­year­old seedlings of populus alba × populus berolinensis, ulmus pumila and betula platyphylla, who reported an increase of water use efficiency. in the same line navarro et al. (2008) on lonicera implexa seedlings, found that pbz was significant for leaf water potential and leaf osmotic potential, and demonstrated that pbz attenuates the effects of deficit irrigation. hatamifar and samani (2017) found that application of paclobutrazol on petunia × grandiflora ‘bravo blue’ increased the number of flowers, aerial parts dry weight and number of lateral branches, and decreased the plant height, root dry weight and el‐ashwah et al. ‐ pbz and ccc enhance drought tolerance in leucophyllum frutescens 227 amount of carotenoids. the above­mentioned findings were confirmed in bougainvillea spp. (ting et al., 2014), later observed in odontonema strictum (rezazadeh et al., 2016), sequoia sempervirens (shu­ ming et al., 2020), and most recently in salvia officinalis (maghsoudi et al., 2023). the principal role of paclobutrazol (pbz), as a triazole compound, is the reduction in plant height by inhibiting gibberellin biosynthesis by blocking the oxidative steps from ent­kaurene to ent­kaurenoic acid, a key step in the gibberellin production pathway (rademacher, 1997). this leads to decreased levels of active gibberellins and subsequently limits stem elongation (tesfahun, 2018). this could explain our results, which showed a reduction in the height of plants treated with pbz. our study showed that application of paclobutrazol (pbz) and cycocel (ccc) significantly reduced plant height by inhibiting gibberellin biosynthesis; they concurrently increased the number of branches and enhanced fresh and dry biomass of leaves and shoots. these results were in agreement with youssef and abd el­aal (2013), who showed that pbz at 50 ppm or ccc at 1000 ppm increased the number of branches/plant as well as leaves and roots fresh and dry weights of tabernaemontana coronaria compared to control untreated plants. these enhancements in branching and biomass likely result from hormonal modulation: pbz suppresses gibberellin synthesis and shifts the isoprenoid pathway toward elevated cytokinin levels and abscisic acid accumulation, thereby reducing stem elongation while promoting lateral and root growth. ccc similarly inhibits gibberellin action, diminishes apical dominance, and encourages lateral bud outgrowth, collectively leading to increased branching and biomass (rademacher, 2000). on the other hand, singh et al. (2016) enumerated the positive role of pbz­treated plants grown under drought conditions. these plants possess a superior defense mechanism against free radicals, allowing them to neutralize harmful oxygen species. pbz treatment enhances stress resistance by boosting the activity of certain antioxidant enzymes. it also protects plants from oxidative damage by either increasing antioxidant levels or reducing the activity of oxidative enzymes. furthermore, pbz treatment stimulates the production of abscisic acid and phytol, hormones that promote plant growth and health. fletcher et al. (2000) added that pbz induces a large elastic module in the cell walls of treated plants, which in turn maintains turgor and thus increases the plant tolerance to drought stress. the elevated proline levels likely result from biosynthesis enzymes ornithine aminotransferase (oat) and pyrroline­5­carboxylate reductase (p5cr) combined with suppressed activity of proline oxidase, the key catabolic enzyme (debnath, 2008). paclobutrazol seems to increase proline content by increasing the enzyme activity of oat, which is involved in the biosynthesis of proline via ornithine (precursors for l­proline biosynthesis) pathway (zhang et al., 2024). pbz blocks the oxidation of ent­kaurene to ent­ kaurenoic acid by inhibiting cytochrome p450­ dependent oxygenases. this inhibition reduces gibberellin (ga) production, causing precursors in the terpenoid pathway to accumulate. these accumulated precursors, such as farnesyl diphosphate (fpp) and geranylgeranyl diphosphate (ggpp), are redirected toward aba biosynthesis, leading to higher aba levels. pbz also inhibits the activity of aba 8’­hydroxylase, an enzyme responsible for aba degradation, further increasing aba concentrations in plant tissues (soumya et al., 2017). pbz can also control stomata movements under drought stress. jack pine seedlings treated with paclobutrazol before a drought showed improved drought tolerance. this was evident in their quicker closure of leaf stomata to conserve water, better water content within the plant, and higher survival rates compared to untreated seedlings (marshall et al., 1991). additionally, minger and sanyu (1999) reported the role of paclobutrazol treatment in decreasing the leaf transpiration rate of satsuma mandarin, and prorogue its reduction during drought stress. abbasi et al. (2015) showed that paclobutrazol minimizes the negative effects of drought stress with evidence of enhancing chlorophyll content and antioxidant enzymes such as alternative oxidase (apx), catalase (cat) and glutathione peroxidase (gpx) that reduces hydrogen peroxide (h2o2) and malondialdehyde (mda) content. as an anti­gibberellin agent, cycocel inhibits the conversion of geranyl geranyl pyrophosphate (ggpp) into ent­kaurene by blocking ent­kaurene synthetases in the mevalonic acid pathway. this results in lower gibberellin levels, ultimately decreasing plant height (rademacher, 1997). adv. hort. sci., 2025 39(3): 215­230 228 applying cycocel (ccc) in plants enhances root growth, water potential, and stomatal resistance in leaves, ultimately improving water­use efficiency by stimulating root activity and reducing transpiration (deotale, 2017). ccc is considered highly effective in mitigating the negative impacts of various abiotic and biotic stresses on crops. ccc plays a key role in the signal transduction pathways of plants under environmental challenges, including drought stress. khondoker et al. (2019) reported the positive role of ccc on the growth and flowering of tuberose subjected to drought stress. in this regard, exogenous application of ccc is believed to influence nutrient uptake and transport, regulate stomatal function, and enhance growth, photosynthesis, chlorophyll synthesis, and transpiration processes (dehghanzadeh and adavi, 2023). the interoperation of the positive role of pbz under drought stress could be applied in the case of cycocel, particularly further enhancing the activities of the key antioxidant enzymes and accumulation of osmolytes, and decreasing the levels of h2o2 (as harmful ros compounds) and malondialdehyde in drought­ stressed plants (dehghanzadeh and adavi, 2023). as mentioned in the case of the effect of pbz on aba synthesis, the accumulated precursors induced by the ccc block of gibberellin synthesis are redirected toward aba biosynthesis, leading to higher aba levels. it is noteworthy that, based on the obtained results, ccc generally exhibited greater effectiveness than pbz. this may be attributed to differences in absorption pathways: paclobutrazol is usually applied either as a foliar spray or through soil drenching, and when applied to foliage, its uptake through mature leaves is relatively limited, with absorption likely occurring via the stem or from droplets reaching the soil. in contrast, cycocel is readily absorbed by both leaves and roots (barrett and bartuska, 1988; rademacher, 2000; maghsoudi et al., 2023). although plants treated with the highest concentrations of pbz (150 ppm) and ccc (3000 ppm) under the lowest irrigation level (50%) did not achieve the best values for most morphological characteristics, wue and sti, these values were still relatively high compared to plants exposed to the three irrigation levels without foliar application of pbz and ccc, except for plant height and the fresh weight of 10 flowers. cycocel at 3000 ppm outperformed pbz at 150 ppm in this aspect, proving more effective 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pumila, and betula platyphylla. ­ scientia silvae sinicae, 42(8): 130­134. untitled 115 1. introduction potato (solanum tuberosum l.) is widely cultivated in italy, with a total field area of 58,398 ha devoted to this crop in 2012, mainly in sicily (11,236 ha), campania (9,467 ha) and calabria (6,115 ha) (istat, 2012). in the southern italian regions of campania, sicily and apulia out-of-season crop production is traditionally practised and the early potato crop covers a total of 14,011 ha (istat, 2012), that is 93.1% of the total acreage under this crop (15.051 ha) in italy. in addition, nearly all of the italian potato production is obtained from conventional farming practice, although in the last nine years there has been a small increase in the area devoted to organic potato production. this latter accounted for about 730 ha in 2003 and 981 ha in 2011 (1.18 and 1.58% of the total area, respectively) (ccpb, 2012). in the mediterranean area, out-of-season potato yield obtained from winter-spring or summer-autumn crop cycles is mainly channelled into the highly profitable export market. however, for both conventional and organic crops the availability of certified seed tubers is a limiting factor. indeed, this crop frequently gives a modest yield because small and/or immature tubers are used (delaplace et al., 2008) which are still conditioned by apical dominance owing to the brief interval between seed tuber harvest and their use for planting. in the case of the summer-autumn potato crop, a possible method to improve yield is to obtain seed tubers from an early crop (winter-spring cycle) in order to harvest the product before aphid proliferation (monti and struik, 1999) and to limit pathogen infections (hospers-brands et al., 2008). under organic farming practice, saucke and döring (2004) found that, in addition to effective weed control, the use of biodegradable mulch films helps reduce aphid infestation on leaves and potato virus y incidence in tubers. moreover, within each microenvironment it is important to choose the planting time which best suits the requirements of the specific cultivar (frusciante et al., 1999; caruso et al., 2010) in order to obtain good results. as a general precaution, the choice of seed tubers of the correct size should be preferred. in fact, seed potatoes should be neither too big, requiring to be cut in order to limit the possible diffusion of infections (franc and banttari, 1984), nor undersized to avoid slow initial growth and tuberization (mustonen, 2004). indeed, shoot vigour effect of mulching and plant density on out-of-season organic potato growth, yield and quality g. caruso1, d. carputo, s. conti, c. borrelli, p. maddaluno, l. frusciante dipartimento di agraria, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: biodegradable mulch, plant leaf area and dry matter, production, solanum tuberosum l., tuber vitamin c. abstract: research was carried out on potato (solanum tuberosum l., cv. spunta) growing in the fi eld in the campania region (southern italy) in 2007 and 2008, adopting organic farming practices, in order to evaluate the effects of two mulching treatments (black biodegradable fi lm and bare soil) and six plant densities (12.5, 10.0, 8.3, 7.1, 6.2 and, as a control, 5.3 plants per m2) on growth, yield and quality of “new potato” winter-spring and summer-autumn crops. only in the case of the summer-autumn crop cycle, mulching resulted in a higher yield, plant dry matter and leaf area compared with the bare soil control, while in both crop cycles this latter treatment induced a delay in harvest. the winter-spring cycle gave a higher production of 40-70 mm tubers, while the summer-autumn cycle resulted in a higher vitamin c content. for the winter-spring crop cycle, the plant density of 8.3 plants·m-2 resulted in the highest yield for food-use tubers, whereas the highest production of seed tubers was obtained with a density of 12.5 plants·m-2. the plant density of 8.3 plants·m-2 also resulted in the highest plant dry matter and leaf area. for the summer-autumn crop cycle, the 10 plants·m-2 density gave the highest production of 40-70 mm calibre tubers, as well as the highest plant dry matter and leaf area. in this cycle, the 6.3 plants·m-2 density resulted in the highest production of 70-80 mm calibre tubers. in terms of cost effectiveness, the choice of biodegradable mulching could save the expense of manual weed control and, in the case of the summer-autumn crop cycle, it is also associated with a higher yield. overall, tuber yield increased with plant density but the fi nal production was also affected by the crop cycle. this may depend on the different environmental conditions and duration which characterized each cultural cycle and, therefore, affected the vegetative development of organic new potatoes. adv. hort. sci., 2013 27(3): 115-121 1 corresponding author: gcaruso@unina.it received for publication 22 march 2013 accepted for publication 2 july 2013 116 and development rate are inversely correlated to seed tuber size, which provides meristematic resources (mackerron et al., 1988): when the latter are limited, scarce shoot emission and hence smaller within-plant resource competition occurs. this leads to the formation of fewer large tubers (hide et al., 1997), which is not desirable for the production of either food-use or seed tubers. crop mechanization (green, 1962), aimed at reducing production costs, has led to an enlargement of between-row and in-row plant spacings. hence, the current trend is to use spacings of 70-80 cm between the rows and 20-30 cm along the rows for conventional potato crops grown from ripe tubers in february-march. in this regard, contrasting results have been published: in some areas both lower and higher in-row spacings resulted in yield reduction (masarirambi et al., 2012) while in other environments a higher plant density resulted in a higher production but reduced tuber calibre (turajizadeh et al., 2011; fontes et al., 2012), the latter effect being desirable for “seed” production. moreover, increasing in-row spacing leads to greater tuber size (tarkalson et al., 2011). in mild climates, winterspring and summer-autumn crops are respectively grown from commercial “seed” potatoes or from locally (re)produced tubers. at the time of planting for out-of-season potato production, the seed tubers from these two crop cycles are still not completely mature and they are conditioned by apical dominance. in these cases, it may be interesting to increase plant density to balance the scarce shooting capacity of physiologically immature propagation material. to our knowledge, no conclusive research has yet been published regarding the use of mulching and its interaction with plant density in organic farming practice in southern italy. thus, we planned our field trials with the aim of testing the effect of biodegradable mulching and of plant density on the production of “seed” and food potatoes from winter-spring crops and on the production of food potatoes only from summer-autumn crops. trials were carried out in the campania region (southern italy) on organic potato crops, cv. spunta. 2. materials and methods research was carried out in san gennaro vesuviano (naples) in 2007 and 2008 on potato cv. spunta, grown in the field under organic farming practices on a sandy-loam soil (table 1); temperature and rainfall values are reported in table 2 as means of the data from the first and second experimental years. twelve experimental treatments were compared. these were obtained from the factorial combination of two mulching treatments (black biodegradable film and bare soil) and six plant densities (12.5, 10.0, 8.3, 7.1, 6.2 and, as a control, 5.3 plants per m2). the experimental treatments were randomized in a split-plot design with three replicates, assigning mulching treatments to the main plots and plant densities to the elementary plots; the latter had a 18.00 m2 (6.00 x 3.00 m) surface area. the soil was prepared by forming raised beds which were mulched with a 15-μm thick biodegradable black film made from corn starch. potatoes were planted in double rows for all treatments except for the control crop which was grown in single rows. the six different plant densities were obtained using the following spacings: 0.40 m between the rows and 0.20, 0.25, 0.30, 0.35 or 0.40 m along the rows for 12.5, 10.0, 8.3, 7.1, 6.2 plants·m-2 treatments; 0.75 m between the rows and 0.25 m along the rows for the 5.3 plants·m-2 treatment (control). table 1 soil characteristics on 100 g of air-dried and 2 mm sieved soil coarse sand g 35.3 fine sand g 45.5 silt g 7.8 clay g 11.4 organic matter (walkley-black method) g 1.18 total nitrogen (n) kjeldhal method g 0.1 available phosphate (p 2 o 5 ) olsen method mg 1.7 available potassium (k 2 o) ammonium acetate method mg 54.0 total lime (dietrich-früling) g traces ph 7.4 table 2 temperature and rainfall values (means of 2007 and 2008) during winter-spring and summer-autumn crop cycles in san gennaro vesuviano (naples) month dates intervals temperature (°c) rainfall (mm)minimum maximum january 21-31 9.3 14.9 13.4 february 1-10 9.5 15.2 42.2 11-20 8.1 14.3 37.1 21-28 10.9 17.3 17.5 march 1-10 11.3 16.0 68.1 11-20 11.3 17.2 33.3 21-31 8.9 16.6 65.2 april 1-10 11.8 19.8 25.7 11-20 13.3 21.3 27.4 21-30 13.7 22.4 21.0 may 1-10 15.2 24.1 10.6 11-20 15.8 24.4 29.4 august 21-31 23.8 33.1 0.0 september 1-10 20.8 30.0 15.7 11-20 18.8 27.6 35.4 21-30 16.2 24.5 23.7 october 1-10 17.6 24.6 28.0 11-20 16.4 24.0 6.0 21-31 15.5 21.6 14.8 november 1-10 14.5 20.4 9.0 11-20 10.6 15.4 24.3 21-30 12.2 16.6 76.4 december 1-10 10.9 15.1 63.0 117 plantings took place on the following dates: 24 january for the winter-spring crop using certified “seed” tubers (50±2 g) and 20 august for the summer-autumn crop using “seed” tubers obtained from the previous crop cycle (3040 mm). tubers were kept at 7°c until use and showed no pre-sprouting at planting time. potato crops were preceded by common bean and fava bean on the same plots. the organic farming system was managed in compliance with ec regulation 834/2007 and the farming practices were: fertilization before planting with 100 kg·ha-1 of n, 85 of p 2 o 5 and 210 of k 2 o with biolsa 6-5-13; drip fertigation with 50 kg·ha-1 of nitrogen as 8.5 n hydrolyzed animal epithelium; weed control by hand; plant protection treatments with copper, azadirachtin and rotenone. new potato tubers were harvested when the aerial part of the plant showed the initial symptoms of senescence (leaf yellowing) and wilting. tubers from the winter-spring crop were harvested on 18 may 2007 and 21 may 2008. the summer-autumn crops were harvested on 4 december 2007 and 2 december 2008. undamaged tubers of regular shape were classified as “marketable” and graded as follows: the 3040 mm and 40-70 mm grades (which are usually addressed to the large scale retail channel and packaged in boxes or bags, respectively) and the 70-80 mm grade (which is usually channelled to the local market). at the time of harvest, the following determinations were made in each plot: number of failures; number of shoots per plant; number and weight of tubers; tuber mean weight on a 50-unit sample; tuber grading and classification into the three calibre classes. tubers falling into the 30-40 mm class were classified as “seed” tubers in the case of the winter-spring crop or as food tubers in the case of the summer-autumn crop. the weight of tubers unsuitable for the market was also recorded in order to monitor total biomass production for each treatment. plant biomass was calculated as the sum of the aboveground plant biomass at the end of the experiment plus the total tuber production. dry residue was assessed after dehydration of the fresh samples in an oven at 70°c under a vacuum until they reached constant weight. leaf area was measured at the end of the cycle, using a bench top li-cor leaf area meter. in order to evaluate the quality of tubers produced both from the winter-spring and from the summer-autumn crop, samples of 20 tubers per plot were randomly collected at harvest time and transferred to the laboratory, where the following determinations were made: dry residue, in an oven at 70°c under vacuum until steady weight; vitamin c content, using a waters 600e hplc system equipped with a waters 486 uv detector set to 410 nm λ and a biorad column mod. hpx87h at 35°c. data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 and 0.01 probability levels, using spss software version 15. 3. results and discussion from statistical processing of the data, no differences were detected between 2007 and 2008, therefore only the mean values of the two research years are shown. in the case of winter-spring crops, mulching did not significantly affect yield but the crop cycle of the mulched crops was four days and a half shorter compared with the bare soil treatments (table 3). similar results were published by boyd et al. (2001) and döring et al. (2005) who reported that mulching did not produce any increase in yield and tuber size but it only caused an increase in soil temperature (xing et al., 2012). conversely, maletta et al. (2006) reported that mulching resulted in increased yield and tuber size in organic potato. plant density significantly affected yield (table 3): maximum production was obtained with a plant density table 3 yield results and growth indices of winter-spring potato crop treatment failures % actual density no.·m-2 shoots no.·pt-1 tubers no.·m-2 tuber mean weight g yield t·ha-1 crop duration days plant growth indices (maximum values) dry matter g·m-2 lai m2·m-2 mulching biodegradable film 4.7 7.9 1.08 25.1 110.6 26.8 114.5 696.3 2.1 bare soil 4.6 7.9 1.09 25.1 107.0 26.0 119.0 663.9 2.0 ns ns ns ns ns * ns ns plant density 5.3 pt·m-2 4.0 5.1 1.10 16.9 f 123.3 a 20.8 d 117.2 546.4 d 1.6 d 6.3 5.3 5.9 1.15 19.8 e 121.5 ab 24.1 c 117.0 622.3 c 1.9 c 7.1 4.6 6.8 1.15 22.5 d 117.5 bc 26.4 b 116.8 681.0 b 2.1 b 8.3 3.7 8.0 1.12 26.1 c 112.0 c 29.2 a 116.7 748.5 a 2.3 a 10.0 5.0 9.5 1.03 29.9 b 97.4 d 29.1 a 116.5 744.0 a 2.3 a 12.5 5.4 11.8 1.00 35.5 a 81.3 e 28.8 a 116.3 738.3 a 2.2 ab ns ns ns * = significant at p≤0.05; ns = not significant; whitin each column, means followed by different letters are significantly different according duncan test at p≤0.05. 118 of 8.3 plants·m-2, though it was not significantly different from 12.5 and 10.0 plants·m-2. the control plant density of 5.3 plants·m-2 gave the lowest yield. similar results were reported by fontes and coworkers (2012), who found that reducing the in-row spacing from 50 to 29 cm led to a higher tuber number and greater yield. however, masarirambi et al. (2012) reported a yield decrease when plant density increased from 3.7 to 7.4 plants·m-2. due to the early plantings, the “seed” tubers used to start the winterspring crops had still not reached their full physiological maturity. consequently, they produced plants with only one shoot and a small number of tubers which did not vary between the treatments. therefore, the productive results were affected both by the number of tubers per square meter and by the tuber mean weight: the highest number of tubers was recorded at the highest plant density while the highest tuber mean weight was obtained with 5.3 plants·m-2. no difference in precocity among the treatments was recorded. total yield increased with increasing plant density up to 8.3 plants·m-2, because the apical dominance effect induced tubers to produce only one shoot, thereby reducing dramatically the competition between plants and within each plant. growth parameters varied similarly to yield in response to mulching and plant density (table 3). dry matter and leaf area were not significantly affected by mulching, while plant density increases up to 8.3 plants·m-2 resulted in the highest values of both dry matter and leaf area, reflecting the mulching effect on total yield. the relative proportion of tubers classified into the different calibre classes (table 4) was not significantly different between the mulched or bare soil treatments, but significant effects were recorded in response to the different plant densities. the highest densities of 12.5 and 10.0 plants·m-2 resulted in the highest incidence of 30-40 mm grade tubers, which are suitable for uncut “seed” use and may therefore be considered the best choice as seed potatoes for the following summer-autumn crop. the plant densities of 6.3 and 7.1 plants·m-2 produced the highest proportion of 40-70 mm grade tubers, which are suitable for the food market, whereas plant density did not significantly affect the incidence of 70-80 mm calibre tubers. these results are in accordance with previously published studies reporting an increase in the smalland mediumtuber size class when the in-row plant spacing was reduced from 50 to 29 cm (fontes et al., 2012) and plant density from 8.0 to 5.3 plants·m-2 (turajizadeh et al., 2011). as a quality indicator of potato tubers, the dry residue was not significantly affected by mulching or by plant density (table 4). vitamin c content was higher in tubers obtained from mulched plots, whereas it did not vary in response to the different plant densities. similar results were published by gram (1951) who found that tubers from straw-mulched crops had significantly higher ascorbic acid content. moreover, in more recent years dvorak et al. (2012) reported no significant effects of mulching on potato tuber ascorbic acid content under different soil or climatic conditions. these contrasting results suggest that the actual ascorbate content of potato tubers may result from an interaction of multiple factors. however, the tuber vitamin c values detected in our research fall in the range reported by brown (2005). after discarding about 10% of virus-infected tubers, the 30-40 mm tubers produced by the winter-spring crops were used as seed potatoes for the following summer-autumn crops. these seed tubers were to be planted about three months after harvest, before they could reach their full physiological maturity, similarly to the commercially available seed potatoes used for the winter-spring crops. as discussed table 4 grading and quality indicators of “seed” and food-use potato tubers produced from winter-spring crop treatment seed-use grade food-use grades tuber quality indicators 30-40 mm 40-70 mm 70-80 mm yield t·ha-1 % of total yield tuber mean weight g potential use z ha % of total yield % of total yield dry residue % vitamin c mg·100 g-1 f.w. mulching biodegradable film 8.9 33.2 49.1 3.4 58.7 8.0 18.5 23.0 bare soil 8.8 34.0 50.6 3.3 58.0 7.9 18.8 22.2 ns ns ns ns ns ns ns * plant density 5.3 pt·m-2 6.7 e 32.2 bc 50.4 ab 2.5 e 59.8 ab 8.0 18.5 22.7 6.3 7.6 d 31.4 c 51.8 a 2.7 de 60.5 a 8.1 18.6 22.5 7.1 8.7 c 32.8 b 51.1 ab 3.2 cd 59.1 ab 8.1 18.7 22.9 8.3 9.8 b 33.4 b 50.3 ab 3.6 bc 58.6 b 8.0 18.9 22.3 10.0 10.3 ab 35.5 a 48.9 bc 4.0 ab 56.6 c 7.9 18.6 22.7 12.5 10.5 a 36.4 a 47.0 c 4.2 a 55.7 c 7.9 18.8 22.5 ns ns ns z using the traditional 75 x 25 cm spacings (5.3 plants·ha-1) in the following summer-autumn crop; * = significant at p≤0.05; ns = not significant; whitin each column, means followed by different letters are significantly different according duncan test at p≤0.05. 119 above, their immature state caused each “seed” to originate only one shoot and, consequently, the number of tubers per plant did not differ among the treatments. therefore, yield in the different plots was only affected by the number of tubers per unit surface area and by the tuber mean weight (table 5). in the summer-autumn cycle, mulching increased crop precocity and affected tuber production favourably, as compared with bare soil treatments (table 5). this result may be explained by the lower failures and higher tuber mean weight recorded in mulched plots, though these variables were not significantly affected by mulching. it was suggested that mulching resulted in higher potato yield due to the reduction of soil nutrient loss caused by runoff (rees et al., 2002) and to the prevention of excessive soil heating (dvorak et al., 2012). plant density significantly affected tuber production (table 5) and the highest yields were recorded with 10.0 and 12.5 plants·m-2, whereas the control plots (5.3 plants·m-2) gave the lowest production. at the same time, tuber mean weight was affected by competition among plants and it increased at lower plant densities. no significant effect of plant density on precocity was recorded. total production increased with plant density up to 10.0 plants·m-2, i.e. even over the 8.3 density threshold recorded for the winter-spring crop. in fact, the latter showed greater plant expansion (tables 3 and 5) due to the longer crop cycle, causing presumably greater plant competition for available resources. moreover, both growth indexes (plant dry matter and leaf area) attained higher values with mulching compared with bare soil, whereas they were not significantly affected by plant density (table 5). tubers harvested in the autumn were graded only with regard for the food market (table 6). mulched plots protable 5 yield results and growth indices of summer-autumn potato crop treatment failures % actual density no.·m-2 shoots no.·pt-1 tubers no.·m-2 tuber mean weight g yield t·ha-1 crop duration days plant growth indices (maximum values) dry matter g·m-2 lai m2·m-2 mulching biodegradable film 14.5 6.9 1.0 20.9 114.1 24.7 105.0 582.1 1.8 bare soil 15.7 7.0 1.0 20.4 112.0 20.7 109.3 496.5 1.5 ns ns ns ns * * * * plant density 5.3 pt·m-2 15.1 4.5 1.0 13.6 f 125.7 a 17.1 e 107.7 372.3 d 1.2 d 6.3 14.9 5.4 1.0 16.2 e 124.5 a 20.2 d 108.4 475.6 c 1.4 c 7.1 15.0 6.0 1.0 18.2 d 118.5 b 21.6 d 108.0 510.2 c 1.5 c 8.3 15.2 7.0 1.0 20.9 c 115.5 b 24.1 c 107.1 570.7 b 1.7 b 10.0 15.3 8.5 1.0 24.7 b 110.0 c 27.1 b 106.4 642.9 a 1.9 a 12.5 15.2 10.6 1.0 30.9 a 84.5 d 26.1 a 105.8 663.8 a 2.0 a ns ns ns * = significant at p≤0.05; ns = not significant; whitin each column, means followed by different letters are significantly different according duncan test at p≤0.05. table 6 grading and quality indicators of food-use potato tubers produced from summer-autumn crop treatment food use tuber grades tuber quality indicators 30-40 mm % of total yield 40-70 mm % of total yield 70-80 mm % of total yield dry residue % vitamin c mg·100 g-1 f.w. mulching biodegradable film 26.4 62.4 11.2 17.8 21.7 bare soil 28.4 59.2 12.4 18.0 21.4 * * * ns ns plant density 5.3 pt·m-2 26.2 d 60.8 bc 13.0 d 17.9 21.5 6.3 26.0 d 58.5 d 15.5 a 18.0 21.6 7.1 27.0 c 59.9 c 13.1 b 17.7 21.4 8.3 27.9 b 61.0 b 11.1 c 17.8 21.7 10.0 28.6 a 62.1 a 9.3 d 18.0 21.5 12.5 28.9 a 62.5 a 8.6 e 17.8 21.6 ns ns * = significant at p≤0.05; ns = not significant; whitin each column, means followed by different letters are significantly different according duncan test at p≤0.05. 120 duced a higher percentage of medium-sized tubers (4070 mm) whereas the crops grown on bare soil produced a higher percentage of small and large tubers (30-40 mm and 70-80 mm, respectively). the highest plant densities of 12.5 and 10 plants·m-2 led to the highest proportion of 30-40 mm calibre tubers, as well as of the 40-70 mm calibre. moreover, the percentage of the largest tubers increased at lower plant densities, whereas this parameter was not significantly affected in the case of the winter-spring crop cycle. the quality indicators of potato tubers (dry residue and vitamin c) were not significantly affected by the experimental factors during the summer-spring crop cycle (table 6). tuber dry residue and vitamin c contents of tubers harvested in the autumn were as much as 4 and 5% lower compared with tubers harvested in the spring. these results were in accordance with previous research (ierna, 2010) reporting higher values of yield and tuber dry residue, but lower tuber mean weight in spring tubers compared to autumn ones. finally, the winter-spring crop cycle resulted in lower tuber mean weight and higher yield, compared with the summer-autumn crop cycle. 4. conclusions in the case of the winter-spring cycles, the use of biodegradable mulching induced an anticipation of the harvest but it was not found to have significant effects on tuber production. the use of biodegradable mulching for the summerautumn crop cycle gave better production results and an anticipation of the harvest; moreover, it also had positive effects on the prevention of water logging caused by abundant rainfall in november. since chemical weed control is not permitted in organic farming, in both crop cycles mulching allowed easier and cheaper weed control than that carried out by hand for bare soil cultivation. unlike conventional “mature potato” crops which are usually grown with a plant density of 5-6 seed tubers per m2, in the case of “new potato” crops a higher yield was obtained by increasing plant density both in the winterspring and summer-autumn cycles. in this respect, the summer-autumn cycle allowed the highest plant density (up to 10.0 plants·m-2) compared with the winter-spring cycle (up to 8.3 plants·m-2) because of the lower plant vegetative growth. in the case of the winter-spring cycle, a plant density of 12.5 plants·m-2 density produced the highest amount of “seed” potatoes (30-40 mm) to be used for the next summer-autumn crop cycle. acknowledgements the 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of selected phalaenopsis cultivars y.k.d.d. thathsarani, j.a.t. tikirikumari, a.i.s. priyadarshan, h.m. herath, s.p. senanayake (*) department of plant and molecular biology, faculty of science, university of kelaniya, sri lanka. key words: anthocyanins, carotenoids, chlorophyll, cultivars, flavonoids, polyphenols. abstract: the genus phalaenopsis in family orchidaceae has gained popularity in the global floriculture market for its value as potted plants and cut flowers. hybridization plays a pivotal role in breeding phalaenopsis, enabling the development of novel cultivars with desirable traits such as diverse floral pigmentation patterns, enhanced longevity and improved growth rate. the selection of suitable parental cultivars is a crucial determinant in the hybridization process, as it plays a key role in defining pigmentation patterns and enhancing desirable traits for the development of superior cultivars. this research investigated the phytochemical composition, including carotenoids, anthocyanins, flavonoids and phenolics contents of selected commercial phalaenopsis cultivars. using uv­vis spectrophotometry, the chlorophylls, flavonoids, carotenoids, anthocyanins and phenolics contents in the flowers, leaves and roots of six cultivars were analyzed quantitatively. the results revealed significant variations in phytochemical properties across the tested cultivars and plant organs. phalaenopsis cvs. taipei gold gold star, red lip 1770, golden sands canary, sogo yukidian v3 and queen beer mantefon exhibited promising phytochemical profiles. notably, cvs. taipei gold gold star, golden sands canary and queen beer mantefon were identified as ideal parental candidates for hybridization due to their potential to develop distinctive floral colorations and robust vegetative traits. these findings provide valuable insights into the floral coloration and the phytochemical richness of vegetative parts in phalaenopsis cultivars. this knowledge can contribute to the development of innovative, high­quality cultivars with enhanced survival rates and greater consumer appeal. 1. introduction the family orchidaceae, one of the largest families of flowering plants, ranks second only to the family asteraceae in its diversity and ecological prominence. comprising over 736 genera, orchidaceae has established a significant presence in the global floriculture market, valued for its use in both potted plants and cut flowers (chase et al., 2015). among its commercially significant genera, phalaenopsis stands out for its refined, (*) corresponding author: priyangi@kln.ac.lk citation: thathsarani y.k.d.d., tikirikumari j.a.t., priyadarshan a.i.s., herath h.m., senanayake s.p., 2025 ­ phytochemical evaluation of selected phalaenopsis cultivars. ­ adv. hort. sci., 39(2): 125­135. orcid: tykdd: 0000­0002­4528­8170 tjat: 0009­0006­3326­5074 pais: 0000­0002­2900­975x hhm: 0000­0001­8387­0420 ssp: 0000­0002­9145­8329 copyright: © 2025 thathsarani y.k.d.d., tikirikumari j.a.t., priyadarshan a.i.s., herath h.m., senanayake s.p. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 3 december 2024 accepted for publication 31 march 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16964 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-4528-8170 http://orcid.org/0009-0006-3326-5074 http://orcid.org/0000-0002-2900-975x http://orcid.org/0000-0001-8387-0420 http://orcid.org/0000-0002-9145-8329 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 125­135 126 elegant aesthetic and extended longevity, captivating both growers and consumers (hsiao et al., 2011). hybridization serves as the primary breeding approach for phalaenopsis, with intergeneric and interspecific hybridizations being the most commonly employed methods to develop novel cultivars. to date, 35,129 phalaenopsis hybrids have been registered with the royal horticultural society, reflecting the immense popularity of the genus (hsu et al., 2018). dynamic and evolving consumer preferences have significantly driven hybridization efforts, making phalaenopsis a focal point in modern orchid breeding. the breeding objectives for phalaenopsis focus primarily on morphology, color, and scent, with color being a key factor in shaping initial consumer impressions (hsu et al., 2018). with the increasing popularity and demand for orchids, commercial cultivars are developed with specific characteristics that enhance their large­scale applicability. key traits include resilience, which reduces the need for inputs such as water, ferti l izers, and pesticides—an essential factor given rising production costs and the growing emphasis on sustainability. modern floriculture, a resource­intensive sector that utilizes energy, water, fertilizers, and propagation materials, faces the challenge of meeting market demands while minimizing environmental impact (darras, 2020; cardoso and vendrame, 2022; cardoso et al., 2023; bhardwaj et al., 2024). phytochemicals play a crucial role in plant growth and development. while primary metabolites support physiological functions, secondary metabolites contribute significantly to plant’s defense mechanisms (thacker and ram, 2020; wani et al., 2022). in orchids, various phytochemicals have been identified, serving both physiological and commercial purposes. notable genera with economic significance include dendrobium (he et al., 2023), phalaenopsis (ling and subramanium, 2007; minh et al., 2016), vanda, bulbophyllum (lalrosangpuii and lalrokimi, 2021), and cymbidium (axiotis et al., 2021). understanding the phytochemical composition of these orchids is essential for developing innovative, sustainable, and high­value cultivars that align with market trends and consumer preferences. the vibrant coloration of phalaenopsis hybrids arises from a complex mechanism of pigment accumulation. flower color is primarily influenced by pigments such as chlorophylls, carotenoids, anthocyanins, and betalains. while many flowers derive their color from a single source of pigment, phalaenopsis exhibits a broader palette through the combination of pigments. yellow to orange shades are mainly due to the accumulation of carotenoids, whereas blue to red shades are typically attributed to anthocyanins (hsu et al., 2018). combinations of purple anthocyanins and yellow carotenoids can result in perceived colors such as brown, bronze, and red in flowers (lightbourn et al., 2008). hence, novel colors can be produced through hybridization, facilitating the combination of different pigments (voegelpoel, 1990). moreover, differential coloration can generate various pigmentation patterns such as blotches, stripes along veins, and irregular markings. these striking patterns and colors increase the aesthetic value of phalaenopsis cultivars, capturing the attention of consumers and expanding their market appeal. the quality of the flower depends on the vegetative growth rate of the plant, as vigorous growth often correlates with superior flower characteristics. photosynthesis, a primary physiological process, drives plant growth by converting light energy into chemical energy, enabling the synthesis of carbohydrates. chlorophyll, the key pigment in photosynthesis, plays an essential role in this process. the two main forms of chlorophyll­chlorophyll a and chlorophyll b­absorb solar energy, facilitate carbon dioxide fixation, and convert energy into carbohydrates, which are vital for plant growth and development. consequently, higher chlorophyll content is directly associated with improved photosynthetic efficiency and plant vigor. in addition to chlorophyll, phenolic compounds also play a crucial role in plant physiology and aesthetics. these compounds, including flavonoids such as anthocyanins, contribute significantly to plant defense mechanisms and pigmentation. phenolic compounds accumulate as an adaptive response to environmental stress, highlighting their role in plant resilience (lattanzio et al., 2012). in phalaenopsis cultivars with desirable traits, flower color intensity is a key selection criterion in tissue culture and breeding programs (ling and subramaniam, 2007). however, the phytochemical profiles of phalaenopsis cultivars remain largely fragmented, limiting the ability to develop hybrids with targeted traits. this lack of comprehensive data highlights the need for in­depth phytochemical studies to support informed breeding decisions. therefore, this study aims to evaluate the thathsarani et al. ‐ phytochemicals in selected phalaenopsis cultivars 127 with different color combinations were obtained from the growers. the details of the selected six commercial phalaenopsis cultivars are presented in table 1. the plants were maintained in the plant house of the department of plant and molecular biology, university of kelaniya, sri lanka. the fresh, full­bloomed flowers, young leaves (the leaf nearest to the flower spike) and roots of each cultivar were collected in the morning and used for phytochemical analysis. the roots and leaves were cleaned with distilled water and wiped. all the samples were frozen at ­80°c immediately after collection. the frozen samples were grounded using mortar and phytochemical properties of selected phalaenopsis cultivars, providing valuable insights for the development of high­value cultivars that align with market trends. 2. materials and methods plant materials this study was conducted to evaluate phytochemical properties of selected phalaenopsis cultivars. six commercially available phalaenopsis cultivars no. name reference for the cultivar name abbreviation 1 phalaenopsis cv. red lip 1770 locally produced cultivar rl1770 2 phalaenopsis cv. golden sands canary (lee et al., 2020) gsc 3 phalaenopsis cv. sogo yukidian v3 (lee et al., 2020) syv3 4 phalaenopsis cv. queen beer mantefon (lee et al., 2020) qbm 5 phalaenopsis cv. taipei gold gold star (lee et al., 2020) tggs 6 phalaenopsis cv. brother strips (lee et al., 2020) bs table 1 ­ list of selected commercial phalaenopsis cultivars adv. hort. sci., 2025 39(2): 125­135 128 pestle. the weights of each powdered plant material were measured and quantified to obtain a standard weight. determination of chlorophyll and carotenoid contents chlorophyll and carotenoid contents of leaves, roots and flowers were measured using the methods described by nguyen et al. (2018) with slight modifications. known volume of dried powder was mixed with a known volume of 80% acetone and kept at 4°c overnight. the mixture was centrifuged at 13,000 g for 5 min to obtain the supernatant. the supernatant was tested to determine the absorbance of chlorophyll a, chlorophyll b and carotenoids in 80% acetone at 664 nm, 647nm and 441 nm respectively using the uv­ visible spectrophotometer (thermo fisher scientific, vantaa, finland). concentrations of chlorophyll a, chlorophyll b and carotenoids were calculated using the following equations (porra, 2002). chlorophyll a = (12.25 x absorbance 664) ­ (2.55 x absorbance 647) x volume of the supernatant (ml)/sample weight (g) chlorophyll b = (20.31 x absorbance 647) – (4.91 x absorbance 664) x volume of the supernatant (ml)/sample weight (g) carotenoids = (4.69 x absorbance 441 x volume of the supernatant (ml)/sample weight (g)) – 0.267 (chlorophyll a ±b) total chlorophyll = chlorophyll a ± chlorophyll b determination of anthocyanin content a known weight of dry powder of leaves, roots and flowers were mixed with a known volume of acidified methanol (99% methanol containing 1% hcl). the mixture was incubated for 24 hours at room temperature followed by centrifugation at 4°c and 3000 rpm for 5 minutes. the obtained supernatant was then subjected to measure the absorbance at 530 nm and 657 nm on the uv­visible spectrophotometer (thermo fisher scientific, vantaa, finland). concentration of anthocyanins were calculated using the following equation. anthocyanin content = (absorbance 530 ­ 0.33 × absorbance 657 / 31.6) x volume of supernatant (ml) / sample weight (g) determination of total flavonoid content the known weight of dry powder of leaves, roots and flowers were mixed with methanol and incubated for 24 hours at room temperature. the supernatant was obtained by centrifugation at 4°c and 3000 rpm for 5 minutes. equal volumes of the supernatant were mixed with 2% aluminum chloride. the mixture was stirred and kept for 15 minutes. the absorbance was measured at 430 nm using the uv­ visible spectrophotometer (thermo fisher scientific, vantaa, finland). quercetin was used as the reference standard. the flavonoid content of each plant part was expressed as milligrams of quercetin equivalents of grams of dry weight (mg qe/g dw) using the following equation. c = c1 (volume of the supernatant (ml))/(dry weight (g)) where c is total flavonoid content of the extract and c1 the quercetin concentration (mg/ml). determination of total polyphenol content the acidified methanol extracts were prepared using the same procedure done for the anthocyanin content analysis. 100 μl of the extract was mixed with 2 ml of 7.5 % na2co3 and allowed to equilibrate for 2 minutes. after 2 minutes diluted folin­ciocalteu reagent was added (1:10 v/v). the absorbance was measured at 765 nm using the uv­ visible spectrophotometer (thermo fisher scientific, vantaa, finland). gallic acid was used as the reference standard. the polyphenol content of each plant part was expressed as milligrams of gallic acid equivalents of grams of dry weight (mg gae/g dw) using the following equation. c = c1 (volume of the supernatant (ml))/(dry weight (g)) where c is the total polyphenol content of the extract and c1 th gallic acid concentration (mg/ml). statistical analysis all the experiments were conducted in triplicates (n=3). the data were expressed as mean ± sd (standard deviation). an analysis of variance (anova) test was performed with tukey’s honest significant difference test (hsd) at p ≤ 0.05 using r software version 4.3.2. 3. results chlorophyll content figure 1 illustrates the chlorophyll a, chlorophyll b, total chlorophyll (a + b), and the chlorophyll a/b ratio in extracts from different parts (leaves, roots, and flowers) of six phalaenopsis cultivars. the analysis revealed that the concentrations of chlorophyll a and b were significantly higher in the thathsarani et al. ‐ phytochemicals in selected phalaenopsis cultivars 129 fig. 1 ­ (a) chlorophyll a, (b) chlorophyll b, (c) chlorophyll a+b contents and (d) chlorophyll ratio in extracts from different plant organs of the six phalaenopsis cultivars tested. bs= phalaenopsis cv. brother strips; gsc= phalaenopsis cv. golden sands canary; qbm= phalaenopsis cv. queen beer mantefon; rl1770= phalaenopsis cv. red lip 1770; syv3= phalaenopsis cv. sogo yukidian v3. leaves compared to the roots and flowers across all tested cultivars. similarly, the total chlorophyll (a + b) content in the leaves was higher than in the roots of the cultivars. additionally, the leaves of all cultivars displayed a higher chlorophyll a/b ratio than that observed in the roots (fig. 1). in this study, the leaves and roots of all tested cultivars, except for the roots of cv. red lip 1770 (0.62±0.01), had a chlorophyll a/b ratio higher than 1. therefore, in most of the tested cultivars, the chlorophyll a/b ratio in leaf and root extracts was below 2.0, while in a few cases, it was approximately 2.0. these findings indicate the predominance of chlorophyll a over chlorophyll b in both leaves and roots, reflecting their physiological roles in photosynthesis. the analysis of chlorophyll content among the tested phalaenopsis cultivars revealed significant variations in both leaf and root tissues. the cv. red lip 1770 exhibited the highest levels of chlorophyll a (1335.81±65.94 mg g⁻¹ dw) and chlorophyll b (695.80±11.00 mg g⁻¹ dw) in the leaves, yielding the highest total chlorophyll content (chlorophyll a + b) of 2031.61±75.83 mg g⁻¹ dw. conversely, cv. golden sands canary reported the lowest levels of chlorophyll a (598.02±65.01 mg g⁻¹ dw) and chlorophyll b (348.59±16.20 mg g⁻¹ dw), resulting in the lowest total chlorophyll content of 946.60±61.35 mg g⁻¹ dw. chlorophyll content in root extracts varied significantly among cultivars. cv. taipei gold gold star had the highest chlorophyll a content (343.53±8.96 mg g⁻¹ dw), while cv. golden sands canary exhibited the highest chlorophyll b content (239.67±1.99 mg g⁻¹ dw). in contrast, cv. sogo yukidian v3 showed the lowest levels of both chlorophyll a (123.49±26.32 mg g⁻ ¹ dw) and chlorophyll b (122.17 ± 28.63 mg g⁻¹ dw). total chlorophyll content in roots followed a similar trend, with cv. taipei gold gold star achieving the highest value (575.53±5.11 mg g⁻¹ dw), while cv. sogo yukidian v3 exhibited the lowest (245.66±14.44 mg g⁻¹ dw). among all the examined phalaenopsis flower samples, cv. golden sands canary exhibited the highest chlorophyll a (563.16±14.80 mg g⁻¹ dw) and chlorophyll b (973.66±25.26 mg g⁻¹ dw) contents. as a result, its total chlorophyll (chlorophyll a+b) content was also the highest in phalaenopsis cv. golden sands canary (1536.82±29.49 mg g⁻¹ dw). conversely, cv. red lip 1770 had the lowest chlorophyll a (2.18±2.10 mg g⁻¹ dw) and chlorophyll b (19.70±2.83 mg g⁻¹ dw) contents, resulting in the (0.024±0.00 mg qe·g⁻¹ dw). these findings highlight the significant variation in flavonoid distribution across different plant organs of the same phalaenopsis cultivar. total polyphenol content figure 3 depicts the total phenolics content in the leaves, flowers and roots of different phalaenopsis cultivars, revealing substantial variation across plant organs within the same cultivar and among different cultivars. flower sample from cv. golden sands canary exhibited the highest phenolics content (10.58±0.109 mg.gae g­1 dw) whereas those from cv. sogo yukidian v3 reported the lowest (1.60±0.01 mg.gae g­1 dw). leaf extracts from cv. golden sands canary again showed the highest phenolics content (6.55±0.49 mg.gae g­1 dw) whereas cv. brother strips had the lowest (1.75±0.01 mg.gae g­1 dw). similarly, root extracts from cv. taipei gold gold star were rich in phenolics (5.68±0.95 mg.gae g­1 dw) whereas the lowest was phalaenopsis cv. brother strips recorded the lowest amount (0.45±0.32 mg.gae g­1 dw). anthocyanin and carotenoid contents anthocyanin and carotenoid contents in different plant parts of phalaenopsis cultivars are shown in figures 4 (a) and 4 (b), respectively. among the tested cultivars, the flower extracts of cv. queen beer mantefon exhibited the highest anthocyanin content 130 adv. hort. sci., 2025 39(2): 125­135 lowest total chlorophyll (a+b) content among all tested flowers. additionally, all flower samples exhibited a chlorophyll a/b ratio of less than 1, except for cv. taipei gold gold star (1.70±0.56) and cv. sogo yukidian v3 (1.18±0.21). total flavonoid content figure 2 illustrates the total flavonoid content in extracts from various parts of phalaenopsis cultivars, highlighting significant variation in flavonoid distribution within the same cultivar. specifically, flower extracts contained significantly higher total flavonoid levels compared to other plant parts. notably, the highest total flavonoid content was observed in cv. brother strips (0.42±0.0 mg qe g­1 dw) whereas cv. queen beer mantefon exhibited the lowest (0.19±0.00 mg qe g­1 dw). on the other hand, root extracts, of cv. queen beer mantefon contained significantly higher levels of flavonoids (0.114±0.04 mg qe.g­1 dw) compared to cv. golden sands canary, which had the lowest value (0.057±0.01 mg qe g­1 dw). fig. 3 ­ total phenolic contents in different plant organs of the six phalaenopsis cultivars tested. bs= phalaenopsis cv. brother strips; gsc= phalaenopsis cv. golden sands canary; qbm= phalaenopsis cv. queen beer mantefon; rl1770= phalaenopsis cv. red lip 1770; syv3= phalaenopsis cv. sogo yukidian v3; tggs= phalaenopsis cv. taipei gold gold star. interestingly, both cv. sogo yukidian v3 and cv. red lip 1770 reported equal total flavonoid levels in root extracts (0.088±0.02 mg qe g­1 dw). moreover, leaf extracts of cv. sogo yukidian v3 exhibited the highest flavonoid content (0.082±0.00 mg qe·g⁻¹ dw), while cv. brother strips had the lowest fig. 2 ­ total flavonoid contents in extracts from different plant organs of the six phalaenopsis cultivars tested. bs= phalaenopsis cv. brother strips; gsc= phalaenopsis cv. golden sands canary; qbm= phalaenopsis cv. queen beer mantefon; rl1770= phalaenopsis cv. red lip 1770; syv3= phalaenopsis cv. sogo yukidian v3; tggs= phalaenopsis cv. taipei gold gold star. thathsarani et al. ‐ phytochemicals in selected phalaenopsis cultivars 131 (10.32±0.06 µmol g­1 dw), significantly surpassing all other cultivars. interestingly, this cultivar had the lowest flavonoid content in flower extracts. in contrast, cv. sogo yukidian v3 showed the lowest anthocyanin content among the flower extracts (0.34±0.00 µmol g­1 dw). although completely yellow cultivars such as taipei gold gold star showed no visible anthocyanin­related pigmentation, small amounts of anthocyanins were detected (0.38±0.01 µmol.g­1 dw). conversely, this cultivar displayed the highest carotenoid content in its flower extracts (260.50±16.97 mg.g­1 dw), significantly exceeding those of the other tested cultivars. flower extracts from cultivars with white tepals and colored labellum such as red lip 1770 and sogo yukidian v3, displayed lower contents of both anthocyanins and carotenoids. furthermore, cv. queen beer mantefon reported significantly higher content of both anthocyanins and carotenoids simultaneously, a pattern not observed in other cultivars. the yellow coloration of the flowers varied across cultivars, with brother strips displaying yellow only in the labellum, fig. 4 ­ the anthocyanin contents (a) and the carotenoids contents (b) in different plant organs of the six phalaenopsis cultivars tested. bs= phalaenopsis cv. brother strips; gsc= phalaenopsis cv. golden sands canary; qbm= phalaenopsis cv. queen beer mantefon; rl1770= phalaenopsis cv. red lip 1770; syv3= phalaenopsis cv. sogo yukidian v3; tggs= phalaenopsis cv. taipei gold gold star. while golden sands canary showing yellow pigmentation throughout the entire flower. despite these color differences, there was considerable variation in both anthocyanin and carotenoid contents among the flower extracts of the tested cultivars. root extracts generally displayed the lowest anthocyanin content among the tested plant organs. the root extracts of cv. taipei gold gold star exhibited the highest anthocyanin content (2.16 ± 0.05 µmol/g dw), while cv. sogo yukidian v3 showed the lowest (0.83±0.01 µmol/g dw). in contrast, leaf extracts displayed more variability, with cv. queen beer mantefon having the highest anthocyanin content (5.57±0.23 µmol/g dw), while cv. brother strips contained the lowest (4.09±0.10 µmol/g dw). the carotenoid content was generally higher in leaf extracts compared to other plant organs, with the exception of cv. taipei gold gold star. in this cv, carotenoid levels were 204.27±19.19 mg.g­1 dw in leaves and 260.50±16.97 mg.g­1 dw in flowers. root extracts consistently displayed lower carotenoid contents compared to other plant parts across all cultivars. among leaf extracts, cv. sogo yukidian v3 exhibited the highest carotenoid content (344.05 ± 32.95 mg g­1 dw), whereas cv. golden sands canary reported the lowest (184.99±29.29 mg.g­1 dw). the highest carotenoid content was found in root extracts from cv. taipei gold gold star (137.19 ± 1.07 mg/g dw), while the lowest was in those from cv. golden sands canary (27.61±2.92 mg/g dw). overall, both anthocyanin and carotenoid levels exhibited significant variation across the different cultivars and plant organs. 4. discussion and conclusions the genus phalaenopsis within the family orchidaceae holds significant prominence in the floriculture industry due to its prolonged blooming, captivating aesthetic, ease of cultivation, adaptability and its success in hybridization. hybridization offers breeders a unique opportunity to develop hybrids with enhanced physiological traits and diverse flower coloration. however, achieving such advancements relies heavily on the selection of parental cultivars with desirable traits, making this step important for creating novel cultivars. breeding strategies, plant tissue culture, and adv. hort. sci., 2025 39(2): 125­135 132 biotechnological advancements play a crucial role in developing novel traits and expanding the global commercialization of orchids (tiwari et al., 2024). understanding the specific phytochemical properties of different plant parts, both floral and vegetative, in phalaenopsis orchids provides valuable insights for informed cultivar selection, ultimately contributing to the development of resilient, high­quality, and commercially viable hybrids. comprehensive knowledge of anthocyanin and carotenoid contents in leaves, roots, and flowers provides insights into pigmentation, which is closely linked to flower coloration, as well as physiological responses such as l ight absorption and stress tolerance. these insights can enhance breeding efficiency and ensure targeted improvements in hybrid cultivars. this approach aligns with the findings of nguyen et al. (2018), emphasizing the importance of exploring the phytochemical composition of orchids to unlock their full potential for breeding and cultivation. by leveraging such information, breeders can innovate more effectively and meet the growing demand for unique and visually appealing phalaenopsis cultivars in the global market. the chlorophyll content analysis of phalaenopsis cultivars revealed that leaves exhibited significantly higher chlorophyll levels than roots, a pattern consistent with findings by trelka et al. (2010). this is expected, as leaves serve as the primary site for photosynthesis in plants. additionally, the chlorophyll a/b ratio was higher in leaves compared to roots, supporting observations by martin et al. (2010), who reported that epiphytic orchids generally have higher chlorophyll a/b ratios in leaves than in roots. notably, the chlorophyll a/b ratios in epiphytes are typically low, often around 2.0 or less. in this study, the chlorophyll a/b ratios in leaves and roots of all tested cultivars, except the roots of red lip 1770 exceeded 1.0. while several ratios were less than 2.0, most were approximately 2.0, which aligns with the characteristic shade adaptation of epiphytic orchids. a reduced chlorophyll a/b ratio, as observed in previous orchid studies, suggests a functional adaptation to low­light environments, allowing these plants to efficiently capture and utilize available light. the results obtained in this study further confirm the shade­adaptive traits of phalaenopsis cultivars. understanding these adaptations enhances the knowledge of their physiological responses and could support targeted breeding and cultivation strategies to optimize growth under various light conditions. higher chlorophylls content serves as a valuable indirect indicator of enhanced photosynthetic efficiency of the plant (lin and hsu, 2004). among the tested cultivars, red lip 1770 exhibited the highest chlorophyll a , chlorophyll b and total chlorophyll levels in its leaf extracts. this suggests that red lip 1770 may have superior photosynthetic efficiency, particularly under shade conditions, making this trait critical during its vegetative phase. interestingly, significant variations in chlorophyll content were observed in the roots. notably, the cultivars with the highest chlorophyll content in their leaf extracts were not the same as those exhibiting the highest chlorophyll content in their roots. this divergence indicates a broader variability in chlorophyll distribution between leaves and roots across the tested cultivars. phalaenopsis cv. golden sands canary demonstrated the highest chlorophyll content in its flower extracts among the tested cultivars. these findings highlight the differential accumulation of chlorophylls among phalaenopsis cultivars, which could be uti l ized for selecting superior parental lines in breeding programs. however, flowers generally reported lower chlorophyll content compared to leaves and roots. this difference could be attributed to the degradation of chlorophyll or the reduced activity of chlorophyll­synthesizing enzymes in flowers. a similar phenomenon has been observed in carnations, where petals contain significant amounts of chlorophyll during early development, which declines as the flower matures (nurcahyani et al., 2021). the presence of chlorophyll in flowers is essential for carbohydrate synthesis during their development, supporting energy requirements and metabolic processes. for phalaenopsis, investigating chlorophyll content at different stages of flower development is essential to gain a deeper understanding of its role and dynamics. such studies could provide insights into optimizing flowering conditions and improving overall plant health and productivity. significant variations in carotenoids and anthocyanin contents were observed in the flowers of studied phalaenopsis cultivars. cultivars with purple flowers such as queen beer mantefon exhibited the highest levels of both anthocyanin and carotenoid contents indicating that these pigments contribute synergistically to the flower’s coloration. thathsarani et al. ‐ phytochemicals in selected phalaenopsis cultivars 133 in contrast, yellow­flowered cultivars like taipei gold gold star contained anthocyanins in minimal quantities, with carotenoids serving as the dominant pigments. notably, flowers with white petals and yellow labellum, such as sogo yukidian v3, displayed lower carotenoid content compared to entirely yellow flowers. interestingly, an inverse relationship was observed between flower and leaf pigment content with cultivars displaying low anthocyanin and carotenoid levels in their flowers while exhibiting higher levels in their leaves. this suggests a potential redistribution or differential regulation of pigment synthesis in different plant parts. therefore, a wide variation in distribution of carotenoid and anthocyanin pigments was observed in different plant parts of the same cultivar. furthermore, some flowers exhibited visible coloration that did not align with their measured pigment content, highlighting the complexity of pigment interactions. as reported by narbona et al. (2021), variations in the type or ratio of pigments can influence flower color, while changes in pigment concentration primarily affect color intensity. the results also underline the intricate patterns and color variations in phalaenopsis flowers, which range from simple monochromatic tones to complex patterns. such variability is typical of orchids and other ornamental species like irises and crowfoots. this study provides valuable insights into the pigment composition and color diversity in phalaenopsis flowers, enhancing our understanding of their aesthetic and physiological characteristics. among the different plant parts analyzed, roots exhibited the highest flavonoid content, while flowers showed the lowest. flavonoids, known for their multifunctionality, play important roles in various physiological and ecological processes. in epiphytic orchids, such as phalaenopsis, the velamen radicum is vital for water and nutrient absorption, storage, and uv­b protection. flavonoids are also integral to leaves and other epidermal tissues, providing photoprotection and antioxidant properties that safeguard plants from environmental stressors (nguyen et al., 2018). in flowers, flavonoids serve as uv­b protectants and as attractants for pollinators, facilitating successful pollination. interestingly, cultivars with white petals, such as red lip 1770 and sogo yukidian v3, exhibited higher flavonoid content despite having minimal anthocyanin and carotenoid levels. this is consistent with findings that white petals, which reflect all wavelengths of visible light, often contain high concentrations of uv­absorbing flavonoids like flavones and flavonols (narbona et al., 2021). the significant variations in flavonoid content among cultivars may emphasize the potential of selecting cultivars with high flavonoid levels in specific plant parts as parental materials for breeding programs. a study on flowers, leaves, and stems of white clover cultivars revealed the potential for selecting cultivars with targeted concentrations of flavonoids (carlsen et al., 2008). hence, applying a similar selection approach in phalaenopsis cultivars could enhance desirable traits, including stress tolerance and aesthetic appeal. plant phenolics, a class of secondary metabolites, play a major role in mitigating oxidative stress by acting as strong antioxidant agents (trelka et al. , 2010). by neutralizing reactive oxygen species (ros), phenolics help minimize oxidative damage in plants, contributing to improved resilience and longevity. additionally, phenolics are involved in delaying senescence, thereby enhancing the functional and aesthetic lifespan of plant organs, as highlighted by cavaiuolo et al. (2013). in this study, golden sands canary exhibited the highest polyphenol content in both leaves and flowers, indicating its superior capacity to combat oxidative stress. conversely, bs showed the lowest polyphenol levels in leaf and root extracts, suggesting a comparatively weaker antioxidant defense. these differences suggest that polyphenol content is a critical determinant of a plant’s ability to endure oxidative stress, which, in turn, influences its storage life and performance as a potted plant or cut flower. selecting and breeding cultivars with high phenolics content could enhance stress tolerance and longevity in ornamental plants. such efforts would not only improve the plants’ defensive mechanisms but also potentially extend their market value and usability. orchid varieties have high phenolics and flavonoids compounds and have higher restraint power of free radicals. moreover, a relationship can be observed between phytochemicals and morphological traits (ebrahimi et al., 2020). the findings underscore the importance of incorporating antioxidant­related traits into breeding programs to produce robust and resil ient phalaenopsis cultivars with superior oxidative stress management capacities. breeding efforts for phalaenopsis have largely adv. hort. sci., 2025 39(2): 125­135 134 focused on producing white, pink, and red hybrids, leading to market saturation. orchid breeders are now shifting their focus toward developing flowers with unique pigmentation patterns distributed across various regions of the flower (hsu et al., 2018). the findings of this study highlighted the potential of leveraging cultivars with differential pigment accumulation to achieve this goal. for instance, combining anthocyanin­ and carotenoid­rich cultivars such as phalaenopsis cv. queen beer mantefon with white cultivars like red lip 1770 and sogo yukidian v3 could result in novel hybrids with diverse and striking pigmentation patterns. the creation of novel flower coloration patterns is critical for maintaining consumer interest in phalaenopsis orchids, as aesthetic appeal remains a key factor driving market demand. among the tested cultivars, queen beer mantefon stood out for its high concentrations of both anthocyanin and carotenoids, while phalaenopsis cv. brother strips and golden sands canary reported the highest flavonoid and polyphenol contents, respectively. these traits indicate potential resilience and extended storage life, adding further value to these cultivars as breeding materials. additionally, the study revealed significant variation in chlorophyll, flavonoid, and polyphenol content across the vegetative parts of different cultivars. these traits are equally important for breeding robust plants with enhanced stress tolerance, longevity, and overall vigor. in conclusion, this study provides insights into floral pigmentation and phytochemical composition of vegetative organs, facilitating the identification and selection of desirable traits for phalaenopsis breeding programs. by selecting parental materials based on pigment profi les and vegetative characteristics, breeders can develop new cultivars with enhanced aesthetic appeal and improved physiological performance. such advancements will not only enhance the ornamental value of phalaenopsis orchids but also strengthen their adaptability and commercial potential. despite these findings, the relationship between phytochemicals and the physiological traits in commercial phalaenopsis cultivars remains unexplored. among the studied commercial cultivars, taipei gold gold star, golden sands canary and queen beer mantefon can be recommended as potential parental candidates for hybridization due to their distinctive floral colorations and robust vegetative traits for adaptability. acknowledgements authors wish to acknowledge the national agricultural research policy for providing financial assistance for this research under the grant no: narp/21/uk/sc/01. references axiotis e., angelis a., antoniadi l., petrakis e.a., skaltsounis l.a., 2021 ­ phytochemical analysis and dermo‐cosmetic evaluation of cymbidium sp. 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university, p.o. box 1247, sana’a,yemen. key words: chilli, germination, salinity, tolerance. abstract: evaluating the genotypes of vegetables is a critical component in establishing effective plant breeding programs. in this study, nine genotypes of yemeni capsicum spp. were collected from various regions in yemen to assess their germination capabilities under different salinity levels (0, 50, 100, 150, 200, and 250 mm). the experiment was conducted using a factorial completely randomized design (crd) with three replicates. results indicated that increasing salinity levels led to a gradual decline in germination percentage (grp), mean germination rate (mgr), germination time (mgt), and seedling dry matter (dm%). additionally, variations in the genotypes’ responses to salt stress were evaluated using four models: the slope of the regression line (b), the integrated evaluation approach (dv), principal components, and the genotypes’ salinity susceptibility index (gssi). all the classified of genotypes was different by analysis models. based on the integrated value (dv), the genotypes were classified into four sensitivity categories: resistant (a, d, and g), moderately resistant (f and v2), sensitive (s and z), and highly sensitive (h and v3) to salinity stress. the findings demonstrate that the slope of the regression line is a reliable indicator for assessing genotype sensitivity to salinity, aligning consistently with the integrated value model (dv). the insights gained from this research are expected to significantly inform breeding strategies aimed at developing salt­tolerant chilli pepper cultivars, which are essential for successful cultivation in challenging environmental conditions. 1. introduction hot peppers (capsicum spp.) are an important vegetable crop cultivated globally in warm and temperate regions for various purposes (comparini et al., 2021).they are highly valued for their nutritional content, particularly their vitamin c and capsaicin levels, which provide notable health benefits (taiti et al., 2024) and antimicrobial activity (serio et al., 2024). this adaptable crop can be consumed fresh, as a spice, or in dried form (taiti et al., 2015; arraf and al­madhagi, 2025). over the past (*) corresponding author: i.madhagi@su.edu.ye citation: al­madhagi i., arraf e., 2024 ­ evaluation of salinity tolerance of yemeni chili pepper genoty‐ pes during germination by using different statical‐ ly models. ­ adv. hort. sci., 38(4): 371­392 orcid: ai: 0000­0001­5568­134x ae: 0009­0009­8252­2323 copyright: © 2024 al­madhagi i., arraf e. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 6 october 2024 accepted for publication 11 december 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16693 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-5568-134x http://orcid.org/0009-0009-8252-2323 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 371­392 372 50 years, global production has steadily increased (fao, 2022). in 2022, yemen contributed approximately 18,22 tons of hot peppers, cultivated on 3,24 hectares, representing roughly 2.3% of global production (fao, 2022). globally, hot peppers were grown on an estimated 689,33 hectares, yielding a remarkable 788,032.04 tons (fao, 2022). salinity poses a significant challenge to agriculture in arid and semi­arid regions due to the accumulation of dissolved salts caused by soil processes, irrigation practices, drainage patterns, and overuse of fertilizers (khondoker et al., 2023). urban expansion and competition for water resources further exacerbate the issue (suarez, 2001; sahbeni et al., 2023). yemen features a range of climates, including semi­humid, semi­arid, and arid tropical types (alhadi et al., 2023). yemen’s extensive coastal region, characterized by a warm climate conducive to pepper cultivation, particularly during the autumn and winter seasons, faces significant challenges related to excessive salinity. an estimated 37,100 hectares of non­desert agricultural land are affected by salinity, while an additional 12 million hectares experience erosion. furthermore, 3.8 million hectares suffer from varying degrees of salinity, with 3­5% of the land at risk of desertification (usaid, 2010; gregory et al., 2018). yemen is home to numerous chilli genotypes (colonna et al., 2019), distributed across regions with diverse climates, altitudes, and soil properties (aldobai and al­shabi, 2010). salinity significantly impairs plant growth through mechanisms such as cell membrane destabilization (hasegawa et al., 2000; mushtaq et al. , 2020), disruption of photosynthesis (momenpour and imani, 2018; zhou et al., 2023), nutrient imbalances (munns, 1993), and cellular damage (hasanuzzaman et al., 2021; ahmad et al., 2022). salt tolerance varies across species, genotypes, and cultivars (khoshsokhan et al., 2012), driven by mechanisms such as ion partitioning and proline synthesis (hasegawa et al., 2000; farooqi et al., 2021). these adaptations, along with oxidative stress management and regulated growth responses (binzel et al., 1985; long et al., 1994; maggio et al., 2007; hasanuzzaman et al., 2021), mitigate stress effects but often reduce overall yield, resulting in smaller plants (greenway and munns, 1980; naeem et al., 2020). furthermore, the response to salt stress is contingent on the growth stage, with certain studies indicating variations in tolerance across different developmental phases (mangal et al., 2023; roșca et al., 2023). notably, vegetable plants, particularly during early life stages, exhibit heightened sensitivity to salt stress, especially during germination and seedling growth (miceli et al., 2021). research on salt stress tolerance in various crops, including pepper (qiu et al., 2017) and fenugreek (al­ maqtary et al., 2024), often focuses on specific salt concentrations and exposure durations. plant responses to salt stress also vary based on climatic conditions and soil characteristics (läuchli and epstein, 1990; munns and gilliham, 2015). elevated salinity levels impede seed germination by reducing water absorption due to osmotic stress, followed by ionic stress. increased salt concentrations in the germination medium negatively affect seed embryo vitality by disrupted ion transport (zowain, 2014). an negative correlation exists between salinity and germination in various vegetable plants, including cucumbers (bolton and simon, 2019), sweet peppers (chartzoulakis and klapaki, 2000; hannachi and van labeke, 2018; karalija et al., 2024), and tomatoes (singh et al., 2012). for example, chilli pepper and tomato seeds failed to germinate at 200 mm nacl (loganayaki et al., 2020). increased salinity prolongs germination time and lowers germination rates (al­swedi et al., 2020; dawd and abdulla, 2020). however, aktas et al. (2006) observed genetic variability in salt accumulation and leaf damage in peppers exposed to 150 mm nacl for 10 days, indicating potential yield discrepancies. different vegetable genotypes exhibit varying levels of resistance to salt stress, as demonstrated in studies of 26 tomato genotypes (devi and arumugam, 2019), 17 chill i pepper genotypes (howlader et al., 2018), and 13 cucurbita genotypes (horuz et al., 2022). the degree of genotype tolerance to salinity depends on inherent resistance mechanisms, including metabolic responses activated during salt stress (horuz et al. , 2022). chill i peppers are classified as either sensitive (lycoskoufis et al., 2005; giorio et al. , 2020; ntanasi et al. , 2024) or moderately salt­tolerant (maas and hoffman, 1977; chartzoulakis and klapaki, 2000; zamljen et al., 2022). among the vegetable plants tested by loganayaki et al. (2020), chilli exhibits greater salinity sensitivity compared to tomatoes and cucumbers. salinity and alkalinity, as critical abiotic stresses, significantly reduce the growth and productivity of pepper plants (chartzoulakis and klapaki, 2000; al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 373 demir and mavi, 2008; amirinejad et al., 2017). ongoing research efforts by institutes and universities aim to develop agricultural techniques to mitigate the adverse effects of salinity on vegetable crop production. these efforts include breeding salt­ tolerant plants (zhu et al., 2000; singla­pareek et al., 2003; yang et al., 2005), employing grafting techniques on vegetables (santa­cruz et al., 2002; edelstein et al., 2005; estan et al., 2005) or fruit (momenpour and imani, 2018), utilizing growth regulators (sakamoto and murata, 2001; abrahám et al., 2003; hamdia et al., 2004; amirinejad et al., 2017), and managing soil salinity through excessive irrigation (semiz et al., 2014; sahbeni et al., 2023; tarolli et al., 2024). exploring genetic diversity and understanding the physiological traits of various vegetable genotypes will provide a foundation for future research, including selective breeding and grafting. therefore, the aim of this study is to evaluate the salinity sensitivity stress of yemeni chilli genotypes. this research could significantly inform breeding strategies for chilli by examining local genotypes based on significant physiological traits. 2. materials and methods chilli seed collection chilli pepper seeds from local genotypes were collected from various regions in yemen (fig. 1, table 1). additionally, the f1 shamakh pepper cultivar, designated as the f code, was included in the study. this cultivar, commonly grown in yemen, was supplied by agro star company, the exclusive agent in yemen for united genetics company (usa) (fig. 2). experimental layout the study was carried out in the horticultural laboratory using a factorial experimental design based on a completely randomized design (crd). the experiment included three replicates, with each replicate comprising 10 seeds. seeds from different genotypes were collected and stored in specially labeled glass containers for future experimental use. fig. 1 ­ the map of yemen shows the geographic origin of the chili pepper genotypes used in this experiment. the sample names reflect the geographic origin of the sam­ ples. table 1 ­ name and origin of nine chili genotypes used in the study * main regions where yemeni chili genotypes were gathered for the research. research code species common name area of distribution (latitude) no. fruit per node fruit attitude spiciness a c. annuum abyani abyan (13° 02' 60.00" n) * 1 hanging sweet lahij (13° 02' 60.00" n) z c. frutescens zaaitri hudaidah (14° 12'00' n)* 1 upright hot taiz (13° 33' 59.99") ibb (13° 58' 0.01" n) h c. frutescens haimi sana'a (15.36 n, 44.191006 2 upright hot d c. annuum dhamari dhamar (15° 39' 59.99" n)* 1 hanging hot ibb (13° 58' 0.01" n) v2 c. chinense jawfi 2 al­jawf ( 16° 46' 59.99" n)* 2 semi upright hot v3 c. chinense jawfi 3 al­jawf ( 16° 46' 59.99" n)* 2 hanging hot g c. annuum hajjai hajjai (15° 41' 59.99" n)* 1 hanging hot s c. frutescens sa'ddi sa'dah (16° 56' 5.39" n)* 1 hanging hot f c. annuum shamakh 1 hanging adv. hort. sci., 2024 38(4): 371­392 374 seed sterilization was performed using a solution containing 10% sodium hypochlorite (naclo), 90% distilled water, and a drop of tween 20. this process lasted for five minutes. post­sterilization, the seeds were subjected to a thorough rinsing regime, involving multiple washes with running water, subsequent rinses with distilled water, and finally, a drying phase. to commence the experimental protocol, 3 milliliters of a sodium chloride (nacl) solution with different concentrations rate: 0, 50, 100, 200, and 250 mm, were dispensed onto filter paper within petri dishes. for control treatments, 3 milliliters of distilled water were added to petri dishes designated for the control group, which did not receive any salinity treatment. parameters of study data on the seed germination process were carefully recorded daily over a 21­day period, beginning from the start of the experiment. furthermore, photographic evidence was collected daily for every treatment and replication under examination. the calculated metrics of the germination data is detailed in table 2. in the germination equations: n, the total number of seeds in each experimental unit; ni , the number of seeds germinated in the ith time; k, the last day of germination evaluation; ti , the period from the commencement of the experiment to the i th observation; gi, the number of seeds germinated in the i th time; and xi , the number of days from sowing; sdg denotes the germination standard deviation. salinity sensitivity index the salinity sensitivity index (ssi) values for the each single parameter were calculated separately as (horuz et al., 2022): ssi = salinity level ­ control x 100 (7) control table 2 ­ the various metrics used to calculate the process of seed germination in the experiment no. measurements unit equation references 1 germination percentage (grp) % 1 (kader, 2005) 2 mean germination time (mgt) day 2 (ranal and santana, 2006) 3 mean germination rate (mgr) day­1 3 (ranal, 1999) 4 germination speed coefficient (gsc) % 4 (ranal, 1999) 5 coefficient of velocity of germination (cvg) % 5 (ranal, 1999) 6 dry matter (dm) % 6 (al­madhagi and al­sharagi, 2019) fig. 2 ­ local chilli pepper genotypes utilised in this experiment. the attitude of the peduncle explains the fruit behaviours, with the down peduncle denoted as v2, h, and z, indicating fruit with upright habits. the length and width of the fruit for different genotypes are shown in cm. the other differences between the fruit of genotypes of chili are clear from colour, size, direction, shape, neck at base of fruit, shape at blossom end, appendage and pedicel with fruit. al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 375 evaluation of salt tolerance by an integrated evaluation system the examination of salt tolerance across all genotypes was comprehensively conducted through the application of subordinate function and standard deviation coefficient techniques, utilizing the stress intensity (si) metric to evaluate the effects of salt stress on nacl concentrations surpassing 100 mm as described by xie et al. (2021). the value of each evaluation index was calculated by the following equations: si = control ­ salinity level x 100 (8) control x(𝑢) x ­ x min (9) x max ­ x min x(𝑢) 1 ­ x ­ x min (10) x max ­ x min (11) (12) (13) (14) firstly, standardization of index data was conducted using the subordinate function as defined in [equations (8) and (9)]. for traits negatively correlated with salinity tolerance (nacl), the dependency value was determined using the inverse subordinate function (equation 9). conversely, for traits positively correlated with salinity tolerance, the dependency value was calculated using equation (8). in this context, x(𝑢) represents the subordinate function value of the μth indicator, x denotes the observed indicator value, while xmax and xmin indicate the maximum and minimum values of the indicator, respectively [equation (10). ̅ 𝑋̅̅𝑗 signifies the average of the jth assessment index, with n denoting the total number of genotypes, and xij referring to the jth evaluation index of the ith genotype [equation (11)]. vj represents the standard deviation coefficient of the jth evaluation index, with xj depicting the jth evaluation index of genotypes [equation (12)]. wj stands for the weighting coefficient of the jth evaluation index [equation (13)]. 𝑢(xj) corresponds to the subordinate function value of the jth evaluation index. dv denotes the aggregated values for salt tolerance in chilli pepper [equation (14)]. a lower in the dv value indicates higher salt tolerance. genotypes salinity susceptible index (gssi) the tolerance genotypes salinity sensitivity index was calculated for germination percentage by the formula (afzal et al., 2022): (15) where gs: an average of certain genotypes under salinity stress conditions, go: an average of genotypes under optimum conditions, ags: an average of all genotypes under salinity stress conditions, and ago: an average of all genotypes under optimum conditions. the criterion for determining the tolerance level to salinity stress was this: if the gssi value is 0.5, then the genotype is tolerant (t), if 0.5 <gssi≤ 1.0, the genotype is moderate (m), and if gssi> 1.0 then the genotype is sensitive (s) (pasaribu et al., 2021). estimating genotype sensitivity to salinity using slope of the regression line the sensitivity of each genotype was evaluated using the r‐square values and slope coefficients calculated for each parameter. the r­square value serves as an indicator of the significance of a trait, with higher values suggesting greater relevance. in this study, the overall r‐square values for each trait were considered a measure of their importance in assessing genotype sensitivity to salinity stress. according to the established hypothesis, genotypes exhibiting lower slope values in the context of sub­ salinity treatments are classified as resistant. this implies that these genotypes maintain their performance despite increasing salinity levels, thereby demonstrating a higher tolerance to salinity stress compared to those with steeper slope values. data analysis the data were analyzed using the statistical analysis program genestat 12, then the means of single factors (genotypes or salinity) were compared 376 adv. hort. sci., 2024 38(4): 371­392 using the least significant difference test (lsd0.05) (p< 0.05). the values of the means of the interactions (genotype × salinity) were compared using a multiple range test (p< 0.05). sas 17 was used for correlation analysis and the principal component, while spss 21 was used for regression analysis for each genotype. 3. results germination percentage (grp) all factors examined, including salinity stress levels, genotype, and their interaction, had highly significant effects on the germination percentage (grp) of chilli genotypes (p<0.001). among the sources of variation, genotype explained approximately 73% of the total variation, while salinity stress accounted for 27% of the observed changes in grp (table 3). the mean grp for the genotypes across all genotypes ranged from a low of 53.9±6.77% for the g genotype to a high of 97.2±1.35% for the d genotype (table 4). these values were significantly different (p < 0.05) from each other except between the f and z genotypes. increasing salinity levels dramatically reduced grp, decreasing from 88.89 ± 2.68% at 0 mm to 62.22 ± 4.90% at 250 mm nacl. the reduction rate was approximately 0.118% for each additional millimole of nacl, as described by the regression equation: grp = 94.97 – 0.118 (nacl), with an r2 of 0.539. among the genotypes, the d genotype exhibited the lowest salinity sensitivity in terms of grp, with the lowest slope value (b = ­0.013, r2 = 0.042), ranking first based on the regression slope value. the remaining genotypes were ranked as follows: a, s, z, v2, f, v3, and g. the h genotype showed the highest sensitivity to salinity (b = 0.25, r2 = 0.62) ranking last (order = 9). notably, certain genotypes maintained higher grp at higher salinity levels (250 mm), with s (83.3%), a (73.3%), and z (73.3%) showing no significant difference from the control treatment (0 mm) (table 4). the interaction between genotype and salinity stress revealed that the d genotype achieved a grp of 100% under control condition, significantly differing from the a and v2 genotypes. at the 50 mm nacl, genotype a exhibited the lowest grp, which was significantly different from the other genotypes. although the g genotype maintained a high grp in the control treatment, its performance declined with salinity levels exceeding 50 mm, with reductions of 30%, 46.67%, and 36.67% at higher salinity concentrations. similarly, the f hybrid cultivar could not maintain a high grp beyond 200 mm nacl (table 4). compared to the control, the d genotype displayed significantly greater salt tolerance for grp across all salinity stress levels, with a positive salt sensitive index (ssi) of 33.3% at 0, 50, 150, and 250 mm nacl. in contrast, the v2, h, s, and a genotypes showed significant salt resistance up to 150 mm nacl. the g, v3, and z genotypes exhibited the lowest salt resistance (ssi) up to 50 mm nacl, while the f genotype showed reduced resistance up to 100 mm nacl (fig. 3). mean germination time (mgt) the mean germination time (mgt) of all chilli genotypes was significantly influenced by salinity stress levels, genotype, and their interaction (genotype × salinity) compared to the control treatment (p<0.001). genotype accounted for approximately 62% of the total effect (100%), while the remaining 38% was attributed to the influence of salinity on mgt (table 3). across the genotypes, mgt varied from the shortest time of 5.82 days for the d genotype to the longest times of 11.52 days and 11.16 days for the g and v2 genotypes, respectively (table 5). these differences were statistically significant (p<0.05). table 3 ­ the predictive capabilities that explaining the contribution of salinity and genotypes to the variation in the total score (100%) that affected the germination parameters. the chosen model is a forward stepwise grp= germination percentage; mgt= mean germination time; mgr= means germination rate; dm%= dry matter, gsc= germination speed coefficient; cvg= coefficient of velocity of germination. factors grp mgt mgr gsc cvg dm% genotypes 73.0 62.0 70.0 70.2 68.0 100 nacl 27.0 38.0 30.0 29.8 32.0 0 al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 377 ta bl e 4 ­ in te ra ct io n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n th e ge rm in at io n pe rc en ta ge (g rp ) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . g en ot yp es n ac l m m m ea n g en ot yp es r2 co ef fic ie nt o rd er * * 0 50 10 0 15 0 20 0 25 0 c b a 9. 28 ± 0 .7 l­ r 7. 64 ± 1 .2 7 r­ w 7. 47 ± 0 .8 3 s­ w 10 .9 3 ± 0. 15 f­ l 11 .7 1 ± 1. 4 d­ i 14 .1 0± 0 .0 5 ab 10 .1 91 ± 0 .6 5 0. 53 7. 34 0. 02 3 7 d 4. 32 ± 0 .6 3 a 4. 23 ± 0 .3 3 a 6. 57 ± 1 .1 1 v­ y 5. 6 ± 0. 38 x ­a 8. 14 ± 1. 16 q ­v 6. 1± 2 .0 1 w ­z 5. 83 ± 0 .4 9 g 0. 21 4. 42 0. 01 1 2 f 8. 47 ± 0 .3 3 p­ u 8. 53 ± 0 .1 8 o­ u 9. 77 ± 0 .2 3 j­q 10 .7 8 ± 0. 57 g ­m 11 .7 6± 0 .4 8 c­ i 14 .1 6± 0 .3 2 ab 10 .5 8 ± 0. 49 b c 0. 86 7. 78 0. 02 2 5 g 9. 85 ± 0 .3 9 j­p 9. 61 ± 0 .4 4 k­ q 12 .3 2 ± 0. 88 c ­g 14 .3 3 ± 2. 19 a 11 .3 7± 0 .6 4 e­ j 11 .6 7 ± 0. 33 d ­i 11 .5 2 ± 0. 55 a 0. 13 7 10 .3 5 0. 00 9 1 h 5. 58 ± 0 .3 3 x­ a 6. 56 ± 0 .8 7 v­ y 8. 82 ± 0 .4 5 n­ t 10 .8 8 ± 0. 33 g ­l 13 .1 1± 1 .3 4 a­ d 12 .9 3 ± 1. 16 a ­e 9. 65 ± 0. 77 d e 0. 82 5. 46 0. 03 3 9 s 4. 44 ± 0 .1 6 za 4. 46 ± 0 .2 9 za 4. 5± 0 .1 0 za 5. 43 ± 0 .5 3 y­ a 7. 91 ± 0 .3 7 r­ v 9. 75 ± 0 .9 8 j­q 6. 08 ± 0 .5 2 g 0. 73 3. 38 0. 02 2 5 v2 9. 14 ± 0 .9 2 m ­s 10 .5 2 ± 1. 23 h ­n 10 .2 2 ± 0. 62 i­ o 11 .0 8 ± 0. 28 f­ k 12 .6 1 ± 1. 56 b ­f 13 .4 1 ± 0. 9 a­ c 11 .1 6 ± 0. 49 a b 0. 46 9. 12 0. 01 6 3 v3 5. 73 ± 0 .0 8 x­ a 6. 94 ± 0 .5 8 u­ y 6. 93 ± 0 .5 5 u­ y 10 .4 1 ± 0. 9 i­n 12 .2 8± 1 .0 2 c­ g 12 .1 4 ± 0. 77 c ­ h 9. 07 ± 0 .6 7 e 0. 79 1 5. 39 0. 02 9 8 z 4. 74 ± 0 .3 9 za 5. 53 ± 0 .3 5 y­ a 8. 12 ± 0 .6 1 q­ v 7. 89 ± 0 .8 8 r­ v 7. 25 ± 0 .3 5 t­ x 11 .2 5± 0 .4 1 e­ k 7. 47 ± 0 .5 4 f 0. 67 7 4. 79 0. 02 1 4 m ea n n ac l 6. 84 ±0 .4 5 e 7. 14 ±0 .4 5 e 8. 30 ±0 .4 6 d 9. 70 4± 0. 59 c 10 .6 8± 0. 50 b 11 .7 2± 0. 53 a m g t= 6 .4 5 + 0. 02 08 (n ac l) ( r 2= 0 .7 75 ) * g en ot yp es n ac l m m m ea n ge no ty pe s r2 co ef fic ie nt o rd er ** 0 50 10 0 15 0 20 0 25 0 c b a 76 .6 7 ± 14 .5 3 d­ g 73 .3 3 ± 6. 67 e ­h 73 .3 3 ± 6. 67 e ­h 76 .6 7 ± 6. 67 d ­g 56 .6 7 ± 8. 82 h ­j 73 .3 3 ± 8. 82 e ­h 71 .6 7 ± 3. 55 e 0. 04 5 76 .1 5 ­0 .0 36 2 d 96 .6 7 ± 3. 33 a ­c 10 0 ± 0a 10 0 ± 0a 10 0 ± 0 a 86 .6 7 ± 3. 33 a ­e 10 0 ± 0a 97 .2 2 ± 1. 35 a 0. 04 2 98 .8 8 ­0 .0 13 1 f 10 0 ± 0 a 10 0 ± 0a 10 0 ± 0a 83 .3 3 ± 6. 67 a ­f 90 ± 1 0 a­ e 56 .6 7 ± 3. 33 h ­j 88 .3 3 ± 4. 14 b 0. 56 10 7. 14 ­0 .1 50 6 g 83 .3 3 ± 6. 67 a ­f 90 ± 5 .7 7a ­e 36 .6 7 ± 18 .5 6 kl 30 ± 0 l 46 .6 7 ± 3. 33 j­ l 36 .6 7 ± 14 .5 3 kl 53 .8 9 ± 6. 77 f 0. 41 80 .3 1 ­0 .2 1 8 h 93 .3 3 ± 3. 33 a ­d 96 .6 7 ± 3. 33 a ­c 96 .6 7 ± 3. 33 a ­c 93 .3 3 ± 3. 33 a ­d 50 ± 5 .7 7 i­k 36 .6 7 ± 13 .3 3 kl 77 .7 8 ± 6. 39 d e 0. 62 10 8. 30 ­0 .2 5 9 s 93 .3 3 ± 6. 67 a ­d 96 .6 7 ± 3. 33 a ­c 93 .3 3 ± 3. 33 a ­d 10 0 ± 0 a 76 .6 7 ± 3. 33 b ­g 83 .3 3 ± 8. 82 a ­f 90 .5 7 ± 2. 62 b 0. 21 97 .9 3 ­0 .0 59 3 v2 66 .6 6 ± 3. 33 f­ i 86 .6 7 ± 8. 82 a ­e 86 .6 7 ± 8. 82 a ­e 86 .6 7 ± 6. 67 a ­e 60 ± 2 0. 82 g ­j 50 ± 1 1. 55 i­ k 72 .7 8 ± 5. 29 e 0. 1 3 84 .4 4 ­ 0 .0 93 5 v3 96 .6 7 ± 3. 33 a ­c 96 .6 7 ± 3. 33 a b 90 ± 0 a ­e 93 .3 3 ± 3. 33 a ­d 63 .3 3 ± 18 .5 6 g­ j 50 ± 1 5. 28 i­ k 81 .6 8 ± 5. 62 c d 0. 48 10 5. 23 ­0 .1 88 7 z 93 .3 3 ± 3. 33 a ­d 10 0 ± 0 a 83 .3 3 ± 12 .0 2 a­ f 83 .3 3 ± 3. 33 a ­f 93 .3 3 ± 3. 33 a ­d 73 .3 3 ± 3. 33 e ­h 87 .7 9 ± 2. 87 b c 0. 24 96 .3 4 ­0 .0 69 4 m ea n n ac l 88 .8 9 ± 2. 68 a b 93 .3 3± 2 .0 6 a 84 .4 4± 4 .3 4b c 82 .9 6± 4 .1 3c 69 .2 6± 4. 43 d 62 .2 2± 4. 90 e g rp = 9 4. 97 – 0 .1 18 (n ac l ) , ( r2 = 0 .5 39 ) * ta bl e 5 ­ in te ra ct io n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n th e m ea n ge rm in at io n tim e (m g t) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . ** t he g en ot yp es a re li st ed in d es ce nd in g or de r a cc or di ng o n th e va lu es o f t he re gr es sio n slo pe li ne (b ). adv. hort. sci., 2024 38(4): 371­392 378 negative sensitivity at 50 mm nacl. in contrast, all other genotypes displayed positive ssi across all salinity levels. the ssi for the h, v3, s , and f genotypes increased linearly with rising salinity levels. the highest ssi for mgt was recorded at the 250 mm salinity level (139.2%) for the z genotype, whereas the lowest ssi was at 100 mm nacl (­19.8%) for the a genotype (fig. 4). germination speed coefficient (gsc) germination speed coefficient (gsc) of chilli genotypes was significantly influenced by salinity stress, genotype, and their interaction (genotype × salinity) compared to the control treatment (p < 0.001). as shown in table 3, genotype accounted for approximately 70.2% of the total variation, with the remaining 29.8% attributed to the effect of salinity on gsc. gsc values between genotypes ranged from the lowest (8.94 ± 0.35%) in genotype g to the highest (19.146 ± 1.42%) in genotype d (table 6). these values were statistically different from other genotypes, except for genotype s , where no significant differences were observed between d and s, or between h and v3.salinity stress led to a significant reduction in gsc across all genotypes as salt concentrations increased beyond 50 mm nacl (p < 0.05). gsc decreased from 16.4 ± 1.09% at 0 mm to 9.5 ± 0.88% at 250 mm nacl. regression analysis indicated that for every 1 mm increase in nacl concentration, gsc declined by approximately 0.030% (gsc = 16.518 ­ 0.030 × nacl, furthermore, the mgt for all genotypes significantly increased (p < 0.05) with rising salt levels compared to the control, escalating from 6.84 ± 0.45 days at 0 mm to 11.72 ± 0.53 days at 250 mm. regression analysis indicated an increase of approximately 0.0202 days for every 1 mm nacl addition to the control level (mgt = 6.45 + 0.0208 nacl) (r2 = 0.775). the genotypes were arranged in descending order of mgt response as follows: g, d, v2, z, f, s, a, v3, and h. genotype g exhibited the least change in mgt (b = 0.009, r2 = 0.137), while the h genotype showed the most substantial change (b = 0.033, r2 = 0.815). the interaction between genotypes and salinity stress showed a variation of results. mgt of the g genotype was significantly higher (9.85 ± 0.39 days) than that of the other genotypes in the control treatment (0 mm). at salinity levels ranging from 50 to 250 mm, the mgt for the g and v2 genotypes was significantly greater than that of the other genotypes (p<0.05). notably, the d genotype consistently exhibited the lowest mgt across all salinity levels. while several genotypes demonstrated increased mgt at the highest salinity levels, genotypes f, h, and s maintained their mgt up to 150 mm nacl, whereas g, v3, and z maintained their mgt up to 100 mm nacl (table 5). salinity sensitivity index (ssi) compared to the control indicated that the a genotype exhibited a negative sensitivity to salinity up to 100 mm. mgt values for both 50 mm and 100 mm nacl were lower than those of the control, the g genotype showed fig. 3 ­ salinity sensitivity index (ssi) for germination percentage (grp) in chili genotypes. values represent the mean of three replicates. different letters denote significant dif­ ferences according to the duncan multiple range test (dmrt). fig. 4 ­ salinity sensitivity index (ssi) for mean germination time (mgt) in chili genotypes. values represent the mean of three replicates. different letters denote significant dif­ ferences according to the duncan multiple range test (dmrt) at p<0.05. al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 379 r2 = 0.758). genotype s exhibited the greatest reduction in gsc (b = ‐0.05, r2 = 0.78), ranking 9th in the rate of change, whereas genotype g exhibited the smallest change (b = ‐0.01, r2 = 0.216), ranking 1st. genotypes v2, f, a, d, v3, z, and h ranked 2nd through 8th, respectively, in the table 6 are reported the values of the genotypes according to their variation rate. in terms of the genotype × salinity interaction, genotype d displayed the highest gsc (24.07 ± 3.15%) at 0 mm nacl (p<0.05), with no significant difference from genotype s . at salinity levels between 50 and 250 mm, genotypes d and s exhibited significantly higher gsc than other genotypes (p<0.05), with genotype d maintaining the highest gsc at 250 mm nacl. the salinity sensitivity index (ssi) analysis for gsc revealed that genotype a exhibited a positive ssi up to 100 mm nacl, exceeding the control values. genotypes d and g also demonstrated positive ssi at 50 mm nacl. conversely, genotypes z, v3, v2, h, and f exhibited negative ssi across all salinity levels, with ssi values for genotypes f, h, s, v3, and v2 decreasing linearly as salinity levels increased. the highest ssi for gsc was observed at 100 mm nacl (25.3%) in genotype a, while the lowest was recorded at 200 mm nacl (­57.97%) in genotype h (fig. 5). mean germination rate (mgr) the mean germination rate (mgr) of chill i ta bl e 6 ­ in te ra ct io n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n g er m in at io n sp ee d co ef fic ie nt (g sc ) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . ** t he g en ot yp es a re li st ed in d es ce nd in g or de r a cc or di ng o n th e va lu es o f t he re gr es sio n slo pe li ne (b ). g en ot yp es n ac l m m m ea n r2 co ef fic ie nt o rd er * * 0 50 10 0 15 0 20 0 25 0 g en ot yp es c b a 10 .9 1± 0. 89 k ­s 13 .8 1± 2. 21 g ­j 13 .7 1± 1. 47 g ­j 9. 15 ±0 .1 3 p­ x 8. 76 ±0 .9 4 q­ x 7. 09 ±0 .0 3 x 10 .5 7± 0. 74 d 0. 39 13 .3 4 ­0 .0 22 4 d 24 .0 7± 3. 15 a 23 .9 3± 1. 97 a b 16 .3 2± 3. 23 e ­g 18 .0 3± 1. 26 d ­f 12 .8 8± 2. 08 h ­l 19 .6 5± 4. 98 c d 19 .1 46 ±1 .4 2 a 0. 2 22 .9 7 ­0 .0 31 5 f 11 .8 5± 0. 45 i­ o 11 .7 3± 0. 25 i­ p 10 .2 5± 0. 24 l­ u 9. 33 ±0 .5 2 o­ x 8. 53 ±0 .3 5 r­ x 7. 07 ±0 .1 6 x 9. 79 ±0 .4 3 de 0. 9 12 .2 5 ­0 .0 2 3 g 10 .1 8± 0. 4 n­ v 10 .4 5± 0. 49 k ­t 8. 24 ±0 .5 9 t­ x 7. 38 ±1 .3 2 w x 8. 85 ±0 .5 1 q­ x 8. 59 ±0 .2 5 r­ x 8. 94 ±0 .3 5 e 0. 21 9. 92 4 ­0 .0 08 1 h 18 .0 5± 1. 04 d ­f 15 .7 4± 1. 85 fg 11 .4 ±0 .5 9 i­q 9. 21 ±0 .2 9 o­ x 7. 78 ±0 .7 3 u­ x 7. 86 ±0 .7 t­ x 11 .6 7± 1. 01 c 0. 81 17 .1 7 ­0 .0 44 8 s 22 .5 7± 0. 83 a b 22 .6 1± 1. 56 a b 22 .2 4± 0. 48 a ­c 18 .7 3± 1. 67 d e 12 .7 1± 0. 58 h ­m 10 .4 7± 1. 09 k ­t 18 .2 2± 1. 25 a 0. 78 24 .9 2 ­0 .0 54 9 v2 11 .1 6± 1. 09 j­ r 9. 77 ±1 .1 5 o­ w 9. 86 ±0 .6 3 n­ w 9. 04 ±0 .2 3 q­ x 8. 22 ±1 .1 6 t­ x 7. 53 ±0 .5 4 v­ x 9. 26 ±0 .4 1 e 0. 46 10 .9 5 ­0 .0 14 2 v3 17 .4 6± 0. 26 d ­ f 14 .5 9± 1. 12 g h 14 .6 2± 1. 15 g h 9. 75 ±0 .8 4 o­ w 8. 25 ±0 .6 6 t­ x 8. 3± 0. 49 s­ x 12 .1 6± 0. 91 c 0. 83 17 .1 4 ­0 .0 4 6 z 21 .3 9± 1. 87 b c 18 .2 2± 1. 13 d ­f 12 .4 5± 0. 89 i­ n 13 .0 4± 1. 64 h ­k 13 .8 5± 0. 67 g ­i 8. 91 ±0 .3 4 q­ x 14 .6 4± 1. 07 b 0. 69 19 .9 9 ­0 .0 43 7 m ea n n ac l 16 .4 ±1 .0 9 a 15 .6 5± 1. 02 a 13 .2 3± 0. 86 b 11 .5 2± 0. 82 c 9. 98 ±0 .5 2 d 9. 5± 0. 88 d *g sc = 1 6. 51 79 17 ­ 0. 03 04 33 (n ac )l (r 2 = 0. 75 8) fig. 5 ­ salinity sensitivity index (ssi) for germination speed coefficient (gsc) in chili genotypes. values represent the mean of three replicates. different letters denote signifi­ cant differences according to the duncan multiple range test (dmrt) at p<0.05. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. adv. hort. sci., 2024 38(4): 371­392 380 genotypes was significantly influenced by salinity stress levels, genotype variations, and their interaction (genotype × salinity) compared to control conditions (p <0.001). the genotype effect accounted for approximately 70% of the total variation (100%), while salinity contributed 30% to the observed changes in mgr (table 3). across the examined genotypes, mgr varied significantly, with the lowest rate recorded for genotypes f, g, and v2 (0.09 seeds/day) and the highest for genotype d (0.19 seeds/day) (table 7). these differences were statistically significant (p<0.05); however, no significant difference in mgr was observed between genotypes d and s. a marked decline in mgr was observed across all genotypes when salinity levels exceeded 50 mm compared to the control treatment (p<0.05). specifically, mgr decreased from 0.16 ± 0.01 seeds/day at 0 mm nacl to 0.09 ± 0.008 seeds/day at 250 mm nacl. regression analysis indicated that for every 1 mm increase in nacl, mgr decreased by approximately 0.0003 seeds/day, represented by the equation: mgr = 0.165153 ­ 0.000304 (nacl) (r2 = 0.758).the genotypic ranking based on the slope (b) of mgr responses to salinity is presented in table 7. genotype s demonstrated the greatest decline (b = ­0.00053, r2 = 0.78), (order 9), whereas genotype g exhibited the least decline (b = ­ 0.00008, r2 = 0.216), while remaining genotypes v2, f, a, d, v3, z, and h were ranked in between of them, respectively. in terms of the genotype × salinity interaction, mgr for genotype d was significantly higher (p < 0.05) than that of other genotypes under control conditions (0 mm nacl), except for genotypes s and z. at salinity levels ranging from 50 to 250 mm, mgr for genotypes d and s was significantly higher than that of the remaining genotypes, with genotype d achieving the highest mgr (0.19 ± 0.05) at 250 mm (table 7). the salinity sensitivity index (ssi) for mgr was positive for genotypes d, g, and s at 50 mm nacl, while genotype a maintained a positive ssi up to 100 mm nacl. in contrast, genotypes z, v3, v2, h, and f exhibited negative ssi values for mgr across all salinity levels. the decline in ssi was linear for genotypes f, h, s, v2, and v3 with increasing salinity. the highest ssi value for mgr (24.9%) was recorded at 100 mm nacl for genotype a, while the lowest ssi (­58.08%) was observed at 250 mm nacl for genotype z (fig. 6). ta bl e 7 ­ ii nt er ac tio n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n th e m ea n ge rm in at io n ra te (m g r) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . ** t he g en ot yp es a re li st ed in d es ce nd in g or de r a cc or di ng o n th e va lu es o f t he re gr es sio n slo pe li ne (b ). g en ot yp es n ac l m m m ea n r2 co ef fic ie nt o rd er * * 0 50 10 0 15 0 20 0 25 0 g en ot yp es c b a 0. 11 ± 0 .0 1 j­q 0. 14 ± 0 .0 2 g­ j 0. 14 ± 0 .0 1 g­ j 0. 09 ± 0. 00 1 n­ s 0. 09 ± 0 .0 1 n­ s 0. 07 ± 0. 00 s 0. 11 ± 0 .0 07 d 0. 39 1 0. 13 4 ­0 .0 00 22 4 d 0. 24 ± 0. 03 a 0. 24 ± 0. 02 a 0. 16 ± 0. 03 f­ g 0. 18 ± 0. 01 d e 0. 13 ± 0 .0 2 h­ l 0. 19 ± 0. 05 b ­d 0. 19 ± 0. 01 4 a 0. 19 9 0. 23 0 ­0 .0 00 30 5 f 0. 12 ± 0 .0 04 i­ n 0. 12 ± 0. 00 2 i­n 0. 10 ± 0. 00 2 k­ s 0. 09 ± 0. 01 m ­s 0. 09 ± 0 .0 03 n ­s 0. 07 ± 0. 00 1 s 0. 09 ± 0. 00 4 de 0. 89 5 0. 12 2 ­0 .0 00 19 3 g 0. 10 ± 0. 00 4 k­ s 0. 11 ± 0 .0 04 k ­r 0. 08 ± 0. 01 o ­s 0. 07 ± 0. 01 rs 0. 09 ± 0 .0 05 n ­s 0. 09 ± 0 .0 02 n ­s 0. 09 ± 0 .0 04 e 0. 21 6 0. 99 ­0 .0 00 08 1 h 0. 18 ±0 .0 10 d e 0. 16 ± 0 .0 2 e­ h 0. 11 ± 0. 01 j­ o 0. 09 ± 0. 00 3 n­ s 0. 08 ± 0. 01 q ­s 0. 08 ±0 .0 1 p­ s 0. 12 ± 0 .0 1 c 0. 80 5 0. 17 2 ­0 .0 00 44 8 s 0. 23 ±0 .0 1 a 0. 23 ± 0 .0 2 a 0. 22 ± 0. 00 5 ab 0. 19 ± 0 .0 2 c­ e 0. 13 ±0 .0 1 h­ l 0. 11 ± 0. 01 k ­r 0. 18 ± 0. 01 3 a 0. 78 1 0. 24 9 ­0 .0 00 53 9 v2 0. 11 ±0 .0 1 j­p 0. 09 ± 0. 01 l­ s 0. 09 ± 0. 01 k ­s 0. 09 ± 0. 00 2 n­ s 0. 08 ±0 .0 1 o­ s 0. 08 ± 0. 01 rs 0. 09 ± 0. 00 4 e 0. 45 9 0. 11 0 ­0 .0 00 13 2 v3 0. 18 ± 0 .0 02 d ­f 0. 15 ± 0. 01 f­ i 0. 15 ± 0 .0 1 f­i 0. 09 ± 0. 01 l­ s 0. 08 ±0 .0 1 o­ s 0. 08 ±0 .0 1 o­ s 0. 12 ± 0. 00 9 c 0. 82 2 0. 17 2 ­0 .0 00 39 6 z 0. 21 ± 0. 02 a ­c 0. 18 ± 0. 01 d e 0. 12 ± 0. 01 i­ m 0. 13 ± 0. 02 h ­k 0. 14 ± 0. 01 g ­j 0. 09 ± 0. 00 3 n­ s 0. 15 ± 0 .0 11 b 0. 68 6 0. 20 0 ­0 .0 00 43 7 m ea n n ac l 0. 16 ±0 .0 1 a 0. 16 ±0 .0 1 a 0. 13 ±0 .0 08 b 0. 12 ±0 .0 08 c 0. 09 ±0 .0 05 d 0. 09 ±0 .0 08 d m g r= 0 .1 65 15 3 ­ 0 .0 00 30 4 (n ac l), (r 2= 0. 75 8) al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 381 coefficient of velocity of germination (cvg) the coefficient of velocity of germination (cvg) for all chilli genotypes was significantly influenced by salinity stress levels, genotype differences, and their interaction (genotype × salinity) compared to the control treatment (p<0.001). genotype alone accounted for approximately 68% of the total variation (100%), while salinity contributed an additional 32% to the cvg (table 3). among the genotypes, cvg ranged from 18.75±1.45 for genotype f to 37.37±3.61 for genotype v3 (table 8), with significant differences observed (p < 0.05). however, no significant differences were noted among genotypes v3, a, d, h, s, and z. furthermore, increasing salinity levels led to a dramatic decrease in cvg compared to the control (0 mm), with values dropping from 38.48±3.65 (at 0 mm) to 25.69 ± 2.10 (at 250 mm). no significant differences were observed between the 50, 100, and 150 mm nacl treatments, or between the 200 and 250 mm nacl treatments (table 8). the regression analysis indicated that for every 1 mm increase in nacl, cvg decreased by approximately 0.0485, as described by the equation: cvg = 37.725 ­ 0.0485 (nacl) (r2 = 0.166). the genotypes were ranked according to their cvg response, with s, h, f, d, a, z, g, v3, and v2 arranged from 1 to 9, respectively. the s genotype exhibited the least impact from salinity (b = ­0.010, r2 = 0.004), ranking first, while the v2 genotype showed the greatest impact (b = ­0.106, r2 = 0.53), placing last fig. 6 ­ salinity sensitivity index (ssi) for mean germination rate (mgr) for chili genotypes. values represent the mean of three replicates. different letters denote significant dif­ ferences according to the duncan multiple range test (dmrt) at p<0.05. genotypes: a= abyani; z= zaaitri ; h= haimi; d= dhamari; v2= jawfi 2; v3= jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. ta bl e 8 ­ in te ra ct io n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n th e co ef fic ie nt o f v el oc ity o f g er m in at io n (c vg ) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . ** t he g en ot yp es a re li st ed in d es ce nd in g or de r a cc or di ng o n th e va lu es o f t he re gr es sio n slo pe li ne (b ). g en ot yp es n ac l m m m ea n r2 co ef fic ie nt o rd er * * 0 50 10 0 15 0 20 0 25 0 g en ot yp es c b a 45 .7 2± 1 2. 31 a ­f 31 .3 6± 8 .7 7 c­ o 24 .7 ± 3. 03 i­ o 36 .8 1± 5 .7 6 a­ k 31 .0 3± 8 .7 3 d­ o 31 .1 2± 1 .3 9 c­ o 33 .4 6± 3 .0 6 ab 0. 05 7 37 .8 7 ­0 .0 35 5 d 40 .8 9± 9 .6 8 a­ j 29 .9 2± 4 .1 3 e­ o 52 .6 8± 1 7. 34 a 23 .7 7± 6 .1 9 j­o 40 .9 6± 5 .7 4 a­ j 28 .4 8± 1 2. 21 f­ o 36 .1 2± 4 .2 4 a 0. 02 6 40 .2 5 ­0 .0 33 4 f 14 .1 7± 4 .7 5 o 20 .0 2± 1 .0 5 k­ o 15 .9 6± 4 .8 7 no 19 .7 7± 4 .8 5 k­ o 21 .6 6± 1 .5 1 k­ o 20 .9 5± 3 .4 9 k­ o 18 .7 5± 1 .4 5 d 0. 12 1 15 .7 0 ­0 .0 24 3 g 43 .0 7± 7 .2 4 a­ h 27 .6 1± 4 .3 5 g­ o 30 .5 5± 0 .1 5 e­ o 18 .0 9± 9 .0 9 l­o 14 .7 9± 2 .2 6 o 21 .8 2± 0 .4 3 k­ o 25 .9 9± 3 .2 1 c 0. 42 37 .2 1 ­0 .0 87 7 h 31 .3 6± 9 .3 7 c­ o 34 .1 9± 1 1. 77 b ­ 50 .3 9± 3 .5 2 ab 43 .6 9± 4 .0 6 a­ g 32 .7 2± 3 .1 2 c­ n 25 .6 4± 7 .3 8 h­ o 36 .3 3± 3 .3 3 a 0. 02 1 39 .1 6 ­0 .0 23 2 s 33 .7 4± 4 .9 2 b­ m 32 .4 9± 6 .2 4 c­ n 19 .7 2± 4 .3 1 k­ o 42 .0 3± 1 5. 31 a ­i 29 .2 3± 3 .2 7 f­o 34 .7 9± 1 0. 11 b ­l 32 ± 3. 32 a b 0. 00 4 30 .7 2 0. 01 0 1 v2 40 .5 3± 6 a ­j 36 .3 7± 6 .3 a ­k 33 .9 8± 5 .3 b ­m 24 .8 4± 6 .3 1 i­o 18 .4 l± 5 .5 3 m ­o 16 .0 1± 5 .6 6 no 28 .3 5± 3 .0 04 b c 0. 53 5 41 .6 1 ­0 .1 06 9 v3 48 .3 3± 1 1. 17 a ­d 52 .5 9± 7 .0 9 a 30 .7 9± 2 .2 2 e­ o 36 .5 3± 5 .6 a ­k 33 .2 ± 11 .5 4 b­ n 22 .7 7± 2 .7 2 k­ o 37 .3 7± 3 .6 1 a 0. 34 9 50 .2 4 ­0 .1 03 8 z 48 .4 8± 2 2. 35 a ­c 35 .3 2± 1 2. 02 b ­l 41 .3 5± 1 .2 a ­i 47 .3 ± 13 .6 4 a­ e 17 .2 ± 1. 79 m ­o 29 .6 6± 4 .8 4f ­o 36 .5 5± 4 .8 7 a 0. 12 46 .7 3 ­0 .0 81 6 m ea n n ac l 38 .4 8± 3. 65 a 33 .3 2± 2. 66 b 33 .3 5± 2. 99 b 32 .5 4± 3. 16 b 26 .5 8± 2. 30 c 25 .6 9± 2. 10 c * cv g = 37 .7 25 ­ 0. 04 85 (n ac l), (r 2= 0. 16 6) adv. hort. sci., 2024 38(4): 371­392 382 dry matter of germinated seed (dm%) the dry matter percentage (dm%) of all chilli genotypes was significantly influenced by salinity stress levels, genotype differences, and the interaction between genotype and salinity stress when compared to the control treatment (p < 0.05). across the genotypes, dm% ranged from 7.77 ± 0.74% for genotype g to 17.95 ± 1.38% for genotype z. no significant differences were observed among genotypes s, h, f, and a (table 9). furthermore, (table 8). interaction between genotype and salinity stress, under control conditions (0 mm), genotype z showed a cvg of (48.48 ± 22.35) higher than the lower values observed for genotypes f (14.17 ± 4.75) and h (31.36 ± 9.37) (table 8). at salinity levels ranging from 50 to 250 mm, the cvg for genotype f was statistically lower than that of the other genotypes. the genotype f showed a positive salinity sensitivity index (ssi) for cvg across all salinity levels. in contrast, genotypes v2, a, and g exhibited negative ssi values for cvg at all salinity levels, with genotype v3 showing negative values above 50 mm nacl. the h genotype maintained a positive ssi up to 200 mm nacl, while genotype z exhibited positive ssi values at the initial two salinity levels. the highest ssi value for cvg was observed at the 200 mm nacl (98.5%) for genotype f, whereas the lowest was recorded at 200 mm nacl (­65.46%) for genotype g (fig. 7). fig. 7 ­ salinity sensitivity index (ssi) of coefficient of velocity of germination (cvg) in chili genotypes. values represent the mean of three replicates. different letters denote sig­ nificant differences according to the duncan multiple range test (dmrt) at p < 0.05. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. ta bl e 9 ­ in te ra ct io n ef fe ct s o f y em en i c hi li ge no ty pe s a nd n ac l l ev el s o n th e pe rc en ta ge o f d ry m at te r ( dm % ) a ft er 2 1 da ys m ea ns c on ta in in g th e sa m e la tin le tt er s ar e no t c on sid er ed s ig ni fic an t, as d et er m in ed b y ls d 0. 05 fo r s in gl e fa ct or s (g en ot yp es o r s al in ity ) o r b y th e m ul tip le ra ng e du nc an te st (m rd t) fo r t he in te ra ct io n (g en ot yp es × sa lin ity ). g en ot yp es : a = ab ya ni ; z = za ai tr i ; h = ha im i; d= dh am ar i; v2 = ja w fi 2; v 3 = ja w fi 3; g = ha jja i; s = sa 'd di ; f = s ha m ak h. th e va lu es fo r r ² a nd th e re gr es sio n co ef fic ie nt s, w he re c = th e in te rc ep t a nd b = sl op e of th e lin e, a re p ro vi de d fo r t he si m pl e re gr es sio n an al ys is of e ac h ge no ty pe . * si m pl e re gr es sio n eq ua tio n w as p er fo rm ed fo r t he m ea n of a ll ge no ty pe s ( df = 1 8) . ** t he g en ot yp es a re li st ed in d es ce nd in g or de r a cc or di ng o n th e va lu es o f t he re gr es sio n slo pe li ne (b ). g en ot yp es n ac l m m m ea n r2 co ef fic ie nt o rd er * * 0 50 10 0 15 0 20 0 25 0 g en ot yp es c b a 10 .9 5± 0 .2 5 g­ o 10 .3 3± 1 .3 3 h­ o 10 .1 ± 0. 89 h ­o 10 .1 9± 0 .6 h ­o 13 .0 1± 0 .2 e ­l 10 .2 4± 0 h ­o 10 .8 ± 0. 45 c d 0. 40 9. 18 0. 01 4 2 d 14 .8 6± 1 .7 2 d­ i 16 .6 7± 1 .8 4 b­ f 15 .0 1± 5 .0 7 d­ h 20 .3 1± 0 .6 2 ab 13 .4 8± 0 .8 7 e­ k 16 .9 ± 1. 18 b ­e 16 .2 ± 1. 04 b 0. 00 4 15 .7 2 0. 00 3 3 f 11 .9 5± 1 .8 3 e­ m 13 .8 7± 0 .9 2 e­ k 9. 49 ± 1. 96 k ­q 15 .5 5± 1 .7 5 c­ g 6. 79 ± 2. 84 n ­r 5± 0 p ­r 10 .4 4± 1 .0 6 d 0. 27 13 .9 4 ­0 .0 26 8 g 9. 98 ± 0. 98 h ­o 10 .3 4± 0 .5 1 h­ o 9. 39 ± 1. 57 k ­q 4. 5± 0 .7 1 r 4. 06 ± 0. 04 r 8. 33 ± 2. 15 l­ r 7. 77 ± 0. 74 e 0. 25 8 10 .0 5 ­0 .0 18 5 h 12 .2 8± 1 .5 1 e­ m 13 .0 5± 1 .9 6 e­ l 11 .7 4± 0 .7 f­ n 13 .3 7± 1 .0 3 e­ l 7. 7± 0 .3 m ­r 4. 83 ± 0 qr 10 .5 ± 0. 86 d 0. 51 14 .1 9 ­0 .0 30 9 s 11 .4 5± 1 .9 g ­o 10 .9 8± 1 .1 6 g­ o 10 ± 2. 13 h ­o 14 .8 2± 3 .1 2 d­ i 6. 53 ± 2. 93 o ­r 4. 67 ± 0. 6 r 9. 74 ± 1. 1 d 0. 21 12 .7 7 ­0 .0 24 7 v2 9. 7± 1 .2 5 j­p 10 .0 2± 0 .6 1 h­ o 11 .1 ± 0. 23 g ­o 11 .5 5± 1 .7 g ­o 14 .7 1± 1 .0 8 d ­ j 15 .9 8± 0 .3 5 b­ g 12 .1 8± 0 .6 7 c 0. 66 8. 89 0. 02 6 1 v3 12 .2 3± 1 .5 2 e­ m 13 .3 7± 3 .2 8 e­ l 14 .3 8± 0 .7 9 d­ k 9. 86 ± 2. 85 i­ o 10 .4 4± 4 .5 h ­o 13 .4 2± 0 e ­k 12 .2 8± 0 .9 9 c 0. 00 8 12 .8 0 ­0 .0 04 4 z 19 .8 1± 6 .0 8 a­ c 22 .1 7± 4 .2 3 a 16 .7 1± 3 .3 9 b­ f 14 .1 8± 0 .7 8 d­ k 19 .0 6± 2 .4 a ­d 15 .7 8± 1 .8 5 b­ g 17 .9 5± 1 .3 8 a 0. 07 4 20 .2 3 ­0 .0 18 5 m ea n n ac l 12 .5 8± 0. 93 a b 13 .4 2± 0. 93 a 11 .9 9± 0. 82 b c 12 .7 ±0 .9 6 ab 10 .6 4± 1. 08 c d 10 .5 7± 1. 07 d *d m % = 13 .2 53 59 9 ­0 .0 10 08 5( n ac l) , ( r2 = 0. 12 ) al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 383 pearson correlation and principal component analysis the principal component analysis (pca) conducted in this study (table 10) classified the variables into two primary components have eigenvalues greater than 1, which together explained 76.2% of the total variance observed. the first principal component (pc1) exhibited an eigenvalue of 2.75, accounting for 55.11% of the overall variance (table 10). the coefficients associated with pc1 indicate higher correlations with: dm% (0.713), grp (0.808), mgt (­0.715), mgr (0.881), and cvg (0.552) (table 11). the second principal component (pc2) demonstrated an eigenvalue of 1.079, explaining 21.585% of the variance in the data (table 11). the biplot diagram (fig. 9) displays both the first and second principal component (pc) scores of the various parameters. strong positive correlation of pc1 with both cvg and dm%, as confirmed by the pearson correlation coefficient (r= 0.44, p> 0.01) (fig. 10). additionally, mgr and grp exhibited a significant positive correlation (r= 0.58, p> 0.01), indicating their increasing salinity levels led to a significant decline in dm%, with values decreasing from 12.58 ± 0.93% at 0 mm to 10.57 ± 1.07% at 250 mm. no significant differences were detected between salinity levels from 0 mm to 150 mm (table 9).the regression analysis indicated the effect of salinity on dry matter, with the order of genotypes ranked as v2, a, d, v3, g, z, s, f, and h from 1 to 9, respectively. according to the r² values, salinity had a minimal influence on genotypes d (b = ­0.003, r2 = 0.004) and v3 (b = ­0.004, r2 = 0.008), while it exerted the most significant effect on genotype h (b = ­0.03, r2 = 0.51) (table 9). regarding the interaction between genotype and salinity stress, the dm% for genotype z was significantly higher than the one of the other genotypes across all salinity levels tested (0, 50, 100, 200, and 250 mm). at 150 mm nacl, genotype d exhibited a significantly higher dm% compared to the other genotypes (table 9). in terms of the salinity sensitivity index (ssi) for dm%, genotypes v2 and d displayed positive ssi values across all salinity levels. genotype a showed a positive ssi value under salinity levels up to 150 mm. in contrast, genotype g exhibited negative ssi values under salinity levels up to 50 mm. the lowest ssi value for dm% was recorded at the 150 mm salinity level (­71%) for genotype g, while the highest ssi value was observed at the 250 mm salinity level (72.7%) for genotype d (fig. 8). fig. 8 ­ salinity sensitivity index for dry matter of seedling (dm%) in chili genotypes. values represent the mean of three replicates. different letters denote significant dif­ ferences according to the duncan multiple range test (dmrt) at p<0.05. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. table 10 ­ the eigenvalue, variability (%) and the cumulative percentage of the principal component for first, second, third, fourth and fifth components number of principal component cumulative percentage eigenvalue percentage 1 55.11 2.755.500 55.11 2 76.69 1.079.260 21.58 3 87.12 0.521455 10.42 4 96.24 0.455748 9.11 5 100.00 0.188037 3.76 table 11 ­ the coefficients of the principal component score (prin) for first to fifth components parameters prin1 prin2 prin3 prin4 prin5 dm% 0.71321 0.37635 0.57006 ­0.15021 ­0.0464 grp 0.80819 ­0.01046 ­0.00483 0.58824 0.02586 mgt ­0.71563 0.62246 0.07473 0.13852 0.27505 mgr 0.88102 ­0.23142 ­0.13593 ­0.23594 0.31001 cvg 0.55258 0.70463 ­0.41521 ­0.1109 ­0.11599 the principal components are based strongly correlated of para­ meters with each component. number with light color means no correlation. adv. hort. sci., 2024 38(4): 371­392 384 effectiveness as prominent indicators of salinity stress resilience. conversely, pc1 exhibited a negative correlation with mgt. the analysis revealed a significant negative correlation between mgt and mgr (r= ­ 0.73, p>0.01), suggesting that genotypes characterized by shorter mgts tend to display higher mgr under saline conditions. a similar negative correlation was observed between mgt and grp (r = ­0.50, p > 0.01), indicating that genotypes with lower mgts achieve higher grp in response to salinity stress. the pca biplot (fig. 9) and data from table 12 show that the genotypes were distributed across all fig. 9 ­ biplot principal component analysis (pca) of various parameters contributing of mgt, cvg, dm% and grp to salinity and genotypes. genotypes: a= abyani; z= zaaitri h= haimi; d= dhamari; v2= jawfi 2; v3= jawfi 3; g=hajjai; s = sa'ddi; f= shamakh. fig. 10 ­the correlation matrix (pearson) displaying the relation­ ships among parameters investigated in the current study. significant correlations are detailed below the diagonal, whereas above the diagonal, correlations between parameters under various treatments. the degree of correlations between these parameters under treatment is shown by varying sizes of circles and shades of color that correspond to different correlation values. factors prin1 prin2 prin3 prin4 prin5 order grope genotypes a ­0.59566 0.32256 ­0.09868 ­0.08734 ­0.09748 6 3 d 189.235 ­0.41852 0.10463 ­0.20911 0.10728 1 1 f ­0.72275 ­0.25615 0.43255 0.80284 0.18231 8 4 g ­177.313 0.0807 ­0.2378 ­0.34424 0.1064 9 4 h ­0.26658 0.25372 ­0.35926 0.03307 ­0.0791 5 4 s 10.002 ­0.95791 ­0.67273 ­0.0669 0.21997 4 1 v2 ­0.85458 0.41669 0.34601 0.11202 ­0.01167 7 3 v3 0.12219 0.29816 ­0.15171 0.02071 ­0.17465 3 2 z 119.796 0.26074 0.637 ­0.26106 ­0.25305 2 2 0 107.166 ­0.23484 ­0.36013 ­0.16135 ­0.05502 salinity 50 100.443 ­0.32537 ­0.01125 0.09954 ­0.01775 100 0.28284 ­0.0954 ­0.11151 0.06181 ­0.10089 150 ­0.09113 0.24656 0.10626 0.19838 ­0.05322 200 ­0.94522 0.11829 0.15719 ­0.02344 0.04212 250 ­132.258 0.29076 0.21944 ­0.17494 0.18476 table 12 ­ the coefficients of the principal component score (prin) for first to fifth components for genotypes and salinity, with ranking of yemeni chili genotypes for salinity tolerance, determined by the cumulative coefficients of the principal component score (prin) for first and second components values the principal components are based strongly correlated of parameters with each component. number with light color means no correla­ tion. order = the rank of genotypes according to summation of values the prin1 and prin 2. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 385 four quadrants, highlighting significant genetic variation among the tested genotypes. genotypes d and s appeared in the quadrant with the highest pc1 and lowest pc2 values, indicating tolerance and a strong correlation with mgr and grp. genotypes z and v3 were positioned in the quadrant with the highest pc1 and highest pc2 values, displaying a strong correlation with cvg and dm, suggesting moderate resistance. in contrast, genotypes a and v2 were located in the quadrant with the lowest pc1 and highest pc2 values, indicating sensitivity and a close correlation with mgt. genotypes f and g were placed in the quadrant with the lowest pc1 and lowest pc2 values, reflecting very high sensitivity and showing no correlation with germination parameters. based on the cumulative pc1 and pc2 scores, the chilli genotypes were ranked from 1 to 9, with genotype d ranked the highest (order 1) and genotype g ranked the lowest (order 9), indicating its heightened sensitivity to salinity stress (table 12). pc1 of nacl showed a positive correlation at concentrations ranging from 0 to 100 mm. however, it was negatively correlated, with no significant effect at 150 mm, and significantly negatively correlated at concentrations of 200 to 250 mm nacl. evaluation of salinity tolerance in yemeni chilli genotypes by integrated value (dv) the salt tolerance levels among the chill i genotypes in this experiment were assessed using the integrated value (dv), as presented in table 12. the weighted coefficients indicate the significance of various parameters in measuring the sensitivity of the genotypes to salinity. notably, germination rate percentage (grp), mean germination time (mgt), mean germination rate (mgr), and germination speed coefficient (gsc) collectively accounted for over 70% of the overall weight in this study. the arrangement of integrated values (dv) ranked the genotypes according to their salt tolerance, with genotype a occupying the top position (rank 1), indicating higher resistance to salinity. in contrast, genotype h ranked last (order 9), reflecting greater sensitivity to salinity. based on the integrated value (dv), the genotypes were categorized into four groups: resistant (d, a, and g), moderately resistant (f and v2), sensitive (s and z), and highly sensitive (h and v3) to salinity (fig. 11). additionally, the genotypes salinity susceptibility index (gssi) was calculated based on grp (table 13). the results indicated that genotypes a and d were tolerant to salinity, while genotypes f, g, h, and v3 were sensitive. genotypes v2 and z are exhibited moderate sensitivity to salinity. interestingly, the ranking of genotypes according to the subordinate function of grp (o grp) slightly differed from that of the gssi, as genotype a ranked fifth in o grp but was classified as tolerant in the gssi assessment. evaluation of salinity tolerance in yemeni chilli genotypes by regression slop in this study, the significance of various traits was assessed through the r­square (r²) values derived from total regression analyses for each genotype across all measured characteristics. higher r² values indicate a greater significance of the trait, while lower values suggest diminished relevance. the r² values for germination percentage (grp), mean germination time (mgt), mean germination rate (mgr), and germination speed coefficient (gsc) were all above 50%, with values of 53, 77, 76, and 76 %, respectively (table 13). these findings demonstrate that salinity significantly affects these traits, providing a reliable measure of the sensitivity of the fig. 11 ­ cluster analysis of 9 chilli genotypes using integrated value (vd). the first group contains salinity­resistant (t) genotypes. the second group contains genotypes that are moderately sensitive (m) to salinity; the third group contains genotypes that are sensitive (s) to salinity; and the fourth group contains genotypes that are very sensitive to salinity (vs). genotypes: a= abyani; z =zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. adv. hort. sci., 2024 38(4): 371­392 386 genotypes employed in this experiment. conversely, the r² values for germination velocity coefficient (cvg) and dry matter (dm) were markedly lower, indicating that these traits are less significant, with r² values below 0.25. table 14 summarizes the slope values obtained from the regression analyses for each genotype across all parameters. genotypes with higher slope values (b) are regarded as being more adversely affected by salinity and thus exhibit lower resistance to salt stress. based on the summation of slope values for each genotype across all parameters, the genotypes were ranked from 1 to 9, with genotype a achieving the highest rank, followed by genotype d. table 13 ­ the values of the subordinate function, integrated value (dv), and order of each chili genotypes under salt stress. grp means germination percentage, mgt means germination time, mgr means germination rate, md% means dry matter, gsc means germination speed coefficient, cvg germination velocity coefficient on the 21th day, gssi genotypes salinity susceptible index and wj is the weighted coefficient the principal components are based strongly correlated of parameters with each component. number with light color means no correla­ tion. order = the rank of genotypes according to summation of values the prin1 and prin 2. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. genotypes grp mgt mgr dm% gsc cvg d order o grp grope (gssi) * a 0.074 0.238 0.059 0.093 0.060 0.099 0.622 1 5 t d 0.036 0.261 0.086 0.091 0.079 0.080 0.634 2 1 t f 0.096 0.251 0.082 0.098 0.085 0.082 0.695 4 6 s g 0.099 0.245 0.071 0.084 0.073 0.098 0.671 3 7 s h 0.125 0.325 0.157 0.086 0.162 0.083 0.937 9 9 s s 0.053 0.314 0.148 0.088 0.153 0.072 0.828 6 3 t v2 0.072 0.264 0.101 0.091 0.104 0.101 0.733 5 4 m v3 0.103 0.328 0.157 0.079 0.162 0.098 0.926 8 8 s z 0.049 0.340 0.163 0.076 0.168 0.080 0.876 7 2 m wj 0.18 0.20 0.20 0.11 0.21 0.11 table 14 ­ the ranking of yemeni chili genotypes for salinity tolerance, determined by the cumulative regression line slope values of seed germination parameters. the r² values represent the strength of association for each parameter the principal components are based strongly correlated of parameters with each component. number with light color means no correla­ tion. order = the rank of genotypes according to summation of values the prin1 and prin 2. genotypes: a = abyani; z = zaaitri ; h= haimi; d=dhamari; v2 = jawfi 2; v3 = jawfi 3; g= hajjai; s = sa'ddi; f = shamakh. genotypes grp mgt mgr cvg dm% gsc ∑ order a ­0.036 0.023 ­0.00022 ­0.035 0.014 ­0.022 ­0.0342 1 d ­0.013 0.011 ­0.0003 ­0.035 0.003 ­0.031 ­0.0343 2 f ­0.15 0.022 ­0.00019 ­0.035 ­0.026 ­0.02 ­0.1892 6 g ­0.21 0.009 ­0.00008 ­0.035 ­0.018 ­0.008 ­0.2541 8 h ­0.25 0.033 ­0.00044 ­0.035 ­0.03 ­0.044 ­0.2824 9 s ­0.059 0.022 ­0.00053 ­0.035 ­0.024 ­0.054 ­0.0965 4 v2 ­0.093 0.016 ­0.00013 ­0.035 0.026 ­0.014 ­0.0861 3 v3 ­0.188 0.029 ­0.00039 ­0.035 ­0.004 ­0.04 ­0.1984 7 z ­0.069 0.021 ­0.00043 ­0.035 ­0.018 ­0.043 ­0.1014 5 r2 0.539 0.775 0.758 0.166 0.12 0.758 al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 387 in contrast, genotype h was ranked last (order 9), indicating its greater susceptibility to salinity stress. 4. discussion and conclusions natural hybridization in peppers plays a vital role in the development of numerous species and varieties; however, it also complicates their classification due to overlapping traits (comparini et al., 2021). of approximately 35 capsicum species found in nature, only five have been domesticated for human use (comparini et al., 2021; swamy, 2023).this study focuses on three yemeni chilli species: c. frutescens, c. annuum, and c. chinense, as presented in table 1. seed germination marks the beginning of the plant’s life cycle and requires specific conditions to ensure successful germination. salinity hinders seed germination by inducing osmotic stress and ionic toxicity (hasanuzzaman et al., 2021; fu and yang, 2023). salinity stress disrupts essential physiological processes in plants, , leading to a reduced k+/na+ ratio and imbalances in ascorbate/dehydroascorbic acid and glutathione/ oxidized glutathione levels (kaya et al., 2020). furthermore, it reduces sugar content, alters organic acid metabolism, and promotes the accumulation of phenolic compounds (zamljen et al., 2022). these physiological disruptions limit germination percentage, delay germination time, and reduce both germination rate and biomass production (gupta and huang, 2014). the results showed a decrease in germination and biomass of hot chilli genotypes under salt stress, with a more pronounced impact observed in salt­sensitive genotypes compared to moderate and salt­tolerant ones. these findings align with the findings of sarkar et al. (2023). this decline in germination parameters can be attributed to disruptions in nutrient uptake and the accumulation of sodium ions, which lead to ion­specific toxicity and increased osmotic pressure, and nutrient imbalances(munns, 1993), as well as damage to plant cells and tissues (hasanuzzaman et al., 2021; ahmad et al., 2022). a lower mgt value indicates faster seed germination (kader, 2005), while a higher gsc value reflects quicker seed germination. in contrast, mean germination rate (mgr), calculated as the inverse of mgt, and represents the rate of seed germination per unit of time. the coefficient of velocity of germination (cvg) is another metric used to assess germination speed; it typically increases with a higher number of germinated seeds and a shorter germination period (talská et al., 2020). the gradual reduction in grp, mgr, cvg, and gsc, but increasing the mgt, is due to salinity’s influence and is inevitable, given the limited tolerance of plants to salt. this reduction in salt tolerance is influenced by the plant’s capacity to absorb salt concentrations and its response to salt stress, whether by enhancing osmosis through the production of organic compounds like proteins, proline, and sugars or by excluding salt via selective ion permeability (wien and stützel, 2020). these response mechanisms vary depending on plant species, varieties, and genotypes (loganayaki et al., 2020; ali et al., 2022). the most significant cvg values were observed in the tolerant genotypes examined in this study. elevated salt concentrations can impede water absorption due to intracellular osmotic pressure, disrupting cell division and elongation, thereby more effectively inhibiting water absorption than reducing seed germination (meyer and boyer, 1981; munns, 1993; hasegawa et al., 2000; mushtaq et al., 2020; hasanuzzaman et al., 2021; ahmad et al., 2022; zhou et al., 2023). simple correlation analyses are commonly used because they are easy to calculate. yet, for complex traits, a basic analysis may not be sufficient. in such cases, principal component analysis, or non­linear pca, can be utilized. the accumulation of dry matter in seedlings indicates the absorption of nacl ions and the genotypes’ response mechanisms to salt stress. the dry matter percentage decreases with increasing salinity levels; a negative correlation was found between dm% and mgt (r= ­0.26, p>0.01), the genotypes that content a higher dry matter percentage at high salinity levels are considered resistant, with a positive correlation with grp (r=0.49, p>0.01) and with mgr (r = 0.48, p>0.01) (fig. 10). genotype d exhibited higher dm% across all salt concentrations compared to the control, while genotype a maintained stable dm% levels. although variations in dm% were observed among genotypes, indicating points of peak resistance, stepwise regression analysis revealed that 100% of the observed effects were attributed to genetic differences (table 3). this suggests that, while dm% can be indicative of salinity tolerance, its overall significance as a trait was relatively limited in this adv. hort. sci., 2024 38(4): 371­392 388 study. these findings highlight the potential of dm% as a useful physiological marker for evaluating salinity tolerance, especially during the germination stage. numerous researchers have also highlighted the presence of genetic variances in salt tolerance among various vegetable crops, such as in tomato (devi and arumugam, 2019) and in pepper (howlader et al., 2018). salt stress exerts adverse effects on seed germination percentage, plant length, root length, root/plant length ratio, as well as fresh and dry weights of seedlings, along with the seedling vigor index (kayacetin, 2022). seed germination and seedling growth represent the plant growth stages most susceptible to salt stress (miceli et al., 2021). in this investigation, the germination rate percentage of most genotypes significantly decreased, but they were able to maintain up to 50% germination even at very high nacl concentrations (250 mm), indicating that the threshold for poor germination among most yemeni hot pepper genotypes was 200 to 250 mm nacl. similarly, the pcs revealed a negative effect at 200 and 250 mm nacl concentrations (table 12). in this study, distinct variations were observed among hot pepper genotypes regarding their salinity tolerance index. genotypes a and d genotypes exhibited superior resistance in terms in both germination percentage and dry matter accumulation. in contrast, these genotypes, showed heightened resistance specifically in terms of germination speed and rate. consequently, the most salt­tolerant genotype was identified based on the slope of the regression line, the integrated value (dv), principal component analysis, and the genotype salinity susceptibility index (gssi) indicators. pca helps identify key traits impacting salinity tolerance (negrão et al., 2016; mubushar et al., 2022). this study used principal component analysis (pca) to evaluate variables, with the first and second pcs explaining the majority of the variation (76.7%). the distribution of genotypes across the four quadrants highlighted distinct groupings. salt­ tolerant genotypes showed high grp and mgr values and low mgt. among all genotypes, d consistently ranked as the most resistant, achieving the first position across all analytical methods. however, the classification of other genotypes differed depending on the analysis model. the cumulative value of the weighted coefficients (wj) for grp, mgt, mgr, and gsc exceeded 76% of the overall weights in this study. and the cumulative percentage of the first and second pcs (table 10) was about 79.69% in which similar to wj . correspondingly, the r­square values for grp, mgt, mgr, and gsc were all greater than 50%, indicating their reliability in assessing the sensitivity of the genotypes to salinity stress. these findings suggest that these four characteristics could serve as fundamental parameters in a framework designed to evaluate the tolerance of chilli pepper germination to salinity stress. additionally, the r­square value may be considered a viable alternative to the weighted coefficient (wj) in this assessment. despite minor discrepancies between the integrated value (dv) and the slope of the regression line in the arrangement of genotypes (tables 12 and 13), we propose that the slope is a more effective metric for evaluating salinity sensitivity. this is primarily because the slope quantitatively represents the extent of decline in each characteristic as salinity levels increase across all genotypes. in contrast, the dv calculation depends on higher salinity levels, which may not fully capture the nuanced responses of genotypes. principal components (pcs) effectively highlight the relationship between variables and their respective impacts, while summated regression slope values provide a comprehensive measure of the overall influence across all traits. when evaluating the impact of salinity on genotypes, the r­squared value is a critical metric for assessing the significance of the parameters. moreover, our hypothesis regarding the efficacy of the regression line slope (b) has been validated. in contrast, the genotype salinity susceptibility index (gssi) model is not recommended, as it evaluates parameters independently rather than offering a comprehensive understanding of the genotypes’ responses to salinity stress. previous scholarly investigations, alongside our findings, indicate that the dv value is a superior metric for assessing salt tolerance (fang et al., 2017; xie et al., 2021). cluster analysis based on the dv value enabled a comparative evaluation of salt tolerance across different genotypes. the results of this study clearly categorized the genotypes into four clusters: the first cluster, comprising a and d, exhibited salt tolerance and thus represents a valuable set of materials suitable for cultivation in al‐madhagi and arraf ‐ salinity tolerance in chilli genotypes 389 saline environments. the analysis effectively delineated varying degrees of salinity sensitivity among the genotypes, classifying them as resistant (d, a, and g), moderately resistant (f and v2), sensitive (s and z), and highly sensitive (h and v3) to salinity. yemeni chilli genotypes exhibit considerable variation in salinity tolerance. results have identified genotypes a and d as promising candidates for cultivation in saline environments, designating them as elite genotypes. these genotypes offer valuable prospects for hybridization with those exhibiting moderate to low salt tolerance, aiming to enhance resilience and productivity. the study underscores the efficacy of using the regression line slope as a robust method for assessing genotypic sensitivity to salinity. these findings are pivotal for advancing the development of salt­tolerant chill i cultivars, optimizing breeding strategies, and promoting sustainable 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yan et al., 2019). among them, there are many reports of growth differences by irradiating red and blue leds with a monochromatic color, mixing red and blue leds, and irradiating them in combination, such as changing the irradiation time. in particular, depending on the quality of the led light used for irradiating the plants, differences in morphological growth such as fresh and dry weight and leaf area, and in the amount of substances such as anthocyanins and vitamins in plants have been reported (bleiss and smith, 1985; jieun et al., 2012; jishi et al., 2016; bian et al., 2018; ishii et al., 2018). it is also known that light quality and quantity not only promotes plant photosynthesis, but also regulates plant growth (chen et al., 2016). however, it is reported that blue lights, which affect photomorphogenesis and red, which affects photosynthesis, are (*) corresponding author: akutsu@shinshu­u.ac.jp citation: kamimaeda h., yoshida y., tamaki k., ichihara m., akutsu m., 2023 ­ effect of far‐red light applied at the end of the day in red and green leaf lettuce cultivars grown under two types of white led. ­ adv. hort. sci., 37(2): 231­ 239. copyright: © 2023 kamimaeda h., yoshida y., tamaki k., ichihara m., akutsu m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 july 2022 accepted for publication 19 may 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12062 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(2): 231­239 232 highly efficient for plant growth (goto, 2005). moreover, far­red (fr) light, along with red light, is known to have a significant effect on plant growth and has been shown to affect seed germination, plant growth, and flowering (hisamatsu et al., 2002). it has been reported that fr led treatment after sunset (end of day­far red: eod­fr) promotes shoot elongation in poinsettia (euphorbia pulcherrima) and chrysanthemum (chrysanthemum sp.) (lund et al., 2007; islam et al., 2014), and also extends the hypocotyl axis in the rootstock of pumpkin (cucurbita maxima) seedlings (yang et al., 2012). in komatsuna (brassica rapa var. perviridis) and pak choi (brassica rapa var. chinensis), eod­fr treatment has been found to increase fresh weight, dry matter weight, and leaf area (akutsu et al., 2017). it has been reported that the fresh and dry weights and leaf length of baby leaf lettuce grown under white light irradiation with fr light increased by more than 10% compared to those grown without the fr light irradiation (li and kubota, 2009). in japan, most indoor plant factories growing lettuce use red or red­ blue mixed leds. however, white leds are used extensively in our daily life and are easily available. if white leds can be used for lettuce production in an indoor plant factory, it may reduce the unit price spent on leds and consequently increase cost effectiveness. in addition, white leds have begun to be used for growing leafy vegetables such as leaf lettuce in plant factories, but it might be difficult to optimize the light wavelength and intensity for plant cultivation as they are originally used for indoor lighting in households. compared to using white led irradiation alone for plant production, it is possible to optimize the light intensity for each wavelength by combining monochromatic led lighting (watanabe et al., 2016), but we confirmed a difference in the growth using white led irradiation and whether changes were made by fr led irradiation. in this study, we investigated the effects of eod­fr treatment on lettuce using 13 leaf lettuce cultivars under irradiation with two types of white leds. plants were grown hydroponically in growth chambers to maintain environmental conditions for air temperature, humidity and concentration of co2 other than light conditions. 2. materials and methods the 13 cultivars of leaf lettuce used in this study are listed in table 1. the seeds of each cultivar were sown on urethane cubes (m hydroponic research co. ltd., aichi, japan) with distilled water and then ger­ minated for one week in a growth chamber (tge­5­ 2l; espec corp., osaka, japan) at 25°c, 70% relative humidity, and 600 ppm co2 for 16 h under continu­ ous illumination at 100 µmol/m2/s cool white fluores­ cent lamps (fhf32ex­d­hx­s; nec corp., tokyo, japan). subsequently, the germinated seeds were transferred into a commercial a treatment nutrient solution suitable for lettuce cultivation (oat agrio co., ltd., tokyo, japan) and grown for an additional week. after that, eight seedlings were transferred to containers (293 mm × 211 mm × 106 mm) with 6 l of commercial a treatment nutrient solution (oat agrio co., ltd., japan). the growth of the seedlings was observed for three weeks under the following four table 1 ­ lettuce cultivars used in this experiment kinds of lettuce cultivar name name of seedling company red leaf lettuce leaf lettuce red sakata seed corp., japan red wave sakata seed corp., japan bancyu sun bright nakahara seed co. ltd.,japan sun bright nakahara seed co. ltd.,japan fancy red nakahara seed co. ltd.,japan red fire takii seed co. ltd., japan bancyu red fire takii seed co. ltd., japan sun marino takii seed co. ltd., japan calbee red nakahara seed co. ltd.,japan green leaf lettuce summer green sakata seed corp., japan fancy green nakahara seed co. ltd.,japan green wave takii seed co. ltd., japan yakiniku lettuce sakata seed corp., japan kamimaeda et al. ‐ effect of eod‐fr light under white leds in lettuces 233 irradiation treatments: white led (white a; 16 h white photoperiod, fluorescent lamp­type led for growing plants, espec corp., osaka, japan), fr led irradiation for 3 h after irradiation with white a (white a + fr; 16 h white + 3 h fr photoperiod, fr led: valore corp., kyoto, japan), another white led (white b; 16 h white photoperiod, fluorescent lamp­ type led for growing plants, espec corp., osaka, japan), and fr led irradiation for 3 h after irradiation with white b (white b + fr; 16 h white + 3 h fr photoperiod). the intensity of irradiation in white led treatments was 100 µmol/m2/s and fr led treat­ ment was 13.2 µmol/m2/s. the wavelengths for all leds are shown in figure 1. during cultivation, to their roots were given sufficient air using an air pump (kotobuki kougei co., ltd., japan) to avoid root rot. once a week, all solutions were replaced with fresh ones, and the electric conductivity (ec) value adjust­ ed to 1.2 ds/m1. twenty­one days after the start of cultivation, all plants were harvested and their fresh weight, root weight, maximum leaf length, number of leaves, and spad value (spad­502; konica minolta holdings inc., tokyo, japan) were measured. after drying for more than three days at 70°c, the dry leaf weight and dry root weight were also measured. the plants on day 14 were alternately harvested, and all cultivation experiments were repeated twice. all data were evaluated by one­way anova (analysis of vari­ ance) using the statcel add­in (oms publishing inc., saitama, japan) in excel (microsoft corp., redmond, wa), followed by tukey’s multiple post­hoc compari­ son test. 3. results among the red leaf lettuce cultivars, the effect of white led irradiation was observed on the fresh total weight, fresh leaf weight, root weight, and dry weight of ‘leaf lettuce red’ (table 2). fresh and dry weights of ‘red wave’, ‘fancy red’, and ‘calbee red’ cultivated under white a and white a + fr irradia­ tion were significantly greater than those cultivated under white b and white b + fr. the fresh total weight, fresh leaf weight, stem weight, and maximum leaf length in ‘bancyu sun bright’ and ‘sun bright’ grown under white a + fr and white b + fr tended to be more, and the fresh leaf weight and dry total weight in ‘sun bright’ and the fresh total weight, fresh leaf weight, and stem weight in ‘sun bright’ were significantly greater than those cultivated under white a and white b. the fresh total weight, fresh leaf weight, and dry total weight of ‘red fire’ and ‘sun marino’ cultivated under white a + fr tended to be more, and the fresh leaf weight in ‘red fire’ and the fresh total weight in ‘sun marino’ were signifi­ cantly greater than those cultivated under the other leds. all investigated items except the number of leaves and spad in ‘bancyu red fire’ grown under white a and white b showed a higher value, espe­ cially for the dry total weight, which was significantly greater than that grown under white a + fr and white b + fr. among the green leaf lettuce cultivars, in ‘summer green’, the effect of white led irradiation was observed on the fresh total weight, fresh leaf weight, and dry total weight (table 3). in ‘fancy green’, the fresh total weight, fresh leaf weight, and fig. 1 ­ wavelength distribution characteristics of various led light sources used in this experiment. a= white a led, b= white b led, c= fr led. (a) it was indicated relative value with the maximum peak taken as 1 against the measured light intensity. adv. hort. sci., 2023 37(2): 231­239 234 maximum leaf length tended to be more when grown under white a + fr and under white b. the main stem was significantly greater than that grown under white a, but the spad was lower than that grown under the other leds. in ‘green wave’, there was no difference in fresh total weight grown under white a, white b, and white b + fr, and the fresh leaf weight and dry total weight tended to be more when grown under white a. in ‘yakiniku lettuce’, there was no dif­ ference in fresh total weight, stem weight, maximum leaf length, and dry total weight when grown under white a + fr, white b, and white b + fr. the root weight, stem weight, number of leaves, and dry total weight of plants grown under white a tended to each value was indicated by mean±standard error (n=8). different letters indicate significant differences by tukey's multiple test with a significance level of 0.05. table 2 ­ the growth difference in red lettuce cultivars by two kinds of white led and fr light treatment cultivars kinds of led fresh total weight (g) fresh leaf weight (g) stem weight (g) root weight (g) maximum leaf length (cm) main stem length (cm) number of leaf spad dry weight (g) leaf lettuce red 1 white a 5.7±1.0 ab 3.7±0.3 b 1.5±0.3 b 0.4±0.0 b 13.3±0.6 18.6±0.4b 8.1±0.5 22.6±2.6a 0.31±0.05 b 2 white a+fr 4.3±0.6 b 3.0±0.4 b 1.3±0.2 b 0.3±0.1 b 13.0±0.4 19.4±0.3b 7.4±0.6 16.8±1.5b 0.23±0.03 b 3 white b 8.7±0.7 a 6.3±0.5 a 2.4±0.2 a 1.0±0.2 a 13.2±0.6 21.2±0.3ab 8.8±0.4 16.9±0.8b 0.50±0.07 a 4 white b+fr 4.3±0.2 b 2.8±0.8 b 2.0±0.5 ab 0.4±0.1 b 13.6±0.8 21.7±0.3a 7.4±0.5 18.2±1.3ab 0.38±0.07 a red wave 5 white a 9.1±1.5 a 8.5±1.4 a 0.6±0.2 1.7±0.3 a 16.7±0.6 3.5±0.7b 6.7±0.3 17.2±2.0 0.50±0.08 a 6 white a+fr 9.0±1.5 a 8.1±1.3 a 0.8±0.2 1.2±0.2 a 17.3±0.7 4.7±0.9b 6.0±0.3 15.1±1.7 0.50±0.08 a 7 white b 7.0±1.3 ab 6.1±1.1 ab 0.9±0.3 0.9±0.1 ab 17.4±0.4 6.8±0.5a 6.5±0.5 17.8±3.1 0.36±0.06 ab 8 white b+fr 3.5±0.7 b 3.1±0.7 b 0.5±0.1 0.4±0.1 b 15.7±0.6 7.4±0.2a 6.1±0.4 12.8±1.2 0.20±0.04 b bancyu sun 9 white a 6.8±1.1 b 6.2±1.0 b 0.6±0.1 b 0.7±0.2 b 16.6±1.2b 8.3±0.3ab 9.1±0.4 17.5±1.7 0.35±0.06 b 10 white a+fr 13.3±2.0 a 12.3±0.9 a 1.0±0.2 a 1.8±0.2 a 20.1±0.4a 6.4±0.3b 9.9±0.5 16.0±1.2 0.51±0.10 a 11 white b 6.5±1.2 b 5.8±1.2 b 0.8±0.1 ab 0.9±0.2 b 19.0±0.5ab 9.5±0.6a 9.5±0.6 14.4±1.7 0.30±0.04 b 12 white b+fr 11.3±0.9 a 10.5±0.8 a 0.9±0.1 a 1.6±0.1 a 20.8±0.7a 7.3±0.7b 10.4±0.5 16.9±1.5 0.54±0.05 a sun bright 13 white a 10.1±0.7 b 8.9±0.6 b 0.9±0.0 b 1.2±0.2 b 15.6±0.5c 7.4±0.4b 9.6±0.3 15.2±1.3b 0.47±0.03 b 14 white a+fr 23.5±1.3 a 18.4±1.1 a 2.0±0.1 a 2.0±0.3 a 20.0±0.6b 9.5±0.7ab 9.6±0.3 17.0±2.4ab 0.62±0.03 a 15 white b 11.3±0.9 b 11.6±0.3 b 1.0±0.1 b 1.1±0.1 b 17.7±0.5 bc 8.7±0.8 b 9.9±0.4 15.9±2.0 b 0.47±0.07 b 16 white b+fr 22.7±3.7 a 15.7±0.9 a 2.6±0.2 a 1.1±0.2 b 26.1±0.9 a 11.5±0.6 a 9.1±0.4 24.0±1.4 a 0.59±0.05 a fancy red 17 white a 9.1±0.6 a 9.3±0.4 ac 0.6±0.2 1.7±0.3 a 16.7±0.6 3.5±0.7 b 8.2±0.3 17.2±1.0 a 0.49±0.10 ab 18 white a+fr 8.9±0.6 a 9.5±0.3 a 0.8±0.2 1.2±0.2 a 17.3±0.7 4.7±0.9 b 8.3±0.5 15.1±0.7 ab 0.52±0.07 a 19 white b 3.5±0.7 b 3.1±0.7 b 0.5±0.1 0.4±0.1 b 15.7±0.6 7.4±0.2 a 8.0±0.4 12.8±1.2 b 0.20±0.03 c 20 white b+fr 6.6±1.1 c 5.3±0.8 c 0.6±0.1 0.9±0.2 a 17.4±0.4 4.9±0.6 b 8.2±0.3 17.8±1.1 a 0.36±0.06 b red fire 21 white a 10.9±0.9 c 9.4±1.0 b 1.5±0.1 b 1.4±0.1 19.2±0.3 b 6.9±0.6 c 6.5±0.3 b 14.4±1.7 0.71±0.08 b 22 white a+fr 20.7±1.3 a 15.1±1.7 a 5.6±0.6 a 2.0±0.6 22.5±0.5 a 19.9±0.8 b 8.3±0.4 a 13.2±0.9 1.17±0.15 a 23 white b 17.9±0.8 bc 13.0±1.2 ab 4.9±0.6 a 2.0±0.4 22.1±0.6 a 22.3±1.3 ab 7.4±0.3 ab 14.9±1.1 0.91±0.11 ab 24 white b+fr 16.7±0.6 b 11.6±1.1 b 5.1±0.6 a 1.3±0.2 21.4± 0.6 a 24.7±1.6 a 8.5±0.3 a 14.3±0.7 0.93±0.10 ab bancyu red fire 25 white a 9.5±0.6 a 8.6±0.4 a 1.0±0.3 a 1.5±0.3 19.1±0.8 ab 7.0±0.7 a 8.4±0.5 16.5±2.3 0.48±0.08 26 white a+fr 5.6±0.7 b 5.4±0.7 b 0.3±0.1 b 0.9±0.1 16.3±0.8 b 3.4±0.6 c 8.1±0.3 19.8±2.4 0.34±0.05 27 white b 8.5±0.6 a 8.0±0.5 a 0.6±0.1 ab 1.2±0.2 20.7±0.8 a 5.3±0.4 bc 8.5±0.7 15.5±2.0 0.47±0.03 28 white b+fr 6.0±0.9 ab 6.0±0.9 ab 0.2±0.0 b 1.0±0.1 15.3±0.8 b 3.1±0.4 c 8.6±0.4 18.8±2.6 0.38±0.06 sun marino 29 white a 3.7±0.3 b 3.6±0.3 ab 0.1±0.0 b 1.1±0.2 10.0±0.3 b 2.5±0.6 8.1±0.3 14.1±1.4 b 0.20±0.02 b 30 white a+fr 6.3±0.8 a 5.5±0.9 b 0.3±0.1 a 0.8±0.2 11.8±0.7 ab 2.8±0.5 8.3±0.5 17.0±1.0 a 0.34±0.03 a 31 white b 4.2±0.6 b 3.6±0.7 ab 0.2±0.0 a 0.8±0.2 12.1±0.3 a 2.5±0.3 7.8±0.3 17.9±1.1 a 0.33±0.05 a 32 white b+fr 2.7±0.2 b 2.7±0.3 a 0.4±0.1 a 0.7±0.1 10.8±0.7 ab 3.5±1.3 7.5±0.3 12.5±1.4 b 0.24±0.06 ab calbee red 33 white a 13.2±1.5 a 8.5±0.9 a 1.0±0.4 b 1.3±0.2 ab 16.5±0.4 b 5.9±1.2 c 7.7±0.2 19.8±0.9 a 0.73±0.10 a 34 white a+fr 13.1±0.9 a 8.7±0.5 a 2.9±0.2 a 1.6±0.2 a 20.3±0.6 a 16.2±0.6 a 7.8±0.3 18.5±0.5 ab 0.64±0.04 a 35 white b 4.6±0.7 b 4.0±0.6 b 1.7±0.3 b 0.9±0.1 ab 17.6±0.4 b 13.8±0.5 b 6.7±0.5 19.4±1.2 ab 0.26±0.03 b 36 white b+fr 7.5±0.6 b 4.6±0.5 b 1.7±0.3 b 0.6±0.1 b 18.0±1.4 ab 17.7±0.7 a 6.6±0.3 16.0±1.14 b 0.38±0.06 b kamimaeda et al. ‐ effect of eod‐fr light under white leds in lettuces 235 decrease, and a significant difference was observed in the stem weight and dry total weight. although the total light intensity was higher in white a + fr and white b + fr than in white a and white b, white b + fr showed no significant decrease in fresh leaf weight and dry weight only in ‘fancy green’ grown under white b, suggesting that fr led had little effect on the total light intensity in this experiment. however, it would be necessary to investigate the effects of similar total light intensity with and with­ out fr irradiation in the future. principal component analysis was conducted to divide the cultivars into red lettuce cultivars and green lettuce cultivars to make it easier to under­ stand the tendency. the average values of various traits for each led treatments for each cultivar were used, and the parameters are shown in tables 4 and 5. the contribution rates of the first principal compo­ nents of the red lettuce cultivars and green lettuce cultivars were 47.34% and 38.23%, respectively. the first principal component in red lettuce cultivars showed a positive factor loading for all traits except the main stem length, and a negative factor loading for only the main stem length. the first principal component in green lettuce cultivars showed a positive factor loading for fresh total weight, fresh leaf weight, stem weight, maximum leaf length, and main stem length, and a negative factor loading for root weight, number of leaves, spad, and dry total weight. furthermore, the second main component in red lettuce cultivars showed a positive factor loading for stem weight and main stem length, and a nega­ tive factor loading for the others. the second main component for green lettuce cultivars showed a posi­ tive factor loading for fresh total weight, and fresh table 3 ­ the growth difference in green lettuce cultivars by two kinds of white led and fr light treatment table 4 ­ eigen value, contribution and factor loading of 1st, 2nd and 3rd principal components in red leaf lettuce culti­ vars each value was indicated by mean±standard error (n=8). different letters indicate significant differences by tukey's multiple test with a significance level of 0.05. cultivars kinds of led fresh total weight (g) fresh leaf weight (g) stem weight (g) root weight g) maximum leaf length (cm) main stem length (cm) number of leaf spad dry weight (g) summer green 37 white a 14.4±0.8 a 12.2±0.6 a 2.1±0.4 ab 1.7±0.2 18.1±0.6 9.5±0.8 b 14.8±1.3 32.2±3.2 0.42±0.09 ab 38 white a+fr 12.7±1.1 b 8.6±1.0 b 2.3±0.3 ab 1.6±0.2 18.3±0.6 10.6±0.5 b 15.3±0.9 39.2±3.0 0.31±0.05 b 39 white b 12.4±0.6 b 10.4±0.4 b 2.0±0.2 b 1.6±0.4 17.7±0.8 11.6±0.5 b 16.1±1.1 36.3±1.9 0.33±0.08 ab 40 white b+fr 13.8±1.1 ab 12.1±0.5 a 3.1±0.4 a 1.6±0.5 19.3±0.3 16.3±1.1 a 14.1±1.0 28.4±4.5 0.63±0.10 a fancy green 41 white a 14.1±0.4 b 11.8±0.7 b 1.6±0.1 ab 1.0±0.1 b 19.3±0.5 b 10.4±0.4 a 10.0±0.4 16.4±1.5 b 0.49±0.04 b 42 white a+fr 17.5±1.3 a 15.2±0.4 a 1.3±0.2 bc 1.5±0.3 ab 23.5±0.7 a 6.4±0.4 b 9.9±0.6 24.9±1.3 a 0.57±0.06 a 43 white b 19.5±0.7 a 16.6±0.7 a 1.9±0.1 ab 1.8±0.2 ab 24.7±0.7 a 7.3±0.5 b 9.1±0.7 27.8±2.7 a 0.63±0.07 a 44 white b+fr 10.4±0.7 c 9.2±0.7 b 0.7±0.1 c 2.1±0.5 a 19.7±0.6 b 5.7±0.6 b 9.8±0.3 30.3±1.4 a 0.49±0.05 b green wave 45 white a 6.2±0.4 a 3.9±0.9 2.3 ±0.5 0.6±0.1 a 21.7±0.8 b 13.5±0.5 7.0±0.5 24.3±2.8 0.57±0.11 46 white a+fr 4.4±0.5 b 2.7±0.3 1.7 ±0.2 0.4±0.1 b 21.5±1.3 b 13.4±0.5 6.7±0.6 22.9±0.9 0.47±0.06 47 white b 5.1 ±1.1 ab 2.8±0.7 2.3±0.5 0.6 ±0.1 a 25.9±1.4 a 13.8±0.6 7.0±0.3 22.3±1.1 0.50±0.09 48 white b+fr 5.1 ±0.6 ab 3.0±0.4 2.1± 0.3 0.4±0.1 b 26.8±1.6 a 14.3±0.5 6.6±0.3 22.3±1.1 0.53±0.03 yakiniku lettuce 49 white a 2.5±0.4 b 1.8±0.3 b 0.3±0.1 b 0.4±0.1 b 10.3±0.5 b 7.4±0.6 b 6.3±0.5 18.6±1.3 a 0.16±0.02 b 50 white a+fr 4.1±0.3 a 3.4±0.3 a 0.7±0.1 a 0.7±0.1 a 12.0±0.4 ab 9.1±0.5 ab 6.5±0.4 20.8±1.9 a 0.39±0.04 a 51 white b 3.4±0.6 ab 2.0±0.3 b 0.7±0.1 a 0.3±0.1 b 13.7±0.8 a 11.1±0.9 a 6.6±0.6 14.8±1.5 b 0.31±0.01 a 52 white b+fr 4.0±0.2 a 3.3±0.2 a 0.7±0.1 a 0.5±0.1 ab 12.9±0.3 a 10.3±0.5 a 6.8±0.6 15.7±1.2 ab 0.32±0.04 a characteristics component no. 1 2 3 fresh total weight (g) 0.460 ­0.011 0.049 fresh leaf weight (g) 0.458 ­0.027 ­0.028 stem weight (g) 0.327 0..541 0.179 root weight (g) 0.411 ­0.154 ­0.234 maximum leaf length 0.333 0.281 0.425 lengh of main stem (cm) ­0.038 0.648 ­0.124 no. of leaves 0.266 ­0.347 ­0.332 spad 0.071 ­0.385 0.738 dry weight (g) 0.337 ­0.051 ­0.244 eigen value 3.287 1.423 0.868 contribution 47.34% 20.49% 12.51% cumulative contribution 47.34% 67.84% 80.34% 236 adv. hort. sci., 2023 37(2): 231­239 leaf weight, stem weight, root weight, maximum leaf length, spad, and dry total weight, and a negative factor loading for the others. the cumulative contri­ bution rates of the first and second principal compo­ nents were 67.84% and 70.13% in the red and green lettuce cultivars, respectively. principal component analysis was performed because the values were considered to be effective. for red lettuce cultivars, the scatter diagram of the types of leds and each cultivar for the first (z1) and second main components (z2) showed that the fresh total weight, fresh leaf weight, stem weight, root weight, and dry total weight in ‘red wave’, ‘bancyu sun bright’, ‘sun bright’, ‘fancy red’, ‘red fire’, ‘sun marino’ and ‘calbee red’ grown under white a + fr was higher (fig. 2). in addition, it showed an opposite tend for the fresh total weight, fresh leaf weight, and dry weight in ‘leaf lettuce red’ and ‘bancyu red fire’ grown under white b. also, for ‘bancyu sun bright’, ‘sun bright’, ‘fancy red’, and ‘calbee red’ grown under white b, the fresh total weight, fresh leaf weight, and dry weight tended to be less. while the maximum leaf length for these cul­ tivars grown under white b tended to be less, the main stem length tended to be more. on the other hand, for green lettuce cultivars, ‘summer green’ grown under white a + fr and white b, ‘yakiniku let­ tuce’ grown under white a, and ‘green wave’ grown under white a + fr showed a tendency for the fresh weight, fresh leaf weight, stem weight, and dry weight and the maximum leaf length to be lower as shown in the scatter diagram for the first and second main components (fig. 3). 4. discussion and conclusions the fresh total weight, fresh leaf weights, and dry total weight cultivated under white a and b with or without fr led in lettuce cultivars tested in this study increased or decreased. it was considered that the fig. 2 ­ the scatter diagram in z1­z2 plane nine characteristics in red lettuce cultivars arranged by the principal compo­ nent analysis. ■: white a irradiation，●: white a + fr irradiation，×: white b irradiation，△: white b + fr irradiation *numbers were shown in table 2. fig. 3 ­ the scatter diagram in z1­z2 plane nine characteristics in red lettuce cultivars arranged by the principal compo­ nent analysis. ■: white a irradiation，●: white a + fr irradiation，×: white b irradiation，△: white b + fr irradiation. * numbers were shown in table 3. table 5 ­ eigen value, contribution and factor loading of 1st, 2nd and 3rd principal components in green leaf lettuce cul­ tivars characteristics component no. 1 2 3 fresh total weight (g) 0.354 0.418 0.059 fresh leaf weight (g) 0.285 0.442 ­0.015 stem weight (g) 0.473 0.188 0.143 root weight (g) ­0.101 0.462 0.330 maximum leaf length 0.449 0.004 ­0.227 lengh of main stem (cm) 0.427 ­0.256 0.196 no. of leaves ­0.030 ­0.189 0.875 spad ­0.357 0.380 ­0.041 dry weight (g) ­0.216 0.368 0.095 eigen value 2.752 2.297 0.890 contribution 38.23% 31.93% 12.37% cumulative contribution 38.23% 70.13% 82.5% kamimaeda et al. ‐ effect of eod‐fr light under white leds in lettuces 237 type of led irradiated in the photoperiod was deeply related to the effect of the fr irradiated in the dark period, as this phenomenon was similar to the elongation of the main stem (tables 2 and 3). for the cultivars ‘leaf lettuce red’, ‘bancyu red fire’, ‘calbee red’, and ‘green wave’, it was considered better to use only white leds without fr treatment because white led irradiation with fr led did not increase leaf weight and the running cost was more. however, white leds that are generally sold have different wavelengths depending on the manufacturer, and hence this inference may not apply to all white leds. in general, it has been reported that fr light treatment promotes the elongation of the main stem or hypocotyl in cucumber (cucumis sativus l.), lily (lilium longiforum thumb.), and chrysanthemum (chrysanthemum moriforlium ramat.) (blom et al., 1995; xiong et al., 2002; hisamatsu et al., 2008). however, in brassicaceae, akutsu et al. (2016) reported that komatsuna and red mustard (b. juncea) were unable to extend the main stem, while pak choi and ‘shimana’ (a cultivation of b. juncea) easily extended the main stem regardless of the cultivation period and the light intensity of fr leds; thus, the effect differed depending on the plant species and cultivars being treated with the fr led. furthermore, the effect of fr treatment on leaf area and main stem in komatsuna and pak choi differed depending on the variety (akutsu et al., 2017). the elongation of the main stem causes quality deterioration in terms of appearance and taste in leafy vegetables such as lettuce. the main stem was elongated regardless of the fr treatment for all lettuce cultivars tested in this experiment. however, it was found that the relationship between the main stem elongation and fr treatment can be divided into several patterns regardless of whether they were green or red lettuce cultivars. under white a irradiation, fr treatment did not promote the main stem elongation in ‘leaf lettuce red’, ‘red wave’, ‘fancy red’, ‘sun marino’, ‘summer green’, ‘green wave’, and ‘yakiniku lettuce’, while it promoted the main stem elongation in ‘sun bright’, ‘red fire’, and ‘calbee red’. in addition, fr treatment suppressed the main stem elongation in ‘bancyu sun bright’, ‘bancyu red fire’, and ‘fancy green’. on the other hand, under white b irradiation, fr treatment did not have an effect on the main stem elongation in ‘leaf lettuce red’, ‘red wave’, ‘bancyu sun bright’, ‘red fire’, ‘sun marino’, ‘fancy green’, ‘green wave’, and ‘yakiniku lettuce’, while it promoted the main stem elongation in ‘sun bright’, ‘calbee red’ and ‘summer green’. in addition, fr treatment suppressed the main stem elongation in ‘fancy green’ and ‘bancyu red fire’. thus, it was considered that the relationship between fr irradiation and main stem elongation depends on the variety and cultivar. in addition, some cultivars such as ‘fancy red’, ‘red fire’, ‘summer green’ and ‘fancy green’, which showed an increase or a decrease in the main stem elongation after fr treatment, depending on if they were cultivated under white a or white b irradiation. there are also reports that fr treatment did not have any effect on main stem elon­ gation in lettuce (mickens et al., 2018; lin et al., 2020). therefore, it was found that the effect of fr treatment on the main stem elongation differs depending on the wavelength of the led irradiated in the photoperiod and on the cultivars. on the other hand, some cultivars such as ‘sun bright’, ‘sun mari­ no’, and ‘fancy green’, which an increase in spad after fr treatment depending on if they were cultivated under white a or white b irradiation, there was no effect of fr irradiation on spad in the other cultivars investigated in this experiment (table 2 and 3). thus, it was considered that no relationship between fr irradiation and spad depends on the variety and cultivar. furthermore, based on the results of the main component analysis for fresh and dry weights, the fresh total weight and the fresh leaf weight increased as the z1 axis (the first principal component) became positive in all lettuce cultivars tested in this experi­ ment, and the dry weight increased as the z2 axis (the second principal component) became positive in red lettuce cultivars and as the z2 axis became nega­ tive in green lettuce cultivars. under white a led irradiation, fresh total weight and fresh leaf weight were increased in all red lettuce cultivars except ‘leaf lettuce red’ and ‘bancyu red fire’ on treatment with fr led, while the fresh total weight and fresh leaf weight were increased in only ‘fancy green’ in green lettuce cultivars. this suggests that the effect of fr treatment cultivated under white a irradiation may be higher in red lettuce cultivars than in green lettuce cultivars. on the other hand, under white b irradia­ tion, fresh total weight and fresh leaf weight were increased in only ‘bancyu sun bright’ and ‘sun bright’ of red lettuce cultivars and in ‘summer green’ and ‘yakiniku lettuce’ of green lettuce cultivars on the treatment fr led. this suggests that the effect of fr treatment when cultivated under white a may be higher than when cultivated under white b in red let­ adv. hort. sci., 2023 37(2): 231­239 238 tuce cultivars. therefore, it is necessary to select let­ tuce cultivars that match the type of white led used as a source of light in the photoperiod, and depend­ ing on the lettuce cultivar used, select whether to treat with fr led. the main stem, which is unsuitable for sale was remarkably elongated under the two kinds of white leds either with or without fr treatment for the 13 leaf lettuce cultivars tested in this experiment. in general, white leds have different intensity, spec­ trum, and shade by adjusting the monochromatic emission of red, yellow, green, and blue (chang et al., 2012), as shown in white a and b, respectively, used in this experiment. also, in lettuce, the effect of fr light is clearly intensity­dependent, and the intensity required for maximum response depends on the trait (zou et al., 2021). furthermore, the effect of fr light has been found to depend on the type of photosyn­ thetic photon flux density (ppfd, 400­700 nm) radia­ tion and light intensity (meng and runkle, 2019). this suggests that it might be possible to obtain the effect of fr light by changing the type and intensity of the white leds used in this experiment, or by changing the intensity of fr light, even in lettuce cultivars that were investigated in this study and did not show the fr light effect. therefore, it would be necessary to investigate the characteristics of the white leds used and the plants before growing plants. furthermore, as used in a previous report (ishii et al., 2018), it would be necessary to investigate the effect of fr light on lettuce cultivars that did not elongate the main stem when they were cultivated under monoclonal red or blue, or mixed red­blue leds. references akutsu m., izsena j., takakura t., 2016 ­ effect of eod‐ fr on the growth and morphology of brassicaceae fam‐ ily plants in each cropping season. ­ hort. res. 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marina, 6, 56010 san piero a grado, pisa, italy, bandinelli.roberto@libero.it. ** ph.d. in agrobiotechnology for tropical production. the first clonal selection works in italy were carried out by breviglieri in 1943, when he noticed an intravarietal variability in the varieties cultivated in tuscany. since then, the study of clones has stimulated research for new genetic materials characterized by high performance. the council directive 68/193/eec of 9 april 1968 (concerning the marketing of material for vegetative propagation of vines) and subsequently the presidential decree of 24 december 1969 (n.1164) entitled “production and marketing of vine pre-multiplication material” gave a positive impulse to promote clonal selection activity. these decrees forced a gradual change from standard (of massal origin) to certified material (obtained through clonal selection activity). the institutes of pomology of the universities of florence and pisa, during the period 1960-1975, promoted research to establish and reorganize tuscany’s ampelographic platform. these studies focused on identifying suitable varieties for cultivation in different tuscan provinces. the clonal materials, identified by both universities, were preserved at the monna giovannella farm (andrenelli, 1984). in 1976 clonal selection activity continued with the national research council project entitled “improvement of crops for food and industry by genetic interventions sub project grapevines to wine grapes”. upon conclusion of the project, a new project was sponsored by the ministry of agriculture and forestry entitled “viticulture: production of vegetative propagation material of grapevine through by clonal selection” (triolo, 1976; pisani and bandinelli, 1990). in tuscany, research is still in progress in this sector, as carried out at public institutions and privately, and through collaborations. early clonal selection activities focused on finding plants with high productivity and vigor. in recent years, the trend has been to select individuals phenotypically characterized by a reduced vegetative growth, moderate fertility and higher tolerance to the most common plant diseases. due to desirable enological and technological aspects, other preferred features include looser clusters, smaller berries, good rate of polyphenols and anthocyanins, early ripening and high sugar content. the health status of selected material is guaranteed thanks to mipaaf (ministry of agriculture, food and forestry) protocols. public interest in the genetic material obtained from clonal selection activities stimulated the creation (on 24th march 1977) of a center for premultiplication of grapevine material, thanks to an agreement signed between the region of tuscany and the university of pisa. the agreement confirms the public interest for the multiplication and spread of plant material obtained through genetic selection of varieties and rootstocks deemed important for our national production chain (triolo, 2010). the research institutions involved provided the first registered clones to this center. nowadays, tos.co.vit. (association of tuscan wine-makers, founded on 29 january 2003) carries on the natural evolution of the activities that began with the center for premultiplication of grapevine. this institution continues premultiplication activities and distribution of the genetic material – selected in tuscany – with regard to the basic category. a total of 63 clones of european grapevine, selected in tuscany, from different projects (both public research institutions and private entities) are managed, such as: 32 clones of sangiovese, three of prugnolo gentile, four of ansonica, three of canaiolo nero, one of ciliegiolo, two of colorino, two of trebbiano toscano, two of vernaccia di san gimignano, two of malvasia bianca lunga, seven of vermentino, one of mammolo, one of aleatico, one of barsaglina, two of moscato bianco. in addition, two clones of hybrid rootstocks are also present. references andrenelli l., 1984 riordinamento ampelografico e selezione clonale delle uve bianche in toscana. università degli studi di firenze, tesi di laurea a.a. 1983-1984, pp. 76-83. breviglieri n., 1943 i principali vitigni relativi ai più diffusi tipi di vino italiano. estratto dal “trattato di enologia” del prof. garoglio. adv. hort. sci., 2013 27(3): 96-97 97 pisani p.l., bandinelli r., 1990 stato attuale della selezione clonale della vite in toscana. atti x incontro su “contributo ed influenza della chimica nella produzione e commercializzazione del vino”. siena, 7 giugno, istituto di chimica organica, università degli studi di siena, pp. 41-50. triolo e., 1976 selezione sanitaria e termoterapia della vite da vino in toscana. l’italia agricola, 3:103. triolo e., 2010 l’associazione toscana costitutori viticoli (to.sco.vit.) e l’impiego dei sistemi a radiofrequenze rfid (radio frequency identification). vivaismo viticolo: nuove performance di tracciabilità e sviluppi per il mercato vivaistico, eds. debatte, italy, pp. 8-13. 225 1. introduction traditional horticultural science has focused on plant production. nowadays, the interaction between the welfare of human beings and plants is receiving widespread interest (relf and lohr, 2003). people-plant interactions are defined as the wide array of human responses (mental, physical, and social) that occur as a result of both active and passive participation with plants (relf, 1992). one example of a positive response to plants is observed in the postexposure recovery from attention fatigue (kaplan and kaplan, 1989; tennessen and cimprich, 1995; lohr et al., 1996; herzog et al., 1997; wells, 2000). in addition, activities that involve direct contact with plants, such as gardening and horticulture, have been used as therapy for different groups of people in various settings to promote health, well-being, and social inclusion (davies, 1998; sempik et al., 2003). the natural environment, which includes plants, affects humans via the five senses and provides stimulation via vision (scenery) and olfaction (aroma of the plant) (tsunetsugu et al., 2010). there are two areas of experimental methods to clarify the interactions between people and plants. the first and predominant area of study is field experiments, clarifying the effects of total environments. the second area is through indoor experiments that test each stimulation on the basis of the five senses; the results of indoor experiments can support the outcome of field experiments. therefore, we focused on indoor experiments based on the physiological effects of plant odor stimulation. the physiological and psychological effects of essential oils have been acknowledged in folk medicine and aromatherapy for a long time (tisserand, 1988). also, essential oils and their components are widely used as constituents of different medical products in the medicine industry, as flavoring additives in the food industry, and as cosmetics and fragrances (cawan, 1999). oranges are a favorite fruit worldwide. in a previous ambient odor study, exposure to orange essential oil in a dentist’s waiting room decreased anxiety and improved mood in female patients (lehrner et al., 2000). the present paper reports an indoor experiment conducted in humans to determine the physiological effects of the odor of orange essential oil by measuring hrv, blood pressure, and pulse rate before and during inhalation of orange essential oil and comparing the values with those obtained before and during inhalation of fresh air (control). physiological effects of orange essential oil inhalation in humans b.-j. park*, k. ono**, y. yoshinami**, y. miyazaki*** (1) * department of environment & forest resources, chungnam national university, 99 daehak-ro, yuseong-gu, daejeon 305-764, republic of korea. ** nissan motor co., ltd. 1-1, takashima 1-chome, nishi-ku, yokohama city, kanagawa prefecture 220-8686, japan. *** center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa city, chiba prefecture 277-0882, japan. key words: aroma therapy, blood pressure, horticulture therapy, heart rate variability, people-plant interaction. abstract: this study was conducted to clarify the physiological and psychological effects of the odor of orange essential oil in humans. thirteen healthy male university students (mean age 23.0±1.1 years) participated. the study was conducted in an artificial climate chamber with temperature 24°c, relative humidity 50%, and illumination 50 lux. the subjects randomly inhaled orange essential oil for 120 s. fresh air inhalation was used as the control condition. heart rate variability (hrv), blood pressure, and pulse rate were continuously measured before (resting time) and during inhalation of the experimental odor. in addition, sensory evaluation and subjective odor intensity were evaluated after inhalation. the high frequency component of hrv was significantly higher, systolic and diastolic blood pressure was significantly lower, and the subjective “feeling of comfort” was significantly greater during inhalation of the orange essential oil than during inhalation of fresh air. these findings indicate that inhalation of orange essential oil effectively induces relaxation in humans. adv. hort. sci., 2014 28(4): 225-230 (1) corresponding author: ymiyazaki@faculty.chiba-u.jp received for publication 17 september 2014 accepted for publication 6 october 2014 226 2. materials and methods subjects thirteen healthy male university students were recruited for the study (mean age 23±1.1 years), which was conducted according to the guidelines of the institutional review committee of the center for environment, health and field sciences, chiba university, chiba, japan. before beginning the experiment, the subjects provided written informed consent after a detailed description of the aim and experimental procedures were provided to them. experimental stimuli and odor delivery system orange essential oil (ogawa & co., ltd., japan) was used as an experimental stimulus. fresh air without any odor was used as a control. the odor stimuli were controlled by an odor delivery system that comprised four parts: 1) a polypropylene odor bag, 2) a container, 3) an air pump with a gauge, and 4) a funnel with a plastic tube. the polypropylene odor bag was filled with 24 l of diluted orange essential oil. the odor of orange essential oil flowed from the plastic odor bag through an air pump set at 2.5 l/min and was delivered through a teflon tube to a funnel located 15 cm from the subject’s nostrils. after inhalation of the odor stimuli, the subjects were asked to evaluate the subjective odor intensity on a 6-point scale, from an “insensible level” (0) to an “unbearable level” (6). the level of subjective intensity was controlled at an “easily sensible level.” experimental design the study was conducted in an artificial climate chamber with temperature 24°c, relative humidity 50%, and illumination 50 lux. the time schedule for the experiment is shown in figure 1. after the attachment of sensors for physiological measurement, the subjects were asked to close their eyes and rest. blood pressure and pulse rates were monitored in real time. after 30 s in a stable state, the subjects were exposed to odor simulation for 120 s. sensory evaluation was conducted after physiological measurements. the subjects were asked to evaluate their subjective feelings and subjective intensity. measurement hrv, blood pressure, and pulse rates were measured as physiological indices. these indices are frequently employed to estimate changes in autonomic nervous activity (tsunetsugu et al., 2010). the time interval between two consecutive r waves (r-r interval) on an electrocardiogram was measured using a portable electrocardiograph (activtracer ac-301a, gms, japan) with three disk electrodes that were attached to the patient’s chest; this data was then analyzed using the maximum entropy method (memcalc/win; gms, japan). two major spectral components of hrv were calculated: low frequency (lf: 0.04-0.15hz) and high frequency (hf: 0.150.4 hz) components (task force of the european society of cardiology and the north american society of pacing and electrophysiology, 1996). hf is considered to reflect parasympathetic nervous activity (cacioppo et al., 1994), which increases under relaxation conditions, while lf/ (lf+hf) is considered to reflect sympathetic nervous activity (weise and heydenreich, 1989), which increases under stressful conditions. blood pressure and pulse rates were measured on the left middle finger (finometer pro, fms ltd. co., netherland). this method is noninvasive, and data is available per second. sensory evaluation was conducted after odor inhalation. the subjects were asked to evaluate and rate their “feeling of comfort,” “feeling of being soothed,” and “feeling of naturalness” on a 13-point scale. statistical analysis a paired t-test was used for comparison of hrv, blood pressure, and pulse rate measurements during inhalation of orange essential oil with those during inhalation of fresh air. the wilcoxon signed-rank test was used to analyze the subjective feelings scores. statistical analysis of physiological data was processed with excel 2003 (microsoft inc., japan). each measured value is represented as mean ± sd. a p-value of <0.05 was considered statistically significant. 3. results the subjective odor intensity ratings revealed the orange essential oil to be at the “easily sensible level”, fig. 1 time schedule for the experiment conducted to investigate the physiological indices of the olfactory effects of orange essential oil odor. 227 which was significantly different from control (fresh air) inhalation. figure 2 reports results of sensory evaluation of subjective feelings during orange essential oil inhalation. with regard to the “feeling of comfort,” the subjects rated orange essential oil as “moderately comfortable,” which was significantly different from control inhalation (fig. 2 a). with regard to the “feeling of being soothed” (fig. 2 b) and “feeling of naturalness” (fig. 2 c), no significant differences were observed between the ratings for orange essential oil and those for fresh air. figure 3 shows the hf component of hrv recorded every 30 s during the 120 s of essential oil inhalation; this was significantly enhanced compared with that during control inhalation (fig. 4). the relative systolic blood pressure, measured every 30 s during exposure, tended to be lower with orange essenfig. 2 changes in subjective feelings after inhalation of an orange essential oil or fresh air as a control. (a) feeling of comfort; (b) feeling of being soothed; (c) feeling of naturalness. n= 13, mean ± standard deviation. **p < 0.01 as determined by the wilcoxon signed-rank test. fig. 3 changes in the high frequency component of heart rate variability during 120 s of inhaling an orange essential oil or fresh air as a control. n= 13, mean ± standard deviation, determined by the paired t-test. fig. 4 changes in the average high frequency component of heart rate variability during 120 s of inhalation of an orange essential oil or fresh air as a control. n= 13, mean ± standard deviation *p < 0.05 as determined by the paired t-test. 228 tial oil inhalation than during control inhalation (fig. 5). the average relative systolic blood pressure (fig. 6), relative diastolic blood pressure (fig. 7), and average relative diastolic blood pressure showed similar results (fig. 8). 4. discussion and conclusions in this study, subjects exposed to the odor of orange essential oil for 120 s showed a significantly greater “feeling of comfort” compared with those who were exposed to fresh air without any odor. the results regarding psychological states indicated that inhalation of orange essential oil has beneficial effects. inhalation of essential oil is believed to produce reliable and predictable effects on psychological state (sanderson and ruddle, 1992), and previous studies have investigated this possibility. the effects of orange essential oil have been demonstrated through relief from anxiety and tension and improvements in mood (lehrner et al., 2000, 2005). subjective relaxation effects have also been found for lavender essential oil (diego et al., 1998; motomura et al., 2001; moss et al., 2003; burfig. 5 changes in the relative systolic blood pressure during 120 s of inhalation of an orange essential oil or fresh air as a control. n= 11, mean ± standard deviation, determined by the paired t-test. fig. 6 changes in the relative average systolic blood pressure during 120 s of inhalation of an orange essential oil or fresh air as a control. n= 11, mean ± standard deviation. *p < 0.05 as determined by the paired t-test. fig. 7 changes in the relative diastolic blood pressure during 120 s of inhalation of an orange essential oil or fresh air as a control. n= 11, mean ± standard deviation, determined by the paired t-test. fig. 8 changes in the relative average diastolic blood pressure during 120 s of inhalation of an orange essential oil or fresh air as a control. n= 11, mean ± standard deviation. *p < 0.05 as determined by the paired t-test, 229 nett et al., 2004) and peppermint essential oil (ilmberger et al., 2001; raudenbush et al., 2001, 2002). the data obtained in this study suggest that the inhalation of orange essential oil has positive effects on autonomic nervous system activity. the test subjects showed a significant enhancement of parasympathetic nervous activity, which has a relationship with relaxation. the results from the present study are consistent with those of a previous study that investigated the hf component of hrv in healthy, young, adult university students (park et al., 2007; tsunetsugu et al., 2007; park et al., 2008; lee et al., 2009; park et al., 2009, 2010; lee et al., 2011). during inhalation of orange essential oil for 120 s, both systolic and diastolic blood pressure were significantly lower than those during control inhalation, indicating that the inhalation of orange essential oil has a significant relaxing effect on the human body compared with the inhalation of fresh air. the results of this study demonstrate that when subjects were exposed to an odor simulating the natural environment, they experienced the environment via the five senses, including olfaction. these findings strongly support the belief that plant odor, such as that of oranges, is a factor for inducing relaxation in humans during exposure to the natural environment. in conclusion, we measured physiological and psychological indices 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(ed.) the role of horticulture in human well-being and social development. timber press. portland, oregon, usa. relf d., lohr v., 2003 human issues in horticulture. hort. science, 38: 984-993. sanderson h., ruddle j., 1992 aromatherapy and occupational therapy. br. j. occup. ther., 55: 310-314. sempik j., aldridge j., becker s., 2003 social and therapeutic horticulture: evidence and messages from research. ccfr, loughborough university. task force of the european society of cardiology and the north american society of pacing and electrophysiology, 1996 heart rate variability: standards of measurement, physiological interpretation and clinical use. circulation, 93: 1043-1065. tennessen c.m., cimprich b., 1995 views to nature: effects on attention. j. environ. psychol., 15: 77-85. tisserand r., 1988 essential oils as psychotherapeutic agents, pp. 167-182. in: van toller s., and g.h. dodd (eds.) perfumery: the biology and psychology of fragrance. chapman and hall, london, uk. tsunetsugu y., park b.j., ishii h., hirano h., kagawa t., miyazaki y., 2007 physiological effects of shinrin-yoku (taking in the atmosphere of the forest) in an oldgrowth broadleaf forest in yamagata prefecture, japan. j. physiol. anthropol., 26: 135-142. tsunetsugu y., park b.j., miyazaki y., 2010 trends in research related to “shinrin-yoku” (taking in the forest atmosphere or forest bathing) in japan. environ. health prev. med., 15: 27-37. weise f., heydenreich f., 1989 effects of modified respiratory rhythm on heart rate variability during active orthostatic load. biomed. biochim. acta, 48: 549-556. wells n.m., 2000 at home with nature effects of “greenness” on children’s cognitive functioning. environ. behav., 32: 775-795. 73 1. introduction persimmons are the edible fruits of deciduous trees in the genus diospyros, many of which originated in china (martínez-calvo et al., 2012), even though some species are native to other parts of the world, including d. lotus, or date plum, which is native to southwest asia and southeast europe. the fruit was called “fire of the gods”, or dios pyros by the ancient greeks, thus the name of the genus. persimmons produce popular and nutritious fruits that have become traditional crops in korea and japan and are found throughout the world (veberic et al., 2010; dembitsky et al., 2011; giordani et al., 2011). they are sweet and are relatively good sources of carotenoids, vitamin c, polyphenols and proanthocyanidins (gu et al., 2008; dembitsky et al., 2011; giordani et al., 2011). moreover, persimmons have been reported to have health benefits in both traditional and western medicine (giordani et al., 2011). for example, persimmons of the triumph variety improved lipid metabolism and atherosclerosis indices in rats that were fed a diet that was high in cholesterol (gorinstein et al., 1998; 2011). the most cultivated species is the d. kaki thunb. (martínez-calvo et al., 2012; usda, 2012), also known as chinese persimmon, japanese persimmon, kaki, caqui and diospyros kaki l. (martínez-calvo et al., 2012; usda, 2012). there is also a species (d. virginiana) that is native to eastern north america (celik and ercisli, 2008). persimmons are climacteric fruits (veberic et al., 2010). that is, the amount of sugars (particularly sucrose) and total carotenoids increase in the final stages of ripeness and firmness, while soluble tannins and titratable acidity levels decrease, resulting in improved flavor (candir et al., 2009). in the northern hemisphere, they are harvested between september and december (dembitsky et al., 2011). on the other hand, the brazilian caqui cultivar called ‘giombo’ is very productive and matures late, with fruits being picked from march to the end of may (martins and pereira, 1989). this relatively short harvest season, coupled with the lack of information about storage, limits its expansion and causes losses in the final processing and marketing of the fruit (donazzolo and brackman, 2002). persimmon cultivars are classified into four groups: pollination-constant astringent, pollinationvariant astringent, pollination-constant non-astringent, and pollination-variant non-astringent (campo-dall’orto et al., 1996; celik and ercisli, 2008). persimmons have also been divided into a volatile-independent group (vig, corresponding to the pollination-constant non-astringent group) and the volatile dependent group (vdg, consisting of the pollination-constant astringent, pollination-variant astringent and pollination variant non-astringent types) (giordani et al., 2011). tannins in the vig type are usually effect of active modified atmospheres on the quality of non-astringent persimmons (caqui giombo) d. kaki thunb. when stored under refrigerated conditions m.r. de moraes*, é.r. daiuto*, r.l. vietes*, n.c. cardoso*, r.e. smith** (1) * faculty of agronomic sciences, unesp botucatu, c.p. 237, botucatu 18610307, sp, brazil. ** u.s. fda, 11510 w 80st, lenexa, ks 66224, usa. key words: caqui giombo, diospyros kaki l., firmness, persimmon, postharvest. abstract: the effects of active modified atmospheres were evaluated in ‘giombo’ persimmons with tannins already removed (non-astringent) and stored at 0ºc and 85-90% relative humidity for 35 days. the goal was to maintain quality and delay ripening. the fruits were picked by hand when they were about 50% green, sanitized and subjected to different mixtures of co2 and o2. fruits were wrapped in thin film plastic made of nylon + polyethylene and analyzed every seven days for weight loss, respiratory activity, coloration, titratable acidity, soluble solids, ratio, ph, firmness, pectin methyl esterase and polygalacturonase enzyme activities, reducing sugars, ascorbic acid and astringency index. refrigerated storage and active modified atmospheres were effective in conserving the quality of ‘giombo’ persimmons. the fruits submitted to the highest co2 concentrations (7 and 8%) had the lowest weight loss and respiratory intensity with a delay in the climacteric peak. adv. hort. sci., 2013 27(1-2): 73-80 (1) corresponding author: robert.smith@fda.hhs.gov received for publication 13 march 2013 accepted for publication 25 may 2013 74 relatively high in molecular weight and soluble in water. moreover, their concentrations are maximum at an early stage of development and are <1% of the fresh weight. on the other hand, the vdg types contain tannins that are usually soluble in water, have a lower molecular weight and are not palatable at harvesting time. the seeds of pollination-variant non-astringent cultivars can exude ethanol, which makes the water-soluble tannins insoluble (giordani et al., 2011). all persimmons are edible when soft, but they can be astringent at harvest time (giordani et al., 2011). parthenocarpic fruits of pollination-variant nonastringent cultivars, and both seeded and parthenocarpic fruits of astringent cultivars are edible only after removing the astringency artificially or when soft, overripe or dried. this happens when low molecular weight, soluble tannins are made water-insoluble, probably by binding with pectins (giordani et al., 2011). astringency can be removed by storing persimmons in a modified atmosphere containing elevated amounts of co 2 or ethanol (del bubba et al., 2009; edagi et al., 2009) and refrigeration can delay the ripening of persimmons picked when only half-ripe (vieites et al., 2012). the heart-shaped ‘hachiya’ cultivar is the most popular pollination-constant astringent persimmon, while the ‘fuyu’ is a popular pollination-constant non-astringent cultivar in japan (celik and ercisli, 2008). the brazilian ‘giombo’ cultivar is in the variable denomination which includes fruits that are yellow and contain tannins. seedless fruits keep their astringency even when ripe, so the tannins must be removed artificially. the biggest inconvenience in accelerating the ripening process to remove tannins is that it diminishes the shelf life (edagi et al., 2009). according to antoniolli et al. (2000), this can compromise the firmness of the pulp when stored for a long period. refrigerated storage is among the practices used to maintain the quality of fruits for a short length of time (vieites et al., 2012); it can prolong the useful storage time, but the majority of workers show that it should not exceed 35 days to remain safe (ben-aire and zutkhi, 1992; brackmann and saquet, 1995; chitarra and chitarra, 2005). other methods have been tested to extend the shelf life of fruits, with modified atmospheres standing out. also plastic films can increase co 2 and decrease o 2 however the concentrations of these gases are not controlled and vary with time, temperature, type of plastic and respiratory rate (sargent et al., 1993). according to ferri et al. (2004) storing ‘fuyu’ persimmons at 0oc maintains the firmness of the pulp for 90 days, but when only using refrigerated storage the shelf life is less than 30 days. another approach is to use a modified active atmosphere in which the initial concentration of gases inside the packaging is controlled. ‘giombo’ persimmons have not yet been tested under these conditions, therefore the objective of this study was to test the effects of a modified active atmosphere on the cold storage of ‘giombo’ persimmons that have had the tannins removed, making them non-astringent. 2. materials and methods ‘giombo’ persimmons were from the sacramento agropastoril ltda, avaré (sp), located at a latitude of 23°05’56”s, longitude 48º55’33”w and altitude of 780 m, with an annual precipitation of 1500-1700 mm yr-1, annual temperature between 20 and 24°c and soil classified as purple oxysoil (structured earth, purple, oxidized). the fruits were collected by hand when they were at stage 3 of maturity, medium-ripe, about 50% green. to remove the tannins, fruits were collected in plastic boxes and exposed to ethanol fumes at a concentration of 6.6 ml kg-1, in chambers at 25°c for 48 h. fruits were then submitted to the following gas mixtures: 0.03% co 2 and 21% o 2 (t1= control; 5% co 2 and 4% o 2 (t2); 6% co 2 and 4% o 2 (t3); 7% co 2 and 4% o 2 (t4) and 8% co 2 and 4% o 2 (t5). the fruits were wrapped in plastic wrappers made of nylon and polyethylene and stored refrigerated at 0±0.5ºc and 8590±5% relative humidity for 35 days, and were analyzed every seven days. for the control group (non-destructive), two whole fruits were analyzed five times and for the destructive group, two fruits were cut into pieces and then analyzed in triplicate. loss of mass (%) the weight of fruits was measured with an analytical balance and the results expressed as a percentage. respiratory activity the liberation of co 2 was measured following the method of bleinroth et al. (1976), using a saturated solution of barium hydroxide and 0.1 n potassium hydroxide. the respiratory rate was calculated using the equation: tco 2 = 2.2 (vo-v1). 10/p.t. where t co 2 = respiratory rate (ml of co 2 . kg-1.h-1); vo= volume of hcl needed to titrate the potassium hydroxide solution before and v1, after absorbing co 2 (ml); p= mass of the fruits; t = time of respiration; 2.2= equivalent weight of co 2 (44/2), multiplied by the concentration of hcl; 10= adjustment for the total amount of koh used. analyses were carried out in triplicate. titratable acidity expressed as gram equivalents of malic acid per 100 g of pulp (g of malic acid 100 g-1), obtained by titrating 5 g of homogenized pulp diluted to 100 ml with distilled water with 0.1 n naoh, using a phenolphthalein indicator, in conformance with odair et al. (2008). soluble solids made using a palette atago pr-32 refractometer and expressed as (ºbrix), in conformance with odair et al. (2008). maturity index (ratio) determined from the ratio of soluble solids to titratable acidity (2008). 75 ph measured using a model 300 ph meter in conformance with odair et al. (2008). sugars measured using the methods of somogy (1945) and nelson (1944) and a micronal b 382 spectrophotometer to measure the absorbance at 535 nm. ascorbic acid determined by adding 30 ml of 4% oxalic acid to 30 g of pulp and titrating with 0.5% dpi-2,6-diclorophenolindophenol with results expressed as ml of ascorbic acid 100 ml-1 of pulp (mapa, 2011). astringency index determined using the method of gazit and levy (1963) and modified by vitti (2009) in which one of the sides of the cut fruit is placed on a piece of filter paper that has been impregnated with a solution of 5% fecl 3 . soluble tannins react and turn the paper dark, which is then analyzed visually on a scale of 1 to 5, with 5 being the darkest and most astringent. coloration measured in a konica minolta (chroma meter, cr 400/410) colorimeter over the spectral region of 380 to 780 nm. the reflectance reading was obtained with an angle of observation of 2º and illumination c. the color was expressed by a system of rectangular coordinates: l* a* and b* in conformance with the cie (comission internatinale de e’clairage), where l* is the percent luminosity (0% = black and 100% = white), a* represents the colors red (+) or green (-) and b* the colors yellow (+) or blue (-). firmness measured using a texture analyzer (stevens – lfra texture analyzer) with a penetration distance of 10 mm and a velocity of 2.0 mm sec-1, and using a ta 9/1000 fixture and a pressure of 15 gram of force per cm2 (gf cm-2). enzyme activity activities of polygalacturonase (pg) and pectin methylesterase (pme) were determined by the methods of albershein et al. (1967) and ahmed and labavitch (1980). the tukey test at a 5% probability level was used to compare results, as recommended by gomes (2000) and by linear regression analysis for weight loss. 3. results and discussion the respiratory activity of ‘giombo’ persimmon fruits increased during the storage periods, as shown in figure 1. concomitantly, there was a steady weight loss, depending on the treatment (table 1). the fruits exposed to 7 and 8% co 2 showed less weight loss than the other treatments. in order to have acceptable surface shrinkage for fresh fruits (finger and vieira, 2002) the maximum tolerated weight loss should be between 5 and 10%, thus the weight losses of ‘giombo’ persimmons found in this study are acceptable. it was also observed that the respiratory rate was less at the highest concentrations of co 2 (6, 7 and 8%) compared table 1 weight loss (%) of ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0°c and 85-90% relative humidity for 35 days storage days control treatment 5%co 2 4%o 2 6%co 2 4%o 2 7%co 2 4%o 2 8%co 2 4%o 2 averages 7 0.105±0.024 aa 0.236±0.189 ca 0.136±0.184 aa 0.038±0.011 aa 0.024±0.093 aa 0.108±0.028 14 0.144±0.09 aa 0.323±0.127 a 0.216±0.112 aa 0.098±0.019 aa 0.093±0.047 aa 0.175±0.053 21 0.186±0.104 aa 0.353±0.226 ca 0.261±0.108 aa 0.123±0.026 aa 0.118±0.059 aa 0.208±0.155 28 0.190±0.023 ab 0.695±0.068 ba 0.270±0.120 ab 0.143±0.046 ab 0.130±0.068 ab 0.286±0.141 35 0.268±0.119 ab 1.041±0.262 aa 0.355±0.220 ab 0.221±0,017 ab 0.222±0.063 ab 0.421±0.353 averages 0.149±0.115 0.441±0.281 0.206±0.098 0.104±0.057 0.098±0.015 small letters compare averages of different treatments on each day. upper case letters compare averages between different days. averages followed by at least one letter in common do not differ statistically. fig. 1 respiratory activity (mg co 2 .kg -1. h-1) of ‘giombo’ persimmons with tannins removed and submitted to modified atmospheres and stored at 0ºc, 85-90% relative humidity for 35 days at different concentrations. 76 to fruits exposed to 5% co 2 and 4% o 2 which reached a peak on the 14th day of storage. the fruits exposed to 6, 7 and 8% co 2 had maximum respiratory activity after 28 days. the elevated concentrations of co 2 inhibited the respiratory activity of the fruits. the color of the fruits was not statistically different in fruits treated differently, as shown in tables 2-4. no darkening was seen on the surface of the fruits. the values of a* in the apical region of the fruits was less than that of the median or basal regions. negative a* values were found only on the first day, indicating the presence of a green color and verifying that the fruits did ripen during storage. this is in agreement with data reported by chitarra and chitarra (2005) who reported that the change in color is associated with ripening, which is a standard attribute for determining fruit quality. the increase in a* and b* color indices each reflected changes from yellowish-green to orangish-red. brackmann et al. (1997) reported that persimmons stored refrigerated at 5ºc did not show an appreciable change in color since low temperatures inhibit the biosynthesis of carotenoids. the differences in color seen in the present study were due to differences at the time of collection. danieli et al. (2002) reported that ‘fuyu’ persimmons that were collected when still yellowish-green eventually changed to red. still, there were no such changes in the treatments in the current study. this is commercially important since coloration is a primary quality standard. the amounts of soluble solids, titratable acidity and the ratio between the two are reported in table 5, which shows that there is no significant change, regardless of dose of co 2 and storage time. working with the same cultivar, antoniolli et al. (2000) found that there was little change in the amount of soluble solids, as confirmed also by the current study. murray and valentini (1998) reported that limits in the precision of the method and the many factors that affect soluble solids make it difficult at times to establish interactions between the process of maturation and the content of soluble solids. thus, the amount of soluble solids serves best as a standard of quality rather than an index that measures the effects of storage. the present study also verified that there is a slow increase in the titratable acidity of the fruits during storage, with values ranging from 0.07 to 0.10 grams equivalents of malic acid per 100 g of pulp. according to costa and balbino (2002), the increase in titratable acidity is due to the formation of galacturonic acid during the process of breaking down cell walls, which occurs during fruit storage. the ratios of soluble solids to titratable acidity varied little during storage. the ph of the samples oscillated between 5.49 and 5.87, as shown in table 6. this is similar to the results reported by blum et al. (2008) who found no change in ph or acidity in ‘giombo’ persimmons that were covered with carnuba wax during cold storage. therefore, the modified atmospheres used in the current study did not affect the ph very much. one of the main concerns about storage is the rapid loss of firmness of the pulp, which makes fruits commercially unacceptable. thus, the fact that the present study shows only small variations in firmness, as shown in table 7, is important, and can be explained as a combination of cold storage in a modified atmosphere, each of which decrease metabolism and prolong shelf life. table 2 luminosity (%) of caquis ‘giombo’ persimmons with tannins removed and submitted to modified atmospheres and stored at 0°c and 85-90% relative humidity for 35 days days of storage luminosity reg. apical reg. mediana reg. basal 0 45.3 ab 48.9 ab 46.6 a 7 44.3 b 48.4 b 45.6 ab 14 44.8 b 48.2 b 45.2 ab 21 45.3 ab 48.8 ab 44.7 b 28 46.2 a 49.3 a 45.2 ab 35 45.5 ab 49.3 a 46.0 ab averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 3 color a* in ‘giombo’ persimmons with tannins removed and submitted to modified atmospheres and stored at 0°c and 8590% relative humidity for 35 days storage days color a* reg. apical reg. media reg. basal 0 -0.1 d 0.5 d 1.4 d 7 2.5 cd 3.0 c 7.5 c 14 2.8 cd 3.1 c 7.3 c 21 6.7 b 9.8 b 11.8 b 28 12.5 a 15.8 ab 15.8 ab 35 15.6 a 18.9 a 18.3 a averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 4 color b* in ‘giombo’ persimmons with tannins removed, submitted to modified atmospheres and stored at 0°c and 85-90% relative humidity for 35 days storage days color b* apical medium basal 0 34.5 a 39.0 a 32.9 a 7 31.2 b 37.7 abc 32.1 a 14 32.6 b 36.7 bc 31.9 a 21 29.0 c 35.0 c 26.7 b 28 32.6 b 37.4 bc 28.1 b 35 32.1 b 38.9 abc 32.3 a averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. 77 table 5 amounts of soluble solids (ºbrix), titratable acidity (g malic acid per 100 g of pulp) and ratio in ‘giombo’ persimmons with tannins removed and submitted to modified atmospheres and stored at 0°c and 85-90% relative humidity for 35 days treatments soluble solids (ss) averagesstorage days 0 7 14 21 28 35 control 17±0.3 15.7±0.2 16.9±1.8 17.4±1.5 14.4±1.9 17.4±0.5 16.5±1.6 5%co 2 4%o 2 17±0.3 16.5±0.9 15.8±0.9 16.6±2.0 15.7±1.0 17.0±0.8 16.5±1.1 6%co 2 4%o 2 17±0.3 16.8±0.2 15.9±0.9 16.4±0.7 15.3±0.5 15.6±0.2 16.2±0.8 7%co 2 4%o 2 17±0.3 18.1±1.0 16.8±0.7 16.8±0.3 15.2±0.3 17.0±0.8 16.8±1.0 8%co 2 4%o 2 17±0.3 15.8±0.6 16.9±0.4 16.9±1.6 15.4±1.0 16.1±0.6 16.4±1.0 average 17a±0.3 16.6a±1.1 16.5a±1.03 16.8a±1.2 15.2b±1.0 16.6a±0.9 titratable acidity (ta) control 0.07±0.01 0.09±0.01 0.07±0.01 0.08±0.05 0.08±0.03 0.08±0.01 0.08±0.02 5%co 2 4%o 2 0.07±0.01 0.06±0.01 0.08±0.01 0.08±0.05 0.10±0.04 0.09±0.01 0.08±0.03 6%co 2 4%o 2 0.07±0.01 0.08±0.01 0.07±0.02 0.07±0.05 0.06±0.02 0.09±0.01 0.07±0.02 7%co 2 4%o 2 0.07±0.01 0.07±0.01 0.06±0.01 0.08±0.03 0.10±0.02 0.14±0.04 0.09±0.03 8%co 2 4%o 2 0.07±0.01 0.07±0.01 0.06±0.01 0.10±0.04 0.08±0.03 0.09±0.01 0.08±0.02 average 0.07b±0.01 0.07b±0.01 0.07b±0.01 0.08ab±0.04 0.08ab±0.03 0.10a±0.03 ratio testemunha 234±27 183±17 250±34 260±114 195±77 230.5±45 225.3±60 5%co 2 4%o 2 234±27 267±48 199±10 279±142 183±75 188.2±27 225.0±71 6%co 2 4%o 2 234±27 221±26 224±32 278±125 265±72 176.4±14 233.2±63 7%co 2 4%o 2 234±27 283±53 300±63 239±73 155±22 131.8±40 223.9±76 8%co 2 4%o 2 234±27 222±24 269±7 220±152 215±77 179.6±29 223.2±66 averages 234ab±23 235ab±48 248.7ab±47.1 255.1a±107.6 202.4ab±68.8 181.3b±42.8 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 6 ph of ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0°c and 85-90% relative humidity for 35 days treatments days of storage averages 0 7 14 21 28 35 control 5.87±0.28 5.50±0.03 5.52±0.16 5.63±0.13 5.83±0.12 5.91±0.24 5.71a±0.23 5%co 2 4%o 2 5.87±0.28 5.49±0.16 5.44±0.04 5.49±0.08 5.48±0.11 5.60±0.08 5.56b±0.20 6%co 2 4%o 2 5.87±0.28 5.50±0.07 5.48±0.08 5.60±0.12 5.52±0.05 5.68±0.14 5.61ab±0.19 7%co 2 4%o 2 5.87±0.28 5.52±0.11 5.46±0.13 5.44±0.09 5.44±0.02 5.54±0.06 5.55b±0.19 8%co 2 4%o 2 5.87±0.28 5.48±0.07 5,57±0.02 5.45±0.06 5.54±0.10 5.53±0.06 5.58ab±0.18 averages 5.87a±0.24 5.50b±0.08 5.49b±0.01 5.52b±0.12 5.56b±0.16 5.65b±0.18 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 7 firmness (gf.cm-2) of ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0ºc and 85-90% relative humidity for 35 days treatment storage days averages 0 7 14 21 28 35 control 671±48 497±26 515±295 568±207 378±204 378±320 501 b±209 5%co 2 4%o 2 671±48 579±86 651±75 786±43 627±76 732±253 674 ab±123 6%co 2 4%o 2 671±47 652±49 780±65 766±38 627±89 691±199 698 a±100 7%co 2 4%o 2 671±47 607±84 634±47 673±169 551±108 647±179 630 ab±1084 8%co 2 4%o 2 671±47 555±13 661±142 684±170 503±91 694±126 628 ab±1203 averages 671ab±404 578ab±735 648ab±157 695a±145 537b±141 628ab±232 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. 78 the activities of the enzymes pme and pg exhibited different tendencies during the 35 days of storage, as shown in tables 8 and 9. in a previous study, enzyme activities decreased, remained constant or increased during maturation, depending on the fruit and method of analysis (lima et al., 2006) for the fruits tested in this study, there was a nearly linear increase in pme activity with storage time. however, there was little difference in the pme activities of fruits that were exposed to 6, 7 and 8% co 2 . a reduction in pg activity was also seen. it has been suggested that this can be explained by the lack of substrate for the enzyme or the existence of other multi-enzyme complexes (abeles and takeda, 1989). enzymes such as β-galatosidase and other cellular proteins could be acting in the destruction of the cell walls of the fruits, causing the extravasation of cellular fluids. it is also possible that proteases could have catalyzed the hydrolysis of pg. antunes et al. (antunes et al., 2006) measured the activities of pg and pme in blackberries (rubus spp.) that were stored in different environments for different times. they concluded that the activity of pme increased during storage for all cultivars and storage conditions, while the activity of pg decreased. this was confirmed in the present study on persimmons. there was a slow decrease in the amount of reducing sugars beginning on the 28th day of storage, as shown in table 10. the fruits had ascorbic acid levels ranging from 15.6 to 40.4 mg per 100 ml, as shown in table 11. the amount of ascorbic acid decreased with time. silva et al., (2011) evaluated the quality of ‘fuyu’ persimmons and verified that covering them with wax did not affect the levels of ascorbic acid. according to chitarra and chitarra (2005) vitamin c tends to decrease during the ripening and stortable 8 pectin methylesterase (ue.min-1.g-1 of fresh fruit) of ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0ºc and 85-90% relative humidity for 35 days treatment days of storage 0 7 14 21 28 35 control 1343af±85 3782ac±145 3320ce±140 3490ad±155 10898aa±385 10675ab±375 5%co 2 4%o 2 1343af±85 2766bd±130 6081aa±415 2814cc±133 2057de±125 4830bb±225 6%co 2 4%o 2 1343af±85 2446db±115 2520da±130 1881ed±114 2074cc±125 1811ee±100 7%co 2 4%o 2 1343af±85 2607ca±125 22389eb±128 1913de±123 2056ec±125 1945dd±105 8%co 2 4%o 2 1343af±85 1751ee±102 3879ba±149 3150bb±150 2174bd±130 2189cc±185 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 9 polygalacturonase (min-1g-1 of fresh fruit) of ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0ºc and 85-90% relative humidity for 35 days treatment storage days 0 7 14 21 28 35 control 890 aa±143 548±95 aa 591±55 ab 341±55 bc 213±40 ac 206±86 ab 5%co 2 4%o 2 890 aa±143 453±60 ab 239±37 bc 473±71 bb 243±72 ab 143±84 bc 6%co 2 4%o 2 890 aa±143 89±4 bc 200±39 bc 648±14 aa 257±88 aa 394±15 aa 7%co 2 4%o 2 890 aa±143 432±268 ab 257±40 bc 374±30 bb 80±33 dd 175±43 cc 8%co 2 4%o 2 890 aa±143 333±25 ab 673±24 aa 241±83 cc 166±132 bc 102±47 bc averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. table 10 reducing sugars (%) in ‘giombo’ persimmons, with tannins removed and submitted to modified atmospheres and stored at 0ºc and 8590% relative humidity for 35 days treatment storage days average 0 7 14 21 28 35 control 13.3±1.8 12.1±2.1 13.9±1.6 13.5±1.1 10.8±0.5 11.1±0.4 12.4±1.7 5%co 2 4%o 2 13.3±1.8 13.5±1.3 13.7±0.9 13.9±2.0 12.4±0.6 10.9±0.6 13.0±1.5 6%co 2 4%o 2 13.3±1.8 15.2±1.2 13.8±0.9 12.7±0.5 11.6±0.4 10.0±1.4 12.8±2.0 7%co 2 4%o 2 13.3±1.8 16.0±1.5 13.6±0.7 13.1±0.8 12.0±0.5 11.1±0.5 13.2±1.8 8%co 2 4%o 2 13.3±1.8 14.1±0.6 14.1±1.1 13.0±1.6 12.2±0.9 10.6±0.4 12.9±1.6 average 13.3±1.8 14.2a±1.9 13.8a±0.93 13.2a±1.2 11.8b±0.8 10.7b±0.8 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. 79 age of many fruits, due to the action of ascorbic acid oxidase, other oxidative enzymes and/or peroxidases. the astringency index of ‘giombo’ persimmons that had their tannins removed and were exposed to modified atmospheres and were stored at 0°c had an index of 1 (no tannins) until the end of storage, which is in agreement with results reported by núñez-delicado et al. (2003) who reported that ethanol dissolves the tannins. refrigerated storage in active modified atmospheres is effective in preserving ‘giombo’ persimmons. fruits exposed to 7 or 8% co 2 gave the best results. this work should not be taken as reflecting fda policy or regulations. references abeles f.b., takeda f., 1989 increased cellulase activity during blackberry fruit ripening. hortsci., 24(5): 851. ahmed e.a., labavitch j.m., 1980 cell wall metabolism in ripening. i cell wall changes in ripening “bartlett” pears. plant physiol., 65: 1009-1013. albersheim p., nevins d.j., english p.d., karr a., 1967 a method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. carbohyd. res.; 5(3): 340-345. antoniolli l.r., castro p.r.c., kluge r.a., scarpare f.j.a., 2000 remoção da adstringência de frutos de caquizeiro ‘giombo’ sob diferentes períodos de exposição ao vapor de álcool etílico. pesq. agropec. bras., 35(10): 2083-2091. antunes l.e.c., gonçalves 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treatment storage days averages 0 7 14 21 28 35 control 38±11 44±4 33±7 34±15 20±9 18±7 31±13 5%co 2 4%o 2 38±11 38±2 37±9 37±19 24±11 13±3 31±13 6%co 2 4%o 2 38±11 36±3 34±7 33±18 21±2 18±2 30±11 7%co 2 4%o 2 38±11 46±13 30±6 43±22 36±20 13±0 34±16 8%co 2 4%o 2 38±11 39±2 41±2 19±2 41±11 16±2 32±12 averages 38ab±9 40a±7 35ab±67 33ab±16 28bc±13 16c±4 averages followed by the same letter, lower case in columns and capital letters in rows do not differ significantly by the tukey test at 5% probability. 80 donazzolo j., brackmann a., 2002 efeito do co 2 em atmosfera controlada na qualidade de caqui (diospyros kaki, l.) cv. fuyu. rev bras. agroci. pelotas, 8: 241-245. edagi f.k., chiou d.g., terra f.a.t., sestari i., kluge r.a., 2009 remoção da adstringência de caquis ‘giombo’ com subdosagens de etanol. ciênc. rural, santa maria, 39: 2022-2028. ferri v.c., rinaldi m.m., danielli r., lucchetta l., rombaldi c.v., 2004 atmosfera modificada na conservação de caquis (diospyrus kaki, l.). cultivar fuyu. rev. bras. agroci., 10: 111-115. finger f.l., vieira g., 2002 controle da perda pós colheita de água em produtos hortícolas. ufv, viçosa, pp. 29. gazit s., levy y., 1963 astringency and its removal in persimmon. israel j. agr. res., 13(3): 125-132. giordani e., doumett s., nin s., del bubba m., 2011 selected primary and secondary metabolites in fresh persimmon (diospyros kaki thunb.): a review of analytical methods and current knowledge of fruit composition and health benefits. food res. intl., 44: 1752-1767. gomes f.p., 2000 curso de estatística experimental. 14. ed. fundação de estudos agrários luiz de queiroz fealq, piracicaba, brazil. gorinstein s., bartinikowska e., kulasek g., zemser m., trakhtenberg s., 1998 dietary persimmon improves lipid metabolism in rats fed diets containing cholesterol. j. nutr., 128: 2023-2027. gorinstein s. leontowicz h., leontowicz m., jesion i., namiesnik j., drzewiecki j., park y.s., ham k.s., giordani e., trakhtenberg s., 2011 influence of two cultivars of persimmon on atherosclerosis indices in rats fed cholesterol-containing diets: investigation in vitro and in vivo. nutr., 27(7-8): 838-846. gu h.-f., li c.-m., xu y.-j., hu w.-f., chen m.-h., wan q,-h., 2008 structural features and antioxidant activity of tannin from persimmon pulp. food res. intl., 41: 208-217. lima m.a.c., alves r.e., filgueiras, h.a.c. 2006 mudanças relacionadas ao amaciamento da graviola durante a maturação pós-colheita. pesq. agropec. bras., 41(12): 1707-1713. mapa, 2011 modified tillman’s method. ministério da agricultura, pecuária e abastecimento http://www.agricultura. gov.br . martínez-calvo j., naval m., zuriaga e., llácer g., badenes m.l., 2012. genet. resour. crop. evol. published on-line. martins f.p., pereira f.m., 1989 cultura do caquizeiro. jaboticabal. funep, pp. 71. murray r., valentini g., 1998 storage and quality of peach fruit harvest at different stages of maturity. acta horticulturae, 465: 455-463. nelson n.a., 1944 a photometric adaptation of somogy method for the determination of glucose. j. biol. chem., 153: 375-380. núñez-delicado e., sojo m.m., garcía-carmona f., sánchez-ferrer a., 2003 partial purification of latent persimmon fruit polyphenol oxidase. j. agric. food chem., 51: 2058-2063. odair z., neus s.p., tiglea p., 2008 métodos físico-químicos para análise de alimentos. physical-chemical methods for analyzing foods. instituto adolfo lutz, são paulo, brasil. sargent s.a., crocker t.e., zoellner j.j., 1993 storage characteristics of ‘fuyu’ persimmons. proc. florida state hort. soc., 106: 131-134. silva m.c., atarassi m.e., ferreira m.d., mosca m.a., 2011 qualidade pós –colheita de caqui ‘fuyu’ com utilização de diferentes concentrações de cobertura comestível. postharvest quality of ‘fuyu’. ciênc, agrotec. lavras, 35(1): 144-151. somogy m., 1945 determination of blood sugar. j. biol. chem., 160: 69-73. usda, 2012 germplasm resources information network (grin). website: http://www.ars-grin.gov/cgi-bin/npgs/ html/taxon.pl?14293 veberic r., jurhar j., mikulic-petkovsek m., stampar f., schmitzer v., 2010 comparative study of primary and secondary metabolites in 11 cultivars of persimmon fruit (diospyros kaki l.). food chem., 119: 477-483. vieites r.l., picanço n.f.m., daiuto é.r., moraes m.r., 2012 optimum temperature and state of maturity for storing persimmons, diospyros kaki l., caqui ‘giombo’. nat. prod. j., 2: 180-187. vitti d.c.c., 2009 destanização e armazenamento refrigerado de caqui «rama forte» em função da época de colheita. phd, escola superior de agricultura “luiz de queiroz”, universidade de são paulo, piracicaba, brasil. impaginato 3 adv. hort. sci., 2024 38(1): 3­11 doi: 10.36253/ahsc­14997 influence of ground cover and tunnels on production of red russian kale in urban gardens m.l. richardson, c.g. arlotta, l. monroe­lord (*) college of agriculture, urban sustainability and environmental sciences, university of the district of columbia, united states of america, 4250 connecticut ave, nw, washington, dc 20008, usa. key words: dietary fiber, hoop house, mulch, nutrients, season extension, small­ scale farming, urban agriculture. abstract: kale, brassica oleracea l. var. acephala, is an important urban crop for human health and may potentially be grown year­round even in a temper­ ate climate. we investigated black plastic and straw mulch compared to bare soil cover in low tunnels at 10 urban garden sites and in low tunnels within a high tunnel in the usa to ascertain the influence on yield and nutrients of red russian kale, soil temperature, air temperature, weed pressure, and aphid abundance. kale had low yield in garden sites, likely because the outside envi­ ronment was too cold for low tunnels to gain and retain heat. cultivating kale in a high tunnel resulted in good yields, especially when paired with a low tun­ nel and plastic or straw mulch, which resulted in the highest air and soil tem­ peratures. the amount of minerals in plants within the high tunnel largely did not vary across combinations of low tunnels and ground covers, except for cop­ per and sulfur, which were lowest in plots with no low tunnel or ground cover. also, dietary fiber was higher when no low tunnel or ground cover was used compared to plots with a low tunnel and no ground cover. weeds were sup­ pressed by straw and black plastic mulch, but none of the ground covers influ­ enced aphid abundance. overall, our work demonstrates that red russian kale can be grown in a temperate climate during winter with some combinations of tunnels and ground covers. 1. introduction as urban agriculture becomes more common worldwide, analyses of its benefits, limitations, and role within sustainable cities and the food supply chain also become more common (orsini et al., 2013; goldstein et al., 2016; azunre, 2019; taylor et al., 2021). urban agriculture is not a replacement for large­scale rural agriculture partly because it cannot meet caloric needs of the growing and urbanizing human population. however, urban agriculture can address nutritional deficiencies in humans by providing a local source of crops that provide important micronutrients (weidner et al., 2019). horticultural crops, such as pulses (*) corresponding author: lmonroelord@udc.edu citation: richardson m.l., arlotta c.g., monroe­ lord l., 2024 ­ influence of ground cover and tunnels on production of red russian kale in urban gardens. ­ adv. hort. sci., 38(1): 3­11. copyright: © 2024 richardson m.l., arlotta c.g., monroe­ lord l. this is an open access, peer reviewed arti­ cle published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 august 2023 accepted for publication 22 december 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14997 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 3­11 4 (cerozi et al., 2022), tubers (richardson and arlotta, 2023), root crops (cerozi et al., 2022), and vegetables (song et al., 2020) that are nutrient­dense, high­ yielding, and easy to cultivate, are among the most suitable crops for urban production (clinton et al., 2018). production of horticultural crops in urban areas also shifts dietary intake toward consumption of locally grown fresh foods (mccormack et al., 2010), which has health benefits, such as preventing chronic disease (boeing et al., 2012). production of horticultural crops in urban areas can also potentially have numerous social and environmental benefits, such as providing educational spaces, connecting people with the food supply chain, reducing food miles, and efficiently using natural resources (specht et al., 2014). kale, brassica oleracea l. var. acephala, is one such crop well­suited to urban areas. it is grown for its high yield of edible leaves that are consumed raw or cooked and is an important staple crop for diversi­ fied market farms, community supported agriculture, and school, community, and home gardens in urban areas. brassicaceous crops, such as kale, are a good source of mineral nutrients, antioxidant phytochemi­ cals, secondary metabolites, and prebiotic carbohy­ drates (manchali et al., 2012; chang et al., 2019). kale is higher in calcium, folate, vitamin k, riboflavin, and vitamin c than other brassicaceous crops. furthermore, kale is a good source of vitamins (a, b1, b2, b6, c and e), folic acid and niacin, fatty acids, and essential minerals (especially k, ca, mg, fe and cu) (jahangir et al., 2009; acikgoz, 2011; thavarajah et al., 2016). young kale sprouts especially contain a high amount of protein and dietary fibers (vale et al., 2015). consumption of kale is linked to several health benefits and it has traditionally been used to treat various diseases, such as gastritis and gastric ulcer (leonti and casu, 2013), diabetes mellitus, rheuma­ tism, bone weakness, ophthalmologic problems, hepatic diseases, anemia, and obesity (lemos et al., 2011; gonçalves et al., 2012; kuerban et al., 2017). kale consumption has shown promising results in prevention of cancers of the reproductive system (han et al., 2014), gastrointestinal system, and lungs (wu et al., 2013). the strong inhibitory activities of kale extracts against glioblastoma cells and liver can­ cer cells, but negligible effects on the metabolites and bioactivity of normal cells, demonstrate its antiproliferative properties against tumorous cells (gonçalves et al., 2012). furthermore, kale extracts demonstrate antigenotoxicity: properties that main­ tain the genetic information within a cell by prevent­ ing damage and mutations (gonçalves et al., 2012). also, people with hypercholesterolemia who con­ sumed 150 ml of kale juice daily for 12­weeks signifi­ cantly increased their hdl­cholesterol and hdl­ to ldl­cholesterol ratio and significantly reduced their ldl­cholesterol (kim et al., 2008). kale can be consid­ ered a functional food since it may be useful for pre­ venting different chronic degenerative diseases. kale is relatively easy to grow, cold hardy, and can provide a steady supply of fresh greens for most of the year if practices for extended season and winter production in temperate climates are identified. extended season and winter production rely, in part, on protective structures (e.g., low tunnels, high tun­ nels) that trap solar radiation as heat in air, soil, and water. these structures are common in urban tem­ perate climates and may provide a suitable environ­ ment for crop growth when temperatures are other­ wise too low (lamont, 2009). protective structures act to reduce the frequency and duration of freezing temperatures that would otherwise kill crops. exposure to freezing temperatures can cause margin­ al necrosis on kale leaves and temperatures between ­14° to ­30°c can kill the plant. protective structures not only serve to reduce the frequency and duration of freezing temperatures but should also optimize the daily duration of temperatures above the thresh­ old for growth. the optimal temperature range for kale growth is 16°­21°c, but growth is possible above 4.4°c (andrews and coop, 2011). mulches can also be a possibility for aiding in sea­ son extension or winter production. organic mulch, like straw or woodchips, may have insulating proper­ ties (richardson et al., 2023) that slow the rate of heat loss in protective structures each night and slow the warming each day. therefore, organic mulches may reduce both the frequency and duration of freezing events as well as the duration of optimal growing conditions. black plastic mulch is known to warm soil, but it is unclear whether this material will act to buffer temperatures to the same extent as organic mulches. in northern climates where freezing temperatures are the norm, it is recommended that crops be grown on bare ground to allow a rapid buildup of heat within structures during the day (coleman, 2009), but the influence of mulches on kale in low and high tunnels is not completely known. providing plants with optimal environmental con­ ditions improves their rate of growth and yield but richardson et al. ‐ urban production of kale in winter 5 may also support the growth and development of weeds and insect pests that negatively affect yield. lepidopteran eggs laid in the summer or fall will yield caterpillars feeding in an extended season crop. aphids will continue to thrive in winter production because the same conditions that are optimal for kale growth are sufficient for aphid survival and reproduction. aphid pest species, such as green peach aphid (myzus persicae sulzer), potato aphid (macrosiphum euphorbiae thomas), and cabbage aphid (brevicoryne brassicae l.), have been reported to continue asexual reproduction at temperatures as low as 5­10°c (barlow, 1962; soh et al., 2018). we researched combinations of protective struc­ tures and ground covers for production of red russian kale through winter in an urban temperate climate. specifically, we investigated the influence of ground cover in low tunnels at 10 urban garden sites and in low tunnels within a high tunnel at the university of the district of columbia’s farm to ascer­ tain the influence on the yield and nutrients of red russian kale, soil temperature, air temperature, weed pressure, and abundance of aphids. 2. materials and methods sites and production systems urban garden sites. we started kale seedlings on 25 september 2020 in a high tunnel at the university of the district of columbia’s 58 ha firebird farm (beltsville, md, usa) before transplanting them into 10 urban garden sites in virginia, maryland, and washington, dc between 21 october through 4 november 2020. each urban site contained three plots which were weeded and randomly assigned a ground cover treatment: bare soil, black plastic mulch, or straw mulch (fig. 1). each plot was approxi­ mately 3 m x 0.6 m with 8­9 kale plants spaced 0.3 m apart. we used 1.0 mil thick black plastic mulch (dubois agrinovation, ontario, canada) and applied straw to a depth of 7.6 cm. we constructed low tun­ nels over each plot using wire hoops (0.9 m high and 0.3 m wide) and 0.8 mil low tunnel clear plastic (agriculture solutions, kingfield, me, usa), the edges of which were secured with pins and weights. ground cover and low tunnels were installed the same day as planting at all locations except one where the initial planting failed. warm ambient tem­ peratures initially required the low tunnels to be left open until 16 november 2020; at this point they were closed for the remainder of the research. high tunnel. red russian kale from the same batch of seedlings used for the garden sites was transplanted 28 october 2020 into a high tunnel (30.48 m long x 9.15 m wide x 4.57 m high) covered with a 6­mil thick double­layered polyethylene film (sun master®, farmtek, dyersville, ia, usa) and locat­ ed at the firebird farm (fig. 2). we established four plots, each of which was 3 m by 0.9 m, in each of four parallel rows (i.e., blocks) and used a randomized complete block design to assign one of four treat­ ments to each plot. the treatments were: 1) bare soil; 2) bare soil inside a low tunnel, 3) black plastic mulch inside a low tunnel; and 4) straw mulch inside a low tunnel. ground cover and low tunnel treat­ ments were set up 20 october 2020 as previously described for the urban garden sites. each plot con­ tained nine kale plants, spaced 0.3 meters apart. warm ambient temperatures initially required the low tunnels to be left open until 3 december 2020, at which point they were closed until 8 march 2021. fig. 1 ­ example layout for growing red russian kale at 10 urban garden sites in the usa. kale is planted in a low tunnel with bare soil in the foreground, black plastic mulch in the middle ground, and straw mulch in the background. adv. hort. sci., 2024 38(1): 3­11 6 environmental data and plant yield we measured air temperatures hourly in all plots at garden sites and in the high tunnel using hobo® pendant mx 2201 data loggers (onset, bourne, ma, usa). they were initially placed flat and 7.62 cm above the ground upon planting kale. if plants in any plot grew large enough to cover the data logger, all sensors at that site were then raised to 33 cm (gar­ den sites) or 40.6 cm (high tunnel) above the ground. we also measured soil temperature in each plot hourly using hobo® pendant mx 2201 data loggers or, in two cases, a hobo mx2303 two external temperature sensors data logger (onset, bourne, ma, usa). data loggers were buried 7.62 cm below the surface of the soil upon planting. data from all loggers were downloaded weekly. we began collecting weed mass biweekly starting 26 november 2020. weed mass was measured by removing all weeds from a plot, shaking dirt from the roots, and weighing the total fresh mass. we scouted for all insect pests bi­weekly from 10 december 2020 to 17 march 2021, but aphids were the only pest reg­ ularly present. to measure the abundance of aphids, we removed the leaf with the most aphids from each plant that had more than five aphids present, stored them in a freezer, and then counted the aphids under a dissecting microscope. after leaves were removed, we treated all plants at a site using 59 ml triple action neem oil (southern agricultural insecticides, inc., boone, nc, usa) per 3.8 l water to reduce pest infestations. biweekly harvest began on or after 10 december 2020 when at least three plants per plot had one or more leaf blades 20.3 cm long. individual leaves 20.3 cm or longer were cut 5 to 7.6 cm from the stalk of the plant and all fresh mass from a plot was weighed. final harvest was 17 march 2021. nutrient analysis due to low yield of kale in garden sites, nutrient analysis could only be conducted with samples from the high tunnel. approximately 12 fully developed leaves from each of three subsamples within a treat­ ment were shipped on ice to waypoint analytical (leola, pa) to quantify mineral content, including cal­ cium (%), magnesium (%), nitrogen (%), phosphorous (%), potassium (%), sodium (%), sulfur (%), boron (ppm), copper (ppm), iron (ppm), manganese (ppm), and zinc (ppm) using the united states environmental protection agency (us epa) sw­846 method and american oil chemists’ society (aocs) official method ba 4e­93 for nitrogen. amounts are reported on a dry matter basis. additionally, we noticed a distinct difference in texture between kale plants in bare soil with no low tunnel versus all other treatments which had a low tunnel, which we sus­ pected was due to fiber content. so, we harvested approximately 100 g of fresh kale leaves per subsam­ ple in the two bare soil treatments (with and without a low tunnel) and mailed them on ice to eurofins microbiology laboratories (lancaster, pa). eurofins microbiology laboratories analyzed total dietary fiber using the association of official analytical chemists (aoac) method 991.43. the percent con­ tent of fiber is reported on a fresh matter basis. leaves harvested for all nutrient analysis were included in our measurements of total yield. data analysis in all analyses, the plot is the replicate and the individual kale plants are subsamples. we used the mean yield per plant, plot, and month in a mixed model, (proc glimmix; sas institute, 2020) to determine whether yield differed among the three ground cover treatments at garden sites, with mean air and soil temperatures as covariates, treatment as a fixed effect, and site as a random effect. we also used separate mixed models (proc glimmix; sas institute, 2020) to determine whether weed mass and the number of aphids per plant differed among fig. 2 ­ red russian kale in a high tunnel in maryland, usa. kale was planted in one of four treatments: bare soil (no ground cover and low tunnel); bare soil + low tunnel (no ground cover and covered with a low tunnel); black plas­ tic + low tunnel (black plastic mulch and covered with a low tunnel); straw + low tunnel (straw mulch and cov­ ered with a low tunnel). sixteen experimental plots are arranged in four rows, with a row of non­experimental plants separating the rows into pairs. richardson et al. ‐ urban production of kale in winter 7 the three treatments at garden sites, with treatment as a fixed effect and site as a random effect. we used the mean yield per plant, plot, and month in a mixed model, (proc glimmix; sas institute, 2020) to determine whether yield differed among the four ground cover treatments in the high tunnel experiment, with mean air and soil tempera­ ture as covariates and treatment as a fixed effect. differences in weeds and most nutrients, except fiber, across treatments in the high tunnel experi­ ment were also analyzed with separate general linear models (proc glm; sas institute, 2020). we used a negative binomial model (proc genmod; sas institute, 2020) to determine whether the total num­ ber of aphids differed among the four treatments. this analysis differs from the aphids at garden sites because the number of plants in each plot was vari­ able, but the number of plants in each plot in the high tunnel was the same, allowing for a count model to be used. differences in dietary fiber between bare soil plots with and without a low tunnel were com­ pared with a t­test. we used base­10 log transforma­ tions prior to some analyses to meet assumptions of normality but means for non­transformed data are presented in the results. we used means separation tests for all analyses to determine which means dif­ fered (p<0.05). 3. results garden sites the amount of kale harvested from garden sites was extremely low and did not differ across ground cover treatments (f2,106 = 0.24, p = 0.78) (table 1). however, yield was higher when mean air (f1,106 = 20.1, p<0.01) and soil temperature f1,106 = 16.6, p<0.01) were higher. the mean marketable yield/plant/treatment generally decreased over time from a maximum of 37.5 g in december to 11.4 g, 3.7 g, and 4.0 g in january, february, and march, respec­ tively. ground cover treatments influenced weed mass, with the greatest mass manually removed from plots with bare ground, a lesser amount from plots with straw, and nearly none from plots with black plastic mulch (f2,18 = 58.1, p < 0.01) (table 1). the number of aphids per plant did not vary across treatments (f2,18= 0.95, p = 0.40) (table 1). high tunnel kale yield varied across treatments (f3,58 = 4.1, p = 0.01), with the greatest yield from plots with straw and plastic mulch and the least yield from plots with bare soil and no low tunnel (table 2). yield was also greater when mean air (f1,58 = 20.2, p < 0.01) and soil temperature (f1,58 = 31.9, p < 0.01) were higher, and this was influenced by treatments. specifically, mean air temperature was 19.1°c in plots with straw table 2 ­ mean yield per plant of red russian kale, mass of weeds, and total number of aphids across four treatments in a high tunnel in maryland from october 2020 to march 2021. there were four plots per treatment each plots within the high tunnel was assigned one of four treatments: bare soil (no ground cover and low tunnel); bare soil + low tunnel (no ground cover and covered with a low tunnel); black plastic + low tunnel (black plastic mulch and covered with a low tunnel); straw + low tunnel (straw mulch and covered with a low tunnel). means with different letters within rows are different (p<0.05). standard devia­ tions are provided in parentheses. table 1 ­ mean yield per plant of red russian kale, mass of weeds, and number of aphids per plant across three treatments at 10 urban garden sites in virginia, maryland, and washington, dc from october 2020 to march 2021. the sample size for each measurement equals the number of garden sites (10) each urban site included three plots with a ground cover treat­ ment: bare soil, black plastic mulch, or straw mulch. means with different letters within rows are different (p<0.05). standard deviations are provided in parentheses. variable ground cover treatment bare soil black plastic straw mulch crop yield (g) 14.1 (30.9) 9.8 (25.6) 5.1 (16.6) weeds (g) 313 (349) a 68.6 (59) b 7.0 (8.8) c no. aphids per plant 48.4 (72.2) 19.7 (20.5) 23.0 (49.1) variable treatment bare soil bare soil + low tunnel black plastic + low tunnel straw + low tunnel crop yield (g) 917 (830) b 1334 (632) ab 1384 (709) a 1390 (577) a weeds (g) 141 (189) b 142 (96) b 2.8 (4.3) b 65 (110) ab no. aphids 231 (285) 118 (141) 66 (90) 65 (121) 8 adv. hort. sci., 2024 38(1): 3­11 mulch, 18.5°c in plots with plastic mulch, and only 15.6°c in plots with bare soil and no low tunnel. mean soil temperature was 12.2°c in plots with straw mulch, 12.5°c in plots with plastic mulch, and only 10.9°c in plots with bare soil and no low tunnel. ground cover treatments influenced weed mass with the greatest mass manually removed from plots with bare soil (with or without a low tunnel) and nearly none from plots with black plastic mulch (f3,12 = 7.6, p<0.01). however, the total number of aphids did not differ across treatments (f3 = 1.75, p = 0.63) (table 2). analysis of 12 plant elements in the high tunnel experiment revealed that b, ca, fe, k, mg, mn, n, na, p, and zn showed no variation across treatments (all p­values > 0.07). however, cu was highest in plants in straw mulch and lowest in those grown in bare soil without a low tunnel (table 3). plants in all treat­ ments with a low tunnel exhibited higher s compared to those without a low tunnel (table 3). furthermore, plants grown in bare soil under a low tunnel dis­ played greater dietary fiber compared to those in bare soil without a low tunnel (table 3). 4. discussion and conclusions one potential benefit of urban agriculture is the possibility of producing local, nutritious foods year­ round, such as red russian kale. however, winter production in unheated structures such as low and high tunnels in temperate environments relies on capturing sufficient solar radiation to support crop production. heat generated within these structures during daylight will dissipate from those structures at night. heat storage and the rate of dissipation are influenced by environmental conditions outside of the tunnel and the size and construction of the pro­ tected structures. for example, secondary covers within a high tunnel may increase the amount of time plants experience optimal growing tempera­ tures during colder months and modulate extreme temperatures (borrelli et al., 2013; drost et al., 2017). small tunnels with a single layer of plastic will experience faster heat loss than larger tunnels with multiple layers of plastic. insulating ground covers may act to slow down heat loss below­ and above­ ground (bhardwaj, 2013). in our study straw and black plastic ground covers combined with low tun­ nels were not sufficient to produce a viable yield of red russian kale at 10 garden sites. yield at these garden sites was higher when air temperature and soil temperature were higher, but these tempera­ tures were likely influenced by geographic location because the ground covers were not associated with differential yield. outside temperature around the low tunnels was likely too cold, and the area within the tunnels too small, to increase or retain sufficient heat for crop production. however, cultivating kale in a high tunnel resulted in good yields, especially when paired with a low tunnel and either plastic or straw mulch; this combination led to the highest air and soil temperatures. our results should not be taken to mean that low tunnels alone cannot be used to pro­ duce kale or that a high tunnel is necessary. these systems may vary in their effectiveness under other environmental conditions or if made from other materials. also, other varieties of kale may perform better in these systems than red russian kale because different varieties, cultivars, and genotypes of a plant species respond differently to cultivation methods and environmental conditions (yoder and davis, 2020; richardson and arlotta, 2021, 2022; richardson et al., 2022). mineral absorption in plants is known to be influ­ enced directly or indirectly by air and soil tempera­ table 3 ­ mean content of nutrients that differed in red russian kale across four treatments in a high tunnel in maryland from october 2020 to march 2021. there were four plots per treatment each plot within the high tunnel was assigned one of four treatments: bare soil (no ground cover and low tunnel); bare soil + low tunnel (no ground cover and covered with a low tunnel); black plastic + low tunnel (black plastic mulch and covered with a low tunnel); straw + low tunnel (straw mulch and covered with a low tunnel). means with different letters within rows are different (p<0.05). nutrient treatment p bare soil bare soil + low tunnel black plastic + low tunnel straw + low tunnel copper (ppm) 9.8 b 11.4 ab 10.7 ab 11.7 a 0.02 sulfur (%) 0.88 b 1.24 a 1.23 a 1.16 a <0.01 dietary fiber (%) 4.4 a 3.4 b na na <0.01 richardson et al. ‐ urban production of kale in winter 9 ture (tachibana, 1982; inthichack et al., 2013). given this understanding, we expected to observe variation in mineral content among the different treatments. however, our results indicate that the use of low tun­ nels and ground covers in the high tunnel did not influence mineral content, except for copper and sul­ fur, which were lower in plots without a low tunnel or ground cover than some other treatments. although yield and mineral content, to an extent, were improved with low tunnels and ground cover in the high tunnel, dietary fiber was higher when no low tunnel or ground cover was used. this may help urban farmers grow kale for instances in which maxi­ mizing dietary fiber is a priority. however, it is impor­ tant to consider that these plants were also much smaller in structure and yielded less, which could potentially affect profitability. beyond the influence on the red russian kale, the ground covers also had an impact on weed growth. as expected, the use of plastic and straw mulch sup­ pressed weed growth. contrary to our expectations, we did not find evidence that these ground covers in combination with low tunnels increased the abun­ dance of insect pests. insect pests were observed in all treatments in garden sites and the high tunnel. it is likely the presence of insect pests in all treatments can be attributed to the warmer air temperature fos­ tered by the low and high tunnels alone, rather than the addition of ground covers. in conclusion, our work demonstrates that red russian kale, a highly nutritious, desired, and appro­ priate crop for urban systems can be grown in tem­ perate regions throughout the winter using specific combinations of tunnels and ground covers. these findings open opportunities for year­round cultiva­ tion of this crop in urban systems. future research could continue to build upon our findings to eluci­ date the best methods that maximize yield and nutri­ ents while not significantly contributing to increased abundance of insect pests. acknowledgements we thank anna wallingford for help with project ideas and funding and an anonymous reviewer for their constructive feedback. we thank our graduate student researcher megan thompson and our under­ graduate intern khrista­lee amore. we thank our collaborators arcadia center for sustainable food and agriculture 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richardson et al. ‐ urban production of kale in winter 11 wu q.j., xie l., zheng w., vogtmann e., li h.l., yang g., ji b.t., gao y.t., shu x.o., xiang y.b., 2013 ­ cruciferous vegetables consumption and the risk of female lung cancer: a prospective study and a meta‐ analysis. ­ ann. oncol., 24: 1918­1924. yoder n., davis j.g., 2020 ­ organic fertilizer comparison on growth and nutrient content of three kale cultivars. ­ horttechnol., 30: 176­184. impaginato 35 adv. hort. sci., 2024 38(1): 35­45 doi: 10.36253/ahsc­14786 “hurdley technologies” utilized to improve postharvest life of asparagus spears (asparagus officinalis l.) m.e. tiznado­hernández 1, a.a. gardea­bejar 2, a. sánchez­estrada 1 (*), j.a. orozco­avitia 1, a.j. ojeda­contreras 1, r. troncoso­rojas 1, r. melendrez­amavizca 3 1 coordinación de tecnología de alimentos de origen vegetal, gustavo enrique astiazarán rosas 46, hermosillo, 83304 sonora, méxico. 2 coordinación guaymas, centro de investigación en alimentación y desarrollo, a.c. (ciad, a.c.), gustavo enrique astiazarán rosas 46, hermosillo, 83304 sonora, méxico. 3 departamento de investigación en física, universidad de sonora, hermosillo, cp 83190, sonora, méxico. key words: asparagus officinalis, metabolic heat, postharvest quality, respira­ tion, uv­c and gamma irradiation, water status. abstract: asparagus (asparagus officinalis l.) has short shelf­life due to the high metabolic activity of the apical meristems. storage at low temperature and high relative humidity is used commercially to keep fresh asparagus spears. techniques denominated “hurdley technologies” (uv­c or gamma irradiation) have been tested in fruits and vegetables to extend postharvest life. these technologies were used to extend postharvest shelf life of asparagus spears by inhibition of meristematic activity. spears were irradiated with uv­c at dosages of 2.46, and 4.93 kj m­2 and gamma irradiation at 1 and 1.5 kgy, before storage at 2°c and 90% relative humidity (rh) for 20 days. metabolic heat (rq) was measured in apical meristems, as well as whole spear respiration, sugars con­ tent, water potential components and color descriptors. metabolic heat and whole spears respiration rate did not show differences due to effect of uv­c treatments, while spears treated with gamma radiation showed a metabolic activity inhibition of 10 and 15% for 1 and 1.5 kgy, respectively, while whole spear respiration rate was not affected. changes in color variables showed a slight reduction in gloss. sugars content in uv­c remained unchanged, while gamma radiation induced a reduction in glucose. an increase in fresh weight loss was noticeable on those treated with gamma irradiation. no changes in water potential components were observed. it was concluded the treatments used did not reported positive benefits in extending asparagus spears shelf life. 1. introduction green asparagus is a high value vegetable, and its consumption dates back to ancient times (anido and cointry, 2008). worldwide demand is increasing because its gourmet features (pegiou et al., 2020) and (*) corresponding author: aestrada@ciad.mx citation: tiznado­hernández m.e., gardea­bejar a.a., sánchez­estrada a., orozco­avitia j.a., ojeda­contreras a.j., troncoso­rojas r., melendrez­amavizca r., 2024 ­ “hurdley tech‐ nologies” utilized to improve postharvest life of asparagus spears (asparagus officinalis l.). ­ adv. hort. sci., 38(1): 35­45. copyright: © 2024 tiznado­hernández m.e., gardea­bejar a.a., sánchez­estrada a., orozco­avitia j.a., ojeda­contreras a.j., troncoso­rojas r., melendrez­amavizca r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 august 2022 accepted for publication 30 august 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14786 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 35­45 36 nutraceutical properties (fan et al., 2015). this produce is harvested while actively growing and continues to grow even after harvest, keeping a high metabolic activity characteristic of apical meris­ tems (aegerter et al., 2011). spears are exposed to different stresses after harvest, such as wounding, dehydration and limited nutrient supply. as a result, they cannot maintain metabolic homeostasis because of their high levels of metabolic activity, res­ piration rate, and carbohydrate consumption. consequently, spears metabolites are rapidly con­ sumed, affecting their quality and accelerating senes­ cence during storage, transport, and retailing (anastasiadi et al., 2020). quality loss is mainly noticed by color changes, such as the loss of a bright green color, besides tip­ ping, bending and feathering, which together decreases postharvest quality (anastasiadi et al., 2020). concomitantly, dehydration adversely affects fresh weight and turgor loss (gardea et al., 2023). low temperatures and high humidity are the condi­ tions commonly utilized in many horticultural prod­ ucts to control senescence and increase shelf life (singh et al., 2014). however, other methods for fresh produce preservation known as “hurdley tech­ nologies” are available now (arvanitoyannis et al., 2009), including cold plasma (jia et al., 2022), uv­c irradiation (haro­maza and guerrero­beltrán, 2013) and gamma ray irradiation (prakash and ornelas­paz, 2019). irradiation based technologies take advantage of plants natural defense capabilities by stimulating plant defense mechanism, including biosynthesis of secondary plant metabolites, as well as defense­ related enzymes activation (ghaouth et al., 2003; arvanitoyannis et al., 2009; chipurura and muchuweti, 2010; bisht et al., 2021). furthermore, it has been demonstrated that uv­c irradiation, both in pre­ and post­harvest can inhibit the growth and sprouting of vegetables (duarte­ sierra et al., 2020; sinha and häder, 2002; verdes­ teodor et al., 2019). the same effect has been observed in some gamma­irradiated vegetables as like potato (blessington et al., 2015; mani and hannachi, 2015), seeds and sprouts (rajkowski and latiful, 2012). uv­c irradiation could induce photomorphogenic responses that encourage growth and development in a distinct way, reaching photoreceptors that utilize signals to activate similar gene expression, and thus, inducing phenotypic changes, such as flowering and stem branching (darras et al., 2012). additionally, gamma irradiation has the potential to destroy dna, stopping plant meristematic activity (arvanitoyannis et al., 2009), and therefore, they can inhibit potato sprouting (cools et al., 2014) or reduce growth rate in fressia hybrid l. (darras et al., 2019). in summary, reduction in spear tip meristematic activity will reduce respiratory and sugar consump­ tion rates, which in turn will cause a reduction in deterioration rate (verlinden, 2014). to our knowl­ edge, the effects of ionizing and non­ionizing irradia­ tions on asparagus spear shelf life has not been explored. microcalorimetry capacity to measure the heat product of metabolism such as changes in respiratory variables can contribute to our understanding of plants metabolic activity in response to external stim­ ulus (wadsó and hansen, 2015). microcalorimetry is an excellent nondestructive tool for detecting small metabolic changes in intact vegetable tissues. gardea et al. (2023) have suggested that calorimetry is a practical tool to detect changes in tip metabolic activ­ ity related to physiological and quality changes that determine spear shelf­life. based on the above mentioned, our objective was to evaluate the potential to increase green spears shelf­life by using “hurdley technologies”, such as uv­c and gamma irradiations. 2. materials and methods plant material and stored treatments asparagus (asparagus officinalis l.) cv. brock was harvested from a commercial asparagus orchard near caborca, sonora, mexico, 30°47’22.32”n, 112°45’51.98”o. spears were of standard quality with dimensions of 18 cm in length and 1.0­1.5 cm in diameter. asparagus were grouped for experiments with non­ionizing and ionizing irradiation. in each, there were 6 groups of 25 spears each and packaged upright into a commercial plastic box (30 cm height x 35 cm length x 30 cm depth) including moisture pads at the bottom. in the first experiment (experiment 1), asparagus spears were exposed to uv­c irradiation, removed from the top cover of the boxes, and placed inside a laminar hood. the uv­c irradiation was carried out using two lamps (unfiltered general electric 15 w g15t8 lamps), 15 cm above the box. time exposure was equivalent to 2.46 and 4.96 kj m2 of energy level. tiznado‐hernándezet al. ‐ hurdley technologies improve asparagus postharvest life 37 during the second experiment (experiment 2), gamma radiation was applied at absorbed doses of 1 and 1.5 kgy using a gammacell 2200 excel irradiator (mds nordion, march road, ottawa, notary, canada) equipped with a 60co source of activity of 14.4 kci, and a chamber for irradiating samples with a maxi­ mum volume of 3.7 l, located at the university of sonora, hermosillo, sonora, mexico. after treatments exposure, spears were stored in a controlled temperature chamber kept at 2°c and 90­95% rh. in each experiment, a non­irradiated group was included as control. every 4 days (d) tip metabolic activity (rq), and the respiration rate of whole spear were assessed; also fresh weight loss, water potential turgor pressure and osmotic poten­ tial, sugar content (sucrose, glucose, fructose), color lightness (l*), hue angle (h°), chroma (c), and change color (δe). metabolic heat [rq, (µwmg‐1)] of asparagus spears apical sections microcalorimetry ability to measure changes in metabolic heat can contribute to our understanding of plants metabolic activity in response to external stimulus. six asparagus spears were sampled for treatment and kept protected in a cooler with moist absorbent paper and ice pack gels to prevent dehy­ dration and transferred immediately to the lab. metabolic heat production [rq, w mg­1 of dry weight (dw)] was determined by isothermal microcalorimetry at 25°c, according to millan­soto et al. (2019). we used a multi­cell differential scanning microcalorimeter csc 4100 (calorimetry science corporation, pleasent grove, utah, usa), equipped with four sealed metal cells of 1 ml. the fourth cell was used as reference and kept empty during mea­ surements. with the aim to prevent condensation inside the instrument, we kept a steady flow of 1.75 mlmin­1 with n2. the temperature in the instrument chamber was kept constant at 15°c using a circulat­ ing­cooling bath (polyscience, niles, illiinois, usa). three samples were measured simultaneously. in total, six asparagus spears tissues with an average fresh weight of 90 mg were used. to prevent sample dehydration during data acquisition, 50 µl of sterile water were added on each cell and one apical meris­ tem was used per cell, with cut side (basal) in contact with water. after placing the samples, the instrument was allowed to stabilize for about 2500 s to achieve a steady­state metabolic heat rate, which usually hap­ pened during the last quarter of this protocol period. co2 (g) + 2oh­(aq) = co3 2­(aq) + h2o (aq) δh = ­108.5 kj mol­1 (orozco et al., 2022) samples were dried in a vacuum oven precision (model 19, thermo electron corp. usa) at 65°c for 48 h. further, the data were baseline adjusted and cal­ culated based on dry weight. whole spears respiration carbon dioxide production was determined according to badillo and segura­ponce (2020). six asparagus spears were placed in a 1 l container (three containers by treatment). air samples (1 ml) were taken from each headspace after incubation for 1 h and injected into a varian 3400cx gas chromato­ graph equipped with a hayesep n column (2 m x 3.17 mm inside diameter, supelco, inc.) coupled to a ther­ mal conductivity detector. the parameters were: 100°c injection temperature, 170°c thermal conduc­ tivity detector, and 120°c flame ionization detector. nitrogen was used as a carrier gas with a flow rate of 25 ml min­1 co2 production was calculated using co2 standards of known concentration according to the algorithm: ml co2 kg­1 h­1 = (spa * stc * hs) / (stpa * w * t) where spa is the sample peak area, stc is the concen­ tration of the reference standard (co2: 0.05 ml l−1), hs is the headspace volume (l), stpa is the standard peak area, w is the sample weight (kg), and t is the incubation time in hours. results were expressed in ml co2 kg­1.h­1. sugars glucose, fructose, and sucrose were quantified according to ma et al. (2014) with some modifica­ tions. briefly, 10 g of tissue were mixed with 50 ml of distilled water and homogenized for 2 minutes with a t25 ultra­turrax homogenizer (ika, staufen, germany) equipped with a dispersing tool (ika, staufen, germany). the samples were filtered, and 2 ml of filtrate were placed in eppendorf vials and cen­ trifuged at room temperature for 15 minutes at 3700 g. the supernatant was purified with gv filters with 0.22 µm pore size. 20 µl of sample filtrate were injected into an hplc (varian prostar 210) equipped with a lc­nh2 column (sigma chemical co., st. louis; 250 m length, 4.6 mm of internal diameter, and 5 μm of particle size) with an lc­nh2 guard column and a refractive index detector (varian prostar 350). the adv. hort. sci., 2024 38(1): 35­45 38 mobile phase used was acetonitrile:water (80:20, v/v) at a flow of 1 ml min­1. quantification was car­ ried out with a mix of standards curves done by injecting into the hplc equipment solutions of glu­ cose, fructose, and sucrose with different concentra­ tions. the r2 for glucose, fructose, and sucrose stan­ dard curves were 0.9544 and 0.9634, and 0.9321, respectively. individual sugars were reported as mg g fw­1 of sample. water status a sartorius balance was used to determine spears fresh weight loss (fwl) every four days. five bunches per treatment were taken and their initial and final weights (at the end of the storage period) were recorded. the results were averaged and expressed in percentages of fwl. in the middle segment of the spear, a cylinder 1.5 cm long was cut to evaluate water potential (ψw). three asparagus spears were used for treatment every 4 d, according to muy­rangel et al. (2004). three spear middle segments were weighed and soaked in sucrose solutions with molalities of 0.1, 0.05, 0.015 and 0.025. the osmotic potential (ψs) component was calcu­ lated by the equation ψs = ­cirt; where ci is sucrose molality, r is the gases ideal constant (0.0083 kg·mpa·mol­1 k­1) and t is the temperature at 273 °k (rodríguez­burgos et al., 2015). sucrose solutions pressure potential (ψp) is zero because the solution is not inside of a container. pressure potential was calculated from the equation ψw = ψp – ψs, taking into account that pressure potential is zero, we con­ cluded that osmotic potential equals tissue water potential ψw = ­ψs (rodríguez­burgos et al., 2015). samples were left to rest for 2.25 h (time required to reach equilibrium between samples and solution). then removed and excess water dried on tissue paper and their weights were recorded. after that, sucrose solution, in which the tissues did not gain or lose weight, corresponds to water potential. the ψs was determined in the middle segment of 3 spears with a steam pressure osmometer wescor model 5520 using the methodology developed by jia et al. (2020). a sap sample obtained from the middle segment was preserved at ­20°c until use. after defrosting at room temperature, 10 µl of exudate was placed on a 0.32 cm2 filter paper disc and placed inside the chamber. the osmometer was calibrated with standards of 100, 290, and 1000 mg kg­1 nacl solution and the results of molality were converted to an osmotic potential with the van’t hoff equation, where ψs = cirt (jia et al., 2020). pressure potential (ψp) was calculated with the difference between osmotic and water potentials according to jia et al. (2020). color the color of spears was measured with a portable colorimeter (minolta cr200, konica minolta, japan). the l*, a*, and b* color space variables were record­ ed in the tip, middle and basal segments of 10 asparagus spears, every four days. differential color parameter (δe) was calculated using the following equation: (δe) = [(l*­l*0)2+ (a*­a*0)2+ (b*­b*0)2]1/2; where: l*0, a*0, and b*0 were the readings of the color space at zero­day; while l*, a*, and b* corre­ spond with the color space variables determined at each sampling day (kohli et al., 2022). hue angle (h°) was calculated from a* and b* color space variables using the following equation: [h°=tan­1 (b*/a*) when a*>0 and b*>0 or h°=180+ tan­1 (b*/a*) when a*<0 and b*>0)], and saturation color named chroma using the following equation: c = (a*2+b*2)1/2 (kohli et al., 2022). statistical analysis analysis of variance (anova) was performed based on a completely randomized design, with a factorial arrangement of treatments, where two irra­ diation protocols, plus controls were established and evaluated on six dates (3 x 6). when main effects sig­ nificant differences (p<0.05) were found, tukey mul­ tiple range tests were carried out. all the analysis were done with the infostat 2017e version (university of cordoba, cordoba, argentina). 3. results and discussion metabolic heat of meristematic apical tissue and res‐ piration rate of whole pears. statistical analysis did not find a significant inter­ action between storage period and uv­c dosages. uv­c irradiated spears showed a metabolic heat (rq) significant reduction during storage at 2°c (p<0.05) (table 1). all samples started at 40 µw mg­1 dw, then steadily decreased until reaching 22 µw mg­1 dw on day 20. it follows the same pattern as that reported by gardea­bejar et al. (2023) which found a constant decrease in metabolic heat by spears stored at 2 and 5°c. regarding uv­c dosages, no significant differ­ tiznado‐hernándezet al. ‐ hurdley technologies improve asparagus postharvest life 39 ences were found between controls and uv­c treat­ ed spears, averaging of 29 µw mg­1 dw. gamma­exposed spears data did not report signif­ icant interactions, but significant main factors. as far as storage period, no differences (p>0.05) were found in the first eight days averaging ca. 39 µw mg­1 dw. however, starting on day 12th, significant reduc­ tions (p<0.05) took place decreasing to 10.9 on day 20 (table 2). as far as gamma irradiated treatments a significant effect was found between controls and 1 and 1.5 kgy averaging values of 34.7, 31.5 and 27.0 kgy (table 3), respectively. thus, implying a severe injury by gamma radiation to meristematic cells, as reported elsewhere (dina et al., 2018). usually, deterioration rate of harvested commodi­ ties is proportional to their respiration rate (kader, 1992). a significant reduction, although somehow erratic, in respiration of uv­c treated spears was found along the 20 days storage, starting at 15.8 ml co2 kg­1 h­1 and eventually leveling to 11.1 units (table 1). slightly higher respiration rate was observed in uv­c treated tomatoes with 15.8­ and 19.8­ ml co2 kg­1 h­1 at 20°c irradiated with at 0.003 y 0.033 kjm­2 (cote et al., 2013). even though aspara­ gus apical meristem shows the highest respiratory activity (anastasiadi et al., 2022) and that is why the respiration rate of the upper segment (apical zone) is the one with the highest rates (verlinden et al., 2014). further, the normal behavior for whole asparagus spears in postharvest is the reduction in respiration rate after harvested (wu and yang, 2016). in cherry (michailidis et al., 2019), and broccoli flo­ rets (costa et al., 2006), uv­c irradiation affected res­ piratory metabolism, resulting in a slower respiratory rate than in non­irradiated plants (yang et al., 2014). as far as uv­c treatments, a slight but significant table 2 ­ effect of the gamma radiation on physiology and quality parameters in asparagus during storage at 2°c, independently of energy level *significant interaction, ** highly significant interaction. means followed by same letters within rows are not statistically significant (tukey, p > 0.05). *significant interaction, ** highly significant interaction. means followed by same letters within rows are not statistically significant (tukey, p > 0.05). table 1 ­ effect of the uv­c radiation over physiology and quality parameters in asparagus during storage at 2°c, independent of level energy variable uv­c storage time (days) 0 4 8 12 16 20 metabolic heat (µw mg­1dw) ** 40.3 e 34.2 d 29.2 c 26.5 bc 23.6 ab 22.4 a respiration (ml co2 kg­1 h­1) ** 15.8 d 12.3 c 10.2 a 10.4 ab 9.7 a 11.1 b fructose (mg g­1 fw)* 14.24 a 10.48 a 9.95 a 8.72 a 7.94 a 7.60 a glucose (mg g­1 fw)* 16.22 b 10.95 ab 10.76 ab 8.51 a 8.16 a 7.58 a chroma (c) 38.6 a 37.8 a 38.2 a 38.3 a 37.5 a 38.1 a hue (°) 114.3 ab 115.0 b 114.7 ab 114.3 ab 113.8 ab 112.9 a l** 47.7 abc 49.0 c 49.0 c 48.04 bc 46.59 ab 45.58 a δe 0.0 a 3.2 b 3.7 b 3.6 b 4.5 b 4.3 b variables gamma storage time (days) 0 4 8 12 16 20 metabolic heat (µw mg­1dw) ** 38.7 d 40.4 d 38.0 cd 33.0 c 27.2 b 10.9 a respiration (ml co2 kg­1 h­1) ** 15.8 c 10.3 b 10.3 b 10.7 b 4.38 a 4.5 a fructose (mg g­1 fw) 10.52 a 8.24 a 8.16 a ­­­­­ ­­­­­ ­­­­­ glucose (mg g­1 fw)* 6.67 a 5.02 a 6.36 a ­­­­­ ­­­­­ ­­­­­ chroma c© 34.5 ab 33.9 a 33.2 a 35.8 b 34.6 ab 34.0 a hue (°) 118.2 a 118.3 a 118.9 a 115.1 a 115.6 a 114.4 a l** 47.7 c 38.7 a 41.5 b 41.8 b 41.7 b 41.3 b δe 0.0 a 15.2 e 12.8 d 9.1 b 12.2 d 11.0 c 40 adv. hort. sci., 2024 38(1): 35­45 change in response to irradiation was found with val­ ues of 11.8, 11.3 and 11.5 for controls, irradiation at 2.4 and 4.96 kjm2, respectively (table 3). we hypoth­ esized that low respiration rate may be caused by reducing succinic dehydrogenase and cytochrome c oxidase activities and higher integrity of mitochondri­ al membrane with the consequence of lower perme­ ability and gas exchange, as was demonstrated in uv­ c irradiated peach during postharvest (yang et al., 2014). table 3 shows that gamma­irradiated spears went through a statistically significant reductions in respi­ ratory activity during storage starting at 15.8 ml co2 kg­1 h­1 on day 0 and ending at 4.5 on day 20th. gamma irradiation dosages induced a significant reduction (p<0.05) with increasing irradiation strength with values of 10.8, 8.7 and 8.4 ml co2 kg­1 h­1 for the 0, 1 and 1.5 kgy treatments (table 3). as reported in several crops, low dosages resulted in increasing responses (lu et al., 2023), while expo­ sures above 0.5 kgy caused decreases, (ali et al., 2015). a clear impact of gamma irradiation on bam­ boo shoots irradiated at 0.5 kgy and stored at 2°c and 90% rh decreased respiratory rate at 60 and 5 % at the beginning and end of the experiment (zeng et al., 2015), preuss and britta (2003) stated that a high dose of gamma radiations affects cell cycle arrest during g2/m phase, inhibiting growth during cell divi­ sion, consequently causing a drop in respiratory rate. sugar content in asparagus spear sucrose was not detected in asparagus spears, while fructose and glucose were present in both experiments. constant declines in fructose and glucose con­ tents were observed along storage, although statisti­ cally significant only for glucose, starting at 16 mg g­1 fw and ending below 7.6 (table 1). such behavior has been reported by davies et al. (1996) in spears of the cultivar limbras with sugar values falling down 30% in the first three days of storage. reductions in sugar content is normal for aspara­ gus during postharvest due to their constant energy needs to maintain their high respiratory activity (sergio et al., 2019). same pattern was found in gamma irradiation spears (table 2), although this experiment last only eight days of storage. since gamma irradiation accelerates spear metabolic activity, glucose is consumed rapidly (king et al., 1990), triggering senescence as a result, in addition to depletion caused by damage to tissue membranes and cell walls (ali et al., 2015). water status spears fresh weight losses (fwl) did not differ between uv­c irradiated treatments and controls. at 4th and 8th days of storage, fwl accounted for 2 and 3% respectively, on days 12th and 16th, increased to 5 and 7%, and finally at day 20 reached 13% (fig. 1a). wang et al. (2019) found results slightly lower in spears wrapped in perforated film, reporting a fwl of only 2 % after 20 days in storage at 2°c at 85% rh. further, wang et al. (2020) reported fwl within a range of 3.0­5.0% in asparagus under modified atmosphere and stored at 4°c. gamma irradiated spears showed significant table 3 ­ effect of different uv­c and gamma irradiation over physiology and quality parameters in asparagus during storage at 2°c. values are the average of six sampling dates * significant interaction, ** highly significant interaction. means followed by same letters within rows are not statistically significant (tukey, p>0.05). experiments uv­c (kj m­2) gamma (kgy) variable 0 (control) 2.4 4.96 0 (control) 1.0 1.5 metabolic heat (µw mg­1dw) ** 30 a 29 a 29 a 34.7 c 31.5 b 27.9 a respiration (ml co2 kg­1 h­1) ** 11.8 b 11.3 a 11.5 ab 10.8 b 8.7 a 8.4 a fructose (mg g­1 fw) 9.97 a 9.99 a 9.55 a 9.35 a 8.87 a 8.70 a glucose (mg g­1 fw)* 10.91 a 10.40 a 9.72 a 10.33 b 3.11 a 4.61 a chroma (c) 38.9 b 37 a 38.2 ab 35.1 b 33.9 a 34.0 a hue (°) 115.1 b 113.7 a 113.7 a 126.4 a 131.7 a 137.3 a l** 47.88 a 47.92 a 47.05 a 45.8 b 40.7 a 39.8 a δe 2.4 a 2.9 a 4.5 b 5.1 a 11.9 b 13.2 c tiznado‐hernándezet al. ‐ hurdley technologies improve asparagus postharvest life 41 effects on spears fwl. after 16 days of storage, fwl accounted for 12% in the irradiated samples, and only 7.5% in controls (fig. 1e). by contrast, lescano et al. (1993) found that fwl increased linearly, without significant differences between controls and irradiat­ ed samples in white asparagus with doses of 1.0, 1.5 and 2 kgy. these data support the rapid senescence of irradiated spears. no differences among treatments in water poten­ tial for both experiments were found on each sam­ pling day. ψw decreased gradually, from ­0.5 mpa at 0 days down to ­2 mpa at 16 days in the uv­c experi­ ment (fig. 1b). gamma­irradiated asparagus decreased at slightly slow rate down to only ­1mpa (fig. 1f). although no significant differences were found within each experiment, uv­c treated spears reached final values around ­1.75mpa, while gamma irradiation caused a mean decrease between ­0.5 and ­1.0 mpa. therefore, as far as water potential, gamma irradiation caused a less aggressive response. osmotic pressures (ψs) remain constant in both experiments without significant changes during the 16 d storage at 2°c (fig. 1c and g). spear turgor potential (ψp) in uv­c fell below the 0 mpa threshold at day 4 and kept decreasing down to ­4 mpa on day 16 (fig. 1d). on the other side, gamma irradiated asparagus remained basically con­ stant at around 0 mpa along the 16 days of storage, subsequently, it was no longer possible to evaluate the water status of the asparagus spears gamma irra­ diation treated after day 16th (fig. 1h). according to siomos (2003), asparagus spears have between 92 and 94% water content, which reduces drastically once harvested, the loss of com­ mercial quality occurs when the fwl exceeds 8%. spears in the uv­c treatment reached such threshold only until day 16; although turgor pressure fell below 0 on day 8 without showing any visual dehydration. when samples were gamma irradiated fwl did exceed 8% on day 12, showing obvious signs of dehy­ dration, a cooked appearance, slippery epidermis, and loss of rigidity because of the effects of gamma irradiation (jeong and jeong, 2018). color parameters table 1 shows the results of c, h°, l* and δe in asparagus spears treated with uv­c kept at 2°c for 20 d. chroma (c), no significant changes were found during the 20 days storage period. however, uv­c at 2.4 kj·m­2 did cause a small, but significant decrease, as compared to controls and the 4.96 kj·m­2 exposure (table 3). the c results of both experiments found in this work agree with data published by costa et al. (2006), who found similar tendencies, suggesting the slight presence of a dull color. in the variable of hue, erratic but significant changes were observed, although after 20 days of storage showed 113 units, similar to initial values. uv­c radiation at both exposures caused a small and significant reduction from 115 to 113.7 units (table 1). these values are slightly lower than reported by wang et al. (2020) in asparagus treated with co2 and jept under modified atmospheres at 4°c with values of 120­122, corresponding to slightly yellow. significant changes were recorded in l, although somehow erratic, increasing on days 4, 8 and 12, fol­ lowing to a decrease to initial values. as far as the fig. 1 ­ changes in fresh weight loss (a and e), water potential (b and f), osmotic pressure (c and g) and turgor potential (d and h) in asparagus spears cv. brock treated with uv and gamma levels as indicated and stored at 2°c and 90% of relative humidity. a, b, c, and d graphics corre­ spond uv­c irradiation experiment; e, f, g and h graph­ ics correspond gamma irradiation experiment. differential color parameter (δe). values increased dur­ ing storage from 0 to 4.3 units at day 20 (table 1). while the highest uv­c dosage caused almost a double­fold sig­ nificant (p<0.05) increase from 2.4 to 4.5 units (table 3). adv. hort. sci., 2024 38(1): 35­45 42 effects by uv­c on l, no significant changes occurred (table 1). these slight color changes observed in green fruits and vegetables treated with uv­c are probably due to chlorophyll degradation induced by irradiation damage to the chloroplast and biosynthesis of some anthocyanins (siomos et al., 2000; villanueva et al., 2005; li et al.; 2006). indeed, in our lab, we investi­ gated the development of yellowish color in cucum­ bers treated with large uv­c irradiation energy levels and observed a color change from intense green to a paler green (data not shown). however, in the uv­c irradiation experiment, the differences in color observed in this experiment probably are enough to cause a significant change as to induce consumer rejection. table 3 shows color­related results of gamma­ irradiated spears at two intensities. a significant, but erratic, response was observed in chroma during storage ending with values similar to the initial 35.5 units. gamma irradiation exposures caused a signifi­ cant, although small, reduction of 1.1 units in c (table 2). gamma irradiation did not cause significant changes in hue during the 20 days storage with val­ ues averaging 114.4 units. (table 2). gamma irradiation did cause a significant and overall reduction in l during storage, ending on 41.3 units as compared to the initial 47.7 (table 2). also, significant reductions were found when exposed to two gamma irradiations, with 40.7 and 39.8 units for 1 and 1.5 kgy, respectively (table 3). gamma irradiat­ ed papayas at 0.75 kgy stored at 11°c and 90 % rh showed similar behavior. indeed, irradiated papaya (c = 40.89 and l* = 51.50) achieved a slightly more intense color, as compared with non­irradiated con­ trols (c = 41.67 and l* = 52.50). borzouei et al. (2013) determined that chlorophyll content in cv. roshan increased after exposure to radiations at 100 kgy, which could correlate with stimulated metabolism of pigment biosynthesis, suggesting that yellow color developed slightly more quickly in controls (pimentel and walder, 2004). peak color differential (δe) was recorded just after 4 d in storage reaching 15.2 units, followed by signifi­ cant decreases reaching 11 units at day 20 (table 2). exposure to gamma irradiation did cause a significant increase in this variable with controls showing 5.1 units and 11.9 and 13.2 units for exposures at 1 and 1.5 kgy irradiation causing a loss of glossy appear­ ance (table 3). such change in the green chlorophyll appearance from bright to dull is associated with decrease of l and slight changes in chroma may result from deformations in thylacoyd structure and damage to chloroplasts (wi et al., 2007; choi et al., 2021). 4. conclusions “hurdley technologies” do not extend postharvest shelf life in asparagus spears cv. “brock”, although the uv­c irradiation with energy levels of 2.46 kj.m­2 and 4.92 kj.m­2 did not adversely affect spear postharvest quality. the levels of uv­c radiation applied did not affect metabolic activity significantly. a slight tendency to develop a yellowish coloration was observed when irradiated with uv­c 4.96 kjm­2. gamma irradiated spears decayed rapidly, as demonstrated by several variables. such as a drastic decrease in metabolic heat production and respira­ tion, as well as development of a glossy color, cooked appearance, slippery epidermis, and losses in fresh weight and turgency. at the intensities tested, hudley technologies do not extend postharvest shelf­ life of asparagus spears. acknowledgements this work was supported by grants from fundación produce sonora a.c., and the authors also are thankful to growers pablo borquez and leopoldo santiago for providing the asparagus spears. references aegerter b., cahn m., koike s., smith r., hartz t., suslow t., 2011 ­ asparagus production in california. ­ agriculture and natural resources, university of california, davis, ca, usa, pub. no. 7234, pp. 1­6. ali h., ghori z., sheikh s., gu a., 2015 ­ effects of gamma radiation on crop production, pp. 27­78. ­ in: hakeem k. 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cultivation in tropical climate ecosystems using floating and non­ floating systems with different doses of cow manure s. susilawati 1 (*), i. irmawati 1, m. umar harun 1, b. ichwan 2 1 faculty of agriculture, universitas sriwijaya. jl. raya palembang‐ prabumulih km 32, ogan ilir 30662, south sumatra, indonesia. 2 faculty of agriculture, universitas jambi jl. jambi ‐ muara bulian no. km. 15, mendalo darat, kec. jambi luar kota, kabupaten muaro jambi, jambi, indonesia. key words: fertilizer, raft, shallot bulb, swamp, yield. abstract: deep swamp is swampland with the longest flooding period, making it challenging for crop cultivation. however, by adopting a floating system, this pro­ longed duration of flooding can be used for shallot growing. thus, this study aimed to ascertain the growth and yield of shallots cultivated in polybags using conventional non­floating and floating systems with the application of different doses of cow manure. the research was located in the experimental field and reservoir of the faculty of agriculture, sriwijaya university (3°13’30.3ʹʹ s; 104°38’55.1” e). non­floating and floating farming systems were utilized with the application of 0, 10, 15 and 20 ton/ha of cow manure. the findings demonstrated that shallots cultivated in the floating system had lower numbers and length of leaf but could produce more bulbs in comparison to the conventional method. the application of 15 ton/ha manure in the floating system resulted in higher weights of fresh and air­dried bulbs per plant, weighing 74.40 g and 64.82 g, respectively, compared to those in the non­floating system (46.77 g and 37.84 g, respectively). in conclusion, the bima brebes shallot variety potentially can be cultivated in a floating system with the application of 15 tons of cow manure per hectare. 1. introduction as one of the strategic commodities widely consumed in indonesia, shallot (allium ascalonicum l.) is a vegetable crop that significantly con­ tributes to the country’s horticultural production and inflation rate. according to the findings of the socio­economic survey in september 2021, indonesians consume an average of 2.49 kg of shallots per person each month. shallots are required for the food sector, where they are processed into ready­to­use seasonings for sprinkling on food dishes, as well as for usage in households as a seasoning for cooking (ministry of (*) corresponding author: susilawati@fp.unsri.ac.id citation: susilawati s., irmawati i., harun m.u., ichwan b., 2024 ­ shallot cultivation in tropical climate ecosystems using floating and non‐floa‐ ting systems with different doses of cow manure. ­ adv. hort. sci., 38(1): 25­34. copyright: © 2024 susilawati s., irmawati i., harun m.u., ichwan b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 july 2023 accepted for publication 29 november 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14940 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 25­34 26 agriculture, 2019; irjayanti, 2022). the amount of shallots needed for household consumption and the food industry continues to increase. the government’s involvement in meeting these needs is through a program to organize and grow shallot production centers outside java island so that production centers are not just concentrated on java. this program aims to realize shallot self­suf­ ficiency in every province in indonesia (ministry of agriculture, 2019; indriyana et al., 2020). south sumatra is one of the provinces targeted by this pro­ gram; this is because shallot production in south sumatra is still low at only 0.057% of national pro­ duction or 1125 tons in 2021 (central bureau of statistics for south sumatra province, 2022; directorate of statistical dissemination, 2022). even though south sumatra is a lowland region that is suit­ able for growing shallots, there are still several chal­ lenges that may affect the shallot growth. one of these is the land’s condition as swampland, particu­ larly lebak swampland. lebak swampland, with its alluvial soil type, has considerable potential to increase the production of food and horticultural crops. however, the use of lebak for crop cultivation is faced with high­water fluctuations that cause flooding in the rainy season and drought in the dry season. the typology of lebak swampland based on the height and duration of standing water is divided into shallow, middle and deep swamp. once or twice a year, rice can be grown in the shallow and middle swamps. during the dry season, horticultural crops, particularly vegetable crops, can also be grown, although there is a risk from drought (djafar, 2013; suprapto, 2016; suryana, 2016; widuri et al., 2016; pujiharti, 2017; simatupang and rina, 2019). deep swamp is an inland swamp area with stagnant water for more than six months and even during the dry season it remains stagnant. as a result, cultivating plants becomes quite challeng­ ing. the deep swamp is mostly left unutilized during the high flooding period. utilizing a floating cultiva­ tion technique is one option for making use of this area (siaga et al., 2018; jaya et al., 2019; lakitan, 2021; susilawati et al., 2023). according to hasbi et al. (2017), a projected floating farming system for the cultivation of vegetables was created based on the statements of farmers who are interested in using the newly introduced floating farming. shallots are one of the many crops that may be grown in the floating system. one of the factors that affect the growth of shal­ lots is the planting medium used. the texture and structure of the soil have significant impact on the production and quality of shallots. applying organic fertilizer will create the fertile, loose soil that shallots need for the development of their bulbs. one of the components that can increase the physical, chemical and biological qualities of the soil to boost the pro­ ductivity of shallot plants while reducing the amount of phosphorus (p) fertilizers added to p­deficient soils is organic fertilizer (noviyanty and salingkat, 2018; susikawati et al., 2018; nguyen et al., 2021). one organic fertilizer that can promote plant develop­ ment is cow manure (sudarsono et al., 2014; atman et al., 2018; musdalifah et al., 2021). thus, this research was conducted with the aim of evaluating the growth response and production of shallot plants cultivated in polybags using non­floating and floating systems with the application of various doses of cow manure fertilizer. 2. materials and methods research gate the research was located in the experimental field and reservoir of the faculty of agriculture, sriwijaya university, indralaya ogan il ir (3°13’30.3ʹʹs; 104°38’55.1”e). figure 1 shows the arrangement dur­ ing the dry season in tropical climate ecosystems of south sumatra, indonesia, from may to august 2022. typical agroclimatic conditions at the outdoor research facilities are shown in figure 2. procedures the shallot bulbs used were of the bima brebes variety originated from the shallot seed farmers in brebes, central java. the experiment was arranged using a factorial randomized block design with two factors and three replicates. the treatments consist­ fig. 1 ­ non­floating (a) and floating (b) farming practices of shallot cultivation. susilawati et al. ‐ shallot cultivation in tropical swampland 27 ed of different farming practices (conventional non­ floating and floating systems) and dosages of cow manure (0, 10, 15 and 20 ton/ha). for all farming practice treatments, the planting media were prepared using the same method: after being completely mixed, they was placed into 35 cm x 30 cm polybags. the planting media were a mixture of alluvial topsoil gathered from lebak swamp combined with cow manure according to the treatments. in the conventional farming technique, the area was prepared by clearing the weeds to make a space for placing the polybags filled with the media. the planting space was 20 cm x 20 cm, follow­ ing the recommendation for shallot cultivation. in the floating cultivation, the polybags were put on a 2 m x 1 m bamboo raft. each replicate was put on one bamboo raft. the planting media were sprayed with bio soil grow booster at a concentration of 4 ml/l of water one week before planting. inorganic fertilizers were also used, with dosages of 69 kg/ha of p2o5, 46 kg/ha of n and 60 kg/ha of k2o. phosphorus fertilizer was applied 7 days before planting, whereas nitrogen and potassium were applied twice, 7 and 25 days after planting, each time in half the prescribed amount. before being planted, the top one­third of the bulb was cut off and the bulb was placed into a planting hole at depth 2­3 cm. shallot plants cultivat­ ed in the non­floating system were watered regularly to ensure sufficient water availability. in the floating system, the plants were not watered because water was continuously supplied through the soil pores by capillary force from the swamp water below. data analysis the variables were growth characteristics such leaf length, leaf number and leaf color, shallot production variables such as bulb number, bulb diameter, fresh weight, dry weight and shrinkage percentage, as well as estimated production per hectare. leaf color was measured using a chlorophyll meter (spad­502, minolta) to estimate leaf green­ ness level correlated to the chlorophyll content (spad value). growth parameters were observed every week, whereas the production data were gath­ ered after the harvest. the collected data were ana­ lyzed using r studio statistical analysis software. the calculated f­value generated from the analysis of variance (anova) was compared to values at p≤0.05 and p≤0.01 for justifying the significant effects of the treatments. furthermore, if the treatment effect was significant for any measured trait, the least significant difference (lsd) test was conducted to determine significant differences among treatment levels for each specified trait. 3. results and discussion leaf length (cm) and leaf number of shallots shallot plants grown in tropical climate ecosys­ tems using two farming practices ­ conventional non­ floating (fig. 1a) and floating (fig. 1b) systems with various doses of cow manure ­ showed differences in growth and yield. rainfall continued to decline from may to august 2022, the period when the study was conducted. the rainfall reduced from 206 mm in may to 63 mm in august (fig. 2a). high rainfall levels at the start of the study provided a favorable environ­ ment for shallot growth in the non­floating cultiva­ tion system (polybags stacked on dry soil). on the other hand, shallots grown in wetlands using the floating system (polybags placed on rafts) did not favor heavy rainfall. from may to august 2022, the temperature and humidity remained relatively stable at 27.47­27.92°c and 84.51­86.36%, respectively (fig. 2b). fig. 2 ­ typical agroclimatic conditions at the outdoor research facilities: (a) rainfall; (b) temperature and relative humid­ ity. source: https://www.bmkg.go.id. adv. hort. sci., 2024 38(1): 25­34 28 anova revealed that the variations in farming practices had a significant impact. the results of the lsd test on leaf length demonstrated that the two farming practices differed significantly. in conven­ tional cultivation, the maximum leaf length was 40.43±1.25 cm at 5 weeks after planting (wap), whereas in the floating system it was only 30.47±0.47 cm at 4 wap. in the floating agriculture system, the position of the plants was adjusted so that they would always be waterlogged to a height of about 3 cm from the base. in that case, water was continu­ ously supplied via the soil pores by capillary force, causing slower oxygen diffusion. additionally, when it rains heavily, the media become more water­saturat­ ed, which lowers the amount of accessible oxygen. oxygen is needed by the roots for respiration and for maintaining healthy cell function (neira et al., 2015; ernest, 2018; jaya et al., 2021; kartika et al., 2021). damage to the root will eventually affect the upper plant growth, as seen from the agronomical features. research by susilawati et al. (2012) on red pepper plants showed that all cultivars experienced varying degrees of root damage as a result of flooding stress. the amount of oxygen present in the planting media is significantly influenced by the height of the water table. research on bean plants has shown that the roots, particularly the process of root respiration, were significantly impacted by water levels that were 10 cm below the surface of the planting media. although organic fertilizer applied to shallots in a floating system did not affect growth, a proper water level and the application of organic matter to shallot plants considerably stimulated growth (susilawati and lakitan, 2019; susilawati et al., 2019, 2022). with regard to leaf length, the application of cow manure showed insignificant results in the first week but sig­ nificant results at 2–8 wap. the longest leaves (40.19 ± 2.96 cm) were obtained at a p3 treatment dose of 20 ton/ha, which is not significantly different from the 40.06 ± 1.69 cm obtained at a p2 dose of 15 ton/ha. the combination of farming practices and cow manure treatments had a significant effect on leaf length only at 6 and 8 wap in the conventional p3 treatment, with lengths of 44.74 ± 1.10 cm and 44.92 ± 1.36 cm, respectively. for leaf number, the difference in farming prac­ tices only had a significant effect in the first three weeks. the average number of leaves was mostly higher in the conventional system, except in the fourth week when the leaf number in the floating system was higher at 24.33 ± 1.92 compared to 23.39 ± 1.33 in the conventional system. the research on eggplant showed that increasing the water content of the substrate from 1 to 3 cm would increase the growth of vegetative organs (jaya et al., 2019). the difference in cow manure dose affected the number of leaves, with the highest leaf number of 31.78 ± 2.65 obtained in p2 treatment at 7 wap (table 1). similarly, research by feriatin et al. (2021) also showed that the use of cow manure would affect leaf number of the lokananta shallot variety. the p3 treatment for conventional cultivation and the p2 treatment for floating cultivation produced the high­ est average leaf length and leaf number when cow manure was applied. in the conventional p3 treat­ ment, the maximum leaf length was 37.45 cm with an average of 21.33 leaves, while in the floating p2 treatment the highest leaf length was 29.80 cm with 23.14 leaves. cow manure is an organic fertilizer that can alter the structure and texture of the soil, making the media crumblier, which explains the difference in the dosage of cow manure between the two cultiva­ tion systems (fig. 3). meanwhile, as a result of the relatively high moisture of the planting media in the floating culture, cow manure already affects the tex­ ture and structure of the media at lower doses (elisabeth et al., 2013; gudugi, 2013; ekwealor et al., 2020; wisdom et al., 2021). spad value in this study, the parameter of leaf greenness ­ which serves as an indicator for chlorophyll content ­ was quantified using the spad tool without damaging the leaves measured from the second to the eighth week. at 4 and 6 wap, the culture technique treatment significantly affected the spad value but had no significant effect at 2 and 8 wap. shallots cul­ tivated in the floating system were recorded to have higher values at 2, 4 and 6 wap (77.69±2.69, 64.10± 4.41 and 48.18 ± 1.25, respectively) compared to those in the conventional system (74.68±3.08, 48.18±1.25 and 48.79±1.43). however, at 8 wap, the spad value in the conventional system was higher than in the floating system (44.78±1.33 vs. 43.22±4.48). the spad value of the two cultivation techniques was at its highest at 2 wap and then con­ tinued to decrease until 8 wap. in comparison to other models, the polynomial model’s regression analysis of the spad value in the shallot cultivation resulted in the largest determination coefficient (r2), which is close to 1: 0.9123 for the conventional sys­ tem and 0.9618 for the floating system. the magni­ susilawati et al. ‐ shallot cultivation in tropical swampland 29 tude of the r2 value indicates that the spad value is affected by the cultivation technique in a quadratic manner, increasing until it reaches the peak before starting to decline (table 2). a study on corn resulted in a similar result, where the spad values would decrease after reaching their peak, mostly affected by the environment (ghozali, 2016; kandel, 2020; szulc et al., 2021). the higher spad values in the floating system, especially at 4 and 6 wap, indicated that the photosynthesis process is going well due to sufficient water availability. in contrast, if there is a deficit of water (moisture stress), photosynthetic activity will be reduced due to chlorophyll damage (pallavolu et al., 2023). the research results of ai nio et al. (2019) showed that the water deficit induced by peg 8000 with media water potential (wp) ­0.25 and ­0.5 mpa reduced the total leaf chlorophyll content, leaf chlorophyll a and leaf chlorophyll b. this study also found that the increase in cow manure dosage increases the spad value, with the fig. 3 ­ effect of cow manure dosage on leaf length (a) and leaf number (b) in the different farming practices. table 1 ­ leaf length (cm) and leaf number of shallot with the application of cow manure (ton/ha) in different farming practices treatment weeks after planting (wap) 1 2 3 4 5 6 7 8 farming practice leaf length (cm) conventional 11.67 ± 0.66 a 26.60 ± 0.79 a 36.43 ± 1.03 a 40.32 ± 1.45 a 40.43 ± 1.69 a 38.39 ± 1.71 a 38.39 ± 1.66 a 36.09 ± 1.95 a floating 8.28 ± 0.32 b 20.04 ± 0.56 b 27.58 ± 1.09 b 30.47 ± 1.43 b 29.45 ± 1.90 b 26.66 ± 2.08 b 25.66 ± 1.82 b 24.88 ± 1.76 b significance ** ** ** ** ** ** ** ** lsd0.05 1.516 1.375 2.209 1.961 1.992 2.208 2.536 4.549 leaf number conventional 7.83 ± 0.39 a 12.38 ± 0.43 a 17.78 ± 0.69 a 22.13 ± 0.93 23.39 ± 1.33 25.69 ± 1.50 26.00 ± 1.43 23.64 ± 1.21 floating 5.88 ± 0.21 b 10.38 ± 0.47 b 15.66 ± 0.61 b 20.94 ± 1.25 24.33 ± 1.92 25.25 ± 2.15 23.61 ± 2.06 20.03 ± 1.91 significance ** ** * ns ns ns ns ns lsd0.05 0.777 1.303 1.548 2.479 2.929 3.831 4.679 4.567 cow manure (ton/ha) leaf length (cm) p0 (0) 9.31 ± 0.90 21.37 ± 1.74 b 27.53 ± 2.37 b 29.07 ± 2.54 b 26.80 ± 2.99 c 24.83 ± 3.09 c 25.61 ± 3.07 c 25.24 ± 2.96 b p1 (10) 10.19 ± 1.23 22.09 ± 1.24 b 30.71 ± 1.86 a 33.59 ± 2.21 b 32.72 ± 2.64 b 29.26 ± 3.12 b 29.86 ± 3.00 b 30.29 ± 2.58 b p2 (15) 9.54 ± 0.87 25.30 ± 1.58 a 35.52 ± 1.81 a 39.69 ± 1.74 a 40.06 ± 1.69 a 38.93 ± 1.33 a 37.84 ± 1.34 a 37.16 ± 1.80 a p3 (20) 10.86 ± 1.12 24.52 ± 1.89 a 34.26 ± 2.31 a 39.23 ± 2.82 a 40.19 ± 2.96 a 37.10 ± 3.46 a 34.76 ± 4.63 a 29.27 ± 4.69 b significance ns ** ** ** ** ** ** * lsd0.05 2.144 1.945 2.189 2.773 2.817 3.122 3.587 6.434 leaf number p0 (0) 6.00 ± 0.40 b 10.33 ± 0.31 14.33 ± 0.41 b 17.55 ± 0.62 b 17.72 ± 0.88 c 19.50 ± 1.64 c 20.50 ± 2.11 b 19.39 ± 2.28 p1 (10) 6.94 ± 0.59 ab 11.33 ± 1.06 16.78 ± 1.11 a 20.39 ± 1.12 b 22.50 ± 1.32 b 25.99 ± 1.47 b 26.28 ± 2.09 ab 23.11 ± 2.04 p2 (15) 6.72 ± 0.58 ab 11.39 ± 0.76 17.22 ± 0.99 a 24.00 ± 1.59 a 29.05 ± 2.38 a 31.78 ± 2.65 a 29.67 ± 2.39 b 25.94 ± 1.88 p3 (20) 7.78 ± 0.68 a 12.50 ± 0.55 18.55 ± 0.59 a 24.22 ± 0.80 a 26.16 ± 1.32 ab 24.61 ± 1.78 bc 22.78 ± 2.14 b 18.89 ± 2.36 significance * ns ** ** ** ** * ns data represent the mean and standard error. values followed by different letters within each column indicate a significant difference at lsd 0.05. 30 adv. hort. sci., 2024 38(1): 25­34 highest spad value of 82.54 obtained in the p3 treat­ ment and the lowest (31.21) in the p0 treatment. the spad value has been widely used to estimate the chlorophyll content of other crops, such as tomatoes (jiang et al., 2017). furthermore, the application of cow manure could also increase leaf chlorophyll, as indicated by the spad value in wheat and rice plants (shah et al. , 2017; atman et al. , 2018). the combination of cultivation techniques and cow manure treatments resulted in no significant effect at 2 and 8 wap, a significant effect at 4 wap and a highly significant effect at 6 wap. the highest spad values were obtained in the floating cultivation with a cow manure dose of 20 ton/ha: 77.01 ± 5.35 at 4 wap and 75.11 ± 3.96 at 6 wap (table 3). bulb number, bulb diameter (mm) and weight of fresh and air‐dried bulbs (g) the yield components include the number of bulbs, bulb diameter and weight for both fresh and table 3 ­ the spad value of shallot in different farming practices with the application of cow manure table 2 ­ regression analysis correlations of the spad value with several mathematical models of farming practices using different doses of cow manure ** significant difference at p < 0.05. farming practice linear model y = ax + b logarithmic model y = a ln x + b polynomial model y = ax2 + bx +c power model y = axb conventional y = -8.9073x + 76.38 y = -21.1ln(x) + 70.877 y = 5.6238x2-37.026x + y = 70.35x0.357 r2 = 0.6917 r2 = 0.8417 r2= 0.9123 r2 = 0.8842 r = 0.8316** r = 0.9174** r = 0.9551** r = 0.9432** floating y =­10.769x + 88.122 y = -22.56ln(x) + 79.122 y = -0.7451x2-7.0429x + y = 80.712x0.375 r2 = 0.9581 r2 = 0.9116 r2 = 0.9618 r2 = 0.8804 r = 0.9788** r = 0.9547** r = 0.9807** r = 0.9383** treatment spad value 2 wap 4 wap 6 wap 8 wap farming practice conventional 74.68 ± 3.08 48.18 ± 1.25 b 48.79 ± 1.43 b 44.78 ± 1.33 floating 77.69 ± 2.88 64.10 ± 4.41 a 59.78 ± 4.55 a 43.22 ± 4.48 significance ns ** ** ns lsd value 7.996 6.040 4.381 4.615 cow manure (ton/ha) p0 (0) 68.16 ± 2.38 45.31 ± 2.88 c 40.90 ± 2.71 c 31.21 ± 4.08 c p1 (10) 75.74 ± 5.19 58.48 ± 5.77 ab 56.21 ± 4.35 b 44.61 ± 2.93 b p2 (15) 78.27 ± 4.09 55.78 ± 4.94 b 55.98 ± 3.73 b 46.60 ± 2.09 b p3 (20) 82.54 ± 2.98 65.00 ± 6.08 a 64.04 ± 5.28 a 53.60 ± 3.80 a significance ns ** ** ** lsd value 11.307 8.542 6.197 6.527 farming practice x cow manure (ton/ha) conventional x p0 68.47 ± 5.10 46.81 ± 1.43 b 43.29 ± 3.72 ef 39.69 ± 3.16 c conventional x p1 74.13 ± 10.00 46.93 ± 1.68 b 48.02 ± 1.74 de 43.42 ± 10.00 bc conventional x p2 74.01 ± 4.26 46.00 ± 1.36 b 50.87 ± 0.68 bc 46.57 ± 4.26 bc conventional x p3 82.13 ± 4.21 52.99 ± 3.47 b 52.98 ± 1.17 cd 49.46 ± 4.21 ab floating x p0 67.86 ± 1.46 43.81 ± 6.11 b 38.52 ± 4.15 f 22.73 ± 1.46 d floating x p1 77.37 ± 5.68 70.03 ± 5.53 a 64.41 ± 4.95 b 45.81 ± 5.68 bc floating x p2 82.54 ± 6.90 65.56 ± 4.94 a 61.10 ± 6.57 bc 46.63 ± 6.90 bc floating x p3 82.97 ± 5.15 77.01 ± 5.35 a 75.11 ± 3.96 a 57.73 ± 5.15 a significance ns * ** ns lsd value 15.991 12.081 8.763 9.231 wap= week after planting. data represent the mean and standard error. values followed by different letters within each column indicate a significant difference at lsd 0.05. susilawati et al. ‐ shallot cultivation in tropical swampland 31 air­dried bulbs. the farming practice had no significant impact on the quantity of bulbs but a very significant impact on the weight of fresh and air­ dried bulb and the bulb diameter. the conventional method produced the greatest number of bulbs, whereas floating cultivation produced the best results in terms of bulb diameter and weight of fresh and air­dried bulbs. the growing media conditions strongly affected how the bulbs were initially formed. since water was constantly accessible from beneath the growing media through capillaries, floating cultivation used growing media that were somewhat moist. there was also intense rainfall during early growth of the shallots, causing the planting media to be very wet. there were about 7.78 ± 0.35 bulbs formed in conventional cultivation and 7.75 ± 0.33 in the floating cultivation. however, the number of bulbs was not linearly correlated with the greater dose of cow manure applied, as the largest number of bulbs was obtained at a dose of 15 ton/ha (table 4). the largest numbers of bulbs (8.33 in convention­ al cultivation and 8.22 in floating cultivation) were obtained from the same manure treatment, which was p2. the lowest numbers (6.78 in conventional cultivation and 6.67 in floating cultivation) were also obtained from the same manure treatment, p0. plant growth can thus be supported by appropriate cultiva­ tion methods (khorasgani and pessarakli, 2019; cahyaningrum et al., 2023). the average diameter of bulbs grown using float­ ing cultivation was 23.51 ± 1.29 mm, which is much larger than the 20.62 ± 1.62 mm average diameter of bulbs grown using conventional cultivation (fig. 4). the largest diameter of bulbs produced as a result of cow manure application was at a dose of 15 ton/ha, and the lowest was at 0 ton/ha (table 4). based on the combination of treatments, p2 treatment (15 ton/ha) in floating cultivation produced the largest bulb diameter of 30.78 mm, and p3 treatment (20 ton/ha) in conventional cultivation produced the largest bulb diameter of 25.36 mm. the two cultiva­ tion methods produced the smallest bulb diameters in the same treatment, p0 (0 ton/ha), with the con­ ventional system producing a diameter of 15.22 mm table 4 ­ shallot yield components for different farming practices with the application of cow manure treatment number of bulbs diameter of bulb (cm) fresh weight of bulb (g) air­dried weight of bulb (g) farming practice conventional 7.78 ± 0.35 20.62 ± 1.76 b 35.36 ± 6.14 b 26.79 ± 5.64 b floating 7.75 ± 0.33 23.51 ± 1.29 a 42.33 ± 3.52 a 35.69 ± 2.94 a significance ns ** * ** lsd value 1.016 1.741 6.419 4.703 cow manure p0 (0) 7.44 ± 0,45 15.44 ± 0.77 c 20.57 ± 2.85 c 14.71 ± 2.12 d p1 (10) 7.44 ± 0.50 19.88 ± 0.84 b 34.46 ± 2.36 b 25.64 ± 1.49 c p2 (15) 8.28 ± 0.53 27.10 ± 1.74 a 58.24 ± 7.76 a 48.22 ± 7.75 a p3 (20) 7.89 ± 0.44 25.84 ± 0.81 a 42.11 ± 2.94 b 36.39 ± 1.84 b significance ns ** ** ** lsd value 1.437 2.461 9.078 6.651 farming practice x cow manure conventional x p0 6.78 ± 0.72 15.22 ± 1.40 e 19.58 ± 4.90 e 13.15 ± 3.18 f conventional x p1 8.22 ± 0.58 18.49 ± 0.98 de 33.01 ± 3.72 cd 24.57 ± 1.99 de conventional x p2 8.33 ± 0.76 23.43 ± 0.81 bc 42.08 ± 1.87 bc 31.61 ± 0.73 bcd conventional x p3 7.78 ± 0.44 25.36 ± 1.03 b 46.77 ± 4.21 b 37.84 ± 0.89 b floating x p0 8.11 ± 0.22 15.66 ± 0.99 e 21.57 ± 3.95 de 16.28 ± 3.15 ef floating x p1 6.67 ± 0.57 21.28 ± 0.80 cd 35.92 ± 3.45 bc 26.71 ± 2.44 cd floating x p2 8.22 ± 0.90 30.77 ± 1.02 a 74.40 ± 6.05 a 64.82 ± 4.92 a floating x p3 8.00 ± 0.88 26.32 ± 1.40 b 37.45 ± 1.96 b 34.96 ± 2.56 b significance ns * ** ** lsd value 2.032 3.481 12.838 9.406 wap= week after planting. data represent the mean and standard error. values followed by different letters within each column indicate a significant difference at lsd 0.05. adv. hort. sci., 2024 38(1): 25­34 32 and the floating system a diameter of 15.66 mm. the high bulb diameter in the floating culture was greatly supported by the conditions of the growing media, where during the growth stages the rainfall contin­ ued to decline, so the media in floating culture was not saturated with water and oxygen was still avail­ able. nutrient absorption, water uptake and root res­ piration are all affected by oxygen availability, which is a crucial component for plant growth. the use of biological fertilizers or a combination of biological and organic fertilizers can increase the shallot bulb diameter compared to controls (neira et al., 2015; xiong et al., 2015; purba et al., 2020; widyastuti et al., 2021). shallot production is highly dependent on the weight of fresh and air­dried bulbs. the floating cultivation system yielded the greatest average data, which was significantly different from conventional cultivation (42.33±3.52 g vs. 35.69±2.94 g (table 4). according to jaya et al. (2019), the availability of water below the plant media in the floating system significantly promotes plant growth and yield. based on the results, conventional farming required a high­ er dosage of cow manure (20 ton/ha) compared to floating farming, which required only 15 ton/ha. in terms of cost and bulb production, paputri et al. (2016) found that a cow manure dose of 20 ton/ha was economically feasible. however, arzad et al. (2017) found that mustard plants needed up to 25 ton/ha of cow manure. the estimated production per hectare was calculated using data on air­dried bulb weight under the assumption of a planting space of 20 cm x 20 cm. the highest estimated production was obtained in the floating system from p2 treatment with 21.61 ton/ha, while the highest production in the conven­ tional system was from p3 treatment with 12.61 ton/ha. the p0 treatment had the lowest estimated yield, at 4.38 and 5.43 ton/ha in the conventional and floating cultivation systems, respectively (fig. 5). fig. 4 ­ shallot bulbs produced by the conventional (a) and floating (b) systems with the application of cow manure. fig. 5 ­ estimated production of shallot from different farming practices with the application of cow manure. based on the results, it was concluded that the bima brebes shallot variety has the potential to be cultivated using the floating technique with the application of 15 ton/ha cow manure, as seen from production estimation reaching 21.61 tons of dry bulb, which is higher than the 9.9 ton/ha of dry bulb reported by the ministry of agriculture (2019). based on this large potential yield, further research has been carried out again in 2023 to obtain more accu­ rate production data. the results obtained will be the basis for considering recommendations for floating shallot development. acknowledgements we would like to express our sincere gratitude to our research team and colleagues at the department susilawati et al. ‐ shallot cultivation in tropical swampland 33 of agronomy, faculty of agriculture, universitas sriwijaya, for their helpful feedback and valuable support for our research. references ai nio s., pirade m., ludong d.p.m., 2019 ­ leaf chloro‐ phyll content in north sulawesi (indonesia) local rice cultivars subjected to polyethylene glycol (peg) 8000‐ induced water deficit at the vegetative phase. ­ biodiversitas, 20(9): 2462­2467. arzad n.h., tambing y., bahrudi n., 2017 ­ the effect of various rates of cow manure application on growth and yield of mustard (brassica juncea l.). ­ agriculture sci. j., 4(1): 42­48. atman a., bakrie b., indrasti r., 2018 ­ effect of cow manure dosages as organic fertilizer on the productivity of organic rice in west sumatra, indonesia. ­ int. j. environ., agric. biotechn. 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côte d’ivoire j.p. dao 1 (*), c. kouakou 2, m. cherif 1, k.t. yeo 3, n.b. guinagui 1, k.a.a. mbo 1, k.l. kouakou 2 1 university of félix houphouët‐boigny, ufr biosciences, laboratory of biotechnology, agriculture and biological resources development, 22 bp 582 abidjan 22, côte d’ivoire. 2 university nangui abrogoua, ufr science de la nature, laboratory of biology and improvement of plant production, 02 bp 801 abidjan 02, côte d’ivoire. 3 national center for agronomic research, korhogo research station, bp 856 korhogo, côte d’ivoire. key words: domestication, cultivation, garcinia kola, grafting, season. abstract: garcinia kola, a high­socioeconomic and multipurpose fruit tree, has been shown to have a low germination and long juvenile phase, which limit its cultivation initiative. our study aimed to determine successful g. kola grafting conditions to promote its cultivation. so trials were carried out in june and august, testing four grafting methods: simple whip, top cleft, side cleft, and chip budding made on 12­ and 36­month­old rootstock. the grafts were unwrapped at 15, 22, and 29 days after grafting. the results showed that the month of june is the favorable period for g. kola grafting. the best grafting success was observed with the chip budding and top cleft grafting methods applied on the 36­ and 12­month­old rootstocks, respectively. the graft unwrapping time at 29 days after grafting promotes the best grafting success. the combined effect showed that the best graft success rate was recorded in june on 36­month­old rootstock (43.33%) with the chip budding and side cleft grafting method and 83.33% for the graft made on 12­month­old rootstock with the top cleft grafting method, respectively. this study is an important step for good plant material for domestication and cultivation of this resource. 1. introduction garcinia kola is a native tree found in the tropical rainforests of west and central africa and belongs to the clusiaceae family. g. kola is recognized as one of the non­timber forest products of great socio­ economic importance (assogbadjo et al., 2017; codjia et al., 2018). the (*) corresponding author: dao.patrick@ufhb.edu.ci citation: dao j.p., kouakou c., cherif m., yeo k.t., guinagui n.b., mbo k.a.a., kouakou k.l., 2025 ­ exploring grafting to propagate and con‐ serve garcinia kola a vulnerable species in côte d’ivoire. ­ adv. hort. sci., 39(1): 45­54 orcid: djp: 0000­0002­6710­1848 kc: 0000­0001­5814­2966 cm: 0000­0003­2857­341x ykt: 0009­0007­0375­6603 gnb: 0000­0002­1740­0305 mkaa: 0000­0001­5519­9270 kkl: 0000­0002­3546­4978 copyright: © 2025 dao j.p., kouakou c., cherif m., yeo k.t., guinagui n.b., mbo k.a.a., kouakou k.l. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 28 january 2025 accepted for publication 31 march 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17232 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-6710-1848 http://orcid.org/0000-0001-5814-2966 http://orcid.org/0000-0003-2857-341x http://orcid.org/0009-0007-0375-6603 http://orcid.org/0000-0002-1740-0305 http://orcid.org/0000-0001-5519-9270 http://orcid.org/0000-0002-3546-4978 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 45­54 46 fruits, seeds, stems, roots, and bark are used for food and medicine for the treatment of several diseases such as diarrhea, laryngitis, gonorrhea, headache, and gastritis (maňourová et al., 2019). the seed trade is an activity of great economic importance (kouame et al., 2016; maňourová et al., 2019). despite the importance of the species, no enhancement program is undertaken. however, g. kola is one of the species identified as a priority agroforestry species to benefit from a domestication program in west africa (franzel et al., 1996). in addition, g. kola is an agroforestry species, which contributes through the exploitation of its fruits and seeds to the diversification of farmers’ income and the reduction of famine (maňourová et al., 2019). however, the cultivation of the species by farmers remains very difficult due to the low germination capacity of the seeds and the time required for the plant to enter into flowering and fruiting, which is between 20 and 30 years (yakubu et al., 2014; agwu et al., 2018). it is therefore urgent to find effective strategies for the regeneration of elite trees that can induce early fruiting of the species. according to sanou et al . (2004) and leakey et al . (2017), domestication and genetic improvement strategies are necessary to conserve genetic material and improve fruit production. leakey and simons (2000) reported the growing interest in the genetic improvement of tropical forest trees aimed at developing tree cultivars with fruits with desirable characteristics. vegetative propagation of plants plays an important role in the production of high­quality plants for the domestication of forest trees (leakey and akinefessi, 2008). cuttings, grafting, budding, and layering are vegetative macropropagation methods mainly used for the regeneration of several species (love et al., 2017). among these methods, grafting is the most used to improve the quality of plants. grafting technique mainly involves joining parts of plants (scion and rootstock) in a manner to unite both parts so they can form a plant. a large number of research works have been carried out on agricultural fruit trees using grafting. akinnifesi et al. (2009) reported that grafted trees of uapaca kirkiana began to produce fruits only after 2­3 years, while those derived from seedlings took 12­15 years. grafting techniques were also successfully used on allanblakia floribunda oliv. (clusiaceae) to reduce the long juvenile phase of about 10­12 years to less than 5 years (asaah et al., 2012). concerning garcinia kola, grafting is a recent area to explore. in nigeria, yakubu and akinyele (2021) indicated the suitability of g. kola for grafting with 52% of grafting success. but these authors investigate only one source of the grafts using a single grafting technique. however, several research studies have shown that the success of grafting depends on several factors such as the grafting technique, the graft ing period, and the age of the rootstock (munjunga et al., 2013; akter et al., 2016; nguyen and yen, 2018). in côte d’ivoire, works on vegetative propagation of g. kola have until now focused on cutting (kouakou et al., 2016; dao et al., 2020). more in­depth investigations to ensure efficient production of quality plants are necessary on g. kola grafting ability. the objective of this study is to determine the conditions favorable to the successful grafting of g. kola. 2. materials and methods site of study the study was carried out from june 2018 to february 2020 in abidjan at the university nangui abrogoua (una) research station (05°23´n, 04°00´w). this site is located in the forest zone where the rainfall pattern is bimodal with two dry seasons (from december to march and from july to august) and two rainy seasons (from april to june and from september to november). mean annual rainfall varies between 1800 and 2000 mm. the mean monthly temperature varies between 27 and 30°c, whereas the mean relative humidity ranges from 70 to 84% (kouakou et al., 2016). plant material plant materials were constituted of 12­ and 36­ month­old seedlings of garcinia kola obtained in the nursery of the experimental research station of una. the average height and collar diameter of the rootstocks were 84.1 ± 10.04 cm and 1.31 ± 0.18 cm for 36­month­old seedlings and 43 ± 5.74 cm and 0.84 ± 0.10 cm for 12­month­old seedlings, respectively. these plants were used as rootstocks in this study. the mature fruiting tree located in ahouabo (06°22´n, 03°82´w) was used for scion’ collection. rootstock preparation and scion collection mature fruits were naturally collected under a dao et al. ‐ vegetative propagation of garcinia kola by grafting 47 single tree in a cocoa field in ananguié village (06°33´n, 03°67´w). the seeds from this fruit were used for the production of seedlings in the nursery. seedlings of 24 and 36 months old were selected based on their vigor for use as rootstocks. the preparation of the rootstock concerned rootstocks aged 36 months old and consisted of reducing the lateral branches one week before the grafting operation in order to allow an accumulation of sap. in fact, the sap allows the scion, once placed, to maintain its vigor and accelerates its fusion with the rootstock. but no reducing branches were made on 12­month­old rootstock because there are no ramifications. the rootstock was watered every day one week before the grafting operation. for scion collection, non­flowering lateral shoots of current season’s growth were cut from a superior mature tree selected according to farmers’ criteria, which included good physical and sanitary characteristics (such as vigorous growth and absence of parasites) as well as the quality of the fruits and nuts (tasty pulp and size of seeds). these shoots (15­ 20 cm long) were collected early in the morning (between 6 and 7 am) using disinfected scissors with 70% alcohol. to avoid dehydration of the shoots, they were stored in a damp, moistened gunny bag and packed in a cooler before transportation to the nursery. they were free from disease and unignified. they were used to provide the scions or buds for grafting onto the rootstocks depending on the graft ing technique being tested. graft ing on rootstocks was carried out early in the morning, the day after the collection of the grafts, due to the long distance between the collection location and the nursery. grafting methods and experimental design in order to determine the most favorable period for successful grafting of garcinia kola, the grafting operation was carried out over two periods. the first trial was carried out in june (main rainy season) and the second in august (short dry season). at each trial period, four grafting techniques were tested in order to identify the optimized grafting success. these techniques were simple whip, top cleft, side cleft, and chip budding and were made according to takoutsing et al. (2014). the grafting operations were made on 36­month­old and 12­month­old rootstock. all the grafting techniques were made on 36­month­ old rootstocks. but only simple whip and top cleft techniques were applied on 12­month­old rootstock due to their small diameter (<1 mm), inapt for chip budding and side cleft technique application. the scions constituted of shoots were used for simple whip, top cleft, and side cleft. while buds were used for chip budding. to determine the time required for the graft to weld for each technique, the graft was unwrapped at 15, 22, and 29 days after grafting. the experimental design adopted was a completely randomized block with two factors: the grafting technique and the unwrapped time for each trial period and the type of rootstock used. for the grafting carried out on 36­month­old rootstocks and at each trial period, 12 treatments were carried out (4 grafting techniques x 3 unwrapping times) and 6 treatments for 12­month­old rootstocks (2 grafting techniques x 3 unwrapping times). however, due to the limited availability of rootstock plants, the treatments were repeated twice. each treatment consisted of 15 grafted plants. over the two trial periods, a total of 720 plants were grafted from 36­ month­old rootstocks (2 periods x 4 graft ing techniques x 3 unwrapping times x 15 grafted plants x 2 replicates) and 360 plants grafted with 12­month­ old rootstocks (2 periods x 2 grafting techniques x 3 unwrapping times x 15 grafted plants x 2 replicates). monitoring and data collection the monitoring consisted of keeping the plot clean and regularly pulling out the weeds in the bags by hand in order to avoid any competition with the rootstocks. the unwrapping process, which consists of removing the ligature strips from the contact zone between the scion and the rootstock, was carried out at 15, 22, and 29 days after grafting in order to determine the time necessary for good welding between the scion and the rootstock. one week after each stripping operation, the rootstocks of the chip budding and the side graft were pruned at 2 cm above the grafting point without leaving any knots (soloviev and gaye, 2004). the data collection included the number of sprouted grafts and the days taken for each graft to sprout. parameters evaluated during the experiment were the graft success, recovery time, and graft survival rate. the graft success rate (gsr) reflects the ratio of the number of successful grafts to the number of grafts performed and is expressed by the following formula: gsr (%) = (number of successful grafts) x 100 (number of grafts performed) adv. hort. sci., 2025 39(1): 45­54 48 the graft recovery time (grt) corresponding to the sum of the recovery times of each graft , compared to the total number of successful grafts. it is expressed by the following formula: grt (day) = (∑tr)/n where tr is recovery time per graft and n the total number of successful grafts. the survival rate of grafted plants was evaluated for 12 months after grafting taking into account the grafting technique and the age of the rootstock. statistical analysis data was subjected to analysis of variance (anova) with respect to grafting season, technique, graft unwrapping time, and their interaction. when a significant difference was observed between the means (p <0.05), the anova is supplemented by the least significant difference (lsd at p≤0.05) for separation of means. all the analyses were processed using the software statistica 7.1. 3. results effect of seasonality on the success of garcinia kola grafting the results indicated in table 1 show that the graft success rate and the average recovery time of grafts carried out on rootstocks aged 36 and 12 months varied depending on the grafting period. the highest success rates for grafts carried out on rootstocks aged 36 months (34.02±2.47%) and rootstocks aged 12 months (48.33±5.00%) were recorded in the month of june. the lowest values of graft success rate were observed in the month of august. as for the average recovery time, the shortest (28.02±0.39 days and 20.72±0.69 days) were observed in grafts carried out in the month of june, respectively, with aged rootstocks of 36 and 12 months old. the month of june favors the success of garcinia kola grafting with a higher success rate and an earlier recovery time. furthermore, rootstocks aged 12 months showed good aptitude for grafting with an early graft recovery, unlike rootstocks aged 36 months old. effect of grafting technique on the success of garcinia kola grafting grafting technique significantly influenced the graft success rate in 36­month­old rootstocks (p= 0.006) and 12­month­old rootstocks (p<0.011). on the other hand, no significant difference was observed in the average recovery time, whatever the technique, in the two types of rootstocks (table 2). table 1 ­ comparison of average values of grafting parameters according to period and age of rootstocks in a column, the values followed by different letters are significantly different at p<0.05. grafting period graft success (%) graft recovery time (day) 36 month old rootstock 12 month old rootstock 36 month old rootstock 12 month old rootstock june 34.02 ± 2.47 a 48.33 ± 5.00 a 28.02 ± 0.39 b 20.72 ± 0.69 b august 21.66 ± 1.46 b 27.77 ± 3.38 b 30.00 ± 0.52 a 23.27 ± 0.59 a f 50.45 47.20 8.55 7.53 p <0.001 <0.001 0.005 0.011 table 2 ­ comparison of average values of grafting parameters according to grafting technique and rootstock age in a column, the values followed by different letters are significantly different at p<0.05. ­ not evaluated. grafting techniques graft success rate (%) graft recovery time (day) 36 month old rootstock 12 month old rootstock 36 month old rootstock 12 month old rootstock simple whip 26.94 ± 2.83 b 32.77 ± 3.11 b 29.33 ± 0.56 a 21.94 ± 0.69 a top cleft 28.88 ± 3.34 b 43.33 ± 5.99 a 29.33 ± 0.48 a 22.05 ± 0.69 a chip budding 32.22 ± 3.66 a ­ 27.83 ± 0.77 a ­ side cleft 23.33 ± 2.80 c ­ 29.55 ± 0.86 a ­ f 4.55 12.44 1.38 0.01 p 0.006 <0.001 0.258 0.906 dao et al. ‐ vegetative propagation of garcinia kola by grafting 49 the highest graft success rate (32.22±3.66%) was obtained with the chip budding technique on rootstocks aged 36 months. while on 12­month­old rootstocks, the highest graft success rate (43.33±5.99%) was recorded with the simple whip technique. concerning the average graft recovery time, it was between 27 and 29 days with grafts carried out on rootstocks aged 36 months, respectively, for the chip budding technique and the other techniques. while for plants aged 12 months, the average recovery time is 21.94±0.69 days for the simple whip technique and 22.05±0.69 days for the top cleft grafting technique. however, no significant difference is observed between these two average recovery times. the chip budding and top cleft grafting techniques are more favorable in rootstocks aged 36 and 12 months, respectively. figure 1 shows an overview of the recovery of grafted plants with the different grafting techniques applied. effect of graft unwrapping time on the success of garcinia kola grafting the results presented in table 3 show that only the graft success rate was significantly influenced (p < 0.001) by the graft unwrapping time. the highest percentages of graft success (38.75±2.71% and 55.83±5.42%) were obtained when the grafts were unwrapped at 29 dag on 36­ and 12­month­old rootstocks, respectively. while the lowest graft success rate (16.66±1.33% and 24.16±3.57%) was recorded when the grafts were unwrapped 15 days after grafting on 36­ and 12­month­old rootstocks, respectively. the average recovery time for rootstocks aged 36 months is between 28 and 29 days, while that of rootstocks aged 12 months is between 21 and 22 days. the graft unwrapping time of 29 days is favorable to the success of grafting garcinia kola in both types of rootstocks. combined effect of seasonality, grafting technique and graft unwrapping time on garcinia kola grafting success the interaction of season, grafting technique, and graft unwrapping time significantly influenced the graft success rate regardless of the age of the rootstock (table 4). on the other hand, no significant difference was observed in terms of graft recovery time. for grafts carried out in the month of june on rootstocks aged 36 months, the highest graft success rates (43.33±8.81% and 43.33±3.33%) were obtained with the chip budding and side cleft techniques, respectively, and were unwrapped at 29 days after grafting. concerning the grafts carried out in the month of june on rootstocks aged 12 months, the best graft success rate (83.33±3.33%) was recorded with the top cleft technique when unwrapped at 29 days after grafting. while the lowest graft success rate (23.33±3.33%) was obtained with the simple whip technique when the grafts were unwrapped 15 days after grafting. the graft recovery time varies from 24 to 30 days and 19 to 22 days after grafting with grafts performed in june on 36­month­old rootstocks and 12­month­old rootstocks, fig. 1 ­ successful grafts from different grafting techniques: (a) chip budding ; (b ) simple whip ; (c) top cleft ; (d) side cleft. table 3 ­ comparison of average values of grafting parameters according to graft unwrapping time and rootstock age in a column, the values followed by different letters are significantly different at p<0.05. graft unwrapping time (day) graft success rate (%) graft recovery time (day) 36 month old rootstock 12 month old rootstock 36 month old rootstock 12 month old rootstock 15 16.66 ± 1.33 c 24.16 ± 3.57 c 29.66 ± 0.58 a 22.41 ± 1.05 a 22 28.12 ± 2.01 b 34.16 ± 4.83 b 28.58 ± 0.47 a 21.58 ± 0.91 a 29 38.75 ± 2.71 a 55.83 ± 5.42 a 28.79 ± 0.71 a 22.00 ± 0.62 a f 53.70 39.03 0.96 0.26 p <0.001 <0.001 0.386 0.767 50 adv. hort. sci., 2025 39(1): 45­54 respectively. while for grafts performed in august, the graft recovery time varies from 28 to 32 days and 20 to 24 days after grafting for grafts performed on 36­month and 12­month rootstocks, respectively, 36­ month­old, regardless of the grafting technique and graft unwrapping time. grafted plants survival rate the survival rate was not significantly influenced by the grafting technique (fig. 2). the grafting techniques tested on both types of rootstocks showed a survival rate of 100% at 12 months after grafting. the figure 3 presented a grafted plant of garcinia kola. table 4 ­ comparison of mean values of grafting parameters depending on the interaction of season, grafting technique, graft unwrapping time, and rootstock age in a column, the values followed by different letters are significantly different at p<0.05. ­ not evaluated. grafting period grafting technique graft unwrapping time (day) graft success rate (%) graft recovery time (day) 36 month old rootstock 12 month old rootstock 36 month old rootstock 12 month old rootstock june simple whip 15 15.00 ± 2.88 d 23.33 ± 3.33 e 28.00 ± 1.15 a 20.66 ± 2.40 a 22 16.66 ± 4.40 d 33.33 ± 3.33 d 28.00 ± 1.15 a 20.66 ± 2.40 a 29 28.33 ± 4.40 c 50.00 ± 5.77 b 28.66 ± 0.66 a 21.00 ± 1.52 a top cleft 15 25.00 ± 2.88 c 40.00 ± 5.77 c 30.00 ± 1.15 a 19.33 ± 1.33 a 22 31.66 ± 6.00 c 56.66 ± 8.81 b 29.33 ± 1.76 a 20.00 ± 2.30 a 29 4.00 ± 5.77 b 83.33 ± 3.33 a 28.00 ± 1.15 a 22.66 ± 0.66 a chip budding 15 23.33±3.33 c ­ 26.00 ± 1.15 a ­ 22 40.00 ± 5.77 b ­ 28.00 ± 1.15 a ­ 29 43.33 ± 8.81 a ­ 24.33 ± 2.02 a ­ side cleft 15 13.33 ± 3.33 d ­ 28.66 ± 0.66 a ­ 22 30.00 ± 5.77 c ­ 28.00 ± 1.15 a ­ 29 43.33 ± 3.33 a ­ 29.33 ± 1.76 a ­ f 12.87 1.18 0.852 0.34 p 0.003 0.013 0.543 0.713 august simple whip 15 10.33 ± 3.33 e 20.00 ± 5.77 d 32.66 ± 1.76 a 23.66 ± 1.45 a 22 23.33 ± 3.33 c 26.66 ± 3.33 c 28.66 ± 0.66 a 22.00 ± 1.15 a 29 30.00 ± 5.77 b 36.00 ± 5.77 b 30.00 ± 1.15 a 23.66 ± 1.45 a top cleft 15 13.33 ± 3.33 d 13.33 ± 3.33 e 30.00 ± 1.15 a 24.00 ± 1.15 a 22 20.00 ± 5.77 c 20.00 ± 5.77 c 28.00 ± 1.15 a 23.66 ± 1.45 a 29 23.33 ± 3.33 c 46.66 ± 6.66 a 30.66 ± 0.66 a 20.66 ± 0.88 a chip budding 15 20.00 ± 5.77 c ­ 32.00 ± 2.30 a ­ 22 26.66 ± 3.33 c ­ 28.66 ± 0.66 a ­ 29 36.66 ± 3.33 a ­ 28.00 ± 1.15 a ­ side cleft 15 13.33 ± 3.33 f ­ 30.00 ± 1.15 a ­ 22 16.66 ± 3.33 e ­ 30.00 ± 3.05 a ­ 29 26.66 ± 3.33 ­ 31.33 ± 4.37 a ­ f 1.10 0.13 0.51 2.95 p 0.038 <0.001 0.788 0.116 fig. 2 ­ survival rate of garcinia kola grafted plants with differents grafting techniques on 36 and 12 month old rootstoks. dao et al. ‐ vegetative propagation of garcinia kola by grafting 51 4. discussion and conclusions in this study, the higher graft success observed in june with 12­ and 36­month­old rootsocks and their earliness in graft recovery time, unlike grafts performed in august, would be due to the optimum humidity in the month of june (karadeniz, 2005). indeed, june was characterized by an abundance of rain (361 mm) with an average temperature of 26°c. these conditions promote the well growth of plants and grafts success. this finding is in agreement with the results of chipojola et al. (2013), who obtained similar results for anarcadium occidentale in malawi. while august is characterized by the short, dry season. the low rainfall and high temperatures during the august season slow down the physiological activity of the rootstock and favor the scions drying. these results are in agreement with those of djaha et al. (2012), who observed a low success of cashew grafting in november in korhogo, in the north of côte d’ivoire. according to these authors, the dry season would not favor the vegetative phase of the plant because of the harmattan and the scarcity of rains. similar observations were made by soloviev et al. (2004), who obtained low success rates on the grafting of african plum (sclerocarya birrea) in the cold dry season and in the middle of the hot dry season. ondo et al. (2018) reported a low success rate in the grafting of two clones (gt1 and pb 217) of hevea brasiliensis (h.b.k.) (muell. arg) during the dry season in gabon. negative effects of dry season on graft success are also reported in mango by sivudu et al. (2014) and in vitex payos (lour.) merr. by bala et al. (2017). the best graft success rate recorded with the chip budding method on 36­month­old rootstocks, unlike the other grafting methods, could be explained, on the one hand, by the presence of latex at the level of the scion and of the rootstock. indeed, notches made on the rootstock during the operation do not affect the wood. it could promote the welding between the bud scion and the rootstock in the chip budding method. concerning the other grafting methods, the contact zone between the rootstock and the scion is largely between the lignified parts. this makes it difficult to weld between the rootstock and the scion. according to takoutsing et al. (2014), the success of the graft ing process depends on the level of l ignification of the rootstock and the graft ing method. these authors reported a 100% graft success rate of garcinia lucida vesque using the top cleft method on six­month­old rootstocks. the graft unwrapping time at 29 days after grafting enhances graft success more than those unwrapped at 15 and 22 days, whatever the rootstock’s age. this means that this duration is sufficient for the establishment of the weld between the rootstock and the scion. indeed, the realization of the anatomical welding is an essential condition for the success of the grafting (scheidecker, 1961). in rubberwood (hevea brasil iensis), grafts were generally unwrapped 21 days after graft ing (udayakumara and seneviratne, 2005; ondo et al., 2018). the formation of new tissues by the rootstock and the scion at the level of the contact zone is done gradually over time. in this study, the average sprouting time differs according to the age of the rootstock, with 22 and 28 days for 12­ and 36­month­ old rootstocks, respectively, regardless of the graft unwrapping time. similar graft recovery time was reported by yao et al. (2019) in shea butter (vittelaria fig. 3 ­ garcinia kola grafted plant with the chip budding technique 12 months after grafting. adv. hort. sci., 2025 39(1): 45­54 52 allanblakia stuhlmannii, with grafting success over 70% (munjunga et al., 2013). the survival rate (100%) of all the grafted plants after 12 months indicates that graft ing is an alternative for the production of quality plants for garcinia kola. however, monitoring must be extended until flowering to confirm the long­term success of this technique. this study has shown that it is possible to propagate elite trees of garcinia kola by using the four grafting methods tested. chip budding and side cleft were better on 36­month­old rootstocks, while the top cleft method is appropriate on 12­month­old rootstocks. the determination of adequate season (in rainy season) and graft unwrapping time (29 days after graft ing) for the graft ing success is very important to promote the utilization of this strategy by farmers for the species domestication and cultivation. the present study is the first in côte d’ivoire to systematically explore grafting techniques for the propagation and conservation of this vulnerable species with significant socio­economic value. results of this study can be exploited by forest management structures to produce high­quality plants for agroforestry programs with several objectives, such as the mitigation of the effects of climate change and the improvement of farmers’ incomes. acknowledgements this study was funded by the international foundation for science (ifs), project ifs garcinia. (d/5274­2). references agwu o.p., bakayoko a., jimoh s.o., stefan p., 2018 ­ farmers’ perceptions on cultivation and the impacts of climate change on goods and services provided by garcinia kola in nigeria. ­ ecological processes, 7(1): 10. akinnifesi f.k., mng’omba a.s., sileshi g., chilanga tg., mhango j., ajayi c.o., chakeredza s., betserai i.n., gondwe f.m.t., ­ 2009 ­ propagule type affects growth and fruiting of uapaca kirkiana, a priority indigenous fruit tree of southern africa. ­ hortsci., 44(6): 1662­1667. akter j., rahim m.a., haque t., hossain m.m., 2016 ­ effect of scion defoliation period and methods of paradoxa), with a graft recovery time between 21 and 30 days. the reestablishing of the connection between the rootstock and the scion is manifested by the budding of dormant buds on the scion. the lower graft success rate observed with grafted plants unwrapped at 15 days after grafting can be explained by an incomplete welding of the two parts. indeed, graft unwrapping leads to the separation of the rootstock and the scion. this opening of the contact zone causes it to dry out on the one hand and the proliferation of several pathogens, such as fungi, on the other hand, which accelerates the death of the graft. the combined effect of season, method, and unwrapping time significantly influenced the graft success rate. the best graft success rate was recorded on grafts made in june on 36­month­old rootstock (43.33%) with chip budding and side cleft grafting methods with grafts unwrapped at 29 days after grafting and 83.33% for grafts made on 12­ month­old rootstock with the top cleft grafting method with grafts unwrapped at 29 days after graft ing. this result would mean that this combination of season, graft ing method, and unwrapping time depending on the age of the rootstock optimizes the success of the grafting. these results could be explained by favorable climatic conditions in june. indeed, the month of june is in the great rainy season. in addition, chip budding and side cleft grafting methods were adapted to older rootstock because this method does not affect the wood (yakubu et al., 2014). while the top cleft method promotes well­grafting to unlignified stems, such as the 12­month­old rootstock apical part. in this study, young g. kola plants, whatever their age, have a good ability for grafting when the grafts are unwrapped at 29 days after grafting. the short graft recovery time observed with 12­month­old rootstocks is in agreement with several authors who reported that younger rootstocks presented good ability for grafting in papaya (nguyen and yen, 2018) and in mango (mahunu et al., 2009; upadhya et al., 2014). other trees, such as garcinia xanthochymus (hook. f.) [syn. g. tinctoria (wight)] (krishnamoorthy et al., 2006), garcinia lucida vesque (takoutsing et al., 2014), and vittelaria paradoxa (sanou et al., 2004), were successfully grafted. the best result obtained by using the top cleft method in this study suggests that this grafting method promotes well­ grafting success. several species were successfully grafted by the top cleft method, such as allanblakia parviflora in ghana (ofori et al . , 2008) and dao et al. ‐ vegetative propagation of garcinia kola by grafting 53 tinctoria (wight)]. ­ j. spices aromatic crops, 15(1): 63­ 64. leakey r.r.b., akinnifesi k., 2008 ­ towards a domestication strategy for indigenous fruit trees in the tropics, 28-49. in: akinnifesi f.k., r.r.b. leakey, o.c. ajaui, g. sileshi, z. tchoundjeu, p. matakala, and f.r. kwesiga (eds.) indigenous fruit trees in the tropics: domestication, uti l ization and commercialization. cab international, oxfor, uk, pp. 427. leakey r.r.b., schreckenberg k., tchoundjeu z., 2017 ­ the participatory domestication of west african indigenous fruits. ­ the international forestry review, commonwealth forestry association, united kingdom, pp. 338­347. leakey r.r.b., simons a.j., 2000 ­ when does vegetative propagation provide a viable alternative to propagation by seed in forestry and agroforestry in the tropics and subtropics, pp. 67­81. ­ in: wolf h., and j. albrecht (eds.) problem of forestry in tropical and subtropical countries: the procurement of forestry seed‐the example of kenya, ulmer verlag, germany. love k., paull r., cho a., kawabata a., 2017 ­ tropical fruit tree propagation guide. ­ college of tropical agriculture and human resources, pp. 1­10. mahunu g.k., adjei p.y., asante, a.k., 2009 ­ comparative studies on ages of rootstock seedlings and graft methods in cashew (anacardium occidentale l.). ­ ghana j. horticulture, 7: 11­17. maňourová a., leuner o., tchoundjeu z., damme p.v., verner v., přibyl o., lojka b., 2019 ­ medicinal potential utilization and domestication status of bitter kola (garcinia kola heckel) in west and central africa. ­ forests, 10(2): 124. munjunga m., kariuki w., njoroge j.b.m., ofori d., jamnadass r., 2013 ­ effect of rootstock type, scion source and graft ing methods on the healing of allanblackia stuhlmannii grafts under two nursery conditions. ­ afr. j. hortic. sci., 7: 1­10. nguyen v., yen c., 2018 ­ rootstock age and grafting season affect graft success and plant growth of papaya (carica papaya l.) in greenhouse. ­ chilean j. agric. res., 78(1): 59­67. ofori d.a., peprah t., henneh s., von berg j.b., tchoundjeu z., jamnadass r., simons a.j., 2008 ­ utility of grafting in tree domestication with reference to allanblackia parviflora a. chev. ­ ghana j. for., 24: 42­48. ondo o.p., kebangoye h­s., medza m.s.d., pamphile n.n., kevers c., dommes j., 2018 ­ facteurs permettant d’améliorer la réussite au greffage des clones gt1 et pb217 d’hevea brasiliensis (hbk)(muell. arg) dans les conditions climatiques du nord gabon. ­ j. animal plant sci., 35(3): 5749­5762. sanou h., kambou s., teklehaimanot z., dembele m., yossi h., sina s., djingdia l., bouvet j.m., 2004 ­ grafting on success and survivability in mango. ­ progressive agric., 27(3): 242­248. asaah e.k., tchoundjeu z, van damme p., 2012 ­ beyond vegetative propagation of indigenous fruit trees: case of dacryodes edulis (g. don) hj lam and allanblackia floribunda oliv. ­ afrika focus, 25(1): 61‐72. assogbadjo a.e., idohou r., chadare f.j., salako v.k., djagoun c.a.m.s., akouehou g., mbairamadji j., 2017 ­ diversity and prioritization of non timber forest products for economic valuation in benin (west africa). ­ african j. rural devel., 2(1): 105­115. bala p., gitehi g., vincent o., omondi s.f., albert l., okeyo m., 2017 ­ effect of seasonality, graft type and scion characteristics on propagation of vitex payos in the drylands of kenya. ­ j. hortic. for., 9(6): 49­58. chipojola f., weston m., mwase f., kwapata m.b., njoloma j.p., bokosi j.m., maliro m.f., 2013 ­ effect of tree age, scion source and grafting season on the grafting success of cashew nut (anacardium occidentale l.). ‐ afr. j. agric. res., 8(46): 5785­5790. codjia s., aoudji a., koura k., ganglo j.c., 2018 ­ systèmes agroforestiers à garcinia kola heckel au sud‐ est du bénin : distribution géographique, connaissances endogènes et retombées financières. ­ european sci. j., 14(12): 188­208. dao j.p., kouakou k.l., kouakou c., cherif m., ouedraogo m.h., koffi k.k., zoro bi i.a., 2020 ­ effect of leafy and leafless greenwood, softwood and hardwood cuttings success of garcinia kola (heckel). ­ agric. sci., 11: 897­911. djaha j­b.a., n’daadopo a.a., koffi e.e., ballo c.k., coulibaly m., 2012 ­ croissance et aptitude au greffage de deux génotypes d’anacardier (anacardium occidentale l.) élites utilisées comme porte‐greffe en côte d’ivoire. ­ int. j. biol. chem. sci., 6(4): 1453­1466. franzel s.c., jaenicke h., janssen w., 1996 ­ choosing the right trees: setting priorities for multipurpose tree improvement. isnar research, report no. 8. the hague. international service for national agricultural research, pp. 100. karadeniz t., 2005 ­ relationships between graft success and climatic values in walnut (juglans regia l.). ­ j. central european agric., 6(4): 631­634. kouakou k.l., dao j.p., kouassi k.i., beugré m.m., koné m., baudoin j.p., zoro bi i.a., 2016 ­ propagation of garcinia kola (heckel) by stem and root cuttings. ­ silva fennica, 50: 17. kouame n.m.t., ake c.b., mangara a. n’guessan k., 2016 ­ analyse de l’intérêt socio‐économique des graines de garcinia kola heckel (clusiaceae) dans la commune de koumassi (abidjan), côte d’ivoire. ­ int. j. biol. chem. sci., 10(6): 2587­2595. krishnamoorthy b., mathew p.a., rema j., divakaran m., jayarajan k., 2006 ­ soft wood grafting of garcinia xanthochymus (hook. f.) [syn. g. adv. hort. sci., 2025 39(1): 45­54 54 vegetative propagation of vitellaria paradoxa by grafting. ­ agrofor. syst., 60(1): 93­99. scheidecker d., 1961 ­ la greffe, ses conditions anatomiques, ses conséquences physiologiques et ses résultats génétiques éventuels. ­ année biologique, 37(3-4): 107­172. sivudu b.v., reddy m.l.n., baburatan p., dorajeerao a.v.d., 2014 ­ effect of structural conditions on veneer graft ing success and survival of mango grafts (mangifera indica cv. banganpalli). ­ plant archives, 14: 71­75. soloviev p., niang t., gaye a., 2004 ­ propagation par greffage du prunier d’afrique [sclerocarya birrea (a. rich) host.] au sénégal. ­ fruits. 59, 275­280. takoutsing b., tsobeng a., tchoundjeu z., degrande a., asaah e., 2014 ­ vegetative propagation of garcinia lucida vesque (clusiaceae) using leafy stem cuttings and grafting. ­ afrika focus, 27(3): 57­71. udayakumara e., seneviratne p., 2005 ­ position of the bud on the bud stick on success of bud graft and growth hevea brasiliensis (muel arg). ­ j. agric. sci., 1(1): 31­40. upadhya b., baral d.b., gautam d.m., shrestha s.m., 2014 ­ influence of rootstock age and pre‐defoliation of scion on the success of epicotyl grafting of mango. ­ int. j. res., 1(7): 172­182. yakubu f.b., adejoh o.p., ogunade j.o., igboanugo a.b.i., 2014 ­ vegetative propagation of garcinia kola (heckel). ­ world journal of agricultural sciences. 10: 85­90. yakubu f.b., akin$yele a.o., 2021 ­ scion source effects on the growth performance of grafted seedlings of garcinia kola heckel. ­ singapore j. sci. res., 11: 10­16. yao s.d.m., alui k.a., kouame n.m.t., ble p.a., kone b., diarrassouba n., 2019 ­ réussir le « greffage en fente simple » et le « greffage de cote dans l’aubier » du karité. ‐ j. appl. biosci., 137: 13961­13972. untitled 102 characteristics of recent released clones selected by disaaa-a in tuscan coast line premultiplied by tos.co.vit g. ferroni*, g. scalabrelli*, g. di collalto** * department of agriculture, food and environment, university of pisa, via del borghetto, 80, 56124 pisa, giuseppe.ferroni@unipi.it ** department of crop, soil and environmental science, university of florence, viale delle idee, 30, 50019 sesto fiorentino (fi). at the conclusion of the research project “enhancement and renewal of viticulture in the province of grosseto”, “sangiovese selection in the area of morellino di scansano” and “enhancement of candidate clones of tuscan grapevines” have been homologated in 2007 four clones of ansonica and five clones of vermentino and in 2011 three clones of sangiovese (scalabrelli and di collalto, 1999; scalabrelli et al., 2004, scalabrelli et al., 2005, scalabrelli et al, 2012). main traits of ansonica clones i-cosa 1. large conical bunches of medium firmness, elliptical-short berry medium size, and greenish-yellow, slightly amber. bud fertility below the average (1.18); bud break medium earliness, slightly more precocious ripening. mediumhigh vigour. pale straw yellow wine, with notes of tropical and fruity floral, light citrus, hazelnuts and honey, sapid and structured, the most appreciated by the panel. quality clone requires control of production. suitable to produce structured white wine, even with aging. i-settefinestre 1. conical bunches, sometimes winged, less than average size, medium sparse and separated, berry green-yellow, amber, slightly elliptical-short, slightly coarser than cosa1 clone. medium bud fertility (1.34). time of bud break and ripening are slightly later than the average of the population. higher vigour of clone cosa 1, production lower than the average. pale straw yellow wine, with notes of almond, slightly fruity and floral, with a good structure, flavor, freshness and agreeableness. clone less productive, suggested for the production of assembled wines, especially with the previous clone. i-settefinestre 2. bunches large, conical, mildly sparse, elliptical-short, medium size, green-yellow, amber. bud fertility (1.46) and yield above the average. average earliness of bud break and ripening. medium-high vigour; pale straw yellow wine tropical fruity, note of floral and slightly spicy, soft nutty and honeyed notes, medium-bodied, balanced and pleasant. clone productive of good quality, able to bring aromatic complexity, suitable to produce also varietal wines. i-settefinestre 3. bunches of medium size, broad conical shaped, of medium size, largest of the clone settefinestre 1 and smaller of clone cosa 1; berry elliptical-short, medium size , yellow, amber. production and fertility of the buds below average (1.20). the earliness of bud break and time of ripening are like the average of the population; medium-high vigour. pale straw yellow wine, with mediterranean spiced and nutty, with hints of fruity and honey, balanced structure and good pleasantness. clone of intermediate level of production and quality, suitable to produce white wines ready to drink and for assemblage. main traits of vermentino clones i-sirena 1. time of bud break like the population mean, average vigour and low productivity, mean bud fertility (1.26), medium-small cluster, short conic shape and medium tightness, medium berry size, spherical shape and, firm skin. earlier ripening compared to the population average (7-10 days), sugar accumulation influenced by territorial environment. straw yellow wine, good structure, floral and fruity, palatable, sapid, with slightly bitter final, suitable for short aging. i-marem 1. bud break timing similar to the population mean. earlier ripening compared to the population average (7 days). medium high plant vigour and fertility (1.40) yield lower than the population average. loose bunch with a wing more evident, the berry is slightly smaller than the average, the skin is firm. it exhibits low sensitivity to the bunch rot. sugar accumulation higher than the reference population. straw yellow wine with notes of ripe fruit, citrus and spicy mediterranean (very pronounced). it maintains high qualities of freshness, good body, suitable for the production of wines with a good structure which can be subjected to a medium aging period. adv. hort. sci., 2013 27(3): 102-103 103 imarem 3. bud break contemporary to the population. earlier ripening than the population average (7days). medium-high vigour and bud fertility (1.46) lower yield to the average. bunch medium-small of truncated cylindrical shape, with a short wing, medium loosen. berry shortmedium ellipsoidal, good tolerance against botrytis. accumulation of sugars compared to the reference population. wine with aromatic complexity, good freshness, texture and excellent balance, suitable for producing structured wines and wines ready to drink. i-sileno 1. bud break slightly earlier than the average of the population. later ripening as compared to the average (about 10 days). low vigorous and very productive, medium-low bud fertility (1.31). very large, composed and tight cluster, large berries. sugar accumulation potential slightly below the average of the population. straw yellow wine, perfume with notes of fresh fruitiness and marked by spicy mediterranean, fresh, light structure, suitable for the grapes and the production of wines ready to drink and sparkling wines. i-sileno 3. bud break slightly earlier than the average of the population. later ripening (about 10 days after the avg.). low vigour and fertility (1.25). very large, composed and tight cluster, large berry, higher yield. accumulation potential of sugars is below the average of the population. straw yellow wine, aroma with notes of fresh fruitiness and marked by spicy mediterranean, fresh, light structure, suitable for the assemblage with other clones for the production of wines ready to drink and sparkling wines. main traits of sangiovese clones (selected in the docg area “morellino di scansano”). i-chi 8. production is on the average, plant vigour slightly below the average, bud fertility above the average. the bunch is medium-small, conical-winged and tightness on the average. berry of spherical shape, uniform black blue color, waxy and thick skin, very rich in polyphenols and anthocyanins. the wine has an intense ruby red color, with intense fruity notes with evidence of red fruits and floral notes, well-structured and balanced suitable to aging. i-chi 10. production and vigour on the average, bunches short, conical, rather compact. berry black blue uniform, ellipsoidal, skin resistant, thick and waxy. it ripens on ii-iii decade of september. ruby red wine with floral notes, spicy and fruity, with a good structure, balanced, suitable to aging. clone suitable to the assemblage with other clones. i-chi 13. bunch of size below the average, conical shape moderate compactness, sometimes winged. berry mediumsmall size, ellipsoidal shape, blue black color, waxy and thick skin, rich in color. ripening time in the second half of september. intense ruby red wine with distinct notes of red fruit, good structure and balance, suitable to the production of assembled red wines to be aged. these new clones of ansonica, vermentino and sangiovese, possess different productive, qualitative and sensorial characteristics, some of which are complementary, therefore, they can respond to several growing and oenological requirements, allowing the establishment of polyclonal vineyards to achieve the wine target using the best combinations. references scalabrelli g., di collalto g., 1999 rinnovamento e valorizzazione della viticoltura della provincia di grosseto. provincia di grosseto. arsia. regione toscana, pp. 5-24. scalabrelli g., ferroni g., borgo m., porro d., di collalto g., 2005 la selezione clonale del “vermentino” vitigno bianco di grande interesse. l’informatore agrario, 35: 67-74. scalabrelli g., ferroni, f., d’onofrio c., borgo m., porro d., stefanini m., 2012 la selezione clonale del vitigno ansonica in toscana. italus hortus, 3-5: 451-455. scalabrelli g., loreti f., ferroni g., 2004 aspetti agronomici e ampelografici della selezione clonale di sangiovese nelle zone doc val di cornia e morellino di scansano. atti convegno “miglioramento qualitativo delle produzioni vitivinicole e del materiale di produzione, quaderno arsia, 1: 27-46. untitled 107 harmonization and validation of diagnostic protocols for the detection of grapevine viruses covered by phytosanitary rules f. faggioli1, f. anaclerio2, e. angelini3, m. g. antonelli1, n. bertazzon3, g. bianchi4, p. bianchedi5, p.a. bianco6, s. botti7, p. bragagna5, m. cardoni7, p. casati6, r. credi8, e. de luca2, g. durante9, c. gianinazzi9, g. gambino10, v. gualandri5, d. luison1, a. luvisi11, u. malossini5, f. mannini10, p. saldarelli12, f. terlizzi8, e. triolo11, n. trisciuzzi13, m. barba1 1 consiglio per la ricerca e sperimentazione in agricoltura, centro di ricerca per la patologia vegetale, via c.g. bertero, 22, 00156 rome, italy. francesco.faggioli@entecra.it 2 vivai cooperativi rauscedo, rauscedo, italy. 3 cra, centro di ricerca per la viticoltura, conegliano veneto, italy. 4 ersa, friuli venezia giulia, pozzuolo del friuli, italy. 5 fondazione edmund mach, istituto agrario san michele all’adige, italy. 6 disa, dipartimento di scienze agrarie e ambientali, produzione, territorio, agroenergia, università degli studi di milano, italy. 7 centro attività vivaistiche, tebano, italy. 8 dipartimento di scienze e tecnologie agroambientali, patologia vegetale, alma mater studiorum, università di bologna, italy. 9 ipad lab, lodi, italy. 10 cnr, istituto di virologia vegetale, uos di grugliasco, italy. 11 department of agriculture, food and environment, università di pisa, italy. 12 cnr, istituto di virologia vegetale, uos di bari, italy. 13 crsa, basile caramia, locorotondo, italy. the italian ministry of agriculture funded in 2009 the “arnadia” project, aimed at producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest and, among them, grapevine viruses. in this framework, the “working group arnadia – grapevine viruses (wg)”, composed of eight universities and research bodies, three accredited private laboratories, one plant health service and one association of grapevine nurseries, was established. the aim of the wg was to produce referenced and validated serological and molecular protocols allowing for the harmonization of diagnosis of eight grapevine viruses: grapevine leafroll-associated virus 1, 2, 3, (glrav 1, 2, 3) grapevine virus a (gva), grapevine virus b (gvb), arabis mosaic virus (armv), grapevine fanleaf virus (gflv) and grapevine fleck virus (gfkv). the validation of a protocol consists of the evaluation of specific parameters designed to determine their suitability to identify the presence of a specific “target”. the parameters that influence the capability of the test to accurately predict the sample’s infection status are the diagnostic sensitivity (ability of the utilized method to detect the presence of the pathogen in the samples truly infected by the pathogen in question true positive) and diagnostic specificity (ability of the utilized method not to detect the presence of the pathogen in samples not infected by the pathogen in question true negative). other parameters that must be considered and which determine the efficiency of a protocol are the analytical sensitivity (the smallest amount of infectious entities that can be identified by the diagnostic method), repeatability or accordance (degree of conformity of the results obtained in replications of the process, made at short time intervals, using the same reference sample and in the same working conditions i.e. equipment, operator, laboratory) and reproducibility or concordance (degree of conformity of the results obtained using the same method with the same reference samples in different laboratories). the latter parameter was defined in collaboration with five laboratories of the regional phytosanitary services. specifically, 122 grapevine samples (varieties, rootstocks and “pools” of five plants, of which only one infected) were analyzed by elisa, using 25 antisera from three commercial companies (agritest, bioreba, sediag) and multiplex rtpcr protocols. for elisa, the tests were conducted carefully following instructions provided by the companies; multiplex rt-pcr was performed using the protocol described by gambino and gribaudo (2006). the tests were performed in 18 laboratories using the same samples (analyzed in blind conditions) and reagents. in each laboratory, results were obtained using the same threshold value calculated on the basis of the spectrophotometer readings for elisa and by analyzing the adv. hort. sci., 2013 27(3): 107-108 108 electrophoretic gels for the multiplex rt-pcr. processing of the obtained results (about 24,000 data points) led to definition of the validation parameters according to uni/en/iso 16140 and 17025 and eppo standards pm7/76 and pm7/98. as reported in table 1, elisa proved to be a highly effective technique, comparable to the molecular method, although the latter turned out, as expected, to be more efficient for some viruses and on some specific samples (rootstocks and “pool”). table 1 summary of validation parameters obtained by the elisa test for each virus and antiserum and comparison with those obtained with the molecular protocol virus diagnostic protocol sensitivity specificity accuracy analytical sensitivity repeatability reproducibility a rmv multiplex 92 % 99 % 98 % 10-2 100% 100 % elisa – a/b/s 64/48/50% 85/95/96% 74/72/72% 10-2 100% 95% gflv multiplex 68 % 100% 90 % 10-3 100% 76% elisa – a/b/s 75/82/77% 96/92/92% 80/84/81% 10-2 100% 90% gfkv multiplex 95% 95% 95% 10-2 100% 95% elisa – a/b/s 90/90/30% 100% 92/92/46% 10-1 98% 88% gva multiplex 96 % 99 % 98 % 10-2 100% 94 % elisa – a/b/s 77/45/87% 100/100/96% 83/58/89% 10-1 98% 82% gvb multiplex 100% 100% 100% 10-2 100% 100% elisa – a/b/s 86/nt/nt% 100% 92% 100 (2-2) 100% 85% glrav 1 multiplex 74 % 100 % 94 % 10-2 100% 70 % elisa – a/b/s 89/94/96% 100% 93/96/98% 10-2 100% 92% glrav 2 multiplex 84% 98% 85% 10-2 95% 83% elisa – a/b/s 86/67/87% 100% 93/96/98% 100 (2-2) 93% 84% glrav 3 multiplex 100 % 93 % 95 % 10-3 100% 100 % elisa – a/b/s 81/90/97% 100% 84/92/97% 10-3 100% 94% a= agritest; b= bioreba; s= sediag. in conclusion, harmonized and validated reference diagnostic protocols for grapevine viruses subjected to phytosanitary rules are, for the first time, available. the efficiency and robustness of the protocols have been proven using a large number of samples in a variety of laboratories. on this basis, both serological and molecular protocols resulted valid, and their use could be as a function of different specific applications. acknowledgements this study was funded by the mipaaf project “aron-arnadia” (2009 -11). references gambino g., gribaudo i., 2006 simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with co-amplification of a plant rna as internal control. phytopathology, 96(11): 1223-1229. 133 1. introduction plants are naturally exposed to ultraviolet (uv) rays mostly at uv-a range (wave length, 320-400 nm), especially on sunny days in summer. in contrast, the level of harmful uv-c (wave length >220 nm) and uv-b (wave length, 280-320 nm) reaching the ground surface which potentially damages living organisms including plants could be effectively blocked and minimized by the presence of ozone layer above the stratosphere (staehelin et al., 2001). however, especially in the southern hemisphere, the area of seasonal depletion of the ozone layer often expands outside the antarctic region, occasionally allowing temporal increases in solar uv-b and relatively long-wave range of uv-c reaching the earth’s surface (staehelin et al., 2001). therefore, from the global point of view, studies on the plant-damaging impact of uv rays have key importance to both biologists and environmental researchers. in general, irradiation with a high dose uv-c results in induction of programmed cell death in living plants. in seedlings and protoplasts of arabidopsis thaliana, a dose of uv-c around 10-50 kj m-2 induces an oligonucleosomal dna fragmentation which is reminiscent of the apoptotic dna laddering often described in mammalian cells (danon and gallois, 1998). in addition, uv-c-dependent cell death development in arabidopsis involves caspase-like activity which is also similar to the events in animal apoptosis (danon et al., 2004). on the other hand, a pulse or low dose of uv-c radiation is frequently used for direct removal of germs from fresh produce (stevens et al., 1998). for instance, exposure of fruit tissue slices of zucchini squash to low dose uv-c impact of uv irradiation in leaves, fruits and suspension-cultured cells of micro-tom, tomato t. hiramatsu,1 l.a. terry,2 t. kadono,1 t. kawano1,3,4 * 1 laboratory of chemical biology and bioengineering, faculty and graduate school of environmental engineering, the university of kitakyushu, kitakyushu, japan. 2 plant science laboratory, cranfield university, bedfordshire cranfield, mk43 0al, united kingdom. 3 kitakyushu research center (linv@kitakyushu), kitakyushu, japan. 4 université paris diderot, sorbonne paris cité, paris 7 interdisciplinary energy research institute (pieri), paris, france. key words: ethylene, pathogenesis, redox, ripeness, salicylic acid, solanum lycopersicum, ultraviolet. abbreviations: acc= 1-amincocyclopropane-1-carboxylic acid; acs1a= 1-amincocyclopropane-1-carboxylic acid synthase; apx= cytosolic ascorbate peroxidise; cia= chloroform and isoamyl alcohol; dnase= deoxyribonuclease; pal= phenylalanine ammonia-lyase; pr= pathogenesis-related; ros= reactive oxygen species; rt-pcr= reverse transcription polymerase chain reaction; sa= salicylic acid; uv, ultraviolet. abstract: the present study aims to understand both positive and negative impacts of ultraviolet (uv) rays in living dwarf tomato plants (solanum lycopersicum l. cv. micro-tom). this paper examines the impact of uv-c (254 nm) and uv-a (365 nm) on induction of cell death and expression patterns of pathogenesis-related (pr), stress-related and redox-related genes, namely, of 1-amincocyclopropane-1-carboxylic acid synthase (acs1a), cytosolic ascorbate peroxidise (apx), phenylalanine ammonia-lyase (pal), and pathogenesis-related genes (pr1 and pr-p2), in leaves, fruits (both green and red), and suspension-cultured cells of micro-tom. effects of short exposure to uv-c, but not to uv-a, on induction of cell death (in cell suspension) and development of lesions accompanied by ion leakage (in the leaves) were observed while no morphological change was observed in the uv-treated green and red fruits. uv-dependent induction of pr genes (pr1 and pr-p2) in these samples suggested that uvs can be used for plant defense activation. in addition, expression of acs1a was shown to be negatively and positively regulated by uv-c and uv-a, respectively. thus uv-dependent postharvest controls of fruit maturity and shelf-life are likely applicable (i.e. retardation and/or acceleration of maturation). adv. hort. sci., 2014 28(3): 133-140 (*) corresponding author: kawanotom@env.kitakyu-u.ac.jp received for publication 25 june 2014 accepted for publication 1 august 2014 134 reportedly lowers the microbial activity and prevents deterioration of fruit quality during subsequent storage at low temperatures, while a burst in respiration rate in the treated tissues is induced (erkan et al., 2001). in addition to direct removal of pathogenic microbes from the plant surface by uv, researchers have reported attempts to use uv radiation for so-called ‘plant hormesis’ by which the susceptibility of plants to pathogens could be minimized and shelf-lives of fresh produce could be extended (stevens et al., 1996, 1998). to date, various fruits and vegetables including sweet potatoes (stevens et al., 1990, 1999), apples (lu et al., 2007), peaches (stevens et al., 1998; lu et al., 2007), and tomatoes (liu et al., 1993) have been tested for uv-mediated disease resistance controls. in most of the cases, resistance against pathogens was induced by uv-c (stevens et al., 1999). it is likely that treatment with uv rays (including uvc) builds up the “immunity” against microbial infection in living plants, thus the uv hormesis effects described above may be, at least partially, attributed to stimulation of plants’ innate immunity. the innate immune system of plants against pathogenic microbes is known to be elicited in response to recognition of pathogen-derived molecules by plant cells, as reviewed elsewhere (dangl and jones, 2001; chisholm et al., 2006; yoshioka et al., 2008). recognition of such elicitors by the host cells’ transmembrane receptors (jones and dangl, 2006; altenbach and robatzek, 2007) or resistance (r) proteins (allen et al., 2004; chisholm et al., 2006; dodds et al., 2006) reportedly initiates the cellular signaling cascades which finally activate the defense mechanisms. micro-tom, a dwarf cultivar of tomato (solanum lycopersicum l.), originally produced for ornamental purposes, has been proposed as preferred plant material for molecular biological research, mainly because of its compact habitat and short life cycle (meissner et al., 1997; eyal and levy, 2002; marti et al., 2006). recently, abuqamar et al. (2009) suggested that micro-tom is a good model for studying the crosstalk between biotic and abiotic stress responses through regulation of gene expression. in the present study, cellular damage (viz. cell death increase in suspension culture and increase in ion leakage at tissue level) and preceding changes in the gene expression patterns, especially those of defence-related, redox-related, dna maintenance-related and fruit maturation-related genes, are shown to be induced by uv-a and uv-c in the leaves, fruits and suspension-cultured cells of micro-tom. the signalling mechanisms contributing to the regulation of uv responses are also discussed by analogy to plant immunity responses. furthermore, the discussion addresses uv-dependent extension of the storage life of tomato fruits through stimulation of plant immunity mechanism by post-harvest exposure to uvs. 2. materials and methods plant materials following the protocols described elsewhere (kadono et al., 2009), seeds of tomato (solanum lycopersicum l., cv. micro-tom) were allowed to germinate on a wet paper towel in a transparent plastic container placed in a light-cycleconditioned incubator (12 h light and 12 h dark at 23°c). immediatly after germination the resulting plantlets were re-planted in plastic pots filled with a standard soil mixture and watered daily in the light-cycle-controlled incubator for three months. from the adult plants, leaflets, green immature and red ripe fruits (minimum size, 15 mm in diameter) were harvested before and after irradiation with uv lights. preparation of cell suspension culture again, following protocols described elsewhere (kadono et al., 2009), cell suspension culture derived from micro-tom was prepared. briefly, the leaf slices were taken from a seedling of micro-tom grown in vitro and placed on a ms agar plate containing 2,4-dichlorophenoxyacetic acid (0.2 μg ml-1) to promote the formation of calli. suspension culture of cells was initiated by addition of the sliced calli into the ms liquid medium (ph 5.8). the cells suspended in 30 ml of media in 100 ml-conical flasks were kept on gyratory shakers (at 130 rpm) at 23°c in darkness, with occasional sub-cultures by innoculating the fresh media with 3 ml of confluent culture. after about six months of continuous propagation of the cells with constant sub-culturing (initially twice a month and later once a week), a stable cell line was obtained. for experimental purposes, the culture was pre-conditioned as follows. the confluent culture was used to inoculate the fresh ms liquid medium (3 ml culture to 30 ml medium) and pre-cultured for three days. then 6.5 ml of each pre-culture was transferred to 100 ml of fresh ms liquid medium (in a 500 ml conical flask) and further cultured for three days. the resultant three-day-old large scale culture (at log-phase) was harvested and used for the experiments. uv treatments intact and/or excised leaves (leaflets and leaf disks), green and red fruits, and suspension-cultured cells of microtom were irradiated with uv-c (254 nm) or uv-a (365 nm) using a handy uv trans-illuminator (sluv-4, as-one, osaka, japan). the intensities of uv-a and uv-c were monitored with uv meters (uvx-25, uvp inc., upland, ca; yk-34uv, lutoron electronic enterprise co., ltd., taipei, taiwan) and the intensity of uv-a and uv-c applied to the surfaces of plant materials were adjusted to 2.2 mw cm-2, therefore the doses of uv irradiation were controlled by altering the length of irradiation time. leaflets and fruits were exposed to uv rays immediately after harvest. to prevent water loss from the leafy samples during uv irradiation and incubation, leaflets and leaf discs were floated on ultrapure water in petri dishes. for treatment of the cultured cells, 1 ml of cell suspension was added to each well on 12-well microplates and used for irradiation with uv rays. irradiation time varied as indicated in the results section. measurement of ion leakage from leaf discs measurements of ion leakage were performed using the discs of leaflets floated on the ultrapure water. leaf discs were irradiated with uv rays for 30 min and further in135 cubated in darkness for up to 24 h. each single well on a 12-well microplate was filled with 5 ml of ultrapure water and used to float three leaf discs (diameter, 9 mm) freshly prepared from the leaflets. using a handheld conductivity meter (cd502a, custom, tokyo, japan), monitoring of the changes in conductivity in the bathing liquid (with 1 h intervals up to 10 h) were carried out following 24 h of post-uv incubation. an increase in conductivity reflects the leakage of ions from the uv-dependently damaged leaves. the extent of ion leakage was expressed as the ratio (percentage) of recorded conductivity to the maximal conductivity obtained after boiling the samples. evaluation of cell death analysis of induced cell death in suspension culture was carried out as described elsewhere (iwase et al., 2014). following uv-treatments, 200 μl-aliquots of cell suspension were sampled and transferred into 1.5 ml tubes and statically incubated in darkness (for 2 h unless indicated). then, 0.1 % (w/v) evans blue was added to the cell suspension and further incubated for an additional 1 h. following repeated washes with fresh media, counting of the stained cells was performed under microscopes (smz800 and labophoto, nikon, tokyo, japan; vhx-100, keyence, tokyo, japan) and the level of cell death was quantified. for statistical analysis, three to four different digital images of cells under the microscope (each covering 50-100 cells to be counted) were acquired and analyzed. rna isolation following uv irradiation isolation of rna followed by reverse-trancriptase polymerase chain reaction (rt-pcr) was carried out basically as described (kunihiro et al., 2011). leaflets and fruits were subjected to irradiation with uv rays for 30 min, and were sampled and frozen in liquid n 2 at 0, 1 and 10 h after irradiation. post-uv incubation was carried out in darkness. cell suspensions on 12-well microplates subjected to 15 min of irradiation with uv rays were further incubated for 1 h in the darkness. cells were then harvested by filtering through 40-μm pore nylon mesh and washed with fresh liquid culture medium. the obtained samples were frozen in liquid n 2 . these frozen samples were ground using a pestle and mortar and transferred to plastic tubes and extracted with the rna extraction buffer containing 100 mm tris-hcl (ph 8), 100 mm ethylenediaminetetraacetic acid, 100 mm licl, and 1% sodium dodecyl sulfate. then aliquots of 1:1 mixture of phenol and cia (chloroform:isoamyl alcohol at 24:1) were added and samples were subjected to centrifugation at 14,000 g for 10 min at 4°c. the upper layer collected in separate tubes were further subjected to extraction with aliquots of phenol and cia and centrifugation. the resultant upper layer was again collected in new tubes and mixed with 1/3 volume of 10 m licl and kept at -30°c for 2 h. following centrifugation, the resultant pellets were collected and re-suspended in 2 m licl and spin-collected. the pellet formed was dissolved in te buffer and added with 1/2 volume of both phenol and cia. the samples were subjected to 10 min of centrifugation and the upper layer was collected and mixed with the aliquot of cia, and further centrifuged. for precipitation of rna, the upper layer was collected and mixed with 1/10 volume of 3 m sodium acetate and 2.5 volume of absolute ethanol, and centrifuged. the pellet washed with 70% ethanol was spin-collected, dried and dissolved in diethylpyrocarbonate-treated water. rt-pcr prior to analysis with rt-pcr, genomic dna concomitantly present in the total rna preparations was eliminated with rnase-free cloned dnase i (takara bio inc., otsu, japan). first-strand cdna synthesis was performed using superscripttm iii reverse transcriptase (invitrogen corporation, ca). the reaction mixtures (20 μl/tube) contained total rna (2 μg), oligo-(dt) 20 (2.5 mm), and dntp mixture (2 mm). the tubes were heated to 65°c for 5 min, cooled and kept at 4°c for 1 min using program temp control system pc-320 (astec, fukuoka, japan). to the reaction mixture, 4 μl of 5x first-strand buffer, 1 μl of 0.1 m dithiothreitol, 1 μl of rnaseout recombinant rnase inhibitor and 1 μl of superscripttm iii reverse transcriptase (200 units μl-1) were added and incubated at 50°c for 60 min. the reaction was terminated by heating at 70°c for 15 min and cooling at 4°c for 15 min. the resultant cdna solution was used for pcr performed with 60 ng of first-strand cdna and takara extaqtm (takara bio inc., otsu, japan). for each sample, 30 cycles of pcr were performed with denaturing at 94°c for 1 min, annealing for 1 min and elongation at 72°c for 1 min. sequences of the primers used and anealing temperatures employed are listed in table 1. table 1 list of primers used for rt-pcr genes studied accession numbers * forward primers reverse primers tm (˚c) actin u60481 cacactgtccctatttacga gtaataacttgtccatcagg 51.3 acs1a u72389 gctttgggttagtttcagctc gttcatgaactcatgatccaatc 56.5 apx dq096286 gagtacctcaaggctgttgacaaatg gagcctcagcaragtcagcaaag 60.0 pr-p2 x58548 ggagagagttaacaagttgtgtg gagtagtattaaaagttagctcg 60.0 pr1 dq159948 cttctcatggtattagcc ccaccatccgttgttgc 50.3 pal m83314 gacacacaagttgaagcatcac cacatcttggttgtgttgctc 56.5 * accession numbers were of ncbi genbank. tm= melting temperature used for annealing. 136 3. results cellular damage on the uv-c-treated leaves, the damaging impact of uv-c at cellular level could be visualized as spotted lesions appeared (fig. 1). such visible damage was not observed on the uv-a-treated leaves. in addition to visible symptoms, uv-c irradiation resulted in a marked increase in ion leakage from leaf disc preparations (fig. 2). leakage of ions indicates that the cellular membrane is one of the targets of the damaging impact of uv-c irradiation. after irradiations with uv rays at 2.2 mw cm-2, suspension-cultured cells of micro-tom showed development of cell death depending on the type of uvs, exposure time and the length of post-exposure incubation (fig. 3). compared to uv-a, impact of uv-c irradiation was shown to be much more severe, requiring shorter irradiation time and post-irradiation incubation. uv-responsive gene expressions the genes examined were 1-amincocyclopropane1-carboxylic acid (acc) synthase gene (acs1a), ascorbate peroxidise gene (apx, cytosolic isoform), pathogenesis-related (pr) genes pr1 and pr-p2, and phenylalanine ammonia-lyase gene (pal) as possible targets of uv impact, and actin gene as non-inducible reference. fig. 1 development of uv-induced symptoms (lesions) on the leaves of micro-tom after the uv-irradiation. leaflets were irradiated with uv rays for 30 min and further incubated in darkness on ultrapure water. lesions on the leaves were observed under microscopes. typical images from three repeated experiments are shown. fig. 2 measurement of ion leakage from the uv-irradiated leaf discs of micro-tom. leaf discs were irradiated with uv rays for 30 min and further incubated in darkness on ultrapure water. the percentage of ion leakage was calculated as a ratio to conductivity after boiling of the leaf samples. each data point and error bar reflect the mean and s.d., respectively (n = 3). fig. 3 uv-induced cell death in suspension cultured cells. (a) effect of irradiation time on the uv-induced cell death. (b) progress of cell death after the uv-a-irradiation. (c) progress of cell death after the uv-c-irradiation. cell death was judged by evans blue staining under microscopes. each data point and error bar reflect the mean and s.d., respectively (n = 3). 137 in the cell suspension culture of micro-tom, expression of apx, pr-p2, pal, and actin were shown to be maintained at active level even prior to the irradiation with uv rays (fig. 4 a, dark control). in the leaf samples of micro-tom, acs1a, apx, pal, pr-1 and actin were shown to be expressed without uv irradiations (fig. 4 b). similarly, mature red fruit samples revealed high expressions of acs1a, apx, pr-1 and actin, prior to the irradiation with uv rays (fig. 5 b). in contrast, in the green fruit samples, only apx and actin were expressed in the dark control (fig. 5 a). among the genes tested, acs1a was the only gene uvdependently activated in the cell suspension culture. expression of acs1a was induced by both uv-a and uv-c (fig. 4 a). uv-a-dependent activations of pr1 in the green fruits and leaves, of acs1a in the green fruits and cell suspension culture, and of pal and pr-p2 in the green fruits were observed (fig. 4 and 5). uv-c-dependent activation of pr1 in green fruits and leaves, of pr-p2 in the leaves, of pal in green fruit tissue, and of acs1a in the green fruits and also in the cell suspension culture were observed (fig. 4 and 5). although pr1 was shown to be responsive to both uv-a and uv-c in green fruit tissue, the temporal profiles of induced gene expression largely differed. in green fruits, uv-a and uv-c were shown to be rapid and slow inducers of pr1 expression, respectively (fig 5). since, most of the genes examined were active in the dark control of red fruit samples, drastic activation of gene expression could not be observed, except for the case of enhancement of pr-p2 expression which was originally active in the dark control. apx was shown to be active in all materials even prior to uv irradiation, thus only the suppressive impacts of uv irradiations could be expected with this gene. suppression of apx expression was observed in the uv-c-treated red fruit samples at 10 h after irradiation. similarly, following irradiation with uv-c, expressions of pr-p2 and pal, which were constitutively active in the cell suspension, were eventually suppressed. expression of acs1a in the leaves and red fruits was shown to be suppressed by uv-a irradiation. expression of acs1a, pr-p2, and pr1, originally active in red fruits, were shown to be suppressed by uv-c treatments. 4. discussion and conclusions cell death or defense activation? a number of researchers have documented toxic impacts of uv rays in living plants including the damages to dna, inhibition of photosynthesis, generation of reactive oxygen species (ros) (roldán-arjona and ariza, 2009). on the other hand, there have been some attempts to develop the uv irradiation protocol for so-called ‘plant hormesis’ by which the susceptibility of plants to invading pathogen is minimized and thus shelf-life of fresh produces is likely extended (lu et al., 2007). for example, kunz et al. (2008) demonstrated that uv-c-induced dna damage in arabidopsis accompanies the doseand time-dependent development of resistance against hyaloperonospora parasitica. interestingly, it has been shown that, even in the absence of uv-c, plant nucleotide excision repair mutants displayed the identical type of resistance to h. parasitica, suggesting a positive role for uv-c-mediated dna damaging events in plant immunity activation. apx, one of the typical antioxidant genes, encodes for the enzyme capable of h 2 o 2 elimination, thus protecting fig. 4 rt-pcr analysis of uv-responsive gene expressions in cell suspension and green leaves. typical rt-pcr profiles of gene expressions in uv-irradiated cell suspension (a) and leaf samples (b) are shown. actin was used as an internal control. note: comparison of the density of bands on several gels were performed after gathering the images of gels on the black background. fig. 5 rt-pcr analysis of uv-responsive gene expressions in green fruits and red ripe fruits. typical rt-pcr profiles of gene expressions in uv-irradiated green fruits (a) and red ripe fruits (b) are shown. actin was used as an internal control. note: comparison of the density of bands on several gels were performed after gathering the images of gels on the black background. 138 the plants from the damaging impacts of ros. pr genes (viz., pr-p2 and pr1) and pal are related to the actions and production of salicylic acid (sa). sa is a hormonelike natural signaling molecule involved in the defense response against infection by pathogens in higher plants. sa acts by stimulating the production of pr proteins through a complex signaling mechanism involving ros, calcium and protein phosphorylation in the early stages (kawano et al., 1998; kawano and bouteau, 2013). in solanaceae plants including tobacco and tomato, the increase in pal expression reportedly results in accumulation of sa (kawano et al., 2004). here, the short exposure to uv-c but not to uv-a resulted in an acute increase in cell death in the cell suspension culture (fig. 3). development of lesions (visible symptoms), (fig. 1) accompanied by ion leakage (sign of membrane damage) (fig. 2) was induced in the uv-c-treated leaf samples. in contrast, no visible damage was observed in the uvtreated green and red fruits. it is noteworthy that uv-a or sub-lethal low dose uv-c was shown to induce the defenserelated genes (pr1 and pr-p2 genes) in the leaves (fig. 4 b) and green fruit tissues (fig. 5 a). the above data imply that cell death (possibly apoptotic, data not shown) and plant protection mechanisms (expression of antioxidant genes and pr genes) are induced by high and low doses of uv-c, respectively. therefore, moderate doses of uv irradiation should be applicable to plants to confer tolerance to a variety of abiotic stresses (chiefly, oxidative stress) and innate plant immunity (represented by the induced expression of pr genes). possible involvement of sa signaling our data are comparable to the work of marco et al. (2008) who reported the ozone-induced gene expression controls in the leaves of three tomato cultivars (viz., nikita, alisa craig and valenciano). similar to our data on uv-treated micro-tom, expression of apx is reportedly suppressed in the ozone-treated nikita tomato (marco et al., 2008). in addition, the tomato leaves chronically exposed to ozone showed activation of redox-related and defense-related genes such as pal (marco et al., 2008). thus, ozone-induced gene expression patterns and the uv-induced gene expression patterns may share a common regulatory mechanism. one of the key pathways that may be common to ozone response and uv response in tomato is the sa signaling pathway. expressions of pr genes and eds1, known factors functioning upstream of sa-dependent expression of pr genes (falk et al., 1999), were induced by ozone in three tomato cultivars (marco et al., 2008). in the present investigation, activation of pal and pr genes in the uvtreated micro-tom leaves (fig. 4b) was observed, suggesting the possible involvement of a sa signaling path. recently, in the culture of arabidopsis cells, we observed that over-expression of bacterial salicylate hydroxylase gene (nahg) and pharmacological reagents targeting either ros or calcium signaling effectively blocked both the cell death induction by high-dose uv-c and induction of defense-related gene expressions by sub-lethal low-dose uv-c (hiramatsu et al., unpublished results). requirements for early calcium signaling, proven by aequorin luminescence and the action of calcium targeted pharmacological reagents, and generation of ros, are commonly observed in various plant models during the responses to sa (kawano et al., 1998; kawano and muto, 2000; kawano and bouteau, 2013), pathogen-derived molecules (kadota et al., 2004), ozone (kadono et al., 2006, 2010; tran et al., 2013), peroxyacetyl nitrate (yukihiro et al., 2012), toxic metal ions (lin et al., 2005; kagenishi et al., 2011; kunihiro et al., 2011), and uv (hiramatsu et al., unpublished results). possible regulation of sa action by uv-c according to the work by nawrath et al. (2002) using arabidopsis thaliana, expression of eds5, a member of multidrug and toxin extrusion transporter family, known to be involved in the accumulation of sa and pr1 transcript, is very low in unstressed plants but strongly induced by attacks by pathogens, treatment with sa, and irradiation with uv-c. eds5 expression induced by pathogen infection and uv-c exposure largely depends on the pathogen response proteins eds1, pad4, and ndr1, suggesting the requirements for the signal transduction pathways commonly employed in uv-c responses and defence responses (nawrath et al., 2002). this defense mechanismdependently induced eds5 transcript reportedly starts accumulating 2 h after exposure to uv-c, and the transcription level likely remains for two days, thus further contributing to the long-lasting sa responses. this view shall be studied in detail in our future experiments. possible role of chlorophylls. our data indicate that the response in green samples (leaves and immature fruits) and non-green samples (ripe fruits and suspension cultured cells) might differ in their modes of responses to uvs. while pr1 expression, one typical measure of stress responses, was induced by uv-c in green tissues, non-green samples (both the ripe fruits and cell suspensions) showed no induction of pr1 expression (fig. 4 and 5). it is tempting to speculate that the chlorophyll-related compounds may behave as secondary active signals for mediating the stress responses. one of such candidate compounds may be pheophorbide a derived from chlorophyll a, which is known to be produced in the ethylene-exposed green tomato fruits (kawano et al., 1999). recently, it was revealed that pheophorbide a plays a key role in both light-dependent and light-independent cell death mechanism in plants (hirashima et al., 2009). this aspect is worth testing in future experiments. regulation of ethylene production acs1a coding for acc synthase is a ripening-related gene responsible for production of ethylene precursor, acc. since ethylene promotes the ripening of fruits and senescence of leaves, uv-dependent changes in acs1a expression may drastically affect the life-cycle of the 139 plants and shelf-life of the fruits. this ethylene biosynthesis-related gene was shown to be activated by both uv-a and uv-c in the cell suspension culture and green fruits, suggesting that uv treatments may be available for postharvest enhancement of fruit maturity. interestingly, uv-a and uv-c irradiation to red fruits resulted in gradual lowering of the acs1a expression level (fig. 5), suggesting that uvs are possibly applicable for retardation and prevention of ethylene-mediated fruit over-ripening and softening. acknowledgements this work was supported by the grant-in-aid from kitakyushu foundation for the advancement of industry, science, and technology (fais) and a grant of regional innovation strategy support program implemented by ministry of education, culture, sports, science and technology 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15(2): 129134. tran d., rossi m., biligui b., kawano t., mancuso s., bouteau f., 2013 ozone-induced caspase-like activities are dependent on early ion channel regulations and ros generation in arabidopsis thaliana cells. plant signal behav., 8: e25170. yoshioka h., bouteau f., kawano t., 2008 discovery of oxidative burst in the field of plant immunity: looking back at the early pioneering works and towards the future development. plant signal. behav., 3: 153-155. yukihiro m., hiramatsu t., bouteau f., kadono t., kawano t., 2012 peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line. plant signal behav., 7: 113-120. impaginato 21 adv. hort. sci., 2025 39(1): 21­30 doi: 10.36253/ahsc­16590 https://oaj.fupress.net/index.php/ahs continuous lighting improves the leaf quality of sweet basil (ocimum basilicum l.) grown in a controlled environment m. puccinelli (*), r. maggini, a. pardossi, l. incrocci department of agriculture, food and environment, university of pisa, via del borghetto, 80, 56124 pisa, italy. key words: artificial light, hydroponics, led, 24­hour photoperiod, vertical farms. abstract: in vertical farms, continuous lighting (cl) with lower light intensity (photosynthetic photon flux density, ppfd) is a method to reduce the investment costs for the lighting system. continuous lighting has both negative and positive effects on crop performance, depending on the plant species. in this study, we investigated the effect of cl on plant growth and leaf quality in sweet basil (ocimum basilicum l. cv. tigullio) cultivated in a growth chamber with light emitting diode (led) (r:b:g=3:1:1). basil plants were grown hydroponically for 14 days with a photoperiod of 16 h d­1 (control) or 24 h d­1 with a ppfd of 220 or 147 µmol m­2 s­1, respectively. the daily light integral was 12.7 mol m­2 d­1 in both treatments. plant growth was not significantly affected by the light regime. compared with the control, cl increased the leaf antioxidant capacity and concentration of total chlorophylls, flavonoids and phenols, and reduced the nitrate level. continuous lighting would slightly increase or decrease electricity costs compared to 16­hour illumination, depending on the daily schedule of the standard lighting regime. 1. introduction in many countries, high­value fresh vegetables and herbs are increasingly produced in plant factories with artificial light, also known as vertical farms. in vertical farms, crop yield is enhanced due to the use of multi­tiered cultivation systems and the maintenance of optimal environmental conditions for plants (zhu and marcelis, 2023). in a controlled environment, plant growth is primarily determined by the daily light integral (dli) (warner et al., 2023), which is the light level plants receive over a day and thus depends on the photosynthetic photon flux density (ppfd) and the photoperiod. generally, crop yield and quality are (*) corresponding author: martina.puccinelli@agr.unipi.it citation: puccinelli m., maggini r., pardossi a., incrocci l., 2025 ­ continuous lighting improves the leaf quality of sweet basil (ocimum basilicum l.) grown in a controlled environment. ­ adv. hort. sci., 39(1): 21­30 orcid: pm: 0000­0001­7571­2393 mr: 0000­0003­2542­6561 pa: 0000­0001­9927­4017 il: 0000­0001­9994­763x author contribution: conceptualization, ap, mp; methodology, ap, mp.; formal analysis, mp, rm; resources, ap, li; data curation, mp; writing ­ original draft preparation, mp, ap; manuscript review and editing, mp, rm, ap, li; supervision, ap. copyright: © 2025 puccinelli m., maggini r., pardossi a., incrocci l. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 10 september 2024 accepted for publication 24 february 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16590 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-7571-2393 http://orcid.org/0000-0003-2542-6561 http://orcid.org/0000-0001-9927-4017 http://orcid.org/0000-0001-9994-763x http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(1): 21­30 22 boosted by increasing dli to a certain level. in vertical farms, typical dli values are 12 to 17 mol m­2 d­1 for leafy greens and herbs, and 15 to 40 mol m­2 d­ 1 for fruiting crops (mechatronix horticulture lighting, 2025). the photoperiod is generally 16 or 18 h d­1 with pffd ranging between 150 and 300 µmol m­2 s­1) (nájera et al., 2022). the main drawback of vertical farms is the high electricity requirement for artificial illumination and air conditioning (zhu and marcelis, 2023). despite the adoption of energy­efficient light­emitting diode (led) fixtures, the lighting system remains one of the main components of the capital costs of vertical farms and accounts for approximately 80% of the total electricity demand (cai et al., 2024). therefore, the key to reducing energy costs and/or enhancing energy use efficiency in vertical farms is innovating lighting systems and strategies (liu et al., 2022). continuous lighting (cl; i.e., 24­hour photoperiod without dark interruption) with lower pffd is a cost­ effective strategy to achieve the same dli in vertical farms, as it reduces the investment costs for the lighting system (fewer fixtures are necessary) and the operational costs for illumination in case of a time­ based pricing scheme for electricity (lanoue et al., 2022). long photoperiod (>18 h d­1) can enhance plant production by increasing leaf photosynthesis. under the same dli, longer photoperiods with lower ppfd increase yield in most species (warner et al., 2023), since crop light use efficiency rises with decreasing irradiance (e.g., palmer and van iersel, 2020; carotti et al., 2021). this explains why many studies have been conducted on the effect of cl in greenhouse crops, which started nearly one century ago (arthur et al., 1930), and more recently in vertical farms (velez­ramirez et al., 2011; shibaeva et al., 2023 a). however, leaf injury and growth inhibition were observed in some species grown under greenhouse conditions with cl for a relatively long period, such as tomato, eggplant, pepper, and cucumber (velez­ ramirez et al., 2011; shibaeva et al., 2023 a). although the mechanism of leaf damage under cl has not yet been completely elucidated, it has been attributed to carbohydrate accumulation in leaf tissues and disruption of endogenous circadian rhythms, which results in early senescence and reduced photosynthesis (velez­ramirez et al., 2011; shibaeva et al., 2023 a). in contrast to fruiting crops, leafy vegetables and herbs have a short growing cycle and generally, they are not negatively affected by long photoperiod or cl (shibaeva et al., 2023 a); positive effects have also been reported (table 1s). for instance, in rocket cl enhanced plant growth and improved leaf quality by reducing nitrate concentration and increasing the concentration of antioxidants (proietti et al., 2021). sweet basil (ocimum basilicum l.) is an economically important herb cultivated worldwide (camlica and yaldiz, 2023) due to its adaptability to different growing conditions and systems. its short cycle, rapid growth, and easy cultivation make basil one of the most used species in vertical farms; basil is the most studied species for controlled environment agriculture after lettuce (dsouza et al., 2023). there exists a reasonable consensus on the optimal dli for basil grown indoors, which ranges between approximately 13 and 15 mol m­2 d­1 (liaros et al., 2016; dou et al., 2018; pennisi et al., 2020), but contrasting results have been found on the response of this species to cl, since positive (islam et al., 2010; lanoue et al., 2022; fayezizadeh et al., 2024), negative (beaman et al., 2009), or no (pennisi et al., 2020) effects have been reported. in this work, the impact of cl on plant growth and leaf quality was studied in sweet basil cultivated hydroponically in a growth chamber with led light under a photoperiod of 16 or 24 h d−1 with the same dli (12.7 mol m­2 d­1). based on the results in the literature and a preliminary experiment, in which only growth parameters were measured, we hypothesized that cl does not affect plant growth but improves leaf quality by reducing nitrate accumulation and increasing the concentration of antioxidant compounds. 2. materials and methods plant material and growing conditions two experiments were conducted with sweet basil (cv. tigullio; franchi sementi, grassobbio, italy) grown in a floating raft system in a growth chamber at the university of pisa. basil seeds were sown in 240­cell trays with stone wool plugs and 41 days after sowing, the seedlings with two pairs of true leaves were transplanted in plastic tanks with 14 litres of aerated nutrient solution. the solution had a ph of 6.0 and an electrical conductivity of 2.39 ds m­1, and contained the following concentration of nutritive elements: n­ no3 10.0 mm, p 1.5 mm, k 9.0 mm, ca 4.5 mm, mg http://oaj.fupress.net/index.php/ahs/article/view/16590/version/18456 puccinelli et al. ‐ continuous light benefits basil 23 2.0 mm, fe 40.0 µm, b 40.0 µm, cu 3.0 µm, zn 10.0 µm, mn 10.0 µm, and mo 1.0 µm. twelve plants were grown in each tank and crop density was approximately 96 plants m­2. air temperature and relative humidity were kept at 24.0°c and 65% ­ 70%, respectively. basil plants were illuminated by red, blue, and green (r:b:g=3:1:1) (fig. 1) led tubes (circular natural indoor, c­led, imola, italy) with a 16­hour photoperiod at 220 µmol m ­2 s ­1 ppfd, ti l l transplantation in hydroponic tanks. afterwards, one group of plants was kept under the same light regime (control) while another group was grown with a 24­ hour photoperiod at 147 µmol m−2 s−1 ppfd (cl). the lowest ppfd was achieved by using fewer lamps and slightly adjusting the distance between lamps and the plant canopies. an opaque plastic screen was used to avoid contamination between the two light treatments, which lasted 14 days. each treatment had four replicates, each consisting of one hydroponic tank. determinations leaf fresh (fw) and dry weight (dw), stem and root dw, plant height, and leaf area were measured in plant samples collected in each replicate; each sample consisted of six individual plants. dry weight was determined in plant samples that were dried in a ventilated oven at 70°c until they were of constant weight. leaf area was measured using a digital planimeter (dt area meter mk2, delta t­devices). leaf area index (lai) was computed as the average leaf area per plant divided by the area occupied by one plant. twelve days after the beginning of the experiment, stomatal behaviour was assessed by measuring the abaxial leaf diffusion conductance (gs) with a diffusion porometer (ap4, delta­t devices, cambridge, uk). the gs was measured on two individual plants in each replicate of both treatments at two times corresponding to midday and midnight of the control treatment. plant water uptake was determined by measuring the weight of each hydroponic tank at the beginning and the end of each experiment. the weight difference was assumed to be equal to the plant water absorption because the tank was covered by the polystyrene floating raft and thus direct evaporation was insignificant. leaf concentration of mineral elements was determined in dried samples whereas the antioxidant capacity and the concentration of total chlorophylls, carotenoids, flavonoids, and phenols were examined in fresh samples. each sample comprised all leaves from six individual plants collected from each tank. to determine leaf mineral concentration, finely ground samples underwent mineralization in a 5:2 v/v mixture of 65% hno3 and 35% hclo4 at 240°c for one hour or were extracted with distilled water at room temperature for two hours. the mineralized samples were uti l ized for assessing the concentrations of k, ca, mg, cu, fe, mn, and zn through atomic absorption spectroscopy. at the same time, p levels were determined spectro­ photometrically with olsen’s method. water extracts were analysed spectrophotometrically for nitrate concentration using the salicylic­sulfuric acid method (puccinelli et al., 2023). methanol (99% v/v) was used to extract fresh samples, followed by a 60­minute sonication (frequency 28­34 khz, power peak 350 w). the samples were stored at ­18°c for 24 hours; afterwards, the concentrations of total chlorophylls, carotenoids, phenols, and flavonoids were determined spectrophotometrically as previously reported (puccinelli et al., 2023). the antioxidant capacity of leaf samples was measured in methanol extracts using the ferric­reducing ability of plasma (frap) assay (benzie and strain, 1996) and the 2,2­ diphenyl­1­picrylhydrazyl (dpph) assay (blois, 1958). electricity costs of light regimes the electricity cost of the light regimes tested in this work was calculated every week using the fig. 1 ­ the spectral distribution of the led tubes (circular natural indoor, c­led, imola, italy) used in the experi­ ments with sweet basil in a growth chamber. the spec­ trum was measured using a portable spectroradiometer (spectrapen lm 510, photon systems instruments, drásov, czech republic). ppfd= photosynthetic photon flux density. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/vacuum-evaporation https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/vacuum-evaporation https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/vacuum-evaporation adv. hort. sci., 2025 39(1): 21­30 24 scheme for time­of­use pricing of electricity in place in italy, which differentiates the prices by single­hour rate and time slot (f), as follows (enel, 2025 a): f1: from 8.00 am to 7.00 pm from monday to friday, excluding national holidays; f2: from 7.00 am to 8.00 am and from 7.00 pm to 11.00 pm from monday to friday, saturday from 07.00 am to 11.00 pm, excluding national holidays; f3: from 11.00 pm to 7.00 am from monday to saturday, sunday, and national holidays. the prices used for calculation were 0.1783, 0.1716 and 0.1485 € kwh ­1 for f1, f2, and f3, respectively. they are mixed costs because they have variable and fixed components. the variable component is the pun index gme (pun is the italian acronym for prezzo unico nazionale, “national single price”), which is the weighted average of electricity prices in different areas of italy and at different times of the day. the fixed part is the contribution paid to the energy provider. we used a fixed component of 0.020 € kwh−1 (enel, 2025 a) while the puns were the mean values recorded in january 2025 for the three time slots (enel, 2025 b). the weekly lighting cost was computed for three light regimes with the same dli (12.7 mol m­2 d­1): 220 µmol m­2 s­1 ppfd for 16 h d­1 from 11:00 pm until 03:00 pm of the next day (lr1) or from 08:00 am until 12:00 pm (lr2); 147 µmol m­2 s­1 ppfd for 24 h d­ 1 (lr3). the consumption of electricity was estimated using a photon efficacy of led lamps of 3.1 µmol j­1. statistical analysis since the two experiments were conducted in the same growing conditions and the results were quite similar, the data were pooled, subjected to 1­ or 2­ way anova, and reported as the mean values (±se) of eight replicates. leaf diffusion conductance data were subjected to 2­way anova, with the time of measurement and light regime as variability factors. data were tested for the normality of the distribution using the shapiro­wilk test and for the homogeneity of variances using levene’s test. statistical analysis was performed using jmp statistical software. 3. results plant growth in our study, the light regime did not affect basil growth as no significant differences (p>0.050) were found between the controls and cl plants regarding plant height, leaf and stem fw (yield), and leaf, stem, root, and total dw (table 1). the production for the market of bunched fresh herbs was 0.96 kg m­2, a noticeable yield for a crop that lasted only two weeks. the leaf area index has an average value of 2.16 and was not significantly affected by the light regime (data not shown) plant water and mineral relations the cumulated water uptake during the experiments was not significantly influenced by the light regime and averaged 34.8±2.1 l m­2. this result is consistent with the absence of significant differences in lai between the controls and cl plants, and the measurements of gs, which did not vary significantly between the two plant groups at both midday and midnight of the control light regime light regime anova significance control cl leaf fw (kg m­2) 0.783±0.044 0.748±0.005 ns stem fw (kg m­2) 0.198±0.018 0.195±0.012 ns leaf dw (kg m­2) 0.059±0.003 0.058±0.003 ns stem dw (kg m­2) 0.015±0.004 0.015±0.004 ns root dw (kg m­2) 0.013±0.001 0.012±0.001 ns total dw (kg m­2) 0.087±0.004 0.084±0.004 ns plant height (cm) 11.9±0.2 11.5±0.3 ns table 1 ­ leaf and stem fresh (fw) and dry weight (dw), root and total dw and plant height, in sweet basil plants grown hydroponically for 14 days in a growth chamber under two led light regimes with the same daily light integral (12.7 mol m­2 d­1): a 16­hour photoperiod and 220 µmol m­2 s­1 ppdf (control); 24­hour photoperiod and 147 µmol m­2 s­1 ppdf (cl) mean values (±se) of eight replicates. ppfd= photosynthetic photon flux density. significance level: ns = not significant. puccinelli et al. ‐ continuous light benefits basil 25 (fig. 2). however, on average gs was significantly higher at midday (290.0±8.6 mmol h2o m­2 s­1) that at midnight (187.7±12.1 mmol h2o m­2 s­1) . in both experiments, no plant revealed evident mineral deficiency symptoms and the leaf concentration of nutritive elements (table 2) was within or above the adequate levels reported for sweet basil (bryson et al., 2014). nonetheless, cl plants showed a significantly lower leaf concentration of mg and mn, and a higher concentration of zn than the control plants. in japanese mugwort, cl induced a lower leaf concentration of k, mn, and zn, in partial agreement with our findings (hata and kawamura, 2023). in our experiments, leaf moisture content was not affected by the photoperiod (fig. 3a). compared to the 16­hour photoperiod, cl significantly increased the leaf concentration of total chlorophylls (+35%), flavonoids (+40%), and phenols (+44%), and the antioxidant capacity, which was measured using both frap (+47%) and dpph (+63%;) assay (fig. 3). conversely, no significant differences in carotenoids concentration were found between the controls and cl plants (fig. 3d) and the nitrate level was slightly but significantly reduced by cl (fig. 3b) a significant positive correlation was found between the total antioxidant capacity measured with the two assays (r2 = 0.856; n=8) and between the antioxidant capacity and the level of phenols (r2=0.756 and r2=0.845 for frap and dpph assay, respectively) or flavonoids (r2=0.956 and r2=0.845). electricity consumption and cost in our simulation, two reference light regimes were compared to cl: from 11:00 pm until 03:00 pm the following day (lr1), or from 08:00 am until 12:00 pm (lr2, table 3). the calculated electricity consumption for artificial lighting was 7.95 kwh m­2 week­1 across all three scenarios, with weekly electricity costs estimated to range from 1.30 to 1.39 € m­2 week­1, based on the time­of­use pricing model in italy (table fig. 2 ­ abaxial leaf diffusion conductance in sweet basil plants grown hydroponically for 14 days in a growth chamber under two led light regimes (lr) with the same daily light integral (12.67 mol m­2 d­1): a 16­hour photoperiod and 220 µmol m­2 s­1 ppdf (control); 24­hour photoperi­ od and 147 µmol m­2 s­1 ppdf (cl). the measurements were taken at midday and midnight of the control light regime 12 days after the beginning of the experiment. mean values (±se) of eight replicates. significance level: *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns = not significant. light regime anova significance control cl ca (g kg­1) 1.138±0.079 0.922±0.122 ns k (g kg­1) 5.938±0.411 5.019±0.575 ns mg (g kg­1) 0.342±0.020 a 0.240±0.025 b ** p (g kg­1) 0.840±0.025 0.771±0.022 ns cu (mg kg­1) 1.812±0.106 1.640±0.084 ns mn (mg kg­1) 6.946±1.177 a 3.687±0.328 b * fe (mg kg­1) 16.233±1.552 14.061±3.366 ns zn (mg kg­1) 4.185±0.268 b 6.254±0.786 a * leaf quality we evaluated basil quality by measuring leaf moisture content, which influences the produce’s response to post­harvest processing and storage (clarkson et al., 2003), and concentration of several substances associated with the visual (chlorophylls), nutritional (antioxidant compounds), or safety (nitrate) quality. table 2 ­ leaf concentration (on a fresh weight basis) of macro and microelements in sweet basil plants grown hydroponically for 14 days in a growth chamber under two led light regimes with the same daily light integral (12.7 mol m­2 d­1): 16­hour photoperiod and 220 µmol m­2 s­1 ppdf (control); 24­hour photoperiod and 147 µmol m­2 s­1 ppdf (cl) mean values (±se; n = 8) flanked by different letters are signifi­ cantly different at the 5% level. significance level: *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns = not significant. ppfd= photosynthetic photon flux density. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/nutrient-deficiencies 26 adv. hort. sci., 2025 39(1): 21­30 3). when evaluating cl against a 16­hour light regime, we found a minor increase (+2.6%) or decrease (­4.4%) in electricity costs, which depended on the daily schedule of the latter system. 4. discussion and conclusions according to beaman et al. (2009), basil needs a dark period for optimal growth since cl may cause leaf chlorosis and necrosis, which were not observed in our experiments. in an experiment conducted in a growth chamber with the same ppfd level (250 µmol m­2 s­1) from led lamps, fresh and dry biomass did not significantly differ in basil plants grown with a photoperiod of 16, 20, or 24 h d­1, notwithstanding the large differences in dli (14.4, 18.0, and 21.6 mol m­2 d­1, respectively) (pennisi et al., 2020). in contrast, in greenhouse­grown basil the supplementation of natural radiation with artificial light (from high­ pressure sodium lamps) for 24 h d­1 increased shoot dw compared to illumination for 16 or 20 h d­1 (islam et al., 2010). islam et al. (2010) used the same ppfd (150 μmol m­2 s­1 from hps lamps) in the three light treatments and therefore the artificial dlis were different (8.64, 10.80, and 12.96 mol m­2 d­1), and this probably accounts for the differences between their and our findings. continuous lighting also increased the biomass of green basil microgreens compared to a 16­hour photoperiod with the same dli (14 or 24 mol m­2 d­1) (lanoue et al., 2022). positive effects of cl on plant growth were also observed in chinese cabbage (kang et al., 2024), rocket (proietti et al., 2021), japanese mugwort (hata and kawamura, 2023), and microgreens of different species (lanoue et al., 2022; shibaeva et al., 2023 b). the significant reduction of gs detected at midnight in both treatments (fig. 2) suggested that a circadian rhythm of stomatal movements was present in plants grown under cl (dodd et al., 2004). similar results were found in chinese cabbage (kang et al., 2024). in fig. 3 ­ leaf moisture content (a) and concentration (on a fresh weight basis) of nitrates (b), total chlorophylls (c), carotenoids (d), flavonoids (e), and phenols (f), and antioxidant capacity measured by frap (g) and dpph (h) assay, in sweet basil plants grown hydroponically for 14 days in a growth chamber under two led light regimes with the same daily light integral (12.67 mol m­2 d­1): 16­ hour photoperiod and 220 µmol m­2 s­1 ppdf (control); ii) 24­hour photoperiod and 147 µmol m­2 s­1 ppdf (cl). mean values (±se) of eight replicates. significance level: *** p≤0.001; ** p≤0.01; * p≤0.05; ns = not significant. abbreviations: pffd, photosynthetic photon flux density; frap the ferric reducing ability of plasma (frap) assay w; dpph, the 2,2­diphenyl­1­picrylhydrazyl assay. table 3 ­ leaf concentration (on a fresh weight basis) of macro and microelements in sweet basil plants grown hydroponically for 14 days in a growth chamber under two led light regimes with the same daily light integral (12.7 mol m­2 d­1): 16­hour photoperiod and 220 µmol m­2 s­1 ppdf (control); 24­hour photoperiod and 147 µmol m­2 s­1 ppdf (cl) light regime ppfd (µmol m­2 s­1) photoperiod (h d­1) daily light integral (mol m­2 d­1) lighting schedule costs (€ m­2 week­1) lr1 220 16 12.7 11:00 pm ­ 03:00 pm 1.30 lr2 220 16 12.7 08:00 am ­ 12:00 pm 1.39 cl 147 24 12.7 continuous light 1.33 ppfd= photosynthetic photon fluence rate. puccinelli et al. ‐ continuous light benefits basil 27 contrast, in soybean (kassai, 2008) and potato (wheeler et al., 2019) grown in a growth chamber, midday gs was lower in plants grown with cl than in those grown with a photoperiod of 10­12 h d­1. compared with the control, cl increased the leaf antioxidant capacity and concentration of total flavonoids and phenols and reduced the nitrate level (fig. 3). in general, our results agree with previous findings (table 1s). for instance, an increase in pigment concentration was observed in rocket plants grown with cl (proietti et al., 2021). in lettuce, cl improved leaf quality by reducing nitrate concentration and increasing the concentration of phenolic compounds, even when it was applied only for 1­3 days before harvest (bian et al., 2018; yang et al., 2022; zhang et al., 2021). in contrast, in several species in the amaranthaceae, the leaf concentration of pigments and total polyphenols, and the antioxidant capacity were invariably lower in cl plants than in those grown with shorter photoperiods (ali et al., 2009). notwithstanding that nitrate may have positive effects on human health, nitrate intake with diet is linked with some health risks (karwowska and kononiuk, 2020). therefore, the nitrate level in drinking water and food is strictly regulated and some agronomic practices have been proposed to reduce nitrate content in vegetables, which represent the primary source of nitrate in the human diet (colla et al., 2018). sweet basil is prone to accumulate high levels of nitrate (corrado et al., 2020). the cl­induced reduction in leaf nitrate concentration observed in our study agrees with previous findings in other crops (bian et al., 2016; proietti et al., 2021; shibaeva et al., 2023 b). it is known that light regulates nitrate reductase (lillo and appenroth, 2001); cl can increase the activity of nitrate reductase (nr) and consequently the reduction of nitrate in plant leaves. in lettuce, the reduction of leaf nitrate concentration induced by short­term (two days) cl was associated with increased activity and gene expression of nr and nitrite reductase (bian et al., 2016, 2018). in contrast, cl augmented leaf nitrate levels in purslane (he et al., 2023) and this was associated with a reduction in nr, compared with a 12­hour photoperiod. continuous il lumination can increase the generation of reactive oxygen species (ros) and induce oxidative stress (haque et al., 2015; huang et al., 2019; liu and liu, 2024). the balance between ros production and the plant’s ability to scavenge these molecules largely determines the extent of cl­ induced plant damage (kumar et al., 2022). if this balance is achieved, injury­free production is possible under cl, with the additional improvement of produce quality resulting from a higher content of antioxidants (e.g. phenolics) and other health­ promoting compounds, as was found in this and other works (bian et al., 2016; hata and kawamura, 2023). as such, applying cl for indoor cultivation of short­cycle crops can be considered an eustressor capable of promoting crop yield and improving produce quality (vázquez­hernández et al., 2019). for the same dli, the use of cl can markedly decrease the number (­33% compared to the 16­hour photoperiod, in this work) and the installation costs of l ight fixtures. moreover, in countries that implement a time­of­use pricing model of the electricity market, the operational costs of lighting are lower during off­peak periods. electricity prices are generally lower early in the day, overnight, and during the weekends and holidays. for basil production in vertical farms, illumination is generally operated for 16 continuous hours per day, switching on leds during the nighttime, when energy prices are lower than in the daytime (avgoustaki and xydis, 2021). according to our simulation, cl would slightly increase or decrease electricity costs compared to 16­hour illumination, depending on the daily schedule of the standard lighting regime (table 3). for microgreens production in vertical farms, compared to a 16­hour photoperiod, cl reduced electricity costs (expressed per unit of fresh biomass) by 8% to 38%, when dlis were the same (lanoue et al., 2022). however, lanoue and colleagues (lanoue et al., 2022) did not clarify when the light was switched on in the control treatment. vertical farms use powerful air conditioning systems to regulate air temperature and humidity for optimal crop production (zhu and marcelis, 2023). continuous illumination at relatively low ppfd reduces the heat generated by led lamps and the moisture released through plant transpiration, thus reducing the demand for air cooling and dehumidification (cai et al., 2024). in addition, cl could provide beneficial effects for pest and disease management. for instance, cl was found to suppress the sporulation of peronospora belbahrii, which is the causal organism of basil downy mildew, one of the most destructive diseases of sweet basil (radetsky et al., 2020). continuous light also reduced whiteflies’ http://oaj.fupress.net/index.php/ahs/article/view/16590/version/18456 adv. hort. sci., 2025 39(1): 21­30 28 and stress factors, pp. 135­163. ­ in: prakash c.s., s. fiaz, m.a. nadeem, f.s. baloch, and a. qayyum (eds.) sustainable agriculture in the era of the omics revolution. springer international publishing, chaem, switzerland, pp. 514. carotti l., graamans l., puksic f., butturini m., meinen e., heuvelink e., stanghellini c., 2021 ­ plant factories are heating up: hunting for the best combination of light 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lighting in vertical farming systems enhances bioactive compounds and productivity of vegetables crops. ­ biol. life sci. forum 16(1): 24. palmer s., van iersel m.w., 2020 ­ increasing growth of lettuce and mizuna under sole‐source led lighting using adv. hort. sci., 2025 39(1): 21­30 30 2021 ­ led light quality of continuous light before harvest affects growth and asa metabolism of hydroponic lettuce grown under increasing doses of nitrogen. ­ plants, 10: 1­14. zhu x., marcelis l., 2023 ­ vertical farming for crop production. ­ modern agric., 1: 13­15. impaginato 83 adv. hort. sci., 2025 39(2): 83­92 doi: 10.36253/ahsc­17201 https://oaj.fupress.net/index.php/ahs enhance the longevity and aesthetic appeal of anthurium andraeanum cv. fire cut leaves by using some natural components m.f. rida (*), a.m. taha ornamental plants research and landscape gardening department, horticulture research institute, agriculture research center, alexandria, egypt. key words: anthurium, arabic gum, chitosan, glossiness, leaf postharvest, rosehip oil. abstract: this experiment was conducted to examine the effects of rosehip oil (rh oil), arabic gum (ag), and chitosan (cs) on the vase life and quality of anthurium andraeanum cv. fire under laboratory conditions. the experimental layout followed a completely randomized design. seven treatments were applied: tap water (control), rh oil (2% or 4%), rh oil (2% or 4%) + ag (5%), and rh oil (2% or 4%) + ag (5%) + cs (500 ppm). the leaves were sprayed using a hand sprayer until runoff occurred. results showed that the highest increases in vase life, final water uptake, chlorophyll a and b content, and the degree of leaf health and glossiness were obtained after the application of 4% rh oil. scanning electron micrographs illustrated that stomata were open in untreated leaves, moderately open after application of 2% rh oil, slightly open after spraying with 4% rh oil, and completely closed after the application of rh oil (2% or 4%) + ag (5%). 1. introduction anthurium andraeanum (flamingo flower) is a very popular plant which grows up to 60 cm tall. it features rich green, elongated, heart­ shaped leaves and waxy white or coral­colored spathes, which are used in wedding arrangements (jack, 1985). its attractive foliage and showy cut flowers make it highly valuable in the global flower market (anand et al., 2017). when the plant is not in flower, the leaves harmonize with those of other tropical plants suitable for shady spots (jane and graham, 1997). in recent years several varieties of anthuriums have been introduced into the egyptian market. most of these varieties have large, glossy leaves, making them suitable for use as cut foliage. unfortunately, anthuriums andraeanum cv. fire has large, pale green leaves, which reduce its economic value. (*) corresponding author: magdeldinhamza2@yahoo.com citation: rida m.f., taha a.m., 2025 ­ enhance the longe‐ vity and aesthetic appeal of anthurium andraea‐ num cv. ‘fire’ cut leaves by using some natural components. ­ adv. hort. sci., 39(2): 83­92. orcid: rmf: 0000­0003­4122­0440 tam: 0009­0009­8547­200x copyright: © 2025 rida m.f., taha a.m. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 19 january 2025 accepted for publication 27 may 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17201 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0003-4122-0440 http://orcid.org/0009-0009-8547-200x http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 83­92 84 to optimize the commercial worth of cut flowers, researchers have concentrated on enhancing their longevity and quality through flower preservation; nevertheless, chemical flower preservation has negative environmental consequences. (moussa et al., 2024). natural ingredients, such as plant extracts and essential oils, are utilized instead of chemicals in cut flower preserving solutions due to their negative impact on human health, particularly those with silver components. numerous researchers have explored the impact of natural materials (sarhan et al., 2023). in this respect hashemabadi et al. (2021) found that adding dill essential oil to dianthus caryophyllus l. cv. yellow candy solution improved vase life and solution uptake compared to pure water. bañuelos­hernández et al. (2017) found that applying 1.0 and 1.5% chitosan coating to heliconia bihai flower stems increased vase life by 10.3 and 7 days, respectively, compared to the control. also, creel (2006) reported that soaking flowers in 10% or 20% acacia gum can extend the vase life of snapdragon. the fruit of rosa canina l. is known as rosehip (rh) or rose haw. it is red to orange in color. it consists of approx. 30­35% seeds and 65­70% pericarp (uggla and nybom, 1998). rosehip oil is rich in polyunsaturated fatty acids, stearic acid (48.11%), linoleic acid (35.38%), palmitoleic acid (33.78%) and eicosadienoic acid (30.57%) (vasić et al., 2020). due to its antioxidant properties, rosehip oli is widely used in pharmaceutical industry (franco et al., 2007; machmudah et al., 2007). acacia senegal and acacia seyal trees are the primary sources of arabic gum (ag), a natural polysaccharide polymer used in the biological industry. recently, arabic gum has gained considerable attention as a postharvest edible coating due to its ability to preserve the quality and extend the shelf life of fresh products. it’s excellent emulsifying, stabilizing, binding, and shelf­life­ extending properties make it an efficient food additive (tiamiyu et al., 2023). the shelf life of various fruits and vegetables, including tomatoes (ali et al., 2010), sweet cherries (mahafaudi and hamdi, 2014), green chilies (chitravathi et al., 2014) and mangoes (khaliq et al., 2015), has been successfully extended by arabic gum coating. chitosan (cs) is a naturally occurring polymer with several advantageous properties, including non­ toxicity, biocompatibility, biodegradability, and the ability to form chelates. these characteristics, along with its versatility, make chitosan a valuable material for various applications (lingait et al., 2024). it can be processed into hydrogels, nanoparticles, pastes, nanofibers, fi lms, membranes, microgranules, sponges, etc. additionally, chitosan can be modified by grafting, crosslinking, ion templating, or blending with other materials to provide a variety of specific properties for new and targeted applications (kluczka, 2024). it’s excellent film­forming ability makes chitosan an effective edible surface coating for fruits and vegetables. this study investigate the effects of rosehip oil, arabic gum, and chitosan on the vase life and visual appeal of anthurium andraeanum cv. fire cut leaves. 2. materials and methods this experiment was carried out at antoniades gardens, ornamental plants research and landscape gardening department, horticulture research institute, agriculture research center, alexandria, egypt, in the years of 2022 and 2023. plant materials and treatments on march 29, 2022, and march 31, 2023, during the first and second seasons, anthurium andraeanum leaves were sourced from a well­known commercial nursery. the leaves were transported to the laboratory under dry conditions, and the petioles were re­cut to a length of 27 cm before treatment. preliminary experiment was done by using several concentrations of rosehip (rh) oil, arabic gum (ag), chitosan (cs) and their combinations. seven treatments with the higher vase life and best appeal beside control were chosen to do this experiment. treatments included: i. tap water (control); ii. rh oil (2%); iii. rh oil (4%); iv. rh (2%) + ag (5%); v. rh oil (4%) + ag (5%); vi. rh oil (2%) (5%) + cs (500 ppm); vii. rh oil (4%) + ag (5%) + cs (500 ppm). all treatments included the addition of tween 80 (2%). the anthurium leaves were sprayed with the different treatments using a hand sprayer until runoff occurred. afterward, the leaves were placed in glass jars containing 500 ml of tap water to complete their shelf­life period. the leaves were maintained at the average rida and taha ‐ quality improvement of anthurium andraeanum cut leaves by natural components 85 temperature of 19o to 23o, average humidity (59­65%) and 24 hours fluorescent light (about 550 ­575 lux). data collection on postharvest characteristics vase life (vl) expressed in days. anthurium leaf was excluded when about 20% of its surface was yellow. this stage was considered the end of the potential valuable longevity of the cut leaf. loss of leaf fresh weight percentage (llfw). it was set at the end of vase life as expressed by the following formula: llfw (%)= (initial leaf fresh weight ­ final leaf fresh weight) × 100 (initial leaf fresh weight ) final water uptake (fwu) expressed in grams. four evaporation control jars [jars which did not contain any leaves] were located between these containg leaves at different places. the evaporation of each jar was taken and the average of the four jars was calculated. it was calculated at the end of the experiment using the following formula: fwu (g) = fwu (g) = [ass ­ase] ­ ed where ass is the amount of solution at the start of the experiment, ase is the amount of the solution remaining at the end of the experiment and ed is the average of evaporation data. leaf fresh weight/leaf dry weight ratio (lwr). at the end of the experiment, the leaves were oven dried at 72oc for 48 hours for a constant weight to get the leaves dry weight. the fresh weight was then divided by the dry weight based on the following equation: lwr = fresh weight per leaf (g) ×100 dry weight per leaf (g) the amount of transpired water from the leaf surface (tw) expressed in grams. after 2, 4 and 6 days from the beginning of experiment, the transpired water from the leaf surface was calculated as given by the formula below: tw (g) = {wf + jw + wvs at (day n )} – {wf +jw+ wvs at day (n+1)})/(ifw) where wf is the weight of the leaf, jw is jar weight, wvs is the weight of vase solution, ifw is initial leaf fresh weight, n = 1, 3 and 5 days and n+1 is the next day. where n = 1, 3 and 5 days and n+1 is the next day. relative fresh weight (rfw). fresh weight of the leaves was set just before the immersion of the leaves into the solutions and recorded on the 2nd, 4th , 6th , 8th, 14th, 20th , 26th and 30th day from the beginning of the experiment. the fresh weight of each leaf was expressed relative to the initial weight to represent the water status of the leaf as follows: rfw = wt ×100 w0 where (wt) the weight of leaf (g) on the 2nd, 4th, 6th, 8th, 14th, 20th, 26th and 30th day from the beginning of the experiment, (w0) the initial fresh weight of the same leaf (g). vase solution uptake rate (vsur). it was measured according to the following formula: vsu rate = (st ­ 1) ­ st x 100 ifw of stem where (st) is the weight of vase solution (g) after 2, 4, 6, 8 and 10 days from the beginning of experiment, (st­1) is weight of the vase solution (g) on the previous day and (ifw) is the initial fresh weight (g). chlorophyll content. samples from each treatment were collected at the end of the vase life of the control treatment, and the amounts of chlorophyll a and b (mg/100 g fresh leaf weight) were determined according to moran (1982). appearances and glossiness. on the 6th day from the experiment start. a jury of twenty members from different age groups evaluated the leaves visually for its glossiness and scored on a scale (not glossy, moderate glossy, glossy and high glossy) and its appearance and scored a scale (bad, good and very good). visualization of stomatal apparatus by scanning electron microscope )sem). at the end of the first experiment season, the treatments which led to the longest vase life were chosen to investigate its effect on stomata structure. anthurium leaves were sprayed with these treatments beside tap water. after 24 hours of the treatment, small pieces of fresh specimens of anthurium leaves were removed and fixed by immersing them immediately in 4f1g (fixative, phosphate buffer solution) ph=7.4 at 4oc for 3 hours. specimens were then post fixed in 2% osmium tetroxide (oso4) in the buffer at 4oc for 2 adv. hort. sci., 2025 39(2): 83­92 86 hours. samples were washed in the buffer and dehydrated at 4oc through a graded series of ethanol. samples of anthurium leaves were dried by means of critical point method , mounted using carbon paste on an al­ stub and coated with gold up to thickness in a sputter coating unit (jfc­1100e). observation of stomata morphology in the coded specimens were performed in a jeol jsm ­ 5300 scanning electron microscope operated between 15 and 20 kev. the examination by electron microscope was done at the electron microscope unit at the faculty of science, alexandria university, egypt (tahmasebi et al., 2015 ) experimental layout and statistical analysis the experimental layout was a complete randomized design (crd). it consisted of seven treatments with three replicates, and each replicate contained four cut leaves. the means of the different variables were compared using the “least significant difference (lsd)” test at 5% level of probability. (snedecor and cochran, 1989). 3. results post harvest parameters the postharvest parameter results are summarized in table 1. vase life data show that all treatments outperformed the control. the most notable increase in vase life occurred with the application of 4% rh oil in both seasons, with values of 33.22 days in 2022 and 33.28 days in 2023. in contrast, the lowest vase life was observed with the combination of 2% or 4% rh oil + 5% ag + 500 ppm cs, which had a significance level value comparable to the control in both seasons. the greatest reduction in llwt percentage was achieved with 4% rh oil, which resulted in 1.62% in 2022 and 1.88% in 2023. the highest increase in lfwt was found with the combination of 2% or 4% rh oil + 5% ag + 500 ppm cs or 4% rh oil + 5% ag, yielding similar significant results in both seasons. the highest final water uptake was recorded with 2% or 4% rh oil, with no significant difference between the two concentrations across both season. additionally, the greatest increase in lwr was observed with 4% rh oil, with values ranging between 5.36 in 2022 and 5.24 in 2023. table 2 revealed that during the first six days of the experiment the greatest significant increase in transpiration rate fluctuated between the control and application of either rh oil (2%) + ag (5%) + cs (500 ppm) or rh oil (4%) + ag (5%) + cs (500 ppm) in both experimental seasons. two days after experiment started, in 2022 season, the application of rh oil (4%) + ag (5%) + cs (500 ppm) resulted in the highest significant increase in transpiration rate, with a recorded value of 0.3391. in 2023 season, the highest significant increase in transpiration rate was obtained following control treatment, which recorded 0.3493, and rh oil (4%) + ag (5%) + cs (500 ppm), which recorded 0.3411 with the same level of significance. four days after the experiment began, the untreated control showed the largest significant rise, with results of 0.2469 and 0.3215 for 2022 and 2023 seasons , respectively. furthermore, the application of rh oil (4%) + ag (5%) + cs (500 ppm) in table 1 ­ effect of foliar application of rosehip oil, arabic gum, and chitosan on vase life, fresh weight loss, final water uptake, and fresh/dry weight ratio of anthurium leaves during the 2022 and 2023 seasons treatment vl (days) llfw (%) fwu (g) lwr (%) 2022 2023 2022 2023 2022 2023 2022 2023 control 10.55 10.78 4.90 4.64 19.95 23.51 4.43 4.51 rh oil (2%) 26.10 23.00 6.07 6.01 47.06 49.95 4.80 4.77 rh oil (4%) 33.22 33.28 1.62 1.88 50.79 57.61 5.36 5.24 rh oil (2%) + ag (5%) 17.87 17.83 7.22 6.27 23.01 28.46 4.81 4.46 rh oil (4%) + ag (5%) 17.89 18.67 10.88 10.77 22.28 26.50 4.89 4.69 rh oil (2%) + ag (5%) + cs (500 ppm) 13.11 14.17 11.56 10.74 25.08 25.03 4.26 4.46 rh oil (4%) + ag (5%) + cs (500 ppm) 12.89 12.00 13.71 12.48 26.97 18.82 4.17 4.37 lsd at 0.05 5.66 5.38 3.23 2.14 12.15 14.53 0.18 0.06 lsd at 0.05 = least significant difference test at 5% level of probability. rh oil = rosehip oil; ag = arabic gum; cs = chitosan. vl = vase life; llfw = loss of leaf fresh weight percentage; fwu = final water uptake; lwr = leaf fresh weight/leaf dry weight ratio. rida and taha ‐ quality improvement of anthurium andraeanum cut leaves by natural components 87 for rfw, figure 1 showed that the untreated leaves had the greatest rise in rfw% on the 2nd day after the experiment began. while the largest drop in rfw was recorded following application of rh oil (4%), ag (5%), and cs (500 ppm). also, figure 1 revealed a minor decline in rfw% following all treatments, with the exception of rh oil (4%) which increased slightly in the season of 2022, but for 2023, all treatments caused a slight decrease in rfw% on the 4th day after the experiment began. the drop in rfw% persisted marginally on the 6th day after start of the experiment for all treatments except rh oil (4%) + ag (5%) + cs (500 ppm), which declined considerably in both seasons. on the 14th day of the experiment, the rh oil (2%) + ag (5%) + cs (500 ppm) and rh oil (4%) + ag (5%) + cs (500 ppm) treatments ended, and the rfw% continued to decline marginally following the use of rh oil (2%) in both experimental seasons. on the 20th day, all treatments were discontinued with the exception of rh oil 2% and 4%, and rfw% decreased gradually in 2022 season and 2023 season. on the 26th and 30th days after the trial began, the reduction in rfw% persisted somewhat. figure 2 shows that the control treatment had the greatest rise in vsur on the 2nd day of the experiment, and this increase persisted on the 4th day of the experiment when compared to the other treatments. the vsur of control treatments declined dramatically on the 6th and reached its lowest point on the 10th of the experiment in the 2022 and 2023 seasons. while the lowest vsur value was observed on the 2nd day following the application of rh oil (4%) treatment, the drop in this value over the first 10 fig. 1 ­ effect of foliar application of rosehip oil, arabic gum, and chitosan on the relative fresh weight (rfw%) of anthurium cut leaves during the 2022 and 2023 seasons, measured at 2, 4, 6, 14, 20, 26, and 30 days after the start of the experiment. table 2 ­ effect of foliar spray with rosehip oil, arabic gum, and chitosan on the amount of water transpired from the surface of anthurium leaves during the 2022 and 2023 seasons, measured at 2, 4, and 6 days after the start of the experiment treatment tw (g) 2022 2023 2 days 4 days 6 days 2 days 4 days 6 days control 0.3320 0.2469 0.1437 0.3493 0.3215 0.1570 rh oil ( 2%) 0.2173 0.1482 0.1350 0.2478 0.1690 0.1447 rh oil (4%) 0.1928 0.1638 0.1075 0.2316 0.1659 0.1343 rh oil (2%) + ag (5%) 0.2574 0.1677 0.1401 0.2781 0.1694 0.1146 rh oil (4%) + ag (5%) 0.1705 0.1287 0.1042 0.1616 0.1190 0.1194 rh oil (2%) + ag (5%) + cs (500 ppm) 0.3228 0.2302 0.1736 0.3343 0.1852 0.1822 rh oil (4%) + ag (5%) + cs (500 ppm) 0.3391 0.1830 0.1425 0.3411 0.1648 0.1482 lsd at 0.05 0.0029 0.0004 0.0003 0.0033 0.0007 0.0002 lsd at 0.05 = least significant difference test at 5% level of probability. rh oil = rosehip oil; ag = arabic gum; cs = chitosan. tw = amount of transpired water from the leaf surface. both seasons produced the greatest significant rise six days after the experiment began, with values of 0.1736 in 2022 season and 0.18822 in 2023 season. 88 adv. hort. sci., 2025 39(2): 83­92 days of the experiment persisted with a gradual rhythm in both experimental seasons. table 3 shows that the control treatment resulted in the lowest significant levels of leaf pigments. in contrast, the application of 4% rh oil led to the most substantial increase in chlorophyll a and b levels in both seasons. leaves appearance and glossiness figure 3 shows that all jury members agreed the glossiness of anthurium cut leaves was high when treated with rh oil (2% or 4%) combined with ag fig. 2 ­ effect of foliar application of rosehip oil, arabic gum, and chitosan on vase solution uptake rate (vsur) of anthurium cut leaves during the 2022 and 2023 seasons, measured at 2, 4, 6, and 10 days after the start of the experiment. table 3 ­ effect of foliar spray with rosehip oil, arabic gum, and chitosan on leaf pigment content in anthurium leaves during the 2022 and 2023 seasons lsd at 0.05 = least significant difference test at 5% level of probability. rh oil = rosehip oil; ag = arabic gum; cs = chitosan. tw = amount of transpired water from the leaf surface. treatment chlorophyll (mg / 100 g fw) a b 2022 2023 2022 2023 control 1.05 1.19 0.63 0.80 rh oil (2%) 2.03 2.11 1.07 1.07 rh oil (4%) 2.44 2.26 1.41 1.59 rh oil (2%) + ag (5%) 2.17 1.96 1.20 1.02 rh oil (4%) + ag (5%) 2.09 1.98 1.04 1.19 rh oil (2%) + ag (5%) + cs (500 ppm) 1.78 2.10 0.91 1.04 rh oil (4%) + ag (5%) + cs (500 ppm) 1.87 1.69 1.06 0.92 lsd at 0.05 0.10 0.11 0.03 0.04 fig. 3 ­ percentage of jury evaluations for glossiness of anthurium leaves following foliar application of rosehip oil, arabic gum, and chitosan, compared to untreated (control) leaves. (5%) and cs (500 ppm), as well as with 4% rh oil alone. in contrast, untreated leaves were rated as not glossy. additionally, figure 3 indicates that 60% of jury members rated the glossiness of leaves treated with 2% rh oil alone as high. figure 4 shows that the overall opinion of the jury regarding the appearance quality of anthurium cut leaves treated with rh oil (2% or 4%) was very good. however, the application of rh oil (2% or 4%) in combination with ag (5%) and cs (500 ppm) resulted in the development of brown patches on the leaves, rida and taha ‐ quality improvement of anthurium andraeanum cut leaves by natural components 89 leading to a poor appearance rating for these treatments, as shown in figures 4 and 5. visualization of stomatal apparatus scanning electron micrographs of the abaxial surface of anthurium leaves (fig. 6) showed that fully open stomata were observed in untreated control leaves. moderately open stomata were seen following application of 2% rosehip oil, while slightly open stomata appeared after spraying with 4% rosehip oil. in contrast, stomata were completely closed after treatment with rosehip oil at 2% or 4% combined with 5% arabic gum. furthermore, close­up scanning electron fig. 5 ­ effect of foliar application of rosehip oil, arabic gum, and chitosan on the glossiness and appearance of anthurium leaves on the 6th day after the start of the experiment, during the 2023 season. fig. 4 ­ jury evaluation percentages of anthurium leaf appear­ ance after foliar treatments with rosehip oil, arabic gum, and chitosan, in comparison to control (untreated) leaves. fig. 6 ­ scanning electron micrographs (sem) of the abaxial sur­ face of anthurium leaves 24 hours after spraying with: (a) control; (b) 2% rosehip oil; (c) 4% rosehip oil; (d) 2% rosehip oil + 5% arabic gum; (e) 4% rosehip oil + 5% arabic gum. micrographs were taken at 600× magnifica­ tion; scale bar = 20 μm. adv. hort. sci., 2025 39(2): 83­92 90 micrographs of individual stomata after different treatments (fig. 7) revealed that the largest stomatal pore (width: 2.18 µm, height: 24.37 µm) was found in untreated leaves. a moderately sized pore (width: 1.438 µm, height: 12.87 µm) was recorded after treatment with 2% rosehip oil, while the smallest pore (width: 1.318 µm, height: 8.038 µm) was observed after treatment with 4% rosehip oil. stomatal closure was evident following application of rosehip oil at 2% or 4% in combination with 5% arabic gum. 4. discussion and conclusions stomata regulate the diffusive conductance of leaves, influencing both carbon assimilation and transpirational water loss. their response is critical in maintaining the balance between water supply and atmospheric demand. under low humidity conditions, for instance, reduced leaf water content triggers stomatal closure to limit transpiration and preserve internal water status (thomas, 2005). in the present study, foliar application of rosehip oil induced partial stomatal closure (figs. 6 and 7), leading to decreased water loss through transpiration (table 2). this effect contributed to improved water­ use efficiency and leaf turgor maintenance, as reflected in the modest reduction in relative fresh weight during the experimental period (fig. 1) and the gradual decline in vase solution uptake rate over the first 10 days (fig. 2). the slight decrease in final fresh weight percentage (table 1) may also be attributed to this stomatal behavior. these findings are consistent with previous reports identifying stomatal regulation as a key determinant of water loss in cut flowers (fanourakis et al., 2016; in et al., 2016; schroeder and stimart, 2005). this stomatal response may be further explained by the chemical composition of rosehip oil, which contains a range of fatty acids ­ including myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidic acids (paladines et al., 2014). arachidic acid, in particular, has been associated with the endogenous synthesis of salicylic acid (sa), a signaling molecule known to induce stomatal closure, enhance membrane stability, and reduce lipid peroxidation (coquoz et al., 1995; hakimeh, 2012). during the first six days, high water loss through transpiration led to a strong drop in relative fresh weight (fig. 1), reduced water uptake, shorter vase life, and faster chlorophyll breakdown (table 1). in untreated leaves, this continuous water loss after detachment caused the flowers to lose freshness earlier and wilt faster. these results confirm that keeping good stomatal control after detachment is important to reduce water loss and maintain vase life fig. 7 ­ scanning electron micrographs (sem) of a single stoma on the abaxial surface of anthurium leaves 24 hours after spraying with: (a) control; (b) 2% rosehip oil; (c) 4% rose­ hip oil; (d) 2% rosehip oil + 5% arabic gum; (e) 4% rose­ hip oil + 5% arabic gum. micrographs were taken at 2000× magnification; scale bar = 10 μm. rida and taha ‐ quality improvement of anthurium andraeanum cut leaves by natural components 91 (salunkhe et al., 1990). treatment with 4% rosehip oil markedly improved leaf longevity and freshness, likely by limiting early transpiration (table 2), sustaining vase solution uptake over time (fig. 2), and reducing fresh weight loss (table 1). the increased chlorophyll a and b contents further supported better color retention and extended vase life, aligning with the findings of rida (2019) on aster new york. application of 4% rosehip oil as a foliar spray on anthurium leaves is recommended, as it enhanced vase life, final water uptake, and chlorophyll a and b levels, while also promoting healthier and glossier leaf appearance. references ali a., maqbool m., ramachandran s., alderson p.g., 2010 ­ gum arabic as a novel edible coating for enhancing shelf‐life and improving the postharvest quality of tomato (solanum lycopersicum l.) fruit. ­ postharvest biol. technol., 58(1): 42­47. anand m., sankari a., 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of solanaceous crops, including potato, tomato, tobacco, and other cultivated or uncultivated solanaceae (cameron et al., 2002). due to the economically important damage it causes, this pest has a long history of exposure to a broad array of synthetic insecticides. not surprisingly, p. operculella has developed resistance to many of these insecticides, including chlorinated hydrocarbons, organophosphates, carbamates, and pyrethroids (dillard et al., 1993; symington, 2003). in the past decade, several classes of conventional insecticides have emerged that show great promise for controlling p. operculella (edomwande et al., 2000; saour, 2008; clough et al., 2010). spinosad is a naturally-derived biorational insecticide with a relatively benign toxicology profile (aydin and gürkan, 2005). it is comprised primarily of two macrocyclic lactones, spinosyn a and d, secondary metabolites produced by the actinomycete, saccharopolyspora spinosa, under natural fermentation conditions (thompson et al., 2000). insects ingesting spinosad experience paralysis caused by rapid excitation of the nervous system through binding to the nicotinic acetylcholine and/or gamma-aminobutyric acid (gaba) receptors (salgado, 1998). currently, spinosad is registered in over 60 countries and is used to control lepidoptera pests in many vegetables, fruits, and field crops (legocki et al., 2010; wang et al., 2013). emamectin benzoate (an epi-methyl amino derivative of abamectin) is a second-generation avermectin analog with exceptional activity against lepidopterous on a variety of vegetable crops worldwide (ioriatti et al., 2009). avermectins (a 16-membered family) are naturally occurring macrocyclic lactones isolated from fermentation products of the soil micro-organism streptomyces avermitilis (ishaaya et al., 2002). emamectin benzoate causes irreversible activation of chloride channels in the nervous system of insects. shortly after contact or feeding exposure, the insect larvae stop feeding, become irreversibly paralyzed and die in three to four days. due to its rapid photodegradation by sunlight, contact activity of emamectin benzoate against insect predators or parasites is limited to a very short period, allowing selective control of some lepidoptera pests (sechser et al., 2003). chromafenozide is a relatively novel insecticide against lepidopteran larvae characterized by a methylchromane moiety in its dibenzoylhydrazine structure. its mechanism of action is similar to nonsteroidal ecdysone agonists known as an insect-specific ecdysis hormone (nakagawa, 2005). although this compound is very toxic to insects, it is safe for mammals and is environmentally benign (european food safety authority, 2013). chromafenozide on plants is ingested orally by insect larvae to exhibit an ecdysis-promoting activity and lead to death. it is effective in controlling various lepidopterous pests biorational insecticides against the potato tuber moth (lepidoptera: gelechiidae) on stored potatoes g. saour, h. ismail, i. jassem, s. tamer department of biotechnology, atomic energy commission of syria, po box 6091, damascus, syria. key words: larvicidal, ovicidal, phthorimaea operculella, potato storage, reduced-risk insecticides. abstract: this study was conducted to evaluate the residual activity and efficacy of spinosad, emamectin benzoate, and chromafenozide on potato tuber moth, phthorimaea operculella. almost 0% egg hatch of 1-1.5 and 4-4.5-day-old eggs occurred when eggs were treated topically with spinosad at a concentration of 216 mg/l. no ovicidal activity was observed when emamectin benzoate and chromafenozide were tested against the eggs at concentrations of 5, 10, 15 and 37.5, 75 mg/l. spinosad and emamectin benzoate were equally highly toxic to larvae (100% mortality) even when they were used at low rates. a relatively small proportion of f1 adults (≈11 to 20%) emerged in the chromafenozide treatment at concentrations of 37.5 and 75 mg/l. one hundred percent larval mortality was noted when potato tubers were sprayed with spinosad and emamectin benzoate and stored for at least 90 days after application. whereas, chromafenozide applied at 75 mg/l was effective in reducing moth emergence, exhibiting activity for 14 days only after application; thereafter a similar number of f1 adults occurred in chromafenozide and control treatments. thus, spinosad and emamectin benzoate could be used to efficiently protect potato tubers from p. operculella infestation for three months in unrefrigerated rustic potato stores. adv. hort. sci., 2014 28(3): 146-152 (1) corresponding author: ascientific@aec.org.sy received for publication 19 june 2014 accepted for publication 13 november 2014 147 (i.e. tortricidae, pyralidae, noctuidae) on vegetables or other agricultural plants (yanagi et al., 2006). in reviewing the literature, no experimental data were found related to the direct contact or residual activity of these insecticides against p. operculella under storage conditions. thus, the current study was performed to determine the potential effects of three insecticides spinosad, emamectin benzoate, and chromafenozide on the embryonic and early larval stages of the potato tuber moth. moreover, this study was designed to evaluate the residual activity of the insecticides tested at various intervals up to 90 days after application. 2. materials and methods insects insects used in the experiments were obtained from a laboratory stock culture, which is supplemented each year with field-collected p. operculella larvae (infested tubers). larvae were reared on wax-coated potato slices placed in plastic containers (40x25x10 cm). the newly emerged moths were collected and confined in 800 ml transparent plastic jars (10-12 pairs in each jar). a band of filter paper was added to the bottom of each jar for oviposition and 10% sucrose solution was presented as a food source. the rearing procedures were conducted at a constant temperature of 25±1°c with 70±5% relative humidity (rh) and a photoperiod of 12:12 h (l:d). insecticides the commercially formulated insecticides used in the present study were spinosad (spintor® 2 sc, 240 mg [ai]/ ml), emamectin benzoate (proclaim® 05 sg, 50 g [ai]/kg), and chromafenozide (matric® 5% sc, 50 g [ai]/l). ovicidal effect after the copulation and oviposition period, the first batch of eggs deposited on the oviposition support (white filter paper) from each pair were removed, counted and approximately divided into two groups. the eggs were counted with the aid of a binocular stereo microscope (kyowa optical sdz-pl, japan). the first group was used for insecticide treatments and the second served as a control. eggs were kept in a small transparent plastic box (4x3x2 cm) and held at a constant temperature of 25±1°c with 70±5% rh, until used. treatments were prepared by diluting each commercial formulation of the aforementioned insecticides in 1 l water. p. operculella eggs aged 1-1.5 and 4-4.5 days deposited on the oviposition support were dipped for 30 s in three different concentrations 72, 144, 216 ml/l and 5, 10, 15 mg/l for spinosad and emamectin benzoate, respectively (0.3, 0.6, 0.9 ml/l of spintor and 0.1, 0.2 and 0.3 g/l of proclaim); while chromafenozide was applied at two concentrations of 37.5 and 75 mg/l (0.75 and 1.5 ml/l of matric). the concentrations used did not exceed the manufacturer’s recommended application rates. a non-ionic organic surfactant (agral® 600 g/l nonyl phenol ethylene oxide condensate) was added at 0.15 ml/l as a wetting agent to ensure good dispersal of the preparation. after treatments, the eggs were air-dried and held for incubation. a 0.15 ml/l surfactant solution was used as a control in the experiments. seven days following treatment the percentage of eggs hatch was recorded. the experiment consisted of three replicates for each concentration of each insecticide with a set of 1000 eggs per replicate for each age group. larvicidal effect experiment 1. healthy, medium-sized potato tubers of 130-150 g weight (n≈150) were sprayed to runoff with spinosad, emamectin benzoate, and chromafenozide including surfactant at the above-mentioned concentrations, while the untreated tubers (control) were misted with surfactant solution. all tubers were then allowed to air dry at room temperature. after treatments, tubers were deposited over a layer of sand in transparent plastic containers (20x12x10 cm, three tubers per container). first-instar of p. operculella larvae (<16 h old) were gently placed on the treated and untreated potato tubers (three larvae per tuber) using a fine camel-hair brush and held at the rearing conditions described above. the number of emerged f 1 adults was recorded. the experiment was conducted three times using a total of 1080 larvae (45x3 larvae for each concentration of each insecticide). experiment 2. the experiment was carried out to determine whether or not larvae hatched from insecticide-treated eggs are able to survive if they are allowed to develop on untreated tubers (from a practical point of view this is only possible when insecticide-treated and untreated potato heaps were stored together). as described above, 1 1.5day-old eggs on the filter paper bands were dipped for 30 s in the respective insecticide preparations, air-dried, cut to small sections containing ten eggs and fixed on the surface of uninfested potato tubers using mini paper pins (one egg section per tuber). eggs of the control were treated with surfactant-added water solution (0.15 ml/l). the tubers were placed in transparent plastic containers (three tubers per container) with a layer of sand completely covering the bottom of the container and held at the rearing conditions. the tubers were visually inspected and the number of emerged f 1 adults was noted. the experiment was repeated three times for each concentration of each insecticide with 120 eggs per replicate. residual activity four heaps (≈50 kg each) of healthy, medium-sized potato tubers were thoroughly sprayed with the respective insecticide preparations at the highest concentrations 216, 15 and 75 mg/l for spinosad, emamectin benzoate, and chromafenozide, respectively. water/surfactant solution was used for the control treatment. after drying, the tubers were stored in the dark at room temperature (≈23°c) in order to reduce tuber weight loss and the accumulation of solanine (haddadin et al., 2001; gachango et al., 2008). 148 newly hatched larvae were placed on the tubers at 0, 7, 14, 21, 28, 35, 42, 49, 60 or 90 days after insecticide applications (three larvae per tuber and three tubers were placed in each plastic container). the number of emerged f 1 adults was recorded. three replicates were retained for each stored period of each insecticide with 45 larvae per replicate. statistical analysis differences in egg hatchability and emergence of f 1 adults were tested by the analysis of variance (anova) at the 5% level (p<0.05). significant anovas were followed by the protected least significant difference (plsd) at α < 0.05. differences in egg hatch between 1-1.5 and 4-4.5-day-old eggs were determined using paired-samples t-test (statview version 4.02; abacus concepts, 1994). data were arcsine transformed prior to analysis to stabilize the variance. dose-mortality responses were estimated by probit analysis (ibm, 2010). percentages of egg hatching and adult emergence were corrected according to schneider-orelli’s (kroschel and koch, 1996) formula: % m = (b k/100 k) x 100 where m = corrected %, b = % observed in the treatment, and k = % observed in the control. 3. results among the insecticides tested, ovicidal activity was observed only in spinosad preparations (table 1). spinosad at the median and high concentrations (144 and 216 mg/l) was very effective in controlling p. operculella egg hatch compared to emamectin benzoate, chromafenozide and control treatments. there were no significant differences in egg hatchability between emamectin benzoate and chromafenozide used at 5 and 37.5 mg/l and the control for 1-1.5-days-old eggs (f= 476.2; df= 8, 261; p< 0.0001). the lc 99s for 1-1.5-day-old eggs were 240.81, 97.17 and 530.32 mg/l for spinosad, emamectin benzoate and chromafenozide, respectively. 1-1.5-dayold eggs were more sensitive to emamectin benzoate and chromafenozide than 4-4.5-day-old eggs (t= 6.3; df= 29; p<0.0001 for emamectin benzoate used at 15 mg/l concentration). a drastic reduction in the percentage of f 1 emerged adults was observed when insecticide-treated tubers were offered to p. operculella neonate larvae (table 2). adult emergence was completely inhibited in spinosad and emamectin benzoate treatments compared with 72.2% in the control (f= 474.2; df= 8, 126; p<0.0001). however, 20.3 and 11.3% of f 1 adults successfully emerged in chromafenozide treatment at 37.5 and 75 mg/l, respectively. significant differences were observed in the mean percentage of f 1 adults emerged from larvae that hatched from insecticide-treated eggs and fed on untreated potatoes compared to the control (f= 1616.5; df= 8, 99; p<0.0001) (table 3). however, no f 1 adults emerged with spinosad treatment at 0.6 and 0.9 mg/l, while 13.4 and 18.3% of adult emergence was noted in emamectin benzoate and chromafenozide at 0.1, and 37.5 mg/l, respectively. the time-dependent efficacy of the tested insecticides against p. operculella is presented in table 4. the residual activity of spinosad and emamectin benzoate used at 216 and 15 mg/l remained unchanged (zero f 1 adults table 1 mean (±se) hatchability of potato tuber moth eggs of two age groups treated topically with spinosad, emamectin benzoate, and chromafenozide insecticides insecticides chemical group active ingredient mg/l % of hatched eggs (z) 1-1.5 day old egg 4-4.5 days old egg (y) control 91.9±4.2aa 94.1±3.9aa spinosad spinosyns 72 12.7±3.9ac 15.8±3.8ac (spintor® 2 sc, 240 mg/ml) 144 0.0±0.0ac 6.0±1.5bd 216 0.0±0.0ac 1.6±0.4ad emamectin benzoate avermectins 5 86.8±5.3abb 93.7±3.3aa (proclaim® 05 sg, 50g/kg) 10 78.6±4.0bab 87.4±4.5aab 15 70.3±5.1bb 86.3±5.1ab chromafenozide non-steroidal 37,5 84.6±5.0bab 91.6±4.1aab (matric® 5% sc, 50g/l) ecdysteroid agonist 75 76.9±3.9bb 84.7±4.8ab (z) means in row for each egg age followed by the same uppercase letter are not significantly different (p < 0.05, t-test); means in column for each egg age followed by the same lowercase letter are not significantly different (p < 0.05, fisher plsd). (y) data were corrected according to schneider-orelli’s formula and arcsine transformed prior to analysis; mean of three replicates, 1000 eggs per replicate for each insecticide and concentration. 149 table 2 mean percentage (±se) of potato tuber moth f 1 adults emerged from spinosad-, emamectin benzoate-, and chromafenozide-treated and untreated potato tubers insecticides chemical group active ingredient mg/l f 1 emerged adults (%) (z) control 72.2±7.84 a spinosad (spintor® 2 sc, 240 mg/ml) spinosyns 72 0.0±0.0 d 144 216 emamectin benzoate (proclaim® 05 sg, 50 g/kg) avermectins 5 0.0±0.0 d 10 15 chromafenozide (matric® 5% sc, 50 g/l) non-steroidal 37,5 20.3±2.1 b ecdysteroid agonist 75 11.3±2.8 c (z) means in column followed by the same letter are not significantly different (p<0.05, fisher plsd); data were corrected according to schneiderorelli’s formula and arcsine transformed prior to analysis; mean of three replicates, 45 larvae per replicate for each concentration of each insecticide. table 3 mean percentage (±se) of potato tuber moth f 1 adults emerged from spinosad-, emamectin benzoate-, and chromafenozide-treated and untreated eggs, the hatched larvae being fed on untreated tubers insecticides chemical group active ingredient mg/l f 1 emerged adults (%) (z) control 68.9±8.4a spinosad spinosyns 72 7.1±3.1d (spintor® 2 sc, 240 mg/ml) 144 0.0±0.0e 216 emamectin benzoate avermectins 5 13.4±1.6c (proclaim® 05 sg, 50g/kg) 10 8.1±2.0d 15 0.0±0.0e chromafenozide non-steroidal 37,5 18.3±4.6b (matric® 5% sc, 50g/l) ecdysteroid agonist 75 13.1±5.1c (z) the treated eggs were at the 1-1.5 day-old egg stage; means in column followed by the same letter are not significantly different (p<0.05, fisher plsd); data were corrected according to schneider-orelli’s formula and arcsine transformed prior to analysis; mean of three replicates, 120 eggs per replicate for each concentration of each insecticide. table 4 mean percentage (±se) of potato tuber moth f 1 adults emerged from spinosad-, emamectin benzoate-, and chromafenozide-treated and untreated potato tubers several days after treatment stored period, days after insecticides application f 1 emerged adults (%) (z) controlspinosad (216 mg/ml) emamectin benzoate (15 mg/l) chromafenozide (75 mg/l) 7 0.0 ± 0.0 c 0.0 ± 0.0 c 11.7 ± 1.5 b 70.3 ± 8.9 a 14 0.0 ± 0.0 c 0.0 ± 0.0 c 23.4 ± 4.1 b 72.1 ± 9.9 a 21 62.2 ± 6.9 a 68.9 ± 7.2 a 28 65.1 ± 6.1 a 67.9 ± 7.8 a 35 68.0 ± 9.9 a 71.0 ± 9.9 a 42 65.7 ±7.8 a 66.9 ± 6.9 a 49 68.2 ± 9.8 a 69.1 ± 9.0 a 60 70.0 ± 9.9 a 71.4 ± 9.7 a 90 0.0 ± 0.0 b 0.0 ± 0.0 b 66.3 ± 7.5 a 68.0 ± 9.6 a (z) means in row followed by the same letter are not significantly different (p<0.05, fisher plsd data were corrected according to schneider-orelli’s formula and arcsine transformed prior to analysis; mean of three replicates, 45 larvae per replicate for each stored period of each insecticide. 150 emergence) for up to 90 days after application. in contrast, a severe loss in efficacy occurred at the end of the two-week test period for tubers treated with chromafenozide. significant differences were obtained when chromafenozide was compared to the other insecticides tested (f= 5714.3; df= 3, 56; p<0.0001 after 14 days of storage), indicating that spinosad and emamectin benzoate were more efficient in protecting potato tubers from p. operculella infestation than chromafenozide even during 14 days after application. 4. discussion and conclusions in general, there are few studies regarding the effect of insecticides on eggs of lepidoptera pests and most publications focus on the control of the larval stage, consequently, little information is available about the effect of insecticides on eggs of p. operculella. regardless of egg developmental stages, nearly 100% mortality (or 0% egg hatch) was obtained when eggs were treated with spinosad at high and medium concentrations (216 and 144 mg/l, respectively), which was not the case for emamectin benzoate and chromafenozide. therefore, it is obvious that spinosad has excellent embryocidal activity and the compound was able to penetrate the chorion (eggshell) and reach the developing embryos. our results concerning the efficacy of spinosad on p. operculella egg hatchability agree with several authors who found an excellent ovicidal activity of spinosad when applied against the eggs of the egyptian cottonworm spodoptera littoralis (boisd.), the cactus moth cactoblastis cactorum (berg), the cranberry fruitworm, acrobasis vaccinii riley and the diamondback moth plutella xylostella (l.) (bloem et al., 2005; temerak, 2005; wise et al., 2010; mahmoudvand et al., 2011). on the other hand, el-barkey et al. (2009) reported that a 52% reduction in percentage of egg hatching was obtained when eggs of the pink bollworm pectinophora gossypiella (saunders) were challenged with radiant sc 12% (the second generation of spinosad) at the lc 50 level. however, according to perez et al. (2007), spinosad showed no ovicidal properties when applied against eggs of the aedes aegypti (l.) mosquito. when p. operculella eggs were topically treated with emamectin benzoate and chromafenozide either at low and medium, or at high concentrations, egg hatchability was not prevented. however, at the highest concentrations, a weak ovicidal effect was noted and eggs hatching were significantly reduced by 8.3 and 10% at the concentrations of 15 and 75 mg/l for emamectin benzoate and chromafenozide, respectively. our data corroborate the results that emamectin benzoate used in field experiments at the rate of 13.5 g ai/ha had no ovicidal effect on the minute pirate bug orius albidipennis (reuter) (sechser et al., 2003). in fact, emamectin benzoate was registered as lepidopteran larvicidal insecticide, which means that the product must be ingested by lepidoptera larvae to be effective. accordingly, scarpellini (2001) reported that all larval stages of the cotton leafworm alabama argillacea hübner died 12 h after they started eating cotton leaves treated with emamectin at 9.6 g ai/ha. not surprisingly, we found no effect of chromafenozide on the mean percentage of egg hatch. chromafenozide, like other inhibitors of ecdysteroid biosynthesis, shows toxic effects against larvae of lepidopteran pests mainly via digestion (yanagi et al., 2006). in this respect, slama (1995) reported that the ecdysone agonists were completely ineffective in all methods of topical application against the ligated larvae of galleria mellonella l., manduca sexta (l.) and pieris brassicae (l.). nonetheless, kandil et al. (2012) reported that the hatchability of 1-d-old eggs of p. gossypiella was 53% when they were treated with chromafenozide at the lc 50 level compared with 97.0% in the control. our data show that the older eggs (4-4.5 days old) were relatively more tolerant to insecticides than the younger ones (1-1.5 days old). in general, the hatchability of lepidoptera eggs following insecticide treatments depends on the compound, dose, and age of eggs (gelbic et al., 2011). the relative sensitivity of newly laid eggs to insecticides compared to those of older age classes can be attributed, at least in part, to chorion hardening during the embryo development that obstructs the penetration of external products (tavares et al., 2011). p. operculella neonate larvae exhibited a high response (100% mortality) to all concentrations tested of spinosad and emamectin benzoate, however low numbers of larvae that challenged chromafenozide-treated tubers achieved their development and emerged as f 1 adults (the percentage of emerged adults was relatively high when the compound was used at the low concentration). these findings agree with the data reported by other researchers regarding the larvicidal efficacy of spinosad, emamectin benzoate and chromafenozide in controlling several lepidopteran pests (kandil et al., 2012; abouelghar et al., 2013; dong et al., 2013; nasir et al., 2013; tong et al., 2013). when p. operculella larvae hatched from spinosad-, emamectin benzoate-, and chromafenozide-treated eggs were fed on untreated tubers, the percentages of f 1 emerged adults were completely inhibited for spinosad and emamectin benzoate used at medium and high concentrations and drastically reduced for chromafenozide treatment. it seems probable that treated-egg chorion retained toxic residues to cause the death of newly hatched l1 larvae which puncture the eggshell and eat their way through while hatching. it’s worth mentioning that when we evaluated the results concerning the emamectin benzoate and chromafenozide treatments, we found that p. operculella neonate larvae had been successful at creating a small opening in the eggshell during the process of exiting the chorion; however, most of the larvae failed to survive and died without feeding in close proximity to the egg. this indicates that although these two compounds do not have ovicidal activity, they have demonstrated ovilarvicidal activity when applied topically after eggs have been laid. our data concerning the ovi-larvicidal activity 151 of emamectin benzoate and chromafenozide are congruent with studies on the effects of these insecticides and other insecticides with ecdysone mode of action on helicoverpa armigera hübner and a. vaccinii (dhadialla et al., 2005; wise et al., 2010; singh and kumar, 2012). determination of residual activity of an insecticide is essential information to protect agricultural products from re-infestation. spinosad and emamectin benzoate provide, under protected environments (complete darkness), a 100% residual control effect up to 90 days after application, while chromafenozide applied at 75 mg/l showed limited residual activity of 7 to 14 days. therefore, p. operculella larvae exposed to chromafenozide-treated tubers at 21 days following treatment continued their development and reached the adult stage. the extended period of residual activity of spinosad and emamectin benzoate could be related to the rate of their photodegradation, since these two compounds are known to undergo relatively rapid photodegradation via photolysis (primary route of degradation) that ultimately affects their residual toxicity (liu et al., 1999; jones et al., 2005; zhu et al., 2011). our insecticide-treated potatoes were stored in total darkness and this could explain the slow degradation of spinosad and emamectin benzoate under our experimental laboratory conditions. on the other hand, ditya et al. 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r² = 0.9694). in conclusion, butterhead lettuce is sensitive to excess water, as shown by morphological changes, and requires two weeks to recover after water stress. 1. introduction the vegetables need of urban communities can be met through the optimization of cultivation in urban areas. agriculture in urban areas is one of the efforts to support food sustainability (abdoellah et al., 2023). the benefits of urban farming to the food resilience of urban populations today are beginning to be recognized, especially after the covid­19 pandemic (o’hara and toussaint, 2021; murdad et al., 2022). in addition, urban farming is an effort to preserve and enhance social space, green infrastructure, and biodiversity (pradhan et al., 2023). the optimization of urban farming is also essential, especially when reviewed from an aesthetic point of view. more thoroughly, nicholas et al. (2023) stated that urban farming was very beneficial, especially in the environmental, social, and psychological contexts. the benefits of urban farming can also be seen from (*) corresponding author: blakitan60@unsri.ac.id citation: muda s.a., lakitan b., ramadhani f., juwinda, 2024 ­ butterhead lettuce growth under shallow water tables and its recovery on tropical urban ecosystem.­ adv. hort. sci., 38(4): 327­337 orcid: msa: 0000­0001­9687­8114 lb: 0000­0002­0403­2347 rf: 0009­0009­9438­3113 j: 0009­0000­9593­5682 copyright: © 2024 muda s.a., lakitan b., ramadhani f., juwinda. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 3 june 2024 accepted for publication 24 september 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16233 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-9687-8114 http://orcid.org/0000-0002-0403-2347 http://orcid.org/0009-0009-9438-3113 http://orcid.org/0009-0000-9593-5682 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 327­337 328 the nutritional security and economic perspectives. lal (2020) reported that beside being beneficial for improving environmental ecosystems, urban farming plays an important role in contributing to food and nutrition security as well as being economically beneficial. in line ebenso et al. (2022) who emphasized that developing urban farming is important in supporting nutritional security. furthermore, yuan et al. (2022) mentioned that urban farming will increase community income thereby bringing economic benefits on both micro and macro scales. climate uncertainty is an issue that must be addressed, especially in tropical ecosystems. according to sheldon (2019), climate change causes climate uncertainty that impacts ecology and evolution. as a result, this condition requires adaptation for several types of activities, one of which is activities related to agriculture. climate change has a significant impact on the availability of water on agricultural land. rainfall with high intensity is an impact of climate change. according to eccles et al. (2019), excess water is one of the impacts of climate change that can occur in the tropics. this condition causes excess water availability, so plants experience excess water stress. in a riparian wet land, similar to this study site, excess water stress can occur through flooding. several cases of excess water that negatively affect plant growth have been reported, such as tomatoes (yin et al., 2023) and brassica napus (guo et al., 2020). the efforts to find vegetable crops and their cultivation techniques under conditions of excess water stress continue to be developed. susilawati and lakitan (2019) reported that chickpea (phaseolus vulgaris l.) plants were able to grow at a water table of 20 cm below the soil surface. meanwhile, in other plants, such as tomato plants, 5 cm and 10 cm below media surface, did not reduce leaf growth rate, specific leaf weight, and leaf water content (meihana et al., 2017). recovery is an effort to restore plant growth performance after experiencing excess water stress. hud et al. (2023) stated that the recovery ability of white cabbage was considered satisfactory after experiencing excess water stress. however, each plant has its period to recover from excess water stress. nazari et al. (2019) emphasized that the longer the recovery period, the better the changes after experiencing the effects of hypoxia, especially in the roots. meanwhile, some plants show a better response after recovering from excess water stress, as has been reported in grass pea (wiraguna et al., 2021). experiments on the effect of a water table on vegetable growth, particularly on butterhead lettuce, have been few and far between. this validates the fact that vegetables are a kind of plant that is susceptible to stress. a shallow water table experiment on butterhead lettuce will provide an understanding, particularly of this lettuce’s level of tolerance to climate uncertainty, particularly in excess water conditions. the study was aimed to evaluate the growth of butterhead lettuce on several shallow water tables as well as its ability to recover afterward. 2. materials and methods research site and agroclimatic conditions the research was carried out at the jakabaring research facility in palembang, south sumatra, indonesia (104°46’44’’e and 3°01’35’s). the study began on july 18, 2023, and ended on september 16, 2023. the study site is a lowland urban area with a tropical ecosystem. the study area has entered the dry season, which is characterized by low rainfall, but air humidity is high, often exceeding 70% (fig. 1). fig. 1 ­ daily rainfall­relative humidity (a) and air temperature­ sunshine duration (b) at the research location during the research was carried out. source: meteorological, climatological, and geophysical agency, 2023. muda et al. ‐ butterhead lettuce exposed to shallow water tables 329 research protocol twenty days old butterhead lettuce seedlings were used in the study. the seedlings were transplanted into pots (27.5 cm of height and diameter). as the growing substrate, the pots were fi l led with topsoil. the plants that had been transplanted to the growing substrate were placed in an open field until 4 weeks after transplanting. fertilization was performed by npk (16:16:16) fertilizer after 3 weeks, and watering was performed regularly in the afternoon when it was not raining. in 4 weeks after transplanting, butterhead lettuce was treated with water maintaining 16.7 cm water tables (wt1), 12.7 cm (wt2), and 9.7 cm (wt3) from the substrate surface (fig. 2). each treatment was repeated 3 times. this stage was carried out in an experimental pond measuring 4 m (length) x 2 m (width) x 0.5 m (height). the pond was equipped with an outlet to allow water to flow out in the event of excessive rain. as a result, water level can be controlled based on the water table treatments. during this stage, the plants get their water from the bottom of the pot via capillary water movement, so no watering is required. after 7 days of water treatments (wt1, wt2 and wt3) butterhead lettuce was return to the open area. as additional treatments, several recovery times were treated, including no recovery, one week of recovery, two weeks of recovery, and three weeks of recovery. during this phase, butterhead lettuce was watered minimally and only when there was no rain for three days in consecutive days. data collection butterhead lettuce growth data was collected consisting of individual leaf growth, canopy diameter, canopy area, fresh weight and dry weight organ. individual leaf growth was monitored daily for length and width, starting when the leaf was fully unfolding. the canopy diameter was measured daily on the cross­sectional widest canopy to track canopy diameter growth. the butterhead lettuce canopy area and leaf number were measured weekly. the canopy area was measured using the image scanner easy leaf area software for android (easlon and bloom, 2014). meanwhile, substrate moisture was measured using a moisture meter (lutron soil moisture meter pms­714). destructive observation was conducted to collect fresh weight and dry weight data of plant organs. to obtain dry weight, each plant organ was thinned and then dried in an oven at 100°c for 24 hours. experimental design and statistical analysis the study used a randomized block design. the shallow water table treatments consisted of 16.7 cm (wt1), 12.7 cm (wt2), and 9.7 cm (wt3) from the substrate surface. all data collected were subjected to analysis of variance (anova), then significance among treatments using the least significant difference (lsd) at p<0.05. the significance of differences among treatments was also tested using independent t­test at p<0.05. the analysis was performed using rstudio (v2023.06.0+421) for windows 10 (rstudio team, pbc, boston, ma, usa). meanwhile, data trend on the selected variables were analyzed using microsoft excel for windows 10 (microsoft inc., redmond, washington, usa). 3. results individual leaf growth butterhead lettuce leaf length increased up to 5 days after leaf unfolding (dau). furthermore, beginning at 8 dau, leaf length gradually stagnated. the shallowest water table (wt3) affected the inhibited leaf length during the treatment (fig. 3). leaf widening was also observed with the shallow water table treatment. butterhead lettuce grown in wt1 produced larger leaves. meanwhile, butterheads planted at the shallowest water table (wt3) experienced inhibited leaf widening, resulting in narrower leaves. the butterhead lettuce leaves, on the other hand, continued to widen until 9 dau. as a result, the width of the leaves stagnated and experienced senescence, which caused the tips of the fig. 2 ­ the illustration of the shallow water table treatments application. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. adv. hort. sci., 2024 38(4): 327­337 330 leaves to dry out, resulting in a decrease in leaf width (fig. 4). the leaf shape is represented by the leaf length­ width ratio. leaves with a length­width ratio over one indicate elongated leaf growth. if the leaf length­ width ratio is less than one, it indicates the leaf growth has widened. there was no difference in the dynamics of changes in the shape of butterhead lettuce leaves in each water table treatment. butterhead lettuce leaves widened as they aged as a whole (fig. 5). different water table treatments influenced the growth canopy of butterhead lettuce. butterhead lettuce with the shallowest water table (wt3) fig. 4 ­ daily leaf width of butterhead lettuce on different shallow water tables. the shallow water tables (wt) consisted of wt1 (a), wt2 (b), and wt3 (c). the measurement was carried out when leaf was fully unfolded. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. fig. 3 ­ daily leaf length of butterhead lettuce on different shallow water tables. the shallow water tables (wt) consisted of wt1 (a), wt2 (b), and wt3 (c). the measurement was carried out when leaf was fully unfolded. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. muda et al. ‐ butterhead lettuce exposed to shallow water tables 331 exhibited lower canopy growth. the stunted growth of leaves in wt3 resulted in a low canopy width. in contrast, better leaf growth in wt1 and wt2, respectively, resulted in a wider canopy (fig. 6). weekly growth of butterhead on different shallow water tables the wt1 exhibited a better trend for butterhead lettuce leaf initiation than the wt2 and wt3. however, statistically, no significant difference was found between the three treatments (wt1, wt2, and fig. 5 ­ daily leaf length­width ratio of butterhead lettuce on different shallow water tables. the shallow water tables (wt) consisted of wt1 (a), wt2 (b), and wt3 (c). the measurement was carried out when leaf was fully unfolded. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. fig. 6 ­ daily canopy diameter of butterhead lettuce on different shallow water tables. the shallow water table consist of wt1 (a), wt2 (b), and wt3 (c). wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. 332 adv. hort. sci., 2024 38(4): 327­337 wt3). the leaf number grows in line with the plant’s age. the increase in leaf number follows a polynomial curve (fig. 7). the canopy area of butterhead lettuce differed among the treatments. during early vegetative growth (1 and 2 wat), wt1 exhibited the most expansive canopy area, indicating significant differences in the canopy area. the canopy of butterhead lettuce showed no difference at later ages. however, when compared to the wt2 and wt3 treatments, the trend of canopy area growth of butterhead lettuce in wt1 remained higher, with canopy area growth following a polynomial curve. there are signs that wt1’s canopy area growth has stagnated, especially after 4 wat, when the wt1 butterhead lettuce canopy area is almost the same as the wt2 treatment (fig. 8). butterhead growth performance during recovery time after recovery from water stress, the production of edible and non­edible butterhead lettuce leaves fluctuated. all treatments showed peak edible leaf production at 2 weeks after recovery (war). at this time, butterhead lettuce in wt3 has the highest edible leaf production. meanwhile, non­edible leaf production was highest in wt2 compared to the fig. 7 ­ leaf number of butterhead lettuce (a) and their trend (b) during on different shallow water table. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. the ns indicated each treatment non­significant at lsd0.05. fig. 8 ­ canopy area of butterhead lettuce (a) and their trends (b) on different shallow water table. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. the different letters on bar indicated each treatment significant different at lsd0.05. the ns indicated each treatment non­significant at lsd0.05. muda et al. ‐ butterhead lettuce exposed to shallow water tables 333 other water table treatments (wt1 and wt3) (fig. 9). additionally, according to shoot fresh weight, butterhead lettuce shoots on all shallow water tables reached their peak growth at 2 weeks after recovery (war). following the recovery time, the wt3 treatment showed improved shoot growth in fig. 9 ­ edible leaf and non­edible leaf of butterhead lettuce (a­b) and their trends (c­d) on recovery from different water tables. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. ns: non­significance different based on independent t­test at p< 0.05; *: significance different based on independent t­test at p<0.05. comparison to wt 1 and wt 2 (fig. 10). leaf estimation the butterhead lettuce leaf has a morphology with pinnate veins. the pinnate leaf blade makes it possible to assign leaf length (ll) and leaf width (lw) fig. 10 ­ fresh weight and dry weight of butterhead lettuce shoot (a­b) and their trends (c­d) on recovery from shallow water table. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. ns: non­significance different based on independent t­test at p< 0.05; *: significance different based on independent t­ test at p<0.05; **: significance different based on independent t­test at p<0.01. adv. hort. sci., 2024 38(4): 327­337 334 4. discussion and conclusions plant growth response under excess water conditions the shallower the water table, the deeper the pot is submerged, hence the less aerobic space available to the plant. aerobic space is incredibly beneficial to as primary predictors. the results showed that the combination of ll x lw using the zero­intercept linear regression type most reliably represented leaf area (r2 = 0.9694) (table 1). the physiological capacity of a leaf is determined by its leaf area. the findings revealed that increased leaf fresh weight was linearly related to increased leaf area (r2=0.8744). this suggests that larger leaves have more biomass and water. the opposite condition occurred in narrow leaves (fig. 11). water status on different water table treatment butterhead lettuce grown in the wt3 treatment receives more water than those grown in wt1 and wt2. this is an indication of excess water, as indicated by the moisture level of the substrate in each treatment. the shallow water table in wt3 causes water to fill the substrate pores faster, resulting in higher substrate moisture than in the other treatments. as a result of this condition, the aerobic space in the wt3 substrate is lower than in the wt1 and wt2 substrates (fig. 12). fig. 11 ­ relation between leaf area and leaf fresh weight of butterhead lettuce. table 1 ­ butterhead leaf estimation involve leaf length (ll), leaf width (lw), and ll x lw as predictors coefficient of determination (r2) indicated strength level of each predictor and regression predictors regression type equation r2 leaf length (ll) linear y= 5.0467(ll)­12.66 0.8674 exponential y= 2.6911e0.2656(ll) 0.7598 logarithmic y= 29.963ln(ll)­32.71 0.7534 quadratic y= 0.1327(ll)2+3.0079(ll)­5.7844 0.8752 power y= 0.6121(ll)1.7939 0.8681 zero intercept linear y= 3.5771(ll) 0.9491 zero intercept quadratic y= 0.2172(ll)2+1.5467(ll) 0.8715 leaf width (lw) linear y= 9.0909(lw)­18.948 0.8464 exponential y= 1.8915e0.4829(lw) 0.6645 logarithmic y= 38.424ln(lw)­33.461 0.7777 quadratic y= 0.118(lw)2+7.908(lw)­16.239 0.8469 power y= 0.6148(lw)2.2685 0.8055 zero intercept linear y= 5.5761(lw) 0.9312 zero intercept quadratic y= 0.7056(lw)2+1.508(lw) 0.8310 ll×lw linear y= 0.5548(ll×lw)+2.9686 0.8813 exponential y= 6.8069e0.0267(ll×lw) 0.6010 logarithmic y= 17.35ln((ll×lw)­34.847 0.7874 quadratic y= ­0.0027(ll×lw)2+0.8335(ll×lw)­2.3609 0.9039 power y= 0.5508(ll×lw)1.0323 0.8780 zero intercept linear y= 0.6076(ll×lw) 0.9694 zero intercept quadratic y= ­0.002(ll×lw)2+0.7422(ll×lw) 0.9026 muda et al. ‐ butterhead lettuce exposed to shallow water tables 335 growth. however, some plants, such as white cabbage, are potentially water­tolerant (hud et al., 2023). thus, excess water is a problem for some crops, including butterhead lettuce. in response to excess water, several approaches have been tried, including enriching co2 in the substrate (pérez­ romero et al., 2019) and utilizing the role of ethylene (khan et al., 2020). recovery as an effort to restore plant growth performance recovery by returning to open areas was aimed to restore the butterhead lettuce growth after experiencing excess stress. plant organ architecture and physiological regulation will improve as a result of recovery (yin and bauerle, 2017). depending on the level of stress, each plant requires a different amount of time to recover and return to average or near­normal growth. our observations indicate that butterhead lettuce takes 2 war to restore its growth performance after being grown in shallow water table treatments, was shown clearly in the wt3. the fresh weight of edible leaf and plant organs indicates this. in another case, nazari et al. (2019) found that even 4 days after recovery, hypoxia did not affect cicer arietinum. after recovery, each plant treated with a different shallow water tables demonstrated a different level of endurance. interestingly, the shallowest water table (wt3), which had stunted growth when treated with shallow water, had the best recovery growth. because of the residual pretreatment, the water availability in wt3 was adequate, resulting in better growth. wt3, the shallowest water table, causes the most water retention in the substrate when compared to wt1 and wt2. plants use excess water during the recovery process since they are rarely watered during this period. according to bateman et al. (2019), substrates with adequate water storage will promote plant growth. leaf area estimation and leaf morphological characterization the role of the length and width of butterhead lettuce leaf as a predictor is essential for a plant with a pinnate leaf shape. these predictors were also tested on leaves with similar leaf shapes, such as citrus (muda et al., 2023) and swiss chard (ria et al., 2023). furthermore, complex leaf shapes, such as amorphaphalus mullieri , can be estimated by considering leaf morphology (nurshanti et al., 2022). plants as a source of oxygen for many kinds of crucial metabolic activities. oxygen plays an essential role in several metabolisms in plants, including respiration, carbohydrate formation, protein synthesis, and nutrient solubility (moreno roblero et al., 2020; xu et al., 2020). oxygen plays an important role in the growth of some soil microorganisms (wichern et al., 2020). as a result, if the amount of oxygen in the pot is insufficient, plant growth will decrease. based on the results, butterhead lettuce grown at the shallowest water table (wt3) showed a stunted growth response. actually, each plant has different tolerance abilities in excess water conditions. as a consequence of excessive water stress, each plant exhibits specific symptoms. in the case of butterhead lettuce, the plants exhibited stunted leaf and canopy growth. on other hand, plant growth performance was inhibited, as evidenced by data trend of fresh weight and dry weight of edible leaf and shoot on 0 week after recovery before recovery (war) (fig. 9 and 10). plants respond to excessive water stress by changing their physiology, anatomy, and morphology (jia et al., 2021; kumar et al., 2022). according to zhou et al. (2020), plants grown under excess water, change metabolic energy, respiration, photosynthesis, and endogenous hormone regulation. hypoxia conditions further hinder plant growth. some leafy vegetables, such as tomatoes (tareq et al., 2020) and broccoli (casierra­posada and peña­ olmos, 2022), have been shown to have stunted fig. 12 ­ substrate’s water status on different shallow water table as indicated by substrate. moisture. the shallow water table consist of wt1, wt2, and wt3. wt1: 16.7 cm below the substrate surface; wt2: 12.7 cm below the substrate surface; wt3: 9.7 cm below the substrate surface. the measurement was conduct at 4 weeks after treatment. adv. hort. sci., 2024 38(4): 327­337 336 furthermore, the choice of regression type influences predictor reliability in predicting leaf area. according to the findings, the zero­intercept linear regression with the ll x lw predictor was the most dependable. the logic behind zero intercept regression is that if the predictor is 0, the leaf area will also be 0 (lakitan et al., 2022). the use of zero intercept regression in estimating leaf area has been confirmed in cassava (lakitan et al., 2023) and chaya (gustiar et al., 2023). butterhead lettuce has been proven to be intolerant of excess water in growing environments, such as at a water table of 9.7 cm from the substrate surface. butterhead lettuce that has experienced excess water stress needs recovery with the most optimal recovery time within 2 weeks. butterhead lettuce has a pinnate leaf morphology, and leaf area can be estimated using the formula y = 0.6076 leaf length x leaf width. references abdoellah o.s., suparman y., safitri k.i., mubarak a.z., milani m., surya l., 2023 ­ between food fulfi l lment and income: can urban agriculture contribute to both? ­ geography sust., 4(2): 127­137. bateman a.m., erickson t.e., merritt d.j., veneklaas e.j., muñoz­rojas m., 2019 ­ water availability drives the effectiveness of inorganic amendments to increase plant 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p., callaghan m., hu y., dahal k., hunecke c., reußwig f., campen h.l., kropp j.p., 2023 ‐ a systematic review highlights that there are multiple benefits of urban agriculture besides food. ­ global food security, 38: e100700. ria r.p., lakitan b., sulaiman f., yakup y., 2023 ­ searching for suitable cultivation system of swiss chard (subsp.(l.) wdj koch) in the tropical lowland. ­ j. hort. res., 31(1): 81­90. sheldon k.s., 2019 ­ climate change in the tropics: ecological and evolutionary responses at low latitudes. ­ annual rev. ecol, evol., systematics, 50: 303­333. susilawati., lakitan b., 2019 ­ cultivation of common bean (‘phaseolus vulgaris’ l.) subjected to shallow water table at riparian wetland in south sumatra, indonesia. ­ australian j. crop sci., 13(1): 98­104. tareq m.z., sarker m.s.a., sarker m.d.h., moniruzzaman m., hasibuzzaman a.s.m., islam s.n., 2020 ­ waterlogging stress adversely affects growth and development of tomato. ­ asian j. crop sci., 2(1): 44­50. wichern f., islam m.r., hemkemeyer m., watson c., joergensen r.g., 2020 ­ organic amendments alleviate salinity effects on soil microorganisms and mineralisation processes in aerobic and anaerobic paddy rice soils. ­ frontiers sustain. food syst., 4: e30. wiraguna e., malik a.i., colmer t.d., erskine w., 2021 ­ tolerance of four grain legume species to waterlogging, hypoxia and anoxia at germination and recovery. ­ aob plants, 13(4): plab052. xu c., chen l., chen s., chu g., wang d., zhang x., 2020 ­ effects of rhizosphere oxygen concentration on root physiological characteristics and anatomical structure at the tillering stage of rice. ­ ann. appl. biol., 177(1): 61­73. yin j., bauerle t.l., 2017 ­ a global analysis of plant recovery performance from water stress. ­ oikos, 126(10): 1377­1388. yin j., niu l., li y., song x., ottosen c.o., wu z., jiang f., zhou r., 2023 ­ the effects of waterlogging stress on plant morphology, leaf physiology and fruit yield in six tomato genotypes at anthesis stage. ‐ veg. res., 3(1): 1­9. yuan g.n., marquez g.p.b., deng h., iu a., fabella m., salonga r.b., ashardiono f., cartagena j.a., 2022 ­ a review on urban agriculture: technology, socio‐ economy, and policy. ­ heliyon, 8(11): e1183. zhou w., chen f., meng y., chandrasekaran u., luo x., yang w., shu k., 2020 ­ plant waterlogging/ flooding stress responses: from seed germination to maturation. ­ plant physiol. biochem., 148: 228­236. untitled 98 sanitary and agronomic selection of tuscan germplasm to improve wine production p. storchi*, f. giannetti*, p. valentini*, i. bazzo**, e. angelini** * consiglio per la ricerca e la sperimentazione in agricoltura, unità di ricerca per la viticoltura, arezzo, paolo.storchi@entecra.it. ** consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la viticoltura, conegliano (tv). clonal selection of grape was launched in 1985 at the operative section in arezzo of the former of experimental institute for viticulture [currently the research unit for viticulture of the council for research in agriculture (c.r.a.)]. studies focused on the search for putative clones of the most popular varieties in central italy, carrying out detailed investigations in old vineyards in different wine-producing districts of tuscany, initially in maremma and later in montepulciano, montalcino, san gimignano and chianti (armanni et al., 2010). beginning in 1990, some experimental vineyards were established with the progenies of the clones, which were tested for their health and evaluated for their vegetative and productive characteristics. the work was carried out in particular on the sangiovese cultivar (calò et al., 1995), but over the years many other varieties have been included, with particular attention to indigenous germplasm for which availability of propagation material with good sanitary status is still lacking (borgo et al., 2009). particular attention was given to the results from experimental vinifications using virus-free clones, allowing expression of their genetic properties without interferences, especially those relating to the phenolic richness and aromatic complexity of the wines. through the research it was possible to evaluate the incidence of viruses in the ranges of selection. the leafroll agents, were very common, even considering that a careful visual preselection control of symptoms was carried out. ampelovirus type 1 was identified in particular in materials from pitigliano and chianti classico areas, while ampelovirus type 3 affected, almost exclusively, selections from maremma. this virus, along with fleck, was found in significant proportions in clones under selection in the vineyards of tuscany. among virus-like diseases of minor importance, the incidence of vein necrosis was very high. the selection activity led to the registration in 2002 in the national catalogue of grape varieties of clones isv-rc1 and isv2 of sangiovese (storchi et al., 2004); in 2011 other clones of sangiovese, aleatico, vermentino, trebbiano toscano and canaiolo were registered. still other clones will be ready soon for application to the ministry of agriculture once agronomic observations and sanitary checks are complete. currently, there are clone comparison vineyards in arezzo and in the district of chianti classico and collaborations are in progress regarding the clonal selection with the “consorzio vivaisti viticoli italiani” and with the main wine cellars of tuscany. since the protocol for the selection of a clone demands a long period of time, one of the next objectives will be to reduce the time needed to secure clone registration in the national catalogue. in this sense, early selection of mother plants with stable and better features, compared to the standard variety, is crucial, as is subsequent rapid propagation of plant material to establish the fields for clone assessment and comparison in different environments. references armanni a.b., randellini l., valentini p., giannetti f., leprini m., storchi p., 2010 recupero, tutela e valorizzazione delle identità viticole dell’italia centrale. rivista di viticoltura e di enologia, 1(4): 31-41. borgo m., costacurta a., egger e., storchi p., scalbrelli g., loreti f., 2009 la selezione clonale del vitigno aleatico. atti aivv, tornata isola d’elba, pp. 99-112. calò a., costacurta a., paludetti g., crespan m., giust m., egger e., grasselli a., storchi p., borsa d., di stefano r., 1995 characterization of biotypes of sangiovese as basis of clonal selection. proc. int. symp. clonal selection, portland, pp. 99-104. storchi p., egger e., borgo m., valentini p., pieri m., 2004 contributo al miglioramento qualitativo del sangiovese in toscana: le selezioni isv. ii simp. sangiovese, firenze, pp. 347-353. adv. hort. sci., 2013 27(3): 98 184 1. introduction to address the food shortages in the near future caused by a decrease in arable land or increase in the world population, greenhouse horticultures, especially hydroponic cultures without soil, are becoming more important. while one advantage of a hydroponic culture is that plants can be cultivated under optimally controlled conditions for nutrients regardless of the soil conditions, a large amount of water is required to grow crops with nutrient solution. new hydroponic systems that enable stable crop production while saving water have been anticipated in recent years. aeroponics is a water-saving hydroponic technique without rooting media (weathers and zobel, 1992; ritter et al., 2001; farren and mingo-castel, 2006). in this culture, liquid fertilizer is periodically sprayed on the roots from nozzles with pipes in the rooting zone. the sprayed nutrient solution that is not absorbed by the roots is usually re-sprayed using a re-circulation system or it is discarded directly. tomato, one of the most important crops world-wide, has recently been cultivated using aeroponics (biddinger et al., 1998; zhao et al., 2010). one of the features of this culture system is nutrient accessibility in which nutrients are absorbed efficiently by the roots. furthermore, root growth in most vegetable crops is expected to increase under the aerobic conditions of the rhizosphere environment because there is no solid phase and less liquid phase compared to the physical composition of soil or any other rooting media (hillel and david, 1988; cherif et al., 1997). dry-fog aeroponics is a novel hydroponic technique that sprays a very fine fog of atomized liquid fertilizer using a specialized nozzle as a double-fluid atomizer with nutrient solution and compressed air (developed and patented by ikeuchi co., ltd., osaka, japan). dry-fog is the finest fog and is less than 10 µm in diameter on average per droplet. the droplets are too small to wet objects, such as roots. currently, dryfog is mainly used to humidify factories producing electronic components or printing to keep the environment static-free and has not yet been applied to crop cultivation in commercial horticulture. in dry-fog aeroponics, dry-fog fertilizer is atomized using a specialized nozzle in the rhizosphere where the plant roots directly absorb water and nutrients from the dry-fog (fig. 1), like an aerial root of epiphytic orchids. because the fine, foggy, nutrient solution is atomized, only one specialized nozzle can occupy the large space for the rooting zone in a chamber (1000×660×300 mm), with a smaller amount of water compared to the other types of predominant spray hydroponic techniques; therefore, dry-fog aeroponics can reduce water usage and the number of nozzles that are needed, resulting in a lower running cost and greater environmental conservation. the dry-fog nutrients that are not being absorbed by the roots accumulate as condensation on the inner wall of the cultivation chamber, and the collected condensation in the nutrient reservoir is atomized again. thus, there a novel aeroponic technique using dry-fog spray fertigation to grow leaf lettuce (lactuca sativa l. var. crispa) with water-saving hydroponics y. hikosaka, m. kanechi(1) , y. uno graduate school of agricultural science, kobe university, kobe 657-8501, japan. key words: ascorbic acid, hydroponics, root respiration, soilless culture, sprayponics. abstract: growth characteristics of lettuce cultivated using a novel “dry-fog” hydroponic technique were investigated and compared to lettuce cultivated using deep flow technique (dft) as the prevailing hydroponic technique. dry-fog hydroponics is an aeroponic technique that sprays a very fine foggy nutrient solution with an average droplet diameter of less than 10 µm into the root zone. the roots extend into the chamber filled with dry-fog of the liquid fertilizer and absorb water and nutrients from the dry-fog that fills the rhizosphere. this soilless culture system needs less water than any other hydroponic technique, and no differences were found in growth and harvest quality of plants between the two tested sytems. for dry-fog culture, root growth was encouraged and root hair significantly developed to catch the foggy nutrient solution efficiently. the contents of ascorbic acid, nitrate nitrogen, ca+ and chlorophyll of leaves were not significantly different between the two hydroponic cultures. however, respiration rate of roots and photosynthetic rate of leaves significantly increased with dry-fog culture. because the amount of water around the roots is less with dry-fog, horticultural crops are expected to grow well with this novel hydroponic technique which optimizes the growth and quality of plants with water-saving hydroponics. adv. hort. sci., 2014 28(4): 184-189 (1) corresponding author: kanechi@kobe-u.ac.jp received for publication 12 june 2014 accepted for publication 29 september 2014 185 is little puddling of the fertilizer solution in the chamber, and the roots hang in the dry fog. an ultrasonic humidifier also can atomize the very fine fog, and has been used for many bioreactors to generate the nutrient fog (mohammad et al., 2000; lin et al., 2003; jing and yunwei, 2013). in contrast, it is believed that the nozzle used for dry-fog aeroponics atomizes a larger amount of fine fog at a low cost and without problems. dry-fog aeroponics has not yet been investigated or established as a commercial cultivation system for horticultural crop production. in the present study, leaf lettuces were cultivated using dry-fog aeroponics in a controlled environment. the growth and leaf constituents were measured and compared to plants cultivated with traditional hydroponic techniques (deep flow technique) to evaluate the efficacy of dry-fog aeroponics. root respiration and leaf photosynthetic rate were also investigated and compared in order to evaluate affects on water and nutrient absorption resulting from differences in the rhizosphere environment in the two hydroponic systems. 2. materials and methods a plastic chamber (1000×660×300 mm) was fitted with a dry-fog atomizing nozzle to establish a dry-fog cultivation system (fig. 1). a double-fluid atomizer nozzle was connected to the nutrient solution reservoir with a plastic tube (6 mm across) and a compressor with teflon tube (8 mm across), with an air pressure of 0.3 mpa. size fractionation of atomized droplets was measured using ldsa-spr (tohnichi computer applications co. ltd., tokyo, japan), which analyzes the droplets by fraunhofer diffraction and calculates the size distribution of the droplets by rosin-rammler distribution. a commercial liquid fertilizer (otsuka chemical co. ltd., tokyo, japan, ec 1.2 ms cm-1 , ph 6.0, n: 130 ppm, p: 60 ppm, k: 200 ppm, ca: 115 ppm, mg: 30 ppm, fe: 1.4 ppm, mn: 0.8 ppm, zn: 0.05 ppm, cu: 0.02 ppm, b: 0.75 ppm, mo: 0.02 ppm) was added to the nozzle from a nutrient solution reservoir equipped at the end of an aeroponic chamber. the nozzle atomized fine foggy nutrients that continuously filled the chamber. each chamber had 48 holes (20 mm across) across the top to hold plants and only the roots remained in the chamber. as a control experiment, a deep flow technique (dft) hydroponic chamber (800×665×310 mm) was filled with 30 l of the same liquid fertilizer. leaf lettuce (lactuca sativa l. cv. okayama saradana, takii co. ltd., kyoto, japan) was sown on sponge blocks (10×10×20 mm) supplied with enough pure water. the air temperature and relative humidity throughout the cultivation were maintained at 26°c and 60% respectively. the photosynthetic photon flux density was 250 µmol m-2 s-1 supplied by cool white tubular fluorescent lamps with a 16-h day length. after germination, the liquid fertilizer (ec 0.6 ms cm-1, ph 6.0) was supplied by bottom irrigation for two weeks, and 48 seedlings that had grown uniformly were transplanted into each hydroponic system and grown for four weeks. every week, plants were sampled from both hydroponic systems and the fresh weight, leaf length, and fresh weight of roots were recorded. after sampling, the leaves and roots were dried in an oven at 80°c for two days and the dry weights were recorded. at three and four weeks after transplantation, intact root samples (1 g fw) were taken from a growing plant that developed new roots under hydroponic conditions, and the rate of root respiration was measured polarographically at 26°c using a clark-type gas-phase oxygen electrode (cb1d, hansatech, norfolk, uk) with incoming humidified air and 21% o 2 under dark conditions. the contents of nitrate nitrogen, calcium ion, ascorbic acid and chlorophyll in the leaves were measured at three and four weeks. leaves (10 g fw) were sampled to determine nitrate nitrogen and calcium contents. the leaves were homogenized in de-ionized water and the nitrate nitrogen and calcium contents in the filtrated fraction were measured using rq-flex (merck millipore, darmstadt, germany). the other leaves (10 g fw) were also sampled and homogenized in a 5% metaphosphoric acid solution, and the content of ascorbic acid in the filtrated fraction was measured using rq-flex. the content of leaf chlorophyll was measured using spad meter (spad-502, konica minolta, inc., tokyo, japan). at four weeks after planting, co 2 assimilation rates (a) of fully expanded mature leaves were measured under different co 2 concentrations (50-1500 µmol mol-1) using an li-6400 portable photosynthesis system fig. 1 a) cutaway drawing of the dry-fog hydroponic chamber. b) system chart of the dry-fog hydroponic technique. 186 (li-cor, lincoln, ne, usa). the measurement conditions were leaf temperature, 20°c, and photosynthetic photon flux densities, 1200 µmol m-2 s-1. the maximum rate (max) and apparent co 2 fixation efficiency (φ) were estimated as parameters of the best-fitted non-rectangular hyperbola for the photosynthetic responses to intercellular co 2 concentrations (ci). all data were subjected to one-way analysis of variance (anova), and the mean differences were compared using the tukey hsd test when the f-test indicated a significant difference at p≤0.01. each data point was the mean of six replicates and a comparison with p≤0.05 was considered significantly different. 3. results and discussion in order to characterize dry-fog spray fertigation conditions, the fog was measured using ldsa-spr of droplets of various sizes (fig. 2). the minimum and maximum diameter of droplets were 1.64 µm and 149 µm, respectively. ldsa-spr analyzed the size distribution of droplets, and the sauter mean diameter, which commonly means the average particle size of powders and liquid droplets, was found to be 8.85 µm by calculation. therefore, the fog in the chamber was certainly dry-fog. there was no significant difference in the fresh and dry weight of leaves between the dry-fog and dft hydroponic cultures (fig. 3). root growth was not significantly different at three weeks after planting, but during the fourth week of dry-fog culture the root fresh weight increased by 29% compared to roots grown using dft. the roots from the dry-fog culture had many lateral roots, and many root hairs developed on the surface. this development was not observed in the roots grown using dft (fig. 4). the root respiration rate at four weeks after planting was significantly high in the dry-fog culture, but there was no significant difference at three weeks between the two hydroponic fig. 4 leaves and roots of the lettuce grown using dry-fog or dft hydroponics at four weeks after transplanting. fig. 2 the particle size distribution of droplets using a double-fluid atomizer nozzle to establish a dry-fog hydroponic technique measured by ldsa-spr. the mean diameter of droplets was analyzed and calculated based on this distribution. fig. 3 changes in fresh and dry weights of lettuce leaves and roots grown using dry-fog or dft hydroponics during the four weeks after transplanting. different uppercase and lowercase letters show a significant difference (p≤0.05), bars represent ±se (n=6). 187 systems (fig. 5). the photosynthetic rates of the mature leaves grown for four weeks dry-fog culture significantly increased in both limited and saturated ci compared to those in dft (fig. 6). in both hydroponic cultures, the photosynthetic rate increased linearly up to approximately 300 µmol mol-1 ci and saturated at approximately 600 µmol mol-1 ci. leaf nutrient contents were compared between the two hydroponic cultures at three and four weeks after transplanting (fig. 7). the nitrate nitrogen content in the leaves grown using dry-fog culture significantly fig. 5 respiration rates of lettuce roots grown using dry-fog or dft hydroponics for three and four weeks after transplanting. different letters show significant difference (p≤0.05), bars represent ±se (n=6). fig. 6 relationships between co 2 assimilation rates (a) and intercellular co 2 concentration (ci) measured for the attached mature leaves of lettuce grown using dry-fog or dft hydroponics for four weeks after transplanting. the saturated rate (max) and the apparent co 2 fixation efficiency (φ) were estimated as parameters of the best-fitted non-rectangular hyperbola response curve to ci. different letters show significant difference (p≤0.05). fig. 7 nitrate nitrogen, ascorbic acid, calcium ion and chlorophyll content of mature lettuce leaves grown using dry-fog or dft hydroponics for three and four weeks after transplanting. different letters show significant difference (p≤0.05), bars represent ±se (n=6). 188 increased by 16% compared to those grown using dft at three weeks after planting, but there was no significant difference at four weeks. the content of ascorbic acid in the leaves grown using dry-fog culture increased by 9% at three weeks and 4% at four weeks, compared to the leaves grown using dft, however there were no significant differences. the calcium content in the leaves grown using dry-fog culture significantly increased at three weeks after planting, but there was no significant difference at four weeks. the chlorophyll content of mature leaves significantly increased with dry-fog culture only at four weeks. to evaluate the efficacy of dry-fog culture as a novel aeroponic system, the growth of lettuce was investigated and compared to dft hydroponic culture. one of the features of dry-fog culture is water conservation. for the systems used in this study, the dft always needed at least 30 l of liquid fertilizer, while dry-fog culture could fill a chamber with only 1 l of solution because the droplets of dry fog are very fine. in addition, the total amount of liquid fertilizer in a whole dry-fog culture system was less than 10 l. this aspect is an advantage not only in terms of water resource depletion but also because the styrofoam chamber culturing unit remains light, making it possible to install the system almost anywhere. chamber units can also be multistaged easily with low costs, meaning this system is suitable for a plant factory or urban farming. furthermore, because the effort to maintain the chamber units and fertigate plants are also greatly reduced, dry-fog aeroponics is expected to be usable not only for commercial crop production but also for welfare and food education. despite using less liquid fertilizer in the rhizosphere for the dry-fog culture, there was no difference in leaf growth in the two different hydroponic techniques, probably due to changes in root morphology and physiology. root hairs that developed only in the dry-fog culture had a normal increase in the efficiency of nutrient absorption from heterogeneous soil due to a significant increase in the root surface area (bates and lynch, 1996; ma et al., 2001). root hairs are not needed in dft hydroponic technique because there is adequate water and liquid nutrients in the root zone. they are not formed in traditional aeroponics because nutrient solutions are sprayed on roots which are always kept wet. in dry-fog culture, because the rhizosphere is almost an air phase, roots have to catch the very fine droplets of nutrient solutions floating in the air in a chamber. importantly, as an adaptation mechanism, root hairs develop to catch more droplets of fertilizer. in the present study, the significant increase in root growth was observed only at four weeks after planting. also the respiration rate of the roots was significantly higher in the dry-fog cultures than in the dft hydroponic system at four weeks (fig. 5). the respiration rate of roots has a close relationship with water and nutrient absorption in the roots (hansen, 1980). indeed, concomitant increases in both root growth and respiration rate in the dry-fog culture were observed, which might result in promotion of water and nutrient absorption and increases of growth and yield. however, no significant increase in leaf growth was observed in dry-fog culture as compared with dft. because the growth and respiration activity of roots significantly increased at four weeks and the morphology of new roots developed in the dry-fog culture changed, it may take longer for plant roots to adapt to the dry-fog culture environment as compared with other hydroponic systems. the ascorbic acid content in the leaves also tended to be slightly higher in the dry-fog culture, but there were no significant differences. leaf ascorbic acid is a secondary metabolite, and its content in leaves increases under environmental stress conditions (robinson and bunce, 2000; reddy et al., 2004, koyama et al., 2012). because there is nearly no extra water in the rhizosphere in dry-fog culture, roots might suffer from slightly drought conditions. the increase in secondary metabolites caused by environmental stresses generally comes with a decrease in growth and harvest (champolivier and merrien, 1996; kirda et al., 2004, koyama et al., 2012). however in the present study, such decreases were not observed. under drought conditions, the development of root hairs and increase in root surface area are countermeasures to growth depression due to a water shortage. in dry-fog cultures, plants develop root hairs to catch the very fine droplets of nutrient solution, and this development is thought to effect secondary metabolism. dry-fog culture might be useful to adjust mild drought conditions in rhizosphere by changing the airflow speed at the frequency of atomization, which affect harvest quality (brix of secondary metabolite content). furthermore, the atomized dry-fog from the nutrient solution in a chamber fills the rhizosphere space immediately, meaning the rhizosphere environment is changed quickly and drastically. this aspect is expected to lead to new cultivation methods that can increase the content of secondary metabolites by controlling environmental stresses without decreasing growth or harvest. with regard to the photosynthetic rates of mature leaves grown in the dry-fog culture, significant increases were shown in both φ and max compared with dft culture. this indicates that the rhizosphere environment of dryfog culture may affect the photosynthetic ability of lettuce leaves by improving co 2 assimilation efficiency of mesophyll cells. the content of leaf chlorophyll was significantly increased in the dry-fog culture at four weeks, and this may be considered to be due to improvement in the activity or quantity of photosynthetic light or dark reaction components. the relationship between enhancement of photosynthetic activity and rhizosphere environment in the dry-fog culture remains unclear. in summary, dry-fog aeroponics enables lettuce plants to grow with less water without sacrificing yield and leaf quality as compared to traditional hydroponics. the lightweight cultivation chamber can decrease the workload for growers and is easy for new growers to maintain. dryfog aeroponics posses potential as a novel hydroponic system to promote high-value crop production and yield more than other hydroponic systems by controlling the rhizosphere environment while saving water and nutrients. it is worth noting that the nozzle used in this study 189 can atomize enough dry-fog to fill a much larger chamber (6000×700×450 mm). more study is needed to determine the utility of a dry-fog culture in horticulture. further research would be helpful to determine rhizosphere environmental conditions (density of dry-fog, frequency of atomizing, flow speed of dry-fog) to maximize plant growth and the amount of secondary metabolites produced. in addition, optimization of the composition and strength of the nutrient solution, especially for dry-fog culture, is needed and could be determined through investigation of the absorption nutrients in the plants. references bates t.r., lynch j.p., 1996 stimulation of root hair elongation in arabidopsis thaliana by low phosphorus availability. plant, cell environ., 19(5): 529-538. biddinger e.j., liu c., joly r.j., raghothama k.g., 1998 physiological and molecular responses of aeroponically grown tomato plants to phosphorus deficiency. j. am. soc. hortic. sci., 123(2): 330-333. champolivier l., merrien a., 1996 effects of water stress applied at different growth stages to brassica napus l. var. oleifera on yield, yield components and seed quality. eur. j. agron., 5(3-4): 153-160. cherif m., tirilly y., belanger r.r., 1997 effect of oxygen concentration on plant growth, lipidperoxidation, and receptivity of tomato roots to pythium f under hydroponic conditions. eur. j. plant pathol., 103(3): 255-264. farren i., mingo-castel a.m., 2006 potato minituber production using aeroponics: effect of plant density and harvesting intervals. am. j. potato res., 83: 47-53. hansen g.k., 1980 diurnal variation of root respiration rates and nitrate uptake as influenced by nitrogen supply. physiol. plant, 48: 421-427. hillel s., david w.b., 1988 effect of dissolved oxygen concentrations in aero-hydroponics on the formation and growth of adventitious roots. j. am. soc. hortic. sci., 113(2): 218-221. jing l., yunwei z., 2013 an automatic aeroponics growth system based on ultrasonic atomization. appl. mech. mater., 288: 161-166. kirda c., cetin m., dasgan y., topcu s., kaman h., ekici b., derici m.r., ozguven a.i., 2004 yield response of greenhouse grown tomato to partial root drying and conventional deficit irrigation. agric. water management, 69(3): 191-201. koyama r., itoh h., kimura s., morioka a., uno y., 2012 augmentation of antioxidant constituents by drought stress to roots in leafy vegetables. horttechnology, 22(1): 121-125. liu c.z., guo c., wang y.c., ouyang f., 2003 comparison of various bioreactors on growth and artemisinin biosynthesis of artemisia annua l. shoot cultures. process biochem., 39: 45-49. ma z., bielenberg d.g., brown k.m., lynch j.p., 2001 regulation of root hair density by phosphorus availability in arabidopsis thaliana. plant, cell environ., 24: 459-467. mohammad a., khan a.g., kuek c., 2000 improved aeroponic culture of inocula of arbuscular mycorrhizal fungi. mycorrhiza, 9(6): 337-339. reddy a.r., chaitanya k.v., vivekanandan m., 2004 drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. j. plant physiol., 161(11): 1189-1202. ritter e., angulo b., riga p., herran c., relloso j., 2001 comparison of hydroponic and aeroponics cultivation systems for the production of potato minituber. potato res., 44(2): 127-135. robinson m., bunce j.a., 2000 influence of drought-induced water stress on soybean and spinach leaf ascorbatedehydroascorbate level and redox status. int. j. plant sci., 161(2): 271-279. weathers p.j., zobel r.w., 1992 aeroponics for the culture of organisms, tissues and cells. biotechnol. adv., 10(1): 93-115. zhao x., li t.l., sun z.p., 2010 effects of prolonged rootzone co 2 treatment on morphological parameter and nutrient uptake of tomato grown in aeroponic system. j. appl. bot. food qual., 83(2): 212-216. 202 1. introduction asparagus (asparagus officinalis l.), of the family asparagaceae, is a perennial crop widely cultivated in the world. the crop is cultivated from northern to southern regions in japan, and is an important vegetable. it is expected that production of the crop will increase due to its high profits in many places. the southwestern region of japan has an advantage because spears emerge earlier in this region than in the northern region. several diseases such as stem blight, leaf spot and brown spot cause serious problems in open field culture in warm regions. therefore, rain protected culture is necessary (kobayashi and shinsu, 1990). among these diseases, stem blight caused by phomopsis asparagi (sacc.) is the most serious disease in japan, korea, china and southeast asia and causes devastating damage to the crop in economically producing fields; the disease is also distributed in australia, greece, italy, new zealand and usa (davis, 2001; elena, 2006; udayanga et al., 2011). recently disease damage was also reported in northern parts of japan (sonoda, personal communication). the causal fungus first forms small lesions or spots on the lower part of the asparagus stem. the lesions continue to expand and produce yellowish-brown spindle shaped spots (sonoda et al., 1997). pycnidia usually appear at the central part of a spot and become secondary sources of infection (sakai et al., 1992). the symptoms are different with aging of stems, and result in systemic symptom, and finally stems are killed (fukutomi, 1993). thus, production of cultivars with strong resistance to stem blight has become an urgent issue. the genus asparagus is composed of about 100-300 (bailey, 1944; dahlgren et al., 1985) species mainly distributed in arid regions of the mediterranean, africa and asia (dahlgren et al., 1985). the species are widely used not only as food source but also for medicinal or ornamental purposes. sonoda et al. (1997, 2001) reported that there were varietal differences of stem blight resistance but there were no strong resistant cultivars in a. officinalis. they also reported that several asparagus species had strong disease resistance, but it was impossible to produce hybrids by interspecific crosses with a. officinalis. kunitake et al. (1996) produced somatic hybrids between a. officinalis (2n=2x=20) and a. macowanii (2n=2x=20) by protoplasts electrofusion for the purpose of introduction of disease resistance characteristics into asparagus. however, the hybrids failed to mature because of their abnormal genome composition. marcellán and camadro (1999) also performed interspecific hybridization between a. officinalis and a. densiflorus (kunth) jessop cv. sprengeri (2n=6x=60), but the stem blight resistance of asparagus kiusianus and its hybrid with a. officinalis m. iwato*, m. kosaza*, y. takeuchi*, m. matsumoto**, m. inada***, y. ozaki**** (1), h. okubo* * laboratory of horticultural science, faculty of agriculture, kyushu university, hakozaki, higashi-ku, fukuoka, 812-8581, japan. ** institute of tropical agriculture, kyushu university, hakozaki, higashi-ku, fukuoka, 812-8581, japan. *** saga prefectural agricultural experiment station, kawasoe, saga, 840-2205, japan. **** laboratory of agricultural ecology, faculty of agriculture, kyushu university, kasuya, fukuoka, 811-2307, japan. key words: asparagus kiusianus, asparagus officinalis, disease resistance, interspecific hybrid, phomopsis asparagi, stem blight. abstract resistance of cultivated asparagus (asparagus officinalis), wild a. kiusianus and their interspecific hybrids to stem blight caused by phomopsis asparagi (sacc.) bubák was evaluated. a. kiusianus had higher resistance than a. officinalis although there were individual variations. the resistance of interspecific hybrids between a. officinalis and a. kiusianus was mostly intermediate, but some individuals had strong resistance equivalent to a. kiusianus. the results suggest that it is possible to introduce the resistance of a. kiusianus to stem blight into a. officinalis through interspecific hybridization for further asparagus breeding. adv. hort. sci., 2014 28(4): 202-207 (1) corresponding author: ozaki@farm.kyushu-u.ac.jp received for publication 17 september 2014 accepted for publication 6 october 2014 203 endosperm failed to develop normally, leading to subsequent embryo abortion. asparagus kiusianus (2n=2x=20) is a dioecious species native to the coastal region of the japan sea from yamaguchi to northern kyushu. it is, therefore, expected that the species is adapted with the climate of these regions with high disease or salt resistance. ito et al. (2011) successfully produced interspecific hybrids between a. officinalis and a. kiusianus and also obtained backcross progenies. the objective of this study is to demonstrate the usefulness of a. kiusianus for producing stem blight disease resistant asparagus cultivars, making cultivation in open field culture in southwest region of japan as well as in other warm climate areas in the world possible. evaluation of disease resistance was performed in a. kiusianus, cross compatibility between a. officinalis and a. kiusianus and disease resistance in the interspecific hybrids was surveyed. 2. materials and methods plant materials asparagus officinalis cv. uc157f 1 , as the control cultivar (sonoda et al., 1997), and a. kiusianus keya line from fukuoka, japan were used for inoculation tests in 2010 to compare the resistance to stem blight in these two species (table 1). uc157f 1 and a. kiusianus (nijinomatsubara line from saga, japan) were used for crossing experiments. uc157f 1 and its intraspecific f 1 hybrids, a. kiusianus (nijinomatsubara line from saga, keya line and uminonakamichi line from fukuoka, japan) and interspecific f 1 hybrids of a. officinalis with a. kiusianus (ok) were used for inoculation tests in 2011 and 2012 (table 1). inoculum phomopsis asparagi strain p1, preserved in saga prefectural agricultural experiment station, was cultured on potato sucrose agar (psa) medium at 25°c for one month. after sporulation, conidiospores were suspended in sterilized water and adjusted to the inoculum density of 2×106 spores/ml for use in the following tests. interspecific cross interspecific crosses between a. officinalis (female) and a. kiusianus (male) and intraspecific cross in a. officinalis cv. uc157f 1 were made by hand pollination in a greenhouse of kyushu university farm in 2010. obtained seeds were germinated at 25°c in plastic petri dishes, and germinated seeds were sown in vermiculite in plastic cell trays in an incubator. they were transplanted in plastic pots six months after seed sowing and transferred. genomic dna was extracted from young cladodes of each plant by modified ctab method (stajner et al., 2002). hybridity of interspecific progenies was confirmed by three ssr markers (caruso et al., 2008), ag2, ag7 and ag10, followed by takeuchi et al. (2012). evaluation of stem blight resistance for inoculation test inoculation tests were performed three times from november 2010 to january 2011 with uc157f 1 and a. kiusianus, keya line grown in plastic pots. seven to 10 plants were used for each test. the plants were inoculated with the vinyl tape and cotton method by reference to the protocols previously described by sonoda et al. (1997, 1999, 2001). absorbent cotton moistened with spore suspension was put around the basal part (2-5 cm below the lowest branching nodes) of the stem, and covered with vinyl tape. the plants were maintained at 25°c with 90% humidity for three days, followed by incubation at 25°c and at 60-70% humidity in a growth chamber. the spears, which emerged two to three weeks after cutting the areal stems of the seedlings, were examined. disease severity was determined by scoring 0-4 grades for each plant weekly until five weeks after inoculation. the disease severity grade (dsg) was: 0 = no lesion; 1 = small-sized lesion (<1 cm in length); 2 = spread lesion; 3 = large-sized lesion (>half of the aerial stem) or defoliation; 4 = aerial part death. percentage of infected plants and disease indices were calculated, as follows: percentage of infected plants = number of infected plants total number of plants employed × 100 disease index (di) = ∑(number of plants classified into each grade × dsg number) total number of plants employed × 4 × 100 the degree of pycnidia formation was also surveyed. resistance in uc157f 1 and its intraspecific f 1 hybrids, a. kiusianus (keya line, nijinomatsubara line and uminonakamichi line) and interspecific f 1 hybrids between them was evaluated by inoculation test three times from august to september and from november to december in 2011 and from october to november in 2012. assessments were performed according to the procedure as described above. table 1 plant materials used for inoculation tests species cultivar and line tested year no. of individuals investigated a. officinalis uc157f 1 2010 14 a. officinalis uc157f 1 , f 1 progenies of ‘ uc157f 1 ’ 2011, 2012 25 a. kiusianus keya line 2010 15 a. kiusianus keya line, uminonakamichi line, nijinomatsubara line 2011, 2012 21 interspecific hybrid hybrids between a. officinalis and a. kiusianus 2011, 2012 25 204 3. results evaluation of the resistance to stem blight in a. officinalis and a. kiusianus most of the plants in both species showed pathogenic symptoms on the stems (table 2). all the inoculated asparagus cultivars began to show blight by one week after inoculation and the aerial parts of all plants were dead within five weeks. in contrast, a. kiusianus showed lower di than a. officinalis and more than half of the plants survived five weeks after inoculation although they exhibited symptoms. there were differences of susceptibility among a. kiusianus accessions, whereas all a. officinalis accessions showed high susceptibility with severe symptoms. pycnidia formation was frequently observed in a. officinalis from two to four weeks after inoculation (data are not shown). a few accessions showed pycnidia formation in a. kiusianus, whereas some other individuals showed no symptoms possessing strong resistance. interspecific crosses between a. officinalis and a. kiusianus fruit set rate and number of seeds in interspecific crosses between a. officinalis (female) and a. kiusianus (male) were lower than those in intraspecific crosses among a. officinalis (table 3). germination rate of the interspecific hybrid was high (76.1%) at 25°c. hybridity of the progenies was confirmed by three ssr markers (caruso et al. 2008), ag2, ag7 and ag10 (data are not shown). the hybrid plants showed intermediate morphology between the parents. evaluation of the resistance to stem blight in the interspecific hybrids most of the hybrid plants and their parents showed pathogenic symptoms (fig. 1, table 4). the aerial stems of all inoculated a. officinalis showed blight by one week after inoculation and most of them faded within four weeks (fig. 2 d). in contrast, the aerial stems of a. kiusianus and the interspecific hybrids survived even four weeks after table 2 development of disease symptoms in a. officinalis and a. kiusianus inoculated with p. asparagi species percentage of infected plants disease index 1 (z) 2 3 4 5 1 2 3 4 5 a. officinalis 100 a 100 a 100 a 100 a 100 a 45 a 54 a 88 a 98 a 100 a a. kiusianus 77 a 85 a 93 a 93 a 93 a 25 b 38 b 54 b 63 b 69 b (z) weeks after inoculation. different letters in a column indicate significant difference according to student’s t-test (p≦0.05). table 3 result of interspecific crosses between a. officinalis and a. kiusianus seed parent pollen parent no. of pollinated flowers no. of obtained fruits (%)(z) no. of obtained seeds (n) (y) no. of germinated seeds (%)(x) no. of obtained seedlings a. officinalis × a. kiusianus 301 27 (9.0) 67 (2.5) 51 (76.1) 42 a. officinalis × a. officinalis 46 25 (54.3) 119 (4.8) 104 (87.4) 45 (z) no. of obtained fruits/no. of pollinated flowers)×100. (y) no. of obtained seeds/no. of obtained fruits. (x) (no. of germinated seeds/no. of obtained seeds)×100. fig. 1 frequency of individuals with their disease severity grades (dsg) in a. officinalis, a. kiusianus and f 1 hybrids screened for resistance to stem blight. tests were performed in august (a) and november (b) in 2011.  0 = no lesion; n 1 = small-sized lesion (<1 cm); n 2 = spread lesion; n 3 = large-sized lesion (>half the size of plant) or defoliation; n 4 = aerial part death. 205 the inoculation (fig. 2 e, f). the di’s in a. kiusianus and the interspecific hybrids were lower than those in a. officinalis although lesion was formed in most of the plants (table 4). the disease severity grade of a. officinalis was high compared with that of a. kiusianus and the interspecific hybrids (fig. 1). most a. kiusianus showed lower grades even five weeks later. the interspecific hybrids showed varied degrees of symptoms and the distribution of dsg was intermediate between the parental species. diseased lesions were observed in all the stems of a. officinalis and numbers of pycnidia were frequently formed four weeks after inoculation (figs. 3, 4). there was little pycnidia formation in a. kiusianus and interspecific hybrids, while lesions were observed in the stems of most plants of a. kiusianus and the interspecific hybrids. the hybrids showed intermediate susceptibility between their parent species and their pycnidia formation rates were as low as the degree of a. kiusianus (fig. 4). table 4 development of disease symptoms in a. officinalis, a. kiusianus and their hybrids inoculated with p. asparagi species percentage of infected plants disease index 1 (z) 2 3 4 5 1 2 3 4 5 a. officinalis 95 a 100 a 100 a 100 a 100 a 30 a 35 a 60 a 90 a 100 a a. kiusianus 81 a 88 a 88 a 88 a 88 a 27 a 31 a 34 b 37 b 40 b hybrids 86 a 92 a 92 a 92 a 95 a 29 a 36 a 40 ab 47 b 59 b (z) weeks after inoculation. different letters in a column indicate significant difference according to turkey’s multiple range test (p≦0.05). fig. 2 disease symptoms of a. officinalis (a, d), a. kiusianus (b, e) and f 1 hybrids (c, f) inoculated with p. asparagi. a-c: 1 week after inoculation, d-f: 4 weeks after inoculation. scale bars 9 cm. fig. 3 lesion on stems of a. officinalis (a), a. kiusianus (b) and interspecific hybrids (c, d) inoculated with p. asparagi. arrows indicate pycnidia. scale bars 1 mm. fig. 4 rates of lesion and pycnidia formation of a. officinalis, a. kiusianus and interspecific hybrids four weeks after inoculation with p. asparagi. bars represent se (n=3). different letters indicate significant difference according to turkey’s multiple range test (p≦0.05). 206 4. discussion and conclusions the symptoms were severer in this study than those reported by sonoda et al. (1997, 1999, 2001). di was nearly 100 in a. officinalis four weeks after inoculation in this study, while it ranged from 42 to 79 in another investigation (sonoda et al., 1997). this might be due to aggressive pathogenicity of the phomopsis strain used or differences in environmental conditions from the previous study (sonoda et al., 1997). the results of the present study show that a. kiusianus had higher resistance than a. officinalis with various degrees of resistance among individuals. although the lesions were observed in most a. kiusianus plants, the resistant accessions displayed reduced disease development and varying levels of resistance. pycnidia were infrequently formed on the stems of a. kiusianus, restricting the spread the pathogen. it is thought of as incomplete (or partial) resistance (poland et al., 2009). the rate-reducing resistance or partial resistance is believed to be effective against a large number of pathogen genotypes and durable resistance since it is non-race specific (niks and rubiales, 2002; poland et al., 2009). therefore, the resistance identified in a. kiusianus will be useful for asparagus breeding programs. there were differences in disease resistance among asparagus species (sonoda et al., 2001). asparagus densiflorus, a. virugatus, a. asparagoides and a. macowanii were highly resistant. they are, however, genetically distant to a. officinalis and extremely difficult to obtain viable interspecific hybrids. in contrast, a. kiusianus is genetically a closer species to a. officinalis than other asparagus species based on the analysis of non-coding cpdna regions (fukuda et al., 2005, 2011; kubota et al., 2011), although a. kiusianus is endemic to japan, far from of the habitat of a. officinalis which is the mediterranean. ito et al. (2011) obtained interspecific crosses between a. officinalis and a. kiusianus and their backcross progenies, suggesting the possibility of producing disease resistant asparagus cultivars. it was indicated in this study that the resistance derived from a. kiusianus was inherited in the interspecific hybrids, although it was variable among individuals. further study with these hybrids and their backcross progenies are needed in order to screen the genotypes with a higher level of resistance to stem blight for breeding disease-resistant asparagus cultivars. references bailey l.h., 1944 asparagus, pp. 406-411. in: bailey l.h. 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sci., 14(1): 21-29. sakai y., ito t., tanaka a., 1992 the primary source of stem blight of asparagus (asparagus officinalis l.) and the disease spread from them. bul. hiroshima prefect agr. res. ctr., 55: 97-107. sonoda t., kaji k., uragami a., oowada m., 1999 studies of resistance to stem blight for asparagus breeding 1. development of novel inoculation method for the pathogenic fungus. bull. of the fukushima prefect agric. exp. stn., 36: 1-6 (in japanese with english summary). sonoda t., uragami a., itoh k., kohmura h., ohwada m., kaji k., 2001 evaluation of asparagus species and comparison between sexes in a. officinalis cul207 tivars for resistance to stem blight. j japan soc. hort. sci., 70: 244-250. sonoda t., uragami a., kaji k., 1997 evaluation of asparagus officinalis cultivars for resistance to stem blight by using a novel inoculation method. hortscience, 32: 10851086. štajner n., bohanec b., javornik b., 2002 genetic variability of economically important asparagus species as revealed by genome size analysis and rdna its polymorphisms. plant sci., 162: 931-937. takeuchi y., sakaguchi y., ozaki y., okubo h., 2012 genotyping of asparagus (asparagus officinalis l.) cultivars with ssr markers. acta horticulturae, 950: 165-172. udayanga d., liu x., mckenzie e.h.c., chukeatirote e., bahkali a., hyde k.d., 2011 the genus phomopsis: biology, applications, species concepts and names of common phytopathogens. fungal diversity, 5: 189-225. impaginato 141 adv. hort. sci., 2024 38(2): 141­153 doi: 10.36253/ahsc­15535 field evaluation of biostimulants on growth, flowering, yield, and quality of snap beans in subtropical environment s.h. brengi 1, i.a. abouelsaad 1, 2, (*), r.m. mahdy 3, a.a. khadr 1 1 horticulture department, faculty of agriculture, damanhour university, damanhour 22516, egypt. 2 faculty of desert agriculture, king salman international university, ras sedr 46618, egypt. 3 horticulture department, faculty of agriculture, tanta university, tanta 31527, egypt. key words: chitosan, potassium silicate, 6­benzylaminopurine, snap bean, tria­ contanol. abstract: the cultivation of snap beans (phaseolus vulgaris l.) in subtropical regions faces environmental challenges leading to potential declines in yield. this study explores the efficacy of biostimulants as a solution, specifically inves­ tigating spraying treatments with 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) on the snap bean cv. paulista. over two growing seasons with late sowing and elevated summer tempera­ tures, the research assesses growth, flowering, yield, and quality. notably, 5 ppm tria demonstrates the most significant impact on plant growth and leaf nutrient content. treatments with 40 ppm 6­ba, 5 ppm tria, or 200 ppm ksi exhibit notable effects on inflorescence flower count and flowers per plant. these treatments prove most effective for crucial green pod yield measures, including the number and weight of marketable pods. moreover, 40 ppm 6­ba or 5 ppm tria significantly enhances pod characteristics, such as length, diame­ ter, and weight, consistently improving over both seasons. particularly, 5 ppm tria outperforms in enhancing the chemical quality of pods throughout the study. overall, the findings suggest that the application of 5 ppm tria offers the most favorable enhancements for the growth, flowering, productivity, and quality of snap bean plants in subtropical field conditions. 1. introduction the global population is now and will continue to exert increased pres­ sure on the need for food. hence, it is essential for farmers to annually increase food production with the existing resources to fulfill this demand. common bean (phaseolus vulgaris l.) is a common vegetable that includes both snap and dry beans (lin et al., 2008). in egypt, farmers dedicate 27363 hectares to green bean cultivation, yielding 284299 tons annually (faostat, 2021).however, snap beans have a notable suscepti­ (*) corresponding author: ibrahim.abouelsaad@ksiu.edu.eg citation: brengi s.h., abouelsaad i.a., mahdy r.m., khadr a.a., 2024 ­ influence of ground cover and tunnels on production of red russian kale in urban gardens. ­ adv. hort. sci., 38(2): 141­153 copyright: © 2024 brengi s.h., abouelsaad i.a., mahdy r.m., khadr a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2023 accepted for publication 7 march 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15535 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 141­153 142 bility to high summer temperatures, particularly when subjected to delayed planting, like in april and may under egyptian field conditions (el­bassiony et al., 2012). while it has been stated that the optimal temperature for bean plants is 23°c (dickson and boettger, 1984). omae et al. (2006) observed that the occurrence of high summer temperatures (26°c min and 30°c max.) during the initiation of the blooming stage had an adverse impact on the quanti­ ty and the weight of pods. along with this, climate models predict a 50% decrease in global cultivated area by 2050 due to global warming (rippke et al., 2016; rama rao et al., 2022). one potential approach to enhancing snap bean production is through the breeding of new cultivars. however, it is important to note that this process often takes a significant amount of time and may provide limited results (xiong et al. , 2022). biostimulants provide a compelling alternative in the context of degraded agricultural regions and the risks associated with climate change. in recent times, there has been increased research focusin the utiliza­ tion of biostimulantsin the form of plant growth reg­ ulators (e.g., 6­benzylaminopurine; 6­ba as a synthet­ ic cytokinin, and triacontanol; tria), chitosan (ch), and trace elements (e.g., silicon; si) that have been found effective in improving plant productivity (du jardin, 2015; yaghubi et al. , 2019; islam and mohammad, 2020; hassan et al., 2021; stasińska­ jakubas and hawrylak­nowak, 2022). although cytokinins (cks) have vital function in controlling plant development, they have also been shown to confer other benefits, such as improving photosyn­ thetic rates, photosynthetic pigments, and nutrient uptake (aremu et al., 2020; li et al., 2021). in a study conducted by mostafa and brengi (2018), it was shown that the application of 6­ba solution on okra leaves resulted in improved yield and chemical com­ position. furthermore, yang et al. (2016) illustrated that the treatment with 6­ba resulted in an improve­ ment in several aspects of wheat grain development, including wheat grain filling and endosperm cell divi­ sion under heated growth conditions. it is a widely recognized that tria is plant growth regulator (islam and mohammad, 2020). triacontanol is a saturated alcohol initially discovered in alfalfa (ries et al., 1977) and is found naturally as a wax coating on a variety of plant species (islam and mohammad, 2020). in addi­ tion to its function in eliciting responses to stresses, tria is participated in plant growth, production, and vital physiological processes (faiz et al., 2024). in this manner, waqas et al. (2016) showed that both nor­ mal growth and heat stress conditions, tria treat­ ment of mung bean plants resulted in improved plant growth, leaf chlorophyll content, nutrients, and pro­ tein content. chitosan is a naturally carbohydrate polymer that has been produced from chitin, a sub­ stance found in the shells of crustaceans (hidangmayum et al., 2019). it is non­toxicand bio­ compatible, making it potentially useful in agriculture and biotechnology (stasińska­jakubas and hawrylak­ nowak, 2022). basically, ch improves physiological responses and reduces the negative effects of abiotic stressors through the secondary messengers (hidangmayum et al., 2019). therefore, ch is thought to be a viable exogenous addition for increasing crop production and overcoming abiotic stress (stasińska­ jakubas and hawrylak­nowak, 2022). apart from this, ch also enhanced the productivity of many crops such as tomatoes (el­tantawy, 2009), cowpea (farouk and amany, 2012) and cucumber (ali et al., 2020). generally, silicon (si)ranks among the most abundant elements found in soil (souri et al., 2021). recently, the connections between si and various biological processes in multiple crops were clarified, and silicon was recognized as one of the vital nutrients required by plants (zargar et al., 2019). silicon is engaged in many biological activities such as photo synthesis, nutrient uptake, and plant adaptation to stress (zargar et al., 2019; souri et al., 2021). potassium sili­ cate (ksi) is usually used as biostimulant and a pro­ ducer of both soluble k and si (yaghubi et al., 2019). it is well recognized that k is a core element and par­ ticipates in a vital function in cell division, protein synthesis, the formation of sugars, and plant growth, as well as vital processes such as plant photosynthe­ sis and stomata movement (ali et al., 2021). although previous studies have examined the individual impacts of these elicitors on plant growth, a comprehensive investigation into their effects specifically on snap bean plants remains lacking. moreover, these studies evaluated different parame­ ters and were conducted in different growing envi­ ronments; consequently, the field evaluation of these biostimulants under a particular subtropical summer conditions are required. thus, this research was cre­ ated to test the beneficial impacts of 6­ba, ch, tria, or ksi on the growth, blooming, productivity, and quality of snap bean plants grown in delayed summer cultivation in a subtropical environment. abouelsaad ‐ field evaluation of biostimulants 143 2. materials and methods snap bean field conditions field trials were undertaken in the sidi ghazy region of kafr el­dawar city, located in the beheira governorate of egypt. these experiments were done during the seasons of 2021 and 2022. the geographi­ cal coordinates of the study area are around 31°07ʹn latitude and 30°08ʹ e longitude. the region has an arid climatic condition characterized by an annual precipitation of about 90­110 mm, mostly in the form of ineffectual showers during the winter. figure 1 presents a summary of the monthly temperatures, measured over the course of two cultivation seasons. the source of this data originates from the egyptian ministry of agriculture and reclamation of soils, bul­ letin of agricultural meteorological data. samples from the trial soil were subjected to drying and then sifted by a two­mm sieve. these samples were then analyzed using the protocols outlined by page et al. (1982). experimental soil had a clay texture (22.5% sand, 35.4% silt, and 42.1% clay) with a ph of 8.14, ec value of 1.19 dsm­1, and an organic material level of 1.75%, as an average over the two seasons. plant material, experimental design, and treatments seeds of the snap bean (phaseolus vulgaris l.) cv. paulista (alsafwa company, egypt) were planted on may 1st and may 2nd in the seasons of 2021 and 2022, respectively. the selection of this cultivar was based on its significant economic worth in both local and for­ eign markets. the experimental site was plowed and leveled adequately before plots were established in accordance with the experimental layout. the experi­ mental treatments were organized as stated by the randomized complete block design (rcbd). the trial consisted of 9 treatmentswith3replicates (plots). the seeds were manually cultivated on a single side of the ridge, with a spacing of 10 cm between each seed. the ridge itself had a dimension of 60 cm and 5 m long. each plot consisted of three ridges, and the total size of each plot was 9 m2.two guard ridges were present between each treatment to prevent spray drift. in this investigation, four biostimulants plus a con­ trol were examined, namely 6­benzylaminopurine (6­ ba, sigma­aldrich, usa) at 20 and 40 ppm, chitosan (ch, sigma­aldrich, usa) at 100 and 200 ppm, triacon­ tanol (tria, sigma­aldrich, usa) at 2.5 and 5 ppm, potassium silicate (ksi, sigma­aldrich, usa) at 100 and 200 ppm, and a control group treated with distilled water. former studies were employed to identify the suitabledose range of 6­ba (abouelsaad and brengi, 2022; zarea and karimi, 2023) ch (ibrahim and ramadan, 2015; stasińska­jakubas and hawrylak­ nowak, 2022), tria (islam and mohammad, 2020), and ksi (ibrahim et al., 2020). foliar treatments were employed 3 times, 15 days after seed sowing, fol­ lowed by further applications every 15 days after­ wards. the plants were subjected to a foliar applica­ tion employing a knapsack sprayer throughout the afternoon until drop­off. all treatments were provided with an equal total quantity of 50 n, 60 p, and 60 k (kg ha−1) fertilizer for the duration of the season. these fertilizers(surface broadcast application) were given in two equal por­ tions, with the first dosage delivered during the third week after seed planting and the second dose applied during the seventh week. nitrogen, p, and k were provided as ammonium nitrate, single superphos­ phate, and potassium sulfate, respectively. all the required practices for cultivating snap beans were properly conducted as required. plant growth, flowering, chlorophyll content and min‐ eral analysis after 50 days of planting, three plants from each fig. 1 ­ monthly means of maximum (t max.) and minimum (t min.) temperature during the two studied seasons (sum­ mer 2021 and 2022) (egyptian ministry of agriculture and reclamation of soils, bulletin of agricultural meteo­ rological data). adv. hort. sci., 2024 38(2): 141­153 144 replicate (nine from each treatment) were taken for measuring the height (cm), branch number (plant­1), and number of leaves (plant­1). the relative content of chlorophyll in snap bean were assessed using the chl meter instrument (spad­502) manufactured by konica minolta sensing in japan. the assessment included measuring the number of inflorescences plant­1, number of flowers in the inflorescence, total number of flowers plant­1, and evaluating the length of the inflorescence (cm) (at 50% flowering according to schwartz and langham, 2010) the fresh weight (g) of shoot was measured, and subsequently, the plants were subjected to oven­drying at a temperature of 60°c till their weights stabilized, leading to the deter­ mination of the dry weight (g) of the shoot. plant leaves area (cm2) was conducted using a mathemati­ cal analysis that examined the relationship between the dry weight of leaves (plant­1) and the dry weight and area of 20 discs taken from fresh leaves using a borer with a known diameter. wallace and munger (1965) established this relationship and presented it in the following formula: leaves area (cm2) = leaves dry weight (g) x 20 discs area (cm2)/20 discs dry weight (g) micro­kjeldahl was employed to assess nin snap bean leaves (sáez­plaza et al., 2013), but p level was quantified by colorimetric techniques (watanabe and olsen, 1965). zinc, fe, mg, and mn were analyzed in leaves using the atomic absorption spectrophotome­ ter model perkin elmer 3100, while k and ca levels of leaf tissue were quantified by a flame­photometer (corning m410) as employed by munns et al. (2010). green pod yield and quality at harvest time (65 days from sowing), observa­ tions were recorded for seven characters viz., the number of pods (plant­1), the weight of pods (g plant­ 1), the number of marketable pods (plant­1), the weight of marketable pods (g plant­1), the mean of pod weight (gm), pod length (cm) and pod diameter (mm). this research considers pods that possess sig­ nificant quality traits that are important for the export market, such as well­formedness, uniformity, straightness, and absence of flaws, as being consid­ ered marketable. total n (%) was determined in green pods using the micro­kjeldahl apparatus as defined by sáez­plaza et al. (2013). subsequently, total protein (%) was calculated using n% (mariotti et al., 2008). the detection of vitamin c in the green pods was carried out at a wavelength of 525 nm, uti­ lizing the methodology previously established by srivastava and singh (1988). to establish a standard curve, the utilization of ascorbic acid (analytical reagent, solarbio) was employed. the quantification of vitamin c is presented in mg100 g­1 fw. the mea­ surements of crude fiber and soluble sugar in pods were conducted using the methodology established by slavin (1987) and rady et al. (2019), respectively. data analysis the data underwent statistical analysis using a one­way factorial design within the framework of a randomized complete block design (rcbd). the costat program (costat program version 6.311, 2005) was used to conduct a statistical analysis, namely the duncan’s multiple range test, with a sig­ nificance threshold of p≤0.05, for the purpose of comparing the means. 3. results and discussion snap bean growth and chlorophyll contents changes in snap bean growth parameters caused by the foliar application of growth elicitors (6­ba, ch, tria, or ksi) are presented in tables 1 and 2. the application of all treatments boosted plant growth, as clarified by the increases in snap bean height, leaves area, shoot fresh and dry weights. in most instances, the applied treatments also resulted in boosted the number of leaves and branches, although in the case of the treatment with 100 ppm ch, both seasons’ values were similar to the control treatment. several studies have provided evidence suggesting that the applications of 6­ba, ch, and ksi have the potential to increase the overall snap bean growth (werner and schmülling, 2009; hidangmayum et al., 2019; yaghubi et al., 2019; gomaa et al., 2021). cytokinins (cks) are often char­ acterized as hormones that stimulate growth; howev­ er, it should be noted that several substances exhibit­ ing ck activity have been discovered to control many features of plant development (haberer and kieber, 2002). cytokinins influence cell multiplication, that in turn influences plant development, and they also promote adventitious buds growth (kieber and schaller, 2014). such substances, including exoge­ nous applications, were used to promote growth in crops and vegetables (yang et al., 2016; mostafa and brengi, 2018; el­areiny et al., 2019; aremu et al., abouelsaad ‐ field evaluation of biostimulants 145 2020; abouelsaad and brengi, 2022). additionally, ch, a biopolymer, employed in crops production primari­ ly owing to its biocompatible and biodegradable nature, together with its notable biological activity (hidangmayum et al., 2019). despite not being a con­ stituent of plant tissues, ch significantly boosts the development and growth of plants (stasińska­ jakubas and hawrylak­nowak, 2022). this was con­ firmed by el­miniawy et al. (2013), who claimed that spraying ch increased the growth (height, leaf area, and weight) of strawberry cv. sweet charlie. in another study, foliar application of ch increased both the growth and nitrate reductase activity of okra (mondal et al., 2012). recently, ample evidence has shown that si is a key nutrient for crops such as grains, legumes, and vegetables (souri et al., 2021). both in vitro and field investigations confirmed the favorable benefits of si in boosting plant develop­ ment, especially in stressful situations (zargar et al., 2019; souri et al., 2021). according to eneji et al. (2008), si has been shown to act as a bioregulator and have the capacity to enhance plant develop­ ment. the usage of ksihas shown a substantial influ­ ence on the growth of several agricultural crops such as maize and strawberry (yaghubi et al., 2019; ibrahimet al., 2020; gomaa et al., 2021). nevertheless, within the range of applied treat­ ments, it was observed that the application of 5 ppm tria had a more pronounced impact on plant growth over both seasons. as average for the two growing seasons, with 5 ppm tria the snap bean height boosted by 17.23%, the leaf area expanded by table 2 ­ the number of branches, number of leaves, and leaves area of snap beans affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons table 1 ­ plant height, shoot fresh weight, and shoot dry weight of snap beanas affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment plant height (cm) shoot fresh weight (g) shoot dry weight (g) 2021 2022 2021 2022 2021 2022 control 44.30 e 45.63 d 293.07 d 301.90 d 29.83 d 30.89 e 6­ba (20 ppm) 50.33 bc 52.33 ab 326.02 c 339.03 c 35.64 bc 37.30 cd 6­ba (40 ppm) 52.30 a 53.83 a 364.40 ab 375.09 ab 37.79 b 39.04 bc ch (100 ppm) 48.43 cd 49.93 c 351.40 b 361.91 bc 34.77 c 35.99 d ch (200 ppm) 47.97 d 51.67 bc 357.60 b 385.00 ab 35.48 bc 38.66 bc tria (2.5 ppm) 49.67 cd 52.80 ab 355.13 b 377.60 ab 36.62 bc 39.31 bc tria (5 ppm) 51.90 ab 53.53 a 373.93 a 386.23 a 41.31 a 42.84 a ksi (100 ppm) 48.17 d 52.17 ab 355.83 b 385.54 a 36.46 bc 40.00 b ksi (200 ppm) 50.10 bc 53.93 a 360.27 ab 387.89 a 35.94 bc 39.16 bc means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment no. of branches plant­1 no. of leaves plant­1 leaves area (cm2) 2021 2022 2021 2022 2021 2022 control 6.67 c 6.67 b 20.00 f 20.67 c 2712.00 e 2793.67 e 6­ba (20 ppm) 7.33 abc 7.33 ab 21.33 cde 22.33 b 2901.33 d 3017.00 cd 6­ba (40 ppm) 8.33 a 8.33 a 23.33 a 24.00 a 3144.67 a 3292.67 a ch (100 ppm) 7.00 bc 7.00 ab 20.33 ef 20.67 c 2845.00 d 2930.33 de ch (200 ppm) 7.33 abc 8.00 ab 21.00 def 22.67 ab 2992.00 c 3222.00 ab tria (2.5 ppm) 7.67 abc 8.33 a 21.67 bcd 23.33 ab 3096.67 ab 3237.33 ab tria (5 ppm) 8.00 ab 8.33 a 22.67 ab 23.67 ab 3130.67 a 3230.67 ab ksi (100 ppm) 7.67 abc 8.33 a 21.33 cde 23.33 ab 2886.67 d 3127.00 bc ksi (200 ppm) 7.67 abc 8.33 a 22.33 abc 24.00 a 3018.00 bc 3250.00 ab 146 adv. hort. sci., 2024 38(2): 141­153 15.54%, the snap bean fresh weight boosted by 27.76%, and the shoot dry weight boosted by 38.58% as compared to the control. triacontanol (tria) is plant growth regulator that has a significant function in facilitating many plants metabolic processes, ulti­ mately resulting in enhanced growth and develop­ ment (naeem et al., 2012; islam and mohammad, 2020). its foliar application at low concentrations stimulates the plant biomass of the crops under both control and stressful circumstances (naeem et al., 2012). a growing body of research has shown that tria is an important factor in controlling a wide range of plant morphological responses. one notable effect is its ability to promote many aspects of plant growth, such as increased height, enhanced biomass, greater leaf number, and expanded leaf area across multiple crop species (naeem et al., 2012). this increase in plant growth might be because tria acti­ vates l (+)­adenosine, a second messenger that sends signals throughout the plant to boost growth by promoting cell expansion and proliferation (masroor et al., 2006; naeem et al., 2012). the growth of plants is greatly impacted by the level of photosynthetic pigments, that is critical for photosynthesis. in the current study, the use of spraying treatments has resulted in enhancements in chlorophyll contents, but these improvements were seen at comparable levels in most instances (table 3). studies have also shown the effect of cks, ch, tria, orksi on increasing the content of photosyn­ thetic pigments. cytokinins can impede or decelerate the process of plant senescence by inhibiting the degradation of chlorophyll, hence preserving the green color of the leaves (werner and schmülling, 2009; kieber and schaller, 2014). meanwhile, treat­ ing wheat leaves with 6­ba has been shown to boost the production of the chlorophyll founder, d­ aminolevulinic acid (wang et al., 2022). also, the spray of ch has been reported to boost the levels of photosynthetic pigments in rice plants (pongprayoon et al., 2013) and creeping bentgrass plants suffering temperature stress conditions (huang et al., 2021). in another study, tria shown a notable increase in pig­ ment content, namely chlorophyll a, b, and carotenoids, by 25.6, 33.9, and 13.0% respectively, in the leaves of basil plants, relative to the control (hashmi et al., 2011). also, masroor et al. (2006) showed similar results in their study, where they noted a substantial rise in chlorophyll and carotenoid content in tomato seedlings that were treated with tria. former research has also verified the beneficial influence of ksi on the chlorophyll levels in plant leaves (yaghubi et al., 2019; zargar et al., 2019; tejada­ruiz et al., 2020; gomaa et al., 2021). elemental analysis nutrients are fundamental for the growth and productivity of agricultural crops. they are needed in varying quantities and play key functions in various biological processes. the application of 6­ba, ch, tria, or ksi contributed to a higher level of macronu­ trients and micronutrients in snap bean leaves, with some exceptions (tables 4 and 5). for instance, the treatments with 100 ppm ch during the first season, 200 ppm ch during the second season, and 100 ppm si throughout both seasons demonstrated a p level comparable to that of the control. additionally, the application of ch at concentrations of 100 and 200 ppm resulted in limited changes to the ca and zn content of the leaves. previous studies have also doc­ umented the positive effects of cks and si on the content and uptake of essential nutrients. cytokinins regulate the plants’ capacity to uptake various ele­ ments, like n, p, and k (argueso et al., 2009). in a study conducted by abouelsaad and brengi (2022), the application of cks through foliar means led to a rise in the n and p levels in potato leaves, relative to the control. haberer and kieber (2002) reported that cks regulate the expression of multiple transporter genes, thereby influencing the plant’s ability to uptake nutrients. from this perspective, some stud­ ies also showed that si treatment boosts macronutri­ table 3 ­ relative chlorophyll content (spad value) of snap bean as affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are consi­ dered significantly different (p<0.05) using the duncan's multiple range test. treatment relative chlorophyll content (spad value) 2021 2022 control 41.33 c 41.67 c 6­ba (20 ppm) 41.67 bc 43.00 b 6­ba (40 ppm) 42.67 a 43.67 ab ch (100 ppm) 41.67 bc 43.33 ab ch (200 ppm) 42.33 ab 43.67 ab tria (2.5 ppm) 42.67 a 43.33 ab tria (5 ppm) 43.00 a 44.00 a ksi (100 ppm) 42.67 a 43.00 b ksi (200 ppm) 42.67 a 43.67 ab abouelsaad ‐ field evaluation of biostimulants 147 ent (e.g., p, k, and ca) and micronutrient (e.g., cu, and fe) absorption in crops (zargar et al., 2019). the results also showed that 5 ppm tria signifi­ cantly raised the average contents of n (9.10%), p (10.2%), k (32.73%), ca (13.33%), mg (38.66%), zn (39.06%), fe (26.8%), and mn (14.23%), compared to the control, throughout the two successive growing seasons (tables 4 and 5). notably, this treatment was the most effective among the spraying treatments for all the examined nutrients. as previously reported, the use of tria demonstrated a significant influence on the levels of n, p, and k in some crops (masroor et al., 2006; naeem et al., 2012; islam and mohammad, 2020). despite limited research on the impact of tria on micronutrient content, it may be inferred that tria induces modifications in plants, resulting in changed nutrient contents. in a manner similar to the 5 ppm tria treatment, the application of 40 ppm of 6­ba revealed the highest content of nutrients, but only for p, fe, zn, and mn (tables 4 and 5). flowering characteristics flowering characteristics (e.g., number of flowers and inflorescences) can have a great influence on the productivity of crops. in this study, the applied treatments had a beneficial effect on the length of the inflorescence in comparison to the control treatment, and the 40 ppm 6­ba treatment achieved table 4 ­ nitrogen (n), phosphorus (p), potassium (k), and calcium (ca) of snap bean leavesas affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment n (%) p (%) k (%) ca (%) 2021 2022 2021 2022 2021 2022 2021 2022 control 3.20 g 3.17 g 0.48 f 0.50 e 2.67 e 2.69 f 2.08 f 2.12 d 6­ba (20 ppm) 3.30 f 3.25 f 0.54 b 0.53 bcd 2.86 d 2.81 e 2.15 def 2.17 c 6­ba (40 ppm) 3.40 bc 3.36 c 0.58 a 0.56 a 3.06 b 2.99 c 2.27 bc 2.22 b ch (100 ppm) 3.31 ef 3.29 e 0.49 ef 0.48 f 2.89 d 2.92 d 2.08 f 2.12 d ch (200 ppm) 3.37 cd 3.36 c 0.51 cde 0.51 e 2.92 cd 2.94 cd 2.12 ef 2.10 d tria (2.5 ppm) 3.43 b 3.42 b 0.51 cde 0.53 cd 3.10 b 3.16 ab 2.31 b 2.33 a tria (5 ppm) 3.48 a 3.47 a 0.53 bc 0.55 ab 3.25 a 3.21 a 2.40 a 2.36 a ksi (100 ppm) 3.31 f 3.33 d 0.50 def 0.52 de 2.95 cd 2.93 cd 2.19 de 2.22 b ksi (200 ppm) 3.36 de 3.38 c 0.52 bcd 0.54 abc 3.02 bc 3.11 b 2.21 cd 2.24 b table 5 ­ manganese (mn), iron (fe), zinc (zn), and manganese (mn) of snap bean leavesas affected by 6­benzylaminopurine (6­ba), chi­ tosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment mg (%) fe (%) zn (%) mn (%) 2021 2022 2021 2022 2021 2022 2021 2022 control 0.36 e 0.39 g 127.67 d 122.33f 37.33 e 40.33 e 48.33 d 50.00 e 6­ba (20 ppm) 0.41 d 0.43 f 151.33 ab 146.33 c 48.00 b 50.67 b 51.00 cd 52.67 d 6­ba (40 ppm) 0.49 ab 0.47 cd 159.00a 154.67 a 54.00 a 52.00 ab 55.33 ab 56.00 a ch (100 ppm) 0.44 cd 0.45 e 137.00 cd 135.00 e 41.00 de 41.67 de 51.33 c 50.00 e ch (200 ppm) 0.48 abc 0.49 bc 141.00 c 139.00 de 43.00 cd 42.00 de 55.67 a 54.00 abcd tria (2.5 ppm) 0.48 ab 0.49 bc 155.67 a 152.33 ab 48.67 b 50.33 b 52.33 c 53.00 cd tria (5 ppm) 0.51 a 0.53 a 160.67 a 156.67 a 53.67 a 54.33 a 57.00 a 55.33 ab ksi (100 ppm) 0.45 bc 0.47 de 139.33 c 142.00 cd 43.00 cd 44.00 cd 52.67 bc 53.67 bcd ksi (200 ppm) 0.49 a 0.50 b 145.67 bc 147.00 bc 47.00 bc 45.33 c 56.67 a 55.00 abc adv. hort. sci., 2024 38(2): 141­153 148 the highest value in both seasons (table 6). additionally, except for 100 ppm ch (first season) and 20 ppm 6­ba (second season), the foliar treatments had a stimulating impact on the number of inflorescences plant­1 (table 6). while there is less documentation on the impact of ch and ksi on promoting vegetable flowering, it has been shown to have positive effects on flower crops (pichyangkura and chadchawan, 2015; amer, 2020). among the applied treatments in this study, the use of 40 ppm 6­ ba, 5 ppm tria, and 200 ppm ksi resulted in the most significant increase in the number of flowers in the inflorescence and number of flowers plant­1, a trend that persisted over both seasons. several studies have shown the role of cks as pivotal regulators of inflorescence morphology in plants, primarily through regulating meristem activity (kieber and schaller, 2014). according to d’aloia et al. (2011), flowering is induced in arabidopsis plants by exogenous cks applied during non­inductive short days. similar findings were reported by rylott and smith (1990), who demonstrated that synthetic cks enhance plant productivity and promote competition between generative and vegetative organs. in the current study, the number of flowers in the inflorescence and the number of flowers plant­1 exhibited respective increases of 22.8% and 50.15% in snap bean treated with 5 ppm tria, relative to the control treatment (fig. 2). the application of tria has been found to exert a positive influence on the flow­ ering process of various crops. this was confirmed by baba et al. (2017), who clarified that tria raised the number of flowers plant­1 while also influencing the timing of flowering in strawberry cv. camarosa. in addition, sharma et al. (2011) tested the effects of tria on olives and found that it enhanced the num­ ber of flowers in relation to the control. however, some treatments, such as the application of 100 ppm ch and 20 ppm 6­ba, did not have any notable influ­ ence on the number of flowers for the inflorescence table 6 ­ number of inflorescences and length of inflorescenceof snap bean as affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment no. of inflorescences plant­1 length of inflorescence (cm) 2021 2022 2021 2022 control 9.00 c 9.33 c 7.50 f 7.96 f 6­ba (20 ppm) 10.33 ab 10.00 bc 10.63 cd 10.87 c 6­ba (40 ppm) 11.33 a 11.67 a 13.53 a 12.93 a ch (100 ppm) 10.00 bc 10.33 b 8.83 e 9.53 de ch (200 ppm) 10.67 ab 11.33 a 9.83 d 9.93 d tria (2.5 ppm) 11.00 ab 11.67 a 10.83 c 11.20 c tria (5 ppm) 11.33 a 11.67 a 12.43 b 11.77 b ksi (100 ppm) 10.33 ab 11.33 a 8.87 e 9.30 e ksi (200 ppm) 11.33 a 11.67 a 9.83 d 9.66 de fig. 2 ­ the number of flowers in the inflorescence and number of flowers plant­1 of snap bean as affected by 6­benzy­ laminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the two studied seasons (summer 2021 and 2022). means (bars) with different letters for each season are considered significantly differ­ ent (p<0.05) using the duncan's multiple range test. data are mean value ± se. abouelsaad ‐ field evaluation of biostimulants 149 and number of flowers plant­1in relation to the con­ trol plants in both cultivation seasons (table 6). snap bean yield the value of crop yield is determined by the mar­ ketable yield, which is a crucial indicator of agricul­ tural productivity. in this study, relative to the con­ trol, it was noted that all the evaluated treatments had a positive impact on the total pod number plant ­ 1, except the treatments with ch at 100 ppm or ksi at 100 ppm for only the first season (table 7). also, the implemented treatments caused a significant increase in the number of marketable pods plant ­1, the weight of total fresh pods plant ­1, and the weight of marketable pods plant ­1. among the treatments used, applying 6­ba at a concentration of 40 ppm was the most effective treatment to achieve the highest yield parameters, a tendency that held across both seasons. this caused an increase in the total pod number by 36.67%, the number of marketable pods by 49.97%, the weight of total fresh pods by 38.23%, and the weight of mar­ ketable pods by 49.49% compared to the control treatment (table 7). according to jameson and song (2016), an elevated concentration of ck throughout the developmental stages of pods and seeds has been identified as a constraining factor in their growth and maturation. a study by nonokawa et al. (2012) also illustrated that ckfor both lupin and soy­ bean crops stopped flower abortion and improved pod set, which ultimately led to a higher yield. nonetheless, the current data showed that the treat­ ments with 200 ppm ch, 5 ppm tria, and 200 ppm ksi had comparable outcomes to the 40 ppm 6­ba treatment in terms of the number and weight of pods suitable for sale in both seasons (table 7). considering the data shown in table 8, except for the application of 6­ba at 20 ppm and ksi at 200 ppm during the first season, all treatments exhibited enhancement in the weight of the pods. the applica­ tion of 6­ba (20 or 40 ppm) and tria at 5 ppm, resulted in statistically significant increases in pod length in both growing seasons. furthermore, the application of ch at 200 ppm resulted in a notable enhancement of the pod diameter during both grow­ ing seasons. moreover, the implementation of 6­ba (20 or 40 ppm), tria (2.5 or 5 ppm), and ksi (100 or 200 ppm) exhibited a significant increase in pod diameter, specifically during the first growing season (table 8). similar studies have shown strong evidence supporting the efficacy of ch, tria, and ksi applica­ tions for improving the yield and yield components of both vegetable and grain crops (artyszak, 2018; kocięcka and liberacki, 2021). green pod quality the value of yield quality extends beyond mere productivity. it encompasses economic, environmen­ tal, social, and health aspects, making it a crucial fac­ tor for agricultural production (abouelsaad et al., 2022). as shown in table 9, the effects of treatments on the quality features (ascorbic acid, fiber, soluble table 7 ­ number of total pods, number of marketable pods, fresh pods weight, and marketable pods weight of snap bean as affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment no. of total pods plant­1 no. of marketable pods plant­1 fresh pods weight (g plant­1) marketable pods weight (g plant­1) 2021 2022 2021 2022 2021 2022 2021 2022 control 19.00 f 17.67 e 9.43 e 9.33 d 100.08 g 92.69 e 49.71 d 48.95 d 6­ba (20 ppm) 22.33 bcd 21.00 d 12.67 bcd 11.67 c 118.65 bcd 110.61d 66.38 bc 61.45 c 6­ba (40 ppm) 25.67 a 24.33 a 14.00 a 14.00 a 137.06 a 129.13 a 73.20 a 74.29 a ch (100 ppm) 20.00 ef 22 bcd 2.00 d 12.00 c 106.73 fg 116.9 bcd 64.04 c 63.76 c ch (200 ppm) 21.00 cde 22 bcd 13.33 abc 13.67 ab 112.14 def 116.68 bcd 71.21 ab 72.48 ab tria (2.5 ppm) 22.67 bc 22.67 bc 12.33 cd 12.67 bc 121.11 bc 120.36 bc 65.90 bc 67.26 bc tria (5 ppm) 23.67 b 23.33 ab 13.67 ab 13.33 ab 126.77 b 123.98 ab 72.22 ab 70.84 ab ksi (100 ppm) 20.67 def 21.67 cd 12.00 d 12.67 bc 109.87 ef 115.19 cd 63.79 c 67.34 bc ksi (200 ppm) 22.00 bcd 23 abc 13.67 ab 13.33 ab 117.99 cde 122.36 abc 75.09 a 70.93 ab adv. hort. sci., 2024 38(2): 141­153 150 sugar, and protein) of snap bean green pods were investigated. except for plants treated with 6­ba at 20 ppm, the ascorbic acid content in the pods of treated plants was significantly increased with respect to the control in both cultivation seasons. also, the treatments with 6­ba (40 ppm), tria (2.5 or 5 ppm), or ksi (200 ppm) significantly increased the amount of soluble sugar in the pods with respect to the control in both cultivation seasons. moreover, it was observed that the application of 6­ba (20 and 40 ppm), ch (200 ppm), tria (2.5 or 5 ppm), orksi (200 ppm) resulted in enhancement of protein content within the pods, with respect to the control plants, across both seasons (table 9).comparable findings also demonstrated the beneficial effects of cks, ch, tria, orksi on the levels of protein, soluble sugar, and ascorbic acid in cereal crops, vegetable, or legumes (naeem et al., 2012; artyszak, 2018; hu et al., 2022). moreover, snap beans should have fleshier green pods with little fiber content where immature pods are eaten as vegetables. in this study, the applied treatments significantly reduced the amount of fiber in the pods relative to the control group (table 9). overall, in both growth seasons, the tria (5 ppm) spraying treatment showed remarkable effi­ cacy across all quality criteria. table 8 ­ average of pod weight, pod length, fresh pods weight, and pods diameter of snap bean as affected by 6­benzylaminopurine (6­ ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment average of pod weight (g) pod length (cm) pods diameter (mm) 2021 2022 2021 2022 2021 2022 control 5.27 b 5.25 c 12.97 b 13.27 cd 6.67d 7.67 b 6­ba (20 ppm) 5.31 ab 5.27 b 13.57 a 13.70 ab 8.00 abc 8.00 ab 6­ba (40 ppm) 5.34 a 5.31 a 13.70 a 13.77 a 8.33 ab 8.33 ab ch (100 ppm) 5.34 a 5.31 a 12.97 b 13.23 d 7.33 cd 8.33 ab ch (200 ppm) 5.34 a 5.30 a 13.10 b 13.30 cd 8.33 ab 8.67 a tria (2.5 ppm) 5.34 a 5.31 a 13.40 ab 13.50 bc 8.00 abc 8.33 ab tria (5 ppm) 5.36 a 5.31 a 13.57 a 13.67 ab 8.33 ab 8.33 ab ksi (100 ppm) 5.32 ab 5.32 a 13.07 b 13.33 cd 7.67 ab 8.00 ab ksi (200 ppm) 5.36 a 5.32 a 13.37 ab 13.47 bcd 8.67a 8.33 ab table 9 ­ the contents of ascorbic acid, fiber, soluble sugar, and protein in snap bean pods as affected by 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), and potassium silicate (ksi) in the 2021 and 2022 seasons means with different letters for each plant parameter are considered significantly different (p<0.05) using the duncan's multiple range test. treatment ascorbic acid (mg 100 g fw­1) fiber (g 100 g fw­1) soluble sugar (g 100 g fw­1) protein (%) 2021 2022 2021 2022 2021 2022 2021 2022 control 17.43 d 18.60 c 3.51a 3.49 a 2.13 d 2.19 c 17.94 f 18.25 e 6­ba (20 ppm) 18.53 cd 19.73 bc 3.36 c 3.35 d 2.21 c 2.19 c 18.69 de 19.31 c 6­ba (40 ppm) 19.57 abc 20.38 ab 3.31 d 3.31 e 2.25 b 2.23 b 19.75 ab 19.44 c ch (100 ppm) 19.70 abc 20.13 b 3.38 bc 3.36 d 2.20 c 2.21 bc 18.06 f 18.81 d ch (200 ppm) 20.70 a 21.30 a 3.39 bc 3.37 cd 2.21 c 2.21 bc 19.13 cd 19.44 c tria (2.5 ppm) 19.31 bc 19.78 b 3.30 d 3.29 e 2.30 a 2.32 a 19.63 abc 19.88 b tria (5 ppm) 20.44 ab 20.61 ab 3.28 d 3.28 e 2.33 a 2.32 a 20.00 a 20.31 a ksi (100 ppm) 19.70 abc 20.07 b 3.40 bc 3.39 bc 2.20 c 2.21 bc 18.50 ef 18.81 d ksi (200 ppm) 19.87 abc 20.07 b 3.42 b 3.41 b 2.21 bc 2.23 b 19.13 cd 19.63 bc abouelsaad ‐ field evaluation of biostimulants 151 4. conclusions the applications of 6­benzylaminopurine (6­ba), chitosan (ch), triacontanol (tria), or potassium sili­ cate (ksi) by foliar spraying have the potential to enhance the development and agronomic characteris­ tics of snap bean plants.specifically, the use of 5 ppm tria demonstrates the most advantageous improve­ ments in growth, blooming, yield, and overall quality. this study could 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cropping system. ‐ 3 biotech, 9(3): 73. impaginato 433 adv. hort. sci., 2023 37(4): 433­449 doi: 10.36253/ahsc­14381 the habit of strawberry flowering is the key for runner propagation, where the photoperiod is the main environmental factor ­ a review i.a.h. al­madhagi department of horticulture and its technologies, faculty of agriculture, foods and environment, sana’a university, sana’a, p.o. box 1247, yemen. key words: chilling, division, flowering, fragaria x ananassa duch, gibberellin, photoperiod, proliferation. abstract: despite the advancement of tissue culture in strawberry plant propa­ gation, the degree of elite for field cultivation depends on forcing the plant to produce runners. the strawberry flower habit [everbearing (eb), seasonal berry (sb), short­day (sd), long­day (ld), and day­neutral (dn)] defines the method of encouraging the plant to generate runners, since the formation of runners is mostly influenced by genetic factors before being influenced by environmental factors. stolon production, which occurs as a result of vying for resources under certain environmental circumstances, is the reverse of blossoming. therefore, any stimulus that encourages stolon formation and vegetative growth limits the development of flower buds, which is necessary for elite propagation. long photoperiod, temperature, chilling hour, or cold storage, and plant growth reg­ ulators (pgr) are cited as these variables. temperature has a significant impact on runner development, although the long daily photoperiod (ld) remains the most crucial component in runner induction. however, when ld interacts with other factors like temperature, cold storage, and gibberellins, its efficiency is increased. thus, based on the cultivars and the seasonal climate of the geo­ graphical location, the best approach for strawberry propagation is identified by optimising the planting date for propagation or adjusting the propagation circumstances. 1. introduction strawberry (fragaria x ananassa duch) is a commercial crop grown worldwide for its nutritional and health benefits. strawberries are con­ sumed as fresh fruit or juice, or processed industrially into jam used in a various of desserts such as candy, milk and ice cream. according to the fao statistics service agency (fao, 2021), strawberry production has increased significantly over the past half­century (from 1960 to 2021). the total global production in 2021 is estimated to be around 9,175,384.43 t, with a cultivated area of about 389,665 ha. china con­ (*) corresponding author: isam.madhagi@gmail.com i.madhagi@su.edu.ye citation: al­madhagi i.a.h., 2023 ­ the habit of straw‐ berry flowering is the key for runner propagation, where the photoperiod is the main environmental factor ‐ a review. ­ adv. hort. sci., 37(4): 433­449. copyright: © 2023 al­madhagi i.a.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 february 2023 accepted for publication 1 august 2023 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-14381 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(4): 433­449 434 tributes for approximately 37% to global production, while the arab nations contribute only 6%, with egypt, morocco, and jordan making up the majority. the development of strawberry production in var­ ious countries depends on the selection of the most suitable cultivars for their annual climate. vegetative propagation is the ideal technique for strawberry propagation since it retains the mother’s characteris­ tics (li et al., 2020). strawberry nurseries, as a result, play an important role in the expansion of strawberry cultivation within a specific geographical area. it is critical for commercial plant production to select the best factors to stimulate the plant to produce run­ ners. in this review, the vegetative growth of strawber­ ry plants will be covered as a technique for contain­ ing blossoms and encouraging the plant to generate runners. blossoming and the development of runners are mutually exclusive. flowers must be controlled in the practical application of runner production, either by eliminating the flowers or by altering the environ­ mental conditions. long photoperiods (dl) and high temperature (ht) are crucial for promoting stoloniza­ tion (smeets, 1955; smeets and kronenberg, 1955; went, 1957; leshem and koller, 1965; smeets, 1980), which is related to increased gibberellin production (tafazoli and vince­prue, 1978). in addition, cold storage of plants promotes the production of runners (hamano et al., 2009; watanabe et al., 2009; al­mad­ hagi et al., 2018). exogenous application of growth regulators such as gibberellins, cytokinins or their combination supports the development of the run­ ners (kender et al., 1971). long photoperiods (ld) are the most essential factors for runnering induction in strawberry, but its effectiveness is enhanced by its interaction with other factors such as temperature, cold storage, and gibberellins. within this context, the primary goal of this paper is to present an overview of the factors that influence strawberry runner yield. strawberry flowering habit and cultivars division genetics is the primary component governing strawberry proliferation. moreover, it pinpoints the best techniques as well as the coefficients of the propagation means. since f. ananassa duch, the cul­ tivar of the strawberry, is a hybrid plant, the varia­ tions in strawberry cultivars may be attributed to variations in its fundamental parents, f. virginiana and f. chiloensis, each of which has a unique bloom­ ing and runnering behaviour. f. chiloensis began to bloom before f. virginiana and its majority of the leaves stayed evergreen throughout the winter. in the meantime, genotypes of f. virginiana seemed to become dormant, and their leaves became brown and withered off in the late fall and winter, outper­ forming f. chiloensis for runner production (darrow, 1966; hancock et al., 2003). the capacity of the bud in cultivars to continue producing inflorescences throughout the growing season accounts for the variation in runner develop­ ment. according to other researches (guttridge, 1985; hytönen and elomaa, 2011), this is connected to the differentiation of the meristem into a leaf rosette, also known as a branch crown or stolon. a branch crown serves as a platform for inflorescences, whereas a runner is a vegetative, extended shoot with a terminal daughter plant that may be employed for clonal multiplication (samad et al., 2021). based on physiological and production features, strawberry plants were split into various classes. as seen in figure 1, each group has a specific function, one for physiological characteristics and the other for productive attributes. because they influence plant­ ing and harvesting dates, as well as seedling repro­ duction and harvesting techniques, physiological fea­ tures are important. for both fresh and processed products, the production features (qualitative and quantitative) are essential to satisfy local consumers’ needs and exporters’ demands. fig. 1 ­ a list of different features that can be taken into account to describe a strawberry cultivar and eventually classify it. strawberry cultivars are classified into physiological and productive traits, with physiological traits playing the most important role in determining runner performance and productivity. sd: short­day, ld: long­day, dn: day­ neutral, eb: everbearing, sb: seasonal berry. al‐madhagi ‐ key of strawberry runnering 435 based on how they react to the photoperiod for flower induction, strawberry cultivars are categorised as short­day (sd), long­day (ld), or day­neutral (dn) (durner et al., 1984). the ideal method for plant mul­ tiplication, blooming behaviour, and fruit production is determined by this division, which is crucial. in the meanwhile, strawberry cultivars exhibit considerable addiction as a result of interactions between pho­ toperiodic temperature. this link has led to the inter­ change ability of the two terms when describing strawberry blossoming behaviour (cai et al., 2017). based on their respective production periods, strawberry cultivars were divided into two groups: everbearing (eb) and seasonal berry (sb). this divi­ sion may be thought of as an implementation of actual cultivar behaviour in response to temperature and photoperiod. while certain places of the world may only see one season of production from differ­ ent strawberry sb kinds, other locations may experi­ ence two seasons. as a result, the split of production cycles is inaccurate globally while being based on the same latitude (table 1). the major gene that regulates blooming and run­ ner production is called perpetual flowering runnering (pfru) (hytönen and kurokura, 2020). retentive, day­neutral (dn), continuous flowering, and long­day plants are all examples of everbearing (eb) cultivars (cai et al., 2017). the words “day­neu­ tral” (dn) and “everbearing” (eb) are interchange­ able and refer to a physiological insensitivity to day­ duration in flower bud initiation and a realistic expec­ tation of strawberry producing. weak, moderate, and strong day­neutral cultivars can be used to categorise the everbearing strawberry cultivars (nicoll and galletta, 1987). eb cultivars can produce multiple crops through­ out the year, regardless of day length, at a signifi­ cantly higher temperature than seasonal berry (sb) cultivars (smeets, 1980). everbearing (eb) strawberry cultivars, in contrast to sb strawberry cultivars, have been linked to early flowering and initiation at shoot tips, resulting in bet­ ter crown branching ability (hytönen and elomaa, 2011). as a result, eb strawberry cultivars tend to produce few stolons on a large scale (darrow, 1966; simpson and bell, 1989; dale et al., 1996), and fewer stolons than sb. since branch crowns are ended by inflorescences, the quantity of branch crowns is essentially correlated with the quantity of inflores­ cences (hytönen et al., 2004; tenreira et al., 2017). in the meanwhile, branch crowns that form from buds in the leaf axils of the crowns of mature plants are divided to create economically viable eb straw­ berry plants. the eb strawberry cultivars are quanti­ tative ld plants at medium temperatures, day neu­ tral only at low temperatures (15°c), and qualitative ld plants at high temperatures (pedraza et al., 2010; samad et al., 2021). short­day (sd) conditions, as seen in figure 2, cause eb cultivars to stop growing and become dwarfed throughout the summer season (darrow and waldo, 1934). for growth, bloom initia­ tion, and stolon formation in eb strawberries, a criti­ cal photoperiod of 15 hours at 18°c and 14 hours at 30/25°c day/night temperature is needed (nishiyama et al., 2006; sønsteby and heide, 2007). seasonal strawberry blooms (sb), which bloom in the spring and produce a fruiting crop in the summer, have been identified as short day (sd), once bloom­ ing, seasonal flowering (sf), seasonal berry (sb), sin­ gle crop, or june­bearing plants (cai et al., 2017). in table 1 ­ shows the differences in runnering and cropping between everbearing (eb) and seasonal blooming (sb) strawberry varieties throughout the year type defined cropping over the year runnering commercially propagated everbearing (eb) remontant a couple of crops only non­ to a few runners dividing of branch crowns day­neutral (dn) perpetual ld plants seasonal flowering (sb) once flowering one fruit crop more runner from the plantlets seasonal flowering (sf) seasonal berry (sb) single cropping june­bearing short­day (sd) adv. hort. sci., 2023 37(4): 433­449 436 response to the seasonally reducing photoperiod and temperature circumstances, sb strawberries begin to bloom in the late summer and fall, the year before blossoming and fruiting. the majority of sb cultivars are now regarded as facultative short­day (sd) plants as it has been established that they are mostly sd plants. at temperatures between 18 and 20 °c, they need sd for bloom induction, although at lower tem­ peratures, the majority of cultivars begin flowering on long days (ld) (ito and saito, 1962; heide, 1977; heide et al., 2013). the crucial photoperiod for sd induction is 14–15 hours (darrow and waldo, 1934; konsin et al., 2001) and the minimum number of sd cycles required for induction depends on the cultivar (heide et al., 2013). the flower­inducing effect of sd, on the other hand, is temperature sensitive, peaking at intermedi­ ate temperatures and decreasing rapidly at tempera­ tures above 21°c (heide et al., 2013). 2. propagation of strawberry sb strawberry plants are commercially propagat­ ed from plantlets that multiply from the runner nodes of mature plants because technique is quicker than seed propagation and daughter plants retain the traits of their mother plant (li et al., 2020). this plantlet is often created by nodes borne by runners or stolons that sprout from buds in the crown’s leaf axils over the summer (darrow, 1966). the runners often is elongated branch which have nodes and internodes running parallel to their length, with the bud at the first node usually being inactive (ahmed and ragab, 2003). it’s length is due to the cell divi­ sion and intermodal elongation in the plant are responsible for runner growth (nishizawa and hori, 1993). therefore, one of the most important metrics is the number of runner and daughter plants pro­ duced by mature plants. 3. environmental factors the environmental conditions are one of the most crucial factors impacting the generation of strawber­ ry runners. in all of the fragaria genotypes exam­ ined, stolon formation and flowering induction can compete for space in the axillary meristems, and both developmental strategies are sensitive to envi­ ronmental factors (brown and wareing, 1965; guttridge, 1985; bradford et al., 2010; hytönen and elomaa, 2011; heide et al., 2013; hytönen and kurokura, 2020). in response to any alteration in the environment that encourages flowering (flowering habit), the strawberry produces a crown or stolon (fig. 2). the photoperiod, chilling periods, and tem­ perature are especially linked to these environmental or seasonal factors, and their interactions may have a major effect on strawberry dispersal (andrés and coupland, 2012; salinas et al., 2017). photoperiod photoperiod is the duration of the daily exposure of an organism to illumination within hour (cammack et al., 2008), it is defined as the period of time within a 24­hour time frame that light is available (lanoue et al., 2019). one of the most crucial environmental factors for plants is light. where, the plant is impacted by the length of the lighting period (photoperiod), the radia­ tion strength, and the type of illumination wave­ lengths (colours). plants employ photosynthesis, a process that uses light as an energy source, to pro­ duce secondary compounds and carbohydrates. additionally, photoreceptors produce light that is utilised to detect and keep track of environmental changes (chen et al., 2004). when it comes to the photoperiodic control of flowering in wild strawber­ ries, phytochromes are crucial photoreceptors (rantanen et al., 2014). the photoreceptors’ main module, the leaves, is capable of detecting a broad range of wavelengths, light intensities, and photoperiods. it controls the fig. 2 ­ a schematic diagram shows the flower habit of strawber­ ry cultivars that flower all year round is the key to propa­ gation. where: everbearing (eb) behave as day­neutral (dn) plants at low temperature and are considered quali­ tative long­day (ld) at medium temperature and quanti­ tative long­day (ld) at high temperature. seasonal flowering (sf) behave like long­day (ld) at lower temper­ ature and like short­day (sd) at medium temperature. al‐madhagi ‐ key of strawberry runnering 437 essential gene proteins that the plant’s developmen­ tal regulatory programme may use to transmit infor­ mation about timing and light (valverde, 2011; shim et al., 2016). photoreceptors also enable plants to accurately monitor ambient light conditions and alter their development, morphology, and metabolic rates, including the start of blooming, in accordance with the particular environment in which they exist (song et al., 2018; roeber et al., 2022). flowering locus t (fvft1) and suppressor of the overexpression of constans1 (fvsoc1), two significant genes in the photoperiodic regulation of blooming and runners in woody strawberries, have provided some information on the photoperiodic control of fvtfl1 in seasonal flowering woodland strawberry. fvtfl1 integrates photoperiod and tem­ perature signals to control flower induction, and higher fvft1 mrna levels are linked to earlier flow­ ering under a variety of environmental conditions including light quality, photoperiod, and tempera­ ture, while turning off this gene significantly delays flowering (hytönen and kurokura, 2020). the long­day photoperiod (ld) is one of the most important environmental elements affecting the growth and development of strawberries (ito and saito, 1962; darrow, 1966; heide, 1977; okimura and igarashi, 1997; robert et al., 1999; heide and sønsteby, 2007; al­madhagi et al., 2011; hasan et al., 2011; li et al., 2020). and in distinguishing strawber­ ry runner axillary buds (hytönen et al., 2009). the effect of photoperiod on strawberry vegeta­ tive development and runner production has been widely discussed and has attracted the attention of numerous studies. petioles length, leaf number, leaf area, and runner number and length all increase with ld photoperiod (darrow, 1966; sung, 1973; plancher and naumann, 1978; nishizawa and hori, 1993; pipattanawong et al., 1996; robert et al., 1999; wiseman and turnbull, 1999; konsin et al., 2002; serçe and hancock 2005; sønsteby et al., 2006; hasan et al., 2011; li et al., 2021 b). ld conditions promote cell division and cell elongation (nishizawa, 1992; nishizawa, 1994), due to an increase in the amount of endogenous gibberellins (gas) that promote bud development in the plant (taylor et al., 1994). the number of hours during the ld photoperiod that the plant must be urged to produce a runner depends on a variety of factors, including cultivars and temperature. the photoperiodic cycle of 10 h light and 10 h dark failed to develop runners, but 14 h light and 14 h darkness did, and runner plants growing in ld were induced to flower provided they remained connected to parent plants growing in sd (hartmann, 1947). as shown in figure 3, the impact of photoperiod (p) on strawberry propagation depends on a number of different parameters, including culti­ var (c), cold storage (cs), temperature (t) and plant growth regulators (pgr). photoperiod × cultivars (p×c) the number and length of runners was influenced not only by photoperiod but also by cultivars or genetics, and the interaction of the two (pipattanawong et al., 1996; serçe and hancock 2005; hasan et al., 2011). in terms of runner produc­ tion, the strawberry parents responded differently to the photoperiod. the study by serçe and hancock (2005) shows the different responses of the wild strawberry genotype, f. chiloensis ‘cfra 0024’ (central chile) and ‘cfra 0368’ (alaska) and f. virginiana ‘eagle 14’ (ontario); they just found that only ‘eagle 14’ and ‘cfra 0368’ produced an appreciable number of runners while ‘eagle 14’ did not show a consistent trend, while ‘cfra 0368’ had the most runners under the 11 hour photoperiod. clear photoperiod responses to vegetative devel­ opment and stolon production were observed in sb cultivars (plancher and naumann, 1978; konsin et al., 2002; sønsteby et al., 2006; hasan et al., 2011), as well as on eb strawberry cultivars (guttridge, 1969; dennis et al., 1970; serçe and hancock, 2005). the difference between eb strawberry cultivars is fig. 3 ­ the response of strawberries to the photoperiod (p) effect on runner development is influenced by a number of other parameters, including cultivar (c), cold storage (cs), temperature (t), and plant growth regulators (pgr). 438 adv. hort. sci., 2023 37(4): 433­449 also evident: cultivars ‘aromas’, ‘tribute’, ‘frederick 9’, and ‘fort laramie’ did not produce runners under either ld (16h) or sd (8 h), but ‘quinalt’ produced 0.2 runner/plant under ld (16h) (serçe and hancock, 2005). flower initiation and runner development occur independently of ld in eb strawberry cultivars (piringer et al., 1958; piringer and borthwick, 1961; guttridge, 1969; dennis et al., 1970). there was also a difference between sb cultivars, where ‘camaroga’ yielded the most plantlets (22.06 per plant) when grown under the 17 hour photoperi­ od (hasan et al., 2011). according to serçe and hancock (2005), the ld photoperiod had a significant effect on runner pro­ duction, with a significant effect of an interaction between the sb cultivar and the photoperiod, with ‘allstar’ and ‘honeoye’ not producing runners in pho­ toperiod ranges of 8 ­11 h, and ‘chandler’ producing runners in photoperiod ranges of 8 , 9, or 11 h. strawberry sb developed more crowns than run­ ners during the sd photoperiod compared to the plant under the ld (18 h) photoperiod on the ‘korona’ sb strawberry (konsin et al., 2001). the number of runners is also differs between cultivars, in strawberry ‘seolhyang’ it increased dramatically after ld (16 h) (li et al., 2020). meanwhile, for sb ‘camarosa’ and ‘camaroga’ cultivars, there was no significant difference between the 15 h and 17 h ld photoperiods, and the ld (15 h) was determined to be extremely efficient (hasan et al., 2011). temperature (t) strawberry flowering habits (eb or sb) have been found to be under either qualitative or quantitative genetic influence (heide et al., 2013). figure 2 shows the behaviour of strawberries under different tem­ perature conditions. wherever, the value of 10°c is a base tempera­ ture of strawberries (al­madhagi et al., 2018). in fragaria vesca, higher temperatures were found to be critical for runner induction (heide and sønsteby, 2007). the temperature variation between day and night at the same average daily temperature is also significant in creation runners; in strawberry ‘seolhyang’, 25/15°c day/night was the best temper­ ature for runners formation (li et al., 2020). lt (11°c) and sd (10 h) enhanced branch crown growth in wild strawberry fragaria vesca, but ht (>18°c) promoted runner initiation independent of photoperiod (bedry, 2017). regardless of temperature, all of the f. x ananas‐ sa eb cultivars showed very low runner counts. temperature has an effect on runner formation in eb strawberries (smeets, 1955; smeets and kronenberg, 1955; serçe and hancock, 2005). controlling temper­ ature alone, as well as cultivating plants at varied temperatures, will not increase eb strawberry runner output (samad et al., 2021). the eb cultivars responded differently to temper­ ature in terms of runner production, with the great­ est number of runner being 0.6 and 0.7 at 30°c in ‘aromas’ and ‘tribute,’ respectively. in contrast, below the t­ range of 18­30°c, neither ‘ogallala’ nor ‘quinalt’ produced a runner (serçe and hancock, 2005). a recent study samad et al. (2021) found that run­ ner production in eb strawberries decreased signifi­ cantly at 20°c, with no statistical difference between 25 and 30°c. runners was almost twice in eb straw­ berries ‘murano’ than in ‘favori’ during the season and it was significantly higher in the plants raised outdoors than in those raised in the greenhouse (sønsteby et al., 2022). the explanation for this is that eb’s runner potential is cultivar dependant, and low temperatures (lt) put more energy into flower­ ing than runner development (rivero et al., 2021 a; sønsteby et al., 2021). photoperiod × temperature (p×t) overall, the effect of photoperiod on runner and crown production was influenced by temperature at the time of photoperiod application. ld and high temperature (ht) have been shown to improve run­ nering in all flowering classes of strawberry cultivars (eb or sb) (serçe and hancock, 2005). when the pho­ toperiod was 12 h or more and the temperature was above 10°c, runners began to multiply (went, 1957; darrow, 1966). the strawberry’s reaction to the photoperiod is affected by temperature (darrow, 1936). actual pho­ toperiod and temperature parameters varied by cul­ tivar (went, 1957). the ld must exceed a certain value at ht for runner development in eb and sb strawberry cultivars (darrow, 1937; went, 1957; smeets, 1980). temperature and photoperiod pro­ mote runnering by inhibiting flower initiation and increasing the activation of vegetative buds on the rosette crown (went, 1957; leshem and koller, 1965). meanwhile, there was no runner development at sd lt, and lt at higher light intensities had to be suppressing flower initiation (went, 1957). strawberries can develop runners at a higher temper­ al‐madhagi ‐ key of strawberry runnering 439 ature than at a lower temperature (smeets, 1955; smeets and kronenberg, 1955; went, 1957; leshem and koller, 1965; smeets, 1980). the ideal temperature for strawberries varies by cultivars. under both ld and sd photoperiods, straw­ berry cultivars differ in the optimal temperature for runner development, and the number of runners produced increased as the temperature rose from 20 to 26°c under 16 h ld, but decreased as the temper­ ature increased from 26 to 29°c (bradford et al., 2010). eb produced many runners during the 15 h ­ 20 h ld photoperiod with a temperature of at least 22.7°c (darrow, 1966; rivero et al., 2021 a), no runners developed in ‘marshall,’ at 10°c but did for 16 hours at 14°c, and for 12 hours at 17°c (went, 1957). when the temperature dropped to 18°c, the ld photoperi­ od factor alone was sufficient for optimal leaf and inflorescence growth and development (sønsteby et al., 2006). photoperiod preconditioned plants pro­ duced significantly more branch crowns than control plants, but cold­stored tray­conditioned plants pro­ duced much fewer crowns (sønsteby et al., 2006). a ld of at least 14 hours was required for runner production in 9 cultivars cultivated at 13, 16, and 21°c in eb strawberry cultivars, where the photoperi­ od ld up to 14 h being the key determinant, a specif­ ic temperature being required for a prolonged runner development duration (darrow, 1936). flower initia­ tion and runner formation in ‘revada’ and ‘rabunda’ occurred at 20 and 26°c regardless of the ld, and the length of runner formation was longer at 20 and 26°c than at 14°c, and at 16 and 24 h than at 8 h (smeets, 1980). runners are formed almost entirely in the vegeta­ tive phase of plant growth in sb cultivars, with ld × ht favouring runner production (darrow and waldo, 1934; heide, 1977; durner et al., 1984; bradford et al., 2010). sb cultivar also behaves like eb plants at lt under ld circumstances (14 h) (darrow and waldo, 1934; darrow, 1936). the honeoye sb cultivar did not develop runners at 14 or 17°c, regardless of pho­ toperiod, and it did not produce runners under sd, independent of temperature (bradford et al., 2010). in the number of runners of f1­hybrid ‘delizzimo’ cul­ tivar was significantly higher at 26°c than at lower 12°c under both sd and ld conditions (samad et al., 2022). addition, higher temperatures increased the concentrations of sugars in the leaves in ld photope­ riod (rivero et al., 2022). the eb trait can also arise when inflorescences are removed during the growing season, leading to the development of latent buds, as in the ld and ht traits causing flowering suppression (sugiyama et al., 2004). as shown in table 2, the runner formation rises at ht × ld in both sb and eb. table 2 shows a rough summary of the influence of photoperiod and temperature interaction on the generation of runners. despite the fact that the criti­ cal value of each variety is different, both groups agreed that the long day (ld) at high temperature is the best condition for the development of runners. chilling hour and cold storage (c) flower initiation in strawberries requires chilling (ito and saito, 1962; darrow, 1966; kinet et al., 1993; lieten, 1997; al­madhagi et al., 2018; al­doubibi et al., 2021). for the best production and berry quality, both types of strawberries (eb and sb) required dif­ ferent amounts of chilling period before planting. the chill­hours are measured in degrees below than 5, 7, or 8°c (yanagi and oda, 1993; risser and robert, 1993; bigey, 2002; gallace et al., 2019). if the natural environment is not favourable, the refrigerator can be used to carry out cold treatments (hamano et al., 2009). chilling stimulates cell division and elongation by breaking dormancy (lee et al., 1970; yanagi and oda, 1989). the effect of chilling (up to zero and less than 5°c) or cold storage (below zero °c) on runner develop­ ment has been connected to the type of strawberry (eb or sb), cultivars, degree of chilling, length of cold storage, and cumulative of natural chilling hours, according to the most recent study. longer cold treatments (more than 500 hours) limit flower development (taghavi and aghajani, *sd (short­day) is less than 14 hours, ld (long day) is more than 14 hours, and dn (day­neutral) is 12 hours. √: producing runner, ×: non­producing runner. low= less than 20°c. table 2 ­ interaction effect of photoperiod and temperature on runnering of strawberry factors cultivars photoperiod temperature eb sb sd* low × × ld low some cultivars × dn low × × sd high × × ld high √ √ dn high some cultivars √ adv. hort. sci., 2023 37(4): 433­449 440 2017), lead to the shorter flower differentiation (lieten, 2006; al­madhagi et al., 2018) and delay the re­initiation of fresh floral primordial in the spring (guttridge, 1958; gallace et al., 2019). strawberry propagation could benefit from this approach. chilling has been shown to increase runners gen­ eration in both eb and sb strawberry cultivars (yanagi and oda, 1990). many runners were formed when the strawberry eb or sb cultivar was subjected to a lot of cooling hours (bringhurst et al., 1960; bailey and rossi, 1965; guttridge, 1969; braun and kender, 1985; kahangi et al., 1992; risser and robert, 1993; lieten, 1997; tehranifar et al., 1998; bigey, 2002; hokanson et al., 2004; taghavi and aghajani, 2017; al­madhagi et al., 2018). the sensitivity of the chilling duration varies between cultivars; sb strawberry cultivars are more sensitive than eb cultivars, and prolonged chilling inhibits blossom production in sb cultivars (yanagi and oda, 1990). after more than 1000 hours of chilling, eb culti­ vars formed runners (hamano et al., 2009; watanabe et al., 2009; al­madhagi et al., 2018). although the cultivar does not develop runners under normal con­ ditions, and does not produce runners when chilled for 0 h, 360 h and 720 h, long chilling hours (1080 h and 1440 h) in a cold room at 2°c will reduce the flower and promote more runner (al­madhagi et al., 2018). for japanese eb strawberry cultivars (‘akihime’, ‘askaruby’, ‘sachinoka’, ‘tochiotome’, ‘toyonoka’, ‘nyoho’, and ‘yumenoka’), cold storage for more than 1000 hours interrupts dormancy, pro­ motes runner development, and increases leaf elon­ gation (watanabe et al., 2009). the duration of the cooling period for current eb strawberries is related to the cultivars. chilling tem­ peratures in eb cultivars start with runner develop­ ment in ‘revada’ and ‘rabunda’ cultivars that have not experienced natural hours of chilling (smeets, 1980), as well as in ‘rabunda’, ‘ostara’, and ‘kletter’ cultivars refrigated at 1°c for 1 and 2 months (yanagi and oda, 1990). flowering degree and stolon production in eb strawberry cultivars delizzimo and favori had little or no effect when chilled at 2°c for six weeks (rivero et al., 2021 a). furthermore, for a one to four weeks of chilling at 1°c increased runners in the cultivar eb ‘pajaro’, with no significant difference in the length of cold storage, while one or two weeks of cold storage resulted in a larger number of daughter plants (taghavi and aghajani, 2017). longer cold storage duration improved runner production in the sb strawberry cultivars ‘hokowase’ (yanagi and oda, 1990), ‘korona’ and ‘elsanta’ (sønsteby and heide, 2006), ‘allstar’, ‘chandler’, ‘latestar’, ‘northeaster’ and usda selection b27’ (hokanson et al., 2004) and ‘sulhyang’ (lee et al., 2020). sb strawberry cultivars stored chilled at 1°c for two months produced more runners than fresh plants that had never been exposed to cold (hokanson et al., 2004). the degree of cold storage also influences runner quality. according to (lee et al., 2020) sulhyang’ plants held at ­5°c produced fewer daughter plants than those stored at ­2°c, and the quantity of daughter plants was modest. long cold storage reduced vigour and glucose stores of mother plants (lieten et al., 1995). plants that have been stored cold for a long period should have a higher starch content and if possible, be culti­ vated in nurseries located at higher altitudes (lópez et al., 2002; al­doubibi et al., 2021). on the other hand, naturally cool night­time temperatures at high­ er elevations help plants collect more starch. the quantity of chilling hours the plant experiences affects runner production; both insufficient chilling and excessive chilling have an effect (hamano et al., 2009). photoperiod × cold storage (p × c) as a result, exposure to prolonged photoperiods and longer cold storage duration improved runner production. the results of the previous study show that cultivars respond differently to photoperiod × cold storage and duration. after determining the cul­ tivar type (eb or sb), this interaction is linked to the length of cold storage lc and ld photoperiod (sønsteby and heide, 2006; hamano et al., 2009; watanabe et al., 2009; rivero et al., 2021 a). due to its insensitivity to the pre­chilling history and day duration, the eb strawberry ‘rabunda’ showed con­ secutive flower development (yanagi and oda, 1989). meanwhile, more efficient runners production can be achieved in eb strawberry cultivars by combining cold storage with ld photoperiod, where ld (16 h) increase runner production by about 10% in plants chilled at 4°c that for 1000 and 1500 hours in com­ parison to unrefrigerated ones (watanabe et al., 2009). the same result was observed in eb strawber­ ry ‘natsuakari’ and ‘dekoruju’ treated with 1000 h chilling under 16 h ld (hamano et al., 2009). eb strawberry cultivars ‘natsuakari’ and ‘dekoruju’ al‐madhagi ‐ key of strawberry runnering 441 chilled for 1500 and 2000 hours (5°c) produced run­ ners above natural day length, but not below natural day length regardless of ld treatment. in contrast, after 5 and 10 weeks of precondition­ ing at 2°c no runners occurred under either ld 10 h or 20 h, while runners were common in sd, particu­ larly at 26°c and with 10 weeks of preconditioning (rivero et al., 2021 a). photoperiod enhanced the condition of fragaria shoot cultures maintained at 4°c in sb strawberries (reed, 2002). in sb strawberry cultivars ‘korona’ and ‘elsanta’, no cooling was required to re­establish nor­ mal leaf and inflorescence elongation and runner development under subsequent ld circumstances (sønsteby and heide, 2006). 4. exogenous hormone gibberellins gibberellins are required for initiation of straw­ berry runners and inhibit ga production with pp333, amo­1618, or prohexadione­calcium (pro­ca) (an inhibitor of the ga3­oxidase enzyme) (rademacher, 2000), causes the formation crown branches and reduces runner development (avigdori­avidov et al., 1977; nishizawa, 1993; reekie and hicklenton, 2002; black, 2004; hytönen et al., 2009; grez et al., 2021). the ga20ox gene is mainly expressed in the axillary meristem dome and primordial, and in developing stolons. runner less strawberries such as the wood­ land diploid strawberry (f. vesca) are caused by a mutation in the active site of a gibberellin 20­oxidase enzyme (ga20ox). as a result, ga3 stimulates run­ ners development in all genotypes and species of strawberries, including the eb types of f. vesca, f. vir‐ giniana , and the eb and sb of f. x ananassa (agafonov and solovei, 1972; solovei, 1972 a; verzilov and mikhteleva, 1974; soetarto, 1979; choma and himelrick, 1984; braun and kender, 1985; deyton et al., 1991; fouad et al., 1991; ra et al., 1996; dwivedi et al., 1999 a, b; paroussi et al., 2002 a, b; tenreira et al., 2017; li et al., 2021 a; godara et al., 2022). overall, the effect of ga3 on runner growth was variable and dependent on ga concentration (solovei, 1972 b; mohammad et al., 1990; rajesh et al., 2008), with ga3 at 50 ppm having no effect on runner growth in ‘sparkle’ (sb) and ‘ozark beauty’ (eb) strawberries (waithaka and dana, 1978). according to agafonov and solovei (1974) ga3 administered to strawberries at a concentration of 0.005% improved the quantity of runners but decreased their quality. ga3 reduced runner growth at concentrations of 100 and 200 mg/l (solovei, 1972 a). application of 50 mg/l ga3 produced runner before flower in sd ‘camarosa’ and ‘camroga’ culti­ vars (al­madhagi et al., 2012). the number of straw­ berry ‘seolhyang’ runners was reduced by ga3 foliar spray, which showed a negative correlation between the concentration and number of runners (li et al., 2020). effect of ga3 on runner growth varies between cultivar (solovei, 1972 a; choma and himelrick, 1984), with ga3 stimulating daughter­ plant formation in the eb cultivar but suppressing it in the sb cultivar (waithaka and dana, 1978; choma and himelrick, 1984). according to kender et al., (1971) the response of three eb cultivars to ga3 at 50 increased runner development in cultivars ‘ozark beauty’ and ‘superfection’, but had no effect on culti­ var ‘geneva’. due to longer internodes, eb strawberry ‘tribute’ and ‘selva’ cultivars treated with ga3 produced fewer daughter plants (dale et al., 1996). compared to naa and ccc, ga3 produced the greatest vegeta­ tive growth and runner production at 90 ppm on ‘sweet charlie’ (rajesh et al., 2008). ga3 use was related to the frequency of applied (tafazoli and vince­prue, 1978; duarte and hermosa, 1998). gas increased runner production when applied prior to the onset of dormancy and during the chill require­ ment stage (honda, 1972), but did not increase the number of runners and hastened flowering when applied about a month before the appearance of flower buds, while hastened fruit maturation when applied at the flowers opening stage. cytokinins exogenous benzyladenine (ba) resulted in a greater numbers of runners in certain studies (kour et al., 2017; liu et al., 2019), while cytokinin and auxin coordinate the dormancy and expansion of axil­ lary buds in strawberries (qiu et al., 2019). the influence of the exogenous hormone cytokinin on vegetative development has also been studied by several researchers, ba­type cytokinin has been observed by several researchers to enhance runner induction (waithaka et al., 1978; waithaka and dana, 1978; kour et al., 2017; liu et al., 2019). 6­ ba also enhanced runner induction, with 50 mg/l being the most effective concentration (li et al., 2020). adv. hort. sci., 2023 37(4): 433­449 442 in ‘sparkle’ (sb) and ‘ozark beauty’ (eb) strawber­ ries, foliar spraying with pba at 200­600 ppm increased runner production (waithaka and dana, 1978). in contrast, ba alone had no effect on the genera­ tion of runners such as eb cultivar ‘geneva’ (kender et al., 1971), sb cultivars ‘pajaro’, ‘queen eliza’, and ‘paros’ (momenpour et al., 2011) and ‘redchief’ (sb) (archbold and strang, 1986). pba caused axillary bud explants to grow into stolons (waithaka et al., 1980). interaction of exogenous hormone on runner devel‐ opment the effect of the combining hormones on straw­ berry runner development is based on a fight between them that prevents flowering. in eb ‘geneva’ the use of both n6b and ga3 had a signifi­ cant impact on runner formation (kender et al., 1971). in ‘ozark beauty’ (eb), a combination of pba and ga3 had a stronger impact on runners and daughter plant development than pba alone, and pba reduced rooting of daughter plants, which ga3 could not overcome (waithaka and dana, 1978). when ba and ga3 were combined, petioles and stolon internodes were less thickened and elongated, resulting in greater leaf area than when pba was used alone (waithaka and dana, 1978). the number of runners in the eb ‘tribute’ and ‘selva’ strawberries treated with ga3 and ba increased linearly when the benzyladenine (ba) con­ centration was increased up to 1800 mg/l, the rec­ ommend that ba at 1200 mg/l + ga3 at 300 mg/l in strawberries, under field or greenhouse conditions for runner formation (dale et al., 1996). application of 6­ba + acc resulted in the maximum number of plantlets (six plantlets per plant) (kirschbaum, 1998). in eb, ga3 at 50 ppm, ba at 50 ppm, or companion boosted the number of runners in ‘miyoshi’ by 2­3 fold, whilst ga3 or ga3 + ba raised the number of runners by up to 8 and 4 times in ‘enrai’ and ‘summer berry’, respectively (pipattanawong et al., 1996). gibberellic acid, when combined with benzylade­ nine, significantly increased runner development in the geneva cultivar, but benzyladenine alone had lit­ tle impact (kender et al., 1971). photoperiod × pgr day length photoperiod and gibberellin alone both increase runner production in strawberry culti­ vars with different genotypes and blooming habits. strawberry plant susceptibility to exogenous gib­ berellins was enhanced by ld photoperiods (tafazoli and vince­prue, 1978; al­madhagi, 2012). the ld photoperiod increased the level of endogenous gib­ berellins, which promoted the growth of plant buds (taylor et al., 1994). meanwhile, the ld photoperiod had the same effect as gibberellin, leading to a greater number of epidermal cells, indicating that cell division and internodes length were increased (nishizawa and hori, 1993; nishizawa, 1994). suppression of gas biosynthesis has been shown to promote crown branching, restrict runner produc­ tion, and improve flowering by increasing the num­ ber of possible sites for floral induction and differen­ tiation (hytönen and elomaa, 2011; tenreira et al., 2017). in a prolonged photoperiod, exogenous ga3 com­ pletely reversed the effect of prohexadione­calcium when transferring ga3­treated plants from short to long days, on the other hand, it restored normal run­ ner development, this did not happen in plants that had not been treated with ga3 (hytönen et al., 2009) the influence of photoperiod and exogenous hor­ mone interaction on the vegetative development of strawberries has been documented mainly with ga3. after exposure to the ld photoperiod, ga3 elicited comparable change in strawberries (paroussi et al., 2002 a). the study by soetarto (1979) discovered that dur­ ing the 24 h photoperiod ga3 at 150 ppm improved the stolon length of cultivar ‘ostara’. sd, dn, and ld photoperiods plus ga3 (50 ppm) resulting in the greatest vegetative growth in the ld photoperiod with 50 ppm ga3 application and great­ est number of crowns/plant when plants in the ld photoperiod and treated with 1000 ppm ccc (dwivedi et al., 1999 a). plants grow faster when treated with ga3 in the ld photoperiod than in the sd photoperiod (paroussi et al., 2002 a). by increasing the level of soluble sugar in ‘seolhyang’ the strawberry cultivar, ld photoperiod (16 h) and 50 mg/l 6­ba break the dormancy of axil­ lary buds and produced runners (li et al., 2020). gibberellins can compensate for the effects of envi‐ ronmental variables however, chilled strawberry plants treated with ga3 in tropical countries (kenya) produced about the same number of runners as those subjected to chill­ ing alone, but plants treated with ba produced signif­ icantly more runners than chilling alone (kahangi et al‐madhagi ‐ key of strawberry runnering 443 al., 1992). when the plant was exposed to chilled conditions in conjunction with the treatment of ba + ga3, the number of runners increased (kahangi et al., 1992). ga3 induced and enhanced vegetative growth, equivalent to the impact of four to six weeks of chilling (tehranifar and battey, 1997). 5. discussion and conclusions the formation of strawberry runners was influ­ enced by the interaction of genetic (cultivars), envi­ ronmental (photoperiod, temperature, chilling hours or cold storage), and internal (hormones and carbo­ hydrate) factors. the most significant factor impacted by long­day photoperiod (ld) is the cultivar in strawberry runner proliferation. for the development of stolons in all flowering strawberry classes, ld and ht must inter­ act. additionally, in order for runners to grow in eb and sb strawberry cultivars, ld must surpass a cer­ tain value at ht (darrow, 1937; went, 1957; smeets, 1980). this may have been connected to the influ­ ence of photoperiod on photosynthesis and the metabolism of carbohydrates, which suggested that the amount of carbohydrates may rise during the cre­ ation of runners and that the amount of soluble sug­ ars was positively correlated with the number of run­ ners (li et al., 2020). everbearing (eb) strawberries absorbed more co2 when temperature and irradi­ ance rose (rivero et al., 2021 b). however, the accu­ mulation of photosynthates was not the only factor that affected the runner induction in cultivated strawberries (li et al., 2021 b). the photoperiod also enhanced the amount of endogenous hormone as well as the synthesis and accumulation of starch, sugar, amino acids, and protein (li et al., 2022). espe­ cially gibberellins that promote runner bud develop­ ment (taylor et al., 1994). and the effect of the appli­ cation of gibberellins or photoperiod is the same result (taylor et al., 1994). meanwhile, more efficient runner production can be achieved in eb and sb strawberries by gibberellins (gas) cytokinin and chilling period (cp) individually or in combination lead to production of stolons. in addition, cp or gas enhances the effect of the pho­ toperiod ld. long cold storage or the chilling (cp) also works on converting starch to soluble sugars (lópez et al., 2002; al­madhagi et al., 2018; al­ doubibi et al., 2021), and increased level of endoge­ nous gibberellins (avigdori­avidov et al., 1977). in fact, greater photosynthesis and respiration under the ld condition imply that more chemicals and energy are produced, which may account for the increased soluble sugar content in strawberry seedlings during runner production (li et al., 2020). in which the respiration produced atp and hydrolyzed the sugar for biosynthesis, resulting in altered levels and ratios of endogenous hormones, maybe with a focus on gibberellins and cytokinin, by transferring more sugar to axillary buds that are in high demand while restricting the quantity of sugar via the apical shoot (mason et al., 2014). in strawber­ ry runners (non­dormant buds), as opposed to dor­ mant buds, the expression of genes involved in sugar metabolism and signalling was also increased (qiu et al., 2019). in order to explain the changes in sig­ nalling between stages of bud release to sustained development, cao et al. (2023) propose a model of apical dominance that combines auxin, sucrose, strigolactones, gibberellins, and cytokinin. the application of cytokinin helped to break the apical dominance and shift the auxin/cytokinin ratio (al­madhagi, 2012; qiu et al., 2019), which led to the growth of axillary buds to runner (li et al., 2020). this cytokinin appears to promote runnering in early development stage, but prolonged, elevated it levels inhibit runnering. the application of ab­6 increased the level of free active of endogenous gibberellins and auxin in straw­ berry seedlings to a value higher than the free active of endogenous cytokinin (al­madhagi, 2012) and increased the soluble sugar (al­madhagi, 2012; li et al., 2020). this ultimately converted polysaccharides into soluble sugars and stimulated axillary buds to produce runners. additionally, the photoperiodic control the two genes (fvft1) and (fvsoc1), that reg­ ulation of blooming and runners of woody strawber­ ries (hytönen and kurokura, 2020). this may be able to explain how the interaction of photoperiod and temperature influences the growth of runners by enhancing photosynthesis, elevating endogenous hormone levels, raising respiration, and raising the amount of soluble sugars. in order to fully develop a runner in sb strawberries or partially generate in eb strawberries, the plant makes advantage of the indi­ rect effects of photoperiod or cold storage as shown in figure 4. it can be concluded that, in all strawberry cultivars (eb or sb), soluble sugars may be required for axillary buds to emerge from their dormant case and pro­ duce runners, when this is impacted by the applica­ adv. hort. sci., 2023 37(4): 433­449 444 tion of photoperiod ld or exogenous hormone. the optimal strategy is determined by optimizing the planting date for propagation or changing the propagation conditions, depending on the cultivars and the seasonal environment (photoperiod × tem­ perature) of the geographic region. those 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and non‐everbearing strawberry cultivars. ­ j. jap. soc. hortic. sci., 59: 357­363. yanagi t., oda y., 1993 ­ effects of photoperiod and chill‐ ing on floral formation of intermediate types between june and everbearing strawberries. ­ acta horticulturae, 348: 339­346. 183 book reviews la cura dei giardini storici. teoria e prassi. michael rohde. edizione italiana a cura di massimo de vico fallani. giardini e paesaggio, vol. 31. leo s. olschki, firenze (italy), 2012. pp. xiii + 569. isbn 978-88-222-6149-6. € 58.00. the author of this interesting volume has, for some time, worked with historic gardens in germany and has asked the important question of how to define professional, sustainable and lasting care of historic gardens and how to put it into practice. michael rohde, director of the garden of the prussion castles and parks of berlin-brandenburg foundation, together with a team of four other authors, has created an important base not only for understanding historic gardens but also for future research about caring for and preserving them. this 570-page book is the result of two research projects undertaken at the department of landscape architecture at the university of hannover. the first part of the book is dedicated to the past: the theory of the art and design, and the care for trees, flowers, paths and waterworks are documented in detail, crossing over all stylistic periods. in the first part, the various members of the working group describe the techniques adopted in the various periods considered. barbara vogt reports about studies on flowers, flowerbed ornamentation and provides a list of flowers used and certification of their origin from renaissance up to the xx century. the chapter by andreas von hoeren documents changes in the use of irrigation and hydraulic systems from the renaissance to the 20th century. the second part of the volume is dedicated to the care and restoration of historic gardens. some well-known parks are taken as example and interventions undertaken and recommended for trees, shrubs, blossoms, paths and waterworks are described, with precise instructions for their care. there are numerous color illustrations of historic plans, engravings, drawings and inventories, adding valuing to the theoretical and practical parts of the work. the volume concludes with results and recommendations to care for historic gardens properly. as massimo de vico fallani points out in the presentation, “reading this book will, for some, be a surprise because it reveals a well-structured reality toward the mastery of curative nature which is not always known, at least not in present times, by those working in italy, and draws attention to maintanence, which is often overlooked.” this book enriches the considerable literature available on historic gardens from historic and cultural and scientific and technical points of view, offering the reader meaning and knowledge about his topic which plays a leading role in the image of our country. francesco ferrini impaginato 197 adv. hort. sci., 2024 38(2): 197­210 doi: 10.36253/ahsc­14261 inhibition of bleaching of stored red hot pepper through appropriate postharvest technologies and practices o.f. etefa (*), s.f. forsido, y.b. tola department of postharvest management, jimma university, jimma, ethiopia. key words: capsicum spp., carotenoids, color fading. abstract: the colour qualities of hot red pepper are the major issue in pepper value chain. due to poorly coordinated scientific evidence, information on the factors causing colour loss and the inhibition mechanism is not well known. therefore, this review paper aimed to summarize the inhibition mechanism of stored red hot pepper bleaching through appropriate postharvest technologies and practices. the information in this paper was gathered from a variety of sources, including journal articles, books, book chapters, workshop proceed­ ings, fao reports, and aoac official methods of analysis. according to these studies, carotenoids, surface colour, and extractable colour (asta value) are the primary colourants that define hot red pepper. the findings demonstrate that low­temperature drying methods, such as open sun drying, are best for preserving the red hot pepper powder’s colour quality, while higher tempera­ tures cause the colour to darken. blanching, the use of desiccants (cacl2), and chemical dipping are pretreatments that preserve the best colour quality by hastening the drying time. similarly, storage of red hot pepper powder at lower temperatures (5oc) resulted in less colour degradation. in other words, materials used for packaging that have a high barrier to light, moisture, and air, such as laminated aluminium, amber or black polyethylene, and high­density polyethylene, maintained a higher level of colour quality. through their influ­ ence on drying and processing times, breeding technologies, varieties, and maturity level also impact colour quality. in conclusion, the colour quality of red hot pepper is highly influenced by environmental, biological, and process­ ing methods. it is, therefore, critical to use appropriate drying and pretreat­ ment techniques, storage time, well­managed storage temperature, appropri­ ate processing methods and packing materials, and improved agronomic prac­ tices for the sustainable management of colour fading and adulteration that can occur throughout the value chain. 1. introduction pepper (capsicumspp.) is one of the oldest and most widely used crops. evidence from prehistoric times show that capsicum species were used as a spice as early as 5500 bc. central and south america are the origins of the capsicum spp., with peru and mexico believed to be the (*) corresponding author: obsemaatii@yahoo.com citation: etefa o.f., forsido s.f., tola y.b., 2024 ­ inhibition of bleaching of stored red hot pepper through appropriate postharvest technologies and practices. ­ adv. hort. sci., 38(2): 197­210. copyright: © 2024 etefa o.f., forsido s.f., tola y.b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 january 2024 accepted for publication 7 march 2024 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-14261 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 197­210 198 second centres of origin, subsequently it spread into the new world tropics before being introduced into asia and africa in 1493 (bosland and votava, 2000). it was introduced to europe by christopher columbus in the 15th century, and it later spread to africa and asia via the flourishing trade routes of spain and portugal at the time. as it spread around the world, it was quickly adopted and used as a spice, giving rise to many regional varieties (jarret et al., 2019). today, hot pepper (capsicum annuum l.) is the world’s most important vegetable cultivated after tomato, and it is used as a vegetable, spice, or condiment in fresh, dried, or processed products (acquaah, 2009). asia accounts for approximately 65% of global pepper production, whereas the united states, europe, and africa each contribute 13.3%, 11.9%, and 10.1%, respectively (fao, 2019). red peppers have been used as food additives for several years. they are used fresh as green, red, and multi­colored whole fruits in sauce, paste, canning, and pickling; dried spice (whole fruits and powder); and as an ornamental (batiha et al., 2020). it is also used as a medicinal plant to treat and relieve pain due to its antioxidant, immune­modulating, antibac­ terial, and anticancer properties (maji and banerji, 2016). it also prevents gastrointestinal problems such as flatulence, loss of appetite, gastroesophageal reflux disease, and gastric ulcers (kim et al., 2014). it also reduces oxidative stress, inflammation, and body weight, and is used to treat dyspepsia, as well as antiatherosclerotic, antidiabetic, and antihyperten­ sive medications (baenas et al., 2019). red pepper carotenoids are also beneficial to humans in a variety of ways, including as sources of natural food color and provitamin a (villa­rivera and ochoa­alejo, 2020). moreover, ground pepper and oleoresins are used to improve the color and flavour of soups, stews, sausage, cheese, snacks, salad dress­ ing, sauces, pizza, confectionaries, and beverages (arimboor et al., 2015). the oleoresin, which is used to replace synthetic food colorants extracted from red carotenoids, is now a source of income for pep­ per­producing countries (melgar­lalanne et al., 2017). it also contains vitamin c, phenolic com­ pounds, proteins, fat, carbohydrate, dietary fibre, sodium, potassium, calcium, magnesium, iron, zinc, copper, and manganese (dobón­suárez et al., 2021). natural food colourants are preferable to synthet­ ic food colourants because they reduce the risk of synthetic food colourants to human health (arimboor et al., 2015). however, fading of the colour in red pepper is becoming a major issue due to a variety of complex reasons; as a result, fraudulent traders are adulterating the red pepper products with other arti­ ficial products such as sudan dyes, water and oil solu­ ble dyes, oils, and rhodamine (osman et al., 2019). the colour fad is also caused by improper post­har­ vest handling and pepper production technologies (hyderabad­avanti et al., 2019). it is also influenced by a lack of sufficient agricultural inputs, postharvest technological equipment, and knowledge, heat gen­ erated during grinding, drying techniques, red pep­ per powder particle size, water activity and interac­ tion with moisture, type of packaging materials, stor­ age periods, maturity stage, relative humidity, and light (kasampalis et al., 2022). several novel drying technologies and postharvest handling systems were developed to preserve the nutritional and colour qualities of hot red pepper products (getahun et al., 2021). perhaps the information on the bleaching of stored red hot pepper, the factors that cause the bleaching, and the inhibition mechanisms are not well summarized for protracted management of the problem and ensuring the nutritional importance of the red hot pepper. this paperis, therefore, aimed to review the appropriate postharvest technologies and practices that inhibit the bleaching of stored red hot pepper. 2. materials and methods literature was gathered between june 2022­ september 2022. this review relied on journal arti­ cles, books, book chapters, workshop proceedings, fao reports, aoac official methods of analysis, bul­ letins, legal papers, and unpublished reports, includ­ ing m.sc. and ph.d. dissertations. we used the nice guidelines: the manual (nice, 2014), systematic reviews: crd’s guidance for undertaking reviews in health care (crd, 2009), the cochrane handbook (lefebvre et al., 2011), and the joanna briggs institute reviewers’ manual (santos et al., 2018) guidelines to conduct our literature search. in total, 1406 pertinent sources of information were found after a thorough investigation of databases and web­ sites. the databases used in this study were food science technology abstracts (fsta), biosis citation index, pubmed (medline), web of science, cab abstracts, cochrane library, science direct, wiley online library, scopus, and google scholar. additionally, theses and dissertations were gathered etefa et al. ‐ inhibition of bleaching of stored red hot pepper 199 from institution websites. the related electronic liter­ ature was obtained using a boolean search tech­ nique. based on their direct relevance to the review’s title, 107 articles from the entire corpus of down­ loaded literatures were used (cooper et al., 2018). 3. major red pepper colors and measurements methods carotenoids carotenoids are yellow­orange­red lipophilic pig­ ments found in photosynthetic plants, algae, and microorganisms (mezzomo and ferreira, 2016). it is a fat­soluble pigment found in animals and plants that contains over 700 compounds that exhibit red, orange, and yellow colours (jaswir et al., 2011). it is a colour basal structure derived from tetraterpenephy­ tane (c40), and any changes to this backbone result in various types of carotenoids (mezzomo and ferreira, 2016). carotenoids such as β ­carotene, zeaxanthin, and lutein are primary carotenoids that are directly involved in photosynthesis, whereas lycopene, α­carotene, and capsanthin are secondary carotenoids that play no role in photosynthesis (arimboor et al., 2015). overall, it is a good natural source of colour and is commercially used as food colourants and feed additives (jaswir et al., 2011). carotenoids such as capsorubin, cryptoxanthin, and zeaxanthin are found in peppers as fatty acid esters. the carotenoids formed in the fruit during ripening are primarily responsible for the colour of red pepper. red pepper carotenoids have over 50 dif­ ferent structures (arimboor et al., 2015). capsanthin, capsorubin, and their isomers are the most impor­ tant pigments, accounting for 30­60% and 6­18% of the total number of carotenoids in peppers, respec­ tively (nadeem et al., 2011). it is also reported that capsanthin contributes 45.27% of total carotenoid, while antherazanthin and capsorubin contribute 8.95% and 11.45%, respectively (ko et al., 2022). the type of carotenoids depends on environmental con­ ditions, ripening stage, cultivar and agro­climatic con­ ditions, and so on (kim et al., 2021). processing methods such as drying, seed removal, and grinding are also cited as major contributors to the carotenoid content of fruits (loizzo et al., 2013). the wide structural diversity, as well as possible isomeric forms and derivatives, have been used to analyse carotenoid content, but the analysis method is difficult (yan et al., 2020). sample preparation, extraction with various solvents, purification, saponi­ fication, separation, detection, and quantification are all common analytical steps. however, the instability associated with carotenoids’ characteristic conjugat­ ed double bond structure necessitates the incorpora­ tion of control measures such as minimizing the pos­ sibility of carotenoid loss during analysis (borba et al., 2019). the precautions include performing laboratory operations in dimmed, yellow, or red light and per­ forming sample preparation, extraction, evaporation, and saponification steps in the presence of antioxi­ dants under a protective nitrogen or argon environ­ ment at a temperature below 40°c, as well as storing the samples/extracts in an inert atmosphere at tem­ peratures around ­20°c (arimboor et al., 2015). surface color surface colour measurements are used to specify colours perceived by the human eye. different sur­ face colour measurement techniques have been developed to lessen the challenge of an object’s colour being perceived differently by various observers. this is due to the fact that various factors, including the observer’s sensitivity, the size of the object, the light source and illumination, the back­ ground colour and contrast, and the angle at which the object is viewed, affect how each observer com­ prehends the colour they observed (yang et al., 2018). since the late 1920s and 1930s, the relative eye’s sensitivity to light was recognized as a standard observer trait (macdougall, 2010). the cielab (international commission on illumination’s lab) colour system is used to ascertain the surface colour of red peppers (pathare et al., 2013). hunter colour parameters have also been used to determine the surface colour of red pepper (sharangi et al., 2022). the hunter lab system has a more uniform colour space than cie. furthermore, the surface colour of the hot red pepper is determined by the variety, maturity stage, pretreatments made during process­ ing and drying techniques used (sharma et al., 2015). extractable color extractable colour is a spectrophotometer­mea­ sured total pigment content expressed in asta units. the current procedures for measuring the extractable colour in dehydrated capsicums and oleoresins were developed by the association of official analytical chemists. higher asta colour units indicate a brighter red colour and product acceptability (babu et al., 2014). however, because the extraction pro­ adv. hort. sci., 2024 38(2): 197­210 200 cess takes 16 hours, the asta method is completely objective, destructive, and time consuming (monago­ maraña et al., 2022).the extractable colour of the pepper is heavily influenced by storage condition­ sand temperature (belović et al., 2014), pretreat­ ment method and maturity stage (bhandari et al., 2013). 4. kinetics of red hot pepper color fading and deter­ minant factors red pepper colour degradation is caused by oxi­ dation caused by singlet oxygen and other reactive species such as o2, h2o2 and oh (ding et al., 2015). the oxidation process results in the complete loss of carotenoids. it is also caused by carotenoid isomer­ ization from trans to cis form, which is accelerated by exposure to relatively higher heat, acids, and light (provesi and amante, 2015). unlike oxidation, iso­ merization results in colour saturation rather than a complete loss of carotenoids (song et al., 2017). the following sections go over some of the factors that contribute to red pepper colour fading. light light has a significant impact on the colour quality of peppers both during cultivation and storage. crop exposure to direct sunlight in the cultivation field has a negative impact on the colour and final product of the pepper. for example, a pepper grown and kept in a greenhouse for less than 200 days revealed a high extractable colour (gómez et al., 1998). similarly, prolonged exposure to sunlight resulted in a decrease in c* values and an increase in h ab values, indicating a decrease in vividness (saturation) and an increase in yellowness in tandem with a decrease in redness, because changes in c* values are directly related to colour stability (pathare et al., 2013). the carotenoid contents in fruits grown in shaded green­ houses were also significantly higher than those grown in unshaded greenhouses and fields (keyhaninejad et al., 2012). open sun drying with long processing times was also detrimental, resulting in losses of 79% capsaicinoids and 24.6% capsaicin (topuz et al., 2011). in comparison to frozen and hot air­dried peppers, chilli peppers dried in the open sun also preserved less bright red colour and ascorbic acid (toontom et al., 2012). similarly, 50% asta colour values were reduced in korean red pepper powders exposed to sunlight for 42 days (kim et al., 2015). in general, higher oxygen exposure and intense vaporisation from the pepper’s surface cause pigment decomposition during open sun drying (sharangi et al., 2022). water activity “water activity” is a thermodynamic measure of water in material that is calculated by dividing the vapour pressure of the water in a sample by the vapour pressure of pure water at a given tempera­ ture, which ranges from 0.1­1 (lewicki et al., 2004).it gauges how effectively the water in the reaction can participate in a chemical or physical process. moreover, it has a negative impact on the colour and safety of red pepper (rhim and hong, 2011). it has been reported that 0.4­0.6 water activity values result in less colour loss (lee, 2012), but higher water activity develops brown and tarnish­black colour, indicating that the degradation of carotenoid pig­ ments, non­enzymatic browning index, asta values, and surface colour (rhim and hong, 2011). in other words, water activity between 0.4 and 0.6 reduces red pepper surface colour deterioration (lee, 2012). moreover, high water activity at high storage tem­ peratures exacerbates the level of colour fading. therefore, storing red pepper powder below 25oc, below medium ranges of water activity, and between 10­14% moisture content to maintain red pepper colour quality (rhim and hong, 2011). for instance, storing red peppers powder at water activity below 0.3, in a nitrogen atmosphere and/or reducing the package free space volume and lowering storage temperature improves the carotenoid content (lee et al., 1992). an increasing trend of colour stability was also observed with increasing water activity values during pepper powder storage, with the greatest sta­ bility observed at a water activity of 0.64 (relative moisture, 14%), but increasing the moisture level above this value caused a significant reduction in colour intensity (kanner et al., 1977). drying and storage temperature temperature is an important factor that con­ tributes to the degradation of red pepper colour quality by causing oxidation of carotenoid pigments due to heat destruction and lipid oxidation at high temperatures (maurya et al., 2018). it also results in the loss of volatile compounds and nutrients like vita­ mins c, capsaicinoids, phenolic compounds, antioxi­ dant capacities, and other physicochemical proper­ ties. for example, colour reduction of pepper powder etefa et al. ‐ inhibition of bleaching of stored red hot pepper 201 is observed to be rapid at high temperatures even when moisture content is low during storage (kim et al., 2004). temperature and relative humidity above ambient storage conditions also degrade the colour of pepper, but pepper powder stored in refrigeration and freeze showed a minimal extractable and surface colour loss (addala et al., 2015). in other words, the drying kinetics of red and yel­ low chilli peppers showed a decreasing trend of red colour decomposition at lower drying air tempera­ tures (less than 55°c) (andrade et al., 2019).for instance, yellow sweet peppers dried in a microwave convective dryer showed a 3.5­fold increase in red colour decomposition with increasing microwave power and temperature due to isomerization of carotenoids at high temperatures (swain et al., 2014). however, carotenoids are more stable at high temperatures in pungent cultivars because capsaici­ noids in pungent varieties affect the chemical inter­ actions of carotenoids in red pepper tissues, provid­ ing significant protection against thermal destruction. thermal destruction and lipid oxidation, on the other hand, can be inhibited by endogenous antioxidants such as phenols, vitamin c, and e. for example, heat has reduced the carotenoid degradation of dried pepper prior to milling to greater than 2 mg/g, but pungency cannot be guaranteed for more than 3 storage months (daood et al., 2006). processing methods the red pigment of red pepper is also affected by processing methods and conditions. furthermore, the kinetics of colour degradation during processing and storage are temperature and water­activity depen­ dent. for example, the colour of ground pepper dete­ riorated faster than that of non­ground pepper during storage, indicating that powdering reduces carotenoid concentration (carnevale et al., 1980). exoquin and irradiation­treated samples also retained more red colour of the pepper powder after six months of stor­ age (addala et al., 2015). similarly, after 12 months of storage, red pepper with 10% seed had higher colour stability than controls (van blaricom and allen, 1953). dry­heat cooking methods such as stir­frying and roasting are also preferred over moist heat cooking methods such as boiling and steaming to retain the nutrient compositions and antioxidant properties of red pepper (wang et al., 2017). pepper seed addition the initial carotenoid concentration of extractable colour is also strongly influenced by the extent of powdering and seed inclusion. the inclusion of seeds reduces the carotenoid content because seeds have little carotenoid content and are highly affected by water activity, packaging atmosphere, storage tem­ perature, and pepper treatment (lee et al., 1992). in other words, adding seed to the flesh slows the rate of colour loss, but because the seed dilutes the initial colour, it reduces the overall shelf life (klieber and bagnato, 1999). the addition of 10% seed to sesame­ treated samples also improved red pepper colour stability even after a 12­month storage period (van blaricom and allen, 1953). variety the studies revealed that cultivar and growing conditions all have an effect on the degree of pepper coloration. for example, a significant difference in total carotenoid content was observed among differ­ ent pepper varieties grown in nigeria, with geno­ types uns3 and nskyre exhibiting the highest total carotenoids content and genotype tatase containing high­carotene content (17.30±0.35 mg/100 g) (abu et al., 2020). color value difference was also observed among three red hot pepper varieties grown in ethiopia, with marakofana having the highest icu value (648331±31673)(kinfe, 2009). the colour of hot red pepper pods also varied significantly among dilla­ grown varieties, with melkazala having a darker­red pod colour than marekofana (teferi et al., 2015). similarly, asta values ranging from 2­296 were reported for local and exotic hot pepper genotypes grown in the same conditions in ethiopia, indicating that variety influences hot red pepper colour (aklilu et al., 2018). fruit ripening at harvest pepper colour varies greatly depending on maturi­ ty stage due to its carotenoids content; unripe fruit can be green, yellow, or white, turning to red, dark red, brown, and sometimes black when mature (mohd­hassan et al., 2019). capsanthin, capsorubin, and cryptocapsin are keto carotenoids that produce a brilliant red colour, whereas ­carotene, zeaxanthin, violaxanthin, and ­cryptoxanthin produce a yellow­ orange colour (arimboor et al., 2015). in other words, red peppers contain β­cryptoxanthin, capsan­ thin, capsorubin, β­carotene, antheraxanthin, violax­ anthin lutein and α­carotene, while yellow colored peppers contains β­carotene, zeaxanthin, antherax­ anthin, violaxanthin lutein and α­carotene, but 202 adv. hort. sci., 2024 38(2): 197­210 brown colored peppers have green color (chlorophll b) and lutein (mohd­hassan et al., 2019). ripe pepper fruits of various varietiesexhibit a wide range of colours ranging from white to deep red (mohd­hassan et al., 2019). for example, total carotenoids content increased with maturity stage, with red sweet pepper having the highest total carotenoids compared to orange, yellow, green, and white cultivars (gnayfeed et al. , 2001). total carotenoids also increased up to 24 fold with increas­ ing ripening, with deep red pepper containing the most total carotenoids, followed by faint red and colour breaks (markus et al., 1999). the apparent colour values of marekofana vari­ eties grown in ethiopia also varied according to maturity stage, with green pepper having the highest l* (38) and b (23.3) values (getahun et al., 2020). similarly, the after­ripened spice paprika had the highest asta colour value when compared to the early and late harvest stages (sipos et al., 2010). the pepper powder produced from fruits left to dry on the plant (40­25% moisture content) had also the highest asta colour value (270 asta unit), but decreased to 160 asta unit in succulent fruits imme­ diately after red­ripening (kanner et al., 1977). however, the green matured stage of hot red pepper (bhandari et al., 2013) cultivars had higher b­ carotene content than the ripe red stage. 5. inhibiting red hot pepper bleaching via posthar­ vest technologies and practices drying method drying red pepper before processing is a common practice around the world to maintain colour quality (yang et al., 2018). open sun drying is the most wide­ ly used drying technique in developing countries and produces the best red pepper colour when compared to hot air and infrared drying, but infrared drying sig­ nificantly improved drying time at the same drying temperature (cao et al., 2016). for example, sun­ dried paparika contained more carotenoids than paparika dried in a hot air oven, refractive window, or freeze drier (topuz et al., 2011). the convectional drying method, on the other hand, resulted in colour darkening and a decrease in vitamins and bioactive compounds (guiné, 2018). in other words, as com­ pared to other unconventional dryers, solar drying is generally more cost­effective, and consistent, and does not rely on weather conditions (tiwari, 2016). in contrast to sun drying, reflection window drying is less expensive, takes less time to dry, and improves product quality (el­hamzy and ashour, 2016). hot air drying is also preferred over sun drying due to its rapid, massive, uniform, and sanitary dry­ ing; however, due to its higher temperature require­ ment, nutrient degradation and a lower rehydration ratio have been reported (guo et al., 2021). similarly, the highest colour retention was found in red pepper dried at 65oc in oven­dried red pepper at the short­ est drying time (480 min), but prolonged sun­dried products had the hardest texture (sharangi et al., 2022). dry heat processing is also believed to pre­ serve the colour of red pepper better than wet pro­ cessing (rybak et al., 2020). drying pretreatments pretreatments are used to reduce drying time, increase energy efficiency, and improve product quality (srimagal et al., 2017). several pretreatment methods were used to reduce drying time while retaining the red colour and other nutritional proper­ ties of red pepper. the most commonly used pre­ treatment techniques in drying red pepper are ethyl oleate and microwave blanching (srimagal et al., 2017), hot water blanching, the use of desiccants (cacl2) (romauli et al., 2021), and chemical dip (guiné, 2018). of these, steam blanching produced more colour and other bioactive compounds than hot water blanching (rybak et al., 2020). similarly, ohmic heat treated pepper and bell pepper blanching in boiling solution containing 1% sodium hydroxide and 0.25% magnesium carbonate for 3 minutes and drying at 50°c air temperature (singh et al., 2000) produced better colour quality than the other meth­ ods. the highest red pigment and drying kinetics were also observed in oh­pretreated pepper powder as compared to hot water blanching because oh pretreatment and drying at 70°c reduce drying time while retaining red pepper quality (incedayi, 2020). the use of 2% ethyl oleate and 5% k2co3 solution dip pretreatments at 50oc during hot air dying of red pepper dried also exhibited the highest hunter l (lightness), *a (redness), and *b (yellowness) values than untreated peppers (máximo et al., 2017). similarly, microwave blanching followed by brine solution dipping improved the drying rate and retained more beta carotene content in the samples (delfiya et al., 2018). the asta and surface colour values of the rehydrated peppers were also greatly improved by pretreatment with a solution containing etefa et al. ‐ inhibition of bleaching of stored red hot pepper 203 calcium, salts (10%), and sodium metabisulfite prior to hot­air, oven (50­80°c), or microwave drying at 60­ 180 w (vega­gálvez et al., 2008). the surface colour of the pepper powder made from sliced chilli pods without the pedicle and pretreated with 0.01% kms (sarker et al., 2012) and bell pepper shreds dried in a solar polytunnel drier after pretreatment with a kms 0.20%+ca 0.50% solution (sharma et al., 2015) was also better than the control. ethoxyquin­treated and irradiated samples also retained a better extractable colour after six months of storage (addala et al., 2015). similarly, red pepper powder blanching in the microwave, infrared, and high­humidity hot air impingement had the highest red pigments when compared to hot water treatment (wang et al., 2017) (table 1). storage time and temperature management lower temperatures are recommended to prevent red pepper colour loss during storage (rhim and hong, 2011). for example, red pepper powders had the same asta colour value, capsanthin content, and redness (a*) when stored at ­5 and ­20oc than 20oc, 2oc, and ­1oc storage temperatures (choi table 1 ­ effect of pretreatment on the colour quality of red hot pepper pretreatments drying method recommendation references sample without pedicle, cut longitudinal­ ly and treated with 0.01% kms; sample without pedicle and sliced; sample with­ out pedicle as a whole; sample with pedi­ cle as a whole sundrying the powder without pedicle, sliced into two parts along the length and treated with 0.01% kms maintained better sensory quality sarker et al. (2012) blanching at 93°c in plain or in 2% nacl solution for 4 min solar dryingand in the open sun blanching followed by drying in natur­ al convection solar dryer scored better color acceptability owusu­kwarteng et al. (2017) submerging for 10 min at 25°c in an aqueous solution of 15% (w/w) nacl, 1.0% (w/w) cacl2 and 0.3% (w/w) na2s2o5; blanching in hot water at 85 °c refractancewindow™ drying; oven drying; sun drying blanching prior to refractance window drying enhanced stability of pepper colorfor 12 months storage period el­hamzy and ashour (2016) dipping in chemical solutions (w/v):2% ethyl oleate;2% ethyl oleate+2% naoh; 2% ethyl oleate+2% naoh+4% potassium carbonate. dipping temperature; 23°c; 60°c greenhouse drying; open sun drying dipping red peppers in 2% ethyle­ oleate + 2% naoh + 4%k2co3 solution at 60oc resulted in best color reten­ tion ergüneş and tarhan (2006) blanching with hot water; blanching with steam at 98◦c for 3 minutes; ultrasonic treatment (us); pulsed electric field treatment (pef); combined methods vacuum drying (10 mpa, 70°c for 24 h) better retention of vitamin c and carotenoids content was observed for the treatment combination based on sonication with a pulsed electric field rybak et al. (2020) control (without any pre­treatment); blanching in boiling water for 3 min; blanching + soaking for 5 min in the fol­ lowing chemical solutions: kms (potassi­ um meta bisulphite) 0.25%; kms 0.35%; ca (citric acid) 0.3%; ca 0.6%; kms 0.2% + ca 0.5%; kms 0.3% + ca 0.25% dehydrator a 58±2°c till constant weight obtained blanching of bell pepper shreds in boiled water followed by pre­ treatments with kms 0.20% + ca 0.50% at 55­60°c resulted higher colour stability sharma et al. (2015) adv. hort. sci., 2024 38(2): 197­210 204 et al., 2018). furthermore, as the storage period increased up to 12 months at high temperature, the overall freshness, redness, hot flavour, moisture release, and edibility decreased (wang et al., 2017). chilli powders stored at 5oc retained vitamin c and colour for up to 6 months without the use of any synthetic preservatives. additionally, chilli pepper powders packed in flexible foil and stored at 5oc retained more vitamin c and colour without the addition of preservatives for up to 6 months as compared to high­density polypropylene storage (al­ sebaeai, 2017). in other words, red pepper lightness (cie l*) and yellow colour intensity (cie b* value) increased, but red colour (cie a* value) decreased, and the hue angle shifted from red orange to orange­ yellow after three years of storage at 21.5oc (belović et al., 2014). for samples stored at 35oc for 42 days, the l*, a*, b*, delta e, visual colour acceptability, and total carotenoids also revealed a decreasing trend in candida red, candida orange, and candida yellow (kim et al., 2015). similarly, minimum (5%) extractable colour and hunter l, a, b values change observed for red pepper stored under refrigeration and freeze conditions for both pretreated and non­treated samples at 6 months storage time compared to the room (22°c) and elevated temperatures (35°c and 80%) (addala et al., 2015). total carotenoid content loss is also facilitated by storing the microencapsulated naec at temperatures above 35oc (guadarrama­lezama et al., 2014). in general, the longer the storage period, the lower the storage temperature required, and vice versa (choi et al., 2018).red pepper can also be stored at low temperatures (0­5oc) for up to 6 months and frozen (­5oc) for longer than 6 months (wang et al., 2017). packaging materials packaging is a method of providing proper environmental conditions for food during storage, and the materials used in packaging are determined by the nature of the product, storage, and handling conditions (temperature, humidity, risk of physical deterioration) (marsh and bugusu, 2007). the air composition inside the package is determined by the nature of the packaging materials, which affects the rate and extent of nutrient loss and microbial activity (amit et al., 2017). natural jute sacks, for example, were the most widely used bags to pack pepper pods in the world, with significant retention of product quality, but red peppers stored in jute bags were vulnerable to aflatox in contamination as compared to pods packed in polyethylene bags (iqbal et al., 2015). hot peppers stored in ldpe film also spoiled faster than unpacked control fruits at 18­20oc and 28­30oc storage temperatures due to excess water vapour accumulated inside the package (mahajan et al., 2016). similarly, 77.4% of the initial amounts of health and nutrition­promoting compounds in pepper pods stored in natural jute at room temperature for five months were preserved, but capsaicinoids and antioxidant levels gradually decreased in dry pepper pods over the long storage period (iqbal et al., 2015). purdue improved crop storage (pics) bags have recently been introduced to store agricultural products because they provide a better barrier to light, air, and moisture. other storage materials used to pack dried peppers include high­density polyethylene (hdpe) bags, which have a high moisture barrier and thus cause less deterioration to dried products than natural jute sacks (sachidananda et al., 2013). five months of storage in synthetic hdpe plastic bags at room temperature preserved 87.3% of the initial amounts of total carotenoids and ascorbic acid (iqbal et al., 2015). similarly, due to the high rate of migration of water vapour from the storage environment into these packaging materials, aluminium foil, and high­ density poly ethylene bag had no effect on water activity for the entire storage period for both red and yellow capsicum pod up to 60 days as compared to polypropylene and poly ethylene bags (sachidananda et al., 2013). the studies also revealed that red pepper pods stored in laminated aluminium for more than 120 days showed the least difference in total carotenoid content and browning index, followed by high­density polyethylene (hdpe) and polypropylene (weil et al., 2017). this is due to the low light transitivity through the laminated aluminium foil, which results in less reduction of the photo­ degradable carotenoid pigments. in general, aluminium foil laminate was generally recommended for storing chilli pepper powder because it is a barrier to light, moisture, and air. as alternatives, amber or black polyethylene, high­density polyethylene, and saran/cello/saran poly laminate pouches are recommended (sachidananda et al., 2013). table 2 also illustrates some packaging­related effects on the colour quality of red hot peppers. etefa et al. ‐ inhibition of bleaching of stored red hot pepper 205 variety and breeding technologies developing breeding programs that effectively preserve the vibrant red color of peppers throughout the drying and processing stages plays a pivotal role in enhancing the appeal and desirability of red peppers. furthermore, to prevent colour loss through breeding programs, the total carotenoid content of dried products from different cultivars should be compared and selected, and the cultivar with the highest total carotenoid content should be chosen (paran and table 2 ­ effect of packaging materials on the colour quality of red hot pepper packaging materials type conservation method recommendation references polypropylene; aluminum laminated pouch; woven polypropylene bags red chilli powder with and without vacuum pumping storage at 5±0.5°c and 26±1°c for 12 months less color loss was recorded at 5±0.5°c and in laminated bags under vacuum at both storage temperature prerna et al. (2019) aluminum; ldpe; hdpe; paper bags; polythene line gunny bags; gunny bags dried red pepperpod storage for two months under ambient conditions laminated aluminium film retained highest color, ascorbic acid and oleoresin anjaneyulu and sharangi (2022) polypropylene packages with thickness of 3, 4, 5, 12.5, 20 and 30 micron ground chilli pepper storage at room temperature (28±2°c) for two months polypropylene films with thicknesses of 30 and 20 microns exhibited enhanced preservation of carotenoid content akusu and emelike (2019) low density polythene pouch; pet jar; laminated film pouch green chilli pepper slice storage for 28 days after 7% k2s2o5 pretreatment + blanching for 1.5 min + dehydration in a hot oven at 53°c pet jar and laminated film poach preserved better visual colour of the slices ansari et al. (2020) sealed polyethylene bags; sealed polypropylene bags sweet peppers pod storage for 28 days at 8°c after dipping in hot water at 50°c for 3 min and 55°c for 1 min polypropylene bags exhibited superior preservation of appearance, carotenoids, and ascorbic acid regardless of the temperature used for dipping said et al. (2013) polypropylene; polyvinyl chloride plastic film ‘yalova charleston’ pepper in normal atmosphere and map storage using 35μ pp and 35μ pvc at 7°c and 90±5% rh samples were successfully stored for 30 days using 35μ pp at 7°c and 90±5% rh akbudak (2008) polyethylene bag; jute sac; banana leaf; refrigerator; ambient condition; evaporative cooling system sweet pepperpod freshsealing combining an evaporative cooling system with a polythene bag proved to be a more effective storage method garuba et al. (2022) adv. hort. sci., 2024 38(2): 197­210 206 fallik, 2011). however, when comparing and selecting cultivars, one should consider not only the variety with the highest total carotenoid content but also the cultivar’s carotenogenic capacity, which is manifested by a higher red­to­yellow isochromatic pigment fraction ratio (r/y) and capsanthin to zeaxanthin ratio (caps/zeax) (hornero­méndez et al., 2000). cultivar mana had the highest total carotenoid content (13208 mg/kg dwt), but the lowest r/y(1.25) and caps/zeax (3.38) ratios, so these parameters should be improved. cultivar negral, on the other hand, had a higher carotenoid content (8797 mg/kg wet), r/y, and caps/zeax ratios, whereas cultivar numex had the highest caps/zeax ratio (7.17) and lower total carotenoid content and should be improved by crossbreeding with mana, which has a higher total carotenoid content (hornero­méndez et al., 2000). additionally, the capsaicin, heat level, and colour quality of known pepper genotypes revealed that genotypes have a significant effect on capsaicin and colour values. furthermore, breeders should consider disease resistance as well as drying characteristics of peppers, as some genotypes are not suitable for drying, with increased pungency and colour loss during the drying process (arpaci et al., 2020). gnayfeed et al. (2001) also reported that k­v2 variety had also the highest total carotenoids followed by f­03, sz­178 and cseresezenye, respectively. the studies also revealed that the maturity stage has a significant impact on red pepper colour, with candida red paprika having higher total carotenoids, b*, and l* values than candida yellow paprika (kim et al., 2015). different varieties considered for the study showed the same ripening­dependent pattern of b­ carotene content increase, with the highest b­carotene content observed in the ripe red stage (bhandari et al., 2013). the total carotenoid content also decreased from 7966 to 1701 g/g in the break, faint red, and deep red stored pepper after three months, with faint red having the highest carotenoid content (markus et al., 1999). in general, breeding technologies, and varieties have a significant impact on the colour and other bioactive compounds of hot red pepper and should be considered in the management of red pepper colour loss (martínez­ispizua et al., 2021). 6. conclusions in conclusion, the findings of this review paper indicated that colour fading hot red pepper is one of the major challenges in pepper production worldwide, which is highly determined by many factors such as genotype, maturity stage, exposure time to sunlight, higher temperature, relative humidity, moisture content and water activity, and oxygen during drying, processing techniques, storage methods, and product particle size. there is a need for significant effort to raise community awareness, engage stakeholders, and prioritize scientific research in order to address the inadequate attention given to preventing the fading of the bright red color in hot peppers. the use of light­ and oxygen­permeable packaging materials, high­temperature drying, prolonged storage of the powder at high temperatures, and early and/or delayed harvesting were all noted in our review paper as contributing factors to the typical colour loss of red hot pepper powder. based on the consulted literature, it is vital to employ techniques like low temperature drying, water blanching, and chemical dipping before the drying process in order to minimize color loss in red hot pepper powder. furthermore, storing the peppers at a low temperature of 5°c, using packaging materials that are resistant to light, moisture, and air, and ensuring that the peppers are harvested at the appropriate maturity level are also essential steps to be taken in consideration. acknowledgements the authors acknowledged all sources cited in this review paper. references abu n.e., chimdi g.o., ojua e.o., 2020 ­ nutritional and anti‐nutritional evaluation of ten genotypes of pepper (capsicum annuum l.) grown in a derived savanna ecology of nigeria. ­ ethiopian j. sci. technol., 13: 17­ 30. acquaah g., 2009 ­ horticulture: principles and practices, 4th edition. ­ pearson publishing, cambridge, uk, pp. 816. addala r., vasavada m., dong j., subramanian s., 2015 ­ effect of 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changes of red pepper (capsicum annuum) dur‐ ing hot air drying. ­ j. food eng., 231: 101­108. impaginato 315 adv. hort. sci., 2024 38(4): 315­326 doi: 10.36253/ahsc­16385 https://oaj.fupress.net/index.php/ahs interaction between sowing date and mulching is important for better growth and productivity of carrot in a weather­ vulnerable area of ethiopia j.s. hussen 1 (*), s.h. muhie 2 1 department of horticulture, college of agriculture and natural resource, mekdela amba university, tuluawlia, ethiopia. 2 department of plant science, college of agriculture, wollo university, dessie, ethiopia. key words: delay sowing, early sowing, grass mulch, marketable yield, mulch, root vegetable, weed suppress. abstract: inadequate agronomic practices and unfavorable weather conditions often hinder carrot cultivation. therefore, this experiment evaluated the effects of sowing date and mulching on the growth and yield of carrots during the 2023/2024 main cropping season at kersole, legambo district. the experi­ ment involved three sowing dates (early, mid, and late in july) and four mulching materials (no mulch, sawdust, straw, and dried grass), utilizing a randomized completely block design (rcbd) with three replications. except main effects of sowing date, and interaction effects on days to 50% emergence and root diameter, all other parameters were significantly (p≤0.05) affected. early sowing combined with either sawdust or dried grass mulch resulted in the highest marketable root yields respectively. late sowing without mulch and with dried grass mulch showed the lowest marketable root yield and minimum weed density respectively. early sowing with sawdust mulch also provided the highest net benefit, while early sowing with dried grass mulch exhibited the highest marginal rate of return. therefore, early sowing with dried grass mulch can be recommended for carrot cultivation in study areas and similar agroe­ cologies. however, for optimal results, it is necessary to carry out the experi­ ment using several mulching materials and various sowing dates across seasons and locations. 1. introduction carrot (daucus carota l.) is a commonly important root crop of the apiaceae family, widely distributed worldwide. carrots are herbaceous dicotyledonous plants that grow upright, reaching a height of 20­50 cm when mature. the fleshy taproot is its primary edible part, which typically (*) corresponding author: jemalseid780@gmail.com citation: hussen j.s., muhie s.h., 2024 ­ interaction between sowing date and mulching is important for better growth and productivity of carrot in a weather‐vulnerable area of ethiopia. ­ adv. hort. sci., 38(4): 315­326. orcid: hjs: 0009­0000­4631­2761 msh: 0000­0002­1144­5668 copyright: © 2024 hussen j.s., muhie s.h. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 20 july 2024 accepted for publication 13 december 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-16385 http://oaj.fupress.net/index.php/ahs http://orcid.org/0009-0000-4631-2761 http://orcid.org/0000-0002-1144-5668 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(4): 315­326 316 exhibits a straight, conical, and cylindrical shape. it was originally wild in different parts of asia and europe. it was primarily domesticated in rich afghanistan, considered the first center of origin, and turkey is believed to be the second center of origin. from these centers of diversity, carrots gradually spread across europe, the mediterranean, and numerous countries in asia. over time, it was culti­ vated and introduced to local wild varieties across the globe (stolarczyk and janick, 2018). carrot roots are highly valued for their abundance of carotenoids, which serve as precursors to vitamin a (tabor and yesuf, 2012). additionally, carrots contain flavonoids, vitamins, and minerals, making them a nutritious crop that contributes to overall health and well­being (zeleke and derso, 2015). china is the leading global producer, with europe emerging as the rapidly advancing carrot market. notably, north america, particularly the usa and canada, boasts the most sig­ nificant shares in the carrot markets. as of 2020, the world consumed 46.3 million metric tonnes of turnips and carrots globally, according to faostat 2021 data. ethiopia has a relatively low production compared to the global average. its production has not been adequately exploited as it faces several constraints such as limited research activities on the topic, unfa­ vorable weather conditions, and poor agronomic practices such as unplanned sowing dates, lack of mulching practices, and improper weeding. in the study area, the quality of carrot roots is also compro­ mised due to suboptimal agronomic practices, thus discouraging farmers from engaging in carrot produc­ tion. typically, farmers in the study area sow carrots on bare beds without applying mulches. similarly, they often sow carrots in late july, thus leading to poor seed germination, inadequate growth, and development as well as exposure to severe winter conditions (soil moisture deficit and lack of rainfall) towards the end of summer. the research by mengistu (2009) reveals that the pre­termination of rainfall, occurring during critical developmental stages of the crop, especially the root initiation stage, results in both quantitative and qualitative reduc­ tions in yield. farmers relying on subsistence agricul­ ture within legambo district have faced recurrent droughts and famines resulting from severe weather occurrences associated with climate change, such as erratic rainfall, frost threats, and hailstorm flooding (cafer and rikoon, 2017). within subsistence agriculture, the occurrences and regularities of climate extremes and variabilities pose significant challenges comparable to average annual shifts. the magnitude and implications of cli­ mate variability are not adequately examined in ethiopia. for subsistence agriculture, occurrences and frequencies of climate extremes and variabilities are equally affecting as of mean annual changes. however, despite its climatic constraints, the region still holds promise as a viable location for cultivating carrots, especially through strategic adjustments in sowing dates and the application of mulching tech­ niques. the importance of adjusting the cropping schedule is appropriate to adapt a weather and cli­ mate variations in the particular region (desta et al., 2020). the timing of sowing is essential as it aligns with favorable climatic conditions and has been demonstrated to impact the growth and yield of car­ rots (gagopale, 2019). moreover, the use of mulch helps to regulate temperature extremes (rajasekar et al., 2020). furthermore, cultivators must consider the appropriate sowing date and mulching as one of the most significant factors to maximize productivity and quality, minimize weed occurrence, and maintain soil health. consequently, this study was conducted to evaluate the effect of sowing date and mulching on the growth and yield of carrots in kersole, legambo district. 2. materials and methods description of the study area the experiment was conducted at kersole kebele, legambo district of agriculture and temperate fruit nursery site, south wollo zone, ethiopia in the 2023/2024 main cropping season (from july to november). the site is located at 10°51’ n and 39°11’ e (fig. 1) with an altitude of 2800 meters above sea level (m a.s.l.). the area is situated 501 kilometers north of addis ababa and 372 kilometers east of fig. 1 ­ map of the study area. hussen and muhie ‐ sowing date and mulching significantly impacts carrot growth and yield 317 bahir dar. the area experiences an annual rainfall ranging from 700 to 1300 millimeters. the site has an average minimum temperature of 3°c and an aver­ age maximum temperature of 18°c. the predomi­ nant soil type in the area is clay soil, characterized by a ph of 5.57 (regassa et al., 2023) (fig. 2). the region is predominantly characterized by its rolling terrain, (regassa et al., 2023). approximately 68.54% of the district features a temperate (dega) agroecology, with the next largest portion comprising alpine (wurch) landscapes at 29.53%. a small percentage of 1.93% and 0.0016% of the district area is covered by subtropical (woinadega) and tropical (qolla) agroe­ cology, respectively. treatments and experimental design the experiment contained two factors (sowing date x mulching material). sowing dates comprised three levels (early ­ july = 10th july (s1); mid ­ july = 20th july (s2) and late ­ july = 30th july (s3)). four lev­ els of mulching materials were: no mulch (m0), saw­ dust (m1), straw (m2) (fig. 3), and dried grass (m3). the experiment arrangement used a randomized completely block design (rcbd) in three replicates. the whole experimental area was 218.3 m2 (29.5 m x 7.4 m) in length and width respectively. this area was divided into three blocks, and each block was further subdivided into 12 plots. as a result, there were a total of 36 unit plots. the treatments were assigned randomly within each plot of the block. each unit plot had a net size of 3.6 m2 (2 m x 1.8 m) which consisted of 9 rows and 40 plants per row. the distance between adjacent blocks was 1.0 meters, while the distance between plots within a block was 0.5 meters. experimental materials and procedures ‘nantes’ variety of carrots was used for the experi­ ment. the seeds of this particular variety were sourced and acquired from the debrezeit agricultural research center. farmers are highly interested in this variety of carrots for their good adaptation, high mar­ keting demand, and better root quality in the study area. the experimental field was readied using tradi­ tional tillage methods and cultivated using oxen to loosen the soil, catering to the deep and well­drained soil requirements favored by carrots. the soil was molded into raised beds to enhance drainage, pro­ mote extensive root growth, ensure uniformity, and minimize soil compaction. after this, a field layout was implemented, and each treatment was assigned randomly to the experimental plots. seeds were sown as per each sowing date time on a 20 cm height raised bed with 20 cm x 5 cm spacing between rows fig. 2 ­ monthly average weather data of the study area during the experimentation period (2023/2024). source: https://power. larc.nasa.gov/data­access­viewer/ fig. 3 ­ pictures of mulch during a field trial. adv. hort. sci., 2024 38(4): 315­326 318 and plants respectively. the straw and dried grass mulch were cut into small pieces approximately 5­10 cm by machete, following the method outlined by olfati et al. (2008). after sowing the seeds on each particular sowing date, mulching materials were applied at about a rate of 4 t ha­1 sawdust and 6 t ha­1 straw and dried grass. fully rate of phosphorus (175 kg p2o5 ha 1) was applied at the time of each sowing date, while the nitrogen in the form of urea (150 kg ha 1) was applied in a split way: half of the rate was applied at the time of each sowing date, and the remaining half was top dressed in the spaces during the active vegetative crop stage, which occurred 5 weeks after emergence (wae). weeds were lifted manually and removed from the crop fields, while harvesting was done at maturity by using a hand cul­ tivator. data collection days to 50% emergence and 90% physiological maturity were obtained by counting the number of days to days when 50% of the seeds emerged and 90% of the plants attained physiologically matured, respectively. carrot plants were physiologically matured when their lower leaves turned yellow and roots were at the harvestable size with their crown attaining a diameter ranging from 2­3.8 cm as described by unece (2018). at physiological maturity stage, the plant height and number of leaves per plant were obtained from ten randomly selected plants from each unit experi­ ment. the height of ten randomly selected plants from the ground level to the end of the uppermost parts was measured using a ruler. after harvesting, the root length and diameter of ten randomly selected marketable carrot roots from each unit experiment was recorded using a digital caliper. the root diameter was obtained approximately two centimeters below the root collar (at middle) accord­ ing to zelalem (2019). then the root fresh weight was recorded by weighing ten randomly selected mar­ ketable carrot roots from each net plot area and each replication after harvesting using a sensitive balance and the mean values were computed for further analysis. however, marketable carrot roots were those that were free from mechanical damage, dis­ ease, insect pest attack, undersized (<50 g), and cracks. the weight of those roots obtained from the designated plot was measured in kilograms using a scale balance and the yield was expressed as a ton per hectare. carrot roots that were diseased, insect pest­damaged, cracked, branched, and undersized (<50 g) were considered unmarketable roots and their weight was measured in kilograms using a scale balance and their yield was also expressed as a ton per hectare. weed density was assessed determining the num­ ber of weeds in each plot during the second and fourth weeks after sowing. samples were collected from a designated area measuring 0.25 square meters (0.5 m x 0.5 m) at two randomly selected points within each plot. the mean values were com­ puted for further analysis and expressed as a quanti­ ty per square meter. data analysis all the collected data were subjected to analysis of variance (anova) using the statistical procedures described by gomez and gomez (1984) with the help of r­software version 4.2 and package agricolae (r institute, 2022). the mean separation was conducted using the least significant difference (lsd) at 5% of the required probability level. a simple correlation analysis was conducted to determine the relationship between growth and yield parameters of carrots as influenced by sowing date and mulching. moreover, partial budget analysis was conducted to evaluate the economic feasibility of sowing date and mulching, following the procedures below described by cimmyt (1988). in brief: total root yield (try): the average yield of each treatment, measured in tons per hectare. adjusted total root yield (ajtry): the average yield was decreased by 10% to accommodate the tendency for experimental yields to exceed what farmers could attain using identical treatments. in economic assessments, farmer yields are typically adjusted to be 10% lower than research findings to align with practical expectations. adjusted total root yield (ajtry) = try x (1 ­ 0.1) gross field benefit (gfb): determined by multiplying the farm/ field gate price received by farmers for the carrots when sold by the adjusted marketable yield. gross field benefit (gfb) = ajtry x price of carrot at farm gate total variable costs (tvc): the expenses for mulch, the application cost of mulch, and labor costs for weeding varied across the treatments. the costs of additional inputs and production practices like land hussen and muhie ‐ sowing date and mulching significantly impacts carrot growth and yield 319 preparation, sowing, fertilizing, and harvesting were either consistent or negligible across the treatments. net benefit (nb): obtained by deducting the total variable costs for inputs (tvc) from the gross field benefit (gfb). net benefit (nb) = gross field benefit (gfb) ­ total variable costs (tvc) marginal rate of return (mrr%): calculated as the division of the change in net benefit by the change in total variable cost. marginal rate of return (mrr%) = [change in net benefit (δnb)]/[change in total variable cost(δtvc)] x 100 3. results phenological parameters the analysis of variance (anova) showed that except for the effects of sowing date on days to 50% emergence, days to 90% maturity were significantly affected (p≤0.05) by sowing date, mulching, and their interactions (sowing date with mulching materials). in the case of mulching materials, sawdust mulch (m1) resulted in a minimum of 13.00 days, while bare soil (m0) led to a maximum of 15.00 days for 50% emergence. a maximum (101.66 days) number of days to achieve 90% maturity was observed from late sowing in july with growing carrots on bare soil (s3m0). whereas, the minimum (97.00 days) number of days to 90% maturity was recorded from early sowing in july with sawdust mulch (s1m1) (table 1). growth and yield parameters anova revealed that all the growth and yield parameters were significantly (p≤0.05) affected by sowing date, mulching, and their interactions. however, root diameter was not significantly influ­ enced by the interaction effects of sowing date and mulching. the maximum plant height (23.94 cm), and number of leaves (9.80) were observed from early sowing with sawdust mulch (s1m1). conversely, min­ imum plant height (18.65 cm) and leaf numbers (6.80) were obtained from late sowing without mulch (s3m0) (table 2). the maximum root length (17.43 cm), and root fresh weight (106.78 g) were observed from early sowing with sawdust mulch (s1m1). while, minimum root length (14.00 cm) and root fresh weight (54.87 g) were obtained from late sow­ ing without mulch (s3m0). moreover, root diameter showed a linear decrease as the sowing date was delayed in which the maximum (2.67 cm) and mini­ mum root diameter (2.39 cm) were recorded from growing carrots on sawdust mulch (m1) and without mulch (m0) respectively (table 2). on the other hand, early sowing with sawdust mulch (s1m1) resulted in the highest marketable root yield (26.19 t ha­1) and the minimum (0.71 t ha­1) unmarketable root yield. following closely, the early sowing with dried grass mulch (s1m3) recorded a marketable root yield of 23.57 t ha­1. in contrast, late sowing without mulch (s3m0) resulted in the lowest marketable root yield of 13.19 t ha­1 and the maxi­ mum (2.12 t ha ­1) unmarketable root yield. furthermore, marketable root yield showed a linear decrease as the sowing date was delayed. conversely, table 1 ­ effects of sowing date and mulching on days to 50% emergency (de) and 90% maturity (dm) asian economic status mean values within rows and columns followed by a different letter(s) are significantly different at a 5% probability level. cv = coefficient of variation. lsd = least significant difference. factors de (no.) dm (no.) sowing dates (s) s1 13.83 a 99.50 c s2 13.91 a 100.25 b s3 14.00 a 101.00 a mulching (m) m0 15.00 a 101.55 a m1 13.00 c 98.44 d m2 13.88 b 100.77 b m3 13.77 b 100.22 c interaction (s x m) s1m0 15.00 a 101.33 a s1m1 13.00 c 97.00 d s1m2 13.66 b 100.00 b s1m3 13.66 b 99.66 b s2m0 15.00 a 101.66 a s2m1 13.00 c 98.33 c s2m2 14.00 b 101.00 a s2m3 13.66 b 100.00 b s3m0 15.00 a 101.66 a s3m1 13.00 c 100.00 b s3m2 14.00 b 101.33 a s3m3 14.00 b 101.00 a lsd (0.05) 0.25 0.84 cv (%) 1.87% 0.49% 320 adv. hort. sci., 2024 38(4): 315­326 unmarketable root yield showed a gradual increase as the date of sowing was delayed (table 3). weed density anova revealed that sowing date, mulching, and their interactions significantly affected weed density (p≤0.01). the density of weeds showed a progressive decrease as the date of sowing was delayed in which the maximum (109.36 n m­2) and minimum (68.00 n m­2) densities of weeds were recorded from early (s1) and late (s3) sowing in july respectively. whereas, in the cases of mulching maximum (179.87 n m­2) and minimum (35.62 n m­2) densities of weeds were recorded from growing carrots on bare soil and dried grass mulch (m3) applications respectively (table 3). correlation the correlation analysis using pearson correlation coefficients (r) was performed to assess the relation­ ship between the growth and yield parameters of carrots, considering the effects of sowing date and mulching. the results showed a significant positive correlation among all the growth parameters of the carrots, indicating a direct relationship where the effect of one parameter depends on another. however, the growth parameters, namely plant height and leaf number, exhibited a negative correla­ tion with weed densities (wd). accordingly, the total yield of carrots positively correlated with plant height (r = 0.77**), number of leaves per plant (r = 0.61**), root length (r = 0.75**), root diameter (r = 0.69**), root fresh weight (r = 0.64**), and marketable root yield (r = 0.6**). furthermore, except for the unmar­ ketable root yield (ury), all the yield parameters were negatively correlated with weed densities (table 4). partial budget analysis the minimum (375 usd ha ­1) and maximum table 2 ­ effects of sowing date and mulching on plant height, number of leaves, root length, root diameter, and root fresh weight mean values within rows and columns followed by a different letter(s) are significantly different at a 5% probability level. cv = coefficient of variation. lsd = least significant difference. factors plant height (cm) leaves (no.) root lenghts (cm) roort diameter (cm) root fresh weight sowing dates (s) s1 21.94 a 8.20 a 16.14 a 2.68 a 80.65 a s2 21.06 b 7.82 b 15.41 b 2.51 b 65.82 b s3 20.30 c 7.49 c 14.86 c 2.45 b 60.40 c mulching (m) m0 19.63 d 7.23 c 14.75 c 2.39 c 60.87 c m1 22.49 a 8.76 a 16.32 a 2.67 a 80.55 a m2 20.89 c 7.50 b 15.35 b 2.53 b 66.13 b m3 21.38 b 7.79 b 15.46 b 2.61 ab 68.28 b interaction (s x m) s1m0 20.25 ef 7.45 c 15.47 c 2.50 cde 63.98 cd s1m1 23.94 a 9.80 a 17.43 a 2.86 a 106.78 a s1m2 21.68 bc 7.65 c 15.76 bc 2.64 bc 73.45 b s1m3 21.88 bc 7.90 bc 15.90 b 2.74 ab 78.37 b s2m0 19.99 f 7.45 c 14.78 e 2.35 ef 63.77 cd s2m1 22.39 b 8.35 b 16.02 b 2.62 bcd 67.62 c s2m2 20.34 ef 7.60 c 15.39 cd 2.48 def 65.29 cd s2m3 21.53 c 7.90 bc 15.44 c 2.60 bcd 66.61 c s3m0 18.65 g 6.80 d 14.00 f 2.32 f 54.87 e s3m1 21.15 cd 8.15 b 15.51 c 2.54 cd 67.24 c s3m2 20.66 def 7.45 c 14.92 e 2.47 def 59.66 de s3m3 20.74 de 7.58 c 15.04 de 2.49 cde 59.84 de lsd (0.05) 0.74 0.49 0.38 0.09 5.57 cv (%) 2.09% 3.75% 1.46% 3.72% 4.93% hussen and muhie ‐ sowing date and mulching significantly impacts carrot growth and yield 321 (863.33 usd ha ­1) total variable cost (tvc) was obtained from early sowing in july with no mulching (s1m0) and late sowing in july with sawdust mulching (s3m1) respectively and all the remaining treatments were confined between these two ranges (table 5). according to the results of the partial bud­ get analysis, early sowing with sawdust mulch (s1m1) yielded the highest net benefits of 11266.67 usd per hectare with a remarkable marginal rate of return (799.4%). following closely was early sowing in july with dried grass mulch (s1m3), which had a net ben­ efit of 10555.67 usd per hectare and the highest marginal rate of return (12,199.7%). on the other hand, late sowing in july with no mulching (s3m0) resulted in the lowest net benefit of 6489.5 usd per hectare and an unacceptable marginal rate of return (mrr%) (table 5). 4. discussion and conclusions phenological parameters despite the sowing date, mulching regulates important seed emergence factors. in line with a study conducted by mengistu and yamoah (2010), most carrot varieties exhibited a typical emergence period ranging from 10 to 15 days. mulching regulates essential factors for seed germination and emergence, including soil moisture, temperature, and air conditions. this optimized environment created by mulching can contribute to faster and table 3 ­ effects of sowing date and mulching on marketable root yield (mry), unmarketable root yield (ury), and weed density (wd) mean values within rows and columns followed by a different letter(s) are significantly different at a 5% probability level. cv = coefficient of variation. lsd = least significant difference. factors mry (t ha­1) ury (t ha­1) wd (n m­2) sowing dates (s) s1 23.40 a 0.96 b 109.36 a s2 21.65 b 1.48 a 86.01 b s3 19.16 c 1.56 a 68.00 c mulching (m) m0 17.60 c 1.75 a 179.87 a m1 23.74 a 0.99 d 46.42 c m2 21.79 b 1.36 b 89.25 b m3 22.49 ab 1.23 c 35.62 d interaction (s x m) s1m0 20.57 cd 1.31 de 231.03 a s1m1 26.19 a 0.71 i 61.35 f s1m2 23.31 b 0.97 gh 93.03 d s1m3 23.57 b 0.85 hi 52.02 g s2m0 19.04 d 1.82 b 172.50 b s2m1 23.25 b 1.07 fg 45.63 g s2m2 22.1 bc 1.62 c 90.40 de s2m3 22.23 bc 1.44 cd 35.51 h s3m0 13.19 e 2.12 a 136.10 c s3m1 21.79 bc 1.20 ef 32.3 h s3m2 19.97 cd 1.49 cd 84.3 e s3m3 21.69 bc 1.42 d 19.33 i lsd (0.05) 2.45 0.19 8.12 cv (%) 6.76% 8.76% 5.46% table 4 ­ correlation analysis of growth and yield parameters of carrot as influenced by sowing date and mulch par = parameters, de = days to 50% emergence, dm = days to 90% maturity, ph = plant height, nl = number of leaves, rl = root length, rd = root diameter, rfw = root fresh weight, mry = marketable root yield, ury = unmarketable root yield, try = total root yield, wd = weed densities, ** = highly significant (p ≤ 0.01), * = significant (p ≤ 0.05), ns = not significant. par de dm ph nl rl rd rfw mry ury try wd de 1 0.77 ** ­0.76 ** ­0.66 ** ­0.67 ** ­0.65 ** ­0.54 ** ­0.72 ** 0.66 ** ­0.70 ** 0.77 ** dm 1 ­0.85 ** ­0.86 ** ­0.84 ** ­0.73 ** ­0.79 ** ­0.60 ** 0.74 ** ­0.56 ** 0.49 ** ph 1 0.84 ** 0.88 ** 0.85 ** 0.81 ** 0.80 ** ­0.85 ** 0.77 ** ­0.50 ** nl 1 0.86 ** 0.70 ** 0.83 ** 0.64 ** ­0.70 ** 0.61 ** ­0.40 * rl 1 0.82 ** 0.85 ** 0.78 ** ­0.86 ** 0.75 ** ­0.32 ns rd 1 0.73 ** 0.72 ** ­0.79 ** 0.69 ** ­0.4 2* rfw 1 0.67 ** ­0.72 ** 0.64 ** ­0.23 ns mry 1 ­0.77 ** 0.60 ** ­0.42 * ury 1 ­0.72 ** 0.40 * try 1 ­0.41 * wd 1 adv. hort. sci., 2024 38(4): 315­326 322 growing carrots without mulch, the practice of mulching generally enhanced the likelihood of achieving early maturity. mulching achieved this by providing organic matter, effectively regulating soil temperature and moisture levels. in agreement with this experiment, singh and jaysawal (2021) observed that sawdust mulching has a significant influence on the harvesting period (maturity). growth and yield parameters the higher plant height and leave numbers observed from the early sowing in july (s1) could be attributed to maximum rainfall and temperature in july and august (fig. 2). moreover, they contributed to improving various vegetative aspects, including plant height and leaf number. additionally, carrots require a sufficient amount of water for proper growth and development consequently, these conditions play a crucial role in accelerating the crop’s physiological processes. considering the combined effect of precipitation and temperature, carrot farmers must plan their planting and cultivation schedules accordingly. sowing during periods of adequate rainfall can improve the chances of successful growth. this result is consistent with kabir et al . (2013), who observed that all environmental conditions, especially temperature, facil itated vegetative growth. furthermore, researchers have observed that early sowing may result in maximal photosynthesis and a longer growth period than late sowing, which encountered more efficient seed emergence. the moisture content should be sufficient for the seeds to absorb water and germinate. low soil moisture content delays or inhibits seed germination in the field, reduces uniformity of seedling performance, and total stand establishment, and ultimately reduces the yield of carrots (muhie et al., 2024). the delayed maturity observed in late sowing (s3) could be attributed to unfavorable weather conditions typically experienced towards the end of summer (august). insufficient rainfall during the critical growth stages such as root development may adversely affect plant phenology. throughout the vegetative growth stages of the late sowing date, minimum temperatures were received. this might have requested a prolonged time to reach 90% maturity. in contrast, the vegetative growth periods (july and august) associated with early sowing in july benefited from sufficient rainfall and relatively favorable temperatures (fig. 2). indeed, this promoted better vegetative growth, which is vital for achieving early 90% maturity. sandler et al. (2015), support the current finding, that a proper sowing date could help to minimize damage from cold, moisture deficit, weeds, pests, and diseases. similarly, the accelerated maturity of carrot roots might be associated with the easy uptake of nutrients as the available water helps the plant to dissolve the nutrients and move through transpiration pull which in turn helps carrot roots to mature early (muhie et al., 2024). in contrast to table 5 ­ partial budget and marginal rate of return (mrr) analysis for a response of carrot to sowing date and mulching d = dominated, selling price of carrot at farm gate = 0.5 usd kg­1, labor cost = 2.5 usd man per day. treatment combinations total root yields (kg ha­1) adjustable total root yield (kg ha­1) growth field benefit (usd) total variable cost (usd) net benefit (usd) marginal rate return (%) rank s1m0 21880 19692 9846 375 9471 ­­ s2m0 20860 18774 9387 387.5 8999.5 d s3m0 15310 13779 6889.5 400 6489.5 d s1m3 24420 21978 10989 433.33 10555.67 12199.7 1 s2m3 23670 21303 10651.5 445.83 10205.67 d s3m3 23110 20799 10399.5 458.33 9941.16 d s1m2 24280 21852 10926 541.66 10384.33 531.8 3 s2m2 23720 21348 10674 554.16 10119.83 d s3m2 21470 19323 9661.5 566.66 9094.83 d s1m1 26900 24210 12105 838.33 11266.67 799.4 2 s2m1 24320 21888 10944 850.83 10093.17 d s3m1 22990 20691 10345.5 863.33 9482.16 d hussen and muhie ‐ sowing date and mulching significantly impacts carrot growth and yield 323 bare soil encounters unfavorable soil conditions, leading to the production of carrots with poor root quality. findings of this experiment align with, shahadot (2021), indicating that the application of sawdust mulch is likely associated with the provision of consistent moisture and nutrients to the root zone. this favorable supply of moisture and nutrients promotes rapid cell division and cell elongation, ultimately leading to the production of long and thicker roots. in addition, consistent findings of the maximum fresh weight of roots were reported in multiple studies using sawdust mulch, including those conducted by ladumor et al. (2020), acharyya et al. (2020), and paunović et al. (2020). figure 2 reveals a decrease in precipitation levels from july to november. carrots require a sufficient amount of water for proper growth and develop­ ment. inadequate rainfall during critical stages, such as root development, can lead to stunted growth and reduced yields. the success of crop establishment, yield, and profitability could be attributed to the favorable precipitation and temperature observed during the vegetative growing periods (july and august) of early­sown crops. an optimal environment for the crops, promotes healthy growth and develop­ ment, ultimately leading to higher yields and increased profitability. furthermore, improved vege­ tative performance, characterized by increased net photosynthetic rate, stomatal conductance, and leaf chlorophyll content, plays a vital role in enhancing root quality. conversely, late­sown crops faced chal­ lenges due to inadequate rainfall and lower tempera­ tures experienced during the vegetative growing periods (august and september) (fig. 2), thus unfa­ vorable conditions during critical growth stages might have negatively impacted crop performance, leading to potential yield reductions. mulching offers a comprehensive supply of essen­ tial resources, thereby enhancing the quality of roots for the market. this study is in line with, hasan et al. (2018), who reported that the use of mulch resulted in the highest marketable root yield. the maximum unmarketable root yield observed in bare soil could be attributed to fluctuating soil moisture, tempera­ ture, and inadequate soil aeration. these contribute to the development of poor­quality roots, character­ ized by branching, cracking, forking, under­sizing, underweight, and green shoulder roots. whereas, minimum non­marketable root yield observed in car­ rot cultivation with sawdust mulch could be attrib­ harsh winter months immediately following sowing and thus diminished growth (lavanya et al., 2017). however, mulching offers vital soil microclimate elements such as moisture, temperature, nutrients, aeration, and weed control, which can enhance crop growth and development. acharyya et al. (2020) verified this result, sawdust mulch maintains ideal soil temperature, which promotes vegetative development and overall crop yields to a satisfactory level. based on the trends shown in figure 2, the fluctuations in precipitation and temperature can impact the enhancement of root growth (root length, diameter, and weight) throughout the entire growing season. crops that were sown early have a greater opportunity to experience relatively optimal temperatures and precipitation throughout their entire growing periods, consequently, this favorable condition of early sowing can result in increased leaf production and canopies, which have the potential to capture more sunlight. this can contribute to improved root growth and ultimately lead to higher crop yields. on the other hand, the root developmental periods of late sowing in july (september and october) were characterized by lower maximum (19.1°c) and minimum (8.6°c) temperatures (fig. 2). these were below the physiological range of temperatures (15­ 20°c), and might potentially hinder root growth by disrupting the normal physiological processes of crops. this experimental result is supported by the findings of different researchers, who reported that carrots are a temperature­sensitive root crop and their root growth was developed under suitable environmental conditions (kabir et al., 2013). the observed maximum root length, root diameter, and root weight in the sawdust mulch could be attributed to several factors such as mulch contributes to the improvement of soil structure and aeration, this creates a favorable environment for root development, allowing roots to penetrate the soil more easily and access nutrients effectively. in addition, the mulch layer helps to maintain the temperature of the soil by providing insulation, which can mitigate extreme temperature fluctuations. this stable soil temperature promotes optimal root growth and function. furthermore, mulch supports beneficial microbial flora in the soil. these contribute to nutrient cycling and availability, promoting nutrient uptake by roots and enhancing root growth. whereas, the cultivation of carrots on adv. hort. sci., 2024 38(4): 315­326 324 uted to the regulating effect of mulching on fluctuat­ ing soil conditions, especially moisture and tempera­ tures. by maintaining more stable soil conditions, mulching reduces the occurrence of root branching and cracking, resulting in roots that are highly desir­ able to both consumers and the market. furthermore, mulching contributes to the production of fewer green shoulder roots by protecting the soil against cracking and direct exposure to light. green shoulder roots, which have a bitter taste, negatively impact root appearance and quality, unsuitable for consumption and market. the present experimental result is consistent with paunović et al. (2020) who revealed that various mulching materials such as sawdust affected the availability of nutrients to the plants. the application of sawdust mulch reduces the loss of phosphorous due to excessive precipitation, thus leading to an increase in the production of quali­ ty roots (sarolia and bhardwaj, 2012). weed density the reason for the density of weeds showing a progressive decrease as the date of sowing was delayed could be, that at the onset of summer (july), there was a higher amount of rainfall and a faster rate of temperature rise compared to the end of summer (fig. 2). consequently, these conditions con­ tribute to the proliferation of weeds, resulting in higher weed growth and densities. the availability of water resources encourages weed species to flourish and compete with desired plants for resources. the result is consistent with singh et al. (2019), who found that weed emergence is comparatively weaker during the latter part of summer and early autumn compared to the early summer and spring periods. the reason for minimum weed density within dried grass mulch might be attributed to its slow decompo­ sition rate, which has the potential to suppress weed growth and promote positive plant growth supported by hayati et al. (2023). among the different mulch materials studied, the straw mulch was the least effective in terms of weed suppression potential. this can be attributed to the loose nature of straw mulch, which does not provide tight coverage of the soil. as a result, straw mulch does not offer effective weed control efficacy when compared to dried grass and sawdust mulch. furthermore, the current investiga­ tion aligns with ossom et al. (2019), who suggested that mulches effectively inhibit weed growth by blocking the penetration of light or excluding specific wavelengths of light required for weed germination and growth. additionally, biswas and das (2019) reported that straw mulch decomposes rapidly, lead­ ing to a short duration of weed control efficiency. correlation this finding suggests that both the application of sowing date and mulching positively impacted the yield of carrots by influencing important yield com­ ponents of the crop. as a result, the yield of carrots was increased. this could be attributed to the fact that increased weed presence leads to a reduction in crop growth and yield. weeds compete with the crop for essential resources such as nutrients, water, space, and light, as supported by the findings of manthy et al. (2020). in general, there was a positive correlation between the total yield of carrots and the growth parameters. enhanced vegetative growth such as plant height and leaf performance, con­ tributed to the production of higher quantities of photoassimilates, consequently leading to increased root yield. this concept is supported by the findings of acharyya et al. (2020), who reported that the use of organic mulching promotes improved vegetative growth, ultimately resulting in increased root yield. partial budget analysis from the economic point of view, all treatments with a marginal rate of return higher than the mini­ mum rate of return are considered advantageous and economically viable. the results indicated that the most economically productive treatment combi­ nation, offering the highest marginal rate of return, was early sowing with dried grass mulch (s1m3), making it an ideal choice for small­scale farmers. additionally, for resourceful cultivators or investors, the application of early sowing in july with sawdust mulch (s1m1) proved to be profitable despite its higher cost, resulting in the highest net benefit among all the treatments. in the study area, characterized by a temperate climate, farmers typically cultivate carrots on bare beds and frequently sow carrots in late july, attrib­ uted to severe winter conditions, such as soil mois­ ture deficit due to insufficient rainfall, at the end of summer. this leads to suboptimal yield and, as a result, farmers are discouraged from engaging in car­ rot cultivation. for this reason, this study focused on implementing management practices such as proper sowing dates and mulching to minimize adverse effects on root yield and suppress weed growth. the result showed that sowing date and mulching had a hussen and muhie ‐ sowing date and mulching significantly impacts carrot growth and yield 325 significant influence on almost all parameters except for days to 50% emergence. early sowing with saw­ dust mulch resulted in the maximum plant height, number of leaves, root length, root fresh weight, and marketable root yield. additionally, early sowing with no mulch (s1m0) resulted in the maximum weed density. while late sowing with dried grass mulch (s3m3) had a minimum weed density. the correla­ tion analysis showed that the growth parameters and the majority of the yield parameters of carrots exhib­ ited positive correlations with both marketable and total root yields. based on the partial budget analysis, early sowing in july with sawdust mulch (s1m1) resulted in the highest net benefit. however, the highest marginal rate of return was recorded from early sowing in july with dried grass mulch (s1m3). this research evidences that early sowing with saw­ dust mulch resulted in the highest marketable root yield and net benefit, despite the associated higher costs. for resource­full producers, it can be recom­ mended as the second­best alternative. however, for the economical production of carrots, a temporary recommendation is to utilize an early sowing in july with dried grass mulch. this particular combination exhibited the highest marginal rate of return, making it the most desirable agronomic management prac­ tice for small­scale farmers in the study area. however, this investigation specifically emphasizes agronomic practices. in addition, it is a 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institutional repository. zeleke a., derso e., 2015 ­ production and management of major vegetable crops in ethiopia. ­ eiar, addis ababa; kopia ethiopian center, addis ababa, ethiopia, pp. 149. 231 1. introduction flower organs are important in plant pollination, with petals and stamen attracting pollinators such as insects and bees (proctor and yeo, 1973; westwood, 1978). since pollination is generally indispensable for successful fertilization and setting, flower organs are important in fruit production as well. however, flowers are occasionally damaged by insects, diseases, and weather disasters such as strong wind and hail. several studies indicate that successful pollination and fruit quality are reduced in damaged flowers. in persimmon, the calyx robes play an essential role in fruit development after setting, and damage or removal of the robes results in inferior fruit growth (nakamura, 1967; yonemori et al., 1996). it was recently reported that removal of the calyx from apple blossoms at flowering time reduced fruit size and weight at harvest (kitahara et al., 2013). however, the roles of flower organs in determining fruit set, development, and quality in most fruit trees are still largely unknown. sweet cherry is one of the most commercially important fruit trees. this tree has tender flowers that bloom from april to may in japan, when hail still occurs in certain regions. in addition, flower organs such as petals and stamen, occasionally suffer damage from feeding insects. it was recently reported that artificial stamen removal (emasculation) at the balloon stage of flowering in sweet cherry trees suppressed ovule development and reduced fruit set (hedhly et al., 2009). damage to other flower organs may also influence fruit set, development, and quality of sweet cherries. in this study, artificial wounds were inflicted to petals, stamen, sepals, and peduncles of flowers in two japanese cultivars of sweet cherry in order to examine the roles of these flower organs on fruit production. 2. materials and methods plant materials adult trees of the sweet cherry (prunus avium l.) cultivars, ‘satohnishiki’ and ‘benishuho,’ planted at the experimental orchard of the shonai laboratory for agriculeffects of wound treatments to flower organs on fruit set, development, and quality in sweet cherry (prunus avium l.) m. yuki *, d. matsumoto *, k. ikeda**, s. taira* (1) * faculty of agriculture, yamagata university, tsuruoka 997-8555, japan. ** university farm, yamagata field science center, faculty of agriculture, yamagata university, tsuruoka 997-8555, japan. key words: anthocyanin concentration, artificial removal, cultivar difference, physical damage. abstract: flower organs are important elements for pollination and fruit production. although several studies on fruit trees have shown that damage to certain flower organs affects the resultant fruit, the influences of damage to flower organs on fruit production are largely unknown for most fruit trees. sweet cherry is one of the most commercially important fruit trees, the tender flowers of which are occasionally damaged by factors such as insects, diseases, and weather disasters. in this study, the flower organs of two different japanese sweet cherry cultivars, ‘satohnishiki’ and ‘benishuho,’ were wounded at flowering time by petal removal, stamen removal, sepal removal, or peduncle wounding, and the effects on fruit set, development, and quality at harvest were examined. all wound types slightly increased the fruit drop in ‘satohnishiki,’ whereas fruit drop in ‘benishuho’ was promoted by petal removal and peduncle wounding, and was suppressed by sepal removal. the fruits resulting from the wounded flowers developed in a similar manner to those of control flowers in both cultivars. fruit weight, total soluble solid concentration, and titratable acidity of the flesh juice were not affected. on the other hand, the anthocyanin concentration in fruit skins was differentially affected depending on the cultivar, increasing in ‘satohnishiki’ fruits but decreasing in ‘benishuho’ fruits. adv. hort. sci., 2014 28(4): 231-235 (1) corresponding author: staira@tds1.tr.yamagata-u.ac.jp received for publication 17 september 2014 accepted for publication 7 october 2014 232 tural production in sakata city of yamagata prefecture were used in this study. seventeen lateral branches were randomly selected from three trees per cultivar. a total of 150 flowers (seven to ten flowers per branch) were used for each treatment as described below. wound treatments four wound treatments were designed: (1) petal removal (all petals were removed), (2) stamen removal (all stamens were removed), (3) sepal removal (all sepals were removed), and (4) peduncle wounding (the middle part of peduncle was pierced using a sewing needle). flowers without wounds were used as controls. treatments were conducted either on the day of flowering or the following day (‘satohnishiki’ 29 april-4 may 2013; ‘benishuho’ 3-7 may 2013). all flowers used in this study were artificially pollinated with the mixed pollen of ‘rockport bigarreau,’ ‘napoleon,’ and ‘jabouley’ sweet cherries immediately after wounding. pollen source flowers were collected from the experimental orchard of the faculty of agriculture, yamagata university in tsuruoka city of yamagata prefecture. fruit thinning was performed on 4 june (37 days after full bloom) in ‘satohnishiki’ and on 18 june (46 days after full bloom) in ‘benishuho’, according to the customary practices in yamagata prefecture, with minor modification. the fruits from the wounded flowers were retained, while all other fruits from the same branch were thinned so that one branch bore two or three pieces of fruit per bouquet spur. fruit drop and fruit development fruit drops were counted twice per week from flowering to harvest. the fruit drop rate was calculated as the number of fruits remaining compared to the number of wounded flowers. fruit development was observed by measuring fruit diameter twice per week from 29 may when fruit set was first established. measurements of fruit quality all fruits were harvested at the optimum time according to each cultivar. the diameter and weight of the fruits, total concentration of soluble solids, titratable acidity of the flesh juice, and anthocyanin concentration of the fruit skin were examined. the total soluble solids concentration was determined using a hand refractometer (atc-1, atago co. ltd., tokyo). titratable acidity was determined by titrating the juice with 0.1 n naoh. the results are expressed as equivalents of malic acid. fruit skin discs were removed using a cork borer. anthocyanin was extracted from the discs by incubation with 1% hcl-meoh for 24 h under refrigeration. the anthocyanin concentration was determined based on the absorbance at 530 nm (uv-150-01 spectrophotometer, shimadzu co. ltd., kyoto) and calculated as equivalents of cyanidin-3-glucoside. 3. results and discussion fruit drop figures 1 and 2 show the time-course of fruit drop and cumulative fruit drop rate in the ‘satohnishiki’ and ‘benishuho’ cultivars, respectively. compared to control fruits, wounding promoted fruit drop in both cultivars, except for sepal removal in ‘benishuho’ which slightly depressed the fruit drop. the wound-induced fruit drop rate was higher in ‘benishuho’ than in ‘satohnishiki’. although fruit drops were normally heavier in ‘satohnishiki’ than in ‘benishuho’ (takahashi and arasawa, 2001), the latter was more sensitive to wounding, and thus led to larger fruit drops. two peaks in fruit drop were observed (figs. 1 and 2). the first large peak was caused by fertilization failure, whereas the second smaller peak was caused by competition for nutrients among the developing fruits (westwood, 1978; fukai, 1995). peduncle wounding commonly promoted fruit drop in both cultivars, but the peak within which the drops occurred differed between the two cultivars. more fruits dropped during the second peak in fig. 1 the effects of wounding of flower organs on fruit set in ‘satohnishiki’ sweet cherry. a, fruit drop rate. b, cumulative fruit drop rate. n=150 flowers for each treatment and control. fr ui t d ro p (% ) c um ul at iv e fr ui t d ro p (% ) days after full bloom days after full bloom 233 ‘satohnishiki,’ while more fruits dropped during the first peak in ‘benishuho’. it is possible that peduncle wounding restricts the translocation of photosynthates from the leaves to the young fruits in ‘satohnishiki,’ whereas it might also disturb fertilization in ‘benishuho’. wounding at flowering did not severely influence fruit set in this study. on the other hand, removal of the stamen at the balloon stage was reported to reduce fruit set in ‘vignola’ and ‘sunburst’ sweet cherry trees (hedhly et al., 2009). in addition, the removal of sepals at the pink bud stage in ‘cox’s orange pippin’ apples was reported to reduce fruit set to approximately 85% of normal (vemmos and goldwin, 1994). taken together, these results suggest that wound stresses at immature stages of flower development are more detrimental to fruit set than wounds occurring at flowering. further studies are necessary to determine whether wound stresses on flower organs at earlier stages of flower development influence fruit set in sweet cherry trees. fruit development figures 3 and 4 show the changes in diameter of ‘satohnishiki’ and ‘benishuho’ fruits, respectively. the growth patterns revealed double sigmoidal curves in both cultivars. fruits from wounded flowers grew in a manner similar to those from non-wounded flowers in both cultivars. this result indicates that fruits from wounded flowers can continue to grow in a similar manner to normal ones. fruit quality fruit weight, total soluble solids concentration, and titratable acidity of the flesh juice at harvest were not affig. 2 the effects of wounding of flower organs on fruit set in ‘benishuho’ sweet cherry. a, fruit drop rate. b, cumulative fruit drop rate. n=150 flowers for each treatment and control. fr ui t d ro p (% ) c um ul at iv e fr ui t d ro p (% ) days after full bloom days after full bloom fig. 4 the effects of wounding of flower organs on fruit development in ‘benishuho’ sweet cherry. vertical bars represents ±sd (petal removal, n = 72; stamen removal, n = 83; sepal removal, n = 93; peduncle wounding, n = 72; control, n = 82). days after full bloom fr ui t d ia m et er (m m ) fig. 3 the effects of wounding of flower organs on fruit development in ‘satohnishiki’ sweet cherry. vertical bars represent ±sd (petal removal, n = 48; stamen removal, n = 43; sepal removal, n = 41; peduncle wounding, n = 43; control, n = 54). days after full bloom fr ui t d ia m et er (m m ) 234 fected by wounding in ‘satohnishiki’ (table 1). none of these parameters were affected in ‘benishuho’ either, except that stamen removal significantly increased the total soluble solids concentration (table 2). on the other hand, anthocyanin concentration in the fruit skin was affected in both cultivars in different ways. in ‘satohnishiki’, all wounds increased fruit skin anthocyanin concentration, with peduncle wounding causing a significant increase in anthocyanin concentration. conversely, in ‘benishuho’, all wound types decreased anthocyanin concentration, among which stamen and sepal removal significantly decreased anthocyanin concentration. these results indicate that the fruit quality of these sweet cherry cultivars at harvest was minimally affected by flower wounding, with the exception of anthocyanin accumulation in the fruit skin. interestingly, the effect on the anthocyanin concentration observed in ‘benishuho’ appeared to be quite opposite to that in ‘satohnishiki’. the reason for this difference is unclear. thus, such cultivar differences should be examined further, and the responses of other cultivars should be identified in the future. anthocyanin accumulation in fruit skin is controlled by both environmental and physiological factors (westwood, 1978). for example, l-phenylalanine ammonialyase (pal) is a key enzyme in anthocyanin biosynthesis in various kinds of fruits (kataoka et al., 1983; farger and chalmers, 1997). increases in ethylene production induced by wounding of plant organs were reported to increase pal activity and promote anthocyanin synthesis (kataoka et al., 1983; arakawa, 1990). further studies are required to clarify the effects of wounding on ethylene production and pal activity, which is possibly related to anthocyanin accumulation in the fruit skin of sweet cherries. 4. conclusions the wounding of flower organs at the time of flowering slightly suppressed fruit set, but did not strongly affect fruit development or fruit quality at harvest in two japanese cultivars of sweet cherry. on the other hand, anthocyanin concentrations increased in the skins of wounded ‘satohnishiki’ fruits, but declined in wounded ‘benishuho’ fruits. in the future, the effects of wound stresses at earlier stages of flower bud development and flowering need to be examined and it would be interesting to explore cultivar differences in response to such stresses. acknowledgements we sincerely thank mr. m. ishiguro, mr. s. akashi, and the other staff members of the shonai laboratory for agricultural production of yamagata prefecture in sakata city for their useful advice and technical assistance. table 2 the effects of the wounding of flower organs on fruit quality at harvest in ‘benishuho’ sweet cherry treatment number of fruits fruit weight (g) total soluble solids (° brix) titratable acidity(z) (g/100 ml juice) anthocianin in fruit skin (y) (µg/cm2) petal removal 72 9.81 a 17.2 ab 0.59 a 22.8 ab stamen removal 83 9.87 a 17.6 a 0.69 a 19.6 bc sepal removal 93 9.83 a 17.3 ab 0.62 a 17.7 c peduncle wounding 72 9.62 a 17.2 ab 0.62 a 22.0 abc control 82 9.62 a 16.9 b 0.57 a 26.4 a (z) as malic acid. (y) as cyanidin-3-glucoside. letters following the means indicate statistical significance by tukey’s test, p <0.05. table 1 the effects of the wounding of flower organs on fruit quality at harvest in ‘satohnishiki’ sweet cherry treatment number of fruits fruit weight (g) total soluble solids (° brix) titratable acidity(z) (g/100 ml juice) anthocanin in fruit skin (y) (µg/cm2) petal removal 48 6.97 a 20.2 a 0.75 a 24.6 ab stamen removal 43 6.80 a 20.4 a 0.83 b 24.4 ab sepal removal 41 7.04 a 20.1 a 0.77 ab 25.5 ab peduncle wounding 43 6.88 a 20.5 a 0.83 b 29.9 a control 54 6.88 a 19.9 a 0.81 ab 21.2 b (z) as malic acid. (y) as cyanidin-3-glucoside. letters following the means indicate statistical significance by tukey’s test, p <0.05. 235 references arakawa o., 1990 ethylene production in apple fruit. agric. hortic. (nogyo-oyobi-engei), 65: 363-366. farger d., chalmers d.j., 1997 regulation of anthocyanin synthesis in apple skin (iii). involvement of phenylalanine ammonia-lyase. aust. j. plant physiol., 4: 133-141. fukai n., 1995 cherry. fruit set, appropriate setting, pp. 63-87. in: fukai n. (ed.) cultivation techniques in sweet cherry and pear production. yokendo, tokyo, japan. hedhly a., hormaza j.i., herrero m., 2009 flower emasculation accelerates ovule degeneration and reduces fruit set in sweet cherry. sci. hortic., 119: 455-457. kataoka i., kubo y., sugiura a., tomana t., 1983 changing in l-phenylalanine ammonia-lyase activity and anthocyanin synthesis during berry ripening of three grape cultivars. j. japan. soc. hortic. sci., 52: 273-279. kitahara y., ogasawara c., taira s., 2013 effect of cutting of calyx lobes and shading of immature fruit on fruit development and quality in apple and persimmon. hort. res. (japan), 12: 475. nakamura m., 1967 physiological and ecological studies on the calyx of the japanese persimmon. res. bull. fac. fac. agric. gifu univ., 23: 1-62. proctor m., yeo p., 1973 insect-pollinated flowers-i, pp. 45. in: huxley s.j., j. golmour, m. davies, and k. mallanby (eds.) the pollination of flowers. collons, london, uk. takahashi k., arasawa n., 2001 difference of fruit drop pattern in sweet cherry cultivars. hort. res. (japan), 70: 229. vemmos s.n., goldwin g.k., 1994 the photosynthetic activity of cox’s orange pippin apple flowers in relation to fruit setting. ann. bot., 73: 385-391. westwood m.n., 1978 temperate-zone pomology, pp. 203-208. w.h. freeman and company, new york, usa. yonemori k., itai a., nakano r., sugiura a., 1996 role of calyx lobes in gas exchange and development of persimmon fruit. j. amer. soc. hort. sci., 121: 676-679. untitled 112 rfid microchips as a tool for traceability in grapevine nurseries: preand post-grafting implants f. mannini, i. gribaudo plant virology institute cnr, grugliasco unit, via l. da vinci 44, 10095 grugliasco (to), italy, i.gribaudo@ivv. cnr.it radio frequency identification (rfid) technology has been used for some years for the identification and traceability of various products. this approach has been successfully adopted also in the agrifood sector to guarantee a reliable traceability system. in grapevine nurseries, the storage of data in a field log is an essential step in the plant production process. specific regulations require the presence of colored labels indicating the type, origin and other features of the material. this system is inexpensive and simple, but has several drawbacks, thus the application of rfid technology in grapevine nurseries has been studied. the insertion of rfid tags (microchip) in grafted and ungrafted cuttings would allow to store and retrieve a large quantity of data concerning the single plant, including the application of hot water treatment against phytoplasma diseases. in march 2011, microchips (0.21 x 1.20 cm) were inserted in the pith of two types of grapevine (vitis spp.) propagation materials: grafted cuttings (a) and grafted rootlings (b), subjected or not to hot water treatment (50 °c for 45 min). in (a) the microchips were inserted in the rootstock cuttings (kober 5bb and s.o.4) after direct drilling of pith, before grafting the cuttings with scions of ‘barbera’ and ‘white muscat’. in (b) the microchips were inserted in grafted rootlings of three ‘nebbiolo’ clones, a few cm below the grafting point, through a “u” cut performed laterally by a specially designed machine. tags were read through a palmtop computer and an appropriate software program allowed management of the stored data. after the first year of cultivation in the nursery, the grafted cuttings (a) were planted in a vineyard in spring 2012. there was some variability in the nursery take among treatments also related to the quality of the plant materials, whereas tagging accuracy (readable microchips) in september 2012 was very good (about 90% successful read rate). the grafted rootlings (b) were planted in a vineyard in spring 2011. in the following summer more than 90% of microchips were readable, whereas this percentage decreased to 50% in summer 2012 under more humid conditions, indicating that microchip insertion in grafted rootlings can encounter some problems. in conclusion, electronic tagging of grapevine propagation materials proved technically feasible, although the cost of microchips (still too high compared to the average price of grapevine rootlings) continues to hinder their routine adoption in nurseries. nevertheless, electronic tagging could be advisable for “basic” material (that used by nurseries to establish their mother-vine vineyards) as it has greater commercial value. adv. hort. sci., 2013 27(3): 112 244 1. introduction haskap (japanese blue honeysuckle; lonicera caerulea l. var. emphyllocalyx nakai), a deciduous berry shrub, growing to 1.5-2.0 m tall, bearing a type of blue-berried honeysuckle about 1 cm in diameter, has recently been listed as one of four promising, emerging berry crops with commercial potential despite its current status of low economic importance (hummer et al., 2012). therefore, these crops have attracted the attention of world agriculturalists and breeders even if the history of their agricultural handling is still brief (hummer et al., 2012). blue-berried honeysuckles, including haskap, are native throughout the cool temperate northern hemisphere; haskap is cultivated in hokkaido, the coldest local region in japan. haskap berry is now known as the earliest fresh fruit harvested in hokkaido prefecture, japan, starting in late june (fu et al., 2011). in the last two decades, an american research team has conducted a survey on the adaptability of various blue honeysuckle plants to the northwestern united states. they have considered qualities from several geographic sources including botanical varieties edulis, kamtchatica, altaica, and boczkarnikovae from russia, edulis and boczkamikovae from northeast china, and emphyllocalyx from hokkaido, japan, and concluded that the japanese variety emphyllocalyx (haskap) has superior adaptability in oregon (thompson, 2006; thompson and barney, 2007). therefore, a breeding program utilizing this variety was initiated in 2003 in the states of oregon and also idaho, aiming for outstanding selections in hopes of identifying superior individuals to release as cultivars as the basis for a new berry industry in the united states. to date, a research team at hokkaido university has surveyed the ploidy level and geographical distribution of wild haskap, based on the flow cytometric analysis revealing the presence of dna diploid and dna tetraploid plants sampled in japan (miyashita et al., 2011). accordingly, chromosomal analysis confirmed that diploid and tetraploid plants showed 2n=2x = 18 and 2n = 4x = 36, respectively. the dna diploid populations were found only in lowland mires, betsukai, bekanbeushi, kushiro and kiritappu located in eastern hokkaido prefecture. on the other hand, dna tetraploid populations were widely distributed in most areas in hokkaido prefecture, and also in mainland japan. in fact, commercial cultivars of haskap have been selected only from wild plants and thus, fruit traits and other agricultural characteristics have been largely limited until recently (miyashita and hoshino, 2010). breeding and plant biotechnological attempts to obtain novel haskap cultivars have followed, resulting in enhanced yield and quality. around a decade ago, selection of haskap wild lines showing notable edible qualities and some horticultural charattempt for postharvest ripening of immature fruits of haskap (lonicera caerulea l. var. emphyllocalyx nakai), an emerging fruit in northern japan y. yamamoto1, y. hoshino2, h. masago3, t. kawano1,3,4,* 1 graduate school of environmental engineering, the university of kitakyushu, kitakyushu, japan. 2 field science center for northern biosphere, hokkaido university, sapporo, japan. 3 linv kitakyushu research center (linv@kitakyushu), kitakyushu, japan. 4 université paris diderot, sorbonne paris cité, paris 7 interdisciplinary energy research institute (pieri), paris, france. key words: agricultural mechanization, fruit softening, postharvest ripening. abstract: haskap or japanese blue honeysuckle (lonicera caerulea l. var. emphyllocalyx nakai) is a deciduous shrub berry crop, which is recently listed as one of promisingly emerging berry crops. in the present short survey, an attempt for postharvest ripening of immature fruits of haskap was testified by examining the changes in fruit hardness, peel color, pigment synthesis and sugar-acid balance during storage at 5, 10, 15 or 20°c. softening and coloring were shown to be induced during postharvest storage, especially at 20°c. the extent of maturation was largely enhanced by longer storage period. it is conclusive that haskap berries can be harvested at premature stage if postharvest maturation was allowed during storage and/or transportation to the markets. adv. hort. sci., 2014 28(4): 244-249 corresponding author: kawanotom@kitakyu-u.ac.jp received for publication 14 october 2014 accepted for publication 20 november 2014 short note 245 acteristics were performed for a further breeding program (takada et al., 2003). accordingly, interspecies crosses between l. caerulea var. emphyllocalyx and lonicera gracilipes var. glabra miquel were examined to increase genetic variability of l. caerulea var. emphyllocalyx (miyashita and hoshino, 2010). furthermore, miyashita et al. (2009) reported the regeneration of haskap plantlets from endosperm culture of selected lines to enhance the breeding strategy. although haskap is one of most promising berry crops in hokkaido, the amount of cultivation and supply to the market is largely limited due to the attention required to handle soft ripe berries. in japan’s small-scale orchards, farmers carefully harvest the haskap berries by hand so as to avoid losing any juice through damage of the delicate peel (fu et al., 2011). to date, conventional agricultural machinery has failed to be introduced for the harvest. there could be two distinct approaches applicable to improve harvesting efficiency: (1) development of novel machinery or devices for automated or semi-automated harvesting with maximal care from aids such as sensors and actuators; and (2) development of novel working algorithms to simplify the overall processes so that conventional agricultural machines or equipment can be readily introduced, as recently proposed for other horticultural crops (kawano et al., 2012). some examples of recent approaches can be found in a series of studies conducted by fu et al. (2011). after testing various combinations of separating, collecting and cleaning methods, they concluded that the harvesting rate for haskap went from 1.45 kg/h (conventional hand picking) to a maximum of 10.36 kg/h. the present study aims to contribute to the documentation, designing a novel harvesting strategy based on the ripening physiology of haskap berries, which could be readily automated with minimal efforts. mature haskap berries are easily damaged by mechanical operations during harvest, selection and transportation. therefore, mature haskap berries are hard to handle by conventional harvesting machines and those harvested in the hokkaido region bear transportation with difficulty over great distances to markets in large cities in mainland japan. to avoid these problems or difficulties which could be attributed to fruit maturity, it is tempting to propose harvesting immature green berries which are less sensitive to mechanical stresses, to then be followed by postharvest forced maturation during storage or transportation. in this brief survey, we report an attempt at postharvest ripening of immature fruits of haskap by examining the changes in fruit hardness, peel color, pigment synthesis and sugar-acid balance during storage at different temperatures. 2. materials and methods plant materials mature and immature haskap berries were picked from two-year-old shrubs cultivated at the experiment farm at hokkaido university. the berries, harvested by hand, were immediately packed for transportation under temperaturecontrolled conditions (exposure to heat or cold was avoided), and used for the experiments within two days after harvest. color measurements as described elsewhere (kawano and shimokawa, 2003), changes in color of the fruit peel were monitored using a handy cielab color reader (cr-13, konica minolta sensing inc., osaka, japan) by measuring the a* and b* values of cie (commission international de l’eclairge) 1976 l*a*b* color space units (cielab system), at 0, 1, 3, and 7 days of storage. as the a* value corresponds to a red-green scale (red, positive; green, negative) and the b* value corresponds to a yellow-blue scale (yellow, positive; blue, negative) (kawano, 2013), we assumed that the a* and b* values represent degreening due to chlorophyll loss and blue color development due to the synthesis of anthocyanin, respectively. hardness changes in fruit hardness during storage were determined using a universal fruit hardness meter (max., 1 kg; model km, fujiwara scientific co. ltd, tokyo). storage as the studied species are native to a cold region, we tested the effect of a low to moderate range of temperature, namely, 5, 10, 15 and 20°c, by keeping the samples in temperature-controllable cool incubators (model cn-25c, mitsubishi electronic engineering co. ltd., tokyo, japan). sugar and acid contents freshly squeezed berry sap was used for determination of sugar content using a pocket sugar meter (model pal-1, atago co. ltd., tokyo, japan) and organic acid content using a portable amperometric acid sensor (model fs-101n, atago co. ltd., tokyo, japan). for determination of sugar and organic acid, 0.5 ml and 0.1 ml of berry sap were used, respectively. quantification of organic acids is based on the voltammetric reduction of 3,5-di-tert butyl 1,2-benzoquinone (in quinone reagent mixture provided by atago co. ltd.) in the presence of acids (kotani et al., 2008). pigment analysis anthocyanins were extracted from 0.1 g fresh weight of homogenates in 20 ml of 50% (w/v) acetic acid kept at 23°c for 24 h. then, 1 ml of crude extract was subjected to centrifugation at 10,000 rpm (8217 x g) at room temperature for 10 min and the resultant supernatant was used for optical reading at 550 nm using a spectrophotometer (u-3310, hitachi, tokyo). total anthocyanin content (expressed as of cyanidine-3-glucoside) was estimated according to matsuzoe et al. (2006). 3. results and discussion appearance of berries before and after the storage prior to and after storage, the color of berry peels was compared with that of mature samples with the naked eye 246 (fig. 1). while the peel of mature berries were apparently highly pigmented with a deep blue color, the immature berry samples lacked the blue pigmentation but were green due to the remaining chlorophylls. after a week of storage at 20°c, the peel of immature berry samples was slightly colored blue (fig. 1 bottom). these observations suggest that postharvest ripening of haskap berries could likely be achieved under controlled conditions. softening and coloring the progress in postharvest maturation in haskap berries kept under various temperature was scored by the changes in mechanical hardness (fig. 2) and peel color (fig. 3). the results suggest that the softening of berries is affected by the storage temperature. at time 0, the difference in hardness between mature and immature samples was significantly large. the hardness of the immature samples kept at 20°c was gradually lowered and finally attained a level comparable to naturally-matured berry samples (fig. 2). similarly, coloring was enhanced in the samples stored at higher temperature. peel color was non-destructively determined by a colorimeter and the changes in peel color were expressed as values in cielab system (fig. 3). at 5°c, neither lightness (l* values), greenness (negative a* values), nor blueness/yellowness (b* values) showed significant change during seven days of storage. in contrast, under higher temperatures, all coloring values were altered with time, suggesting that chlorophyll degradation and anthocyanin biosynthesis were induced. loss of chlorophylls (de-greening) is one key feature of fruit ripening found in various fruits with ethylene-producing climacteric (kawano fig. 1 images of typical haskap berries at three different stages of maturation. (a) mature berries. (b) immature berries prior to storage. (c) semi-colored berries obtained after storing the immature samples for 1 week at 20°c. fig. 2 changes in mechanical hardness of haskap berries during storage under various temperatures. vertical bars on the graph indicate se (n=4). fig. 3 colorimetric changes in peel color in haskap berries during storage under various temperatures. peel color was determined by colorimeter and expressed as values in cielab system, namely l* (a), a* (b), and b* (c). vertical bars on the graph indicate se (n=4). 247 and shimokawa, 1994; kawano et al., 1999; kawano and shimokawa, 2004, 2005 a) and non-climacteric natures (kawano and shimokawa, 2003, 2005 b). thus, further investigation of the de-greening mechanism is encouraged. anthocyanin content as the decrease in b* value in the peel color (fig. 3 c) reflects the increase in blue pigments, the changes in anthocyanin content during postharvest storage were monitored (fig. 4). the mature samples were rich in anthocyanin (fig. 4 a-i) and immature samples contained no detectable anthocyanin (fig. 4 a-ii). among the samples which were subjected to postharvest maturation, only the samples kept at 20°c showed signs of induced pigmentation (fig. 4 a-vi). similarly to the colorimetrically determined blueness (fig. 3), the blue pigmentation in the samples stored at 20°c was shown to be linearly increased with time (fig. 4). based on the observed tendency, we can further expect that enhanced pigmentation can be manifested by longer storage. sugar/acid contents it is well known that both the absolute values and balance in brix index and acidity in the berry sap largely determine the consumption quality of fresh haskap berries (takada et al., 2003). during postharvest maturation under various temperatures, no significant increase in sugar content in the immature berries was observed (fig. 5 a). instead, compared to green immature samples examined prior to storage, there was significant decrease in acidity over the seven days of storage in most samples kept at different temperatures (fig. 5 b), thus contributing, although slightly, to the increase in sugar/acid ratio (fig. 5 c). in the end, significance between the sugar acid ratio in mature samples and that in stored samples was lost, suggesting that sugar/acid ratio was amended by the postharvest maturation process employed. texture, taste, flavor, and attractive components softening of the berries is one of the important factors determining the texture of haskap berries. as described above, the mechanical hardness of berries was significantly lowered during postharvest incubation at 20°c and attained a level comparable to naturally-ripened samples (fig. 2). as shown in figure 5 a, the postharvest maturation approach was barely successful for enhancing the sweetness increase, although sweetness and sourness represented by the contents of sugar and organic acids are major factors determining the tastes of berries (takada et al., 2003). however, due to the induced decrease in total acid fig. 4 changes in anthocyanin content in haskap berries during storage under various temperatures. (a) typical spectral profiles of extracts from different samples. (i) mature berries. (ii) immature berries prior to storage. (iii, iv, v, vi) immature berries subjected to oneweek-long storage under 5, 10, 15, 20°c, respectively. (b) increase in anthocyanin content in haskap berries during storage under various temperatures. (i) comparison of anthocyanin content between mature samples and immature samples subjected to storage. (ii) data in (i) were enlarged for ease of comparison among the samples stored under different temperatures. vertical bars in (b) indicate se (n=4). fig. 5 changes in sugar and acid contents in haskap berries during storage under various temperatures. (a) sugar content (percentage by weight). (b) organic acid content (percentage by weight). (c) sugar/acid ratio are compared. vertical bars on the graph indicate se (n=4). 248 level (with limited extent, fig. 5 b), the sweetness/acidity balance might be enhanced reaching a level relatively close to the level of naturally-matured samples (sugar/acid ratio in fig. 5 c). a preliminary organo-lip test performed by laboratory members is in support of the data on sugar/ acid balance, but it is still early to provide any conclusion from such a limited survey. one of major uses of haskap in hokkaido is in bakery goods as sour taste accents and as color-attractive toppings. therefore, rather than sweetness, production of pigments, chiefly anthocyanin, is of more importance from this point of view. it is notable that haskap is considered a new berry crop with high antioxidant capacity due to its wealth in anthocyanins and related substances: anti-oxidative scores for haskap varieties were the highest among commercial fruits examined through multiple methods such as ferric reducing antioxidant power (frap) assay, oxygen radical absorbance capacity (orac) assay, the 1,1-diphenyl-2picrylhydrazyl (dpph) free radical scavenging assay, the aluminum chloride colorimetric method and the folin-ciocalteu method (rupasinghe et al., 2012). more recently, takahashi et al. (2014) examined the effects of dietary intake of anthocyanin-rich phenolic phytochemical (containing 13.2% anthocyanin) purified from haskap fruit on postprandial serum triglyceride and blood glucose levels in rats, concluding that a decrease in postprandial blood lipids and blood glucose by short or long-term haskap phytochemical ingestion is due to anthocyanin and other polyphenols contained in the haskap phytochemical. reports on the antioxidant capacity of haskap and other health-related studies regarding haskap-derived pigments have been reviewed elsewhere (celli et al., 2014). in addition to pigments, the flavors or aromas of haskap berries are of commercial importance and have recently gained attention from food industries. for instance, haskap residues after juice extraction have been used preliminary as natural flavoring for teas (sakamoto et al., 2012). thus, the impact of postharvest maturation of haskap berries on the production of flavors and aromas should be documented in future studies. 4. conclusions two key parameters of fruit maturation, namely softening and coloring, were significantly amended by postharvest storage at moderate temperature and the extent of maturation is likely to be enhanced by a longer storage period. as enhanced coloring represents an increase in anthocyanin content, valued for its antioxidant action, the postharvest maturation approaches presented here may contribute to the market quality of this crop. however, the sugar content in the berry sap could not be altered during storage and the change in sap acidity induced during storage was limited. in conclusion, haskap can be harvested at premature stage if postharvest maturation is allowed during storage and/or transportation to the markets. acknowledgements tk was supported by a grant of regional innovation strategy support program implemented by ministry of education, culture, sports, science and technology (mext), japan. references celli g.b., ghanem a., brooks m.s.l., 2014 haskap berries (lonicera caerulea l.) a critical review of antioxidant capacity and health-related studies for potential valueadded products. food bioprocess technol., 7: 1541-1554. fu s., okamoto h., hoshino y., esaki y., kataoka t., shibata y., 2011 efficient harvesting of japanese blue honeysuckle. eng. agric. environ. food, 4(1): 12-17. hummer k.e., pomper k.w., postman j., graham c.j., stover e., mercure e.w., aradhya m., crisosto c.h., ferguson l., thompson m.m., byers p., zee f., 2012 emerging fruit crops. fruit breeding. handbook of plant breeding, 8: 97-147. kawano t., 2013 primitive optical computing model with films: boolean conjunction of the square matrix-arrayed color codes. j. adv. comput. intellig. intellig. informat., 17: 791-798. kawano t., adachi m., kurata h., azuma r., shimokawa k., 1999 calcium-dependent catabolism of phaeophorbide a in tomato fruit. j. japan. soc. hort. sci., 68: 810-816. kawano t., kawano a., kawano m., 2012 simplified exercise for mechanical 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organ. cult., 98: 291-301. rupasinghe h.p.v., yu l.j., bhullar k.s., bors b., 2012 haskap (lonicera caerulea): a new berry crop with high antioxidant capacity. can. j. plant sci., 92: 1311-1317. sakamoto m., arakawa y., miyoshi k., kanazawa y., moriya k., 2012 production and sensory evaluation of hokkaido haskap tea. nippon shokuhin kagaku kogaku kaishi, 59: 456-464. takada m., hoshino y., nakako h., sato h., 2003 evaluation of eating qualities and some horticultural characteristics for selection of elite lines in lonicera caerulea l. res. bull. univ. farm hokkaido univ., 33: 21-38. takahashi a., okazaki y., nakamoto a., watanabe s., sakaguchi h., tagashira y., kagii a., nakagawara s., higuchi o., suzuki t., chiji h., 2014 dietary anthocyanin-rich haskap phytochemicals inhibit postprandial hyperlipidemia and hyperglycemia in rats. j. oleo sci., 63: 201-209. thompson m.m., 2006 introducing haskap, japanese blue honeysuckle. j. of the amer. pomol. soc., 60: 164-168. thompson m.m., barney d. l., 2007 evaluation and breeding of haskap in north america j. of the amer. pomol. soc., 61: 25-33. 167 1. introduction japan is located between 24° and 46° north latitude and has four distinct seasons, spring, summer, autumn, and winter. it has a very mountainous topography and steep landforms that are poorly suitable for cultivation of horticultural crops. however, despite the rough mountainous terrain and poor soils, the japanese have still managed to be successful in growing many crops. japan enjoys mostly a temperate zone climate which varies considerably in different parts because the country stretches from north to south and is surrounded by the sea. the main horticultural crops include apples, grape, cherries, melon, onions, sweet corn, carrots, tomatoes, japanese radish, chinese yams and beans. several types or floricultural crops are also produced and exported to other countries (araki, 2002). tomato, cucumber, welsh onion, strawberry, watermelon and spinach are mostly produced in greenhouses and high tunnels. strawberries are usually grown on high beds for comfortable work and lower labor costs whereas, tomatoes are produced in glasshouses and high tunnels in order to prevent crop damage by rain (araki, 2002). japan uses 69% of its total greenhouse area for vegetable production, only 17% for flowers, and 13% for fruit tree production. only 3% of greenhouses use the modern facilities suitable for crop production through hydroponics (nichols, 2008) although a gradual change has been taking place in greenhouse production. more sustainable horticulture practices in greenhouses are desirable and possible through the use of hydroponic methods that allow only small amounts of water to drain off and usually offer a higher yield. commercial hydroponic systems have proved to be more productive than conventional systems of agriculture. hydroponics has averaged around 20 to 25% higher yields than conventional soil cultivation. this could truly benefit japan’s current gdp in agriculture while feeding more people with sustainable and fresh horticultural crops. conventional hydroponic techniques use culture solution with bare root systems and do not use growing media. on the other hand, soilless cultivation is a very sustainable crop production practice because many of the components in modern media such as perlite and vermiculite take many years to decay and go back into the environment. the improved and consistent vegetable quality is second to none because of the elimination of pesticides and herbicides. this keeps toxic chemicals away from the culture system and it will in turn help keep japan’s groundwater and oceans free from contamination. hydroponics is currently a managed culture technique horticultural research in japan. production of vegetables and ornamentals in hydroponics, constraints and control measures t. asao*(1), md. asaduzzaman**, mondal f. md.* * department of agriculture, faculty of life and environmental science, shimane university, 2059 kamihonjo, matsue, shimane 690-1102, japan. ** olericulture division, horticulture research centre, bangladesh agricultural research institute, joydebpur, grazipur 1701, bangladesh. key words: activated charcoal, allelochemicals, autotoxicity, control of minerals, human health, hydroponics, plant factory, vegetables and ornamentals. abstract: japan has a very mountainous topography, steep landforms, and varied climate making it less suitable than other countries for growing horticultural crops. however, good crop production technologies make it possible to grow high quality produce in japan. the major horticultural crops are mainly produced in greenhouses and high tunnels however, a vast majority of greenhouse systems are outdated. horticultural production practices could be more sustainable through the use of hydroponics in the greenhouse. commercial hydroponic systems have proved more productive (20-25% higher) than conventional systems of agriculture. a number of research projects have been conducted to better understand production constraints in hydroponics, such as autotoxicity, and to identify suitable cultivars and supplementation techniques. this review discusses greenhouse production techniques for vegetables and ornamentals in hydroponics, as well as constraints and means for sustainable production. adv. hort. sci., 2014 28(4): 167-178 (1) corresponding author: asao@life.shimane-u.ac.jp received for publication 17 september 2014 accepted for publication 1 october 2014 168 that is largely used for the production of vegetables as it is capable of being sustainable with low amounts of water loss and use of soilless media. therefore, advanced management of this technique has been practiced throughout japan with an aim toward yield maximization and higher quality assurance. however, constraints have been found and several researchers have tested strategies to overcome them. this review discusses greenhouse production techniques of vegetables and ornamentals in hydroponics, and the constraints and means for sustainable production. 2. hydroponics for higher yield and production of specialty horticultural crops hydroponics is a method for growing plants using mineral nutrient solutions, in water, without soil. in this system plants can be grown with their roots in the mineral nutrient solution only orin an inert medium. it is possibly the most intensive method of crop production providing efficient use of water and mineral nutrients with a minimal use of space. it has been used successfully by commercial growers for fast-growing horticultural crops such as lettuce, strawberries, tomatoes, cucumbers and ornamentals in japan. this technology enables a more precise control of growth conditions, making it easier to study the variable factors or parameters. one specialty of this technique is the vigorous development of root systems and the efficient uptake of the essential nutrients from culture solution resulting in a better crop yield. therefore, this technology has gained popularity for producing high value crops with high quality and specialty production providing human health benefits. however, there are limits due to self-toxicity from root exudates in recycled nutrient solutions. finding suitable control measures to overcome these constraints could result in sustainable horticultural crop production in greenhouses. in the following sections, hydroponic production techniques for root crops (carrot), production of functional foods for kidney dialysis patients (melon), and enhanced crop quality (turnip) will be discussed. growing carrots hydroponically using perlite substrate root vegetables are often discouraged to grow by hydroponics possibly because of poor root development. carrot [daucus carota l.] is a root vegetable which forms numerous hairy roots with a reduced tap root in the nutrient solution. moreover, inside nutrient solution this storage organ form hypertrophy due to the ample supply of water and nutrient resulting decrease in length and weight compared to non-submerged condition (terabayashi et al., 2008). therefore, appropriate moisture content of the growth medium is crucial for optimum growth and development of the storage root (eguchi et al., 2008). it has been found that the use of suitable soilless media can increase both marketable yield and quality of root crops by many folds (hanna, 2009). perlite is widely preferred as soilless media, as it encourages faster root development, reduces risk of damping off, avoids water logging and provides an optimum balance of air and water. its strong attraction for water automatically draws up solution from the reservoir at the same rate that the plants remove water, leaving excess solution in the reservoir. therefore, an optimum moisture level can be maintained around the root, and this is a significant advantage over rockwool with less capillary action. recently perlite has emerged as an excellent medium with versatile use. it has been widely used to grow many horticultural crops including tomatoes, cucumber, melon, peppers, lettuce and rose (szmidt et al., 1988; cantliffe et al., 2003; hochmuth and hochmuth, 2003; fascella and zizzo, 2005; frezza et al., 2005; rodriguez et al., 2006). suitable perlite size, along with optimal concentration of culture solution, has been suggested for maximizing carrot yield in hydroponics (asaduzzaman et al., 2013 a). it was found that carrot plants grown in 0.6 mm perlite supplied with 100% nutrient solution produced significantly higher root yield compared to larger perlite particles and higher concentrations of nutrient solution. interestingly it was observed that when carrots are grown in 0.3 mm perlites, shorter roots are produced which are wider near the proximal end and whitish in the distal end due to excessive water content causing oxygen deficiency. this ultra fine perlite can hold excessive water causing oxygen deficiency in the substrate air zone and as a result roots become whitish with a reduced amount of carotenoids. in the study, the feasibility of growing carrot in used perlites from previous culture was also evaluated. the residual nutrients available in the reused perlite lowers in turn the demand of nutrients in the second culture where carrot plants can uptake about 50% residual nitrogen. therefore, it was recommended to use 0.6 mm perlite and 100% (for first culture) or 75% (for second culture) ‘enshi’ nutrient solution for growing carrots hydroponically for maximum yield and higher quality. production of low potassium content melon fruits in hydroponics through managing nutrient solution potassium is crucial for the normal functioning of muscles, heart, and nerves in the human body. it is one of the main electrolytes and is concentrated within the body cell. about 90% of body k is normally excreted by the kidneys but patients with kidney dysfunction suffering from chronic kidney disease (ckd) cannot completely excrete it and thus it accumulates. abnormally elevated levels of k in the blood (hyperkalemia) can cause adverse effects (kes, 2001). this author also reported that a normal kidney has the capacity to excrete in excess of 400 m mol k day-1, and it is unlikely that an individual will become chronically hyperkalemic without some degree of chronic renal impairment. sometimes hyperkalemia causes arrhythmias, muscle weakness, disturbed consciousness, heart failure, and can even lead to sudden death (spital and stems, 1988; putcha and allon, 2007). the ckd patient population is increasing year by year and it is expected that the total number of patients will continue to increase progressively. therefore, preventive measures have been stressed to divert the prevalence of this disease in a de169 creasing trend. restricted diets are mainly used as a means of treatment for ckd patients. as a primary control measure, foods with high k content are restricted but a normal daily diet including fruits such as melon, fresh vegetables, seaweed, beans and potatoes are rich in k (weiner and wingo, 1998). k is a major nutrient, essential for normal growth and development of plants (schachtman and liu, 1999). plants absorb more k than any other mineral element with the exception of n (tisdale and nelson, 1975; mäser et al., 2002; britto and kronzucker, 2008; szczerba et al., 2009). it is the only monovalent cation that is essential for all higher plants and it is involved in three major functions: enzyme activation, charge balance, and osmoregulation (mengel, 2007; szczerba et al., 2009). it is inevitable that reduced k supply will inhibit plant growth and yield. therefore, investigations on minimal requirements of k in plants to maintain their normal growth and development are important. in the above context, investigations have been conducted for the production of melon fruits with low k content to provide supplementary diet to dialysis patients (asao et al., 2013). in japan greenhouse cultured raw melon generally has higher k content: 340 mg 100 g-1 fresh weight (mext, 2011). this amount of k was decreased considerably, improving the diet of dialysis patients (asao et al., 2013). the researchers applied a hydroponic method which enabled a more precise control of growth conditions and made it easier to study the variable factors or parameters. regular nutritional testing can be conducted in hydroponics, making it possible to determine if the desired amount of nutritional content is present in the plants or not. precise control over the concentration and composition of culture solution allows the production of either mineral enriched or deficient fruits or vegetables. results indicated that a general trend of decreasing k content in fruit with a decrease of kno 3 concentration in the nutrient solution. in spring 2009, the k content of fruits harvested from plants grown in the nutrient solution with 1/4th kno 3 was about 39% lower compared to those harvested from plants grown in standard nutrient solution, while it was about 35% and 43% lower in fruits from plants grown with 1/16th and without kno 3 in the spring 2010 and 2011, respectively. the content of na in fruits however increased progressively with the decrease of k in the nutrient solution. a consistent antagonistic relation with fruit k concentration was found due to the reduced levels of kno 3 . compared to control plants, about 83% (spring 2010) and 51% (spring 2011) increased na were found in fruit harvested from plants grown in nutrient solution with 1/6th or without k, respectively. low k content melon fruit can provide human health benefits to dialysis patients but the higher na content would likely cause hyperpiesia and edema. therefore, it is necessary to evaluate the benefits of the reduction of k against the risks of the increased na intake by the dialysis patients, whose k intake should be restricted to 1500-2000 mg day-1 (agondi et al., 2011) and nacl intake (equivalent to 2000-3200 mg na) should be restricted to 5000-8000 mg day-1. na intake must be limited to 1.3-1.6 times of k intake. however, the benefits of reducing the intake of k are greater than the risks of increasing the intake of na. nutrient level changes in anthocyanin and nitrate-n contents in hydroponically grown turnip “tsuda-kabu” (brassica rapa l. rapifera group) is a local turnip cultivar popularly grown in shimane prefecture in japan. the cultivar is red with a comma-shaped root. the crop is normally sown in september and harvested in december. harvesting turnip in early december is profitable mainly since many people in japan send it as a year-end gift. however, sowing turnip in september in field conditions is often delayed due to incessant rainfall. moreover, turnip in soil culture requires extra labor for harvesting and washing. in hydroponics, turnip cultivation seems to be less influenced by environmental changes and harvesting, and processing is also convenient. another problem with soil cultivation is that most of this turnip root remains above the ground and only a few centimeters of the root remains in the soil. this red turnip has large amounts of anthocyanin in the root, adding quality for processing and delicious foods. previous research reported that nitrate inhibited the sucrose-induced accumulation of anthocyarine in grapevine (faust, 1965; pirie and mullins, 1976). thus, an optimum level of nutrients in the culture solution is a precondition for harvesting quality products with maximum yield through hydroponic culture. in this context, asao et al. (2005) investigated the influence of different nutrient solution concentrations on the growth of turnip in hydroponics. it was found that no 3 --n contents in the shoots and roots of turnip were decreased with decreasing no 3 --n levels in the nutrient solution. on the other hand, anthocyanin concentration in the root was inversely proportional to the content of no 3 --n in the same. however, when no 3 --n content at 50% nutrient solution concentration was changed from full (8 m mol-1) to half (4 m mol-1), no 3 --n content and anthocyanin contents in the turnip root were not affected. these results indicate that the effects of other nutrients except no 3 --n might be associated with the formation of anthocyanin in turnip. therefore, they suggested that the medium range (50%) nutrient solution with 4 m mol-1 no 3 --n may be treated as optimum concentration for production of quality turnip by hydroponic culture. 3. production constraints in recycled hydroponic culture autotoxicity in cucumber (cucumis sativus l.) under closed hydroponics autotoxicity in cucumber plants was reported in previous research as due to accumulation of phytotoxic phenolic compounds in the substrates used for long-term cultivation (politycka et al., 1984; yu and matsui, 1994). root exudates from the cucumber cv. shougoin-aonaga-fushinari gave maximum inhibitory effect on seedling growth of its own cultivar, as well as other test cultivars of cucumber 170 (asao et al., 1998 a). the growth of tomato (yu and matsui, 1993 a, b) and cucumber plants (yu and matsui, 1994) increased upon addition of activated charcoal (ac) to the nutrient solutions. yu and matsui (1993 c, 1994) collected the root exudates of cucumber cv. tokiwa from hydroponic culture through a continuous trapping technique (tang, 1986) and identified a number of autotoxic chemicals. fruit yield of cucumber was improved by the addition of ac to the nutrient solution (asao et al., 1998 b) because the added charcoal adsorbed the phytotoxic root exudates from the nutrient solution and thus favored cucumber growth. it was also found that the fruit yield of cucumber plants decreased significantly at late reproductive stage (2 weeks ahead of final harvest) while this inhibition was recovered if nutrient solutions were renewed biweekly or supplemented with ac to the nutrient solution (asao et al., 1998 a). characteristic shrunken cucumber fruits were harvested from the plant grown in non-renewed culture solution. autotoxicity of cucumber was also found to be different among cultivars (asao et al., 1998 b). fruit harvesting of a susceptible cucumber cultivar grown in a closed nutrient flow system was prolonged by grafting onto a non-autotoxic cultivar (asao et al., 1999 b). thus, cucumber root exudates from a closed hydroponic system were analyzed and 2,4-dichlorobenzoic acid (dcba) was found to be the strongest inhibitor among a number of growth inhibitors detected (asao et al., 1999 c; pramanik et al., 2000). autotoxicity in strawberry (fragaria × ananassa duch.) under closed hydroponics in japan, closed hydroponics have been considered feasible for strawberry cultivation (takeuchi, 2000; oka, 2002; koshikawa and yasuda, 2003), however it was reported that a yield reduction, caused by unknown factors, occurred in this production system for strawberry (oka, 2002). many researchers showed that root exudates can be removed by adding ac to the nutrient solution (koda et al., 1977; asao et al., 1998 a, 1999 c; sato, 2004). subsequently, kitazawa et al. (2005) identified potential chemicals in root exudates of strawberry and investigated the effects of non-renewed nutrient solution, and on vegetative and reproductive growth of strawberry with the addition of ac. non-renewed nutrient solution resulted in a significant decrease in the growth of strawberry plantlets compared to growth when the nutrient solution was renewed. the number of flower clusters, flowers and fruits harvested all decreased in non-renewed nutrient solution. this phenomenon was also evident in cucumber (asao et al., 1998 a), mitsuba (koda et al., 1980) and rose (sato, 2004). gc-ms analysis of strawberry root exudates revealed the presence of lactic, benzoic, succinic, adipic, and p-hydroxybenzoic acids. these acids showed phytotoxicity during bioassay and benzoic acid was found to be the strongest inhibitor of vegetative and reproductive growth in strawberry plantlets. therefore, it was concluded that reduction in performance of strawberry grown in closed hydroponic systems occurred thorough autotoxic root exudates from the strawberry plant itself. autotoxicity in some leaffy vegetables when grown in recycled hydroponics yield reduction due to continuous culture of numerous crops has been demonstrated by researchers around the world. allelopathic effects from crop residues and root exudates have been extensively studied in crops such as alfalfa (miller, 1983; nakahisa et al., 1993, 1994; chon et al., 2002, chung et al., 2011), asparagus (young, 1984; young and chou, 1985; hartung et al., 1990), cucumber (yu and matsui, 1994, 1997), watermelon (kushima et al., 1998; hao et al., 2007), taro (asao et al., 2003), strawberry (kitazawa et al., 2005), tomato (yu and matsui, 1993 b), lettuce (lee et al., 2006) and so on. thus, autotoxicity is considered to be one of the causes of growth retardation on the successive culture of vegetables. it was found that many leaffy vegetables demonstrated autotoxicity when grown in recycled hydroponics (asao et al., 2001 a). in another study autotoxic substances from eight leaffy vegetables were identified as lactic, benzoic, m-hydroxybenzoic, p-hydroxybenzoic, adipic, succinic, and vanillic acids (asao et al., 2004 b). benzoic acid was the strongest inhibitor overall for leaffy vegetables among the substances tested. these results confirm that some unidentified compounds inhibited growth of leaffy vegetables in hydroponic culture (asao et al., 2001 b). autotoxicity in taro (colocasia esculenta schott.) under closed hydroponics yield of taro plants decreased when cultivated consecutively for several years on the same land (takahashi, 1984). rotation with other crops for at least three years (miyoshi et al., 1971), in combination with organic matter and soil disinfectants (murota et al., 1984), has been suggested to improve the yield of taro. however, even in a fixed crop rotation system, there was a great difference in the growth and yields of taro plants. miyaji and shirazawa (1979) found that taro residues in soils after harvest were inhibitory to its growth. asao et al. (2003) identified the chemicals exuded by taro roots and evaluated their phytotoxicity on growth and yield of taro using a hydroponic system. gc-ms analysis of taro root exudates detected methyl esters of lactic, benzoic, m-hydroxybenzoic, p-hydroxybenzoic, vanillic, succinic, and adipic acids. among them benzoic and adipic acids significantly inhibited the growth of taro plantlets at concentrations ranging from 0 to 400 mm l-1. benzoic acid affected growth of taro plants even at 50 mm l-1. their conclusion was that the decline in yield in successive culture of taro appeared to be related to the allelochemicals exuded from taro plants. autotoxicity in some beans grown with non-renewed culture solution edible beans are grown as vegetables and intensively cultivated in the same farmland year after year. the production of these common bean plants and other perennial legumes declines in replanting conditions owing to autotoxicity, a form of intraspecific allelopathy that occurs when 171 a plant species releases chemical substances that inhibit or delay germination and growth of the same plant species (putnam, 1985; miller, 1996; singh et al., 1999). allelopathy has been investigated in some beans such as pisum sativum (kato-noguchi, 2003), mucun pruriens (fujii et al., 1991), glycine max (huber and abney, 1986; xiao et al., 2006; yan and yang, 2008), and cicer arietinum (yasmin et al., 1999). in field experiments, it has been reported that residues and extracts of pea plants suppressed the growth and population size of several plant species (purvis, 1990; schenk and werner, 1991; tsuchiya and ohno, 1992; akemo et al., 2000). phytotoxic substances in pisum sativum root exudates have been reported by several researchers (hatsuda et al., 1963; yu and matsui, 1999) and, recently, pisatin has been identified as an inhibitory chemical from its shoots (kato-noguchi, 2003). removal of the inhibitory chemicals from soils or culture solution can permit continued crop cultivation on the same land for several years. hydroponic culture technique has the ability to trap and isolate the chemicals released through plant roots. elimination of these growth inhibitors from recycling culture solution is desirable from the viewpoint of conservation-oriented agriculture. thus, identification of the allelochemicals from bean root exudates, evaluation of their phytotoxicity, and their removal would facilitate the maintenance of profitable crop production. asaduzzaman and asao, (2012) studied autotoxicity in pisum sativum, phaseolus vulgaris, and vicia faba, and their responsible allelochemicals using hydroponics. they also evaluated phytotoxicity of the identified allelochemicals using seedling growth bioassay of the test plants. results indicated that yield of these beans decreased greatly (over 50%) when grown in non-renewed culture solution, however addition of ac in the culture improved the yield significantly. the identified allelochemicals were benzoic, salicylic, and malonic acids in root exudates of p. vulgaris and lactic, benzoic, phydroxybenzoic, vanillic, adipic, succinic, malic, glycolic, and p-hydroxyphenylacetic acids in v. faba. bioassay of the identified allelochemicals revealed that benzoic, salicylic, and malonic acids significantly reduced the growth of p. vulgaris even at low concentrations. autotoxicity in some ornamental plants evidenced in recycled hydroponics asao et al. (2007 a,b) investigated autotoxicity in some selected ornamentals along with a possible remedial measure to overcome growth inhibition. among the 37 plants under study, growth of lily, prairie gentian, corn poppy, farewell-to-spring, rocket larkspur, and carnation was drastically reduced in the absence of ac, compared with those in the presence of ac in the nutrient solution. in this study the added ac adsorbed phytotoxic root exudated from nutrient solution leading to improved plant growth. thus use of ac showed potentiality of overcoming autotoxicity under recycling hydroponics. root exudates of some plants were analyzed and several organic compounds were detected. strong growth inhibitors such as lactic acid in pot marigold, benzoic and p-hydroxybenzoic acids in lily, o-hydroxyphenylacetic acid in rocket larkspur, benzoic and p-hydroxybenzoic acids in sweet pea, and maleic and benzoic acids in prairie gentian were detected in the root exudates. the reduced growth of prairie gentian after prolonged cultivation in a field suggested that it could be avoided by amending the soil with ac at a rate of 60 kg 10 a–1. 4. causes of crop yield reduction in non-recycled hydroponics decrease of cucumber yield in non-renewed culture solution investigations were conducted to clarify the reasons for fruit yield reduction during a late growing period of cucumber cultured in hydroponic nutrient solution which was not renewed completely (asao et al., 1998 a). it was found that vegetative growth was unaffected by biweekly renewed or non-renewed nutrient solution whereas, fruit yield decreased when nutrient solutions were restored during culture as compared to total renewed or supplemented with ac. influence of isolated phenolics on fruit yield in cucumber phenolics isolated from the nutrient solution growing cucumber plant had significant influence on fruit yield (asao et al., 1999 a, 1999 c). the researchers isolated and identified growth inhibiting substances of unknown origin in the nutrient solution culturing cucumber plants. the growth inhibitors were adsorbed on the ac and extracted by an organic solvent. the active substances were analyzed by gc-ms as benzoic acid, p-hydroxybenzoic acid, 2, 4-dichlorobenzoic acid (dclba) and phthalic aicd. among these substances, dclba exhibited the strongest inhibitory activity toward cucumber seedlings and thus it was considered the most effective allelochemical of cucumber. it was also found to cause growth inhibition of cucumber seedlings in a seedling growth bioassay. therefore, it was assumed that this phenol was one of the compounds responsible for the inhibition of cucumber growth in nonrenewed hydroponic culture solution. other researchers have also shown that combinations of certain phenolics can have interaction effects on the germination of various crops and weed species (williams and hoagland, 1982). combination of dclba (20 µmol liter-1) with benzoic acid, p-hydroxybenzoic acid, and phthalic acid resulted in growth suppression of cucumber (asao et al., 1999 a). the effects of dclba on the number of harvested cucumbers were evaluated by split-root system hydroponic culture (asao et al., 2001 c). dclba applied in the nutrient solution at 10 µmol liter-1 severely damaged the roots by disrupting the integrity of epidermal cells, and remarkably inhibited the uptake of no 3 -, h 2 po 4 -, and k+ ions leading to a decreased number of harvested cucumber fruits. dclba is a herbicide and also a synthetic auxin, and it stunts cucumber plants at low concentration but kills them at higher levels. this allelochemical was applied through nutrient solution to cucumber seedlings grown hydroponically and exposed to two microorganisms (ts22 and ts-29) and one rhizoplane aci, isolated from soil 172 and cucumber roots (asao et al., 2001 b). it was found that cucumber seedlings not exposed to dclba and different microorganisms grew vigorously, whereas those exposed to this chemical were stunted. effect of temperature and photoperiod on phytotoxins exudation in cucumber light and temperature have profound effects on the quality and quantity of exudates because they affect the processess of photosynthesis, translocation, and respiration in plants (hale et al., 1971; hale and moore, 1979). an increase in exudation at high temperature has been reported for many crops (rovira, 1959, vancura, 1967; rovira, 1969; hale et al., 1978). the effect of temperature and photoperiod has been assessed on the quality and quantity of growth inhibitors exuded from the root of cucumber (pramanik et al., 2000). the researchers found that exudation rate varied greatly with the kind of acids, temperatures, and photoperiods, ranging from 0.2 to 4.17 µg day-1 plant-1. exudation tended to increased with plant growth and maximum exudation rate was recorded with high temperature and long photoperiod, and minimum with low temperature and short photoperiod. 5. possible measures to control autotoxicity under closed hydroponics selection of suitable crop cultivars screening for differential autotoxic potential of cucumber cultivars has been conducted in a closed hydroponic system using seedling growth bioassay (asao et al., 1998 b). in this study, some commercial cucumber cultivars were classified into four groups, such as pi 169391, encore i, hokushin and aodai. the cultivars showed intermediate, high, intermediate and low sensitivity to growth retarding substances in culture solutions once used for different cultivars, while growth reduction was not found, except in pi 169391. these findings indicate that there are intraspecific variations in the autotoxic potential of cucumber. thus, cucumber cultivars with less growth reduction of seedlings in culture solution once used for the same cultivars would be most suitable for culturing in closed hydroponics. species differences in autotoxicity susceptibility in another study species differences in susceptibility to autotoxicity among sixteen leaffy vegetables were studied in hydroponics (asao et al., 2001 a). among the species, parsley inhibited most severely in the absence of ac followed by celery, edible burdock, garland chrysanthemum, kale, curled lettuce, pak-choi, head lettuce and mitsuba, whereas komatsuna, chinese cabbage, radish, takana, welsh onion, perilla and spinach were found to be unaffected. use of autotoxin-tolerant plant as rootstock fruit yield of cucumber in a closed nutrient flow system has been increased by grafting “shogoin-aonagafushinari” on “hokushin” or “aodai” seedlings (asao et al., 1999 b). the number of harvested fruit of “shogoinaonaga-fushinari” in the summer crop was increased by grafting on the rootstock of “hokushin” or “aodai”. the decreased weekly number of harvested fruits in the late harvest period on ungrafted plants was not observed on plants grafted on “hokushin” or “aodai” seedlings, thereby extending the harvest season. there was no evidence found that rootstocks influence vegetative growth. use of bloomless rootstocks to increase number of harvested cucumber fruits the number of harvested fruits of cucumber cultivar “shogoin-aonaga-fushinari” grafted on bloomless rootstocks such as “hikari-power” and “kitora” was increased slightly when ac was not added, but increased greatly when ac was added in the culture solution (asao et al., 2000). these results indicate that root exudates influence the harvested fruit number, which can be limited in bloomless rootstocks. use of ac in addition to dissolved oxygen levels to increase cucumber fruit number investigation on the effects of dissolved oxygen levels and addition of ac has revealed a significant influence on fruit yield of cucumber in closed hydroponics (asao et al., 1999 d). addition of ac had no influence on the dissolved oxygen concentration (1.7-3.1 ppm and 5.0-7.5 ppm after 6 and 24 h aeration, respectively) while fruit yield per plant increased significantly. in this study, aeration hours or dissolved oxygen did not show any effect on yield of cucumber. addition of ac to adsorb allelochemicals from recycled culture solution and/or replanting soil activated charcoal, with its large surface area, pore volume and polarity, has tremendous adsorption capacity for many organic compounds. in cucumber, tomato, and asparagus, increased productivity has been observed after using ac in non-renewed culture solution or replanting soil (yu et al., 1993; yu and matsui, 1994; asao et al., 2003; motoki et al., 2006). mat-rush does not grow well if it is cultivated consecutively for years in the same land. the continuous cultivation of mat-rush accumulates toxic allelochemicals from roots in soil and inhibits subsequent plant growth. fujitomi et al. (1999) reported an approximate 45% yield reduction in second year cultivation of mat-rush. asao et al. (2007 b) reported that mat-rush seedlings grown in soils collected from fields consecutively cropped with mat-rush for three years had lower shoot dry weight compared to plants grown in soils amended with ac (138 and 165% lower dry weight compared to coarse and fine ac, respectively). mitigation of autotoxicity using microbial strain microorganisms can degrade chemical substances in soil and water (sundin and waechter-kristensen, 1994) and phytotoxins, both autotoxins and microbial toxins (caspersen et al., 2000; asao et al., 2003, 2004 a; chen et al., 2011). sim173 ilarly, many isolates from suppressive soils or others can degrade autotoxins in the rhizosphere of continuously cropped plants (asao et al., 2004 a; chen et al., 2011). growth and yield of cucumber plants significantly decreased with addition of dclba (the strongest growth inhibitor released during reproductive stage) to the recycled nutrient solution but growth recovered upon addition of microbial strains. it is suggested that microorganisms, if added to nutrient solution at the reproductive stage of cucumber, can catabolize autotoxic compounds from root exudates into non-toxic compounds and thus increase fruit yield. mitigation of autotoxicity by supplementation of 2,4-dichlorophenoxyacetic acid (2,4-d) and 1-naphthaleneacetic acid phenolic compounds disrupt the endogenous hormonal balance in plants (rice, 1984; asao et al., 2001 c). benzoic acid or similar structural compound substitution has been considered as anti-auxinous (keitt and barker, 1966; karabaghli-degron et al., 1998). callis (2005) reported that fruit enlargement and maturation of strawberry depends on auxin. thus, the effects of foliar applications of 2,4-d and naa on growth of strawberry were investigated to mitigate the autotoxicity in growing plants in closed hydroponic systems (kitazawa et al., 2007). supplementation of 5.4 µm naa was found to be the most effective treatment for alleviating autotoxicity of strawberry and increasing fruit yield. mitigation of autotoxicity by supplementation of amino acids amino acids are the nitrogenous compounds which form the basic component of all living cells (furuya and umemiya, 2002). therefore, they have great potential for use in culture techniques: recently they were applied as foliar spray to improve the growth, yield and quality of several crops (mazher et al., 2011; takeuchi et al., 2008). similarly, supplementation of amino acids has been investigated to recover growth and yield of strawberry plants with autotoxicity in closed hydroponics. in greenhouse experiments, 22 water soluble amino acids were sprayed on strawberry plants at 2 ml per plant and among them glutamic acid and hydroxy-proline spray produced 50% greater fruit yield compared to control water spray in plants grown under nonrenewed culture (mondal et al., 2013). mitigation of autotoxicity through electrodegradation of root exudates in recycled hydroponic culture, significant growth inhibition of strawberry has been observed due to accumulation of toxic root exudates in the nutrient solution; benzoic acid was found to be the most potent growth inhibitor in root exudates (kitazawa et al., 2005). electrochemical methods have also been applied for degradation/oxidation of phenols and their derivatives from organic waste or pollutants by several researchers. phenolic compounds, including phenols, catecol and hydroquinone in aqueous solution and even benzene were found to decompose when treated by electro-degradation (fleszar and ploszynka, 1985; comninellis and pulgarin, 1991; feng and li, 2003). these compounds are oxidized rapidly at the anode and decompose to co 2 . thus, electrodegradation may cause the decomposition of allelochemicals, including benzoic acid exuded in the nutrient solution from plants, offering a possibly useful tool to mitigate autotoxicity in strawberry. based on these results, electrodegradation has been attempted for the decomposition of benzoic acid in the culture solution (asao et al., 2008). it was found that benzoic acid exogenously added to nutrient solution was almost completely decomposed within 24 h. electrodegradation of nutrient solution could mitigate autotoxicity in strawberry plants grown in closed hydroponic culture and it recovered fruit yield up to 71% of control. the appropriate timing and intensity of electrodegradation of nutrient solution has also been investigated and it was recommended that application of electrodegradation to non-renewed culture solution for 2 h at four-week intervals can recover fruit yield completely (99%) compared to non-renewed culture solution without electrodegradation (asaduzzaman et al., 2012). selection of ideal succeeding crops after asparagus, taro and beans successive culture of the same crop on the same land for years causes soil sickness or replanting injuries (rice, 1984; tsuchiya, 1990) resulting in reductions in both crop yield and quality. among the possible reasons for this complex natural phenomenon self-allelopathy, or autotoxicity, has often been suggested (asao et al., 2003; asaduzzaman and asao, 2012). this phenomenon has been evidenced in asparagus (hartung and stephens, 1983; young and chou, 1985; lake et al., 1993), taro (takahashi, 1984) and several beans (putnam, 1985; miller, 1996; singh et al., 1999). therefore, growth performances of 67 vegetable crop cultivars were evaluated through seedling growth bioassay using once used nutrient solution of asparagus and also replanting soil of asparagus, taro and three beans (asaduzzaman et al., 2013 b). strategies to overcome this problem in replanting soil or reuse of hydroponic culture solution have been suggested by many researchers. bioassays using asparagus with used nutrient solution, with or without ac, suggest cucumber, garden pea, komatsuna, melon, pak-choi cv. ‘tyoukou’, parsley, soybean (except cv. ‘tankurou’), cabbage cv. ‘early ball’ and lettuce cv. ‘shato’ as possible succeeding crops. while, bioassays using replanting soil, with or without ac, have suggested that most of the cultivars tested can be planted after asparagus, taro, and three beans (vicia faba l., pisum sativum l. and phaseolus vulgaris l.) with little adverse effects. among the three methods of bioassay used (i.e. nutrient solution, direct seed sowing and seedling transplanting in replant soil) the nutrient solution bioassay proved more sensitive than replanting soil bioassay. however, results of nutrient solution bioassay may not be reproducible in field conditions. therefore, the seedling transplanting method can be used as an easy and practical bioassay approach to select succeeding crops for fields with replanting problems. 174 6. future challenges and endeavors in horticultural research in japan currently japan faces problems of an aging population, mainly in rural areas. in fact, many researchers are trying to breed new high quality cultivars to develop labor-saving and environmental-friendly technologies for horticultural cultivation. tomato production in japan is an especially up and coming market that needs to be looked at much more closely. in this regard, outdated hydroponic systems should be up-scaled with modern facilities. a number of favorable resources are found in japan, such as rivers, the ocean, and a relatively temperate climate, and they can make greenhouse productions much more sustainable. through the use of wind, solar and geothermal power these new greenhouses can be brought into sustainable horticultural production. recent advances in solar technology have created an outstanding 17% conversion efficiency in solar panels. these free-standing towers can be set up on the south end of greenhouses on an automatic swivel to rotate with the movement of the sun to ensure optimal light intensity at all times of the day. this solar energy collected during the day can be used to heat water tanks under the greenhouse for use during the night to heat the greenhouses as needed. through the generation of electricity with wind and solar power, heating and electricity needs beyond these systems will be minimal, greatly reducing greenhouse electricity consumption from less sustainable sources. therefore, development of plant factory supported facilities with this geothermal energy system should be expanded throughout japan. according to “japanese society of nephrology” about 13.3 million people suffer from ckd (i.e. one in every eight adults) and it expected that the total number of patients will continue to increase progressively. therefore, preventive measures should be stressed to divert the prevalence of this disease toward a decreasing trend. ckd patients are restricted from consuming k rich foods but our typical daily diet includes fruits, fresh vegetables, seaweed, beans and potatoes with high k content, making it difficult for sufferers to eat their usual diet with other family members. this restriction impacts on their quality of life 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atc = automatic temperature compensation; b = blue; bc = biostimulant complex; c = cyan; ec = electrical conductivity; f­c = folin­ciocalteu; ftir = fourier transformed infrared; g = green; gae = gallic acid equivalent; glm = general linear model; gm = genetic modification; m­ir = mid­ infrared; o = orange; pca = principle component analysis; pi = pink; pls­da = par­ tial least squares­discriminant analysis; pu = purple; qe = quercetin equivalent; r = red; rwc = relative water content; spls­da = sparse partial least squares­dis­ criminant analysis; ssc = soluble solids content; tp = total phenolics; uatr = uni­ versal attenuated total reflectance; w = white; y = yellow. abstract: strawberry is a popular functional food due to the presence of antiox­ idant and anti­inflammatory phytochemicals. enhancing this functional food value is an opportunity to improve consumer health, but strategies to do so cannot compromise yield or organoleptic properties, which are highest priori­ ties for farmers and consumer, respectively. one promising strategy is the sup­ plementation of fertiliser regimens with biostimulants, which are non­nutritive substances associated with species­specific improvements to crop growth, yield, and quality. accordingly, the impacts of a biostimulant complex (bc) con­ taining molasses, aloe vera extract, and fish­hydrolysate is characterised herein for its potential to impact strawberry growth, yield, quality, and functional food value. results indicated that bc treatment significantly increased (p < 0.05) plant biomass and canopy area (growth), total fruit count and weight per plant (yield), fruit aroma and colour (quality), and antioxidant potential (func­ tional food value). the results presented highlight the potential utility of bios­ timulants to the strawberry sphere, providing a strategy to enhance the fruit to the benefit of both farmers and consumers. (*) corresponding author: jamie@nutrifield.com citation: wise k., selby­pham j., simovich t., gill h. 2024 ­ a biostimulant complex comprising molas‐ ses, aloe vera extract, and fish‐hydrolysate enhances yield, aroma, and functional food value of strawberry fruit. ‐ adv. hort. sci., 38(1): 47­62. copyright: © 2024 wise k., selby­pham j., simovich t., gill h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 august 2023 accepted for publication 21 september 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15033 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 47­62 48 1. introduction the genus fragaria is comprised of 25 species of small flowering plants known as strawberries, which are widely cultivated for their edible fruits (hirakawa et al., 2014). of these 25 species, the hybrid octo­ ploid fragaria x ananassa, is the most popular vari­ ety, accounting for 60% of the worlds strawberry fruit production (amil­ruiz et al., 2011). the global strawberry industry is a profitable and growing indus­ try, with world production outputs in 2020 equal to 40.76 million tonnes (fao, 2022). whilst strawberry fruits are primarily consumed as a fresh fruit, they are also popular additions to processed foods such jams, juices, dairy products, and flavoured drinks, making strawberries one of the most popular and versatile global agricultural products (moraga et al., 2006; basu et al., 2014). the drivers of consumer perception of strawberry fruit quality are their physical features, organoleptic properties, nutritional value, and added secondary health benefits (functional food properties) such as antioxidant, anti­inflammatory, and anti­hyperten­ sive activities (basu et al., 2014). improving these measures is therefore desirable to consumers who would experience higher quality produce and poten­ tially added health benefits from increased functional food properties. the antioxidant activity of strawber­ ry fruits is a particular driver of the fruit’s popularity, as this functional food property is associated with a range of benefits including improvements to cardio­ vascular health (giampieri et al., 2015), neurodegen­ eration (esposito et al., 2002), cancer (zhang et al., 2008), and type 2 diabetes (da silva pinto et al., 2010). the antioxidant activity of the fruit is attribut­ ed to the high concentrations of polyphenols includ­ ing flavonoids, anthocyanins, and ellagitannins, and vitamins such as ascorbic and folic acid, which have been shown to vary in concentration in fruits depending on cultivar, storage, processing, and culti­ vation system (basu et al., 2014; afrin et al., 2016). in addition to improving the functional properties of the fruit, strawberry yield and organoleptic improve­ ments are also significant opportunities to benefit the strawberry industry. processing (moraga et al., 2006), plant breeding (diamanti et al., 2012), and cultivation practises (akhatou et al., 2014) have all been shown to impact fruit sensory properties such as firmness and taste, however, breeding is slow and costly, and processing is unsuitable to the fresh fruit sector. therefore, modifications to farming practises could be potential­ ly fast and versatile interventions to strawberry culti­ vation, to improve fruit quality metrics ­ functional food potential and organoleptic desirability ­ and thereby enhance customer perception of quality and value returned to farmers. accordingly, implementa­ tion of biostimulants continues to generate interest as a fast and environmentally friendly improvement to cultivation practises, as these inputs are non­nutri­ tive compounds or substances which beneficially impact plant growth, development, or yield (du jardin, 2015). various biostimulants have been asso­ ciated with improvements to fruit quality in a range of species, however effects are plant­species and dosage dependent, requiring further exploration of these effects in target crops such as strawberry (rodrigues et al., 2020; wise et al., 2020). molasses is a by­product from the sugar industry, which is rich in simple and complex carbohydrates, proteins, amino acids, organic acids, and maillard reaction by­products (melanoidins) (najafpour and shan, 2003; chandra et al., 2008). it has been used as an additive in agriculture and has demonstrated bios­ timulant effects including increasing yield in beetroot (nadeeka and seran, 2020), sugar cane (srivastava et al., 2012) and spinach (pyakurel et al., 2019), improved biomass growth in maize (shahzad et al., 2018) and sorghum (suliasih and widawati, 2016), enhanced salinity tolerance in thyme (koźmińska et al., 2021), and improvements to disease resistance in various species (welbaum et al., 2004). similarly, molasses distillery effluent, which is more dilute but similarly comprised to molasses, also has demon­ strated biostimulant benefits such as increased yield in banana (thakare et al., 2013) and sweet pepper (gaafar et al., 2019), increased nutrient uptake in radish (hatano et al., 2016), improved growth and development of rapeseed (li et al., 2020), altered antioxidant activity in cabbage (bimova and pokluda, 2009) and black bean (elayaraj, 2014), as well as increased heavy metal uptake by common reed and sedge for potential applications to bioremediation (nagy et al. , 2020). accordingly, utilisation of molasses as a biostimulant input for strawberry farm­ ing may be expected to positively impact a range of yield, growth, and quality measures. aloe vera extracts are also complex mixtures con­ taining plant nutrients, vitamins, enzymes, amino acids, sugars, hormones, and hormone­like com­ pounds (ishartati et al., 2019; cortés et al., 2021). the biostimulant effects associated with application wise et al. ‐ biostimulant complex improves strawberry quality 49 of aloe vera extracts are varied, including increased propagation efficiency and growth of populus tree clones (el sherif, 2017), eucalyptus tissue cultures (hendi, 2021), and grape vine cuttings (uddin et al., 2020), improved growth, biomass, and oil content of sweet basil (hamouda et al., 2012), increased growth, yield, oil content, and nutritional content of caraway (khater et al., 2020), improved growth and chlorophyl content of fenugreek (al­yasiri et al., 2021), increased leaf growth and terpene content in lavender (el sherif et al., 2020), enhanced fruit yield and nutritional content of okra (hemalatha et al., 2018 a, b), and dose­dependent positive and nega­ tive effects on cereal germination (baličević et al., 2018). accordingly, aloe vera extracts are utilised as fertiliser additives to improve plant growth and have the potential to improve a range of attributes when added to strawberry plants during cultivation. agricultural amino acids can be purified or com­ plex mixtures, which are often extracted (hydrolysed) from animal, plant, or microbial products (calvo et al., 2014). legume­derived hydrolysates are common sources of amino acids and have a range of beneficial effects associated with their use during cultivation including increased vegetative growth, yield, and sec­ ondary metabolite production in capsicum (ertani et al., 2014), and increased yield (colla et al., 2017), nutritional content (colla et al., 2017; rouphael et al. , 2017), firmness (organoleptic property) (mirabella et al., 2021), and antioxidant activity (caruso et al., 2019) in tomato. furthermore, amino acids derived from fish­hydrolysate have demonstrat­ ed increased yield in tomato (garcía­santiago et al., 2021), pig blood­hydrolysate increased phenolic and antioxidant properties of lettuce (zhou et al., 2022), and a commercial amino acid product was shown to increase antioxidant activity in the leaves of aloe vera (ardebili et al., 2012). amino acids are also asso­ ciated with improved nutrient uptake in plants, either directly through provision of organic­n or indi­ rectly through stimulation of soil microbes or chela­ tion of nutrients (callahan et al., 2007; halpern et al., 2015). accordingly, addition of amino acids during strawberry cultivation may demonstrate biostimulant effects to improve a range of measures and thereby benefit consumers and/ or farmers. noting the potential benefits of molasses, aloe vera extract, and fish­hydrolysate as biostimulants during strawberry cultivation, the aim of this study was to explore impacts to strawberry growth, yield, and quality associated with the supply of a complex of these biostimulants. this involved the hydroponic growth of strawberry plants with application of the biostimulant complex, followed by temporal assess­ ment to impacts to fruit yield, in addition to end point fruit quality measures such as fruit antioxidant potential, and fruit sensory profile. characterisation of the effect of these biostimulants to strawberry has the potential to benefit both strawberry farmers and consumers by providing a cost efficient and easily implemented farming strategy to enhance the value of strawberry yields. 2. materials and methods plant materials and growth conditions stock tubes of strawberry plants (fragaria x ananassa ‘albion’), supplied by sunny ridge strawberry farm pty ltd. (boneo, vic, australia), were planted into 15 cm pots in coco perlite sub­ strate (nutrifield pty ltd., melbourne, vic, australia) and maintained in indoor growth rooms for 5 weeks with coco a&b nutrients (nutrifield pty ltd., melbourne, vic, australia) at ph = 5.8, and ec = 1.0. fertigation was delivered via a flood and drain sys­ tem, wherein trays containing potted plants were filled with fertigation liquid to 75% the height of the pots and subsequently drained. plants were grown under 315w ceramic metal­halide horticultural lamps (315w cmh pro 4200k, indoor sun, melbourne, vic, australia), with a recom 315w ballast (lucius, melbourne, vic, australia), and light:dark (l:d) pho­ toperiod of 12 hours day and 12 hours night. environmental conditions were restricted to day tem­ perature and relative humidity of 21.5°c, and 70%, respectively, and night temperature and relative humidity of 18°c, and 51%, respectively. after 5 weeks, plants were separated into treatment groups and re­potted into 30 cm square pots with coco per­ lite substrate and a top layer (2 cm) of hydro clay (nutrifield pty ltd., melbourne, vic, australia). treatment and fertigation programme twelve plants were split into 2 groups (n = 6): con­ trol group, receiving coco a&b nutrients as per usage instructions, and treatment group, receiving coco a&b nutrients as per usage instructions plus the bios­ timulant complex (bc) comprising molasses (10% w v­ 1), aloe vera extract (2.5% v v­1), and fish­hydrolysate (5% v v­1) at 2 ml l­1 during weeks 10­18. fertigation was delivered to plants via a recirculating drip­irriga­ adv. hort. sci., 2024 38(1): 47­62 50 tion system (4 × 4 l h­1 dripper­1 plant­1) as described in table 1. the elemental composition, phytohor­ mone profile, and metabolite profile of the biostimu­ lant complex is provided in supplemental tables s1, s2, and s3, respectively. vegetative measurements leaf colour (l*a*b*) was measured using a cr­ 400 chroma meter colourimeter (konica minolta, tokyo, japan). canopy area was measured using the smartphone application easy leaf area free (easlon et al., 2014). at the conclusion of the harvest period leaf and crown number were counted, and final fresh­ and dry­weight measurements were taken for leaf and non­leaf tissues. vegetative tissues were dried in an ed 53 oven (binder, tuttlingen, germany) at 70°c for 3 days. harvesting and fruit measures ripe fruit ­ defined as bbch = 87 according to wise et al. (2022) ­ were harvested, immediately measured (weight and length), and placed into a dt5600 food dehydrator (sunbeam, fl, usa) at 55°c for 7 days. strawberry fruit length at harvest was measured as the perpendicular distance from the centre of the calyx to the tip of the receptacle. fruit width at harvest was measured by image analysis using imagej (schneider et al., 2012). in short, image global pixel scale was set based on known fruit length and the ‘measure’ feature within the roi manager was utilised to measure the widest fruit diameter. determination of ph and brix of crude fruit extract a crude extract was prepared by pressing fresh strawberry fruits through four layers of muslin cloth and subsequently passing the filtrate through an additional single layer of muslin cloth. an automatic temperature compensation (atc) portable refrac­ tometer (sugar/brix refractometer 0­32% 300001, super scientific ltd., scottsdale, az, usa) was used to measure brix (°bx) of the pure crude extract. a 1/1000 dilution of the crude extract in water was used to measure ph (sension+ mm 374 glp 2 chan­ nel laboratory meter with sension + 5014t ph liquid combination electrode with silver ion barrier, hach, loveland, co, usa). fruit phytochemical extraction whole fruit extraction was carried out with adap­ tations to the method described in chandra et al. (2014). in short, the dried strawberry fruits were pul­ verised in a ‘multigrinder ii’ (sunbeam, fl, usa) and then extracted in 8 ml ethanol (100%) per g pul­ verised fruit. the extraction was carried out in a soni­ cator at 40°c for 10 min and then filtered through 7­ 10 µm membrane filter paper with 0.1% ash content (westlab, ballarat, vic, australia). the ethanol filtrate was evaporated in a 100°c water bath to achieve a dried extract, which was then resolubilised in 5 ml 5% methanol in a sonicator at 40°c for 10 min. determination of total phenolic and flavonoid con‐ tent total phenolic (tp) and flavonoid content was determined as per the folin­ciocalteu (f­c) method, and the aluminium chloride colorimetric method, respectively, which were adapted from those described in chandra et al. (2014). in short, tp con­ tent was determined by combining 100 µl of resolu­ bilised extract with 100 µl f­c reagent, 300 µl 8% w v­1 saturated sodium carbonate solution, and 1.5 ml distilled water. solutions were reacted under light in a ps­10a sonicator (jeken, dongguan, china) at 40°c for 30 min. the absorbance was measured at 765 nm and phenolic content was calculated as gallic acid equivalent per gram dry fruit (gae g g­1 d.w.). total flavonoid content was determined by combining 1 ml of the resolubilised extract with 1 ml 10% (w v­1) aluminium chloride. the solutions were reacted at room temperature for 1 h, and then absorbance was measured at 420 nm. the results were calculated as quercetin equivalent per gram dry fruit (qe mg g­1 d.w.). all absorbance measurements were analysed using the dr 5000™ uv­vis spectrophotometer (hach, loveland, co, usa). sensory perception testing a blinded test was conducted to explore if the biostimulant treatment was associated with changes to sensory perception of fruits. participants (n = 6) were asked to score the fruits (on a 9­point scale) table 1 ­ fertigation programme for strawberry plants during treatment (weeks 6­18) weeks fertigation programme (split evenly throughout the day) 6­11 3 × 10 min 12­13 4 × 10 min 14­18 6 × 10 min wise et al. ‐ biostimulant complex improves strawberry quality 51 based on their texture, taste, and aroma (table 2). plant image colour analysis colour data of strawberry fruit images was extracted as per wise et al. (2022), wherein individu­ al pixels were categorised as either achromatic (a, light grey­black), blue (b), cyan (c), green (g), orange (o), pink (pi), purple (pu), red (r), white (w), or yel­ low (y), based on maximal similarity to predefined colours. mid‐infrared (m‐ir) analysis infrared spectra of the dried strawberry fruits were collected using a spectrum 2 ftir spectropho­ tometer (perkinelmer inc., waltham, ma, usa) equipped with a universal attenuated total reflectance (uatr) accessory with diamond crystal. spectra were collected with a 4 cm­1 resolution over the 500­7000 cm­1 range with four accumulations to produce an averaged spectrum. for data analysis the 4000­7000 cm­1 range was excluded. spectral data were standardised to 1875 cm­1 prior to analysis, cor­ responding with the region of the sample spectra which has minimal influence from the presence of water as indicated by the water absorption spectra (nist, 2022), presented in the nist chemistry webbook (linstrom and mallard, 2001). statistical analysis analyses implemented during exploration of treatment effects on individual measures included general linear model (glm), tukey’s test 95% confi­ dence grouping analyses, anderson­darling normality test, and mood’s median test in the minitab 19 sta­ tistical software package (minitab inc., state college, pa, usa). analyses implemented to explore treat­ ment effects on the profiles of plant measures (and fruit m­ir spectra) were heatmap analysis, dendro­ gram, principle component analysis (pca), partial least squares­discriminant analysis (pls­da), sparse pls­da (spls­da), and fold change (1.3­fold thresh­ old), using the web­tool metaboanalyst 5.0 (chong et al., 2019). the profiles of plant measures were nor­ malised within metaboanalyst using the cube root transformation and pareto scaling functions, while m­ir data was transformed by auto­scaling. mean changes in sensory perception was assessed by a repeated­measures t­test, performed in minitab 19. replicates per analysis are presented in supplemen­ tal table s1. 3. results profile analyses (vegetative, yield, and quality) a range of growth, yield, and quality measures were taken per plant (27 measures total) to assess the impact of the bc treatment on strawberry plants (table s4). principal component analysis (fig. 1a) and pls­da (fig. 1b), identified clearly distinct 95% confi­ dence regions in the trait profiles between bc and control treated plants, which is consistent with the clustering of sample profiles by treatment within the dendrogram (fig. 1c) and heatmap (fig. s1). heatmap analysis (fig. s1 and table s5) identified two clusters within the 27 plant measures (13 vege­ tative, 7 yield, and 8 quality), one cluster with mini­ mal difference between treatments (6 vegetative, 5 table 2 ­ sensory perception scoring matrix sensory perception poor acceptable optimal aroma ­ desirability 1 2 3 4 5 6 7 8 9 taste ­ desirability 1 2 3 4 5 6 7 8 9 low moderate high mouthfeel ­ firmness 1 2 3 4 5 6 7 8 9 mouthfeel ­ juiciness 1 2 3 4 5 6 7 8 9 none moderate strong aroma ­ intensity 1 2 3 4 5 6 7 8 9 taste ­ sweet 1 2 3 4 5 6 7 8 9 taste ­ sour/acid 1 2 3 4 5 6 7 8 9 taste ­ intensity 1 2 3 4 5 6 7 8 9 52 adv. hort. sci., 2024 38(1): 47­62 yield, and 4 quality), and one cluster of measures with large difference between treatments (7 vegeta­ tive, 2 yield, and 4 quality). the cluster associated with large differences was comprised of the mea­ sures: leaf count (number), leaf fresh weight (g f.w.), leaf dry weight (g d.w.), above ground (non­leaf) fresh weight (g f.w.), above ground (non­leaf) dry weight (g d.w.), canopy area (cm2), and leaf colour­a (v2, v3, v4, v6, v7, v9, and v11, respectively), total fruit harvested (g) and fruit harvested (number) (y5 and y6, respectively), and fruit gae per fruit dry weight (g·g­1 d.w.), brix %, fruit ph, and fruit water % (q1, q2, q3, and q5, respectively). vegetative analyses treatment with bc had significant impacts on sev­ eral vegetative measures (table s4). the greatest effect was seen for leaf measures including: leaf number (p < 0.001) which increased by 107.4% (over two­fold) from 15.67 in control to 32.5 in treatment (fig. 2a), leaf dry weight (p = 0.002) which increased by 66.7% from 7.4 g d.w. in control to 12.4 g d.w. in treatment (fig. 2b), and canopy area (p = 0.001) which increased by 51.8% from 401.4 cm2 in control to 609.3 cm2 for treatment (fig. 2c). whilst vegeta­ tive measures tended to increase, no significant change (p = 0.205) was identified for leaf water con­ tent (table s4). furthermore, the bc treatment increased (p = 0.001) non­leaf aerial dry weight by 64% from 5.7 g d.w. for control to 9.4 g d.w. for treatment (table s4), and with marginal significance (p = 0.057) increased crown number by 35% from 3.3 to 4.5 (fig. 2d), whilst not significantly affecting leaf fig. 1 ­ effect of biostimulant complex (bc) on strawberry (fragaria x ananassa ‘albion’) trait profiles. (a) principal component analysis (pca), and (b) partial least squares­ discriminant analysis (pls­da), with shading indicating 95% confidence regions. c) dendrogram indicating hie­ rarchical clustering (ward clustering algorithm) based on euclidean distance of plants based on measured traits. trait values measured from 6 biological replicates. fig. 2 ­ effect of biostimulant complex (bc) treatment on vegeta­ tive growth measures of strawberry (fragaria x ananassa ‘albion’). a) number of leaves, (b) leaf dry weight, (c) canopy area, and (d) number of crowns compared between control and bc treated plants. data presented mean ± standard deviation of 6 biological replicates. significant differences are indicated by ‡ for p < 0.1; * for p < 0.05; ** for p < 0.01; *** for p ≤ 0.001 calculated by student’s t­test. wise et al. ‐ biostimulant complex improves strawberry quality 53 colour measures l*, a*, and b* (table s4). yield analyses the biostimulant complex significantly increased the yields of strawberry plants in terms of total weight of fruits per plant (p = 0.038), and number of fruits per plant (p = 0.035), whilst not significantly (p = 0.666) affecting individual fruit weight (table s4). the average weight of total fruits harvested per plant increased by 50.7% from 128.3 g for control to 193.3 g for treatment (fig. 3a), while the average number of fruits harvested per plant increased by 56.9% from 12.0 for control to 18.8 for treatment (fig. 3b). whilst no changes were observed for individual fruit weight, changes to fruit shape were observed wherein bc treatment significantly (p = 0.013) increased fruit length (table s4) from 30.33 mm to 35.48 mm (17% increase), whilst fruit width was unchanged (p = 0.446). harvest timing was normally distributed for control plants (p = 0.268) but not for treatment plants (p = 0.038), however no significant difference between treatments was identified in median fruit harvest timing (p = 0.971, fig. 3c). fruit quality analyses chemical analysis of strawberry fruits identified that bc treatment significantly increased fruit water content (p = 0.001) wherein fruit water content from treatment plants was 90.2%, whilst fruits from con­ trol plants had 89.06% water content (fig. 4a). additionally, the bc treatment resulted in significant changes to fruit image colour profiles, wherein r% increased from 29.66% for control to 39.22% (p = 0.001), g% increased from 0.38% for control to 1.01% (p = 0.001), and o% decreased from 28.90% for control to 20.79% (p = 0.006), whilst y% (p = 0.371) and a% (p = 0.186) were not significantly impacted (fig. 4b). the fruit colour measures b, pi, c, w, and pu accounted for on average less than 0.01% of pixels within images and so were not explored dur­ ing analyses. no significant difference was observed for brix content (p = 0.941) between treatment or control plants (fig. 4c). the biostimulant complex treatment resulted in a marginally (defined as 0.1 < p < 0.05) significant (p = 0.055) increase to ph of dilut­ ed crude fruit extract, from 4.2 to 4.4 (table s4). it was identified that the bc treatment had signifi­ cant affects (p = 0.029) on tp content (fig. 4d) with a 32% increase from 0.0139 gae g·g­1 d.w. for control to 0.0184 gae g g­1 d.w. for treatment, whilst not signifi­ cantly (p = 0.534) affecting flavonoid content (fig. 4e). fruit quality perception was analysed by a blinded sensory perception test on a 9­point scale, which identified that the bc treatment significantly (p = 0.043) increased fruit mouthfeel firmness (‘firmness’), from 4.2 for control to 5.5 for treatment, whilst not significantly impacting mouthfeel juiciness (‘juici­ ness’), or fruit taste measures (table s6). fig. 3 ­ effect of biostimulant complex (bc) treatment on yield and harvest timing of strawberry fruits (fragaria x ananassa ‘albion’). fruit yield per plant by (a) weight, and (b) count compared between control and bc treated plants. a­b) data presented as mean ± standard deviation of 6 biological replicates. significant differences are indicated by * for p < 0.05 calculated by student’s t­ test. c) histogram of harvest timing from 72 fruits from control plants and 97 fruits from bc treated plant. adv. hort. sci., 2024 38(1): 47­62 54 nent analysis (pca: fig. s2) and pls­da (fig. s3) did not identify significant differences between the pro­ file of spectral bins between treatment groups, while spls­da (fig. 4h) indicated significant differences in a subset of the wavelength’s measured. comparison of the average spectra of each treatment (fig. s4), iden­ tified a region between 1024­1048 cm­1 with the high­ est fold change (>1.3) between treatments (fig. s5). furthermore, the bc treatment significantly (p = 0.040) increased fruit aroma intensity from 3.5 to 5.2, and had a marginally significant (p = 0.067) effect to aroma desirability which increased from 5.0 to 6.7 (fig. 4f). mid­infrared spectrometry analysis between 500­ 4000 cm­1 identified biostimulant induced changes to fruit chemical composition (fig. 4g). principle compo­ fig. 4 ­ effect of biostimulant complex (bc) treatment on quality and sensory perception of strawberry (fragaria x ananassa ‘albion’) fruits. a) fruit water (22 and 27 biological replicates for control and bc, respectively), (b) colour profile (22 and 27 biological replicates for control and bc, respectively with each colour represented as their respective colour), (c) brix (6 and 13 biological replicates for control and bc, respectively), (d) total phenolics (22 and 27 biological replicates for control and bc, respectively), and (e) total flavonoids (22 and 27 biological replicates for control and bc, respectively) compared between control and bc trea­ ted plants. a, c­e) data represented as mean ± standard deviation. f) comparison of mean scores (6 participants) for blind sen­ sory perceptions of fruit from control and bc treated plants. a­e) solid bars represent control and dashed bars represent bc treatment. a, c­f) significant differences are indicated by ‡ for p < 0.1; * for p < 0.05; ** for p < 0.01; *** for p ≤ 0.001 calculated by student’s t­test (a­e), and repeated­measures t­test (f). g) mid­infrared (m­ir) spectra of dehydrated strawberry fruits analy­ sed between 500­4000 cm­1 (7 and 11 biological replicates for control and bc, respectively). h) sparse partial least squares­discri­ minant analysis (spls­da) of m­ir spectra with shading indicating 95% confidence regions. wise et al. ‐ biostimulant complex improves strawberry quality 55 was observed from application of the bc. changes to fruit size and shape can be impactful to farmer sales due to the compliance standards imposed by super­ markets. for strawberry fruits, compliance is general­ ly determined according to diameter (usda, 2006; woolworths supermarkets ltd, 2010), which is also the highest correlating size measurement (r2 = 0.93) with consumer preference, however, length is the second highest correlation (r2 = 0.77) (lewers et al., 2020), suggesting that longer fruits of unchanged width may be considered preferable by consumers. accordingly, application of this biostimulant complex to strawberries during growth can benefit farmers by improving yield and improve customer perceptions of quality through altered fruit size. whilst the aforementioned changes to fruit size are likely to be impactful to consumer perception of fruit quality, organoleptic properties and colour fea­ tures are also highly correlative with strawberry qual­ ity perception (lewers et al., 2020). organoleptic measures include taste, texture, mouthfeel, and aroma, which are conferred to the fruit through its chemical composition (saliba­colombani et al., 2001). common measures associated with taste include sol­ uble solids content (ssc), titratable acidity (wozniak et al., 1996), and ph (gunness et al., 2009). herein no significant change was detected for brix (p = 0.941), a measure of ssc (saranwong et al., 2003), which is consistent with the results from the sensory assess­ ment wherein no significant change (p = 0.822) was observed in the correlated measure, sweetness (jouquand et al., 2008). similarly, sour perception may have been expected to change with ph (jouquand et al., 2008), and whilst a marginally sig­ nificant change was observed for fruit ph (p = 0.055), no significant change was reported from panellists for the sensory measure sour (p = 1.000). this may be explained by the apparently small change of 0.11 ph of diluted extract, which is consistent with the find­ ings of harker et al. (2002) wherein a minimum shift of 0.14 ph of apple extract was required for partici­ pants to perceive a change in apple acidity. these results suggest that utilisation of the bc during straw­ berry cultivation may increase yield without compro­ mising quality. noting that a growing point of con­ sumer dissatisfaction is the reduction in food flavour and aroma due to the prioritisation of more prof­ itable crop attributes such as yield and visual aesthet­ ic (klee, 2010; tieman et al., 2017), these results sup­ port the utilisation of biostimulants as being advanta­ geous to both farmers and consumers. 4. discussion and conclusions crop productivity is an important factor in food production when considering the growing global pop­ ulation and uncertainties associated with climate change (lobell and gourdji, 2012). accordingly, new strategies to increase crop outputs are highly sought after, with a focus on fast acting benefits which do not contribute to environmental degradation. whilst genetic modification (gm) continues to benefit many crop sectors, the costs, time, and resources required to develop approved gm food crops is a significant hurdle. accordingly, biostimulants are becoming increasingly popular additives during plant growth due to their benefits to crop productivity, natural ori­ gin, cost, and ease of use (parađiković et al., 2019). herein a naturally derived biostimulant complex comprising molasses, aloe vera extract, and fish­ hydrolysate exemplifies these beneficial effects by increasing the growth and yield of strawberry. application of the bc was shown to increase vege­ tative biomass and canopy area measures (fig. 2a­c), potentially associated with the provision of zeatin (cytokinin) ­ the only phytohormone detected in both the bc concentrate and its associated reservoir solu­ tion (table s2) ­ which has been shown to increase shoot and root growth when applied exogenously to strawberry (debnath, 2006). additionally, the bc treatment resulted in a marginally significant (p = 0.057) increase to crown number (fig. 2d). the crown is the central node of the strawberry plant from which roots, leaves, inflorescence, and addition­ al crowns form (savini et al., 2005; poling, 2012). as crowns are the base of future inflorescence forma­ tion, their number correlates strongly with fruit yield (strik and proctor, 1988; kadir et al., 2006) and is therefore an important factor for strawberry cultiva­ tion. additionally, application of the bc was observed to increase both yield­weight (fig. 3a, p = 0.038) and yield­number (fig. 3b, p = 0.035) per plant ­ poten­ tially associated with the increased crown number ­ which are crucial measures of profitability for farm­ ers. accordingly, the increases in yield outputs reported herein (fig. 3a and 3b) support the utilisa­ tion of these biostimulants by the strawberry indus­ try and thereby presents as a low­cost, effective, and easily integrated farming strategy to improve growth and yield. furthermore, changes to strawberry shape ­ increased length (fig. s6a, p = 0.013) but not weight (fig. s6b, p = 0.666) or width (fig. s6c, p = 0.446) ­ adv. hort. sci., 2024 38(1): 47­62 56 fruit mouthfeel and texture are associated with cell wall composition (caner et al., 2008) and thick­ ness (szczesniak and smith, 1969), water content (cordenunsi et al., 2002), and ph (plotto et al., 2010). additionally, salentijn et al. (2003) and wang et al. (2021) have shown that increased expression of genes associated with lignin production is associated with increased firmness of strawberry fruit. accordingly, the significant increase in mouthfeel­ firmness (p = 0.043) may relate to the presence of caffeic acid in the bc (table s3), which may internally translocate via the phloem and xylem (zhang and hamauzu, 2004; ishimaru et al., 2011) and has been shown to increase lignin production in soybean (bubna et al., 2011). richter (1978) identified that plant cell turgidity is highly sensitive to changes in relative water content (rwc), with flaccidity (loss of turgidity) to full turgor occurring over the narrow range of 5% rwc. accordingly, as fruit firmness is impacted by turgidity (szczesniak and smith, 1969; raharjo et al., 1998), the 1% increase in fruit water content (p = 0.001) identified from the bc treatment may explain the observed increase in the sensory measure for firmness. whilst this apparently minor change in fruit water content (fig. 4a) may have impacted perceived firmness (fig. 4f), it is however not surprising that this small change in fruit water volume (150 µl, based on 1% of 10.5 g average fruit fresh weight) was below sensory perception thresh­ olds to impact perceived juiciness, as juiciness is gen­ erally considered as the amount of liquid released during chewing (roger harker et al., 2003; harker et al., 2006). aroma ­ also referred to as odour ­ is the detec­ tion and recognition of compounds within the olfac­ tory system and is conferred by the presence of volatile compounds (el hadi et al., 2013). in strawber­ ry, aroma is predominantly attributed to esters, fura­ nones, terpenes, and sulfur compounds (yan et al., 2018). as with fruit flavour, aroma is often seen by consumers as a sacrifice for higher yields (klee, 2010; tieman et al., 2017), which necessitates the need for methods to improve aroma, or improve yields with­ out compromising this measure. herein a significant difference (p = 0.040) was observed for aroma inten­ sity and a marginally significant (p = 0.067) difference was observed for aroma desirability (fig. 4f), sug­ gesting that the bc treatment may have altered the volatile contents or profiles of the fruits. the m­ir analysis presented in figure 4g revealed a narrow region between 1024–1048 cm­1 with a high fold change, which has been associated with chemicals in the classes of phenolic alkyl­aryl ethers, aryl phenolic ester tannins (abbas et al., 2017), pyranose rings (saccharides), alkyl amines, and alcohols (lingegowda et al., 2012). furthermore, of these classes of com­ pounds, esters are one of the most abundant volatiles in strawberries (yan et al., 2018) and have been shown to correlate strongly with strawberry fruit liking (fan et al., 2021). whilst the scoring of odour desirability alone herein was only marginally significant, this association with overall fruit liking combined with the other changes reported herein, is likely to contribute to an overall improvement to fruit quality perception from the bc treatment. accordingly, bc treatment may have resulted in changes to ester levels to enhance the sensory aroma properties of strawberry fruits, which is also reflected by m­ir profile changes over a narrow region. these outcomes address consumer concerns for losses in aroma associated with prioritisation of more profitable traits, by demonstrating that bc treatment increases both yield and aroma. finally, fruit appearance, which includes colour (crisosto et al., 2003) and damage (jaeger et al., 2018), is a major impactor to consumer perception of quality and purchasing decision, as it is the first impression of a fruit. biostimulant complex treat­ ment resulted in significant increases to the colour measures for red (p = 0.001) and green (p = 0.001) and reductions in orange (p = 0.006). strawberry colour is conferred by the presence (amount and types) of anthocyanins, which have a strong ph­ colour relationship (holcroft and kader, 1999). the predominant anthocyanins in strawberry are pelargonidins and cyanidins (andersen et al., 2004) which appear red at low ph and with increasing ph change to colourless, yellow, or blue forms which affects the overall appearance of the fruit (holcroft and kader, 1999). whilst a marginally significant (p = 0.055) increase of 0.11 ph in diluted fruit extract was associated with the bc treatment, this degree of change is small relative to the change observed in wang et al. (2015) wherein ph shifts of 1.0 were associated with noteworthy changes to colour. accordingly, ph is l ikely not the driver of the observed changes in fruit colour, which may instead be attributed to changes in the concentrations or ratios of anthocyanins present (yoshida et al., 2002). nevertheless, strawberry colour is a driver of con­ sumer preference, as evidence by wang et al. (2017) wherein an ‘ideal red’ colour was the preference for wise et al. ‐ biostimulant complex improves strawberry quality 57 fresh strawberry fruit, and by wendin et al. (2019) which showed that red colour intensity had a signifi­ cant positive impact to consumer preference for woodland strawberries. these studies suggest that the increased red from bc treatment reported herein (fig. 4b) for common garden strawberries may also be associated with increased consumer preference. unlike organoleptic measures and colour features which are directly detectible by consumers and therefore impactful to quality perception and prefer­ ence, other properties such as nutritional and func­ tional food value should also be considered as targets for improvement during production and cultivation as their increased presence may benefit consumer health (selby­pham et al., 2017; topolska et al., 2021). due to the presence of many beneficial polyphenols and vitamins, strawberries are consid­ ered to be a functional food which can reduce hyper­ tension, postprandial oxidative stress, inflammation, and hyperglycaemia when consumed (giampieri et al., 2015). furthermore, agricultural practises such as fertiliser form (tomic et al., 2016), cultivation system (d’evoli et al., 2010), and beneficial microbes (rahman et al., 2019) have been shown to impact phytochemical profiles and antioxidant activities in strawberries. accordingly, the results presented herein are similar to these observations, wherein altered cultivation conditions through application of the bc was shown to impact phytochemical concen­ trations which are associated with functional activity when consumed. herein two methods were utilised to measure functional compounds in strawberry, and whilst the aluminium chloride method has relatively good speci­ ficity for flavonoid quantification (mabry et al., 1970), the f­c method is a non­specific method, which quantifies total reducing capacities (antioxidant activ­ ity) rather than specific classes of compounds (magalhães et al., 2008). accordingly, the 32% increase (p = 0.029) in tp and unchanged (p = 0.534) flavonoid contents (fig. 4d and 4e, respectively) reported herein indicates that the biostimulant treat­ ment increased the antioxidant activity of the fruits in the non­flavonoid portion of the phytochemical profile. aaby et al. (2007) identified that the largest contributors to strawberry antioxidant activity were ascorbic acid, and the polyphenolics ellagitannins, and anthocyanins, which accounted for 24%, 19%, and 13% of strawberry antioxidant capacities, respec­ tively. anthocyanins are also the class of compounds conferring the majority of strawberry colour (yoshida et al., 2002), which also changed in response to the biostimulant application (fig. 4b), discussed above. as noted, changes in anthocyanin concentrations or ratios may explain the changes in colour observed for biostimulant treated fruits, and changes to antho­ cyanins may also affect antioxidant activities of the fruit extracts (cerezo et al., 2010). the distinguishing feature of anthocyanins, is their multiple aromatic rings with hydroxyl groups (phenolic) structure, derived from the flavylium ion (khoo et al., 2017). whilst the carbon bonds of these aromatic rings (c=c) and the hydroxyl groups (oh) are associated with ir absorption at 1654 cm­1 and 677 cm­1, and 3385 cm­1, respectively, the c­o bond connecting the hydroxyl to the aromatic ring is associated with wavenumber 1029 cm­1 (wahyuningsih et al., 2017), which is contained within the range of wavenumbers (1024­1048 cm­1) identified herein as having increased from the bc treatment (fig. 4h and s5). accordingly, it appears that the bc treatment induced changes in the strawberry anthocyanin con­ tent or profile, which would be consistent with the changes observed for tp (fig. 4d), colour (fig. 4b), and m­ir spectra (fig. 4g). furthermore, the changes in tp (antioxidant activity) correspond with improved functional food potential of these fruits (giampieri et al., 2015), which may impart greater health benefits than control strawberries when consumed. implementation of this complex is therefore a promising improvement to strawberry cultivation practises and may be an additional tool available to farmers to improve yields and quality of produce for consumers. biostimulants are an exciting development in agriculture which have the potential to improve crop yields and quality, whilst not requiring signifi­ cant time and money to substantially alter crop out­ puts, by contrast to alternative strategies such as genetic modification and selective breeding. however, species­specific efficacies of popular bios­ timulants even when applied to commonly grown food crops are often not well understood. accordingly, this project characterised the impacts of a complex containing the biostimulants molasses, aloe vera extract, and fish­hydrolysate when applied to strawberry in a hydroponic, environmental­con­ trolled growth system. the results demonstrated that application of the complex increased crop yield, vegetive growth, and fruit quality measures includ­ ing aroma and functional food value. accordingly, utilisation of biostimulants within farming practises adv. hort. sci., 2024 38(1): 47­62 58 has demonstrated potential to increase crop out­ puts for 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6 1 hasanuddin university, faculty of agriculture, agrotechnology study program, makassar, south sulawesi, indonesia. 2 hasanuddin university, faculty of agriculture, department of agronomy, makassar, south sulawesi, indonesia. 3 west sulawesi university, faculty of agriculture and forestry, agroecotechnology program, majene, west sulawesi, indonesia. 4 regional technical implementation unit of the agricultural extension and human resources development center, food, crops and horticulture office, east kalimantan, indonesia. 5 lambung mangkurat university, agroecotechnology program, agriculture faculty, banjarbaru, indonesia. 6 muhammadiyah enrekang university, faculty of science and technology, agrotechnology program, makassar, south sulawesi, indonesia. key words: early growth, micronutrient, seed treatment. abstract: shallot (allium ascalonicum l.) is a vital horticultural commodity in indonesia, valued for its culinary and medicinal properties. seed priming, especially with boron (b), represents a promising approach to enhance germination performance. this study investigated the effects of various boron concentrations on the germination performance of shallot seeds. the experiment was conducted under screen house conditions at the faculty of agriculture, hasanuddin university, in may 2024, using a randomized block design with 11 boron concentrations (0­100 mg l­1) and three replications. priming with 100 mg l­1 resulted in the best performance across several germination parameters, including as germination percentage (gp), germination speed index (gsi), the time required for 10% of seeds to germinate (t10), the time required for 50% of seeds to germinate (t50), mean germination time (mgt), mean germination rate (mgr), and germination speed coefficient (gsc). regression analysis showed a strong positive linear relationship between boron concentration and both final germination percentage (r² = 0.84) and germination speed index (r² = 0.76). these findings suggest that boron priming, especially at 100 mg l­1, significantly enhances germination performance of shallot seeds and may as an effective method for improving seedling vigor. (*) corresponding author: muhfaried@agri.unhas.ac.id citation: zaiful z.k., faried m., syam’un e., mantja k., putri w., jalil a., wijaya p., cennawati c., 2025 ­ effect of boron priming on germination traits of shallot (allium ascalonicum l.). ­ adv. hort. sci., 39(3): 205­214. orcid: zzk: 0009­0009­8750­0991 fm: 0000­0001­6326­1724 se: 0000­0001­5875­118x mk: 0000­0001­6522­1689 prw: 0000­0003­1591­668x ja: 0009­0000­5435­0717 wp: 0009­0003­0604­4100 cc: 0000­0003­4949­4016 copyright: © 2025 zaiful z.k., faried m., syam’un e., mantja k., putri w., jalil a., wijaya p., cennawati c. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 22 march 2025 accepted for publication 28 may 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17508 http://oaj.fupress.net/index.php/ahs mailto:muhfaried@agri.unhas.ac.id http://orcid.org/0009-0009-8750-0991 http://orcid.org/0000-0001-6326-1724 http://orcid.org/0000-0001-5875-118x http://orcid.org/0000-0001-6522-1689 https://doi.org/10.36253/ahsc-13616 http://orcid.org/0009-0000-5435-0717 http://orcid.org/0009-0003-0604-4100 http://orcid.org/0000-0003-4949-4016 http://orcid.org/0000-0003-4949-4016 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(3): 205­214 206 1. introduction shallot (allium ascalonicum l.) is a horticultural commodity extensively cultivated by farmers across various regions in indonesia. in addition to its wide­ spread use as a culinary spice, shallots contain numerous bioactive compounds with potential medi­ cinal properties, such as quercetin, flavonoids, saponins, tannins, glycosides, polyphenols, and alka­ loids (devika et al., 2021). the ongoing development of shallot cultivation is driven by its various advan­ tages, including meeting national consumption needs, providing a source of income for farmers, and its potential to contribute to foreign exchange earn­ ings. these attributes position shallots as a strategi­ cally important commodity with high economic value. shallot production in indonesia reached 1,985,233 tons in 2023, an increase from 1,982,360 tons in 2022 but a decrease from 2,004,590 tons in 2021 (c e n t r a l b u r e a u o f s t a t i s t i c s o f i n d o n e s i a , 2024). shallot production still experiences fluctua­ tions, often resulting in imbalances between supply and demand in the market. national demand for shallot has been increasing at a rate of approximately 5.0­6.67% annually (shrestha et al., 2019). therefore, efforts to boost production must continue, including improving the quality of planting materials by transi­ tioning to true shallot seed (tss). however, chal­ lenges are often encountered during the seedling process when cultivating shallots from tss. the nurs­ ery process, which typically lasts 30­45 days before planting, extends the time required for crop produc­ tion and delays harvest (nciizah et al., 2020). additionally, shallot seeds are highly susceptible to losings viability due to various factors during storage, leading to reduced germination ability and speed (simatupang and pangaribuan, 2022). germination is a crucial stage in the plant life cycle that determines the success of subsequent growth stages (atabakia et al., 2022). seed priming is an effective method for producing high­quality seedlings (tanjung et al., 2021). this technique can improve initial seed germination and seedling growth (shimizu et al., 2023), accelerate germination, and enhance germination performance even under extreme environmental conditions and poor soils (chookhampa et al., 2023). several factors influence seed priming, including water potential, priming agents, duration, temperature, seed vigor, and storage conditions (farooq et al., 2011; faisal et al., 2023). among these, the choice of priming agent is crucial for the success of seed priming. micronutrients, such as boron (b) can be used as priming agents to enhance plant productivity and quality (mansouri et al., 2022). boron priming enhances seed vigor and promotes better growth and seedling development compared to untreated seeds (chakraborty and dwivedi, 2022). as a priming agent, boron contributes to improving and stabilizing cell wall structure and has a positive impact on reducing disease severity (pangestuti et al., 2021). research by rehman et al. (2022) reported that seed priming with 0.01% boron significantly acceler­ ated seedling emergence and the time to reach 50% seedling emergence, improved germination index, leaf number, fresh and dry root weight, chlorophyll content, various yield parameters, and increased car­ bohydrate, protein, and fiber content in mung bean plants (vigna radiata l.). research by nciizah et al. (2020) also revealed that seed priming with 0.01% boron reduced the number of days to seedling emer­ gence by 94% and increased seedling fresh and dry weight, as well as the chlorophyll index by 29%, 47%, and 58%, respectively, compared to the control in maize plants (zea mays l.). mansouri et al. (2022) found that seed priming with boron enhanced chlorophyll and carotenoid pigment content and increased the levels of enzymatic and non­enzymatic antioxidants such as anthocyanin, superoxide dismu­ tase, and peroxidase in wheat seeds. based on the above reviews, this study aims to determine the effect of different boron concentrations as a priming agent on the germination performance of shallot seeds. 2. materials and methods research location and experimental design this research was conducted under screen house conditions at the experimental farm of the faculty of agriculture, hasanuddin university, in may 2024. the study was designed as a randomized block experi­ ment with a single treatment factor. the treatment factor was boron concentration, which included 11 levels: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg l­1. each treatment level was replicated three times, resulting in a total of 33 experimental units. priming and germination assay the seed variety used in this study was ‘maserati’. zaiful et al. ‐ influence of boron as a priming agent 207 rstudio version 4.2.1 (wickham, 2016; shimizu et al., 2023; r core team, 2024). 3. results germination parameters the germination parameters of shallot seeds exhibited a significant response to increasing boron concentrations (table 2 and table 3). the fgp rose from 80.67% at 0 mg l⁻¹ to 96.00% at 100 mg l⁻¹, reflecting a 15.33% increase, which highlights boron’s positive influence on germination efficiency. similarly, gsi improved by 63.79%, from 6.96 to 11.40, indicating enhanced metabolic activity. t10 decreased from 4.03 days to 2.40 days, showing a 40.45% reduction, while t50 decreased by 33.39%, from 6.08 days to 4.05 days. mgt also improved, dropping by 28.25% from 6.30 days to 4.52 days, reflecting faster and more uniform germination under higher boron concentrations. the application of boron significantly influenced several germination parameters of shallot seeds. mgr and gsc increased significantly with higher boron concentrations, with the highest values observed at 100 mg l⁻¹, indicating faster germination compared to the control. although vgt and gs were not significantly affected (p > 0.05), lower vgt values at 20, 50, 80, and 100 mg l⁻¹ suggest more uniform germination, while gs tended to improve at 20, 50, and 80 mg l⁻¹. the cvgt was significantly reduced at 20 and 50 mg l⁻¹, reflecting more consistent germina­ tion timing. overall, boron concentrations between 50 and 100 mg l⁻¹ were most effective in enhancing germination performance by improving speed, rate, and uniformity. regression analysis the impact of boron concentration on various ger­ mination parameters of shallot seeds is depicted in the series of linear regression analyses (table 4, fig. 1). the analysis demonstrates that boron application significantly enhances several key germination para­ meters. for instance, fgp shows a strong positive correlation with boron concentration (r² = 0.84), indicating that higher boron levels lead to a substan­ tial increase in germination efficiency. this suggests that boron plays a vital role in breaking seed dorman­ cy and supporting metabolic processes essential for successful germination. similarly, the gsi exhibited a positive trend (r² = the seeds were primed with a boron solution follow­ ing the specified treatment protocols. they were then placed in plastic jars containing the solution at a seed­to­solution ratio of 1:5, with an aerator added to ensure an adequate oxygen supply. the priming process lasted for 20 hours at room temperature (ghassemi­golezani et al., 2010; farahzety et al., 02023; katriani et al., 2023; faried et al., 2024). afterward, the seeds were removed and air­dried at room temperature for 48 hours before further use. germination was evaluated using a soil and com­ post growth medium in a 1:1 (v/v) ratio. the medium was thoroughly mixed and placed into transparent plastic boxes with drainage holes at the bottom. each plastic box had a volume of 500 cm³ and was filled with 500 g of the growth medium. the primed seeds were then planted by creating furrows 1 cm deep. each plastic box contained 50 shallot seeds. after sowing, the seeds were lightly irrigated twice daily (morning and evening) with approximately 30 ml of water per application to maintain adequate soil mois­ ture under the warm ambient conditions of the experiment. watering volumes were carefully con­ trolled to prevent waterlogging while providing opti­ mal conditions for germination. the seedling emer­ gence was observed daily until day 14. data collection and analysis the parameters evaluated in this study included final germination percentage (fgp) (%), germination speed index (gsi), the time required for 10% of seeds to germinate (t10) (d), the time required for 50% of seeds to germinate (t50) (d), mean germination time (mgt) (d), mean germination rate (mgr), variance of germination time (vgt), coefficient of variation of germination time (cvgt), germination synchrony (gs), and germination speed coefficient (gsc) (table 1). these ten parameters were calculated using observational data on the number of seeds germinat­ ing daily. seeds were considered to have germinated when the plumule emerged above the surface of soil. an analysis of variance (anova) was conducted to test significant differences among treatments. when significant effects were found, duncan’s multiple range test was applied to separate means. additionally, regression analysis was conducted to assess the relationship between boron concentration and the germination parameters. calculations for the parameters were performed using the seedcalc pack­ age, while data analysis and visualization were con­ ducted using the ggplot2 and agror packages in adv. hort. sci., 2025 39(3): 205­214 208 0.76), indicating that higher boron concentrations accelerated germination rate. the reductions in t10 (r² = 0.43) and t50 (r² = 0.47) further supported this, as boron shortened the time needed to reach early and intermediate germination stages. mgt showed a negative correlation (r² = 0.45), reflecting a decrease table 1 ­ overview of the measured parameters related to seed germination no parameters formula 1 final germination percentage (fgp) fgp = (n/n) x 100 n is the number of seeds germinated, and n is the total number of seed. 2 germination speed index (gsi) n is the number of seeds germinated on each day of daily counting up to the last count, and t is the number of days after the beginning of the test in each count. 3 t10 germination (t10) days at which 10% cumulative germination is reached. 4 t50 germination (t50) n is the final number of seeds germinated, and ni and nj are the total number of seeds germinated in adjacent counts in time ti and tj, respectively, when 5 mean germination time (mgt) ni is the number of seeds germinated per day (not the accumulated number, but the number corresponding to the i­th observation), and ti is the time since the beginning of the germination test up to the i­th observation. 6 mean germination rate (mgr) mgr = covg / 100 = 1/t t is the mean germination time, and covg is the germination speed coefficient. 7 variance of germination time (vgt) t is the mean germination time, ti is the time between the beginning of the experiment and the i­th observation (day or hour), ni is the number of seeds germinated in time i, and k is the last count of the germination test. 8 coefficient of variation of germination time cvgt= (st / t) 100 (cvgt) st is standard deviation of the germination time, and t is mean germination time. 9 germination synchrony (gs) sinc = ∑cni, 2 / n cni, 2 = ni (ni – 1) / 2 n = ∑ni (∑ni ­1) / 2 cni is the combination of the seeds germinated in time i, two by two, and ni is the number of seeds germinated in time i. 10 germination speed coefficient (gsc) fi is the number of newly germinated seeds on day i, and xi is the number of days from sowing. zaiful et al. ‐ influence of boron as a priming agent 209 in total seedling emergence time, while mgr increased (r² = 0.47), highlighting improved metabol­ ic efficiency and faster development. in contrast, vgt (r² = 0.01), cvgt (r² = 0.12), and gs (r² = 0.03) all revealed weak or negligible correlations, suggesting that boron had limited effects on uniformity, consis­ tency, and synchronization of germination. conversely, gsc demonstrated a moderate posi­ tive correlation (r² = 0.47), emphasizing boron’s role in enhancing the speed of germination completion. overall, these findings suggested that boron effec­ tively improved germination performance, particular­ ly in terms of percentage and speed, while having minimal impact on uniformity and synchronization. correlation analysis the correlation matrix illustrates the relationships between different germination parameters of shallot seeds under varying boron concentrations. gp showed a strong positive correlation with gsi (r = table 2 ­ germination performance parameters of shallot seeds under different boron concentrations (x) different letters within the same column indicate statistically significant differences according to duncan's multiple range test. boron concentration (mg l­1) final germination percentage (%) x germination speed index x gemination time t10 (d) x gemination time t50 (d) x mean germination time (d) x 0 80.67 d 7.26 de 3.52 bc 5.19 bc 5.92 ab 10 82.00 d 7.42 de 3.39 bcd 5.57 ab 5.86 abc 20 82.00 d 8.10 d 3.34 bcd 4.94 bcd 5.22 bcdef 30 82.00 d 6.96 e 3.66 ab 6.08 a 6.3 0a 40 82.00 d 8.10 d 3.18 cde 4.67 cde 5.36 bcde 50 86.00 c 7.95 de 4.03 a 5.00 bcd 5.5 9bcd 60 88.67 c 9.65 bc 2.84 efg 4.34 de 4.89 def 70 88.67 c 9.40 c 2.82 efg 4.23 de 5.21 bcdef 80 92.67 b 10.52 ab 2.67 fg 4.23 de 4.65 ef 90 95.33 a 9.99 bc 2.95 def 4.41 cde 5.14 cdef 100 96.00 a 11.40 a 2.40 g 4.05 e 4.52 f p‐value <0.01 <0.01 <0.01 <0.01 <0.01 table 3 ­ metabolic and synchronization parameters of shallot seed germination under different boron concentrations (x) different letters within the same column indicate statistically significant differences according to duncan's multiple range test. boron concentration (mg l­1) mean germination rate (x) variance of germination time coefficient of variation of germination germination synchrony germination speed coefficient (x) 0 0.169 de 2.38 25.04 bc 0.24 16.95 de 10 0.170 de 2.11 24.73 bc 0.25 17.05 de 20 0.191 bcd 0.87 17.91 c 0.34 19.12 bcd 30 0.158 e 2.57 25.31 bc 0.22 15.88 e 40 0.187 cd 2.09 24.81 bc 0.25 18.79 cd 50 0.179 cde 1.04 17.42 c 0.39 17.98 cde 60 0.204 abc 1.74 26.31 abc 0.30 20.48 abc 70 0.192 bcd 3.05 32.00 ab 0.24 19.26 bcd 80 0.215 ab 1.06 22.27 bc 0.33 21.59 ab 90 0.194 bcd 4.07 36.78 a 0.29 19.48 bcd 100 0.220 a 1.64 27.81 abc 0.28 22.08 a p‐value <0.01 >0.05 <0.01 >0.05 <0.01 210 adv. hort. sci., 2025 39(3): 205­214 table 4 ­ regression analysis of parameters no. paramaters equation r2 1 final germination percentage (%) y = 78.6 + 0.165 0.84 2 germination speed index y = 6.77 + 0.040 0.76 3 time to 10% seed germinated y = 3.69 + 0.010 0.43 4 time to 50% seed germinated y = 5.55 ­ 0.015 0.47 5 mean germination time y = 5.99 – 0.013 0.45 6 mean germination rate y = 0.166 + 0.000466 0.47 7 variance of germination time y = 1.82 + 0.00482 0.01 8 coefficient of variation of germination time y = 21.4 + 0.0819 0.12 9 germination synchrony y = 0.27 + 0.000365 0.03 10 germination speed coefficient y = 16.6 + 0.0466 0.47 fig. 1 ­ regression analysis of each germination trait to boron concentration. zaiful et al. ‐ influence of boron as a priming agent 211 0.90), suggesting that higher germination percent­ ages are associated with faster germination rates. however, gp negatively correlates with t10 (r = ­ 0.64), t50 (r = ­0.69), and mgt (r = ­0.71), indicating that increased boron concentration reduces the time needed for germination (fig. 2). gsi also exhibited significant positive correlations with mgr (r = 0.93) and vgt) (r = 0.93), showing that faster germination is linked to higher consistency in seed performance. conversely, gsi has a strong neg­ ative correlation with mgt (r = ­0.91), emphasizing that a higher speed index leads to shorter germina­ tion times. t10, t50, and mgt parameters displayed high intercorrelations, with t10 and t50 showing r = 0.78 and t50 and mgt showing r = 0.87. parameters related to uniformity, such as vgt and gs, showed weaker and mostly non­significant correlations with the other considered parameters. 4. discussion and conclusions seed priming is a crucial technique in agriculture aimed at enhancing seed germination and seedling establishment under various stress conditions. by promoting pre­germination metabolic activity through controlled hydration and drying, seed prim­ ing can significantly improve germination rates and seedling vigor, helping plants to overcome abiotic stresses such as salinity and drought (chatterjee et al., 2018; hameed et al., 2025; macdonald and mohan, 2025). the positive impacts of seed priming are not limited to increasing germination and vigor, but also extend to improving crop yields and quality, making it an essential practice for sustainable agricul­ ture (gour et al., 2023; jarrar et al., 2024; zhang et al., 2025). the significant improvement in shallot seed ger­ mination parameters in response to increasing boron concentrations highlights boron’s essential role in seed physiology. the germination percentage increased by 15.33%, from 80.67% at 0 mg l⁻¹ to 96.00% at 100 mg l⁻¹, demonstrating boron’s positive impact on germination efficiency. this improvement is likely due to biochemical and physiological changes induced by priming, which enhance water uptake, activate metabolic pathways, and initiate early growth processes. bajwa et al. (2018) similarly found that priming wheat seeds increased alpha­amylase activity, facilitating carbohydrate breakdown and providing energy for embryo development. a compa­ rable enzymatic activation may contribute to the enhanced germination observed in shallot seeds. boron also accelerated germination times, as indi­ cated by the 40.45% reduction in time to 10% germi­ nation, a 33.39% decrease in time to 50% germina­ tion, and a 28.25% reduction in mean germination time. the shortest durations were recorded at 2.40 days (t10), 4.05 days (t50), and 4.52 days (mgt), all significantly lower than the control. these findings align with previous research by rehman et al. (2022), which demonstrated that boron priming accelerates the germination process. however, it is important to consider that at certain concentrations, boron can become toxic to plants (hussain et al., 2011; atique­ ur­rehman et al., 2020). additionally, boron priming enhanced the germi­ nation speed index. at 100 mg l⁻¹, the highest gsi (11.40) was recorded, whereas the control treatment had the lowest value (6.96). this suggests that boron plays a crucial role in expediting germination, likely due to its involvement in cell wall formation, mem­ brane integrity, and metabolic regulation. similar results were reported by farooq et al. (2011), who found that boron priming significantly increased the germination index of rice seeds. kaya and ergin (2023), in a study conducted on carthamus tinctorius seeds, also observed a linear increase in germination index with higher boron concentrations, reinforcing boron’s beneficial effects on seed germination. interestingly, the coefficient variation of germina­ tion time increased by 105.46%, reflecting improved fig. 2 ­ correlation analysis among parameters. synchronization of the germination process. this sug­ gests that boron not only accelerates germination but also promotes more uniform seedling establish­ ment. however, the lack of significant differences in germination synchrony and the stability of the germi­ nation speed coefficient indicate that boron’s impact is more pronounced in accelerating germination rather than reducing variability. chookhampa et al. (2023) also reported that seed priming on andrographis paniculata, sesamum indicum, and abelmoschus esculentus improves the germination performance, leading to more synchronized germina­ tion. the limited effect of boron on germination uni­ formity may be influenced by inherent genetic fac­ tors and environmental conditions. beyond improving germination rates and speed, priming has also been linked to enhanced seedling vigor and quality (biradar et al., 2023; faried et al., 2023). overall, this study demonstrates that increas­ ing boron concentrations positively influence germi­ nation percentage, speed index, and synchronization, while also accelerating the overall germination process. these findings underscore boron’s potential as a seed priming agent to enhance germination effi­ ciency and metabolic activity in shallot seeds. however, the effects observed are indeed influenced by multiple factors. the success of seed priming depends on the choice of priming agent, its concen­ tration, the duration of priming, and the specific response of the plant species or variety (siyar et al., 2020; corbineau et al., 2023; fu et al., 2024; jarrar et al., 2024). moreover, priming cannot overcome issues arising from genetic factors; seeds lacking in genetic purity, will result in poorly growing plants (reed et al., 2022). priming with 100 mg l­1 produced the most favor­ able outcomes on several germination parameters, such as final germination percentage, germination speed index, time required for 10% of seeds to ger­ minate, time required for 50% of seeds to germinate, mean germination time, mean germination rate, and germination speed coefficient. regression analysis showed a strong positive linear relationship between boron concentration and final germination percent­ age (r² = 0.84) as well as germination speed index (r² = 0.76). the study concludes that boron priming, par­ ticularly at 100 mg l­1, significantly improves germi­ nation performance in shallot seeds, highlighting its potential to enhance seedling vigor and productivity. the results of this research offer valuable insights for both shallot researchers and farmers. adv. hort. sci., 2025 39(3): 205­214 212 acknowledgements the authors express their sincere gratitude to the agrotechnology program for providing the necessary facilities and support for this research. we also extend our appreciation to colleagues and technical staff for their valuable assistance. references atabakia z.m., gherekhlooa j., ghaderi­fara f., ansaria o., hassanpour­bourkheilia s., pradob r.d., 2022 ­ the effect of environmental factors on seed germination and emergence of cut leaf geranium. ­ adv. weed sci., 41: 1­8. atique­ur­rehman, qamar r., hussain a., sardar h., sarwar n., javeed h.m.r., maqbool a., hussain m., 2020 ­ soil applied boron (b) improves growth, yield and fiber quality traits of cotton grown on calcareous saline soil. ­ plos one, 15: 1­13. bajwa a.a., farooq m., nawaz a., 2018 ­ seed priming 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malaysia. key words: identification, mycorrhizal, orchid. abstract: orchids are a diverse and widespread family of flowering plants, with over 25,000 known species and more than 100,000 hybrids and cultivars. orchids are characterised by their often showy and highly specialised flowers and have unique and intricate floral. orchids are known to be highly dependent on their mycorrhizal fungi for nutrient uptake, especially during the early stages of their development. orchid seeds lack the endosperm present in most other seeds, which means they cannot germinate without a source of nutrition. the relationship between orchids and mycorrhiza is known as orchid mycorrhizae or orchid mycorrhiza. in orchid mycorrhiza, the orchid plant forms a mutualistic relationship with certain species of fungi that are able to penetrate the orchid’s roots and colonise its tissues to provides the orchid with essential nutrients. orchid mycorrhizal fungi are often highly specific, meaning that they can only form partnerships with certain orchid species, and vice versa. the importance of mycorrhizal fungi in the orchid life cycle is crucial from both evolutionary and ecological standpoints. therefore, it is essential to acquire a thorough comprehension of this relationship and develop methodologies for isolating, identifying, and preserving significant fungal strains that are associated with different orchid species. in recent years, there has been a considerable increase in research concentration on mycorrhizal interactions in orchids. however, certain inquiries remain unresolved pertaining to the fungal communities associated with orchids as well as the divergences notices across different species and geographical locales. the present paper provides a through, and extensive analysis of the fungal life associated with orchids. this article presents a succinct overview of the molecular techniques utilised by researchers globally to isolate and identify peloton­forming fungi in both temperateterrestrial and tropical orchids. the review begins by proving a concise introduction to the background material regarding the wide range of fungal species that are linked with orchids. it then proceeds to explores the topic of orchid mycorrhizal fungi (omf) and orchid non­mycorrhizal fungi (onf). the subsequent analysis explores the crucial function that orchid mycorrhizal fungi play in the processes of seed germination and development. moreover, the study elaborates on the methodologies utilised for isolating fungi, extracting fungal dna, selecting primers, amplifying dna and subsequent analysis sequence data. this article considers several molecular identification approaches that are used in studying orchid endophytic mycorrhizal. using molecular approaches, orchid mycorrhizal can be further explored and identified. (*) corresponding author: azizun@ums.edu.my citation: shamsudin n.a., seelan j.s.s., gansau j.a., rusdi n.a., 2024 ­ a review: molecular identifica‐ tion of orchid mycorrhiza. ­ adv. hort. sci., 38(1): 97­116. copyright: © 2024 shamsudin n.a., seelan j.s.s., gansau j.a., rusdi n.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 july 2023 accepted for publication 6 november 2023 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-14952 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(1): 97­116 98 1. introduction the orchidaceae family is considered the second largest among flowering plants, with its size exceeded only by the asteraceae family (givnish et al., 2015). according to govaerts et al. (2017), the number of recognised orchid species is estimated to be 29,199. the decision to classify all orchids under appendices i and ii by the convention on international trade in endangered species of wild fauna and flora (cites) in 2017 effectively prohibited the illicit trade of these plants (hinsley et al., 2018). based on the evaluations made on a total of 1770 species of orchids, it has been determined that approximately 46.5% of these species are classified under the categories of vulnerability, endangered, or critical endangered as reported by the international union for conservation of nature (iucn, 2021). this precarious state remains due to a variety of variables, including their difficult germination process and human intervention, such as overcollection caused by economic and horticultural needs (pujasatria et al., 2020; suresh et al., 2023). orchids are extraordinarily important for biodiversity, conservation, and producing a vast array of therapeutic substances, nutritious foods, and ornamental plants (hinsley et al., 2018). orchid conservationists strive to manage market needs and biodiversity on a global scale, which would need large­scale production (pujasatria et al., 2020). numerous species encounter the peril of extinction; however, orchids adopt two distinct evolutionary strategies, namely sympodial growth and monopodial development, which are regulated by a diverse array of endophytic fungus species. these techniques serve to extend the longevity of orchids as herbaceous plants. (srivastava, 2018). orchid endophyte has a different way of penetrating and colonising their host, which makes them different from another fungal pathogen. for example, orchid fungi endophytes enter through stomata laterally in the anticlinal epidermal cell. they remain intracellularly in the shoot without colonising the cell. in contrast, pathogen fungi enter directly from the cell wall and typically grow extracellularly, potentially causing harm to the host (sarsaiya et al., 2019). diverse fungal taxa include mutualistic mycorhiza, endophytic fungi and considerably diverse as well as non­mycorrhizal fungal associates. the role of the root­allied fungi is not well understood. according to lee and yeung (2018), some of these fungi may supply organic carbon, nutrients, and water to the orchid, but the degree of this transfer is typically unknown. numerous report on specific mycorrhizal fungi also shows the ability to stimulate the embryo’s development and supply it with necessary nutrients, allowing the orchid seeds to germinate (liu et al., 2010; zhang et al., 2016; shao et al., 2017; herrera et al., 2019; shah et al., 2019; suresh et al., 2023). in recent years, the has been a significant transformation in the application of molecular techniques. the identification of fungi within roots has been accomplished through the application of polymerase chain reaction (pcr) techniques, employing fungal­specific primers (gardes and bruns, 1993). such methods have been used to characterize mycobionts of orchidaceae, taylor and bruns (1999) employed these techniques to characterise mycobionts of orchidaceae, thereby removing the laboratories process of culturing. the region that is most frequently studied is the nuclear ribosomal internal transcriber spaces (its). therefore, there is a want for molecular techniques capable of discerning distinct fungal species in cases where numerous fungal species are present in a single plant. the present review has provided an overview of the principal discoveries and methodologies utilised in the discipline, underscoring the significance of molecular techniques such as fungal dna extraction, primer selection, polymerase chain reaction (pcr), and high throughput sequencing (hts) in discerning the taxonomy of mycorrhizal fungi and elucidating the underlying molecular mechanisms that regulate these symbiotic relationships. furthermore, the utilisation of molecular method has provided researchers with enhanced capabilities to explore the extensive range of orchid mycorrhizal variety. the investigation has not only shown evolutionary relationship but has also yielded significant insights into ecological and conservation concerns. a thorough comprehension of mycorrhizal connections is essential for the efficient preservation of orchid species. in addition, the exploration of orchid mycorrhizal fungus in the fields of biotechnology and agriculture has resulted in the identification of new and important mycorrhizal fungi. in the context of identifying orchid mycorrhizal fungi, many methodologies are routinely applied, encompassing the isolation and cultivation of fungi, microscopic analysis and molecular studies. the ongoing refining shamsudin et al. ‐ molecular identification of orchid mycorrhiza 99 and improvement of these techniques play a crucial role in further our understanding and fascinating associations between orchids and their mycorrhizal fungus. 2. orchid and its fungi diversity orchids form a unique symbiotic relationship with the plant and animal species present in forest habitats in order to acquires nutrients, facilitate their own development, and facilitate the process of pollination. the mycorrhizal fungi, which exhibit symbiotic germination, are if significant importance in facilitating embryo development and supply vital nutrients within the natural environment. this symbiotic relationship is crucial in the effective germination of orchid seeds (liu et al., 2010; herrera et al., 2019). fungi have a crucial role as the principal provider of essential nutrients for developing orchidaceae plants, especially in setting characterised by low nutrients availability (long et al., 2022). orchids interact with a smaller number of mycorrhizal fungi than other mycorrhizal plants, with greater specificity for orchid mycorrhizal fungi than ectomycorrhizae, arbuscular mycorrhizae, and even ericoid mycorrhizal fungi. in symbiotic connection, fungi provide plants with water and mineral nutrients (especially phosphorus) while protecting them from biotic and abiotic stresses. in exchange, plant hosts provide carbon from photosynthesis to the fungi (rasmussen, 1995; tedersoo et al., 2017). many orchid species cannot commence germination or grow without their compatible symbiotic fungus (rasmussen, 1995; davis et al., 2015; fay, 2018; attri, 2022), as their specificity of mycorrhizal connections that permit in situ symbiotic seed germination in orchids is frequently so rigorous. the aforementioned circumstance has stimulated inquiries into the importance of fungus in symbiotic relationships that are equally crucial and beneficial for the ex‐situ preservation of orchids, specifically in the context of reintroduction endeavours. the first recorded evidence of a mycorrhizal fungus in an orchid may be traced back to the year 1824, as documented by the renowned german naturalist heinrich link. nevertheless, the specific function of the fungus remained ambiguous until the early 1900s when nöel bernard established a scientific correlation between filamentous fungi and the process of seed germination (arditti and pridgeon, 1997). following this, in the early 1900s, the study of orchid endophytes emerged as a significant area of interest within the field of orchid biology research. chand et al. (2020) conducted a comprehensive investigation wherein they isolated and identified many orchid endophytes, and thoroughly evaluated their probable role in orchid symbiosis. orchids frequently establish symbiotic relationships with fungus that display substantial evolutionary and ecological variability. epiphytic orchids exhibit a prevalence of both basidiomycota and ascomycota in their aerial roots as well as subterranean roots or rhizomes, while chytridiomycota, glomeromycota, zygomycota, or mucoromycota are present in comparatively smaller quantities (waud et al., 2014; cevallos et al., 2017; egidi et al., 2018; novotná et al., 2018). the classification of orchid fungus is determined by the existence or absence of functional pelotons within cortical cells, leading to the categorization of orchid mycorrhizal fungi (omf) or orchid non­mycorrhizal fungi (onf) (li et al., 2021). 3. orchid mycorrhizal fungi (omf) and orchid non­ mycorrhizal fungi (onf) the phenomenon referred to as “orchid mycorrhiza” pertains to the symbiotic relationship established between the orchid plant and many fungal species that are capable of cohabiting within its root system. the germination of an orchid mycorrhizal fungus (omf), and these seeds rely on one or more omf’s for sustenance during their whole life (bidartondo and read, 2008). within the realm of fungi, a subset of these organisms can be classified as transient, denoting their inability to maintain a sustained presence within the developing and maturing tissues of orchids. conversely, there exist other fungi that establish more long­lasting associations with these plants. according to lee and yeung (2018), during the maturation process of orchids, specific fungi that play a role in facilitating germination persist as “permanent residents” whereas other fungi initiate germination and are subsequently replaced by different fungal partners. the identification of coiled pelotons within cortical root cells is recognised as a characteristic feature of orchid mycorrhizal fungus (omf), as examiner in research undertaken by dearnaley et al. (2016) as well as rasmussen (1995). in contrast, adv. hort. sci., 2024 38(1): 97­116 100 orchid mycorrhizal fungi (onf) pertain to a distinct classification of endophytic fungi that reside within the roots or other tissues or orchids at specified phases of their l ife cycle. nevertheless, it is imperative to elucidate that oligotrophic nitrogen­ fixation bacteria (onfs) are devoid of peloton­like structures and do not elicit any noticeable pathogenic consequences in the host plants. the aforementioned phenomenon has been emphasised in scientific inquiries conducted by sisti et al. (2019) and selosse et al. (2018). several investigations, like those conducted by herrera et al. (2019) and waterman et al. (2011), have shown empirical evidence indicating the involvement of certain orchid mycorrhizal fungi (omfs) in the process of decomposition. the observed mycorrhizal fungi (omfs) have been documented to facilitate the decomposition of nearby substrates and provide essential nutrients to orchids. it is important to acknowledge that specific obligatory mycoheterotrophic fungi (omfs) may have experienced evolutionary shifts from ancestral obligate non­photosynthetic fungus (onfs), gradually developing mycorrhizal capacities. the aforementioned phenomenon has been thoroughly investigated in scholarly studies conducted by selosse et al. (2018) and wang et al. (2021). the classification of orchid mycorrhizal fungus (omf) has a wide range of fungal species, consisting of at least 17 families from the basidiomycetes group and five families or genera from the ascomycetes group, as documented by dearnaley (2007) and dearnaley et al. (2012). within this set, there are several noteworthy groups, namely ceratobasidiaceae (cantharellales), tulasnellaceae, and serendipitaceae, which were previously referred to as the sebacinales clade b. the classification of these groupings as rhizoctonia­type basidiomycetes is largely acknowledged in the scientific community, as evidenced by multiple research (rasmussen, 1995; bayman and otero, 2006; sisti et al., 2019; selosse et al., 2018; jędryczka et al., 2023). basidiomycetes and ascomycetes, which are widely distributed in terrestrial ecosystems and cultivated plants globally, have notable associations with orchids (trivedi et al., 2020; wang et al., 2019). the significance of orchid mycorrhizal fungi (omf) in specific microenvironments cannot be understated, as they play a crucial role in promoting the germination of orchid seeds and the subsequent growth of orchid seedlings. as a result, geographical areas that display a significant occurrence of orchid mycorrhizal fungi (omf) tend to showcase a higher range of orchid species, as documented by li et al. (2021). in their study, hemrová et al. (2019) conducted germination tests and developed species distribution models that integrated multiple habitat parameters. the results of their study emphasized the crucial significance of fungal symbionts in influencing the spatial distribution of orchids on a large geographical scale. furthermore, mccormick et al. (2019) and other scientific inquiries have provided substantial data supporting a strong and positive association between the prevalence of mycoheterotrophic orchids, which depend on fungi for nourishment, and the existence of omf. the cumulative evidence suggests that omf has a significant role in shaping the population dynamics of orchids. 4. orchid mycorrhizal and its roles in seed germination and development in general, asymbiotic or symbiotic procedures can be used to germinate orchid seeds (yam and arditti, 2009). it has been demonstrated that asymbiotic seed germination is an effective method for producing plantlets of numerous orchid species for both commercial and conservation. it was believed that root orchid mycorrhizal fungi are the actual source of seed­germinating orchid mycorrhizal fungi (rasmussen, 1995). root fungal endophytes are seen as advantageous plant residents that may increase their productivity and eventually support ecological functions. roots of mature plants have provided fungi that have been isolated and tested, with several successes have been attained employing these fungi (nontachaiyapoom et al., 2011; sebastián et al., 2014). the in situ/ex‐situ seed baiting technique has been increasingly popular in recent years as a means of obtaining efficacious fungi that facilitate seed germination. according to previous studies conducted by zhou and gao (2016) and rasmussen and whigham (1993), it has been observed that fungus obtained from naturally occurring protocorms or seedlings possess the capacity to induce seed germination and facil itate the subsequent development of seedlings (table 1). shao et al. (2020) conducted a conservation project with the objective of protecting dendrobium species shamsudin et al. ‐ molecular identification of orchid mycorrhiza 101e= epiphytes; t= terrestrial. table 1 a ­ list of orchid mycorrhizal fungi that had been identified and their roles in orchid micropropagation orchid fungi sp. roles references vanda wightii (e) ceratobasidium sp. seed germination suresh et al. (2023) paphiopedilum barbigerum (t) epulorhiza sp. seed germination and seedling development tian et al. (2022) serapias vomeracea (t) tulasnella calospora seed germination and seedling development ghirardo et al. (2020) epidendrum secundum (t) ceratobasidium sp., sebacina vermifera seed germination and seedling development durán­lópez et al. (2019) dactylorhiza majalis (t) piriformospora indica seed germination shah et al. (2019) chloraea gavilu (t) tulasnella sp. seed germination herrera et al. (2017) aerides multiflora (e) ceratobasidium sp. seed germination bhatti et al. (2017) dendrobium friedericksianum (e) tulasnella sp., seed germination and seedling development agustini et al. (2016) tulasnellaceae rigidoporus vinctus, polyporales ceratobasidium sp., tulasnellaceae flavodon flavus, polyporales nigroporus vinosus, polyporales coriolopsis retropicta, polyporales valsa eugeniae, diaporthales. paphiopedilum villosum (e) tulasnella sp., seed germination khamchatra et al. (2016) tulasnellaceae dendrobium lancifolium (e) rhizoctonia sp. seed germination agustini et al. (2016) liparis japonica (t) rhizoctonia sp. seed germination ding et al. (2014) dendrobium aphyllum (e) tulasnella sp., seed germination zi et al. (2014) trichoderma sp. dendrobium aphyllum (e), dendrobium devianum (e), and cymbidium manni (e) tulasnella sp., epulorhiza sp. seedling growth zi et al. (2014) dendrobium officinal (e) tulasnella sp. seed germination and seedling growth ming et al. (2014) dendrobium nobile (e), dendrobium chrysotoxum (e), dendrobium falconer (e), dendrobium aphyllum (e) xyalariaceae sp. seed germination chen et al. (2013) dendrobium crumenatum (e) guignardia endophyllicola seed germination mangunwardoyo et al. (2011) pecteilis susannae (l.) epulorhiza sp. seed germination and development chutima et al. (2011) 102 adv. hort. sci., 2024 38(1): 97­116 e= epiphytes; t= terrestrial. table 1 b ­ list of orchid mycorrhizal fungi that had been identified and their roles in orchid micropropagation orchid fungi sp. roles references dendrobium nobile (e) leptodontidium seedling development hou and guo (2009) cymbidium eburneum (e) alternaria sp., chaetomium sp, vegetative growth zhao and liu (2008) fusarium sp. gastrodia elata (t) mycena osmundicola seed germination kim et al. (2006) cymbidium goeringii (t) rhizoctonia sp seedling development jianrong et al. (2005) gastrodia elata (t) mycena osmundicolor seed germination hong et al. (2002) paphiopedilum armeniacum (t) phacodium sp. seedling development ming and zhou (2001) cypripedium reginae (t) fusarium sp. seed germination warcup (1981) dendrobium discolor (e), tulasnella cruciate, seed germination warcup (1981) tulasnella irregularis, tulasnella allantospora calochilus sp. (t), tulasnella asymmetrica, seed germination warcup (1981) diuris maculata sm. (t), tulasnella cruciate, spiranthes sinensis (t) tulasnella irregularis, tulasnella violea, tulasnella allantospora diuris sulphurea. r.br. (t) tulasnella asymmetrica seed germination warcup (1981) orthocersa strictum (t) tulasnella asymmetrica, seed germination warcup (1981) tulasnella cruciate, tulasnella irregularis, tulasnella violea thelymitra ixoides (t) tulasnella asymmetrica, seed germination warcup (1981) tulasnella cruciata thelymitra flexuosa (t) tulasnella irregularis, seed germination warcup (1981) tulasnella cruciata thelymitra media (t) tulasnella violea, seed germination warcup (1981) tulasnella asymmetrica thelymitra carnea (t) tulasnella allantospora, seed germination warcup (1981) tulasnella violea shamsudin et al. ‐ molecular identification of orchid mycorrhiza 103 that have been excessively harvested. they effectively isolated and obtained fungi that enhance germination for several dendrobium species using the seed baiting approach, as described by huang et al. (2018). 5. fungal dna extraction there are various methodologies commonly employed for the isolation of orchid mycorrhizal fungus from orchid plants. these methodologies encompass the isolation of complete tissue or tissue segments, in situ seedings, trapping isolation, and isolation from a solitary peloton. among these methods, the technique of isolating a single peloton, which involves micromanipulation­based isolation from host cells, is widely regarded as the most reliable and precise approach for extracting endophytic mycorrhizal fungi (zettler et al., 2003; batty et al., 2006; zi et al., 2014; zettler and corey, 2018). the prevailing conventional method for molecular identification of orchid mycorrhizal fungus generally entails the extraction of dna from agar plates or liquid cultures, as opposed to direct extraction from orchid roots (zettler and corey, 2018). the fungal cell wall primarily consists of around 80­90% polysaccharides, inorganic ions, lipids, polyphosphates, and proteins, which together form the matrix that binds the wall. this type of cell wall also can be characterized by microfibrillar components like chitin, β­glucan, and/or cellulose, which pose challenges in dna extraction (turzhanova et al., 2018). moreover, the presence of a substantial quantity of secondary metabolites, such as melanin, can impede subsequent reactions (fernandez et al., 2016; janowski et al., 2019) this become a major challenge in dna extraction of fungi as it has a robust cell walls that are resist to lysis method (jiang et al., 2011). the isolating nucleic acids from fungi, often necessitates the incorporation of additional lysis steps, which can include enzymatic lysis, mechanical homogenization, sonication, or the use of potentially harmful chemicals (turzhanova et al., 2018). dna samples were gathered over a period of 15 years, during which a diverse range of extraction procedures were utilised to extract fungal dna. nevertheless, the extraction of dna from the various types of fungi encountered does not have a universally optimised approach. the standard procedure for the extraction of fungal dna typically encompasses several sequential stages. these stages involve the cultivation of fungi in either liquid or solid growth media, disruption of the fungal cell wall, elimination of proteins using phenol and chloroform, and subsequent isolation of dna through precipitation with ethanol or isopropanol (faggi et al., 2005). even though the presence of polysaccharide and polyphenolic compound in the fungi may inhibit the activity and effect of dna polymerase, but they can be easily removed by either using a vacuum or spin column and by mixing the sample with bovine serum albumin (bsa), β­mercaptoethanol (βме), n­ trimethyl ammonium bromide (ctab) and polyvinylpyrrolidone (pvp) (tripathy et al., 2017). a variety of methodologies have been devised to isolate dna from fungal tissues, and the most efficacious dna extraction procedures frequently integrate physical methodologies (such as microwave treatment, freeze/thaw cycles, homogenization using glass beads, and grinding in liquid nitrogen) with enzymatic approaches (including gluconases, chitinases, and proteases) (zhang et al., 2010). the exists variety of ways for extracting dna and among them, the ctab approach (gardes and bruns, 1993) is frequently utilised. additional alternatives for fungal genomic dna isolation kits are the omega fungal e.z.n.a kit (manufactured by omega biotech, doraville, ga, usa), the qiagen plant dneasy kit, genomic tip kits (qiagen, valencia, cam usa), or sangin biotech rapid fungi genomic dna isolation kits (long et al., 2022). in order to ascertain the effectiveness of a dna extraction technique, it is important to evaluate both the quality and quantity of the dna obtained. the concentration of dna in the samples was assessed by employing spectrophotometry at wavelength of 260 nm, with measurement expressed in units of nanograms per microliter (ng/µl). in addition, the assessment of dna purity was conducted by determining the a260/a280 ratio and a260/280 ratio utilising either a uv­vis spectrophotometer or nanodrop devise (thermo electron scientific instruments llc, usa). generally, the a260/a280 ratio exceeded 1.8 suggesting that the dna was largely devoid of proteins. in terms of the a260/a230 ratio, if it was approximately 2, that indicate the samples did not contain significant impurities such as carbohydrates, peptides, phenols, salts, or aromatic compounds (turzhanova et al., 2018). furthermore, the quality of the dna also can be evaluated through adv. hort. sci., 2024 38(1): 97­116 104 electrophoresis after pcr amplification of the genomic dna, using gene­specific primers (tripathy et al., 2017). the standard ctab phenol­chloroform extraction procedure has proven effective across a wide range of species (strugnell et al., 2006; reineke et al., 1998) and produce a high purity of dna (zettler and corey 2018). study by turzhanova et al. (2018) on optimization of dna extraction methods of fungi has shown that ctab­method and dneasy plant mini kit (qiagen) resulted a highest dna quality, while sds method resulted in the lowest sample yields and quality. however, ctab­method uses toxic chemicals and requires a significant amount of bench time, both limiting its applicability when scaling up for big comparative research (schiebelhut et al., 2017). nowadays, commercial dna extraction kits are more desirable since they reduce exposure to toxic chemicals and allow for faster extraction periods. these kits could offer a range of low­ to high­ throughput processing, vary in price from quite inexpensive to highly costly, and may require some specialist gear. table 2 below shows a l ist of extraction methods and kits used in the extraction method of dna orchid fungi. a large percentage of orchid mycorrhizal fungi are mycelia sterilia. conventional techniques have led to a paraphyletic taxonomy in which unrelated fungi are grouped together, requiring molecular techniques for accurate identification, phylogenetic inference, and genetic relatedness (sen et al., 1999; otero et al., 2002; shan et al., 2002; yagame et al., 2008). molecular sequencing, microscopic examination, and biochemical analysis were among the most used methods to identify mycorrhizal fungi. for fungi identification by morphological characterisation, it table 2 ­ types of manual dna extraction protocol used to extract mycorrhizal fungi dna * modified method. protocol name abbreviation chemistry/mechanism kits/supplies required dna extraction time references cetytrimethyl ammo­ nium bromide (ctab)­ phenol­chloroform ctab ctab lysis, followed by phenol chloroform purification step all reagents are made in­house 1 hour 30 min sambrook et al. (2001); dawson et al. (1998) sodium dodecyl sulfate sds lysis, followed by phenol chloroform purification step sds and mercaptoetha­ nol lysis, followed by chloroform purification step all reagents are made in­house 1 hour 5 min turzhanova et al. (2018) phenol chloroform isoamyl alcohol extraction method pci buffer lysis. followed by phenol/chloroform /isoamyl alcohol purification step all reagents are made in­house 2 hour 10 min varma and kwon chung (1991) ezna sp fungal dna omega fungal ezna silica based purification system omega fungal ezna kit (omega biotek, doraville, ga, usa) 45 min omega (2019) qiamp mini kit (qiagen) qiaamp mini kit silica based purification system qiamp mini kit (qiagen) 35 min turzhanova et al. (2018) fungi/yeast genomic dna isolation (norgen)* fungi/yeast genomic dna isolation silica based purification system fungi/yeast genomic dna isolation kit (norgen) more than 2 hours kumar and mugunthan (2018) shamsudin et al. ‐ molecular identification of orchid mycorrhiza 105 can be conducted alone or in combination with molecular analysis, and usually, most research will use both combination methods in identifying mycorrhizal fungi. however, the orchid fungus is notoriously difficult to be determined at the species level because they do not sporulate readily on cultures (boddington and dearnaley, 2008; ko et al., 2011; ma et al., 2015). 6. primer selection for fungal amplification after the completion of dna extraction from orchid mycorrhizal fungi, the subsequent step involves the amplification of fungal dna through the utilisation of a polymerase chain reaction (pcr) technique. this amplification process necessitates the use of primers that are specifically designed to target the ribosomal dna (rdna) region. the rdna cluster consists of several components, including 18s rdna, 5.8s rdna, 28s rdna, the external transcribed spacer (ets), and internal transcribed spacer 1 and 2, which are generally referred to as its1 and its2. the utilisation of the its region for molecular identification of fungi can be traced back to the early 1990s, as shown by horton and bruns (2001) and seifert (2009). the utilisation of the its region for molecular identification is of great significance in fungal identification, principally owing to the inclusion of two remarkably variable spacers, namely its1 and its2, which frequently exhibit species­specific characteristics either independently or in conjunction. moreover, it includes the 5.8s gene, which is renowned for its exceptional level of conservation. the high degree of sequence conservation observed in the adjacent genes, along with their designation as the region undergoing the most rapid evolution and the existence of multiple copies of the ribosomal operon, facilitates the efficiency of primer design and pcr amplification for the its region (bengtsson palme et al. , 2013; fajarningsih, 2016; raja et al., 2017). these two spacers are copied from the ribosomal dna, and when the ribosomal rnas complete, they are removed from the rrnas. since the spacers are not used in the final structure of the ribosome, they are not strongly selected against mutations. therefore, the identification of mycorrhizal fungus is considered efficient by using a region­specific to eukaryotes (tedersoo and nilsson, 2016). the nuclear ribosomal rna genes, including the small subunit (ssu) (18s) and large subunit (lsu) (28s) are commonly utilised in scientific investigations pertaining to aquatic fungus and arbuscular mycorrhizal fungi. nevertheless, in the case of ascomycetes and basidiomycetes, these markers generally offer taxonomic insights primarily at level beyond the species, and occasionally at the genus level. this problem is caused by the fact that the ssu and lsu sequences of the many species that belong to these fungal groupings have only minute to nonexistent differences between them. because of this, precise distinction becomes a challenging obstacle. according to the findings of the research carried out by nilsson et al. (2019), the ability of ssu, lsu, and protein­coding genes like the rna polymerase gene rpb2 to be aligned across different fungal phyla is a significant benefit offered by these types of genes. this makes it possible to analyse large­scale phylogenetic relationships at the phylum and order levels, which is something that the its region normally has difficulty accomplishing without very identical reference sequences (větrovský et al., 2016). because the its region often ranges in length from 500­700 bases, the majority of high­throughput sequencing (hts) studies concentrate on the shorter its1 or its2 subregions, which typically range in length from 250­400 bases. this constraint is the result of the fact that the its region is normally between 500­700 bases in length. according to tedersoo et al. (2015), the its2 subregion in particular stands out due to the fact that it exhibits lesser length fluctuations and more universal primer sites. this, in turn, results in reduced taxonomic bias. the its1 and its2 subregions have demonstrated their suitability for second generation high­ throughput sequencing (hts) techniques. however, third generation methodologies, such as those utilising pacbiosciences (pacbio) and oxford nanopore platforms, provide the ability to target the complete its region, as well as segments or even the entire adjacent rrna genes (nilsson et al., 2019). targeting the entire internal transcribed spacer (its) area, rather than its subregions has several advantages, including improved taxonomic accuracy and less amplification of non­viable organism. nevertheless, one limitation of this methodology is its reduced efficacy when utilised on materials of subpar quality, such as ancient herbarium specimens, which degrade to a degree where doing its dna sequencing becomes impractical (tedersoo et al., adv. hort. sci., 2024 38(1): 97­116 106 2017). according to study conducted by nilsson et al. (2019), it is recommended to allocate a significant amount of effort to the analysis and selection of primers, this is due to the fact that only a limited number of primers have the capability to amplify over 90% of fungal groups. additionally, the process of primer selection necessitates meticulous examination of the target taxa, as highlighted by tedersoo et al. (2015). the following table 3 and 4 show an illustrative depiction of its primers together with their corresponding sequences. 7. identified fungal from orchid root by using internal transcribe region gardes and bruns (1993) and white et al. (1990) have produced well recognised primers in the field of fungal ecology for species­level identification based on sequencing. these primers, namely its1, its2, its3, its4, its1f, its86f, and cnl2f, are considered to be broad­spectrum primers. the its1 and its4 primers are commonly employed as standard primers in numerous laboratories (fajarningsih, 2016). the list of endophytic fungi that have been isolated and identified from orchid roots is presented in table 5. this was accomplished by employing a broad­ spectrum primer (its1 and its4). however, some primers are designed to be specific. for example, the its86f primers are used primarily for medically important fungal pathogen, but they are rarely used in mycorrhizal identification, especially fungi communities from environmental samples. in orchid mycorrhizal fungi identification, the its1­f is one of the most effective primers for the its region amplification, especially for eumycota. for example, the primer its1­f and its4 always used in pair to identified an fusarium sp. as in the study by sukarno et al. (2023), where they manage to identified several species of fusarium using this primer combination. the its1­f and its4­b primer were designed to be specific basidiomycetes (gardes and bruns, 1993). besides that, both primers can minimise plant sequence amplification (taylor and mccormick, 2008). however, this primer is ineffective in amplifying some species of tulasnellaceae that belong to basidiomycota phylum, as their nuclear ribosomal is evolving rapidly and some primers are typically conserved along the eumycota are not maintained in tulasnellaceae table 3 ­ list of recommended primer for identification of orchid mycorrhizal primer sequence (5’­3') references modified its1ngs tccgtaggtgaacctgc oja et al. (2014) modified its1fngs ggtcatttagaggaagtaa oja et al. (2014) modified its4ngs tcctscgcttattgatatgc oja et al. (2014) its4tul2 ttcttttcctccgctgawta oja et al. (2014) tw14ngs ctatcctgrgrgaaayttc tedersoo et al. (2014) fits9 gaacgcagcraaiigyga ihrmark et al. (2012) gits7 gtgartcatcgartctttg ihrmark et al. (2012) fits7 gtgartcatcgaatcttc ihrmark et al. (2012) its4tul ccgccagattcacacattga taylors and mccormick (2008) its1­of aactcggccatttagaggaagt/aacttggtcatttagaggaagt taylors and mccormick (2008) its4­of gttactaggggaatccttgtt taylors and mccormick (2008) its86f gtgaatcatcgaatctttgaa turenne et al. (1999) its1f cttggtcatttagaggaagtaa gardes and bruns (1993) its4b caggagacttgtacacggtccag gardes and bruns (1993) its1 tccgtaggtgaacctgcgg white et al. (1990) its2 gctgcgttcttcatcgatgc white et al. (1990) its3 gcatcgatgaagaacgcagc white et al. (1990) its4 tcctccgcttattgatatgc white et al. (1990) ns7 gaggcaataacaggtctgtgatgc white et al. (1990) cnl2f gtttcccttttaacaatttcac white et al. (1990) 107 table 4 ­ list of recommended primer pair for sequencing of orchid mycorrhizal partners primer pair primer name (forward/ reverse) sequence (5’­3') target region annealing temp (oc) target clade (orchid specific group fungi) references its1/its4 its1 (f) tccgtaggtgaacctgcgg its1/its2 53 all basidiomycota white et al. (1990) its4 (r) tcctccgcttattgatatgc its1/ its4­ tul its1 (f) tccgtaggtgaacctgcgg its1/its2 54 tulasnella taylor and mccormick (2008) its4­tul ccgccagattcacacattga its1­of/its4­of its1­of (f) aactcggccatttagaggaagt its1/its2 60 all basidiomycota taylor and mccormick (2008) its1­of (f) aacttggtcatttagaggaagt its4­of (r) gttactaggggaatccttgtt ssu1318­tom/lsu­tom4 ssu1318­tom (f) cgataacgaacgagaccttat ssu/lsu 62 thelephoraceae taylor and mccormick (2008) lsu­tom4 gccctgttccaagagactta its86f/its4 its86f (f) gtgaatcatcgaatctttgaa its­2 59 both ascomycota and basidiomycota white et al. (1990) its4 (r) tcctccgcttattgatatgc (including some tulasnella) its3/its4of its3 (f) gcatcgatgaagaacgcagc its­2 62 all basidomycota white et al. (1990) its4of (r) gttactaggggaatccttgtt taylor and mccormick (2008) 5.8s­tulngs/its4­tul2 5.8s­tulngs cattcgatgaagaccgttgc its­2 57 all basidiomycota (inc. serendipitaceae and rammitsu et al. (2021) its4­tul2 ttcttttcctccgctgawta tulasnellaceae) oja et al. (2014) ns7/its1of­rc ns7 (f) gaggcaataacaggtctgtgatgc ssu 62 some ascomycota and basidiomycota (including some tulasnella) white et al. (1990) its1of­rc­g (r) acttcctctaaatggccgagtt waud et al. (2014) its1of­rc­a (r) acttcctctaaatgaccaagtt waud et al. (2014) its1of/its2 m its1­of (f) aactcggccatttagaggaagt its­1 62 some ascomycota and basidiomycota (including some tulasnella) taylor and mccormick (2008) its1­of (f) aacttggtcatttagaggaagt its 2 m tcgctgcgttcttcatcga its1f/its2 its1f (f) cttggtcatttagaggaagtaa its­1 62 both ascomycota and basidomycota (including gardes and bruns (1993) its2 (r) gctgcgttcttcatcgatgc some tulasnella) white et al. (1990) its4of­rc/cnl2f its4of­rc (f) aacaaggattcccctagtaac lsu 59 some ascomycota and basidiomycota (including waud et al. (2014) cnl2f (r) gtttcccttttaacaatttcac some tulasnella) white et al. (1990) shamsudin et al. ‐ molecular identification of orchid mycorrhiza adv. hort. sci., 2024 38(1): 97­116 108 table 5 ­ list of endophytic fungi that has been isolated and identified from root by using a broad­spectrum primer (its1 and its4) orchid species endophytic fungal (accession no./taxonomic affiliation) type of primer country references vanda wightii ceratobasidium_wyd1 (mw59578) its1 and its4 india suresh et al. (2023) dendrobium longicornu alternaria sp. (mn256650), its1 and its4 nepal shah et al. (2022) cladosporium sp. (mn256649), coniochaeta sp. (mk225602), penicillium sp. (mn256653), fusarium sp. (mn256645), fusarium sp. (mn256647), fusarium sp. (mn256646). aerides rosea tulasnellaceae sp. (jf691200) its1 and its4 china zhao et al. (2021) dendrobium nobile tulasnella deliquescens (lc175331) its1 and its4 china zhao et al. (2021) dendrobium cucullatum tulasnella sp. strain sscdo­4 (mh348613 its1 and its4 china zhao et al. (2021) epigeneium amplum tulasnella sp. 140 (ay373281) its1 and its4 china zhao et al. (2021) gastrochilus calceolaris ceratobasidium sp. gc (gq369961), its1 and its4 bangladesh hossain (2019) ceratobasidium sp. fpub 168 (ef536969), rhizoctonia sp. abn1b (aj318432), rhizoctonia sp. onv6 (aj318436) aerides multiflora ceratobasidum sp. (jx913820), ceratobasidum sp. (jx913820), its1 and its4 india bhatti et al. (2017) paphiopedilum villosum (lindl.) stein. tulasnella sp. (ay373281)/tulasnellaceae rigidoporus vinctus (hq400710)/ polyporales ceratobasidium sp. (hm117643)/tulasnellaceae flavodon flavus (jq638521)/polyporales nigroporus vinosus (ab811859)/polyporales coriolopsis retropicta (kc867403)/polyporales valsa eugeniae (ay347344)/diaporthales its1 and its4 thailand khamchatra et al. (2016) aerides multiflorum ceratobasidium sp. (eu605733) its1 and its4 western himalayas hossain et al. (2013) rhynchostylis retusa ceratobasidium sp. (eu605732) its1 and its4 western himalayas hossain et al. (2013) pecteilis susannae (l.) epulorhiza sp. gq856216 its1 and its4 thailand chutima et al. (2011) epulorhiza sp. gq856215 epulorhiza sp. gq856214 fusarium sp. gq862347 epulorhiza sp. fj882028 epulorhiza sp. gq862346 epulorhiza sp. fj940903 epulorhiza sp. fj873174 shamsudin et al. ‐ molecular identification of orchid mycorrhiza 109 (taylor and mccormick 2008). to address this issue, the its4­tul primer has been designed to study only tulasnella species, thereby minimising the amplification of other taxa. two primers that are tulasnellla specific which is its4­tul and its4r are designed from the 3­end of its2 (suárez et al., 2006). the its4­tul primer is a perfect or near­perfect match for some of the core species of tulasnella but their mismatches with the majority of other fungi make them a specific primer. its4­tul has been used widely as a primer, especially for the identification of orchid mycorrhizal primarily targeted tulasnellaceae, which are mostly reported to have the ability to promote seed germination (oja et al. , 2014; mccormick et al., 2021; suetsugu et al., 2021). meanwhile, its1­of and its4­of is nowadays are increasingly used in characterising orchid fungal symbionts as they are designed to be a broad spectrum basidiomycete specific primer (currah and sherburne, 1992; taylor and mccormick, 2008; jacquemyn et al., 2010). a study on identification of fungi identification of terrestrial orchid mycorrhizal by using broad spectrum fungal taxa primer (its86f/its4) by waud et al. (2014) has outperformed the other primer pair. the study also assessed the efficacy of several type of broad­spectrum primer and specific primer for orchid mycorrhizal fungi to understand and characterized orchid mycorrhizal communities and suggested several suitable primer pairs. other study also uses the broad­spectrum primer pair its86f/its4 to investigate the orchid mycorrhizal community in both epiphytic and terrestrial orchid (cevallos et al., 2017; johnson et al., 2021). however, the use of broad­ spectrum primer for identification of orchid mycorrhizal fungi is constrained by a primer bias, which arise from the inability of the primer to identify a specific fungus within a sample due to the mismatch during pcr. while tulasnellaceae fungi are commonly associated with orchids (dearnaley et al. 2012), their molecular detection poses challenges due to mismatches with universal fungal primers (suárez et al., 2006; taylor and mccormick 2008; waud et al., 2014; rammitsu et al., 2021). moreover, previous comprehensive investigations conducted through sanger sequencing­based methodologies have indicated distinctions between the mycorrhizal communities associated with epiphytic orchids and those associated with terrestrial orchids (martos et al., 2012; xing et al., 2019). the utilising of tulasnellaceae­specific primers for the assessmen of orchid mycorrhiza; networks by metabarcoding analysis is highly recommended, particurlay in the context of epiphytic orchids, as emphasised in the research conducted by rammitsu et al. (2021). the commonly used broad spectrum primer, its86f/its4 effectively identified ceratobasidiaceae and serendipitaceae fungi but proved inadequate in detecting the diversity of tulasnellaceae fungi (rammitsu et al., 2021). due to significant primer biases present within the tulasnellaceae family, which plays a crucial role as mycorrhizal symbionts in the majority of orchid species, it is imperative to exercise caution in selecting primers and thoroughly assess potential biases (oja et al., 2014). 8. sequencing when it comes to fungi, morphology is often the method of choice for performing the fundamental function of species distinction. however, distinguishing species based on their morphology can be difficult, particularly for fungi that do not have complex fruiting bodies, as is the case with the three families of rhizoctonia species that are linked with orchids (gardes and bruns, 1993). conventionally, it has been thought that the ‘rhizoctonia’ complex, which includes species from three different fungal families (tulasnellaceae, ceratobasidiaceae, and serendipitaceae), makes up the bulk, if not the entirety, of orchid mycorrhizal fungus. however, recent research suggests that this may not be the case. septal ultrastructure is a defining characteristic that separates the various clades within rhizoctonia (currah and sherburne, 1992), but careful inspection is still required to distinguish sebacinaceae and tulasnellaceae (andersen, 1996). this problem is compounded by the fact that when the cryptic, resupinate fruiting structures are seldom observed. basidial morphology offers suitable identification of orchid­associated rhizoctonia species at the morphospecies level (warcup and talbot, 1967). however, orchid isolates are rarely induced to fruit in culture as some fungi cannot be produced in artificial circumstances (currah et al., 1990). in order to expand knowledge of fungal variety, culture­independent technologies (sequencing and cloning) have been created. morphological identification methods are conventional identification method that involves evaluating the adv. hort. sci., 2024 38(1): 97­116 110 morphological and microscopic features of fungi on different culture media and under different conditions. this method can be accompanied by other identification methods to help identify fungi more accurately. other methods, such as microscopic examination or biochemical screening, can be performed alone or in conjunction with molecular analysis. with the recent development of advanced molecular techniques (e.g., next­generation sequencing), the spectrum of fungi discovered at the species level has expanded significantly, allowing for more precise ecological inferences (peay, 2014). high­throughput sequencing (hts) technologies provide a number of benefits, including the capability to identify fungi at trace levels, quick microbial community structure analysis, and cost­effectiveness (cruz et al., 2014; tedersoo and nilsson, 2016; nilsson et al., 2019). these benefits can be found in hts technologies. according to nilsson et al. (2019), a typical hts metabarcoding process consists of several important stages, including dna extraction, marker­based pcr amplification, dna sequencing, sequence processing, and data analysis. these processes are listed in the order as follows: sampling then dna extraction. however, one potential downside of these technologies is that they may potentially result in the spread of pollutants and mycorrhizal fungi that are not specific to orchids. research methodology and sequencing carried out on high­throughput platforms are the two components of the most typical approaches to molecular identification. dna microarrays, clone libraries, denaturing gradient gel electrophoresis, fluorescence in situ hybridization, and gene chip approaches are some of the other methods that can be utilised for the identification of fungi (dearnaley, 2007). however, these technologies have short­ comings such as limited throughput, time­consuming processes, and lower accuracy. additionally, they have been overshadowed by the growing popularity of alternative methods such as the miseq pe300 and hiseq pe250 platforms (julou et al., 2005). furthermore, alternative methods, such as using an illumina novaseq/hiseq sequencer and the application of shotgun metagenomic technology, provide access to functional gene information from all microorganisms within a community through genomic dna analysis (bahram et al., 2018; fadiji and babalola, 2020). these methods were developed by bahram et al. (2018) and fadiji and babalola (2020). an important step forward in orchid mycorrhiza research has been taken thanks to the development of this technique and the growing availability of orchid and reference orchid mycorrhizal fungal genomes (zhang et al., 2016). because of their ability to simultaneously sequence a mixed dna template across numerous samples with a high sequencing depth (nilsson et al., 2019), next­generation sequencing (ngs) approaches have become practically widespread in mycorrhizal research in recent years. this is partly owing to the fact that ngs methods have grown more affordable in recent years. in contrast, sequencing dna from individual mycorrhizal root tips may be ideal for sanger sequencing when it comes to detecting shifts in regularly occurring fungus species (shemesh et al., 2020). this was found by shemesh and colleagues. in contrast to next­generation sequencing technologies, which can process millions of dna fragments simultaneously, the sanger sequencing method only processes one dna fragment at a time (slatko et al., 2018). this makes the sanger sequencing method superior in terms of sequencing volume. this distinction has the ability to bring forth different conclusions regarding the make­up of the community. 9. conclusions this review provides an overview of the most significant literature in orchid mycorrhizal fungi from about 2002­2023. the molecular identification of orchid mycorrhiza represents a significant advancement in our understanding of the complex relationships between orchids and their mycorrhizal fungal. in addition, finding the most appropriate extraction method and choosing a suitable primer for amplification is essential to ensure accurate identification. moreover, the utilization of molecular techniques compliments morphology­based identifications offers a reliable, unbiased, and frequently more precise tools for confirming species. it is particularly beneficial for cryptic species, hybrids, morphological variables organism such as mycorrhizal, or situations when usual identification methods fail. based on the review, the its regions prove to be a great primer in the field of mycorrhizal studies to its inherent variability, widespread applicability and straightforward amplification process and compatibility with established databases. this technique enables researchers to accurately identify the specific fungal species shamsudin et al. ‐ molecular identification of orchid mycorrhiza 111 associated with a particular orchid species and to investigate the functional tole of these fungi in orchid growth and development. with the advancement of molecular techniques, it is now possible to examine the genetic diversity of these fungi and understand the evolutionary relationship between different orchid mycorrhizal fungi. these may lead to the development of new conservation strategies for these unique and valuable plant species. acknowledgements we would like to thank to sabah 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principal component analysis, trait association. abstract: this study evaluated the genetic diversity of indian long­day garlic genotypes based on agro­morphological and biochemical traits. a significant variation was observed across 23 traits, indicating high genetic diversity. key traits such as bulb weight and 20­clove weight, leaf thickness, and clove showed substantial variability and antioxidant properties highlighted the potential for developing high­quality garlic varieties. hierarchical cluster analysis grouped 94 genotypes into four clusters based on key traits, emphasizing their utility for breeding programs. genotypes vgs­55 and vgs­43 excelled in growth traits, while vgs­51 and vgs­49 demonstrated superior biochemical content. principal component analysis (pca) revealed that the first two components accounted for 40.73% of the total variability, with yield­ promoting traits dominating pc1 and biochemical traits influencing pc3. trait association studies indicated strong positive correlations between bulb yield and traits like bulb weight (r=0.97 ***) and equatorial diameter (r=0.73 ***), whereas no significant association was observed between bulb yield and biochemical traits. these findings underscore the immense genetic potential within indian garlic germplasm for breeding programs targeting higher yields and improved biochemical traits, catering to the increasing demand for both bulbs and fresh garlic leaves in india. 1. introduction genus allium includes about 918 species distributed all over the world (the plant list, 2013), of which only 30 to 36 have been distributed in the indian plains and himalayan region (santapau and henry, 1973; karthikeyan et al., 1989). among this garlic (allium sativum l.) is one of the oldest and strongest flavoured allium species. owing to its typical (*) corresponding author: rahul2iari@gmail.com citation: dev r., hedau n.k., santhiya s., pal r.s., paschapur a., kant l., 2025 ­ agro‐ morphological characterization of indian garlic (allium sativum l.) germplasm under mid hill of northwest himalaya. ­ adv. hort. sci., 39(2): 137­ 148. orcid: dr: 0000­0001­9182­8169 hnk: 0000­0002­9796­3588 ss: 0009­0003­9358­0269 prs: 0000­0003­3185­4466 pa: 0000­0002­3152­0602 kl: 0000­0001­8964­6438 copyright: © 2025 dev r., hedau n.k., santhiya s., pal r.s., paschapur a., kant l. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 6 february 2025 accepted for publication 29 may 2025 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-17277 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0001-9182-8169 http://orcid.org/0000-0002-9796-3588 http://orcid.org/0009-0003-9358-0269 http://orcid.org/0000-0003-3185-4466 http://orcid.org/0000-0002-3152-0602 http://orcid.org/0000-0001-8964-6438 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2025 39(2): 137­148 138 flavor, garlic is mostly used as a spice worldwide in the form of fresh or stored bulbs (block, 2010). in many regions, people also enjoy fresh garlic leaves in salad (koch and lawson, 1996). additionally, there has been a recent increase in the consumption of dehydrated garlic (ogar et al., 2021). as a result of its versatility and popularity, garlic has become the second most important species in the amaryllidaceae family, following onions, for both culinary and medicinal uses (kamenetsky and rabinowitch, 2001). garlic is also an excellent source of vitamins and minerals (selenium), flavonoids, antioxidants, lectins, several enzymes, and amino acids (pizzorno and murray, 2005). it contains about 33 sulphur compounds (albrecht et al., 2017), of which allicin (diallyl­dithiosulfinate) is an important constituent. it is formed when non­proteinogenic acid alliin (s­allyl cysteine sulfoxide) is exposed to enzyme alliinase upon tissue damage (fesseha and goa, 2019). allicin is responsible for garlic’s characteristic pungent odour and medicinal value. however, composition and concentration of these compounds mainly depend on cultivar types, place of origin, and growing environments (baghalian et al., 2005; khar et al., 2011). garlic consumption helps improve health by enhancing immunity (percival, 2016), reducing cholesterol and triglycerides (yeh and liu, 2001), lowering blood pressure levels (ried et al., 2013), curing skin allergies (lee and park, 2003), and reducing cancer risk (sengupta et al., 2004). the numerous health benefits associated with this versatile plant highlight its significance in supporting human well­being. despite its health benefits, there exists a notable demand­supply gap in garlic production, particularly in india. this gap arises from the limited production in tropical regions, where cultivation faces constraints such as suboptimal climatic conditions and lower yields compared to temperate countries. garlic requires a cold period (vernalization) for proper bulb development. the mild winters in much of india are less effective for vernalization compared to the prolonged and colder winters in temperate countries. additionally, garlic cultivation in india is largely concentrated in specific regions, with the indian himalayan region contributing minimally to the overall supply. this geographical limitation contrasts sharply with high­yielding temperate countries, where garlic production is more prevalent and efficient (lawande et al., 2009). india is second only to china in terms of area and production of garlic, but the national average productivity is only 5 t/ha (faostat, 2017) and ranks 74th in the world (faostat, 2010). garlic plants are sensitive to photoperiod and temperature and thrive in temperate conditions. fertile flowers and true seed formation in garlic are observed only in its primary diversity centre (hong et al., 2000). garlic flowers are hermaphrodites and entomophilous and are pollinated primarily by bees, butterflies and moths. flowering is controlled by several genetic factors such as photoperiod and temperature (kamenetsky et al., 2004). however, in a country like india, which is close to the place of origin, garlic clones often form bulbils (aerial bulbs) instead of flowers (kamenetsky and rabinowitch, 2001). therefore, it is essentially regenerated by cloves or bulbils in the region. such asexual propagation methods are generally favourable for maintaining the true­to­type identity and uniformity of a variety or accession because there is no segregation of alleles. despite its sexually sterile nature, garlic has differences in various characteristics such as: morphological (maturity, plant growth) characteristics (panthee et al., 2006; wang et al., 2014) and biochemical characteristics (bhusal et al., 2019; chadha et al., 2019; barboza et al., 2020; benke et al., 2021), reproductive (maturity, bolting behaviour) (kamenetsky and rabinowitch, 2001), and bulb characteristics (bulb shape, bulb size, bulb colour, storage life) (bradley et al., 1996; wang et al., 2014). this diversity is thought to be due to sexual reproduction in the wild plant (maab and klaas, 1995), phenotypic plasticity (bradley et al., 1996), and extensive somatic mutations (ata, 2005), mainly due to their apomictic nature. however, this diversity presents challenges, particularly when considering the adaptation of garlic varieties to different climatic conditions. garlic from temperate climates does not grow well in tropical and subtropical areas, making it difficult to compare different varieties of garlic in similar climate conditions. although numerous studies have been conducted on genetic variation in garlic germplasm at various levels, there remains a significant gap in research focused on evaluation, selection and development of long­day garlic varieties or strains specifically suitable for indian conditions. bridging this gap requires a strategic effort to expand garlic production into underutilized regions (i.e. himalayan dev et al. ‐ genetic diversity analysis in indian garlic 139 intercultural operations, were maintained throughout the cropping period. nitrogen, phosphorus, potash, and sulfur (npks) at 110:50:50:50 kg/ha were used as fertilizers. the first half of the n dose and the total doses of p and k were given at the time of transplantation, with the remaining n dose given 30 and 45 days later. each genotype was planted in two rows, with row spacing of 15 cm and plant spacing of 10 cm, over a row length of 2 m. cloves were directly sown in shallow line at a depth of 3­4 cm during the winter seasons of october 2020 and 2021. watering was provided as required to prevent leaf wilting. the crop was harvested when the leaves reached senescence or necks fell off. all morphological traits were recorded on five random plants in each replication. data were recorded from five random plants in each replication for each 94 genotype to assess morphological, biochemical differences. these germplasms consist of varieties, landraces, improved materials, and cultivars (sm table 1s). the observations were recorded on sixteen morphological and seven biochemical parameters. morphological traits including number of leaves, fourth leaf width (mm), fourth leaf length (cm), pseudostem length (cm), pseudostem diameter (mm), plant height (cm), and neck thickness (mm) were recorded at 80 days of planting after completion of vegetative growth when the crop was in the field. while, other bulb traits viz. number of cloves per bulb, average weight of bulb (g), bulb polar diameter (mm), bulb equatorial diameter (mm), and weight of 50 cloves (g) were recorded after harvest at neck fall. clove samples were randomly taken from the harvested crop to quantify the above biochemical and antioxidant traits. total soluble solids (tss) were determined immediately after manual juice extraction from the macerated sample using a cotton cloth using a handheld digital refractometer model pal­3 (atago, japan) and expressed in °brix. the qualitative characteristics of the genotypes were recorded according to the descriptors of the plant varieties and farmers’ rights authority, government of india. the biochemical properties, namely total soluble solids (tss), 2,2’­azino­bis (3­ethylbenzo­ thiazoline­6­sulfonic acid (abts), 2,2­diphenyl­1­ picryl­hydrazyl­hydrate (dpph), total antioxidant activity (taa), ferric reducing antioxidant power (frap), and total polyphenols (tpp) were estimated in the quality laboratory of the institute. states), thereby ensuring wider availability of this health­enhancing crop. this highlights the need for targeted research to address the unique climate challenges faced by garlic farmers in india. to delve deeper into these aspects, this study aims to assess the genetic diversity and population structure of 94 garlic germplasms in the mid­hill regions of uttarakhand, with special emphasis on morphological and biochemical traits. this information serves as a starting point for improving breeding strategies and developing high­yielding, regionally adapted long day garlic varieties. ultimately, this research aims to address the challenges of low productivity and promote garlic crop improvement in indian temperate conditions. 2. materials and methods to identify suitable garlic material for the long day climatic conditions in mid­himalayan region, an experiment was conducted using a total of 94 garlic accessions collected from the indian himalayan region and distributed under the all­india network research project (ainrp­ onion and garlic) and was maintained at institute genebank. the current study was conducted over two consecutive years, during the rabi seasons of 2020­21 and 2021­22, at the experimental farm of icar­vivekananda parvatiya krishi anusandhan sansthan, located in hawalbagh, almora, uttarakhand (latitude 29°35’ n, longitude 79°39’ e, and an elevation of 1250 meters above sea level). the region experiences a warm, temperate climate characterized by substantial but unpredictable rainfall, averaging 1132.5 mm annually. summer lasts from late june to september, with an average yearly temperature of 23.5°c. the annual maximum temperature ranges between 20.0°c and 38.1°c, while the minimum temperature varies from 6.6°c to 25.2°c (dev et al., 2015). as per the köppen climate classification, the area lies in the northern hemisphere and is classified under the “cfa” subtype, indicating a humid subtropical climate. ninty­four garlic genotypes were cultivated in finely prepared soil with a planting depth of 20–30 cm. prior to sowing, the experimental field was enriched with 20 t/ha of well­decomposed farmyard manure (fym), which was thoroughly mixed into the soil. consistent field management practices, including nutrient application, irrigation, and http://oaj.fupress.net/index.php/ahs/article/view/17277/13662 adv. hort. sci., 2025 39(2): 137­148 140 chemicals and reagents all of the solvents employed in the research were of analytical purity, whereas the water was hplc pure. the solvents and reagents were procured from merck­sigma (bangalore, india) and used without further purification. the extraction and analysis were carried out using uv­vis spectroscopy. different biochemical observations were made on the harvested fruits. the experiments were done in triplicate, and the data are shown as mean values ± standard deviations. preparation of extracts methanolic extracts of garlic were used to measure antioxidant metabolites and activities. fresh samples (2 g) were ground in a pestle and mortar in 20 ml of 80% methanol. the methanolic extracts of the samples were placed in an orbital shaker overnight (16 hours) to completely extract antioxidant metabolites. the methanolic extract was centrifugated at 5000 rpm for 10 minutes, and the supernatant was kept at 4°c for further analysis. methanolic pure (80%) extract was used for the study. all assays were performed out in triplicate, and results are expressed as mean ± standard deviations. total polyphenolic content and antioxidant activities total polyphenolic content. the folin­ciocalteau reagent was used to calculate total phenolic compounds (singleton and rossi, 1965). the final solution was vigorously stirred in a vortex mixer after adding 0.9 ml of distilled water, 0.5 ml of folin­ ciocalteau reagent and 2.5 ml of sodium carbonate solution to the freshly obtained extract. the absorbance was measured at 725 nm after keeping the reaction at 300°c for 40 min. the standard curve for gallic acid was prepared by taking a different concentration of gallic acid (10­100 µg). determination of 2,2‐diphenyl‐1‐picryl‐hydrazyl‐ hydrate (dpph•). with some alterations, the dpph test was carried out by detecting the reduction in absorbance of a methanolic dpph solution at 515 nm in the presence of the extract (brand­williams et al., 2005). the stock solution was made by dissolving 24 mg of dpph in 100 ml methanol which was then stored at ­20°c until needed, while, working solution was made by mixing 10 ml stock solution with 45 ml methanol to achieve an absorbance of 1.17±0.02 units at 515 nm. garlic extracts (150 µl) were allowed to react for 24 hours in the dark with 2850 µl of dpph working solution, and the absorbance was measured at 515 nm. the radical scavenging activity of dpph• was calculated as a percentage of dpph• discolouration using the equation: radical scavenging (percent) = (a control – a sample) / a control × 100 where, a sample is the absorbance of the solution recorded while adding extract/reference at a specific level. a control is the absorbance of the dpph solution without adding extract. determination of scavenging effect on abts•+ radicals the abts test was carried out by detecting the reduction in methanolic abts solution absorbance at 745 nm in the presence of the extract (arnao et al., 2001). the workable solution wa made by combining two stock solutions in equal amounts and allowing them to react for 12 hours at room temperature in the dark. the solution was then diluted to attain an absorbance of 0.9 0.02 units at 745 nm by combining 1 ml abts solution with 3 ml methanol. garlic extracts (200 litres) were allowed to react for 30 minutes in the dark with 2000 liters of newly made abts solution, and absorbance was measured at 745 nm. the percentage inhibition was calculated using the equation: radical scavenging (percent) = (a control – a sample) /a control × 100 where, a sample is the absorbance of the solution recorded when adding extract/reference at a specific level, and a control is the absorbance of the abts solution without extract. determination of total antioxidant activity the total antioxidant activity of the methanolic extracts of both samples was determined using a phosphomolybdenum technique (prieto et al., 1999), which is based on the reduction of mo (vi) to mo (v) by the sample analyte and the creation of green phosphate / mo (v) compounds. a sample extract of 0.3 ml was mixed with 2.7 ml of the reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the sample was sealed and incubated for 90 minutes in a boiling water bath at 95°c. the absorbance was measured at 695 nm after the samples had cooled to room temperature. total antioxidant activity was measured in trolox dev et al. ‐ genetic diversity analysis in indian garlic 141 equivalents (mm/g of extract). determination of ferric‐reducing antioxidant power (frap) the frap test was performed with few changes according to benzie and strain. 300 mm acetate buffer (3.1 g c2h3nao2.3h2o and 16 ml c2h4o2), ph 3.6, 10 mm tptz (2, 4, 6­ tripyridyl­s­triazine) solution in 40 mm hcl, and 20 mm fecl3.6h2o solution were produced as stock solutions. 25ml acetate buffer, 2.5ml tptz solution, and 2.5ml fecl3.6h2o solution were mixed and warmed to 370c before use to make a fresh working frap solution. methanolic extracts of samples (150 l) were allowed to react with 2850 l of frap solution in the dark for 30 minutes, and the coloured product (ferrous tripyridyltriazine complex) was measured at 593 nm. the frap value was calculated by drawing a standard curve formed by adding ferrous sulfate heptahydrate (20­200 mm) to the frap reagent, with the results represented in mm equivalent to feso4.7h2o. statistical analysis the experiment employed a randomized complete block design with two replications, using a mean value over two years for each trait in the diversity analyses. using multivariate methods, such as principal component analysis, agro­morphological diversity was analysed (jolliffe, 1986). r software was used to do multivariate cluster analysis based on pca charts and ward’s technique (ward, 1963). biplot­ pca, variables­pca, and individuals­pca graphics of pca visualisations were created using the r packages “devtools,”“ggplot2,” and “factoextra.” trait correlations were computed using the “metan” package in r, while principal component scores and descriptive analysis were obtained via past04 software (hammer et al., 2001). 3. results genetic diversity based on agro‐morphological and biochemical traits the results showed a huge variation among the characters of interest of indian long­day garlic in terms of the twenty­three morphological and biochemical characters as given in supplementary materials table 1s and table 2s. descriptive data analysis revealed that most traits, except for a few variables, exhibit high genetic diversity. the most variable traits were stem thickness, leaf thickness, bulb weight, bulb diameter, clove width, 20­clove weight and dpph (% inhibition) with cv values greater than 35 percent. among the 20 agronomic traits, abts (percent inhibition) (cv=9.33%) achieved the lowest cv value. yield characteristics differed significantly between garlic genotypes, with bulb weight ranging from 6.50 to 35.13 g (cv= 34.60%) and 20 clove weight ranging from 9.0 to 68.0 g (cv= 27.08%). the number of cloves per bulb varied between 5.80 and 39.60. the polar diameter ranged from 2.17 to 4.10 cm (cv= 11.54%), while the equatorial diameter varied from 2.27 to 5.07 cm (cv= 15.73%).the average clove length was 26.90 mm, ranging from 16.33 to 41.50 mm, while clove width varied from 2.83 to 39.33 mm, with an average of 12.77 mm. genotypes vgs­43 and vgs­103 were noted for their clove length and width. leaf thickness ranged from 0.70 mm (vgs­98) to 1.85 mm (vgs­55), length from 13.00 cm (vgs­51) to 45.13 cm (vgs­96), and width from 1.15 cm (vgs­91) to 3.75 cm (vgs­ 43), with coefficients of variation of 14.08%, 17.49%, and 23.66%, respectively. plant height varied from 14.80 cm (vgs­82) to 44.80 cm (vgs­55), and the number of leaves ranged from 4.20 (vgs­49) to 9.0 (vgs­95), with coefficients of variation of 19.03% and 17.49%, respectively. the increasing demand for fresh garlic leaves in india highlights the potential of these diverse genotypes for producing leafy garlic varieties. additionally, there was considerable variation in quality metrics l ike tss and total carbohydrates, with tss being crucial for assessing bulb quality in the tested accessions. among 94 genotypes, 43 genotypes had tss values in­between 16.16 and 43.90°brix, with vgs­67 having the highest at 43.90°brix, followed by vgs­19 at 43.60°brix, and vgs­5­2 with the lowest at 16.16°brix. garlic cloves primarily consist of carbohydrates, making up 33.06% of the total carbohydrate content (usda national nutrient database).the study found that carbohydrate content in the genotypes ranged from 14.39% (vgs­ 8) to 28.81% (vgs­11b), with an average of 21.42%. important antioxidant properties suggested potential for developing healthier varieties. total polyphenols varied between 0.30 and 1.25 mg gae/g dw, with a standard deviation of 0.15 and a coefficient of variation of 29.90%. the dpph inhibition ranged from 10.08% to 49.23%, with a standard deviation of 10.08 and a coefficient of variation of 49.23%.the free radical inhibition percentage against abts in garlic http://oaj.fupress.net/index.php/ahs/article/view/17277/13662 http://oaj.fupress.net/index.php/ahs/article/view/17277/13752 142 adv. hort. sci., 2025 39(2): 137­148 accessions ranged from 31.65% to 83.42%, averaging 62.14%, indicating low genotypic variation. the total antioxidant activity varied between 12.71 and 66.43 mm trolox equivalent/g dw, with a standard deviation of 6.43 and a coefficient of variation of 24.97%. the frap values ranged from 95.04 to 269.32 mm feso4 equivalent/g dw, with a standard deviation of 32.77 and a coefficient of variation of 21.26%. vgs­51 had the highest frap values among the genotypes tested (table 1). hierarchical cluster analysis cluster analysis using ward’s minimum variance (ward, 1963) identified four main groups among 94 genotypes, explaining 29.78%, 51.06%, 17.02%, and 2.12% of total germplasm, respectively (fig. 1). cluster 1, consisting of 28 genotypes, showed associations with higher clove numbers (e.g., vgs­32, vgs­33), pst (vgs­33), pd (bhima omkar, vgs­06, vgs­103), bw (vgs­89), and ty (vgs­89). all released varieties, except swarna­9, are in this cluster. the second cluster, the largest with 48 genotypes, is characterized by maximum plant height (vgs­55), lt (vgs­55), and higher tss (vgs­35, vgs­36, vgs­37, vgs­13 and vgs­14), taa (vgs­11­b), and (vgs­11­ b). this cluster is advantageous for selecting genotypes with enhanced growth and bulb quality. the third cluster included thirteen genotypes with fig. 1 ­ dendrogram of cluster analysis of garlic germplasm based on morphological and biochemical traits (ward method). table 1 ­ summary statistics morphological and biochemical characteristics of garlic accessions characteristics abbreviation min max mean se variance sd cv (%) plant height (cm) ph 14.80 44.80 31.39 0.62 35.70 5.97 19.03 leaf length (cm) ll 13.00 45.13 32.01 0.58 31.35 5.60 17.49 leaf width (cm) lw 1.15 3.75 1.93 0.05 0.21 0.46 23.66 no. of leaves/plant ln 4.20 9.00 5.65 0.09 0.74 0.86 15.27 pseudostem thickness (mm) pst 6.20 18.58 10.76 0.32 9.86 3.14 29.19 leaf thickness (mm) lt 0.70 1.85 1.33 0.02 0.03 0.19 14.08 polar diameter (cm) pd 2.17 4.10 3.25 0.04 0.14 0.37 11.54 equatorial diameter (cm) ed 2.27 5.07 3.62 0.06 0.32 0.57 15.73 20 clove weight (gm) cw­20 9.00 68.00 25.41 1.18 131.02 11.45 45.04 clove length (mm) cl 16.33 41.50 26.90 0.40 14.94 3.87 14.37 clove width (mm) cw 2.83 39.53 12.77 0.49 22.85 4.78 37.44 no of clove layer/ bulb cnl 1.00 3.00 2.38 0.04 0.19 0.43 18.23 no of clove/ bulb cn 5.80 39.60 17.92 0.65 39.35 6.27 35.01 bulb weight (gm) bw 6.50 35.13 21.35 0.60 33.43 5.78 27.08 total yield (q /ha) ty 32.50 175.67 105.67 3.09 899.52 29.99 28.38 plant weight (g) pw 8.00 52.67 23.99 0.87 71.52 8.46 35.25 tss (°brix) tss 16.16 43.90 38.27 0.46 19.77 4.45 11.62 total polyphenols (mg gae/g) tpp 0.30 1.25 0.51 0.02 0.02 0.15 29.90 dpph (% inhibition) dpph 10.08 49.23 27.49 1.01 96.64 9.83 35.76 abts (% inhibition) abts 31.65 83.42 62.14 0.60 33.62 5.80 9.33 taa (mm trolox equivalent/g dw) taa 12.71 66.43 25.74 0.66 41.32 6.43 24.97 frap vale (mm feso4 equivalent/g frap 95.04 269.32 154.13 3.38 1074.06 32.77 21.26 total carbohydrate (%) tca 14.39 28.81 21.42 0.35 11.76 3.43 16.01 dev et al. ‐ genetic diversity analysis in indian garlic 143 distinctive leaf traits such as leaf length (vgs­96), leaf width (vgs­43), leaf number (vgs­95), and several yield­related traits such as bulb weight (vgs­96, vgs­ 105, swarna­9) and total yield (vgs­39). in contrast, the fourth cluster contained only two genotypes, vgs­51 and vgs­49, which were characterized by their high biochemical content, including tpp, dpph, abts, and frap. trait association study the total bulb yield showed significant positive correlations with bw (0.97***), ed (0.73***), cw (0.24*), ln (0.39***), ll (0.29**), lw (0.24*), pst (0.48***), pw (0.66***), 20­cw (0.61***), and pd (0.25*). however, no association was found between bulb weight and various biochemical traits, including taa, abts, total phenolics, and dpph activities, suggesting that bulb weight has no influence on bulb quality traits. furthermore, bulb weight, tss, and bulb diameter had no relationship with plant height (fig. 2). conversely, tss was negatively correlated with 20­clove weight (­33**), clove abts (­0.29**), and dpph activity (­0.27**), while there was a non­ significant negative correlation with clove phenol content (tpp), taa, and frap values. significant positive correlations were observed among dpph (0.66**), taa (0.39***), abts (0.55***), and frap fig. 2 ­ pearson correlation matrix for morphological and biochemical traits in indian long day garlic germplasm. (0.60***), indicating a complex gene action mechanism involving garlic quality parameters. principal component and biplot analysis prior to performing principal component analysis (pca), pearson correlation analysis was conducted to identify and remove highly correlated variables (bw and ed), as such variables can disproportionately influence the pca results. the first principal component (pc1) accounts for 22.77% of the total variation, with the highest eigenvalue of 4.78. it includes features such as cw­20, pst, pw, tpp, dpph and lw, which have positive contributions, while cn, cnl and tss have negative effects (table 2). the second principal component (pc2) has an eigenvalue of 3.22 and explains 15.31% of the variation. positive contributions come from traits such as pd, ty, and pw, whereas most of the biochemical traits i.e., tpp, dpph, abts, taa, and frap have negative contributions. the third principal component (pc3) explained 8.38% of the total variation, mainly due to biochemical features such as frap, dpph (% inhibition), tpp, abts, taa and a morphological trait i .e., ph. the fourth principal component (pc4) accounted for 7.16% of the variation and was primarily influenced by clove length (cl), clove width (cw) and plant diameter (pd). leaf thickness (lt) had the highest loadings in the fifth principal component (pc5), while total carbohydrates (tca) and total soluble solids (tss) dominated in the sixth principal component (pc6), highlighting their importance. yield­ promoting traits were mainly captured in pc1 and pc2, while pc3 focused on biochemical traits and highlighted their role in phenotypic variation. tpp, dpph, abts, lw, and frap made the main positive contributions based on the squared cosine (cos2), while cw­20, st, bw, ty, and pw made notable negative contributions (fig. 3). genotypes vgs­10a, vgs­84, vgs­82, vgs­79, vgs­54, and vgs­70 had the highest square cosine values, while vgs­96, vgs­ 109, vgs­105, vgs­108, vgs­76, vgs­42, vgs­49, vgs­51, and swarna­9 were the least contributions (fig. 4). a biplot of pc1 and pc2 revealed distinct trait groups, including cnss, ty, and various biochemical traits (tpp, dpph, abts, taa, frap). genotypes vgs­96, vgs­43, vgs­34, vgs­51, vgs­ 49, vgs­44, vgs­12, vgs­14, vgs­97, vgs­104, and vgs­89 were in gaps, highlighting their uniqueness (fig. 5). 4. discussion and conclusions the present study highlighted the significant genetic diversity among indian long­day garlic adv. hort. sci., 2025 39(2): 137­148 144 table 2 ­ principal component loadings for morphological and biochemical traits of garlic traits pc 1 pc 2 pc 3 pc 4 pc 5 pc 6 pc 7 pc 8 pc 9 pc 10 ph ­0.20 0.18 0.34 0.05 0.24 ­0.16 ­0.32 ­0.14 ­0.36 0.01 ll ­0.01 0.26 0.18 ­0.28 ­0.15 ­0.23 0.49 0.29 ­0.26 0.24 lw 0.32 0.03 ­0.27 ­0.12 0.20 0.02 0.01 ­0.09 ­0.06 ­0.22 ln 0.09 0.27 0.25 ­0.18 0.33 0.12 0.00 ­0.06 ­0.31 ­0.06 pst 0.30 0.26 ­0.05 ­0.14 0.00 0.09 0.17 0.02 0.04 ­0.02 lt 0.04 ­0.06 ­0.08 0.12 0.63 ­0.12 ­0.06 0.44 0.14 0.41 pd ­0.14 0.32 0.12 0.41 ­0.04 0.12 0.22 0.03 0.17 ­0.10 cw­20 0.36 0.25 ­0.04 ­0.04 ­0.01 0.03 ­0.20 0.01 0.03 0.11 cl 0.09 0.15 ­0.01 0.68 0.00 ­0.03 0.15 ­0.01 ­0.16 ­0.03 cw 0.26 0.10 ­0.11 0.33 0.16 ­0.04 0.19 ­0.14 0.01 0.18 cnl ­0.27 0.16 0.29 0.06 ­0.11 ­0.10 0.10 0.19 0.37 ­0.20 cn ­0.33 0.07 0.25 ­0.01 0.23 0.09 ­0.09 ­0.07 0.01 ­0.06 ty 0.16 0.41 0.17 ­0.11 0.04 0.00 ­0.26 ­0.06 0.16 ­0.04 pw 0.21 0.37 0.04 ­0.06 ­0.19 0.02 ­0.17 ­0.01 0.37 0.04 tss ­0.22 0.02 ­0.01 ­0.09 0.18 0.48 0.17 ­0.48 0.27 0.37 tpp 0.29 ­0.25 0.33 0.05 0.07 0.15 ­0.03 ­0.09 0.10 ­0.02 dpph 0.26 ­0.17 0.28 0.09 0.05 0.00 0.16 ­0.25 ­0.18 ­0.33 abts 0.18 ­0.26 0.28 0.07 0.08 ­0.07 ­0.23 0.27 0.31 ­0.16 taa 0.13 ­0.11 0.28 0.16 ­0.44 0.22 ­0.28 0.09 ­0.24 0.52 frap 0.18 ­0.21 0.37 ­0.14 0.08 0.20 0.42 0.13 0.11 0.05 tca ­0.07 0.09 ­0.15 0.03 0.02 0.71 ­0.08 0.48 ­0.22 ­0.27 eigen value 4.78 3.22 1.76 1.50 1.32 1.12 0.98 0.90 0.81 0.75 variance 22.77 15.31 8.38 7.16 6.26 5.35 4.67 4.29 3.85 3.57 cumulative variance 22.77 38.08 46.47 53.63 59.89 65.24 69.91 74.20 78.05 81.63 fig. 3 ­ contribution of different variables towards principal component analysis based on cos2 value. fig. 4 ­ contribution of different garlic accessions toward principal component analysis based on the cos2 value. dev et al. ‐ genetic diversity analysis in indian garlic 145 genotypes based on agro morphological and biochemical traits. the wide range of mean and coefficient variations observed between traits highlights the potential for improving existing genotypes through clonal selection and paves the way for the development of new, tailored varieties suitable for high­altitude regions. previously, benke et al. (2020) reported similar genetic differences in garlic genotypes. our results confirm the large variability found in morphological traits such as leaf width, stem thickness, bulb weight, 20­clove weight, clove width, and biochemical traits such as total phenolic content, dpph inhibition, total antioxidant activity, and frap, and highlight their potential for selection and improvement. this is consistent with the findings of bhusal et al.(2019); narayan et al. (2019), who also found significant variability in yield­ determining and biochemical traits in garlic genotypes and demonstrated the usefulness of such traits in the clonal selection highlighted programs. the variability of yield characteristics and biochemical properties offers opportunities for targeted selection. for example, features such as bulb equatorial diameter (cv = 56.54%) and clove length (cv = 34.82%), as reported by ayed et al. (2019) indicate scope for clonal selection to develop new garlic varieties. furthermore, studies by jabbes et al. (2012) and baghalian et al. (2005) identified significant differences in plant height, number of cloves per bulb and bulb dimensions, consistent with the results of the current study. this variability forms the basis for improving both the agronomic and phytochemical properties of garlic. present study also identified fig. 5 ­ segregation of 94 accessions of garlic according to morphological and biochemical traits determined by pc score and loadings. genotypes such as vgs­11b (high taa and tca content) and vgs­51 (high total phenolic content, dpph, abts and frap activities) as candidates for developing healthier garlic varieties. compared to existing commercial varieties, these genotypes showed significantly improved antioxidant and phenolic profiles, suggesting that they have the potential to meet growing consumer demand for nutrient­dense and health­promoting garlic products. these results echo bhusal et al. (2019), who identified superior genotypes for antioxidant activity and suggested their use for health­oriented breeding programs. the analysis of the tss (total soluble solids) values in this study indicates a range of 16.16 to 43.90 °brix, which relates these results to existing research and shows that they are slightly lower than those, observed in certain indian genotypes from the western region (bhusal et al., 2019) and slightly higher than the values recorded for egyptian garlic genotypes (moustafa et al., 2011).this variation highlights the potential for breeding programs aimed at growing garlic varieties with specific tss characteristics. such tailored varieties, particularly those with medium tss and low pungency, can be particularly beneficial for improving post­harvest storage and processing, allowing growers to optimize quality and shelf l ife. genotypes with high antioxidant activity and different tss values are suitable for leaf purposes because tss is not a crucial trait for selecting leaf genotypes. our study found that vgs­55 (leaf thickness), vgs­43 (leaf width), vgs­95 (number of leaves) and vgs­96 (leaf length) are superior in leaf characteristics. furthermore, bhusal et al. (2019) identified pgs­105 as a genotype with higher antioxidant activity supporting improved health benefits in leaf production. despite the clonal propagation and inherent sterility of garlic, significant genetic variation has been observed, likely due to mutations, soma­clonal variations, or genetic transformation variations arising from sexual reproduction in the wild plant (novak, 1990; bradley et al., 1996; wang et al. 2014). these sources of variation are not unique to garlic and have also been reported in other clonally propagated crops such as potatoes (zaag, 1987) and bananas (ortiz and vuylsteke, 1996). however, the extent and impact of such variations may vary depending on the genetic architecture and propagation method of each crop. the use of pca and cluster analysis revealed four major groups conversely, the lack of association between bulb weight and biochemical traits indicates the need for independent selection strategies for yield and quality traits. supporting this perspective, jabbes et al. (2012), imani and shamili (2017) and benke et al. (2021) found a strong positive correlation between marketable yield and yield­contributing traits in different garlic accessions. panthee et al. (2006) reported comparable results in various nepalese garlic accessions. this study identifies the first two principal components (pcs) that encompass essential yield and biochemical traits, including bulb length, number of cloves, leaf count, plant weight, total soluble solids (tss), clove length, total antioxidant activity (taa), and total carotenoid content (tca). therefore, these pcs can aid in selecting genotypes with favorable horticultural and yield characteristics (useche­carrillo et al., 2021). this collective evidence indicates that garlic’s production potential is closely linked to its vegetative development, making these traits valuable for direct selection in garlic cultivation. the study showed significant genetic diversity among 94 indian garlic genotypes based on both agro­morphological and biochemical traits. large differences were observed in traits such as bulb weight, clove size, leaf characteristics and biochemical parameters such as total polyphenols, antioxidant activity and total soluble solids (tss). notably, genotypes vgs­43 and vgs­103 had superior clove properties, while vgs­51 had the highest antioxidant properties. the cluster and principal component analyses identified distinct groups based on yield­promoting traits and biochemical content, providing a basis for selecting genotypes with specific desired traits. yield­related traits such as bulb weight and 20­clove weight were positively correlated with several morphological traits but showed no association with biochemical traits such as total antioxidants, suggesting that these quality traits are independent of yield. the study highlights the potential of these different genotypes for breeding programs aimed at increasing both the yield and quality of garlic production, particularly improving antioxidant content and bulb quality. future efforts should focus on integrating molecular markers into phenotypic assessments, which can help accelerate the development of high­ yielding, nutrient­dense garlic varieties suitable for regional cultivation. adv. hort. sci., 2025 39(2): 137­148 146 within the 94 genotypes, reflecting the diversity of morphological and biochemical traits. these clusters were dominated by the group of genotypes that were better in some traits. fruit­related traits, for instance, were the most effective in differentiating across mango cultivars (samsampour et al., 2020). however, overlapping features were also recorded. similarly, benke et al. (2020) reported overlap in both qualitative and quantitative characteristics. these findings align with egea et al. (2017) and wang et al. (2014), oyetunde et al. (2021) on biochemical, color traits, and phenotypic diversity. traits such as tpp, dpph, and abts significantly influenced clustering patterns, emphasizing their role in defining genetic diversity, as seen in chinese (hassan et al., 2015), polish (bozin et al., 2008), and serbian (kim et al., 2013) garlic genotypes. other studies also showed clustering based on various traits. for example, bhusal et al. (2019) identified two clusters among 26 indian garlic genotypes based on antioxidant and quality traits, wang et al. (2014) clustered 212 garlic accessions into six groups based on morphological traits while barboza et al. (2020) classified argentinean genotypes into four groups using organo­sulfur and ssr markers. these studies highlight the value of clustering techniques in understanding genetic diversity and informing breeding programs. furthermore, clustering patterns were unrelated to the geographic origins of the accessions, as noted by benke et al. (2020). the clustering patterns and pca results emphasize the importance of traits like tss, antioxidant activity, and phenolic content in defining genetic diversity. the high variability and significant correlations observed among yield and biochemical traits highlight the potential for using diverse genotypes in breeding programs. for example, strong correlations between bulb weight and clove dimensions could prioritize selection for these traits to enhance overall yield. meanwhile, weak or non­significant correlations between yield and biochemical traits suggest that independent selection strategies might be necessary to balance productivity with quality improvements. this understanding enables breeders to design more focused and efficient strategies tailored to specific breeding goals. the total bulb yield showed strong positive correlations with traits like bulb weight, bulb diameter, and clove width, suggesting their importance in yield improvement. dev et al. ‐ genetic diversity 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doi: 10.36253/ahsc­15769 https://oaj.fupress.net/index.php/ahs effectiveness of kmno4 and activated carbon on the quality and storage properties of mango fruit t.t. tran 1, 2 (*), t.t.t. le 3, h.p. nguyen 1, 2 (*) 1 department of plant physiology, university of sciences, ho chi minh city 7000, vietnam. 2 vietnam national university, ho chi minh city 7000, vietnam. 3 center for business incubation of agricultural high technology, ho chi minh city 7000, vietnam. key words: cat hoa loc, fruit quality, mangifera indica l., postharvest, shelf life, vitamin c. abstract: cat hoa loc mango (mangifera indica l.) is a well­known variety in vietnam because of its distinct taste and aroma. however, it has a short shelf life and can suffer post­harvest losses if not handled and packaged correctly. the demand for fresh cat hoa loc mangoes has been increasing worldwide, and this has led to the development of effective handling methods to extend their shelf life. to address this issue, this study was conducted to evaluate the effectiveness of kmno4 and activated carbon in preserving the quality and shelf life of cat hoa loc mangoes in vietnam. the cat hoa loc mango variety in cao lanh, dong thap province, was chosen for the study. the study used five replications of a completely randomized block design. six different treatments with kmno4 and activated carbon (1:1 ratio) were tested, including 0; 4; 8; 12; 16; 20 g/box. six mangoes were stored in perforated cartons (36x26x9 cm) at room temperature (28­30°c) during the study period. the study evaluated several parameters to assess the quality and shelf life of the mangoes, including weight loss, fruit firmness, browning index, respiration, ethylene release rate, soluble sugar, and vitamin c. the results showed that the quality of the mangoes was extended when treated with 12 g of kmno4 and activated carbon per box. this treatment resulted in the lowest physiological weight losses, respiration, and ethylene release rate. furthermore, this treatment showed the highest fruit firmness, soluble sugar, and vitamin c content, as well as the longest shelf life at the end of the storage period. 1. introduction cat hoa loc mango (mangifera indica l.) is a popular tropical fruit variety known for its unique flavor, aromatic fragrance, and vibrant color. it is highly valued for its nutritional content, abundant vitamins, and minerals (athoo et al., 2024). cat hoa loc mangoes have gained (*) corresponding author: trtthang@hcmus.edu.vn hoangphuc0421.hcmus@gmail.com citation: tran t.t., le t.t.t., nguyen h.p., 2024 ­ effectiveness of kmno4 and activated carbon on the quality and storage properties of mango fruit. ­ adv. hort. sci., 38(3): 233­238. orcid: ttt: 0000­0002­9322­2424 tttl: 0009­0006­0167­579x hpn: 0009­0008­1272­6796 copyright: © 2024 tran t.t., le t.t.t., nguyen h.p. this is an open access, peer reviewed article published by firenze university press (https://www.fupress.com) and distributed, except where otherwise noted, under the terms of cc by 4.0 license for content and cc0 1.0 universal for metadata. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no conflict of interests. received for publication 8 february 2024 accepted for publication 27 june 2024 ahs advances in horticultural science ahs ­ firenze university press issn 1592­1573 (on line) ­ 0394­6169 (print) http://doi.org/10.36253/ahsc-15769 http://oaj.fupress.net/index.php/ahs http://orcid.org/0000-0002-9322-2424 http://orcid.org/0009-0006-0167-579x http://orcid.org/0009-0008-1272-6796 http://www.fupress.com http://creativecommons.org/licenses/by/4.0/legalcode http://creativecommons.org/publicdomain/zero/1.0/legalcode adv. hort. sci., 2024 38(3): 233­238 234 significant recognition both domestically and internationally and are widely exported to various markets. despite its popularity and economic importance, cat hoa loc mango faces challenges related to its postharvest shelf life. the limited shelf life poses a considerable threat to its preservation and exportation (nguyen et al., 2024). to overcome this challenge, researchers and experts have focused on developing postharvest preservation methods to minimize quality deterioration and extend the shelf life of cat hoa loc mangoes. previous research focused on mango preservation has employed essential oils derived from four aromatic plant species, namely thymus vulgaris, salvia mirzayanii, artemisia persica, and rosmarinus officinalis. the objective has been to impede the proliferation of aspergillus niger, thus prolonging the fruit’s storage viability (javadpour et al., 2018). in addition to essential oils, a spectrum of chemical agents has been extensively utilized for fruit preservation. for instance, potassium phosphite has been employed in the preservation of citrus clementina, while a composite of alginate and cyclea barbata leaf powder has been implemented for guava preservation (strano et al., 2015; utama et al., 2022). in the various preservation methods, the use of potassium permanganate (kmno4) and activated carbon has gained attention. kmno4 has been utilized in the postharvest management of peaches and mangoes to uphold their quality (alonso­salinas et al., 2023; fatima et al., 2023). the research findings indicate that the application of 30 g kmno4 is optimal for preserving and enhancing the color, taste, aroma, firmness, total sugar, ph, and total soluble solids of the fruits, while minimizing weight loss and waste percentage over a 20­day storage period (fatima et al., 2023). potassium permanganate is a powerful oxidizing agent with antimicrobial properties. when used in postharvest preservation, kmno4 can effectively inhibit the growth of pathogens and spoilage microorganisms that contribute to the deterioration of fruits (alonso­salinas et al., 2023). the antimicrobial action of kmno4 is attributed to its ability to oxidize the cellular metabolism of microorganisms. kmno4 can interfere with the vital biochemical processes of microbes, such as respiration and energy production, which are essential for their survival and proliferation. the strong oxidizing properties of kmno4 can target and oxidize key enzymes and other critical cellular components involved in these metabolic pathways. this oxidative damage can impair the microorganism’s ability to carry out normal metabolic functions, ultimately disrupting its ability to function and survive (rudra et al., 2013). by inhibiting microbial growth, kmno4 treatment helps to extend the shelf life of cat hoa loc mangoes and maintain their quality during storage and transportation. however, the oxidation of ethylene by kmno4 requires time, so it is necessary to supplement with some ethylene­adsorbing carriers with porous structures and large surface areas to facilitate the redox reaction. in food preservation, activated carbon has a high capacity for adsorbing ethylene, especially in the form of granular activated carbon. activated carbonis a highly porous material with a large surface area, which gives it excellent adsorption properties (roopa et al., 2023). when applied in postharvest preservation of fruits, activated carbon acts as a purification agent by adsorbing and removing harmful substances such as ethylene gas, volatile compounds, and toxins (nooun et al., 2023). ethylene is a natural plant hormone that accelerates the ripening process in fruits. by adsorbing ethylene, activated carbon helps low the ripening process, thus extending the shelf life. additionally, activated carbon can also adsorb volatile compounds responsible for off­flavors and odors, thereby preserving the sensory quality of the fruit. therefore, the objective of this research is to assess the effectiveness of potassium permanganate and activated carbon treatments on various quality parameters of postharvest cat hoa loc mango. by studying the physiological and biochemical changes that occur during the ripening process under the influence of kmno4 and activated carbon, this research aims to optimize postharvest treatments to improve fruit quality, reduce losses, extend shelf life, and ensure a higher yield of marketable cat hoa loc mangoes. the findings of this study will contribute to a better understanding of the preservation techniques for cat hoa loc mangoes, enhancing their market value and global competitiveness. 2. materials and methods plant material and experimental design the cat hoa loc mangoes were harvested from a commercial orchard located in cao lanh city, dong thap province, vietnam. on may 25, 2023, mango fruits were collected from homogenous plants using tran et al. ‐ kmno4 and activated carbon to prolong cat hoa loc mango shelf life 235 a randomized block pattern. the fruits, with an average weight of about 450 g, were picked precisely 85 days after the fruit set and were carefully hand­picked. after being picked, the mangoes were transported to the university of science, located in ho chi minh city. a total of 180 mango fruits were used and distributed into five replications using a completely randomized block design. each replication consisted of six mangoes that were stored in perforated cartons (36 cm x 26 cm x 9 cm). to test the effects of kmno4 and activated carbon, a bag containing a mixture of the two substances in a 1:1 ratio was placed in each carton. the weight of the mixture ranged from 0, 4, 8, 12, 16, and 20 g per box. the mangoes were stored at a constant temperature of 28­30°c and ambient humidity of 70­80% throughout the study. the research evaluated several parameters, including weight loss, fruit firmness, color, browning index, respiration rate, ethylene release rate, soluble sugar content, vitamin c content, and the shelf life of the mangoes. determination of weight loss and browning index weight loss is determined by recording the initial weight of the fresh sample. after the storage period, the final weight is determined. the percentage of difference between the initial and final weight to initial weight represents the physiological weight loss (workneh et al., 2012). the color of the outer layer of the fruit was determined using the l*a*b* (cielab) color space of a digital color meter from apple inc. to calculate the browning index (bi), the formula proposed by ruangchakpet and sajjaanantakul (2007) was used: [100 (x – 0.31)]/0.17. in this formula, x is calculated as (a* + 1.75 l*)/(5.645 l* + a* −0.3012b*). determination of fruit firmness, respiration rate, and ethylene release rate the fruit firmness was evaluated using a fruit firmness testing device (gy­3, jiangsu, china) equipped with a cylindrical probe. for the assessment of the fruit’s respiration rate in a sealed chamber, a co2 analyzer with a non­dispersive infrared sensor was utilized (thang et al., 2022). furthermore, the release of ethylene gas was determined by utilizing an ethylene gas analyzer with an electrochemical sensor (sky2000­c2h4, safegas, china) connected to the same sealed chamber. determination of soluble sugar and vitamin c to determine the total sugar content, fresh fruit flesh (1 g) was finely ground and mixed with 10 ml of 96% ethanol. following this, the mixture was heated in a water bath for 15 min and subjected to centrifugation at 10,000 rpm for 10 min to obtain the supernatant. then, 1 ml of the extracted solution was combined with 1 ml of a 5% phenol solution and 5 ml of concentrated h2so4. the resultant mixture was allowed to react, and the optical density was measured at a wavelength of 490 nm. the total sugar content was then calculated using a sucrose standard curve as a reference (dubois et al., 1956). to quantify the amount of vitamin c, 1 g of the sample was ground and mixed with 10 ml of a methanol solution. subsequently, the mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. next, 1 ml of the extracted solution was combined with 2 ml of 1% sodium nitroprusside, 1 ml of 1% potassium dichromate, and 1 ml of concentrated sulfuric acid. the resultant mixture underwent a reaction, and the optical density was measured at a wavelength of 564 nm. the content of vitamin c was then determined by comparing it to a corresponding standard curve (saeed et al., 2018). statistical analysis the collected data was subjected to an analysis of variance (anova) to determine the significant differences among the means at a 5% probability level. duncan’s multiple range test was then employed using spss 20.0 to identify the significant differences. the results were presented as the mean values together with their corresponding standard deviations, and the ‘ns’ indicates that the differences were not statistically significant. 3. results the changes in weight loss and browning index the utilization of kmno4 and activated c in mango preservation has yielded noteworthy results. the experiment revealed that on days 8 and 10, the control group and treated groups of 4, 12, 16, and 20 g/box did not exhibit significant differences in weight loss. however, the treated group with 8 g/box demonstrated a noticeable reduction in weight loss adv. hort. sci., 2024 38(3): 233­238 236 percentage. moreover, the treatment with kmno4 and c significantly enhanced the color changes in the mangoes. the treated fruit manifested a lower browning index than the control group. the flesh of the treated mangoes retained a vibrant, fresh yellow color, while the skin remained a bright green (figs. 1 and 2). the changes in fruit firmness, respiration rate, and ethylene release rate throughout the course of the analysis period, the fruit’s firmness gradually decreased. however, treatments utilizing kmno4 and activated carbon proved effective in maintaining the fruit’s firmness across all three­time points analyzed. of the treatments tested, kmno4 and activated carbon at a concentration of 12 g/box delivered the most effective outcomes in preserving fruit firmness. similarly, the utilization of kmno4 and activated carbon treatments allowed for the extension of both the ethylene peak and respiration rate. in the control fig. 2 ­ the changes in weight loss and browning index during the post­harvest ripening process of mango. values with different letters are significantly different according to duncan’s test (p=0.05). fig. 1 ­ the variations in fruit color among different treatments using kmno4 and c with various concentrations after a period of 12 days. group, respiration intensity and ethylene release were high on the eighth day, decreasing gradually on days 10 and 12. in contrast, the fruits treated with kmno4 and activated carbon exhibited two peaks of ethylene release and respiration rate, occurring on day 10 (fig. 3). the changes in soluble sugar and vitamin c throughout the process of mango ripening, the control group demonstrated a significant increase in total soluble sugar content, while the vitamin c content remained stable. upon comparison of the control group with the kmno4 and activated c treatments, it was observed that treatment groups receiving 12, 16, and 20 g/box were instrumental in maintaining the highest level of total soluble sugars on day 12. however, no significant difference was found in vitamin c content between the control group and the kmno4 and activated c treatments, as noted in figure 4. fig. 4 ­ the changes in soluble sugar and vitamin c during the post­harvest ripening process of mango. values with dif­ ferent letters are significantly different according to duncan’s test (p=0.05). ns= not significant. fig. 3 ­ the changes in fruit firmness, respiration rate, and ethy­ lene release during the post­harvest ripening process of mango. values with different letters are significantly dif­ ferent according to duncan’s test (p=0.05). tran et al. ‐ kmno4 and activated carbon to prolong cat hoa loc mango shelf life 237 4. discussion and conclusions the preservation of mangoes post­harvest can be extended by utilizing kmno4 and activated carbon. the treated group, receiving 8 g/box of kmno4 and activated carbon, demonstrated a significant reduction in weight loss percentage compared to the control, as shown in figure 2. kmno4 and activated carbon act to preserve the natural color of mangoes by inhibiting the activity of enzymes, such as polyphenol oxidase, responsible for enzymatic browning (mope et al., 2024). this enzymatic reaction occurs when the fruit’s phenolic compounds react with oxygen, resulting in a brownish discoloration. by inhibiting this enzymatic activity, kmno4 and activated carbon help preserve the fruit’s natural color, rendering it visually appealing and marketable for a longer duration. the maintenance of firmness in mangoes is another critical aspect of preservation. kmno4 and activated carbon inhibit the activity of cell wall­degrading enzymes, such as pectinase and cellulase (chen et al., 2021; kumar et al., 2023). these enzymes break down the cell walls of the fruit, leading to softening and a loss of firmness. by inhibiting these enzymes, kmno4 and activated carbon help preserve the structural integrity of the fruit and maintain its firm texture over a longer period of time. kmno4 and activated carbon treatments delay the onset of senescence and over­ripening by extending the ethylene peak and respiration rate. the control group showed a progressive decline in respiration intensity and ethylene release on days 10 and 12, which peaked on the eighth day. in contrast, fruits treated with kmno4 and activated carbon showed two peaks in the respiration rate and ethylene release on day 10, as shown in figure 3. ethylene is a natural plant hormone involved in the ripening process. kmno4 physically absorbs the surrounding ethylene through a porous medium, oxidizing it to produce co2, manganese oxide, potassium hydroxide, and water (kumar et al., 2023; meena et al., 2024). activated carbon, with its porous structure, can adsorb and remove volatile compounds, including those responsible for producing off­flavors. by reducing the presence of these compounds, activated carbon helps maintain the fruit’s quality and freshness (nooun et al., 2023; roopa et al., 2023). by reducing the peak production of ethylene and respiration rate, kmno4 and activated carbon slow down the ripening process, allowing the fruit to maintain its desirable qualities for a more extended period. slowing down respiration helps fruits ripen more slowly, and as a result, the carbohydrate metabolism process occurs at a slower rate, allowing fruits to maintain a higher sugar content (fig. 4). furthermore, kmno4 acts as an antimicrobial agent by releasing oxygen and oxidizing organic matter. it helps inhibit the growth of microorganisms on the fruit’s surface, reducing the risk of spoilage and extending the shelf life (alonso­ salinas et al., 2023). the treatment regimens of kmno4 and activated carbon at levels ranging from 12 to 20 g/box each exhibited significant efficacy. however, the most optimal treatment for enhancing fruit quality and extending postharvest shelf­life was observed at the 12 g/box dosage. at this level, minimal weight losses were recorded, and essential fruit attributes such as firmness, soluble sugar, and vitamin c content were well­preserved. notably, elevating the concentration of the kmno4 and activated carbon mixture beyond 12 g/box did not yield additional benefits in terms of prolonging storage life. conversely, it resulted in escalated costs due to the higher treatment dosage. the current study elucidates the physiological changes that occur during the ripening of mango fruit and the efficacy of kmno4 and activated carbon treatment in this regard. kmno4 and activated carbon (12 g/box) treatment can effectively delay the ethylene climacteric, which is responsible for the rapid deterioration of fruits. this delay in ethylene production and subsequent ripening processes significantly extends the storage period, consequently enhancing the flexibility in handling and distributing mango fruits for farmers and distributors alike. the treatment also aids in preserving the firmness and weight of the fruit, ultimately enhancing the nutritional quality of the fruit through increased sugar content and vitamin c. these outcomes enable consumers to enjoy mango fruits that have an extended shelf life and improved nutritional value. acknowledgements the authors are grateful to master vo k.p. for her plant material support. references alonso­salinas r., lópez­miranda s., gonzález­ báidez a., pérez­lópez a.j., noguera­artiaga l., 238 adv. hort. sci., 2024 38(3): 233­238 núñez­delicado e., carbonell­barrachina á., acosta­motos j.r., 2023 ­ effect of potassium permanganate, ultraviolet radiation and titanium oxide as ethylene scavengers on preservation of postharvest quality and sensory attributes of broccoli stored with tomatoes. ­ foods, 12(12):2418. 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daniel et al., 1955; defrance, 1951).this method consists of three types of mixing including cool-cool season turf, cold-warm season turf and rarely warm-warm season turf seed mixtures. mixture of cool-warm season turf may be useful for areas with inconsistent environmental conditions because each component in the mixture excels in a specific environment (davis, 1958), especially in a transition zone (akbari et al., 2011; salehi, 2008). for example, in missouri (classified as a transition zone) bermudagrass and kentucky bluegrass are grown together (dunn et al., 1994). bermudagrass dominates in the summer, while kentucky bluegrass in the cooler months of fall and spring (daniel et al., 1955). early investigations to mix bermudagrass with cool season species were often unsuccessful because of the dominance of bermudagrass during summer months (beard, 1973; davis, 1958). this problem could be solved by: i) using warm season grasses with lower dominance in order to get a lower competition between the species. there is no known report on this method (salehi, 2008). ii) using more aggressive cool-season turf may lessen the advantage for bermudagrass in summer and may make a permanent, balanced, cool-warm season mixture more practical than in past years (davis, 1958; turgeon, 1991). iii) using growth inhibitors for warm-season turfs in the first summer after planting to prevent exceeding growth or using accelerator hormones in the first spring and fall after planting for more growth of cool season turfs (salehi, 2008). comparison of tall fescue (festuca arundinacea schreb.) and common bermudagrass (cynodon dactylon [l.] pers.) turfgrasses and their seed mixtures m.r. salehi, h. salehi (1) department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: combined seeds, lawn, management practices, visual quality. abstract: with dense shoots above ground, a well-developed root system and large amounts of biomass underground, turfgrass provides many environmental benefits, including moderating soil erosion, water runoff and leaching, contributing to carbon sequestration, moderating temperatures, and reducing noise, glare, and visual pollution. in this investigation, (cynodon dactylon [l.] pers.) tall fescue (festuca arundinacea schreb.) and common bermudagrass were compared in monoculture and different mixtures of 0 to 100%, based on the number of seeds used. perennial ryegrass (lolium perenne l.), a common lawn in shiraz, was used as control. the experiment was conducted in a split block design (season as main plot and turfgrass types as subplot) and each treatment had four replications. data were analyzed with mstatc software and means were compared using tukey’s test at 5% level. turfgrasses were compared by measuring visual quality after winter and summer, rooting depth, verdure and/ or root fresh and dry weight, tiller density, and clippings fresh and dry weight. results showed that, with the exception of mean rooting depth and chlorophyll index after summer, spring sowing is better than fall sowing. however, it can be concluded that the 80% tall fescue and 20% bermudagrass treatment is the best treatment, or has not significant differences with the other good treatments, except with regard to tiller density. this type can be used alternatively in overseeding programs in areas with soil and environmental conditions similar to the present investigation site. adv. hort. sci., 2013 27(1-2): 81-87 (1) corresponding author: hsalehi@shirazu.ac.ir received for publication 11 april 2013 accepted for publication 3 may 2013 82 misiha (1991) compared different turfgrass seed mixtures and demonstrated that the mixture of poa pratensis l. ‘entopper’ with festuca rubra l. ‘hareld’ had the best establishment rate and highest chlorophyll content, plant density, and clipping dry weight. newell et al. (1996) indicated that lolium perenne l. and festuca rubra l. seed mixture had the best wear tolerance. salehi and khosh-khui (2004) used lolium perenne l., cynodon dactylon (l.) pers., poa pratensis l. and festuca rubra l. in mixtures of 1:1 (by weight) and a 1:1:1:1 (by weight) and two sport turfgrass cultivars, bar11 (barenbrug co.) and mm (mommersteeg co.). the seeds were sown in march and october in two years. the turfgrasses were compared by measuring visual quality, chlorophyll index after winter and summer, rooting depth, verdure and/or root fresh and dry weight, tiller density, and clippings fresh and dry weight. they showed that fall sowing was superior to spring sowing and resulted in greater root growth, clippings yield, and chlorophyll content. poa+cynodon seed mixture was the best treatment and showed high tiller density, root growth, and chlorophyll content. they concluded that the cool-warm season seed mixture (poa+cynodon) can be used alternatively in overseeding programs in transition zone areas similar to shiraz, iran. following the study by salehi and khosh-khui (2004), akbari et al. (2011) compared poa and cynodon turfgrasses and their seed mixtures. in this research turfgrasses kentucky bluegrass (poa pratensis l. ‘merion’) and common bermudagrass (cynodon dactylon [l.] pers.), in monoculture or in mixtures of 0 to 100%, based on number of seeds, were used. perennial ryegrass (lolium perenne l. ‘barball’), a common turf in the campus of shiraz university, was used as control. the seeds were sown in october in two years. the turfgrasses were compared by measuring visual quality after winter and summer, chlorophyll index after winter and summer, rooting depth, verdure and/or root fresh and dry weight, tiller density, and clippings fresh and dry weight. poa monoculture showed high tiller density, root fresh and dry weight and total fresh and dry weight. lolium monoculture showed high rooting depth after winter and clippings fresh and dry weights. cynodon monoculture quality was poor with regard to many characteristics, mainly due to fall sowing. the seed mixture composed of 20% cynodon + 80% poa was the best treatment and resulted in the highest rooting depth after summer, verdure fresh and dry weights, chlorophyll index after winter and summer, visual quality after winter and summer, and a good turf according to the other characteristics. based on good characteristics of recently coming cool season festuca arundinacea schreb. cultivars to shiraz, we proposed to test the performance of cool-warm season seed mixtures of a cultivar of this species with common bermudagrass in the same location as previous report. to the best of our knowledge there is no report of testing the seed mixture of this species with bermudagrass. 2. materials and methods characteristics of experimental location studies were conducted at the experimental farm of the department of horticultural science, college of agriculture, shiraz university, shiraz, iran, at bajgah, 1810 m above mean sea level, 52°32’ e and 29°36’ n, with daneshkadeh soil series (fine, mixed, mesic, calcixerollic xerochrepts, ph=8), from 2007 to 2008. the meteorological data for the experimental site is shown in table 1. long-term averages of maximum and minimum temperatures are 38°c and -9°c, respectively and yearly precipitation at this site is 400 mm. treatments the species used in this study were festuca arundinacea schreb. ‘starlet’ and cynodon dactylon [l.] pers. (california origin) in monoculture or in different mixture (by number of seed) and lolium perenne l. cultivar squire in monostand. in this investigation perennial ryegrass was used as control (table 2). turfgrass plots were established by directly sowing the seeds at two times, april and october, in 2007. management practices during investigation turfgrasses were irrigated every week in spring, every four days in summer, and every ten days in fall and every month in winter. it was enough to moisten the soil without overwatering. turfgrasses were mowed at 5 cm height with an electrical mower. weeds were manually removed. in this study no fertilizer was used. table 1 monthly average of temperature and precipitation (from april 2007 to august 2008) year month precipitation (mm) minimum t (°c) maximum t (°c) average t (°c) 2007 april 138.5 -3 24.5 11.56 may 3 2 30.5 17.37 june 0 7.8 38 22.07 july 2.5 12.4 38 25.86 august 0 9 36 24.16 september 0 6 36 21.26 october 0 3 29.5 15.79 november 0 -5 26.5 11.34 december 0 -12.3 18.5 3.4 2008 january 34 -14.8 12.6 0.5 february 35 -13.7 20 3.5 march 0 -9.6 22.2 7.5 april 3.5 -3 29 14 may 0 1.4 32.5 17.3 june 0 5 38 22.4 july 0 10.6 39 25.1 august 0 13 38 24.2 83 measurements tiller density, verdure fresh and dry weight, clippings fresh and dry weight, root fresh and dry weight and total fresh and dry weight were measured after summer (october). chlorophyll index and mean root depth and visual quality were measured after summer and winter (october and march, respectively). visual quality was assessed periodically throughout and after each growing season using a ranking scale from 0 to 9, 0= no live turf; 9= ideal shoot density, winter and summer color, and uniformity. clippings and verdures were collected 20 days after mowing from 5 cm above the ground. to measure dry weight, the materials were dried out at 60°c for 48 h. all data were measured for 100 cm2 subsamples in each plot. a random subsample of each plot was collected using a 10×10×50 cm metal block inserted into the soil. then, the samples were soaked in tap water and soil was removed. after cleaning and air drying, plant samples were transferred to the laboratory for further measurements. chlorophyll index (mg 100 cm-2) was measured by spectrophotometric methods (by spectronic 20d device) at 645 and 663 nm wavelengths (salehi and khosh-khui, 2004). experimental design and data analysis experiments were conducted in 2×12 split block design with factorial arrangements, sowing season (spring and fall) as main plot and seed mixtures as subplot, and four replications in each treatment. individual plots measured 4 m2 (1×4 m). data were analyzed using mstat-c program and the mean comparisons were made following tukey’s test at p ≤0.05. 3. results average root depth after summer results show that with spring sowing, there was no significant difference among all the seed mixtures’ average root depth after summer. however, with fall sowing, the increase of tall fescue percentage led to greater root depth in the soil. the deepest root system was observed in treatment 1 with fall sowing composed of 100% seeds of tall fescue. comparing averages of spring and fall sowing data, it is clear that the average root depth with fall sowing is greater than spring sowing (table 3). average root depth after winter with spring sowing the deepest root system belonged to the treatment composed of 100% tall fescue seed; decreasing the percentage of tall fescue seed, the root depth decreased. the deepest root system with fall sowing was found in lolium monoculture, followed by the mixture with 90% tall fescue and 10% bermudagrass. by decreasing the percentage of tall fescue seeds, root depth decreased, except between treatments 4 and 5, in fall sowing and after winter. averages of spring and fall sowing data indicate that the highest proportion of the root system grows near the soil surface with fall sowing (table 3). tiller density the average tiller density with spring sowing was found to be higher than with fall sowing. in spring, the mixture composed of 60% tall fescue and 40% bermudagrass had the highest tiller density and bermudagrass monoculture had the lowest; in fall, lolium monoculture had the highest and bermudagrass monoculture had the lowest (table 3). root fresh and dry weight according to the results obtained, with the spring sowing, the mixture composed of 70% tall fescue and 30% bermudagrass produced the heaviest root system. with a decreasing percentage of tall fescue seeds, root system weights decreased. therefore, the lowest root weight with spring sowing was observed in bermudagrass monoculture. with the fall sowing, the mixture composed of 80% tall fescue and 20% bermudagrass produced the heaviest and the 10% tall fescue and 90% bermudagrass mixture produced the lightest root systems (table 3). clippings fresh and dry weight maximum values for clippings fresh and dry weights were found with the spring sowing of 60% tall fescue and 40% bermudagrass mixture. however, there was no significant difference (p≤0.05) for treatments 1 to 6. furthermore, increasing the percentage of bermudagrass seeds (up to 40%), clippings weight increased and then decreased. with the fall sowing, the maximum clippings weight belonged to the mixture with 90% tall fescue and 10% bermudagrass. however, by decreasing the percentage of tall fescue seeds the weight decreased. indeed, the lowest clippings weight was found with bermudagrass monoculture. averages of spring and fall sowings data table 2 seeding rate of turfgrasses used (by percentage and weight) treatment number cynodon (%) lolium (%) festuca (%) cynodon (g m-2) lolium (g m-2) festuca (g m-2) 1 0 0 100 0.0 0 46.0 2 10 0 90 0.7 0 41.4 3 20 0 80 1.4 0 36.8 4 30 0 70 2.1 0 32.2 5 40 0 60 2.8 0 27.6 6 50 0 50 3.5 0 23.0 7 60 0 40 4.2 0 18.4 8 70 0 30 4.9 0 13.8 9 80 0 20 5.6 0 9.2 10 90 0 10 6.3 0 4.6 11 100 0 0 7.0 0 0.0 12 0 100 0 0.0 50 0.0 84 showed that clipping weights (both fresh and dry) of fall sowing was more than spring sowing (table 4). verdure fresh and dry weight spring sowing results reveal that the maximum verdure fresh and dry weights belonging to the mixture composed of 60% tall fescue and 40% bermudagrass. however, it was not significantly different in treatments 4 and 6. with fall sowing, the 70% tall fescue and 30% bermudagrass mixture produced the maximum verdure weight. however, it was not significantly different in treatments 2 to 7 and 12 and tall fescue monoculture. furthermore, averages of spring and fall sowing data show that verdure fresh and dry weights were greater in spring sowing than fall sowing (table 4). total fresh and dry weight the maximum total fresh and dry weights with spring sowing were found in treatment 5 (60% tall fescue and 40% bermudagrass). however, the values were not significantly different from treatment 4. the minimum total weight with spring sowing was observed in bermudagrass monoculture. with fall sowing, maximum and minimum total weights were noted in treatments composed of 80% tall fescue and 20% bermudagrass and bermudagrass monoculture, respectively. furthermore, the average total weight of spring sowing was more than fall sowing (tables 4 and 5). chlorophyll index after winter with regard to chlorophyll, results showed that with spring sowing, the maximum index belonged to the 70% tall fescue and 30% bermudagrass mixture. a decrease in tall fescue seeds percentage led to a decrease in chlorophyll index. with fall sowing, the maximum index was found with the 90% tall fescue and 10% bermudagrass mixture (treatment 2). however, there was no significant difference between this treatment and treatments 3 and 4 (table 5). chlorophyll index after summer as for the chlorophyll index after summer, with spring sowing, treatment 3 gave the maximum chlorophyll index, however there was no significant difference between this treatment and treatments 1 and 2; the minimum index was table 3 effects of different seed mixtures and sowing times on some biological characteristics of the turfgrasses used sowing season treatment number average root depth after summer (cm) average root depth after winter (cm) tiller density (no 100 cm-2) root fresh weight (g) root dry weight (g) spring 1 26.25 c-f* 33.37 a 175 de 53.75 cd 22.40 bc 2 25.37 c-f 33.25 a 180 cd 52.50 cd 21.00 c 3 23.75 e-h 31.50 ab 186 bcd 51.80 cde 20.34 cd 4 23.25 e-h 29.62 bc 193 ab 59.75 bc 22.98 bc 5 22.50 fgh 29.00 bcd 194 ab 57.87 bc 22.26 bc 6 25.00 c-g 28.75 bcd 179 cde 44.60 def 16.86 de 7 23.25 e-h 28.75 bcd 155 fg 42.50 efg 15.74 ef 8 24.75 d-g 26.87 c-f 141 hi 40.75 fg 14.87 efg 9 24.62 d-g 27.12 cde 129 ij 34.25 gh 12.32 fgh 10 24.50 d-g 25.87 def 124 jk 22.75 ij 7.84 ij 11 23.75 e-h 24.12 efg 116 kl 19.87 ij 6.56 j 12 26.75 cde 32.25 ab 178 cde 35.70 fgh 11.57 gh average 24.48 b 29.21 a 162.8 a 43.02 a 16.22 a fall 1 33.62 a 23.50 fg 183 bcd 66.00 ab 27.50 a 2 33.25 a 24.50 efg 185 bcd 66.87 ab 26.96 a 3 31.75 ab 24.12 efg 190 abc 69.87 a 27.95 a 4 31.75 ab 22.12 gh 177 de 64.12 ab 24.85 ab 5 28.75 bc 24.00 efg 167 ef 57.62 bc 21.83 bc 6 27.75 cd 19.62 hi 152 gh 35.60 fgh 13.49 e-h 7 25.00 c-g 19.37 hi 148 gh 34.37 gh 12.88 fgh 8 24.00 c-g 16.87 ij 130 ij 29.12 hi 10.48 hi 9 23.37 e-h 14.12 jk 105 lm 19.00 j 6.78 j 10 21.12 gh 12.75 k 96 m 13.75 j 4.79 j 11 20.00 h 11.62 k 77 n 14.37 j 4.79 j 12 31.75 ab 25.12 efg 198 a 42.32 fg 12.81 fgh average 27.68 a 19.81 b 151 b 42.75 b 15.27 b * in each column, means followed by the same letter(s) (small letters for means and capital letters for main averages) are not significantly different at 5% level according to tukey’s test. 85 found with treatment 11. with fall sowing, maximum and minimum chlorophyll indices were found with treatments 2 and 11, respectively. averages of spring and fall sowing data showed that the chlorophyll index for spring sowing is significantly greater than fall sowing (table 5). visual quality after summer with spring sowing, treatment 5 gave grasses with the best visual quality. however, there was no significant difference between treatments 2 to 7, and tall fescue, bermudagrass and lolium monocultures. with fall sowing, treatment 3 gave the best visual quality; there was no significant difference between this treatment and treatments 2 to 5, and tall fescue and lolium monocultures. furthermore, the average visual quality of spring sowing was greater than fall sowing (table 5). visual quality after winter the best average of visual quality with spring sowing was found with the treatment composed of 100% lolium and, generally, by decreasing the percentage of tall fescue seeds the visual quality decreased. however, visual quality after winter increased between treatments 2 and 3, and also between 4 and 5. with fall sowing, lolium monoculture gave the best average visual quality. in the mixtures, the 80% tall fescue and 20% bermudagrass mixture gave the best visual quality and the worst was found with bermudagrass monoculture. averages of spring and fall sowings data showed that visual quality with spring sowing is more than fall sowing (table 5). 4. discussion and conclusions average root depth after summer there were no significant differences among different seed mixtures’ root depth with spring sowing, maybe because their growth took place at the same time in spring. maximum root depth in spring belonged to lolium, based on its powerful germination and growth at the beginning of culture (christians 2004; salehi 2008; akbari et al., 2011). with fall sowing, by increasing the tall fescue seed pertable 4 effects of different seed mixtures and sowing times on some biological characteristics of the turfgrasses used sowing season treatment number clippings fresh weight (g) clippings dry weight (g) verdure fresh weight (g) verdure dry weight (g) total fresh weight (g) spring 1 8.00 de* 1.68 efg 16.75 d-g 5.46 def 78.50 de 2 7.95 def 1.69 efg 23.37 b 7.30 ab 83.82 cd 3 7.95 def 1.69 efg 21.62 bc 6.36 bcd 81.45 d 4 8.17 de 1.77 def 24.87 ab 7.23 ab 92.80 abc 5 8.60 cd 1.91 cde 27.00 a 7.71 a 93.47 abc 6 7.90 def 1.76 d-g 23.75 ab 6.74 abc 76.27 de 7 6.97 efg 1.58 fgh 19.12 cde 5.31 def 68.60 efg 8 6.42 gh 1.48 gh 16.87 d-g 4.60 f-i 64.05 fgh 9 5.00 ij 1.18 ij 15.50 fgh 4.17 g-j 54.75 hi 10 4.17 ijk 1.01 jk 14.50 ghi 3.87 h-k 41.42 jk 11 3.20 kl 0.80 kl 10.77 jk 2.83 kl 33.85 kl 12 6.90 efg 1.37 hi 17.37 d-g 4.96 efg 60.02 fgh average 6.77 b 1.49 b 19.29 a 5.54 a 69.08 a fall 1 10.65 ab 2.26 ab 16.25 e-h 5.41 def 92.60 abc 2 11.20 a 2.38 a 17.12 d-g 5.38 def 95.20 ab 3 11.15 a 2.36 a 18.62 c-f 5.85 cde 99.65 a 4 9.75 bc 2.13 abc 19.62 cde 5.85 cde 93.50 abc 5 9.12 cd 1.99 bcd 18.62 c-f 5.46 def 85.37 bcd 6 8.37 d 1.88 cde 18.62 c-f 5.46 def 62.62 fgh 7 6.60 fg 1.50 fgh 17.00 d-g 4.83 e-h 57.97 ghi 8 5.12 hi 1.19 ij 13.12 hij 3.70 ijk 47.37 ij 9 3.75 jkl 0.88 kl 11.75 ij 3.25 jk 34.50 kl 10 2.52 lm 0.61 lm 10.87 jk 2.93 kl 27.15 l 11 1.72 m 0.43 m 8.10 k 2.16 l 24.20 l 12 7.95 def 1.59 fgh 19.77 cd 5.63 def 70.05 ef average 7.32 a 1.60 a 15.79 b 4.58 b 65.87 b * in each column, means followed by the same letter(s) (small letters for means and capital letters for main averages) are not significantly different at 5% level according to tukey’s test. 86 centage, the average root depth increased because tall fescue had good growth in early autumn while at the same time bermudagrass growth was very weak as it is a warmseason turfgrass. average root depth after winter with spring sowing, maximum root depth belonged to tall fescue monoculture. moreover, with increased tall fescue seed percentage in spring sowing, the growth of bermudagrass became slow with the beginning of the fall season, however lolium and tall fescue grew continuously. with fall sowing, maximum root depth was found with lolium because of its power to germinate. an increase in tall fescue seed percentage led to root depth increases because tall fescue goes into dormancy slowly and with increased temperature growth starts early. tiller density with both sowing times, minimum tiller density was found with bermudagrass, which confirms the results of akbari et al. (2011). furthermore, with increasing tall fescue seed percentage, tiller density increased, which may refer to its power of tiller production. root fresh and dry weight increasing bermudagrass seed percentage led to decreases in root weight for both the sowing times, confirming the results of akbari et al. (2011). clippings and verdure fresh and dry weight with spring and fall sowing times, maximum weight was found in treatment 5 (60% tall fescue) and treatment 2 (90% tall fescue), respectively for clippings; and treatment 5 60% (tall fescue) and treatment 4 (70% tall fescue), respectively for verdure weight. because tall fescue has good shoot growth, increasing its seed percentage increases clippings weight. however, this character is not suitable because it means increased mowing. total fresh and dry weight maximum total weights were found among treatments composed of higher percentages of tall fescue seeds; with table 5 effects of different seed mixtures and sowing times on some biological characteristics of the turfgrasses used sowing season treatment number total dry weight (g) chloropyll index after winter** chlorophyll index after summer** visual quality after summer visual quality after winter spring 1 29.53 cd* 2.10 cd 9.38 ab 8.25 ab 8.00 a-d 2 29.99 cd 2.07 cd 9.41 a 7.75 a-d 7.75 a-e 3 28.40 de 2.07 cd 9.47 a 8.37 ab 8.12 a-d 4 31.99 bcd 2.28 abc 9.05 b 8.37 ab 8.12 a-d 5 31.89 bcd 1.94 def 8.60 c 8.87 a 8.37 abc 6 25.35 ef 1.74 fg 8.27 cde 8.37 ab 7.00 b-f 7 22.64 fg 1.41 h 8.00 ef 8.00 ab 6.37 def 8 20.97 gh 1.07 i 7.79 fg 7.12 b-e 6.00 efg 9 17.67 hi 0.74 j 7.54 gh 6.62 cde 5.25 fg 10 12.73 hi 0.45 k 7.29 hij 7.12 b-e 2.87 hi 11 10.19 kl 0.09 l 7.04 j 7.75 a-d 0.00 k 12 17.90 hi 1.61 gh 8.19 de 8.62 a 8.87 a average 23.27 a 1.47 b 8.34 a 7.93 a 6.39 a fall 1 35.17 ab 2.24 bc 9.32 ab 8.50 a 8.37 abc 2 34.74 ab 2.47 a 9.36 ab 8.50 a 8.50 abc 3 36.12 a 2.37 ab 9.30 ab 8.87 a 8.75 ab 4 32.84 abc 2.32 ab 8.60 c 8.50 a 8.12 a-d 5 29.84 cd 2.00 de 8.41 cd 7.87 abc 7.87 a-d 6 20.74 gh 1.79 efg 8.06 def 6.25 ef 6.75 c-f 7 19.20 ghi 1.52 h 7.83 fg 5.87 efg 5.37 fg 8 15.38 ij 1.05 i 7.53 gh 5.00 fgh 4.50 gh 9 10.39 kl 0.84 j 7.39 hi 4.87 gh 1.87 ij 10 8.34 l 0.35 k 7.16 ij 4.37 h 1.00 jk 11 7.38 l 0.03 l 6.54 k 6.50 de 0.00 k 12 20.03 gh 1.82 ef 8.22 de 8.78 a 9.00 a average 22.51 b 1.57 a 8.14 b 6.99 b 5.84 b * in each column, means followed by the same letter(s) (small letters for means and capital letters for main averages) are not significantly different at 5% level according to tukey’s test. **mg 100 cm-2. 87 increasing bermudagrass seed percentage, total weight decreases, especially with fall sowing. chlorophyll index after winter and summer after winter, the chlorophyll index in bermudagrass was very low because it is a warm season turf and in winter its shoots become chlorotic. however, tall fescue and lolium shoots remain green during winter. after summer, all the mixtures increased the chlorophyll index of shoots in all the seed mixtures. furthermore, by lowering the plant density of tall fescue, light can penetrate more easily and thus the chlorophyll index becomes greater than tall fescue monoculture. visual quality after summer and winter visual quality has a direct correlation with chlorophyll index: as chlorophyll index increases, so does visual quality. furthermore, visual quality depends on weed density and in mixtures with more bermudagrass the number of weeds increases, particularly with fall sowing. among festuca and cynodon seed mixtures, 80%f + 20%c was selected as an excellent turfgrass mixture in this study, with regard to all the positive characteristics except tiller density. this seed mixture established a fine, green color throughout the year. furthermore it can be used alternatively in overseeding programs in areas with soil and environmental conditions similar to the present investigation site. additional studies are needed to investigate the best cultural conditions of this selected seed mixture. references akbari m., salehi h., khosh-khui m., 2011 coolwarm season poa-cynodon seed mixtures and their turf growth and quality. acta agric. scand., section b soil plant sci., 61(6): 559-564. beard j.b., 1973 turfgrass: science and culture. prenticehall, englewood cliffs, nj, usa, pp. 704. christians n., 2004 fundamentals of turfgrass management. john wiley and sons inc., nj, usa, pp. 359. corman j.f., 1955 home lawns. new york agr. exp. sta. ext. bul., no. 922. daniel w.h., hull r.b., lee o.c., lehker g.e., sharvelle e.g., 1955 the lawn-how to establish and maintain. indiana agr. exp. sta. ext. bul., no. 245. davis r.r., 1958 the effect of other spices and mowing height on the persistence of lawn grasses. agron. j., 50: 671-673. defrance j.a., 1951 how to make a lawn. quart. rev., rhode island agr. exp. sta. dunn j.h., minner d.d., fresenburg b.f., bughrara s.s., 1994 bermudagrass and cool season turfgrass mixtures: response to simulated traffic. agron. j., 86: 1016. misiha a., 1991 effect of cool season turfgrasses seed mixtures on lawn characteristics. bul. facult. agr. univ. cairo, 42: 401-414. newell a.j., crossley f.e.m., jones a.c., 1996 selection of grass species, cultivars and mixtures for lawn tennis courts. j. sports turf. res. inst., 72: 42-60. niehaus m.h., 1976 effect of cultivar, seeding rate, and nitrogen fertilization on kentucky bluegrass-perennial ryegrass turf mixtures. agron. j., 68: 955-977. salehi h., khosh-khui m., 2004 turfgrass monoculture, cool-cool, and cool-warm season seed mixture establishment and growth responses. hortscience, 39: 17321735. salehi m.r., 2008 comparison between tall fescue and common bermudagrass turfgrasses and their seed mixtures. msc thesis, shiraz university, iran. turgeon a.j., 1991 turfgrass management. prentice-hall, englewood clifs, nj, usa, pp. 392. 153 1. introduction mangosteen (garcinia mangostana l.), the “queen of fruits” is a successful introduction into kerala and flourishes well under the warm humid tropics (yusuf and kurien, 2012). it has high export potential but is limited by its long gestation period (wiebel et al., 1992, 1995). however, this long period of 10-15 years (lim, 1984; richards, 1990; wiebel et al., 1995) can be reduced by resorting to vegetative propagations; the problem of slow growth only gets magnified and the consequent low canopy volume leads to lower yield. hence, mangosteen related work that will lead to activation of growth should get top priority in research. the knowledge of symbiotic associations of mycorrhizal fungi with roots of vascular plants is a century old (mohandas, 1993). root infections by arbuscular mycorrhizal (am) fungi have been reported in many perennial fruits such as grapes citrus, and apple. root inoculated perennials with am fungi effectively enhances the growth of plants such as plant height, number of leaves and leaf area. inoculation of am fungi in mangosteen resulted in significant changes in length-related characteristics (masri et al., growth studies in mangosteen (garcinia mangostana l.). ii. activation of seedling growth in mangosteen using arbuscular mycorrhizal fungi and azospirillum l.m. yusuf 1, s. kurien 2*, k. surendragopal 3, a. augustin 4 1 bhabha atomic research centre, vishakapatnam, 530012 india. 2 department of science and technology, kerala agricultural university (kau), 680656 thrissur, kerala, india. 3 department of agricultural microbiology, kerala agricultural university (kau), 680656 thrissur, kerala, india. 4 centre of plant biotechnology and molecular biology, kerala agricultural university (kau), 680656 thrissur, kerala, india. key words: arbuscular mycorrhizal fungi, azospirillum, garcinia mangostana, glomus fasciculatum, glomus mosseae. abstract: an experiment was undertaken in the central orchard at the main campus of the kerala agricultural university to address the slow growth in mangosteen, a highly potential crop of the humid tropics. glomus mosseae, glomus fasciculatum and azospirillum individually and in combinations, as well as a control, formed the treatments. the treatments were adequately replicated in a completely randomized design. the best treatments for activating seedling growth were the combinations of glomus fasciculatum (5 g) + azospirillum (10 g) + single super phosphate (10 g) followed by glomus fasciculatum (5 g) + azospirillum (20 g) + single super phosphate(10 g) per plant. a rhythmic pattern was observed with the treatments giving the best seedling growth also yielding higher values of nitrogen, phosphorus, potassium, crude protein, chlorophyll a, b, total chlorophyll, total phenol total carbohydrates and abscisic acid content; treatments with intermediate growth recording also gave intermediate values except in the case of sodium. control plants gave values that fell beween those of intermediate and the least growth. the highest spore count was observed in plants inoculated with glomus fasciculatum (20 g) + single super phosphate (10 g) followed by glomus fasciculatum (20 g) + azospirillum (20 g) + single super phosphate (10 g). with regard to root infection, plants inoculated with glomus fasciculatum (5 g) + azospirillum (20 g) + single super phosphate (10 g) per plant and glomus fasciculatum (20 g) + azospirillum (10 g) + single super phosphate (10 g) revealed the maximum percentage of infection. the azospirillum population was highest in the plants inoculated with glomus fasciculatum (5 g) + azospirillum (10 g) + followed by glomus mosseae (20 g) + azospirillum (20 g) + single super phosphate (10 g). the standard procedure for identification and quantification of abscisic acid was modified, as clear banding patterns were not obtained. using the modified procedure, the characteristic-banding pattern corresponding to standard abscisic acid was obtained and confirmed when standards of abscisic acid were also simultaneously used with samples. banding patterns and quantification of samples of the treatment with arbuscuar mycorrhizal fungi and azospirillum-inoculated plants were also successfully obtained and are presented. growth measurements at the end of the first year revealed that all characters recorded were far superior to the established selection indices for the purpose. adv. hort. sci., 2014 28(3): 153-163 (*) corresponding author: sajanalice@gmail.com received for publication 8 september 2014 accepted for publication 12 november 2014 154 1998). other similar reports exist on increased growth in various crop plants including root growth enhancement as reported and reviewed by gerdemann (1968) and cherian (2001). the role of am fungi in increasing the mobilization and uptake of p and thereby the productivity of many crops is well documented and reviewed by gerdemann (1968), mosse, (1973), meenakumari (1987), nelsen (1987) and bhandari et al. (1990). there are numerous reports of am fungi increasing the n concentration in plant shoots and aiding in stimulating nodulation (carling et al., 1978). am fungi directly enhances the uptake of micronutrients, viz. zn, cu and fe (gildon and tinker, 1983; kucey and tanzen, 1987). zinc deficiency can also be corrected by inoculating plants with an endomycorrhizal fungus (gilmore, 1971). it was observed that am fungi association resulted in a higher uptake of micronutrients in various plants, which was brought about by selective uptake and better utilization of n, cu, zn and s in various crops (bhandari et al., 1990) with regard to azospirillum, there is only scanty information available on its influence in perennial crops (rao, 1982). studies have been made in the rhizosphere and rhizoplane of cocoa and pepper (govindan and nair, 1984; govindan and chandy, 1985). however, most of the studies with azospirillum are on field crops (rao et al., 1979). rao and dass (1989) found that soil inoculation with pure cell suspension of azospirillum brasilense or azotobacter chrocaccum resulted in growth enhancement of ber and pomegranate. enhanced root elongation, root hair development and branching in a number of crops have been reported following azospirillum inoculation (kapulnik et al., 1983). high crude protein content was noticed in inoculated plants (patel et al., 1993). however, there are also reports on the dual inoculation of am fungi and azospirillum and its growth response in plants. combined inoculation of azospirillum and am fungi significantly increased shoot biomass in mulberry (nagarajan et al., 1989); plant height, shoot and root weight in pepper (bopaiah and khader, 1989); and shoot growth, and thereby biomass production, in tectonia grandis (sugavanam et al., 1998). greater root colonization resulting in higher n and p and micro nutrients like fe, cu, zn and mn have been reported in coffee (kumari and balasubramanian, 1993). sonowane and konde (1997) revealed that co-inoculation of am fungi and azospirillum or azotobacter resulted in the highest leaf area in studied grape vines and comparison between the two revealed that azospirillum was superior when used in conjunction with a mixed culture of am fungi (sugavanam et al., 1998). the probable reason for this increased dry weight was attributed to a higher photosynthetic rate (estrada-luna, 2000), or perhaps the production of growth promoting compounds namely auxin, gibberellins and cytokinins or vitamins (miller, 1971; crafts and miller, 1974; slankis, 1975). masri et al. (1998) observed that arbuscular mycorrhiza enhanced the growth and reduced the nursery period of mangosteen (garcinia mangostana l.) seedlings. in mangosteen (garcinia mangostana l.), alteration of root system characteristics and nutrient uptake in response to am fungal inoculation have been studied. arbuscular mycorrhizal inoculation induced significant changes in root characteristics and this was accompanied by a tremendous increase in nutrient uptake. uptake of p was increased by 67-88% in inoculated seedlings (masri and azizah, 1998). the present study was undertaken with the prime objective of ascertaining as to whether the growth rate in mangosteen can be increased through symbiotic association of am fungi and azospirillum. however, its presence and beneficial effects in cultivated crops of kerala have been reported by various workers (potty, 1978; sivaprasad et al., 1982, 1984; girija and nair, 1985; nair and girija, 1986). 2. materials and methods the experimental site experiences a warm humid tropical monsoon climate. it is situated at 12°32’n latitude and 74°20’e longitude at an altitude of 22.5 m above mean sea level. the study was carried out at the kerala agricultural university central orchard, thrissur. the area receives an average rainfall of 2150 mm distributed over a year’s period. the mean maximum temperature ranged 28-36°c and the mean minimum temperature 12.8-20.6°c. the relative humidity was 90-98% with a mean of 94%. the soil type is typical sandy clay loam with a ph of 5.4, ec of 1.25 dsm-1 and belongs to the order ultisols with 8 ph ranging from 5.5-5.8. the soil is low in available n and p 2 05 and high in k 2 o. fruits were collected from plants belonging to the same age group (25-50 years) from the pariyaram area of the thrissur district in kerala and seeds were extracted. the seeds were sown in black polythene bags (45x30 cm) filled with potting mixture comprised of farmyard manure, sand and cow dung in the ratio 2:2:1. the weight of the potting media was 5 kg, which was uniformly maintained. seedlings were subjected to a secondary selection for uniformity in growth. three months after germination of the seeds, the treatments were inoculated in the potting media and a uniform dose of 10 g of single super phosphate was added to the potting media of each polybag. the treatments were as follows: 1. glomus mosseae (g.m.) 5 g 2. glomus mosseae 10 g 3. glomus mosseae 20 g 4. glomus fasciculatum (g.f.) 5 g 5. glomus fasciculatum 10 g 6. glomus fasciculatum 20 g 7. azospirillum (az.) 5 g 8. azospirillum 10 g 9. azospirillum 20 g 10. azospirillum 5 g + glomus mosseae 5 g 11. azospirillum 10 g + glomus mosseae 5 g 155 12. azospirillum 20 g + glomus mosseae 5 g 13. azospirillum 5 g + glomus mosseae 10 g 14. azospirillum 10 g + glomus mosseae 10 g 15. azospirillum 20 g + glomus mosseae 10 g 16. azospirillum 5 g + glomus mosseae 20 g 17. azospirillum 10 g + glomus mosseae 20 g 18. azospirillum 20 g + glomus mosseae 20 g 19. azospirillum 5 g + glomus fasciculatum 5 g 20. azospirillum 10 g + glomus fasciculatum 5 g 21. azospirillum 20 g + glomus fasciculatum 5 g 22. azospirillum 5 g + glomus fasciculatum 10 g 23. azospirillum 10 g + glomus fasciculatum 10 g 24. azospirillum 20 g + glomus fasciculatum 10 g 25. azospirillum 5 g + glomus fasciculatum 20 g 26. azospirillum 10 g + glomus fasciculatum 20 g 27. azospirillum 20 g + glomus fasciculatum 20 g 28. single super phosphate (ssp) 10 g alone 29. control the morphological observations recorded were the plant height, girth at collar, total number of leaves, number of new flushes per year, number of leaves/flush and total leaf area and survival rate of the seedlings at oneyear stage after germination stage. leaf area was calculated by standardizing a common factor (0.6727) and then multiplying the length breadth with this factor (i.e. l×b×factor). the leaf area was expressed as cm2. the factor was pre-standardised taking 100 leaves and measuring the length and breadth. the leaf area of the corresponding leaf was measured using a leaf area meter to work out the factor value. thus, the factor value (0.6727) was derived using the formula factor = (leaf area/length x breadth) using the factor value, the leaf area of a leaf/whole plant was calculated. survival rate was calculated by counting the established plants and expressing it as a percentage of the total number of seedlings observed after germination. shoot and root fresh weight and dry weight, root to shoot dry weight ratio, length of longest root, number of primary, secondary and tertiary roots, and total number of roots were also recorded. to estimate fresh weight of shoot and root, the seedlings were uprooted one year after germination. the plants were immediately cut and separated into shoots and roots. the fresh weights were recorded separately and the average expressed in grams. to obtain the dry weights, the samples collected to determine the fresh weights were dried in an oven maintained at 60°c till the weight of the samples remained constant. dry weights were recorded separately and the average expressed in grams. dry weight ratio of root to shoot was calculated as follows: dry weight ratio = (root dry weight/shoot dry weight) length of the longest root (tap root) was measured from the collar region to the growing tip using a scale and expressed in centimeters. for biochemical studies, leaf samples from seedlings were collected one year after germination; leaf samples from mother plants were also collected. the third leaf from the tip was collected and oven dried at 60°c, ground and used to estimate the content of n, p, k and na. the following methods were applied, as described by jackson (1973): for total nitrogen the microkjeldhal method was used and the average expressed as percentage; phosphorus content was determined using di-acid extract method; potassium content was determined with di-acid extract, then read in an eel flame photometer, at 548 nm, and the average expressed in percentage; nitrogen content was estimated by microkjeldhal method; the value of nitrogen content was multiplied by the factor 6.25 to obtain the crude protein content and the average expressed in percentage; sodium content was determined with di-acid extract, then read in an eel flame photometer at 598 nm and the average expressed in percentage. the chlorophyll content (total chlorophyll, chlorophyll a and chlorophyll b) was estimated in leaf samples using arnon’s acetone method (sadasivam and manickam, 1996) and the average expressed in milligrams. the total sugars were estimated using standard procedure (aoac, 1980), the total carbohydrates using anthrone method (dubois et al., 1951), and total phenol content using folin-ciocalteau method (sadasivam and manickam, 1996), all expressed in milligrams. the procedure adopted for quantification of abscisic acid was a modification of the standard method of little et al. (1972). the modification became imperative as bands were not obtained. the procedure was standardized and bands were obtained corresponding to the standard abscisic acid. further quantification was carried out using a u-v spectrophotometer and standards of known concentration from which a standard graph was obtained. microbial observations estimation of the spore population in the rhizosphere was carried out (gerdemann and nicolson, 1963) and expressed as number of spores/100/g soil. percent infection by am fungi was calculated as described by philips and hayman (1970). the infection percentage was worked using the standard formula: percent infection= (number of infected root segments/ total number of root segments observed) x 100. the azospirillum population in the rhizosphere was estimated using serial dilution technique. the population was then calculated using an mpn table or chart (cochran, 1950). the study of all morphological and biochemical characters using a combination of arbuscular mycorrhizal fungi (amf) + azospirillum + single super phosphate was carried out as a completely randomized block design using analysis of variance techniques. another set of plants receiving identical treatments were maintained for destructive analyses for taking root, shoot characters and biochemical analysis. the significance was tested by f test and the treatments were compared by duncan’s multiple range test (snedecor and cochran, 1983). 156 3. results morphological characters of seedlings all mean data on morphological characters of seedlings are presented in table 1. at the twelve-months stage, maximum height was observed in the plants inoculated with g.f. (5 g )+ az. (10 g )+ ssp (10 g), which was significantly superior to all other treatments except the next best combination of inoculation, g.f. (5 g) + az. (20 g) + ssp (10 g). the greatest significant increment in height was recorded in plants inoculated with the same treatment as at the six-months stage, followed by plants treated with ssp (10 g) alone. these treatments were statistically superior in terms of rate of height increment compared to other treatments. maximum girths at twelve months were recorded in the same treatment combinations as above, with both treatments statistically significant. also at the twelvemonths stage, the greatest increment was observed in the treatment combination g.f. (5 g) + az. (20 g) + ssp (10 g), which was significantly superior to g.m. (10 g) + az. (20 g) + ssp (10 g). these two treatments were significantly superior to all other treatments including the control. with regard to the total number of leaves at the twelvemonths stage, maximum leaf count was recorded with table 1 morphological characters of twelve-month-old mangosteen (garcinia mangostana l.) seedlings inoculated with arbuscular mycorrhizal fungi (amf) and azospirillum treatments (g) twelve month stage (nine months after inoculation) height (cm) increment in height (cm) girth (cm) increment in girth (cm) total number of leaves increment in total number of leaves new flushes/ year (no) leaves/ flush (no) total leaf area (cm2) survival rate (%) g.m. 5 10.13 c 2.43 bcd 1.31 d 0.09 d 12.67 abcde 2.00 abcd 1.57 ab 2.00 82.61 bc 100.00 a g.m. 10 7.73 c 1.33 d 1.62 abcd 0.11 cd 8.00 e 1.33 abcd 1.12 b 2.00 18.93 c 100.00 a g.m. 20 11.23 bc 1.33 d 1.77 abcd 0.25 abcd 12.33 abcde 3.33 ab 1.67 ab 2.00 31.66 c 100.00 a g.f. 5 8.93 c 1.27 d 1.38 cd 0.08 d 11.00 abcde 1.67 abcd 1.54 ab 2.00 42.43 bc 100.00 a g.f. 10 11.57 bc 1.67 d 1.68 abcd 0.21 abcd 14.33 abcd 1.67 abcd 2.00 a 2.00 107.00 abc 100.00 a g.f. 20 8.43 c 1.60 d 1.52 abcd 0.23 abcd 8.67 de 0.67 cd 1.22 ab 2.00 40.29 c 100.00 a az. 5 10.50 c 2.75 bcd 1.65 abcd 0.24 abcd 11.00 abcde 1.33 abcd 1.60 ab 2.00 111.10 abc 100.00 a az. 10 10.47 c 2.07 d 2.13 abcd 0.50 abcd 9.67 bcde 0.33 d 1.42 ab 2.00 84.49 bc 100.00 a az. 20 10.33 c 2.30 bcd 1.60 abcd 0.18 bcd 10.33 abcde 1.67 abcd 1.55 ab 2.00 47.77 bc 100.00 a az. 5 + g.m. 5 8.70 c 3.63 bcd 1.51 abcd 0.18 bcd 9.00 cde 1.00 bcd 1.21 ab 2.00 20.00 c 100.00 a az. 10 + g.m. 5 11.23 bc 3.10 bcd 1.76 abcd 0.30 abcd 13.00 abcde 2.00 abcd 1.80 ab 2.00 89.48 abc 100.00 a az. 20 + g.m. 5 8.83 c 2.70 bcd 1.54 abcd 0.20 abcd 11.33 abcde 2.00 abcd 1.68 ab 2.00 63.95 bc 100.00 a az. 5 + g.m. 10 11.73 abc 4.00 abcd 1.80 abcd 0.31 abcd 14.33 abcd 1.67 abcd 1.93 ab 2.00 119.80 abc 100.00 a az. 10 + g.m. 10 9.33 c 2.00 d 1.64 abcd 0.23 abcd 12.00 abcde 2.00 abcd 1.75 ab 2.00 76.21 bc 100.00 a az. 20 + g.m. 10 14.10 abc 3.90 abcd 2.18 abc 0.58 ab 16.33 a 3.00 abc 2.00 a 2.00 181.90 abc 100.00 a az. 5 + g.m. 20 13.27 abc 4.00 abcd 1.91 abcd 0.38 abcd 14.67 abcd 2.00 abcd 1.64 ab 2.00 205.20 abc 100.00 a az. 10 + g.m. 20 13.07 abc 3.57 bcd 1.91 abcd 0.37 abcd 14.67 abcd 2.00 abcd 1.68 ab 2.00 168.50 abc 100.00 a az. 20 + g.m. 20 11.00 bc 3.00 bcd 1.43 bcd 0.14 cd 12.00 abcde 1.67 abcd 1.34 ab 2.00 70.01 bc 100.00 a az. 5 + g.f. 5 11.70 abc 2.97 bcd 1.82 abcd 0.41 abcd 12.33 abcde 3.00 abc 1.43 ab 2.00 133.40 abc 100.00 a az. 10 + g.f. 5 18.30 a 6.47 abc 2.32 a 0.52 abcd 15.00 abc 3.00 abc 2.00 a 2.00 287.50 a 100.00 a az. 20 + g.f. 5 17.77 ab 7.77 a 2.28 ab 0.63 a 15.33 ab 2.33 abcd 2.00 a 2.00 211.60 abc 100.00 a az. 5 + g.f. 10 11.43 bc 3.00 bcd 1.81 abcd 0.32 abcd 14.00 abcde 2.00 abcd 1.99 a 2.00 106.90 abc 100.00 a az. 10 + g.f. 10 11.80 abc 2.57 bcd 1.67 abcd 0.25 abcd 13.00 abcde 2.33 abcd 1.83 ab 2.00 246.60 ab 100.00 a az. 20 + g.f. 10 9.47 c 2.23 cd 1.64 abcd 0.13 cd 11.33 abcde 1.33 abcd 1.75 ab 2.00 78.29 bc 100.00 a az. 5 + g.f. 20 9.47 c 1.10 d 1.59 abcd 0.23 abcd 11.00 abcde 3.00 abc 1.54 ab 2.00 143.70 abc 66.67 b az. 10 + g.f. 20 13.17 abc 4.00 abcd 2.23 abc 0.53abc 14.33abcd 3.67 a 2.00 a 2.00 223.30 abc 100.00 a az. 20 + g.f. 20 10.23 c 1.90 d 1.44 bcd 0.27abcd 11.67abcde 1.33 abcd 2.00 a 2.00 54.21 bc 100.00 a ssp10 alone 14.50 abc 6.53 ab 2.08 abcd 0.52abcd 14.00abcde 3.33 ab 1.68 ab 2.00 123.90 abc 100.00 a control 12.30 abc 2.63 bcd 1.83 abcd 0.32abcd 12.33abcde 2.33 abcd 1.12 b 2.00 169.60 abc 100.00 a mean values 11.41 3.03 1.76 0.30 12.40 2.03 1.66 2.00 115.19 97.70 cd. (p <0.05) 5.63 3.51 0.69 0.35 5.03 1.96 0.69 ns 166.20 24.78 numbers followed by the same letter do not differ significantly at 5% level. g.m.= glomus mosseae, g.f.= glomus fasciculatum, az.= azospirillum. 157 g.m. (10 g) + az. (20 g) + ssp (10 g), which was significantly higher than g.f. (5 g) + az. (20 g) + ssp (10 g). these two treatments were significantly superior to all other treatments including the control. the maximum increment at this stage was observed in the treatment g.f. (20 g) + az. (10 g) + ssp (10 g). the greatest number of new flushes/year was recorded in treatment plants inoculated with g.f. (10 g) + ssp (10 g), g.m. (10 g) + az. (20 g) + ssp (10 g), g.f. (5 g) + az. (10 g) + ssp (10 g), g.f. (5 g) + az. (20 g) + ssp (10 g), g.f. (20 g) + az. (10 g) + ssp (10 g) and g.f. (20 g) + az. (20 g) + ssp (10 g) which produced the same number of flushes, followed by g.f. (10 g) + az. (5 g) + ssp (10 g) that were statistically at par with each other. all the above treatments were significantly superior to the other treatments. there were no variations observed among the treatments with regard to the number of leaves/flush and all the treatments gave an average of two leaves. the maximum leaf area was recorded in the plants inoculated with g.f. (5 g) + az. (10 g) + ssp (10 g), followed by g.f. (10 g) + az. (10 g) + ssp (10 g). these two treatments were at par with each other and were relatively superior to all other treatments, including the control. a 100% survival rate was observed in all treatments except g.f. (20 g) + az. (5 g) + ssp (10 g), which had only a 66.67% survival rate. biomass and root characters all mean data related to the characters of shoots and roots of one-year-old mangosteen seedlings are presented in table 2. the maximum fresh weight of shoot was recorded table 2 morphological characters of twelve-month stage mangosteen (garcinia mangostana l.) seedlings inoculated with arbuscular mycorrhizal fungi (amf) and azospirillum treatments (g) shoot fresh weight (g) root fresh weight (g) shoot dry weight (g) root dry weight (g) root to shoot dry weight ratio root length (cm) primary roots (no) secondary roots (no) tertiary roots (no) total roots (no) g.m. 5 6.00 ij 1.00 lmn 3.13 kl 0.65 ij 0.21 b 10.2 k 12.0 kl 16.0 g 9.0 no 37.0 l g.m. 10 3.05 lmn 0.89 mno 0.97 r 0.35 mno 0.36 b 23.2 efgh 21.0 fghijk 36.0 d 15.0 jklm 72.0 ghij g.m. 20 4.79 jkl 1.08 lm 0.22 t 0.51 jklm 2.62 a 22.8 efgh 31.0 abcde 43.0 cd 19.0 ijk 93.0 efgh g.f. 5 3.09 lmn 0.70 op 1.20 q 0.31 nop 0.26 b 20.7 hi 14.0 jkl 31.0 def 12.0 lmno 57.0 ijkl g.f. 10 10.54 f 2.05 g 4.74 h 0.86 g 0.18 b 23.2 efgh 22.0 efghij 38.0 cd 10.0 mno 70.0 ghijk g.f. 20 7.54 hi 1.56 i 3.27 k 0.68 hi 0.21 b 24.6 cdefgh 26.0 defghi 39.0 cd 26.0 gh 91.0 efgh az. 5 1.53 n 0.42 qr 0.67 s 0.21 opq 0.30 b 17.6 ij 19.0 hijkl 28.0 defg 11.0 lmno 58.0 ijkl az. 10 3.94 klm 1.06 lm 1.81 op 0.50 jklm 0.28 b 21.2 ghi 20.0 ghijk 32.0 def 15.0 jklm 67.0 hijkl az. 20 3.36 lm 0.58 pq 1.19 q 0.17 pq 0.14 b 20.3 hi 15.0 jkl 19.0 efg 8.0o 42.0 jkl az. 5 + g.m. 5 5.57 jk 1.13 kl 1.94 o 0.38 lmn 0.20 b 18.3 ij 20.0 ghijk 32.0 def 14.0 klmn 66.0 hijkl az. 10 + g.m. 5 8.32 gh 1.83 h 3.77 j 0.90 g 0.24 b 21.5 ghi 23.0 defghij 38.0 cd 24.0 hi 85.0 fghi az. 20 + g.m. 5 6.42 ij 1.37 ij 2.55 m 0.55 ijk 0.22 b 14.6 j 19.0 hijkl 52.0 bc 30.0 g 101.0 defg az. 5 + g.m. 10 11.04 ef 2.51 f 4.79 h 1.10 f 0.23 b 20.3 hi 29.0 bcdefg 86.0 a 46.0 de 161.0 b az. 10 + g.m. 10 5.56 jk 1.49 ij 2.21 n 0.57 ijk 0.26 b 26.8 bcde 36.0abc 94.0 a 58.0 ab 188.0 a az. 20 + g.m. 10 12.52 de 2.40 f 5.90 f 1.16 f 0.20 b 27.4 bcd 30.0bcdef 59.0 b 38.0 f 127.0 cd az. 5 + g.m. 20 2.49 mn 0.26 r 0.92 r 0.11 q 0.11 b 16.2 j 10.0 l 18.0 fg 12.0 lmno 40.0 kl az. 10 + g.m. 20 4.08 klm 0.83 no 1.68 p 0.36 lmno 0.21 b 21.7 fghi 22.0 efghij 33.0 def 14.0 klmn 69.0 hijk az. 20 + g.m. 20 8.47 gh 1.95 gh 3.69 j 0.81 gh 0.22 b 28.4 bc 16.0 jkl 44.0 cd 16.0 jkl 76.0 ghi az. 5 + g.f. 5 7.72 hi 1.30 jk 3.00 l 0.52 ijkl 0.17 b 22.5 fgh 20.0ghijk 34.0 de 20.0 ij 74.0 ghi az. 10 + g.f. 5 19.72 b 6.31 a 10.55 b 3.03 a 0.29 b 36.4 a 40.0 a 58.0 b 60.0 a 158.0 b az. 20 + g.f. 5 18.60 b 3.32 de 6.20 e 1.42 e 0.23 b 23.2 efgh 18.0 ijkl 28.0 defg 30.0 g 76.0 ghi az. 5 + g.f. 10 11.36 ef 2.49 f 5.39 g 1.36 e 0.25 b 25.8 cdef 28.0 cdefgh 40.0 cd 47.0 de 115.0 cde az. 10 + g.f. 10 19.65 b 2.49 f 8.76 c 2.15 c 0.25 b 18.2 ij 15.0 jkl 33.0 def 42.0ef 90.0 efgh az. 20 + g.f. 10 9.80 fg 3.97 c 4.49 i 1.82 d 0.41 b 23.2 efgh 16.0 jkl 30.0 defg 18.0 jk 64.0 hijkl az. 5 + g.f. 20 16.95 c 4.01 c 6.92 d 1.67 d 0.24 b 26.8 bcde 32.0 abcd 43.0 cd 50.0 cd 125.0 cd az. 10 + g.f. 20 25.53 a 3.39 d 11.09 a 2.56 b 0.23 b 30.2 b 30.0 bcdef 42.0 cd 53.0 bc 125.0 cd az. 20 + g.f. 20 13.91 d 5.23 b 5.43 g 1.35 e 0.25 b 23.8 defgh 29.0 bcdefg 38.0 cd 43.0 ef 110.0 def ssp10 alone 16.27 c 3.15 e 6.89 d 1.79 d 0.26 b 30.4 b 38.0 ab 44.0cd 58.0 ab 140.0 bc control 10.54 f 3.91 c 3.65 j 0.45 klmn 0.12 b 25.4 cdefg 28.0 cdefgh 32.0 def 18.0 jk 78.0 ghi mean values 9.60 2.16 4.04 0.98 0.31 22.93 23.41 40.0 28.138 91.552 c.d. (p< 0.05) 1.63 0.19 0.19 0.15 0.33 3.60 8.17 13.08 4.903 26.15 numbers followed by the same letter do not differ significantly at 5% level. g.m.= glomus mosseae, g.f.= glomus fasciculatum, az= azospirillum. 158 in the plants inoculated with g.f. (20 g) + az. (10 g) + ssp (10 g), which was significantly superior to all other treatments. this was followed by g.f. (5 g) + az. (10 g) + ssp (10 g), g.f. (10 g) + az. (10 g) + ssp (10 g) and g.f. (5 g) + az. (20 g) + ssp (10 g). these three treatments were at par with each other and significantly superior to all the other treatments including the control. the highest fresh weight of the root was recorded in the treatment g.f. (5 g) + az. (10 g) + ssp (10 g) followed by g.f. (20 g) + az. (20 g) + ssp (10 g). the differences between the treatment means were significant and both these treatments were also significantly superior to all other treatments including the control. maximum dry weight of shoot and root were recorded in plants inoculated with g.f. (20 g) + az. (10 g) + ssp (10 g) and g.f. (5 g) + az. (10 g) + ssp (10 g), followed respectively by g.f. (5 g) + az. (10 g) + ssp (10 g) and g.f. (20 g) + az. (10 g) + ssp (10 g). in both cases (i.e. dry weight of shoot and root), the means of the two treatments which gave maximum dry weight not only significantly differed between them but was also superior to all other treatments. a critical analysis revealed that only the relative positions of the best and second-best treatments in the case of root and shoot dry weight inter changed. maximum dry weight ratio of roots and shoots were recorded in plants inoculated with g.m. (20 g) + ssp (10 g), which was significantly superior to all other treatments, which were at par with each other including the control. the maximum root length was observed in plants inoculated with g.f. (5 g) + az. (10g) + ssp (10 g), which was significantly superior to all other treatments. this was followed by plants treated with ssp (10 g) alone and g.f. (20 g) + az. (10 g) + ssp (10 g), which were statistically at par. these treatments produced better root length, giving significantly superior results compared to all other remaining treatments including the control. maximum and significantly higher primary root count were recorded in the plants inoculated with g.f. (5 g) + az. (20 g) + ssp (10 g) followed by ssp (10 g) alone. the greatest number of secondary roots was observed in the plants inoculated with g.m. (10 g) + az. (10 g) + ssp (10 g), which was at par with g.m. (10 g) + az. (5 g) + ssp (10 g), the next best treatment and significantly superior to all other treatments. this was followed by g.f. (5 g) + az. (10 g) + ssp (10 g) and g.m. (5 g) + az. (20 g) + ssp (10 g). the means of the latter two treatments were at par with each other. the highest tertiary root count was observed in the treatment g.f. (5 g ) + az. (10 g) + ssp (10 g), which was statistically at par with the treatment combination g.m. (10 g) + az. (10 g) + ssp (10 g) and ssp (10 g) alone. the latter two treatments were at par with g.f. (20 g) + az. (10 g) + ssp (10 g). the above treatments were significantly higher than all other treatments including control. the total number of roots was greatest in plants inoculated with g.m. (10 g) + az. (10 g) + ssp (10 g) , which was superior to all other treatments. this was followed by g.m. (10 g) + az. (5 g) + ssp (10 g), which was at par with g.f. (5 g) + az. (10 g) + ssp (10 g) and ssp (10 g) alone. the means of these treatments were significantly superior to the means of all other treatments including the control. biochemical characters of seedling leaves for the purpose of analyzing the biochemical characters, the treatments were categorized as those showing the best growth, intermediary growth and the least growth (fig. 1). typical treatments for each group were selected and are presented in table 3: best growth, g.f. (5 g) + az. (10 g) + table 3 biochemical characters of the leaf samples in one-year-old mangosteen (garcinia mangostana l.) seedlings treated with arbuscular mycorrhizal fungi and azospirillum amf and azospirillum inoculated plants n (%) p (%) k (%) crude protein (%) na (%) chlorophyll a (mg/g) chlorophyll b (mg/g) chlorophyll a/b ratio total chlorophyll (mg/g) total phenols (mg/g) total carbohydrates (mg/g) abscisic acid (mg/g) best (az. 10 + g.f. 5) 2.35 0.19 a 0.34 14.67 a 0.34 0.95 a 0.42 a 2.30 1.37 a 0.64 7.85 a 0.21 a intermediate (g.m. 10 g) 2.08 0.05 b 0.30 12.99 b 0.34 0.66 b 0.29 a 2.38 0.95 ab 0.46 6.59 ab 0.16 ab least (az. 10 + g.m. 20) 1.79 0.04 b 0.28 11.22 c 0.34 0.17 c 0.11 b 1.88 0.27 b 0.47 4.85 b 0.08 b control 1.94 0.04 b 0.29 12.10 bc 0.34 0.41 c 0.20 ab 2.05 0.61 ab 0.46 5.72 ab 0.14 ab mean values 2.04 0.08 0.30 12.75 0.34 0.55 0.25 2.15 0.80 0.51 6.25 0.15 c.d. (p<0.05) ns 0.09 ns 1.46 ns 0.25 0.17 ns 0.80 ns 2.13 0.096 numbers followed by the same letter do not differ significantly at 5%. g.m.= glomus mosseae, g.f.= glomus fasciculatum, az= azospirillum. fig. 1 seedling of treatments, showing the best, the intermediate and the least growth response to arbuscular mycorrhizal fungi (am fungi) and azospirillum in mangosteen (garcinia mangostana l.). 159 ssp (10 g); intermediate growth, g.m. (10 g) + ssp (10 g); least growth, g.m. (20 g) + az. (10 g) + ssp (10 g). the nitrogen content was highest in the maximum growth treatment g.f. (5 g) + az. (10 g) + ssp (10 g). a distinct trend was noticed, with maximum values found in treatments with the best growth, the intermediary values being recorded in treatments showing intermediate growth, and the lowest values in treatments showing the least growth. however, there were no significant differences observed between the treatment means. in the case of phosphorus, a pattern nearly similar to that of nitrogen was observed. however, the mean content in the treatment, which showed the best growth, was significantly superior to other treatments including the control. the potassium content also revealed a similar trend: the treatment with maximum growth also had the highest potassium content. however there were no significant differences observed between the various treatment means including the control. the control treatment gave the values between intermediate growth and least value treatments. the highest crude protein content was also observed in the treatment which showed best growth. it was significantly superior to other treatments and also to the control. the treatments which showed intermediate growth also showed higher values for this parameter and they were significantly higher than the treatment with least growth and control plants. treatments showing the best, intermediate and least growth, as well as the control, all showed the same level of sodium content. maximum chlorophyll a and b and total chlorophyll content were observed in the treatment that recorded the maximum growth. in the case of chlorophyll a, this was significantly higher than the means observed in plants of other categories and the control. the content of the plants with the least growth was at par with control. in the case of chlorophyll b content , the trend observed was similar to that of growth. the treatment with maximum growth had the highest chlorophyll b content, which was on par with the treatment showing intermediate growth. these two levels were significantly superior to the level observed in the plants with the least growth, but it was statistically at par with the control. contrary to what is commonly believed, the total phenol content was highest in the treatments with maximum growth (fig. 2). however, there were no significant differences observed between treatment means and the control. the greatest amount of total carbohydrate was found in the treatment that had maximum growth. this was at par with the treatment means that showed intermediate growth and also with the control. the content of abscisic acid, which normally goes hand in hand with growth inhibition, was also contrary to normal lines of thought. the treatments which revealed more growth also had the highest abscisic acid content. the means of the group with the highest abscisic acid content were also at par with those of the treatment with intermediate growth and the control, but the treatment means of the first group was significantly superior to the category which showed the least growth (fig. 1, 2, and 3). microbial population and percent root infection the mean data related to microbial population and the percent root infection by am fungi are presented in table 4. total spore population of arbuscular mycorrhizal fungi (amf) the highest number of spores was recorded in the rhizosphere of plants inoculated with g.f. (20 g) + ssp (10 g), followed by g.f. (20 g) + az. (20 g) + ssp (10 g). these two treatments were on par with all other treatments except the control, proving the superiority of the treatment in colonization. percent root infection root infection, which gave a clear picture of colonization, was also studied. the highest percentage of root infection was recorded in plants inoculated with g.f. (5 g) + az. (10 g) + ssp (10 g) and g.f. (20 g) + az. (10 g) + ssp (10 g), with both registering the highest values of mean infection percentage. these treatments were at par with all other treatments except g.m. (5 g) + ssp (10 g), fig. 2 total phenols and abscisic acid content of the am fungi and azospirillum treated plants showing best, intermediate and least growth in mangosteen (garcinia mangostana l.). fig. 3 banding pattern observed in tlc used to quantify abscisic acid content in various treatments using arbuscular mycorrhizal fungi (am fungi) and azospirillum in mangosteen (garcinia mangostana l.) 160 g.f. (5 g) + ssp (10 g), all the azospirillum + ssp alone treated plants, g.m. (5 g) + az. (5 g) + ssp (10 g), ssp (10 g) and also the control. azospirillum population in soil the maximum azospirillum population was recorded in the rhizosphere of plants inoculated with g.f. (10 g) + az. (20 g) + ssp (10 g), which was on par with all other treatments except the combination of g.m. (5 g) and (10 g) alone with ssp (10 g) absolute control of ssp (10g) alone and the control plants. 4. discussion and conclusions the best treatments in case of microbial inoculation was a combination of g.f. (5 g) + az. (10 g) + ssp (10 g) followed by g.f. (5 g) + az. (20 g) + ssp (10 g). critical analysis of the data revealed that the percentage of am fungi infection was the highest in treatments with maximum growth. the next best in order of growth also showed a high infection percentage. though the total spore count was not the highest the treatments were on par with the treatments that yielded the highest spore count namely g.f. (20 g) treated plants. these two-treatment combinations also showed a high azospirillum population. these three characters proved, beyond a doubt, that the combination was best among the treatments for maximum growth. this is a reflection of mycelial mat formation on absorbing roots, which in turn leads to higher p, n and k uptake as observed in the study. the effects were most pronounced with regard to p as the content registered a three-fold increase. this should have been due to two factors, namely the external apply of ssp (10 g) and a proven concept that mycorrhiza gives out an organic acid secretion which is capable of solubilising as well as mobilizing the acid soluble phostable 4 microbial population in seedlings rhizosphere and infectivity in roots after one year of inoculation in mangosteen (garcinia mangostana l.) treatments total amf spores (no./100 g of soil) root infection by amf (%) azospirillum population (x 106 cfu/g of soil) g.m. 5 198.00 ab 40.00 cdef 0.22 bc g.m. 10 214.00 ab 55.00 abcdef 0.24 bc g.m. 20 219.00 ab 65.00 abcde 0.26 abc g.f. 5 206.00 ab 40.00 cdef 0.27 ab g.f. 10 222.00 ab 60.00 abcdef 0.28 ab g.f. 20 228.00 a 70.00 abcd 0.30 ab az. 5 136.00 bc 35.00 def 0.34 ab az. 10 142.00 abc 40.00 cdef 0.35 ab az. 20 148.00 abc 30.00 ef 0.37 ab az. 5 + g.m. 5 200.00 ab 45.00 bcdef 0.31 ab az. 10 + g.m. 5 213.00 ab 55.00 abcdef 0.36 ab az. 20 + g.m. 5 215.00 ab 50.00 abcdef 0.37 ab az. 5 + g.m. 10 204.00 ab 50.00 abcdef 0.33 ab az. 10 + g.m. 10 218.00 ab 65.00 abcde 0.37 ab az. 20 + g.m. 10 222.00 ab 70.00 abcd 0.34 ab az. 5 + g.m. 20 206.00 ab 60.00 abcdef 0.32 ab az. 10 + g.m. 20 220.00 ab 75.00 abc 0.38 ab az. 20 + g.m. 20 224.00 ab 55.00 abcdef 0.39 ab az. 5 + g.f. 5 204.00 ab 80.00 ab 0.32 ab az. 10 + g.f. 5 216.00 ab 90.00 a 0.36 ab az. 20 + g.f. 5 219.00 ab 85.00 ab 0.40 a az. 5 + g.f. 10 210.00 ab 80.00 ab 0.33 ab az. 10 + g.f. 10 220.00 ab 85.00 ab 0.37 ab az. 20 + g.f. 10 224.00 ab 85.00 ab 0.38 ab az. 5 + g.f. 20 213.00 ab 85.00 ab 0.37 ab az. 10 + g.f. 20 224.00 ab 90.00 a 0.35 ab az. 20 + g.f. 20 226.00 ab 80.00 ab 0.39 ab ssp10 alone 150.00 abc 30.00 ef 0.23 bc control 90.00 c 25.00 f 0.12 c mean values 201.10 61.21 0.32 c.d. (p< 0.05) 73.55 32.69 0.14 numbers followed by the same letter not differ significantly at 5% level. g.m= glomus mosseae, g.f= glomus fasciculatum. 161 phate (i.e. semi-soluble phosphate) into soluble form. some of the previous reports substantiate these findings (bartlett and lewis, 1973). increases in selective uptake of major nutrients have also been reported by several workers (hatch, 1937; umesh et al., 1988; rizzardi, 1990). the capability of transferring it into the soluble form has also been reported (bolan et al., 1984). another possible reason is that they can enhance the storage capacity, and the continuous disintegration of arbuscules leads to the availability of more mineral nutrients to the host (gerdemann, 1968). the higher levels of p, almost three times, should have been the reason for quantitative improvement of the root characters. this can be observed in the best treatment which induced the highest fresh and dry weight of roots, as well as characters such as length of longest root and improved characters of number of roots. in mangosteen, masri and azizah (1998) reported alternation of root characters such as root density and branching density, which led to 67-88% higher uptake of p. studies on alteration of rooting density are of paramount importance in mangosteen owing to the fact that the crop produces only magnolioid roots, an unique feature of mangosteen among other fruit crops. due to lack of production of root hairs, absorption of nutrients and water is less and even survival at nature’s mercy or benevolence. as such any treatment, which improves the qualitative aspects of rooting such as root length, root branching and root density will certainly influence all aspects of growth and productivity and hence should be the prime consideration in crop management. the production of growth promoting substances by glomus mosseae and glomus fasciculatum is well documented (miller, 1971; crafts and miller, 1974; slankis, 1975). greater nutrient uptake and growth promoting substances should have been the reason for the increased chlorophyll content and higher leaf area, which together accounted for higher carbon assimilation (estrada-luna et al., 2000) leading to higher carbohydrate content as observed in the study. higher crude protein content also points to the level of protein synthesis due to am fungi inoculation, which is a new area worth to be probed further. the greater nutrient uptake and carbohydrate accumulation resulted in greater shoot and root biomass and dry weight content. differences observed in growth increment during intervals of observation are basically due to infectivity and colonization of am fungi on feeder roots. this is partly an efficiency factor of the fungi and secondly is influenced by soil ecology factors as well as the host. in mangosteen, masri et al. (1998) observed increased growth due to am fungi inoculation and thereby reduced nursery period for mangosteen. as in the case of activation of growth using bioregulators, here also the content of inhibitors namely phenols and aba were higher in the best treatment and a general decrease was observed in the treatment showing the least growth. this can also be argued only on the lines of correlative inhibition. the higher content did not inhibit the growth, as the balance of various growth regulators should have been more towards the plant growth promoters than growth inhibitors. more detailed investigation encompassing the whole endogenous levels of plant hormones at the critical stages of bud dormancy, bud activation, flushing and post flushing can only answer this vital question. the present study convincingly proves the efficacy of the two treatments in activating growth of mangosteen. the mean values of growth characters obtained in the best treatments are far superior to the selection indices standardized for seedling growth (yusuf and kurien, 2012). this investigation is of great practical application and gains more importance as mangosteen is a crop which lacks fine root hairs (richards, 1990) while the study revealed that colonization of am fungi and azospirillum takes place on the network of fine ramified roots enabling better uptake of nutrients and thereby growth. references aoac, 1980 official methods of analysis of the association of official analytical chemists. association of official analytical chemists, 13th ed., washington d.c., usa, pp. 1098. bartlett e.m., lewis d.h., 1973 surface phosphatase activity of mycorrhizal roots of beech. soil biol. biochem., 5: 249-257. bhandari s.c., somani l.l., gulati i.j., 1990 symbiotic vesicular arbuscular mycorrhizas, pp. 295-311. in: somani l.l., s.c. bhandari, s.n. saxena, and k.k. vyas (eds.) biofertilizer. scientific publishers, jodhpur, india. bolan n.s., barrow, n.j., aylmore l.a.g., 1984 specific activity of phosphorus in mycorrhizal and non mycorrhizal plants in relation to the availability of phosphorus to plants. soil biol. biochem., 16: 299-304. bopiah b.m., khader k.b.a., 1989 effect of biofertilizers on growth of black pepper (piper nigram l.). indian. j. agril. sci., 59(10): 682-683. carling j.e., riehle w.g., brown m.f., johnson d.r., 1978 effects of a vesicular-arbuscular mycorrhizal fungus on nitrate reductase and nitrogenase activities in nodulating and non-nodulating soybeans. phytopathology, 68: 1590-1596. cherian s.a., 2001 effect of azospirillum and va mycorrhiza on the growth of cocoa seedlings and incidence of seedling blight disease. m.sc. 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subhadrabandhu s., 1992 mangosteen (garcinia mangostana l.) a potential crop for tropical northern australia. acta horticulturae, 321: 132-137. yusuf l.m., kurien s., 2012 preliminary studies on selection indices for activating seedling growth in mangosteen (garcinia mangastana. l.). adv. hort. sci., 26(3-4): 115-130. impaginato 155 adv. hort. sci., 2024 38(2): 155­167 doi: 10.36253/ahsc­15691 pigment composition and physico­ chemical parameters of bittergourd (momordica charantia l. cv. jadeite) during postharvest period as influenced by illumination colors j.g. ponteras 1 (*), j.d.c. quisil 2 , f.m. salas 3 1 agriculture department, institute of agricultural technology and entrepreneurial studies, southern philippiness, agribusiness and marine and aquatic school of technology, malita, davao occidental, philippines. 2 college of agriculture, central mindanao university, university town, musuan, maramag, bukidnon, philippines. 3 department of pure and applied chemistry, college of arts and sciences, visayas state university, baybay city, leyte, philippines. key words: bittergourd, led, pigmental composition, postharvest quality. abstract: fruits and vegetables that exhibit a higher chlorophyll content, as reflected in their visual appearance, are the preferred choice of consumers. the study aimed to evaluate the effects of light emitting diodes (leds) on the physical, chemical, and pigmentation quality of bitter gourd using white, blue, and red at 1.5 w/135 lumens (foshan electrical and lighting co., ltd [fsl], china). bitter gourd, with a short postharvest life of 4­5 days due to physical and chemical disorders, was harvested weighing 300–400 g and 25 x 5 cm from the farm and subjected to varying illuminations within a 4­hour period for five days, with measurements taken daily. statistical differences between treatments were observed in physicochemical parameters such as fruit shrivelling, yellowing, visual appearance, weight loss, dry matter content, total chlorophyll, ph values, and ta. the quality and shelf­life of bitter gourd fruits were found to be improved by the white led. the visual appearance was maintained, and fruit shrivelling and yellowing were delayed, with lower weight loss observed. slight changes in chlorophylls and carotenoids, vitamin c, and a shelf­life of 5 days were recorded. 1. introduction momordica charantia l. is known as bitter gourd, with its good nutritional and medicinal properties, is grown in approximately 340,000 ha (dhillon et al., 2016) annually in tropical asia, including eastern asia, india, and china, which is its center of origin (behera et al., 2010; (*) corresponding author: ponterasjohn@gmail.com citation: ponteras j.g., quisil j.d.c. , salas f.m., 2024 ­ pigment composition and physico‐chemi‐ cal parameters of bittergourd (momordica cha‐ rantia l. cv. jadeite) during postharvest period as influenced by illumination colors. ­ adv. hort. sci., 38(2): 155­167. copyright: © 2024 ponteras j.g., quisil j.d.c. , salas f.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 january 2024 accepted for publication 31 january 2024 ahs advances in horticultural science https://doi.org/10.36253/ahsc-15691 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2024 38(2): 155­167 156 prajapati et al., 2021 a). the production volume in the philippines has increased by 1.8% to 32.05 thousand metric tons (psa, 2022). its primary demand arises from its anti­diabetic properties (rosario and macusi, 2009). the presence of phenols, terpenes, and flavonoids in bitter gourd contributes to its bitter taste and antioxidant properties (dutta et al., 2021), which are desired by consumers (behera et al., 2010; taiti et al., 2017) and can help prolong its shelf­life (salas et al., 2015). the metabolites found in bitter gourd are affected by cultivars and cultural practices such as light, temperature, soil, and nutrition (valyaie et al., 2021). the importance of light in postharvest produce cannot be overstated, as plants perceive stimulus for growth and development through light. recent studies have discovered that even after plants are harvested, their light­dependent processes continue. to ensure the longevity of postharvest fruits and vegetables and maintain their metabolite levels, various treatments ­ both chemical and non­chemical ­ can be applied. for example, bittergourd fruits can be stored for up to five days under ambient conditions without ripening, yellowing, or losing their bitterness (salas et al., 2015; prajapati et al. , 2021 a). as a means of preserving postharvest produce, researchers have begun exploring the use of light emitting diodes (leds). in fact, supplemental lighting from leds has been shown to enhance the market value of harvested sweet peppers by inducing colour break (jones, 2018). researchers have therefore explored the use of light emitting diodes (leds) to prolong shelf­life and maintain metabolites in various crops (ma et al., 2014; d’souza et al., 2015; bantis et al., 2018; loi et al., 2020; poonia et al., 2022). the findings of these studies have been corroborated by the upregulation of vitamin c, antioxidants, anthocyanins, and the physical appearance of fresh produce towards market acceptability. for example, white led lighting facilitates the accumulation of phenols in harvested vegetables (poonia et al., 2022), which are known to have antioxidant and anti­inflammatory properties and may enhance the health benefits of these crops. it was observed that the leds had a drying effect, leading to a rapid increase in transpiration, as reported by chua et al. (2021). various studies have shown that different types of light can have varying effects on different types of vegetables. for example, white and blue leds can modulate the stomata opening and number of stomates (zhan et al., 2013), resulting in weight loss of certain vegetables such as brassica oleracea l. var. italica plenck (favre et al., 2018), brassica oleracea var. chinensis lei (zhou et al., 2020), and freshly­cut leaves of amaranthus dubius l. (jin et al., 2021). on the other hand, dark­ stored celery has a lower dry matter content compared to fresh­cut celery due to lower total soluble solids, ascorbic acid, and chlorophylls (zhan et al., 2013; florkowski et al., 2014). in another study, freshly sliced cherry tomatoes exposed to white, blue, and green leds showed a transitory increase in vitamin c one day after slicing, while those exposed to red light remained stable (kong et al., 2020). it is worth noting that the effect of led lighting on postharvest crops varies depending on the type of crop, led intensity, and duration of exposure. for example, in previous studies, broccoli exposed to 9.5 and 19.0 w m­2 white led illumination for three hours per day showed delayed chlorophyll degradation and lower weight loss (pintos et al., 2020). okra fruits treated with white and blue leds (17.28 w m­2) for 8 h showed an increased total phenolics content (thilini deepashika perera et al., 2022), while berry grape treated with 41 and 42 w m­2 of led showed an increased anthocyanin content at 24 h, and blue led decreased fungal infections in citrus fruits at a fluence rate of 120 w m−2 and 700 w m­2 for 18 h under 25°c (nassarawa et al., 2020). in addition, bitter gourd exposed to uv­c led for 40 min at 10°c and 85­95% rh had a prolonged shelf­life of up to 16 d (prajapati et al. , 2021 b). while chemical­based treatments have been the focus of many researchers to improve the postharvest life of vegetables, there is a growing interest in using led treatments to increase shelf­life and enhance metabolites. it is hypothesized that varying illuminations of white, blue, and red led with 1.5 wattage (w) can delay pigmental degradation, preserve organic compounds, and extend shelf­life in harvested bitter gourd. this study tried to assess how the quality of bitter gourd change during post­ harvest by applying varying illuminations color led (white, blue, and red) with 1.5 wattage (w). 2. materials and methods fruit samples and sample preparation the cultural practices and harvesting process of sixty bitter gourds from a commercial vegetable farm ponteras et al. ‐ postharvest bittergourd as influenced by illumination colors 157 in barangay buenavista, baybay city, leyte (lat. 10° 39’ 34.0” n, long. 124° 50’ 27.6” e) are described in this study. organic and inorganic fertilizers were applied, including chicken dung through the basal method and soil drenching for urea and complete fertilizers during the vegetative and reproductive stages. the fruits, weighing 300­400 g and measuring 25 x 5 cm, were harvested at the dark green stage and were carried out carefully to minimize mechanical injuries. fruits with defects were excluded from the study. the farmer­harvester, an expert in harvesting, chose the fruits for marketing based on the local market standard from the city mentioned above. the fruits were then transported from the farm to the department of horticulture crop physiology laboratory for postharvest assessment from october 28 to november 03, 2022. the shelf­life of the fruits was 5 days (d), and the treatment application was done after 24 hours (h) to acclimatize and equilibrate the fruits at ambient conditions. the amount and time of fertilizer application were not included in the present study, as it was based on the farmer’s feedback during the harvest since the study utilized fruits from the farm instead of from the wet market. treatment preparation the cardboard boxes with aluminum foil were arranged on the surface and equipped with 1.5 w/135 lumens (foshan electrical and lighting co., ltd [fsl], china) led lights, measuring 56 [l] x 45 [w] x 24 [h] cm. the led source was positioned at a distance of 19 cm from the fruits. in the current study, each treatment was subjected to white, blue, and red leds for a duration of 4 h, with five (5) fruit samples per treatment. after incubation, the lights inside each cardboard box were switched off. however, the irradiated fruit samples were transferred and placed in plastic trays (40 × 30 cm) under ambient conditions (26­28°c). each tray represented a replicate exposed to lights that remained switched on continuously for 8 h daily, including non­incubated fruit samples for storage and postharvest evaluation. enhanced and stable metabolites were demonstrated during storage, resulting in a prolonged shelf­life from exposure to uv­c led for 40 minutes (min), blue and red leds for 24 h, white and blue light for 8 h, and white led for 3 h daily, as shown in previous studies (nassarawa et al., 2020; pintos et al., 2020; prajapati et al., 2021 b; thilini deepashika perera et al., 2022). experimental design in a completely randomized design (crd), the study had five samples per treatment replicated three times. the treatments were designated as follows: t1 ­ control (room light condition), t2 ­ incubated 4 h with white led at 1.5 watt/135 lumen (fsl, china), t3 ­ incubated 4 h with blue led at 1.5 watt/135 lumen (fsl, china), t4 ­ incubated 4 h with red led at 1.5 watt/135 lumen (fsl, china). data collection physico‐chemical parameters. the fruits were assessed before and after gathering using a digital weighing scale (general master, japan), with the parameters being determined for each fruit from the five samples in replication manually every day for six days using different indices. the cumulative weight loss from the five samples from each replication was determined by weighing the initial weight and daily as known storage period (prajapati et al., 2021 b). the fruit shrivelling index was assessed with slight modifications (benitez et al., 2015; lualhati and del carmen, 2018) using a 4­point scale ranging from 1 to 4 (where 1 indicated no shrivelling, 2 indicated slight shrivelling (1­25% fruit surface affected), 3 indicated moderate shrivelling (26­50% fruit surface affected), and 4 indicated severe shrivelling (more than 50% fruit surface affected). the visual quality rating (vqr) of bitter gourd was evaluated daily as reported by valida et al. (2018). in brief, vqr was assessed using a 9­point scale, where 9 indicated excellent, field fresh or no defects, 7 indicated good, defects minor, 5 indicated fair, defects moderate, limit of marketability, 3 indicated poor, defects serious, l imit of edibility, and 1 indicated non­edible under usual condition. the degree of yellowing was rated manually daily as reported by valida et al. (2018) using a 5­point scale, where 1 indicated full green, 2 indicated 1­10% surface yellowing, 3 indicated 11­30% surface yellowing, 4 indicated 31­50% surface yellowing, and 5 indicated extensive yellowing/discoloration. the dry matter content (%) was determined by subjecting the samples (50 g) to oven drying at 70°c for 24 h until they reached a constant weight. the remaining weight of the samples after drying served adv. hort. sci., 2024 38(2): 155­167 158 as input to calculate the percent dry matter content as a percentage of the wet sample (gonzales and benitez, 2019). the pigment composition (mg g­1) was determined by soaking a gram of each representative bitter gourd fruit in 10 ml of 95% ethanol overnight. the absorbance of the filtrates at wavelengths of 666, 653, and 470 nm was measured using an ultraviolet­ visible spectrophotometer at the vsu­casl (salas et al., 2020). the shelf life was determined when sample fruits reached a vqr of 5, which is fair with moderate defects and limited marketability (salas et al., 2015; valida et al., 2018). chemical parameters. for the examination of chemical parameters, one fruit from each replication was randomly selected as a representative. the protocol appears to have been based on the report of gonzales and benitez (2019) and salas et al. (2020). the fresh­cut samples were homogenized in 10 g per 50 ml distilled water for 10 min using a homemade blender (camel®), and the filtrates were measured for potential hydrogen (ph), electrical conductivity (ec) and total dissolved solids (tds) using a smart combined meter (milwaukee, mw 802) total soluble solid (°brix) was measured using a hand­held refractometer (atago n1, japan) by placing 1­3 drops of juice on the instrument prism and taking the reading. titrable acidity (%) was determined by adding 5 ml extract with two drops of 1% phenolphthalein indicator into a volumetric flask containing 4 g naoh diluted with 1 l distilled water, followed by titration with 0.1% naoh until a faint pink colour was obtained. finally, for vitamin c analysis (mg 100 g fresh fruit­1), fresh cut (12 g) was subjected to 5 min with 120 ml distilled water in a blender until supernatant filtered. an aliquot (1 ml) extract was mixed in a 125 ml erlenmeyer flask containing 50 ml distilled water and three drops of starch solution as an indicator. iodometric titration and volumetric techniques were employed for the analysis. statistical analysis after the analysis of variance (anova) was conducted, the treatment mean was compared and separated by the honest significant difference (hsd) using the statistical tool for agricultural research (star) program, which had been developed by the international rice research institute (irri). 3. results and discussion fruit shriveling table 1 shows a gradually increasing index of bitter gourd fruit shriveling. at 3 days (d), delayed shriveling for those fruits with white and blue light treatments. consequently, shriveling was slight to moderate until 6 d of storage. endalew (2020) states that shriveled fruit reduces consumer acceptability and marketability. during the transport, the cushioning materials such as newspaper, foam nets, leaves, and other local materials then packed in cardboard boxes or plastic trays could help prevent fruits from bruising and touching, which leads to morpho­physiological disorder and increased entropy (ahmad and siddiqui, 2015; valida et al., 2018; hussein et al., 2020). table 1 ­ fruit shrivelling of bittergourd as influenced by illumination colors during storage data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test. ns= not significant. cv= coefficient of variation. treatments fruit shrivelling 2 d 3 d 4 d 5 d 6 d control (no leds) 1.00±0.00 ns 1.06±0.12 ab 1.78±0.57 ns 2.42±0.24 ns 2.72±0.05 ns white leds 1.00±0.00 ns 1.00±0.00 b 1.27±0.12 ns 2.40±0.40 ns 2.60±0.35 ns blue leds 1.00±0.00 ns 1.00±0.00 b 1.33±0.42 ns 2.40±0.40 ns 2.78±0.71 ns red leds 1.00±0.00 ns 1.21±0.03 a 1.50±0.30 ns 2.33±0.42 ns 3.13±0.23 ns cv (%) 0.00 5.56 26.28 15.51 14.74 ponteras et al. ‐ postharvest bittergourd as influenced by illumination colors 159 yellowing index bitter gourd is a climacteric fruit that ripens and turns yellow during storage due to ethylene production (yahia et al., 2019). as shown in table 2, there is a 1­10% (yi=2) yellowing after the third. fruits exposed to white leds (wl) and blue leds (bl) maintained the greenness at 3 d of storage but compared to control. as the days passed until 6 d, the yellowing progressed to 11­30% (yi=3), and the quality began deteriorating. several factors, including senescence, free radicals, energy, metal ions, and some secondary metabolites in fruits and vegetables, can cause postharvest yellowing (luo et al., 2019). diaz et al. (2006) observed that chlorophyll degradation and anthocyanin accumulation cause the yellowing of arabidopsis thaliana leaves. according to other studies, light treatment prevents some fruit postharvest problems, for example: (a) the use of red led light delays yellowing and reduces ethylene production in broccoli inflorescences (ma et al., 2014); (b) white and blue leds of 20 molm­2 s­1 controls yellowing and maintains the green color of the outer and inner leaves of brussels sprouts during storage (hasperué et al., 2016); (c) artificial lighting modulates stomata opening in green tissues keeps fruits from dehydration and delays degreening by delaying cell aging and disorganization (pintos et al., 2020); (d) continuous exposure to white and blue leds under 5 and 22°c results to have higher chlorophylls a and b which remains stable and green during storage (hasperué et al., 2016 a, b). it is in line with the findings of loi et al. (2019), which the broccoli heads increased the chlorophylls resulted from metabolic activity exposed with bl, 467 nm, 4.1 w/m and wl, 31 lm/w. visual quality rating in the selection of fruits and vegetables, visual characteristics are frequently used by consumers. visual quality loss with moderate defects after 5 d of storage was delayed by white and blue light, as shown in table 3. poor quality with serious defects was observed in those fruits without led and red led table 2 ­ yellowing index of bittergourd as influenced by illumination colors during storage data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test;ns= not significant. cv= coefficient of variation. treatments yellowing index 2 d 3 d 4 d 5 d 6 d control (no leds) 1.00±0.00 ns 1.06±0.12 ab 1.91±0.38 ns 3.14±0.43 ns 3.33±0.76 ns white leds 1.00±0.00 ns 1.00±0.00 b 1.40±0.40 ns 2.45±0.40 ns 3.01±0.52 ns blue leds 1.00±0.00 ns 1.13±0.12 ab 1.64±0.34 ns 2.75±0.43 ns 3.03±0.29 ns red leds 1.00±0.00 ns 1.28±0.10 a 1.98±0.23 ns 2.83±0.76 ns 3.50±0.50 ns cv (%) 0.00 8.64 19.70 18.86 16.91 table 3 ­ visual quality rating of bittergourd as influenced by illumination colors during storage. data represent mean ± deviation stan­ dard data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test; ns= not significant. cv= coefficient of variation. treatments visual quality rating 2 d 3 d 4 d 5 d 6 d control (no leds) 9.00±0.00 ns 7.84±0.73 ns 6.18±0.84 ns 3.85±0.13 b 3.06±0.82 ns white leds 9.00±0.00 ns 8.73±0.23 ns 6.47±0.61 ns 6.24±0.80 a 4.13±0.81 ns blue leds 9.00±0.00 ns 8.73±0.46 ns 6.53±0.50 ns 5.50±0.87 ab 3.97±0.29 ns red leds 9.00±0.00 ns 8.00±0.20 ns 5.93±0.31 ns 3.71±0.92 b 3.28±1.49 ns cv (%) 0.00 5.52 9.48 15.55 26.45 160 adv. hort. sci., 2024 38(2): 155­167 treatments, leaving them unfit for consumption (fig. 1). the deterioration of visual quality over time following harvest has been observed in various studies. however, the visual appearance and shelf life of fresh produce can be improved by postharvest lighting using leds. the effect of white­blue leds on the outer and inner leaves of brussels sprouts during a 10 d storage period at 22°c was investigated. lower respiration rates and better visual quality were found than in the controls (poonia et al., 2022). the levels of vitamin c (35 mg 100 g fw­1) and soluble carbohydrates (8.3 mg g fw­1 sucrose on the fourth day) in fresh­cut lettuce were increased by postharvest lighting. on the initial day, light at 50 % falls to 15 mg 100 g fw­1 vitamin c and 0.2 mg g fw­1 sucrose, respectively (witkowska, 2013). the observed improved visual appearance and, eventually, longer shelf life can be attributed to this (bantis et al., 2018). as demonstrated by freshly cut leaves amaranth, photosynthesis is unsustainable with insufficient light intensity (jin et al., 2021). as a signal driving this process, the conversion of sugars via gluconeogenesis is driven by low irradiance, producing glucose through the tca cycle (wolter and seifu, 2015). fresh­cut celery had a higher total dissolved solids than dark­stored celery (zhan et al., 2013). it indicates that photosynthesis continued functioning, allowing postharvest crops to remain viable. because stored organic acids such as malic and citric are undissociated from the vacuoles, preservation of titratable acidity is a good indicator of fruit quality, reflecting shelf­life (utama et al., 2022). furthermore, the activity of polyphenol oxidase, responsible for cell integrity breakdown, can be inhibited by ascorbate, making it a universal antioxidant that can help preserve the freshness of fruits and vegetables (toivonen and brummell, 2008). weight loss (%) in table 4, weight loss (%) is shown in increasing order. the acceptable limit (1­11%) for bitter gourd is observed in the present findings; if it exceeds, it leads to severe fruit shriveling and undesirability (lualhati and del carmen, 2018). as a result, significant weight loss after three days of storage is prevented in bitter fig. 1 ­ a visual quality rating (vqr) of bitter gourd fruit ‘jadeite’ cultivar at 5th day of storage. (a) t1­ control (no leds), the fruits were poor with serious defects and limit edibili­ ty; (b) t2­ white leds, the fruits were good with minor defects; (c) t3­ blue leds, the fruits were fair with mod­ erate defects and limit marketability; (d) t4­ red l e d s , the fruits were poor with serious defects and limit edibili­ ty. table 4 ­ weight loss (%) and dry matter content (%) of bittergourd as influenced by illumination colors during storage data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test; ns= not significant. cv= coefficient of variation. treatments weight loss (%) dry matter content (%)3 d 4 d 5 d 6 d control (no leds) 1.95±0.23 ab 4.48±0.68 ns 7.42±1.23 ns 11.41±1.42 ns 20.87±0.64 b white leds 1.58±0.12 b 4.04±0.19 ns 6.52±0.08 ns 9.99±0.17 ns 20.67±0.31 b blue leds 2.15±0.20 a 4.64±0.39 ns 7.46±0.78 ns 11.26±1.11 ns 21.40±1.31 b red leds 1.88±0.12 ab 4.83±0.49 ns 7.79±0.80 ns 12.17±0.77 ns 20.47±0.46 b initial data ­ ­ ­ ­ 26.00±0.00 a cv (%) 9.08 10.44 11.39 8.77 3.19 ponteras et al. ‐ postharvest bittergourd as influenced by illumination colors 161 gourd treated with white and red light. the findings are consistent with li’s (2016) study on strawberries, which found that fruits illuminated with blue light lost more weight than fruits in white and dark treatments due to increased calyx transpiration under blue light. bitter gourd wilts and shrivels due to weight loss, which lowers its market value and consumer acceptability (prajapati, 2021 a). furthermore, shelf life is extended by slowing the water absorption rate, as with pepper (wills et al., 1998). weight loss in fresh produce is caused by water loss due to transpiration and respiration (zhu et al., 2008). throughout the storage period, the percentage of weight loss increases. typically, weight loss during fruit storage results from the respiratory process, humidity transfer, and some oxidation processes (ayranci and tunc, 2003). quality parameters were preserved, and storage was prolonged from 15 to 20 d by treating bitter gourd fruits with 100 mmol l­1 /mm calcium lactate (cl) at 10°c and 85­95 % rh, 1­mcp, edible coating with carnauba wax (1.0 %), uv­c led 40 min, modified atmospheric packaging using ldpe (100 microns) (prajapati et al., 2021 b). the enhanced membrane integrity was due to calcium stabilizing the cell membrane and turgor pressure. in uv­c led exposure, the epicuticular wax morphology changed by reducing microcrystalline structure, which gave a better protective layer and thus reduced weight loss (prajapati et al., 2021 a). dry matter content (%) the data presented in table 4 clearly demonstrate an inverse relationship between the moisture content and dry matter content of eggplant, both during storage and after harvest. the high respiration rate of eggplant fruit results in high water loss and low dry matter content, which can lead to shrivelling and reduced consumer acceptability, unless proper postharvest treatments are applied. in this regard, fruits treated with 1% alginate and lacking an edible coating showed a lower dry matter content on the initial day, decreasing from 8.4% to 7.5%. despite the positive results reported by huang et al. (2017) on the dry biomass of oyster mushrooms under blue led light, our study found that the dry matter content was unaffected by this type of light. ripening is a complex process that involves the accumulation of various metabolites, such as carbohydrates, sugars, vitamins, and other compounds, which contribute to the dry matter content and the conversion into other products. the fresh­cut celery treated with leds revealed a higher dry matter content than dark­stored celery, due to the higher levels of total soluble solids, ascorbic acid, and chlorophylls, as reported by zhan et al. (2013) and florkowski et al. (2014). finally, the results are consistent with those of bantis et al. (2018), who found that high levels of blue led light exposure can increase the dry matter content and enhance radical scavengers and soluble sugars in mushrooms, thanks to the higher metabolic activity in synthesizing other products as a result of respiration. pigmental composition (mg g‐1) bittergourd fruits before treatment contained chlorophyll a (6.57 mg g­1), b (3.61 mg g­1), and total (10.19 mg g­1), respectively. table 5 shows that the fruits at 5 d storage had maintaining levels of pigments but were degrading. fruits exposed to data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test; ns= not significant. cv= coefficient of variation. table 5 ­ pigmental composition (mg g­1) and shelf­life (d) of bittergourd as influenced by illumination colors during storage treatments chlorophyll and carotenoid contents (mg g­1) shelf­life (d) chl a chl b t chl t carns control (no leds) 3.06±0.36 b 1.58±0.35 b 4.65±0.71 b 1.80±0.34 ns 4.67±0.58 ns white leds 5.66±2.01 ab 2.64±1.08 ab 8.30±3.07 ab 2.53±0.58 ns 5.00±0.10 ns blue leds 4.91±1.21 ab 2.34±0.55 ab 7.25±1.75 ab 1.95±0.51 ns 5.33±0.58 ns red leds 4.56±0.85 ab 2.20±0.27 ab 6.76±1.16 ab 1.99±0.24 ns 4.67±0.58 ns initial data 6.57±0.00 a 3.61±0.00 a 10.19±0.00 a 2.48±0.00 ns ­ cv (%) 22.73 23.28 22.71 18.15 14.38 adv. hort. sci., 2024 38(2): 155­167 162 white, blue, and red leds had better pigmental composition than those exposed to control. between the two pigments, chlorophyll a contained higher levels than chlorophyll b, resulting in reduced amounts during storage. it agrees with prajapati et al. (2021 a), who found that total chlorophyll decreased with storage treatments. this event is considered a tug­of­war between two pigments during the yellowing process. high chlorophyll is sought after by consumers. the chlorophyll content decreased when the broccoli started yellowing (loi et al., 2019). in contrast, there were carotenoid and chlorophyll syntheses in brussels sprouts under low light intensity using wb led for 10 d. however, photooxidation will destroy the carotenoid at higher light intensity (hasperué et al., 2016). the delayed decline of carotenoid contents was due to exposure to white led at 1.4 w m−2 for 8 d (nassarawa et al., 2020). the carotenoid primary function is to act as an accessory light­harvesting system, which aids in light absorption. it is considered to be photoprotective by inhibiting free radicals in chloroplasts (jones, 2018; salas et al., 2019). it acts as an antioxidant by donating hydrogen to neutralize singlet oxygen (gorni et al., 2021). the phytochrome family perceives red light between 600 and 750 nm. at the same time, the blue light (320­500 nm) spectrum includes cryptochromes and phototropins under the zeitlupe/adagio family. similarly, the stability of cryptochrome circadian regulator 1 (cry1) depends on the il lumination, but the cryptochrome circadian regulator 2 (cry2) receptor breaks after light exposure (jones, 2018). although red leds (rl) did not induce chlorophyll accumulation, when combined with blue leds (bl), the chlorophyll content of a non­heading chinese cabbage increased (fan et al., 2013; bantis et al. 2018). led illumination improved crop pigmental composition while lowering reactive oxygen species activity (kong et al., 2020; loi et al., 2020). the green pigments found in fresh vegetables and fruits originated from chlorophyll in the chloroplast, which would then destroy psii by decreasing the chlorophyll contents during yellowing (luo et al. , 2019). both chl a and chl b have collaborated to widen the light spectrum associated with photoreceptors to harvest light emissions (salas et al., 2019). before yellowing, nyc1 levels increased, but after 5 d of storage, they decreased (luo et al., 2019). the intensity of rl at 50 molm­2 s­1 increased the carotenoid content of satsuma mandarin after 6 d of storage. at the same time, bl did not improve carotenoid accumulation (ma et al., 2014; bantis et al., 2018). shelf‐life it was found that the use of different illuminations did not have a significant impact on the shelf life of bitter gourd, as per the study that was discussed (table 5). regardless of whether leds were used or not, the fruits were only lasting for around 4­5 d during storage (salas et al., 2015). however, it was suggested that future research could explore the possibility of increasing led duration and lowering temperatures in order to prolong the shelf life of bitter gourd. it was noted that fruits are generally highly perishable when stored at room temperature, which can result in significant postharvest losses (pott et al., 2020). as such, extending the shelf life of such crops can be considered a significant breakthrough in research. bitter gourd, in particular, degrades quite rapidly due to various factors, such as the presence of protruding ridges, excessive seed development, tissue softening, yellowing, and ripening, which can make it challenging to market (prajapati et al., 2021 b). previous studies have shown that leds can have a positive impact on the metabolites and postharvest life of various crops, including bitter gourd. for example, it was found that the shelf life of crops like m. charantia (prajapati et al., 2021 a), brassica oleracea l. var. italica (loi et al., 2019), and broccoli (hasperue et al., 2016; pintos et al., 2020) was prolonged significantly when exposed to certain types of leds. it was found that the study did not yield significant results on the shelf­life of bitter gourd. however, as argued by d’souza et al. (2015), the shelf­life and quality of horticultural products can be influenced by postharvest light through the potential increase of soluble carbohydrates, which serve as the substrate for respiration during postharvest storage, and through the enhancement or preservation of visual appeal through pigment accumulation, such as lycopene, carotenoids, and anthocyanins. significant potential has been demonstrated by led technology for promoting the growth and synthesis of beneficial compounds and extending the shelf­life of fruits and vegetables during postharvest storage, as reported by loi et al. (2020). it was observed that fruits did not experience any prevention from shrivelling, ponteras et al. ‐ postharvest bittergourd as influenced by illumination colors 163 yellowing, and weight loss when exposed to blue and red led. additionally, there was no influence on visual quality with red led on the fifth day. however, an increased content of total chlorophyll, vitamin c, ph, and total phenolics in cabbage was observed when exposed to continuous lights using white, blue, green, and red leds. green tomatoes’ delayed ripening and softening resulted from exposure to blue and red leds for 21 d, while an increase in contents of sugars, chlorophyll, and carotenoids in broccoli at 5°c and 22°c was observed when exposed to white and blue led with a fluence rate of 20 w m−2 (reviewed by nassarawa et al., 2020). chemical parameters fruit cell walls containing polysaccharides solubilized and hydrolyzed into simple sugars, which gives rise to the soluble solids (ayu et al., 2020; mirshekari et al., 2020; suriati et al., 2022) and are affected by room temperature (mutua et al., 2021). however, the decrease in soluble solids resulted from respiration during fruit storage (de paula et al., 2020). the enhanced shelf­life of fruits indicates slow utilization of soluble solids (nassarawa et al., 2020). one of the critical metabolites is vitamin c, an antioxidant against free radicals and a nutrient source in fruits and vegetables (loi et al., 2019; mirshekari et al., 2020). as a precursor, glucose synthesis is necessary for synthesizing vitamin c via the l­ galactose pathway and d­galacturonic acid (fernandez and de guzman, 2022). using organic acids in respiration increases ph over storage (gonzales and benitez, 2019). the increase in ph values was concomitant with a reduction in vitamin c and ta, which concurs with salas et al. (2020). cucumber’s shelf life has been extended due to the preservation of ta over time (zapata et al. 2008, as cited by gonzales and benitez 2019). based on the study results, the electrical conductivity (ec) increased with storage time. conforming to the salas et al. (2020) study, eggplant fruits with a high amount of electrolytes deteriorate quickly. this is due to the gradual breakdown of cell membrane integrity mediated by lower lipoxygenase (cai et al., 2006), resulting in ion imbalance and electrolyte leakage as well as excessive shriveling and softening (tesfay and magwaza, 2017; cheema et al., 2018). table 6 shows significant results in bitter gourd chemical characteristics such as ph and ta as influenced by the varying illumination. however, the result of this study yielded insignificance in terms of ec, tds, tss, and vitamin c. it agrees with the findings of d’souza et al. (2015) that the white led (500­700 nm) imposed did not influence the ascorbate production. it can deduced that light treatments with 1.5 w in 4 h have insufficient doses to bring significant improvement in the said metabolite. exposure to continuous white and blue leds resulted in a slight increase in ascorbic acid (asc) content at the end of broccoli storage (hasperué et al., 2016). sweet peppers with blue light at 450 nm in 8 h per day had minimum changes in ascorbic acid content (thilini deepashika perera et al., 2022). blue led at a lower intensity, 20 μmol m−2 s−1 under 5°c, decreased the asc content of broccoli while maintaining the levels at a higher intensity, 50 μmol m−2 s−1 (loi et al., 2019). red, blue, and green lights at 20, 40, and 60 w m−2 increased the tss of table 6 ­ chemical parameters of bittergourd as influenced by illumination colors during storage. data represent mean ± deviation stan­ dard data represent mean ± deviation standard. mean values (n = 5) from three replicates in each column of sampling times followed by different letters are significantly different from each other at 5% tukey’s test; ns= not significant. cv= coefficient of variation. treatments chemical parameters ph value ec ms tds ppm tss °brix ta % vit. c mg 100 g­1 control (no leds) 7.83±0.06 a 3.12±0.25 ns 2153.33±142.24 ns 1.83±0.14 ns 0.49±0.11 b 0.14±0.02 ns white leds 7.73±0.12 a 3.26±0.55 ns 2233.33±390.68 ns 1.92±0.14 ns 0.38±0.08 b 0.12±0.04 ns blue leds 7.16±0.12 b 3.05±0.18 ns 2086.67±128.97 ns 1.83±0.14 ns 0.47±0.14 b 0.18±0.03 ns red leds 7.16±0.06 b 2.94±0.53 ns 2013.33±355.29 ns 1.75±0.25 ns 0.47±0.07 b 0.19±0.05 ns initial data 4.60±0.00 c 2.40±0.00 ns 1640.00±0.00 ns 2.00±0.00 ns 0.86±0.00 a 0.18±0.00 ns cv (%) 1.18 12.49 12.41 8.47 17.34 19.98 adv. hort. sci., 2024 38(2): 155­167 164 fruits (nassarawa et al., 2020). a single application of rl to harvested vegetables increased the sugar, soluble protein, and vitamin c content (loi et al., 2020; poonia et al., 2022). bitter gourd has a low tss (°brix) compared to other fruits with high sugar content, such as pitaya, which contains several monosaccharides that contribute to sweetness. in addition, the sugar content of the skin of grape berries was enhanced after red and blue light exposure at 50 μmol m−2 s−1 (d’souza et al., 2015). cell wall breakdown and postharvest decay were delayed by blue light emission within 2 h at 25°c, which decreased monosaccharides such as glucuronic acid in pitaya fruits (pott et al., 2020). 4. conclusions the current study aimed to investigate the effects of white, blue and red light emitting diodes (leds) on the pigment composition, physical and chemical parameters of bittergourd (momordica charantia l.). irradiated and non­irradiated fruit samples were at ambient temperature with an 8 h continuous light source from the room ceiling. based on the present findings, the white and blue leds delayed fruit shrivelling and yellowing on the third day and maintained an excellent visual appearance on the fifth day compared to the control and red leds. white leds had lower weight loss, followed by red and no leds on the third day. among the treatments, the dry matter content decreased from the initial day. the white, blue, and red leds did not prevent fruits from yellowing, but there was a slight change compared to the control with the lowest amount of total chlorophyll a and b, whereas the carotenoids remained stable. regardless of treatments, the ph values (neutral) increased from the initial day (acidic). however, the blue and red leds had lower ph values compared to white and control. irrespective of treatments, the ec, tds, and vitamin c remained stable during storage. in contrast, ta lowered from the initial day. white and blue reached a shelf­life of 5 d, whereas red led and control until four days, numerically. generally, leds with 1.5 w had the potential to improve the quality and shelf­life of bitter gourd fruits. among the treatments, the white led positively affected the physical and chemical parameters. increasing the wattages or light intensities under continuous or intermittent lighting with lower temperatures makes it possible to achieve preserved fruit quality beyond five days of shelf­life. based on the results, it would suggests potentiality of leds for the future studies, which can be a useful information towards preserving the physico­chemical attributes of bittergourd and other postharvest crops. acknowledgements the authors would like to acknowledge the support of the commission of higher education (ched) region viii, tacloban city, leyte, for a scholarship and the department of horticulture and office of the graduate school of the visayas state university, visca, baybay city, leyte, philippines, for the laboratory tools. references ahmad m.s., siddiqui m.w., 2015 ­ factors affecting postharvest quality of fresh fruits, 7­32. ­ in: ahmad m.s., and m.w. siddiqui postharvest quality assurance of fruits. 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characterized by the hatch-slack pathway (c 4 ), evolved a rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) type that more efficiently utilizes high co 2 at high temperature, and have an optimum growth temperature range of 27 to 35°c, which is approximately 10°c higher than in c 3 plants (leegood, 1993). c 4 grasses are best adapted to the transition, warm-arid, and warm-humid climatic zones and usually have the ability to acquire thermotolerance by exposure to acute heat stresses (dipaola and beard, 1992). plants compensate for their sessile nature by developing specific responses to abiotic conditions. exposure to sub-lethal temperatures results in changes in physiological, biochemical, metabolic, and molecular processes, developing an active regulatory mechanism for maintaining cellular homeostasis, as well as enhanced tolerance (kislyuk et al., 2004, 2007; guy et al., 2008; kumar et al., 2013). the severity of damage to cellular and subcellular structures mainly depends on the intensity, duration, and rate of temperature increase. changes in protein metabolism have been correlated with the thermotolerance mechanism. in particular, transcription and translation of specific heat shock proteins (hsps) are induced or enhanced when plants are exposed to supraoptimal temperature, playing a crucial role in adaptive mechanism. most of the hsps are molecular chaperones, functioning by binding and stabilizing proteins at intermediate stages of folding, assembly, degradation, and translocation across membranes (xu et al., 2011). furthermore, carbohydrate metabolism was found to be affected by heat shock on arabidopsis, with an accumulation of maltose, sucrose, galactinol, myo-inositol, raffinose and monosaccharide cell-wall precursors (rizhsky et al., 2004). induction of the triose phosphate and starch hydrolytic pathways of carbohydrate metabolism provides precursors, leading to raffinose biosynthesis and accumulation of galactinol and raffinose. soluble sugars are known osmolytes that are beneficial during heat stress conditions, providing protection of cell membranes during stress exposure (diamant et al., 2001). bermudagrass (cynodon spp. rich.) and zoysiagrass (zoysia spp. willd.) provide excellent surfaces for functional, recreational, and ornamental areas, including golf course fairways, ornamental lawns and parks. the ability of these perennial, warm-season grasses to tolerate heat physiological responses of c4 grasses to prolonged heat stress a. pompeiano(1)*, m. volterrani**, and l. guglielminetti** * laboratory of plant physiology, center of agricultural sciences, federal university of alagoas, maceio, br 104, km 85, s/n, rio largo, al 57100-000, brazil. ** department of agriculture, food and environment, university of pisa, italy. key words: cynodon, heat shock proteins, total soluble sugars, zoysia. abstract: c4 grasses are best adapted to the transition, warm-arid, and warm-humid climatic zones and have the ability to acquire thermotolerance by exposure to acute heat stress. exposure to sub-lethal temperatures results in changes in physiological, biochemical, metabolic, and molecular processes. the response of two warm-season grasses to prolonged heat stress was investigated. plants of hybrid bermudagrass (cynodon dactylon × c. transvaalensis ‘tifway’) and japanese lawn grass (zoysia japonica steud. ‘meyer’) were exposed for 168 h to supraoptimal temperature conditions (47°c) in controlled-environment chamber. compared with zoysiagrass, bermudagrass showed greater damage. metabolite profi les were affected by prolonged heat exposure, with signifi cant differences between these species. consistent differences were found in total soluble sugars accumulation over the study period and severity of plant organ senescence. bermudagrass roots were more affected, as compared to leaves. leaf proteins expression determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed an early degradation in zoysiagrass, as thermal exposure proceeded. a signifi cant net decline in protein content was observed after 48 h of exposure, while in bermudagrass an analogous decline was not detected until 96 h of treatment. although heat stress is not considered a detrimental factor to c4 grass species, the two species showed signifi cant differences in their physiological response to continuous high temperatures. adv. hort. sci., 2013 27(3): 127-132 1 corresponding author: onaiepmop@gmail.com received for publication 2 may 2013 accepted for publication 17 september 2013 128 stress is reported to be excellent compared to cool-season grasses (beard, 1973). in an investigation, conducted to determine the relative effects of drought and heat on coolseason tall fescue (festuca arundinacea schreb.) and manilagrass [zoysia matrella (l.) merr.], the superior heat and drought tolerance recorded for zoysiagrass has been associated with its ability to maintain photochemical activity and cellular membrane stability (du et al., 2008). this characteristic was also associated with maintenance of more active antioxidant enzymes and lower membrane lipid peroxidation (du et al., 2009). additionally, comparing the ability of three warm-season turfgrass species to mitigate heat accumulation during a prolonged 60-day summer drought, zoysiagrass increased rates of leaf damage and maintained significantly higher canopy temperatures during drought conditions, as compared to bermudagrass and st. augustinegrass (stenotophrum secundatum (walt.) kuntze), suggesting these latter species have an enhanced heat dissipation through greater evapotranspiration (steinke et al., 2009). amongst the species, few significant differences were observed during cultivar comparisons, although manilagrass tended to accumulate and retain heat more quickly than cultivars of japanese lawn grass (zoysia japonica steud.). the differential heat tolerance after prolonged stress, exhibited by bermudagrass as compared to poa pratensis (l.), was attributed to a higher accumulation of organic acids, amino acids, soluble sugars, and inositol (du et al., 2011). differences in photosynthetic response in c 4 plants do exist. rubisco in cynodon dactylon constantly exhibited a higher catalytic turnover rate at 16, 28, and 40°c than in zoysia japonica, with analogous activation energy of 50.1-51.2 kj mol-1 (sage, 2002). however, it exceeded the photosynthetic capacity above 25°c, and was not a limiting factor at warm temperature. limited work has been done to examine and compare physiological responses in protein induction and degradation, as well as carbohydrate metabolism under heat stress in c 4 grasses. the objectives of the present research were (i) to evaluate the heat tolerance of bermudagrass and zoysiagrass standard cultivars using controlled environment and heating procedures, and (ii) to determine differences in stress responsive metabolite accumulation in c 4 standard cultivars to shortand longterm heat stress. 2. materials and methods experimental conditions the research was carried out at the department of agriculture, food and environment, university of pisa, italy. on 22 february 2011, plants of cynodon dactylon × c. transvaalensis burtt. davy cv. ‘tifway’ and zoysia japonica steud. cv. ‘meyer’, selected as standard cultivars, were collected and clonally propagated as phytomers (1to 2-cm segments of stolon obtained from mature plants, containing root tissue, crown, and shoot material) into a sphagnum moss peat-based growing medium, mixed with volcanic sand, into 160-hole seed trays, with a cell volume of 5 cm3. plants were established at 23°c (±4°c) day/night temperatures for 32 weeks in a greenhouse. during the active growing season a mineral solution (8n-7p-19.9 k + 4 ca-2 mg) at 1.3 g l-1 was supplied three times per week. the fertilization program was periodically adjusted according to the physiological age and state of the grass. irrigation was applied as needed to prevent wilting and plant material was maintained at a cutting height of 2.5 cm throughout the entire pre-treatment phase. mature plants were then acclimated in a growth chamber for four weeks before treatments were applied, and maintained at 22±1°c with a 12-h photoperiod and a light intensity of 90 μmol m-2s-1. all treatments were performed in parallel. the control was maintained in a growth chamber, and stress was applied by exposing plants to 47±1°c with 45% relative humidity and 12-h photoperiod of 90 μmol m−2s−1 photosynthetically active radiation. temperature was monitored with a datalogger (campbell scientific, logan, ut, usa), and plants exposed to heat stress were subirrigated daily to avoid drought stress. for each species, five plants were removed from the heat chamber at target times (set at 6 h, 24 h, 48 h, 96 h, and 168 h), and finally transferred again to the growth chamber for assessment of vitality after three weeks. biometric response to heat stress was evaluated as fresh weight (fw) (expressed in percentage compared to t0= 100%) regrowth in the growth chamber for three weeks. analysis of carbohydrates samples (0.5 g fw) were ground to a powder and extracted as described by tobias et al. (1992) in order to quantify glucose, fructose, and sucrose (total soluble sugars, total soluble carbohydrates). samples were assayed by coupled enzymatic assay methods (guglielminetti et al., 1999) measuring the increase in a 340 . the accuracy of the method was tested using standards with known amounts of carbohydrates. incubations of samples and standards were carried out at 37°c for 30 min. the reaction mixtures (1 ml) were as follows. glucose: 150 mm tris-hcl, ph 7.6, 3 mm mgcl 2 , 2 mm atp, 0.6 mm nadp, 1 unit glc6p dehydrogenase, 1 unit hexokinase; fructose was assayed as described for glucose plus the addition of 2 units of phosphoglucose isomerase; the increase in a 340 was recorded. sucrose was first broken down using 85 units of invertase (in 50 mm na-acetate, ph 4.6) and the resulting glucose was assayed as described above. recovery experiments evaluated losses taking place during the extraction procedures. two tests were done for each metabolite by adding a known amount of authentic standards to the samples prior to the extraction. the concentration of the added standards were similar to those estimated to be present in the tissues in preliminary experiments. the percentage of recovery ranged between 94 and 107% depending on the sugar. data were corrected on the basis of the recovery percentages obtained for 129 each sample, and expressed as μmoles hexoses equivalent g-1 fw. analysis of pigments pigments were extracted by incubating tissues (50-100 mg fw) in 1.5 ml 80% acetone for one week at 4°c in darkness. the absorbance of extracts was measured spectrophotometrically at 470.0, 663.2, and 646.8 nm. these absorbance values were used for calculation of chlorophyll a, chlorophyll b, and carotenoids contents in accordance with pompeiano et al. (2013). protein extraction and separation leaves were collected at the experimental target times previously reported and extracted in 50 mm tris-hcl buffer (ph 7.6 containing 10 mm dtt and 10% glycerol). protein quantification was performed according to guglielminetti et al. (1997). equal amounts of protein (2 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on 12.5% polyacrylamide gels followed by conventional silver nitrate staining. statistical analysis the experiment was replicated for a total of four experimental replications. the statistical analyses of biometric and growth traits were performed using one-way analysis of variance (anova) to determine whether significant differences among cultivars and groups existed. when significant differences were found, the means were compared using the least significant difference (lsd) test. significant differences for all statistical tests were evaluated at the level of p= 0.05. all computations were performed with r 2.15.0 (r development core team, 2012) and r package agricolae (de mendiburu, 2012). for the photosynthetic pigments and soluble carbohydrates data, the student-newman-keuls (snk) test was used for a posteriori multiple comparison of means. 3. results heat tolerance both species exhibited an increasing susceptibility, expressed as percentage of fresh weight canopy regrowth relative to the control, following prolonged exposure to heat stress (fig. 1). no significant difference between the two species was detected after 6 h of treatment. bermudagrass showed significant (p< 0.001) higher resistance to heat stress than zoysiagrass at all target times after the first 6 h of imposed stress. moreover, our data showed that, after 6 and 24 h of exposure at sub-lethal temperature, recovery of ‘tifway’ was greater as compared to t0, although these differences were not significantly different vs. the control. no significant difference was observed until 96 h of heat stress for this species. a prolonged exposure to sub-lethal temperature revealed a significant decline, although canopy recovery at the last target time, 168 h, still displayed 49.8% of regrowth as compared to t0. in contrast, a rapid, sharp decline was clearly evident in ‘meyer’ after 6 h of treatment, with 36.3% of regrowth as canopy recovery. no vitality was detected following 96 h of exposure. photosynthetic pigments clear differences in photosynthetic pigments were noticed between control and heat shock-stressed plants (fig. 2). under heat stress conditions, significant (p < 0.05) changes in chlorophyll a, b and carotenoid contents were observed, although the species had different behaviors. in bermudagrass, a progressive decline was detected from the initial hours of stress, particularly evident in chlorophyll b levels. chlorophylls were completely degraded after 168 h of exposure, while the degradation of carotenoids was less severe at the end of the treatment (-64.7% compared to the control). in contrast, zoysiagrass pigments showed a sharp increase and attained a peak level at 48 h of heat exposure, resulting in a significant (p< 0.001) difference between treatments. thereby indicating a significant decline under increasing heat stress as compared with the control. in bermudagrass, changes in photosynthetic pigments did fig. 1 changes in canopy recovery after heat treatments as percentage of living fresh tissues at control time. error bars represent standard error of the mean (n=4). fig. 2 leaf photosynthetic pigments in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoysiagrass heat shock observed over time. error bars represent standard error of the mean (n=4). 130 not lead to any significant difference in the carotenoids-tochlorophylls ratio at 48 h exposure, whereas in zoysiagrass the ratio significantly (p< 0.01) increased under heat stress. soluble carbohydrates analysis of total soluble sugars data showed differential responses to heat stress in the two warm season grasses (figs. 3, 4). with few exceptions, total soluble sugars (tss) levels in the controls remained constant throughout the experiment. under control and heat stress conditions, sucrose comprised the majority of the total sugar concentration in all the tissues analyzed. in bermudagrass leaves, heat stress stimulated a significantly (p< 0.001) greater tss production in the initial 48 h as compared with the control (fig. 3). although a pronounced peak after 24 h of stress was observed (with a concentration of 49.8 μmol g-1 fw), tss declined gradually thereafter till 16.0 μmol g-1 fw, 42.2% lower than the control (fig. 3b). glucose and fructose concentrations generally remained constant throughout the investigation, and no significant differences were detected between the control and treated samples. zoysiagrass leaves reduced significantly their tss content soon after the beginning of the heat stress, with a sudden contraction (-52.4%) found at the 6 h sampling. a gradual decrease in tss was observed when treatment was prolonged; the minimum concentration of 9.8 μmol g-1 fw at 96 h (-74.7% compared to the control) was reached, and a plateau level was attained at the last target time. bermudagrass roots contained significantly (p< 0.001) higher tss than zoysiagrass under both control and heat stress conditions. overall, tss content decreased considerably in plants exposed to heat stress. the averages of all the independent observations were 64.6 and 32.7% lower than those of the control plants in bermudagrass and zoysiagrass, respectively (fig. 4). under heat conditions, sucrose levels showed a pronounced decline (-79.0% vs. the control) 6 h after the beginning of treatment. this metabolite significantly increased during the first 24 h to 36.8 μmol g-1 fw, but later decreased constantly with the treatment (fig. 4 b). significant decreases in glucose and fructose contents were recorded in bermudagrass roots exposed to stress. in ‘meyer’, tss content decreased considerably in both treatments compared to bermudagrass. moreover, levels of tss showed a sharp decline after 48 h of exposure to heat stress. protein expression during heat stress in both c 4 grasses, soluble protein expressions were significantly affected in response to heat stress. in bermudagrass leaves, protein synthesis mostly ceased after 96 h of heat shock exposure, whereas in japanese lawn grass degradation occurred after 48 h of treatment (fig. 5). however, a few bands of zoysiagrass leaf protein sdspage persisted till the end of the treatment period. degradation of a large band, corresponding to the large subunit of rubisco (about 50 kd), occurred after 48 h of heat exposure in zoysiagrass, while in bermudagrass it was no longer detectable at 96 h of heat stress. 4. discussion and conclusions in open fields usually plants are simultaneously exposed to combined drought and heat effects, two interacting abiotic stresses that limit growth and quality. since initial reports, ‘meyer’ has been identified as a heat and drought tolerant species (forbes and ferguson, 1947; dunn, 1989). in the midwest transition zone, it was observed to lose color in response to extreme midsummer heat, yet still leaving a playable surface (dunn, 1998). however, a lower dehydration rate and drought resistance were attributed to zoysiagrass when compared to cynodon spp., due to a limited root system, higher et rate, and slower rate of epicuticular wax production under stress conditions (beard and sifers, 1997). the present study indicates that zoysiagrass has a moderate tolerance to prolonged heat stress compared to bermudagrass. after the initial 6 h period of stress, zoysiagrass canopy recovery exhibited a sharp decline, while bermudagrass maintained an unaltered plant regrowth response until 96 h of exposure. fig. 3 glucose, fructose, and sucrose (as hexose equivalents) leaf contents in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoysiagrass heat shock over time. error bars represent standard error of the mean (n=4). fig. 4 glucose, fructose, and sucrose (as hexose equivalents) root contents in (a) bermudagrass control, (b) bermudagrass heat shock, (c) zoysiagrass control, and (d) zoysiagrass heat shock over time. error bars represent standard error of the mean (n=4). 131 physiological characterization of c 4 plants subjected to prolonged heat stress revealed significant differences between the species. while in bermudagrass a gradual net decline of photosynthetic pigments was observed throughout the experimental period, zoysiagrass enhanced chlorophyll synthesis during the first 48 h of heat stress, followed by a severe leaf senescence induced by a prolonged exposure. the increasing chlorophylls and carotenoids contents might reflect an adaptive physiologic response to the abiotic stress. it may be associated with a higher photosynthetic performance, as well as with a better dehydration tolerance of ‘meyer’ in comparison to ‘tifway’ (kim, 1987). moreover, under heat stress conditions, carotenoids resulted more stable than chlorophylls in both species, as shown by the chlorophyll a, b:carotenoids ratios in the treated plants, in agreement with wahid (2007) observations. changes in tss content exhibited consistent differences in the timing and severity of plant organ senescence induced by the heat treatment. compared with bermudagrass, zoysiagrass had greater damage. moreover, bermudagrass root tissues were more affected than leaves, which had a peak of soluble sugars after 24 h of exposure. previous studies showed similar results in soybean (djanaguiraman and prasad, 2010; djanaguiraman et al., 2011) and, as observed in our study, this effect occurred despite a concomitant loss of chlorophyll pigments, usually attributed to membrane damage. causes of this significant accumulation of soluble sugars are unknown, although degradation of starch (geigenberger et al., 1998) could be involved. examination of the pattern of leaf proteins subjected to sds-page showed an early degradation in zoysiagrass as thermal exposure proceeded. a significant net decline was observed after 48 h of exposure, while in bermudagrass an analogous decline in soluble protein was not detected until 96 h of treatment. overall, bermudagrass showed a greater ability to cope with high-temperature stress, as indicated by the persistence of the band, corresponding to the large subunit of rubisco. in accordance with previous reports (hashimoto et al., 1989; veerasamy et al., 2007), our results showed that chlorophyll breakdown was related to protein degradation, as a progressive decline of both occurred under prolonged stress conditions. in summary, although heat stress is not a detrimental factor for c 4 grass species, which usually respond positively to complex and simultaneous environmental conditions occurring in the field, the two species showed significant differences in their physiological response to continuous exposure to high temperature. compared to bermudagrass, zoysiagrass showed a greater susceptibility to heat stress: differences in chlorophyll breakdown, tss content and proteins expression revealed a different 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